Tap chi Cong nghe Sinh hoc 12(4): 615-621, 2014 T30 DOING, BIEU HI) N VA TINH S3CH NHAN TO TANG TRUONG NGUYEN BAO S01 (FGF-1) NG1Td1 TRONG ESCHERICHL4 COLI Ding Tit Truirng, Trinh Minh Thwyng, Trin Vin Hien Truang DO hoc Khoa hoc At nhien, DO hoc Quoc gia pha Ha Chi Minh Ngiy nhin bii: 02.4.2014 Ngiy ratan ding: 30.9.2014 TOM TAT FGF-1 (fibroblast growth factor 1) la mit protein da chic nang Protein ci khit fling kich thich phin bao, hoot his si,r sang cia to bao, tham gia vao nhieu qua trinh sinh hoc nhtr su phat trien phoi thai, sire chila cac mo, tai tao to bao, thic day su hinh mach mau va su phat trien cue nguyen bao sqi fron; qua trinh chea lanh vet thuang Vii flitting tiem nang nhu vtty, hien FGF-I da duqc ap din vao rat nhieu linh vtrc nhu san xuat m9 phalli, chum tri cac benh el) lien quan den tim mach, chum limit vet thucrng Nhim to protein FGF-I tai to hqp vii gilt hqp 19 cho nhimg Ong dung i Vi4t Nam, chain tai tien hint' tao ding E coli ci kha nang bieu hien protein tai to hqp FGF-1 Gen fgfl (423 bp) duqc khutch dal tO plasmid pIDT-fgf/ bAng phuong phap PCR va ding his vao plasmid pET-His Plamid tai to hqp mang gen fgfIdAt duii su kiem soat cia promoter T7 duqc dAt ten la pET-fg/7 Gen fg/1 plamid pET-fgf/dtrqc kiem tra bing PCR, enzyme giii hon va giai trinh tu Sau di, plasmid pET-fgf/dtrqc hien nap vao chimg E coli BL21(DE3) 'Chi nuoi cay lac chant tai to hcrp mei truing LB, be sung IPTG de cam Ong T7 promoter, FGF-1 duqc bieu hien dui% dang the vii to bao chit cia E coli vii ham lucrnt chiem lchoang 22% tang protein sau 16 gib cam Ong phan tich bang SDS-PAGE, lai Western va phan mem Quantity-One The vii china FGF-1 duqc hoa tan bang dung dich Guanidin 4M va tai gap cuen bang bang phuong phap pha loing nhanh, thu nhan duqc FGF-1 clang holt tan Phuong phap sic ky lac vii heparin duqc sir dung de tinh soch FGF-1 Ket qua cho they FGF-1 duqc thu b phan down dung ly vii nang cla NaCI I,2M va vii nang di NaCI 0,4M c6 c6 de tinh soch Wong Ong la 93,4% va 50,1% Chi c6 protein i Rhin doon ming de mutii cao c6 hoot tinh tang sinh in vitro ding nguyen bao sqi chuit NIH 3T3 xap xi sun phim FGF-1 among mai, vii E1350 = 657 pg/ml khea: FGF-1, th'j vui, tinh ch" r i Igc voi heparin thu hoot tinh MO DAU Nhan to tang truing nguyen bac sqi (fibroblast growth factor (FGF)-1) hay cern goi la nhan to tang truing nguyen bio sqi c6 tinh acid (acidic fibroblast growth factor — aFGF) la met protein da chic nang FGF-1 c6 kha nang kich thich phan bio, hoot h6a su song sot to bio, tham gia vao nhieu qui trinh sinh hoc nhu su phat trien phoi thai, sira china cic mo vi tit too to bio, thic day su phat trien to No.