Thiết kế vector chuyển gen biểu hiện kháng nguyên vi rút PRRS trong tế bào thực vật

 K20 

Luận văn thạc sĩ 2011 – 2013 1

ĐẠI HỌC QUỐC GIA HÀ NỘI
TRƯỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN




Lê Thi ̣ Thanh



THIẾT KẾ VECTOR CHUYỂN GEN BIỂU HIỆN
KHÁNG NGUYÊN VI RÚT PRRS TRONG TẾ BÀO THỰC VẬT


LUẬN VĂN THẠC SĨ KHOA HỌC





Hà Nội – 2013
 K20 

Luận văn thạc sĩ 2011 – 2013 2

ĐẠI HỌC QUỐC GIA HÀ NỘI
TRƯỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN

Lê Thi ̣ Thanh


THIẾT KẾ VECTOR CHUYỂN GEN BIỂU HIỆN
KHÁNG NGUYÊN VI RÚT PRRS TRONG TẾ BÀO THỰC VẬT


Chuyên ngành: Sinh học thực nghiệm
Mã số: 60420114

LUẬN VĂN THẠC SĨ KHOA HỌC

NGƯỜI HƯỚNG DẪN KHOA HỌC:
TS. Nguyễn Tường Vân
PGS. TS. Võ Thị Thương Lan


Hà Nội – 2013
 K20 

Luận văn thạc sĩ 2011 – 2013 3


Hi chng ri lon sinh s   p  ln (Porcine Reproductive and
nh truyn nhim nguy hi
kh t hi nng n .  Vit Nam bnh
c gn mc bng b xung huyt  tai,

 sa bng sy thai  l
a hou chng bnh v c bi ln con cai sa.
Bn l Hoa K t hin 
nhip: Canada (1987); Nht Bn

T  dch ln  nhic Bc M
n tht ln cho ngh  gii.  M, thit hi
kinh t (trc tip) ca bnh ln tai xanh trong nh
ln nht so vi thit h lc
M phu nhng tn tt do bng 560 triu USD do vi
hu ln chn ng d ng.
 Vit Nam, bn ln
nhp t M . T ch bnh nng ch 
p 17 tnh t hi nng n nh
i. n nay b 








r thc tp  Vit Nam.
  ng bnh ln tai xanh, bi   c s d  
cxin kt hn  t s ng hn ch 
vcxin bt hoc cho bnh ln tai xanh s dng c
hou. cxin dt s cxin bt
hot hiu qu bo v min d


 ng cao, 


m bo v ti vng n
 li. Hin nay  ng Vii vcxin chng
 K20 

Luận văn thạc sĩ 2011 – 2013 4

PRRSV dhiu qu li thp h  
u tr u cho v ng vt min nhim vi vi
.
Vcxin thc v





 c (edible vaccinet trong
nhng sn phm ca   sinh hc hi i. Thc cht vcxin thc v 
vcxin tin v c sn xut d thng thc v 
n. So v thng sn xut vcxin truyn thng, vcxin thc
vt  u  m : d    n xu   i; c
biu hin trong thc vt nh  nhi  bo qu
s dng; c ng mic nt, c;
an; c bit vn xu ca h th
mc rut hiu qu 
T nh n v khoa hc tin
  t k vector chuyn gen biu hi
t c v t d  hc t v KH&CN theo ngh nh

c Vit Nam  c BNghiên cứu biểu hiện gen mã hóa
Glycoprotein vỏ (GP5) của virus gây bệnh lợn tai xanh trong thuốc lá và đậu
tương” n 2011- c tit T 
Thc vt, Vi   sinh hc, Vi   t Nam vi mc
: i) Thit k vector chuyn gen  cho mt s   ca vi 
PRRt thc vu qu s dng c
qua vic  biu hin ca  thu-2.



