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研究生:吳建緯
研究生(外文):Chien-Wei Wu
論文名稱:自鮑氏不動桿菌噬菌體中設計新型抗菌胜肽
論文名稱(外文):Design of novel antimicrobial peptide derived from Acinetobacter baumannii phage
指導教授:張凱誌
指導教授(外文):Kai-Chih Chang
學位類別:碩士
校院名稱:慈濟大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:58
中文關鍵詞:鮑氏不動桿菌噬菌體抗菌胜肽
外文關鍵詞:Acinetobacter baumanniiphageantimicrobial peptide
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由於抗生素的濫用,使多重抗藥性菌株面臨無藥可醫的情形。鮑氏不動桿菌 (Acinetobacter baumannii, AB)就是其中之一。病人若是感染多重抗藥性鮑氏不動桿菌 (multiple drug resistance Acinetobacter baumannii, MDRAB),將面臨無藥可醫的情形。有鑑於此,我們想要找尋新的殺菌劑來殺死多重抗藥性菌株。最近的文獻指出,抗菌胜肽 (antimicrobial peptide)在生物體扮演抵抗部份外來入侵為生物的角色。本研究首先以生物資訊學軟體分析本實驗室分離之鮑氏不動桿菌噬菌體的溶菌酶 (lysin)及 Holin 胺基酸序列,並藉以設計出具有不同特性 (二級結構、帶電性、疏水性及疏水性矩)的六條胜肽來進行殺菌實驗。在殺菌實驗結果顯示當靜電荷為+6、疏水性為-0.99及疏水性矩為0.49的 LysAB2 113-145 G1對於多重抗藥性鮑氏不動桿菌具有最好的殺菌效果,其最低抑菌濃度為8 μM,而最小殺菌濃度為16 μM。在溶血實驗試驗中顯示這六條抗菌胜肽的溶血率隨著胜肽的濃度增加而升高,顯示出這六條抗菌胜肽具有部分細胞毒性。在協同作用試驗中發現當 LysAB2 113-145 G1結合 LysAB2 113-145 G3,或者以 LysAB2 113-145 G1結合HolAB2 18-37 G1不具有協同作用且不為拮抗作用,但所需的抗菌胜肽濃度降低,但此時仍有部分細胞毒性。經掃描式電子顯微鏡 (scanning electron microscope, SEM)觀察到抗菌胜肽能直接破壞鮑氏不動桿菌。在細菌細胞膜通透性試驗中發現,抗菌胜肽可以造成細菌細胞膜通性改變。由以上實驗結果得知,本實驗所設計的胜肽是一種有效對抗細菌的抗菌胜肽,並有潛力作為殺菌劑之潛力。未來我們希望此研究能應用在新型抗菌藥劑之研發上。
The emergence of multidrug-resistant strains of Acinetobacter baumannii (MDRAB) constitutes a serious threat to public health. The increasing problem of antibiotic resistance among pathogenic bacteria requires the development of new antimicrobial agents. This study was designed to test the possibility that antimicrobial peptides could be derived from the genomic sequences of phage lysin and Holin. Base on A. baumannii phage2 lysin and Holin amino acid sequences, we designed and produced six putative peptides (LysAB2 113-145, LysAB2 133-145 G1, LysAB2 113-145 G2, LysAB2 113-145 G3, HolAB2 18-37 and HolAB2 18-37 G1). Synthetic these six peptides displayed potent activity against A. baumannii (minimum inhibitory concentration, MICs ranging from 8-128 μM; minimum bactericidal concentration, MBCs ranging from 8-128 μM) while displaying hemolytic activity against human erythrocytes (HC50=32 μM). We investigated antimicrobial peptides for potential synergy with each other against clinical strains of multidrug resistance A. baumannii. The bactericidal test results show that these peptides can effectively restrain parts of Gram negative bacteria. Under the Scanning Electron Microscope, it was observed that these peptides can directly destroy A. baumannii. Treating bacteria with antimicrobial peptides clearly enhanced permeation of the bacterial cytoplasmic membrane. The possible relevance of these results showed these peptides may be used to design new potent antimicrobial candidates much needed in face of the ever threatening drug resistance problems.
目錄 I
中文摘要 III
英文摘要 IV
第一章 前言 1
1.1鮑氏不動桿菌 (Acinetobacter baumannii)之特性 1
1.2 噬菌體之特性及應用 1
1.3 溶菌酶及 Holin之特性及應用 3
1.4 抗菌胜肽簡介 7
1.5 抗菌胜肽結構分類 8
1.5.1 雙親性和疏水性α-螺旋胜肽(amphipathic and hydrophobic α-helices) 8
1.5.2 β-褶板抗菌胜肽 10
1.5.3擁有特殊胺基酸組成的抗菌胜肽 11
1.6 抗菌活性機制 12
1.6.1抗菌胜肽目標特異性和選擇毒性的機制 12
1.6.2膜的組成、疏水性作用力和極性 12
1.6.3抗菌胜肽作用機制 14
1.6.4 結構參數 22
1.7 實驗目的 26
第二章 材料與方法 27
2.1 培養基 27
2.2 本實驗所用鮑氏不動桿菌菌株 28
2.3 人工合成抗菌胜肽設計 29
2.4 殺菌試驗 29
2.5 最低抑菌濃度測定 (Minimal Inhibitory Concentration;MIC) 30
2.6 最小殺菌濃度測定 (Minimum bactericidal concentration;MBC) 30
2.7 抗菌胜肽協同作用試驗 30
2.8 紅血球溶血試驗 31
2.9掃描式電子顯微鏡 31
2.10細菌細胞膜之通透性試驗 32
第三章 結果 33
3.1 抗菌胜肽設計和胺基酸序列分析結果 33
3.2 抗菌胜肽殺菌實驗結果 34
3.3 抗菌胜肽協同作用實驗結果 34
3.4紅血球溶血試驗結果 35
3.5 掃描式電子顯微鏡結果 35
3.6 細菌細胞膜之通透性試驗結果 36
第四章 討論 37
第五章 圖表 40
圖一、溶菌酶序列之分析結果 40
圖二、Holin序列跨膜區之分析結果 41
圖三、Holin序列分析結果 42
圖四、抗菌胜肽二級結構分析結果 43
圖五(a)、抗菌胜肽協同作用測試之結果 44
圖五(b)、抗菌胜肽協同作用測試之結果 45
圖六、抗菌胜肽溶血活性測試之結果 46
圖七、抗菌胜肽殺菌試驗之結果圖 47
圖八、抗菌胜肽殺菌於高倍率掃描式電子顯微鏡之結果 48
圖九、細菌細胞膜之通透性試驗結果圖 49
表一、本實驗所合成之抗菌胜肽簡介 50
表二、抗菌胜肽對鮑氏不動桿菌的最低抑菌濃度與最小殺菌濃度之結果 51
表三、抗菌胜肽對不同細菌的最低抑菌濃度與最小殺菌濃度之結果 52
第六章 參考文獻 53
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