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系統識別號 U0026-0708201216522500
論文名稱(中文) iLOV 螢光蛋白在厭氧環境下的生物應用
論文名稱(英文) The Study of iLOV Fluorescent Protein for Biological Application under Hypoxia
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 100
學期 2
出版年 101
研究生(中文) 席欣瑜
研究生(英文) Hsin-Yu Hsi
學號 s16991045
學位類別 碩士
語文別 中文
論文頁數 103頁
口試委員 指導教授-張敏政
口試委員-張明熙
口試委員-張文粲
口試委員-鄭宏祺
中文關鍵字 iLOV  螢光共振能量轉移  厭氧  蛋白交互作用 
英文關鍵字 iLOV  FRET  hypoxia  protein-protein interaction 
學科別分類
中文摘要 螢光蛋白像是GFP及其衍生物,已被廣泛的運用在活體中觀察蛋白表現及功能性的分析,並且,螢光蛋白可做為蛋白質複合物的功能性分析,像是在活細胞中利用螢光共振轉移(FRET)技術觀察蛋白質的交互作用,儘管GFP及其變異衍生物已被視為是活體內非侵入性的影像技術的重要發現,GFP及其家族蛋白仍存在一個主要缺點,即是在個別的螢光發色基團成熟過程必須要有氧分子的參與, 相較於GFP,我們實驗室先前研發出的藍色螢光蛋白dBP6,由於他利用輔因子NADPH的結合做為螢光發色基團,所以在有氧及無氧環境皆可以發出藍色的螢光,為了進一步擴展dBP6的應用性,我們應用了一個與FMN (flavin mononucleotide)結合的綠色螢光蛋白iLOV(約11kDa),由於iLOV的發光機制與dBP6類似,是一有潛力與dBP6做為無氧下FRET配對的螢光蛋白,另外,根據先前文獻,iLOV還未被應用在哺乳類細胞當中,我們的目標則是要評估在哺乳類細胞中,iLOV是否能夠用於蛋白質標記及有氧和無氧下的觀察,利用改變iLOV的密碼子使用,我們成功的將iLOV在哺乳類細胞中有氧及無氧環境中表現,並且發現有做為融合蛋白功能。在分析iLOV及dBP6的吸收波長之後,發現iLOV的最大激發波長及dBP6的散射波長區域有重疊,接著我們將dBP6和iLOV連接在一起,進一步觀察在細胞中FRET的現象,但是結果顯示iLOV在350nm時也會被激發,與dBP6的激發波長重疊,為了要解決此問題,我們測量了dBP6及iLOV 不同的激發波長,每10nm為間距由320nm到350nm,結果顯示縮短激發波長可能有辦法解決iLOV上述的問題,有鑑於激發波長的改變,我們可以設計讓330nm以下波長激發光通過的特製濾鏡供無氧的應用。螢光蛋白的螢光強度仍然需要再改良,這個研究可以帶給我們在厭氧環境下偵測蛋白交互作用一個新的方向。
英文摘要 Fluorescent proteins (FPs), like green fluorescent protein(GFP) and its derivatives, have been used for various applications as in detecting protein expression and conducting functional studies in living systems. Moreover, complex functional studies can be performed using FPs such as visualizing protein-protein interactions in living cells by FRET (Fluorescence Resonance Energy Transfer) detection technique. Although GFP and its variants have become an invaluable tool in the field of non-invasive in vivo imaging technology, a major drawback of all members of GFP family-their strict requirement for molecular oxygen as a cofactor for the synthesis of their respective chromophores. In contrast to GFP, our laboratory developed blue fluorescent protein-dBP6, which emits blue fluorescence under normoxia and hypoxia due to its cofactor NADPH as the fluorescent chromophore. To further extend the use of dBP6, we utilized a FMN (flavin mononucleotide) based green fluorescent protein iLOV (≈11kDa) that may potentially become FRET pair with dBP6 under hypoxia since its illuminated mechanism is similar to that of dBP6. In addition, studies regarding to iLOV application in mammalian cells have not been explore yet. Our goal is to evaluate whether iLOV can be utilized for protein labeling and detection under hypoxia and normoxia in mammalian cells. By changing the iLOV codon usage, we successfully expressed iLOV in mammalian cells under normoxia and hypoxia and found that it can function as a fusion protein. After checking the spectrum of iLOV and dBP6, we found the overlaping region between iLOV maximum excitation peak and dBP6 emission peak. Next, we linked the dBP6 and iLOV together to further investigate FRET application in cells. However, the results showed that iLOV could be excited at the spectra of 350nm overlapped with that of dBP6(350nm). In order to solve this problem, we measured different excitation wavelengths of dBP6 and iLOV every 10nm in between 320nm to 350nm. The result indicated that lowering the excitation wavelength of dBP6 might avoid the above problem of iLOV. Due to the results of excitation wavelength shift, we can design the filter specific for the wavelength below 330nm for further application under hypoxia.The fluorescent intensity still have to be optimized. This study provides us a new direction of detecting protein protein interaction under hypoxia.
