THÔNG TIN TÀI LIỆU
• Nghien cu'u - Ky thuot Chi&I luvc viec bleu hien erythropoietin (EPO) tren dung to bao CHO-DG44 bang vector tricistronic Nguy& Anh Xuan', Lao Dire Thuan3, Doan Ngoc HAP, Do Minh Si u* Khoo Sinh hoc, Truang Dai hoc Khoa hoc Ter nhien — Dai hoc Quoc gia Tp Ho Chi Minh COng iv TNHH CNSH Dirac NANOGEN ' Khoa Ging ngh "(3 sinh hoc, Mtrang Dai hoc Mir Tp 114 Chi Minh 'Vin Stir khoe nghe nghiep vd Moi trtrang E-mail: minhsi@nanogenpharma.com Summary Erythropoietin (EPO) gene was cloned into the expression vector pDIGF-EPO, which carries other two expression cassettes (GFP and DHFR) The dihydrofolate reductase deficiency (DHFR-) CHO (DG44) cell line was transfected by this vector using lipofectin method The GFP-based system was used to shorten the cloning duration and enhance the opportunity to select super clones The DHFR-based system was used for gene amplification The transfected cells were selected and processed through cycles of FACS followed by gene amplification and single cloning by limited dilution method GFP gene from the single cell clone was removed by recombination An optimized fed-batch was developed to culture the EPO expressing cells The cells could survive for 14 days; the protein titer was 902.6 ± 19.96 mg/mL (comparable to the previously reported values for EPO production in pilot scale) The purified recombinant erythropoietin from the fed-batch had the molecular weight within the range of 30-40 kDa as checked by SDS-PAGE Its isoform pattern by isoelectric focusing (IEF) electrophoresis satisfied the requirement of isoforms issued by the European pharmacopoeia 7.0 (EP 7.0) with ratios in appropriate ranges Peptide mapping and sialic acid content analysis strongly passed the requirement stated in EP 7.0 Biological activity of the obtained EPO-alfa versus the 3rd WHO International Standard for Erythropoietin for bioassay was 112.545 ILI/mg protein Keywords: Recombinant human erythropoietin (rHuEpo), CHO-dh&-e cell, GFP, single clone, gene amplification, recombination Dat van de EPO to nhien hoan toan giong ye trinh tu' acid amin va CO chu6i oligosaccharid giong c5u talc hydrat carbon EPO tai to hop CO hoat tinh sinh hoc twang tu' nhu' EPO nOi sinh Erythropoietin (EPO), mot nhan to tang troang kith thich tao hong ceu, giup cai thien clang 1(6 ch5t luvng cuOc song cue benh nh5n mac chOng suy th5n ho5c chOng thieu mau tem tong EPO Bong vai trO quan trong so tang sinh va biet h6a cue cac to bao tier, than hong chu tOy xuang 111 Ph5n Ian EPO duvc tao chu yeu bai th5n de dap Ong vai tinh tang thieu oxy mo, mot phen nha (10%-14%) gan tong hop (gan la ca quan chinh san xuet EPO a bao thai) 141 Trong nhOng nam gen day, viec Ong dung cue EPO tai to hop dieu tri benh tang ph6 Nen ten toan the gi&i EPO tai to hap va NhOng to bao dOng vat nhu, CHO duvc Ira chon de san xuet cac protein sinh duvc nhu' EPO bai cac bien doi sau dich ma dm to bao phu hop vai ceu