本文選題：結(jié)核分枝桿菌 + 恥垢分枝桿菌��； 參考：《大連醫(yī)科大學(xué)》2016年碩士論文

【摘要】：結(jié)核分枝桿菌(Mycobacterium tuberculosis,M.Tb)是引起結(jié)核病(Tuberculosis,TB)的病原體。目前結(jié)核病的防治形勢(shì)依然嚴(yán)峻。2015 WHO統(tǒng)計(jì)報(bào)告指出,2014年的結(jié)核病新增病例超過(guò)960萬(wàn),死亡病例達(dá)150萬(wàn),呈增加趨勢(shì)。這主要是由于結(jié)核病的傳播途徑為空氣的飛沫傳播,易于傳染;此外,近年出現(xiàn)了多重耐藥性及極度耐藥性M.Tb菌株的感染,使對(duì)結(jié)核病患者的治療困難。因此要治愈結(jié)核病,克服耐藥的M.Tb感染,有必要尋找有效的抗結(jié)核新藥或新的藥物靶點(diǎn)。M.Tb細(xì)胞壁結(jié)構(gòu)特殊,對(duì)分枝桿菌的生存和繁殖極為重要,因而影響細(xì)胞壁完整性的因素,常為尋找和設(shè)計(jì)抗結(jié)核新藥的理想靶點(diǎn)。分枝桿菌細(xì)胞壁核心結(jié)構(gòu)為mAGP(Mycolic acid-Arabinogalactan-Peptidoglycan),是由分枝菌酸、聚阿拉伯半乳糖和肽聚糖三種組分共價(jià)連接形成。肽聚糖是其中重要的組成成分,其完整性影響細(xì)菌的增殖和生存。本文研究的靶向分子DdlA(D-丙氨酰-D-丙氨酸連接酶)為影響肽聚糖生物合成過(guò)程中的供體D-丙氨酰-D-丙氨酸的供給。D-丙氨酰-D-丙氨酸是以D-丙氨酸為原料,由DdlA催化形成的。因此選取M.Tb基因組中的ddlA基因(Rv2981c)作為研究對(duì)象,因其所編碼的DdlA直接影響肽聚糖的多肽鏈的合成,進(jìn)而影響肽聚糖的交聯(lián)與成熟。2003年,Sesseti的轉(zhuǎn)座子突變結(jié)果表明ddlA基因?yàn)镸.Tb生長(zhǎng)必需基因;此外,目前應(yīng)用于臨床的二線抗結(jié)核藥物D-環(huán)絲氨酸即以DdlA為作用靶點(diǎn),但該藥物由于嚴(yán)重的神經(jīng)系統(tǒng)副作用而使其在治療結(jié)核病的應(yīng)用中頗為受限。綜上表明,DdlA是良好的抗結(jié)核藥物作用靶向位點(diǎn),對(duì)其進(jìn)行研究具有重要的理論和實(shí)踐意義。本文的研究策略:1.獲取rv2981c基因序列信息,體外表達(dá)tb-ddla蛋白,并建立能應(yīng)用于篩選抑制劑的酶分析體系。在大腸桿菌表達(dá)體系中誘導(dǎo)、表達(dá)并純化ddla蛋白及鑒定其d-丙氨酰-d-丙氨酸連接酶酶活性;建立酶活性分析體系,為ddla抑制劑的篩選提供基礎(chǔ);同時(shí)以ni-nta親和層析純化的蛋白制備其多克隆抗體,以期用于對(duì)sm-ddla反義下調(diào)表達(dá)模型的驗(yàn)證。2.通過(guò)比對(duì)分析獲取rv2981c(ddla)在恥垢分枝桿菌中的同源序列m.smeg-2395(ddla),構(gòu)建其反義rna下調(diào)表達(dá)的恥垢分枝桿菌菌株模型,以強(qiáng)力霉素誘導(dǎo),繪制經(jīng)反義rna下調(diào)表達(dá)載體調(diào)控的恥垢分枝桿菌生長(zhǎng)曲線;應(yīng)用制備的多克隆抗體驗(yàn)證所構(gòu)建的下調(diào)表達(dá)模型;觀察sm-ddla基因下調(diào)表達(dá)對(duì)細(xì)菌生長(zhǎng)及形態(tài)的影響,為闡明特異性的ddla抑制劑的作用機(jī)制提供理論參考。本文所獲得的實(shí)驗(yàn)結(jié)果:1.構(gòu)建了pcold-tb-ddla表達(dá)載體及ddla在大腸桿菌中的表達(dá)體系;提取、純化tb-ddla蛋白,并鑒定其d-丙氨酰-d-丙氨酸連接酶酶活性;建立了應(yīng)用d-氨基酸氧化酶體系對(duì)酶活性進(jìn)行分析的elisa方法,并確定了最適反應(yīng)條件為:底物丙氨酸的濃度10mmol/ml,緩沖液為含有5mmol/mlatp的100mmhepes(ph8.0)37℃保溫1h;制備抗tb-ddla蛋白的多克隆抗體,并用酶聯(lián)免疫吸附試驗(yàn)以及免疫印跡法證實(shí)了所獲得的抗體具有高效價(jià)性與特異性。2.構(gòu)建了sm-ddla反義表達(dá)載體pmind-sm-ddla-as及恥垢分枝桿菌sm-ddla反義rna下調(diào)表達(dá)模型,以強(qiáng)力霉素誘導(dǎo)pmind-sm-ddla-as在恥垢分枝桿菌中的表達(dá),生長(zhǎng)曲線結(jié)果表明最佳的誘導(dǎo)條件為:強(qiáng)力霉素濃度,20ng/ml;培養(yǎng)時(shí)間,36h左右。本研究中以pmindinsmeg的恥垢分枝桿菌菌株作為對(duì)照進(jìn)行研究3.提取恥垢分枝桿菌pmind-sm-ddla-as反義下調(diào)表達(dá)菌株在20ng/ml強(qiáng)力霉素誘導(dǎo)生長(zhǎng)36h時(shí)細(xì)菌總蛋白,以制備的抗ddla多克隆抗體檢測(cè)細(xì)菌中ddla蛋白的表達(dá),westernblot結(jié)果顯示在反義下調(diào)表達(dá)菌株中ddla蛋白呈下調(diào)表達(dá)。透射電子顯微鏡對(duì)pmind-sm-ddla-as反義下調(diào)表達(dá)菌株的形態(tài)觀察結(jié)果表明:ddla蛋白下調(diào)表達(dá)后細(xì)菌的形狀變長(zhǎng),胞內(nèi)物質(zhì)密度不均一。本研究中以pmindinsmeg的恥垢分枝桿菌菌株作為對(duì)照。未來(lái)的研究方向:1.以建立的DdlA酶促反應(yīng)體系篩選能特異地作用于M.Tb DdlA的化合物。2.通過(guò)制備型HPLC提取、分離并純化ddlA基因反義下調(diào)表達(dá)的恥垢分枝桿菌的肽聚糖,將提取的肽聚糖與RAW264.7巨噬細(xì)胞共培養(yǎng),觀察D-丙氨酰-D-丙氨酸連接酶缺陷的恥垢分枝桿菌細(xì)胞壁對(duì)細(xì)胞感染的可能影響。本實(shí)驗(yàn)將以pMind in Smeg的恥垢分枝桿菌菌株細(xì)胞壁作為對(duì)照。3.檢測(cè)反義下調(diào)菌株對(duì)一二線抗結(jié)核藥物的敏感性4.基于本課題的結(jié)果制備大量sRNA,并利用虛擬篩選尋找抗結(jié)核藥物。
