摘要
构建以绿色荧光蛋白 (Greenfluorescenceprotein ,GFP)为报告基因的重组表达质粒AngRem10 4 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AngRem10 4 EGFP融合蛋白在细胞中的分布和定位 ,用RT PCR和Westernblot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AngRem10 4 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AngRem10 4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。Westernblot的结果也确证了AngRem10 4 EGFP融合蛋白的表达。提示新基因AngRem10 4 /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 gRem10 4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。
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.
出处
《基础医学与临床》
CSCD
北大核心
2003年第1期30-34,共5页
Basic and Clinical Medicine
基金
国家自然科学基金 (39970 92 9)
北京大学人类疾病基因中心科研基金 (A 2 1)
