抗P185工程抗体表达与纯化研究

ChinaBiotechnology,2006,26(9):32~37P185*乔婧娟刘海波朱娟娟程联胜刘兢**(230027)人类的原癌基因Her2编码一个185kDa的跨膜受体酪氨酸激酶,也称为p185,是肿瘤相关蛋白,在多种癌细胞,特别在乳腺癌中有高表达构建了抗p185/HER2的二价的嵌合抗体(scFvFc),并证明其保留了亲本抗体的特异结合活性,进而建立了高表达的细胞株对该嵌合抗体进行滚瓶大规模培养,并对培养产物的纯化条件进行了摸索,建立了亲和层析和阳离子交换层析两步法最终产物的纯度在95%以上,回收率为63%摸索了嵌合抗体的冻干保存条件,抗体可以保持其稳定性和结合活性一年以上嵌合抗体纯化工艺和保存条件的确定,为进一步的临床研究奠定了基础嵌合抗体亲和纯化离子交换纯化稳定性Q819:20060427:20060623*863(2004AAZ15260),(30570362)**,:[e-mail]185erbB2,,,,P185[1~4]P185p185,,HerceptinFDAP185,P185,[5~8]P185scFv,scFvIgG1Fc,scFvFcscFv,Fc[9],,[10,11],scFvFcCHO,,,30mg/L[12],,,[13],,,,11.1P185CHO,(GreinerBioone,),3L800ml:DMEM(GIBCO)5%(v/v)(GIBCO),60mg/ml,35mg/ml1%37!CO2,7d,7d,24h,2006,26(9):P1851.220L0.22mA(0.05mol/L,4.0mol/L,pH9.0)25mlProteinA(rProteinASepharoseFF,Amersham),AProteinA,B(0.05mol/L,0.05mol/L,0.3mol/L,pH3.0)A1∀2.51∀1,UV280,1ml;10%(1.0mol/LTrisHC,lpH8.8),pH5,4!;,SDSPAGE,,ELISALowry1.3C(20mmol/L,pH5.0),0.22mC50mlSP(SPSepharoseFF,Amersham),C,D(20mmol/L,0.4mol/L,pH5.0)C1∀41∀1,,SDSPAGE,,ELISAProteinA,1.51.4HPLC4~5mg/m,l200l1mlC4(Waters,USA)(0.1%),1.5ELISAFc(Fizegiald)(F96,Nunc),HRPFcHER2T617(P185),,HER23T3(P185),HeceptinHer20.80.40.20.1mg/L,50.045.12#10-78,1.5,OD490,Kaff(M1)=1/(2[Ab∃]t[Ab]t)[14],[Ab∃]t[Ab]tODProteinA,,HRPProteinA(Sigma)ProteinABSA1.6Lowry,ProteinAssayKit(Sigma)(TCA)BSA,Lowry,Folin,750nm1.7MTTHer2T6175#10496,100,l5#1021#1025#1031#1030.5#104,,Her2Heceptin9637!,5%CO272h,MTT(CellCountingKit8,Dojindo)10l/,2h,OD450,(ODmaxODmin)/ODmax[15]1.8,:60mmol/L,100mmol/L,0.01%,pH5.5,1.0mg/ml2ml,,,4!,SDS-PAGE,ELISA22.1,CHOELISA,,(1),,7d,,5d33ChinaBiotechnologyVo.l26No.9200610%,30mg/L1CHOFig.1TheeffectofsodiumbutyrateontheexpressionofchimericantibodyinrecombinantCHOcells2.2,2,ELISA(3),a,b,,,2ProteinAFig.2AffinitychromatogramofrProteinASepharoseFF(AmershamBiosciences)column2.3ProteinA,,ProteinA,SP(b)4,:cd,ELISA(5)dProteinAELISA(6),,ProteinA3ELISAProteinAFig.3ELISAassayofbindingactivityofdifferentelutedcollectiontubesofProteinAcolumn4SPFig.4CationexchangechromatogramofSPSepharoseFF(AmershamBiosciences)column5ELISASPFig.5ELISAassayofbindingactivityofdifferentelutedcollectiontubesofSPcolumn2.4SDS-PAGE(7)HPLC(342006,26(9):P1856ELISAProteinAI:ProteinAb;II:SPdFig.6ELISAassayofthecontentoftheProteinArelic8)(Scion),,90%,,95%ELISALowry,7SDSPAGE1:ProteinA;2:SP;3:ProteinA;4:SPFig.7CoomassiebulestainedSDSPAGEresultsofpurifiedchimericantibody2.519S,3:9.0#107,8.9#1077.9#107M1,8.6#107M1Kaff,KD11.6nM2.610MTTHer2,(10g/ml),chA2132%,Heceptin46%8C4HPLC(a)(b)Fig.8EluatecurveofC4reversecolumninHPLCsystem