BCG与嵌合ESAT-6重组鞭毛蛋白的联合黏膜免疫及其特性分析

发布时间：2018-04-21 16:16

本文选题：结核病 + 结核分枝杆菌　；参考：《扬州大学》2010年硕士论文

【摘要】： 结核病(tuberculosis ,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,M.tb)复合物引起的一种慢性传染病,目前全球有1/3人口感染了M.tb,每年大约有300万人死于结核病,且大部分是在发展中国家。接种疫苗被认为是控制结核病的手段之一,当前使用的卡介苗(Bacille Calmette and Guérin, BCG)能够有效预防儿童和新生儿患病,但是对成年人活动性结核病保护效果有限。因此,迫切需要研制一种新型有效的疫苗或对现有疫苗进行改良。 M.tb一些分泌抗原的保护性作用已经得到确认,在这些分泌蛋白中,6 kD早期分泌靶抗原(6-kDa early secreted antigenic target, ESAT-6)的作用相当重要。ESAT-6存在于许多致病性分枝杆菌中,但是在BCG和大部分环境分枝杆菌中缺失。它作为一种T细胞抗原,能够有效激发细胞免疫应答,被建议作为诊断试剂用于区分M.tb感染和先前的BCG接种。因此,在本研究中,以沙门菌鞭毛蛋白为载体嵌合运送ESAT-6蛋白,并将其与BCG联合应用,以黏膜滴鼻方式免疫小鼠,探讨诱导的免疫应答特性。 1. BCG与大肠杆菌表达fliC/esat蛋白联合应用的黏膜免疫应答规律研究从大肠杆菌表达系统中表达并纯化fliC/esat蛋白,Western-blotting分析其与野生型鼠伤寒沙门菌血清或ESAT-6单抗(Ms mAb to ESAT-6)作用后情况,结果都出现了特异性的目的条带。制备小鼠的骨髓树突状细胞(bone marrow dendritic cells,BMDC),培养至第6天时用fliC/esat蛋白刺激,24 h后收集细胞分别标记CD11c-FITC及CD80-Biotin、CD86-Biotin、MHC-Ⅰ-Biotin、MHC-Ⅱ-Biotin,FACS检测各分子的表达情况,发现fliC/esat蛋白能够诱导BMDC的成熟,不同程度的上调共刺激分子CD80、CD86、MHC-Ⅰ、MHC-Ⅱ的表达水平。通过滴鼻免疫途径,将BCG与fliC/esat蛋白联合免疫C57BL/6(H-2b)小鼠,结果显示,血清和支气管肺泡灌洗液(bronchoalveolar lavage fluid,BAF)中IgA抗体水平显著高于单独免疫BCG或fliC/esat蛋白时抗体水平,说明能够诱导产生一定水平的黏膜抗体;运用淋巴细胞增殖试验、ELISPOT、夹心ELISA等免疫学方法检测脾脏淋巴细胞免疫应答情况后显示,细胞经纯化的结核杆菌素(PPD)和ESAT-6(1-20aa)多肽刺激后能够明显增殖,产生IFN-γ的分泌细胞数显著上升并与产生IL-4的分泌细胞在数量上差异显著,诱导以Th-1为主的免疫应答,且免疫应答水平要高于单独免疫BCG或fliC/esat蛋白组。动态实验持续观察BCG与fliC/esat蛋白联合免疫小鼠后2-14周的免疫应答变化情况,发现IgA抗体水平、早期活化分子CD69的表达、淋巴细胞增殖及细胞因子的分泌都能持续到8-10周后才呈现下降趋势。因此,BCG与fliC/esat蛋白联合免疫的策略在小鼠模型上能够诱导更强和更持久的黏膜免疫应答和细胞免疫应答,可为结核病的预防提供新的途径和方法。 2. BCG与沙门菌重组鞭毛蛋白联合黏膜免疫诱导的免疫应答研究用P22噬菌体介导LB5000(fliC/esat)向都柏林沙门菌SL5928转导,利用PCR、动力学检测及血清凝集实验鉴定并获得转导菌SL5928(fliC/esat)。提取转导沙门菌SL5928(fliC/esat)鞭毛蛋白,Western-blotting显示其与野生型鼠伤寒沙门菌血清或ESAT-6单抗(Ms mAb to ESAT-6)作用后都能出现特异性的目的条带。制备小鼠的BMDC,培养至第6天时用SL5928(fliC/esat)鞭毛蛋白刺激,6 h后收集BMDC,利用RT-PCR技术检测Toll样受体5 (TLR-5)的表达,结果能够表达TLR-5 mRNA,而对照组(未刺激)未见表达。将SL5928(fliC/esat)鞭毛蛋白与BCG通过滴鼻免疫途径联合免疫C57BL/6(H-2b)小鼠,检测结果显示,血清和BAF中IgA抗体水平显著高于单独免疫BCG或SL5928(fliC/esat)鞭毛蛋白时水平,说明能够产生一定水平的黏膜抗体;CD4+、CD8+ T细胞表面分子CD69表达提高,T细胞得到活化;运用淋巴细胞增殖试验、夹心ELISA等免疫学方法检测淋巴细胞免疫应答情况后显示,脾脏淋巴细胞经PPD或ESAT-6(1-20aa)多肽刺激后能够明显增殖;IFN-γ的分泌水平与IL-4的分泌水平在含量上差异显著,趋向于Th-1为主的免疫应答,且免疫应答水平要高于单独免疫BCG或SL5928(fliC/esat)鞭毛蛋白;肺脏淋巴细胞呈现同样趋势,且分泌的IFN-γ和IL-4含量要高于脾脏淋巴细胞。综上,SL5928(fliC/esat)鞭毛蛋白可以作为潜在的结核病疫苗候选株,与BCG联合应用,为结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.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【引证文献】