Tgp chi Cong nghe Sinh hoc 13(1): 47-53, 2015 TAO DONG, BIEU HL$N, TINH Cid VA XAC DINH HOAT TINH CiJA TAC NHAN KICH THICH TAO QUAN LAC DAI THIX BAO Tif CHUQT TRONG H$ THONG ESCHERICHIA COLI Nguyen Thanh Thio, Nguyen Bich Thio, Tren Van Hieu Trtremg Dgi hoc Khoa hoc tit nhien, Ali hoc Quac gia Thanh phd Ho Chi Minh Ngay nhan bai: 19.3.2014 Ngay nhan ding: 30.9.2014 TOM TAT Granulocyte macrophage-colony stimulating factor (GM-CSF) la Man to kich thich tao quin the bao tham gia vao qui trinh dip Ong mien dich nhu bach au, dal thin bao, duOng bao (4 bao mast), to bao TK, nguyen bao sqi DO la met cytokin c6 kha ning tic deng len nhieu lopi to bio mau va ci nhieu tiem ning ring Truic c6 the duqc Ong dung nhu dieu tri neutropenia, ay ghip tay xuong hoc lam ti duqc dung tren ngtrai, GM-CSF an duqc thu nghiem tucmg dump tren m8 hinh deng vet Trong nghien dm nay, mGM-CSF ducrc bleu hien Escherichia coil (E coli) Whim tao nguon nguyen lieu cho nhang thir nghiem tren mo hinh chuet Gene mgm-csf mi h6a protein mGM-CSF (ITN khuech dpi to plasmid pIDT-GM-CSF bang phucmg phip PCR, xir Ii bing enzyme at giii han vi duqc gin chin vao plasmid pET28a Plasmid tii to hqp pET-mGM sau d6 duqc bier' nap vao chung bleu hien E coil BL21(DE3) Sau qua trinh cam irng bieu hien yeti IPTG, ket qua phin tich bing SDS-PAGE cho they mGM-CSF bieu hien duei clang the vii to bao chit ctia E coil BL21(DE3) Ket qua lai Western voi khing the dic hieu khing mGM-CSF khing dinh protein duqc bleu hien vugrt mirc chinh la protein mGM-CSF Protein mGM-CSF thu nhan duqc tit ding the vii kh6ng ci host tinh to bao E coli dime tii gip cuen vi tinh sach bang sic Id trio deli cation Cac ket qua phan tich bang lai Western va dinh luqng bang phan mem xic dinh dim de cho they di thu nhan duqc mGM-CSF v6i de tinh sach dot 94,9% Cuei cing, mGM-CSF duqc dinh gia hoot tinh sinh hoc daa tren kha ang kich thich tang sinh ding to bao FDC-P1 va cho ket qua ED50= 3022 pg/ml Tit khoa: mGM-CSF, tai gap cuen, FDC-P1, thfr hoot tinh M6 DAU Granulocyte macrophage-colony stimulating factor (GM-CSF) met cytokin c6 Wing tic Ong len nhieu loci to bito miu N6 ci ning kich thich tang sinh in vitro va biet hoi cic to bio miu tien than tra thinh bio bach cau ho va dai thin bio, tic Ong len chic nang ctia bao mien dich, tir di tang phin ling mien dich bam sinh cling nhu mien dich dac bieu (Enzler, Dranoff, 2003) Do di, GMCSF c6 nhieu tiem ning ins dung, dac Wet ho trq dieu tri cac khiem khuyet ye miu, ning cao hoot deng di too to bio miu sau hia tri lieu hoc ciy ghip ttiy xucmg (Armitage, 1998), he trq mt.& va qui trinh dieu tri berth bach cAu ac tinh ding (Richard et al., 1995), nu8i ciy to bio tua img dung dieu hi ung thu (Shurin, Salter, 2009) va lim ti duqc vaccine (Jones et al., 1994; Hamilton and Anderson, 2004) Vii tiem ning ling dung reng rii cilia GM-CSF, ching Vol mong mu& xay dung met qui trinh sin xuAt GM-CSF gia thinh re nuic Tnrac c6 the duqc ling dung tren ngtroi, nhing nghien cuu ve GM-CSF can phil triti qua nhimg thin nghiem tua ng duang