Y Hpc VigT NAM THANG 10 • s 6 DAC BlgT/2013
BIEU HIEN KHANG NGUYEN CUA VIRUS H5N1 TRONG THUC VAT
Nguyen Thu Hien", Nguyin Thu Giang", Chu Hoang Hd**, Chu Hodng M^u*
TOM TAT
Cum la mpt can benh nguy hiem gay t i i vong cao tren khap the gidi. Hang nSm dich cum da lam cho nita ty ngu'di mSc benh, chu yeu la tre em. Hien nay, cum H5N1- chiing gia cam nguy hiem nhat hien nay da lan truyen tren 40 quoc gia d chau A, Trung dong, chau Au va chau Phi.Diihg tnTcfc nguy cd ve mpt dai djch cum ldn Ihi viec phong benh bang vaccine la mot chien liTtfc hffu hieu va cap thiet nhat. Hien nay, cd mot so vaccine khac nhau du'dc san xuat, nhu'ng cac nha khoa hpc tren the gidi van dang tiep tuc phat trien nhutig loai vaccine mdi co tfnh uXi viet hdn, re hdn, de bao quan va phan phoi hdn, an toan va hieu qua hdn. Vaccine thu'c vat la mpt loai vaccine du'di ddn vj hay protein tai to hdp, 6\Ia vao cd the theo du'dng tieu hoa, san xuat trong he thong thu'c vat.Vaccine an du'dc co kha nang kfch thfch cd the san xuat khang the hieu qua hdn cac loai vaccine tiem. Vi vaccine an du'dc qua du'dng mieng se cam Lfng he thong mien djdi, san xuat cac khang the chong lai cac tac nhan gay benh, do day la ndi tap trung cac mo CO kha nang dap uhg mien djch cao nhat cua cd lhe.Trong bai bao nay, chung toi da suf dung gen HAI cua virus H5N1 phan lap tiy gia cam Viet Nam de du^ gen vao vector bieu hien trong thu'c vat. K^ qua cho thay vector hoat dpng tot, protein HAI da du'dc tong hdp trong hat dau tu'dng giong BT12 vdi mii'c do cao (ki hieu la hslop). Day se la cd sd de cho bi/dc thu" nghiem phan u'ng mien djch d gia cam, tien tdi san xuat vaccine.
Tu'khoa: Bieu hien, khang nguyen, H5N1, J ^ ^ ^ t t ^ vat, thu'c vat chuyen gen
Bat iioc Thai Nguyen. " Vien cong ngh? sinh HQC. VAST.
Plian tiign khoa hgc: PGS.TS. Ngo Giang Lien
S U M M A R Y
EXPRESSION OF H 5 N 1 VIRUS A N H G E N HA I N PLANT
The avian influenza virus H5N1 causes disease in avian of many countries, and has already caused more than $10 billion in losses to poultry farmers. This virus genome consists of eight segments of negative-stranded RNA, which code for 11 proteins. In there, matrix proteins (M), hemagglutinin (HA) and neuraminidase (NA) are important proteins, which are targets for vaccine production. Vaccination is considered as the most important measure to prevent pandemics. Although different types of Influenza vaccines including "inactivated" vaccines, vaccines with immunopotentlators, live attenuated vaccines and DI\tA vaccines have been developed, scientists around the world are developing better vaccines, which are less expensive, easier to store and deliver, safer and more efficacious. Plant produced oral vaccines are particularly attractive because they are economical, easily adaptable to large-scale production, present a minimal risk of contamination with animal or human pathogens, and are stable at room temperature for a long time allowing them to be used in remote areas.
This paper, halop gene have successful constructed in plant expression vector. Protein HAI fused a mucosal adjuvant pnDtein LTB and C-myc tag is expressed In high level in Arabldopsis seeds. These results are the base fiar immunological evaluation and protection of orally administered seed produced antigens. Morever, these resuls will be applied for other antigen of virus.
