人血管内皮生长因子基因cDNA的克隆及其在COS-7细胞中的表达

目的：克隆和在COS－7细胞中表达人血管内皮生长因子165（VEGF165）。方法：利用RT－PCR方法，从新鲜卵巢癌组织中扩增到人VEGF165的全长cDNA，并测定其核酸序列；利用基因重组技术构建VEGF165的真核表达载体pcDNA3.1-VEGF165；应用脂质体介导的基因转移术将这一表达载体导入COS－7细胞后，免疫组化（SP法）检测瞬时表达的产物。结果：经酶切鉴定和基因测序证实，克隆的基因片段为人VEGF65cDNA，重组质粒pcDNA3．1－VEGF165转染COS－7细胞后，免疫组化检测有VEGF表达，结论：成功克隆人VEGF165基因，并在COS－7细胞获得表达。

Cloning of human vascular endothelial growth factor cDNA and its expression in COS-7 cells

Objective: To clone vascular endothelial growth factor ( VEGF ) cDNA gene, construct its eukaryotic expression vector and to express this recombinant plasmid in COS-7 cells. Methods: Human VEGF165 cDNA was amplified by RT-PCR from human ovarian carcinoma. After DNA sequenced, the VEGF165 cDNA was inserted into eukaryotic expression vector pcDNA3.1(-) . The recombinant plasmid pcDNA3.1-VEGF165 containing VEGF165 cDNA was identified by enzyme digestion and transferred into COS-7 cells mediated by liposome. The transient expression of VEGF was detected by immunohistochemical staining. Results: The cloned VEGF165 cDNA was confirmed by enzyme digestion and DNA sequence analysis. The immunohistochemical results showed that the VEGF165 protein was expression in COS-7 cells 72 h after gene transfer. Conclusion: VEGF165 cDNA gene successfully cloned and expressed in COS-7 cells.