猪流行性腹泻病毒S蛋白受体识别特性与NSP9蛋白结构研究

发布时间：2018-03-22 16:49

本文选题：PEDV　切入点：pAPN蛋白　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)是α冠状病毒属的一个成员,它能感染各种年龄的猪。近年来,猪流行性腹泻(porcine epidemic diarrhea,PED)的暴发给全球养猪业造成了巨大的经济损失。PEDV基因组能编码16个非结构蛋白(NSP1-NSP16)、ORF3、S、M、E、N蛋白,其各个蛋白在病毒生活周期中发挥了重要作用。PEDV S蛋白是病毒表面的膜糖蛋白,它在调控其与宿主细胞表面受体蛋白的相互作用中发挥了至关重要的作用,S蛋白上的抗原表位及受体结合域可以作为疫苗和抗病毒药物研发的靶点。NSP9蛋白具有核苷酸结合特性,它是病毒复制复合体的一员,在病毒复制等过程发挥了重要作用。本研究以PEDV S和NSP9蛋白为研究对象,探索了S蛋白的受体识别特性、NSP9蛋白的结构及其核苷酸结合特性,具体内容如下:1.研究了PEDV S蛋白与其蛋白受体pAPN及其辅助受体糖的识别特性。为鉴定PEDV S1蛋白中的受体结合域,构建了5个不同截短片段的S1蛋白及pAPN蛋白,流式细胞及ELISA实验结果显示PEDV受体结合域位于S1蛋白的C端区域(氨基酸253-638),其受体结合域比其它冠状病毒更宽。CHGD-01 S253-638和CV777S249-634片段序列相似度很高,流式细胞实验结果显示它们结合pAPN的能力相似,表明PEDV经典弱毒株和变异毒株受体结合力没有明显差异。PEDV S1C-terminus(S477-629)与TGEV、HCoV-NL63受体结合域在结构和序列上相似,推测PEDV S1 C-terminus结构中突出于β-barrel区域顶端的3个loop也可能参与了受体的结合,为此,将CHGD-01 S477-629片段的3个loop分别突变为HCoV-NL63对应序列或“SGSGS”,ELISA、点杂交、pull-down实验结果显示突变体蛋白没有完全丧失pAPN结合力,说明预测的3个loop在PEDV S-pAPN互作中并不发挥关键作用,因此,PEDV展现出不同于TGEV、PRCV及HCoV-NL63的受体识别模式。为确定PEDV S蛋白是否具有糖结合能力,构建了全长S1及7个截短蛋白,ELISA实验结果证明S1蛋白的N端具有糖结合能力,同时,CHGD-01 S1-324蛋白结合mucin的能力强于CV777 S1-320蛋白,表明PEDV变异毒株的糖结合力强于经典弱毒株。2.深入研究了PEDV NSP9蛋白的结构及其核苷酸结合特性。经过蛋白的表达、纯化,晶体的初筛、优化等过程,解析了NSP9蛋白的晶体结构,其结构显示NSP9蛋白单体包含7个β-strand和1个位于C端的α-helix。PEDV NSP9蛋白有2种二聚体形式:一种由C端的α-helix平行互作形成,氨基酸Gly95、Gly99和Gly102之间形成了主要的疏水性相互作用;另一种由二硫键及β-strand 4和5之间形成的分子间氢键、疏水性相互作用形成。Crosslinking和AUC实验结果显示NSP9蛋白在溶液中主要以单体和二聚体形式存在。SDS-PAGE及AUC实验结果证实NSP9C59A突变体为单体状态,而NSP9G95E,G99E,G102E突变体状态与野生型NSP9一致,说明二硫键在NSP9蛋白二聚体形成中发挥了关键作用,解析的NSP9C59A突变体的晶体结构进一步证实了以上结果。分析发现大量带正电荷的氨基酸分布于NSP9蛋白结构表面,二聚体界面或单体中间区域展示出很强的带正电荷的能力,表明这是一个可供核苷酸结合的界面。为验证NSP9蛋白中氨基酸Cys59、Gly95、Gly99、Gly102、Lys10、Arg68、Lys69、Arg106是否与其核苷酸结合能力相关,构建了一系列相关突变体,EMSA及MST实验结果表明NSP9蛋白中参与二聚体形成的关键氨基酸Cys59、Gly95、Gly99、Gly102对其核苷酸结合力没有显著影响,而NSP9蛋白结构表面带正电荷的氨基酸在核苷酸结合中发挥了重要作用,同时,PEDV NSP9蛋白与核苷酸结合力的强弱与核苷酸的长度无关。PEDV S蛋白和NSP9蛋白在结构及功能上展现出异于其它冠状病毒的特性,因此,PEDV拥有其独特的入侵、复制、致病机制。本文为阐明PEDV的致病机理奠定了理论基础,为抗病毒药物或疫苗的研发提供了新思路。
[Abstract]:Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV) is a member of alpha coronavirus, it can infect pigs of all ages. In recent years, porcine epidemic diarrhea (porcine epidemic, diarrhea, PED) in the swine industry worldwide has caused tremendous economic losses to the genome of.PEDV 16 non structural protein encoding (NSP1-NSP16), ORF3, S, M, E, N protein, the protein in the viral life cycle plays an important role in the.PEDV S protein is a membrane glycoprotein on the surface of the virus, it plays a crucial role in the regulation of its interaction with the host cell surface receptor protein, S protein antigens on the table and the receptor binding domain as a target for vaccines and antiviral drugs.NSP9 protein has the nucleotide binding properties, it is a member of the viral replication complex, plays an important role in virus replication process in this study. PEDV S and NSP9 protein as the research object, explores the receptor recognition of S protein, NSP9 protein structure and nucleotide binding properties, the specific contents are as follows: 1. to study the identification characteristics of PEDV S protein and its receptor protein pAPN receptor and its auxiliary sugar. For identification of PEDV S1 protein in the receptor binding domain, construct 5 different truncated S1 protein and pAPN protein, flow cytometry and ELISA assay results showed that PEDV receptor binding domain in the C terminal region of S1 protein (253-638 amino acids), its receptor binding domain than other coronavirus S253-638 and.CHGD-01 wide CV777S249-634 fragment sequence similarity is very high, the