【摘要】 目的:构建人线粒体超氧化物歧化酶2(SOD2)的真核细胞表达载体,探讨其在人脐静脉内皮细胞(HUVEC)中的表达效果。方法:以HUVEC mRNA为原始模板,应用RT-PCR技术,扩增编码线粒体SOD2全长的cDNA片段。利用分子生物学技术,将扩增的cDNA片段与真核细胞表达载体pcDNA3.0的多酶切位点相连接,构成重组质粒并转染到HUVEC,将细胞分为HUVEC母细胞组、转染pcDNA3.0空载组和转染pcDNA3.0-SOD2组。应用RT-PCR方法检测SOD2在HUVEC中的表达情况。结果:将表达SOD2全长cDNA片段定向克隆至质粒pcDNA3.0,测序结果表明成功构建人线粒体SOD2的真核细胞表达载体pcDNA3.0-SOD2。该载体稳定转染HUVEC后,在细胞内获得了稳定表达。与母细胞组和空载组比较,转染pcDNA3.0-SOD2组SOD2mRNA表达水平明显增加。结论:成功构建SOD2的真核细胞表达载体pcDNA3.0-SOD2,并在人HUVEC中成功表达。本研究为SOD2对细胞保护作用研究提供了实验模型。更多还原

【Abstract】 Objective To construct the recombinant eukaryotic expression vector of mitochondrial superoxide dismutase 2(SOD2)and to explore its expression efficacy in human umbilical vein endothelial cells(HUVEC). Methods SOD2gene full-length cDNA was amplified from HUVEC by RT-PCR and the target gene SOD2was connected with the plasmid pcDNA3.0vector.The recombinant plasmid was constructed and transfected into HUVEC.The cells were grouped into HUVEC,pcDNA3.0and pcDNA3.0-SOD2transfected groups.The expression of SOD2in HUVEC was detected by RT-PCR.Results The SOD2 gene full-length cDNA was cloned into pcDNA3.0successfully.The pcDNA3.0-SOD2eukaryotic expression vector was constructed successfully confirmed by double enzyme digestion and sequencing analysis.After the vector was transfected into HUVEC,the vector expressed stably in the cells.Compared with HUVEC and pcDNA3.0 groups,the mRNA level of SOD2in pcDNA3.0-SOD2transfected group was increased apparently(P<0.05).Conclusion The eukaryotic expression vector of SOD2 is constructed successfully,and expresses in HUVEC successfully.更多还原