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研究生:陳惠恩
研究生(外文):Hui-En Chen
論文名稱:利用海鱺血清分析巴斯德桿菌抗原
論文名稱(外文):Evaluation of antigens from Photobacterium damselae subsp. piscicida using cobia (Rachycentron canadum) antiserum
指導教授:楊惠郎
指導教授(外文):Hui-Lang Yang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
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
論文頁數:67
中文關鍵詞:全菌蛋白海鱺免疫分析細胞外產物巴斯德桿菌
外文關鍵詞:Photobacterium damselaeimmunoassaycobiawhole cell bacterinextracellular products
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本實驗室曾從疾病流行過後死亡的海鱺身上分離出Ph.d. p (Photobacterium damselae subsp. piscicida),並對海鱺進行巴斯德桿菌攻毒測試。結果發現其LD50介於107~106之間,對海鱺而言為一致死病原菌。
基於預防甚於治療的前提下,疫苗的使用也就日漸重要。過去有使用死菌疫苗免疫的方式,雖具有一定程度保護力,但其成效並不令人滿意,推測可能還有其他可促進免疫反應之物質未被有效應用。於是Magarinos在死菌疫苗中添加細菌細胞外產物(extracellular product,ECP)為疫苗,結果發現其效果比死菌疫苗好。但對於海鱺之防範Ph.d. p感染是否適用須先行評估。
疫苗的成效主要依賴動物體對抗原所產生的免疫反應,在不同種類的魚群存在不同程度的差異。疫苗研發之初,有必要對於抗原在動物體內所引起的免疫反應加以探討。因此本實驗首先依照Liu所發表的方法稍加修飾收集ECP,同時也收集Ph.d. p全菌蛋白,分別以海鱺抗Ph.d. p血清(注射Ph.d. p死菌疫苗)、海鱺抗Ph.d. p血清(巴斯德桿菌攻毒測試殘存者)及控制組未給予任何Ph.d. p抗原的海鱺血清,及兔抗巴斯德桿菌血清四組為探針,利用西方點墨法加以分析Ph.d. p全菌蛋白及ECP。
結果發現就ECP而言,海鱺各組血清對53kD均有反應,而兔抗Ph.d. p血清(注射Ph.d. p死菌疫苗)則除了81kD外還對53kD有強免疫反應。因而推測此53kD為一重要免疫原。
就Ph.d. p全菌蛋白而言,海鱺抗Ph.d. p血清而言,不論注射Ph.d. p死菌疫苗或巴斯德桿菌攻毒測試殘存者血清皆在49.8kD有較明顯免疫反應。或許,49.8kD為免疫成功與否的指標。另一方面,從Ph.d. p全菌蛋白及ECP之免疫評估發現,海鱺血清及兔子血清對一39kD抗原有明顯的免疫反應。而本實驗依據Hirono所發表的序列所表現之蛋白質以兔子血清測試發現,兔子血清稀釋倍數需25倍。因此猜測它對兔子而言是一弱抗原性物質的抗原。因此在39kD附近可能含有其他免疫物質。雖然此39kD抗原是一個弱抗原性物質,或許對其他抗原有佐劑之效果。不過這尚屬推論,尚須進一步以動物實驗加以評估。
Pasteurellosis or pseudotuberculosis caused by the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida according to 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data. Pasteurellosis is one of the main bacterial diseases lethal to many fish. In Taiwan, it is a common bacterial disease in cobia aquaculture, and it usually causes more than 30% mortality. People have been using chemotherapeutic agents extensively to eliminate infections; however, this also brought infections caused by drug-resistant pathogens into cultured fish. For this reason, immune-prophylaxis has become the best way for controlling the disease.

A number of studies examined the effectiveness of vaccination as a preventive mean against pasteurellosis. The majority of the vaccines being tested contained formalin-killed bacteria, but it didn’t give a satisfactory result. Some other studies showed that an addition of extracellular products in killed vaccines tends to give higher protection level than killed vaccines. Unknown components in extracellular products which increase the protection level of killed vaccines worth further investigation.

In this experiment, whole cell bacterin and its extracellular products were harvested first, and then four probes, rabbit anti-Ph.d.p serum(inoculated killed bacteria), cobia anti-Ph.d.p serum(inoculated killed bacteria), cobia anti-Ph.d.p serum(survival after challenge with Ph.d.p), and cobia serum(pre-inoculated Ph.d.p) were used for western blot analysis.

According to the results of western blotting of ECP, all sets of cobia serum can interact with 53 kD. The western blotting of cell bacterin showed that Cobia anti-Ph.d.p serum had a major band in 49.8 kD; therefore, 49.8 kD antigenic substance could possibly be used as an indicator of successful vaccination. Furthermore, the cobia anti-Ph.d.p antiserum and rabbit anti-Ph.d.p antiserum can interact with a 39kD protein. This experiment was performed based on antigenic substances screened from genomic DNA library published by Hirono et al in 1997. The gene of interest was cloned in order to express the antigenic substances; however, the expressed antigenic substance only interacted weakly with rabbit anti-Ph.d.p antiserum and cobia anti-Ph.d.p antiserum. For this reason, this antigenic substance is not suitable for vaccine development maybe due to its poor antigenity. To confirm the inference metioned above, further animal experiments should be performed.
目 錄
英文摘要………………………………………………3
中文摘要………………………………………………5
名詞縮寫表……………………………………………9
第一部分 緒論
  第一章 前言……………………………………10
  第二章 文獻探討………………………………12
第二部分 材料與方法………………………………21
  第一章 實驗設計………………………………22
  第二章 實驗材料………………………………25
  第三章 實驗方法………………………………32
第三部分 結果與討論
  第一章 結果……………………………………45
  第二章 討論……………………………………49
第四部分 圖表………………………………………51
第五部份 參考文獻…………………………………61
第六部份 附錄………………………………………67
參考文獻
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