布鲁氏菌疫苗株104M的蛋白质组学研究与保护性抗原筛选

发布时间：2018-04-23 07:40

  本文选题：布鲁氏菌 + 104M株　； 参考：《中国人民解放军军事医学科学院》2017年博士论文

【摘要】：布鲁氏菌是一种胞内寄生的革兰氏阴性球杆菌,具有多个种型,它所引起的布鲁氏病是一种人畜共患的国家乙类传染病。近年来,我国的布鲁氏病疫情非常严峻,感染人数迅速攀升,带来较大危害。此外,布鲁氏菌具有在环境中存活能力强、易被气溶胶化等特点,可能被用作生物恐怖剂。布鲁氏菌对我国的人民健康和生物安全带来严重威胁,对其进行深入研究具有重要意义。疫苗是防控布鲁氏病的有效手段。目前,兽用布鲁氏菌疫苗已获得广泛使用,在控制布鲁氏病疫情方面起到重要作用,而人用布鲁氏菌疫苗研制较为困难,国外尚未有疫苗上市,我国唯一批准人用的布鲁氏菌疫苗为减毒活疫苗104M株。人用布鲁氏菌疫苗研制困难的主要原因,可能在于布鲁氏菌存在多个种型,且保护性抗原谱复杂,单一抗原很难起到完全保护作用。随着组学以及生物信息学的发展,反向疫苗学逐渐成为研制新型疫苗的重要方法。反向疫苗学以组学和生物信息学为基础进行抗原预测,并随后对预测的候选抗原进行验证评价,进而获得有效的保护性抗原用于新型疫苗研制。国内外利用反向疫苗学筛选布鲁氏菌抗原已有一些研究,但相关的组学数据还不完善。近年来,随着质谱技术的迅速发展,获得高覆盖率的蛋白质组并采用质谱数据反向注释基因组成为可能,即蛋白质基因组学。同时,蛋白质组学方法也是研究布鲁氏菌本身生理代谢与致病机制的重要工具。本研究首先通过蛋白质基因组学方法来完善布鲁氏菌104M株的组学数据。随后通过定量蛋白质组技术研究布鲁氏菌在模拟胞内应激条件下的差异蛋白质组,进而为了解布鲁氏菌的胞内寄生机制提供线索。在此基础上,建立综合组学与生物信息学的保护性抗原筛选新策略,对布鲁氏菌保护性抗原进行预测,筛选并验证疫苗候选抗原,为新型人用布鲁氏菌疫苗设计提供依据。在蛋白质基因组学方面,本研究建立了多策略蛋白质组质谱鉴定方法,分别采用SDS-PAGE和液相色谱预分离方法,对104M全蛋白质组与膜蛋白质组进行全面的质谱鉴定,并对104M基因组进行反向重注释,获得了较高覆盖率的104M株蛋白质组数据库。共鉴定到蛋白1729个(总注释蛋白3072个),覆盖率达56.3%。鉴定蛋白在等电点、分子量、疏水性、跨膜区等方面分布情况与104M注释基因相符。鉴定蛋白覆盖了几乎全部COG功能分类(20/22),体现出功能分布的全面性。本研究还鉴定到17个重要已知保护性抗原和14个关键毒力基因在104M中大量表达,为了解104M免疫保护性与残余毒性提供了依据。同时,验证了218个假想蛋白的存在,并对假想蛋白的功能进行了分析预测,其中26个为膜或分泌蛋白,57个为潜在保护性抗原或毒力基因。对104M基因组重注释,发现了6处新基因并纠正了3处基因注释错误,并利用RT-PCR方法对这些基因进行了进一步验证。此外,在蛋白质组水平发现了104M大小染色体在蛋白丰度上的差异,提示大染色体上蛋白表达量可能高于小染色体,并初步探讨了导致差异的机理。在定量蛋白质组学方面,本研究采用Label-free定量蛋白质组技术,通过设置多组应激条件模拟胞内生存环境,探究多应激综合作用下布鲁氏菌生理与代谢的改变机制。共设置1个正常体外培养条件对照组(TSB),7个单一应激条件组:血清、营养限制、理化应激、氧化/氮化应激、缺氧、铁缺乏、抗菌肽,以及1个综合应激组来模拟胞内生存环境。结果显示,与正常培养条件相比,104M在不同应激条件下生存率不同(3.2%-73.2%),营养限制、氮化/氧化应激以及缺氧环境对104M的生存具有较大影响。对各应激条件下生长的104M菌株蛋白进行Label-free定量质谱鉴定,共鉴定到定量蛋白2272个,其中1221个蛋白在应激条件下表达与对照组相比具有显著差异。这些差异表达蛋白主要富集在氧化磷酸化、ABC转运蛋白、双组分系统、次生代谢物的生物合成、卟啉和叶绿素代谢、甘油磷脂代谢、柠檬酸循环(TCA循环)、硫胺代谢、氮代谢、碳代谢等与布鲁氏菌胞内生存和环境压力适应密切相关的代谢通路上,为深入了解布鲁氏菌的胞内生存机制提供了线索。在保护性抗原筛选方面,本研究总结了与保护性抗原预测相关的多种因素,最终选定了6个关键因素,包括蛋白亚细胞定位、保护组相似性、抗原性、表位、毒力基因、粘附素,进行预测打分,将打分数据综合后排序,建立了“多因素保护性抗原预测法”(Multi-factor Prediction of Protective Antigens,MPPA)。通过已知的保护性抗原数据库与非保护性抗原数据库对MPPA进行方法验证,证明其可以有效区分保护性抗原与非保护性抗原,预测效果良好(灵敏性0.783、特异性0.940),优于国外报道的预测筛选方法。应用MPPA筛选布鲁氏菌104M株保护性抗原,共获得高概率保护性抗原26个,其中包含9个已知布鲁氏菌保护性抗原,如Omp19、Omp25、Omp31、SodC和Invasion protein B等,进一步验证了筛选方法的可行性。同时还筛选到多个潜在的新保护性抗原,包括外膜脂蛋白LipA、Ⅰ型分泌系统蛋白HlyD、Ⅳ型分泌系统蛋白VirB8、外膜外排蛋白TolC和受体蛋白TonB等,为新型人用布鲁氏菌疫苗的设计提供了基础数据。在候选抗原保护性验证方面,本研究选择了4个代表性保护性抗原Omp19、VirB8、HlyD和LipA进行验证评价。分别通过大肠杆菌表达系统重组表达和纯化获得了Omp19、VirB8、HlyD和LipA重组蛋白抗原,在小鼠模型上进行免疫原性和保护效力评价。血清学检测结果显示,Omp19、VirB8和HlyD免疫后,能够刺激小鼠产生较强的体液免疫反应,总抗体IgG滴度分别为8.2×105,3.0×105与3.3×106。Omp19与VirB8抗体亚类以IgG1为主,为Th2偏向;HlyD抗体亚类以IgG2a为主,为Th1偏向。通过分离各组免疫后小鼠脾细胞,在体外用对应抗原刺激培养,检测细胞因子TNF-a、IFN-γ、IL-6和IL-10的分泌水平,结果显示Omp19、VirB8和HlyD能够较好的刺激机体产生以TNF-α、IFN-γ为特征的Th1型免疫反应,和以IL-6、IL-10为特征的Th2型免疫反应。布鲁氏菌A19攻毒实验结果显示,Omp19、VirB8和HlyD免疫小鼠后,可以显著降低脾脏与肝脏中细菌定值数,具有较好的保护效果。综上所述,本研究通过串联质谱方法对布鲁氏菌疫苗株104M进行了蛋白质基因组学研究,获得了迄今为止最高覆盖率的布鲁氏菌蛋白质组数据库,并完善了基因组注释;通过Label-free定量蛋白质组方法研究了布鲁氏菌在模拟胞内应激条件下的差异蛋白质组,发现了涉及布鲁氏菌胞内生存和环境压力适应的多个重要通路,为深入了解布鲁氏菌的胞内生存机制提供了重要线索;在组学研究基础上,建立了“多因素保护性抗原预测法”(MPPA),对104M进行了保护性抗原预测与筛选,获得了多个潜在的新保护性抗原;对4个候选保护性抗原进行了免疫验证,证实3个抗原Omp19、VirB8和HlyD能够刺激小鼠产生较强的特异性体液免疫反应和细胞免疫反应,且在攻毒实验中显示较好的保护效果,为新型人用布鲁氏菌疫苗的设计提供了依据。
