Nghiên cứu phát triển vaccine thực vật dùng qua đường miệng cho gia cầm phòng chống bệnh H5N1 ở Việt

Header Page of 133 B KHOA HC V CễNG NGH NHIM V HP TC QUC T V KHOA HC V CễNG NGH THEO NGH NH TH VI VNG QUC B (Giai on 2007 2009) - BO CO TNG HP KT QU KHOA HC CễNG NGH TI Nghiờn cu phỏt trin vaccine thc vt dựng qua ng ming cho gia cm phũng chng bnh H5N1 Vit Nam (Development of a plant-based veterinary oral vaccine to combat avian influenza in Vietnam) Ch nhim ti: TS Lờ Vn Sn 8032 H Ni, 12/2009 Footer Page of 133 Header Page of 133 B KHOA HC V CễNG NGH NHIM V HP TC QUC T V KHOA HC V CễNG NGH THEO NGH NH TH VI VNG QUC B (Giai on 2007 2009) - BO CO TNG HP KT QU KHOA HC CễNG NGH TI Nghiờn cu phỏt trin vaccine thc vt dựng qua ng ming cho gia cm phũng chng bnh H5N1 Vit Nam (Development of a plant-based veterinary oral vaccine to combat avian influenza in Vietnam) Ch nhim ti: TS Lờ Vn Sn C quan ch trỡ: Vin Cụng ngh sinh hc a ch: Nh A10, 18 Hong Quc Vit, H Ni in thoi: 37562368 E-mail: levanson@ibt.ac.vn H Ni, 12/2009 Footer Page of 133 Header Page of 133 Bỏo cỏo tng kt ti Li cm n LI CM N hon thnh ti nghiờn cu ny, ch nhim ti xin chõn thnh cm n: B Khoa hc v Cụng ngh Vit Nam ó cp kinh phớ thc hin Lónh o Vin Cụng ngh sinh hc, Ban K hoch ti chớnh Vin Khoa hc v Cụng ngh Vit Nam ó giỳp v to iu kin tt ti thc hin ỳng tin Phũng thớ nghim trng im Cụng ngh gen - Vin Cụng ngh sinh hc ó thc hin vic gii trỡnh t gen Tri sinh hc thc nghim C Nhu - Vin Cụng ngh sinh hc ó h tr cõy ngoi nh li Phũng Min dch hc Vin Cụng ngh sinh hc ó thc hin vic kim tra mc ỏp ng dch trờn ng vt Ch nhim ti TS Lờ Vn Sn Thi gian thc hin 2007-2009 Footer Page of 133 i Header Page of 133 VIN KHOA HC V CễNG NGH VIT NAM VIN CễNG NGH SINH HC CNG HO X HI CH NGHA VIT NAM c lp - T - Hnh phỳc H Ni, ngy thỏng nm 20 BO CO THNG Kấ KT QU THC HIN TI I THễNG TIN CHUNG Tờn ti: Ting Vit: Nghiờn cu phỏt trin vaccine thc vt dựng qua ng ming cho gia cm phũng chng bnh H5N1 Vit Nam Ting Anh: Development of a plant-based veterinary oral vaccine to combat avian influenza in Vietnam Thuc: Nhim v hp tỏc quc t v khoa hc v cụng ngh theo ngh nh th vi Vng quc B Ch nhim ti: H v tờn: Lờ Vn Sn Ngy, thỏng, nm sinh: 22/01/1967 Nam/ N: Nam Hc hm, hc v: Tin s Chc danh khoa hc: Chc v: in thoi: T chc: 043 7562368 Mobile: 0915570750 E-mail: levanson@ibt.ac.vn Tờn t chc ang cụng tỏc: Vin Cụng ngh sinh hc a ch t chc: 18 Hong Quc Vit, Cy Giy, H Ni a ch nh riờng: xúm 4B, C Nhu, T Liờm, H Ni Footer Page of 133 Header Page of 133 T chc ch trỡ ti/d ỏn: Tờn t chc ch trỡ ti: Vin Cụng ngh sinh hc, Vin Khoa hc v Cụng ngh Vit Nam in thoi: 37562790 Fax: 38363144 E-mail: tnhai@ibt.ac.vn Website: http://www.ibt.ac.vn a ch: 18 Hong Quc Vit, Cu Giy, H Ni H v tờn th trng t chc: PGS TS Trng Nam Hi S ti khon: 931.01.064 Ngõn hng: Kho bc nh nc Ba ỡnh, H Ni Tờn c quan ch qun ti: B Khoa hc v Cụng ngh II TèNH HèNH THC HIN Thi gian thc hin ti/d ỏn: - Theo