 K20 

Luận văn thạc sĩ 2011 – 2013 5

- 
            (PORCINE
REPRODUCTIVE AND RESPIRATORY SYNDROME, PRRS)

Hi chng ri lon sinh s   p  ln (Porcine Reproductive and
Respnh truyn nhim nguy hi
lan kh  i mm b
cht t ln nhim bnh t hi nng n  [54, 59]. 
Vit Nam b c g  n mc bng b
xung huyt   sa bng
sy thai  la hou chng bnh v c bi
 ln con cai sa [61].
B l Bc M  
nh
1998, bn  cc, Nht Bn, Trung Qu [7, 59].
Thc 

nhau [59, 60]:
 Bm  ln (Mystery Swine Disease, MDS)
 Bnh tai xanh (Blue Ear Disease, BED)
 Hi ch    y thai  ln (Porcine Endemic Abortion &
Respiratory Syndrome, PEARS)
 Hi ch       ln (Swine Infertility & Respiratory
Syndrome, SIRS)
i ngh quc t v sc kh chh
gii (OIE) 





 i chng ri lon sinh s
p  l and Respiratory Syndrome, PRRS) [7]. K t
 c ca bnh.
 K20 

Luận văn thạc sĩ 2011 – 2013 6

 Vit Nam, bn nhp t M.
n nay, bkhp  c [28].
1.1.2. Triu chng bnh
B xy ra  tt c a tung biu hin  hai tr:
sinh sp [28, 61, 62]:
 Ri lon sinh s ng l

 t hoc cht trong
-35%), ln con sinh non chim 10% ng thi chng bit sa 

l





n con chc cai sa.
 Ri lop: ln th dc, th dn dp, khi kii cho thy
i, hoi t phi rt nng.
NH LN TAI XANH
lystad, nh
ln tai xanh,  [59, 61] lo
 nh l 


Arterivirus, h Arteriviridae, b Nidovirales,  [18].

PRRSV 
45  55 nm, nucleocapsid  30  35 nm. 
ARN
Arterivirus [18, 60, 61].




1.1. ca  P



 [61].

1.2.2. Cn
 K20 

Luận văn thạc sĩ 2011 – 2013 7

H gen ca  PRRS , c khong 15 kb
c m (ORF - open reading frame): -7. Trong
        protein phi c   replicase, polymerase
n ca -7 u
 g glycoprotein (GP2, GP3, GP4, E) protein mng
i   (N) [18] 

1.2. 

ch gen  [61].
Dt PRRS:
c chng ci ph 
gc chng
Bc M (v-2332) [18, 62]. Gt bin th ca  II 
c lc cxut hin u tn tht   [28, 61]. M
triu ch  
cu cho thy gia hai  t ln v b gen (m ng ch
55-80% ng min dch cc lc  [7, 42]u 
vcxin  mt chng PRRSV s  bo v 
n.
 K20 

Luận văn thạc sĩ 2011 – 2013 8



amin 
 -
76% (ORF2), 72% (ORF3), 80% (ORF4  [7, 54].
Pvi  . Gi

GP2, 3, 4, E, M, N)   
m 
VR-2332 [9, 54].
             
 -

  

-232 
 26, 28, 54].
                 
PRRSV ng: dn 
- 
 [54].
1.2.3
     nh ca PRRSV v  c hi   
u ch thy rng  c bit vi thc bit
i thc ng min dch t  b mt
54, 60]. p,  s sao i thc 
ph to ra nhiu  mng th  
ng min d mi tn t i thi
i th      c n   i
 K20 

Luận văn thạc sĩ 2011 – 2013 9

tht  gm c  [54, 60 cho thy s
i th khi nhim PRRSV.

A
B
: i th nhim PRRSV. i th
ng; i th y [54].
Mi th y, PRRSV hi b h
thng bo v c [54]. u kin thun li cho nhng vi khun
ng h ng
in mc bng b bi nhim v
dch t ln, t huy  bnh do xon khun Leptospira spp,
bnh do Steptococcus spp, Mycoplasma spp, E.coli nh k 
 cht nhiu  ln mc bnh PRRS [7, 18, 54].
1.2.4
Trong nh    n vcxin chng l  c ghi
nhn trong hai thp k qua, vic thit k vcxin ti, vic t ng
min dch t ch th c bi

   
n vic bo v chng lm, lng sn
xu c hii vi protein N. M
bin nh , 

  
ng min dch bo v l




 protein N ch
 chu







cxin ti [15].
n cu to c  protein cu
  (bao g  protein
 K20 

Luận văn thạc sĩ 2011 – 2013 10

   xy ra
mnh v


 thit k v











 . GP5 

 c s
th  lt v then cht ca min dch dch th [19, 23, 39].
u cho tht vi protein mi M
[32, 34, 55]. 



