I HC Y DC TP.

HCM
Khoa Dc
















SINH HC PHN T
(TI LIU GING DY SAU I HC)







Bin son:
PGS.TS. Trn Ct ng
TS. Nguyn T Anh












2012


3
Mc lc
Bi 1. Nghin cu sinh hc trong th k 21 7
1.1. S bin chuyn ca sinh hc phn t 7
1.2. H gen hc - Genomics 7
1.2.1. Khi nim................................................................................................. 7
1.2.2. Mt s cng c......................................................................................... 8
1.2.3. ng dng ................................................................................................. 9
1.3. H protein hc - Proteomics 10
1.3.1. Khi nim............................................................................................... 10
1.3.2. Mt s cng c....................................................................................... 11
1.3.3. ng dng proteomics trong nghin cu pht trin thuc...................... 14
1.4. H t bo hc - Cytomics 16
1.5. Sinh hc h thng 16
Bi 2. ng dng sinh hc h thng trong tm kim thuc mi 19
2.1. Khi qut 19
2.2. Omics: To v khai thc d liu quy m ln 20
2.3. Cc m hnh my tnh: t cc qu trnh sinh hc n sinh l bnh 22
2.4. S dng cc h thng t bo phc tp th nghim v to m hnh sinh
hc 24
2.5. Tng lai 28
Bi 3. Tin sinh hc 30
3.1. Lch s 30
3.2. Cc nh ngha 31
3.3. Cc lnh vc nghin cu chnh 31
3.3.1. Phn tch trnh t.................................................................................... 31
3.3.2. To m hnh cc h thng sinh hc ....................................................... 35
3.3.3. Phn tch hnh nh hiu nng cao .......................................................... 35
3.4. Cng c 35
Bi 4. K thut xc nh kiu gen 37
4.1. i cng 37
4.1.1. ngha v lch s ca vic nh typ vi sinh vt.................................... 37
4.1.2. Tiu ch ca mt phng php nh typ tt........................................... 37
4.1.3. Phn loi cc phng php nh typ phn t......................................... 38
4.1.4. La chn gen phn t bng phng php phn t............................ 38
4.1.5. Nhc im ca phng php phn t.................................................. 39

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4.2. Du n da trn PCR ca nhim sc th vi khun 39
4.2.1. K thut chung .......................................................................................39
4.2.2. Phng php nh typ ribosom..............................................................41
4.2.3. Rep-PCR.................................................................................................41
4.2.4. Th nghim RAPD.................................................................................43
4.3. Cc phng php ct gii hn b gen 44
4.3.1. Cc vn chung....................................................................................44
4.3.2. Phng php RFLP kt hp Southern blot.............................................45
4.3.3. PCR-RFLP..............................................................................................46
4.3.4. AFLP ......................................................................................................47
4.3.5. CFLP.......................................................................................................48
4.3.6. Phn tch PFGE nhim sc th ...............................................................48
4.4. nh trnh t DNA 50
4.5. Cc kha cnh thc tin ca vic nh typ bng phng php phn t 51
Bi 5. ng dng cng ngh sinh hc trong tm kim thuc mi 53
5.1. Gii thiu 53
5.1.1. Qui trnh tm kim thuc mi .................................................................53
5.1.2. Pht hin thuc mi bng phng php sng lc hiu nng cao ...........54
5.2. Pht trin thuc mi da trn ng dng cng ngh sinh hc 55
5.2.1. K thut genomic ...................................................................................56
5.2.2. K thut proteomic.................................................................................57
5.2.3. K thut metabolomic ............................................................................58
5.2.4. Cng ngh sinh hc ng dng k thut nano .........................................59
5.2.5. Cc phng php sng lc......................................................................59
5.2.6. Antisense ................................................................................................60
5.2.7. iARN.......................................................................................................61
5.2.8. Biochip v microarray trong tm kim thuc .........................................62
5.2.9. ng dng tin sinh hc trong tm kim thuc..........................................63
5.3. Kt lun 63
Bi 6. Hp cht thin nhin v ng dng k thut sinh hc phn t trong nghin
cu 65
6.1. Hp cht thin nhin 65
6.1.1. Khi nim...............................................................................................65
6.1.2. Ngun gc...............................................................................................65
6.1.3. Bn cht ha hc ....................................................................................67
6.2. Kh khn trong sn xut hp cht thin nhin 69
6.2.1. Kh khn trong chit xut ......................................................................69


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6.2.2. Kh khn trong nui trng..................................................................... 69
6.2.3. Kh khn trong tng hp....................................................................... 70
6.3. Sn xut hp cht thin nhin bng cng ngh sinh hc 70
6.3.1. ADN ton phn...................................................................................... 70
6.3.2. Thnh lp th vin gen .......................................................................... 71
6.3.3. Sng lc gen chu trch nhim sinh tng hp hp cht thin nhin...... 71
6.3.4. D biu hin hp cht thin nhin.......................................................... 72
6.4. Kt lun 72
Bi 7. ADN ti t hp v tnh an ton sinh hc 73
7.1. Gii thiu 73
7.1.1. ADN ti t hp (Recombinant DNA = rDNA) ..................................... 73
7.1.2. ng dng ADN ti t hp ..................................................................... 73
7.1.3. Phn loi mc an ton sinh hc i vi phng th nghim.............. 73
7.2. nh gi mc an ton sinh hc ca ADN ti t hp 74
7.2.1. i tng cn nh gi mc an ton................................................ 75
7.2.2. nh gi mc an ton....................................................................... 75
7.2.3. nh gi mc nguy him ca sinh vt bin i gen......................... 76
7.2.4. Qui trnh nh gi mc nguy him................................................... 77
7.3. Cc bin php ngn chn nhm m bo an ton sinh hc 77
7.4. Cc th nghim phi tun th qui nh an ton sinh hc 78





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B B i i 1 1. . N Ng gh hi i n n c c u u s si in nh h h h c c t tr ro on ng g t th h k k 2 21 1
Mc tiu:
- Trnh by c vai tr, nhim v v s pht trin ca sinh hc phn t trong th k 21
- Trnh by c mt s phng php nghin cu ca sinh hc phn t hin i
1.1. S bin chuyn ca sinh hc phn t
Sinh hc lc khi u l mt khoa hc m t, cc nh sinh hc ch yu quan st v
ghi nhn, so snh, phn loi v gii thch cc hin tng sinh hc n l, t mang tnh khi
qut. n th k 19, Charles Darwin vi thuyt tin ha v sau Gregor Mendel vi cc
nh lut di truyn th sinh hc mi bt u c ci nhn su rng hn vo c ch ca s sng
mc h thng. n u th k 20, cc nh sinh hc bt u quan tm n cc quan st c tnh
nh lng v nghin cu quan h nhn-qu trong sinh hc. Tuy nhin, sinh vt l mt h
thng phc tp, nht l khi t n trong mi trng sng, cc quan h nhn-qu khng c
kt ni mt cch trc tip m trong mt mng li a chiu vi cc kt ni chng cho nhau.
Do vy, lm sng t v hiu c cc quan h phc tp ny cc nh sinh hc bt u
vi cc h thng n gin, trong chui quan h nhn qu ngn hay trc tip. Cch lm vic
vi cc h thng n gin hay chia nh cc quan h phc tp thnh tp hp cc quan h 1:1,
c tnh lp li cao gip lm sng t nhiu qui lut t nhin trong sinh hc nh sao chp
ADN, m di truyn, phin m, dch m,
Tuy nhin, khi hu ht cc qui lut t nhin c lm sng t v b gen ca mt s
sinh vt c gii m hon ton, ban u ch l cc vi sinh vt, nhng n nay b gen ngi
cng c gii m xong, th cc nh sinh hc nhanh chng nhn ra rng cc h thng sng
vi c ch iu ha nhiu lp v iu ha ngc tr nn qu phc tp c th c hiu
theo kiu khoa hc chnh xc ca sinh hc phn t. Chng hn m di truyn ca sinh vt
c chng minh l ph qut, nhng vic suy din kt qu thu c vi sinh vt cho cc sinh
vt bc cao hn vn c th dn n nhiu sai lm, v cc sinh vt bc cao phn ln trnh t
ca ADN tuy khng m ha cho protein nhng li c vai tr iu ha. Do nghin cu sinh
hc cn cch tip cn c tnh ton th hn. iu may mn l t cui th k 20 n nay cng
ngh in ton pht trin mnh m, cho php s dng my tnh phn tch cc quan h
phc tp, nhiu tham s m trc y khng th thc hin c.
S pht trin ca cng ngh in ton v s b tc ca ha sinh v sinh hc phn t khi
vn dng cc qui lut gii quyt vn mc t bo dn n s ra i v pht trin ca
cc ngnh nghin cu mi vi cch tip cn c tnh h thng nh h gen hc, h protein hc,
h t bo hc v sinh hc h thng.
1.2. H gen hc - Genomics
1.2.1. Khi nim
B gen (genome) l tp hp y tt c cc gen ca mt sinh vt. Trc y khi ni
n b gen tc l ni n cc nhim sc th nguyn vn, tuy nhin vi s ra i ca k thut
nh trnh t gen vo nm 1975, ngi ta bt u lm vic vi cc on ADN c th hay cc
gen. n nm 1997 b gen ca Bacillus subtilis v Escherichia coli c gii trnh t v
cng b. Gen ngi u tin c to dng vo nm 1976, cc k thut knock-out v
knock-in gen c a ra t 1986, n nm 1996, cc c s d liu pht trin n mc
c th ng dng trong vic nghin cu b gen v nm 2006 trnh t ca nhim sc th cui
cng ca ngi c cng b. Khoa hc v b gen, h gen hc (h gen hc) cng bt

8
u cng vi s tch ly trnh t cc gen c gii m. H gen hc nghin cu vic lp bn
, gii trnh t, phn tch v ch gii b gen.
Vic nghin cu b gen c th c chia thnh hai nhnh: cu trc v chc nng. H
gen hc cu trc l pha khi u ca vic phn tch b gen, n kt thc vi vic xy dng cc
bn v vt l, di truyn v phin m ca mt sinh vt. Cch tip cn ny tp trung vo vic
tm hiu cch thc s thay i kiu gen dn n thay i kiu hnh, da trn bn vt l v
di truyn v s a hnh v trnh t ADN. H gen hc chc nng (biu hin) da trn b su
tp cc cADN. Cch tip cn ny cho php pht trin cc phng php h thng tm kim
gen cn quan tm v xc nh chc nng ca chng. Cc gen c to dng v trnh t ADN
tng ng ca chng cung cp cng c xc nh c im biu hin mt cch ton din
ca mt h gen, v cc nghin cu thc nghim c h thng v chc nng ca cc sn phm
gen. Do , h gen hc chc nng s dng cc thng tin ca h gen hc cu trc.
1.2.2. Mt s cng c
1.2.2.1. Gii trnh t b gen
Trnh t b gen ca nguyn mt sinh vt c gii m, lp ghp v xy dng bn l
bc u tin trong vic nghin cu b gen. i vi cc b gen n gin nh vi sinh vt, vic
gii trnh t c th thc hin bng k thut shot gun, trong b gen c ct thnh cc
mnh nh enzym ct gii hn, tng mnh c gii trnh t ring v vic lp ghp cc mnh
li thnh b gen hon chnh da trn vic phn tch cc trnh t trng nhau gia cc mnh.
Vi cc b gen ln v d ca ngi, cc nhim sc th s c ct thnh cc mnh khong
150.000 nucleotid v cc mnh ny li c ct v to thnh cc BAC (Bacterial Artificial
Chromosome), sau cc BAC s c gii trnh t theo kiu shot gun v lp ghp ngc
ln ti to b gen hon chnh. Cch tip cn ny rt tn thi gian.
Gn y nh cc tin b v in ton, ngi ta c th gii trnh t v lp ghp cc b gen ln
bng k thut shot gun.
1.2.2.2. ADN microarray
ADN microarray c th c s dng nghin cu s thay i v mc biu hin ca
gen, pht hin cc a hnh nucleotid n (single nucleotide polymorphism, SNP) , nh typ
hay nh trnh t cc b gen t bin,
Nguyn l chnh ca ADN microarray da trn k thut Southern blot, tc s lai ha
gia cc ADN si n nh c tnh b sung cp base c hiu. Tuy nhin, thay v thc hin
tng phn ng Southern blot n l ADN microarray s dng mt s lng ln cc on d
khc nhau c gn trn mt vi chip theo trt t nht bit trc to thnh vi chip ADN, mu
c lai ha vi vi chip ADN v cc v tr c xy ra lai ha s c c v gii m theo v tr
(nh tnh) v cng tn hiu (nh lng). Mu s dng c th l ADN hoc ARN.

Hnh 1.1. Tn hiu ADN microarray


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1.2.3. ng dng
1.2.3.1. H gen hc dc
H gen hc dc nghin cu s lin h gia kiu gen ca mt c th v kh nng
chuyn ha cc cht ngoi lai ca ngi . N c phm vi kh rng v tng t nh y hc
phn t n nhm pht hin, theo di v x l cc nguyn nhn mc phn t ca bnh. H
gen hc dc ng dng cc k thut nghin cu h gen nh nh trnh t gen, di truyn hc
thng k v phn tch biu hin gen i vi thuc trong qu trnh pht trin v th nghim
lm sng. V nhiu bnh pht ra do hu qu t s hot ng khng chnh xc ca mt nhm
gen, h gen hc dc c th nhn din cc gen hay locus c lin quan khi xc nh s p ng
vi thuc. Theo cch ny, c im di truyn ca mt dn s bnh nhn tr thnh mt phn
tch hp ca tin trnh khm ph v pht trin thuc. H gen hc dc c th da trn cc hiu
bit mi mc phn t ny pht hin cc ch tr liu v can thip mi v gp phn lm
sng t nhm gen quyt nh tnh hiu qu v c tnh ca thuc.
1.2.3.2. nh hng i vi vic pht minh thuc v nghin cu lm
sng
Vic ng dng h gen hc dc trong cc c s th nghim tin lm sng c th gip
khi u sng lc cc hp cht vi sai s thp nht gia cc c th. Nu gen ch c la
chn, ngi ta c th tm c hp cht c tc dng tt nht vi tt c cc typ ca gen v
chn th nghim su hn. Do , vic chn thuc s thay th cho vic chn bnh nhn,
gim ri ro do p ng khc bit gia cc bnh nhn khi lp h s ng k v khi thng mi
ha, cng nh gim nhu cu sng lc v di truyn. H gen hc cng c th c s dng d
bo tc dng ph trc khi a vo th nghim lm sng. V d, kiu biu hin gen gan ca
ng vt dng thuc c th ch ra cc chui gen c lin quan n c tnh c c khi ng
hay khng. S bin ng v mc biu hin gen cng hu ch nh bin d di truyn trong
vic d on p ng thuc bt k giai on th lm sng no v cng l mt du hiu chn
on. D liu di truyn dc rt quan trng trong qu trnh pht trin mt hp cht c gii
hn tr liu hp hay c chuyn ha t tin thuc, cc thng tin ny c th nh hng n
quyt nh nn tip tc pht trin thuc hay khng hay thit k th nghim nhm nghin
cu p ng lm sng cc i tng c s a hnh i vi enzym lin quan.
Trong giai on tin lm sng, c nhiu vn thit yu cn c xem xt. Cc vn
v ha hc ca thuc, bo ch thuc c hp thu thch hp, kiu chuyn ha, phn b, v
o thi vn cn mang tnh kinh nghim. D sao, cc thuc phn t nh tc ng trn cc ch
c pht hin bi h gen hc s nhanh chng chim a s cc thuc c a ra th trng
trong thi gian ti.
H gen hc dc hu ch trong nhiu giai on pht trin thuc. N nh hng ng k
n thit k th nghim, ch yu thng qua vic ci thin cc tiu ch chp nhn/ loi b v
nh gi chnh xc hn p ng ca bnh nhn. Cc gen c lin quan n chuyn ha thuc
trong nghin cu tin lm sng c th c xc nh bnh nhn tham gia th nghim giai
on I. Kiu gen c lin quan n tc dng ph cng c th c dng sng lc bnh nhn
lin quan trong cc th nghim tip sau. Hn na, nu d liu v hiu qu c thu thp trong
th nghim giai on I, s a hnh ca gen ch tc ng ca thuc c th c xc nh
bnh nhn tham gia th nghim nh gi hay bin ng v p ng thuc. Phn tch ny c
th c hiu chnh trong th nghim giai on II v cho php cng ty tin hnh th nghim
giai on III trong mt nhm nh bnh nhn p ng tt v t gp tc dng ph. Kt qu l
thuc khng ch c hiu qu tt hn m cn c h s an ton r hn.
mc lm sng, trong khi cc triu chng ca bnh thng nht qun, cc khc bit
v gen c th dn n hiu qu iu tr khc nhau gia cc bnh nhn. H gen hc dc c th

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xc nh quan h gia s khc bit v gen vi s p ng thuc khc nhau, qua c th tng
tc vic pht minh thuc mi mt cch ng k. Mt s thuc khng t yu cu trong cc
th nghim trc kia c th c xem xt li s dng i vi mt nhm bnh nhn khc.
1.3. H protein hc - Proteomics
1.3.1. Khi nim
H protein hc, proteomics, c hnh thnh t nhng nm 1990, l ngnh khoa hc
nghin cu h protein hon chnh (proteome) ca mt sinh vt hay mt t bo, tc nghin cu
v kho st c im ca tt c protein bao gm s tng hp, bin i, chc nng v s tng
tc ca chng. Mt cch c th hn proteomics:
1. Xc nh cu trc v chc nng ca tt c cc protein c to ra bi b gen ca mt
sinh vt, bao gm cc bin i hu dch m
2. Xc nh tt c cc tng tc ca cc protein ny vi phn t ln hay nh khc
3. Xc nh s biu hin ca cc protein ny theo thi gian v iu kin sinh l
4. Phi hp cc thng tin ny thnh mt bn m t thng nht v nht qun ca sinh vt
mc t bo, c quan hay sinh vt hon chnh.
Vic xc nh cc protein c to ra bi t bo (proteome) c th phc tp v n qu
ln. Cch n gin nht l nh cp 1:1 gia tng protein v gen ca n. iu ny c th thc
hin d dng nhn nguyn thy v khng c ct-ni ARNm, nhng vn kh thi nhn tht.
Cng cn lu rng mi gen c th khp vi nhiu protein do s bin i hu dch m hoc
s ct-ni theo nhiu cch. Vic xc nh chc nng ca protein th phc tp hn. C th thc
hin theo cch truyn thng nh t bin hay phn lp protein. Tuy nhin khi s protein
bit r chc nng v cu trc tng ln, ngi ta c th s dng cc cng c tin sinh hc d
on v do rt ngn qu trnh. Nghin cu s biu hin ca protein mc tng th,
proteomics biu hin, hin nay cn gii hn vic chp nh proteome sau khi kch thch t
bo mt cch thch hp. Vic phn tch cc nh chp ny tp trung trong 2 kha cnh: xc
nh s thay i v mc (tng hay gim) v thay i v bin i hu dch m, thng l s
phosphoryl ha protein. Cui cng cc thng tin c kt ni nh cc cng c in ton.
S pht trin ca cc k thut phn tch v nhn din dn n s hnh thnh nhiu
lnh vc c th hn ca proteomics nh proteomics biu hin v proteomics tng tc. Hn
na, cc ng dng c th cng c hnh thnh nh proteomics lm sng v proteomics
c tnh.
Proteomics biu hin
Proteomics biu hin c nh ngha l s xc nh, nh lng v phn tch mc
ton b cc protein c biu hin ca mt sinh vt, m, hay t bo trong iu kin bnh
thng hay cc iu kin khc nh bnh tt hay sau khi c x l vi mt cht no . Khi
u, lnh vc ny ch yu da trn k thut in di 2 chiu (2-DE), nhng gn y cc k
thut khng s dng gel c pht trin nh LC-MS v protein array.
Proteomics tng tc
Proteomics tng tc nghin cu s tng tc gia cc protein hay gia protein vi cc
cht khc v nghin cu vai tr ca cc phc hp protein trong cc qu trnh sinh hc. Cc
phng php c s dng nh l h thng lai i nm men (yeast two-hybrid system), hin
th phage (phage display), protein array, v tinh ch i lc cc phc hp ri phn tch bng
khi ph c p dng thnh cng trong nghin cu proteomics tng tc.
Proteomics lm sng and Proteomics c tnh
y l cc lnh vc ng dng ca proteomics biu hin trong lm sng. Proteomics lm
sng bao hm vic xc nh cc du n sinh hc (biomarker) pht hin bnh hay tin trin


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hay thm ch l s hi phc sau khi iu tr. Proteomics c tnh tp trung vo vic lm sng
t tc ng c hay gy bnh tim tng ca cc cht hay yu t mi trng.
1.3.2. Mt s cng c
1.3.2.1. in di gel hai chiu (2D Gel Electrophoresis, 2-DE)
2-DE l k thut c s dng rng ri nht trong nghin cu proteomics tch v
phn tch protein. Trong 2-DE, protein c tch v hin hnh bng cc php nhum trc
hay sau in di, nhum theo loi protein (v d protein c phosphoryl ha hay glycosyl
ha), nhum hunh quang hay t x k vi cc protein c nh du phng x. Thng
thng, 2000 n 3000 protein ca c phn gii trn mt gel 2-DE chun (Hnh 1.2). K
thut ny c tnh lp li tt, nh lng c v kh n gin c th thc hin vi s mu
phn tch ln.

Hnh 1.2. Phn tch protein bng in di gel polyacrylamid hai chiu
V k thut vic in di c tin hnh 2 ln, ln th nht mu c tch da trn in
di ng in (isoelectric focusing, IEF), kt qu ca ln chy th nht c dng lm mu cho
ln chy in di th 2 da vo k thut tch theo kch thc.
1.3.2.2. Khi ph
Mt trong cc th thch ca nghin cu proteomics l xc nh trnh t protein c
tch ra. Do s protein trong 1 sinh vt ln n hng nghn nn cc k thut nh trnh t
protein thng thng khng p dng c. S pht trin ca k thut khi ph (mass
spectrometry, MS) trn cc i phn t sinh hc gn y gii quyt vn ny vi
nhy, chnh xc v kh nng x l s lng mu cao. Cc c im u vit ny cng vi
s m rng ca d liu genomics, to ra mt cuc cch mng trong vic pht hin v xc
nh protein. MS c dng xc nh protein c tch bi in di hay sc k (Hnh
1.3).

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V k thut, protein cn phn tch bng khi ph s c ct thnh cc peptid ngn nh
trypsin, cc peptid ny sau c ion ha dng kh, s khi ca cc ion c o v sau
d liu s c phn tch da trn s khi ca cc mnh v d liu genomics vi s gip
ca my tnh. Vic ion ha cc peptid c thc hin bng phng php ion ha mm, tc
chuyn peptid thnh ion m khng lm gy cc lin kt ha hc ca n. Hin c hai k thut
ion ha mem c s dng rng ri l phun in t (electrospray ionization, ESI) v gii
hp bng laser c h tr bi cht nn (matrix-assisted laser desorption ionization, MALDI).
Tch bng gel /sc k Mu protein Thy gii protein
Tm vi c
s d liu
Tm vi c
s d liu
MALDI: du n
khi ph ca peptid
MS/MS: thng tin
trnh t v s khi
Lp li n ln

Hnh 1.3. Xc nh protein t gel bng khi ph
Vt protein quan tm trn gel c ct ra v x l vi trypsin thu c cc mnh peptid. C hai
phng php c dng xc nh mt protein t cc petid thu c. Phng php th nht, s khi
ca cc peptid c th c xc nh bng du n khi v so snh vi d liu b gen hin din trong c s
d liu tin sinh hc. Phng php khc, tng ion peptid s c chn phn mnh - khi ph lin tip
(tandem MS, MS/MS), v to ra thng tin trnh t mi. S t hp cc thng tin trnh t v s khi ca
peptid c dng xc nh chnh xc protein bng cch so snh vi d liu b gen.
Cc mu sinh hc thng cc k phc tp do d phn tch, khi ph thng c
ghp vi sc k. Thng thng, sc k lng hiu nng cao pha o (reverse phase high
performance liquid chromatography, RP-HPLC) c s dng ghp vi khi ph (LC-
MS). Khi ph s nhn v phn tch cc peptid c gii hp tun t t ct sc k. LC-MS
hot ng chm hn so vi MALDI, nhng cho d liu c cht lng tt hn. phn tch
cc hn hp rt phc tp vi phn gii cc k cao ngi ta thng s dng cc k thut
sc k hai ln kt hp vi khi ph lin tip (LC/LC-MS/MS), i khi cng c gi l k
thut xc nh protein a chiu (multidimensional protein identification technology, MudPIT),
has been established. Ln sc k th nht thng l trao i ion (IEC) v theo sau bi RP-
HPLC.
1.3.2.3. M ng v
Ngi ta c th s dng cc ng v phng x nh du protein, peptide nhm phn
bit chng khi phn tch khi ph. Vic nh du c th thc hin trong qu trnh nui cy
hay khi ct protein bng trypsin trc khi phn tch. Cch tip cn ny cho php nh lng
phn bit cc protein m phng php phn tch c khng lm c.


13
1.3.2.4. H thng lai i nm men
H thng lai i nm men (yeast two-hybrid system) gip xc nh cc tng tc tim
nng ca protein do cho php tm hiu su cc qu trnh sinh hc. Trong k thut ny, gen
m ha cho protein hay on peptid cn nghin cu (cu) c ni vo vng m ha cho
on gn ADN ca yu t phin m nm men (thng dng GAL4 hay LexA). Vng mu
ha cho mt protein (mi) hay mt th vin protein t cc m khc nhau (th vin mi)
c ni vo on hot ha phin m ca cng yu t phin m trn. Mt gen tn hiu c
t di s kim sot ca yu t phin m ny. Nu cu v mi tng tc vi nhau (cn
cu th yu t hot ha gn c vo trnh t hot ha kch hot s biu hin gen tn hiu.
Cc gen tn hiu c s dng l cc enzym hay yu t tng trng do c th nhn bit
kiu hnh d dng.

