增强型绿色荧光蛋白融合人核糖核酸酶抑制因子包装细胞系的建立及鉴定

Identification of the expression of recombinated plasmid pLNCX-EGFP-C1-hRI in PA317 packaging cell line

目的:建立及鉴定产生绿色荧光蛋白融合人核糖核酸酶抑制因子(hRI)的PA317包装细胞系。方法:采用脂质体转染法将pLNCX-EGFP-C1-hRI质粒转染至PA317包装细胞,分为空白细胞组(未转染组)、pLNCX-EGFP-C1转染组(对照组)、pLNCX-EGFP-C1-hRI转染组(实验组),每组设3个复孔,转然后用RT-PCR和Western blot方法检测egfp-hRI基因在PA317细胞的表达。结果:RT-PCR和Western blot方法显示,转染后在PA317细胞中检测到egfp-hRI基因表达;用脂质体转染法获得了G418阳性的PA317包装细胞克隆。结论:实验成功建立了产生绿色荧光蛋白融合人核糖核酸酶抑制因子的PA317包装细胞系,为后续试验奠定了基础。

Objective： To identify the expression of plasmid pLNCX-EGFP-C1-hRI targeting the gene of Human ribonuclease inhibitor（hRI） in PA317 cells which is capable of expression in mammalian cells. Methods： The vector of pLNCX-EGFP-C1-hRI was transfected into PA317 cells by Lipofectamine 2000 and then the expression of recombinated plasmid was verified in living cells by observing the transcription level of egfp-hRI fusion gene mRNA with RT-PCR method and the expression level of egfp-fusion hRI protein with western blotting method respectively. Results： Both RT-PCR and western blotting showed the egfp-hRI fusiongene was obviously expression in PA317 cells. Conclusion： The plasmid of pLNCX-EGFP-C1-hRI targeting hRI is successfully constructed and the protein of hRI can be expressed in PA317 cells correctly.