人胚肾细胞SGLT2基因异源表达体系的构建

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-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。

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

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 cultured human HEK293 cells was evaluated with the uptake test of glucose analogue. Results SGLT2-GFP fusion protein was expressed in cultured human HEK293 cells. 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 transfected 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.