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研究生:陳瀅如
研究生(外文):Ying-Ju Chen
論文名稱:紅螢光蛋白DsRed與綠螢光蛋白EGFP融合蛋白之研究
論文名稱(外文):Biochemical Characterization of DsRed and EGFP fusion protein
指導教授:吳宗遠
指導教授(外文):Tzong-Yuan Wu
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物科技研究所
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:2002
畢業學年度:90
語文別:中文
論文頁數:58
中文關鍵詞:綠螢光蛋白紅螢光蛋白螢光共振能量轉移發光團
外文關鍵詞:Green fluorescent proteinGFPDiscosoma species RedDsRedFluorescence resonance energy transferChromophore
相關次數:
  • 被引用被引用:3
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中文摘要
螢光蛋白(fluorescent protein, FP)不論在基因表現、蛋白的交互作用及運輸上都可作為一個良好的標記。綠螢光蛋白(green fluorescent protein, GFP)是以monomer的型式存在;紅螢光蛋白(DsRed)則是呈現tetramer的型式,其共同的特性是不需任何的co-factor或substrate作用就可產生螢光訊號。本篇論文主要是針對DsRed/EGFP fusion protein之特性來探討:一、觀察此fusion protein在細胞內是以monomer或是tetramer的形式存在。二、探討此fusion protein的mature time是否會因此而有所變化。
所以本實驗構築了三個載體,分別為pDsRed-EGFP (pD-E)、pEGFP-DsRed (pE-D)、pEGFP-His-DsRed (pE-H-D) 。並分別將其與pDsRed及pEGFP在哺乳細胞cos-7中表現,以螢光顯微鏡進行觀察,之後再以Western blot分析其結果。
由實驗結果得知其EGFP-DsRed/DsRed-EGFP fusion protein紅螢光出現的時間較DsRed來的早,但是EGFP-His-DsRed fusion protein不管紅螢光或是綠螢光出現的時間均較其他的protein晚。此三種不同的螢光融合蛋白,和DsRed蛋白類似,在cos-7 cell中均以tetramer的形式存在,其中EGFP-His-DsRed fusion protein在cos-7 cell中較DsRed-EGFP fusion protein及EGFP-DsRed fusion protein易出現aggregation的現象。在transfection後約16hr,這三個constructs所產生的螢光蛋白會有螢光能量共振轉移的現象產生(fluorescence resonance energy transfer, FRET),因而使cos-7細胞呈現黃螢光。所以這三個fusion protein會進行顏色的轉換(color shift),由綠色至紅色,最後呈現黃色,故所構築的fusion protein可作為一fluorescent timer,應用在研究細胞生物學和發育生物學上,作為觀察基因表現的工具之一。
Abstract
Fluorescent proteins (FPs) have become an invaluable marker for gene expression, protein interaction and trafficking. The green fluorescent protein, GFP, exists as a monomer, and the red fluorescent protein, DsRed, exists as a tetramer. The common property is that they both do not need any co-factor or substrate for their production of fluorescent signal.
The main objectives of this dissertation is to characterize the properties of DsRed/EGFP fusion protein, including (1) whether this fusion protein exists as a monomer or a tetramer in mammalian cells, and (2) whether the monomer or tetramer have different mature times for this fusion.
Three vectors, pDsRed-EGFP (pD-E), pEGFP-DsRed (pE-D), and pEGFP-His-DsRed (pE-H-D) were constructed, and were transfected to cos-7 cells. Then, we observed fluorescence in cos-7 cells by fluorescence microscopy. SDS-PAGE and Western blot were further used to confirm the molecular weight of the fusion proteins.
We demonstrated that red fluorescence of DsRed-EGFP/EGFP-DsRed fusion proteins were developed faster than that of EGFP-His-DsRed. Green or red fluorescence of EGFP-His-DsRed fusion protein is developed slower than other proteins. The structure of EGFP-DsRed, EGFP-His-DsRed and DsRed-EGFP fusion proteins is tetramer, which is similar to DsRed protein. More protein aggregation is in EGFP-His-DsRed fusion protein than in EGFP-DsRed and DsRed-EGFP fusion proteins in cos-7 cells. Sixteen hours after transfection, fluorescence resonance energy transfer (FRET) was detected in EGFP-DsRed, EGFP-His-DsRed, and DsRed-EGFP fusion proteins, therefore yellow fluorescence was detected in cos-7 cells. These fusion proteins switch their fluorescence from green to red and to yellow over time, and accordingly it can be “fluorescent timer” that can be applied in monitoring gene expression in cellular and developmental biology.
目錄
英文摘要(abstract)…………………………………………1
中文摘要……………………………………………………..3
序論…………………………………………………………..5
材料和方法…………………………………………………..9
結果………………………………………………………… 17
討論………………………………………………………….23
參考文獻…………………………………………………….28
圖表………………………………………………………….34
附錄………………………………………………………….55
參考文獻
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