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論文名稱 Title |
開發以胰蛋白酶-磁性奈米粒子在高溫下對蛋白質進行快速消化反應之技術及以微酯體包覆受質結合質譜分析技術應用於極微量蛋白質的偵測 Fast Protein Digestion with the Assistance of Magnetic Nanoparticle Coated with Trypsin and Detection of Trace Protein with Assistance of Liposome encapsulated signal material and MALDI-TOF |
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系所名稱 Department |
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畢業學年期 Year, semester |
語文別 Language |
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學位類別 Degree |
頁數 Number of pages |
77 |
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研究生 Author |
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指導教授 Advisor |
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召集委員 Convenor |
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口試委員 Advisory Committee |
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口試日期 Date of Exam |
2006-06-16 |
繳交日期 Date of Submission |
2006-06-26 |
關鍵字 Keywords |
胰蛋白酶、微酯體、磁性奈米粒子、基質輔助雷射脫附游離質譜法、蛋白質 MALDI, Liposome, Protein, Magnetic Nanoparticle, Trypisn |
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統計 Statistics |
本論文已被瀏覽 5675 次,被下載 3591 次 The thesis/dissertation has been browsed 5675 times, has been downloaded 3591 times. |
中文摘要 |
生物體內許多參與結構組成、新陳代謝、訊息傳導、轉錄因子(transcription factor)、調節、細胞週期調控的單位均以蛋白質為主。所以要了解生命的奧秘,首先就要對蛋白質(包括結構、功能等)有深入的了解。在蛋白質體中,MALDI-TOF MS為辨認、分析蛋白質的質譜工具之ㄧ(另一主要工具則是電噴灑游離質譜法)。目前此技術已被廣泛應用於決定蛋白質與胺基酸的序列、結構、純度、與後轉錄修飾等。雖然MALDI具有高靈敏度,高準確性,樣品需求量少,及快速分析能力等優點,但對於分析物種中若有濃度低或分子量大的蛋白質,往往會因離子壓抑(ion suppression)和質量歧視效應(mass discrimination),導致分析物中濃度低及分子量大的分子在質譜圖上無法表現出來。因此一般生化樣品,若未經適當的前處理(如層析及電泳分離等)就無法以上述質譜法得到相關蛋白質的消息。然而這些樣品處理步驟,一般而言均是繁瑣且耗時的,而且要如何將一些蛋白質有效的自這些分離系統中還原也是一個很棘手的問題。 每個蛋白質都具有獨特的序列,利用酵素消化蛋白質之後,將所得到的胜肽片段,進行資料庫比對即可鑑定出蛋白質的身份,但是傳統的消化反應耗時,所以近年來有許多學者利用不同的改良方式以縮短消化反應的時間。本研究第一部份將利用奈米磁性粒子(Magnetic Nanoparticles,MNPs)與特定酵素(如胰蛋白酶,trypsin)結合後簡稱為T-MNP(Trypsin-Magnetic Nanoparticles),在高溫下快速消化蛋白質並利用磁鐵將磁性奈米粒子收回,即可減少因胰蛋白酶自我分解對生化質譜分析所產生的訊號干擾,並將蛋白質進行消化後所得到的胜肽片段,經過資料庫比對,鑑定蛋白質。 本研究第二部分將利用微酯體(liposome)包覆各式蛋白質(如酵素或標記蛋白),並在微酯體外圍塗覆特定抗原,此微酯體在溶液中抓取到抗體後,以化學的方式,釋放出原先包覆在微酯體的酵素或標記蛋白,再直接或間接地偵測具大分子量的蛋白質抗體。因為微酯體可以包覆的蛋白質數目與其所能抓到的抗體數目比較,是相差非常大的,由此策略可以放大一個微弱的化學或生化訊號偵測到極微量的蛋白質分析物。如此可以解決大分子在質譜上不易被偵測的缺點。 |
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目次 Table of Contents |
目錄 論文摘要...................................................................................Ⅰ 目錄...........................................................................................Ⅱ 圖目錄………………………………………………………...Ⅳ 表目錄………………………………………………………...Ⅹ 壹、 緒論...................................................................................1 一、 前言..............................................................................1 二、 基質輔助雷射脫附游離質譜法(MALDI)...................3 三、 磁性奈米粒子簡介………………………………......5 四、 酵素及胜肽質量指紋簡介…………………………..5 五、 影響酵素催化速率之因素…………………………..9 1.時間……………………………………………………9 2.溫度…………………………………………………………..10 3.pH值……………………………………………………….....10 4.酵素之濃度………………………………………………….. 12 六、 微酯體(Liposome)的簡介…………………………...17 七、 免疫球蛋白簡介……………………………………..22 八、 抗體與抗原的定義及結合力......................................24 九、 酵素免疫分析..............................................................24 十、 論文目標…………………………………………....27 貳、 實驗.................................................................................30 一、 儀器裝置.....................................................................30 二、 實驗藥品及樣品…………………………………….30 三、 Trypsin-Magnetic Nanoparticle (T-MNP)配製……...32 四、 微酯體(Liposome)合成……………………………...34 五、 實驗流程…………………………………………......34 六、 直接免疫分析法的實驗流程及質譜偵測..................39 參、 結果與討論......................................................................41 肆、 結論……………………………………………………..72 伍、 參考資料..........................................................................73 |
參考文獻 References |
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