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系統識別號 U0026-1408201217504800
論文名稱(中文) 利用第三型纖維黏結蛋白的第十個模組設計對整合素αvβ3具專一性的拮抗劑
論文名稱(英文) Design and characterization of integrin αvβ3-specific antagonist using the tenth module of fibronectin type III
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 100
學期 2
出版年 101
研究生(中文) 劉欣宜
研究生(英文) Hsin-Yi Liu
學號 S16991100
學位類別 碩士
語文別 中文
論文頁數 106頁
口試委員 指導教授-莊偉哲
口試委員-鄭宏祺
口試委員-鄭文義
中文關鍵字 整合蛋白  第三型纖維黏結蛋白 
英文關鍵字 integrin αvβ3  fibronectin type III 
學科別分類
中文摘要 整合蛋白是一種細胞表面黏著受器,其功能是影響細胞的黏著、遷移跟生長。纖維黏結蛋白是一種細胞外基質的糖蛋白,會與整合蛋白作用。纖維黏結蛋白的組成是由三種不同類別的重複性單元所組成,包括12種第一型、2種第二型、15到17種第三型。特別的是,第三型纖維黏結蛋白的第十個模組對於整合蛋白αvβ3具有低的親和力。我們若要利用第三型纖維黏結蛋白設計對於整合蛋白的藥物,必須使其具有高穩定性、選擇性以及活性。因此我們利用第三型纖維黏結蛋白的第十個模組作為鷹架蛋白,去設計對於整合蛋白αvβ3具有專一性的拮抗劑。在本實驗中,我們將:(1)套用實驗室對於去整合蛋白的研究出的整合蛋白特殊辨識序列或者是之前研究篩選出的對於整合蛋白的特殊辨識序列到第三型纖維黏結蛋白的第十個模組上,(2)並增加一對雙硫鍵於RGD loop上,並改變其雙硫鍵的鍵結模式,(3)接著進行對整合蛋白αvβ3具有專一性的第三型纖維黏結蛋白第十個模組蛋白突變株作特性的分析,(4)最後也將第三型纖維黏結蛋白第十個模組的蛋白突變株應用於癌症的研究上。現在我已利用大腸桿菌系統成功表達20種以上的第三型纖維黏結蛋白第十個模組蛋白突變株,並得到純的蛋白產物。在功能分析上顯示所有的蛋白突變株對於抑制血小板凝集能力是低的,顯示其較不會引起血小板缺乏症。在抑制細胞黏著試驗中,證明了套用去整合蛋白序列的FN-III10(PRGDMPD)突變株對於整合蛋白αvβ3具有高度的親和力。當加入一對雙硫鍵鍵結在RGD loop上,顯示整合蛋白αvβ3傾向於喜好CX7C的patterns,而非CX4C 及CX8C patterns。而且FN-III10(PRGDMPD)選擇性地抑制整合蛋白αvβ3,其IC50值為101 nM。利用量熱儀以及硫酸胺鹽沉澱法測試其熱穩定度及溶解度,分別顯示Tm質與溶解度:FN-III10(CPRGDMPDC)(85℃)(27.8 mg/ml) > FN-III10(81℃)(7.3 mg/ml) > FN-III10(PRGDMPD)(74℃)(5.3 mg/ml),表示當加入一對雙硫鍵鍵結於RGD loop上,可以增加第三型纖維黏結蛋白第十個模組蛋白突變株的熱穩定性和溶解度。再來,利用NMR技術分析證明了FN-III10(CPRGDMPDC)具有正確的摺疊以及動力學分析指出其RGD loop具高穩定性,因此較能辨識整合蛋白αvβ3。此外,在癌症研究上,由體外試驗證明了FN-III10(CPRGDMPDC)可以抑制A375細胞的遷移能力以及HUVEC細胞的tube生成。在體內試驗中,FN-III10(CPRGDMPDC)在減少Lewis lung cancer cells在C57/B6老鼠身上的腫瘤生長上的結果沒有顯著的意義,但仍可以延長老鼠的壽命。總結來說,我們已經成功的將第三型纖維黏結蛋白第十個模組蛋白改變成對於整合蛋白αvβ3具有高度親和力、選擇性,並且具有高的熱穩定性和溶解度。我們的結果顯示我們對於整合蛋白αvβ3專一性的第三型纖維黏結蛋白第十個模組蛋白突變株,較先前所發表的FN-III10(PRGDWNEG)好。此外,如何應用這個專一性的蛋白突變株於整合蛋白αvβ3相關的疾病的研究,也正在進行中。
英文摘要 Integrins are cell-surface adhesion receptors that affect cell adhesion, migration and proliferation. Fibronectin (FN) is an extracellular matrix glycoprotein that binds to integrins. FN is composed of three different types of homologous repeating domains, including FN-I, FN-II, and FN-III. FN contains 12 of FN-I, 2 of FN-II, and 15-17 of FN-III repeats. In particular, FN-III10 can bind to integrin αvβ3 with low affinity. To use FN-III10 as integrin drugs, it is essential to engineer them to have high stability, selectivity, and potency. Therefore, we propose to use FN-III10 as the scaffold to design integrin αvβ3-specific antagonists. In this study I: (1) incorporate the integrin-specific recognition sequences into FN-III based on the results of our study on