抗乙型肝炎病毒表面抗原T7噬菌体抗体库的构建.pdf

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1、84微生物学杂志2012年1月第32卷第1期JOURNALOFMICROBIOLOGYJan．2012Vo1．32N0．1抗乙型肝炎病毒表面抗原，I’7噬菌体抗体库的构建刘兵，马晓宇，李响。，罗恩杰(1．中国医科大学基础医学院病原生物教研室，辽宁沈阳110001；2．中国医科大学96期七年制临床专业，辽宁沈阳110001；3．沈阳医学院奉天医院药剂科，辽宁沈阳110024)摘要构建1_7噬茵体单链抗体(scFv)库筛选抗乙型肝炎病毒表面抗原抗体。从抗一HBs阳性患者外周血淋巴细胞中提取总RNA，反转录合成eDNA第1条链，PCR分别扩增抗体重链可变区基

2、因(V)和轻链可变区基因(V)，经重叠延伸拼接(SOE)PCR组成seFv基因，并将其与1_7噬茵体载体的2个臂相连接。体外包装后，在宿主茵BLT5403中。扩增重组噬茵体抗体库。以乙型肝炎病毒表面抗原进行4轮“吸附一洗脱一扩增”的筛选，酶免疫实验检测抗体活性。所建抗体库库容为1．53X10，扩增后初级库滴度为2．42×10pfu／mL。以乙型肝炎病毒表面抗原筛选后抗体出现特异性富集，经酶免疫实验鉴定，得到2株与HBsAg抗原特异结合的噬茵体抗体，成功构建了抗HBsAg蛋白1_7噬茵体抗体库。关键词乙型肝炎病毒表面抗原；scFv；T7噬茵体抗体库中图分

3、类号R373．21文献标识码B文章编号1005—7021(20t2)01—0084—04ConstructionofaT7PhageAntibodyLibraryofAnti-HepatitisBSurfaceAntigenLIUBing，MAXiao．yu，LIXiang，LUOEn．jie‘(1．Dept．ofPatho．Bio1．，BasicMed．Col1．，2．Clin．Med．，96th7一year’sSch1．，ChinaMedicalUniversity，Shenyang110001；3．Dept．ofMedicament，Fengti

4、anHosp．Afil．toShenyangMed．CoU．，Shenyang110024)AbstractAT7phageantibodylibraryofsingle—chainfragmentofvariableregion(seFv)wasconstructedforscreen—ingantibodiesofanti—hepatitisBsurfaceantigen(HbsAg)．TotalRNAwasextractedfromperipherallymphocyteofpatients，whosedetectedanti—HBsantibo

5、dywaspositive．Thefirsts~andofcDNAwassynthesizedbyreversetran—scription．Variableregionofheavychain(VH)geneandvariableregionoflightchain(VL)genewererespectivelyamplifiedbyPCR；theseFvgenewasconstructedbysplicingovedapextension(SOE)PCR，andthenligatedthemontothe2armsofT7phagevector．A

6、fterpackaginginvitro，theT7phageantibodylibrarywereamplifiedandrecom-binedwithinhoststrainBLT5403．Phagesfromthelibrarywerescreened4roundsof“absorption—elution—amplifica—tion”withHbsAgandindividualcloneswereassayedbyenzymeimmunoassay．Phagedisplaylibrarywithsizeof1．53X10wasobtained

7、．Thestoragecapacityofthephagelibrarywas2．42×10pfu／mLaftertheamplification．SpecificphagewasenrichedafterscreeningwithHbsAgand2cloneswithabilityofspecificbindingtoHBsAgwereidentifiedbyenzymeimmunoassay．TheresultsdemonstratedthattheT7phageantibodylibraryofanti—HbsAgproteinwascon—st

8、ruetedsuccessfully．KeywordsHbsAg；scFv；3"7phagea