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研究生:鐘彩華
研究生(外文):Tsai-Hwa Jong
論文名稱:利用YeastTwo-Hybrid的方法尋找Eps8N端序列可能的結合蛋白質
論文名稱(外文):Identification of N-terminal Eps8 binding proteins by Yeast Two-Hybrid Method
指導教授:呂增宏
指導教授(外文):Tzeng-Horng Leu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:74
中文關鍵詞:酵母菌蛋白質
外文關鍵詞:Eps8Yeast Two-Hybridprotein
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之前我們實驗室已經證實在老鼠纖維母細胞中過量表達p97Eps8會造成細胞的轉型及促進腫瘤的產生,所以可以知道p97Eps8在細胞生理功能上扮演著重要的角色。由胺基酸序列的分析得知p97Eps8具有:(1) N端的split PH domain □ (2) 一個SH3 domain □ (3) 四個proline rich regions □ 及 (4) 一個putative nuclear targeting sequence。為了進一步了解p97Eps8和腫瘤之間的關係,我們希望能有系統的分析出p97Eps8的結合蛋白質。在此我們利用yeast two-hybrid的方法尋找可以和N-terminal p97Eps8 結合的蛋白質。從human brain cDNA library我們篩選出246個可以製出和N-terminal p97Eps8相互結合之蛋白質的cDNA。其中的NEBP1(N-terminal Eps8 Binding Protein 1) 由GenBank的基因庫比對為BAIAP2。NEBP2為TLE2。
由於先前實驗室的研究結果指出p97Eps8會被non-receptor tyrosine kinase Src所磷酸化,同時97Eps8的proline rich regions在in vitro可以和Src的SH3 domain結合。所以我們也利用yeast two-hybrid的方法定出p97Eps8真正可以和Src的SH3 domain結合的胺基酸序列。實驗結果我們發現完整的wt-p97Eps8及261-Eps8可以和Src的SH3SH2 domain結合,而SrcSH3SH2 domain無法與其他truncated p97Eps8結合。
Previously, we demonstrated that cells overexpressing p97Eps8 not only exhibited the ability of focus formation in cell culture but also promoted the tumor formation in mice as compared to controls. However the N-terminal truncated 261-p97Eps8 has lost such transforming ability. Thus, the N-terminal domain may play an important role in Eps8-mediated cellular transformation. Protein sequence analysis revealed the following domains existing in the N-terminal p97Eps8 that might contribute to its cellular functions : a putative nuclear targeting sequence, a split PH domain and two proline-rich regions. In this study we utilized yeast two-hybrid method to screen a human brain cDNA expression library to search for N-terminal p97Eps8 binding proteins (NEBP). We have obtained 246 cDNA clones encoding the potential N-terminal p97Eps8 binding proteins. Sequence comparison with the information derived from GenBank, NEBP1 and NEBP2 were identified BAIAP2 and TLE2 respectively. How these proteins interact with p97Eps8 and play a role in Eps8-induced cellular transformation needs further investigation.
Previously, there was evidence indicating that Src can phosphorylate p97Eps8 and the proline rich regions of p97Eps8 can interact with the Src SH3 doamin in an in vitro binding assay. To substantiate this interaction in vivo, we also utilize yeast two-hybrid method to define the Src SH3 domain binding sequence of p97Eps8. We find that SrcSH3SH2 domain can interact with wt-p97Eps8 and 261-p97Eps8, but not the other truncated p97Eps8.
目錄
中文摘要1
英文摘要3
縮寫檢索表5
第一章 緒論7
第二章 實驗材料及方法15
第一節 實驗材料16
第二節 實驗方法21
第三章 實驗結果32
第四章 討論38
圖表43
參考文獻64
參考文獻 (References)
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林妲妮 建立持續性表達附著於細胞膜且不具完整Pleckstrin Homology p97Eps8的細胞株及其對細胞轉型的影響。 國立成功大學藥理學研究所碩士論文。中華民國九十年七月。
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