Construction of a recombinant vector based on AAV carrying human endothelial nitric-oxide synthase gene  

携带人内皮一氧化氮合酶基因的AAV重组表达载体的构建(英文)

作　　者：贺晨霞[1] 缪朝玉[2] 姚纪花[1] 陈浩明[1] 卢大儒[1] 苏定冯[2] 薛京伦[1] 

机构地区：[1]复旦大学生命科学学院遗传学研究所遗传工程国家重点实验室 [2]第二军医大学基础部药理学教研室

出　　处：《Acta Pharmacologica Sinica》2003年第7期637-640,724,共5页中国药理学报（英文版）

基　　金：Project supported by the National Natural Science Foundation of China, № 30100102.

摘　　要：AIM: To construct an AAV based vector carrying human endothelial nitric-oxide synthase (eNOS) cDNA and study its expression in vitro for future gene therapy. METHODS: eNOS cDNA was inserted into the EcoR I site of pSNAV-1 containing the cytomegalovirus (CMV) promoter and inverted terminal repeat sequences of adeno-associated virus. The constructed vector was transfected into BHK and C2C12 cells, eNOS cDNA and mRNA were detected by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), respectively. RESULTS: By restriction enzyme digestion analysis, it was proved that eNOS cDNA was inserted into pSNAV-1 in a proper direction. PCR detection demonstrated that pSNAV-eNOS was transferred into both BHK and C2C12 cells. RTPCR analysis showed that these pSNAV-eNOS transfected cells could express eNOS mRNA. CONCLUSION: pSNAV-eNOS was successfully constructed with the ability to express human eNOS mRNA in cultured mammalian cells.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。

关 键 词：nitric oxide nitric-oxide synthase plasmids gene therapy adeno-associated virus 

分 类 号：R346[医药卫生—基础医学]