tren mo hinh dOng vat Mat khic, GM-CSF la kh8ng c6 phan ling cite° gift cic loii, GM-CSF cita nguai kh8ng c6 tic dung len to bio chuOt vi ngtrqc lei (Enzler, Dranoff, 2003) Deng thud, su duing his khong &lb huang ten host tinh dui mGM-CSF vii bang chimg la mGM-CSF tai to hqp tir E coli du kh8ng duqc glycosyl hoi van c6 hoot tinh sinh hoc cao ham protein hr nhien thin nghiem in vitro (Schrimsher et al., 1987; Wingfield et aL, 1988) Nhiun cung cap nguyen lieu cho nhitng thin nghiem GM-CSF ten mo hinh chuet sau nhu thin nghiem lam ti duqc vaccine, gitip hoi phuc he thong to bio miu mu h6a hay xa tit chung to i nen hinh ding h6a gen mgm-csf ma hos mGM-CSF vao plasmid bieu hien pET28a di tao thinh plasmid tii to hqp pET-mGM Plasmid tii to hqp tiep tuc duqc bien nop vat) E.coli BL21(DE3) nham too chung ce kha ning bleu hien 47 Nguyen Thanh Thao et aL mGM-CSF Protein mGM-CSF thu nhan dual dang the duqc tai gip cuen, tinh sach va thu nghiem hoat tinh sinh h9c in vitro tren dang to bito FDC-Pl VAT LIEU VA PHUONG PHAP Ching chi vi plasmid Chung vi lchuin E coli DH5a [F- endA hsdR17 (rk-/mk) supE44 thi A," recA gyrA96 dlacU169 080 lacZ 2u1115)] duqc sir dung de nhan ban plasmid E coli BL21(DE3) [F-, ompT, hsdS (rema), gal (DE3)] duqc sir dung lam to bao chi bieu hien mGM-CSF tii to hqp Plasmid pET-28a (Novagen, M9), kich think 5369 by mang promoter T7, gen khang kanamycin (Kan) dirge ding de lam vector bieu hien Plasmid pIDT-GM-CSF (cung dip bei Be mon Ong nghe Sinh h9c Phan tir Ara Mei truing, Truing DH Khoa hcc Ttr thien TP HCM) duqc sir dung lam vector cung cap nguon gen mgm-csj Cic phtrang phip sinh hqc phin trlr Nhu tach chiet plasmid, too to bao kha hien, bien nap plasmid vao to bio E coli dirge nen hanh theo Sambrook & Russell (2001) Thiet ke plasmid tai hqp pET-mGM mang gen mgm-csf Gen mgm-csj c6 kfch thtroc 389 by (GI:109734154) mi h6a cho protein mGM-CSF duac khuech dai tir plasmid pIDT-GM-CSF bang phan img PCR yeti cep mOi doe hieu cho gen co mang trinh tv cat cia hai enzyme cat giei han Xhol vi Ncol Sin pliant PCR duqc tinh the bang be kit EZ-10 Spin Column DNA Gel Extraction (BioBasic, Canada) theo huang dAn cint nha sin xuat va duqc xir H vai hai enzyme Xhol vi Ncol Sau cat bang enzyme han che, gen mgm-csf ductc not trio pET-28a di duce cat bang hai enzyme de too vector tai to hqp pET-mGM mang gen mgm-csf Sing 19c the hien nap vi kiem tra plarnid tai to Ito Vector di to hqp pET-mGM duqc bien nap vac, to bao E coli DH5a The hien nap mqc thinh khuan lac tren mei truing LB ca bo sung kanamycin 30 vg/m1 duqc sang I9c de chqn ding mnc tieu bang PCR khuan lac yeti cap moi d#c bleu cho gen mgmcsf theo chuang trinh: 95°C/5 phut; 30 chu kSr: 94°C/45 giiy, 55°C/45 giay, 72°C/ 45 giiy; ket thfic, 72°C/10 pita Sin phiun PCR duqc kiem tra bing 48 dien di tren gel agarose 1% Plasmid tir the bien nap cho ket qua sang 19c duang tinh tit') tuc duqc kiem tra bang PCR vei c#p mOi bit cOp tren vector nim ngoai vung MSC la T7 promoter va T7 terminator Plasmid tai to hqp cho ket qua ducmg tinh