Tgp chi Cong nghi Sinh hoc 1(2): , 2009 BIEU HIEN GEN HAS MA HOA KHANG NGUYEN HEMAGGLUTININ (HA) CUA VIRUS CUM A/H5N1 TRONG ESCHERICHIA COLI D6

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1 Tgp chi Cong nghi Sinh hoc 1(2): , 2009 BIEU HIEN GEN HAS MA HOA KHANG NGUYEN HEMAGGLUTININ (HA) CUA VIRUS CUM A/H5N1 TRONG ESCHERICHIA COLI D6 Thi HuyenS Bui Hong Van^ Van Thi Nhu- Ngoc', Truong Van Dung^ Truong Nam Hai' ' Viin Cong nghi sinh hoc ^ViinThuy '' - ^Truong Dgi hoc Khoa hoc Tunhiin, Hd Noi TOMTAT :,:. V :-. ;, -,.:- f, Benh ciim gia cam gay ra bai virus the doc luc cao la mpt benh truyen nhiem nguy hiem do co toe do liy lan nhanh va ty le gay chet cao trong dan gia cam nhiem benh. Hien nay, chiing virus ciim A/H5N1 la chung virus gay ra dich benh chu yeu a nhieu quoc gia tren the giai, trong do co Viet Nam. Trong nghien ciiu nay, chiing toi tien hanh bieu hien toan bo gen hao ma hoa cho khang nguyen HA5 ciia virus cum A/H5N1 trong te bao E. coli BL21. Gen ha5-0 dugc thiet ke dung hgp vdi gen ma hoa cho protein thioredoxin trong vector pet22trx ya dudi hai dang: 1) Toan bo gen ha5-0 trii bo ba mo dau va phan tin hieu tiet; 2) Thay the doan doan-peptide noi giiia hai tieu phan HAI va HA2 bang mpt doan polylinker (ha5-l-2). Kha nang bieu hien cua chiing tai to hop E. coli BL21 mang cac dong gen ha5-0 va ha5-l-2 dupc khao sat trong moi truang LB vdi nhan to chpn lpc ampicillin va chat cam iing IPTG. Anh huang ciia nong dp chat cam iing IPTG dugc khao sat d cac gia tri 0; 0,05; 0,1; 0,2; 0,4; 0,6; 0,8; 1; 1,5 va 2 mm. Kha nang bieu hien cua cac dong te bao tai to hgp dugc khao sat d cac nhiet dp 22, 25, 28, 30 va 37 C. Ket qua thu dupc cho thay, protein dung hpp TrxHa5-0 va TrxHa5-l-2 co kich thuac khoang 66 kda da dugc bieu hien tot nhat trong te bao E. coli dudi su dieu khien cua T7 lac promoter vdi chat cam iing IPTG va d nhiet dp cam iing la 25''C. Protein tai to hgp khdng dupc tong hpp khi trong moi trudng khong c6 chat cam iing IPTG. Tuy nhien, nong dp chat cam iing khong anh hudng nhieu den miic dp bieu hien gen cua te bao, protein tai to hpp dugc cam iing bieu hien tot ngay a ndng do chat cam ling rat thap la 0,05 mm va tang len khong dang ke d cac nong dp cao hon. Ben canh do, protein TrxHa CO miic dp bieu hien va kha nang tan cao han so vdi protein TrxHa5-0 khi dupc bieu hien d cung dieu kien nhiet dp va ham lugng chat cam iing. Ty le tan cua protein TrxHa5-l-2 dat tdi tren 70% protein tong sd khi dupc bieu hien d nhiet dp 25 C. Cau tnic gen dimg hgp nay se dupc dua vao vector ppic9 de bieu hien gen trong nam men P. pastoris nham nghien ciiu tao vaccine tai td hgp dua vao chung nam men. Tie khoa: E. coli BL21, ha5-l, pet22trx, protein tdi to hap, vaccine ;,:.:<;,> MO DAU Su thay doi thuang xuyen va manh me ciia cac khang nguyen be mat (HA va NA) dan den su thay Dich ciim hang nam gay ra boi virus ciim gia doi ve dgc tinh cua virus ciim va dong thai ciing la cim th6 dgc luc cao la benh truysn nhilm nguy nguyen nhan chu yeu gay ra tinh mua vu, mgt trong hiem, toe do lay lan nhanh, ty le gay chet cao trong lihiing dac tinh nguy hiem cua dich benh nay dan gia cam va c6 kha nang lay lan sang nguai va (Laemmli, 1970; Nayak et al, 1985). Khang gia siic din d8n tii vong (Bridges et al, 2002, nguyen HA bao gom 2 tieu phin noi vai nhau bai Simonsen et al, 2000; Thompson et al, 2003). S6 mgt doan 6 amino acid. Su thay d6i ve trinh tu ciia nguoi tii vong do cac dai dich ciim trong lich sii doan amino acid nay dan den su thay doi ve dgc dugc uac tinh la cao hon