pEGFP-N1/MnSOD重组质粒的构建及体外表达

Construction of recombinant plasmid pEGFP-N1/MnSOD and its express in vitro

目的:构建大鼠源性锰超氧化物歧化酶(MnSOD)基因真核表达载体pEGFP-N1/MnSOD,并检测其在原代培养的大鼠耳蜗血管纹边缘细胞中的表达。方法:从大鼠心肌组织中扩增出SOD基因,克隆入融合表达载体pEGFP-N1中,用脂质体法转染大鼠耳蜗血管纹边缘细胞,激光共聚焦显微镜观察绿色荧光蛋白及目的基因的表达,流式细胞仪检测转染效率。结果:构建了大鼠源性MnSOD基因真核表达载体pEGFP-N1/MnSOD,并观察到转染/感染48h后,绿色荧光蛋白及目的基因在大鼠耳蜗血管纹边缘细胞中表达,目的基因的转染效率为23.47%。结论:成功构建了大鼠源性MnSOD基因真核表达载体pEGFP-N1/MnSOD,并使目的基因成功在大鼠耳蜗血管纹边缘细胞中表达,为内耳抗氧化基因治疗奠定了基础。

Objective： To construct eukaryotic expression plasmid of rat Mn-superoxide dismutase（MnSOD） gene pEGFP-N1/MnSOD and express it in primary culture system of marginal cells （MC） of the rats. Method： The aimed segments were obtained from rat myocardial tissue and were inserted into a eukaryotic expression plasmid pEGFP-N1/MnSOD. The recombinant expression plasmid pEGFP-N1/MnSOD was transfected into MC by lipo-fectamine2000-mediated gene transfer method, which were observed through co-Focus Fluorescence microscopy. The percentage of transfection was determined by fluorescence activated call sorting （FACS） analysis. Result： Rat MnSOD cDNA eukaryotic expression plasmid pEGFP-N1/MnSOD have been successfully constructed. Green fluo-rescent protein（GFP） and MnSOD protein could be contected in the transfected MC 48 hours after transfection. The percentage of transfection was 23.47 %. Conclusion： Rat MnSOD cDNA eukaryotic expression plasmid pEGFP- N1/MnSOD have been successfully constructed and expressed successfully in MC. The research paved the way for antioxidant gene therapy of inner ear.