臺灣博碩士論文加值系統

整合蛋白們（integrins）是位於細胞表面異質雙體的接受器，其功能為細胞移動、黏附上不同的細胞或是不同的細胞基質，整合蛋白的配體是利用RGD ( Arg-Gly-Asp )、ILDV或是短的序列作為它們與整合蛋白黏結的基本結構。從蛇毒中純化出去整合蛋白為含有保留性的RGD與 cysteine-rich的蛋白家族。很多研究指出RGD motif 與 RGD motif 相鄰的胺基酸對於整合蛋白與配體間的辨識扮演很重要的角色，而且這類蛋白主要是與整合蛋白 β1 和 β3 家族作用。在本研中，我們以Rhodostomin（簡稱Rho）為鷹架，去探討 RGD motif 與 RGD motif 相鄰的胺基酸對於整合蛋白與配體間的辨識， Rho 為去整合蛋白中的一員，它含有68個胺基酸，以及6對雙硫鍵鍵結，並且在序列48∼53上含有PRGDMP的序列。在我們先前研究中發現到在RGD motif 上R與D之間的殘基，或是在相接於RGD motif C-terminally 含有 DL （Asp-Leu）的殘基，兩者皆具有對於整合蛋白αVβ3有特異性。結合以上兩著的概念，我們設計出 48PRXDDL53 motif，藉此了解在R與D之間的殘基對於整合蛋白辨識所造成的影響。除了實驗室先前表現PRGDDL與PRLDDL，我已經利用 P. pastoris 系統表現出十七株Rho 的突變蛋白，各為PRADDL 、 PRDDDL 、 PREDDL 、 PRFDDL 、 PRHDDL 、 PRIDDL 、PRKDDL 、 PRMDDL 、 PRNDDL 、 PRPDDL 、 PRQDDL 、 PRRDDL 、PRSDDL 、 PRTDDL 、 PRVDDL 、 PRWDDL 和 PRYDDL 並均質純化，這些Rho 的突變蛋白在P. pastoris 表系統的產率為4.3∼16.5mg/L，其突變株之預估分子量與實驗值誤差小於1 Da，暗示著在 Rho 和其突變株有六對雙硫鍵的形成。利用血小板凝集和細胞黏著試驗結果發現Rho的含有48PRXDDL53突變對αIIbβ3與α5β1親和性差，其中PRMDDL、 PRPDDL、 PRWDDL與PRLDDL能夠特異性的抑制整合蛋白αVβ3，其IC50數值分別是175、188、387與342 nM。此研究，可以去探討整和白與去整合蛋白結構與功能間的關係，提供未來設計專一性抑制整合蛋白αvβ3的去整合蛋白。

Integrins are heterodimeric cell surface receptors required for cell trafficking and for adhesion to other cell types and to constituents of the extracellular matrix. The ligands of integrins utilize RGD, ILDV, or short sequences as a key structural component for their integrin-binding site. Disintegrins are a family of RGD-containing and cysteine-rich proteins isolated from snake venoms. Many reports show that the RGD motif and the amino acid residues flanking the RGD sequence of disintgrins play an important role in recognizing integrins. They mainly interact with the β1 and β3 families of integrins. In this study we used rhodostomin (Rho) as the scaffold to study the roles of the RGD motif and the amino acid residues flanking the RGD sequence of disintegrins in recognizing disintegrins. Rho is a disintegrin that contains 68 amino acids including 6 disulfide bonds and a PRGDMP sequence at the positions of 48-53. Our previous study showed that Rho containing either the residue flanking the R and D residues or the DL residues C-terminally adjacent to RGD motif had high potency and specificity to integrin αVβ3. Therefore, we incorporated the 48PRXDDL53 motif into Rho to study the effect of the residue flanking the R and D residues in recognizing integrins. In addition to PRGDDL, PRLDDL, and PRIDDL mutants, I have expressed seventeen mutants PRHDDL, PRYDDL, PRADDL, PRCDDL, PRDDDL, PREDDL, PRFDDL, PRKDDL, PRMDDL, PRNDDL, PRPDDL, PRQDDL, PRRDDL, PRSDDL, PRTDDL, PRVDDL and PRWDDL in Pichia pastoris and purified them to homogeneity. The mutant proteins produced in P. pastoris were 4.3-16.5mg/L. The experimental molecular weights of the mutants deviate <1 Da when compared with calculated values, indicating the formation of six disulfide bond in these mutants. The analysis of platelet aggregation and cell adhesion assays showed that Rho mutants-containing the PRXDDL motif has low potency to integrin α5β1 and αIIbβ3. The mutants PRMDDL, PRPDDL, PRWDDL, and PRLDDL can selectively inhibit integrin αvβ3 with the IC50 value of 175, 188, 387, and 342 nM, respectively. This study may serve as the basis for exploring the structure and function relationships of integrins and disintegrins and for designing integrin αvβ3-specific disintegrins.

中文摘要 I
英文摘要 II
誌謝 IV
目錄 V
表目錄 VIII
圖目錄 IX
縮寫檢索表 X
儀器 XII

第一章 緒論 1
1-1 整合蛋白 (Integrin) 之介紹 1
1-2 去整合蛋白 (Disintegrin) 之介紹 4
1-3 馬來蝮蛇去整合蛋白 (Rhodostomin；簡稱Rho) 之介 6
1-4 酵母菌Pichia pastoris (P. pastoris) 表現系統之介 7
1-5 論文研究動機與目標

第二章 材料與方法 11
2-1 Rho與突變株重組蛋白之製備 11
2-1-1 實驗菌株、質體與培養基配方 11
2-1-2 重組基因之構築 13
2-1-3 重組蛋白之表現與純化 16
2-1-4 重組蛋白之質譜鑑定 22
2-2 Rho與突變株重組蛋白抑制血小板凝集功能之研究 23
2-2-1 血小板之製備 23
2-2-2 重組蛋白抑制血小板凝集之功能分析 24
2-3 Rho與突變株重組蛋白抑制細胞黏著之研究 24
2-3-1 細胞培養 25
2-3-2 Fibronectin之製備 27
2-3-3 重組蛋白抑制細胞黏著之功能分析 28

第三章 結果 30
3-1 Rho與突變株重組蛋白之製備與鑑定 30
3-2 Rho與突變株重組蛋白抑制血小板凝集的結果 31
3-3 Rho針對αVβ3 設計突變株重組蛋白抑制細胞黏著的結 32
3-3-1 測試抑制細胞黏著條件的結果 32
3-3-2 Rho針對αvβ3設計突變株重組蛋白對整合蛋白αIIb3、αvβ3 和α5β1的影響 33
3-3-3 Rho針對αvβ3設計突變株重組蛋白增加對整合蛋白αIIb3和α5b1的特異性 34
3-3-4 Rho針對αvβ3設計突變株重組蛋白增加對整合蛋白αvβ3特異性與親和性 34

第四章 討論 35
4-1 Rho突變株49RXDDL53重組蛋白對於整合蛋白的影響 35
4-1-1 Rho突變株48PRXDDL53重組蛋白增加對整合蛋白αIIbβ3和α5β1的特異性的討論 37
4-1-2 Rho針對αVβ3設計突變株重組蛋白對整合蛋白αVβ3的討論 38

第五章 結論 39
參考文獻 40
表 46
圖 58
附錄 73
自述 96

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