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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/10484

Title: 增強魚類超氧歧化酵素基因轉殖應用之評估
Enhance Fish Anti-Oxidative Stress with Copper/Zinc Superoxide Dismutase by Gene Transfer
Authors: 林棋財
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Date: 2001-02
Issue Date: 2011-06-28T07:45:12Z
Publisher: 行政院農業委員會
Abstract: 摘要:本實驗的目標在於利用重組DNA的方法, 增加魚類銅鋅型超氧歧化酵素( SOD )的穩定性, 並利用此高穩定性的酵素增加魚類抗氧化能力, 同時利用基因轉殖法將外源性SOD基因轉入魚體中, 建立穩定遺傳特性的基因轉殖魚, 以增加魚類抗氧化能力.以顯微注射法將魚類銅鋅型SOD酵素基因與水母綠螢光嵌合基因一同轉殖進入魚胚胎中, 利用螢光顯微鏡可觀察SOD酵素之表現, 將表現綠螢光的仔魚養至成熟, 利用PCR方法可快速分析外來基因嵌入與否.以此具有外源性SOD基因的成魚交配產生F?子代, 篩選具備外來基因遺傳的魚加以飼養.預期在此活體分析外來基因轉殖的技術建立下, 可獲得SOD基因轉殖魚, 並期望此轉殖魚具備遺傳特性, 可將SOD基因傳給子代.在養殖基因轉殖魚的過程中, 可利用基因定點突變法將魚類SOD基因進行定點突變, 與大腸桿菌表現載體pET-20b( + )接合, 送入大腸桿菌表現, 並以含鎳金屬離子管柱層析法獲得較純的突變型SOD, 分析此突變蛋白的活性、耐熱性、耐酸鹼性等, 生化特性, 預期獲得穩定性高的突變型SOD, 並以此作為未來基因轉殖應用的考量.預期產生具有外源性Cu/Zn-SOD基因嵌入的仔魚, 以進行抗氧化壓力的評估; 利用基因突變法篩選穩定性高之Cu/Zn-SOD, 並進行蛋白質浸泡或基因轉殖之抗氧化能力相關研究.
Abstract:The objective of this experiment is to increase fish anti-oxidative stress ability by using copper/zinc superoxide dismutase ( SOD )gene transfer and enzyme immersion.The SOD gene will be fused with green fluorescence protein ( GFP )gene, then microinjected into fish embryos.The SOD gene expression can be monitored by GFP gene expression.Using PCR technique to analyze foreign gene integration, the positive lines will be raised till mature.After cross mating, we can select the germ line transmission fish as F?, then raise to mature.Meanwhile, we will design two primers for SOD gene point mutation, the mutant SOD gene was ligated with expression vector pET-20b ( + ), and transformed into E.coli expression host AD494( DE3 )pLysS.The mutant SOD protein can be purify by Ni-NTA column chromatography.In addition, the purified mutant SOD was subjected to demonstrate the biochemical properties such as different temperature, pH.We hope to obtain the Cu/Zn-SOD gene germ line transmission F?and more stable mutant SOD protein for future anti-oxidative stress application.
Relation: 90農科-2.1.1-漁-F1(3)
URI: http://ntour.ntou.edu.tw/ir/handle/987654321/10484
Appears in Collections:[水產養殖學系] 研究計畫

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