, 







 3 


 , lo













 mc cao [54].
NH L GI VIT NAM

Ma nh bnh song v
   nh c ghi nhn  M vi
nhng triu ch sinh sn kt hp vi chng
 t hin t
kh [61, 62].
 dch ln  nhic Bc
Mn tht ln v kinh t cho ngh 
gii [61, 62]. 

 [59, 62]


 [59].  Mi
t hi kinh t (trc tip) ca bnh ln tai xanh trong
nhn nht so vi thit h lc
c M phu nhng tn tt do bng 560
triu USD do vi ln chn ng d 
 K20 

Luận văn thạc sĩ 2011 – 2013 11

ng [59, 62].  a dch bnh khong 2 triu ln 
cht [59, 62].  t 

- 
 - 73,1% [59].

007 cho 

do nh
 [28]

 amin trong
gen ) [28]
Liaoning, Ji
 [28]


 [28, 34].


 [28]. D
 2007 [59].  
 
  
 [59].

tai xanh c 
[26, 28]. t
 [59].
 K20 

Luận văn thạc sĩ 2011 – 2013 12

 khnh rng Hi chng ri lon sinh sp  ln
t trong nhng ng   ra nhiu tn tht nng n  
n ca nhiu qu gii.

 Vit Nam, bn ln
nhp t M 10 trong s 
v y ngay  [28]. T n
 dn. Vic xy
 dch lu  ch  c
 chc quc tch xy ra
ng  mt s c trong khu vc [59, 62]. T nh xut
hich ti nhi tn tht ln v kinh t i
 th 
  dt hin tc 65 huyn ca 18 t.
Tng s ln mc b ln ch  [1].
  dch xut hin tng, thuc 103 huyn ca 26 tnh,
. Tng s ln mc b ln chc ph
 [2].
  dch xy ra  c 26 huyn ca 13 t: Bn
ng Nai, Ti ng Ninh, Bc
Giang, Qung Nam, Gia Lai, Ba - k Lk vi 7.030
ln mc bn buc ph [3].
  dt hin ti ng, th trn thuc 286 qun,
huyn ca 49 t. Tng s ln mc b ln ch
buc ph  [4].
 K20 

Luận văn thạc sĩ 2011 – 2013 13

 ch ln tai xanh x s  dch  s

ln mc bnh, s ln b y gim rt nhiu (gn 20 ln c v ph
dch  y) so v [5]. C th:
+ t 1: Tng s ln mc b
5.346 con ln thn con; tng s ln ph
n ln thn con [5].
+ t 2: Tng s ln mc ba
ch; tng s ln ph [5].
 dch xy ra ti 453 /ng, th trn ca 95 qun/huyn thuc
28 tnh. Tng s ln mc b90.688, tng s ch14.065 con, tng s ln
ph51.761 con [6].
Bng 1.1. Thng k ch ln tai xanh ti Vit Nam 2012 [1, 2,
3, 4, 5, 6]

Số tỉnh
có dịch
Số huyện
có dịch
Số xã, phường
có dịch
Số lợn
mắc bệnh
Số lợn chết,
tiêu hủy

18
65
324
70.577
20.366


26
103
956
309.586
300.906

13
26
69
7.030
5.847

49
286
1.978
812.947
442.699

15
49
264
42.317
26.519

28
95
453
90.688
51.761

Kt qu a  nh ti Vit Nam cho
thy thuc chng Bc M [28], tt c 
mng cao so vi  PRRS chc lc cao ca Trung Quc ( 99-
99,7%) [18, 28].
 K20 

Luận văn thạc sĩ 2011 – 2013 14

T nhng th thy rng nhng thit hi chng
ri lon sinh sn p  ln. Bnh  t
hi nng n v kinh t c tin  Vit Nam cho th li nhiu
hu qu  i, nhi v
i sng ch yu dn.
1.4. U TR BNH LN TAI XANH
c nhng din bin phc tt hi to li chng ri lon
sinh sn  hp n ca nhiu qu gin
nay nhiu v n dch b.
c bi v  nh, triu chng, b
a dch bnh. Cng ch bnh
ng  nhii hiu qu n.
Mt s     p v 
dng k thun kit ch con ging ch
 nh ln tai xanh, kt qu n ch c thit hp
knh vn tn tu thit hi v
kinh t [54].
Hin nay vc tr bnh ln tai xanh hn ch thit hi
do b ng s dng mt s thu     Enrofloxacine
nhm u tr nh k ng s n bi
thuc tr lc (B.comlex, vitamin ) kt hu tr triu ch
st, chng a chi [61u qu 