[Abstract]:Brucella is a kind of intracellular parasitic gram negative bacilli with multiple species. Brucellosis caused by it is a kind of national B infectious disease. In recent years, Brucella disease in China is very severe, the number of infected people is rising rapidly, and it brings great danger. In addition, Brucella has a strong ability to survive in the environment. It is easy to be used as an aerated gelation, and may be used as a bioterrorism agent. Brucella poses a serious threat to the health and biosafety of our people. It is of great significance to study it in depth. Vaccine is an effective means to prevent and control Brucella disease. The main reason for the development of Brucella vaccine in China is that the human Brucella vaccine has not been listed in foreign countries. The only authorized Brucella vaccine in our country is the 104M strain of the live attenuated vaccine. The main reason for the development of Brucella vaccine is that Brucella is stored in many species, and the protective antigen spectrum is complex and single resistance. It is difficult to complete protection. With the development of histology and bioinformatics, reverse vaccine has gradually become an important method for developing new vaccines. Reverse vaccine studies antigen prediction based on histology and bioinformatics, and then tests the predicted candidate antigens to obtain effective protective antigens. In the development of new vaccine, there have been some studies on the screening of Brucella antigen at home and abroad, but the related data are not perfect. In recent years, with the rapid development of mass spectrometry technology, it is possible to obtain high coverage protein groups and use mass spectrometry data reverse annotated gene composition as possible, that is, protein genomics. Meanwhile, eggs The white matter method is also an important tool for the study of the physiological metabolism and pathogenesis of Brucella itself. In this study, we first perfected the data of the 104M strain of Brucella by means of protein genomics, and then studied the differential proteome of Brucella in the simulated intracellular stress by quantitative proteomic technique. To provide clues to the intracellular parasitism mechanism of Brucella, based on this, a new strategy for screening and screening of protective antigen of Brucella, screening and verification of vaccine candidate antigens is established, and the basis for the design of new human Brucella vaccine is provided. In this study, a Multi Strategy proteome mass spectrometry identification method was established. The SDS-PAGE and liquid chromatography pre separation methods were used to identify the 104M whole protein group and the membrane protein group, and the 104M genome was re annotated, and a high coverage 104M protein group database was obtained. 1729 proteins were identified. (total annotated protein 3072), the coverage rate reached 56.3%. identification protein at isoelectric point, molecular weight, hydrophobicity, transmembrane region and other aspects. The identification protein covered almost all COG functional classification (20/22), reflecting the overall function distribution. This study also identified 17 important known protective antigens and 14 key points. The key virulence genes were expressed in 104M, which provided a basis for understanding the protective and residual toxicity of 104M. At the same time, the presence of 218 hypothetical proteins was verified, and the function of hypothetical proteins was analyzed and predicted, of which 26 were membrane or secretory proteins, 57 were potentially protective antigens or virulence genes. The 104M genome was re annotated and distributed. 