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

摘要

目的 构建携带增强绿色荧光蛋白基因的人14-3-3σ真核表达载体pEGFP-GV142-SFN并转染至人乳腺癌MCF-7细胞,鉴定所表达的蛋白活性.方法 通过逆转录-聚合酶链反应从胎脑组织中获得编码14-3-3σ的全长cDNA,定向克隆至真核表达载体GV142中,鉴定正确后与带有增强绿色荧光蛋白的pEGFP-N1-SFN酶切后连接,重组携带14-3-3σ的表达载体pEGFP-GV142-SFN.将pEGFP-GV142-SFN质粒转染至乳腺癌MCF-7细胞,观察荧光蛋白的表达.结果 经限制性内切酶酶切分析、聚合酶链反应和DNA序列测定证实目的 基因已插入重组质粒,荧光显微镜下观察发现:转染24 h后,乳腺癌MCF-7细胞内可见增强绿色荧光蛋白表达,48 h表达最稳定,72 h可见荧光蛋白淬灭,细胞形态逐渐消失.结论 成功构建了14-3-3σ真核表达载体pEGFP-GV142-SFN并转染至乳腺癌MCF-7细胞,观察和检测到绿色荧光蛋白表达.%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.