鼠疫菌质粒间互作及其与致病性的关系研究

发布时间：2018-03-16 20:11

  本文选题：鼠疫耶尔森氏菌　切入点：质粒相互作用　出处：《安徽医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：鼠疫是由鼠疫耶尔森氏菌(以下简称鼠疫菌)引起的一种自然疫源性烈性传染病。在人类历史上,曾经发生过3次世界鼠疫大流行,给人类带来过深重灾难。新中国成立后，鼠疫疫情虽得到有效控制，但近年来，世界鼠疫疫情呈上升趋势，2000年世界卫生组织将鼠疫列为重新抬头的传染病。我国鼠疫自然疫源地分布广泛，动物鼠疫流行面积不断扩大，人间鼠疫病例不断增加。此外，鼠疫菌又是一种典型的生物战剂和生物恐怖剂。因此，对鼠疫菌致病机制的研究对国民健康、社会经济发展和国家安全具有重要意义。 鼠疫菌是由假结核耶尔森氏菌（以下简称假结核菌）O:1b血清型进化而来。典型的鼠疫菌含有3个毒力质粒（pMT1、pPCP1和pCD1），其中pCD1是从假结核菌遗传下来的固有质粒，而pMT1和pPCP1则是通过水平转移从外界获得的质粒。鼠疫菌引起严重的致死性疾病，而假结核菌只引起温和的肠道疾病，这说明新获得质粒与鼠疫菌的强致病性密切相关。那么，新获得质粒与固有质粒之间是否存在相互作用呢？若存在相互作用，其对致病性的影响又是如何呢？为了回答上述两个问题，本文基于田鼠型鼠疫菌201质粒缺失突变株，通过毒力分析、基因敲除和蛋白表达分析等策略，研究了质粒间相互作用及其与致病性的关系。 鼠疫菌201株，除含有pPCP1、pMT1、pCD1外，还含有一个隐性质粒pCRY。本研究基于鼠疫菌201株及其15个不同质粒缺失株，通过生存分析评价了它们的毒力。结果表明，pCD1质粒是鼠疫菌致病必不可少的，pMT1质粒对小鼠有一定的毒力，pPCP1和pCRY单独或共存时毒力很低。脏器存活分析结果显示，所有含pCD1质粒的鼠疫菌株pCD1+pMT1+pCRY+、pPCP1+pCD1+pMT1+、pPCP1+pCD1+pCRY+、pPCP1+pCD1+、pCD1+pCRY+、pCD1+在肝、脾脏器中能分离到活菌，但pCD1+pMT1+例外，这个结果表明pMT1可能对pCD1质粒具有抑制作用，而pPCP1或pCRY却能抵消pMT1的这种抑制作用。此后，我们结合SDS-PAGE电泳和质谱分析的方法，对pCD1质粒编码的三型分泌系统外膜蛋白（Yops）的分泌情况进行了测定，发现pCD1+pMT1+与其它含pCD1质粒的菌株相比，YopN和LcrV的表达明显受到抑制，，再次证明pMT1对pCD1质粒有抑制作用，而这种抑制作用会因其它质粒的存在而消失。 为了研究质粒间互作与致病性的关系，我们采用基因敲除的策略构建了pPCP1+pCD1+pMT1+pla、pPCP1+pMT1+pla、pCD1+pMT1+ymt和pMT1+ymt等基因缺失突变株。通过比较这些突变株与未突变株的毒力，证明在pCD1质粒缺失的情况下，鼠毒素起主要的毒力作用，同时pla具有降解鼠毒素的作用。pPCP1+pCD1+pMT1+pla与pPCP1+pCD1+pMT1+相比，在小鼠脏器中存活能力明显降低，说明了pPCP1促进鼠疫菌在宿主体内的感染能力主要由pla负担。pCRY除可能抵消pMT1的抑制作用外，其他功能尚不清楚，但pPCP1、pMT1和pCD1却是鼠疫菌保持完整毒力所必需的。
[Abstract]:Yersinia pestis is a natural infectious disease caused by Yersinia pestis. In human history, there have been three world plague pandemics, which have brought serious disasters to mankind. After the founding of New China, Although the plague epidemic situation has been effectively controlled, in recent years, the world plague epidemic situation is on the rise. In 2000, the World Health Organization listed the plague as a newly rising infectious disease. In China, the plague natural foci are widely distributed, and the epidemic area of animal plague is constantly expanding. In addition, Yersinia pestis is a typical biological warfare agent and bioterrorist agent. Therefore, the study of the pathogenic mechanism of Yersinia pestis is of great significance to national health, social and economic development and national security. Yersinia pestis evolved from the serotype O: 1b of pseudotuberculosis. Typical Yersinia pestis contains three virulent plasmids, pMT1, pPCP1 and pCD1, in which pCD1 is an inherent plasmid inherited from pseudotuberculosis. PMT1 and pPCP1 are plasmids obtained from outside through horizontal transfer. Yersinia pestis causes serious fatal diseases, while pseudotuberculosis causes mild intestinal diseases, which indicates that the newly obtained plasmids are closely related to the strong pathogenicity of Yersinia pestis. Is there any interaction between the newly obtained plasmids and the inherent plasmids? If there is interaction, how does it affect pathogenicity? In order to answer the above two questions, the relationship between plasmids interaction and pathogenicity was studied by virulence analysis, gene knockout and protein expression analysis based on plasmids deletion mutants of Yersinia pestis 201. 201 strains of Yersinia pestis, in addition to pPCP1, pMT1, pCD1, also contained a recessive plasmid pCRY. based on 201 strains of Yersinia pestis and 15 different plasmid deletion strains, The virulence of pCD1 plasmid was evaluated by survival analysis. The results showed that pCD1 plasmid was essential to Yersinia pestis. The virulence of pPCP1 plasmid to mice was very low when pPCP1 and pCRY co-existed, and the organ survival analysis showed that the virulence of pCD1 plasmid was very low. All Yersinia pestis strains containing pCD1 plasmids pCD1 pMT1 pCRY pPCP1 pCD1 pMT1 pPCP1 pCD1 pCRY pPCP1 pCD1 pCD1 pCRY pCD1 can be isolated from the organs of liver and spleen, except pCD1 pMT1. The results indicate that pMT1 may have inhibitory effect on pCD1 plasmid. However, pPCP1 or pCRY could counteract the inhibition of pMT1. After that, we determined the secretion of the outer membrane protein of the three type secretory system encoded by pCD1 plasmids by SDS-PAGE electrophoresis and mass spectrometry. It was found that the expression of YOPN and LcrV in pCD1 pMT1 was significantly inhibited compared with other strains containing pCD1 plasmids. It was proved that pMT1 had inhibitory effect on pCD1 plasmids, and this inhibition would disappear due to the existence of other plasmids. In order to study the relationship between plasmids interaction and pathogenicity, pPCP1 pCD1 pMT1 plapPCP1 pMT1 plapCD1 pMT1 ymt and pMT1 ymt were constructed by gene knockout strategy. The results showed that the murine toxin had the main virulence in the absence of pCD1 plasmid, and that pla could degrade the mouse toxin. Compared with pPCP1 pCD1 pMT1, the survival ability of pCD1 pMT1 pla was significantly lower than that of pPCP1 pCD1 pMT1. The results showed that the ability of pPCP1 to promote the infection of Yersinia pestis in the host was mainly affected by pla. The other functions were not clear except that pla might counteract the inhibitory effect of pMT1, but pPCP1, pMT1 and pCD1 were necessary for Yersinia pestis to maintain their complete virulence.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R516.8

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