6 new genes were presented and 3 gene annotation errors were corrected. The RT-PCR method was used to further verify these genes. In addition, the difference in protein abundance of 104M size chromosomes was found at the level of proteome, suggesting that the protein expression on large chromosomes may be higher than that of small chromosomes, and the mechanism leading to the difference was preliminarily discussed. In quantitative proteomics, the Label-free quantitative proteome technique was used to explore the physiological and metabolic mechanisms of Brucella under multiple stress conditions by setting a number of stress conditions to simulate the intracellular survival environment. A total of 1 normal culture conditions control groups (TSB) were set up, and 7 single stress conditions group: serum, battalion Restriction, physical and chemical stress, oxidation / nitriding stress, anoxia, iron deficiency, antibacterial peptide, and 1 integrated stress groups to simulate the intracellular environment. The results showed that the survival rate of 104M was different under different stress conditions (3.2%-73.2%), nutrition restriction, nitriding / oxidative stress and hypoxia environment were larger for the survival of 104M. A total of 2272 quantitative proteins were identified by Label-free quantitative mass spectrometric identification of 104M strain proteins under stress conditions. The expression of 1221 proteins in stress conditions was significantly different from those of the control group. These proteins were mainly enriched in oxidative phosphorylation, ABC transporter, dual component system and secondary metabolism. Biosynthesis of substances, porphyrin and chlorophyll metabolism, glycerol phospholipid metabolism, citric acid cycle (TCA cycle), thiamine metabolism, nitrogen metabolism, carbon metabolism, etc. are closely related to the intracellular survival and environmental pressure adaptation of Brucella. It provides a clue for understanding the intracellular survival mechanism of Brucella. This study summed up a variety of factors related to the prediction of protective antigen, and finally selected 6 key factors, including protein subcellular localization, protection group similarity, antigenicity, epitopes, virulence genes, adhesion elements, forecasting scores, sorting the score data, and establishing a "multi factor protective antigen prediction method" (Multi-factor Pred). Iction of Protective Antigens, MPPA). Through the known protective antigen database and the non protective antigen database to verify the method, it is proved that it can effectively distinguish between the protective antigen and the non protective antigen, and the prediction effect is good (sensitivity 0.783, specificity 0.940), superior to the foreign reported prediction