以酿酒酵母为替代宿主研究嗜肺军团菌效应蛋白的功能

发布时间：2018-01-31 06:03

本文关键词： 嗜肺军团菌效应蛋白酿酒酵母 LegS2 线粒体定位　出处：《中山大学》2011年硕士论文　论文类型：学位论文

【摘要】：嗜肺军团菌是军团菌病(Legionnaires' disease)的病原,目前已成为胞内病原菌-宿主相互作用研究的理想模型。嗜肺军团菌实现胞内增殖的关键在于吞噬泡中的军团菌能通过IV(B)型分泌系统分泌大量的效应蛋白,改变巨噬细胞的内吞途径,阻止溶酶体与吞噬泡的融合,使自己免于被消化,并通过抑制巨噬细胞的蛋白合成和凋亡等,实现胞内的增殖和扩散。近年来,多项研究致力于揭示嗜肺军团菌效应蛋白的功能,但由于大部分效应蛋白功能冗余并且缺乏有效的检测手段,目前对这些效应蛋白的功能还知之甚少。酿酒酵母(Saccharomyces cerevisiae)是具有类似许多真核生物中保守的基础的生化和细胞生命过程的一种单细胞生物,其易于培养、遗传背景清楚和基因操作方便等特点,使之成为分子遗传学和细胞生物学等研究的经典模型。目前的研究发现,许多效应蛋白在酵母胞内的表达都可引起酵母细胞表型的改变,而这些蛋白的胞内定位也为了解其功能提供有益的线索。另外,作为研究蛋白-蛋白相互作用技术方案之一的酿酒酵母多拷贝校正筛选系统也可以在筛选和鉴定军团菌效应蛋白相应的宿主靶标蛋白的研究中提供新的途径。在本论文中,我们选择了几个军团菌效应蛋白Ceg20 (即lpg1137)、LegK1 (即lpg1483)、RalF (即lpg1950)和LegS2 (即lpg2176),通过相关的技术方案来研究这些蛋白的胞内定位和在酿酒酵母细胞中表达的表型变化,发现了这些效应蛋白在酵母细胞中的表达均能引起酵母细胞不同程度的生长缺陷,并发现了定位于酵母细胞线粒体的效应蛋白LegS2,效应蛋白LegS2还能降低酵母细胞的耗氧量,提示该效应蛋白可能通过改变宿主线粒体的功能而起作用。以上结果为阐明这些效应蛋白在酿酒酵母细胞中的功能提供了有用的数据。另外,本文也对应用酵母多拷贝校正筛选系统来筛选军团菌效应蛋白的酵母靶标蛋白作了初步的尝试。
[Abstract]:Legionnaires pneumophila is the pathogen of Legionnaive 'disease. It has become an ideal model for the study of intracellular pathogen-host interaction. The key to the intracellular proliferation of Legionella pneumophila lies in the ability of phagocytic Legionella to pass through IVB. The secretory system secretes a large number of effector proteins. To change the endocytosis pathway of macrophages, to prevent the fusion of lysosome and phagocytic vesicles, to prevent themselves from being digested, and to realize the proliferation and diffusion of macrophages by inhibiting the protein synthesis and apoptosis of macrophages. Many studies have been devoted to revealing the function of Legionella pneumophila effector proteins, but because most of them are redundant and lack effective detection methods, little is known about the functions of these effector proteins. Saccharomyces cerevisiae is a single-celled organism that has a conserved basis of biochemical and cellular life processes similar to that of many eukaryotes. It is easy to culture, genetic background is clear and easy to operate, making it a classical model of molecular genetics and cell biology. The expression of many effector proteins in yeast cells can cause phenotypic changes in yeast cells, and the intracellular localization of these proteins also provides useful clues for understanding their functions. As one of the techniques to study protein-protein interaction, the Saccharomyces cerevisiae multi-copy correction screening system can also provide a new way to screen and identify the corresponding host target proteins of Legionella. In this paper. We have selected several Legionella effector protein Ceg20 (lpg1137) and LegK1 (lpg1483). RalF (i.e. lpg1950) and LegS2 (lpg2176). The intracellular localization and phenotypic changes of these proteins in Saccharomyces cerevisiae cells were studied by relevant technical schemes. It was found that the expression of these effector proteins in yeast cells could cause the growth defects of yeast cells to varying degrees, and the effector protein LegS2 located in yeast cell mitochondria was also found. The effector protein LegS2 can also reduce the oxygen consumption of yeast cells. These results provide useful data for elucidating the function of these effector proteins in Saccharomyces cerevisiae cells. The yeast target protein of Legionella effect protein was screened by yeast multiple copy correction screening system.
【学位授予单位】：中山大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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