抗SARS冠状病毒S1蛋白N端249至667的单克隆抗体的制.docx

抗SARS冠状病毒SI蛋白N端249至667的单克隆抗体的制抗SAKS冠状病毒S1.蛋白N端249至667的单克隆抗体的制备与鉴定作者：温坤车小燕东广州510282；1；梅亚波1（1；丘立文1：廖志勇1；袁国勇2：1第一军医高校珠江医院中心试验室，广2香港高校微生物学系，香港）摘要：目的在获得了具有免疫原性的SARS冠状病毒S1.蛋白片段的基础上，制备和鉴定特异性抗该段S1.蛋白单克隆抗体（mAb）.方法原核表达含S蛋白受体结合区的SARS冠状病毒S1.蛋白片段S1.c（N端249-667氨基酸残基），其免疫原性经SARS病人复原期血清鉴定后免疫BAI.B/C小鼠，按常规方法制备单克隆抗体，并采纳E1.1.SA间接法、免疫荧光和免疫印迹进行筛选和鉴定。结果筛选出3株特异性针对SARS冠状病毒S1.蛋白N端249-667的mAb杂交瘤细胞株，IgC亚类鉴定1株为IgG1.,2株为1.gG2a,经免疫荧光鉴定与人冠状病毒株229E和0C43无交叉反应。结论获得3株抗SAKS冠状病毒S蛋白受体结合区特异性单克隆抗体，为建立新的SARS冠状病毒检测方法的和进一步探讨S蛋白的功能奠定了基础。关键词：SARS冠状病毒；单克隆抗体：刺突蛋白：受体结合区Preparationandcharacterizationofmonoc1.ona1.antibodiesagainstS1.domainatN-termina1.residues249to667ofSRS-associatedcoi'onavirusS1.proteinWENKun1.：MEIYa-bo1.；Q1.U1.i-wcn1.；1.IAOZhi-yong1.；YuenKwok-yung2;CHEXiao-yan1.!Centra1.1.aboratory,ZhujiangHospita1.,FirstMi1.itaryMedica1.University,Guangzhou510282,China;2DepartmentofMicrobio1.ogy,UniversityofHongKong,HongKong,China.Abstract:ObjectiveToprepareandcharacterizemonoc1.ona1.antibodies(mbs)againstS1.proteinofsevereacuterespirato-rysyndrome(SARS)-associatedcoronavirus(SARS-CoV).Methods6-His-taggedrecombinantfragmentatN-termina1.residues249to667ofSARS-CoVS1.proteininc1.udingSproteinreceptor-bindingdomainwasexpressedinE.co1.i.Theim-munogenicityofthisS1.domainwasidentifiedandusedtoimmunizeBA1.B/cmicefortheproductionofhybridomas.TheidentificationofthemAbsagainstthisS1.domainwasperformedusingindirectenzyme-1inkedimmunosorbentassay(E1.ISA),indirectimmunof1.uorescenceassay(IF)andWesternb1.otting,respective1.y.Resu1.tsThreehybridomasproducingmAbsspe-cifictotheS1.domainwasobtained,withare1.ativemo1.ecu1.armassof48500.Noneofthe3mAbswerereactivewithhumancoronaviruses229Eand0C43.TwooftheInAbSwereIgG2aisotype,andtheotherwasIgG1.Conc1.usionThisisthefirstre-portofnbsproducedagainstSproteinreceptor-bindingdomainofSARS-CoV.The3S1.-spccificmAbsmaybeusefu1.forfurtherstudyofthefunctionoftheSproteinandfordiagnosisofSARS-CoVinfection.Keywords:severeacuterespiratorysyndrome-associatedCOrOnaVirus;SARS-CoV;monoc1.ona1.antibody：spikeg1.ycoprotein;receptor-bindingdomainReceived:2004-01-03ThisworkissupportedastheKeyMedica1.ResearchProjectforSARSPreventionsponsoredbytheMinistryofScienceandTechno1.ogyofChinaandbyGuangdongProvinceCorrespondingauthor:CHEXiao-yan,M.D.,ProfessorintheCentra1.1.aboratoryofZhujiangHospita1.,Te1.:86-20-61643592,Fax:86-20-61643592,E-mai1.:IinChe©,chexiaoyanThetwoauthors,WENKunandMEIYa-bo,havemadeequa1.contribu-tionstothiswork.nove1