Do  ng bnh ln
tai xanh c lc cao, bic s dcxin kt h
bi t s ng hn ch cxin bt hoc
c cho bnh ln tai xanh s dng c hou. Vcxin
d t s  cxin bt hot hiu qu bo v min dch


 ng cao,    

   bo v t i v 
chng  li [18, 21, 24, 38].
 K20 

Luận văn thạc sĩ 2011 – 2013 15

Hin na ng Vii vcxin chng PRRSV d
v(4ng/liu hiu qu ch  
kh u tr  u ch   o v ng vt min
nhim vi  [18]. n nay, th gii duy nhc sn xuc
vcxin chng c lc cao [18], tuy  vn 


xut khu lo.
1.5.  
1.5 
Vic sn xu hn dng ng dng
c sinh h mng trong vic ng bnh
ng vt. Mt h thng biu hin protein hiu qu  tin v
n xut. H thng biu hin trong thc
vn nhm thay th h thng biu hin trong t ng vt hay

vi sinh vt  v n
xut. C th  [18, 25, 38]:
 T c v i sau d
t n bc cao, m bo vcxin i vi
ng vt.
 Vcxin c t   u ki       
sinh hnh.
 T c vm bnh cng v
  hp vi t c cng vt.
Thc cht vcxin thc vcxin ti, c sn xut d
thng thc v n. t xu th 
trin mi c   sinh hc. Nhi        
khun, vc chuyc vu hin vi hiu qu cao. c
vt chuyn gen biu hic s dng trc ticxin
 K20 

Luận văn thạc sĩ 2011 – 2013 16

i hoc ng vng. So vi vcxin truyn thng,
vcxin thc vc t tr [18, 21, 24, 38]:
 u hin trong thc vt nh  nhi 
d o qu dng.
  ng mit, c).
 c sn xut trong thc vcxin ti
kh ng min d h gen ca  
vcxin gic lc hay bt hocxin d

 nh cho
ng vng thm mm b
vcxin sn xut trong t ng vt.

 n xu ca h thc
rut hiu qu c t, hu hu
 qua l nhy ca ng ting tit
niu. 

 

  ng min d
co v ng li nhng vi sinh v
n vcxin trong thc vu mc ngot quan trng 
c sn xut vcxin da  ngun,
r tin  i vn. Rt nhiu quy
n xut vcxin trong thc vc vt chuyn
gen, thc vt mang lc lp chuyn gen, thc vt chuyn gen tm thi, thc vt
nhim  y t c vt [18]. n xut vcxin
thc v   ng cho c, h         ng vt
c ch biu hin v s do qun. nh vic
tng bic sn xut vcxin trong t c v
cy in vitro u ha hi vic s dng
  cht v c cao, nh,
 thuu kim bnh t
 [18].
 K20 

Luận văn thạc sĩ 2011 – 2013 17

y t u dy r ph, khi,
t   u c ch tp
i tn k thuy t ng huy
c kh thi cao nht trong vic t n xu u

khic chuy tii d 
Trong nhing hp, vic sn xut vcxin thc vt gp phi tr ngi ln
khi phi mt vi hing dung nng ming. T min d
nhy cc thit k ng min dch vi th
tiy, mt s loc vng
 c h min dy nh c li, mt
s "th  n ng tc thi hong ming
ng tn ti voxin B subunit - c t u nhit ca
vi khun E. coli) ng min dch v  mc caoy,
 ng c s d   t cho vic chuyn t   
ng ming [20, 31, 37].
1.5.2. S dng t ng BY   thng biu hin
T     c to ra t nhi  c v   
Arabidopsis thaliana, Taxus cuspidate, Catharanthus roseus ng quan
trng          [18]. 
 dng d  BY-2  thng
biu hin. T - 
hc Nht Bn chn lc t Nicotiana tabacum
- ng nht
n, r tin vi t 