Hp ng ó ký kt: t thỏng 06 nm 2007 n thỏng 05 nm 2009 - Thc t thc hin: t thỏng 06 nm 2007 n thỏng 12 nm 2009 - c gia hn : - Ln 1: t thỏng 05 nm 2009 n thỏng 12 nm 2009 (Quyt nh s 2312/BKHCN-XHTN, ngy 14/9/2009 ca B Khoa hc v Cụng ngh) Kinh phớ v s dng kinh phớ: a) Tng s kinh phớ thc hin: 950 tr., ú: + Kớnh phớ h tr t SNKH: 950 tr. + Kinh phớ t cỏc ngun khỏc: tr. b) Tỡnh hỡnh cp v s dng kinh phớ t ngun SNKH: Footer Page of 133 Header Page of 133 S TT Theo k hoch Thi gian (Thỏng, nm) Thc t t c Kinh phớ (Tr.) Thi gian (Thỏng, nm) Ghi chỳ (S ngh quyt toỏn) Kinh phớ (Tr.) 2007 500 2007 463, 383 524 463, 383 524 2008 450 2008 388, 216 476 388, 216 476 2009 98, 400 000 98, 400 000 Cng 950 950 c) Kt qu s dng kinh phớ theo cỏc khon chi: n v tớnh: Triu ng S Ni dung TT cỏc khon chi Theo k hoch Tng SN KH Ngun khỏc Thc t t c Tng SN KH Ngun khỏc Tr cụng lao ng (khoa hc, ph thụng) 193,7 193,7 213,700000 213,700000 Nguyờn, vt liu, nng lng 469,8 469,8 497,518075 497,518075 Thit b, mỏy múc 0 0 0 Xõy dng, sa cha nh 16,0 16,0 16,000000 16,000000 Chi khỏc 270,5 270,5 222,781925 222,781925 950 950 950 950 Tng cng - Lý thay i (nu cú): iu chnh tin on ra, b sung cho nghiờn cu nc Cỏc bn hnh chớnh quỏ trỡnh thc hin ti/d ỏn: S TT S, thi gian ban hnh bn Tờn bn Footer Page of 133 Ghi chỳ Header Page of 133 95/Q-BKHCN ngy 18/1/2007 Q thnh lp Hi ng KHCN xột duyt cng NC 823/Q-BKHCN ngy 22/5/2007 Q phờ duyt cỏc nhim v hp tỏc QT 2007 29/823/2007/HNT ngy 6/12/2007 H v Phiu thm nh thc hin nhim v hp tỏc KHCN theo Ngh nh th 1116/KHCNVNHTQT ngy 8/8/2007 Thay i ch nhim nhim v hp tỏc KHCN theo Ngh nh th 2312/QBKHCN-XHTN ngy 14/9/2009 Gia hn thi gian thc hin nhim v hp tỏc KHCN theo Ngh nh th 2976/QBKHCN-XHTN ngy 26/11/2009 Thay i ni dung-kinh phớ ca nhim v hp tỏc KHCN theo Ngh nh th 3157/QBKHCN-XHTN ngy 19/12/2009 iu chnh kinh phớ mua vt t húa cht ca nhim v hp tỏc KHCN theo Ngh nh th 564a/Q-CNSH 25/12/2009 Thnh lp hi ng nghim thu quy trỡnh KHCN 564/Q-CNSH 25/12/2009 Thnh lp hi ng nghim thu c s ca ti T chc phi hp thc hin ti, d ỏn: S TT Tờn t chc Tờn t chc ó ng ký theo tham gia thc hin Thuyt minh Trung tõm thỳ Phũng Min dch y vựng Cn hc, Vin Cụng ngh Th/ TT kim sinh hc nghim thuc thỳ y T Footer Page of 133 Ni dung tham gia ch yu Sn phm ch yu t c Kim tra hiu qu Kim tra sn phm trờn gia dch trờn gia cm cm Header Page of 133 Trng i Trng i hc T hc T do (VUB), Vng (VUB), Vng quc B quc B - Thit k vector o tao, hp tỏc - Chuyn gen nghiờn cu - Phõn tớch cõy chuyn gen - Kim tra hiu qu sn phm Trung tõm Thỳ Trung tõm Thỳ y v Kim tra hiu qu y v Nụng Nụng nghip (VAR- sn phm trờn gia nghip (VAR- CODA-CERVA), cm CODAVng quc B CERVA), Vng quc B - Lý thay i (nu cú): Cỏ nhõn tham gia thc hin ti, d ỏn: (Ngi tham gia thc hin ti thuc t chc ch trỡ v c quan phi hp, khụng quỏ 10 ngi k c ch nhim) S TT Tờn cỏ nhõn ng ký theo Thuyt minh Tờn cỏ nhõn ó tham gia thc hin Ni dung tham gia chớnh Sn phm ch yu t c TS