傳統微陣列晶片技術不適合用來分析具有多個結合位之蛋白質，因為晶片表面的配基密度和間距沒有辦法被有效控制與確定。因此近年來許多團隊引進了輔助性分子到微陣列晶片上希望藉此控制配基的密度和分布狀況；但由於大多被使用的是具彈性的骨架，配基間距沒有辦法被有效控制。在這篇論文中引進了脯胺酸多肽PPII的結構來作為分子骨架，使得配基間的距離能夠控制在9 Å、18 Å以及27 Å。另一方面，我們也引進了全氟烷基到脯胺酸多肽骨架上，一方面利用氟與氟之間的作用力連接到微陣列晶片上，一方面控制PPII螺旋結構在表面的位相。本論文中對LecA、WGA以及2G12抗體做測試，分別以半乳醣、N-乙醯葡萄糖胺以及Man4作為配基接到骨架上。LecA以及2G12對27 Å的骨架具有較高的專一性，這個結果對應到了LecA結合位置的間距26 Å以及2G12的35 Å；而WGA則對三種骨架都有不錯的專一性，9 Å以及18 Å的骨架應到了13-14 Å的結合位置間距，27 Å的骨架則對應到29 Å的結合位置間距。最後，我們針對2G12抗體，做出同樣以脯胺酸多肽為骨架的2G12抑制劑，並藉由表面電漿共振實驗得到解離常數。結果發現以27 Å骨架所做的抑制劑的解離常數最小，此結果與微陣列晶片實驗的結論相符。這些實驗結果證明此種新的微陣列晶片設計可以初步但精確地得到多價性蛋白質的結合位置資訊，相信配合著微陣列晶片的優點，能夠對於多價性蛋白質抑制劑的開發有相當大的幫助。

Conventional microarray systems are not suitable for analyzing the multivalent binding properties of proteins, due to the uncertainty of the distances between the distributed ligands on the surface. Recently, many groups introduced supportive molecules to control the distribution of the ligands on the surface. However, flexible scaffolds are often used, causing the distances between ligands not precisely controlled. Here we introduced PPII structure as supportive scaffold to adjust the distance between glycan ligands attachment sites at 9 Å, 18 Å and 27 Å on the peptide scaffold. Also, perfluoroalkyl groups were introduced to the scaffold for immobilization to the microarray surface via fluorous interaction to control the orientation of the helical scaffolds. Using LecA, WGA and 2G12 antibody as model protein, LecA and 2G12 showed binding preference to the 27 Å scaffold which match the binding distance of 26 Å between two galactose binding sites of LecA and 35 Å between two high-mannose binding sites of 2G12 antibody. On the other hand, WGA has indistinguishable binding affinity to three of the scaffold due to the multi-distances of 13-14 Å and 29 Å between GlcNAc binding sites. Finally, surface plasmon resonance assay of 2G12 to Man4 conjugated polyproline inhibitors were also done to confirm the result of microarray, which exactly matched the result of SPR. This novel strategy could provide a preliminary but precise information of the distance between binding sites of multivalent binding proteins, which is essential to the development of multivalent inhibitor to these proteins.

摘要 1
Abstract 2
目錄 4
圖目錄 6
表目錄 8
式目錄 8
流程目錄 8
縮寫表 9
第一章、緒論
1.1. 前言 12
1.2. 醣類與蛋白質之交互作用 13
1.2.1. LecA 13
1.2.2. WGA 14
1.2.3. HIV中和抗體─2G12 16
1.3. 多價交互作用 18
1.4. 微陣列晶片 20
1.4.1. 表面官能基化 21
1.4.1.1. 非共價性固定 21
1.4.1.2. 共價性固定 22
1.4.2. 全氟烷微陣列 23
1.5. 脯胺酸多肽的特性及應用 25
1.5.1. 脯胺酸多肽的結構與特性 25
1.5.2. 脯胺酸多肽在生物研究上的應用 26
1.6. 研究動機 27
1.7. 實驗設計 30

第二章、結果與討論
2.1. 固相多肽合成構築單元合成 34
2.2. 氟脯胺酸多肽合成 37
2.3. 全氟烷晶片製備 39
2.4. Cy3標記之氟脯胺酸多肽在氟晶片上的測試 41
2.5. 氟脯胺酸多肽在氟微珠上的測試 45
2.5.1. 全氟烷微珠製備 45
2.6. 全氟烷醣脯胺酸多肽合成 48
2.6.1. 炔基氟脯胺酸多肽合成 48
2.6.2. 醣類配基合成 50
2.6.3. 氟脯胺酸多肽與醣簇之銅催化疊氮─炔環化加成 50
2.7. 醣基化氟脯胺酸多肽在氟晶片上測試凝集素與配基的選擇性結合 52
2.7.1. Cy3-LecA與多肽24-26之專一性測試 53
2.7.2. Cy3-WGA與多肽27-29之專一性測試 56
2.7.3. Cy3-2G12與多肽30-32之專一性測試 58
2.8. 以表面電漿共振測試2G12對多肽33-36之解離常數 60
2.9. 結論 63

第三章、實驗材料與方法
3.1. Synthesis of Peptide Building Blocks 65
3.2. Peptide Synthesis 81
3.3. Cy3 Coupling and Glycan Conjugation 84
3.4. Flourous Quartz Beads Preparation 93
3.5. CD Measurement and Assay 94
3.6. Fluorous Slide Preparation 95
3.7. Microarray Printing 95
3.8. Micaoarray: Cy3-Peptide Washing Test 96
3.9. Protein Labelling 98
3.10. Micaoarray: Protein Binding Test to Glycopeptide 99
3.10.1. LecA 101
3.10.2. WGA 104
3.10.3. Antibody 2G12 107
3.11. Microarray Scanning and Data Processing 109
3.12. Surface Plasmon Resonance 110

第四章、參考文獻 113

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