Genes of M. tuberculosis Induced During Human Infection

人类感染期间诱导的结核分枝杆菌基因

• 批准号: 6736413
• 负责人: Jeffrey D. Hillman
• 金额: $ 10万
• 依托单位: ORAGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6736413
• 关键词: Escherichia coli; Mycobacterium tuberculosis; SDS polyacrylamide gel electrophoresis; bacteria infection mechanism; bacterial antigens; bacterial genetics; clinical research; computer assisted sequence analysis; functional /structural genomics; gene expression; gene induction /repression; genetic library; genetic screening; genome; host organism interaction; human tissue; molecular cloning; nucleic acid sequence; open reading frames; pathologic process; plasmids; tissue /cell culture; transfection /expression vector; western blottings

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.

描述（由申请人提供）：结核病是世界上传染源导致死亡的主要原因。世界卫生组织 (WHO) 估计每年新增病例 800 万例，死亡人数为 2 至 300 万人。目前的诊断和治疗方法，特别是多重耐药菌株的诊断和治疗方法是不够的。 1993年，世界卫生组织采取了史无前例的步骤，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，是一种很难用传统方法研究的微生物。因此，关于它引起疾病的方式还有很多有待了解。在本提案中，我们描述了一种新颖的专有技术（我们称之为体内诱导抗原技术（IVlAT））在结核分枝杆菌研究中的应用。我们的目的是在第一阶段 SBIR 中为疫苗、诊断和抗生素策略确定一组可申请专利的结核分枝杆菌靶标。 IVlAT 平台技术可识别在人类感染期间特异性表达的病原体基因。 IVlAT 已在很多方面证明了自己优于其他相关技术，包括它不依赖动物模型、它很容易适用于基本上任何细菌或真菌病原体，并且它可以识别瞬时表达的基因。由于这些原因，IVlAT将用于鉴定结核分枝杆菌菌株CSU93的体内诱导基因。这将通过两个具体目标来实现。在第一个具体目标中，来自代表广泛疾病状态的 10 名结核病患者的混合血清将被体外生长的完整结核分枝杆菌细胞和细胞提取物彻底吸附。所得血清将用于菌落提升方法，以探测大肠杆菌中结核分枝杆菌的基因组表达文库。在具体目标 2 中，将对反应性克隆中的克隆 DNA 插入片段进行测序和分析，以确定负责体内诱导抗原表达的克隆开放阅读框 (ORF)。相关的 ORF 将被亚克隆到适当的表达载体中，并使用聚丙烯酰胺凝胶和蛋白质印迹方法对克隆进行最终确认。在第一阶段 SBIR 中鉴定的体内诱导抗原将在第二阶段进一步表征和分析，以确定最适合作为诊断、疫苗和抗生素治疗靶点的抗原。