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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2011, Vol. 31 Issue (11): 51-57    
研究报告     
Ad-Ang1-IRES-VEGF165腺病毒载体的构建及其促血管形成作用
刘铁连1,2, 陈佑明2, 黄敬2, 李华2, 杨吉成1
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
Construction of Adenovirus-mediated Ang1 and VEGF165 Co-expression Adenovirus Vector and Its Proangiogenesic Activity
LIU Tie-lian1,2, CHEN You-ming2, HUANG Jing2, LI Hua2, YANG Ji-cheng1
1. Cell and Molecular Biology Institute, College of Medicine, Soochow University, Suzhou 215123,China;
2. Third Affiliated Hospital of Southern Medical University, Guangzhou 510630,China
 全文: PDF(708 KB)   HTML
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。

关键词: 血管生成素1血管内皮生长因子腺病构建    
Abstract:

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 4×1010 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.

Key words: Angiogenin-1    Vascular endothelial growth factor    Adenovirus    Construction
收稿日期: 2011-05-03 出版日期: 2011-11-25
ZTFLH:  Q81  
基金资助:

国家"973"计划资助项目(2005CB623906)

通讯作者: 杨吉成     E-mail: jcyang@suda.edu.cn
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引用本文:

刘铁连, 陈佑明, 黄敬, 李华, 杨吉成. Ad-Ang1-IRES-VEGF165腺病毒载体的构建及其促血管形成作用[J]. 中国生物工程杂志, 2011, 31(11): 51-57.

LIU Tie-lian, CHEN You-ming, HUANG Jing, LI Hua, YANG Ji-cheng. Construction of Adenovirus-mediated Ang1 and VEGF165 Co-expression Adenovirus Vector and Its Proangiogenesic Activity. China Biotechnology, 2011, 31(11): 51-57.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I11/51


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