结核分枝杆菌蛋白质组芯片筛选血清标志物的研究

发布时间：2018-01-24 02:31

本文关键词： 蛋白质组芯片血清标志物结核潜伏感染活动性结核病卡介苗接种　出处：《北京市结核病胸部肿瘤研究所》2017年博士论文　论文类型：学位论文

【摘要】：据统计,全球约三分之一的人感染结核分枝杆菌(Mycobacterium tuberculosis,Mtb),大部分人处于潜伏感染状态(Latent tuberculosis infection,LTBI),即宿主和病原体之间保持相对平衡,但当这种平衡一旦被打破,大约5%至10%的人会最终发展成活动性结核病。因此,及早发现潜伏感染者,并给予适当的预防和治疗,可以降低结核病的发病风险,特别是对高危人群的预防和治疗更加有意义。目前诊断潜伏感染的方法主要是结核菌素皮肤实验(Mantoux Tuberculin Skin Test,TST)和γ-干扰素释放实验(Interferon-gamma release arrays,IGRAs),而这些方法都存在一定的局限性。在本文第一部分中,我们利用结核分枝杆菌蛋白质组芯片对潜伏感染者和活动性结核病人的血清样本进行检测,对芯片结果进行生物信息学分析后,发现众多抗原的特异性IgG或IgM抗体的响应程度在两组人群间具有明显差异。后续我们扩大样本量,选择差异较大的IgG抗体组的12个抗原,应用酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)方法进行验证,受试者操作曲线(Receiver operating characteristic curve,ROC曲线)分析显示单个抗原Rv2031c和Rv1408在潜伏感染者和活动性结核病患者中具有较好的诊断效果。此外,我们通过二元逻辑回归分析,建立了一个回归模型,该模型的诊断效果高于任何一个单一抗原以及Rv2031c、Rv1408和Rv2421c三个抗原的融合效果,其敏感性和特异性均较高,分别为88.04%和96.77%,在利用血清学方法鉴别潜伏感染和活动性结核病方面具有潜在的应用价值。卡介苗(Bacille Calmette Gurrin,BCG)是世界卫生组织(World health organization,WHO)公布的唯一预防结核病的疫苗,据统计目前累积已有超过30亿人口接种过卡介苗。尽管卡介苗的应用如此广泛,但是其接种后的免疫保护效果的评价一直都没有一种明确的方法,传统使用的是观察BCG接种瘢痕和结核菌素皮肤实验(TST),这两种方法都有各自的局限性。同时,对于卡介苗接种后刺激机体产生保护性免疫的具体作用机制仍然有很多未知之处,因此也限制了对疫苗保护效果评价方法的研究。在本研究第二部分中,我们利用结核分枝杆菌蛋白质组芯片,对健康人群和卡介苗成功接种人群的血清样本进行检测,结果显示有142个抗原对IgG抗体有较明显的差异响应,91个抗原对IgM抗体有较明显的差异响应,提示这些抗原可引起较强的体液免疫应答,可为深入理解卡介苗的保护机制提供一定的基础数据。后续,我们选择6个抗原,通过扩大样本量进行ELISA验证,结果显示Rv2728c、Rv2150c和Rv2716抗原在卡介苗接种组中对应的IgG抗体水平明显高于健康人对照组,ROC分析证实这些抗原均有较好评价效果。二元逻辑回归分析建立的回归模型显示,相比于单个抗原具有更高的敏感性和特异性,分别为95.16%和98.39%,为未来建立一种用来检测卡介苗接种效果的评价方法奠定基础。
[Abstract]:According to statistics, about 1/3 people in the world are infected with Mycobacterium tuberculosisus (Mtb). Most people are in latent tuberculosis infection, that is, the relative balance between the host and the pathogen. But when this balance is broken, about 5% to 10% people will eventually develop active TB. Therefore, early detection of latent infections and appropriate prevention and treatment. Can reduce the risk of tuberculosis. Especially for the prevention and treatment of high-risk groups. The current diagnosis of latent infection is mainly tuberculin skin test (. Mantoux Tuberculin Skin Test. TST and Interferon-gamma release arraysme IGR As. In the first part of this paper, we use the proteome chip of Mycobacterium tuberculosis to detect the serum samples of latent and active TB patients. After bioinformatics analysis of the microarray results, it was found that the response of specific IgG or IgM antibodies to many antigens was significantly different between the two groups. Twelve antigens from different IgG antibody groups were selected, and Enzyme-linked immunosorbent assay was used by enzyme-linked immunosorbent assay (Elisa). Erisa method was used to verify the receiver operating characteristic curve. ROC curve) analysis showed that single antigen Rv2031c and Rv1408 had good diagnostic effect in latent infection and active tuberculosis patients. In addition, we used binary logistic regression analysis. A regression model was established and the diagnostic effect of the model was higher than that of any single antigen and the fusion of Rv2031cncnrv1408 and Rv2421c antigens. Its sensitivity and specificity were 88.04% and 96.77%, respectively. It has potential application value in identifying latent infection and active tuberculosis by serological method. Bacille Calmette Gurrin. BCG is the only vaccine against TB published by the World Health Organization (WHO). According to statistics, more than 3 billion people have been vaccinated with BCG vaccine. Although BCG vaccine is widely used, there has been no clear method to evaluate the immune protection effect of BCG vaccine. The traditional method is to observe the scar and tuberculin skin test of BCG, both of which have their own limitations. For the specific mechanism of BCG vaccination to stimulate the body to produce protective immunity, there is still a lot of unknown, so it also limits the study of vaccine protective effect evaluation methods. In the second part of this study. We detected the serum samples of healthy people and successful BCG vaccinated population by using Mycobacterium tuberculosis proteome microarray. The results showed that 142 antigens were significantly different in response to IgG antibodies. The 91 antigens were significantly different in response to IgM antibody, suggesting that these antigens could induce a strong humoral immune response and provide some basic data for further understanding the protective mechanism of BCG vaccine. We selected 6 antigens and verified by ELISA by expanding the sample size. The results showed that Rv2728c. The corresponding IgG antibody levels of Rv2150c and Rv2716 antigens in BCG inoculation group were significantly higher than those in healthy control group. ROC analysis confirmed that these antigens had good evaluation effect. The regression model established by binary logistic regression analysis showed that these antigens were more sensitive and specific than single antigens. It was 95.16% and 98.39, respectively, which laid a foundation for the evaluation of BCG inoculation effect in the future.
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R378.911

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