本文選題：TEM-1 + β-內(nèi)酰胺酶　； 參考：《西北大學》2016年博士論文

【摘要】：抗生素耐藥問題是目前人類醫(yī)療活動中面臨的重大社會問題,解決這一問題的主要途徑包括抗生素的合理使用、新型抗生素的研發(fā)和抗生素與其體內(nèi)靶點的相互作用研究等。p-內(nèi)酰胺類抗生素是臨床上最常用的抗菌藥物,其產(chǎn)生耐藥性的主要原因在于：細菌編碼產(chǎn)生抗藥靶蛋白p-內(nèi)酰胺酶,催化水解p-內(nèi)酰胺類抗生素。TEM-1β-內(nèi)酰胺酶(TEM-1)是最常見的超廣譜p-內(nèi)酰胺酶,其催化水解β-內(nèi)酰胺類抗生素的首要環(huán)節(jié)是識別并結(jié)合抗生素。本論文選擇青霉素G、頭孢氨芐和頭孢西丁三種β-內(nèi)酰胺類抗生素為對象,首先采用位點靶向性分子模擬技術(shù)研究TEM-1與這三種抗生素的分子識別,并應用熒光光譜法和親和色譜法研究它們相互作用的機理。該研究能從分子水平上闡明TEM-1與β-內(nèi)酰胺類抗生素的相互作用機制,對解決抗生素耐藥問題和研制新型高效抗生素藥物具有重要意義。全文共分3章,主要工作和內(nèi)容如下：1.采用分子對接技術(shù)對TEM-1與青霉素G、頭孢氨芐和頭孢西丁的分子識別過程進行了模擬,并采用光譜法探查TEM-1與三種抗生素發(fā)生分子識別時該酶自身構(gòu)象的變化,證明分子識別是其相互作用的關鍵環(huán)節(jié)。結(jié)果表明：TEM-1分子和三種抗生素分子分別形成6,5和3個氫鍵,通過氫鍵生成1：1的復合物；TEM-1與三種抗生素相互作用的主要推動力是氫鍵和范德華力。紫外吸收光譜結(jié)果表明：隨著抗生素濃度的增大,紫外吸收值增大,吸收峰紅移。碘化鉀(KI)猝滅實驗結(jié)果表明：KI對TEM-1-抗生素復合物的猝滅常數(shù)高于對TEM-1的猝滅常數(shù),且形成復合物后,TEM-1表面的發(fā)色氨基酸殘基的數(shù)目減少。同步熒光光譜結(jié)果表明：在Λλ=60 nm時和Λλ=15 nm時,隨著抗生素濃度的逐漸增大,TEM-1的同步熒光強度逐漸下降,其Λλ=60 nm時下降更顯著。二階導數(shù)熒光光譜結(jié)果表明：激發(fā)波長為278 nm時,酶與酶-抗生素復合物均有兩個負峰,形成復合物后峰強度和位移均有變化：激發(fā)波長為295 nm時,酶與酶-抗生素復合物均只有一個負峰,形成復合物后峰強度降低。2.采用熒光光譜法和親和色譜法研究TEM-1與三種抗生素分子相互作用的機制。結(jié)果表明,TEM-1與三種抗生素以1：1的比例發(fā)生相互作用,猝滅機制為靜態(tài)猝滅。熱力學研究結(jié)果表明：焓變ΛH、熵變AS和自由能變化ΛG均0,說明TEM-1與三種抗生素的作用過程為自發(fā)的放熱過程,主要作用力為氫鍵和范德華力。另一方面,TEM-1和三種抗生素相互作用的表觀活化能均為負值,表明結(jié)合過程沒有能壘,是一個自發(fā)的過程。根據(jù)Forster能量轉(zhuǎn)移理論計算得到TEM-1與三種抗生素的結(jié)合距離r均小于7 nm,且滿足0.5R01.5R0,說明能量轉(zhuǎn)移過程為非輻射轉(zhuǎn)移。親和色譜法的研究結(jié)果表明,固定化TEM-1仍然保留其生物活性,能特異性的識別三種抗生素分子,兩者相互作用過程符合單朗格繆爾模型。前沿色譜分析測定TEM-1與青霉素G、頭孢氨芐和頭孢西丁相互作用過程的結(jié)合常數(shù)分別為：3.54×104 M-1,1.89×104 M-1和1.23×104 M-1,結(jié)合常數(shù)大小順序為青霉素G頭孢氨芐頭孢西丁,與熒光光譜法測定結(jié)果一致,證明固定化TEM-1可用于研究TEM-1與抗生素類藥物的相互作用。
[Abstract]:The problem of antibiotic resistance is a major social problem in human medical activities. The main ways to solve this problem include the rational use of antibiotics, the development of new antibiotics and the study of the interaction between the antibiotics and the targets in the body..p- lactam antibiotics are the most commonly used antibiotics in the bed, and they produce drug resistance. The main reason is that bacteria encode the production of anti drug target protein p- lactamase, and catalyze the hydrolysis of p- lactam antibiotic.TEM-1 beta lactamase (TEM-1) as the most common super broad-spectrum p- lactamase. The first link to catalyze the hydrolysis of beta lactam antibiotics is to identify and bind antibiotics. This paper chooses penicillin G, cefampicin Three kinds of beta lactam antibiotics, cefoxitin, are used to study the molecular recognition of TEM-1 and the three antibiotics using site targeting molecular simulation technique. The mechanism of their interaction is studied by fluorescence spectroscopy and affinity chromatography. This study can elucidate the phase of TEM-1 and beta lactam antibiotics at the molecular level. The interaction mechanism is of great significance for the solution of antibiotic resistance and the development of new efficient antibiotic drugs. The full text is divided into 3 chapters. The main work and contents are as follows: 1. the molecular recognition process of TEM-1 and penicillin G, cefamaxin and cefoxitin was simulated by molecular docking, and three species were detected by spectral method. The change in the conformation of the enzyme at the time of