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题名: 应用噬菌体展示技术对对虾白斑综合症病毒(WSSV)结构蛋白及感染相关蛋白的研究
作者: 肖囡
答辩日期: 2007-06-08
导师: 戴和平
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 对虾白斑综合症病毒(WSSV) ; 噬菌体展示 ; 单链抗体(scFv) ; 淘选 ; 对虾感染相关蛋白 ; WSSV基因组文库 ; WSSV cDNA文库
其他题名: Studies on the structural proteins of White Spot Syndrome Virus (WSSV) and shrimp functional proteins involved in the infection utilizing Phage Display Technology
摘要: 对虾白斑综合症病毒(White Spot Syndrome Virus, WSSV)自1992年引起对虾白斑病爆发以来,给全球对虾养殖业带来了毁灭性的打击。该病毒传播快,致死率高,已经给对虾养殖经济造成巨大损失。WSSV在分类学上已经被鉴定为一个新的病毒家族中的新种,同已知的病毒之间无明显同源性,因此对其感染宿主的分子机理的研究难以借鉴已知病毒的研究结果。同时由于对虾细胞系尚未成功建立,为该病毒结构、功能蛋白以及感染机制的研究造成很大困难。为此,本研究利用噬菌体展示技术的独特优点,对WSSV结构蛋白以及与感染相关对虾功能蛋白进行了研究。 本实验室在过去的研究中构建了抗WSSV的噬菌体展示单链抗体cDNA库,并从中筛选到了一系列能和WSSV结合的单链抗体,为了能够深入的了解WSSV表面抗原结构及其结构蛋白,揭示其致病机理,本研究分别构建了T7展示WSSV cDNA文库和M13展示WSSV基因组文库,并利用单链抗体分别对这两个文库进行“反向淘选”,筛选出单链抗体所对应的病毒抗原。由于构建T7展示WSSV cDNA文库的cDNA来源于感染了WSSV的南美白对虾,该文库中大部分cDNA为对虾的cDNA,所以此文库也可以看作是感染了WSSV的南美白对虾T7展示对虾cDNA文库 本研究首先利用纯化的抗WSSV单链抗体A1对T7展示WSSV cDNA文库进行淘选, 筛选到了能和单链抗体A1特异结合的病毒抗原WSSV388,免疫胶体金电镜观察显示WSSV388为WSSV核衣壳蛋白。 本研究对另一种特异性识别WSSV的单链抗体P1A6的基本性质进行了分析,并利用该单链抗体对M13展示WSSV基因组文库进行淘选。经过两轮筛选后,得到一个能够特异和单链抗体P1A6结合的M13噬菌体, 编号为B11。测序结果表明展示在噬菌体B11上的蛋白氨基酸序列与WSSV一开放阅读框WSSV217(WSSV-TW, AF440570)上的一段完全同源。 本研究还利用完整的WSSV病毒粒子对感染了WSSV的南美白对虾T7噬菌体展示对虾cDNA文库进行淘选,从而获得与病毒感染相关的对虾功能蛋白。经过四轮淘选得到了一个能够与WSSV病毒粒子特异结合的T7噬菌体,编号为SP4G7。由于SP4G7噬菌体与WSSV病毒粒子的结合能被对虾血细胞所影响,因此推测展示于T7噬菌体SP4G7上的外源蛋白可能代表了对虾血细胞的一个功能蛋白,并且参与了宿主细胞和WSSV病毒的相互作用。本实验为研究对虾宿主细胞与WSSV病毒相互作用提供了一种全新的试验方法。
英文摘要: White Spot Syndrome, causing high mortality and economic damage, has become a major threat to shrimp culture all over the world since its outbreak in 1992. White Spot Syndrome virus (WSSV), the viral pathogens of the white spot syndrome, has been erected as a new spices that belongs to a new family, and its primary structure is different from other known virus, therefore, it is difficult to use references of known viruses to infer its molecular mechanisms of infection. Since the shrimp continuous cell line has not been established yet, it is very difficult to study the function of the virus protein and the molecular mechamism of its infection. In this study, the advantage of phage display technology was used to identify the structural protein of WSSV and the Penaeus vannamei infection-related protein. A phage display single chain fragment variable regions antibody (scFv) cDNA library against WSSV has been constructed in previous works. In order to further study the WSSV antigen and structural protein, explore the infection mechanism, we constructed the T7 phage display WSSV cDNA library and M13 phage display WSSV genome library. After panning these two library against scFv antibodies, the target antigens were selected. T7 phage display WSSV cDNA library was constructed utilizing cDNA isolated from shrimp infected with WSSV. This implies that the majority of the components in this library are shrimp cDNA, so this library can also be used as a WSSV-infected Penaeus vannamei T7 phage display shrimp cDNA library. In T7 phage display WSSV cDNA library, phages displayed the candidate epitope were selected with scFv A1. After several selection rounds, phages displayed a fragment of WSSV388 were enriched. The immune electron microscopy revealed that WSSV388 located on the nucleocapsid. Another scFv P1A6, which has a high affinity for WSSV, was charactered and its target antigen was selected using the M13 phage display WSSV genome library. After two selection rounds, one phage named B11 showed specific binding properties to P1A6 by enzyme-linked immunosorbent assay (ELISA). The sequence of peptide displayed on the phage B11 mathced a fragment of ORF WSSV217. In order to study the WSSV infection mechanism and explore the interaction between the virus and host cells, a T7 phage display cDNA library, which was constructed from WSSV-infected Penaeus vannamei, was used to pan against the intact WSSV virion. After four selection rounds, a positive phage colony SP4G7 that specifically interacted with the WSSV virion was isolated and identified by ELISA. Since the interaction between phage SP4G7 and WSSV virion could be interfered by the shrimp hemocyte, the protein translated by the cDNA fragment fused on the suface of phage SP4G7 may be a functional protein on the shrimp’s hemocyte and involves in the interaction between host cell and WSSV virion. This study provides a new method to uncover interaction mechanism between the host cells and WSSV virion.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12268
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
应用噬菌体展示技术对对虾白斑综合症病毒(WSSV)结构蛋白及感染相关蛋白的研究.肖囡[d].中国科学院水生生物研究所,2007.20-25
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