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THIT K VECTOR MANG GEN HA1 CA VIRUS H5N1 S DNG TRONG CHUYN GEN THNG QUA VI KHUN A.RHIZOGENES Nguyn Huy Hong*, V Th Nh Trang B mn Sinh hc - i hc Y dc Thi Nguyn
TM TT H5N1 l mt bin chng ca virus cm A, l nguyn nhn gy ra dch cm gia cm. ngn nga s ly nhim virus, phng php ph bin nht l tim phng vaccine. Hng nm, Vit Nam cn mt lng vaccine rt ln tim chng phng bnh. Hin nay, chuyn gen m ha cc khng nguyn nhm sn xut vaccine ti t hp vo thc vt, c th l vo cc cy trng l ngun thc n chnh cho con ngi, ng vt nui l mt trong nhng hng i c trin vng. Tuy nhin, xu hng s dng h thng nui cy sinh khi t bo sn xut cc dc phm sinh hc ti t hp cng c bt u quan tm nghin cu v ng dng. Trong nghin cu ny, nhm to c s cho vic sn xut vaccine A/H5N1 ra mi trng nui cy r t thuc l chuyn gen, chng ti tin hnh thit k vector chuyn gen HA1 biu hin khng nguyn b mt ca virus cm A/H5N1 mang on trnh t tn hiu gip cho protein HA1 c th tit ra mi trng nui cy. on gen HA1 c tch dng v ni vi on trnh t tn hiu, cu trc ny c ghp ni thnh cng vo vector chuyn gen pK7WG2D bng k thut lai Gateway. T kha: biu hin gen, HA, vaccine thc vt, H5N1, r t. T VN Cm g (avian influenza) l mt bnh truyn nhim cp tnh ca cc loi chim, k c gia cm v thu cm, do cc subtype khc nhau thuc nhm virus cm A gy nn. Do c sc khng t nhin tt nn mt s loi chim mang virus gy bnh, nhng khng c biu hin ca bnh. y l mi nguy him lan truyn bnh cho cc loi gia cm khc. Ngoi ra, chng cn l ngun tng tr bin i ngun gen to nn cc subtype mi. Cc subtype virus cm A gy bnh trn ngi u c ngun gc tin ho bin th v bin chng t ng vt v sau khi thch ng trn ngi th gy bnh, trc y to nn nhng v dch thm khc, ri bin mt sau mt thi gian li ti hin v gy nn i dch mi [1, 3]. phng chng s ly lan mnh m ca i dch cm gia cm, hng nm Vit Nam cn mt lng vaccine rt ln tim chng cho hng trm triu con g, vt. Ngoi nhng cng ngh sn c, Vit Nam ang tm kim nhng cng ngh sn xut vaccine mi c ngha kinh t, an ton v hiu qu cao. Vaccine n c c ngun gc t thc vt s l ngun vaccine p ng c cc tiu ch , ng thi c xem l hng nghin cu mi v mc ch chm sc sc khe cng ng ph hp vi nhng nc ang pht trin nh Vit Nam. Chuyn gen m ha cc khng nguyn ti t hp vo thc vt c th l vo cc cy trng l ngun thc n chnh cho con ngi, ng vt nui l mt trong nhng hng i chnh hin nay. Tuy nhin bn cnh , mt xu hng cng c bt u quan tm nghin cu v ng dng l s dng h thng nui cy sinh khi t bo sn xut cc dc phm sinh hc ti t hp. So snh vi cy trng chuyn gen, nui cy to sinh khi t bo thc vt c u im ln l khng i hi din tch canh tc, khng chu nh hng ca kh hu, ma v, sn phm thu c khng mang cc yu t c hi t thuc tr su, thuc dit c v rt ngn c rt nhiu thi gian trng trt. Thm vo , nui cy t bo thc vt d dng, mi trng nui cy n gin, r tin, ch ng, c th sn xut mt lng sinh khi ln trong khong thi gian ngn. c bit hn c l h thng nui cy

