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目的:构建一个新型的携带有人内皮一氧化氮合酶(eNOS)cDNA的质粒载体并研究其体外表达,以用于基因治疗。方法:eNOS cDNA插入到腺相关病毒质粒pSNAV-1的EcoR Ⅰ位点。该质粒的启动子为CMV启动子,并携带有腺相关病毒的末端重复序列。构建好的质粒转染到两种哺乳动物细胞BHK和C2C12细胞中,通过PCR和RT-PCR分别检测eNOS cDNA和mRNA。结果:限制性内切酶分析证明,eNOS cDNA以正确的方向插入到pSNAV-1质粒中。PCR检测表明pSNAV-eNOS被转入BHK和C2C12细胞中。RT-PCR检测表明转染有pSNAV-eNOS的细胞可表达eNOS mRNA。结论:成功构建的pSNAV-eNOS可在体外培养的哺乳动物细胞中表达人eNOS mRNA。
Objective: To construct a novel plasmid vector carrying human endothelial nitric oxide synthase (eNOS) cDNA and study its expression in vitro for gene therapy. Methods: eNOS cDNA was inserted into EcoR I site of adeno-associated virus plasmid pSNAV-1. The plasmid’s promoter is the CMV promoter and carries the terminal repeat of adeno-associated virus. The constructed plasmids were transfected into two mammalian cells, BHK and C2C12 cells, and eNOS cDNA and mRNA were respectively detected by PCR and RT-PCR. Results: Restriction endonuclease analysis demonstrated that eNOS cDNA was inserted into pSNAV-1 plasmid in the correct orientation. PCR detection showed that pSNAV-eNOS was transfected into BHK and C2C12 cells. The results of RT-PCR showed that the cells transfected with pSNAV-eNOS could express eNOS mRNA. Conclusion: The constructed pSNAV-eNOS can express human eNOS mRNA in cultured mammalian cells.