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  • 范丽1 ,徐增辉2 ,金华君2 ,徐凤青1 ,丁娜1 ,严淋2 ,钱其军1,2*.应用杆状病毒系统制备8型腺相关病毒[J].第二军医大学学报,2011,32(2):134-138    [点击复制]
  • FAN Li1,XU Zeng-hui2,JIN Hua-jun2,XU Feng-qing1, DING Na1,YAN Lin2, QIAN Qi-jun1,2*.Preparation of adeno-associated virus serotype 8 using baculovirus system[J].Acad J Sec Mil Med Univ,2011,32(2):134-138   [点击复制]
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应用杆状病毒系统制备8型腺相关病毒
范丽1,徐增辉2,金华君2,徐凤青1,丁娜1,严淋2,钱其军1,2*
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(1.浙江理工大学生命科学学院新元医学与生物技术研究所,杭州 310018; 2.第二军医大学东方肝胆外科医院病毒基因治疗实验室,上海 200438)
摘要:
\[摘要\]目的建立应用杆状病毒系统制备8型腺相关病毒(adeno-associated virus, AAV)的技术体系,并初步检测所制备病毒的感染活力。方法采用杆状病毒生产系统包装rAAV8-EGFP,经高效液相色谱法提取并纯化病毒,实时荧光定量PCR测定病毒滴度,通过观察绿色荧光蛋白的表达强度来检测rAAV8-EGFP对HEK-293细胞的感染活力。结果实时荧光定量PCR显示成功制备高滴度rAAV8-EGFP,滴度可达1.5×1012 vg/ml,100 ml摇瓶共得到1.5×1013 vg rAAV8-EGFP病毒颗粒;感染后的HEK-293细胞具有较强的绿色荧光蛋白表达。结论应用杆状病毒系统成功制备高滴度、高活力的重组腺相关病毒,为后续研究奠定了基础。
关键词:  杆状病毒系统  腺相关病毒  昆虫细胞
DOI:10.3724/SP.J.1008.2011.0134
投稿时间:2010-11-30修订日期:2011-01-04
基金项目:国家“十一五”新药创制重大专项(2009ZX09103-687),国家自然科学基金(30772477).
Preparation of adeno-associated virus serotype 8 using baculovirus system
FAN Li1,XU Zeng-hui2,JIN Hua-jun2,XU Feng-qing1, DING Na1,YAN Lin2, QIAN Qi-jun1,2*
(1. Xinyuan Institute of Medicine and Biotechnology, School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, Zhejiang, China; 2. Laboratory of Virus and Gene Therapy, Eastern Hepatobiliary Surgical Hospital, Second Military Medical University,Shanghai 200438, China)
Abstract:
\[Abstract\]ObjectiveTo set up a baculovirus system for preparing recombinant adeno-associated virus serotype 8 (rAAV8) and to preliminarily examine the infection viability of the prepared virus in vitro. MethodsThe rAAV8-EGFP was prepared using baculovirus system and was subsequently extracted and purified by high performance liquid chromatography. The viral titer was determined by real-time quantitative PCR and its infection viability for HEK-293 cells was examined by observing the EGFP reporter. ResultsReal-time quantitative PCR showed that high titer of rAAV8-EGFP was prepared (1.5×1013 vg/bottle\[100 ml medium\]) and strong expression of EGFP was observed in HEK-293 cells infected with rAAV8-EGFP. ConclusionThe baculovirus system is capable of producing rAAV8 with high titer and infection viability, paving a way for future research.
Key words:  baculovirus system  adeno-associated virus  insect cell