草鱼呼肠孤病毒混合感染及其致细胞病变效应的蛋白组学分析-临床兽医学专业论文.docxVIP

上海海 上海海洋大学硕士学位论文 II II 在特定部位高表达；NS16 分布于细胞质中，在特定部位高表达；NS38、VP7 和 VP6 均匀分布于细胞质中；单一转染 5 个重组质粒均不产生明显的细胞病变效应。 本研究首次报道了 GCRV 的混合感染，为混合感染的检测和草鱼出血病流行 病学调查提供新方法；对目前免疫防控策略的制定和疫苗的研发提供新思路，对 GCRV 与宿主细胞相互作用机理和各编码蛋白的生物学功能研究提供参考。 关键词：GCRV，FLAC，混合感染，宿主细胞，蛋白组学分析，亚细胞定位 PAGE PAGE IV ABSTRACT Grass carp reovirus (GCRV), also named as Grass carp hemorrhage virus, is the most important pathogen of Grass carp, which causes haemorrhagic disease. The viral genome is composed of 11 segments of double stranded (ds) RNA, and encodes 7 structural proteins and 5 non-structural proteins. Cell membrane permability, apoptosis and stress granule formation can be induced by GCRV infection. Full-length amplification of cDNA technology was developed for sequencing full- length cDNA of double-stranded RNA virus , and it was also widely used to amplify any uncharacterized Aquareovirus genomes with accuracy and time-saving . Two dimensional electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry were used to analyze the proteomic changes and mechanisms during virus infection. FLAC technology, real-time RT-PCR, Two dimensional electrophoresis and MALDI-TOF/TOF mass spectrometry assay were employed in the study. The results were as follows: Two GCRV strains were isolated from diseased grass carp collected in Jiangxi province. Partial of full-length cDNAs were sequenced by FLAC, indicating that co-infection of two grass carp reovirus, named GCRV-JX01 and GCRV-JX02 which shared high homology with GCRV-873 and GCRV-GD108, respectively, were revealed in the same diseased sample through Blast method. The results demonstrated that there were two GCRV strains prevalent in major grass carp farms, and the ratio of co-infection (55%) was higher than single-infection (30%). Infinite dilution method and RT-PCR were used to purify the two virus strains in 96-wells. Quantitative real-time PCR assay was carried out to monitor the replication efficiency of both virus strains in either CIK cells or infected cell super