VIRULENCE DETERMINANTS OF MYCOBACTERIUM TUBERCULOSIS

结核病，人类的一大祸害，正在重新成为一种 美国的重大健康问题，最常困扰美国人 老年人、无家可归者、艾滋病患者和来自各国的移民 结核病发病率高的地方。虽然结核病的病原体， 结核分枝杆菌，是一个世纪前发现的，关于 基本的生理能力，遗传学，特定的毒力 这些细菌致病的决定因素和致病机制(S)几乎是 根本不存在。 因此，我们建议鉴定和鉴定M。 在这种生物的致病性中起重要作用的结核病 通过对基因组DNA和重组人的分子和功能分析 基因组文库中的分子。具体地说，我们将(I)确定 结核分枝杆菌H37Rv独有的染色体DNA区域 H37Rv与H37Ra基因组DNA的消减杂交 H37Rv与卡介苗之间，(Ii)互补DNA(CDNA)文库的构建 从人巨噬细胞纯化的结核分枝杆菌H37Rv 感染后不同时间和肉汤中生长的H37Rv杆菌 培养，(Iii)确定在不同阶段表达的基因 巨噬细胞DNA之间的消减杂交感染- 培养和培养的结核分枝杆菌H37Rv，(Iv)将基因亚克隆到 合适的载体并将其导入H37Ra或卡介苗以确定 克隆的基因是否会提高小鼠的存活和/或生长 巨噬细胞中的无毒衍生物，以及(V)表征独特的 通过核苷酸序列测定和计算机进行区域和基因 分析。

Tuberculosis, one of the great scourges of mankind, is reemerging as a significant health problem in the U.S., most frequently afflicting the elderly, the homeless, individuals with AIDS, and immigrants from nations where the incidence of TB is high. Although the causative agent of TB, Mycobacterium tuberculosis, was identified a century ago, knowledge about the fundamental physiological capabilities, genetics, specific virulence determinants, and mechanism(s) of pathogenicity of these bacilli is almost nonexistent. We therefore propose to identify and characterize genes from M. tuberculosis that are important in the pathogenicity of this organism through molecular and functional analyses of genomic DNA and recombinant molecules from genomic libraries. Specifically, we will (i) identify regions of chromosomal DNA that re unique to M. tuberculosis H37Rv by subtractive hybridizations between genomic DNAs from H37Rv and H37Ra and between H37Rv and BCG, (ii) prepare complementary DNA (cDNA) libraries from M. tuberculosis H37Rv bacilli purified from human macrophages at different times after Infection and from H37Rv bacilli grown in broth culture, (iii) identify genes that are expressed at different stages of infection by subtractive hybridizations between cDNAs from macrophage- grown and broth-grown M. tuberculosis H37Rv, (iv) subclone genes into appropriate vectors and introduce them into H37Ra or BCG to determine whether or not the cloned genes will enhance survival and/or growth of the avirulent derivatives in macrophages, and (v) characterize the unique regions and genes by nucleotide sequence determination and computer analysis.