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研究生:王智安
研究生(外文):Wang, Chih-An
論文名稱:探討魚精蛋白序列中具抗菌潛力的胜肽片段
論文名稱(外文):Potential Antibacterial Peptides in Protamine Sequences
指導教授:張立乾
指導教授(外文):Chang, Li-Chien
口試委員:張立乾陳金順楊啟裕
口試委員(外文):Chang, Li-ChienChen, Jin-ShuenYang, Chi-Yu
口試日期:2014-05-16
學位類別:碩士
校院名稱:國防醫學院
系所名稱:藥學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:75
中文關鍵詞:多重抗藥性抗菌胜肽低分子量魚精蛋白
外文關鍵詞:Multidrug-resistantAntibacterial peptidesLow molecular weight protamine
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在過去的數十年當中,多重抗藥性細菌持續出現的問題,已經威脅到大眾的健康,所以迫切地需要尋找替代療法。抗微生物胜肽的發展被認為是有潛力的策略,而魚精蛋白對革蘭氏陽性菌和革蘭氏陰性菌都具有顯著抑菌效果。文獻指出低分子量魚精蛋白明顯地降低原來的毒性、抗原和免疫反應。因此,本研究的目的是在探討低分子量魚精蛋白作為新一代抗菌胜肽的潛力。
在此研究中,將salmine和clupeine經由酵素thermolysin分解成兩種型態的低分子量魚精蛋白衍生物,分別為thermolysin-digested salmine protamines (TDSP)和thermolysin-digested clupeine protamines (TDCP),這些胜肽的特徵都富含有精氨酸,分子量範圍約在700至1,800之間。從TDSP主要片段中分離出TDSP3 (PRRRRSSSRRP) 和TDSP5 (VSRRRRRRGGRRRR)胜肽,並利用基質輔助雷射脫附游離飛行時間質譜儀作身分鑑定。TDSP5對革蘭氏陰性菌包括大腸桿菌(Escherichia coli)、綠膿桿菌(Pseudomonas aeruginosa)和鮑氏不動桿菌(Acinetobacter baumannii)有高的抗菌活性表現;另一方面,所有魚精蛋白胜肽對人類紅血球有非常低的溶血毒性。
綜合兩者結果,短鏈魚精蛋白胜肽(如TDSP5)顯示具有發展為新一代抗菌胜肽的潛力,可開發出廣效性之低溶血性魚精蛋白衍生物,以用來對抗革蘭氏陽性菌和革蘭氏陰性菌感染。
Over the past few decades, the emergence of multidrug-resistant bacteria continuously threatening public health has created an urgent need for alternative therapeutics. While the development of antimicrobial peptides (AMPs) have been considered as a promising strategy, protamine possessing prominent efficacy against a range of Gram-positive and Gram-negative bacteria shall be a potential target. A review of literatures indicates that low molecular weight protamines (LMWP) have significantly reduced toxicity, antigenicity and immunogenicity. Therefore, the aim of this study is to investigate the potential of LMWP as a new class of antibacterial peptides.
In this study, two types of LMWPs derived from salmine and clupeine via enzymatic digestion, denoted thermolysin-digested salmine protamines (TDSP) and thermolysin-digested clupeine protamines (TDCP), were prepared. They were characterized as arginine-rich sequences with molecular weights ranging from 0.7-1.8 kDa. While the major portion of TDSP was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to compose of TDSP3 (PRRRRSSSRRP) and TDSP5 (VSRRRRRRGGRRRR) peptides, TDSP5 showed high potency against Gram-negative strains including Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii. On the other hand, all protamine peptides exhibited very little hemolytic toxicity to human red blood cells.
Taken together, the chain-shortened protamine peptide such as TDSP5 showed the potential to develop as a new class of antibacterial peptide with broad-spectrum activity and low cytotoxicity against Gram-positive and Gram-negative bacteria.
第一章 緒言
第一節 研究動機
壹、 多重抗藥性細菌的出現
貳、 藥物不良反應事件的頻傳
參、 核准可上市的新抗生素數量減少
第二節 研究目的
第三節 抗菌胜肽之簡介
壹、 概要介紹
貳、 抗菌胜肽結構特徵
參、 抗菌胜肽作用機轉
第四節 魚精蛋白與其低分子量衍生物之發展現況
壹、 來源與特徵
貳、 生物活性之臨床應用
參、 魚精蛋白之抗菌活性研究
第二章 實驗設計與材料方法
第一節 實驗設計流程圖
第二節 研究材料
壹、 藥品與試劑
貳、 實驗耗材
參、 儀器設備
肆、 各種溶液之製備
伍、 菌種
第三節 研究方法
壹、 低分子量魚精蛋白的製備
貳、 低分子量魚精蛋白的分離與除鹽
參、 質譜儀與胜肽身分鑑定
肆、 鄰苯二甲醛(o-Phthalaldehyde)螢光蛋白質定量分析法
伍、 建立抗菌及溶血活性之預測方法
陸、 抗菌活性試驗
柒、 溶血毒性試驗
第三章 結果
第一節 低分子量魚精蛋白分離與身分鑑定
壹、 胜肽的分離
貳、 胜肽質量的指紋分析
參、 選作抗菌胜肽的結構特徵
第二節 抗菌與溶血活性預測模型
壹、 抗菌活性預測模型
貳、 溶血活性預測模型
參、 預測魚精蛋白及衍生胜肽的抗菌及溶血活性
第三節 抗菌活性
壹、 最小抑菌濃度(MICs)
貳、 最小殺菌濃度(MBCs)
第四節 溶血毒性
第四章 討論
第一節 低分子量魚精蛋白定性與定量分析
壹、 定性分析
貳、 定量分析
第二節 魚精蛋白及其低分子量衍生物的抗菌活性
第三節 魚精蛋白及其低分子量衍生物的溶血毒性
第四節 抗菌與溶血活性預測模型的發展應用
壹、 比較抗菌與溶血活性之預測模型與實驗結果
貳、 運用預測模型發展魚精蛋白衍生物之抗菌胜肽
第五章 結論
第六章 參考文獻

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