| 应用AdEasy腺病毒载体系统构建血管内皮生长因子165重组腺病毒 |
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| 引用本文： | 高全文,宋慧锋,柴家科,柳春明. 应用AdEasy腺病毒载体系统构建血管内皮生长因子165重组腺病毒[J]. 组织工程与重建外科, 2008, 4(5): 263-266 |
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| 作者姓名： | 高全文 宋慧锋 柴家科 柳春明 |
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| 作者单位： | [1]中国人民解放军总医院第一附属医院烧伤整形科,北京市100037 [2]中国人民解放军总医院,北京市100853 |
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| 摘 要： | 目的利用AdEasy腺病毒载体系统构建血管内皮生长因子165（VEGF165）重组腺病毒，并在293T细胞中扩增。方法将PCR获取的VEGF165基因酶切并插入到pAdTrack-CMV中，构建成腺病毒穿梭质粒pAdTrack-VEGF165，经PmeI酶切线性化后，采用电穿孔转化到事先电转化腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌感受态细胞中，挑选同源重组菌落。提取质粒并用Pac I酶切鉴定，线性化重组质粒pAdEasy-VEGF165转染293T细胞，包装成重组腺病毒颗粒，荧光显微镜下观察绿色荧光表达，并扩增收集重组腺病毒，测定病毒滴度。结果经限制性内切酶检测、基因测序及和绿色荧光观察证实成功构建了携带VEGF165基因的重组腺病毒，并扩增出10^9pfu／mL的高滴度重组腺病毒。结论成功构建了携带VEGF165基因的重组腺病毒载体，为研究骨组织工程血管化局部基因治疗奠定了基础。
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| 关 键 词： | 腺病毒 血管内皮生长因子165 基因工程 |
Construction of Recombinant Adenovirus Vector Carrying the Vascular Endothelial Growth Factor 165 Using the AdEasy System |
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| Gao Quanwen SONG Huifeng CHAI Jiake LIU Chunming. Construction of Recombinant Adenovirus Vector Carrying the Vascular Endothelial Growth Factor 165 Using the AdEasy System[J]. , 2008, 4(5): 263-266 |
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| Authors: | Gao Quanwen SONG Huifeng CHAI Jiake LIU Chunming |
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| Affiliation: | Gao Quanwen SONG Huifeng CHAI Jiake LIU Chunming.1 Department of Burns , Plastic Surgery,First Affiliated Hospital of PLA General Hospital,Beijing 100037,China.2 Department of Plastic Surgery,PLA General Hospital,Beijing 100853,China.Corresponding author:LIU Chunming. |
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| Abstract: | Objective To construct recombinant adenovirus vector carrying vascular endothelial growth factor 165 (VEGF165) and amplify the adenovirus vector in 293T cells.Methods VEGF165 obtained by PCR was digested and inserted into adenovirus shuttle plasmid pAdTraek-CMV to generate recombinant plasmid pAdTrack-VEGF165,and then the Pme I-linearized plasmid pAdTrack-VEGF165 was electroporated into E.coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1.The identified recombinant plasmid ... |
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| Keywords: | Adenovirus Vascular endothelial growth factor 165 Gene engineering |
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