具有ELR-CXC的趨化素在許多急慢性免疫或發炎疾病佔有重要的地位，這類的趨化素可以誘發嗜中性球的免疫功能，並造成免疫系統的激活。介白素8又稱為趨化素CXCL8，是一個由72個氨基酸所組成的蛋白。在急慢性發炎患者體內，趨化素CXCL8的濃度會上升，並可能造成過度的免疫反應，引發傷害。先前的研究中指出，將介白素8的離氨酸11與甘氨酸31同時突變成精氨酸11與脯氨酸31將使介白素8變成其受體CXCR1及CXCR2的擷抗劑。因此，本研究針對此一擷抗劑的生產與純化過程提出一個簡單易於執行，且可提供高純度蛋白質的方法；此外本研究利用核磁共振光譜解出該蛋白在水溶液中的結構，並與原生的介白素8作一結構上的比較，以了解擷抗劑作用機制。本研究同時利用旋光光譜儀、微卡計滴定法、超高速分析式離心等方法，測定出該擷抗劑的各種解離常數與其他物化特性。經由本研究的成果，提供了生產該擷抗劑的有效方法，不僅可大量生產，更可輕易將生產過程中所沾染的內毒素去除。同時，本研究結果也指出，該擷抗劑的擷抗作用主要來自脯氨酸31突變所造成結構變化所導致與觸發其受體訊號傳遞的影響。本研究結果將有助於未來該擷抗劑臨床實驗的進行，並對於了解與開發新的擷抗劑提供一有用的資訊。

The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8) binds to both the CXCR1 and CXCR2 receptors with high affinity and the expression levels of CXCL8 are elevated in many inflammatory diseases. Recently, an analogue of human CXCL8, CXCL8(3–72)K11R/G31P (hG31P) has, been developed. It has been demonstrated that hG31P is a high affinity antagonist for both CXCR1 and CXCR2. To obtain large quantities of hG31P, this study has successfully constructed and expressed hG31P in Escherichia coli. Moreover, a new protocol for high-yield purification of hG31P and for the removal of lipopolysaccharide (LPS, endotoxin) associated with hG31P due to the expression in E. coli has been developed. The solution structure of hG31P is also demonstrated in this study. In addition, the structural properties of hG31P were studied by circular dichroism (CD), ultracentrifuge, isothermal titration calorimetry (ITC), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy. The results demonstrated that the possible mechanism on hG31P to inhibit signal transduction is contributed from the perturbation of the G31P loop. Furthermore, this study also indicated that this purification protocol is very simple and easy to amplify at a large scale. The results of this study will provide good explanation for the antagonist according to the structure basis, and it also provides effective route to produce large quantities of hG31P for future clinical studies.

誌 謝 I
中 文 摘 要 II
Abstract III
Chapter 1 Introduction 1
Chapter 2 Material and Methods 9
Construction expression plasmid 9
Expression of CXCL8(3-72)K11R/G31P recombinant protein 10
Expression of CXCL8(3-72) K11R/G31P protein with 15N label 11
Expression of CXCL8(3-72) K11R/G31P protein with 15N and 13C label 11
Lysis of cells and purification of recombinant CXCL8(3-72)K11R/G31P 12
Endotoxin (LPS) removal and detection 14
Neutrophil chemotaxis assay 15
Assay of intracellular Ca2+ flux 16
Circular Dichroism (CD) spectroscopy 16
Mass Spectrometry 17
Fast performance liquidchromatography (FPLC) analysis 17
Isothermal titration calorimetry (ITC) 17
Fluorescence spectroscopy 18
Analytical ultracentrifugation 19
NMR spectroscopy 20
Data Processing and Analysis 22
Structure Calculation 22
Chapter 3 Results 24
Extraction and purification of hG31P 24
Lipopolysaccharide (LPS) removal 26
Activity assay 28
Identify the purified hG31P via heating and LPS removal procedure with CD and NMR 29
Three dimensional structure of hG31P in solution 29
Equilibrium of monomer and dimmer 31
Binding of the N terminal peptide of CXCR1 to hG31P 32
Chapter 4 Discussion 35
Chapter 5 Conclusion 39
References 77

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