本文選題：紅斑丹毒絲菌 + 細(xì)胞壁相關(guān)蛋白　； 參考：《華中農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：紅斑丹毒絲菌感染豬導(dǎo)致豬丹毒,主要引起豬的敗血癥、心內(nèi)膜炎和多發(fā)性關(guān)節(jié)炎。2010年以來(lái),我國(guó)多個(gè)省份均出現(xiàn)了豬丹毒的大范圍流行,給豬場(chǎng)造成了巨大的經(jīng)濟(jì)損失。雖然紅斑丹毒絲菌成為嚴(yán)重危害養(yǎng)豬業(yè)的傳染病病原,但關(guān)于其致病機(jī)制和免疫機(jī)制的研究還相對(duì)滯后。紅斑丹毒絲菌的致病機(jī)制不明確成為防控豬丹毒最大的阻礙,新毒力(相關(guān))因子及毒力調(diào)控基因的發(fā)掘以及功能研究是目前紅斑丹毒絲菌致病機(jī)理研究的關(guān)鍵。本研究力圖系統(tǒng)性發(fā)掘紅斑丹毒絲菌新的毒力(相關(guān))因子,為進(jìn)一步研究紅斑丹毒絲菌致病機(jī)制奠定基礎(chǔ)。鑒于革蘭氏陽(yáng)性菌的細(xì)胞壁相關(guān)蛋白在細(xì)菌的粘附、侵入等致病過(guò)程中發(fā)揮重要作用,本研究以紅斑丹毒絲菌的細(xì)胞壁相關(guān)蛋白為研究對(duì)象,利用i TRAQ結(jié)合LC-MS/MS的方法鑒定了強(qiáng)弱毒菌株差異表達(dá)的細(xì)胞壁相關(guān)蛋白,并對(duì)鑒定到的潛在的毒力相關(guān)因子Sbp進(jìn)行了驗(yàn)證,評(píng)價(jià)了Sbp制備的亞單位疫苗對(duì)仔豬的免疫保護(hù)效果。在90年代初期,我國(guó)通過(guò)疫苗免疫等防控手段,使得豬丹毒疫情得到控制。如今豬丹毒這一“老病”新發(fā),也提出了許多問(wèn)題:是否現(xiàn)有疫苗預(yù)防不理想?或是臨床流行的紅斑丹毒絲菌耐藥性發(fā)生了變化?為了給當(dāng)前防控豬丹毒提供科學(xué)的用藥指導(dǎo),本研究對(duì)紅斑丹毒絲菌臨床分離株進(jìn)行了21種常用藥物的耐藥譜檢測(cè)。并進(jìn)一步對(duì)紅斑丹毒絲菌耐喹諾酮藥物的機(jī)制進(jìn)行了初步探索。本研究的主要成果總結(jié)如下:1.紅斑丹毒絲菌比較蛋白質(zhì)組學(xué)研究本研究以紅斑丹毒絲菌的強(qiáng)毒菌株和弱毒菌株的細(xì)胞壁相關(guān)蛋白為研究對(duì)象,利用i TRAQ結(jié)合LC-MS/MS的方法鑒定差異表達(dá)蛋白,共鑒定到100個(gè)差異表達(dá)的細(xì)胞壁相關(guān)蛋白。強(qiáng)毒菌株中高表達(dá)蛋白為57個(gè),主要為ABC轉(zhuǎn)運(yùn)蛋白和粘附相關(guān)蛋白;低表達(dá)蛋白為43個(gè),主要為壓力反應(yīng)蛋白。強(qiáng)毒菌株中高表達(dá)的蛋白可能跟細(xì)菌毒力相關(guān),本研究選取強(qiáng)毒菌株中高表達(dá)的部分蛋白,利用原核表達(dá)系統(tǒng)表達(dá)重組蛋白,純化后免疫小鼠制備多克隆抗體,通過(guò)Western Blot檢測(cè)其在強(qiáng)弱毒菌株中的實(shí)際表達(dá)情況,其表達(dá)情況與i TRAQ結(jié)果一致。對(duì)強(qiáng)毒菌株高表達(dá)的蛋白進(jìn)行分析,發(fā)現(xiàn)糖轉(zhuǎn)運(yùn)蛋白Sbp在強(qiáng)毒菌株中表達(dá)量為弱毒菌株的1.73倍。Sbp蛋白被報(bào)道參與細(xì)菌雙精氨酸轉(zhuǎn)位通路,是一些細(xì)菌重要的毒力因子。為了驗(yàn)證Sbp在紅斑丹毒絲菌致病過(guò)程中發(fā)揮的作用,本研究利用同源重組原理構(gòu)建了sbp基因缺失突變菌株。sbp基因缺失后,紅斑丹毒絲菌粘附侵入豬髖動(dòng)脈內(nèi)皮細(xì)胞(PIEC)的能力顯著下降,其在健康豬全血中的存活率也顯著下降,同時(shí)其抵抗小鼠吞噬細(xì)胞吞噬的能力也降低。動(dòng)物感染實(shí)驗(yàn)結(jié)果顯示:與野生菌相比,sbp基因缺失菌株對(duì)豬的致病力下降。這些結(jié)果表明Sbp是紅斑丹毒絲菌重要的毒力相關(guān)因子。同時(shí),病原菌表面的毒力相關(guān)蛋白通常也是其重要的保護(hù)性抗原,所以本研究利用仔豬模型評(píng)價(jià)Sbp蛋白的保護(hù)效力。在本研究中,純化的重組Sbp蛋白能夠誘導(dǎo)仔豬產(chǎn)生高水平的抗體,并保護(hù)66.7%的仔豬抵抗致死劑量的紅斑丹毒絲菌人工感染。抗體跟蹤檢測(cè)表明Sbp誘導(dǎo)的抗體可持續(xù)最少6個(gè)月。結(jié)果表明Sbp是一個(gè)有效的疫苗候選蛋白。2.紅斑丹毒絲菌耐喹諾酮藥物機(jī)制初探本研究用藥敏紙片法檢測(cè)了紅斑丹毒絲菌臨床分離株對(duì)21種常用藥物的耐藥譜,旨在了解當(dāng)前流行株的耐藥情況,為臨床用藥提供參考。進(jìn)一步利用E-test藥敏紙條檢測(cè)臨床分離株對(duì)環(huán)丙沙星和左旋氧氟沙星(喹諾酮類藥物)的MIC值,根據(jù)CLSI標(biāo)準(zhǔn),判定臨床分離的紅斑丹毒絲菌對(duì)環(huán)丙沙星的耐藥率為48.8%,且不同菌株之間對(duì)環(huán)丙沙星藥物的MIC值呈現(xiàn)多樣性。進(jìn)一步通過(guò)測(cè)序分析喹諾酮耐藥相關(guān)基因DNA解旋酶(Gyr A、Gyr B)和拓?fù)洚悩?gòu)酶Ⅳ(Par C、Par E)的突變情況,結(jié)合各菌株對(duì)環(huán)丙沙星藥物的MIC,發(fā)現(xiàn)了2個(gè)耐藥相關(guān)基因突變位點(diǎn):Gyr A(90D→N)和Par C(81 S→I)。進(jìn)一步對(duì)這兩個(gè)位點(diǎn)進(jìn)行了定向點(diǎn)突變實(shí)驗(yàn),結(jié)果發(fā)現(xiàn)Gyr A90位點(diǎn)的突變能使紅斑丹毒絲菌對(duì)環(huán)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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.6

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