results of flow cytometer show their pAPN binding ability similar to that PEDV classic and variant strains of attenuated receptor binding force and there is no significant difference in.PEDV S1C-terminus (S477-629) and TGEV, HCoV-NL63 receptor binding domain in structure and sequence. Like that, 3 loop PEDV S1 C-terminus structure protruding from the top of the beta -barrel region may be involved in receptor binding, therefore, the CHGD-01 S477-629 fragment of 3 loop were mutated to HCoV-NL63 corresponding sequence or "SGSGS", ELISA, dot blot, pull-down results showed that the mutant protein without the complete loss of pAPN binding Force 3, loop forecast does not play a key role in the PEDV S-pAPN interaction so PEDV show different from TGEV, PRCV and HCoV-NL63 receptor recognition pattern. In order to determine whether the S protein PEDV with sugar binding capacity, constructed a full-length S1 and 7 truncated ELISA protein, the experimental results show that the S1 protein of N end with a sugar binding ability, at the same time, the CHGD-01 S1-324 mucin protein binding ability to CV777 S1-320 protein showed that PEDV mutant strain of sugar binding is stronger than the classical attenuated strain.2. research PEDV NSP9 protein The structure and nucleotide binding properties. After the protein expression, purification, crystal screening, optimization process, analysis the crystal structure of NSP9 protein, its structure showed that NSP9 protein monomer contains 7 beta -strand and 1 in C terminal of -helix.PEDV alpha NSP9 protein has 2 kinds of forms: a two dimer a -helix end of the parallel interaction formed by C Gly95, amino acid, mainly the hydrophobic interaction formed between Gly99 and Gly102; another by two disulfide bonds and -strand beta 4 and 5 between the formation of intermolecular hydrogen bonds, hydrophobic interactions to form.Crosslinking and AUC experimental results showed that NSP9 protein in solution mainly to the monomer and dimer forms of two.SDS-PAGE and AUC experimental results confirmed that the NSP9C59A mutant as a single state, while NSP9G95E, G99E, G102E mutant and wild type NSP9 state, two disulfide bonds in NSP9 protein dimer formation in two play off The key role of the crystal structure of NSP9C59A mutant analysis further confirmed the above results. A large number of positively charged amino acid distribution in NSP9 protein found on the surface of structure analysis, two dimer interface or intermediate region monomer show strong positive charge ability, that this is an available nucleotide binding interface. Validation of the NSP9 protein amino acid Cys59, Gly95, Gly99, Gly102, Lys10, Arg68, Lys69, Arg106 and nucleotide binding ability, constructs a series of related mutants, EMSA and MST results showed that the NSP9 protein involved in key amino acid Cys59 two dimer formation of Gly95, Gly99, Gly102 had no significant effect on the binding force the nucleotide, amino acid and NSP9 protein structure positively charged surfaces play an important role in nucleotide binding and nucleotide binding PEDV NSP9 protein and the strong and weak nucleotides in length Independent of.PEDV S protein and NSP9 protein in the structure and function show characteristics different from other coronavirus. Therefore, PEDV has its unique invasion, replication, pathogenesis. This paper lays a theoretical foundation for the pathogenesis of PEDV is clarified, provides new ideas for the development of antiviral drugs or vaccines.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.651

【相似文献】