【摘要】：結核分枝桿菌(M tuberculosis)是一種傳染性很強的病原菌,能夠感染人畜引發(fā)結核病,通常為肺結核。結核分枝桿菌有極強的抗逆性,能夠長期潛伏在活體內,它們通過合成降解藥物蛋白(如內酰胺酶)、使藥物靶標發(fā)生突變等機制產生抗藥性。本論文研究在結核分枝桿菌中發(fā)現(xiàn)了Rv2752c同時兼有內酰胺酶活性和RNase活性等雙重功能,具體結果如下： (1)生物信息學分析表明,Rv2752c屬于金屬內酰胺酶家族中P-CASP亞家族,同時含有RMMBL結構域,可能兼有RNase和內酰胺酶的雙重功能；通過氨基酸序列比對發(fā)現(xiàn)Rv2752c與RNaseJ具有很高的同源性。(2)針對該基因通過系列點突變和缺失突變,表達純化得到了野生型和突變蛋白,發(fā)現(xiàn)Rv2752c同時具有內酰胺酶和RNase雙重活性。兩個殘基D184和H397被證實參與金屬離子的結合而且對于蛋白的雙重活性是必須的。C-末端的108個氨基酸殘基的缺失也導致Rv2752c的內酰胺酶和RNase活性的喪失。(3)通過細菌雙雜交篩選和PULL-DOWN證實Rv2752c能與Rv2373c相互作用,進一步分析發(fā)現(xiàn)Rv2373c能夠抑制Rv2752c的雙重活性。 這是第一次在結核分枝桿菌中發(fā)現(xiàn)并證實一個蛋白同時兼有內酰胺酶和RNase雙重功能,這不僅提高了我們對于結核分枝桿菌中mRNA的成熟加工與耐藥機制的理解,而且該研究對于闡明微生物的內酰胺酶的結構與功能及其與細菌抗藥之間的關系等具有重要意義。
[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.
【學位授予單位】：華中農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R378

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本文編號：2362874