Expression and Identification of Single Chain Antibody of Prokaryotic Expression Vector pOPE101-8E5
原核表达载体pOPE101-8E5的改构及单链抗体的表达鉴定

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 KG1a cells lysate simultaneously. The binding function can be detected by the binding of core-streptavidin and biotin directly.