【作者】 乔凤；

【导师】 于雅琴； 陈泽良；

【作者基本信息】 吉林大学 ， 医学基因组学， 2009， 博士

【摘要】 布鲁氏菌病是一种危害严重的人兽共患病,在世界范围内有广泛流行,给人类健康和经济发展带来巨大损失。布鲁氏菌是一种胞内寄生菌,具有比较独特的胞内生存机制和免疫机制。它的致病机制以胞内生存为主要特征,而免疫机制则是在与宿主免疫系统斗争中逃避免疫反应而得以生存繁殖。针对布鲁氏菌的有效的免疫反应以细胞免疫为主,并且是多种抗原综合作用的结果。深入探讨布鲁氏菌的胞内生存机制和免疫保护机制,对于布鲁氏菌致病机制的理解、疫苗的保护机制以及新型疫苗的研发等具有重要意义。外膜蛋白在稳定细菌外膜的结构、适应胞内外环境和抵抗胞内杀菌机制等方面起着重要作用,与细菌的毒力有密切关系。外膜蛋白位于细菌的表面,容易被免疫系统识别,在与宿主的相互作用中发挥作用。另一方面,很多外膜蛋白都具有免疫原性,其中很大一部分是免疫保护抗原。因此,很多有关细菌致病机制和免疫反应机制的研究都集中在外膜蛋白的研究上。布鲁氏菌病的控制重在预防,大规模接种疫苗是有效控制疾病的重要措施。事实证明,尽管经历了很多对其它疫苗形式的尝试,如亚单位疫苗、基因工程疫苗和核酸疫苗等,但是减毒活疫苗仍是目前预防布鲁氏菌病最为有效的疫苗形式。减毒活疫苗之所以能够发挥有效的免疫保护作用,是否与其外膜的成分,特别是外膜蛋白有关?或者说外膜蛋白在减毒活疫苗中发挥了什么样的作用?这些问题的回答,有利于解释布鲁氏菌疫苗的作用机制和保护机制,也有利于进一步开发新型的疫苗。为研究布鲁氏菌主要外膜蛋白在布鲁氏菌毒力和免疫保护中的作用,本研究中,我们首先利用蛋白质组技术,分离鉴定布鲁氏菌的外膜蛋白,明确外膜蛋白的组成。然后,选取主要的外膜蛋白,构建其缺失突变株,并比较分析这些突变株的胞内外的毒力表型,探讨其与毒力的关系。用M5及缺失突变菌株免疫小鼠,然后用M5本身和羊种布鲁氏菌强毒株16M进行攻击,观察M5及突变菌株的免疫保护性,探讨主要外膜蛋白在疫苗株免疫保护中的作用及其机制。最后,体外克隆表达主要外膜蛋白,分析其免疫原性,探讨其作为诊断抗原的可行性。通过以上研究,将初步了解羊布鲁氏菌外膜蛋白的组成,认识主要外膜蛋白在M5的毒力及免疫保护中的作用,并探索了主要外膜蛋白作为诊断抗原的可行性。更多还原

【Abstract】 Brucellosis, which caused by Brucella, is one of the most important bacterial zoonoses endemic in the world, especially in developing countries. These pathogens can affect a broad range of mammals and cause serious economic losses. Three Brucella species, B. melitensis, B. abortus and B.suis, are virulent for humans, causing a chronic, debilitating disease with severe and sometimes fatal outcomes. Moreover, as Brucella spp. can be easily aerosolized, this pathogen could be used to develop biological weapons.Brucella is an intracellular bacterium. Research on Brucella and its interaction between host reveals that Brucella activate reduced immune response. The virulence of Brucella depends upon its ability to survive and replicate in host cells. They replicate in endoplasmic reticulum (ER) and escape from the immune response. Effective immune response is mainly mediated by cellular immunity. Outer membrane proteins (OMPs) play important roles in stabilizing the structure of the outer membrane, adapting to external and intracellular environments, and Brucella virulence. OMPs are located on surface of bacteria and can be easily recognized by host immune system, therefore, some of them represent important immunoantigen.Although many attepts have been tried for development of different vaccine types, live attenuated vaccine is the most efficient vaccine for Brucellosis for the present. In the present study, to analyze the roles of major OMPs in Brucella virulence and immunological protection, outer membrane proteome was separated by 2D eletrophoresis and the proteins were identified by MS. A total of 67 proteins were identified to be possibly membrane proteins. Nine of these protein spots are products of Omp25 and seven were those of Omp31. Omp25 is a member of the OmpA protein family, which have immunogenicity. Outer membranes proteins play important roles in stabilizing the structure of the outer membrane and are relative to the virulence. To predict its function, the sequence of omp25 was analyzed by bioinformatics methods. In the genome of Brucella, there are four omp25 genes(BMEI1249(omp25)、 BMEI1007(omp25b)、BMEI1829(omp25c) and BMEI1830(omp25d). Among the four genes, only omp25 (BMEI1249) have been shown to be related with brucella virulence .To further investigate the function of omp25, the mutants of the four omp25 genes were constructed by homologous recombination, and then their virulence phenotypes were tested. Firstly, the kanamycin gene of pBBR1MCS-2 was PCR amplified and cloned at the multicloning site of pUC19 to generate a new suicide plasmid pUC19K, which was then used to construct the mutant strains. By using resistance replacement method, mutants of the four omp25 genes were successfully constructed, named BMΔ1007, BMΔ1249, BMΔ1829 and BMΔ1830.In vitro growth curve assays showed that the four mutants and BM have similar growth curves. However, the mutants showed higher growth rate than the wild type strain at logarithmic phase. At stationary phase, BMΔ1007 showed the highest cell density. This implyied that omp25 negatively regulate growth of Brucella under the assaying conditions. When grow into stationary phase, BMΔ1249 autoaggregated to form clamps in culture, which were not observed in the other three mutant strains. Survival under stress conditions showed that the four mutants showed different sensitivity to the stress simulating intracellular environments, including high salt, high osmosis, low pH, heat shock and oxidative stress. Sensitivity of these mutants to polymyxin B and sodium deoxycholate were also increased. These data indicated that omp25 plays important roles in Brucella adaptation to hostile environments and maintaining integrity of membrane structure. Intracellular survival showed that the omp25 mutants could invade macrophage, but were rapidly cleared by host cell, implying that omp25 is important for Brucella intracellular replication and chonic infection. Balb/c mice was infected with M5 and omp25 mutants, and then challenged with BM and 16M to test their protection capability. When challenged with M5, no great differences were observed in protection efficiency between three mutants and M5, but the BMΔ1829 showed better protection. Then, BMΔ1249, BMΔ1829 and M5 was used to immunize mice and challenged with 16M. The mutant showed reduced protection than M5. The mice immunized with omp25 mutants showed lower antibody titer and IFN-v those infected with M5, implying that omp25 plays important roles in protection efficency of M5. At last, the four omp25 proteins were expressed in E. coli. Western blot result showed that all the proteins could react with brucellosis sera. Only omp25 protein could differentiate sera of M5 from that of the mutant strain.The results above showed that the omp25 play important roles in brucella adaptation to hostile environments, intracellular survival and maintaining integrity of outer membrane. These data also showed that omp25 proteins play important roles in protection of live attenuated strain. These findings expand our knowledge of the function of Omp25 in Brucella survival both in vivo and in vitro, virulence phenotypes and immuno-protection, providing important clues for the understanding of the molecular mechanism of intracellular survival and protection mechanism of Brucella.更多还原