Thuc Vat Chuyen Gen - Vaccine Thuc Vat

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vn
I HC QUC GIA THNH PH H CH MINH
TRNG I HC BCH KHOA
KHOA CNG NGH HA HC
B MN CNG NGH SINH HC
N MN HC CHUYN NGNH
TH C V T CHUY N GEN
VACCINE TH C V T
CBHD: TS NGUY N TH Y H NG
SVTH : NGUY N TH
AN ANH
MSSV : 60604018
TpHCM, thng 6 nm 2010
Tai lieu chia se tai: wWw.SinhHoc.edu.vn

MC C
DANH MC BNG .....................................................................................................iv
DANH MC HNH ....................................................................................................... v
CH NG 1: M U ................................................................................................. 1
CH NG 2: VACCINE V CC KHI NIM ....................................................... 3
2.1
2.1.1
n v
n m ................................................................................3
.......................................................................................3
2.1.2
2.2
v t ............................................................................................... 5
Vaccine th
2.2.1
2.2.2
C s
.........................................................................5
2.2.3
.......................................................... 6
u nh
2.2.4
2.2.5
................................................3
..................................................10
........................................................................................ 13
M
u trong n
i............................................18
CH NG 3: SN UT VACCINE TH C V T ................................................. 24
3.1
p n p
u n
n .......................................................................24
3.1.1
Agrobacterium ................................ 25
3.1.2
.................................................................................31
3.1.3
........................................................ 31
3.1.4
.................................................................34
n
3.2
n n u n v l
n tn
trn ......................... 37
3.3
Ti sinh cy .....................................................................................................37
3.4
N n
3.5
P n t
n
v
trn .....................................................................................40
m tr qu trn
3.5.1
3.5.2
3.5.3
u n
n ...............................................40
.................40
RN ..................................................................45
) ......45
CH NG 4: SN UT VACCINE VIM GAN B .............................................. 50

ii

4.1
B n v m
n B............................................................................................. 50
4.1.1
Virus vim gan B ...................................................................................... 52
4.1.2
Vaccine vim gan B ..................................................................................55
4.2 S to dn v b u
n
n n u n b mt v m n B (HBsA ) v o
t u l ...............................................................................................................57
4.2.1
HB
4.2.2
HB
4.3
l .............................................58

.........69
Kt lu n ...........................................................................................................79
TNG KT .................................................................................................................. 83
TI LIU THAM KHO .......................................................................................... 85
iii

DANH MC BNG
Bn 4 1 N
Bn 4 2 M
d d
in vitro
HB
ELIS
...... 66
..................................................................................................................... 67
Bn 4 3 M
HB
ELIS . ............................................................................................................ 69
iv

DANH MC HNH
Hn 2 1 B
..................................................................................................................... 18
Hn 3 1 M
Hn 3 2
................................................ 24
T plasmid .......................................................................................... 26
Hn 3 3 T
Agrobacterium ........................................................................................................ 28
Hn 3 4 S
Hn 3 5 S
Agrobacterium tumefaciens ............................... 29
Agrobacterium .................................................. 30
Hn 3 6 S
d ............................................................................ 32
Hn 3 7 My xung gen .................................................................................................... 32

Hn 3 8 S
Hn 3 9 S
.................................................... 33
...................................................................................................... 34
Hn 3 10 S
Hn 3 11 Q
.................................................. 35
........................................................ 36
Hnh 3.12
in
vitro .................................................................................................................................... 37

Hnh 3.13
....................................................................... 38
Hnh 3.14 S
Hnh 3.15 S
Hnh 3.16 S
Hnh 3.17 S
Hnh 3.18 S
......................... 42
........................................... 43
P R ........................................................................................ 45
W
............................................................................. 47
(
d

.................................. 47
Hnh 3.19 S
ELIS
Hnh 4.1 G
................................................................................................... 50
Hnh 4.2 B
...................................................................................... 49
HBV
...................................... 51
v

Hnh 4.3
HBV ...................................................................................................... 53
Hnh 4.4
HBV
Hnh 4.5 Vng T DN

Hnh 4.6 S
.............................................................. 55
( ) HBS, ( ) HER, (c) pEFEHBS, (d) pEFEHER. ........ 60

P R DN
Hnh 4.7 Phn tch PCR Southern b
Hnh 4.8 Phn tch RT P R
.................... 63
............................ 64
HB
gen ..................................................................................................................................... 65
Hnh 4.9 T
HB
ng ................................................................................................................................ 67
Hnh 4.10 Vng T DN
pHER100 v pHBs100 .................................................... 71
Hnh 4.11 S
i ........................................................................................................................ 755
Hnh 4.12 S
NT I
Hnh 4.13 P
EcoRI
P R DN
d
. ........................................................................................................... 76
.K
10 DN
699
BamHI
pHER100 ........................................................................................................................... 77
Hnh 4.14 L
HB
NT I
ELIS
............................................. 78
Hnh 4.15 P W
d
pHBs100
v pHER100 ...................................................................................................................... 78
Hnh 4.16 M

d
HB

cc
...................................................................................................... 79
vi
C n 1: M u
CH NG 1: M U
V
1980,
d d
.
d
Vaccine t
.L
(d
)
.Y
1990,
( q
)
q
,
, , d
q
d
,
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vong sau khi tim phng vaccine.
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(gi thnh cao,
).
DN
:
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1
C n 1: M u

d
S
2005,
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2
D w
C n 2: Vaccine v cc khi ni m
CH NG 2: VACCINE V CC KHI NIM

2.1 V
n v
2.1.1 K
n m
n mv
d
d
lo v
.
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n
Edward Jenner,
u ma, J
b
.D
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u b,
, J
N
J
. 48
T G
1796, chu u
.
,
n v n m
d
b
min nhim
Vaccine
b
i Anh. N
. L
V
2.1.2 C
d
P
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P
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P
,
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c ngy nay

:
[10].
80
1885, L
P
d
M
,
q
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d
3
J
.
g trong
[10].
H
[10]:
Mt l vaccine bt hot l vaccine cha vi sinh v t gy b

gi t ch t
bi nhi t hoc tc nhn ho h c. V d
n hnh ca lo i ny l vaccine nga
cm, t, dch h ch v vim gan siu vi A. Hu h t cc vaccine lo

ng min dch khng hon ton v ng n h n, cn phi tim nhiu ln.
Hai l vaccine sng, gim c lc l vaccine cha vi sinh v
y u. Nhng chng vi sinh v
c nui c
u ki
gi
c tnh ca chng hoc l nhng chng vi sinh v
c lm
c bi t lm
chng vi sinh v
bi
c tnh y
. L i vaccine ny
gy phn ng min d
d
ng s d
i l n. V d cho
lo i ny l vaccine nga b nh s t vng da, si, rubella v quai b.
Ba l vaccine cha c t bt hot ca vi sinh vt gy bnh (toxoid).
Chng h n vaccine nga u n vn, b ch hu.
Bn l vaccine cha cc tiu phn ca vi sinh vt gy bnh: Vaccine ny ch
cha nhng phn gy phn ng min dch m khng cha vi sinh v t gy b nh.
V d
HBV ( a vim gan siu vi B) ch cha protein v ca
virus Hepatitis, PLV ch ng HPV ch cha protein capsid ch ch t.
N
,
.V
d
.T

(
(
). V
,
,
DN
7
10 ). T
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q
d
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B. T
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4
q
(S
K
q
1,5
, 2003).
2,7 USD. T
10 20
,
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. V

V m t trong nhng l i th ca chng ta l ngnh Cng ngh

,
kha vng
pht tri
c cng ngh sinh h c. Nhng thnh t u v cy

trng bi
i gen trn th gi i khng ch gi i h
c nng nghi p, t o
ra nhng ma mng b i thu, gii quy t n
u m cn c th sn xut ra
cc lo i bi d c ch
c cc b nh hi
i. ng dng cng
ngh sinh h
sn xut nh d c ph m tng h p trong th c v
n
m nh vo nh
1990, ng h n: cc thnh phn c
t cu t ,
albumin, cc khng th , hormone, cc y u t
c sn xut t cy thu c
l bi
i gen (Goldstein v Thoman, 2004). Nhi

v
chuy n gen vo cy tr
t o vaccine th c ph
:
nh vim
gan B (Thanavala v c ng s 1995), v vaccine phng b nh t
c sn xut t cy
khoai ty (Arakawa v c ng s 1997). Kang v c ng s (2003), Kim v c ng s
(2004) dng Agrobacterium tumefaciens
chuy n gen CTB (cholera toxin
B subuint) vo cy thu c l (Nicotiana tabacum) v gen HIV-1 gp120 V3 vo
khoai ty. Daniell v c ng s (2001)
n gen CTB vo cy thu c l b ng
d i bom (bombardment) [2].

2.2 V
n t
2.2.1 K
H
v t
n mv
n t
v t
:
5
V
.G
(d
),
. N
"
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2.2.2 C s
N
n t
v t
,
3
2.2.2.1
T
2/3 d

)
Y
3
d
T
. N
(d
.
,
Bacillus thuringiensis (B )
( w
.
.P
.
d
,
)
d
6
)d
[3].
d
.N

.
T

(
d .
.
d
g tim hay qua
. S

.

