臺灣博碩士論文加值系統

伴刀豆球蛋白(Con A)是能與甘露糖(mannose)及葡萄糖(glucose)結合的外源凝集素，本論文分別以共沉澱法及檸檬酸鈉還原法合成表面修飾檸檬酸之氧化鐵(Fe3O4)奈米粒子與金(Au)奈米粒子，或再進一步在其表面鍵結聚乙二醇(PEG)製得Fe3O4-PEG與Au-PEG奈米粒子。這些粒子藉碳二醯胺活化作用將Con A固定化於其表面，可製得磁性可操控之Fe3O4-ConA、Au-Con A、Fe3O4-PEG-Con A及Au-PEG-Con A奈米載體，用於N-醣蛋白的分離及與流感病毒H1N1的結合。
表面修飾檸檬酸之Fe3O4奈米粒子平均粒徑約11.7 ± 3.9 nm，Fe3O4-ConA及Fe3O4-PEG-Con A奈米粒子鍵結之Con A可由FTIR分析確認，其鍵結量經熱重分析與蛋白質分析得知分別約為10%及9.5%。相似地，表面修飾檸檬酸之Au奈米粒子平均粒徑約13.4 ± 3.1 nm，Au-Con A及Au-PEG-Con A奈米粒子鍵結之Con A可由FTIR分析確認，其鍵結量經熱重分析與蛋白質分析得知分別約為10 %及8.6 %。
將Fe3O4-Con A奈米粒子用於N-醣蛋白的分離，證實非N-醣蛋白之牛血清蛋白無法被吸附，而屬於N-醣蛋白之乳鐵蛋白則可被吸附，並遵循Langmuir恆溫吸附模式。在25 ℃、pH 7.4、0.02 M Tris-HCl緩衝溶液中乳鐵蛋白在Fe3O4-Con A奈米粒子上的最大吸附量為59.2 mg/g，Langmuir吸附平衡常數為0.0103 L/mg。藉由電泳分析，Fe3O4-Con A奈米粒子可選擇性分離N-醣蛋白的能力可進一步獲得確認。
此外，將上述鍵結Con A的奈米粒子用在與A型流行性感冒病毒的結合。透過酵素免疫分析法及穿透式電子顯微鏡分析可知，固定化在奈米粒子上之Con A仍能保留其活性並捕捉病毒。且Fe3O4-ConA及Au-Con A奈米粒子與A型流行性感冒病毒間的解離常數(Kd)分別為9.5 × 10-9 M與1.1 × 10-7 M，但Fe3O4-PEG-Con A及Au-PEG-Con A奈米粒子與A型流行性感冒病毒間的解離常數則分別降至2.1 × 10-9 M與2.88 × 10-8 M，顯示其捕捉A型流行性感冒病毒的效果較佳。此結果將有助於抗流感病毒藥物與疫苗的發展。