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lng cc m t bo thc vt vic biu hin dc phm sinh hc ti t hp ra mi trng nui cy n gin hn rt nhiu [5]. Khc vi nui cy m, nui cy sinh khi t bo thc vt khng nh hng to ra cy hon chnh m ch pht trin cc dng t bo nh nui cy m so, huyn ph t bo, r t to sinh khi ln cc sn phm. Trong , r t c gy to ra bi qu trnh tng tc gia vi khun t A. rhizogenes v t bo vt ch. u im trong nui cy r t l c th sinh trng, pht trin tt trn cc mi trng nui cy khng b sung cc cht kch thch sinh trng, kh nng phn nhnh cao, k thut nui cy v chuyn gen d dng. Hn na, cc r t c th c nui cy to sinh khi lin tc, rt c ngha trong dy chuyn sn xut cc cht th cp hay cc dc phm sinh hc ti t hp c chng ti quan tm nghin cu. Vi mc ch to c s cho vic sn xut vaccine A/H5N1 n c, chng ti tin Bng 1. Cc cp mi v trnh t nucleotide s dng trong nghin cu
Tn HA1_SacI_F HA1_HindIII_R Trnh t DNA 5 GGGGAGCTCGATCAGATCTGCATTGGAT 3 5 GGAAGCTTTTACCTTCTTTCTCTCTGTGG 3 Ch thch Cp mi nhn on gen HA1, thm trnh t ct enzyme hn ch SacI vo mi xui v HindIII vo mi ngc on tn hiu Calreticulin (Cal - 81 bp) c tng hp nhn to, thm trnh t gn mi ca attB1 u 5 v trnh t gn mi ca attB2 u 3 Cp mi nhn on Calreticulin ng thi to v tr ti t hp attB, on gen ny s dng ghp ni vo vector pDONRTM221 bng k thut Gateway MCS mang trnh t ct SacI, XbaI, HindIII

hnh nghin cu thit k vector mang gen HA1 biu hin khng nguyn HA ca H5N1 v bc u to dng r t chuyn gen cy thuc l. y se l ngun nguyn liu c s bin nap va biu hin khang nguyn cua virus trong cy trng. VT LIU V PHNG PHP Chng vi sinh vt v h gen Chng E. Coli DH5 (Invitrogen) c s dng nhn dng gen. Vector pUC18/Haop mang gen HA biu hin khng nguyn b mt virus cm A, gen ny c chuyn m ti u cho biu hin thc vt. Vector pDONRTM221 (Invitrogen) c s dng to dng gen bng k thut Gateway. Vector chuyn gen thc vt pK7WG2D [6]

Calreticulin

5 AAGAAGGAGATATATACCATGGCTACTCAACGA AGGGCAAACCCTAGCTCTCTCCATCTAATTACTGT ATTCTCTCTGCTCGTCGCTGTCGTCTCCGCTGAG CTCTCTAGAAAGCTT 3 5GGGGACAAGTTTGTACAAAAAAGCAGGCTCATT TAACTTTAAGAAGGAGATATATACC GGGGACCACTTTGTACAAGAAAGCTGGGTCAAGC TTTCTAGAGAGCTC 3

attB1_SP attB2_SP_MCS

Trnh t cc on nucleotide c tng hp ti cng ty MWG, CHLB c v cng ty Epoch Biolabs, Hoa K. Tng hp on Calreticulin trnh t tn hiu

Da trn trnh t gen Calreticulin trn GenBank (m s Z71395.1), chng ti tng hp nhn to on trnh t nucleotide m ha on peptide tn hiu Calreticulin (81 nucleotide) v thm trnh t gn mi ca attB1 v attB2 u 5 v 3 tng ng (bng 1).

