English  |  正體中文  |  简体中文  |  Items with full text/Total items : 26994/38795
Visitors : 2388350      Online Users : 50
RC Version 4.0 © Powered By DSPACE, MIT. Enhanced by NTU Library IR team.
Scope Adv. Search
LoginUploadHelpAboutAdminister

Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17532

Title: 抗氧化酵素2-Cys 和 1-Cys Peroxiredoxin的研究
Study on the antioxidant enzymes 2-Cys and 1-Cys Peroxiredoxin
Authors: Hui-Ming Huang
黃慧明
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 抗氧化酵素;特性分析
2-Cys Peroxiredoxin;1-Cys Peroxiredoxin
Date: 2004
Issue Date: 2011-07-04
Abstract: 本論文由聚合酶連鎖反應, 選殖出樟芝子實體的2-Cys peroxiredoxin (簡稱2-Cys Prx) 和1-Cys peroxiredoxin (簡稱1-Cys Prx) cDNA。全長分別為958 bp 和837 bp,轉譯區分別為567 bp 和672 bp, 分別譯出188 個胺基酸及223 個胺基酸。2-Cys Prx 在Cys-48、165 位 置和1-Cys Prx 在Cys-46 位置,分別有兩個及一個高保守區的半胱胺 酸(Cys)。 2-Cys Prx 以pAVD 10 作為表現載體,以E.coli BL21 (DE3) pLysS 作為表現宿主,經由IPTG 的誘發,即可表現出大量具有活性的重組蛋 白質。此蛋白質在15 % SDS–PAGE 下,以單元體及雙倍體的形式同時 存在。其在60 ℃處理2 min 後,活性還有68 %。經不同的pH 處理, 顯示在pH 5.4-11 環境中比較安定。若以2 % SDS 處理下,可使2-Cys Prx 單元體及雙倍體解離,活性還有57 % 。以0.4 M imidazole 處理下, 活性還有47 % 。以chymotrypsin 在37 ℃反應3 h 後,會使2-Cys Prx 單元體解離,活性剩下42 %。以trypsin 處理後,2-Cys Prx 較具有抵 抗能力不易被分解。另外,將2-Cys Prx 在Cys-48 、 165 、 48 / 165 位置上的Cys 突變成Ser 後,這些蛋白質仍具有部分活性。 1-Cys Prx 以pET-20b (+) 作為表現載體,以E.coli BL21 (DE3) pLysS 作為表現宿主,經由IPTG 的誘發,即可表現出大量具有活性的 重組蛋白質。此蛋白質在15 % SDS–PAGE 下,只以單元體形式存在。 其在60 ℃ 處理16 min 後,活性還有37 %。經不同的pH 處理,顯示 pH 9-10 中比較安定。但在SDS 處理後,1-Cys Prx 變為無活性。以0.4 M imidazole 處理下,會使1-Cys Prx 的單元體解離,活性還有68 % 。 II 以chymotrypsin 在37 ℃處理20 min 後,會使其單元體解離,活性剩 下15 %。以trypsin 在37 ℃處理40 min 後,會使其單元體解離,活性 剩下9 %。
The purpose of this thesis is to study antioxidant enzyme. A full length cDNA of 958 bp encoding a putative 2-Cys Peroxiredoxin (2-Cys Prx) from Antrodia camphorata (A. cam.) fruit body was cloned by polymerase chain reaction (PCR). Nucleotide sequence analysis of this cDNA revealed that it comprised a complete open reading frame coding for 188 amino acid residues. Computer analysis of its sequence revealing the two cysteines (48 and 165) that form a disulfide bond, was well-conserved among the reported 2-Cys Prx sequences. To further characterize the A. cam. 2-Cys Prx, the coding region was subcloned into an expression vector pAVD 10 and transformed into E.coli BL21 (DE3) pLysS. The expression of the 2-Cys Prx was purified by Ni2+ -nitrilotriacetic acid Sepharose superflow column. The purified enzyme showed two forms by 15 % SDS-PAGE. The enzyme retained 68 % activity after heating at 60 ℃ for 2 min. The enzyme activity was inhibited under acidic pH (pH 2.2). The enzyme activity was only a little decrease under 1 % SDS treatment. The enzyme retained 47 % activity under 0.4 M imidazole treatment. The enzyme showed 42 % activity after 3 h of incubation at 37 ℃ with chymotrypsin. Furthermore, the dimeric enzyme was much resistant to proteolysis after 3 h of incubation at 37 ℃ with trypsin. In addition, the mutants of A. cam. 2-Cys Prx at position 48 、165 or 48 / 165 from Cys to Ser showed lower activity. IV A full length cDNA of 837 bp encoding a putative 1-Cys peroxiredoxin (1-Cys Prx) from A. cam. fruit body was cloned by PCR. Nucleotide sequence analysis of this cDNA revealed that it comprised a complete open reading frame coding for 223 amino acid residues. Computer analysis of its sequence revealing cysteine (46) that hethered with the other subunit to form a disulfide bond was well-conserved among the reported 1-Cys Prx sequences. To further characterize the A. cam. 1-Cys Prx, the coding region was subcloned into an expression vector pET-20b (+) and transformed into E.coli BL21 (DE3) pLysS. The expression of the 1-Cys Prx was purified by Ni2+ -nitrilotriacetic acid Sepharose superflow column. The purified enzyme showed one form by 15 % SDS-PAGE. The enzyme retained 37 % activity after heating at 60 ℃ for 16 min. The enzyme was stable under basic pH (above pH 9.0). The enzyme was inhibited in the present of SDS (above 1 %). The enzyme retained 68 % activity at 0.4 M imidazole. The enzyme retained 15 % activity after 20 min of incubation at 37 ℃ with chymotrypsin. The enzyme retained 9 % activity after 40 min of incubation at 37 ℃ with trypsin.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M92360007
http://ntour.ntou.edu.tw/ir/handle/987654321/17532
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

Files in This Item:

File Description SizeFormat
index.html0KbHTML202View/Open


All items in NTOUR are protected by copyright, with all rights reserved.

 


著作權政策宣告: 本網站之內容為國立臺灣海洋大學所收錄之機構典藏,無償提供學術研究與公眾教育等公益性使用,請合理使用本網站之內容,以尊重著作權人之權益。
網站維護: 海大圖資處 圖書系統組
DSpace Software Copyright © 2002-2004  MIT &  Hewlett-Packard  /   Enhanced by   NTU Library IR team Copyright ©   - Feedback