红斑丹毒丝菌新型毒力相关因子鉴定及其耐喹诺酮药物机制初探

发布时间：2018-05-01 21:23

本文选题：红斑丹毒丝菌 + 细胞壁相关蛋白　；参考：《华中农业大学》2017年博士论文

【摘要】：红斑丹毒丝菌感染猪导致猪丹毒,主要引起猪的败血症、心内膜炎和多发性关节炎。2010年以来,我国多个省份均出现了猪丹毒的大范围流行,给猪场造成了巨大的经济损失。虽然红斑丹毒丝菌成为严重危害养猪业的传染病病原,但关于其致病机制和免疫机制的研究还相对滞后。红斑丹毒丝菌的致病机制不明确成为防控猪丹毒最大的阻碍,新毒力(相关)因子及毒力调控基因的发掘以及功能研究是目前红斑丹毒丝菌致病机理研究的关键。本研究力图系统性发掘红斑丹毒丝菌新的毒力(相关)因子,为进一步研究红斑丹毒丝菌致病机制奠定基础。鉴于革兰氏阳性菌的细胞壁相关蛋白在细菌的粘附、侵入等致病过程中发挥重要作用,本研究以红斑丹毒丝菌的细胞壁相关蛋白为研究对象,利用i TRAQ结合LC-MS/MS的方法鉴定了强弱毒菌株差异表达的细胞壁相关蛋白,并对鉴定到的潜在的毒力相关因子Sbp进行了验证,评价了Sbp制备的亚单位疫苗对仔猪的免疫保护效果。在90年代初期,我国通过疫苗免疫等防控手段,使得猪丹毒疫情得到控制。如今猪丹毒这一“老病”新发,也提出了许多问题:是否现有疫苗预防不理想?或是临床流行的红斑丹毒丝菌耐药性发生了变化?为了给当前防控猪丹毒提供科学的用药指导,本研究对红斑丹毒丝菌临床分离株进行了21种常用药物的耐药谱检测。并进一步对红斑丹毒丝菌耐喹诺酮药物的机制进行了初步探索。本研究的主要成果总结如下:1.红斑丹毒丝菌比较蛋白质组学研究本研究以红斑丹毒丝菌的强毒菌株和弱毒菌株的细胞壁相关蛋白为研究对象,利用i TRAQ结合LC-MS/MS的方法鉴定差异表达蛋白,共鉴定到100个差异表达的细胞壁相关蛋白。强毒菌株中高表达蛋白为57个,主要为ABC转运蛋白和粘附相关蛋白;低表达蛋白为43个,主要为压力反应蛋白。强毒菌株中高表达的蛋白可能跟细菌毒力相关,本研究选取强毒菌株中高表达的部分蛋白,利用原核表达系统表达重组蛋白,纯化后免疫小鼠制备多克隆抗体,通过Western Blot检测其在强弱毒菌株中的实际表达情况,其表达情况与i TRAQ结果一致。对强毒菌株高表达的蛋白进行分析,发现糖转运蛋白Sbp在强毒菌株中表达量为弱毒菌株的1.73倍。Sbp蛋白被报道参与细菌双精氨酸转位通路,是一些细菌重要的毒力因子。为了验证Sbp在红斑丹毒丝菌致病过程中发挥的作用,本研究利用同源重组原理构建了sbp基因缺失突变菌株。sbp基因缺失后,红斑丹毒丝菌粘附侵入猪髋动脉内皮细胞(PIEC)的能力显著下降,其在健康猪全血中的存活率也显著下降,同时其抵抗小鼠吞噬细胞吞噬的能力也降低。动物感染实验结果显示:与野生菌相比,sbp基因缺失菌株对猪的致病力下降。这些结果表明Sbp是红斑丹毒丝菌重要的毒力相关因子。同时,病原菌表面的毒力相关蛋白通常也是其重要的保护性抗原,所以本研究利用仔猪模型评价Sbp蛋白的保护效力。在本研究中,纯化的重组Sbp蛋白能够诱导仔猪产生高水平的抗体,并保护66.7%的仔猪抵抗致死剂量的红斑丹毒丝菌人工感染。抗体跟踪检测表明Sbp诱导的抗体可持续最少6个月。结果表明Sbp是一个有效的疫苗候选蛋白。2.红斑丹毒丝菌耐喹诺酮药物机制初探本研究用药敏纸片法检测了红斑丹毒丝菌临床分离株对21种常用药物的耐药谱,旨在了解当前流行株的耐药情况,为临床用药提供参考。进一步利用E-test药敏纸条检测临床分离株对环丙沙星和左旋氧氟沙星(喹诺酮类药物)的MIC值,根据CLSI标准,判定临床分离的红斑丹毒丝菌对环丙沙星的耐药率为48.8%,且不同菌株之间对环丙沙星药物的MIC值呈现多样性。进一步通过测序分析喹诺酮耐药相关基因DNA解旋酶(Gyr A、Gyr B)和拓扑异构酶Ⅳ(Par C、Par E)的突变情况,结合各菌株对环丙沙星药物的MIC,发现了2个耐药相关基因突变位点:Gyr A(90D→N)和Par C(81 S→I)。进一步对这两个位点进行了定向点突变实验,结果发现Gyr A90位点的突变能使红斑丹毒丝菌对环丙沙星的MIC值升高3倍。
[Abstract]:Erysipelas infected pigs lead to swine erysipelas, which mainly caused the septicaemia, endocarditis and multiple arthritis of pigs for.2010 years. The large epidemic of porcine erysipelas has emerged in many provinces in China, causing huge economic losses to pig farms. Although erysipelas has become a serious infectious disease causing swine industry, but it is about it The pathogenesis of the pathogenic mechanism and immune mechanism is still lagging behind. The pathogenesis of erysipelas is not clear as the biggest hindrance to the prevention and control of swine erysipelas. The key to the study of the pathogenesis of erysipelas erysipelas is the key to the research of the new virulence (related) factors and virulence regulation genes and the function research. The new virulence factor of filamentous bacteria lays the foundation for further research on the pathogenesis of erysipelas erysipelas. In view of the important role of cell wall related proteins in Gram-positive bacteria in bacterial adhesion and invasion, the cell wall related protein of erysipelas erysipelas is used as the research object, and I TRAQ is used to combine LC-MS/M with the cell wall related proteins. S method identified the differentially expressed cell wall related proteins of the strong and weak strains, and verified the potential virulence related factor Sbp identified, and evaluated the immune protection effect of the subunit vaccine prepared by Sbp. In the early 90s, the epidemic of porcine erysipelas was controlled by means of vaccine immunity and other control methods. The recent development of this "old disease" of swine erysipelas has also raised a number of questions: whether the existing vaccine prevention is not ideal? Or the drug resistance of the clinical epidemic erysipelas has changed? In order to provide scientific guidance for the current prevention and control of swine erysipelas, the drug resistance of 21 common drugs in the clinical isolates of erysipelas erysipelas was studied in this study. Preliminary exploration on the mechanism of quinolone resistant