.
, ,
,
Th nht,
c ca vector cng nh cng t t, v d xm nh p vo t
, ng thi thu n l i trong qu trnh sao chp.
7
Th hai, vector phi c kh
th
n, nh DN
i c
c duy tr trong qun th t bo.

c tnh cho php pht hi n d d
Th ba, mang nh
c m ho
bi cc gen ch th ch n l ,
a gen ch th thao tc trong E.coli v
gen ch th
ti n hnh ch n l c th c v . T
ng l cc gen
khng v i khng sinh, nh
l a ch
c t bo mang gen chuy n
(vector) t qun th l n cc t bo.
Th t, cha t nht 1 v tr nh n bi t ca enzyme gi i h
lm v tr ghp DNA trong t o th ti t h p.
V
. V
,
.N
DN
s dng
DN
2.2.2.2
H
,
,
q
d
d . K
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d
d
)
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a.
c
n vaccine, ph
ch ph
nh khng nguyn ca
n y u t quy
. Y u t quy
c ch
nh khng nguyn chnh
l thnh phn protein khng nguyn c trn b mt ca tc nhn gy b nh,

hay trn b mt ca ch ph m vaccine ca chnh tc nhn gy b
.
Vaccine truyn th ng hay vaccine ti t h p l cc ch ph m sinh h c ca vi
c lm gi
c l c, khng cn kh
iv
.K
u c kh
sinh min d . K
sinh v
ng nh
,
chu s kch thch ca khng nguyn, s cm ng sn xut ra m t lo i

protein m i c ch
o v g i l khng th (antibody) [2].
nh ngha : Nhng ch
s
hi
ng min d
c g i l khng nguyn.
Th ba, cu trc phn t phc tp :

d
.
(
q
,
Th hai, trng lng phn t ln : K

10.000 d
(
).
C khng n
.K

gy ra
Th nht, n phi c tnh l :
:
(
).
Phn loi : N i ta chia khng nguyn vi sinh v t ra cc lo i sau:

Khng nguyn vi khu n, bao gm:
Th nht, khng nguyn vi khu n ho tan l cc enzyme ngo i
bo.
9
Th hai, khng nguyn vi khu n khng ho tan l thnh phn t

bo ch y u n m trn b mt t bo, 2 lo
c quan tm nht l
khng nguyn lng (k hi u H), v khng nguyn thn (k hi u O).

Th ba, cc ch
c (toxin) khi b m
c tnh tr thnh gi
t (
d)
khng nguyn cn quan tm.
c
t
Khng nguyn virus, gm:

Th nht, khng nguyn V : bao gm 1 phn ca virus hoc c
virus.
Th hai, khng nguyn S : l khng nguyn ho tan c th l v
glycoprotein hay nucleic acid ca virus.
b.
K
d q
.K
d ,
I (I
: I G, I M, I D, I
2.2.3 C
u n m
n).
. 5
, IgE [4].
v
n t
v t
2.2.3.1
S
, accine
ph m c
u
sau: gi
thnh r, , d n xut trn quy m
, d qun l, khng n tinh
s h, bo qun lu, d n chuy
An ton: V
d
.D
,

. D ,
.
Quy m sn xut:

,d
d d
10
.V
Tnh n nh cao, d bo qun v s dng:

d
q
.N
)
d
d
.T
.D
,
G
q
d d
c phn phi qua ng ming: N
),
Vaccine n c kch thch sn xut khng th ca h thng th dch hiu

qu hn vaccine tim: T
.K
d
d
d
d
.K
,
,
E.coli,
d .
d d
Chi ph khc: S d
d d
cc chi ph khc.
V
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V d: I G
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500 USD/ ,
600 USD/ . M
I G
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f f
500
50

,
2.2.3.2
T
(0.01 2%)
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. V d
d
d .
q /
. V d, q
.
3 .
,
B 10 100
E. coli
.H
q
.
gii quyt nhng hn ch ny mt s k thut, bin php c s

dng:
T
khng nguyn bi u hi n trn th c v t.

Bi u hi n trn cc plastid.
S dng virus th c v
chuy n gen.
Dung h p protein.
12
S dng cc promoter, enhancer hay trnh t k t thc ly t virus th c v t.

u khi
Gen khng nguyn k t h p v i cc nhn t
u khi n s bi u
hi n protein, thi gian bi u hi n trong chu trnh pht tri n ca cy v b ph n

bi u hi n.
S d
d
,
c s dng l LT B, CT B, tc
u khi n min dch c

ng v t v virus, h p cht th cp ca th c v t.
Khi khng th bi
i trong th c v t th khng th

,
dng vaccine khoai ty khi khng bi

c
ng protein khng nguyn tch lu
,
t v nan gii.
T
i v i m t s lo i cy nh nh nh nghin c
m t
cch l c th thi t k chng thnh nhng vin thu c hay nhng mi ng bnh v i
.
ng chnh xc protein khng nguyn tron

2.2.4 An to n s n
.N
GMO).
q
2.2.4.1
R
.G
S pht tn c
ng.
Chuy n gen t th c v t sang vi sinh v t.
Chuy n gen t th c v t sang virus th c v t.
Ngoi ra cy chuy n gen cn c th gy
ng cho cc sinh v t khc.
2.2.4.2
S
.K
q
,
13
.T
1000
(1987)
.T
.D
.
d
[5].
f w) ,
.V
d
.N

g).
(
,
2.2.4.3
(
d
q
f )
(
.

,
,
.N
c [5].
DN
. V d,

.P
f )
d

)
),
,
.
14
cng
.T
, d
t [5].
d
d
. V d:
B,
cc retrovirus
,
. N
z
,
.
d
, q

IDS
. MV
men, cn trng v E. coli.

d
.
S
php c
d
d
.
DN
,
15
,
khc trong geno
ng khc
[5].
d
chuy n gen t th c v t vo vi sinh v t.
u qu sinh thi.

DN
.N
Tm hi
.T
. V
DN
d .V
[5].
2.2.4.4
K
d
d

)
.D

1986.

.N

, d
[5].
2.2.4.5
T
1999,
M
M
.B
16
.M
(M )
d
q
.T
2.2.4.6
V
d
d

. S d
.N
.D
:V
. N

i vaccine ny [5].
d
ny khng
17
Hn 2 1 B
D
m v yu cu dn nhn trn cc sn ph m
cng ngh sinh h c [16].
:d
do chnh protein khng nguyn.

N
.D
2.2.5 M t s t
d
.
n t u tron n
v trn t
2.2.5.1
Trn Th gi i tnh cho t i hi n nay c rt nhiu thnh t u v
xut vaccine t th c v ,
t vi v d n hnh:
c sn
H T
N
, H G f
)
NI S (J
16
18
( TB).
,d d
d
d
d
50 /
;
.
d
.
GM
GM
.H
[17].
Sn xu t khng nguyn HIV t cy chuy n gen

V

HIV
T
.G
q 100
IDS
HIV 24,
d
HIV,
N f
d
[18].
24 N f
.
24
(HIV)
V
d
vaccine.
IDS. M d
20
40%
[18].
19
NSH,
j ff
,P
Variola
.N
.L
;q

[19].
. B
,M ,
,
d

N
,

d
d

L
X ,
. Y. pestis

Y. pestis
, d
Yersinia pestis,
P ,
d
q
,
.M
S. M
.T
2
1
q

Y.pestis
,
.L
d
:
F1,
Y.pestis, khng nguyn V, c tham gia
F1
V.
2
Agrobacterium
[20].

d
100%
tumefaciens. S
nguy
, 3
;
, 3
; ,
Y pestsis
d
20
21
Vaccine ng a d ch t t g o
N
d
.M
.V
, d q ,
Theo

[21].
d
.