Hnh 1.4. Nguyn l h thng lai i nm men

Hnh 1.5. Protein array

14
1.3.2.5. Protein Array
Cc protein, khng th hay phn t sinh hc khc nhau bit trc c gn theo trt t
nht nh trn mt b mt rn rt nh to thnh protein chip, mu cha cc protein cn phn
tch c cho tng tc vi protein chip theo nguyn l tng t ELISA (Hnh 1.5). Protein
array c th dng phn tch s biu hin, tng tc hay chc nng ca protein.
1.3.3. ng dng proteomics trong nghin cu pht trin thuc
Trong lch s, dc phm c pht trin ch yu thng qua vic phn tch ngu nhin
hng ngn hp cht tng hp v sn phm t nhin. Phng php nghin cu dc phm
truyn thng da trn l thuyt sinh ha v dc l v mt bnh no . Cc mc tiu c
xc nh da trn l thuyt ny v hng nghin cu l mt s ngu nhin. Nghin cu c
in bao gm phn lp v xc nh c im ca cc sn phm t nhin cng vi vi hot tnh
sinh hc. Cc hp cht ny sau c tinh chnh li bng cch thit k li cu trc phn t
ca n to ra cc cht mi c hot tnh sinh hc cao hn v t tc dng ph hn. Hn ch
ch yu ca mt phng php nh vy l vic nghin cu sn phm t nhin vi hot tnh
sinh hc xc nh v c bn l may ri v do n thiu mt c s hp l.
Tin b ch yu u tin trong nghin cu dc phm l ha t hp, bao gm vic dng
cc k thut c hiu nng cao chun b mt s lng ln cc hp cht cho vic sng lc
trn cc i tng sinh hc khc nhau. Nhm khc phc cc kh khn trong nghin cu
truyn thng, genomics c dng nh l mt phng thc tng cng hiu bit v bnh
tt. Ngi ta hi vng rng nhng hiu bit ton din v cu trc di truyn ca sinh vt s dn
n nhng nghin cu hiu qu hn. Mc d c ch, vic phn tch trnh t DNA n l
khng gip xc nh mc tiu mi hiu qu, bi v ngi ta khng th d dng kt lun chc
nng cc sn phm ca gen, hay protein, v cc con ng sinh tng hp protein t trnh t
DNA. Cc giai on chnh trong nghin cu pht trin dc phm vi s tham gia ca
genomics, proteomics v tin sinh hc gm cc bc nh sau:
- Xc nh cc ch tc dng ca thuc mi t cc c s d liu trnh t protein
- Xc nh c im chc nng sinh hc ca cc protein ch
- nh gi liu php thch hp cc chc nng protein ch
- Xc nh cc phi t bng cch tm kim cc tng tc protein-phi t
- Pht trin cc th nghim in vitro
- Sng lc tm cht c hot tnh (cc hit, cht c tim nng)
- Chuyn cc cht tim nng thnh u thuc (lead)
- Ti u ha cc lead bng cu trc 3D ca ch v/hoc thit k thuc da trn lead
- nh gi cc lead ti u bng cc m hnh
- La chn cc ng vin tin lm sng
Pht minh thuc l mt qu trnh lu di v tn km vi s gii hn cc lead c trin
vng. Genomics v proteomics chc nng cung cp mt s lng ln cc ch tc dng
mi ca thuc. Sng lc hiu nng cao v cc th vin hp cht c to ra bi ha t hp
lm tng s lng cc lead mi. Thch thc hin ti l lm tng hiu qu ca cc th nghim
hiu lc v c lc. Trong thc t, iu ny khng d dng bi v v s cc gen, protein v
cc phn t khc tng tc vi nhau qua cc con ng truyn tn hiu nh hng chc
nng ca t bo. Nh nhng tin b ca k thut proteomic, n ngy cng c ng dng
nhiu ci thin qu trnh pht minh thuc. Bi v hu ht cc thuc u tc ng ln
protein, do vic tp trung nghin cu mc ny rt quan trng.
1.3.3.1. Protein v tc dng thuc
Nhiu h thng iu ha quan trng i vi thuc hot ng thng qua protein (sau dch
m). Nhiu loi thuc hot ng bng cch gn vi cc protein. VD mt loi thuc c ch


15
protease c thit k bt hot enzyme protease (mt protein gip virus nhn ln). Mt
loi thuc c cu trc thch hp c th gn ln b mt ca protein protease, v ngn cn hot
ng ca n. Nu protease b bt hot, virus khng th nhn ln, do gii hn s tn cng
ca n. tm kim cc hp cht c li nh cht c ch protease virus, cc nh khoa hc cn
hiu r cu trc v chc nng ca c hp cht v protein m n tc ng.
a s ch tc dng ca thuc l cc protein c m ha bi cc gen biu hin trong
cc m b nh hng ca mt bnh no . Ngi ta d on l c khong 10.000 enzym,
2.000 th th lin kt protein G, 200 knh ion v 100 th th hormon nhn c m ha trong
b gen ngi. Cc protein ny l nhng thnh phn chnh trong cc con ng chuyn ha
lin quan n bnh v do c kh nng l mt ngun cung cp ch tc dng ca thuc
phong ph. Cc ch tc dng ca thuc c th nghim c nhng c im m ch c
th c xc nh bng cch hiu r cc mc biu hin ca chng trong t bo ch khng
th xc nh bng trnh t gen ca chng. Cc ch tc dng ca thuc (1) ch yu thng
c biu hin trong cc m chuyn bit, cho php chn la tc dng thuc v lm gim nguy
c phn ng ph v (2) thng biu hin mc thp trong t bo. Do , mt h thng
pht hin ch tc dng hiu qu s kch hot vic d tm cc gen m ha cho protein biu
hin trong cc m chuyn bit mc thp, t cho php nhanh chng xc nh cc
protein c kh nng l ch tc dng cho s pht trin phng php chn on v cc liu
php. V nhng l do , thng tin nghin cu xc nh cc protein mi chuyn t cc t
bo dng sang s pht hin cc protein thay i trong cc m ngi thc s.
1.3.3.2. S tng tc ca protein
Vic xc nh c tng tc hay gn kt ca mt protein cha bit chc nng vi mt
protein khc bit ri chc nng hay c lin quan n mt qu trnh no s cung cp
thng tin xc nh chc nng ca protein cha bit v hon thin cc qu trnh sinh hc.
Vic lm sng t cc qu trnh sinh hc gip hiu r c ch bnh v do c th gip pht
trin cc phng php iu tr hay thuc thch hp.
1.3.3.3. Cc du hiu sinh hc protein trong nghin cu lm sng v
tin lm sng
Cc du hiu sinh hc (biomarker) l cc biu hin c th o c c ca cc qu trnh
sinh hc. Cc du hiu ny c th mc h thng, t bo hay phn t. Nghin cu proteomics
thc y qu trnh xc nh cc biomarker protein ca nhiu qu trnh sinh hc khc nhau.
Cc du hiu ny c th dng trong chn on, tin lng bnh, nh gi hiu qu iu tr v
nh gi tc dng ph hay c tnh ca thuc.
1.3.3.4. Proteomics trong pht minh thuc tr ung th
K thut proteomic c kt hp vi cc cch tip cn genomic cho nghin cu dc
phm tr ung th v nh gi ch tc dng. Trong phn ln cc k thut proteomic c cho
mc ch ny, k thut quan trng nht l s xc nh cu trc 3D ca protein, protein
microarray, vi phu bng laser v nghin cu cc tng tc ca protein-protein v protein-
thuc. Cc biomarker ung th v mt vi con ng chuyn ha t bo l ch tc dng quan
trng ca thuc. Mt vi k thut c s dng c trong lnh vc nghin cu cng nh cng
nghip v c kt qu ng khch l trong nghin cu dc phm ung th.
1.3.3.5. Proteomics trong pht minh thuc tc ng trn CNS
K thut proteomic thch hp cho nghin cu m no v c th p dng cho vic khm
ph ch tc dng ca thuc cha cc ri lon v thn kinh. Cc bnh c bnh hc lin
quan n protein nh bnh Alzheimer c bit thch hp vi cch tip cn ny. Cc receptor

16
quan trng cho cc loi thuc CNS (h thn kinh trung ng) bao gm cc protein nh cc
receptor cp i vi protein G, receptor ca N-methyl-D-aspartate v cc protein kinase. Cc
chn on phn t c th c da trn cc protein c pht hin trong dch no ty, v cc
protein tng t c th s dng nh l ch tc dng ca thuc. Proteomics s b sung cho
pharmacogenomics v to iu kin cho s pht trin ca dc phm chuyn bit cho cc ri
lon v thn kinh.
1.4. H t bo hc - Cytomics
B gen xc nh c im di truyn ca mt sinh vt, nhng khng phi ton b khng
gian v thi gian ca t bo u c huy ng p ng vi cc thay i ca mi trng.
Do ty tng thi im v iu kin mi trng m t bo s c tnh trng ng hc
(cytome) khc nhau. Cytome c th c nh ngha nh l cc h thng t bo v cc thnh
phn chc nng v tiu h thng ca c th. H t bo hc (cytomics) l khoa hc nghin cu
kiu hnh phn t ca mt t bo n pht xut t kiu gen v di s nh hng ca mi
trng vi kh nng truy xut thng tin mnh nh tin sinh hc. S iu ha cc chc nng t
bo cc loi t bo khc nhau l mt qu trnh c bit ha cao v khng gian, thi gian v
ng hc. Cc loi t bo khc nhau s dng cc c ch iu ha nhiu cp khc nhau
ty bin cc thit lp ca b gen theo s thay i ca mi trng.
Bnh tt hay thuc s can thip vo cc chc nng ca t bo v nh hng gy ra trn
t bo khc nhau ty thuc loi v tnh trng chc nng ca n (v d s bit ha, chu k t
bo, dao ng ngy-m). Trong nhng nm gn y, t bo c nghin cu nhiu nh l
mt cng c iu tr cc bnh phc tp qua cc nghin cu v t bo gc v liu php gen.
Nghin cu cytomics gip hiu c ng hc hnh vi ca t bo v nh ci thin c hi
thnh cng.
Vic sng lc cc ch chc nng trong pht minh thuc i hi iu kin mi trng
ni bo nht nh. Chc nng ng ca mt protein ni bo ty thuc vo vic x l n v
mi trng. Do tnh khng ng nht ca cc loi t bo v s khc bit v chc nng v cu
trc gia cc t bo (v d t bo gan, t bo thn knh, ) tnh trng khe mnh v bnh
tt, ta cn phi xem xt tnh khng ng nht ny v cu trc v chc nng. Kiu hnh nn
trong mt protein c biu hin cn c xem xt khi nghin cu chc nng ca n trong
qu trnh pht minh thuc.
Cc tin b mi trong k thut o t bo da trn hnh nh v dng chy cho php
nghin cu cc qu trnh t bo vi nhiu chi tit v t thu c nhiu thng tin v chc
nng v cu trc ca t bo. Cc knh hin v laser qut v trng rng cho php nghin cu
s nh v v ng hc ca cc phn t trong t bo v m. c im tc ng ca thuc c
th c nghin cu mt cch chi tit nh knh hin vi k thut s nh lng. Trong nhng
nm gn y, chng ta thy s pht trin ca cc nghin cu t bo nh lng qui m
ln nh. Vic lp ghp cc c s d liu ca tin trnh t bo c thit lp qua D n H t
bo Ngi (Human Cytome Project - HCP) c th gip chng ta ci thin hiu bit v tin
trnh bnh v ng hc v khng gian, thi gian ca chng mc t bo. iu ny s to ra
mt nn tng vng chc cho vic nghin cu pht trin v khm ph thuc mi.
1.5. Sinh hc h thng
Sinh hc h thng nghin cu mt sinh vt nh l mt h thng cc gen, protein v cc
phn ng sinh ha thng nht v tng tc vi nhau to ra s sng. Thay v phn tch cc
thnh phn hay kha cnh ring r ca sinh vt, nh s chuyn ha ng hay nhn t bo,
cc nh sinh hc h thng tp trung vo tt c cc thnh phn v cc tng tc gia chng, tt


17
c nh l thnh phn ca mt h thng. Nhng tng tc ny chu trch nhim cui cng i
vi hnh dng v chc nng ca mt t bo. V d, h min dch khng phi l kt qu ca
mt c ch hay mt gen n l. ng hn l do s tng tc ca nhiu gen, protein, c ch v
mi trng bn ngoi sinh vt to ra cc p ng min dch chng li s nhim trng v
bnh tt.
Sinh hc h thng xut hin nh l h qu ca "catalog" di truyn hc c cung cp
bi d n b gen ngi (Human Genome Project), v s tng cng hiu bit v cch cc gen
v sn phm protein ca chng to ra hnh dng v chc nng sinh hc. Vic nghin cu sinh
hc h thng c tr gip ca internet cho php cc nh nghin cu lu tr v phn phi
mt khi lng thng tin khng l, nhng tin b ca cc k thut nghin cu mnh m, v
s kt hp gia cc nh khoa hc thuc cc lnh vc khc nhau nh cc nh khoa hc my
tnh, nh ton hc, vt l, k s.

Ti liu tham kho:
1. Szathmary, E., F. Jordan, and C. Pal, Molecular biology and evolution. Can genes explain
biological complexity? Science, 2001. 292(5520): p. 1315-6.
2. Kellenberger, E., The evolution of molecular biology. EMBO Rep, 2004. 5(6): p. 546-9.
3. Westerhoff, H.V. and B.O. Palsson, The evolution of molecular biology into systems
biology. Nat Biotechnol, 2004. 22(10): p. 1249-52.
4. Rheinberger, H.-J., Epistemology. What happened to molecular biology? B.I.F. FUTURA,
2007. 22(4): p. 218-223.





19
B B i i 2 2. . n ng g d d n ng g s si in nh h h h c c h h t th h n ng g
t tr ro on ng g t t m m k ki i m m t th hu u c c m m i i
Mc tiu:
- Nm c xu hng tm kim thuc mi hin nay.
- Trnh by cch tip cn h thng trong tm kim thuc mi.
2.1. Khi qut
Trong Th k 20 chng ta c chng kin nhng thnh tu ni bc trong hiu bit v
c im ca cc vt cht (vt l, ha hc v my mc) v s ha thng tin (khoa hc my tnh).
Tuy nhin, nhiu ngi trong chng ta vn b bnh tiu ng, ung th v thoi ha thn kinh
bi v y hc hin ti vn cha pht trin mt chin lc hiu qu tin on v ngn chn. y
l s tht v nguyn nhn gy bnh chnh xc ca nhng bnh l phc tp ny, v l do ti sao
mt vi ngi li d mc bnh vn cha hiu r.
Th k 21 bt u vi vic gii trnh t hon ton b gen ngi, mt thnh tu to ra nn
tng cho cho cuc cch mng sinh hc. Cc nh sinh hc c th c c m ngun ca bt k
loi no. S gii m gen ca mt loi l im bt u cho sinh hc h thng. Nhng th nghim
qui m ln vi nm men, rui gim, chut ch c th tng tng cch y vi thp k by gi
c th c thc hin thng qui v chng ta bit c trnh t b gen.
Tuy nhin, hy vng chuyn gen thnh thuc mt cch nhanh chng khng thnh v
trn thc t sinh hc v bnh tt qu phc tp v vic pht trin thuc phi cn n s hiu bit
su sc v cc p ng sinh hc. Sinh hc h thng hng n vic miu t v hiu c s hot
ng ca h thng sinh hc phc tp v cui cng l pht trin m hnh d on bnh ngi.
Mc d m hnh y ngha cp phn t v chc nng t bo v m ngi cn xa vi,
nhng cc n lc ca sinh hc h thng ang nh hng n vic khm ph thuc.
Vic pht minh thuc t bui s khai song hnh vi sinh hc h thng: cc thuc c
truyn, thuc dc liu c khm ph nh s quan st quan h nhn qu ngi mang
bnh. T th k 19-20, nh s tin b ca ha hc hng nghn hp cht t nhin hay dn xut
ca n v cc cht tng hp mi c sng lc nhm pht minh thuc mi. Tuy nhin, sng
lc thuc thi k ny vn c thc hin trong cc h thng phc tp, tc s dng ton b sinh
vt, nhng thay con ngi bng ng vt th nghim. Hu ht cc thuc m ta c ngy nay u
trc tip hay gin tip c pht minh nh cch tip cn sinh hc h thng ny. Nh nhng
hiu bit mi v c ch ca cc qu trnh sinh hc v nhu cu tng tc sng lc, ngi ta thay
th m hnh ng vt bng cc m hnh m (v d trng lc ca c mch mu v c kh qun),
ri n cc m hnh da trn t bo (s phn bo hay to cytokin) v gn y nht l cc sng lc
hiu nng cao hay siu nng cho php sng lc hng trm nghn hp cht mi ngy.
Cch tip cn theo kiu p o s lng ny rt hu hiu khi ch tc ng c bit r
(ngha l cc ch ca thuc c), nhng vi cc ch mi, t c hiu r hn v mt sinh hc (v
d t b gen) th rt ng tht vng v ch rt t thuc mi c khm ph. Tham vng khai thc
kho tng b gen phi i mt vi thch thc mi l vic bit r ch khng ging nh bit
hot ng ca ch. Thc t, mc d vi s u t khng l vo genomics v k thut sng lc
hn 20 nm qua, gi thnh ca vic khm ph thuc mi tip tc gia tng trong khi t l c cp
php li gim. Vic sng lc da trn ch-ng vin tht bi do tnh phc tp ca bnh l. Do
cn phi quay li vi sinh hc h thng, d nhin vi cch tip cn mi, hin i hn.
Mc tiu ca sinh hc h thng hin i l hiu chc nng sinh l hc v bnh t mc con
ng ha sinh phn t, mng li iu ha, cc t bo, m, c quan v cui cng l c c th.
Hin ti, sinh hc h thng bao hm rt nhiu cch tip cn v m hnh khc nhau khm ph

20
v hiu s phc tp ca sinh hc, v nghin cu cc sinh vt t vi khun n ngi. V hc thut
ch yu tp trung vo pht trin nhng cng c tin sinh hc v nn tng in ton tch hp cc
d liu n gin (d liu v biu hin gen tng th, proteomic v metabolomic) vo cc m hnh
mng li iu ha v hot ng t bo. Do tnh phc tp ca h thng sinh hc l mt hm m
ca s thnh phn ca h thng v s tng tc gia chng v chng tng ln theo mc t
chc (Hnh 2.1), nn cc kt qu hin b gii hn trong nhng sinh vt n gin hay vi cc con
ng ha sinh c th trong mt t bo v iu kin mi trng rt c th cc sinh vt phc
tp hn. Mt khc ngay khi c d liu mc phn t th gii hn ca cng ngh my tnh
cng hn ch vic tch hp chng hiu mc t bo hay m. Do cn c phng php
lc d liu.
2.2. Omics: To v khai thc d liu quy m ln
Cch tip cn omics i vi sinh hc h thng tp trung vo cc thnh phn cu to nn
cc h thng phc tp (gen, protein v cc cht chuyn ha). Cch tip cn ny c ngnh
cng nghip dc phm s dng trit nhm b sung cho cch tip cn truyn thng trong vic
xc nh v thm nh ch thuc, cho vic pht sinh gi thuyt v cho cc phn tch thc nghim
trong cc phng php da trn gi thuyt truyn thng. V d cc omics c th c dng
tm ra gen, protein hay trng thi phosphoryl ha no ca protein c biu hin hay iu ha
vt mc trong tin trnh bnh, a n cc gi thit c th kim chng v cc kiu iu ha
quan trng trong vic cm ng hay tin trin ca bnh (Bng 2.1). S tch hp cc d liu
genomic, proteomic v metabolomic trong iu kin gen c kim sot hay s ri lon do thuc
trn t bo phc tp v ng vt (v trn d liu lm sng) l nn tng cho cc n lc ca sinh
hc h thng ti mt s cng ty thuc bao gm Eli Lilly, ni h tng tc vic nghin cu cc tin
trnh sinh l phc tp nh s bin dng ca xng.
S phn loi omics v trng thi bnh c th a n nhiu ch tc dng hiu qu hn hay
thm ch c th c nhn ha cc liu php bng cch xc nh qu trnh phn t c trng trong
cc trng thi bnh nht nh v cc bnh nhn c th. Mt ng dng quan trng khc ca k
thut l xc nh cc du hiu (marker) i din pht hin bnh, hoc theo di cc liu php
iu tr. Mc d, tip cn omics tng tc s pht trin cc gi thit v c ch v thu hiu v lm
sng, hiu bit mc h thng khng t nhin xut hin.
Ngi ta n lc rt nhiu hiu cc con ng ha sinh chnh v p ng mc sinh
vt bng cch da trn cc c tnh biu hin gene ton cc v d liu biu hin protein (ngha l
c tnh ton h thng khng th c d on t cc b phn). cc sinh vt tng i n
gin, cc nghin cu hp nht vic phn tch d liu hoc mARN b gen theo thi gian, phn
tch stress v xc nh cc thnh phn c iu ha cng nhau trn din rng, v nghin cu
tng tc protein-protein dn n nhim hiu bit mi v cc chc nng ca con ng ha
sinh v s t chc ca mng li tn hiu trong cc tin trnh sinh hc c th, chng hn s tng
sinh t bo hay p ng vi cc stress chuyn ha. D s phc tp trong bnh tt ca ngi cng
nh cc gii hn v kinh t v in ton hn ch mnh vic ng dng omics nh l mt tip
cn c lp hiu bit mc h thng, k thut omics s ng vai tr quan trng trong vic
xy dng cc c s gip xc nh v gii hn cc pathway v kt ni kh d ca mng li tn
hiu t bo trong m hnh t trn xung.


21



M hnh ha
Th nghim h thng t bo phc
Omics
Phn t Con ng T bo M Ngi
Mt
Giy

Hnh 2.1. Cc tip cn n sinh hc h thng trong ngnh dc
Omics (kiu tip cn t di ln) tp trung vo vic xc nh v o c mc ton cc cc thnh phn phn
t. M hnh ha (kiu tip cn t trn xung) to cc m hnh tch hp (xuyn qui m) v sinh l ngi v
bnh, tuy nhin, vi cng ngh hin c, cc m hnh ny tp trung vo cc vn kh c th mt qui m
no v d mc con ng ha sinh hay c quan. Kt ni hai cch tip cn ny c th l cc d liu lc
ha (v d lc ha hot ng a thnh phn sinh hc, Biologically multiplexed activity profiling,
BioMAP) ly t cc th nghim hiu nng cao c thit k hp nht tnh phc tp sinh hc nhiu mc
: cc con ng hot ng a tng tc, cc kiu t bo giao tip a hng v nhiu kiu mi trng khc
nhau. Cch tip cn h thng t bo phc tp nh vy nhm n nhu cu d liu v cc p ng t bo i
vi cc kch thch sinh l v vi tc nhn thuc nh l mt b sung i vi ngi lm m hnh, v cng l
mt cch tip cn thc t n sinh hc h thng mc mng li tn hiu t bo v qui m tng tc gia
cc t bo.
Bng 2.1. ng dng v thch thc ca mi cch tip cn sinh hc h thng

Omics
Cc h thng t
bo phc tp*
M hnh ha
Hiu su mc h thng +/- + +
Hnh thnh gi thuyt + + +
Th nghim m hnh - + -
Thm nh/xc nh ch + + +/-
Thm nh hp cht +/- + -
S ti u ha u thuc/SAR - + -
Du hiu bnh/ thit k th nghim - +/- +
Nng sut x l Chm Nhanh Rt chm
Cc thch thc
S lng d liu
Cht lng d
liu
Cn trong bi
cnh sinh hc
S sn c ca
cc loi t bo
M hnh ha b
gii hn ca cc
tc ng c tnh
h thng
D liu b b
qua hoc c sai
s
Kh khn trong
thm nh m
hnh

22
-: Khng gii quyt vn ; +: Gii quyt c vn ;
+/-: Gii quyt c trong iu kin nht nh
* xem BioMAP (Hnh 2.3)
2.3. Cc m hnh my tnh: t cc qu trnh sinh hc
n sinh l bnh
Mc ch ca vic m hnh ha trong h thng sinh hc l cung cp nn tng cho vic
to gi thuyt v tin on da trn m phng in silico v sinh hc bnh ca ngi. n nay,
mt m hnh tch hp tng qut v hnh vi t bo ngi vn cn xa vi, nhng cng c cc
tin trin tht s trong n lc pht trin v th nghim cc phng php thc nghim v tnh
ton i vi sinh hc h thng in silico nhiu cp khc nhau (Bng 2.2). Hn na, trong
pht trin thuc, khng cn n cc m hnh ton din. Hin nay, cc m hnh chuyn su, nh
hng n cc vn c th c nh hng n vic thm nh ch tc ng v cc quyt
nh pht trin trn lm sng (Bng 2.1).
Bng 2.2. Mt s v d m hnh in ton lin quan n sinh hc bnh ngi
Cch tip cn H thng Nhn xt
Sinh l hc bnh Tim Cc m hnh nh lng ca tim t gen n sinh l
Diabetes M hnh bnh tiu ng
Asthma M hnh ton hc trn my tnh ca bnh hen mn
tnh d on p ng ca bnh nhn vi cc
liu php khc nhau
M hnh hen mn tnh d on s km hiu qu
ca s c ch IL-5
M hnh t bo tch
hp
Ung th M hnh mng li cha 1.000 gen/protein, 3.000
thnh phn c d on tc ng ca gim biu
hin gen (gene knockdown)
T bo c tim Kt ni cc m un (chuyn ha trung gian, sinh l
in hc v c) pht trin m hnh in ton t
bo c tim
M hnh con ng
ha sinh
a h thng Cc c tnh tng ha (khong thi gian tn hiu
c ko di, cc hnh vi ngng) ca s truyn
tn hiu trong cc m hnh mng li
EGFR/MAPK Cc m hnh in ton ca truyn tn hiu EGFR
Tc thay i nng phi t so vi i lc trong
kt xut xui dng
Cc m t m hnh lun l thc thi c ca mng
li EGFR
NF-B Cc c tnh chm ca c ch nhn tn hiu v c
tnh nhy cm ca cc thnh phn tn hiu
Chng minh vai tr ca cc ng ng IB trong
c tnh x l tn hiu song phng ca con ng
truyn tn hiu NF-B
M hnh trong pht minh v pht trin thuc phi m phng cc p ng qui m phc
tp ca t bo, m hay c quan, tc l qui m biu hin ca bnh tt. ng thi m hnh cng
phi chi tit truy xut cc im can thip cho vic pht minh thuc v c th m un ha in
silico d on kt xut mc c quan. V d, m hnh gi lp s co tht c tim phi c lin


23
kt gia knh Na
+
/Ca
2+
v s co tht c d on cc thuc tc ng trn cc knh ny. Kh
khn xut hin khi m hnh c gng tch hp mt cch hiu qu cc mc t phn t n t bo
v c quan. Bn cnh kh khn v cng ngh tnh ton, cc gii hn v kin thc t di ln
v hiu bit v cc con ng ha sinh, cu trc mng li v cc tng tc cng nh thiu kin
thc chun v cc p ng mc t bo v c quan i vi cc kch thch sinh hc c th
thm nh m hnh l cc vn c bn v lu di, cn c gii quyt trc khi xem xt cc m
hnh tch hp a qui m.
Mt cch tip cn thc t gii quyt vn tnh ton l a vo mt khung mc c
quan v gia tng tnh phc tp ca m hnh theo dng m un. V d, m hnh vim c th c
bt u vi xem xt giao tip t bo-t bo qua mng li cytokin v tip n thay cc l trng
bng cc m phng hnh vi t bo c m hnh ha t cc m un li (v d m hnh tnh di
ng ca b khung t bo, cc p ng cytokin hay tng sinh) v cui cng thay cc m un con
ng ha sinh cn trng bng cc tip cn t di ln.
Cng ty Entelos pht trin cc m phng sinh l bnh phc tp s dng khung cc
phng trnh vi phn quyt nh da trn d liu thc nghim ngi. Trong cc m hnh ny,
cc con ng tn hiu ni b khng c m hnh ha mt cch r rng, t bo hay thm ch
m c th hin di dng l trng p ng vi d liu nhp vo bng cch cung cp cc d
liu xut c th thay i theo thi gian. S dng khung mc c quan nh vy, ngi pht
trin cc m hnh ton hc ca bnh hen mn tnh tch hp cc tng tc gia cc t bo v mt
vi mc phc tp cc p ng ca chng vi nhau v vi mi trng. Cc tham s ca m
hnh c th c iu chnh t c cc im tham chiu trng thi n nh no , v d
trng thi hen mn tnh (bao gm vim t bo a acid mn tnh, tc nghn ng th mn tnh,
tng p ng ng th v tng mc IgE) hay trng thi tc nghn ng th do vn ng. p
ng hen c m phng nh d on i vi nhiu thuc gm ch vn 2, glucocorticoid v i
vn leukotrien. Hn na, bng cch m phng s gim interleukin 5 (IL-5) ph thuc khng th
(cht dn d bch cu a acid trong bnh hen) m hnh ny c th d on s gim bch cu a
acid trong ng h hp nhng t ci thin v thng ca ng th, cc d on ny ph hp
vi th nghim lm sng s dng khng th chng IL-5 ngi ha trong bnh hen.
Cc m hnh qui m t bo v c quan tng t v chuyn ha v cn bng ni m
glucose xut pht t quan h n gin gia glucose v mc insulin trong tun hon v c pht
trin thnh cc m hnh phc tp hn, tch hp cc p ng a m v vai tr ca chng trong
chuyn ha glucose. M hnh ny c ng dng trong thit k th nghim pha I cho thuc
tr tiu ng type 2. Nh c m hnh cc phn khc liu cng nh s bnh nhn cn thit c
gim xung v do vy gim chi ph v tng tnh chc chn ca kt qu th nghim.
Nhng hiu bit chi tit hn v hnh vi h thng trong cc con ng tn hiu lin bo,
chng hn xc nh cc nt quan trng hay cc im iu ha trong mng li hay hiu r hn
s lin h gia cc con ng, cng c th gip hiu ng ch tc ng ca thuc v ngha i
vi sinh l mc c quan hay sinh vt. Theo hng ny, nhiu n lc tp trung vo cc con
ng truyn tn hiu trong t bo (Bng 2.2). Cc m hnh ny c li t s lng ln cc d
liu l thuyt v vic ha hn rng cc n lc v omics c th cung cp cc lin kt trn nhng
qu trnh. Nh cc m hnh mc t bo v c quan, cc m phng v mng li truyn tn hiu
ng vt c v thng da trn cc phng trnh vi phn ph thuc thi gian v lp m hnh
con ng ha sinh trong trng thi c lp v di nhng iu kin rt c th (v n gin). Mc
chi tit k tip tng cng tnh thit thc ca cc m hnh con ng ha sinh ny l s lin
h gia cc con ng. Khi m hnh ha cc m un tn hiu v ngi ta nhn thy rng s kt
hp cc m un gin n dn n cc p ng khng tuyn tnh hay cc c tnh tng ha ca
h thng. Cc kt qu khng th nhn thy ny da trn tnh khng tuyn tnh ca con ng ha
hn cho vic nhn dng v sp xp theo mc u tin cc im can thip trong mng li
truyn tn hiu.

24
Kin trc ca cc con ng truyn tn hiu th hin s bo tn ng k trong qu trnh
tin ha, kin thc ang c s dng xc nh v hiu v cc con ng truyn tn hiu
trong t bo ng vt c v da trn tnh tng ng ca nhng con ng c xc nh r
rng cc ng vt bc thp, v nm gia cc con ng lp li do tin ha ngi. Mc d cc
tng ng ca con ng c th cung cp gi v s bo tn ca cc im then cht cho s
can thip ha hc trong truyn tn hiu, tuy nhin, s phn ha cc chc nng ca con ng v
cc tng tc iu ha l ph bin, do cui cng th khng th thay th cc nghin cu trong
nhng h thng phc tp ngi.
Cc n lc v m hnh d on hin ti d thnh cng n u, chng vn thch thc vic
thm nh bng thc nghim (theo l thuyt, ch kh thi vi d liu ca ngi) v cc chu trnh
ci tin tha hng n lc m hnh ha (Hnh 2.2). T quan im pht trin thuc, bt k thnh
cng no cho n nay cng c th xem l giai thoi v cho n khi mt m hnh no chng t
c kh nng d on thnh cng ngi, vic da vo m hnh a ra quyt nh pht
trin c th l mo him. Tuy nhin, cc d on da vo m hnh chc chn s l mt trong
nhiu tham s a ra quyt nh trong qu trnh pht trin thuc ca cng nghip dc.