phan img PCR duqc giai trinh ding ty Macrogen (Han Quoc) Sr Wang ding giea trinh ar gen plasmid vi trinh to thiet ke duqc kiem tra bang phan mem Jellyfish Cam ling bieu hiOn mGM-CSF E coil BL21(DE3)/pET-mGM duqc nuoi mei truing LB c6 be sung kanamycin yeti nOng de 30 µg/ml, lac 250 v/p, 37°C den ODA Qat 0,6 0,8 thi tien hanh be sung IPTG 0,5 mM de cam Ong str bieu hien gen Sinh khei to bao duqc thu sau gib cam Ong va duce pha va bang sang sieu am sir dung may Ultrasonic Cell Disruptor (M9) de thu protein tong so Cir ml djch huyen phi to bao se duqc sieu am 4°C vOi xung 15W, mei xung dai 30 giay Protein pha tan va khong tan duqc phlin tach bang ly tam vOi tot d6 13000 Ong/phut 20 phut, 4°C Protein di to hqp duqc kiem tra bang SDS-PAGE vi Western Blot sir dung khang the dac hieu khang mGM-CSF (Santacruz, Mj,) vi &Km phat hien bang ECL kit (GE Healthcare, Anh) Tii gap cuOn mGM-CSF The vii sau thu nh#n duqc nia sach va hoa tan dung djch Urea 2M, pH 12,5 vii ti l'e 0,5 g the yid : 20 ml dich has tan Khuiy i nhiet de phOng 10 phut, sau d6 pha ling tn,rc tiep vao dung djch tii gap cuen (25mM Tris base, 2M Urea, 0,1% Tween 80, 0,5 mM Cystine, 2,5 mM Cystein, pH 9,5) de n6ng di) protein cu6i cling dat khoing 100 — 200 sau d6 khuiy nhe 16-18 tiOng i 10°C Protein sau tai gAp cuen dtrqc 19c hoc ly tam loci be bao quan 4°C de phut vu cho cac thi nghiem ve sau Tinh sach mGM-CSF Protein mGM-CSF c6 diem ding dien pI=6 (Schrimsher et al., 1987) vi v#y thing OH sir dung phtrcmg phip tinh sach bang sic kjI trao di cation tren hg dicing sic AKTA Explorer (GE Healthcare, Anh) vOi cet sic ky Hitrap SP FF ml Qua trinh tinh sach duqc nen hanh nhu sau: (1) can bang OA bang 10 ml dung djch NaOAc 20 mM, pH 5; (2) nap 10 ml mAu vao cet; (3) rea cac phan gin kh6ng at hieu bang 10 ml dung djch NaOAc 25mM, pH 5; (4) dung ly bang 60% dung djch NaOAc 20 mM, pH 5, 40% dung djch NaOAc 20 Tgp chi Cong nghe Sinh hpc 13(1): 47-53, 2015 mM, NaC1 1M pH Cac phin down protein thu 'thin duqc kiem tra bing dien di SDS-PAGE a cac dieu kien c6 vi khong c6 chit kit* ceu not disulfide nhu 2-mercaptoethanol hay DTT 37°C va doc ket qua mit (10 quang a btrac sang 450nm KET QUA VA THAO LUAN Thir nghiem host tinh mGM-CSF in vitro Thiet ke plasmid pET-mGM Hoot tinh sinh hoc cita mGM-CSF duqc xfic djnh thong qua Idle nang dap 1mg tang sinh cia clang bao FDC-P1(ATCC CRL-12103) Te bac) duqc nuoi cey tong mei truang DMEM 10% FBS, mit dO bao 2.105 bao/ml, ti ii 37°C, 5% CO2, (10 Am 85% Mk protein an tit* nghiem duqc pha loing bic 10 tir neng dp 2x106 pg/ml xueng 2x10' pg/ml Ching toi tien hanh thin nghiem vai 50p1 mau protein/gieng va 50111 djch bao/gieng Sau 48 Oa, kha nang tang sinh cue to bio duqc Vic djnh bing cfich bo sung 100 CCK-8/gieng, u them gia by Gen mgm-csf (389 bp) ma hem cho protein mGMCSF di duqc khuech d3i cong tir plasmid pIDTGM-CSF bang phial img PCR vai moi dic hieu (Hinh 1A) Sin phem not cua gen v.11 plasmid (Urge bien nap vao to bao E coli DH5a The hien nop duqc sang loc bang khang sinh kanamycin va phuang phip