人源化绿色荧光蛋白基因真核表达载体的构建与表达检测

目的: 构建人源化绿色荧光蛋白(humanized greenfluorescent protein,hGFP)基因的真核表达栽体pcDNA3GFP,并观察其在MDCK细胞中的表达情况. 方法: 以Not I切取GFP后插入真核表达载体pcDNA3,以BamH I为鉴定方向,以Lipofect AMINE PLUSTM将pcDNA3GFP转染至培养的MDCK,经G418筛选GFP阳性克隆,于荧光显微镜下观察. 结果: 成功构建了含hGFP cDNA的真核表达载体 pcDNA3GFP,并成功转染MDCK细胞,在荧光显微镜下阳性克隆呈绿色. 结论: GFP基因可在MDCK细胞中成功表达,并发出绿色荧光,是一良好的报告基因和筛选标记.

Construction and expression detection of enkaryotic expression vector of humanized green fluorescent protein gene

Objective:To construct the eukaryotic expression vector pcDNA3GFP for determination of expression of green fluorescent protein(GFP).Methods:GFP cDNA was obtained from plasmid pCIGFP with single Not I digestion and was inserted into the same restriction site of pcDNA3GFP. The upright insertion was determined with BamH I digestion.Results:With transfection of pcDNA3GFP encapsulated by Lipofect AMINE PLUS TM into the cultured MDCK cells,G418 was used for selection of positive clones expressing green fluorescence. GFP positive clones of MDCK cells formed 20 days after transfection with fluorescent microscopy.Conclusion:It was demonstrated that GFP was successfully expressed in MDCK cells,implying that GFP was a good reporter and selection marker molecule in mammalian cells.