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研究生:陳逢叡
研究生(外文):Ferng-Ruey Chen
論文名稱:溶藻弧菌蛋白分解酵素特性及其對斑節蝦之致病機制研究
論文名稱(外文):Studies on the characteristics and pathogenesis of protease produced by Vibrio alginolyticus on kuruma prawn, Penaeus japonicus
指導教授:李國誥李國誥引用關係
指導教授(外文):Kuo-Kau Lee
學位類別:博士
校院名稱:國立海洋大學
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:1999
畢業學年度:87
語文別:中文
論文頁數:92
中文關鍵詞:溶藻弧菌蛋白分解酵素斑節蝦
外文關鍵詞:Vibrio alginolyticusproteasekuruma prawn
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本論文探討分離自罹病斑節蝦病原弧菌Vibrio alginolyticus Swy之蛋白分解酵素特性及其對斑節蝦之致病機制。
本研究首先從Swy菌株之細胞外產物中分離純化出蛋白分解酵素,再進行特性分析及致死試驗。所純化出之蛋白分解酵素以hide power azure (HPA)為酵素受質所測得的活性會被phenylmethane sulfonyl fluoride (PMSF)及antipain所抑制,在pH值8至11、溫度50至55℃之間酵素活性最高,其分子量經以SDS-PAGE及膠過濾層析法估算為33 kDa,以IEF電泳估算pI值為4.3。此蛋白分解酵素能使斑節蝦迅速死亡,其LD50值為0.29 g protein/g body weight,且其對斑節蝦的毒性幾乎完全被PMSF所抑制,顯示此由Swy菌株所生產的蛋白分解酵素為具致死性的絲胺酸型蛋白分解酵素(alkaline serine protease)。
Swy菌株所生產的蛋白分解酵素以水溶性受質(azoalbumin及azocasein)來進行抑制試驗時可完全被EDTA,EGTA,但若以非水溶性受質(azocoll 及HPA)進行抑制試驗時僅部份被EDTA,EGTA抑制。但以此四種酵素受質進行抑制試驗時皆不受1,10-phenanthroline所抑制,顯示Swy菌株所生產的蛋白分解酵素非屬金屬型蛋白分解酵素。因此為了避免被誤導,進行酵素種類判定時需慎選酵素受質及抑制劑
為了解不同來源的V. alginolyticus菌株其細胞外產物是否具有共通性,因此進行比較五株不同來源菌株的細胞外產物。各種來源的V. alginolyticus菌株的主要酵素活性皆為蛋白分解酵素,由LD50之結果顯示分離自斑節蝦之菌株Swy及分離自九孔之菌株H-11其細胞外產物之致死性比其他菌株強。對所有菌株細胞外產物進行抑制,毒性中和試驗,結果顯示PMSF對所有菌株的蛋白分解酵素活性皆有抑制效果,酵素殘存活性越高的菌株其細胞外產物的致死率則越高,因此V. alginolyticus的蛋白分解酵素對斑節蝦的致病性應扮演著重要的角色。兔子抗Swy菌株細胞外產物或致死蛋白分解酵素之抗血清對各菌株之細胞外產物僅具部份毒性中和效果。但不論是抗細胞外產物或抗致死蛋白分解酵素抗血清最高僅有50%之保護性,其原因有待進一步釐清。
以交叉免疫電泳(CIE)觀察Swy菌株活菌、細胞外產物、致死蛋白分解酵素三者對斑節蝦血漿之影響,結果發現細胞外產物及致死蛋白分解酵素在體外很明顯會對斑節蝦血漿中的凝結蛋白造成影響,使其在CIE膠片上發生電泳快速移動現象,且造成蝦血漿顏色由藍轉紅褐及粉紅。而且細胞外產物尚可影響斑節蝦血漿中其它所有的成分。在體外處理組中活菌及細胞外產物攻擊蝦體後會使斑節蝦血漿之凝結蛋白含量明顯降低,但致死蛋白分解酵素處理組則較不明顯。
This thesis investigated the characteristics of protease and pathogenesis of Vibrio alginolyticus strain Swy in kuruma prawn.
An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy was purified using the AKTA purifier system with hydrophobic interaction chromatography, anion exchange and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl methylsulphonyl fluoride (PMSF), antipain and shows maximal activity at pH 8 to 11(HPA used), having a pI of 4.3 and a molecular weight of 33 kDa. The purified protease was lethal for kuruma prawn at an LD50 of 0.29 g protein/g body weight. The addition of PMSF completely inhibited the lethal toxicity of the purified protease, indicating that this serine protease was a lethal toxin produced by the bacterium.
Four chromogenic substrates for characterizing serine protease of strain Swy were evaluated. The protease activity of bacterial extracellular products (ECP), or the 33 kDa purified protease, was completely inhibited by EDTA, EGTA using water-soluble substrates (azoalbumin and azocasein). Since chelating agents and 1,10-phenanthroline are commonly employed as inhibitors to identify metalloprotease, the two water-soluble substrates may not be appropriate for this purpose, except for using 1,10-phenanthroline as an inhibitor.
