中国全科医学2013，Vol.16Issue(12)：1342-1345,4.DOI:10.3969/j.issn.1007-9572.2013.04.083
携带增强绿色荧光蛋白基因的人14-3-3σ真核表达载体的构建及其在转染乳腺癌MCF-7细胞的表达
Construction of Eukaryotic Expression Vector of Human 14-3-3σ Containing Enhanced Green Fluorescence Protein Gene and Its Expression in Transfection of Human Breast Cancer Cell MCF-7
摘要
Abstract
Objective To construct the eukaryotic expression vector of human 14-3 -3σ containing enhanced green fluorescence protein gene and to transfer it into humman breast cancer cell MCF - 7 in order to identify activity of expression protein. Methods The full - length human 14 -3 - 3σ cDNA was obtained by RT - PCR and was then inserted into GV142 vector. After identification it was connected with pEGFP - N1 - SFN containing enhanced green fluorescent protein to recombine expression vector pEGFP - GV142 - SFN with 14 - 3 - 3σ. The pEGFP - GV142 - SFN plasmid was transfected to breast cancer MCF - 7 to observe the expression of green fluorescent protein. Results Amplified ribosomal DNA restriction analysis and PCR and DNA sequencing showed that the target gene was inserted into the recombined plasmid. Fluorescence microscope showed that after 24 h transfection, expression of enhanced green fluorescent protein could be seen in MCF - 7 , and the expression was the most stable at 48 h, and the protein quenched and cell morphology started to disappear at 72 h. Conclusion The construction of the eukaryotic expression vector pEGFP - GV142 - SFN is transfected into breast cancer MCF -7 cells successfully to observe the expression of green fluorescent protein.关键词
乳腺肿瘤/基因/绿色荧光蛋白质类Key words
Breast neoplasms/ Genes/ Green fluorescent proteins分类
医药卫生引用本文复制引用
郝苗,齐凤杰,马玲..携带增强绿色荧光蛋白基因的人14-3-3σ真核表达载体的构建及其在转染乳腺癌MCF-7细胞的表达[J].中国全科医学,2013,16(12):1342-1345,4.基金项目
辽宁省科技基金项目(2010225034) （2010225034）