論文目次 中文摘要 I
AbstractIII
致謝V
目錄VI
圖表目錄VIII
縮寫表X
第1章緒論
1-1螢光生物分類1
1-2綠色螢光蛋白(GFP)簡介1
1-3非傳統螢光性的藍色螢光蛋白dBP6簡介2
1-4螢光蛋白iLOV 簡介4
1-5影響螢光蛋白表現的因子5
1-6螢光蛋白在生物醫學上的應用7
研究動機16
第2章材料與方法
2-1實驗菌株、載體與培養基配方17
2-1-1實驗菌株17
2-1-2載體17
2-1-3培養基配方17
2-2質體建構18
2-2-1 聚合酶連鎖反應 ( PCR ) 18
2-2-2 構築PCR片段於質體中19
2-2-3 E coil 形質轉形20
2-2-4 小量質體製備21
2-3細胞培養方法23
2-3-1 實驗細胞株23
2-3-2 細胞解凍23
2-3-3 細胞繼代培養24
2-3-4 細胞計數24
2-3-5 細胞保存25
2-3-6 細胞轉染26
2-4蛋白質分析27
2-4-1 蛋白質萃取27
2-4-2 蛋白質定量28
2-4-3 SDS-PAGE 蛋白質電泳28
2-4-4 西方墨點法30
2-5螢光蛋白LOV及iLOV基本特性分析31
2-5-1利用Luminescence Spectroscopy 分析LOV及iLOV吸收光譜31
2-5-2 利用LDH cytotoxicity assay分析表現iLOV螢光蛋白細胞的毒性試驗32
2-6 螢光蛋白LOV及iLOV在有氧及厭氧環境下的螢光表現33
2-6-1 利用螢光顯微鏡觀察原核系統iLOV螢光蛋白表現33
2-6-2 利用螢光顯微鏡觀察真核系統LOV及iLOV螢光蛋白表現34
2-6-3 利用Luminescence Spectroscopy分析LOV及iLOV螢光強度分析35
2-6-4利用西方墨點法分析LOV及iLOV螢光蛋白在有氧及厭氧環境下的蛋白質表現35
2-7 螢光蛋白D7及iLOV做為FRET配對螢光蛋白分析37
2-7-1 利用Luminescence Spectroscopy個別分析D7及iLOV不同激發波長的散射波鋒37
2-7-2 利用Luminescence Spectroscopy分析D7- iLOV在特定激發波長下之散射波鋒38
2-7-3 利用SDS-PAGE分析D7-linker-iLOV蛋白質表現39
第3章實驗結果
3-1 利用改變密碼子使螢光蛋白iLOV 在原核及真核中表現41
3-2 iLOV螢光蛋白的基本特性分析41
3-3 綠色螢光蛋白iLOV在生醫上的基礎應用44
3-4 iLOV螢光蛋白在厭氧環境下的表現45
3-5 藍色螢光蛋白dBP6及綠色螢光蛋白iLOV做為FRET配對螢光蛋白設計46
3-6 評估不同激發波長下iLOV及D7表現情形47
3-7 利用BiFC原理設計dBP6及iLOV的fluorescent complex49
第4章實驗討論51
參考文獻56
實驗結果圖表63
附錄93
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