Omics Y vn
M hnh
in silico
Phn tch
BioMAP
Thm nh
Nghin cu ex vivo
M hnh ng vt
Nghin cu lm sng
D liu mc
hp phn
L thuyt /
M hnh
D liu p ng
mc h thng
Xc nh kin trc
mng li chc nng
Biu hin
gen v protein
Tng tc thc th
Sai
ng
M hnh d
on c

Hnh 2.2. S pht trin ca m hnh in silico
Hnh 2.2 minh ha chu trnh pht trin ca m hnh in silico tch hp s dng d liu p
ng mc hp phn v h thng. Cc m hnh tch hp ca bnh c th c pht sinh t d
liu l thuyt cng nh s biu hin protein v b d liu tng tc, c tim nng nhn thng tin
bng cc tin on v t chc mng li chc nng v p ng t bo da trn cc th nghim
phc tp trn t bo ngi (xem v d Hnh 2.3). Cc m hnh c th nghim lp i lp li
v ci tin bng cch so snh cc d on vi cc p ng mc h thng (t bo, m hoc c
th) o c bng thc nghim thng qua cc th nghim truyn thng hay t cc d liu c
c t hn hp cc t bo ngi phc tp c hot ha trong nhng iu kin mi trng
khc nhau. D liu omics mc hp phn c th cung cp khung, gii hn s m hnh kh d
mc phn t.

2.4. S dng cc h thng t bo phc tp th
nghim v to m hnh sinh hc
M hnh ha cc qu trnh sinh hc cho n gi vn cha lin kt tt vi sinh hc bnh h
thng c nh hng ng k trn pht trin thuc. M hnh t trn xung mc t bo - c
quan v c th c nhiu tim nng, nhng li ph thuc nhiu vo d liu p ng t bo theo


25
hon cnh. Hn na, lp khong trng gia omics v m hnh ha, cn phi c d liu sinh
hc t bo dng tch hp s phc tp v cc c tnh tng ha ca h thng iu ha t bo v
tt nht l c th lp li v thch hp lu tr thnh c s d liu, c th chia s v phn tch
nh lng.
Ti mt mc , cc p ng m ca ngi t chng c th c o ex vivo, mt cch
tip cn c chng minh l hu dng trong vic xc nhn cc hp cht v cc mc tiu nht
nh, mc d c gii hn v tnh c sn ca m ngi v v lp li. Tuy nhin, cc tip cn c
lp li cao hay t ng ha i vi sinh hc t bo c gp phn vo cc phn tch chc nng
gen v hp cht quy m ln m cng nghip dc mong mun v cn thit nh mt nn tng
cho cc n lc m hnh ha. Tht vy, nhng h thng sng lc hiu nng cao da trn t bo,
thng da trn cc th nghim v dng t bo pht tn hiu, c nhiu cng ty s dng mt
cch c hiu qu xc nh cc hp phn ca nhng qu trnh sinh hc, sng lc cc hp cht
c hot tnh v lp d liu thuc da trn tc ng ca chng ln qu trnh hay cc biu hin p
ng kch thch n gin. Tuy nhin, nhng th nghim ny ni chung c thit k c lp cc
qu trnh n l v gii hn tnh phc tp sinh hc v v th khng tn dng c cc thun
li cng nh cung cp ci nhn thu sut vo cc c tnh tng ha ca h thng t bo. Kiu
sinh hc thuc h thng ny tp trung vo cc qu trnh c n gin ha v th khc bit vi
kiu sinh hc h thng tp trung vo cc c tnh tng ha v phc tp.
Cng lc , mt vi nhm ang bt u nh gi cao tm quan trng ca cc c tnh tng
ha trong pht trin thuc. V d nh cc nh nghin cu CombinatoRx (Boston, MA, USA)
tm kim cc liu php kt hp mi bng cch li dng u th t hai tc nhn kch thch (phorbol
myristate acetate - cht hot ha ca chui p ng protein kinase C, v ionomycin - cht hot
ha truyn tn hiu ph thuc Ca
2+
). Cc kch thch ny khi ng cc qu trnh phc tp trong
nhng t bo s cp tm kim cc cp hp cht c hot ng i khng khi kt hp (v d nh
yu t hoi t khi u (TNF)-cht tit t t bo T hot ng), nhng khng i khng khi s dng
ring l. ni khc, Rosetta Inpharmatics (Seattle, WA, USA) o c hng nghn gen u
biu hin nm men, dng cc d liu p ng gen c c t cc ri lon do di truyn hay ha
cht xc nh bng cch no cc gen tc ng ln s tng trng li khp vi cc con ng
ha sinh v tm ra c ch hot ng ca cc hp cht. Nhng cch tip cn da vo thc
nghim ny bt u c sc mnh ca sinh hc h thng, nhng cc h thng c nghin cu
vn cn n gin mt cch c tnh, ch tp trung vo mt s u vo hoc u ra (CombinatoRx)
hay mt trng thi sinh l n gin trn mt sinh vt m hnh (Rosetta). S phc tp l mt sn
phm th cp, khng phi l thit k ca nhng cch tip cn ny.
Cc c tnh phc tp v tng ha trong sinh hc bt ngun t mt vi thuc tnh: th nht,
cc u vo phc tp kch hot nhiu qu trnh; th hai, nhiu u ra hp nht cc p ng mng
li i vi cc u vo; th ba, s tng tc gia cc loi t bo; th t, nhiu bi cnh v mi
trng cho mi loi t bo hay s kt hp cc loi t bo. Cng nghip pht hin thuc u t
hng t USD vo cng ngh nh gi cc u ra, nhng kt hp cc s phc tp c lin
quan n bnh vo trong khm ph thuc, cc n lc c ch cn phi c thc hin nghin
cu cc t bo trong s phi hp gi lp tng tc t bo - t bo c tnh quyt nh n cc
mng li iu ha in vivo v th nghim cc t bo trong nhng hon cnh mi trng phc
hp khc nhau (trong cc kt hp khc nhau ca cc qu trnh c hot ha). Phn tch hon
cnh song song hay a h thng quan trng bi v protein v cc qu trnh c tin ha kt
hp cc u vo v u ra t nhiu hon cnh, v th hiu c tc ng ca thuc (hay ch),
d liu phi c bt ngun t cc p ng t bo trong nhiu mi trng.

26
Thuc v/hay Gen
Cc kt hp t bo s cp ngi
Cc hon cnh bnh phc
Hot tnh trn nhiu con ng
c tnh tng ha
Nhiu
u vo
Cc kt hp t
bo v kch thch
Nhiu
u ra
Cc h thng a hon cnh + t bo
H 1 H 2 H 3 H 4
Cc trng thi
thuc hay di truyn
Tng quan cp v
nh gi a chiu
Thuc
Cc statin
Glucocorticoid
PI3K
pathway
Thuc tc ng trn
pathway thng gp
Cht c ch
p38 MAPK
Thuc tc ng
trn ch
thng gp
Cht c ch
Hsp90
Pathway th
th t bo T
Tng quan cp v
nh gi a chiu
Gen
Chia nhm theo chc
nng gen
NF-B
IFN-
PI3K
Kin trc mng li v kt ni
ng dng: Xc nh v thm nh ch
T chc con ng/mng li
Quan h cu trc tc dng
ng dng: Thm nh ch v hp cht
Ti u ha u thuc
Thng tin con ng
Chia nhm theo chc
nng thuc
Th th
Cytokin
Chemokin
Enzym
Cht nn

Hnh 2.3. B d liu hot ng a sinh hc (BioMAP) trong pht minh thuc
BioSeek (Burlingame, CA, USA) pht trin cc th nghim da trn t bo ngi nhm
tch hp tnh phc tp nhiu mc , dng s thm d song song ca cc h thng t bo
c chun ha (cc t bo cng vi mi trng) c thit k gi lp tnh phc tp sinh l
bng cch bao gm mt hay nhiu kiu t bo s cp cng nh s kt hp cc t bo v cc con
ng ang hot ng (Hnh 2.3a). Cc h thng t bo c thit k bao hm cc p ng
lin quan n bnh cho vic phn tch chc nng sinh hc, m hnh ha v pht hin thuc. V
d, mt nhm gm bn h thng t bo (cc kt hp ca cc t bo ni mc v cc t bo mu
n nhn trong bn mi trng vim phc hp khc nhau) chng t n bao hm h sinh hc
phc tp phn nh nhiu hp phn ring bit ca cc ch dc l lin quan n chng vim.
Cc d liu c to thnh t khong 24-40 thng s protein (bao gm cc cytokin, chemokin,
th th kt nh v cc cht trung gian gy vim khc) nh cc gi p ng ca cc h thng
phc hp ny c th phn bit v phn loi hu ht cc con ng v c ch b tc ng bi cc
cht iu bin bit ca chng vim, cng nh mt dy ng ngc nhin ca cc th nghim
thuc v cc con ng khc (Hnh 2.3b). Quan trng hn, cc b d liu to ra t nhng hn
hp phc tp t bo hot ha ny c tnh lp li, cho php lu tr trong c s d liu v tm kim
v phn tch t ng bi tnh tng t ca b d liu hay cc c tnh khc (v d nh cc tc
ng ln nhng thng s chnh lin quan n bnh).
Hnh 2.3 minh ha cch thc hot ng ca B d liu hot ng a sinh hc (BioMAP)
p dng vo chc nng gen, cu trc mng v cc mi quan h hot ng ca thuc. (a) Cc t


27
bo s cp (v d nh t bo ni mc v/hoc t bo bch cu) c kt hp v cho tip xc vi
cc kch thch (v d nh cc cytokin, cc yu t tng trng hay cc cht trung gian ha hc)
trong cc kt hp c lin quan n sinh hc bnh ang c quan tm (v d nh hin tng
vim). Cc gi tr c c dng o c cc p ng ca h thng c th l cc protein, cc
trng thi hot ha ca protein, gen hay cc hp phn a bo khc, hay cc c tnh chn lc lin
quan n bnh (v d nh cc cytokin, cc yu t tng trng, cc th th kt dnh, l cc cht
trung gian cui cng ca giao tip t bo v chc nng trong trng hp bnh) v s p ng vi
mi trng v cc u vo dc l (ni dung thng tin). Cc ri lon trong cc h thng song
song xc nh nhng d liu hot ng sinh hc ca thuc hay gen ang nghin cu. Vic kt
hp nhiu kiu t bo v nhiu con ng hot ha to ra s iu ha mng li phc v cc
c tnh tng ha lm tng nhy cm v kh nng ca h thng phn bit cc tc ng ln
gen v thuc n nht. (b) Mt vi h thng t bo ngi phc hp (cc t bo hay cc kt hp
t bo trong mi trng c lin quan n bnh) c xem xt cng vi cc gen (nh biu hin
vt mc hay siRNA) hay thuc ang c quan tm v tc ng ln nng cc protein ch th
la chn c xc nh, to ra mt b d liu c s dng nh l du n a h thng ca
chc nng ca tc nhn th nghim. Cc o c thng k s ging nh ca d liu (ngha l cc
tc nhn no cm ng cng mt p ng a h thng) c th c s dng nhm cc gen
hoc thuc theo chc nng, v to ra mt miu t dng biu v mi lin h cc chc nng
ca chng vi nhau). V d, tp hp cc b d liu cm ng bi biu hin qu mc gen (bn tri
di) biu l cc mi quan h con ng chnh (v d Ras/MAPK, phosphatidyl inositol 3-
kinase (PI3K), interferon- (IFN-y), v cc nhm lin kt NF-B) cng nh cc tng tc con
ng-con ng trong mng li tn hiu kim sot p ng t bo ni mc trong s hin din
ca cc cytokin vim khc nhau. Tp hp b d liu cm ng thuc t nhng h thng m hnh
vim (bao gm cc s kt hp c hot ha ca cc t bo ni mc v cc t bo n nhn mu
ngoi vi) pht hin v phn bit ng ca ca hu ht cc yu t bit ca chng vim cng
nh mt lot cc mc tiu thuc v con ng khc, bao gm cc glucocorticoid, cht khng
cytokin, v cht c ch ca HMG-CoA reductase, calcineurin, inosin monophosphat
dehydrogenase, phophodiesterase, th th hormon nhn, phosphatidyl inositol 3 kinases, protein
sc nhit 90, casein kinase 2, kinase 2 mt hot ha, v p38 MAPK (c minh ha pha trn
bn phi). Thuc c hiu cho cc ch thng gp hoc cho cc ch trong con ng thng
gp c nhm vi nhau, nhng cc hp cht c cc hot tnh ngoi ch khc nhau th c
pht hin d dng (v d, cc b d liu ca 3 cht c ch JAK vi hot tnh th cp c
bit; nh du hoa th). Tp hp cc d liu hot tnh t mt lot u thuc ha hc c th xc
lp mi quan h cu trc - tc dng c hiu cho hp cht dng ti u ha u thuc (bn
phi pha di; cc ng ng khc nhau c mu khc; kch thc vng trn phn nh nng ).
Trong v d ny, tp hp BioMAP xc lp 2 loi hot tnh chc nng t cc cht c ch p38
MAPK c lin quan v cu trc.
Cch tip cn ny, c tn l lp d liu hot ng a sinh hc (BioMAP), a vo s
dng thnh cng trong cc nghin cu m hnh cho thy kh nng ng dng ca n trong mt s
giai on ca qu trnh khm ph thuc (Bng 2.1). i vi xc nh v thm nh mc tiu, cc
cch tip cn tin hc da trn tnh tng ng ca cc b d liu a h thng c lu tr trong
c s d liu cho thy kh nng lin h nhanh chng gia gen hay hot ng ca thuc vi
cc con ng bit hay mi, v d on cc con ng chc nng hay cc tng tc mng
li (Hnh 2.3b). Cc b d liu a h thng c cm ng bi biu hin vt mc gen trong t
bo ni mc trong bn mi trng cytokin khc nhau (v bn cht, cc du n a h thng ca
chc nng gen) t ng kt cm thnh cc nhm phn nh cc mi quan h con ng bit
vi tin cy ng ngc nhin. Hn th na, th biu din ca cc mi quan h tng t v
chc nng (Hnh 2.3b) ch ra cc vai tr ring bit ca hai sn phm gen, MyD88 v IRAK, trong
cc tng tc trung gian gia cc con ng yu t nhn B (NF-B) v protein kinase hot ha
bi Ras/mitogen (MAPK). MyD88, trc y c bit l truyn tn hiu thng qua NF-B,
c xc nh sau trong cc nghin cu v sinh ha l cng c vai tr kch hot con ng
MAPK, lm c ch s hot ha ca NF-B trong vng phn hi m hot ha bi IL-1 m

28
khng phi bi TNF-. Cc b d liu p ng a h thng v kt cm, trong cc h thng
c thit k bt cc c tnh tng ha, v th c th gip xc nh cu trc chc nng ca cc
mng li truyn tn hiu, thng tin quan trng (trong s kt hp vi b d liu thng thng)
cho vic thit k v th nghim cc m hnh in ton.
i vi vic m t c tnh hp cht, cc b d liu gii hn, vic t ng ha v khi qut
chc nng c th lm cho cc b d liu xut pht t cc hn hp t bo hot ha, phc tp tr
thnh mt cch hiu qu sng lc cc th vin trng im cho cc tc ng ln sinh hc lin
quan n h sinh hc lin quan n bnh phc tp, v quan trng hn na xp th t u tin
kt qu t trong sng lc hiu nng cao truyn thng. Trong cc nghin cu m hnh, chng ti
dng b d liu trong bn h thng phn loi cc kt qu t v u thuc theo hot tnh
sinh hc ca chng, xc nh cc hp cht vi hot tnh trt ch (c th l ng mong mun
hoc khng ng mong mun), phn bit dy u thuc p ng tt cho cc p ng sinh
hc nht qun v theo di cc mi lin h chc nng cu trc nh mt hng dn ti u
ha u thuc.
Mt u th khc ca cch tip cn a h thng l cc h thng song song c th c thit
k bt mt dy rng cc hot ng suy din ca con ng ha sinh v sinh hc (lin quan
n bnh); v th, hiu qu ca thuc hay gen c th c nh gi ng thi i vi cc p
ng sinh hc phc tp lin quan n nhiu bnh khc nhau v c th c dng sng lc cc
ch th liu php iu tr mi. iu ny tri ngc vi hu ht cc n lc m hnh ha v thm
ch cc th nghim ng vt hoc lm sng, vn thng c thit k cho mt mc tiu bnh
duy nht. V d nh cc m hnh h thng t bo phc tp ca chng vim (Hnh 2.3) pht hin
d dng cc hot ng ca nhng cht c ch 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA)
reductase (v d cc statin) trong truyn tn hiu vim. iu ny t ra cu hi rt ng quan tm
l liu vic s bao hm cc phn tch h thng sinh hc phc tp trong s pht trin cc statin c
tng tc s pht hin vai tr tim nng ca chng trong cc ri loi vim v t min hay khng?
Omics c th v chc chn nn c p dng vo cc h thng t bo c thit k kt
hp tnh phc tp sinh hc. Tuy nhin, nhiu nghin cu cho thy cc du n v chc nng c
tnh thng tin cao i vi gen v cc tc ng ca thuc c th c to ra t s lng rt nh
(mi) cc thng s quan trng sinh hc, khi chng c th nghim trong mt vi s kt hp t
bo v mi trng phc tp khc nhau. iu ny cho thy tnh phc tp sinh hc c th n cha
cc thng tin hu ch v chc nng protein v thuc, v n c th lm n by cho vic lp d
liu chc nng quy m cng nghip mt cch thng minh hn, nhanh hn v r hn.
T gc nhn kh st thc t, cc cch tip cn ny to ra c hi tch hp sinh hc h
thng hiu qu v tit kim hn trong qu trnh khm ph thuc. T gc nn tng, cc c s
d liu ca cc p ng sinh hc nh lng t bo ngi i vi cc bin i gen v thuc,
di cc iu kin c chun ha v c th lp li c thit k bao hm tnh phc tp lin
quan n bnh v nm bt cc c tnh tng ha, c th hu ch trong vic d on cu trc chc
nng ca mng li iu ha phc v s cung cp mt cu ni cn thit cho vic tch hp d liu
omics vo m hnh in silico ca hnh vi h thng t bo, cng nh mt bc kim tra cho nhng
m hnh ny khi chng pht trin (Hnh 2.2).
2.5. Tng lai
Trong qu trnh pht trin thuc, cc quyt nh hng triu USD phi c thc hin
thng xuyn s dng cc tiu ch khng hon chnh da trn kin thc sinh hc khng y :
v d, mc tiu c u tin bi v chng c iu ha tng mc gen trong trng thi bnh
(d iu ny khng ng vi nhiu ch bit r); cc hp cht c la chn do c hiu ha
sinh (d thc t nhiu thuc c tc dng tt li khng c hiu); cc m hnh ng vt c xem
nh yu t cn thit (d chng c bit d on thnh cng trn lm sng km). Hiu bit tt
hn v sinh hc, nht l kin thc c lin quan n bnh ngi v c kh nng kt hp vo tin
trnh khm ph thuc, rt cn a ra quyt nh. Mc d cc tip cn sinh hc h thng c


29
phc tho y vn cn s si, chng ng gp vo cc quyt nh pht trin thuc c
ngha bng cch tng tc sinh hc nh hng gi thit, bng vic m hnh ha cc vn sinh l
c th trong thm nh ch hoc trong sinh l lm sng v bng cch cung cp cc m t c
tnh nhanh v din gii cc p ng lin quan n bnh mc t bo v h thng t bo.
Cc tip cn ny hin nay c theo ui cc phng th nghim v cng ty ring l, r
rng l chng b sung ln nhau v cui cng th chng phi c tch hp vi nhau sinh hc
h thng pht huy c tim nng ca n. Cc nghin cu chc nng sinh hc v sinh hc t bo
vn a chiu v a bin l nhng thch thc i vi tin sinh hc, vic tiu chun ha ca thit k
th nghim v d liu l thit yu trc khi to c t ph trong sinh hc h thng. t
c iu ny, cn c cc ngn ng nh du cho d liu biu hin gen, pht trin c s l lun
mi chia s v tch hp cc loi d liu khc nhau ca omic v sinh hc thng thng, pht
trin cc th nghim sinh hc h thng hiu nng cao c tiu chun ha v cc tip cn cng
ngh thng tin lin quan.

Ti liu tham kho:
1. Debouck, C. and B. Metcalf, The impact of genomics on drug discovery. Annu Rev
Pharmacol Toxicol, 2000. 40: p. 193-207.
2. Ideker, T., T. Galitski, and L. Hood, A new approach to decoding life: systems biology. Annu
Rev Genomics Hum Genet, 2001. 2: p. 343-72.
3. Ward, S.J., Impact of genomics in drug discovery. Biotechniques, 2001. 31(3): p. 626, 628,
630, passim.
4. Cho, C.R., et al., The application of systems biology to drug discovery. Curr Opin Chem
Biol, 2006. 10(4): p. 294-302.
5. Zhu, J., B. Zhang, and E.E. Schadt, A systems biology approach to drug discovery. Adv
Genet, 2008. 60: p. 603-35.




30
B B i i 3 3. . T Ti in n s si in nh h h h c c
Mc tiu:
- Hiu c cc khi nim c bn veef tin sinh hc
- Trnh by mt s lnh vc nghin cu chnh ca tin sinh hc
- Bit v s dng c mt s cng c ca tin sinh hc trong nghin cu.
3.1. Lch s
T u nhng nm 1980, khi nim s dng my tnh thc hin cc tnh ton phc v
nghin cu cc phn t sinh hc bt u, nhng khi nim tin sinh hc vn cha hnh thnh.
Lc by gi cc nhm nghin cu v cu trc protein hay tin ha phn t m ch yu l nhng
ngi tham gia v cc nhm nh trnh t ADN, c gng ng dng my tnh vo cng vic
ca h, mc d cc n lc ny cn rt h , nhng cng cho thy tim nng to ln ca my tnh
trong vic phn tch trnh t v dng m hnh phn t. Nhm cc nh sinh hc ny cng vi cc
nh ton hc, tin hc, tinh th hc v vt l hc to ra cng ng tin sinh hc u tin.
Nm 1985, Tp ch khoa hc u tin v lnh vc bt u xut bn (Cc ng dng ca My
tnh trong cc Khoa hc Sinh hc Computer Applications in the Biosciences). Cc bi bo ch
yu l v tin sinh hc, do vi nm sau tp ch i tin thnh Tin sinh hc. Lc ny Th
vin d liu trnh t EMBL Heidelberg hot ng c 4 nm v mt th vin khc ca
M, Ngn hng Gen (GeneBank), cng bt u. Nhng c s d liu trnh t u tin c n
hnh di dng sch hoc bng t, v cho thy hon ton v dng v khng kh thi v thng tin
v trnh t c tch ly qu nhanh.
Trong thp nin 90, Tin sinh hc chuyn mnh mnh m, vt ngoi d on nh mt
lot cc tin b. Trc ht l do s ra i v cng nhn ca internet nh l mt mng my tnh
ton cu. Lc ny vic phn tch, dch v, c s d liu, c th thc hin d dng qua cc
trang web. Th hai, cc tin b v k thut nh trnh t lm cho vic nh trnh t c mt loi
tr nn kh thi, thng tin v b gen hon chnh qu phc tp v ch c th c tnh ton v lp
ghp bi my tnh, do vy cch duy nht truy cp thng tin ny l dng my tnh. V thi i
ca tin sinh hc bt u.
Thng tin trnh t tng ln gp i sau mi 18 thng, do cc h thng my tnh cng
nh thut ton phn tch cng phi pht trin tng ng. Gi phn mm Staden c pht
trin bi Roger Staden, LMB, Cambridge (UK) c s dng lp ghp v phn tch trnh t
ti phng th nghim ca Fred Sanger.
Vic tm kim cc trnh t cng tr nn kh khn hn khi s lng thng tin tng ln. Ban
u cc d liu trnh t c phn phi di dng cc tp n gin, khng c cu trc (flat file),
cng vi cc thng tin ch thch nh loi, t kha. Do vy cch thc tm kim trnh t cn quan
tm l dng cc trnh so snh chui hoc tm bng mt da trn bn in ra. Cc chng trnh tm
kim da trn c s d liu flat file nh ACNUC, c pht trin vo gia nhng nm 80, v
gn y hn l SRS.
Mt vn khc do khi lng thng tin ln l thi gian tm kim. Cc thut ton c
a ra t nhng nm 70 bi Smith v Waterman, chy trn cc main frame (my tnh ln), chy
rt chm m khng phi ai cng c th s dng c v khng c cc main frame t tin. Nm
1985, thut ton mi c pht trin trong chng trnh FASTA, do David Lipman v Bill
Pearson (US) pht trin da trn phng php ca Wilbur v Lipman vi thut ton do Dumas
v Nino cng b. FASTA to ra mt cuc cch mng trong vic tm kim trnh t, v n r v
nhanh, li c th chy trn cc my tnh c nhn r tin. Do cc nh khoa hc c th s dng
d dng tm kim chc nng ca mt trnh t mi da trn cc protein c bit trc.


31
Nhng n 1990, FASTA cng khng nhanh, mc d cng ngh my tnh pht trin
c cc trm lm vic ngy cng mnh hn. Mt chng trnh mi, BLAST (Basic Local
Alignment Search Tool), ra i. N c vit bi cc nh sinh hc, ton hc v tin hc Trung
tm Quc gia Thng tin Cng ngh Sinh hc (National Center for Biotechnology Information,
NCBI - Hoa K). N chy nhanh hn FASTA nhiu kha cnh, ng thi cho php xp hng
tm quan trng ca cc trnh t tm c.
Cc chng trnh cng c pht trin d on v xy dng cu trc protein da trn
thng tin v trnh t v c s d liu cc cu trc bit.
Ngy nay khi m nhiu sinh vt c gii m di truyn hon chnh v to nn cc c s
d liu lin thng gia nhiu trung tm trn th gii, vic tm kim v phn tch thng tin ca
mt trnh t mi tr nn d dng hn bao gi ht. Vi mt trnh t no , ngi ta c th nhanh
chng xc nh c chc nng, thm ch cu trc phn t da trn cc thng tin hin c trong
ngn hng d liu. Nh m s pht trin ca khoa hc sinh hc c tng tc ln rt nhiu
ln so vi thi k u.
Hin nay v trong tng lai gn tin sinh hc s pht trin mnh c th thc hin
khng ch tm kim phn tch thng tin sinh hc m cn c th cho php tin hnh cc th
nghim in silico vi tin cy cao, tit kim nhiu thi gian v tin bc cho cc th nghim in
vitro v in vivo, gp phn y mnh tc pht trin khng ch sinh hc m cn cc khoa hc
ng dng ca n nh y hc, dc phm.
3.2. Cc nh ngha
Tin sinh hc hay sinh hc in ton bao hm vic s dng cc k thut ca ton hc ng
dng, tin hc, thng k hc v khoa hc my tnh gii quyt cc vn sinh hc. Nghin cu
v tin sinh hc thng chong lp vi cc nghin cu v sinh hc h thng. Cc cng vic
nghin cu chnh gm c ging trnh t (sequence alignment), tm kim gen, lp rp thng tin b
gen, ging cu trc protein, d on cu trc protein, d on biu hin gen v tng tc
protein-protein, v dng m hnh tin ha. Thut ng tin sinh hc v sinh hc in ton thng
c s dng nh ng ngha, tuy nhin, t sau nhn mnh v vic pht trin thut ton v cc
phng php in ton, trong khi tin sinh hc bao hm c sinh hc, ton hc v khoa in ton.
Mt s thut ng hp hn ch cc lnh vc nh trong sinh hc in ton nh b gen hc in ton
(computational genomics), ha sinh hc in ton (computational biochemistry) v l sinh hc
in ton (computational biophysics).
Tin sinh hc (bioinformatics): Nghin cu, pht trin, hay ng dng cch tip cn v cng
c in ton khai thc thng tin, d liu sinh hc, y hc, hnh vi hay sc khe bao gm thu
nhn, tch ly, t chc, lu tr, phn tch, hay biu din cc thng tin ny.
Sinh hc in ton (computational biology): Pht trin v ng dng cc phng php l
thuyt v phn tch thng tin, dng m hnh ton hc v k thut m phng in ton nghin
cu cc h thng sinh hc, hnh vi v x hi.
3.3. Cc lnh vc nghin cu chnh
3.3.1. Phn tch trnh t
Vic xc nh cc motif bo tn trong cc trnh t c th cung cp cc thng tin v cu trc
v chc nng ca protein, v xc lp cc cn c phn loi. Thng tin t c s d liu s cp
c hp l ha v gn thnh cc h. Cc c tnh chnh ca h c lc ra thnh cc bn m t
trnh t c cha trong c s d liu th cp, bn m t ny c dng d on cc trnh t
trong tng lai. Vic xc lp c s d liu th cp rt quan trng v c s d liu s cp tng ln
rt nhanh v tr nn rt kh phn bit cc quan h xa vi cc nhiu nn.

