發(fā)布時(shí)間：2018-06-25 09:20

  本文選題：金黃色葡萄球菌 + 一氧化氮自由基��； 參考：《天津醫(yī)科大學(xué)》2009年博士論文

【摘要】： 研究背景：金黃色葡萄球菌(Staphylococcus aureus)是最常見(jiàn)的革蘭陽(yáng)性致病菌，可以引起人體種類(lèi)眾多的感染。而且，隨著抗生素的廣泛使用，耐藥性問(wèn)題越來(lái)越嚴(yán)重。萬(wàn)古霉素作為治療耐藥金黃色葡萄球菌感染的最后一道防線，也出現(xiàn)了敏感性降低，甚至完全耐藥的現(xiàn)象。其實(shí)，人類(lèi)的自身先天免疫系統(tǒng)是有效抗擊外來(lái)微生物入侵的第一道防線。其中一種重要的機(jī)制就是誘導(dǎo)性一氧化氮合成酶系統(tǒng)，其所合成的NO~*可以直接抑制微生物的有氧呼吸能量代謝，并抑制微生物的DNA復(fù)制。而金黃色葡萄球菌區(qū)別于其他非致病性葡萄球菌的一個(gè)主要特征，就是可以抵抗NO~*的毒性，在外源性NO~*壓力下仍能生長(zhǎng)和繁殖。其N(xiāo)O~*解毒機(jī)制一方面與黃素血紅蛋白(hmp SACOL0220)行使的NO~*雙氧化酶功能有關(guān)，可以將毒性NO~*轉(zhuǎn)化為無(wú)毒的硝酸；另一方面與hmp同處于一個(gè)操縱子中的金黃色葡萄球菌乳酸脫氫酶-1(ldh-1 SACOL022)的誘導(dǎo)高表達(dá)密切相關(guān)。ldh-1與金黃色葡萄球菌中持續(xù)表達(dá)的同功酶ldh-2可以改變金黃色葡萄球菌在NO~*生存環(huán)境下細(xì)菌的能量代謝途徑，從而使金黃色葡萄球菌可以在NO~*環(huán)境壓力下正常生長(zhǎng)和繁殖。研究思路：理論上ldh-1蛋白可以作為一個(gè)抗金黃色葡萄球菌的潛在靶點(diǎn)，任何對(duì)ldh-1具有選擇性抑制作用的小分子物質(zhì)，即只抑制金黃色葡萄球菌ldh-1但對(duì)人類(lèi)的乳酸脫氫酶同功酶無(wú)明顯抑制作用，就可能成為一種新的抗生素。研究方法：本研究首先將金黃色葡萄球菌中編碼ldh-1和ldh-2的基因全長(zhǎng)克隆到表達(dá)載體pET-28a中，大量表達(dá)重組蛋白，再利用重組蛋白的Histag，以親和層析法和分子篩兩步聯(lián)合方法純化重組蛋白純度超過(guò)95％。進(jìn)而，，在不同理化條件下促使蛋白結(jié)晶，然后用所得到的單晶進(jìn)行X射線衍射以解析蛋白的三維機(jī)構(gòu)。研究結(jié)果：l；dh-1蛋白結(jié)晶，并收集高質(zhì)量衍射數(shù)據(jù)。初步分析屬于C2空間群，晶胞參數(shù)為a=131．36，b=74．362，c=103．243。ldh-1的結(jié)構(gòu)最終可以被精修到2．2 (?)，R-Factor為17％，Rfree-factor為23．4％。使用分子置換法解析出其三維結(jié)構(gòu)，分辨率達(dá)2．2埃。每個(gè)晶胞中有兩個(gè)不對(duì)稱(chēng)的乳酸脫氫酶分子。每個(gè)ldh-1分子中包含兩個(gè)結(jié)構(gòu)域。大的結(jié)構(gòu)域由22-160和248-264號(hào)氨基酸形成具有典型Rossmann特征的折疊。通過(guò)蛋白一級(jí)結(jié)構(gòu)氨基酸序列的多重序列比對(duì)，活性位點(diǎn)的關(guān)鍵氨基酸序列保守，但活性環(huán)區(qū)上翹，使活性位點(diǎn)更加暴露。本研究還對(duì)ldh-1、ldh-2、人類(lèi)骨骼肌ldh-A和心肌ldh-B的酶催化活性進(jìn)行了比較，發(fā)現(xiàn)ldh-1與底物和輔酶的親和性，和反應(yīng)效率均明顯高于人類(lèi)的同功酶。通過(guò)對(duì)金黃色葡萄球菌中1dh結(jié)構(gòu)、功能的研究，希望能以酶結(jié)構(gòu)為基礎(chǔ)，使用工作站篩選相關(guān)類(lèi)別的小分子數(shù)據(jù)庫(kù)，發(fā)現(xiàn)潛在的結(jié)構(gòu)抑制劑。
[Abstract]:Background: Staphylococcus aureus is the most common gram-positive bacteria, which can cause a variety of human infections. Moreover, with the widespread use of antibiotics, the problem of drug resistance is becoming more and more serious. Vancomycin, as the last line of defense in the treatment of drug-resistant Staphylococcus aureus infection, also has the phenomenon of reduced sensitivity and even complete drug resistance. In fact, the human innate immune system is the first line of defense against the invasion of foreign microorganisms. One of the important mechanisms is the inducible nitric oxide synthase system, which can directly inhibit the aerobic respiration energy metabolism of microorganisms and inhibit the DNA replication of microbes. One of the main characteristics of Staphylococcus aureus differs from other non-pathogenic staphylococci is that it can resist the toxicity of NOA * and can still grow and reproduce under the pressure of exogenous NOA *. The detoxification mechanism is related