truc protein cua ngudi ye mat chac nal-1g cOng nhu' duvc dOng hoc 16 ViOC sir dung to bao CHO cho nuoi cay san xuet protein vol quy me Ian tang ph6 bien bai cac (long to bao c6 the thich Ong vai viec phat trier) mai twang huyen ph0 khOng chive huyet Ngoai ra, cac clang to bao duvc tong nh5n la cac to bao an toan cho viec san xuAt protein tai to TAP CHI DUCK HQC - 11/2015 (SO 475 NAM 55) 25 '91 • Nghien civu - Ky thuOt hap m Tuy nhien, de Iva chgn disc d6ng to bao dOng vat the hien cac protein muc tieu (bien dOi sau dich ma) veri hieu suet cao, met s6 yen de 41 dircyc giai quyet nhir hO the -1g bieu hien kern hieu dO qua, thol gian chuyen gen va tao d6ng, bieu hien protein the') & quy m6 Ian Day le nhCrng khO khan gap phai hau het cac dong to bao hien 18.91 Trong nghien ctru tnyac day, Meng va cong sv da phat then he thong dong bieu hien gen muc tieu vcil gen chon loc DHFR (ma hoe cho enzym dihydrofolat reductase) va gen GFP (ma hoe cho protein phat hujmh quang xanh) de Iva ch9n d6ng to bao bieu hien protein tai to hcyp muc tieu vai bleu suet cao nhat [121 Deng to bao tot nhat dvgc chgn 19c dva ten he thong chon I9c DHFR va he thong phan tech to bao dva ten tin hieu hujmh quang cue GFP (FACS) Trong nghien cull nay, chimg t6i diva mot chitin lucyc mai cho viec tao dong to bao bieu hien On dinh protein EPO tai to hap He thong vector tricistronic gem gen muc tieu, gen GFP (tao thuan Igi cho viec rut ngan th6i gian tao d6ng va tang kha nang chgn 19c d6ng to bao tOt nhet) va gen DHFR (dung ch9n loc ten dong to bao khuyet cluVng DHFR CHO-DG44) Biyac tiep theo cue nghien CCPU le lc* be gen GFP bang phvang phap tai to hgp sau chon duvc to bao don dong bieu hien protein muc tieu tOt nhat Qua trinh se giiip giam hay loai be su' bieu hien die GFP VOt lieu, phvang phap nghien ciru Thief ke vector Trong nghien ciru nay, plasmid mang gen ma hoa EPO tai to hgp ngueri (EPO), gen GFP (ma hoe protein GFP), gen khang Zeocin va gen chon loc DHFR (ma hea dihydrofolat reductase) Trinh tcy Hu-EPO duvc thiet ke va tong hcyp bai hang Genscript (Piscataway, M9) Gen eGFP duvc phan tech tip plasmid pEGFP-N1 (Clontech, M9) Gen DHFR duvc phan tech tiv plasmid pSV2-dhfr (ATCC, M9) Promoter CMV duvc sir dyng de Trinh dieu hoa bieu hien EPO tai to hgp tcy intron dm gen beta-globin tren the (GenBank: V00882.1) va \rung CpG cica gen HNRPA2B1 ngt.rol ma hea cho loai protein hnRNP A2 va B1 (GenBank: D28877.1) duvc be sung de 26 tang cv6ng kha clang bieu hien gen muc tieu Ket thuc qua trinh bieu hien GFP bang phvang phap tai to hgp Hai vi tri tai to hgp tuvng dOng FRT duvc them vao truac promoter va sau trinh tty gen GFP De dam bao cho GFP bieu hien thap km gen ma hoe EPO, trinh to IRES duvc chen vao truac promoter dm GFP VOng promoter (SV40) ducyc sir dyng de dieu h6a bieu hien DHFR Plasmid ducyc d6ng hoa vao to bao vi khuen E co/i Top1OF theo hvang den cica nha san xuet (Invitrogen, Mg) Plasmid bleu hien duvc