新型结核病疫苗免疫组分和诊断分子标识候选物的初步筛选及鉴定

发布时间：2018-08-18 15:07

【摘要】：结核病(tuberculosis,TB)一直是一个严重的全球公共健康问题,其死亡率居于单一细菌感染致死亡性疾病之首。传统的结核病检测方法有痰涂片,痰培养,PCR,ELISPOT, TST,胸片等,但是它们存在明显的不足,比如有的耗时较长,有的假阳性率高,而且不能对结核分枝杆菌菌阴的病人进行正确诊断。因此需要寻找一种准确、快速、新的诊断方法,为后期临床治疗提供依据。现今细胞免疫检测方法使用的抗原有ESAT-6, CFP10和TB7.7,血清学检测使用的抗原是38kDa(Rv0934)。目前临床上使用的唯一预防性结核病疫苗-卡介苗对儿童结核性脑膜炎具有保护作用,但是对青少年和成人的肺结核保护力为0-80%,具有可变性。因此迫切需要研制新型有效的结核病疫苗。本研究通过筛选结核分枝杆菌的分泌蛋白和膜蛋白,寻找潜在的新型结核病疫苗免疫组分和诊断分子标识候选抗原。首先使用生物信息学软件对结核分枝杆菌H37Rv菌株全蛋白质组进行预测分析确定研究对象。然后将入组的膜蛋白和分泌蛋白进行重组表达。对表达获得的重组蛋白逐一进行Western Blot和ELISPOT筛选,根据第二轮细胞免疫筛选结果选取5个蛋白进行动物实验,验证5个蛋白在C57BL/6小鼠体内的免疫原性。最终结果如下：经生物信息学软件对结核分枝杆菌H37Rv菌株的3989个开放阅读框编码的蛋白预测确定有240个开放阅读框编码的膜蛋白和242个开放阅读框编码的分泌蛋白入组。本研究利用Gateway系统和一步克隆法成功构建了467个表达克隆,通过两轮表达得到219个膜蛋白、141个分泌蛋白。将所有纯化得到的蛋白使用离子轨道肼质谱验证重组蛋白的蛋白序列是否正确,经验证360个蛋白序列全部正确,正确率100%。经过使用229个活动性肺结核病病人血清进行三轮蛋白质免疫印迹实验筛选得到18个膜蛋白、26个分泌蛋白反应强度与阳性对照Rv0934的峰面积比值大于1。每个蛋白使用3个活动性肺结核病人PBMC进行ELISPOT,初次筛选使用73个活动性肺结核病人PBMC。经过初步筛选得到8个膜蛋白、9个分泌蛋白平均反应强度达到阳性对照ESAT-6的50%以上。将Western Blot初步筛选得到的17个蛋白使用健康人和病人血浆进行复筛,得到5个蛋白反应在健康人和病人血浆中反应具有差异。将ELISPOT初步筛选结果得到的17个蛋白使用15个ESAT-6阳性病人 PBMC进行复筛,得到5个蛋白反应强度达到阳性对照ESAT-6的40%以上,可以作为目标候选抗原；6个ESAT-6阴性病人PBMC进行复筛,3个蛋白可能与ESAT-6互为补充,与ESAT-6组合使用可能会提高活动性肺结核检测的吻合率。蛋白免疫小鼠实验结果显示S43蛋白能刺激小鼠CD4+、CD8+T淋巴细胞产生IFN-γ, CD8+T淋巴细胞产生IL-2；S4蛋白能刺激小鼠CD8+T淋巴细胞产生IFN-yo M62, M15, M1蛋白能刺激小鼠CD8+T淋巴细胞产生IL-2。M15, M1, S43, S4, S71作为抗原刺激脾细胞时,可以观察到CD4+T, CD8+T淋巴细胞的增殖现象,但是与PBS对照组相比没有统计学意义。当M15, M1, S43, S4, S71作为抗原刺激脾细胞时,可以检测到不同细胞因子不同程度的分泌。本研究已经获得了10个候选蛋白的体液免疫和细胞免疫、及免疫原性的初步结果。这10个蛋白的应用价值需要进一步研究验证。综上所述,我们首次对结核分枝杆菌H37Rv的膜蛋白和分泌蛋白进行系统研究,得到了一系列可以用作疫苗候选物或是诊断标识的蛋白。由于结核分枝杆菌的复杂性,大规模的蛋白质组学研究无论是发现新的结核病疫苗免疫组分还是寻找新的诊断标识都具有深远影响,改善现有的结核病的预防和诊断现状。因此,这种蛋白质组学研究具有良好的发展前景,或许还可以推广到寻找生物体的新抗原的研究中。
[Abstract]:Tuberculosis (TB) has always been a serious global public health problem with the highest mortality rate of a single bacterial infection. Traditional TB detection methods include sputum smear, sputum culture, PCR, ELISPOT, TST, chest X-ray, etc. But they have obvious shortcomings, such as time-consuming and high false positive rate. Therefore, it is necessary to find an accurate, rapid and new diagnostic method to provide the basis for the later clinical treatment. The antigens used in cell immunoassay are ESAT-6, CFP10 and TB7.7, and the antigens used in serological detection are 38kDa (Rv0934). Bacillus Calmette-Guerin (BCG), the only preventive tuberculosis vaccine, has a protective effect on tuberculous meningitis in children, but the protective power against tuberculosis in adolescents and adults is 0-80%. Therefore, a new and effective tuberculosis vaccine is urgently needed. Immunocomponent and diagnostic molecule marker candidate antigens of type B tuberculosis vaccine. First, the whole proteome of Mycobacterium tuberculosis H37Rv strain was predicted and analyzed by bioinformatics software. Then the membrane proteins and secreted proteins were recombined and expressed. ELISPOT screening, according to the results of the second round of cellular immune screening, five proteins were selected for animal experiments to verify the immunogenicity of five proteins in C57BL/6 mice. In this study, 467 expression clones were successfully constructed using Gateway system and one-step cloning method. 