Lờ Vn Sn TS Lờ Vn Sn Ch nhim ti Tng hp phõn tớch kt qu GS TS Lờ Trn Bỡnh GS TS Lờ Trn Bỡnh Phõn tớch cõy chuyn gen Chn cỏc dũng chuyn gen TS Nguyn Trung Nam TS Nguyn Tng Võn Chuyn gen vo arabidopsis Cõy arabidopsis chuyn gen CN Phan Trng Hong CN Phan Trng Hong Chuyn gen vo thuc lỏ Cõy thuc lỏ chuyn gen TS Nguyn Bỏ Thnh KS Bựi Phng Tho Ti u quy trỡnh chuyn gen u tng Quy trỡnh chuyn gen Prof Dr Geert Angenon TS Chu Hong H Thit k vector Cỏc vector chuyn gen Dr Tran Thanh Thu CN Ngụ Th Thu Hng Phõn tớch cõy chuyn gen Phõn tớch cỏc dũng cõy c.gen Dr Thierry van ThS on Th Kim tra hiu qu ỏnh giỏ hiu Footer Page of 133 Header Page of 133 den Berg Thanh Hng sn phm trờn gia cm lc khỏng nguyờn ca ht chuyn gen Prof Dr Geert Angenon Ch nhim phớa B Tng hp phõn tớch kt qu 10 Dr Tran Thanh Thu Chuyn gen v phõn tớch cõy c gen Cõy arabidopsis chuyn gen - Lý thay i ( nu cú): Tỡnh hỡnh hp tỏc quc t: S TT Theo k hoch Thc t t c 2007: 01 cỏn b sang VUB, B 2007: 01 cỏn b sang VUB, B thc nghiờn cu, kinh phớ i li thc nghiờn cu, kinh phớ i li phớa VN cp phớa VN cp 2008: 03 cỏn b sang VUB, B 2008: 01 cỏn b sang VUB, B thc v hi tho, kinh phớ i li hi tho, kinh phớ i li phớa VN cp phớa VN cp 2008: 04 chuyờn gia B sang VN 2008: 04 chuyờn gia B sang VN trao i ti, kinh phớ sinh hot trao i ti, kinh phớ sinh hot phớa VN cp phớa VN cp 2009: 02 chuyờn gia B sang VN 2009: 01 chuyờn gia B sang VN trao i ti, kinh phớ sinh hot trao i ti, kinh phớ sinh hot phớa VN cp phớa VN cp - Lý thay i (nu cú): Phia i tỏc khụng b trớ c thi gian Tỡnh hỡnh t chc hi tho, hi ngh: S TT Theo k hoch Hi tho v thc hin ti ti H Ni Thc t t c Hi tho tho lun kt qu thc hin ti ti phũng CNTBTV, Vin CNSH - Lý thay i (nu cú): Footer Page of 133 Header Page 10 of 133 Túm tt cỏc ni dung, cụng vic ch yu: S TT Thi gian Cỏc ni dung, cụng vic ch yu Theo k hoch Thc t t c Ngi, c quan thc hin Thit k vector dựng cho chuyn gen 6/200712/2007 6/200712/2007 Vin CNSH Xõy dng ti u quy trỡnh chuyn gen vo Arabidopsis v cõy u tng 6/20073/2008 6/20073/2008 Vin CNSH To cỏc dũng cõy chuyn gen 4/200812/2008 4/20084/2009 Vin CNSH Phõn tớch mc phõn t cỏc dũng cõy chuyn gen 10/200812/2008 1/20098/2009 Vin CNSH ỏnh giỏ s b hiu lc ca khỏng nguyờn 1/20095/2009 5/200911/2009 Phũng Min dch hc -Vin CNSH - Lý thay i (nu cú): Thi v cõy trng khụng thun li v phớa bn (i tỏc B) cú s thay i v t chc nờn khụng theo ỳng k hoch c III SN PHM KH&CN CA TI, D N Sn phm KH&CN ó to ra: a) Sn phm Dng I: (Mu (model, maket); sn phm (l hng hoỏ, cú th c tiờu th trờn th trng); vt liu; thit b, mỏy múc; dõy chuyn cụng ngh; ging cõy trng; ging vt nuụi v cỏc loi khỏc) S TT Tờn sn phm v ch tiờu cht lng ch yu n v o Cỏc vector chuyn gen cú vector mang cỏc gen ca H5N1 Cỏc dũng u tng dũng Footer Page 10 of 133 S lng Theo k hoch Thc t t c 7 1-2 Header Page 164 of 133 Bỏo cỏo túm tt kt qu thc hin ti M Hỡnh 17 Kt qu phõn tớch Southern cỏc dũng cõy T0 chuyn gen 3 M (-) Hỡnh 18 