人C-Src蛋白酪氨酸激酶真核表达、纯化及活性检测  

Eukaryotic Expression,Purification and Activity Testing of Human C-Src Protein Tyrosine Kinase

作　　者：徐岚[1] 肖斌[1] 陈慧芹[1] 李晓荣[1] 郝文波[1] 

机构地区：[1]南方医科大学抗体工程研究所,广州510515

出　　处：《生物技术通报》2016年第5期75-81,共7页Biotechnology Bulletin

基　　金：国家高技术研究发展计划("863"计划)(2014AA020904)

摘　　要：旨在构建人C-Src蛋白酪氨酸激酶(Csk)基因真核表达系统。从He La细胞中提取总的RNA,通过RT-PCR获得Csk基因全长c DNA序列,并将其克隆至真核表达载体p ENTER中,构建重组质粒p ENTER-Csk-his。重组质粒转染293T细胞48h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,hispulldown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p ENTER-Csk-his正确没有突变,SDSPAGE及Western blot均可检测到大小约为51 k D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p ENTER-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。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 He La cells. The full-length c DNA sequence of Csk gene was isolated and amplified via RT-PCR,and cloned into a eukaryotic expression vector p ENTER,the recombinant p ENTER-Csk-his plasmid was constructed. The recombinant plasmid was transfected into 293 T 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 p ENTER-Csk-his was constructed properly without mutation. Both SDS-PAGE and Western blot detected a 51 k D protein,indicating that Csk protein was expressed successfully in the 239 T cells. The indirect immunofluorescence confirmed the expression of Csk protein in cytoplasm. Finally,the purified protein Csk by nickel chelate beads interacted with the IGF1 R and SHC1 by his-pulldown and CO-IP. Conclusively,this study successfully acquired the full-length sequence of Csk,the eukaryotic expression vector p ENTER-Csk-his was constructed,and the gene was expressed efficiently in 293 T cells,moreover,the expressed protein presented bioactivity.

关 键 词：Csk基因 293T细胞 真核表达 纯化 活性鉴定 

分 类 号：Q78[生物学—分子生物学] Q55