以痘苗病毒为辅助病毒生产慢病毒载体的探究  

Application of A Vaccinia Replication System for Manufacturing of Lentiviral Vectors

作　　者：马强[1] 李明[1] 吴英松[1] 

机构地区：[1]南方医科大学生物技术学院,广州510515

出　　处：《热带医学杂志》2006年第5期503-506,F0003,共5页Journal of Tropical Medicine

基　　金：国家自然科学基金(No.30300316)

摘　　要：目的建立新型、高产量的慢病毒衍生载体的生产体系。方法构建主框架质粒pVECRNA、包装质粒pGAGPOL及包膜质粒pVSVG。通过脂质体将这三个质粒共转染至BHK21细胞,再用含有T7RNA聚合酶基因的重组痘苗病毒vTF-3感染细胞,培养4d后,收集培养上清,0.22μm滤膜过滤。结果将培养上清进行RT-PCR反应,结果能扩增出96bp长度的目的条带,将培养上清感染BHK21细胞,检测到BHK21细胞中GFP的表达,测定病毒载体滴度为(1.45±0.30)×107tu/ml。结论已初步生产出慢病毒载体,为下一步生产系统建立,系统产量优化奠定良好基础。Objective To establish a high titer production system for Lentiviral vector. Methods In this system, BHK2t Cells were co-transfected by a mixture of plasmids containing the transducing plasmid pVECRNA, the packaging plasmid pGAGPOL, and the envelop plasmid pVSVG with Lipofectamin2000^TM followed by infection with the vaccinia vTF-3 which contains the bacteriophage T7 RNA polymerase gene. 4 days after transfection culture supematant was collected and filtered with 0.22μm filter membrane. Results As judged from the size of PCR products （96bp）, lentiviral vectors were found in the culture supernatant. Infection of BHK2t ceils with the culture supernatant resulted in the expression of GFP. The titers were about （1.45 ± 0.30）×10^7 tu/ml. Conclusion Lentiviral vector can be produced with this new method. It provides the basis for the future development.

关 键 词：慢病毒载体 痘苗表达系统 基因治疗 

分 类 号：R394[医药卫生—医学遗传学]