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目的克隆人血管内皮生长因子(hVEGF)基因VEGF165片段,构建pcDNA3.1/hVEGF165,观察其在COS-7细胞中的表达,为基因治疗缺血性心脏病的研究奠定基础。方法从合法引产的胎儿心肌组织中提取总核糖核酸(RNA),应用逆转录-聚合酶链反应(RT-PCR)方法获得hVEGF165基因,将其重组入T载体,聚合酶链反应(PCR)法鉴定并测序,双酶切后克隆入真核表达载体pcDNA3.1/myc-his-B中,构建pcDNA3.1/hVEGF165重组体。用脂质体介导将其转染COS-7细胞,蛋白印迹(Westernblotting)法检测rhVEGF165的表达蛋白。结果用RT-PCR方法从胎儿心肌组织中获得了正确的hVEGF165基因序列,并成功构建pcDNA3.1/hVEGF165,且实现转染COS-7细胞的瞬时表达。结论构建的pcDNA3.1/hVEGF165转染真核细胞COS-7能够表达rhVEGF蛋白。
Objective To clone VEGF165 gene of human vascular endothelial growth factor (hVEGF) gene and construct pcDNA3.1 / hVEGF165 to observe its expression in COS-7 cells, which will lay the foundation for the gene therapy of ischemic heart disease. Methods The total RNA was extracted from the fetus myocardium of the legitimate induced labor. The hVEGF165 gene was obtained by reverse transcription - polymerase chain reaction (RT - PCR), and then was recombined into the T vector. The polymerase chain reaction Identified and sequenced, double digested and cloned into the eukaryotic expression vector pcDNA3.1 / myc-his-B to construct pcDNA3.1 / hVEGF165 recombinant. The recombinant plasmid was transfected into COS-7 cells by lipofectamine. Western blotting was used to detect the expression of rhVEGF165 protein. Results The correct hVEGF165 gene sequence was obtained from fetal myocardial tissue by RT-PCR and pcDNA3.1 / hVEGF165 was constructed successfully. Transient expression of COS-7 cells was also achieved. Conclusion The constructed pcDNA3.1 / hVEGF165 transfected eukaryotic cells COS-7 can express rhVEGF protein.