nhieu so vai so nguai chet tlnh ciia virus (Ross et al, 2000; Saelens et al, trong chign tranh th6 giai (Potter, 1998). Tinh tu nam 1999). Hau het vaccine phong ciim dang dugc sii 2003 d6n thang 5 nam 2008, s6 ca nhap vien vi benh dung la vaccine bit hoat dugc san xuit tren phoi ga, ciim tren toan thl giai la 383, trong do s6 ca tu vong trong do khang nguyen HA dugc chiing to la dich la 241, dai da s6 dugc xac dinh la nhilm virus cum tac dgng chu yeu de tao ra dap iing miln dich A/H5N1 ( Cung vai do la iihiing thiet phong ve (Skehel et al, 2000; Wright et al, 2001). hai to lan ve kitih tl, mgt s6 Ion gia cim thuang Mac du loai vaccine nay dugc cong nhan la toan xuyen bi tieu hiiy khi co dich benh xuit hien gay anh dien va hieu qua, nhung doi hoi thai gian san xuat huang nghiem trgng den nganh chan nuoi va dai dai do do khong dap ung dugc nhu cau sii dung sdng cua nguai dan. vaccine phong ciim tren toan the giai. De dap iing 185

2 D6 Thi Huyen et al dugc nhu cau nay, nhieu doi tugng va phuang thiic tiep can khac nhau da dugc trien khai thuc hien (Ghendon et al, 2005; Kistner et al, 2007; Pau et al, 2001). Vaccine duai dan vi la mgt trong nhiing phuang thiic tiep can mai, cho phep san xuat dugc vaccine trong mgt thai gian ngan va de dang ma rgng quy mo san xuat. Cac doi tugng dugc ap dung nhieu trong nghien ciiu san xuat vaccine duai dan vi phong ciim la te bao con tning (Nwe et al, 2006; Treanor et al, 2006), te bao thuc vat (Karasev et al, 2005; Williamson et al, 2005) va te bao vi sinh vat (Davis et al, 1983; Deltito et al, 1995; Rong et al, 2007; Saelens etal, 1999). Trong nhiing nam gan day, nhieu loai protein, enzyme va khang nguyen da dugc bieu hien thanh cong trong te bao vi sinh vat va nhieu trong so do da dugc ap dung rgng rai trong thuc te cugc song nhu dung lam thuoc, thuc pham chiic nang... Khang nguyen HA cua mgt so virus ciim A nhu, HONl, H3N2, H7N9 va H5N1 da dugc nghien ciiu bieu hien trong te bao vi sinh vat nhu E. coli, Baculorvirus, nam men P. pastoris va S. cerevisiae (Jabbar et al, 1985; Yi-Ming et al, 2006). Nam 1981, Davis va dong tac gia da tien hanh bieu hien khang nguyen HA ciia virus ciim A/HONl trong E. coli duai dang dung hgp vai protein p-galactosidase. Protein HA tai to hgp dugc chiing to la chiia quyet dinh khang nguyen do dugc nhan biet mien dich bai khang the khang virus ciim, tuy nhien miic do bieu hien la rat thap (Davis et al, 1981). Nam men P. pastoris da dugc su dung lam te bao bieu hien khang nguyen HA cua mgt so subtype trong nghien ciiu cua Yi-Ming Xu va dong tac gia nam Cac protein HA tai to hgp trong nghien ciiu do dugc tihan biet mien dich bai khang the khang virus ciim va dugc coi la doi tugng tiem nang de san xuat vaccine duai dan vi phong benh ciim. Trong cac nghien ciin truac (Nguyen Phuoc Hai et al, 2006; Van Thi Nhu Ngoc et al, 2007), chiing toi da tach dong va bieu hien thatih cong tung tieu phan rieng re ciia khang nguyen HA cua virus ciim A/H5N1 (HA5-1 va HA5-2) trong E. coli. Trong nghien ciiu nay, chiing toi tien hanh bieu hien toan bg khang nguyen HA5 trong E. coli duai dang HA5-l-linker-HA5-2 (trinh tu n6i giiia hai tieu phan khang nguyen dugc thay the bing mpt doan polylinker), dong thai tach dong gen va bilu hien toan bg cau tnic nguyen ven ciia khang nguyen HA5-0. VAT LIEU VA PHUONG PHAP Vat lieu Chung vi sinh vat: E. coli DH5a [end Al rec Al hsd R17 sup E44 gyp A96 thi-1 relala lac U169 ((j)80 laczm15)] dugc su dung de chgn dong va nhan dong gen. Chung E. coli BL21 [F-omp hsd SB (rbmb) gel dem (DE3) plyss (Caml)f dugc sii dung lam te bao chu cho bieu hien gen. Plasmid: Plasmid pcr2.1 (Invitrogen) dugc su dung lam vector tach dong trong te bao E. coli DH5a. Plasmid pet22trx, pet22trxhal va pet22trxha2 mang gen ha5-l va ha5-2 do Phong Ky thuat di truyen, Vien Cong nghe sinh hgc thiet kl. Vector pcrh5 mang gen ha5 va huyet thatih ga chiia khang the khang virus cum A/H5N1 do PGS. TS. Dinh Duy Khang, Phong Vi sinh vat hgc phan tu, Vien Cong nghe sitih hgc cung cap. Phuong phap Ky thudt PCR vd tdch ddng gen Gen ha5-0 dugc nhan len bang RCR voi chu trinh nhu sau: Bien tinh DNA khuon dien ra a 94 C trong 4 phiit, sau do thuc hien tiep vai 25 chu ky, moi chu ky gom 3 buac: Buac 1: Bien tinh sgi DNA khuon a 94 C trong 1 phiit; buac 2: Moi ket cap bo sung vai doan gen tuong dong tren sgi khuon o 55 C trong 1 phiit; buac 3: Tong hgp, keo dai chuoi a 72 C trong 1 phiit 30 giay. Phan iing ket thiic a 72 C trong 8 phiit va u mau a 4 C. Khuon DNA la dong gen pcrh5 dugc tach ttr he gen virus trong cac nghien ciiu truac. Moi nhan gen do hang Amersham Pharmacia Biotech tong hgp, trong do moi xuoi chiia trinh tu ciia Ncol (chii gach chan) va moi ngugc c6 trinh tu cua //indlll (chii gach chan) voi trinh tu tihu sau: Moi ha5-0f: tccatggatgagaaaatagtgcttcttc; Moi ha5-0r: taagcttaatgcaaattctgcattg Cac phuang phap ve sinh hgc phan tu nhu cat, noi cac doan gen, bien nap plasmid vao te bao E. coli, dien di kiem tra san pham tren gel aagarose, polyacrylainid... dugc tien hanh theo phuang phap chuan (Sambrook, Russell, 2001). Thiet ke vector bieu Men Gen ha5-0 dugc nhan Ien tu dong pcrh5 bing cap m6i dac hieu co chiia hai trinh tu cua enzyme han chi Ncol va HinAlll. San phim PCR dugc nii true tilp vao vector tach dong pcr2.1. Trong 186

3 Tgp chi Cong nghi Sinh hoc 1(2): , 2009 vector tach dong, chiing toi tiln hanh kiem tra va dgc trinh tu gen. Sau do gen ha5-0 dugc cit bing hai enzyme han che tren va dua vao vector bieu hien pet22trx dl tao thanh vector pet22trxha5-0. Protein thioredoxin ma hoa tu gen trx giiip cho protein tai to hgp bieu hien 6n dinh hon va cugn xoan chinh xac han, han chi l i trong qua trinh cugn xoan. Plasmid pet22trxha5-0 dugc bien nap vao te bao E. coli BL21 de bieu hien gen. Gen ha5-l-linker-ha5-2 (ha5-l-2) dugc thiet ke dua tren hai dong gen ha5-l va ha5-2 da thiet ke trong cac nghien ciiu truac. Gen ha5-2 dugc cat ra tu vector pet22trxha5-2 bang cap enzyme han che BamHl va Hindlll, dong thai, vector pet22trxha5-l ciing dugc cat ma vong bang chinh cap enzyme han che nay. Sau khi titih che, cac san pham cat dugc lai ghep voi nhau va sang lgc trong te bao E. coli DH5a. Dong plasmid dugc lua chgn tiep tuc bien nap vao te bao. co/!bl21de nghien ciiu bieu hien.. ;;;-i Bieu Men gen has, ha5-l-2 Cac chiing E. coli BL21 mang gen ha5, ha5-l-2 dugc nuoi cay trong moi truang LBA den khi ODgoo dat khoang 0,6-1,0. Sau do, te bao dugc cam iing bang 1 M IPTG de kich thich sinh tong hgp protein tai to hgp. Sau 4 h nuoi cay trong moi truang cam ling, te bao dugc thu lai bang ly tam va dugc hoa lai bang dem PBS sao cho OD^oo =10. Sau do, te bao dugc pha bang dem pha mau de kiem tra protein tong so bang phuang phap dien di SDS-PAGE. Protein pha tan dugc chuan bi bang each sieu am dich te bao bang may sieu am den khi dich tra nen trong, khong nhat. Ly tam rpm trong 10 phiit de thu pha protein tan. Can sau khi ly tam dugc hoa lai trong dem PBS vai the tich bang the tich truac khi ly tam va dugc xu ly bang dem xu ly mau. Protein dugc hoa rd tu can la pha protein khong tan. KET QUA VA