牛分枝杆菌BCG菌株3-甲基腺嘌呤糖基化酶AAG的调控机制研究

发布时间：2018-03-06 02:06

  本文选题：卡介苗　切入点：糖基化酶　出处：《华中农业大学》2014年硕士论文　论文类型：学位论文

【摘要】：碱基切除修复(BER)是生物体内广泛存在的一种DNA损伤修复机制,能够使机体有效应对各种内源或外源因素引发的碱基损伤。3-甲基腺嘌呤DNA糖基化酶AAG能够识别并切除受损碱基,在碱基切除修复系统中发挥重要作用。然而,作为引起结核病的结核分枝杆菌,其AAG的调控机制仍然不是很清楚。牛分枝杆菌BCG菌株(Bacillus Calmette Guerin,卡介苗菌株)在过去的七十多年里广泛作为预防结核病的疫苗,是对抗结核病的重要武器,同时它与病原菌结核分枝杆菌基因组具有高度同源性,因此被广泛作为研究结核分枝杆菌基因调控的模式菌株。本研究综合运用多种技术手段,重点研究了BCG菌株中AAG的双重调控机制,主要包括以下内容： (1)通过细菌单杂交筛选发现和鉴定了一个TetR家族的转录因子BCG0878c,并进一步用EMSA和ChIP实验证明BCG0878c能够直接结合牛分枝杆菌aag基因(mbaag)的启动子。运用实时定量PCR实验和半乳糖苷酶实验证明BCG0878c能够下调mbaag基因的表达,同时促进自身基因表达。用启动子分段截短EMSA实验和DNase Ⅰ足迹实验进一步鉴定了BCG0878c所识别的保守DNA序列。 (2)采用细菌双杂交、免疫共沉淀、表面等离子共振、pull-down等技术手段证明BCG0878c蛋白质能够与MbAAG相互作用。BCG0878c能够促进MbAAG结合受损DNA,同时亦能促进MbAAG对受损碱基的切割。而MbAAG反过来能够促进BCG0878c的DNA结合活性。 (3)在BCG中超表达mbaag和BCG0878c都会抑制BCG菌株生长,但是抑制程度有所不同。在亚硝酸胁迫下,mbaag表达上调,但是BCG0878c表达水平不变,而且损伤剂亚硝酸不能影响BCG0878c对启动子的结合。 本研究结果为转录因子的调控机制提供了新的视角,同时增加了我们对分枝杆菌糖基化酶AAG调控的理解,填补了相关基础研究领域的空白。
[Abstract]:Base excision repair (ber) is a widespread mechanism of DNA damage repair in organisms, which can effectively respond to the base damage caused by various endogenous or exogenous factors. AAG can recognize and remove the damaged bases. Plays an important role in the base excision repair system. However, as Mycobacterium tuberculosis, the cause of tuberculosis, The regulatory mechanism of its AAG is still unclear. The BCG strain Bacillus Calmette Guerin (Bacillus Calmette Guerin) has been widely used as a vaccine against tuberculosis in the past 70 years and is an important weapon against tuberculosis. At the same time, it has high homology with the pathogen Mycobacterium tuberculosis genome, so it is widely used as a model strain to study the gene regulation of Mycobacterium tuberculosis. The double regulation mechanism of AAG in BCG strain was studied, including the following contents:. (1) A transcription factor BCG0878cof TetR family was identified by single hybridization screening, and the promoter of aag gene of Mycobacterium bovis was proved by EMSA and ChIP experiments. Galactosidase assay showed that BCG0878c could down-regulate the expression of mbaag gene. At the same time, the promoter truncated EMSA test and DNase 鈪，

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