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研究生:蔣柏皇
研究生(外文):Bo-Huang Jiang
論文名稱:台灣鯛鏈球菌雙價疫苗效力評估
論文名稱(外文):Evaluation of the effectiveness of Streptococcus spp. bivalent vaccine in tilapia
指導教授:陳石柱陳石柱引用關係
指導教授(外文):Shih-Chu Chen
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:獸醫學系所
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:82
中文關鍵詞:無乳鏈球菌瓶鼻海豚鏈球菌福馬林不活化鏈球菌雙價疫苗鳥氨酸氨甲酰轉移酶磷酸激酶
外文關鍵詞:Streptococcus agalactiaeStreptococcus iniaeformalin inactivated Streptococcus spp. bivalent vaccineornithine carbamoyltransferase (OCT)phosphoglycerate kinase (PGK)
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無乳鏈球菌Streptococcus agalactiae以及瓶鼻海豚鏈球菌Streptococcus iniae屬於革蘭氏陽性菌。其宿主範圍相當廣泛,對哺乳動物、爬蟲動物、兩棲動物、魚類都具有致病性。在台灣對鏈球菌具感受性的養殖魚類包括吳郭魚、烏魚、金目鱸魚、條紋鱸魚,並好發在夏季,常造成高死亡率的嚴重經濟損失。再加上抗生素治療衍生的藥物殘留及抗藥性等問題,研發有效預防鏈球菌的疫苗有其必要性。本研究探討福馬林不活化鏈球菌雙價疫苗可有效抵抗台灣鯛無乳鏈球菌及瓶鼻海豚鏈球菌的感染。實驗魚隻分別以混合油質佐劑及氫氧化鋁膠佐劑之雙價疫苗,以腹腔注射方式進行免疫,並於兩週後進行同等劑量補強注射。免疫後一個月,油質佐劑雙價疫苗組、氫氧化鋁膠雙價疫苗組及油質佐劑單價疫苗組以無乳鏈球菌進行攻毒後,其相對存活率以油質佐劑雙價疫苗組最高75.0%、油質佐劑單價疫苗組其次45.0%、氫氧化鋁膠雙價疫苗組則為0%;另,以瓶鼻海豚鏈球菌進行各組之攻毒後,油質佐劑雙價與單價疫苗組相對存活率皆為100.0%、氫氧化鋁膠雙價疫苗組則為90.0%。免疫後三個月同組別再次分別以無乳鏈球菌及瓶鼻海豚鏈球菌進行攻毒,相對存活率分別為,油質佐劑雙價疫苗組:52.6及28.9%、氫氧化鋁膠雙價疫苗組0及20.0%、油質佐劑單價疫苗:36.8及3.3%。此外為要了解評估鏈球菌雙價疫苗對台灣鯛較長時間的免疫保護力,因此以三重複的動物試驗再次重複前述疫苗注射實驗並延長期程至六個月。油質佐劑雙價疫苗及氫氧化鋁膠雙價疫苗組別免疫後一個月以無乳鏈球菌攻毒三重複之平均相對存活率為63.4及1.4%、免疫後三個月平均相對存活率為37.6及9.3%、免疫後六個月均相對存活率為31.3及3.7%;以瓶鼻海豚鏈球菌攻毒免疫後一個月之平均相對存活率為87.4及81.1%、免疫後三個月之平均相對存活率為79.0及63.3%、免疫後六個月之平均相對存活率為67.7及54.4%。實驗結果顯示,油質佐劑之雙價不活化菌苗其免疫保護效力確實優於氫氧化鋁膠佐劑,而疫苗之保護效力無論油質佐劑或是氫氧化鋁膠佐劑均隨免疫後時程逐漸下降,但對瓶鼻海豚鏈球菌之保護效力仍達54.4%以上,確實極具潛力之魚用疫苗。本研究亦進行無乳鏈球菌的外膜蛋白(鳥氨酸氨甲酰轉移酶OTC及磷酸激酶PGK)選殖重組蛋白表現,以福馬林不活化無乳鏈球菌免疫實驗兔,再以兔抗血清做為西方墨點法的抗體,其結果證明重組OTC及PGK確實具有無乳鏈球菌表面抗原特性,在魚用鏈球菌疫苗之範疇中將可提供未來包括次單位疫苗研發或免疫原性毒力強弱等研究之參考。
Streptococcus agalactiae and S. iniae, are gram-positive bacteria, and well know as a causative agent of mammals, reptiles, amphibians, and fish. In Taiwan, Streptococcsis has been usually infected spreads out in tilapia (Oreochromis sp), grey mullet (Mugil cephalus), seabass (Lates calcarifer), striped bass (Morone saxatilis) during summer has resulted in high mortality and serious economic losses. In this study, animal trial design, vaccination and challenge to tilapia, was used to test the hypothesis, that formalin inactivated Streptococcus spp. bivalent vaccine is useful in helping to protect fish from S. agalactiae and S. iniae infection. Fish were immunized by intraperitoneally injection with formalin inactivated bivalent vaccine mixed oil adjuvant or aluminum hydroxide gel group and monovalent vaccine. They challenged by S. agalactiae at 1 month post immunization, the RPS of non-mineral oil, aluminum hydroxide gel and monovalent vaccine group were 75.0, 0 and 45.0%; challenged by iniae group were 100.0, 90.0 and 100.0%. At 3 month post immunization, the RPS values were 52.6, 0 and 36.8%, challenged by S. agalactiae; 28.9, 20.0 and 3.3%, challenged by S. iniae. Further studies, experiments were repeated in triplicate for each group, following the same experimental design, challenged at one, three and six months after immunization, respectively. The average RPS of non-mineral oil and aluminum hydroxide gel group challenged by S. agalactiae at 1 month post immunization were 63.4, 1.4%; challenged by S. iniae were 87.4, 81.1%. At 3 month post immunization, they were 37.6, 9.3 and 79.0, 63.3%, then 31.3, 3.7 and 67.7, 54.4% at 6 monrh post immunization. This study has revealed inactivated Streptococcus spp. bivalent vaccine with oil adjuvant candidates to induce the protective immune response. In addition we amplify the S. agalactiae outer surface protein ornithine carbamoyltransferase (OCT) and phosphoglycerate kinase (PGK) gene by using polymerase chain reaction (PCR), focus on the molecular cloning, and expression of these proteins. On the other hand, sear taken from rabbits immunized with formalin inactivated S. agalactiae were prepared and used for recombinant OCT and PGK antigenicity comparisons via Western blotting assay.
