Ad-Ang1-IRES-VEGF165腺病毒载体的构建及其促血管形成作用  

Construction of Adenovirus-mediated Ang1 and VEGF165 Co-expression Adenovirus Vector and Its Proangiogenesic Activity

作　　者：刘铁连[1,2] 陈佑明[2] 黄敬[2] 李华[2] 杨吉成[1] 

机构地区：[1]苏州大学医学部基础医学与生物科学学院,苏州215123 [2]南方医科大学第三附属医院,广州510630

出　　处：《中国生物工程杂志》2011年第11期51-57,共7页China Biotechnology

基　　金：国家"973"计划资助项目(2005CB623906)

摘　　要：目的:构建人血管生成素1(Ang1)和血管内皮生长因子VEGF165(VEGF165)的共表达腺病毒载体Ad-Ang1-IRES-VEGF165(简称Ad-AV),为研究Ad-AV转基因细胞表达产物血管诱生活性提供实验依据。方法:采用IRES介导的Ang1和VEGF165双基因腺病毒共表达模式,通过常规的基因克隆和重组技术,构建Ad-AV双基因共表达腺病毒载体,经感染人胚肾QBI-293A细胞(293A)进行扩增和效价测定后,再感染WI-38人胚肺成纤维细胞,均用ELISA法检测VEGF、Ang1目的基因的表达,并采用鸡胚尿囊膜血管形成实验(CAM)法分析其对血管形成的影响。结果:扩增的Ad-AV腺病毒效价可达4×1010pfu/ml;Ad-AV不仅能在293A细胞中成功表达目的基因Ang1、VEGF,而且在WI-38成纤维细胞也能成功表达,其表达产物具有显著的促进CAM上血管生成的活性。结论:成功构建并获得了Ad-Ang1-IRES-VEGF165重组病毒子,目的基因均能在人胚肾和人胚肺成纤维细胞中表达,其表达产物具有诱导血管形成的功能。Objective： To construct a recombinant adenoviral vector co-expressing angiogenin-1（Ang-1） and vascular endothelial growth factor（VEGF） and to provide experimental evidence for the effect of the transgenic fibroblasts on inducing angiogenesis.Methods： The co-expressing adenoviral vector Ad-Ang1-IRES-VEGF165 was constructed by cloned sequence and recombined technology.The recombinant adenoviruses were transfected into QBI-293A cells and tested it＇s titer.Then the WI-38 cells were transfected by the adenovirus Ad-AV and the expression of Ang1 and VEGF were identified by enzyme-labeled immunosorbent assay（ELISA）.Meanwhile,the vessel forming test of chorioallantoic membrane（CAM） of enbryonated eggs was made to observe the growth of vessels.Results： The recombined adenovirus Ad-AV could be constructed and it was successfully packaged with the titer being as high as 4×10^10 pfu/ml.The aim gene Ang-1 and VEGF165 could be effectively express in 293A cells and WI-38 cells.The CAM blood vessel of test group added with culture supernatantwere was more than that in control group（P〈0.01）.Conclusion： The recombined adenovirus Ad-AV can be constructed successfully.Both Ang-1 and VEGF165 effectively express in target cells and the target gene had a marked proangiogenesic activity.

关 键 词：血管生成素1 血管内皮生长因子 腺病 构建 

分 类 号：Q81[生物学—生物工程]