THAO LUAN ThiSt ks vector bisu hien gen Bing phuang phap PCR su dung cap moi dac hieu, gen ha5-0 da dugc khuech dai thanh cong tii DNA khuon la gen ha5 trong vector pcrh5 theo chu trinh nhiet da mo ta a phan vat lieu va phuang phap. San phim PCR tren gel la mgt bang dac hieu, CO kich thuac khoang 1,5 kb dung tihu kich thuac ciia gen ha5-0 theo tinh toan ly thuyet. Ket qua giai trinh tu gen cho thay, dong gen lua chgn c6 trinh tu tuang d6ng 100% vai gen ha5-0 da dugc dang ky ti-en Ngan hang gen ma s6 AJ Dong gen nay sau do dugc chuyen vao vector bieu hien pet22trx (ket qua khong trinh bay) va nghien ciiu bieu hien trong te bao E. coli BL21. Ben canh do, dong gen ha5-l-2 dugc thilt kl nhu Mo ta trong phan doi tugng va phuang phap. Dong plasmid lua chgn dugc cat kiem tra bang mgt so enzyme han che (Hinh 1). Ket qua tren dien di do cho thay cac bang DNA thu dugc co kich thuac diing nhu tinh toan ly thuyet. Trinh tu DNA xac dinh dugc cua dong gen nay da khang dinh dong plasmid lua chgn mang gen ha5-l-2 mong muon. Ncol * Hjndlll BamHl + Hindlll Ncol + BamHl Hinh 1. San pham cat kiem tra cac dong plasmid tai to hgp pcrha5-1-2 tren gel agarose 0,8%. 1-3: Cac dong plasmid so 1, 2, 3 tuang ung; d/c: Doi chung la vector pcr2.1; M: Thang DNA chuan 1 kb (Fermentas). Dong gen thu dugc tiep tuc chuyen sang vector pet22b(-l-)trx (ket qua khong tritih bay) va nghien ciiu bieu hien trong te bao E. coli BL21. Bieu hien gen/r«5., Cac dong E. coli BL21 tai to hgp mang plasmid pet22trx mang gen ha5-0/pet22trxha5-l-2 dugc nuoi cay a 37 C qua dem trong 2 ml moi truang LB CO bo xung ampicillin 100 (xg/ml (LBA). Te bao sau do dugc cay chuyen sang moi truang LBA mod sao cho mat do te bao ban dau dat ODgoo «0,1 va dugc nuoi tiep a cung dieu kien cho den khi ODgoo dat khoang tu 0,5 den 0,8 (pha log). Protein tai to hgp dugc cam iing bieu hien bang IPTG nong do 1 mm, a nhiet do 30 C trong khoang thai gian la 4 h. Protein tong so thu dugc tu dich chiet te bao bieu hien dugc kiem tra tren SDS-PAGE. Ket qua dugc trinh bay tren hinh 2 va 3. Ket qua tren hinh 2 va 3 cho thay, tren duang chay cua cac cac dong te bao mang plasmid tai to hgp xuat hien mgt protein la co kich thuac khoang 66 kda, tuong duang vai kich thuac du doan cua protein HAS dung hgp voi thioredoxin theo tinh toan ly thuylt. Miic do tong hgp cua ca HA5-0 va HA

4 D6 Thi Huyen et al 2 la khong cao, tuy nhien, HA5-1-2 dugc tong hgp manh han (chiem 5%) protein t6ng s6) so vai HA5-0 (chiim 1,5%) protein t6ng so). Trong cac nghien ciiu truac day (Nguyen Phuac Hai et al. 2006; Van Thi Nhu Ngge et al, 2007), khi khang nguyen HAS dugc bieu hien thanh 2 tieu phan rieng re HA5-1 va HAS-2, miic do bieu hien ciia khang nguyen tang len dang ke, chiem tii IS.- 18%) protein tong so ciia te bao. Do do, miic dp bieu hien thap han cua cac khang nguyen tai to hgp trong nghien ciiu nay c6 the mpt phan dugc giai thich do kich thuac lan han ciia khang nguyen. Ngoai ra, ty le cao cua ma hiem trong gen ha5, te bao chu khong nhan biet dugc do do dan den miic do bieu hien. Trong nghien ciiu cua Deltino va dong tac gia (199S) ciing thu dugc ket qua bieu hien rat thap khi dua gen hal-0 vao bieu hien trong E. coli. Trong nghien ciiu nay, khi gen ha5 dugc bieu hien duoi dang dung hgp voi thioredoxin miic do bieu hien gen da dugc cai thien, dac biet khi doan peptide noi hai tieu phan khang nguyen dugc loai bo. Cac chung tai to hgp dugc khao sat bieu hien a cac dieu kien len men khac nhau nham thu dugc du lugng khang nguyen cho cac nghien cuu tiep theo. Toi uu