HGF基因真核表达质粒的构建及其细胞表达

目的:构建真核表达质粒p EGFP-N1-HGF并观察干细胞生长因子(hepatocyte growth factor,HGF)在人皮肤成纤维中的表达。方法:从扩增人的间充质干细胞中提取全长RNA为模板,设计合成引物,用RT-PCR法扩增出HGF基因片段,然后将HGF基因片段连接于真核表达质粒p EGFP-N1中。双酶切鉴定及测序分析后,用脂质体包裹转染体外培养的人的皮肤成纤维细胞,通过荧光显微镜观察其转染效果,酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测HGF蛋白的表达。结果:经酶切鉴定及测序验证p EGFP-N1-HGF构建正确,经转染的人皮肤纤维细胞中观察到较强的绿色荧光并且可以表达一定量的HGF。结论:成功构建了真核表达质粒p EGFP-N1-HGF,为基因治疗脱发疾病提供实验基础。

Construction of HGF Eukaryotic Expression Plasmid and Expression in Fibroblasts

Objective:To construct eukaryotic expression plasmid pEGFP-N1-HGF and to detect the expression of hepatocyte growth factor (HGF) in human skin fibroblasts.Methods:Total RNA was extracted from human marrow mesenchymal stem cells (MSCs), specific primers were designed and HGF gene was amplified by RT-PCR. And then the gene fragment was constructed into eukaryotic expression plasmid pEGFP-N1. The recombinant plasmid was identified by digestion with restriction enzyme and gene sequencing. Human skin fibroblasts (HSF) were transfected with the recombinant plasmid using lipofectamine. The transfection efficiency was analyzed with fluorescence microscopy and enzyme-linked immunosorbent assay (ELISA) was applied to detect the expression of HGF.Results:Restriction enzyme digestion and gene sequencing confirmed the correct construction of recombinant eukaryotic expression plasmid pEGFP- N1- HGF. Bright fluorescence and a certain amount of HGF were detected in human skin fibroblasts (HSF).Conclusion:The recombinant eukaryotic expression plasmid pEGFP-N1-HGF can be used to express HGF and will provide a foundation for the gene therapy of alopecia.