Genes of M. tuberculosis Induced During Human Infection

描述（由申请人提供）：结核病是世界上由传染性病原体导致死亡的主要原因。世界卫生组织（世卫组织）估计，每年有800万新病例，200万至300万人死亡。目前的诊断和治疗方法，特别是对多重耐药菌株的诊断和治疗方法是不够的。1993年，世卫组织采取了前所未有的步骤，宣布结核病为全球紧急情况。结核分枝杆菌是结核病的病因，是一种难以用常规方法研究的微生物。因此，关于它引起疾病的方式还有很多东西要了解。在这个建议中，我们描述了一种新的专有技术，我们称之为体内诱导抗原技术（IVlAT）的应用，以研究M。结核我们在第一阶段SBIR中的目的是确定一组可申请专利的M。结核病疫苗、诊断和抗生素战略的目标。平台IVIAT技术鉴定在人类感染期间特异性表达的病原体的基因。IVIAT已经在许多方面证明了其自身优于其他相关技术的上级，包括其不依赖于动物模型的事实，其容易适用于基本上任何细菌或真菌病原体，并且其可以鉴定瞬时表达的基因。基于这些原因，IVIAT将用于鉴定M.结核菌株CSU 93。这将通过两个具体目标来实现。在第一个具体目标中，来自代表广谱疾病状态的10名结核病患者的合并血清将被体外生长的完整M.结核病细胞和细胞提取物。所得血清将用于菌落提升法以探测M的基因组表达文库。大肠杆菌中的结核病。在具体目标2中，将对反应性克隆中的克隆DNA插入物进行测序和分析，以确定负责体内诱导抗原表达的克隆开放阅读框（ORF）。将相关ORF亚克隆到适当的表达载体中，并使用聚丙烯酰胺凝胶和Western印迹方法获得克隆的最终确认。在第I阶段SBIR中鉴定的体内诱导抗原将在第II阶段进一步表征和分析，以鉴定最适合作为诊断、疫苗和抗生素治疗靶点的抗原。

DESCRIPTION (provided by applicant): Tuberculosis is the world's leading cause of death from an infectious agent. The World Health Organization (WHO) estimates that there are 8 million new cases annually and two to three million deaths. Current methods for diagnosis and treatment, particularly of multiple drug resistant strains, are inadequate. In 1993, WHO took the unprecedented step of declaring tuberculosis a global emergency. Mycobacterium tuberculosis, the cause of tuberculosis, is a difficult microorganism to study using conventional methods. Consequently, there is still much to be learned about the ways it causes disease. In this proposal we describe the application of a novel proprietary technology that we call in vivo induced antigen technology (IVlAT) to the study of M. tuberculosis. It is our intention in this Phase I SBIR to identify a select group of patentable M. tuberculosis targets for vaccine, diagnostic and antibiotic strategies. The platform IVlAT technology identifies genes of a pathogen that are specifically expressed during a human infection. IVlAT has proven itself superior to other related technologies in a number of ways, including the fact that it does not rely on animal models, it is readily applicable to essentially any bacterial or fungal pathogen, and it can identify transiently expressed genes. For these reasons, IVlAT will be employed to identify in vivo induced genes of M. tuberculosis strain CSU93. This will be accomplished in two specific aims. In the first specific aim, pooled sera from 10 tuberculosis patients representing a broad spectrum of disease states will be exhaustively adsorbed with in vitro grown whole M. tuberculosis cells and cell extracts. The resulting serum will be used in a colony lift method to probe a genomic expression library of M. tuberculosis in Escherichia coli. In specific aim 2, the cloned DNA inserts in reactive clones will be sequenced and analyzed to determine the cloned open reading frames (ORFs) responsible for expression of the in vivo induced antigens. The pertinent ORFs will be subcloned into an appropriate expression vector and final confirmation of the clones will be obtained using polyacrylamide gel and Western blot methods. The in vivo induced antigens identified in this Phase I SBIR will be further characterized and analyzed during Phase II to identify ones best suited to serve as targets for diagnostics and vaccines and antibiotic therapies.