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时间:2020-05-14
《抗乙型肝炎病毒表面抗原T7噬菌体抗体库的构建.pdf》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
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
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