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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/16867

Title: 養殖海鱺血漿中蛋白質分解酵素抑制物alpha2 macroglobulin之分離純化與特性分析
Purification and characterization of alpha2 macroglobulin proteinase inhibitor from plasma of cobia, Rachycentron canadum.
Authors: Chia-Yu Hung
洪佳鈺
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 海鱺;蛋白質分解酵素抑制物
alpha2 macroglobulin;cobia
Date: 2008
Issue Date: 2011-06-30T08:43:32Z
Abstract: 本論文主要藉由聚乙二醇 6000 沉澱、親合性層析 concanavalin A SepharoseTM 4B、疏水性層析以及離子交換法等分離純化方式,純化海鱺(Rachycentron canadum)血漿中蛋白質分解酵素抑制物,α2 -macroglobulin(α2 M),及其相關特性分析研究。以 Natie PAGE 進行電泳分析並以 Coomassie Brilliant Blue R-350 染色後,在分子量大小為 360kDa 處出現一條蛋白帶,在溫和還原下於 SDS PAGE 中的分子量大小約為 180kDa,可知海鱺血漿中 α2 M 為二聚體,以 dithiothreitol(DTT)破壞其鍵結後,可以得到兩個不同分子量大小的片段,分別約為 93 及 87kDa。以醣蛋白染色確定其為醣蛋白,並藉由交叉免疫電泳及西方墨點分析鑑定其純度。 海鱺血漿及純化之 α2 M 以 0.2 M methylamine 處理30分鐘後,發現兩者對 methylamine 皆有敏感性。Methylamine 與 α2 M 結合後將會抑制α2 M與其他蛋白質分解酵素結合。純化之 α2 M(0.07 mg protein∕ml)對 Vibrio alginolytius 細胞外產物(0.364 mg protein∕ml)具有 50% 以上之抑制能力;對 Streptococcus iniae 細胞外產物(0.097 mg protein∕ml)作用時亦有 50% 以上之抑制能力。並可發現以連續稀釋處理細菌細胞外產物後,α2 M 抑制能力有上升之情形,可推測細胞外產物蛋白質分解酵素的濃度會影響 α2 M 抑制效力。 以不同溫度條件處理之下,可發現其於溫度高於 50℃ 時,抑制活性會大幅降低,屬於熱不安定型;在不同 pH 值處理下,發現 α2 M 處於 pH 9 的環境下其抑制活性最高,並可發現其於鹼性環境下有較好的抑制能力。
A protease inhibitor, α2 macroglobulin(α2 M), was purified from cobia (Rachycentron canadum)plasma by a four-step procedure:poly ethylene glycol fractionation, concanavalin A sepharoseTM 4B chromatography, hydrophobic interaction chromatography and ion exchange chromatography on Fast Protein liquid chromatography system. It migrated as one band and its molecular mass was about 360 kDa in the native state, whereas in SDS PAGE it was about 180 kDa under nonreducing condition. This result revealed that the native protein was a dimer. In addition, it was cleaved into two different fragments of molecular mass about 93 and 87 kDa when reduced by dithiothreitol(DTT). The α2 M was a glycoprotein as stained a Glycoprotein Staining kit. It was a purified protein as further checked Western blot and crossed immuno- electrophoresis(CIE). The protease inhibitory activities of cobia plasma or purified α2 M were sensitive to the addition of 0.2 M methylamine for 30 min. When incubated with methylamine, α2 M could not bind to other proteases. The purified α2 M(0.07 mg protein∕ml)exhibited more than 50% inhibitory activity against Vibrio alginolytius ECP(0.364 mg protein∕ml) while it exhibited more than 50% inhibitory activity against Streptococcus iniae ECP(0.097 mg protein∕ml). It seems that the ECP protease activity may affect the inhibitory activity of the α2 M. The anti-protease activity of the purified α2 M was apparently decreased as temperature elevated. The α2 M exhibited highest inhibitory activity at pH 9. The results indicate that the α2 M is a heat-labile and alkaline protease inhibitor.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M95330016
http://ntour.ntou.edu.tw/ir/handle/987654321/16867
Appears in Collections:[水產養殖學系] 博碩士論文

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