dieu Idf n bieu hien gen Nhiet do len men Te bao E. coli sinh truang toi im a 37 C, tuy nhien trong qua trinh bieu hien gen, te bao E. coli tai to hgp thuang dugc nuoi cay 6 nhiet do thap hon nhiet do nay (Davis et al, 1983; Nguyen Phuac Hai et al, 2006; Van Thi Nhu Ngoc et al, 2007). Vl mat nguyen ly, dieu nay dugc giai thich la khi sinh truang a nhiet do toi thich protein ngoai lai do dugc dieu khien bdi promotor manh se dugc tong hgp 6 at mgt lugng rat lan trong mgt thai gian ngan. Trong khi do, cac thanh phan protein khac tham gia vao cac cau tnic bac cao hon ciia protein lai khong dugc tong hgp vai toe do tuong iing, do do anh huong din ciu tnic thii cap ciia protein ngoai lai va thong thuang lam cho protein dugc tong hgp ra duoi dang khong tan,co hoat tinh thap hoac tham chi mit hoat tinh. D6ng thai, 37 C la nhiet do t6i thich cho cac protease ngi bao hoat dgng, do do cac protein ngoai lai se bi phan cat sau khi tong hgp, anh huong din hieu xuat bieu hien va chat lugng protein. Trong thi nghiem nay, dl khao sat anh huang cua nhiet do doi vai qua trinh t6ng hgp khang nguyen HA5-0 va HAS-1-2 tai t6 hgp, chiing toi tiln hanh nuoi cay cam iing a cac nhiet do 22, 25, 30 va 37 C. Sau 4 h nuoi cay cam iing, te bao,dugc thu lai de kiem tra miic do bieu hien va tinh chat cua protein tai to hgp. Dich chilt va phin can tl bao dugc kiem ti-ati-ensds-page. Kit qua tiinh bay tien hinh 4. Kit qua cho thiy, miic do bieu hien TrxHA5-0 cua tl bao tang len khi dugc nuoi cay cam ling a nhiet dp cao han, a 30 C protein ngoai lai dugc t6ng hgp nhieu nhat. Tuy nhien, kha nang tan cua TrxHA5-0 tai to hgp lai tang len khi nhiet do nuoi cay cam iing giam. Ci 22 C, ty le tan cua protein la tien 70%), tiong khi do ty le nay chi con khoang 50% a 30 C C, TrxHA5-0 tai to hgp dugc tong hop a miic do tuang doi cao voi ty le protein tan khoang 70%. Thi nghiem tien hanh tuang tu doi vai dong te bao mang plasmid pet22trxha5-l-2. Ket qua thu dugc (Hinh S) cho thay, protein TrxHAS-1-2 ciitig dugc tong hgp tot va co ty le tan cao a 25 C. Theo cac nghien ciiu cua Davis va dong tac gia (1981, 1995), khi dupc bieu hien duai dang dung hgp vai protein Tip, toan bp protein TprHl-0 dugc tong hgp tiong te bao E. coli ton tai a dang khong tan. Protein nay chi tan mgt phan rat nho khi dugc bien tinh tiong dung dich dem Tris chiia Ure vai nong do 8 M. Trong nghien ciiu nay, protem dung hgp TrxHAS-O va TrxHA5-l-2 dugc tong hgp vai ty le tan cao hon nhieu so voi cac nghien cuu truac day. Dong thai, khi doan peptide dung hgp dugc loai bo, TrxHA5-l-2 tai to hgp co ty le tan cao hon so vai TrxHA5-0. Nong do chdt cdm umg IPTG Protein ngoai lai dugc dieu khien ting hgp boi promoter T7 tien vector pet22trx. Promoter nay dugc cam iing ba su co mat ctia IPTG tiong moi truang nuoi cay. Nong do chat nay anh huang den miic do bieu hien protein ngoai lai ciia te bao, a nong do qua cao no gay dgc cho te bao, tiong khi a nong do qua thip protein ngoai lai thuang dugc ting hgp kem. Chiing toi tien hanh khao sat len men cac chung tai tl hgp a cac nong dp IPTG la 0; 0,05; 0,1; 0,2; 0,4; 0,6; 0,8; 1 va 2 mm vai nhiet dp cam iing la 25 C. Protein tong so dugc kilm tia bing SDS- PAGE 12,6%. Ket qua thu dugc tien hinh 6 cho thay, khi khong co mat IPTG tiong moi truang, TrxHAS-0 va TrxHAS-1-2 tai to hgp khong dugc ting hgp trong te bao. Tuy nhien, khi moi truang dugc bo xung IPTG thi nong do chat nay khong anh huang nhieu den miic do tong hgp protein ngoai lai, lugng protein tai to hgp thu dugc la gan tuang duang nhau a cac nong do chat cam iing khac nhau. Day la mpt tiong nhiing ylu to gop phin lam giam chi phi khi ma rgng quy ino bieu hien frong buih tam giac lan han va tiong noi len men. 188

