VEGF基因表达载体的构建及其表达

Expression of recombined plasmid encoding vascular endothelial growth factor

为构建血管内皮生长因子(VEGF)基因的表达载体,以人胎肝cDNA库为模板,应用多聚酶链反应(PCR)技术,扩增全长的VEGF165cDNA序列,定向连接进质粒pcDNA3.1/Zeo(+)的多克隆位点中,以重组质粒转染体外培养的293细胞,收集不同时相细胞裂解液进行蛋白质的聚丙烯酰胺凝胶电泳,以抗人VEGF165单克隆抗体行Westen blotting检测,观察VEGF基因的表达情况。结果显示选择合适的退火温度和引物,可以自人胎肝cDNA库中特异性地扩增VEGF165的cDNA序列。所构建的pcDNA3.1/Zeo(+)-VEGF165真核表达载体系统能较好地转染体外培养的293细胞,Westen blotting检测到特异性表达条带。提示重组的VEGF165基因表达载体能够成功转染哺乳动物细胞,并表达目的基因,为解决组织移植中血运重建问题提供了一条基因治疗途径。

To construct a recombined plasmid encoding vascular endothelial growth factor 165 (VEGF165),which can be used to promote angiogenesis in the tissue transplantation by gene therapy,VEGF165 cDNA wasamplified by polymerase chain reaction (PCR) from the human fetal liver cDNA libraries. The fragment withcorrect DNA sequence was selected, and then linked into a mammalian expression vector pcDNA3. 1/Zeo (+).Preparation and purification of pcDNA3. 1/Zeo (+)-VEGF165 was operated under the protocol of QIAGENendofree...