抗河豚毒素单链抗体基因的构建、表达及活性分析  

Cnstruction,expression and activity analysis of ScFv against TTX

作　　者：孟宪梅[1,2] 周玉[1] 卢士英[1] 李岩松[1] 张俊辉[1] 李研东[1] 张磊[1] 柳增善[1] 

机构地区：[1]吉林大学人兽共患病教育部重点实验室畜牧兽医学院,长春130062 [2]吉林工商学院,生物工程分院,长春130062

出　　处：《中国卫生检验杂志》2009年第1期14-16,178,共4页Chinese Journal of Health Laboratory Technology

基　　金：国家自然科学基金资助项目(30671762)

摘　　要：目的:构建抗河豚毒素(TTX)单链抗体表达载体并进行活性分析。方法:提取抗TTX单克隆抗体3D4杂交瘤细胞总RNA,根据鼠源单克隆抗体设计轻、重链可变区基因VL和VH引物并进行RT-PCR扩增,借助Linker(G1y4Ser)3连接成单链抗体TTX-ScFv(VH-Linker-VL)基因,克隆至表达载体pET-22b(+)后转入E.coliBL21(DE3)诱导表达,SDS-PAGE鉴定表达产物,ELISA检测表达蛋白的反应活性。结果:测序结果表明TTX-ScFv基因全长744 bp,经NCBI blast分析氨基酸序列符合鼠抗体可变区特征,其中VH366 bp,VL333 bp,Linker 45 bp,SDS-PAGE与薄层扫描分析显示表达产物相对分子量为26.045ku,表达蛋白以包涵体形式存在,表达量占菌体总量的42%;ELISA检测表明TTX-ScFv可与TTX特异结合。结论:成功构建了抗TTX单链抗体表达载体,表达产物具有一定的免疫学结合活性。Objective：To construct the expression vector for ScFv against TTX and analyze antigen binding activity of ScFv.Methods：Total RNA from the hybridoma cell line 3D4 was extracted.The VH and VL genes,which were amplified by RT-PCR according to the primers designed by the variable region gene of the mice antibody,were linked via a linker（G1y4Ser）3 to construct ScFv（VH-Linker-VL） gene by SOE-PCR.The ScFv gene was cloned into vector pET-22b（＋） and expressed in E.coli BL21（DE3）.The expressed proteins were detected by SDS-PAGE and thin scanning analysis and antigen binding activity was analyzed by ELISA.Results：DNA sequencing indicated that ScFv gene was 744 bp（VH 366 bp,VL 333 bp,linker 45 bp）.And NCBI blast analysis confirmed that ScFv had the characteristics of the mouse variable region gene.The molecular weight of the expressed protein,in the form of inclusion body,was about 26.045 ku and expressed protein possessed 42% of total somatic protein.ELISA assay indicated that the expressed protein showed specific binding to TTX.Conclusion：The expression vector for ScFv against TTX has been successfully constructed to express protein having specific TTX binding activity.

关 键 词：河豚毒素 单链抗体 表达 活性 

分 类 号：TQ78[化学工程]