重组人pEGFP-C2-NPHS2表达载体的构建及其在人胚胎肾293细胞中的表达

Construction of human recombinant pEGFP-C2-NPHS2 and its expression in human embyo kidney 293 cell lines

目的:构建人pEGFP-C2-NPHS2表达载体,并观察其在人胚胎肾(HEK)293细胞中的表达。方法:应用基因重组技术,构建增强绿色荧光蛋白与NPHS2的融合表达载体,经PCR、限制性内切酶酶切和测序鉴定,应用基因转染技术将其转入HEK293细胞中,荧光显微镜和免疫印迹检测NPHS2的表达产物podocin。结果:经鉴定目的基因NPHS2全长插入重组质粒,HEK293细胞不表达NPHS2基因,转染后可以检测到绿色荧光蛋白分布于细胞的膜周部,免疫印迹证实为编码蛋白podocin。结论:成功构建重组人pEGFP-C2-NPHS2表达载体,有助于进一步研究podocin在足细胞疾病中的作用。

Objective To construct the pEGFP-C2-NPHS2 expression vector and observe its expression in HEK293 cells. Methods Technique of gene recombination was used to construct the expression vector pEGFP-C2- NPHS2. PCR, restriction enzyme digestion, and DNA sequencing were used to identify the correct vector. The recombinant expression vector was transfected into HEK293 ceils and detected by fluoroscopy and Western blot. Results The target gene NPHS2 was inserted into the vector pEGFP-C2. Green fluorenee located on HEK293 cell membrane.The protein podocin expression of NPHS2 was detected by western blot. Conclusion The pEGFP-C2- NPHS2 expression vector was successfully constructed and would contribute to the further study on the role of podoein in podoeyte disease.