本文選題：卡介苗　切入點(diǎn)：糖基化酶　出處：《華中農(nóng)業(yè)大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：堿基切除修復(fù)(BER)是生物體內(nèi)廣泛存在的一種DNA損傷修復(fù)機(jī)制,能夠使機(jī)體有效應(yīng)對(duì)各種內(nèi)源或外源因素引發(fā)的堿基損傷。3-甲基腺嘌呤DNA糖基化酶AAG能夠識(shí)別并切除受損堿基,在堿基切除修復(fù)系統(tǒng)中發(fā)揮重要作用。然而,作為引起結(jié)核病的結(jié)核分枝桿菌,其AAG的調(diào)控機(jī)制仍然不是很清楚。牛分枝桿菌BCG菌株(Bacillus Calmette Guerin,卡介苗菌株)在過(guò)去的七十多年里廣泛作為預(yù)防結(jié)核病的疫苗,是對(duì)抗結(jié)核病的重要武器,同時(shí)它與病原菌結(jié)核分枝桿菌基因組具有高度同源性,因此被廣泛作為研究結(jié)核分枝桿菌基因調(diào)控的模式菌株。本研究綜合運(yùn)用多種技術(shù)手段,重點(diǎn)研究了BCG菌株中AAG的雙重調(diào)控機(jī)制,主要包括以下內(nèi)容： (1)通過(guò)細(xì)菌單雜交篩選發(fā)現(xiàn)和鑒定了一個(gè)TetR家族的轉(zhuǎn)錄因子BCG0878c,并進(jìn)一步用EMSA和ChIP實(shí)驗(yàn)證明BCG0878c能夠直接結(jié)合牛分枝桿菌aag基因(mbaag)的啟動(dòng)子。運(yùn)用實(shí)時(shí)定量PCR實(shí)驗(yàn)和半乳糖苷酶實(shí)驗(yàn)證明BCG0878c能夠下調(diào)mbaag基因的表達(dá),同時(shí)促進(jìn)自身基因表達(dá)。用啟動(dòng)子分段截短EMSA實(shí)驗(yàn)和DNase Ⅰ足跡實(shí)驗(yàn)進(jìn)一步鑒定了BCG0878c所識(shí)別的保守DNA序列。 (2)采用細(xì)菌雙雜交、免疫共沉淀、表面等離子共振、pull-down等技術(shù)手段證明BCG0878c蛋白質(zhì)能夠與MbAAG相互作用。BCG0878c能夠促進(jìn)MbAAG結(jié)合受損DNA,同時(shí)亦能促進(jìn)MbAAG對(duì)受損堿基的切割。而MbAAG反過(guò)來(lái)能夠促進(jìn)BCG0878c的DNA結(jié)合活性。 (3)在BCG中超表達(dá)mbaag和BCG0878c都會(huì)抑制BCG菌株生長(zhǎng),但是抑制程度有所不同。在亞硝酸脅迫下,mbaag表達(dá)上調(diào),但是BCG0878c表達(dá)水平不變,而且損傷劑亞硝酸不能影響B(tài)CG0878c對(duì)啟動(dòng)子的結(jié)合。 本研究結(jié)果為轉(zhuǎn)錄因子的調(diào)控機(jī)制提供了新的視角,同時(shí)增加了我們對(duì)分枝桿菌糖基化酶AAG調(diào)控的理解,填補(bǔ)了相關(guān)基礎(chǔ)研究領(lǐng)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，

本文編號(hào)：1572849