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研究生:魏秀如
研究生(外文):Wei, Hsiu Ju
論文名稱:顯著增加短鏈抗菌肽抗內毒素之效益
論文名稱(外文):Boosting Anti-Endotoxin Effects of short Antimicrobial Peptides
指導教授:程家維
指導教授(外文):Cheng, Jya-Wei
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:55
中文關鍵詞:抗內毒素萘基丙胺酸尾端修飾短鏈抗菌肽
外文關鍵詞:anti-endotoxin effectβ-naphthylalanineend-taggingshort antimicroial peptides
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敗血症一直是各國之間非常重要的死亡原因之一.在美國每年約有75萬人罹患敗血性休克,而其中導致死亡的人數更是高達二十二萬五千人.引發敗血症的原因主要是革蘭氏陰性細菌外膜組成成分之一的內毒素(又稱作脂多醣,LPS),其會在細菌死亡,細胞膜破裂、損壞時,釋放到人體血液或組織中.雖然抗菌肽與細菌細胞膜之間的構效關係(structure-activity relationship)已被研究多年,然而對於抗菌肽與細菌細胞膜上內毒素的作用關係卻很少科學家討論.
在之前我們已經建立一個簡易提升抗鹽性與血清穩定性的方法,藉由抗菌肽的尾端加上非自然性胺基酸-萘基丙胺酸(β-naphthylalanine)作為修飾.在研究中,短鏈富含色胺酸的抗菌肽在高鹽濃度下活性會受到抑制,然而末端以萘基丙胺酸修飾的抗菌肽活性則沒有受到高鹽濃度的影響.萘基丙胺酸末端修飾可以有助提升抗菌肽與細菌或真菌細胞膜作用,而讓抗菌肽更有效破壞細胞膜,並且即使位在高鹽濃度中仍保有抗菌活性.因此,我們希望延續此研究,探討利用萘基丙胺酸尾端修飾的抗菌肽是否也能有效抑制細菌細胞膜上內毒素的活性,這將可以幫助我們設計更符合臨床使用的抗菌肽.

In America, there are about 750,000 cases of septic shock each year. Among these cases, 225,000 of them are fatal. The major cause of sepsis is from endotoxin (also referred as lipopolysaccharide or LPS). Endotoxin constitutes the major component of the outer leaflet of Gram-negative bacteria membrane and is released to blood or tissue upon lysis of bacteria. While structure-activity relationships have been extensively studied with regard to antimicrobial peptide and bacteria membrane interactions, less is known about the anti-endotoxin effects of antimicrobial peptides.
Previously, we have developed an easy strategy to boost salt resistance and serum stability of short antimicrobial peptides by adding the non-nature bulky amino acid β-naphthylalanine to their C-termini. The activities of the short salt-sensitive tryptophan-rich peptide S1 were diminished at high salt concentrations, whereas the activities of its β-naphthylalanine end-tagged variants were less affected. β-naphthylalanine end-tagging may help these peptides to penetrate deeper into the bacterial and fungal cell membranes, hence making these peptides more efficient at disrupting the membranes even under high salt conditions. Herein, we have extended this study to characterize the anti-endotoxin effects of β-naphthylalanine end-tagged short antimicrobial peptides. The results will be used to help us design more clinical feasible antimicrobial peptides.

Contents
致謝 vii
摘要 viii
Abstract ix
Chapter 1. Introduction 2
1.1 From bloodstream infection to Sepsis 2
1.2 Pathogenicity of Gram negative bacteria in sepsis 3
1.3 Current treatments of sepsis 4
1.4 Antimicrobial peptides 4
1.5 Endotoxin neutralizing peptide 5
1.6 Tryptophan-rich peptides 6
Chapter 2. Materials and methods 8
2.1 Peptides synthesis 8
2.2 Quantization of the peptides 8
2.3 Limulus amebocyte lysate(LAL) assay 8
2.4 Circular Dichroism (CD) 10
2.5 Tryptophan blue-shift assay 12
Experiment procedure 13
2.6 Tryptophan quenching 13
2.7 Dynamic light scattering (DLS) 15
2.8 Isothermal titration calorimetry (ITC) experiments 16
Chapter 4 Results 19
4.1 RP-HPLC 19
4.2 Limulus amebocyte lysate chromogenic assay 19
4.3 Circular Dichroism 19
4.4 Tryptophan blue-shift assay 20
4.5 Quenching 20
4.6 Dynamic light scattering (DLS) 21
4.7 Isothermal titration calorimetry (ITC) experiments 21
Chapter 5 Discussion 23
Chapter 6 Conclusion 26
References 52


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