大鼠谷氨酰胺转运蛋白SNAT2-EGFP融合蛋白的表达与鉴定  

Expression and Identification of Rats Glutamine Transporters SNAT2-EGFP Fusion Protein

作　　者：孟雯[1] 王函[1] 董晓云[1] 李洋[1] 张舟[1] 

机构地区：[1]上海师范大学生命与环境科学学院,上海200234

出　　处：《中国细胞生物学学报》2011年第11期1248-1253,共6页Chinese Journal of Cell Biology

基　　金：国家自然基金(No.30870560);上海市科学技术委员会(No.10540503400);上海市教育委员会科研创新项目(No.09ZZ139);上海市重点学科建设项目(No.S30406);上海师范大学细胞生物学重点学科建设项目(No.DZL808)资助项目~~

摘　　要：谷氨酰胺转运蛋白是中枢神经系统中一种重要的中性氨基酸转运蛋白,对谷氨酰胺的跨膜转运十分重要。为了更方便地研究大鼠谷氨酰胺转运蛋白2(SNAT2)在细胞膜上的表达与定位,利用亚克隆技术将增强型绿色荧光蛋白(EGFP)构建于SNAT2的C端,通过菌液PCR、酶切和DNA测序鉴定重组真核表达质粒;将测序正确的重组质粒瞬时转染人胚胎肾细胞(HEK293T cells),用Western blot和激光共聚焦电子显微镜荧光检测技术鉴定SNAT2-EGFP的表达与亚细胞定位。结果表明,SNAT2-EGFP融合蛋白重组质粒在细胞中表达并正确定位于细胞膜上。SNAT2-EGFP融合蛋白重组质粒的成功构建为今后深入研究SNAT2的结构和功能提供了一个有效的工具。Sodium-coupled neutral amino an important role in transporting small, neutral acid transporters （SNATs） in the central neural system played amino acids, such as glutamine and alanine, across cellular membranes. In order to detect expression and localization of SNAT2 on cell membranes conveniently, an EGFP protein sequence was subcloned into SNAT2＇s C-Terminus in the eukaryotic expression vector pBK-CMV-（△[ 1 098-1 300]）-SNAT2-myc. After pBK-CMV-（△[1 098-1 300]）-SNAT2-EGFP was transiently transfected into HEK293T cells, expression of SNAT2-EGFP was detected by laser scanning confocal microscope and Western blot using anti-GFP antibody. The results showed that SNAT2-EGFP was successfully expressed and localized on cell membranes. The eukaryotic expression plasmid pBK-CMV-（△[1 098-1 300]）-SNAT2-EGFP constructed successfully is an effective tool for studying structure and function of SNAT2 in the future.

关 键 词：谷氨酰胺转运蛋白 增强型绿色荧光蛋白 融合蛋白 表达 

分 类 号：Q78[生物学—分子生物学]