disintegrin or reported sequences; (2) modify disulfide bond pattern (CXnC) within RGD loop; (3) characterize integrin αvβ3-specific FN-III10 variants; and (4) study anti-tumor activity of integrin αvβ3-specific FN-III10 variants. Now I have expressed > 20 FN-III10 variants in E. coli and purified them to homogeneity. Functional analysis showed that all FN-III10 variants had lower activity in inhibiting platelet aggregation, suggesting that they exhibited low thrombocytopenia effect. Cell adhesion inhibition study demonstrated that FN-III10(PRGDMPD) mutant incorporating disintegrin sequence had higher affinity to integrin αvβ3. The incorporation of disulfide linkage into the RGD loop showed that integrin v3-specific FN-III10 variants preferred the CX7C but not CX4C and CX8C patterns, and FN-III10(CPRGDMPDC) selectively inhibited integrin αvβ3 with an IC50 value of 93 nM. Differential scanning calorimetry analysis and solubility measurements by ammonium sulfate precipitation showed that their Tm values and solubility FN-III10(CPRGDMPDC) (85 oC; 27.8 mg/ml) > FN-III10 (81oC; 7.3 mg/ml) > FN-III10(PRGDMPD) (74 oC; 5.3 mg/ml). These results indicate that the incorporation of a disulfide bond into the RGD loop of FN-III10 can increase its thermostability and solubility. NMR and dynamics analyses demonstrated that FN-III10(CPRGDMPDC) has the correct folding and the RGD loop of FN-III10(CPRGDMPDC) exhibited higher rigidity with preference for integrin v3 binding. Integrin αvβ3-specific FN-III10 (CPRGDMPDC) protein can inhibit A375 cell migration and HUVEC tube formation in vitro. The xenograft mouse model showed that it only slightly reduced the growth of Lewis lung cancer cells in C57/B6 mice; however, it can prolong their survival in vivo. In conclusions, we have successfully engineered FN-III10 scaffold to produce an integrin αvβ3-specific variant with high potency, selectivity, thermostability, and solubility. Our results also showed that this integrin αvβ3-specific FN-III10 variant is superior than the reported variant, FN-III10(PRGDWNEG). The further optimization of integrin v3-specific FN-III10 variant for the treatment of integrin αvβ3-related diseases is ongoing.
論文目次 中文摘要 I
Abstract II
誌謝 III
目錄 V
表目錄 VIII
圖目錄 IX
附錄目錄 X
縮寫檢索表 XII
儀器 XIII
第1章 緒論 1
1-1 背景資料 1
1-1-1 整合蛋白(Integrin)的介紹 1
1-1-2 整合蛋白αvβ3的介紹5
1-1-3 纖維黏結蛋白與第三型纖維黏結蛋白 6
1-1-4 第三型纖維黏結蛋白的第十個模組之介紹 8
第2章 研究目標與策略10
第3章 材料與方法 12
3-1 Fibronectin突變株重組蛋白之製備 12
3-1-1 實驗菌株、質體和培養基配方12
3-1-2重組基因之構築13
3-1-3重組蛋白之表現與純化16
3-1-4重組蛋白之質譜鑑定20
3-2 FNIII10與突變株重組蛋白抑制血小板功能之研究21
3-2-1 血小板的製備21
3-2-2重組蛋白抑制血小板凝集功能之分析21
3-3 細胞株及培養方法22
3-3-1 細胞培養22
