应用杆状病毒系统制备8型腺相关病毒

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。

Preparation of adeno-associated virus serotype 8 using baculovirus system

[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.