to the function of hmp SACOL0220, which can transform noxotoxin into nontoxic nitric acid. On the other hand, the induced overexpression of staphylococcus aureus lactate dehydrogenase 1 (ldh-1 SACOL022) in the same operon with hmp was closely related to the sustained expression of isoenzyme ldh-2 in Staphylococcus aureus, which could change the expression of Staphylococcus aureus. The energy metabolism pathway of bacteria in the living environment, Thus Staphylococcus aureus can grow and reproduce normally under environmental pressure. Research idea: theoretically, ldh-1 protein can be used as a potential target against Staphylococcus aureus, any small molecule with selective inhibitory effect on ldh-1. It may become a new antibiotic to inhibit only Staphylococcus aureus ldh-1 but no obvious inhibition of human lactate dehydrogenase isoenzyme. Methods: in this study, the full-length gene encoding ldh-1 and ldh-2 in Staphylococcus aureus was cloned into the expression vector pET-28a to express a large number of recombinant proteins. The purity of recombinant protein was more than 95% by affinity chromatography and molecular sieve. Furthermore, the protein was crystallized under different physicochemical conditions, and then X-ray diffraction was carried out with the obtained single crystal to analyze the three-dimensional mechanism of the protein. Results the protein was crystallized and high quality diffraction data were collected. The preliminary analysis belongs to the C2 space group, and the unit cell parameter is a 131.36 / 74.362C ~ (-1) 103.243.ldh-1 structure which can be refined to 2.2 (?) R-factor 17 ~ (th) Rfree-factor = 23.4 ~ (th) ~ (th). The three-dimensional structure was analyzed by molecular replacement method with a resolution of 2.2 A. There are two asymmetric lactate dehydrogenase molecules in each unit cell. Each ldh-1 molecule contains two domains. The large domain consists of 22-160 and 248-264 amino acids to form folding with typical Rossmann characteristics. The key amino acid sequence of the active site is conserved by multiple sequence alignment of amino acid sequence of the primary structure of protein, but the active ring region is upwarped, which makes the active site more exposed. The enzyme catalytic activity of ldh-1 ldh-2, human skeletal muscle ldh-A and myocardial ldh-B was compared. It was found that the affinity and reaction efficiency of ldh-1 to substrate and coenzyme were significantly higher than that of human isozyme. Through the study of the structure and function of 1dh in Staphylococcus aureus, it is hoped that based on the enzyme structure, a workstation can be used to screen the relevant small molecular databases and to find potential structural inhibitors.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R378;R515

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