大肠杆菌EF-Tu多蛋白复合物诱导THP-1细胞免疫应答的研究

发布时间：2018-04-05 02:02

本文选题：EF-Tu　切入点：OmpA　出处：《厦门大学》2008年硕士论文

【摘要】： 细菌延伸因子Tu(elongation factor Tu,EF-Tu)是一种高丰度蛋白,在蛋白质生物合成的延伸过程中起到了关键性的作用。近来研究发现EF-Tu可以作为一种新的病原相关分子模式(Pathogen-associated molecular patterns,PAMPs)分子而被宿主识别。作为一个具有高度保守性的细菌蛋白质,它是如何被宿主细胞所特异性地识别并进而引发宿主的哪些免疫应答反应。这无疑具有着非常重要的生物学意义。本实验首先采用分子克隆和抗体制备技术,以模式生物大肠杆菌K12为实验材料从其基因组中克隆出细菌延伸因子EF-Tu并制备其抗体。然后在本实验室之前研究EF-Tu多蛋白复合物的基础上表达并纯化了EF-Tu多蛋白复合物中OmpA、EF-Tu和OmpF三个蛋白组份。接着采用Western blotting和激光共聚焦证明了OmpA、EF-Tu和OmpF三个蛋白与人单核巨噬细胞THP-1有相互作用并被细胞膜受体识别而定位于细胞膜上。最后运用酶联反应吸附试验技术对EF-Tu多蛋白复合物所引发人单核巨噬细胞THP-1细胞的免疫反应进行研究,发现EF-Tu多蛋白复合物中各蛋白组份可以明显促使THP-1细胞细胞因子IL-Iβ、IL-8、IL-10、IL-12和TNF-α分泌量的增多。同时应用流式细胞术发现EF-Tu多蛋白复合物中各蛋白组份可以显著提高人单核巨噬细胞THP-1细胞的噬菌能力并具有剂量依赖效应。上述研究首次发现了EF-Tu多蛋白复合体可以作为一种新型病原相关分子模式通过细胞膜受体而被宿主免疫细胞所识别。阐明了其与人单核巨噬细胞THP-1细胞所引起的免疫学反应。这些研究结果对深入阐明EF-Tu病原相关分子模式的生物学功能具有主要作用。
[Abstract]:Bacterial extension factor (Tu(elongation factor Tu-EF-TuS) is a high abundance protein, which plays a key role in the extension of protein biosynthesis.Recently, it has been found that EF-Tu can be recognized by the host as a new pathogen-associated molecular pattern pattern (PAMPs) molecule.As a highly conserved bacterial protein, how is it specifically recognized by host cells and then triggered by host immune responses.There is no doubt that this has very important biological significance.In this experiment, the bacterial extension factor (EF-Tu) was cloned from the genome of Escherichia coli K12 and its antibody was prepared by molecular cloning and antibody preparation techniques.Three protein components of EF-Tu polyprotein complex OmpAF-Tu and OmpF were expressed and purified on the basis of the previous study in our laboratory.Then, Western blotting and laser confocal technique were used to demonstrate that OMP EF-Tu and OmpF interact with human mononuclear macrophage THP-1 and are recognized by cell membrane receptor and located on the cell membrane.Finally, the immunoreaction of human mononuclear macrophage THP-1 cells induced by EF-Tu polyprotein complex was studied by enzyme linked reaction adsorption assay (Elisa).It was found that the protein components of EF-Tu polyprotein complex could significantly increase the secretion of IL-10 IL-12 and TNF- 伪 in the THP-1 cytokines IL-I 尾 -IL-8, IL-10, IL-12 and TNF- 伪.At the same time, it was found by flow cytometry that each protein component of EF-Tu polyprotein complex could significantly improve the bacteriophage ability of human mononuclear macrophages THP-1 cells and had a dose-dependent effect.These studies have shown for the first time that EF-Tu polyprotein complexes can be recognized by host immune cells as a novel pathogen-associated molecular pattern through cell membrane receptors.The immunological reaction induced by human mononuclear macrophage THP-1 cells was elucidated.These results play an important role in elucidating the biological functions of EF-Tu pathogen-associated molecular models.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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