100 lt tun. Mi-ng tt
vn gen bng vi khun Agrobacterium [18]. Do vy, t -2
 c vt  t cho vic sn xut nhiu loi
 hp.
 K20 

Luận văn thạc sĩ 2011 – 2013 18

1.6. CHUY   C V    KHUN Agrobacterium
tumefaciens [12, 17]
t A. tumefaciens t s  
n mt phn nh  c vt, o khi u.
Khi u to ng, ng thng hp mt s cht dinh
ng i cho vi khun (opine)y, A. tumefaciens c hi thut
i mo ra i h n ADN
ca A. tumefaciens c ng d gen hii [12, 17].
Vic s dng A. tumefaciens bu t n vi
khu i u  m b c gi
u c r 4). Tng A. tumefaciens to kht plasmid rt
ln vi c khong 200-800 kb. T nhng chng A.
tumefaciens 
cn thit cho vic to khi u. Do vc g-plasmid (tumor
inducing-plasmid) [12, 17].

 .4. Vi khun Agrobacterium tumefaciens  i u. a: Vi khun A.
tumefaciens dn t; b: Khi u   kht hin chi mt
  [12].
Ti-Plasmid gm b            c
chuyn sang t c v-DNA (Transferred DNA). T-DNA mang
2 h gen: Mt h i u cho thc vt 3 gen tng h
phytohormonn vic to 
 K20 

Luận văn thạc sĩ 2011 – 2013 19

  thu ng c gen th hai
t s u khin tng hn xut ca amino acid hay
ng gm kh

n s tip hc (virulence region)
vir, gi ng trong vic chuyn T- gen
a t c vi Ti-
T-i opine cung cp ngu
vi khun [12, 17].

 -plasmid [12].
-c khong 10 kb  20 kb, nm gia 2 tr t lp
li gm 25 bp g   phi (right border  
ng yu t cn thi ng cho s chuyn ADN. S m
c 10 bp cu n s chuyn ca T-DNA.
  n T-DNA b u t v      v    nh
t quan trng, nu thiu yu t c chuyn gen s y ra
hou qu.
Ho ng c       ng cho vic chuyn T-DNA
sang t c vc khom 6 operon:
n thit cho vic tn
vic ti u.  chung, gen virA biu hin  mu kin; gen virC
biu hin  kh u hin mnh  dch chit t 
 K20 

Luận văn thạc sĩ 2011 – 2013 20

 n. Hong ct ch n s 
mt c  p ch  c tit ra t    n c   vt
             p ch  
-hydroxyacetosyringone. Chvt
ng dn d vi khup, lt ch
hovir thuc Ti- cn T-ADN (t
 ph gh gen thc vt.

a gen vir n gc gi
virA t nhn hp cht phenol  t 
 nhn bit chn s hon phm gen virG
l  biu hin ca tt c gen vir. S hoa protein
thuc gen virG  do s vn chuyn nhc
   gen virD2  virE2 quan tr i vi vic chuyn T-DNA. Protein
  t endonuclease, ct s  c hiu  b ph   a T-DNA,
Protein virE2 gn lp tn s gn li vi Ti-
1.6     tng hp sa cha DNA, t s   i trong Ti-
plasmid xut hin li mt T-t protein virD2 g
a T-DNA si c ct ra b. B
to ra mt phc h DNA-c chuy c v ca
  gen
virB tt l hng, qua l nc h DNA-c vn chuy
thc vn t c vt phc h  
virE2 ch nh v c hic h 
gc vt m nhng ch i ca DNA ca
t c v     h   c hiu, bao gm
nh rt ngn 5-t hc thc hin nh 
thc v  tham gia ca protein virD2 [7, 12, 17].
 K20 

Luận văn thạc sĩ 2011 – 2013 21


H minh hn T-DNA ca Ti-plasmid. 1: T-DNA
vi b ph -plasmid; 2: Sc ct ra nh c
i gen virD2; 3: Sa T-c git hp vi protein do
virD2 vvirE2  t  s c tng hp b sung; 4: Ly ch
trng trong Ti-ng gch nm). Si T-DNA t c vn chuy

c vt  dng phc h DNA-protein [12].
T nh thy Agrobacterium tumefaciens t h
thng chuyn gen t . p cho vic ng
d gen  nh:
- Do to kh e mnh t
t n np gen.
- S tng h
cn thit.
- DNA l (ngo --DNA. Nhng
m.
 K20 

