血管内皮生长因子(VEGF165)真核表达载体的构建与鉴定

Construction of adenoviral vector encoding human VEGF cDNA and its expression in vitro

目的 构建人VEGF16 5腺病毒表达载体 ,探讨应用血管内皮生长因子 (VEGF)基因治疗脑梗死的可行性。方法 采用分子克隆技术 ,以质粒hVEGF16 5·SR为模板 ,扩增hVEGF16 5成熟cDNA。经穿梭质粒PShuttle克隆到腺病毒质粒上 ,构成Adeno VEGF16 5腺病毒重组质粒 ,经酶切及测序鉴定正确后 ,包装成为重组Adeno VEGF16 5腺病毒 ,并进行电镜观察及滴度测定。用免疫印迹及免疫组化方法检测VEGF蛋白的表达。结果 经酶切鉴定及基因测序证实重组腺病毒质粒构建成功 ,电镜显示包装细胞中有病毒存在 ,包装的病毒滴度为 8× 10 1 0 pfu ml,免疫印迹及免疫组化检测有VEGF16 5蛋白的表达 ,而对照未见VEGF16 5转录和表达。结论 构建的VEGF16 5腺病毒表达载体可在体外表达 。

Objective To construct the adenoviral vector bringing VEGF cDNA for evaluation of the possibility of VEGF gene therapy in ischemic disease. Methods Human vascular endothelial growth factor cDNA obtained form the plasmid VEGF was cloned into plasmid pshuttle and further cloned to Adeno X Viral DNA. The recombinant adenoviral plasmid was identified and them transferred to the adenoviral packaging cell HEK293 by lipofectamine mediated gene transfer method to pack the virus. Results The recombinant Adeno VEGF was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. After MSCs were transfected by the virus, RT PCR showed that VEGF mRNA was transcripted from the VEGF gene. Conclusion The recombinant adenoviral vector bringing VEGF was successfully constructed.