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研究生:黃淑萍
研究生(外文):Shu-Ping Huang
論文名稱:與Poloboxdomain作用之蛋白質的鑑定
論文名稱(外文):Identification of the Polo box domain interacting proteins
指導教授:李沁李沁引用關係
指導教授(外文):Chin Li
學位類別:碩士
校院名稱:國立中正大學
系所名稱:分子生物研究所
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:2006
畢業學年度:94
語文別:中文
論文頁數:57
中文關鍵詞:細胞週期細胞核運送訊息序列
外文關鍵詞:nuclear localization signal(NLS)cell cyclePolo box domainPolo-like kinase
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哺乳類動物的polo-like kinase 1 (Plk1) 與果蠅的polo具有相同的弁遄A是一個含有絲胺酸(serine)及酥胺酸(threonine)的蛋白質激酶,其弁鄍D要是在調節細胞的有絲分裂。就結構上來說,Plk1 在胺基端(N-terminal)含有一個kinase domain,而在羥基端(C-terminal)則有一個常見的結構稱之為polo box。為了要更進一步的瞭解Plk1生物性的弁遄A我們先利用在試管的(in vitro) pull down實驗再藉由質譜儀分析以全面性的去鑑定Plk1的受質。在我們初步的實驗中已經發現了有四個蛋白質會跟Plk1的Polo box domain結合,分別是熱休克蛋白質90 beta (Heat-shock protein 90β),人類的轉譯延長因子2 (human translation elongation factor 2),核苷酸還原酶1 (ribonucleotide reductase motif 1),及beta 微管蛋白(β-tubulin)。同時我們也利用在試管的pull down實驗來確認Plk1與熱休克蛋白質90 beta或beta微管蛋白之間的作用關係。
另一方面,在整個細胞週期中Plk1同時會分佈在細胞核與細胞質中。雖然之前有報導認為在Plk1的kinase domain帶有具有弁鄋槓ipartite nuclear localization signal (NLS),但是我們的結果指出polo box domain對於Plk1在細胞核內的位置可能扮演著很重要的角色。因此,我們利用β-galactosidase當作位置的reporter,去觀察與各種不同的Plk1片段結合時Plk1的進核效率。接著,我們將細胞質與細胞核分離來偵測帶有β-galactosidase的polo box domain在HeLa細胞內的分佈,而我們的結果證明了polo box domain具有新的NLS的能力。
Polo-like kinase 1 (Plk1), a mammalian ortholog of Drosophila
polo, is a serine-threonine protein kinase implicated in the regulation of
multiple aspects of mitosis. Plk1 contains a kinase domain in the
amino-terminal region and a common structure motif called polo boxes
domain in the carboxyl-terminal region. In order to further understand the biological functions of Plk1, we performed in vitro pull down assay followed by mass spectrometry to identify the Plk1 substrates comprehensively. We have identified four proteins interacting with the Polo box domain of Plk1 in our preliminary experiments, and these are heat shock protein 90β (hsp90β), human translation elongation factor2 (eEF2), ribonucleotide reductase motif 1 (RRM1), and β-tubulin. We also used in vitro pull down assay to confirm the interaction of Plk1 with hsp90β and with β-tubulin.
On the other hand, Plk1 is distributed both in the nucleus and in the cytoplasm throughout the cell cycle. Although it was reported that a functional bipartite nuclear localization signal is located in the kinase domain, our data indicated that the polo box domain may also play an important role in Plk1 intracellular localization. For this purpose, we used β-galactosidase as the localization reporter to examine the efficiency of nuclear import when fused to various Plk1 fragments. By isolating cytoplasmic and nuclear fraction of HeLa cells transiently expressing β-galactosidase fusion Polo box domain, our result showed that the Polo box domain is able to function as a novel NLS.
中文摘要……………………………………………………………….…i
英文摘要………………………………………………………………...iii
目錄...........................................................................................................iv
圖目錄…………………………………………………………………..vii
第一章 序論..............................................................................................1
第二章 材料與方法……………………………………………………..7
2-1 實驗材料………………………………………………………..7
2-1.1 實驗藥品………………………………….…………………..7
2-1.2 培養液配方………………………………….………………..7
2-1.3 緩衝溶液配方………………………….……………………..8
2-1.4 SDS-PAGE濃度配方………………………...….…………..11
2-2 實驗方法…………….………………………………………...11
2-2.1a 聚合酶連鎖反應(polymerase chain reaction:PCR)…...…11
2-2.1b 聚合酶連鎖反應(polymerase chain reaction:PCR) (eEF2與RRM1適用)…………………………………………………….......12
2-2.2 質體pHTPP2 -Polo box的構築………………………….…12
2-2.3 質體pHTPP2 – E4 Plk1的構築……………….……………12
2-2.4 質體pFlag-E4 Plk1-LacZ的構築…………………………..13
2-2.5 DNA的補齊反應(fill in)…………………….………………13
2-2.6 質體pCEP4e-myc-E4 Plk1-HA的構築…………….………14
2-2.7 真核生物的轉譯延長因子2 (eEF2)的構築……….……….14
2-2.8 核糖核苷酸還原酶motif1 (RRM1)的構築…….…………..14
2-2.9 質體轉型 (plasmid Transformation)……….……………….14
2-2.10 抽取小量質體DNA (Miniprep plasmid extraction)…….…15
2-2.11 DNA片段的回收或純化…………………...………………15
2-2.12a 純化帶有GST-tag的重組蛋白質…………………….….16
2-2.12b純化帶有His-tag的重組蛋白質…….…………………….17
2-2.13a in Vitro GST-Pull down assay…………….……………….18
2-2.13b in Vitro Pull down assay (HeLa cells)……………………..18
2-2.14 轉染 (Transfection)…………….………………………….19
2-2.15 Double thymidine blocking……………………….………...19
2-2.16 置備細胞質與細胞核的萃取物(Preparation of nuclear extract from a single plate)…………………….…………………...20
2-2.17 免疫螢光染色(Immunostaining)…………….…………….20
2-2.18 西方轉漬法(Western Blotting)………………………….…21
第三章 實驗結果………………………………………………………23
3-1 蛋白質的純化…………………………….…………………...23
3-1.1 Polo box融合蛋白質的純化………….……………………..23
3-1.2 E4 Plk1融合蛋白質的純化………………………………….23
3-2 in vitro Pull down assay……………………….……………….24
3-3 Flag-E4 Plk1-LacZ融合蛋白質在HeLa細胞中的位置…………………………………………………………………...24
第四章 討論……………………………………………………………27
第五章 參考文獻….…………………………………………………...47
附圖……………………………………………………………………..30
附錄……………………………………………………………………..45
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