重组人碱性成纤维细胞生长因子工

Q55610183097120152000.5.302000.6..9.StudyonhighdensityfermentationandpurificationprocedurefortheE.colihavingrecombinanthumanbasicfibroblastgrowthfactor32cellgrowthfactorhighdensityfermentationpurificationhumanbasicfibroblastgrowthfactor,hbFGFpUChbFGFbFGF160mg/LOD60040.8bFGF2.6bFGF55ED500.45ng/ml2.2106UmgDNADNAbFGFDNAAbstractHumanbasicfibroblastgrowthfactor(hbFGF)isanimportanceoneincell.Itcannotonlypromotethegrowthofvascularendothelial,nervouscells,cornealepithelialandchondrocytes,butalsopossessgreatpotentialofclinicalapplication.Inthisexperiment,theE.colihavingrecombinantplasmidpUC-hbFGFwereexpressedfusionbFGFproteinbyhighdensityfermentation.It’sexpressionlevelofbFGFandopticaldensity(OD)were160mg/Land40.8respectively.Theexpressedproductswereseparatedfromcytoplasmbyultrasonicationandpurifiedbyionexchangeandaffinitychromatography.Theresultsshowedthatproteinrecoverywere2.6%intotalproteinsofE.coliand55%inbFGFproteins,respectively.Fromdetectingresult,the50%effectivedose(ED50)offinalproductwere0.45ng/ml,thespecificactivityreached2.2106u/mg.Othercharacterizations,suchasthepurity,molecularweightandresidualDNAetc.alsocameuptoStandardfortheControlofRecombinantDNAproducts.Thepreparationofbio-activebFGFprovideafoundationforbothscaleproductionandclinicalapplication.Keywords:hbFGFHighDensityFermentationFusionproteinRecombinantDNAProducts.125712212525262930bFGFbFGFDNA30Trowell70Gospodarowicz,fibroblast,growthfactor,FGFFGF,SepharoseFGFGospodarowicaCM-SephadexC-50Sepharose90FGF[1]FGFFGF(acidicFGF,aFGF)FGFbasicFGF,bFGF;aFGF57bFGF9.6FGFcDNA15418KD55[2]bFGFN[3]FGFN108154CN[45]bFGFcDNAbFGF[6]bFGF21992Zhux[67]bFGF34AspGlyArgbFGF4[811]bFGFbFGFbFGF[1217]bFGFmRNA[1824]bFGFbFGF[25]2~4[26][27]bFGFFGF[28]bFGF1~2[29]bFGFFGF[30]bFGF[3135]bFGFbFGFbFGF[3640]bFGFbFGFbFGFbFGFbFGFbFGF200bFGFbFGF1E.coliJM103PUCbFGFLacZbFGFcDNAIPIGAmp2BALBC3T33bFGFBioRadBioRad4DMEMGIBCO5Bio-Rex70BioRadHeparinHyperDBioRad6BoehringerMammbeim7bFGFPromegaIgG-HRP8IPTGACRBISTFAMTTPBSNC1NBSG252NBS40L3BeckmanJ2-MC4SLS705ISCOUA66302SRL7BioRadMini8BioRadGS7009CO2REVCDRCD300010BioRadModel45011HPLCVarisCDS40112UV26513PIBioRadModelbFGF111.01.00.50.4NaC10.25K2HPO40.1KH2PO4pH7.2~7.42425ml0.25mlAmp(10mg/ml)25l37200634500ml5mlAmp(10mg/ml)25ml372008211.61.60.50.4NaC10.25K2HPO40.1KH2PO4pH7.2~7.4240L20LAmp100ug/ml1037PH7.0~7.5IPTG50ug/ml12400rpm34500rpm58400rpmpH20gIPTG350ug/ml13400rpm46500rpm78600rpm910500rpmPHPHIPTG61OD600bFGF(ELISA)pHDO23410000rpm/min4bFGF80.1MNaC1,10mMEDTA20mMPB415000rpm/min30minbFGF51XK5030Bio-Rex700