登革病毒复制子反式包装系统的建立

发布时间：2018-09-17 12:44

【摘要】： 目的：利用反向遗传学操作构建登革病毒复制子的反式包装系统；在此基础上,制备登革病毒单轮感染性颗粒,为登革热新型疫苗的研究奠定基础。方法： 1.在登革1型病毒广州分离株基因组全长感染性克隆的基础上,利用重叠PCR扩增获得病毒基因组5’端与GFP融合片段,将其插入病毒基因组中以替换病毒的绝大部分结构基因,并获得带有绿色荧光蛋白的病毒复制子DV1-EGFP-RP。同步构建其致死性突变复制子DV1-EGFP△GDD-RP作为对照。通过报告基因和RT-PCR的方法鉴定病毒复制子的复制特性。 2.利用重叠PCR扩增获得DV/JEV嵌合型prME片段,将其插入pcDNA3.1质粒构建相应真核表达载体,转染BHK21细胞后,通过RT-PCR和免疫荧光鉴定登革病毒prME的胞内表达特性。利用超速离心对上清中的病毒样颗粒进行纯化,通过电镜和western blot对其进行鉴定。 3.为制备获得复制子包装颗粒,首先将病毒复制子RNA电转BHK-21细胞后,再利用脂质体转染方法将辅助包装质粒导入细胞,以使病毒结构和非结构蛋白共表达于宿主细胞中。利用超速离心获得细胞培养上清中的病毒样颗粒,利用RT-PCR、western blot方法对其复制子包装颗粒的生化组分进行鉴定。最后将该病毒样颗粒接种BHK-21细胞,明确其单轮感染性特征。结果： 1.获得了高效表达的含绿色荧光蛋白报告基因的登革病毒复制子载体。 2.成功构建了登革病毒prME嵌合质粒pcDV/JEV prME,并制备了分泌性病毒样颗粒。 3.制备了具有单轮感染性的登革病毒病毒样颗粒。结论：成功建立了基于登革病毒复制子的反式包装系统,制备了具有单轮感染性的病毒样颗粒,为下一步的研究工作打下良好的基础。
[Abstract]:Aim: to construct a trans-packaging system for dengue virus replicators by reverse genetic operation, and to prepare dengue virus single round infectious particles, which will lay a foundation for the study of new dengue vaccine. Methods: 1. Based on the full-length infectious cloning of the genome of Guangzhou isolate of dengue 1 virus, the fusion fragment of 5 'terminal and GFP of the virus genome was obtained by overlapping PCR amplification. The fusion fragment was inserted into the genome of the virus to replace most of the structural genes of the virus. The viral replicon DV1-EGFP-RP. with green fluorescent protein was obtained. The lethal mutant replicon DV1-EGFP GDD-RP was constructed simultaneously as control. Identification of replication characteristics of virus replicators by reporter gene and RT-PCR. 2. The chimeric prME fragment of DV/JEV was amplified by overlapping PCR amplification and inserted into pcDNA3.1 plasmid to construct the eukaryotic expression vector. After transfection into BHK21 cells, the intracellular expression characteristics of dengue virus prME were identified by RT-PCR and immunofluorescence. Virus-like particles in supernatant were purified by ultracentrifugation and identified by electron microscope and western blot. In order to prepare the replicon packaging particles, firstly, the viral replicon RNA was electrotransferred into BHK-21 cells, then the auxiliary packaging plasmid was introduced into the cells by liposome transfection, so that the virus structure and non-structural proteins could be co-expressed in the host cells. The viral particles in the supernatant of cell culture were obtained by ultracentrifugation and the biochemical components of the replicon packaging granules were identified by RT-PCR,western blot method. Finally, the virus-like particles were inoculated into BHK-21 cells to identify their single-round infectious characteristics. Results: 1. A highly expressed dengue virus replicon vector containing green fluorescent protein reporter gene was obtained. 2. 2. The dengue virus prME chimeric plasmid pcDV/JEV prME, was successfully constructed and secreted virus-like particles were prepared. Dengue virus-like particles with single-round infection were prepared. Conclusion: the trans-packaging system based on dengue virus replicon was successfully established, and virus like particles with single round infection were prepared, which laid a good foundation for further research.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

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