32
T khi Phage -X174 c gii trnh t vo nm 1977, cc trnh t DNA ca nhiu loi
sinh vt c gii m v lu trong c s d liu in t. D liu ny c phn tch xc
nh nhng gen no m ha cho protein, cng nh nhng trnh t thng thng. S so snh cc
gen trong cng mt loi hay gia cc loi khc nhau c th cho thy s tng ng gia cc
chc nng ca protein, hay mi quan h gia cc loi (s dng phng php phn loi sinh hc
xy dng cy pht sinh loi). Vi s gia tng lng d liu, ngi ta mt thi gian kh lu
phn tch trnh t DNA bng cch th cng. Ngy nay, cc chng trnh vi tnh c dng
tm kim hng ngn gen ca cc sinh vt, cha hng t nucleotid. Nhng chng trnh ny c th
a vo cc t bin, (thay i, mt hoc thm base) trong trnh t DNA, nhm xc nh cc
trnh t c lin quan, nhng khng ging. Mt bin th ca vic ging cc trnh t c dng
trong qu trnh gii trnh t ca n. V d trong k thut gii trnh t shotgun (c s dng bi
Vin nghin cu b gen (Institute for Genomic Research) gii trnh t gen vi khun u tin,
Haemophilus influenza) kt qu thu c khng phi l cc danh sch lin tip ca trnh t
nucleotid, m l trnh t ca hng ngn mnh DNA nh (mi mnh c chiu di 600 - 800
nucleotid). Cc u tn ca nhng mnh ny chng ln nhau v khi ging li ng cch th to
thnh trnh t hon chnh. Gii trnh t theo kiu shotgun cho d liu trnh t nhanh chng,
nhng cng vic lp ghp cc mnh c th rt phc tp i vi nhng b gen ln hn. Trong d
n Bn gen ngi, mt khong vi thng x l my tnh (dng my tnh loi DEC Alpha vo
nm 2000) lp ghp cc mnh. Gii trnh t theo kiu shotgun l phng php c la chn
gii m cc b gen hin nay, v nhng thut ton lp rp b gen l mt lnh vc quan trng
ca nghin cu tin sinh hc.
Mt lnh vc khc ca tin sinh hc trong phn tch trnh t l t ng tm kim cc gen v
cc trnh t iu ha trong b gen. Khng phi tt c cc nucleotid trong b gen l gen. Trong b
gen ca cc sinh vt bc cao, phn ln cc thnh phn ca DNA khng mang ngha no c. Cc
trnh t ny c gi l DNA tp, tuy nhin c th cha cc yu t c chc nng cha c bit.
Tin sinh hc c th b tc cc thiu st gia nhng nghin cu v gen v protein, v d nh dng
trnh t DNA xc nh protein.
3.3.1.1. Ch gii b gen: tm kim gen
Trong phm vi ca khoa b gen hc, ch gii gen l qu trnh xc nh cc gen v cc c
tnh sinh hc khc trong trnh t DNA. H thng phn mm ch gii b gen u tin c thit
k bi Owen White vo nm 1995, l thnh vin ca nhm gii trnh t v phn tch b gen ca
sinh vt hon chnh u tin c gii m, vi khun Haemophilus influenzae. Tin s White
to mt phn mm tm gen (phn trnh t DNA m ha cho mt protein), RNA vn chuyn,
v cc c tnh khc, v gn chc nng s b cho nhng gen ny. Hu ht cc h thng ch gii
b gen hin ti lm vic tng t nhau, nhng nhng chng trnh c th phn tch DNA ang
lun thay i v ci tin.
3.3.1.2. Sinh hc tin ha trn my tnh
Sinh hc tin ha l ngnh nghin cu ngun gc v th h ca loi, cng nh s bin i
ca loi theo thi gian. Ngnh tin hc gip cho cc nh sinh hc nghin cu v tin ha, n c
kh nng:
- theo du s tin ha ca mt s ln sinh vt bng cch xc nh s thay i trong DNA,
hn l ch dng phn loi theo hnh thi hay quan st sinh l hc.
- gn y hn, so snh cc b gen, cho php nghin cu nhiu bin c tin ha phc tp
hn, chng hn nh s lp gen, s chuyn gen ngang, v tin on nhn t hnh thnh
loi vi khun.
- xy dng cc m hnh dn s phc tp trn my tnh tin on kt xut ca h theo
thi gian
- tm v chia s thng tin v mt lng ln loi v sinh vt
Trong tng lai l xy dng li cy tin ha phc tp hn by gi.


33
Cc nghin cu trong khoa hc my tnh c s dng thut ton di truyn i khi b nhm
ln vi sinh hc tin ha trn my tnh. Thut ton di truyn bao hm vic s dng phn mm
my tnh c bit pht trin cc cng thc, thut ton, hay cc tit k mc tch hp ly tng
t cc nguyn l tin ha nh l nhn bn, s a dng ha do s ti t hp hay t bin, ph hp,
s sng st do s chn lc, v tc ng qua li, ni chung gi l c my Darwin.
3.3.1.3. Nghin cu tnh a dng sinh hc
a dng sinh hc ca h sinh thi c th c nh ngha nh l tng cc thch nghi v mt
b gen t tt c cc loi hin din i vi mt mi trng c th bt k mi trng l mt
mng sinh thi hay m khai thc b hoang, mt git nc bin, mt nm t, hay ton b sinh
quyn ca hnh tinh Tri t. C s d liu c dng thu thp tn loi, m t, s phn b,
thng tin v gen, tnh trng v s lng dn c, mi trng sng cn thit, v cch mi sinh vt
tc ng qua li vi loi khc. Cc chng trnh phn mm c bit c dng tm kim, hin
th v phn tch thng tin, v quan trng nht l trao i thng tin vi ngi khc. Cc kiu m
hnh trn my tnh nh ng hc qun th, hay tnh ton s tch ly gen tt ca tp hp con
ging (trong nng nghip) hay qun th sp tit chng (trong bo tn). Mt trin vng ng quan
tm trong lnh vc ny l trnh t ton b DNA, hay b gen ca nhng loi nguy c tuyt chng
c th c lu gi trong my tnh (in silico) v c th c s dng trong tng lai, thm ch
nu loi b mt i.
3.3.1.4. Phn tch biu hin gen
Biu hin ca nhiu gen c th c xc nh bng cch o lng mRNA vi nhiu k
thut nh microarray, EST (expressed cDNA sequence tag), SAGE (serial analysis of gene
expression), MPSS (massively parallel signature sequencing), hay nhng ng dng khc ca s
lai ti ch a gi. Tt c cc k thut ny c mc nhiu tp rt cao v/hoc gy ra cc sai s cho
php o sinh hc, v mt lnh vc nghin cu chnh ca tin sinh hc l pht trin cc cng c
thng k tch tn hiu ra khi nhiu tp trong cc nghin cu biu hin gen theo kiu hong
lot (high-throughput). Nghin cu kiu hng lot thng c s dng xc nh nhng gen
lin quan n cc ri lon: v d c th so snh d liu microarray t t bo biu m ung th vi
d liu t t bo bnh thng xc nh protein iu ha dng hay m trong cc t bo ung
th.
3.3.1.5. Phn tch iu ha
S iu ha l s phi hp phc tp ca nhiu s kin bt u vi mt tn hiu bn ngoi
bo v cui cng dn n tng hoc gim trong hot ng ca mt hay nhiu phn t protein. K
thut tin sinh hc c ng dng khm ph cc cp khc nhau trong qu trnh ny. V
d, phn tch promoter gm vic tm ra v nghin cu cc m thc trnh t vng xung quanh
vng m ha ca gen. Cc nh hng ca nhng m thc ny th hin thng qua s phin m
ca gen thnh mRNA. D liu v biu hin gen c th c dng suy ra qui lut iu ha
gen: ngi ta c th so snh d liu microarray t nhiu trng thi khc nhau ca sinh vt
hnh thnh gi thuyt v gen c lin quan mi trng thi. sinh vt n bo, ngi ta c th so
snh cc giai on ca chu trnh t bo, song song vi nhng iu kin khc nhau (sc nhit,
thiu dinh dng, ). Sau ngi ta c th ng dng thut ton t hp i vi d liu biu
hin xc nh gen no c ng biu hin. Phn tch su hn c th theo nhiu hng khc
nhau: mt nghin cu (2004) phn tch cc trnh t promoter ca cc gen ng biu hin tm
cc yu t iu ha chung v dng k thut hc kinh nghim my xc nh cc yu t
promoter lin quan n vic iu ha tng cm gen.

34
3.3.1.6. Phn tch biu hin protein
Microarray ca protein v khi ph nhanh (HT MS - high throughput mass spectrometry)
c th phn tch nhanh v s hin din cc protein trong mu sinh phm. Tin sinh hc c s
dng rt nhiu trong vic phn tch d liu microarray ca protein v HT MS.
3.3.1.7. Phn tch s t bin trong ung th
Nhiu n lc ln ang c tin hnh xc nh trnh t nhng t bin im cc gen
ung th khc nhau. Khi lng d liu ln thu c cn h thng t ng c trnh t, v so
snh kt qu nhng trnh t vi trnh t bit ca b gen ngi, k c a hnh nguyn thy
bit.
Microarray oligonucleotid, bao gm s lai ha vi b gen so snh v dy a hnh nucleotid
n (single nucleotid polymorphyism array), c th d cng mt lc vi trm ngn v tr trn b
gen ang nghin cu xc nh s tng hay gim ca nhim sc th trong ung th. Phng
php phn tch im thay i v m hnh Hidden Markov ang c pht trin suy ra s bn
bin i thc t d liu cha thng nhiu tp.
3.3.1.8. D on cu trc
D on cu trc protein l mt ng dng quan trng na ca tin sinh hc. Trnh t amino
acid ca mt protein, gi l cu trc s cp, c th xc nh d dng t trnh t trn gen m ha
n. phn ln trng hp, cu trc s cp ny quyt nh cu trc c nht ca protein trong
mi trng t nhin. (d nhin, c ngoi l, v d nh trong bnh b in - prion bnh c cu trc
khc cu trc t nhin). Bit c cu trc ny th mi hiu chc nng ca protein. Thng tin v
cu trc thng c phn loi theo cu trc bc hai, ba v bn. Gii php chung ng tin cy
d on vn cn l vn cha gii quyt. Cho n nay, hu ht cc kt qu c c u da
vo kinh nghim.
Mt trong nhng cch gii quyt chnh trong nghin cu tin sinh hc l khi nim v tnh
tng ng. Trong phn ngnh b gen ca tin sinh hc, tnh tng ng c dng d on
chc nng ca gen: nu trnh t ca gen A, m chc nng bit, tng ng vi trnh t gen B,
chc nng cha bit, ngi ta c th suy ra rng B c th mang chc nng ca A. Trong phn
ngnh cu trc ca tin sinh hc, tnh tng ng c dng xc nh phn no ca protein l
quan trng trong vic hnh thnh nn cu trc v tng tc qua li vi cc protein khc. Trong k
thut gi l dng m hnh tng ng, thng tin ny dng d on cu trc ca protein khi
cu trc ca protein tng ng bit. K thut ny hin nay vn l cch duy nht ng tin cy
d on cu trc protein.
V d cho k thut ny l tnh tng ng protein gia hemoglobin ngi v hemoglobin
cy h u (leghemoglobin). C hai gi cng chc nng vn chuyn oxy vo sinh vt. Mc d
hai protein ny c trnh t amino acid khc nhau, cu trc protein ca chng th gn nh ng
nht, phn nh cc chc nng gn ging nhau.
Nhng k thut khc cho d on cu trc protein gm c dng m hnh da vo protein
threading v de novo t thng tin vt l.
3.3.1.9. Khoa b gen hc so snh
Ct li ca phn tch b gen so snh l vic thit lp s tng ng gia cc gene hay cc
c tnh b gen khc cc sinh vt khc nhau. Chnh cc bn so snh gia cc b gen ny cho
php chng ta truy tm qu trnh tin ha chu trch nhim i vi s phn ha ca hai b gen.
Nhiu s kin tin ha hot ng nhiu mc t chc khc nhau quyt nh s tin ha.
mc thp nht, cc t bin im nh hng n tng nucleotid. mc cao hn, cc on
nhim sc th ln b lp, b chuyn ngang, nghch o hay chuyn v, b mt hay chn. Cui
cng, c b gen tham gia qu trnh lai to, s to th a bi v ni cng sinh, thng thng dn


35
n s hnh thnh loi mi nhanh chng. Tnh phc tp ca tin ha b gen l nhng th thch
ln i vi vic pht trin cc m hnh ton hc v thut ton.
Nhiu nghin cu trong s ny da trn vic pht hin tnh tng ng v tnh ton cc h
protein. Cc nhm PhIG (Phylogenetically Inferred Group - Nhm suy lun v pht sinh loi)
gn y pht trin phng php tch hp cc tn hiu sinh loi vo vic xy dng cc cm gen
s dng trong b gen hc so snh.
3.3.2. To m hnh cc h thng sinh hc
Sinh hc h thng bao gm vic s dng cc m phng my tnh cc h thng di t bo
(nh l mng li cht trao i v cc enzym to thnh s trao i cht, cc con ng truyn tn
hiu v mng li iu ha gen) phn tch ln biu din cc mi quan h phc tp ca cc qu
trnh t bo ny. S sng nhn to hay s tin ha o ang n lc hiu r v cc qu trnh tin
ha thng qua vic m phng trn my tnh nhng th sng n gin (nhn to).
3.3.3. Phn tch hnh nh hiu nng cao
K thut my tnh cng c dng tng tc hay t ng ha vic x l, nh lng v
phn tch lng ln hnh nh sinh y hc c mang nhiu thng tin. H thng phn tch hnh nh
hin i to kh nng o c hnh nh ln v phc tp, do tng s chnh xc, tnh khch quan, v
tc . H thng phn tch c pht trin hon chnh c th hon ton thay th ngi quan st.
Trong khi nhng h thng ny khng phi ch lin quan n hnh nh sinh hc, nhng ng dng
ca chng trong sinh hc vn cung cp cc gii php v nhng thch thc, do nhiu ng dng
phn tch nh c coi nh ng dng ca tin sinh hc. Cc h thng ny ang tr nn quan trng
hn trong chn on v nghin cu.
3.4. Cng c
Cng c cho sinh hc my tnh ni ting c l l BLAST, mt thut ton tm kim d liu
trnh t ln (protein, DNA). NCBI cung cp cng c thng thng tm kim d liu trnh t
ca h.
Ngn ng my tnh (Perl, Python) thng dng phn tch d liu sinh hc v phn tch
d liu ra t cc chng trnh tin sinh hc.
Nhng phng tin tm kim tin sinh hc ln (Entrez, Bioinformatic Harvester) gip tm
kim thng tin t mt s c s d liu.
Nhng ngi thit k chng trnh cho m ngun m nh EMBOSS, Bioconductor,
BioPerl, BioLinux, BioPython, BioRuby, v BioJava m c th pht trin v sp xp cc i
tng (nh program modules) v cc cng c chia s d dng phn tch hn.
VigyaanCD l mt phn mm min ph mnh cho tin sinh hc v sinh hc my tnh.
Cc b phn thng mi nh DNAStar (Lasergene), Informatix, Discovery One, Virtual
Cloning, cung cp kh nhiu cng c phn tch hu ch v tin dng, tuy nhin gi thnh
ca chng l mt vn ln, ch nhng phng th nghim ln mi c th mua v s dng c.
Mt s a ch website hu ch v tin sinh hc:
- NCBI (Trung tm CNSH Quc gia M): http://www.ncbi.nlm.nih.gov/
- GenBank (NCBI) - Ngn hng gen:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide
- Ngn hng gen Nht: http://www.ddbj.nig.ac.jp/
- BLAST (NCBI) - so snh, tm kim trnh t : http://www.ncbi.nlm.nih.gov/BLAST/
- SubtiList - tm kim gen ca Bacillus subtilis : http://genolist.pasteur.fr/SubtiList/

36
- Signal Peptide Server - tm kim trnh t xut ca protein:
http://www.cbs.dtu.dk/services/SignalP/
- LipoP Server - tm kim trnh t xut ca protein v lipoprotein:
http://www.cbs.dtu.dk/services/LipoP/
- CLUSTALW (so snh trnh t ADN v protein): http://align.genome.jp/
- CLUSTALW (so snh trnh t protein):
http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl
- Bng s dng m: http://www.kazusa.or.jp/codon/
- C s d liu trnh t iu ha: http://wwwmgs.bionet.nsc.ru/mgs/systems/geneexpress/


Ti liu tham kho:
1. Bansal, A.K., Bioinformatics in microbial biotechnology--a mini review. Microb Cell Fact,
2005. 4: p. 19.
2. Teufel, A., et al., Current bioinformatics tools in genomic biomedical research. Int J Mol
Med, 2006. 17(6): p. 967-73.
3. Glossary of bioinformatics terms. Curr Protoc Bioinformatics, 2011. Appendix 2: p. A 2 1-
13.
4. Aliferis, C.F., et al., Trends and developments in bioinformatics in 2010: prospects and
perspectives. Yearb Med Inform, 2011. 6(1): p. 146-55.





37
B B i i 4 4. . K K t th hu u t t x x c c n nh h k ki i u u g ge en n
Mc tiu:
- Trnh by c cc tiu ch ca mt phng php nh typ tt.
- Trnh by v vn dng c mt s phng php nh typ da trn acid nucleic.
4.1. i cng
S bng pht ca cc bnh truyn nhim thng xut pht t vic tip xc vi mt ngun
ly chung ca tc nhn gy bnh. Ni chung, tc nhn gy dch li xut pht t mt c th m
con chu ca chng c s tng ng hoc c quan h gn v mt di truyn. Theo thut ng dch
t hc, cc vi sinh vt lin quan n cn dch c quan h dng, hay ni cch khc chng c
cng ngun gc. Cc vi sinh vt c quan h dng l thnh vin ca cng mt loi c chung cc
thuc tnh sinh ha, yu t c lc v c tnh di truyn. Tuy nhin, c s a dng mc loi
cc vi sinh vt phn lp c t cc ngun khc nhau, thi gian khc nhau v v tr a l
khc nhau c th c phn bit hay phn loi thnh cc typ hay chng.
4.1.1. ngha v lch s ca vic nh typ vi sinh vt
Vic nh typ c ngha quan trng trong dch t hc nhn bit cn dch, pht hin s
ly truyn cho ca tc nhn trong bnh vin, nhn din cc chng c c lc c bit v theo
di cc chng trnh tim chng.
T thi ca Pasteur, cc tip cn dch t hc lin quan n vic nh typ v phn bit cc vi
khun u da ch yu vo cc phng php kiu hnh hay hiu sinh hc. Cc phng php ny
c gng xc nh v sp xp vi khun theo mt trt t kiu Linne. Tuy nhin, quan im loi
c in dng nh khng thch hp phn loi vi khun. Gn y, cc nh ngha loi bng
hnh thi hc c thay th hon ton bi vic so snh trnh t ca 16S rADN. Chng thng
c hiu trong ng cnh ca s a dng v nucleotid khng c hiu c lin quan n mt s
kiu hnh (v d tnh trng v kh nng gy bnh). Du sao, cc phng php kiu hnh vn cn
rt quan trng i vi cc phng th nghim vi sinh hc. Mt s phng php ny hin nay c
bao gm c du n v chuyn ha, phn tch v thnh phn acid bo, cc bin th cu trc trong
cc c tnh v hnh thi trn b mt vi khun. Phng php nhum Gram c s dng xc
nh v phn bit vi khun, tuy nhin, vi nhiu l do n tr nn khng ng tin cy. Cc thay
i nh trong iu kin nui cy, s thiu ht mt s c tnh kiu hnh v s hi t tin ha
gia cc c tnh ny c th hy hoi cc kt lun v dch t hc ca mt v dch.
4.1.2. Tiu ch ca mt phng php nh typ tt
Ni chung cc phng php nh typ phi p ng mt s tiu chun nht nh c th
c p dng rng ri. Trc ht tt c cc vi sinh vt trong mt loi u phi c th nh typ
c bng phng php c chn. i vi mt s phng php, v d da vo khng th hay
s hin din ca th th thc khun, th cc c tnh ny c th khng c trong tt c cc chng
ca loi. Typ huyt thanh c gn cho chng ch khi c s hin din ca cc du hiu huyt
thanh hc c th no . V d, 10 - 15% cc chng Mycobacterium avium khng th nh typ
huyt thanh c v chng khng phn ng vi bt k khng huyt thanh no hin c. Tng t,
typ thc khun cng thng c dng cho Staphylococcus aureus, S. aureus khng c th th
ca thc khun th khng th b nhim c v do khng th nh typ c.
Cc phng php nh typ cng phi c kh nng phn bit cao. N phi c kh nng phn
bit r rng cc chng khng c lin quan, v d cc chng c ngun gc a l hon ton khc,
ng thi phi chng minh c quan h ca cc chng c phn lp t cng mt ngun gc.

38
lp li c bit quan trng khi xy dng c s d liu ca cc chng bit dng so
snh vi cc chng cha bit. S biu hin kiu hnh khng n nh, v d biu hin gen c lc
khng lin tc, c th gy kh khn cho vic lp li
Cui cng tnh d dng v gi thnh ca phng php cng c vai tr quan trng khi la
chn phng php inh typ.
Cc phng php nh typ da trn kiu hnh ni chung khng p ng c ht cc yu
cu trn, trong khi cc phng php da trn trnh t ADN hay kiu gen cho php khc phc
cc yu im ny. Cc k thut ny c nhiu u im so vi phng php truyn thng nh
trnh c nhu cu v mi trng nui cy, tng nhy v n nh ca cc th nghim, rt
ngn thi gian. Cui cng, vic nh typ phn t xut pht t du n ADN hay trnh t
nuclceotid thu c nhiu thng tin. V d, php phn tch du n vi cc mi ERIC, REP v
BOX c th to ra hng chc sn phm khuch i cha thng tin v mt chng c th, trong khi
trnh t ca mt gen ompA c th cung cp hng trm c im a hnh.
4.1.3. Phn loi cc phng php nh typ phn t
Cc phng php nh typ phn t c th c chia lm ba nhm:
a) Phn tch du n nhim sc th s dng k thut khuch i gen
b) Ct gii hn nhim sc th vi khun
c) Khuch i mt gen v phn tch trnh t
Trong qu trnh thc hin, c nhiu ci tin hay bin th ca cc chin lc c bn trn. V
d, cc phng php du n da trn PCR c th khai thc nhiu nhm mi lp khc nhau nh
REP, cc trnh t xen gia rARN, cc trnh t ADN a hnh khuch i ngu nhin (RAPD).
Chin lc da vo s a hnh di cc on ct gii hn (RFLP) c th c phn tch gin
n vi gel agarose nhum ethidium bromid hay c th c chuyn ln mng v lai vi cc
on d nh du. Vic khuch i mt gen v phn tch trnh t c th khc nhau do gen c
chn khc nhau, hay do vic s dng thng tin trnh t thu c trc tip hay gin tip thng qua
vic dch thnh protein.
Cc k thut du n da trn PCR hin ang s dng phn bit cc chng gm du n
PCR vi mi lp (REP-PCR), PCR ca vng xen gia 16S v 23S rDNA (ribotyping), v PCR
ADN a hnh khuch i ngu nhin (RAPD-PCR).
4.1.4. La chn gen phn t bng phng php phn t
Vic phn tch chng t cc trnh t nucleotid hin mnh phn bit cc vi khun
c th, khi phi hp vi PCR mt on gen vi nh trnh t t ng, n c th cung cp thng
tin chi tit v kiu gen mt cch nhanh chng. Vic phn tch trnh t nucleotid tr thnh mt
nhnh ph ca khoa h thng v phn loi hc vi khun. Cc trnh t ADN ribosom c
chng minh l hu hiu trong vic phn bit cc nhnh sinh loi ca cc h vi khun c quan h
xa. Cc trnh t rARN ca tiu n v nh, c chng minh rt hu ch trong vic gii quyt
cc quan h tin ha trong ngnh khun lam. Tuy nhin, tc thay th cc k chm trong cc
gen rARN nh tin cho vic s dng chng trong vic phn gii cc n v phn loi c
quan h gn (v d loi v chng). Do , mt s gen m ha cho protein vi khun c
nghin cu s dng lm cy sinh loi ca cc chng c quan h rt gn. Tnh hiu qu trong vic
phn bit ca cc gen s dng trong nh typ cc chng khc nhau thng c xc nh mt
cch thc nghim trong mt h vi khun c th. Trong mt s trng hp, vi locus khc nhau
c th c dng pht hin lm tng chnh xc ca vic nh danh v tng phn gii
gia cc chng. Cc tip cn kiu ny c p dng hiu qu trong vic nh typ cc vi khun
ng rut. v d, trnh t nucleotid ca gapDH v ompA chng minh cc chng phn lp
hin nay ca Escherichia vulneris khng c cng mt nhnh phn loi, nh trc y, v do
khng nn xem xt cc chng ny trong cng mt loi. Ni chung, c nhiu yu t bn trong cc


39
gen m ha ca vi khun c th dn n vic xem xt chng hu dng nh l du ch phn t
phn bit chng:
a) Mc bin thin ca trnh t nucleotid vt xa cc trnh t rADN tiu n v nh v
ln.
b) Protein c m ha c chc nng c bo tn xuyn sut trong nhiu chi, chng t
gen s ton vn trong cc loi c lin h xa.
c) V tr vt l ca gen trn nhim sc th vi khun chng t c s iu hng gia cc
n v phn loi khc xa nhau.
4.1.5. Nhc im ca phng php phn t
Mc d cch tip cn di truyn phn t t ra mt cng c phn tch hiu qu nht xc
nh v theo di cc chng vi khun, chng khng phi khng c sai lm. Vic s dng cc on
d phn t v cc locus nhim sc th khc nhau phc ha kiu gen ca vi khun c th pht
sinh sai st. Gen c s dng phn bit loi ny c th khng cha trnh t bin thin cn
thit phn bit vi cc chng ca mt loi khng lin quan khc. Vic xc nh hiu qu nh
typ thc nghim i vi mt n v phn loi c th i khi rt tn km v thi gian v tin bc.
V d, cc trnh t rADN tiu n v c dng hiu qu trong vic phn bit cc Pseudomonas
nhng li thiu cc on cn thit phn bit vi cc loi khc trong ng rut nh l mt s
Escherichia hay Erwinia. Hn na, c tnh siu nhy ca cc k thut nh typ phn t, nht l
cc k thut c lin quan n PCR, lm cho vic phng trnh ngoi nhim tr thnh mt vn
ln.
Vi cc tin b gn y v k thut phn tch trnh t ADN v lai ha, hu ht cc phng
php hin dng u khng c nh du bng phng x nh trc. Mt s qui trnh ngy nay
cho php nh danh v phn bit hiu qu, kinh t v an ton cc chng dng trong th y, y hc,
nng nghip.
4.2. Du n da trn PCR ca nhim sc th vi khun
4.2.1. K thut chung
C nhiu phng php lm du n b gen vi khun da trn phn ng khuch i.
Phn tch du n bng PCR mi lp, rep-PCR, da vo mt s nhm trnh t c bo tn
ca cc yu t ADN lp c phn b trong nhim sc th vi khun. Tng t nh phn tch
VNTR nhn tht, cc oligonucleotid c s dng trong rep-PCR pht hin s a hnh kch
thc bng cch khuch i cc vng nhim sc th nm gia cc yu t lp. Bng cch ny,
s cc mnh khuch i lm mt hm s ca v tr vt l ca yu t lp trn nhim sc th.
Phn tch PCR ca vng xen gia 16S v 23S, ribotyp, cng cho thng tin v kiu gen ca
chng di dng s a hnh kch thc. Mi oligonucleotid c thit k t u 3 ca 16S
rADN v u 5 ca 23S rADN khuch i vng xen gia (IGS) hai tiu n v rARN.
Phn tch ADN a hnh khuch i ngu nhin (RAPD-PCR), s dng phn ng khuch i
cc phn bt k ca ADN nhim sc th nh mt hay nhiu on mi ty ngn. Phn tch
trn gel agarose cho mt ph cc sn phm khuch i thng c trng cho mt chng c
th.
S a hnh chiu di on khuch i (AFLP) l mt k thut hiu qu khc phn bit cc
chng vi khun c lin quan gn da trn PCR. AFLP khuch i ADN sau khi ct vi
enzym gii hn. Mt cch vn tt, ADN vi khun c ct trc vi enzym gii hn (v d
EcoRI v MseI) to ra mt hn hp cc mnh ADN c chiu di khc nhau. Cc on ni
oligonucleotid si i lc ny c ni vo cui cc mnh ADN trn. Cc on mi c hiu
vi on ni vi cc nucleotid chn lc 3 khc nhau c dng khuch i mt s mnh
t hn hp. AFLP khc vi RAPD-PCR ch bc khuch i khng hon ton ngu nhin
v khc vi REP-PCR ch n khng da vo trnh t chung bit ca vi khun. Ni cch

