大肠杆菌EF-Tu多蛋白复合体的研究

发布时间：2018-06-12 09:51

本文选题：EF-Tu + 多蛋白复合物　；参考：《厦门大学》2007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。近年来,EF-Tu却是作为一种新发现的病原相关分子模式(Pathogen-associated molecular patterns, PAMPs)分子而受到高度重视。作为一个具有高度保守性的菌细胞内的蛋白质,它是如何从细胞内跨膜运动到细胞外,并且被宿主细胞特异性地识别进而引发宿主的免疫反应的。同时,在此过程中又有哪些膜蛋白起了重要的作用。这无疑具有着相当重要的生物学意义。试验首先采用Ni-NTA柱纯化和生物质谱技术,以模式大肠杆菌为试验材料,发现了内膜蛋白MdtE、外膜蛋白OmpF和具有4个跨膜片段的外膜蛋白OmpA与浆蛋白EF-Tu之间存在着蛋白质相互作用,形成了1个多蛋白复合体,被命名为EF-Tu多蛋白复合体。同时采用Western blotting、Far-Western blotting和免疫共沉淀等技术,充分证实了上述结论。并提出了该多蛋白质复合体的分子结构模型。在此基础上,研究这4种蛋白在胞浆中的结合情况。结果发现,他们以EF-Tu·OmpA,OmpF·OmpA和MdtE·OmpA 3种两两结合的形式存在。进一步探讨该复合体在细胞分泌性蛋白中的状态,发现该多蛋白质复合体缺少了MdtE,而以EF-Tu、OmpA和OmpF蛋白复合体形式分泌至胞外。最后,对EF-Tu多蛋白复合体与人单核/巨噬细胞结合情况进行了研究,发现EF-Tu、OmpA和OmpF仍然以一个多蛋白复合体的形式和细胞发生了相互作用,其中EF-Tu和OmpA为直接结合分子。这些结果提示OmpA和OmpF在EF-Tu被宿主细胞识别和引起免疫反应的过程中发挥了重要作用。上述研究首次发现了EF-Tu多蛋白复合体,揭示了该复合体从胞浆至胞外的形成和运输特征,阐明了其与动物细胞作用形式。这些研究结果对深入了解EF-Tu病原相关分子模式的生物学功能具有重要作用。
[Abstract]:The bacterial elongation factor Tub (EF-TuS) is a high abundance protein, which plays a key role in the extension of protein biosynthesis. Recently, EF-Tu has attracted much attention as a novel pathogen-associated molecular pattern (PAMPs) molecule. As a highly conserved intracellular protein, it moves from the intracellular membrane to the outside of the cell and is specifically recognized by the host cell to trigger the host's immune response. At the same time, which membrane proteins play an important role in this process. There is no doubt that this is of great biological significance. Firstly, Ni-NTA column purification and biomass spectrometry were used in the experiment, and the model Escherichia coli was used as the experimental material. The protein interaction between membrane protein MdtE, outer membrane protein OmpF and outer membrane protein OmpA with four transmembrane fragments and plasma protein EF-Tu was found, resulting in the formation of a polyprotein complex named EF-Tu polyprotein complex. The results were confirmed by Western blotting Far-Western blotting and immunoprecipitation. The molecular structure model of the polyprotein complex was proposed. On this basis, the binding of these four proteins in the cytoplasm was studied. The results showed that they existed in the form of EF-Tu OmpF OmpA and MdtE OmpA. Further study of the state of the complex in cell secretory protein showed that the polyprotein complex lacked MdtE, but secreted to the extracellular in the form of EF-Tu OmpA and OmpF protein complex. Finally, the binding of EF-Tu polyprotein complex to human mononuclear / macrophage was studied. It was found that EF-Tu and OmpF still interacted with cells in the form of a polyprotein complex, in which EF-Tu and OmpA were direct binding molecules. These results suggest that OmpA and OmpF play an important role in the recognition and immune response of EF-Tu by host cells. The interaction with animal cells was elucidated. These results play an important role in understanding the biological functions of EF-Tu related molecular models.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

【引证文献】