一种新型慢病毒载体制备体系的初步建立

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4天后,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-timePCR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白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1.1×1012/ml,培养上清原始滴度达到1.3×108tu/ml,高出目前常用制备体系产量1个数量级.上述结果表明,新型慢病毒载体制备体系已初步建立,为今后该系统的大规模应用提供客观的科学依据.

The Initial Establishment of a New Poxviral/Lentiviral Hybrid System for Efficient Lentiviral Vector Production

Efficient gene delivery and sustained gene expression are required for successful human gene therapy. Although viral vectors are considered the most efficient vehicles for gene transfer, currently available viral vectors have not fully achieved these two requirements. Lentiviral vectors (LVs) can integrate into host chromosomes, allowing long-term gene expression, in addition, these vectors are non-toxic and minimally immunogenic since no viral genes are encoded in the vector genome , but are still limited to in vitro or ex vivo gene delivery because of their relatively low titers using transient transfection experiments. In order to develop an efficient transient transfection method for large-scale production of high titer lentiviral vector stocks, a minimal lentiviral vector producing system based on vaccinia virus that synthesizes T7 RNA polymerase was developed. BHK21 was co-transfected by three main plasmids containing the transducing plasmid pVECRNA, the packaging plasmid pGAGPOL and the envelope plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamin2000^TM. After 4 days, the culture supernatant of lentiviral vectors was collected, the RNA from the supernatant was examined by the RT-PCR, the protein from the supernatant was examined by Western blot, and the supernatant was used to transfect normal 293T , HepG2 and Vero, which were observed by the immunofluorescence microscopy. The type of cell lines, plasmids dosage and the MOI (the proportion between cell numbers and virus copies) were considered so critical to the output of this system that 3×3×3 factorial design was used to explore the yield optimization of this system. As judged by the results of RT-PCR and the Western blot, lentiviral vectors were found in the culture supernatant; as judged by immunofluorescence with microscopy, 293T, HepG2 and Vero which were transfected by the supnantant expressed the report protein - green fluorescent protein(GFP), the results confirmed the valid infectivity of the lentiviral vector produced by the system. Eventually, the best titers of lentiviral vector stocks was up to 1.3×10⁸ tu/ml, which is one order of magnitude higher than the output of classical manufacture system. 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.