* mo vi to chic vot 19 dm to bao nei mo, thic day su hinh thinh mach miu vi su phit trien ctia nguyen bao sqi qui trinh china linh vet thucmg (Langer et al., 1993, Zakrzewska et al., 2008) Vii nhOng chic nang nhu viy, hien FGF-1 da duqc irng citing vao rat rthieu linh vac nhu sin xuttt m9 pham, chtra trj cic benh c6 lien quan den tim mach, china linh vet thucmg Trong y hoc, nhting nghien ciru ye FGF-1 da duqc chimg minh la ci nang kich thich su tang sinh mach vi sira china cic hu hong, ca tim Sau duqc dua vao ca tim, FGF-1 thic day su hinh thinh mach cic mo thieu miu cic be (ho(ic thieu oxy) nheng benh nhan dang giai dog' cu6i Do viy, FGF-1 Bang duqc nghien cia thir nghiem lam sang pha I vi II de chira tri cic benh ye tim mach (Cuevas et al., 1997, Htun et al., 1998, Schumacher et al., 1998) Ngoii ra, FDA can dua khio sit mei di dinh gia FGF-1 dieu trj viem loit mo tieu FGF-1 c6 the duqc sir dung dieu trj cic vet thuang man tinh vi chan thwmg bao gem loit Cr miu tinh mach, vet sera paw thuit va b6ng Trong m9 pham, FGF-1 li met tic nhan duqc sir dung phi bien giap tri met lin da khoe, cheng leo hOa vi tre hia lin da c6 tic dung kich thich su gia tang dm cac nguyen bio sqi da; tang truing to bio sung, sin xuAt chAt nen ngoai bio, kich thich sin xuAt cic yeti to tang truing an thiet khic di phuc hei da; vi kich thich tic tang truing Vii riffling uu 615 DOng Tat Twang et al diem nhu vay, FGF-1 dirge sir dung tang nhieu cac serum mg pham chi:mg lao h6a Truck nherng tiem nang ung dung dui FGF-1 y hoc va mg phim, chimg toi tien hanh to clang to bao E coil c6 kha nang bieu hien vucrt mac FGF-1 lam nguen nguyen lieu va tinh such protein cung cap cho viec img dung FGF-1 tai Viet Nam a VAT LIEU VA PHIYONG PHAP Chung chu va plasmid Chung vi khuin E coli DH5a [F- endA hsdR17 recAl gyrA96 NacU169 (rk-/mk) supE44 thi (080 lacZ 4M15)] (Takara) duqc sir dung de nhan ban plasmid E coli BL21(DE3) [F-, ompT, hsdS (re- ma) gal (DE3)] duqc sir dung lam to bao chu bieu hien FGF-1 nguai tai to hqp Plasmid pET-His (Novagen), kich thy& 4636 by china promoter T7 duce cam img bing IPTG (isopropyl fl-D thiogalactoside), gen khang ampicillin (Amp) dirge dung de too plasmid pETfgfl bieu hien FGF-1 Ck phuang phap sinh hoc ph'in ter nhu tach chiet plasmid, ghep n6i gen, cat kiem tra gen bang enzyme ban che, to to bao E colt kha bien va bien nap plasmid vao to bit° E coli duqc thvc hien theo Sambrook va cong stir (Sambrook et al., 2001) Thiet ke plasmid tai to hop pET-fgf/ mang gen fgfl ma hoa cho protein FGF-1 Gen fgfl ma h6a cho protein FGF-1 (ma se tren ngfin hang gen?, c6 kich thuerc bao nhieu, lay ca gen hay met down gen de bieu hien) duqc khuech dui tar plasmid pIDT-fgf/ (cung cap beri Be mon COng nghe Sinh hoc Phan tir va Mei tnrerng, Twang DH Khoa hoc Ttir nhien TPHCM) bing PCR vai Op mei bit Op dfic hien vol hai dill gen FGF1F/FGF1R San phim PCR duqc xir Ijr vai enzyme cat han che NdeI va BamHI va tinh che bing EZ-10 Spin Column DNA Gel Extraction kit (BioBasic, Canada) theo Imams dAn cua nha san xuat Plasmid pET-His dirge cat mer yang bang cep enzyme NdeI Ara BamHI, bit hogt enzyme bang