本文選題：大環(huán)內酯 + rRNA��； 參考：《華中農業(yè)大學》2017年碩士論文

【摘要】：大環(huán)內酯類抗生素在臨床廣泛應用,主要用于治療革蘭氏陽性菌引起的感染,而細菌對大環(huán)內酯耐藥現(xiàn)象日益嚴重。甲基化酶erm基因是導致大環(huán)內酯高水平耐藥的重要機制之一。文獻認為erm催化23S rRNA 2058位腺嘌呤核苷酸的修飾,使其單甲基化或二甲基化。甲基化后的空間結構使細菌與大環(huán)內酯的結合位點被隱藏,阻斷了大環(huán)內酯等抗生素與細菌核糖體之間的結合,從而導致大環(huán)內酯、林可酰胺及鏈霉殺陽菌素耐藥。但是erm甲基化作用機制及其對細菌耐藥程度的貢獻在國際國內尚未見報道。本實驗室于臨床發(fā)病雛鴨腦和脾臟中分離的解沒食子酸鏈球菌巴氏亞種(S.gallolyticus subsp.pasteurianus)中發(fā)現(xiàn)耐藥基因ermB和ermT,且這兩種erm基因介導這些分離株大環(huán)內酯高水平耐藥。此外,我們還發(fā)現(xiàn)不同分離株中ermB單核苷酸多態(tài)性(Single Nucleotide Polymorphism,SNP)引起ermB突變,且其大環(huán)內酯MIC存在差異。為了探討erm甲基化作用機制及其對大環(huán)內酯耐藥的貢獻,本研究優(yōu)化了ermB和ermT表達體系,選取了ermB進行研究,并基于生物信息學分析及臨床分離株中的SNP構建了32種ermB突變體,首次利用微量稀釋法對這32種突變體進行篩選,選取了六個突變體,分別檢測了它們對23S rRNA的甲基化活性和對大環(huán)內酯及林可酰胺的最小抑菌濃度(MIC)。研究結果如下:1、erm B、ermT甲基化酶表達載體的構建與純化及23S rRNA的分離純化將ermT耐藥基因分別構建到pET28a、pET15b載體上,ermB耐藥基因分別構建到pET21b、pET28a載體上,比較蛋白分離純化結果,選擇ermB、ermT構建至pET28a載體的克隆,利用親和層析純化出ErmB、ErmT蛋白、MBP-MS2蛋白以及S.gallolyticus subsp.pasteurianus 23S r RNA。2、erm B、ermT的甲基化活性比較用3H標記S-腺苷甲硫氨酸為甲基供體,23S r RNA為底物,對ErmB、ErmT進行酶促動力學試驗。兩個蛋白的甲基化活性通過液閃儀檢測轉移到23S rRNA上3H的放射性強度完成。試驗結果表明,ErmB和ErmT均可以甲基化修飾S.gallolyticus subsp.pasteurianus 23S r RNA,但ermB甲基化活性要低于ermT。3、erm B突變體構建結合實驗室前期解沒食子酸鏈球菌分離株中ermB的SNP,我們選取ermB進行研究。運用生物信息學方法分析erm及ermB同源序列,對ermB保守區(qū)域部分氨基酸殘基進行突變,構建了32個突變體。4、erm B、ermB突變體MIC值檢測粗篩對ermB及其32個突變體進行MIC值檢測。結果表明,與野生型ermB的MIC值相比,32株突變體中對紅霉素MIC降低的突變體包括:突變菌株40(K40A)、69-2(E69D)、83-1(D83A)、139(R139A);對林可酰胺MIC降低的突變體包括:突變菌株40(K40A)、69-2(E69D)、116-1(E116A)、139(R139A);對克拉霉素MIC降低的突變體包括:突變菌株58-1(E58A)、58-2(E58D)、60-2(E69D)、83-1(D83A)、139(R139A)、165(K165A)。5、微量稀釋法測定6株突變體的MIC值通過ermB、ermB突變體MIC值檢測粗篩選擇6株ermB突變體進行精確MIC值檢測,檢測結果顯示,突變菌株83-1(D83A)對紅霉素、克拉霉素、林可酰胺的MIC值分別為:512μg/mL、64μg/mL、512μg/mL;突變菌株139139(R139A)對三種抗生素的MIC值分別為:512μg/mL、64μg/mL、128μg/mL;突變菌株165165(K165A)對三種抗生素的MIC值分別為:1024μg/mL、128μg/mL、128μg/mL;突變菌株4040(K40A)對三種抗生素的MIC值分別為512μg/mL、128μg/mL、256μg/mL;突變菌株37(G37A)對三種抗生素的MIC值分別為:1024μg/mL、256μg/mL、512μg/mL;突變菌株39(G39A)對三種抗生素的MIC值分別為:1024μg/mL、512μg/mL、1024μg/mL。6、erm B及erm B突變體甲基化活性比較用3H標記S-腺苷甲硫氨酸為甲基供體,23S rRNA為底物,對野生型及突變體ErmB的甲基化活性進行檢測。試驗結果表明,突變體37(G37A)、39(G39A)酶活性高于野生型ermB甲基化酶活性,突變體40(K40A)、83-1(D83A)、139(R139A)、165(K165A)酶活性小于野生型ermB甲基化酶活性。7、erm B甲基化酶生物進化樹構建利用Mrbays3.1.2生物信息學軟件對ermB不同物種進行同源性分析,構建了ermB甲基化酶的系統(tǒng)發(fā)育樹。結論:ermB、ermT基因構建到pET28a載體上蛋白純化效果較好。構建的32個ermB突變體與野生型ermB的MIC值相比,對紅霉素MIC降低的突變體包括:突變菌株40(K40A)、69-2(E69D)、83-1(D83A)、139(R139A);對林可酰胺MIC降低的突變體包括:突變菌株40(K40A)、69-2(E69D)、116-1(E116A)、139(R139A);對克拉霉素MIC降低的突變體包括:突變菌株58-1(E58A)、58-2(E58D)、60-2(E69D)、83-1(D83A)、139(R139A)、165(K165A)。突變菌株37(G37A)、39(G39A)獲得的酶活性大于野生型ermB,突變菌株40(K40A)、83-1(D83A)、139(R139A)、165(K165A)獲得的酶活性小于野生型ermB,與MIC值變化趨勢相符。以上結果說明,氨基酸K40、D83、R139及K165對ermB甲基化活性具有重要作用,這些氨基酸殘基進一步影響大環(huán)內酯耐藥。本研究為揭示大環(huán)內酯耐藥機制提供了信息,首次建立了MIC值與erm酶活性的關系,為解決核糖體甲基化產生的抗生素耐藥問題提供了新的方向。
