重组人质粒pEGFP-人内皮一氧化氮合酶的构建

Construction of recombinant human plasmid pEGFP/human endothelial nitricoxide synthase plasmid

目的:构建含有增强绿色荧光蛋白报道基因的人内皮一氧化氮合酶重组质粒,为细胞转染提供实验依据。方法:实验于2005-05/10在首都医科大学宣武医院完成。用EcoRⅠ酶切pBluescriptⅡSK-人内皮一氧化氮合酶质粒,获得人内皮一氧化氮合酶基因,设计引物进行聚合酶链反应扩增。将人内皮一氧化氮合酶与pMD18-Tsimple平滑载体连接后,行EcoRⅠ和XbaⅠ双酶切,回收人内皮一氧化氮合酶,然后将其与经过酶切处理的含有增强绿色荧光蛋白报道基因的载体pEGFP-C1进行连接,形成8.4kb的质粒,经EcoRⅠ和XbaⅠ双酶切进行电泳筛选、鉴定和测序。结果:①目的基因片段人内皮一氧化氮合酶cDNA的获得:pBluescriptⅡSK-人内皮一氧化氮合酶质粒经EcoRⅠ酶切后,电泳可见3kb及3.7kb处各出现一条条带,与质粒图谱所示相符,证明质粒正确。加入上下游引物行聚合酶链反应扩增后,电泳可见3.7kb处出现一条条带,证明人内皮一氧化氮合酶cDNA的获取成功。②人内皮一氧化氮合酶cDNA的酶切处理:人内皮一氧化氮合酶与pMD18-Tsimple平滑载体连接后,EcoRⅠ和XbaⅠ双酶切,电泳分析可见3.7kb和2.7kb处各出现一条条带,证明两者连接成功。③pEGFP-人内皮一氧化氮合酶质粒的筛选:载体pEGFP-C1与人内皮一氧化氮合酶cDNA的连接后,EcoRⅠ单酶切电泳可见8.4kb处出现一条条带,再用EcoRⅠ和XbaⅠ双酶切,电泳可见4.7kb和3.7kb处各出现一条条带,证明连接正确。④pEGFP-人内皮一氧化氮合酶质粒的序列测定:序列测定结果与pEGFP-C1插入点序列及目的基因片段人内皮一氧化氮合酶cDNA两端序列对比相同,证明重组质粒构建成功。结论:pEGFP-人内皮一氧化氮合酶重组质粒的成功构建,可用于细胞转染,为人内皮一氧化氮合酶基因转移治疗在心血管疾病中的应用提供实验基础。

AIM： To construct a plasmid which has an enhanced green fluorescent protein （EGFP） reporter gene for the role of human endothelial nitricoxide synthase （heNOS）in cell transfeetion. METHODS： The experiment was carried out in Xuanwu Hospital , Capital University of Medical Sciences from May to October 2005.HeNOS was cut by restricted enzyme EcoR Ⅰ from plasmid pBlueseript Ⅱ SK- heNOS, then designed primer for polymerase chain reaction amplification and inserted to pMD 18-T simple vector. The plasmid was cut by restricted enzyme EcoR Ⅰ and Xba Ⅰ,then heNOS DNA was reclaimed. Then the heNOS DNA was connected with the plasmid pEGFP-C1 and a 8.4kb plasmid was obtained. Then EcoR Ⅰ and Xba Ⅰ were used for eleetrophoresis screening, identification and sequencing. RESULTS：①To obtain EGFP reporter gene segment： pBlueseript Ⅱ SK- heNOS was cut by restricted enzyme EcoR Ⅰ and eleetrophoresis showed that a band appeared at 3 kb and 3.7 kb respectively , which was in accorded with plasmid map , proving the sequence of the pEGFP-beNOS plasmid was correct. Up-and-down stream primers were added for polymerase chain reaction, and eleetrophoresis showed a band appeared at 3.7 kb area, proving heNOS cNDA was obtained successfully. ② enzyme treatment of heNOS cDNA ： heNOS was connected with pMD 18-T simple vector, and treated with EcoR Ⅰ and Xba Ⅰ. Electrophoresis showed the a band appeared at 3.7 kb and 2.7 kb, respectively , proving the two was connected successfully. ③Screening of pEGFP-heNOS plasmid： pEGFP-C1 was connected with heNOScDNA, and was cut with EcoR Ⅰ . Eleetrophoresis showed that a band appeared at the 8.4kb area. Then EcoR Ⅰ and Xba I were used and electrophoresis showed there was a band at 4.7 kb and 3.7 kb , proving the connection was right. ④ Sequencing of pEGFP-heNOS plasmid： sequencing was the same as that of pEGFP-C1 inserting point and target gene segment heNOS cDNA, proving the construction of recombinant plasmid was successful. CONCLUSION： The recombinant plasmid pEGFP-heNOS has been successfully constructed and it could be used as a transgenic plasmid in the research of cardiovascular diseases.