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研究生:潘志建
研究生(外文):Zhi-Jian Pan
論文名稱:海鱺血清中抑制弧菌蛋白質分解酵素物質之純化及特性分析
論文名稱(外文):Purification and Characterization of Vibrio protease inhibitors from the serum of cobia (Rachycentron canadum)
指導教授:劉秉忠
指導教授(外文):Ping-Chung Liu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:117
中文關鍵詞:蛋白質分解酵素抑制物純化
外文關鍵詞:proteaseinhibitorpurification
相關次數:
  • 被引用被引用:2
  • 點閱點閱:173
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摘要

本論文進行海鱺血清中具有抑制細菌細胞外產物蛋白質分解酵素活性之物質的分離純化及特性分析。實驗菌株為溶藻弧菌V. alginolyticus C11a及巴斯德桿菌Photobacterium damselae subsp. piscicida Cp。
以硫酸銨沈澱法將海鱺血清區分成飽和度30~50 % (A組)及50~70 % (B組)兩組,再利用快速蛋白質液態層析系統(FPLC)中的親和力層析管柱、疏水性層析管柱、陰離子交換法等管柱來分離及純化血清中蛋白質分解酵素抑制物。以Native-PAGE、交叉免疫電泳(CIE)及SDS-PAGE電泳分析鑑定其純度及分子量,發現兩組在Native-PAGE膠片各出現一條蛋白帶,在CIE膠片各出現一個沈澱弧,分子量分別為185 kDa及74 kDa,再以zymogram電泳確認此蛋白帶具有抑制蛋白質分解酵素活性。
兩組部份純化之蛋白質分解酵素抑制物,以 60℃處理20分鐘後,其抑制蛋白質分解酵素活性的抑制活性皆降低至15%以下,屬於熱不安定型之酵素。以0.2 M methylamine處理30分鐘後,其抑制蛋白質分解酵素活性的抑制活性也明顯的受到影響,以及其等電點值各介於pI 4.15~4.55及pI 3.75~4.55之間,因此推測海鱺血清中的兩種蛋白質分解酵素抑制物應該屬於α 2-macroglobulin類。
Abstract

The thesis investigates the anti-vibrio protease component(s) present in the serum of cobia, Rachycentron canadum, including isolation, purification and characterization. Strains of Vibrio alginolyticus C11a and Photobacterium damselae subsp. piscicida Cp were used for assays.
Cobia serum was precipitated by ammonium sulfate and seperatated into parts A (30~50%) and B (50~70%). The protease inhibitor(s) were isolated and purified from A or B parts by using affinity chromatography column, hydrophobic interaction chromatography column and anion exchange column on the Fast Liquid Chromatography System. After purification, an analysis for the homogeneity and molecular weight of the protease inhibitor(s) were determined by using Native-PAGE、crossed immunoelectrophoresis (CIE) and SDS-PAGE. One protein band was observed on Native-PAGE and one precipitation arc was observed on CIE gel using fractions purified from parts A and B, respectively. The protein band on Native-PAGE exhibiting anti-protease avtivity was confirmed by zymogram. The molecular weight of the protease inhibitors was 185 kDa and 74 kDa, respectively.
More than 85% of the anti-protease activity in the two partially purified fractions was inhibited after incubation at 60℃ for 20 minutes prior to the assay indicating that the two protease inhibitors were heat-labile. The anti-protease activity of the two protease inhibitors was both apparently inhibited by 0.2M methylamine (incubated 37℃ for 30 mintues) together with pI values estimated between 4.15-4.55 and 3.75-4.55, respectively. Therefore, the two protease inhibitors in cobia serum were suggested to be α2-macroglobulin-like proteins.
目錄