[Abstract]:Macrolide antibiotics are widely used in clinical application, mainly for the treatment of gram positive bacteria infection, and bacterial resistance to macrolide is increasingly serious. Methylation enzyme ERM gene is one of the important mechanisms leading to the high level of macrolide resistance. It is believed that ERM catalyzes the modification of 23S rRNA 2058 adenine nucleotides to make them Monomethylation or two methylation. The spatial structure of methylation causes the binding sites of bacteria and macrolides to be hidden, blocking the combination of macrolides and bacterial ribosomes, leading to the resistance to macrolides, linacamide and streptomycin. But the mechanism of ERM methylation and the tribute to the degree of resistance to bacteria In the laboratory, the drug resistant gene ermB and ermT were found in S.gallolyticus subsp.pasteurianus isolated from the brain and spleen of ducklings, and these two ERM genes mediate the high level resistance of these isolates, and we also found different isolates. The ermB single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) in the plant causes ermB mutation and its macrolide MIC is different. In order to explore the mechanism of ERM methylation and its contribution to the resistance to macrolide, this study optimized the ermB and ermT expression system, selected ermB to study, and based on bioinformatics analysis and clinical practice. 32 ermB mutants were constructed by SNP in the isolated strain. The 32 mutants were screened by microdilution method for the first time and six mutants were selected. The methylation activity of 23S rRNA and the minimum inhibitory concentration (MIC) for macrolide and linlacamide were detected respectively. The results were as follows: 1, ERM B, ermT methylation enzyme expression vector Construction and purification and separation and purification of 23S rRNA were constructed to pET28a, pET15b vector, and ermB resistant genes were constructed on pET21b, pET28a vector, and compared with the results of protein separation and purification. ErmB, ermT were constructed to the clone of pET28a carrier, and ErmB, protein, protein, and purified protein were purified by affinity chromatography. The methylation activity of yticus subsp.pasteurianus 23S R RNA.2, ERM B, ermT was compared with 3H labeled S- adenosine methionine as the methyl donor and 23S r as the substrate, and the enzyme catalyzed kinetic test. The methylation activity of the two proteins was transferred to the radioactive intensity by the liquid flicker detection. S.gallolyticus subsp.pasteurianus 23S R RNA can be modified by methylation and ErmT, but ermB methylation activity is lower than ermT.3 and ERM B mutants are constructed in conjunction with ermB SNP of Streptococcus gallate isolates in the early laboratory. Partial amino acid residues in the region were mutated, and 32 mutant.4, ERM B, ermB mutant MIC values were detected for ermB and 32 mutants to be detected by MIC values. The results showed that, compared with the MIC value of the wild type ermB, the mutants of erythromycin MIC decreased in 32 mutants include: