人血管内皮生长因子基因的克隆及真核表达质粒的构建

CLONING OF HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR GENE AND THE CONSTRUCTION OF ITS EXPRESSIVE PLASIMAD

目的 :克隆人血管内皮生长因子基因 VEGF1 6 5片段 ,构建 pc DNA3/ VEGF1 6 5真核表达质粒。方法 :采用逆转录聚合酶链式反应 (RT- PCR)技术 ,从人肝癌细胞株 SMMC- 772 1中扩增出血管内皮生长因子 VEGF1 6 5基因片段 ,通过 DNA重组技术将该基因片段重组于 pc DNA3真核表达载体上 ,构建成 pc DNA3/ VEGF1 6 5重组质粒 ,分别用 PCR扩增、酶切电泳分析及 DNA测序的方法对重组 DNA进行鉴定。结果 :经 PCR扩增、酶切电泳分析和 DNA测序证实 ,本实验构建的重组质粒目的基因片段为人 VEGF1 6 5c DNA。结论 :本实验成功地克隆了 VEGF1 6 5基因并构建了其真核表达质粒 ,为进一步研究用 VEGF基因转染的方法来恢复或增强放疗后组织的血管生成能力奠定了基础。

Objective:The aim of this study is to clone VEGF gene and to construct the expressive plamsid pcDNA 3/VEGF 165Methods:Human vascular endothelial growth factor gene was amplified by RT-PCR method from human cells SMMC-7721 strains and constructed into the expressive plamsid pcDNA 3/VEGF 165. The gene in the expressive plamsid pcDNA 3/VEGF 165 is identified by PCR amplifcation, enzyme digestion and DNA sequencing.Result:The cloned DNA was confirmed to be VEGF 165 gene.Conclusion:In this study we successfully cloned VEGF 165 gene and constructed its expressive plamsid pcDNA 3/VEGF 165, which provided the foundation of using VEGF gene therapy to resume or build up the angiogenesis of ischemia tissues in vivo.