结核分枝杆菌中金属内酰胺酶的功能鉴定及其与分子伴侣蛋白的相互作用研究
发布时间：2018-11-28 12:50
【摘要】：结核分枝杆菌(M tuberculosis)是一种传染性很强的病原菌,能够感染人畜引发结核病,通常为肺结核。结核分枝杆菌有极强的抗逆性,能够长期潜伏在活体内,它们通过合成降解药物蛋白(如内酰胺酶)、使药物靶标发生突变等机制产生抗药性。本论文研究在结核分枝杆菌中发现了Rv2752c同时兼有内酰胺酶活性和RNase活性等双重功能,具体结果如下： (1)生物信息学分析表明,Rv2752c属于金属内酰胺酶家族中P-CASP亚家族,同时含有RMMBL结构域,可能兼有RNase和内酰胺酶的双重功能；通过氨基酸序列比对发现Rv2752c与RNaseJ具有很高的同源性。(2)针对该基因通过系列点突变和缺失突变,表达纯化得到了野生型和突变蛋白,发现Rv2752c同时具有内酰胺酶和RNase双重活性。两个残基D184和H397被证实参与金属离子的结合而且对于蛋白的双重活性是必须的。C-末端的108个氨基酸残基的缺失也导致Rv2752c的内酰胺酶和RNase活性的丧失。(3)通过细菌双杂交筛选和PULL-DOWN证实Rv2752c能与Rv2373c相互作用,进一步分析发现Rv2373c能够抑制Rv2752c的双重活性。 这是第一次在结核分枝杆菌中发现并证实一个蛋白同时兼有内酰胺酶和RNase双重功能,这不仅提高了我们对于结核分枝杆菌中mRNA的成熟加工与耐药机制的理解,而且该研究对于阐明微生物的内酰胺酶的结构与功能及其与细菌抗药之间的关系等具有重要意义。
[Abstract]:Mycobacterium tuberculosis (M tuberculosis) is a highly infectious pathogen that can infect humans and animals and cause tuberculosis, usually tuberculosis. Mycobacterium tuberculosis has a strong resistance to stress and can lurk in vivo for a long time. By synthesizing and degrading drug proteins (such as lactamases), Mycobacterium tuberculosis causes drug resistance by mutagenesis of drug targets. In this paper, we found that Rv2752c has the dual functions of both lactamase activity and RNase activity in Mycobacterium tuberculosis. The results are as follows: (1) Bioinformatics analysis shows that Rv2752c belongs to the P-CASP subfamily of the metal lactamases family and contains the RMMBL domain, which may have the dual functions of RNase and lactamases. Amino acid sequence alignment showed that Rv2752c and RNaseJ had high homology. (2) the wild-type and mutant proteins were purified by a series of point mutations and deletion mutations. It was found that Rv2752c had double activities of both lactamases and RNase. The two residues D184 and H397 were confirmed to be involved in the binding of metal ions and necessary for the double activity of proteins. The loss of 108 amino acid residues at the C-terminal also resulted in the loss of Rv2752c lactamases and RNase activities. (3) Bacterial two-hybrid screening and PULL-DOWN confirmed that Rv2752c could interact with Rv2373c. Further analysis showed that Rv2373c could inhibit the double activity of Rv2752c. This is the first time that a protein has been found in Mycobacterium tuberculosis with both lactamase and RNase functions, which not only improves our understanding of the mechanism of mRNA maturation and drug resistance in Mycobacterium tuberculosis. This study is of great significance in elucidating the structure and function of microbial lactamases and the relationship between lactamases and bacterial resistance.
【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：2362874
[Abstract]:Mycobacterium tuberculosis (M tuberculosis) is a highly infectious pathogen that can infect humans and animals and cause tuberculosis, usually tuberculosis. Mycobacterium tuberculosis has a strong resistance to stress and can lurk in vivo for a long time. By synthesizing and degrading drug proteins (such as lactamases), Mycobacterium tuberculosis causes drug resistance by mutagenesis of drug targets. In this paper, we found that Rv2752c has the dual functions of both lactamase activity and RNase activity in Mycobacterium tuberculosis. The results are as follows: (1) Bioinformatics analysis shows that Rv2752c belongs to the P-CASP subfamily of the metal lactamases family and contains the RMMBL domain, which may have the dual functions of RNase and lactamases. Amino acid sequence alignment showed that Rv2752c and RNaseJ had high homology. (2) the wild-type and mutant proteins were purified by a series of point mutations and deletion mutations. It was found that Rv2752c had double activities of both lactamases and RNase. The two residues D184 and H397 were confirmed to be involved in the binding of metal ions and necessary for the double activity of proteins. The loss of 108 amino acid residues at the C-terminal also resulted in the loss of Rv2752c lactamases and RNase activities. (3) Bacterial two-hybrid screening and PULL-DOWN confirmed that Rv2752c could interact with Rv2373c. Further analysis showed that Rv2373c could inhibit the double activity of Rv2752c. This is the first time that a protein has been found in Mycobacterium tuberculosis with both lactamase and RNase functions, which not only improves our understanding of the mechanism of mRNA maturation and drug resistance in Mycobacterium tuberculosis. This study is of great significance in elucidating the structure and function of microbial lactamases and the relationship between lactamases and bacterial resistance.
【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
【参考文献】
相关期刊论文 前1条
1 郑卫;β-内酰胺酶及其抑制剂研究进展[J];国外医药(抗生素分册);2001年02期
，本文编号：2362874
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2362874.html
最近更新
教材专著