. S
,
vaccine d
.K q

d
d d ,
, Vibrio Cholerae, vo trong
30
d .
d
,
.
2 tng:
[21].
T
,d
K
,
.T
Nhm nghin
H
T
. T
K

,
,
: T
d
;
,
d
. D ,
21
d d
P ,M L
,N
nng d
[21].
2.2.5.2
H
,V
9/10
UNI EF
.N
.
q

V
S
N
B
cy c chua.
TP.H M
Q
V
q
q
N

.M
, ...
10 - 15% d
B (HBV). T
(
q ,

HB

, ...
).
.V
D T N
Agrobacterium. N
d
d
,
d
( )

,
W ff
HB
22
10
,
G d
q , ,
, ...
Agrobacterium
tumefaciens,
V
S
N

a mang gen m ho khng nguyn
B (HB
) [22].
23
C n 3: Sn xut vaccine th c v t
CH NG 3: SN UT VACCINE TH C V T
Hn 3.1 M hnh minh h a sn xut vaccine th c ph m [3].
3.1 C
p n p
u n
DN
d
ny.
d
:
24
DN
( d
,
.T
. K
),
q

.M
),
.T
:
).
d
),
DN
d-DNA (liposome), vector

Agrobacterium,
),
d
... T
3.1.1 C u n
3.1.1.1
n t p b n v
: Agrobacterium
:V
Ngnh : Proteobacteria
L
:
u n Agrobacterium
Agrobacterium
P
G
B : Rhizobiales
H : Rhizobiaceae
G
: Agrobacterium
: Alpha Proteobacteria
Agrobacterium
Agrobacterium
tumefaciens v Agrobacterium rhizogenes
,
d
. Agrobacterium
. T
. Agrobacterium
A. tumefaciens v A.
25
rhizogenes
d
R
A. tumefaciens
d.
.K
T DN
T DN . N
.
T DNA
.G
3.1.1.2 Ti plasmide
Ti
q
.T
.
d(
d)
200 250 kb,
.T
T
d
Agrobacterium,
TDNA v vng
TDN
Hn 3.2 Cu t o Ti plasmid [24]

3.1.1.3 T DNA
T DN
7
T

.T
DN
25 - 35
25
, B,
,
D, E, G,
,
,
T - plasm d, T DN
26
H
RB
J.
LB. Ngoi T DNA, trn Ti
d,
DN
),
ho opine.
3.1.1.4
. K
Agrobacterium
H
d
G. S

TDN
A.tumefaciens q
,

.K
d A. tumefaciens
1
DN T plasmid

.
M d
Agrobacterium
.
,
tumefaciens [1].
(T DN )
3.1.1.5 G
V
d
E2,

DN
Agrobacterium rhizogenes
T DN A.rhizogenes

,
auxin,
.K
TDN

d d
2

A.
DNA
B,
,
D, ...
T-DNA, bao

.
Vai tr cc gen vir c th tm lc nh sau:
27
virA : Nh n bi t tn hi u t bo b t
t virG.
virG : Ho t ho phin m gen vir.

virD1 : C
VirD2 :
n TDNA.
H ng dn TDN
ng nhn t bo th c v t
Bo v 5 a TDNA
Gip TDNA g n vo nhn t bo th c v t.
virE2 :
Bo v TDNA
H tr v n chuy n TDNA
virB211 : T o knh chuy n TDNA.
virD4 : T o knh chuy n TDNA.
virF : H tr g n TDNA vo nhn t bo.
Hn 3.3 T o th c v t chuy n gen b
khu n Agrobacterium [3]
n gen gin ti p nh vi
28
Hn 3.4 S chuy n gen thng qua Agrobacterium tumefaciens [5]
29
Hn 3.5 S ly nhim ca vi khu n Agrobacterium [5]

P
.T
Agrobacterium
d

,
tumefaciens d
d
[1].
A.
30
3.1.2 C u n
T
n tr
t p
DN
3.1.3 C u n
.T
DN
.Q
n c vector [1].
.T
q
)
2
),
.P
DN
,d
pect

q ,
PGE
Agrobacterium
,
.V
Japonica (Datta, 1990), ng (Doon, 1990), la m (Vassil, 1992).

nb n
t u t un n
n (electroporation) l m
c s dng
phn c c vo trong t bo ch qua mng t
.T
php ny, m
n cao th trong khonh kh c (vi phn nghn giy) c kh
i lo n cu trc mng kp phospholipid, t o ra cc l thng t m thi cho

php cc phn t DNA ngo i lai t
ng xm nh p vo bn trong t bo [1].
K thu
Nhiu k thu t nghin cu trong sinh h c phn t yu c

c protein
ngo i lai vo trong t bo ch. V l p phospholipid kp ca mng sinh cht c m t
31
c pha ngoi v m
c c no, bao gm c DN
c pha trong, nn bt k phn t phn

, u khng c kh
cch t do (Farabee, 2001).
Hn 3.6 S mng phospholipid kp [1]

S ny cho thy cc thnh phn ha h c ca mng sinh ch .
c phn c
c c khng th
Nhi
ng v
yn

ng v pha
cng bm gi mng. Cc phn t phn
h tr bn ngoi.
c pht tri n
t qua ro c
,
DNA v cc phn t khc vo trong t bo
c nghin cu. M t trong nhng
thu
n.
K thu
n d a trn tr ng thi
i y u c
ng tc k
c
ca phospholipid kp v kh
ph p
l i m t cch t
ng ca n sau khi b r i
lo n (Purves, 2001). V v y, m t xung
Hn 3.7 My xung gen (Gene pulser)

(Hng Biorad) [1]
n ch p nhong c th gy ra r i lo n cc v tr ca mng m t cch nht thi,

lm cho cc phn t phn c c c th
q ,
i
nhanh chng v t bo khng b
ng g c.
Cc t bo ch v DNA ngo
trong m t cuvette nh
n c c.
c t o thnh dch huyn ph v cho vo
32
t
n cao th trong m t thi gian ng n n

(
b g
my ny c th
)(
). Q
c trnh by b
i ta s dng m t thi t
n din ra bn trong
S ny cho thy m
bn cung c
cng t c th nh ,
nn
n
vo v tch m
n p cao. Khi cng
n cho k thu
n Khi
t c th
,
n p ny phng qua
dch huyn ph t bo. M
n
cn thi t cho k thu
ng l
khong 10.000-100.000 /
(
i
c ca t bo) trong vi
phn tri
n m t phn ngn giy.

X
n ny lm r i lo n phospholipid
kp ca mng t bo v t o ra cc l t m
thi. Kh
n qua mng t bo
0,5 1,0V v v y cc
phn t
cn
n di [1].
Hn 3.8 S b tr m
n ca
n [1]
bo thng qua cc l b ng cch thc
L DN
bo khng th quan st th d i knh hi
,
v ny cho thy khi ni

n ca s t o thnh cc l trn mng m DNA c
th
q .
K
n v cc phn t q
, mng t
n
v cc l
i m t cch nhanh chng v phospolipid kp phc hi l i cu

(W
, 1995). L
mong mu
trong t bo v
chng
c s dng cho cc nghin cu ti p theo.
P
s d
i v i g
t c cc lo i t bo ca cc
.L
c s d
chuy n gen vo cc t
ng
v t c v, v sau cho c t bo th c v t d ng protoplast ... V i m t s cy m t l
mm quan tr ng (loi la ph Japonica, ng, la m) m khng th th th c hi n
cb
n gen gin ti p nh Agrobacterium
thnh cng v
. H u qu bi n n p cao. Trong m t nghin cu

E.coli, 80% s t bo nh
c DNA ngo i lai (Miller v Nickoloff, 1995).
L ng DNA ngo i lai cn thi

(W
,
1995). P
th c hi n v i cc m in vivo cn nguyn vn
33
(Weaver, 1995). o n DNA ngo
c bi n n
v chi d
tr nn qu l n hoc b hng khng th
c l n. Tuy nhin
t s l ca t bo s
i sau khi t
n, lm cho
t bo b t
c b thng (Weaver, 1995). M t h n ch na l s v n

chuy n DNA ngo i lai vo v ra khi t bo trong su t th
n bi n n p l
c hi u. iu ny d
n k t qu l khng cn b ng ion m sau
lm r i lo n ch
3.1.4 C u n
a t bo v t bo ch t (Weaver, 1995) [1].
nb n s n b n
Sng b n gen (Gene gun) l m t thi t b s d

t
n vo
,
c thi t k u tin cho bi n n p DNA ngo i lai vo t bo th c v t v
c pht tri
u th p nin 1980 do cc nh th c v t h c i h c Corrnell
cng v i cc nh nghin cu Corrnell Nanofabrication Facility, Newyork, USA.

Sng b
c bn trn th
1990. n s dng cho lo i sng
ny l cc h t kim lo i n

c bao b c DN . T

ca sng b n gen l h th ng phn ph i h t biolistics (biolistic particle delivery
system) v k thu
cg im
n l biolistics (s k t
h p gia hai thu t ng biology (sinh h c) v ballistics (s b n tung)). Mc d c
nhiu thi t k k thu
s dng p l c xung c
gia t c cc h t [1].
Hn 3.9 Sng b n gen (Hng Biorad) [1]
Sng b n gen bao gm hai bung b ng thp khng g, kch th

6 7 10
n i v
. DN
c g n vo cc h t tungsten c
34
ng knh rt nh, kho
c s d
ng xuyn do gi c
t). Cc h
t trn
mt bn trong ca sng. S bng n kh helium 1000psi lm cho
n v
cv it
1300 f d/ ,
n ri khi nng sng. M t tm ch n lm d
c phng v pha cc t
.
it
khi m t
i v cc h t vng hay
q
bo v
phng thch cc phn t DNA (Hnh 2.21). Sng b n gen s dng k thu t DNA ti
t h
h p nht s bi u hi

i. Cc t bo bi
i di truyn
c th
c s d
t o th c v t bao gm c s s i di truyn mong mu n
trong tt c cc t bo ca chng (Voiland, 1999) [1].
Hn 3.10 S nguyn l ho
ng ca sng b n gen [1]
m l thao tc d dng, c th chuy n gen vo nhiu

lo i t bo v m, cc t
c bi n n p c t l s
gen vo t bo v tr mong mu n ...
35
Hn 3.11 Quy trnh chuy n gen b ng sung b n gen [5]