[Abstract]:Mycobacterium tuberculosis (M.Tb) is the pathogen causing Tuberculosis (TB). The current situation of the prevention and control of tuberculosis is still severe, the serious.2015 WHO statistics report indicates that in 2014, more than 9 million 600 thousand new cases of tuberculosis were added, and the death cases were 1 million 500 thousand, increasing. This is mainly due to the spread of tuberculosis. Air droplets are spread easily, and in addition, in recent years, the infection of multi resistance and extreme resistance M.Tb strains makes the treatment of tuberculosis patients difficult. Therefore, to cure tuberculosis and overcome the resistance of M.Tb infection, it is necessary to find effective anti tuberculosis drugs or new drug targets,.M.Tb cell wall structure special, to Mycobacterium The survival and reproduction are very important, so the factors that affect the integrity of cell wall are the ideal targets for the search and design of new anti tuberculosis drugs. The core structure of the cell wall of Mycobacterium is mAGP (Mycolic acid-Arabinogalactan-Peptidoglycan), which is covalent connected by three components of Mycobacterium acid, polygalactose and peptidoglycan. Sugar is an important component of it, its integrity affects the proliferation and survival of bacteria. The target molecule DdlA (D- alanyl -D- alanine ligase) is a source of.D- alanine -D- alanine, which affects the donor D- alamyl -D- alanine in the biosynthesis of peptidoglycan biosynthesis, which is based on D- alanine and is catalyzed by DdlA. The ddlA gene (Rv2981c) in the M.Tb genome is selected as the research object, because its encoded DdlA directly affects the synthesis of peptide chain of peptidoglycan, and then affects the cross-linking of peptidoglycan and the mature.2003 years. The result of the transposon mutation of Sesseti indicates that the ddlA gene is the essential gene for M.Tb growth. In addition, the current application of the second line anti tuberculosis in the clinic The drug D- cyclic serine is used as the target of DdlA, but the drug is quite limited in the treatment of tuberculosis due to the serious side effects of the nervous system. It is shown that DdlA is a good target site for anti tuberculosis drugs, and it is of great theoretical and practical significance to study it. The research strategy of this article: 1. to obtain rv298 1C gene sequence information, in vitro expression of tb-ddla protein, and establish an enzyme analysis system which can be used to screen inhibitors. Induce, express and purify ddlA protein and identify d- alanine -d- alanine ligase activity in Escherichia coli expression system; establish an enzyme activity analysis system to provide a basis for screening ddlA inhibitors; and Ni-NTA The protein purified by affinity chromatography was prepared for the polyclonal antibody, in order to verify the antisense expression model of sm-ddla antisense.2., and to obtain the homologous sequence m.smeg-2395 (ddlA) of rv2981c (ddlA) in Mycobacterium foul by comparison analysis and construct its antisense RNA down expression of Mycobacterium foul Mycobacterium strain model, induced by doxycycline. Antisense RNA downregulates the growth curve of Mycobacterium tumefaciens regulated by expression vector; the down-regulation model constructed by polyclonal antibody preparation is used to observe the effect of down regulation of sm-ddla gene on the growth