219 membrane proteins and 141 secretory proteins were obtained through two rounds of expression. All purified proteins were identified by ion-orbital hydrazine mass spectrometry. The results showed that the 360 protein sequences were correct, and the correct rate was 100%. 18 membrane proteins were screened by three rounds of immunoblotting test using 229 active tuberculosis patients'serum. The peak area ratio of 26 secretory proteins to positive control Rv0934 was greater than 1. Each protein used 3 active tuberculosis. ELISPOT was used to screen 73 active pulmonary tuberculosis patients for the first time. After preliminary screening, 8 membrane proteins were obtained. The average reaction intensity of 9 secretory proteins reached more than 50% of the positive control ESAT-6. Western Blot screened 17 proteins using healthy and patient plasma for screening, and 5 protein reactions were obtained. The results of ELISPOT preliminary screening showed that 17 proteins were screened by 15 ESAT-6 positive patients with PBMC, and 5 proteins with a reaction intensity of more than 40% of the positive control ESAT-6 could be used as the target candidate antigen; 6 ESAT-6 negative patients with PBMC were screened, and 3 proteins were possible. The results showed that S43 protein could stimulate CD4 +, CD8 + T lymphocytes to produce IFN - gamma, CD8 + T lymphocytes to produce IL - 2, and S4 protein could stimulate CD8 + T lymphocytes to produce IFN - yo M62, M15, M1 protein to spine. The proliferation of CD4+T and CD8+T lymphocytes was observed when stimulating spleen cells with IL-2.M15, M1, S43, S4 and S71 as antigens, but there was no significant difference compared with PBS control group. Secretion. Preliminary results of humoral and cellular immunity and immunogenicity of 10 candidate proteins have been obtained. The application value of these 10 proteins needs further study and verification. In summary, we have systematically studied the membrane proteins and secretory proteins of Mycobacterium tuberculosis H37Rv for the first time, and obtained a series of vaccines that can be used as vaccines. Because of the complexity of Mycobacterium tuberculosis, large-scale proteomics studies have far-reaching implications for both discovering new immune components of tuberculosis vaccines and finding new diagnostic markers to improve the current status of tuberculosis prevention and diagnosis. Good prospects for development may also be extended to search for new antigens in organisms.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

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