猪繁殖与呼吸综合征病毒GP4蛋白单克隆抗体制备与抗原表位筛选

发布时间：2018-02-21 04:13

  本文关键词： 猪繁殖与呼吸综合征病毒 GP4蛋白 单克隆抗体 噬菌体展示技术 抗原表位　出处：《东北农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)属于套式病毒目(Nidovirales)动脉炎病毒科(Arteriviridae)动脉炎病毒属(Arterivirus),临床感染主要表现为发热,早产,流产,死胎和木乃伊胎等猪群的繁殖障碍和呼吸道症状。GP4蛋白是病毒粒子囊膜的组成部分,是该病毒的次要结构蛋白之一,是由ORF4基因编码的糖基化蛋白,少量存在病毒粒子中,由183或178个氨基酸残基组成,有4个糖基化位点,GP4能够诱导机体产生中和抗体,而且在细胞受体识别病毒粒子以及在病毒变异等方面具有重要意义。噬菌体展示技术研究抗原表位不仅可以证明抗原分子的具体结构与功能,还可以揭示抗原抗体的反应机制,已广泛用于研发多种多肽疫苗和新型药物。本实验以PRRSV-HUN4株为研究对象,制备了其GP4蛋白单克隆抗体并对其抗原表位进行了筛选和初步研究。本实验以原核表达的PRRSV GP4蛋白为免疫原免疫Balb/C小鼠,通过常规方法进行细胞融合,3轮亚克隆后,成功筛选出1株抗PRRSV GP4蛋白的单克隆抗体,经亚类鉴定为Ig G2b型,该株抗体的稳定性与特异性较好,不仅能够与重组GP4蛋白反应,而且能够与PRRSV有良好的反应性。应用噬菌体随机肽库对制备成功的5F12单抗进行4轮生物淘选,得到10株阳性噬菌体,测序后结果为:10个噬菌体中有4个氨基酸序列相同,其12肽序列分别为AKFEVCSPVVLG、GVNQENMLHFSF、NPRIRLNFIRIG和SGVYKVAYDWQH,命名为phage-Ⅰ-Ⅳ。序列比对发现phage-Ⅰ和Ⅱ的12肽序列分别与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。噬菌体竞争抑制试验与ELISA方法证实筛选出的亲和噬菌体与PRRSV及其GP4蛋白有很高的反应性,可以用于检测PRRSV及相关表位疫苗的研究,这为PRRSV病毒特性及其临床防治研究奠定理论和实验基础。
[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，

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