有氧胁迫下幽门螺杆菌球形变异相关蛋白的鉴定及功能研究

发布时间：2018-06-19 15:19

本文选题：幽门螺杆菌 + 球形体　；参考：《第三军医大学》2006年博士论文

【摘要】： 幽门螺杆菌(Helicobacter pylori, Hp)是人类慢性活动性胃炎和消化溃疡的重要原因,与胃癌和胃淋巴样组织淋巴瘤的发生也极度相关。目前人类对其生物学特点、致病机制及其与宿主的关系等方面均尚未完全阐明。随着对Hp在分子水平上的研究不断深入,人们发现Hp较其它细菌表现出更高的生物多样性,提示该菌面对复杂的定居环境,具有高度的变异性和独特的适应性机制。大量研究发现,Hp在各种不利因素的作用下可发生形态变异,由螺旋体转变为球形体。Hp作为一种专性微需氧菌,在有氧胁迫下更容易发生适应性变异,转变为一种有活力但不能培养成活(viable but noculturable stage,VNC)的Hp球形变异体,其在Hp感染的传播途径及其相关消化系统疾病的复发中可能发挥着重要作用。研究Hp在有氧胁迫下发生适应性球形变异的变异特征及其变异本质,解析Hp的适应性调节元件,将为揭示Hp可能的传播途径、阐明Hp的适应性变异机制,以及创新性地开展基因工程改造Hp菌株耐氧培养的研究提供有力的实验依据。Hp菌26695株和J99株全基因组测序工作的完成,为后续的蛋白质组学研究提供了条件。近几年,国内外研究者采用蛋白质组学技术相继在Hp的遗传变异、生理、免疫靶点筛选及毒力因子确定等方面进行了一系列的研究,而在有氧胁迫下Hp发生适应性球形变异方面却未见相关研究报道。本研究选择Hp临床分离株,采用高氧浓度的培养条件,有氧胁迫Hp发生球形变异,建立稳定的球形体模型,进而对Hp球形体的超微结构、菌体代谢、酶活特性、毒力因子mRNA水平表达差异、体内外回复定植力等变异特征进行实验研究。同时,采用目前蛋白质组学研究中的核心技术———双向电泳,对适应性变异前后的Hp螺旋体和球形体进行全菌蛋白质表达谱的比较分析,再通过MALDI-TOF质谱技术对差异蛋白进行初步鉴定分析,最后通过基因克隆技术构建差异蛋白相关基因的Hp高表达菌株,验证该基因在有氧胁迫条件下对Hp氧耐受性的作用。具体研究结果如下:
[Abstract]:Helicobacter pylori (HP) is an important cause of chronic active gastritis and peptic ulcer, which is also closely related to the occurrence of gastric cancer and lymphatic tissue lymphoma. At present, the biological characteristics, pathogenetic mechanism and its relationship with the host have not been fully elucidated. With the further study of HP at molecular level, it has been found that HP exhibits higher biodiversity than other bacteria, suggesting that HP has a high degree of variability and unique adaptive mechanism in the face of complex settlement environment. A large number of studies have found that the morphological variation of HP can occur under the action of various unfavorable factors, from spirulina to spheroid.Hp, as a specific microaerobic bacterium, is more susceptible to adaptive variation under aerobic stress. It may play an important role in the transmission of HP infection and the recurrence of digestive diseases associated with HP infection. To study the variation characteristics and nature of adaptive spherical variation of HP under aerobic stress, to analyze the adaptive regulatory elements of HP, to reveal the possible transmission pathway of HP, and to clarify the adaptive variation mechanism of HP. And the innovative research on the oxygen tolerance culture of genetically engineered HP strain provides a powerful experimental basis. The complete genome sequencing of 26695 and J99 strains of HP strain provides the conditions for further proteomics research. In recent years, researchers at home and abroad have carried out a series of studies on the genetic variation, physiology, immune target screening and virulence factor determination of HP by proteomics. However, there were no related studies on the adaptive globular variation of HP under aerobic stress. In this study, the clinical isolates of HP were selected. Under the condition of high oxygen concentration, the spherical variation of HP was observed under aerobic stress, and a stable spheroidy model was established, and the ultrastructure, cell metabolism and enzyme activity of HP spheroidium were further studied. The expression of virulence factor mRNA and the ability of colonization in vivo and in vitro were studied experimentally. At the same time, the protein expression profiles of Helicobacter pylori and spheroids before and after adaptive mutation were compared and analyzed by two-dimensional electrophoresis, which is the core technique in proteomics research. The differential protein was identified and analyzed by MALDI-TOF mass spectrometry. Finally, the high expression strain of the differential protein related gene was constructed by gene cloning technique to verify the effect of the gene on the oxygen tolerance of HP under aerobic stress. The specific findings are as follows:
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R378

【引证文献】