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研究生:林坤廷
研究生(外文):Kuen-Tyng Lin
論文名稱:臺灣龜殼花毒腺Mucrosobin融合蛋白的功能性表達
論文名稱(外文):Functional Expression of a Recombinant Mucrosobin Protein of Trimeresurus mucrosquamatus(Taiwan habu)
指導教授:郭耀文郭耀文引用關係
指導教授(外文):Yaw-Wen Guo
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:57
中文關鍵詞:臺灣龜殼花功能性表達再折疊活化蛇毒蛋白纖維蛋白原分解
外文關鍵詞:Mucrosobinrefoldingfunctional expressionTrimeresurus mucrosquamatusTaiwan habusnake venomfibrinogenase
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Mucrosobin是本實驗室過去利用免疫篩選的方法,從屬於響尾蛇亞科,為臺灣六種常見陸地性毒蛇之一的臺灣龜殼花蛇毒腺 cDNA library 中所篩選出來的。由於此 mucrosobin cDNA 和 batroxobin 有很高的相似性,因此推測 mucrosobin 應具有分解纖維蛋白原 (fibrinogen) 的生物活性。為了要了解其生物活性表現,本實驗則選用 E. coli BL21 (DE3) 作為表達系統。但利用大腸桿菌所表達出來結果,其融合蛋白會聚集形成不具生物活性的內含體 (inclusion bodies),因此,本研究的目的便是要重新溶解這些內含體,並且提供一個適合 mucrosobin 再折疊活化的條件,使其恢復生物活性的表現。首先將表達蛋白純化,以西方轉印法及蛋白質微量基酸序列分析,確認所表達出來的融合蛋白是分子量為 28 kDa,屬於臺灣龜殼花蛇毒蛋白一員的 mucrosobin 融合蛋白。接著以含有 8M urea, 1mM oxididize glutathion 及 2mM reduced glutathion 且 pH 值為 8.0 的變性劑先將融合蛋白的雜亂結構完全打散變成一級結構,接著再以含有 20% glycerol, 0.75M NaCl, 1mM oxididize glutathion 及 2mM reduced glutathion 且 pH 值為 8.0 的再折疊緩衝液 I 與含有 1% glycerol, 5mM NaCl 且pH 值為 7.5 的再折疊緩衝液 II 的先後各透析 24 小時後,使融合蛋白再折疊活化出具生物活性的三級結構。而由本實驗所提供的分解纖維蛋白原的生物活性分析的結果,顯示成功再折疊活化的 mucrosobin 融合蛋白的確和電腦活性比對分析結果相近,具有分解纖維蛋白原 B-beta 多鏈的生物活性。

Mucrosobin was cloned and sequenced from the venom gland cDNA library of Trimeresurus mucrosquamatus (Taiwan habu), one of the common land venomous snakes in Taiwan and belonging to the subfamily of Crotalinae. Sequence comparison of the deduced amino acid shows high sequence homology between mucrosobin and batroxobin suggested that it might have fibrinogenolytic activity. In order to investigate the biological function of mucrosobin, E. coli BL21 (DE3) was chosen as the expression system for mucrosobin protein. After in-frame expression and purification, the results of Western blot and protein microsequenc show that the recombinant protein with an apparent molecular weight of 28 kDa and with antigenic specificity for Taiwan habu snake venom. However, the recombinant protein in inclusion bodies are the main forms of expressed protein in E. coli with no biological activity. In order to produce functional proteins, the expressed proteins in inclusion bodies was transform into a soluble form and reestablish its biological function by refolding. First the inclusion bodies was solubilized in denature solution (pH 8.0, 8M urea, 1mM oxidize glutathion and 2mM reduced glutathion), then dialysis in refolding buffer I (pH 8.0, 20% glycerol, 0.75M NaCl, 1mM oxidize glutathion and 2mM reduced glutathion) for 24 hrs and dialysis again in refolding buffer II (pH 7.5, 1% glycerol, 5mM NaCl) for another 24 hrs. Biological activities was characterized by fibrinogenolytic activity analysis. In this study, a successful refolding condition was established for the functional expression of a well-characterized recombinant mucrosobin protein, with specific beta-fibrinogenolytic activity.

