Abstract:The objective of this work is to construct a eukaryotic expression vector for the C-Src tyrosine kinase（Csk）gene from human. Total RNA was extracted from HeLa cells. The full-length cDNA sequence of Csk gene was isolated and amplified via RT-PCR，and cloned into a eukaryotic expression vector pENTER，the recombinant pENTER-Csk-his plasmid was constructed. The recombinant plasmid was transfected into 293T cells，after 48 hours，the expression levels of Csk protein were determined by SDS-PAGE and Western blot. The localization of Csk protein was detected via indirect immunofluorescence，and the protein Csk was purified by nickel chelate beads；in addition，the activity of the protein was measured via his-pulldown and CO-IP. Results showed that：Double digestion and sequencing reveled that recombinant plasmid pENTER-Csk-his was constructed properly without mutation. Both SDS-PAGE and Western blot detected a 51 kD protein，indicating that Csk protein was expressed successfully in the 239T cells. The indirect immunofluorescence confirmed the expression of Csk protein in cytoplasm. Finally，the purified protein Csk by nickel chelate beads interacted with the IGF1R and SHC1 by his-pulldown and CO-IP. Conclusively，this study successfully acquired the full-length sequence of Csk，the eukaryotic expression vector pENTER-Csk-his was constructed，and the gene was expressed efficiently in 293T cells，moreover，the expressed protein presented bioactivity.