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研究生:黃信儒
研究生(外文):Hsin-Ru Huang
論文名稱:抗菌胜肽之二級結構與其殺菌過程中產生之氫氧自由基之關係研究
論文名稱(外文):The relationship between secondary structure of antimicrobial peptide and hydroxyl radical productions during bactericidal treatments
指導教授:劉哲文劉哲文引用關係
指導教授(外文):Je-Wen Liou
學位類別:碩士
校院名稱:慈濟大學
系所名稱:微生物學免疫學暨生物化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:英文
論文頁數:61
中文關鍵詞:氫氧自由基抗菌胜肽結構
外文關鍵詞:Hydroxyl radical productionpeptide secondary structure
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  • 下載下載:31
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隨著細菌的抗藥性越來越強,發展新的可以在臨床使用的新抗生素就變得很重要。其中抗菌胜肽就是新抗生素一個很好的來源。
在先前的研究中已經知道殺菌類抗生素在殺菌過程中會伴隨著氫氧自由基的產生。在我們的實驗室中也發現到在抗菌胜肽的殺菌過程中細菌的氫氧自由基產生量會提高,而此一現象可能和胜肽的的結構有關。因此本研究的目的為探討胜肽的α-螺旋含量和殺菌過程中細菌的氫氧自由基產生量的關係。
我們利用馬蹄蟹的Sushi 3 胜肽做為模板,將這段34個胺基酸的胜肽做點定位突變,得到一系列的序列後利用生物資訊的軟體做二級結構預測。從預測結果中挑選具有不同α-螺旋比例的胜肽來合成,並用傅利葉轉換紅外光光譜鑑定這些胜肽的二級結構。α-螺旋比例最高的I8G15P18 胜肽具有最佳的殺菌能力,及最高的氫氧自由基產生量。由實驗數據我們可知道α-螺旋比例和在胜肽的殺菌過程中,細菌所產生的氫氧自由基具有相關性。
目前的研究中普遍認為α-螺旋類抗菌胜肽比β-平板類具有更好的殺菌能力,在我們的研究中發現,這可能和α-螺旋類抗菌胜肽在殺菌過程中,會導致細菌產生氫氧自由基有關。
In order to combat the contentious developments of bacterial antibiotic resistance, searching and developing new antibacterial drugs has become a vital task for clinical applications. Antimicrobial peptides are one of the most important approaches for this purpose.
Previous studies indicated that hydroxyl radical productions are the common mechanism during the antibiotic treatments of bacteria. Our research group also found that the antimicrobial peptides were also capable of induce the hydroxyl radical productions during the treatments, and this effect might be associated with the secondary structures of the peptides. This study attempted to link the secondary structure contents and the quantity of the hydroxyl radical productions during the antimicrobial treatments.
Bioinfomatic approach was applied to predict the secondary structure contents of the mutant peptide derived from horseshoe crab Sushi 3 peptide via series of point mutations in its sequence. The predicted mutant peptides were chemically synthesized, and structurally examined using biophysical techniques Fourier transform infrared spectroscopy (FT-IR). The FTIR results indicated that the Sushi mutant I8G9A, G9A, I8G15P18A peptides had different helix contents, and the highly helix containing peptide I8G15P18A showed the best bactericidal ability. Peptides with higher helix contents also resulted in higher hydroxyl radical productions during bactericidal treatments as measured by 3’-(p-hydroxyphenyl) fluorescein dye and flowcytometry. The results of this study will provide valuable information for the design of new and effective antimicrobial peptides.
碩士論文審核通過書…………………………………………………………………1
致謝……………………………………………………………………………………2
Abstract………………………………………………………………………………..3
中文摘要………………………………………………………………………………4
Contents………………………………………………………………………………..5
List of tables…………………………………………………………………………...6
List of figures………………………………………………………………………….6
Abbreviations………………………………………………………………………….7
1. Introduction…………………………………………………………………………8
2. Aim of the study……………..…………………………………………………......9
3. Materials and methods…………………………………………………………….10
3.1 Peptide secondary structure prediction using bioinformatic methods…………10
3.2 Hydrophobicity of peptide………………………………………………...…..10
3.3 Peptide synthesis and purifications……………………………………………11
3.4 Peptide secondary structure measurement…………………………………….12
3.5 Hydroxyl radical production measurement by flowcytometry……………...…15
3.6 Bactericidal ability of peptides………………………………………………...17
4. Results……………………………………………………………………………..18
4.1 Prediction results of selected peptides………………….……………………...18
4.2 Peptide secondary structure measurements using FTIR……………………….18
4.3 Relationship of peptide α-helix contents (%) and amphipathicity…………….21
4.4 Bacteria hydroxyl radical production and peptide bactericidal ability…...……22
5. Discussion…………………………………………………………………………25
6. Conclusion…………………………………………………………………………29
7. Reference…………………………………………………………………………..39
8. Appendix…………………………………………………………………………..42
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