人血管内皮细胞生长因子165基因真核表达载体的构建及其在大肠杆菌的表达

Construction and expression of eukaryotic expression vector containing Human vascular endothelial growth factor 165 gene

目的 获得人血管内皮生长因子 (hVEGF165)cDNA ,构建真核表达载体 ,并研究其在大肠杆菌中的表达情况。方法 采用PCR方法 ,从HL6 0细胞中扩增出hVEGF165cDNA ,将其克隆至 pcDNA3 真核表达载体上 ,构建成为 pCD hVEGF165重组质粒。将重组质粒转染感受态的大肠杆菌BL2 1(DE3)中进行表达 ,用Westernblot检测表达产物。结果 经酶切鉴定及基因测序证实hVEGF165cDNA基因克隆成功 ,并建立了高效表达的 pCD hVEGF165重组质粒。Westernblot检测表明 ,组建了具有高效表达hVEGF165的大肠杆菌菌株。结论 成功地克隆和表达出了hVEGF165基因 ,为进一步建立VEGF转基因动物模型 。

Objective To clone human vascular endothelial growth factor 165 (hVEGF_(165)), construct its eukaryotic expression and to study the expression of hVEGF_(165) in E.coli.Method The hVEGF_(165) cDNA was amplified by PCR method from the HL60 cells and cloned into expression vector pcDNA_3.The pCD-hVEGF_(165) recombinant plasmids were transformed to E.coli BL21 (DE3) cell.The expressed product was detected by Western blot.Results The cloned cDNA was confirmed to be hVEGF_(165) cDNA and the expression of human VEGF gene was detected distinctly 72h after transferring. The Western blot result showed that the hVEGF_(165) protein was expressed in E.coli BL21 (DE3).Conclusion The hGEF_(165) gene was cloned and expressed successfully,which provides the further foundation of the model of VEGF transgenic animal and makes a basis for the further study in retinal neovascularization.