PCR Ket plasmid tech chiet tir khuin 10c dugt quit.giai trinh tir tuyen chon cho they trinh ttr gen mgm-csfIrong plasmid tai to hqp tuang (long 100% yeti trinh tu thiet ke (Hinh 1B) Nhu voy plasmid pET-mGM di duqc thiet ke cong 11 21 41 31 51 61 71 =1111-91T - 5TOCCACCCACCC3C9CACCCATUCTITTMCCGOCCTFOGNIOCATCTLGAGGCCAMJAZALGCCCTOJIACCMT 011097331 36 ATOCCACCUCCC6C19.ACCCATCACTOICACCCOGCCT9013■039.41011GA0GCCAT911604•00CCTS3ACCICCT 20X0 C40443300 supicecceacCeperssecc4c440141eaccc0/941100449.414114041109051444.060reeu4.acetcc t 5000 01-CST 19-6 603TGALATGCCTUTCACAT941.5103•0300TAGAAGIVOTVITTAALTAGITCITCRUACAALCTIACATOTOTGC 01 011011734156 3000 91 101 Ill 131 131 141 151 GGATGACATGCMTC/ICA1TOAATGAA(1/009,146.40TCOTCTCTAACGICTTCTCCTITIJAAALCTWATISTOTGC C33346994 1103144.4144C140.0494 ccomtga03449c449540140141eueeflagmcceccum03•034 tow 41419199 161 171 101 191 301 211 221 231 O01-CST 0-0 AOACCCOCCTGUZATATTCOACCA04101=ACO0000.41T9CACCAAACTCA4GGGCGCCTTOAACATGACAOCCAOC 500 011007 36134 AOACCCGCCTGA•GATERCOAOCA0009CTAC3000C541.919,ACCAAACTCA5GGGCGCCTIWACATGAMGCCAOC Consmaus 440690099941440•1411.404994030101490930944cmaceasscr.46409;e9eoccg sat la scapec ago 241 • •11••••• 251 261 271 201 291 301 311 5011-CA v-4 TACTACCAGACATAGTOCCCCCCABC7tC003114C00511430,UACKAAGITACCAGCTATOCOGATTITATACACAG 500 — 1111110 600 — await )00— —311819 0/109736136 -1107TACCACCTATGCNIATITCATAGACAG TALTALCAGACJIMACTOCCCCCGIACTCCOOJAACOGICTOTILYAC1.9 Cowmwn 10.4099340aucige.eeesaet.0040seqp.w.4664440443tcaecoccr 0e9eattcc•u0.49 231 331 141 351 361 371 361 391 O101-9339 -13 0C7110405CT9TC-9301-114917CCCC1119.0.470CAA1AWC4611T.A.4104 CCITAAAACCTTITMACTLITATCCCCTITOUTOCAAWACCIATCWJAL Canne6499 e411marece.90900902a50ceecettc40331419403644096010044444 A B Hinh Kat qua thu nhan gen mgm-csf (A) San phArn khuyech dal gen mgm-csf 1: thang DNA 1kb plus, 2: gen mgm-csf, (B) Kat qua sap gieng cat trinh tu, gen mgm-csf thu nhan v61 trinh to cong b6 tren ngan hang gen (GI: 109734154) Bien hiOn protein mGM-CSF to bho E coli BL21(DE3)/pET-mGM Ket qua cam (mg bieu hien protein tren ban dien di SDS-PAGE cho they, so vai khong duqc cam ang (Hinh 2A, gieng 2), chimg E coli BL21(DE3)/pET-mGM sau duqc cam 1mg bai IPTG di sinh tong hqp mGM-CSF vai luqng kha Ian, c6 kich thy& khoing 14,4 kDa (Hinh 2A, gieng 4) va phen Idm protein duqc bieu hien a clang the vii (Hinh 2A, gieng 5) Ket qua phi hqp vai cong bt) cue DeLamarter et aL, (1985) Tuy nhien nghien ciru nay, Itrqng protein mGM-CSF chi chiem 10% tong luqng protein to bao Trong Q6, protein mGM-CSF cue ching toi duqc bleu hien intim dO cao, chiem 26,9% tong Wong protein to bao (kat qua khong trinh bay) Protein mGM-CSF tai to hqp da duqc khing djnh bang lai Western vai khang the chuyen biet khang mGM-CSF Cfic protein tai to hqp mGM-CSF dung djch protein tong so, protein pha khOng tan (the vii) va pha tan deu c6 kha nang nhin biet vi lien ket tot vai khang the khang mGMCSF (gieng 4, 5, Hinh 2B) Nhtr v4y, c6 the khing djnh protein mGM-CSF duqc cam (mg bieu hien chting E coli BL21(DE3)/pET-mGM