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目的 克隆人血管内皮生长因子基因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奠定基础。方法 从胎儿心肌组织中提取总RNA,应用RT PCR方法获得hVEGF165基因,将其重组入T载体,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,WesternBlotting方法检测rhVEGF165的表达蛋白。结果 RT PCR方法从胎儿心肌组织获得正确的hVEGF165基因序列,成功构建pcDNA3·1 /hVEGF165且实现转染COS 7细胞的瞬时表达。结论 该实验构建的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 and construct pcDNA3.1 / hVEGF165 to observe the expression of VEGF165 in COS7 cells, which will lay the foundation for the gene therapy of ischemic heart disease. METHODS: The total RNA was extracted from the fetal heart tissue. The hVEGF165 gene was obtained by RT-PCR. The hVEGF165 gene was recombined into T vector, identified by PCR and sequenced. After double enzyme digestion, the recombinant plasmid was cloned into eukaryotic expression vector pcDNA3 1 / myc his B 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 myocardium by RT-PCR. The pcDNA3.1 / hVEGF165 was successfully constructed and transiently transfected into COS-7 cells. Conclusion The constructed pcDNA3.1 / hVEGF165 transfected eukaryotic COS 7 can express rhVEGF protein.