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研究生:鄭宇軒
研究生(外文):Jheng, Yu-Hsuan
論文名稱:探討斑馬魚 (Danio rerio)餵食epinecidin-1經由電穿孔轉殖豐年蝦以增強抗Vibrio vulnificus感染與免疫相關基因表現變化之研究
論文名稱(外文):Zebrafish fed on transgenic Artemia expressing recombinant epinecidin-1 exhibit increased survival and altered expression of immunomodulatory genes upon Vibrio vulnificus infection
指導教授:陳志毅陳志毅引用關係
指導教授(外文):Chen, Jyh-Yih
口試委員:陳志毅許祖法潘婕玉胡清華龔紘毅
口試委員(外文):Chen, Jyh-YihHui, Cho-FatPan, Chieh-YuHu, Chin-HwaGong, Hong-Yi
口試日期:2014-07-01
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
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:2014
畢業學年度:102
語文別:中文
論文頁數:81
中文關鍵詞:抗菌胜肽豐年蝦電穿孔epinecidin-1
外文關鍵詞:Epinecidin-1Antimicrobial peptideArtemiaZebrafishVibrio vulnificus
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豐年蝦的無節幼蟲被廣泛用於養殖漁業中,作為幼魚及蝦類、貝類的活體餌料。抗菌胜肽epinecidin-1於2005年由點帶石斑魚體內發現,對諸多細菌如Staphylococcus aureus、Pseudomonas aeruginosa、Streptococcus pyogenes、以及Vibrio vulnificus都有顯著的抗菌效果。本研究係以巨細胞病毒啟動子 (Cytomegalovirus,CMV),啟動下游經修飾過的水母綠螢光蛋白 (EGFP) 和epinecidin-1之質體DNA (CMV-EGFP-epi),利用電穿孔 (Electroporation) 的方式將質體DNA轉殖進入經去殼處理過的豐年蝦卵內。經由測試不同的卵數 (Cysts weight (0.2 g))、質體DNA濃度 (DNA concentration (50 μg/100μl)) 和不同的電穿孔轉殖條件如電壓 (Voltage (150 V))、波長 (Pulse length (20 ms))、脈衝次數 (Number of pulse (2)) 得到最好的轉殖效率為49.84 %、其中孵化率為41.15 %、螢光強度為47.46 A.U.,並結合quantitative RT-PCR得到EGFP-epi在第120小時開始大量表達。藉西方墨點分析法 (Western blot) 與抑菌圈 (Zone of inhibition) 測試證實轉殖豐年蝦確實有產生EGFP-epinecidin-1的protein並有抑制創傷弧菌 (Vibrio vulnificus 204) 的效果。之後將斑馬魚餵食基因轉殖的豐年蝦,待餵食7到21天後將斑馬魚做對V. vulnificus的抗菌實驗,並藉由分別餵食野生型 (Wild type) 豐年蝦和一般飼料 (Fodder) 的斑馬魚比較其存活率,得到epinecidin-1可以藉由基因轉殖進入餌料生物後,利用攝食的方式進入魚類體內,達到抗菌的效果,並於餵食21天後可提高斑馬魚15 %的存活率,同時降低體內肝臟與腸道的含菌量。接著再利用quantitative RT-PCR偵測各個時間點斑馬魚的免疫基因表現量變化,證實當斑馬魚攝取轉殖豐年蝦後會改變其體內免疫基因的表現量,使個體抵抗V. vulnificus的能力及存活率增加。此研究對於養殖漁業極力尋找有效抗菌的藥物及疫苗,提供一個解決問題的方向。
Artemia has been used extensively in aquaculture as a fodder for larval fish, shrimp, and shellfish. Epinecidin-1, an antimicrobial peptide, was isolated from grouper (Epinephelus coioides) in 2005. Epinecidin-1 has been previously reported to possess antimicrobial activity against several Gram-positive and Gram-negative bacterial species, including Staphylococcus coagulaes, Pseudomonas aeruginosa, Streptococcus pyogenes, and Vibrio vulnificus. In this study, we used electroporation to introduce plasmid DNA encoding a green fluorescent protein (EGFP)-epinecidin-1 fusion protein under the control of the cytomegalovirus (CMV) promoter into decapsulated Artemia cysts. Optimization of various properties (including cysts weight (0.2g), plasmid concentration (50 μg/100 μl), pulse voltage (150 V), length (10 ms), and number (2)) resulted in a hatching rate of 41.15 %, a transfection efficiency of 49.81 %, and a fluorescence intensity (A.U.) of 47.46. The expression of EGFP-epinecidin-1 was first detected by quantitative RT-PCR at 120 hours post-electroporation, and protein was identified by Western blot at the same time. Furthermore, the EGFP-epinecidin-1 protein inhibited the Vibrio vulnificus (204) growth, as demonstrated by zone of inhibition studies. Zebrafish fed on transgenic Artemia expressing CMV-EGFP-epi combined with commercial fodder were resistant to infection by Vibrio vulnificus (204): survival rate was enhanced by over 70 % at 7, 14, and 21 days post-infection, and bacterial numbers in the liver and intestine were reduced. In addition, feeding of transgenic Artemia to zebrafish affected the immunomodulatory response to Vibrio vulnificus (204) infection; expression of immune-responsive genes, including hepcidin and defbl2, was altered, as shown by qPCR. These findings suggest that feeding transgenic Artemia expressing CMV-EGFP-epi to larval fish has antimicrobial effects, without the drawbacks of introducing drug residues.
