THANH DOAN So KHOA HOC CONG NGHE THANH PHO HO CHI MINH THANH PHO HO CHI MINH CHLIONG TRINH VUOM UdM SANG TAO KHOA HOC CONG NGI4 THE Bao cao nghiem thu d tali: THU NH8P KHANG NGUYEN TAI TO HOP CUA VIRUS CUM A PHAN TYPE H5 (DA Dli(iC DONG GOP 12- KIEN VA CHINH SfIA) Chu nhiem a tai: ThS. Tran thi Ngoc Tuydn Cd quan chit tri cid tai: Trung tam phat tridn KH&CN Tre. Nam 2007 MUC LUC PHAN I: TOM TAT CAC NOI DUNG CHiNH CUA DE TAI 1 1. CO SO KHOA HOC 2 1.
Cac phuang phap phat hien virus cum 8 1. Vaccine virus cum 9 1. Protein HA va cac nguyen dru ve protein hemagglutinin 10 1. Nguyen tL mot so phuang phap dung trong de tai 12 PHAN II: VAT LIEU VA PHVONG PHAP THVC HIEN DE TAI 17 2.
THIET BI, HOA CHAT VA VAT LIEU 17 2. Thu nhan gen ma hoa cho protein HA dm virus cum A phan type H5 32 2. Tao plasmid tai to hap pTZ-HA 33 2. Tao plasmid tai to hop pG-HA va pE-HA 33 2.
Bi6u hien protein tai to hop HA-GST va HA-His. coli va xac nhan su bleu hien dm cac protein tai to hgp 35 2. Thu nhan va tinh sach protein HA tai to hop: HA-GST va HA-His. Gay dap Crng mien dich chuOt bang HA tai to hgp va thu nhan khang huy6f thanh 40 2.
Kim tra kha nang phan irng cUa khang huye't thanh khang HA vai khang nguyen HA-His.Nus va HA-GST 40 PHAN III. KET QUA TH1YC HIEN DE TAI 42 3. THU NHAN GEN MA HOA CHO PROTEIN HA CUA VIRUS CUM A PHAN TYPE H5 42 3. TAO PLASMID TAI TO HOP PTZ-HA 42 3.
TAO PLASMID TAI TO HOT PG-HA VA PE-HA 46 3. Tao plasmid tai to hop pG-HA 46 3. Tao plasmid tai to hop pE-HA 50 3. BIEU HIEN PROTEIN TAI TO HOP HA-GST VA HA-HIS.
COLI VA XAC NHAN Sif BIEU HIEN CUA CAC PROTEIN TAI TO HqP 52 3. BiL hien va xac nhan protein HA-GST 52 3. BiL hien va xac nhan protein tai to hop HA-His. THU NHAN VA TINH SACH PROTEIN TAI TO HOP HA-GST VA HA- HIS.
Thu nhan va tinh sach protein tai to hop HA-GST 56 3. Thu nhan va tinh sach protein tai to hgp HA-His. KIEM TRA TINH KHANG NGUYEN CiJA HA-HIS.NUS BANG ELISA 58 3. KIEM TRA TINH CHUYEN BIET KHANG THE 60 PHAN IV: KET LUAN VA DE NGIII 61 TAI LIEU THAM KHAO 63 TD TP.
HCM So KHCN TP. HCM PHAN I: TOM TAT CAC NO DUNG CHINH CiJA DE TAI 1. MO DAU Virus clam A dang la nguyen nhan gay ra nhieu dich cum Ian tren th6 gioi, gay tir vong cao a nguai va gia cAm. Tren nen tang huy6t thanh hoc va virus phan lap tir nam 1933, chi c6 virus phan type HI, H2, H3 la c6 kha nang xam nhfe'm va gay benh tren nguai.
Tuy nhien nhimg nam gAn day, virus cum A chUng H5N1 dang la m6i de doa d6n sdc khOe con nguai vi virus nay khang nhung gay dich tren gia cam va con c6 the gay benh a nguai [40]. Virus cum A c6 1)6 gen la RNA soi don mach am, luan O. clang 8 manh va c6 khang nguyen be mat rat da dang (16 phan type HA va 9 phan type NA). Tinh chAt dm virus cum A la su bin d6i khang nguyen, dac biet su bin do'i tren cac khang nguyen be mat (hemagglutinin, neuraminidase, protein M2), nhung nhiL nghien ciru ve thu6c tri virus hay vaccine dna tren cac khang nguyen nay va tieu biL la khang nguyen hemagglutinin (protein HA) [36], [42].
