本文選題：WhiB2 + 細胞分裂　； 參考：《西南大學》2012年碩士論文

【摘要】：結(jié)核病死灰復燃,是一種嚴重危害人類健康的傳染病。2010年,估算全球新發(fā)結(jié)核病患者880萬例,未感染艾滋病的結(jié)核病患者死亡人數(shù)為110萬,另有35萬死于艾滋病病毒感染相關(guān)結(jié)核病。隨著人類社會和藥物治療的發(fā)展,人們對結(jié)核病的致病菌——結(jié)核分枝桿菌(Mycobacterium tuberculosis)的生物學特性有了進一步的研究,繼而研發(fā)了鏈霉素、異煙肼、利福平等治療性藥物,使得人類對結(jié)核病的控制取得了可喜的成績,遏制了結(jié)核病的蔓延和傳播。1995年至2010年間,在實施了現(xiàn)代結(jié)核病控制策略/遏制結(jié)核病策略的結(jié)核病防治規(guī)劃中,5500萬例結(jié)核病患者得到了治療,4600萬例患者獲得成功治療,這些治療挽救了近700萬人的生命。然而由于化學藥物的不合理應(yīng)用、艾滋病與結(jié)核病共感染、人口流動的增加以及耐藥結(jié)核病菌的流行,使得近年來結(jié)核病疫情下降緩慢,在部分地區(qū)甚至有所回升。當前,在結(jié)核病控制工作中急需有效的預防性、治療性疫苗和新型藥物。尋找新的藥物作用靶點和開發(fā)新型抗結(jié)核藥物已成為當前最為關(guān)注的焦點。 whi基因普遍存在于放線菌屬和分枝桿菌屬中,因其突變能導致天藍色鏈霉菌菌落呈白色而得名。whiB是whi基因中的一類,其編碼的蛋白功能涉及孢子形成、細胞分裂、致病性以及營養(yǎng)缺陷等壓力傳感。經(jīng)Protein Sequence Analysis (PSA) server分析顯示W(wǎng)hiB樣具有helix-turn-helix (HTH)基序,是可能的轉(zhuǎn)錄調(diào)控因子。結(jié)核分枝桿菌具有7個WhiB-like蛋白(WBL),為WhiB1～7。序列分析顯示這些WBL都具有四個保守的半胱氨酸殘基,且排列為"C-X19-36-C-X-X-C-X5-7-C", C-X-X-C可以和[2Fe-2S]配位。通過同源性分析,發(fā)現(xiàn)除了WhiB5和WhiB6,結(jié)核分枝桿菌中其它5個WhiB蛋白在分枝桿菌屬中的同源性都很高。但是結(jié)核分枝桿菌7個WBL蛋白的功能還沒有完全詮釋。與胰島素共孵育檢測蛋白的二硫鍵還原功能,結(jié)果表明除了WhiB2,其他6個WhiB蛋白都能夠還原胰島素的二硫鍵。WhiB1是唯一的在結(jié)核分枝桿菌中被發(fā)現(xiàn)的受cAMP受體蛋白CRP調(diào)控的蛋白,能夠與結(jié)核分枝桿菌體內(nèi)最佳生長所必需的基因glgB相互作用。WhiB3與組織損傷有關(guān),且結(jié)核分枝桿菌調(diào)節(jié)whiB3的表達來應(yīng)對體內(nèi)的環(huán)境信號.M. tuberculosis H37Rv的whiB7無效突變菌株對多種抗生素(四環(huán)素,林可霉素,紅霉素)高度敏感,表明WhiB7蛋白與壓力傳感有關(guān)。在M. tuberculosis CDC1551中檢測7個WbL在不同抗分枝桿菌因子和其它的壓力條件下的表達情況,結(jié)果表明作用于核糖體的氨基苷類—鏈霉素和卡那霉素—刺激whiB7的表達。在PH4.5的酸性條件下,whiB6和whiB3的表達特異性增高 在恥垢分枝桿菌中過表達WhiBTM4,導致恥垢分枝桿菌表現(xiàn)出WhiB2敲除表型,即阻礙隔膜的形成,表明WhiBTM4下調(diào)WhiB2。研究表明結(jié)核分枝桿菌WhiB2蛋白與恥垢分枝桿菌WhmD蛋白的功能是相同的。whmD是必需基因,恥垢分枝桿菌染色體上whmD的缺失導致不可逆的,分枝生長呈現(xiàn)減少的隔膜形成,說明該基因和細胞分裂有關(guān),但是與細胞分裂相關(guān)的具體機制目前尚不清楚。為了研究WhiB2與細胞分裂的機制及其功能,本研究將結(jié)核分枝桿菌whiB2基因克隆到穿梭質(zhì)粒pNIT-myc中,得到重組質(zhì)粒pNIT-myc-whiB2,再用電穿孔的方法將重組質(zhì)粒轉(zhuǎn)入恥垢分枝桿菌中得到過表達WhiB2蛋白的恥垢分枝桿菌重組菌株。然后研究whiB2的過表達對恥垢分枝桿菌的生長,對過氧化氫的敏感性及對與細胞分裂有關(guān)基因的表達的影響,檢測了抗分枝桿菌抗生素對恥垢分支桿菌的MIC。結(jié)果表明whiB2的過表達對恥垢分枝桿菌的生長并沒有什么影響,卻使恥垢分枝桿菌增加了對異煙肼和卷曲霉素的敏感性,而對過氧化氫的耐受性明顯增加。RT-PCR結(jié)果顯示W(wǎng)hiB2的過表達使與細胞分離有關(guān)的基因MSEMG_4225和ftsZ的表達量增加。熒光顯微鏡分析顯示W(wǎng)hiB2蛋白的過表達使得細胞分裂加快�？偠灾�,我們的研究表明了結(jié)核分枝桿菌WhiB2與抗生素和氧化物過氧化氫的關(guān)系,進一步揭示了WhiB2與細胞分裂的機制,為了解結(jié)核分枝桿菌的生理生化現(xiàn)在提供了進一步的依據(jù)。
[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.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378

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