SARS冠状病毒RNA依赖的RNA聚合酶的原核和真核表达  

Prokaryotic and eukaryotic expression of RNA dependent RNA polymerase of SARS

作　　者：张蕾[1] 张海婧[1] 段素素[1] 赵艳[1] 车昌燕[1] 张云[1] 王树蕙[1] 刘力[1] 

机构地区：[1]中国医学科学院基础医学研究所病原微生物学系,北京100005

出　　处：《中国卫生检验杂志》2008年第10期1974-1976,共3页Chinese Journal of Health Laboratory Technology

基　　金：国家自然科学基金资助项目(30671944)

摘　　要：目的:在大肠杆菌中表达和纯化重组RDRP蛋白,观察RDRP蛋白在293细胞中的表达及亚细胞定位。方法:构建原核表达质粒pGEX-4T-1-RDRP,实现插入基因的融合表达,经GST亲合层析纯化蛋白。构建真核表达载体pCMV-myc-RDRP瞬时转染293细胞,转染48 h后免疫荧光染色,共聚焦显微镜观察蛋白表达及定位以及WesternBlotting检测。结果:成功构建了表达RDRP融合蛋白的原核表达载体,并在大肠杆菌内高效表达,纯化后得到分子量约为121 kDa的融合蛋白。RDRP蛋白在293细胞中高效表达,主要分布在细胞质。结论:获得了重组GST-RDRP融合蛋白,为进一步探讨RDRP的功能奠定基础,在真核细胞中过表达的RDRP蛋白主要定位在细胞质。Objective：To express and purify the RNA dependent RNA polymerase（RDRP） fusion protein in E.coli and to observe RDRP expression and subcellular localization in 293 cells.Methods：RDRP was cloned into pGEX-4T-1 vector.The GST-RDRP fusion protein was expressed in E.coli and purified by GST affinity chromatography.The plasmid pCMV-myc-RDRP was transiently transfected into 293cell.The expression and localization of RDRP protein were observed by immunostaining approach.Results：The GST-RDRP fusion protein was expressed and then successfully purified from E.coli cells.RDRP protein was highly expressed in transfected 293 cells,and distributed in cytoplasm.Conclusion：The recombinant GST-RDRP fusion protein had been isolated and could be used for further antibody production and functional characterization.RDRP protein is highly expressed in transfected 293 cells.

关 键 词：RNA依赖的RNA聚合酶 原核表达 真核表达 蛋白纯化 

分 类 号：Q55[生物学—生物化学]