【作者】 李嵚；

【导师】 陈英杰； 杨丰； 张景海；

【作者基本信息】 沈阳药科大学 ， 药学， 2003， 博士

【摘要】 对虾白斑综合症病毒(White Spot Syndromic Virus, WSSV)，又称为白斑杆状病毒(White Spot Bacilliform Virus, WSBV)，是严重危害对虾养殖业的主要病原之一。本论文从WSSV宿主广泛性出发，围绕着病毒致病关键蛋白，特别是病毒早期蛋白，以及病毒蛋白与宿主蛋白间的相互作用机制，从结构功能基因组学和蛋白质组学角度对该病毒快速致死性的原因和机制进行了深入的研究。 本文内容主要包括下列工作：(1)以卤虫(Artemia franciscana)为实验对象简单阐述了其在WSSV宿主链中的作用；(2)在WSSV感染过程中起关键作用的病毒早期蛋白，如：病毒类胶原蛋白(Collagen-like Protein，CLP)和胸腺嘧啶核苷酸合成酶(Thymidylate Synthase，TS)的结构功能作了深入的研究；(3)通过病毒基因组同源重复区特异性结合蛋白的纯化鉴定，对病毒早期基因表达产物与宿主蛋白间的相互作用和在病毒感染和复制过程的作用做了初步的分析。 实验结果显示：□卤虫是一种acute host和latent host之外的，非致病性的载体宿主(passive carrier)，病毒进入宿主体内并不增殖，宿主无大量死亡和其他典型的病理特征，病毒染色体DNA可随着宿主繁殖而传递到受精卵中，并随着子代个体的发育成熟逐渐消失；□作为一种双链DNA动物病毒，在WSSV中发现了独特的类胶原蛋白(WSSV-CLP)和高保守的病毒胸腺嘧啶核苷酸合成酶(WSSV-TS)，这两种蛋白在病毒感染宿主早期开始转录表达，其中WSSV-clp基因编码一个病毒糖蛋白，该蛋白位于病毒粒子核衣壳和病毒包膜之间，并可形成多聚体结构。WSSV-CLP是第一个被证实糖基化修饰的结构蛋白。WSSV-ts基因编码表达WSSV基因组中最为保守的一个蛋白，体外表达的重组TS具有特异的底物结合能力。虽沈阳药科大学博士学位论文摘要然没有明显的poly(A)加尾信号(AATAAA)，3‘一RAcE发现该基因在病毒感染过程中可以正常转录，其mRNA含有poly(A)尾巴;口通过改进的亲和层析技术，SDS一PAGE电泳及GMSA分析等手段的相互结合，纯化得到特异结合于病毒同源区(homologous repeat regions，hrrs)高保守结构域(highly eonserved domain，HCD)的一组蛋白。这些蛋白与病毒感染宿主，并启动宿主已封闭基因的重新表达，及病毒复制增殖相关的信号传导系统有关。 结果表明，WSSV一CLP是病毒结构蛋白，该蛋白经过糖基化修饰，可能在病毒感染宿主细胞，与宿主细胞表面受体结合，并促进宿主细胞膜融合的过程中起重要的作用。包括病毒胸腺嗜睫核昔酸合成酶在内的一系列由病毒早期基因编码表达的，与DNA代谢密切相关的酶组成的独立于宿主之外的病毒胸腺啼睫核昔酸合成系统，可能是WSSV感染宿主导致快速死亡的主要原因之一。病毒人尹脚特异性结合蛋白的发现，将为研究病毒核酸复制，表达调控提供了大量的信息。更多还原

【Abstract】 Shrimp White Spot Syndrome Virus (WSSV), which has also deen referred to as White Spot Bacilliform Virus (WSBV), is a major pathogen of cultivated shrimp but its host range includes a large number of crustaceans. This paper is concerned on the possible pathogen related gene, expecially on the viral early gene products and DNA-specific binding proteins, for studying the infection machenism of WSSV on functional proteomics level.In this thesis, three main works are included. (1) Using Anemia franciscana as experimental animals, we briefly expatiate on the host wideness of WSSV. (2) The structure and function of two infection- and pathogen-related proteins of WSSV, an unique viral collagen and a highly conserved viral thymidylate synthase, was identified and characterized. (3) Isolation and identification of DNA-specific binding proteins of WSSV and the modification and regulation of these cofactors in process of viral transcription and translation by using a combination of DNA-affinity chromatography, SDS-PAGE and mass spectrographic analysis.The results we obtained in this thesis showed, D As a member of crustaceans, Artemia fransicana was predicted to be passively non-infectious carrier of WSSV. In Artemia, WSSV was not proliferation and can be transmited from nauplii to adult. WSSV was removed during the hatching of the reproductive cysts and the offspring Artemia was WSSV PCR-negative. D As the largest DNA virus which sequence has been completely sequenced, WSSV-clp and ts gene was transcribed at the early stage of infection. WSSV-CLP was an envelope protein and located between the double-layer membranes of WSSV and can form a network structure under transmission electron microscopy. WSSV-CLP was the first envelope protein that discovered to beglycosylated by using the treatment of N-glycosylationase F. WSSV-te encodes a highly conserved protein, and the recombination TS maintained substrate-specific binding activity. Without obviously polyadenylation signal [poly(A)], result of 3’-RACE indicated that ts was transcribed during infection and the mRNA was found contain a poly A at the 3’terminer. D Through the recombination of improved affinity chromatography, SDS-PAGE and gel mobility shift assay (GMSA), the ///rs-specific binding protein comples were isolated and purified. This protein fraction was thought to be involved in the gene replication of WSSV and interaction with host proteins.The results suggested that WSSV-CLP was a WSSV envelope protein that is N-glycosylated. It may play important roles in infection of WSSV as other glycoproteins found in the envelope of Baculoviridae. Together with a seriers of early genes, WSSV-TS constructed a de novo dTMP synthses system, which plays important roles in viral DNA replication. The hrrs-specific binding protein complex showed us important information on the replication of WSSV DNA. This suggest that when the virus enters the host cells, viral early genes is expressed and form complexes in the immediately early stage of infection with the regulation of host proteins which make it possible to consider this as the main causes for the rapid death of infected shrimp. Results in these investigations lead us directions for the prevention and for the therapy of WSSV.更多还原