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研究生:謝佩珊
研究生(外文):Pei-Shan Hsieh
論文名稱:利用酵母菌雙雜合系統及蛋白質截短技術研究一個具有類似腫瘤胚胎表現型的未知基因--LRRC16B
論文名稱(外文):Yeast two-hybrid analysis and truncational study of an oncofetal-like gene - LRRC16B
指導教授:何中良何中良引用關係
指導教授(外文):Chung-Liang Ho
學位類別:碩士
校院名稱:國立成功大學
系所名稱:醫學檢驗生物技術學系碩博士班
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:2009
畢業學年度:97
語文別:中文
論文頁數:109
中文關鍵詞:細胞核入核訊號c-SrcLRR蛋白酵母菌雙雜合系統Expressed Sequence Tags 資料庫腫瘤胚基因
外文關鍵詞:Expressed Sequence Tagsoncofetal genesYeast two-hybrid systemLRR proteinsnuclear localization signalsc-Src
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腫瘤胚基因 (oncofetal gene) 係為表現於胚胎時期的基因,隨著發育為正常成體會降低表現或是不表現,而後當腫瘤形成時,其腫瘤胚基因又再次大量表現。一般較為人熟知的腫瘤胚基因有 α-fetoprotein (AFP)及 Glypican-3 (GPC3),這類基因在肝癌上是很好的癌症診斷指標,基於這樣的概念,本實驗室從 Unigene、ORESTES 等資料庫中,建立屬於我們自己的資料庫,藉由這個資料庫,我們鎖定一個功能未知基因— LRRC16B (leucinerich repeat containing 16B),將之命名為 Embryonic Gene Re-express in Tumor–Unknown 5 簡稱 EGRT-U5。藉由一連串的篩選,我們也確認了EGRT-U5具有類腫瘤胚基因的表現型。利用生物資訊軟體分析發現此未知蛋白之 N端具有 LRR domain,而 C端含有數個細胞核定位訊號 (NLS),為了瞭解這兩個蛋白模組對EGRT-U5功能上的角色,我們將 EGRT-U5 分成兩段進行酵母菌雙雜合實驗,依序分別為具有LRR domain之U5 (1-991) 與含有 NLS 之U5 (991-1370) 。以酵母菌雙雜合實驗分析,找到4 個可能與U5 (1-991) 以及5 個可能與U5 (991-1370) 有交互作用的蛋白質,分別有��-actin、Protein phosphatase 3 (PPP3)、c-Src以及importin α等。以免疫沉澱和免疫螢光染色確認篩選結果,我們發現EGRT-U5與c-Src有交互作用。而且,先前的實驗結果也顯示,EGRT-U5具有促進細胞增生以及腫瘤形成的能力,我們推測可能是由於與c-Src的交互作用而造成。此外我們也針對c-Src下游的訊息傳導路徑做初步探討,結果顯示JNK pathway表現以及磷酸化程度都有顯著性增加。總結目前的實驗結果,EGRT-U5在促進細胞生長以及腫瘤形成的過程中有其生物意義,作用機制可能藉由與c-Src交互作用而調控JNK pathway活化,進而導致細胞增生以及腫瘤形成。
Oncofetal proteins, such as alpha-fetoprotein (AFP) and glypican-3 (GPC-3), are proteins expressed in embryonic stage, down-regulated in adult normal tissues but re-expressed in tumors. Recent studies showed that these proteins may be useful in cancer diagnosis and prognosis. Previously, we found LRRC16B (EGRT-U5, Embryonic Gene Re-express in Tumor-Unknown 5) presenting oncofetal-like patterns that was expressed in many tumor and fetal tissues including liver and colon but down-regulated in normal and non-tumor tissues. According to the bioinformatics analysis, EGRT-U5 has several leucine rich repeats (LRR) in N-terminus and nuclear localization signals in C-terminus. A number of recent studies revealed that the LRR domains probably were related to protein–protein interactions and the protein containing LRR domain may be associated with many biologically important processes, morphology and dynamics of the cytoskeleton and early mammalian development. In order to further determine protein domains of EGRT-U5 which are responsible for interaction, we had cloned two truncated constructs pGBKT7-U5 (1-991) and pGBKT7-U5 (991-1370). By using these truncated constructs as baits, we screened a testis cDNA library to sift protein-protein interaction by yeast two-hybrid system. Presently, we found 4 proteins may interact with U5 (1-991) and 5 proteins may interact with U5 (991-1370), including beta-actin, Protein phosphatase 3 (PPP3), c-Src tyrosine kinase and importin alpha among others. Using immunoprecipitation and immunofluorescence to verify these results, we observed the EGRT-U5 could interact with c-Src. Combined with the data before, it revealed the ability of cell proliferation and tumor formation may associated with the interaction between EGRT-U5 and c-Src. Moreover, we also detected the c-Src downstream signaling pathway expression levels, only the JNK pathway showed the dramatic change. In conclusion, we suggested the oncofetal-like protein, LRRC16B (EGRT-U5) may play a role in tumorgenesis and medieated by its interaction with c-Src to regulate the JNK pathway which results in tumor formation.