誌謝.....................................................I
中文摘要..................................................II
英文摘要..................................................III
目次.....................................................IV
圖目次...................................................VII
表目次...................................................VIII
壹、 前言...............................................1
貳、 文獻整理............................................5
一、豐年蝦簡介...............................................5
二、斑馬魚簡介...............................................6
三、抗菌胜肽簡介..............................................7
四、抗菌胜肽epinecidin-1.....................................8
五、魚類免疫反應概述..........................................9
5.1 魚類先天性免疫系統....................................9
5.2 Toll-like receptors (TLRs) 概述....................9
5.3 Cytokines概述.....................................10
5.3.1 Interleukin 1................................11
5.3.2 Interleukin 6................................11
5.3.3 Interleukin 10...............................11
5.3.4 Turmor necrosis factor-α.....................12
5.3.5 Interferons..................................12
5.3.6 Chemokines...................................12
參、 實驗材料與方法......................................14
一、實驗材料................................................14
1.1 實驗生物 (Experimental animals)....................14
1.1.1 豐年蝦 (Artemia francisca)....................14
1.1.2 斑馬魚 (Danio rerio)..........................14
1.2 實驗菌株(Experimental bacterial)...................15
1.3 細胞株 (Cell line).................................15
1.4 引子 (Primers)....................................15
1.5 實驗儀器與器材......................................17
1.6 實驗試劑與藥品......................................18
1.7 實驗抗體 (Antibodys)...............................19
1.8 使用軟體...........................................19
二、實驗方法................................................20
2.1 質體DNA之大量製備...................................20
2.2豐年蝦卵去殼 (Decapsulation of Artemia cysts ).......20
2.3 豐年蝦卵之電穿孔轉殖 (Electroporation)...............20
2.4 孵化率計算 (Hatching rate).........................21
2.5 轉殖效率計算 (Transfection efficiency)..............21
2.6 螢光強度計算 (Fluorescence intensity)...............21
2.7 RNA萃取 (RNA extraction)..........................22
2.7.1豐年蝦RNA萃取..................................22
2.7.2斑馬魚RNA萃取..................................22
2.8 RNA反轉錄 (Reverse Transcription)..................22
2.9 即時定量聚合酶鏈鎖反應 (Quantitative real-time PCR, qPCR)..23
2.9.1 標準曲線之繪製................................23
2.9.2豐年蝦EGFP-epi表現量偵測 (EGFP-epi expression detection)..23
2.10 豐年蝦蛋白質萃取 (Artemia protein extraction).......24
2.11 豐年蝦蛋白質定量 (Atemia protein quantification.....24
2.12 豐年蝦螢光蛋白定量 (Artemia EGFP protein quantification..24
2.13 西方轉漬法 (Western blot).........................25
2.13.1 蛋白質凝膠電泳 (SDS-PAGE).....................25
2.13.2 西方轉漬法...................................25
2.14 創傷弧菌 (Vibrio vulnificus 204) 之培養............26
2.15 創傷弧菌之檢測.....................................26
2.16 抑菌圈檢測 (Zone of inhibition)...................26
2.17 小鼠巨噬細胞RAW264.7之培養..........................27
2.18 細胞存活率實驗 (MTS assay).........................27
2.19 斑馬魚細菌攻擊實驗 (Bacterial challenge)............27
2.20 斑馬魚腸道含菌量檢測................................28
2.21 斑馬魚餵食7天後未注射V. vulnificus 的免疫基因與抗菌胜肽表現量偵測..28
2.22 斑馬魚餵食後注射V. vulnificus的免疫基因表現量偵測......28
肆、 實驗結果...........................................30
一、豐年蝦卵的抗菌胜肽epinecidin-1電穿孔轉殖....................30
1.1 豐年蝦卵去殼與孵化率之關係............................30
1.2豐年蝦的電穿孔轉殖....................................30
1.2.1 轉殖豐年蝦螢光偵測..............................30
1.2.2 卵數與質體DNA濃度對孵化率、轉殖效率與螢光強度之影響...31
1.2.3 電穿孔脈衝條件對孵化率、轉殖效率與螢光強度之影響......31
二、轉殖豐年蝦偵測...........................................32
2.1 轉殖豐年蝦EGFP-epi表現量偵測.........................32
2.2 豐年蝦total protein偵測............................32
2.3 豐年蝦protein定量與EGFP-epi含量偵測..................33
2.4 轉殖豐年蝦total protein與epimecidin-1抗菌測試........33
2.4.1 轉殖豐年蝦 total protein抑菌實驗................33
2.4.2 轉殖豐年蝦protein細胞毒性實驗....................34
三、斑馬魚抗菌實驗...........................................35
3.1 斑馬魚存活率實驗.....................................35
3.2 斑馬魚肝臟與腸道含菌量檢測............................35
3.3 斑馬魚攝取轉殖豐年蝦未注射V. vulnificus的表現量變化......36
3.4 斑馬魚免疫基因與自身抗菌胜肽表現量檢測...................36
伍、 討論..............................................38
一、去殼溶液的濃度與浸泡時間會影響豐年蝦卵的孵化率以及電穿孔實驗......38
二、電穿孔轉殖豐年蝦的偵測....................................38
三、進行電穿孔時,豐年蝦 EGFP-epi表現量取決於轉殖效率的高低........39
四、豐年蝦EGFP-epi protein定量與對V. vulnificus的抑菌能力定....40
五、轉殖豐年蝦protein並不會對正常細胞造成細胞毒性................41
六、斑馬魚攝取轉殖豐年蝦可以增加對V. vulnificus的抵抗力..........41
七、斑馬魚攝取轉殖豐年蝦後可以降低V. vulnificus的含菌量..........42
八、斑馬魚攝取轉殖豐年蝦後會改變自身免疫基因與抗菌胜肽的表現量.......42
陸、 結論..............................................45
柒、 參考文獻...........................................46
捌、 圖與表.............................................57
玖、 附錄..............................................79











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