原核表达载体pOPE101-8E5的改构及单链抗体的表达鉴定  

Expression and Identification of Single Chain Antibody of Prokaryotic Expression Vector pOPE101-8E5

作　　者：许静[1] 刁世勇[1] 张磊[1] 徐洁[1] 孟磊[1] 

机构地区：[1]中国医学科学院中国协和医科大学血液学研究所实验血液学国家重点实验室,天津300020

出　　处：《中国生物工程杂志》2007年第4期18-22,共5页China Biotechnology

基　　金：国家自然科学基金资助项目(30170349);天津市科技发展计划资助项目(003119611)

摘　　要：利用基因重组技术将链亲和素(core-streptavidin)cDNA插入原核表达载体pOPE101-8E5的3′端,并用单链抗体scFv-C4的重链和轻链可变区cDNA取代其scFv-8E5,构建重组表达载体pOPE101-C4∷core-streptavidin。将该表达载体转化入在大肠杆菌中进行诱导表达,用聚丙烯酰胺凝胶电泳和免疫印迹法分析表达产物的表达量和产物活性。结果提示成功地获得一个分子量约为45kDa的scFv-C4∷core-streptavidin的融合蛋白,它可结合KG1a细胞裂解物中分子量约为60kDa、45kDa的蛋白带,且其结合功能可以通过融合蛋白中的链亲和素基因直接测定。In the present study, we inserted the core-streptavidin cDNA into downstream of multi-cloning site of plasmid pOPE101-8E5 by DNA gene recombination technology. And then, the variable fragments of heavy and light chain of the scFv-8E5 were replaced by the scFv-C4 variable fragments to construct the expression vector pOPE101-C4 ∷ core-streptavidin. After transformed the vector pOPE101-C4 ∷core streptavidin into E. coil, the fusion protein C4 ∷core streptavidin-His-tag can be expressed by inducing with IPTG, and the expression level and activity of the expressed fusion protein analyzed by SDS-PAGE and Western blot. The results show that a scFv-C4 ∷ core-streptavidin fusion protein of 45kDa was obtained, which can bind proteins of 60kDa ＆ 45kDa from the KG1 a cells lysate simultaneously. The binding function can be detected by the binding of core -streptavidin and biotin directly.

关 键 词：链亲和素 单链抗体 融合蛋白 表达载体 

分 类 号：Q786[生物学—分子生物学]