本文選題：布魯氏菌 + 104M株　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年博士論文

【摘要】：布魯氏菌是一種胞內(nèi)寄生的革蘭氏陰性球桿菌,具有多個(gè)種型,它所引起的布魯氏病是一種人畜共患的國家乙類傳染病。近年來,我國的布魯氏病疫情非常嚴(yán)峻,感染人數(shù)迅速攀升,帶來較大危害。此外,布魯氏菌具有在環(huán)境中存活能力強(qiáng)、易被氣溶膠化等特點(diǎn),可能被用作生物恐怖劑。布魯氏菌對(duì)我國的人民健康和生物安全帶來嚴(yán)重威脅,對(duì)其進(jìn)行深入研究具有重要意義。疫苗是防控布魯氏病的有效手段。目前,獸用布魯氏菌疫苗已獲得廣泛使用,在控制布魯氏病疫情方面起到重要作用,而人用布魯氏菌疫苗研制較為困難,國外尚未有疫苗上市,我國唯一批準(zhǔn)人用的布魯氏菌疫苗為減毒活疫苗104M株。人用布魯氏菌疫苗研制困難的主要原因,可能在于布魯氏菌存在多個(gè)種型,且保護(hù)性抗原譜復(fù)雜,單一抗原很難起到完全保護(hù)作用。隨著組學(xué)以及生物信息學(xué)的發(fā)展,反向疫苗學(xué)逐漸成為研制新型疫苗的重要方法。反向疫苗學(xué)以組學(xué)和生物信息學(xué)為基礎(chǔ)進(jìn)行抗原預(yù)測,并隨后對(duì)預(yù)測的候選抗原進(jìn)行驗(yàn)證評(píng)價(jià),進(jìn)而獲得有效的保護(hù)性抗原用于新型疫苗研制。國內(nèi)外利用反向疫苗學(xué)篩選布魯氏菌抗原已有一些研究,但相關(guān)的組學(xué)數(shù)據(jù)還不完善。近年來,隨著質(zhì)譜技術(shù)的迅速發(fā)展,獲得高覆蓋率的蛋白質(zhì)組并采用質(zhì)譜數(shù)據(jù)反向注釋基因組成為可能,即蛋白質(zhì)基因組學(xué)。同時(shí),蛋白質(zhì)組學(xué)方法也是研究布魯氏菌本身生理代謝與致病機(jī)制的重要工具。本研究首先通過蛋白質(zhì)基因組學(xué)方法來完善布魯氏菌104M株的組學(xué)數(shù)據(jù)。隨后通過定量蛋白質(zhì)組技術(shù)研究布魯氏菌在模擬胞內(nèi)應(yīng)激條件下的差異蛋白質(zhì)組,進(jìn)而為了解布魯氏菌的胞內(nèi)寄生機(jī)制提供線索。在此基礎(chǔ)上,建立綜合組學(xué)與生物信息學(xué)的保護(hù)性抗原篩選新策略,對(duì)布魯氏菌保護(hù)性抗原進(jìn)行預(yù)測,篩選并驗(yàn)證疫苗候選抗原,為新型人用布魯氏菌疫苗設(shè)計(jì)提供依據(jù)。在蛋白質(zhì)基因組學(xué)方面,本研究建立了多策略蛋白質(zhì)組質(zhì)譜鑒定方法,分別采用SDS-PAGE和液相色譜預(yù)分離方法,對(duì)104M全蛋白質(zhì)組與膜蛋白質(zhì)組進(jìn)行全面的質(zhì)譜鑒定,并對(duì)104M基因組進(jìn)行反向重注釋,獲得了較高覆蓋率的104M株蛋白質(zhì)組數(shù)據(jù)庫。共鑒定到蛋白1729個(gè)(總注釋蛋白3072個(gè)),覆蓋率達(dá)56.3%。鑒定蛋白在等電點(diǎn)、分子量、疏水性、跨膜區(qū)等方面分布情況與104M注釋基因相符。鑒定蛋白覆蓋了幾乎全部COG功能分類(20/22),體現(xiàn)出功能分布的全面性。本研究還鑒定到17個(gè)重要已知保護(hù)性抗原和14個(gè)關(guān)鍵毒力基因在104M中大量表達(dá),為了解104M免疫保護(hù)性與殘余毒性提供了依據(jù)。