3-4 FNIII10與突變株重組蛋白抑制細胞黏著之研究26
3-4-1 Fibronectin 之製備 26
3-4-2 重組蛋白抑制細胞黏著之分析28
3-5 FNIII10與突變株重組蛋白抑制細胞遷移能力之分析30
3-6 FNIII10與突變株重組蛋白抑制臍靜脈內皮細胞血管新生能力31
3-7 FNIII10與突變株重組蛋白蛋白溶解度的測定32
3-8 FNIII10與突變株重組蛋白熱穩定的測定33
3-9 FNIII10與突變株重組蛋白應用於老鼠癌症模式34
第4章 結果35
4-1 FNIII10與突變株重組蛋白之製備與鑑定35
4-2 FNIII10與突變株重組蛋白對於抑制血小板凝集的結果35
4-3 FNIII10與突變株重組蛋白抑制細胞黏著的結果 36
4-4 FNIII10與突變株重組蛋白抑制細胞遷移的結果 40
4-5 FNIII10與突變株重組蛋白抑制臍靜脈內皮細胞血管新生的結果41
4-6 FNIII10與突變株重組蛋白蛋白溶解度的測定的結果41
4-7 FNIII10與突變株重組蛋白熱穩定的測定結果 42
4-8 FNIII10與突變株重組蛋白應用於老鼠癌症模式 42
第5章 討論 43
5-1 增加一對雙硫鍵對於FNIII10突變株蛋白之性質與功能之影響 43
5-1-1 對於整合蛋白αvβ3之親和力的影響 43
5-1-2 對於溶解度及熱穩定度的影響 43
5-2 FNIII10(CPRGDMPDC)與FNIII10(PRGDWNEG)之比較 44
5-3 不同鷹架蛋白的序列交換對於整合蛋白親和力的轉換 44
5-4 與使用Rhodostomin作為蛋白鷹架設計之對於整合蛋白αvβ3具有專一性之蛋白做比較46
5-5 FNIII10突變株蛋白-- FNIII10(CPRGDMPDC)應用於癌症老鼠模式 47
5-6 未來展望 47
第6章 結論 49
參考文獻 51
表 58
圖 63
附錄 86


表目錄
表2-1 各種突變蛋白的突變引子(primer)序列57
表4-1 FNIII10與突變株重組蛋白對於不同整合蛋白的抑制活性(IC50)值以及血小板凝集抑制的總表 58
表4-2 FNIII10(CPRGDMPDC) 對於不同整合蛋白與血小板凝集抑制的IC50值 59
表4-3 FNIII10與突變株重組蛋白熱穩定度與溶解度測定結果 60
表5-1 FNIII10(CPRGDMPDC)與FNIII10(PRGDWNEG)之比較:對於不同整合蛋白的親和力 60
表 5-2 FNIII10(CPRGDMPDC)與FNIII10(PRGDWNEG)之比較:熱穩定度以及溶解度 61
表 5-3 FNIII10突變株蛋白與Rho突變株蛋白對於不同整合蛋白以及血小板凝集之IC50值 62

圖目錄
圖2-1 FNIII10突變蛋白 63
圖3-1 Fibronectin突變株重組蛋白之製備流程圖 64
圖3-2 重疊延伸方式產生定點突變之示意圖 65
圖3-3 抑制血小板凝集試驗 66
圖3-4 細胞黏著抑制實驗之流程圖 67
圖4-1 突變株重組蛋白定序以及胺基酸序列比對的結果 68
圖4-2 FNIII10突變株重組蛋白以Ni+-chelating chromoatography純化及HPLC純化的結果 70
圖4-3 FNIII10突變株重組蛋白以質譜儀分析的結果 71
圖4-4 FNIII10與其突變株重組蛋白質以reverse phase HPLC精純化的結果 72
圖4-5 FNIII10突變株重組蛋白血小板凝集抑制的分析結果75
圖4-6 套用去整合蛋白、噬菌體展現技術篩選出的integrin-specific於
FNIII10 F-G loop之IC50值條狀圖 76
圖4-7 以條狀圖顯示突變株重組蛋白對整合蛋白αvβ3的IC50值,並以卡通圖呈現突變蛋白的結構 77
圖4-8 FNIII10(CPRGDMPDC)抑制細胞遷移能力的結果及條狀圖 79
圖4-9 FNIII10(CPRGDMPDC)抑制臍靜脈內皮細胞血管新生的結果 80
圖4-10 FNIII10與突變株重組蛋白蛋白溶解度的測定的結果 81
圖4-11 FNIII10與突變株重組蛋白熱穩定的測定結果 82
圖4-12 FNII10突變蛋白株對Lewis lung cancer cell腫瘤生長以及Kaplan-Meier 存活曲線 83
圖5-1 FNIII10(CPRGDMPDC)與FNIII10(PRGDWNEG)之比較 84
圖5-2 不同鷹架蛋白的序列交換對於整合蛋白親和力的轉換 85

附錄目錄
附錄表1-1 整合蛋白的受質與分布 87
附錄表1-2 整合蛋白與其可辨識序列 88
附錄表1-3 整合蛋白與其相關癌症 89
附錄表1-4 整合蛋白拮抗劑應用於臨床癌症治療 90
附錄表1-5 纖維黏結蛋白結合之整合蛋白及其辨認序列 91
附錄表1-6 利用FNIII10作為蛋白模板設計的藥物 92
附錄圖1-1 整合蛋白家族次單元的 93
附錄圖1-2 整合蛋白之一級結構、二級結構及整合蛋白v3胞外區之三級結構 94
附錄圖1-3 整合蛋白與受質結合方式 95
附錄圖1-4 整合蛋白家族 96
附錄圖1-5 整合蛋白結構變化模擬圖 97
附錄圖1-6 整合蛋白構型與受質親和力 98
附錄圖1-7 整合蛋白αvβ3與RGD peptide結合圖 99
附錄圖1-8 纖維黏結蛋白結構圖 100
附錄圖1-9 免疫球蛋白多變重鏈(VH)區域與第三型纖維黏結蛋白模組的結構圖 101
附錄圖1-10 第三型纖維黏結蛋白結構對應序列比對 102
附錄圖4-1 第三型纖維黏結蛋白的第十個模組之結構圖 103
附錄圖4-2 第三型纖維黏結蛋白第十個模組(FNIII10)與蛇毒蛋白Rhodostomin利用NMR分析的結果 104
附錄圖4-3 人類黑色素瘤細胞--A375 cell 以流式細胞儀分析之三種整合蛋白表現量以及Transwell migration Assay的結果 105
附錄圖5-1 整合蛋白αvβ3與FNIII10 F-G loop結合表面分析圖 106
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