Bac-to-Bac杆状病毒昆虫表达系统介导的重组腺相关病毒制备  

Production of adeno-associated virus mediated by Bac-to-Bac baculovirus insect expression system

作　　者：陈雅宁[1] 王春荣[1] 杨宗灿[1] 姬朝霞[1] 张延瑞[1] 韩双印[1] 

机构地区：[1]郑州大学人民医院中心实验室,450003

出　　处：《中华实验外科杂志》2012年第7期1363-1366,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81172415）;河南省重点科技计划攻关项目（102102310065）;河南省医学科技计划攻关项目（201-1020117）

摘　　要：目的建立高效的临床级重组腺相关病毒（rAAV）制备方法。方法将rAAV的反向末端重复序列（ITR）及目的基因表达框（2053bp）从pAAV—MCS剪切引入杆状病毒载体中构建顺式载体，rAAV血清型2的衣壳和复制蛋白基因在反式载体pFastBacDual中利用真核基因遗漏扫描原理表达，两者分别制备成杆状病毒，共感染昆虫细胞sf9包装rAAV。增强绿色荧光蛋白（EGFP）作为报告基因验证其可行性。结果顺式和反式杆状病毒载体分别在DH10BacTM中转座形成杆粒bacmid、昆虫细胞sf9中制备成杆状病毒，病毒滴度可达1×10^8～3×10^8v．g．／ml。二杆状病毒共感染昆虫细胞sf9，完成rAAV拯救、复制和包装过程，经rAAV—EGFP感染293T细胞测试具有良好的生物学活性。结论该系统具有高效、安全、简便等特点，为临床级rAAV制备和基因治疗奠定了基础。Objective To establish an efficient and safe method for production of recombinant ad- eno-associated viruses （rAAV） of clinical scale. Methods The 2053 bp of rAAV inverted terminal repeat （ITR） and foreign gene expression cassette was cloned into baculovirus vector as cis-acting vector. The capsid and replication proteins of rAAV serum type 2 were expressed in a trans-acting baculovirus vector （pFastBacDual） by leaky scanning mechanism of eukaryotic gene transcription. Both constructs were made into baculovirus which infected insect cells sf9 for packaging rAAV. Enhanced green fluorescent protein （EGFP） was used as reporter gene to verify the feasibility of system. Results Transposition of cis-acting and trans-acting vector into bacmid was carried out in DH10BacTM respectively. Two baculoviruses were prepared in insect cells sf9 and the liters were about 1 × 10s-3 × l0s v. g./mL, rAAV were rescued, repli- cated and packaged by co-infection of two baculoviruses into sfg. The biological activity of rAAV-EGFP was proved by infecting 293T cells. Conclusion This system provides an experimental data for making rAAV of clinical scale and a good basis for future gene therapy.

关 键 词：杆状病毒昆虫表达系统 腺相关病毒 SF9细胞 

分 类 号：R3[医药卫生—基础医学]