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Dng ha on trnh t tn hiu Cal vo pDONRTM221 bng k thut Gateway K thut Gateway l mt k thut dng ha ph bin, da vo im c hiu trong h thng ti t hp ca phage I. K thut Gateway cho php chuyn on DNA gia cc vector tch dng khc nhau trong khi vn duy tr nh hng chnh v cu trc c. K thut ny l mt phng php c hiu qu cao cho vic tch dng c nh hng ca cc sn phm PCR. K thut ny gm c hai loi phn ng LR v BP (Invitrogen). Trong nghin cu ny s dng cp mi attB1_SP v attB2_S_MCS nhn on trnh t tn hiu Cal to mang trnh t ti t hp attB1&2. Phn ng BP c thc hin gia sn phm PCR trn v vector pDONRTM221 mang di s xc tc bi hn hp enzym BP ClonaseTM, kt qu to vector tip nhn pENTR 221/cal. Phn ng c thc hin theo hng dn ca b kit Gateway Cloning (Invitrogen) c ci tin cho ph hp vi iu kin phng th nghim. Ghp ni on gen HA1 vo vector tip nhn pENTR221/cal on gen HA1 c nhn ln bng PCR t plasmid pUC18/HAop vi cp mi c hiu HA1_Sacl_F v HA1_HindIII_R theo chu trnh: 95oC - 3 pht, 30 chu k ( 95oC 30 giy, 570C - 30 giy,72oC - 1 pht 30 giy), 72oC - 10 pht v phn ng kt thc sau khi mu c lm lnh n 10oC. Sn phm tng hp gen trn v pENTR221/cal c ct to u sole bng 2 enzyme gii hn (SacI & HindIII), ni ghp to pENTR221/cal/HA1 v bin np vo t bo kh bin E.coli DH5. Vector ti t hp pENTR221/cal/HA1 c kim tra bng phng php colony-PCR v ct kim tra bng enzyme hn ch SacI & HindIII. Xc nh trnh t gen HA1 bng my phn tch trnh t t ng ABI PRISM 3100 Avant Genetic Analyzer theo nguyn l ca Sanger vi b kit BigDye Terminator v. 3.2 Cycle Sequencing. Thit k vector chuyn gen thc vt bng k thut Gateway Phn ng LR c thc hin gia vector tip nhn pENTR221/cal mang trnh t ti t hp attL1&2 v vector ch pK7WG2D mang trnh t ti t hp attR1&2 di s xc tc bi hn hp enzym LR ClonaseTM II theo hng dn ca b kit Gateway Cloning (Invitrogen). Vector chuyn gen pK7WG2D/cal/HA1 (hnh 1) c kim tra bng phng php PCR v ct enzyme gii hn. KT QU V THO LUN Kt qu dng ha on trnh t tn hiu Cal vo pDONRTM221 bng k thut Gateway Vi mc ch dn protein ti t hp tit ra mi trng nui cy lng r t cy thuc l, chng ti thit k on trnh t tn hiu tit calreticulin ni vi gen HA1 u 5. on trnh t tn hiu ny c phn lp t Nicotiana plumbaginifolia h hng gn vi thuc l (Nicotiana tabacum) - cy nhn gen chuyn, iu ny nhiu kh nng lm tng hiu qu ca qu trnh tit protein ti t hp [2]. to iu kin cho thit k vector chuyn gen thc vt bng k thut Gateway cc th nghim tip theo, on trnh t tn hiu Cal c to v tr ti t hp attB bng phng php PCR s dng cp mi attB1_SP v attB2_SP_MCS (Bng 1). on trnh t trn c ghp ni vo vector pDONRTM221 bng k thut Gateway theo nguyn l v phng php nh miu t phn phng php nghin cu. Kt qu ca qu trnh ghp ni on gen Cal vo pDONRTM221 c xc nh bng bin np sn phm ni ghp gen vo t bo E.Coli. Sau , chn cc khun lc kim tra bng phng php colony-PCR vi cp mi M13. Nu vector mang gen ngoi lai, kim tra sn phm PCR s nhn c bng DNA c kch thc khong 0,4bp. Kt qu kim tra sn phm PCR khng nh

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on trnh t Cal c ghp ni vo pDONRTM221 to vector tip nhn pENTR221/cal. Thit k vector pENTR221/cal/HA1 HA1 l mt trong ba tiu phn cu to nn protein HA, cho php nhn bit t bo ch ca ng vt c xng sng v hon thnh qu trnh nhn bit bng cch gn kt vi th th cha sialic acid ca nhng t bo ny [4, 7]. on gen HA1 c nhn ln t pUC18/HAop bng phng php PCR vi cp mi c hiu HA1_SacI_F v HA1_HindIII_R. Kt qu in di thu c on DNA c hiu, r, c kch thc khong 1000 bp ng theo tnh ton l thuyt (978 bp) khi thit k mi (hnh 1A).

M 1

M 2

2,5bp kb 1kb kb

Hnh 1. Kt qu nhn on gen HA1 bng PCR (A) v kim tra cu trc vector pENTR221/cal/HA1 (B). M: thang DNA chun 1kb; 1: sn phm PCR ca HA1; 2: sn phm ct bng enzyme SacI/HindIII

10 kb 1 kb 1 kb 0,5 kb bp
A B

Hnh 2. in di kim tra vector ti t hp pK7WG2D/cal/HA1 bng PCR (A) v ct bi enzyme ct hn ch SacI/HindIII (B). M: thang chun 1kb; ging 1 - 10: mu vector ti t hp tch t cc dng khun lc. to iu kin ghp ni gen HA1 trc tip vo pENTR221/cal, sn phm PCR trn c tinh sch loi b cc thnh phn khng mong mun trc khi ct bng cp enzyme hn ch SacI v HindIII. on gen HA1 ny c ni ghp vo vector

