真核表达载体pIRES2-EGFP-Nurr1的构建及鉴定

目的:构建带有增强型绿色荧光蛋白报告基因EGFP及目的基因Nurr1的真核表达载体pIRES2-EGFP-Nurr1,并检测其在293T细胞中的表达。方法:采用反转录-聚合酶链式反应(RT-PCR)方法从大鼠黑质中获取Nurrl基因,连接T载体测序正确后与真核空载体pIRES2-EGFP一起,经Nhe1和Xho1双酶切,T4 DNA连接酶连接,构建pIRES2-EGFP-Nurr1;真核表达载体pIRES2-EGFP-Nurr1测序正确后采用脂质体法将其转染293T细胞,倒置荧光显微镜下观察转染效率,PCR检测Nurr1基因mRNA水平的表达情况,免疫印迹试验(Western Blot)检测Nurr1蛋白的表达水平。结果:酶切及测序鉴定证实成功构建了重组真核表达载体pIRES2-EGFP-Nurr1;293T细胞转染pIRES2-EGFP-Nurr1后可以高度表达绿色荧光,有效转录Nurr1基因并正确的高表达Nurr1蛋白。结论:成功构建Nurr1真核表达载体且在293T细胞中高水平表达,为进一步转染大鼠骨髓间充质干细胞(BMSCs),基因治疗帕金森病奠定基础。

Objective:To construct the eukaryotic expression vector pIRES2-EGFP-Nurr1 containing enhanced green fluorescent protein report gene(EGFP) and target gene Nurr1 and observe its expression in 293 T cells. Methods:Nurr1 gene was amplified from the rat substantia nigraby using reverse transcription-polymerase chains reaction(RT-PCR). By TA cloning and sequencing, pIRES2-EGFP-Nurr1 plasmid was constructed through the NheⅠ and XhoⅠdouble digestion and T4 DNA ligase conjunction. The plasmid transfected 293 T cells by Lipofectamine after the sequencing. The transfection efficiency was observed under the inverted fluorescence microscope and expression of Nurr1 in 293 T cells was assayed by PCR and Western blot. Results:Enzyme digestion and sequencing confirmed recombinant eukaryotic expression vector pIRES2-EGFP-Nurr1 was constructed successfully. Green fluorescence is highly expressed in the cytoplasm of transfected cells under fluorescence microscope. Nurr1 gene can be transcribed and translated effectively in 293 T cells. Conclusions:the eukaryotic expression vector containing Nurr1 has been constructed successfully and expressed highly in 293 T cells. This laid the foundations for the future transfection of Bone marrow mesenchymal stem cells(BMSCs) of rat and gene therapy for Parkinson's disease.