219
CHUYgN pg: HiNH THAI HQC VA C 6 N G NGHg T H 6 N G TIN
Keywords: Antigen, expression, transgenic piant, piant vaccine
H5N1,
I. T6NG QUAN
Cum gia cAm (Avian Influenza, AI) Id b$nh truyen nhiem c^p tinli cua gia ckm. do nhdm virus cum A, thu0c hp Orthomyxoviridae gay ra. Day \i nh6m virus c6 bien dp chu rOng, dupe phan chia thanh nhi^u phan type khac nhau dua tren khang nguyen HA va NA co trSn be m5t capsid cua hat virus (de Wit, Fouchier, 2008). Nhom virus cum A co 16 phan type HA (tir HI d^n H16) va 9 phan type NA (tir Nl d6n N9), vi su tai to hpp (reassortment) gi&a cac phan type HA va NA, v^ mat ly thuyet, se tao ra nhieu phan type khac nhau ve dpc tinh vi kha nang gay benh. Mat khac, virus ciim A e6 dac tinh quan trpng la dl dang dot bidn trong gen/he gen (dac biet 6 gen NA va HA), hoac trao doi cac gen khang nguyen voi nhau, trong qua trinh xam nhiem va t6n tai lay truyen giiia cac loai vat chu. Doi vai dich cum tren ngucri, nghien cira ph4t trien vaccine khong nhihig ngan ngira Iam giam dupe benh a gia cam, ma con khdng chi nguon truyen lay ciia loai virus nguy hiSm nay sang nguoi. Khang thi dSc hieu co th6 dupe CO the sinh ra do kich thich cua khang nguyen trong vaccine, va do la ciSc khang thi khang HA, NA, MA va nhiSu Ioai hinh khac cua virus duong nhiem, gop phSn vo hieu hoa virus cum dung doi tupng khi chung xam nhap vao. Co nhiSu loai khang the, nhung truoc hk chi khang the khang HA (H5) co vai tro tien quydt quan trpng trong qua trinh trung hoa virus cua hai Ioai vaccine chinh hien nay. Vaccine an dupe o thuc vat la vaccine tieu phan protein dupe san xuSt dua
tren h? thdng thyc v^t de thu dupe protein lam miln dich thi dich va mien djch td bao.
Vaccine nay ben vimg trong dich tieu hda, di qua dudng tieu hda ma khdng bi phan hiiy.
Chuyen gen vac cay trdng vdi muc dich tao ra protein khang nguyen de san xuat vaccine phdng chong benh eho ngucri va gia c3m dang thu hut sy quan tam cita nhidu nha khoa hpc tren thd gidi, nhidu protein khang nguydn da dupe bidu hidn thanh cdng d thuc vat nhu;
khdng nguyen be m^t virut vidm gan B (Kapusta et al. 1999), protein G cua virut dji (Rojas-Anaya el al.. 2009); protein cua E.coli (Kang et a.,l 2003); protein CTB cua Vibrio cholerae (Arkawa et al, 1997; Oszvald et al., 2008). Vi vay. vide san xuat vaccine dua vao thyc vat la mdt phucmg phap day hiia hen,an toan thuan Ipi, gid thanh thdp va dl su dyng.
Nhdm tao ra ngudn nguydn lieu cho vide thir nghidm khd nang dap ring mien dich 6 gia cdm chung tdi da nghien cilu bidu hien khdng nguyen HA ciia H5N1 trong hat ciia cay dau tuong. Kdt qud Iai Western blot cho thay khdng nguydn HA dd dupe bidu hien cao trong hat ciia cay ddu tuong chuydn gen dthShdTI.
II. VAT UEU V A P H U O N G PHAP NGHIEN
cCru
2.1. Vat li|u
Cdc chiing vi khudn Agrobacterium, chiing £ coli mang vector chiia cdu tnic gen HAI cua virut H5N1 do phdng Cdng nghe te bao thyc vdt cung cdp.
Gidng ddu tuomg DT12 do Tning tam
Nghien cira Phat tridn cdy ddu dd, Vien Cay
luong thyc va Cay thyc phdm cung dp,
khdng thd khdng HA do phdng Vi sinh phan
tir cung cdp.
