凋亡抑制因子Survivin的克隆、高效表达与纯化

Survivin蛋白抑制细胞的凋亡并参与调控细胞的分裂，在绝大多数肿瘤细胞中均有过量表达。本实验以人肝癌细胞系MHCC-97L总RNA为模板，应用RT-PCR的方法得到survivin cDNA，并构建了重组原核表达载体pET-21b(+)-survivin，导入BL21（DE3）菌株进行表达，表达产物以包涵体形式存在，表达量超过总蛋白的60%。Western blot结果表明表达产物与抗人survivin抗体发生特异性反应，经凝胶过滤层析后纯度达到95%以上,为进一步研究靶向Survivin的诊断试剂与抑制剂奠定了基础。

Cloning, High Level Expression and Purification of Human Survivin

Survivin is a protein that inhibits apoptosis and regulates cell division. In our study, The cDNA sequence of survivin was amplified by RT-PCR and sub-cloned into the prokaryotic expression vector pET-21b(+), followed by transformation into E.coli strain BL21(DE3) and induction with IPTG. The recombinant survivin protein fusing with 6xHis tag was expressed in E.coli in the form of inclusion body at the expression level over 60% of the total cell protein. Results of Western blotting showed that recombinant survivin reacted specifically with anti-human survivin antibody. After gel filtration, the recombinant protein reached the purity over 95%, which facilitate the study of diagnosing and inhibitor agents targeting survivin.