40
khc, AFLP da vo s khuch i chn lc mt phn cc mnh c trnh t tng ng vi
mi PCR.
4.2.1.1. Chun b ADN nhim sc th
C nhiu phng php n gin v nhanh chun b ADN nhim sc th dng trong cc
qui trnh du n da trn PCR. ADN dng cho PCR khng i hi tinh khit cao nh cc th
nghim sinh hc phn t khc. Do vy cc qui trnh chit tch ADN chy PCR thng nhanh
v r tin. Hin c nhiu sn phm c thng mi ha chit tch nhanh ADN t vi khun
Gram m nh Instagene (Bio-Rad), Wizard (Promega) v DNeasy (Qiagen). Cc sn phm ny
thng s dng nha tinh ch c th gn khng hi phc vi protein t bo v cc thnh phn
khc c th c ch phn ng khuch i.
ADN ca cc vi khun Gram dng c th c tch chit bng cc kit nh Puregene
(Gentra Systems) v Isoquick (Orca Research). Tuy nhin, thng thng khng cn n cc kit
nh trn v cng c nhiu phng php n gin thc hin iu ny. Thng thng
chit ADN ca vi khun ngi ta ly gii t bo phng thch ADN vo mi trng. Vi khun
Gram m thng ly gii hn v ch cn x phng hay un si, trong khi vi khun Gram dng
cn cc phng php ph v nng tnh c hc hn.
4.2.1.2. iu kin phn ng
Cc iu kin phn ng ni chung ging cc phn ng PCR thng thng, trong tng
lng mi s dng khong 50 pmol, Taq polymerase 2,5 UI, nng MgCl
2
i khi cn c
xc nh li ty theo tng trng hp c th. Cc ng phn ng c t vo my lun nhit
c lm nng trc 94
o
C. Cc chng trnh chy cho cc loi mi khc nhau c th tham
kho trong bng bn di.
Bng 4.1. Mi thc hin du n da trn PCR v cc thng s hot ng
Mi / Trnh t mi
Phng
php
Nhit
gn mi
Nhit
ko di
Thi gian
ko di
S chu
k
16SF / TTGTACACACCGCCCTCA Ribotyping 55 72 1,5 35
23SR / GGTACCTTAGATGTTTCAGT
REP1R-I / IIIICGICGICATCIGGC REP-PCR 40 65 8,0 35
REP2-I / ICGICTTATCIGGCCTAC
ERIC1R/ ATGTAAGCTCCTGGGGATTCAC REP-PCR 52 65 8,0 35
ERIC2 / AAGTAAGTGACTGGGGTGAGCG
BOXA1R/ CTACGGCAAGCGGACGCTGACG REP-PCR 52 65 8,0 35
MBO1/ CCGCCGTTGCCGCCGTTGCCGCCG REP-PCR 54 65 8,0 35
GTG5 / GTGGTGGTGGTGGTG REP-PCR 40 65 8,0 35
OPA-1/ CAGGCCCTTC RAPD-PCR 35 72 2,0 35
Bc3/CCGGCGGCG RAPD-PCR 35 72 2,0 35
Bc4/CGGCCCCTG RAPD-PCR 35 72 2,0 35
Bc5 / CGGCCACTGT RAPD-PCR 30 72 2,0 35
4.2.1.3. in di sn phm khuch i
hin th nhanh hnh nh du n t kt qu nh typ ribosom, Rep-PCR, RAPD-PCR,
sn phm c phn tch trn gel agarose 1,5%, kch thc gel khong 7,5x10 cm. Nu cn
phn tch k hn, nn s dng gel ln hn v d 20x24cm. Gel nn c chy vi m TAE vi
dng in 5V/cm. Sau khi chy gel c th nhum bng ethidium bromide v chp hnh di UV.
4.2.1.4. Phn tch v bin gii kt qu du n PCR
Kt qu c th c bin gii t nh chp gel. Kiu gen c th c ghi mt cch n
gin vi thng tin v kch thc bng da vo cc thang chun ADN. S dng nhiu thang ng


41
thi s lm tng chnh xc. V hu ht cc k thut ny u cho sn phm c kch thc t 0,2
- 2 kp, do nn dng thang c kch thc ngn hn. v d 100 bp.
Vic theo di cc chng vi khun nh kiu gen ca chng l mt phng php hiu qu v
tin cy theo du s phn tn ca tc nhn gy bnh. Cc kiu gen ny n nh v lp li
nu c tin hnh trong cc iu kin xc nh. iu ny c chng minh bng thc nghim
khi ly mt s chng gy nhim v ti phn lp t m k ch.
Ngoi vic p dng theo du cc chng, cc thng tin thu c bng k thut du n
PCR cng c dng phn bit v di truyn v pht sinh loi ca nhiu chng cng lc. Cc
ma trn nh phn (0 v 1) c th c xy dng t gel kt qu do s hin din hay khng ca cc
bng v tr c th. Ma trn ny sau c th c dng tnh ton theo cp cc khong cch
v di truyn gia cc chng vi thut ton thch hp. Ngoi ra cc ma trn nh phn cng trc
tip dng trong phn tch s phn nhnh.
4.2.2. Phng php nh typ ribosom
Nh trn ni, gen rARN c th dng pht hin cc khc bit di truyn gia cc n
v phn loi cao hn ca vi khun. Gn y, cc vng xen gia 16S v 23S c s dng
nhiu hn phn bit vi khun. Vng ny c tnh a hnh v kch thc rt ln v do c
tn dng phn bit cc vi khun c quan h gn. Phng php nh typ ribosom ban u c
ng dng theo di dch t hc trong cc c s lm sng do tnh kh thi i vi hu ht cc
chng vi khun v tnh lp li ca kt qu. Tuy nhin, k thut ny cng mnh phn bit
cc loi v chng vi khun ng rut khc. S phn b cc bng trong hnh 1 th hin hai kha
cnh quan trng. u tin, mi loi u c cc bng c th ring. Th hai, cc bin th di
truyn c th c pht hin gia cc chng trong loi. K thut c p dng cho nhiu loi
vi khun gy bnh v trong nng nghip. Mi n hin sn c thc hin du n cho vng xen
gia (IGS), trnh t, nhit gn mi v nhit /thi gian ko di c cho trong bng 1. Sn
phm khuch i ca phng php nh typ ribosom c kch thc 200 bp n 2 kb v bao gm
cc bng c tnh n nh cao cho d cht lng ADN tng i km.
4.2.3. Rep-PCR
Rep-PCR l phng php du n di truyn vi khun c thc hin bng cch khuch i
ADN nhim sc th vi cc mi bt cp vi cc trnh t lp trong b gen vi khun. Do s phn
b ca cc trnh t ny trong b gen khc nhau gia cc chng khc nhau nn sn phm khuch
i c s a hnh v kch thc. Hai yu t lp c s dng cho trong Rep-PCR. Cc yu t i
xng ngoi gen lp (Repetitive Extragenic Palindromic - REP), l cc trnh t di khong 38 bp
bao gm su v tr thoi ha v nt tht 5 bp thay i gia mi pha ca mt nhnh i xng
c bo tn. Cc trnh t REP c thy nhiu vi khun ng rut v c cho l c vai tr
iu ha trong cc vng khng dch m ca operon vi khun.
Cc trnh t chung ngoi gen lp ca vi khun ng rut (Enterobacterial Repetitive
Intergenic Consensus - ERIC) l loi yu t lp th hai c v c s dng thnh cng trong
nh typ bng ADN. Cc trnh t ERIC gm cc yu t di 126 bp cha mt on lp nghch o
tm bo tn cao v cng hin din trong cc vng khng dch m ca nhim sc th. Cc trnh
t ERIC khng c s tng ng no vi REP. Chng c m t da trn cc trnh t thu c
t E. coli v Salmonella typhimunium.
Ngoi cc trnh t REP v ERIC c s dng nhiu nht trong nh typ bng ADN, cn
c mt yu t lp khc, trnh t BOX, cng c dng phn bit cc Streptococcus
pneumoniae. Yu t BOX nm trong cc vng xen gia cc gen v cng c th to cu trc nt
tht do tnh cht i xng cp. Chng l cc yu t lp khm cu to gm cc phi hp khc
nhau ca ba n v trnh t boxA, boxB, v boxC. Ba n v ny c chiu di tng ng l 59,
45, v 50 nucleotid. Yu t BOX khng c quan h trnh t vi c REP ln ERIC. Yu t BOX
c tm thy nhiu vi khun.

42
Cc mi REP ban u c thit k t nhnh bn phi v tri ca trnh t i xng bo tn
v hng nghch nhau khuch i hng ra theo chiu 3 tnh t tm ca vng i xng. Theo
cch ny, cc sn phm khuch i l trnh t nm gia cc vng i xng (kch thc 200 bp
n 4 kb). Trong nhiu trng hp ca mi lp, ngi ta dng insosine lm tng tnh thoi
bin ca mi v nhy ca phn ng. iu ny dn n s bng to ra ln hn v do tng
kh nng phn bit chnh xc. Nhiu phn ng PCR mi lp to ra du n c tnh c hiu chng
cao ch vi mt mi n nh BOXA1, GTG5 v MBO1. Mi v cc thng s hot ng c
ghi trong bng 1.
Rep-PCR c th c thc hin vi ADN c chit t khun lc hay chit th t t bo.
Phn ng khuch i REP hay ERIC c th c thc hin vi mi n, hay mt b cc mi
n, hay hay nhiu b mi n. Thng thng kt qu khuch i vi mi ERIC t phc tp hn
so vi mi REP, nhng c hai u c kh nng phn bit tt n mc chng. Vic p dng c hai
mi cho cng mt mu lm tng kh nng phn bit cao hn cc phng php khc s dng c
lp.

Hnh 4.1. Rep-PCR trn loi Bacillus sphaericus
Rep-PCR nhanh chng tr thnh phng php c s dng rng ri nht nh typ bng
ADN. Rep-PCR vi on mi REP v ERIC c s dng thnh cng phn bit cc chng
Bartonella, Bacillus subtilis, Citrobacter diversus. Enterobacter aerogenes, Rhizobium meliloti,
Staphylococcus aureus khng methicillin, Streptococcus pneumoniae, Acinetobacter baumannii,
Burkholderia cepacia, Legionella pneumophilia, Helicobacter pylori, Neisseria gononnhoeae v
N. meningitidis. K thut ny d thc hin v c th p dng trn s lng mu phn lp nh hay
rt ln. Rep-PCR th hin kh nng p dng rng ri trn cc loi khc nhau hn v c nng lc
phn bit cao hn cc phng php du n b gen hay profile plasmid. Rep-PCR cng c nng
lc phn bit cao hn k thut phn tch gii hn gen 16S rRNA hay vng xen gia 16S v 23S.
Hn na, cc nghin cu so snh Rep-PCR vi cc phng php khc nh in di enzym a
locus, sinh ha, hay nh typ ribosom u cho thy Rep-PCR u vit hn. Cui cng, mt s
nghin cu chng minh Rep-PCR c tnh tng quan tt vi kt qu PFGE nhng ni chung
c nng lc phn bit hi thp hn.
Rep-PCR cng c thit k t ng ha vi mi c nh du hunh quang v phn
tch vi my nh trnh t kiu hunh quang. Phng php ny cho kt qu n nh v ng thi
kt qu cng c s ha nn vic phn tch sau o d dng hn. Cc chng cha bit c th
c so snh vi c s d liu s ha nhm mc ch nh danh. Nh vy, cng nh PFGE,
Rep-PCR phn no c chun ha v cho php so snh kt qu gia cc phng th nghim
khc nhau.


43
4.2.4. Th nghim RAPD
Th nghim ADN a hnh khuch i ngu nhin (Random Amplified Polymorphic DNA -
RAPD), cn gi l PCR vi mi ty tin. RAPD da vo vic s dng cc on mi ngn c
trnh t ngu nhin di 8-10 nucleotid ca th lai ha vi i lc vi ADN nhim sc th
nhit thp v do c th khi u cho vic khuch i cc vng ADN ca b gen vi khun.
Nu hai mi RAPD bt cp cch nhau vi kilobase theo hng thch hp, vng ADN gia chng
s c khuch i thnh sn phm PCR vi di tng ng. S lng v v tr ca cc im
bt cp ny khc nhau gia cc chng trong cng mt loi, do sn phm khuch i khi c
phn tch trn gel agarose s c s a hnh c trng cho mi chng. Ni cch khc, cc chng
ch c mt vi nucleotid khc bit c phn bit da vo s hin din hay khng ca mt s sn
phm khuch i. Ngha l do c s d bit v di truyn, s lng v kch thc ca sn phm s
khc nhau gia cc chng.
RAPD c s dng lm cng c lp bn di truyn ca thc vt v lm c s hiu s
phn ha ca cc chng nm gy bnh. RAPD-PCR c nhiu trin vng phn bit v di truyn
cc chng vi khun v c s dng phn bit cc vi khun Gram m cng nh Gram
dng. V k thut ny kh nhy, chng c bit hu dng lm du n cc loi khi nghin cu
v dch t hc trong cc cn dch trong bnh hay bnh ly qua thc n. Tuy nhin, mt iu bt
tin ca k thut l tnh tin cy v lp li khng cao. Cc yu t nh hng n lp li ca
RAPD bao gm: cht lng v nng ADN, loi Taq polymerase c dng v nng
MgCl
2
. Vic chn la ha cht v thit k th nghim phi ht sc cn thn. c bit, ADN
khun mu phi t tinh khit cao nht c th.
Mi ca RAPD-PCR thng di 8-10 bp v khng mang trnh t ca bt k trnh t c bn
no trn nhim sc th vi khun. Hu ht on mi c thit k giu GC (70-100%), v do kch
thc nh nn thng c nhit bt mi rt thp. Mi RAPD c xu hng hot ng hiu qu
ty theo loi, do nhiu loi mi khc nhau s c th trn cng mt khun mu ADN xc
nh mi thch hp nht. Mi OPA-1 ni chung hot ng tt trn vi khun, trong khi cc mi
Bc thng dng cho Bacillus v cc vi khun Gram dng khc. Gn y, mi l cc trnh t
bo lu ca phage M13 c s dng lm mi cho RAPD, iu ny cho php chun ha mt
s kha cnh ca th nghim.
Nhiu nghin cu p dng RAPD thnh cng phn bit cc chng ca loi nh
Acinetobacter baumannii, Candida albicans, Helicobacter pylori, Proteus minabilis,
Histoplasma capsulatum, Haemophilus somnus, Leptospira, Staphylococcus aureus v
Legionella pneumophilia.

Hnh 4.2. RAPD trn Bacillus sphaericus khuch i bng mi AP21

44
Khi so snh vi cc phng php khc, RAPD ca kh nng phn gii tt hn RFLP ca
16S rARN hay vng xen gia 16S v 23S rARN nhng km hn Rep-PCR. Hn na, nhiu vn
c ghi nhn vi RAPD nh lp li km v khng c tiu chun ha. V cc on mi
khng tng ng vi bt k locus di truyn no nn s bt cp xy ra khng hon ho v do
qu trnh khuch i b nh hng mnh bi s thay i d nh ca nhit gn mi. Cc on
mi c thit k theo kinh nghim v c iu kin hot ng v ha cht ring lm cho vic
chun ha gp nhiu kh khn.
4.3. Cc phng php ct gii hn b gen
Phn tch bng ct gii hn ADN nhim sc th hay phn tch s a hnh chiu di cc
on ct gii hn (RFLP) v s dng on d khng ng ng c p dng trn cc allen
bin thin nhim sc th ngi, v d phn bit cc c th vi qun th. Gn y hn, vic
ct gii hn nhim sc th vi khun l mt phng php nhy cm v c lp li cao pht
hin cc bin d trong trnh t ADN v phn bit cc chng vi khun. K thut ny da trn
tin l ADN ca hai chng vi khun khc nhau s c trnh t nucleotid khc nhau, do vy khi
b ct vi cng enzym s to cc hn hp mnh ct khc nhau. Do nhim sc th vi khun ch
bng 1/1000 kch thc nhim sc th ngi, nn c th phn tch gii hn ton b nhim sc th
trong mt ln v phn tch bng in di m khng cn nh du bng phng x hay enzym vi
cc on d c hiu cho cc vng c hiu trn b gen. Phng php ny rt nhy cm vi cc
bin ng v trnh t v c th xc nh cc dng ring bit ca vi khun.
4.3.1. Cc vn chung
Vi cc nhim sc th n gin nh ca vi khun, RFLP c th thc hin vi vic phn
tch kt qu trc tip t gel m khng cn thng qua bc lai phn t hay s dng on d. iu
ny trnh cho phng php nhiu im bt li thng gp khi lai phn t. u tin, n loi b
nhu cu to dng v xc nh cc trnh t ADN khi cc on d c mc bin thin cao c s
dng. Th hai, s hin din ca cc ADN lp khng gy ra kh khn no v on d khng c
s dng. Th ba, vic khng s dng on d lm cho th nghim c tnh lp li cao gia cc
phng th nghim khc nhau. V cui cng, gi thnh ca th nghim c gim i nhiu ln
khng khng dng on d.
Mt s vi khun c cc yu t di truyn nm ngoi nhim sc th nh plasmid hay thc
khun. Cc chng ny thng th hin s khc bit trn kt qu RFLP khi so vi cc chng
tng t nhng khng c yu t di truyn ngoi nhim sc th. Cc bng khc bit ny thng
khng c m nh l ca nhim sc th, do t l v s mol khc nhau. Trong trng hp ny,
cc chng c mang ADN ngoi nhim sc th cn c kho st s hin din ca cc plasmid
hay virus bt thng.
4.3.1.1. Chun b ADN b gen phn tch
Cht lng ADN dng phn tch vi enzym ct gii hn thng cao hn nhiu so vi
cc th nghim du n nhanh hay PCR khc. Hin nay c nhiu kit thng mi tinh ch ADN
sch dng cho RFLP. Ni chung cc phng php ny da trn vic loi mui, sc k ct
hay chit bng dung mi hu c.
4.3.1.2. Enzyme
Vic quyt nh chn enzym no phn tch l mt vn mang tnh thc nghim. C
nhiu yu t cn xem xt khi chn enzym: chng cn phn tch, kch thc nhim sc th, t l
GC v gi c enzym. S khc bit v gi gia cc enzym rt ln, thng thng cc enzym r
cng to c kt qu ti u.
Cc chng trong h Pseudomonadaceae, vi cc bin th c sc t hunh quang hay khng
nh Pseudomonas, Aeromonas, v Xanthomonas, c t l GC cao th HaeIII v MspI cho kt qu


45
tt. C hai enzym ny nhn din cc motif GC c hiu di 4 nucleotid. Ngc li, h
Enterobacteriaceae, gm a s cc chng gy bnh trong bnh vin hay qua thc n, c nhiu
loi v chng c b gen ln hn nhng t l GC thp hn (50 -55%), th enzym HinfI, nhn din
GANTC, cho kt qu tt trong nhiu trng hp. Mt s trng hp cn dng phi hp nhiu
enzym mi phn bit c v khi cn lu chn cc enzym c m hot ng tng thch
nhau.
Phn ng ct sau khi hon thnh thng c tip theo bi bc chit tch vi dung mi
hu c lm ngng phn ng ng thi cho php bo qun hn hp sn phm lu di trc khi
phn tch trn gel acrylamide.
4.3.1.3. in di gel polyacrylamide (PAGE)
Gel polyacrylamide (5%) thng c dng phn tch sn phm ct. Polyacrylamide c
nhiu u im so vi agarose. u tin, lng ADN c th np ln hn do cc bng d nhn
thy hn. Th hai, vic nhum gel polyacrylamide c mc tng phn cao hn nn d phn tch.
V cui cui cng, nng lc phn gii ca PAGE vt xa gel agarose. PAGE gel c th phn gii
s khc bit n tng nucleotid, trong khi gel agarose khng th lm c. Khong phn gii ti
u ca PAGE gel l 40 - 4000 bp. V y cng l kch thc ca hu ht cc mnh ct nn
PAGE l s lu chn thch hp nht phn tch RFLP.
Vic nhum gel c th tin hnh n gin vi ethidium bromide, hay nhum hunh quang
vi kit Gelstar (FMC Bioproducts) c nhy cao hn. ny cao nht c th t c vi
phng php nhum bc. K thut nhum bc kh phc tp, tuy nhin, hin cc kit thng mi
gip cng vic tr nn d dng hn.
4.3.1.4. Phn tch v bin gii d liu
Cng ging nh d liu du n PCR, cc mnh ct cng c ghi nhn theo kch thc v
s hin din cc v tr c th. V cc thang ADN chun 100 bp, 1 kb cng cn nhn nh
chnh xc kch thc cc mnh. Cc mnh ct gii hn thng khng c mc a hnh cao nh
Rep-PCR, nhiu chng trong cng mt loi c kiu ct. V l do ny, cc enzym hay phi hp
enzym khc nhau cn c kho st t c mc phn bit mong mun.
Cng nh trn, d liu nh phn cng c dng phn tch s phn nhnh hay pht sinh
loi. Mc d vic phn tch RFLP tn kh nhiu cng sc nhng kt qu c lp li cao v
c ng dng trong nhiu nghin cu.
4.3.2. Phng php RFLP kt hp Southern blot
Southern blot c s dng t rt lu pht hin v nh v cc trnh t ca b gen
trn cc sinh vt nhn nguyn thy v nhn tht. pht hin gen, ton b b nhim sc th ca
loi quan tm c ct bng cc enzym gii hn, tip theo chng c phn tch trn gel
agarose. Nhng mnh DNA c phn tch ny sau c chuyn t gel agarose sang mt
mng nylon hoc nitrocellulose bng k thut Southern blot. Cc acid nucleic trn mng s c
lai vi mt hay nhiu on d c nh du tng ng vi on gen cn kim tra. Cc on d c
th c nh du bng nhiu cch khc nhau nh c cht cho phn ng enzyme o mu hoc
enzyme pht quang ho hc. Phng php ny thc hin phn bit cc chng vi khun da
trn c s v tr nhn din ct ca enzym gii hn trong cc locus di truyn c tnh a hnh gia
cc chng, do kt qu ct s to c cc bng c kch thc khc nhau gia nhng chng
khc nhau. Tnh cht a hnh v v tr ct gii hn trong cc on m di truyn xc nh c gi
l s a hnh chiu di on ct gii hn (rectriction fragment length polymhayphism - RFLP).
Ch c nhng on DNA no c lai vi on d th mi c th nhn thy bng RFLP, do
vic phn tch c n gin ha rt nhiu.
Cc mi c hiu gen c s dng c hiu qu nh typ cc loi Brucella,
Legionella pneumophila v P. aeruginosa. Hn na, nh typ ribosom, mt bin th ca RFLP-

46
Southern blot trong on d xut pht t cc gen 16S v 23S rARN, cng c p dng thnh
cng trong nhiu nghin cu phn bit chng vi khun. nh typ ribosom cho kt qu vi t
bng, nn vic phn tch n gin hn, tuy nhin, iu ny cng gii hn kh nng phn bit cc
chng c quan h gn.

Hnh 4.3. PCR-Southern Blot trn 9 chng Trichophyton rubrum sau khi ct bng
enzym EcoRI vi cc mu d nh du thch hp
Southern blot c th c s dng vi nhiu on d cng lc cho nhiu gen kh nng
nh typ vi khun. V d Grundmann v cng s dng phi hp on d ca gen toxA vi
on d 16S v 23S nh typ cc mu phn lp ca P. aeruginosa. Phi hp ny cho php thu
c cc thng tin v quan h tin ha gia cc chng c kho st, nhng n khng t hiu
qu phn bit cc chng cao nh PFGE. Trong khi RFLP-Southern blot vn hu ch trong mt
s nghin cu nh typ chng, do nhng phc tp v k thut nn hin nay a s chng c
thay th bi phng php PCR-RFLP (RFLP c hiu locus da trn PCR).
4.3.3. PCR-RFLP
Vi PCR cc locus di truyn c th c th c khuch i d dng v phn tch cc du
hiu khc nhau ca cc chng hay s hin din ca gen khng khng sinh. Cc locus ny
c khuch i vi cc mi c hiu ri c phn tch RFLP. Cc mnh ADN sau c
phn tch trn gel agarose hay gel polyacrylamide nh, kiu RFLP c hin th bng cch
nhum vi ethidium bromide.
PCR-RFLP c p dng trong nhiu trng hp. Shhaytridge v cng s s dng k
thut ny trn gen ureC chng minh s da dng di truyn ca cc chng Helicobacter pylori.
Cc gen 16S, 23S v vng xen gia 16S v 23S cng c dng. Trong bin th ny ca k
thut nh typ ribosom, ADN ribosom c khuch i v ct vi enzym gh, cc mnh c
phn tch trn gel v nhn kt qu bng cch nhum ethidium bromide, do khng cn n k
thut Southern blot. nh typ ribosom c p dng thnh cng phn bit cc chng vi khun
c mc d bit ln trong operon rARN.
Cockerill v cng s p dng PCR-RFLP pht hin t bin im trn gen katG ca
Mycobacterium tuberculosis, c lin quan n mc khng vi isoniazid. Tuy nhin, nng
lc phn bit ca PCR-RFLP ni chung khng tt nh cc phng php khc, ch yu do vng
gen m n kho st b gii hn.
PCR-RFLP c nhiu p dng quan trng trong cc nghin cu dch t hc ca virus vim
C (HCV). HCV c chia lm su kiu gen chnh c nh s 1 n 6. Davidson v cng s
pht trin mt phng php n gin xc nh kiu gen ca HCV da trn PCR-RFLP ca
vng 5 khng dch m ca b gen HCV. Vic ct gii hn c thc hin vi enzym ring l
hay phi hp ca HaeIII-RsaI, MvaI-HinfI, BstI v ScrFI cho php phn bit cc m phn lp
virus thnh cc nhm 1a, 1b, 2a, 2b, 3a, 3b, 4, 5 v 6. Phn tch RFLP ca vng 5 khng dch
m gip ch rt nhiu trong cc nghin cu v phn b a l ca kiu gen virus, lch s t
nhin ca bnh, v s tng quan gia kiu gen v p ng vi interferon.