9ELISA0.045.12#1078Fig.9ELISAresultsofchA21bindingtodifferentconcentrationsofcoatedHer2antigen2.7SDSPAGE(11),;ELISA(12),,,3,P185scFv-Fc,105kDa,35ChinaBiotechnologyVo.l26No.9200610MTTFig.10MTTresultsofchA21restrainningtheT617cancercellsexpressingHer2onmembrane11SDSPAGE1~12:1~12Fig.11CoomassiebluestainedSDSPAGEresultsofchimericantibodyinstorage12ELISA2~12Fig.12ELISAassayofbindingactivitiesoftheantibodieshavingbeenstoredfor2~12months,,,,Fc,CHO,60L,26mg/L,1gProteinAFc,ProteinA[16],90%,70%,ProteinA,ProteinA,,ProteinA,,SP,8.1,ProteinA,SPpH5.0,90%,63%,95%,ProteinA,,,,8.6#107M1,Heceptin5.8#108M1();32%,Herceptin46%Herceptin,,,,,,[1]DrebinJA,LinkVC,SternDF.Inhibitionoftumorgrowthbyamonoclonalantibodyreactivewithanoncogeneencodedtumor362006,26(9):P185antigen.ProcNatlAcadSc,i1986,96:9129~9133[2]SlamonDJ,GodolphinW,JonesLA,eta.lStudiesoftheHer2/neuprotooncogeneinhumanbreastandovariancancers.Science,1989,244:707~712[3]DohertyJK,BondC,JardimA,eta.lTheHer2/neureceptortyrosinekinasegeneencodesasecretedautoinhibitor.ProcNatlAcadSc,i1999,96(19):10869~10874[4]ParkJW,HongK,CarterP,eta.lDevelopmentofantip185HER2immunoliposomesforcancertherapy.ProcNatlAcadSc,i1995,92(5):1327~1331[5]DavidWM.UpdateonHer2asatargetforcancertherapyHerceptinintheclinicalsetting.BreastCancerRes,2001,3:380~384[6]SchiffR,ChamnessGC,BrownPH.Advancesinbreastcancertreatmentandprevention:preclinicalstudiesonaromataseinhibitorsandnewselectiveestrogenreceptormodulators.BreastCancerResearch,2003,5:228~231[7]LiP,LiY,LiJ,eta.lCharacterizationandutilizationoftwonovelantierbB2monoclonalantibodiesindetectionofsolubleErbB2forbreastcancerprognosis.CancerLetters,2003,193:139~148[8]AgusDB,AkitaRW,FoxWD,eta.lTargetingligandactivatedErbB2signalinginhibitsbreastandprostatetumorgrowth.CancerCel,l2002,2:127~137[9]ChengL,LiuA,YangJ,eta.lConstruction,expressionandcharacterizationoftheengineeredantibodyagainsttumorsurfaceantigen,p185cerbB2.CellResearch,2003,13:35~48[10]DornerAJ,WasleyLC,KaufmanJ.IncreasedsynthesisofsecretedproteinsinducesexpressionofglucoseregulatedproteinsinbutyratetreatedChinesehamsterovarycells.JBioChem,1989,264(34):20602~20607[11]MalmB.Amethodsuitablefortheisolationofmonoclonalantibodyfromlargevolumesofserumcontaininghybridomacellculturesupermatants.JImmunolMethods,1987,104:103~109[12],,,.CHO.,2005,25(10):12~16MaJ,DongYQ,ZouM,eta.lChinaBiotechnology,2005,25(10):12~16[13]WuA,TanG,ShermanM,eta.lMultimerizationofachimericantiCD20singlechainFvFcfusionproteinismediatedthroughvariabledomainexchange.ProteinEngineering,