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pENTR221/cal tin x l vi cng cp enzyme trn v nhn dng trong t bo E.coli DH5. Kt qu ca qu trnh bin np c kim tra bng phng php tch chit plasmid ct kim tra (hnh 1B). Kt qu trn in di cho thy 2 bng DNA co kich thc khoang 978bp va 2544 bp, tng ng vi kich thc cua gen HA1 va vector ng nh tnh ton l thuyt. Bn cnh , kt qu gii trnh t gen cho thy, dng gen la chn c mang chnh xc trnh t on tn hiu Cal ni vi trnh t gen HA. Thit k vector chuyn gen Cu truc gen cha HA1 va trnh t tn hiu Cal c chuyn vao vector pK7WG2D [6] bng k thut Gateway theo m t phn phng php v vt liu nghin cu. Kt qua kim tra vector tai t hp bng PCR va ct enzyme han ch SacI/HindIII cho thy san phm PCR vi cp mi c hiu HA1_SacI_F/HA1_HindIII_R co kich thc khoang 978 bp (hnh 2A), sn phm ct vector pK7WG2D/cal/HA1 bng SacI/HindIII thu c 4 oan gen kich thc khoang 434bp, 978 bp, 1201bp v 11159 bp kich thc nay phu hp vi tinh toan theo ly thuyt (Hinh2B). Nh vy, cu trc gen HA1 c thit k thnh cng vo vector chuyn gen thc vt pK7WG2D. Dng plasmid s 1 c la chn cho bin np vo Agrobacterium rhizogenes ATCC15834 to dng r t chuyn gen. KT LUN thit k thnh cng vector chuyn gen thc vt pK7WG2D/cal/HA1 mang cu trc gen HA1 ni vi trnh t tn hiu Cal dn HA1 ti t hp tit ra mi trng nui cy r t cy thuc l chuyn gen. Vector chuyn gen c bin np vo A.rhizogenes ATCC 15834 to r t cy thuc l chuyn gen. TI LIU THAM KHO 1. Alexander, D.J. (2007), Summary of avian influenza activity in Europe, Asia, Africa, and Australasia, 2002-2006. Avian Dis, 51(1 Suppl): p. 161-6. 2. Borisjuk NV, Borisjuk LG, et al. (1999). "Production of recombinant proteins in plant root exudates." Nat. Biotechnol. 17(5): 466469. 3. Bosch, F., M. Orlich, H. Klenk, and R. Root (1979), The structure of the hemagglutinin: a determinant for the pathgencity of Influenza virus, Virology, 95: p. 197-207. 4. Chen, L., C. Davis, H. Zhou, N. Cox, and R. Donis (2008), Genetic compatibility and virulence of reassortants derived from contemporary avian H5N1 and human H3N2 influenza A viruses. PLoS Pathog, 4(5): p. e1000072. 5. Doran, P.M. (2000), Foreign protein production in plant tissue cultures. Curr Opin Biotechnol, 11(2): p. 199-204. 6. Karimi M, Inze D, et al. (2002). "Gateway vectors for Agrobacterium-mediated plant transformation." Trends Plant Sci. 7(5): 193195. 7. Zhao, Z., K. Shortridge, M.G. M, Y. Guan, and X. Wan (2008), Genotypic diversity of H5N1 highly pathogenic avian influenza viruses. J Gen Virol, 89(9): p. 2182-2193.

CONSTRUCTION OF BINARY VECTOR CARRYING HA1 OF H5N1 VIRUS USING IN PLANT TRANSFORMATION VIA A.RHIZOGENES Nguyn Huy Hong*, V Th Nh Trang Thai Nguyen University of Medicine and Pharmacy SUMMARY

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H5N1 is a sulotyspe of influenza A, commonly called avian flu or bird flu. To prevent the viral infection, the most common method is vaccination. Currently, there have been many flu A vaccines available. However, plant-based oral vaccine is targeted by many researchers around the world because this subunit vaccine can be eaten, easy to administer and more effiective than other injection vaccines. In this study, we aimed establish a method to transfer HA1 gene encoded antigens HA from H5N1 into tobacco plant as a very first stage of develop an plantbased oral vaccine. With the aim of creating facilities for the production of H5N1 vaccine, creating materials to produce transgenic plants likeness of virus antigens in the crop. We have studied gene transfer vector design HA1 expression of surface antigen HA H5N1 virus carrying the signal sequence may help the HA1 protein secreted outside the culture medium Gateway hybrid technique. Keywords: H5N1, gene expression, plant vaccine, HA, hairy root.