T
Gen Bt
. V
(B
d
d , 1997).
d
. Bacillus thuringiensis
1 )
.G
, DN
Bt.
, 1994). Trong khi
(R
Bt. S
DN
36
d
d .
3.2 L a chn khng nguyn v l a chn tng cy trng

T
. T
. T
q ,
.T
q . D
.
.
T
,
. D q
. P
.K
. D
d
3.3 Ti sinh cy
Hnh 3.12
c ph bi
pht tri n cy hon chnh trong nhn gi ng in vitro:
c ph bi
pht tri n cy hon chnh trong nhn gi ng in

vitro [3]
37
T
in vitro
.T
Hnh 3.13 Callus (A) v ti sinh chi t callus (B) [3]

q
M th c v t :
ng tng libe m , q
bo di p nhc l v m ti
ng t .
thi k
ng m nh nh
d tr, tr b r,
,
m, t
c ch n ly m phi l cy s ch b nh,
m bo m c sc s ng cao nht.
Kh trng: Mu cy l nhng m s ng nn ph
c kh trng b ng cc dung
dch kh
gi
c tnh sinh h c ca n. Cc dung dch kh trng
ng l: hypochlorite calcium, hypochlorite sodium, chlorur thy ngn, H2O2
Chun b mu: trong qu trnh t o m so t l, m s
ng xut hi n v t c t,
a l hay d c theo h th ng m . D
ct o
v t c t ngang h th ng m ch (gn chnh l).
T o m so: mu sau khi t o v t c
c chuy
ng pht sinh callus.

D
ng ca cc ch
ng, cc t bo s
phn phn ho, phn chia hn lo n t o thnh m so. Trong qu trnh nui cy m so
c phn c
ng xuyn v cy chuy
ng m
n
c s
d
chuy n gen. Ty theo tng lo i mu m s t o m so cn hay khng cn nh
ng h p s t o m s
ng t i t
. iv i
l khoai ty, nn t o m s
u ki
u ki n t ,
i v i l cy
thu c l, m c khoai ty nn t o m s
u ki n chi u sang.
Trong qu trnh pht sinh m so, ch
c bi t
quan tr ng. Ch
c s dng l auxin. Tu thu c vo

v t li u nui cy m lo i v n
auxin s d
.
c s
38
d
kh
5M). i v
u v duy tr m so gm IAA (1010 105M) v NAA (1010 10

cc mu cy hai l m
t o m so cn s dng ph i h p auxin
v cytokinin.
Ti sinh chi: S
ng t o chi. Ch
d i). Trong m t s
c chuy n gen, m s
c chuy
c t o thnh mt m so ti p xc v
ng h
ng nui cy m so ch
ng cm
ng (mt
c t o ra
pht tri n chi cn phi chuy n m
ng ti sinh chi. Qu trnh

ti sinh ch
ng l n, nhi
cao v cn chi u
sng. S dng ch
ng trong ti sinh chi kh phc t p. Nhn chung
hu h t cc loi th c v t c
ph i h p v i n
auxin th
cm ng s t o chi. N
cytokinin cao
q
ng trong s t o chi nhiu lo i
th c v t khc nhau. Khi chi ln v i m
c nh nh tu theo loi th c v t,
i ta ti n hnh tch cc ch
ng ti sinh r invitro.
Ti sinh r invitro:
n ny ko di 2 4 tu . mu c th cm ng ra r
c cn chuy n m
ng chuyn bi . M
ng ra r
ng phi c
auxin v khng c cytokinin. Tuy nhin m t s loi th c v t s ra r cn c s c mt
c
.
c b
ng ra r
ng l IAA (0,1
10mg/l), NAA (0,05 1,0 mg/l) v IBA (0,5 3,0 mg/l). R hnh thnh trong mi
pht tri n tro
ng c auxin, nn
trong m t s
ng h p ch
ng c auxin ri chuy n sang
ng ra r. N
ng gim xu ng m t na
so v
. ng vn cn cho s ra r,
f
sucrose. S d
ng l
ra r t
c. T t nht l nn cho
ra r trn m t qu cu giy c
ng lng. Trong m t s
ng h p
khng nht thi t phi chuy n mu ra kh
ng ti sinh ch dng h t dinh
d ng m ta c th b
ng ti sinh r lng vo bnh nui cy n u mi

ng ti sinh ch
c. Trong qu trnh ti sinh r nn h n ch

ng
ca nh sng do nh sng cn s ra r. Ti sinh r s din ra t
nhi
mi
ng cao.
Ti sinh r invivo:
c chuy n ra khi bnh nui cy, ch
c cy vo m t
hn h
t
gip cho s ra r. Hn h
t c th l than bn, v cy,
khong ch ,
,
, , t v c th tr n thm m
ng nh phn bn. Mi
ng cho s ra r invivo l c pH trung tnh ho

d,
i thng thong. Khi cy pht tri n hon chnh c th ti n hnh cc th

nghi m ki m tra s h p nht ca gen khng nguyn vo genome.
39
3.4 Nhn ging cy trng
N
T
50 100 . K
.Q
.N
.G
3.5 Phn tch v ki m tra qu trnh chuy n gen

3.5.1 Ki m tra s c mt c a c a gen ti t hp genome th c v t
T
,
DN
DN
P R
d d
DN
DN (1
= 1/1000
.T
,
P R
),
.P
3.5.1.1 P
DN
,
P R
Nguyn t c ca k thu t ny l d a vo s lai phn t, d

u ki n
thch h p hai chu
d o thnh m t phn t lai. Phn ng lai ph
thu c rt nhiu vo m
ng ca hai trnh t nucleotide. Vi c t o

thnh phn t s
th xy ra ch y u thng qua lin k t hydrogen gia

cc base G v i C, v A v i T. Thnh phn v s phn b ca cc base khng
gi ng r r t v i cc phn t nucleic acid cho k t qu cc tnh cht lai khc nhau,
v th lin k t hydrogen (hoc lai phn t gi
)
c
40
ghp cp thch h
p m t cng c c gi tr
nh cc chui
ng nht v i chui nucleic acid quan tm (mu d).
lin quan ho
Trong s cc k thu t khc nhau s dng lai phn t
phn tch nucleic
acid, th k thu
c s dng ph bi n nht l lai m d
du v
d
c c nh trn m t v
r ,
mng nitrocellulose hoc nylon.
Trnh t ca k thu t lai Southern blot bao g
c sau [6]:
u tin, ph
n c t h n ch ca genomic DNA b
agarose gel.
(
Th hai, chuy n DNA t

na kh (a)
Th ba,
d
n di
m tch
m tch mao dn (b))
d
c
ng l
32
cc nucleic
41
Hnh 3.14 S minh h

Q
(
DN
S
:
d )
d
d ( )
)
,
DN
Xq
(
, DN
d
q
B). DN
d
32
d,
.S
n di agarose gel [6]
d
.
,
d,
B),
DN (
d
d
.T
,
,
DN
d
.T
.
DN
B

42
z
.S
. N
[6].
Hnh 3.15 S k thu t c
S
v genomic DNA trong hnh ny
c c t b ng EcoRI
n di trn agarose gel)
3.5.1.2 P
DN
n b ng
P
olymerase)
Nguyn tc ca PCR : Taq polymerase l m t lo i enzyme DNA

polymerase chu nhi t (c vi khu n chu nhi
cao Thermus aquaticus)
d
tng h
n DNA m
ng c b n lo i
d
d (d TP, d TP, dGTP
dTTP)
,

khun mu ca m
n DNA nh
t ho
t trnh t . Cc
n DNA m i hnh thnh l
c s dng lm khun mu. Sau nhiu chu k,
s
DN
c nhn ln gp nhiu ln, nh v y c th s
43
tch ra, phn tch trnh t hoc t o dng... Primer

trn s
DN 3 5
ng trn s
ng
c g i l primer thu n (k hi u l F). Primer bn phi
DN 5 3
cg
c (k hi u l R) [6].
T chu k th hai Taq DNA polymerase (g i t t l Taq pol) b

u t o ra
n DNA c chi d
.
ng l m t oligonucleotide
tng h p c khong 10 20 nucleotide ho . N u bi t trnh t c
n
gen cn khu
i th c th tng h p nhn t

th c
hi n PCR v tch chng ra b ng k thu
d.P R
ng ti n hnh khong
-6
25 35 chu k, q
10
DN
u c th khu
i ln t i trn 1
mg (khong 2 kb).
Mi chu k PCR bao g
n c nhi
khc nhau:
Gy bin tnh (denaturation) 90 95oC

