EGFP和ALR融合基因表达载体构建及其在HeLa细胞中的表达

Construction of Expressing Vector of Fusion Gene of EGFP and ALR and Its Expression in Hela Cells

从人血液中提取总DNA,利用PCR技术扩增人肝细胞再生增生因子基因,将其插入表达载体pEGFP-C1的多克隆位点中,构建增强绿色荧光蛋白(enhanced green fluorescence protein gene,EGFP)和人肝细胞再生增强因子(human augmenter of liver regeneration gene,ALR)融合基因表达载体pEGFP/ALR,并将其转染Hela细胞系,用含G 418的DMEM/F12培养液筛选转基因细胞,然后利用PCR和聚丙烯酰胺凝胶电泳检测转基因细胞中ALR基因的存在及其表达,用荧光显微镜检测EGFP基因的表达.结果显示:得到了构建正确的EGFP和ALR融合基因表达载体;在转基因细胞中,PCR扩增得到1.7 Kb的ALR条带,蛋白电泳得到57 KD大小条带,与ALR和EGFP融合蛋白大小相符;荧光显微镜下观察到发绿色荧光的Hela细胞.在转基因细胞中,EGFP和ALR同时存在并表达,绿色荧光蛋白可作为报告基因指示目的基因的表达,从而简化了目的基因繁琐的检测手段.

Human ALR gene were amplified from human genomic DNA using PCR and inserted into the MDS of pEGFP--C1 plasmid to construct ALR and EGFP co-expression vector pEGFP/ALR. It was transferred into the Hela cells and selected by G418. ALR gene of transgenic cells was tested by PCR and SDS--PAGE and EGFP were observed with the confocal laser scanning microscope. The result showed that both PCR and enzyme digestion analysis confirmed constructed plasmids were correct,the ALR gene were detected in these positive cells. SDS--PAGE results indicated that there was specific 57 KD protein band in the positive cells,which matches the expression of EGFP as well. Therefore,EGFP can be used as a marker for target gene in the screening of transgenic cells.