phenol/chloroform va tinh che bing EZ-10 Spin Column PCR Products Purification kit (BioBasic, Canada) Sau san pham PCR va plasmid da duqc xir ljr bang NdeI va BamHI dirge nei vOi de tao plasmid pET-fgfl 616 h Sing Iric the Wen flop va giai trinh tkr DNA Gen fgfl va plasmid pET-His sau xir IS/ bang enzyme NdeUBainHI duqc nei v6i bang enzyme T4 ligase Sau qua trinh nei, sari pham Wei duce bien nap vao to bao E coil DH5a va tien hanh sang lac dung vi khan mang plasmid tai to hqp tren mei truerng thach LB china ampicilin 100 tg/ml Tiep theo, khuan lac mac duqc ten mei Mrerng china khang sinh dirge nu8i cay va tach chi& plasmid tai to hqp Plasmid tai to hqp duqc kiem tra bing cach cat gi&i han yeti hai enzyme NdeUBamHI va PCR vai cep moi FGF1F/FGFIR theo chucmg trinh: 95°C/5 phut; 30 chu kg: 94°C/45 phut, 55°C145 &ay, 72°C/45 phut; ket chic, 72°C/10 phut Deng tiled, gen fgfl duqc giai trinh tv c8ng ty Macrogen (Han Quec) Viec phan tich trinh tkr gen duqc tien hAnh tren phin mem Jellyfish Dieu hitn protein FGF-1 Ching E coil BL21(DE3)/pET-fgf/ duqc nuoi mei truang LB-Amp100, lac 250 v/p, 37°C den OD600 dot 0,6 - 0,8 thi tien hanh be suns IPTG den ming de) 0,5 mM de cam Ong stir bieu hien FGF-I, tiep tic nueoi lic them 16 gia Sinh khei to bao dirge thu loi va huyen phi dem lanh (10mM Tris-HCI, pH7, ImM EDTA), d#t di 15 phut Te bao dirge pha von bing Ultrasonic Cell Disruptor (Mg) sau de) ly tam 13000 v/p, 4°C, 10 phut de thu phin doon protein tan (dich noi) va protein khang tan (can) Protein tong so dung cho SDS_PAGE duce chum bi bang each them dung dich xir 1Y man vao huyen phi to bao, gia nhiet phut 95°C, ly tam 13000 v/p, 4°C, 15 phut de loci be Phin tich SDS-PAGE va lai Western Sr bleu hien FGF-1 duqc phan tich bing SDSPAGE 15% Protein duce phat hien bing Coomassie Brilliant Blue, khei luring phan tir FGF-1 duqc so veri thang phan tir luring thap (Amersham) Sr hien dien cua FGF-1 duqc kiem chimg bing phuang phip lai Western vai khang the khang dac hien FGF-1 (R&D) Ham luring cua FGF-1 to bio E coil duqc xic dinh tuang dei bang phin mem Quantity One (Bio-rad) Tai gip cu#n FGF-1 hr the vii The vii (pha protein khOng tan) thu nhan t6 E coli BL21(DE3)/pET-fg/7 duqc rira Ian bang dung dich rira the vii (Tris-HCI 20mM, pH 8,0) c6 bo sung Triton-X100 ding de 1% de loci met s6 protein tap, sau de rira lin bing dung dich rira the vii khang be Top chi COng nghe Sinh hoc 12(4): 615-621, 2014 sung Triton X-100 The vai sau dra duqc h6a tan dung djch Gu-HCI 4M de neng da protein dot sau duqc pha bang nhanh bang dung djch tai gap cuOn (Tris-HC120mM, urea 2M, sucrose 10%, tween80 0.1% pH 7.5) de nOng d'6 protein dot 0,2 mg/ml, khuay deu 4°C yang 14 gib giai hart deu dinh tuang ung vai gen fgfl, san phim co kich thuac 4601 bp 20000 by Tinh chi FGF-1 bing sic kj, Isrc vIri heparin C6t HiTrap Heparin HP (Ge Healthcare, They SI) duqc can bang bang dung