5 Tgp chi Cong nghi Sinh hoc 1(2): , 2009 TrxHA5-0 TrxHA D/c M kda Trx Trx Hinh 2. Protein tong so tu cac ddng te bao E coli BL21 mang plasmid tai to hgp pet22trxha5-0 tren SDS-PAGE 12,6%. 1-6: Cac ddng te bao tai to hgp; D/c: ddng te bao doi chu'ng chi mang vector pet22trx; M: Thang protein chuan (Fermentas). Hinh 3. Protein tong so tcf cac ddng te bao E. coli BL21 mang plasmid tai td hgp pet22trxha5-1-2 tren SDS-PAGE 12,6%. 1-3: Cac ddng te bao tai to hgp; 0/c: ddng te bao do! chu'ng chi mang vector pet22trx; IVl: Thang protein chuan (Fermentas). 25''C Hinh 4. Protein tu cac ddng te bao. coli BL21 mang plasmid tai to hgp pet22trxha5-0 dugc nudi cay cam u'ng d cac nhiet dp khac nhau tren SDS-PAGE 12,6%. 1: Protein tong so; 2: Protein khdng tan; 3: Protein tan; IM: Thang protein chuan (Fermentas). D/c: Ddng te bao mang plasmid pet22trx. D/c 22 C 25 C D/c 30''C 37 C M M ^4 TrxHA1-lk-HA2 W 66 kda I» J^^_ Trx 25 kda»4 i» *»fc iiiliifliiniiii J Hinh 5. Protein tai td hgp tu cac ddng te bao E coli BL21 mang plasmid tai to hgp pet22trxha5-1-2 dugc nudi cay cam Cmg d cac nhiet dp khac nhau tren SDS-PAGE 12,6%. 1: Protein tong so; 2: Protein khdng tan; 3: protein tan; M: Thang protein chuan (Fermentas). D/c: Ddng te bao mang plasmid pet22trx. 189

6 D6 Thi Huyin et al 1 M TrxHAO Trx Hinh 6. Protein tong so tcr cac ddng te bao E. coli BL21 mang plasmid tai to hgp pet22trxha5-0 dugc nudi cay d cac nong dp chat cam ung khac nhau tren SDS-PAGE 12,6%. 1-9: Nong dp chat cam ung IPTG tuang iing la: 0; 0,05; 0,1; 0,2; 0,4, 0,6; 0,8; 1 va 2 mm. M: Thang protein chuan (Fermentas). 8 9 TrxHA1-lk-HA2 Trx Hinh 7. Protein tong so tcf cac ddng te bao E. coli BL21 mang plasmid tai to hgp pet22trxha5-1-2 dugc nudi cay a cac ndng dp chat cam iing khac nhau tren SDS-PAGE 12,6%. 1-9: Nong dp chat cam i>ng IPTG tuang ung la: 0; 0,05; 0,1; 0,2; 0,4; 0,6; 0,8; 1 va 2 mm. M: Thang protein chuan (Fermentas). KET LUAN Nhu vay, gen ha5 da dugc bieu hien thanh cong tiong te bao E. coli BL 21 a dang dtmg hgp vai thioredoxin va duai hai thilt ke khac nhau la ha5-0 va ha5-l-2. Viec loai bo doan peptide dung hgp giiia hai tieu phan ciia khang nguyen HAS giiip cho te bao tang kha nang bieu hien gen ciing nhu lam tang ty le protein tan dugc tong hgp. Ket qua nay la co so dl chiing toi tien hanh thiet ke dua gen vao bilu hien tiong te bao nam men P. pastoris. Loi cam on: Bdi bdo duac thuc hien bdng kinh phi di tdi: "Nghiin cuu sdn xudt vaccine tdi to hap phdng benh cum gia cdm bdng ky thudt bieu hien gen md hda khdng nguyin HA cua chung virus cum A/H5N1 Viet Nam trong ndm men Pichia pastoris." do Viin Thu y chu tri vd Du dn thuoc Quy nghiin cwu Viet Nam - Thuy Diin cdp cho PGS. TS. Truang Nam Hdi. Ddng gen pcrh5 sic dung trong nghiin cuu ndy duoc cung cdp bai PGS. TS. Dinh Duy Khdng. Cong trinh duac thuc hiin nho trang thiit bi cua Phdng thi nghiem trong diim Cong nghi gen, Vien Cdng nghi sinh hoc. TAI LIEU THAM KHAO Bridges CB, Harper SA, Fukuda K, Uyeki TM, Cox NI, Singleton JA (2002) Prevention and control of influenza. Recommendations of the Advisory Committee on 190

