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题名: 应用噬菌体展示技术研究对虾白斑综合症病毒(WSSV)的结构蛋白
作者: 王玉珍
答辩日期: 2008-06-10
导师: 戴和平
专业: 水生生物学
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) ; 噬菌体展示技术 ; 单链抗体(scFv) ; VP28 ; 线性抗原表位 ; 构象抗原表位 ; 中和实验 ; 加强型绿色荧光蛋白 ; 流式细胞技术 ; 肽库
其他题名: Using Phage Display Technology to Study Structural Proteins of White Spot Syndrome Virus (WSSV) of Shrimp
摘要: 对虾白斑综合症病毒(White Spot Syndrome Virus, WSSV)是目前对虾养殖业最主要的病毒性病原之一。自上世纪90年代初发现以来,该病毒以其宿主范围广、毒力强、致死亡率高、传播速度快等特点,给全世界的对虾养殖业造成了巨大的损失,并严重威胁世界水环境。因为该病毒与目前已知的病毒同源性极低,属于一类新的病毒家族,使得已知病毒的研究成果不能借鉴。另外,对虾的细胞系尚未成功建立,这也给WSSV结构蛋白以及感染的分子机理的研究带来极大的困难。因此,目前虽然对WSSV基因组及蛋白质组的研究已较为深入,但仍无有效的防治措施。为此,我们应用噬菌体展示技术,力图开辟一条新的研究途径, 对WSSV结构蛋白进行研究。 VP28是WSSV最主要的囊膜蛋白之一,可能在病毒感染过程中起着关键性的作用。在本研究中,应用噬菌体展示技术,成功构建抗VP28单链抗体噬菌体展示文库,并且通过运用各种淘选方法,从中淘选到6种具有不同重链CDR3区的单链抗体,且这些单链抗体分别识别VP28 的N端、C端和中间区域。对单链抗体进一步研究发现,其中有四种单链抗体识别的为线性抗原表位,另外两种单链抗体识别的为构象抗原表位。以分离得到的单链抗体为工具,利用胶体金免疫电镜技术,对单链抗体对应抗原表位在WSSV上进行定位,结果显示这些抗原表位均位于病毒囊膜表面,且其中有部分抗原表位位于VP28蛋白三聚体结构内部。同时,对这些单链抗体进行体内和体外中和实验分析,实验结果显示它们都不能阻止或延缓病毒感染宿主,暗示这些单链抗体对应的抗原表位不是病毒感染宿主的吸附位点。 同时,在本研究中,我们还以加强型绿色荧光蛋白(Enhanced green fluorescent protein, EGFP)为报告基因,在原核表达载体中构建并表达VP28与EGFP的融合蛋白,利用流式细胞仪和荧光显微镜技术研究WSSV囊膜蛋白VP28与宿主细胞间是否有直接的相互作用。遗憾的是在研究结果中,我们发现原核表达的重组VP28并不能直接吸附于宿主细胞表面,与其他研究者的结果不一致。所以,通过本实验室对VP28的研究,我们认为对于该蛋白在病毒感染过程中所行使的功能以及其作用的机理仍有待进一步探讨。 为了能对WSSV上更多的未知结构蛋白进行研究,在本研究中,以变性的纯化WSSV为免疫原免疫小鼠,并成功构建抗变性WSSV单链抗体噬菌体展示文库。通过对各种不同淘选方法的探索,建立了能有效筛选更多识别WSSV上不同抗原的单链抗体的方法,并分离鉴定出6种能特异识别WSSV的单链抗体,对这些单链抗体的性质进行初步研究发现其中有一个单链抗体识别线性表位,能用于Western blot分析。同时,利用胶体金免疫电镜技术,对这些单链抗体对应抗原表位进行定位分析。该研究为获取识别多种WSSV抗原的抗体提供了新的方法路线。 单链抗体P75E8为分离得到的能特异识别VP28上线性抗原表位的单链抗体。为了建立应用噬菌体展示技术高通量淘选单链抗体对应抗原表位的方法,以单链抗体P75E8为研究对象,分别采取传统的抗原表位研究方法和对噬菌体展示随机十肽文库进行淘选的方法,来研究单链抗体P75E8对应的抗原表位。以传统的方法我们确定P75E8对应的抗原表位氨基酸序列为:SDAQMKEEDA。通过对十肽文库的淘选,虽然得到一些阳性克隆,但是并不能从这些阳性克隆对应的多肽确定单链抗体抗原结合位点对应的表位。进一步研究发现这是由于非定向化固定的单链抗体由于构象发生改变或抗原结合位点被掩盖,使极大部分单链抗体失去结合特定抗原的能力。所以,要建立以单链抗体分离鉴定抗原表位的方法,首先要解决单链抗体定向固定的问题。该研究为进一步研究以单链抗体筛选抗原表位的方法奠定了基础。
英文摘要: White spot syndrome virus (WSSV)is one of the major pathogen of shrimp culture with a wide host range and high mortality rate. Since it first appeared in the 1990s, WSSV has spread around the world rapidly, caused large damage to shrimp aquaculture and serious threat to world water environment. WSSV with a low homology to the known virus has been classified into a new virus family. So it is difficult to infer the molecular mechanism of WSSV systemic infection from the known viruses. Since the shrimp cell line has not been established yet, it is difficult to study the structural proteins of WSSV and its infection mechanism using conventional methods. To date, there is no efficient method to protect shrimp from WSSV despite of more progress on the study of genome and proteome of WSSV. In this work, phage display technology was used to probe into a new way to study the structural proteins of WSSV. VP28 is one of the major envelope proteins of WSSV and may paly a key role in WSSV infectivity. In this study, using phage display technology, a single-chain fragment variable (scFv) antibody library displayed on phages was constructed using spleen from the mice immunized with VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies with different CDR3 in their heavy chain specifically binding to the epitopes in the N-terminal, middle and C-terminal regions of VP28 respectively were isolated from the library. Four of the scFvs recognize linear epitopes and the other two recognize conformational epitopes. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy and some of the epitopes were located in the inner of the trimer of VP28. Neutralization assay with these antibodies in vitro and in vivo showed that none of the scFvs could inhibit or delay WSSV infectivity. The result suggests that these epitopes may not be the attachment site of WSSV to host cell receptor. At the same time, a recombinant protein VP28 fused with enhanced green fluorescent protein (EGFP) was constructed and expressed in E. coli. Using EGFP as reporter, the interaction between VP28 and host cell was investigated by flow cytometry and fluorescent microscopy. It is a pity that the result indicated that no direct interaction between VP28 and host cell was appeared, which is different from that of other researcher. After the work on VP28 in our lab, we think that further study on the function and mechanism of VP28 in infection is very necessary. In order to study the function of more structural proteins of WSSV, a scFv antibody phage display library was constructed using spleen from mice immunized with denatured WSSV. After investigation of several panning methods, the efficient methods to generate more different scFvs binding to different epitopes of WSSV were established and six scFvs specifically against WSSV were isolated and characterized. One of the scFvs recognizes a linear epitope and can be used in Western blot assay. The location of the epitopes bound by the isolated scFvs on WSSV was performed by mmuno-electron microscopy. This study provides a new strategy to obtain more different antibodies specifically binding to WSSV. The isolated scFv P75E8 recognizes a linear epitope in VP28. In order to establish a high throughout method to identify the epitopes bound by scFvs using phage display technology, two methods were adopted to identify the epitope bound by scFv P75E8: 1) using conventional method, ELISA assay of binding between P75E8 with several truncated VP28 proteins to determine the epitope; 2) using phage display technology, a panning against a phage display random decapeptide library was performed using purified P75E8 as an immobilized target. The epitope was determined with a amino acid sequence: SDAQMKEEDA using method 1). But we could not infer the epitope recognized by P75E8 using method 2) despite a lot of positive clone were isolated. Further study indicated that P75E8 immobilized randomly lost the ability to bind to VP28. So it is very important to establish efficient way to directionally limmobilize scFv before panning. The work lays the groundwork for further research on epitope recognized by scFv.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12266
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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