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研究生:許台慶
研究生(外文):Tai-Ching
論文名稱:草蝦幾丁質結合蛋白之定性分析
論文名稱(外文):Characterization of Penaeus monodon Chitin Binding Protein
指導教授:陳威仁陳威仁引用關係
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生物醫學科學學系碩士班
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:45
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草蝦是台灣的重要水產養殖物種,在1980年代養殖業達到高峰,卻在1990年代遇上了高致死率的蝦白點病毒 (White spot syndrome virus, WSSV),導致台灣嚴重的經濟損失。在先前的研究中以酵母菌雙雜合法 (Yeast two hybrid, Y2H) 用WSSV的封套蛋白質VP53A釣出草蝦的幾丁質結合蛋白 (Penaeus mondon chitin binding protein, PmCBP)。而CBP在植物中最先被徹底調查出在抗真菌及細菌感染中扮演重要的抑制角色,保護植物能夠順利在環境壓力中生長;無脊椎動物中也存在類似的蛋白質並且也和抗病菌有相關連。在本研究中使用釣到的部分PmCBP序列,經由和其它物種作序列比對發現擁有高度CBP序列的保留特性。於是透過免疫的實驗想來瞭解一個擁有抗病毒能力的草蝦蛋白質和病毒封套蛋白之間有什麼樣的關係。接著用部分的序列將蛋白質轉譯,並製造出抗體,之後運用免疫實驗分別在離體 (in vitro) 以及活體 (in vivo) 中先找出PmCBP可能所在的位置,以西方墨點法去確認草蝦本身擁有這個蛋白質的存在,再進一步用草蝦血球的免疫螢光實驗,來觀察VP53A和PmCBP在草蝦感染WSSV的血球中,具有共同聚集 (colocalization) 的現象,而兩者之間的分子機制仍須做進一步的實驗證明。

Tiger prawn (Penaues monodon) is an important aqua-cultural species in Taiwan. It reaches its highest amount during the 1980s’. It suffered a serious loss resulted from high mortality rate of White spot syndrome virus (WSSV) infection during the 1990’s. Previous research indicated WSSV viral envelope protein, VP53A could interact with shrimp protein P. monodon chitin binding protein (PmCBP) through the yeast two hybrid method. CBP was first investigated in plant and the specialists found that CBP has both antifungal and antimicrobial activities. It could help the plant to resist the environmental stress and stay healthy. In invertebrate animals, they have the same protein which works to prevent the viral infection and bacterial attachment. In this research, PmCBP partial sequence was compared with other species and it has high similarity between them. And I try to understand the relationship between the antimicrobial PmCBP and the virus envelope protein VP53A through immune experiments. Western blotting was applied to detect the protein expression in shrimp tissues. In in vivo experiment, using WSSV infected shrimp hemocytes to react with immunofluorescence and observe the colocalization between PmCBP and VP53A. More detailed experiments are needed to discover the molecular mechanism.

目錄

摘要 1
ABSTRACT 2
序論 3
1. 蝦白點病毒WSSV (White spot syndrome virus) 3
2. 草蝦Penaeus monodon 5
3. 幾丁質(Chitin)與幾丁質結合蛋白 (Chitin Binding Protein, CBP) 7
4. 研究目的 9
材料方法 10
1. 實驗動物 11
2. 勝任細胞 (Competent cell)製作 11
3. PmCBP質體建構以及大腸桿菌基因表現系統 12
4. PmCBP蛋白質純化 13
5. 抗體備製 14
7. 草蝦組織萃取 15
8. 蛋白質定量(BIO-RAD) 15
9. 草蝦RNA萃取 16
10. 5’RACE for PmCBP (Ambion Catalog #1700) 17
11. 膜質分離 (PIERCE) 19
12. 西方墨點法 20
13. PmCBP在血球細胞中的位置 21
15. 共軛焦螢光顯微鏡的使用 24
16. 序列分析軟體的使用 23
結果 24
1. 草蝦幾丁質結合蛋白質部分序列分析 24
2. 草蝦幾丁質結合蛋白質和其它物種之間的序列比較及親源關係 …………………………………………………………………24
3. 草蝦幾丁質結合蛋白質抗體的備製及測試 25
4. 草蝦幾丁質結合蛋白質在各組織之間的分佈 26
5. 草蝦幾丁質結合蛋白質在昆蟲細胞SF9轉染之後表現的狀況 …………………………………………………………………26
6. 草蝦血球免疫螢光尋找草蝦幾丁質結合蛋白質的位置 27
討論 28
參考文獻 33
表與圖 36



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