M.smeg-2395基因反义下调表达对耻垢分枝杆菌生长影响的研究

发布时间：2018-05-11 03:34

本文选题：结核分枝杆菌 + 耻垢分枝杆菌　；参考：《大连医科大学》2016年硕士论文

【摘要】：结核分枝杆菌(Mycobacterium tuberculosis,M.Tb)是引起结核病(Tuberculosis,TB)的病原体。目前结核病的防治形势依然严峻。2015 WHO统计报告指出,2014年的结核病新增病例超过960万,死亡病例达150万,呈增加趋势。这主要是由于结核病的传播途径为空气的飞沫传播,易于传染;此外,近年出现了多重耐药性及极度耐药性M.Tb菌株的感染,使对结核病患者的治疗困难。因此要治愈结核病,克服耐药的M.Tb感染,有必要寻找有效的抗结核新药或新的药物靶点。M.Tb细胞壁结构特殊,对分枝杆菌的生存和繁殖极为重要,因而影响细胞壁完整性的因素,常为寻找和设计抗结核新药的理想靶点。分枝杆菌细胞壁核心结构为mAGP(Mycolic acid-Arabinogalactan-Peptidoglycan),是由分枝菌酸、聚阿拉伯半乳糖和肽聚糖三种组分共价连接形成。肽聚糖是其中重要的组成成分,其完整性影响细菌的增殖和生存。本文研究的靶向分子DdlA(D-丙氨酰-D-丙氨酸连接酶)为影响肽聚糖生物合成过程中的供体D-丙氨酰-D-丙氨酸的供给。D-丙氨酰-D-丙氨酸是以D-丙氨酸为原料,由DdlA催化形成的。因此选取M.Tb基因组中的ddlA基因(Rv2981c)作为研究对象,因其所编码的DdlA直接影响肽聚糖的多肽链的合成,进而影响肽聚糖的交联与成熟。2003年,Sesseti的转座子突变结果表明ddlA基因为M.Tb生长必需基因;此外,目前应用于临床的二线抗结核药物D-环丝氨酸即以DdlA为作用靶点,但该药物由于严重的神经系统副作用而使其在治疗结核病的应用中颇为受限。综上表明,DdlA是良好的抗结核药物作用靶向位点,对其进行研究具有重要的理论和实践意义。本文的研究策略:1.获取rv2981c基因序列信息,体外表达tb-ddla蛋白,并建立能应用于筛选抑制剂的酶分析体系。在大肠杆菌表达体系中诱导、表达并纯化ddla蛋白及鉴定其d-丙氨酰-d-丙氨酸连接酶酶活性;建立酶活性分析体系,为ddla抑制剂的筛选提供基础;同时以ni-nta亲和层析纯化的蛋白制备其多克隆抗体,以期用于对sm-ddla反义下调表达模型的验证。2.通过比对分析获取rv2981c(ddla)在耻垢分枝杆菌中的同源序列m.smeg-2395(ddla),构建其反义rna下调表达的耻垢分枝杆菌菌株模型,以强力霉素诱导,绘制经反义rna下调表达载体调控的耻垢分枝杆菌生长曲线;应用制备的多克隆抗体验证所构建的下调表达模型;观察sm-ddla基因下调表达对细菌生长及形态的影响,为阐明特异性的ddla抑制剂的作用机制提供理论参考。本文所获得的实验结果:1.构建了pcold-tb-ddla表达载体及ddla在大肠杆菌中的表达体系;提取、纯化tb-ddla蛋白,并鉴定其d-丙氨酰-d-丙氨酸连接酶酶活性;建立了应用d-氨基酸氧化酶体系对酶活性进行分析的elisa方法,并确定了最适反应条件为:底物丙氨酸的浓度10mmol/ml,缓冲液为含有5mmol/mlatp的100mmhepes(ph8.0)37℃保温1h;制备抗tb-ddla蛋白的多克隆抗体,并用酶联免疫吸附试验以及免疫印迹法证实了所获得的抗体具有高效价性与特异性。2.构建了sm-ddla反义表达载体pmind-sm-ddla-as及耻垢分枝杆菌sm-ddla反义rna下调表达模型,以强力霉素诱导pmind-sm-ddla-as在耻垢分枝杆菌中的表达,生长曲线结果表明最佳的诱导条件为:强力霉素浓度,20ng/ml;培养时间,36h左右。本研究中以pmindinsmeg的耻垢分枝杆菌菌株作为对照进行研究3.提取耻垢分枝杆菌pmind-sm-ddla-as反义下调表达菌株在20ng/ml强力霉素诱导生长36h时细菌总蛋白,以制备的抗ddla多克隆抗体检测细菌中ddla蛋白的表达,westernblot结果显示在反义下调表达菌株中ddla蛋白呈下调表达。透射电子显微镜对pmind-sm-ddla-as反义下调表达菌株的形态观察结果表明:ddla蛋白下调表达后细菌的形状变长,胞内物质密度不均一。本研究中以pmindinsmeg的耻垢分枝杆菌菌株作为对照。未来的研究方向:1.以建立的DdlA酶促反应体系筛选能特异地作用于M.Tb DdlA的化合物。2.通过制备型HPLC提取、分离并纯化ddlA基因反义下调表达的耻垢分枝杆菌的肽聚糖,将提取的肽聚糖与RAW264.7巨噬细胞共培养,观察D-丙氨酰-D-丙氨酸连接酶缺陷的耻垢分枝杆菌细胞壁对细胞感染的可能影响。本实验将以pMind in Smeg的耻垢分枝杆菌菌株细胞壁作为对照。3.检测反义下调菌株对一二线抗结核药物的敏感性4.基于本课题的结果制备大量sRNA,并利用虚拟筛选寻找抗结核药物。
[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.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R378.91

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