正文目錄.......................................................I
圖次目錄......................................................IV
縮寫表.........................................................V
中文摘要....................................................VIII
英文摘要......................................................IX
第一章緒言..................................................1
第一節臺灣常見毒蛇分類簡介..................................1
第二節毒腺分怖及蛇毒毒性簡介................................2
壹. 負責阻斷神經肌肉電位傳導作用(neuromuscular transmission) 的
蛇毒蛋白...................................................4
貳. 干擾血液凝集作用 (blood coagulation) 的蛇毒蛋白............5
參. 造成溶血作用 (hemolysis) 的蛇毒蛋白........................8
肆. 導致內出血 (hemorrhage)、傷口組織壞死 (necrosis) 及水腫
(edema) 等作用的蛇毒蛋白...................................8
伍. 影響心臟血管機能 (cardiovascular effects)的蛇毒蛋白........9陸. 刺激獵物釋放出具有藥理活性物質 (liberation of
pharmacologically active substances) 的蛇毒蛋白...........10
第三節 臺灣龜殼花毒腺 cDNA 基因庫的建立及基因篩選.............12
第四節 Batroxobin 蛇毒蛋白簡介................................14
第五節 蛇毒之分子生物層面研究的進展...........................16
第六節 實驗動機及目的.........................................17
第二章 材料與方法.............................................19
第一節 實驗材料...............................................20
壹. 實驗動物..................................................20
貳. 實驗菌種、載體與抗體......................................20
參. 實驗藥品與試劑............................................20
肆.實驗儀器.............................................22
第二節 實驗方法...............................................23
壹. 實驗流程..................................................23貳. Mucrosobin 融合蛋白的表現與純化...........................23
一. Mucrosobin 融合蛋白的誘導表現與親和純化 (affinity
purification).............................................23
二. 蛋白質膠體電泳分析........................................24
三. 西方轉印法 (Western bolt) 分析............................26
參. 蛋白質微量基酸序列分析 (protein micro-sequencing).......26
肆. 蛋白質濃度測定............................................27
伍. Mucrosobinb 融合蛋白的變性 (denaturation)與再折疊活化
(refolding) 步驟..........................................27
陸. 再折疊活化 mucrosobin 融合蛋白分解纖維蛋白原的活性
(fibrinogenolytic activity) 分析..........................29
第三章 結果...................................................31
第一節 Mucrosobin 融合蛋白的表達與西方轉印法分析..............31
第二節 Mucrosobin 融合蛋白的微量基酸序列分析................31
第三節 Mucrosobin 融合蛋白的變性與再折疊活化分析..............32
第四章 討論...................................................42
第一節 以遺傳工程技術來大量製備及研究微量蛇毒蛋白.............42
第二節 Mucrosobin 融合蛋白的西方轉印法分析....................43
第三節 Mucrosobin 融合蛋白的微量基酸序列分析................44
第四節 Mucrosobin 融合蛋白的變性與再折疊活化分析..............45
第五節 再折疊活化 mucrosobin 融合蛋白分解纖維蛋白原的活性分析.46
第六節 未來展望...............................................47
第五章 結論...................................................48
第六章 參考文獻...............................................49
圖次目錄
圖1. 臺灣龜殼花蛇毒腺 mucrosobin 融合蛋白功能性表達的實驗流程.33
圖2. Mucrosobin 基因表達融合蛋白的電泳分析及西方轉印檢測......34
圖3. Mucrosobin 融合蛋白經電泳分離轉印至 PVDF 膜的結果分析....35
圖4. Mucrosobin 融合蛋白氨基端微量基酸序列分析結果..........36
圖5. Mucrosobin 融合蛋白與 mucrosobin cDNA 轉譯區基因片段所推
衍出來的氨基端基酸序列分析比較.........................39
圖6. 未經再折疊活化 mucrosobin 融合蛋白與纖維蛋白原在不同作用
時間下的電泳膠體分析.....................................40
圖7. 再折疊活化 mucrosobin 融合蛋白與蛇毒液在不同作用時間下,
分解纖維蛋白原活性的電泳膠體分析.........................41

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