T
n bi n tnh, phn t DNA khun mu d ng xo n kp
c tch thnh hai s
. Tt c cc phn
z
n
u b dng l i (v d: phn ng tng h p DNA t chu k
).
o
Gn mi 40 65 C
T
n ny cc primer g n vo cc v tr c trnh t
DNA khun mu. Cc primer b l c nh chung quanh do chuy
ng
ng
Brown v th cc lin k
c t o thnh v b t gy lin tc gia
primer s
DN
u s
.
t ion n
t o thnh m
n nh (
)
n
nh DNA s
(
u v primer) Taq pol c th b u qu trnh

sao chp khun mu.
n ny ph m vi nhi
c s dng c th
rt r ng ty thu c vo trnh t nucleotide c
,
ng khong
55o ,
35oC ho
n 68oC.
Ko di phn t nhi
70 72oC.
ng nhi
t i thch cho Taq pol ti n hnh tng h p DNA
b ng cch b sung cc dNTP b
u t cc v tr c primer theo chi 5
3.
t vi base g n vo khun mu c m i lin k t ion m nh
c ph v cc lin k t ny s khng b t gy. Cc primer cc v tr

khng b t cp chnh xc l i b ri ra (do nhi
cao) khi khun m
khng tng h
c DNA.
44
Hnh 3.16 S phn ng PCR [23]

3.5.2 Ki m tra s tn ti c a mRNA
RN
RN
.T
P
d
...)
RN
. P
d
RN

(
RN d
RN
RN
d
d d ,
(d
q d
d ). T
RN
.
,
nh m bi u hi n c a protein khng th (protein ngoi lai)
3.5.3
Thng th
,d
d

q
. T
45
B
.N
,
35
[ S]
d
35
100 C
[ S]M
d SDS polyacrylamide gel
[6].
Phn tch Western blot b ng cch dng cc khng th lin k

c hi u v i
q
c th m tch ln mng nitrocellulose sau khi th c hi n di n

di SDS PAGE.
N u m
bi u hi n th
t gen lacZ
ng v
c bi u
hi . N
y, n u s phin m hoc dch m h n ch bi u hi n ca gen th nhng
i trong h th ng bi u hi n c th
ho t tnh c galactosidase.
i trong
c ki m sot b ng nh
3.5.3.1 Western blot

P
,
K
khng nguyn
d

d )
.N
d
), d
,
ngu
)
W
.N
d SDS-P GE,
) q
) (H I.2.17).
I.2.18. S

. V
.
-d
d
d
K
q
d
.
-rad
.S
d )
[6].
46
Hnh 3.17 S k thu t Western blot [6]
Hnh 3.18 S m t lin k t protein (khng nguyn) v i khng th th nht

c hi u v khng th th
du enzyme trong Western blot [6]

S
in situ (in situ
dz
47
W
.N
( ff
). K
d
ELIS .
( z
3.5.3.2 ELISA
ELIS
nguyn
d
d
.M
,

z
(
( nh I.2.19) [6].
d
(
ELIS
). M
,
.
W
D ELIS
). K
)
(
(
khng
48
),
Hnh 3.19 S k thu t ELISA [6]
49
C n 4: Sn xut vaccine vim gan B
CH NG 4: SN XUT VACCINE VIM GAN B

4.1 B nh vim gan B
T trung tm nghin cu v
ng y h c Brown Providence cho bi t
ung
u m t bo gan l m t v
q
T gi i, v t l
M . Khong 90 95% nhng kh i u ny l k t qu t nhng
h u qu sinh h c do virus vim gan B (HBV) v i kh
n t i cao v s nhim
virus vim gan C (HCV). C khong 450 tri u b nh nhn m c b nh m n tnh do HBV
v 200 tri
i m c b nh do HCV. Chui lm sng chung ca nhng s vi c ny l

,
s ti p xc HBV s m trong cu c s
v phc hi l i, s pht tri n ca ch
n tnh, t
.N
n ca ung
bo bi u m gan vo khong 25 35 ln v i nhng b nh nhn b nhim HBV

m
i khng b nhi ,
ng 130 ln ca
nhng b nh nhn b nhim v i c HBV v HCV
i khng b nhim.
S
i ch
d
nh gan
q
n HBV trn ton Th gi i l
khong 1.2 tri
i [25].
B nh vim gan B l m
B (HBV)
.K
nh t
.
nh ny do siu vi vim
i l n v tr em nhim HBV, h min dch ca h
c th ch ng l
nh ny. H b nhim vin gan B cp tnh trong thi gian ng n.
Nhi
i b nhi
ng khng cm thy c tri u chng g v th m ch khng
bi t khng bi t l mnh b nhi . N
ng 5% s
i l n v 90% tr nh ti p
xc v i HBV khng th lo i b
nh ny.
Hnh 4.1 Gan b
(d
i)
50

V
,
G
Bd
40%
B (HBV)
1/3 d
98%
350
.N
[11].
Hnh 4.2 B
phn b nh
c b nhim HBV m n tnh [15]
Vi t Nam l m t trong nh
c v i t l vim gan B cao nht th gi i. D a
theo th
i trn Current Probl. Cancer 6, c 4

i Vi t th c m
i
m vi khu
B(
ng 25% tng s dn chng c th ly

b nh c
i khc, m khng h hay bi t). Theo m t trong nhng th ng

k m i nht th t l
i mang b nh l khong 17%. Tuy trong nh
a qua,
khi chch nga b nh vim gan B t i Vi N
c ng dng m
q
,
con s b nh nhn b b nh vim gan B, ch thuyn gim m t cch ch m ch p. D a theo
m t nghin cu c H
The American Journal of Tropical Medicine

and Hygiene (2003), trong lc th nghi m khong 1350 dn chng s
a h t
Thanh Ha, c khong 12.5%
( 9 n 18 thng), 18.4% tr em (t 4 n 6
tui), 20.5% thanh nin, thi u n (t 14 n 16 tu ) 18.8%
i l n (t 25 n 40
tu )
nhim trng bi vi khu
B. d a theo ti li u ny, s c
51

khong 15 25% b nh n
ng 3.5 tri
ng t i Vi t Nam s la trn v nhng bi n chng ca b nh ny. y l m t con

s r
i cho m
V N
q
c xp x 100 tri u dn [13].
HB
,q
,d
d
,
HBV
d HBV
.N
,
HBV

,
,q
d ,
,V
HBV
4.1.1 Virus vim gan B

HB
4.1.1.1
Virus vim gan B (siu vi trng HBV) thu c lo i siu vi trng

Hepadnaviridae v i kh
n t i cao. HBV l virus mang DNA hai s i
khng khp kn, c tr
ng phn t 2 x 106 D
,
c cu t o bi 3200
base, v
i xng hnh kh ,
c khong 27 nm, v ngoi bao
kho 7
c cu t o bi 3 protein cu trc: protein l n, protein trung bnh
v protein nh; v bao t o cho virus c hnh c
ng knh 42 nm.
H
HBV
enzyme polymerase. Trn
P (P
)
4 ,

ring: Terminal protein, Spacer, RT domains v RNAase H [14].
T
RT
ny
q
(RT d
DN
:
7
)
RN
RN
B
[14].
DN
G. V
52
DN
HBV
,
HBV
HBV
.S
RN
trung gian (DNA RNA DNA).

N DN
, HBV
DN
)
, DN
d
D
RN .
RN
DN
. P DN
d
Hnh 4.3 Cu t o HBV [26]
DN
virus.
HBV bn vng v i nhi
: 100oC th virus s
n s ng t i 20 . HBV
c 30 pht, 20oC
t ho t trong formalin (foocmon).
Xt nghi m mu c th c 3 lo i HBV v
c kch c 22 nm, 42nm,

v 22 200 nm. Khng nguyn HBcAg ch c
c 42 nm. Trong
mu b nh nhn c c h t nhim v khng nhim (virion). Cc h t khng nhim
khng c genome ca virus (dsDNA) nn khng c kh
nh. Nng
10
cc h t khng nhim c th ln t i 10 virion/ml. V v y c t i 65% b nh
nhn c HBsAg khng c tri u chng b nh, 35% c cc tri u chng ca vim
gan.
Genome gm m t DNA c phn g
antigen:
ng 3,2 kbp, t o nn cc
HBsAg (khng nguyn b mt) : thu c l p v ca HBV dng trong xt

nghi
bi HBV
.K

13
17 25
,
10
/1 .
(
) ,
20
d
,
11

10
/1 .H

53
1012/
HB
HB
[27].
HBcAg (khng nguyn li) : thu c l p li ca HBV d
bi t HBV
n.
HBeAg (khng nguyn n i sinh) : n u c trong mu b

kh
t cao.
.
Gen X c th l nguyn nhn t

M
.N
B
B
B. N
HB
HB
HB
HBsAg
d
B. N
q
HB

HBs

d
.S
HB
d
HB
4.1.1.2
C ch: HBV
.S
DN
DN

d
RN
.N
DN

, DN HBV
,
,
. T
DN ,
HB
[28].
54
Hnh 4.4
xm nh p ca HBV vo t bo [28]
,
DN
HBV
d
( DN ).
DN
(
0.7 ; 2.1 ; 2.4
RN . RN d
DN
3.5 ),
RN
d
B (HBV)
,
DN

ent
4
RN
,
.N
q
o [25].
4.1.2 Vaccine vim gan B

Vim gan B l m t trong nh
khong 350 tri

i m c b nh mn tnh trn th gi
ng trn ton cu. C

c tnh c 75 100
55

i trong s
tri
ch
u m t bo gan. S
u tr l kh t n km v t l cha khi b
Vaccine vim gan B (Engeix-B) 1981. N 3
90%
.N
.T
,
cine
M
HBV d
.M
d
N wE
T Y
dJ
vaccine Engerix-B
MS. M
B
1991
1998, 18

q .
DN
.T
(MS) -
1990
e vim gan B. Vo thng 2 2001,
e
B
MS
fM d
.T
,
H
d

q
[12]. T
MS
q
e vim
(SIDS). T
B,
e
,
K t qu ca s xut hi n ca cng ngh DNA ti t h p trong vi c pht tri n

vaccine rDNA s dng n
t h th ng bi u hi n. Chi ph cho
vaccine phng nga ny bao gm cc
m chng m r
, c bi t l
trong cc qu
n. Sn xut vaccine d a trn th c v t, t
trong cc ph
q , c th cho php pht tri n hi u qu gi thnh
v l
a l c th chp nh
c vaccine.