djch A (Tris-HC1 20 mM, pH 7,5) Dung djch the vai tai gap cuan duqc nap vac cat HiTrap Heparin HP vai tic d6 2,5 sau d6 cat duct tai can bang bang dung djch A Tien Wirth dung ly cac protein barn cat bang bon hqp dung djch A va dung djch B (Tris-HCI 20 mM, NaCI 2M, pH 7,5) vai ti la 20%, 40%, 60%, 80%, 100% dung djch B, tot da 2,5 ml/phut Cac phan down protein duqc thu nhan, dian di SDSPAGE, xic djnh da tinh sach bang phan mem Quantity One (Bio-rad) va djnh luqng FGF-1 bang phuang phi!) Bradford Dinh gii host tinh tang shill in vitro ctia FGF-1 sau doh chi Hoot tinh sinh hqc cua FGF-1 sau tinh the duqc danh gia thong qua tiler nghiam tang sinh in vitro tren d6ng nguyen bao sqi chuat Te bao NIH 3T3 duqc nu8i moi tnemg DMEM/F12 vai 10% huyet (fetal bovin serum) den dot da bao phu khoang 80% be mat binh nuoi thi den hinh tech va thu nhan to bito don bang enzyme trypsin 0,25% Sau 46, to bio NIH 3T3 duqc huyen phu moi tru6ng DMEM/F12 vai 10% huyet va bo sung vac dia 96 gieng a mat d0 5x103 to bio/gieng, ti a 37°C, CO2 5% Sau 24 gia u, mei tru6ng cu duqc but b6 va thay bang moi truong DMEM/F12 duqc b.& sung FGF-1 tinh the vii n6ng 80 tang bac 10 tar 10 pg/ml den 10 Sau 48 gib, moi gieng nu8i city duqc 1:46 sung 10 al MTT mg/ml va d6 hap the quang a btrac song 550 nm KET QUA VA BI$N LUAN Thlit ki plasmid tai to hqp pET-fg/7 Gen fgfl (423 bp) ma h6a FGF-1 da duqc khuech dal plasmid pIDT-fgf/ bang phuang phap PCR (Hinh 1) va xir IS, too deu dinh bang cap enzyme Ndel va BamHI Plasmid pET-His cans duqc cat m6 Ong bang cimg mat cap engzyme cat 500 b 400 by 423 bp 75bp Hinh PCR khuech dai gen fgll 1: Thang chuan DNA kb plus; 2: San pharn PCR gen fgfl San pham not gen fgfl va plasmid pET-his duqc bien nap vac* vi khuan E coli DH5a, sau d6 tien 14nh sing lqc (long E coli mans plasmid tai to hqp bang khang sinh ampicilin va kiem tra plasmid tai to va giai trinh ar Ket qua hqp bang PCR, cat giai PCR plasmid tai to hqp yeti moi FGF1F/FGFIR va hai enzyme Ndel, BamHI deu cho sin phan co kich thy& 423 by tucmg img vai kich thuac dm gen fgfl duqc then vac) (Hinh 2) Eking thei, genie/ duqc giai trinh ttr va so sixth vai trinh ttr thiet ke bang phan mem Jellyfish, ket qua so sanh cho they gen fgfl plasmid tai to hop khong co sai sot ten trinh tu nucleotide, va ding khung djch ma tren plasmid pET-His (ket qui kh6ng trinh bay a day) Bien hiin gen fgfl ma hos FGF-1 E coli BL21(DE3)/pET-fgf/ Nhiun danh gia sir bieu hian va tim hieu sir hien dian cua FGF-1 a clang the vai hay clang h6a tan to bio chit, chimg E coli BL2I(DE3)/pETfel duqc nu8i city lac LB-Atup100 va cam Ung bang IPTG Sinh Ishoi to bao duqc thu nhan sau 16 gib cam img va tien hanh phi ming to bao de phan tich ink protein tong so, phan down tan (chill FGF-1 tan) va phan down killing tan (chira FGF-1 dung the Ket qua phan tich SDS-PAGE cho they, duqc cam mg bai IPTG, thing tai to hqp di tong hqp