Kt qu phõn tớch Western cỏc dũng cõy T0 chuyn gen bit c kh nng biu hin ca gen chuyn, chỳng tụi s dng khỏng th n dũng c-myc phõn tớch kh nng tng hp protein bng western blot Protein sau c tỏch t ht cõy chuyn gen, ó c chy in di trờn polyacryamide v chuyn lờn mng lai Sau lai vi khỏng th c-myc v khỏng th chỳng tụi cú kt qu cho thy protein tỏi t hp biu hin mnh cỏc dũng SLHEP v SLHEP-M1(hỡnh 18), cu trỳc cha gen SLHEP-HA cú mc biu hin rt yu Da vo kt qu ny cho thy cỏc dũng cõy chuyn gen u biu hin protein cỏc mc khỏc i vi dũng cõy M1 c phõn tớch thy protein tỏi t hp c tng hp cao Cũn vi cỏc dũng cõy SLHEP cú dũng dũng biu hin rừ nht Trong cỏc th h tip theo, quỏ trỡnh phõn ly s to mt s dũng cõy khụng mang gen chuyn loi tr nhng dũng cõy ny, ht ca cõy T1 tip tc c chn lc riờng r trờn mụi trng cha khỏng sinh chn lc v cho cỏc th h tip theo T2, T3 (Hỡnh 19) th h T3, cõy ng hp t gen chuyn cú t l cao nờn s biu hin ca gen chuyn t mc ng u tt c cỏc dũng Ht ca th h ny s c th tinh sch v phõn tớch kh nng ỏp ng dch trờn ng vt Hỡnh 20 cho thy kt qu ca vic tinh sch protein tỏi t hp SLHEP bng kit c-myc Sn phm tinh sch ny cú lng v tinh sch tng i cao Hỡnh 19 Ht ca cỏc dũng cõy A thaliana chuyn gen th h T1 Hỡnh 20 Kt qu tinh c chn lc trờn mụi trng cha khỏng sinh v trng trờn bu sch protein tỏi t hp t SLHEP Thi gian thc hin 2007-2009 Footer Page 164 of 133 18 Header Page 165 of 133 Bỏo cỏo túm tt kt qu thc hin ti 3.3.2 Bin np vo cõy thuc lỏ Cỏc cu trỳc pCB301Kan-35S-HAx100ELP, pCB301Kan-35S-M1x100ELP v pCB301Kan-35S-NAx100ELP c bin np vo cõy thuc lỏ bng Agrobacterium thụng qua lõy nhim cỏc mnh lỏ (Hỡnh 21) Cỏc cõy tỏi sinh sau chn lc trờn mụi trng cha Kan s c trng trờn giỏ th thớch hp v tin hnh thu lỏ kim tra s cú mt ca gen chuyn bng PCR Kt qu phõn tớch PCR cho thy cỏc dũng cõy thu c sau chn lc u dng tớnh (Hỡnh 22) Hỡnh 21 Mt s hỡnh nh chuyn gen vo cõy thuc lỏ Hỡnh 22 PCR kim tra cõy thuc lỏ chuyn gen HA, NA v M1 Hỡnh 23 Biu hin protein tỏi t hp lỏ cõy thuc lỏ bin np khng nh cỏc cu trỳc ny cú c biu hin protein hay khụng, chỳng tụi chn 20 dũng cõy chuyn gen ny tip tc c kim tra Western blot Kt qu kim tra lỏ ca cỏc cõy ny th hin trờn hỡnh 23 u chng t u biu hin protein cỏc mc khỏc Trong ú cú dũng biu hin protein tỏi t hp khỏ cao gm dũng HA, dũng NA v dũng M1 c gi li phõn tớch tip 3.3.3 Bin np vo cõy u tng Thớ nghim c tin hnh vi tng s 1386 mu c s dng cho chuyn gen SLHEP, 396 mu cho chuyn gen HA v 386 mu cho chuyn gen M1 Kt qu cho thy cú s khỏc bit gia cỏc cu trỳc c chuyn i vi vector mang gen SLHEP, cú 75 chi kộo di trờn mụi trng chn lc SEM cha 50 mg/l kanamycin, ú 55 chi cú kh nng r v thu c 43 cõy T0 phỏt trin trờn giỏ th So sỏnh vi vic chuyn vector mang gen HA, thu c 11 cõy T0 phỏt trin trờn giỏ th Vic chuyn cu trỳc M1 vo nỏch lỏ mm u tng v tỏi sinh cõy gp nhiu khú khn nht, tng s 386 mu bin np kt qu thu c 10 cõy T0 cú kh nng sng trờn giỏ th Thi gian thc hin 2007-2009 Footer