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研究生:王筱婷
研究生(外文):Hsiao-ting Wang
論文名稱:石斑皮膚菌Aeromonashydrophila之特性研究
論文名稱(外文):Study on the characteristics of Aeromonas hydrophila infecting on the skin of grouper
指導教授:林全信林全信引用關係
指導教授(外文):Chan-shing Lin
學位類別:碩士
校院名稱:國立中山大學
系所名稱:海洋資源學系研究所
學門:自然科學學門
學類:海洋科學學類
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:133
中文關鍵詞:產氣單胞菌蛋白質分解酵素石斑魚
外文關鍵詞:Aeromonas hydrophilagrouperprotease
相關次數:
  • 被引用被引用:4
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數年來石斑魚細菌皮膚及肌肉的出血性潰瘍,推測為病原菌之胞外蛋白酶分解肌肉所造成的。因此本研究調查海洋菌之胞外蛋白酶活性,並分離石斑致病菌,探討其特性。海水背景菌相調查的採樣點為台東外海,使用MB2216 及PPESⅡ兩種培養基。細菌胞外蛋白酶活性分析則採用milk seawater (MS)培養基、azocasein及azoalbumin三種方法,其活性為1~5 U/mg。由MB2216所分離的細菌菌株普遍比PPESⅡ之菌株有較高的平均蛋白酶活性。以azocasein及azoalbumin法來分析蛋白酶活性,所得到的趨勢大致相似,而MS 透明環之結果變異度大。自養殖場石斑魚皮膚所分離FI-02致病株,經過16S rDNA序列分析及生化鑑定後判定為Aeromonas hydrophila,該菌可以生長在含0~4% NaCl的LB中,此菌不被ampillicin、cefazolin、erythomycin、sulfisoxazole所抑制,而對gentamicine、streptomycin、doxycline、norfloxacin、cefotazime、oxolinic acid及tetracycline具感受性。在FI-02的胞外蛋白之蛋白酶活性研究,分別用四種培養基培養FI-02細菌,酵素活性皆在4 U/mg以上,其中以生長在LB培養基上的菌株酵素活性最高,約為6.08 U/mg。以27, 37, 42及56℃加熱二十分鐘後,測定胞外蛋白酶之活性,其結果並無顯著的差異。在魚類抗菌免疫力的研究時,由於血清成分複雜,尚無法找出對抗石斑抗體IgM之二級抗體,仍待將來進一步改進之。
In recent years, the skin and muscle hemorrhagic ulcer occurred in grouper hatchery, suggesting that the bacteria extracellular protease was involved. In this study I surveyed the extracellular protease activity of marine bacteria and investigated the characteristics of the pathogen isolated from diseased grouper. The sampling sites of background survey were located along the coast of Taitung. The cultured media were MB2216 and PPESII. The extracellular protease activity was measured using milk seawater medium (MS) and dye-modified proteins of azocasein and azoalbumin. The extracellular protease activities of the marine bacteria were 1~5 U/mg. The average protease activities of the bacteria from MB2216 media were higher than those from PPESII media. The extracellular protease activity assay using azocasein and azoalbumin had similar tendency but the result by the clear zone measurement on the MS medium was of high variations. The FI-02 isolated from the diseased grouper skin was identified as Aeromonas hydrophila by the biochemicial characteristics and 16S rDNA sequence analysis. FI-02 was able to grow on the LB broth with 0~4% NaCl. The FI-02 was resistant to ampillicin, cefazolin, erythomycin, sulfisoxazole, whereas it was sensitive to streptomycin, doxycline, norfloxacin, cefotazime, oxolinic acid, and tetracycline. When FI-02 growing in the four media, the extracellular protease activities were higher than 4 U/mg. The highest protease activity was 6.08 U/mg when the bacteria grew on the LB medium. After heating on 27, 37, 42 and 56℃ for 20 mins, the protease activities remained the same. In the investigation of the fish immunity, owing to the complex compositions of the fish serum, no conclusion can be drawn on the specificity of the antibody against the bacterium.
謝誌 ----------------------------------------Ⅰ
中文摘要 ------------------------------------Ⅱ
Abstract ------------------------------------Ⅲ
目錄 ----------------------------------------Ⅳ
表目錄 --------------------------------------Ⅴ
圖目錄 --------------------------------------Ⅵ
壹、前言
一、 台灣養殖石斑魚概況 ----------1
二、 魚病的診斷 ------------------3
三、 海洋常見致病菌 --------------6
四、 研究目的 --------------------21
貳、材料與方法
一、 海洋菌之培養 -----------------22
二、 石斑魚致病菌之分離 -----------22
三、 細菌胞外產物之製備
(extracellular products, ECP)----23
四、 蛋白酶 (protease) 活性測定 ----23
五、 蛋白質濃度定量 ----------------24
六、 革蘭氏染色 --------------------25
七、 菌種鑑定 ----------------------25
八、 細菌生理特性分析 -------------28
九、 細菌對石斑魚之攻擊試驗 -------30
十、 石斑魚免疫球蛋白之偵測 -------31
參、結果 ----------------------------------------38
肆、討論 ---------------------------------------45
伍、參考文獻 ---------------------------------52
陸、圖表 -------------------------------------76
附錄A ---------------------------------------107
附錄B ---------------------------------------108
附錄C ---------------------------------------112
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