结核分枝杆菌Rv0901基因功能研究

发布时间：2018-01-17 00:05

本文关键词：结核分枝杆菌Rv0901基因功能研究　出处：《四川大学》2007年博士论文　论文类型：学位论文

【摘要】： 结核病是由结核分枝杆菌引起的一种世界性传染病。世界卫生组织2004年的世界卫生报告指出结核病已经成为可治疗的感染性疾病当中的头号致死性疾病。WHO感染性疾病的死亡登记和监管记录表明：2004年全球新增结核病人890万，其中390万例痰涂片阳性，而2004年全球结核病死亡人数则达到了170万。随着人口流动增加、HIV与结核分枝杆菌伴发感染以及结核分枝杆菌多重耐药性菌株的出现等原因，结核分枝杆菌的传播呈逐年稳步增长趋势。近年来研究发现，，结核病与多种慢性疾病有密切相关，如糖尿病，营养不良和由吸烟及大气污染导致的呼吸系统疾病等等。因此，探索结核分枝杆菌的致病机制和新的防治策略显得十分重要。而结核分枝杆菌H37Rv基因组测序的完成和更新，为结核分枝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分枝杆菌假想蛋白基因，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分枝杆菌细胞壁的重要构件，其中尤以Rv0901基因最为引人注目。该基因在1998年基因组测序完成时被划分为编码未知蛋白类及保守的假想蛋白类基因，于2002年基因组更新时被重新划入了细胞壁和细胞突(cell一wall andcell processes category)类基因。结核分枝杆菌细胞壁的特殊结构和成分在其与宿主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分枝杆菌毒力、致病机制的关系，我们首先将该基因528bp片段从基因组中扩增出，在原核融合表达载体pET32a(+)中成功表达了pET—Rv0901融合蛋白并制备了蛋白的多克隆抗体，为研究该基因功能奠定了基础。继而将Rv0901基因片段构建进入真核表达载体PcDNA3．1，在真核细胞中得以成功表达，再将该基因片段转入小鼠腹腔巨噬细胞，研究该基因对小鼠腹腔巨噬细胞活性的影响。进一步通过穿梭表达载体pMV261经电转化将Rv0901基因片段重组入不含该基因序列的无毒耻垢分枝杆菌(mc~2 155)，并在重组耻垢分枝杆菌(Rmc~2 155)中成功表达了19kDa的Rv0901编码蛋白。将经诱导表达的重组耻垢分枝杆菌和未重组的耻垢分枝杆菌同时以相同复数感染人单核巨噬细胞THP-1和BALB／c小鼠，比较重组株和未重组株的细胞学和动物学作用。结果发现，Rv0901基因片段转入小鼠腹腔巨噬细胞后，小鼠腹腔巨噬细胞的凋亡增加，其培养液上清中的NO(一氧化氮)和IFN—γ(γ干扰素)释放量增加。Rmc~2 155诱导THP-1细胞的凋亡率高于mc~2 155，其感染THP—1细胞后细胞的存活率低于mc~2 155的感染，且细胞培养液中NO的产生高于mc~2 155的感染。Rmc~2 155感染BALB／c小鼠后小鼠脾淋巴细胞增殖水平高于mc~2 155的感染，小鼠血清中IFN—γ和NO的释放量均增加，但是较mc~2 155没有显著差异。以上结果提示Rv0901可能对于结核分枝杆菌在宿主细胞的存活以及调控宿主细胞凋亡以维持其生存环境的稳定性中有一定作用。
[Abstract]:Tuberculosis is a worldwide infectious disease caused by Mycobacterium tuberculosis. The WHO in 2004 World Health report pointed out that TB has become infectious diseases can be treated in the most deadly disease.WHO infectious disease death registration and supervision records show that in 2004 the global new 8 million 900 thousand TB patients, including 3 million 900 thousand cases of sputum smear positive, and in 2004 the global TB deaths reached 1 million 700 thousand. With the increase of population mobility, HIV and Mycobacterium tuberculosis infection with Mycobacterium tuberculosis and multidrug-resistant strains have other reasons, the transmission of Mycobacterium tuberculosis showed an increasing trend. In recent years, the study found that there are closely related, tuberculosis and many chronic diseases such as diabetes, nutrition bad and caused by smoking and air pollution in the respiratory system diseases and so on. Therefore, exploring Mycobacterium tuberculosis Pathogenic mechanisms and new strategies for prevention and treatment are very important. The completion and update of H37Rv sequencing of Mycobacterium tuberculosis provide important and accurate data for the study of Mycobacterium tuberculosis genomics and proteomics.
The preliminary study shows that a group of Mycobacterium tuberculosis has not yet determined the biological characteristics and function of the hypothetical protein gene, which may be related to its virulence mechanism. These genes are located in the cell wall and cell activity gene and regulation protein encoding gene downstream by the same operating control, its encoding product is an important component of Mycobacterium tuberculosis the cell wall, especially in the Rv0901 gene is most conspicuous. The gene in 1998 to complete genome sequencing is divided into encoding unknown protein and conserved hypothetical protein gene, gene group in 2002 when the update was re assigned to the cell wall and cell process (cell wall andcell processes category) gene with special structure. And the components of the mycobacterial cell wall plays an important role in its interaction with the host cell and its pathogenicity. In order to study the Rv0901 gene The function and virulence of Mycobacterium tuberculosis and the pathogenic mechanism of the relationship, we will first, the 528bp gene fragment was amplified from the genome, in the prokaryotic fusion expression vector pET32a (+) successfully expressed pET Rv0901 fusion protein and polyclonal antibody protein was prepared, which laid the foundation for the research of the gene function and then Rv0901 gene were constructed into eukaryotic expression vector PcDNA3.1 was successfully expressed in eukaryotic cells, then the gene fragment into mouse peritoneal macrophages, study the genes that influence the activity of mouse peritoneal macrophages. The shuttle expression vector pMV261 by avirulent Mycobacterium smegmatis transformed Rv0901 gene fragment into plasmid does not contain the gene sequence (mc~2 155), and the recombinant Mycobacterium smegmatis (Rmc~2 155) in the expression of Rv0901 encoding protein 19kDa. The expression of recombinant. Mycobacterium smegmatis and recombinant Mycobacterium smegmatis infection of human monocyte macrophage THP-1 and BALB / c mice with the same number of recombinant strains and non recombinant strains of cytology and animal studies. Results showed that the Rv0901 gene fragment into mouse peritoneal macrophages, apoptosis of murine peritoneal macrophages increased in the culture medium in the supernatant of NO (nitric oxide) and IFN (interferon gamma) increased release of.Rmc~2 155 induced apoptosis of THP-1 cells was higher than that of mc~2 155, the infection of THP 1 cells after the cell survival rate was lower than 155 mc~2 infection, and NO in the cell culture medium of.Rmc~2 infection is higher than that of mc~2 155 BALB 155 infection / c mice after proliferation of spleen lymphocytes in mice was higher than that of mc~2 155 infection, release IFN gamma and NO in serum were increased, but no significant difference compared with mc~2 155. The above results suggest that Rv0901 may be on It has some effect on the survival of the host cell and the regulation of the host cell apoptosis in order to maintain the stability of its living environment.

【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R378

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