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研究生:莊惟
研究生(外文):Wei Chuang
論文名稱:CTEN 於細胞核中的功能探討
論文名稱(外文):Studies on the functions of human C-terminal tensin-like (CTEN) protein in the nucleus
指導教授:廖憶純
指導教授(外文):Yi-Chun Liao
口試委員:黃楓婷謝淑貞賴韻如
口試日期:2015-07-22
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:46
中文關鍵詞:CTEN蛋白質純化染色質免疫沉澱
外文關鍵詞:CTENprotein purificationChIP
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CTEN (C-terminal tensin-like) 蛋白質位於集中附著點,屬於 tensin family 中的一員,並且在細胞的附著、移動和癌症發生過程中扮演著重要角色。先前的研究顯示 CTEN 在大腸癌中除了有過量表現之情形外,同時在細胞核內也能偵測到高表現量的 CTEN,而這樣的現象促使我們欲研究 CTEN 在細胞核中的功能。其他的研究指出,某些 focal adhesion 蛋白質會進入至細胞核內作為 coregulatory 或 coactivator,因此我們假設 CTEN 是一個能與 DNA 結合之蛋白質,在癌細胞中會進入至細胞核內參與調控促進腫瘤發生的基因表現。本論文先利用線上資料庫分析 CTEN 的胺基酸序列和蛋白質結構,但是並沒有發現可與 DNA 結合之高度保守性序列,因此另一方面我們想以實驗進一步證實 CTEN 是否具有與 DNA 結合之能力。首先本論文成功選殖出可在大腸桿菌中表現 CTEN 的重組質體 pET-28a-CTEN,想要利用純化的 CTEN 蛋白質測試其與 DNA 結合的能力,嘗試不同的表現與純化條件發現部分重組蛋白質 CTEN 會形成不可溶體,無法得到大量的可溶性 CTEN 蛋白質。同時,本論文利用 CTEN 抗體進行 ChIP assay 再經由毛細管電泳分析,實驗結果發現表現 CTEN 蛋白質之組別其 DNA 片段含量有較高之情形,符合 CTEN 是一個能與 DNA 結合的蛋白質之假設,此結果為 CTEN 於癌細胞核中累積之現象提供了一可能解釋。

C-terminal tensin-like (CTEN) protein is localized at focal adhesion complex. It is a member of the tensin protein family, and also plays a crucial role in cell adhesion, migration, and the development of malignancies. Previous studies have indicated that the expression levels of CTEN is elevated in colon cancer, accompanied with the detection of high population of CTEN in the nucleus. It prompts us to study the functions of human CTEN protein in the nucleus. Some of focal adhesion proteins are able to enter the nucleus and function as coregulators or coactivators. Therefore, we hypothesize that CTEN is a DNA binding protein, and could enter the nucleus in cancer cells to regulate the gene expression contributing to tumorigenicity. In this work, we firstly use the online database to analyze the amino acid sequence and the protein secondary structure of CTEN. It turns out that no highly-conserved DNA binding domain is found. On the other hand, we want to determine whether CTEN possesses the ability to bind DNA. First, the recombinant plasmid pET-28a-CTEN, which can express CTEN in E.coli, is successfully constructed. Then we plan to use the purified recombinant CTEN protein to examine its ability to bind DNA. However, high levels of the recombinant protein form inclusion bodies, and the yield of soluble CTEN is low. Moreover, ChIP assay followed by capillary electrophoresis is used to analyze whether CTEN is able to bind DNA. Our results show that the amount of DNA after ChIP is increased in the cells expressing CTEN protein. This result supports the hypothesis that CTEN functions as a DNA binding protein, and provides one potential explanation for the accumulation of CTEN in the nucleus in cancer cells.

目錄.....i
縮寫表.....iv
摘要.....vi
Abstract.....vii
1. 本論文之研究基礎.....1
1.1 Focal Adhesion.....1
1.2 Tensin 蛋白質的結構與功能.....2
1.3 CTEN 蛋白質的功能研究.....3
1.4 CTEN 於細胞核內之分佈與其功能探討.....4
1.5 本論文之研究目的 .....5
2. 材料與方法.....6
2.1 菌種.....6
2.1.1 大腸桿菌 DH5alpha.....6
2.1.2 大腸桿菌 BL21 (DE3).....6
2.2 表現載體 DNA.....6
2.2.1 原核表現系統載體 .....6
2.2.2 真核表現系統載體 .....6
2.3 DNA 分析與質體建構.....7
2.3.1 CTEN 表現質體 DNA 的建構.....7
2.3.2 聚合酶連鎖反應 (polymerase chain reaction, PCR).....7
2.3.3 DNA 片段純化.....8
2.3.4 以限制酶截切載體與 PCR 產物.....8
2.3.5 DNA 接合法 (DNA ligation).....8
2.3.6 重組質體之轉型與篩選.....9
2.3.7 質體純化 .....9
2.3.8 DNA 瓊脂糖膠體電泳法.....9
2.4 重組蛋白質之表現與純化.....10
2.5 細胞實驗.....11
2.5.1 細胞繼代.....11
2.5.2 細胞計數.....12
2.5.3 細胞轉染.....12
2.6 蛋白質分析.....13
2.6.1 蛋白質定量.....13
2.6.2 樣品製備.....13
2.6.3 聚丙烯醯胺膠體電泳.....13
2.6.4 Coomassie Brilliant Blue R (CBR) 染色法.....14
2.6.5 蛋白質轉印.....14
2.6.6 免疫呈色法.....14
2.7 細胞核與細胞質分離 (Fractionation).....15
2.8 染色質免疫沉澱
(Chromatin Immuno-precipitation assay, ChIP).....15
2.8.1 sonication 條件之超音波細胞破碎儀.....16
2.8.2 sonication 條件之超音波均質機.....16
2.9 毛細管電泳 (Capillary electrophoresis).....16
2.10 生物資訊學分析.....17
2.10.1 胺基酸序列分析.....17
2.10.2 蛋白質二級結構......17
3. 研究結果.....18
3.1 分析 CTEN 之胺基酸序列是否具有 DNA binding domain.....18
3.2 重組蛋白質 CTEN 的表現與純化.....19
3.2.1 CTEN 表現質體之建構.....19
3.2.2 探討重組蛋白質 CTEN 的最佳表現條件......20
3.2.3 重組蛋白質 CTEN 之純化......21
3.3 利用染色質免疫沉澱法探討 CTEN 是否有和 DNA 結合之能力......22
3.3.1 利用細胞核與細胞質分離實驗確認 CTEN 會進入至細胞核內......22
3.3.2 利用染色質免疫沉澱法探討 CTEN 是否會與 DNA 結合......22
3.3.3 超音波均質機之條件測試和應用在染色質免疫沉澱法中...... 23
4. 討論與未來方向......25
5. 參考文獻 ......31
6. 圖與表.....34



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