Giao Trinh Nhap Mon CNSH - Nguyen Hoang Loc

PGS. TS.
Nguyn Hong Lc

Gio trnh
Nhp mn Cng ngh sinh hc

Nh xut bn i hc Hu
Nm 2007

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Nguyn Hong Lc

Ch bn vi tnh:
Nguyn Hong Lc

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Li ni u

Cng ngh sinh hc l ngnh khoa hc ng dng hiu bit ca con
ngi v cc h thng sng s dng cc h thng ny hoc cc thnh
phn ca chng cho cc mc ch cng nghip. y l mt ngnh mi nhn,
hin ang c c th gii quan tm do c tc pht trin nhanh chng v
ang to ra mt cuc cch mng sinh hc trong nng nghip, cng nghip
thc phm, y-dc, bo v mi trng, vt liu
T cc sn phm cng ngh ln men truyn thng n cc sn phm
ca cng ngh sinh hc hin i nh: sinh vt bin i gen, ng vt nhn
bn, nui cy t bo gc, cng ngh sinh hc nan... cho thy phm vi
nghin cu v ng dng ca cng ngh sinh hc ngy cng m rng v a
dng, hng n mt s pht trin mi l nn cng nghip cng ngh sinh
hc. iu ny cho thy cng ngh sinh hc chnh l s phi hp ca khoa
hc v cng ngh khai thc nhng kin thc v cc h thng sng cho
cc ng dng thc hnh.
nay cng ngh
trong nc v trn th gii. Gio trnh Nhp mn cng ngh sinh hc ny
cung cp nhng kin thc c bn cho sinh vin v cng ngh DNA ti t
hp, cng ngh ln men vi sinh vt, cng ngh sinh hc thc vt, cng ngh
sinh hc ng vt, cng ngh protein cng nh mt s ng dng ca chng
trong lnh vc nng nghip, y hc v mi trng.
Gio trnh ny mi c xut bn ln u tin nn kh trnh khi
thiu st hoc cha p ng c yu cu bn c. V th, chng ti mong
nhn c nhiu kin ng gp ln xut bn sau c hon thin hn.
Chng ti chn thnh cm n Qu Nng cao cht lng-D n Gio
dc i hc h tr chng ti bin son gio trnh ny, PGS. TS. L Trn
Bnh c bn tho v gp nhiu kin qu bu.

Tc gi

5

Phn I

Cc khi nim v nguyn l c bn

6
Chng 1

M u

I. nh ngha cng ngh sinh hc
1. nh ngha tng qut
C nhiu nh ngha v cch din t khc nhau v cng ngh sinh
hc ty theo tng tc gi, nhng tt c u thng nht v khi nim c bn
sau y:
Cng ngh sinh hc l qu trnh sn xut cc sn phm trn quy m
cng nghip, trong nhn t tham gia trc tip v quyt nh l cc t bo
sng (vi sinh vt, thc vt, ng vt). Mi t bo sng ca c th sinh vt
hot ng trong lnh vc sn xut ny c xem nh mt l phn ng nh.
u nhng nm 1980, bt u hnh thnh cng ngh sinh hc hin
i l lnh vc cng nghip s dng hot ng sinh hc ca cc t bo
c bin i di truyn. Cng ngh sinh hc hin i ra i cng vi s
xut hin k thut gen. C s sinh hc c p dng y bao gm sinh
hc phn t, sinh hc t bo, ha sinh hc, di truyn hc, vi sinh vt hc,
min dch hc, cng cc nguyn l k thut my tnh...
C hai cch nh ngha cng ngh sinh hc mt cch tng qut nht:
- Do UNESCO (1985) nh ngha: Cng ngh sinh hc l cng ngh
s dng mt b phn hay t bo ring r ca c th sinh vt vo vic khai
thc sn phm ca chng.
- Do Trng Lut Stanford (1995) nh ngha: Cng ngh sinh hc l
cng ngh chuyn mt hay nhiu gen vo sinh vt ch nhm mc ch khai
thc sn phm v chc nng ca gen .
S khc bit r rt nht ca hai nh ngha trn thuc v i tng tc
ng ca cng ngh sinh hc: UNESCO xem c quan, b phn, t bo v
chc nng ring r ca sinh vt l i tng, trong khi Trng Lut
Stanford li coi gen l i tng tc ng ca cng ngh.
T cc nh ngha trn, c th phn bit c hai nhm cng ngh
sinh hc l:

7
1.1. Cng ngh sinh hc truyn thng (traditional biotechnology)
Bao gm:
+ Thc phm ln men truyn thng (food of traditional fermentations)
+ Cng ngh ln men vi sinh vt (microbial fermentation technology)
+ Sn xut phn bn v thuc tr su vi sinh vt (production of
microbial fertilizer and pesticide)
+ Sn xut sinh khi giu protein (protein-rich biomass production)
+ Nhn ging v tnh bng nui cy m v t bo thc vt (plant
micropropagation)
+ Th tinh nhn to (in vitro fertilization)

1.2. Cng ngh sinh hc hin i (modern biotechnology)
Bao gm:
+ Nghin cu genome (genomics)
+ Nghin cu proteome (proteomics)
+ Thc vt v ng vt chuyn gen (transgenic animal and plant)
+ ng vt nhn bn (animal cloning)
+ Chip DNA (DNA chip)
+ Liu php t bo v gen (gene and cell therapy)
+ Protein bit dc (therapeutic protein)
+ Tin sinh hc (bioinformatics)
+ Cng ngh sinh hc nano (nanobiotechnology)
+ Hot cht sinh hc (bioactive compounds)

2. Ni dung khoa hc ca cng ngh sinh hc
Cng ngh sinh hc cng c th c phn loi theo cc kiu khc
nhau. Xt v gc cc tc nhn sinh hc tham gia vo qu trnh cng ngh
sinh hc, c th chia thnh cc nhm sau:
- Cng ngh sinh hc thc vt (plant biotechnology)
- Cng ngh sinh hc ng vt (animal biotechnology)
- Cng ngh sinh hc vi sinh vt (microbial biotechnology)
8
- Cng ngh sinh hc enzyme hay cng ngh enzyme (enzyme
biotechnology)
Gn y, i vi cc nhn t sinh hc di t bo cn hnh thnh khi
nim cng ngh protein (protein engineering) v cng ngh gen (gene
engineering). Cng ngh protein v cng ngh gen xuyn sut v tr thnh
cng ngh cha kha nm trong cng ngh sinh hc thc vt, cng ngh sinh
hc ng vt v cng ngh sinh hc vi sinh vt. Nh k thut c trnh t
gen v k thut DNA ti t hp, cng ngh gen t c nhng thnh tu
ht sc to ln mang tnh quyt nh, m ra nhng giai on pht trin mi.
l nghin cu v ton b genome ca nhiu sinh vt, ng ch l vic
gii m genome ca con ngi v ca cy la. l vic hnh thnh c mt
phng hng nghin cu, ng dng v kinh doanh cc sinh vt bin i
gen (gentically modified organism-GMO) v cc thc phm bin i gen
(gentically modified food-GMF). Cng ngh protein c tim nng ng dng
rt ln trong vic sn xut ra cc protein ti t hp (recombinant protein)
dng lm dc phm iu tr cc bnh him ngho nh interferon,
interleukin, insulin...
Mt khc, ty vo i tng phc v ca cng ngh sinh hc, c th
chia ra cc lnh vc cng ngh sinh hc khc nhau nh:
- Cng ngh sinh hc nng nghip (biotechnology in agriculture)
- Cng ngh sinh hc ch bin thc phm (biotechnology in food
processing)
- Cng ngh sinh hc y dc (biotechnology in medicine-
pharmaceutics)
- Cng ngh sinh hc mi trng (environmental biotechnology)
- Cng ngh sinh hc vt liu (material biotechnology)
- Cng ngh sinh hc ha hc (biotechnology in chemical production)
- Cng ngh sinh hc nng lng (biotechnology in energy
production)...
Mt s tc gi cho rng loi ngi p dng cng ngh sinh hc t
rt lu vo cc hot ng sn xut, v d: cng ngh sn xut ung (ru,
bia...) hoc cng ngh sn xut thc phm (men bnh m, nc mm, tng,
chao...). Do , vic nh ngha v phn loi cng ngh sinh hc trong giai
on pht trin ban u c mt ngha rt quan trng c nhng chnh
9
sch u t hp l v u tin cho cng ngh sinh hc. Di y l cc lnh
vc ng dng cng ngh sinh hc hin nay ang c quan tm hng u.

3. Cc lnh vc ng dng ca cng ngh sinh hc
3.1. Cng ngh sinh hc trong nng nghip
Lnh vc nng nghip tuy khng phi l mc tiu pht trin hng u
ca cng ngh sinh hc nhiu nc cng nghip trn th gii, nhng trn
thc t nhng hot ng nghin cu v pht trin, sn xut v thng mi
ha lnh vc ny cng c nhiu tp on ln quan tm. C th nu ba
lnh vc chnh l:
- Ging cy trng v vt nui nhn v tnh v chuyn gen mang nhng
c im nng-sinh qu gi m cc phng php truyn thng khng to ra
c, ng thi li c bo v thng qua bn quyn tc gi.
- Cc ch phm sinh hc dng trong bo v cy trng vt nui, nh:
vaccine, thuc tr su bnh v phn bn vi sinh.
- Cng ngh bo qun v ch bin nng-hi sn bng cc ch phm vi
sinh v enzyme. Gi tr nng sn c nng ln nhiu ln v quy trnh cng
ngh i km trang thit b l mt dng hng ha trong kinh doanh chuyn
giao cng ngh.
Ngoi ra c th lit k thm mt s lnh vc khc:
- Cng ngh sinh hc ch bin thc phm: Cc enzyme (amylase,
rennin, -galactosidase, invertase, gluco-isomerase, pectinase), cc cht ph
gia thc phm (cc cht to ngt, hng v, to mu, bt n v lm n nh,
cc vitamin, cc amino acid, cc cht chng oxy ha, cc cht bo qun, cc
cht hot ha b mt...).
- Cc loi thc n b sung cho chn nui (khng sinh mi...).
- Cc loi thuc tr su, dit c vi tnh c hiu tng ln (cc sn
phm Bt, cc baculovirus, tuyn trng k sinh...).
- Cc hormone sinh trng thc vt (cc cytokinin...).
- Cc ha cht chn on bnh cho ng-thc vt.

10
3.2. Cng ngh sinh hc trong y dc
C l thnh tu cng ngh sinh hc c th hin r nt nht l lnh
vc y hc. Hin nay, hu ht cc sn phm quan trng sau y u c sn
xut trn c s cng ngh sinh hc, bao gm cc ng dng sau:
- Cc loi khng sinh v cc cht dit khun, cc loi vitamin v cht
b dng, cc loi amino acid v hn hp ca chng trong dch truyn, cc
loi vaccine v cc loi hormone cha bnh.
- Cc b kit chun dng trong chn on bnh v chn on ha sinh
trong y dc.
- Cy trng v vt nui c cy chuyn nhng gen sn sinh ra cc
loi protein tr liu ang l mc tiu u t ca kh nhiu cng ty y dc
hng u trn th gii hin nay.
C th l nghin cu v sn xut cc dc phm, cc khng th n
dng, interferon, cc hormone (hormone sinh trng, insulin, erythropoietin,
thrombopoietin...), cc enzyme (urokinase, heparinase, alcohol
dehydrogenase), cc protein khc (cc khng nguyn c hiu, albumin,
antithrombin, fibronectin...), cc khng sinh, thuc v vitamin mi, cc
dc phm c bn cht protein, cc loi vaccine vim gan B, C, HIV, cm,
st rt, vim no, t v cc tc nhn gy bnh tiu chy, cc kit chn on
nh: chn on s c mt HIV, virus vim gan B v C trong mu, mt s
chn on thai..., liu php gen: iu tr cc gen gy bnh di truyn.
Hin nay, cc cng ty cng ngh sinh hc y dc hng u th gii
ang tp trung vo nghin cu to ra sn phm chng li cc cn bnh
nh HIV/AIDS, cc loi bnh ung th, tiu ng, cc bnh tim mch, cc
bnh truyn nhim...

3.3. Cng ngh sinh hc cng nghip v ch bin thc phm
Cng ngh sinh hc cng nghip bao gm cc lnh vc sn xut cc
loi enzyme nh amylase, cellulase v protease dng trong cng nghip dt,
cng nghip x phng v m phm, cng nghip bnh ko, ru bia v nc
gii kht
Sau y l cc loi sn phm ca cng ngh sinh hc cng nghip:
- Cng nghip ha cht: Cc ha cht thng dng (v d: acrylamide)
u c th sn xut bng cng ngh sinh hc. Cng nghip ha hc s c
11
hiu qu hn nu dng cc cht xc tc sinh hc (enzyme), ti sinh v x l
cc dung mi bng con ng sinh hc.
- Qu trnh ch bin tinh bt: Dng cc enzyme do cng ngh sinh hc
to ra dch ha v ng ha tinh bt thnh glucose v chuyn ha thnh
fructose.
- Cng nghip lm sch: Cc cht git ty hin i uc b sung
protease v cc enzyme khc lm sch cc vt bn protein, tinh bt v cht
bo.
- Cng nghip bt g v giy: Nhu cu ca th trng v bo v mi
trng ngy cng ln i vi giy t cha cc hp cht chlorine gy
nhim. Qu trnh sn xut bt giy hin nay gy nhim rt nng. Cng
ngh sinh hc a ra gii php sinh hc sn xut bt giy khng gy
nhim bng cch s dng cc loi nm phn hy lignin-cellulose to
bt. Cc enzyme cng c dng nng cao cht lng si v cht lng
giy.
- Cng nghip khai khong v pht hin khong sn. C hai cng
ngh: lc sinh hc/oxy ha sinh hc cc kim loi, x l nhim kim loi v
ti sinh. Cng ngh lc kim loi dng cc vi sinh vt c th thu c cc
kim loi qu nh ng, km v cobalt. Cng ngh x l sinh hc nhim c
th p dng i vi cc kim loi nng.

3.4. Cng ngh sinh hc mi trng
Tuy l lnh vc kh mi nhng s pht trin v ng dng ca cng
ngh sinh hc mi trng rt ng k. Mi qu trnh x l cht thi nu
khng khp kn bng x l sinh hc th kh c th thnh cng trn vn.
Cc hot ng chnh ca cng ngh sinh hc mi trng ang c
ch trng l:
- Cng ngh phn hy sinh hc: Dng cc c th sng phn hy cc
cht thi c to nn cc cht khng c nh nc, kh CO
2
v cc vt liu
khc. Bao gm, cng ngh kch thch sinh hc: b sung cht dinh dng
kch thch s sinh trng ca cc vi sinh vt phn hy cht thi c sn trong
mi trng, cng ngh b sung vi sinh vt vo mi trng phn hy cht
nhim, cng ngh x l nhim kim loi v cc cht nhim khc bng
thc vt v nm.
12
- D phng mi trng: Pht trin cc thit b d v theo di nhim
mi trung, c bit trong vic d nc v kh thi cng nghip trc khi
gii phng ra mi trng.

II. S lc lch s hnh thnh cng ngh sinh hc
Cng ngh sinh hc pht trin cho n ngy nay, qua ba giai on
chnh:
- Cng ngh vi sinh.
- Cng ngh t bo (nui cy m v t bo ng-thc vt...).
- Cng ngh sinh hc hin i, tc cng ngh gen.
Cng c tc gi gn qu trnh pht trin nu trn vi ba cuc cch
mng sinh hc.
- Cch mng sinh hc ln th nht (u th k 20): s dng qu trnh
ln men sn xut cc sn phm nh acetone, glycerine, citric acid,
riboflavin...
- Cch mng sinh hc ln th hai (sau th chin th 2): sn xut khng
sinh, cc sn phm ln men cng nghip nh glutamic acid, cc
polysaccharide; trong c cc thnh tu v t bin, to cc chng vi sinh
vt cho nng sut v hiu qu cao, pht trin cc qu trnh ln men lin tc
v pht hin phng php mi v bt ng enzyme s dng nhiu ln...
- Cch mng sinh hc ln th ba (bt u t gia thp nin 1970): vi
cc pht hin quan trng v enzyme ct hn ch, enzyme gn, s dng
plasmid lm vector to dng, t nn mng cho mt nn cng ngh sinh hc
hon ton mi l cng ngh DNA ti t hp.
Hai giai on u, cng ngh vi sinh v cng ngh t bo, s dng
hot ng sinh hc ca cc t bo tch bit, nhng cha bin i c cu
trc di truyn ca chng, nn c xem l hai giai on ca cng ngh sinh
hc truyn thng. Phi n cuc cch mng sinh hc ln th ba nh nu
trn, th mi ra i nn cng ngh sinh hc hin i, giai on pht trin cao
nht ca cng ngh sinh hc, m ra k nguyn mi ca sinh hc.
Cng c th chia lch s hnh thnh v pht trin cng ngh sinh hc
theo cc giai on sau:

13
1. Giai on th nht
hnh thnh t rt lu trong vic s dng cc phng php ln men
vi sinh vt ch bin v bo qun thc phm, v d sn xut pho mt, dm
n, lm bnh m, nc chm, sn xut ru bia Trong , ngh nu bia c
vai tr rt ng k. Ngay t cui th k 19, Pasteur cho thy vi sinh vt
ng vai tr quyt nh trong qu trnh ln men. Kt qu nghin cu ca
Pasteur l c s cho s pht trin ca ngnh cng nghip ln men sn xut
dung mi hu c nh aceton, ethanol, butanol, isopropanol vo cui th
k 19, u th k 20.

2. Giai on th hai
Ni bt nht ca qu trnh pht trin cng ngh sinh hc trong giai
on ny l s hnh thnh nn cng nghip sn xut thuc khng sinh
penicillin, khi u gn lin vi tn tui ca Fleming, Florey v Chain
(1940). Trong thi k ny xut hin mt s ci tin v mt k thut v
thit b ln men v trng cho php tng ng k hiu sut ln men. Cc th
nghim x l cht thi bng bn hot tnh v cng ngh ln men ym kh to
biogas cha ch yu kh methane, CO
2
v to ngun phn bn hu c c gi
tr cng c tin hnh v hon thin.

3. Giai on th ba
Bt u t nhng nm 50 ca th k 20, song song vi vic hon thin
cc quy trnh cng ngh sinh hc truyn thng c t trc, mt s hng
nghin cu v pht trin cng ngh sinh hc hnh thnh v pht trin
mnh m nh mt lot nhng pht minh quan trng trong ngnh sinh hc
ni chung v sinh hc phn t ni ring. l vic ln u tin xc nh
c cu trc ca protein (insulin), xy dng m hnh cu trc xon kp ca
phn t DNA (1953). Tip th -
(Bng 1.1).

14

.
Y t Dng enzyme to cc b cm bin sinh hc trong cc thit
b phn tch y t. S dng t bo vi sinh vt, t bo ng-
thc vt trong sn xut thuc (v d: steroid) v tng hp
cc loi khng sinh mi. S dng enzyme trong cha tr
bnh.
Cng nghip
thc phm
enzyme
.
Gim st mi
trng . cht
cht ).

(citric acid, itaconic acid, acetic
acid...), sn xut .
Nng lng
.

4. Giai on th t
Bt u t nm 1973, khi nhng th nghim khi u dn n s ra
i ca k thut DNA ti t hp c thc hin v s xut hin insulin-sn
phm u tin ca n vo nm 1982, cng vi th nghim chuyn gen vo
cy trng cng thnh cng vo nm ny. n nay, cng ngh sinh hc hin
i c nhng bc tin khng l trong cc lnh vc nng nghip (ci
thin ging cy trng...), y dc (liu php gen, liu php protein, chn on
bnh...), cng nghip thc phm (ci thin cc chng vi sinh vt...)... Nhng
thnh cng ny s c trnh by chi tit hn trong Phn II-Cc ng dng
ca cng ngh sinh hc.
15
III. Mt s kha cnh v khoa hc v kinh t ca cng ngh sinh
hc hin i
Cc phng tin thng tin i chng ng ti khng t cc kin
phn i ng dng mt s thnh tu cng ngh sinh hc trong sn xut,
thm ch i vi nhng thnh tu c gii khoa hc nh gi l sng chi.
Tht vy, cng ngh sinh hc cng nh khoa hc ht nhn, bn cnh cc
ng dng to ln cho li ch v pht trin ca loi ngi, c th cn mang li
nhiu him ha khng th lng trc c hu qu. Gn y, khi cc nh
khoa hc xc nhn k thut nhn bn cu Dolly hon ton c th p dng
cho vic nhn bn con ngi, khp cc nc dy ln mt ln sng phn
i vic nhn bn ngi, c ni cm hon ton hng nghin cu ny. Sau
y chng ta s tm hiu cc him ha tim tng ca cng ngh sinh hc.

1. V khoa hc
S d dt trong s dng cc sn phm chuyn gen lm thc phm cho
ngi v gia sc do nhiu l do khc nhau, nhng tu trung c th chia
thnh hai nhm sau:
- B my di truyn ca sinh vt mang tnh hon thin rt cao v tin
ha qua hng trm triu nm, nhng gen mi c gn thm vo cho cy
trng v vt nui tng nng sut hoc cht lng nng sn, bit u c
th ph v tnh hon thin, tnh cn bng ca s sng cc sinh vt ny. V
v th, con ngi khng th yn tm vi vic hng ngy nut vo c th mt
s lng ln cc sn phm thiu tnh hon thin, cn bng hay ni cch khc
l c th c d tt.
- Cho n nay trong vic to ra cc GMO, cc gen khng khng sinh
nh kanamycin, ampicillin hoc hygromycin thng c s dng km theo
lm gen ch th chn lc. Chng tn ti trong sn phm ca cc GMO v
c th c nh hng trc tip hoc gin tip thng qua dy chuyn thc n
ca sinh quyn n con ngi. Mc d kh nng ny l v cng thp, thm
ch khi mt gen khng sinh c pht tn sang mt sinh vt khc th tc
ng ca vic ny cng khng ng k do cc gen ch th chn lc c s
dng trong sinh vt chuyn gen c ng dng rt hn ch trong th y v y
hc. Tuy nhin, lm du nhng lo lng ca x hi, cc nh nghin cu
c yu cu trnh s dng cc gen khng khng sinh trong sinh vt chuyn
gen. Vic s dng gen ch th thay th khc ang c nh gi v pht
16
trin. Hin nay, ngi ta ang tm cch thay th cc gen ch th chn lc c
bng cc gen c v t hi hn nh gen m ha protein pht hunh quang
mu xanh lc (green fluorescence protein-GFP). Gen GFP c coi l mt
gen ch th tt, v n lm cho cc GMO pht sng xanh rc r khi t di
tia t ngoi. Nhng d sao s nghi ngi vn cn, v gen GFP c ngun gc
t mt loi c Bc Bng Dng, ch khng t mt ng vt c ngun gc
gn vi ngi.

2. V kinh t
2.1. Nhng cng ty a quc gia v cng ngh sinh hc
T chc quc t nng nghip tin b RAFI (Rural Advancement
Foundation International) l mt t chc phi chnh ph Canada hot ng
nhm hn ch nh hng ca cc cng ty a quc gia v ging. Theo RAFI,
cc cng ty a quc gia v cng ngh sinh hc s hot ng rt mnh trong
th k 21, hin nay nhng cng ty ny ang pht trin nhanh chng nh
thu tm cc cng ty nh hn v trc ht nh li nhun khng l thu c
trong c quyn bn cc sn phm GMO.
Chng hn cch y hn 15 nm, cng ty Monsanto ch chuyn v cc
sn phm ha du, thuc tr su v tr c. Tuy nhin, thi gian gn y
Monsanto u t rt ln v trin khai cng ngh gen thc vt to ra
cc ging GMO v ang tr thnh cng ty ging ln nht th gii. RAFI gi
Monsanto l mt Microsoft cng ngh sinh hc v t nm 1996 n nay
Monsanto mua li nhiu cng ty trc y vn l ngi khng l trn th
trng ht ging.

2.2. S l thuc vo cc cng ty a quc gia v cng ngh sinh hc
RAFI tin on ngi nng dn hu ht cc nc trn th gii, k c
cc nc cng nghip pht trin, dn dn s b l thuc vo mt nhm nh
cc cng ty cng ngh sinh hc a quc gia.
Vi quy ch ngt ngho v quyn tc gi IPR (Intellectual Property
Right) hin hnh trong quan h kinh t th gii, ngi nng dn s b tc
b hon ton quyn t do trng cy g trn mnh t ca mnh v bn cho ai
sn phm ca mnh. L do cc cng ty nh Monsanto c c nhiu
quyn hn nh vy chnh l s tin b ca cng ngh sinh hc.
17
Chng hn, gen terminator c c quan ng k bn quyn ca M
chnh thc cp bng pht minh cho cng ty Delta Pine (3/1998). Khi chuyn
gen vo bt c mt ging cy no, ht bn ra s ch ny mm trong mt th
h duy nht. Nu ngi nng dn ly ht trng v sau, gen ny s to ra
mt hp cht git cht mm, v th ht hon ton khng ny mm c. Vi
gen terminator trong tay, cc cng ty a quc gia s bt nng dn cc nc
hng nm phi mua ht ging ca h.
Mt khc, cc cng ty ging ang thn tnh dn cc cng ty ch bin
lng thc, thc phm l u ra ca nng sn. Va c quyn ht ging
GMO li va nm cc cng ty ch bin nng sn, cc cng ty a quc gia
cng ngh sinh hc s khng cha mt li thot no cho nng dn cc nc
ang pht trin.

IV. Cc vn php l ca cng ngh sinh hc hin i
Cng ngh DNA ti t hp gip cc nh khoa hc thay i c ch
tin ha ca t nhin, sng to ra sn phm ca gen, to ra cc dng sinh vt
mi. Ngy cng c nhiu bng chng hin nhin v li ch ca cng ngh
DNA ti t hp. Tuy nhin, cng phi cn nhc n nhng nguy c tim
tng ca n, v thc t cng ny sinh mt s vn php l quan trng
buc chng ta phi xem xt li mt cch thn trng.
Chng hn, chng ta c th tham kho h thng qun l i vi cc
sn phm cy trng ca cng ngh sinh hc hin i M, ni m lnh vc
cng ngh sinh hc c u t v pht trin tt nht trn th gii.
H thng qun l ca M l mt b phn quan trng nhm m bo an
ton lng thc. Phi hp vi B Nng nghip M (USDA) v Cc Bo v
Mi trng (EPA), Cc qun l Thc phm v Dc phm (FDA) ng vai
tr qun l cc loi lng thc c ngun gc thc vt c to ra nh cng
ngh sinh hc. Theo o lut Lng thc, Dc phm v M phm
(FD&C), FDA c thm quyn bo m an ton ca tt c cc lng thc
trong nc v nhp khu cho ngi v ng vt trn th trng M. Ngoi
tr tht gia sc-gia cm v mt s sn phm trng, nhng hng mc ny
thuc phm vi iu tit ca USDA. Tuy nhin, an ton ca d lng
thuc th y trong tht gia sc, gia cm v thy sn l do FDA qun l. Thuc
tr su li ch yu do EPA iu tit. C quan Kim tra Sc kho Thc vt
v ng vt ca USDA (APHIS) c chc nng gim st an ton nng
18
nghip v an ton mi trng trong trng trt v th nghim ti hin trng
cc ging cy trng c to ra nh cng ngh sinh hc.
Cc loi lng thc v thnh phn lng thc c to ra nh cng
ngh sinh hc phi p ng nhng tiu chun an ton tng t nh cc tiu
chun m o lut FD&C p dng i vi cc cy trng c to ra theo
phng php lai ging thng thng. iu ny c ngha l cc sn phm
cng ngh sinh hc cng phi an ton ging nh cc sn phm truyn thng
trn th trng. FDA c quyn loi tr mt loi lng thc khi th trng
hoc trng pht nhng ngi bun bn loi lng thc nu n gy ra ri
ro i vi sc khe cng ng. Cn lu rng o lut FD&C quy nh
nhng ngi p dng cng ngh sinh hc phi chu trch nhim php l
nhm m bo rng nhng lng thc m h bn cho ngi tiu dng phi
an ton v p ng tt c cc yu cu v php l.

1. An ton sinh hc
1.1. S chuyn gen bng ht phn
Cho ti nay khng c ht phn ca loi cy trng bin i gen no
c hn ch kh nng pht tn. Cc phng thc qun l nh cch ly
khng gian v thi gian c th hn ch s lu chuyn gen (gene flow) gia
cy trng, hn ch ht st li trong t v cy st li sau khi thu hoch. Vic
s dng vng cch ly, ro cn cy trng v cc ro cn thc vt khc gia
ngun to v ni nhn ht phn cng c th gim mc pht tn ht phn.
Thi gian ht phn trong khng kh cng kh di, do c th pht tn
n khong cch kh xa. Tuy nhin, iu kin thi tit v mi trng thay
i c th gy ra s pht tn nhng khong cch xa hn na. Cc bin
php cch ly sinh hc ang c pht trin nhm xc nh liu s sinh sn
cy trng c th kim sot c hay khng trnh s giao lu gen qua ht
hoc ht phn.
c bit cc ging hoc dng c cy bt dc c, s xy ra hin
tng lai xa vi ging bin i gen hu th mt tn s cao hn v khong
cch xa hn so vi ging truyn thng. S tch ly gen (gene stacking)
c quan st cy trng v ngi ta d on l cy trng mang gen a
khng s tr nn ph bin sau khi cy trng chuyn gen c php a vo
th trng, v v vy cy mc hoang bin i gen s phi cn cc bin php
dit c khc.
19
Cc nghin cu cho thy phn ln s th phn cho xy ra khong
cch ngn v kh nng th phn thnh cng gim theo hm m so vi
khong cch t ngun pht ra ht phn. Nhng trn phm vi nng tri vn
c s lu chuyn gen, mc d mc xy ra rt thp mt khong cch
kh xa, v vy s tch bit hon ton v mt di truyn l rt kh duy tr.
Trong khi ht phn ng vai tr quan trng trong s pht tn theo
khng gian th ht ging ng vai tr quan trng trong s pht tn theo thi
gian. Do , khi cch ly cy trng chuyn gen vi cy trng khng chuyn
gen phi tnh n chuyn trc cy trng chuyn gen c c trng trn
cng mnh t khng v tp qun canh tc c gy ra s di chuyn cc ht
gia cc mnh rung hay khng.
Ngoi ra, s lu chuyn gen gia cy bin i gen v h hng ca n
cn ty thuc vo loi tnh trng gen chuyn quy nh, c im sinh hc
ca cy (th phn cho hoc t th phn) v bi cnh nng nghip (h thng
cy trng, t chc khng gian gia cc tha rung).

1.2. S bn vng ca DNA trong t
DNA ca cy chuyn gen c th c phng thch vo mi trng t
cc nguyn liu thc vt gi hoc mc nt. Vn ny c kho st
mt s cy chuyn gen nh thuc l (aacC1), hoa d yn (NOS-nptII) v
c ci ng (bar/TR1, TR2/nptII, 35S/BNYVV-cp). S bn vng ca cu
trc DNA trong t c pht hin bng cch tch chit DNA trc tip t
t, sau khuch i cu trc ny bng k thut PCR. Chn lc primer
thch hp cho php pht hin r rng cu trc chuyn gen bn cnh cc gen
xut hin t nhin. Vi phng php ny s hin din ca cu trc DNA c
th c pht hin nhng khng c thng tin no v s hin din ca n
trong nguyn liu thc vt mc nt, c th do DNA t do c hp th
vo b mt t. DNA ca cy c ci ng chuyn gen c pht hin trong
mu t v tr khng s dng 6, 12 v 18 thng sau khi cy c ci
ng b cy lp trong t. Ngi ta cng tm thy DNA cy thuc l
chuyn gen trong t sau hn 1 nm thu hoch. Trong khi DNA ca
hoa d yn chuyn gen ch c th pht hin vo thi im 2 thng sau khi
cy c cy lp trong t.
20
Mc d ch c mt vi kho st v s bn vng ca DNA cy chuyn
gen trong t, nhng s bn vng ca cu trc trong mt thi gian di c
th c chng minh r rng.

1.3. Chuyn gen ngang t thc vt vo vi sinh vt t
Chuyn gen ngang (horizontal gene transfer) l hin tng chuyn cc
gen hoc nguyn liu di truyn trc tip t mt c th ring bit vo mt c
th khc bng cc qu trnh tng t s gy nhim. Phn bit vi mt qu
trnh bnh thng l chuyn gen dc (vertical gene transfer)-t b m vo
con ci-xut hin trong qu trnh sinh sn. Chuyn gen ngang trong phn
ny cp n DNA ngoi lai ca cy chuyn gen hin din trong t, vi
khun pht trin kh nng nhn gen ny v cui cng, cc trnh t ny
c hp nht trong genome ca vi khun.
Nguy c ca cng ngh di truyn l lm tng tim nng ca s
chuyn gen ngang qua cc loi khng h hng. Cc c ch t bo cho php
cc gen ngoi lai xen on vo genome ca mt loi no . Cc gen khng
thuc dit c hoc khng khng sinh ca vi khun thng c s dng nh
l cc ch th chn lc i vi cy chuyn gen. V th, chuyn ngang t thc
vt vo vi sinh vt ca cc gen khng nh th thng c xem nh l mt
hiu ng tim tng khng mong mun gia cy chuyn gen v cc vi sinh
vt t.
Tuy nhin, cho n nay cha c bng chng r rng v vic chuyn
gen t thc vt vo cc vi sinh vt. Hin nay, cc nghin cu an ton sinh
hc (biosafety) v chuyn gen ngang t cy chuyn gen vo vi sinh vt (vi
khun v nm) c hai hng chnh l tm hiu c ch chuyn gen t thc vt
vo vi sinh vt v nh gi cc hu qu sinh thi ca n.
C ch ch yu ca vic chuyn gen t thc vt vo vi sinh vt l qu
trnh bin np t nhin i hi s hp th DNA t do. Vi khun t c th
bin np t nhin v hp nht DNA ngoi lai trong genome ca mnh.
chuyn gen t thc vt vo vi sinh vt iu kin ng rung, khng phi
ch c c ch cho php hp th v sao chp trong mt vt ch mi m s
chn lc vt ch biu hin mt tnh trng mi l quan trng nht. Pht
hin chuyn gen ngang c th thc hin bng cch phn tch vi khun t
sau giai on nui cy u tin.

21
1.4. Chuyn gen t thc vt vo virus
Kt qu u tin v cy chuyn gen biu hin protein v ca virus
khm thuc l (TMV) ngn chn s pht trin ca bnh xut hin trong
nm 1986. Phng thc ny sau c s dng to ra tnh khng
cho cc loi virus khc nhau, tuy nhin cc nh di truyn hc t cu hi
v s an ton ca cy trng chuyn gen ngay t nhng ngy u tin. Nguy
c r rt nht l tim nng to ra cc virus gy nhim mi bng s ti t
hp, v d: gen chuyn ca virus (viral transgene) lin kt hoc trao i cc
phn vi nucleic acid ca cc virus khc. Do v protein khng ngn c
virus xm nhp vo t bo thc vt, gen chuyn (transgene) s c tip xc
vi cc nucleic acid ca nhiu virus c mang ti thc vt bi cc vector
cn trng (insect vector).
Mt s nghin cu chng minh rng cc virus thc vt c th tn
cng mt lot cc gen virus khc nhau t cy chuyn gen. Chng hn:
- Virus gy bnh khm hoi t cy c ba l mu (red clover
necrotic mosaic virus-RCNMV) dng khim khuyt thiu gen cho php
n chuyn t t bo ny n t bo khc (v th khng gy nhim c)
ti t hp vi mt bn sao ca gen trong cy thuc l chuyn gen
Nicotiana benthamiana, v sinh sn cc virus gy nhim.
- Cy ci Brassica napus chuyn gen VI, mt nhn t hot ng dch
m, ca virus khm sp-l (cauliflower mosaic virus-CaMV), ti t hp
vi phn b sung ca virus thiu mt gen , v to ra virus gy nhim
trong 100% cy chuyn gen.
- S ti t hp gia CaMV dng hoang di v dng chuyn gen VI
c chng minh trong N. bigelovii. t nht mt trong s cc virus ti t hp
c c tnh hn dng hoang di.
- Cy N. benthamiana biu hin mt on gen protein v ca virus
CCMV (cowpea chlorotic mottle virus) ti t hp vi virus khim khuyt
thiu gen .
Nhiu kho st cho thy trong cc th nghim c CaMV tn s ti t
hp cao hn nhiu so vi cc virus khc. Trong khi CCMV ti t hp c
phc hi t 3% cy chuyn gen N. benthamiana, th CaMV ti t hp c
phc hi t 36% cy chuyn gen N. bigelovii. Ngi ta nghi ng rng s t
gy DNA si i c th xy ra trong trng hp ti t hp CaMV do thc
t l DNA chuyn gen bao gm c promoter CaMV 35S.
22
2. An ton thc phm
Cc ging cy trng chuyn gen ngy cng c pht trin nh vo
cc cng c ca cng ngh sinh hc hin i. Cng chnh v vy m nhiu
ngi bn khon rng liu cc thc phm ny c an ton bng cc loi thc
phm c c nh s dng cc phng php nng nghip truyn thng hay
khng. Vy s khc bit gia lai ging thng thng v cng ngh sinh hc
thc vt l g. Thc ra c hai u c cng mt mt mc tiu l to ra cc
ging cy trng c cht lng cao vi nhng c tnh c ci thin gip
chng pht trin tt hn v ngon hn. S khc bit l ch mc ch ny
t c bng cch no.
Lai ging truyn thng i hi s trao i hng ngn gen gia hai cy
c c tnh trng mong mun. Trong khi , nh cng ngh sinh hc
hin i, chng ta c th la chn mt c tnh mong mun v chuyn ring
n vo ht ging. S khc bit gia hai k thut ny l rt ln. Phng php
cng ngh sinh hc hp l hn, c hiu qu cao v em li kt qu rt tt.
Cc k thut s dng trong cng ngh sinh hc hin i cung cp cho
nhng nh lai to ging nhng cng c chnh xc cho php h chuyn
nhng c tnh mong mun vo cy trng. Hn th na, h c th lm iu
ny m khng b chuyn thm cc tnh trng khng mong mun vo cy nh
vn thng xy ra, nu s dng lai ging truyn thng.
Thc phm c ngun gc t cy trng chuyn gen phi tri qua nhiu
th nghim hn bt k loi thc phm no trong lch s. Trc khi c
a ra th trng, chng phi c nh gi sao cho ph hp vi cc quy
nh do mt vi t chc khoa hc quc t a ra nh T chc Y t Th gii,
T chc Nng Lng, T chc Hp tc v Pht trin Kinh t Nhng quy
nh ny nh sau:
- Cc sn phm chuyn gen cn c nh gi ging nh cc loi thc
phm khc. Cc nguy c gy ra do thc phm c ngun gc t cng ngh
sinh hc cng c bn cht ging nh cc loi thc phm thng thng.
- Cc sn phm ny s c xem xt da trn an ton, kh nng
gy d ng, c tnh v dinh dng ca chng hn l da vo phng php
v k thut sn xut.
- Bt k mt cht mi no c a thm vo thc phm thng qua
cng ngh sinh hc u phi c cho php trc khi a ra th trng,
23
cng ging vic cc loi cht ph gia mi nh cht bo qun hay mu thc
phm cn phi c cho php trc khi thng mi ha.
Mt s nhn nh trong vn an ton thc phm hin nay nh sau:
- Mc n ton ca thc phm chuyn gen t nht cng tng ng
vi cc thc phm khc bi v qu trnh nh gi an ton i vi thc phm
chuyn gen k lng hn nhiu so vi vic nh gi cc thc phm khc.
Qu trnh nh gi an ton thc phm m bo rng thc phm chuyn gen
mang li tt c cc li ch nh thc phm thng thng v khng c thm
mt tc hi no.
- Cha c bng chng no cho thy thc phm chuyn gen hin ang
c trn th trng gy ra bt c lo ngi no v sc kho con ngi hay c
bt k kha cnh no km an ton hn so vi cy trng to c nh lai
ging truyn thng.
- Mt im c trng ca k thut chuyn gen l n a vo mt hay
nhiu gen c xc nh r. iu ny gip cho vic th nghim c tnh
ca cc cy trng chuyn gen d thc hin hn so vi cc cy trng bnh
thng.

2.1. Cc cht gy d ng
Mt trong nhng mi quan tm ln nht v thc phm chuyn gen l
cht gy d ng (mt protein gy ra d ng) c th c chuyn vo thc
phm. n nay cc nh khoa hc bit rt nhiu v cc thc phm gy ra
d ng tr nh v ngi trng thnh. Khong 90% s d ng thc n l
c lin quan ti tm thc phm v nhm thc phm-ng vt c v (tm,
cua, s, hn), trng, c, sa, lc, u tng, qu hch v la m. Nhng loi
thc phm ny v rt nhiu cht gy d ng khc c xc nh rt r v
do vy kh tin rng chng c th c a vo thc phm chuyn gen.
Tuy vy, vic kim tra tnh d ng vn l mt khu quan trng trong
vic kim tra an ton trc khi mt ging cy trng c a ra lm thc
phm. Hng lot cc th nghim v cu hi phi c xem xt k quyt
nh liu thc phm ny c lm tng s d ng hay khng.
Cc cht gy d ng c nhng c tnh chung nh khng b phn hy
trong qu trnh tiu ha, c xu hng khng b phn hy trong qu trnh ch
bin thc phm, v thng c rt nhiu trong thc phm. Cho n nay,
24
khng c loi protein no c chuyn vo thc phm chuyn gen c
thng mi ha li mang nhng c tnh ni trn. Chng khng c tin s
v kh nng gy d ng hay c tnh, cng khng ging vi cc cht gy d
ng hay cc c t bit v ni chung chc nng ca chng c bit
r. Nhng protein ny c mt hm lng rt thp trong thc phm chuyn
gen, nhng nhanh chng b phn hy trong d dy v c kim tra
an ton trong cc nghin cu v thc phm cho ng vt.
Cc gen m ha thng tin di truyn c mt trong tt c cc loi thc
phm v vic n chng khng gy ra bt k nh hng xu no. Khng c
tc hi di truyn no xy ra khi tiu ha DNA c. Trn thc t, chng ta
lun nhn DNA mi khi n do n c mt tt c thc vt v ng vt.

2.2. nh gi an ton ca cc thc phm
Bt k mt sn phm chuyn gen no trc khi c a ra th trng
phi c th nghim ton din, c cc nh khoa hc v cc gim nh
vin nh gi c lp xem c an ton v dinh dng, c tnh v kh nng
gy d ng hay khng. Cc kha cnh khoa hc thc phm ny da trn
nhng quy nh ca cc t chc c thm quyn ca mi nc, bao gm: mt
hng dn s dng sn phm, thng tin chi tit v mc ch s dng sn
phm, cc thng tin v phn t, ha sinh, c tnh, dinh dng v kh nng
gy d ng. Cc cu hi in hnh c th c t ra l: (1) Cc thc phm
chuyn gen c c to ra t thc phm truyn thng c cng nhn an
ton hay khng. (2) Nng cc c t hay cht gy d ng trong thc
phm c thay i hay khng. (3) Hm lng cc cht dinh dng chnh c
thay i hay khng. (4) Cc cht mi trong thc phm chuyn gen c m
bo tnh an ton hay khng. (5) Kh nng tiu ha thc n c b thay i hay
khng. (6) Cc thc phm c c to ra nh cc quy trnh c chp
nhn hay khng.
Ngay khi cc cu hi ny v cc cu hi khc v thc phm chuyn
gen c tr li, vn cn nhiu vic phi lm trong qu trnh ph chun
trc khi thc phm chuyn gen c thng mi ha. Thc t, thc phm
chuyn gen l loi sn phm c nghin cu nhiu nht trong cc loi
c sn xut.

25
3. o c sinh hc
o c sinh hc (bioethics) l mt phm tr phc tp m cch nhn
nhn ty thuc vo c im dn tc v vn ha khc nhau. Cho nn, nhng
vn c coi l hp vi o c ni ny c th l tri o c ni
khc. Thut ng ny c l bt ngun M vo nhng nm 1970, khi cc k
thut thao tc gen (gene manipulation), cn gi l k thut di truyn hay
cng ngh DNA ti t hp, c p dng.
Phm tr o c sinh hc bao hm cch nh gi li ch v ri ro lin
quan ti s can thip ca con ngi, c bit l cng ngh mi, xem xt lm
cn i s theo ui quyn t do c nhn vi trch nhim php l. o c
sinh hc i hi phi nh gi cng ngh tht k, trong c nh gi nh
hng n x hi v c nhn.
Cng vi thi gian, vn ny ngy cng tr nn su sc. Trc
nhng xo trn do s pht trin ca di truyn hc, ngi ta t hi mnh ang
tin ti loi x hi no v s cn bng mi no trn hnh tinh s c thit
lp.
o c sinh hc khng gii hn suy ngh v mi quan h gia khoa
hc v x hi. N gn lin quan h gia con ngi vi t nhin trong tnh a
dng sinh hc ca n, k c bn cht ca chnh con ngi. Mt khc, o
c sinh hc l mt cch suy ngh v tng lai v gi tr ca chng ta. N
gip cho gii chuyn mn i thoi vi nhng ngi ra quyt nh v ngi
dn, cng quan tm n s tn ti ca x hi loi ngi.
Ngy 25/7/1978, b gi c th tinh trong ng nghim (Louise
Brown) ra i Anh. T n nay, k thut ny to ra khng bit
bao nhiu em b nh vy trn th gii, k c Vit Nam. Mc ch u tin
ca cng vic ny l hon ton lnh mnh. Trong trng hp ca Louise
Brown, ngi m b v sinh do khuyt tt vi trng nn gip b c con,
ngi ta ly t bo trng ca b th tinh trong ng nghim vi chnh tinh
trng ca chng b, ri cy hp t vo ngay t cung ca b. V mt sinh
hc v php l, em b l con ca h v iu ny cng khng t ra vn g
v o c hay vi phm mt iu lut no.
Nhng mt vn t nh v phc tp khc li c t ra nu mt ph
n khng th hoc khng mun mang thai, ngh mt ph n khc nhn
trng c th tinh ca mnh v mang h ci thai , vy a con s l ca
ai. Cho n nay, nhng nc c dch v mang thai h pht sinh nhiu
26
v kin, v ngi c thu nhiu khi ph hp ng, khng mun tr li a
con cho ngi thu na.
Mt biu hin ca ch ngha u sinh di dng mi, l ngi ta hy
vng c c nhng a con thin ti bng cch xin hoc mua tinh trng
ca cc nh bc hc c gii thng Nobel, cho th tinh vi trng ca
nhng ph n tr p v thng minh ri cy phi vo nhng ph n ny.
Nhng cch lm ny khng chc chn tuyt i do quy lut phn ly di
truyn v a con sinh ra vn c th thuc loi tm thng. Sau thnh cng
nhn bn cu Dolly, ngi ta hy vng khc phc c vn trn bng
cch nhn bn cc thin ti nh chnh t bo ca h. Nh ta bit, nhn
bn ngi l mt vn rt kh v hin nay hu nh b cm trn th gii.
V li ng nht di truyn khng c ngha l ng nht bn sc c nhn. Xt
v mt lun l v o c vic lm trn khng th chp nhn c, cn v
mt khoa hc cng kh hin thc: thin ti ch biu hin mt tui no
v nu nh cho ra thin ti theo cch ny cng kh v hnh dng v th
cht ca ngi m khc trc. Li cng kh thc hin nu thng qua mt
ph n xa l khng phi l m mnh, v h gen ca t bo cht trong trng l
cng c nh hng v s khng pht huy c nh ca chnh m mnh.
Hin nay, s pht trin nhanh chng ca cng ngh sinh hc t
cho cc y ban o c v lut php trn th gii nhng vn sau y:
- C nn cho php thay i chng trnh di truyn ca ngi hay
khng; v nu cho php th mc no, cho d vic lm ny c bin
minh l cha cc bnh di truyn.
- C nn chp nhn vic chn on trc khi sinh la chn gii
tnh ca a tr hay khng.
- C nn bt buc thc hin cc chng trnh pht hin di truyn phc
v li ch sc kho ca ngi dn hay mi c nhn nhn xt c hi da
vo cc th nghim m kt qu c th tri ngc, nh hng ti h v ngi
thn ca h (v d vic sinh ra mt a con c th c ri ro khuyt tt hay
khng).
- C nn cm liu php gen (gene therapy) nhm vo cc t bo sinh
dc hay khng. Theo Suleiman, gio s nghin cu cc vn quc t v
gim c ca y ban nghin cu chu u (i hc Princeton, M) th Nh
nc cn xc nh mc can thip vo nghin cu khoa hc qua tranh
lun cng khai nhm hp php ha hnh ng cng nh ngi dn kim
27
sot cc hnh ng ny. Tm li, nh nc cn hp tc vi cng ng khoa
hc m bo t do nghin cu v ng dng hp o c cc kt qu t
.

4. Quyn tc gi v s hu tr tu
4.1. Quyn tc gi
Mc d c rt nhiu cuc tranh lun cc din n quc t v
quyn tc gi ca cc nc c ngun gen qu him c phng Ty s
dng trong cng ngh to ging nhng n nay vn cha em li mt kt
qu tht s no. 169 nc ng k vo cng c Quc t v a dng
sinh hc (Convention on Biological Diversity) v cng c ny c hiu lc
t 12/1993, trong quy nh cng chia s quyn li gia cc nc c
ngun gen vi cc cng ty phng Ty s dng ngun gen . Tuy nhin, t
n nay cc nc c ngun gen qu him vn tip tc b mt dn ti sn
quc gia ca mnh m quyn li c chia s th khng ng k.
Chng hn, nm 1994 hng ArgEvo phn lp c gen PAT
(phosphinothricin acetyltransferase) t dng vi khun Streptomyces
viridochromogens c trong mu t ly t Camerun. Gen PAT cho php to
ra cc ging cy trng khng thuc dit c nhm glufosinate, ng gp quan
trng vo doanh s 2,3 t USD ca AgrEvo nm 1995. Tuy nhin, hng ny
t chi khng tr cho Camerun mt khon tin no v quyn tc gi.
Ngy 16/1/1996, Bn quyn s hu s 5.484.889 ca M c cp
cho Gio s Sylvia Lee-Huang (i hc New York) bo v quyn tc gi
ca ng v mt loi protein chit t mt ging mp ng (Momordica
charantia) c ngun gc t min Nam Trung Quc. Ging mp ng ny
l thnh phn chnh ca mt bi thuc dn gian c truyn ca Trung Quc
chng nhim trng. Lee-Huang cho rng nh cng ngh DNA ti t
hp, t nay ng khng cn phi mua ht mp ng t Trung Quc na, v
cc protein ti t hp sn xut trong phng th nghim ca ng hon ton
ging nh protein chit t qu mp ng trc y.
Cc trng hp trn cho thy cc cng ty ln cc nc pht trin
nh vo tim nng khoa hc v ngun vn di do ca mnh thng
lng v bn quyn tc gi vi t th ca k mnh. S thua thit ca cc
nc yu v cng ngh sinh hc s cn ko di.
28
4.2. S hu tr tu
Mt trong nhng nt c trng ca cng ngh sinh hc hin i l s
gia tng tnh s hu ca n. Hin nay, ngnh cng ngh sinh hc c bo
v bi cc bng sng ch v cc quyn v s hu tr tu (IPR).
Nh chng ta bit, s hu tr tu i din cho cc sn phm ca tr tu.
Chng l cc tng c chuyn thnh dng hu hnh. V d ca s hu
tr tu bao gm: cc sng ch, phn mm my tnh, n phm, bng a ca
nhc, ging cy trng-vt nui... to ra nhng sn phm nh vy thng
i hi mt khong thi gian di v mt ngun vn u t ln. Do vy, cc
nh sng ch thng tm cch thu hi cc ngun u t bng cch s dng
IPR. IPR cho php cc sng ch gii hn quyn s dng s hu tr tu,
khng mt c nhn hoc t chc no c php s dng sn xut, nui
trng, bn hay ngh sng ch m khng c cho php. C mt s
hnh thc bo v cc tc gi bao gm: quyn tc gi, sng ch, b mt
kinh doanh, nhn hiu hng ha, quyn bo h ging cy trng-vt nui...
Cc bng sng ch, quyn bo h ging cy trng-vt nui v cc
nhn hiu hng ha c ban hnh bi chnh ph ca tng quc gia v s
bo h ch c hiu lc trong cc nc m s hu tr tu (IP) c ban hnh.
Do vy, nhn c s bo h nhiu nc, cc quyn ny phi c p
dng v thng qua mi nc. Cn quyn tc gi v b mt kinh doanh
khng c trng theo quc gia. Hin nay, nhiu cng ngh mi nhn c
s dng to ra cc sn phm cng ngh sinh hc nng nghip dng nh
khng c bo h cc nc ang pht trin. Chng hn, cc bng sng
ch i vi promoter CaMV 35S ch c cp v c hiu lc Hoa k v
Chu u (v Nht Bn ch c mt n xin ng k cp bng). Do , hin
nay cha c IP nghim cm cc nc ang pht trin s dng cng c ny
trong nghin cu.
Hn na, cc t chc v c nhn c th s dng cc cng ngh trong
to ging cy trng bao gm trin khai, sn xut v tiu th cc nc m
cng ngh sn xut ny cha c IP bo h. Tuy nhin, cc vn lin quan
n IP s pht sinh cc nc c nhng cng ngh c bo h bi IPR.
Thi gian pht trin sn phm cng cn c cn nhc k lng v cc bng
sng ch c th c cp trong nc cng thi im pht trin sn phm.
Do vy, cc nh khoa hc cc nc ang pht trin cn phi bit v cc
vn lin quan n IP v c phng n gii quyt thch hp.
29
Cy trng c canh tc s dng bn vng cc nc ang pht
trin v cc cng ngh c p dng to ra cc cy trng ny ang nhn
c rt t s quan tm thng mi ca khu vc kinh t t nhn. Trn thc
t, cc cng ngh ny v ang c chuyn giao nhm tng nng sut
ma v. Tuy nhin, cc nh khoa hc cc nc ang pht trin cn thn
trng v chuyn giao cng ngh lin quan n nhiu vn , khng ch l k
kt cc hp ng chuyn giao nguyn liu v cp giy php s dng cho
mt sn phm. C bn chuyn giao v bn tip nhn cng ngh phi thn
trng vi cc IPR lin quan n cng ngh v iu ny l cn thit cho cc
i tc to s tin tng ln nhau gia cc bn tham gia.
Cc nc ang pht trin lun thiu nng lc v ngun lc qun l IP
tin hnh cc phn tch v nh gi v s cho php s dng cng ngh
nhm pht trin sn phm nhp khu, s dng hoc xut khu sn phm. Do
vy, gip chuyn giao cc cng ngh ng dng trong nng nghip cho
cc nc ang pht trin, vic xy dng kh nng qun l IPR l rt quan
trng cho c bn chuyn giao v bn tip nhn cng ngh. Cy trng c
canh tc s dng bn vng cc nc ang pht trin v cc cng ngh
c ng dng to ra cc cy trng ny r rng nhn c t s quan tm
thng mi ca khu vc kinh t t nhn. Trn thc t cc cng ngh ny
v ang c chuyn giao nhm tng nng sut ma v.
Trong lnh vc cng ngh sinh hc nng nghip, sng ch c th bao
gm: cc phng php chuyn gen thc vt, cc vector, cc gen... Cc
sng ch gi vai tr quyt nh nht trong bo h cng ngh sinh hc nng
nghip v c nh gi l cng c mnh nht trong h thng IP. Cc sng
ch tm thi, thng c bo h trong khong 20 nm v ty thuc vo
mi quc gia.
Ni chung, cc c quan nghin cu khoa hc ca chnh ph cn xy
dng nng lc qun l s hu tr tu m h nhn c hay to ra. Kin thc
v IPR s gip cc nh khoa hc ca cc nc ang pht trin xc nh c
cc thng tin v mt cng ngh nht nh thuc quyn s hu cng cng
v h c quyn s dng. Hn na, IP do cc khu vc kinh t nh nc to ra
c th c xem xt l ti sn c trao i vi cc cng ty t nhn hoc
c s dng lm hng ha trong cc m phn chuyn giao cng ngh. S
hp tc gia cc khu vc kinh t nh nc v t nhn trong pht trin cng
30
ngh nh chia s b quyt sn xut v IP s thc y s chuyn giao cng
ngh cng nh em li li ch cho c hai bn.

Ti liu tham kho/c thm
1. Nguyn Ngc Hi. 2005. Sinh hc mo him. NXB Thanh nin, H Ni.
2. Nguyn Vn Uyn v Nguyn Tin Thng. 1999. Nhng kin thc c
bn v cng ngh sinh hc. NXB Gio dc, H Ni.
3. Bains W. 2003. Biotechnology from A to Z. Oxford University Press Inc.
New York, USA.
4. Borm A, Santos FR and Bowen DE. 2003. Understanding
Biotechnology. Prentice Hall PTR, New Jersey, USA.
5. Ratledge C and Kristiansen B. 2002. Basic Biotechnology. Cambridge
University Press, UK.
6. Shantharam S and Montgomery JF. 1999. Biotechnology, Biosafety,
and Biodiversity: Scientific and Ethical Issues for Sustainable Development.
Science Publisher Inc. USA.

Nhp mn Cng ngh sinh hc 31
Chng 2

Cng ngh DNA ti t hp

I. M u
Cng ngh DNA ti t hp c hnh thnh t nhng nm 1970 nh
s pht trin ca cc phng php v k thut dng trong nghin cu cc
qu trnh sinh hc mc phn t. T , cho php phn lp, phn tch v
thao tc trn cc nucleic acid theo nhiu phng thc khc nhau, gip hiu
bit su sc cc lnh vc mi ca sinh hc nh cng ngh sinh hc, bo ch
cc loi thuc mi, y hc phn t v liu php gen.
S khm ph ra cc enzyme hn ch (restriction endonuclease) trong
nhng nm u 1970 l s pht trin then cht (key development) khng ch
cho kh nng phn tch DNA hiu qu hn, m cn cung cp kh nng ct
cc phn t DNA to ra cc on DNA ti t hp mi, mt qu trnh m
ngy nay c gi l to dng gen (gene cloning). Phng thc to dng
ny bo hiu mt k nguyn mi trong thao tc, phn tch v khai thc
cc phn t nucleic acid.
To dng gen cho ra nhiu pht minh quan trng v cung cp
nhng hiu bit gi tr trong cu trc, chc nng v s iu ha hot ng
ca gen. T nhng ng dng u tin ca chng, cc phng php xy dng
th vin gen (gene library) c hnh thnh v pht trin, v hin nay c
xem nh l nn tng c s cho nhiu th nghim ha sinh v sinh hc phn
t. Mc d phn ng chui polymerase (polymerase chain reaction-PCR)
khuch i gen cho php phn tch gen nhanh hn nhng trong nhiu trng
hp k thut to dng gen vn cn hu ch v l mt yu cu tuyt i.
Nhng phn di y cung cp mt bc tranh tng qut v cc qu trnh c
bn ca cng ngh DNA ti t hp.

II. Phn lp on DNA/gen
Mt s phng php phn lp gen thc hin k thut ti t hp
DNA c s dng l:

1. Tch cc on DNA t genome
Phng php ny c thc hin nh sau: DNA h gen (genomic
DNA) ca mt sinh vt c ct thnh cc on nh di khong 20 kb (kch
thc thch hp ty thuc vo loi vector nhn chng) bng enzyme hn ch
ri gn vo vector xy dng th vin genome (genomic library).
Escherichia coli Saccharomyces cerevisiae
.
Thng th
4 bp, v d nh Mbo - -
.
(clone).
.
(physical mapping).

2. Sinh tng hp cDNA t mRNA
on DNA c tng hp da trn khun mu mRNA c gi l
cDNA (complementary DNA). ba
nh sau:
- .
- - 2 (Mg
2+
.
- .
mRNA-cDNA (khi tng hp si th E.
coli
(
E. coli
5
.
- 1.
. Si
bacteriophage (cDNA library).

3. Phn lp on DNA bng phng php PCR
Ngoi hai phng php trn, hin nay ngi ta s dng rt ph bin
phng php PCR phn lp mt trnh t nucleotide (gen) t genome ca
sinh vt da trn cc primer c hiu cho trnh t . Phng php PCR n
gin v t tn thi gian hn hai phng php trn, m hiu qu vn rt cao.

2.1.

PCR l mt k thut c s dng ph bin trong cng ngh sinh hc
hin i v ng gp rt ln cho nhng tin b v sinh hc phn t, nh
du mt b
cc enzyme hn ch v k thut Southern blot (phn tch DNA).
AAAAAA 3 mRNA
AAAAAA 3 mRNA
TTTTTT 5 si cDNA th nht

TTTTTT 5

AAAAAA 3
TTTTTT 5
AAAAAA 3
TTTTTT 5 cDNA si i
Nuclease S1
DNA polymerase I Tng hp si cDNA th hai
Bin tnh nhit v x l
RNase H ph hy si
mRNA
Tng hp si cDNA th
nht trn khun mu
mRNA
Reverse transcriptase
Gn oligo(dT) primer
5
5
5
3
u 3 ca cDNA to
thnh vng cp tc
5
3
AAAAAA 3 mRNA
TTTTTT

M
(v d: no)
Sinh tan t bo
v tinh sch mRNA
in vitro
-
20 nucleotide.

Nguyn tc ca PCR
Taq polymerase l mt loi enzyme DNA polymerase chu nhit (c
vi khun chu nhit cao Thermus aquaticus
tide (dATP,
dCTP, dGTP v dTTP) v hai primer, trn c s khun mu ca mt on
DNA nht
, nh vy c th s lng
tch ra, phn tch trnh t hoc to dng. P
DNA 3 -
5
- ).
Nguyn tc ca PCR c trnh
u to ra cc on DNA c chiu di xc
nh. Nu bit trnh t ca on gen cn khuch i th c th tng hp nhn
to cc primer tng ng thc hin PCR v tch chng ra bng k thu
- , qua t 10
-6
g
DNA ban u c th khuch i ln ti trn 1
PCR bao gm ba giai on c nhit khc nhau:
- Gy bin tnh (denaturation) 90-95
o
C. Trong giai on bin tnh,
phn t DNA khun mu dng xon kp c tch thnh hai si n
(single strands). Tt c cc phn ng enzyme trong giai on ny u b
dng li (v d: phn ng tng hp DNA t chu k trc ).
- Gn primer (annealing) 40-65
o
C. Trong giai on ny cc primer
gn vo cc v tr c trnh t tng ng DNA khun mu. Cc primer b
lc nh chung quanh do chuyn ng Brown v th cc lin kt ion c to
thnh v b t gy lin tc gia primer si n v DNA khun mu si
n. Cc lin kt ion n nh h on nh (cc primer
) v trn cc i (khun mu v primer)
enzyme Taq polymerase c th bt u qu trnh sao chp khun mu.

Hnh 2.2. S phn ng chui polymerase (PCR)

- Ko di phn t (extension) 70-72
o
C. y l khong nhit ti
thch cho Taq polymerase tin hnh tng hp DNA bt u t cc v tr c
primer theo chiu 5 3. Cc primer
c mi lin kt ion mnh hn cc lc ph v
. Cc primer cc v tr khng bt cp chnh xc li b ri ra (do
nhit . Enzyme Taq
polymerase b sung cc dNTP t 5 3.

III. To dng (gn) on DNA/gen vo vector
1. Enzyme hn ch
Vic pht hin ra cc enzyme hn ch (restriction enzyme) ca vi
khun ct DNA nhng trnh t c trng, gip cho vic thao tc gen d
dng hn, v n c th gim chiu di ca cc phn t DNA thnh mt tp
hp bao gm cc on ngn hn.
Cc enzyme hn ch hin din trong hu ht cc t bo vi khun
ngn cn DNA ngoi lai (ca bacteriophage) tip qun b my tng hp
Gen ch
DNA khun mu
Khuch i theo hm m
Chu k 35
2
2
= 4 2
3
= 8 2
4
= 16 2
36
= 68 t bn sao
bn sao bn sao bn sao
protein ca t bo. DNA ca chnh chng s c bo v khi tc dng ca
enzyme hn ch, nh s c mt ca cc enzyme ni bo c th methyl ha
(methylation) cc nucleotide c bit, v th cc nucleotide ny khng th b
nhn bit bi cc enzyme hn ch.
Mi enzyme hn ch nhn bit v ct mt trnh t DNA c trng
cha 4 hoc 6 cp nucleotide. V d: enzyme EcoRI chit t E. coli ct trnh
t GAATTC, enzyme TaqI ca Thermus aquaticus ct trnh t TCGA (Hnh
2.3). Hin nay, c trn 900 enzyme hn ch khc nhau c tinh sch t
hn 230 chng vi khun. Cc enzyme hn ch ct gy cc phn t DNA si
i theo hai cch khc nhau nh trnh by hnh 2.3:

PvuII (Proteus vulgaris) EcoRI (Escherichia coli)

(A1) (A2)
RsAI (Rhodopseudomonas sphaeroides) TaqI (Thermus aquaticus)

(B1) (B2)
Hnh 2.3. Cc kiu ct v nhn bit trnh t nucleotide ca enzyme hn ch.
(A1): to ra u bng v (A2): to ra u so le vi trnh t 6 nucleotide. (B1): to
ra u bng v (B2): to ra u so le vi trnh t 4 nucleotide.

- Ct trn mt ng thng i xng to ra cc phn t u bng
(Hnh 2.3, A1 v B1).
- Ct trn nhng v tr nm i xng quanh mt ng thng i xng
to ra nhng phn t u so le (u dnh) (Hnh 2.3, A2 v B2).
V mt enzyme hn ch nhn bit mt trnh t duy nht, cho nn s v
tr ct trn mt phn t DNA c bit thng l nh. Cc on DNA c
ct bi enzyme hn ch c th c phn tch theo kch thc bng in di
agarose gel nghin cu. Do s tng t ca t chc phn t trong tt c
cc c th, cho nn DNA vi khun, DNA thc vt v DNA ng vt c v
tng hp nhau v cu trc. V th, mt on DNA t mt dng sng ny c
th d dng c pha trn vi DNA ca mt dng sng khc. S tng t
ny cng ph hp i vi plasmid, nhn t di truyn ngoi nhn c tm
thy trong nhiu loi vi khun khc nhau. Chng l nhng phn t DNA
mch vng ng-si i c dng lm vector mang cc on DNA ngoi
lai (foreign DNA) dng trong k thut ti t hp DNA.

2. Cc vector c dng to dng cc on DNA
in vitro
5-PO
4
.
.
Trong thc t, cc on DNA c gn vi nhau to ra phn t
DNA ti t hp thng khng phi l cc on ngu nhin ca DNA h gen,
m thay vo l mt trong cc on DNA mang mt gen t sinh vt
eukaryote hoc vi khun v mt on DNA khc thng l mt phn t
vector (vector molecule) hot ng nh l mt vt truyn chuyn gen
quan tm vo trong mt vi khun hoc mt t bo eukaryote. Hai loi vector
c s dng ph bin nht l mt loi c ngun gc t cc plasmid ca vi
khun v mt loi khc c ngun gc virus xm nhim vo t bo vi khun
(bacteriophage, vit tt l phage).

2.1. Plasmid vector
1-
.
c
.
i) hay multiple cloning sites (vng

3-
:
- . 2.600 bp, mang gen
Amp
r
v gen lacZ lacZ
.
N c u im k
lacZ
.
- . 3.000 bp, mang gen
Amp
r
, gen lacZ
.
- .
2.6).

2.4. Plasmid vector pUC19. Vng to dng (t 396-447) c gn vo gen
lacZ, nhng khng can thip vo chc nng ca gen.

2.5. Plasmid vector pGEM -T Easy. Loi vector ny c m sn
vng to dng trn gen lacZ mang hai u T, thch hp cho vic gn cc sn phm
PCR do chng c mang hai u A.

AGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCATAATCATGGTCAT
EcoRI SacI KpnI BamHI XbaI HincII PstI SphI HindIII
SmaI
XmaI
AccI
SalI
1
lacZ ThrIleMetThr(Met)
400 420 440 460

2.6. Plasmid vector pBluescript II SK (+/-). Vector ny mang vng to
dng (multiple cloning sites-MCS) v tr t 598-826 trn gen lacZ, v cc v tr
gn primer khc nhau dng cho phn tch trnh t on DNA ngoi lai.

, DNA ca plasmid c
in vitro
- (
ca plasmid (ligation).
TTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGAC

GGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTC

CAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCC
M13-20 primer binding site T7 primer binding site KS primer binding site

KS primer binding site SK primer binding site

T3 primer binding site M13 reverse primer binding site
BssHII T7 Promoter KpnI DraII XhoI SalI
ApaI
EcoO1091
ClaI HindIII EcoRV EcoRI PstI SmaI BamHI SpeI XbaI EagI BstXI SacII SacI
Bsp1061 NotI
T3 Promoter BssHII -gal -fragment
2.2. Bacteriophage vector
C hai loi bacteriophage thng c s dng l v M13, tuy
nhin chng ny ch trnh by loi ph bin hn l bacteriophage .
Bacteriophage
cos
cos (R-cos v L-cos)
(lysis)

).

2.7. genome. Cc gen lin quan n
cc chc nng khc nhau c trnh by trn s . cIII, N, cI, cro v cII: cc
gen lin quan n hot ng iu ha, min dch tin phage, siu nhim. O v P:
cc gen tng hp DNA. Q: gen iu ha chc nng mun. S v R: cc gen phn
gii t bo vt ch. P
L
: promoter bn tri, P
R
: promoter bn phi, L-cos: u dnh
bn tri, R-cos: u dnh bn phi.

(recipient). Phage vector
L-cos
A W B C D E FI FII Z U V G T H M L K I
J
u ui
P
L
P
R
ori
AJ b att int xis red gam cIII N cI cro cII O P Q S R
Cc gen hnh
thnh u,
ui v pht
sinh hnh thi
Vng hp nht v ti t
hp (vng m)
Vng iu ha v
min dch
Vng sao
chp DNA
Vng phn
gii vt ch
Vng iu ha
chc nng mun
R-cos
-
.
khong 1/3 chiu di ca phage
phage
. Trn
phage vector
in vitro (in vitro
(Hnh 2.8).

Hnh 2.8. Vector EMBL 3. Vector ny c thit k t phage mang cc v tr
nhn bit cho ba enzyme SalI, BamHI v EcoRI.

3. Gn on DNA vo vector
3.1. Gn cc on cDNA
, chng hn nh: b
S
a
l
I

B
a
m
H
I

E
c
o
R
I

E
c
o
R
I

B
a
m
H
I

S
a
l
I

Nhnh tri (20 kb) Vng m (14 kb) Nhnh phi (9 kb)
5GGATC TGGGT CGACG GATCC GGGGA ATTCC CAGAT CC3
SalI BamHI EcoRI
(homopolymetric tailing)
(linkers v adapter)
.

vector. Si
(v d: on Klenow ca DNA polymerase
I ca E. coli
ase 3 -
(polymerase 5 3
.
.

3.1.2. Cc adapter
vector DNA .


2.9. .
on Klenow to u bng cho cDNA si i v enzyme nuclease S1 ct vng cp
tc. Cc linker th nht v th hai c gn tun t vo hai u ca cDNA, sau
on cDNA ny s c ct cng enzyme hn ch vi vector to dng c v tr
nhn bit trn hai linker nhn to. Cui cng on cDNA c hai u tng ng
c gn vi vector v bin np vo E. coli.

cDNA si i
Sa cha bng cch x l vi on Klenow
B sung linker th nht
Ct bng nuclease S1
Sa cha bng cch x l vi on Klenow hoc
bng DNA polymerase ca bacteriophage T4

B sung linker th hai

Gn vi plasmid vector

Bin np vo t bo kh bin E. coli

Ct bng enzyme hn ch

3.2. Gn cc sn phm PCR
Trong mt s trng hp nht nh c th thay th phng php to
dng truyn thng bng phng php PCR, v PCR cho php sn xut ra
mt lng ln on DNA mong mun v sau c th to dng on DNA
ny trong cc vector plasmid hoc phage thch hp. Phng thc to dng
cho cc sn phm PCR hon ton ging to dng cc on DNA thu c t
nhng thao tc DNA truyn thng, v c th c to dng vi u bng
hoc u kt dnh (so le). DNA polymerase n nhit nh Taq polymerase
khuch i cc sn phm PCR c gc A li ra u 3. Nh vy, c th to
dng sn phm PCR vo trong cc dT vector (c gi l to dng dA:dT).
iu ny cho thy vic b sung cc gc A vo u cui c th gip gn
thnh cng sn phm PCR vi vector c chun b cc gc T li ra
(Hnh 2.10). Phn ng c xc tc bi DNA ligase, nh trong mt phn
ng gn truyn thng.

Hnh 2.10. To dng cc sn phm PCR bng phng thc to dng dA:dT

Ngi ta cng c th to dng u dnh vi cc sn phm PCR. Trong
trng hp ny cc oligonucleotide primer c thit k vi mt v tr ct
hn ch c kt hp cht ch trong chng. Do s b tr ca cc primer cn
thit l tuyt i u 3, nn thng thng u 5 ca primer l vng nh
Phn ng gn T4 DNA ligase
Vector (dT) + on chn PCR

Sn phm PCR c khuch i bng Taq polymerase
A
A
Vector (dT)
T
T
v ca v tr ct hn ch. iu ny cn phi c thit k vi s ch thn
trng do hiu sut ct DNA bng mt enzyme hn ch nht nh s gim
nu cc nucleotide b sung thm cho s nhn bit ca enzyme li thiu
u 5. Trong trng hp ny cc phn ng ct v gn ging nh cc phn
ng truyn thng.

4. Bin np vector ti t hp vo vi khun/t bo vt ch
Sau khi to c vector ti t hp mang gen ngoi lai, vic tip theo l
bin np n vo t bo vt ch. Trong trng hp ny t bo vt ch thng
c s dng l vi khun E. coli khuch i mt lng ln DNA ca
plasmid ti t hp dng cho cc phn tch v sau.
Hai phng php c dng bin np vector ti t hp vo E. coli
l in bin np (electroporation transformation) v ha bin np (chemical
transformation).

4.1. in bin np
y l k thut hiu qu nht bin np vi khun. Hai thng s quan
trng ca phng thc ny l loi t bo vi khun v tn s xung in cn
thit. Th tch ca dung dch t bo thng c dng l 30 L (tng ng
vi nng 10
10
t bo E. coli/mL) c b sung 5 ng plasmid trong mt
cuvette c khong trng in cc (electrode gap) 0,1 cm. Hiu sut bin np
ca phng thc ny ln hn 1 10
9
th bin np/g plasmid siu xon v
khong 1 10
8
th bin np/g plasmid c dng trong phn ng gn. Tn
s bin np khong 0,02 cho c hai loi plasmid. Tn s bin np thp
ngn cn c s ng bin np (co-transformation) vo vi khun ca hai
hoc nhiu phn t plasmid.
Phng php in bin np c mt s u im sau:
- Hiu sut bin np cao.
- C th dng mt th tch dch t bo nh. Th tch dch t bo
khong 20 L c th cho hiu sut khong 10
9
th bin np.
- Phng php chun b t bo bin np rt n gin, khng s dng
cc k thut phc tp v tn thi gian. Hn na, cc t bo dng bin np
c th c chun b trc v bo qun v hn nh m khng mt tnh kh
bin.
- Tn s in bin np vi DNA siu xon v DNA mch vng l
ging nhau. Do , khng cn thit phi dng vector c tinh sch cao
trong cc phn ng gn.
- Hiu sut bin np phn t cho DNA mch vng rt cao i vi cc
plasmid c kch thc ln n 50 kb.
Nhc im ca phng php ny l i hi thit b bin np t tin.

4.2. Ha bin np
y l phng thc kinh in bin np plasmid vo t bo E. coli.
Cc t bo c trong dung dch CaCl
2
tr thnh t bo kh bin gip
cho chng d tip nhn plasmid. Plasmid c a vo bng cch shock
nhit nhanh (40-50 giy), cc t bo bin np sau c chn lc bng
phng php chn lc dng tnh trn a agar cha mi trng LB vi
khng sinh thch hp. Mi khun lc trn a khng sinh i din cho mt
th bin np n. Cc t bo cha plasmid mang DNA ngoi lai c th xc
nh bng mt trn a mi trng c b sung thm c cht nhim sc th
cho -galactosidase (X-gal) v chng l cc khun lc khng mu do s kh
hot tnh ca enzyme bng cch chn on DNA ngoi lai.
Phng php chun b v bo qun t bo kh bin trong ha bin np
cng rt n gin. Hiu sut bin np ca phng php ny trong khong
10
4
-10
6
th bin np/g plasmid, ty thuc vo kch thc ca on DNA
chn (DNA ngoi lai) v chng vi khun c s dng. Hiu sut ny thch
hp cho cc phng thc to dng truyn thng. i vi cc phng thc
cn hiu sut bin np cao hn (v d: xy dng th vin cDNA, xy dng
th vin phn tch trnh t DNA...) th tt hn ht l dng phng php in
bin np. Tuy nhin, nu khng c sn thit b bin np bng in th vn c
th thu c hiu sut bin np cao bng cch dng cc chng vi khun
thch hp hn cho mc ch ny v c th thu c hiu sut 10
9
th bin
np/g plasmid.

IV. Chn dng mang DNA ti t hp
Trong th nghim to vector ti t hp, hn hp vector v mt lng
ln phn t DNA ct cng mt enzyme hn ch c gn li vi nhau bng
enzyme DNA ligase. Kt qu, hn hp ny c plasmid ti t hp ln cc
plasmid khng c gen ngoi lai chn vo v chng c trn ln vi cc t
bo vi khun thc hin bin np. Sau , ngi ta chuyn tt c ln mi
trng dinh dng chn lc chng pht trin thnh cc dng tc cc
khun lc vi khun. Do cch tin hnh th nghim trong mt hn hp khng
ng nht nh vy nn cc dng vi khun mc ln c ba loi nh sau:
- T bo vi khun khng nhn plasmid.
- T bo vi khun nhn plasmid khng c gen ngoi lai chn vo.
- T bo vi khun nhn ng plasmid ti t hp.
V vy, vic xc nh ng cc dng vi khun cha plasmid ti t hp
phi mt nhiu cng sc. C ba phng thc chnh xc nh cc dng
DNA ti t hp l lai khun lc v vt tan, kh hot tnh bng chn on, v
to dng nh hng.

1. Lai khun lc v vt tan
DNA c to dng trong plasmid sn xut ra cc khun lc khi cc
nui cy bin np c dn mng trn a agar cha mi trng sinh trng
v nui cy di nhng iu kin thch hp. Cc bacteriophage sinh tan t
bo b chng xm nhim v sn xut ra cc plaque (vt tan) c dng hnh
trn chu vi khong 2-3 mm c mu sng trn thm vi khun (bacterial lawn)
ca lp agar nh [trong trng hp ny ngi ta chun b a agar c hai
lp: lp agar nh (top agar) c nng agar thp bacteriophage d sinh
tan t bo vi khun, v lp agar y (bottom agar) c nng agar cao hn].
Cc vector, nh m t trn, thng cha gen ch th cho php chn
lc nhng t bo vt ch mang vector (th bin np). Cc ch th ny thng
l gen khng khng sinh v cc t bo bin np sinh trng trn mi trng
cha khng sinh tng ng.
Ngoi ra, cc vector cn cha cc gen ch th b sung phn bit cc
t bo bin np cha on chn ca DNA ngoi lai vi cc t bo cha cc
vector t ti to li vng. V d: gen lacZ m ha enzyme -galactosidase.
Mt dng cha cc chui DNA quan tm c bit c th c xc
nh bi lai khun lc hoc vt tan. Mt lng nh khun lc bin np hoc
vt tan c chuyn ln mng nitrocellulose hoc nylon bng cch ph
(overlay) mng ny ln trn a agar. DNA c bin tnh v c nh trn
mng bng cch un nng (baking) hoc chiu tia t ngoi (UV-
crosslinking), v sau c lai trong m cha probe nh du ng v
phng x c trnh t b tr mt phn hoc ton b ca chui c xc nh.
V d: c th mt oligonucleotide tng hp nhn to, c ngun gc t DNA
h gen tng phn, cDNA hoc trnh t protein hoc sn phm PCR. Mt i
khi probe c th c thit k da trn trnh t bt ngun t gen tng ng
ca cc loi khc v thng c lai vi cng lc thp. Cc probe tha
c ra khi mng vi phim X-quang. Theo hng ca phim (sau khi
ra) so snh vi a agar gc, chng ta c th i chiu cc khun lc/vt
tan thc t vi cc khun lc/vt tan lai dng tnh (positive clones) tng
ng trn phim X-quang.

(v d: Amp
r
, lacZ ca ct bi
E. coli
-galactosidase ca gen lacZ
-
-gal s c mu trng do on DNA ngoi lai chn vo gia gen lacZ
lacZ khng b mt hot tnh (Hnh 2.11).

Hin Bam
ca vector
Bam Hin
E. coli
(protruding ends) Hin Bam
E. coli
- .

Hnh 2.11. Kh hot tnh bng chn on. Amp
r
: gen khng ampicillin, lacZ: gen
lacZ m ha enzyme -galactosidase.

V mt l thuyt, k thut ti t hp DNA cho php a bt k mt
on gen no t sinh vt ny vo sinh vt khc. Vn quan trng tip theo
l lm sao cc gen l c s biu hin.

Amp
r

lacZ
Plasmid vector DNA ngoi lai
BamHI BamHI BamHI

Amp
r

Amp
r

Gen lacZ mt hot tnh
lacZ
on DNA
ngoi lai
chn gia
gen lacZ lm
gen lacZ mt
hot tnh
Amp
r
DNA ligase
Bin np vo E. coli v cho
sinh trng 37
o
C trn mi
trng c Amp v IPTG+X-gal
Vi khun E. coli cha vector ti to
vng pht trin thnh khun lc c
mu xanh
Vi khun E. coli cha vector ti t
hp pht trin thnh khun lc c
mu trng
BamHI

Hnh 2.12. To dng nh hng. Tet
r
: gen khng tetracycline, Tet
s
: gen khng
tetracycline b khuyt on v mt hot tnh.

