本文選題：結(jié)核病 + 結(jié)核分枝桿菌��； 參考：《揚州大學(xué)》2010年碩士論文

【摘要】： 結(jié)核病(tuberculosis ,TB)是由結(jié)核分枝桿菌(Mycobacterium tuberculosis,M.tb)復(fù)合物引起的一種慢性傳染病,目前全球有1/3人口感染了M.tb,每年大約有300萬人死于結(jié)核病,且大部分是在發(fā)展中國家。接種疫苗被認(rèn)為是控制結(jié)核病的手段之一,當(dāng)前使用的卡介苗(Bacille Calmette and Guérin, BCG)能夠有效預(yù)防兒童和新生兒患病,但是對成年人活動性結(jié)核病保護效果有限。因此,迫切需要研制一種新型有效的疫苗或?qū)ΜF(xiàn)有疫苗進行改良。 M.tb一些分泌抗原的保護性作用已經(jīng)得到確認(rèn),在這些分泌蛋白中,6 kD早期分泌靶抗原(6-kDa early secreted antigenic target, ESAT-6)的作用相當(dāng)重要。ESAT-6存在于許多致病性分枝桿菌中,但是在BCG和大部分環(huán)境分枝桿菌中缺失。它作為一種T細(xì)胞抗原,能夠有效激發(fā)細(xì)胞免疫應(yīng)答,被建議作為診斷試劑用于區(qū)分M.tb感染和先前的BCG接種。因此,在本研究中,以沙門菌鞭毛蛋白為載體嵌合運送ESAT-6蛋白,并將其與BCG聯(lián)合應(yīng)用,以黏膜滴鼻方式免疫小鼠,探討誘導(dǎo)的免疫應(yīng)答特性。 1. BCG與大腸桿菌表達fliC/esat蛋白聯(lián)合應(yīng)用的黏膜免疫應(yīng)答規(guī)律研究 從大腸桿菌表達系統(tǒng)中表達并純化fliC/esat蛋白,Western-blotting分析其與野生型鼠傷寒沙門菌血清或ESAT-6單抗(Ms mAb to ESAT-6)作用后情況,結(jié)果都出現(xiàn)了特異性的目的條帶。制備小鼠的骨髓樹突狀細(xì)胞(bone marrow dendritic cells,BMDC),培養(yǎng)至第6天時用fliC/esat蛋白刺激,24 h后收集細(xì)胞分別標(biāo)記CD11c-FITC及CD80-Biotin、CD86-Biotin、MHC-Ⅰ-Biotin、MHC-Ⅱ-Biotin,FACS檢測各分子的表達情況,發(fā)現(xiàn)fliC/esat蛋白能夠誘導(dǎo)BMDC的成熟,不同程度的上調(diào)共刺激分子CD80、CD86、MHC-Ⅰ、MHC-Ⅱ的表達水平。通過滴鼻免疫途徑,將BCG與fliC/esat蛋白聯(lián)合免疫C57BL/6(H-2b)小鼠,結(jié)果顯示,血清和支氣管肺泡灌洗液(bronchoalveolar lavage fluid,BAF)中IgA抗體水平顯著高于單獨免疫BCG或fliC/esat蛋白時抗體水平,說明能夠誘導(dǎo)產(chǎn)生一定水平的黏膜抗體;運用淋巴細(xì)胞增殖試驗、ELISPOT、夾心ELISA等免疫學(xué)方法檢測脾臟淋巴細(xì)胞免疫應(yīng)答情況后顯示,細(xì)胞經(jīng)純化的結(jié)核桿菌素(PPD)和ESAT-6(1-20aa)多肽刺激后能夠明顯增殖,產(chǎn)生IFN-γ的分泌細(xì)胞數(shù)顯著上升并與產(chǎn)生IL-4的分泌細(xì)胞在數(shù)量上差異顯著,誘導(dǎo)以Th-1為主的免疫應(yīng)答,且免疫應(yīng)答水平要高于單獨免疫BCG或fliC/esat蛋白組。動態(tài)實驗持續(xù)觀察BCG與fliC/esat蛋白聯(lián)合免疫小鼠后2-14周的免疫應(yīng)答變化情況,發(fā)現(xiàn)IgA抗體水平、早期活化分子CD69的表達、淋巴細(xì)胞增殖及細(xì)胞因子的分泌都能持續(xù)到8-10周后才呈現(xiàn)下降趨勢。因此,BCG與fliC/esat蛋白聯(lián)合免疫的策略在小鼠模型上能夠誘導(dǎo)更強和更持久的黏膜免疫應(yīng)答和細(xì)胞免疫應(yīng)答,可為結(jié)核病的預(yù)防提供新的途徑和方法。 2. BCG與沙門菌重組鞭毛蛋白聯(lián)合黏膜免疫誘導(dǎo)的免疫應(yīng)答研究 用P22噬菌體介導(dǎo)LB5000(fliC/esat)向都柏林沙門菌SL5928轉(zhuǎn)導(dǎo),利用PCR、動力學(xué)檢測及血清凝集實驗鑒定并獲得轉(zhuǎn)導(dǎo)菌SL5928(fliC/esat)。提取轉(zhuǎn)導(dǎo)沙門菌SL5928(fliC/esat)鞭毛蛋白,Western-blotting顯示其與野生型鼠傷寒沙門菌血清或ESAT-6單抗(Ms mAb to ESAT-6)作用后都能出現(xiàn)特異性的目的條帶。制備小鼠的BMDC,培養(yǎng)至第6天時用SL5928(fliC/esat)鞭毛蛋白刺激,6 h后收集BMDC,利用RT-PCR技術(shù)檢測Toll樣受體5 (TLR-5)的表達,結(jié)果能夠表達TLR-5 mRNA,而對照組(未刺激)未見表達。將SL5928(fliC/esat)鞭毛蛋白與BCG通過滴鼻免疫途徑聯(lián)合免疫C57BL/6(H-2b)小鼠,檢測結(jié)果顯示,血清和BAF中IgA抗體水平顯著高于單獨免疫BCG或SL5928(fliC/esat)鞭毛蛋白時水平,說明能夠產(chǎn)生一定水平的黏膜抗體;CD4+、CD8+ T細(xì)胞表面分子CD69表達提高,T細(xì)胞得到活化;運用淋巴細(xì)胞增殖試驗、夾心ELISA等免疫學(xué)方法檢測淋巴細(xì)胞免疫應(yīng)答情況后顯示,脾臟淋巴細(xì)胞經(jīng)PPD或ESAT-6(1-20aa)多肽刺激后能夠明顯增殖;IFN-γ的分泌水平與IL-4的分泌水平在含量上差異顯著,趨向于Th-1為主的免疫應(yīng)答,且免疫應(yīng)答水平要高于單獨免疫BCG或SL5928(fliC/esat)鞭毛蛋白;肺臟淋巴細(xì)胞呈現(xiàn)同樣趨勢,且分泌的IFN-γ和IL-4含量要高于脾臟淋巴細(xì)胞。綜上,SL5928(fliC/esat)鞭毛蛋白可以作為潛在的結(jié)核病疫苗候選株,與BCG聯(lián)合應(yīng)用,為結(jié)核病疫苗研究提供新思路。
