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

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

目的 :克隆和在 COS- 7细胞中表达人血管内皮生长因子 16 5 (VEGF16 5 )。方法 :利用 RT- PCR方法 ,从新鲜卵巢癌组织中扩增得到人 VEGF16 5的全长 c DNA,并测定其核酸序列 ;利用基因重组技术构建 VEGF16 5的真核表达载体 pc DNA3.1- VEGF16 5 ;应用脂质体介导的基因转移技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 VEGF16 5 c DNA,重组质粒 pc DNA3.1- VEGF16 5转染 COS- 7细胞后 ,免疫组化检测有 VEGF表达。结论 :成功克隆人 VEGF 16 5基因 ,并在 COS- 7细胞中获得表达。

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.