V. Biu hin ca gen c to dng
Mun gen to dng c biu hin tng hp protein cn cu to vector
c cc yu t phin m v dch m. Cc vector ny c gi l vector
Cc th bin np c
dn mng trn mi
trng c Amp

Tet
s

Amp
r

H

B
Hiu sut bin np thp
Tet
s

Amp
r

H

B
Hiu sut bin np cao
Plasmid vector DNA ngoi lai
T4 DNA ligase
HindIII BamHI HindIII

H B B H
Amp
r

Tet
r

Amp
r

Tet
s

H

B
H B
Amp
r

H

B
Tet
s

BamHI
HindIII
in di agarose gel
biu hin. Nu gen khng nm gia promoter v terminator, n s khng
c phin m. Cc gen c tng hp ha hc hay t cDNA khng c
promoter nn phi gn chng cnh promoter th mi c th biu hin phin
m. s dch m c thc hin, mRNA cn phi mang u 5 trnh t
RBS (ribosome binding sites-vng lin kt ribosome). on gen ngoi lai
thiu im bm ca ribosome (RBS), do mun c dch m th n phi
gn vo v tr nm sau promoter v RBS.
mt s gen ca sinh vt eukaryote, s dch m i hi qu trnh
splicing tc l ct b cc on intron khi tin thn mRNA thng tin
(premature mRNA) v ni cc exon li vi nhau to thnh mRNA hon
chnh (mature mRNA).
Mc ch vic to dng cc gen ca ng vt c v, nht l ca ngi,
l nhm to ra cc sn phm ng nh trong c th vi s lng ln v c
gi tr thng mi. S biu hin ca cc gen eukaryote trong t bo vi khun
nhiu khi gp tr ngi, do cn phi thit k cc vector biu hin thch hp
cho php gen ngoi lai biu hin mc cao.

1. Vector biu hin
E. coli
E. coli
. biu hin tt c cc gen ngoi lai trong E.
coli phi bt u bng vic gn on gen ngoi lai vo trong vector biu
hin (thng l plasmid). Vector ny phi c cc cu trc cn thit sau:
- Cc trnh t m ha gen ch th chn lc (selectable marker) m
bo duy tr vector trong t bo.
- Mt promoter kim sot phin m (v d: lac, trp hoc tac) cho php
sn xut mt lng ln mRNA t cc gen c to dng.
- Cc trnh t kim sot dch m nh vng lin kt ribosome c b
tr thch hp v codon khi u ATG.
- Mt polylinker a gen ngoi lai vo trong mt hng chnh xc
vi promoter.
Ch khi c cu trc y nh th, cc vector biu hin mang gen
ngoi lai mi c bin np vo chng E. coli thch hp.
Tuy nhin, cn lu l s lng cc bn sao ca vector phi hp l
i vi mt t bo vt ch v n c s n nh lu di, ng thi cn trnh
s thy phn sn phm protein (proteolysis) do cc enzyme ca t bo. Hnh
2.13 m t mt loi vector biu hin prokaryote.

Hnh 2.13. Vector biu hin pRSET A prokaryote (E. coli) c thit k da
trn h thng biu hin promoter T7. S biu hin ca cc gen ch to dng
trong vector c cm ng bng cch sn xut T7 RNA polymerase trong t bo
vt ch E. coli chng BL21(DE). P
T7
-promoter mnh ca bacteriophage T7 cho
php biu hin gen mc cao, RBS-vng lin kt ribosome, ATG-m khi u,
N-terminal polyhistidine (6His) tag-cho php tinh sch nhanh protein bng nickel
resin v pht hin bng khng th Anti-HisG, N-terminal Xpress
TM
epitope-cho
php pht hin protein bng khng th Anti-Xpress
TM
, EK-enterokinase ct im
loi b u dung hp, MCS-vng to dng, Stop-gen kt thc phin m terminator,
f1 ori ca phage dng si (filamentous phage)-sn xut DNA si n cho php
phn tch trnh t v pht sinh t bin d dng, Ampicillin-gen chn lc khng
khng sinh. Nhm pRSET bao gm 3 vector A, B v C. Mi loi vector c trnh t
m ha N-terminal tag trong mt khung c khc nhau lin quan vi vng MCS
n gin ha s to dng trong khung ca gen quan tm.
2. Xc nh mc biu hin ca gen c to dng
Ni chung c ba cch thng c dng nh gi mc biu hin
protein ngoi lai ca gen c to dng:
- in di polyacrylamide gel xc nh protein c kch thc thch
hp c sn xut mc cao trong cc t bo mang vector biu hin.
Thng thng, protein quan tm c th quan st bng cch nhum gel vi
Coomassie Brilliant Blue hoc bng thuc nhum bc. Nu khng c bng
protein mi c thy khi dng cc thuc nhum ny, th nh du s trao
i cht vi 100 Ci ca [
35
S]Met hoc [
35
S]Cys trn 1 mL dch nui cy
trong 5 pht. K thut SDS-PAGE
1
v phng x t ghi c th cho php pht
hin protein quan tm.
- Phn tch Western blot bng cch dng cc khng th c hiu lin
kt vi protein quan tm c thm tch ln mng nitrocellulose sau khi
thc hin k thut SDS-PAGE.
- Nu mc biu hin thp th nn t gen lacZ cng hng vi gen
c biu hin. Nh vy, nu s phin m hoc dch m hn ch biu hin
th nhng thay i trong h thng biu hin c th c kim sot bng
nhng thay i trong hot tnh ca -galactosidase.

Phn tch Western blot
- K thut SDS-PAGE
in di trn polyacrylamide gel vi s c mt ca SDS cho php phn
ly cc phn t protein c khi lng khc nhau. SDS c in tch m rt ln
v c kh nng lin kt vi mch peptide. Nh vy, s lng SDS tng tc
vi protein t l vi kch thc phn t protein v in tch ca SDS bm
vo c th lm bt c phn t protein no cng chuyn ng trong in
trng t cc m sang cc dng. Do , bng phng php in di, c th
phn tch ring bit cc protein c khi lng phn t khc nhau (Hnh
2.14). Ngoi ra, c th in di cc protein ty theo im ng in
(isoelectric point-IEP) ca chng. Phng php ny c gi l in di tp
trung ng in (isoelecric focusing-IEF). Trong dung dch m c pH bin
thin lin tc (gradient pH), cc protein s phn ly n v tr tng thch vi

1
SDS-PAGE: in di SDS-polyacrylamide gel (sodium dodecyl sulfate-
polyacrylamide gel electrophoresis).
im ng in ca chng. Hai phng php in di theo khi lng phn
t v im ng in c th kt hp vi nhau to nn k thut in di 2
chiu (2-D electrophoresis). in di protein trn polyacrylamide gel cho
php phn on, xc nh khi lng phn t v phn lp protein. Ngoi ra,
khi lng phn t ca protein cn c xc nh chnh xc bng phng
php sc k khi ph.

Hnh 2.14. Hnh nh in di SDS-PAGE. WM: chun khi lng phn t ca
protein. Cc ng t 1-5: mu protein c in di.

- Phn ng lin kt khng nguyn-khng th
Phn ng lin kt khng nguyn-khng th c tnh c hiu rt cao. V
vy, c th p dng phn ng ny pht hin s c mt v tinh sch
protein. Khng th (antibody) c sn xut khi a khng nguyn vo th
v c tinh sch t mu th sau khi gy nhim. Nhng khng th to ra
bng cch ny l nhng khng th a dng (polyclonal antibodies-do cc t
bo lympho khc nhau tit ra), do chng c kh nng nhn bit mt s
khng nguyn. Ngc li, khng th n dng (monoclonal antibodies) ch
tng tc vi mt khng nguyn nht nh.
Khng th c nh du bng enzyme (hoc bng cht pht hunh
quang) pht hin protein c hiu (thng c thm tch ln mng
nitrocellulose sau khi chy in di SDS, v c nh ) thng qua k thut
Western blot (hoc immunoblot) (Hnh 2.15). Nguyn l ca phn ng lin
WM 1 2 3 4 5
kt khng nguyn-khng th c trnh by hnh 2.16. Sau khi protein trn
mng nitrocellulose gn vi khng th th nht c hiu v tip n l
khng th th hai c nh du enzyme (v d: alkaline phosphatase, horse-
radish peroxidase) th phc hp ny s c lin kt vi c cht to
mu. S hin din ca protein ngoi lai (sn phm dch m ca gen ngoi lai
c chuyn vo t bo vt ch) s c pht hin nh s xut hin mu
ca phn ng lin kt.
S phn b ca protein c hiu trong t bo v t chc m cng c
th pht hin bng k thut lai in situ (in situ hybridization) vi nguyn tc
tng t Western blot. Ngoi ra, khng th cng c s dng tinh sch
protein c hiu bng kt ta min dch hoc sc k i lc (affinity
chromatography). Khng th nh du cn c dng nh lng khng
nguyn trong k thut xt nghim hp th min dch lin kt enzyme
(enzyme-linked immunosorbent assay) gi tt l ELISA.

Hnh 2.15. S k thut Western blot
in di SDS protein
tng s v thm tch
ln mng nitrocellulose
Bc 1. Ngn chn cc
phn ng khng c
hiu
Bc 2. B sung khng
th 1 c hiu (khng
th n dng)
Bc 3. Ra mng
Bc 4. B sung khng
th 2 nh du enzyme
Bc 5. Ra mng
Bc 6. B sung c cht
bt mu
Bc 7. Dng phn ng
bt mu

Hnh 2.16. S phn ng lin kt khng nguyn vi khng th th nht c
hiu v khng th th hai c nh du enzyme trong Western blot

1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith
JA and Struhl K. 2002. Short Protocol in Molecular Biology. Vol 1 and 2. 5
th
ed.
John Wiley & Sons Inc. USA.
2. Glick BR and Pasternak JJ. 2003. Molecular Biotechnology: Principles
and Applications of Recombinant DNA. 3
rd
ed. ASM Press, USA.
3. Maniatis T, Fritsch EF and Sambrook J. 1989. Molecular Cloning-A
Laboratory Manual. Cold Spring Habor Laboratory, USA.
4. Ohman DE. 1989. Experiments in Gene Manipulation. Prentice Hall,
New Jersey, USA.
5. Primrose SB, Twyman R and Old RW. 2001. Principles of Gene
Manipulation. 6
th
ed. Blackwell Science, Oxford, UK.
6. Rapley R and Walker JM. 1998. Molecular Biomethods Handbook.
Humana Press Inc. New Jersey, USA.
7. Walker JM and Rapley R. 2002. Molecular Biology and Biotechnology.
4
th
ed. The Royal Society of Chemistry, Cambridge, UK.

Alkaline phosphatase
Khng th th hai (th)
Khng th th nht (th)
Khng nguyn

Thm tch (blot)
Chng 3

Cng ngh ln men vi sinh vt

I. M u
Cc c th vi sinh vt c kh nng sinh trng trn nhiu loi c cht
(mi trng dinh dng) khc nhau v c th sn xut nhiu sn phm
thng mi. Gn y, vic p dng cc k thut di truyn in vitro m
rng phm vi cc sn phm c sn xut bi vi sinh vt v cung cp cc
phng php mi tng sn lng ca nhng sn phm . Khai thc
thng mi s a dng ha sinh (biochemical diversity) ca cc vi sinh vt
thc y pht trin cng nghip ln men, v cc k thut di truyn
thit lp mt nn cng nghip u th to c hi pht trin cc qu trnh mi
v ci thin nhng qu trnh ang c.
Thut ng ln men (fermentation) trong cng ngh vi sinh c ngun
gc t ng t Latin fervere ngha l un si, m t s hot ng ca nm
men trn dch chit ca tri cy hoc cc ht ng cc c to mch nha
(malt) trong sn xut ung c ethanol. Tuy nhin, s ln men c cc
nh vi sinh vt hc v ha sinh hc gii thch theo cc cch khc. Theo cc
nh vi sinh vt hc thut ng ln men c ngha l qu trnh sn xut mt sn
phm bng nui cy sinh khi vi sinh vt. Tuy nhin, cc nh ha sinh hc
li cho rng l qu trnh sn sinh ra nng lng trong cc hp cht
hu c hot ng vi vai tr va l cht cho ln cht nhn in t, l qu
trnh ym kh m nng lng c sn xut khng cn s tham gia ca
oxygen hoc cc cht nhn in t v c khc.
Trong chng ny thut ng ln men c s dng theo ngha rng
ca n, gc vi sinh vt hc.

II. Sinh trng ca vi sinh vt
Sinh trng ca vi sinh vt c th to ra s trao i cht, nhng sn
xut mt cht trao i nh mong mun th c th ca chng phi c sinh
trng di nhng iu kin nui cy c bit vi mt tc sinh trng
c trng.
Nu vi sinh vt ch c a mt ln vo mi trng sinh trng, th
nui cy ban u (innoculated culture) s tri qua mt s giai on v h
thng ny c gi l nui cy m (batch culture). u tin, s sinh trng
khng xut hin v qu trnh ny c xem nh l pha lag, c th coi y l
thi k thch nghi. Tip theo l khong thi gian m tc sinh trng
ca t bo tng dn, cc t bo sinh trng vi mt tc cc i v khng
i, thi k ny c xem l pha log hoc pha sinh trng theo hm m v
c m t bng phng trnh:
x
dt
dx
=
(1)

Trong : x l nng t bo (mg/mL), t l thi gian nui cy (gi),
v l tc sinh trng c trng (gi). T phng trnh tch phn (1) ta
c:

t
t
e x x

0
=
(2)

Trong : x
0
l nng t bo thi im bt u nui cy v x
t
l
nng t bo sau mt khong thi gian t (gi).
Nh vy, ng cong logarithm t nhin ca nng t bo theo thi
gian t c dc bng tc sinh trng c trng. Tc sinh trng c
trng trong sut pha log t cc i cc iu kin nui cy thng thng
v c m t nh l tc sinh trng cc i c trng (
max
). Phng
trnh (1) v (2) b qua trng hp s sinh trng s lm tiu hao cc cht
dinh dng v tng tch ly c t ca sn phm. Tuy nhin, trong thc t
khi cht dinh dng b hao ht v cc sn phm c c tch ly, th tc
sinh trng ca t bo s khng t cc i v cui cng lm ngng qu
trnh sinh trng, lc ny nui cy i vo pha tnh v sau mt thi gian s i
vo pha cht, dn n gim s lng t bo sng st (Hnh 3.1).
Nh trnh by, hin tng ngng sinh trng trong nui cy m l
do hao ht thnh phn dinh dng hoc tch ly sn phm c. Tuy nhin,
c th khc phc iu ny bng cch b sung mt lng ti thiu mi
trng sch (mi) vo bnh nui. Khi mi trng mi c b sung lin tc
mt tc thch hp (h nui cy lin tc-continuous culture), th sinh
trng ca t bo trong h ny c iu chnh bng s sinh trng gii hn
v thnh phn ca mi trng, v vy h thng ny c xem nh l mt
chemostat (th n nh ha tnh). H thng nui cy lin tc cho php t
ti trng thi n nh (steady-state) v vic hao ht sinh khi t bo qua
dng chy ra (output) s c b p bi s sinh trng t bo trong bnh
nui.

Hnh 3.1. ng cong sinh trng c trng ca cc c th n bo trong
nui cy m

Dng chy mi trng qua h thng iu chnh vo bnh nui c
m t bi thut ng tc pha long (dilution rate), k hiu l D, bng tc
b sung mi trng trn th tch lm vic ca bnh nui. S cn bng
gia sinh trng ca t bo (growth) v s hao ht ca chng t h thng
ny c th c m t nh sau:
= dt dx/ growth output
hoc:
Dx x dt dx = /
Di cc iu kin trng thi n nh:
0 / = dt dx
v v th, Dx x = v D =

N
n
g

s
i
n
h

k
h
i

P
h
a

s
i
n
h

t
r
n
g

n
h
a
n
h

P
h
a

s
i
n
h

t
r
n
g

c
h
m

Pha
lag
Pha
log
Pha
tnh
Pha
cht
Thi gian
K t y, tc sinh trng ca vi sinh vt c iu chnh bng tc
pha long, v y l mt bin thc nghim. iu ny lu rng di cc
iu kin nui cy m, mt c th s sinh trng tc sinh trng cc
i c trng ca n. V th, nui cy lin tc ch c th hot ng cc tc
pha long pha di tc sinh trng cc i c trng. Nh vy, trong
cc gii hn nht nh, tc pha long c th c dng iu chnh tc
sinh trng ca nui cy chemostat.
C ch v hiu qu iu chnh tc pha long l mi quan h gia
(tc sinh trng c trng) v s (nng c cht gii hn trong
chemostat) c chng minh bi Monod vo nm 1942:

s K
s
s
+
=
max

(3)

Trong : K
s
l hng s s dng hoc bo ha, bng gi tr ca nng
c cht khi bng 1/2 ca
max
. trng thi n nh, =D, v th:
s K
s
D
s
+
=
max

Trong : s l nng c cht trng thi n nh trong chemostat,
v:
D
D K
s
s
=
max

(4)

Phng trnh (4) cho thy nng c cht c xc nh bng tc
pha long. Trong thc t, iu ny xy ra do sinh trng ca t bo lm
tiu hao c cht ti mt nng cn thit tc sinh trng bng tc
pha long. Nu c cht b hao ht di mc cn thit th tc sinh trng
ph thuc tc pha long v mt lot cc kh nng c th xy ra nh sau:
- Tc sinh trng ca t bo km hn tc pha long v chng s
b ra tri khi bnh nui mt tc ln hn tc m chng ang c
sn xut, kt qu l lm gim nng sinh khi t bo.
- Nng c cht trong bnh nui s tng ln do cc t bo c li
t hn trong bnh nui tiu th n.
- Nng c cht c tng ln trong bnh nui s cho kt qu cc t
bo sinh trng mt tc ln hn tc pha long v nng sinh khi
s tng.
- Trng thi n nh s c thit lp tr li.
Nh vy, chemostat l h thng nui cy t cn bng c gii hn
cht dinh dng, c th duy tr trng thi n nh trong mt phm vi rng
ca cc tc sinh trng cc i c trng.
Nui cy m c cung cp dinh dng (fed-batch culture) c xem l
h thng trung gian gia qu trnh nui cy m (batch) v nui cy lin tc
(continuous). Thut ng nui cy m c cung cp dinh dng c dng
m t cc nui cy m c cung cp dinh dng lin tc (hoc ni tip
nhau) bng mi trng mi m khng loi b dch nui cy c. Nh vy,
th tch ca loi nui cy ny tng ln theo thi gian. Pirt (1975) m t
ng hc ca h thng ny nh sau: Nu sinh trng ca mt c th b gii
hn bi nng ca c cht trong mi trng th sinh khi pha tnh, x
max
,
s c m t bi phng trnh:
R
YS x =
max

Trong : Y l yu t hiu sut, bng khi lng t bo c sn xut
trn mt gram c cht c s dng, v S
R
l nng ban u ca c cht
gii hn s sinh trng. Nu mi trng mi c b sung vo bnh nui
tc pha long km hn
max
th sau hu nh tt c c cht s c s
dng khi n i vo h thng:
Y
X
FS
R
=
Trong : F l tc dng chy v X l sinh khi tng s trong bnh
nui, v d: nng t bo c nhn ln bi th tch nui cy.
Cho d khi sinh khi tng s (X) trong bnh nui tng ln theo thi
gian th nng t bo (x) hu nh vn khng i; v vy 0 / = dt dx v
D = . Mt h thng nh th c xem l trng thi gn nh n nh
(quasi-steady-state). Khi thi gian v th tch nui cy tng ln, th tc
pha long li gim. Nh vy, gi tr ca D c a ra nh sau:
t
F V
F
D
+
=
0

Trong : F l tc dng chy, V
0
l th tch nui cy ban u, v t
l thi gian. ng hc Monod d bo rng khi D h xung th nng c
cht cn tha cng s gim v kt qu l lm tng sinh khi. Tuy nhin, trn
phm vi cc tc sinh trng hot ng th s tng sinh khi s khng c
ngha. S khc nhau gia trng thi n nh ca chemostat v trng thi
gn nh n nh ca fed-batch ch trong chemostat th D (k t y l )
l hng s cn fed-batch th D (k t y l ) li gim theo thi gian. Tc
pha long trong fed-batch c th c gi khng i bng cch tng
(theo hm m) tc dng chy nh s dng mt h thng iu chnh thng
qua computer.

III. Sinh khi vi sinh vt v cng ngh ln men
S ln men vi sinh vt c th c phn loi theo cc nhm chnh sau:
- Sn xut cc t bo vi sinh vt (sinh khi) nh l sn phm.
- Sn xut cc cht trao i ca vi sinh vt.
- Sn xut cc enzyme vi sinh vt.
- Sn xut cc sn phm ti t hp.

1. Sinh khi vi sinh vt
Cng ngh thu sinh khi vi sinh vt l cc qu trnh nui cy cc
chng thun khit hoc hn hp vi chng thu c khi lng t bo
sau khi sinh trng vi cc mc ch:
- Sinh khi giu protein dng lm thc phm cho ngi v thc n
cho gia sc l nhng t bo vi sinh vt (k c sinh khi to) sy kh v
cht, giu protein, cc vitamin nhm B v cht khong. Ngun sinh khi
ny c gi l protein n bo.
- Sinh khi nm men l nhng t bo sng dng trong cng nghip
bnh m-men bnh m, sinh khi vi khun lactic sng c hot tnh enzyme
tiu ha sn xut cc thuc h tr tiu ha nh biolactovin
- Sinh khi c nh m lm phn bn vi sinh, cc loi phn bn vi
sinh vi vi khun sng t do trong t v sng cng sinh vi cy h u.
- Sinh khi vi khun sinh c t i vi cc loi su thn mm ph
hoi rau mu, sn xut thuc tr su vi sinh.
- Sinh khi vi sinh vt c h enzyme phn gii cc cht hu c k c
thuc tr su v hydrocarbon sn xut cc ch phm vi sinh x l nc
thi v nhim trong bo v mi trng.

2. Qu trnh ln men
Hnh 3.2 minh ha cc phn ca mt qu trnh ln men tng qut.
Phn trung tm ca h thng l h ln men, trong c th c sinh
trng di cc iu kin ti u to thnh sn phm. Trc khi s ln
men bt u, mi trng phi c pha ch v kh trng, h ln men v
trng, v nui cy khi u phi c mt s lng vi sinh vt va trong
mt trng thi sinh l ph hp cy truyn vo h ln men sn xut. Kt
thc qu trnh ln men cc sn phm phi c tinh sch v x l thm.

Hnh 3.2. S chung ca mt qu trnh ln men

Cc c th vi sinh vt c th sinh trng trong kiu nui cy m (Hnh
3.3), nui cy m c cung cp dinh dng v nui cy lin tc. u im ca
nui cy lin tc i vi sn xut sinh khi l qu r rt (c th xem
nhng tnh ton sau) nhng i vi cc sn phm vi sinh khc th nhc
im ca n li ln hn u im k thut l c kh nng iu chnh ci
thin qu trnh ln men.

Nui cy
trong bnh
tam gic
c lc
Pht trin nguyn liu vi sinh vt
Nui cy
mu gc
H ln men kt ht
Kh trng mi trng
Pha ch mi trng
Nguyn liu chun b mi trng
H ln men sn xut
X l
cht thi
Dch
nui cy
Phn tch
t bo
Sinh khi
Th ni
v bo
Tch chit
sn phm
Tinh sch
sn phm
ng gi
sn phm

Hnh 3.3. Cu hnh c bn ca mt h ln men m

Hiu sut ca nui cy m c th c m t bi phng trnh:

ii i
0 max
batch
t t
x x
R
+
=
(5)

Trong : R
batch
l sn lng nui cy trong gii hn nng sinh
khi/gi, x
max
l nng t bo cc i t c pha tnh, x
0
l nng t
bo ban u lc gy nhim, t
i
l thi gian c th sinh trng
max
v t
ii
l
thi gian m c th khng sinh trng
max
bao gm pha lag, pha gim tc
, v cc thi k ca tng m, kh trng v thu hoch.
Hiu sut ca nui cy lin tc c th c biu din nh sau:
R
cont
=
|
.
|
\
|

T
t
x D
iii
1
(6)

Trong : R
cont
l sn lng ca nui cy trong gii hn nng t
bo/gi, t
iii
l thi gian trc khi thit lp trng thi n nh bao gm thi
gian chun b bnh nui, kh trng v hot ng trong nui cy m trc khi
hot ng lin tc. T l thi gian m cc iu kin trng thi n nh chim
u th, v x l nng t bo trng thi n nh.
4 x vch ngn
B phn phun kh
Sensor nhit
Cnh khuy
Trc khuy
Motor
in cc pH
in cc O
2
Sn lng cc i ca sinh khi trn mt n v thi gian (v d hiu
sut) trong mt chemostat c th t ti bng cch hot ng tc pha
long cao nht ca x D , gi tr ny c xem nh l D
max
. Hiu sut ln
men m, nh m t trong phng trnh (5), l mt gi tr trung bnh cho
thi gian tng s ca s ln men. Do dx/dt = x, nn hiu sut ca nui cy
tng ln theo thi gian, v nh vy, phn ln sinh khi trong qu trnh nui
cy m c sn xut gn phn kt thc ca pha log. Trong chemostat
trng thi n nh, hot ng (hoc gn) D
max
cho hiu sut duy tr khng
i, v t cc i cho s ln men ton phn. Cng nh vy, mt qu trnh
lin tc c th c hot ng mt thi gian rt lu sao cho thi k khng
sn xut, t
iii
trong phng trnh (6), c th khng c ngha. Tuy nhin, yu
t thi gian khng sn xut cho nui cy m l rt c ngha, c bit khi
h ln men c thit lp li nhiu ln trong sut thi gian vn hnh, v v
th t
ii
s ti din nhiu ln.
Bn cht ca qu trnh lin tc trng thi n nh cng c thun li
do n d dng iu chnh hn h ln men m. Trong sut thi gian ln men
m, sn lng nhit, s sn xut kim hoc acid, v s tiu th oxygen s
bin thin t cc tc rt thp lc bt u ti cc tc rt cao trong
sut pha log mun. V vy, iu chnh mi trng ca mt h thng nh th
kh hn nhiu so vi qu trnh lin tc m trng thi n nh cc tc
sn xut v tiu th l hng s.
Nhc im thng xuyn ca h thng nui cy lin tc l s mn
cm ca chng vi s nhim bn bi cc c th bn ngoi. Ngn cn s
nhim bn l vn hng u khi thit k h ln men, xy dng v vn
hnh, v phi c khc phc bi mt cng ngh tt.
Sn xut cc sn phm ph c kt hp vi s sinh trng (v d
nh ethanol) s hiu qu hn trong nui cy lin tc. Nhng vic ng dng
nui cy lin tc sn xut cc sn phm sinh tng hp ca vi sinh vt
(ngc vi s d ha) gp nhiu hn ch. Mc d, v l thuyt c kh
nng ti u mt h thng lin tc c th tng hiu sut ca s trao i
cht, tuy nhin kh nng n nh trong mt thi gian di ca cc h thng
nh th l rt kh khn do s thoi ha ca chng vi sinh vt. Kho st v
ng hc ca nui cy lin tc cho thy rng h thng ny l s chn lc
cao v thch hp cho vic nhn ging nhng c th thch nghi tt nht trong
nui cy. S thch nghi tt nht trong phm vi ny c xem l i lc ca c
th i vi c cht c gii hn tc pha long ang hot ng.
Mc d cng nghip ln men min cng chp nhn nui cy lin
tc sn xut cc cht trao i ca vi sinh vt, nhng nhng tin b rt
ng k li thu c trong s pht trin cc h thng nui cy m c cung
cp dinh dng. Nui cy m c cung cp dinh dng c th c s dng
t ti mt mc rt ng k ca s iu chnh qu trnh v m rng
thi gian sn xut ca qu trnh nui cy m truyn thng m khng c cc
nhc im c hu ca nui cy lin tc c m t trn. u im
chnh ca cung cp thnh phn mi trng vo nui cy l cht dinh dng
c th c duy tr nng rt thp trong sut qu trnh ln men. Nng
cht dinh dng thp c th thun li trong mt s mt sau:
- Duy tr cc iu kin nui cy trong phm vi kh nng thng kh ca
h ln men.
- Loi b cc nh hng khc nghit ca cc thnh phn mi trng,
v d nh s dng nhanh cc ngun nitrogen, carbon v phosphate.
- Trnh cc hiu qu c ca thnh phn mi trng.
- Cung cp mt mc gii hn cht dinh dng cn thit cho cc
chng d dng.

IV. Cc sn phm ln men vi sinh vt
1. Ln men ru
Ru c con ngi sn xut v s dng rt lu, vo khong
6.000 nm trc cng nguyn. Do nhu cu v li ch ca sn phm ny nn
n nay vic nghin cu v m rng sn xut chng ngy cng c quan
tm. C rt nhiu loi ru v mi loi u c thnh phn v quy trnh sn
xut khc nhau, c th tm chia thnh ba loi ch yu sau: Ru trng
(ethanol), ru vang (wine) v ru mi (liquor).

1.1. Ru trng
Ru trng c sn xut bng hai phng php chnh: phng php
ln men vi sinh vt v phng php ha hc. Tuy nhin, phng php ln
men vi sinh vt l phng php ch yu. y l qu trnh ln men ru ca
nm men v mt s vi sinh vt khc, trong nm men l i tng chnh
c s dng sn xut ru quy m cng nghip (Hnh 3.4). Ln men
ru l mt qu trnh phc tp chuyn ng thnh ru, c s tham gia
ca nm men trong iu kin ym kh. Phng trnh tng qut ca ln men
ru nh sau:
C
6
H
12
O
6
2C
2
H
5
OH + 2CO
2
+ 27 kcal
Quy trnh sn xut ru trng bng phng php ln men ru bi
nm men c thc hin qua cc bc sau: Ch bin nguyn liu thnh dch
ng, ln men bin ng thnh ru, chng ct v tinh ch ethanol.
Trong , ln men bin ng thnh ru l giai on quan trng nht trong
sn xut ru, quyt nh cht lng sn phm to thnh. Sau khi dch
ng ha c x l, ngi ta b sung thm mt s thnh phn cung
cp thm vitamin v amino acid nh mui ammonium, mui phosphate,
dch thy phn nm men. Mi trng c thnh phn nh trn c th s dng
ln men. Ging c s dng ch yu trong ln men ru l cc chng
nm men Saccharomyces cerevisiae c tc pht trin mnh v hot lc
ln men cao, ln men c nhiu loi ng khc nhau v c tc ln
men nhanh, c kh nng chu c ethanol cao t 10-12%.

Hnh 3.4. Nh my sn xut ethanol quy m nh

Mi trng ln men sau khi c kh trng cn c ng t 90-
120 g/L v pH trong khong 4,5-4,8. Thi gian ln men t 65-72 gi, trong
10 gi u c sc kh nm men sinh si ny n, sau cho ln men
tnh (ym kh). Qu trnh ln men ru qua cc bc sau: ng v cc cht
dinh dng ca mi trng ln men c hp th vo trong t bo nm men
qua mng t bo v tham gia vo qu trnh trao i cht, ru ethanol v
CO
2
to thnh lin thot ra khi t bo, ru ethanol tan tt trong nc do
vy n khuch tn rt nhanh vo mi trng chung quanh. Kt thc ln men
ru, sau khi loi b t bo nm men, mun c ru tinh khit cn
chng ct dch ln men loi b tp cht. K thut chng ct ru nh
hng rt ln n cht lng ru thu c.

1.2. Ru vang
,
- .
c (Hnh 3.5).

Hnh 3.5. Mt dy chuyn sn xut ru vang

2
ngn cn
cc phn ng
.
Sac.
ellipsoideus, Sac. cerevisiae, Sac. oviformis
bao gm ba g
, b
.
, gn
. Qu trnh
gn lc v ln men ph c th lp li nhiu ln c dung dch trong sut.
. ,
, do cn h th ru ni mt mt thi gian lu
ru c chn v c cht lng hon ho.

2. Sn xut enzyme
ng dng thng mi chnh ca cc enzyme vi sinh vt l trong cng
nghip thc phm v sn xut bia mc d enzyme c tha nhn trong
cc ng dng phn tch v chn on bnh, cng nh trong sn xut bt
git. Hu ht cc loi enzyme c tng hp trong pha log ca nui cy m
v c th, v th, c xem nh cc cht trao i s cp. Tuy nhin, trong
mt s trng hp amylase (Bacillus stearothermophillus) c sn xut
bi nui cy idiophase v th c th xem l tng ng vi cc cht trao
i th cp. Cc enzyme c th c sn xut t ng-thc vt cng nh
cc ngun vi sinh vt, nhng sn xut bng ln men vi sinh vt l phng
php kinh t v thch hp nht. Hn na, hin nay nh cng ngh DNA ti
t hp ngi ta c th chuyn gen vo cc t bo vi sinh vt sn xut cc
enzyme ca ng-thc vt (Hnh 3.6).
Cc tin b ca cng ngh DNA ti t hp m rng phm vi cc
sn phm ln men tim tng ca vi sinh vt. C kh nng a cc gen t cc
c th bc cao vo cc t bo vi sinh vt nh l cc t bo nhn tng hp
cc protein (bao gm enzyme) ngoi lai. Cc t bo vt ch dng trong
nhng trng hp ny l E. coli, Sac. cerevisiae v mt s loi nm men
khc.

2.1. Cc loi enzyme vi sinh vt
Trong qu trnh sinh trng, cc enzyme c hnh thnh trong t bo
v mt s c tit ra mi trng xung quanh. Trong sn xut ch yu l
sn phm ca enzyme ngoi bo, cn nu mun tch enzyme ni bo th
phi ph v t bo. Cc vi sinh vt c dng trong sn xut enzyme gm
c vi khun, nm mc, nm men v x khun.
Cc ch phm enzyme c sn xut t vi sinh vt c ng dng
trong nhiu ngnh cng nghip khc nhau, ch yu l cc enzyme thy
phn: amylase, protease, pectinase, cellulase

Hnh 3.6. Sn xut enzyme quy m cng nghip

2.1.1. Amylase nm mc
Nhiu chng nm mc c kh nng sn xut enzyme amylase.
Amylase nm mc c cc loi sau:
- o-amylase c tc dng thy phn tinh bt thnh maltose, glucose v
cc dextrin c phn t lng khc nhau.
- Glucoamylase c tc dng thy phn tinh bt, glycogen v
polysaccharide. Enzyme ny c dng trong sn xut ru, chuyn nhng
dextrin c phn t lng cao khng ln men thnh nhng hp cht ln men
c v do nng cao c hiu sut nu ru t cc nguyn liu l tinh
bt.
- o-glucosidase thy phn maltose thnh glucose.
- Dextrinase thy phn isomaltose, panose v dextrin thnh nhng loi
ng c th ln men c.

2.1.2. Amylase vi khun
Mt s vi khun c kh nng sinh ra nhiu enzyme o-amylase.
Amylase vi khun ch c kh nng phn hy tinh bt mnh v to thnh
nhng o-dextrin phn t lng cao bt mu vi iodine. Enzyme o-amylase
vi khun c dng trong sn xut ng mt ng v chocolate, trong sn
xut bia, ch bin dextrin vi dch ng sn xut thc n cho ngi gi
v tr em, trong sn xut nc qu v trong y hc.
Dextrinase nm mc v amylase vi khun cn c s dng rng ri
trong cng nghip dt v giy.

2.1.3. Protease
Protease l nhm enzyme thy phn cc lin kt peptide trong phn t
protein hoc cc polypeptide.
- Protease thy phn protein thnh cc peptide c phn t lng nh
(peptone v polypeptide). Tip theo l s phn hy cc peptide trn thnh
cc amino acid t do di tc dng ca peptidase.
- Protease c dng nng cao gi tr dinh dng ca tht c, thy
phn protein ca sa ch bin nhng mn n king c bit, c dng
trong thuc da, sn xut bt git, phim nh, t si, len d v trong y hc.
Protease vi sinh vt c th s dng cng vi amylase trong ch bin thc n
gia sc.

2.1.4. Pectinase
L nhm enzyme thy phn pectin to thnh galacturonic acid,
glucose, galactose, arabinose, methanol Pectinase c nhiu loi, nhng c
hai loi c nghin cu nhiu hn c l pectinesterase v
polygalacturonase.
- Pectinesterase c tc dng thy phn cc lin kt ester trong phn t
pectin, tch nhm metocyl to thnh methanol v polygalacturonic acid.
- Polygalacturonase thy phn pectinic acid v cc polygalacturonic
khc, tch cc gc D-galacturonic acid t do.

2.1.5. Cytolase
Vi sinh vt (c bit l nm mc) sn sinh ra h enzyme c hot tnh
cao c th phn hy hemicellulose, pentozan, lignin Cc enzyme ny
c gi chung l cytolase (bao gm cellulase, hemicelllulase, pentosinase).
Cellulase tc dng phn hy cellulose thnh cellobiose, ri sau tip
tc thy phn ti glucose. Vic phn lp cc chng vi sinh vt sn xut
cellulase c hot tnh cao v tch enzyme ny ra di dng tinh khit vn
cn gp nhiu kh khn. V vy, hin nay cha sn xut c enzyme ny
quy m cng nghip, song vic s dng n trong cc ngnh kinh t v cng
nghip c nhiu tim nng. V d cytolase c th dng trong cng nghip
bia phn hy cc v ht khng phi v mch, trong sn xut nc qu,
trong ch bin bnh m, trong cc qu trnh gia cng thc phm nng cao
gi tr dinh dng, cng nh trong sn xut thc n gia sc.

2.1.6. Invertase
Invertase ca nm mc v nm men u thy phn saccharose, nhng
c ch tc dng ca chng hon ton khc nhau. Invertase ca nm mc l
glucosidase, tc dng ln u glucose ca saccharose. Cn invertase ca
nm men l fructosidase, tc dng ln u fructose ca saccharose.
Invertase l enzyme ni bo v ch thot ra mi trng khi t bo b
phn hy. Enzyme ny c dng rng ri trong sn xut bnh ko, ru
mi, kem, mt ong nhn to. N lm tng v ngt khi thy phn ng
saccharose thnh fructose v glucose, lm tng ha tan ca saccharose
trong sn phm.

2.1.7. Enzyme oxy ha glucosooxydase-catalase
Glucosooxydase l enzyme oxy ha kh, ch tc dng ln |-D-glucose
khi c mt oxygen, n oxy ha glucose thnh gluconic acid v H
2
O
2
. Di
tc dng ca catalase (mt enzyme hay i cng vi glucosooxydase) H
2
O
2

s b kh thnh H
2
O v O
2
.
Glucosooxydase-catalase c th loi b oxygen khng kh khi mi
trng. V vy, chng c dng bo v nhng nguyn liu, vt liu
khc nhau trnh oxy ha bi khng kh. S dng nhng enzyme ny cho
php ko di thi gian bo qun thc phm (cc dch c c, cht bo, bia,
ru vang, nc ung, sa). ng thi chng cng c s dng rng ri
trong y hc t nm 1950 cha bnh.

2.2. Sinh tng hp enzyme cm ng
yme
v .
Asper. oryzae
phng p
-
-amylase.
Asper. awamori trn
-
.
-
a .
:
.
.
Mun tng hp c enzyme cm ng cn phi c bn iu kin:
- C gen tng ng trong th nhim sc ca t bo.
- C y cc nguyn liu xy dng cc phn t enzyme (cc
amino acid v cc hp cht coenzyme nu enzyme gm hai cu t).
- Nng lng cn thit dng cho vic tng hp enzyme.
- Cht cm ng, nu khng c cht cm ng th d c ba iu kin
trn cng khng th tng hp c enzyme.
Nh vy, c th coi vic c cht cm ng l iu kin rt cn thit
thu c nhng enzyme mong mun. Trong cng nghip sn xut enzyme
cn phi la chn nhng cht cm ng thch hp v xc nh nng ti u
ca n trong mi trng c hiu sut sinh tng hp cao nht.

2.3. Nhng phng php nui cy vi sinh vt sn xut enzyme
Cng ngh sn xut enzyme hin nay trn th gii ng dng hai
phng php: nui cy b mt v nui cy chm.
Trong nui cy b mt, vi sinh vt mc trn b mt mi trng rn
(Hnh 3.7) hoc lng. Cc mi trng rn trc khi nui cy vi sinh vt cn
c lm m. Vi sinh vt pht trin s ly nhng cht dinh dng trong mi
trng v s dng oxygen phn t ca khng kh h hp. m bo
cho vi sinh vt mc u trn b mt mi trng v s dng c nhiu cht
dinh dng sinh ra enzyme, nhng lp mi trng rn cn phi mng (ch
dy khong 2-5 cm). iu ny dn n mt nhc im c bn ca phng
php ny cn phi c mt bng sn xut ln v chi ph lao ng chn tay
nhiu.
Trong nui cy chm, vi sinh vt hiu kh ch s dng c oxygen
ha tan trong mi trng, v vy trong qu trnh nui cy phi sc kh v
khuy lin tc. Phng php nui cy chm hin i hn, d c kh ha v
t ng ha, vic t chc quy m ln tng i d dng v n gin. Vi
phng php ny c th dng cc chng vi sinh vt t bin c kh nng
sinh tng hp enzyme cao v la chn cc thnh phn mi trng thch hp,
cc iu kin nui cy ti u.
Phng php nui cy b mt trn mi trng rn cng c mt s u
im so vi phng php nui cy chm, l: nng enzyme to thnh
mi trng rn cao hn nhiu ln, khng cn cc thit b phc tp, ch yu
nui trn khay v bung nui gi nhit v m thch hp, qu trnh
sn xut tiu tn t nng lng. Trong phng php nui cy b mt vi sinh
vt c nui cy trong iu kin khng v trng tuyt i. Nu c vi sinh
vt tp nhim th ch cn loi b phn . Cn trong nui cy chm cn phi
gi v trng tuyt i trong tt c qu trnh, nu b nhim th h hng ton
b v c th phi b i hon ton. Khi nui cy chm khng nhng ch cn
v trng qu trnh nhn ging, ln men, m cn phi m bo v trng i
vi khng kh thi vo mi trng.

Hnh 3.7. Ln men trn mi trng rn. A: ln men k kh trong ni bng t
nung, B: ln men hiu kh.

2.3.1. Phng php nui cy b mt
Nui cy nm mc v mt s vi khun theo phng php b mt
sn xut enzyme thng dng mi trng rn, mt s trng hp c th
dng mi trng lng.
Mi trng rn thng l cc nguyn liu t nhin nh cm, i khi
dng go tm, ng, b bia, b c ci ng, khoai ty, li ng hoc hn
hp nhng nguyn liu ny. Mi trng lng thng l r ng, dch thy
phn t thc mm, nc b ru c pha thm mui khong.
m bo cc cht dinh dng trong mi trng ngi ta c th
b sung cc ngun N, P, K hoc cc cht sinh trng (nc khoai ty, cao
A B
ng). m 58-60% tng i thch hp vi nhiu chng nm mc nui
cy b mt trn khay h. Tuy nhin, m 60% vi khun d pht trin, d
gy tp nhim, kh thng kh. Trng hp m t 45-50%, khi nui cy
mi trng s kh nhanh, sinh bo t yu v lm gim hot tnh ca enzyme
to thnh. Trong thi gian nui cy, nn gi m ca mi trng 50-
60%, mun vy m khng kh phng nui cy phi khong 90-100%.
Tuy rng, nui cy b mt khng cn iu kin v trng tuyt i
nhng mi trng (c bit trong qu trnh nhn ging) cng cn c v
trng cho ging pht trin bnh thng nht l giai on u. Trong sn
xut cn phi v trng mi trng rn 1-1,5 atm bng hi nng trong 45-
60 pht. Nu mi trng trc khi v trng c trn vi chlohydric acid
hoc sulfuric acid n pH thch hp, hay thm mt t formalin hoc mt s
cht st trng khc th ch cn v trng di 0,2-0,3 atm. Thm acid v
gi mi trng pH nht nh s gip cho mt vi enzyme to thnh c
nhiu hn.
Mi trng c dn mng ra cc khay v trng dy khong 2-
2,5 cm, ngui ti 30
o
C th tin hnh cy ging. Ging c nhn cng
theo phng php b mt hoc bng bo t thu c theo phng php tch
bo t khi mi trng nhn ging v cha vo cc bnh nt kn hoc trong
cc ti polyethylene. Trong nui cy nhn ging thng mc pht trin
n gi sinh ra nhiu bo t. T l nhn ging khong 0,2-2%. Mi gram
bo t mc c th cy vo 10 kg mi trng. Cc khay c mi trng
cy mc c t vo phng nui c sn cc gi. Phng nui c th iu
chnh c nhit , m v c thng gi. Nhit thch hp vi a s
mc l 30-32
o
C, nu nhit xung di 24
o
C nm pht trin chm, sinh
bo t yu, thi gian nui cy di dn n gim kh nng sinh tng hp
enzyme. Thi gian nui cy nm mc khong 36-60 gi.
Qu trnh nui cy nm mc trn b mt mi trng bao gm ba thi
k:
- Khong 10-14 gi u. Bo t bt u ny mm, thi k ny cha
hnh thnh enzyme khng i hi phi thng kh nhiu, ch cn lm thong
khong 2-3 th tch khng kh/th tch phng/gi. Ging rt nhy cm vi
nhit nhng gi ny, nhit bung nui cn gi 29-31
o
C .
- Thi k gia khong 14-18 gi. Mc pht trin nhanh, h hp
mnh. Si nm c th quan st thy bng mt thng, lc u lp lng c
mu trng xm v ngy cng r, lm mi trng kt bnh li. C th phi lt
mi trng, b nh ra si nm mc tt hn. Cc cht dinh dng trong
mi trng tiu hao nhanh phc v cho qu trnh trao i cht trong t
bo v ging h hp mnh ta ra mi trng chung quanh 80-90 kcal/gi,
lm nhit mi trng c th tng ln n 37-40
o
C hoc hn. Thi k ny
cn phi thng kh mnh, ti 60 th tch kh/th tch phng/gi cung cp
O
2
cho mc v ui CO
2
ra khi mi trng, ng thi lm gim nhit
bung nui. Nhit bung nui giai on ny cn gi 28-29
o
C v
m trong phng khong 100%.
- Thi k cui khong 10-20 gi. Cc qu trnh trao i cht vn tip
tc nhng yu dn, nhit mi trng gim xung v vic to thnh
enzyme ca t bo vn tip tc. Nhit lng ta ra khong 15-30
kcal/kg/gi. Thng kh khng qu 20-25 th tch khng kh/th tch
phng/gi, gi nhit bung nui 30
o
C.
Ty thuc vo c tnh sinh l ca tng loi mc, thi gian nui cy
c th kt thc ti im m lng enzyme to thnh ti a.

2.3.2. Phng php nui cy chm
Nui cy vi sinh vt sinh enzyme theo phng php chm c thc
hin trong cc bnh ln men c cnh khuy v sc kh lin tc (Hnh 3.8 v
3.9). Qu trnh tng t nh trong sn xut amino acid, khng sinh
Khng th c mi trng nui cy chung cho tt c cc chng vi sinh
vt, v vy cn phi chn thnh phn mi trng, t l cc cht dinh dng
sao cho thch hp vi tng chng, c bit phi ch ti cht cm ng cn
thit cho vi sinh vt sn sinh ra enzyme mc ti a. Trong nhiu mi
trng nui cy chm,
Aspergillus,
.


Hnh 3.8. Ln men bng phng php nui cy chm trong mi trng lng
quy m phng th nghim (5 L)

Thnh phn khong trong mi trng cng rt c ngha. Trong mi
trng nui cy mt s chng Asper. oryzae ngoi tinh bt v nitrate cn
cn thm MnSO
4
. Nu thiu MnSO
4
mc vn pht trin bnh thng, nhng
amylase khng c to thnh (trong phn t amylase c cha nhng amino
acid mang S v Mn). Mi trng c v trng trong thit b ring hoc
trong bnh ln men 121-125
o
C/45-60 pht. Mi trng trc khi v trng
cn c dch ha s b trnh tnh trng tinh bt h ha lm mi trng
c st hoc c dnh cao.
Sau khi lm ngui mi trng n nhit thch hp s tin hnh tip
ging. Ging c cy t ng nghim qua cc bnh tam gic, t trn my
lc, ri nui bnh nhn ging c th tch bng 5-10% th tch bnh ln men
t 24-36 gi. Nh vy, n
. Cy ging mc bo
t theo phng php chm s ko di thi gian ny mm v cng ko di
ton b qu trnh nui cy. Mi trng nhn ging c th dng cc hp cht
nitrogen d tiu i vi vi sinh vt m trong qu trnh nui cy vn nng cao
c hot tnh sinh tng hp. T l tip ging nm trong khong 2-5%,
nhng mt s chng t l ny thp hn nhiu (0,5-0,6%).
Sinh tng hp enzyme theo phng php nui cy chm thng
khong t 2-4 ngy. a s cc enzyme thy phn do nm mc, x khun to
thnh c tit ra mi trng xung quanh, phn cn li trong h si sau ba
ngy nui cy khong 10-15%. pH mi trng c mt ngha rt ln,
pH thch hp cho sinh tng hp o-amylase l 7-8, glucoamylase l 4,5-5.
Khi dng cc mui ammonium lm ngun nitrogen, qu trnh pht trin vi
sinh vt s acid ha mi trng cn khi dng nitrate lm ngun nitrogen
mi trng s b kim ha.

Hnh 3.9. Ln men bng phng php nui cy chm trong mi trng lng
quy m pilot (200 L)

S sc kh khng nhng nh hng n sinh trng ca vi sinh vt m
cn nh hng n s to thnh enzyme. Tc s dng oxygen cao nht
ca nm mc sau khong 24 gi nui cy ri gim dn. Tng nng tt c
cc cht dinh dng v oxygen ha tan trong mi trng c th nng cao
c kh nng sinh tng hp o-amylase.

2.4. Tch v tinh sch ch phm enzyme
2.4.1. Ch phm enzyme t mi trng nui cy b mt
chit rt enzyme t mi trng rn ngi ta dng nc, cc dung
dch mui trung tnh, cc dung mi hu c (ethanol, acetone). Nhiu nghin
cu cho thy dng nc c kt qu tt v d p dng trong sn xut. C th
chit c lng enzyme trn 90-95% v trong nc chit khng cha cc
tp cht khng tan. Nc thng dng khuch tn ha tan nhit 25-
28
o
C. trnh tp nhim nn thm vo nc mt t formalin hoc cht st
trng khc. Dch chit thu c c mu nu sm, kh trong, cha 10-15%
cht kh ha tan v c lm lnh kp thi xung 10-12
o
C.
Dch chit c c c chn khng ti 50-55% cht kh ha tan. Dch
m c ny c th bo qun lu di m khng mt hot tnh v rt d ha
tan. Dch chit c th khng cn phi c c m a ngay vo my sy phun
v s thu c sn phm dng bt.
Phng php tch chit v lm sch enzyme c s dng rng ri
nht hin nay l phng php kt ta enzyme bng dung mi hu c
(ethanol, isopropanol v acetone). Cc dung mi hu c ny lm gim hng
s in mi ca mi trng. Nh ta bit, lc ht tnh in t l nghch
vi hng s in mi. V vy, cc enzyme-protein cng nh cc cht c
phn t lng thp trong h dung dch nc-dung mi hu c s kt ta v
lng xung. ha tan ca enzyme vo dung dch ethanol-nc ph thuc
vo nng ethanol, nhit , pH, lc ht ion ca dung dch v tnh cht
protein ca enzyme. Thng thng, ngi ta thm 3-4 th tch ethanol vo
mt th tch nc chit enzyme. trnh mt hot tnh ca enzyme tt c
phi c lm lnh xung 3-5
o
C. Khi trn phi khuy mnh, khi cc
enzyme kt ta v lng xung dui cn tch ly tm ngay. Enzyme c ra
2-3 ln bng ethanol cao , ri a vo bnh ht m hoc my ng kh
chn khng, sn phm thu c s c dng bt. Dng isopropanol kt ta
enzyme ch cn 1,5-2 th tch dung mi/1 th tch dch chit. Cc enzyme
tch ra s dng sa c qunh rt kh sy. Dng acetone vi t l nh khi
dng isopropanol, nhng k thut phng trnh chy n trong sn xut l rt
kh khn.
Phng php th hai tch enzyme l dng mui trung tnh kt
ta. Ch phm enzyme thu c c hot lc cao hn so vi vic tch bng
dung mi. Mui trung tnh thng dng l (NH
4
)
2
SO
4
vi t l 50-66% (c
khi cao hn) so vi dch chit enzyme. (NH
4
)
2
SO
4
pha thnh dung dch bo
ha ri cho vo dch ln men. Dch ln men c th s b c c trong thit
b chn khng v nh vy s cn dng mt lng (NH
4
)
2
SO
4
t hn. Ch
phm enzyme thu c c ln (NH
4
)
2
SO
4
, v vy mun s dng rng ri cn
phi loi mui bng cch thm tch qua mng bn thm.

2.4.2. Ch phm enzyme t dch nui cy chm
Dch nui cy chm sau khi lc sinh khi vi sinh vt v cc tp cht
rn khng tan cn khong 1-3% cht kh ha tan, trong c cc enzyme.
V nguyn tc tch enzyme t dch lc nui cy chm cng tng t nh
tch t dch chit trong mi trng rn nui cy b mt. Dch lc cn phi
c gim th tch t 4-10 ln trong iu kin chn khng 25-30
o
C, ri
tin hnh tch enzyme.
Ngoi phng php c chn khng, c th tin hnh theo phng
php hp ph qua nha trao i ion hoc cc cht c hot tnh b mt, sau
li tin hnh phn hp ph. Tin hnh nhiu ln v dch thu c cha o-
amylase ca nm mc v vi khun c th c hp ph li bng tinh bt
khoai ty hoc ng c x l s b bng nhit. Trc khi cho hp ph
cn cho thm 20% (NH
4
)
2
SO
4
nng cao kh nng hp ph ca tinh bt.
Tr amylase c hp ph cn cc enzyme khc s li trong dch. Tinh
bt c amylase c sy kh (khng cn phn hp ph) v em s dng
trong k thut sn xut cc sn phm cha tinh bt.
Hin nay, cn mt s phng php tng i phc tp khc kt
tinh v tch enzyme nh lc gel (gel filtration), in di (electrophoresis),
siu ly tm (ultracentrifuge)

3. Sn xut khng sinh
3.1. Penicillin
Penicillin l khng sinh c tm ra u tin v c sn xut sm
nht dng cha mt s bnh nhim khun vo nhng nm u ca Th
chin th 2.
Nhng vi sinh vt sinh penicillin thuc cc ging nm mc Penicillum
v Aspergillus. Nhng cc chng ca Penicillum notatum v Pen.
chrysogenum c hot lc cao v c dng trong cng nghip khng sinh.
Tuy nhin, nhng chng Penicillum c hot lc cao li thng km n nh.
Do , mt vn kh khn c t ra l to c kh nng sinh khng
sinh cao nht, gi c n nh trong qu trnh nghin cu v sn xut.
Nhim v ny c mt ngha rt ln trong cng nghip. Ngy nay, nh k
thut di truyn hc ngi ta to c nhng ging n nh, t nht sau
su th h vn khng gim hot tnh khng sinh.
Qu trnh ln men penicillin thuc vo loi ln men hai pha: pha sinh
trng v pha sinh penicillin. Ngun carbon trong ln men penicillin bng
nm Pen. chrysogenum c th l glucose, saccharose, lactose, tinh bt,
dextrin, cc acid hu c (lactic, acetic, formic), cc amino acid... Tuy nhin,
ng lactose cho hiu sut penicillin cao nht v thng c dng trong
cng nghip. Nhng do nm s dng ng lactose chm, v vy trong thc
t lactose c dng phi hp cng vi ng khc (glucose, saccharose...)
trong mi trng dinh dng.
Trong pha ln men th nht ging pht trin mnh, s dng glucose
v lactic acid ca cao ng. Sau , lactose mi c s dng (ch yu trong
pha th hai to thnh penicillin). Khi trong mi trng cn lactose v khng
b sung cc cht dinh dng, h si nm s bt u t phn, nu tip tc ln
men nng penicillin s gim.Trong thc t sn xut cn phi kt thc ln
men trc thi im ny.

Phng php sn xut penicillin
Sn xut penicillin cng nh cc ch phm sinh hc khc, da trn c
s nui cy vi sinh trn mi trng rn hoc lng. Trong qu trnh nui cy,
ging vi sinh vt pht trin s tch t cc sn phm trao i cht trong mi
trng hoc trong sinh khi. Quy trnh cng nghip sn xut penicillin da
trn nm mc Pen. chrysogenum c kh nng sinh penicillin cao, theo hai
phng php:
- Ln men b mt. Phng php ny c p dng trong thi gian u
ca cng nghip khng sinh. Mi trng nui cy b mt c th l cc c
cht rn hoc lng. (1) C cht rn c th l cm hoc cc loi ht, cm
c lm t ri tri ln khay mt lp dy khong 2 cm, ging nm mc
c trn vo mi trng c m 50-60% v trng ngui ti 30
o
C.
Thi gian ln men 6-7 ngy 24-28
o
C trong cc bung c iu chnh
nhit , m v thng gi. Ni chung, phng php ny ging nh ln
men cc enzyme bng nm mc. (2) Mi trng lng dng ngun carbon l
lactose, cao ng v mt s nguyn t khong. Ging c cy vo mi
trng ri ln men 24
o
C khong 6-7 ngy t hiu sut khong
193 unit/mL penicillin. Ngy nay, phng php ln men chm thay th
phng php ln men b mt.
- Ln men chm. Thnh phn mi trng gm c cao ng, glucose,
lactose v cc mui khong. Ging dng trong cng nghip thng dng
bo t. Bo t c nui trn cc bnh nhn ging c cnh khuy v sc kh
36-50 m
3
/gi h si nm pht trin, sau chuyn vo cc bnh ln men.
Qu trnh ln men penicillin bng nm mc Pen. chrysogenum 261
o
C
trong khong 120-125 gi. Trong qu trnh ln men pha th nht nm pht
trin h si mnh, sinh khi tng nhanh, cc ngun carbon d ng ha
(glucose, saccharose) cng cc ngun nitrogen c tiu hao nhanh, cng
h hp tng dn n cc i, pH tng v penicillin c to thnh t.
Sang pha th hai h si nm pht trin chm, lactose c tiu hao dn, pH
tng n khong 7-7,5 v penicillin c to thnh ch yu trong pha ny.
Nu ngun carbon trong mi trng cn v sinh khi nm mc bt u t
phn th pH c th tng ti 8 hoc hn, lng penicillin c to thnh
trong mi trng s gim. V vy, qu trnh ln men cn c kt thc trc
thi im h si nm mc bt u t phn. Nm mc sinh penicillin rt hiu
kh, cho nn qu trnh nui cy (nhn ging v ln men) cn phi sc kh v
khuy mi trng m bo ha tan oxygen cn bng vi nhu cu sinh
l ca chng. Nu khng oxygen hiu sut penicillin c th gim ti hai
ln.

3.2. Streptomycin
Streptomycin l mt khng sinh dng ph bin trong y hc, th y v
bo v thc vt. Schatz v cs (1944) pht hin ra streptomycin t dch
nui cy mt chng x khun Streptomyces griseus (cn gi l Actinomyces
streptomycin).
Ging x khun sinh streptomycin khi nui cy chm pht trin thnh
hai pha:
- Pha th nht (pha sinh trng mnh). Cc bo t ny chi v mc
thnh si sau 6-8 gi, mi bo t mc mt chi, khun ty thng thng v
phn nhnh rt yu, t bo cht a kim.
- Pha th hai (khun ty khng pht trin). Cui ngy th ba si x
khun b chia nh v bt u t phn.
Nhng ging sinh streptomycin rt khng n nh. Do , trong tng
lai cn c s can thip ca k thut di truyn to ra nhng ging c hot
lc cao v n nh a vo sn xut. Gi bo t dng ng kh trong
khong nm nm c th cn 96-99% hot lc, trong ct thch anh ti ba
nm, trn mi trng thch nc u 5
o
C ti mt nm. Cc ngun carbon
m ging Streptomyces c th ng ha c v sinh khng sinh l glucose,
tinh bt, dextrin, maltose, fructose, galactose, manose. Trong thc t,
glucose v tinh bt c dng lm ngun nguyn liu trong sn xut
streptomycin.

Phng php sn xut streptomycin
Ln men streptomycin c thc hin theo phng php nui cy
chm. Qu trnh ln men ny cng ging nh ln men cc loi khng sinh
khc, bao gm cc giai on: Nhn ging v ln men chnh.
- Nhn ging. Ging x khun c bo qun dng bo t. Cy bo
t vo mi trng nhn ging trong bnh tam gic lc 180-220 vng/pht
26-28
o
C/30-70 gi, sau cho tip vo cc bnh nhn ging (c sc kh v
khuy), nui tip cho pht trin sinh khi 20-40 gi. Nhim v chnh trong
giai on nhn ging l to ra mt khi lng ln khun ty x khun a
kim c kh nng pht trin mnh trong giai on ln men chnh v to
thnh mt lng ln khng sinh.
- Ln men. Ln men streptomycin l qu trnh ln men hai pha in
hnh. Nhit ln men khong 26-28
o
C, thi gian ln men 96 gi. Trong
thi gian ln men cn phi thng kh v khuy trn mi trng. Lng
khng kh thi qua mi trng trung bnh l 1 th tch kh/1 th tch mi
trng. Khuy trn mi trng lin tc trong sut c qu trnh ln men (k
c khi nhn ging) nu ngng khuy ch trong mt thi gian ngn s lm
gim hiu sut streptomycin. pH trong nhng gi u c gim cht t sau
tng dn.

3.3. Tetracycline
Tetracycline l mt dy cc cht khng sinh c cng mt nhn chung
tetracycline (v d: tetracycline, chlotetracycline, oxytetracycline,
dimethyltetracycline) v mt s nhm chung c trong phn t (v d
nhm dimethylamino -N(CH
3
)
2
, nhm amide CONH
2
). Tetracycline c
dng rng ri trong y hc v th y. Tetracycline c th c sn xut bng
ln men x khun Streptomyces aureofaciens. Tetracycline c tm thy
vo nm 1953 bng cch kh halogen trong phn t chlotetracycline. Lc
u phng php ny c dng trong cng nghip nhng gi thnh sn
phm rt t, sau ngi ta tm thy cht khng sinh ny c trong dch
nui cy x khun sinh chlotetracycline l Strep. aureofaciens.
Ging x khun c kh nng tng hp tetracycline v chlotetracycline
l Strep. aureofaciens, cn ging sinh oxytetracycline l Strep. rimosus.
Ngun carbon dng trong nui cy Strep. aureofaciens l glucose (tch t
nhiu tetracycline), cn Strep. rimosus cho nhiu oxytetracycline trn mi
trng maltose.
Trong qu trnh ln men, pha th nht cc cht dinh dng tiu hao
nhanh. Trong khong 24-48 gi nui cy khi khun ty c 70-80%
mc ti a v 60-80% cc cht dinh dng c s dng. Bc sang pha
ln men th hai cc ging x khun ny u pht trin chm li, tc s
dng cc cht dinh dng gim i rt nhiu, pht trin khun ty chm li
dn, t ti mc cc i v n nh ri bc vo giai on t phn.
Khng sinh tch t ti a 110-120 gi.

Phng php sn xut tetracycline
Ln men tetracycline (tetracycline, chlotetracycline, oxytetracycline,
dimethyltetracycline) theo phng php nui cy chm. Qu trnh ln men
y ging nh ln men cc ch phm khc, bao gm cc giai on: nhn
ging v ln men.
- X khun Strep. aureofaciens dng trong ln men tetracycline v
chlotetracycline hoc cc halogentetracycline khc. Cy bo t vo mi
trng nhn ging trong bnh tam gic pH 6,8-7,0 lc 220-250 vng/pht
khong 24-40 gi. Sau , c tip tc nhn ging trong ni nh ri
chuyn vo mi trng ln men. Ln men tetracycline v chlotetracycline l
ln men hai pha in hnh.
- Ging Strep. rimosus c nhn ging bnh tam gic lc 220-250
vng/pht 27-28
o
C/48-72 gi, sau nhn tip tc trong ni c sc kh v
khuy ri chuyn sang mi trng ln men c iu kin tng t nhng ko
di t 5-7 ngy.

4. Sn xut acid hu c
4.1. Acetic acid
Acetic acid (CH
3
COOH) c th thu c bng phng php ln men
vi khun acetic. Acid ny (cn gi l dm n) c dng trong ch bin
thc phm, p chua rau qu. Qu trnh ln men nh vi khun acetic oxy
ha ru ethanol thnh acetic acid (Hnh 3.10).
C trn 20 loi vi khun c kh nng ln men acetic, chng c gi
mt tn chung l vi khun acetic. Trong mi trng ru ethanol (5-
13%) th sn phm ch yu l acetic acid, nu nng ru thp hn cc vi
khun acetic s oxy ha trit ru thnh CO
2
v H
2
O.
Vi khun acetic l bn a m v rt hiu kh, c tc sinh trng rt
nhanh t mt t bo sau 12 gi c th pht trin thnh 12 triu t bo. Trong
qu trnh sinh trng v pht trin chng to thnh acetic acid v nng
acid thp li kch thch s sinh trng ca chng. V vy, trong sn xut
dm c th dng ru khng cn v trng c b sung mt t acetic
acid ha mi trng, nhit ln men khong 25-32
o
C v sc kh mnh.

Hnh 3.10. Qu trnh ln men acetic acid

Cc loi vi khun acetic c gi tr nh: Acetobacter aceti, Ace.
pasteurianum, Ace. orleaneuse, Ace. xylium, Ace. schiitzenbachii, Ace.
curvum, Ace. suboxydans.
Ngun c cht ch yu trong ln men acetic l ethanol c b sung
thm mt t ng, ngun nitrogen v c hoc hu c, v mt s cht
khong khc. C ba phng php ln men acetic: (1) Phng php chm
cn gi l phng php Orlean hoc phng php Php, dng nc hoa qu
lm nguyn liu vi vi khun Ace. orleaneuse. (2) Phng php nhanh cn
gi l phng php c, phng php ny c p dng ch yu trong
Alcohol-dehydrogenase
CH
3
CH
2
OH CH
3
CHO + 2H
(Ethanol) (Acetaldehyde)
OH

CH
3
CH CH
3
COOH + 2H
(Acetic acid)
OH
(Acetaldehyde)
Aldehyde-dehydrogenase
cng nghip sn xut dm n trn th gii, vi khun c s dng l Ace.
schiitzenbachii hoc Ace. curvum. (3) Phng php ln men chm, y l
kiu ln men bn lin tc s dng cc chng vi khun ca loi Ace.
suboxydans.

4.2. Citric acid
Citric acid hay limonic acid (C
6
H
8
O
7
) c nhiu trong thin nhin, c
bit trong cc loi cy n qu c mi (h cam chanh-Rutaceae) c dng
ch yu trong ch bin thc phm v dc phm. Citric acid cng c th
c sn xut quy m cng nghip bng phng php ln men, nm mc
s chuyn ha ng thnh citric acid.
C ch sinh tng hp citric acid vi sinh vt c th biu din bng
phng trnh tng qut nh sau:
2C
6
H
12
O
6
+ 3O
2
2C
6
H
8
O
7
+ 4H
2
O
Cc nm mc sinh citric acid hiu kh, nhit thch hp cho pht
trin v ln men l 30-32
o
C. Ngun carbon tt nht i vi Asper. niger l
saccharose, cn i vi Citromyces l maltose. Nng ng trong mi
trng 10-20% l thch hp hn c. Cc ngun nitrogen v c dng trong
ln men citric acid tt nht l nitrate cn nitrogen hu c l nc chit u
nnh. Trong mi trng ln men cn ch cc nguyn t khong P, Mg, K,
Fe v Zn.
C hai phng php c dng sn xut citric acid l ln men b
mt (trn mi trng lng hoc rn) v ln men chm.
- Phng php ln men b mt trn mi trng lng. Phng php
ny c dng rng ri trong cng nghip sn xut citric acid. Lp vng
nm pht trin trn cc khay ln men cha mi trng dinh dng l dch
ng s chuyn ha ng thnh citric acid.
- Phng php ln men chm. Qu trnh ln men ging nh sn xut
khng sinh, c thc hin trong cc bnh ln men cha mi trng dinh
dng v ging nm mc. Sau khi kt thc ln men dng H
2
SO
4
chuyn
calcium citrate thnh citric acid.

V. Cng ngh ti t hp vi sinh vt
Khng c mt lnh vc no ca cng ngh sinh hc thc nghim li
pht trin nhanh chng nh cng ngh di truyn (genetic engineering) hay
cn gi l cng ngh DNA ti t hp (DNA recombinant technology), cng
khng c mt lnh vc no khc c th a ra nhiu loi sn phm mi v
hu ch n nh vy. Nguyn l c bn ca cng ngh ny l thao tc c
nh hng v c ch (a vo hoc loi b) DNA v cc loi nguyn liu
di truyn khc nhm lm thay i c tnh di truyn ca c th sinh vt.
Hu ht cc k thut u bt u bng s la chn mt gen mong
mun, tip theo l phn lp n v ct n bng cc enzyme hn ch. Gen ny
c gn vo mt vector to dng (plasmid) v sau a vo mt vt ch,
n s c dch m thnh mt protein c bit.

1. Cc vi sinh vt ti t hp
Mt trong nhng ng dng u tin ca cng ngh di truyn l to ra
mt chng Pseudomonas syringae. Chng hoang di (wild type) ca vi
khun ny thng thng cha mt gen to bng, gen ny kch thch s to
bng trn cc b mt lnh v m. S bin i gen ny to ra mt th ti t
hp c kh nng ngn nga s to thnh bng gi (frost). c to ra di
tn gi l Frostban, vi khun ny mang li mt thnh cng khim tn
trong vic chng li vic to thnh bng gi trn cc cy du ty v khoai
ty ngoi ng rung. Trong mt th nghim khc, mt chng virus c kh
nng dit su o bp ci c th vo mt tha rung bp ci. N c
thit k c th t ph hy sau mt thi k nht nh. Pseudomonas
fluorescens c ti t hp vi cc gen sn sinh cht dit cn trng sinh
hc Bac. thuringiensis. Cc vi khun ny c th vo t, chng s bm
vo r v gip tiu dit cc cn trng ang tn cng.
Ni chung, mi s phng thch cc th ti t hp vo mi trng u
phi c cc c quan bo v mi trng chun y v c gim st cht
ch. n nay, nhiu nghin cu cho thy cc vi sinh vt ny khng sng
st hoc sinh si trong mi trng v chc chn khng gy ra nhiu him
ha. Cc virus c thit k di truyn c bit c ch trong cc ng dng y
hc, chng hn sn xut vaccine.

2. Cc ng dng trong cng ngh vi sinh
Mt iu lm cng ngh di truyn tr nn c bit hp dn l cc k
thut ca n c th kt hp vi cc k thut ca cng ngh ln men sn
xut ra nhng s lng ln cc cht ging khng sinh hoc steroid. Hu ht
cc protein ti t hp ang bn trn th trng hin nay u rt hu ch trong
y hc. Di y l mt s trong cc sn phm ny:
- Insulin, cht thay th hormone dng trong iu tr bnh tiu ng
type I.
- Hormone sinh trng ca ngi, c s dng iu tr nhng tr
em b bnh ln hoc bnh gi trc tui.
- Interferone, mt cht min dch c s dng iu tr mt s loi
ung th, vim gan mn tnh.
- Interleukin-2, mt cht hot ha t bo T v B c dng trong iu
tr ung th.
- Erythropoietin (EPO), mt cht kch thch cc t bo ty sng sinh
hng cu, dng iu tr mt s bnh thiu mu.
- Hot t plasminogen ca m (tPA), mt enzyme tham gia vo qu
trnh lm tan cc cc mu ng (huyt khi).
- Nhn t VIII, mt protein gy ng mu cho nhng ngi a chy
mu.
- Cc vaccine ti t hp cho bnh vim gan, v bnh vim mng no
do Hemophilus influenza B gy ra.
- Octolon, mt cht c ch min dch dng cho cc bnh nhn cy
ghp ni tng.

1. Trn Th Thanh. 2003. Cng ngh vi sinh. NXB Gio dc, H Ni.
3. Asenjo JA and Merchuk JC. 1995. Bioreactor System Design. Marcel
Dekker Inc. New York, USA.
New York, USA.
5. Klefenz H. 2002. Industrial Pharmaceutical Biotechnology. Wiley-VCH
Verlag GmbH, Weinheim, Germany.
6. Lee JM. 2000. Biochemical Engineering. Prentice Hall Inc. USA.
8. Shuler ML and Kargi F. 2002. Bioprocess Engineering-Basic Concepts.
2
nd
ed. Prentice Hall Inc. New Jersey, USA.
Chng 4

Cng ngh sinh hc thc vt

I. M u
Nui cy m thc vt l mt trong nhng lnh vc ng dng t nhiu
thnh cng ni bt ca cng ngh sinh hc thc vt. Bng cc k thut nui
cy trong iu kin v trng cc b phn tch ri ca c th thc vt, ngi
ta nhn ging in vitro thnh cng nhiu loi cy trng c gi tr m trc
y cc phng thc nhn ging truyn thng gp nhiu kh khn. Bn
cnh , mt s k thut khc cng c ng dng c kt qu nh: nui
cy n bi (1n) to dng thun chng phc trng ging cy trng, dung
hp protoplast gip m rng ngun gen to ra nhiu loi thc vt mang tnh
trng mi hu ch, chn dng bin d soma v bin d giao t c kh nng
chng chu cc iu kin bt li ca ngoi cnh nh nng-lnh, phn-mn,
kh hn, su-bnh, v cui cng sn xut cc cy trng sch bnh virus t
nhng c th nhim bnh virus.
, thit k cc vector
biu hin cao v xy dng cc k thut chuyn gen hin
1980,
.
. Hin nay, nhiu hp

cht t nhin dng lm dc phm hoc ph gia thc phm c sn
xut thnh cng bng phng thc nui cy t bo trn quy m cng nghip
cho hiu sut rt cao. c bit, vic sn xut cc protein ngoi lai iu tr
bnh trong h thng t bo thc vt ang c ch do chng an ton cho
ngi hn cc protein c ngun gc t t bo ng vt, bi v cc cht
nhim bn v virus thc vt khng phi l tc nhn gy bnh ngi.

II. Nui cy m v nhn ging in vitro
1. Thut ng hc (terminology)
Nui cy m (tissue culture) l thut ng dng ch qu trnh nui
cy v trng in vitro cc b phn tch ri khc nhau ca thc vt. K thut
nui cy m dng cho c hai mc ch nhn ging v ci thin di truyn (v
d: ging cy trng), sn xut sinh khi cc sn phm ha sinh, bnh hc
thc vt, duy tr v bo qun cc ngun gen qu Cc hot ng ny c
bao hm trong thut ng cng ngh sinh hc.
Thut ng nhn ging in vitro (in vitro propagation) hay cn gi l vi
nhn ging (micropropagation) c s dng c bit cho vic ng dng
cc k thut nui cy m nhn ging thc vt, bt u bng nhiu b
phn khc nhau ca thc vt c kch thc nh, sinh trng iu kin v
trng trong cc ng nghim hoc trong cc loi bnh nui cy khc.
Trong thc t, cc nh vi nhn ging (micropropagators) dng thut
ng nhn ging in vitro v nui cy m thay i cho nhau ch mi
phng thc nhn ging thc vt trong iu kin v trng. Thut ng ng
ngha l nui cy in vitro (in vitro culture).
Nhn ging in vitro v nui cy m bt u bng cc mnh ct nh
ca thc vt, sch vi sinh vt, v c nui cy v trng. Thut ng u tin
dng trong qu trnh nhn ging l explant (mu vt) tng ng vi cc
phng thc nhn ging khc l cutting (cnh gim), layer (cnh chit),
scion (cnh ghp) hoc seed (ht).
Nm thut ng khc c dng ch cc loi ti sinh sinh dng
(vegetative or somatic regeneration) c bn trong nhn ging in vitro v
nui cy m:

1.1. Nui cy nh phn sinh (meristem-tip culture)
Phng thc nhn ging bng cch dng cc phn rt nh ca nh
chi (shoot-tip) bao gm m phn sinh nh ring r (single apical
meristem) v mm l non (young leaf primordia) ko di chi (shoot
elongation) ngay sau . Kiu nui cy ny c dng ln u tin lm
sch virus (virus-free) thc vt. Nu dng nh phn sinh khng th sng
st v to r mt cch c lp, th c th thay th bng phng thc vi ghp
(micrografting).
Thnh cng in hnh trong phng thc ny l nhn ging cc cy
mt l mm nh hoa lan, da, hu v chui (protocorm hoc cm chi)...
hoc cy hai l mm nh khoai ty, c chua v cc (ko di chi)...

1.2. Sinh sn chi nch (axillary shoot proliferation)
Kiu nui cy ny s dng chi ca cc im sinh trng bn v ngn
ni m s ko di ca chi tn cng (elongation of terminal shoot) b km
hm v s sinh sn chi nch c y mnh. S iu khin ny cho php
nhn nhanh c cc chi in vitro (microshoots), l cc chi c th tch ra
v to r in vitro hnh thnh cy trong ng nghim (microplants), hoc n
c th c ct ra ring bit to thnh cc cnh gim in vitro (microcuttings)
to r bn ngoi in vitro.
Phng thc ny thng c p dng cho cc i tng hai l mm
nh cc, c chua, thuc l...

1.3. To chi bt nh (adventitious shoot induction)
Loi nui cy cho php hnh thnh cc chi bt nh hoc trc tip
trn mu vt hoc gin tip t m callus, m m callus ny hnh thnh trn
b mt vt ct ca mu vt. H thng nui cy ny c nhng yu cu tng
t vi nui cy m phn sinh nh, n ch khc v ngun mu vt v ngun
gc bt nh ca cc chi mi. Mt s loi mu vt c dng nh l on
thn (thuc l, cam, chanh, c chua, bp ci), mnh l (thuc l, c chua, bp
ci, c ph, ca cao), cung l (thy tin), cc b phn ca hoa (sp l, la
m, thuc l), nhnh c (h hnh, h lay n, h thy tin), on mm (mng
ty)...

1.4. Pht sinh c quan (organogenesis)
Thut ng ny dng m t qu trnh ti sinh cc chi hoc/v r bt
nh t cc khi t bo callus. Qu trnh ny xy ra sau thi im m mu
vt c t vo mi trng nui cy v bt u cm ng to callus. i
vi mc ch nhn ging in vitro, nu ti sinh c cy hon chnh trc tip
t mu vt nui cy ban u th khng nhng nhanh chng thu c cy m
cc cy cng kh ng nht v mt di truyn. Tuy nhin, trong nhiu trng
hp m nui cy khng ti sinh cy ngay m pht trin thnh khi callus. T
bo callus khi cy chuyn nhiu ln s khng n nh v mt di truyn.
trnh tnh trng nht thit phi s dng loi callus va pht sinh, tc l
callus s cp ti sinh cy th hy vng s thu c cy ti sinh ng nht.

1.5. Pht sinh phi v tnh (somatic embryogenesis)
Thut ng ny dng cho s pht trin ca cc phi hon chnh t cc
t bo sinh dng c sn xut t cc ngun mu vt khc nhau sinh
trng trong nui cy in vitro. Thut ng tng ng i vi s pht trin
phi thc vt sinh trng trong iu kin t nhin l pht sinh phi hu
tnh (zygotic embryogenesis) v pht sinh phi v tnh (apomitic
embryogenesis). Phi v tnh c cu trc tng t phi hu tnh ca thc
vt sinh trng trong iu kin t nhin. im khc nhau c bn gia phi
hu tnh v phi v tnh l phi hu tnh lun lun i km vi ni nh l c
quan d tr nng lng v cht dinh dng phc v cho qu trnh ny mm,
cn phi v tnh hon ton khng c ni nh. Phng thc to phi v
tnh c ng dng rt hiu qu trong sn xut ht nhn to (synthetic seed).

2. Nhn ging in vitro v cc h thng nui cy m
Phng php nhn ging in vitro thc cht l mt tin b vt bc ca
cc phng php nhn ging v tnh c in nh gim cnh, gim chi,
chit, ghp, tch dng y gi tr thc tin ca cc tin b khoa hc k
thut l bin nhng phng thc c in thnh nhng phng thc
hon ton mi v cht cho php gii quyt nhng kh khn m phng php
c in khng th vt qua. V d: k thut gim cnh ch c th ng dng
thnh cng mt s cy trng nht nh, v rng vi kch thc 5-20 cm
kh nng to r ph ca vng m tng tng gn vt ct hoc kh nng nh
thc chi ph vn b cc vng t bo ln cn v ton b phn cn li ca
on gim khng ch. Nu tin hnh nui cy mu m vi kch thc
5-10 mm, tc l lm gim th tch khi m xung 10
3
ln th r rng mi
tng tc gia cc t bo v cc loi m s n gin i rt nhiu, hiu qu
tc ng ca cc bin php nui cy s phi cao hn. Sau y l mt s
phng thc nhn ging in vitro:

2.1. Ti sinh cy mi t cc cu trc sinh dng
2.1.1. Nui cy m phn sinh nh hay nh phn sinh
Phng thc ny s dng cc b phn nh nht ca nh chi hay
nh sinh trng (apex) lm mu vt nui cy. N bao gm m phn sinh
nh v cc mm l non. Khi nim m phn sinh nh (ngn) ch ng khi
mu vt c tch t nh sinh trng c kch thc trong vng 0,1-
0,15 mm tnh t chp sinh trng. Trong thc t mu vt c tch vi kch
thc nh vy ch khi no ngi ta tin hnh nui cy vi mc ch lm
sch virus cho cy trng. Thng s gp kh khn ln trong vic nui thnh
cng cc m phn sinh nh ring r c kch thc nh nh vy. Do ,
trong khun kh nhn ging in vitro ngi ta thng nui cy c nh chi
hoc nh sinh trng. Ph bin nht cc i tng nh phong lan, da,
ma, chui nh sinh trng c tch vi kch thc t 5-10 mm, ngha
l ton b m phn sinh nh v mt phn m xung quanh.
Tng quan gia ln ca chi nui cy, t l sng v mc n
nh v mt di truyn ca chi c biu hin nh sau: Nu ln tng th
t l sng v tnh n nh tng, nu ln gim th t l sng v tnh n
nh gim. Nhng xt v hiu qu kinh t nui cy (th tch bnh nui,
lng dung dch mi trng dinh dng): Nu ln tng th hiu qu kinh
t gim, nu ln gim th hiu qu kinh t tng. Do , phi kt hp hi
ha c cc yu t trn tm ra phng thc ly mu ti u.
Mt nh sinh trng nui cy iu kin thch hp s to mt hay
nhiu chi v mi chi s pht trin thnh mt cy hon chnh. Xt v
ngun gc ca cc cy c ba kh nng:
- Cy pht trin t chi nh (chi ngn).
- Cy pht trin t chi nch ph ng.
- Cy pht trin t chi mi pht sinh, v d nui cy on tr di
mm (hypocotyl) ca cy mng cu (Annona squamosa) s cho xut hin rt
nhiu mm (buds) trn m nui cy, mt s mm sau s pht trin thnh
chi (shoots) v tr thnh cy in vitro hon chnh (plantlet). Tuy nhin,
thng kh phn bit c chi ph ng v chi pht sinh mi. Cc phng
thc pht trin cy hon chnh t nh sinh trng nui cy nh sau:
- Pht trin cy trc tip
Ch yu cc i tng hai l mm (dicotyledon) nh khoai ty,
thuc l, cam chanh, hoa cc v d khoai ty (Solanum tuberosum):
Mm (nh sinh trng) Chi nch Cy (Hnh 4.1)

Hnh 4.1. S pht trin cy trc tip. A: mm khoai ty. B: s ko di mm
khoai ty trong nui cy v pht sinh chi nch, cc on thn mang chi nch
s c tch ra v cy chuyn trn cng mi trng nhn nhanh.