Page 165 of 133 19 Header Page 166 of 133 Bỏo cỏo túm tt kt qu thc hin ti kim tra s cú mt ca gen chuyn, sau cõy khong thỏng, mu lỏ ca cỏc dũng cõy T0 c thu tỏch chit DNA tng s v tin hnh phn ng PCR vi cỏc cp mi c hiu cho gen SLHEP v gen HA Kt qu in di sn phm PCR cho thy ó thu nhn c 29 cõy dng tớnh vi phn ng PCR nhõn gen SLHEP, chim t l 2,1% tng mu bin np v cõy dng tớnh vi phn ng PCR nhõn gen HA (Hỡnh 25), chim t l 0,3% Cỏc dũng cõy ny ang c tip tc trng thu ht v ỏnh giỏ s biu hin ca gen chuyn cỏc th h sau Riờng i vi gen M1, thớ nghim c tin hnh sau nờn cỏc dũng cõy T0 hin mi ang c trng trờn giỏ th v chun b thu lỏ tin hnh kim tra bng phn ng PCR M WT M Hỡnh 24 Hỡnh nh minh quỏ trỡnh chuyn gen WT 10 11 12 13 14 15 + + Hỡnh 25 Kim tra cõy u tng T0 chuyn gen SLHEP (hng trờn) v HA (hng di) bng PCR Nhng dũng cõy dng tớnh sng sút c trng nh li, nhng ch cú dũng cõy chuyn cu trỳc SLHEP v dũng cõy HA cho ht, vi lng 1-2 ht/dũng cõy õy cú th iu kin nh li hoc chm súc cõy u tng in vitro cha c phự hp th h tip theo T1, cỏc ht c gieo trờn t, cỏc dũng cõy u tng phỏt trin bỡnh thng (Hỡnh 26) Cỏc dũng cõy ny tip tc c sng lc bng PCR vi cỏc cp mi c hiu Kt qu thu c 3/7 dũng cõy T1 dng tớnh (Hỡnh 27) Hỡnh 26 Cỏc cõy u tng chuyn gen th h T1 c trng nh li Hỡnh 27 Kim tra cõy u tng T1 chuyn gen SLHEP bng PCR õy l ngun nguyờn liu tip tc sng lc cỏc v tip theo T1, T2, T3, loi tr cỏc dũng khụng mang gen chuyn to quỏ trỡnh phõn ly Kt qu thu c s l cỏc dũng ng hp t, biu hin protein tỏi t hp cao Thi gian thc hin 2007-2009 Footer Page 166 of 133 20 Header Page 167 of 133 Bỏo cỏo túm tt kt qu thc hin ti 3.4 KIM TRA MC P NG MIN DCH TRấN NG VT 3.4.1 Kim tra kh nng ngng kt hng cu ca khỏng nguyờn Ht Arabidopsis chuyn gen c nghin v tinh ch thụ, sau ú kim tra kh nng ngng kt hng cu trờn cỏc ging thớ nghim ELISA Nng cỏc protein ban u khong 0,0544 àg v h s pha loóng mu cỏc ging tip theo l Hỡnh 28 Kim tra kh nng ngng kt hng cu ca protein ht cõy chuyn gen Kt qu ny cho thy cu trỳc SLHEP v SLHEP-HA cú kh nng ngng kt hng cu, vi h s tng ng l 24 v 27 Nh vy dũng cõy chuyn cu trỳc gen ny s c s dng cho thớ nghim kim tra ỏp ng dch trờn ng vt Vi protein ht cõy chuyn gen SLHEP-M1 l protein vựng m (matrix) nờn khụng cú kh nng lm ngng kt hng cu, kt qu ging vi protein ht cõy khụng chuyn gen 3.4.2 Kim tra s b kh nng ỏp ng dch trờn chut Ht cõy chuyn gen th h T3 cú mc biu hin n nh v ng u s c s dng lm thc n cho chut thớ nghim õy ht ca Arabidopsis chuyn cu trỳc gen SLHEP ỏp ng s lng v tiờu chun tin hnh phõn tớch s b Ht dũng cõy ny c nghin nh trn nc v cho chut ung ln theo quy trỡnh gõy dch Mi ln cho n vi liu lng 10 mg ht/ngy ngy Ln gõy dch th hai lp li sau tun so vi ln th nht, huyt c thu sau tun tip ú Hỡnh 29 Kt qu c ELISA khỏng th SLHEP huyt chut 3.4.3 Kim tra kh nng ỏp ng dch trờn g G c nuụi lụ thớ nghim nh chut, huyt c thu c giai on khỏc nhau: g ngy tui, trc cho n ht chuyn gen gõy dch v sau Thi gian thc hin 