[Abstract]:Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M.tb) complex. At present, the population of 1/3 is infected with M.tb, and about 3 million people die of tuberculosis every year, and most of them are in developing countries. Vaccination is considered to be one of the means to control tuberculosis. Bacille Calmette and Gu e Rin (BCG) can effectively prevent children and newborns from being ill, but it has limited protection effect on adult active tuberculosis. Therefore, it is urgent to develop a new type of effective vaccine or improve the existing vaccine.
The protective effects of M.tb secretory antigens have been confirmed. In these secretory proteins, the role of the target antigen (6-kDa early secreted antigenic target, ESAT-6) in the early 6 kD is quite important in many pathogenic mycobacteria, but is missing in BCG and large part of the environmental Mycobacterium. It is a T cell. Antigen, which can effectively stimulate the cellular immune response, is suggested to be used as a diagnostic reagent to distinguish M.tb infection and previous BCG inoculation. Therefore, in this study, ESAT-6 protein was transported with Salmonella flagellin as a carrier, and it was combined with BCG to immunize mice with mucous nasal drops to explore the induced immune response.
Study on mucosal immune response in combination of 1. BCG and fliC/esat protein expressed by Escherichia coli
FliC/esat protein was expressed and purified from the Escherichia coli expression system, and Western-blotting was used to analyze its effect on the sera or ESAT-6 monoclonal antibody (Ms mAb to ESAT-6) of the wild type of Salmonella typhimurium (Ms mAb to ESAT-6). The result showed a specific target band. The mouse bone marrow dendritic cells (bone marrow dendritic cells, BMDC) were prepared and cultured to sixth It was stimulated by fliC/esat protein at the time. After 24 h, the cells were collected to mark CD11c-FITC and CD80-Biotin, CD86-Biotin, MHC- I -Biotin, MHC- II -Biotin, FACS to detect the expression of each molecule, and found that fliC/esat protein could induce the maturation of BMDC. The immunization of BCG and fliC/esat protein in C57BL/6 (H-2b) mice showed that the level of antibody in serum and bronchoalveolar lavage fluid (bronchoalveolar lavage fluid, BAF) was significantly higher than that in individual immune BCG or fliC/esat protein, indicating that it could induce a certain level of mucosal antibody and use lymphatic fines. Immunological methods, such as cell proliferation test, ELISPOT and sandwich ELISA, detected the immune response of spleen lymphocytes, and showed that the cells could proliferate obviously after the purified PPD and ESAT-6 (1-20aa) peptides were stimulated, and the number of secretory cells of IFN- gamma was significantly increased and the number of secretory cells produced by IL-4 was significantly different. The immune response was Th-1 dominated, and the immune response level was higher than that of the single immune BCG or fliC/esat protein group. The dynamic experiment