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目的:构建人铜锌超氧化物歧化酶(SOD1)基因真核表达载体,并在HeLa细胞中进行表达。方法:应用RT-PCR从人外周血中扩增SOD1基因开放阅读框(ORF),采用TA克隆技术,将目的片段插入到pUCm-T载体中进行鉴定,重组的质粒命名为pUCm-T-SOD1。随后,将SOD1进一步克隆到真核表达载体pTracer-CMV/Bsd中。用脂质体将经过测序、验证的重组pTracer-CMV/Bsd-SOD1质粒转染HeLa细胞,荧光显微镜下观察绿色荧光蛋白的表达,转染HeLa细胞,经杀稻瘟菌素(Blasticidin)筛选4周,用RT-PCR、Western blot检测SOD1的表达。结果:成功构建了pTracer-CMV/Bsd-SOD1真核表达质粒,转染HeLa细胞,发现转染成功的细胞均发绿色荧光;RT-PCR及Western blot检测结果表明,所转染的SOD1在HeLa细胞中成功表达。结论:成功构建了能够同时表达绿色荧光蛋白和SOD1的真核表达质粒pTracer-CMV/Bsd-SOD1,为进一步研究SOD1在基因治疗中的作用奠定了基础。
Objective: To construct eukaryotic expression vector of human copper-zinc superoxide dismutase (SOD1) gene and express in HeLa cells. Methods: The open reading frame (ORF) of SOD1 gene was amplified from human peripheral blood by RT-PCR. The target fragment was inserted into pUCm-T vector by TA cloning technique. The recombinant plasmid was named pUCm-T-SOD1 . Subsequently, SOD1 was further cloned into the eukaryotic expression vector pTracer-CMV / Bsd. The recombinant plasmid pTracer-CMV / Bsd-SOD1 was transfected into HeLa cells by lipofectamine. The expression of green fluorescent protein (GFP) was observed under a fluorescence microscope and transfected into HeLa cells. After Blasticidin screening, The expression of SOD1 was detected by RT-PCR and Western blot. Results: The eukaryotic expression vector pTracer-CMV / Bsd-SOD1 was successfully constructed and transfected into HeLa cells. All the transfected cells showed green fluorescence. The results of RT-PCR and Western blot showed that the transfection of SOD1 in HeLa Cells were successfully expressed. CONCLUSION: The eukaryotic expression plasmid pTracer-CMV / Bsd-SOD1 that can express green fluorescent protein and SOD1 at the same time has been successfully constructed, which lays the foundation for further study on the role of SOD1 in gene therapy.