2007-2009 Footer Page 167 of 133 21 Header Page 168 of 133 Bỏo cỏo túm tt kt qu thc hin ti tun gõy dch ln Thớ nghim c thc hin ti phũng Min dch hc - Vin Cụng ngh sinh hc Kim tra khỏng nguyờn bng phn ng ngng kt hng cu (HA) Khỏng nguyờn c s dng cho thớ nghim l khỏng nguyờn H5 B mụn Virus hc, Vin Thỳ y cung cp Kt qu thớ nghim cho thy hiu giỏ HA ca khỏng nguyờn l hiu giỏ 1/640 (ngng kt n ging s 7) Kim tra khỏng th H5 ca cỏc mu huyt bng phn ng ngn tr ngng kt hng cõu (HI) Pha khỏng nguyờn: Theo qui trỡnh ca T chc Thỳ y th gii (OIE) khỏng nguyờn H5 phi c pha hiu giỏ n v HA thc hin phn ng HI Kt qu chun kim tra khỏng nguyờn trờn cho thy: hiu giỏ HA ca khỏng nguyờn l 1/640, khỏng nguyờn pha cho HI l n v HA = 640:8 = 80 Nh vy, trn phn khỏng nguyờn vi 79 phn nc sinh lý 0,9% NaCl s t c dung dch khỏng nguyờn l HA Chun kim tra khỏng nguyờn HA: tin hnh nh phn ng HA Mc ớch kim tra l m bo khỏng nguyờn HA phi chc chn cú kh nng kt hp (ngng kt) vi hng cu g 0,5% Kt qu trờn cho thy, n pha loóng 1/8 (23) tc l HA, khỏng nguyờn cú kh nng ngng kt vi hng cu g Kt qu phỏt hin khỏng th khỏng H5 bng phn ng HI: Kt qu cho thy, 02 mu huyt i chng (lỳc g ngy tui: DOA-15.1.10, DOB-15.1.10); mu huyt trc thớ nghim (g 17 ngy tui: G1 1/1.2.10 n G7 1/1.2.10); mu huyt g khụng n ht (g 43 ngy tui: G1 2/26.2.10 n G3 2/26.2.10); v 01 mu huyt g n ht khụng chuyn gen (g 43 ngy tui: G7 2/26.2.10) õm tớnh, tc l hon ton khụng cú khỏng th khỏng li protein H5 i vi 05 mu huyt g n ht chuyn gen, cú 02 mu (G4 2/26.2.10 v G6 2/26.2.10) cú biu hin dng tớnh kim tra; 02 mu G5 2/26.2.10 v G8 2/26.2.10 cú biu hin nghi ng; v mu G7 2/26.2.10 cho kt qu õm tớnh Kt qu phỏt hin khỏng th khỏng H5 bng phn ng ELISA Qua kim tra ELISA cỏc mu huyt cho thy, cú 02 mu ca g n ht chuyn gen thớ nghiờm cú biu hin dng tớnh nh (G6 2/26.2.10 v G8 2/26.2.10) cú giỏ tr S/P tng ng bng 0,513 v 0,521; 01 mu g n ht chuyn gen G4 2/26.2.10 dng tớnh cú giỏ tr S/P l 0,566; phự hp mu cú kt qu dng tớnh bng phn ng HI; s cũn li tt c u õm tớnh Thi gian thc hin 2007-2009 Footer Page 168 of 133 22 Bỏo cỏo túm tt kt qu thc hin ti Header Page 169 of 133 TNG QUT HểA V NH GI KT QU TI Qua hn hai nm thc hin, ti ó thu c nhng kt qu nh ng ký ban u, ng thi ó cú mt s kt qu vt mc v m rng Cỏc kt qu chớnh c thng kờ qua bng nh sau: TT Ni dung ng ký ó t c Kt qu Cỏc vector chuyn gen cú mang cỏc gen ca H5N1 vector vector t Cỏc dũng chuyn gen 1-2 dũng dũng Vt ng ký dũng arabidopsis dũng arabidopsis dũng thuc lỏ Vt ng ký u tng Cỏc dũng cõy chuyn gen Quy trỡnh chuyn gen vo cõy u tng 1 t Bi bỏo chuyờn ngnh 1-2 Vt ng ký Bỏo cỏo hi ngh quc t Vt ng ký Kt qu phõn tớch kh nng gõy dch ca sn phm trờn i tng gia cm 1 t o to 1-2 Thc s Thc s Tin s Vt ng ký Mt s ỏnh giỏ v hiu qu ti mang li Hiu qu v khoa hc v cụng ngh - a cụng ngh to cõy chuyn gen thnh mt cụng c h tr c lc cho cụng tỏc to ging truyn thng cho cõy trng, ng thi nõng cao nng lc tip cn cỏc lnh vc khoa hc hin i lnh