基于精准蛋白质基因组学的结核分枝杆菌复合群特异性新基因的鉴定及功能研究

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB), has been the most deadly infecting disease worldwide. Although a lot of achievements have been made, the TB clinical diagnosis and treatment is still a challenge because of MTB cross infection with other mycobacteria and respiratory bacteria, slow grow and difficult culture. Total 22 novel genes were identified and verified from MTB by precision proteogenomics, which achieved the reannotation of MTB genome. More importantly, 11 genes were Mycobacterium tuberculosis complex (MTBC) specific genes among these novel genes by comparative genomics, which provides technical support for MTB rapid and accurate diagnosis. To further understand the potential function of the unannotated proteins, we proposed the label-free based deep coverage quantitative proteomics and high resolution interaction proteomics for analyzing their interacted proteins. To identify the potential application of the unannotated proteins, we planned to investigate their immunogenicity by ELISA and protein chip technologies. High immunogenic proteins will provide original innovative markers in new MTB-specific antigen development. Further function analysis for one of the most important proteins will help clarify the internal connection of “unannotated gene-MTBC specific-virulence-immunogenicity”.

结核分枝杆菌（MTB）所致的结核病（Tuberculosis, TB）是致死人数最多的感染性疾病。尽管研究众多，但由于MTB与其它分枝杆菌、呼吸道感染病菌的交叉感染难于区分，加上MTB菌生长慢，培养难，使得TB的诊治仍是难题。我们利用深度覆盖和精准蛋白质基因组学技术从MTB鉴定并验证了22个新编码基因，实现了MTB菌的基因组重注释；比较基因组学发现其中11个新基因是结核分枝杆菌复合群特异基因，为结核病临床快速诊治提供了技术支撑。本项目旨在利用深度覆盖精准定量蛋白质组技术和高分辨精准相互作用蛋白质组技术鉴定漏注释蛋白的相互作用蛋白；通过ELISA和蛋白芯片技术筛选漏注释蛋白的免疫原性，为结核新型特异性抗原的开发提供原始创新性成果；基于结核分枝杆菌复合群特异新基因、毒力蛋白及强免疫原性蛋白，开展1个重要漏注释蛋白的功能研究，揭示“漏注释-结核分枝杆菌复合群特异性-毒力-免疫原性”的内在关系。

结核分枝杆菌（Mycobacterium tuberculosis, MTB）所致的结核病（Tuberculosis, TB）是致死人数最多的感染性疾病。尽管研究众多，但由于MTB与其它分枝杆菌、呼吸道感染病菌的交叉感染难于区分，加上MTB菌生长慢，培养难，使得TB的诊治仍是难题。了解MTB生物学、新的优势诊断标志物和复杂的MTB-宿主相互作用的研究进展需要准确和完整的结核分枝菌编码基因的基因组注释。我们利用深度覆盖和精准蛋白质基因组学技术对MTB进行重新注释，共鉴定了2794个注释基因产物（≈70%），确定了49个新ORF和102个N端校正的全基因编码区，并对MTB的TuberuList、NCBI和UniProt数据库注释进行了矫正。与两个TB的经典标志物相比(Rv3874, CFP-10; Rv3875, ESAT-6)，比较蛋白基因组学筛选到33种物种特异性的全新编码基因，包括14个高特异性ORF和19个较高特异性ORFs。TB临床标本评估表明，物种特异性新编码基因可用于结核分枝杆菌菌株鉴别，3个编码新基因可用于组织免疫组织化学（IHC）染色，2个新基因可用于质谱（MS）检测结核感染者的血浆外泌体。因此，物种特异性新ORF可以显著提高结核分枝杆菌的鉴别和结核感染病例的检测，甚至更早地检测结核分枝杆菌感染，提供结核分枝杆菌生物学见解，并促进新诊断技术的发展。