副猪嗜血杆菌外膜囊泡的组分分析及免疫原性研究

发布时间：2018-03-16 07:21

本文选题：副猪嗜血杆菌　切入点：外膜囊泡　出处：《河南农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：副猪嗜血杆菌(Haemophilus parasuis,HPS)属于巴斯德杆菌科、嗜血杆菌属,为一种多形性革兰氏阴性短杆菌,多存在于猪上呼吸道,是一种共栖菌。该菌可引起以多发性浆膜炎、关节炎、脑膜炎为主要特征,有较高死亡率的猪格氏病。已鉴定出该菌有15个血清型,但还有部分菌株不能定型,且各菌株之间缺乏交叉免疫保护性,因此研发新型有效的副猪嗜血杆菌病疫苗已成为当务之急。外膜囊泡(Outer membrane vesicle,OMV)是革兰氏阴性菌生长到稳定阶段后,外膜向外周环境中释放的包括整个外膜上的相关蛋白和脂多糖(Lipopolysaeeharide,LPS)成分的微小囊泡,不仅是致病性病原菌的毒力因子之一,而且具有很好的免疫原性,是极具潜力的新型疫苗抗原。副猪嗜血杆菌是革兰氏阴性菌,但目前对其OMV了解甚少。为此,本研究开展了OMV主要组分及生物学特性初步研究。首先采用超速离心法提取血清5型HPS菌株OMV,通过密度梯度离心进行纯化,SDS-PAGE实验分析其主要蛋白组分,同时与同种菌株包含的胞质蛋白(CP)、周质蛋白(PP)及外膜蛋白OMP进行比较,并用BCA法测定OMV的蛋白含量;通过PicoGreen法定量OMV中DNA浓度;进一步通过动物试验研究其免疫原性和保护作用。在此基础上,通过对纯化的OMV做蛋白酶K处理,接种人喉癌上皮细胞,测定细胞上清中IL-8含量,初步研究OMV的免疫保护机制及分子基础。研究结果表明:(1)副猪嗜血杆菌OMV的主要蛋白成分与外膜蛋白OMP的相似,并且其所含的蛋白主要分布于32~57KD;(2)DNA主要位于OMV腔内,并且含量远多于囊泡表面含有的DNA;(3)小鼠免疫OMV后,7天即产生高水平的ELISA抗体,二次免疫后能产生一定的攻毒保护;(4)OMV诱导上皮细胞产生细胞因子IL-8,且呈一定的剂量依赖性。这些生物学特性研究表明,OMV是HPS潜在的一种毒力因子和新型疫苗抗原,为副猪嗜血杆菌OMV疫苗的相关研究提供了依据。
[Abstract]:Haemophilus parasuisus (HPSs) belongs to the family Pasteulidae, Haemophilus, a pleomorphic gram-negative short bacilli, mostly found in the upper respiratory tract of pigs, is a common bacteria that can cause polyserositis and arthritis. Meningitis is the main characteristic of swine Grignard disease with high mortality. It has been identified that there are 15 serotypes of the strain, but some of the strains can not be typed, and there is no cross-immunity protection between the strains. Therefore, it is urgent to develop a new and effective vaccine against Haemophilus parasuis. Outer membrane vesiclev (OMV) is a Gram-negative bacterium that has grown to a stable stage. The microvesicles released by the outer membrane into the peripheral environment, including the proteins associated with the whole outer membrane and the lipopolysaccharide Lipopolysaeeharide LPSs, are not only one of the virulence factors of pathogenic bacteria, but also have good immunogenicity. Haemophilus paraswine is a gram-negative bacteria, but its OMV is little known. In this study, the main components and biological characteristics of OMV were preliminarily studied. First, OMV of serotype 5 HPS was extracted by ultracentrifugation, and its main protein components were purified by density gradient centrifugation. At the same time, the protein content of OMV was determined by BCA method, and the concentration of DNA in OMV was determined by PicoGreen, compared with cytoplasmic protein, pericytoplasmic protein and outer membrane protein OMP of the same strain. The immunogenicity and protective effect of the purified OMV were further studied by animal experiments. On this basis, the purified OMV was inoculated with human laryngeal carcinoma epithelial cells and the content of IL-8 in the supernatant was determined by the treatment of protease K. The immunological protective mechanism and molecular basis of OMV were preliminarily studied. The results showed that the main protein components of OMV of Haemophilus parais were similar to those of outer membrane protein OMP, and the protein mainly distributed in the cavity of OMV. Moreover, the content of ELISA antibody was much higher than that of DNA-3 on the surface of vesicle) 7 days after immunization with OMV, high level of ELISA antibody was produced in mice. After secondary immunization, OMV could induce the production of IL-8 in epithelial cells in a dose-dependent manner. These biological characteristics indicate that OMV is a potential virulence factor and a new vaccine antigen of HPS. It provides the basis for the research of Haemophilus parais OMV vaccine.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

【相似文献】