血管内皮生长因子重组DNA的构建及体外表达

目的:以质粒PCNDA3.0为载体,研究血管内皮生长因子(VEGF)目的基因在体外真核细胞中的表达.方法:将编码人可溶性VEGF165的目的基因克隆于带有CMV启动子/增强子的PCNDA3.0载体上,重组为PCNDA3.0-VEGF165质粒载体.直接以质粒DNA形式应用脂质体介导的方法转染人脐静脉内皮细胞ECV304,收取细胞上清,用ELISA和Western blot方法检测上清中VEGF165可溶性表达产物.结果:VEGF165基因在CMV启动子/增强子的调控下,可在ECV304细胞高效表达,且在转染后72 h表达量明显高于转染后48 h的表达.结论:以质粒PCNDA3.0为载体,VEGF165基因可以在体外真核细胞中表达,为治疗缺血性疾病提供一个新的基因治疗途径.

Construction of recombinant DNA encoding human vascular endothelial growth factor and it＇s expression in vitro

Objective: To construct a plasmid vector encoding human vascular endothelial growth factor (VEGF) and analyse its expression in eukaryotic cells in vitro. Methods: A soluble human VEGF 165 gene was cloned into the vector PCNDA3.0 which contained the human cytomegalovirus (CMV) promotor/enhancer. The recombined PCNDA3.0 VEGF vector was used directly to transfect to endothelial cells of the human umbilical vein ECV304. The cells were transfected with liposome. The supernatant then was collected and the soluble protein of VEGF gene expression in the supernatant was analysed with ELISA and Western blot.Results: The recombined plasmid vector might express VEGF in ECV304 cell line under the regulation of human CMV promotor/enhancer. Conclusion: This vector with PCNDA3.0 VEGF can express VEGF in eukaryotic cells in vitro and may be useful in the treatmant of ischemic disorders.