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研究生:陳喬富
論文名稱:第一部份斑馬魚熱休克因子HSF2之分子選殖,表現及其活性分析,第二部份有機重金屬三丁基錫對斑馬魚胚胎之核酸配對錯誤修補辨識蛋白MSH2及MSH6mRNA表現之影響
論文名稱(外文):Molecular cloning, expression and characterization of zebrafish (danio rerio) HSF2
指導教授:許濤許濤引用關係
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產生物技術研究所
學門:生命科學學門
學類:生物學類
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:81
中文關鍵詞:熱休克反應熱休克因子有機重金屬三丁基錫斑馬魚核酸配對錯誤修補辨識蛋白MSH2及MSH6之mRNA表現量
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第一部份
在熱休克反應 (Heat shock response) 中,藉由熱休克因子 (HSF, Heat shock factor) 對熱休克蛋白 (HSP, Heat hock protein) 之基因進行調控,使生物體在遭受熱休克的環境壓力下,產生HSPs以保護會因熱休克導致折疊錯誤之蛋白 ; 一般生物體中的HSFs所辨識的HSE (Heat shock element) 序列,具有以5’-nGAAnnTTCnnGAAn-3’ 反向重複之排列特性 (Inverted repeated arrays)。
在本研究中,依據斑馬魚之熱休克因子HSF2全長序列設計專一性引子,以成熟斑馬魚抽取而來的總量RNA合成出的cDNA進行PCR,獲得一段1470 bp之完整cDNA轉譯區 (Open reading frame),可轉譯出490個氨基酸,其氨基酸序列與大白鼠、小白鼠及人類的HSF2之相似度分別達67%、67%及68%,且發現所選殖出的斑馬魚HSF2蛋白之N端第5~110個之氨基酸序列和其它物種的HSF2之N端HSE辨識區域 (DNA binding domain) 有著高達90%以上的相似性。將構築完成之表現載體成功送入宿主細胞,以IPTG誘導斑馬魚重組蛋白HSF2大量表現後,獲得大量之斑馬魚重組蛋白HSF2,且多數以可溶性蛋白之形式存在,加上其N端His-tag後之分子量估計約為61 kDa。將經His-tag 的resin管柱進行純化後的重組蛋白以凝膠抗阻試驗進行活性分析,發現此斑馬魚重組蛋白HSF2對於辨識一般生物體中之HSE sequences具有極高之專一性,反之,對於其他control sequences間的親和力則相當低。
第二部分
環境毒物往往因其毒性而影響生物體內的基因表現,而有機重金屬三丁基錫 (TBT,Tributyltin) 便是一種常見的環境污染物,常被使用於船身作為抗生物附著劑 (Antifouling paints),利用其毒性避免如貝類等濾食性生物吸附於船身 ; 由於TBT帶有三個正丁基 (-CH2CH2CH2CH3),具有脂溶性,提高了對細胞的通透能力,TBT便可藉由環境荷爾蒙的形式,進入生物體內對其基因表現造成影響 ; 由於斑馬魚屬於水生動物,在其發育過程中也可能處於受TBT污染的環境,故針對TBT對胚胎發育時期之斑馬魚核酸配對錯誤修補辨識蛋白MSH2及MSH6之mRNA表現量會有什麼樣的影響進行探討。
藉由斑馬魚發育初期胚胎對TBT耐受性之測試,確認在選定之不同TBT濃度處理下的斑馬魚胚胎由外觀上皆呈現健康狀態後,進行MSH2及MSH6之mRNA表現量的分析,發現經不同TBT濃度處理之受精後10小時的斑馬魚胚胎中MSH2及MSH6 的mRNA表現量並無明顯之差異。

中文摘要 1
英文摘要 3
壹﹑前言 5
貳﹑實驗材料與方法
第一部份
一﹑材料 11
二﹑方法
Ⅰ﹑斑馬魚之飼養 18
Ⅱ﹑斑馬魚之熱休克因子 HSF2 之選殖
A﹑斑馬魚總量核糖核酸 ( total RNA ) 之抽取 18
B﹑斑馬魚熱休克因子 HSF2 基因專一性寡核甘酸引子 18
之設計 ( gene specific primer )
C﹑反轉錄-聚合脢鏈鎖反應 ( RT-PCR ) 19
D﹑DNA 洋菜膠體電泳法 20
E﹑DNA 分子之萃取 20
F﹑DNA 接合 21
G﹑質體轉型 21
H﹑微量質體DNA之抽取 22
I ﹑目標質體之確認 22
Ⅲ﹑斑馬魚之熱休克因子 HSF2 之表現
A﹑質體轉型 23
B﹑目標質體之確認 23
C﹑蛋白表現的誘導 24
D﹑菌體超音波破碎 24
E﹑蛋白質濃度測定 25
F﹑蛋白質樣品分析 25
G﹑蛋白質純化 27
H﹑西方點墨分析 28
I﹑ 活性分析 29
第二部份
一﹑材料 32
二﹑方法
Ⅰ﹑斑馬魚之飼養及受精卵之收集 36
Ⅱ﹑有機重金屬三丁基錫之處理
A﹑有機重金屬三丁基錫之前處理 36
B﹑以有機重金屬三丁基錫處理斑馬魚胚胎 36
Ⅲ﹑斑馬魚胚胎總量核糖核酸 ( total RNA ) 之抽取 37
Ⅳ﹑RNA 洋菜膠體電泳法 38
Ⅴ﹑斑馬魚之核酸配對錯誤修補辨識蛋白MSH2及MSH6
之探針製備及放射線標定
A﹑斑馬魚之核酸配對錯誤修補辨識蛋白之探針製備 38
B﹑斑馬魚之核酸配對錯誤修補辨識蛋白之放射線標定 39
Ⅵ﹑北方點墨分析 39
參﹑結果 41
肆﹑討論 47
伍﹑參考文獻 50
陸﹑附圖 58

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