在免疫防禦機制中，人類受到外在或是內在病原的侵襲後，會使人體產生趨化因子，吸引相關免疫細胞進行防禦。然而，過度的免疫細胞浸潤會進而造成組織二度傷害。在此我們探討嗜中性白血球的趨化蛋白，介白素8，為72個胺基酸組成的ELR-CXC趨化素。介白素8可與嗜中性白血球膜上受體CXCR1以及CXCR2結合從而誘導發炎反應。在先前研究中，已經設計出介白素8的類似物，RP4，並經過研究證實可以有效抑制CXCR1/CXCR2所引起的趨化作用。實驗室團隊近年也成功表現膜蛋白GST-CXCR1以及GST-CXCR2。所以在此研究中，我們完成GST-CXCR1切除GST的步驟，並將完整膜蛋白重構在脂質上形成蛋白脂質體。初步以動態光散射儀以及穿透式電子顯微鏡分析蛋白脂質體的物理特性，得到初始蛋白脂質體粒徑以及型態。利用ELISA親和力實驗分析CXCR1與IL-8以及RP4的解離常數。本研究結果顯示，在CXCR1無重構的情況下，RP4比IL-8具有比較好的親和力。並且在重構後的情況，也有相同的結果。我們也發現在重構後的膜蛋白比沒重構的蛋白具有更好的親和力，證明重構膜對膜蛋白有一定的影響。本研究結果將有助於未來該類似物在結構生物學上的分析，對研究未來其他膜蛋白與其配體將提供可靠的資訊。

In the immune defense mechanism, humans are attacked by pathogens, causing the body to produce chemokines, and attracting relevant immune cells to defend. However, excessive immune cell infiltration cause tissue second-degree damage. Here we discuss the neutrophil chemoattractant protein, Interleukin-8, consisting of 72 amino acids ELR-CXC chemokine. Interleukin 8 can bind to the receptors CXCR1 and CXCR2, inducing an inflammatory response. In our previous studies, an analogue of interleukin 8 which named RP4 had been designed, and it had been determined that it could effectively inhibit the chemotaxis which is caused by CXCR1/CXCR2 pathway. In recent years, we had purified GST-CXCR1 and GST-CXCR2. In order to study on structure of protein, we completed the subsequent purification of cleaving GST from GST-CXCR1 and reconstituted the CXCR1 to form proteoliposomes. The physical properties of protein liposomes were analyzed with dynamic light scattering (DLS) instrument and transmission electron microscope (TEM) to obtain proteoliposome shapes and particle sizes. Using ELISA affinity experiments analyzed the dissociation constants of reconstituted CXCR1 with IL-8 and RP4. Our results showed that in the case of CXCR1 absence of reconstitution, RP4 had a better affinity than IL-8 and had the same results after reconstitution. The results also showed that the membrane protein after reconstitution had better affinity than the non-reconstituted protein. This proved that reconstituted had a definite influence on the activity of membrane proteins. This study will provide effective route to the future analysis of structure on other membrane proteins.

Table of content
中文摘要 3
Abstract 4
Table of content 5
Chapter 1 Introduction 6
1.1 CXC Chemokines 6
1.2 The role of interleukin-8 (IL-8/CXCL8) 6
1.3 Design of the analogues of interleukin-8 7
1.4 Receptors of ELR+ CXC Chemokine, CXCR1 and CXCR2 8
1.5 Proteoliposome 9
Chapter 2 Material and Methods 10
2.1 Chemical and Reagents 10
2.2 Expression and purification of recombinant IL-8 and RP4 11
2.3 Expression and purification of recombinant CXCR1 12
2.4 Determination of CXCR1 by Western blotting 14
2.5 Reconstitution of membrane protein 14
2.6 Characterization of proteoliposomes 15
2.7 Determination of dissociation constants by ELISA 15
2.8 Statistical analysis 18
Chapter 3 Results 18
3.1 Cleave GST tag to purify full length CXCR1 18
3.2 Reconstitute CXCR1 by detergent-mediated method 19
3.3 DLS and TEM show the particle size and shape 20
3.4 RP4 binds to reconstituted CXCR1 has better binding affinity than IL-8 21
Chapter 4 Conclusion and Discussion 23
Tables & Figures 25
Abbreviation Index 49
References 50

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