.1MNaC1,20mMPB(pH7.0)0.3MNaC1,20mMPB(pH7.0)0.6MNaC1,20mMPB(pH7.0)20ml/min2XK5030HeparinHyperD0.6MNaC1,20mMPB(pH7.0)1.2MNaC1,20mMPB(pH7.0)2.0MNaC1,20mMPB(pH7.0)15ml/minbFGFbFGF1lowry[41]2MTTBALBC3T3ED50ED501BALB/C3T35000100ul10DMEM375CO22420.4DMEM243bFGF3645mg/mlMTT50ul/450.04N100ul/RT15’OD57031SDSPAGE[42]154.5[43]2HPLCHPLCC180.1TFAv/v0.1TFA0.1TFA100l0.2mg/ml,1.0ml/min,220nm;60060v/v)4SDSPAGE5190320nm6DNADNA[44]DNA7CNBr[45.46]phastSystem(pharmacia)10.5ml48hrs237010mg/ml0.1ml424hrs45dH2O20ul10ulSDS-PAGE620ul10ul7565min820SDS-PAGE1ulphastSystem9phastSystem8B1O-RADMODEDL111MINIIEFCELL91SDS-PAGE2NC30V15hrs33BSA24hrsbFGFMcAb3730min4IgG-HRP,3730min50.05DAB0.01H2O225mTris-0.14MNaC1PH7.610minbFGFOD6000.64,OD6006.8,1pHOD60025bFGFOD600bFGF6IPTG3OD60013.6bFGF133OD600bFGF5.07.420L680gSDSPAGE11920KDbFGF1h6101010(g/L)01089pH6.5-7.56.5-7.57.0-7.27.0-7.2OD6006.825.735.940.8bFGF(mg/L)19.028.068.6160.0(mg/L.OD600)2.81.11.93.91SDS-PAGE12bFGF50g2130mgbFGF5g2.6160mg/LbFGF50gbFGF235mgbFGF55234~62bFGF(ml)(mg/ml)bFGF(%)bFGF(mg)(%)ED50(ng/ml)(u/mg)(u)bFGF4286.018.64748.01.31053.2108bFGF2761.570.9289611.367.351053.0108bFGF1321.098.3130450.452.21062.91082bFGF3bFGF4bFGF5bFGF6bFGFbFGF1798.36HPLC988713bFGF48HPLC(bFGF)29SDSPAGElgRfbFGF19016D9124bFGF310214nm277nm10190~320nm(bFGF)4DNA11DNA500pg200pg100pg50pg20pgbFGF100ulDNA20pg100ul11DNA(bFGF)512CNBr121524bFGF613PI10.05,9.30,8.65,8.15,7.35,bFGFPI9.61312bFGF714141bFGF2bFGF3hbFGFbFGFbFGF(mass)pHbFGFbFGFlac[47]pHbFGFpHbFGFbFGFBio-Rex705.0cm20ml/minPH7.0bFGFPH7.0bFGFHeparinHyperDHeparinHyperD1,000cm/hr0.1MNaOH,,.bFGFNbFGF108154CNN40[48]bFGFNbFGFMW19KDED500.45ng/ml2.2106u/mgbFGFcDNA3CNBr383654bFGFCNBr,bFGF3000bFGF5[49]bFGFbFGFbFGF[50]bFGFbFGF1bFGF2bFGF3DNA4bFGF[1]AbrahamJa,etal.Recentdevelopmentsinthecellbioloqyotbasictibroblastgrowthtactor.JCellDiol.1989Jul;109(1):16[2]AbrahamJA,etal.Humanbasictibroblastgrowthtactor:nucleotidesequenceandgenomicorganization.EMBOJ.1986Oct;5(10):25238[3]SommerA,etal.Atormofhumanbasictibroblastgrowthtactorwithanextendedaminoterminus.BiochemBiophysResCommun.1987Apr29;144(2):54350[4]FlorkiewicaRZetal.Recentdevelopmentsinthecellbiologyotbasictibroblastgrowthtactor.JCellBiol.1989Jul;109(1):16[5]UenoNetal.Isolationotanaminoterminalextendedtormotbasictibroblastgrowthtactor.BiochemBiophysResCommun.1986Jul31;138(2):5808[6]ZhangJD,etal.Three-dimensionalstructureothumanbasictibroblastgrowthtactor,astructuralhomologofinterlenkin1beta[publishederratumappearsinprocNat1AcadSciU.S.A1991mn15;88(12):5477]PROC-Nal-Acad-Sci-U-S-A.1991Apr15