鉤狀螺旋體屬的致病螺旋體分佈廣泛，經常能引起一種嚴重的人畜共通的鉤狀螺旋體病。儘管最近幾年，隨著對致病螺旋菌體研究的深入，人們對於致病螺旋體感染宿主的致病機理愈加愈清晰。但是，對於其一毒素因子LipL32脂蛋白的研究尚不完整。LipL32是一種在致病螺旋體外膜上大量存在的脂蛋白，當在它的lipobox上發生脂化後，便會轉移到菌體的膜上進而發揮毒素因子的作用。致病螺旋體膜外蛋白與宿主的細胞外基質組分(ECM: extracellular matrix molecules)的接觸會導致致病螺旋體病的發生。纖維連接蛋白(Fibronectin)是細胞外基質組分中重要的組成部分之一，也是病菌與宿主發生相互作用的媒介之一。由我們實驗室之前的研究，通過stains-all CD, ELISA(Enzyme-Linked Immunosorbent Assay)實驗證明第一型纖維連接蛋白的肝素結合區域 (F30) 能結合LipL32，而且鈣離子能對F30結合LipL32進行調節。第三型纖維連接蛋白12-13片段(FN1213)也具有重要的肝素結合域。通過分子結合模擬的方法預測，經由FN1213蛋白的表面分佈的正電荷(R98/R99/R101/R115/K117/R146)，能與LipL32蛋白表面D-loop(142DDDDD146, D146/D149)分佈的負電荷相互作用，LipL32可與FN1213 結合成一可能的複合物。在這次實驗中，我們利用Native-PAGE，化學交連反應(chemical cross linking)和等溫滴定量熱議(ITC)等實驗檢試LipL32與FN1213相互作用的可能，發現LipL32與FN1213相互作用相互作用力極其微弱，以至於很難形成複合物晶體。於複合物晶體成長實驗，我們只得到單獨FN1213的晶體而非LipL32與FN1213複合物晶體。通過這個研究，我們提供生化實驗討論LipL32與FN1213相互作用的研究，希望有利於進一步瞭解這兩個蛋白質的特性以及鉤狀螺旋體感染宿主的機制。

Spirochetes of the genus leptospira often cause, with worldwide distribution, the serious zoonotic disease leptospirosis. There have been numerous advances in the understanding of the molecular pathogenesis of the infection the host, yet the mechanism of a viral factor of leptospira, LipL32, remains unclear. LipL32 is a lipoprotein and abundant on the outer membrane of leptospira. Leptospirosis occurs through the leptospira outer membrane proteins including LipL32 interact with the extracellular matrix (ECM). Fibronectin (FN) is a prominent component of the extracellular matrix molecules with which bacterial cell surface proteins interact. The binding of F30 to LipL32 has been observed by stains-all CD and enzyme-linked immunosorbent assay experiments. And Ca2+ can modulate fibronectin binding to LipL32. The heparin-binding site, fibronectin type III repeats 12-13 (FN1213), contains FN12 and FN13 in which takes a clump of positively charged residues. The structural analyses of LipL32 indicate that it possesses a large area of negative electricity. We have predicated the possible interaction of LipL32-FN1213 complex by a docking program. We provide several biochemistry methods such as Native-PAGE, chemical cross linking and ITC (Isothermal Titration Calorimetry) experiments to study the protein-protein interaction using LipL32 and FN1213 as an example. There may be indeed interaction between LipL32 and FN1213 by non-covalent hydrophobic interaction through the D-loop (142DDDDD146, D146/D149) of LipL32 and the positive region (R98/R99/R101/R115/K117/R146) of FN13. From this study we suggest the interaction between LipL32 and FN1213 should be week and non-specific hydrophilic interactions.

Abstract I
中文摘要 II
Acknowledgements III
Chapter 1 Introduction 1
Chapter 2 Materials and Methods 4
2.1 Expression and purification 4
2.2 Molecular docking 5
2.3 Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry 6
2.4 Circular dichroism spectroscopy 6
2.5 Protein decalcification 7
2.6 SDS-PAGE and native-PAGE 7
2.7 N-termial aminal acid sequencing 8
2.8 Size exclusion chromatography (SEC) 8
2.9 Chemical cross linking 9
2.10 Isothermal Titration Calorimetry (ITC) 10
2.11 Crystallization 10
2.12 X-ray data collection 11
2.13 Structure determination and refinement 11
Chapter 3 Results and Discussion 12
3.1 Characterization of LipL32 and FN1213 12
3.2 Bioinformatic analyses of LipL32 and FN1213 13
3.3 The molecular wight of LipL32 and FN1213 15
3.4 The secondary structures of LipL32 and FN1213 16
3.5 LipL32 and FN1213 interaction confirmed by Native-PAGE 17
3.6 LipL32 and FN1213 interaction confirmed by Isothermal Titration Calorimetry (ITC) 20
3.7 LipL32 and FN1213 interaction confirmed by chemical cross linking and SEC 21
3.8 Crystallization, X-ray data collection and structure determination 23
3.9 FN1213 structure 24
Chapter 4 Conclusion 26
Tables and Figures 28
Reference 57

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