同時(shí),驗(yàn)證了218個(gè)假想蛋白的存在,并對(duì)假想蛋白的功能進(jìn)行了分析預(yù)測,其中26個(gè)為膜或分泌蛋白,57個(gè)為潛在保護(hù)性抗原或毒力基因。對(duì)104M基因組重注釋,發(fā)現(xiàn)了6處新基因并糾正了3處基因注釋錯(cuò)誤,并利用RT-PCR方法對(duì)這些基因進(jìn)行了進(jìn)一步驗(yàn)證。此外,在蛋白質(zhì)組水平發(fā)現(xiàn)了104M大小染色體在蛋白豐度上的差異,提示大染色體上蛋白表達(dá)量可能高于小染色體,并初步探討了導(dǎo)致差異的機(jī)理。在定量蛋白質(zhì)組學(xué)方面,本研究采用Label-free定量蛋白質(zhì)組技術(shù),通過設(shè)置多組應(yīng)激條件模擬胞內(nèi)生存環(huán)境,探究多應(yīng)激綜合作用下布魯氏菌生理與代謝的改變機(jī)制。共設(shè)置1個(gè)正常體外培養(yǎng)條件對(duì)照組(TSB),7個(gè)單一應(yīng)激條件組:血清、營養(yǎng)限制、理化應(yīng)激、氧化/氮化應(yīng)激、缺氧、鐵缺乏、抗菌肽,以及1個(gè)綜合應(yīng)激組來模擬胞內(nèi)生存環(huán)境。結(jié)果顯示,與正常培養(yǎng)條件相比,104M在不同應(yīng)激條件下生存率不同(3.2%-73.2%),營養(yǎng)限制、氮化/氧化應(yīng)激以及缺氧環(huán)境對(duì)104M的生存具有較大影響。對(duì)各應(yīng)激條件下生長的104M菌株蛋白進(jìn)行Label-free定量質(zhì)譜鑒定,共鑒定到定量蛋白2272個(gè),其中1221個(gè)蛋白在應(yīng)激條件下表達(dá)與對(duì)照組相比具有顯著差異。這些差異表達(dá)蛋白主要富集在氧化磷酸化、ABC轉(zhuǎn)運(yùn)蛋白、雙組分系統(tǒng)、次生代謝物的生物合成、卟啉和葉綠素代謝、甘油磷脂代謝、檸檬酸循環(huán)(TCA循環(huán))、硫胺代謝、氮代謝、碳代謝等與布魯氏菌胞內(nèi)生存和環(huán)境壓力適應(yīng)密切相關(guān)的代謝通路上,為深入了解布魯氏菌的胞內(nèi)生存機(jī)制提供了線索。在保護(hù)性抗原篩選方面,本研究總結(jié)了與保護(hù)性抗原預(yù)測相關(guān)的多種因素,最終選定了6個(gè)關(guān)鍵因素,包括蛋白亞細(xì)胞定位、保護(hù)組相似性、抗原性、表位、毒力基因、粘附素,進(jìn)行預(yù)測打分,將打分?jǐn)?shù)據(jù)綜合后排序,建立了“多因素保護(hù)性抗原預(yù)測法”(Multi-factor Prediction of Protective Antigens,MPPA)。通過已知的保護(hù)性抗原數(shù)據(jù)庫與非保護(hù)性抗原數(shù)據(jù)庫對(duì)MPPA進(jìn)行方法驗(yàn)證,證明其可以有效區(qū)分保護(hù)性抗原與非保護(hù)性抗原,預(yù)測效果良好(靈敏性0.783、特異性0.940),優(yōu)于國外報(bào)道的預(yù)測篩選方法。應(yīng)用MPPA篩選布魯氏菌104M株保護(hù)性抗原,共獲得高概率保護(hù)性抗原26個(gè),其中包含9個(gè)已知布魯氏菌保護(hù)性抗原,如Omp19、Omp25、Omp31、SodC和Invasion protein B等,進(jìn)一步驗(yàn)證了篩選方法的可行性。