抗SARS冠状病毒S1蛋白N端249至667的单克隆抗体的制备与

抗SARS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1；梅亚波1；丘立文1；廖志勇1；袁国勇2；车小燕1(1第一军医大学珠江医院中心实验室，广东广州510282；2香港大学微生物学系，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S1c(N端249-667氨基酸残基)，其免疫原性经SARS病人恢复期血清鉴定后免疫BALB/c小鼠，按常规方法制备单克隆抗体，并采用ELISA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，IgG亚类鉴定1株为IgG1，2株为IgG2a，经免疫荧光鉴定与人冠状病毒株229E和OC43无交叉反应。结论获得3株抗SARS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研究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PreparationandcharacterizationofmonoclonalantibodiesagainstS1domainatN-terminalresidues249to667ofSARS-associatedcoronavirusS1proteinWENKun1；MEIYa-bo1；QIULi-wen1；LIAOZhi-yong1；YuenKwok-yung2；CHEXiao-yan11CentralLaboratory,ZhujiangHospital,FirstMilitaryMedicalUniversity,Guangzhou510282,China;2DepartmentofMicrobiology,UniversityofHongKong,HongKong,China.Abstract:ObjectiveToprepareandcharacterizemonoclonalantibodies(mAbs)againstS1proteinofsevereacuterespirato-rysyndrome(SARS)-associatedcoronavirus(SARS-CoV).Methods6-His-taggedrecombinantfragmentatN-terminalresidues249to667ofSARS-CoVS1proteinincludingS-proteinreceptor-bindingdomainwasexpressedinE.coli.Theim-munogenicityofthisS1domainwasidentifiedandusedtoimmunizeBALB/cmicefortheproductionofhybridomas.TheidentificationofthemAbsagainstthisS1domainwasperformedusingindirectenzyme-linkedimmunosorbentassay(ELISA),indirectimmunofluorescenceassay(IFA)andWesternblotting,respectively.ResultsThreehybridomasproducingmAbsspe-cifictotheS1domainwasobtained,witharelativemolecularmassof48500.Noneofthe3mAbswerereactivewithhumancoronaviruses229EandOC43.TwoofthemAbswereIgG2aisotype,andtheotherwasIgG1.ConclusionThisisthefirstre-portofmAbsproducedagainstS-proteinreceptor-bindingdomainofSARS-CoV.The3S1-specificmAbsmaybeusefulforfurtherstudyofthefunctionoftheSproteinandfordiagnosisofSARS-CoVinfection.Keywords:severeacuterespiratorysyndrome-associatedcoronavirus;SARS-CoV;monoclonalantibody;spikeglycoprotein;receptor-bindingdomainReceived:2004-01-03ThisworkissupportedastheKeyMedicalResearchProjectforSARSPreventionsponsoredbytheMinistryofScienceandTechnologyofChinaandbyGuangdongProvinceCorrespondingauthor:CHEXiao-yan,M.D.,ProfessorintheCentralLaboratoryofZhujiangHospital，Tel:[phone],Fax:[phone],E-mail:[e-mail],[e-mail],WENKunandMEIYa-bo,havemadeequalcontribu-tionstothiswork.Anovelcoronavirushasbeenidentifiedasthema-jorcauseofseveracuterespiratorysyndrome(SARS)[1][2][3],butthebiologicalfunctionsofthisSARS-associatedcoronavirus(SARS-CoV)remaincurrentlypoorlychar-acterized.Recentstudiesreportedthatangiotensin-con-vertingenzyme2(AEC2)couldbeafunctionalreceptorforSARS-CoVandthereceptor-bindingdomain(RBD)wasidentifiedintheN-terminalresidues17to274oftheS1spikeproteinofthevirus[4].SubsequentstudiesdemonstratedthattheRBDwaslocatedintheN-termi-nalresidues303to537[5]orresidues318to510[6].TheS1proteinisalsofoundtobeatargetforinducingneu-tralizingantibodyresponsestoSARS-CoVinfection[7].InspiteofthefactthatthefunctionofSproteinisnotfullyunderstood,S1proteinseemstoplayakeyroleintheinitialvirusinfection.Basedonthisunderstanding,themonoclonalantiodies(mAbs)targetedatS1domaincanbeapotentiallyusefultoolforthestudyofthefunc-tionofSproteinandforthediagnosisofSARS.Fur-thermore,theavailabilityofblockingantibodiesmaybemeaningfulinthetreatmentofSARS.Inthispaper,wereportthecloning,expressionandantigeniccharacterizationofthevariousfragmentsofS1domainaswellasthepreparationandcharacteriza-tionofmAbsagainsttherecombinantproteinS1cde-rivedfromtheS1proteincontainingtheN-terminalresidues249to667.MATERIALSANDMETHODSVirusandcelllinesHumancoronavirusstrains229E(No.VR740),OC43(No.VR759)andcelllinesVeroE6(No.CRL-1586),MRC-5(No.CCL-171),BS-C-1(No.CCL-26)wereallpurchasedfromAmericanTypeCultureCollection(ATCC).PlasmidsTheplasmidencodingS1proteingene,pET-28b(+)/S1(40-2001bp,HKU-39849),waskindlyprovid-edbytheDepartmentofMicrobiology,UniversityofHongKong.TheexpressionvectorpQE-30andM15strainofEscherichiacoliwerepurchasedfromQIA-GEN.ReagentsTherestrictionendonucleasesBamHIandHindIIIwerepurchasedfromNewEnglandBiolabs,andKpnI,PstI,ExTaq,dNTPandDNAladderwerefromTaKaRa.NiresinwasobtainedfromQIAGEN.Freund'sadju-vant,50%PEG1450solution,HATandHTwerepur-chasedfromSigmaAldrich.Goatanti-mouseIgM,IgG,IgG1,IgG2a,IgG2bandIgG3peroxidaseconjugates,goatanti-humanIgGperoxidaseconjugates,goatan-ti-mouseIgGFITCconjugateandaminoethylcarbazole(AEC)singlesolutionweretheproductsofZYMED.RPMI1640culturemediumandfetalbovineserumwerefromGibco-BRL.Allthechemicalreagentsusedinthisstudywereofanalyticalgrade,andpreparedwithion-depletedwater.SeraandanimalsSerumsamplesofSARSpatientsforthisstudywereserologicallyconfirmedbyindirectimmunofluo-rescenceandenzyme-linkedimmunosorbentassay(ELISA)[8],andtheserafromhealthydonorswereusedascontrolforthesameassay.Five6-week-oldfemaleBALB/cmicewereprovidedbytheExperimentalAni-malCenterofFirstMilitaryMedicalUniversity.CloningS1fragmentsintotheexpressionvectoranditsexpressionS1geneencodingtheaminoa