凋亡抑制因子Survivin的克隆、高效表达与纯化  

Cloning,High Level Expression and Purification of Human Survivin

作　　者：李海[1] 彭宇[1] 黎晓天[1] 吴文言[1] 

机构地区：[1]中山大学生命科学学院生物医药中心,广州510275

出　　处：《中国生物工程杂志》2007年第10期12-16,共5页China Biotechnology

基　　金：广东省科技攻关资助项目(2005B10401046;2006B35501002);广州市科技攻关资助项目(2006Z3-E4101)

摘　　要：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survivinc DNA,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。Survivin is a protein that inhibits apoptosis and regulates cell division. The cDNA sequence of survivin was amplified by RT-PCR and sub-cloned into the prokaryotic expression vector pET-21 b（ ＋ ）, followed by transformation into E. coil strain BL21 （ DE3 ） and induction with IPTG. The recombinant survivin protein fusing with 6 x His 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 antihuman survivin antibody. After gel filtration, the recombinant protein reached the purity over 95%, which facilitate the study of diagnosing and inhibitor agents targeting survivin.

关 键 词：SURVIVIN 克隆 原核表达 纯化 

分 类 号：Q789[生物学—分子生物学]