BMP-7/GFP融合基因在人肾小管上皮细胞的表达与定位  

Expression and localization of bone morphogenetic protein-7-GFP fused gene in human renal tubular epithelial cell line

作　　者：李娅[1] 陈楠[1] 陈佳韵[1] 王伟铭[1] 

机构地区：[1]上海第二医科大学瑞金医院肾脏科,上海200025

出　　处：《上海第二医科大学学报》2004年第8期621-623,631,共4页Acta Universitatis Medicinalis Secondae Shanghai

基　　金：上海市科委重大项目基金(03JC14084);上海市卫生局领先专业重点学科基金(983009)资助项目.

摘　　要：目的 构建骨成形蛋白-7(BMP-7)/绿色荧光蛋白(GFP)融合基因,观察其在人肾小管上皮细胞的表达与定位。方法 将小鼠BMP-7全长cDNA与GFP融合,连接进入真核细胞表达质粒pEGFP-C1中,以脂质体介导的方法将重组表达质粒pEGFP-C1-BMP-7转染入人肾小管上皮细胞。采用Western blot方法检测BMP-7在肾小管上皮细胞的表达;激光共聚焦荧光显微镜观察BMP-7在人肾小管上皮细胞中的定位情况。结果 限制性酶切以及DNA测序结果均说明所构建的重组质粒为BMP-7/GFP融合基因表达质粒。瞬时转染人肾小管上皮细胞BMP-7蛋白表达量较空载体转染组明显增加;GFP转染的细胞荧光均匀分布在整个细胞中,而pEGFP-C1-BMP-7转染的细胞荧光主要集中在细胞浆和细胞膜表面。结论 重组BMP-7/GFP融合蛋白具有GFP自发荧光的特性,且不影响BMP-7在细胞内的正确表达。Objective To study the expression and localization of bone morphogenetic protein-7 ( BMP-7 ) - green fluorescent protein (GFP) tagged gene in a human renal tubular epithelial cell line. Methods The full length BMP-7cDNA was linked into an eukaryotic expression vector pEGFP-C1. The accuracy of BMP-7 constructed and se-lected plasmids was confirmed by restriction enzymatic analyses and DNA sequencing. The recombinant plasmid pEGFP-C1-BMP-7 was then transiently transfected into cultured human renal tubular epithelial cells facilitated by li-pofectin. The expression level of BMP-7 protein was determined by Western blot . The distribution of BMP-7-GFP in intact renal proximal tubules was also investigated by confocal laser scanning microscopy. Results The recombinant plasmid pEGFP-C1-BMP-7 was successfully constructed. The confocal laser scanning microscopy showed that fluorescence light was spread over a broader cellular distribution both in the nucleus and cytoplasm in all of renal tubular epithelial cells transfected with pEGFP-C1 , but fluorescence light was focused on the cellular membrane and cytoplasm of renal tubular epithelial cells transfected with pEGFP-C1-BMP-7. Conclusion The GFP tag does not in-terfere with the natural assembly and transport of BMP-7 protein.

关 键 词：BMP-7 GFP融合基因 肾小管 上皮细胞 基因表达 定位 绿色荧光蛋白 

分 类 号：R692[医药卫生—泌尿科学]