一种新型慢病毒载体制备方法的建立

A Novel System for Producing Lentiviral Vectors

摘要:

为了建立新型、高产量的慢病毒载体制备体系，将构建好的主框架质粒pVECRNA、包装质粒pGAGPOL及包膜质粒pVSVG通过脂质体共转染至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，提取培养上清的RNA，进行RT-PCR反应；将培养上清进行免疫印迹鉴定；将培养上清感染正常的293T细胞、HepG₂细胞、Vero细胞，荧光显微镜下观察细胞GFP的表达情况；采取3*3*3析因分析方法，优化系统产量，Real-time PCR方法测定细胞培养上清中病毒载体的拷贝数，利用流式细胞术检测病毒载体滴度。RT-PCR及p24免疫印迹结果均提示在细胞上清中存在慢病毒载体；通过荧光显微镜观察到感染组293T细胞、HepG2细胞、Vero细胞均表达绿色荧光蛋GFP，说明此系统制备出的慢病毒载体具有感染性；系统经优化后，培养上清中慢病毒载体拷贝数达到（11.71±0.80）×10¹¹ copies/mL，培养上清原始滴度达到（1.3±0.18）×10⁸tu/mL，高出目前常用制备体系产量1个数量级。初步建立了新型慢病毒载体制备体系，为今后该系统的大规模应用提供客观的科学依据。

Abstract:

The aim was to develop a cell culture system capable of producing high titer lentiviral vector stocks with recombinant vaccinia viruses as helpers. BHK₂₁ was co-transfected by three main plasmids containing the transducing plasmid pVECRNA, the packaging plasmid pGAGPOL and the envelop plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T₇ RNA polymerase gene using Lipofectamine2000^TM. After 4 days incubation, the culture supernatant of lentiviral vectors was collected and judged by RT_PCR and the Western blot,the results showed that lentiviral vectors were found in the culture supernatant; approximately （11.71±0.80）×10¹¹ copies of lentiviral vector RNA were present per mL of cell culture supernatant, as detected by Real_time PCR; the vector stocks with titers was up to（1.3±0.18）×10⁸ tu/mL, as detected by flow cytometry , which is one order of magnitude higher than the output of classical manufacture system. These results suggest that the new poxviral/lentiviral hybrid system for efficient lentiviral vector production was initially established .It provides the basis for the future development of industrial application.