人VEGF-C基因荧光真核表达载体的构建和表达

目的:构建含有绿色荧光蛋白基因的人血管内皮生长因子C基因的真核表达载体,检测该基因在真核细胞中的表达,研究VEGF-C基因在淋巴管生成中的作用.方法:含有绿色荧光蛋白基因的质粒pEGFP-1通过末端平滑化,酶切获得5'(粘端)BamHI-EGFP-XbaI(平端)3'片断,已经构建的VEGF-C表达载体pcDNA3.1/VEGF-C通过末端平滑化,酶切获得5'-(粘端)BamHI-pcDNA3.1-目的基因-KpnI(平端)3',两片段连接构建含有绿色荧光蛋白的真核表达载体,琼脂糖凝胶电泳并测序检测插入片段的正确性.将该载体通过脂质体转染到真核细胞CHO中,观察绿色荧光蛋白在CHO细胞中的表达情况.通过G418筛选阳性转染细胞,提取总RNA,行RT-PCR,PCR产物琼脂糖凝胶电泳观察VEGF-C基因在CHO细胞中的表达情况.结果:琼脂糖凝胶电泳证实了插入EGFP片段的大小,测序结果证实了插入片段的正确性,CHO细胞中看到绿色荧光蛋白的表达,RT-PCR产物中含有VEGF-C cDNA.结论:成功构建了含有绿色荧光蛋白基因的VEGF-C真核表达载体并检测到在真核细胞CHO中的表达.

Construction and expression of eukaryotic fluorescent expressing vector of VEGF-C gene in CHO cell

Objective:To construct eukaryotic expressing vector of VEGF-C gene with EGFP gene and test its expression in CHO cells.Methods:cDNA of EGFP in plasmid of pEGFP-1 was inserted into eukaryotic expressing vector pcDNA3.1/VEGF-C by DNA recombinant technology and further transfected into CHO cell with lipofectin.Expression of EGFP in transfectants was examined by fluorescence microscope.The expression of VEGF-C gene was examined by RT-PCR.Results:Agarose gel electrophoresis proved the insert fragment was EGFP gene.It was proved further by DNA sequence.After being selected by G418,one clone was obtained.mRNA of exogenous VEGF-C gene could be detected by RT-PCR.Conclusion:The eukaryotic expressing vector of VEGF-C with EGFP gene was constructed successfully and could be expressed in CHO cell.