Y HOC VI$T NAra T H A N G 1 0 - S O P A C B l g T / 2 0 1 3
2.2. Phirong p h a p
Thiit ki vector chuyin gen thirc vat Gen HAI dupe nhan ldn bdng PCR vdi cap mdi djc hieu ^ o / - H A / / / ! n d l l I - H A theo chu trinh: 94°C/5 phut, 30 chu ki (94°C/30 giay, 54T/30 gidy, 72°C/1 phut 30 giay), 72°C/7 phut va 4°C/30 phut. Cdc phuong phap ghep ndi vdo vector theo Sambrook va Russell (2002) vd theo quy trinh Gateway kit cua Invitrogen. DNA plasmid dupe bidn nap vao E. coli theo phuang phdp sdc nhiet cua Cohen va ddng tac gid (1972) vd bidn nap vao Agrobacterium bdng phucmg phdp cua Hotgen va ddng tdc gid (1988). DNA
1 dupe tdch chidt vd lam sach theo phap cua Sambrook va Russell (2002). DNA tdi td hpp dupe kidm tra bdng phuong phap PCR vdi cap mdi dac hidu H.o/-HA/a«dlIl-HA.
Biin nap gen
Cdu true gen HAI dupe chuySn vao giong dau tuong DTI2 thdng qua vi khuan Agrobacterium theo phuang phdp cua Topping cd cai tidn (1998). Chuan bi cac manh la co kich thudc khoang 1 cm^ sau do dat len moi trudng GM trong 2 ngdy, cac manh la sau 2 ngay nudi cam ling trdn mdi tniong GM dupe nhiing vao dung dich huyen phil cd chira Agrobacterium cd ndng do 0D~ 0,8. Khoang 10 phut sau chuyen manh cdy ldn gidy thdm tiet triing, tham khd va cdy Ien mdi trudng GM khdng cd khang sinh trong 2 ngay, chuydn mdnh cay sang ni6i tnrdng GM cd bd sung khdng sinh cefotaxim 400 mg/I vd 30 mg/l kanamycine, sau 2-3 tudn cdc chdi hinh thanh dupe 'kuyen sang mdi truang GM chira khdng sinh cefotaxim 400 mg/l vd 50 mg/l
kanamicine. Khi chdi dai 2-3 cm cdt vd chuydn sang mdi trudng ra rd RM cd bd sung khang sinh cefotaxim 400 mg/l va 30 mg/l kanamicine. Khi cdc cdy con cao 5-7 cm cdt chdi chuyen sang mdi trudng RM cd chiia khang sinh 50 mg/I kanamicine, sau dd ra cay trong bdu dkt d nha kinh.
Kiem tra cay chuyen gen bdng phdn ung PCR
DNA ciia cay dupe tach chiet theo phuong phap cua Edwards (1991).Tien hanh phdn img PCR vdi cap mdi ddc hidu cda gen HAI theo chu ki nhiet: 94°C / 5 phut 30 chu ki (94°C/30 giay, 54°C/30 giay,72''C/l phut 30 gidy 72°C/7 phut va 4°C/30 phiit. San phim PCR dupe dien di tren gel agarose 0,8%
Phdn tich protein bdng Western blot:
Protein tdng sd dupe chay trdn gel SDS polyacrylamide 12.5% theo phuong phdp ciia Laemmli (1970), sau dd dupe chuydn Ien mang nitrocellulose Hybond™ (Amersham).
Sau khi da chuydn mang thdnh cdng tidn hdnh phil mdng bdng cdc khang thd. Trong cdu tnic bidu hien cd gdn phan dudi c-myc nen chung tdi da sir dung khang thd 1 la khdng the khang c-myc dd phii mang trong 2 gid. Khang the ndy se gdn dac hieu vdi c-myc. Sau dd rira hdt nhiing phdn khdng thd 1 khdng gdn kdt va cac chdt bdn. Tidp tuc phu khang thd 2 la khdng thd chupt cdng hpp horseradish peroxidase trong vdng 1 gid sau dd rira dd Ioai khang the 2 khdng gdn kdt. Dd mdng khd sau dd djt mdng len 1 tdm nilon mdng. Trdn 2 co chat Reagent 1 (3 ml) va reagent 2 (3ml) rdi do ddu ldn mdng trong vdng 70 gidy sau do tham khd. Dat them mpt Idp nilon mdng trdn mang rdi cho vdo hop X-ray cassette rdi hidn phim trong phdng tdi.
