MafA真核表达载体的构建及在人肝癌细胞中的表达

Construction of eukaryotic expression vectorinsertedby MafA and its expression in HepG-2 cells

目的:构建肌腱膜纤维肉瘤肿瘤基因同系物A(MafA)真核表达载体pEGFP-N2/MafA,pEGFP-C1/MafA,观察MafA在人肝癌细胞HepG-2细胞内的表达。为研究Mafa基因功能和作用机制奠定基础。方法:提取胰腺总RNA,经RT-PCR获得MafA基因片段,在两端分别引入Hind Ⅲ和Sal Ⅰ的酶切位点,重组至有增强绿色荧光蛋白标记的真核表达载体pEGFP-N2,pEGFP-C1中,用脂质体方法转染至人肝癌细胞HepG-2细胞中,通过荧光显微镜观察荧光蛋白的表达,通过RT-PCR检测MafA基因和insulin Ⅱ基因的表达。结果:通过RT-PCR获取了MafA基因并成功克隆入载体,转染的细胞有绿色荧光蛋白表达及MafA基因表达,但没检测到insulin Ⅱ基因表达。结论:成功构建质粒pEGFP-N2/MafA和pEGFP-C1/MafA,并成功表达MafA基因,但没有insulin Ⅱ基因的表达。

Objective：To construct eukaryotic expression vectors pEGFP-N2/MafA and pEGFP-C1/MafA,encoding the mouse musculus v-maf musculoaponeurotic fibrosarcoma oncogene family,protein A（MafA）gene,for the further research of its expression in HepG-2 cells.Methods：pRNA of the mouse MafA gene was distilled by RT-PCR from the mouse,and inserted into Hind Ⅲand SalⅠrestriction sites by PCR,then cloned into pEGFP-N2 and pEGFP-C1 vectors to obtain plasmids pEGFP-N2/MafA and pEGFP-C1/MafA.Human liver cancer cells（HepG-2） were transfected with formed plasmids by means of lipidosome.The MafA-EGFP fused protein was viewed directly with fluorensce microscope,and expression of MafA and insulin Ⅱ was detected by RT-PCR.Results：The mouse MafA gene was amplified through RT-PCR and successfully cloned into transfer vectors.The favorite gene sequence could be expressed and was completely consistent with that reported in genebank,but the insulin Ⅱ gene expression was not detected.Conclusion：The recombinant plasmids pEGFP-N2/MafA and pEGFP-C1/MafA were successfully constructed.But the insulin Ⅱgene was not expressed.