论文部分内容阅读
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 the expression vector of vascular endothelial growth factor (VEGF) gene, the human fetal liver cDNA library was used as a template to amplify the full-length VEGF165 cDNA sequence by polymerase chain reaction (PCR) technology and to be directed into the plasmid pcDNA3.1 / Zeo +) Were transfected into 293 cells in vitro by recombinant plasmids. Cell lysates from different phases were collected and subjected to polyacrylamide gel electrophoresis. The anti-VEGF165 monoclonal antibodies were detected by Westen blotting and observed VEGF gene expression. The results show that the cDNA sequence of VEGF165 can be specifically amplified from human fetal liver cDNA library by selecting appropriate annealing temperature and primers. The constructed pcDNA3.1 / Zeo (+) - VEGF165 eukaryotic expression vector system can be well transfected 293 cells cultured in vitro, Westen blotting detected specific expression bands. It suggested that the recombinant VEGF165 gene expression vector could successfully transfect mammalian cells and express the target gene, which provided a gene therapy approach to solve the problem of revascularization during tissue transplantation.