and morphology of bacteria, which provides a theoretical reference for elucidating the mechanism of specific ddlA inhibitors. The results obtained in this paper: 1 The expression system of pcold-tb-ddla expression vector and ddlA in Escherichia coli was constructed, the tb-ddla protein was extracted, purified and the activity of d- alanine -d- alanine ligase enzyme was identified. A ELISA method for the analysis of the enzyme activity by the d- amino acid oxidase system was established, and the optimum reaction condition was determined as the concentration 10mm of the substrate alanine. Ol/ml, the buffer solution is 1H containing 5mmol/mlatp 100mmhepes (ph8.0) 37 C, and the polyclonal antibody of anti tb-ddla protein is prepared. It is confirmed by enzyme linked immunosorbent assay and immunoblotting that the obtained antibody has high potency and specific.2. to construct sm-ddla antisense expression vector pmind-sm-ddla-as and Mycobacterium foul Mycobacterium tuberculosis sm-ddla. The expression model of antisense RNA was downregulated and pmind-sm-ddla-as was induced in Mycobacterium tumefaciens by doxycycline. The results of growth curve showed that the optimal induction conditions were: doxycycline concentration, 20ng/ml, culture time, and 36h. In this study, the strain of Mycobacterium humilis in pmindinsmeg was used as control to study 3. disgrace branching rods The bacterial pmind-sm-ddla-as antisense strain down regulated the total protein of the bacteria when 20ng/ml was induced by doxycycline, and the anti ddlA polyclonal antibody was used to detect the expression of ddlA protein in bacteria. The result of Westernblot showed that the ddlA protein was down regulated in the antisense down-regulation strain. The antisense regulation of pmind-sm-ddla-as was down regulated by the electron microscope. The morphological observation of the expressed strains showed that the shape of the bacteria was longer and the density of the intracellular substance was not uniform after the downregulation of ddlA protein. In this study, pmindinsmeg was used as control. The future research direction: 1. the screening of M.Tb DdlA compound.2. through the system of DdlA enzymatic reaction system The peptidoglycan was isolated and purified by the preparation of HPLC, and the antisense expression of ddlA gene was purified and purified. The extracted peptidoglycan was co cultured with RAW264.7 macrophages, and the potential effect of the cell wall of Mycobacterium tumefaciens cell wall on the D- alanine -D- alanine ligase deficiency was observed. This experiment will be based on the pMind in Smeg's shameful branch. The cell wall of the bacteria strain was used as the control.3. to detect the sensitivity of the antisense down-regulation strain to the second line anti tuberculosis drug 4. based on the results of this project to prepare a large number of sRNA, and to use the virtual screening to find anti tuberculosis drugs.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R378.91

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 徐苗,陳保文,沈小兵,蘇城,羅永艾,王國(guó)治;恥垢分枝桿菌作為免疫調(diào)節(jié)劑的研究[J];中華微生物學(xué)和免疫學(xué)雜志;2005年09期

2 湛W歐，

本文編號(hào)：1872249