人C-Src蛋白酪氨酸激酶真核表达、纯化及活性检测

摘要

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.%旨在构建人C-Src蛋白酪氨酸激酶（Csk）基因真核表达系统。从HeLa细胞中提取总的RNA，通过RT-PCR获得Csk基因全长cDNA序列，并将其克隆至真核表达载体pENTER中，构建重组质粒pENTER-Csk-his。重组质粒转染293T细胞48 h后通过SDS-PAGE、Western blot检测Csk蛋白表达情况，间接免疫荧光进行蛋白定位，通过镍螯合的磁珠法纯化Csk蛋白，his-pulldown及CO-IP检测表达蛋白的活性。结果显示，经双酶切及测序鉴定，真核表达载体pENTER-Csk-his正确没有突变，SDS-PAGE及Western blot均可检测到大小约为51 kD的目的蛋白，说明Csk蛋白在293T细胞中表达成功，间接免疫荧光定位重组Csk蛋白在细胞质中表达，通过磁珠纯化得到Csk蛋白，最后通过his-pulldown及CO-IP发现Csk能够与IGF1R、SHC1相互作用，说明表达的Csk蛋白具有生物学活性。成功获得Csk基因全长序列，并构建重组pENTER-Csk-his真核表达质粒，在真核细胞293T的细胞质中获得高效表达且表达的蛋白具有生物学活性。