47

Hnh 4.4. AFLP sau khi khuch i gen EcoA and MseC trn Acinetobacter
4.3.4. AFLP
S a hnh di on khuch i (Amplified Fragment Length Polymorphism - AFLP) l
mt k thut du n b gen da trn s khuch i chn lc mt tp con cc on ADN pht sinh
bi vic ct gii hn b gen. Ban u n c p dng kho st b gen thc vt, nhng nay
AFLP c p dng trong nh typ vi khun. C hai bin th ca AFLP, mt c thc hin
vi hai enzym ct gii hn khc nhau v dng hai on mi khuch i. Bin th th hai ch
s dng mt on mi v mt enzym gii hn. Cch hay c s dng nht l ADN vi khun
c chit tch v ct vi hai enzym v d nh EcoRI v MseI. Cc mu ct sau c ni vi
mt on ni cha v tr nhn din ct v mt trnh t tng ng vi v tr gn on mi. Mi
PCR c s dng khuch i mang trnh t ADN tng ng vi on ni v mang mt hay
hai base chn lc u 3. V d, on mi chn lc i vi v tr EcoRI c th c trnh t nh
sau 5-GAATTCAA-3, trong su base u tin b sung vi trnh t nhn din ca EcoRI v
hai Adenosin u 3 dng chn lc v n ch cho php khuch i cc v tr nhn din EcoRI
vi trnh t 3-CTTAAGTT-5. Chng s khng khuch i v tr EcoRI c trnh t 3-
CTTAAGTC-5, nh vy, cc nucleotid chn lc ch cho php khuch i mt tp hp con ca
cc on ct gii hn. nhn thy kt qu, mt trong cc on mi c th c nh du phng
x hay hunh quang hoc gel c nhum vi ethidium bromide.
Nhiu nghin cu cho thy AFLP c tnh lp li cao v c nng lc phn bit cc chng
tt. Nng lc phn bit ca AFLP c v cao hn cc phng php nh typ ribosom da trn
PCR, tuy nhin n cha snh c vi Rep-PCR hay PFGE. Cch thc hin AFLP tn nhiu
cng sc hn Rep-PCR, nhng nhanh hn PFGE. Cch lm vi nh du phng x khng thc
tin trong lm sng, nhng vi cch nh du hunh quang v phn tch kt qu trn my nh
trnh t th c th nhiu phng th nghim cha trang b. Nhiu kit thng mi s dng
phng php pht hin bng bng enzym, tuy nhin, n l i hi phi chuyn cc on ADN
ln mng nn do li mt nhiu thi gian v cng sc hn. D sao, lp li cao, kh nng so

48
snh kt qu vi ngn hng d liu v kh nng phn bit cao nn phng php ny cng c
s dng nhiu cc phng th nghim c trang b tng i mnh.
4.3.5. CFLP
K thut a hnh di on ct bng cleavase (cleavase fragment length polymorphism -
CFLP) da vo c im hot ng ca Cleavase I, l mt endonuclease ti t hp bn nhit bao
gm nhm chc nng 5-nuclease (amino acid 1 n 281) ca DNA polymerase ca Thermus
aquaticus. Trong khi cc enzym gii hn nhn din cc trnh t ADN c hiu th cleavase I
nhn din cc cu trc c hnh thnh bi cc cp base nhit cao. ADN c bin tnh
bng cch un ln 95
o
C, sau khi ngui xung khong 50-60
o
C, ADN si n bt u tin
trnh t bt cp ti to si ADN b sung, trong qu trnh ny cc cu trc kp tc c trng
vi mi si c hnh thnh trc khi bt cp vi ADN b sung. Qu trnh ny to ra mt lot
cc cu trc nt tht c ni vi nhau bi cc vng si n ko bt cp. Cleavase I nhn din
cc cu trc nt tht v ct ADN u 5 ca cp base u tin vng tip gip si n v si
i. V l thuyt, trong bt k dung dch ADN no, cc si n b bin tnh cng mang mt hn
hp tt c cc cu hnh c th c ca si n. thc hin phn ng cleavase, mt sn phm
PCR c nh du mt u s c ct khng hon ton sao cho tt c cc sn phm c th
c u hin din trong hn hp phn ng. Hn hp thu c tng t nh phn ng nh trnh t
dideoxy trong cha mt tp hp cc ADN si n ct ngu nhin c nh du vi chiu di t
u b nh du n v tr ct.
S khc nhau trong du n cu trc gia cc sn phm PCR khng ging nh xut hin
nh s c hay mt mt bng hay s dch chuyn mt bng hay thay i m ca bng nht
nh. S xut hin hay mt hay dch chuyn c th c pht hin d dng, trong khi s thay i
v mt bng rt kh nhn ra v d gy sai st.
Nng lc phn gii ca k thut a hnh di on ct bng cleavase ph thuc vo trnh
t ADN c phn tch. V d, nu s dng cleavase ct 16S rDNA s thu c nhiu bng,
trong khi rt t sn phm c to thnh khi phn tch gen rpoB ca M. tuberculosis. Hn na,
mi si ADN c th c mc phn bit khc nhau khi ct. V d, nu s khc nhau v trnh t
nucleotid ca vng xen gia cc gen ca cc typ huyt thanh 1, 2 v 8 ca Shigella dysenteriae
c th hin trong du n cu trc ca si phin m, du n thu c t si khng phin m
khng th phn bit c cc chng ny. Khi so snh kh nng phn bit ca CFLP v k thut
phn tch tnh a hnh cu dng si n (single-strand conformation polymorphism) trong pht
hin t bin im, k thut sau pht hin c 6 trng hp c t bin, trong khi CFLP ch pht
hin c ba. Do , kh nng p dng ca CFLP i vi mt ADN no khng th d on
trc m phi xc nh mt cch thc nghim. Mt s phn t DNA cho kt qu xut sc v
phng din phn bit, trong khi mt s khc li khng cho kt qu no, m nguyn nhn th
khng xc nh c. CFLP cng b gii hn v kch thc, ngha l, s khc nhau v du n cu
trc ca mnh ADN ln hn 1 kb khng th phn bit c bng in di.
CFLP c p dng phn bit kiu gen ca cc mu phn lp HCV. Tuy nhin, trong
khi kt qu c lp li rt tt trong cng mt phng th nghim th lp li gia cc phng th
nghim rt km.
Cc vn nh phi xc nh tnh kh dng bng thc nghim, s kh khn trong bin gii
kt qu, s phc tp ca k thut, llapj li gia cc phng th nghim km d hn ch vic
p dng k thut ny trong thc tin. Bn cnh kh nng phn bit ca n so vi cc phng
php khc cng cha c chng minh.
4.3.6. Phn tch PFGE nhim sc th
in di trng xung (Pulsed-field gel electrophhayesis - PFGE) thng c coi l tiu
chun vng ca cc phng php nh typ phn t. thc hin PFGE, cc mu phn lp vi
khun c nui trong canh thang hay thch c kt hp vi agarose un chy ri vo


49
cc khun nh cho ng li. Khi agarose ng li thu c nt thch cha vi khun. Vi khun
ny s c ly gii in situ bng enzym-x phng, v sau ADN phng thch ra c ct vi cc
enzym him. Nt thch sau khi x l c nhi vo bn thch agarose vo c in di trong
thit b in di trng xung. Trng xung cho php tch cc mnh ADN c kch thc rt ln
trong khong 10 n 800 kb. Kt qu in di c pht hin bng cch nhum gel vi mt thuc
nhum hunh quang nh ethidium bromide v chp hnh. D liu c lu tr v phn tch bng
mt trong cc phn mm ca Applied Math, Bio-Rad, BioSystematics, Media Cybernetics, hay
Scanalytics.
4.3.6.1. Cc vn chung
Mt s yu t ni ti hay khch quan c th lm thay i phn gii ca PFGE. S bin
thin ca tnh cht di chuyn ca ADN ty theo nhit , gc ca in trng, thi gian gian ca
xung, v cu trc s cp, th cp ca ADN. Yu t ngoi lai quan trng nht l nng agarose.
Khi nng agarose tng ln, tnh di ng ca cc mnh ADN gim xung, nhng phn gii
li tng. Bn cnh kh nng phr cc ADN di gim xung khi nng agarose tng ln.
4.3.6.2. Cht lng ADN
Ging nh trong k thut RFLP, cht lng ADN cn thc hin PFGE phi cao. Cc
enzym him c s dng trong PFGE cn ADN nguyn vn v sch, v mi enzym ch ct
ADN nhim sc th ti vi im. Nu ADN b phn hy mt phn s dn n mt cc mnh
c kch thc ln hay to ra cc mnh nh gi to, lm cho vic bin gim kt qu b sai. K
thut chung c c ADN thch hp l nhi mu vo mt nt thch rn bo v ADN.
4.3.6.3. Cc enzym c s dng trong PFGE
Hu ht cc enzym s dng l cc enzym c v tr nhn din him gp, gi l enzym
him, ch ct nhim sc th ti vi im. c im him ny ph thuc vo di ca trnh t
nhn din, ni chung trnh t ny cng di th xc sut xut hin cng thp. Mt s th d nh
BlnI, SpeI, SpeI, SwaI, XbaI, v XhoI c dng thnh cng ct nhim sc th ca cc vi
khun ng rut v Pseudomonas. Mt s enzym khc nh DraI, NaeI, NotI, SfiI, v SmaI. S
lng chnh xc v tr ct c nhn din bi cc enzym ny ph thuc vo loi hay chng c
phn tch, nhng khng c enzym no hon ho cho tt c cc loi. Mt s nh nghin cu
ghi nhn cc kt qu khng ti u cho mt s kt hp enzyme-nhim sc th nh ch ct mt
phn, mnh ct qu nh, bng b khuch tn. Do , cn xc nh enzym mt cch thc nghim
cho mi loi cn kho st.
4.3.6.4. iu kin in di
iu kin in di c th ty thuc vo h thng c s dng, kch thc ca mnh cn
phn tch v kch thc ca gel. Ni chung, in trng khong 5-6 V/cm trong 16-22 gi
nhit 15
o
C c ghi nhn l tt. Hn na, thi gian gia cc xung c lin quan n cht
lng phn gii cc mnh c di nht nh. V d cc mnh t 8-100 kb c phn gii tt
ch xung 1-5 giy, ADN 50-240 kb nn chy ch 1-20 giy v cc mnh 200-800 kb
chy tt nht 40-80 giy.
4.3.6.5. Phn tch v bin gii
Cng nh cc k thut phn tch gii hn khc, PFGE cng ghi nhn kt qu da vo kch
thc bng v s hin din hay khng ca mt s bng nht nh. Tuy nhin, thang ADN s
dng PFGE phi ln hn nhiu v thng c nhi sn vo nt thch. V d phc bi ca
DNA thc khun (c c 48,5 kb n 1 Mb vi cc bc tng 48,5 kb) v cc nhim sc th ca
Saccharomyces cerevisiae (225 kb n 2,2 Mb). Cng nh RFLP, PFGE c mc a hnh thp
hn cc phng php du n PCR. Nu a hnh khng t yu cu, cn phi thc nghim vi
cc phi hp enzym khc nhau n khi tm c enzym ti u.

50
Tenover v cng s xut mt h thng chun ha vic bin gii kt qu PFGE vi
s quan h gia cc chng. Theo h thng ny, cc mu phn lp vi khun c cng kiu PFGE
(pulsotyp) c coi nh cng mt chng. Cc mu phn lp khc nhau ch mt im v di
truyn, ngha l c s khc bit t mt n ba bng, c coi l c quan h gn. Cc mu c s
khc bit bn n su bng, th hin hai s bin i v di truyn c lp, c th c quan h. Cc
mu c khc bit t su bng tr ln, hay c t ba bin i v di truyn tr ln, c coi l
khng c quan h. Cc tiu chun ny kh thi i vi cc nghin cu nh, c tnh cc b trong
s a dng v di truyn c gi nh l thp.

Hnh 4.5. PFGE ca loi Shigella flexneri
n nay c nhiu nghin cu c thc hin xc nh cc kiu bng ca nhiu vi
khun khc nhau c ct vi enzym khc nhau. PFGE chng t nhiu u im so vi cc
phng php nh typ ha sinh hay phn t khc. N c nng lc phn gii cao v c u th hn
cc phng php khc trong phn tch cc chng Escherichia coli, cc enterococci khng
vancomycin, S. aureus, Acinetobacter, Pseudomonas aeruginosa, v M. avium. PFGE c hiu
qu cao hn cc phng php khc trong vic phn bit cc chng S. aureus khng methicillin.
PFGE cng c nng lc phn gii cao hn Rep-PCR khi phn tch cc chng trong phc hp
Acinetobacter calcoaceticus-Acinetobacter baumannii, enterococci khng vancomycin,
Neisseria gonorrhoeae, v P. aeruginosa.
Vi s h tr ca cc phn mm qut gel, ngi ta c th xy dng ngn hng mu PFGE
cho tt c cc loi vi sinh vt, v dng lm d liu tham chiu cho cc chng mi.
Mt trong cc yu t hn ch ca PFGE l thi gian phn tch qu di, mt n 2 - 3 ngy,
do hn ch kh nng phn tch mt lng mu ln.
4.4. nh trnh t DNA
Ni cho cng, cc phng php di truyn phn t phn bit cc typ vi sinh vt u da
trn s khc nhau v trnh t ADN, do vy phng php nh trnh t v l thuyt l cch trit
nht. Tuy nhin, thc t khng phi nh vy.
C nhiu vn cn xem xt trc khi p dng nh trnh t trong vic nh typ. u tin,
v do yu cu v tnh kh thi trong cc phng th nghim, vic nh trnh t ADN ch c thc
hin trn mt on ADN ngn, vic nh trnh t ca nhiu gen hay mt vng ln ca nhim sc
th l khng thc tin. Do , ngc li vi cc k thut nh PFGE, Rep-PCR, hay RAPD, trong
ton b nhim sc th c s dng phn tch, nh trnh t ADN ch kho st mt phn
rt nh cc v tr c tim nng khc bit gia cc chng. Nn kh nng c s a hnh phn bit
thp hn.
Hn na, vng ADN c chn nh trnh t phi p ng mt s yu cu. V cu trc
n phi cha vng trnh t bin thin cao nm gia cc vng trnh t c tnh bo tn cao, ndf cn


51
thit thit k mi c kh nng khuch i cho tt c cc chng trong loi hay cho nhiu loi
khc nhau. Vng bin thin trong trnh t phi phn bit cc chng trong tp hp cn kho
st. Ngoi ra, trnh t c chn phi khng di chuyn d dng sang cc chng khc trong loi.
Trn thc t i vi vi khun v vi nm rt t trnh t p ng c cc yu cu trn. Nu
gen 16S rRNA c dng xc nh loi mi th n li km bin thin c th dng phn
bit cc chng trong loi. Vng xen gia gen 16S v 23S rRNA cng c s dng vi cc kt
qu khng nht qun. V c bn cc locus c s dng nh trnh t c xc nh mt cch
thc nghim.
Cc gen m ha cho protein mng ngoi c dng phn bit vi khun n mc loi
hay di loi. V d, gen ompA, m ha cho protein mng ngoi 3A ca vi khun ng rut,
c s dng phn bit cc nhnh c quan h gn trong cc chi vi khun gy bnh nh
Escherichia, Vibrio, v Chlamydia. Cc protein ny nm b mt t bo v ng nhiu vai tr
quan trng t n nh cu trc mng vi khun n vn chuyn xuyn mng. Tc tin ha
nhanh ca cc gen ny tr thnh th mnh khi xc nh kiu gen ca cc vi khun gy bnh.
Ngc li, vic nh trnh t ADN li c coi l tiu chun vng trong vic nh typ
virus. nh trnh t acid nucleic c nng lc phn gii cao hn khi dng nh typ HCV v l
phng php phn t duy nht xc nh chnh xc cc t bin lin quan n khng thuc
HIV.
Ngoi ra, vic bin gii kt qu nh trnh t phi ht sc cn thn. Cn phi lu cc yu
t gi to lm cho vic c kt qu tr nn rc ri v d mc li, v d nh mt nucleotid b lp
trong trnh t c th dn n bng b dn li v c c nh mt nucleotid n v ko theo vic
c sai phn trnh t cn li. Tt nht c hai si ADN nn c nh trnh t v so snh vi nhau
gim thiu sai st.
Mc d phn tch trnh t tn km hn cc phng php dng enzym, kt qu ca n c
nhiu u th hn cc phng php du n khc. Trc ht, v tt c cc k t di truyn u c
xem xt nn mc a hnh rt cao. iu ny lm cho vic xc nh cc chng chnh xc v tin
cy hn nhiu. Th hai, trong trng hp cc chng ng nht v kiu hnh v di truyn th vic
phn tch trnh t nucleotid pht hin cc bin d phn bit kh nng tn ti ca chng
trong t nhin. Cui cng, kiu gen phn tch t d liu trnh t r rng v khng cn n cc
bin php th cp nh kch thc c kt lun. iu ny khng phi lc no cng t c vi
cc phng php du n v hai bng c cng kch thc cha chc xut pht t cng v tr trn
nhim sc th.
Tuy nhin, trong thc tin ng dng, phng php nh trnh t ADN rt tn km v phc
tp v k thut. Hn na vic u t thit b ban u c th ln n hng trm nghn USD cng
lm vic trin khai phng php tr nn kh khn i vi nhiu phng th nghim.
4.5. Cc kha cnh thc tin ca vic nh typ bng
phng php phn t
Vic chn la mt phng php c th thc hin da vo nng lc phn bit cn t,
lp li, mc phc tp ca k thut v cch bin gii kt qu, chi ph trin khai, k nng ca
ngi thc hin v tnh trng trang b ca phng th nghim. Cc bng tng hp bn di cung
cp cc so snh v tng kt thc tin gip lm cn c quyt nh vic chn la phng php.
Bng 4.2. Tm tt c im cc phng php phn t
Phng php S dng
Phn
tch kt
qu
Kh
nng
phn
Thi
gian thu
kt qu
lp
li trong
1 PTN
lp
li gia
cc PTN
Chi ph
u t
ban u
Chi ph
cho mt
ln th

52
bit (ngy)
PFGE
Bnh
thng
D Cao 3 Tt Tt
Va
phi
Va phi
PCR-RFLP D D
Trung
bnh
1 Tt Tt
Va
phi
Thp
Rep-PCR D D Cao 1 Tt
Trung
bnh
Va
phi
Thp
RAPD D D Cao 1
Trung
bnh
Yu
Va
phi
Thp
CFLP
Bnh
thng
Bnh
thng
Trung
bnh
2 Tt Yu
Va
phi
Cao
AFLP
Bnh
thng
D Cao 2 Tt Tt Cao Va phi
Sequencing Kh
Bnh
thng
Cao 2 Tt Tt Cao Cao

Bng 4.3. Tm tt qui trnh v thng tin s dng ca phng php
Phng php PFGE Lc-RFLP Rep-PCR RAPD AFLP nh trnh t
Nhi VSV
vo thch
PCR PCR PCR RE PCR
Protease RE in di in di Gn linker in di
RE in di Nhum Nhum PCR Phn tch
in di Nhum Kt qu Kt qu in di Kt qu
Cc bc
tin hnh
Kt qu Kt qu Kt qu
Gi thnh $20 $14 $11 $11 $20 $40
phc tp Va D D D Va Kh
c kt qu D D D D D Va
phn gii Cao Va Cao Cao Cao Cao
Chi ph u t
(x1000 USD)
10-20 8-10 8-10 8-10 45-120 45-120

Ti liu tham kho:
1. Blanc, D.S., et al., Molecular typing methods and their discriminatory power. Clin Microbiol
Infect, 1998. 4(2): p. 61-63.
2. Olive, D.M. and P. Bean, Principles and applications of methods for DNA-based typing of
microbial organisms. J Clin Microbiol, 1999. 37(6): p. 1661-9.
3. Gil-Lamaignere, C., et al., Molecular typing for fungi--a critical review of the possibilities
and limitations of currently and future methods. Clin Microbiol Infect, 2003. 9(3): p. 172-85.




53
B B i i 5 5. . n ng g d d n ng g c c n ng g n ng gh h s si in nh h h h c c
t tr ro on ng g t t m m k ki i m m t th hu u c c m m i i

Mc tiu:
- Hiu c tm quan trng ca nh hng pht trin thuc mi da trn cng ngh sinh hc
- Bit c qui trnh tng qut sng lc thuc hiu nng cao da trn cng ngh sinh hc
- Trnh by c mt s phng php sinh hc phn t s dng trong tm kim thuc mi
5.1. Gii thiu
5.1.1. Qui trnh tm kim thuc mi
Theo truyn thng, thuc mi c pht hin bng cch tm kim ngu nhin t hng ngn
cht ha hc c ngun gc tng hp hay t thin nhin. Nghin cu mt hp cht ha hc s
dng lm dc phm l qu trnh lu di cn nhiu thi gian, chi ph, cng sc. Theo s liu ca
Hip hi cc nh sn xut dc phm ti M, t giai on nghin cu u tin n khi thuc lu
hnh trn th trng mt khong 11- 15 nm v tn 500 800 triu USD, ty theo mc ch tr
liu, loi thuc v cc nghin cu lm sng cn trin khai. Nhm p ng nhu cu cung cp
thuc phc v iu tr, cc cng ty dc u t ~ 20% doanh s cho lnh vc nghin cu v
pht trin (R&D) thuc mi. Tuy nhin, kt qu t c rt kh xc nh, gp nhiu tr ngi khi
nghin cu tc dng ca hot cht trn lm sng.
Cc giai on trong qui trnh tm kim v pht trin mt thuc mi:
- Pht hin nghin cu c bn:
o Tng hp v chit xut: l qu trnh xc nh hp cht mi c tim nng lm thay
i c ch ch h thng sinh hc
o Sng lc hot tnh sinh hc v th tc dng dc lc: nghin cu hot tnh dc lc
v kh nng tr liu

Xc nh mc tiu
nh gi mc tiu
Trin khai sng lc
Xc nh hot cht tim
nng, cht a vo giai on
th nghimlmsng
nh gi cht a vo giai
on th nghim lm sng
5

6

n

m
Xc nh mc tiu
nh gi mc tiu
Trin khai sng lc
Xc nh hot cht tim
nng, cht a vo giai on
th nghimlmsng
nh gi cht a vo giai
on th nghim lm sng
5

6

n

m


Hnh 5.1. Qu trnh pht hin thuc mi

54
- Th nghim tin lm sng
o Th nghim c tnh v tnh an ton: xc nh nguy c tim n i vi con ngi v
mi trng, th nghim trn ng vt, nui cy t bo,...
o Xy dng liu dng v n nh: nghin cu dng bo ch v liu s dng trn
ngi.
- Np h s nghin cu thuc mi
o H s xin th nghim trn ngi, M c gi l IND (Investigational New Drug)
o Th nghim lm sng giai on I
o Th nghim hot cht mi trn 20 80 ngi tnh nguyn khe mnh xc nh
kh nng dung np, tc dng dc l, hp thu, phn b, chuyn ha v thi tr
- Th nghim lm sng giai on II
o Th nghim trn 100 300 bnh nhn chn lc nh gi hiu qu iu tr v c
tnh cp tnh
- Th nghim lm sng giai on III
o Th nghim trn 1.000 5.000 bnh nhn nh gi li ch lm sng v tc dng
ph
- Xy dng qui trnh sn xut v qun l cht lng
o Xy dng qui trnh sn xut trn qui m ln nhm m bo n nh, ng
nht v cht lng
- nh gi sinh kh dng
o Tin hnh trn ngi tnh nguyn khe mnh vi mc ch so snh gia dng s
dng trong nghin cu v dng sn xut
o Np h s xin lu hnh thuc
o H s xin lu hnh thuc trn th trng, M c gi l NDA (New Drug
Application)
- Giai on IV
o Sau khi lu hnh, tip tc pht hin tc dng ph v c tnh trng din
5.1.2. Pht hin thuc mi bng phng php sng lc hiu nng
cao
Pht hin thuc mi l mt trong nhng giai on u tin ca qu trnh pht trin mt
dc phm. Thut ng sng lc hiu nng cao (HTS High Throughput Screening) cp n
phng php th nghim t ng cc cht c tim nng lm dc phm vi hiu sut > 10,000
cht / 1 tun. HTS l mt cng c hu hiu trong vic pht hin thuc mi do kh nng xc nh
mi lin quan gia hot tnh sinh hc v hp cht kho st mt cch nhanh chng, cc th
nghim c tin hnh ng thi. Cht nghin cu c th l cht c ch enzym, hay cnh tranh
v tr gn vi th th,...HTS gip rt ngn thi gian pht hin thuc mi do tng tc sng lc
cc th vin mu c th ln n hng trm ngn mu. t chnh xc v lp li, HTS
thng c thc hin bng h thng t ng hay robot. Mc ch ca giai on ny l tin hnh
th nghim trn 1 s lng ln cc cht tm kim kt qu sng lc dng tnh, gi l cc hot
cht tim nng (HIT) nhm xc nh cht c th tip tc c nghin cu tin lm sng (LEAD).
Sau khi la chn c LEAD, c th tng hp thm cc dn cht c cng thc ha hc tng t
to ra dn xut mi c kh nng c hot tnh dc lc. Trung bnh, sng lc khong 300.000
hp cht s thu c 200 300 hot cht tim nng v 1 2 cht c th nghim tin lm sng.


55
Nhng sng lc qui m ln hn, ~ 1.000.000 cht c th tin hnh trong vi thng tm ra
khong 5 cht dng trong th nghim. Cng vi s pht trin ca nhiu lnh vc cng ngh lin
quan nh k thut sinh hc phn t, cng ngh gen, ha tng hp c bit l tin sinh v tin ha,
tc sng lc tng t 10.000 cht / 1 nm trong nhng nm 1980 ln n > 100.000 cht
/ngy hin nay (uHTS ultra High Throughput Screening), cung cp mt lng ng k cc
LEAD. Nh vy, t nhng nm 1990, qu trnh tm kim thuc mi c thc hin theo mc
tiu xc nh, khng tin hnh mt cch ngu nhin nh trc y.
Hai yu t chnh nh hng n qu trnh sng lc hiu nng cao: cc th nghim sng lc
v thu thp mu (th vin mu).
- Th nghim sng lc: xc nh dng hot tnh sinh hc cn tm kim. Th nghim
khng ng s dn n kt qu khng chnh xc, tuy nhin th nghim ng cng c th
khng a n kt qu tt. a s sng lc hiu nng cao tin hnh da trn sng lc cc
phn ng sinh ha nh gi tng tc gia hp cht v protein ch. Cng vi thnh
cng trong vic xc nh c ch bnh l, thng tin v chc nng gen t d n b gen
ngi, c rt nhiu protein c chng minh l mc tiu tc dng ca qu trnh tr
liu. Th nghim sng lc c chia thnh 2 nhm chnh: th nghim khng ph thuc
t bo (cell-free) th nghim sinh ha v th nghim t bo (cell-based). Kt qu giai
on ny c th c s khc bit so vi th nghim trn sinh vt sng v gy kh khn
khi thit k th nghim lm sng.
- Th vin mu: mu c thu thp qua nhiu nm, t nhiu chng trnh sng lc, c
th t tng hp ha hc hay c ngun gc thin nhin. Cng ngy th vin mu cng
gia tng v s lng cng nh s a dng v cng thc, cu trc ha hc ca cc hp
cht .
- Hin nay, HTS c p dng trong sng lc th vin ha hc, gen, protein v peptid.
Bt k phng php sng lc no cng gm cc bc c bn sau:
- Xc nh mc tiu th nghim: c khong 500 mc tiu ang c dng trong th
nghim gm c th th mng t bo ch yu l protein G (chim 45%), enzym (28%),
hormon (11%), cha r (7%), knh ion (5%), th th nhn (2%) v ADN (2%)
- Thnh lp th vin sng lc: l bn mng gm cc ging nh cha hot cht dng
kh hay ng kh. S lng ging trn mi bn t 96 1.536, vic tng s lng nhng
gim th tch ging gip tng tc sng lc v gim chi ph.
- Thit k th nghim:
o Th nghim giai on 1: c lp li 2 ln ti 1 nng duy nht (~ 1- 10 mM i
vi cht ha hc)
o Th nghim giai on 2: nu th nghim giai on 1 cho kt qu dng tnh, tin
hnh nh lng, xc ng IC50
5.2. Pht trin thuc mi da trn ng dng cng ngh
sinh hc
Thuc ra i cng vi s xut hin ca con ngi. Tuy nhin ngnh dc phm hin i
mi ch bt u t cui th k 19 v thc s pht trin vo th k 20. Qu trnh pht trin thuc
c th chia thnh 5 giai on:
- 1820 1880: khm ph tc dng tr liu t sn phm thin nhin, sn phm ln men,
xut hin thuc h st, thuc an thn, thuc gim au.

56
- 1880 1930: tr liu th nghim v ha tr liu. S dng cht nhum mu hu c tng
hp pht hin vi sinh vt gy bnh, bt u sn xut thuc khng n bo, huyt
thanh, c t, vacxin.
- 1930 - 1960: gii thiu nhm thuc sulfa, khng sinh, khng histamin, vitamin,
corticosteroid, hormon sinh dc.
- 1960 1980: gii thiu cc thuc tc ng trn h tim mch nh thuc cao huyt p;
thuc tc ng trn h thn kinh trung ng nh thuc chng lo u, chng trm cm;
khng sinh bn tng hp penicillin, cephalosporin; thuc khng vim khng steroid.
- 1980 hin nay: protein tr liu, thuc khng ung th, khng virus. Tr liu t bo, tr
liu gen, cc thuc mi v xt nghim chn on da trn ADN ti t hp v khng th
n dng. S ra i ca genomic, proteomic v cng ngh sinh hc nano, iARN
(interference ARN), cht nh du sinh hc thc y qu trnh pht trin thuc giai
on cui th k 20 v th k 21.
Trong khong 10 nm nay, ngy cng c nhiu thuc ra i da trn cng ngh sinh hc.
Thnh tu ny t c do ng dng k thut sng lc hiu nng cao, chip sinh hc, ngi my
cng vi s pht trin cng ngh trong lnh vc genomic, proteomic v metabolomic.
Mt s k thut cng ngh sinh hc thc y qu trnh tm kim thuc mi:
- K thut omic: genomic, proteomic, metabolomic
- Microarray v chip sinh hc (biochip)
- Khng th n dng (Monoclonal antibody - MAb)
- Antisense ARN
- iARN (interference ARN)
- Cng ngh sinh hc ng dng k thut nano (Nanobiotechnology)
- nh du sinh hc (Biomarker)
- Tin sinh (Bioinformatic)
5.2.1. K thut genomic
Nh cp, vi kin thc v genomic hin nay, ~ 500 mc tiu tr liu c xc
nh. Trong tng lai, genomic m ra c hi iu tr cho hn 100 bnh quan trng c lin quan
n 500 1.000 gen gy bnh v 3.000 10.000 thuc mi. Genomic gip phn tch chc nng
cc gen bng phng php hiu nng cao kt hp vi phn tch thng k v x l d liu trn
my tnh. nh hng ca k thut genomic l t nghin cu trn tng gen hay protein ring l
n nghin cu trn tt c cc gen hay protein cng mt lc, mang tnh h thng .
5.2.1.1. SNP v pht hin thuc
Single-nucleotid polymorphism (SNP) l s khc nhau ti 1 nucleotid trn 2 trnh t ADN.
V d 2 allel: Trnh t 1: ATTCGATC
Trnh t 2: ATTTGATC
Nghin cu SNP l cng c hu hiu lm sng t vai tr phn t ch trong cc bnh l.
Trong lnh vc pht hin thuc mi, SNP gip:
- Xc nh ch iu tr. SNP c dng nh nhng cht nh du sinh hc khi gen nhy
cm vi 1 loi bnh c to dng gn v tr SNP, to ra tnh trng tng t bnh l.
- Xc nh c im ch iu tr. Mc khc nhau v trnh t nucleotid ca ch iu
tr rt quan trng, nh hng n chc nng gen cng nh kh nng tng tc vi
thuc. c bit khi p dng phng php sng lc hiu nng cao, tin hnh trn 1 s


57
lng ln cc hp cht t th vin mu, th vic xc nh ch iu tr mang tnh i
din rt quan trng. Hn th, SNP cn nh hng n cu trc v chc nng ca
protein. ch iu tr ca thuc c sng lc mc acid nucleic.
- Xc nhn ch iu tr. C rt nhiu ch tc dng ca thuc nhng khng phi tt c
cc ch ny ng vai tr gy bnh. Kho st tnh di truyn s gip xc nh c cc
ch va l mc tiu tc dng ca thuc ng thi c vai tr gy bnh.
- Dc l di truyn (pharmacogenetic). Nghin cu nh hng ca di truyn n tc dng
v chuyn ha ca thuc. S a dng ca enzym chuyn ha l nguyn nhn gy ra cc
tc dng ph khng mong mun liu iu tr. y l c s pht trin loi thuc
dng theo c a bnh nhn.
- Dc l b gen (pharmacogenomic). X l thng tin di truyn a ra cc quyt nh
iu tr ph hp vi bnh nhn. Thng tin di truyn ny c th l ca c th vt ch
(bnh thng hay mc bnh) hay ca yu t gy bnh. Dc l b gen c vai tr trong
tt c cc giai on pht trin mt thuc mi t pht hin thuc mi n th lm sng.
y cng l c s nn tng ca loi thuc dng theo c a bnh nhn.
5.2.1.2. Mc biu hin ca gen
Phn tch mu gen biu hin t ngn hng d liu EST (Expressed Sequence Tag) l cc
on cADN c gii trnh t - s cung cp thng tin quan trng v ch iu tr ca thuc
cng nh cht nh du dng trong chn on. Vic thu thp, lu gi, qun l, phn tch v trnh
by th vin d liu biu hin ca cADN c thc hin nh k thut tin sinh v h thng my
tnh. So snh s biu hin gen gia m bnh thng v m bnh l s bit chc nng ca gen.
5.2.1.3. Hn ch ca k thut genomic
Mc d hu ch nhng nu ch phn tch trnh t ADN ca b gen s khng th bit chc
nng ca protein hay sn phm ca gen v vy s gp kh khn trong vic xc nh ch iu tr
mi cho cc bnh l ngi.
V vy, nu kt hp k thut genomic v proteomic s gip pht hin mt lng ln ch
tc dng ca thuc. Cng vi phng php sng lc hiu nng cao v th vin cc hp cht ha
hc, s kt hp ny lm gia tng s lng cc cht c a vo giai on th nghim lm sng.
Vn t ra l lm th no tng hiu qu cc th nghim nh gi tc dng v c tnh ca
thuc. Phng php truyn thng th c tnh ca thuc trn ng vt th nghim da vo ch s
huyt hc, sinh ha v cc thng s c bn khc khng th p ng yu cu ny. Nghin cu
trn gen v s biu hin protein sau qu trnh iu tr s xc nh c cc thay i sinh ha lin
quan n hiu qu v c tnh ca thuc. Kt qu s thay i ny c dng lm tiu chun nh
gi trong sng lc hiu nng cao.
5.2.2. K thut proteomic
Proteomic l s biu hin protein t b gen (PROTEin expressed by a genOME) v l phn
tch cc d liu protein mang tnh h thng. Khi nim proteome ch tt c protein t mt
chng, tng ng vi khi nim genome bao gm tt c cc gen. Proteomic rt quan trng trong
qu trnh pht hin thuc do nhiu h thng iu ha dc phm hot ng thng qua cc
protein. a s thuc tc dng dng kt hp vi protein. V d, thuc c ch protein c tc dng
ngn cn hot ng ca enzym protease l enzym cn thit cho qu trnh nhn ln ca virus.
Thuc s gn kt ln b mt ca enzym protease nn enzym b bt hot, gip ngn chn s
lng virus gia tng. Proteomic trong pht trin thuc mi c gi l pharmacoproteomic v
gp phn trong vic hnh thnh thuc theo c a bnh nhn.

