| 新型重组AAV5/5载体及包装系统 |
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| 引用本文： | 董小岩,田文洪,袁振华,谭淑萍,吴小兵.新型重组AAV5/5载体及包装系统[J].生物工程学报,2010,26(5):679-686. |
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| 作者姓名： | 董小岩 田文洪 袁振华 谭淑萍 吴小兵 |
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| 作者单位： | 1. 中国疾病预防控制中心病毒病预防控制所,病毒基因工程国家重点实验室,北京,100052;复旦大学生命科学学院遗传学研究所,遗传工程国家重点实验室,上海,200433;北京五加和分子医学研究所有限公司,北京,100176
2. 中国疾病预防控制中心病毒病预防控制所,病毒基因工程国家重点实验室,北京,100052 |
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| 基金项目： | 国家高技术研究发展计划 (863计划) (No. 2007AA021202)，病毒基因工程国家重点实验室自主课题 (No. 2008-s-0008) 资助。 |
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| 摘 要： | 本研究组建了一种可用于规模化生产的以重组单纯疱疹病毒为辅助病毒的AAV5/5载体包装系统。首先，将5型腺相关病毒 (AAV5) 的rep和cap基因插入I型单纯疱疹病毒 (HSV-1) 基因组非必需基因UL2中，获得重组病毒rHSV1-rep5cap5。其次，构建一种携带AAV5 ITR的通用型载体质粒pAAV5neo，将报告基因EGFP插入pAAV5neo中，得到pAAV5neo-EGFP质粒。将pAAV5neo-EGFP质粒导入BHK-21细胞，用G418选择培养，挑选出表达EGFP并在重组病毒rHSV1-rep5cap5感染下能高效产生rAAV5/5-EGFP的单克隆载体细胞株C020。用rHSV1-rep5cap5感染C020细胞制备rAAV5/5-EGFP，用“氯仿处理-聚乙二醇/氯化钠-氯仿抽提”方法粗纯化rAAV5/5-EGFP。用100 kDa分子量截流超滤方法进一步纯化和浓缩，获得高纯度的rAAV5-EGFP。SDS-PAGE电泳分析可见3条特征性外壳蛋白带。电镜分析显示病毒颗粒以实心颗粒为主。用rAAV5/5-EGFP病毒按1×105 vg/cell感染体外培养的HEK293细胞，可见30％细胞呈现绿色荧光。本研究提出了一种高效AAV5/5载体生产系统和纯化方法，为重组AAV5载体的进一步应用提供了基础。
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| 关 键 词： | 5型腺相关病毒 (AAV5)，I型单纯疱疹病毒 (HSV1)，病毒载体，包装系统 |
| 收稿时间： | 1/3/2010 12:00:00 AM |
A novel packaging system of recombinant AAV5/5 vector |
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| Xiaoyan Dong,Wenhong Tian,Zhenhua Yuan,Shuping Tan and Xiaobing Wu.A novel packaging system of recombinant AAV5/5 vector[J].Chinese Journal of Biotechnology,2010,26(5):679-686. |
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| Authors: | Xiaoyan Dong Wenhong Tian Zhenhua Yuan Shuping Tan and Xiaobing Wu |
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| Institution: | State Key Laboratory for Molecular Virology and Genetic Engineering, Institute for Viral Disease Prevention and Control, Chinese Center for Disease Prevention and Control, Beijing 100052, China; State Key Laboratory of Genetic Engineering, Institute of Ge;State Key Laboratory for Molecular Virology and Genetic Engineering, Institute for Viral Disease Prevention and Control, Chinese Center for Disease Prevention and Control, Beijing 100052, China;State Key Laboratory for Molecular Virology and Genetic Engineering, Institute for Viral Disease Prevention and Control, Chinese Center for Disease Prevention and Control, Beijing 100052, China;State Key Laboratory for Molecular Virology and Genetic Engineering, Institute for Viral Disease Prevention and Control, Chinese Center for Disease Prevention and Control, Beijing 100052, China;State Key Laboratory for Molecular Virology and Genetic Engineering, Institute for Viral Disease Prevention and Control, Chinese Center for Disease Prevention and Control, Beijing 100052, China |
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| Abstract: | We developed a scalable AAV5/5 vector packaging system by using replication competent recombinant herpes simplex type 1 virus as helper virus. The fragment containing rep and cap genes of AAV5 was inserted into the non-necessary gene (UL2) of HSV1 genome, resulting in the helper virus rHSV1-rep5cap5. An AAV5/5 vector pAAV5neo carrying two AAV5 ITRs was constructed by inserting a neo gene expression cassette into the plasmid backbone of pAV5CMV-GFP. pAAV5neo-EGFP was constructed by inserting EGFP gene into p... |
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| Keywords: | Adeno-associated virus type 5 (AAV5) herpes simplex virus I virus vector production system |
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