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

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1、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：温坤梅亚波丘立文I廖志勇袁国勇2；车小燕1C第一军医大学珠江医院屮心实验室，广东广州510282；彳香港大学微生物学系，香港）摘要：EI的在获得了具有免疫原性的SARS冠状病毒S1蛋门片段的基础上，制备和鞭定特异性抗该段S1蛋白单克隆抗体（mAb）0方法原核表达含S蛋门受体结合区的SARS冠状病毒S1蛋门片段Slc（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，按常规方法制备社克隆抗体，

2、并采JIJELISA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SAKS冠状病毒S1蛋门N端249-667的mAb杂交瘤细胞株，IgG亚类鉴定1株为IgGl,2株为IgG2a,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Preparationandcharactcrizationofmo

3、noclonalantibodiesagainstSIdomainatN-tcrminalresidues249to667ofSARS-associatodcoronavirusSIproteinWENKun1；METYa-bo1；QTULi-wen1；LIAOZhi-yong1；YuenKwok-yung2；CHEXiao-yan1"CentralLaboratory,ZhujiangHospital,FirstM订itaryMedicalUniversity,Guangzhou510282,China;"De

4、partmentofMicrobiology,UniversityofHongKong,HongKong,China.Abstract:ObjectiveToprepareandcharacterizemonoclonalantibodies(mAbs)againstSIproteinofsevereacuterespirato-rysyndrome(SARS)-associatedcoronavirus(SARS-CoV).Methods6-His-taggedrecombinantfmgmentatN-ter

5、minalresidues249to667ofSARS-CoVSIproteinincludingS-proteinreceptor-bindingdomainwasexpressedinE.coli.Theim-munogenicityofthisSIdomainwasidentifiedandusedtoimmunizeBALB/cmicefortheproductionofhybridomas.TheidentificationofthemAbsagainstthisSIdomainwasperformed

6、usingindirectenzyme-1inkedimmunosorbentassay(ELISA),indirectimmunofluorescenceassay(TFA)andWesternblotting,respectively.ResuItsThreehybridomasproducingmAbsspc-cifictotheSIdomainwasobtained,witharelativemolecularmassof48500.Noneofthe3mAbswerereactivewithhumanc

7、oronaviruses229Eand0C43.TwoofthemAbswerelgG2aisotype,andtheotherwasIgGl-ConclusionThisisthefirstre-portofmAbsproducedagainstS-proteinreceptor-bindingdomainofSARS-CoV.The3Sl-specificmAbsmaybeusefulforfurtherstudyofthefunctionoftheSproteinandfordiagnosisofSARS-

8、CoVinfection.Keywords:severeacuterespiratorysyndrome-associatedcoronavirus;SARS-CoV;monoclonalantibody;spikeglycoprotoin;receptorebindingdomainReceived:2004-01-03ThisworkissupportedastheK