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研究生:楊昀珮
研究生(外文):Yun-Pei Yang
論文名稱:弧菌奈米化菌苗搭配重組蛋白於石斑魚之保護效力
論文名稱(外文):Protective immunity against Vibrio harveyi in Grouper induced by nano-bacterin plus recombinant protein GAPDH
指導教授:楊忠達
指導教授(外文):Chung-Da Yang
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:動物疫苗科技研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:87
中文關鍵詞:Vibrio harveyi (V. harveyi)石斑魚glyceraldehyde-3-phosphate dehydrogenase (GAPDH)奈米化疫苗
外文關鍵詞:Vibrio harveyi (V. harveyi)Grouperglyceraldehyde-3-phosphate dehydrogenase (GAPDH)nano-vaccine
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點帶石斑魚(Epinephelus coioides)在東南亞(例如中國,台灣)是一個重要的經濟養殖魚類。但在養殖過程中常常因為疾病的感染而造成重大損失。由於台灣養殖漁業密集飼養,造成養殖魚類感染性疾病劇增,使得養殖魚貝類大量死亡,直接影響到國家重大經濟損失。其中魚類感染性疾病的病原體,弧菌是最嚴重疾病之一,尤其Vibrio harveyi 為海洋魚類中的主要病原菌,因此有關水產養殖魚類的弧菌症的研究備受重視。感染V. harveyi 的魚隻在臨床病徵有腸胃炎和眼睛病變,且會造成高死亡率。接種疫苗是目前公認可控制弧菌症的有效方法,但目前並沒有商業化的疫苗來控制V. harveyi,目前的研究顯示致病菌的外膜蛋白(OMP)可引起宿主的保護性免疫反應,而Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 為其中之一,因此本研究利用大腸桿菌表現系統大量表現重組GAPDH (rGAPDH),經免疫轉漬法的分析,顯示來自石斑魚的V. harveyi感染血清可以清楚辨認純化的rGAPDH。之後我們進行石斑魚試驗,經腹腔注射免疫PBS、rGAPDH (50 μg/fish)、奈米不活化菌苗(microfluidizer nano-bacterin, MF) (50 μg/fish)、奈米不活化菌苗(50 μg/fish) 混合rGAPDH (50 μg/fish)、奈米不活化菌苗(150 μg/fish) 混合rGAPDH (50 μg/fish)及福馬林不活化V. harveyi菌苗,再隔三星期後進行石斑魚的二次腹腔注射免疫,並在第一次免疫後的14及35天收集血清,並利用ELISA確認血清中的抗體和V. harveyi全菌蛋白質的結合能力,結果顯示特異性抗體力價明顯增加於免疫rGAPDH (50 μg/fish)、奈米不活化菌苗(microfluidizer nano-bacterin, MF) (50 μg/fish)、奈米不活化菌苗(50 μg/fish)混合rGAPDH (50 μg/fish)、奈米不活化菌苗(150 μg/fish) 混合rGAPDH (50 μg/fish)及福馬林不活化V. harveyi菌苗的魚隻。補強注射後三星期將魚隻以4.45× 106 CFU V. harveyi (AOD99094-1)進行攻毒,並監測死亡率21天,且計算每一組的相對存活率(RPS),結果顯示免疫rGAPDH (50 μg/fish) 組魚隻的相對存活率為9.1%、免疫MF (50 μg/fish) 組魚隻的相對存活率為93.75%、MF (50 μg/fish) + rGAPDH (50 μg/fish) 組魚隻的相對存活率為93.75%、MF (150 μg/fish) + rGAPDH (50 μg/fish) 組魚隻的相對存活率為87.51%、免疫福馬林不活化V. harveyi菌苗組魚隻的相對存活率為45.5%。綜合免疫分析及攻毒試驗的結果證明免疫奈米不活化菌苗或奈米不活化菌苗混合rGAPDH皆可以提供石斑魚強大的保護力來抵抗V. harveyi的感染。總之,本研究成功應用奈米不活化菌苗搭配重組蛋白於開發石斑魚弧菌的疫苗。
Orange-spotted grouper (Epinephelus coioides) is an economically important farmed fish in southeast Asia, including China and Taiwan. Infecting with diseases is causing huge loss in aquaculture industry. In Taiwan, the disease rate of farming fish has increased tremendously due to the way of high-density farming fishery, which resulted in high death rate of fishes and shells. The economic consequence has been devastating. Vibriosis is one of the most serious diseases, especially Vibrio harveyi, a major pathogen of marine fish in this region, research related to vibrosis of farming fish has been intensively carried out worldwide. Clinical signs induced by V. harveyi included gastroenteritis and eye lesions. The infection causes high mortality in fish. The vaccination against Vibrio species is a viable strategy for controlling vibriosis. However, no commercial vaccine is currently available. Current studies have shown that the outer membrane proteins (OMPs) of pathogenic Vibrio bacteria are protective antigens to induce effective protection against Vibrio infection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one OMP of