中文摘要----------------------------------------------------------------------------------------Ⅰ
英文摘要----------------------------------------------------------------------------------------Ⅲ
謝辭----------------------------------------------------------------------------------------------Ⅴ
表目錄-------------------------------------------------------------------------------------------Ⅸ
圖目錄-------------------------------------------------------------------------------------------Ⅸ
一、 前言-----------------------------------------------------------------------------------------1
二、 文獻整理-----------------------------------------------------------------------------------5
三、 材料方法----------------------------------------------------------------------------------21
3.1海鱺血清之製備------------------------------------------------------------------------21
3.1.1海鱺(Rachycentron canadum)來源------------------------------------------21
3.1.2海鱺血液之取----------------------------------------------------------------------21
3.1.3海鱺血清之處理保存-------------------------------------------------------------21
3.2弧菌、桿菌及其細胞外產物-----------------------------------------------------------22
3.2.1菌株來源及保存-------------------------------------------------------------------22
3.2.2細菌細胞外產物之製備及保存-------------------------------------------------23
3.3海鱺血清中蛋白質分解酵素抑制物質之分離純化------------------------------23
3.3.1硫酸銨沈澱法(Ammonium sulfate precipitation)-------------------------23
3.3.2親和力層析法(Affinity chromatography)----------------------------------24
3.3.3疏水性管柱層析法(Hydrophobic interaction chromatography, HIC)-----24
3.3.4陰離子交換法-RESOURCE Q(Pharmacia)管柱層析法----------------25
3.3.5陰離子交換法-MONO Q(Pharmacia)管柱層析法------------------------25
3.4蛋白質分解酵素抑制物質純度(Homogenetity)之鑑定及分子量(Molecular weight)之估算-------------------------------------------------------------------------26
3.4.1電泳及染色-------------------------------------------------------------------------26
3.4.2海鱺血清中蛋白質分解酵素抑制物質之鑑定-------------------------------27
3.4.3交叉免疫電泳(Crossed immunoelectrophoresis)-------------------------28
3.5兔子抗血清之製備---------------------------------------------------------------------28
3.5.1兔子抗海鱺全血清抗血清之製備----------------------------------------------28
3.5.2兔子抗海鱺血清蛋白質分解酵素抑制物抗血清----------------------------29
3.6部分純化之海鱺血清蛋白質分解酵素抑制物質之特性分析------------------29
3.6.1蛋白質含量之測定----------------------------------------------------------------29
3.6.2海鱺血清蛋白質分解酵素抑制物之抑制活性測定-------------------------30
3.6.3等電點(isoelectric point, pI)之判定--------------------------------------------31
3.6.4 Methylamine對海鱺血清蛋白質分解酵素抑制物抑制細菌細胞外產物蛋白質分解酵素活性之影響---------------------------------------------------31
3.6.5溫度對海鱺血清蛋白質分解酵素抑制物抑制活性之影響----------------31
3.6.6 pH值對海鱺血清蛋白質分解酵素抑制物抑制活性之影響---------------32
3.6.7海鱺血清蛋白質分解酵素抑制物對不同稀釋度之細菌細胞外產物的抑制活性測定------------------------------------------------------------------------32
3.6.8海鱺血清蛋白質分解酵素抑制物對不同菌種之細胞外產物的抑制活性測定---------------------------------------------------------------------------------32
四、 結果----------------------------------------------------------------------------------------34
4.1海鱺血清蛋白質分解酵素抑制物之分離純化------------------------------------34
4.1.1硫酸銨沈澱法(Ammonium sulfate precipitation)----------------------------34
4.1.2親和力層析法(Affinity chromatography)-------------------------------------34
4.1.3疏水性管柱層析法(Hydrophobic interaction chromatography, HIC)-----35
4.1.4陰離子交換法-RESOURCE Q(Pharmacia)管柱層析法------------------35
4.1.5陰離子交換法-MONO Q(Pharmacia)管柱層析法------------------------36
4.1.6蛋白質分解酵素抑制物質純度之鑑定及分子量之估算-------------------37
4.1.7交叉免疫電泳分析部分化之蛋白質分解酵素抑制物質-------------------38
4.1.8海鱺血清部分純化之蛋白質分解酵素抑制物之等電點值(pI value)---39
4.2海鱺血清蛋白質分解酵素抑制物之特性分析------------------------------------39
4.2.1 Methylamine對海鱺血清蛋白質分解酵素抑制物抑制細菌細胞外產物蛋白質分解酵素活性之影響---------------------------------------------------39
4.2.2溫度對海鱺血清蛋白質分解酵素抑制物抑制活性之影響----------------40
4.2.3 pH值對海鱺血清蛋白質分解酵素抑制物抑制活性之影響---------------41
4.2.4海鱺血清蛋白質分解酵素抑制物對不同稀釋度之細菌細胞外產物的抑制活性測定------------------------------------------------------------------------42
4.2.5海鱺血清蛋白質分解酵素抑制物對不同菌種之細胞外產物的抑制活性測定---------------------------------------------------------------------------------43
五、 討論----------------------------------------------------------------------------------------45
5.1海鱺血清中蛋白質分解酵素抑制物之種類探討---------------------------------46
5.2 pH值對海鱺血清蛋白質分解酵素抑制物抑制活性之影響探討--------------52
5.3交叉免疫電泳分析蛋白質分解酵素抑制物質及海鱺全血清------------------52
5.4海鱺血清中蛋白質分解酵素抑制物之電荷異質性(charge heterogeneity)--53
5.5探討海鱺血清中蛋白質分解酵素抑制物在抵抗細菌性疾病中可能的角色----------------------------------------------------------------------------------------------------53
六、 參考文獻----------------------------------------------------------------------------------56
表-------------------------------------------------------------------------------------------------76
圖-------------------------------------------------------------------------------------------------77
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