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研究生:林積陽
論文名稱:養殖海鱺細菌性病原之分離及巴斯德桿菌之致病性研究
論文名稱(外文):Isolation of bacterial pathogens in cultured cobia, rachycentron canadum and pathogenesis of photobacterium damsela subsp. piscicida infection
指導教授:劉秉忠
學位類別:碩士
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:2002
畢業學年度:90
語文別:中文
中文關鍵詞:海鱺巴斯德桿菌致病性研究
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  • 被引用被引用:9
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本研究針對分離自罹病養殖海鱺(Cobia, Rachycentron canadum)腫脹後腎之Photobacterium damsela subsp. piscicida(strain C.P.)進行其對海鱺魚體致病機制之研究,並與分離自罹病養殖海鱺前腎之Vibrio alginolyticus (strain C3c)及V. carchariae(strain C3d)比較其菌體對海鱺魚體之毒性。
在菌體對海鱺魚體(10g)之毒性試驗中,Ph. damsela subsp. piscicida(strain C.P.)之LD50約為1.03 × 104 CFU / g fish body weight;V. alginolyticus (strain C3c)之LD50約為3.28 × 104 CFU / g fish body weight;而V. carchariae(strain C3d)之LD50約為7.48 × 104 CFU / g fish body weight,所以此三者中又以Ph. damsela subsp. piscicida(strain C.P.)對海鱺之毒性最強。而在細胞外產物對海鱺(10g)之毒性試驗中,strain C.P. 之LD50約為1.26 *g protein / g fish body weight。
以Ph. damsela subsp. piscicida(strain C.P.)之細胞外產物在快速液體層析儀(FPLC)經陰離子交換層析管柱(Q Sepharose High Performance)、RESOURCE Q層析管柱及Mono Q層析管柱作蛋白質分解酵素之分離純化。所得部分純化物質在SDS-PAGE鑑定其分子量約為7.2 KDa與34.3 KDa,而利用酪蛋白膠片分析後確認只有34.3 KDa處之物質具有蛋白質分解酵素活性,並可造成海鱺死亡。該部分純化物質對海鱺血球具溶血能力(210 haemolytic units / mg protein),對casein具有分解能力,熱不安定(60℃,30分鐘)且其最適酸鹼值為pH 6∼8;其蛋白質分解酵素活性受到L-cysteine、EDTA、EGTA、1-10 phenanthroline、TPCK及TLCK等抑制劑之明顯抑制,顯示其應為金屬型蛋白質分解酵素,而SDS亦抑制70%的蛋白質分解酵素活性;此蛋白質分解酵素在巴斯德桿菌感染海鱺的致病機制中可能扮演重要角色。
This study investigated the pathogenesis of Photobacterium damsela subsp. piscicida (strain C.P.), isolated from swollen kidney of diseased cobia (Rachycentron canadum) in the fish. Virulence of the bacterial cells was compared to that of Vibrio alginolyticus (strain C3c) and V. carchariae (strain C3d) isolated from kidney of diseased cobia as well.
The virulence tests were conducted in cobia (10g) and the LD50 values of Ph. damsela subsp. piscicida (strain C.P.), V. alginolyticus (strain C3c) and V. carchariae were 1.03 × 104, 3.28 × 104 and 7.48 × 104 CFU / g fish body weight, respectively, Ph. damsela subsp. piscicida (strain C.P.) was more virulent than the other two vibrio species. The LD50 value of extracellular products (ECP) from strain C.P. in cobia (10g) was 1.26 *g protein / g fish body weight.
Protease was partially purified from ECP of Ph. damsela subsp. piscicida (strain C.P.) by using anion exchange columns (Q Sepharose High Performance, RESOURCE Q and Mono Q) on Fast Protein Liquid Chromatography (FPLC). Partially purified protease fraction contained 7.2 KDa and 34.3 KDa protein band on SDS — PAGE with the latter band band exhibiting protease activity. The fraction was lethial to cobia, haemolytic (210 haemolytic units / mg protein) against erythrocytes of cobia, caseinolytic and heat-labile with optimal pH values between 6 and 8. As protease activity in the fraction was completely inhibited by the presence of L-cysteine, EDTA, EGTA, 1-10 phenanthroline, TPCK and TLCK, it was suggested to be a metallo-protease. However, about 70% of protease activity in the fraction was also inhibited by SDS. This metallo-protease may play an important role in the pathogenesis of Ph. damsela subsp. piscicida infection in the cobia.