vc to ging v khoa hc cõy trng núi chung - Gúp phn a sinh hc phõn t kt hp vi cỏc phng phỏp dch enzyme phũng chng dch bnh, qun lý cõy chuyn gen v qun lý dch bnh trờn gia cm Thi gian thc hin 2007-2009 Footer Page 169 of 133 23 Header Page 170 of 133 Bỏo cỏo túm tt kt qu thc hin ti - o to cỏc cỏn b khoa hc tr nm vng cỏc k thut vic nghiờn cu chuyn gen thc vt cng nh vic sn xut vaccine n c theo ng thc vt - a nn khoa hc nc ta tng bc hi nhp khu vc v quc t trờn cỏc lnh vc v cụng ngh sinh hc thc vt núi chung v cụng ngh sinh hc cõy trng núi riờng Hiu qu v kinh t xó hi - Vic sn xut vaccine cho gia cm chng virus H5N1 trờn cõy trng a li hiu qu kinh t ni bt nh sn xut n gin, giỏ thnh thp, hiu qu cao phũng bnh v cú an ton cao, vaccine sn xut t thc vt c xem l hng i phự hp vi nhng nc ang phỏt trin vỡ mc ớch chm súc sc kho cng ng - Vaccine cỳm gia cm H5N1 trờn cõy trng chuyn gen s l ngun vaccine c hiu chng cỳm gia cm Vit Nam, luụn ch ng cụng tỏc phũng chng dch cỳm, iu ú s em li hiu qu kinh t cao, gim thiu chi phớ Vic sn xut vaccine cỳm gia cm trờn cõy trng chuyn gen s thỳc y v lm c s phỏt trin cỏc loi vaccine khỏc - Hn ch v khng ch dch cỳm H5N1 l bo v sc kho vt nuụi v ngi Ht cõy trng chuyn gen H5N1 s l ngun thc n, nú s gúp phn gim thit hi, phũng trỏnh tỏi phỏt v lu cu tỏc nhõn gõy bnh mụi trng - a nc ta hi nhp bỡnh ng vo tro lu phỏt trin nụng nghip cụng ngh cao ca khu vc v th gii thụng qua gim s dng hoỏ cht v tng cng cht lng sn phm Thi gian thc hin 2007-2009 Footer Page 170 of 133 24 Header Page 171 of 133 Bỏo cỏo túm tt kt qu thc hin ti KT LUN V NGH KT LUN Tỏi sinh thnh cụng cõy u tng in vitro thụng qua a chi t nỏch lỏ mm ht chớn v xỏc nh c nng thớch hp ca mt s cht iu hũa sinh trng cho cỏc mụi trng: mg/l BAP cho mụi trng to a chi, 0,5 mg/lGA3 + 0,1 mg/l IAA cho mụi trng kộo di chi v 0,1 mg/l IBA cho mụi trng r ng thi cng xỏc nh c giỏ th thớch hp cho vic cõy: tru hun : cỏt vng ó ti u quy trỡnh tỏi sinh phc v chuyn gen cho ging u tng T12 v xỏc nh c ngng nng cht chn lc kanamycin cho hai giai on quan trng ca quỏ trỡnh tỏi sinh cõy u tng thụng qua a chi: 75 mg/l cho giai on to a chi, 50 mg/l cho giai on kộo di chi Thit k c vector chuyn gen biu hin c trng ht v vector chuyn gen biu hin ton b cõy; bao gm: pPhaso-SLHEP, pPhasoSLHEP-HA, pPhaso-SLHEP-NA, pPhaso-SLHEP-M1, pCB301Kan-35SHAx100ELP, pCB301Kan-35S-M1x100ELP v pCB301Kan-35S-NAx100ELP Cỏc cu trỳc biu hin ton b cõy 35S-HAx100ELP, 35S-M1x100ELP v 35S-NAx100ELP, ó c chuyn vo cõy thuc lỏ dũng cõy chuyn gen thu c cú biu hin protein tỏi t hp cao Cỏc cu trỳc biu hin c trng ht SLHEP, SLHEP-HA, SLHEP-NA v SLHEP-M1 ó c bin np vo cõy Arabidopsis, thu c s dũng cõy bin np cu trỳc SLHEP v SLHEP-M1 ln hn cu trỳc SLHEP-HA Mt s dũng cõy bin np SLHEP v SLHEP-M1 ó c chn lc n th h T3 Mc biu hin khỏng nguyờn ht ca dũng cõy bin np cu trỳc gen SLHEP v SLHEP-M1 cao hn cỏc dũng cõy mang cu trỳc gen HA Sn phm protein SLHEP tinh sch