mat luqng len protein co kich thtroc khoang 16 617 Ding Tit Tnrong et al kDa (Hinh 3A, gieng 3), protein khong co dich tong kitting cam img IPTG (Hinh 3A, gieng 2) So sanh veri khoi lucmg phan tir ly thuyet cua FGF-1 khoang 15,8 kDa, cho 111'4 vach protein dam hien dien dich tong dtrqc cam Crn4 co the la aFGF tai to hop bleu hien tUpET-fg1/ Nham khang dinh vach protein duce bieu hien la FGF-1, chung ten phan tich them bang lai Western vii khang the dac hieu khang FGF-1, tren ban phim xuat hien vach tin hieu tucmg frng vari vi tri cita vach protein xuat hien dich tong protein duqc cam img IPTG (Hinh 3B, gieng 3), kat qua 20000 by kiting dinh vach protein dam xuat hien duqc cam ung chinh la FGF-I Ben canh do, phan tich phan doan khong tan va tan dich to bao da duce phi ming cho protein FGF-1 chi duqc bieu hien dang the the viti trong to bao E coli (Hinh 3A, B, gieng 4) Dinh lung bang phan mem Quantity-One, ch6thay FGF-I bieu hien chiem 22% tong protein dm to bao E coli BL2 I (DE3)/pET-fgf/ (Hinh 3C, gieng 4) Nhang ket qua ten chirng to ch6ng t6i da cong viec tao chimg E toll BL21(DE3)/pET7/ie7 bieu hien FGF-1 ngtr&i tai to hqp 20000 by 500 by 400 by 423 by 5000 by 4000 by 4601 by 500 by 400 by 423 by 75bp 75 bp A B Hinh Keit qua kiern tra gen fgfl plasmid tai to hqp pET-fgf1 PCR vdri cap mdi FGF1F/FGF1R (Hinh 2A); Cat bang hai enzyme Ndel, BamHI (Hinh 2B) 1A: Thang DNA kb plus, 2A: chirng am, 3A: gen fgfl, 4A: san pham PCR, 18: Thang DNA kb plus, 2B: plasmid tai to hqp tuft cat, 3B: plasmid tai t6 hop sau cat bang Ndel/BamHl kDi 97,0 66,11 -45,0 — 3(1,0 — 20.1 1I 4IP • 14,4 — Hlnh Str bieu hian FGF-1 E coli BL.21(DE3)/pET-fgf1 Dit)n di SDS-PAGE (A); lai Western yeti khang 014 dOC hiOu khang FGF-1 ngtrtri (B); dinh krqng bang Quantity-One (C); 1: thang protein; 2: protein tong so K mAu kh6ng cam UN IPTG; 3: protein tong s6 to mhu cam Ong IPTG: 4: phan doan protein kh8ng tan cam Ong IPTG; 5: phan doan tan cam Ong IPTG 618 Tgp chi Cong nghe Sinh hoc 12(4): 615-621, 2014 TM gip cuOn FGF-1 tu the vfii Guanidin IA chit hien manh lam cho cac protein bj ket cum the vAi du& va tan dung djch Sau pha loang bang dung djch tai gap cuOn, ming dO Guanidin giam xuting tao,dieu kien cho cac protein tai ciu trAc tar lei Met so chit nhu urea, sucrose, tween80 duqc bo sung de qua trinh tai gip cuOn dien tot hen, ngan chAn stir ket coin tra lei cua cac protein 4, gieng 2) khang swat hien nheng vach protein nay; lieu cluing to dung dich guanidin 4M da 116a tan hill het protein the viii Sau tai gip cuen, pha protein tan xuat hien vita protein Wang img vai FGF-1 (Hinh 4, gieng 4), (Icing th6i khang co protein bj ket cum qua trinh tai gip cuOn (Hinh 4, gieng 5) Nhu vAy, FGF-1 da duce tai gap cuOn va ton tai b clang tan sau tai gap men va sin sang cho nhAng 'mac tiep theo Tinh chi FGF-1 bing sic Ic* iii lrc voi heparin kDa T 97,066,045,0- L : 30,020,114,4 - I