中文摘要 II
Abstract IV
謝誌 VI
目錄 VII
圖表目錄 XI
第1章 前言 1
第2章 文獻回顧 4
2.1 吳郭魚簡介與養殖現況 4
2.2鏈球菌(Streptococcus spp.) 4
2.2.1 魚類鏈球菌症 4
2.2.2 無乳鏈球菌(Streptococcus agalactiae)概述 5
2.2.3 瓶鼻海豚鏈球菌(Streptococcus iniae)概述 5
2.2.4 形態學及生化特性 6
2.2.5 鑑定及診斷 7
2.2.6 臨床症狀及病灶 8
2.2.7 流行病學 8
2.3.防治 11
2.3.1魚類鏈球菌症治療方法 11
2.3.2魚類鏈球菌症預防方法 12
2.4. S. agalactiae外膜蛋白 13
第3章 材料與方法 15
3.1 實驗用菌株 15
3.2 微生物分離及鑑定 15
3.2.1 菌株培養分離純化 15
3.2.2 菌株DNA純化萃取 15
3.2.3 菌株特異性引子對選擇 16
3.2.3.1 S. agalactiae特異性引子對選擇 16
3.2.3.2 S. iniae特異性引子對選擇 16
3.2.4 特異性引子對聚合酶連鎖反應(polymerase chain reaction;PCR) 17
3.2.4.1 PCR材料 17
3.2.4.2 S. agalactiae PCR條件 17
3.2.4.3 S. iniae PCR條件 17
3.2.5 電泳分析 17
3.3 鏈球菌S. agalactiae、S. iniae疫苗保護力試驗 18
3.3.1 福馬林不活化全細胞菌苗製備 18
3.3.2 台灣鯛 18
3.3.3 台灣鯛之疫苗免疫 18
3.3.3.1 不同佐劑之鏈球菌疫苗三個月保護力試驗魚隻免疫 18
3.3.3.2不同佐劑之鏈球菌疫苗六個月保護力試驗魚隻免疫 19
3.3.4 標的疫苗之保護力試驗 19
3.3.4.1 不同佐劑之鏈球菌疫苗短期三個月保護力試驗 19
3.3.4.2 不同佐劑之鏈球菌疫苗長期六個月保護力試驗 19
3.3.4.3魚隻觀察及試驗終止 19
3.3.5 相對存活率(relative percent survival;RPS)分析 20
3.3.6 血清凝集反應試驗(agglutination assays) 20
3.4無乳鏈球菌S. agalatiae 外膜蛋白重組表現及純化 20
3.4.1 引子對 20
3.4.1.1 無乳鏈球菌S. agalactiaee之外膜蛋白ornithine carbamoyltransferase (OCT) 引子對 20
3.4.1.2 無乳鏈球菌S. agalactiaee之外膜蛋白phosphoglycerate kinase (PGK) 引子對 21
3.4.2 聚合酶連鎖反應(polymerase chain reaction;PCR ) 21
3.4.2.1 PCR材料 21
3.4.2.2 OCT PCR反應條件 21
3.4.2.3 PGK PCR反應條件 21
3.4.3 DNA及序列比對 21
3.4.4 蛋白質重組及表現 22
3.4.4.1 接合反應(ligation) 22
3.4.4.2 轉形作用(transformation) 22
3.4.4.3 蛋白質表現 22
3.4.5 重組蛋白質純化 23
3.4.6 重組蛋白質濃度測定 23
3.4.7 重組蛋白質電泳分析 23
3.5 重組蛋白抗原性分析 24
3.5.1 西方墨漬法(Western blot) 24
第4章 結果 25
4.1 菌株鑑定 25
4.2 疫苗保護力試驗 25
4.2.1 不活化菌苗製備 25
4.2.2不同佐劑之鏈球菌疫苗短期三個月保護力試驗 25
4.2.2.1 一個月保護效力試驗 25
4.2.2.2三個月保護效力試驗 26
4.2.3不同佐劑之鏈球菌疫苗長期六個月保護力試驗 26
4.2.3.1 一個月保護效力試驗 26
4.2.3.2 三個月保護效力試驗 27
4.2.3.3 六個月保護效力試驗 28
4.2.4 細菌再分離率 28
4.2.5 血清凝集反應試 29
4.3 S. agalactiae 外膜蛋白重組表現 29
討論 61
參考文獻 66
附錄一、Tilapia annual output value and yield, from 2006~2011 in Taiwan. 79
附錄二、pet151dtopo_map 80
附錄三、Schematic representation of the three different principle strategies for the production of combined adsorbed vaccines with three different antigens A–C (e.g. DT, TT and PT) adsorbed on an adsorbent (Ad). 81
作者簡介 82

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