tinh sach bang b0 kit tech chiet DNA (Qiagen, M9) Quy trinh chuyen gen Te bao CHO-DG44 (ngan hang cGMP, Invitrogen, M9), dong to bao mang dOt Nen gen reductase dihyrofolat (DHFR) nem troll nhiern sac the se 2, duvc Sir dung rOng rai san xuet protein tai to hgp th6ng qua phvang phap chon loc khuyet dveyng DHFR Te bao CHO-DG44 duvc nu6i cey m6i truteng PowerCHO2 (Lonza, M9) co be sung hypoxanthin va thymidin (HT, Sigma-Aldrich, Mg) 37°C, 5% CO2 De chuyen plasmid vao to bao CHODG44, nghien cOu sir dung WO kit chuyen gen Lipofectamine LTX with Plus Reagent kit (Invitrogen, Mg) Quy trinh thvc hien duvc tien hanh theo Wang den cue nha san xuet Nghien cipu ben hanh chuyen vector tricistronic vac) to bao CHO-DG44 Sau 48 gi6 chuyen gen, to bao chuyen gen Neu hien GFP duvc quan sat bang kinh hien vi hujmh quang va sv bieu hien protein EPO dich nuoi cey to bao duvc dinh luvng bang bi) kit human EPO ELISA (Stem Cell technology, Canada) Chon loc va tao clang bieu hien EPO tai to hop to bac) CHO-DG44 Qua trinh chon 19c citycyc tien hanh bang cach nu6i to bao de' chuyen gen m6i trirevng chon 19C be) sung 400 pg/ml zeocin, 50 nM methroxat (MTX) ve khuyet cluOmg HT (Sigma-Aldrich, M9) Sau ch9n loc, to bao mang plasmid 'Jinx nu6i cey tang sinh moi tupteng tri c6 be sung 50 pg/mI zeocin Te bao chuyen gen bleu hien manh GFP dirgc chon 19c len ten he thong may FAGS Calibur (BD Biosciences, M9) va sir TAP CHI DUOC HOC - 11/2015 (SO 475 NAM 55) • Nghien civu - Ky thUOt dung cho nhirng nghien ciyu tiep theo Te bao sau lkya chon bang GFP tiep tuc duvc ben hanh chon IQC thong qua khuech dai gen Te bao duvc cam irng mei trtyang nuoi cey bO sung MTX vai ruing dO tang dan tO, 50nM den 500 nM Sau dO, to bao duvc phyc hoi va tiep tuc nuoi tang sinh DOng to bao dun (single clone) bieu hien On dinh protein muc tieu tiep tuc duvc tao bang phiyang phap pha bang tai han Sang loc nhiyng dong to bad dun co bleu hien GFP (quan sat dkrai hien vi hu9nh quang) va EPO (dinh luvng bang phiyang phap ELISA) cao nhat Cu6i cOng, su' bieu hien GFP duvc loaf bó bang cach chuyen plasmid pOG44 mang gen ma hoa recombinase (Invitrogen, M9) vac) d6ng to bao Ikra ch9n Hieu suet tai to hap cluvc clanh gia bang he thong may FACS Calibur Huai ch tang sinh to bac) san xuAt EPO tai to hcrp tren quy mo this, nghi§rn Doing dan to bao sau chon I9c theo quy trinh neu ten (bleu hien manh nhat EPO tai to hap) duvc sir dung de nuoi chy tang sinh bang m6i tru,Crng PowerCHO2 (Lonza) Trkrac tier), to bao duvc nuoi cay Ong nuoi cey lac 50 ml (BD Biosicence, M9), sau dO du,cyc nuoi cey tang sinh len the tich cao hon (600m1) he thong nuoi cey Tube spin flask (TPP, Thyy ST) ket hcyp vai tO nuoi cey lac (Kuhner, Thuy ST) Cu6i ding, qua trinh nuoi cey to bao san xuet EPO tai to hap duvc thy,c hien binh nuoi cey lac L (Thompson, M9) Te bao duvc nuoi bey vai mat dO ban clau 0,5 x 106 to bao/mL L m6i tnyOng san xuet O' 37°C, 5% CO2, tOc de lac 100 vong/phOt