I • Illnh Phan tich protein qua trinh UM gap cuOn FGF1 tren SDS-PAGE T: Thang protein chuan; 1: Th6 viti trtr6c h6a tan; 2: COn sau hoa tan the viii; 3: Dung dich the viii h6a tan bAng Guanidin 4M; 4: DO protein sau tai gap cu6n; 5: C6n sau tai gap cuOn The viii truac hoa tan (Hinh 4, gieng 1) va dung djch the viii boa tan bang guanidin 4M (HInh 4, • gleng 3) co cfic vach protein tuong img vai nhau; do, phan cAn sau hoa tan the yin (Hinh FGF-1 co hi luc manh vai heparin, twang tic gicra FGF-1 Ira heparin lien ket ion gala polyanion tren heparin (nhorn sulfate) v6i Lys113, Arg119, Lys128, Asn18, Asn114 va GIn127 tren be mAt cua phan tir FGF-1 (Fan et al., 2012, Zakrzewska et al., 2009) Khi dung djch mutii co ntmg de cao, lien ket gift FGF-1 NIA heparin bj pha va Phan door, 20% (Hinh 5A, gieng 4) va 60% dung djch B (Hinh 5A, gieng 5) deu co protein co khoi luqng phan tin I9 thuyet cua FGF-1 Ichoang 15,8 kDa Cac protein FGF-1 tai to.hqp cac phan doan da duqc khang djnh bang lai Western (Hinh 5B, gieng va 5) Ben canh d6, phan tich phan doan dung ly 20% va 60% dung djch B bang phAn mem Quantity-One, ket qua cho thiy FGF-1 thu nhAn duqc chiem tuong Ang IA 50,1% va 93,40% luqng protein tinh sach duqc (Hinh 5C, gieng Ira 5) Hieu suit tinh sach va hieu suet thu hei cua toin qua trinh &rot trinh bay bang Ket qua phi hqp vai ding b8 cua Moringlane va dOng tic gia (1990), FGF-1 co boat tinh duqc thu nhan dung ly voi dung djch china NaCI 1,2 M tinh the sac k' lvc heparin (Moringlane et al., 1990) Hinh Phan tich protein cac phan doan tinh the Ivc heparin Dien di SDS-PAGE (A): lai Western vii khang the dac hieu khang FGF-1 ngu*i (B): Phan tich tinh sach bang phan mem Quantity One (C) T: Protein chuen: 1: Dung dich tru.eic qua cot: 2: Dung djch khoi cot sau nap meu: 3: Dung dich riya cot: 4: Phan doan 20% dung dich B: 5: Phan doan 60% dung djch B 619 Dang TAt Trtrimg et al Bang Phan tich Wang FGF-1 va hiau suet thu hei FGF-1 caa town qua trinh tinh che Luang protein t6ng (mg) Ty la FGF-1 (%) Ltmng FGF-1 (mg) Meu the vui ttrai ban au 12,00 49,30 5,92 Phan doan 20% dung djch B 0,65 50,10 0,32 5,41 Phan doan 60% dung dich B 2,26 93,40 2,11 35.64 Dinh gia host tinh tang sinh in vitro ctia FGF-1 sau tinh the Do thj danh gia hog tinh tang sinh cua FGF-I c6 dong dac trung cho mgt thin nghiem tang sinh Ket qua cho thay c6 su tuang quan giira ruing dO FGF-1 va su tang sinh clang to bao NIH 3T3, ,khi gia tang nOng dO FGF-I moi twang nuoi cay thi mat dO to bat) NIH 3T3 c6 su gia tang tuang img Tir thj suy duot gia tri ED50 cua cac phan doan Ket qua cho they mAti FGF-1 thu (kw tir phan doan 60% dung dich B c6 EDso = 657 pg/ml cao ham mau FGF-1 thucrng mai voi ED50 = 535 pg/ml Nguyen nhan khien gia tri ED50 cua Tau FGF-1 thu Mau suet thu hal (%) dtrqc tir phan (loan 60% dung djch B cao hen meu thucrng mai co the de tinh sach ctia mau FGF-I phan doan 60% dung