基于组学研究对致病性钩体毒力因子及免疫靶标的分析

发布时间：2018-02-10 01:26

本文关键词： 钩端螺旋体基因组学表面暴露蛋白质组毒力因子硫醇过氧化物酶　出处：《上海交通大学》2015年博士论文　论文类型：学位论文

【摘要】：钩端螺旋体病是由致病性钩端螺旋体引起的一种全球范围内的人兽共患病,感染钩端螺旋体可引起黄疸、发热、出血以及急性的多器官衰竭等危害,其严重威胁着人类的健康,与此同时也对畜牧业造成了极大的损失。由于钩体特殊的进化地位以及有效遗传操作系统的缺乏,人们对于钩体及钩体病的研究进展一直十分缓慢,钩体的主要致病因子及致病机制仍不明确,亦缺乏相应的诊断及疫苗靶标。直到2003年第一株钩体全基因组测序数据的报道,基于全基因组测序技术的相应研究不断为人们对于钩体生理、代谢以及致病机制的研究和认识提供新的方法和视野。本论文基于对我国致病性钩体主要血清群代表株的基因组测序研究,初步探讨了钩体不同血清群泛基因组的组成,并以核心基因组为基础进一步分析了这些血清群间的进化关系。另外,通过454焦磷酸测序并结合Sanger测序技术,完成了我国钩体疫苗株56610(401)株的全基因组测序研究,鉴定出了其染色体外环状质粒的存在以及特异的O抗原编码区域。结合课题组已经完成全基因组测序的另一株钩体疫苗菌株56603(Gui44)株以及国际上已公布的6株钩体(4株强毒及2株腐生型钩体)全基因组测序数据,我们也对8株不同毒力的钩体全基因组进行了比较分析。通过对致病性菌株特有基因的筛选,预测到20个潜在的核心毒力编码基因。在全基因组测序的基础上,我们完成了问号钩体56601株、56603(Gui44)株及56610(401)株的表面暴露蛋白质组质谱鉴定,结合优化的表面暴露蛋白预测策略共筛选的81个核心表面暴露蛋白、61个非必需表面暴露蛋白以及122个特异表面暴露蛋白。另外,我们在分泌蛋白质组内筛选到一个保守存在的蛋白-硫醇过氧化物酶。通过实验证实,由基因LA0862编码的钩体硫醇过氧化物酶可以通过非典型的分泌途径分泌到胞外,在钩体与宿主相互作用的过程中,保护钩体免受外源过氧化物压力的威胁。重组表达的钩体硫醇过氧化物酶刺激产生的抗体与致病性钩体具有较好的交叉免疫反应,钩体硫醇过氧化物酶和钩体免疫的动物血清也可以发生反应,这说明LA0862可以做为钩体的疫苗及诊断的靶点。
[Abstract]:Leptospirosis is a worldwide zoonosis caused by pathogenic leptospirosis. Infection with leptospirosis can cause jaundice, fever, bleeding and acute multiple organ failure. At the same time, it has caused great losses to animal husbandry. Due to the special evolutionary status of leptospirosis and the lack of effective genetic operating system, the progress of research on leptospirosis and leptospirosis has been very slow. The main pathogenic factors and pathogenetic mechanism of Leptospira were still unclear, and the corresponding diagnosis and vaccine target were lacking. Until 2003, the first Leptospira genome sequencing data were reported. The corresponding research based on the whole genome sequencing technology has been used for the study of Leptospira physiology. The study and understanding of metabolism and pathogenesis provide a new method and perspective. Based on the genome sequencing of the main serogroup representative strains of leptospira in China, the composition of pangenome of different serogroups of Leptospira was preliminarily discussed. Based on the core genomes, the evolutionary relationships among these serogroups were further analyzed. In addition, the whole genome sequencing of Chinese leptospirosis vaccine strain 56610 / 401 was completed by 454 pyrosequencing and Sanger sequencing. The exocyclic plasmids and the specific O antigen coding region were identified. Another Leptospira vaccine strain 56603 Gui44, which has been sequenced by our research group, and 6 Leptospira Leptospira strains published internationally, have been identified. Complete genome sequencing data of two strains of saprophytic leptospirosis. We also compared and analyzed the whole genome of eight different virulence leptospira strains. By screening the specific genes of pathogenic strains, we predicted 20 potential core virulence coding genes. We have completed the surface exposure proteome mass spectrometry identification of 56601 strains of Leptospira interrogans, 56603, Gui44, and 5661010401. A total of 81 core surface exposure proteins, 61 non-essential surface exposure proteins and 122 specific surface exposure proteins were screened in combination with the optimized surface exposure protein prediction strategy. We screened a conserved protein-mercaptoperoxidase (mercapto peroxidase) from the secretory proteome. It was proved by experiments that the leptospirase encoded by the gene LA0862 can be secreted to the extracellular via atypical secretory pathway. During the interaction between Leptospira and host, Leptospira was protected from the threat of exogenous peroxidase. The antibody produced by recombinant Leptospira mercaptoperoxidase stimulated by Leptospira had a good cross-immune response to pathogenic Leptospira. Leptospira mercaptoperoxidase and Leptospira immunized animal serum can also react, which suggests that LA0862 can be used as a vaccine and a diagnostic target for leptospirosis.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R514.4

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