- Pht trin cy thng qua giai on protocorm
Ch yu gp cc i tng mt l mm (monocotyledon) nh phong
lan, da, hu Cng mt lc nh sinh trng to hng lot protocorm
(proembryo) v cc protocorm ny c th tip tc phn chia thnh cc
protocorm mi hoc pht trin thnh cy hon chnh. Bng phng thc ny
trong mt thi gian ngn ngi ta c th thu c hng triu c th, v d
Hoa lan (Orchidaceae):
nh sinh trng Protocorm Cy (Hnh 4.2)
mm
A B

Hnh 4.2. Pht trin cy qua giai on protocorm. A: nh sinh trng. B:
Protocorm hnh thnh t nui cy nh sinh trng.

Cc i tng hoa lan mang li hiu qu kinh t c bit cao. Sau
nhng kt qu u tin chi Cymbidium ca Morel (1966), n nay ngi
ta thu c kt qu rt tt khong 30 chi khc nhau ca h ny. S d
nhn ging v tnh hoa lan t c thnh cng ln v c ng dng rng
ri nh vy l v hoa lan c phng thc sinh sn qua protocorm. Nh c
phng thc nhn ging nhanh v r tin m hoa lan vn t tr nn c gi
phi chng v c nhiu ngi a chung. Nhng thnh cng h lan
khng nhng ch l bng chng m cn m ng cho vic ng dng k
thut ny i vi cc loi cy khc.
Lnh vc ng dng mi y nht cng bt u c kt qu l cc cy
n qu v cy lm nghip, trong c cc cy qu nh c ph, to, l,
thng, b Tng s c trn 30 chi khc nhau c nui cy thnh
cng. Do cc cy trng rng v cc cy n qu l nhng cy trng lu nm
nn mi chi ph ban u trong nhn ging in vitro u c th chp nhn
c.
- Ghp nh chi (shoot apex grafting) hay vi ghp
V nguyn tc, vi ghp l nui cy nh sinh trng, nhng thng qua
dinh dng t nhin ca gc ghp. nh sinh trng dng lm mc ghp c
kch thc khong t 0,2-0,5 mm c tch t bp non ang sinh trng
mnh ca cy m trng thnh, gc ghp l mm gi mi ny mm t ht
ca ging hoang di, ton b cy ghp c nui dng trong iu kin ng
nghim v trng. Phng thc ny thng dng to ra cc ging cy n
qu sch bnh virus nhm cung cp mt ghp v cnh chit u dng lm
nguyn liu nhn ging cho sn xut i tr. Phng thc ny cho php thu
M phn sinh nh
Mm l
nh phn sinh c
tch ra nui cy
A B
c cy hon ton sch bnh v mang c im di truyn ca cy m cho
mt ghp.
C nhiu cch ghp khc nhau, chng hn: (1) Ghp ln mt ct: t
mt ghp trc tip ln b mt lt ct, trn vng tng tng. (2) Ghp ch T-
ngc: dng u nhn ca li dao ct l ghp hnh ch T-ngc, chn ch
T l mt ct d bc l vng tng tng. (3) Ghp hm ch: khot trn
thn mm cch mt ct 5 mm mt vt lm hnh hm ch, chiu su vt lm
bng chiu dy lp v. t mt ghp vo y hm ch (Hnh 4.3).

Hnh 4.3. V tr mt ghp trong ba kiu vi ghp khc nhau

2.1.2. Nui cy chi bt nh (adventitious shoot culture)
H thng nui cy ny c nhng yu cu tng t vi nui cy m
phn sinh nh, n ch khc v ngun mu vt v ngun gc bt nh ca
cc chi mi. nh chi bt nh mi c th pht trin hoc trc tip trn
mu vt hoc gin tip t m callus, m m callus ny hnh thnh trn b
mt vt ct ca mu vt (Hnh 4.4). Mt s loi mu vt c dng nh sau:
- on thn: thuc l, cam, chanh, c chua, bp ci
- Mnh l: thuc l, c chua, bp ci, c ph, ca cao
- Cung l: thy tin
- Cc b phn ca hoa: sp l, la m, thuc l
- Nhnh c: h hnh, h lay n, h thy tin
1. Ghp ln mt ct

V
Tng tng

Li

2. Ghp vo vt T-ngc

3. Ghp vo l trn thn
- on mm: mng ty

Hnh 4.4. Ti sinh chi bt nh v nhn ging in vitro cy Hylotelephium
sieboldii. A: Chi bt nh. B: Cy in vitro hon chnh.

S pht sinh chi bt nh trc tip bt u bng cc t bo nhu m
(parenchyma cells) nm trong biu b hoc ngay pha di b mt ca
thn; mt s t bo ny tr thnh m phn sinh v cc ti nh gi l th
phn sinh (meristemoids) pht trin. Cc th phn sinh ny r rng c ngun
gc t cc t bo n. Tuy nhin, chiu hng phn ng ca thc vt cng
ty thuc vo nng phytohormone. Nghin cu s to chi m nui
cy ca cy linh sam Douglas cho thy cytokinin (BAP 5 M) cn thit cho
s pht sinh chi bt nh, nhng c ba kiu phn ng khc nhau c kt qu
ty thuc vo nng ca auxin c cung cp. Nng auxin thp (NAA
5 M) ch c chi pht trin. Khi nng auxin cao hn (NAA 5 M) l
mm to ra c callus v nhiu chi. Khi cung cp ch ring auxin (NAA =
5 M) th ch c callus c to thnh.
S pht trin cc chi bt nh gin tip u tin qua giai on hnh
thnh callus c s (basal callus) t cc chi c tch trong nui cy. Cc
chi sau pht trin t ngoi vi m callus v khng c quan h ban u
vi cc m c mch dn (vascular tissue) ca mu vt.

2.2. Nhn ging thng qua giai on callus
Trong khun kh ca mc ch nhn ging in vitro nu ti sinh c
cy hon chnh trc tip t mu vt nui cy ban u th khng nhng
nhanh chng thu c cy m cc cy cng kh ng nht v mt di truyn.
A B
Tuy nhin, trong nhiu trng hp m nui cy khng ti sinh cy ngay m
pht trin thnh khi callus (Hnh 4.5).

Hnh 4.5. Callus (A) v ti sinh chi t callus (B) ca chi Lilium

T bo callus khi cy chuyn nhiu ln s khng n nh v mt di
truyn. trnh tnh trng nht thit phi s dng loi callus va pht
sinh, tc l callus s cp ti sinh cy th hy vng s thu c cy ti sinh
ng nht. Thng qua giai on callus cn c th thu c nhng c th
sch virus nh trng hp ca Kehr v Sehaffer (1976) thu c ti.

2.3. Nhn ging thng qua pht sinh phi v tnh-cng ngh ht nhn to
2.3.1. Phi v tnh
Mt phng thc nhn ging v tnh na l to phi v tnh t t bo
callus. Nm 1958, Street v Reinert l hai tc gi u tin m t s hnh
thnh phi v tnh t cc t bo n ca c rt (Daucus carota). n nm
1977, Murashige cho rng phi v tnh c th tr thnh mt bin php nhn
ging in vitro. mt s loi, s pht sinh phi v tnh hnh thnh trc tip
t nhng phi bt nh (adventitious embryos) nm trong phi tm (nucellar
embryos). n nay, cng ngh phi v tnh c coi l cng ngh rt c
trin vng cho nng nghip trong th k 21.
Phi v tnh l cc c th nhn ging (propagules) c cc tnh bt
ngun t cc t bo soma (Hnh 4.6 v 4.7). Chng rt ging phi hu tnh
(zygotic embryo) hnh thi, qu trnh pht trin v sinh l, nhng do
khng phi l sn phm ca s th tinh gia giao t c v giao t ci, v v
vy khng c qu trnh ti t hp di truyn (genetic recombination), cc
A B
phi v tnh c ni dung di truyn ging ht vi cc t bo soma sinh ra
chng.
trng hp phi hu tnh, s kt hp giao t c v ci cho ra hp
t (zygote). Hp t phn chia nhiu ln to nn phi hu tnh c cu trc hai
cc: r v ngn. Khi hp t pht trin, min sinh trng r v min sinh
trng ngn cng pht trin v cui cng to thnh cy hon chnh, qua cc
giai on phi hc nh sau:
- Trng hp cy hai l mm:
Dng cu dng thy li dng c l mm

- Trng hp cy mt l mm:
Dng cu dng scutellar dng dip tiu

Hnh 4.6. Cc phi v tnh (A) v cy mm pht sinh t phi v tnh (B)

Hnh 4.7. Phi v tnh ca chui (A) v nui cy pht trin phi v tnh (B)

A B
A B
Bng 4.1. Mt s cy trng c gi tr kinh t c nhn ging bng phng
thc pht sinh phi v tnh in vitro

Stt Tn khoa hc Tc gi
1 Citrus Stevenson (1966), Rangan et al.
(1968), Jumin et al. (1996)
2 Theobroma cacao Litz (1986), Alemanno et al. (1996)
3 Coffea arabica Sondahl and Sharp (1977), Boxtel et
al. (1996)
4 Coffea canephora Berthouly et al. (1996), Boxtel et al.
(1996)
5 Hevea brasiliensis Carron and Enjalsic (1985)
6 C. cogiensis C. canephora Boxtel et al. (1996)
7 Eugenia Litz (1984)
8 Camellia sinensis Ponsamuel et al. (1996)
9 Medicago suffructicosa Li et al. (1996)
10 Saccharum officinarum Aftab et al. (1996)
11 Docynia indica Litz (1985)
12 Malus domestica Eichholtz (1979)
13 Picea sitchensis Moorhouse et al. (1996)
14 Mangifera indica Litz (1982), Pliego-Alfaro et al.
(1996)

rt nhiu cy, ngi ta nhn thy cc t bo ang phn chia v t
chc to nn callus khi nui cy. C th thay i hng pht trin ca
chng to ra cc phi v tnh vi cc bc pht sinh hnh thi rt ging
vi trng hp phi hu tnh. im khc nhau c bn gia phi hu tnh v
phi v tnh l phi hu tnh lun lun i km vi ni nh l c quan d tr
nng lng v cht dinh dng phc v cho qu trnh ny mm, cn phi
v tnh hon ton khng c ni nh. S khc nhau ny khng ch ng ch
v mt khoa hc m cn l mt yu t rt quan trng trong cng ngh phi
v tnh.
Kh nng to phi v tnh trong nui cy m thc vt, ngoi cc iu
kin vt l, ha hc thun li cho s to phi, cn ph thuc rt ln vo
loi, vo cc ging (cultivars), dng (strains) trong cng mt loi. Kh nng
ny c chng minh l do mt hoc mt vi gen ph trch. V vy, bng
bin php lai to c th chuyn kh nng to phi v tnh cao t cy ny qua
cy khc.

2.3.2. Cng ngh ht nhn to
Ht nhn to (artificial seed hoc synthetic seed) l phi v tnh bc
trong mt lp v polymer nh agar, agarose, alginate Trong cu trc li
ca cc lp v , nc, cht dinh dng v cht sinh trng c cung cp
thay cho ni nh, gip cho phi v tnh c th ny mm tr thnh cy hon
chnh (Hnh 4.8).

Hnh 4.8. Ht nhn to ca ging to M.26

Trong vic sn xut cc ht nhn to thng qua phi v tnh t nui
cy dch lng, th ni phn ng sinh hc (bioreactor) l thit b khng th
thay th c.
Do phi v tnh cng c th ny mm v pht trin thnh cy hon
chnh, nn k thut ht nhn to c nghin cu v ng dng thnh
cng nhiu nc. Ht nhn to gm c ba phn:
- Phi v tnh
- V bc polymer (alginate)
- Mng ngoi (calcium alginate)
C nhiu loi polymer t nhin c th nghim dng cho cng
ngh phi v tnh, trong alginate c coi l tt nht. Alginate l mt
polymer sinh hc, c chit t rong bin m ch yu l cc loi thuc chi
Sargassum. Aliginate do cc phn t manuronic acid gn vi nhau to
thnh, ging nh cc phn t glucose to nn cellulose. c im quan
trng nht ca alginate l chng dng ha tan trong nc khi kt hp vi
cc ion ha tr mt (monovalent) nh: Na
+
, K
+
,
4
NH v lp tc chuyn
sang dng khng tan trong nc khi kt hp vi cc ion ha tr hai
(divalent) hoc a ha tr (polyvalent) nh: Ca
2+
, Mg
2+
, Al
3+
, Nu nh
mt git dung dch sodium alginate vo dung dch CaCl
2
th sodium alginate
phn din tch ngoi ca git s chuyn ha ngay thnh calcium alginate
v to nn mt mng khng thm nc hnh thnh cc vin alginate.

2.3.3. Nhn ging trong cc ni phn ng sinh hc
Trc y, cc ni phn ng sinh hc hay cn gi l bnh ln men
(fermenter) ch yu c dng cho cng ngh vi sinh. Trn c s cc thit
b , vi mt s ci tin, nhiu tc gi nhn ging thnh cng nhiu loi
phi v tnh v cc th chi, cm chi hoc c nh.
Phi v tnh c ph c sn xut thnh cng Brasil trn cc ni
phn ng sinh hc dung tch t 2-4 lt. Ni vn hnh theo cc nguyn tc
ca mt bnh ln men (c th khng dng cnh khuy m ch dng bt kh
thc hin vic sc kh v truyn nhit). Mi m c th thu c 4-5 triu
phi v tnh c ph. Indonesia, cm chi da c a vo sn xut thnh
cng vi bnh ln men 10 lt. im ng ch trong cng ngh ny l thay
v bm kh vo ni phn ng, dch lng nui cy (mi trng mi) c
bm vo ni v ht ra (mi trng c) theo chu k ngn, nh vy m v t
bo thc vt c oxy v cht dinh dng pht trin mnh. Phng thc
nui cy ny c gi l nui cy th n nh ha tnh (chemostat culture).
C siu bi (microtuber) c th trng quc t cng nhn l dng
khoai ty ging ca th k 21. C khoai ty siu bi c kch thc bng hoc
nh hn ht ng, hon ton sch bnh virus c cng ty Microtuber Inc.
(M) sn xut trong cc ni phn ng l cc on thn khoai ty nhn ging
bng nui cy m theo phng php kinh in. Trong ni phn ng, cc
on thn c kch thch ra r v to c nh. Hin nay, Microtuber Inc. c
th trng Bc M v H Lan. Ni phn ng hng Microtuber Inc. l cc
ng nha kn chu nhit, ng knh 15 cm, di 50 cm, qu trnh to c hon
ton khng cn chiu sng.
Hnh 4.9 m t cc phng thc ph bin pht trin cy hon chnh
trong nhn ging in vitro.

Hnh 4.9. Cc phng thc to cy hon chnh in vitro

3. Cc giai on trong quy trnh nhn ging v tnh in vitro
Quy trnh nhn ging v tnh in vitro c thc hin theo ba (hoc
bn) giai on ty theo cch phn chia ca tng tc gi:
- Cy gy
- Nhn nhanh
- Chun b v a ra ngoi t

3.1. Giai on I-cy gy
a mu vt t bn ngoi vo nui cy v trng phi m bo nhng
yu cu sau:
- T l nhim thp
Cy trng
thnh mi
Cy trng
thnh
Nui cy
t bo
Nui cy
callus
Nui cy
c quan
Nui cy
phi
- T l sng cao
- Tc sinh trng nhanh
Kt qu bc cy gy ny ph thuc rt nhiu vo cch ly mu. Quan
trng nht vn l nh sinh trung, chi nch, sau l on hoa t, hoa,
on thn, mnh l, r
Chn ng phng php kh trng s cho t l sng cao v mi
trng dinh dng thch hp s t c tc sinh trng nhanh.
Mt s dng mi trng dinh dng ph bin:
- Mui khong: Theo White (1943), Heller (1953), Murashige v
Skoog (1962) (Bng 4.2).
- Cht hu c:
+ ng saccharose 1-6 %.
+ Vitamin: B1, B6, myo-inositol, nicotinic acid.
+ Amino acid: Arg, Asp, Asp-NH
2
, Glu, Glu-NH
2
, Tyr.
+ Phytohormone:
Nhm auxin: IAA, IBA, NAA, 2,4-D
Nhm cytokinin: BAP, kinetin, 2-iP, zeatin...
Nhm gibberellin: GA
3
.
Ty theo tng loi, tng b phn nui cy v tng mc ch nui cy
m b sung cc hm lng v thnh phn phytohormone khc nhau.

3.2. Giai on II-nhn nhanh
giai on ny ngi ta mi kch thch to c quan ph hoc cc cu
trc khc m t cy hon chnh c th pht sinh. Nhng kh nng to cy
l:
- Pht trin chi nch
- To phi v tnh
- To nh sinh trng mi
Trong giai on ny cn nghin cu cc tc nhn kch thch phn ha
c quan, c bit l chi nh:

Bng 4.2. Thnh phn mi trng Murashige v Skoog (1962)

Thnh phn

Nng
(mg/L)
Thnh phn

Nng
(mg/L)
1. Cc nguyn t a
lng
MgSO
4
.7H
2
O
KH
2
PO
4

KNO
3

NH
4
NO
3

CaCl
2
.2H
2
O

370
170
1900
1650
440
FeSO
4
.7H
2
O
Na
2
EDTA.2H
2
O

3. Ngun carbon
Sucrose
27,8
37,3

30000
2. Cc nguyn t vi
lng
H
3
BO
3

MnSO
4
.4H
2
O
ZnSO
4
.7H
2
O
Na
2
MoO
4
.2H
2
O
CuSO
4
.5H
2
O
CoCl
2
.6H
2
O
KI

6,2
22,3
8,6
0,25
0,025
0,025
0,83
4. Ph gia hu c
- Cc vitamin
Thiamine.HCl
Pyridoxine.HCl
Nicotinic acid
myo-inositol

- Cc cht khc
Glycine

0,5
0,5
0,5
100

2

- B sung t hp phytohormone mi (tng cytokinin gim auxin).
Tng t l auxin/cytokinin s kch thch m nui cy to r v ngc li s
kch thch pht sinh chi.
- Tng cng thi gian chiu sng 16 gi/ngy, ti thiu 1.000 lux.
Trong thc t nghin cu, ngi ta nhn thy kh tch bit nh hng ca
chu k chiu sng khi nh hng ca cng chiu sng. nh sng tm
l thnh phn quan trng kch thch phn ha mnh. nh sng c nh
hng ging cytokinin (cytokinin-like effect), n to nn s tch ly
cytokinin trong m ca mt s loi, chnh lng cytokinin ny gp phn
kch thch qu trnh pht sinh c quan v to chi t nhng m nui cy in
vitro.
- Bo m ch nhit trong khong 20-30
o
C. Trng hp nhng
loi c ngun gc nhit i, nhit nui cy thch hp vo khong t 32-
35
o
C. Ngc li, i vi nhng loi hoa vng n i nhit thch hp
cho qu trnh to cm chi phi 30
o
C.
Mc tiu quan trng nht ca giai on ny l xc nh c phng
thc nhn nhanh nht bng mi trng dinh dng v iu kin kh hu ti
thch.

3.3. Giai on III-chun b v a ra ngoi t
y l giai on quan trng bao gm vic to r, hun luyn thch
nghi vi thay i nhit , m, s mt nc, su bnh v chuyn t trng
thi d dng sang t dng hon ton. Giai on ny thng b b qua mt
cch thiu cn c.
Cc nghin cu v cu trc ca l khoai ty nui cy in vitro v so
snh vi l cy khoai ty trng bn ngoi cho thy chng rt khc nhau.
iu chng t phi tin hnh thch nghi dn dn cy nhn ging in vitro
vi iu kin t nhin. Qu trnh thch nghi y phi c hiu l qu
trnh thay i nhng c im sinh l v gii phu ca bn thn cy non .
Thi gian ti thiu cho s thch nghi l 2-3 tun, trong thi gian ny cy
phi c chm sc v bo b trc nhng yu t bt li sau:
- Mt nc nhanh lm cho cy b ho kh.
- Nhim vi khun v nm gy nn hin tng thi nhn.
- Chy l do nng.

4. Nhn ging in vitro v vic s dng ging u th lai
Trong chn nui, vic s dng ging vt nui mang u th lai th h
F
1
tr thnh bin php tng nng sut quan trng bc nht.
ngnh trng trt, ging u th lai mi ch c ng dng mt s
i tng nh: ng, c chua, la, ci du, bp ci, hnh ty, mng ty, v
c bit l cc ging hoa
S dng u th lai khng nhng lm tng nng sut t 20-40%, m
ging lai cn c c im l rt ng u so vi ging b m. Tnh ng
u ca ging l tin quan trng cho sn xut theo phng thc cng
nghip. sp-l chng hn, phng thc sn xut cng nghip i hi phi
thu hoch ton b din tch bng c gii vo mt thi im. iu ny ch
c thc hin khi s dng ging u th lai F
1
. Nu dng ging thun
chng theo phng thc t phi th khng m bo, v cc ging rau h
ci (Brassicaceae) thng xut hin hin tng bt t th. V vy, phng
php nhn ging v bo qun ging trong ng nghim i vi mt s ging
rau v ging hoa c mt ngha kinh t cao.
Vn t ra hin nay l phi nghin cu cc quy trnh nhn ging in
vitro ti u cho tng loi cy trng v ci tin quy trnh gim ti mc
i a cc tn km v nhn cng lao ng trong cc cng on nui cy v
a cy con ra ngoi t.

5. Nhn ging in vitro v cc c im khng di truyn
Bn cnh nhng c im di truyn thc vt ngi ta cn pht hin
c hng lot c im (hnh thi v sinh l) khng di truyn, l cc c
im epigenetic. Qu trnh nhn ging in vitro c nh hng ti cc c
im khng di truyn ny.

5.1. Hin tng cc c im epigenetic c lu li
Nghin cu nhn ging v tnh v cy Phyllanthus amarys (cy ch
) cho thy:
Phyllanthus l mt loi cy c c thn thng ng v cnh ngang
ging l kp nhng li nch l v c hoa cho nn vn tm coi nh cnh
ngang. L thn ng xp theo kiu xon c, nhng cnh ngang l tng
i so le.
nch l c cnh ngang, chi nch pht trin mnh thnh cnh thng
ng nh thn. Nu ct cnh ngang m th s thu c cnh ngang di
hng mt. Cnh ngang pht trin v hn theo mt chng trnh ngang
nh trc. Nu m cnh thng th s thu c cy thng ng. Nh vy,
m phn sinh ca cnh khc m phn sinh ca thn v khi nhn ging v
tnh cc b phn khc nhau s thu c cc dng cy khc nhau. y m
phn sinh nh iu khin m phn sinh cnh pht trin theo hng ngang.
Nu ct b m phn sinh nh sm th cnh ngang s pht trin thnh cnh
ng.
T bo sinh trng nh (organisator) iu khin s hot ng ca cc
t bo khc (tng t nh ng vt). Hin tng iu khin hng pht
trin ny c mt ngha thc tin rt ln.
Cc cy c ph v ca cao cng c c im xp cnh tng t nh
vy, do khi tin hnh nhn ging v tnh cc loi cy ny phi ch n
hin tng iu khin hng pht trin nh Phyllanthus.

5.2. Hin tng cc c im epigenetic khng lu li
Nhn ging v tnh in vitro ging da Cayen khng gai thng qua
phn chia protocorm ngi ta thu c mt t l ng k cc c th c gai.
Bnh thng trong k thut trng da ngi ta s dng hom da c
kch thc kh ln 15-30 cm. c im khng gai c chng trnh ha
trong cc t c kch thc ln nh vy v vn da trng nh vy u
khng c gai. V sao khi tch nh sinh trng v nui cy thnh protocorm
v cy da non th gai li xut hin.
C th giai on sinh l sm ca protocorm v qu trnh nui cy
nh sinh trng phn lp, m phn sinh ca nhng cy da non tng lai
c gii phng mt phn khi nh hng ca t chc iu khin v v
th chng pht trin t do theo c im c gai ca th h xa xa. Hin
tng tng t chng ta cng bt gp trng hp cy cam khng gai. Khi
gieo ht hoc nui cy phi v tnh t m phi tm s thu c nhng c
th c gai.
Hin nay, vn ny ang c quan tm v mt s c tnh chng
chu nh chu mn, chu bnh, chu lnh vn thng l nhng c tnh
epigenetic v liu thng qua nhn ging in vitro cc c tnh c cn c
gi li hay khng.

III. Cc k thut chuyn gen thc vt
Chuyn gen
.
chuyn gen v mt cch hiu qu
(selectable marker
gene
la (screenable marker gene
.
: trnh t (promoter),
-
trnh t kt
. Hai prom
: promoter CaMV 35S
promoter ubiquitin
.
(
.
, k
Agrobacterium tumefaciens
ng sinh. Agrobacterium
Agrobacterium
-
-
...
n
. C nhiu phng php
chuyn gen khc nhau thc vt, nhng y ch trnh by mt vi phng
php ch yu:

1. Chuyn gen gin tip thng qua Agrobacterium
1.1. Vi khun Agrobacterium
Agrobacterium tumefaciens Agrobacterium rhizogenes
. A. tumefaciens
-
A.
tumefaciens
A. tumefaciens -plasmid (T-DNA)
.

1.2. Ti-plasmid
-
. vi kh -
.
Agrobacterium
cis v trans. y l hai dng vector rt thun li
ti t hp gen ngoi lai v chuyn vo t bo thc vt. Dng cis ch s
dng Ti-plasmid v t bo vt ch l Agrobacterium tumefaciens m khng
c s tham gia ca cc plasmid v vi khun khc. Vng T-DNA ca Ti-
plasmid c thit k li gn nhng gen ngoi lai mong mun, cc phn
cn li ca Ti-plasmid vn c gi nguyn. Agrobacterium tumefaciens
c dng lm t bo vt ch nhn ln nhiu bn sao ca Ti-plasmid v
chuyn gen. Dng trans hay binary l dng s dng hai hay nhiu loi
plasmid v vi khun cng lc, v d: vi khun E. coli v Agrobacterium,
plasmid trong trng hp ny thch ng vi c E. coli v Agrobacterium.
Trc tin, plasmid ca E. coli cha on T-DNA c gii hn bi b phi
(right border-RB) v b tri (left border-LB) mang gen ngoi lai c thit
k v nhn ln trong vi khun E. coli. Tip n plasmid mang gen ngoi lai
c chuyn np vo vi khun Agrobacterium nh mt helper plasmid thc
hin s tip hp thng qua qu trnh giao phi b ba (triparental matting).
Vi khun Agrobacterium mang sn mt loi plasmid khc cha vng vir
(virulence region) c chc nng quan trng trong qu trnh chuyn gen
ngoi lai. S tn ti song song hai plasmid ny tng tc ln nhau trong
vic chuyn gen vo t bo thc vt. Nh vy, gen ngoi lai v vng DNA
gip qu trnh chuyn gen (vng vir) khng nm trn cng mt plasmid nn
h chuyn gen ny c gi l h trans.

1.3. Vng T-DNA
-
, cytokinin,
opine - -
- -
(vng vir
4.10).
- -
vir vir
virE2, virB, virD, virD2, vir
-
.
A. tumefaciens -
- A. rhizogenes
- A.
rhizogenes
.
, Agrobacterium
-
Agrobacterium .


g Ti-plasmid. A: dng t nhin ban u, B: dng
cis, C: dng trans.

Agrobacterium
tumefaciens
Agrobacterium
-
A.
T-DNA
cytokinin
auxin opine B tri
B phi
vir
ori
chuyn ha opine
A

B

T-DNA
B tri
B phi
vir
ori
chuyn ha opine
gen ch

ori
vir
C

T-DNA
B tri
B phi
ori
gen ch
tumefaciens A. rhizogenes
.
gn T-DNA vo t bo thc vt, u tin vi khun A. tumefaciens
phi tip xc vi thnh t bo thc vt b tn thng. Qu trnh ny c
thc hin nh cc gen chvA v chvB. Gen chvB m ha mt protein lin
quan n hnh thnh -1,2 glucan mch vng, trong khi gen chvA xc
nh mt protein vn chuyn, nh v mng trong ca t bo vi khun.
Protein vn chuyn gip vn chuyn -1,2 glucan vo khong gia thnh t
bo v mng sinh cht. -1,2 glucan gi vai tr quan trng vi khun
Agrobacterium tip xc vi thnh t bo thc vt. Nu khng c s tip xc
ny, s khng c s dn truyn T-DNA.
Cc sn phm protein ca vng vir c tc dng cho vic dn truyn T-
DNA t vi khun vo t bo thc vt. Cc loi protein rt cn thit cho
qu trnh ct T-DNA khi Ti-plasmid, cm ng thay i mng t bo thc
vt m chng tip xc, tham gia di chuyn phn T-DNA qua mng vi khun
ti t bo cht ca t bo thc vt, vn chuyn ti nhn ri cui cng xm
nhp vo genome ca cy ch.
Thc cht ch ring T-DNA ca Ti-plasmid c chuyn vo genome
t bo thc vt, m khng cn phn no khc. Qu trnh dn truyn ch do
sn phm ca cc gen vir (vng vir) v gen chv quyt nh m khng lin
quan n cc gen khc trn T-DNA. Tuy nhin, chui DNA 25 bp (RB v
LB ca T-DNA) c vai tr l v tr cm ng cho cc sn phm ca t hp
cc gen vng vir, c bit l protein t gen virE mang chng dn truyn vo
t bo thc vt. Chng hot ng nh cc tn hiu nhn bit v khi ng
qu trnh dn truyn. Trc ht gen virA trong t hp gen vng vir c
phosphoryl ha nh tc ng ca cc hp cht phenol nh acetosyringone
gii phng ra t cc t bo thc vt tn thng. Sn phm ca qu trnh ny
li tip tc phosphoryl ha gen virG. Sn phm ca gen virG lin tip lm
hot ha ton b cc gen vir cn li, m hai gen cui cng c hot ha l
gen virB v virE. Trc , khi gen virD c hot ha, sn phm ca n
cm ng nhn bit RB v LB ca T-DNA v lm t phn T-DNA ra khi
DNA ca Ti-plasmid thnh cc si n. ng thi qu trnh phosphoryl ha
ny cng lm thay i thm xut mng t bo thc vt, mng t bo b mm
ra v b thng. Cc si n T-DNA c gn vo protein do gen virE tng
hp v dch chuyn v pha mng t bo vi khun. Ngay sau , si T-DNA
c trt t vi khun vo t bo thc vt. Cu ni chnh l s tip hp
(conjugation) gia hai t bo do cm ng sn phm gen virB m thnh. Khi
T-DNA c chuyn giao vo t bo thc vt, chng nhanh chng hp
nht (integration) trong genome t bo thc vt c n nh v di truyn
nh cc gen bnh thng khc (Hnh 4.11).

Hnh 4.11. Phng thc chuyn T-DNA vo genome ca thc vt

2. Cc gen ch th chn lc v gen ch th sng lc
-
glucuronidase (gus
hunh quang mu xanh lc ( .
promoter
Agrobacterium , gen
ngo
Vng vir
S cm ng
ca cc gen vir
Si T-DNA v
cc protein Vir

VirE2
VirD2
VirB

Agrobacterium T bo thc vt

T-DNA
Nhim sc th
Nhn
Hp nht ca T-DNA
Thnh t bo
?
Ti
T-DNA
ca
.
.
: gus A, npt II, lux, cat,
nos v bar 4.3).
- Gen npt II. Gen m ha cho
.
- Gen bar. Gen bar
phosphinothricin acetyltransferase (PAT),
. Gen bar
Streptomyces hygroscopicus
bar
.
- Gen gus A. G -
- -
-
gen gus - - - - - - - - -
- -
chuy .
- Gen lacZ. Gen m ha cho enzyme -
116 kD. Gen lac E. coli
- lac
lac
lac .

A. Mt s gen ch th chn lc

gen

npt II Neomycin phosphotransferase Kanamycin
hyg Hygromycin phosphotransferase Hygromycin
gent Gentamycin acetyl transferase Gentamycin
aat Streptomycin phosphotransferase Streptomycin
bleo Bleomycin
bar Phosphinothricin acetyltransferase Phosphinothricin
bxn Bromoxynil nitrilase Bromoxynil
B. Mt s gen ch th sng lc

gen

gus A -glucuronidase X-Gluc
lacZ -galactosidase X-Gal
luc Lumis Phos
lux Lumi Phos
cat Chloramphenicol acetyltransferase
nos Nopaline synthase Nopaline

- Gen cat. c phn lp v to dng t dng vi khun Tn9, l gen
gy kh nng khng chloramphenicol vi khun ni chung. Gen cat
c dng rng ri trong cng ngh gen ng vt v thc vt v n m ha
cho enzyme chloramphenicol acetyltransferase (CAT). Enzyme ny xc tc
phn ng acetyl ha hai v tr trn phn t chloramphenicol v lm n bt
hot.

3. Chuyn gen bng vi n
.
-
-
(g
.

Hnh 4.12
L thng hi
M hoc t bo thc vt
Li chn vin n ln
Vi n
Vin n ln
Bung np thuc sng
Kim ha

Hnh 4.13. Thao tc chuyn gen bng phng thc di bom

4. Cc ng dng ca cng ngh chuyn gen
4.1. Mt s kt qu bc u
ng dng ca cc k thut chuyn gen trong cng ngh sinh hc thc
vt c mt tim nng rt ln, vt qua cc tin b k thut quan trng trong
cuc cch mng sn xut nng nghip trc y. N ch mt bn nm cho
s pht trin thng mi cc cy trng chuyn gen Bc M v t ti 52%
i vi u tng, 30% vi ng, v 9% cho c hai loi bng v canola (nm
1999) cng vi vic tng sn lng trn nhiu loi cy trng chuyn gen
khc nh: la, la m, la mch, la min, ma, c ci ng, c chua,
khoai ty, hng dng, u phng, u , cc loi cy g v cc loi cy
hoa nh cm chng Trong s 27,8 triu hecta cy trng chuyn gen
c canh tc trong nm 1998, th 74% c trng M, 15% Argentina,
10% Canada v 1% c. Cc tnh trng chuyn gen c tp trung ch
yu l chng chu cht dit c (71%), khng cn trng (28%) v 1% cho cc
tnh trng khc.
Cc cy chuyn gen ang c thng mi ha hin nay l th h u
tin ca cc cy trng chuyn gen, v ba th h cy trng chuyn gen c th
c d on trc l :
- Th h th nht. Cc tnh trng sn xut (v d chng chu cht dit
c, khng bnh/cn trng).
Nng sng
Thuc sng
Hp n
Cc vi n c
bc DNA
M thc vt
- Th h th hai. Cc gen xp thnh chng cho nhiu tnh trng (v d
t hp ca cc gen khng bnh cng vi cc tnh trng cht lng).
- Th h th ba. Cc tnh trng khc nhau c p ng cho vic s
dng c bit cui cng (v d thc phm, si, nhin liu, du nhn, nha,
dc phm v cc nguyn liu th cho cc qu trnh cng nghip).
Vic sn xut cc cy trng chuyn gen th h th hai ang tin trin.
Tuy nhin, hng ti cc li ch tim tng ca cng ngh sinh hc thc
vt chuyn gen, c nhiu nhn t b sung cn xem xt bao gm: s kim
sot cng ngh sinh hc, s hu tr tu, an ton thc phm, s chp nhn
ca cng ng, tnh cht gy d ng, nhn hiu, s chn la, mi trng, s
phn bit ca cc sn phm chuyn gen v mu dch quc t. Tm quan
trng y l vic ng dng v cc li ch tim tng ca cc k thut
chuyn gen. Trong khi mi k thut ang pht trin, th mc ch cn t
c l ti a cc li ch v gim thiu cc ri ro.

4
.
.
.

5. Cng ngh di truyn trong khng cht dit c
c tnh khong 10% tng sn lng lng thc hng nm trn th
gii b mt mt do c di, mc d tiu tn khong 10 t USD v s dng
trn 100 loi ha cht khc nhau dit tr. Hn na, dng thuc dit c
di vn b hn ch v nhiu loi thuc khng phn bit c c di v cy
trng. Glyphosate (phosphonomethyl glycine) l mt loi thuc dit c
nhiu loi c di nhng chng cng c th lm cht cy trng. V vy, nu
to c cc ging cy chuyn gen chng chu c glyphosate th n s
c dng ph bin dit c. Mt trong nhng hng nghin cu l
chuyn gen sn xut d tha 5-enolopyruvyl-shikimate-3-phosphatase
(EPSP), mt enzyme b c ch bi thuc dit c glyphosate. Ngha l nu
cy sn xut c nhiu enzyme EPSP chng c th chng chu c
glyphosate.
Cht dit c l phng php c chn la kim sot c di trong
hu ht cc h thng nng nghip quy m ln. Chng ng mt vai tr quan
trng trong vic tng sn lng cy trng bng cch gim thiu s cnh
tranh gia cy trng vi c di v khng gian, nh sng, nc v cht dinh
dng. C di cng c th hot ng nh mt ngun cung cp cc tc nhn
gy bnh cho cy trng.
Do cc gen khng cht dit c cng l cc gen ch th chn lc hiu
qu trong trng trt, nn y l tnh trng chuyn gen u tin c sn xut
v thng mi ha, v cc th (variety) chng chu cht dit c vn ang l
cc cy trng chuyn gen sinh trng rng ri nht.
Da trn c s hoc l s biu hin ca gen khng mn cm cht dit
c, s thoi bin ca cht dit c hoc s biu hin mnh ca sn phm gen
ch ca cht dit c, m tnh khng c chuyn gen thch hp trong mt
phm vi rng cc cht dit c nh 2,4-D, glyphosate, glufosinate,
protoporphyrinogen oxidase inhibitors, imidazalonones, chlorsulfuron/
sulfonylureas, bromoxynil, triazines v isoxazoles.
Hin nay, c nhng bng chng tt cho thy chng nhng khng tng
s dng ca cc cht dit c, m s iu chnh cc cy trng chuyn gen
khng cht dit c c cung cp cho nng dn cho kt qu gim s
dng glyphosate ti 33% trn cc ging u tng Roundup Ready, v gim
s dng glufosinate khong 20% i vi ging canola Liberty Link.

6. Cng ngh di truyn trong khng su-bnh
6.1. Khng cn trng
S dng ha cht phng tr su b cn trng va t tin va tc
ng xu n mi trng. Cc cy trng nh bng, ng v khoai ty chuyn
gen ang c sinh trng thng mi biu hin c t ca Bacillus
thuringiensis (Bt) to ra tnh khng i vi cc cn trng nhm nhai-
nghin (chewing insects). B. thuringiensis tng hp cc protein -endotoxin
tinh th c m ha bi cc gen Cry. Khi cn trng n vo bng, cc
prototoxins b t gy trong d dy kim ca cn trng to thnh c t
hot ng. Cc lin kt ny to ra cc receptor c trng trong cc t bo
biu m rut lm thnh cc l chn lng v cui cng l gy cht cn trng.
Mt s u im ca c t Bt nh sau :
- Tnh c hiu, mi protein Cry ch hot ng chng li mt hoc
mt vi loi cn trng.
- S a dng, nhiu protein Cry khc nhau c nhn bit.
- Cc nh hng khng bt li hoc b gim c xc nhn trn cc
cn trng khng phi ch hoc cc ch th t nhin ca cn trng.
- c tnh vi ng vt c v l rt thp.
- C th thoi bin d dng.
Kt qu nghin cu cho thy im mu cht l gen chu trch nhim
tng hp protein tinh th tr su ca vi khun B. thuringiensis chng
kurstaki, cha c bit biu hin r cy trng. nng cao hiu qu ca
c t, cc nh nghin cu ct bt gen ch tng hp phn protein c
hot tnh cha c t m khng cn cc phn gen ph khc. Gen Cry c
ct bt biu hin tng hp protein gp 500 ln so vi gen t nhin. Hin
nay, hn 40 gen khc nhau mang tnh khng cn trng c hp nht
trong cy trng chuyn gen vi mt vi ging c thng mi ha cc
nc khc nhau nh M v c.
a ra li ch ca cc c t ca Bt i vi s kim sot cn trng,
cc phng thc qun l khc nhau phi c chp nhn lm chm s
pht trin ca tnh khng cn trng i vi Bt. Nhng ci bao gm :
- B tr cc vng bn cnh trng cy bng khng chuyn gen Bt lm
ni tr n gim p lc chn lc hng ti vic khng cn trng.
- Trin khai cc gen khng cn trng khc nhau (v d: protease
inhibitors).
- Dng cc loi c t Bt cho cc receptors ch khc nhau.
- Dng cc promoter khc nhau iu chnh s biu hin ca cc gen
Bt.
- Dng cc promoter c trng m (tissue-specific promoter) nh th
cn trng c th n m khng tn hi n kinh t trn cc b phn t quan
trng ca thc vt.
C cc hng khc pht trin tnh khng cn trng cho cy chuyn
gen da trn c s: protease inhibitors, -amylase, lectins, chitinases,
cholesterol oxidase, cc virus ca cn trng c to dng, tryptophan
decarboxylase, anti-chymotrypsin, anti-elastace, nhn t c ch trypsin
tuyn ty ca b v nhn t c ch l lch.

6.2. Khng cc virus thc vt
Cc virus gy ra nhng thit hi ng k trong hu ht cc cy trng
lng thc v cy cho si trn phm vi th gii. Nhiu phng thc c s
dng kim sot s xm nhim virus bao gm cc x l ha hc git
cc vector virus, chuyn vo cy trng cc gen khng t nhin t cc loi
lin quan, s dng chn on v ch dn m bo nhn ging cc vt liu
khi u sch virus (v d: ht, c). Tuy nhin, s pht trin chnh khai
thc tnh khng xut pht t cc tc nhn gy bnh, v d s dng cc trnh
t xut pht t virus c biu hin trong cc cy chuyn gen cung cp
tnh khng i vi cc virus thc vt. Hng ny da trn c s cc nghin
cu v s gy nhim (inoculation) hay xm nhim (infection) thc vt
khi u vi cc chng virus nh cung cp s bo v chng li s gy
nhim tip theo vi cng loi chng virus hoc cc virus lin quan gn gi.
Tnh khng bt ngun t tc nhn gy bnh nh vy i hi s chuyn np
gen thc vt vi cc trnh t xut pht t virus; tnh khng vt ch xut hin
cho kt qu t hai c ch khc nhau: (1) s bo v c dn xp bi s biu
hin ca cc protein virus t nhin hoc bin i (v d protein v, replicase,
v replicase khim khuyt), v (2) s bo v c dn xp mc phin
m (RNA-mediated resistance), i hi s phin m ca RNA hoc t cc
chui hon chnh hoc tng phn xut pht t virus ch (bao gm cc gen
cho protein v, replicase, replicase khim khuyt, protease, protein vn
ng).
Trng hp cc phn t lm nn tng cho tnh khng xut pht t
virus l i tng ca nghin cu chuyn su. C s ca tnh khng virus
c sp t bi RNA (RNA-mediated) v s im lng ca cc gen hu dch
m c kh nng tng t, phn nh cc hot ng c bn trong trong cc t
bo thc vt pht hin, bt hot v o thi cc DNA hoc cc RNA
ngoi lai. V d: cc gen ni sinh ca thc vt c chn vo virus nh
PVX c th biu hin im lng ca gen ni sinh ca thc vt.
to ging cy trng chng chu virus, hin nay c cc hng:
- Bo v cho.
- S dng RNA v tinh.
- S dng enzyme replicase.

6.2.1. Bo v cho
L bin php li dng hin tng lp v protein ca virus th nht
cn tr s xm nhp ca virus th hai, ngha l cho cy nhim loi virus c
c lc va phi s chng c s xm nhim ca virus c c lc cao hn.
tng cng kh nng biu hin gen protein v ngi ta gn thm vo
ui phn promoter 35S t virus khm sp l (CaMV) v a vo genome
cy trng nh h thng Ti-plasmid ca Agrobacterium. Thnh cng u tin
theo hng ny l khng bnh virus khm thuc l (TMV) cy thuc l,
nh s biu hin gen cp. Th nghim ng rung u tin v cy c chua
biu hin gen cp khng bnh TMV tin hnh nm 1987 ti M v cho kt
qu khng bnh cao. Sau hng lot kt qu chng t gen cp c hiu lc
i vi tt c cc loi virus gy bnh 20 loi cy khc nhau k c cc loi
cy trng nh c chua (1987), da hu (1987), la (1990), khoai ty (1989,
1990), c ci ng Qua hng lot th nghim ng rung, B Nng
nghip M cng nhn ging quc gia cho ging b xanh (Freedom II)
khng virus (1995). Cc ging khoai ty, da chut, c chua chng bnh
virus v ang c tip tc cng nhn.

6.2.2. S dng RNA v tinh
Trong mt s qun th virus c cc thc th ging virus cha cc phn
t RNA nh hn gi l RNA v tinh (sattelite RNA). RNA v tinh dng
nh khng m ha cho bt c protein no nhng c nhn ln nh enzyme
ca RNA t virus bnh thng v hp thnh cc tiu phn ging virus c v
protein. Mt s RNA v tinh c th c ch rt mnh s nhn bn ca virus
bnh thng. cDNA t RNA v tinh c tng hp vi s tham gia ca
promoter 35S CaMV ri a vo cy thng qua h thng Ti-plasmid ca
Agrobacterium. Cc cy c chuyn gen kiu ny th hin tnh khng virus
thuc l v virus khm da chut kh tt. Tuy nhin, so vi tnh khng theo
phng php bo v cho, tnh khng ny kh t hn v phi cn nng
virus rt cao. Bn cnh , ngi ta vn lo ngi d xy ra hin tng t
bin t phn t RNA v tinh thnh mt loi gy c cho chnh cy ch.

6.2.3. S dng enzyme replicase
Replicase l enzyme tham gia qu trnh tng hp nucleic acid ca
virus, hot ha cho s nhn ln ca DNA hoc RNA theo c ch b sung.
Cy trng chng virus cng c th c to ra bng chuyn cc gen
replicase vi nhiu on b bin i hoc b ct bt, kt qu cho thy khng
virus rt cao. V d, thuc l theo phng php chuyn gen biu hin gen
replicase b ct bt cho kh nng khng virus khm thuc l rt tt (1990).
Hin nay, vn cha thnh cng vi enzyme replicase ca virus khm t c
alfalfa i vi bnh virus khm thuc l. Protoplast i mch c chuyn
gen replicase t virus khm brome cng cha khng c bnh ny i
mch. Tuy th, t khi tm thy hng ng dng gen replicase, ngi ta vn
ang hy vng vo hng ny.

6.3. Khng cc bnh nm
Nm bnh gy hi cy trng rt nng, nht l cc nc nhit i c
m cao. Ci to ging chng nm hi da trn nguyn l a gen m ha
mt loi enzyme no c tc dng c ch trc tip hoc gin tip n s
pht trin ca nm hi. Enzyme lm thoi ha cc thnh phn chnh ca v
t bo nm chitin v -1,3 glucan l loi ang c ch . Khi c gen
chitinase chuyn vo, cy thuc l chuyn gen tng cng hot tnh
chng nm hi. S biu hin ng thi ca c hai gen chitinase v glucanase
trong thuc l lm cho cy c tnh khng nm hi cao hn cy c mt gen
c lp. Cng tng t, c chua cho tnh khng nm Fusarium cao hn hn,
sau khi c chuyn giao c hai gen ni trn.
Protein c ch ribosome (ribosome inhibitive protein, RIP) cng biu
hin tnh khng nm kh quan. Cy thuc l cho tnh khng nm rt tt, khi
cy c chuyn giao ng thi gen RIP v chitinase.

6.4. Khng cc bnh vi khun
i vi vi khun, hng nghin cu to ging cng ngh sinh hc
mi bt u. V c bn c ba hng :
- Dng gen m ha enzyme lm thoi ha thnh t bo vi khun, v d
gen lysozyme t cc ngun t bo ng vt hoc gen lysozyme t thc
khun th T4 a vo cy thuc l v khoai ty. Cc gen ny biu hin rt
cao hot tnh lysozyme v cc t bo c kh nng phng tr vi khun
Erwina carotovora rt tt.
- Gen m ha -thionin-cystein c chuyn giao sang cy thuc l
cng phng nga c vi khun Pseudomonas syringae.
- Cy gen sn xut protein lm gim c t ca vi khun, l hng c
nhiu ha hn. Cc gen ny ch yu sn xut cc loi enzyme phn hy c
t ca vi khun, do vy v hiu ha tc hi ca chng.

IV. Sn xut cc dc liu sinh hc
1. Cc hp cht t nhin
Thc vt l ngun cung cp cc hp cht ha hc dng lm dc liu
rt c gi tr. Nhng sn phm ny, c bit nh l cc cht trao i th
cp (secondary metabolites), thng c sn xut vi mt lng rt nh
(dng vt) trong thc vt v khng c chc nng trao i cht r rng.
Chng dng nh l sn phm ca cc phn ng ha hc ca thc vt vi
mi trng chung quanh, l s thch nghi vi stress ca mi trng hoc l
s bo v ha hc chng li vi sinh vt v ng vt.
sn xut cc sn phm th cp t thc vt, cc m thc vt ngoi
sinh t cy hon chnh c nui cy dch huyn ph (suspension culture)
trong iu kin v trng (Hnh 4.14). C s ca k thut nui cy m thc
vt da trn tnh ton th ha sinh (biochemical totipotency) duy nht ca t
bo thc vt. Nhiu sn phm trao i cht c th c sn xut t nui cy
dch huyn ph c cht lng cao hn trong cy hon chnh. C nhiu bng
chng cho thy c mi quan h ngc (feedback) gia tc sinh trng v
sn xut cc cht th cp. Khi tc sinh trng cao, cc qu trnh s cp
ca t bo l phn chia t bo v sn xut sinh khi t bo. Trong pha tnh,
khi s sinh trng gim n mc ti thiu, s sn xut v tch ly cc cht
th cp s tng ln.

Hnh 4.14. Nui cy t bo dch huyn ph thc vt trong h ln men 100 L
Cc cht trao i th cp hay cn gi l cc cht th cp c th xp
trong ba nhm chnh: alkaloid, tinh du v glycoside.
Cc alkaloid c dng tinh th l cc hp cht cha nitrogen, c th
c tch chit bng cch dng dung dch acid. Alkaloid c hot tnh sinh l
trn tt c ng vt v c s dng trong cng nghip dc. H alkaloid
bao gm: codein, nicotine, caffeine v morphine. Cc tinh du cha hn hp
terpenoid v c s dng nh l cht mi, cht thm v dung mi.
Glycoside bao gm cc phenolic, tanin v flavonoid, saponin v cc
cyanogenic glycoside, mt s trong chng c s dng lm cht nhum,
cc cht mi thc phm v dc phm.

1.1. Cc alkaloid
Ngi ta cng c th thu c cc cht nh caffein t nui cy t bo
cy Coffea arabica, betalain trong callus c ci ng, berberin t t bo
cy Coptis japonica (loi cy ny phi trng t 4-6 nm mi thu c hm
lng ng k berberin trong r, so vi hm lng ny c th thu c sau 4
tun bng phng php nui cy t bo) Nhng cht ny c s dng
rng ri trong cng nghip hng liu v trong y hc.
Cht reserpine c tc dng cha bnh cao huyt p v cc bnh ri
lon tun hon cng c sn xut bng phng php nui cy t bo cy
Rauwolfia serpentina. Nui cy t bo ca cy ny trong 30 ngy h ln
men quy m ln c th sn xut c 3.500 kg reserpine, tng ng vi
lng hng nm ca c th gii thu c t r cy .
Cc nh nghin cu thuc t hp dc phm Gibageigy (Based, Thy
S) sn xut c loi alkaloid l scopolamine t t bo cy Hyoscyanus
aegypticus nui cy trong h ln men khng c cnh khuy. Bng cch chn
lc cc dng t bo cao sn nh k thut t bin t bo trn, bin d n
dng v k thut gen, ngi ta tng c sn lng scopolamine ln gp
hng ngn ln.
Nhiu nghin cu cho thy nui cy callus v t bo ca cy
Catharanthus roseus c hm lng serpentin ngang vi cy dc liu bnh
thng. Mt s nghin cu phn lp c cc dng t bo
Cantharanthus sn xut serpentin v ajmalacine t nui cy in vitro. Bng
loi mi trng sn xut c bit ngi ta a c sn lng alkaloid
ca hai dng t bo tt nht ln mt mc cao hn na, trong mt dng
to c 162 mg/L serpentin, cn dng kia to c 72 mg/L serpentin
cng vi 264 mg/L ajmalacine. Mi y ngi ta hon thin c cng
ngh nui cy t bo ca cy Catharanthus roseus sn xut viblastine v
vincristine l hai cht khng ung th rt mnh, hin ang c nhu cu rt cao
v chng c s dng cha ung th mu.
Sikuli v cs (1997) sau khi gy nhim cy Datura stramonium vi
Agrobacterium rhizogenes nhn thy hm lng hyoscyamine r t
cc i sau 6 tun nui cy <100 mg/L.

1.2. Cc steroid
Trong lnh vc steroid v chuyn ha steroid, cc dng t bo c
nng sut cao c Kaul v cs cp n t nm 1969. H nui cy
thnh cng t bo ca cy Dioscorea deltoidea sn xut diosgenin, l
nguyn liu th ch yu sn xut cc steroid chng th thai v cc
hormone tuyn thng thn.
Qu trnh chuyn ha cc hp cht glycoside tim (cardiac) bng nui
cy t bo ca cy Digitalis lanata cng c nghin cu. Ngi ta
nhn thy, mc d cc t bo Digitalis ngng sn xut glycoside tim
nhng chng vn c kh nng hydroxyd ha digitoxin nguyn t
12
C
to ra digoxin. Digoxin l mt hp cht c ngha y hc ln hn
digitoxin. Qu trnh hydroxyd ha xy ra trong nui cy t bo rt nhanh
v rt hiu qu khi a vo mi trng nui cy cht -methyl-digitoxin.
Sau 12 ngy, ngi ta thu c 4 g -methyl-digitoxin trong mt bnh
nui dung tch 20 L.

1.3. Mt s cht khc
Th d in hnh nht l cng ngh sn xut shikonin, mt loi sc t
c kh nng dit khun, c trong r ca cy Lithospermum
erythrorhizon. Bnh thng shikonin tch ly khng nhiu trong r. Tuy
nhin, cc nh khoa hc Nht to c dng t bo r cy
Lithospermum c kh nng tch ly n 15% shikonin v hon chnh
cng ngh nui cy t bo sn xut shikonin. Cng ngh ny cho php
trong mt chu k nui cy thu hoch ti 5 kg hot cht v gip gim rt
nhiu gi thnh ca shikonin.
Hm lng tng i cao ca ubiquinone-10 c tm thy trong t
bo thuc l nui cy in vitro v ca L-dopa trong mi trng nui cy t
bo Mucuma pruriens. Nui cy t bo ca cy Panax pseudoginseng
cho hm lng saponin kh cao. Nui cy t bo ca cy Glycyrrhiza
glabra thu c hm lng glycyrrhizin t 3-4% khi lng kh.
Hm lng cht th cp cao nht c tm thy trong nui cy t bo
ca cy Coleus blumei l cht rosmarinic acid chim 13-15% khi lng
kh trong chu k nui 13 ngy, ln gp 5 ln so vi hm lng trong cy
trng iu kin t nhin. Trong nhng nm 1980, ngi ta cng sn
xut rt c hiu qu ginsengoside l hot cht ch yu ca nhn sm Panax
ginseng. Cc anthraquinone l mt nhm cc sn phm t nhin quan trng
c vi khun, nm, a y v thc vt bc cao c cc hot tnh sinh hc nh:
khng khun, khng nm, gim huyt p, gim au, chng st rt, chng
oxy ha, khng bnh bch cu v cc chc nng t bin. thc vt bc
cao, chng c tm thy rt nhiu h thc vt khc nhau, chng hn
Rubiaceae, Rhamnaceae, Polygonaceae, Leguminosae... Nui cy t bo cc
loi ca h Rubiaceae cho php thu c mt lng ln anthraquinone
thm ch trong mt s trng hp vt qu hm lng anthraquinone
cy b m.

2. Cc protein ti t hp
Protein ti t hp (protein ngoi lai) l protein t nhin c bin i
bng cng ngh DNA ti t hp nhm nng cao hoc thay i hot tnh ca
chng. Nui cy t bo thc vt c s dng sn xut cc sn phm
t nhin cch y hn 20 nm v gn y hn chng c dng sn xut
cc protein ti t hp. Cc t bo thc vt rt thch hp cho cc nguyn liu
ti t hp do chng c th sinh trng trn mi trng tng i n gin
khng cn b sung protein. Nu protein ngoi lai c sn xut trong nui
cy t bo v c tit ra trong mi trng, nhiu hn phn c tch ly
trong t bo, th vic thu hi v tinh sch sn phm c th c tin hnh
m khng c nhiu protein nhim bn. Cc protein c ngun gc thc vt an
ton cho ngi hn cc protein c ngun gc t t bo ng vt bi v cc
cht nhim bn v virus thc vt khng phi l tc nhn gy bnh ngi.
Ngoi ra, nui cy t bo thc vt cng l mt cng c thc nghim thun
li cho vic kho st s sn xut protein ngoi lai trong cy hon chnh.
Bng 4.4. Sn xut cc protein ti t hp bng nui cy t bo thc vt

Protein Loi thc vt
Hormone sinh trng ngi Nicotiana tabacum
Albumin huyt thanh ngi N. tabacum, Solanum tuberosum
Nhn t sinh trng biu m ngi N. tabacum
Nhn t sinh trng c hi N. tabacum
-interferon ngi Oryza sativa
Hirudin (chng ng mu) N. tabacum
Erythorpoetin N. tabacum
and haemoglobin ngi N. tabacum
Human muscarinic cholinergic receptors N. tabacum
GM-CSF chut N. tabacum
Interleukin-2 v Interleukin-4 N. tabacum
Alkalinephosphatase nhau thai ngi N. tabacum
1
-antitrypsin ngi
O. sativa
Hormone sinh trng ngi N. tabacum
GM-CSF ngi N. tabacum, O. sativa
Thc vt chuyn gen hin nay c xem l h thng sn xut rt
kinh t cho vic sn xut cc protein ngoi lai nh khng th, enzyme v
hormone. Sn xut thng mi mt s protein ca vi khun v ng vt
c tin hnh bng thc vt. Yu t quan trng nh hng n hiu qu
kinh t ca sn xut protein da trn c s thc vt l hiu sut ca protein
ngoi lai hoc nng ca sn phm c tch ly trong sinh khi. Theo ,
ngi ta ch ci thin s biu hin gen ngoi lai trong cy chuyn gen
thng qua vic pht trin cc promoter tt hn, chn lc cc dng chuyn
gen n nh, v c ch gen im lng (silence gene). Tuy nhin, mt yu t
quan trng l s t gy protein ngoi lai lm gim nng ca sn
phm chc nng trong m thc vt sau khi cc phn t c tng hp v lp
rp. S t gy protein ngoi lai lm bn sn phm vi cc on protein
mt hot tnh, v ngi ta cng gp kh khn khi loi b cc protein t gy
ny trong cc hot ng thu hi protein chc nng sn xut quy m ln.
Tm hiu chi tit v v tr v c ch ca s t gy ni v ngoi bo l rt
cn thit c th pht trin phng php sao cho gim thiu c s tn
tht protein sau dch m.

3. Vaccine thc phm (edible vaccine)
Cho n thi gian gn y ngi ta vn s dng vaccine sng nhc
c lm khng nguyn kch thch to khng th cn thit trong c th ngi
v vt nui. Vaccine kiu ny c mt s hn ch nh: c kh nng quay tr
li dng c hoc hot lc ca n gim kh nhanh trong c th ngi v vt
nui. Hin nay, nh cng ngh DNA ti t hp ngi ta sn xut c
protein v ca mt s loi virus nh virus bnh l mm long mng, bnh
di v vim gan B. Tuy nhin, vaccine c sn xut theo cc phng php
trn c gi thnh cao, iu kin bo qun v vn chuyn nghim ngt, cn
c k thut vin tin hnh tim chng.
Vaccine thc phm l mt m hnh l tng cho cc nc ang pht
trin, v n gip khc phc c cc kh khn ni trn ca vaccine c sn
xut theo phng php truyn thng hoc DNA ti t hp. Nguyn l c
bn ca qu trnh ny l chuyn mt loi gen c bit vo t bo thc vt.
Loi gen ny hot ng trong c th thc vt, s bin thnh ni sinh ra
protein khng nguyn. Khi nhng khng nguyn ny i vo c th ngi
thng qua n ung (di dng ti sng khng nu chn, nu khng s lm
mt hot tnh khng nguyn), h thng min dch ca ngi s t ng sinh
ra khng th chng li khng nguyn. Nh vy l thay vic tim chng
vaccine bng vic n nhng hoa qu hoc rau xanh c khng nguyn.
Vaccine thc phm c mt s u im sau: gi thnh r, n nh, d
sn xut trn quy m ln, d qun l, khng cn tinh sch, bo qun lu, d
vn chuyn
Mt s kt qu nghin cu bc u ca vaccine thc phm:
- Sn xut vaccine chng bnh infectious bursan desease virus (IBDV)
g trong c Arabidopsis chuyn gen.
- Chuyn gen orf2 ca virus gy bnh vim gan E vo cy c chua, v
cy Pichia pastoris.
- Sn xut vaccine vim gan B trong cy chui chuyn gen, cy
Physalis ixocarpa, u lupin vng, rau dip v c chua.
- Chuyn gen ltb ca E. coli (B subunit of E. coli heat-labile
enterotoxin) gy bnh ng rut vo khoai ty.
- Chuyn gen ctb (cholera toxin B subunit) gy bnh t ca vi khun
Vibrio cholerae v gen ltb vo cy thuc l

Hnh 4.15. M hnh pht trin vaccine thc phm

Virus si
RNA gy bnh si
Hemagglutinin (H) protein
A: Chn khng nguyn lm
vaccine v xc nh trnh t m
ha
Vector mang gen ca protein H
Plasmid
Agrobacterium

B: Chuyn trnh t m ha vo
vector (plasmid) v bin np vo
Agrobacterium
Mnh l

C: ng nui cy Agrobacterium
v m thc vt
Cy chuyn gen Phn tch Western blot xc
nh s hin din ca protein H
D: Ti sinh cy hon chnh t cc
t bo c chuyn gen v phn
tch s biu hin ca khng
nguyn bng Western blot
Tim Cho n Nghin cu ng
vt linh trng
E: Xc nh kh nng sinh min
dch khng nguyn trong ng vt
m hnh
Mt nghin cu c cng b gn y trong lnh vc sn xut
vaccine t thc vt, l gy min dch trong c th ngi bng vaccine
thc phm iu tr bnh vim gan B. Loi cy trng c s dng
chuyn gen vim gan B ln ny l khoai ty. Ngi ta hy vng rng khi n
loi khoai ty ny, cht khng nguyn s gy ra mt phn ng min dch nh
trong c th ngi. T , c th ngi s to ra cht min dch c th i
vi cn bnh ly nhim vim gan B. 42 nhn vin chm sc sc khe
tui 25-58 tham gia vo cuc nghin cu. Trong , 33 ngi c ch
nh n khoai ty chuyn gen m khng c t dc, mt cht lm tng kh
nng phn ng min dch. Chun khng nguyn khng virus vim gan B
trong huyt thanh c o trong mt s ln nht nh mi ngy. Kt qu cho
thy i vi nhng ngi n khoai ty khng chuyn gen cc chun
khng tng, trong khi 19 trong s 33 ngi n khoai ty chuyn gen th
chun tng 57,6%, trong khi vaccine hin c trn th trng c tc dng
ti 90% i tng, k c khi c cha cht t dc.

1. Ammirato PV, Evans DA, Sharp WR and Bajaj YPS. 1990. Handbook
of Plant Cell Culture. Vol 5, McGraw-Hill Publishing Company. USA.
2. Chrispeels MJ and Sadava DE. 2003. Plants, Genes, and Crop
Biotechnology. 2
nd
ed. Jones and Bartlett Publishers, Massachusetts, USA
3. Cutler SJ and Cutler HG. 2000. Biologically Active Natural Products:
Pharmaceuticals. CRC Press LLC, USA.
4. Jain SM, Gupta PK and Newton RJ. 1994. Somatic Embryogenesis in
Woody Plants. Vol 3. Forestry Sciences 44, Kluwer Academic Publishers,
Netherland.
6. Narayanaswamy S. 1994. Plant Cell and Tissue Culture. Tata McGraw-
Hill Publishing Co. Ltd. New Delhi, India.
7. Ramawat KG and Merillon JM. 1999. Biotechnology: Secondary
Metabolites. Science Publishers Inc. USA.
9. Razan MK. 1994. An Introduction to Plant Tissue Culture. Oxford and
IBH Publishing Co. Pvt. Ltd. New Delhi, India.
10. Roberts MF and Wink M. 1998. Alkaloids: Biochemistry, Ecology,
and Medicinal Applications. Plenum Press, New York, USA.
11. Trigiano RN and Gray DJ. 2000. Plant Tissue Culture Concepts and
Laboratory Exercises. CRC Press, New York, USA.
Chng 5

Cng ngh sinh hc ng vt

I. M u
T bo ng vt c th sinh trng trn cc loi mi trng dinh
dng tng hp bn ngoi c th, v th chng c nui cy cho cc
mc ch sau:
- Nghin cu cc t bo ung th, phn loi cc khi u c tnh, xc nh
s tng hp ca m trong cy ghp, nghin cu cc t bo c bit cng s
tng tc ca chng, sn xut t bo gc
- ng dng sn xut cc hp cht ha sinh quan trng dng trong
chn on nh cc hormone sinh trng ca ngi, interferon, hot t
plasminogen m, cc viral vaccine v cc khng th n dng (monoclonal
antibodies). Theo phng php truyn thng cc hp cht ha sinh ny c
sn xut bng cch s dng cc ng vt sng hoc c tch chit t xc
ngi cht. Chng hn, cc khng th n dng c th c sn xut bng
cch nui cy cc t bo hybridoma trong cc khoang mng bng
(peritoneal cavity) ca chut, hoc hormone sinh trng dng cha bnh
ci (dwarfism) c th c tch chit t xc ngi cht. Tuy nhin, s
lng thu c t cc phng php ny rt hn ch v th vic ng dng
rng ri chng trong iu tr cn gp nhiu kh khn.
Chuyn gen vo ng vt to ra ngun thc phm c gi tr l mt
trong nhng ng dng c ngha ca cng ngh sinh hc ng vt. Tuy
nhin, hng nghin cu ny vn cn mt vi hn ch trn ng vt c v
ln do chng sinh sn mi ln rt t trng, vic cy phi tr vo m mang
phc tp, mi m mang ch nhn c mt t phi, trng ca a s ng vt
nui c t bo cht rt c nn kh nhn thy tin nhn chuyn gen vo
Mc tiu ca chuyn gen l nhm a vo vt nui nhng tnh trng c hiu
qu kinh t cao nh s dng trit thc n, nhiu tht t m, sinh trng
nhanh, khng bnh Mc d, cn gp mt s kh khn v tht bi nhng
ngi ta cng c c mt vi thnh cng bc u nh to ra loi g
khng bnh do avian leukosis virus gy ra hay cu cho nhiu lng Cc kt
qu ny cho php hy vng s t c nhng bc tin mi trong tng lai.
Nhn bn v tnh (to dng) i vi cc vt nui c nng sut cao
nhng cc th h con ca n li khng c nh vy cng c mt vi
thnh cng nht nh, k thut ny cho php ti to cc vt nui c y
phm cht nh ban u bng phng thc v tnh. Thnh cng vang di
trong lnh vc ny l kt qu ca Wilmut v cng s (1996) cho ra i
ch cu Dolly. Cu Dolly khng c b m hiu theo ngha thng thng m
c to ra bng cch sao y mt con cu trng thnh. Ngoi ra, k thut
ny cng c p dng trong nhn ging cc ng vt chuyn gen, cc ng
vt ny khi sinh sn hu tnh c th th h con khng nhn c gen ch,
do s can thip ca nhn bn v tnh trong trng hp ny l rt cn
thit. Bn cnh cc ng dng trong sn xut, hin nay vic ng dng nhn
bn v tnh bo tn cc ngun gen v ng vt qu him cng ang c
ch trng c bit.

II. Nui cy t bo ng vt c v
1. Cc u im v hn ch ca nui cy t bo ng vt
1.1. Cc u im ca nui cy t bo ng vt
- H thng t bo ng vt l cc nh my t bo thch hp cho vic
sn xut cc phn t phc tp v cc khng th dng lm thuc phng bnh,
iu tr hoc chn on (Bng 5.1).
- Cc t bo ng vt p ng c qu trnh hu dch m chnh xc
i vi cc sn phm protein sinh-dc (biopharmaceutical protein).
Chuyn gen ca ng vt c v cng c th c sn xut bi h thng vi
khun bng cch dng cng ngh DNA ti t hp. Tc sinh trng
nhanh, thnh phn mi trng n gin v r tin ca nui cy t bo vi
khun khin chng c nhiu u im hn so vi nui cy t bo ng vt c
v. Tuy nhin, vi khun thiu kh nng bin i hu dch m (post-
translational modifications) bao gm vic phn gii protein, lin kt tiu n
v (subunit), hoc nhiu phn ng kt hp khc nhau nh glycosylation,
methylation, carboxylation, amidation, hnh thnh cc cu ni disulfide hoc
phosphorylation cc gc amino acid. Nhng sa i ny rt quan trng nh
hng n hot tnh sinh hc ca sn phm. V d qu trnh glycosylation
c th gip bo v protein chng li s phn gii chng, duy tr kh nng n
nh cu trc v bin i khng nguyn.

Bng 5.1. Cc sn phm quan trng ca nui cy t bo ng vt

Enzyme Urokinase, hot t plasminogen m
Nhm I Hormone Hormone sinh trng (GH)
Cc nhn t sinh
trng
Cc cytokine khc
Nhm II Vaccine Bnh di, bnh quai b, bnh si
ngi
Veterinary-FMD vaccine, New Cattles
Disease ...
Nhm III Khng th n dng Cc cng c chn on
Nhm IV Virus cn trng Thuc tr su sinh hc cho Baculovirus
Nhm V Cc cht iu ha
min dch
Interferon v interleukin
Nhm VI Cc t bo nguyn
vn
Th nghim c cht hc

- Sn xut cc viral vector dng trong liu php gen (bin np mt gen
bnh thng vo trong t bo soma mang gen tng ng b khim khuyt
cha bnh do s khim khuyt gy ra). Cc mc ch chnh ca liu php
ny l cc bnh ung th, HIV, chng vim khp, cc bnh tim mch v x
ha u nang.
- Sn xut cc t bo ng vt dng nh mt c cht in vitro trong
nghin cu c cht hc v dc hc.
- Pht trin cng ngh m hoc pht sinh c quan sn xut cc c
quan thay th nhn to-sinh hc/cc dng c tr gip, chng hn:
+ Da nhn to cha bng.
+ M gan cha bnh vim gan.
+ o Langerhans cha bnh tiu ng.

1.2. Mt s hn ch ca nui cy t bo ng vt
Mc d tim nng ng dng ca nui cy t bo ng vt l rt ln,
nhng vic nui cy mt s lng ln t bo ng vt thng gp cc kh
khn sau:
- Cc t bo ng vt c kch thc ln hn v cu trc phc tp hn
cc t bo vi sinh vt.
- Tc sinh trng ca t bo ng vt rt chm so vi t bo vi sinh
vt. V th, sn lng ca chng kh thp v vic duy tr iu kin nui cy
v trng trong mt thi gian di thng gp nhiu kh khn hn.
- Cc t bo ng vt c bao bc bi mng huyt tng, mng hn
nhiu so vi thnh t bo dy chc thng thy vi sinh vt hoc t bo
thc vt, v kt qu l chng rt d b v.
- Nhu cu dinh dng ca t bo ng vt cha c xc nh mt
cch y , v mi trng nui cy thng i hi b sung huyt thanh
mu rt t tin.
- T bo ng vt l mt phn ca m c t chc (phn ha) hn
l mt c th n bo ring bit nh vi sinh vt.
- Hu ht cc t bo ng vt ch sinh trng khi c gn trn mt b
mt.

2. Cc dng t bo ng vt c v v cc c im ca n
Cc t bo ng vt c v l t bo eukaryote, chng c lin kt vi
nhau bi cc nguyn liu gian bo to thnh m. M ng vt thng
c phn chia theo bn nhm: biu m (epithelium), m lin kt
(connective tissue), m c (muscle) v m thn kinh (nerve). Biu m to
thnh lp ph v lp lt trn cc b mt t do ca c th, c bn trong v
bn ngoi. m lin kt, cc t bo thng c bao bc trong th gian bo
rng (ko di), c th l cht lng, hi rn hoc rn. Cc t bo m c
thng thon di v c gn vi nhau thnh mt phin hoc mt b bi m
lin kt. M c chu trch nhim cho hu ht chuyn ng ng vt bc
cao. Cc t bo m thn kinh gm c thn bo cha nhn v mt hoc nhiu
phn m rng di v mnh c gi l si. Cc t bo thn kinh c kch
thch d dng v truyn xung ng rt nhanh.

2.1. Cc t bo dch huyn ph
T bo hng cu (blood) v bch huyt (lymph) l cc m lin kt
khng in hnh dng th lng. Cc t bo mu hoc dch bch huyt l cc
t bo dch huyn ph (suspension cells), hoc khng dnh bm khi chng
sinh trng trong nui cy in vitro. Cc t bo khng dnh bm khng i
hi b mt sinh trng.
Chng hn, cc t bo bch huyt (lymphocytes) bt ngun t m
bch huyt l cc t bo khng dnh bm v c hnh cu ng knh t 10-
20 m. Chng c th c nui cy trong mi trng dch lng theo
phng thc tng t vi khun.

2.2. Cc t bo dnh bm
Hu ht cc t bo ng vt bnh thng l cc t bo dnh bm, v th
chng cn c b mt gn vo v sinh trng. Trong cc ng dng, ngi
ta s dng rng ri cc loi t bo dnh bm l t bo biu m v nguyn
bo si (fibroblast). Cc t bo dnh bm cn c mt b mt m sinh
trng nh l thy tinh hoc plastic. a petri hoc cc chai trc ln l cc
loi c s dng rng ri nht. Cc chai c t nm trn mt trc ln
quay trn chm trong t m. Chai c dung tch mt lt cha khong 100 mL
mi trng l thch hp cho cc t bo va sinh trng trn thnh chai va
tip xc vi mi trng v khng kh. Tuy nhin, chai trc ln ch dng cho
quy m phng th nghim v din tch b mt trn mt n v th tch ca
chai nui cy kh nh (500 cm
2
/L).
T l din tch/th tch c th c tng ln khi cc t bo sinh trng
trn cc gi th l polymer bt bin (spongy), th gm (ceramic), cc si
rng, microcapsule, hoc trn cc ht nh c kch thc hin vi gi l
microcarrier.

3. Cc sn phm thng mi ca nui cy t bo ng vt c v
Cc sn phm sinh hc c sn xut bng t bo ng vt c v ch
yu l cc glycoprotein. Bng 5.2 gii thiu mt s sn phm tiu biu. S
phc tp v chi ph cao ca cc qu trnh nui cy t bo ng vt cho thy
sn xut protein bng t bo ng vt c v ch tht s kinh t i vi nhng
sn phm c gi tr cao (>USD 10
6
/kg). V th, cc sn phm protein ca t
bo ng vt c v l nhng sn phm ch yu dng lm dc phm.
Khng th n dng (monoclonal antibodies-Mabs) l sn phm nui cy t
bo ng vt c v c gi tr nht hin nay. Cc tnh cht lin kt c hiu
cao ca Mabs c th c dng trong chn on (c y hc ln th y), phn
tch hnh nh (ung th v bnh tim), tinh sch sn phm (sc k i lc) v
nh l cc nhn t tr liu. Cc protein c c tnh dc liu khc sn xut
bng nui cy t bo ng vt c v c hng ti s dng trong iu tr
ung th, bnh tim, cc bnh v mu v ri lon hormone.
- Qu trnh glycosyl ha mt phn t protein (hu dch m) xy ra
mng li ni sinh cht (endoplasmic reticulum-ER) v phc hp Golgi ca
t bo eukaryote, v ph thuc vo s hin din ca cc enzyme c hiu:
glycosyltransferase v glycosidase.
- Vi khun hoc khng cha cc c quan t ny hoc khng cha cc
enzyme v th khng th thc hin s bin i hu dch m ny.
- Nm men v nm si (eukaryote) c th glycosyl ha cc protein t
cc t bo ng vt c v nhng thc hin kh khn hn.
- Mt s protein dng lm dc phm khng c glycosyl ha hoc
khng cn c glycosyl ha cho chc nng thch hp nh insulin hoc
hormone sinh trng ngi, albumin huyt thanh ngi v haemoglobin,
c th c sn xut vi gi thnh thp hn nhiu hn nh vi khun, nm
men hoc nm si.

4. Glycosyl ha protein (glycosylation)
- Trong khi qu trnh tng hp protein c hng dn bi cc khun
mu DNA v RNA, th vic b sung ng vo protein l mt qu trnh
khng cn khun mu. V th, c th tm thy nhiu bin th trong cc cu
trc oligosaccharide ca cc glycoprotein (cc protein c glycosyl ha).
- Cc glycoprotein c trnh t amino acid ging nhau, nhng cc cu
trc oligosaccharide khc nhau c gi l cc glycoform. Cc cu trc
oligosaccharide c lin kt ng ha tr vi protein hoc nguyn t
nitrogen (N-glycosylation) hoc nguyn t oxygen (O-glycosylation). Hai
dng glycosylation ny khc nhau khng ch v tr gn vo ca ng m
cn loi ng v s lng ng c b sung.

Bng 5.2. Mt s protein dng lm dc phm c sn xut bng nui cy t
bo ng vt c v

Protein dc liu Chc nng Loi
glycosylation
Hot t plasminogen
m (tPA)
Tc nhn phn gii fibrino-gen
(cht to t huyt)
Lin kt N
Erythropoietin (EPO) Tc nhn chng thiu mu Lin kt N v O
Nhn t VII, VIII, IX
v X
Cc tc nhn gy cc mu, bnh
mu long kh ng
Lin kt N v O
Hormone kch thch
nang non (FSH), kch
dc t mng m
ngi (hCG)
iu tr v sinh Lin kt N v O
Interleukin-2 Chng ung th, iu ha min
dch, iu tr HIV
Lin kt O
Interferon-alpha
(IFN- )
Chng ung th, iu ha min
dch
Lin kt N v O
Interferon-beta
(IFN- )
Chng ung th, tc nhn chng
virus
Lin kt N
Interferon-gamma
(IFN- )
Tc nhn chng ung th, iu
ha min dch
Lin kt N
Nhn t kch thch
khun lc bch cu ht
(G-CSF)
Chng ung th Lin kt O
Khng th n dng Tr liu v chn on Lin kt N

- Hu ht cc protein hin din trn b mt t bo, virus, v trong mu
ca cc ng vt c glycosyl ha, v v th n c xem ging nh mt
s dc phm sinh hc cng s c glycosyl ha c cng chc nng
nh cc bn sao t nhin ca chng.
- Vi khun khng glycosyl ha cc protein ca chng (hoc ng hn
l c cc loi lin kt peptide-ng hon ton khc vi ng vt), v th
cc k thut ca cng ngh di truyn c pht trin cho nm men v cc t
bo eukaryote l nhng loi c glycosyl ha. D nhin, chng khng lun
lun glycosyl ha trong mt phng thc chnh xc nh cc t bo ngi
thc hin.
- ng c th c lin kt vi protein thng qua cc nhm amide
ca asparagine (Asn) trong chui peptide ngn Asn-X-Ser/Thr (trong X
i din cho mi amino acid ngoi tr proline), hoc him khi hn, thng
qua nhm hydroxyl ca serine (Ser) v threonine (Thr).
- Glycosyl ha l mt dng ca s bin i hu dch m, tc l bin
i ha hc ca protein sau khi protein c dch m t RNA. Mt kiu
glycosyl ha protein khc l theo phng thc ha hc, n xy ra bt c khi
no mt protein nm trong cc dung dch ng mt thi gian lu. Phng
thc ny cng c gi l glycosylation.
- Mt t bo c th thu c mt hn hp cc glycoform khc nhau.
Cc glycoform khc nhau c cc tnh cht v chc nng khc nhau trong
nhiu trng hp, v c nhn bit bng h thng min dch. Cc t bo
ung th thng sn xut cc glycoform khc nhau t nhng t bo bnh
thng t khi glycosyl ha cc protein b mt ca chng. Nhiu ch th khi
u trn thc t l cc du hiu phn bit glycoprotein c trng cho cc t
bo ung th, v do l phng thc c nhiu tim nng trong chn on
ung th hoc sn xut cc dc phm ch cho n.

5. Mi trng nui cy t bo ng vt c v
Nhu cu dinh dng ca cc t bo ng vt c v ln hn vi sinh vt
do, khng ging cc vi sinh vt, ng vt khng trao i cht nitrogen v
c. V th, nhiu amino acid v vitamin cn phi c b sung vo mi
trng. Mi trng c trng dng trong nui cy t bo ng vt bao gm
cc amino acid, cc vitamin, cc hormone, cc nhn t sinh trng, mui
khong v glucose. Ngoi ra, mi trng cn c cung cp t 2-20% (theo
th tch) huyt thanh ca ng vt c v. Mc d huyt thanh c thnh phn
cha c xc nh y , nhng nhiu nghin cu cho thy n rt cn
thit cho s pht trin v tn ti ca t bo trong nui cy. Bng 5.3 trnh
by thnh phn v hm lng ca cc cht trong mi trng Eagle (Eagle
1959), y l mt trong nhng mi trng c s dng ph bin.

