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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/10487

Title: 抗菌peptide在水產養殖之應用研究
Application of Antimicrobial Peptides in Aquaculture
Authors: 林正輝
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Date: 2001-02
Issue Date: 2011-06-28T07:45:13Z
Publisher: 行政院農業委員會
Abstract: 摘要:
計畫目標: 首先, 我們將由現有已知的抗菌peptide, 篩選最適之抗菌peptide, 並利用化學合成抗菌peptide.以輻射擴散分析法或最小抑制濃度檢測化學合成之抗菌peptide對水產病源菌之活性.由最適抗菌peptide之基因序列, 設計適當之primer, 利用化學合成後, 將其接到基因表現載體上, 利用大腸桿菌大量製造.架構( 重要工作項目 ): 菌種: 主要為水產病源菌: Vibrio carchariae ( 分離自石斑魚 ), Vibrio alginolyticus( 分離自石斑魚、黃鰭鯛、斑節蝦或草蝦 ), Vibrio harveyi( 分離自黃鰭鯛、斑節蝦或草蝦 ), Vibrio cholera( 分離自九孔 ), Vibrio parahaemoliticus( 分離自九孔 ), Aeromonas sobria( 分離自虹鱒、九孔或錦鯉 ), 還有其他菌種正在收集或購買中.輻射擴散分析法( radial diffusion assay ): 實驗前一天先將菌株劃至TSA培養液中, 在25-30℃培養18-22小時.把培養隔夜的菌液倒到1X phosphate buffer solution中, 將菌液均勻混合, 測OD???的吸光值, 且調整為1.0左右.將滅好菌的TSA培養液冷卻至45-50℃, 再把菌液加入; 均勻搖晃後倒到TSA培養基上.等培養基冷卻後, 以電動吸取器及吸管進行打洞.將各種抗菌蛋白( 1μl )分別注入TSA培養基之洞中, 放到25-30℃的溫箱中靜置培養, 隔天檢視抑制環之產生.最小抑制濃度( minimal inhibitory concentration )測定: 實驗前一天先將菌株劃至TSA培養液中, 在25-30℃培養18-22小時.把培養隔夜的菌液倒到1X phosphate buffer solution中, 將菌液均勻混合, 測OD???的吸光值, 且調整為0.001左右.在microtiter plate上, 每個well均加入25μl菌液, 在分別加入100μl不同濃度或不同的抗菌peptide.不同濃度的抗菌peptide是以兩倍稀釋.最小抑制濃度是以介於細菌生長的最高濃度及抑制的最低濃度表示之.預期效益: 完成各種抗菌peptide之化學合成, 篩選最適之抗菌peptide, 完成抗菌peptide表現載體之構築.
Abstract:Goals of plan: Firstly, We will screen the optimal antimicrobial peptides from known antimicrobial peptides.The antimicrobial peptides will be chemically synthesized and the activities of antimicrobial peptides will be determined by radical diffusion assay and minimal inhibitor concentrtation.The oligonucleotides coded for antimicrobial peptide will be synthesized and cloned into expression vector.Then recombinant antimicrobial peptide will be produced in E.coli.Working plan: Strains: Vibrio carchariae ( isolated from grouper ), Vibrio alginolyticus ( isolated from grouper、yellow fin porgy、penaeus japonicu or penaeus monodom ), Vibrio harveyi ( isolated from yellow fin porgy、penaeus japonicu or penaeus mondom ), Vibrio cholera ( isolated from Haliotis divorsicolor ), Vibrio parahaemoliticus ( isolated from Haliotis divorsicolor ), Aeromonas sobria ( isolated from rainbow trout、Haliotis divorsicolor or carp ).Radial diffusion assay: A single colony will be inoculated into TSA medium and cultured overnight at 18-22℃.The OD???of the culture will be adjusted to 1with 1x phosphate buffer solution.The fresh TSA medium will be added into diluted overnight culture and poured into petri dish.For monitoring the activities of antimicrobicrobial peptide, 1ml of each sample will be pipetted onto a bacterial plate, and the plate will be examined for clear zones in the bacterial lawn after 18-20hours of incubation at 18-22℃.minimal inhibitory concentration ( MIC ): A single colony will be inoculated into TSA medium and cultured overnight at 18-22℃.The OD???of the culture will be adjusted to 0.001with 1x phosphate buffer solution.Twofold dilutions of the working of antimicrobial peptide will be fresh prepared with microtiter plate.The wells finally containing 100ml of culture medium with antimicrobial peptide.Twenty five ml of bacterial culture will be added to each well.After incubation at 18-22℃, the MIC will be calculated.predictions: synthesized the antimicrobial peptides, find the optimal antimicrobial peptides, construction of antimicrobial peptide expression plasmid.
Relation: 90農科-2.1.1-漁-F1(11)
URI: http://ntour.ntou.edu.tw/ir/handle/987654321/10487
Appears in Collections:[水產養殖學系] 研究計畫

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