人源MAP3K7克隆及其GST融合蛋白的表达和鉴定  

Cloning of Human MAP3K7 Gene and Expression and Identification of GST-MAP3K7 Fusion Protein

作　　者：冯艳玲[1] 刘彩红[1] 朱亚勤[1] 

机构地区：[1]中国医科大学基础医学院细胞生物学教研室,卫生部细胞生物学重点实验室,细胞病理生物学研究室,沈阳110001

出　　处：《中国医科大学学报》2011年第8期688-690,共3页Journal of China Medical University

基　　金：辽宁省自然科学基金项目(20092123);辽宁省教育厅高校科研计划(2009S108)

摘　　要：目的构建MAP3K7蛋白GST的融合表达载体,高效表达和纯化GST-MAP3K7融合蛋白,为GST Pulldown实验做准备。方法通过PCR扩增MAP3K7全长编码基因,经双酶切定向克隆至表达载体pGEX-5X-1,构建原核重组表达载体pGEX-5X-1-MAP3K7,通过酶切和测序鉴定后,导入E.coli BL21宿主菌中,IPTG诱导表达融合蛋白,经谷胱甘肽琼脂糖凝胶纯化表达产物,SDS-PAGE和Western blot分析鉴定。结果经测序鉴定成功构建了原核重组表达载体pGEX-5X-1-MAP3K7,诱导后的GST融合蛋白经SDS-PAGE和Western blot检测到分子量正确的特异性蛋白条带,经纯化后,得到了高纯度的融合蛋白。结论成功构建了MAP3K7全长的GST重组表达载体,确定了GST-MAP3K7融合蛋白表达的最佳条件,诱导成功并得到了高纯度的融合蛋白,为进一步研究MAP3K7蛋白的生物学功能奠定了基础。Objective Constructing GST-MAP3K7 fusion protein expression vector and purifying GST-MAP3K7 fusion protein to prepare for the further study of GST Pull down experiment.Methods MAP3K7 gene segments were amplified by polymerase chain reaction（PCR）and cloned into the expression vector pGEX-5X-1.Recombinant prokaryotic expression vector pGEX-5X-1-MAP3K7 was identified by restriction enzyme digestion and sequencing.Then they were transformed into E.coli BL21,induced by IPTG and purified by glutathione beads.The purified products were analyzed by SDS-PAGE and Western blot.Results The recombinant prokaryotic expression vector pGEX-5X-1-MAP3K7 had been constructed successfully.A correct specific molecular weight protein band could be seen by SDS-PAGE after GST fusion protein expression was induced.After purification,we got fusion protein with high purity.Conclusion The human MAP3K7 full length gene segments were successfully cloned into prokaryotic expression vector.The adaptive conditions for inducing the GST-MAP3K7 target protein expression were confirmed and the GST-MAP3K7 target proteins with high purity were obtained.This study provides the basis for further studies on the biological function of MAP3K7 protein.

关 键 词：人MAP3K7基因 基因克隆 融合蛋白 原核表达 

分 类 号：Q291[生物学—细胞生物学]