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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
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
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).
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
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