胃癌SGC-7901细胞中人RIPX—GFP融合蛋白载体构建及表达定位  

Construction of Eukaryotic GFP-RIPX Expression Vector and Localization of the GFP-RIPX Fusion Protein in Gastric Cancer SGC-7901 Cells

作　　者：王桂玲[1] 王孝会[1] 周颖[1] 陈薇[1] 姜佩佳[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,沈阳市110001

出　　处：《医学分子生物学杂志》2011年第1期36-39,共4页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30871294）

摘　　要：目的构建真核表达载体pEGFP—RIPX，并鉴定RIPX在胃癌细胞中的定位，并检测外源性RIPX的表达。方法以pBluescriptR—RIPX为模板，采用PCR法进行人RIPX编码序列（CDS）的扩增，并将其克隆至真核表达载体pEGFP—C1中。将pEGFP—RIPX质粒瞬时转染到胃癌SGC-7901细胞中，用Western印迹方法检测GFP—RIPX融合蛋白的表达；用激光扫描共聚焦显微镜观察RIPX的定位。结果人的RIPX编码序列被成功克隆至真核表达载体pEGFP—C1中；证实了GFP—RIPX融合蛋白的表达；转染pEGFP．RIPX后，在胃癌SGC-7901细胞胞质内可见明亮的绿色荧光。结论成功地构建了pEGFP-RIPX载体，鉴定了外源性RIPX的表达；同时证实在胃癌SGC-7901细胞中RIPX主要定位于细胞质，为进一步研究RIPX的生物学特性及其信号转导通路奠定了基础。Objective To construct an eukaryotic expression vector pEGFP-RIPX and identify the expression and localization of exogenous RIPX in gastric cancer cells. Methods Using pBluescript R-RIPX as template, we obtained human RIPX coding sequence by PCR amplification and cloned it into the eukaryotic expression vector pEGFP-C1, pEGFP-RIPX was then transiently transfected into gastric cancer SGC-7901 cells. Expression of GFP-RIPX fusion protein was analyzed by Western blot. Subcellular localization of RIPX was determined by laser scanning confocal microscope. Results hRIPX coding sequence was correctly cloned into pEGFP-C1 eukaryotic expression vector. After transfection of pEGFP-RIPX plasmid, GFP-RIPX fusion protein was efficiently expressed and localized in the cytoplasm of gastric cancer SGC-7901 cells. Conclusion The established pEGFR-RIPX construct may be used for further study of biological features and signal transduction pathways of RIPX.

关 键 词：RIPX 绿色荧光蛋白 蛋白表达及定位 胃癌SGC-7901细胞 

分 类 号：R349.7[医药卫生—基础医学]