臺灣博碩士論文加值系統

摘要
本論文探討巴斯徳桿菌Photobacterium damselae subsp. Piscicida對養殖海鱺之致病機制，所使用之試驗菌株strain 9205是分離自南部屏東養殖海鱺魚苗（ 3∼30 g ）之肝、脾、頭腎、後腎及腹水等。菌體及其細胞外產物對海鱺魚體（5 g ）具毒性其LD50分別約為3.28 × 104 CFU及 3.25 µg protein / g fish body weight。 strain 9205細胞外產物中磷脂質分解酵素用高速液體層析儀（ HPLC ）經Q Sepharose High Performance、RESOURCE Q後以切膠方式得到純化物，純化物質在Native – PAGE及SDS-PAGE鑑定其分子量約為27KDa及58KDa，其pI值在pI 3.65 ( 27KDa ) 及5.20 -5.30 ( 58KDa )，以含0.3% egg yolk膠片覆蓋Native – PAGE後發現27KDa有相對的磷脂質分解酵素活性存在，58KDa不明顯，覆蓋SDS - PAGE中發現不具有磷脂質分解酵素活性存在。 27KDa及58KDa經薄層層析、Phospholipase C Assay Kit及Phospholipase A2 Assay Kit後判斷分別為Phospholipase C ( PLC ) ( 27KDa )及Phospholipase A2 ( PLA2 )( 58KDa )，對海鱺紅血球都具溶血能力。PLC ( 27KDa ) 對海鱺 ( 5 g ) 的LD50約為0.91 µg protein / g fish body weight明顯比ECP 之LD50來的低，PLA2 ( 58KDa ) 之LD50約為7.06 µg protein / g fish body weight，而蛋白質分解酵素之LD50約為6.8µg protein / g fish body weight，且在加入脂多醣 ( LPS ) 後發現無明顯加成作用。另外，在毒性中和實驗中證實PLC ( 27KDa ) 應為其細胞外產物中之主要毒素。 以PLC ( 27Kda ) 及PLA2 ( 58Kda ) 及蛋白質分解酵素與海鱺抗( Strain 9205 )細菌細胞外產物之抗血清作用後，結果皆有反應，顯示魚體皆會對此三種重要毒素產生免疫反應，此結果對將來疫苗開發上應能有所貢獻。

Abstract
This thesis investigated the pathogenesis of Photobacterium damselae subsp. piscicida in cultured cobia ( Rachycentron canadum ). Strain 9205 originally isobated from livers , spleen , kidney and ascites moribum cobia ( 3-30 g ) was used in the study. The bacterial cell and extracellular products ( ECP ) were lethal to the cobia ( 5 g ) with LD50 values of 3.28 x 104 CFU and 3.25µg protein / g fish ,respectively. Extracellular phospholipase were partially purified by using various columns ( Q Sepharose HP and RESOURCE Q ) on HPLC system. Two protein bands ( 27 and 58Kda ) exhibited phospholipase actiuities on Native – PAGE but on SDS-PAGE ( by 0.3% egg yolk agar-overlay ) ware excised and eluted as the final purification step. The 27KDa and 58KDa factions were determined and identified as Phospholipase C ( PLC ) and Phospholipase A2 ( PLA2 ), respectively , by using PLC Assay Kit and PLA2 Assay Kit﹒Both of the two phospholipase were hemolytic against red blood cells of cobia. The LD50 values of PLC ( 27Kda ) and PLA2 ( 58Kda ) were 0.91 µg protein (lower than ECP ) and 7.06 µg protein / g fish ( higher than ECP ) in cobia, respectively. In addition, the LD50 value of 33Kda protease was 6.8 µg protein / g fish in cobia. The combination of LPS with PLC, PLA2 or protease did not evhance the lethal toxicity remarkably. Furthermore, the PLC ( 27KDa ) was confirmed to be a major extracellular toxin by a toxicity – neutraligation test. The PLC and PLA2 and protease were immunoreactive against cobia sera pre-immunized with formalinized ECP. The result indicates that three toxins are immunogenic in cobia and may by very useful for future vaccine development.

