鉤端螺旋體病是一種好發於亞熱帶地區的傳染性疾病，屬於人畜共通感染的疾病，鉤端螺旋體菌主要感染於人類的腎臟並造成急性腎衰竭。而鉤端螺旋體菌為病原菌的一種並且是造成鉤端螺旋體病發生的原因，其外觀呈現細桿螺旋狀並含有鞭毛。LipL32脂蛋白是在鉤端螺旋體菌外膜上表現量最多的脂蛋白，並含有一段高度保留性的[LVI][ASTVI][GAS][C]區域稱之為lipobox，此lipobox區域上的cysteine胺基酸通常會經由脂肪酸的修飾增加蛋白與細胞膜的結合性。之前我們實驗室已經決定LipL32脂蛋白結構(PDB code: 2WFK)。然而目前對於鉤端螺旋體菌感染宿主的機制仍然不完全了解。先前已經有文獻發現cathelicidin類的抗菌胜肽BMAP-28，SMAP-29和LL-37對於鉤端螺旋體菌有著抑制其移動和生長的能力。本篇研究想要知道抗菌胜肽(BMAP-28，SMAP-29和LL-37)跟LipL32脂蛋白交互作用的機制為何，我們從定性的生物素蛋白沉澱實驗(biotin pull-down assay)和定量的等溫滴定微量熱儀(isothermal titration calorimetry)的分析結果發現SMAP-29抗菌胜肽與LipL32脂蛋白有交互作用。並利用陽離子交換層析管柱分離得到SMAP-29與LipL32脂蛋白複合物。藉由結晶學方法進行結構與功能的研究探討SMAP-29和LipL32脂蛋白之間可能的交互作用，這些結果將有助於未來在治療鉤端螺旋體病的研究發展。

Leptospirosis is widespread, zoonotic and cause human acute renal failure. The pathogen, Leptospira, which cause leptospirosis, is a thin and spiral-shaped bacterium with flagella. LipL32 is the most abundant lipoprotein of Leptospira, and the cysteine of lipobox “[LVI][ASTVI][GAS][C]” is lipidated by fatty acids. The three-dimensional structure of LipL32 has been solved in our laboratory (PDB code: 2WFK). However, the infection mechanism of Leptospira is still unclear. Recently, report indicated that Leptospira growth can be inhibited by cathelicidin-derived antimicrobial peptides (AMPs) which originally found in mammals, such as BMAP-28, SMAP-29 and LL-37, etc. In this study, the antimicrobial peptides (BMAP-28, SMAP-29 and LL-37) were selected to study the possible interaction with LipL32. We have successfully found that SMAP-29 may interact with LipL32 by biotin pull-down assay and isothermal titration calorimetry (ITC) assay. The SMAP-29-LipL32 complex was isolated by cation exchange chromatography but the mount of complex is few. Based on the crystallization or structural and functional studies, the specific interaction of SMAP-29 and LipL32 can be identified. However, BMAP-28 and LL-37 show no interaction with LipL32. From these results, SMAP-29 would be more benefited in drug development for treating leptospirosis.

中文摘要 I
Abstract II
誌謝 III
Chapter 1 1
Introduction 1
Chapter 2 7
Materials and methods 7
2.1 Expression and purification 7
2.2 Antimicrobial Peptides 8
2.3 Size-exclusion chromatography 8
2.4 Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry 9
2.5 Circular dichroism spectroscopy 9
2.6 Biotin pull-down assay 10
2.7 Western blotting analysis 11
2.8 Isothermal Titration Calorimetry (ITC) 12
2.9 Ion exchange chromatography 12
2.9.1 Anion exchange chromatography 12
2.9.2 Cation exchange chromatography 13
2.9.3 Ion exchange chromatography analysis 14
2.10 Molecular docking 14
Chapter 3 16
Results and Discussion: 16
3.1 Characterization, Expression and Purification of LipL32 16
3.2 Characterization of antimicrobial peptides 17
3.3 Biotin pull-down assay analysis of interaction between LipL32 and AMPs 18
3.4 SMAP-29 and LipL32 interaction confirmed by Isothermal Titration Calorimetry (ITC) 20
3.5 SMAP-29-LipL32 complex separated by ion exchange chromatography 21
3.6 The structure of SMAP-29-LipL32 complex by molecular docking 23
Chapter 4 26
Conclusions 26
Tables and Figures 30
Reference 51

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