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研究生:蔡淳光
研究生(外文):Chung-Guang Tsai
論文名稱:N-甲基化之胜肽類緣物的設計、合成、純化及其生物活性分析
論文名稱(外文):Design, Synthesis, Purification, and Bioactivity Analysis of N-Methylated Peptide Analogs
指導教授:龍鳳娣
指導教授(外文):Feng-Di Lung
口試委員:顧野松駱碧秀
口試委員(外文):Yesong GuBih-Show Lou
口試日期:2015-06-30
學位類別:碩士
校院名稱:東海大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:87
中文關鍵詞:抗微生物胜肽比古昔素細菌胜肽甲基化
外文關鍵詞:antimicrobial peptideIxosin-BbacteriaN-methylated peptide
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由於具有抗菌性的病原體廣泛地在全球出現,重新引起研發新穎的抗微生物製劑的興趣。先前的研究中,我們發現了兩個抗微生物胜肽,MAP-04-03與MAP-04-04,為胜肽Ixosin-B-amide ( QLKVDLWGTRSGIQPEQHSSGK ) 的類緣物,具有抗菌效果。
許多研究指出甲基化的胜肽具有高選擇性以及低細胞毒性。因此,本論文以MAP-04-03及 MAP-04-04作為模板,在不同特定的端點上修飾,設計合成一系列甲基化胜肽。所有的胜肽均應用固相胜肽合成法進行合成以甲基化之化學修飾是經由硫酸二甲酯反應進行。
每一條合成的胜肽,透過RP-HPLC進行純化之後,應用MALDI-TOF-MS 進行鑑定。分子量確認後的胜肽進行抗微生物活性、抗癌活性、溶血性的分析。
在本研究中,我們發現了一條具有潛力的抗微生物胜肽MAP-04-03-W7,其顯示出具有對抗大腸桿菌、金黃色葡萄球菌及綠膿桿菌的功效,最小抑制濃度 (MIC) 分別為5 μM、5 μM及10 μM,並且其溶血的副作用也降低。
研究結果有助於研發具有高選擇性及低副作用之新型的抗微生物製劑,並可應用於治療細菌引起的疾病。
Because of the emergence of antibiotic-resistant pathogens worldwide, it has made an urgent request for the development of novel antimicrobial agents. In our previous studies, two antimicrobial peptides, MAP-04-03 and MAP-04-04 which are analogs of Ixosin-B-amide (QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY) were found to exhibit antibacterial activity. Many research showed that N-methylated peptides exhibited high selectivity and low cell-toxicity. Therefore, we used MAP-04-03 and MAP-04-04 as the template for modifications at specific sites of peptides to design a series of N-methylated peptides.
All of the peptides were synthesized by solid-phase method, and chemical methylation modification was performed by dimethyl sulfate reaction, followed by RP-HPLC purification and MALDI-TOF-MS characterization of synthesized peptides. Characterized peptides were analyzed for their antimicrobial activities , anticancer activities, and hemolytic activities. In the present study, we discovered a potential antimicrobial peptide, MAP-04-03-W7, which exhibited antibacterial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa with the MIC value of 5 μM, 5 μM, and 10μM, respectively, and it displayed a reduced hemolytic activity.
Results of these studies should be useful for developing antibacterial agents with higher selectivity and the least side effects for the treatment of bacteria-caused diseases.

第一章 前言 1
第二章 文獻回顧 3
2.1 新型抗菌胜肽之重要性 3
2.2 抗菌胜肽 4
2.3 抗微生物胜肽的結構分類 4
2.4 細菌分類與外膜之簡介 5
2.5 抗菌胜肽的抗癌作用 6
2.6 抗菌胜肽的化學修飾 8
第三章 實驗流程與材料方法 9
3.1 抗微生物胜肽之設計 11
3.2 固相胜肽合成法原理 13
3.2.1 固相胜肽合成法的實驗流程 15
3.2.2 甲基化胜肽之合成方法 19
3.2.3 Ninhydrin test 原理 23
3.3 逆相高效能液相層析法 25
3.3.1 實驗原理 25
3.3.2 實驗方法 26
3.4 基質輔助雷射脫附游離飛行時間質譜儀 27
3.4.1 實驗原理 27
3.5 細菌之培養及抗菌功效測試方法之建立 29
3.5.1 細菌的接種(固態培養皿之劃碟法) 30
3.5.2 細菌的大量培養(液態培養) 31
3.5.3 細菌數目的測量 32
3.5.4 抗微生物胜肽活性的評估方法 MIC的制定 32
3.6 溶血性分析(Hemolysis assay) 33
3.6.1 實驗原理 33
3.6.2實驗方法 34
3.7 胜肽之抗癌活性分析 36
3.7.1 細胞培養之常用藥品試劑及設備 36
3.7.2 細胞培養液的配製 37
3.7.3 冷凍細胞的解凍方法 37
3.7.4 人類乳癌細胞之培養以及胜肽毒性測試實驗之方法 38
3.7.5 胜肽抗癌細胞增生之評估方法 39
3.7.6 細胞冷凍保存的方法 40
3.8 應用圓二色光譜探討胜肽之二級結構 41
3.8.1 實驗原理 41
3.8.2 圓二色光譜實驗方法 43
第四章 實驗結果與討論 44
4.1 甲基化之設計與合成 44
4.2 細菌活性測試 45
4.3 人類紅血球之溶血性分析結果 47
4.4 胜肽對人類乳癌細胞株( MCF-7 )細胞存活率之結果 49
4.5 圓二色光譜探討二級結構變化 52
第五章 結論 56
第六章 未來展望 57
參考文獻 58

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