Bng 5.3. Thnh phn mi trng Eagle (1959)

Thnh phn Nng
(mg/L)
Thnh phn Nng
(mg/L)
1. L-Amino acid
Arginine
Cystine
Glutamine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Tyrosine
Valine

2. Carbohydrate
Glucose
Serum

105
24
292
31
52
52
58
15
32
48
10
36
46

1000
5-10%
3. Vitamin
Choline
Folic acid
Inositol
Nicotinamide
Pantothenate
Pyridoxal
Riboflavin
Thiamine

4. Mui
NaCl
KCl
CaCl
2

MgCl
2
.6H
2
O
NaH
2
PO
4
. 2H
2
O
NaHCO
3

1
1
2
1
1
1
0,1
1

6800
400
200
200
150
2000

Huyt thanh dng trong mi trng nui cy khng ch t tin m
cn l ngun nhim bn virus v mycoplasma. Do bn cht ha hc ca
huyt thanh cha c xc nh y nn trong mt s trng hp c th
nh hng xu n kt qu nui cy. S hin din ca nhiu protein khc
nhau trong huyt thanh cng c th lm phc tp cc qu trnh phn tch v
tinh sch u ra (downstream processing). V l do , nhiu nghin cu
c thc hin xy dng cng thc mi trng khng c huyt thanh.
Nhng cng thc ny cha cc hormone v cc nhn t sinh trng c
tinh sch thay th cho huyt thanh.

6. Nui cy t bo ng vt c v trn quy m ln
6.1. Cc iu kin chung
H thng ln men (fermenter) c s dng trong nui cy vi
khun v nm men t rt lu. u tin, s ln men (fermentation) l thut
ng dng cho sn xut ethanol. Sau , cc nh vi sinh vt hc ng dng
cc nguyn tc trn tch chit cc vitamin, cc acid hu c v cc khng
sinh Kt qu dn n s pht trin nhanh chng cc phng php v cc
h thng ln men khc nhau.
Cc nguyn l tng t sau c ng dng cho nui cy sinh khi
t bo ng vt v thc vt. Tuy nhin, nui cy cc t bo ng vt v thc
vt kh khn hn nhiu so vi vi sinh vt, ci chnh l do qu trnh trao i
cht trong cc loi t bo ny din ra chm, iu ny cng phn nh tc
sinh trng chm ca t bo. Cc t bo ng vt c nhu cu dinh dng
phc tp hn so vi vi khun v nm men, chng khng c thnh t bo nh
vi khun v th chng rt d bin dng v v. Do , cc h thng khuy v
sc kh c thit k khc vi nui cy vi khun. Mt t bo thp s cho
nng sn phm thp. Mc d c mt s im khng thun li, nhng h
thng ln men c s dng nui cy t bo ng vt t nht cng
vi chc nm (Hnh 5.1). Cc dng t bo khc nhau nh BHK-21, LS, cc
t bo Namalwa c sinh trng trong h ln men theo phng thc
nui cy chm ngp trong mi trng sn xut cc viral vaccine v cc
sn phm khc.
- c im d bin dng v d v ca t bo ng vt c khc
phc bng cch:
+ S dng h ln men c cnh khuy hnh mi cho.
+ Cung cp kh trc tip c th to ra bt kh d lm v t bo, v th
cn cung cp kh bng cch khuch tn thng qua ng silicone.
+ Mi trng cha nhiu protein huyt thanh c kh nng gy ra hin
tng to bt nn cn khuy chm v nh. i vi nui cy mt cao, cn
cung cp thm oxygen. Phng php dng ng silicone sc kh c nhiu
u im do khng to ra bt kh v tc truyn oxygen l tha ng. Tuy
nhin, khc ln ca tube d v, y l kh khn v hn ch i vi cc h
ln men quy m nh dng trong phng th nghim.
- Nh vy, cc h ln men vi sinh vt c ci tin thch hp c th
dng nui cy sinh khi cc t bo ng vt sinh trng trong dch huyn
ph.
- Nu mun nui cy mt dng t bo dnh bm th nn dng mt h
thng cht mang nh l microcarrier.

Hnh 5.1. Nui cy t bo ng vt trong h ln men 50 L

6.2. Nui cy m
Trong nui cy m (batch culture), cc t bo cy gy (cc t bo
c tip vo-inoculum cells) c b sung vo th tch tng s ca mi
trng nui cy (Hnh 5.2). T bo s s dng ht cht dinh dng trong
mi trng v tit ra cc sn phm ph (by-product) trong sut qu trnh
sinh trng. S sinh trng ch dng li khi c cht b s dng ht hoc sn
phm ph t n mt nng c th c ch t bo. Tuy nhin, trong
nhiu trng hp nguyn nhn lm ngng sinh trng t bo vn cha c
lm r.
- Nui cy t bo ng vt quy m phng th nghim. Cc t bo
ng vt c v c duy tr bng cch cy chuyn vi mt s lng n nh
trong cc chai bt bng nha c y nng (c gi l T-flask hoc Roux-
bottle) cha t 10-100 mL mi trng (Hnh 5.3):
+ Cc t bo dnh bm s gn vo y chai, v nhng ln cy chuyn
sau phi dng trypsin (mt loi protease ha tan c cc protein bc cu)
tch ri t bo.
+ Cc t bo dch huyn ph gn lng lo hn t bo dnh bm v c
th ly ra bng cch lc bnh nui cy. Cn lu l lng mu a vo
khng c qu t, mt thng c s dng l khong 2 10
5
t bo/mL
hoc hn cng vi mt t mi trng gn ht cht dinh dng ca ln
nui cy trc (spent medium), trong mi trng ny c th cha cc
nhn t cha bit c tc dng kch thch sinh trng t bo. Trong mt s
trng hp khc, mi trng s dng gn ht cht dinh dng phi c
loi b bng cch ly tm trnh cc sn phm ph gy c ch c mt trong
mi trng.
- Nui cy t bo ng vt c v quy m ln. Th tch mi trng
thng s dng l khong 200 L, th tch ny p ng c yu cu sn
xut cho cc protein tr liu c gi tr cao. Nhng d vy, qu trnh nui cy
vn i hi mt s bc trung gian, bc u tin chuyn t bo t nui cy
tnh ti bnh nui c lc hoc bnh nui xoay (spinner flask) (Hnh 5.3).
Bnh nui xoay, c trang b cnh khuy t tnh treo xung t np bnh m
khng tip xc vi y, c pht trin u tin to ra s khuy trn nh
cho nui cy microcarrier, nhng hin nay cng c dng cho nui cy
dch huyn ph. H s chia (scale-up factor) t nui cy tnh, hoc cc
bnh lc khng iu chnh pH, thng nh hn 5, ngha l mt th tch cy
gy t nht l 20% phi c dng. Trong h ln men, ni c mt t bo
cao hn, th h s chia c th ln ti 10 (ngha l cy gy 10% v/v hoc t
hn).
Mt nui cy m c trng, chng hn nui cy t bo hybridoma
trong h ln men, ko di t 3-5 ngy t ti mt t bo l 2-5 10
6
t
bo/mL. Tc sinh trng cc i c trng () ca cc t bo hybridoma
v myeloma khong 0,05/gi. Lng khng th n dng c sn xut
trong nui cy m ca t bo hybridoma nm trong khong t 10-
100 mg /L. quy m ln, sn xut m ca khng th n dng c tin
hnh h ln men thng khuy loi 1 m
3
, trong mt t bo ln ti
5 10
6
t bo/mL thu c sau 3,5 ngy. Sn xut thng mi u tin vi
cc t bo dnh bm c thc hin trong chai quay (Hnh 5.2). Cc chai
quay c gi trong mt chuyn ng khng i bng cch quay trn v
cc t bo dnh bm sinh trng trn b mt chai. Mt b mt c trng t
750-1.500 cm
2
vi 200-500 mL mi trng s cho sn lng 1-2 10
8
t
bo. Mt din tch b mt ln hn c th thu c bng cch dng
microcarrier trong cc h ln men thng khuy.

6.3. Nui cy m c cung cp dinh dng
Nui cy m c cung cp dinh dng (fed-batch culture), trong mt
ngha chnh xc, c iu khin cng mt phng thc nh nui cy th
n nh ha tnh (chemostat culture), ngha l tc sinh trng t bo b
hn ch bi tc pha long v c cht gii hn s sinh trng. L do s
dng k thut nui cy m c cung cp dinh dng (c gii hn c cht)
l v s gii hn O
2
v chuyn ha bi tit qu mc b ngn cn trong qu
trnh nui cy, kt qu l mt t bo cao hn nhiu so vi nui cy m.
Mc d nui cy m c cung cp dinh dng (c gii hn glucose v
glutamine) cng gii quyt c vn chuyn ha bi tit qu mc trong
t bo ng vt c v, nhng n khng lm tng mt t bo ln
mt cch ng k. Bng cch cung cp mt hn hp cn bng cc cht dinh
dng, mt t bo v nng sn phm cng c ci thin hn 10 ln
so vi nui cy m. Nui cy m c cung cp dinh dng c th ko di t
nht ti mt thng, cc qu trnh nui cy quy m 15 m
3
c kho st
v mt t bo trong khong 1-1,4 10
7
t bo sinh trng/mL cng
c thng bo.

6.4. Nui cy th n nh ha tnh
Nui cy chemostat l kiu nui cy c s b sung lin tc mi trng
sch v s chy ra ca cht lng nui cy, ng thi gi th tch nui cy
khng i (Hnh 5.2). Trong nui cy vi sinh vt trng thi n nh
(steady-state), mi quan h gia tc pha long (D) v tc sinh trng
c trng () c biu din bng ng thc D. Tuy nhin, trong nui
cy t bo ng vt c v, s sinh trng ca nui cy cn phi c tnh
ton. S sinh sn ca t bo khng ch thay th s hao ht cc t bo sng
st (ang sinh trng) b cun theo dng chy ra, m cn thay th cho cc t
bo b cht trong qu trnh nui cy. Do , c th m t trng thi n nh
cho tc sinh trng nh sau:
1
v t
N N D
Trong : N
t
l nng t bo tng s (t bo cht cng vi t bo
sng st) v N
v
l nng t bo sng st. T quan h ny cho thy rng
ln hn D khi hin tng cht t bo xut hin trong h thng.
Trong nui cy t bo ng vt c v, mi trng cha nhiu ngun
carbon v nitrogen, do thit lp s sinh trng trng thi n nh c
gii hn bi mt cht dinh dng l kh khn. Mc d, mt trong cc ngun
nng lng (glucose hoc glutamine) c th gii hn hiu sut sinh khi
trong nui cy trng thi n nh, nhng tc tiu th cc cht dinh dng
khc c th ph thuc vo mc cung cp ngun nng lng, hoc vo
nng ca mt cht dinh dng ring r.
Nhiu hng nghin cu tp trung ti u mi trng v sinh l hc
ca t bo ng vt c v, chng hn nh nh hng ca ln s to thnh
sn phm v nh hng ca nng O
2
ha tan, pH, nng glucose v
glutamine, nng cc vitamin v amino acid ln sinh trng v to thnh
sn phm, c kho st bng cch dng h nui cy chemostat. Cc qu
trnh sn xut chemostat vi reactor (bnh nui) c th tch ln ti 2 m
3
cng
c kho st.
Nui cy chemostat cho cc mc ch sn xut c mt vi nhc
im. Thi gian nui cy di ngy (t nht l nm tun) tng ng k
nguy c nhim bn, v thi gian cn thit ti thit lp mt nui cy trng
thi n nh sau khi s nhim bn xut hin lu hn vic ti khi ng cc
qu trnh nui cy m hoc m c cung cp cht dinh dng. Hn na, gi
tr ca mt qu trnh sn xut da trn nui cy lin tc mun c tha
nhn phi chng minh c rng dng t bo ang s dng l n nh trong
thi gian nui cy.


Hnh 5.2. Cc phng php nui cy t bo ng vt c v. Cc mi tn trng
ch dng chy mi trng, mi tn en dy ch dng chy ca dch nui cy c
sinh khi, mi tn xm nht ch dch nui cy tch sinh khi ra.

6.5. Nui cy perfusion
Trong nui cy perfusion, sinh khi c tch ly khi t bo c gi
li trong reactor nh b phn thu hi, trong khi mi trng sch c a
vo v mi trng hao ht cht dinh dng b loi b. Theo cch ny,
mt t bo ln ti 3 10
7
t bo/mL v c th t c nng sn phm
cao hn trong nui cy m. Cc b phn phn tch t bo t dch lng nui
Nui cy m
H ln men
(fermenter)
Chai quay
(Roller bottle)
Nui cy m c
cung cp dinh
dng
Nui cy th n
nh ha tnh
Nui cy
perfusion
Phn tch t bo
in situ
Phn tch t bo
bn ngoi

H si rng
cy c th c t bn trong hoc bn ngoi reactor (Hnh 5.2). Mt vi h
thng perfusion c th c phn bit, da trn phng php phn tch t
bo v mi trng:
- B lc xoay (spin-filter) s dng bung quay c li kim loi
(ng knh l 5-75 m). Nhc im ca spin-filter l d lm tc nghn,
dn n gim tc dng chy mi trng qua h lc v cui cng bt kn
tt c mc li ca mng lc.

Hnh 5.3. Cc loi bnh nui cy t bo ng vt. (A) Bnh T-flask. (B) Bnh
spinner loi 0,5 L.

- Mt chn la khc l h lc si rng (hollow fibre) c th c dng
phn tch t bo t dch lng nui cy. Vic lm tc nghn h lc cng
c th xut hin nhng c th khc phc bng cch dng tia nc ngc.
Cc thit b lng thu sinh khi cng c pht trin trong trng hp
phn tch t bo t dch lng nui cy c mt cao.
- Mt thit b c bit dng trng lc hp dn gi t bo trong
reactor l h lc m thanh (acoustic filter). H thng ny dng sng m
thanh tnh tp trung cc t bo trong dng chy. Cc t bo tch ly trong
cc giao im (node) ca sng v lng ngc xung y cht lng trong
nui cy, ngc li vi dng chy ln (up-flowing effluent stream). Sau
cng, s dng phng php ly tm thu hi t bo cho cc qu trnh sn
xut quy m ln.
Cc h thng nui cy si rng c th c xem l mt loi c bit
ca nui cy perfusion trong t bo c phn tch vt l khi dng chy
mi trng (Hnh 5.2). Cc t bo c sinh trng trong mt khi khng
A B
gian siu mao dn (extra capillary space), trong mi trng sch c
cung cp thng qua mt s lng ln cc si rng ca mng (s chuyn
khi). C th t ti mt 10
8
t bo/mL trong khng gian siu mao dn
v mi trng dng chy trong khng gian ny cha mt nng cao ca
sn phm. Tuy nhin, khi nng cc cht dinh dng tng dn v sn
phm c to thnh trn khp cc si s gii hn kh nng ca cc khi si
rng c thit k cho cc bnh nui (reactor) quy m sn xut ln. Mc d
vy, cc khi si rng vn d dng s dng v c ng dng thnh cng
trong cc qu trnh sn xut thng mi.

6.6. S lng v cht lng sn phm
Mt sn phm c tinh sch t nui cy t bo ng vt c v khng
th c 100% hot tnh sinh hc m ty thuc vo nhng bin i trong kiu
glycosylation hoc s phn gii protein. Hai thng s ny chu nh hng
bi cc iu kin mi trng. Phng thc glycosyl ha trong phn ng ph
thuc vo nhiu nhn t nh kiu nui cy, pha sinh trng ca nui cy
m, t bo c sinh trng trong cc microcarrier hoc trong dch huyn
ph, nng glucose, nng ammonium, hiu qu ca cc hormone trong
mi trng, s hin din ca huyt thanh, hm lng ca protein v lipid
trong mi trng, pH, v nng O
2
ha tan. V vy, vic chn la cc iu
kin sinh l thch hp trong mt qu trnh sn xut l rt quan trng c
c s glycosyl ha chnh xc ca mt protein dc phm.
Khng nhng cht lng ca cc sn phm m hiu sut ton phn
ca nui cy t bo ng vt c v cng chu nh hng ca nhiu thng s
nh pH, nng cc ion ammonia/ammonium v lactate, nng huyt
thanh, phng php nui cy, tui t bo nui cy, lng mu cy gy v
thnh phn mi trng. Do s phc tp ca sinh l t bo ng vt c v,
nn s phi hp gia cc mi trng v phng php nui cy khc nhau
thng phi c s dng, v nu tch ring nh hng ca tng nhn t
c trng s gp nhiu kh khn. Tuy nhin, tc sinh trng vn l thng
s chnh nh hng r rt ln hiu sut c trng ca sn phm t bo
ng vt c v.
Hiu sut c trng cng c th c ci thin bi cc hp cht khng
phi l thnh phn bnh thng ca mi trng nui cy t bo. Nui cy
mt s dng t bo ng vt c v cho thy chng c hiu sut c trng
cao hn trong mi trng m p lc thm thu c tng ln t mc
bnh thng l 330 mosmol ti hn 400 mosmol. Mc d cha hiu c
y , nhng ngi ta nhn thy nh hng ny ph thuc vo dng t bo
v mi trng c bn c s dng.
S lng sn phm c sn xut trong qu trnh nui cy c th biu
din bng phn trm ca lng protein tng s c sn xut. Trong mt s
trng hp, khi tc sinh trng tng ln th phn trm ca sn phm s
gim xung mt lng ng k (t l nghch). V d: tc sn xut c
trng ca protein trong dng t bo hybridoma c thng bo l
1,5 mg/10
9
t bo gi tc sinh trng c trng 0,02/gi. Lng sn
phm c sn xut tng ng vi 28% protein tng s. Nhng mt dng
t bo tng t c tc sn xut c trng thp hn nhiu (0,2 mg/10
9
t
bo gi) tc sinh trng 0,058/gi, th lng sn phm ch chim 1%
ca protein tng s. Trong mt s trng hp khc, dng t bo myeloma
sn xut khng th ti t hp th phn trm ca protein sn phm tng ln t
18%-29% quan st c khi tc sinh trng tng ln t 0,016/gi n
0,042/gi (t l thun).
Nui cy t bo ng vt c v thnh cng nht (nng v hiu sut)
l sn xut khng th n dng vi cc t bo hybridoma hoc myeloma.
Nh trnh by trn, tim nng sn xut ca cc t bo ng vt c v l
khng gii hn, nhng iu c quan tm hn c l nng sinh khi
c th t c. p ng yu cu ny, nui cy m c cung cp cht dinh
dng v reactor si rng (hollow fibre reactor) c s dng hng
ti cc nui cy c mt t bo cao ca hybridoma v myeloma. S gii
hn glucose v glutamine c phi hp vi vic cung cp amino acid v
huyt thanh, cho kt qu l nng t bo tng s xp x 5 10
7
t bo/mL
(trong t hn mt na l sng st) sau hn 550 gi, v nng cui cng
ca khng th l 2,4 g/L, vi sn lng 0,1 g/L ngy. Sn xut thng mi
cc khng th n dng trong cc reactor si rng c th cho sn lng
khong 700 g sn phm/thng (khong 2 g/L). Mi ln nui cy ko di
khong ba thng nhng ln nui cy u tin l khng sn xut do thi gian
ny c yu cu cho vic xy dng sinh khi trong khng gian siu mao
dn. Hiu sut trong h thng ny l 0,3 g/L ngy trong sut thi gian thu
hoch.

III. Cng ngh di truyn ca cc t bo ng vt c v
Chuyn np v biu hin DNA ngoi lai trong t bo eukaryote nui
cy in vitro c bt u cch y hn 30 nm. Phng php chuyn nhim
m u cho mt chui da dng v phc tp ca cc k thut chuyn gen.
Hu nh ng thi vi s pht trin cc quy trnh chuyn np l vic khm
ph ra enzyme phin m ngc (reverse transcriptase) t c th to ra
cc bn sao DNA b sung (cDNA) ca mi mRNA. S pht hin enzyme
ct hn ch m ra k nguyn ca cng ngh DNA ti t hp. Nhn chung,
vic pht trin cc k thut thao tc DNA to ra cng ngh di truyn thc
vt, ng vt v liu php gen ngi tr thnh hin thc. Cc k thut
hin i ci thin hiu qu chuyn DNA, tng s a dng ca vector
iu ha v to thun li cho biu hin ca gen trong mt phm vi rng cc
loi t bo ch (target cell).
Chuyn gen c nh ngha nh l vic a DNA bn ngoi vo
genome, sao cho n n nh v duy tr cng vi di truyn ca vt ch. Hn
15 nm qua, vic chuyn gen vo genome ca ng vt c v tr thnh
mt cng c thc nghim c lm u n v ang tng tm quan trng
trong cng nghip cng ngh sinh hc. Thng thng, DNA ngoi lai c
a vo trong phi mt t bo bng phng php vi tim v cc phi sng
st sau c cy vo con ci th tinh gi v cho php pht trin ti k
hn. Trong mt s phi c cung cp, DNA hp nht trong genome
trc khi phn chia t bo ln th nht, th chuyn gen s c chuyn qua
nhng ln sinh sn tip theo thng qua phi.
Cc k thut chuyn gen c tim nng ng dng rt ln trong nghin
cu. mc phn t chng cho php xc nhn cc trnh t cis-acting
DNA quan trng trong s biu hin gen c trng m v/hoc pht trin
nh hng, v thao tc c bit ca s biu hin gen in vivo. Vi s ra i
ca k thut t bo mm phi (embryo stem-ES), v s pht trin cc
phng php gip t c s ti t hp tng ng, cc nghin cu hin
nay c kh nng tm hiu v chc nng ca mt gen c bit v xc nh
chc chn cc nh hng in vitro ca nhng bin i c bit n chc nng
gen. S i mi ny c nhiu gi quan trng cho nhiu lnh vc nghin
cu sinh-y bao gm thit k cc m hnh bnh, s dng m nh l cc h ln
men cho vic sn xut cc protein tr liu ngi v, cui cng, sa cha
nhng sai st bm sinh ca s chuyn ha bi gen ch.
1. Cc phng php chuyn gen
C nhiu phng php thch hp chuyn DNA ngoi lai vo trong
cc t bo eukaryote, chng hn nh: chuyn nhim (ha bin np) bng
calcium phostphate hoc diethylaminoethyl-dextran (DEAE-dextran), xung
in, lipofection, liposome, viral vector (k c cc tiu phn phage), vi tim,
bn gen (vi n)
Chn phng php chuyn gen ph thuc vo mc biu hin c
mong i, biu hin trong thi gian ngn hoc biu hin n nh; loi t bo
ch, nh t bo dch huyn ph hoc t bo dnh bm, cc dng t bo
thch nghi hoc phn ha. Mi phng php u i hi s ti u cao, bao
gm cc yu t nh: s lng t bo, nng DNA, cc vector biu hin.
Tng quan tm tt mt s phng php chuyn gen thng dng di y
m t k thut c bn, hiu qu biu hin gen, v cc ci tin gn y minh
ha cho cc ng dng lm sng.

1.1. Phng php chuyn nhim (transfection)
Dng calcium phostphate kt ta DNA, c phm vi hiu qu t 1-10
4

khun lc/10
6
t bo/g DNA. S hp nht ca DNA ngoi lai trong DNA
t bo mang tnh ngu nhin. DNA c chuyn nhim thng ti t hp
trc khi hp nht lm cho th hi nhp mang nhiu bn sao DNA trong t
bo. Hiu qu chuyn np c th tng mt vi dng t bo c x l
dimethyl sulfoxide (DMSO) hoc glycerol trong mt thi gian ngn (4-6
gi) sau khi chuyn nhim. X l sc sau chuyn nhim bng
chloroquindiphosphate gy c cao. Mc c thay i gia cc dng t
bo, c bit cc t bo nui cy dch huyn ph v cc t bo phn ha
giai on cui. Khi thay i calcium phosphate bng DEAE-dextran th
chuyn nhim DNA c th t c hn vi mi x l sc sau chuyn nhim
t bo nui cy dch huyn ph v t bo phn ha.

1.2. Phng php lipofection
S dng cc lipid trung tnh hoc mang cation to thnh cc
liposome. Phc lipid hp nht vi mng huyt tng s phng thch DNA
dnh bm vo trong phn bo tan (cytosol). Phng php ny cho hiu sut
chuyn np cao hn chuyn nhim bng DEAE-dextran hoc calcium
phostphate. Tnh ng nht ca thnh phn lipid gia mng t bo vt ch
v lipofectant lm tng hiu qu dung hp v tng kh nng xm nhp ca
DNA gn km. Cc liposome mang cation (lipofectin) thch hp cho chuyn
np gen in vitro vi hiu sut trn 90% mt s loi t bo nui cy.
Lipofectin c s dng bc cc virus. Vic to ra cc liposome cha
protein virus trong lp lipid ci thin hiu qu ca vector vi cc t bo
c bit nh t bo gan (liver hepatocytes). Ch c mt s phng php
thch hp chuyn DNA qua mng huyt tng bng thc m bo
(endocytosis) v y mnh cc qu trnh ni th gy thoi bin v sp xp
li DNA. Liposome c dng phn phi in vivo v ex vivo cc gen
ngi ti t bo ch thch hp.

1.3. Phng php xung in (electroporation)
- Phng php ny cng c s dng cho chuyn gen thc vt.
Tuy nhin, t bo thc vt phi c ph b thnh cellulose mi cho hiu
sut chuyn np cao.
- Yu cu nghim ngt cc thng s ca thit b to xung in (Hnh
5.4) lin quan n hiu sut xm nhp ca DNA. Dng in c s dng
to ra cc t bo treo trong dung dch DNA cc l thng trn mng huyt
tng v qua DNA theo gradient mt chui vo trong phn bo tan.
Phng php ny c s dng rng ri trong nui cy dch huyn ph
lymphocyte (lympho bo).
- Chuyn np gen bng xung in c khuynh hng to ra cc th hi
nhp mang bn sao DNA n v thng c s dng chuyn gen vo
cc t bo mm phi. Vic chuyn np gen thng qua in trng cc t
bo mu (hematopoietic cells) v cc th h t tin ca chng l phng
thc thch hp cho cc viral vector cn h thng ng gi phc tp.
- Cc t bo ty xng tin thn (bone marrow progenitor cells) c
nui trong mi trng c cytokine, interleukin-3 s tng tn s chuyn
nhim v biu hin gen cc t bo to bch cu ht (granulopoietic) v t
bo hng cu tin thn (erythroid). Cc gen chn lc tri nh l pSVNeo ghi
m cho cc gen ca prokaryote, neomycin phosphatransferase (neo) mang
trnh t khi ng (promoter) v trnh t tng cng (enhancer) ca Simian
Virus 40 (SV40), cho php phn lp cc t bo khng neomycin bng cch
dng mt dng ng ng ca neomycin, G418, trong mi trng b sung.
Chuyn np gen pSVNeo bng xung in vo trong t bo mm ty xng
cho kt qu tt, v c th ng dng chuyn np cDNA ca yu t ng
mu (factor IX) vo trong t bo m (stromal cells) ngun gc tu xng.

Hnh 5.4. H thng chuyn gen bng xung in

1.4. Phng php vi tim (microinjection)
- y l phng php chuyn DNA trc tip nht v c hiu qu cao.
Tuy nhin, s lng t bo th nghim gii hn ch trong vi trm. K thut
tinh xo v thit b t tin ca vi tim (Hnh 5.5 v 5.6) hn ch vic s
dng rng ri phng php ny. Tin b ln nht ca phng php vi tim
l kh nng gim st biu hin ca DNA ngoi lai cc t bo ring r.
- Hn na, trong khi chuyn nhim v chuyn gen bng xung in c
th v cng c th vi tim phn phi DNA trc tip vo trong nhn t bo
m khng gy nguy him n s nguyn vn ca mng t bo. gim c
tnh ti mc ti thiu, th tch DNA phi c hn ch nh hn 10% th
tch nhn.
- c trng ca vi tim l cung cp phng thc to ra cc ng
vt c chuyn gen, nh gi biu hin gen c to dng trong cc t bo
phi, cung cp phng php trc tip to cc t bin thm on
(insertional mutants), xc nh cc nhn t iu ha biu hin gen c trng
m v c trng t bo, v phn lp cc dng provirus (cc virus DNA c
hp nht trong DNA nhim sc th ca t bo vt ch) nguyn vn ca cc
retrovirus (cc virus mang RNA si n c ti bn thng qua DNA si
i trung gian) tin hnh phn tch bnh l hc.

Hnh 5.5. Thit b vi tim

Hnh 5.6. Vi tim DNA vo t bo

Xc nh phng php chuyn np thch hp cho cc gen ngoi lai c
trng l cn thit thc hin liu php sa cha gen in vivo. Mt vi bnh
di truyn xut hin do thiu cc gen chc nng hoc sn phm gen, v vy
thay th mt gen khim khuyt bng mt gen bnh thng c th sa cha
c khuyt tt v trnh cc t bin tim tng cc locus ngu nhin. Gen
thay th phi c thit k thch hp thc hin ti t hp tng ng
gia DNA ngoi lai v DNA ni sinh. Tn s ti t hp tng ng locus
ch (target locus) trung bnh l 1/10
3
t bo nu dng phng php vi tim.
chut chuyn gen, gen c trng c nghin cu cung cp mt h
thng m hnh in vivo cho php nh gi hiu qu ca bin php thay th
gen. M hnh ng vt cho hi chng Lesch-Nyhan to thun li nh
gi cc quy trnh chuyn np gen in vivo, cho d bnh hc ca ng vt
him khi tng t nh ngi.

1.5. Phng php dng sng bn gen (gene gun)
S dng cc ht kim loi nh tungsten hoc vng lm vi n. Vi n
c bc bng DNA v i vi mt vn tc thch hp xm nhp vo t
bo ch. Hiu qu ca phng php ny tng ng vi phng php
chuyn nhim. S biu hin thnh cng ca DNA ngoi lai trong t bo
chut NIH 3T3, t bo kh COS 7 v mt dng i thc bo (macrophage)
c thng bo. Sng bn gen v phng php tim trc tip DNA trn
ca plasmid b hn ch i vi tim v cc t bo c xng ca ng vt. Ch
c 1-3% t bo nhn DNA v sn xut mt lng nh protein c ghi m.
Cc phng thc hin hnh hu ht u thch hp cho cc mc ch pht
trin liu php DNA vaccine trong vi mt lng nh protein l
to ra mt phn ng min dch (xem thm chng 4).

1.6. Phng php dng cc viral vector
y l cc cu trc ti t hp trong mt hoc nhiu gen ca virus
c thay th bi DNA to dng. Chuyn np gen c trng m hoc t bo
c thc hin bng cch chn lc cc im th cm ca t bo c hiu
virus. Virus mang DNA ti t hp xm nhim vo t bo ch c trng v
phn phi ti trng di truyn vo trong chng. Mt c im hp dn ca
viral vector l iu ha biu hin gen khi c mt promoter v enhancer ca
virus. Cc viral vector c ngun gc t hu ht cc virus DNA, bao gm
SV40, cc virus to u dng nh (papilloma virus) ngi v b, nhm virus
DNA ca parvoviridae (adeno-associated virus, AAV), adenoviruses (cc
virus mang DNA si i), cc virus gy bnh mn gip (herpes), v virus
bnh u ma (vaccinia). Kch thc ca on chn (insert DNA) thay i
t 2-3 kb cc papovavirus ti > 50 kb vaccinia. Cc viral vector c th
cho php biu hin trong thi gian ngn v gn nh 100% iu kin in
vitro. DNA ca provirus c sn xut nh phin m ngc RNA ca
retrovirus hp nht nh l mt bn sao n trong nhim sc th vt ch,
gim ti mc ti thiu s phin m gen. Cc viral vector c bit c thit
k phn phi DNA ngoi lai vo trong cc t bo phn ha, cc t bo
mm phi, cc lymphocyte. Mc d cc retrovirus c s dng
chuyn gen nhng cng c mt vi kh khn do provirus ca n thiu kh
nng hp nht vo cc t bo th ng, bi v DNA ch hp nht khi t bo
tri qua thi k phn bo. iu ny dn ti tht bi khi biu hin mc
cao cc gen chuyn np. c kh nng ti t hp, kch thc ti a ca
insert DNA ch khong 7 kb.
Chn la mt phng php chuyn gen thch hp ph thuc vo mc
ch th nghim v loi t bo ch (dnh bm hay dch huyn ph), chng l
nhng t bo s cp hay thch nghi, phn ha hay c nhiu tim nng.
Th nghim biu hin ca DNA chuyn nhim c th l tm thi (trong thi
gian ngn) hoc bn vng (n nh) vi cc mc biu hin c bn hoc
c th suy din. Cc dng t bo dnh bm d thao tc bng cc phng
php chuyn gen khc nhau v cho biu hin gen thnh cng sau . Khi
c tnh ca phng php chuyn nhim loi tr kh nng biu hin gen, th
mt trong cc phng php khc c th cho php thit k th nghim thnh
cng. Khi phng php chuyn nhim, xung in v viral vector tht bi th
phng php vi tim c th s thch hp. Chuyn gen vo cc t bo khng
dnh bm kh thc hin nhng s dng viral vector v chuyn nhim
DEAE, cng nh liposome c th l gii php hp l. i vi cc t bo
khng dnh bm th c th s dng phng php vi tim chuyn gen.
Nng DNA dng trong th nghim chuyn gen thay i t 1 ng n
10 g cho 10
5
-10
6
t bo nhn. Trong vi tim, mt vi trm t bo c tim
trc tip DNA nng t 1 pg n 10 g/L tng ng vi 1-10
2
bn
sao ca cu trc ti t hp. S lng bn sao a vo trong cc t bo nhn
(t bo vt ch) t l vi kch thc ca vector, on chn v nng DNA.
Cc cu trc mch thng hp nht thng qua ti t hp cao hn khong 10
ln cc cu trc mch vng. Chuyn nhim thng qua liposome tr thnh
mt k thut ph bin nh c tnh thp hn v hiu qu chuyn np cao.
Trong khi phng php xung in i hi mt s lng ln DNA (10-
40 g) v trung bnh s git cht mt na t bo nhn.

2. Cc iu kin cn thit cho biu hin gen ngoi lai
Sau s vn chuyn hiu qu DNA vo trong nhn ca t bo nhn,
biu hin ca DNA ngoi lai ty thuc vo cc nhn t iu ha: enhancer
v promoter trnh t cis 5 v cc nhn t phin m tc ng trans (trans-
acting) ca t bo vt ch. Promoter v enhancer l cc nhn t c th c
chuyn vo trong vector iu ha biu hin gen sau khi hp nht vo
trong DNA t bo. Hiu bit c ch iu ha ca gen chuyn np s chn
c t bo vt ch cho php cc nhn t phin m thch hp hin din v
hot ng. Cu trc mt vector chuyn np gen thch hp bao gm cc nhn
t iu ha eukaryote v prokaryote cn thit thc hin khuch i trong
mi trng prokaryote v biu hin sn phm protein mong i trong vt
ch eukaryote.
S iu ha ca prokaryote c kt hp vi s khuch i DNA ti
t hp trong E. coli hoc cc t bo vt ch prokaryote khc. iu kin ti
thiu l phi c vng khi u sao chp (ori) vi khun, promoter vi khun
v gen ch th chn lc hoc sng lc phn lp DNA ti t hp c
khuch i. Cc gen ch th c s dng nh gen neomycin transferase
(neo), thymidine kinase (tk), xanthineguanine phosphoribosyl transferase
(xgprt), khng methotrexate nh dihydofolate reductase (dhfr), khng
hygromycin (hgr) v khng tetracycline (tet). Cc gen ch th l cc gen
prokaryote c to dng trong cc vector biu hin cc eukaryote cho
php th nghim biu hin tm thi ca DNA ngoi lai nhng khng ph
thuc vo mi trng chn lc. Cc gen ch th l -galactosidase (-gal),
luciferase (luc), chloramphenicol acetyl transferase (cat) v green
fluorescent protein (gfp). Cc sn phm protein ca mi gen tng ng l B-
GAL, LUC, CAT v GFT.
Ni chung, cc promoter eukaryote rt phc tp, i hi hai hoc
nhiu trnh t cis m nhn t phin m lin kt thnh hng RNA polymerase
II phin m. Cc nhn t promoter ti thiu bao gm cc lp protein lin
kt DNA thng gp AP-1 v SP-1 c xem l cc nhn t bo th tm
thy nhiu trong promoter ca eukaryotic virus. To dng tr li
(subcloning) promoter 5 ca virus vo trong cc gen m ha cc sn phm
protein chn lc s to iu kin cho mt s vector biu hin. Cc promoter
ca virus hot ng mnh trong mt s loi t bo nui cy c bit nhng
li hot ng khng ging nhau cc t bo phn ha hoc cc t bo phi.
Nhiu gen eukaryote quan tm bt ngun t mRNA thng qua phin m
ngc sn xut cDNA khng cha cc nhn t iu ha. To dng cDNA
vo trong cc vector mang promoter mnh biu hin trong t bo nhn.
Cc promoter SV40 v CMV (cytomegalovirus) cng cha cc enhancer l
nhn t lm tng hiu qu ca promoter virus trong hu ht cc t bo
eukaryote. Cc enhancer c ngun gc t virus c tc dng tng hiu qu
phin m ca promoter.
Vng khi u sao chp DNA ca eukaryote c th c gn vo
vector tng s sao chp vi hng triu DNA t bo. Trong thc t, tt c
cc vng khi u sao chp DNA ca eukaryote c ngun gc t h gen ca
virus. S sao chp ph thuc vo DNA polymerase v accessory protein ca
cc t bo cng nh replicase c trng ca virus. duy tr n nh DNA
ngoi lai i hi hoc l DNA c hp nht trong nhim sc th ca t bo
vt ch hoc l tn ti s b tr c trng gia episome v protein tc ng
t ngoi (trans-acting protein).
Phin m eukaryote khi u ti hng ca promoter nhng li kt
thc khng chnh xc. Phc nhn RNA-hnRNA d hp (heterogenenous
nuclear RNA) l th phin m s cp (5 3), mt bn sao chnh xc ca
si DNA khun mu (3 5), v tng ng vi si m ha ca DNA
(5 3). Khi c mt intron, cc spliceosome s loi intron ranh gii c
trng exon-intron gn cng vi chui m ha c biu hin ca gen trong
mt khung c m dch m. Cc bc ca qu trnh ny l c trng ca
t bo eukaryote v khng xut hin trong cc t bo prokaryote. S iu
ha biu hin ca gen de novo trong eukaryote phc tp hn s iu ha
dch m v cc bc ca qu trnh sau dch m. S iu ha tip theo c th
bao gm kh nng n nh ca mRNA, cc tn hiu ch th (maker signals)
trong vng khng dch m 5 hoc 3 (UTR) ca mRNA, cc chui mang b
ba m ha khi u ti u Cc hiu bit ban u v c ch phc tp v
tinh vi ca s iu ha gen l c s chuyn np gen thnh cng iu
kin in vitro v in vivo. Liu php gen ngi i hi thng tin chi tit v
gen bm sinh v xc nh cc nhn t iu ha biu hin gen thch hp.

3. Cc iu kin thc nghim ti u
Cc nhn t cn thit ti u thc nghim bao gm s lng t bo,
nng DNA, iu ha biu hin gen trong cc t bo nhn, v chn la
cc h thng phn tch. C th xc nh c trng iu ha ca gen trong
cc mi trng t bo khc nhau bng cch phn tch biu hin tm thi nh
CAT, B-GAT, hoc GFP. phn tch biu hin n nh bn vng c th s
dng cc gen ch th nh -gal, hoc cc gen nh du chn lc nh neo.
Chi tit ca mi th nghim phn tch c trnh by trong cc b kit chun
v cc hng dn cn k trong chn la nhng u im v nhc
im ca tng phng php.
Cc plasmid mang gen ch th khng chn lc nh biu hin LUC
hoc GFP rt hu ch khi lm vic trn mi trng khng c p lc chn
lc. Gen m ha GFP (gfp) c to dng t loi sa pht sng sinh hc
(Aequorea victoria). c tnh hp dn nht l s n gin ca phng php
pht hin do ch cn knh hin vi hunh quang chun v khng cn c cht
hoc cc cofactor khc. Cc phn tch ha min dch v min dch hunh
quang cho php pht hin cc t bo nhn bng mt thng v quan st cht
ch s hp nht ca cu trc t bo. GFP l gen duy nht trong s cc gen
ch th c th pht hin bng knh hin vi hunh quang cc t bo, c th
theo di s s dng v biu hin ca cc protein ch th trong phn chia t
bo giai on tip theo v phn loi cc t bo biu hin bng cch s
dng thit b sng lc cc t bo c hot tnh pht hunh quang.
Gen gfp c to dng tr li trong cc retrovirus vector mang on
lp li tn cng di (long terminal repeat-LTR) ca virus, cc v tr to dng,
v gen khng neo ngc hng ca LTR mang u tn cng 3. Chuyn cc
vector GFP vo trong cc t bo COS-7 biu hin mc hunh quang ca
protein c th pht hin trong 24-48 gi m khng cn c nh. Cc
retrovirus vector mang GFP cho php chuyn nhim dng c ng gi
v sau 24 gi cc t bo c th c phn lp bng thit b phn loi t bo
c pht hunh quang (fluorescence-activated cell sorter-FACS) v tin hnh
nui cy. Thit b m bng phng php nhum propidium iodide s pht
hin cc t bo cht v tch chng ra khi t bo sng biu hin GFP trong
qu trnh phn tch FACS.

4. ng chuyn np gen ch th v gen th nghim
Chuyn np gen bng cch dng hai plasmid, mt mang DNA quan
tm v mt mang gen ch th, nh neo, -gal, luc hoc gfp, vo trong cc t
bo ng vt c v thng nh du cc t bo biu hin DNA ngoi lai.
Th nghim ng chuyn np u tin bt u nm 1979 vi cc gen -
globin v tk ca herpes virus. Tip , nhiu qu trnh ti t hp gen chn
lc v khng chn lc c s dng bng nhiu phng php chuyn
gen v thu c nhiu thnh cng khc nhau. Nu gen ch th hoc gen
chn lc c biu hin trong t bo nhn, th gen th hai (gen quan tm)
cng s c biu hin. Khi gen chn lc c s dng phn lp cc
qun th t bo biu hin gen nh du tri, th cc t bo tng t s c
theo di cho kiu hnh ca gen th hai. Chng hn, gen ch th tri pSVneo
cho php chn lc cc t bo trn mi trng G418 trong gen quan tm
th hai c th c theo di cho kiu hnh c trng. Vi tim pSVneo vo t
bo NIH3T3 sn xut 35% khun lc khng neo. Vi tim gen ras (mt
loi oncogene) c to dng, c ngun gc t ung th bng quang ngi,
th c t 10-20% t bo biu hin kiu hnh c chuyn np. Vi tim cc t
bo NIH3T3 vi c hai gen ras v neo cho php chn lc cc t bo khng
neo, trong 50% ca chng c kiu hnh oncogene.
H thng chn lc kp vi hai gen chn lc c s dng nh
hygromycin v neomycin, i hi xc nh cn thn cc iu kin th
nghim. c tnh kt hp vi s chn lc trong G418 v hygromycin
gii hn kh nng sng st ca t bo, c bit cc dng t bo s cp v
phn ha. Phng php vi tim mn cm hn 100 ln so vi phng php
chuyn nhim khi chuyn gen ras vo cc nguyn bo si d bi ca ngi
c bt t bi khng nguyn SV40T. Mt t bo, c-rasSVneo-HAL,
c hnh thnh bng k thut vi tim pSV2neoT24 sau khi khng thnh
cng vi cc k thut lipofection, chuyn nhim v xung in. Tn s biu
hin ras p21 km hn 10% trong th nghim tm thi v 1% cho biu hin
n nh.
Vi tim trc tip vo 100-200 t bo cho c hi quan st cc phn ng
ni bo ring r ti cc i phn t. Antisense oligodeoxynucleotide (ODN)
v peptide nucleic acid (PNA) cung cp mt phng thc mi cho liu php
gen trong tng lai. nh gi phn ng tr li si i ngha (antisense) ca
gen ch (target gene) c thc hin bng cch dng phng php vi tim
h thng m hnh. M hnh xy dng phn tch hiu qu ODN v PNA
c thit k c bit. Gen ch l n v phin m sm ca DNA SV40
m ha cho khng nguyn T ln. Cc t bo cha bn sao hp nht n ca
DNA SV40 vi khng nguyn T ln mn cm nhit (tsA8) c dng
nh gi s km hm ca khng nguyn SV40 sau khi vi tim cc cu trc
PNA v ODN ca si khng c ngha (missense) v si i ngha. ng vi
tim vi -gal cho php pht hin cc t bo c vi tim ring r nh phn
tch min dch hunh quang kp cho khng nguyn T v -gal. Hai chrome
hunh quang (fluorochromes) ring bit cho php xc nh s phn trm t
bo biu hin -gal c hoc khng biu hin khng nguyn T nh l kt qu
ca s c ch si i ngha. Vi tim PNA c t 15-20 b vo nhn c ch
hon ton biu hin c bn ca khng nguyn T SV40. S c ch c hiu
c chng minh trn c s khng c s kh trong gen -galactosidase
c ng vi tim. PNA ca si khng c ngha khng kh khng nguyn T
SV40 m cng khng biu hin -gallactosidase.
Tip n cc t bo thn thch nghi ca kh (CV-1) c vi tim
plasmid biu hin khng nguyn T ca SV40 v mt plasmid phn chia
cha -gal c iu ha bi promoter CMV. Phng php min dch
hunh quang kp c dng xc nh s lng biu hin ca -gal nh
mt nhn t ch th ca mt t bo vi tim v s c mt hoc vng mt
khng nguyn T c xem nh mt n v o s lng ca hiu sut c ch
gen bi si i ngha ODN hoc PNA. Mt h thng th nghim iu khin
cao nh th cho php so snh trc tip hiu qu ca tnh c hiu chui
oligonucleotide, s bin i chiu di v nng , trong s so snh vi
peptide nucleic acid nh l cc lp phn chia oligomer antisense.

5. K thut t bo mm phi, chuyn gen v ti t hp tng ng
Mt phng thc khc rt hu hiu trong chuyn gen l a DNA
ngoi lai vo cc t bo mm phi (embryonic stem-ES) cn gi l t bo
gc (Hnh 5.7a). Thit lp mt dng t bo ES, cc t bo c cy chuyn
t khi t bo bn trong ca ti phi (blastocyt) ang pht trin ln cc tng
nui dng hoc vo mi trng c hot tnh c ch phn ha
(differentiation inhibiting activity-DIA), duy tr trng thi khng phn
ha ca chng. DNA ngoi lai c th c a vo trong cc t bo ES
bng mt s phng thc sau: xung in, chuyn nhim hoc vi tim. Sau
, cc t bo chn lc c a tr li vo trong ti phi v c dnh vo
trong con ci mang thai gi cho php pht trin ti k hn sinh . S khc
bit quan trng gia cc con ca mt s ng vt thu c theo phng
thc ny so vi nhng con vt thu c bng k thut vi tim vo phi mt
t bo (one-cell embryo) l chng to ra cc th khm v cc t bo mang
gen c bin np ch chim mt t l nht nh ca sinh khi t bo bn
trong ca ti phi. Tuy nhin, phng thc ny cung cp cc t bo chuyn
gen ng gp vo dng t bo mm (germline), sau mt phng thc
chn ging thch hp s cho php thit lp mt dng chuyn gen.

Hnh 5.7. Chuyn gen ng vt. (a) Sinh sn ca cc ng vt chuyn gen bng
cc phng thc thao tc t bo ES. (b) Tip theo vic chuyn DNA, cc t bo ES
mang th ti t hp tng ng c th c chn lc trc khi a vo trong ti
phi.

Vic a DNA ngoi lai vo trong t bo ES c ngha hn phng
php vi tim vo phi mt t bo nh u im thc t l DNA ngoi lai c
th c thit k ti t hp tng ng vi bn sao ni sinh ca n (Hnh
5.7b). S chn lc tip theo, cc dng ch chnh xc c th c nhn din,
hoc bng Southern blot hoc phn tch PCR, v a tr li vo trong cc
Phn lp mt s t bo
ni sinh t ti phi
Cc dng t bo ES
(b)
(a)
Chuyn DNA ch
Chn lc v/hoc
sng lc PCR
Nhn dng cc con chuyn gen
Gy ging th khm
Chuyn cc t bo ES
vo trong ti phi nhn
X
ti phi vt ch sinh sn cc th khm chuyn gen mong mun. Mt s
phng thc c pht minh cho chn lc dng tnh cc th ti t hp
tng ng phi hp vi chn lc m tnh da theo s hp nht ngu nhin.
Chn lc dng tnh c th hng ti bng cch ngt qung cc chui tng
ng trong vector ch vi marker chn lc, nh l gen neo khng
neomycine ca vi khun. Nu vector hp nht trong genome mt kiu
tng ng, th sau gen neo
r
s c kt hp cht ch trong germline.
Mi chui b sung ca vector m khng c vng tng ng s b mt.
Bng cch t gen HIV-1-tk

trong vng khng tng ng ca vector ch,
mi t bo duy tr gen ny thng qua cc trng hp hp nht ngu nhin s
b git cht trong s hin din ca cc nucleoside nhn to thch hp.

IV. Nui cy t bo mm sn xut c quan ngi
Kh nng s dng t bo ES trong nghin cu y sinh hc h m
mt lnh vc ng dng mi l y hc ti to bt ngun t tnh a nng
ca ca ES do chng c kh nng t phn chia v i mi v hn, hoc
phn ha thnh nhiu loi t bo, khng ch l loi bt ngun t ba qun th
t bo sng lp (ngoi b, trung b v ni b) phi t s sinh ra tt c cc
t bo ca c th, m cn c cc loi m chuyn ha h tr phi trong t
cung.
Nm 1981, Martin v Evans cng lc pht hin thy rng cc t bo
ES ca chut c nui cy trong cc iu kin c bit, bt u phn chia
v c th nhn ln v hn m khng phn ha, c nh giai on pht trin
phi sm ny. Tuy nhin, ngay c sau khi to ra nhiu th h trong nui cy,
nhng t bo ES ny vn nh chng c chng trnh ha v ci g, v gi
kh nng phn ha thnh bt c loi t bo chuyn ha no ca c th.
Nm 1998, Thomson v cng s phn lp c t bo ES t phi
ngi v nhn thy chng khc vi t bo gc ca chut nhiu im,
chng hn nh kh duy tr v nhn ln trong nui cy. Nm 2000, Pera v
cng s chng minh cc t bo ES ngi cng c th phn ha trong iu
kin in vitro thnh cc loi t bo trng thnh khc nhau. Chng hn, h
phn lp c t cc t bo ES nui cy in vitro mt qun th tin t bo
thn kinh thun khit, v trong mt s iu kin chng c th phn ha
thnh cc t bo thn kinh thnh thc.


Hnh 5.8. Nui cy t bo mm sn xut c quan ngi

Hai tnh cht ca t bo ES ngi a nng l kh nng sinh sn v hn
trng thi phi nguyn thy trong iu kin in vitro, v kh nng phn ha
tht s thnh bt k loi t bo trng thnh no. V vy, c rt nhiu lnh
vc ng dng chng, nhng tu trung c cc hng chnh sau:

1. ng dng t bo mm phi trong nghin cu c bn
Trn thc t, nhng t bo ES l mt cng c qu gi hiu r hn
s pht trin ca phi ngi v nhng d dng khc nhau c th lm ri lon
s pht trin ny. Cc t bo ES cung cp mt phng tin nghin cu cc
s kin ch yu nh s tng sinh cc t bo a nng, s tham gia ca chng
vo cc con ng phn ha c th, cng nh cc nhn t iu khin
chng. Vic nghin cu dng t bo ES mang ba nhim sc th nh th ba
21 s gip cc nh nghin cu nh gi hu qu ca nhng tai bin di truyn
ny n s pht trin. Ngoi ra, t bo ES l nim hy vng trong cuc u
tranh chng li nhiu loi u tr em, nh u nguyn bo thn, bt ngun t
cc qun th t bo phi, nhng du hiu sinh bnh kh nghin cu. T bo
ES s gip chng ta xc nh tc dng ca cc gen gy ung th v cc gen
Phi sm ca ngi giai on
blastocyst (tng ng phi nang
ca ng vt c v)
T bo gan
T bo mm phi

HOC

Tu xng

T bo mm trng thnh

Nui cy
t bo mm
Cc iu kin nui
cy khc nhau
T bo thn kinh
T bo c tim
Cc loi t bo c
phn ha khc nhau
ngn chn ung th cc qun th t bo ch sinh ra cc khi u pht trin
ny.
Nhng t bo ES v con chu phn ha ca chng, l tin thn ca cc
loi t bo nht nh, s c dng xc nh tc dng ca cytokine v cc
nhn t sinh trng khc n s tng sinh cc qun th t bo phi m cho
n nay vn cha tip cn c nghin cu. V phng din iu tr,
tng t nh cc nhn t c xc nh v tc dng kch thch sinh trng,
cc t bo ES mu c ng dng lm sng trong trng hp ghp ty,
nhng nhn t sinh trng v phn ha c xc nh nh nui cy t bo
ES c th c ng dng trong vic ti to cc m khc.

2. Nghin cu chc nng gen
Vi thnh tu xc nh c b gen ca ngi, ngi ta c th xt
nghim nhanh trong ng nghim xc nh chc nng ca cc gen. V cc
t bo ES nui cy c th d x l di truyn, nht l chu nhng bin i
nh v ti cc v tr nhim sc th c th nh cch ti t hp cng ngun,
mi hoc bit, trong cc t bo phi hoc cc t bo trng thnh
phn ha.

3. Nghin cu cc m hnh bnh l
Theo nguyn l, t bo ES l mt ngun in vitro i vi bt k loi t
bo no ca c th, v th chng c vai tr quan trng trong nghin cu c
cht hc v qu trnh tm ra cc loi thuc. Kh nng sn xut vi s lng
ln mt loi t bo c bit ca ngi trong ng nghim, t nhng t bo
tin thn m ngi ta c th bin i di truyn, s gip to ra cc m hnh
bnh l do khuyt tt ca mt hoc nhiu gen mt m c bit. Nhng t
bo ngi ny bt ngun t cc t bo ES c bin i di truyn s l
nguyn liu l tng chn lc trong ng nghim cc phn t iu tr.
Chng ta s khng cn n m hnh ng vt, m nhng m hnh ny c th
thiu nhng c im quyt nh s pht trin bnh ngi.

4. ng dng trong y hc ti to
Mc d cc ng dng tng lai ca t bo ES trong nghin cu l
quan trng, nhng ng dng hp dn nht l c th dng cc t bo ny lm
ngun m ghp. Nhiu bnh lm ngi suy yu do hin tng t bo
cht, hoc thoi ha do cc qu trnh sinh l-bnh l mn tnh hoc tn
thng nghim trng. Trong nhng trng hp nh vy, vic thay th cc
m tn thng bng mt m lnh l hy vng cha khi nht. T nhiu nm
nay, dch v cy ghp phi ng u vi tnh trng khng cc m v
c quan cng hin. Cho nn v nguyn tc, t bo ES cng c quan tm,
v chng l ngun i mi v hn cho tt c cc loi t bo ca c th, bn
cnh cc t bo mm trng thnh. Trn thc t, trong nhiu tnh hung
lm sng, vic ghp t bo c kh nng ti to m c hiu qu tt hn so vi
ghp c quan trn vn. Nu kt hp cng ngh t bo ES vi cng ngh
nhn bn ng vt c v, ngi ta c th xem xt vic sn xut t bo ES
bt ngun t chnh cc t bo ca ngi bnh, nhm vo mt loi t ghp,
trnh hin tng thi loi. Ch cn dung hp mt t bo ca ngi bnh
vo mt non bo tch nhn (nh trong trng hp nhn bn cu Dolly),
ri cho trng thu c t pht trin ti giai on phi bo phn lp t
bo ES, kch thch cc t bo ny phn ha m cn ghp. Phng php
ny, c gi l l nhn bn iu tr, c th to ra cc m lnh hon ton
ph hp, v c cng thnh phn di truyn nh ca ngi bnh, cng du
chun tng hp m. Chng hn, ngi ta thnh cng khi sn xut t bo
ES chut t mt phi bo c nhn bn, sau nhng t bo ny khi
c tim li vo mt phi chut, phn ha nh mong i, thnh rt
nhiu loi m.
Nhng phi nhn mnh rng, tt c cc ng dng t bo ES ngi,
trc khi ra i, s cn phi hiu r hn cc qu trnh sinh hc k cn, cng
nh ci tin cc h thng nui cy t bo. Hin nay, hiu bit ca chng ta
v t bo ES a nng cn kh hn ch, k c chut, cho nn vn cn nhiu
iu phi bit v t bo ES ngi trc khi c th nui cy chng vi quy
m ln v x l chng v mt di truyn. Cng phi cn hiu cch iu khin
s phn ha ca chng thnh cc t bo tin thn ca cc m khc nhau, v
cch nui cy cc t bo ny. V d: trong trng hp tin thn ca t bo
thn kinh, c nhng h nui cy rt tt gip chng ta nhn chng ln, ri
chng bin i thnh t bo thn kinh thnh thc. Ngi ta cng phn
ha c trng cc t bo ES chut thnh cc t bo thn kinh c hot tnh
dopamin, m nu thoi ha s gy bnh Parkinson. Tic rng, i vi nhiu
dng t bo khc, ngi ta vn cha lm ch c cc giai on ny. Cui
cng, trc khi nhn bn iu tr t ti giai on ng dng lm sng, vn
cn nhiu vic phi lm ci tin hiu qu ca giai on nhn bn, v
chng minh rng cc phi c nhn bn c th sinh ra cc m hot ng
bnh thng. Tuy c rt nhiu tr ngi nhng hiu bit ca con ngi v s
pht trin phi tin b rt nhanh trong nhng nm gn y. Nu cng
ng khoa hc c ng h c gng nghin cu n cng, th t bo gc
ngi a nng c th s ng gp ch yu vo y hc th k 21 ny.

V. Cng ngh phi ng vt c v
1. Cy truyn hp t
Cng ngh cy truyn hp t hay cn gi l cng ngh chuyn phi
(embryo transfer technology) l nhng kt qu ca nhng bc tin ln
trong sinh hc pht trin v sinh hc phn t. Cc vi thao tc trn phi ca
cc loi vt nui u c tin hnh trong iu kin in vitro, sau khi ly
phi ra t ng vt cho (donor) v trc khi nui cy phi vo ng vt
nhn (recipient), bao gm cc thao tc nh:
- Tch phi thnh hai hay nhiu phn.
- Phi hp hai hay nhiu phi thnh mt th khm.
- Bin i cc thnh phn trong t bo ca phi khi mi pht trin.
K thut vi thao tc phi bt ngun t cc thao tc trn ng vt th
nghim, da trn c s cc pht hin v c ch phn ha phi sm, cc c
ch pht trin c th ng vt khng xng sng v sau ng vt c
xng sng bc thp v cui cng l ng vt c v.
Cc thc nghim u tin chng minh c nhng t bo phi tch
t phi hai t bo (two-cell embryo) c kh nng c pht trin thnh phi
hon chnh, ng thi kt hp hai hp t chut c th hnh thnh nn mt
c th dng khm. Thc nghim vi thao tc phi ng vt c v c
tin hnh u tin trn th.

2. Bo qun phi
y l giai on tin hnh trc khi cy truyn phi vo ng vt
nhn, to iu kin m bo c th vn chuyn phi i xa, v khng cn
phi gy ng dc ng thi cho con vt nhn ngay sau khi vi thao tc ly
phi ra t vt cho. Trong thc tin cy truyn phi, bo qun lnh phi
nitrogen lng (-196
o
C) i vi tt c cc loi vt nui (tr ln). b, k
thut ng lnh phi c th cho kt qu th thai khong 80%, trong
trng hp phi nguyn cho kt qu cao hn phi thao tc do tc ng ca
cc yu t thao tc khi tch phi.

3. Nui cy tm thi phi trong c th sng
Ngi ta tin hnh nui cy tm thi phi gia sc, vt nui trong
cc h thng khc nhau: ng dn trng ca cu, chut, th; t cung ca b
ci; xoang mng bng (phc mc, periton) ca chut; xoang i phi g.
Hu ht cc h thng ni trn u c s dng cng vi cc vi thao
tc, v u cn n k thut bc phi trong mt loi cht bo v (thng l
agar) bo v mng trong sut ca phi khng b tn thng.

4. K thut bc phi bng agar
S dng dung dch agar trong 0,9% NaCl. Trc khi thao tc, cho
nng chy agar, sau lm ngui agar 39
o
C, va khng qu nng i vi
phi, nhng cn c th nh git. Vic bc agar quanh phi phi tin hnh
rt nhanh, agar cha kp ng li.
Trc khi bc agar, cc phi m lp mng trong sut phi c
nhng vo huyt thanh b c un m. Mc ch ca thao tc nu trn l
khng lm thng tn t bo phi.

5. K thut nui cy tm thi phi trong ng dn trng
Ly mt hoc hai khi agar bc phi, chuyn vo ng dn trng ca
cu ci, km mt lng ti thiu mi trng. Cu ci phi c tm ngng
sinh dc, khng ng dc hoc khng cha trong thi gian nui cy. Cc
iu kin nu trn c th p ng nu trc ngy thao tc cy phi, ngi ta
tim vo t cung progestagen. Cc phi bnh thng s i vo t cung giai
on tm n mi su t bo.
ng dn trng ca th c s dng rng ri cy phi ng vt,
cy cc hp t c th tinh trong ng nghim. cy vo ng dn
trng th, cc phi cn bc bi mt lp mng muxin. Thng ngi ta dng
ng dn trng ca th nui cy tm thi phi ln.
Ngoi ra, k thut nui cy tm thi phi trong t cung ca b c
s dng nghin cu cc pha sm trong qu trnh hnh thnh th khm
phi b. Xoang i ca g c s dng nui cy phi ca chut, d v
mo. Phi c bc trong cc khi agarose sau chuyn vo xoang i ca
g.
VI. Nhn bn v tnh ng vt c v
1. Khi nim c bn
Nhn bn v tnh (hay cn gi l to dng v tnh) l qu trnh to ra
mt mt tp hp cc c th ging ht nhau v mt di truyn v ging b
(hoc m) ban u bng phng thc sinh sn v tnh. Nhn bn v tnh da
trn quan im cho rng mi t bo ca mt c th a bo u xut pht t
mt t bo hp t ban u qua phn bo nguyn nhim, do nhn ca
chng hon ton ging ht nhau v mt di truyn.
Sinh sn v tnh l s sinh sn khng km theo ti t hp di truyn,
c thc hin theo c ch phn bo mitose, trong genome c ti bn
nguyn vn. Khc vi sinh sn v tnh, sinh sn hu tnh (tin ha hn) c
km theo ti t hp di truyn v c thc hin theo c ch phn bo mitose
v meiose. Ti t hp di truyn l nguyn nhn dn n s a dng sinh hc
(biodiversity).
Sinh sn hu tnh l hnh thc sinh sn ph bin cc sinh vt bc
cao. Trong khi sinh sn v tnh ch tn ti nhng c th c cu trc
tng i n gin v gp nhiu thc vt (thc vt cng c hnh thc sinh
sn hu tnh). ng vt bc cao sinh sn v tnh ch tn ti giai on
pht trin sm hoc di hnh thc bin dng ca sinh sn hu tnh nh
hnh thc n tnh sinh.

2. Nhn bn v tnh ng vt
C th hiu mt cch n gin y l k thut nhn nhiu c th t
nhng t bo v tnh (somatic cell) (Hnh 5.9).
Nhn bn v tnh ng vt c nh gi l mt phng thc tng s
lng cc ng vt thng mi v khoa hc quan trng. u tin, s pht
trin hon chnh chut ch thu c khi tin nhn (pronucleus) hoc nhn
(nucleus) t cc phi chut giai on sm hai t bo c chuyn vo non
bo ph b nhn (enucleated oocyte). Nhn t cc giai on sau c
xem nh khng kh nng ti chng trnh ha h tr s pht sinh
phi. Ngi ta cng thy rng s hot ha nhn to (bng cch kch thch
in) cho php nhn t cc phi chut c tm t bo c chuyn vo cc t
bo cht giai on MII, v vic chuyn theo tng chui (sinh trng cc
trng c khi phc li trong mi trng cha cytochalasin B, sn xut
hai nhn ging tin nhn, v chuyn tun t nhng ci ny vo cc phi
mt t bo c th tinh ph b nhn trc ) ci thin vic sn xut cc
dng chut. Bng cch dng phng thc chuyn nhn, cu c to
dng t cc t bo c ngun gc t phi v t cc dng t bo soma c
ngun gc t thai v tuyn v. Gn y hn, to dng cc phi b bng cch
chuyn a nhn cng c thng bo.

Hnh 5.9. S nhn bn v tnh ch

3. Nhn bn v tnh cu Dolly
y l cng trnh ca Wilmut, Campbell v cc cng s (Ecosse,
Anh). i tng nghin cu y khng cn l nhng t bo phi nang, m
l nhng t bo ly t tuyn v mt cu ci Finn Dorset, su nm tui, lng
trng, thi k ba thng cui t khi cu ci mang thai, l thi k t bo
tuyn v c phn ha (differentiation) cao v pht trin.

Phi ch hoc
nng nc (u trng)
T bo trng ch
Nhn
Nhn b hy
Nng nc
Phi 8 t bo
Cy nhn
T bo rut
Nhn
UV


Hnh 5.10. Cu Dolly v cu m

em nui cy in vitro cc t bo tuyn v, 5 ngy trong mt mi
trng nui cy rt ngho huyt thanh, vi mc ch lm cho chu k t bo
gim t t cho ti khi ngng hon ton. Giai on ny c gi l G
0
. Sau
, lm lnh trc khi a mi t bo tuyn v vo mt non cha th tinh,
rt nhn ca mt cu ci khc, u en. Kt qu, mt t bo mi c
hnh thnh, pht trin v to thnh phi.
y c s phi hp ca hai k thut, mt l hot ha trng ly
nhn ra ca cu en, hai l lm ngng chu k sng ca t bo tuyn v cu
trng, tc l nhng t bo soma c bit ha cao , tch t mt c th
trng thnh v ngi ta thnh cng trong k thut dung hp t bo.
Thnh cng ny vt ln cc cng trnh trc , t 1992 tht bi ch
yu l do cc t bo phi s dng chuyn nhn khng c nh v giai
on G
0
(nh trng hp to cu Dolly), m pht trin ti G
2
(pha tng
trng) hoc S (pha ti bn, tng hp DNA), cn tr s dung hp t bo.
Cu Dolly sinh ngy 5/7/1996, c trng lng bnh thng khng c
biu hin d dng nh cc th nghim trc . Sng trong t cung ca m
nui h lng en, nhng cu Dolly vn c lng trng, cc phn tch kim
tra di truyn xc nhn cu Dolly l bn sao ca cu Finn Dorset, cu
cung cp t bo tuyn v.


Hnh 5.11. S nhn bn v tnh cu Dolly

Sau Wilmut v Campbell cn to ra ba cu con t nhng t bo
mt bo thai 26 ngy v bn cu con t nhng t bo mt phi mi hnh
thnh c 9 ngy.
Thnh cng nu trn chng t mt ng vt c v ln c th c
nhn bn t t bo soma m khng cn c tc ng g ca t bo sinh dc,
Cu cho t bo
tuyn v
Nui cy t bo
tuyn v. Chu
k t bo c
ngng li
(phage G
0
)
Cu cho
t bo trng
T bo trng
t non
Loi b nhn
Dung hp t bo
T bo sinh
trng trong
nui cy
Nhn ca t bo
tuyn v
Phi sm
Cy truyn vo t
cung ca con cu
th ba
Cu m thay th
Pht trin phi
Cu non (Dolly) ging ht
nhim sc th vi cu cho t
bo tuyn v
ngoi sinh cht ca mt non bo. Ngi ta cng nhn bn thnh cng
kh v ln, v gn y l trn c ngi dng cc phi non (14 ngy) cung
cp nguyn liu cy ghp cha bnh.
V cht lng, ni chung nhn bn t t bo soma c th to c c
th c hoc ci u vit theo mun. Tuy nhin, vn cn mt vn cn
tip tc kim tra, l vai tr ca t bo cht ca trng (non) khi dung hp
vi t bo soma (tuyn v). T bo cht ca trng tip nhn nhn chuyn
vo khi ng cho s pht trin ca phi. Tuy nhin, c ch ca qu trnh
chuyn genome m vo genome phi vn cha c sng t v vai tr ca
t bo cht ca non trong th nghim dung hp cng cha c bit y .

1. Nguyn Ngc Hi. 2005. Sinh hc mo him. NXB Thanh nin, H Ni.
2. Phan Kim Ngc v H Hunh Thy Dng. 2001. Sinh hc ca s
sinh sn. NXB Gio dc, H Ni.
3. Phan C Nhn. 2001. Di truyn hc ng vt. NXB Khoa hc v K
thut, H Ni.
New York, USA.
5. Coleman WB and Tsongalis GJ. 1997. Molecular Diagnostics-For The
Clinical Laboratorian. Humana Press Inc. Totowa, New Jersey, USA.
8. Marshak DR, Gardner RL and Gottlieb D. 2001. Stem Cell Biology.
Cold Spring Harbor Laboratory Press, USA.
10. Mather JP and Roberts PE. 1998. Introduction to Cell and Tissue
Culture: Theory and Technique. Plenum Press, New York, USA.
11. Pollard JW and Walker JM. 1997. Basic Cell Culture Protocols.
Humana Press Inc. Totowa, New Jersey, USA.
13. Shuler ML and Kargi F. 2002. Bioprocess Engineering-Basic
Concepts. 2
nd
ed. Prentice Hall Inc. NJ, USA.
Chng 6

Cng ngh protein

I. M u
Cng ngh protein l mt hng nghin cu quan trng ca cng ngh
sinh hc, c tim nng ng dng rt ln. Trn c s cng ngh DNA ti t
hp hin nay nhiu loi protein nguyn th (protein th h th nht), protein
c sa i hoc mi (protein th h th hai) ang c sn xut nh
cc sinh vt prokaryote nh vi khun E. coli hoc eukaryote nh nm men
Sac. cerevisiae. Do tc sinh sn nhanh nn vi sinh vt c th sn xut cc
protein vi s lng ln trong mt thi gian ngn, sn phm c tinh sch
khng c nguy c nhim bn virus nh cc trng hp c tch chit t
ngi. i vi mt s protein c cu trc phc tp khng th biu hin vi
khun hoc biu hin vi hiu sut thp nm men, ngi ta ang c xu
hng chuyn gen m ha ca n vo thc vt. L do l v h thng thc vt
c nhiu u im nh c th trng trn quy m ln nh nng lng mt tri
nn t tn km hn, cc virus thc vt khng phi l tc nhn gy bnh cho
ngi, cc gen biu hin m to du thc vt nn rt d tch chit v thu
nhn protein.
Cng ngh protein l qu trnh xy dng cc phn t protein mi,
bng cch thit k mt phn t protein da trn cc nguyn l c bn nht,
hoc sa i cu trc ca mt protein ang c, nhm mc ch:
- Nghin cu qu trnh lp rp ca cc protein v cc nhn t ca
chui s cp tham gia vo s cun xon, n nh v th hin chc nng ca
chng. Cc c im ny c th c kho st bng cch sa i mt hoc
nhiu amino acid c trng theo mt kiu c nh hng trong protein
v quan st kt qu sau khi sn xut phin bn c sa i. Thng
thng cc protein c trnh t tng t khng ging nhau hon ton, tn ti
trong t nhin s c cc tnh cht hi khc nhau v ngi ta c th da vo
cc trnh t khc nhau ny tin hnh nhng sa i sau .
- Sn xut cc phn t protein n nh cho mt mc ch cng ngh
c bit, tuy nhin cc phin bn c tm thy trong t nhin khng c cc
tnh cht ti u cn thit. V d: mt enzyme c th c xem nh mt phn
ca mt qu trnh cng nghip nhng mt im c trng ca enzyme,
chng hn n nh nhit hoc l pH ti u cho hot tnh xc tc
khng th tng thch vi qu trnh . Cc thay i amino acid c th bin
i enzyme ny sao cho n thc hin chc nng tt hn trong mi trng
mi. C nhiu minh ha khc nhau v protein c sa i gip cho
chng ph hp tt hn vi cc hot ng cng ngh v thng mi, v mt
s trong c trnh by mc IV-Mt s ng dng ca cng ngh
protein.
Mun cng ngh ha mt protein cn phi hiu bit v cc nguyn l
cu trc ca protein v c im ca nguyn liu thit k hp l, hoc
sa i cc tnh cht mong mun. Hn na, cng ngh ny phi c c cc
cng c sn xut v phn tch protein mong mun. Cc cng c v nguyn
l c bn hin nay ang c pht trin song song.
Cc phn di y cung cp nhng kin thc c bn ca cng ngh
protein v tm tt mt vi pht trin ca lnh vc ny trong thi gian gn
y.

II. Cu trc protein
Cc nghin cu v cu trc ca protein cho thy c th phn bit
cu trc ca phn t protein thnh bn bc nh sau: Cu trc bc mt (cu
trc s cp) l trnh t sp xp cc amino acid trong chui polypeptide. Cu
trc ny c gi vng nh cc lin kt peptide (lin kt cng ha tr). Cu
trc bc hai (cu trc th cp) l tng tc khng gian gia cc gc amino
acid gn nhau trong chui polypeptide, hay ni cch khc l dng cun
xon cc b (local fold) ca tng phn trong chui polypeptide. Cu trc
ny c gi vng nh lin kt hydrogen c to thnh gia cc lin kt
peptide gn k nhau, cch nhau nhng khong xc nh. Cu trc bc ba l
tng tc khng gian gia cc gc amino acid xa nhau trong chui
polypeptide, l dng cun xon trong khng gian ca ton chui polypeptide
(overall fold), y l hnh dng chung ca chui polypeptide. Cc lin kt
nh lin kt Van der Waals, lin kt tnh in, lin kt hydrogen gia cc
mch bn ca cc gc amino acid u tham gia gi vng cu trc bc ba.
Cu trc bc bn xut hin nhng phn t protein bao gm hai hay nhiu
chui polypeptide hnh cu (bc ba), tng tc khng gian (s sp xp) gia
cc chui ny trong phn t gi l cu trc bc bn. Mi chui polypeptide
ny c gi l mt tiu n v (subunit). Chng gn vi nhau nh cc lin
kt hydrogen, lc Van der Waals gia cc nhm phn b trn b mt ca
cc tiu n v (Hnh 6.1).
Tuy nhin, n nay nhiu vn c bn v cc tnh cht ca protein
vn cha c gii quyt, v d c ch cun xon protein vn cn l ch
ca mt s cuc tranh lun.
Phn tip theo di y s m t cc cng c c bn ca cng ngh
protein v mt lot cc v d thnh cng trong lnh vc ny.

Hnh 6.1. Cc bc cu trc ca mt phn t protein

III. Cc cng c
1. Nhn dng trnh t
Hin nay, nhn dng trnh t (sequence identification), bng phn tch
trnh t gen hoc protein l mt qu trnh tng i khng kh khn. Cc c
s d liu ln hin c cha nhiu ngn trnh t khc nhau. Hn na, cc d
n phn tch trnh t genome ang cung cp cc trnh t mi v cc khung
c m (open reading frame) vi mt tc tng ln lin tc. Cc chc
nng ca nhiu trnh t ny c bit, t cc d liu di truyn hoc ha
sinh, hoc bng s tng ng trnh t i vi cc trnh t cha bit khc.
iu ny gip cho cng ngh protein mt phng php tip cn vi s
thit k hp l.
Cu trc bc 1
(cu trc s cp)
Cu trc bc 2
(cu trc th cp)
Cu trc bc 3

Cu trc bc 4

Cc gc amino acid Xon Chui polypeptide Cc tiu n v c lp rp
Lys
Lys
Gly
Gly
Leu
Val
Ala
His
2. Xc nh cu trc v m hnh ha
Cc thng s v cu trc c phn gii cao, c xc nh bng tia
X hoc tinh th hc in t (electron crystallography) hoc k thut cng
hng t ht nhn (nuclear magnetic resonance-NMR), l im ct li ca
s hiu bit v ha sinh protein. S lng protein c cu trc phn gii
cao ang ngy cng nhiu, nhng vn cn mt s ln cc trnh t c
xc nh l cha bit. Trong lc ch i lm y ch trng trong c s d
liu cu trc, khi c s d liu v cu trc ton din ny ang c lp rp,
th nhng c gng khc vn ang c tin hnh bng nhiu phng thc
d bo cc cu trc ca protein.
D bo cc cu trc protein t trnh t s cp (cu trc bc mt) c
mt lch s lu di v c thc hin bng cch m hnh ha cc trnh t
mi da trn cc cu trc bit ca cc trnh t hoc cc tiu trnh t (sub-
sequence) tng ng hoc gn tng ng bng k thut xu kim thnh
chui (threading), trong mt trnh t mi c so snh trc tip vi cc
kiu cu trc bit.

3. Bin i trnh t
Bin i mt protein ang tn ti bng cch sa i trnh t gen hin
nay l mt cng vic bnh thng v lnh vc ny by gi l phn t kh
khn nht ca qu trnh cng ngh. T u 1980, cc phng php bin i
trc tip protein bng pht sinh t bin im nh hng oligonucleotide
(oligonucleotide-directed site mutagenesis) theo phng thc tng hp gen
hon chnh t cc oligonucleotide primer hoc dng phn ng chui
polymerase (PCR) tr nn thng dng.
Cc phng php ni trn ch gii hn cho cc amino acid c m
ha bi gen. Tuy nhin, nhiu trnh t cng c th c to ra bng cch
tng hp protein theo phng php ha hc trong iu kin in vitro, phng
thc ny cng cho php hp nht cc amino acid khng c m ha trong
chui protein.

Cc phng php pht sinh t bin im nh hng
C hai phng php c bn sa i mt trnh t m ha ang tn
ti ca protein mc DNA. C hai phng php u i hi gn () mt
hoc nhiu oligonucleotide (t nht l tm thi) trn DNA si n (single
strand DNA, ssDNA), sau hot tnh DNA polymerase in vitro s xc tc
m rng cc oligonucleotide ny. Hai phng php l:

- Phng php khng dng PCR
cc phng php khng dng PCR, trnh t DNA c bin i
lin kt vi gc ti bn (v d plasmid, bacteriophage hoc phagemid), cho
php khuch i in vivo kiu gen b m hoc kiu gen c sa i. Mt
oligonucleotide tng hp mang t bin cn thit c gn vi khun mu
ssDNA mch vng (v d genome ca phage c ssDNA nh M13, hoc
X174 hoc dng ssDNA ca phagemid (Hnh 6.2a), hoc vi mt khun
mu dsDNA c si n ha tng phn (Hnh 6.2b). S dng nhiu
phng php enzyme khc nhau c th to ra khun mu ssDNA tng phn.

Hnh 6.2. Phng thc pht sinh t bin ssDNA khng dng PCR

Cc oligonuleotide c hot ng nh mt primer cho s tng hp
in vitro DNA bng cch dng enzyme DNA polymerase, thng dng l
DNA polymerase T4 hoc T7, cng vi s hin din ca enzyme DNA
Oligonucleotide primer
t bin
Trnh t c
bin i
ssDNA vector
DNA polymerase
+ DNA ligase
heteroduplex
DNA mch vng ng.
Si ti bn in vitro mang
t bin
Vt ch bin np
thch hp, vd: E. coli.

Phn bit in vivo cc
trnh t ca b m
v ca t bin

Chn lc hoc sng
lc trnh t t bin

ligase, cc phn t DNA si i (dsDNA) mch vng ng s c to ra
(Hnh 6.2c). DNA si i c a vo trong cc t bo vt ch thch hp
ti bn v phn chia. Sau , s bin i mong mun c xc nh bng
mt trong s phng thc sng lc hoc chn lc.

- Phng php da trn PCR
Phng php khng dng PCR bit k cng v c ti u ha
trong nhiu nm. Tuy nhin, gn y cc phng php da trn c s PCR
c ng dng rng ri hn, c bit ti sp xp nhanh cc vng protein
ni m cc v tr ct hn ch ph bin thng khng c sn.
Phng php da trn c s PCR cho php thc hin s bin i
mong mun v khuch i in vitro bng cch DNA ch (target DNA)
c bin tnh vi mt oligonucleotide mang t bin cn thit c th hot
ng nh mt primer cho s ti bn si DNA b sung (Hnh 6.3).
C nhiu cch thc khc nhau trong phng php da trn PCR, mt
s trong chng cn cc v tr ct hn ch cc oligonucleotide t bin.
iu ny c th lm gim s lng cc phn ng. V d: nu hnh 6.3, cc
primer A v D c cc v tr ct hn ch hu ch trong cc trnh t v D v
tr l t 3 ti trnh t t bin, th cc sn phm phn ng u tin c th
c to dng trc tip ngay sau . on DNA c khuch i s lin kt
vi gc ti bn ca vi khun trong plasmid hoc phage v c to dng
bng cch xm nhim vo trong vi khun.
Mt cch thc khc ca phng php da trn PCR cho php cc trnh
t ring bit m ha cho cc vng ca cc protein khc nhau c th lin kt
vi nhau, hoc ti t chc li cc vng trong mt protein. Trong phng
thc ny (Hnh 6.4), cc primer C v D l cc th lai cha cc trnh t c th
vi c hai vng (hai trnh t nucleotide). Sau , cc phn ng u tin s
to ra cc on dsDNA chng ln nhau theo trnh t v sn phm mong
mun c th c to ra trong phn ng th ba bng cch dng cc primer
A v B. Trong tt c trng hp, thit k cc trnh t oligonucleotide v cc
iu kin phn ng cn phi c xem xt cn thn cng vi s chn la
chnh xc ca DNA polymerase n nhit. Tuy nhin, cc phng thc ny
cho php tin hnh rt nhanh v c hiu qu rt cao.


