生物工程学报

206200411ChineseJournalofBiotechnologyVol.20No.6November2004:2004204227,:2004208202:(No.2003J12C0171)(No.40376030)3Tel:[phone];Fax:[phone];E2mail:[e-mail]113221(,510632)2(458,510601)HBsAgFabHBVHBsAgFab,FabScFv3HBsAgFab,FabFab,9618%,,30%40%ScFvFab,9715%,75%85%Fab,97%,75%85%,ScFvHBsAgFab,,FabHBsAg,Fab,,Q78A100023061(2004)0620884206,,,,[1]Fab,[2-4],,[5]HBsAgFabHBV[6],[7,8]HBsAgFab[9],(Pichiapastoris)[10-12]Fab[10],HBsAgFab,,,HBsAgFab1111HBsAgFabGS115PFab,ScFv14F7,5mLHiTrapTMNHSactivatitedSepharoseHP5mLHiTrapTMDEAE2sepharoseFastFlow5mLHiTrapTMCM2sepharoseFastFlow30mLAmershamPharmaciaDEAE2sepharoseCM2sepharoseAmershamPharmacia,AKTA(FPLC)Pharmacia11211211HBsAgFab:YPDHBsAgFabGS115PFab10mLBMGY,30OD60010,200mLBMGY,30OD60010©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.10%5L,Fed2batch96h6000rPmin25min,,40%(NH4)2SO4011molPLTrisHCl(pH810),424h11212FabFab:Fab(Sigma)(012molPLNaHCO3,015molPLNaCl,pH813)(1mgPmL),5mLHiTrapTMNHSactivatitedSepharoseHP,310mL1mmolPLHCl,015mLPmin,510mLFab,2530minA(015molPL,015molPLNaCl,pH813)B(015molPL,015molPLNaCl,pH410),pH,4FabA(011molPLNaAc,015molPLNaCl,pH415)B(011molPLTris2HCl,015molPLNaCl,pH810)25mL,2mLPmin011molPLTrisHCl,pH810(3040mL)Fab,HBsAgFab20mL,1mLPmin011molPLTrisHCl,pH810(3040mL),0101molPLTrisHCl,pH810(2030mL),2mLPmin011molPLGly2HCl115mLPmin,SDS2PAGEWestern2Blot11213ScFvFab:ScFv14F7BalbPC,[13]FabScFv:FabFabELISA,4,ScFv,371h,IgG,371hOPD,A450P630ScFv(1mgPmL),5mLHiTrapTMNHSactivatitedSepharoseHPFabAB25mL,2mLPmin011molPL(011molPLNa2HPO4,011molPLNaH2PO4,pH810)ScFv,2030mL,1mLPmin011molPLpH810(3040mL),0101molPLpH810(2030mL),2mLPmin011molPLpH215Gly2HCl115mLPmin,SDS2PAGEWestern2blot11214Fab:011molPL(011molPLNa2HPO4,011molPLNaH2PO4,pH615)5mLHiTrapTMDEAE2sepharoseFastFlow,0102molPL2mLPmin20mL115mLPmin1molPLNaCl115mLPmin011molPL5mLHiTrapTMCM2sepharoseFastFlow,0102molPL2mLPminDEAE115mLPmin1molPLNaCl120mL,115mLPmin,SDS2PAGEDEAE2SepharoseCL26BCM2SepharoseCL26B,,,1molPLNaOH1molPLHCl30min(),()0101molPL30mL(2cm20cm)Fab5mL11215Fab:HBsAgFab[20]HBsAgFab[12]HBsAgFabIn2HakChoi[14]ELISAHBsAg(100ngPmL),4,2%BSA,,HBsAgFab,371h,,5886:HBsAgFab©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.Fab,1h,,,A540P6302211FabFabHBsAgFab,Fab,SDS2PAGE1Fab,Fab,,Fab9618%FabFab,,30%40%1FabFabSDS2PAGEFig.1SDS2PAGEanalysisofpurifiedFabfragmentbyFabaffinitychromatographycolumn212ScFvFabHBsAgScFvHBsAgFabELISA1,FabScFv,1FabScFvELISATable1AffinityanalysisofrecombinantFabantibodyandScFvmAbbyELISAConcentrationofScFvMab110001100001500001