molecular recognition shows that molecular recognition is the key link in its interaction. The results show that TEM-1 and three kinds of antibiotic molecules form 6,5 and 3 hydrogen bonds respectively, and the complex of 1:1 through hydrogen bonds; the main driving force for the interaction between TEM-1 and three kinds of antibiotics is hydrogen bond and Fan De The UV absorption spectra showed that the UV absorption value increased with the increase of antibiotic concentration, and the absorption peak was red shift. The results of the quenching experiment of potassium iodide (KI) showed that the quenching constant of KI to TEM-1- antibiotic complex was higher than that of the quenching constant to TEM-1, and the number of amino acid residues on the TEM-1 surface decreased after the formation of complex. The results of the step fluorescence spectrum showed that the synchronous fluorescence intensity of TEM-1 decreased gradually with the increasing of the concentration of the antibiotics at the lambda =60 nm and the lambda =15 nm. The two order derivative fluorescence spectra showed that when the excitation wavelength was 278 nm, the enzyme and the enzyme antibiotic complex had two negative peaks, forming a complex. The intensity and displacement of the post peak were changed: when the excitation wavelength was 295 nm, the enzyme and enzyme antibiotic complex had only one negative peak, and the peak intensity of the complex was reduced by.2.. The interaction mechanism between TEM-1 and three kinds of antibiotic molecules was studied by fluorescence spectroscopy and affinity chromatography. The results showed that the proportion of TEM-1 and three antibiotics was in the proportion of 1:1. The quenching mechanism is static quenching. The thermodynamic study shows that the enthalpy change a H, the entropy change AS and the free energy change a G are all 0, indicating that the action process of the TEM-1 and the three kinds of antibiotics is spontaneous exothermic process, the main force is hydrogen bond and Fan Dehua force. On the other hand, the apparent activation energy of the interaction of TEM-1 and three kinds of antibiotics are all Negative value indicates that the binding process has no energy barrier and is a spontaneous process. According to the Forster energy transfer theory, the binding distance between TEM-1 and three antibiotics is less than 7 nm and satisfies 0.5R01.5R0, indicating that the energy transfer process is non radiative transfer. The results of affinity chromatography still retain the biological activity of immobilized TEM-1. The interaction process of three kinds of antibiotic molecules can be specifically identified. The interaction process of the two is consistent with the single Langmuir model. The binding constants of the interaction process between TEM-1 and penicillin G, cefamaxin and cefoxitin are respectively: 3.54 x 104 M-1,1.89 x 104 M-1 and 1.23 x 104 M-1, and the binding constant size sequence is penicillin G head Ceftazidime, which is consistent with the results of fluorescence spectrometry, proves that immobilized TEM-1 can be used to study the interaction between TEM-1 and antibiotics.
【學位授予單位】：西北大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R978.1

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