抗H5N1亚型高致病性禽流感猴源噬菌体scFv的构建和表达.pdf

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1、第32卷第4期中国预防兽医学报Vo1．32．No．42010年4月ChineseJournalofPreventiveVeterinaryMedicineApr．2010抗H5N1亚型高致病性禽流感猴源噬菌体scFv的构建和表达陈樱，邓国华，陈化兰(中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室／农业部动物流感重点开放实验室，黑龙江哈尔滨150001)摘要：为探索治疗禽流感的方法，本实验从免疫H5N1亚型禽流感疫苗的猕猴淋巴细胞中提取总mRNA并扩增重链和轻链可变区(VH、V和V)，用重叠延伸PCR方法构建vH．Linker．V和VH．Linker—V形式的

2、单链抗体(scFv)，采用噬菌体展示技术构建了噬菌体抗体库，从2．2×10的噬茵体库中淘选得到具有较高亲和力的H6株scFv，并通过ELISA、SDS—PAGE、westernblot、间接免疫荧光(IFA)和竞争抑制ELISA对其进行鉴定，证实所获得的scFv为32ku，并且能与病毒发生特异性结合。IgBlast序列分析发现，所得到的scFv基因v与人免疫球蛋白胚系基因同源性达91．5％，而v同源性为75％。本研究所得到的猴源抗体可变区为人源化抗体的构建以及禽流感的治疗奠定了基础。关键词：猴源scFv；重叠延伸PCR；噬菌体抗体库；间接免疫荧光中图分类号：$852．

3、65文献标识码：A文章编号：1008—0589(20l0)04．0249—05Expressionofphagedisplayedsingle—chainantibodyofrhesusmacaqueagainstH5N1highlypathogenicavianinfluenzavirusCHENYing，DENGGuo—hua，CHENHua—lan(AnimalInfluenzaLaboratoryoftheMinistryofAgricu1ture／StateKeyLaboratoryofVeterinaryBiotechnologyHarbinVeterin

4、aryResearchInstitute，ChineseAcademyofAgriculturalSciences，Harbin150001，China)Abstract：Inordertoconstructasubtractivesingle—chainantibody(scFv)againstH5N1highlypathogenicavianinfluenzavirus．thevariablecDNAfragmentofHandLchainoftheantibodywasamplifiedfromthetota1mRNAofimmunizedrhesusmacaq

5、uelymphocytes．Thesingle—chainvariablefragments(VmLinker—VandVn-Linker—V)wereconstructedbysplicingoverlap—extension(SOE)PCRanddisplayedonthephagesurface．Panningofthephagelibraryof2．2X10clonesbyELISAallowedtheisolationofH6，ahighaffinityscFvantibody．ThescFvwasfurtherconfirmedbySDS—PAGEandw

6、esternblot，anditsactivitywasanalyzedbyindirectimmunofluorescenceassay(IFA)andcompetitiveinhibitionELISA，ThescFVwasabout32kuwhichcouldspecificallybindthevirus．IgBlastresultsrevealedthattheVgenesharing91．5％identitywiththoseofhumanimmunoglobulingermlinegenes，whiletheVHgenesharedonly75％iden

7、tity．ThescFvofrhesusmacaquereportedhereprovidedafoundationforconstructionofthehumanizedantibody，andthisapproachofgeneratingantibodyfragmentscouldbesuitableforprophylaxisandtherapeutics．Keywords：rhesusmacaquesingle—chainantibody；SOEPCR；phageantibodylibrary；IFA}Correspondingautho