空肠弯曲菌peblA和cadF的克隆表达及多克隆抗体制备与纯化

发布时间：2018-10-29 21:57

【摘要】：空肠弯曲菌(Campylobacter jejuni)是一种导致人类发生急性肠炎与食物中毒的、危害仅次于沙门氏菌和志贺氏菌的人畜共患病原菌。建立空肠弯曲菌快速的检测方法,是有效预防和控制其疾病发生的重要手段。通常检测空肠弯曲菌的方法有分离培养方法、生化检测、免疫学检测、分子生物学方法等。免疫学快速筛查方法在病原微生物的检测中具有重要的应用价值,目前在空肠弯曲菌的快速检测方面,相关产品及技术甚少。获得针对空肠弯曲菌的特异性抗体,是建立空肠弯曲菌的免疫学快检方法的首要前提。为此,本文开展了以下研究。本文首先在大量文献调研的基础上,确定了空肠弯曲菌的外膜蛋白PEB1、CadF作为特征性抗原。其编码的基因序列相对保守,且在不同的血清型空肠弯曲菌中均有表达,提示可作为检测抗原。采用分子生物学的技术手段,本文成功克隆表达了PEB1和CadF两种外膜蛋白,利用镍离子亲和柱纯化了表达产物。以PEB1和CadF两种纯化蛋白免疫新西兰长耳兔,分别制备了相应的多克隆抗体,其效价分别为1:1024000和1：128000。本文利用斑点杂交的方法,发现PEB1抗血清与绿脓假单胞菌、金黄色葡萄球菌、保加利亚乳杆菌、福氏志贺菌等存在交叉反应,CadF抗血清与普通变性杆菌、副溶血弧菌、单增李斯特菌、金黄色葡萄球菌和短双歧杆菌等存在交叉反应。为此,本文采用辛酸硫酸铵沉淀法,去除了血清中的杂蛋白。进一步采用磁珠法,消除了两种抗血清与上述微生物的交叉反应,获得了高纯度和高特异性的抗体。本文研究结果将为建立空肠弯曲菌的免疫学检测方法,奠定技术基础、提供实验材料。
[Abstract]:Campylobacter jejuni (Campylobacter jejuni) is a zoonotic pathogen that causes acute enteritis and food poisoning in humans and is second only to Salmonella and Shigella. To establish a rapid detection method for Campylobacter jejuni is an important method to prevent and control the occurrence of Campylobacter jejuni. The methods for detection of Campylobacter jejuni are isolation, biochemistry, immunology, molecular biology and so on. The immunological rapid screening method has important application value in the detection of pathogenic microorganisms. At present, there are few related products and techniques in the rapid detection of Campylobacter jejuni. Obtaining specific antibodies against Campylobacter jejuni is the first prerequisite for rapid immunological detection of Campylobacter jejuni. Therefore, this paper carried out the following research. Based on a large number of literatures, the outer membrane protein (PEB1,CadF) of Campylobacter jejuni was identified as the characteristic antigen. The coding gene sequence was relatively conserved and expressed in different serotypes of Campylobacter jejuni, suggesting that it could be used as an antigen for detection of Campylobacter jejuni. Two kinds of outer membrane proteins, PEB1 and CadF, were successfully cloned and expressed by molecular biological techniques, and the expressed products were purified by nickel ion affinity column. Two purified proteins, PEB1 and CadF, were used to immunize New Zealand long-eared rabbits, and the corresponding polyclonal antibodies were prepared, the titers of which were 1: 1024000 and 1: 128000, respectively. In this paper, the cross reaction between PEB1 antiserum and Pseudomonas aeruginosa, Staphylococcus aureus, Lactobacillus bulgaricus, Shigella flexneri, CadF antiserum and denatured bacillus vulgaris, Vibrio parahaemolyticus were found by dot blot hybridization. Listeria monocytogenes, Staphylococcus aureus and Bifidobacterium brevis have cross reactions. In this paper, ammonium octanoate precipitation method was used to remove the miscellaneous proteins in serum. Furthermore, the cross reaction of two antisera with the above microorganisms was eliminated by magnetic beads method, and high purity and high specificity antibodies were obtained. The results of this study will provide experimental materials for the establishment of immunological detection method for Campylobacter jejuni.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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