continued to observe the changes in the immune response in the 2-14 weeks after the combined immunization of BCG and fliC/esat protein in mice, and found the level of IgA antibody, the expression of the early activated molecular CD69, the proliferation of lymphocyte and the secretion of cytokines. Therefore, the combined immunization strategy of BCG and fliC/esat protein can induce a stronger and more lasting mucosal immune response and cellular immune response in the mouse model, which can provide new ways and methods for the prevention of tuberculosis.
Immune response induced by 2. BCG combined with Salmonella recombinant flagellin combined with mucosal immunity
P22 phage was used to mediate LB5000 (fliC/esat) to SL5928 of Salmonella Dublin. PCR, kinetic detection and serum agglutination test were used to identify and obtain SL5928 (fliC/esat) of transduced bacteria. The protein of SL5928 (fliC/esat) flagellin was extracted and transduced, and Western-blotting showed that it was with the sera or ESAT-6 monoclonal antibody of Salmonella typhimurium. SAT-6) can produce a specific target strip after the action. The BMDC of mice was prepared, stimulated with SL5928 (fliC/esat) flagellin at sixth days, BMDC was collected after 6 h, and RT-PCR technique was used to detect the expression of Toll like receptor 5 (TLR-5), and the TLR-5 mRNA was expressed, while the control group (unstimulated) was not expressed. The results showed that the level of IgA antibody in serum and BAF was significantly higher than that of BCG or SL5928 (fliC/esat) flagellin in serum and BAF. The results showed that the level of IgA in serum and BAF was significantly higher than that of BCG or SL5928 (fliC/esat) flagellin alone, indicating that the level of mucosal antibodies could be produced; CD4+, CD8 + T cell surface molecular CD69 expression was enhanced and the cells were activated. Lymphocyte proliferation test, sandwich ELISA and other immunological methods were used to detect lymphocyte immune response. The spleen lymphocytes proliferated obviously after PPD or ESAT-6 (1-20aa) peptide stimulation, and the secretion level of IFN- gamma was significantly different from that of IL-4, which tended to Th-1 based immune response and immune response water. The level of BCG or SL5928 (fliC/esat) flagellin is higher than that of individual immunization; the lung lymphocytes present the same trend, and the content of IFN- gamma and IL-4 secreted is higher than that of the spleen lymphocytes. In conclusion, SL5928 (fliC/esat) flagellin can be used as a potential candidate for tuberculosis vaccine and can be combined with BCG to provide new ideas for the study of tuberculosis vaccine.

【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

【引證文獻】

相關(guān)碩士學(xué)位論文 前1條

1 孟闖;結(jié)核分枝桿菌抗原蛋白的表達、免疫特性分析及在牛結(jié)核病檢測中的初步應(yīng)用[D];揚州大學(xué);2011年

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本文編號：1783167