Tinh stich mGM-CSF mGM-CSF a clang the vii sau tai gip cuOn de 49 Nguyen Thanh Thao et al thu nhAn dang tan duct tien hinh cho qua cOt sic ki trao doi cation Dung ly de thu nhAn mGM-CSF bAng each dung dung dich NaOAc 20mM, NaCI 1M, pH de thay doi luc ion Khi dung dich dung ly di qua cot, cation Na+ se barn vao chat nen tich dien am va day cac protein ngoai Ket qua dien di cho thay kohan doan khong gan cOt (Hinh 3A, gieng 3) chira mOt phan protein mGM-CSF va cac protein khic c6 phan dap truerc qua cOt (Hinh 3A, gieng 2) Nguyen nhan ducrc du doan la prong mlu nap vuo qua kha nang bat cua cOt hoc mOt phan tir mGM-CSF chua hinh clang cau hinh clan den tich dien khac di nen khong the On len cOt MAt khac, a buac nia eot (Hinh 3A, gieng 4) kh6ng xuat hien vach protein nao Trong d6, a phan clop 40% dung dich ly giai, tuang ling 400mM NaCI (Hinh 3A, gieng 5), chang toi da thu duqc vach tuang ling protein mGM-CSF Sir dung phan mem Quantity One cho thay protein thu nhOn &roc c6 dO tinh sach dat 94,9% (Hinh 3B, gieng Nhu viy, chi vii mOt bulk tinh such bang sac IcY trao dal cation, chang tot da thu nhar&roc protein mGM-CSF yen dO tinh such cao, tuang ducmg vii ding bo cua Schrimsher (1987) Tuy nhien, nghien ciru cua cac tic gia nay, protein mGM-CSF phai trai qua hai burc tinh such bang sac i(51 IQC gel va trao doi anion Dieu lam gia tang ding ke chi phi cling nhu lam giant hieu suit thu nhAn mGM-CSF tinh sach Protein rnGM-CSF co hai cau not disulfide nOi phan tir DE kiem tra stir hinh cau not disulfide dm mGM-CSF tai to hop cac phan (loan thu nhan ducrc qua trinh tinh che, chung Col tion hinh dien di SDSPAGE cac phan doan protein vii dung dich bien tinh mau khong be, sung tic nhan khir cau not disulfide nhu DTT hay 2-mercaptoethanol kDa 45 20 — 19A —t 9.5 A Hinh Ket qua bleu hien mGM-CSF Dien di SDS-PAGE (A); lai Western veri khong the OC hieu khong mGM-CSF (B) 1: thang protein phan tir Itygng thep, 2: protein pha tong E coli BL21(DE3ypET-mGM; 3: protein pha tong E colt BL21(DE3)/IPTG; 4: protein pha tong E coli BL21(DE3)/pET-mGMAPTG, 5: protein pha khong tan E coli BL21(DE3)/pETmGM4PTG, 6: protein pha tan E coli BL21(DE3YpET-mGMAPTG I'D 97,0 011,0 45,0 30,0 20,1 14,4 A B Hinh Kiem tra protein cac phan doen tinh such mGM-CSF Dien di SDS-PAGE (A); Dinh luvng bang Quantity One (B) 1: Thang protein phan tir luvng thep, 2: phan doan trtierc qua cet, 3: phan doan khong cOt, 4: phan doan rira got, 5: phan doan dung ly Trong phan doan truoc qua cOt (Hinh 4A, gieng 4), kDa va 20,1 kDa cua thang phin tti luong thAp) co hai vi tri cua protein muc tieu mGM-CSF (giira vi to 14,4 vach, du doan tuang Ung vii hai clang kh6ng co va co 50 • Top chi Cong nghe Sinh hoc 13(1): 47-53, 2015 caunal disulfide caa mGM-CSF tai to hqp Phan doan khong barn cpt (Hinh 4A, giang 3) chaa vach phia tat, de phan doan dung ly (Hinh 4A, gieng 2) chua vach phia dual caa protein vatic tie'u Chang toi du doin, ck protein hinh duqc cau n6i disulfide da gin len cet va duqc thu nhfin qua trinh dung ly, de cac protein khong