Protein HA &mg vai tro quan trong giiip cho virus c6 kha nang nhan dien va barn ket t6 bao dich de xam nhiem vao 14 chu, va cling chinh protein nay quyet dinh dOc luc chung virus. Khi bi virus xam nhi6m, he mien dich dm 14 chil ducfc huy cl6ng a ch6ng lai su xam nhie'm nay, trong d6 mien dich dich tht tao khang the trung him virus tap trung chu y6u tan cong vao protein HA dm virus. HA la mot khang nguyen gay dap irng mien dich manh. NhiL phuang phap mien dich dna ten khang the khang HA da cluov phat trien a phat hien virus dim nhu phan irng HAI (phan ling irc ch6 tinh ngung ket h6ng cAu), phan ling NT (phan irng trung him virus) [11], [26].
MK TIEU De chuAn bi d6i ph6 vai nguy co. xay ra su bung phat dich cum do virus H5N1 tren gia cAm va gay benh a nguai, nhieu bien phap dang ducfc trier' khai bao g6m nhanh chOng phat triL, san xuAt vaccine phang benh, thu6c tri benh va kiem soat dich benh Hien nay, Viet Nam van phai nhap cac b6 haa chAt mien dich a phat hien virus clam A. Ben canh do chin co cong trinh nghien ciru )(ay dung quy trinh phat hi'ri virus cum A phan TT PT KH&CN THE 1 TD TP. HCM SO KHCN TP.
HCM type H5N1 tren gia cam bang phuong phap mien dich. Mac khac, de ph6ng ngira dich benh cho gia cam, hien nay chimg to van nhap vaccine virus cum A (H5) bat hoat de tiem cho gia cam. Tuy nhien, viec sir dung vaccine nay chira dung nguy co tai nhi.em dich cum virus. De dam bao tinh an toan cao, hien nay nhieu cong trinh nghien ciru tao vaccine tieu phAn da thuc hien va cho ket qua ban du clang tin clay.
Can tim co so thuc nghiem cho cac img dung xay dung quy trinh va b6 sinh pham xet nghiem virus cum A bang phtrang phap mien dich hay de phat trien vaccine tieu phan tai to hgp nen muc tieu cila de tai nay là: 1. To khang nguyen HA tai to hop bAng ky thu'at gen 2. Kim tra tinh tinh mien dich ctia kiting nguyen cua khang nguyen HA tai to hop thu nhan 1. NOI DUNG Vai muc tieu tren, de tai g6m co cac not dung chinh nhu sau: — Thu nhan va d6ng hoa gen ma hoa hemagglutinin (HA) tir virus dim A phan type H5N1.
— Thiet ke vector bieu hien protein tai to h9p, thu nhan va tinh the HA tai to hgp. — Gay dap img mien dich vai khang nguyen tai to hop tren chuOt va thu nhan khang huyet thanh. — Kiem tra kha nang tinh dac hieu cua khang huy6t thanh thu dugc vai cac khang nguyen HA tai to hgp. CO SO KHOA HOC 1.
Dich cum Cum la benh nhiem triing cap tinh do nhiern virus cum, thu6ng xuat hien hau het cac noi tren the giai vao mita Tong. Virus cum gay benh tren moi lira tu6i va lay nhiern rat nhanh. Hang nam ten the giai co khoang 20% tre em va 5% ngutri lon bi nhi8m virus cum. D6i tugng chua timg tiep xitc voi virus cum thi kha nang bi tir vong cang cao khi nhfem phai phan type virus cum d6c luc cao [1], [7].
TT PT KH&CN THE 2 TD TP. HCM SO KHCN TP. HCM Virus cum da gay ra nhang tan dai dich Ion, trong do virus cum A c6 the icarn nhigrn tren nguai, tren mot s6 dOng vat him nhCi khac va ca tren gia cam. MOt se phan type virus cum A dugc phan lap tir dOng vat him nha, va mot s6 dugc phan lap tir chim hoang da (dac biet la loci chim nuac di cu).