Concanavalin A (Con A), a lectin with mannose and glucose binding specificity, can be used for the targeted binding of certain oligosaccharide structures of N glycosylated proteins. In this thesis, citrate-modified Fe3O4 nanoparticles and Au nanoparticles were synthesized by co-precipitation method and sodium citrate reduction method, respectively. Their surface was also further bound with polyethylene glycol (PEG) to yield Fe3O4-PEG and Au-PEG nanoparticles. After the immobilization of Con A via carbodiimide activation, the magnetically manipulable Fe3O4-ConA, Au-Con A, Fe3O4-PEG-Con A, and Au-PEG-Con A nanocarriers were obtained and used for the separation of glycoprotein and the binding with influenza A virus.
The citrate-modified Fe3O4 nanoparticles had a mean diameter of 11.7 ± 3.9 nm. The binding of Con A on Fe3O4-ConA and Fe3O4-PEG-Con A nanoparticles could be demonstrated by FTIR analysis. By thermogravimetric analysis and protein assay, the binding amount of Con A were determined to be 10 %及9.5 % for Fe3O4-ConA and Fe3O4-PEG-Con A nanoparticles, respectively. Similarly, the citrate-modified Au nanoparticles had a mean diameter of 13.4 ± 3.1 nm. The binding of Con A on Au-Con A and Au-PEG-Con A nanoparticles also could be demonstrated by FTIR analysis. By thermogravimetric analysis and protein assay, the binding amount of Con A were determined to be 10 %及8.6 % for Au-Con A and Au-PEG-Con A nanoparticles, respectively.
Fe3O4-Con A nanoparticles were used for the separation of glycoprotein. They could not adsorb bovine serum albumin but could adsorb lactoferrin effectively, obeying Langmuir isotherm model. At pH 7 and 25 C, the maximum capacity and equilibrium constant for lactoferrin adsorption were 59.2 mg/g and 0.0103 L/mg, respectively. By SDS-polyacrylamide gel electrophoresis, the capability of Fe3O4-Con A nanoparticles for the selective separation of glycoprotein could be further confirmed.
In addition, the above Con A-bound nanoparticles were used for the binding with influenza A virus. By enzyme-linked immunosorbent assay (ELISA) and TEM analysis, it was demonstrated that Con A bound on the nanoparticles retained its capability for the capture of virus. Moreover, the dissociation constants for Fe3O4-Con A and Au-Con A with influenza A virus were 9.5  10-9 M and 1.1  10-7 M, respectively. However, the dissociation constants for Fe3O4-PEG-Con A and Au-PEG-Con A with influenza A virus were lowered to be 2.1  10-9 M and 2.88  10-8 M, respectively, revealing better capability for the capture of virus. This result will be helpful for the development of anti-influenza virus drugs or vaccines.

中文摘要 Ⅰ
英文摘要 ⅠⅠⅠ
誌謝 Ⅴ
總目錄 VII
表目錄 X
圖目錄 XI
符號 XV

第一章 緒論
1.1 病毒與疫苗 1
1.2 伴刀豆球蛋白 (Concanavalin A) 4
1.2.1 伴刀豆球蛋白的基本性質 4
1.2.2 伴刀豆球蛋白的應用 6
1.3 醣蛋白 (Glycoprotein) 10
1.4 金奈米粒子載體與磁性載體之生醫應用 12
1.5 研究動機與內容 17

第二章 基礎理論
2.1 磁性理論 20
2.1.1 磁性來源 20
2.1.2 磁性體分類 21
2.1.3 磁區與磁滯曲線 25
2.2 吸附理論 29


第三章 實驗方法
3.1 實驗藥品、儀器及材料 33
3.1.1 實驗藥品 33
3.1.2 實驗儀器 36
3.1.3 實驗材料 37
3.2 奈米粒子及奈米粒子表面被覆Con A之製備 38
3.2.1 氧化鐵奈米粒子之製備 38
3.2.2 金奈米粒子之製備 40
3.2.3 表面被覆Con A之氧化鐵奈米粒子的製備 43
3.2.4 表面被覆Con A之金奈米粒子的製備 43
3.3 特性分析 45
3.4 表面被覆Con A之氧化鐵奈米粒子分離乳鐵蛋白的方法 49
3.5 表面被覆Con A之奈米粒子結合病毒之量測 52

第四章 結果與討論
4.1 氧化鐵奈米粒子及表面被覆Con A後之基本特性 54
4.1.1 氧化鐵奈米粒子之基本特性 54
4.1.2 氧化鐵奈米粒子表面被覆Con A後之特性分析 57
4.1.3 氧化鐵奈米粒子表面被覆Con A後之定量分析 64
4.2 金奈米粒子及表面被覆Con A後之基本特性 68
4.2.1 金奈米粒子之基本特性 68
4.2.2 金奈米粒子表面被覆Con A後之特性分析 71
4.2.3 金奈米粒子表面被覆Con A後之定量分析 78
4.3 表面被覆Con A之氧化鐵奈米粒子在分離乳鐵蛋上之應用 80
4.4 表面被覆Con A之奈米粒子與病毒結合之研究 86
4.4.1 表面被覆Con A之氧化鐵奈米粒子結合病毒之研究 86
4.4.2 表面被覆Con A之金奈米粒子結合病毒之研究 90
4.4.3 改善表面被覆Con A之奈米粒子結合病毒的能力 94
第五章 結論 104
參考文獻 106

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