.coronavirushasbeenidentifiedasthema-jorcauseofseveracuterespiratorysyndrome(SARS)1.23,butthebio1.ogica1.functionsofthisSARS-associatedcoronavirus(SARS-CoV)remaincurrent1.ypoor1.ychar-acterized.Recentstudiesreportedthatangiotensin-con-vertingenzyme2(AEC2)cou1.dbeafunctiona1.receptorforSARS-CoVandthereceptor-bindingdomain(RBD)wasidentifiedintheN-termina1.residues17to274oftheS1.spikeproteinofthevirus4.SubsequentstudiesdemonstratedthattheRBDwas1.ocatedintheN-tcrmi-na1.residues303to5375orresidues318to5106.TheS1.proteinisa1.sofoundtobeatargetforinducingneu-tra1.izingantibodyresponsestoSARS-CoVinfection7.InspiteofthefactthatthefunctionofSproteinisnotfu1.1.yunderstood,S1.proteinseemstop1.ayakeyro1.eintheinitia1.virusinfection.Basedonthisunderstanding,themonoc1.ona1.antiodies(mbs)targetedatS1.domaincanbeapotentia1.1.yusefu1.too1.forthestudyofthefunc-tionofSproteinandforthediagnosisofSARS.Fur-thermore,theavai1.abi1.ityofb1.ockingantibodiesmaybemeaningfu1.inthetreatmentofSARS.Inthispaper,wereportthec1.oning,expressionandantigeniccharacterizationofthevariousfragmentsofS1.domainaswe1.1.asthepreparationandcharacter!zationofmAbsagainsttherecombinantproteinS1.cde-rivedfromtheS1.proteincontainingtheN-termina1.residues249to667.MATERIA1.SANDMETHODSVirusandce1.11.inesHumancoronavirusstrains229E(No.VR740),0C43(No.VR759)andce1.1.1.inesVeroE6(No.CR1.-1586),MRC-5(No.CC1.-171),BS-C-I(No.CC1.-26)werea1.1.purchasedfromAmericanTypeCu1.tureCo1.1.ection(ATCC).P1.asmidsThep1.asmidencodingS1.proteingene,pET_28b(+)/SI(402OO1.bp,HKU-39849),waskind1.yprovid-edbytheDepartmentofMicrobio1.ogy,UniversityofHongKong.TheexpressionvcctorPQE-30andM1.5strainofEscherichiaco1.iwerepurchasedfromQ1.A-GEN.ReagentsTherestrictionendonuc1.easesBamHIandHindIIIwerepurchasedfromNewEng1.andBio1.abs,andKpn1.,PstI,ExTaq,dNTPandDN1.adderwerefromTaKaRa.NiresinwasobtainedfromQIAGEN.Freund,Sadju-vant,50%PEG1450so1.ution,HTandHTwerepur-chasedfromSigmaA1.drich.Goatanti-mouseIgM,IgG,IgG1.,IgG2a,IgG2bandIgG3peroxidaseconjugates,goatanti-humanIgGperoxidaseconjugates,goatan-ti-mouseIgGFITCconjugateandaminoethy1.carbazo1.e(EC)sing1.eso1.utionweretheproductsofZYMED.RPMI1640cu1.turemediurnandfeta1.bovinoserumwerefromGibco-BR1.1.1.thechemica1.reagentsusedinthisstudywereofana1.ytica1.grade,andpreparedwithion-dep1.etedwater.Seraandanima1.sSerumsamp1.esofSARSpatientsforthisstudyweresero1.ogica1.1.yconfirmedbyindirectimmunof1.uo-rescenceandenzyme-1inkedimmunosorbentassay(E1.ISA)8,andtheserafromhea1.t