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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 翁啟惠 | |
| dc.contributor.author | Yen-Ta Wu | en |
| dc.contributor.author | 巫衍達 | zh_TW |
| dc.date.accessioned | 2021-06-13T00:18:33Z | - |
| dc.date.available | 2007-07-30 | |
| dc.date.copyright | 2007-07-30 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-07-27 | |
| dc.identifier.citation | Albrich,W.C., Monnet,D.L., and Harbarth,S. (2004). Antibiotic selection pressure and resistance in Streptococcus pneumoniae and Streptococcus pyogenes. Emerg. Infect. Dis. 10, 514-517.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28705 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T00:18:33Z (GMT). No. of bitstreams: 1 ntu-96-R94b46022-1.pdf: 1316693 bytes, checksum: 447a56b4f7884d2f37b4df789e54403f (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | Acknowledgement I
Chinese abstract II English abstract III Table of contents IV Figures VII List of abbreviations IX Chapter 1. INTRODUCTION 1 1. The emergence of antibiotics resistance 1 2. Clostridium 3 C. perfringens 3 C. difficile 4 3. Three stages of peptidoglycan biosynthesis 5 4. Penicillin-Binding-Protein (PBP) 7 5. Moenomycin 8 Chapter 2. MATERIALS and METHODS 11 Materials 11 Methods 11 1.Cloning of PBP expression vectors 11 Polymerase chain reaction 12 DNA electrophoresis 13 DNA purification from agarose gel 13 DNA ligation 14 Preparation of competent cell 14 Transformation 14 Plasmid preparation 15 2.Protein expression and extraction 15 3.Protein purification by nickel affinity chromatography 16 4.Protein analysis by sodium dodecyl sulfate- Polyacrylamide gel electrophoresis(SDS-PAGE) 17 5.Western blotting 17 6.Protein analysis by proprietary fluorescent dye, vista- green 18 7.Determination of protein concentration 18 8.Measurement of moenomycin binding by Fluorescent Polarization (FP) assay 19 9.Immobilization of his-tagged protein on Ni2+ magnetic nanoparticles 19 10.Detection of transglycosylase activity of immobilized PBP with fluorescein-labelled lipid II using TLC analysis 20 11.Elution of moenomycin from immobilized his-tagged protein 20 12.Detection of moenomycin by Matrix-assisted Laser Desorption Ionization mass spectrometry (MALDI) 21 13.Determination of minimum inhibitory concentration (MIC) 21 14.Synthesis and purification of the substrate of PBPs, lipid II 22 Preparation of Micrococcus flavus vesicles 22 Lipid II were synthesized with membrane vesicles23 Chapter 3. RESULTS and DISCUSSION 24 1.Characterization of Penicillin-binding proteins from Clostridium 24 1.1 Expression and purification of Clostridium difficile PBP1b and Clostridium perfrigens PBP1A/1B using pET- 15b vector 24 1.2 Moenomycin binding of recombinant PBPs of C. difficile and C. perfrigens 25 2.Immobilization of PBPs using SNAP technology 25 3.Immobilization of PBPs using Ni2+ magnetic nanoparticles 26 4.Detection of transglycosylase activity of immobilized PBPs in TLC analysis 27 5.MALDI analysis of moenomycin that binds to immobilized PBPs on magnetic nanoparticles 28 5.1 Optimization of MALDI analysis for moenomycin detection 28 5.2 Detection limit of moenomycin using MALDI analysis 29 5.3 Detection of moenomycin that can bind to immobilized PBPs 29 5.4 Detection of lipid II that can bind to immobilized PBPs 30 6.MIC determination of moenomycin that binds to mmobilized PBPs on magnetic nanoparticles 31 Chapter 4. CONCLUSION 32 REFERENCES 35 | |
| dc.language.iso | zh-TW | |
| dc.subject | 盤尼西林結合蛋白 | zh_TW |
| dc.subject | lipid II | en |
| dc.subject | penicillin-binding protein | en |
| dc.subject | MALDI | en |
| dc.subject | moenomycin | en |
| dc.title | 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 | zh_TW |
| dc.title | An Assay Platform to Identify Potential Antibiotics with Immobilized Penicillin-binding Proteins | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 鄭婷仁 | |
| dc.contributor.oralexamcommittee | 鄭偉杰,馬徹 | |
| dc.subject.keyword | 盤尼西林結合蛋白, | zh_TW |
| dc.subject.keyword | penicillin-binding protein,moenomycin,lipid II,MALDI, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-07-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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