Virulence of ECPs and the lethality attributes of serine proteases secreted by five pathogenic V. alginolyticus strains from various sources in kuruma prawn were studied. The ECPs of organisms originally isolated from diseased kuruma prawn or small abalone Haliotis diversicolor supertexta was more virulent (LD50 value of 0.48 or 0.41 g protein/g body weight) than those from diseased tiger prawn P. monodon, yellowfin porgy Acanthopagrus latus or horse mackerel (LD50 value of 0.98-1.17 g protein/g body weight). All the ECPs manifested strong, weak and no activities against gelatin, sheep erythrocytes and chitin, respectively. The higher inhibition of serine protease activity resulted in lower mortality rate of the ECP injected into the prawns suggesting that the protease is the lethal factor secreted by V. alginolyticus. The toxicity of the respective ECPs to kuruma prawns was only partially neutralized by rabbit antiserum to the formalinized Swy ECP and/or rabbit antiserum to the formalinized Swy serine protease.
The haemolymph withdrawn from the moribund prawns injected with the ECP or toxic protease was unable to clot. The coagulogen was fast migrated in CIE following in vitro toxic protease and ECP treatment, but the amount of all components were reduced only in ECP treatment except coagulogen. After these treatment the plasma colour change from blue to pink was recorded. The amount of coagulogen was reduced in CIE following in vivo bacteria and ECP treatment, however that of toxic protease treatment was not remarkable.
封面
中文摘要
Abstract
目錄
前言
第一章 文獻整理
1.1 弧菌症的簡介
1.2 弧菌症的控制
1.2.1 診斷服務
1.2.2 緊迫(stress)的管理
1.2.3 疫苗的使用
1.2.4 化學治療法
1.3 微生物的致病性
1.4 vibrio alginolyticus菌株生理及生化特性
1.5 vibrio alginolyticus的細胞外酵素
1.6 甲殼類的防禦機制
第二章 vibrio alginolyticus swy主要外毒素之分離純化與生化特性
2.1 摘要
2.2 前言
2.3 材料與方法
2.3.1 菌株
2.3.2 細胞外產物之製備
2.3.3 蛋白質含量測定
2.3.4 蛋白分解酵素(protease)活性測定
2.3.5 細胞外產物蛋白分解酵素之分離純化
2.3.6 電泳及染色
2.3.7 分子量估算
2.3.8 兔子抗血清的製備
2.3.9 交叉免疫電泳(CIE)
2.3.10 不同酸鹼度下酵素活性
2.3.11 最適溫度
2.3.12 熱安定性
2.3.13 抑制劑的影響
2.3.14 二價金屬離子的影響
2.3.15 毒性試驗
2.4 結果
2.4.1 細胞外產蛋白分解酵素之純化
2.4.2 電泳
2.4.3 膠過濾液相層析法估算分子量
2.4.4 交叉免疫電泳(crossed immunoelectrophoresis,CIE)
2.4.5 蛋白分解酵素的特性
2.4.6 抑制劑作用
2.4.7 毒性試驗
2.5 討論
第三章 以不同受質來比較vibrio alginolyticus swy細胞外蛋竹日分解酵素的酵素特性
3.1 摘要
3.2 前言
3.3 材料與方法
3.3.1 菌株
3.3.2 細胞外產物之製備
3.3.3 蛋白質含量測定
3.3.4 蛋白分解酵素(protease)活性測定
3.3.5 抑制劑的影響
3.4 結果
3.4.1 酵素活性
3.4.2 抑制劑的影響
3.5 討論
第四章 比較五株不同來源溶藻弧菌細胞外產物生化特性及毒性
4.1 摘要
4.2 前言
4.3 材料與方法
4.3.1 菌株株來源
4.3.2 細胞外產物
4.3.3 蛋白質含量測定
4.3.4 蛋白分解酵素(protease)活性測定
4.3.5 細胞外產物酵素及溶血活性測定
4.3.6 兔子抗血清的製備
4.3.7 免疫擴散試驗
4.3.8 細胞外產物毒性抑制試驗
4.3.9 細胞外產物毒性中和試驗
4.4 結果
4.4.1 vibrio alginolyticus各菌株的細胞外產物之酵素活性和溶血活性
4.4.2 免疫擴散試驗
4.4.3 細胞外產物之致死試驗
4.4.4 細胞外產物毒性抑制試驗
4.4.5 細胞外產物毒性中和試驗
4.5 討論
第五章 vibrio alginolyticus swy活菌、細胞外產物、致死蛋白分解酵素對斑節蝦血漿的影響
5.1 摘要
5.2 前言
5.3 材料與方法
5.3.1 菌株
5.3.2 細胞外產物之製備
5.3.3 蛋白質含量測定
5.3.4 細胞外產蛋白分解酵素之分離純化
5.3.5 兔子抗斑節蝦血淋巴(hemolymph)抗血清的製備
5.3.6 交叉免疫電泳
5.3.7 斑節蝦血漿的取得
5.3.8 V.alginolyticus細胞外產物、致死蛋白分解酵素於體外對斑節蝦血漿的影響
5.3.9 V.alginolyticus活菌、細胞外產物、致死蛋白分解酵素對斑節蝦血漿體內的影響
5.4 結果
5.4.1 體外的影響(in vitro)
5.4.2 體內的影響(in vivo)
5.5 討論
第六章 總結
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