screening method. The application of MPPA sieve. A total of 26 protective antigens of Brucella strain 104M were selected, which included 9 known protective antigens of Brucella, such as Omp19, Omp25, Omp31, SodC and Invasion protein B, and further verified the feasibility of the screening methods. At the same time, several potential new protective antigens, including the outer membrane lipoprotein LipA, were also screened. Type I secretory system protein HlyD, type IV secretory protein VirB8, outer membrane protein TolC and receptor protein TonB provide basic data for the design of new human Brucella vaccine. In the protection verification of candidate antigens, 4 representative protective antigens, Omp19, VirB8, HlyD and LipA, were selected and evaluated. The recombinant protein antigen of Omp19, VirB8, HlyD and LipA was obtained by recombinant expression and purification of the Escherichia coli expression system. The immunogenicity and protective efficacy were evaluated on the mouse model. The serological test results showed that Omp19, VirB8 and HlyD immunization could stimulate the mice to produce a stronger humoral immune response, and the total antibody IgG titer was 8.2, respectively. The subclasses of X 105,3.0 * 105 and 3.3 x 106.Omp19 and VirB8 are IgG1, Th2 bias, and HlyD subclasses are IgG2a dominated and Th1 biased. By isolating the spleen cells of mice immunized from each group, the secretion of cytokines TNF-a, IFN- gamma, IL-6 and IL-10 is detected by corresponding antigen stimulation in vitro. It is better to stimulate the organism to produce the Th1 type immune response characterized by TNF- alpha and IFN- gamma, and the Th2 type immune response characterized by IL-6 and IL-10. The results of A19 toxicity test of Brucella A19 show that the number of bacteria in the spleen and liver can be significantly reduced after Omp19, VirB8 and HlyD are immune to mice. The protein genomics of Brucella vaccine strain 104M was studied by tandem mass spectrometry, and the highest coverage rate of Brucella proteome database was obtained to date and the genome annotation was perfected. The difference protein of Brucella in simulated intracellular stress was studied by Label-free quantitative proteome method. In the group, many important pathways involved in the intracellular survival of Brucella and the adaptation of environmental pressure were found, which provided an important clue to understand the intracellular survival mechanism of Brucella. On the basis of the study of the group study, the "multi factor protective antigen prediction method" (MPPA) was established, and the protective antigen of 104M was predicted and screened, and many of them were obtained. The potential new protective antigen, 4 candidate protective antigens were immunized, which proved that 3 antigens Omp19, VirB8 and HlyD could stimulate the mice to produce strong specific humoral immune response and cell immune response, and showed good protective effect in the attack test, which provided the basis for the design of new human Brucella vaccine. According to it.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392

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