qua ct c-myc cú hm lng v tinh sch tng i cao ó thu c dũng cõy T0 u tng chuyn cu trỳc SLHEP v SLHEP-HA cú ht v dng tớnh vi PCR th h T1 cho dũng cõy dng tớnh vi gen SLHEP Kh nng ngng kt hng cu ca cỏc khỏng nguyờn biu hin ht cho thy SLHEP-HA cú mc cao hn SLHEP Khỏng nguyờn SLHEP-M1 cú kt qu tng t vi protein ht khụng chuyn gen Thi gian thc hin 2007-2009 Footer Page 171 of 133 25 Header Page 172 of 133 Bỏo cỏo túm tt kt qu thc hin ti Kt qu kim tra s b v kh nng ỏp ng dch vi khỏng nguyờn SLHEP trờn chut nghiờn cu l khỏ rừ rng chut n ht chuyn gen cú khỏng th ca epitope SLHEP cao hn chut i chng hoc chut n ht khụng chuyn gen 10 Thớ nghim v kim tra mc ỏp ng dch trờn i tng gia cm (g) cho thy khụng phi tt c cỏc mu huyt thớ nghim dch vi g n ht chuyn gen cú kt qu dng tớnh kim tra bng cỏc phng phỏp ngn tr ngng kt hng cu HI v ELISA; nhng ớt nht cng cú 02-03 mu cú cha hm lng khỏng th H5 v cho kt qu dng tớnh Nh vy, ht cú cha khỏng nguyờn H5 mi cú th kớch thớch sinh khỏng th, hm lng khụng u tt c cỏc mu 11 ti ó cụng b c bi bỏo chuyờn ngnh nc v bỏo cỏo hi ngh quc t, ó v ang o to thc s v tin s NGH Trờn c s kt qu t c, cú th tip tc phỏt trin cỏc kt qu nghiờn cu, chỳng tụi xut mt s kin ngh sau: Tip tc chn cỏc dũng u tng bin np ó thu c Phõn tớch kim tra kh nng ỏp ng dch gia cm trờn quy mụ ln hn Nghiờn cu tip tc bin np gen HA y Thi gian thc hin 2007-2009 Footer Page 172 of 133 26 Header Page 173 of 133 Bỏo cỏo túm tt kt qu thc hin ti TI LIU THAM KHO Bardiya N and Bae JH (2005) Influenza vaccines: recent advances in production technologies Appl Microbiol Biotechnol 67: 299305 Biemelt S, Sonnewald U, Galmbacher P, Willmitzer L, Mueller M (2003) Production of Human Papillomavirus Type 16 Virus-Like Particles in Transgenic Plants J Virol 77: 9211-9220 Binh LT and Trang CH (2005) Research and development of plant edible vaccines J Biotech 3: 133-143 Bolivar FM, Wright R, Funk V, Sentz D, Barrroso L, Wilkins TD, Petri W, Cramer CL (2003) A non toxic lectin for antigen delivery of plant based mucosal vaccine Vaccine 21: 997-1005 Dinh DK, Le TH, Nong VH, Phan VC, Nguyen BN, Nguyen DT and Le TB.Molecular analysis of the avian influenza virus H5N1 isolated in poultry in 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Merrill] Plant Cell Rep 22: 478-482 Thi gian thc hin 2007-2009 Footer Page 178 of 133 vi ... tài: Tiếng Việt: Nghiên cứu phát triển vaccine thực vật dùng qua đường miệng cho gia cầm phòng chống bệnh H5N1 Việt Nam” Tiếng Anh: “Development of a plant-based veterinary oral vaccine to combat... (Giai đoạn 2007 – 2009) - BÁO CÁO TỔNG HỢP KẾT QUẢ KHOA HỌC CÔNG NGHỆ ĐỀ TÀI Nghiên cứu phát triển vaccine thực vật dùng qua đường miệng cho gia cầm phòng chống bệnh H5N1 Việt. .. hình sở số thành công tạo chuyển gen số trồng, hợp tác với trường Đại học Tự (Vương quốc Bỉ) tiến hành thực nhiệm vụ: Nghiên cứu phát triển vaccine thực vật dùng qua đường miệng cho gia cầm phòng