djch B dot 93,4% thap hen so voi met' FGF-1 thuang mai c6 de tinh sach > 97% Ket qua ding cho they !lieu FGF-1 thu duqc tir phan doan 20% dung dich B cho gia tri ED50 cao hen Ian matt thucrng mai Ket qua chUng minh rang mall FGF-I thu dugc tir phan clop 60% dung dich B (twang img voi 1,2M NaCI) c6 hog tinh tang sinh to bao va FGF-1 thu duac tir phan doan 20% dung dich B (twang img yeti 0,4M NaC1) kitting co hog tinh tang sinh to beo twang tu vai nghien cfru cua Moringlane va thing tic gia (Moringlane et al., 1990) FGF-1 thucrng mai _e_ FGF-1 pltht doan 60%B FGF- I phiut doan 20%B C -2 I Log([FGF- I]) (pg/ml) KET LUAN Chung toi de too throe chOng E coli BL21(DE3)/pET-fe bieu hien FGF-1 ngtrai tai to hqp khi, cam ang bang IPTG FGF-1 ton tai dtrai dong the yid kitting tan to bao chat dutyc tai gip cuqn bang phuang phap pha bang nhanh giitp thu nhan dtrgc FGF-1 a dung tan Till qua qua 620 trinh tinh sach bing sac kj, luc voi heparin FGF-1 co hoot tinh tang sinh to bao NIH 3T3 dtrac thu nhan a phan doan 1,2M NaCI co dO• tinh sach 93,4% Day la nghien ciru dau tien ye bieu hien ye thu nhan FG.F-1 c6 hoot tinh sinh hoc Viet Nam ye IA tien de cho cac nghien cuu quy trinh cong nghe sin xuat FGF-1 tai to hop sau cho nhiing ang dung y hoc ye my pham tai Viet Nam Top chi Ging nghe Sinh hQc 12(4): 615-621, 2014 Lirl cim Cong trinh dux thitc hien tit kink phi hang nam caa truang Doi hQc Khoa hoc Ty nhien, Doi hQc Quoc gia HCM cap ltng nam cho Be mon Cong nghe Sinh hoc Pita h - Mai truang TAI THAM KHAO Cuevas P, Reimers D, Carceller F, Martinez-Coso V, Redondo-Horcajo M, Saenz de Tejada 1, Gimenez-Gallego G (1997) Fibroblast growth factor-1 prevents myocardial apoptosis triggered by ischemia reperfusion injury Eur J Med Res 2(11): 465-468 Fan W, Yan W, Xu Z, Ni H (2012) Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique Colloids Surf B Biointerfaces 90: 21-27 Htun P, Ito WD, Hoefer IE, Schaper J, Schaper W (1998) Intramyocardial infusion of FGF-1 mimics ischemic preconditioning in pig myocardium J Mo! Cell Cardiol 30(4): 867-877 Langer R, Vacanti JP (1993) Tissue engineering Science 260(5110): 920-926 Moringlane J, Spinas R, Bohlen P (1990) Acidic fibroblast growth factor (aFGF) is present in the fluid of brain tumour pseudocysts Acta Neurochirurgica 107(3-4):88-92 Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Labo-atoty Press Schumacher B, Pecher P, von Specht BU, Stegmann T (1998) Induction of neoangiogenesis in ischemic myocardium by human growth factors: first clinical results of a new treatment of coronary heart disease Circulation 97(7): 645-650 Zakrzewska M, Marcinkowska E, Wiedlocha A (2008) FGF- I : from biology through engineering to potential medical applications Crit Rev Clin Lab Sci 45(1): 91-135 Zakrzewska M, Wiedlocha A, Szlachcic A, Krowarsch D, Otlewski J, Olsnes S (2009) Increased protein stability of FGF1 can