Luận văn thạc sĩ 2011 – 2013 22

Khc phc nhc c
s i Ti-  -     c t  
nhc tip theo gen tng hc cthay th bng nhng gen
ch th,  [12, 17, 18]i ta s dng h
th c g    n (binary vector),   ch  a Ti-
c thc hin qua hai plasmid. Plasmid lvir 
mang b   i ca T-   a T-DNA duy nht cn
thit cho vic vn chuyc vt. Gia b pht gen chn lc
ng gen l  [12] cu to ca mt vector hai nguc
minh ha  m c thc hin vi
plasmid nh. Tt c nhc t  thc hin trong
nhng t  E. coli        c chuy  A.
tumefaciens. n bin np c  vi vi khun A. tumefaciens
khuc loi b vnh [12. 17].

7.  minh ha h thng vector hai ngun bin np  c

vt bng vi khun A. tumefaciens. o khng hc loi ra, T-
DNA mang m chn lc trong thc vt A.t
ori: Kh A. tumefaciens; E.c. ori: Kh
 K20 

Luận văn thạc sĩ 2011 – 2013 23

   E. coli; KmR     chn lc trong E. coli  A.
tumefaciens; P1, P2: Promoter; T1, T2: Terminator; vir
1.7. THIT K VECTOR CHUYN GEN THC VT BNG K THUT
GATEWAY
 vector chuyn gen thc vt c rt ln (t 8  15 kb)
bn n ca m hn, v a
m ca enzyme gii hn, gen chn lc/ch th , vector chuyn gen cn mang mt
s   chuyn, biu hin ca gen cn chuyn trong h gen ca t 
vt ch. : (promoter),  ng (enhancer), 
T    t k vector chuy  ng s d 
n phn ng ct ADN nh enzyme gii hn  ni ADN
nh enzyme ni       tn ti mt s c
m sau:
- Do vector chuyn gen thc vc rt li






 (single recognition sides  SRS) ca mt loi enzyme gii hn  ct
ng thng rt, ADN
cha 4 loi nucleotide A, T, G, C. Theo quy lu  t th  t l 6

nucleotide bc 4 lop xp m
6
(ty, c 4069 bp s bt gp mt  lp li gm 6 nucleotide.
Mgii hn s dng ph bin trong chuyn gen 
t nhn bit gm 6 nucleotide [14], a mt s c vi sinh vt s
dng ph bit bt gy, vi nhng
vector chuyn khoc  nhn bit 
ca mt enzyme gii hn  v   r t
thi gian. Mu bi
t.
- S d   t - ni ADN bng enzyme gii h  enzyme
ADN ligase k   c chiu c     vector trong
ng hp s dng mt loi enzyme. S dng hai enzyme gii h
 K20 

Luận văn thạc sĩ 2011 – 2013 24

th khc phc hn ch c hin phc tp, tn thi
gian.
S  p ca him tan ca phage
 n   cho s i k thut chuyn gen bng
 thut GateWay. a k thu dng
c hiu cho phn  hp gi 
ADN (g   [30]:
- Trong phn ng BP, gen cn chuyn mang hai v  hai
 hp mc hiu vi hai v attP2 trong
c cung cp trong b i s a enzyme BP clonase.
 hn chuyu
ng thi mang hai v 1.8).

1.8 minh ha phn ng BP [30].
- Tip theo, gen cn chuy   c chuyn sang
vector chuyn gen (vector nhn  n 
hc hiu gii s a enzyme LR
clonase.  h to ra vector chuyn gen mang gen cn
chuy1.9).

1.9 minh ha phn ng LR [30].
 K20 

Luận văn thạc sĩ 2011 – 2013 25

T nh    thc hi  t k vector chuyn gen
biu hi   c v  
chuy 
       ng BY-    n Agrobacterium
tumefaciens.   d c t v KHCN theo ngh
  a Vit Nam   c B Nghiên cứu biểu hiện gen mã hóa
Glycoprotein vỏ (GP5) của vi rút gây bệnh lợn tai xanh trong thuốc lá và đậu
tương”c thc hin t   T c vt, Ving ngh
sinh hc, Vit Nam.