Vibrio species and its gene was cloned into pET24a plasmid to express recombinant GAPDH (rGAPDH) in BL21 (DE3) Escherichia coli. After immunoblotting assay, we found that sera from grouper infected with V. harveyi obviously recognized the purified rGAPDH. Six groups of 25 grouper each were peritoneally vaccinated twice at a 21-day interval with PBS, rGAPDH (50 μg/fish), microfluidizer nano-bacterin (MF) (50 μg/fish) only, MF (50 μg/fish) plus rGAPDH (50 μg/fish), MF (150 μg/fish) plus rGAPDH (50 μg/fish), formalin-killed V. harveyi. Fish sera were collected on days 14 and 35 after the first vaccination. ELISA was used to determine serum responses against V. harveyi lysate. Results showed that specific antibody titers were significant increased in fish vaccinated with rGAPDH (50 μg/fish), microfluidizer nano-bacterin (MF) (50 μg/fish) only, MF (50 μg/fish) plus rGAPDH (50 μg/fish), MF (150 μg/fish) plus rGAPDH (50 μg/fish), formalin-killed V. harveyi. Three weeks after booster, fish were challenged with 4.45× 106 CFU V. harveyi (AOD99094-1) and then monitored for mortalities for 21 days. The relative percent of survival (RPS) for each group was calculated. Fish vaccinated with rGAPDH (50 μg/fish) showed 9.1% of RPS. Fish vaccinated with MF (50 μg/fish) showed 93.75% of RPS. Fish vaccinated with MF (50 μg/fish) plus rGAPDH showed 93.75% of RPS. Fish vaccinated with MF(150 μg/fish) plus rGAPDH (50 μg/fish) showed 87.51% of RPS. Fish vaccinated with formalin-killed V. harveyi showed 45.5% of RPS. These results demonstrated that vaccination with MF or MF mixed with rGAPDH could provide a potent protection against V. harveyi infection in grouper. In conclusion, we successfully applied microfluidizer nano-bacterin on the Vibrio vaccine development in grouper.
摘 要 I
Abstract II
謝誌 IV
目錄 VI
圖表目錄 X
壹、緒言 1
貳、文獻回顧 4
2.1.1石斑魚養殖情況及現況 4
2.1.2石斑魚常見疾病 5
2.1.2.1細菌性疾病 6
2.1.2.2病毒性疾病 7
2.1.2.3寄生蟲疾病 7
2.2魚類免疫系統概述 8
2.3弧菌症 9
2.3.1V. harveyi歷史 10
2.3.2 V. harveyi型態與特性 11
2.3.3臨床症狀及病理變化 12
2.3.4鑑定及診斷方法 12
2.3.5治療方法 13
2.3.6疫苗的發展 13
2.4奈米化的特徵及優點 17
2.5研究目的 17
參、材料方法 19
3.1V. harveyi菌株來源 19
3.2基因轉殖所用之載體及菌株 19
3.2.2 pET-24a 19
3.3重組蛋白的製備 19
3.4核酸定序 20
3.5重組蛋白之表現和分析 20
3.5.1 GAPDH菌株再篩選和重組蛋白大量表現 20
3.5.2十二烷基硫酸鈉聚丙烯醯胺膠體電泳(Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE) 分析確認重組蛋白的表現 20
3.5.3重組蛋白的標記蛋白6x His確認 21
3.5.4重組蛋白質的純化 22
3.5.5重組蛋白質的復性 23
3.5.6西方轉漬法分析重組蛋白的抗原性 23
3.6 V. harveyi福馬林不活化奈米菌苗之製備 24
3.6.1細菌培養 24
3.6.2 Colony Forming Units (CFU)計算 25
3.6.3福馬林不活化及雜菌檢測 25
3.6.4收菌 25
3.6.5奈米化不活化菌苗製備 26
3.7.1魚隻免疫 26
3.7.2攻毒之細菌培養與製備 26
3.7.3免疫反應之測試 27
3.7.3.1免疫血清酵素結合免疫吸附法(Enzyme-linked immunosorbent assay; ELISA) 27
3.7.3.2淋巴球增殖試驗 28
3.7.3.3免疫血清西方轉漬法(western blot)分析 29
3.7.3.4存活率分析 30
肆、結果 31
4.1 V. harveyi菌株來源 31
4.2重組蛋白的製備 31
4.2.1 重組蛋白的分子選殖 31
4.2.2重組蛋白GAPDH (rGAPDH) 的再篩選與大量表現 31
4.2.3 rGAPDH的標記蛋白6x His確認 31
4.2.4 rGAPDH的純化 31
4.2.5 rGAPDH的復性 32
4.2.6 rGAPDH之抗原性分析 32
4.2.6.1 Western blot 分析rGAPDH蛋白質之抗原性 32
4.3 V. harveyi奈米不活化菌苗之製備 32
4.4疫苗效力試驗 33
4.4.2石斑魚淋巴球增殖試驗 33
4.4.3石斑魚免疫血清西方轉漬法(Western blot)分析 34
4.4.4石斑魚存活率分析 34
伍、討論 57
陸、參考文獻 64
附錄 75
作者簡介 87

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