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1、第30卷第4期扬州大学学报(农业与生命科学版)Vol.30No.42009年12月JournalofYangzhouUniversity(AgriculturalandLifeScienceEdition)Dec.2009抗牛病毒性腹泻病毒单克隆抗体的研制3孙宏进,蒋颖,林燕清,朱军,姚丰华,刘振华,朱国强(扬州大学兽医学院,江苏扬州,225009)摘要:将含有牛病毒性腹泻病毒1型(BVDV1)囊膜糖蛋白E2编码基因的真核表达质粒pEC143免疫BALB/c小鼠,应用淋巴细胞杂交瘤技术,以重组E2蛋白纯化产物为检测抗原,经间接ELISA法筛选和3次有限稀释法克隆,得到1株能稳定分泌抗BVDV1
2、和BVDV2单克隆抗体的杂交瘤细胞株。该3D8株单抗能识别BVDV1标准毒株(NADL、OregonC24V)、BVDV2标准毒株(890、104)、2株BVDV1分离株和3株BVDV2分离株,而不能识别牛轮状病毒、牛疱97疹病毒1型,显示出良好的特异性。阳性杂交瘤细胞的上清液、腹水ELISA抗体效价分别为2和10×2,为IgM亚类。其单克隆抗体杂交瘤细胞株的获得,为建立单抗介导的检测BVDV1和BVDV2抗原和抗体的血清学方法和实现标准化诊断奠定基础。关键词:牛病毒性腹泻病毒;基因型;E2囊膜糖蛋白;基因免疫;单克隆抗体中图分类号:S852.65+3文献标识码:A文章编号:167124652
3、(2009)0420001205ThedevelopmentofahybridomacellstrainsecretingmonoclonalantibodyagainstBVDV1andBVDV2SUNHong2jin,JIANGYing,LINYan2qing,ZHUJun,YAOFeng2hua,LIUZhen2hua,ZHUGuo2qiang(CollofVetMed,YangzhouUniv,Yangzhou225009,China)ABSTRACT:TheglycoproteinE2frombovineviraldiarrheavirusgenotype1orBVDV2isanim
4、munodominantantigeninducingtheneutralizingantibodiesafternaturalinfectionorfollowingimmunizationwithliveorkilledvaccines.Inthisstudy,E2glycoproteinwasfocusedandtheBALB/cmicewereimmunizedwiththerecombinanteukaryoticplasmidpEC143encodingasecretedformofE2glycoproteinaccordingtotheimmuneprotocolformakin
5、gmonoclonalantibody.Usinglymphocytehybridomatechnique,themousespleencellswerefusedwithmyelomacells.AhybridomacellstrainwhichcansecretestablymonoclonalantibodyagainsttheE2glycoproteinoftheBVDV1andBVDV2,wasobtainedbyindirectELISAusingthecoatedantigenfromthepurifiedexpressionproductsofrecombinantprokar
6、yoticplasmidofpET28a2BE2andwasclonedthroughthreetimeslimitingdilution.Thehybridomacellstrainwasnamed3D8.Themono2clonalantibodysecretedby3D8strainreactedwithnotonlytheNADLandOregonC24VreferencestrainsandtwoisolatesofBVDV1,butalsothe890and104referencestrainsandthreeisolatesofBVDV2,butnotcrossreactionw
7、ithBRVandBHV21.ThehybridomacellstrainbelongstoIgMsubset.ELISAtiterofmonoclonalantibodywhichwasexistentinasci279tesofmiceandsupenatantsofcultureofhybridomacellswas10×2and2,respectively.Theaboveprelimin
