FXR1基因与增强型绿色荧光蛋白融合载体的构建及表达

Construction and Expression of Enhanced Green Fluorescent Protein and FXR1 Co-expression Vector

目的:构建携带人脆性X相关基因1(Fragile X related genel,FXR1)与增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)的融合表达载体并进行表达,为研究FXR1基因在脆性X综合征中的作用机制奠定基础。方法:以重组质粒pYESTrp3/FXR1为模板,PCR扩增FXR1基因,将扩增片段双酶切后连接到质粒pEGFP-N1中,形成重组表达载体pEGFP-N1/FXR1,并用脂质体法转染人胚肾细胞,荧光显微镜下观察GFP在细胞内的表达及Western Blotting检测FXR1的表达情况。结果:成功构建融合表达载体pEGFP-N1/FXR1,在人胚肾细胞实现表达,检测到融合蛋白EGFP/FXR1。结论:本实验成功构建EFGP和FXR1重组共表达载体并可在真核细胞中表达,为进一步研究FXR1在脆性X综合征中的作用机理奠定基础。

Objective：To construct EGFP and FXR1 co-expression vector and detect its expression in the cultured HEK 293T cells. Methods：A pair of primers of FXR1 with EcoR Ⅰ and Xho Ⅰwere designed, PCR was performed. The FXR1 gene fragment obtained from PCR products that was included EcoR Ⅰ and Xho Ⅰ , and then inserted into pEGFP-N1 that was digested with EcoR Ⅰ and Xho Ⅰ . The recombinant pEGFP-N1/FXR1 was identified by restriction analysis. The recombinant plasmid pEGFP-N1/FXR1 was transfected into HEK 293T cells mediated by liposome reagent. The expression of EGFP in cell was observed by fluorescence microscopy. FXR1 protein was detected by Western Blotting. Result The recombinant expression plasmid was successfully constructed and transferred into HEK 293T cells observed by enzyme cutting and fluorescent microscope. The effective expression of FXR1 was also testified by Western Blotting. Conclusion：The recombinant co-expression vector of EGFP-N1/FXR1 was successfully constructed and effectively expressed in HEK 293T cells.