VEGF165-tPA双基因真核表达载体的构建和体外表达

Construction and in vitro Expression of Human Vascular Endothelial Growth Factor 165 and Tissue-type Plasminogen Activator Gene Recombinant Eukaryotic Expression Vector

目的构建人血管内皮细胞生长因子165(VEGF165)和人组织型纤溶酶原激活因子(tPA)双基因的新型真核表达载体pIRES-VEGF165-tPA,并通过转染人脐静脉内皮细胞系EA.hy926细胞,验证载体的体外表达情况。方法将目的基因片段VEGF165和tPA插入到真核表达载体pIRES质粒中,体外瞬时转染人脐静脉内皮细胞系EA.hy926细胞,通过实时荧光定量RT-PCR和Western blot分别检测mRNA和蛋白水平的表达。结果成功构建pIRES-VEGF165-tPA双基因真核表达载体,测序结果与预期相符;荧光计数显示体外瞬时转染人脐静脉内皮细胞系EA.hy926细胞的转染效率约为(15.6±3.1)%;实时荧光定量RT-PCR和Western blot证实转染pIRES-VEGF165-tPA质粒组细胞在mRNA和蛋白水平的表达均高于各对照组(均P<0.01)。结论成功构建pIRES-VEGF165-tPA双基因真核表达载体,并且实现其在人脐静脉内皮细胞系EA.hy926细胞中的表达,为进一步探索其在机械瓣膜抗凝方面的作用奠定了基础。

Objective To construct a recombinant eukaryotic expression vector pIRES-VEGF165-tPA containing functional region of human vascular endothelial growth factor 165（VEGF165）and tissue-type plasminogen activator（tPA）gene,and investigate their in vitro expression in transfected human umbilical vein endothelial cells（EA.hy926）.Methods The human VEGF165 and tPA genes were inserted into the eukaryotic expression vector pIRES to construct the recombinant plasmid that was transfected into human umbilical vein endothelial cells（EA.hy926）subsequently.The transcription and expression of VEGF165 and tPA genes were detected by real-time fluorescent quantitative RT-PCR and Western blot respectively.Results The sequencing of pIRES-VEGF165-tPA approved that the genes of VEGF165 and tPA were inserted into the eukaryotic expression vector correctly.The transfection efficiency was about（15.6±3.1）%,detected by counting the positive cells of green fluorescence.The mRNA and protein expression levels of VEGF165 and tPA in the pIRES-VEGF165-tPA transfected group were significantly higher than those in the control groups（P0.01）.Conclusion The recombinant eukaryotic expression vector pIRES-VEGF165-tPA is successfully constructed and can be expressed in transfected EA.hy926 cells,which lay a foundation for the subsequent study of its anticoagulant function in patients with mechanical heart valves.