Mutant strain 40 (K40A), 69-2 (E69D), 83-1 (139), 139) 139A); mutants that reduce the MIC of linclamides include mutant strain 40 (K40A), 69-2 (E69D), 116-1 (E116A), 139 (R139A), and mutants of clarithromycin MIC, including mutant 58-1 (E58A), 58-2 (E58D), 60-2 (E69D), 83-1 (D83A), 139 (R139A), 165 The results showed that the MIC values of the mutant strain 83-1 (D83A) to erythromycin, clarithromycin and linclamides were 512 mu g/mL, 64 mu g/mL, 512 micron g/mL, and the MIC values of three mutant strains 139139 (R139A) to three antibiotics were 512 mu g/mL, 64 mu g/mL, 128 mu g/mL, and mutant isolates 165165. (K165A) the MIC values for three kinds of antibiotics were 1024 mu g/mL, 128 mu g/mL, 128 mu g/mL, and the MIC values of the mutant 4040 (K40A) to three antibiotics were 512 mu g/mL, 128 micron and 256 micron g/mL respectively. The MIC values of the mutant 37 (G37A) to three antibiotics were respectively 1024 mu, 256, and 512 mu; The methylation activity of g/mL, ERM B and ERM B mutants was compared with 1024 mu, 512 mu, 1024 mu g/mL.6, ERM B and ERM B, and 3H labeled S- adenosine methionine as the methyl donor and 23S rRNA as the substrate to detect the methylation activity of the wild type and mutant ErmB. The experimental results showed that the mutant 37 was higher than the wild type methylation enzyme activity. Sex, the mutant 40 (K40A), 83-1 (D83A), 139 (R139A), 165 (K165A) enzyme activity is less than the wild type ermB methylation enzyme activity.7, ERM B methylation enzyme biological evolution tree construction uses the Mrbays3.1.2 bioinformatics software to analyze the homology of ermB species, and constructs the phylogenetic tree of ermB methylation enzyme. The protein purification effect on the 8A carrier is better. Compared with the MIC value of the wild type ermB, the 32 ermB mutants for erythromycin MIC include mutant strain 40 (K40A), 69-2 (E69D), 83-1 (D83A), 139 (R139A), and mutant strain 40 (K40A), 69-2 (E69D), 116-1 (139), 139 (139)); Mutants reduced by mycophenin MIC, including mutant strain 58-1 (E58A), 58-2 (E58D), 60-2 (E69D), 83-1 (D83A), 139 (R139A), 165 (K165A), mutant strain 37 (G37A), and 39 (G39A) were more active than wild type ermB, mutant strain 40 (K40A), 83-1 (D83A), 139, and 165 (139), which were less than wild type, and were consistent with the trend of variation. These results suggest that amino acid K40, D83, R139 and K165 have an important role in the ermB methylation activity. These amino acid residues further affect the resistance of macrolides. This study provides information for revealing the mechanism of macrolide resistance and the first establishment of the relationship between the MIC and the activity of the ERM enzyme, in order to solve the antibiotic resistance produced by the ribosome methylation. The problem provides a new direction.
【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】：2017
【分類號】：S859.7

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