论文部分内容阅读
目的构建胰岛素启动子肌腱膜的纤维肉瘤肿瘤基因同系物A(MafA)基因的真核表达质粒,并观察MafA基因在人肝癌细胞HePG-2中的表达及其作用,为糖尿病基因治疗的研究奠定基础。方法提取小鼠胰腺组织总RNA,经RT-PCR扩增MafA基因片段,克隆至真核表达载体pEGFP-N2和pEGFP-C1中。用脂质体将重组表达质粒转染至人肝癌细胞HePG-2中,荧光显微镜观察绿色荧光蛋白的表达,RT-PCR检测转染细胞中MafA和insulinⅡ基因的转录水平,Western blot检测融合蛋白EGFP-MafA的表达。结果重组表达质粒pEGFP-N2/MafA和pEGFP-C1/MafA经PCR、双酶切和测序证明构建正确;重组质粒转染的HePG-2细胞有绿色荧光蛋白表达;经RT-PCR可检测到MafA基因表达,但未检测到insulinⅡ基因表达;经Westernblot检测,在相对分子质量约70 000处可见特异性反应条带。结论已成功构建了MafA基因真核表达质粒pEGFP-N2/MafA和pEGFP-C1/MafA,以其转染的HePG-2细胞可表达MafA基因,并翻译成蛋白,但未表达insulinⅡ基因。
Objective To construct the eukaryotic expression plasmid of fibronarcoma tumor gene homolog A (MafA) of the insulin membrane of the aponeurosis of insulin promoter and to observe the expression of MafA gene in HepG-2 human hepatoma cell line and its role in the gene therapy of diabetes mellitus basis. Methods Total RNA was extracted from pancreatic tissues of mice. The MafA gene fragment was amplified by RT-PCR and cloned into eukaryotic expression vector pEGFP-N2 and pEGFP-C1. The recombinant plasmid was transfected into HepG-2 cells by lipofectamine. The expression of green fluorescent protein (GFP) was observed by fluorescence microscopy. The transcriptional levels of MafA and insulinⅡ in transfected cells were detected by RT-PCR. The expression of EGFP -MafA expression. Results The recombinant plasmids pEGFP-N2 / MafA and pEGFP-C1 / MafA were confirmed by PCR, restriction enzyme digestion and sequencing. The recombinant plasmids transfected HepG-2 cells showed the expression of green fluorescent protein. MafA However, the expression of insulin Ⅱ gene was not detected. Western blotting showed that the specific reaction band was observed at about 70 000 molecular weight. Conclusion MafA gene eukaryotic expression plasmids pEGFP-N2 / MafA and pEGFP-C1 / MafA have been successfully constructed. The transfected HePG-2 cells can express MafA gene and translate into protein but not insulin II gene.