發(fā)布時(shí)間：2018-02-21 04:13

  本文關(guān)鍵詞： 豬繁殖與呼吸綜合征病毒 GP4蛋白 單克隆抗體 噬菌體展示技術(shù) 抗原表位　出處：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：豬繁殖與呼吸綜合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)屬于套式病毒目(Nidovirales)動(dòng)脈炎病毒科(Arteriviridae)動(dòng)脈炎病毒屬(Arterivirus),臨床感染主要表現(xiàn)為發(fā)熱,早產(chǎn),流產(chǎn),死胎和木乃伊胎等豬群的繁殖障礙和呼吸道癥狀。GP4蛋白是病毒粒子囊膜的組成部分,是該病毒的次要結(jié)構(gòu)蛋白之一,是由ORF4基因編碼的糖基化蛋白,少量存在病毒粒子中,由183或178個(gè)氨基酸殘基組成,有4個(gè)糖基化位點(diǎn),GP4能夠誘導(dǎo)機(jī)體產(chǎn)生中和抗體,而且在細(xì)胞受體識(shí)別病毒粒子以及在病毒變異等方面具有重要意義。噬菌體展示技術(shù)研究抗原表位不僅可以證明抗原分子的具體結(jié)構(gòu)與功能,還可以揭示抗原抗體的反應(yīng)機(jī)制,已廣泛用于研發(fā)多種多肽疫苗和新型藥物。本實(shí)驗(yàn)以PRRSV-HUN4株為研究對(duì)象,制備了其GP4蛋白單克隆抗體并對(duì)其抗原表位進(jìn)行了篩選和初步研究。本實(shí)驗(yàn)以原核表達(dá)的PRRSV GP4蛋白為免疫原免疫Balb/C小鼠,通過(guò)常規(guī)方法進(jìn)行細(xì)胞融合,3輪亞克隆后,成功篩選出1株抗PRRSV GP4蛋白的單克隆抗體,經(jīng)亞類鑒定為Ig G2b型,該株抗體的穩(wěn)定性與特異性較好,不僅能夠與重組GP4蛋白反應(yīng),而且能夠與PRRSV有良好的反應(yīng)性。應(yīng)用噬菌體隨機(jī)肽庫(kù)對(duì)制備成功的5F12單抗進(jìn)行4輪生物淘選,得到10株陽(yáng)性噬菌體,測(cè)序后結(jié)果為:10個(gè)噬菌體中有4個(gè)氨基酸序列相同,其12肽序列分別為AKFEVCSPVVLG、GVNQENMLHFSF、NPRIRLNFIRIG和SGVYKVAYDWQH,命名為phage-Ⅰ-Ⅳ。序列比對(duì)發(fā)現(xiàn)phage-Ⅰ和Ⅱ的12肽序列分別與PRRSV Hu N4株GP4蛋白第112至123位和第84至95位氨基酸序列具有很高的相似性。噬菌體競(jìng)爭(zhēng)抑制試驗(yàn)與ELISA方法證實(shí)篩選出的親和噬菌體與PRRSV及其GP4蛋白有很高的反應(yīng)性,可以用于檢測(cè)PRRSV及相關(guān)表位疫苗的研究,這為PRRSV病毒特性及其臨床防治研究奠定理論和實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Porcine reproductive and respiratory syndrome virus (PRRSVV) belongs to Arteriviridae (Nidoviralesae) Arteriviridaevirus. Reproductive disorders and respiratory symptoms of dead fetus and mummies. GP4 protein is a component of viral particle capsule and one of the secondary structural proteins of the virus. It is a glycosylated protein encoded by ORF4 gene. Consisting of 183 or 178 amino acid residues, four glycosylation sites (GP4) can induce neutralizing antibodies. Phage display technique can not only prove the specific structure and function of antigen molecules, but also reveal the reaction mechanism of antigen and antibody. Has been widely used in the development of a variety of polypeptide vaccines and new drugs. In this study, PRRSV-HUN4 strain as the research object, Monoclonal antibodies against GP4 protein were prepared and their epitopes were screened and preliminarily studied. In this study, the prokaryotic expressed PRRSV GP4 protein was used as immunogen to immunize Balb/C mice. A monoclonal antibody against PRRSV GP4 protein was successfully screened, which was identified as Ig G2b by subclass. The antibody was stable and specific, and could not only react with recombinant GP4 protein. The phage random peptide library was used to prepare 5F12 monoclonal antibody for 4 rounds of biological panning and 10 positive phages were obtained. The results showed that four amino acids of the 10 phages were the same. Its 12-peptide sequences were AKFEVCSPVVLGG, GVNQENMLHFSFN, NPRIRLNFNFIRIG and SGVYKVAYDDQH, named phage- 鈪，

本文編號(hào)：1520956