本文選題：PEDV　切入點：pAPN蛋白　出處：《華中農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：豬流行性腹瀉病毒(porcine epidemic diarrhea virus,PEDV)是α冠狀病毒屬的一個成員,它能感染各種年齡的豬。近年來,豬流行性腹瀉(porcine epidemic diarrhea,PED)的暴發(fā)給全球養(yǎng)豬業(yè)造成了巨大的經(jīng)濟損失。PEDV基因組能編碼16個非結(jié)構(gòu)蛋白(NSP1-NSP16)、ORF3、S、M、E、N蛋白,其各個蛋白在病毒生活周期中發(fā)揮了重要作用。PEDV S蛋白是病毒表面的膜糖蛋白,它在調(diào)控其與宿主細胞表面受體蛋白的相互作用中發(fā)揮了至關(guān)重要的作用,S蛋白上的抗原表位及受體結(jié)合域可以作為疫苗和抗病毒藥物研發(fā)的靶點。NSP9蛋白具有核苷酸結(jié)合特性,它是病毒復(fù)制復(fù)合體的一員,在病毒復(fù)制等過程發(fā)揮了重要作用。本研究以PEDV S和NSP9蛋白為研究對象,探索了S蛋白的受體識別特性、NSP9蛋白的結(jié)構(gòu)及其核苷酸結(jié)合特性,具體內(nèi)容如下:1.研究了PEDV S蛋白與其蛋白受體pAPN及其輔助受體糖的識別特性。為鑒定PEDV S1蛋白中的受體結(jié)合域,構(gòu)建了5個不同截短片段的S1蛋白及pAPN蛋白,流式細胞及ELISA實驗結(jié)果顯示PEDV受體結(jié)合域位于S1蛋白的C端區(qū)域(氨基酸253-638),其受體結(jié)合域比其它冠狀病毒更寬。CHGD-01 S253-638和CV777S249-634片段序列相似度很高,流式細胞實驗結(jié)果顯示它們結(jié)合pAPN的能力相似,表明PEDV經(jīng)典弱毒株和變異毒株受體結(jié)合力沒有明顯差異。PEDV S1C-terminus(S477-629)與TGEV、HCoV-NL63受體結(jié)合域在結(jié)構(gòu)和序列上相似,推測PEDV S1 C-terminus結(jié)構(gòu)中突出于β-barrel區(qū)域頂端的3個loop也可能參與了受體的結(jié)合,為此,將CHGD-01 S477-629片段的3個loop分別突變?yōu)镠CoV-NL63對應(yīng)序列或“SGSGS”,ELISA、點雜交、pull-down實驗結(jié)果顯示突變體蛋白沒有完全喪失pAPN結(jié)合力,說明預(yù)測的3個loop在PEDV S-pAPN互作中并不發(fā)揮關(guān)鍵作用,因此,PEDV展現(xiàn)出不同于TGEV、PRCV及HCoV-NL63的受體識別模式。為確定PEDV S蛋白是否具有糖結(jié)合能力,構(gòu)建了全長S1及7個截短蛋白,ELISA實驗結(jié)果證明S1蛋白的N端具有糖結(jié)合能力,同時,CHGD-01 S1-324蛋白結(jié)合mucin的能力強于CV777 S1-320蛋白,表明PEDV變異毒株的糖結(jié)合力強于經(jīng)典弱毒株。2.深入研究了PEDV NSP9蛋白的結(jié)構(gòu)及其核苷酸結(jié)合特性。經(jīng)過蛋白的表達、純化,晶體的初篩、優(yōu)化等過程,解析了NSP9蛋白的晶體結(jié)構(gòu),其結(jié)構(gòu)顯示NSP9蛋白單體包含7個β-strand和1個位于C端的α-helix。PEDV NSP9蛋白有2種二聚體形式:一種由C端的α-helix平行互作形成,氨基酸Gly95、Gly99和Gly102之間形成了主要的疏水性相互作用;另一種由二硫鍵及β-strand 4和5之間形成的分子間氫鍵、疏水性相互作用形成。Crosslinking和AUC實驗結(jié)果顯示NSP9蛋白在溶液中主要以單體和二聚體形式存在。SDS-PAGE及AUC實驗結(jié)果證實NSP9C59A突變體為單體狀態(tài),而NSP9G95E,G99E,G102E突變體狀態(tài)與野生型NSP9一致,說明二硫鍵在NSP9蛋白二聚體形成中發(fā)揮了關(guān)鍵作用,解析的NSP9C59A突變體的晶體結(jié)構(gòu)進一步證實了以上結(jié)果。分析發(fā)現(xiàn)大量帶正電荷的氨基酸分布于NSP9蛋白結(jié)構(gòu)表面,二聚體界面或單體中間區(qū)域展示出很強的帶正電荷的能力,表明這是一個可供核苷酸結(jié)合的界面。為驗證NSP9蛋白中氨基酸Cys59、Gly95、Gly99、Gly102、Lys10、Arg68、Lys69、Arg106是否與其核苷酸結(jié)合能力相關(guān),構(gòu)建了一系列相關(guān)突變體,EMSA及MST實驗結(jié)果表明NSP9蛋白中參與二聚體形成的關(guān)鍵氨基酸Cys59、Gly95、Gly99、Gly102對其核苷酸結(jié)合力沒有顯著影響,而NSP9蛋白結(jié)構(gòu)表面帶正電荷的氨基酸在核苷酸結(jié)合中發(fā)揮了重要作用,同時,PEDV NSP9蛋白與核苷酸結(jié)合力的強弱與核苷酸的長度無關(guān)。PEDV S蛋白和NSP9蛋白在結(jié)構(gòu)及功能上展現(xiàn)出異于其它冠狀病毒的特性,因此,PEDV擁有其獨特的入侵、復(fù)制、致病機制。本文為闡明PEDV的致病機理奠定了理論基礎(chǔ),為抗病毒藥物或疫苗的研發(fā)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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.651

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