7 Tgp chi Cdng nghi Sinh hoc 1(1): , 2009 Immunization Practices (ACIP). MMWR Recomm Rep 51(RR-3): 1-31 Davis AR, Nayak DP, Ueda M, Hiti AL, Dowbenko D, Kleid DG (1981) Expression of antigenic determinants of the hemagglutinin gene of a human influenza virus in Escherichia coli. Proc Nad Acad Sci USA 78(9): Davis AR, Bos T, Ueda M, Nayak DP, Dowbenko D, Compans RW (1983) Immune response to human influenza virus hemagglutinin expressed in Escherichia coli. Gene 21(3): Deltito BJ, Ward JM, Hodgson J, Gershater CJL, Edwards H, Wysocki LA, Watson FA, Sanesh S, Kan JP (1995) Effect of a minor isoleucyl trna on heterologous protein translation va. Escherichia coli. J Bacterial 177(24): Ghendon YZ, Markushin SG, Akopova II, Koptiaeva IB, Nechaeva EA,Mazurkova LA, Radaeva IF, Kolokoltseva TD (2005) Development of cell culture (MDCK) live coldadapted (CA) attenuated influenza vaccine. Vaccine 23(38): Jabbar MA, Sivasubramanian N, Nayak DP (1985) Influenza viral (A/WSN/33) hemagglutinin is expressed and glycosylated in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci USA 82: Karasev AV, Foulke S, Wellens C, Rich A, Shon KJ, Zwierzynski I, Hone D, Koprowski H, Reitz M (2005) Plant based HIV-1 vaccine candidate: Tat protein produced in spinach. Vaccine 23(15): Kistner O, Howard MK, Spruth M, Wodal W, Briihl P, Gerencer M, Crowe BA, Savidis-Dacho H, Livey I, Reiter M, Mayerhofer I, Tauer C, Grillberger L, Mundt W, Falkner FG, Barrett PN (2007) Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-rotectiveimmuneresponses. Vaccine 25(32): Laemmli EK (1970) Cleavage of structure proteins during assembly of the heat of bacteriophage T4. Nature 227: Nayak DP, Davis AR, McQueen NL, Bos TJ, Jabbar MA, Sivasubramanian N, Lionelli G (1985) Biological and immunological properties of haemagglutinin and neuraminidase expressed from cloned cdnas in prokaryotic andeukaryoticcells. Vaccine3(3 Suppl): Nguyen Phude Hai, Van Thi Nhu Ngpc, Nguyen Thanh Nhan, Trin Ngpc Tan, Do Thi Huyen, Truang Nam Hai (2006) Bieu hien gen ha2 ma hda tieu phan khang nguyen hemagglutinin (HA) cua virus ciim A/H5N1 trong Escherichia coli. Tgp chi Cong nghe Sinh hoc 4(3): Nwe N, He Q, Damrongwatanapokin S, Du Q, Manopo I, Limlamthong Y, Fenner BJ, Spencer L, Kwang J (2006) Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture. BMC Microbiol 24: Pau MG, Ophorst C, KoldijkMH, Schouten G, Mehtali M, Uytdehaag F (2001) The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines. Vaccine 19(17-19): Potter CW (1998) Chronicle of influenza pandemics. In: Nicholson KG, Webster RG, Hay AJ, editors. Textbook of influenza. Maiden: Blaekwell Scientific Publication: 3-18 Rong J, Jiang T, Cheng T, Shen M, Du Y, Li S, Wang S, Xu B, Fan G (2007) Large-scale manufacture and use of recombinant VP2 vaccine against infectious bursal disease in chickens. Vaccine 25(46): Ross TM, Xu Y, Bright RA, Robinson HL (2000) C3d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge. Nature Immunity 1(2): Saelens X, Vanlandschoot P, Martinet W, Maras M, Neirynck S, Contreras R, Tiers W, Jou WM (1999) Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin. Eur J Biochem 260: Sambrook J, Russell D (2001) Molecular Cloning: a Laboratory Manual 2nd ed. Cold Spring Harbor Press, NY. Simonsen L, Fukuda K, Schonberger LB, Cox NJ (2000) The impact of influenza epidemics on hospitalizations. J Infect Dis \U(3):i3\-%37. Skehel JJ, Wiley DC (2000) Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69: Thompson WW, Shay DK, Weinfraub E, Brammer L, Cox N, Anderson LJ, Fukuda K (2003) Mortality associated with influenza and respiratory syncytial virus in the United Sta.tes. JAMA 289(2): ,.; Treanor JJ, Sehiff GM, Couch RB, Gate TR, Brady RC, Hay CM, Wolff M, She D, Cox MM (2006) Dose-related safety and immunogenicity