同時(shí)還篩選到多個(gè)潛在的新保護(hù)性抗原,包括外膜脂蛋白LipA、Ⅰ型分泌系統(tǒng)蛋白HlyD、Ⅳ型分泌系統(tǒng)蛋白VirB8、外膜外排蛋白TolC和受體蛋白TonB等,為新型人用布魯氏菌疫苗的設(shè)計(jì)提供了基礎(chǔ)數(shù)據(jù)。在候選抗原保護(hù)性驗(yàn)證方面,本研究選擇了4個(gè)代表性保護(hù)性抗原Omp19、VirB8、HlyD和LipA進(jìn)行驗(yàn)證評(píng)價(jià)。分別通過大腸桿菌表達(dá)系統(tǒng)重組表達(dá)和純化獲得了Omp19、VirB8、HlyD和LipA重組蛋白抗原,在小鼠模型上進(jìn)行免疫原性和保護(hù)效力評(píng)價(jià)。血清學(xué)檢測結(jié)果顯示,Omp19、VirB8和HlyD免疫后,能夠刺激小鼠產(chǎn)生較強(qiáng)的體液免疫反應(yīng),總抗體IgG滴度分別為8.2×105,3.0×105與3.3×106。Omp19與VirB8抗體亞類以IgG1為主,為Th2偏向;HlyD抗體亞類以IgG2a為主,為Th1偏向。通過分離各組免疫后小鼠脾細(xì)胞,在體外用對(duì)應(yīng)抗原刺激培養(yǎng),檢測細(xì)胞因子TNF-a、IFN-γ、IL-6和IL-10的分泌水平,結(jié)果顯示Omp19、VirB8和HlyD能夠較好的刺激機(jī)體產(chǎn)生以TNF-α、IFN-γ為特征的Th1型免疫反應(yīng),和以IL-6、IL-10為特征的Th2型免疫反應(yīng)。布魯氏菌A19攻毒實(shí)驗(yàn)結(jié)果顯示,Omp19、VirB8和HlyD免疫小鼠后,可以顯著降低脾臟與肝臟中細(xì)菌定值數(shù),具有較好的保護(hù)效果。綜上所述,本研究通過串聯(lián)質(zhì)譜方法對(duì)布魯氏菌疫苗株104M進(jìn)行了蛋白質(zhì)基因組學(xué)研究,獲得了迄今為止最高覆蓋率的布魯氏菌蛋白質(zhì)組數(shù)據(jù)庫,并完善了基因組注釋;通過Label-free定量蛋白質(zhì)組方法研究了布魯氏菌在模擬胞內(nèi)應(yīng)激條件下的差異蛋白質(zhì)組,發(fā)現(xiàn)了涉及布魯氏菌胞內(nèi)生存和環(huán)境壓力適應(yīng)的多個(gè)重要通路,為深入了解布魯氏菌的胞內(nèi)生存機(jī)制提供了重要線索;在組學(xué)研究基礎(chǔ)上,建立了“多因素保護(hù)性抗原預(yù)測法”(MPPA),對(duì)104M進(jìn)行了保護(hù)性抗原預(yù)測與篩選,獲得了多個(gè)潛在的新保護(hù)性抗原;對(duì)4個(gè)候選保護(hù)性抗原進(jìn)行了免疫驗(yàn)證,證實(shí)3個(gè)抗原Omp19、VirB8和HlyD能夠刺激小鼠產(chǎn)生較強(qiáng)的特異性體液免疫反應(yīng)和細(xì)胞免疫反應(yīng),且在攻毒實(shí)驗(yàn)中顯示較好的保護(hù)效果,為新型人用布魯氏菌疫苗的設(shè)計(jì)提供了依據(jù)。
[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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392

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