hinh eau not disulfide di bj iota nap mau Protein mGM-CSF tinh such tiep tic duce kiem tra bang phucmg phip SDS-PAGE va lai Western v6i khang the chuyen Wet khang mGM-CSF (Hinh 4B) Kit qua cho thay, tren ban phim c6 vach tin hieu hien re Wang irng v6i each c6 kich thuOc nim giaa hai vach 14,4 kDa vi 20,1 kDa tren bin dien di SDSPAGE Nhu v#y mau protein thu nh#n duqc sau qua trinh tinh such chinh la mGM-CSF 4 kDa 07,0 56,0 45,0 10,0 30,1 14,4 A Hinh Kiem tra protein cac phan doan tinh the mGM-CSF dieu kien then di SDS-PAGE khong CO tac nhan khir c5u nOi disulfide (A) 1: Thang protein phan tir luvng th5p; 2: ph5n doan dung ly: 3: ph5n doan khOng grin Get: 4: phan doan turac qua cot Dien di SDS-PAGE va lai Western blot (B) 1,3: mGM-CSF tai t6 hap: 2,4: thang protein phan tir lung thAp Dinh gii host tinh mGM-CSF Hoat tinh sinh hoc in vitro caa protein mGMCSF duqc dinh gia dua ten kha nang kich thich tang sinh d6ng brio FDC-P1, bei day la clang bac phi thuec mGM-CSF Chung tei tien faith &nth gil kha rang tang true:rig caa to brio day neng de mGMCSF to 10'2 den 106 pg/ml 0.4 0.3 • 0.2 Ket qua phan tich qua phAn mem Graphpad Prism (Hinh 5) cho d6 thj c6 hinh clang dac thing sigma cart met thu nghiem tang sinh, dieu cho dray protein mGM-CSF di kich thich duqc su tang sinh va tri stir sang ctia doll bio FDC-Pl Clc thong se phan tich cho thay to bio dap img vi bat din phis trien manh Ming de 103pg/ml, tang On djnh den neng de 106 pg/ml Thir nghiem cho gia trj ED50 = 3022 pg/ml Gil trj ED50 la cao ham so v6i cac nghien ciru di cong be try& ye hoat tinh sinh hoc dm protein mGM-CSF thu nhOn to E coli hay brio deng 141 (Burgess et al., 1977; Schrimsher et al., 1987) Tuy nhien, viec djnh luqng hoot tinh sinh hoc met cytokin c6 the thay doi b tong he thong thir nghiem va timg dieu kien nuoi cay Vi vily, can c6 nhang thu nghiem so sinh vai matt c6 the &nit gia chinh Vic hoot tinh sinh chart hoc cart mGM-CSF tai to hqp (Meager, 2006) a 0.1 0.0 l'o Log[mGM-C SF) Hinh 06 thi hiring quan giCra tang tarang t6 bao FDCP1 va nOng dO mGM-CSF Day la c8ng be clau tien Viet Nam ve vi'ec to clang vi thu nhim mGM-CSF c6 hoot tinh sinh hoc tir he thong vi khan E coli Tren the gioi, mGM-CSF tai to hqp tir E coli di duqc bieu hien IA dlnh gil hoot tinh sinh hoc cong be caa DeLamarter et al., (1985) Trong nghien dm dm chang till, gen 51 Nguyen Thanh Thao et al ma hem protein mGM-CSF duqc bieu hien E coli BL21(DE3) Protein mGM-CSF tai to hqp duqc bieu hien =iv dO cao va phin 16n protein duqc bieu hien dm% dung the viii Sau bu6c tai gip cuOn va tinh such, ch6ng toi da thu nhen duqc mGM-CSF v6i dO tinh such cao doting thin dA loui be duqc nhiing phan ter mGM-CSF hinh khong &mg ciu n61 disulfide Protein mGM-CSF sau duqc tai Op cuOn va tinh such di' the hien hog tinh sinh hoc, duqc khan djnh qua kha nAng kich thich tang sinh dong bao FDCPI Ket qua cita nghien ciao la ca sof cho cac this nghiem tren m8 hInh chuOt sau KET LUAN Gen ma h6a mGM-CSF di duqc bieu hien ding chin% E coli BL21(DE3) mGM-CSF duqc bieu hien