人胚肾细胞SGLT2基因异源表达体系的构建  

Establishment of Na＋-glucose cotransporter 2 heterologous expression system in HEK293 cells

作　　者：于磊[1] 周绪杰[1] 吕继成[1] 丁嘉祥[1] 朱厉[1] 张宏[1] 

机构地区：[1]北京大学第一医院肾内科北京大学肾脏病研究所卫生部肾脏疾病重点实验室,100034

出　　处：《中华肾脏病杂志》2011年第8期606-610,共5页Chinese Journal of Nephrology

基　　金：国家自然科学基金（30971371）;内蒙古自然科学博士基金（2010BS1102）;高等学校博士学科点专项科研基金（200800010086）

摘　　要：目的构建钠依赖的葡萄糖转运蛋白2（SGLT2）基因异源表达体系，为探讨突变引起SGLT2蛋白功能及表达异常的分子机制提供实验依据。方法利用RT—PCR法从人肾组织中获得SGLT2基因，将该基因克隆到真核表达载体PEXL—GFP（绿色荧光蛋白）中，将携带有SGLT2基因的PEXL载体转染人胚肾细胞系（HEK293细胞），获得目的蛋白的瞬时表达。应用Western印迹及激光共聚焦显微镜检测融合蛋白在HEK293细胞的表达和分布，并通过摄取实验进一步验证SGLT2蛋白的转运功能。结果SGLT2-GFP蛋白可在HEK293细胞表达，激光共聚焦显微镜观察发现，SGLT2一GFP蛋白在细胞膜上呈点状分布，并且与细胞膜标记物（DiI）有良好的共定位，转染SGLT2-GFP质粒的HEK293细胞的转运活性较对照组（未转染及转染空质粒细胞组）强约3．5倍（P〈0．01）。结论成功构建了SGLT2真核表达载体，为探讨SGLT2基因表达、功能及SGLT2突变在家族性肾性糖尿发病的遗传机制提供了重要的依据。Objective To establish heterologous expression system of Na＋-glucose cotransporter 2 （SGLT2） gene. Methods Human SGLT2 cDNA from normal kidney, generated by RT-PCR, was subcloned into PEXL-GFP vector and transfected into HEK293 cells. After 24 hours of incubation, the expression of SGLT2-GFP fusion protein was detected by Western blotting and laser confocal microscopy. Transport activity of SGLT2-GFP fusion proteins in cuhured human HEK293 cells was evaluated with the uptake test of glucose analogue. Results SGLT2-GFP fusion protein was expressed in cultured human HEK293 ceils. Furthermore, confocal microscopy using green fluorescent protein （GFP） revealed a punctate membrane pattern of SGLT2. Glucose analogue uptake increased in HEK293 cells transfected with SGLT2-GFP at least by 3.5 folds compared with HEK293 cells transfeeted with GFP vector only （P〈0.01）. Conclusion Heterologous expression of SGLT2 gene in HEK293 cells is successfully established, which provides valuable approach for the functional and pathological study of SGLT2 gene.

关 键 词：钠-葡萄糖转运体2 糖尿 肾性 转染 家族性肾性糖尿 异源表达体系 

分 类 号：R363[医药卫生—病理学]