Hnh 6.3. Pht sinh t bin PCR bng s m rng chng lp n

4. Pht trin phn t (molecular evolution)
Trong nhiu trng hp s bin i c nh hng ca mt trnh t
khng phi l phng thc thch hp thu c kt qu mong mun, bi
v thng khng xc nh c cc amino acid ch nm u v bin i
chng thnh ci g. V th, mt s phng thc khc c pht trin
sn xut v th nghim cc th vin ln hoc cc tp hp bin th
(repertoires of variants) ca mt trnh t c bit.
Cc phng thc ny thng da vo ba c im chnh: Th nht,
l nucleic acid m ha cho trnh t protein quan tm duy tr lin kt vt l
vi protein. Lin kt ny c c nh s hin din ca protein trn b mt
ca bacteriophage hoc vi khun hoc t bo eukaryote v trnh t m ha
nm trong phage hoc t bo, hoc trn polysome m mRNA v
protein mi c dch m vn cn c lin kt nh ribosome.

PHN NG 1 Khun mu + primer A+D
PHN NG 2 Khun mu + primer B+C
KHUCH I PCR
A
B
C

D
Sn phm AD
Sn phm BC
PHN NG 3
Phn lp cc sn phm AD+BC
Trn v b sung d tha cc primer A v B
KHUCH I PCR
CT SN PHM AB CC V TR X V Y
PHN LP V GN VO VECTOR
X Y

Hnh 6.4. Dung hp vng PCR

Th hai, mt phng php c pht trin to ra mt s lng ln
cc bin th bng cch a cc on oligonucleotide thoi bin (degenerate
oligonucleotide) vo trong trnh t m ha theo phng thc chn on
cassette hoc dng k thut PCR, hoc bng phng thc pht sinh t bin
in vitro. Tuy nhin, vi cc phng thc nh th th mi ln ch c mt
on nh protein c sa i. Cc th vin b gii hn bi kh nng tip
nhn cc thnh vin ring r ca t bo vt ch, v trong trng hp ny th
10
12
-10
14
c xem l mt s lng ln.
Th ba, cc phng php ny i hi mt phng thc sng lc hoc
chn lc t th vin cc trnh t protein mi c kiu hnh quan tm. Phng
thc sng lc bao gm vic gn vi mt phi t c th d dng thc hin.
Phng thc ny cng i hi phi c s xc tc v kt qu l cc protein
PHN NG 1 Khun mu + primer A+D
PHN NG 2 Khun mu + primer B+C
A
X
Y B
C
D
KHUCH I PCR
Sn phm AD
Sn phm BC
PHN NG 3
Phn lp cc sn phm AD + BC
Trn v b sung d tha cc primer A v B
KHUCH I PCR

X Y
CT SN PHM AB CC V TR X V Y
PHN LP V GN VO VECTOR
xut hin c gi li trn mt gi th rn. Cc h thng chn lc thng
bao gm s b sung mt chc nng cn thit trong c th vt ch.
Nhng protein hu ch c phn lp t cc phng thc trn c
th c khuch i bng cch nhn (sinh sn) phage hoc t bo vi khun
mang trnh t gen ca n, hoc bng cch khuch i trc tip cc trnh t
ca chnh gen nh k thut PCR lm giu trnh t mong mun. Cc
phng php loi ny nhanh chng tr thnh k thut quan trng cho cng
ngh protein v c gi bng thut ng pht trin nh hng (directed
evolution).

5. Thit k trnh t de novo
Thit k de novo protein l mt cng vic rt phc tp. V nguyn tc,
i vi mt protein bt k c (n) gc amino acid th kh nng s c 210
n

trnh t khc nhau. Cc c s d liu v cu trc v trnh t protein cho thy
nhiu trnh t s cun xon c th c iu chnh tng t nhau v nh
vy chng c th thc hin cc chc nng nh nhau. V th, phng php
tip cn ngc li chn la s cun xon thch hp v sau xc nh
trnh t no cn thit to ra s cun xon v chc nng mong mun c
th l thch hp hn c. Dahiyat v Mayo (1997) m t cc phng php
my tnh thit k cc trnh t ca vng peptide. Ch khi c trnh t
peptide th protein mi c th c xy dng hoc bng tng hp peptide
(nu trnh t c kch thc va phi) hoc bng tng hp gen. Gn y, cc
phng php khuch i PCR tng hp gen thng c s dng lm
y v khuch i tng phn cc oligonucleotide chng ln nhau (overlap
extension).

6. Biu hin
Khi mt trnh t mi c xc nh cn phi cho n biu hin
chng minh chc nng ca protein. S biu hin ca cc protein ti t hp
l mt cng vic v cng phc tp v cc h thng biu hin vt ch trong
phm vi t vi khun (E. coli c s dng ph bin nht), ti nm men
(chng hn Sac. cerevisiae v Pichia pastoris), ti cc t bo cn trng v
cc nui cy t bo ng vt c v, v cui cng ti ng-thc vt chuyn
gen. Quy m sn xut protein cng thay i khc nhau. cc giai on u
ca qu trnh pht trin mt protein mi, v d mt phn t protein c
nhn dng trong mt th vin th hin, th ch mt lng nh (g) ca
protein l xc nhn mt c tnh sinh hc. Trong giai on hai, cn c
mt lng nguyn liu tinh sch ln hn (t 10 n 100 mg) thu c
cc thng tin v cu trc c trng v thc hin thm mt s th nghim in
vitro v in vivo. Trong mt vi trng hp, c th cn lng nguyn liu
c tinh sch ln hn (t vi g n vi kg) bng cc phng thc nghim
ngt (v thng l tn km) nu protein c sn xut s dng cho cc
mc ch thng mi (v d cc enzyme cng nghip).
Thng thng, ngi ta phi th nghim mt s phng php khc
nhau tm kim mt vt ch biu hin thch hp, v mt protein c sa
i s khng th biu hin trong cng mt kiu nh trnh t ca b m
(thnh thong l tt hn, nhng thng l khng). Cc yu t ny tr nn
quan trng hn khi nhiu phng thc da vo cc h thng th vin th
hin, trong cc thnh vin ca th vin c th b mt hoc khng c
miu t ng mc do biu hin km hoc do cun xon khng ng. c
rt nhiu th nghim ci thin s biu hin v iu chnh s cun xon
protein ca cc h thng vt ch, c bit l E. coli. khc phc mt s
vn ny, cc h thng phin m-dch m in vitro cng c s dng
v ang c ti u ha cho sn xut quy m nh mt cch hiu qu cc
protein mi vi cc s lng thch hp cho phn tch.

7. Phn tch
Cn phi c nhiu phng php khc nhau phn tch c im ca
cc protein c sa i. Khi mt chc nng c sa i (a vo, bin
i hoc loi b) th mt phng php th nghim sinh hc thch hp c th
c pht trin xc nh kh nng ca protein mi c sn xut. Trong
, phi m bo rng php th nghim c th tin hnh vi mt lng rt
nh ca nguyn liu, chng hn cc nguyn liu thu c t phng thc
khuch i th vin.
Trong nhiu trng hp, vic t c v chng minh chc nng mi
ca protein l quan trng nht. Tuy nhin, thm vo cc php th nghim
chc nng, gii thch cc kt qu thng i hi mt s hiu bit y
cu trc c sa i, c bit c c s hiu bit su sc sau ny v s
cun xon ca protein. Vic nh gi s lng trung bnh ca protein mi
c th c tin hnh, cc tnh cht th c th c pht hin bng knh
quang ph (spectroscope) hoc cc php o quy m ln khc, v d: cc tnh
cht lng hng sc vng (circular dichroism), c (turbidity), ethalpy,
lng ng (sedimentation) hoc sc k (chromatography). Tuy nhin,
thng tin cu trc chi tit ca sn phm cui cng vn cn l bc hn ch
trong thit k v trin khai cng ngh protein thch hp. Gn y, kt qu
ca Casimiro v cng s (1997) cho thy s tng hp gen da trn c s
PCR, biu hin trong E. coli ca mt lng protein (mg) c nh du
ng v phng x v ph NMR c th c thc hin trong mt khong thi
gian ngn.

IV. Mt s ng dng ca cng ngh protein
1. Cc t bin im
Cc t bin im ring r trong cc protein c th thu c ch khi c
trnh t gen thun li, bng cch dng cc k thut c trnh by trn.
Di y l mt vi v d in hnh:

1.1. Betaseron/Betaferon (Interferon -1b)
Mt trong nhng kt qu u tin ca cng ngh dc phm protein l
sn xut interferon -1b. Protein mi ny c to ra bi s thay th ca
cysteine (Cys) cho serine (Ser) gc th 17 ca phn t interferon di 154
amino acid. Protein c tng hp biu hin trong E. coli mt hot tnh c
trng gn vi interferon t nhin c ngun gc t fibroblast. Phn t ny
c ng k bn quyn t 1993 s dng cho vic lm gim tn s v
mc khc lit ca s ti pht nhng bnh nhn i li c c s ti
pht yu bnh a x cng.

1.2. Humalog (Lispro Insulin)
Humalog l mt dng bin i gen ca insulin ngi trong hai gc
C-terminus ca chui B, proline (Pro) v lysine (Lys) cc v tr 28 v 29,
tng ng, c o ngc th t ca chng. Humalog l mt dng ng
ng hot ng nhanh ca insulin, c thit k bt chc tc p
ng insulin t nhin ca c th i vi thc phm. S bin i C-terminus
c thit k da trn c s cu trc v s tng ng trnh t vi nhn t
sinh trng 1 ging insulin (insulin-like growth factor 1, IGF-1) v vic o
ngc th t gim s nh trng ha (dimerization) ca tiu n v B.
Humalog c ng k bn quyn s dng trc 1996.

1.3. Cc t dc vaccine mi (adjuvants)
Gn y, cc phng php tip cn mi trong vic thit k vaccine
gip cho lnh vc y hc ny c nhng pht trin quan trng. Nhiu loi
vaccine hin nay l sn phm cng ngh sinh hc rt c gi tr. Cng ngh
protein ang c s dng xy dng cc phn t protein mi cung cp s
h tr min dch (adjuvant) gy ra mt p ng min dch i vi mt
khng nguyn ng phn phi (co-administered antigen). Cc t dc
protein mi ny ang c quan tm c bit nhm kch thch cc phn ng
min dch mucosal sau khi chng nga bng cch ung (oral immunization)
trnh vic s dng phng php tim phng vaccine (injection for
vaccination).
Nhng vaccine c kho nghim tt l c t ca Vibrio cholerae
(CT) v c t khng bn nhit ca E. coli (LT). Cc cu trc tinh th ca
CT v LT c lm sng t. Cc th nghim pht sinh t bin im nh
hng da trn cu trc ca LT gip c c s hiu bit y v mi
quan h ca tiu n v A c hot tnh enzyme n vi 5 tiu n v B ca
cc c t heterohexameric ny, cc v tr lin kt enzyme v cofactor trong
tiu n v A, cng cc im ct hot ng trong chng. Cc nghin cu
ny cho php gii thch ti sao cc c t t bin vn n nh lp rp
trong holotoxin, nhng c tnh in vitro ch cn mc ti thiu trong khi
vn gi li cc c tnh min dch v t dc ca chng. V d: t bin Ser
thnh Lys gc 63 trong tiu n v A ca LT c ch s lin kt vi c
tnh ca NAD cofactor cho hot tnh ADP ribosyltransferase. t bin
S63K ny cho thy cng c tnh gim su ln trong khi cc c tnh
vn duy tr kh nng min dch v t dc. Mt trng hp khc, t bin
gc 192 (arginine-Arg thnh glycine-Gly) trong tiu n v A ca LT cng
c thc hin. t bin ny xy ra v tr m ti tiu vng A1 b
phn gii khi tiu vng A2 v y l bc u tin trong hot ng chc
nng enzyme ca tiu n v A. t bin cng to ra c t bt hot enzyme
duy tr cc c tnh t dc. Cc c t t bin ny hin nay ang c
th nghim trong cc nghin cu l thuyt v lm sng nh gi li ch
ca chng.

2. Sp xp li vng (lin kt, trao i v xa b)
Hu ht cc protein ln do cc vng cun xon c lp nh hn to
thnh. Cc vng ny thng c lin kt cng vi cc chui peptide ngn
v trong nhiu trng hp, nhng khng phi tt c, cc vng ny c th
xc nh nh l cc exon phn chia trong trnh t gen. S dng cc k thut
sinh hc phn t, ngi ta c th b sung, loi b hoc trao i cc vng t
mt protein ny ti protein khc xy dng li cc phn t protein.

2.1. Cc vng lin kt
2.1.1. Cc dung hp vng cho t bo ch
Mt trong cc minh ha u tin ca cng ngh protein l vng c v
tr lin kt ca khng th c lin kt di truyn vi enzyme. n v khng
th c bn l mt cu trc c dng Y hoc T bao gm hai chui nng c
khi lng phn t 50 kDa v hai chui nh c khi lng phn t 25 kDa,
vng lin kt khng nguyn c gi l vng Fab (antigen binding), gm c
cc u tn cng N ca chui nng v chui nh. Cc u tn cng C ca
hai chui nng cng phi hp to thnh vng d kt tinh gi vng Fc
(crystallisable). Vng sau lin quan vi nhng tng tc gia cc t bo
khc nhau ca h min dch v vi b th, cng l yu t quan trng trong
vic xc nh chu k bn phn r (half-life) huyt thanh ca cc khng th,
t nht l loi IgG.
Dung hp gen enzyme-khng th c thc hin bng cch dng trnh
t DNA m ha cho thnh phn chui nng ca Fab ca khng th lin
kt n vi trnh t m ha cho nuclease ca khun t cu hoc trnh t m
ha cho DNA polymerase ca E. coli. Cc gen dung hp sau khi c
chuyn vo t bo sn xut chui nh ca khng th biu hin v khi
phc li c hai hot tnh enzyme v lin kt khng nguyn. Cc nghin cu
u tin theo hng ny cung cp c s cho nhng hng nghin cu
khc, trong mt chc nng lin kt (khng th, cytokine, nhn t sinh
trng hoc vng lin kt phi t ngoi bo (ECD) ca th th) c gn
vi mt chc nng ca c quan phn ng li kch thch (c t, enzyme,
cytokine). Mt vng lin kt khng phi khng th (non-antibody) c th
c gn vi vng Fc ca mt khng th (tn dng u im ca chu k bn
phn r di huyt thanh ca cc khng th) ci thin cc c tnh ng
hc dc phm (pharmacokinetic) ca mt phn t c thit k.
Chng hn, Enbrel
TM
(etanercept) gn y c ng k bn quyn
s dng cho ngi. Enbrel
TM
gm c cc vng th th ngoi bo cho nhn
t gy hoi t khi u (TNF) c dung hp di truyn vi cc vng Fc
ca IgG dng trong vic ngn chn hot tnh ca TNF. Enbrel
TM
hin
nay ng k bn quyn s dng cho iu tr lm gim cc du hiu v
triu chng ca bnh vim khp t va phi n khc lit cc bnh nhn
c cc phn ng khng y i vi mt hoc nhiu loi thuc chng
vim khp.

2.1.2. Cc cytokine c dung hp
Cc cytokine khc nhau thng c cc chc nng chng cho nhau
(overlapping function), hoc c th hot ng hp lc trn cng t bo
gy ra mt thay i sinh l i vi t bo . Lin kt cc cytokine bng
cch dung hp cc gen trong khung (in-frame) buc hai nhm chc nng li
vi nhau v c th v nguyn tc dn n hiu qu sinh hc mong mun
cc liu thp hn nu c thc hin ring r. Cc dung hp kiu ny
c thc hin trng hp interferon cch y hn mt thp k. Gn y
hn PIXY321, mt dng dung hp ca GM-CSF v IL-3 cng c kho
st. Trong trng hp ny, cc gen GM-CSF v IL-3 c sa i loi
b cc v tr N-glycosylation ca ng vt c v v sau c lin kt vi
nhau nh b sung mt on ni gm 15 amino acid linh hot gia C-
terminus ca GM-CSF v N-terminus ca IL-3. Nm men biu hin
PIXY321 cho thy i lc th th c tng cng, hot tnh sinh sn v
hot tnh kch thch to khun lc cng c so snh vi mt trong s cc
protein khi u monomer.

2.2. Trao i cc vng protein
Mt phng thc n gin khc c dng trong cng ngh protein l
trao i cc vng hon chnh, trong cc vng tng t ca cc ngun
khc nhau c trao i trong mt protein a vng cung cp mt chc
nng mi.

2.2.1. Cc khng th khm ngi-chut
y cc vng lin kt khng nguyn khng th n dng ca
chut c gn vi cc vng khng thay i (ni cung cp cc chc nng
phn ng li kch thch min dch v iu khin chu k bn phn r sinh
hc) ca mt khng th ngi. S chuyn i (switching) ny c th lm
gim r rt kh nng to min dch (immunogenicity) khng mong mun so
vi khng th gc ca chut. Ti nay c bn sn phm khng th khm
c ng k bn quyn l cc dc phm, k c phc hp chng tiu
huyt cu (anti-platelet) ReoPro
TM
.

2.2.2. Xa cc vng
Hot t plasminogen m (tPA) l mt serine protease tch chit t cc
t bo mng trong. Sau khi lin kt vi fibrin, tPA hot ha plasminogen
thnh plasmin y l yu t khi u phn gii huyt khi cc b (local
thrombolysis). Reteplase l mt dng bin th m 3 trong 5 vng ca tPA
b xa. Mt trong cc vng c kh nng chn lc fibrin v vng xc tc
c gi li. Reteplase c ng k bn quyn nh l Retavase dng cho
vic iu tr chng nhi mu c tim cp tnh ci thin dng chy ca mu
(blood flow) trong tim.

3. Sp xp li ton b protein
Nhiu protein tn ti nh l cc thnh vin ca mt h ln, trong
nhng protein tng ng th hin cc hot tnh sinh hc hi khc nhau.
thi k u ca cng ngh protein, cc gen lai c to ra bng cch dung
hp cc trnh t nucleotide thng qua cc v tr ct hn ch thng dng, hoc
nh s ti t hp in vivo gia cc gen tng ng trong mt kiu ngu nhin
hn. Cc phng php ny sn xut mt s lng nh cc gen mi c th
c kim tra ring r. Gn y hn, cc dng tng ng ca protein
c s dng to ra cc th vin bin th mi rt ln. Theo phng thc
ny, cc gen i din cho hai hoc nhiu thnh vin ca h c phn on
ngu nhin bng enzyme DNase I. Cc on ny sau c dng nh cc
PCR primer to ra cc trnh t gen mi bng cch lai ngu nhin gia cc
on. Cc bin th mi sau c th c nhn bit bng phng thc chn
lc hoc sng lc nh nu trc y. Mt s trng hp c to ra
theo phng thc ny l cc enzyme, cytokine v cc v tr lin kt khng
th.
4. Cc tng tc protein-phi t
4.1. Bin i enzyme
Trong cng ngh protein, nhiu nghin cu c thc hin theo
hng thay i enzyme kim tra v bin i cc tng tc enzyme-c
cht. Cc loi thay i bao gm: tng hot tnh xc tc; bin i tnh c
hiu c cht, k c s pht sinh de novo cc chc nng xc tc mi; bin i
cc profile ca pH mt enzyme c th hot ng trong cc iu kin
khng sinh l (non-physiology); ci thin s chng oxy ha bng cch thay
th cc amino acid nhy cm vi s oxy ha nh Cys, tryptophan (Trp) hoc
methionine (Met) bng cc amino acid khng th oxy ha c cu trc khng
gian tng t (sterically similar) nh Ser, phenylalanine (Phe) hoc
glutamate (Glu), tng ng; ci thin kh nng n nh i vi cc kim loi
nng bng cch thay th cc gc Cys v Met v cc nhm carboxyl b mt;
loi b cc kiu phn ct ca protease; loi b cc v tr m sn phm
xc tc c th lin kt theo mt kiu khc cm ng s c ch ngc khc
v tr (allosteric).

4.2. Cc cht ch vn hormone (hormone agonist)
S pht trin cc cht siu ch vn (super-agonist) c hot tnh sinh
hc tng ln r rt c th lm tng i lc lin kt ca cc hormone vi cc
receptor ca chng. Grossmann v cng s (1998) m t s thit k cc
bin th ca TSH ngi (human thyroid-stimulating hormone) c hot tnh
tng ln 1.300 ln. Cc bin th c thit k da trn s tng ng vi
hCG (kch dc t mng m ca ngi-human chorionic gonadotrophin),
mt loi glycoprotein hormone khc chia s mt tiu n v chung. S
thay th cc nhm tch in dng trong mt vng mc (loop region) ca
TSH phi hp tng hot tnh ca chng.

4.3. Thay th cc lin kt c hiu
Thay th cc lin kt c hiu c thc hin bng cch chuyn vng
c hiu cho mt phi t t mt protein ny n mt protein khc. Mt s
trng hp i hi nhng thay i nh, mt s khc i hi vic chuyn i
quy m ln ca nhiu gc. V d tnh c hiu ca hormone prolactin
c bin i bng cch thay th 8 amino acid receptor lin kt b mt sao
cho hormone c bin i s lin kt vi receptor ca hormone sinh
trng.
Cc phn t protein thng c cc vng loop ni gia cc kiu cu
trc th cp khc nhau (-helix, -strand). Trong mt s trng hp chc
nng ca cc gc protein trong cc loop ny c th l mc tiu cho cc th
nghim thay th tng i n gin. V d: c trng ca nhn t sinh
trng c tnh base ca fibroblast c bin i thnh loi c tnh acid bng
cch thay th mt vng loop c bit.

V. Sn xut protein trn quy m ln
Nui cy cc vi sinh vt trn quy m ln l phng php kinh t nht
do c cc u im sau:
- S dng cc mi trng n gin, r tin.
- Cc chu k ln men ngn ngy.
- C th kim sot trng thi sinh l ca vi sinh vt trong qu trnh ln
men v m bo c tnh ng nht ca cc m ln men.
- C th k hoch ha vic thu hoch protein mt cch hp l, ph
hp vi cc qu trnh phn tch v tinh sch u ra.
- Hiu sut protein c th c tng ln nhiu ln bng cch ci thin
cc chng truyn thng v pht trin qu trnh ln men, cng vi vic s
dng thm cng ngh DNA ti t hp.
- Cc k thut ti t hp DNA cng m ra cc c hi cho vic sn
xut sinh khi enzyme t vic nui cy nhng loi vi sinh vt i hi iu
kin sinh trng khc khe nh: mi trng dinh dng t tin hoc phi b
sung cc nhn t cm ng (inducers)...
- Cc enzyme ca cc dng vi sinh vt sng iu kin khc nghit
(sinh trng cc iu kin cc oan ca nhit , mui, p lc thm thu,
kim) nh k thut ti t hp DNA nay c th sinh trng thun li trong
cc nui cy mesophilic (a nhit trung bnh 20-40
o
C), v c th sn xut
cc enzyme vi cc c im chu nhit hoc chu mui nng cao.

1. Ln men E. coli ti t hp
Cc enzyme c ngun gc t cc sinh vt prokaryote c th c sn
xut d dng trn quy m ln vi hiu sut cao trong cc vt ch E. coli ti
t hp. Cc qu trnh ln men ny c th c tin hnh quy m t 3.000-
6.000 L, v khng i hi cc bc cy gy (inoculum cuture) phc tp.
Mt cu trc vt ch thch hp cho qu trnh ln men c trng c th nh
sau:
Vt ch E. coli mang (1) plasmid vector c gen m ha cho enzyme
cn thit, (2) cng vi gen ch th khng khng sinh thch hp nh
ampicillin hoc neomycin, v (3) mt nhn t cm ng nh l TAC (b ba
c trng cm ng lactose hoc isopropylthiogalactose).
Hiu qu sn xut cao nh mong mun c th t c bng cch ln
men m c cung cp dinh dng, trong mi trng nui cy m cha cc
thnh phn cho s sinh trng ban u ca vi khun, v d: glucose 2%,
dch chit nm men (yeast extract) 1%, phosphate 1% v cc loi mui khc
cng vi khng sinh c chn. Sau khi s sinh trng ban u c thit
lp, cc cht dinh dng b sung c cung cp cc t l thch hp m
bo ngun carbon v nitrogen.
Ngun carbon thun li l glucose, v nitrogen c th l mt phc hp
t nhin (chng hn nh dch chit nm men, dch thy phn casein), sir
ng (corn sirup), mt loi mui ammonium n hoc urea.
Nui cy E. coli trong mi trng c b sung cc hn hp amino acid
cho kh nng sinh trng v sn xut protein nhanh hn, tuy nhin vic s
dng cc ngun dinh dng n gin hn (chng hn ngun nitrogen), mc
d c th i hi thi gian ln men lu hn, vn c th to ra s sinh trng
ca t bo v hiu sut protein cao tng t.
Sn xut enzyme c cm ng thun li bng cch b sung
isopropylthiogalactoside t 30-300 mg/L, hoc lactose t 1-10 g/L. Vic
quyt nh thi gian v tn s b sung cht cm ng l rt quan trng thu
c hiu sut enzyme cc i.
Mt ti u trn 200 ca OD
600nm
(50 g khi lng kh ca t
bo/L) v s biu hin protein khong 10 g/L (30% ca protein tng s) l
mt kt qu ln men mong mun. Qu trnh ln men thng thc hin trong
hai ngy nhit thp hn nhit sinh trng ti u ca c th, ngha l
t 24-28
o
C.

2. Ln men nm
Cc loi nm vt ch nh Aspergillus v Fusarium, v nm men
hng methyl (Pichia) thch hp cho sn xut cc protein glycosyl ha t
cc ngun ng vt hoc nm. DNA ca protein c hp nht trc tip
trong DNA nhim sc th cng vi h thng promoter iu ha biu hin
gen.
Cc vt ch ti t hp c th c ln men trong mt kiu tng t
nh cc nui cy khng ti t hp v ln men mt cao ca t bo c th
d dng thu c bng cch dng cc phng php ca k thut sinh hc
truyn thng.
Cc vt ch Aspergillus hoc Fusarium c th c sinh trng trn
cc nguyn liu th r tin nh bt u tng c b sung thm cht dinh
dng l sir ng. Nm men, nh Saccharomyces v Pichia, c th sinh
trng trn dch chit nm men hoc cc dch thy phn protein v sir ng.
Qu trnh nui cy c th cho sinh khi t bo cao bng cch s dng
cc mi trng c xc nh y c b sung xen k thm mt vi
cht dinh dng khc. Ln men c trng ko di t 4-8 ngy. S biu hin
protein vt qu 10 g/L thu c quy m cng nghip.

3. Cc enzyme vi sinh vt thay th cc enzyme ng-thc vt
Mt s enzyme cng nghip vn c tch chit t cc ngun ng
vt nh b tim n nguy c ca s nhim bn bnh no dng xp ca b
(bovine spongiorm encephalopathy). Chng hn renin, sau khi thu t d dy
ca b con mi sinh s c s dng lm phomt. N vn khng c
m bo liu c hin din bt k ri ro no cho sc kho ca ngi tiu
dng hay khng, tuy nhin renin ti t hp ca b con hin nay c th c
sn xut nh ln men vi sinh vt l hon ton an ton.
Mt cch khc, bng phng thc tch chit enzyme truyn thng t
cc ngun vi sinh vt v vic s dng k thut sng lc thch hp, nhiu
enzyme mi ging nh enzyme ng-thc vt hin nay c sn xut. V
d:
- Enzyme protease ca Mucor trong nhiu trng hp thay th
renin t d dy ca b.
- Protease ca Aspergillus v Bacillus thay th cc enzyme dng
trong thuc da.
- Rt nhiu loi amylase vi sinh vt c pht trin thay th hoc
b sung cho cc amylase thc vt trong to malt ca la mch hoc la m.
- Hn na, mt s enzyme c ngun gc ng vt (nh hot t
plasminogen ca m) cng c sn xut trong cc vi sinh vt ti t hp v
c th cung cp chui protein c ti cun xon. Cc enzyme glycosyl ha
ca ng vt c v c th c biu hin trong cc c th eukaryote nh
Saccharomyces v Aspergillus. y, vt ch s glycosyl ha enzyme
cung cp hot tnh y cho d cc ng glycosyl ha y c th khc
ng vt c v.
Cc enzyme c th c sn xut trc tip bng nui cy t bo ng
vt c v, tuy nhin gi thnh s rt cao khong t 1.000-5.000 USD/gram,
mt mc gi ch c th chp nhn cho vic sn xut cc enzyme tr liu c
bit nh hot t plasminogen ca m hoc cc loi protein lin quan.

4. Cc nguyn tc ha sinh c bn
Hiu sut ln men enzyme vi sinh vt c th c ci thin bng cc
k thut kinh in, tng t cc k thut dng ci thin hiu sut trong
ln men khng sinh. C hai s ci thin di truyn v qu trnh ln men
dn n s pht trin kh nng ln men trong sn xut enzyme ln ti
20 kg/m
3
. S hiu bit v iu ha di truyn ca qu trnh tng hp enzyme
l rt quan trng trong vic chn lc cc chng c ci thin v ti u
cc qu trnh ln men. C nhiu nhn t quan trng c th nh hng n
sn xut enzyme nh sau:

4.1. S cm ng
S tng hp enzyme thng b c ch (iu kin gip bo tn nng
lng t s tng hp protein khng cn thit) tc l enzyme s ch c sn
xut trong s hin din ca mt cht cm ng, thng l c cht ca n.
Mc cm ng (induction level) c th l rt mnh (tng ln hn 1.000 ln
so vi khng cm ng) v hot ng bng cch gy cn tr s iu ha nhn
t c ch. Nhiu enzyme d ha c th c cm ng, nh:
- Sucrose cn thit cho sn xut invertase.
- Tinh bt cho sn xut amylase.
- Galactoside cho sn xut -galactosidase.
Trong mt s trng hp, mt sn phm hoc mt sn phm trung
gian cng c th hot ng nh mt cht cm ng, v d:
- Phenylacetate cm ng penicillin G amidase.
- Cc acid bo cm ng lipase.
- Xylobiose cm ng cc xylanase.
S cm ng sn phm thng xy ra trong qu trnh tng hp cc
enzyme ngoi bo cn thit cho vic thy phn cc polymer c khi lng
phn t ln chng c th i vo t bo v tip tc gy ra s cm ng.
Cc coenzyme cng c th hot ng nh cc cht cm ng, v d
thiamine cm ng pyruvate decarboxylase. Hn na, c hiu qu trong
sn xut enzyme, s cm ng l iu kin cn thit quyt nh hp l
thi gian sn xut enzyme trong h ln men m bo thu c lng sn
phm cao nht.
Tuy nhin, trong thc t s cm ng thng c tin hnh bng hn
hp cc tc nhn cm ng t tin, c kh trng v b sung cc thi
gian c bit thit lp s ln men. khc phc cc vn ny cc t
bin iu ha c th c sn xut khng ph thuc vo cc nhn t cm
ng v v vy c gi l cc t bin cu thnh.

4.2. c ch ngc
- c ch ngc (feedback repression) l tc dng c ch bng cch c
nh tc ng d lp th ca mt cht ln hot tnh ca t nht mt trong
nhng enzyme xc tc cc bc chuyn ha dn n sinh tng hp ring
ca cht .
- Tng hp enzyme cng c iu chnh bi s c ch ngc xut
hin cc enzyme cn thit cho qu trnh sinh tng hp cc cht c phn t
lng thp (chng hn l mt loi amino acid), trong s tch ly ca sn
phm cui cng c th gy c ch s tng hp cc enzyme c bit, thng
thng l enzyme u tin trong con ng sinh tng hp. C ch ca hin
tng ny c trnh by hnh 6.5.

Hnh 6.5. S chui phn ng ha sinh tng hp cht (X)

- Ngi ta nhn thy sn phm cui cng ca qu trnh sinh tng hp
mt cht c kh nng gy ra s c ch qu trnh tng hp ca chnh n. Sn
phm cui cng d c t bo tng hp hay thu nhn t mi trng bn
ngoi, khi nng d tha so vi nhu cu ca c th vi sinh vt s nh
hng n enzyme u tin trong con ng sinh tng hp.
Enzyme u tin (a) l mt enzyme d lp th. N c c im thay
i cu hnh khng gian khi c mt sn phm cui cng nhm gim bt hot
tnh xc tc ca mnh. enzyme ny, ngoi v tr gn vi c cht A (trung
tm xc tc), n cn c mt hay nhiu v tr gn vi sn phm cui cng X
gi l trung tm d lp th. Trung tm xc tc v trung tm d lp th tch
bit nhau v khng gian v khc nhau v cu trc. Trng thi hot ng ca
enzyme ny c c trng ch n c kh nng gn vi c cht A v nu
bn cnh c cht A cn c s hin din ca X mc d tha so vi nhu
cu ca c th vi sinh vt, th s xy ra s bao vy ca trung tm d lp th,
lm cho trung tm xc tc b bin i cu hnh khng gian n mc khin
cho enzyme (a) khng th gn c vi c cht A m ch gn vi X. Nh
vy, enzyme (a) s khng c hiu lc trong vic chuyn ha A thnh B.
Chui tng hp X s b gin on do X s b gim s lng. V d minh
ha c ch ngc i vi tryptophan hnh 6.6.
Cc t bin c ch ngc c th thu c bng cch chn lc cc
nui cy khng cc c t l cc cht ng ng ca sn phm. Cc dng t
bo sng st s mt s mn cm ngc i vi sn phm. Cc t bin
tng t c th thu c bng cch phn lp cc t bin khuyt dng
(nutritional auxotrophs), cc t bin ny khng th to ra sn phm cui
cng, m ph thuc vo s b sung hp cht ny t mi trng bn ngoi
A B C X
(a) (b) (c)
sinh trng bnh thng. S cung cp cht dinh dng c iu chnh ny
s hn ch cc nng trong ni bo ti mc s c ch ngc xy ra t
hn.

Hnh 6.6. C ch c ch ngc i vi tryptophan

4.3. c ch dinh dng
Tng hp enzyme cng c th c iu chnh bng s c ch dinh
dng c trng bi carbon, nitrogen, phosphate hoc sulphate. Nhng c
ch ny tn ti duy tr s sn xut ca cc enzyme khng cn thit.
Dn chng tt nht l s iu chnh nh hin din ca glucose m
trong carbohydrate ny c th lm ngng sn xut cc enzyme cn thit
cho s chuyn ha ca cc hp cht lin quan v khng lin quan (hiu ng
glucose). c ch glucose l mt khi nim chnh trong nui cy sinh khi t
bo quy m ln (nguyn liu th c gi tr kinh t nht ca qu trnh ln
men). S c ch d ha glucose c th l rt mnh v thng km hm hiu
qu ca cht cm ng. Cc ngun carbon khc nh lactate, pyruvate,
HO
HO
HO
COOH
COOH
COOH
HO
HO
NH
2
CH
2
-CH-COOH

NH
2

N
Shikimate

Chorismate

Anthranilate

Tryptophan
Anthranilate synthetase
Sn phm c ch ngc
succinate v citrate, cng l cc cht c ch hiu qu trong mt s vi sinh
vt. Thm ch citrate cng c th c ch s chuyn ha ca glucose mt s
vi khun.
S c ch d ha glucose cng c gii quyt v mt di truyn bng
cch chn lc cc th t bin chng li hin tng ny. Cc t bin c th
chn lc d dng t mi trng nui cy cha glucose v c cht ca
enzyme cn thit, v d: hn hp glucose/aspartate cho php chn lc cc
dng sn xut aspartate khng b c ch glucose (aspartate c cung cp
y ch l ngun nitrogen). Sn xut penicillin G amidase trong E. coli c
tng ln nhiu ln bng cch chn lc cc t bin c kh nng sinh trng
trn amide nh l mt ngun nitrogen duy nht trong s hin din ca
glucose. Kt qu to ra cc dng vi khun c kh nng sn xut mnh v
khng d b c ch bi glucose. S dng dng ng ng ca glucose, 2-
deoxyglucose, cng l mt phng thc hiu qu chn lc cc t bin
khng c c ch glucose.
4
NH
l mt ngun nitrogen rt c gi tr, cc mui ammonium l
ngun nitrogen rt r so vi cc hn hp amino acid, nhng s hin din ca
cc amino acid thng cho php vi sinh vt sinh trng mnh v nhanh.
Mt s t bin khng b c ch bi ngun nitrogen cng c th c chn
lc da trn c s c im chng chuyn ha ammonium (methyl ha
ammonium, methylammonium).

5. Cng ngh di truyn
Kh nng ng dng ca k thut ti t hp DNA hn 10 nm qua
c mt hiu qu rt ln trong sn xut protein vi sinh vt. Vt ch E. coli
thch hp cho s biu hin cao ca protein, cung cp protein khng c
glycosyl ha. Cc loi Bacillus thch hp cho sn xut cc protein ngoi bo
khng c glycosyl ha. Cc loi Aspergillus cng sn xut rt nhiu cc
protein ngoi bo v hn na c th sn xut cc protein c glycosyl ha.
Mt s im cn lu khi xy dng cc vi khun v vi nm ti t hp
thch hp cho sn xut protein quy m ln. l:
- Chn la vt ch l vn then cht, trong vt ch phi c kh
nng sinh trng mnh, khng khuyt dng v khng c cc h thng
enzyme khng mong mun trong sn phm protein cui cng, chng hn
khng c hot tnh -lactamase trong vt ch c dng sn xut
penicillin G amidase.
- Cu trc vector biu hin phi cng n gin cng tt. V d: Hnh
2.13. (Chng 2) minh ha vector biu hin cho gen ngoi lai trong t bo
vt ch E. coli di s kim sot ca promoter T7 ca bacteriophage.
Sau khi tng hp, protein ti t hp c th hoc tch ly trong t bo
cht (cytoplasm) hoc c chuyn ti cht gian bo (periplasm). Sn xut
d tha protein ngoi lai ny trong cytoplasm mt i khi c th to ra cc
th vi (thng cha protein ti t hp trong trng thi khng cun xon,
hoc x l b hng). Kt qu ny thng khng c mong mun v n gy
nhiu kh khn tch chit v ti cun xon cc protein.
Qu trnh tch chit t cc th vi ch c gi tr kinh t khi sn xut
cc protein c gi tr cao nh insulin v hot t plasminogen m. Kt qu
thng c mong mun hn l protein c vn chuyn vo trong cht
gian bo. hin tng ny xut hin cn thit phi c cc chui tn hiu.
Nhng chui ny c nh v u tn cng amino ca protein (vng
ngc hng, upstream) v c th c chuyn i d dng nh cc
peptidase tn hiu. mt s trng hp nht nh, s tit protein vo mi
trng ngoi bo trong nui cy E. coli b hn ch. Trong trng hp sn
xut cc protein ngoi bo, th Bacillus spp., nm men v Aspergillus spp. l
cc vt ch thch hp c chn.
Cc plasmid phi hin din mt s lng bn sao nhiu trong vt ch
v, lm iu ny, cn phi c mt s p lc chn lc v mt t bo vt
ch khng c plasmid s pht trin nhanh hn vt ch c plasmid. Phng
thc thch hp nht chc chn c s bin np plasmid vo t bo vt ch
l thit k mt gen khng khng sinh trn plasmid v b sung khng sinh
tng ng trong mi trng sinh trng chn lc th bin np. Cc h
thng thng c s dng l kanamycin phosphotransferase, ampicillin -
lactamase, v chloramphenicol acetyltransferase. Nng khng sinh trn
30 mg/L duy tr s hin din ca plasmid. Ni chung, s biu hin ca
cc protein ti t hp trong nm (nh Asper. niger) i hi c s hp nht
ca gen v promoter ca nm cng vi mt c ch chn lc phi hp trc
tip (khng khng sinh hoc mt gen dng hoang di khc phc c nhn
t khuyt dng ca vt ch) vo trong DNA nhim sc th.
- Cc promoter c bn, nh phosphoglycerate kinase v
glyceraldehyde phosphate dehydrogenase c tc ng rt mnh v thng
c chn la. Cc promoter c chn vo ngc hng vi gen protein
mong mun ti u ha sn phm. Chng hn nh: promoter tinh bt c
th c dng nh l mt tn hiu thch hp cho sn xut -amylase, hoc
promoter phenoxyacetic acid c th c dng sn xut penicillin V
amidase. Thng thng cht cm ng hot ha promoter khng lin quan
n protein, v d: methanol c dng kch thch promoter alcohol
dehydrogenase trong Pichia, l loi c th c kt hp vi nhiu gen
protein khc nhau.
- Cht cm ng c chn phi c duy tr trn mt nng ti hn,
tuy nhin vic quyt nh thi gian b sung cht cm ng khng phi l vn
then cht.
- Pht sinh t bin im nh hng. Cc protein bit trnh t
amino acid v cu trc ba chiu c th c bin i bng t bin im
nh hng. V tr hot ng ca amino acid hoc cc vng quan trng khc
c coi l mc tiu thay i thnh cc amino acid khc. iu ny c
thc hin d dng bng cch thay i cc m b ba c trng trong gen m
ha protein v biu hin ca protein c phn tch bng phng php to
dng truyn thng. Cc protein mi d dng sn xut bng cch dng cc
phng thc sn xut ging nh c pht trin cho cc dng t nhin,
v thay i cc amino acid ny khng nh hng ln sinh trng ca vt ch
hoc s biu hin ca protein. Nhng thay i amino acid nh th c
tng ln mt cch n nh.

VI. Cc qu trnh tch chit v tinh sch protein
Qua nhiu nm, vic gia tng s dng vi sinh vt nh l mt ngun
cung cp protein, c bit l cc enzyme, ci thin ng k hiu qu sn
xut v sn phm c to ra nhiu hn. Phn ln enzyme s dng trong
cng nghip l cc protein ngoi bo t cc c th nh Aspergillus sp. v
Bacillus sp., bao gm: -amylase, -glucanase, cellulase, dextranase,
protease v glucoamylase. Nhiu loi trong s ny vn cn c sn xut t
cc chng gc t nhin ca vi sinh vt. Tuy nhin, trong sn xut protein
s dng cc lnh vc chn on lm sng v cho cc ng dng tr liu,
cng ngh protein v cng ngh DNA ti t hp th hin mt vai tr
ngy cng quan trng. Cng ngh DNA ti t hp, ngoi vic cho php ci
thin hiu sut cao, n cng cho php chuyn cc vt liu di truyn t ng
vt vo vi khun vt ch. Theo phng thc ny, cc protein vn ch c mt
lng nh t m ng vt by gi c th c sn xut trong mt lng gn
nh v hn t cc vi khun sinh trng d dng. Mt v d in hnh l
hormone sinh trng ngi, cht ny c sn xut mi ln vi mt lng
rt nh t tuyn yn ca ngi cho n khi n c tha nhn hin din mt
ri ro tim tng i vi bnh nhn t s nhim bn prion l yu t c m
ch trong hi chng Creutzfeld-Jacob. Hormone sinh trng ngi by gi
c sn xut vi mt lng ln hn rt nhiu t vi khun E. coli v n
hon ton sch khng b nhim prion khng mong mun.
Cc protein hoc enzyme c sn xut bng vi sinh vt c th ni
bo, khoang gian bo hoc tit vo mi trng nui cy. i vi cc
enzyme ngoi bo, mc tinh sch cn thit thng l ti thiu, khi sn
phm cui cng c dng trong cng nghip v khng cn tinh sch
cao. Cc qu trnh quy m ln nh th c th cho sn lng protein ln n
hng tn.
Nhiu loi protein khc c sn xut trong cc hn hp ni bo phc
tp gy ra nhiu kh khn trong qu trnh tinh sch chng. Nhng protein
i hi phi tinh sch ny thng c sn xut s dng trong iu tr,
do phi c gng hng ti cc tiu chun tinh sch rt cao, v c c
iu ny ngi ta cn phi pht trin cc quy trnh tinh sch phc tp. Mt
ln na, cng ngh DNA ti t hp c ng gp hiu qu trong lnh vc
ny. Trong trng hp cc protein tr liu, thng c nhng thun li nu
protein quan tm c th c tit hoc vo khong gian bo hoc vo trong
mi trng. iu ny gip gim rt ln nhim bn ca protein v cc i
phn t khc, sn phm tinh sch nh th thng thu c ch trong hai
hoc ba bc tinh sch. Chn la h thng biu hin thch hp c th cho
hiu qu cao hn trong h ln men, c lin quan ti vic ci thin hot tnh
c bit ca nguyn liu khi u. Cng c kh nng b sung cc nhm
chc vo protein gip cho s tinh sch, to ra cho n cc tnh cht c
bit v sau loi b cc nhm chc ny khi chng khng c yu cu lu
hn.
Trong thit k quy trnh tinh sch quy m ln th s lng cc bc,
v s thu hi sn phm mi bc nh hng quan trng ln sn lng
ton phn. S thu hi c trng ca mt bc sc k l trong khong 80%
v 90%, v s tinh sch phc tp i hi nhiu bc do sn lng ton
phn nhiu khi ch bng 10% ca nguyn liu khi u. iu ny khng
thnh vn trng hp tinh sch quy m phng th nghim, nhng nu
tinh sch quy m ln th l vn rt quan trng cn quan tm ti
u ton b qu trnh t h thng biu hin hoc s ln men n bc tinh
sch cui cng, gim thiu s bc tinh sch cn thit.

1. Thu hi protein
S thu hi v tinh sch cc protein v enzyme cng quan trng nh
cc giai on ln men xt theo gc kinh t ca qu trnh sn xut. Thch
thc chnh trong cc bc thu hi l gim thiu s mt hot tnh ca protein.
Trong phn ny s trnh by cc bc thu hi v tinh sch truyn thng cho
protein.

1.1. Thu hi cc protein ngoi bo
Cc protein ngoi bo tng i d thu hi v tinh sch. T bo v
nng ca dung dch hot ng c loi b mt cch n gin, v c th
cung cp trc tip protein th thch hp cho mt s ng dng.
Dung dch protein tng i sch c th thu c bng cch cho cc
nui cy sinh trng trn mi trng n gin c thnh phn xc nh. Cc
bc thu hi v tinh sch ging nh cc bc dng cho protein ni bo
sau khi ph v t bo.

1.2. Thu hi cc protein ni bo
tch chit cc protein ni bo t cc ngun ng-thc vt, m phi
c ph v gii phng chng.

1.2.1. Ph v t bo
a. Nguyn l chung
Lm kh m l phng php thun li n nh v ph v t bo
ng-thc vt. Phng php ng kh khng thch hp ph v m nhng
trnh c s t gy protein, mc d n v cng t trong sn xut quy
m ln. M c th c lm kh trong iu kin chn khng hoc trong
khng kh, hoc kt ta bng dung mi trn vi nc. Sau , thu dch chit
enzyme t cc nguyn liu kh bng cch hydrate ha tr li nguyn liu
trong mt dung dch m thch hp. Phng php ng lnh n gin cng
c tc dng ph v mt t m, mc d y l phng php khng thch hp
cho vic tng quy m sn xut.
Mt s nguyn liu ng-thc vt cn c qu trnh ng ha trc ,
sao cho m c ph thnh nhng mnh nh v trn ln vi nhau, sau s
dng phng php c hc ph v t bo. Mt khi protein c ha tan,
cc v t bo cht c loi b d dng bng phng php lc. Ly tm tc
thp cng c th c s dng. mt s m c s hin din ca cht
bo, vic loi b lp cht bo bng ly tm gp nhiu kh khn. Cc cht bo
ch c loi b d dng bng tch chit dung mi; kt ta acetone l
phng thc thch hp tch protein ra khi cc nguyn liu lipid. Mt
phng thc khc l cc dung mi trn nc nh hexane, cng c th c
s dng.
Ph v cc t bo vi sinh vt thng gp nhiu kh khn hn cc t
bo ng thc vt. Cc t bo vi sinh vt thng dai hn v c kch thc
nh (khong 0,2-10 m) nn phi c phng php ph v c bit. Nhiu
enzyme t nm men v cc vi khun Gram m c th c chit bng cch
dng cc dung mi khng trn nc, nh toluen hoc chloroform, c th
ph v mng t bo v gii phng enzyme. Cc dung mi ha tan nc, nh
ethanol v 2-propanol, c dng tch chit enzyme t khong gian bo,
nhng s dng cc dung mi ny i hi mc an ton cao trong sn xut
quy m ln. Cc cht ty thch hp c th c dng tch chit cc
phn t enzyme nh (khi lng phn t di 70.000 Da).
Trong mt s trng hp enzyme n nh gi tr pH cao, c th s
dng dung dch kim phn gii cc t bo vi khun. Thnh t bo vi
khun c phn gii bng lysozyme, tuy nhin gi thnh ca enzyme ny
l rt cao. Tng t, nm men c th c phn gii bng -glucanase. Hin
tng t phn gii c th xut hin trong nm men, nhng cn c thi gian
di v th s gy kh khn khi iu chnh hoc ti u qu trnh ln men.
Cc t bo vi sinh vt c th c ph v d dng hn bng cc
phng thc vt l. Siu m l k thut thch hp v rt hiu qu quy m
phng th nghim, nhng kh ng dng quy m sn xut ln. Ph v t
bo bng cch dng p sut cao y nguyn liu (dch huyn ph t bo
di dng bt nho c lm ng -20
o
C) qua cc l hp ca my nn th
t bo s b ph v do s thay i pha v thay i th tch cng nh do lc
ct ca cc tinh th . Phng php ny c th s dng ph v cc t
bo nui cy quy m ln 100-1.000 L/gi. Nhiu loi t bo c th b ph
v bng khuy (rung) nhanh hoc trn ln vi cc ht thy tinh nh hoc
cc ht gm (ceramic). quy m phng th nghim, phng thc ny rt
thch hp. quy m sn xut ln ngi ta s dng phng php nghin
bng qu cu thp. T l v hiu sut gii phng enzyme ph thuc vo vn
tc lc v kch thc ca cc loi ht cng nh ng knh ca thit b. Vi
cng mt th tch ht th s dng mt lng ln cc ht nh s hiu qu hn
mt lng tng i nh cc ht ln, v n lm tng s va chm gia cc
ht v cc t bo.
C ba phng php chnh gii phng cc protein ni bo khi vi
sinh vt l phng php enzyme, ha hc v vt l. Tuy nhin, khng
phi tt c cc k thut c sn l thch hp s dng trn quy m ln.
quy m ln, ngi ta thng gp kh khn trong vic thit k cng sut cn
thit cho th tch ln v loi b nhit c sinh ra trong qu trnh ph v t
bo.

b. Cc phng php enzyme
Lysozyme, mt enzyme c sn xut thng mi t lng trng trng
g, thy phn cc lin kt -1,4-glycosidic trong mucopeptide ca thnh t
bo vi khun. Cc vi khun Gram dng c thnh t bo rn chc nh vo
mucopeptide l loi mn cm vi lysozyme nht. Khi phn gii vi khun
Gram m, t khi ngi ta s dng mt mnh lysozyme, m thng b sung
thm EDTA to chelate vi cc ion kim loi s d dng lm tan t bo
(lysis). Mc d qu trnh thao tc n gin v nh nhng, nhng k thut
ny khng c s dng cho vic tch chit quy m ln cc enzyme ca
vi khun, v l do gi thnh tng i cao ca lysozyme v kh nng a
vo cc tc nhn gy nhim bn. Ch c trng hp ngi ta dng lysozyme
quy m ln l gii phng aryl acylamidase khi Pseudomonas
fluorescens.

c. Cc phng php ha hc phn gii t bo
- X l kim. X l bng kim c s dng thnh cng trong tch
chit quy m nh v ln cc protein vi khun. V d enzyme tr liu, L-
asparaginase, c th c gii phng khi Erwinia chrysanthemi bng cch
t bo pH t 11-12,5 trong 20 pht. Thnh cng ca phng php ny l
nh vo kh nng n nh ca sn phm mong mun. Gi tr pH cao c th
lm bt hot protease.
- Cht ty ra. Cc cht ty, hoc l ion v d nh sodium lauryl
sulphate hay cn gi l sodium dodecyl sulphate, sodium cholate (anion) v
cetyl trimethyl ammonium bromide (cation) hoc khng phi ion nh
Trixton X-100, X-450 v Tween, c dng phn gii t bo, thng c
phi hp vi lysozyme. Cc cht ty ion c hot tnh mnh hn cc cht ty
khng phi ion, v c th dn n s bin tnh ca nhiu protein. S hin
din ca cc cht ty cng c th nh hng n cc bc tinh sch tip
theo, c bit kt ta mui. iu ny c th c khc phc bng cch s
dng phng php sc k trao i ion hoc siu lc.

d. Cc phng php vt l phn gii t bo
- Shock thm thu. Shock thm thu cng c dng gii phng
enzyme v protein khi khong gian bo ca a s vi khun Gram m.
Phng php ny bao gm ra t bo trong dung dch m lm sch
chng khi mi trng dinh dng, v sau to dch huyn ph trong mi
trng u trng sucrose 20%. Sau khi t ti s cn bng thm thu, t
bo c thu hi v ti huyn ph nhanh trong nc khong 4
o
C. Trung
bnh ch khong 4-8% protein tng s ca vi khun c gii phng bi
shock thm thu, nhng nu enzyme mong mun hin din trong khoang
gian bo th c cho php tch chit mt lng ln hn t 14-20 ln v rt
sch so vi cc k thut tch chit khc.
K thut shock thm thu rt nh nhng v khng lm bin tnh cc
protein, nhng do c nhiu vi sinh vt chng chu c shock thm thu nn
ngi ta ch s dng n cho cc vi khun Gram m (v d nh E. coli)
tch chit cc enzyme thy phn c trong khoang gian bo. Tuy nhin, c ba
l do hn ch p dng shock thm thu quy m ln, l: th tch lm
vic ln (400 dm
3
cho 10 kg bt nho t bo), nhiu giai on ly tm, v
lun phi duy tr nhit thp.
- Nghin. Trc y k thut ny c nhiu hn ch khi nghin hn
hp bt nho ca t bo trong ci vi bt gy xt, nh l bng thy tinh,
alumina hoc t to ct (kieselguhr). Sau , ngi ta pht trin bng
cch s dng cc my nghin m. Mt sn phm c trng l Dynomill
(WA Bachofen, Switzerland) c th c dng gii phng protein khi
rt nhiu loi vi sinh vt khc nhau. N bao gm mt bung cha cc ht
thy tinh v cc a khuy quay trn. Dch huyn ph t bo c bm vo
bung, sau khuy nhanh ph v thm ch c cc t bo vi khun dai
nht. Bung phn hy phi c lm lnh loi b s sinh nhit. Mt m
hnh quy m phng th nghim, vi bung 600 mL c th thc hin ti 5 kg
vi khun trn mt gi, v cc m hnh quy m sn xut thch hp vi
bung c th tch ln ti 250 L.
Mt s nhn t c th nh hng n t l t bo b ph v, nh l
kch thc v nng ca cc ht thy tinh, loi, nng v tui ca t
bo, tin x l ha cht, tc khuy, tc dng chy qua bung, nhit
, v s sp xp ca cc a khuy, v nhng yu t ny c kho st
nm men v vi khun.
- Trt rn. Phng php ph v t bo bng trt rn c dng
kh lu quy m nh. Nguyn tc ca phng php ny l y nguyn liu
t bo ng lnh qua mt l hp p sut cao v mt nhit thot ra
ngoi khong -20
o
C. Phng php ny t c s dng quy m cng
nghip, do n khng th dng mt lng ln nguyn liu ph v t bo.
- Trt lng. Trt lng l k thut c chn la ph v cc t
bo vi sinh vt quy m ln, c ng dng rng ri trn th gii trong c
hai: cc qu trnh cng nghip v nghin cu. Phng php ny c bit
thun li cho ph v t bo vi khun v nm men.
Tng t nh phng php trt rn, cc t bo trong dch huyn ph
c chuyn qua mt l hp nn di p sut cao. Trng hp quy m
nh hn, ngi ta s dng thit b French Press (Hnh 6.7). quy m ln
thng dng thit b ng ha (homogenizer) l loi c pht trin to
th sa trong cng nghip b sa. Nhit tng ln t nht 10
o
C trong mt
rnh n l thng xy ra, v th cn phi lm lnh dch huyn ph t bo
trc khi ng ha. Thit b ng ha trt lng thng c hot ng
m t bo khong 20%.
Trong trng hp quy m ln th thit b ng ha Manton-Gaulin
(APV Ltd. Crawley, UK) c s dng rng ri nht. N bao gm mt my
bm kiu piston c mt van thot hi c hn ch, c th iu chnh p
sut hot ng cn thit, ti 95 MPa. Thit b ng ha Manton-Gaulin
(Hnh 6.8) loi nh nht, 15-8TA, c th a nguyn liu vo khong
50 L/gi p sut 55 MPa. Mt phin bn ln hn, loi MC-4, c th a
nguyn liu vo 300 L/gi cng p sut 55 MPa.

Hnh 6.7. Thit b ng ha
French Press
Hnh 6.8. Mt ct ngang ca thit b
ng ha Manto-Gaulin

Tc ph v t bo v gii phng protein ph thuc vo mt s nhn
t nh: loi t bo, iu kin ln men, nng v tin x l (chng hn
ng lnh, v cc t bo vi sinh vt thng d v hn nhiu nu chng c
lm lnh trc)... Ngi ta cng nhn thy rng s hin din ca cc th vi
gip cho cc t bo E. coli d dng b ph v hn.
T l protein gii phng khi cc t bo nm men c th c m t
bng mt phng trnh c xy dng da trn kinh nghim nh sau:
KnP
R R
R
m
m
Log

(1)
Trong :
m
R l lng protein ha tan cc i c gii phng theo l
thuyt, R l lng protein c gii phng thc t, K l hng s ph thuc
nhit , n l s rnh (number of passes), P l p sut ngc hot ng v
l hng s ph thuc vo c th.
Gi tr ca s m khc nhau ty thuc vo c th, i vi nm men
n khong 2,9 v E. coli khong 2,0. C nhiu v d v vic s dng thit b
ng nht Manton-Gaulin ph v t bo vi sinh vt quy m ln. Chng
hn: -galactosidase c gii phng khi E. coli, v carboxypeptidase khi
Pseudomonas spp. Mt s ln enzyme c phn lp t vi khun a nhit
Van
Van iu chnh
kch thc khe
Van iu chnh

Bacillus stearothermophilus, bao gm glycerokinase v mt hexokinase c
trng glucose. Trong mt qu trnh nht nh, cc iu kin ph v t bo
phi c thit k ti u mt cch cn thn, v cc bin thin khc nhau
trong mc ph v t bo v gii phng protein c th c cc nh hng
mt cch ngha ln cc bc tinh sch tip theo.

1.2.2. Phn lp cc enzyme ha tan
Phng thc quan trng phn tch enzyme ha tan nhanh v hiu
qu sau khi ph v t bo l lm lnh, dng dung dch m thch hp, v c
s hin din ca cc tc nhn bo v enzyme nh mercapthoethanol (cn
thit n nh mt s dung dch enzyme).
Thng cc nhn t c ch protein phi c b sung lm gim
nh hng phn hy ca protease. Cc enzyme ha tan c th c thu thp
bng phng php lc qua mng hoc bng cch ly tm. Phng php sau
tng i d thc hin quy m phng th nghim vi cc lc ly tm tc
cao. Cc lc g cao nh th khng th t c cc thit b quy m sn
xut ln. Cc my ly tm ln thng c cu to t cc hp kim titanium
t tin chu ng cc lc g cao, nhng mc d th ch c th thu c
cc lc g khng cao lm m thi. khc phc iu ny, cc cht kt ta
nucleoprotein v protein, nh polyethyleneimine, c b sung vo cc cht
ng ha t bo kt bng cc nguyn liu khng mong mun v gim
thi gian lng xung nhanh hn.
S dng phng php lc nh dng t diatomit (diatomaceous earth),
c th l bin php thch hp thu c cc enzyme ha tan v pht trin
d dng quy m ln. Cc phng thc b sung c cng s dng tng
tc lc. Thng thng mt vi tc nhn kt ta c th c cng s dng
gim cc bc tip theo ca qu trnh. Cc phng tin tr lc c th
c dng thu thp cc t bo hon chnh v cung cp mt k thut ph
v t bo m khng da vo cc thit b ng ha c hc. V d cc t bo
trong hn hp lc c th c phn gii ha hc, enzyme hoc phng thc
vt l bng cch khuy nh kh nng gy xc t bo ca diatomit. Cc
enzyme ha tan c gii phng c th thu thp thun li bng phng
php lc n gin.
Siu lc l cng ngh rt ton din, d dng cho quy m ln v, vi s
chn la chnh xc xp ca mng, cc enzyme c th c thu thp mt
cch chn lc theo khi lng phn t ca chng. a v khung, v cc si
rng (hollow fibres) c s dng trong mt thi gian di. Cc mng
ceramic hin nay c s dng ph bin hn do c im d lm sch v
d kh trng ca chng, v chng c th chu c nhit cao di cc
iu kin cht ty v kim cao.
Thng th dung dch enzyme ha tan khng yu cu x l thm na
ngoi tr nng , hoc di p sut gim hoc bng phng php lc
mng, sn xut mt dung dch protein c c c (10-50% cht rn).
Cc cht n nh nh ammonium sulphate c th c b sung nu cn
thit. Cc t dc nh lactose, dextrin cng c th c b sung vi vai tr
l cc cht n nh enzyme.

2. Tinh sch s b
Tinh sch protein l mt bc rt cn thit, tuy nhin thng ch thc
hin i vi nhng protein c gi tr ng dng cao. Quy m ca qu trnh
tinh sch s quyt nh s chn la k thut phn tch, v mt s k thut
gp nhiu kh khn khi tin hnh trn quy m ln.

2.1. Loi b cc mnh v ca t bo
Sau khi ph v t bo, bc u tin trong qu trnh tinh sch protein
ni bo l loi b cc mnh v t bo. S phn tch cc vt rn khi cht
lng l hot ng c bn rt quan trng trong phn lp protein, v thng
c tin hnh bng phng php ly tm hoc lc. Nhiu quy trnh c b
sung mt lng nh DNase giai on ny, ph v thm cc chui DNA
c th lm cho dch chit tr thnh dng st (gelatinous).

2.2. Ly tm m
Ly tm m thch hp vi cc dung tch ly tm trong khong t nh
hn 1 mL n mt vi lt, v c kh nng s dng mt lc ly tm (rotational
centrifugal force, RCF) tng i ln ln ti 100.000 g (hng s hp dn).
Tuy nhin, loi b cc t bo vi khun, mnh v t bo v cc kt ta
protein, th lc ly tm ch cn t khong 20.000 g. Nhiu thit b ly tm
loi ny ph hp cho cc qu trnh tch chit quy m trung bnh.

2.3. Ly tm dng chy lin tc
Ly tm dng chy lin tc thng c s dng cho qu trnh tinh
sch protein quy m ln loi b cc cht dng ht. C ba kiu ly tm
chnh thch hp hn c l: ly tm thng rng (hollow bowl), ly tm thng c
nhiu bung (multi-chamber) hoc a (dics), v ly tm thng (basket).
- Ly tm thng rng. Bao gm mt rotor hnh ng cung cp mt
ng chy di cho dch chit c bm vo trong y v chy ln qua
thng. Cht lng b bn vo thnh ca thng, v dch chit c gn s
chuyn ln ra khi thng vo trong bnh thu. Khi ly tm bt u, ng
knh thc t ca thng gim, v th lm gim ng lng v lc ly tm.
Tc dng chy phi c xc nh theo kinh nghim v n rt khc nhau
gia cc loi dch chit, nhng tc dng chy thch hp cho cc my ln
l khong 60 L/gi.
- Ly tm a. Thng ly tm cha mt dy a xung quanh mt hnh
nn gia. Khi dch chit i vo, cht ht b bn ra ngoi, chm vo cc a
hnh nn v cht lng tp trung trn thnh ca thng. Phng php ny cung
cp mt ng chy khng i, v th hiu sut ly tm t b nh hng.
Nhc im ca kiu ly tm ny l c hao ht mt lng nh sn phm
trong sut qu trnh chy ra ngoi ca dch chit. Phng thc ny cho php
t ti mt lc ly tm khong 8.000 g v c sc cha ln n 20 kg cht
lng.
- Ly tm thng. c thit k hot ng cc lc ly tm thp hn
nhiu, c th ch 1.000 rpm, v c bn l cc b lc ly tm. Thng c
c thng l v thng c lt bng vi lc. ng dng chnh ca ly tm
ny l thu thp cc ht nguyn liu ln; trong phm vi tinh sch enzyme
thng l cc nguyn liu trao i ion c dng cho hp ph theo m
protein mong mun.

2.4. Lc bng mng
Lc l mt phng php khc gn cc dch chit t bo. Tuy nhin,
dch nui cy vi sinh vt v cc dch chit c khuynh hng tr thnh dng
st t nhin thng gp kh khn khi lc bng cc phng php truyn
thng, tr khi din tch mng lc c s dng l rt ln.
C th khc phc iu ny bng cch dng phng php lc dng
chy ngang (cross-flow) hoc tip tuyn (tangential). Trong phng php
ny dch chit chy gc phi theo hng lc, v s dng tc dng chy
cao s c khuynh hng gim s tc nghn bng cc hot ng t lm sch.
Chng hn: thu hi quy m ln L-asparaginase t Erwinia
chrysanthemi ngi ta s dng mt mng lc c din tch 1 m
2
dng thu
hoch t bo t 100 L ca cht lng nui cy trong 2,5 gi lc nng
cc cht rn phn c gi li trn mng tng ln t 0,55%-22% khi
lng kh. Sau , mt mng ging nh th c dng gn lc dch chit
thu c bng phn gii kim cc vi khun ny. Cc s liu ny cho thy
rng thu hoch t bo t 500 L nui cy trong 2,5 gi i hi mng phi
c din tch 7,5 m
2
v chi ph cho qu trnh ny t hn so vi phng php
ly tm.
Do cc hn ch ca ly tm quy m ln nn k thut lc mng thng
c phi hp s dng m bo rng dch chit tht s sch cho bc
sc k tip theo.

3. H phn tch hai pha nc
Mt phng php khc vi ly tm v lc l phng php phn tch
hai pha nc (aqueous two-phase separation), hay cht lng-cht lng. Cc
h hai pha nc c trng c to ra bng cch trn cc dung dch
polyethylene glycol (PEG) v dextran hoc PEG v cc loi mui nh
potassium phosphate hoc ammonium sulphate to thnh hai pha ring
bit. Cc protein v mnh v t bo c kh nng ha tan khc nhau gia hai
pha, v th k thut ny c th c dng cho c hai trng hp: phn tch
protein khi mnh v t bo v phn chia (partitioning) protein trong sut
qu trnh tinh sch. S phn chia chnh xc ca mt protein ty thuc vo
cc thng s nh khi lng phn t v in tch ca chng, nng v
khi lng phn t ca cc polymer, nhit , pH v lc ion ca hn hp v
s hin din ca cc mui a tr nh phosphate hoc sulphate. Cc iu kin
ti u cho mt protein c bit thng c tm thy theo kinh nghim.
Mc d, cc iu kin cn thit t c s phn tch va c th c
xc nh chnh xc, nhng c ch ca s phn chia hai pha hin nay vn
cha c hiu y .
Cc pha c th c phn tch trong mt thng lng, nhng phn
tch nhanh v hiu qu hn c th s dng phi hp vi phng php ly
tm. V phn tch cc cht lng c mt khc nhau d dng hn phn tch
cc cht rn ra khi cc cht lng trn quy m ln, nn phng thc ny c
th c dng h tr cho qu trnh tinh sch enzyme quy m ln. Mc
d gi thnh kh thp ca h thng PEG/mui khin cho n tr nn thch
hp hn cho vic s dng quy m ln, nhng h thng PEG/dextran
(dextran c gi thnh kh cao) phn tch hiu qu hn cng l mt phng
php kinh t nu so snh vi cc phng php tinh sch khc. S dng h
phn tch hai pha nc khng b hn ch i vi cc nguyn liu c ngun
gc vi sinh vt, v phng php ny c dng thnh cng phn lp cc
nguyn liu khi c hai ngun thc vt v ng vt, bao gm c -L-
antitrypsin ca ngi c biu hin trong sa chuyn gen. Trong trng
hp ny tinh sch s b ca protein tng i cao, tc l sau khi phn
tch hai pha n th protein mong mun c tinh sch ti 73%.
S phn tch hai pha nc bng polyethylene glycol, dextran hoc
polyamines c th to ra cc pha phn chia cht lng trong cc protein v
enzyme mong mun c th c tp trung li. X l kiu ny cho php thu
hi li cc polymers s dng. Cc phng php ny c ng dng d
dng i vi ton b dch nui cy c protein hoc enzyme ha tan (enzyme
ngoi bo).
S phn tch hai pha nc c th c ci tin nhm to s phn chia
c trng sinh hc bng cch gn cc phi t vo polymer thay i s
phn chia ca protein. Tt c cc polymer to pha c th c cc phi t c
gn ng ha tr vo chng, v mt phm vi nhiu phi t nh th c
kho st. Do lin kt ha hc n gin ca chng, cc cht nhum phn ng
c s dng thng xuyn nh cc phi t. Ngoi cc thuc nhum ny,
mt s phi t khc cng c nghin cu. Nhng phi t ny bao gm
cc cofactor, nh pyridine nucleotide c s dng thnh cng trong phn
chia i lc cc dehydrogenase.
quy m ln, phng php i lc c s dng cho tinh sch
formate dehydrogenase khi 10 kg nm men Candida bodinii, bng cch
dng thuc nhum triazine, Procion Red HE-3B, c bt ng trn PEG.
Mc d phn tch hai pha nc l phng php c th pht trin d
dng quy m ln sn xut, nhng dng nh n khng c dng
thng xuyn tinh sch quy m cng nghip.
4. Cc phng php kt ta
Thng thng cn tin hnh giai on kt ta m u tin lm gim
th tch tng s ca dung dch enzyme. Cc enzyme c th c kt ta n
gin, tun t hoc phi hp vi ammonium sulphate, sodium sulphate,
polyethyleneimine v polyallylamine, hoc vi cc dung mi hu c nh l
isopropanol, ethanol v acetone.
Streptomycin sulphate, polyethyleneimine v cc polyamine khc c
th kt ta cc cht c tnh cht acid nh nucleic acid v cc nucleoprotein.
Ammonium sulphate v cc dung mi hu c c th kt ta mt cch chn
lc enzyme mong mun vi hot tnh thu hi t 80-90%.
Kt ta n gin cng c th gip loi b protease. Li ch chnh ca
bc ny l lm gim th tch hot ng tng s, thng bng mt tha s
ca 20. Do s gim mt cch ngha th tch ca nha trao i ion. Thay
i pH v nhit cng c th to ra kt ta chn lc v loi b cc protein
khng mong mun.

4.1. Kt ta bng ammonium
Phng php x l mui cc protein c s dng trong nhiu
nm, v t c c hai mc ch tinh sch v c c. Loi mui c s
dng ph bin nht l ammonium sulphate, do kh nng ha tan ca n,
khng c c tnh cho hu ht cc enzyme, v gi thnh thp.
Kt ta protein bng mui ph thuc vo nhiu nhn t nh: pH, nhit
, nng protein, v loi mui c s dng. Nng protein l thng
s c bit quan trng khi tng quy m, bi v hu ht s tinh sch quy m
ln c tin hnh cc nng protein cao hn s tinh sch quy m
phng th nghim. iu ny c th nh hng su sc ln nng ca mui
cn thit kt ta protein mong mun.

4.2. Kt ta bng cc dung mi hu c
Vic b sung cc dung mi hu c vo cc dung dch nc lm gim
kh nng ha tan ca protein do gim hng s in mi ca mi trng. Cc
dung mi hu c khc nhau c dng kt ta protein nh ethanol,
acetone, v 2-propanol l quan trng nht. Thng cc protein b bin tnh
bi cc dung mi hu c nn cn thit lm vic nhit di 0
o
C.
Do bn cht d chy ca cc dung mi hu c nn cn phi s dng
thit b chu la v gi thnh cao, v th cc dung mi hu c thng khng
c dng trong tinh sch enzyme quy m ln. Ngoi tr trng hp x
l mu (blood processing field), trong kt ta ethanol l phng php
chnh tinh sch albumin; v trn thc t phng php ny ang c pht
trin trong mt h thng c iu khin t ng bng computer.

4.3. Kt ta bng cc polymer khi lng phn t cao
Cc cht kt ta hu c khc c th c dng phn on cc
protein l cc polymer ha tan trong nc nh PEG. Cht ny c u im l
khng c c tnh, khng d chy v khng gy bin tnh cc protein. N
c dng ch yu trong lnh vc x l mu.

4.4. Kt ta bng nhit
Khi protein bn kh b bin tnh, th x l nhit c th cung cp
mt mc tinh sch cao c xem nh l bc u tin. V d: quy m
ln, 55% protein khng mong mun c loi b trong mt bc ring r
bng cch lm nng dch chit E. coli mang protein A ca Staphylococcus
ti t hp 80
o
C trong 10 pht. Nu protein ti t hp c tinh sch khi
mt c th a nhit th kt qu ca x l nhit u tin c th l hiu qu
hn nhiu. V d: Khi dch chit E. coli cha malate dehydrogenase ti t
hp ca Thermus aquaticus c lm nng ti 80
o
C trong 20 pht, th
enzyme thun nht c trong th ni l khong 90%.

5. Cc phng php sc k
Thut ng sc k ch cc k thut phn tch v iu ch cho php
tch bit cc hp phn khc nhau ca mt hn hp. Php phn tch sc k
da vo s di chuyn khc nhau trong mt pha ng ca cc cht ha tan
c gn trn mt pha tnh trng thi rn. Ngi ta thng chn cc cht
c kh nng gn kt c vi cc cht (ha tan) nh phn tch lm pha
tnh. Tng tc gia cht ha tan v pha tnh c th l tng tc hp ph,
tng tc ion (trao i ion), tng tc k nc, tng tc kiu ry phn t
hoc tng tc c hiu sinh hc.
Tinh sch protein bng phng php sc k l mt thc hnh chun
phng th nghim trong nhiu nm. tinh sch cc sn phm c th tch
nh v gi tr cao, cc protein tr liu hoc chn on c hiu, th phng
php sc k l phng php c s dng rng ri nht. Sc k ch l
phng php, vi kh nng chn lc c yu cu, tinh sch mt protein
ring r khi hn hp phc tp ca cc protein ti mt s tinh sch cui
cng ln hn 95%.
Cc k thut phn tch bng sc k c s dng ph bin, bao gm
trao i ion, tng tc k nc, ngn chn kch thc, i lc v i lc gi
(sc k vi phi t l thuc nhum).
Bc lm sch thm c th c thc hin bng cch dng sc k
ngn chn kch thc. Tuy nhin, k thut ny kh thc hin quy m ln,
v thng khng cn thit phi tinh sch enzyme ti 99%. Nu cn mc
tinh sch ln hn 95% c th tin hnh sc k hp ph/kh hp ph thm
mt ln na s gip thu c enzyme tinh sch hn. Cc k thut tinh sch
nh th khng lm tng hot tnh ca enzyme, y l mt yu t phi c
xem xt khi kt ta enzyme c tinh khit cao.
Cc kt ta enzyme tinh sch cao thng c ng kh bo qun
v vn chuyn. Cc tc nhn bo v lnh ng polyhydroxy
(cryoprotectants), cc dung dch m, cc cht kh v cc cht khng khun
c th c b sung nu cn thit.

5.1. Sc k lc gel
Trong sc k lc gel, s phn tch protein c da trn c s kch
thc phn t (Hnh 6.9). Pha tnh cha cc ht gel (polymer) xp c nhng
l nh li ti c bao quanh bi pha dung mi chuyn ng. Khi dung dch
cha cc protein chy qua ct th cc phn t ln ca hn hp c ng
knh ln hn l ca ht gel nn khng th khuch tn vo bn trong ht (qua
cc l nh) v v th chng i qua cc k h ca ct v b ra khi ct trc
cng vi th tch dch ra bng th tch gia cc ht gel V
o
. Cc phn t nh
hn s i qua cc l nh khuch tn vo trong cc ht gel v c ra
khi ct sau, th tch dch ra ca chng bng tng th tch (pha dung mi)
ca ct V
t
. Tng th tch ca ct c th biu din nh sau:
m i o t
V V V V
(2)
Trong : V
t
l tng th tch ca ct, V
o
l th tch ca dung mi bn
ngoi cc ht, V
i
l th tch ca dung mi bn trong cc ht, v V
m
l th
tch b chim bi khun.
Th tch ra ca protein v vy c th thay i gia V
o
v V
i
, v c th
tnh ton h s phn chia hiu qu (K
av
) c gi tr gia 0 v 1:
o t
o e
av
V V
V V
K
(3)
Trong : V
e
l th tch ra ca cht ha tan.
i vi cc protein hnh cu, kinh nghim cho thy gi tr ca K
av
t
l nghch vi logarithm ca khi lng phn t tng i.

Hnh 6.9. S ca sc k lc gel

Cc ht polymer
xp c l nh
Hn hp protein c b
sung vo ct cha cc
polymer lin kt cho
Cc phn t protein phn tch theo kch thc,
cc phn t ln hn i qua ct t do hn v xut
hin trong cc phn on u tin
1 2 3 4 5 6
Cn lu khng c s tng tc gia khun v cht ha tan, v th
mi trng lc gel l tng phi tr hon ton. c sc cha cc i ht
gel phi rn v c xp cao. Trng hp quy m ln, rn c l l
thng s quan trng nht v n quyt nh tc dng chy cao nht c th
t c.
Cc nguyn liu lc gel truyn thng da trn c s dextran lin kt
cho (Sephadex), hoc polyacrylamide (BioGel P). Nhng nguyn liu ny
l tr nhng ( cc xp thch hp khc nhau phn on hu ht cc
protein) li qu mm nn kh s dng trn quy m ln.
Cc loi gel rn hn da trn c s thay i nhng vt liu khc nhau
c a vo s dng, v thch hp hn cho ng dng trn quy m ln.
V d: Sephacryl (Amersham Pharmacia Biotech) trn c s dextran v
polyacrylamide; Superdex (Amersham Pharmacia Biotech) trn c s
dextran v agarose, Superose (Amersahm Pharmacia Biotech) trn c s
agarose lin kt cho cao. Tt c nhng nguyn liu ny thch hp cho cc
kch thc ca ht sao cho s phn on c duy tr cc tc dng
chy cao nh rn c tng ln ca ht.

5.2. Sc k trao i ion
Sc k trao i ion c s dng rng ri nht, c th bao gm c trao
i cation v anion mnh v yu. Cc protein v enzyme ni chung c
hp ph lc ion thp hoc cc gi tr pH trong in tch ion tng s
mnh tng tc vi in tch i ca nha trao i ion. S tch ra
protein c thc hin d dng bng cch thay i pH hoc tng lc ion ca
dung dch ra (Hnh 6.10). Nhng nha trao i ion nh th c th c
dng hoc nh mt phng thc hp ph tch cc v tch ra chn lc
enzyme mong mun, hoc nh vo s hp ph n gin ca cc nguyn liu
protein khng mong mun.
Nha trao i ion l mt khung vt liu rn khng ha tan c gn vi
cc nhm ion ha bng lin kt ng ha tr. Cc nhm mang in tch ny
li c lin kt vi cc ion i (opposite ions) v cc ion i li c th trao
i thun nghch vi cc ion tri du. Mt khung c mang cc nhm tch
in dng v ion i tch in m, c gi l nha trao i anion (anion
exchange resin). Ngc li, mt nha trao i cation s mang in tch m.
Ngoi ra, cc nha trao i thng khc nhau v bn cht ha hc ca
gi khung (polysaccharide gel hay nha tng hp) cng nh v lc acid,
base ca nhm ion ha. Ba loi nha trao i ion c trnh by trong bng
6.1.

Hnh 6.10. S sc k trao i ion

Cc protein ca mt hn hp cn phn tch thng c cc nhm bn
ion ha khc nhau, do c pH khc nhau. mt gi tr pH nht nh cc
protein s c mt in tch khng ging nhau, do chng c gi nhiu
hay t bng tng tc ion trn mt nha trao i ion cho v vi mt pha
di ng cho.
Trong thc t ngi ta thng dng cc dung dch m phosphate,
acetate, borate v citrate chit protein ra khi ct. Cc protein khc nhau
s c chit ra khi ct theo tng phn on chit khc nhau, trong
phn on protein cn thu c nng cao nht.
Cc ht polymer mang cc
nhm chc tch in m
Hn hp protein c b sung vo
ct mang cc cht trao i cation
Cc phn t protein chy qua ct vi cc tc xc
nh bi in tch thc ca chng pH c s
dng. Trng hp cc cht trao i cation, cc
protein c in tch m thc ln hn s chy qua ct
nhanh hn v c dung ly trc.
in tch dng thc ln
in tch dng thc

in tch m thc

in tch m thc ln

1 2 3 4 5 6

Bng 6.1. Bn cht ha hc ca cc loi nha trao i ion

Nha trao
i
Bn cht ha hc ca
gi khung
Nhm ion ha Kh nng
trao i
DEAE-
Sephadex
Cc mch dextran c
lin kt cho

Cht trao i
anion yu
Ambelit E-
IR120
Polystyren c lin kt
cho bng divinylbenzen

Sulfonate
Cht trao i
cation mnh
CM-
Sepharose
Agarose c lin kt
cho bng 2,3-
dipromopropanol

Carboxylmethyl
Cht trao i
cation yu

Mt s nha trao i ion t cc dn xut ca cellulose ang c s
dng nh sau:

Nha trao i cation Nha trao i anion
- Carboxyl methyl cellulose
(CM-Cellulose)
- Phosphor cellulose
- Sulfo ethyl cellulose
- Sulfo methyl cellulose
- Diethylaminoethyl cellulose
(DEAE-Cellulose)
- Triethylaminoethyl cellulose

Qu trnh phn tch trn ct bao gm hai giai on:
- Hp ph thun nghch protein cn tinh sch (v cc protein c in
tch gn ging) vo nha trao i ion.
- Kh hp ph cc protein bng cch: (1) thay i pH ca dch ra s
dn n thay i ion ha v do thay i in tch tng ca protein,

-OCH
2
CH
2
NH
+

C
2
H
5
C
2
H
5
-CH
2
COO
-
-SO
3
-
hoc (2) tng lc ion v tng nng ion i cnh tranh. Cc protein no c
i lc vi nha trao i ion yu nht s b y ra trc tin v ngc li.
Khi s dng gradient lc ion hoc/v gradient pH thng lm tng cht
lng ca php phn tch protein.
Vi cc enzyme c tnh acid, ngi ta thng s dng cc anionic
cellulose tch v tinh sch. Cc nha i t cc dn xut ca cellulose c
kch thc l ln, s dng rt thch hp tch, tinh sch enzyme quy m
cng nghip. Tuy nhin, do h s nn cao nn phn no gy kh khn khi
tin hnh sc k. khc phc nhc im trn, ngi ta s dng cc dn
xut ca agarose lin kt cho nh: Sepharose CL-6B hoc polymer tng
hp: trisacryl c hiu sut cao, h s nn khng ng k, khng thay i th
tch theo pH v cng ion, c th ti to li nha m khng cn tch khi
ct.
Trong sc k trao i ion, vic gn mt protein vo nha trao i ion
s ph thuc vo trng thi ion ha ca n, cng nh trng thi ion ha ca
nha trao i, pH, lc ion v nhit . i vi nhng protein khng b bin
tnh pH cao hn im ng in ca chng th c th dng DEAE-
cellulose (nha trao i anion) cn i vi cc protein khng b bin tnh
pH thp hn im ng in ca chng th c th s dng CM-cellulose
(nha trao i cation).

