GFP基因真核表达质粒的构建及其在人胚肾293细胞中的表达

目的克隆绿色荧光蛋白(GFP)基因并构建真核表达载体,观察其在人胚肾293(下称293细胞)细胞中的表达和分布,为制备自行设计的以λ噬菌体为载体的人类免疫缺陷病毒(HIV)核酸疫苗的阳性对照奠定基础。方法以克隆好的pUC18/GFP为模板,根据Genbank中GFP的核苷酸序列设计引物,并在引物的5′端分别引入BamH及Xho酶切位点,特异性扩增GFP基因。TA克隆后经双酶切、测序等鉴定重组质粒,再经双酶切、连接构建含GFP编码基因的真核表达载体,酶切鉴定分析后,将该重组质粒通过脂质体介导,转染293细胞。荧光显微镜观察GFP在细胞内的表达和分布。结果重组质粒经BamH、Xho双酶切成5.4kb与0.7kb的片断,表明表达载体pcDNA3.1(+)中插入了GFP基因片断,测序结果表明编码框正确,并在293细胞中获得了表达,分布均匀。结论pcDNA3.1(+)/GFP真核表达载体已成功构建,并可在293细胞中表达。

Objective.. To construct a eukaryotic expression plasmid containing green fluorescent protein gene and observe its expression in 293 cells, which lays the foundation for further development of positive control as DNA vaccine against HIV. Methods： According to the published green fluorescent protein gene sequence in Genbank,a pair of primers were respectively designed and synthesized. After amplification with polymerase chain reactlon,the product was cloned into pMD-18T vector using TA cloning followed by BamH Ⅰ and Xho Ⅰ digestion and sequencing. The target gene was then subcloned into a highly efficient eukaryotic expression vector pcDNA3. 1（＋ ）. After identification with restrictive endonuclease analysis, the recombinant plasmid was transfected into 293 cells with the cationic liposome DOTAP as the transfection reagent. The expression was observed under a fluorescent microscope. Results： Restrictive endonuclease assay and sequence ananlysis verified thesuccessful construction of the recombinant vector pcDNA3.1 （＋）/GFP,and GFP protein was highly efficiently expressed in 293 cells. Under fluorescent microscope,green fluorescence was seen homogeneously distributed in the entire cell body of the cells transfected by the recombinant vector pcDNA3.1 （＋）/GFP. Conclusion： The GFP eukaryotic expression plasmid was successfully constructed and expressed in 293 cells.