趨化因子(chemokine)，也稱做趨化激素，是屬於細胞因子家族的其中一員的小分子蛋白，具有趨化免疫細胞到發炎組織周圍的能力。CCL5(又稱 RANTES)是一個重要的趨化因子，會誘發白血球附著及移轉位置至發炎部位。CCL5與許多重要的疾病有關，而CCL5在臨床上或細胞上的研究大多以老鼠做為一個重要的動物模型，因此我們的目標是確認老鼠CCL5與人類CCL5兩者間的相似性，證明老鼠CCL5可以利用老鼠模型進行各項在人體試驗前的研究。老鼠CCL5與人類CCL5胺基酸序列上十分相似，然而老鼠CCL5尚未有任何結構資訊且尚未與人類CCL5做比較。因此我們建立CCL5在兩個跨物種之間的比較。首先，我們利用大腸桿菌做為表現系統，成功建立改良的方法純化老鼠CCL5。接著我們確認老鼠CCL5的結構特性並和人類CCL5比較。利用核磁共振實驗結果發現老鼠和人類CCL5具有相似的二級結構分布，同時地，影響人類CCL5多聚體形成的重要氨基酸E66突變成Serine後在老鼠模型中也具有相同的影響。這個研究改良CCL5的純化與製備方式並且建立老鼠CCL5的結構用以在老鼠模型中藥物的測試及人體試驗前的研究。

Chemokines are a family of small cytokines, also called chemotactic cytokines. Chemokines have the ability to induce chemotaxis for immune cells. CCL5 (also known as RANTES) is an important chemokine that demonstrates a feature to induce leukocytes adhesion and transmigration in the site of inflammation. CCL5 plays an important role in many proinflammatory diseases. Most of the trials of CCL5 uses mouse as the model to conduct clinical or cell tests. Thus, we aim to understand if mouse CCL5 (mCCL5) is suitable to mimic human CCL5 (hCCL5) in the mouse model. mCCL5 contains sequence similar to hCCL5. However, there is no information related to mCCL5 structure, even no structural comparison between the two chemokines. Therefore, we established the comparison between the two cross-species CCL5s. Firstly, using E. coli as the expression system, we established an improved method for mCCL5 preparation. Subsequently, we determined structural property of mCCL5 and compared to hCCL5. By using NMR method, human and mouse CCL5 share similar secondary structural elements and meanwhile, oligomerization behavior that the residue E66 involved in hCCL5 self-aggregation also involved in mCCL5 aggregation. Thus, this research confirms mCCL5 and mouse model can be a good system for testing drug candidates in prior of human trials. The established method also benefits for rodent CCL5 production in the future.

Content
ABSTRACT 1
中文摘要 2
CONTENT 3
ABBREVIATIONS 5
CHAPTER 1 6
1.1 Chemokine 6
1.2 CCL5 (RANTES) 7
1.3 CCL5 is associated with a wide range of immune-mediated diseases 8
1.3.1 CCL5 and atherosclerosis 8
1.3.2 CCL5 and HIV-1 9
1.4 Methionylated CCL5 (Met-CCL5) 10
1.5 Oligomeric state of human CCL5 11
1.5.1 Monomer 11
1.5.2 Dimer 12
1.5.3 High-order oligomer 13
1.6 Aims of research 14
CHAPTER 2 20
2.1 Cloning constructs of recombinant mCCL5 20
2.2 Point mutation of mCCL5 to E66S-mCCL5 20
2.3 Strategies for expression and purification of mCCL5 22
2.4 Expression and purification of E66S-mouse CCL5 24
2.5 Cloning constructs of recombinant Met-mCCL5 24
2.6 Point mutation of Met-mCCL5 to Met-E66S-mCCL5 25
2.7 Strategies for expression and purification of Met-mCCL5 26
2.8 Expression and purification of Met-E66S-mCCL5 27
2.9 15N labeled and 15N, 13C labeled protein for a series of NMR experiments 27
CHAPTER 3 38
3.1 human CCL5 NMR structures 38
3.2 Met-mCCL5 for structural investigation by NMR 39
3.3 NMR 1H-15N HSQC for studying a protein 39
3.3.1 Temperature-dependency 1H-15N HSQC of Met-mCCL5 39
3.3.2 pH-dependency 1H-15N HSQC of Met-mCCL5 40
3.3.3 Salt-dependency of 1H-15N HSQC of Met-mCCL5 41
3.4 Backbone assignment of Met-mCCL5 41
3.5 Backbone assignment of Met-E66S-mCCL5 42
3.6 The chemical shift variations of Met-mCCL5 and Met-E66S-mCCL5 43
3.7 Comparison of secondary structure prediction between Met-mCCL5 and Met-E66S 44
3.8 Met-mCCL5 assignment compares with Met-hCCL5 assignment 44
3.9 Dihedral angle prediction of Met-mCCL5 by TALOS+ 45
3.10 NMR assignments and structure determination 46
3.10.1 NMR 3D side chain experiments 47
3.10.2 NMR 3D NOESY experiments 47
CHAPTER 4 64
4.1 Biological test of mCCL5 and Met-mCCL5 64
4.2 Size-determination of MetmCCL5 and Met-E66S-mCCL5 64
CHAPTER 5 65
5.1 The two strategies improved CCL5 protein quality and yield 67
5.2 Human and mouse CCL5 share similar structural properties 68
5.3 Human and mouse CCL5 share similar aggregation properties 68
5.4 Structure determination of CCL5 69
REFERENCES 71

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