血管内皮生长因子165真核基因转染载体转染的骨骼肌细胞

Construction of vascular endothelial growth factor 165 gene eukaryotic vectors for transfection of skeletal muscle cells

背景:血管内皮生长因子基因转染治疗组织损伤的研究倍受关注,构建稳定可靠的人血管内皮生长因子真核表达载体有重要意义。目的:克隆人血管内皮生长因子基因血管内皮生长因子165片段,构建pcDNA4-HisMax-C/VEGF165真核表达质粒,并验证其转染大鼠骨骼肌细胞的可靠性。方法:采用反转录-聚合酶链反应技术,从人卵巢癌患者外周血中提取并扩增出血管内皮生长因子165基因片段,通过DNA重组技术将该基因片段重组于pcDNA4-HisMax-C真核表达载体上,构建成pcDNA4-HisMax-C/VEGF165重组质粒,聚合酶链反应扩增,分别用酶切电泳分析和DNA测序的方法对提取和重组DNA进行鉴定。pcDNA4-HisMax-C/VEGF165重组质粒转染骨骼肌细胞1周后反转录-聚合酶链反应提取血管内皮生长因子基因并酶切电泳鉴定。结果与结论:构建的重组质粒目的基因片段为人血管内皮生长因子165cDNA,对大鼠骨骼肌细胞转染后检测到血管内皮生长因子165基因片段。提示成功地克隆了血管内皮生长因子165基因并构建了其真核表达质粒,能以此为载体转染至骨骼肌细胞,并已整合到骨骼肌的基因组参与转录,证明了其转染的有效性。

BACKGROUND：While researcher became more and more interesting in treating tissue damage by transfection of vascular endothelial growth factor （VEGF）, and construction of human VEGF （hVEGF） gene expressive plasmid is of significance. OBJECTIVE：To clone VEGF gene and to construct the expressive plasmid pcDNA4-HisMax-C/VEGF165, and to observe its expression in rat skeletal muscle cells. METHODS：hVEGF gene was amplified by reverse transcription-PCR method from human peripheral blood and constructed into the expressive plasmid pcDNA4-HisMax-C/VEGF165. The gene in the expressive plasmid pcDNA4-HisMax-C/VEGF165 was identified by PCR amplification, enzyme digestion and DNA sequencing. pcDNA4-HisMax-C/VEGF165 was transfected into skeletal muscle cells. After 1 week, the VEGF gene cloning was identified. RESULTS AND CONCLUSION：The cloned DNA was confirmed to be VEGF165 gene. After VEGF165 gene transfection, the cloned DNA from skeletal muscle cells was confirmed to be VEGF165 gene. In this study we successfully cloned VEGF165 gene and constructed its expressive plasmid pcDNA4-HisMax-C/VEGF165, and proved the efficiency of transfection preliminarily.