Sau nuoi cey, 500 mL dung Bich feed (bao Om Feed tiy Excellgene, Galactose, Manose, Insulin,Transferrin va h6a chat khac de tang qua trinh gen dkveyng cua EPO tO, Sigma) duvc b6 sung hang Qua trinh nuoi cey to bao san xuet EPO ket th0c ti le phan tram to bao sting dual 70% (khoang 12-14 ngay) Dich nuoi cey to bao sau dO duvc thu nhan va tinh the bang he thong sac ki Akta - Mabselect Sure (GE Healthcare, Anh) theo hu,deng dAn cua nha san xuet Protein sau tinh sach duvc kiem tra, danh gia cac chi tieu nhu' ceu truc h6a hoc, c10 tinh sach, nOng dO protein, hopt tinh sinh hoc dkya ten cac quy trinh de" duvc cong b6 [Jelkmann, TAP CHI DUOC HQC - 11/2015 (SO 475 NAM 55) Lee, Breidbach] Ket qua va ban luan Vector bieu hiOn EPO tai to hap 1111M111111 Hinh 1: Ca'u truc vector pDIGF-EPO Chien Vac bleu hien protein EPO ten dong to bao CHO-DG44 nghien ciyu mang lei hieu qua cao xem xet troll khia canh thOi gian tao d6ng va lu,cyng protein duvc tao Wing bleu hien rHu-EPO bao Om gen muc tie(' (Hu-EPO) duvc dieu hoa biyi promoter/enhancer CMV Diya ten ket qua cong b6 cua patent WO 2004067701A2, viing CpG dm gen HNRPA2B1 ma h6a cho protein hnRNPA2 va B1 duvc sir dyng nhu' le yeu to tang twang bleu hien gen (enhancer) [3] Viing CpG duvc chipng minh nhu, la yet, CO tang cu,ang bieu hien gen ngopi lei va co nhieu thuan Igi thiet ke vector cho viec tao dong bieu hien [11) Nghien ciyu cua Buchman va cong (la chOng minh sky hien dien cua vOng intron thuOc gen beta-globin b tho cO kha nang tang ckyo'ng su' bieu hien gen cao Op den 500 len 12] Ket hap cac yeu to tang cuteng tren, su' bieu hien cua GOI tang len dang ke (dir lieu khong trinh bay) Trinh ty, IRES (internal ribosome entry site) le trinh tu' cluvc tim they virus co kha nang huy dOng ribosome de bieu hien protein mot each clOc lap va dOng vai tr6 quan tong qua trinh phat then cac vector polycistronic VOng IRES thu nhan tO, virus Encephalomyocarditis (EMCV) hien diyac sir dung chu yeu heu het cac vector Tuy nhien, luvng protein bieu hien diyai su' dieu flea cua EMCV IRES thuteng kh6ng du de dat duvc cac yeu cau thkyc nghiem hay chiya tri Nghien 27 • Nghien civu - Ky thulat Tao &mg to bao CHO-DG44 bieu hiOn EPO tai to hqp cue Licursi va ding sy, cho they viec sir dung vOng IRES cue nhan to tang biking nOi mach (vascular endothelial growth factor) va protein cam irng collagen loai (type collagen-inducible protein) tang cutrng kha nang bleu hien gen cao han so vai cac vector china EMCV IRES 11°1 Trong nghien ciru nay, chimg tai sir dung trinh ty, de dieu h6a sy' bieu hien cue GFP (marker chon 19c) Cau triic cua vector pDIGF-EPO du'o'c m6 to hinh Sau 48 gib' chuyen gen, su' bieu hien EPO va GFP tam tho'i duvc kiem tra Ian lout bang phutrng phap ELISA va phuvng phap quan sat mOu dutri kinh hien vi huknh quang DO, lieu tit, su' bieu hien GFP cho they hieu suet chuyen gen dat 50 - 70% va to bao bleu hien EPO & mirc 2,3 pg protein/te bao/ngay (bang 1) Bang 1: Sy, bieu hien EPO va GFP cua to bao CHO-DG44 Ham luvng EPO (pg/te bao/ngay) Ti le bleu hien GFP (%) Sau ihn sorting Sau ihn sorting 2,08 ± 0,46 6,82 ± 0,83 9,01± 0,62 12,30 ± 1,07 0,02 14,78 ± 3,27 32,57 ± 6,33 78,22 ± 8,96 94,66 ± 9,13 Sau chuyen gen, to bao duvc chip 19c bang m6i truirrig nuoi cey khuyet dutrng HT-, b6 sung khang sinh va MTX nham ch9n cac to bao chuyen gen On dinh Protein GFP duvc chirng minh la huru ich he thong chon 19c beri protein GFP cho phap tao mot phuvng thirc ch9n I9c nhanh, dun gian va hieu qua ten cac to bao da duvc chuyen gen th6ng qua phkrang phap thing chuyen Dkra ten k9 thuet phan tech va thu nhen to bao co chon loc thong qua tin hieu huknh quang (FAGS), nghien ciru sir dung chien luvc de phat hien cac to bao bieu hien manh protein myc tieu Cac dong to bao c6 tin hieu GFP cao nhat moi trutmg duvc cho la co kha nang bieu hien protein myc tieu EPO vai hieu suet tirang tu' Trong nghien ciru nay, sau hai Ian phan tech va thu nhen to bao bang k9 thuet FAGS, khoang 94% to bao hieu hien GFP cho tin hieu vai cutrng (10 khac (hinh 2) Ket qua cho phap nghien ciru loai 136 cac to bao bieu hien thap protein myc tieu EPO Ngobi ra, cac to bao chuyen gen On dinh cho they sy, tang len dang ke luvng EPO duvc tao Bien hiOn On dinh 1)61 cluing 00 pm Dieu hi#n on dinh Bleu hiOn tam thol DOi Thong sei 100 pm 100 ma FACS Ian FACS lOn 100 pm Hinh 2: Su' bleu hien GFP cua to bao CHO-DG44 truoc va sau chuyen gen Ti le to bac, phat huknh quang truoc va sau chuyen gen, phan tech to bao ducr xac dinh bang kSi thuet FACS Tin hieu phat huknh quang cua t6 bao duvr kiem tra duial kinh hien vi huknh quang Cac to bao sau Itra chon theo quy trinh neu ten tiep tuc nuoi cey tang sinh va tao dOng to bao don bang phuvng phap pha loang tai han nharn chon i9C dong to bao bieu hien On dinh nhet Dich huyen phu tip dia 96 gieng duvc thu 12 sau chuyen to bao vao dia nuoi ch 48 dong to bac tot nhat duvc nuoi ten dia 24 gieng bang cach lye ch9n cac to bao tir die 96 gieng chuyen qua dia 24 gieng Sau nuoi, dich huyen phO tip cac gieng cua dia nuoi cey 24 gieng duvc phan tich Sau dO, 12 dong to bao tot nhet duvc chon va chuyen vao die nuoi cey gieng de tiep tuc nuoi cey tang sinh Dina tren kat qua phan tich tip dich nuoi cey to bao, &mg 28 TAP CHI INICIC HOC - 11/2015 (SO 475 NAM 55) • Nghien an' - Ky thUOt to bao bieu hien tot nhat (EPO - 6, bang 2) duvc Rya chon va nuoi cay tang sinh sir dyng he thong lac On nhiet (Kuhner) Dora Von ket qua thu nhan duvc tir nuoi cay 16c (rn't to bao ban dau x 105 to bao/mL, the Lich 100 mL mai tru,o'ng nuoi cay), d6ng to bao bleu hien tot nhat (EPO 5) duvc Iu'u tiff, ngan hang giOng va kY hieu EP00414 Kat qua danh gia hiOu su6t san xuOt protein muc tieu va duirng cong tang tru,O'ng cho cac clang to bao tot nhat duvc trinh bay bang Bang 2: Hiau suat caa cac dOng to bao don bieu hiOn EPO Ngay Deng M bao NO), Mat d6 Ham lurrng protein Luang