重组蛋白Cox7a2原核表达及鉴定  

Construction of pGEX4T-1-Cox7a2 and Expression,Purification and Identification of the Recombinant Protein

作　　者：陈亮[1] 辛钟成[1] 姜学军[2] 田龙[1] 袁亦铭[1] 刘刚[1] 宋卫东[1] 郭应禄[1] 

机构地区：[1]北京大学第一医院男科中心,北京100009 [2]中国科学院微生物研究所,北京100080

出　　处：《中华男科学杂志》2006年第9期794-797,共4页National Journal of Andrology

基　　金：北京大学医学部"十五""211"工程建设项目(219)

摘　　要：目的:从原代培养的小鼠睾丸Leyd ig细胞中克隆Cox7a2基因,构建pGEX4T-1-Cox7 a2载体,表达和鉴定重组蛋白。方法:应用RT-PCR方法从原代培养的小鼠睾丸Leyd ig细胞中克隆Cox7 a2,利用BamH I和EcoR I酶切位点把Cox7a2克隆到pGEX4T-1载体,酶切和测序鉴定后,诱导重组蛋白的表达,进行纯化后,利用蛋白免疫印迹进行鉴定。结果:从原代培养的小鼠睾丸Leyd ig细胞克隆到Cox7a2完整的编码序列,构建了pGEX4T-1-Cox7 a2载体,表达了预期相对分子质量的融合蛋白,并经免疫印迹检测鉴定。结论:成功克隆Cox7a2,表达纯化了其融合蛋白,为基因的功能研究奠定了前期基础。Objective： To clone and express Cox7a2, one mitochondrial respiratory chain related gene, and to identify its recombinant protein. Methods ： The coding region of CoxTa2 was amplified from primary cultured mouse Leydig cells by RT-PCR. The PCR product was cloned into pGEX4T-1 vector by BamH I and EcoR I sites, and confirmed by DNA sequencing. The recombinant fusion protein vector was transformed and expressed into BL21. The recombinant fusion protein was identified by Western blotting. Results： The entire coding region of Cox7a2 was cloned and expressed. The fusion protein was identified by anti-GST monoclonal antibody using Western blotting. Conclusion ： The cloning of CoxTa2 and the expression of the recombinant protein would help to study the detailed function of CoxTa2, one respiratory chain related and highly differently expressed gene in the tissues of aging testes.

关 键 词：COX7A2 线粒体 pGEX4T-1 重组 睾丸 小鼠 

分 类 号：Q78[生物学—分子生物学]