结核分枝杆菌转录因子WhiB2蛋白的功能和转录调控研究

发布时间：2018-07-03 16:06

  本文选题：WhiB2 + 细胞分裂　； 参考：《西南大学》2012年硕士论文

【摘要】：结核病死灰复燃,是一种严重危害人类健康的传染病。2010年,估算全球新发结核病患者880万例,未感染艾滋病的结核病患者死亡人数为110万,另有35万死于艾滋病病毒感染相关结核病。随着人类社会和药物治疗的发展,人们对结核病的致病菌——结核分枝杆菌(Mycobacterium tuberculosis)的生物学特性有了进一步的研究,继而研发了链霉素、异烟肼、利福平等治疗性药物,使得人类对结核病的控制取得了可喜的成绩,遏制了结核病的蔓延和传播。1995年至2010年间,在实施了现代结核病控制策略/遏制结核病策略的结核病防治规划中,5500万例结核病患者得到了治疗,4600万例患者获得成功治疗,这些治疗挽救了近700万人的生命。然而由于化学药物的不合理应用、艾滋病与结核病共感染、人口流动的增加以及耐药结核病菌的流行,使得近年来结核病疫情下降缓慢,在部分地区甚至有所回升。当前,在结核病控制工作中急需有效的预防性、治疗性疫苗和新型药物。寻找新的药物作用靶点和开发新型抗结核药物已成为当前最为关注的焦点。 whi基因普遍存在于放线菌属和分枝杆菌属中,因其突变能导致天蓝色链霉菌菌落呈白色而得名。whiB是whi基因中的一类,其编码的蛋白功能涉及孢子形成、细胞分裂、致病性以及营养缺陷等压力传感。经Protein Sequence Analysis (PSA) server分析显示WhiB样具有helix-turn-helix (HTH)基序,是可能的转录调控因子。结核分枝杆菌具有7个WhiB-like蛋白(WBL),为WhiB1～7。序列分析显示这些WBL都具有四个保守的半胱氨酸残基,且排列为"C-X19-36-C-X-X-C-X5-7-C", C-X-X-C可以和[2Fe-2S]配位。通过同源性分析,发现除了WhiB5和WhiB6,结核分枝杆菌中其它5个WhiB蛋白在分枝杆菌属中的同源性都很高。但是结核分枝杆菌7个WBL蛋白的功能还没有完全诠释。与胰岛素共孵育检测蛋白的二硫键还原功能,结果表明除了WhiB2,其他6个WhiB蛋白都能够还原胰岛素的二硫键。WhiB1是唯一的在结核分枝杆菌中被发现的受cAMP受体蛋白CRP调控的蛋白,能够与结核分枝杆菌体内最佳生长所必需的基因glgB相互作用。WhiB3与组织损伤有关,且结核分枝杆菌调节whiB3的表达来应对体内的环境信号.M. tuberculosis H37Rv的whiB7无效突变菌株对多种抗生素(四环素,林可霉素,红霉素)高度敏感,表明WhiB7蛋白与压力传感有关。在M. tuberculosis CDC1551中检测7个WbL在不同抗分枝杆菌因子和其它的压力条件下的表达情况,结果表明作用于核糖体的氨基苷类—链霉素和卡那霉素—刺激whiB7的表达。在PH4.5的酸性条件下,whiB6和whiB3的表达特异性增高 在耻垢分枝杆菌中过表达WhiBTM4,导致耻垢分枝杆菌表现出WhiB2敲除表型,即阻碍隔膜的形成,表明WhiBTM4下调WhiB2。研究表明结核分枝杆菌WhiB2蛋白与耻垢分枝杆菌WhmD蛋白的功能是相同的。whmD是必需基因,耻垢分枝杆菌染色体上whmD的缺失导致不可逆的,分枝生长呈现减少的隔膜形成,说明该基因和细胞分裂有关,但是与细胞分裂相关的具体机制目前尚不清楚。为了研究WhiB2与细胞分裂的机制及其功能,本研究将结核分枝杆菌whiB2基因克隆到穿梭质粒pNIT-myc中,得到重组质粒pNIT-myc-whiB2,再用电穿孔的方法将重组质粒转入耻垢分枝杆菌中得到过表达WhiB2蛋白的耻垢分枝杆菌重组菌株。然后研究whiB2的过表达对耻垢分枝杆菌的生长,对过氧化氢的敏感性及对与细胞分裂有关基因的表达的影响,检测了抗分枝杆菌抗生素对耻垢分支杆菌的MIC。结果表明whiB2的过表达对耻垢分枝杆菌的生长并没有什么影响,却使耻垢分枝杆菌增加了对异烟肼和卷曲霉素的敏感性,而对过氧化氢的耐受性明显增加。RT-PCR结果显示WhiB2的过表达使与细胞分离有关的基因MSEMG_4225和ftsZ的表达量增加。荧光显微镜分析显示WhiB2蛋白的过表达使得细胞分裂加快。总而言之,我们的研究表明了结核分枝杆菌WhiB2与抗生素和氧化物过氧化氢的关系,进一步揭示了WhiB2与细胞分裂的机制,为了解结核分枝杆菌的生理生化现在提供了进一步的依据。
[Abstract]:The resurgence of tuberculosis is a kind of infectious disease that seriously endangers human health in.2010, 8 million 800 thousand cases of global new TB patients are estimated. The number of people who have not infected with AIDS is 1 million 100 thousand, and another 35 million deaths from HIV infection related tuberculosis. With the development of human society and drug treatment, people have the cause of tuberculosis. The biological characteristics of the bacteria, Mycobacterium tuberculosis (Mycobacterium tuberculosis), have been further studied, and then the research and development of streptomycin, isoniazid, and benefit equal therapeutic drugs have made a gratifying achievement in the control of tuberculosis, curbing the spread and spreading of TB between.1995 and 2010, in modern times. In the TB control strategy / TB control strategy, 55 million cases of TB patients were treated and 46 million patients were treated successfully. These treatments saved nearly 7 million lives. However, due to the irrational use of chemical drugs, AIDS and tuberculosis co infection, population mobility and drug resistance were increased. The epidemic of tuberculosis bacteria has caused a slow decline in the epidemic situation of tuberculosis in recent years and even in some areas. At present, effective preventive, therapeutic vaccines and new drugs are urgently needed in the work of tuberculosis control. Finding new drug targets and developing new anti tuberculosis drugs has become the focus of the most attention.