protein/ (108 M bao/m/) (pg/m1) to bao/ngay Thai gian nhan dOi (gia) Mat c16 M bao (106 to bao/m1) Ham luring protein 019/m0 EPO 2,23 ± 0,25 135,2 ± 6,33 20,2 ± 0,97 33,38 ± 0,13 4,24 ± 0,31 365,8 ± 10,25 EPO 2,21 ± 0,39 122,1 ± 7,49 18,4 ± 1,23 33,58 ± 0,25 4,37 ±0,27 371,2 ± 11,36 EPO 2,37 ± 0,45 136,3 ± 5,01 19,2 ± 0,85 32,07 ± 0,36 4,62 ± 0,39 312,9 ± 15,26 EPO 3,09 ± 0,51 141,7 ± 8,23 15,3 ± 0,98 27,40 ± 0,24 4,91 ± 0,52 344,8 ± 14,33 EPO 5* 3,13 ± 0,62 198,6 ± 7,22 21,2 ± 1,51 27,21 ± 0,19 5,23 ± 0,68 405,6 ± 17,66 EPO 2,61 ± 0,47 153,7 ± 9,16 19,6 ± 0,98 30,20 ± 0,27 4,31 ± 0,29 311,2 ± 12,38 (*) D6ng to bao clan &Frio Iva chon de tgo ng5n hang M bac): EP00514 Hien nay, cac enzym tai to hap tai cac vi tri chuyen Net (site specific recombinases) duvc phat trien nhirng cong cu sir dung k9 thuat thao tac ten gen vi chi:mg co the xec tac chinh xac cho cac phan irng cat, cher), dao doan hoac chuyen vj tri dm DNA thong qua cac trinh ty, nhan biet dac hieu [5] Nghien ciru sir dung he thong Flp-In (Invitrogen) chira vi trf tai to hgp Flp (Flp Recombination Target site - FRT) de Doi clukng loai 136 gen GFP Su' bleu hien GFP da giam Bang ke du'a he thong (vector pOG44) vao to bao CHO-DG44 bieu hien GFP Theo hinh 3, kat qua cho thay to bao EP00414 sau loai be GFP van bieu hien EPO vai ham luvng tuvng ty, Trong d6, miyc de bleu hien GFP giam dang ke (8,98%) so vai truirc chuyen vector pOG44 (93,64%) Te bao sau loai be GFP duvc lu,u trir va ky hieu EP00514 Tnr■fre (Ai to hop Sat) kit( tai to ivy') 100 mm Hinh 3: Sy bgti hiOn GFP ctla t6 bao CHO-DG44 tnferc va sau tai to h9p Tile to bao phat huknh quang trubv va sau tai to hop duryc xac Binh bang ksi thuOt FACS Tin hiOu phat huknh quang cCia to bao dupy ki4rn tra dtpai kinh hidn vi huknh quang Nu8i cay to bao va san xuAt EPO tai to hoop Hieu su'At cua EP00514 duvc danh gia nuOi cay theo phirang phap nuOi cay fedbatch (quy mo L) vol mai tru,teng duvc toi u'u (mei truing du'o'c be sung theo theri gian, the tich cuOi 2500 mL) sir dung binh nuoi cay L (Thomson) va he thong to cay lac (Kuhner) Luvng protein du,gc xac dinh bang phuvng phap ELISA sir dung be kit human Erythropoietin (EPO) ELISA (Stemcell Technologies) Kat qua TAP CHI DU6C HOC - 11/2015 (SO 475 NAM 55) 29 • Nghien CCPU - K9 thu.at duvc trinh bay bang Voi phiyang phap nue' cey fed-batch duvc tai tyu [lea, EP00514 cO the tri vong 14 Ham luvng protein dat 902,6 ±19,96 µg/mL, twang &rung vai cac Gong be truVc dO ve san xuet EPO Bang 3: Ket qua nuOi cey d6ng to bao EP00514 theo phuung phap nu6i cey Fed-Batch Ngay Mat d6 (106 to bao /m1) Ti le 16 bao song (%) Ngay 0,55 99,21 ± 0,25 2000 mL MOi throng Power CH02 Ngay 0,66 ± 0,09 98,51 ± 0,16 40 mL Feed dupc136 sung mai Ngay 1,43 ± 0,11 98,62 ± 0,22 Ngay 2,32 ± 0,25 95,87 ± 0,31 Ngay 2,84 ± 0,23 97,26 ± 0,25 Ngay 3,65 ± 0,37 94,19 ± 0,47 Ngay 3,53 ± 0,26 93,07 ± 0,51 + Insulin + Transferrin N-glucosamin Ngay 3,52 ± 0,38 95,65 ± 