潜伏性结核感染体液免疫应答蛋白抗原信息库的构建及新型抗原的诊断价值评估

There are still no effective diagnostic methods for latent tuberculosis infection (LTBI) at present. In our previous studies, using the Mycobacterium tuberculosis functional proteome microarrays, recently developed by Chinese scientists, we constructed the database of chip hybridization with sera from healthy controls, LTBI people and tuberculosis (TB) patients. We found that the samples were clustered into three groups by clustering analysis, and some protein hybridization signals had significant rise in group LTBI. Therefore, we hypothesize that some M. tuberculosis antigens can induce the production of specific antibodies in LTBI people. If we find and validate these antigens, new LTBI diagnostic biomarkers will be discovered. In this study, firstly we will screen proteins with significantly increased signals in LTBI group compared with healthy people based on our hybridization database. Then we build the comprehensive LTBI-specific protein antigens information database based on humoral immunity through analyzing their comparative genomics, cellular localizations, functions, pathways, antigenic epitopes, and marking signal differences between LTBI and TB groups. Secondly, these proteins will be expressed in yeast expression system, purified and spotted on microarrays. After microarrays hybridization with the three groups of sera (LTBI, TB and healthy controls) and statistical analysis, we will get candidate LTBI diagnostic biomarkers. Finally, enzyme linked immunosorbent assay (ELISA) with large sera samples will be used to further evaluate the definite diagnostic values of new antigens following their abundant prokaryotic expression and purification. This study will make creative exploration in LTBI diagnosis, contributing to its large-scale screening and early prophylactic treatment. Meanwhile it will also provide new clues to understand humoral immunity mechanism of latent tuberculosis infection and active tuberculosis.

目前仍缺乏有效的潜伏性结核感染（LTBI）诊断方法。我们前期应用新型结核分枝杆菌全蛋白质组芯片与健康人、LTBI和结核病人血清杂交，发现三组样本可分别聚簇，一些蛋白杂交信号在LTBI组显著升高。据此假设结核分枝杆菌某些抗原能诱导LTBI人群产生特异性抗体，找到和验证这些抗原即可发现新的诊断标志物。本课题拟首先基于杂交数据库系统筛选出杂交信号在LTBI组比健康组显著增高的蛋白，进行比较基因组学、细胞定位、功能、通路及抗原表位分析，标记LTBI/结核病组杂交信号差异，全面构建LTBI特异性体液免疫应答蛋白抗原信息库；然后表达纯化蛋白、制备小型蛋白芯片，与三组血清杂交分析后获取候选诊断抗原；最后通过抗原大量表达和ELISA大样本验证，评估新型抗原对LTBI的诊断价值。本研究结果将在LTBI诊断方面做出创新性探索，为其大规模筛查及预防性治疗奠定基础，并为结核潜伏和致病的体液免疫机理研究提供新思路。

数目庞大的潜伏性结核感染（LTBI）人群是新发结核病病例的重要来源，但目前仍缺乏有效的LTBI诊断方法。项目组前期应用新型结核分枝杆菌全蛋白质组芯片与健康人、LTBI和结核病人血清杂交，发现三组样本可分别聚簇，一些蛋白质杂交信号在LTBI组显著升高。据此假设结核分枝杆菌某些抗原能诱导LTBI人群产生特异性抗体，找到和验证这些抗原即可发现新的诊断标志物。.本研究首先基于芯片血清杂交数据库筛选出杂交信号在LTBI组比健康组显著增高的结核分枝杆菌蛋白质142个，通过比较基因组学、细胞定位、功能、通路及抗原表位分析，以及LTBI组/结核病组杂交信号差异标记，全面构建了LTBI特异性体液免疫应答蛋白抗原信息库。然后调取蛋白质对应的酵母表达菌株，诱导表达和纯化蛋白质，点制小型蛋白芯片；将芯片与346份三组血清样本杂交后进行信号差异分析，共获得21个LTBI候选诊断抗原。最后对其中6个候选抗原进行了原核大量表达和纯化，继而包被平板对616份三组血清样本实施盲法ELISA 特异性IgG和IgM检测，结果表明，有3个蛋白质的IgM检测值在LTBI组显著高于健康组和结核病组，提示它们在结核分枝杆菌潜伏感染过程中可能发挥重要作用；进一步将其中差异明显的2个蛋白质的IgM检测值进行ROC曲线分析，结果发现，这2个蛋白质血清IgM单独检测和联合检测的AUC值均在0.6-0.7之间，提示两者用于LTBI和健康人鉴别诊断时区分能力处于较低水平。.综上，本研究在潜伏性结核感染方面做出一些创新性探索，发现了一批在LTBI人群血清中抗体水平显著升高的蛋白分子，并阐明了6个蛋白质血清学抗体检测对LTBI的诊断价值。以上研究结果为结核分枝杆菌潜伏和致病的免疫机理研究提供了新的思路。