◆中文摘要 --------------------------------------------------Ⅰ
◆英文摘要 --------------------------------------------------Ⅲ
◆表目錄 ----------------------------------------------------Ⅴ
◆圖目錄 ----------------------------------------------------Ⅵ
◆第一章 前言 -----------------------------------------------1
◆第二章 文獻整理 -------------------------------------------4
◆第三章 材料與方法------------------------------------------18
壹、試驗菌株之採樣分離、來源、確認與保存 --------------------18
貳、細菌生理生化特性之鑑定分析 ------------------------------19
一、革蘭氏染色 ------------------------------------------19
二、葡萄糖的利用 ----------------------------------------20
三、MacConkey agar之生長能力-----------------------------20
四、對NaCl之耐受性試驗 ----------------------------------20
五、藥物敏感性試驗 --------------------------------------21
六、溶血能力測定 ----------------------------------------21
七、API 20E system -------------------------------------21
八、Biolog system --------------------------------------22
九、BIONOR MONO-Pp rapid diagnostic test ----------------22
參、Photobacterium damsela subsp. piscicida(strain C.P.)細胞
外產物之基本特性分析 ----------------------------------- 23
一、細胞外產物(extracellular products, ECP)之製備-23
二、細胞外產物之特性分析 ---------------------------23
1、蛋白質含量的測定-----------------------------23
2、蛋白質分解酵素(protease)活性測定-----------24
3、不同培養基對細胞外產物蛋白質分解酵素活性之影響- ---------------------------------------------24
4、溫度對細胞外產物蛋白質分解酵素活性之影響-----25
5、pH值對細胞外產物蛋白質分解酵素活性之影響-----25
6、抑制劑對細胞外產物蛋白質分解酵素活性之影響---25
7、二價金屬離子對細胞外產物蛋白質分解酵素活性之
影響---------------------------------- ------26
8、細胞外產物對casein、gelatin、egg yolk、tween80
分解試驗-------------------------------------26
9、溶血活性測定---------------------------------26
肆、Photobacterium damsela subsp. piscicida(strain C.P.)、
Vibrio alginolyticus(strain C3c)與Vibrio carchariae
(strain C3d)對海鱺之毒性攻擊試驗 -----------------------27
一、Ph. damsela subsp. piscicida(strain C.P.)、V.
alginolyticus(strain C3c)與V. carchariae(strain C3d)
菌體對海鱺之毒性試驗 ---------------------------------27
二、Ph. damsela subsp. piscicida(strain C.P.)細胞外產物對
海鱺之毒性試驗 ---------------------------------------27
伍、Photobacterium damsela subsp. piscicida(strain C.P.)細胞
外產物毒性抑制試驗 ------------------------------------28
陸、Photobacterium damsela subsp. piscicida(strain C.P.)細胞
外產物中蛋白質分解酵素之部分分離純化與特性分析 ----------28
一、細胞外產物中蛋白質分解酵素之部份分離純化 -------------28
1、陰離子交換層析法-HP管柱層析 -----------------------28
2、陰離子交換法-RESOURCE Q管柱層析 -------------------29
3、陰離子交換法-Mono Q管柱層析------------------------30
二、電泳及染色 -------------------------------------------30
三、電泳中蛋白質分解酵素之測定 ---------------------------31
四、Mono Q部份純化蛋白質分解酵素的特性分析 ---------------31
五、Mono Q部份純化蛋白質分解酵素對海鱺之毒性攻擊試驗 -----32
◆第四章 結果 ----------------------------------------------33
壹、試驗菌株之採樣分離 --------------------------------------33
貳、細菌生理生化特性之鑑定分析 ------------------------------34
參、Photobacterium damsela subsp. piscicida(strain C.P.)細胞
外產物之基本特性分析 ------------------------------------35
肆、Photobacterium damsela subsp. piscicida(strain C.P.)、
Vibrio alginolyticus(strain C3c)與Vibrio carchariae
(strain C3d)對海鱺之毒性攻擊試驗------------------------37
一、Ph. damsela subsp. piscicida(strain C.P.)、V.
alginolyticus(strain C3c)與V. carchariae(strain C3d)
菌體對海鱺之毒性試驗--------------------------------- 37
二、Ph. damsela subsp. piscicida(strain C.P.)細胞外產物對
海鱺之毒性試驗----------------------------------------37
伍、Photobacterium damsela subsp. piscicida(strain C.P.)細胞
外產物毒性抑制試驗 --------------------------------------38
陸、Photobacterium damsela subsp. piscicida(strain C.P.)細胞
外產物中蛋白質分解酵素之部分分離純化與特性分析 ----------38
一、細胞外產物中蛋白質分解酵素之部份分離純化 -------------38
1、陰離子交換層析法-HP管柱層析 -----------------------38
2、陰離子交換法-RESOURCE Q管柱層析 -------------------39
3、陰離子交換法-Mono Q管柱層析------------------------39
二、電泳及染色 -------------------------------------------39
三、電泳中蛋白質分解酵素之測定 ---------------------------40
四、Mono Q部份純化蛋白質分解酵素的特性分析 ---------------40
五、Mono Q部份純化蛋白質分解酵素對海鱺之毒性攻擊試驗 -----42
◆第五章 討論------------------------------------------------43
◆參考文獻 --------------------------------------------------50
◆表 --------------------------------------------------------63
◆圖 --------------------------------------------------------76
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