.N
,
.K
56
,
M
, 1992; K
, 1999; R
HB
, 1995). HB
d
N
HB
, 2003). HB
,
, 2001).
HB
, 1999). T
M
, 1992;
(2005)
,T

khoai t
d

I G
, 2000; S

d
T
62.5% [7a].
4.2 S to dng v bi u hi n khng nguyn b mt vim gan B (HBsAg) vo cy

thuc l
T
B (HB
)
d
z

. HB
ff
d
d (
)

d
22
HB
. B HB
. X
HB

RN .
d

d
HB
HB

HB
d
[8].
57
. B
P R
q
/
EFEHBS
HBS
q
HB
3.12 . S
P R
ELIS
d
HB
d ,
EFEHER.
P R
EFEHER
. M
EFEHBS d
d d

)
,
M
.T
(RT P R). M
(19.4 /
(9.5
S
ELIS
4d
ER. V
[7 ].
HB
. HB

d
I G
N
HB

HB

.K
.T
(2003)
HB

,
. G , K
HB
.
,S
HB

HB
.
(22
)
K
NT I
[7b].
4.2.1 S bi u hi n c a HBsAg trong cy thuc l

4.2.1.1
a.
Nicotiana tabacum L. var. Havana 425

T
,R j
d , .

34
MS
58
(Murashige v Skoog, 1962).
MS
.S
Agrobacterium [7a].
b. X
G
t Arabidopsis ho
ethylene t chu i (EFE
c t o dng nh promoter ubiquitin3 (ubq3)

u khi n qu trnh hnh thnh enzyme t o
c cung cp bi Ti n s G.D. May, Vi n nghin
cu cy trng Inc Boyce Thompson, Ithaca, Newyork, USA) c hoc khng

c tn hi u duy tr ER. S bi u hi n ca 4 lo i pHBS, pHER, pEFEHBS,
EFEHER
c xy d ng v bn ch
ca Sunil Kumar
(2003). HBS (
B
u C duy tr tn hi u k t thc
ER)
c t o dng nh promoter ubq3
xy d ng tng lo i pHBS v
HER. T
, pEFEHBS
EFEHER
c xy d ng b ng cch t o
d
n HBS v HER vo v tr BamHI v SacI ca pEFEGUS (m t
vector bi u hi n th c v t d a trn pBI121 v i promoter ca chu i) bi s
thay th GUS b ng HER hoc HBS. Phn T DNA ca 4 vector bi u hi n
c m t trong hnh 4.5 [7a].
59
Hnh 4.5 Vng T DNA ca (a) pHBS, (b) pHER, (c) pEFEHBS, (d)
EFEHER. RB
LB
2 u bin tri v phi, Npt II l neomycin
phosphotransferase, UBQ3 l promoter ubiquitin t Arabidopsis, EFE l

u khi n enzyme t o ethylene chu ; HBS
a
, HER
a HBsAg v i tn hi u duy tr ER v Nos l trnh
t k t thc nos [7a]
HB
c.
N
105 (
H
H d, 1993)
(1985).
d

mg/l) + Cefotaxime (400
/ ).
K
(100
/ ). N
MS
N
knh [7a].
d
(
HB
).
Agrobacterium tumefeciens EHA

.N
MS + B
/)+N
4 6
d
MS
12
(1
(0.1
.P R
Agrobacterium
(1
q

/)+N
(0.1
/ ).
.T
,
d
d.
Phn tch PCR

T
DN
CTAB (Stewart v Via, 1993). 50

100
), T q DN
P R
100
DN
94o
(
)
P R
(
(1.0 ), 200 M dNTP, 1

.
P R
5
35

60
: 94oC trong 1
pht, 55oC trong 1 pht v 72o
10
1. 5 T
P R
3
G
(Hybond N+; Amersham P

(
S
K
, 2003)
blot trn mng nylon

).
B HI 681
HB
32
681
TGT T
TGG G
2. 5 T T G G T TT
[7 ]:
TGG T
. S
(1%).
HB
d TP P d
d
BSHER
d
BRIT (
d
.V
(1989) [7a].
S
RT P
T
RN
d
R
M
.L
1 RN
DN d
RT PCR cMaster (Eppendorf,
d
P R
P R
P R.
d
d
M ). 5 DN
d
Phn tch ELISA

T
d
K ,
ELIS (
d
M
, 1992). S
HB
d
ELIS (
ELIS ) (HB
EI
, P L )
d d
ELIS ) (B E
HB
, BIOKIT, T B N ). S
(
) (HB
)
(
)(

)
d . T %
[7 ]:
61

%
ELIS *100% /
=L
HB
ELIS .
e.
0.4M
10
250
, 250
.S
ELIS
. N
HB
. T
Agrobacterium tumefaciens c
(100
d
HB
(hnh 4.7) [7a].
P R
3 d

HB
( 4.6). S
681
/ ). N

.N
d
P R
). N
P R
DN
. K
3 [7a].
4.2.1.2
L
1
2

d
6, 24, 48
. T
HB
46
. 10
681
P R
62
Hnh 4.6 S khu

i PCR ca DNA b gen t cy thu c l chuy n gen. 1,
c t HindIII v EcoRI ca DNA b gen phage Lambda; 2, ki m sot
d ( BS HER); 3, K m sot m (DNA b gen t cy ki m sot khng bi n
i); 4 13, nhng cy thu
c chuy
i pHBS; 14 23, nhng cy
thu
c chuy
i pHER; 24 33, nhng cy thu
c chuy
i
pEFEHBS; 34 43, nhng cy thu
c chuy
i pEFEHER. Ch s
khu
i c
n 681 bp trong ki
d ( 2)
ng cy thu c
l chuy n gen (cc ln 4 43) [7a]
63
Hnh 4.7 Phn tch PCR Southern blot ca cy thu c l chuy n gen. 1, cp
t Lambda HindIII v EcoRI; 2, Ki
d ( BSHER); 3, K m sot
m (sn ph m PCR t cy ki m sot khng bi
i); 4 6, cy thu
c
bi
i pHBS; 7 9, cy thu
c bi
i pHER; 10 12, cy thu c l
c bi n i pEFEHBS; 13 15, cy thu
tn hi u lai c
n 681 bp trong ki
c bi
d (
i pEFEHER. Ch
2)
c l
chuy n gen (cc ln t 4 15) [7a]

P
RT P
(681
4
:
R
d
)
d
,
( II.2.1.4).
ELIS
.
q
EFEHBS
19.4
(F.W)
.G
(
) 17.65 / F.W
HBS

d d
II.2.1.1). M
HB
d 4

d
ER.
.
HB
,
ELIS
64
Hnh 4.8 Phn tch RT PCR ca s bi u hi n ca HBsAg trong cy thu c l

. 1,
1 ; 2,
d ; 3, DN cy ki m sot
i; 4, cDNA t cy thu
c chuy n pHBS; 5, cDNA t cy
c chuy n pHER; 6, cDNA t cy thu
c chuy
i
chuy
khng bi
thu
pEFEHBS; 7, cDNA t cy thu

G
, EFEHBS
. M
EFEHBS
.S
HB
q
3d

B dd
, 1992). M
,
HB
EFEHER),
,

(
G
z, 1997). T

EFE ( EFEHBS
HB
q
pEIEHBS v pEFEHER. N
,
.K q
c chuy n pEFEHER [7a]
z
EFE
[7a].
65

d
Bng 4.1 Nhng khng th
chuy
d a trn phn tch ELISA ca cy thu c l
u ki n in vitro v nhng cy pht tri
trong nh knh [7a]

L
HB
cy in vitro
HB
trong nh knha (ng /g F.W
/ F.W
l)
pHBS
14.38 0.05
17.65 0.03
pHER
12.40 0.01
16.42 0.02
pEFEHBS
19.40 0.02
8.41 0.05
pEFEHER
19.24 0.03
10.31 0.01
12 d
T
F.W
S
,
(
M
(2005)
(1999)
R
(2000)
nh
EFEHER

