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研究生： 莊硯鈞
Chuang, Yen-Jun
論文名稱： 利用電泳晶片和親和性探針進行轉錄活化分析
Transcription activation assays by affinity probe microchip electrophoresis
指導教授： 陳淑慧
Chen, Shu-Hui
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2005
畢業學年度： 93
語文別： 中文
論文頁數： 93
中文關鍵詞： 轉錄 、去氧核醣核酸 、蛋白質 、親和力 、晶片 、電泳
外文關鍵詞： transcription, DNA, protein, affinity interaction, chip, electrophoresis
相關次數： 點閱：153  下載：1 
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    • 　　電泳遷移改變分析法(EMSA)是傳統分析蛋白質和DNA鍵結的工具。然而，電泳遷移改變分析法耗時且不適合做定量分析，所以在此發展搭配螢光偵測法的電泳晶片分析平台來研究蛋白質和DNA的鍵結作用力。在本研究中，以雌激素接受器(ER)和雌激素回應基因(ERE)當作模型並研究其鍵結。ER是一個配位基誘導的轉錄活化因子，透過與ERE的作用調控下游基因表現。利用遷移時間改變，建立了ERE和ERs的解離常數。公認的雌激素回應基因(cERE)對ER-alpha (Kd=2.55nM)的親和力大於對ER-beta (Kd=3.68nM)的親和力。另外，選用一段序列與cERE序列相似度為60%的ERE1576做研究，其對ER-alpha (7.5nM)的親和力，仍大於對 ER-beta (10.4nM)的親和力。比較cERE與ERE1576對ER的親和力，結果顯示cERE對ER的親和力比ERE1576強。在轉錄活化分析中，以不同藥物刺激A549細胞，並萃取藥物刺激後溶胞產物，ER-beta，研究此核蛋白萃取物對ERE1576產生的親和力，而其親和力的強弱順序為Diethylstilbestrol (DES, Kd= 0.143nM)> Genistein (GEN, Kd= 0.797nM) >空白實驗 (無藥物刺激的空白實驗，Kd= 2.12) > 4-hydroxytamoxifen (4OHT, Kd= 10.47nM)，而這些結果與文獻值一致。此外，這個分析系統具有快速分析和低樣品消耗量的優點，因此，此分析系統展現了應用於體外鍵結分析的可行性。

    　Electrophoretic mobility shift assay (EMSA) is a conventional separation tool for the analysis of protein-DNA binding. However, EMSA is relatively slow, time-consuming and not suitable for quantitative analysis. We are developing a microchip electrophoresis system utilizing fluorescence detection as an improved platform technology for the study of protein-DNA binding. In this study, the binding of estrogen receptor (ER) and estrogen -response binding element (ERE) was investigated. ER is a ligand-activated transcription factor, and it acts through binding to ERE located in the regulatory region of downstream genes. In this method, the dissociation constants were determined based on the shift of the migration time of ERE molecules that are labeled with fluorescence probes. The consensus sequence of ERE (cERE) was seen to bind to ER-alpha with a calculated Kd value around 2.55nM, which is higher than the Kd value, 3.68nM, determined for ER-beta. Moreover, a derived sequence, ERE1576 that has 60% homology to cERE, was also investigated and the result shows that ERE1576 binds more tightly for ER-alpha (Kd= 7.5nM) than for ER-beta (Kd= 10.4nM). Moreover, cERE was found to have a higher affinity with ER than ERE1576. In the transcription activation assay for the agonist/ antagonist test, the affinities of ERE1576 to the ER-beta extracted from the nuclear fraction of A549 cell lysates were investigated. The effect of some estrogenic compounds on ERE binding was also examined and the binding strengths obtained was found to sequence as the following: diethylstilbestrol (DES, Kd= 0.143nM)> genistein (GEN, Kd= 0.797nM) > blank (Kd= 2.12nM)> 4-hydroxytamoxifen (4OHT, Kd=10.47nM), and these results are consistent with literature reports. This analytical system has many advantages including the rapid assay and minimal sample consumption; therefore, it will be useful for conducting in-vitro binding assays.

    目錄 英文摘要.....................................................................I 中文摘要...................................................................III 主文目錄....................................................................IV 參考文獻....................................................................82 附錄........................................................................90 表目錄.....................................................................VII 圖目錄.....................................................................VII 主文目錄 第一章 序論 1.1 前言.....................................................................1 1.2 親和性作用力.............................................................2 1.3 生醫電泳晶片.............................................................3 1.4 類固醇調控基因表現.......................................................4 1.5 雌激素接受器(Estrogen Receptor)與雌激素回應基因(ERE) ....................5 1.6 研究目標.................................................................6 第二章 利用晶片式電泳分析DNA和蛋白質的親和力 2.0 前言.....................................................................9 2.1毛細管電泳原理............................................................9 2.2親和性毛細管電泳.........................................................13 2.3蛋白質與DNA之解離常數（Kd）計算..........................................20 2.4 實驗部分................................................................24 2.4.1 晶片式電泳儀及晶片樣式................................................24 2.4.2 藥品和試劑............................................................29 2.4.3 樣品和緩衝液的配置....................................................32 2.4.4 蛋白質螢光染料標示法..................................................33 第三章 晶片的共價性修飾 3.0前言.....................................................................35 3.1 毛細管管道表面修飾......................................................35 3.2 晶片修飾法的選擇........................................................37 3.3 共價鍵的晶片修飾........................................................38 3.4 表面性質分析............................................................40 3.4.1 電滲流量測方法........................................................40 3.4.2 化學分析電子術........................................................41 3.4.3 紅外線光譜儀..........................................................43 3.5實驗部分 3.5.1晶片修飾步驟...........................................................43 3.5.2 在X光光電子能譜儀和紅外線光譜儀使用時的晶片樣品製備...................44 3.5.3 儀器..................................................................45 3.6 實驗架構................................................................46 第四章 結果與討論 4.0 電泳緩衝液的選擇及樣品前處理............................................47 4.1未修飾與共價性修飾電泳的比較.............................................48 4-2 ER和ERE的親和性分析.....................................................57 4-3 ERE和藥物刺激的核蛋白親和性分析.........................................66 4.4 電泳晶片和EMSA的比較....................................................75 4.5 共價性鍵結表面修飾......................................................75 4.5.1 紅外線光譜分析........................................................76 4.5.2 XPS光譜...............................................................76 4.5.3電滲流量測.............................................................79 第五章 結論及未來展望 5.0 結論....................................................................80 5.1 未來展望................................................................81

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