摘 要....................................................1
Abstract..................................................3
致 謝....................................................5
目 錄....................................................7
表 目 錄.................................................10
圖 目 錄.................................................10
附 圖 目 錄..............................................12
第一章 緒 論...........................................13
1.1腫瘤胚基因 (Oncofetal genes)..........................13
1.2 表現序列標幟 (Expressed Sequence Tag,EST)...........15
1.3 找尋新的腫瘤胚基因– LRRC16B (EGRT-U5)...............17
1.4 LRRC16B (EGRT-U5)....................................19
1.5 酵母菌雙雜合系統 (yeast two-hybrid system)...........21
1.6 Src..................................................22
1.7 實驗目的.............................................24
第二章 實驗材料及方法..................................26
2.1 溶液配製.............................................26
2.2 細胞的培養程序.......................................30
2.3 構築質體方法.........................................32
2.4 構築實驗所需質體.....................................37
2.5 酵母菌雙雜合實驗 (yeast two-hybrid assay)............38
2.6 免疫沈澱法 (immunoprecipitation, IP).................44
2.7 西方墨點法...........................................44
2.8 XTT proliferation assay..............................47
2.9 免疫螢光染色 (Immunofluorescence)....................47
第三章 實驗結果........................................49
3.1 針對過度表現EGRT-U5 的細胞做功能性探討...............49
3.1.1 構築表現U5全長以及不同片段的各種表現質體...........49
3.1.2 過度表現U5全長以及不同片段的綠色螢光融合蛋白在HEK293T細胞的分布情形...........................................50
3.1.3 西方墨點法分析各種表現質體之蛋白表現...............51
3.1.4 探討過度表現U5全長以及不同片段之綠色螢光融合蛋白以及Myc tag對於HEK293T 細胞生長的影響........................52
3.2 酵母菌雙雜合實驗.....................................53
3.2.1 構築U5不同片段在酵母菌AH109中之表現載體............53
3.2.2 西方墨點法分析各種表現質體在酵母菌AH109中之蛋白表現.......................................................53
3.2.3 酵母菌雙雜合實驗結果...............................54
3.3 探討EGRT-U5與c-Src之間的相關性.......................55
3.3.1 利用免疫沉澱法 (immunoprecipitation)以及免疫螢光染色(Immunofluorescence) 確認酵母菌雙雜合實驗結果............55
3.3.2 西方墨點法分析在穩定表現EGRT-U5以及不同片段之綠色螢光融合蛋白以及Myc tag細胞群中,c-Src的表現量以及磷酸化程度.57
3.3.3 西方墨點法分析其c-Src下游相關細胞訊息傳導路徑......58
第四章 討論............................................59
第五章 參考文獻........................................67
第六章 表..............................................78
第七章 圖..............................................79
作 者 自 述.............................................109
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周靖恆,結合生物資訊的方法探討腫瘤相關基因在人類腎臟癌中的表現,國立成功大學分子醫學研究所碩士論文,民國九十四年。
吳宗樺,研究一個具有腫瘤胚胎表現型的功能未知基因-LRRC16B,國立成功大學分子醫學研究所碩士論文,民國九十六年。
林照蓉,研究一個具有腫瘤胚胎表現型的功能未知基因-LRRC16B,國立成功大學分子醫學研究所碩士論文,民國九十七年。
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