221
CHUYgN Bt: HJNH THAI HpC VA C 6 N G NGHg T H 6 N G TIN
III. KET QuA VA BAN LUJfiN
3.1. Thiet ke vector tdi to hpp chiia ciu true gen HAlop
Dya trdn trinh ty gen HAI ciia virus A/H5N1 sau khi thay ddi mpt sd md bd ba giiip bieu hidn mirc dp cao amino acid trong cdc cdy tr6ng gen nay dupe k^ hidu Id halop (Nguyen Thu Hien vd c^ng sy 2010), cdp moi ddc hi$u ATioI- HAJHind III-HA1 dupe thidt kd nhy bang 2 dd nhan do?n halop. Trong dd cdc nucleotid in nghidng dam la trinh ty nhan bidt eua enzyme cdt han che Xhol vi HindlU, cac nucleotid in thudng Id trinh ty tuong ddng vdi gen.
Bdng 2. Trinh ty c^p moi
XhdL-Hk CCffCTCC>l GATCTGCATrGGATACCACGCT
C C G M CCT7CTCGTTAGAGTGATGGTATCCGT
Gen halop dupe khudch dai bdng cdp mdi ddc hidu ATiol-HA/Wndlll-HAI, sdn phdm PCR dien di cd kich thudc khodng 1,2 kb.Sdn phdm PCR sau dd dupe xu Ii cdt ddng thdi 2 enzyme Xhol/HindlU vi dupe ndi vdo vector pDONN201 chira cdc gen ma hda peptide chirc ndng bao gdm peptide tin hidu (2S2), mdt protein nidm mac rupt (LTB), mpt doan peptide phue vu nhdn bidt (C-myc) va mpt trinh ty nhdn dang cua mang ludi npi chdt (KDEL).Vector pBeta-Phaso-dest la vector mang promotor phasoline didu khidn tdng hpp protein dac tnmg trong hat. Cdu tnic
gen chira halop vi cic gen ma hda cac peptide chiic ndng dupe chuydn vac vector pBeta-phaso- dest bdng phan ling LR Gateway.
Kdtqud kidm tra vector tdi t6 hpp: cdt vector bdng EcoRI thu dupe 3 doan gen kich thudc khodng 2.2,5 va 10 kb kich thirdc nay phu hpp vdi tinh todn theo Iy thuydt (Hmhl).
Nhu vdy, cdu tnic gen HA da dupe thidt kd thdnh cdng vao vector bieu hidn thyc vat pBeta-Phaso-dest. Cdu tnic vector bieu hidn mang gen HAlop sau dd dupe bien nap vao vi khuan A. tumefaciens vd chuydn vdo cay ddu tuong.
Hinh I : Phdn tich sdn phdm cdt vector tdi td hpp mang gen halop bdng enzyme han ehd £coRI (B). M: Thang chudn 1 Kb; 1, 2, 3:
mau vector tdi td hpp tach tit cac ddng khudn
lac.
Y HPC VigT NAM T H A N G 10 • S 6 P A C BlgT/2013
3.2. Chuyin cac cau triic halop v i o nach la mam gi6ng dau tuong
Tidn hdnh thi nghidm vdi tdng sd 650 mau cho chuydn gen halop , cd 65 chdi keo dai Iren moi tardng chpn Ipc SEM chira 50 mg/I kanamycin, trong dd 45 chdi cd khd ndng ra r l vithu nhan dupe 32cay TO phdt t i d n trdn gid thd . Su dyng 3nl sdn pham DNA da dupe tach cliidt,chiing tdi tidn hdnh phdn iing PCR vdi cdc cdp moi ddc hidu . K& qud dien di sdn phdm PCR cho thdy da thu nhdn dupe 8 cdy duong tinh vdi phdn img PCR (hinh 2)..Cdc ddng cay nay tidp tyc dupe theo ddi vd thu hat de trdng the hd T l .
Hinh 2. Phan tich cay dau tircmg TO chuyen gen halop .M: Thang DNA chuan Ikb; -: Doi chumg am; WT: Cay khong chuySn gen; 1 - 8 : Cdc d6ng TO dvtgc phan tich; +; Doi chung dirong (plasmid).
J I ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ J ^-ITBSbp
J.J. Bdnh gid su cd mat cua gen halop & the he Tj
Chung toi thu nglu nhien la cua cac dong cay cr giai doan T l , tach chiet DNA va tien hanh PCR vcri cap m6i dac hieu Xhol-HA/ Hindlll-HA. Hinh 3 cho thay co 1 dong cay c6 kdt qua duong tinh vai phan ung PCR. San phAm PCR la doan DNA c6 kich thudc diing voi kic thuoc gen halop. Nhu vay c6 the khang dinh gen halop da dugc bien nap thanh cong vao cay d(iu tirong vk c6 di truyen cho the he sau. Vai cac dong cay khac chung toi chua thu dugc ket qua nhu mong muon nen nhieu kha nang gen halop hi phan ly.
