【作者】 杨艳玲；

【导师】 王兴龙；

【作者基本信息】 吉林大学 ， 预防兽医学， 2011， 博士

【摘要】 布鲁氏菌病(简称布病)是由布鲁氏菌引起的一种人兽共患的传染病,是重要的人畜共患病,在世界各地都有广泛流行,尤其是在发展中国家。该病自1887年Bruce发现羊布鲁氏菌以来呈现了复发的趋势,布病的地理分布在不断的改变,新的疫源地不断涌现,数据显示全世界因布病在动物产品以及公共卫生方面的经济损失是巨大的。当前控制和根除布病最关键的问题在于其致病机制不完全清楚以及没有安全有效的疫苗。布鲁氏菌属于胞内寄生菌,其强毒株和疫苗株的外膜结构存在着很大的差异,进而导致它们的胞内寄生过程存在着明显的差异。为此,本研究利用比较蛋白质组学技术对羊布鲁氏菌强毒株16M和疫苗株M5的外膜蛋白进行了比较分析研究,并用半定量RT-PCR技术对这些差异蛋白进行转录水平的分析,从而了解了布鲁氏菌两个不同毒力菌株外膜蛋白的差异表达情况。通过生物信息学数据检索和相关文献数据查询,对这些差异蛋白在细菌生命活动中所发挥的生物学作用以及亚细胞定位等相关信息进行分析,以此来阐释它们与布鲁氏菌的致弱机制、毒力以及胞内寄生等过程中发挥的重要作用。在布病高发地区,所有易感动物的免疫和感染动物的根除是防控布病唯一手段。目前使用的疫苗仍然是以畜用减毒活疫苗为主,如S19,Rev.1,M5,S2等,但他们在安全性和有效性方面存在很多问题,比如容易引起人的感染,干扰血清学诊断,免疫后引起孕畜流产以及毒株扩增等缺点。至今还没有一种安全、有效的人用布病疫苗。然而能克服上述缺点的新型疫苗的研制如亚单位疫苗等备受青睐,成为了当前控制人畜布病的迫切需求。本研究利用免疫蛋白质组学技术在羊布鲁氏菌16M全菌蛋白中进行了免疫候选抗原的筛选鉴定,结合分子生物学和疫苗学验证了其免疫功能,以打破布病新型疫苗研制的瓶颈,为布病防治提供新的依据。首先,利用免疫蛋白质组学技术,对羊布鲁氏菌强毒株16M全细胞蛋白进行了提取并进行双向电泳分离,用临床上采集到的自然感染布鲁氏菌的人,牛羊的布病血清进行Western-blotting分析,从中获得了被三种血清共同识别的免疫候选抗原,即共同抗原。这些蛋白的发现为研制布鲁氏菌亚单位疫苗提供了候选抗原。其次,为了进一步验证这些免疫候选抗原的免疫原性和免疫保护能力,我们对这些蛋白进行了原核表达,并在小鼠体内进行了免疫原性和免疫保护能力的评价,进一步为筛选确实有效的布鲁氏菌亚单位疫苗候选抗原奠定基础。通过以上实验研究,我们得到了如下结果：1.羊布鲁氏菌强毒株16M和弱毒株M5的膜蛋白比较蛋白质组学结果显示,一共有33个差异蛋白点,经质谱鉴定和生物学信息检索后发现它们代表了26个开放阅读框。其中有22种蛋白在布鲁氏菌强毒株16M上高表达,4种蛋白在疫苗株M5上高表达。通过生物信息学数据检索以及相关文献数据显示,在强毒株内高表达的蛋白在布鲁氏菌的能量代谢,脂肪代谢,蛋白质及氨基酸的合成以及细菌外膜多糖的合成等生物过程中发挥着重要作用。同时在M5株中高表达的蛋白是细菌的重要免疫原性蛋白。半定量RT-PCR结果也显示了部分蛋白在转录水平上也存在着差异,如wbkC (spot16), UTP-glucose-1-hosphate-ridylyltransferase spot 35), purH (spot 20), Omp10 (spot 33), sucB (spot 27)。这些差异蛋白的发现将为我们解释布鲁氏菌的致弱机制提供了线索,同时也获得了一些与毒力和免疫相关的分子。2.羊布鲁氏菌标准毒株16M全细胞蛋白免疫蛋白组学结果显示,利用布鲁氏菌感染的人、牛、羊血清,筛选出56种免疫候选蛋白,其中有11种蛋白是三种血清共同识别的抗原。3.候选抗原的原核表达结果显示,7种蛋白被成功表达。辅以佐剂免疫小鼠,体液免疫、细胞免疫以及攻毒保护性实验结果显示,7种候选抗原中有4种候选抗原,即蛋白ahcY (spot 5), RSβ(spot 6), RSα(spot 12)和Isovaleryl-CoA dehydrogenase (spot 17),能够诱导高水平IL-2和IFN-γ为特征的Th1型细胞免疫反应,并能够抗羊布鲁氏菌毒力株16M的攻毒,提供的保护力分别为2.13,1.12,1.12和1.24。其中蛋白ahcY提供的保护力(2.13)与疫苗株M5提供的保护力相当(2.34)。总之,本研究为揭示布鲁氏菌致弱机制,分析外膜蛋白在布鲁氏菌胞内寄生以及毒力等方面发挥的作用提供了依据。同时我们的方法结合了免疫蛋白组学技术和免疫候选抗原的免疫原性和保护能力的评价方法,为布病亚单位疫苗候选抗原的筛选提供了新的策略。更多还原

