大肠埃希氏菌外膜蛋白OmpW抗先天性免疫研究

发布时间：2018-06-25 21:30

本文选题：Red重组 + OmpW　；参考：《浙江理工大学》2012年硕士论文

【摘要】：根据革兰氏染色我们将细菌分为革兰氏阳性细菌和革兰氏阴性细菌。革兰氏阳性细菌的外壁主要为肽聚糖，其蛋白质含量是很低的；但是革兰氏阴性细菌的外壁比较复杂，，除细胞质膜外还包括周质空间和外膜。历史上爆发的许多大规模传染病都是由革兰氏阴性菌引起的。先天性免疫是人和动物抗微生物感染的重要防御机制之一。它主要包括皮肤、粘膜和胎盘的屏障作用；吞噬细胞的吞噬作用；补体对微生物的杀伤作用。同时，细菌经过长期的选择进化也产生了针对人和动物先天性免疫作用的对抗机制。最近几年，人们对革兰氏阴性菌的外膜和外膜蛋白进行了较多的研究，发现其具有抵抗人体杀菌的功能。其中，外膜蛋白OmpW广泛存在于革兰氏阴性菌中，由8条β折叠链组成，形成一个长长的窄的疏水通道。OmpW参与细菌的多种抗性，包括耐药性、对盐浓度的耐受性等，并且OmpW的表达变化与一些疾病有关，如炎症性肠病、反应性关节炎等。由此可见，OmpW对于细菌抵抗外界环境起着重要的作用。正是由于OmpW的多种重要功能，运用Red重组技术构建了大肠埃希氏菌OmpW敲除菌（ΔOmpW），通过比较正常菌K12和ΔOmpW的抗巨噬细胞吞噬和抗血浆补体的杀菌作用来研究OmpW在抗先天性免疫中的作用。体内和体外实验均表明ΔOmpW易被巨噬细胞吞噬，且易被血浆补体杀灭。通过提取正常菌和巨噬细胞内存活菌的膜蛋白经SDS-PAGE分析发现OmpW的表达量升高。因此，认为OmpW在细菌抵抗宿主的先天性免疫过程中起着重要的作用。小鼠的主动保护实验是对小鼠进行OmpW免疫，对免疫组小鼠和未免疫组小鼠注射正常菌K12。结果免疫组小鼠都存活，未免疫组小鼠都死亡。说明针对OmpW的抗体能保护小鼠免受细菌的感染。也从另一角度反映了OmpW对于抵抗宿主免疫的重要性。本研究初步揭示了OmpW的抗先天性免疫功能，为进一步研究OmpW在抗吞噬和抗补体杀菌过程中的作用机制奠定基础，对感染性疾病的控制、治疗具有重要的理论和实践意义。
[Abstract]:According to Gram staining, bacteria are divided into Gram-positive bacteria and Gram-negative bacteria. The outer wall of Gram-positive bacteria is mainly peptidoglycan, and its protein content is very low, but the outer wall of Gram-negative bacteria is more complicated, including the periplasmic space and outer membrane in addition to the cytoplasmic membrane. Many large-scale infectious diseases in history have been caused by gram-negative bacteria. Innate immunity is one of the important defense mechanisms against microbial infection in humans and animals. It mainly includes the barrier of skin, mucosa and placenta, phagocytosis of phagocytes and the killing effect of complement on microorganism. At the same time, bacteria have evolved over a long period of time to produce a mechanism of antagonism against the innate immunity of humans and animals. In recent years, many studies have been carried out on the outer membrane and outer membrane proteins of Gram-negative bacteria. OmpW, an outer membrane protein, is widely found in Gram-negative bacteria and consists of eight 尾 -fold chains, forming a long, narrow hydrophobic channel. OmpW is involved in a variety of bacterial resistance, including drug resistance, tolerance to salt concentration, and so on. And the expression of OmpW is related to some diseases, such as inflammatory bowel disease, reactive arthritis and so on. It can be seen that OmpW plays an important role in bacterial resistance to the external environment. Because of the many important functions of OmpW, Using Red recombination technique, the OmpW knockout bacteria of Escherichia coli (螖 OmpW) were constructed. By comparing the anti-macrophage phagocytosis of normal bacteria K12 and 螖 OmpW and the bactericidal effect of anti-plasma complement, the role of OmpW in anti-congenital immunity was studied. In vivo and in vitro experiments showed that 螖 OmpW was easily phagocytized by macrophages and easily killed by plasma complement. The expression of OmpW was increased by SDS-PAGE analysis of membrane protein extracted from normal bacteria and macrophage survival bacteria. Therefore, OmpW plays an important role in bacterial innate immunity against host. Mice were immunized with OmpW, and K12 was injected into immunized and unimmunized mice. Results all the immunized mice survived and the unimmunized mice died. It showed that the antibody against OmpW could protect mice from bacterial infection. It also reflects the importance of OmpW in resisting host immunity. This study initially revealed the anti-congenital immune function of OmpW, which laid a foundation for further study of the mechanism of OmpW in the process of anti-phagocytosis and anti-complement sterilization, and had important theoretical and practical significance for the control and treatment of infectious diseases.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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