drugs of erysipelas erysipelas. The main achievements of this study were summarized as follows: 1. the comparative proteomics of erysipelas erysipelas in this study was based on the cell wall phase protein of the virulent and weakly toxic strains of erysipelas erysipelas, and I TRAQ combined with LC-MS A total of 100 differentially expressed cell wall related proteins were identified by the /MS method. The high expression protein of the highly toxic strain was 57, mainly ABC transporter and adhesion related protein; the low expression protein was 43, mainly pressure reactive protein. The high expression protein in the virulent strain may be related to the bacterial virulence. The recombinant protein expressed in the highly toxic strain was selected, the recombinant protein was expressed by the prokaryotic expression system, and the polyclonal antibody was prepared from the purified mice. The actual expression of the protein in the strong and weak virulence strain was detected by Western Blot. The expression of the recombinant protein was in accordance with the I TRAQ results. The 1.73 times.Sbp protein expressed in the virulent strain of the strain Sbp is reported to be involved in the bacterial double arginine transposition pathway and is an important virulence factor of some bacteria. In order to verify the role played by Sbp in the pathogenesis of erysipelas erysipelas, this study constructed the SBP gene deletion mutation strain.Sbp by the principle of homologous recombination. After gene deletion, the ability of erysipelas to adhere to the endothelial cell (PIEC) of the porcine hip artery decreased significantly, and the survival rate in the whole blood of healthy pigs decreased significantly, while the ability to resist phagocytic phagocytosis was also reduced. The results of animal infection experiments showed that the pathogenicity of SBP gene missing strains to pigs compared with the wild bacteria. These results suggest that Sbp is an important virulence factor of erysipelas. At the same time, the virulence related proteins on the surface of the pathogen are also important protective antigens. Therefore, this study used the piglet model to evaluate the protective effect of Sbp protein. In this study, the purified recombinant Sbp protein can induce high levels of piglets. Antibody tracking and protection of 66.7% piglets against lethal dose of erysipelas artificial infection. Antibody tracking detection showed that the Sbp induced antibody lasted for a minimum of 6 months. The results showed that Sbp was an effective candidate protein for vaccine.2., a preliminary study on the mechanism of quinolone resistance to red erysipelas. The drug resistance spectrum of 21 commonly used drugs was designed to understand the drug resistance of current epidemic strains and to provide reference for clinical use. The MIC value of clinical isolates to ciprofloxacin and levofloxacin (quinolone) was detected by E-test drug sensitive strips, and the clinical isolation of erythematous erythematous erythematous drugs was determined according to the standard of CLSI. The resistance rate of ciprofloxacin to ciprofloxacin was 48.8%, and the MIC value of ciprofloxacin was diversity among different strains. The mutation of the quinolone resistance related gene DNA (Gyr A, Gyr B) and topoisomerase IV (Par C, Par E) was further analyzed by sequencing, and 2 resistance were found in combination with the MIC of each strain on ciprofloxacin. The mutation loci of drug related genes: Gyr A (90D to N) and Par C (81 S to I). The directional point mutation experiments were carried out on these two loci. The results showed that the mutation of Gyr A90 loci could increase the MIC value of ciprofloxacin by 3 times.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.6

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