结核分枝杆菌Lsr2蛋白的功能及其调控研究
发布时间：2018-11-13 07:17
【摘要】：结核病是一种由结核分枝杆菌引起的传染病。Lsr2(Rv3597c)编码一种结核分枝杆菌的重要抗原,担负类似“组蛋白”的作用。已有的研究表明Lsr2与结核分枝杆菌的多种基因调控功密切相关,但目前关于它是如何被调控及相关的调控蛋白等尚未鉴定。在本研究中筛选到了与Lsr2相互作用的两个蛋白Rv2250A(假想黄素蛋白)与Rv3463(未知功能蛋白),进一步鉴定了它们之间功能性的相互作用,主要取得了以下研究结果： (1)采用细菌双杂交技术探测到了蛋白Rv2250A与Rv3463和Lsr2蛋白的之间能够发生相互作用；采用Pull-Down技术证实体外这些蛋白之间确实存在物理相互作用。(2)采用质粒DNA-TopA松弛螺旋实验发现蛋白Rv2250A与Rv3463能够促进Lsr2蛋白对TopA酶活性的抑制作用；采用琼脂糖迁移实验发现蛋白Rv2250A与Rv3463能够与Lsr2蛋白形成复合体,产生滞后带,进一步确定蛋白Rv2250A和Rv3463与Lsr2蛋白之间能够相互作用,促进Lsr2蛋白与质粒DNA的结合。(3)在耻垢分枝杆菌中,证明了蛋白Rv3463(耻垢分枝杆菌同源蛋白MSMEG_1514)与Rv2250A(耻垢分枝杆菌同源蛋白MSMEG_4334)与Lsr2(MSMEG_6092)的同源蛋白之间的也存在相互作用。采用质粒DNA-TopA松弛螺旋实验发现蛋白MSMEG_4334与MSMEG_1514能够促进Lsr2蛋白抑制TopA松弛螺旋酶的活性；采用Co-IP技术证实了这些同源蛋白和Lsr2蛋白在耻垢分枝杆菌体内能够发生相互作用。(4)通过随机突变鉴定了MSMEG_4334与MSMEG_1514中与Lsr2蛋白发生相互作用的必需氨基酸残基,鉴定了关键的蛋白相互作用位点。 Lsr2蛋白已经证实参与结核分枝杆菌的几个重要代谢途径,其中包括抗生素诱导基因和相关的诱导多耐药性的基因的调控。本研究发现和鉴定了Rv2250A与Rv3463能够与Lsr2蛋白的相互作用,并且证实了这种相互作用对于拓扑异构酶活性的调控影响。因此,本研究成果为进一步揭示lsr2多样化的调控功能提供了重要线索。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
本文编号：2328393
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
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1 何杨;结核分枝杆菌Lsr2蛋白的功能及其调控研究[D];华中农业大学;2010年
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