【作者】 吴悦涵；

【导师】 鲍朗；

【作者基本信息】 四川大学 ， 病理学与病理生理学， 2004， 硕士

【摘要】 结核病是世界上感染性疾病导致的死亡率最高的疾病之一,其致病及免疫保护机制尚未完全阐明。耐药结核杆菌的出现使结核病的防治变得复杂化,因此探索结核杆菌的致病机制和新的防治策略十分重要。结核分枝杆菌H37Rv基因组测序的完成和更新,为结核杆菌基因组学和蛋白组学的研究提供了重要而更加精确的资料。 初步研究表明,一组尚未确定生物学特征和功能的结核杆菌假想蛋白基因,可能与其毒力致病机制有关。这些蛋白基因皆位于细胞壁和细胞活动基因及其调节蛋白编码基因的下游,受同一操纵子控制,其编码产物是构成结核杆菌细胞壁的重要构件,尤以Rv0901基因最为引人注目。该基因在1998年基因组测序完成时划分为编码未知蛋白类及保守的假想蛋白类基因,于2002年基因组更新时重新划入了细胞壁和细胞突(cell-wall and cell processes category)一类。研究发现,结核杆菌细胞壁的特殊结构和成分在结核杆菌与宿主细胞(主要是巨噬细胞)的相互作用及其致病中发挥着重要的作用。为研究Rv0901基因的功能及其与结核杆菌毒力、致病机制的关系,我们首先将该基因528bp片段首次从基因组中扩增出,并在原核融合表达载体pGEX-1λT中成功表达了GST—更多还原

【Abstract】 Tuberculosis remains a leading cause of infectious death worldwide, yet the pathogenesis and the immunoprotecive mechanism of the causative pathogen have not been completely clarified. The emergence of multiple-drug-resistant Mycobacterium tuberculosis isolates complicates the treatment and control, underscoring the need to explore its pathogenesis and develop new strategies for prevention and treatment of tuberculosis. The complete genome sequence and annotation of Mycobacterium tuberculosis H37Rv was published in 1998,then the annotation was updated to be more accurate in 2002.This has provided researchers important information to gain insight into the genomics and proteomics of M. tuberculosis.Preliminary studies suggested that a group of hypothetical proteins, the biological feature and function of which has not been identified, was possibly associated with the virulence of M. tuberculosis. The genes encoding these proteins have been found to be located downstream to those belonging to the cell-wall and cell processes category and those encoding their regulons. They are supposed to beimportant components controlled by the same operons and associated with the structure of the cell-wall of M. tuberculosis, especially the Rv0901. This protein-encoding gene Rv0901 was annotated as unknown and conserved hypothetical in 1998 but has been transferred to the cell-wall and cell processes category in 2002. Central to understanding the pathogenesis of tuberculosis is the interaction between the pathogen and phagocytes, and various mycobacterial cell wall components may be involved in these processes. With this in mind, we amplified the gene Rv0901 from the genome of H37Rv for the first time to make an attempt to investigate the function of it. A recombinant fused expression vector pGEX-Rv0901 was constructed. The recombinant plasmid could express the fusion protein GST-Rv0901 stably, thus provided the basis for the further study of the gene Rv0901. After careful analysis with Bioinformatics and confirmation in experiments, Rv0901 was proved to be a unique sequence in the M.tuberculosis Complex. A recombinant plasmid bearing Rv0901 was constructed using the shuttle vector pMV261. Then it was electroporated into avirulent Mycobacterium smegmatis mc~2155 which is lack of the sequence. The transformants were induced to express a predicted protein of Rv0901 and used to infect mouse bone marrow macrophage line Ana-1. The viability of the cells was evaluated as well as the intracellular survival assay of the mycobacteria was performed at different times postinfection. As expected, neither M. smegmatis nor the recombinants infection of macrophages led to a cytopathic effect at any period of time, but thetransformant containing Rv0901 showed a survival advantage and a lower speed to be eliminated by macrophages than M.smegmatis. Furthermore, we compared Ana-1 cells apoptosis following infection by these two strains and analyzed by flow cytometry for Annexin V staining. By 5 days postinfection, both kinds of the Ana-1 cells underwent obvious apoptosis compared with normal cells as control, and M.smegmatis induced significantly more Ana-1 apoptosis than the recombinant ones bearing the gene Rv0901.These findings suggest a possible role for Rv0901 in the intracellular survival of M. tuberculosis and in the maintenance of a supportive environment for bacterial growth by modulating the apoptotic response of macrophages.更多还原