5.3. Sc k i lc (affinity chromatography)
Phng php ny da vo kh nng lin kt c hiu v thun nghch
ca mt protein vi mt phn t khc (phi t), c gn bng lin kt
ng ha tr vo mt cht mang khng ha tan cha trong ct sc k. Khi
cho mt hn hp c cha protein cn lm sch i qua th ch c protein quan
tm b gi li, cn tt c cc protein khc khng tng tc c vi phi t
(ligand) s b ra tri ra khi ct (Hnh 6.11). Tip , protein s b ra gii
ra bng cc phng php khc nhau.
Phng php sc k i lc rt hiu qu trong vic tinh sch cc
enzyme, cho php thu c enzyme c sch cao (hn sc k trao i ion
khong 10 ln) ch bng mt giai on v trong mt thi gian ngn.
Cc nhn t nh cht mang, phi t, phng php gn kt phi t
cng nh cc iu kin ra gii enzyme u c vai tr quan trng trong sc
k i lc.

Hnh 6.11. S sc k i lc

5.3.1. Cht mang (pha tnh)
Phi c mt s tnh cht sau:
- Hon ton khng ha tan trong pha di ng.
- C bn v ha hc v sinh hc.
- C cng c hc, c tnh ho nc v tnh thm.
- Khng c cc tng tc phi c hiu.
Hn hp protein
Hn hp protein
c b sung vo
ct cha mt
polymer lin kt vi
phi t c hiu cho
protein quan tm
Cc protein
khng mong
mun c
ra tri qua
ct
Cc protein
quan tm c
dung ly bng
cch ha tan
phi t
Ha tan
phi t
Protein quan tm
Phi t
Phi t c gn
vi ht polymer
- C cha nhiu nhm chc c kh nng bin i khi hot ha trong
cc iu kin nh nhng.
Cc cht mang thng l dn xut ca cellulose, cc dextran gel, thy
tinh xp... Tuy nhin, ngi ta hay dng agarose hoc cc gel hn hp
agarose v polyacrylamide v chng c thm kh nng lc gel.

5.3.2. Phi t
Phi t thng l nhng cht tng t c cht ca enzyme mun tinh
sch, cht km hm hoc cofactor. Phi t ni chung phi c hiu vi
protein v phi c mt i lc trung bnh vi n. Nng phi t cng phi
c chn thch hp v nu tha phi t s gy ra nhng n ng khng gian
ng k. Trng hp khi phi t l mt phn t nh hoc khi phn t
enzyme qu ln c th gy ra s cng knh khng gian th phi t s c
ni di thm bng mt on cnh tay n n d tip cn vi tm hot
ng ca enzyme. Cnh tay n thng l mt mch carbohydrate nh chc
di t 6-8 carbon.

5.3.3. Hot ha cht mang
Phi t (v cnh tay n) phi c gn ln cht mang. Cht mang
trc tin phi c hot ha vi cyanogen bromide (CNBr). CNBr s phn
ng vi cc nhm hydroxyl ca agarose, chng hn to ra mt
imidocarbamate v imidocarbamate d dng tc dng vi mt amine bc
nht ca cnh tay n.

5.3.4. Ra gii
Sau khi loi b cc protein khc, enzyme cn tinh sch c th c
ra gii ra khi ct sc k nh phc hp ca n vi phi t c bn cht phi
ng ha tr v thun nghch. Dung dch ra gii thng phi c: (1) hoc
c cha mt phi t t do ca enzyme c kh nng cnh tranh vi phi t
ang trng thi lin kt vi enzyme, (2) hoc do pH ca mnh c th gy
bin tnh thun nghch enzyme do lm bin dng tm hot ng ca
enzyme. C th lm yu tng tc c hiu sinh hc bng cch thay i lc
ion. Phi t cnh tranh (hoc dung dch m lm bin tnh) sau c loi
b bng phng php thm tch.
5.4. Sc k tng tc k nc
Sc k tng tc k nc s dng tnh cht k nc ca protein b
mt nh l mt c im chn lc. Loi phn tch ny thch hp khi
c tin hnh tip theo bc kt ta ammonium sulphate, v khng cn
thit phi loi b mui trc khi thc hin bc sc k ny. Bng cch dng
cc k thut hp ph v kh hp ph b mt s lm gim mnh th tch ca
cc dung dch protein v lng protein cn tinh sch s tng ln t 90-95%.
Cht lng ny cho hu ht cc ng dng ca protein c bit.
Cc protein s ln lt c phn tch ra ty theo tng tc ca chng
vi mt cht mang c cha cc nhm k nc (a bo). Cc protein cha
cc nhm k nc trn b mt, cht mang k nc v dung mi a nc to
thnh mt h ba thnh phn v tng tc c vi nhau. H ny c th b
ri lon khi thay i nhit , pH hoc lc ion.
Ni chung, cc tng tc s mnh nu ta tng lc ion (v d: dung
dch NaCl 4 M). Cc protein b gi li, tip c th c ra gii mt cch
chn lc bng cch gim lc ion ny hoc bng cch gim phn cc ca
dung mi ra (thm ethylene glycol hoc mt cht ty ra hoc tng pH
dch ra). C th gn cc nhm khc nhau ln cht mang. V d
octylsepharose (R) CL-4B v phenylsepharose CL-4b, cc nhm octyl v
phenyl c nh ln cc n v monosaccharide ca agarose bng lin
kt ester khng tch in v bn ha hc. nhng cht mang khc th c th
s dng nhng nhm k nc khc.

5.5. Cc k thut sc k lng hiu sut cao (high performance liquid
chromatographic techniques)-HPLC
Cn c gi l sc k lng cao p (high pressure) hay sc k lng gi
thnh cao (high price). y l phng php phn tch cc cht bng cch
dng p sut y nhanh dung dch qua ct sc k vi mt hiu sut cao.
u tin, k thut ny c thit k phn tch cc phn t hu c nh
ha tan trong cc dung mi khng phi nc, sau k thut ny nhanh
chng pht trin thnh mt phng thc thch hp phn tch cc protein
trong cc dung mi nc. Tuy nhin, a s cc ng dng c cng b
vi k thut sc k ny u ch gii hn quy m phng th nghim.
HPLC s dng mt loi ct cha cc ht nh rt ng nht, c tc
dng ci thin s n nh vt l v ha hc v phn tch nhanh hn cc gel
mm truyn thng. Ct sc k cha cc vt liu m kn c phn gii cao
(8, 15 hoc 40 m) cho php sn xut cc phn on c c hn. Cc ht
nh c sc bn cao i vi dng chy ca cht lng thit b c thit k
hot ng p sut tng i cao. Dung mi c phn phi vo ct bng
bm vi dng khng c xung, khng thay i p sut ngc cao. Ct c
kh nng chu ng s tng p sut. u d (detector) c thi gian phn ng
nhanh v cc nh protein c th tri qua trong mt vi giy. u im chnh
ca h thng HPLC l c thi gian chy nhanh hn nhiu so vi cc phng
php sc k khc nhng nhc im ca h thng ny l t tin nn kh p
dng quy m ln.

Hnh 6.12. H thng HPLC

6. Siu lc
Siu lc tr thnh mt k thut tiu chun ca phng th nghim
c c cc dung dch protein di cc iu kin rt n ha. Phng php
ny c s dng trong trng hp thm tch hoc lc gel kh mui
hoc trao i m. Bng cch dng cc cht kt ta i lc tng khi
lng phn t ca protein mong mun, phng php ny cng c th c
dng nh mt k thut tinh sch.
H siu lc thng s dng mng lc c b mt nhn hoc mng lc
h si rng. Cc si ny c c im tng t vi cc b mt nhn, nhng
i vi cc qu trnh quy m ln th n to ra mt din tch b mt ln
hn th tch cho. trng hp hot ng quy m pilot, h siu lc
thch hp vi din tch mng ln ti 6,4 m
2
, cho tc siu lc ln ti
200 L/gi, ty thuc vo nng ca protein. Cc h ln hn thch hp vi
cc tc siu lc ca hng trm lt/gi. S dng phng php ny c th
ng dng cho hu ht mi quy m hot ng.

7. Thit k cc protein tinh sch
Cc nhn t qu trnh chun b c cht v nguyn liu sn xut
(upstream processing) c th nh hng n s pht trin phng thc tinh
sch protein sau ny, cng ngh DNA ti t hp cng c mt nh hng
quan trng ln s tinh sch protein. Bng cch dung hp mt gen quan tm
vi mt trnh t promoter hiu qu, th mt protein ngoi lai c th c
biu hin trong c th vt ch t 10 ti 40% protein tng s ha tan ca t
bo. iu ny c th so snh vi s biu hin ca nhiu protein nguyn th
(ch chim khong 0,01 ti 4% protein tng s ha tan ca t bo). V vy,
qu trnh tinh sch tip theo ca protein c n gin ha. i vi cc
protein c biu hin trong mt dng ha tan, cc k thut di truyn c th
c dng hng ti vic protein c tng hp mi trong gian bo,
hoc thm ch trong mi trng nui cy. iu ny c th lm tng s n
nh ca protein c biu hin (v ch hai trong s tm protease c bit
ca E. coli l hon ton trong gian bo) v n gin ha s tinh sch (v
ch khong 8% ca tt c protein ca E. coli l khoang gian bo).

7.1. Cc th vi (inclusion)
Cc mc biu hin cao nh th sn xut c rt nhiu protein,
dn n xut hin cc ht khng ha tan c gi l cc th vi. Trng hp
ny c quan st nhiu protein ti t hp, bao gm urogastrone,
interleukin-2, prochymosin v cc interferon. Sau khi ph v t bo, cc ht
nh th c th c lng xung y vi mt lc ly tm (RCF) tng i
thp, sn xut nguyn liu khng ha tan cha hn 50% protein mong
mun.
Nguyn nhn to thnh cc th vi cha c bit y , v khng
phi tt c protein biu hin mc cao u to thnh th vi. T bo vt
ch cng th hin mt vai tr quan trng. Cu trc chnh xc ca protein ti
t hp cng c th nh hng s to thnh cc th vi. Mt nghin cu
c thc hin bng cch dng -interferon ngi ti t hp cho thy rng
ch mt vi thay i amino acid cng c th nh hng n s chuyn ha
gia cc biu hin ca protein ha tan v khng ha tan trong E. coli.
Thng thng, cc th vi c ha tan trong urea hoc guanidinium
chloride (thng gi tr pH cao) v mt vi trng hp c th b sung cht
ty ra. Mt khi c ha tan, protein phi c ti cun xon thnh mt
cu hnh t nhin. Trong nhiu trng hp, ch cn pha long n gin ca
dch chit ha tan trong mt m thch hp l . Nu protein cha cc cu
ni disulfide, ngi ta cn phi tin hnh oxy ha v kh glutathione
cung cp mt mi trng thch hp gip cho chng c to thnh chnh
xc. Thnh thong cn b sung ng dung mi (co-solvent), nh PEG, hoc
cc cht ty ra nh Triton X-100, Tween 20 hoc Zwittergent 3-16. Trong
mt s trng hp, cc protein ha tan c th rt kh ti cun xon, v
ngi ta thy rng vic b sung chaperonins c th gip ch cho cc qu
trnh ti cun xon. Chaperonins l cc protein cn m bo cun xon
chnh xc cc in vivo protein, vi kh nng thun li ca chaperonin ti t
hp chng c dng xc tc cho qu trnh ti cun xon chnh xc ca
cc protein in vitro.

7.2. Cc ui i lc
Khi nim ui i lc xut hin khi thit k di truyn vi mc ch
gip cho s tinh sch protein hoc enzyme hiu qu hn. Gen ca protein
quan tm c dung hp vi chui DNA m ha cho mt s trnh t amino
acid s n gin ha qu trnh tinh sch protein, bng cch bin i cc tnh
cht ca n trong mt kiu c th d on. Mt trong cc trng hp u
tin l dung hp di truyn ca mt s gc arginine vi C-terminus ca
urogastrone. Gen ny s sn xut mt protein lin kt mnh vi khun trao
i ion cho cation. ui polyarginine sau c loi b bng cch dng
enzyme bt ng carboxypeptidase A.

Bng 6.2. Cc phng php loi b cc ui i lc

Trnh t lin kt Phng php
phn ct
Cc iu
kin

Hydroxylamine pH 9; 45
o
C
Acid 10% acetic
acid; 55
o
C

CNBr 70% formic
acid; 20
o
C

CarboxypeptidaseB pH 8; 37
o
C

CarboxypeptidaseA pH 8; 37
o
C

Thrombin pH 7-8;
37
o
C

Factor X
a
pH 7-8;
37
o
C

Enterokinase pH 8; 37
o
C

PreCission Protease pH 7; 5
o
C

Kh khn ch yu cc dung hp i lc tinh sch protein l phi
loi b thnh cng ui i lc v cc tc nhn c s dng. Vn ny c
th c gii quyt tng phn bng s bin np di truyn cc im c hiu
cao cho protease hoc cho s ct b cc lin kt acid khng bn ch ni
ca protein ti t hp v ui i lc. Nhiu phng php phn ct c
gi (Bng 6.2). S dng cc protease c hiu l thch hp hn c, bi v
-Asn-Gly-
-Asp-Pro-
-Met-Xxx-
-Xxx-(Arg)
n
-
-Xxx-(His)
n
-
-Gly-Val-Arg-Gly-Pro-Arg-Xxx
-Ile-Glu-Gly-Arg-Xxx
-Asp-Asp-Asp-Lys-Xxx
-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-
c th ng dng trong cc iu kin nh nhng, ch yu l thrombin,
enterokinase v Factor X
a
. Tt c nhng protein ny hot ng 37
o
C v c
th phn ct cc v tr bn trong protein quan tm, hoc do mt tnh c
hiu hoc t s nhim bn cc protease. Cui cng, cc bc sc k tip
theo s c yu cu loi b ui i lc c phn ct v protease.

Bng 6.3. Cc phng php tinh sch protein bng ui i lc

ui i lc Phi t/khun Cc iu kin
lin kt
Cc iu kin
tch ra
Oligo arginine S-Sepharose pH 4-8 NaCl gradient
Oligo histidine Iminodiacetate-
Sepharose (Ni
2+
)
pH 7-8
guandinium
chloride
m imidazole
hoc gim pH
gradient
guandinium
chloride
Flag
TM
antigenic
peptide
Anti-Flag
antibody-
Sepharose
0,15 M NaCl,
1 mM CaCl
2
,
pH 7,8
10 mM EDTA,
pH 7,4
-galactosidase TPEG-Sepharose 1,6 M NaCl,
pH 7
0,1 M sodium
borate
Chloramphenicol
acetyl transferase
-amino-
chloramphenicol-
Sepharose
0,3 M NaCl,
pH 7,8
5mM
chloramphenicol
Protein A IgG-Sepharose pH 7,6 0,5 M acetic acid
Glutathione-S-
transferase
Glutathione-
Sepharose
pH 7,3 Glutathione

Trong s pht trin gn y ca lnh vc ny, ngi ta s dng
dng ti t hp ca protease 3C t rhinovirus ca ngi. Protease ny c
kch thc nh (20 kDa) v c mt tnh c hiu rt hn ch (Bng 6.3). N
c biu hin nh l mt protein ti t hp c dung hp vi glutathione-
S-transferase. iu ny c mt vi u im, bn thn protease c th c
tinh sch bng sc k i lc trn glutathione-Sepharose. Nu protein ch
c biu hin nh l mt s dung hp vi glutathione-S-transferase th n
c th c tinh sch trn ct glutathione-Sepharose. Sau khi x l vi
protease, protein ch c th c phn tch khi ui glutathione-S-
transferase v protease bng cch chuyn qua ct glutathione-Sepharose th
hai.
Mt cch khc, phn ng phn ct c th c t trn ct
glutathione-Sepharose th nht bng cch b sung protease vo m ca
ct, v th trnh c s cn thit cho mt ct th hai. Protease ny c sn
dng thng mi di tn PreCission protease do Amersham Pharmacia
Biotech sn xut (Bng 6.2).

1. ng Th Thu, L Ngc T, T Kim Anh, Phm Thu Thy v Nguyn
Xun Sm. 2004. Cng ngh enzyme. NXB Khoa hc v K thut, H Ni.
New York, USA.
4. Glick BR and Pasternak JJ. 2003. Molecular Biotechnology: Principles
and Applications of Recombinant DNA. 3
rd
ed. ASM Press, USA.
2
nd
8. Walker JM. 2002. The Protein Protocol Handbook. 2
nd
ed. Humana Press
Inc. NewJersey, USA.
4
th
237

Phn II

Cc ng dng ca Cng ngh sinh hc

238
Chng 7

Cc ng dng trong nng nghip

I. M u
y l lnh vc cng ngh sinh hc c nhiu ng gp quan trng.
Cc sn phm cng ngh sinh hc mi trong nng nghip cha ng trin
vng ha hn i vi ngi tiu dng v nng dn. Hin nay, cc ng dng
cng ngh sinh hc trong nng nghip ang tp trung vo cc hng: chn
lc v bin i di truyn cy trng c c cc c im mong mun
(nng sut cao, phm cht tt, thch nghi vi cc iu kin ngoi cnh bt
li...), nui cy m v t bo thc vt nhn nhanh ging cy trng, sn
xut cc khng th n dng phc v chn on cc bnh thc vt v
ng vt, th tinh trong ng nghim v cy chuyn phi vt nui, ci thin
nng sut v cht lng ca ng vt, nui trng thy sn, ch bin thc
phm...
Nhn chung, trong nhng nm qua cng ngh sinh hc c nhng
tc ng rt tch cc trong sn xut nng nghip, to ra mt cuc cch mng
su sc trong lnh vc ging cy trng, vt nui v ch bin thc phm.
Nhiu kt qu nghin cu c ng dng trong sn xut v em li
nhng gi tr kinh t ln lao. Chng hn, nhiu ging cy trng mang gen
khng su, khng bnh, khng cht dit c c a ra th trng nh
bng, ng, khoai ty, la mch, la nc, c chua, c ci ng... Nhiu
loi vt nui c th tinh trong ng nghim v cy chuyn phi, s
dng hormone sinh trng tng nhanh sc ln v sn lng sa tru,
b, k c sn lng thc phm v cc cht ph gia sinh hc...

II. Ci thin v nhn nhanh ging cy trng
Hng nghin cu c tp trung nhiu nht ci thin v nhn
nhanh ging cy trng l nui cy m v t bo thc vt (plant cell and
tissue culture). y l k thut nui cy v trng in vitro cc b phn tch
ri khc nhau ca thc vt. Ngoi mc ch nhn ging v ci thin di
239
truyn ging cy trng, nui cy m v t bo thc vt cn ng gp vo
vic sn xut sinh khi cc sn phm ha sinh, bnh hc thc vt, duy tr v
bo qun cc ngun gen qu him Cc hot ng ny c bao hm trong
thut ng cng ngh sinh hc nng nghip (biotechnology in agriculture).
Lnh vc nhn ging v ci thin ging cy trng c bn hng chnh:
- Nhn ging trong ng nghim (nhn ging v tnh in vitro) bng k
thut nui cy t bo, m v c quan ca thc vt. Vi k thut ny trong
mt thi gian rt ngn c th sn xut mt lng ln cy con ging ht nhau
v gi nguyn kiu di truyn ca cy m ban u.
- Sn xut cy n bi (1n) bng cch nui cy bao phn hoc ht
phn cho php to ra cc dng thun (ng hp t) phc trng ging cy
trng b thoi ha sau mt thi gian di canh tc. Hoc tm kim cc tnh
trng ln d hp t u vit thu c trong qu trnh chn ging.
- Lai v tnh (somatic hybridization) hay cn gi l dung hp t bo
trn (protoplast fusion) gia cc loi xa nhau v quan h h hng m trong
thc t khng th tin hnh bng phng php lai hu tnh, nh m ra
kh nng to ra nhng ging cy hon ton mi.
- ng dng k thut chn dng t bo bin d soma (somaclonal
variation) trong nui cy in vitro to ra cc ging mi chng chu cc
bnh vi khun, virus v vi nm, chu c cc iu kin canh tc khc
nghit nh hn hn, ngp mn, nng v lnh...

1. Nhn ging v tnh in vitro
Nhn ging in vitro l k thut nhn ging cy trng bng cch s
dng nhiu b phn khc nhau ca thc vt, c kch thc nh v sinh
trng iu kin v trng trong ng nghim hoc trong cc loi bnh nui
cy khc cha mi trng dinh dng nhn to.
Trn quan im ng dng, k thut nhn ging in vitro c ng dng
nhm phc v cc mc ch sau:
- Duy tr v nhn nhanh cc kiu gen qu him lm vt liu cho cng
tc to ging.
- Nhn nhanh vi hiu qu kinh t cao cc loi hoa v cy cnh khng
trng bng ht.
- Nhn nhanh v duy tr cc c th u dng tt cung cp ht ging
cc loi rau, cy cnh v cc cy trng khc.
240
- Nhn nhanh v kinh t cc kiu gen qu ca ging cy ly g trong
lm nghip v gc ghp trong ngh trng cy n qu, cy cnh.
- Nhn nhanh iu kin v trng, cch ly ti nhim kt hp vi lm
sch bnh virus.
- Bo qun cc tp on ging nhn ging v tnh v cc loi cy giao
phn trong ngn hng gen.

2. Sn xut cy n bi in vitro
>
,
.
in vitro
.
:
- .
- .
- (dng thun).
Mt s phng php c s dng to th n bi nh sau:

in vivo
in vivo
241
: s
. Nhn chung, cc k thut ny cho hiu
sut to cy n bi thp.

2.2. Phng php to th n bi in vitro
in vitro
, Poaceae,
Ranunculaceae
.

,
(Hnh 7.1). K thut dung hp protoplast cho php khc phc c hin
tng bt th thng xy ra khi lai khc loi (lai xa) m rng ngun gen,
to ra cc ging cy trng mi mang cc c tnh di truyn u vit.
[Lycopersicum esculentum
258.

242

Hnh 7.1. Dung hp protoplast. A: cc protoplast. B: hai protoplast dung hp
trong mt cp. C: cc protoplast c th dung hp trong th 3 (bn phi nh) hoc
nhiu hn, c khi ti 6 protoplast.

3.1. Dung hp protoplast bng ha cht
Phng php ny dng NaNO
3
hoc polyethylene glycol (PEG)
kch thch s dung hp ca hai protoplast.

3.2. protoplast (electrofusion)
-
trong n
-
A B

C
243
-
.

4. Chn dng bin d soma
in
vitro :
-
- - ...
-

- (overproduction)
...
-
(genetic markers)...

Hnh 7.2. S c Helminthosporium maydis ng
Chn lc trn mi trng cha c t ca
nm bnh r st (Helminthosporium maydis)
Ging ng bt dc c
Nui cy callus
Dng callus khng H. maydis

Ti sinh cy

Kim tra tnh khng H. maydis

244
in
vitro
) , khoai
ty).
-
- ). Tuy nhin, t
.

5. Chuyn gen vo cy trng
.

hn
protoplast.
245

Stt Loi Phng php chuyn gen Th nghim trn
ng rung
1 Chui Bn gen/Agrobacterium -
2 La mch Bn gen Khng vi rus
3 u ty Bn gen -
4 Canola Bn gen/Agrobacterium Chng chu cht dit c,
iu khin s th phn
5 Sn Bn gen/Agrobacterium -
6 Ng Bn gen/Agrobacterium Khng cn trng, chng
chu cht dit c
7 Bng Bn gen/Agrobacterium Khng cn trng, chng
chu cht dit c
8 u Bn gen/Agrobacterium Khng virus
9 u phng Bn gen/Agrobacterium Khng virus
10 Bch dng Bn gen/Agrobacterium Chng chu cht dit c
11 Khoai ty Agrobacterium Khng cn trng, khng
virus, chng chu cht
dit c
12 La Bn gen/Agrobacterium Chng chu cht dit c
13 u tng Bn gen/Agrobacterium Chng chu cht dit c
14 B Bn gen/Agrobacterium Khng virus
15 C ci ng Agrobacterium Chng chu cht dit c
16 Ma Bn gen -
17 Hng dng Bn gen -
18 C chua Agrobacterium Qu chn mun, khng
virus
19 La m Bn gen -
246

Hnh 7.3. Mt s cy trng chuyn gen. A: ng khng cn trng. B: la mch
khng virus. C: c chua cho qu chn mun . D: khoai ty chng chu cht dit c.

).
Agrobacterium
.

A B
C D
247
.

bar gus
bar
.

N
mt cy m h
.
Agrobacterium
c Agrobacterium
Agrobacterium
gus
.
248
- -
(Southern b
.

Phaseolus
Agrobacterium
gusA, bar
gus
gus .

5.6. Cy bng
Agrobacterium
tumefaciens
soma
.

249
5.7. C nh m
Qu trnh c nh m din ra r ca cc loi cy h u nh mt s
loi vi khun cng sinh c kh nng hp th nitrogen ca khng kh v to
ra cht m cho cy. Vi khun chnh tham gia qu trnh c nh m l
Rhizobium. S c mt ca n dn n hnh thnh cc nt sn trn r cy
nhim khun. Cc vi khun sng trong nt sn s thc hin qu trnh c nh
m. Phng trnh tng qut c dng sau:

N
2
+ 10H
+
+ 8e
-
+ 16ATP 2NH
4
+
+ 16ADP + 16Pi + H
2

Hnh 7.4. M hnh bin np gen nif

tng mc cung cp m t nhin cho cy trng, ngi ta chn
gii php chuyn gen nif (nitrogen fixation-gen m ha enzyme nitrogenase)
vo c th thc vt v vi sinh vt sng t do hoc sng cng sinh vi thc
vt (Hnh 7.4). V trong t nhin, gia cc c th vi sinh vt vn xy ra s
Nitrogenase

Rhizobium Plasmid
M vng DNA ca plasmid
Phn lp gen nif
RE
DNA ligase
Protoplast La
Bin np
Protoplast mang
plasmid c gen nif
Callus
Ti sinh cy
mang gen nif
250
trao i thng tin di truyn, nh vy v mt k thut vic chuyn gen nif vo
c th vi sinh vt s d dng hn. Vn cn li l lm th no gen nif
hot ng sau khi c chuyn vo c th vi sinh vt ch. Tng t, vic
chuyn gen nif vo t bo thc vt c th c thc hin thng qua viral
vector hoc vi khun Agrobacterium tumefaciens. Tuy nhin, y l mt
cng vic kh khn v c l trong mt tng lai gn vn ny vn cha
gii quyt c.

III. Chn nui v th y
1. K thut cy chuyn phi
K thut c ng dng kh rng ri hin nay l cy chuyn hp t
b. Nguyn l ca k thut ny l gy rng trng b ci c cc c im
m ngnh chn nui cn n v cho th tinh vi tinh trng ca b c cng
mang nhng c im mong mun. Cc hp t hay phi thu nhn bng cch
ra d con. ng lnh phi v bo qun chng trong nitrogen lng
-179
o
C, phi iu kin ny c vn chuyn d dng n ni cn thit.
Sau , phi c cy vo b ci khc mang thai h. B con pht trin t
cc phi ny s ra i trong mi trng sng ca n v khng gp phi
nhng bt li v mt mi trng nh i vi cc gia sc nhp ni.
K thut cy phi cn cho php to ra gia sc sinh i hoc sinh ba
(hoc nhiu hn na) hon ton ging nhau v mt di truyn. Nguyn tc ca
k thut ny l chia non bo th tinh ca gia sc thnh hai hoc ba phn
(hoc hn na) ging nhau. Sau , cy ngc li vo con ci cha h.
Mt khc, k thut cy chuyn phi cn gip xc nh gii tnh ca
vt nui, vic ny em li hiu qu kinh t cao. Chng hn, ngi ta ch
quan tm ly phi b c to b ly tht, trong khi phi b ci c dng
to b ly sa, hoc bo v nhng ging b u vit. Nguyn tc ca
k thut ny da trn c s phn chia tinh dch thnh hai nhm: nhm cha
nhim sc th X to ra b ci, v nhm cha nhim sc th Y to ra b c.
Ngi ta ch cn chn mt trong hai nhm th tinh trong ng nghim ri
sau chuyn phi c to ra vo b m.

2. To ch phm phng trnh bnh cho ng vt
Cng ngh sinh hc trong nhng nm gn y c nhng ng gp
khng nh, ch yu l to ra cc vaccine th h mi nh p dng cng ngh
251
DNA ti t hp, chng hn nh vaccine phng bnh l mm long mng
(Foot and Mouth Disease Virus-FMDV), bnh Theileriosis (bnh st b
bin ng-East Coast Fever) gia sc, bnh st ln Chu Phi, bnh
Newcastle, bnh cu trng gia cm
Bnh toi g do virus gy ra gia cm, c bit l g. Virus ny thuc
nhm Paramyxovirus, bao gm c virus quai b, thuc h Paramyxoviridae
(h ny bao gm c virus gy bnh ln go). Virus c kh nng lm ngng
kt hng cu, thng qua mt loi protein F ca n. Loi protein ny c kh
nng gy min dch v c dng lm vaccine.
Bnh l mm long mng l loi bnh do virus, ly lan rt nhanh v
gy bnh cho khong 30 loi ng vt mng guc, c bit nguy him i
vi tru, b, ln v cu. Khi c dch, trnh bnh ly lan, phi tiu dit
hng lot gia sc nhim bnh, thc hin cc bin php cch ly nghim ngt.
Ngi ta thnh cng trong vic dng enzyme trypsin x l v protein v
nhn thy mt trong s cc protein v ca n c th to c min dch,
kch thch sn sinh khng th. Nm 1985, cc nh khoa hc xc nh
c gen m ha loi protein ny v to dng n trong E. coli, bin E. coli
thnh nh my sn xut protein ni trn v khi tim vo b, ln th chng
tr nn min dch i vi loi virus gy bnh l mm long mng.

3. Chuyn gen vo ng vt
Mt trong nhng th nghim chuyn gen u tin c tin hnh l
gn gen m ha cho hormone sinh trng ca chut cng v promoter
methallothionein ca chut nht vo plasmid vector, sau vector ny c
tim vo t bo trng ca chut nht th tinh. Kt qu l chut nht
chuyn gen ny ln nhanh hn chut nht bnh thng. Kim tra m chut
nht chuyn gen thy rng promoter methallothionein iu ha s biu hin
gen hormone sinh trng trong gan nhiu hn trong tuyn yn mc d
chut thng th tuyn yn l chnh. iu ny cho thy vic chuyn gen
thot khi s kim tra iu ha ca tuyn yn, dn n vic sn xut mt
lng ln hormone sinh trng.
Ln chuyn gen tng hp hormone sinh trng ca ngi tuy khng
ln hn v kch thc nhng li c lng tht nc nhiu hn, t m hn v
tiu tn thc n t hn t 20-30%. Tuy nhin, cc ng vt chuyn gen d b
252
thp khp, stress v mt kh nng sinh sn cng nh nh hng chuyn
ng.
Nh chng ta bit cystein cn cho s pht trin lng, nhng do cu
thiu kh nng tng hp amino acid ny v do cc vi sinh vt ng rut
ca cu s dng phn ln cystein sn c nn nh hng xu n sn
lng lng cu. khc phc iu ny, ngi ta to dng hai gen vi
khun m ha cho enzyme chuyn ha serine thnh cystein. S n nh ca
cc gen ny trong cu chuyn gen sau gip cu t tng hp c
cystein lm tng s pht trin ca b lng.
Hin nay, cc nh khoa hc cng c nhng thnh cng bc u
trong vic chuyn cc gen ngi vo ln nhm mc ch ly ph tng ca
chng (tim, gan, thn...) cy ghp, thay th cho cc b phn b h hng
ca ngi. Nh vy, cng ngh gen m ra cho y hc mt kh nng to ln
trong lnh vc cy ghp cc b phn cho ngi v mt trin vng gii quyt
vn min dch cy ghp.
Bn cnh vic ch i nhng thnh cng mi ca cng ngh chuyn
gen trc tip cho vt nui, ngi ta cng ang tm kim cc bin php mi
tng nng sut vt nui bng cng ngh DNA ti t hp. Chng hn, mt
loi hormone tng trng ti t hp ca b (recombinant bovine growth
hormone) c chuyn vo E. coli sn xut thnh cng loi hormone
trn, v c th kch thch tng sn lng sa b ln ti 15% m khng cn
phi gia tng thm khu phn thc n hng ngy. Hoc gen sn xut
hormone sinh trng ca g cng c a vo E. coli, v ngi ta
chng minh c rng hormone c sn xut bng k thut trn cho php
tng kh nng sinh trng ca g ln khong 15%.

IV. Ch bin thc phm
y l lnh vc cng ngh sn xut thc phm t nng sn c s tham
gia ca vi sinh vt, cng nh s ng gp ca cng ngh sinh hc hin i
trong vic nng cao hiu sut ca cc quy trnh sn xut ni trn. Thc
phm c to ra bi vi sinh vt rt a dng, t cc sn phm truyn thng
c ngun gc xa xa nh men bnh m, phomt, sa chua, ru vang, ru
ct, n nhng sn phm mi xut hin nh protein nm (mycoprotein)...
Trc y, trong cng nghip thc phm cc nghin cu cng ngh
sinh hc c s dng ch yu hon thin cc quy trnh cng ngh ln
253
men truyn thng. Cn hin nay, cc nghin cu cng ngh sinh hc ch
yu lin quan n vic to ra cc chng mi c nng sut sinh hc cao v
vic p dng chng vo cc cng ngh ln men hin i.
Theo nh gi chung, hin nay mi ch khong 15% thc phm trn
th gii c sn xut bng cc quy trnh cng ngh sinh hc. Do vy, nh
hng ca cng ngh sinh hc hin i trong lnh vc ny thc s cha r
nt. Nguyn nhn c th l do ngnh cng nghip ch bin thc phm hin
nay vn mang nng tnh th cng, cng ngh cha hin i, ngnh cng
nghip ch bin thc phm c quy m to ln nhng li c li nhun nh, do
vy kh c kh nng ti u t.
Tuy vy, trong tng lai khi cng ngh ch bin thc phm truyn
thng khng p ng c nhu cu ngy cng gia tng ca con ngi, th
nh hng v vai tr ca cng ngh sinh hc hin i s gia tng rt mnh.

1. Sn xut sa
Cc sn phm sa quen thuc i vi chng ta u c to ra trong
qu trnh ln men ca mt s nhm vi khun nh Lactobacillius,
Streptococcus, Leuconostos... Trc kia ngi ta thng s dng nhng
nhm vi khun t nhin c mt trong sa ln men, do vy qu trnh ln
men ni chung kh kim sot v hiu sut khng cao. Ngy nay, nh vic
to ra c cc ging, chng vi khun vi cc tnh cht xc nh, ngi ta
c kh nng iu khin c qu trnh ln men ni trn mt cch c nh
hng. Cc sn phm ch yu t sa l phomt, sa chua, b, kem sa...

1.1. Sn xut sa chua
Trong sa c sn mt h vi sinh vt phong ph, l vi khun lactic
(gy ln men lactic), vi khun acetic (ln men acetic), vi khun ng rut,
vi khun gy thi (phn gii cht hu c thnh cc mi thi), nm men, nm
mc...
Trong sn xut sa chua bng ln men lactic, vi khun dng ln
men chnh l mt s chng thuc Lactobacterium bulgaricum... c sn trong
sa. Trong cng nghip sn xut sa chua, ngi ta tin hnh thanh trng
sa ri cy vi khun lactic vo.
Quy trnh sn xut sa chua theo quy m cng nghip nh sau:
254
1.1.1. Nguyn liu
Nguyn liu duy nht l sa (c th b sung thm ng). Sa dng
trong sn xut sa chua gm nhng loi sau:
- Sa ti nguyn hay sa tch bo.
- Sa kh nguyn hay sa sy phun khng bo.
- Sa c nguyn hay sa c khng ng.
Cc loi sa trn trc khi a vo s dng cn phi x l qua cc
khu:
- iu chnh hm lng lipid thch hp thng t 3,2-6%.
- Nu trn b mt sa xut hin vng sa th tin hnh qu trnh ng
ha tr thnh mt th ng nht.
- Tit trng sa 85-90
o
C trong 15-20 pht. ngui sa n nhit
cn thit cho s ln men cy ging vo, thng t 40-50
o
C.

1.1.2. Ging
Cc chng lactic thun khit thng c dng sn xut l:
Streptococcus thermophilus, Lactobacterium bulgaricum, Lactobacterium
acidophilum, Lactobacillus delbrueckibulgaricus. Thng hai chng u
c cng s dng vi s lng bng nhau. Nu Streptococcus
thermophilus c s lng nhiu hn, lc sa chua thu c s qu chua
v km mn.

1.1.3. Ln men to sa chua
Quy trnh sn xut sa chua qua hai giai on: giai on ng t sa
v giai on gi chn sa chua.
- Giai on ng t sa
Sa sau khi tit trng c cy ging vo v khuy u ln men
bng phng php b hoc chai. giai on ny qu trnh ln men c
tin hnh 29-35
o
C. Qu trnh ln men lactic xy ra mnh m, casein sa b
kt ta im pH ng in, sa c ng t v t chua cn thit.
Thng cn c vo cc ng ca sa v chua kt thc qu trnh ln
men. Cc ng phi c, ng nht, khng c hin tng nc-sa tch ri
255
nhau ra. chua phi t 60-80
o
T. Thi gian ln men (thng 8-12 gi) c
th thay i khc nhau ty thuc vo chng vi khun v nhit ln men.
Khi sa c chua cn thit th kt thc qu trnh ln men bng phng
php lm lnh nhanh (6-8
o
C) trnh tnh trng chua tng v nc s
tch ra khi sa.
- Giai on gi chn sa chua
Trong giai on ny qu trnh ln men lactic vn cn xy ra mc
yu, sa tip tc c ng t. Lc ny lipid trong sa tr nn rn, nc t
do lin kt vi protein lm sa ng c thm. Giai on ny cn xy ra qu
trnh to hng cho sa lm cho sa c mi rt c trng bng cch b sung
vaniline, hng v tri cy t nhin (du, cam, da...) hay hng tng hp,
mu thc phm. Thi gian ca giai on ny t 12-14 gi.

1.2. Sn xut phomt
Phomt l sn phm ln men c ch bin t sa (sa b, sa d...)
vi s tham gia ca mt s nhm vi sinh vt. y l mt thc phm c gi
tr dinh dng cao, bo qun c lu. Trong phomt cha 20% protein
(di dng peptone, amino acid), 30% lipid, cc mui khong, vitamin...
Phomt c mi v thm ngon, kch thch qu trnh tit dch tiu ha,
lm tng kh nng ng ha thc n cho c th. Quy trnh sn xut phomt
c tin hnh qua bn giai on. Cng nh sn xut sa chua, nguyn liu
chnh y l sa.
- Giai on lm ng sa
T nguyn liu sa ban u, sa c lm ng li. V mt ha l
l giai on lm kt ta: nhng micelle ca casein dnh li vi nhau hnh
thnh mt gel c, rn cha huyt thanh sa bn trong. Sau khi c kh
trng 85-90
o
C trong 15-20 pht, c x l bng rennin vi mt lng
nh va , sau mt thi gian nht nh sa s ng li thnh mt khi nhy
ph gelatin, mm do, khng thm nc.
lm ng t sa, ngoi vai tr ca rennin ngi ta cn s dng vi
khun lactic. Vi khun lactic c to iu kin hot ng 30
o
C. Khi tin
hnh lm ng t sa, ngi ta cy vi khun lactic vo mi trng sa. Qu
trnh ln men lactic c tin hnh, chuyn ng lactose ca sa thnh
lactic acid. Kt qu l qu trnh ny cng gy ra s ng t sa nh trnh
256
by trn (cc si micelle ca casein sa li kt thnh cc rn cha huyt
thanh sa bn trong). Khi qu trnh ln men lactic xy ra, pH mi trng
s gim, pH hng v mi trng acid l pH thch hp cho hot ng ca
enzyme ng t sa. iu ny dn n s ng t sa cng xy ra nhanh
hn. giai on ny, di tc dng ca rennin, casein v paracasein ca
sa s b phn gii to peptone v amino acid (tyrosine, tryptophan...) tp
trung li trong cc ng. Huyt thanh sa bn ngoi cc ng tn ti
dng dung dch.
- Giai on kh nc
Giai on ny p cc sa tch huyt thanh ra khi cc ng sa.
Qu trnh ny xy ra 35-50
o
C trong 20-24 gi. Trong thi gian ny qu
trnh ln men lactic vn tip tc. Phomt lc ny c thnh phn ch yu l
casein v lipid.
- Giai on mui phomt
Ngay sau khi p cc ng tch huyt thanh, phomt c ngm vo
b nc mui NaCl nng 24% trong vi ngy tng v mn cho
phomt, to s ng nht v thnh phn cho khi phomt v ngn chn s
pht trin ca vi sinh vt c hi, ch yu l trc khun ng rut. Kt qu
ca qu trnh ngm mui l cc cht b mt ca khi phomt nh ng,
mui khong s khuch tn ra ngoi, ngc li NaCl t ngoi dung dch
ngm s thm vo bn trong khi phomt. Mui n thm vo lp b mt ca
phomt, to lp bo v chng li s xm nhp ca vi sinh vt c hi.
- Giai on chn pho mt
Phomt c a vo hm lm chn 50-57
o
C c m l 80-90%.
Qu trnh lm chn phomt ko di kh lu t vi thng n hng nm, bao
gm nhiu khu chuyn ha ha sinh phc tp, c nhiu nhm vi sinh vt
khc nhau tham gia cng vi men ng t sa. Hot ng ca vi khun
lactic v rennin c mi lin h mt thit vi nhau dn n lm tng cht
lng ca phomt trong giai on ny.
Sau khi ch bin xong, trc khi c ct ra v ng gi bng giy
nhm, oliofilm, rilsan, phomt c kh trng bng cch chiu tia t ngoi.
Vic ng gi c tin hnh trong iu kin v trng, cui cng thu c
thnh phm.

257
2. Ch bin tinh bt
Sn phm truyn thng t bt m rt a dng v c to ra trong
nhng quy trnh sn xut khc nhau. Tuy nhin, chng c mt im chung
l s dng nm men bnh m Sac. cerevisiae ln men. Cho n nhng
nm 1950, tinh bt ch yu c thy phn bng cng ngh s dng acid.
Vo nhng nm 1960, bng cng ngh thy phn acid kt hp vi x l
enzyme v sau ny ch yu bng enzyme.
Thng ngi ta thy phn tinh bt bng -amylase v
amyloglycosidase, l hai enzyme c tc thy phn cao, khng gy
nhim mi trng v to ra sn phm c h s ng lng dextrose DE
(dextrose equivalent) cao. H s DE phn nh mc thy phn tinh bt
thnh ng glucose (dch ng glucose nguyn cht c DE = 100, cn
dch tinh bt c DE = 0). Hin nay, ngi ta to c mt s chng vi
khun, trong c Bacillus licheniformis v Bac. amyloliquefaciens, c kh
nng tng hp -amylase chu nhit hot ng c nhit ti 100
o
C.
iu ny cho php qu trnh h ha thc hin c trit , to iu kin
cho giai on ng ha tip theo, to ra dch ng c DE gn ti 100.
Thng thng, ngi ta tin hnh ng ha bng glucoamylase nhn c
t nm Aspergillus niger.
Dch c hm lng glucose cao c s dng vo nhiu mc ch
khc nhau, trong ch yu l lm nguyn liu ln men. Tuy nhin, gn
y mi xut hin hng sn xut sir fructose-glucose t dch glucose.
Hin nay, sn phm ny ang c s dng rng ri trong ngnh ch bin
thc phm cc nc pht trin thay th ng saccharose truyn thng
t ma v c ci ng. Thng thng, sir fructose-glucose c to ra t
glucose ca dch thy phn tinh bt c DE gn 100, thng qua qu trnh kh
ion v ng phn ha dch glucose bi glucose isomerase do Streptomyces,
Bac. coagulans v Actinoplanes missouriensis to ra. Trong , qua mt ln
x l ngi ta nhn c sir cha khong 51% glucose, 42% fructose v
7% oliogosaccharide. Nu x l tip s c th nhn c sir cha ti 90%
fructose.

3. Sn xut nc ung ln men
Cc loi nc ung ln men c cn ni chung u c sn xut t
nguyn liu cha ng. Trong , qu trnh to ethanol bng ln men ca
cc chng nm men thuc ging Saccharomyces l qu trnh chung, cn
258
tng loi nc ung ln men c cn li c quy trnh sn xut ring ca
mnh.

3.1. Sn xut bia
Bia l nc gii kht cha nc, CO
2
, ru, cc cht chit xut,
carbohydrate... C s khoa hc ca ln men sn xut bia l s trao i cht
trong qu trnh sinh trng, pht trin ca mt s loi vi sinh vt trong iu
kin ym kh.
Qu trnh sn xut bia ch yu da vo s ln men ng ca mm
i mch (hin nay c th thay bng ng, go, k...). Bia sn xut t mm
i mch bao gm cng on ch yu l 67
o
C enzyme t nhin trong
mm thy phn tinh bt ca ht i mch, b sung hoa bia v dch thy
phn, dch ln men bia, cui cng tch men bia v gi bia ti mt thi
gian bia chn. Bia hi l loi bia s dng ngay khng qua kh trng. Bia
chai v bia lon bo qun c lu v qua kh trng 55-60
o
C hoc qua
lc, ng chai hoc ng hp bng cc thao tc v trng.
Cc chng nm men c s dng ch yu trong sn xut bia l Sac.
cerevisiae v Sac. calsbergensis. Trong Sac. cerevisiae c s dng
cho c ln men b mt v ln men chm sn xut cc loi nc gii kht
ln men. Cn Sac. calsbergensis dng sn xut bia nh.
Qu trnh ln men bia gm 2 giai on: ln men chnh v ln men ph.

3.1.1. Ln men chnh (ch yu dng ln men chm)
giai on ny t bo nm men sinh trng mnh, mt lng ln c
cht c chuyn thnh ethanol, CO
2
v H
2
O. Thi gian ln men khong 6-
10 ngy. Nhit yu cu t 28-30
o
C, pH mi trng 5,3-6. Trong giai
on ny c hin tng to bt do kh CO
2
to ra lc u tan trong dung
dch, sau vi liu lng cao s tch ra to thnh ti kh, to c s cho s to
bt. Kt thc giai on ln men chnh to ra c sn phm gi l bia non.
Bia non c c im cn c, c mi v c trng ca bia nhng cha thch
hp cho vic gii kht.

3.1.2. Ln men ph
Khc vi ln men chnh xy ra trong cc thit b ln men h, ln men
ph thc hin trong cc bnh kn nhit 0-5
o
C. giai on ny qu trnh
ln men din ra chm, dch ln men c lng ng v bo ha CO
2
. Giai
259
on ny cn gi l qu trnh chn bia. Thi gian ln men ph ko di t
vi tun n vi thng. Kt thc giai on ny thu c sn phm dng
dung dch bo ha CO
2
, c hng v thm ngon, d chu nh nhng qu
trnh chuyn ha xy ra nhit thp. Trong giai on ln men ph
nhit thp, xy ra qu trnh ng t nha hoa hublon, cc hp cht tanin-
protein, t bo men lng xung y bnh v tip tc ln men t t. Kt qu
lm bia trong dn. Nh vy, qu trnh ly tm v lc bia thnh phm sau ny
s d dng hn.
Mun lm trong bia thng ngi ta s dng phng php lc (dng
cht bt tr lc diatomite). C th dng phng php ly tm nhng sau khi
ly tm cn lc bia qua lc ng, lc khung bn khng cn cht tr lc th bia
mi tht s trong sut. Trong qu trnh sn xut bia ngi ta s dng cc ti
cht do hay cao su thu hi CO
2
thot ra, sau em bm vo cc bnh
chu p lc cao. S kh ny c dng np li vo trong bia, bia thnh
phm c CO
2
.
tng bn ca bia trong thi gian di ngi ta kh trng 60-70
o
C
trong 30 pht ngn chn s pht trin ca vi sinh vt v cc qu trnh
bin i trong bia. Bia thnh phm c mu vng, trong sut, hng v thm
ngon, bt nhiu, c v ng cht nh c trng. Bia thnh phm c bo
qun trong kho lnh 0-10
o
C.

3.2. Sn xut ru vang
3.2.1
Trong
.
- .
(nho c
).
260
- ).
.
- .
: (1) S , q
, n
. (2) S
2
O
2
:
2
-
-
( ,
du da...
+
.
+
.
1
...
261
65-70
o
.

- . :
+
men thng c s dng: Sac. ellipsoideus, Sac. cerevisiae, Sac.
oviformis
+
.
- . :
+
.
+ .
20-30
o
n.
+ .
O
2
.
- . C ba giai on:
.
-
.
. Sau giai on mt, t
.
262
-
.
-
-
.

3.3. Sn xut ru trng (cn)
Quy trnh sn xut ru trng bng phng php ln men ru bi
nm men c thc hin qua cc bc sau: ch bin nguyn liu thnh dch
ng, ln men bin ng thnh ru, chng ct v tinh ch ethanol.

3.3.1. Ch bin nguyn liu thnh dch ng
- Nguyn liu. c dng ph bin hin nay l loi nguyn liu c
sn ng (r ng) v nguyn liu c tinh bt (bp, sn, khoai lang, bt
go...).
- Cc phng php ng ha. C ba phng php ph bin nht l
ng ha bng bnh men, maltase v myco-malt.
+ Phng php bnh men. L phng php sn xut ru th cng.
Nm mc (c h enzyme amylase phn gii tinh bt) c nui cy v pht
trin trn mi trng c tinh bt sng. Tinh bt sau khi c chuyn sang
dng d tan (dng nhit cao 130-140
o
C) m khng nht thit phi h ha
thnh dung dch, th amylase ca nm mc s d dng phn hy tinh bt.
Nhc im ca phng php ny l tinh bt khng dng dung dch nn
hn ch tc dng ca amylase v vic s dng bnh men d gy nhim
263
khun, nn phng php ny to ra nhiu sn phm trung gian v hiu sut
tng thu hi thp.
+ Phng php maltase. S dng ch yu l cc enzyme v -
amylase ca ht i mch hoc tiu mch ny mm (malt) chuyn ha
tinh bt ( h ha) thnh ng ln men. u im ca phng php ny
l: thi gian h ha tinh bt ngn, cht lng ru khng b nh hng m
thng to ra nhng hng v c trng d chu, t b nhim khun. Tuy
nhin, phng php ny cng c mt vi nhc im: hiu sut ng ha
khng cao v khng trit v phc h amylase trong mm thc khng hon
chnh, t l malt s dng so vi hm lng tinh bt c trong nguyn liu
tng i cao (8-20%), gi thnh sn phm cao.
+ Phng php myco-malt. y l phng php c dng ph bin
hn c. Qu trnh ng ha y dng enzyme ca vi sinh vt (ch yu l
ca nm mc). a s nm mc c s dng thuc chi Aspergillus nh:
Asper. niger, Asper. oryzae, Asper. flavus, Asper. awamori. Cc chng nm
mc c s dng ng ha tinh bt c nui cy theo hai phng
php: nui cy b mt (thng dng cho Asper. awamori v Asper. oryzae)
v nui cy chm trong h ln men (thng dng cho cc chng Asper. niger
v Asper. batatae).
Thi gian thc hin qu trnh ng ha di hay ngn ty thuc vo
chng ging nm mc v nguyn liu c s dng, thng thng t 24-40
gi. Cht lng ca qu trnh ng ha c nh gi theo hot lc ca
amylase, dextrinase, gluco-amylase. Kt qu, qu trnh ng ha cho cc
sn phm gm hn hp dextrin, maltose, glucose. Trong , glucose chim
t l cao nht.

3.3.2. Ln men bin ng thnh ru
y l giai on quan trng nht trong sn xut ru, quyt nh cht
lng sn phm to thnh. Sau khi dch ng ha c x l, ngi ta
b sung thm mt s thnh phn cung cp thm vitamin v amino acid
nh mui ammonium, mui phosphate, dch thy phn nm men. Mi
trng c thnh phn nh trn c th s dng ln men.
- Ging. Ch yu l cc chng ca nm men Sac. cerevisiae. Cc
chng nm men dng trong sn xut phi c nhng c im c bn sau:
264
+ C y c im c trng ca nm men.
+ Tc pht trin mnh, hot lc ln men cao.
+ Ln men c nhiu loi ng khc nhau v t c tc ln
men nhanh.
+ Chu c cn cao t 10-12%.
+ Thch nghi c vi nhng iu kin khng thun li ca mi
trng, c bit l i vi cht st trng.
- Qu trnh ln men. Mi trng ln men sau khi c kh trng,
kim tra ng t 90-120 g/L v pH 4,5-4,8 th c th cy ging vo.
Thi gian ln men t 65-72 gi, trong 10 gi u c sc kh nm men
sinh si ny n, sau cho ln men tnh (ym kh). Qu trnh ln men ru
xy ra nh sau: ng v cc cht dinh dng ca mi trng ln men c
hp th vo trong t bo nm men qua mng t bo v tham gia vo qu
trnh trao i cht, ru ethanol v CO
2
to thnh lin thot ra khi t bo,
ru ethanol ha tan mnh trong nc do vy n khuch tn rt nhanh vo
mi trng chung quanh.

3.3.3. Chng ct v tinh ch ethanol
Khi kt thc ln men ru, sau khi loi b t bo nm men, mun
c ru tinh khit cn chng ct dch ln men loi b tp cht. K
thut chng ct ru nh hng rt ln n cht lng ru thu c. ng
thi, t l tp cht v cht lng ru li chu nh hng bi nguyn liu
nui cy. Qu trnh chng ct ru din ra theo cc giai on sau:
- Chng ct dch ln men. Qu trnh ny cho php thu c ru th
bng cch tch cn cng cc cht d bay hi ra khi dch ln men.
- Tinh ch ru. L qu trnh tch cc tp cht ra khi cn th thu
cn tinh khit. Trong cn th ngoi ethanol cn c nhiu tp cht. Da vo
khi lng phn t v kh nng bay hi ngi ta chia lm ba nhm:
+ Tp cht u (c nhit si thp hn ethanol: aldehyde acetic,
ethyl acetate, methyl acetate, methanol) c ly ra giai on u ca qu
trnh tinh ch, c gi l ru u hay cn cng nghip.
+ Tp cht cui (c nhit si cao hn ethanol v kh bay hi, l
ru cao phn t: isoamylic, isobutylic) loi ny t tan trong nc, c
gi l du fusel hay ru tp.
265
+ Tp cht trung gian, ty thuc vo nng ru v tnh cht vt l
ca cc tp cht m n s bay hi cng vi tp cht u hay li vi tp
cht cui. S tp cht ny kh tch khi ethanol khi tinh ch, chng hn:
isobutyrate, ethyl isovalerianate. Sau khi tinh ch, tch ba loi tp cht trn,
ta c ru tinh khit. Hn hp ru-nc l hn hp ng ph (c im si
chung) nn vi phng php chng ct thng thng khng th tinh ch
c ru ethanol c nng ru >95,5% (th tch). V vy, mun c
ethanol tuyt i (>99%) ta phi tinh ch thm (v d: bng phng php
chng luyn di p sut thp p 0,0525 atm) hn hp ru-nc khng
c im si chung v cui cng s thu c ethanol tuyt i.

4. Cc sn phm cha protein
4.1. Thc phm ln men truyn thng giu protein
Bao gm cc sn phm khc nhau giu protein nh phomt, u ph,
nc mm... v cc hnh thc bo qun thc phm nh ln men acid nh
trong sn xut cc loi gi ch, xc xch, c p, c mm... Tuy nhin, cho
n nay cc ng dng ca cng ngh sinh hc hin i trong vn ny ni
chung khng ng k tr ngnh sn xut phomt. Ngc li cc ng dng
ca cng ngh sinh hc li th hin rt r nt trong vic to sinh khi vi sinh
vt v ch bin chng thnh cc dng thc phm khc nhau.

4.2. Protein n bo (single cell protein-SCP)
Protein n bo l mt thut ng c gi theo quy c dng ch
vt cht t bo vi sinh vt c s dng lm thc n cho ngi v ng vt.
Thut ng ny hon ton khng chnh xc, v sn phm c to ra thng
khng phi l mt protein thun khit m l cc t bo c x l thuc
nhiu loi vi sinh vt khc nhau bao gm c vi khun, nm men, nm si v
to.
Sinh khi t bo vi sinh vt lun c hm lng protein cao. Do vy,
trong nhng nm gn y lnh vc cng ngh sinh hc rt ch nghin cu
cng ngh sn xut v kh nng ng dng ca protein vi sinh vt. Qu trnh
sn xut SCP gn lin vi cng ngh ln men, do vy nng sut to protein
ca n rt cao (30-80% khi lng kh). Mt nhc im quan trng ca
SCP l thng cha hm lng nucleic acid cao, c bit l vi khun,
266
nhng cng c nm men v nm si. Nu cc loi SCP ny c s dng
cho ngi th l mt vn , v ngi thiu uricase xc tc cho s oxy ha
uric acid thnh allantoin ha tan hn. n nhiu cc dn xut ca purine s
lm tng hm lng uric acid trong mu, acid ny s kt ta v to thnh
tinh th trong cc khp v ng gp vo vic to nn cc vin si trong
ng niu.

4.2.1. Protein t ngun carbohydrate
Cellulose l cht hu c thng gp nht trn tri t v hng nm
c ti to vi mt khi lng khng l. Cc loi rm r cha ti 30-45%
cellulose. Cellulose cng gp nhiu trong b ma, nc thi cng nghip g,
cng nghip dt v cc cht thi ca cc ngnh cng nghip thc phm. Sn
xut SCP t c cht cellulose l mt hng c nhiu trin vng. Cc vi sinh
vt thch hp cho vic s dng cellulose l x khun a nhit, vi khun t d
c ca cc ng vt nhai li v nhiu loi nm si khc... Trong t nhin, t
gp cellulose thun khit m n thng nm di dng lin kt vi cc
polymer khc nh lignin, pectin, hemicellulose... Lignin l mt polymer
c to nn nh s ngng t ca cc gc ru. Lignin bao quanh cc si
cellulose bng mt mng li ba chiu v do vy ngn cn s phn gii
cellulose nh enzyme. Ring vic lm gim ln ca ht cho php tng
ng k s phn gii cellulose.
n nay, mi ch c sn phm protein t nm (mycoprotein) l protein
vi sinh vt duy nht c cho php s dng lm thc phm cho ngi v gia
sc. Mycoprotein l dng thc phm cha si nm Fusarium graminearum.
Sinh khi c to ra trong qu trnh nui cy nm lin tc trn mi trng
cha glucose v mui ammonium. Sau khi qu trnh ln men kt thc, ngi
ta tin hnh x l nhit i vi sinh khi nm thu c nhm mc ch gim
lng ribonucleic acid ca n. Cui cng tch si nm bng cch lc chn
khng. Quy trnh cng ngh ny c nng sut to sinh khi giu protein cao
hn rt nhiu ln so vi chn nui.

4.2.2. Protein t vi khun lam c nh m v vi to
Vi to (microalgae) l tp hp nhng loi to c kch thc nh b v
c th thch hp c vi vic s dng cc phng php nui cy i vi vi
sinh vt. Vi khun lam (cyanobacteria) trc y c gi l to lam, hay
267
to lam-lc. Trong phn ny, chng ta xp hai loi ny chung vi nhau v
gia chng c nhiu c im sinh l khi nui cy ging nhau. Do , qui
trnh sn xut thu sinh khi ca chng v c bn l khng khc nhau.
Ngay t nhng nm 1940, ngi ta bit mt s vi to c kh nng
trong mt thi gian ngn to ra mt lng sinh khi rt ln cha ti 40-60%
protein khi lng kh (Chlorella sp.) v 50-55% protein khi lng kh
(Scenedesmus). vi khun lam, c bit l chi Spirulina hm lng protein
ln n 60-70% khi lng kh. Hm lng cc amino acid ca nhng
protein vi to v vi khun lam kh cn i gn vi quy nh ca protein
tiu chun. c bit l lng amino acid khng thay th trong protein rt
cao c khi ln n 42%.
n u nhng nm 1960, to lam c nh m cng c bt u
nui trng rng ri trn th gii, bnh to c s dng lm thc phm cho
ngi dn, cn bt to c s dng rng ri lm thc phm b sung cho tr
em suy dinh dng v tng tch sa cho sn ph. Chlorella c nui trng
i tr t thp nin 1950. nui trng quy m cng nghip vi to v vi
khun lam, ngi ta s dng hai h thng c bn l h thng kn v h.
- H thng kn. L mt h ln men, dng nh sng nhn to c cng
cao v c sc kh CO
2
. u im ca phng php ny l khng ph
thuc kh hu thi tit, iu kin nui cy c kim tra, khng ch mt
cch ch ng. Nhng phng thc ny c gi thnh cao nn kh p dng
rng ri.
- H thng h. L cc b nui s dng nh sng t nhin. Nhc
im ca h ny l cc lp t bo phn y b nui thc t s khng c
tip xc vi nh sng mt tri tin hnh qu trnh quang hp (Hnh 7.5).
Vic thu hoch sinh khi vi khun lam v vi to l cng on rt quan
trng nh hng n gi thnh sn phm. C nhiu phng php thu sinh
khi khc nhau nh: ly tm, vt, lng kt ha hc, lng kt bng in
trng, t lng kt... Trng hp cc vi to nh Chlorella, Scenedesmus...
c kch thc t bo nh th ch yu dng phng php ly tm thu sinh
khi. Vi Spirulina ngi ta s dng phng php lc. Thu hoch sinh khi
cc vi khun lam ny bng mng lc nghing kt hp vi ht chn khng.
Sau khi thu hoch, sinh khi c sy kh bng cc cch nh sy ng kh,
sy chn khng, sy hnh trng... c c thnh phm.

268

Hnh 7.5. Nui to Spirulina Thi Lan v bt to c ng vin

5. Ch bin rau qu
Rau qu c bo qun lu khng bin cht v trong mt s trng
hp gi tr dinh dng ca chng cn c gia tng nh c x l thng
qua ln men lactic nh vi khun Leuconostoc mesenteroides v
Lactobacillus plantarum.
Ch bin v to ra cc thc phm c gi tr t u tng nh ln men
vi sinh vt c bit t rt lu. Ph bin hn c l cc loi nc chm v
u ph t u tng nh ln Asper. oryzae v Asper. tamari.
sn xut u ph trc ht phi ngm u, sau nghin u
thnh bt v lc qua vi. To kt ta t dch ni trn bng mui Ca hoc
Mg, sau ng thnh bnh u. Trong thi gian , nm si trng pht trin
rt mnh trn b mt bnh u v to hng v c bit cho n.
Hin nay, trong cng nghip sn xut nc qu ngi ta s dng rng
ri cc loi enzyme pectinase, cellulase, hemicellulase, amylase, v
protease. Ch yu x l lm trong nc qu, gim nht, gip qu trnh
lc v n nh ca cht lng ca nc qu p. Trong , qu trnh thy
phn pectin nh pectinase, pectateliase, polygalacturonidase v pectin
esterase c vai tr quan trng bc nht. Chng ct cc lin kt glycoside
trong phn t pectin v do vy, lm tng hiu sut to nc qu cng nh
cht lng ca n.

1. i Duy Ban v L Thanh Ha. 1996. Cng ngh sinh hc i vi vt
nui v cy trng. NXB Nng nghip, H Ni.
269
4. Chrispeels MJ and Sadava DE. 2003. Plants, Genes, and Crop
Biotechnology. 2
nd
ed. Jones and Bartlett Publishers, Massachusetts, USA.
5. Narayanaswamy S. 1994. Plant Cell and Tissue Culture. Tata McGraw-
Hill Publishing Co. Ltd. New Delhi, India.
7. Razan MK. 1994. An Introduction to Plant Tissue Culture. Oxford & IBH
Publishing Co. Pvt. Ltd. New Delhi, India.
8. Trigiano RN and Gray DJ. 2000. Plant Tissue Culture Concepts and
Laboratory Exercises. CRC Press, New York, USA.
Chng 8

Cc ng dng trong y dc

I. M u
Cho n nay, c l thnh tu cng ngh sinh hc c th hin r nt
nht l lnh vc y hc nh liu php protein v liu php gen cha tr
mt s bnh him ngho (ung th, nhim virus v hin ang th nghim
cha tr bnh AIDS...) cng nh chn on bnh (vim gan, st xut
huyt, sn l gan...) v phng bnh (vaccine). Ngy nay, vi nhng cng c
ca k thut gen, ngnh y khng ch da vo cc triu chng lm sng m
cn c kh nng tc ng thng vo cc nguyn nhn su xa ca bnh l
s bt thng ca gen. Cng ngh sinh hc xm nhp vo hu nh mi
lnh vc ca y hc, trong ng k nht l lnh vc chn on v phng
nga vi vic to ra cc b kit chn on bnh bng phng php PCR v
cc DNA vaccine c hiu qu cao. Lnh vc sn xut thuc cha bnh nh
interferon, insulin, interleukin, hormone sinh trng ngi... ngy cng
pht trin mnh v tr thnh mt ngnh cng nghip quan trng. c bit,
liu php gen mc d thnh tu cn t nhng m ra nhng trin vng to
ln trong vic cha tr nhng bnh di truyn v bnh nan y.

II. Vaccine
Trong sn xut vaccine, cho n thi gian gn y, ngi ta vn s
dng vaccine bt hot hoc vaccine sng nhc c lm khng nguyn kch
thch to khng th cn thit trong c th ngi v vt nui. Nhng vaccine
c sn xut theo cch ny c mt vi hn ch, chng hn vaccine sng
nhc c c kh nng quay tr li dng c hoc hot lc ca n gim kh
nhanh trong c th ngi v vt nui.
n nay, nh cng ngh DNA ti t hp ngi ta sn xut c
protein v ca mt s loi virus nh virus bnh di v vim gan B. Sn xut
vaccine k thut gen l mt lnh vc pht trin mnh hin nay ca cng
ngh DNA ti t hp. y l loi vaccine c bo ch t vi khun c
chuyn gen m ha tng hp mt protein khng nguyn ca mt loi virus
hay mt loi vi khun gy bnh no . Hin nay, cc loi DNA vaccine ti
t hp c s dng cho ngi bao gm vaccine vim gan B, vaccine di
kiu mi, vaccine t kiu mi, vaccine st rt v vaccine bnh phong. Virus
vim gan B c v ngoi lypoprotein. Khng nguyn b mt l protein chnh
ca v ngoi, c pht hin trong mu ngi b nhim. Ngi ta bin np
gen tng hp khng nguyn ca virus vim gan B vo vi khun E. coli sau
sn xut sinh khi quy m ln cc vi khun E. coli mang gen ti t hp
ny, bin E. coli thnh nh my sn xut khng nguyn lm vaccine.
Bn cnh , m hnh sn xut vaccine da trn c s thc vt
(vaccine thc phm) cng c tim nng ng dng rt ln. Bng cch chuyn
mt loi gen khng nguyn ca virus hoc vi khun vo t bo thc vt, gen
ny s hot ng trong c th v bin thc vt thnh ni sinh ra khng
nguyn. Khi nhng khng nguyn ny i vo c th ngi th h thng min
dch ca ngi s t ng sinh ra khng th c hiu tng ng. Nh vy,
thay v tim chng theo phng thc thng thng ngi ta c th n nhng
hoa qu c khng nguyn c s dng lm vaccine.

1. Cc phng thc tim chng vaccine hin nay
1.1. Cc vaccine bt hot
Cc vaccine bt hot c sn xut t cc virus gy bnh bng cch
ph hy c tnh ca chng bng -propiolactone hoc formalin nhng vn
duy tr mt kh nng sinh min dch y . Cc vaccine c sn xut theo
phng thc ny tng i an ton v kch thch cc khng th chng li
cc protein b mt ca tc nhn gy bnh. Cc vaccine tiu n v c xem
l mt dng vaccine bt hot nhng c mc thp hn. Trong trng hp
ny, mt phn ca tc nhn gy bnh (nh l protein b mt) c s dng
gy to khng th nhm trung ha tc nhn gy bnh.
S dng rng ri khng nguyn b mt ca virus vim gan B c
tinh sch t mu ca ngi bnh hoc gn y hn t nm men ti t hp l
mt phng thc rt hiu qu to min dch. Khng nguyn b mt ca
virus vim gan B (sn phm ca gen n) kch thch to ra s bo v v gn
nh hon ton chng li s vim nhim virus vim gan B.
Mt v d khc ca loi vaccine tiu n v l s dng bin c t ca
vi khun. Nhiu loi vi khun sn xut c t ng vai tr quan trng trong
s pht trin ca bnh. V vy, mt s tc nhn mang c t (v d bnh un
vn v bnh bch hu) c bt hot bng formaldehyde lin kt vi mt
cht t dc (adjuvant) pht trin thnh vaccine. C th to min dch
bo v bng cch kch thch khng th trung ha cc nh hng ca c t.
Mt loi vaccine khc cng thuc loi tiu n v l cc vaccine v
polysaccharide. Chng hn, nhng vaccine chng li Haemophilus
influenzae v vim mng no do no m cu. Trong trng hp ny dch
chit v ngoi polysaccharide ca vi khun c s dng nh l mt
vaccine v c khi c lin kt vi protein ci thin kh nng gy min
dch. Khng th bn b trong mt vi nm v c th bo v chng vi khun.

1.2. Cc vaccine sng nhc c
Cc vaccine sng c hiu lc (nh mt s vim nhim t nhin)
thng to ra mt s min dch lu di. Hu ht cc vaccine nhc c hin
nay c sn xut mt cch kh kinh nghim, theo cch qu trnh nui cy
c cy chuyn nhiu ln cho ti khi nhn thy c s mt c lc
(virulence) ca tc nhn gy bnh b m. c lc ny c xt nghim
trong cc h thng ng vt m hnh trc khi th nghim trn nhng ngi
tnh nguyn. Chng hn, vaccine dng to min dch chng li bnh lao
c bt ngun sau 13 nm cy chuyn trong mi trng c cha mt bi
Calmette v Guerin (v vy c tn l BCG-Bacille Calmette-Guerin).
Thc t cho thy l c nhiu hng khc nhau sn xut vaccine cho
mt loi bnh, in hnh l vaccine bnh thng hn hin nay. Anh c ba
loi vaccine khc nhau c cp bn quyn. Mt l vaccine t t bo
hon chnh b git cht, loi th hai l dch chit v polysaccharide ca bnh
thng hn, v loi th ba l chng Salmonella typhi sng nhc c.

2. Vai tr ca cng ngh DNA ti t hp trong nhn dng, phn tch v sn
xut vaccine
2.1. Nhn dng v to dng cc khng nguyn c tim nng vaccine
Nhiu tc nhn gy bnh gn nh khng c kh nng nui cy bn
ngoi vt ch t nhin ca chng v iu ny gy cn tr cho cc cch
tip cn truyn thng pht trin liu php vaccine. V d: virus vim gan
B (HBV), tc nhn gy bnh giang mai ngi (Treponema pallidum) v vi
khun gy bnh phong (Mycobacterium leprae) khng th sinh trng trong
iu kin in vitro mc d chng c th sinh sn trong cc loi ng vt m
hnh. Bi vy, ngi ta khng th to ra cc vaccine sng nhc c hoc
cc vaccine bt hot bng cch nui cy cc tc nhn ny.
Cng ngh DNA ti t hp cho php chuyn thng tin di truyn t
nhng c th kh tnh ny vo nhng vt ch d bo hn nh l E. coli,
nm men hoc t bo ng vt c v. Khng phi tt c cc gen khng
nguyn u d nhn dng, to dng v biu hin nh gen khng nguyn b
mt ca vim gan. Trnh t nguyn vn ca genome HBV c kch thc nh
hn 10 kb. Bi vy, tng i n gin khi thit lp khung c m biu
hin. Tuy nhin, trng hp bnh st rt th li khc. T lu cc nh khoa
hc bit rng cc thoa trng (sporozoite) st rt b chiu x c th bo v
chng li bnh st rt. Nhng giai on thoa trng trong chu k sng ca k
sinh trng st rt ch sinh trng vi mt lng nh, nn ngi ta cn phi
ng dng cng ngh DNA ti t hp sn xut vaccine. Tuy nhin,
genome ca k sinh trng st rt li ln hn genome ca HBV hng ngn
ln, v th ngi ta gp rt nhiu kh khn trong vic thu thp cc thng
tin v trnh t gen thch hp v sn phm ca n to ra bo v min dch.
im khi u ca cng ngh DNA ti t hp l xy dng th vin
DNA ca c th c nghin cu trong E. coli. Th vin cDNA hoc
genomic DNA c th cung cp cc phng thc c bn nhn dng v
phn lp cc gen quan tm. Vi cc thnh tu trong k thut phn tch trnh
t gen th thng tin trnh t bt ngun t cc genome hon chnh hoc cc
nhim sc th ring bit tr nn cng ngy cng quan trng.

2.2. Phn tch cc khng nguyn vaccine
2.2.1. Nhng yu t quyt nh khng nguyn t bo B (B-cell epitopes)
Phn tch cu trc ca khng nguyn vaccine c th thu c cc
thng tin gi tr trong vic pht trin vaccine. Chng hn, hiu bit v nhng
epitope (yu t quyt nh khng nguyn) chng li cc khng th trung ha
c th cho php nghin cu loi vaccine peptide thch hp. Cc epitope c
th lin tc hoc gin on. Cc epitope lin tc l cc peptide dng xon
ngu nhin cc huyt thanh min dch cho epitope phn ng vi phn t
hon chnh t trnh t m n c bt ngun. Cc epitope gin on lp
ghp cc phn t c nhm li do cu trc th cp ca protein. Mt vi
epitope trung ha dng lin tc trong khi nhng epitope khc l gin on.

2.2.2. Nhng yu t quyt nh khng nguyn t bo T (T-cell epitopes)
Cc T lymphocyte c th nhn din cc khng nguyn ngoi lai nh
cc peptide c to thnh trong s phi hp vi phn ngoi bo ca phn
t MHC. Cc t bo T tr gip (helper T-cell) CD4+ nhn din c
khng nguyn tip hp vi MHC loi II, trong khi cc t bo T c hi t
bo (cytotoxic T-cell) CD8+ (CTLs) nhn din khng nguyn lin kt vi
cc phn t MHC loi I. a hnh di truyn ca cc phn t MHC loi I v
II xc nh s c hiu v i lc ca lin kt peptide trong s nhn din
ca t bo T.

2.3. Sn xut cc vaccine tiu n v (subunit vaccine)
Ni chung, nui cy t bo ng vt c v l phng php thch hp
sn xut cc vaccine chng li cc tc nhn gy bnh c sao chp
trong cc t bo eukaryote. T bo E. coli khng n nh tin hnh mt
vi bin i hu dch m ca mt s vaccine quan tm. Bi v, cc h thng
vi khun khng th b sung carbohydrate l yu t quan trng trong c tnh
khng nguyn v cu trc ca nhiu khng nguyn bo v ca virus.
Mt iu khc cng rt ng quan tm, l hiu sut kh ngho ca
khng nguyn b mt trong vi khun v protein hon ton thiu kh nng
cun xon chnh xc lp rp trong cc tiu th c ng knh 22 nm, nm
men Sac. cerevisiae cng l mt trong nhng h thng thch hp cho biu
hin gen do n khng nhng cho hiu sut protein hp l m cn to thnh
cc tiu th 22 nm. iu ny, cng vi kinh nghim sn xut quy m ln
trong cng ngh ln men, to ra mt h thng hp dn c bit, v d cho
vic sn xut cc tiu th HbsAg bo ch vaccine trong nm men vi mt
hiu sut cao. Tuy nhin, vaccine sn xut theo phng thc ny vn cn
khong 1% khng c p ng min dch.
Tip theo s thnh cng ca vaccine vim gan v do kh nng c sn
ca cc cng ngh c thit lp cung cp mt lng ln khng
nguyn, th hng nghin cu tiu n v l mt trong nhng phng php
thch hp nht pht trin cc vaccine mi. Qu trnh glycosyl ha protein
l rt quan trng gy ra phn ng min dch ca HbsAg, v th nui cy
t bo ng vt c v c th l thch hp hn. Thc t l mt trong nhng
vaccine vim gan B cp bn quyn c sn xut trong t bo ng vt
c v.
3. Ci thin v sn xut cc vaccine sng nhc c mi
3.1. Ci thin cc vaccine sng nhc c
Cc k thut sinh hc phn t cho php phn tch c tnh v c tnh
khng nguyn mc phn t, nh vy y l c s thun li trin
khai mt hng nghin cu thch hp hn trong vic sn xut cc c th
nhc c cng nghip ha vaccine sng vi cc tnh cht mong mun.
Cc virus loi DNA v cc vi sinh vt khc c th c chuyn gen trc tip
hoc gin tip. Tuy nhin, cc virus loi RNA mt mc no kh gii
quyt hn, mc d c mt vi thnh cng vi virus bi lit (poliovirus)
v virus bnh cm (influenza), do rn chc ca genome virus.

3.2. Cc vector sng ti t hp
3.2.1. Cc th ti t hp ca viral vaccine
Cc viral vaccine cho bnh u ma c s dng hn 150 nm
di dng vaccine sng nhc c. Gi thnh r, sn xut n gin, bo
qun khng cn lnh, kh nng n nh khi tim chng v kch thch p
ng khng th trung gian t bo (cell-mediated) l tt c u im ca loi
vaccine ny.
Tuy nhin, nhng u im ny ch tht s hp dn bi cc th ti t
hp vaccine biu hin gen ngoi lai (Hnh 8.1). Hn 1.000 th ti t hp
vaccine khc nhau biu hin cc gen ca virus, vi khun v cc tc nhn gy
bnh k sinh trng c cng b. a s trong chng cho thy kh
nng bo v cc h thng ng vt m hnh chng li cc tc nhn gy
bnh. Th ti t hp vaccine biu hin glycoprotein v gp160 ca HIV-1
c th nghim trn ngi cho thy cm ng cc p ng min dch i
vi HIV gp160. Tuy nhin, cc bin chng kt hp vi liu php vaccine v
vic tng s lng cc c th ring l mang s thiu ht min dch c th
gii hn hu ch ca cc th ti t hp i vi liu php vaccine ngi.

3.2.2. Cc vaccine ti t hp BCG
Vaccine BCG (Bacillus gy bnh lao xng ca avirulent) l loi
vaccine c s dng rng ri nht trn th gii. T 1948 n nay hn 5 t
liu vaccine c tim chng.


Hnh 8.1. Virus u ma c ti t hp trong t bo ng vt c v vi mt
gen ngoi lai. Cc virus u ma c sinh sn bao gm c hai dng: hoang di v
ti t hp. Cc virus ti t hp c phn lp v s dng pht trin thnh mt
loi vaccine an ton v hiu qu.
Vaccine BCG ti t hp c mt s u im khc bit hn hn cc
hng vaccine a tr khc, trc tin l nh kinh nghim thu c vi
vaccine BCG gc (parent). u im thc t l ch hai vaccine BCG v
vaccine polio ung mi c WHO khuyn co dng cho tr em mi sinh
v ngi tr tui, nhng ngi m liu php vaccine c th khi u tt hn
cho cc c hi thnh cng trong cc chng trnh tim chng vaccine.
Chng nga ring r vi BCG to ra mt min dch trung gian t bo bn
Virus bnh
u ma
Gen ngoi lai
c chn
vo plasmid
vector
T bo ng
vt c v
Virus sinh
sn: dng
hoang di v
th ti t hp
mang gen
ngoi lai
Vt tan ca dng hoang
di v th ti t hp
Cc vt tan ca th ti t
hp c chn lc pht
trin vaccine
vng i vi bnh lao l rt hiu qu vi t hn mt bin chng trn mt
triu liu. Mc d, cc vector loi phage v plasmid c s dng c mt
vi thnh cng nhng nhng nghin cu tip theo vn cn c tin hnh
c th hng ti cc mc biu hin cao v cho php h thng c thao
tc d dng hn.

3.2.3. Cc chng Salmonella nhc c (vaccine vi khun sng)
Cng ngh DNA ti t hp c kh nng a ton b cc t bin hoc
on khuyt vo cc chng vi khun khc nhau lm yu c tnh ca
chng. Cc vaccine nhc c c thit k hp l cng c th dng nh l
cc cht mang cho cc khng nguyn c to dng t cc c th gy bnh
khc. Cc chng Salmonella nhc c c xem l mt ng c vin tt
cho hng ny bi v chng c th c dng nh mt vaccine ung
kch thch cc phn ng min dch ni bo v bi tit trong vt ch. Chng
hn, gen ca tiu n v B khng n nhit (heat labile B subunit)
enterotoxic E. coli c a vo trong chng Salmonella nhc c AroA.
Vi khun Salmonella ti t hp ny c th cm ng cc khng th IgG v
IgA i vi tiu n v B enterotoxic (cng nh Salmonella) trong cc ng
vt c chng nga vaccine.

3.2.4. Cc th khm ca virus bi lit (poliovirus)
Poliovirus chng Sabin type 1 sng nhc c l mt vaccine rt an
ton v hiu qu, kch thch cc p ng khng th tun hon v bi tit. S
hiu bit v cu trc tinh th ca virus cng vi kh nng to virus t cc
phn t cDNA cho php cc vng khng nguyn ca cc tc nhn gy bnh
khc c hp nht chnh xc trong tiu th virus hu ht cc v tr khng
nguyn. V d: DNA m ha cho v tr khng nguyn ch yu ca chng
Sabin type 1 c thay bng DNA ca chui peptide t HIV-1. Huyt thanh
min dch i vi peptide c xem nh l tiu th poliovirus ti t hp v
ngi ta nhn thy trong cc nghin cu min dch virus ti t hp c th
cm ng rng ri cc khng th trung ha anti-HIV.