compensate for its reduced affinity for heparin J Biol Chem 284(37): 25388-25403 CLONING, EXPRESSION AND PURIFICATION OF HUMAN FIBROBLAST GROWTH FACTOR (FGF-1) IN ESCHERICHIA COLI Dang Tat Truong, Trinh Minh Thuong, Tran Van Hien University of Science, Vietnam National University HoChiMinh City SUMMARY FGF-1 (Fibroblast growth factor-I) is a pluripotent growth factor which can possess broad mitogenic and cell survival activities, and is involved in a variety of biological processes, including embryonic development, cell growth, tissue repair, cellular regeneration, angiogenesis, and so on With such potentials, FGF-1 has now been applied to many fields such as cosmetics, treatment to cardiovascular diseases, wound healing In this study, we proceeded to overexpress recombinant protein FGF- I in E coli for low-price FGF-1 production towards applications in Vietnam Thefgfl gene of 423 by encoding for human FGF-1 was synthesized by PCR using plasmid pIDT-fgf/ as template and cloned into plasmid pET-His under the control of strong T7 promoter The gene in correction of recombinant plasmid, termed pET-fgfl, was confirmed by PCR, restriction enzyme digestion and DNA sequencing After that, plasmid pET-fgf/ was transformed into E coli BL21(DE3) to establish the recombinant E coli BL21(DE3)/pET-fg/7 When the recombinant E coli BL21(DE3)/pET-fgf/ was shakily cultured in LB medium containing ampicillin, subsequently added with IPTG to induce the T7 promoter, FGF- I was over-expressed and accumulated as inclusion body in cytoplasm with content accounted for 22 percent of total protein after 16 h induction using SDS-PAGE, Western blot, and Quantity-One softwave analyses Inclusion bodies harboring FGF-1 was solubilized by Guanidin 4M and was refolded using diluted method to isolate solubilized FGF-1 Heparin-affinity chromatography was conducted to purify FGF-1 Results indicated that the recombinant protein was eluted at NaCI 1.2M and NaCI 0.4M with the purity of 93.4% and 50.1%, respectively The former purity had in vitro NIH 3T3 stimulating capacity equal to that of commercial product, having EDso = 657 pg/ml Keywords: FGF-I inclusion bodies, heparin-affinity chromatography, bio-assay Author for correspondence: E-mail: tvhieuQhcmus.edu.vn 621 ... Ndel/BamHl kDi 97,0 66 ,11 -45,0 — 3 (1, 0 — 20 .1 1I 4IP • 14 ,4 — Hlnh Str bieu hian FGF -1 E coli BL. 21( DE3)/pET-fgf1 Dit)n di SDS-PAGE (A); lai Western yeti khang 014 dOC hiOu khang FGF -1 ngtrtri (B);... tic gicra FGF -1 Ira heparin lien ket ion gala polyanion tren heparin (nhorn sulfate) v6i Lys 113 , Arg 119 , Lys128, Asn18, Asn 114 va GIn127 tren be mAt cua phan tir FGF -1 (Fan et al., 2 012 , Zakrzewska... 20% dung djch B 0,65 50 ,10 0,32 5, 41 Phan doan 60% dung dich B 2,26 93,40 2 ,11 35.64 Dinh gia host tinh tang sinh in vitro ctia FGF -1 sau tinh the Do thj danh gia hog tinh tang sinh cua FGF-I