of a trivalent baculovirusexpressed influenzavirus hemagglutinin vaccine in elderly adults. J Infect Dis 193: Van Thi Nhu Ngpc, Dd Thi Huyen, Nguyen Thanh Nhan, Nguyen Phude Hai, Truang Van Dung, Truang Nam Hai (2007) Bieu hien gen ha5-l ma hda tieu phan khang nguyen Hemagglutinin (HA) cua virus ciim A/H5N1 trong Escherichia coli. Tgp chi Cong nghe Sinh hoc 5(3): Webster DE, Thomas MC, Huang Z, Wesselingh SL (2005) The development of a plantbased vaccine for measles Vaccine 23(15): Williamson ED, Flick-Smith HC, Lebutt C, Rowland CA, Jones SM, Waters EL, Gwyther RJ, Miller J, Packer PJ, Irving M (2005) Human immune response to a plague vaccine comprising recombinant Fl and V antigens Infect 191

8 Do Thi Huyin et al Immun 73(6): Hui-Jun LU, Song HAN, Kuo-Shi, Guo-Dong LIANG Air u* n 117 u * n/^ /'.AA1^ r^_.u T (2006) Expression of AIV subtype H5HA, H7HA and Wnght P, Webster RG (2001) Orthomyxoviruses. In: rrnxta u i. r> ;! r^i.- r _,." '..- j,_^ ;, T>... r..,, H9NA hemagglutinin gene in Pichia pastoris. Chin J Knipe H, Griffin, Marton, Lamb, Roizman, editors. Fie/iM. n-. A 99C9V 9'?1 9^fi virology, vol. 1. Philadelphia: Lippincott, Williams, and Wilkins: ,. ' r,,, Yi-Mmg Xu, Ning-Yi JIN, Zhi-Ping XIA, Ming-Xiao MA, ses_table_2008_05_28/en/index.html EXPRESSION OF HAS GEN CODING FOR HEMAGGLUTININ (HA) ANTIGEN OF INFLUENZA VIRUS A/H5N1 IN ESCHERICHIA COLI Do Thi HuyenS Bui Hong Van^ Van Thi Nhu Ngoc', Truong Van Dung^, Truong Nam Hai'' * Institute of Biotechnology i.,' ^Institute of Veterinary Research ^Natural Science University, Hanoi SUMMARY : ", Influenza virus is a highly contagious and acute respiratory disease with a high degree of morbidity and mortality. The highly pathogen subtype of H5N1 is currently circulating throughout many countries in the world. In this research, we studied on expression of the ha5-0 gene coding for hemagglutinin of avian influenza A/H5N1 in E. coli BL21 cell. The ha5-0 gene was fiised with trx gene coding for Thioredoxin in pet22trx and was constructed in two different forms: 1) The whole native ha5-0 gene except codon ATG and signal peptide; 2) Replaced the cleavage site between HAI and HA2 by the artificial polylinker (ha5-l-2). The expression of recombinant strains habouring gene ha5-0 or ha5-l-2 was studied in LB medium with the addition of ampicillin and IPTG. The effects of concentration of induction factor IPTG (0; 0,05; 0,1; 0,2; 0,4; 0,6; 0,8; 1; 1,5 and 2 mm) and temperature (22, 25, 28, 30, and 37 C) were investigated. The obtained results have showed that, the fusion protein of TrxHa5-0 and TrxHa5-l-2 with the molecular mass of 66 kda were synthesized successfiiuy in E. coli BL21 cells at 25 C with the regulation of T7 promoter which is induced by IPTG. These recombinant proteins were not observed when IPTG was not added into the medium and it was synthesized when IPTG was added even with a very low concentration (0.05 mm). However, the expression level was not increased linearly with the increasing of concenfration of IPTG. Interestingly, TrxHa5-l-2 was expressed at the higher level and higher proportion of soluble form in compare with TrxHa5-0. The solubility of TrxHa5-l-2 was over 70% as expressing at the temperature of 25 C. This gene construct will be integrated into the genome of P. pastoris in order to create a sub unit vaccine protect poultry against influenza virus. Keywords: E. coli BL21, has-l, pet22-trx, recombinant protein, sub-unit vaccine * Author for correspondence: Tel: ; Fax: ; tnhai(d),hn.vnnvn 192