Hinh 3. Ket qua dien di san pham PCR voi cap m6i dac hi?u Xhol-UA/ Hindlll-UA voi cac dong cay d^u tucmg chuyen gen the h?
T]. M: Marker, (-) doi chiing am, (+) doi chtog duang,( wt) dong cky kh6ng chuyen gen H1-H9: dong cay thS he T |
3.4. Phan tich bieu hien protein tai t6 hqrp & hat cua cay dau tuffng chuyen gen M 1 2 -
Hinh 4. Phan tich protein H A l o p trong n^t d^u tuong chuyen gen bang Western blot.M: Protein chukn; (-): cay khong chuydn gen; 1-2; cac mau h^t thu duac cua cay dau tacfngHU
223
CHUYgN pg; HJNH THAI HQC VA CflNG NGHg THONG TIN
Protein tong so cua hat d^u tucmg chuyen gen a the h? Tl dugc tach chiet va kiem tra v6i k h ^ g the HA bang ky thu^t lai Western blot. Ket qua ciio thay HA I op dugc phdt hi?n 6 kich thudc 40 kDa tuong ung kich thu6c cua protein H A l o p theo ly thuyet.
Nong dp protein tai to hgp HAI dugc bieu hi^n trong hat khd thap (hinh 4). Nhu v§y, buac dau chung toi khdng djnh rdng protein HAI dk bieu hi^n dugc trong hgt dau tucmg chuyen gen. Day s6 la nguon nguyen li^u de danh gia hoat tinh mien djch cua protein tdi to hgp vcri virus A/H5N1 tren gia cam.
IV. KiT LUJEiN
Dd tao dugc 32 dong cay chuyen gen halop the h? TO, trong do co 8 d6ng ducmg tinh vai PCR bdng cap m6i ddc hieu.
Phan tich bieu hien gen bidn ngp a thd he Tl cho thay halop dk dugc tong hgp trong hat dau tuang. Tuy nhien dd xac dinh dugc nong do chinh xdc cua chung tich luy bao nhieu phan trdm protein tong s6 thi cdn phdi tidn hanh thi nghiem tidp theo
TAI UEU THAM KHAO
/. Arakawa, O., K. Kanno, A. Kanetsuka, and Y. Shiozaki. 1997. Effect of girdling and bark inversion on tree growth and frmt quality of apple. Proc. 6. Int. S ymp. on Integrating Canopy. Acta Hort. 451: 579-586.
, Capua, I., and D. J. Alexander. 2008 Ecology, epidemiology and human health implications of avian influenza viruses.
. Jimenez- Bremont J F (2007) Expression of Escherichia coli heat- labile enterotoxin h subunit (Itb) in carrot (Daucus carota L).
Plant Cell Reports 26: 969-976.
Kang, K.H., Jang, J.K., Pham, TJl, Moon, H., Chang, LS., Kiin,B.H., 2003. A microbial fuel cell with improved cathode reaction asa low biochemical oxygen demand sensor. Biotechnol. Lett. 25 (16),1357-i36l.
Kapusta J, Modelska A, Figlerowicz M, Pniewski T, Leteilier MJLisowa 0, Yusibov V, Koprowski H, PlucienniczakA and Legocki AB (1999) A plant-derived edible vaccine against hepatitisB virus. FAS EBJ 13: 1796-1799.
Oszvald M., Tomoskozi A., Tamds L., Bekes F. (2008): Role of rice and added whea protein m the mixing properties of different rice flours. Acta Alimentaria, 37: 399-408.
Rosales-Mendoza S, Soria-Guerra RE, Olivera-Flores MTD, Lopez-Revilla R, Argullo-Astorga GR, E. De Wit & R.A.M.
Fouchier, J Clin Virol (2008), Avian influenza A virus. - Adaptation.
Streatfield, S.J. 2006. Mucosal immunization using recombinant plant-based oral vaccines. Methods 38:150-157 Subbarao K, Luke C (2007) H5N1 viruses and vaccines. PLoS Pathog 3(3): e40.