真核双表达载体pIRES-hVEGF121 cDNA/hBMP-4的构建与鉴定

目的构建人血管内皮生长因子(hVEGF121)和人骨形态发生蛋白-4(hBMP-4)的真核双表达质粒,转染COS-7细胞并表达。方法将hVEGF121 cDNA和hBMP-4通过非定向和定向克隆至真核双表达质粒pIRES中,重组质粒pIRES-hVEGF121cDNA/hBMP-4,经酶切鉴定后,转染COS-7细胞,提取总RNA和总蛋白,进行RT-PCR和Western blot检测。结果真核双表达载体pIRES-hVEGF121cDNA/hBMP-4构建成功且hVEGF121cDNA和hBMP-4在COS-7细胞中得到了有效表达。结论hVEGF121和hBMP-4在COS-7细胞中成功表达,为进一步研究打下良好基础。

Construction and identification of eukaryotic coexpression vector pIRES-hVEGF121 cDNA/hBMP-4

Objective To construct the eukaryotic coexpression plasmid encoding vascular endothelial growth factor (VEGF_(121) cDNA) and human bone morphogenesia protein-4(hBMP-4), and detect the expression of the plasmid in vitro. Methods VEGF121 cDNA and BMP-4 were cloned into an eukaryotic expression plasmid internal ribosme entrysite(pIRES).The constructed plasmids pIRES-hVEGF121 cDNA/hBMP-4 were confirmed by restriction enzymolysis and then fransfected into COS-7.Then the total RNA and total proteins extracted from the resulting combination were detected by RT-PCR and Western blot.Results The eukaryotic coexpression plasmid pIRES-VEGF121 cDNA/BMP-4 was constructed successfully and VEGF121 cDNA and hBMP-4 the plasmid could coexpress coexpressed efficiently in COS-7.Conclusion The fransfection of the target gene into eukaryotic expression plasmid by non-direct cloning and direct cloning technology offers the possibility of a novel approach of local combination gene therapy of bone regeneration.