49.3%
HB
d . S
HB
16.5
, 1992),
19.4
.T

K
v theo
3
ER
ELIS
HB
d
F.W
EFEHBS.
150 / F.W
16 /
II
,
[7a].
HB
d
vector
/
M
cy t
F.W
ER. N
(
) 9.5
d ( 4.2).
d
HB
EFEHER. V
HB
66
HBS 48
HB
HER, EFEHBS, EFEHER (
F.W
ELI
4.9) [7a].
Bng 4.2 M
bi u hi n ca HBsAg phn ng khng th
thu c l chuy n gen [7a]
L
.T
HBsAga
pHBS
4.5 0.2
31.2
pHER
6.76 0.8
54.5
pEFEHBS
7.84 0.9
40.4
pEFEHER
9.5 0.3
49.3
HB
d d
Hnh 4.9 T
V
ng s bi u hi n ca HBsAg trong l cy thu c l b ng cch

[7a]
67
Cramer, 1999).
T
HB
v
T
d fd
ER
HB
37% HB

th
EFEHER. N

,d
(
(
fd (
HB
W
)q
, 2002)

, 2002). N
S
HB
d
, 2002) [7a].
. N
22
, 2003). N
q
d
HB
d
HB
d .
.S
(2003)
d
49.3%
ER
,
22
(P
, 1990; S
, 1996). ER
d fd (
I
, 1989;
B
, 1996). N

ER
(S F )
(
F d , 1997). M
(1995)
B
(LT-B)
trong th

microso
[7a].
68

HB
S
HB
(6
F.W). S
HB
HER
HB
(

4.3) [7a].
K q
z
. H
fd
HB
, 2000). K
.G 4
otein (theo Storger, 2005).
.
Bng 4.3 M
bi u hi n ca HBsAg trong h t ca cy thu c l chuy n gen
ng b ng ELISA [7a].
F.W
pHBS
1.0 0.02
pHER
6.0 0.023
pEFEHBS
1.5 0.019
pEFEHER
4.0 0.023
4.2.2 S bi u hi n c a HBsAg trong nui cy huyn ph t bo thuc l

4.2.1.1
a.
Dng t bo NT I
c ly t Vi n nghin cu cy trng Inc Boyce
Thompson, Ithaca, New York, M . Cc m s
d
ng
bn r
c t o ra t
ng MS c mu i, 230 mM 2-(Nmorpholino)
69
ethanesulfonic acid, 132mM KH2PO4, 55mM Myoinositol, 3 M thiamine

HCl, 10 M of 2,4 D,
kho
5.7
87.7 M
ng sucrose. pH c
c t o gel v i 0.2% Phytagel (sigma). T bo nui cy
cb
u nui cy v i khong 200 mg m s
50
ng
lng gi ng v
ch n. Nhng t bo nui c
c pht tri n
trn my l c trn 80 100 vng / pht trong t i v cy chuyn 10 ngy 1
i, t bo sau cy chuyn 4 6
ln. V i nhng s bi
[7b].
b.
T
ng mu cy (v d
c s dng
ng m s
c s d
nui cy t bo) l khc nhau t 0.1 t i 5 g kh

ng t
(F.W) a
m s
c c
50
ng lng NT I. Th nghi
c lp
l i 3 ln cho m
c trn my l c trn 100 vng/pht trong
t i. Sau 10 ngy, s l
kh
ng t
c ki m tra cho
m
ng mu cy v s li
c phn tch b ng th ng k [7b].
c.
V

d
d
HER100
HB 100
MSI164,
MSI 99
B HI
S . G HB
d

HER100
HB 100

d

3
[7b].
d
HB
ER
l:
(1) 5 T TGG T
TGG G
T
3.
(2) 5
TGT T
G G
G
3.
(3) 5 GT TT T GG
T TGT T
G 3.
(4) 5 T T G G T TT
G T GT TT T GG
T 3.
(5) 5 T T G G T TT
TGT T
G G
G
M 1

d
TG HB
BamHI
2, 3, 4
K z
70
3.

d
3 HB
. M 5
ER
tr SacI.
HBV
d
d
P R
K q
SacI
HER100,
HB
BR322 (
2, 3, 4
5
BamHI K z
d
ER HB
.
3 P R
BS HER
DN
d
3
1
.
3
SmaI
DN
d
ER 3
HB
. BS HER
BamHI v SacI
HER 699
d
BamHI v SacI
MSI164
pHER100 (hnh 4.10A).
Hnh 4.10 Vng T DNA ca pHER100 (A) v pHBs100 (B). RB v LB l

2 vng bin tri v phi, Npt II l neomycin phosphotransferase, UBQ3 l
promoter ubiquitin t Arabidopsis, HB
a HBsAg, HER l gen
a HBsAg v i tn hi d
ER N
u cu i nos. [7b]
d
HBV
HB 100,
HB
P R d
1
5. S
DN
5
71

BamHI - K z
3 SacI HB
SK+
BS HB .
BamHI v SacI
SacI
P R
(100
681
BS HB
MSI164
dNTP, 1
trong 1 pht v 72o

[7b].
BamHI v
pHBs100 (hnh II.2.2.1B). T

50

(MBI F
, US ) (1.0
), Pf DN
), 200 M
P R :
SmaI
P R,
100
.
o
3
94
30

o
: 94 C trong 1 pht, 55oC
10
HBS100
d.
Agrobacterium tumefaciens EH 105

HER100
d
.N
(4 6
, 0.5
30
Agrobacterium
q
YENB (chi
)
7.5 /
50 /
(400
/)
8.0 / , H
NT I, v duy tr trong 3 ngy v t

f
(50
100
/
NT I
[7 ].
.S
,
.S
d ,
/ ). N

(300
/)
e.
P
S
phn tch S
d
. DN

. T
, 2
HB
[7 ]:
(1) 5 T
TGG T
DN
TGG G
P R
T B
681
3
72

(2) 5 T T G G T TT
50
P R
DN
(1.0
DN
TGT T
), 200 M
100
dNTP, 1
d
10
DN
, UK). M
699
), Taq
P R
100
72oC trong 10 pht.

EcoRI,
H
P R
94
35
,
o
o
: 94 C trong 1 pht, 55 C trong 1 pht, v 72o
B
DN
G G
n di trn gel
d N+ (
Pharmacia
BS HER
d
.
.
q
.N
d
BamHI
[7b].
Phn tch Southern blot
T
20
20
(100 M
X 100
2
4o
30
2 40000
, H 7.4, 100 M
, 0.1% T
M
f
f
d )
q

. K q
30000
d

40
.S
, 270
/
d
d
B
70 T
55000 /
18 .
d

d
HB
ELIS .
HB
,
f
8000
3
.K q
400
pH 6.8, 10% glycerol, 2% SDS, 1% 2
,
100 M d
d
W
. HB
d
.H
1:1000)
d
3:1:2
1
(50 M T HCl,
mercaptoethanol, 0.0025 mM
)
20

HB
(
1
73

I G HRP
20000). B
E L
2(
1:
d .
[7b].
Phn tch Western

Phn tch ELISA
P
HB
(HB
( d
3
(
),
). M
HER100, HB 100
HB
[7b].
HB
.1

S T
HBsAg (Sh

d
HB
W
.K q
,

W
. T
(E
H dR f
4.2.1.2
. N

HB
B
,
,
d
,T
,N
ELIS d
). P
B ) [7b].
S to dng gen s ca HBsAg vo cc vector bin i thc vt: G

HB
d
(SKDEL)
HER100
d 3
3 3

P R.
d
3 ER d

HER100 (d
). HB
d
microsomal du
[7b].
74
S pht trin ca dng t bo thuc l chuyn gen: N
(0.1 5 F.W)

NT I. N
d HB 100
Agrobacterium tumefaciens c ch
HER100
NT I
50
0.2
d
NT I. Sau 2
.N
43.28

. 10
NT I pht
.N
50
10 ngy (hnh 4.11).
[7 ].
PCR v phn tch Southern blot ca nhng dng t bo chuyn gen: DNA
d
P R.
681
HB
Hnh 4.11 S t
kh
ng t
DN
HB 100
d
,
ng mu c
c s l
. K ng 0.1 5.0 g mu c
HER100
(
d
[7b].
4.12). S
50
75
t v
ng lng NT I v i vi c lp l i 3 ln v s l
ng t
t v kh i
c quan st l mc 0.2 g mu cy sau 10 ngy. [7b]
Hnh 4.12 S khu ch i PCR ca DNA b gen t nhng dng t

c
bi
i NT I ca thu
. L 1:
1 ;
2:
d
(pHER100); ln 3 : ki m sot khng bi
i (DNA b gen t nhng t bo
khng bi
i); ln 4 : dng t
c chuy
i pHBs100 v ln 5 : dng
t
i pHER100. Ch s khu
c chuy
ki
EcoRI
DN
HB
d 699
2)
ng dng t
[7b]
i c
c chuy
n 681 bp trong
i (ln 4 v 5)
947
T DN
BamHI
pBS HER (hnh II.2.2.4).
76
Hnh 4.13 Phn tch Southern blot. Khong 10 g DNA b gen

c gi i
h n v i EcoRI
d t
n 699 bp b ng cch c t bi
BamHI c HER100. L 1 ;
1 ;
2:
n 699 bp t pHER100
(ki
d );
3 : DN
gen t nhng t bo khng chuy
i NT
I (ki m sot m); ln 4 : t
c chuy
i pHBs100; v ln 5 : nhng t
c chuy
P
M
ELIS . M
HB 100
HER100
d
N d
d
HB
i pHER100 [7b]
1 HB