58
5.2.2.1. Protein l ch tc ng ca thuc
a s ch tc ng ca thuc l protein c m ha bi gen trong cc m b nh hng
ca bnh l. c tnh b gen ngi m ha cho khong 10.000 enzym, > 2.000 th th protein
G, 200 knh ion v 100 th th hormon nhn. Nhng protein ny l thnh phn then cht trong
nguyn nhn gy bnh, v vy s l ngun di do cho ch tc ng ca thuc. Cc ch tc
ng ny ch c hiu r da trn mc biu hin ca chng trong t bo, khng th ch da
trn trnh t ADN. ch tc ng:
- c biu hin trn m chuyn bit chn lc tc dng dc l v gim tc dng ph
tim n.
- c biu hin mc thp t bo ca c quan tng ng
H thng pht hin ch tc ng c hiu qu gip xc nh cc gen m ha protein biu
hin ti m chuyn bit mc thp.
Sau y gii thiu mt s k thut proteomic ng dng trong tm kim thuc:
5.2.2.2. Bn biu hin protein trn gel 2D
in di trn gel 2D v phn tch hnh nh c dng trong nghin cu cc thay i v biu
hin protein m, t bo hay dch c th. K thut ny gip nh lng protein v xc nh cc
bin i xy ra sau dch m nh s glycosyl ha hay phosphoryl ha. Trong cng mt th
nghim, ti cng mt thi im, hnh nh ca hng ngn protein thu c v c phn tch.
Mc biu hin protein thay i l bng chng v vai tr ca chng trong c ch gy bnh.
5.2.2.3. Sc k lng
Protein c ct nh c th phn tch mc peptid bng phn php sc k lng
(liquid chromatography LC). Thay v tch cc protein ring l trn gel 2D, hn hp protein
c phn tch sau khi tch phn on bng LC a chiu. u tin, dng trypsin x l hn
hp protein, peptid to thnh s tch bng LC v o bng ph khi (mass chromatography MS)
5.2.2.4. Tng tc protein protein
Th nghim tng tc gia cc protein c th thc hin in-vivo. c bit vic pht hin
protein pht hunh quang xanh gp phn quan trng trong vic nghin cu tng tc protein trn
t bo sng. Cu trc ca i phn t protein rt phc tp, gy nhiu tng tc protein nhng
kh khn khi nghin cu trn in-vitro, vn c th phn tch c. K thut nghin cu tng tc
protein trn i tng sng gip hiu r mng li t bo, bao gm cch thc truyn tn hiu,
nhm pht trin v ti u ha dc phm c tc dng iu ha tng tc protein.
Mng li tng tc protein-protein gi l mng li interactome. Interactome l bn
bao gm tt c cc tng tc xy ra trong c th sinh vt, gia tt c cc protein tt c cc t
bo, m mi la tui v di tc ng ca mi iu kin mi trng. Da vo c s d liu
ny, cc nh dc hc s loi bt ch tc ng c nhiu tng tc.
5.2.3. K thut metabolomic
Metabolomic nghin cu cc phn t nh v tng tc ca chng trong c th sinh vt,
ng vai tr quan trng trong qu trnh tm kim thuc. Metabolomic pht hin thy phn ln
trong tng s ~ 6.000 gen ca nm men Saccharomyces cerevisae v tng t cc sinh vt
khc, m ha cho protein khng r chc nng. Rt nhiu trong s nhng gen ny l gen im
lng, khng th hin thnh kiu hnh. nh lng s thay i nng nhiu cht chuyn ha c
lin quan n thay i nng cht chuyn ha ch nh, t c th xc nh v tr tc ng.
Ngoi ra, kt hp gia proteomic v metabolomic s gip xc nh v nh gi cht nh
du sinh hc v chuyn ha, nhm pht trin cc thuc an ton v hiu qu. S kt ni gia d
liu nh gi mc biu hin gen (xc nh mc hot ng gen ca sinh vt ti mt thi


59
im nht nh), mc chuyn ha (nh tnh v nh lng cht ha hc ca sinh vt ti mt
thi im nht nh) v kiu hnh biu hin (xc nh c tnh vt l v ha hc ca sinh vt ti
mt thi im nht nh) c phn tch bng tin sinh, xy dng thnh m hnh b gen chc
nng.
5.2.4. Cng ngh sinh hc ng dng k thut nano
K thut nano cc phn t nano cng nh cc lnh vc ng dng nano khc nh chip
sinh hc nano (nanobiochip), cm bin sinh hc nano (nanobiosensor) thc y pht trin qu
trnh tm kim thuc mi. S dng phn t lng vi kch thc nano (nanofluidics) c th to ra
hng lot v tr nh lng trn mt bn nh. Cc phn t nano nh phn t nano vng v chm
lng t ang c quan tm nhiu do ng dng to ln ca chng trong tm kim thuc.
5.2.4.1. Xc nh ch tc dng
Gn kt a din gia phn t nh v phn t nano lm gia tng i lc gn kt c hiu v
bc l nhiu tnh cht sinh hc mi ca vt liu nano. Thuc a tr liu c nghin cu c
tc dng khng virus v khng vim. Cng vi th vin mu c, phn t nano c gn vo
phn t nh gip lm phong ph thm th vin mu. Sng lc th vin mu ny trn cc dng t
bo khc nhau c th khm ph hng lot phn t nano c chuyn bit cao vi t bo mng
trong, kch hot i thc bo hay t bo ung th ty. K thut a din c th ng dng pht
trin vt liu nano s dng trong cc dng t bo khc nhau, pht hin cc trng thi t bo khc
bit hay loi t bo ch chuyn bit. V do vy, chng c tim nng p dng trong tm kim
hiu nng cao, xc nh ch tc dng, chn on v iu tr.
5.2.4.2. Thit k thuc da trn cng ngh nano mc t bo
thuc c tc ng trn mt bnh l, mt trong nhng vn u tin l phi lm sng
t c bng cch no mt t bo hay nhm t bo lin h vi cc t bo khc v phn ng vi
v s phn t sinh hc xung quanh. u d nano, u d qut ph khi (scanning mass
spectrometry SMS), c th thu c thng tin v tnh cht sinh ha v cu trc ca c cc i
tng sinh hc phc tp. u d SMS c th xc nh bn mi lin h phc tp gia cc t
bo bng cch thm d hot ng t bo trong mi trng t bo t nhin, do chn on bnh
s hiu qu hn v thuc c thit k mc t bo.
5.2.5. Cc phng php sng lc
5.2.5.1. H thng sng lc da trn t bo
Khng ging phng php truyn thng, phng php sng lc da trn t bo cung cp
cc d liu sinh hc (thi im, v tr v hot ng) v tc ng sinh l ca cht nghin cu ti
ch tc ng trn t bo sng chuyn bit. Kt qu ny cung cp thng tin quan trng v tc
dng cng nh c tnh ca cht nghin cu trn t bo, trc khi tin hnh th nghim trn
ng vt hay th nghim lm sng ngi, do vy s tit kim nhiu thi gian v chi ph.
Phng php sng lc da trn t bo rt hiu qu i vi cc yu t iu ha knh ion.
Bi v v tr gn kt cha bit, nn nu thit k th nghim theo phng php sinh ha thng
thng hay phng php thay th ni lin kt s gp nhiu kh khn. Cng vi k thut nh
lng quang hc s dng cht pht hunh quang hay thit b c quang hc, sng lc da trn t
bo c th p dng i vi hu ht ch tc ng, bao gm c cc knh ion. Hn th, u d m
ha di truyn c trang b cm bin sinh hc c th p dng cho phn ng sinh ha ni bo, xc
nh v tr ch tc ng v tng tc protein.
5.2.5.2. Th th tip nhn: m ngi v m ng vt
Trc y, thuc c sng lc trn m ng vt cha rt nhiu th th. Nhng hin nay
nhiu cng ty tin hnh trn m ngi do mt s u im sau:

60
- Do phn lp c th th l ch tc ng, cc nh ha hc c th thit k hp cht
ch c tc ng trn th th chuyn bit, khng tc ng trn th th c kh nng gy tc
dng ph.
- Trnh c s khc bit khi so snh gia hot ng ca thuc trn ngi v trn ng
vt.
- Chi ph hp l do c th loi b hoc thit k li cc hp cht c tc dng km trn th
th tip nhn, trc khi bc vo giai on tn nhiu chi ph l th nghim tin lm
sng v lm sng.
5.2.5.3. Sng lc m
So vi th nghim trn ng vt hay nm men, th nghim trc tip trn m ngi c th
thu c thng tin quan trng lin quan n hot ng ca gen trong h thng c th ngi. T
m hnh ng vt, rt kh suy on c ch tc dng ca thuc trn ngi; v vy kt qu th
nghim trn m ngi quyt nh i tng c chn cho giai on tip theo.
5.2.6. Antisense
Antisense l on ADN hay ARN tng hp, c gi l oligonucleotide (c chiu di t
nht 15 nucleotide), c trnh t b sung vi mARN do vy qu trnh dch m protein b ngng
tr. Vic s dng antisense ngn cn tng hp cc protein bt thng lin quan n bnh l
gi l tr liu antisense. Mt ng dng khc ca antisense l dng ribozyme ct ARN v vy
protein khng c tng hp. Acid peptide nucleic (PNA) - phn t ging ADN l cc tc
nhn antisense hay khng nguyn tim nng. Aptamer l chui nucleotide tng hp gn trc tip
vi protein ch, c ch hot tnh ca protein, cng c xem l hp cht antisense. ng ng
c cu trc tng t ADN, c kh nng kha acid nucleic (locked nucleic acid LNA) cng c
nhiu tnh cht ca antisense. Phc hp LNA/ADN c hot tnh antisense c s dng trong h
thng nh lng.
5.2.6.1. ch tc ng l antisense oligonucleotide
K thut antisense da trn thng tin di truyn thit k cht km hm gen ch. Do trnh
t c hiu nn chng ch tc ng trn gen ch, khng tc ng trn gen c lin quan. V vy,
cht c ch antisense c kh nng xc nh chc nng ca mt gen n chnh xc hn bt k
phng php no khc.
ADN microarray c th lng gi hng ngn gen cng mt lc. Antisense oligonucleotide
d dng km hm mARN ch v s biu hin protein cng nh cch thc di truyn. K thut
antisense l la chn hu hiu trong qu xc nh hiu nng cao chc nng gen v ch tc ng
ca thuc.
Acid peptide nucleic km hm biu hin gen. Trnh t b sung ca PNA c kh nng c
ch ARN ch vi khun, do m ra hng pht trin th h khng sinh mi l cc antisense
PNA.
5.2.6.2. Aptamer
Aptamer l si n ADN hay ARN c i lc cao v c hiu vi phn t ch do chng c
cu trc 3D c bit, v vy c th c ch chc nng sinh hc ca phn t ch. Th vin biu
hin aptamer khng ph thuc vo thng tin trnh t b gen, c dng tm cc peptide c
hot tnh sinh hc mong mun bng cch kch hot hay km hm yu t t bo.
5.2.6.3. ch tc ng l ARN
ARN c cu trc phc tp, cnh tranh vi cu trc ca protein v do c th l ch tc
ng ca cc thuc phn t nh. Cn thc hin nhiu bc pht hin c ARN l ch tc


61
ng. Tt c protein u c tng hp t khun mu ARN nn chng c th b c ch nu km
hm qu trnh dch m ca mARN. Mt s u im khi ch tc ng l ARN:
- Thuc gn kt vi ARN c tc dng chn lc hn gn kt vi protein. V d, thuc gn
kt trn mt vng no ca ARN tng ng vi m ch, khng nh hng n ARN
ca m khc.
- Kh khn khi chit tch v lm tinh sch protein, trong khi ARN dng c tng
hp v dng trong nh lng.
- C th tng hp ARN vi s lng ln nhng vn cn hn ch khi sn xut protein vi
qui m ln.
5.2.6.4. Ribozyme
Ribozyme c bn cht l ARN, va c th hot ng nh cht xc tc va c th nh phn
t di truyn. Trong t nhin, ribozyme c nhim v phn hy ARN v ct ni ARN trong qu
trnh x l ARN. Ribozyme ngy cng c s dng nhiu vi mc ch km hm s biu hin
protein ca mt trnh t gen c hiu bng cch ct mARN tng ng. Ribozyme c thit k
c kh nng ct ARN ti mt v tr mong mun, c th ng dng trong nghin cu c bn hay
chn on. Khc vi dc phm truyn thng, ribozyme lm gin on qu trnh di truyn,
khng c ch chc nng protein.
5.2.7. iARN
iARN l c ch iu ha biu hin gen v qu trnh nhn ln ca virus, dng si i
ARN (dsARN). trong t bo, dsARN b x l thnh cc on dsARN ngn gm 21 23
nucleotide, gi l siARN (small interfering ARN) c vai tr nhn bit v ph hy si ARN c
trnh t b sung. Trong t nhin c nhiu lai ARN nh nh siARN, miARN (microARN) v
rasiARN (repeat-associated siARN). iARN dng nghin cu chc nng gen sinh vt n
gin v dng trong iu tr hay tm kim ch tc ng ca thuc.
5.2.7.1. S dng th vin siARN trong xc nh ch iu tr l gen
Kh nng iARN c th loi b d dng qu trnh biu hin gen m ra hng thnh lp
th vin iARN nh gi tm quan trng ca hng trm hay hng ngn gen m s biu hin
ca chng lin quan n hin tng bnh l.
5.2.7.2. S dng iARN trong nh lng
iARN l rt hu ch trong vic nh lng, la chn HIT v cc th nghim chuyn bit.
iARN c dng nh chng dng trong vic nh gi kt qu nh lng da trn mc
mARN hay protein b nh hng. Tc dng ca hp cht c th nghim trn nh hng km
hm bit ca tc nhn iARN. S dng iARN c hiu vi tng mARN c th d on tc
dng ca hp cht.
5.2.7.3. Nhng thch thc khi s dng iARN trong tm kim thuc
- siARN phi m bo n nh dng cng thc thuc hay cc thay i v mt ha
hc. bn vng ca siARN i vi exo v endonuclease c khc phc bng cc
bin i ha hc.
- Hnh thc thu nhn siARN hiu qu, an ton, kh thi
- Sn xut siARN vi qui m ln
- Kt qu nh lng iARN da trn t bo c khuynh hng khng n nh do t bo
cc ging ngoi ca a tng trng vi tc khc vi cc t bo ging pha bn
trong, v vy khng nn thc hin th nghim cc ging pha ngoi.

62
5.2.7.4. Vai tr microARN trong tm kim thuc
MicroARN (miARN) l nhng phn t nh, hu ht khng m ha cho sn phm gen, c
ngun gc t cc on ln precusorARN hin din tt c sinh vt a bo. miARN l bn sao
gm khong 21 25 nucleotid, c kh nng km hm chc nng ca gen do tng tc vi
mARN ch. miARN iu ha t nht 1/3 s biu hin ca gen v ng vai tr quan trng trong
biu hin tnh trng ca nhiu bnh l nh ung th, ri lon chuyn ha hay nhim trng. V vy
miARN c xem l loi ch tc ng mi ca nhiu dc phm. Vic tm kim miARN ch
v nh gi chc nng ca chng c th s dng phng php di truyn c bn hay sng lc b
gen vi h tr ca my tnh. miARN v gen chu s iu ha ca chng l i tng ca qu
trnh pht trin cc phng php iu tr mi.
5.2.8. Biochip v microarray trong tm kim thuc
Chip sinh hc (biochip) l khi nim dng ch vic s dng cng ngh vi mch
(microchip) trong lnh vc sinh hc phn t. Biochip bao gm cc dy nhng phn t sinh hc
chn lc c c nh trn mt b mt. ADN microarray l phng php nhanh dng gii
trnh t v phn tch gen, mi dy bao gm cc mu c sp xp theo mt trt t nht nh v
ng knh cc im np mu < 200 m. H thng gm c u d ADN c dng vi lng
nh, dng c np mu (robot t ng), c kt qu (my scan) v phn tch s liu (cng c tin
sinh).
5.2.8.1. Tm kim hp cht LEAD
Vi nhim v tm kim thuc mi, cc chip tp trung vo vic xc nh chc nng gen hn
l gii trnh t nhng gen ny, nhm pht hin cc hp cht LEAD. T bo b x l bng cht
nghin cu v kt qu biu hin gen c so snh vi qui trnh trc y, nh vy nhn bit
c s thay i protein hay ch tc ng. Th nghim xc nh ch tc ng in vitro gip gii
quyt nhng kh khn trong qu trnh pht trin thuc mi bi v cc th nghim trn ng vt
tn nhiu chi ph, thi gian
5.2.8.2. Microarray cADN
Phn tch hiu nng cao cc biu hin ca gen ng vai tr quan trng trong tin trnh tm
kim thuc. iu ny yu cu thc hin kho st v so snh nhanh chng mc biu hin gen
gia cht nghin cu v cht i chiu. Vi nhim v ny, microarray c th xc nh c
trong s cc trnh t bit v thu thp, trnh t no p ng yu cu lm ch tc ng ca
thuc. K thut nh lng vi th tch mu gim ti a v s dng phn t lng c kch thc
nh m ra trin vng mi cho sng lc hiu nng cao.
5.2.8.3. S dng d liu biu hin gen trong tm kim ch tc ng
mi
Da trn d liu phn tch biu hin gen, x l s liu bng h thng my tnh cng vi
sinh vt bin i gen, gip lm sng t nhiu chc nng ca gen trc y cha bit. C s
d liu c xy dng da trn h thng s liu biu hin gen ca t bo vi hng lot kiu hnh
khc nhau, nh vy c th xc nh chc nng mi ca gen cng nh hiu c bn cht ha hc
ca c ch tc ng ca thuc. D liu ny nhn mnh s khc bit gia gen bnh thng - gen
gy bnh v nh hng ca thuc n chc nng ca gen.
Phn tch d liu biu hin gen l k thut mi u tin p dng trong qui trnh tm kim
thuc. Microarray c p dng kim tra s biu hin b gen hoang di, sng lc hin
tng a hnh v thnh lp s . K thut ny cho php lng gi cc yu t tham gia biu hin
gen cc trng hp bnh l v xc nh ch tc ng mi. Chip gen cng gip d on tc
dng ph ca thuc v do vy c th chn la nhng thuc t hoc khng c tc dng ph.


63
5.2.9. ng dng tin sinh hc trong tm kim thuc
Nhim v ca tin sinh l xc nh cc i tng c th dng lm thuc t hng lot ng
vin. Tin sinh hc c th:
- Phn loi nhm protein
- Cung cp thng tin v tnh cht sinh ha v hot ng t bo ca ch tc ng
- Pht trin cc ch tc ng
Khi nim in silico biology gip nh nghin cu nhanh chng tin hnh phn tch in
silico bng thao tc n gin chn bm trn my tnh.
5.3. Kt lun
Cng ngh sinh hc ang c p dng rng ri trong cng cuc tm kim thuc mi. C
rt nhiu nghin cu nhm pht trin cc loi thuc mi, thuc theo c a bnh nhn da trn
dc l di truyn, dc l b gen v dc l protein. Cc omics khc nhau, c bit l s kt
kp genomic v proteomic ng vai tr quan trng trong tin trnh ny; tin sinh gip lu gi v
phn tch kho d kiu khng l. Vi s phi hp ng b ca cc lnh vc trn, qu trnh tm
kim thuc mi c th p ng yu cu iu tr trong tng lai.
Ti liu tham kho
1. Molecular biology and biotechnology, John M. Walker, Ralph Rapley
2. High-throughput screening in drug discovery, Jrg Hser




65
B B i i 6 6. . H H p p c ch h t t t th hi i n n n nh hi i n n v v n ng g d d n ng g k k t th hu u t t
s si in nh h h h c c p ph h n n t t t tr ro on ng g n ng gh hi i n n c c u u

Mc tiu:
- Nu c ngun gc, vai tr ca hp cht thin nhin
- Trnh by c gi thuyt cng sinh
- Tm tt c qu trnh sn xut cc cht c ngun gc thin nhin bng phng php
ti t hp gen
6.1. Hp cht thin nhin
6.1.1. Khi nim
Hp cht thin nhin l cc cht ha hc do sinh vt sng sn xut trong t nhin. T hng
ngn nm nay chng c s dng trong nhiu lnh vc ca cuc sng; c bit ngn nga
v iu tr mt s bnh l ngi. Do c hot tnh sinh hc a dng nn hp cht thin nhin
ng vai tr l ngun nguyn liu quan trng cho ngnh cng nghip dc phm. ng ch ,
khong 70% thuc khng sinh v 60% thuc khng ung th ang c s dng trong iu tr l
cc hp cht thin nhin hay c ngun gc t nhin.
6.1.2. Ngun gc
Hp cht thin nhin c tm thy t ba ngun cung cp chnh: thc vt v ng vt trn
cn, vi sinh vt v sinh vt bin.
6.1.2.1. Ngun gc thc vt v ng vt
T khong 2.700 nm trc cng nguyn, con ngi bit s dng cy c iu tr mt
s bnh nh cam tho (Glycyrrhiza glabra), nha thm (loi Commiphora), thuc phin
(Papaver somniferum),...v cho n nay vn c tip tc dng nh nhng v thuc truyn
thng. Nhiu v thuc qu t ng vt c lu truyn, c gi tr s dng cao nh mt gu,
rn, nhung hu,...Ngoi ra, cng vi s pht trin ca cc ngnh khoa hc khc m cc n
cht t cy c c chit xut v tinh ch nh morphin, codein, noscapin (narcotin), v
papaverin t P. somniferum. Hay hemisuccinate carbenoxolone sodium, dn cht bn tng hp
t acid glycyrrhetic c trong cam tho, c tc dng u tr vim d dy t trng. Chnh kinh
nghim iu tr lu i ca cc dc liu gip chng ta phn lp c cc hot cht tinh khit s
dng lm nguyn liu trong ngnh dc phm hin i. Hin nay, rt nhiu hp cht mi tip tc
c tm thy t ngun dc liu c hot tnh dc lc, sau khi tin hnh th nghim lm sng
c php lu hnh. V d, apomorphin dn xut ca morphin chit xut t Papaver
somniferum c dng trong iu tr bnh parkinson. Galantamine l mt alkaloid c
Galanthus nivalis c tc dng di vi bnh Alzheimer. Hay arteether l thuc st rt, dn cht
ca artemisinin t Artemisia annua.


66

Hnh 6.1. Mt s hot cht mi c phn lp t thc vt trn cn
6.1.2.2. Ngun gc vi sinh vt
Cho n u nhng nm 1940, a s thuc c ngun gc thin nhin u chit xut t thc
vt trn cn. Nm 1928, Alexander Fleming tnh c pht hin penicicillin t nm mc
Penicillium notatum c kh nng khng khun m ra mt nh hng mi tm kim cc
dc cht do vi sinh vt sn xut. Vi sinh vt trn cn l ngun cung cp di do mt lng ln
cc hot cht vi s phong ph, a dng v cng thc ha hc cng nh tnh cht sinh hc. c
bit, chng ng vai tr khng th thay th trong ngnh sn xut khng sinh nh cc penicillin,
cc cephalosporin, cc aminoglycosid hay cc polyketid, vancomycin. Ngoi ra, cc cht tm
thy vi sinh vt cn c nhiu tc dng dc l khc c ng dng trong iu tr lm sng,
v d kh nng c ch min dch ca cyclosporin v rapamycin, h cholesterol ca lovastatin v
mevastatin, h ng huyt ca acarbose hay iu tr ung th ca pentostatin, peplomycin, v
epirubicin.
O
H
3
CO
O
OCH
3
HO
O
O
N
O
O
HO
OCH
3
O
O
H
O
HO O

(1) (2)

O
O
O
OH
OH
OH
OH
O
O O
Cl Cl
HN
N
H
H
N
N
H
O
H
N
N
H
OH
OH
HO
O
O O
H
O
O
NH
2
HO
HOOC
O
H
NHCH
3
H
H
2
N
H
OH
H
H
H
3
C
N
H
3
C
H
3
C
N
CH
3
CH
3
N
H
N
CH
3
N
O
CH
3
CH
3
O
O
CH
3
O
O
H
3
C
CH
3
H
N
O
H
3
C
CH
3
N
H
H
N
N
H
N
CH
3
O
CH
3
O
H
3
C
CH
3
CH
3
O
O
CH
3
N CH
3
HO
O
CH
3
H
3
C
CH
3
H
3
C

(3) (4)
Hnh 6.2. Mt s hot cht c phn lp t vi sinh vt
(1) Polyketides FK506 t vi khun Streptomyces tsukubaensis (2) lovastatin t nm Aspergillus terreus (3)
vancomycin t vi khun Amycolatopsis orientalis, v (4) cyclosporin A t Tolypocladium spp


67
6.1.2.3. Ngun gc sinh vt bin
Khng c lch s pht trin lu i nh dc liu, cc sinh vt bin mi c khai thc v
ng dng trong y hc thi gian gn y. Nhng hin nay, trong ba ngun chnh cung cp hp
cht thin nhin, chng tr thnh ngun ch lc do cha rt nhiu hot cht c tim nng hot
tnh sinh hc. Hn th na, mt vn cn lu l khong 70% b mt tri t c bao ph
bi cc i dng, sng ngi v l ni c tr ca trn 80% s loi thc vt v ng vt. Tuy
nhin cc nghin cu v hot tnh sinh hc mi ch tin hnh trn khong 10% s sinh vt bin
ny. Do vy, trong vi thp k gn y, rt nhiu nghin cu tp trung trn i tng ny v
khong 14.000 cht ha hc c phn lp vi nhiu cht c cng thc ha hc khc bit so vi
cc hp cht ca sinh vt trn cn.
Trn mt t, thc vt l ngun cung cp ch yu cc sn phm thin nhin; trong khi
mi trng bin, vai tr ny do cc ng vt khng xng sng m nhn, nh: sa, to , da
gai, v c bit l bt bin. Hng lot cht t bt bin v ang th nghim in-vitro hay in-vivo
v mt s hot cht ang trong giai on th nghim tin lm sng, lm sng hay c s dng
trong iu tr.
Bng 6.1. Mt s hp cht t bt bin c th nghim lm sng hay c php lu
hnh
Hot tnh Hot cht Ngun Tnh trng
Khng siu vi Analogs of spongeothymidine,
spongeouridine
Cryptotethia crypta

Lu hnh
(Ara A)
Khng vim IPL512,602 Petrosia contignata G II
Topsentins Topsentia genitrix, Hexadella
sp., v Spongosorites ruetzleri
TLS
Khng ung th KRN7000 Agelas mauritianus G I
LAF389 Jaspidae sp G I
Dictyodendrins Dictyodendrilla verongiformis TLS
Hemiasterlins Auletta sp., Siphonochalin sp. TLS
Lasonolides Forcepia sp. TLS
Laulimalide Cacospongeia mycofijiensis TLS
Peloruside A Mycale hentscheli TLS
Salicylihalimides A and B Haliclona sp. TLS
Khng Alzheimer Debromohymenialdisine
(DBH)
Stylotella aurantium G I
Khng nm Bengazole, bengamide Pachastrissa sp. TLS
Khng khun Psammaplin A Aplysinella rhax TLS
Khng st rt, lao,
HIV
Manzamine A Haliclona sp. TLS
G: giai on TLS: tin lm sng
6.1.3. Bn cht ha hc
Hp cht thin nhin c th l cc cht chuyn ha s cp, cht chuyn ha th cp hay
cc phn t c khi lng ln nh protein, cellulose, lignin.

