人Nephrin -GFP融合蛋白真核表达载体的构建和鉴定

目的 构建人19号染色体长臂Nephrin基因和绿色荧光蛋白(green fluorescence protein,GFP)真核表达质粒,为进一步研究肾小球裂隙隔膜分子复合物构成与功能提供基础.方法 设计引物,扩增真核表达质粒pEGFP N3中的GFP基因片段,将其插入nephrin原核表达载体pcDNA3.1 nephrin V5-His,重组质粒经酶切鉴定后测序,并转染至COS-7细胞观察表达情况及生物学特性.结果 成功构建pcDNA3.1 nephrin-GFP重组质粒,并将Nephrin -GFP融合蛋白成功表达于COS-7细胞,进一步经交联实验证明Nephrin-GFP融合蛋白具有正确的细胞膜表达.结论 利用pEGFP N3和pcDNA3.1 nephrin V5-His可成功重组Nephrin-GFP表达质粒,为进一步研究肾小球裂隙隔膜分子复合物构成及其功能提供有利工具.

Construction and Identification of Human GFP-Nephrin Fusion Protein Expression Vector

Objective To construct an eukaryotic expression vector efficiently co-expressing Nephrin and GFP.Methods The GFP gene was amplified from pEGFP N3 vector by PCR and inserted into pcDNA3.1 nephrin V5-His vector.The constructed recombination plasmid was analyzed by sequencing.Transfection and cross-link experiments were performed to observe expression and functional features of Nephrin-GFP fusion protein expression in COS-7 cell.Results The prokaryotic cell expression vector pcDNA3.1 nephrin-GFP was successfully constructed.Nephrin-GFP fusion protein was highly expressed in COS-7 cell with a correct topology and biological function.Conclusion The recombination plasmid pcDNA3.1 nephrin-GFP was established and might be used to further study the molecular complex function of slit diagram of glomerular disease.