重组病毒rAAV_2-IRES-EGFP/MnSOD的构建及其表达

Construction of Recombinant Plasmid rAAV_2-IRES-EGFP/MnSOD and Its Expression in vivo and in vitro

目的构建含有大鼠源性锰超氧化物歧化酶(MnSOD)基因的重组病毒rAAV2-IRES-EGFP/Mn-SOD,检测其在大鼠耳血管纹边缘细胞和耳蜗组织内的表达。方法将大鼠心肌组织来源的SOD基因克隆入融合表达载体pEGFP-N1中,再以限制性酶切方法克隆入真核表达载体pSNAV2.0-IRES-EGFP中,最后将其包装为rAAV2-IRES-EGFP/MnSOD病毒颗粒,用激光共聚焦显微镜、流式细胞仪、RT-PCR及Western印迹检测MnSOD基因的表达。结果真核表达载体pEGFP-N1/MnSOD和pSNAV2.0-IRES-EGFP/MnSOD经酶切、PCR和测序鉴定均检测到MnSOD基因的完整序列,被rAAV2-EGFP/MnSOD感染的耳边缘细胞和内耳组织中MnSOD蛋白的表达均高于空白对照耳。结论成功构建了大鼠源性MnSOD基因的重组病毒rAAV2-IRES-EGFP/MnSOD,并使其在大鼠耳边缘细胞和大鼠耳蜗组织中成功表达,为抗氧化基因治疗内耳疾病的研究奠定了基础。

Objective Eukaryotic expression plasmid of rat Mn-superoxide dismutase （MnSOD ） gene rAAV2-IRES-EGFP/MnSOD was constructed and its expression was examined in primary cultured marginal cells （MCs） from the stria vascularis （SV） and cochlear tissue of the rats. Methods The targeted segments were obtained from rat myocardial tissue and were inserted into an eukaryotic expression plasmid pEGFP-N1/MnSOD or rAAV2-IRES-EGFP/MnSOD. Then the recombinant adeno-associated viral vector 2-IRES-EGFP/MnSOD （rAAV2-IRES-EGFP/MnSOD） particles were obtained. The expression of gene of interest was detected by confocal fluorescence microscopy, fluorescence activated call sorting （FACS）, RT-PCR and Western blotting. Results Rat MnSOD cDNA eukaryotic expression plasmid pEGFP-N1/MnSOD and rAAV2-IRES-EGFP/MnSOD were successfully constructed. MnSOD protein could be detected in the MCs and cochlear tissue after transfection. Condusion Rat MnSOD cDNA eukaryotic expression plasmid rAAV2-IRES-EGFP/ MnSOD was successfully constructed and expressed in MCs and cochlear tissue of the rat. This results provide a basis for the antioxidant gene therapy for inner ear diseases.