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vn
TRNG I HC Y - DC
BO CO TNG KT TI NCKH CP B

THIT K VECTOR MANG GEN M HO PROTEIN B MT CA VIRUT H5N1 PHC V NGHIN CU CHUYN GEN TO VACCINE THC VT M s:B2010-TN04-01
Ch nhim ti: ThS. Nguyn Thu Hin

Thi Nguyn, thng 05 nm 2012

Ti liu chia s ti: wWw.SinhHoc.edu.vn NI DUNG BO CO

1 2 3 4 5 6
M U MC TIU - NI DUNG NGHIN CU

TNG QUAN TI LIU VT LIU V PHNG PHP

KT QU V THO LUN

KT LUN V KIN NGH

Ti liu chia s ti: wWw.SinhHoc.edu.vn M U

Ti liu chia s ti: wWw.SinhHoc.edu.vn PHNG CHNG

* Vaccine nhc c (attenuated vaccine) * Vaccine bt hoat (inactivated vaccine) * Vaccin th h mi: - vaccine tai t hp (recombination vaccine) - vaccine thc vt (plant-base vaccine), ...

Ti liu chia s ti: wWw.SinhHoc.edu.vn VACCINE THC VT

Vi mc ch tao c s cho vic sn xut vaccine A/H5N1 n c, chng ti tin

hnh chn ti: Thit k vector mang gen

m ho protein b mt ca virut H5N1 phc v nghin cu chuyn gen to vaccine thc vt .

Ti liu chia s ti: DUNG NGHIN CU wWw.SinhHoc.edu.vn MC TIU, NI

1. Mc tiu nghin cu
- Tao vector ti t hp mang gen m ha protein b mt ca virut H5N1. - Tao chng vi khun Agrobacterium mang vector ti t hp

cha gen m ha protein b mt ca virut H5N1

2. Ni dung nghin cu
- Thit k gen m ha protein b mt ca virus H5N1 vo vector

thch hp chuyn gen thc vt.

- Chuyn gen m ha protein b mt virus H5N1 vo cy thuc l v cy u tng

Ti liu chia s ti: wWw.SinhHoc.edu.vn VT LIU V PHNG PHP NGHIN CU

1. Vt liu
Nguyn liu thc vt: Ging thuc l Nicotiana tabacum K326 t Phng Cng ngh T bo Thc Vt-Vin Cng ngh snh hc.

Chng vi khun v cac nguyn liu DNA:

- Cc chng vi khun E.coli v Agrobacterium do vin Cng

ngh sinh hc cung cp.

- Vector pBeta-Phaso-dest mang promotor chuyn dng c cung cp bi Trng ai hc Ghent, Vng quc B.

Ti liu chia s ti: wWw.SinhHoc.edu.vn

VT LIU V PHNG PHP NGHIN CU

2. Phng php nghin cu - K thut PCR,colony PCR - Cc phng php bin nap: sc nhit,xung in

- Tao vector chuyn gen bng k thut Gateway

- Cc phng php chuyn gen v sng lc cy chuyn gen

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TNG QUAN TI LIU

c tnh khng nguyn ca virus cm A

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Cu trc protein Hemagglutinin

M hnh cu trc protein 3 chiu ca HA

Ti liu chia s ti: wWw.SinhHoc.edu.vn

Khng nguyn HA

Cu trc protein Hemagglutinin

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Khng nguyn NA(neuraminidase)

Cu trc protein neuraminidase

c tnh khng nguyn ca virut cm A

Khng nguyn NA cng vi khng nguyn HA ca virus l cc ch ch yu ca c ch bo h min dch ca c th vi virus cm A, v l c s iu ch cc vaccine phng cm hin nay cho ngi v gia cm, nhm ngn chn dch cm gia cm v han ch ly truyn sang ngi .

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Ti liu chiaSNti: wWw.SinhHoc.edu.vn QUY TRINH s XUT VACCINE THC VT

Thit k vector cha gen quan tm Chuyn vector vao khun Agrobacterium Chuyn gen quan tm vao t bao thc vt Phn tich biu hin gen trong cy Kim tra kha nng min dich ng vt. Tinh sach va san xut.

Ti liu chia s ti: wWw.SinhHoc.edu.vn KT QU

THIT K VECTOR

June 12

Ti liu chia s ti: wWw.SinhHoc.edu.vn CU TRC GEN NHN TO

DNA
GGGGACAAGTTTGTACAAAAAAGCAGGCTTC CTC TGC TTC CTC CTC ACC AAC GCT TAC AGG AAC ACT CAG ATC TAC ACC GGC AAG AGG GAG ATG GTG ATC ATC GGC TCA CAA CAC ATC GAT TCC CAG ACC TAC CTC ACT GAG ACC AAG ATT ATT GCT GCC ATC AGC ATG GAG AAC GAA TGG TCT TAC ATt GTt GAG AAG GAA TGC CCC AAg TAT GTG AAg TCA CTT GAA CAG AAA CTC ATC TCA GAA CTTTCTTGTACAAAGTGGTCCCC ATG GTC ATC ACC AAG GAC TCA GCt AAC GAG GCA GAC AAT TTC AAG AAG GAC AAg AGA GAT AAC GCT GAC AAG GCC CTC CCA CCA TTg CTG AAG CCA AAG TCA ATT TGT CAC GTt GTC AAT TTG CAA ATC GGA GAG GTG GTG AAg CTT GGT TTC TCC CTC GCC AGG TGG CCA GAC GCt TCC CTC ATC TCT ACT ATG AAC AAC CTC ACT AAA GTC ACT TAC TTC AAG AAC GTC TGT GGG GAC TGC GAG ACT CAG GAC AAG GAC TAC CTC GAA GCA CTT GAG GTG ACC ACT CTC CCA AGA CTC GCT TGC AGC GAG TTG CCA GAG GGa AAg TGA CTC GCT TCT GAG ATG GCT GTT CCA AGG ATC AAC TCC GAA TTC GGC GGT AGC AAG GACCCAG