1,33 + Galactose + Mannose Ngay 4,36 ± 0,52 92,37 ± 3,51 Ngay 12 2,76 ± 0,63 90,64 ± 4,73 Ngay 13* 2,62 ± 0,61 73,44 ± 4,33 Ham lucmg EPO (lig/m1) 221,3 ± 12,11 Mei trutmg + Feed + Galactose + Mannose + Non essential amino acid + Uridin + L-Glutamin + Insulin + Transferrin 425,1 ± 13,24 668,7 ±21,12 + NEM + Uridin + Uridin + L-Cystin, + NEM + N-glucosamin + Pluronic + Insulin + Transferrin + NEM + N-glucosamin + Uridin 902,6 ±19,96 (*) thu hoach vao 14 Phan tich EPO tai to tiger) Protein tai to Nip EPO tinh sach tip d6ng to bao EP00514 nu6i cey fed-batch c6 luvng phan tir khoang tip 30 - 40 kDa duvc kiem tra bang phiyang phap SDS-PAGE (hinh 4) KM qua phi) hap vat tieu chuan duvc &Fa DLyo'c dien Chau Au (European pharmacopoeia 7.0 - EP 7.0) -. .^ Hinh 4: Ket qua phan tich mAu protein EPO (A) Ket qua phan tich peptid cua meu EPO bang phuung phap peptid mapping Mau EPO (tren) va md'u chain qu6c to EPO (EDQM, duet) (8) Ket qua phan tich isoform cua EPO bang phuung phap than di diem clang dien (IEF) Gieng 1: AA& EPO, gidng 2: Thang chuan pl, gieng 3: Mau EPO chuan qu6c te (C) Ket qua phan tich lucmg phan ter EPO bang phurtng phap SDS-PAGE Gieng 1: Mau EPO; gidng 2: Mau EPO chuan quOc td, gieng 3: Thang chuan protein Ket qua khao sat ti le isoform bang phiyang phap dien di diem deng dien (IEF) cho they EPO tao dam bao cac yeu cau ve isoform vai cac t9 le nem khoang giO1 Flan Cu the, isoform khong duvc phat hien they, dO isoform chiern phan Ian lirong isoform (bang 4) KM qua phe sec 1(9 peptide mapping cho they EPO ce phe peptid twang veyi EPO chuan guoc te Ham luvng acid sialic cua protein sau tinh such la 14,90 ± 0,30 mol sialic acid/mol EPO (du•a ten phtycvng phap dm EP 7.0) Kat qua ming th6a man cac you cau dm EP 7.0 10 mol sialic acid/mol EPO) 30 TAP CHI DUVC HOC - 11/2015 (SO 475 NAM 55) Hoat tinh sinh hoc cua EPO tao duvc darth gia ten m6 hinh chuOt cai khoe manh • Nghien cU'u - K9 thuOt (20 — 22 g) va so sanh vai EPO chug!' quOc to theo tieu chuen c6a WHO danh cho EPO (3rd WHO International Standard for Erythropoietin— ma NIBSC: 11/170) Haat tinh sinh h9c cua EPO duvc tinh Wan dkra tren chi so hOng cau cOa chuOt tiem EPO Ket qua cho they EPO tao co hopt tinh 112.545,00 ± 6,32 Ill/mg protein Ket qua them man yeu cau dm EP 7.0 100.000 111/mg protein) Bang 4: Ti le isoforms caa EPO Insoform Ti4 phhn tram chun (%) Wet qua (%) Insoform 0-15 Insoform 0-15 Insoform -20 4.9 Insoform 10 - 35 12.7 Insoform 15 -40 23 Insoform 10 - 35 29.5 Insoform 5-25 23.9 Insoform 0-15 4.9 Nghien aru sir dung he thong chon loc mai giup giam clang ke thai gian tao dong (so Iu'Q'ng the he cua d6ng duvc chon 19c la 30) va tang twang kha nang thu nhan cac dong to bao bieu hien manh protein EPO Ter d6, cac d6ng to bao co the duct thiet lap va san xuet protein tri lieu vai do, twang dirang so vai cac he thOng ch9n loc hien Dec biet vai nganh tong nghiep can tOc tao cac nguyen lieu cho cac nghien ciru lam