100000PurifiedFab4101931150219232126131232215382140721597Fermentationsupernatnat3123221834119921198731032210491186811506Negativecontrol01032ScFvHBsAgFabSDS2PAGE2,Fab,,ScFvFab9715%,70%85%2ScFvFabSDS2PAGEFig.2SDS2PAGEanalysisofpurifiedFabfragmentbyScFvMabaffinitychromatographycolumn213Fab,FabHBsAgFab715398,81105pH5mLHiTrapTMDEAE2sepharoseFastFlowHiTrapTMCM2sepharoseFastFlow,FabSDS2PAGE3,,,,NaClFab,30mLSDS2PAGE4,,Fab97%75%85%214FabWestern2blot5,,68820©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.35mLFabSDS2PAGEFig.3SDS2PAGEanalysisofpurifiedFabfragmentby5mLionexchangechromatographycolumns430mLFabSDS2PAGEFig.4SDS2PAGEanalysisofpurifiedFabfragmentusing30mLionexchangecolumnHBsAgFabMALDI2TOF2MS50678149D,HBsAgFab(47914165D),2763184Fab(15),FabN2(Asn2X2SerPThr)O2Fab26,Fab,Fab,Fab(pPICZa2AGS115,HBIGHBsAg5FabWestern2blotFig.5Western2BlotanalysisofpurifiedFabfragment2FabELISATable2ActivityanalysisofpurifiedrecombinantFabantibodyBlankcontrolPurifiedrecombinantFabantibodyFabcolumnScFvMabcolumnIon-exchange3columnFermentsupernatantPositivecontrol(HBIG)NegativecontrolAbsorbence010002117441184211757115162114101040A450P630010004118901186711874116752105701034Ratiotonegativecontrol3613436199361313119141198(Theratiowhichis211timeslargerthenthenegativecontrolwastreatedaspositive.Thevalueofnegativecontrolwhichislowerthen0105wastreatedas0105)6FabELISAFig.6CompareofpurifiedrecombinantFabantibodybyELISAanalysisA:negativecontrol;B:Fabcolumn;C:ScFvMabcolumn;D:ion-exchangecolumn;E:fermentsupernatant;F:positivecontrol3Fab,FabFab[15-17]3FabFabFab7886:HBsAgFab©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,Fab,Fab9618%,Fab,,,Fab,,FabFab30%40%,,HBsAgFabScFv,ScFvScFv,Fab,ScFvHBsAgFab,HBsAgFabLinker,,[18,19]ScFv,,ScFv,HBsAgFab,,FabHBsAgFab,,,HBsAgFabREFERENCES()[1]KttnerG,KramerA,SchmidtkeGetal.Characterizationofneutralizinganti2pre2S1andanti2pre2S2(HBV)monoclonalantibodiesandtheirfragments.MolecularImmunology,1999,36(10):669-683[2]LevyR,MillerRA,Tumortherapywithmonoclonalantibodies.FedProc,1983,42(9):2650-2656[3]TamMR,GoldsteinLC.InManualofClinicalMicrobiology,(LennetteEH,BalowsA,HauslerWJetaleds)4thED.pp905-909Am.SocMicrobiol.WashingtonDC,1985[4]JohnstoneA,ThorpeR,ImmunochemistryinPractice(Blachwell,OXford),1982,pp.905-909[5]SowyerLA.Antibodiesforthepreventionandtreatmentofviraildisease.AntiviralReasearch,2000,47:57-77[6]WeYM,XuXH,HuDCetal.HepatitisBvaccineandanti2HBscomplexasapproachforvaccinetherapy.Cancer,1995,16(3):155[7]DitzelHJ,BinleyJM,Moor