chit yeu to bao chit cita E coli BL21(DE3) duai dung the yid The vOi duqc sit dung de tai gip cuOn va tinh the nhAm thu nhin mGM-CSF co hoot tinh yeti dO tinh such cao Sau qua trinh tinh such chung toi thu nhin duqc mGM-CSF yeti dO tinh such 94,9% Ket qua dui nghiem hoot tinh cho thAy mGMCSF co kha rang ldch thich tang sinh dong bao FDCPI voi gia tri ED50 3022 pg/ml Lei cam an: Cling trinh duac tlurc hien tit kink phi hang num cda Throng Dai hQc Khoa hoc to nhien, Dai hpc Qudc gia HCM clip hang num cho BO mon Cong nghe sinh h9c phan to viz mai truang Cam on GS Nicos Nicola Wien nghien cult Y khoa Walter and Eliza, Uc) cung cap dong to bao FDC-Pl Burgess AW, Camakaris J, Metcalf D (1977) Purification and properties of colony-stimulating factor from mouse lung-conditioned medium J Biol Chem 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Nguyen Thanh Thao, Nguyen Bich Thao, Tran Van Hieu2 University of Science, Vietnam National University HoChiMinh City SUMMARY Granulocyte macrophage-colony stimulating factor (GM-CSF) is a cytokine that stimulates a variety of blood cells and therefore has many potential applications, such as chemotherapy side-effect reduction, bonemarrow cell immobilization, vaccine adjuvant and so on Before GM-CSF can be used in human, it is required an initial trial on animal model In this study, mGM-CSF was expressed in Escherichia coli (E coil) to provide material for testing in mouse model A gene mgm-csf coding for mGM-CSF protein was amplified from pIDTGM-CSF plasmid by PCR method, digested with restriction enzymes and inserted into pET28a plasmid to create a recombinant plasmid pET-mGM The gene in the obtained plasmid was checked by PCR and sequencing The plasmid was then transformed into the expressing strain E coli BL21(DE3) After induction using IPTG, GM-CSF was over-expressed in the form of inclusion body in the cytoplasm of E coli BL21(DE3) and was confirmed by Western blot with anti-GM-CSF antibodies The inclusion bodies was then refolded and subjected for cation chromatography purification The purified recombinant protein was mGMCSF with the purity up to 94.9 percent of total purified fraction Finally, mGM-CSF bioactivity was assessed via the proliferation of FDC-P1 Result showed that the EDse of mGM-CSF was 3022 pg/ml Keywords: Expression, mGM-CSF, refolding, FDC-PI, bio-assay Author for correspondence: Tel: +84-8-38307079; E-mail: tvhieu@hcmus.edu.vn 53 11 ... c6 hoot tinh sinh hoc tir he thong vi khan E coli Tren the gioi, mGM-CSF tai to hqp tir E coli di duqc bieu hien IA dlnh gil hoot tinh sinh hoc cong be caa DeLamarter et al., (1985) Trong nghien... chin% E coli BL21(DE3) mGM-CSF duqc bieu hien chit yeu to bao chit cita E coli BL21(DE3) duai dung the yid The vOi duqc sit dung de tai gip cuOn va tinh the nhAm thu nhin mGM-CSF co hoot tinh yeti... thu nhin mGM-CSF co hoot tinh yeti dO tinh such cao Sau qua trinh tinh such chung toi thu nhin duqc mGM-CSF yeti dO tinh such 94,9% Ket qua dui nghiem hoot tinh cho thAy mGMCSF co kha rang ldch