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標題: | 建立一個利用固定化盤尼西林結合蛋白之分析平台以篩選可能之抗生素 An Assay Platform to Identify Potential Antibiotics with Immobilized Penicillin-binding Proteins |
作者: | Yen-Ta Wu 巫衍達 |
指導教授: | 翁啟惠 |
關鍵字: | 盤尼西林結合蛋白, penicillin-binding protein,moenomycin,lipid II,MALDI, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 抗生素的普遍使用已經造成細菌嚴重的抗藥性問題。其中,具有甲氧苯青黴素(methicillin)抗藥性的金黃葡萄球菌(S. aureus),以及具有萬古黴素(vancomycin)抗藥性的腸球菌(enterococci)和困難腸梭菌(C. difficile),均已對大多數的抗生素具有抗藥性,對人類的健康造成了嚴重的威脅。因此,新型抗生素的研發著實是刻不容緩。
在這份研究中,我們著重在盤尼西林結合蛋白(Penicillin-binding protein) ,並建立了一個篩選盤尼西林結合蛋白抑制物的全新平台。盤尼西林結合蛋白是細菌細胞壁成分中胜肽聚糖(peptidoglycan,亦稱作胞壁質)的合成是不可或缺的,因此可作為抗生素研發的標的之一。A種類的盤尼西林結合蛋白是一個同時具有轉糖基酶(transglycosyalse)與轉胜酶(transpeptidase)雙重功能的蛋白。目前細菌普遍對乙內醯胺類(β-lactam)等針對轉胜酶活性的抗生素已有很普遍的抗藥性,因此轉糖基酶提供了另一可作為新型抗生素研發的潛在標的。 我們首先表現以及純化困難腸梭菌(C. difficile)與產氣莢膜梭狀芽胞桿菌(C. perfrigens)的盤尼西林結合蛋白,並透過偏極光分析法來探討這兩種重組盤尼西林結合蛋白對moenomycin的親合性。我們首先利用薄膜層析法與帶有螢光之lipid II來分析固定化盤尼西林結合蛋白之轉糖基酶活性。再者,固定化之盤尼西林結合蛋白具有與轉糖基酶抑制物moenomycin結合的能力,藉由基質輔助雷射脫附游離飛行時間質譜(MALDI TOF mass spectrometry)可以判定與盤尼西林結合蛋白結合的小分子,並利用96孔多孔盤研究其最小抑制濃度(MIC)。這個平台為篩選可能之轉糖基酶或轉胜酶抑制物提供了一個便捷的管道。 另一方面,我們利用生物合成以及管住層析法可以順利製備出豪克等級量的lipid II,因此為建立有關轉糖基酶對lipid II活性方面的分析方法提升了不少便利性。 The widespread use of antibiotics has generated serious drug resistance problems. These problems are more serious in hospitals and nursing home where the weak patients often need sustained treatment of antibiotics and thus are easily colonized with drug-resistant bacteria. Among them, methicillin-resistant Staphylococcus aueus, vancomycin-resistant enterococci, and Clostridium difficile, which are resistant to most antibiotics, have posted serious threats to public health. There is an unmet medical need to novel and new generation of antibiotics. Herein, we focused on penicillin-binding protein (PBPs) and developed a novel assay platform to facilitate identification of potential PBP inhibitors. Penicillin-binding proteins (PBPs) are essential for peptidoglycan (also called murein) biosynthesis and hence are one of the drug targets for antibiotics development. Class A PBPs are bifunctional proteins that have both transglycosylase and transpeptidase activity. Since the resistance against several β-lactam antibiotics that are targeting transpeptidase activity of class A PBPs has emerged, the transglycosylation represents a new target for potential therapeutics. As the first step, the PBPs from C. difficile and C. perfrigens were expressed and purified. The moenomycin binding activities to these two recombinant proteins were characterized using fluorescence polarization assay with fluorescent moenomycin. The transglysosylase activity of immobilized PBPs with fluorescent lipid II was analyzed by TLC analysis. A novel assay platform was designed so that immobilized PBPs is able to specifically ”fish out” transglycosylase inhibitors such as moenomycin or transglycosylase substrate, lipid II. The bound moenomycin can be easily identified using MALDI analysis and exerted their MIC activity in a 96-well microtiter plate. Thus this novel platform can be used to facilitate the identification of potential inhibitors for transglycosylase and transpeptidase. On the other hand, the biosynthesis of lipid II was realized and further purified by short pass chromatography, therefore milligram quantities of lipid II can be easily obtained. This can facilitate the development of lipid II-based assay of transglycosylase. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28705 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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