3.2.5. Cc chng E. coli ti t hp
Chng enterotoxigenic E. coli (ETEC) gy bnh i chy ln con v
mt vi trng hp ngi (E. coli type huyt thanh O 157). Cc vi khun
ny bm cht vo rut ca vt ch qua fimbrae lin kt b mt tit c t
n nhit (ST-toxin) v khng n nhit (LT-toxin). Fimbrae l khng nguyn
mnh v cc vaccine u tin chng ETEC gm c cc t bo hon chnh
hoc cc dch chit v bo c lm giu chng li fimbrae. Cc vaccine
c bo ch t cc chng ETEC sinh ra cc phn ng bt li ng k do
cc nng cao ca lipopolysaccharide v cc khng nguyn v trn b mt
ca chng ETEC hoang di. Chng vi khun E. coli K12 gy ra phn ng
bt li t hn nhiu v c dng nh l mt vt ch xy dng plasmid
vector biu hin mt hoc nhiu loi khng nguyn khc nhau ca fimbrae.
Tuy nhin, sn xut mt vaccine c phm vi bo v rng th cn a vo
cc thnh phn khng c t. Cc plasmid vector c xy dng bng cch
s dng promoter mnh ca prokaryote biu hin cao LT toxin B subunit.

4. Cc hng tip cn khc trong sn xut vaccine
4.1. Cc DNA vaccine (min dch di truyn)
Mt iu kh ngc nhin l cc plasmid mang cc gen c hiu cho
mt hoc nhiu protein khng nguyn c th c dng to min dch
(chng nga). Cc plasmid c phn phi bng cch tim (thng vo c)
a gen trc tip vo trong mt s t bo v c hp thu bi cc t bo ln
cn ni kim tim c a vo. Ngoi ra, plasmid cng c th c phn
phi bng sng bn gen (cc vin n vng c bc DNA ca plasmid)
bng cch y cc plasmid vo trong cc t bo gn b mt c th, c trng
cho loi ny l t chc da hoc mng nhy. Mt khi vo bn trong t bo,
mt vi plasmid ti t hp s i vo nhn v do gen c iu ha bi mt
promoter mnh ca eukaryote, cc t bo s tng hp cc khng nguyn
c m ha bi plasmid.

Ti u cc p ng min dch
Cc p ng i vi DNA vaccine thng cn mt lng nht nh
ca plasmid DNA ( 50 g trn mt ln tim) v hiu qu min dch khng
bng tim chng t nhin vi tc nhn gy bnh hoc c kch thch bi
cc vaccine sng nhc c. Tuy nhin, cc p ng min dch c th c
ci thin bng cc phng php khc, chng hn dng sng bn gen a
DNA vo t bo.
Phn ng min dch i vi khng nguyn b nh hng bi DNA ca
plasmid mang gen. Do DNA ca plasmid (c ngun gc t vi khun) c t
l CG ln hn so vi DNA trong ng vt c xng sng, hn na cc n
v CG trong plasmid ca vi khun c xu hng khng methyl ha (khng
gn cc nhm methyl) trong khi ng vt chng thng c methyl ha.
Vic tng s lng cc chui kch thch min dch trong plasmid c th s
khuch i tt kh nng sinh min dch ca cc khng nguyn c m ha
trong DNA vaccine.

4.2. Cc peptide vaccine
Trong nhng nm gn y, ngi ta c th phn lp cc t bo nhnh
(dendritic cells) t ty xng hoc mu ngoi bin ca ngi bnh v nui
cy chng trong iu kin in vitro. Cc t bo ny sau c nhi peptide
(hoc mt ngun khng nguyn khc) v a tr li vo trong ngi bnh.
Cc t bo hin din khng nguyn chuyn nghip ny sau s to ra mt
p ng min dch hiu qu m khng th to ra bng cch khc. Liu php
min dch ny c tp trung nghin cu iu tr ung th v c th lin
quan vi mt vi loi tc nhn gy nhim. Hn ch chnh ca hng k
thut cao ny l cn phi c bit kin nhn v v th rt t tin.

4.3. Khng cc kiu gen c th (anti-idiotypes)
Anti-idiotype (anti-Id) cng c th to ra cc vaccine hiu qu, v cc
khng th t chng cng hot ng nh l cc cht khng nguyn
(immunogens). Mt p ng min dch tng ln chng li v tr lin kt
khng nguyn n nht (unique antigen combining site) ca mt khng th
c gi l mt phn ng anti-idiotype v c th tng ng cu trc vi
khng nguyn gc u tin. Khi hin tng ny xut hin th khng th anti-
idiotype (n dng hoc a dng) c th gy ra mt p ng khng th nhn
dng khng nguyn gc v do hot ng nh mt vaccine. Cc anti-
idiotype cho thy mt kh nng bo v trong cc h thng ng vt m hnh
khc nhau. Th d in hnh v tim nng ca hng ny l vic bo v con
tinh tinh khi bnh phi hp HBV (HBV-associated disease) bi min dch
trc vi anti-Id.

III. Khng th n dng
Cng ngh sn xut khng th n dng da trn nguyn l s dng t
bo lai (gia t bo ung th myeloma vi t bo lympho B ca h min dch
ng vt hoc ngi) sn xut khng th. Khng th n dng c rt
nhiu ng dng quan trng trong y hc: pht hin khng nguyn, c ch
phn ng o thi khi ghp c quan, chn on s hnh thnh khi u, nh
hng thuc cha bnh bng khng th n dng, s dng khng th n
dng tinh sch protein

1. Sn xut hybridoma bng cch dung hp t bo sinh dng
Trc y, ngi ta cng to ra khng th bng k thut nui cy t
bo, nhng theo phng php ny, ngi ta phi nh k lm li sau mi ln
thu c khng th, v iu kin nui cy trong ng nghim, cc t bo
thng ch tin hnh mt s ln phn bo nht nh ri b hy.
Trong cng ngh sinh hc, k thut t bo lai hybridoma (lai gia t
bo myeloma vi t bo lympho B) m ra mt phng thc mi trong
min dch hc, sn xut vaccine hng lot. Ngi ta to ra mt loi t
bo lai c th phn bo lin tc trong iu kin nui cy, ng thi li c
kh nng sn sinh ra khng th, t khng th c sn xut ra vi khi
lng rt ln. T bo ung th c c im rt quan trng l loi t bo c
tim nng phn chia lin tc (di cn). Kh nng sinh sn lu di v phn bo
lin tc ca t bo ung th nui cy c lin quan vi kh nng tng sinh ca
cc t bo ung th trong c th sng. Ngi ta chn mt loi t bo c kh
nng sinh ra khng th, hin nay ang s dng t bo lch ca chut nht
c min dch chng mt loi khng nguyn nht nh. Tin hnh lai gia
t bo lch chut nht vi t bo ung th ca ty xng. T bo lai em
nui cy, phn chia lin tc, sn sinh ra mt lng rt ln khng th. Khng
th ny c trng cho mt dng t bo, nn c gi l khng th n dng.
Qu trnh trn chnh l hin tng lai t bo, tc l kh nng dung hp gia
mt loi t bo c kh nng ti bn, sinh sn pht trin khng ngng vi mt
loi t bo c chc nng to ra khng th (Hnh 8.2).


Hnh 8.2. S sn xut khng th n dng

2. Sn xut khng th n dng bng cng ngh DNA ti t hp
2.1 . Phn lp cc gen immunoglobulin
Nh k thut PCR vn tr nn tng i n gin khi phn lp v
to dng cc gen immunoglobulin t cc t bo B (thun thc hoc cha
thun thc) v cc t bo hybridoma bng cc cp primer c hiu. Nguyn
liu khi u thng l mRNA hn l DNA, v mRNA ch cha cc exon
m ha, cc intron b ct b (spliced out) trong sut qu trnh phin m. Hu
ht s quan tm u tp trung vo vic phn lp v to dng cc vng
immunoglobulin c th thay i (immunoglobulin variable regions) ca
chui nng (V
H
) v chui nh (V
L
) cha cc vng b tr xc nh
(complementarity determining regions-CDRs) cn thit trong lin kt khng
nguyn. Bng cch lin kt cc sn phm ca cc gen V
H
v V
L
cc on
chui n c th thay i (scFV) s c to ra, cc sn phm protein ca
chng c kh nng lin kt vi khng nguyn. Cc cu trc scFV ny
Sn xut khng th c
cm ng trong chut
bng gy to min dch
Cc t bo gii
phng khng th
c phn lp
Cc t bo khi u
sinh trng in vitro
Cc t bo gii phng khng th
v cc t bo khi u c dung
hp to thnh hybridoma
Cc hybridoma gii phng
khng th c to dng
Cc khng th n dng c phn lp
c th c a vo trong cc phagemid vector th hin b mt cc
bacteriophage dng si kt i vi mt protein v khng quan trng hin
din mt u ca phage. Cc th vin gen khng th ca ngi c
xy dng theo phng thc ny t cc gen c phn lp ca cc t bo B
trong mu ngoi vi ca c nhng ngi b nhim HIV v nhng ngi mnh
khe.

2.2. Biu hin scFV trn b mt bacteriophage
Cc th vin ca phage trong E. coli TG1 c sinh trng trong mi
trng cha glucose v helper phage (v d M13KO7) c b sung gy
nhim ln th hai (superinfect) cc vi khun. Cc t bo b gy nhim ln
hai sau c ly tm nhanh v cho sinh trng qua m trn mi trng
khng c glucose nhng c b sung ampicillin v kanamycin, trc tin
cm ng gy ng gi phagemid biu hin scFV phi hp vi protein ca
gen III v sau to thnh phage. Cc vi khun nguyn vn c to tiu
th bng ly tm v phage c kt ta t cc th ni vi PEG. Cc bc ny
cho php tinh sch v c c phage thnh stock dng trong chn lc theo
vng trn trn khng nguyn

3. Khng th n dng trong nghin cu Sinh-Y
Cc khng th n dng l nhng tc nhn phn ng cn thit
phn lp, nhn din v xc nh v tr ca cc sn phm c trng ca gen
trong t bo v tr gip trong vic xc nh cu trc i phn t ca
chng. Ni chung, kh c th tho lun chi tit v nhiu ng dng ca
khng th n dng trong nghin cu c bn. Thay vo , chng ta tp
trung chnh v cc ng dng trong chn on v iu tr.
- Cc khng th n dng da theo cc ch th t bo cht v b mt t
bo c mt nh hng khng ch trong nghin cu c bn m cn cho php
pht trin cc phng thc trong iu tr. Chng hn, s biu hin d tha
ca th th i vi nhn t sinh trng biu m (EGF) v cc th th lin
quan, mt sn phm ca proto-oncogene c-erbB-1, c tm thy l ch th
ca s tin lng ngho nn trong cc bnh nhn mang ung th biu m t
bo vy hoc u tuyn. Cng nh vy, nhiu cht ch th b mt ca t bo
c pht hin trn leukocyte c s dng nhn dng cc u c tnh v
quyt nh phng php iu tr.
- Phn tch min dch bng phng php RIA v ELISA, da trn
nguyn tc dng khng th n dng pht hin khng nguyn tng ng
ca n trong cc mu phn tch nh phn ng gn rt c hiu ca khng th
n dng vi khng nguyn.
- c ch thi loi khi ghp c quan bng cch s dng khng th n
dng chng khng nguyn c hiu ca t bo lympho T, nhm loi b phn
ng thi loi khi ghp cc c quan ca ngi vi nhau.
- Chn on hnh thnh khi u, khi c s hnh thnh khi u trong mu
s xut hin mt s tc nhn nh du. Do , c th s dng cc phng
php phn tch min dch pht hin cc tc nhn trn trong mu hoc trn
khi u.
- nh hng thuc cha bnh, li dng kh nng cc phn t khng
th gn rt c hiu vi t bo v tr xc nh no ca c th (trong
c t bo ung th) nh hng thuc cha bnh hoc c cht ti trc
tip khi u hoc ni cn cha tr nh vy s tng hiu qu cha tr ln nhiu
ln.

4. Khng th n dng trong chn on v iu tr bnh
Khng th n dng c cng dng c bit quan trng, c s dng
chn on bnh truyn nhim, bnh di truyn, d ng, ri lon min dch.
Dng khng th n dng l phng tin rt nhy v n gin chn on
c thai, cc bnh lao, phong...
Khng th n dng cng c s dng chng th thai, lm trit
sinh gia sc. Khng th n dng cn c dng chn on di cn ung
th nu c gn thm ng v phng x.
Phng php s dng khng th n dng nhanh chng thay th
cc phng php min dch hc v huyt thanh hc truyn thng trong cc
xt nghim, nh:
- Xc nh lng hormone nh gi chc nng ni tit.
- Pht hin mt s protein c ngha chn on khi u.
- Xc nh vi khun v virus gy bnh.
- Pht hin trong mu cc cu th, cc cht kch thch b cm s dng,
kim tra nng thuc trong mu v cc t chc c th nhm bo m liu
thuc dng khng qu ngng gy c.
Khng th n dng cn c dng c ch phn ng o thi khi
cy ghp c quan (ghp thn, truyn ty...). Gn y nht, khng th n
dng cn c dng pht hin bnh AIDS (hi chng suy gim min
dch).
Ngi ta thnh cng khi tim khng th n dng cho b ci, cu
ci, v cu c hng triu sc vt s sinh khi cht do vi khun
Colibacillus enteritis.

IV. Liu php gen
i vi loi bnh di truyn, bnh do t bin gen ngi ta phi cn
n s can thip ca liu php gen, mt hng cha bnh gn lin vi cc
k thut tin tin trong lnh vc cng ngh sinh hc hin i nh cc vi thao
tc gen, sa i thay th gen...
Liu php gen (gene therapy) thc cht l phng php cha bnh
bng gen. C nhiu khi nim khc nhau v liu php gen, nhng cch hiu
chung nht l tp hp cc bin php s dng cc gen cn thit (cn gi l
gen tr liu) nhm mc ch cha bnh cho con ngi (Hnh 8.3).
Trong , gen tr liu c th l:
- Cc gen hot ng bnh thng (gen lnh) c th a vo t bo
thay th gen hng, gen mt chc nng, khi phc hot ng bnh thng
ca t bo v s sng ca c th.
- L nhng gen c kh nng m ha mt protein c hiu khi a
vo t bo sng c th to nn cc loi protein c hiu. Cc protein c
hiu c th c ch hot ng ca mt gen khc trong t bo, km hm kh
nng phn chia ca t bo hoc gy cht cc t bo b bnh.
- Nhng gen khi a vo t bo hot ng ng thi vi cc gen
bnh (gen b t bin trong t bo) lm hn ch tc ng ca gen bnh hoc
h tr, b p cho cc gen b hng.
- Gen liu php cn l cc gen bt hot c a vo t bo thay th
cho mt gen lnh no , nhm hn ch sn phm khng cn thit ca gen
lnh hoc to ra cho t bo mt trng thi mi, c tc dng chng li bnh
tt.
- Gen tr liu c th l nhng on oligonucleotide c tc dng km
hm hot ng ca gen b hng, b t bin trong t bo.

Hnh 8.3. M hnh chung ca liu php gen

1. Cc loi liu php gen
Liu php gen c chia lm hai nhm phng php c bn l liu
php gen soma v liu php gen giao t (cn gi l liu php t bo mm).

1.1. Liu php soma (somatic gene therapy)
L phng php iu tr thay th hoc sa cha gen hng, gen bnh
ca cc t bo soma trong c th ngi bnh, gip ngi bnh c th c
cuc sng bnh thng. Liu php gen soma c th s dng mt s loi t
bo nh: t bo lympho (lymphocyte), nguyn bo si (fibroblast), t bo
gc (stem cells), t bo gan (hepatocyte), t bo biu b (keratinocyte)...
Liu php gen soma n nay cha v iu tr khi hoc hn ch biu hin
cho mt s kh ln ngi bnh mang cc bnh him ngho nh: thiu ht
min dch phi hp trm trng (SCID), ung th, thiu mu hng cu lim,
x nang...

1.2. Liu php gen t bo mm (germline gene therapy)
L phng php iu tr, sa cha hoc thay th gen hng cho cc
giao t (tinh trng hoc t bo trng) nhm to ra th h sau bnh thng.
T bo ngi
Gen thay th
Virus mang
gen thay th
Nhn
Cc protein ca
gen thay th
Liu php gen t bo mm hin nay cn l vn ang c tranh lun
nhiu nc trn th gii ch yu v l do o c v nhn o, chng hn
vn nhn bn ngi.
2. Cc ng dng ca liu php gen trong cha bnh
2.1. Bnh thiu ht min dch phi hp trm trng (severe combined
immuno deficiency-SCID)
Bnh SCID l bnh thiu ht min dch phi hp trm trng ngi,
gy gim st sc khe hoc c th gy cht do d b mc cc bnh truyn
nhim khc. Bnh SCID do ri lon di truyn, gy mt chc nng t bo
min dch lympho T, lympho B, t bo NK (t bo git t nhin, natural
killer cell)... C nhiu biu hin bnh SCID khc nhau, ty theo mc sai
hng ca cc gen (defective gene), dn n ri lon chc nng ca cc loi
t bo lympho gy thiu ht min dch c th ngi. Bnh SCID ngi
c chia lm hai nhm: bnh SCID lin kt nhim sc th gii tnh X (X-
SCID, chng hn: sai hng gen c nm locus 13.1 trn cnh di ca
nhim sc th gii tnh X ca ngi) v bnh SCID do sai hng cc gen
(ADA, PNP, CD
3
v ZAP 70...) nm trn nhim sc th thng.
2.1.1. Liu php gen cha bnh ADA-SCID
Gen m ha enzyme adenosine deaminase b hng, dn n ri lon
chc nng ca cc lympho T v B gy bnh ADA-SCID. Liu php gen
ex vivo gm c bn bc c bn sau:
- Ly cc t bo lympho T t mu ca bnh nhn ADA-SCID.
- Thit k vector liu php retrovirus mang gen ADA bnh thng.
- Trn vector liu php vi t bo lympho T thc hin qu trnh
chuyn gen vo t bo lympho T.
- a cc t bo lympho T mang gen liu php tr li c th ngi bnh.
Kt qu sau khi iu tr liu php gen t 5-6 thng s t bo min dch
lympho T tng dn ln, sau hai nm kh nng min dch ca bnh nhn
c phc hi.
2.1.2. Liu php gen cha bnh X-SCID
Liu php gen iu tr loi bnh ny c thc hin bng phng
php ex vivo ghp t bo ty xng. T bo ty xng ca bnh nhn
c ly ra kt hp x tr, sau chn lc nhng t bo khe chuyn gen
c bnh thng nh retroviral vector. Kt qu sau khi iu tr t 3-13 thng
th 10/11 bnh nhi khi phc c cc t bo lympho T, B v NK, kh
nng min dch tng ln rt dng k.
Liu php gen SCID thnh cng , Israel, Anh, Php v M.

2.2. Bnh ung th
Bnh ung th cho n nay vn c coi l mt trong nhng bnh nan
y, lm gim st sc khe con ngi, v l loi bnh c t l gy t vong
cao. Ung th l s pht trin khng bnh thng ca t bo, do sai lch hoc
t bin mt hoc mt s gen trong t bo. Cc t bo ung th c qu trnh
phn chia v t chc, khng chu s kim sot ca s cht theo chng trnh
(apoptosis), to nn cc dng khi u. C hai nhm nguyn nhn ch yu gy
ung th l: do t bo v c th b nhim virus gy ung th v t bin gen
do nh hng ca cc tc nhn vt l v ha hc (tia UV, cht gy phng
x, khi thuc l, cht c ha hc...).
iu tr ung th c th thc hin bng x tr, ha cht, cc dng c t
min dch hoc liu php gen. Trong iu tr ung th nhiu cytokine c
ch v th nghim, trong interleukin 2 (IL-2) c hiu qu iu tr ung
th cao, thi gian iu tr ngn, t gy cc tc dng ph nh iu tr bng
ha cht v x tr. Liu php gen (in vivo v ex vivo) l mt trong nhng
phng php mi iu tr ung th c hiu qu. Bng k thut vi tim, ngi
ta a thng vo gia khi u cc vector mang gen tr liu tng ng, c
ch hoc tiu dit cc t bo ung th.
Trong liu php gen iu tr ung th ngi ta s dng nhiu loi
vector liu php khc nh retrovirus, adenovirus v plasmid.
Liu php gen iu tr ung th bng cch tc ng trc tip vo khi
u, c th c thc hin theo nhiu phng thc khc nhau:
- c ch pht trin ca khi u bng cch thc hin cc liu php ex
vivo hoc in vivo vi cc viral vector mang cc gen liu php nh p53,
pRb...
- Kch hot t bo cht theo chng trnh bng cch: s dng cc gen
liu php Bax tng cng gy kch hot cht theo chng trnh, hoc s
dng gen Bcl-2 ngn chn cc phn ng chng apoptosis (anti-apoptosis).
Trng hp apoptosis cc t bo co li, tch khi cc t bo xung quanh,
cc gen ced (gen t st-suicide gene) c hot ha lm cho t bo teo dn
ri cht.
- Thc hin cc liu php thay gen nhm phc hi cc protein b hng,
phc hi kh nng min dch ca t bo.
- S dng liu php antisense km hm hot ng ca cc oncogen,
hoc c ch tc dng cc sn phm ca oncogen.
- Hn ch s pht trin ca khi u bng vi tim cc protein tin
apoptosis (pro-apoptosis protein) nh cc protein BAX (sn phm ca gen
Bax) hoc interferon (TNF), kt hp s dng x tr hoc iu tr ha cht.
- S dng cc gen t st ced v cc sn phm ca n tim thng
vo khi u, chng hn tim ganciclovir-sn phm ca gen m ha enzyme
thymidin kinase c tc dng tiu dit cc t bo ung th.
- Bin php tc ng trc tip vo t bo ung th bng bao vy thnh
mch (anti-angiogenic). y l bin php chn mi lin h v mt dinh
dng ca t bo ung th, lm cho t bo ung th thiu dinh dng v cht
dn dn. Hin nay, pht hin hn 40 hp cht t nhin c chc nng ny
nh: endostatin, angiostatin...

2.3. Bnh thiu mu hng cu lim
Bnh thiu mu hng cu lim SCA (sickle cell anemia) (Hnh 8.4) l
bnh do t bin gen globin lm thay i chc nng phn t protein, xut
hin hemoglobin S. Khi cc phn t hemoglobin S trao i oxygen vi cc
t bo, c th lin kt vi nhau to cc cu trc hnh que, lm cho hng cu
bin dng thnh hnh lim. Hng cu lim khng c hnh lm hai mt nh
hng cu bnh thng, trn km khng chui qua c cc mao mch,
thng xp li thnh m gy tc mao mch gy cn tr qu trnh lu thng
ca mu. Sau mt thi gian s lng t bo hng cu bnh thng gim dn,
do cc t bo hng cu mi sinh ra khng b p kp s t bo hng cu
cht i, trong mu thiu mt lng hng cu ngy cng ln, gi l bnh
thiu mu hng cu lim.


Hnh 8.4. T bo hng cu bnh thng v t bo hng cu lim

Cc nghin cu trn ngi, chut v cc ng vt c v khc u xc
nh r nguyn nhn gy bnh thiu mu hng cu lim do t bin gen
globin. im t bin v cc dng t bin c nghin cu rt k. Tuy
nhin, s dng liu php gen cha bnh SCA ngi thu c kt qu rt
hn ch.

2.3.1. Vector liu php s dng trong iu tr bnh SCA
Trong cc loi vector liu php, lentivirus vector c nh gi l loi
vector c hiu qu nht, v lentivirus gip cho cc gen liu php ha nhp
rt tt. Tuy nhin, nhiu loi virus trong h lentivirus l nguyn nhn gy
nhiu bnh him ngho ngi nh AIDS, lao, ung th... nn s dng
lentivirus lm vector liu php cn nhiu hn ch. Cc lentivirus c th bin
i rt mnh v nhanh, nn c th gy cc hu qu khng lng trc cho
c th ngi. V vy, ngi ta thng s dng cc retrovirus quen thuc
thit k vector liu php nhm hn ch bt cc ri ro.

2.3.2. Cc bc iu tr bnh SCA bng liu php gen
- Tch t bo ty xng ca ngi bnh v chiu x chn cc t
bo khe mnh, trn chung vi retrovirus vector mang gen globin bnh
thng to cc t bo ty xng ti t hp.
T bo hng cu
bnh thng T bo hng cu lim
- X tr v iu tr ha dc tiu dit bt cc t bo ty xng
mang gen t bin.
- Chuyn cc t bo ty xng mang gen globin bnh thng vo
bnh nhn. Sau mt thi gian cc t bo ny sinh trng v bnh thiu mu
gim dn.
- Do lng t bo mang gen t bin (
S
globin) vn tip tc c
hnh thnh trong c th ngi bnh, nn sau mt thi gian nht nh mc
bnh thiu mu li tng ln, do phi lp li cc bc iu tr gy nhiu
tn km.
Hin nay, cc th nghim liu php gen khc nhau trn chut cho kt
qu rt tt. V th, hy vng trong mt tng lai khng xa bnh thiu mu
hng cu lim ngi s c cha khi bng liu php gen.

2.4. Bnh x nang
Bnh x nang (cystic fibrosis-CF) xut hin do dt bin gen trn
nhim sc th thng, gy ri lon trong chuyn ha cc ion Cl
-
trong t bo
dn n chng vim phi, vim ty, vim gan... gy gim st sc khe, c
th dn ti t vong do b suy h hp nng. Bnh x nang l mt bnh di
truyn, c t l mc bnh l 1/2.500 ngi. Bnh x nang do mt loi
protein mng (CFTR) b mt chc nng, khng c kh nng a cc ion Cl
-
tha ra khi t bo, lm mt cn bng gia nc v mui trong t bo. Bnh
x nang thng hnh thnh mt lp dch nhy gy ri lon chc nng
protein mng, lp dch nhy ngy cng c cc m t bo b bnh CF, gy
ra cc chng bnh v phi, gan, ty v rut...
iu tr bnh x nang ngi bng liu php gen thu c nhng
kt qu tng i tt. Cc xu hng s dng liu php gen hin nay bao
gm:
- S dng vector liu php mang gen tr liu cDNA (do kch thc gen
CF rt ln nn ch tng hp cDNA t mRNA m ha protein CFTR v mt
s trnh t c hiu) lm cho gen liu php gn vi nhim sc th nhng
im c hiu.
- Thc hin phng thc sa cha gen bng vector mang cc
oligonucleotide sa cha t bin trn gen CF.
- S dng k thut episomal, ngha l chuyn cc on cDNA cn
thit vo t bo, sao cho cDNA vo nhn dnh vi h gen mt im no
v tn ti nh mt n v c lp trong t bo.

2.5. Bnh HIV/AIDS
Bnh AIDS (acquired immuno deficiency syndrome) l hi chng suy
gim min dch mc phi do nhim virus HIV (human immuno-deficiency
virus), lm mt sc khng ca c th vi cc tc nhn gy bnh, c th
dn ti ung th hoc t vong.
- Cc xu hng iu tr nhim HIV/AIDS bng liu php gen.
Trong nhiu nm qua, nhiu loi khng sinh v bit dc (starudine,
lamirudine, nerirapine, azathioprine, antiretrovir...) c nghin cu v th
nghim trong iu tr chng nhim HIV/AIDS, c nhng kt qu tt gp
phn ko di cuc sng cho nhiu bnh nhn AIDS. Tuy nhin, trong nhiu
trng hp nhim HIV iu tr bng cc loi thuc trn khng c hiu lc
do mt s chng HIV c th c t bin thch ng vi thuc.
hn ch nhim HIV v i dch AIDS, trong nhng nm gn y
mt hng mi trong iu tr AIDS c ch nghin cu v th nghim l
liu php gen:
+ S dng vector liu php chuyn cc gen tr liu vo t bo nhm
hn ch kh nng xm nhim v ti bn ca ca HIV. Trong trng hp ny
ngi ta s dng c hai loi vector: viral virus (adenovirus, AAV v HIV-
1), non-viral virus (oligonucleotide, polycation, liposome...).
+ Kch hot h thng gen ca t bo, tng cng min dch chng s
pht trin ca HIV/AIDS.
+ S dng cc gen t st ced bin i, lm cho t bo b cht khi c
HIV xm nhim. Khi HIV mi xm nhim t bo, bng liu php a cc
gen t st (ced) thymidin kinase (tk) phi hp s dng thuc chng virus
ganciclovir (GCV), to thnh phc hp ganciclovir-phosphate. Ganciclovir-
phosphate c ch hot ng ca enzyme DNA polymerase, lm cho qu
trnh ti bn gen ca t bo b ngng, s phn chia t bo b gin on, t
bo co li v cht dn. T bo nhim HIV b cht, s hn ch kh nng ly
lan ca HIV trong c th.
+ To cc DNA vaccine phng chng nhim HIV. DNA vaccine c
xem l mt chin lc hiu qu ca liu php gen phng chng HIV/AIDS.
DNA vaccine HIV-1 v ang c th nghim trn chut c hiu qu.
Hin nay, Trung Quc va bt u th nghim DNA vaccine phng chng
HIV/AIDS trn ngi, hy vng s c nhng thnh cng qu gi.

V. Protein tr liu
Nhiu cht c hot tnh sinh hc dng lm dc phm c to thnh
trong qu trnh nui cy t bo ng-thc vt nguyn vn trong h ln men
(fermenter/bioreactor). Chng hn: tng hp interferon trong qu trnh nui
cy t bo nguyn bo bch huyt (lymphoblast); k thut to khng nguyn
lm nguyn liu sn xut vaccine v to khng th n dng; sn xut
hormone... Tt c chng u l nhng sn phm y hc cao cp v c tng
lai rt ln.

1. Sn xut hormone
1.1. Hormone sinh trng ngi (human growth hormone-HGH)
Hormone sinh trng ngi l protein cha khong 191 amino acid,
thiu n c th ngi s b ln. Trc y hormone pht trin c tch t
tuyn yn ca ngi cht. Hin nay, hormone pht trin c sn xut bng
cng ngh DNA ti t hp thng qua E. coli (c insulin v hormone sinh
trng ngi l cc protein tng i n gin v khng b glycosyl ha v
hoc bin i nhiu sau dch m, nn c th dng E. coli lm t bo vt ch
c sn xut chng). Hormone sinh trng ngi ti t hp khc vi
hormone sinh trng ngi bnh thng mt amino acid l do E. coli
khng c kh nng loi b gc methionine khi u, thng thng amino
acid ny ch b loi sau khi dch m trong t bo ngi.
Hormone sinh trng ngi l mt trong nhng protein c sn
xut bng cng ngh DNA ti t hp sm nht. Trc y, mun c ch
phm ny ngi ta phi tch chit t tuyn yn ca ngi cht. Mi t thi
cho khong 4-6 mg HGH v mun cha khi cho mt ngi ln phi cn
lng HGH thu c t 100-150 t thi. iu ny cho thy mt tr ngi rt
ln khi cha tr chng ln cho tr em. Bng cng ngh DNA ti t hp,
ngi ta c th thu nhn mt lng ln HGH t vi khun E. coli ti t
hp (1 lt dch ln men ca E. coli thu c mt lng HGH tng ng
vi lng thu c t 60 t thi).

1.2. Somatostatin
Somatostatin l loi hormone c bit, thng c tng hp trong
no ng vt v ngi vi mt hm lng v cng thp. Somatostatin c vai
tr iu ha hormone sinh trng v insulin i vo mu, kim tra s tng
hp hai loi hormone ny. Quy trnh sn xut loi sn phm sinh hc c
bit qu ny cng bao gm cc bc ch yu sau y:
- Phn lp gen m ha somatostatin hoc tng hp trong ng nghim.
- To dng gen somatostain bng cch gn gen ny vo plasmid vector
chuyn gen vo E. coli. Mi m 7,5 L vi khun E. coli nui cy s cho ra
5 mg somatostatin nguyn cht. Khi lng hormone ny trc y mun
c phi tin hnh c nm trn nguyn liu ly t na triu no cu.

2. Sn xut enzyme
Trong mt tng lai gn enzyme s c s dng rt rng ri trong y
hc lm u d cho cc thit b phn tch y khoa v cha bnh. Hin
nay, mt s enzyme nh: glucooxydase, hexokinase, esterase, urease,
cholesteroloxydase, alcoholdehydrogenase c s dng kh rng ri
trong y hc. in hnh nht l s dng glucooxydase c nh trn b mt
in cc platinum trong thit b phn tch hm lng glucose trong mu.
Trong y hc, enzyme cn c s dng cha tr mt s bnh lin quan
n s thiu ht ca mt s enzyme trong c th ngi, hoc dng enzyme
loi b nhng cc tht, m d tha gy nguy him cho mt s b phn
ca c th ngi. V d dng enzyme streptokinase v urokinase lm tan
mu ng gy tc nghn mch mu.

3. Sn xut thuc nh cng ngh DNA ti t hp
y l cng ngh thay i b my di truyn ca t bo bng cch a
gen ngoi lai vo v bt n hot ng. C ngha l bt n to ra mt s
lng ln protein hoc peptide cn thit, m trc bn thn t bo khng
tng hp c. C th ni l cng ngh ny to ra mt bc ngot ln
trong lnh vc sinh hc. Cc sn phm do n to ra a dng v mt s hin
ang c sn xut mc cng nghip.

3.1. Sn xut insulin
Insulin l protein cha 51 amino acid c tng hp tuyn ty ca
ngi. Vo u nhng nm 80 hng Eli Lilly v Gentech Inc. (M) a
ra quy trnh sn xut insulin ngi bng cng ngh DNA ti t hp. l
protein u tin to c bng cng ngh gen v nh cng ngh ny m
hin nay insulin c sn xut i tr vi gi thnh r hn nhiu so vi
trc kia.
Trong c th ngi bnh thng, tuyn ty tit ra mt loi hormone c
chc nng iu ha glucose trong mu. Khi nng glucose trong mu ln
qu ngng, th insulin tc ng ln t bo gan tng cng chuyn ha
ng glucose tha trong mu thnh glycogen, l loi ng d tr. Trng
hp insulin tng hp t nhin trong c th khng , glucose phi thi ra
qua ng nc tiu, gy nn bnh tiu ng. Ngi b bnh tiu ng
ch c cu sng nu c b sung vo c th mt lng insulin nht
nh. Trc kia insulin t v t, kh p ng nhu cu ca nhng ngi b
bnh tiu ng. Ngy nay, vi cng ngh sinh hc hin i, ngi ta
khc phc c kh khn ny p ng nhu cu cho nhng ngi b bnh.
Cng ngh sinh hc hin i sn xut insulin trong vi khun E. coli
bng cch:
- Tch gen ph trch tng hp insulin trong c th sng, c th t
ngi hoc t chut. Gen insulin cng c th c tng hp nhn to trong
ng nghim.
- To dng gen insulin bng cch gn gen insulin vo plasmid vector
sau chuyn plasmid ny vo t bo vi khun nhn (E. coli) sn xut
mt lng sinh khi ln phc v cho vic tch chit insulin.

3.2. Sn xut interferon
Interferon l nhm protein cha khong 146-166 amino acid, chng
xut hin trong t bo ng vt c xng sng khi b nhim virus v gip t
bo khng li virus. Cho n thi gian gn y, interferon ch yu vn c
sn xut t t bo bch cu hay nguyn bo bch huyt ca ngi b nhim
virus. Tuy nhin, bt u t nm 1980 ngi ta phn lp c cc gen
m ha tng hp ba loi interferon trn v xy dng c cng ngh sn
xut interferon t t bo E. coli v nm men.
Interferon l loi protein tng i n gin, chu c nhit ti
65
o
C, l sn phm ca t bo sng, nn bn thn n khng c. Chng
trnh sinh tng hp t nhin interferon c m ha trong gen, nhng s
tng hp ny ch xy ra khi c virus xm nhp c th.
Cc thnh tu ca cng ngh sinh hc hin i m ra mt cuc
cch mng trong nghin cu cu trc v hot ng ca interferon. C ba loi
interferon l , v khc nhau bi s amino acid v hot tnh:
- -interferon do bch cu sn xut khi c virus xm nhp.
- -interferon do nguyn bo si sn xut khi c virus xm nhp.
- -interferon do bch cu sn xut qua phn ng ca h min dch.
V c ch, interferon khng tc ng trc tip ln virus, m tc ng
ln t bo, cc t bo ny tng hp ra loi protein c bit, c kh nng
km hm s pht trin ca virus xm nhp c th. Hin nay, ngi ta pht
hin c nm loi protein ny. Khi virus xm nhp t vo c th, nh khi
c nn dch virus, th interferon t nhin khng chng li virus. Trc
y, ngi ta phi dng mu tch interferon t bch cu dng trong iu
tr vim gan do virus, nhng rt t. Nu tch t mu ra ly interferon,
th d c tch t mu ton b loi ngi cng khng cha bnh cho
mt ngi.
Bng cng ngh sinh hc hin i, ngi ta tch gen interferon t
c th sng, to dng trong plasmid vector v chuyn gen ny vo vi khun
E. coli, vi khun ny sn xut interferon vi hiu sut ln, do thnh
tu ni trn tr thnh cuc cch mng v interferon.
-interferon hin nay c dng c hiu qu nht, iu tr bnh
vim gan C, nhng cn qu t. Cng vi vim gan do virus A v B, ngy
nay vim gan do virus C (tc vim gan C) ang l mi tai ha ln trn th
gii. Trong , phn ln bnh nhn b vim gan C, virus lm t lit h
thng min dch, sau c th dn ti x gan, v mt s trng hp tr
thnh ung th gan.

3.3. Sn xut interleukin
Tng t interferon, mt sn phm khc cng gip c th tng kh
nng min dch l interleukin cng c sn xut theo cng ngh DNA
ti t hp. Interleukin l mt lymphokine bao gm interleukin-1 (IL-1) v
interleukin-2 (IL-2), c kh nng iu ha min dch v chng ung th.
Hin nay, interleukin v interferon ang c th nghim cng vi mt s
bit dc khc cha tr bnh ung th v bnh AIDS.
Interleukin-2 (IL-2) l mt cytokine quan trng nht i vi s pht
trin v p ng min dch thch ng. IL-2 c vai tr chnh trong hot ha
cc lympho T, v d cc t bo c tnh lympho T bc l qu mc cc th
th IL-2, nn c th ngh rng IL-2 l yu t sinh trng ca t bo. Ngc
li, vic s dng tng cng chc nng ca IL-2, cng lm tng p ng
min dch chng li cc khi u. Liu php tim trc tip IL-2 vo mch mu
cng lm tng lng t bo lympho, lm tng t bo NK v lm tng hot
ng ca cc dng t bo kiu LAK. Liu php ny ang c s dng cho
mt s bnh ung th, nh ung th da c tnh (melanoma) v ung th biu
m thn.

VI. Chn on bnh can thip sm
1. Chn on sm gii tnh ca thai
Nh chng ta bit, st bn trong mng nhn t bo ca n c mt
th nhum mu m, m t bo ca nam khng c. T nm 1948, Murray
Barr pht hin vt th gii tnh ny (th Barr), gi l vt th gii tnh v
n gip phn bit nam v n. ng vt c v cng tng t, con ci th c
cn con c th khng.
Th Barr ny c cc loi t bo khc nhau nh t bo nim mc da,
nim mc m o, t bo chn tc, t bo cc tuyn... n nay, ngi ta c
th dng k thut chc i xt nghim th Barr, chn on sm gii tnh
v chn on d hnh, qui thai, nu cn phi s dng cc liu php can
thip sm.
nhim sc th gii tnh ca n v ng vt c v ci c XX, nam
v ng vt c v c c XY. Nhim sc th Y v cn bn, rt t gen. S
lng gen lin kt gii tnh ch yu nm trong X. c gen, th tt yu c
cc sn phm ca gen tng hp. Trong X, c cc gen tng hp nhiu loi
protein enzyme nh glucose-6-phosphatedehydrogenase, -globulin... Theo
suy lun thng thng, nu c hai X n hot ng, sinh tng hp protein,
th hm lng cc loi enzyme trn n s gp i nam. Tuy nhin, cc
th nghim ha sinh xc minh hm lng cc loi enzyme nu trn nam
v n u tng ng nhau.
ng vi hin tng cn bng di truyn nu trn, ngi ta khng
nh th Barr chnh l mt trong hai X ca n, chuyn sang trng thi d
nhim sc cht, khng hot ng di truyn, bt mu m. n v ng vt
c v ci, hai X ny c ngun gc mt t b v mt t m. Trong c th n
v ng vt c v ci, dng t bo ny th X t b khng hot ng, dng t
bo kia th X t m khng hot ng, hon ton theo tnh cht ngu nhin.
Trc y, ngi ta xt nghim t bo phi (hp t) pht hin th
Barr t 12-14 ngy sau khi th thai. Gn y, vi k thut tin tin hn,
pht hin c th Barr t ba ngy u sau th thai, phi cn mang c
im lng tnh, cha phn r nam, n nhng t ngy th ba sau th thai,
c th phn bit gii tnh thai nh k thut chc i.
Trong trng hp c bnh di truyn do gen trn nhim sc th gii
tnh (ch yu l trn X), vi k thut chc i xt nghim sm t bo phi
non, s dng phng php chn on enzyme, dng enzyme hn ch, ngi
ta c th xc nh gen t bin gy bnh. V gen bnh ny lin kt gii tnh
(nm trn X), cho nn k thut enzyme hn ch c th gip cho cc b m
c thai c phng hng quyt nh, hoc chp nhn liu php sy thai,
hoc khng, hy vng vo may ri, v trong trng hp ny kh nng thai d
hnh, qui thai c th khng xy ra, m cng c th l 100%, hoc ch 50%.
V d: mt ph n bnh thng nhng c mang gen mu khng ng dng
th d hp, c mt con trai b bnh mu khng ng, nay mun sinh con
tip. V khng c k thut chn on trc khi sinh i vi bnh ny, ngi
ta phi chp nhn chc i chn on gi, trai. Nu l gi, th yn tm sinh
, v nu chng l ngi bnh thng, th con gi ch mang gen bnh
dng th d hp, v vn l bnh thng, cn nu l thai nam, mun thc s
yn tm, th r rng phi no thai, nhng theo hng ny, cng c th c
n 50% trng hp thai nam no i vn l thai bnh thng.

2. Chn on sm d hnh, qui thai trc khi sinh
Tin hnh k thut chc i, kt hp ng thi phn tch mu b m
bng enzyme hn ch, ngi ta c th chn on sm trc khi sinh (v s
dng enzyme hn ch c kh nng phn bit c gen t bin vi gen bnh
thng). Cc on DNA ct ra, c phn tch qua phng php in di,
em lai vi cc mu d DNA hoc RNA nh du phng x bng
32
P.
nh phng x t ghi cho ta thy cc on DNA c lai vi cc mu d.
Cc on ny c tch ra nghin cu v xc nh t bin hay bnh
thng bng enzyme hn ch, v mt s t bin di truyn c nh hng n
cc v tr dnh cho enzyme hn ch.
V d: bnh thiu mu hng cu lim l do mt t bin ca gen quy
nh cu trc ca hemoglobin. Gen t bin ny dn ti bin i mt amino
acid trong chui ca phn t hemoglobin, t glutamic acid ngi bnh
thng (Hb
A
) thay bng valine ngi bnh (Hb
S
). Chu b b bnh ny
(Hb
S
Hb
S
) cht s sinh, c th c sinh ra t b, m b ngoi bnh thng,
nhng l th d hp v gen t bin (Ss Ss). Cp v chng ny li tip tc
c thai, mun bit ln ny con sinh ra c li b bnh khng, ngi ta dng
k thut chc i, xt nghim t bo thai non. Phn tch mu b, m bng
enzyme hn ch ct on, xc nh DNA ca b v m, u c mang
c on ngn v on di trong khi y DNA ca chu b s sinh do b bnh
hng cu lim ch c on di. Cng bng enzyme hn ch, ngi ta thy
DNA t bo thai non cng c c on ngn v on di. Kt qu ny cho
php chn on thai ln ny cng mang gen bnh, nhng bnh khng biu
hin (khng b bnh) v dng th d hp nh b m.
ngi bnh ao, cp nhim sc th 21 c n 3 chic (khng phi 2
nh bnh thng), b d hnh, si n bm sinh, ln, c rt, m ph, khng c
kh nng sinh dc. V chng c con b bnh ao nay li tip tc c thai c
th nh k thut chc i, chn on sm trc khi sinh v kiu hnh di
truyn t bo, bit c nn sinh hay phi dng liu php sy thai.

3. Chn on pht hin cc tc nhn gy bnh ngoi lai
ng dng cc k thut sinh hc phn t trong lnh vc ny c nhiu
thun li hn cc phng php c in. Th nht, c th rt ngn c thi
gian nh ng dng k thut PCR, chng hn chn on bnh HIV-1, dng
phng php PCR ch mt mt ngy so vi 3-4 tun nui cy ca phng
php truyn thng. Th hai, i vi cc tc nhn gy bnh kh nui cy hay
khng th nui cy th k thut sinh hc phn t l gii php duy nht,
chng hn Chlamydia, Brucella, vim gan B... Th ba, vic to dng mt
gen dng lm probe n gin hn vic sn xut khng th c hiu, hn na
vi mt probe ngi ta c th pht hin tt c cc kiu huyt thanh
(serotype) ca tc nhn gy bnh, iu m ch mt khng th khng th lm
c. Cui cng, nu trc kia phi cn n nhiu k thut (quan st di
knh hin vi, nui cy trn mt lot mi trng, min dch hc...) th hng
chn on bng sinh hc phn t ch cn mt k thut (lai phn t hoc
PCR). Ngoi ra, chng ta c th to dng ngay chnh gen gy bnh sn
xut ra mu th tng ng.
i vi tc nhn l virus, cc k thut lai phn t v PCR cho php
chn on nhiu nhm nh papillomavirus, HBV, HIV. c bit phng
php lai ti ch cn cho php xc nh trc tip virus trn lt ct sinh thit.
i vi cc tc nhn vi khun, c nhiu b kit chn on sinh hc
phn t c thng mi ha cho cc loi: Mycobacterium tuberculosis,
Mycobacterium pneumoniae, Streptococcus pneumoniae, Haemophilus
influenzae, Salmonella, ...
i vi cc tc nhn k sinh trng, ngi ta cng thnh cng trong
chn on Plasmodium, Schistosoma, Trypanosoma, Toxoplasma,
Leishmania bng lai phn t. Phng php PCR cn cho php pht hin tc
nhn ngay trong vt trung gian truyn bnh.
Phng php chn on bng lai phn t v PCR khng ch gii hn
trong y hc m n cn c ng dng rng ri trong lnh vc thc vt. Chn
on cc virus gy bnh thc vt c ngha quan trng trong bo v cy
trng. V d: pht hin cc viroid thc vt, v chng khng c v protein
nn khng th pht hin bng cc k thut min nhim nh ELISA.

4. Chn on cc bnh di truyn
Cho n nay, ngi ta bit c hng trm bnh di truyn do gen
ln. Cc bnh ny hu nh cha c cch cha tr, hin ch hy vng liu php
gen.
Chin lc hin nay nhiu nc l d phng. Trc kia, vic pht
hin gen ln c cha ln m u d hp t rt kh thc hin v ch bit c
khi a tr ra i. Hng gii quyt trong cc trng hp ny l chn on
sm bnh thai nhi trong bng m cho sy thai sm.
Phng php PCR lm tng ng k hiu qu chn on phn t.
Nh s khuch i nucleic acid khng cn to dng, nn chn on phn t
c th thc hin thm ch vi mt t bo thi.
Corner v cng s (1983) xy dng phng php phn tch c hiu
qu v trc tip xc nh t bin cc gen hemoglobin v . Hai on
oligonucleotide (gn 19 nucleotide mi on) c tng hp, mt trnh
t b sung vi on gen c u NH
2
ca -globin (A) bnh thng v on
kia b sung cho -globin (S) bnh hng cu hnh lim. Cc
oligonucleotide c nh du phng x v s dng phn tch Southern
blot. Trong cc iu kin thch hp cc mu c th phn bit gia allele bnh
thng v bnh. DNA ca c th ng hp t bnh thng ch lai vi mu
A v d hp t lai vi c hai mu, ngi bnh hng cu hnh lim ng
hp t lai vi mu S. Cc kt qu ny cho thy cc mu lai oligonucleotide
c th phn bit mt bt cp sai ca on lai. Phng php ny c th p
dng cho nhiu chn on khc nhau, chng hn hi chng nhim sc th X
d gy (X-Fragile syndrome).
Hin nay, trong y hc lm sng nhiu bnh di truyn v sai hng bm
sinh c chn on cc b m mang thai hoc tr s sinh. Tuy nhin, tin
b k thut lm ny sinh nhiu vn x hi lin quan n o c sinh hc
(bioethics). V d: gi s khi pht hin c locus tnh dc ng tnh
(homosexuality) th c nn cho sy thai hay khng?

5. In du DNA (DNA fingerprinting)
In du DNA c s dng trong phn tch ph h ch, mo v trong
lai to ging. ngi, n c dng xc nh ngun gc v s di c ca
cc qun th ngi c. Tuy nhin, k thut ny cn c s dng c bit
nhiu trong php y (forensis science). Phng php in du DNA s dng
trong xc nh ti phm c nhiu u th:
- Vt mu, lng hay tinh trng trn o trc nhiu nm c th dng
xc nh nhn thn.
- Mu bn vn s dng c.
- Tnh c hiu cao.
Ln u tin phng php ny c s dng Anh v n nay bt
u lp th vin in du DNA ca nhiu ngi. Sau , M v nhiu nc
phng Ty khc s dng rng ri. C nhiu v d rt r v tim nng ca
phng php ny:
Chng hn, nm 1984 mt a b ngi Ghana b t chi nhp c vo
Anh v nghi n khng phi l con, m l chu mt ngi n b c php
nhp c. Vic phn tch protein huyt tng, cc khng nguyn v enzyme
ca mu khng xc nh c l con hay chu. Sau , phng php in du
di truyn c thc hin. Kt qu phn tch bng in di cho thy trong 61
on DNA hin du, a b c 25 on c hiu ging vi nhng on ca
ngi n b. Phn tch in du DNA chng minh chc chn a b l con
vi xc sut trng hp cc on DNA rt cao.

1. H Hunh Thy Dng. 1998. Sinh hc phn t. NXB Gio dc, H
Ni.
2. Khut Hu Thanh. 2004. Liu php gen-nguyn l v ng dng. NXB
Khoa hc v K thut, H Ni.
New York, USA.
4. Coleman WB and Tsongalis GJ. 1997. Molecular Diagnostics (For The
Clinical Laboratorian). Humana Press Inc. Totowa, New Jersey, USA.
6. Marshak DR, Gardner RL and Gottlieb D. 2001. Stem Cell Biology.
Cold Spring Harbor Laboratory Press, USA.
7. Pollard JW and Walker JM. 1997. Basic Cell Culture Protocols.
Humana Press Inc. Totowa, New Jersey, USA.
2
nd
10. Walker JM and Rapley R. 2002. Molecular Biology and
Biotechnology. 4
th
Chng 9

Cc ng dng trong mi trng

I. M u
Nh chng ta bit, mi trng sng hin ang b nhim trm trng,
gii quyt nn cht thi vn l vn ht sc nan gii, c bit l cht thi
cng nghip nh hng rt ln n mi sinh. Ngoi ra, mt khi lng ln
cht thi dn dng, rc thnh ph, ph phm nng nghip... cng v ang
gy nhim nng n mi trng sng. Dn s ca th gii li khng ngng
tng, nhu cu tiu th hng ha cng ln, khi lng hng ha phi sn xut
ra ngy cng nhiu, v th lng rc do sn xut v tiu th thi vo mi
trng l khng nh.
Cng vi s pht trin ca khoa hc-k thut, loi ngi ang phi bt
u tm cch gii quyt vn bo v mi trng bng nhiu bin php
khc nhau. Trong , cc bin php cng ngh sinh hc ngy cng t ra u
vit hn so vi cc bin php khc. Ni chung, hin nay vn bo v mi
trng c gii quyt theo ba hng sau: 1) Phn hy cc c cht v c
v hu c; 2) Phc hi cc chu trnh trao i cht ca C, N, P v S trong t
nhin; 3) Thu nhn cc sn phm c gi tr dng nhin liu hoc cc hp
cht hu c.
Hin ti c hai vn c bn c t ra: 1) Gii quyt tiu hy mt
khi lng khng l cc loi cht thi ngy cng nhiu m khng nh hng
n mi trng. 2) V hiu ha cc loi cht c sinh ra trong qu trnh
phn hy cc loi cht thi cng nghip, gy nhim nghim trng t,
nc, khng kh, v gy nguy him n sc khe con ngi v ng vt.
Vi s pht trin ca cng ngh sinh hc ni chung v cng ngh
DNA ti t hp ni ring, vic nghin cu v ng dng thnh tu cng ngh
sinh hc gii quyt vn mi trng c t ra cp bch v thc s
ang c ng dng ngy cng rng ri.

II. X l nc thi
Cho n nay, cc cht thi trong hot ng sng ca con ngi u
c x l mt cch t nhin nh vi sinh vt. Nhn chung, qu trnh ny bao
gm mt s giai on sau: 1) Loi b phn rn, ta v cn. 2) Phn hy cc
cht hu c tan trong nc nh vi khun hiu kh to ra bn non (cn gi
l bn hot tnh), bn non sau b loi b hoc c a vo b phn ng
ti s dng. 3) To ta v tch P v N (giai on ny c khi khng cn
thit). 4) X l bn to ra giai on 1 v 2 bng phn hy ym kh. Qu
trnh ny lm gim th tch cn, s lng vi sinh vt gy bnh, lm mt mi
hi v to ra nhin liu sinh hc cha kh methane, cn gi l biogas.

1. X l hiu kh bng h thng bn hot tnh
Cng ngh x l cht thi bng bn hot tnh c cng sut ln hn
nhiu so vi x l bng b lc thm. Tuy nhin, n cng c mt s nhc
im sau: do cng s dng ln hn, nn mc tiu th nng lng
khuy trn khng kh cng nh s lng sinh khi c to ra ln hn
(Bng 9.1). Cng ngh ny thch hp x l nc thi cho cc im dn
c ng c, v n chim t din ch hn so vi h thng x l theo cng
ngh lc thm.
Bng 9.1. So snh cc qu trnh x l nc thi khc nhau

X l hiu kh X l ym kh
Bn hot tnh MBR UASB
BOD cn li Thp Rt thp Cao
N v P cn li Thp Thp Cao
Sn xut bn Cao Rt thp Rt thp
Nng lng Cao Cao Thp
Din tch y Ln Rt nh Rt nh
tin cy Phn ln l bn Th Ni ht nh
Ch thch:
MBR: b phn ng sinh hc mng (membrane bioreactor)
UASB: b phn ng kiu lp ph bn thi ym kh chy ln (upflow
anaerobic sludge blanket reactor).
Thc t c kh nhiu h thng x l theo cng ngh ny. Thit k ca
chng khc nhau ty thuc vo h s thng kh v cch thc cung cp kh
(Hnh 9.1). Bn non trong h thng x l ny cha nhiu nc hn so vi
bn non trong b lc thm, v vy cha t chng loi vi sinh vt hn. qu
trnh x l t kt qu, vic xc nh thnh phn v nng cc cht trong
nc thi l vn quan trng bc nht nhm to iu kin cho nhiu nhm
vi sinh vt tham gia qu trnh x l. Cc vi sinh vt quan trng nht l nhm
vi khun t dng cht hu c nh: Achromobacter, Flavobacterium,
Pseudomonas v Moraxella. Trng hp nc thi c hm lng cht v c
cao, th cn s c mt cc loi vi khun nh: Thiobacillus, Nitrosomonas,
Nitrobacter v Ferrobacillus, chu trch nhim oxy ha S, NH
3
v Fe.

Hnh 9.1. S dng chy ca bn hot tnh vi s loi b N sinh hc

2. X l ym kh
Trong qu trnh x l ym kh cht thi (ln men to kh methane) c
ba nhm vi khun tham gia: 1) Nhm vi khun chu trch nhim thy phn
v ln men. 2) Nhm vi khun to H
2
v acetic acid. 3) Nhm vi khun to
kh methane t dng s dng H
2
. nng cao hiu sut ca qu trnh ln
men, hin nay ngi ta vn tip tc hon thin cc loi ging, chng vi
khun ln men ym kh bng bin php chn lc t nhin hoc nh phng
php cng ngh DNA ti t hp. c bit, v mt cng ngh ngi ta cn
phi ch khc phc cc yu t gii hn tc phn hy c cht c mt
trong cht thi nh cellulose, tinh bt... v tc to kh methane. Cn lu
Cht
lng
Bn thi
Thu hi nitrate
Thu hi bn
Khng kh
B lng B hiu kh B thiu oxygen
Nc thi
mt s sn phm cui ca qu trnh ln men nh H
2
, CO
2
v H
2
S

thng c
tc ng c ch ngc lm gim hot ng ca vi khun to kh methane.
Trong thc t c rt nhiu kiu thit b ln men ym kh. T nhng
dng cu to n gin hon ton th cng, cho n cc h thng x l c
cu to kh phc tp hin ang hot ng mt s nc pht trin. Di
y l kiu thit b ln men ym kh tng i in hnh v ang c s
dng kh rng ri (Hnh 9.2).

Hnh 9.2. S ca b phn ng UASB

3. Thu hi nc (water recycling)
Hin nay, trn th gii vn thiu nc ngy cng tr nn trm
trng, do vic s dng li nc thi sau khi c x l s l mt hng
u tin trong cc thp k ti.
cc nc thuc th gii ba vic s dng nc thi th ca chn nui
ti trc tip cho cy trng lng thc kh ph bin. Mt h thng vng
khp kn nh th c kh nng gy nhim bn rt ln cc cy trng lng
thc vi cc virus gy bnh hoc prion. y cng l mt thch thc ln
trong vic thc hin cc cng trnh c gi tr kinh t nhm sn xut nc
ti an ton v sinh m khng loi b cc cht c th s dng lm phn bn
Lp bn ym kh
(vng phn ct)
Cht lng chy vo
Cht lng chy ra
Biogas
nh l N v P. Trong trng hp ny, s phn hy ym kh l rt c trin
vng.
Mt hot ng s dng nc rt nhiu na l sn xut cng nghip,
chng hn cng nghip thc phm, kim loi, dt v giy. Hin nay, cc hot
ng sn xut ny ang pht trin cc h thng x l mi cho php thu hi
nc thi ca h trong mt h thng vng kp kn. c trng, l mt h
thng cc quy trnh sn xut nc cht lng cao t nc thi ca qu trnh
sn xut. Quy trnh ny thng phi hp cc x l sinh hc giai on u
v cc x l ha-l bc cui cng. V d: nh my sn xut chip khoai
ty s dng mt chui cc qu trnh bao gm x l hiu kh v ym kh, lc,
kh trng bng kh ozone v thm thu ngc. Mt h thng x l phc tp
nh th cn thit c thc hin loi b hon ton cc carbohydrate,
thuc dit c v vi sinh vt.
Thc t cho thy, sn xut mt tn thp ngi ta cn 280 tn nc.
Trong trng hp ny, nhng c gng thu hi nc t cc l luyn than cc
thng qua x l bn hot tnh b e da bi s nhim c bn thi nhanh
khi hn 50% nc x l c ti s dng. Nguyn nhn do s tch ly
cc hp cht hu c c tnh cao, v vy n cng ch ra s cn thit phi
nghin cu cn thn v cc hp cht hu c cn tha v thm ch cc sn
phm vi sinh vt lm tng qu trnh chuyn ha khng hon ton. Nhiu
thit b x l t nguyn liu dt hin nay ang nng cp cc h thng x l
nc thi ca n thu hi nc. Do thnh phn ha hc ca cc cht thi
lng thay i rt ln (ty thuc vo loi vi v thuc nhum ang c x
l), cho nn t c hai nh my dt cng p dng mt phng thc x l
nc thi ca h (Hnh 9.3).

III. Phn hy bn hu c
Sn xut cc loi bn hu c, chng hn nh bn thi hoc phn ng
vt, ang tng ln nhiu ni trn th gii v chng thng c s dng
mt cch ty tin, v d nh cc ng dng ca chng ln t nng nghip.
V th, hin nay s lng cc nc c lnh cm s dng ty tin ang tng
ln do s nhim ca nc ngm. Cc qu trnh x l thn thin vi mi
trng hn cho cc loi bn hu c li c gi thnh cao v/hoc hiu qu
km.


Hnh 9.3. S dng chy x l trong cng nghip dt. Nc sau khi x l
c dng ra, ty sch, ty trng, nhum v in. X l sinh hc kt hp vi x
l ha-l t c s tinh sch cn thit.

Qu trnh x l cho hiu qu tt i vi cc bn thi v phn ng vt
l s phn hy ym kh trong cc b phn ng c trn u (completely-
mixed reactor). Trong sut qu trnh ny, khong 50% cht rn c bin
i thnh biogas, trong khi phn cn li hu nh c n nh. Sn lng
biogas ca cc cht phn hy ym kh c th c tng ln bng cch ng
phn hy (co-digesting) phn ng vt hoc bn thi vi 10-20% cc cht
thi rn t cng nghip thc phm v nng nghip, v d: cht thi ca l
m gia sc, dc phm, nh bp, cht thi ln men hoc cht thi th.

Bn hot tnh
Lm ng/kt bng
Lc ct
H lc sinh hc
bng carbon hot
tnh dng ht

Nc x l sch

Nc thi
Cc b phn ng c trn u x l cc bn thi hu c c tc
np thp, khong t 2-5 kg cht hu c/m
3
b phn ng trong mt ngy, do
cc cht hu c dng ht phi c ha tan trc khi chng c th thc hin
bin i ym kh (Bng 9.2). Tc ha tan cc cht thi hu c c th kh
thp, chng hn cn phi mt 15 ngy mi thy phn c 90%. V th, cn
duy tr thi gian t nht l 20 ngy, c khi ln ti 60 ngy hoc lu hn.

Bng 9.2. Cc thng s thit k cho cc loi b phn ng ym kh

B phn
ng
UASB
B phn
ng c
trn u
B phn
ng trng
thi rn
Cht lng c x l Nc thi Bn hu
c
Cc cht
thi rn
Nng cht rn trong b phn ng
(g/L)
< 50 50-100 200-400
Tc np (kg cht hu c/m
3
ngy) 10-30 2-5 20-40
Thi gian gi nc (ngy) 0,3-1 20-40 10-20
Thi gian gi cht rn (ngy) >20 20-40 10-20

Thc hin s phn hy cc mc nhit cao cng c th ci thin
c hiu sut do tc thy phn ca cc cht dng ht tng ln cng
vi vic tng nhit . Nh hiu bit y v qu trnh phn hy a
nhit ngi ta xy dng c mt s h phn hy a nhit quy m ln
x l phn bn ng rung. Khi thc hin nhit cao, cc b phn
ng ny cho php thu c cht lng khng c tc nhn gy bnh, khng
ging nh trng hp phn hy a nhit trung bnh, thng tht bi do
cn li cc tc nhn gy bnh c ngun gc t phn. Trc y, mt s
hn ch lm cho s phn hy a nhit kh tr thnh thng dng, v d
cc kh khn ca s khi ng v nhy i vi mt vi nhn t stress
no nh NH
3
v H
2
S. t st bentonite c th c dng loi b s
c ch NH
3
. Mt khc, H
2
S c th b ph hy bng cch a vo cc cht
nhn in t, v d oxygen hoc nitrate trong b phn ng.
Phng php kt ta thng qua iu chnh pH bng vi l gii php
hu hiu v pH cao cng c th gip loi b ammonium. Phng php
ny cng c th c phi hp vi vic b sung mui nhm hoc mui st,
tt nht l t mt ngun r tin nh loi nc thi giu nhm/st t cc thit
b sn xut nc ung. Vn cn mt phng php na ti u cc iu
kin kt ta MgNH
4
PO
4
l thng qua lm lnh v tch CO
2
.
Cc cng ngh hin nay nh n nh ym kh hoc hiu kh, nghin
t (land disposal) v t cc bn thi hu c cung cp cc phng thc lm
mt nc (lm kh) bn thi t 2-5% ti 25-40% cht kh c th c pht
trin r tin v hiu qu hn. Thch thc chnh i vi cng ngh sinh hc
mi trng l pht trin cc enzyme, cc sn phm v phng thc x l
cho php lm mt nc ca sinh khi vi sinh vt (bn d) ti mc thch
hp. Cc pht trin mi ang c ng dng thng mi da vo s sinh
nhit trong qu trnh hu x l hiu kh bc hi lng nc tha. Qu
trnh lm kh sinh hc ny i hi t nng lng hn cc k thut lm kh
bng nhit. Tuy nhin, cn mt kh khn rt nhy cm l s sinh ra mi
thi lm ngng hot ng ca mt s nh my.

IV. X l cht thi rn
Cc phng php x l cht thi rn thng c s dng l chn
trong t (landfilling) hoc t. Hin nay, phng php chn trong t ngy
cng t c la chn do chng ngn cn s thu hi cc sn phm c th
dng li c (plastic, giy, cc vt liu xy dng...) v chng khng hiu
qu lm trong vic thu hi nng lng (biogas). Hn na, chn trong t
ngm c th gy ra s bc mi ca kh gy nhim mi trng. Tng t
nh th, cc l t khng cho php thu hi nguyn liu mc d chng c th
c thit k thu hi nng lng t cht thi. Cc l t c nhiu hn ch
nh gi thnh cao v ngoi ra h thng kh ca ng khi cn c thit k
tinh vi trnh nhim mi trng.
Hin nay, mt phng thc x l cht thi rn cng nghip v th
khc ang c thc hin nh vo thit b phn tch v sn xut phn .
y l nhng thit b rt ln v phc tp, c nng sut cao (100.000 ti
300.000 tn cht thi/nm), c thit k mt h phn tch vt l thu hi
cc vt liu khc nhau t cc vt b i, nh:
- Ct v si bn li nh vt liu xy dng.
- St bn li cho cng nghip luyn kim.
- Nhm v cc kim loi khng cha st khc c gi tr bn li cao.
- Giy v giy cartoon bn cho cng nghip giy.
- Cc plastic cng v mm c dng li hoc t.
- Cc cht hu c c th phn hy sinh hc c bin i thnh phn
v biogas.
Phn on hu c ca cht thi rn th c dng lm phn bng
phng php hiu kh hoc ym kh. Trong khi sn xut phn hiu kh l
mt cng ngh c ng dng t lu, th nhng pht trin gn y trong sn
xut phn ym kh cng c mt vi tin b (Bng 9.3).

Bng 9.3. So snh cc loi phn hiu kh v ym kh

Phn hiu kh Phn ym kh
Chi ph 60 bng Anh/tn (m) 75 bng Anh/tn (m)
Din tch y Ln Nh
Cn bng nng lng Tiu th nng lng Sn sinh nng lng
Mi thi C vn Khng c vn
Cht lng phn
Hm lng phn Cao (c) Thp
Cc tc nhn gy bnh C Khng

Cc cng ty mi trng khc nhau thit k cc h phn hy ym
kh khc nhau cho cht thi rn nh:
- Nng cht rn trong b phn ng: 50-400 g/L.
- Nhit thch hp t nhit trung bnh (35
o
C) ti nhit cao
(55
o
C).
- S giai on ln men (mt hoc hai).
Mt thit k nh th c thc hin l qu trnh DRANCO (dry
anaerobic composting) dng nhit cao (55
o
C) nng cht rn ln
(200-400 g/L) trong ln men mt giai on. Thc t, y l mt qu trnh
tng t vi qu trnh phn hy t nhin cht thi chn trong t, ch khc
ch n c tin hnh trong b phn ng kn c iu chnh tt v tc
phn ng ln hn nhiu. Cc tc phn ng rt cao c th t ti gip cho
n c kh nng hon thnh qu trnh phn hy trong hai tun (Bng 9.2)
thay cho 20 nm trong t. Vn ct li ca qu trnh ny l nhit cao
v cng lc phi trn thng qua s tun hon khp kn cho php tc
phn ng cao hn nhiu v cung cp cht rn trc tip vo trong b phn
ng khng cn b sung nc pha long. Do khuy c hc khng kh
nng lm kh, nn sn phm ca b phn ng c thu hi vi ln, vi vic
b sung nguyn liu sch cung cp mi ln (passage) (Hnh 9.4). Vng thu
hi m bo phi trn y v cho php a nguyn liu cung cp vo.
Sn phm cui cng l t mn c dng lm phn hiu kh rt tt
trong trng trt. Nguyn nhn cc phn hiu kh c th gy c i vi
cy trng l do hm lng mui cao ca chng, trong khi cc phn ym
kh hu nh nh t mui do thc t l khong mt na trong s chng b o
thi bng nc trong my nn lc (Hnh 9.4). Hn na, cc phn ym kh
cha t ht c di v cc tc nhn gy bnh vi sinh vt hn so vi phn
hiu kh. Tuy nhin, gi tr th trng ca cc phn kh thp v hu x l
c trng s phi c tm kim cho cc ng dng khc nhau. Vn sau
c th c thc hin bng cch b sung cc vi sinh vt hu ch nh l c
nh nitrogen v cc vi khun kch thch sinh trng thc vt, mycorrhizae
hoc cc vi sinh vt iu khin sinh hc (biocontrol). S phc hi cc t b
nhim cng c th hu ch nh b sung phn v chng c th gip phn
hy cc hp cht xenobiotic trong cc vng t ny.

V. X l kh thi
1. Loi b cc hp cht v c d bay hi (volatile organic compounds-
VOCs)
Cc phng php x l ha-l truyn thng cho cc kh thi cng
nghip gy nhim, nh l t hoc dng cc b lc than hot tnh, c
khuynh hng thi nhiu nng lng v to ra s nhim th cp. t
chy cc kh ny (vi nng gy nhim l 100 mL/m
3
) trong cc l t,
t nht cn 50 L methane c b sung/m
3
m bo ph v hon ton
cu trc. Trong hu ht trng hp, mt b phn ng sinh hc c th thc
hin oxy ha cc VOCs bng cch cho tip xc khp kn vi cc vi sinh vt
phn hy, O
2
, H
2
O v cc cht dinh dng.

Hnh 9.4. S dng chy ca h sn xut phn trn ym kh

Cc cht gy nhim c tc phn hy sinh hc khc nhau:
- Phn hy sinh hc nhanh: alcohols, ketones, aldehydes, cc acid
hu c, N hu c.

H thy phn
Dranco

2450 m
3
La

D tr
kh
My n +
My pht in
Kh trong t
Nhit thi
in
Biogas
Tin x l
cht thi
sinh hc
n v
liu lng
Bm
Hi nc
m
My lc
p
Nc p
Ly tm
m cht
lng
My c
Hi nc Ngng hi
My to
hi nc
Nhit thi
C
Hi nc
My lc
hi t
Hi nc
ti bm
Kh nhim
Kh sch
H lc
sinh hc
Hon thin
hiu kh
Humotex
- Phn hy sinh hc chm: phenols, hydrocarbons, cc dung mi (v
d: chloroethene).
- Phn hy sinh hc rt chm: cc hydrocarbon a halogen v a
nhn thm.
Cc thit k b phn ng khc nhau c s dng x l kh bng
phng php sinh hc (Hnh 9.5A). Trong cc h lc sinh hc, khng kh
nhim bn chy chm qua mt mi trng xp m (phn , than bn, v
dm bo) lm gi th (vt ) cho qun th vi sinh vt phn hy sng trong
mng nc mng bc vt liu gi th rn. Dng chy ca lp khng kh b
mt khc nhau t 1-15 cm/s. Tc ny cho hiu sut thi gian tip xc
(vi mt m nhi c trng cao t 1-3 cm) t 10-100 s. i vi cc hp
cht phn hy sinh hc bnh thng, c th t c hiu sut loi b ti
90% vi tc np t 0,1-0,25 kg cc cht hu c/m
3
b phn ng trong
mt ngy. Cc u im ca h lc sinh hc l:
- Thit k n gin v r tin (vt liu gi th c thay th sau 2-4
nm).
- Din tch mt trong cao lm cho h lc sinh hc thch hp mt cch
l tng loi b cc cht nhim km ha tan, chng hn cc
hydrocarbon.
- C kh nng a vo cc vi khun thch nghi c bit ph v cc
hp cht xenobiotic, chng hn chloromethane.
Vn kh khn nht l kim sot pH trong cc h lc sinh hc v
H
2
S s b oxy ha thnh H
2
SO
4
, NH
3
thnh HNO
3
, v cc cht hu c cha
chloro thnh HCl. Nhc im ln nht ca h lc sinh hc l:
- Cn mt khng gian nn ln.
- Khng c kh nng iu chnh cc iu kin ca qu trnh, chng
hn nh pH.
- Cc vt liu gi th nh phn t chng sn sinh ra cc mi.
Cc nhc im ny ca h lc sinh hc c th khc phc c trong
my lc hi t sinh hc (bioscrubber) (Hnh 9.5B). Mt my lc hi t
truyn thng chuyn mt cht hin din trong dng kh ti dng cht lng
bng cch phun cht lng trong bung qua b phn m kh c chuyn
qua. Trong my lc hi t sinh hc, cc cht lng c phun l dch huyn
ph ca cc vi sinh vt quay vng ngc v hng ti trc gia bung
phun v mt h x l nc thi bng phn hy sinh hc. Cc thng s ca
qu trnh nh cung cp cht dinh dng thch hp v pH c iu chnh d
dng hn nhiu (trong cht lng tun hon) so vi h lc sinh hc, dn ti
cc tc phn ng nhanh. Trong khi cc h lc sinh hc i hi mt din
tch y ln v chiu cao ca chng khng c vt qu 1 m trnh cng
knh, th cc my lc hi t sinh hc i hi mt khng gian t hn nhiu
bi v b phn hy sinh hc c th cao mt vi mt.

Hnh 9.5. H lc sinh hc v my lc hi t sinh hc c dng loi b
VOCs khi kh thi
B. My lc hi t sinh hc
iu chnh pH v
cht dinh dng
Bn hot tnh
Khng kh
H x l
nc thi c
Bung phun
(my lc hi t)
Kh thi
Kh sch
A. H lc sinh hc
Nc
Kh sch
Vt liu gi th
H lc sinh hc
Kh thi
My gi m
My lc hi t sinh hc l loi thch hp nht cho cc dng chy kh
ln do p sut ngc thp v kch thc nh ca chng. Tuy nhin, chng
c th ch c dng loi b cc kh ha tan hon ton v tc chuyn
khi trong bung phun ca chng km hn trong h lc sinh hc. Trng
hp nng cc cht nhim bn thu c trong kh thi l qu cao, th
mt my lc hi t sinh hc th hai c a vo c b sung cc vi sinh
vt c kh nng phn hy nng cht nhim bn thp hn.
Hin nay, cc nghin cu ang tp trung thit k mt h thng c th
phi hp s hp th kh trn b mt rn (v d: than hot tnh) v phn hy
sinh hc cc hp cht sorbic. Cc mng lc sinh hc loi dng chy nh git
(bio-trickle filters) l cc tm plastic hoc mi trng gi th cho cc vi sinh
vt khc c treo trong dng chy khng kh b nhim bn. Cc tm ny
c thm t lin tc bng dng chy hi lu ca nc cha cc cht dinh
dng cho vi sinh vt. Cc mng lc sinh hc nh git c u im l tit
kim c khng gian.

2. Loi b cc hp cht sulphur v nitrogen t kh ng khi bng phng
php sinh hc
Cc nitrogen oxide (NO
x
) v sulphur oxide (SO
2
) l cc cht nhim
khng kh chnh c to thnh trong lc t than v du gii phng
trong cc loi kh ng khi. C th pht trin cc phng php cng ngh
sinh hc c gi tr kinh t v hiu qu loi b ng thi cc cht gy
nhim khng kh ny, v cc k thut ha-l truyn thng hoc l rt t
hoc l khng hiu qu. Mt h thng mi c xut l kh ng khi
c dn qua mt my lc hi t m trong >95% SO
2
v >80% NO
x

ha tan trong dung dch NaHCO
3
v Fe(II)-EDTA (hp cht sau c th tng
kh nng ha tan NO
x
). Dung dch y N v S c sinh ra trong ba giai
on sinh hc tun t (Hnh 9.6). Giai on u tin bao gm b phn ng
thiu oxygen trong NO c bin i thnh kh N
2
tr thng qua qu
trnh kh nitrogen sinh hc.
2Fe
II
(EDTA) (NO) + cht cho in t 2Fe
II
(EDTA) + N
2
+ CO
2
+ H
2
O
Mt cht cho in t, chng hn methanol hoc ethanol, cn c b
sung duy tr phn ng. Trong hai giai on tip theo, H
2
SO
3
c kh
sinh hc tun t thnh H
2
S v cui cng oxy ha li tng phn thnh lu
hunh nguyn t dng rn:
H
2
SO
3
+ 3H
2
H
2
S + 3H
2
O
H
2
S + 1/2O
2
S
o
+ H
2
O

Hnh 9.6. Mt qu trnh sinh hc mi c pht trin ng thi kh
sulphur ha (desulphurisation) v loi b NO khi cc kh ng khi c sn
xut trong cc thit b nhit

Phn ng kh H
2
SO
3
xy ra trong b phn ng UASB (Hnh 9.2)
c kt ht vi cc vi khun kh sulphate. Cc polymer gy kt bng
c b sung, cng vi cc cht dinh dng cn thit v cc ng lng
My lc hi t
B kh nitrogen
khng c oxygen
UASB
L phn ng sinh
hc c oxygen
Lc
Kh i vo
H
2
SO
3
, NO
Kh i ra
Cht cho in t

N
2
H
2

Khng kh
Lu hunh nguyn
t dng rn
kh (ethanol hoc H
2
), iu chnh t l phn t BOD/H
2
SO
3
mt gi tr
khng i. Trong b phn ng sinh hc th ba cc vi khun hiu kh oxy
ha sulphide thnh lu hunh nguyn t (S
o
) dng rn (sn phm cui). S
oxy ha thm S
o
thnh H
2
SO
3
v H
2
SO
4
b ngn cn bi mt lng gii hn
O
2
. Ton b qu trnh l t ng hon ton vi khong 120 thng s c
phn tch lin tc, a s trong chng l trc tuyn. Nc cng c thu hi
lin tc.

VI. Phn hy cht rn (solid remediation)
1. Kch thch sinh hc (biostimulation) v tng sinh hc
(bioaugenzymetation)
Cc vi sinh vt c kh nng phn hy sinh hc cc cht gy nhim
thng hin din sn trong cc t nhim bn hoc nc ngm. V vy,
trong nhiu trng hp, s phn hy sinh hc cht rn hoc nc ngm
c thc hin bng cch kch thch sinh hc cc vi sinh vt, ni c cht
dinh dng hoc cc nhn t sinh trng cn thit khc ca chng. Cc cht
hot ng b mt cng c th c b sung thun tin cho vic chuyn
khi cc hydrocarbon ha tan km vo trong pha nc ni m cc vi sinh
vt sng. V d ca kch thch sinh hc l a methane vo trong tng ngm
nc b nhim cc dung mi c kh trng bng chloro hoc a
benzoic acid vo trong tng ngm nc b nhim bi polychlorobiphenyls
(PCBs). Cc ngun carbon c a vo nh methane v benzoic acid
kch thch sinh trng ca cc vi sinh vt c hiu sn xut cc enzyme lm
t gy c cht c a vo v cht gy nhim ang hin din.
Gii php thch hp l c th a cc qun th vi sinh vt c hiu
(tng sinh hc) vo trong cc v tr b nhim nht nh khi cht gy
nhim l mt phn t phc tp ch c th b ph v bi mt t hp c bit
ca cc vi sinh vt rt c hiu (c gi l consortium). Cc cht gy
nhim nh th thng l polyaromatic hydrocarbons (PAHs), cc hp cht
hu c c halogen ha, cc thuc tr su nht nh, thuc n TNT,
polychlorobiphenyls (PCBs)... Cc iu kin v cc chng vi sinh vt thch
hp cho thy chng c nh hng n s phn hy sinh hc ca cc hp
cht ny khi nghin cu quy m phng th nghim. V d: s phn hy
ca cc cht thm c kh trng n gin bng chloro trong t v cc
thit b x l nc thi c th pht huy hiu qu hn bng cch a vo nui
cy thun khit cc vi sinh vt c chn lc trong phng th nghim. Cc
cht nhim phc tp hn, v d PCB, c th i hi hot ng phi hp
ca mt s chng vi sinh vt. Trong trng hp c bit ny, vic kh
chloro nh cc consortium pht trin t cc trm tch b nhim bn c
thc hin bng cch nui cy sinh khi trong dng ht cc b phn ng
UASB sinh methane. Cc ht ny cho thy chng gip tng nhanh hn s
phn hy sinh hc ca PCB in situ trong t v cc trm tch.
i vi nhng ng dng c bit, s tng sinh hc c th c tin
hnh nh cc k thut bin i di truyn vi sinh vt thng qua cng ngh
DNA ti t hp. Bin i di truyn vi sinh vt c th gip ngn cn s to
thnh cc sn phm trung gian c gy mt n nh qun th v c ch cc
qu trnh phn hy sinh hc. Thch thc ln nht trong k thut bin i di
truyn vi sinh vt l tng kh nng sng st ca cc chng gy nhim. Theo
hng c mong i ny, cc c hi sng st thng ln hn khi chng
xm nhim c phn lp t v tr c tng sinh hc.

2. Cc k thut phn hy cht rn
C nhiu k thut khc nhau ca cng ngh sinh hc x l cht
rn b nhim. Di y l mt s k thut ph bin nht:

2.1. Phn hy sinh hc ti ch (in situ bioremediation)
y l k thut s dng vi sinh vt v cc cht dinh dng gii
quyt cc cht thi nguy him, chng hn nh t v cc a im sn xut
b nhim. Phn hy sinh hc c nhiu li ch quan trng so vi cc
phng php kh b cht thi truyn thng; n c th c thc hin ti
ch, trnh phi vn chuyn v ct gi cc cht thi nguy him v c hi.
C nhiu vi sinh vt khc nhau c kh nng phn hy cc cht nhim cng
nghip c trng bao gm cc hydrocarbon mch thng, cc hydrocarbon
nhiu gc thm, cc phenol chloro ha v cc phn t hu c phc tp khc.