目錄
◆ 中文摘要 ---------------------------------------Ⅰ
◆ 英文摘要----------------------------------------Ⅱ
◆ 目錄------- ------------------------------------Ⅲ
◆ 表目錄 -----------------------------------------Ⅶ
◆ 圖目錄 -----------------------------------------Ⅷ
◆ 第一章 前言 ------------------------------------ 1
◆ 第二章 文獻整理 -------------------------------- 4
◆ 第三章 材料與方法-------------------------------21
3.1 試驗菌株之採樣分離、來源、確認與保存-----------------21
3.2細菌生理生化特性之鑑定分析 ---------------------------22
3.2.1 革蘭氏染色 --------------------------------------- 22
3.2.2 葡萄糖的利用---------------------------------------22
3.2.3 MacConkey agar之生長能力--------------------------22
3.2.4 對NaCl之耐受性試驗 ------------------------------- 23
3.2.5 藥物敏感性試驗 ----------------------------------- 23
3.2.6 溶血能力測定 ------------------------------------- 23
3.2.7 API 20E system --------------------------------- 23
3.2.8 Biolog system ---------------------------------- 24
3.2.9 BIONOR MONO-Pp rapid diagnostic test ------------ 24
3.3 Photobacterium damsela subsp. piscicida（strain 9205）細胞外產物之基本特性分析 --------------------------------- 24
3.3.1 菌株強化-------------------------------------------24
3.3.2 細胞外產物（extracellular products, ECP）之製備 ---25
3.3.3 細胞外產物之特性分析 ------------------------------25
1、蛋白質含量的測定------------------------------- 25
2、蛋白質分解酵素（protease）活性測定------------- 25
3、磷脂質活性初步測定----------------------------------- 26
4、溶血活性測定----------------------------------- 26
3.4 對海鱺之毒性攻擊試驗-------------------------------- 26
3.4.1菌體對海鱺之毒性試驗--------------------------------26
3.4.2細胞外產物對海鱺之毒性試驗 ------------------------ 27
3.4.3細胞外產物毒性抑制試驗 ----------------------------27
3.5 細胞外產物中磷脂質分解酵素之分離純化與特性分析------ 27
3.5.1細胞外產物中磷脂質分解酵素之部份分離純化------------27
1、陰離子交換層析法－HP管柱層析 -------------------------27
2、陰離子交換法－RESOURCE Q管柱層析 ---------------------28
3 、切膠-------------------------------------------------28
3.5.2 電泳及染色 ----------------------------------------28
3.5.3 電泳中磷脂質分解酵素之測定-------------------------29
3.5.4 等電點（ isoelectric point ，pI ）-----------------29
3.5.5 薄層層析（Thin-layer chromatography，TLC）-------- 30
3.5.6最後純化磷脂質分解酵素的特性分析--------------------30
1、 磷脂質分解酵素 C 活性測定----------------------------30
2、 磷脂質分解酵素 A2 活性測定---------------------------31
3、溫度之影響--------------------------------------------31
4、 pH 之影響--------------------------------------------32
5、二價金屬離子之影響------------------------------------32
3.5.7最後純化磷脂質分解酵素對海鱺之毒性攻擊試驗----------32
1、脂多醣體Lipopolysaccharide（LPS）之萃取---------------32
2、細胞外產物中蛋白質分解酵素之純化----------------------33
3、磷脂質分解酵素、脂多醣體及蛋白質分解酵素對海鱺之毒性攻擊試驗-----------------------------------------------------33
3.6 兔子抗細胞外產物及最後純化物之抗血清製備-------------33
3.6.1 兔子抗細胞外產物及最後純化之血清的製備-------------33
3.6.2 交叉免液電泳(Crossed immunoelectro-phoresis)-------34
3.6.3 抗血清中抗體力價分析-------------------------------34
3.6.4 毒性中和實驗---------------------------------------35
3.7 海鱺對純化物之免疫反應-------------------------------35
3.7.1 海鱺抗細胞外產物之抗血清製備-----------------------35
3.7.2 採血及血清的製備-----------------------------------35
3.7.3 海鱺抗細胞外產物之抗血清對純化物的免疫反應---------36
◆ 第四章 結果 -----------------------------------37
4.1 試驗菌株之採樣分離-----------------------------------37
4.2 細菌生理生化特性之鑑定分析---------------------------37
4.3 對海鱺之毒性攻擊試驗.--------------------------------38
4.3.1菌體對海鱺之毒性試驗--------------------------------38
4.3.2細胞外產物對海鱺之毒性試驗--------------------------38
4.3.3細胞外產物（ECP）毒性抑制試驗-----------------------38
4.4 細胞外產物中磷脂質分解酵素之分離純化與特性分析-------38
4.4.1 細胞外產物中磷脂質分解酵素之部份分離純化-----------39
1. 陰離子交換層析法－（Q Sepharose High Performance, HP）（pharmacia）管柱層析-------------------39
2. 陰離子交換法－RESOURCE Q管柱層析----------------39
4.4.2 最後純化之電泳及染色-------------------------------39
4.4.3 電泳中磷脂質分解酵素之測定-------------------------40
4.4.4 磷脂質分解酵素之等電點-----------------------------40
4.4.5 薄層層析-------------------------------------------40
4.4.6 磷脂質分解酵素的特性分析---------------------------41
4.5 磷脂質分解酵素、脂多醣及蛋白質分解酵對海鱺之毒性攻擊試驗-------------------------------------------------------42
4.5.1 磷脂質分解酵素對海鱺之毒性試驗---------------------42
4.5.2 脂多醣(LPS)----------------------------------------42
4.5.3 蛋白質分解酵素-------------------------------------42
4.5.4 磷脂質分解酵素及蛋白質分解酵-----------------------43
4.5.5 磷脂質分解酵素及蛋白質分解酵加脂多醣---------------43
4.6 細菌細胞外產物蛋白質分解酵素之免疫分析---------------43
4.6.1 交叉免疫電泳---------------------------------------43
4.6.2 兔子抗Strain 9205之ECP及PLA2(58KDa)血清力價之測定--43
4.6.3 毒性中和實驗---------------------------------------44
4.7 海鱺對純化物之免疫辨識-------------------------------44
◆ 第五章 討論-------------------------------------45
◆ 參考文獻 ---------------------------------------54
◆ 表 ---------------------------------------------72
◆ 圖 ---------------------------------------------86

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