WHI gene is commonly found in actinomycetes and mycobacteria. Because its mutation can lead to the white of Streptomyces of Streptomyces azure, the name.WhiB is one of the WHI genes. The function of the encoded protein involves the pressure transmission of spores formation, cell division, pathogenicity and nutritional defects. Protein Sequence Analysis (PSA) server analysis The WhiB sample has helix-turn-helix (HTH) motif and is a possible transcriptional regulator. Mycobacterium tuberculosis has 7 WhiB-like proteins (WBL). The analysis of WhiB1 ~ 7. sequence analysis shows that these WBL have four conserved cysteine residues and are arranged as "C-X19-36-C-X-X-C-X5-7-C", C-X-X-C and [2Fe-2S] coordination. In addition to WhiB5 and WhiB6, the other 5 WhiB proteins in Mycobacterium tuberculosis were found to have high homology in the Mycobacterium genus. But the function of 7 WBL proteins of Mycobacterium tuberculosis was not fully interpreted. The two sulfur bond reduction function of the protein was detected with insulin, and the results showed that the other 6 WhiB proteins could be reduced in addition to WhiB2. The two sulfur bond.WhiB1 of insulin is the only protein regulated by the cAMP receptor protein CRP found in Mycobacterium tuberculosis. The interaction of glgB, which is essential for the best growth of Mycobacterium tuberculosis, is associated with tissue damage, and Mycobacterium tuberculosis regulates the expression of whiB3 to respond to the environmental signal.M. tube in the body. The whiB7 ineffective mutant strain of rculosis H37Rv was highly sensitive to a variety of antibiotics (tetracycline, lincomycin, erythromycin), indicating that WhiB7 protein was associated with pressure sensing. The expression of 7 WbL in M. tuberculosis CDC1551 was detected under different anti Mycobacterium factors and other pressure conditions, and the results showed that the amino group was acted on the ribosome amino group. Glycosides streptomycin and kanamycin stimulate the expression of whiB7. Under the acidic condition of PH4.5, the expression of whiB6 and whiB3 increased.
The overexpression of WhiBTM4 in Mycobacterium tumefaciens led to WhiB2 knockout phenotype, which hinders the formation of septum, indicating that WhiBTM4 down-regulation WhiB2. studies show that the function of WhiB2 protein of Mycobacterium tuberculosis and Mycobacterium foul WhmD protein is the same.WhmD is the essential gene, the deletion of whmD on the chromosome of Mycobacterium foul Mycobacterium It is irreversible that branching growth presents a reduced septum formation, indicating that the gene is related to cell division, but the specific mechanism associated with cell division is not yet clear. In order to study the mechanism and function of WhiB2 and cell division, this study cloned the whiB2 gene of Mycobacterium tuberculosis to the shuttle plasmid pNIT-myc to get the recombinant Plasmid pNIT-myc-whiB2, then the recombinant plasmid was transferred into Mycobacterium foul mycobacteria by electroporation, and the recombinant strain of Mycobacterium foul expressed WhiB2 protein was obtained. Then the overexpression of whiB2 was studied for the growth of Mycobacterium foul, the sensitivity to hydrogen peroxide and the expression of genes related to cell division. The MIC. results of Mycobacterium tumefaciens showed that the overexpression of whiB2 had no effect on the growth of Mycobacterium tumefaciens, but the sensitivity of Mycobacterium tumefaciens to isoniazid and Aspergillus fumigatus was increased, and the tolerance to hydrogen peroxide was significantly increased by.RT-PCR results, which showed that the overexpression of WhiB2 resulted in the separation of the cells from the cells. The expression of related genes MSEMG_4225 and ftsZ increased. The fluorescence microscope analysis showed that the overexpression of WhiB2 protein made cell division faster. In short, our study showed the relationship between Mycobacterium tuberculosis WhiB2 and antibiotics and oxide hydrogen peroxide, further revealing the mechanism of WhiB2 and cell division, to understand the tuberculosis points. The physiology and biochemistry of mycobacteria now provide further evidence.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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