2.2. Landfarming
C th loi b cc vt du loang bng k thut landfarming. K thut
ny c thit lp trn c s phn hy vi sinh vt (Hnh 9.7) v c th c
nng cp mt t bng cch trn t vi cc gc hu c mi (phn ). Nhit
cao, hot tnh v s a dng ca vi sinh vt tng tc phn ng. Hn
na, cc cht ng tc dng c bit gip cho s ng chuyn ha xy ra
d dng hn. Cc h thng landfarming cng c th c nng cp bng
cch tin x l ym kh, v d cc ng hm ym kh c s dng kh
cc hp cht nh trinitrotoluene, bng cch b sung cc cht dinh dng v
cc cht ng tc dng cho cc vi khun ti ch. giai on th hai hiu
kh, cc cht chuyn ha hoc c khong ha hon ton hoc polymer
ha v bt ng trong khun rn. Phng thc ny cng c ng dng
thnh cng kh s nhim bn cc vng t b nhim chloroethene v
cc cht thm BTX (hn hp ca benzen, toluene v xylene).

Hnh 9.7. Mt ct ngang ca h thng landfarming

2.3. Cc b phn ng sinh hc pha bn (slurry-phase bioreactors).
C th c hng ti cng mt mc lm sch trong thi gian
ngn. Trong trng hp ny, cc t khai qut b nhim c x l di
cc iu kin ti u c kim sot, m bo s tip xc hiu qu gia cht
nhim bn v cc vi sinh vt. Yu t sau, trong hu ht trng hp, l cc
Lp ct
t b nhim
Mng (ng dn)
Lp lt tho ra c
Cc cht dinh dng
nui cy c hiu vi sinh vt thch nghi. Vi ton b tc phn hy trong
phm vi 0,2-2 g du/kg t/ngy, thi gian lu ca cht rn l 30 ngy
p ng cc mc lm sch.

VII. X l nc ngm
1. S phc hi hot ng
Phng thc phc hi nc ngm c ng dng nhiu M v chu
u l k thut bm v x l (pump-and-treat). Hng ny s dng ch
yu cc k thut ha-l loi b cc cht nhim trong cc h x l trn
mt t, v d thng qua hp th khng kh v carbon hot tnh, trong khi
cc b phn ng sinh hc c dng t hn 10% trng hp (Hnh 9.8).
Vic s dng hn ch cc x l sinh hc c th l do kinh nghim v
cc s liu chng minh cn qu t, s tip nhn hn ch k thut, nhng
cng c th do khng thch hp trong vic hng ti cc mc lm sch
nh mong mun. n nay, ngi ta thu c nhiu kinh nghim khi ng
dng k thut phn hy sinh hc quy m hon chnh trong iu kin ex
situ v in situ phn hy cc hydrocarbon ca du m, bao gm t cc
chui mch thng v mch nhnh, c bo ha, khng bo ha v cc cht
bo mch vng ti cc hydrocarbon mt, hai v a nhn thm. Tuy nhin,
thi gian gn y cc kiu thit k b phn ng sinh hc mi c pht
trin loi b cc dung mi c polychloro ha v cc cht thm. Chng
hn, cc b phn ng UASB kt ht vi cc bn thi dng ht sinh methane
c kh nng kh hon ton chloro ca tetrachloroethylene (>99%) hin din
vi 4 mg/L trong nc ngm b nhim. Acetate c dng lm ngun
carbon v cht cho in t. Cng ngh b phn ng UASB cng ang c
nng cp vi bn thi dng ht phi hp c hai loi vi khun hiu kh v
ym kh.
Tuy nhin, phng thc bm v x l khng th lm sch trong mi
trng hp v hn na n i hi mt thi gian lm sch di ngy. Trong
77 im x l theo phng php trn (di s kim sot ca US National
Research Council-NRC) vo nm 1992, ch c 8 im c thng bo l t
mc tiu lm sch (lin quan n o lut nc ung an ton). Trong 8
im thnh cng, th 6 im b nhim bi hydrocarbon du ha cng c
o thi thng qua s phn hy t nhin (natural degradation).


Hnh 9.8. Cng ngh phc hi bm v x l
Khng kh
B phn ng sinh hc
Strip column
H lc carbon
hot tnh
Bm v x l
Mt nc
Mt t
Phun tt
Thu hi tt
Nc ngm
Vng nhim
Cc cht dinh dng
Mt t
Mt nc
Phun tt
Nc ngm sch
Vng hot tnh sinh hc
Nc ngm nhim
Hng dng chy
Chn sinh hc
2. S phn hy t nhin v s gim st (monitoring)
Trong thi gian gn y, mt vi vn to ra nhiu quan tm cho
cc cng c gim st mi. Mt trong s l thc t cc k thut phn hy
thng khng p ng c mc tiu lm sch tht s. Hn ch ny buc
cc nh lm lut phi nh gi li cc mc nhim ban u v tnh ton
li vic s dng cc im cui (sau khi x l) da trn s ri ro (risk-based
end-points). Khi nim mi v cc im cui da trn s ri ro i hi pht
trin cc cng c phn tch mi nh gi li gi tr sinh hc, ng hn l
khi nim nng nhim tng s. Cc cng c mi c trng ny da vo
cc xt nghim sinh hc bi v cc phng php phn tch truyn thng
khng th phn bit gia cc cht gy nhim l nhng yu t c gi tr i
vi cc h thng sinh hc v cc cht gy nhim l nhng yu t ang tn
ti trong cc dng khng c gi tr hoc phc tp hoc tr. Mt loi xt
nghim sinh hc c da trn s c ch pht hunh quang sinh hc t
nhin ca sinh vt bin Photobacterium phosphoreum. Tuy nhin, cc xt
nghim ny khng c hiu do s c ch nh sng s xut hin khi tip xc
vi mi cht c. Hn ch ny c khc phc nh mt loi biosensor vi
khun mi c trng cho cc loi cht c nht nh. V d: biosensor c
th pht hin cc kim loi c gi tr sinh hc, c xy dng bng cch gn
gen ch th lux ca Vibrio fischeri vi cc gen khng kim loi nng trong vi
khun Alcaligenes eutrophus. Cc chng ti t hp khi c phi trn vi
t b nhim kim loi hoc nc s pht sng t l vi nng cc kim
loi c gi tr sinh hc c trng. Ngi ta c th d dng o c s pht
sng bng phng php quang ph.
Mt yu t khc c th p ng cho s quan tm gn y trong cc
cng c gim st mi l gi thnh cao v tc chm ca cc k thut phn
hy. Mt hng phn hy thc t hn, c gi l s phn hy t nhin
(hoc phn hy sinh hc ni ti-intrinsic bioremediation), ang c ng h
bi US Environmental Protection Agency. S phn hy sinh hc ni ti da
vo cc qu trnh t nhin loi b, c lp hoc kh c cc cht gy
nhim m khng c s can thip ca con ngi. S phn hy sinh hc ni
ti c quan st thng xuyn nht vi nc ngm b nhim bn cc
hydrocarbons. Nu bng chng gi rng mt a im ang c ci thin
do s phn hy sinh hc ni ti, v rng s nhim khng a ra mt e
da no i vi sc kho ca con ngi, th c quan qun l mi trng c
th cho php t tnh trng ch gim st (monitoring only). Phng thc
ny yu cu n gin ha vic gim st t xa (remote monitoring) theo
di cc nng nhim bn lp di b mt trong iu kin in situ. Gim st
t xa c th c tin hnh vi mt radar nhn xuyn qua mt t kim
tra s ph v nhim trong lp di b mt da trn c s tng dn ca
dung dch, km theo vic ph v cc hydrocarbon hoc cc dung mi c
chloro ha.
Mt k thut khc c dng trong gim st t xa l s dng k thut
bin i di truyn cc vi sinh vt phn ng vi s hin din ca cc cht
nhim bn c bit. Khi cc vi sinh vt ny c gn vo mt t bo quang
in kt ni vi mt con chip sng v tuyn (radio chip), th cc tn hiu nh
sng c bin i trong cc sng v tuyn c pht hin mt khong
cch xa. Cc b cm bin (sensor) ny c th c ri khp cc a im b
nhim gim st tin trin ca s ph v cc cht gy nhim.

1. Trn Vn Nhn v Ng Th Nga. 2002. Gio trnh Cng ngh x l
nc thi. NXB Khoa hc v K thut, H Ni.
4. Arora M. 1998. Biological Control of Environmental Pollution. Vol 1,
Anmol Publications PVT, Ltd. New Delhi, India.

Ph lc

Mt s thut ng c bn
Adapter. , nhng c mt
u bng v mt u li 5 tng ng vi mt v tr ct hn ch
vector c
u tng ng (xem thm linker).
Amino acid. L mt phn t nh mang mt gc amine (-NH
3
) v mt
gc carboxyl (-COOH) lin kt vi cng mt nguyn t carbon. Amino acid
l n v cu trc c s ca chui polypeptide. C 20 amino acid khc nhau
trn cc chui polypeptide
.
Ampicillin (Amp).
.
Amylase. Enzyme xc tc thy phn cc loi carbohydrate d tr (v
d tinh bt) trong thc n thc vt hay glycogen trong thc n ng vt.
BAC (bacteria artificial chromosome). Nhim sc th nhn to ca
vi khun da trn c s plasmid F-factor, c s dng lm vector to dng.
BAC c th ti bn trong E. coli vi cc on chn DNA c kch thc ln
n 300 kb.
Bn ct hn ch (restriction map). Trnh t cc v tr nhn bit
(recognition sites) ca tt c cc enzyme hn ch (restriction enzyme hay
restriction endonuclease, RE) trn mt phn t DNA.
(analog base).
-

- -C.
Baz nit (nitrogen base).
, guanine, cyt
.
Bt cp b sung (complementary base pairing). S kt hp thnh
tng i gia cc nitrogen base nm trn hai mch n ca chui xon kp
DNA-DNA, DNA-RNA hoc RNA-RNA thng qua cc mi lin kt
hydrogen. S bt cp mang tnh c hiu: guanine bt cp vi cytosine,
cn adenine bt cp vi thymine trn DNA hoc uracil trn RNA.
Bt ng (immobilization).
.
Bin i hu dch m (post-translational modification). S thay i
cc lin kt ha tr xy ra trong chui polypeptide, sau khi chui polypeptide
tch khi ribosome v trc khi tr thnh protein hot ng thc s.
Bin np (transformation). L qu trnh truyn DNA ngoi lai vo
mt t bo nhn, chng hn sphaeroplast hoc protoplast, v c th hp nht
trong nhim sc th nh s ti t hp tng ng hoc c bin i trong
mt n v sao chp t tr (autonomous replicon). S bin np c th xut
hin trong cc iu kin t nhin mt s vi khun (v d: Bacillus,
Haemophilus, Neisseria v Streptococcus), nhng nhiu vi khun (v d E.
coli) v cc c th sinh vt eukaryote s bin np ch c th xut hin
nhng t bo thm c DNA bng cc phng php nhn to nh: ha
bin np, in bin np...
Bin np bng in (electroporation). K thut dng xung in to
ra cc l thng tm thi trn mng sinh cht a DNA ngoi lai vo bn
trong t bo vt ch.
Bin tnh (denaturation). L hin tng chuyn t dng mch kp sang
dng mch n ca DNA v RNA thng do nhit gy nn. Bin tnh ca
protein l hin tng chuyn t cu hnh hot ng thnh dng khng hot ng.
Biu hin ca gen (gene expression). L cc qu trnh phin m
(transcription) v dch m (translation) ca mt gen to ra sn phm
protein ca n.
Cm ng (induction). Lin quan n kh nng tng hp mt s
enzyme (sn phm ca gen) vi khun ch khi c mt c cht (substrate)
ca cc enzyme ny trong mi trng. Khi s dng cho khi nim iu ha
biu hin ca gen, thut ng ny c ngha l s khi ng qu trnh phin
m do tng tc gia mt cht cm ng (inducer) vi protein iu ha. V
d E. coli,
).
Cp base (base pair, bp). L lin kt A-T hoc C-G trn mt phn t
DNA mch kp, v l n v o chiu di ca mt phn t DNA.
Cu trc bc mt hay cu trc s cp (primary structure). L trnh
t sp xp cc amino acid trong chui polypeptide.
Cu trc bc hai hay cu trc th cp (secondary structure). L
tng tc khng gian gia cc gc amino acid gn nhau trong chui
polypeptide, hay ni cch khc l dng cun xon cc b ca tng phn
trong chui polypeptide.
Cu trc bc ba (tertiary structure). L tng tc khng gian gia
cc gc amino acid xa nhau trong chui polypeptide, l dng cun xon
trong khng gian ca ton chui polypeptide, y l hnh dng chung ca
chui polypeptide.
Cu trc bc bn (quaternary structure). Xut hin nhng phn
t protein bao gm hai hay nhiu chui polypeptide hnh cu (bc ba). Mi
chui polypeptide ny c gi l mt tiu n v (subunit) v tng tc
khng gian gia cc tiu n v trong phn t gi l cu trc bc bn.
Cu disulphite (disulphite bridge). Lin kt ng ha tr to thnh
gia hai chui polypeptide qua trung gian ca mt gc cystine.
Cht cm ng (inducer). Mt hp cht ha hc
.
Cht trao i s cp (primary metabolite). c sn xut vi s
lng ln hn cc cht th cp v c cc chc nng trao i c bit. Cc
cht s cp thu t sinh vt c s dng nh l thc phm, cc ph gia thc
phm, v cc nguyn liu th trong cng nghip nh l cc carbohydrate,
du thc vt, protein v cc acid bo.
(secondary metabolite).
.
Cht c ch (repressor). Sn phm protein ca mt gen c ch
(gen m h .
Chiral. Mt vi phn t c dng tay phi v tay tri ring bit,
mc d chng mang cc nguyn t ging nhau v lin kt trong cng mt
kiu nhng tnh cht vt l li khc nhau. Mt cht nh th c gi l hp
cht chiral, v hai (hoc nhiu) dng hn th mi dng c gi l cht i
hnh (enantiomer) hoc cht ng phn quang hc (optical isomer).
Chu trnh sinh tan (lylic cycle). Mt kiu chu trnh sng ca thc
khun th (bacteriophage) khi n xm nhim vi khun, iu khin cc hot
ng sinh sn v sinh trng bng cc gen ca n v sinh ra cc
bacteriophage th h con
.
C (lysogenic cycle). L hin tng h gen ca
bacteriophage hin din trng thi n nh v khng sinh tan trong t bo
vt ch sng ca n. Cc t bo vt ch c th tip tc sinh trng v phn
chia, v s sao chp ca h gen bacteriophage (prophage) c phi hp vi
nhim sc th ca vt ch sao cho khi t bo phn chia th prophage cng
c chuyn vo trong c hai t bo con. Prophage c duy tr bng cch
hoc hp nht trong nhim sc th vt ch (v d bacteriophage ,
bacteriophage 105) hoc nh l mt plasmid bn ngoi nhim sc th (v
d bacteriophage P1 v bacteriophage F116). T bo vt ch c th hoc
khng th biu hin ra mt kiu hnh bin i.
(transgenic).
(foreign DNA) bng cc k thut khc nhau (Agrobacterium, vi tim, bn
gen, xung in...) vo mt c th vt ch (vi sinh vt, ng vt hoc thc
vt).
Chuyn nhim (transfection). K thut a DNA phage hoc DNA
virus vo cc t bo vt ch.
Coenzyme. Mt cht hu c thng l mt vitamin hay mt cht tng
hp t vitamin tc dng ln mt cofactor (mt cht phi protein nh: Cu,
Fe hoc Zn hay mt phn t hu c) gip enzyme xc tc mt phn ng trao
i cht.
Cosmid.
cos ) c .
Cng ngh ACCELL (ACCELL technology). Cng ngh phn phi
gen da trn c s thay i cng phng in thng qua git nc nh,
v vy to ra mt s thay i p sut khng kh rt ln lm tng gia tc
ca cc vin n vng bc DNA.
Cng ngh DNA ti t hp (DNA recombinant technology). H
thng cc phng php phng th nghim cho php ct on DNA t mt
sinh vt ghp ni vo DNA ca mt sinh vt khc to ra phn t DNA ti
t hp. Phn t ny c a vo cc sinh vt khc nhau to ra nhng
ging chng vi sinh vt, thc vt v ng vt mi c nhng phm cht c
bit, p ng nhu cu ngy cng cao ca sn xut v i sng con ngi.
Cng ngh ny c ng dng rng ri trong y hc, dc hc, nng nghip v
nhiu ngnh cng nghip khc.
Cng ngh protein (protein engineering). L cc k thut cho php
nghin cu v bin i cu trc ba chiu ca phn t protein. S bin i
ny c thc hin bng phng php ha hc, enzyme hoc gy t bin
im nh hng ln gen m ha cho protein ny, nhm thu c mt cch
hiu qu cc protein c tnh cht l-ha v xc tc thch hp hoc c tnh
cht mi. Mt trong nhng lnh vc ca cng ngh protein l m hnh ha
hoc thit k protein.
Cng ngh sinh hc (biotechnology). Theo ngha rng l cc qu
trnh cng nghip c s dng vi sinh vt hoc cc t bo ng vt v thc
vt (cng ngh sinh hc ). Theo ngha ph bin hin nay l
nhng qu trnh sn xut s dng cc ging sinh vt mi, c to ra bi
cng ngh DNA ti t hp (cng ngh sinh hc ).
Trong cng ngh sinh hc

gh sinh hc.
.
...
Deoxyribonucleotide triphosphate (dNTP).
)
).
Deoxyribonuclease (DNase). (phn
hy) DNA si i hoc DNA si n.
Deoxyribonucleic acid (DNA).
p
nucleotide
base .
.
(transcrip
.
Nm 1962, Watson (M) v Crick (Anh) chia s Gii Nobel vi
Wilkins (Anh) v pht minh ra cu trc khng gian ca DNA v ngha ca
n trong vic truyn thng tin di truyn. iu ng tic l Franklin, ngi
c nhng ng gp ng k cho pht minh ny mt trc . Theo qui
nh th Gii Nobel khng dc php tng cho ngi mt.
Deoxyribose. Phn t .
(nick translation).
[ -
32
P]dCTP nh enzyme DNA polymerase I ca E.
coli.
(translation). . Qu
trnh chuyn thng tin di truyn trong trnh t base ca mRNA sang trnh t
amino acid ca chui polypeptide trong t bo cn gi l qu trnh sinh tng
hp protein.
Dideoxyribonucleotide triphosphate (ddNTP).
(sequencing).
(complementary DNA, cDNA).
trn khun mu mRNA nh qu trnh (reverse
transcription)
dng th vin cDNA (cDNA library).
DNA khun mu (template DNA).
.
DNA polymerase.
.
Nm 1959, hai nh khoa hc ngi M l Kornberg v Ochoa c
nhn Gii Nobel v nhng nghin cu lm sng t c ch c bn ca qu
trnh sao chp DNA lin quan n DNA polymerase I.
Dng (clone). Tp hp cc t bo hoc phn t ging ht nhau cng
bt ngun t mt t bo hay phn t ban u.
(germ line).
.
(end labelling).
4 polynucleotide kinase.
(blunt end).
5 nh ra (protruding ends).
(cohesive ends hoc sticky ends).
.
u tn cng C (C terminus). Gc
.
u tn cng N (N terminus). Gc amine (NH
2
) v tr tn cng ca
mt phn t protein hoc chui polypeptide. Tt c cc polypeptide u
c tng hp bt u t u ny, n gc carboxyl tn cng.
(nick). .
in di trn gel (gel electrophoresis). K thut dng phn tch
cc phn t nucleic acid hoc protein da vo s dch chuyn ca chng trn
gi th dng gel (agarose hoc polyacrylamide) di nh hng ca in
trng. S dch chuyn ca cc phn t ny ph thuc vo in tch, cu
hnh, kch thc v khi lng phn t ca nucleic acid hoc protein cng
nh dung mi v nng ca cht dng lm gi th.
(Klenow fragment).
76.000) ca E. coli 5 3.
on nhi (stuffer fragment). Cn gi l vng m hay vng trung
tm. L mt phn ca phage c th c loi b v thay th bng on
chn DNA (insert DNA) m khng nh hng n kh nng sinh sn ca
phage trong t bo vt ch.
ng du (replica plating). Phng php chuyn nguyn mu cc
khun lc hoc vt tan t mt a thch gc sang a thch mi bng cch
dng mng nylon (v d mng Hybond-N+) va kht p ln mt thch ca
a gc dnh ly cc t bo trong cc khun lc (colony) hoc vt tan
(plaque) ca a gc, ri a mng ny p ln mt thch mi.
Tesner (Tesner degree). 1
o
T = s mL NaOH hoc KOH cn
trung ha 100 mL sa.
(mutation).
.
t bin gen (gene mutation). Cn gi l t bin im, xy ra do
nhng bin i ca tng nucleotide nh mt, thm hay thay i v tr ca
nucleotide trn phn t DNA. L nguyn nhn ca s hnh thnh protein
khng bnh thng. Phn ln t bin l c hi nhng chng vn thng
c gi li trong qun th v thng l ln v do c th c duy tr
trong kiu gen m khng nh hng n sc sng ca sinh vt. Tc t
bin t nhin thp, nhng tn s t bin c th tng nh cc tc nhn nh
phng x ion ha v cc ha cht gy t bin gy ra.
t bin khuyt dng (auxotrophic mutation). Cn gi l t bin
ha sinh. t bin lm mt kh nng tng hp mt cht cn thit cho s sinh
trng ca t bo.
ui polyA (polyA tail). on trnh t di 50-200 nucleotide adenine
c b sung vo u 3 ca hu ht cc mRNA eukaryote sau khi phin
m.
E. coli (Escherichia coli). Vi khun thng c trong rut non ca
ng vt c xng sng. E. coli c coi nh sinh vt mu cho vic nghin
cu hot ng ca t bo. y l vi khun Gram m c kch thc genome
khon 410
6
base-pair. Cc qu trnh biu hin gen (phin m v dch m)
i i vi nhau, sinh ra si mRNA c tng hp mi v c s dng
ngay cho qu trnh dch m. Khng c hin tng bin i hu dch m
(post-translation). V th, E. coli c xem l mt trong nhng t bo vt
ch n gin nht. Rt nhiu th nghim to dng gen ang c thc hin
hng ngy ti cc phng th nghim u s dng E. coli lm vt ch vi
nhiu chng khc nhau v mt di truyn v cho nhng ng dng c bit.
Endonuclease. L e
. Nuclease thy phn nhng lin kt phosphodiester gia cc
nucleotide ca mt phn t nucleic acid. Cc nuclease c th c hiu i
vi DNA (deoxyribonuclease) hoc c hiu i vi RNA (ribonuclease).
Enzyme. Cht xc tc sinh hc
.
Enzyme gn DNA (DNA ligase).
5-
PO
4
- .
Enzyme gn RNA (RNA ligase).
-
.
(restriction enzyme, RE).
.
Enzyme hn ch c pht hin vo nm 1970, chng tn ti trong t bo vi
khun, c tc dng ct DNA ngoi lai (v d: DNA ca phage) ti nhng
im xc nh, tiu dit DNA ny. Cho n nay hn 900 enzyme hn ch
c tm thy. Cc enzyme hn ch c s dng rng ri trong cc
phng th nghim thao tc gen nh nhng chic ko ct DNA ti nhng
im c hiu. V tr ct ph thuc vo loi enzyme hn ch c la chn.
Nm 1978, Arber (Thy S), Nathans (M) v Smith (M) c
nhn Gii Nobel nh pht minh ra enzyme hn ch v nhng ng dng ca
chng gii quyt nhiu vn quan trng ca di truyn hc phn t. Cc
enzyme ny l nhng chic ko phn t c th ct DNA thnh nhng on
xc nh, m ra mt thi k pht trin mi ca sinh hc hin i-Thi k
thao tc gen.
(reverse transcriptase).
-
in vitro.
Epitope. Yu t quyt nh khng nguyn, l mt trnh t cu trc
nm trn phn t khng nguyn c nhn bit bi cc khng th hoc cc
th th ca khng nguyn. Cc epitope ca t bo B l nhng quyt nh
khng nguyn c cc t bo B nhn bit. Cc epitope ny c cu trc gin
on, in hnh ngha l c cu trc khng gian nht nh. Epitope ca t
bo T l mt trnh t peptide ngn t mt khng nguyn c bn cht protein
c lin kt vi phn t MHC v c nhn bit bi mt t bo T ring
bit.
Eukaryote. Sinh vt c t bo mang nhn in hnh (nhn tht) ngha
l nhn c bao bc bi mng nhn v tham gia vo hai c ch phn bo
quan trng l nguyn phn v gim phn.
Exon. Cc on DNA trong gen c chc nng phin m. Exon tn ti
c sinh vt prokaryote v eukaryote. Ring sinh vt eukaryote cc exon
nm xen k vi cc on intron. Cc intron chim ti 90% tng s DNA ca
t bo eukaryote v khng c chc nng phin m.
Exonuclease. Loi enzyme nuclease ch tc ng vo u tn cng
ca phn t nucleic acid, ct ra tng nucleotide mt theo thi gian. Chng
c th chuyn ha theo u 5 hoc 3 ca si DNA.
Ex vivo. Thut ng dng ch cc th nghim thc hin trn t bo
nui cy, cc t bo ny sau s c a vo mt c th sng.
-galactosidase. Enzyme c m ha bi gen lacZ. Enzyme ny
thy phn lactose thnh glucose v galactose.
Gy t bin nh hng bng oligonucleotide (oligonucleotide-
directed mutagenesis). Cn gi l gy t bin nh hng im (site-
directed mutagenesis), l qu trnh to ra mt bin i xc nh trn DNA
bng cch s dng mt oligonucleotide tng hp (primer) c mt vi bin
i trong trnh t nucleotide.
Gen (gene). L n v di truyn, yu t quyt nh mt tnh trng c
th. Thng tin di truyn ca cc gen c m ha trong DNA quyt nh
tnh bin d ca c th v ca loi. DNA l mt chui bao gm cc n v
nucleotide, c bn loi nucleotide mang bn nitrogen base khc nhau l
adenine (A), guanine (G), cytosine (C), v thymine (T). Trnh t cc
nucleotide ca mt gen xc nh mt polypeptide hoc mt RNA. Gen c
kh nng b t bin. Cc gen ch yu nm dc theo nhim sc th trong
nhn t bo. Mi gen chim mt v tr xc nh trn nhim sc th gi l
locus. Gen c th tn ti nhiu dng gi l cc allele. Cc gen biu hin
thng qua cc phn t do chng sinh ra l RNA (trong qu trnh phin m)
v protein (trong qu trnh dch m).
Gen cu trc (structural gene) hay gen m ha (coding gene). L
trnh t m ha sn phm protein.
Gen ch huy (operator). on DNA ngn nm u operon, k st
promoter ni m protein c ch c th bm vo, c tc dng m hoc
ng operon cho cc gen cu trc trong operon hot ng hoc ngng
hot ng.
Gen ch th (reporter gene). L mt gen m ha m sn phm ca n
c trc nghim mt cch d dng (v d chloramphenicol
acetyltranferase), n c th c gn vi bt c mt promoter no sao cho
s biu hin ca gen ny c dng th nghim chc nng ca promoter.
Gen iu ha (regulatory gene). Mt gen m sn phm protein ca
n tham gia vo s iu ha biu hin ca mt gen khc. V d gen m ha
mt protein km hm.
Gen gy cht (death gene). Gen lm cht c th mt giai on no
trong qu trnh pht trin ca chng.
Gen lacZ. Gen ca E. coli m ha -galactosidase thch hp cho chn
lc th bin np bng khun lc xanh ( -galactosidase s kt hp vi IPTG
v X-gal c b sung trong mi trng nui cy) v trng (on DNA
ngoi lai xen vo gia gen lacZ lm cho gen ny mt hot tnh v th khng
sn xut c -galactosidase).
Gradient. Bin thin ca mt i lng theo mt hng no . Mt
gradient mt c xc lp trong mt s trng hp ly tm. Mt gradient
proton hoc ion c to ra qua mt mng nh s vn chuyn tch cc i
hi nng lng.
H gen (genome). L tp hp cc gen c trong mt t bo n bi
eukaryote, trong mt t bo prokaryote hoc trong mt virus.
-
.
H ln men hay (fermentor or bioreactor).
. Tuy
.
Hiu ng glucose (glucose effect). Hin tng kh hot tnh ca
operon cm ng vi khun khi c mt glucose k c khi hin din cht cm
ng operon.
Histone. Nhm protein kim nm trong phc hp vi DNA cc
nhim sc th eukaryote v ng vai tr quan trng trong vic xc nh cu
trc ca nhim sc th.
Hot t plasminogen m (tissue plasminogen activator, tPA).
.
Hormone. L cc hp cht do cc tuyn ni tit to ra. Cc cht tit
ny c truyn i trong c th, tc dng ln cht nhn c hiu b mt
cc t bo ch m n c th kim sot hot ng ng vt.
Hormone thc vt (phytohormone). L cc cht kch thch sinh
trng thc vt.
Hp t (zygote). Sn phm n bo sinh ra do s kt hp cc giao t
c v giao t ci. N pht trin thnh phi thng qua hng lot nguyn
phn lin tip.
Hybridoma. Dng t bo c hnh thnh t s dung hp mt t bo
ung th ty xng (myeloma) v mt t bo bch cu (lymphocyte),
hybridoma c kh nng sn sinh cc khng th n dng (monoclonal
antibody) mt cch vnh vin.
Huyt thanh (serum). Cht lng ca mu cn li khi mu ng c
tch ra bng phng php ly tm (ngha l huyt tng khng c cc nhn
t lm ng mu).
Hunh quang (fluorescence). Hin tng pht mt sng nh sng c
bc sng khc vi bc sng c hp th trc . Mt s phn t
c gi l th hunh quang (v d enzyme luciferase con om m) do
c c tnh ny.
In du DNA (DNA fingerprinting) hay in du di truyn (genetic
fingerprinting). L phng php dng cc mu d phng x hoc dng k
thut PCR nhn dng cc bng DNA c cc on lp li vi tn s cao.
Bn mu hnh cc bng DNA l duy nht i vi mi c th, v do vy c
th dng xc nh c trng c th hoc quan h huyt thng.
In du chn DNA (DNA footprinting). Phng php nhn dng cc
vng DNA m cc protein iu ha bm vo.
Invertase. Enzyme xc tc phn ng thy phn cc disaccharide
thnh cc monosaccharide, c bit thy phn saccharose thnh dextrose v
levulose.
Interferon. Protein c sinh ra t bo ng vt nh l phng tin
chng li virus. N c ch s nhn ln ca cc virus. Nh cng ngh
DNA ti t hp, hin nay interferon c sn xut ra t cc vi sinh vt ti t
hp vi mt s lng ln thng qua k thut chuyn gen interferon ca
ng vt vo vi sinh vt.
Gilbert (1980) ot gii thng Nobel nh thnh cng trong vic
thu nhn interferon t E. coli c ti t hp gen m ha interferon. n
nm 1981, i hc Washington (M) thnh cng trong vic thu nhn
interferon t nm men S. cerevisiae c hiu sut cao gp 10.000 ln so vi
t bo E. coli ti t hp.
Intron. Nhng on DNA nh sinh vt eukaryote khng mang
thng tin m ha amino acid, phn b ri rc dc theo phn t DNA. Sau
khi thng tin t DNA c phin m sang mRNA th cc intron trn mRNA
b ct b, cc on mRNA cn li gm ton cc exon c ni li vi nhau
v chuyn n ribosome dng lm khun mu cho qu trnh dch m.
Intron khng thy c sinh vt prokaryote.
In vitro v in vivo.
(in vitro (in vivo
in silico.
Ko di on mi (primer extension). S tng hp mt bn sao
nucleic acid bt u t on mi. c s dng nh du phng x on
DNA lm mu d hoc khuch i mt on DNA bng k thut PCR.
Khng nguyn (antigen). Phn t thng tm thy trn b mt t bo,
c tc dng kch thch s to thnh khng th. Do vy, n c dng gy
nn mt phn ng min dch.
Khng th (antiboby). Mt protein (immunoglobulin) do bch cu
lympho B ca h thng min dch sn sinh, c tc dng nhn bit mt khng
nguyn ngoi nhp c hiu v gn vi n, nu khng nguyn nm trn b
mt t bo th vic gn kt ny s dn ti s kt cm t bo v lm bt hot
khng nguyn.
Khng th n dng (monoclonal antibody).
. Th
.
Khuch i (amplification.) S sn xut nhiu bn sao ca mt trnh
t DNA nh k thut PCR.
Khuyt on (deletion, deficiency). t bin nhim sc th dn n
lm mt mt on vt cht di truyn v thng tin di truyn cha trong n ri
khi nhim sc th.
Kiu hoang di (wild type). Dng thng thy nht ca mt gen
trong qun th hoang di. Allele kiu hoang di c k hiu bng mt ch
in hoa hoc thm du cng sau ch vit thng, v d: A hay a
+
. Allele kiu
hoang di thng l tri v cho kiu hnh bnh thng.
Kilobase (kb). 1000 base (hoc cp base), c dng nh n v
o hoc xc nh chiu di ca cc phn t DNA hoc RNA.
K thut di truyn (genetic engineering). Cn gi l cng ngh
DNA ti t hp. Bao gm h thng cc phng php di truyn phn t dng
thao tc vt cht di truyn, vi ba bc chnh gm ba khu chnh. 1)
Tch chit DNA t nhng sinh vt khc nhau; 2) Ct v ni DNA nhng
im c hiu to ra DNA ti t hp (DNA mang cc gen c ngun gc
khc nhau), v d: DNA plasmid c mang gen ca ngi; 3) a DNA ti t
hp vo hot ng trong cc t bo hoc c th sng sinh ra nhng sn
phm c bit cn thit cho con ngi, v d DNA plasmid mang gen to
insulin ca ngi c a vo vi khun E. coli sn xut.
Lai khun lc (colony hybridization). in situ
.
Lai phn t (molecular hybridization). Qu trnh trong hai mch
nucleic acid b sung (A-T, G-C) bt cp hnh thnh nn mt mch kp. y
l mt k thut hu ch pht hin mt trnh t nucleotide chuyn bit.
Lai ti ch (in situ hybridization). Qu trnh bt cp gia mu d (l
mt trnh t DNA si n hay RNA) vi DNA ca t bo c c nh trn
lam knh.
LAK (lymphokine activated liller). T bo git c hot ha bng
lymphokine. Liu php t bo LAK (liu php IL-2 gin tip, c thc
hin bng cch tch cc t bo lympho (lymphocyte) t mu ngoi vi ca
bnh nhn ung th sau hot ha v nhn in vitro cc t bo ny cng vi
IL-2 qua mt s ngy. Mt dng lympho khng phi T s c pht trin c
hnh thi lympho ht ln, cc lympho ny c c tnh rt cao chng li cc
khi u thc nghim (gi l cc t bo LAK) c chng minh lm thuyn
gim mt phn hoc thuyn gim hon ton trn 20% bnh nhn b mt s
bnh ung th, c bit l ung th da c tnh v ung th biu m thn.
Land farming. Qu trnh x l bng k thut phn hy sinh hc
(bioremediation) c thc hin lp t mt hoc trong cc x l sinh
hc (biotreatment). t b nhim bn, cht cn hoc bn c trn li trn
b mt lp t v sau nh k s c lt li thng kh.
Landfilling. K thut chn ct cht thi, l cch thng dng nht
kh b cc cht thi sinh hot, thng mi v cng nghip. Khong 80-90%
lng cht thi trn th gii s c kh b bng cch ny trong nhng nm
ti. Nhng a im c s dng chn ct cht thi c th l nhng ni
khai thc m kt thc, cc m b, cc vng t thp, cc thung
lng... Cc ni chn ct cht thi cn c la chn v qun l tht cn thn
trnh nhng s c nh lm nhim nc ngm, thot ra cc kh c
mi kh chu, gy n tng xu v cnh quan...
Ln men (fermentation). L qu trnh bin i sinh hc khi s dng
cc vi sinh vt. cng l nhng bin i bng con ng sinh hc ca mt
cht nn, k c cc hin tng sinh tng hp v chuyn ha sinh hc. Hin
tng ln men cng dng ch phng thc chuyn ha bng cch phn
gii cc cht hu c m c th sng v t bo s dng to ra nng lng
ha hc di dng ATP nhng khng cn n oxygen.
Lin kt peptide (peptide bond). Mi lin kt ng ha tr trong
chui polypeptide, ni nhm -carboxyl ca mt amino acid vi nhm -
amine ca mt amino acid k tip.
Lin kt phosphodieste (phosphodieste bond). Mt phn t cha hai
alcol, ester ha vi mt phn t phosphoric acid lm cu ni gia chng vi
nhau. V d chui polynucleotide c ni bi lin kt phosphodieste
5 3 gia hai nucleotide cnh nhau.
Liu php gen (gene therapy).
chn gen ln .
(germline and
somatic cell gene therapy).
.
Linker. Mt oligonucleotide tng hp c hai u bng,
bacteriophage trc
E. coli to
u bng. Cc linker sau khi gn vi hai u bng ca on cDNA nh
DNA ligase s c ct hn ch to ra u so le tng ng vi hai u
ca vector. Phn ng gn gia on cDNA c mang linker hai u vi
vector cng c xc tc nh DNA ligase.
Ly tm theo gradient mt (density gradient centrifugation). K
thut tch cc hp cht da vo s khc nhau v mt ca chng c
thc hin bng phng php ly tm lm lng cc cht qua mt gradient
nng ca saccharose hoc CsCl.

Lymphokine. L nhng cytokine c sn xut t cc t bo lympho.
Lysosome. Mt bo quan c mng bao bc trong t bo cht ca
nhng t bo eukaryote. Lysosome cha nhiu enzyme thy phn.
M di truyn (codon). Nhm ba nucleotide nm k nhau (b ba) trn
phn t mRNA xc nh mt amino acid trn chui polypeptide, hoc l tn
hiu kt thc vic tng hp polypeptide.
M thoi bin (degenerate codon). M di truyn m mt amino
acid c quy nh bi mt s b ba nitrogen base, ch khng phi ch bi
mt b ba. Thoi bin l c im vn c ca m di truyn tn ti ph bin
sinh gii.
(nonsense codon) hay m dng (stop codon).
(UGA)
Maturation. Qu trnh trong cc mRNA va c phin m tri
qua mt s bin i ha hc tr thnh mRNA hon chnh sn sng lm
khun mu cho vic tng hp protein.
My m nhp nhy (scintillation counter). My dng xc nh
hot tnh phng x trong mt mu th nghim.
Mt quang (optical density). Thng s cho php o hp th
nh sng mt bc sng no ca mt mi trng hoc dung dch.
Mu d DNA (DNA probe). Mt on RNA hay DNA chuyn bit
c nh du bng ng v phng x hay bng ha cht (cht pht hunh
quang hoc enzyme), dng nh v mt trnh t nucleic acid nht nh
thng qua k thut lai phn t (xem Northern blot, Southern blot...)
Micelle. Ht dng huyn ph trong mt dung dch keo, c cu
thnh t mt tp hp cc ht nh hoc i phn t.
Monomer. L cc phn t n v nh, c th lin kt vi cc phn t
n v ging n hnh thnh nhng phn t ln hn (polymer). V d cc
nucleotide l cc monomer ca nucleic acid v cc amino acid l monomer
ca protein.
Mi trng LB (LB medium). Mi trng nui cy E. coli, thnh
phn bao gm (cho 100 mL): 1 g bacto-trypton; 0,5 g dch chit nm men v
1 g NaCl. Nu pha ch mi trng rn nui cy trn a petri th b sung
thm 1,5 g bacto-agar.
Mi (primer). Mt trnh t DNA hay RNA ngn, bt cp vi mt
mch ca DNA khun mu v c mang mt u 3-OH t do gip DNA
polymerase bt u tng hp mt chui DNA mi.
Mucopeptide. D polymer i phn t cha hai loi ng amine v
mt s amino acid. Ngi ta nhn thy mucopeptide gn lin vi thnh t
bo ca prokaryote.
Myeloma.
.
Nm men hng methyl. L nhng nm men ch s dng cht dn
xut methane nh l ngun carbon v nng lng chuyn ha.
Nm men Saccharomyces cerevisiae. L mt vi sinh vt nhn tht
c s dng nhiu trong cng ngh DNA ti t hp. Genome ca nm men
S. cerevisiae khong 1,3510
7
base-pair nhiu hn E. coli khong 3,5 ln.
Nm men thng c dng lm t bo vt ch biu hin nhng protein
c cu trc phc tp cn qu trnh hu dch m m vi khun E. coli khng
th p ng.
Northern blot. K thut chuyn v c nh RNA t formaldehyde
agarose gel (sau khi c phn on bng in di) ln mng lai bng nylon
hoc nitrocellulose lai vi mu d (probe) c nh du ng v phng
x [ -
32
P]dCTP hoc digoxigenin-dUTP. Tn hiu lai sau c pht hin
trn phim X-quang (trng hp [ -
32
P]dCTP) hoc trn mng lai (trng
hp digoxigenin-dUTP).
Nucleic acids. Nhng polynucleotide sinh hc thin nhin, trong
nhng n v nucleotide c kt hp vi nhau bi nhng lin kt
phosphodieste thnh trnh t DNA hoc RNA ring bit.
Nucleotide. Mt nucleoside phosphoryl ha vi mt trong nhng
hydroxyl ca pentose. Phn t ng vai tr cu trc c s ca DNA v
RNA, gm ba phn: ng pentose (ribose trong RNA, deoxyribose trong
DNA), nitrogen base v gc phosphate.
Nui cy m v t bo (tissue and cell culture). K thut cy v
nui v trng m v t bo ng vt hoc thc vt trong mi trng nhn
to bn trong ng nghim hoc bnh thy tinh. V nguyn l, ging nh
nui cy t bo vi sinh vt. K thut ny c nhiu ng dng thc tin, v d
thc vt c dng nhn ging nhanh nhng cy sinh trng chm
hoc b phn ly khi sinh sn hu tnh, ngoi ra cng c s dng trong
chn ging; ng vt k thut ny c dng nui m phc v cho
cng tc nghin cu v sn xut cc cht c hot tnh sinh hc.
Oliofilm. Cao su x l bng HCl.
Oligo. Tip u ng c ngha l t, v d: oligonucleotide
(polynucleotide) c t nucleotide hoc oligopeptide (polypeptide) c t
peptide.
Oligo(dT)-cellulose. Mt on ngn gm cc gc deoxy-thymidine
lin kt vi c cht cellulose, c s dng tinh sch mRNA eukaryote
bng phng php sc k i lc.
Oligomer. Thut ng chung ch mt on ngn cc monomer.
Oligonucleotide. Mt on ngn cc monomer l nucleotide, thng
t 20-30 nucleotide.
Operon. Nhm cc gen vi khun chu s iu khin chung ca mt
gen iu ha. Cu trc operon bao gm: 1) Nhm gen cu trc m hot ng
ca chng chu s iu khin chung, 2) Gen iu ha sn sinh ra protein
iu ha, 3) Vng ch huy (operator) v trnh t khi ng (promoter) chu
tc ng ca protein iu ha. Protein iu ha c tc dng bm vo vng
ch huy lm cho n b ng, do vy nhm gen cu trc ngng hot ng.
Khi trong mi trng xut hin c cht ca cc gen cu trc th c cht ny
bt hot ha protein iu ha khin n khng bm vo vng ch huy c
na, vng ch huy c m v nhm gen cu trc li hot ng.
Phage. Vit tt ca bacteriophage (thc khun th), l loi virus xm
nhim v sinh sn bn trong vi khun. Phage thng c v bc protein, phc
hp bao gm phn u c hnh a din cha nucleic acid (DNA hoc RNA)
v ui m qua nucleic acid xm nhp vo vi khun ch. Sau qu trnh
nhn ln ca nucleic acid ca phage, t bo vi khun ch thng b tan bin.
Loi phage lun lun lm tan t bo vi khun khi chng xm nhim vi
khun gi l phage c, v d phage T4. Ngc li, cn c phage n ha,
khi xm nhim vi khun n gy nn phn ng tim tan, ngha l h gen ca
phage gn vo nhim sc th vi khun v c sao chp cng vi nhim sc
th . H gen ca phage trng thi gn nh vy vi nhim sc th vi
khun gi l prophage.
Phn ng chui polymerase (polymerase chain reaction, PCR).
Phng php dng trong phng th nghim khuch i cc on DNA
c bit ln hng triu ln trong vng vi gi thng qua 20-30 chu k nhit,
mi chu k bao gm ba mc nhit : bin tnh 90-95
o
C, bt cp vi mi
40-65
o
C hoc hn v tng hp mch mi nh DNA polymerase chu nhit
(Taq polymerase) 70-72
o
C. PCR c ng dng rng ri trong chn on y
hc, phn tch s a dng sinh hc, chn ging v trong nhiu lnh vc
khc.
Nh khoa hc M (Tin s Mullis) ngi pht minh ra k thut PCR
nhn gii Nobel nm 1993. Cng chia s Gii Nobel vi Mullis l Smith
(Canada) do c nhng ng gp mang tnh nn tng cho vic gy t bin
im nh hng, da trn cc oligonucleotide v vic pht trin chng
trong cc nghin cu protein.
Phn ha hay bit ha (differentiation). Mt kha cnh ca s pht
trin bao gm s hnh thnh cc loi t bo, cc loi m, cc loi c quan
khc nhau t mt hp t ban u di s iu khin c bit ca cc gen.
(bioremediation).

.
Phn tch trnh t gen (gene sequencing). L k thut xc nh trnh
t theo cu trc bc mt ca chui cc nucleotide trong mt phn t nucleic
acid. Phn tch trnh t ca DNA c cc phng php ha hc ca Maxam-
Gilbert v phng php enzyme ca Sanger. Trong nhng nm gn y, mt
s phng php xc nh trnh t mi nh s h tr ca my tnh xut
hin. Bn cnh k thut thng thng s dng cc polyacrylamide gel
phn ly cc phn t DNA c di khc nhau, cc k thut mi lin quan
n pht hin hunh quang ca cc nucleotide c nh du, phn tch
trnh t DNA bng khi ph, in di mao dn hoc lai vi cc on
oligonucleotide c tng hp nhn to cng ra i.
Nm 1980, Sanger (Anh) v Gilbert (M) c trao gii Nobel do
c nhng ng gp quan trng v phng php xc nh trnh t cc
nucleotide trong phn t DNA. ng gp ny l mc lch s to ln trong
sinh hc phn t, l nguyn l ca tt c cc my xc nh trnh t DNA t
ng ang s dng hin nay trn khp th gii.
Phin m ngc (reverse transcription). Qu trnh tng hp DNA
t khun mu mRNA nh enzyme phin m ngc (reverse transcriptase).
Phng x t ghi (autoradiography). K thut pht hin cc phn t
c nh du phng x thng qua hiu ng to nh ca cc phn t ny trn
phim X-quang.
Phosphoryl ha (phosphorylation). Phn ng to thnh mt dn xut
phosphate ca mt phn t sinh hc di tc dng xc tc ca mt enzyme.
Phosphatase kim (alkaline phosphatase). Enzyme loi b cc
nhm 5-PO
4
t u ca cc phn t DNA v li cc nhm 5-OH.
Phi t (ligand). Mt phn t hoc mt ion kt hp vi mt protein,
v d hormone c th kt hp vi th th (receptor) c hiu ca n.
Plasmid. c cu trc mch vng kp, nm trong t
bo cht v ngoi nhn, c kh nng sao chp c lp i vi nhim sc th
ca t bo. Tn ti c sinh vt prokaryote v eukaryote. Ngy nay, cc
plasmid thit k nhn to c s dng rng ri nh l cc vector dng
trong cc k thut to dng v biu hin gen.
Polyacrylamide. L polymer ca acrylamide v bisacrylamide c cu
trc gm cc lin kt cho to ra mt mng xp (ging bt bin). Cc cht
phi chui vo l gel mi ra c, v vy nhng cht no c khi lng phn
t nh s ra trc v ngc li.
Polymer. Mt on di cc monomer.
Polynucleotide. Trnh t nhng nucleotide ni ng ha tr vi nhau,
trong v tr 3 ca pentose ca mt trong nhng nucleotide c ni vi
mt lin kt phosphodieste v tr 5 ca pentose ca nucleotide tip theo.
Polypeptide. Mt chui di nhng amino acid ni vi nhau bi nhng
lin kt peptide.
Prokaryote. Sinh vt n bo khng c nhn t bo in hnh, DNA
nm trong t bo cht khng c mng bao bc, khng c nguyn phn v
gim phn; i din in hnh l vi khun.
Prophage. Phage n ha xen vo nhim sc th ca vi khun tim
tan. N sao chp ng thi vi nhim sc th ca t bo vi khun ch.
Protein. Mt phn t ln gm mt hoc nhiu chui polypeptide, mi
chui c mt trnh t amino acid v mt khi lng phn t c trng.
Protein l hp cht quan trng bc nht i vi c th sng. V cu trc,
protein l phn t mch di gm cc n v cu trc nh l cc amino acid
ni vi nhau qua mi lin kt peptide. Khi lng phn t ca protein t vi
nghn n vi triu. C khong 20 loi amino acid. Cc loi protein phc tp
hn c lin kt thm vi cc nhm b sung.
Protein dung hp (fusion protein). L mt protein ti t hp lai
c m ha bi mt gen lai (fusion gene) do s dung hp in vitro cc on
gen khc nhau trn plasmid vector v sau bin np vo vi sinh vt ch
(chng hn E. coli). V vy, protein dung hp s mang trnh t amino acid
ca hai protein khc bit c tng hp t u N ca vector biu hin.
Protein nguyn th (native protein). L mt protein ti t hp c
m ha bi mt gen ngoi lai (foreign gene) trong vi sinh vt ch. Khc vi
protein dung hp, protein nguyn th c tng hp t u N ca n ch
khng phi t u N ca vector.
Purine. Mt hp cht d vng, kim, c nitrogen, l thnh phn ca
nhng nucleotide v nucleic acid. Purine cha mt nhn pyrimidine kt hp
vi mt nhn imidazol.
Pyrimidine. Mt nitrogen base d vng c trong cc nucleotide
v nucleic acid.
Qu trnh chun b c cht v nguyn liu sn xut (upstream
processing). Giai on thit k mi trng dinh dng (c cht) v s dng
chng t bo (nguyn liu sn xut) thch hp cho mt qu trnh sinh hc
(bioprocessing).
Qu trnh phn tch v tinh sch u ra (downstream processing).
Gia
.
Retrovirus. L loi virus RNA cha enzyme reverse transcriptase v
sinh sn di dng DNA mch kp. Chng c kh nng xm nhim t bo
vt ch cao. Khi xm nhim n c kh nng gn h gen ca virus vi h gen
ca t bo vt ch, l c s thit k cc vector liu php gen hiu qu.
Ribonuclease. Enzyme xc tc c hiu vic phn hy RNA bng
cch ct cc mi lin kt phosphodiester trn RNA.
Ribonucleic acid (RNA). Thng l phn t a phn mch n gm
cc n v cu trc c s l ribonucleotide. V mt ha hc RNA rt ging
vi DNA. RNA l v
.
Ribonucleotide. n v cu trc c s ca RNA, gm ba thnh phn:
ng ribose, nitrogen base v nhm phosphate.
Ribosome.
. Ngi ta cng thy
ribosome trong ty th, c s tng hp mt s protein ty th.
Ribozyme
nh enzyme.
RNA b sung (complementary RNA).
.
RNA kch thc nh ca nhn (small nuclear RNA, snRNA).
.
RNA polymerase.
-
.
RNA ribosome (ribosomal RNA, rRNA). L
5S; E. coli
.
RNA thng tin (messenger RNA, mRNA). Mt loi RNA c
phin m t mt trnh t DNA. mRNA truyn thng tin di truyn t nhim
sc th ti ribosome
ribosome.
(transfer RNA, tRNA)
.
Sng lc (screening). K thut nhn dng mt dng DNA trong mt
th vin h gen (genomic library) hoc th vin cDNA (cDNA library) bng
mt phng php lai mu d c nh du [ -
32
P]dCTP vi cc vt tan
(trng hp dng bacteriophage lm vector to dng v cho xm nhim
vo vi khun E. coli) hoc khun lc (dng plasmid lm vector to dng)
ca cc th vin trn mng nylon hoc nitrocellulose. Tn hiu lai c
pht hin bng phng x t ghi trn phim X-quang.
Sao chp (replication). S nhn bn vt cht di truyn trong chu trnh
phn bo hoc s tng hp DNA ca phage khi phage sinh sn trong t bo
vi khun.
Sephadex. L mt loi dextran c lin kt cho gia cc mch
polysaccharide vi nhau to thnh mt cu trc mng li ba chiu.
Sephadex trung ha in tch nn khng c tng tc cation cng nh anion,
n l mt loi bt kh, khng tan trong nc, nhng khi ngm nc s
trng ra v to thnh gel. Mt li ca cc sephadex gel thng to nh
khc nhau, ty theo mc lin kt. Nu gia cc chui polysaccharide c
t lin kt th gel s c mt li ln, ngm nc nhiu v ngc li.
Sephadex bn trong mi trng acid yu v kim yu, trong mi trng acid
mnh v kim mnh th cc lin kt glucoside trong gel s b thy phn. Da
vo lin kt ngi ta chia sephadex lm nm loi: sephadex G-25, G-50,
G-75, G-100 v G-200.
Sinh hc phn t (molecular biology). Khoa hc nghin cu cc
hin tng sng mc phn t. Lnh vc khoa hc tr tui ny l im
gp nhau ca cc khoa hc kinh in nh di truyn hc, ha sinh hc, t bo
hc, vt l hc, ha hc hu c v ha l. Theo cch hiu ph bin hin nay,
sinh hc phn t l khoa hc nghin cu cc gen v hot ng ca chng
mc phn t, bao gm phin m, dch m, sao chp, iu ha biu hin
gen, ti t hp v chuyn gen...
Sinh tng hp protein (protein synthesis). Phn ng ha hc din ra
trn ribosome to nn cc phn t protein t cc amino acid trn c s thng
tin di truyn nhn c t trong nhn t bo thng qua mRNA.
Somatotropin.
.
Southern blot. K thut chuyn v c nh DNA bin tnh t
agarose gel (sau khi c phn on bng in di) ln mng lai bng nylon
hay nitrocellulose lai vi mu d (probe) c nh du ng v phng
x [ -
32
P]dCTP hoc digoxigenin-dUTP. Tn hiu lai sau c pht hin
trn phim X-quang (trng hp [ -
32
P]dCTP) hoc trn mng lai (trng
hp digoxigenin-dUTP).
Ti t hp (recombination). Qu trnh m trong nhim sc th
hay phn t DNA t ra ri cc phn t c ni li theo mt t hp mi.
Qu trnh ny c th xy ra trong t bo sng (qua s trao i cho trong
phn bo gim nhim) hay trong ng nghim nh cc enzyme ct v ni
DNA.
To dng (cloning). Cn gi l nhn dng, tch dng hay dng ha,
l s sn sinh nhiu bn sao ca mt phn t DNA, thng l phn t DNA
ti t hp trong plasmid vector, bng cch sao chp phn t trong mt vt
ch thch hp chng hn vi khun E. coli.
Terminal transferase. Enzyme b sung cc gc nucleotide vo u 3
ca oligonucleotide hoc polynucleotide.
T bo kh bin (competent cell). Cc t bo vi khun c kh nng
tip nhn DNA ngoi lai trong qu trnh bin np.
T bo lympho B (B lymphocyte). Pht trin trong ty xng (bone
marrow) c k hi .
T bo lympho T (T lymphocyte). Pht trin v thun thc tuyn
c (thymus) nn k hiu T.
T bo Hfr (high frequency recombination cell). T bo gii tnh
c E. coli, c mang nhn t F gn lin vi nhim sc th vi khun. Khi
nhn t F thc y s tip hp ca t bo Hfr vi t bo ci (F
-
) th cc gen
ca vi khun c truyn sang t bo ci vi tn s cao.
T bo NK. L nhng t bo lympho c ht ln, c kh nng nhn
bit cc t bo b ung th, cc vi sinh vt xm nhim v tiu dit chng nn
gi l t bo git t nhin, c k hiu l NK hay T
NK
(natural killer).
T bo mm phi (embryonic stem cell). T bo phi cha bit ha,
a th chut, c th c nui cy trong mt thi gian di m vn gi
c tnh a th, ngha l kh nng bit ha theo nhiu hng pht trin
thnh cc t bo khc nhau nh t bo c tim, t bo thn kinh, t bo gan...
T bo trn (protoplast). T bo thc vt b mt thnh cellulose
do x l cc enzyme (cellulase, hemicellulase v pectinase), thng c
dng trong cc th nghim dung hp t bo (protoplast fusion) hay cn gi
l lai t bo sinh dng (somatic hybridization) m rng ngun gen to
ra cc ging cy trng mi mang cc c tnh u vit.
Th Barr. Th Barr l nhim sc th X b bt hot v kt c c trong
nhn t bo ca n gii, khng c nam gii.
Th bin np (transformant). T bo hoc sinh vt nhn c gen
ca mt sinh vt khc trong qu trnh bin np v biu hin chc nng ca
gen ra kiu hnh.
Th t bin (mutant). Sinh vt (hoc gen) mang t bin di truyn.
Th khm (mosaic). Phi hoc c th c cc t bo mang cc h gen
khng ging nhau.
Th ti t hp (recombinant). Cc c th hoc t bo mang cc t
hp gen khc vi cha m ca chng do cc qu trnh ti t hp di truyn
sinh ra.
Thng tin di truyn (genetic information). Thng tin c lu tr
trong cc phn t DNA ca sinh vt dng trnh t sp xp ca bn
nucleotide A, T, C v G ng vai tr nh nhng ch ci ca ngn ng
di truyn. Trong ngn ng ny, mi t ch c ba ch ci gi l mt b ba.
Ngha ca mi t l mt amino acid c mt trn phn t protein tng ng.
Mi cu ca ngn ng di truyn l mt gen cha ng thng tin di truyn
m nhim mt chc nng trn vn. Mi chc nng l mt c tnh sinh
l, hnh thi hay cu trc ca s sng. Do c ch sao chp theo kiu na bo
ton ca DNA m thng tin di truyn c truyn chnh xc t th h n
sang th h kia hu nh khng thay i.
Th vin cDNA (cDNA library). Tp hp cc dng DNA c to ra
t mRNA ca mt t bo hoc mt m c th trong bacteriophage vector,
i din cho thng tin di truyn m cc t bo biu hin.
Th vin h gen (genomic library). Tp hp tt c cc on DNA
c to ra t phn ng ct hn ch genome trong bacteriophage vector, i
din c cho ton b cho thng tin di truyn ca mt h gen.
Tnh ton th (totipotency). L kh nng sinh sn hoc ti sinh mt
c th hon chnh t mt b phn ca n.
Trnh t dn u (leader sequence). Mt trong ba phn ch yu ca
mt phn t mRNA. Trnh t ny nm u 5 ca mRNA v mang thng
tin ribosome v cc protein c hiu nhn bit bt u qu trnh tng hp
polypeptide, trnh t dn u khng c dch m thnh trnh t cc amino
acid.
Trnh t iu ha (regulatory sequence). Mt trnh t ca DNA
tham gia vo qu trnh iu ha ca gen. V d promoter hoc operator.
Trnh t khi ng (promoter). Trnh t nucleotide c hiu nm
trong thnh phn operon, c chc nng iu ha hot ng ca operon, ni
RNA polymerase bm vo bt u qu trnh phin m. Trnh t c trng
ca promoter c khong 20-200 nitrogen base.
Trnh t Shine-Dalgarno (Shine Dalgarno sequence, SD). Cn gi
l vng lin kt ribosome (RBS), l mt phn ca trnh t nucleotide u
5 ca mt mRNA prokaryote c th kt hp b sung cp base vi u 3
ca 16S rRNA, dng lm tn hiu cho s khi u dch m.
Trnh t tng cng (enhancer). Trnh t nucleotide dng cis lm
tng cng phin m ca promoter trong gen eukaryote. N c th nm
cch promoter hng ngn cp base v hot ng theo c hai hng bt k
v tr no so vi promoter.
Trypsin. Enzyme xc tc phn ng thy phn cc lin kt peptide
trong protein v thy phn tng phn protein, dn xut t trypsinogen dch
ty tit vo rut non, c enzyme rut non enterokinase tc ng chuyn
thnh.
Vt ch (host). T bo dng nhn cc phn t DNA ln nhiu ln.
Vector. L cc phn t DNA v biu
hin gen, o vt ch (E. coli
. V
.
Vector biu hin (expression vector).
, v d: E.
coli. biu hin cc gen ngoi lai trong E. coli phi bt u bng vic gn
n vo trong vector biu hin (thng l plasmid). Vector ny phi c
cc cu trc cn thit sau: (1) Gen ch th chn lc (selectable marker)
m bo duy tr vector trong t bo. (2) Mt promoter kim sot phin m
cho php sn xut mt lng ln mRNA t cc gen c to dng. (3) Cc
trnh t kim sot dch m nh vng lin kt ribosome c b tr thch hp
v codon khi u AUG. (4) Mt polylinker a gen ngoi lai vo trong
mt hng chnh xc vi promoter.
Vector liu php (therapeutic vector).

C th s dng viral vector
hoc khng phi virus. Cc viral vector thng dng l adenovirus, adeno-
associated virus (AAV), retrovirus, lentivirus v paramyxovirrus. Cc vector
khng phi virus l cc phc hp cationic liposome nh GL67, PEI,
plasmid, SecR, Integrin...
Vector to dng (cloning vector). Phn t DNA mch kp c kh
nng t sao chp trong t bo vt ch. C th gn vo phn t ny mt on
hoc mt vi on DNA khc ngun to nn phn t DNA ti t hp dng
nhn dng.
Vt tan (plaque). Vng trn trong sut xut hin trn thm c ca
cc vi khun mc trn mi trng thch c, do s tan v lp li nhiu chu
k ca cc t bo vi khun b bacteriophage xm nhim v sinh tan.
Vi khun hiu kh (aerobe). Vi khun sng trong khng kh v s
dng oxygen t do sinh trng.
Vi khun k kh (anaerobe). Vi khun sng trong iu kin khng c
khng kh hay oxygen t do, ngc li vi hiu kh. Trong cc qu trnh k
kh, H
2
S c th c to ra gy mi kh chu.
Virus. Phc hp cha nucleic acid (DNA hoc RNA) nm trong mt
v bc protein, c kh nng gy nhim v ti bn bn trong t bo vt ch
c hiu, to ra nhiu virus, lan truyn t t bo ny sang t bo khc. Virus
l dng sng khng c cu trc t bo, c kh nng xm nhp vo cc t bo
sng xc nh v ch sinh sn bn trong cc t bo . Ging nh tt c
cc sinh vt khc, virus c b my di truyn ca ring mnh, m ha vic
tng hp cc ht virus t cc cht c trong t bo vt ch. Nh vy, virus l
nhng vt k sinh ni bo. Virus phn b khp ni trong t nhin, xm
nhp vo tt c cc nhm sinh vt. Ngi ta bit khong 500 loi virus
xm nhp ng vt mu nng, 300 loi xm nhp thc vt bc cao. Mt s
khi u ung th ng vt v ngi c th do virus. Virus tn ti hai
dng: dng ngh hay ngoi bo v dng sinh sn hay ni bo. Kch thc ca
cc ht virus t 15-350 nm, chiu di ca mt s loi virus c th t ti
2000 nm. Phn ln virus ch nhn thy c qua knh hin vi in t. Cht
mang thng tin di truyn ca virus l nucleic acid: DNA hoc RNA. V vy,
c th phn virus thnh hai loi: loi mang DNA v loi mang RNA.
Vi tim (microinjection). K thut a DNA vo nhn hoc vo t
bo cht ca t bo bng kim mao dn v bm p lc. Ton b thao tc
c tin hnh trn knh hin vi ngc pha (inverted microscope).
V tr cos (cos site).
phage .
V tr lin kt khng nguyn n nht (unique antigen combining
site). V tr lin kt vi khng th nm trn mt phn t khng nguyn.
Vng cp tc (hairpin loop). Vng chui n b sung to np gp
cha cc cp base to thnh xon kp,
.
Vng cng hng (downtream region). cp n v tr ca mt
on trnh t no nm pha u 3 ca gen hoc mt on gen quan
tm.
Vng a ni (polylinker hay polycloning site). Mt trnh t DNA
mch kp c tng hp nhn to c mang mt lot cc v tr nhn bit ca
cc enzyme hn ch. Trnh t ny c gn vo cc vector dng trong k
thut to dng gen (nh vng to dng).
Vng lin kt ribosome (ribosome binding site, RBS).
(xem
trnh t Shine-Dalgarno).
Vng ngc hng (upstream region).V tr ca mt trnh t
nucleotide no nm pha u 5 ca phn t DNA so vi gen quan tm.
Vng to dng (multiple cloning sites, MCS).
t s enzyme ct hn ch
thng dng, c thit k chn on DNA ngoi lai vo y.
Western blot. K thut chuyn protein tng s c phn tch
bng in di SDS trn polyacrylamide gel ln mng nylon hoc
nitrocellulose lai vi khng th th nht c hiu v sau l khng th
th hai c nh du enzyme nhm pht hin protein khng nguyn tng
ng ca n.
Xenobiotic. Mt loi hp cht c tm thy trong c th sng nhng
khng c sn xut bnh thng hoc mong i c mt trong c th. N
cng c th bao gm c cc cht hin din vi nng cao hn bnh thng
nhiu ln. c bit, cc loi thuc nh khng sinh c xem l xenobiotic
ca ngi bi v c th ngi khng t sn xut chng. Tuy nhin, trong
lnh vc mi trng thut ng ny thng c s dng ch cc cht gy
nhim nh cc dioxin, cc polychlorinated biphenyl..., v cc nh hng
ca chng ln khu h sinh vt (biota).
X-gal (5-bromo-4-chloro-3indolyl- -D-galactopyranoside). C cht
sinh mu vi -galactosidase cho ra sn phm c mu xanh lam.
YAC (Yeast artificial chromosome). Nhim sc th nhn to ca
nm men, c dng lm vector to dng nhng on DNA c kch
thc rt ln trong nm men.
Yu t tc ng cis (cis-acting element). on trnh t DNA ch biu
hin hiu qu trn chnh phn t DNA m n tc ng. V d: hp CAAT l
mt phn t tc ng cis i vi qu trnh phin m cc sinh vt
eukaryote.
Yu t tc ng trans (trans-acting element). Yu t di truyn c th
biu hin hiu qu m khng cn nm trn cng mt phn t vi on trnh
t ch. Thng yu t m ha cho mt sn phm protein (c th l mt
enzyme hay mt protein iu ha) v sn phm ny c th khuch tn n
im tc ng.

1. Ban T in-NXB Khoa hc v K thut. 2002. T in Bch khoa
Sinh hc. NXB Khoa hc v K thut, H Ni.
New York, USA.
3. Lawrence E. 1995. Hendersons Dictionary of Biological Terms. 7
th
ed.
Longman Group Ltd. Singapore.
5. Singleton P and Sainsbury D. 2001. Dictionary of Microbiology and
Molecular Biology. 3
rd
ed. John Wiley & Sons, Ltd. UK.
4
th

356
MC LC

Trang
LI NI U
Phn I. CC KHI NIM V NGUYN L C BN 5
Chng 1. M U 6
I. nh ngha cng ngh sinh hc 6
1. nh ngha tng qut 6
1.1. Cng ngh sinh hc truyn thng 7
1.2. Cng ngh sinh hc hin i 7
2. Ni dung khoa hc ca cng ngh sinh hc 7
3. Cc lnh vc ng dng ca cng ngh sinh hc 9
3.1. Cng ngh sinh hc trong nng nghip 9
3.2. Cng ngh sinh hc trong y dc 10
3.3. Cng ngh sinh hc cng nghip v ch bin thc phm 10
3.4. Cng ngh sinh hc mi trng 11
II. S lc lch s hnh thnh cng ngh sinh hc 12
1. Giai on th nht 13
2. Giai on th hai 13
3. Giai on th ba 13
4. Giai on th t 14
III. Mt s kha cnh v khoa hc v kinh t ca cng ngh sinh
hc hin i
15
1. V khoa hc 15
2. V kinh t 16
2.1. Nhng cng ty a quc gia v cng ngh sinh hc 16
2.2. S l thuc vo cc cng ty a quc gia v cng ngh
sinh hc
16
IV. Cc vn php l ca cng ngh sinh hc hin i 17
1. An ton sinh hc 18
1.1. S chuyn gen bng ht phn 18
1.2. S bn vng ca DNA trong t 19

357
1.3. Chuyn gen ngang t thc vt vo vi sinh vt t 20
1.4. Chuyn gen t thc vt vo virus 21
2. An ton thc phm 22
2.1. Cc cht gy d ng 23
2.2. nh gi an ton ca cc thc phm 24
3. o c sinh hc 25
4. Quyn tc gi v s hu tr tu 27
4.1. Quyn tc gi 27
4.2. S hu tr tu 28
Ti liu tham kho/c thm 30

Chng 2. CNG NGH DNA TI T HP 31
I. M u 31
II. Phn lp on DNA/gen 31
1. Tch cc on DNA t genome 32
2. Sinh tng hp cDNA t mRNA 33
3. Phn lp on DNA bng phng php PCR 33
III. To dng (gn) on DNA/gen vo vector 36
1. Enzyme hn ch 36
2. Cc vector c dng to dng cc on DNA 38
2.1. Plasmid vector 39
2.2. Bacteriophage vector
42
3. Gn on DNA vo vector 43
3.1. Gn cc on cDNA 43
3.2. Gn cc sn phm PCR 46
4. Bin np vector ti t hp vo vi khun/t bo vt ch 47
4.1. in bin np 47
4.2. Ha bin np 48
IV. Chn dng mang DNA ti ti t hp 48
1. Lai khun lc v vt tan 49
2. Kh hot tnh bng chn on 50
3. To dng nh hng 50

358
V. Biu hin gen c to dng 52
1. Vector biu hin 53
2. Xc nh mc biu hin ca gen c to dng 55

Chng 3. CNG NGH LN MEN VI SINH VT 59
I. M u 59
II. Sinh trng ca vi sinh vt 59
III. Sinh khi vi sinh vt v cng ngh ln men 64
1. Sinh khi vi sinh vt 64
2. Qu trnh ln men 65
IV. Cc sn phm ln men vi sinh vt 68
1. Ln men ru 68
1.1. Ru trng 68
1.2. Ru vang 70
2. Sn xut enzyme 71
2.1. Cc loi enzyme vi sinh vt 72
2.2. Sinh tng hp enzyme cm ng 75
2.3. Nhng phng php nui cy vi sinh vt sn xut
enzyme
76
2.4. Tch v tinh sch ch phm enzyme 82
3. Sn xut khng sinh 83
3.1. Penicillin 83
3.2. Streptomycin 85
3.3. Tetracycline 86
4. Sn xut acid hu c 87
4.1. Acetic acid 87
4.2. Citric acid 89
V. Cng ngh ti t hp vi sinh vt 89
1. Cc vi sinh vt ti t hp 90
2. Cc ng dng trong cng ngh vi sinh 90

359

Chng 4. CNG NGH SINH HC THC VT 93
I. M u 93
II. Nui cy m v nhn ging in vitro 94
1. Thut ng hc 94
1.1. Nui cy nh phn sinh 95
1.2. Sinh sn chi nch 95
1.3. To chi bt nh 95
1.4. Pht sinh c quan 96
1.5. Pht sinh phi v tnh 96
2. Nhn ging in vitro v cc h thng nui cy m 96
2.1. Ti sinh cy mi t cc cu trc sinh dng 97
2.2. Nhn ging thng qua giai on callus 101
2.3. Nhn ging thng qua pht sinh phi v tnh-cng ngh
ht nhn to
102
3. Cc giai on trong quy trnh nhn ging v tnh in vitro 107
3.1. Giai on I-cy gy 107
3.2. Giai on II-nhn nhanh 108
3.3. Giai on III-chun b v a ra ngoi t 110
4. Nhn ging in vitro v vic s dng ging u th lai 110
5. Nhn ging in vitro v cc c im khng di truyn 111
5.1. Hin tng cc c im epigenetic c lu li 111
5.2. Hin tng cc c im epigenetic khng lu li 112
III. Cc k thut chuyn gen thc vt 112
1. Bin np gin tip thng qua Agrobacterium 114
1.1. Vi khun Agrobacterium 114
1.2. Ti-plasmid 115
1.3. Vng T-DNA 116
1.4. Chuyn DNA ngoi lai vo t bo v m thc vt nh
Agrobacterium tumefaciens
117
2. Cc gen ch th chn lc v gen ch th sng lc 119
3. Chuyn gen bng vi n 122

360
4. Cc ng dng ca cng ngh chuyn gen 123
4.1. Mt s kt qu bc u 123
4.2. Trin vng v hng pht trin 124
5. Cng ngh di truyn trong khng cht dit c 125
6. Cng ngh di truyn trong khng su-bnh 126
6.1. Khng cn trng 126
6.2. Khng cc virus thc vt 127
6.3. Khng cc bnh nm 129
6.4. Khng cc bnh vi khun 130
IV. Sn xut cc dc liu sinh hc 130
1. Cc hp cht t nhin 130
1.1. Cc alkaloid 132
1.2. Cc steroid 132
1.3. Mt s cht khc 133
2. Cc protein ti t hp 134
3. Vaccine thc phm 136

Chng 5. CNG NGH SINH HC NG VT 140
I. M u 140
II. Nui cy t bo ng vt c v 141
1. Cc u im v hn ch ca nui cy t bo ng vt 141
1.1 Cc u im ca nui cy t bo ng vt 141
1.2. Mt s hn ch ca nui cy t bo ng vt 143
2. Cc dng t bo ng vt c v v cc c im ca n 143
2.1. Cc t bo dch huyn ph 143
2.2. Cc t bo dnh bm 144
3. Cc sn phm thng mi ca nui cy t bo ng vt c
v
144
4. Glycosyl ha protein 145
5. Mi trng nui cy t bo ng vt c v 147
6. Nui cy t bo ng vt c v trn quy m ln 149

361
6.1. Cc iu kin chung 149
6.2. Nui cy m 150
6.3. Nui cy m c cung cp cht dinh dng 152
6.4. Nui cy th n nh ha tnh 152
6.5. Nui cy perfusion 154
6.6. S lng v cht lng sn phm 156
III. Cng ngh di truyn ca cc t bo ng vt c v 158
1. Cc phng php chuyn np gen 159
1.1. Phng php chuyn nhim 159
1.2. Phng php lipofection 159
1.3. Phng php xung in 160
1.4. Phng php vi tim 161
1.5. Phng php dng sng bn gen 163
1.6. Phng php dng cc vector virus 163
2. Cc iu kin cn thit cho biu hin gen ngoi lai 165
3. Cc iu kin thc nghim ti u 167
4. ng chuyn np gen ch th v gen thc nghim 167
5. K thut t bo mm phi, chuyn gen v ti t hp tng
ng
169
IV. Nui cy t bo mm sn xut c quan ngi 171
1. ng dng t bo mm phi trong nghin cu c bn 172
2. Nghin cu chc nng gen 173
3. Nghin cu cc m hnh bnh l 173
4. ng dng trong y hc ti to 173
V. Cng ngh phi ng vt c v 175
1. Cy truyn hp t 175
2. Bo qun phi 175
3. Nui cy tm thi phi trong c th sng 176
4. K thut bc phi bng agar 176
5. K thut nui cy tm thi phi trong ng dn trng 176
VI. Nhn bn v tnh ng vt c v 177
1. Khi nim c bn 177

362
2. Nhn bn v tnh ng vt 177
3. Nhn bn v tnh cu Dolly 178

Chng 6. CNG NGH PROTEIN 182
I. M u 182
II. Cu trc protein 183
III. Cc cng c 184
1. Nhn dng trnh t 184
2. Xc nh cu trc v m hnh ha 185
3. Bin i trnh t 185
4. Pht trin phn t 188
5. Thit k trnh t de novo 190
6. Biu hin 190
7. Phn tch 191
IV. Mt s ng dng ca cng ngh protein 192
1. Cc t bin im 192
1.1. Betaseron/Betaferon 192
1.2. Humalog 192
1.3. Cc t dc vaccine mi 193
2. Sp xp li vng 194
2.1. Cc vng lin kt 194
2.2. Trao i cc vng protein 195
3. Sp xp li ton b protein 196
4. Cc tng tc protein-phi t 197
4.1. Bin i enzyme 197
4.2. Cc cht ch vn hormone 197
4.3. Thay th cc lin kt c hiu 197
V. Sn xut protein trn quy m ln 198
1. Ln men E. coli ti t hp 199
2. Ln men nm 200
3. Cc enzyme vi sinh vt thay th cc enzyme thc vt 200

363
4. Cc nguyn tc ha sinh c bn 201
4.1. S cm ng 201
4.2. c ch ngc 202
4.3. c ch dinh dng 204
5. Cng ngh di truyn 205
VI. Cc qu trnh tch chit v tinh sch protein 207
1. Thu hi protein 209
1.1. Thu hi cc protein ngoi bo 209
1.2. Thu hi cc protein ni bo 209
2. Tinh sch s b 216
2.1. Loi b cc mnh v ca t bo 216
2.2. Ly tm m 216
2.3. Ly tm dng chy lin tc 217
2.4. Lc bng mng 217
3. H phn tch hai pha nc 218
4. Cc phng php kt ta 220
4.1. Kt ta bng ammonium 220
4.2. Kt ta bng cc dung mi hu c 220
4.3. Kt ta bng cc polymer khi lng phn t cao 221
4.4. Kt ta bng nhit 221
5. Cc phng php sc k 221
5.1. Sc k lc gel 222
5.2. Sc k trao i ion 224
5.3. Sc k i lc 227
5.4. Sc k tng tc k nc 230
5.5. Cc k thut sc k lng hiu sut cao 230
6. Siu lc 231
7. Thit k cc protein tinh sch 232
7.1. Cc th vi 232
7.2. Cc ui i lc 233


364

Phn II. CC NG DNG CA CNG NGH SINH HC 237
Chng 7. CC NG DNG TRONG NNG NGHIP 238
I. M u 238
II. Ci thin v nhn nhanh ging cy trng 238
1. Nhn ging v tnh in vitro 239
2. Sn xut cy n bi in vitro 240
2.1. Phng php to th n bi in vivo 240
2.2. Phng php to th n bi in vitro 241
3. Dung hp protoplast hay lai v tnh t bo thc vt 241
3.1. Dung hp protoplast bng ha cht 242
3.2. Dung hp protoplast bng in 242
4. Chn dng bin d soma 243
5. Chuyn gen vo cy trng 244
5.1. Cy la 244
5.2. Cy la m 246
5.3. Cy la mch 247
5.4. Cy u tng 247
5.5. Cy u ty 248
5.6. Cy bng 248
5.7. C nh m 249
III. Chn nui v th y 250
1. K thut cy chuyn phi 250
2. To ch phm phng trnh bnh cho ng vt 250
3. Chuyn gen vo ng vt 251
IV. Ch bin thc phm 252
1. Sn xut sa 253
1.1. Sn xut sa chua 253
1.2. Sn xut phomt 255
2. Ch bin tinh bt 257
3. Sn xut nc ung ln men 257
3.1. Sn xut bia 258

365
3.2. Sn xut ru vang 259
3.3. Sn xut ru trng 262
4. Cc sn phm cha protein 265
4.1. Thc phm ln men truyn thng giu protein 265
4.2. Protein vi khun n bo 265
5. Ch bin rau qu 268

Chng 8. CC NG DNG TRONG Y-DC 270
I. M u 270
II. Vaccine 270
1. Cc phng thc tim chng vaccine hin nay 271
1.1. Cc vaccine bt hot 271
1.2. Cc vaccine sng nhc c 272
2. Vai tr ca cng ngh di truyn trong nhn dng, phn tch
v sn xut vaccine
272
2.1. Nhn dng v to dng cc khng nguyn c tim nng
vaccine
272
2.2. Phn tch cc khng nguyn vaccine 273
2.3. Sn xut cc vaccine tiu n v 274
3. Ci thin v sn xut cc vaccine sng nhc c mi 275
3.1. Ci thin cc vaccine sng nhc c 275
3.2. Cc vector sng ti t hp 275
4. Cc hng tip cn khc trong sn xut vaccine 278
4.1. Cc DNA vaccine 278
4.2. Cc peptide vaccine 279
4.3. Khng cc kiu gen c th 279
III. Khng th n dng 280
1. Sn xut hybridoma bng cch dung hp t bo sinh dng 280
2. Sn xut khng th n dng bng cng ngh DNA ti t
hp
281
2.1. Phn lp cc gen immunoglobulin 281

366
2.2. Biu hin scFV trn b mt bacteriophage 282
3. Khng th n dng trong nghin cu Sinh-Y 282
4. Khng th n dng trong chn on v iu tr bnh 283
IV. Liu php gen 284
1. Cc loi liu php gen 285
1.1. Liu php soma 285
1.2. Liu php gen t bo mm 285
2. Cc ng dng ca liu php gen trong cha bnh 286
2.1. Bnh thiu ht min dch phi hp trm trng 286
2.2. Bnh ung th 287
2.3. Bnh thiu mu hng cu lim 288
2.4. Bnh x nang 290
2.5. Bnh HIV/AIDS 291
V. Protein tr liu 292
1. Sn xut hormone 292
1.1. Hormone sinh trng ngi 292
1.2. Somatostatin 292
2. Sn xut enzyme 293
3. Sn xut thuc nh cng ngh DNA ti t hp 293
3.1. Sn xut insulin 293
3.2. Sn xut interferon 294
3.3. Sn xut interleukin 295
VI. Chn on bnh can thip sm 296
1. Chn on sm gii tnh ca thai 296
2. Chn on sm d hnh, qui thai trc khi sinh 297
3. Chn on pht hin cc tc nhn gy bnh ngoi lai 298
4. Chn on cc bnh di truyn 299
5. In du DNA 300

Chng 9. CC NG DNG TRONG MI TRNG 302
I. M u 302

367
II. X l nc thi 302
1. X l hiu kh bng h thng bn hot tnh 303
2. X l ym kh 304
3. Thu hi nc 305
III. Phn hy bn hu c 306
IV. X l cht thi rn 309
V. X l kh thi 311
1. Loi b cc hp cht v c d bay hi 311
2. Loi b cc hp cht sulphur v nitrogen t kh ng khi
bng phng php sinh hc
315
VI. Phn hy cht rn 317
1. Kch thch sinh hc v tng sinh hc 317
2. Cc k thut phn hy cht rn 318
2.1. Phn hy sinh hc ti ch 318
2.2. Landfarming 318
2.3. Cc b phn ng sinh hc pha bn 319
VII. X l nc ngm 320
1. S phc hi hot ng 320
2. S suy gim t nhin v s gim st 322

Ph lc. MT S THUT NG C BN 324

MC LC 356


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