AngRem104基因与绿色荧光蛋白融合基因表达载体的构建及在COS-1细胞中的表达定位

构建以绿色荧光蛋白（Green fluorescence protein,GFP)为报告基因的重组表达质粒AngRem104-pEGFP-N1，利用脂质体转染体外培养的COS-1细胞，在活细胞状态下用荧光显微镜直接观察AngRem104-EGFP融合蛋白在细胞中的分布和定位，用RT-PCR和Western blot方法验证其mRNA和蛋白的表达。结果表明在空载体pEGFP-N1转染组中，COS-1细胞内绿色荧光呈弥散分布；重组质粒AngRem104-pEGFP-N1转染组中，绿色荧光集中在细胞核中，随着表达量的增高，绿色荧光在细胞核中聚集成团块状，颗粒状，RT-PCR的结果表明AngRem104在重组质粒转染组中的表达明显高于空载体和空白对照组。Western blot的结果也确证了AngRem104-EGFP融合蛋白的表达。提示新基因AngRem104/pEGFP融合基因真核表达载体在真核细胞COS-1中获得了表达，细胞所表达的融合蛋白具有An-gRem104和EGFP的双重活性，可用荧光显微镜直接观察其表达情况及亚细胞定位，为基因功能研究提供了线索。

Construction of AngRem104/EGFP fusion gene expression vectorand its expression in COS-1 cells

To construct novel gene AngRem104/EGFP fusion gene in eukaryotic expression vector and to investigate its expression in COS 1 cells, human AngRem104 ORF was amplified from pGEM T AngRem104 by PCR and inserted into plasmid pEGFP N1. Using lipofectin method, the recombinant expression plasmid AngRem104 pEGFP N1 was transfected into COS 1 cells. The results showed that AngRem104/EGFP expressed a fusion protein with green fluorescence protein in COS 1 cells as defined by Western blot analysis. This fusion protein was localized in the nucleus of COS 1 cells as detected by fluorescence microscopy. The use of GFP for cell marking in novel gene AngRem104 demonstrated a new technology for functional study of novel gene AngRem104.