小鼠β_2m反义核酸重组荧光蛋白表达载体的构建及表达研究

本研究应用分子生物学技术 ,构建小鼠 β2 m反义核酸重组荧光蛋白表达载体pEGFP β2 mAN ,经过酶切、PCR鉴定 ,以及序列分析证实克隆的正确性。然后用脂质体转染NIH3T3细胞 ,经过RT PCR及Westernblot检测 ,观察重组质粒对细胞β2 mmRNA和蛋白表达的影响 ,同时在荧光显微镜下直接观察细胞转染的结果。结果证明pEGFP β2 mAN已成功导入NIH3T3细胞中 ,且有效表达 ,在荧光显微镜下可见梭形绿色荧光细胞 ;与同步转染的小鼠 β2 m正、反义核酸表达载体pcDNA3 β2 mSN、pcDNA3 β2 mAN的转染结果一起进行分析 ,转染pcDNA3 β2 mSN细胞的 β2 mmRNA和蛋白表达水平升高 ,而转染pcD NA3 β2 mAN和pEGFP β2 mAN的细胞 β2 mmRNA和蛋白表达水平降低。小鼠 β2 m反义核酸重组荧光蛋白表达载体pEGFP β2 mAN的转染结果显示 :它不但可以降低NIH3T3细胞 β2 m基因的表达 ,而且为观察脂质体转染效果提供了直观、即时的方法

Construction and Expression of the Murine β2m Antisense RNA Recombinant Green Fluorescent Protein Vector

Molecular cloning technique was used to construct the murine β 2m antisense RNA recombinant green fluorescent protein vector pEGFP β 2mAN,and it was identified by restriction analysis and PCR with its inserting gene sequenced as well Then the pEGFP β 2mAN was transfected into NIH3T3 cells with lipofectamine.The expression of β 2m on the cells was measured by RT PCR and Western blot,together with cells transfected with the murine sense and antisense RNA pcDNA3 β 2mSN and pcDNA3 β 2mAN As a result of that,the expression of β 2m on the cells transfected with pEGFP β 2mAN was found to be decreased,while pcDNA3 β 2mSN up regulated the expression of β 2m on the cells This result indicates that pEGFP β 2mAN not only has the function of down regulating the expression of β 2m,but also provides a direct way to observe the results of transfection