68
6.1.3.1. Cht chuyn ha s cp
L cc cht ng vai tr then cht trong hot ng sng bao gm chuyn ha v sinh sn
ca tt c cc t bo. Chng c th l acid nucleic, acid amin v ng.
6.1.3.2. Cht chuyn ha th cp
Khc vi cht chuyn ha s cp, cht chuyn ha th cp ch hin din mt s loi sinh
vt nht nh v c vai tr nh hng n sinh vt khc. Chnh nhng cht chuyn ha th cp
ny c bn cht ha hc rt a dng, nh vy chng c nhiu kh nng sinh hc khc nhau v l
i tng ca qu trnh sng lc sinh hc nhm mc ch tm kim cc hot cht mi ng dng
cho lnh vc y hc, chn nui hay nng nghip. Cht chuyn ha th cp c th phn thnh cc
nhm cht chnh: alkaloid, terpenoid v steroid, phenylpropanoid, polyketid v acid bo, mt s
acid amin v hydratcarbon c bit.
Gi thuyt cng sinh
Gi thuyt cng sinh cho rng hp cht thin nhin l cht chuyn ha th cp ca h vi
sinh vt cng sinh vi vt ch, khng phi l sn phm chnh vt ch. Gi thuyt ny c a
ra da trn s ging nhau v cu trc ha hc gia hp cht thin nhin vi cc cht phn lp
c t vi sinh vt. V d cht polyketid rhizoxin khng do nm Rhizopus sn xut m do vi
khun cng sinh Burkholderia; hoc cc peptid vng patellamid l sn phm ca vi khun
Cyanobacteria cng sinh Prochloron didemni, khng phi ca vt ch l loi hi tiu
Lissoclinum patella.
Bng 6.2. Mt s cht chuyn ha ca bt bin tng t vi cht chuyn ha ca vi
khun
Cht chuyn ha ca
bt bin
Ngun Cht chuyn ha ca
vi khun
Ngun
Arenastatin A
Discodermide
Dysidea arenaria
Discodermia dissoluta
Cryptophycin
Alteramide A
Nostoc sp.
Alteromonas sp.
Jaspamide
Keramamide A
Mimosamycin
Jaspis spp.
Theonella sp.
Petrosia sp.
Chondramide D
Ferintoic acid A
Mimosamycin
Streptomyces sp.
Chondromyces crocatus
Microcystis aeruginosa
Motuporin
Renieramycin E
Theonella swinhoei
Reniera sp.
Nodularin
Safracin B
Nodularia spumigena
Pseudomonas fluorescens
Salicylihalamide A
Swinholide A
Misakinolide A
Haliclona sp.
T. swinhoei
Theonella sp.
Saframycin A
Apicularen A
Tolytoxin
Scytophycin C
Streptomyces lavendulae
Chondromyces sp.
Tolypothrix sp.
Scytonema sp.



69
O
N
H
OH
O
O
O
OH
O
OH O
OH
HN
O

Salicylihalamide A Apicularen A
t bt bin Haliclona sp. t vi khun Chondromyces sp.

O OH
NH
OH
H
N
O
O
O
HO
NH
H
N
O
HO
OH
O
O

Discodermide Alteramide A
t bt bin Discodermia dissoluta t vi khun Alteromonas sp.
Hnh 6.3. Mt s cht chuyn ha ca bt bin tng t vi cht chuyn ha ca vi
khun
6.2. Kh khn trong sn xut hp cht thin nhin
Phng php ch yu sn xut hp cht thin nhin bao gm nui trng dc liu chit
xut, tng hp hay bn tng hp. Tuy nhin, i vi hp cht khng do sinh vt vt ch sn xut
m do h vi khun cng sinh chuyn ha th vn khai thc chng s dng trn qui m ln
gp phi mt s kh khn.
6.2.1. Kh khn trong chit xut
Kh khn ln nht l hm lng cc cht ny trong t nhin rt thp. chit tch 1,4 g
bryostatin 1 mt tc nhn khng ung th cn khong mt tn ng vt dng ru ti Bugula
neritina. Hoc t mt tn bt bin Lissodendoryx sp. ch thu c 300 mg halichrondin l mt
cht khng t bo. Nh vy, nu halichrondin c s dng trong tr liu ung th th nhu cu
hng nm khong 1-5 kg, tng ng vi vic khai thc 3.000-15.000 tn bt bin. Hu qu ca
qu trnh khai thc l lm mt cn bng h sinh thi, mt s loi sinh vt c th b tuyt chng.
Ngoi ra, vic thu hoch, x l v chit xut mt lng ln ngun nguyn liu thin nhin trn
thc t khng th thc hin c v cng khng mang li hiu qu kinh t.
6.2.2. Kh khn trong nui trng
Mc d c nhiu n lc nhm ti u ha iu kin nui trng cc sinh vt cng nh nui
cy h vi sinh vt cng sinh nhng vn cha c thnh cng no ng k. Vi iu kin nui
trng nhn to, hm lng cc cht b gim ng k v i khi b mt hon ton. Vn ny c
th do mi trng sng thay i dn n qu trnh chuyn ha sinh vt b nh hng. Hn th
na, yu t mi trng c tc ng quan trng n cc vi sinh vt cng sinh, mt s chng vi
khun rt kh tng trng trong mi trng nui cy phng th nghim. Ngoi ra, xc nh
v phn lp c chng vi khun c kh nng sn xut cc cht c hot tnh l cng vic ht sc
kh khn, phc tp.

70
6.2.3. Kh khn trong tng hp
Mt s hp cht thin nhin c tng hp hay bn tng hp thnh cng tuy nhin
khng t hiu qu kinh t. Nguyn nhn do cc cht chuyn ha th cp thng c cu trc ha
hc ln, cng knh nn qu trnh tng hp phi tri qua rt nhiu giai on vi hiu sut phn
ng thp. V d, tng hp mycalamide B cn thc hin 25 phn ng ha hc vi hiu sut t
18%-79%.
6.3. Sn xut hp cht thin nhin bng cng ngh
sinh hc
Sn xut hp cht thin nhin bng cng ngh sinh hc bao gm cc bc chnh sau:
- Chit tch ADN ton phn ca mu cha hot cht nghin cu
- Thnh lp th vin gen vi ADN c chit tch
- Sng lc th vin gen tm v phn lp gen chu trch nhim sinh tng hp hp cht
thin nhin
- To ADN ti t hp bao gm gen ch v vector biu hin
- To dng vi khun mang ADN ti t hp
- D biu hin hp cht thin nhin dng vi khun ti t hp
- Thu hoch v tinh ch
6.3.1. ADN ton phn
Cc nh khoa hc rt thnh cng khi nghin cu trn tng vi sinh vt ring l. Tuy
nhin, i vi h vi khun cng sinh, c n 99% khng th nui cy trong phng th nghim.
khc phc tr ngi ny, phng php nghin cu ADN ton phn (metagenomic ADN) - bao
gm ADN ca sinh vt vt ch v ca cc vi sinh vt cng sinh c bt u p dng t
nhng nm 1990. N. R. Pace tin hnh th nghim u tin theo phng php ny, ng pht
hin s a dng cc trnh t 16S rARN ca cc sinh vt khng nui cy c trong mu ly t
mi trng. Nm 1998, J. Handelsman a ra khi nim v ADN ton phn, ADN ton phn
gip chng ta nhn thy nhng g m trc y ta khng th thy trong th gii vi sinh vt.
Thnh tu ny tr li c cu hi Ci g? ang lm g? v Lm nh th no ca h vi sinh
vt cng sinh. Bng phng php nghin cu trn ADN ton phn, nhiu gen sinh tng hp cc
hp cht thin nhin c tm thy nh nhm gen sinh tng hp patellamid t Lissoclinum
patella, bryostatin t Bugula neritina hay erdacin t mu t sa mc.

Chit tch ADN
Xy dng th vin
ADN
Sng lc gen ch
D biu hin vi
khun nui cy c
Phn tch ADN
ADN ton phn
Nhmgen sinh tng hp
Ngun cung cp hp
cht thin nhin
Mu
Chit tch ADN
Xy dng th vin
ADN
Sng lc gen ch
D biu hin vi
khun nui cy c
Phn tch ADN
ADN ton phn
Nhmgen sinh tng hp
Ngun cung cp hp
cht thin nhin
Mu

Hnh 6.4. Sn xut hp cht thin nhin bng phng php ADN ton phn


71
6.3.2. Thnh lp th vin gen
Cc bc tin hnh thnh lp th vin gen:
- ADN ton phn ca sinh vt sau khi chit tch s c ct thnh tng on nh bng
phng php vt l nh nng lng sng m (sonication) hay phng php ha hc
nh enzym ct gii hn.
- Cc on ADN sau s c ni ghp vi cc vector to dng. Vector s dng trong
th vin gen thng l cosmid hay fosmid do chng c kh nng mang nhng on
ADN c kch thc ln ~ 25-40 kb.
- ng gi ADN lai ghp vo thc khun th lambda v chuyn vo vi khun ch,
thng l E. coli.
ADN ton
phn
Cc on
ADN
Ct ADN
ton phn
Ti t hp
vi vector
(a) (b)
ADN ton
phn
Cc on
ADN
Ct ADN
ton phn
Ti t hp
vi vector
(a) (b)

Epicenter
Hnh 6.5. (a) Qu trnh ni ghp vi fosmid vector (b) Fosmid PCC1FOS
Vy, th vin gen l tp hp dn s vi khun cha ton b h gen ca mt sinh vt, trong
mi vi khun mang mt on gen.
6.3.3. Sng lc gen chu trch nhim sinh tng hp hp cht thin
nhin
Trnh t mi xui v mi ngc c thit k nhm tm kim trnh t c hiu ca gen
ch bng phng php PCR (Polymerase Chain Reaction). Do cc gen ch thng c kch
thc ln (> 10 kb) nn phi p dng phng php primer walking bc l dn tng on gen
ngn ca ton b nhm gen ch.
ADN ton phn
Ct bng enzym ct
gii hn
ng gi cc on
ADN
ADN ton phn
Ct bng enzym ct
gii hn
ng gi cc on
ADN

Hnh 6.6. Qu trnh thnh lp th vin gen



72
on gen 1
Ton b nhmgen ch
on gen 2
on gen 3
on gen 4
on gen 5
on gen 1
Ton b nhmgen ch Ton b nhmgen ch
on gen 2
on gen 3
on gen 4
on gen 5

Hnh 6.7. Phng php primer walking

6.3.4. D biu hin hp cht thin nhin
Gen ch sau khi phn lp s c ghp ni vi vector biu hin ph hp v bin np vo
vi khun vt ch c php s dng trong cng ngh sinh hc. Mt trong s cc phng php
thng c p dng to vector ti t hp l ti t hp tng ng.

Gen ch
Gn hai u tn cng c trnh
t tng ng vi vector
Gen ch c ti t hp
trong vector biu hin
Gen ch
Gn hai u tn cng c trnh
t tng ng vi vector
Gen ch c ti t hp
trong vector biu hin

Hnh 6.8. Ti t hp tng ng

Hp cht thin nhin sau khi d biu hin s c chit tch v tinh ch, phc v cho
nghin cu hay iu tr lm sng.
6.4. Kt lun
p dng k thut sinh hc phn t xc nh c nhm gen chu trch nhim sinh tng
hp hp cht thin nhin. Kt qu ny khng nhng gip chng ta hiu r con ng sinh tng
hp cc cht trong t nhin m cn to th ch ng trong sn xut cc cht c hot tnh sinh
hc. Bn cnh , nhiu dn cht mi c th c to ra do gy t bin trn cc gen phn lp
c. Phn tch trnh t gen ch cn xc nh c vt ch sn xut hp cht t ngun mu t
nhin a dng, phc tp, t c th pht trin thm cc nh hng nghin cu trong tng lai.
Ti liu tham kho
1. Dewick, P. M., Medicinal Natural Products: A Biosynthetic Approach. 3rd ed. 2009:
John Wiley and Sons.
2. Cseke, L. J., Kirakosyan, A., Kaufman, P. B., Warber, S. L., Duke, J. A and Brielmann,
H. L., Natural products from plants. 2nd ed. 2006: CRC Press.
3. Leeper, F. J., Vederas, J. C and Croteau, R., Biosynthesis: Aromatic Polyketides,
Isoprenoids, Alkaloids. 2000: Springer.
4. Van Broekhoven, A., Shapiro, F and Ann, J., Novel frontiers in the production of
compounds for biomedical use. 2001: Springer


73
B B i i 7 7. . A AD DN N t t i i t t h h p p v v t t n nh h a an n t to o n n s si in nh h h h c c
Mc tiu:
- nh ngha c ADN ti t hp v sinh vt cha ADN ti t hp
- Lit k cc th nghim vi ADN ti t hp phi tun th hng dn an ton sinh hc
7.1. Gii thiu
7.1.1. ADN ti t hp (Recombinant DNA = rDNA)
Trong phng th nghim, ADN ti t hp c to ra bn ngoi t bo sng bng cch kt
hp hai hay nhiu trnh t ADN c ngun gc tng hp hay ngun gc t nhin. Cc rADN ny
khng tn ti trong t nhin. rADN c kh nng sao chp thnh nhiu bn khi a vo c th
sinh vt sng v to ra cc sn phm ti t hp.
ADN tham gia ti t hp c th t nhiu loi sinh vt khc nhau, v d ADN thc vt c
th kt hp vi ADN vi khun, ADN ngi kt hp vi ADN nm. Trn l thuyt, bt k rADN
cng c th c to ra v a vo bt k c th sinh vt sng no. Sinh vt cha rADN c
gi l sinh vt bin i gen (Genetically Modified Organism GMO).
K thut ti t hp ADN u tin c thc hin bng cch to dng vi khun cha trnh
t ADN ch nhm sn xut cc sn phm c hiu ca gen. rADN cng c dng to nn
cc ging cy trng v vt nui bin i gen.
7.1.2. ng dng ADN ti t hp
rADN c s dng trong nghin cu xc nh trnh t, chc nng gen cng nh trong
nhiu lnh vc nh chm sc sc khe bao gm dc phm, chn on y khoa, dinh dng; chn
nui; trng trt; mi trng,...
7.1.2.1. Dc phm
Insulin ngi l sn phm u tin trn th trng ra i bng k thut ti t hp ADN,
thay th cho insulin t heo trong iu tr bnh i tho ng. Hin nay, rt nhiu dc phm
c sn xut t rADN nh cc acid amin; hormon tng trng hGH; cc yu t to mu, ng
mu; thuc khng virus interferon; thuc iu tr ung th; vaccin vim gan B, cm, t,...Sn xut
dc phm bng phng php ny gip gia tng kim sot cht lng, nng cao tinh khit,
tnh an ton ca sn phm cng nh nng sut.
7.1.2.2. Nng nghip
Trong nng nghip, cy trng v vt nui bin i gen c to ra ngy cng nhiu nhm
mc ch:
- Gia tng gi tr dinh dng
- Gia tng kh nng chu tc ng ca mi trng nh lnh; ha cht, thuc tr su,
dit c v cc bnh
- Gim s ph thuc vo phn bn, ti tiu
- Tng sn lng
7.1.3. Phn loi mc an ton sinh hc i vi phng th nghim
Da vo kh nng gy bnh v ly nhim trong cng ng, t chc y t th gii (WHO)
phn cc vi sinh vt gy nhim thnh 04 nhm nguy c 1, 2, 3 v 4 (Bng 7.1). Da theo phn
loi ny, phng th nghim lm vic vi nhm vi sinh vt no s phi tun th mc an ton

74
sinh hc tng ng: mc an ton sinh hc 1, 2, 3 v 4 (Bng 7.2). Mc an ton sinh hc
c quyt nh da trn nhiu yu t nh chc nng hot ng ca phng th nghim, k thut
trin khai, thit b an ton, nguy c gy bnh v ly nhim tim n,...
Bng 7.1. Phn loi vi sinh vt theo nhm nguy c gy bnh
Nguy c
Nhm nguy c
C nhn Cng ng
Kh nng gy bnh
I Thp Thp C th khng
II Trung bnh Gii hn
- C th gy bnh ngi v gia sc
- Khng gy bnh nghim trng cho nhn vin phng
th nghim, cng ng, mi trng
- Cc bnh c th iu tr v phng nga gii hn
kh nng lan rng
III Cao Thp
- Gy bnh nghim trng ngi
- Khng ly nhim
IV Cao Cao
- Gy bnh nghim trng ngi v gia sc
- Ly nhim trc tip hay gin tip

Bng 7.2. Mi lin quan gia cc nhm nguy c v mc an ton sinh hc, thao
tc v trang thit b phng th nghim
Nhm
nguy c
Mc an ton
sinh hc
Nhim v phng TN Thao tc Thit b an ton
I C bn, I Ging dy, nghin cu K thut vi sinh tt Khng
II C bn, II Dch v y t ban u:
chn on, nghin cu
K thut vi sinh tt, qun
o bo v, k hiu nguy
him sinh hc
Bung an ton sinh
hc
III Ngn chn, III Dch v chn on c
bit, nghin cu
Nh mc 2, qun o c
bit, kim sot lung kh
Bung an ton sinh
hc
IV Ngn chn ti
a, IV
Nghin cu tc nhn
gy bnh nguy him
Nh mc 3, kha lung
kh thot, vi sen, x l
rc thi
Nh mc 3, lc kh,
p sut dng
7.2. nh gi mc an ton sinh hc ca ADN ti t
hp
Vo u nhng nm 1970, c nhiu kin tranh lun v tnh nguy him sinh hc ca
rADN do chng c th mang nhng c tnh khng mong mun, khng d on c vo mi
trng t nhin. Do vy, nu thot khi phng th nghim, chng c kh nng gy bnh cho
ngi, ng vt v gy nguy him cho mi trng. Nm 1974, Vin sc khe quc gia ca M
(National Insitute of Health - NIH) thnh lp y ban t vn ti t hp (Recombinant Advisory
Committee RAC) xem xt cc vn ny. Thng 02/1975, ti hi ngh khoa hc t chc ti


75
Asilomar, California (M), cc hng dn u tin v rADN c ban hnh. Hi ngh kt
lun:
- Cc th nghim trn rADN c th tip tc thc hin sau khi nh gi c mc an
ton
- Cc nguy c tim n cn c nh gi bng cc th nghim khc nhau
- Cc vi khun v plasmid c xem l an ton khi khng th sng st trong mi trng
nu ra khi phm vi phng th nghim, vn tip tc c nghin cu
- Mt s th nghim trn rADN cha nh gi c cn dng li
Cng vo nm 1975, hng dn u tin ca NIH ra i. Trong nhng nm gn y,
cc th nghim chng minh cng ngh di truyn vn c th thc hin an ton nu cc nguy c
c lng gi y bng phng php ph hp. n nm 1981, a s th nghim to dng
trn E. coli K12, mt s chng B. subtilis v Saccharomyces cerevisiae khng cn tun theo
hng dn ca NIH.
7.2.1. i tng cn nh gi mc an ton
- rADN
- Sinh vt hay virus cha cc rADN
Tt c cc th nghim lin quan n vic chuyn rADN hay ADN, ARN t cc rADN vo
c th con ngi u phi xin php NIH hay c quan c thm quyn.
Sinh vt cha rADN c to ra bng cch chuyn mt on ADN t sinh vt cho vo
sinh vt nhn (vt ch). Do vy, tnh cht ca sinh vt cho, ca trnh t ADN c chuyn,
ca sinh vt nhn s c dng nh gi mc an ton sinh hc cn p dng i vi cc
GMO.
7.2.2. nh gi mc an ton
rADN v GMO cn c xc nh kh nng gy bnh cng nh cc nh hng khng
mong mun c th xy ra i vi ngi trc tip lm vic trong phng th nghim, n sc khe
cng ng, n mi trng khi chng ra khi phm vi phng th nghim mt cch v tnh hay c
ch ch hay sau mt thi gian s dng chng.
7.2.2.1. i vi h thng biu hin sinh hc
H thng biu hin sinh hc bao gm vector v vt ch. H thng ny phi chng minh
tnh hiu qu v an ton khi s dng v c cp giy chng nhn ca c quan c thm quyn,
v d NIH. H thng biu hin phi bo m gim thiu:
- Kh nng tn ti ca vector trong vt ch mi trng ngoi phng th nghim
- S chuyn vector t vt ch s dng sang vt ch khc
Theo NIH, t bo nhn nguyn thy c 02 h thng biu hin: h thng 1 c an ton
cao v h thng 2 c an ton thp hn khi rADN c th chuyn sang sinh vt khc vi t l <
1/10
8
. V d h thng biu hin gm plasmid pUC18 v vt ch E. coli K12 c xp vo mc
an ton sinh hc cp 1 do pUC18 c gii trnh t v cc gen khng cn thit b loi
b; E. coli K12 l chng khng gy bnh, khng c kh nng nh c trong ng tiu ha ca
ngi v ng vt.
7.2.2.2. i vi vector biu hin
Mc an ton sinh hc cn c nng ln khi:

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- Trnh t ADN c ngun gc t sinh vt gy bnh, c kh nng gia tng lc c ca
GMO
- Tnh cht ca trnh t ADN cha c lm r
- Sn phm ca gen c kh nng c hot tnh dc hc
- Sn phm ca gen l cht gy c
7.2.2.3. i vi vector virus
Mt s vector virus nh vector adenovirus c th c dng chuyn gen sang t bo
khc. Cc vector ny thiu gen t nhn i ca virus nn s nhn ln trong dng t bo. Lu gi
v bo qun vector virus cng mc an ton sinh hc vi virus gc.
7.2.2.4. i vi ng vt bin i gen v knock-out gen
ng vt mang yu t di truyn ngoi lai (ng vt bin i gen) s c p dng mc
an ton tng ng vi c tnh ca sn phm gen ngoi lai.
ng vt b lm mt gen chc nng (ng vt b knock-out gen) thng khng gy nguy
him v mt sinh hc.
7.2.2.5. Thc vt bin i gen
Tnh an ton ca thc vt bin i gen c kh nng chu c thuc dit c hay chng li
cn trng ang gy nhiu tranh ci. Cc nh khoa hc lo ngi v mc an ton thc phm cng
nh nh hng lu di n h sinh thi khi trng cc loi cy ny.
Thc vt mang gen c ngun gc t ngi hay ng vt to ra dc phm hay sn phm
dinh dng c nh gi mc an ton sinh hc da trn cc sn phm ny.
7.2.3. nh gi mc nguy him ca sinh vt bin i gen
nh gi mc nguy him ca GMO da trn c tnh ca sinh vt cho v sinh vt
ch.
7.2.3.1. Sinh vt cho
Vic nh gi cn thc hin khi sn phm ca gen ngoi lai c hot tnh sinh hc hay hot
tnh dc hc v c th gy tng mc gy hi, v d: cht gy c, hormon, yu t iu ha
biu hin gen, yu t gy hay gia tng lc c, gy ung th, khng khng sinh, d ng. Ngoi
ra, phi lng gi mc biu hin nhng gen ny sn phm ca chng c th gy tc dng
sinh hc.
7.2.3.2. Sinh vt ch
Cc yu t cn xem xt:
- Tnh nhy cm ca vt ch
- Kh nng gy bnh ca vt ch: lc c, kh nng nhim v sn xut c t
- Bin i vt ch
- Tnh trng min dch
7.2.3.3. Thay i cc yu t gy bnh
Nhiu bin i gen khng c sn phm gy nguy hi, tuy nhin tc dng khng mong
mun c th do s bin i cc yu t gy bnh hay khng gy bnh sn c. S thay i cc gen
bnh thng c th lm thay i kh nng gy bnh. xc nh mi nguy hi tim n, cc vn
sau cn c nh gi:


77
- C lm tng nguy c gy nhim hay kh nng gy bnh?
- Gen ngai nhim c m ha cho yu t gy bnh sinh vt khc?
- Nu gen ngai nhim c kh nng gy bnh, c th d on kh nng gy bnh
GMO?
- C kh nng iu tr?
- Tnh nhy cm ca GMO vi khng sinh hay phng php iu tr khc?
- C th loi b GMO?
7.2.4. Qui trnh nh gi mc nguy him
Qu trnh nh gi thng gm 02 giai on:
- nh gi ban u: phn loi sinh vt th nghim da trn 04 nhm nguy c (Bng 7.1)
- nh gi ton din: tip theo nh gi ban u, nh gi ton din c thc hin:
o Cc yu t lin quan n sinh vt th nghim nh lc c, kh nng gy bnh, liu
gy nhim, s bn vng trong mi trng, ng ly nhim, vaccin phng nga,
thuc iu tr. Bt k chng no gy nguy him hn chng hoang di s c p
dng mc an ton cao hn. Cc chng gim hay mt lc c s c xp
vo nhm nguy c thp hn so vi chng hoang di.
o Thao tc thc hin: xem xt thn trng i vi mt s tc nhn thuc nhm nguy
c cao. V d, virus st xut huyt thuc nhm nguy c 2 s c nui cy iu
kin an ton sinh hc mc 2 nhng nu virus ny c dng tim chng
ng vt th mc an ton cao hn phi c p dng.
7.3. Cc bin php ngn chn nhm m bo an ton
sinh hc
Ti phng th nghim, cc bin php hiu qu nhm m bo an ton sinh hc bao gm
bin php ngn chn c hc v chn la qui trnh thao tc ph hp trn rADN. Cc bin php
ny c th phn thnh 2 nhm:
- Qui nh tiu chun: p dng vi phng th nghim vi sinh
- Qui nh c bit v qui trnh, dng c, b tr phng th nghim nhm mc ch to
hng ro bo v c hc cc cp khc nhau ty theo nguy him sinh hc.
An ton sinh hc cp 4 c kh nng ngn chn mnh nht v cp 1 ngn chn t nht.
Th nghim tin hnh trn thc vt. C 4 mc an ton sinh hc
- Mc 1: kh nng ngn chn km v cc th nghim chng minh rADN khng c kh
nng tn ti, lan nhim trong mi trng hoc khng c nguy c gy hi tim n i
vi mi trng.
- Mc 2: kh nng ngn chn ln hn do rADN c th tn ti, lan truyn tuy nhin
hu qu gy ra khng nguy him v d on c.
- Mc 3 v 4: kh nng ngn chn c bit do c th gy thit hi nng n n mi
trng v h sinh thi t nhin.
Th nghim tin hnh trn ng vt. C 4 mc an ton sinh hc
- Mc 1: p dng i vi ng vt c chuyn rADN giai on phi thai.
Nhng ng vt bin i gen ny khng c kh nng truyn h gen bin i t b m
sang th h con.

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- Mc 2: p dng i vi ng vt bin i gen v khng c kh nng truyn gen t
th h ny sang th h khc v gia cc loi, kim sot s chuyn gen do loi chn t.
- Mc 3 v 4: p dng i vi ng vt bin i gen c kh nng gy nguy him.
7.4. Cc th nghim phi tun th qui nh an ton sinh
hc
Theo hng dn ca NIH, c 6 loi th nghim lin quan n rADN phi tun th cc qui
nh hng dn.
- Cc th nghim cn s chp thun ca Institutional Biosafety Committee (IBC),
Recombinant DNA Advisory Committee (RAC), c gim c NIH xem xt v ng
trc khi tin hnh.
o V d, th nghim lin quan n s truyn tnh khng thuc, gy nh hng n
vic iu tr ngi, thuc th y hay nng nghip
- Cc th nghim cn s chp thun ca IH/OBA (Office of Biotechnology Activities, 1
t chc ca NIH) v IBC.
o V d, th nghim to dng sn xut cc cht c c LD50 < 100 ng/kg
- Cc th nghim cn s chp thun ca IBC v Institutional Review Board, sau RAC
xem xt trc khi c th bt u.
o V d, th nghim lin quan n s chuyn rADN hay ADN, ARN c ngun gc t
rADN trong cc nghin cu trn ngi
- Cc th nghim cn s chp thun ca IBC.
o Th nghim s dng sinh vt thuc nhm nguy c 2, 3, 4 lm h thng biu hin
vector/vt ch
o Th nghim s dng ADN c ngun gc t sinh vt thuc nhm nguy c 2, 3, 4, to
dng h thng biu hin vector/vt ch c ngun gc t t bo nhn nguyn thy
khng gy bnh hay nhn tht bc thp
o Th nghim s dng ADN hay ARN ca virus ly nhim hay b suy yu trong h
thng nui cy m
o Th nghim trn ng vt
o Th nghim trn thc vt
o Th nghim tin hnh vi > 10 lt mi trng
o Th nghim trn virus cm
- Cc th nghim c tin hnh ng thi vi bo co gi IBC.
o Th nghim to rADN cha khng qu 2/3 b gen ca bt k virus no t bo
nhn tht
o Th nghim trn loi gm nhm bin i gen
- Cc th nghim lin quan n rADN khng gy nguy him cho sc khe v mi
trng, khng phi tun theo hng dn ca NIH.
Ti liu tham kho:
1. Laboratory biosafety manual - Third edition WHO, 2004
2. NIH guidelines for research involving recombinant DNA molecules, 2011