人细胞因子受体样因子3在真核及原核细胞中的表达  

The expression of human cytokine receptor-like factor 3 in mammalian and E.coli cells

作　　者：赵艳[1] 段素素[1] 车昌艳[1] 张蕾[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：《细胞与分子免疫学杂志》2008年第1期27-29,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30671944)

摘　　要：目的:观察CRLF3蛋白在293T细胞中的表达及亚细胞定位,并在大肠杆菌中表达和纯化了重组CRLF3蛋白。方法:将真核表达载体pCMV-myc-CRLF3瞬时转染293T细胞,转染24 h和48 h后免疫荧光染色,共聚焦显微镜观察蛋白表达及定位;构建原核表达质粒pGEX-4T-1-CRLF3,实现插入基因的融合表达,经GST亲合层析纯化蛋白。结果:CRLF3蛋白在293T细胞中高效表达,主要分布在细胞质和细胞膜;成功构建了表达CRLF3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Mr约为74 000的融合蛋白。结论:在真核细胞中过表达的CRLF3蛋白主要定位在细胞质和细胞膜;获得了重组GST-CRLF3融合蛋白,为进一步探讨CRLF3的功能奠定基础。AIM： Cytokine receptor-like factor 3 （CRLF3） is a novel gene cloned from K562 cell line. The aim of the current study is to observe CRLF3 expression and subcellular localization in 293T cells and to express and purify the CRLF3 fusion protein in E. coil METHODS： The plasmid pCMV-myc-CRLF3 was transiently transfected into 293T cell. The expression and localization of CRLF3 protein was observed by immunostaining approach. CRLF3 was also cloned into pGEX-4T-1 vector. The GST-CRLF3 fusion protein was expressed in E. coil and purified by GST affinity chromatography. RESULTS, CRLF3 protein was highly expressed in transfected 293T cells. CRLF3 protein was distributed in both cytoplasm and cell membrane. The GST- CRLF3 fusion protein was expressed as a Mr 74 000 protein and then successfully purified from E. coil cells. CONCLUSION： Overexpreseed CRLF3 is a cytoplasmic protein that distributes in both cytoplasm and cell membrane in mammalian cells. The recombinant GST-CRLF3 fusion protein had been isolated and could be used for further antibody production and functional characterization.

关 键 词：人细胞因子受体样因子3 真核表达 原核表达 蛋白纯化 

分 类 号：Q786[生物学—分子生物学]