sang giai dopn I het sin quan tong, he thong chon loc duvc de xuet nghien ciru hi vong tao dirgc scr khac biet nhet dinh Ket kfan Da nghien va phat trien tong dong to bao CHO-DG44 bieu hien On dinh protein EPO tai to hgp vai hieu suet dat 902,6 ±19,96 mg/mL Cac ket qua dm nghien dm cho they EPO tai to hgp di 'c tao co luvng phan ter, ti le isoform, cau tr6c peptid krang EPO chuen quOc to theo Duvc dien Chau Au (EP 7.0) Ngoai ra, hopt tinh sinh hoc cua EPO dat 112.545 IU/mg, twang &rang vai EPO chuen quOc to WHO (3rd WHO International Standard for Erythropoietin) Tai liOu tham khao Ananth G S (2008), "The role of recombinant erythropoietin in childhood cancer", The Oncologist, 13, 157-166 Buchman A R., Berg P (1988), "Comparison of intron-dependent and intron-independent gene expression", Mol Cell Biol., 8, 4395-4405 Crombie R L., Irvine A S., Lipinski K S., Williams S G (2004), Improved genetic elements providing high levels of expression, Patent WO 2004067701 A2 Diane M R., Fitzroy D., Elliott P (1994), "Erythropoietin: A review", J Nati Med Assoc., 86, 129-135 5.Dymecki S M (1996), "Flprecombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice", Proc Natl Acad Sci U S A, 93, 6191-6196 Estes S., Melville M (2014), "Mammalian Cell line developments in speed and efficiency", Adv Biochem Eng Biotechnol., 139, 11-33 Florian M W., Hacker D (2011), "First CHO genome", Nature biotechnology, 9, 718-720 Jelkmann W (2007), "Control of erythropoietin gene expression and its use in medicine" Methods Enzymol., 435, 179-197 Kim J Y., Kim Y G., Lee G M (2012), "CHO cells in biotechnology for production of recombinant proteins: current state and further potential", Appl Microbiol Biotechnol., 93, 917-930 10 Licursi M., Christian S L., Pongnopparat T., Hirasawa K (2011), "In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression", Gene Ther., 18, 631636 11 Mariati, Yeo J H., Koh E Y., Ho S C., Yang Y (2014), "Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells", Biotechnol Prog., 30, 523-530 12 Meng Y G., Liang J., Wong W L., Chisholm V (2000), "Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells", Gene, 242, 201207 (Ngay nhOn bai: 13/06/2015 - Ngay duyOt (fang: 02/11/2015) TAP CHt DUQC HQC - 11/2015 (SO 475 NAM 55) 31 ... technology, Canada) Chon loc va tao clang bieu hien EPO tai to hop to bac) CHO- DG44 Qua trinh chon 19c citycyc tien hanh bang cach nu6i to bao de' chuyen gen m6i trirevng chon 19C be) sung 400... Bang ke du'a he thong (vector pOG44) vao to bao CHO- DG44 bieu hien GFP Theo hinh 3, kat qua cho thay to bao EP00414 sau loai be GFP van bieu hien EPO vai ham luvng tuvng ty, Trong d6, miyc de bleu... rOng rai san xuet protein tai to hgp th6ng qua phvang phap chon loc khuyet dveyng DHFR Te bao CHO- DG44 duvc nu6i cey m6i truteng PowerCHO2 (Lonza, M9) co be sung hypoxanthin va thymidin (HT, Sigma-Aldrich,
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