应用杆状病毒系统制备8型腺相关病毒  

Preparation of adeno-associated virus serotype 8 using baculovirus system

作　　者：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] 

机构地区：[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

出　　处：《第二军医大学学报》2011年第2期134-138,共5页Academic Journal of Second Military Medical University

基　　金：国家"十一五"新药创制重大专项(2009ZX09103-687);国家自然科学基金(30772477)~~

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1.5×1012vg/ml,100ml摇瓶共得到1.5×1013vg 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。Objective To set up a baculovirus system for preparing recombinant adeno-assoeiated virus serotype 8 （rAAV8） and to preliminarily examine the infection viability of the prepared virus in vitro. Methods The 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. Results Real-time quantitative PCR showed that high titer of rAAVS-EGFP was prepared （1.5X 10^13vg/bottle[100 ml medium]） and strong expression of EGFP was observed in HEK-293 cells infected with rAAV8-EGFP. Conclusion The baculovirus system is capable of producing rAAV8 with high titer and infection viability, paving a way for future research.

关 键 词：杆状病毒系统 腺相关病毒 昆虫细胞 

分 类 号：Q782[生物学—分子生物学]