【摘要】：結(jié)核病是一種由結(jié)核分枝桿菌引起的傳染病。Lsr2(Rv3597c)編碼一種結(jié)核分枝桿菌的重要抗原,擔(dān)負(fù)類似“組蛋白”的作用。已有的研究表明Lsr2與結(jié)核分枝桿菌的多種基因調(diào)控功密切相關(guān),但目前關(guān)于它是如何被調(diào)控及相關(guān)的調(diào)控蛋白等尚未鑒定。在本研究中篩選到了與Lsr2相互作用的兩個(gè)蛋白R(shí)v2250A(假想黃素蛋白)與Rv3463(未知功能蛋白),進(jìn)一步鑒定了它們之間功能性的相互作用,主要取得了以下研究結(jié)果： (1)采用細(xì)菌雙雜交技術(shù)探測(cè)到了蛋白R(shí)v2250A與Rv3463和Lsr2蛋白的之間能夠發(fā)生相互作用；采用Pull-Down技術(shù)證實(shí)體外這些蛋白之間確實(shí)存在物理相互作用。(2)采用質(zhì)粒DNA-TopA松弛螺旋實(shí)驗(yàn)發(fā)現(xiàn)蛋白R(shí)v2250A與Rv3463能夠促進(jìn)Lsr2蛋白對(duì)TopA酶活性的抑制作用；采用瓊脂糖遷移實(shí)驗(yàn)發(fā)現(xiàn)蛋白R(shí)v2250A與Rv3463能夠與Lsr2蛋白形成復(fù)合體,產(chǎn)生滯后帶,進(jìn)一步確定蛋白R(shí)v2250A和Rv3463與Lsr2蛋白之間能夠相互作用,促進(jìn)Lsr2蛋白與質(zhì)粒DNA的結(jié)合。(3)在恥垢分枝桿菌中,證明了蛋白R(shí)v3463(恥垢分枝桿菌同源蛋白MSMEG_1514)與Rv2250A(恥垢分枝桿菌同源蛋白MSMEG_4334)與Lsr2(MSMEG_6092)的同源蛋白之間的也存在相互作用。采用質(zhì)粒DNA-TopA松弛螺旋實(shí)驗(yàn)發(fā)現(xiàn)蛋白MSMEG_4334與MSMEG_1514能夠促進(jìn)Lsr2蛋白抑制TopA松弛螺旋酶的活性；采用Co-IP技術(shù)證實(shí)了這些同源蛋白和Lsr2蛋白在恥垢分枝桿菌體內(nèi)能夠發(fā)生相互作用。(4)通過隨機(jī)突變鑒定了MSMEG_4334與MSMEG_1514中與Lsr2蛋白發(fā)生相互作用的必需氨基酸殘基,鑒定了關(guān)鍵的蛋白相互作用位點(diǎn)。 Lsr2蛋白已經(jīng)證實(shí)參與結(jié)核分枝桿菌的幾個(gè)重要代謝途徑,其中包括抗生素誘導(dǎo)基因和相關(guān)的誘導(dǎo)多耐藥性的基因的調(diào)控。本研究發(fā)現(xiàn)和鑒定了Rv2250A與Rv3463能夠與Lsr2蛋白的相互作用,并且證實(shí)了這種相互作用對(duì)于拓?fù)洚悩?gòu)酶活性的調(diào)控影響。因此,本研究成果為進(jìn)一步揭示lsr2多樣化的調(diào)控功能提供了重要線索。
[Abstract]:Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Lsr2 (Rv3597c) encodes an important antigen of Mycobacterium tuberculosis and plays a role similar to histone. Previous studies have shown that Lsr2 is closely related to many genes of Mycobacterium tuberculosis, but how it is regulated and its related regulatory proteins have not been identified. In this study, two proteins Rv2250A (pseudoflavin protein) and Rv3463 (unknown functional protein) interacting with Lsr2 were screened, and their functional interactions were further identified. The main results are as follows: (1) the interaction between protein Rv2250A and Rv3463 and Lsr2 proteins was detected by bacterial two-hybrid technique. Pull-Down technique was used to confirm the physical interaction between these proteins in vitro. (2) plasmid DNA-TopA relaxation helix test showed that protein Rv2250A and Rv3463 could promote the inhibition of TopA enzyme activity by Lsr2 protein; Agarose migration assay showed that protein Rv2250A and Rv3463 could form complex with Lsr2 protein and produce hysteresis band. It was further confirmed that protein Rv2250A and Rv3463 could interact with Lsr2 protein. Promote the binding of Lsr2 protein to plasmid DNA. (3) in Mycobacterium smeareus, The interaction between protein Rv3463 (MSMEG_1514) and Rv2250A (MSMEG_4334) and Lsr2 (MSMEG_6092) homologous proteins was also demonstrated. Plasmid DNA-TopA relaxation helix assay showed that protein MSMEG_4334 and MSMEG_1514 could promote the inhibition of TopA relaxation helicase activity by Lsr2 protein. The interaction between these homologous proteins and Lsr2 proteins was confirmed by Co-IP technique. (4) the essential amino acid residues of MSMEG_4334 interacting with Lsr2 protein in MSMEG_1514 were identified by random mutation. The key protein interaction sites were identified. Lsr2 proteins have been shown to be involved in several important metabolic pathways of Mycobacterium tuberculosis, including the regulation of antibiotic induced genes and related genes that induce multidrug resistance. In this study, the interaction of Rv2250A and Rv3463 with Lsr2 protein was identified, and the effect of this interaction on topoisomerase activity was confirmed. Therefore, the results of this study provide an important clue for further revealing the diversified regulatory functions of lsr2.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378

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1 何楊;結(jié)核分枝桿菌Lsr2蛋白的功能及其調(diào)控研究[D];華中農(nóng)業(yè)大學(xué);2010年

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本文編號(hào)：2328393