Trong the kS, 20 da c6 4 tan dich cum lon xay ra ten nguiyi va tran dich cum lan nhat the vao nam 1918 — 1919 gay chet han 40 trieu nguai chet ten toan au. Hien nay, dang lo sq la virus cum A phan type H5N1 yen la virus chi gay benh tren gia cam nay c6 dec lirc cao va c6 kha nang tranh dugc he mien dich cua nguai [12], [29], [41]. Theo thong ke ctla To chdc Y Te the giai, virus cam gia cAm H5N1 bung Oat vao cuei nam 2003 da lam han 173 nguai bi nhiern cum, gay tir vong it nhat 93 nguai va han 200 trieu gia cAm bi tieu htly [23]. Den nam nay (4/4/2006), virus dim gia cAm dugc phat hien tren 35 nu& ten the gi6i, da gay ra it nhat 191 truang hgp bi nhi&n benh cum gia am va 108 truang hop tir vong [11], [39].
Viet Nam mot trong nhung nuOc chiu thiet hai Ong ne ve nguai va kinh to do virus H5N1. 12/2003 den 3/2005, Viet Nam da xay ra 3 do:t benh cum a nguiyi va dich tren gia cam [39]: Dgt 1 (12/2003 den 13/2004): 16 truang hop ten nguai bi tir vong do nhigm virus. Dgt 2 (17/2004 den 8/2004): 29 truang hop ten nguai nhigm virus, trong do 20 truang hgp bi tir vong. Dgt 3 (12/2004 den 3/2005): 61 truang hop ten nguai nhigm virus, trong do c6 19 truang hgp bi tir vong.
Trong 3 do:t dich, tong so gia cAm bi ch&. va tieu 114 hon 60 trieu con 1. floc diem sinh hoc ciia virus aim A Virus aim A thuOc ho Orthomyxoviridae, chi influenzavirus. Hai glycoprotein xuyen mang hemagglutinin (HA) va neuraminidase (NA) la 2 khang nguyen chinh cila virus cum A.
Dua tren su khac biet ve tinh khang nguyen dm 2 protein nay ma virus cum type A dugc chia thanh cac phan type H (1 — 16) va N (1 — 9) khac nhau [19], [22]. TT PT KH&CN THE 3 TD TP. HCM SO KHCN TP. HCM Ten goi day du cua mot chUng virus dim dugc phan lap phai bao gom cac tfianh phan la: type virus (A hoac B), loci k9 chu, vi tri dia 19, se') thir ttr, nam phan lap, H va N.
Vi du ten mot chung virus cum A H5N1 dugc phan lap teen vit tai Viet Nam nhu sau: Aiduck/V ietnam/166/04(H5N1) [20], [36]. floc diem cau tnic va bd gen Virus corn A có the hinh cau hoac hinh oval, duong kinh khoang 80 — 120nm, chieu dai có the dat 2000nm. Sir thay d6i hinh clang hay kich thuac phu thuOc vao loci to bao k9 chO va dac diem di truyen cua chUng virus (Ifinh 1. Thanh phan cau tao dm virus Om khoang 70% protein, 20% lipid, 5 — 8% carbohydrate va 1% RNA.
MO't virion hoan chinh gom mot mang bao kep phospholipid có ngu6n gif)c tir to bao chu, bao b9c nucleocapsid ben trong. Tren mang bao virus có cac glycoprotein dInh xuyen mang nhu hemagglutinin (a dang trimer, dai 135A°), neuraminidase (clang tetramer, dai 60A°), va protein M2. Mat trong cua mang bao dugc phii bang protein nen Ml. Nucleocapsid bao g6m 8 manh RNA, m8i manh RNA dugc bao boc bOi cac nucleoprotein (NP) tao nen phirc hgp RNA — nucleoprotein (RNP).
Moi phirc hgp RNP chira 3 tieu phan cua RNA polymerase la protein PA, protein PB1 va protein PB2. Ngoai cac RNP, virus con chira cac protein khong cau trOc NS1 va NS2 (Ifinh 1. BO gen virus cum type A la RNA sgi don mach am, g6m 8 doan RNA (7 doan o. type C) ma hoa cho 10 protein.
Dau 5' va 3' dm moi doan co trinh tkr rat bao ton (Bang 1. Protein PB1 la RNA polymerase. PB2 116 trg cho hog dOng dm RNA polymerase.