四株宋内氏志贺菌的比较研究

发布时间：2018-03-15 06:05

本文选题：宋内氏志贺菌　切入点：生物学特性　出处：《西南大学》2011年硕士论文　论文类型：学位论文

【摘要】：志贺氏菌属(Shigella spp.)是一类能够通过侵袭肠上皮细胞引起患者典型性痢疾症状(发热、腹痛、腹泻)的食源性细菌。宋内氏志贺菌(S. sonnei)是痢疾流行的主要病原菌之一。由于各个地区环境不同,宋内氏志贺菌呈现很多差别。本课题通过四株来源于不同的志贺菌(S09071、S09072、S09076、S09077)的比较研究,对志贺菌致病临床防疫以及流行病学的研究提供一定的指导意义。通过毒力志贺氏菌大质粒缺失突变体库构建可以更好的研究毒力大质粒与染色体基因组之间的相互作用,也同时为志贺氏菌致病机理的研究打下基础。论文首先对四株宋内氏志贺菌(S. sonnei) S09071、S09072、S09076、S09077从形态、血清凝集反应、毒力评价、生长特征、生理生化特性、耐药性六个方面做了详细比较研究；其次我们做了毒力基因检测、双向电泳、全菌蛋白表达谱的比较,从分子的角度进一步比较研究了四株菌；最后对蛋白表达图谱的差异蛋白做了MALDI-TOF/TOF质谱鉴定。另一方面本论文还建立了志贺菌毒力大质粒大片段敲除的方法。通过这些研究,取得了以下主要结果： (1)四株宋内氏志贺菌形态呈直杆状,无芽胞、荚膜、鞭毛及菌毛,菌体表面形态凹凸不平,大小为0.5～0.7×1～2μm。菌落在LB培养基上呈大圆形、扁平、光滑湿润、无色、半透明、边缘较整齐,大小约为2.5 mm。四株菌菌体和菌落形态几乎无差别。 (2)生长曲线比较发现S09071生长最快,S09076、S09077其次,S09072菌株生长最慢。 (3)生化特性略有差别,S09076、S09077可以利用鼠李糖、L-岩糖、熊果甙、七叶灵发酵而S09071、S09072不能利用。 (4)耐药性比较发现S09071、S09072比S09076、S09077两株菌有更强的耐药性,其中S09072耐药性最强。 (5)毒力评价实验发现只有S09072具有上皮细胞侵袭能力,而S09071、S09076、S09077则没有侵袭力。 (6)毒力基因检测发现S09071、S09076、S09077部分毒力相关基因发生丢失；只有S09072毒力相关基因完整。 (7)通过比较蛋白质组学研究发现S09071、S09072全菌蛋白表达图谱比较一致,而S09076、S09077全菌蛋白表达图谱也趋于一致。S09071、S09072与S09076、S09077之间有48个明显的蛋白差异点,质谱鉴定得到47个分值有效的蛋白点,对应了43个蛋白,如GadA、GadB、OmpA、TatB等,这些蛋白在细菌代谢、毒力发挥以及耐受不同的环境等过程中扮演重要作用。 (8)成功建立了志贺氏菌毒力大质粒大片段敲除方法,为志贺菌毒力大质粒的功能以及志贺氏菌的致病机理研究奠定了基础。
[Abstract]:Shigella spp.is a group of typical dysentery symptoms (fever, abdominal pain) that can be caused by the invasion of intestinal epithelial cells. Shigella sonneiensis is one of the leading pathogens of dysentery. There are many differences in Shigella sonnei. A comparative study was conducted on four strains of Shigella s09071, S09072, S09076 and S09077, respectively. It can be used to study the interaction between virulence plasmids and chromosomal genomes by constructing large plasmid deletion mutants of Shigella virulent. It also lays a foundation for the study of the pathogenic mechanism of Shigella. In this paper, four strains of Shigella sonneiensis S09071C S09072C S09076N S09076S09077 were studied in detail in terms of morphology, serum agglutination reaction, virulence evaluation, growth characteristics, physiological and biochemical characteristics, and drug resistance. Two dimensional electrophoresis, the comparison of the whole bacterial protein expression profile, from the molecular point of view, further comparative study of four strains of bacteria; Finally, the differential protein of protein expression map was identified by MALDI-TOF/TOF mass spectrometry. On the other hand, the method of large plasmid fragment knockout of Shigella virulence was established. (1) the four strains of Shigella sonnei showed straight rod shape, no spores, capsule, flagella and fimbriae, and the surface morphology of the bacteria was uneven, and the size of the bacteria was 0. 5 0. 7 脳 1. 2 渭 m. The colony on LB medium was large, flat, smooth, moist, colorless and translucent. The edges were neat and the size was about 2.5 mm. there was almost no difference in the morphology of the four strains of bacteria and colony. The growth curve of S09071 was compared with that of S09071. The growth rate of S09071 was the fastest, and that of S09076-S09077 was the slowest. (3) the biochemical characteristics of S09076 and S09077 could be used to ferment rhamnose L-, arbutin and aescectin, but S09071 and S09072 could not be used. The drug resistance of S09071 / S09072 was stronger than that of S09076 / S09077, and the resistance of S09072 was the strongest. 5) virulence evaluation showed that only S09072 had the ability of invasion of epithelial cells, while S09071 and S09076 of S09076-S09077 had no invasiveness. The virulence genes of S09071and S09076S09076-S09077 were found to be lost, but only S09072 was intact. (7) through comparative proteomics studies, we found that the protein expression profiles of S09071 and S09072 were consistent, while that of S09076-S09077 was similar to that of S09071-S09072 and S09076-S09077.There were 48 distinct protein differences between S09071-S09072 and S09076-S09077, and 47 effective protein spots were identified by mass spectrometry. They correspond to 43 proteins, such as GadAl, GadBX, OmpAMP TatB, which play an important role in bacterial metabolism, virulence and tolerance to different environments. The large fragment knockout method of Shigella virulent plasmids was successfully established, which laid a foundation for the function of virulent plasmids of Shigella and the pathogenic mechanism of Shigella.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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