【Abstract】 Brucellosis, which is caused by Brucella, is one of the most important bacterial zoonoses endemic in many countries, especially in developing countries. Brucellosis has been an emerging disease since the discovery of Brucella melitensis by Bruce in 1887. The geographical distribution of brucellosis is constantly changing, with new foci emerging or re-emerging. All data suggest that worldwide economic losses due to brucellosis are extensive not only in animal production but also in public health.The most critical problem that controlling and eradicating brucellosis in animals and human beings is that the investigation in its pathogenic mechanism is not fully clear and have no safe and effective brucellosis vaccines. Brucella is an intracellular bacterium.and the intracellular survival mechanism of the virulent strains are completely different from the vaccine ones, which maybe caused by the different structure in their outer membranes.Therefore, in the present study,the outer membrane proteins of the two strains were analyzed by comparative proteomitics, and these different expressed proteins were further analyzed in transcriptional level by the semiquantitative RT-PCR, and the related outer membranes were identified, which provide new clues for the understanding on the role of outer membrane proteins in Brucella virulence and intracellular survival.In regions with high prevalence of the disease, the only way of controlling and eradicating this zoonosis is by vaccination of all susceptible hosts and elimination of infected animals. They remains used to vaccinate the animals, although live, attenuated vaccine, such as S19,Rev.1,M5,S2 have some disadvantages:they can be infectious for humans; they can interfere with diagnosis; they may result in abortions when administered to pregnant animals; and the vaccine strain can spread in the region. Howerver, have no a safe and effective vaccine against human brucellosis. It will be favor that a new vaccine overcoming the above-mentioned drawback,such as subunit vaccine, which will be urgent needs for controlling the brucellosis in animals and humans.In the present study, the immunogenic candidate antigens were identified by immoproteomics from B.melitensis 16M soluble proteins. And tne immunologic function were further evaluated by combining the molecular biology and vaccinology with the aim to design new-type vaccines against Brucella.First, the immoproteomics strategy was used to screen novel immunogenic candidate proteins for the development of brucellosis subunit vaccine.The soluble proteins of Brucella melitensis were extracted and separated by two-dimensional electrophoresis (2-DE), and the Western blotting analysis were performed probing with the Brucella-infected serm from humans, cattles and goats.The immunodominant proteins were identified by Liquid chromatography tandem mass spectrometry (LC-MS/MS).Some common immunodominant proteins recognized by the three different serum will be new immunogenic candidate proteins for the development of brucellosis subunit vaccine.Then, these immunogenic candidate proteins were expressed in E.coli for evaluating their immunogenicity and protective ability in the mice model, which laid a foundation for obtaining really effective vaccine antigens candidates.The corresponding results were obtained based on the above studies:1、33 differentially expressed proteins from Brucella melitensis virulent strain 16M and vaccine strain M5 outer membrane proteins were found.The results of LC-MS/MS and bioinfermatics indicated that they represented 26 open reading frames. And 22 and 5 proteins were up-regulated in 16M and M5, respectively. The data of bioinfermation retrieval and related references showed that the up-regulated proteins in 16M mainly participated in energy metabolism, fat metabolism, protein, amino acid and polysaccharides biosynthesis and the one in M5 were important immunogenic proteins.The results of semi-quantitative RT-PCR showed that 5 Brucella memebrane proteins, which are wbkC (piont16), UTP-glucose-1-phosphate-uridylyltransferase (piont 35), purH (piont 20), Omp10 (piont 33) and sucB (piont 27), are also difference in transcription level, which basically participate in bacteria cell membrane biosynthesis.2、The 56 immunodominant proteins were successfully identified by LC-MS/MS. Among them,11 common antigens were recognized by the serum from Brucella-infected humans, cattles and goats serum. 3、The results from the evaluation of immunogenicity and protective ablity in mice model showed that 4 of 11 immunodominant proteins, they are ahcY(piont 5), RSβ(piont 6), RSα(piont 12), Isovaleryl-CoA dehydrogenase (piont 17), could significantly induce the Th1 cell immnune response characterized by high level of IFN-γand IL-2. And they provided partially protective ability against B.melitensis 16M chanllenge, They provided the log unit protection of 2.13,1.12,1.12 and 1.24, respectively.And the protein ahcY can confer the similar profective ability(2.13) to the vaccine strain M5 (2.34).Finally, our study provided the basis to reveal that the role of Brucella membrane protein in Brucella virulence and intracellular survival and our approaches may provide a new strategy for screening vaccine candidates in combination with immunoproteomics and analysis of the immunogenicity and protective ability in a mouse model.更多还原