Protein

MANKLFLVCAALALCFLLTNALDAPQSITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGATFQVEVPGSQHIDSQ KKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMENSDPHVPNLDLEEFEWSYIVEKANPVNDLCYPGGGECPKYVKS NRLVLATGLRNSKDEQKLISEEDLNGSKDEL.

RE1 attB1 Size (bp) 31 352S2 S 63 LTB 6 372 L 21 18 RE2 RE3

B epitope
57

G
6

T epitope
60

C-myc KDEL attB2 35 S 6 39 12 31

June 12

Ti liu chiavector tai t hp cha cu trc gen HAop Bc 1:Thit k s ti: wWw.SinhHoc.edu.vn
Tn mi XhoI-HA HindIII-HA Trnh t CCGCTCGAGATCTGCATTGGATACCACGCT CCCAAGCTTGCAGATCCTGCACTGAAGAGAT

1.7kb

Kt qua PCR nhn oan gen HAop bng XhoI-HA/HindIII-HA

Ti liu chia vector tai t hp cha cu trc gen HAop s ti: wWw.SinhHoc.edu.vn Bc 1:Thit k

Ti Bc 1:Thit k vector tai t hp cha cu trc gen HAop liu chia s ti: wWw.SinhHoc.edu.vn

Ct pDONN201-HAop bng XhoI/HindIII

Ti liu chia s ti:vector c trng trong ht Bc 2: Thit k wWw.SinhHoc.edu.vn

TiBc 2: Thit k vector c trng trong ht liu chia s ti: wWw.SinhHoc.edu.vn

in di kim tra vector tai t hp pPhaso-HAop bng PCR (A)v ct bi enzyme hn ch EcoRI (B). M: Thang chun 1 Kb; 1, 2, 3: mu vector tai t hp tach t cac dong khun lc.

Ti liu chia npti: wWw.SinhHoc.edu.vn Bc 3: Bin s vector tai t hp vo chng vi

khun Agrobacterium

Kim tra dong A. tumefaciens CV58 mang gen bin nap bng PCR

Ti liu chia s ti: wWw.SinhHoc.edu.vn

Kt qu bin np cu trc gen vo cy thuc la

Ti liu chia s ti: wWw.SinhHoc.edu.vn Kt qu bin np cu trc gen vo cy thuc la

Kim tra gen chuyn HAop trong cy thuc la. M: Thang chun 1 Kb; 1: mu cy thuc la khng chuyn gen; 2, 3, 4, 5, 6 va 7: mu cy thuc la chuyn gen HAop.

Ti liu chia s ti: wWw.SinhHoc.edu.vn Kt qu bin np vo cy u tng

Hat u tng

Hat sau kh trng 3 ngy

Gy nhim 40

Cm ng tao chi trn SIMKm50/5d

Ra cy trn ct/tru

Ra r trn RM/4 tun

Ko di chi trn SEM-Km50/ 4 -8 tun

Chn lc trn SIM-Km75/4 tun

Ti liu chia s ti: wWw.SinhHoc.edu.vn

Kt qu chuyn cac cu trc HA1 vo nch l mm ging u tng T12
LTN S mu bin nap S mu tao chi S chi ko S chi ra di r S cy sng trn gia th

1
2 3

250
220 180 650

80
70 55 205

30
25 20 65

16
25 14 45

12
10 10 32

Tng

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Kt qu chuyn cac cu trc HA1 vo nch l mm ging u tng T12

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Cac dong u tng chuyn gen th h T0

Ti liu chia s lun wWw.SinhHoc.edu.vn Kt ti: v kin ngh

KT LUN
1.Bng vic s dng ngun gen HA ca virus H5N1 phn lp Vit Nam, chng ti

thit k thnh cng vector mang cu trc gen ph hp biu hin thc vt.
2. thu c cy thuc la mang cu truc HAop y la ngun nguyn liu cho phn tch kh nng biu hin gen chuyn va c s cho bin nap vao cac cy trng khac.

3. S dng phng php chuyn gen thng qua nch l mm nh vi khun

A.tumefacien, bc u thu c 8 dng cy T0 mang gen HA.1 y l tin quan trong cho vic tao c cc dng u tng mang gen m ho cho cc khng

nguyn ca virus H5N1 lm vacine n c cho gia cm phng chng bnh H5N1
Vit Nam.

Ti liu chia s lun wWw.SinhHoc.edu.vn Kt ti: v kin ngh

KIN NGH 1. Tip tc chn lc cac dng u tng bin nap thu c. 2. Phn tch kim tra kh nng ap ng min dch gia cm.

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XIN TRN TRNG CM N!