/ F.W
2 / F.W
q
( 4.14). q

HB
d .
HER100
10 /
HBS100
5 / ( 4.14).
100o
1 )
d
z
(
) (d
). B
ELIS
HB
d
[7b].
77
Hnh 4.14 L
HB
c bi u hi n trong n i bo v cc hnh thc ti t
ra t nhng t bo thu c l NT I
hi n trong n i bo c HB
M
ti HB
Phn tch Weste

d
HB
HB 100
HB
HER100
(hnh 4.15
nh b
ELIS . () S bi u
c tnh theo ng/g F.W ca m s . ()
/
HB
dng [7b]
B). K
HB
d 24
Hnh 4.15 Phn tch Western blot ca dng t bo thu

c chuy
i
pHBs100 (A) v pHER100 (B). Ln 1 : ki m sot m (nhng t bo khng
c chuy
i); ln 2 : ki
d ( HB
n g c t nm
men); ln 3 : nhng t
c chuy
i pHBS100; ln 4 : nhng t bo
c chuy
i pHER100; (C) Phn tch Western blot ca nh
n
gradient t tr ng CsCl t nhng t bo thu
c chuy
i pHBs100. Ln
78

d
1 : ki
( HB
n g c t nm men); ln 2 : ki m sot m
(nhng t bo kh
HB
. P
d
HB

HB 100. M
Hnh 4.16 M
. T
7
W
d HB

40
(1
ELIS
HB
40
i) [7b]
c chuy
6
4.15 C).
c chuy
i trong
HB
1.102 1.087 g/ml (hnh 4.16).
ca HBsAg t nhng t bo thu

cc phn ca gradient CsCl [7b]
4.3 Kt lu n
T
1
d

;1

2000
1999). H ,
d d
40
/ (
d
d
. N
)/
.Q
. N
10%
79
d
,
.
H
d
d
.
d
.S
.
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,
q
G
d
d fd
(
M , 2003; H , 2004). G
)
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d ,
).
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q
d (
)
( GLP). N d
d d
d
N
.

.
80

T
(Nicotiana tabacum)
. M d

.N
d
q
d
.N
d
,
. H
/H1N1 ,
. T
81V9
100
q
Agrobacterium
.T
, d

. M
G
, 2001). T
(
d
T
q

q .T
.T
.T
,
20
81

p
. T
sinh d
. N
.
. M
d
82
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TNG KT
V
ring

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,
q
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,
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,
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.
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vaccine.
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,

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83
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vaccine
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,
84

TI LIU THAM KHO
[1]
T Q
H .
[2]
, 2006. Cng ngh chuyn gen (ng vt, thc vt). NXB
L , 2006. Vaccine thc phm loi vaccine th h mi. NXB
H .
[3]
L , 2007. Nhp mn cng ngh sinh hc. NXB
[4]
N L
Minh.
[5]
T T L , 2006. Gio trnh cng ngh gen trong nng nghip.
[6]
N
Q
[7a]
G.B. Sunil Kumar, L. Srinivas, T.R. Ganapathi & V.A. Bapat, 2006. Hepatitis
B surface antigen expression in transgenic tobacco (Nicotiana tabacum) plants
using four different expression cassettes. Plant Cell, Tissue and Organ Culture,
, 2006. Sinh hc i cng,
2. NXB HQG
H .
H
H .
H
L , 2009. Gio trnh cng ngh DNA ti t hp. NXB
T H M .
84:315323.
[7b]
G.B. Sunil Kumar, T.R. Ganapathi, C.J. Revathi, K.S.N. Prasad, v V.A. Bapat,
2003. Expression of hepatitis B surface antigen in tobacco cell suspension
cultures. Protein Expression and Purification, 32:1017
[8]
Hugh S. Mason, Dominic Man Kit Lam, v Charlies J.Arntzen, 1992.

Expression of hepatitis B surface antigen in transgenic plants. Proc. Nati. Acad.
Sci. USA, 89:11745-11749.
[9]
Reynald Tremblay, David Wang, Anthony M. Jevnikar, Shengwu Ma, 2010.

Tobacco, a highly efficient green bioreactor for production of therapeutic
proteins. Biotechnology Advances 28:214221
[10]
http://vi.wikipedia.org/wiki/V%E1%BA%AFc-xin
[11]
http://suckhoedoisong.vn/20090415042934696p0c61/hiem-hoa-tu-virut-viemgan-b.htm
[12]
http://www.nhakhoalananh.com/vn/print.php?ceoseek=news&cat=65&post=57
8
85

[13]
http://www.ungthu.org/cacbenhungthu/ut_gan/Hep_B.asp
[14]
http://giangduongykhoa.wordpress.com/2009/02/22/xac-d%E1%BB%8Bnhki%E1%BB%83u-gen-virus-viem-gan-b/
[15]
http://www.dshs.state.tx.us/idcu/disease/hepatitis/hepatitis_b/faqs/
[16]
http://www.sacbee.com/static/live/news/projects/biotech/imgs/grfx_c5_2.gif
[17]
http://tinkhoahoc.blogspot.com/2008/11/cy-la-bin-i-gen-to-vaccine-chng-ligiun.html
[18]
http://www.agbiotech.com.vn/vn/?mnu=preview&key=2682
[19]
http://www.agbiotech.com.vn/vn/?mnu=preview&key=1554
[20]
http://www.isb.vt.edu/news/2006/news06.apr.htm#apr0602
[21]
http://www.khoahoc.com.vn/doisong/yhoc/suc-khoe/15612_Bao-che-vac-xinngua-dich-ta-tu-gao.aspx
[22]
http://www.ovsclub.com.vn/show_article.php?aid=13762&lg=vn
[23]
http://biommed.lsu.edu/staticpages/index.php?page=RQPCR
[24]
http://commons.wikimedia.org/wiki/File:Ti_Plasmid.jpg
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http://www.natap.org/2004/HBV/100804_02.htm
[26]
http://www.drthuthuy.com/reseach/HBVGenotype.html
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[28]
http://bestpractice.bmj.com/bestpractice/monograph/127/basics/pathophysiology.html
86

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Tai lieu chia se tai: wWw.SinhHoc.edu.

TpHCM, thng 6 nm 2010

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CH NG 4: SN UT VACCINE VIM GAN B .............................................. 50

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Virus vim gan B ...................................................................................... 52

Vaccine vim gan B ..................................................................................55

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Agrobacterium tumefaciens ............................... 29

Hn 3 7 My xung gen .................................................................................................... 32

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Hnh 4.5 Vng T DN

( ) HBS, ( ) HER, (c) pEFEHBS, (d) pEFEHER. ........ 60

Hnh 4.7 Phn tch PCR Southern b

Hnh 4.8 Phn tch RT P R

pHER100 v pHBs100 .................................................... 71

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vong sau khi tim phng vaccine.

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CH NG 2: VACCINE V CC KHI NIM

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Mt l vaccine bt hot l vaccine cha vi sinh v t gy b

cm, t, dch h ch v vim gan siu vi A. Hu h t cc vaccine lo

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V m t trong nhng l i th ca chng ta l ngnh Cng ngh

c cng ngh sinh h c. Nhng thnh t u v cy

ra nhng ma mng b i thu, gii quy t n

d i bom (bombardment) [2].

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g tim hay qua

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Th hai, vector phi c kh

c duy tr trong qun th t bo.

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nh khng nguyn chnh

l thnh phn protein khng nguyn c trn b mt ca tc nhn gy b nh,

chu s kch thch ca khng nguyn, s cm ng sn xut ra m t lo i

Th ba, cu trc phn t phc tp :

Th hai, trng lng phn t ln : K

Th nht, n phi c tnh l :

Phn loi : N i ta chia khng nguyn vi sinh v t ra cc lo i sau:

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Th hai, khng nguyn vi khu n khng ho tan l thnh phn t

khng nguyn lng (k hi u H), v khng nguyn thn (k hi u O).

khng nguyn cn quan tm.

Khng nguyn virus, gm:

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Tnh n nh cao, d bo qun v s dng:

c phn phi qua ng ming: N

Vaccine n c kch thch sn xut khng th ca h thng th dch hiu

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gii quyt nhng hn ch ny mt s k thut, bin php c s

khng nguyn bi u hi n trn th c v t.

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S dng cc promoter, enhancer hay trnh t k t thc ly t virus th c v t.

Gen khng nguyn k t h p v i cc nhn t

hi n protein, thi gian bi u hi n trong chu trnh pht tri n ca cy v b ph n

u khi n min dch c

dng vaccine khoai ty khi khng bi

ng chnh xc protein khng nguyn tron