碩士 / 國立臺灣海洋大學 / 水產養殖學系 / 92 / Abstract
The thesis investigates the anti-vibrio protease component(s) present in the serum of cobia, Rachycentron canadum, including isolation, purification and characterization. Strains of Vibrio alginolyticus C11a and Photobacterium damselae subsp. piscicida Cp were used for assays.
Cobia serum was precipitated by ammonium sulfate and seperatated into parts A (30~50%) and B (50~70%). The protease inhibitor(s) were isolated and purified from A or B parts by using affinity chromatography column, hydrophobic interaction chromatography column and anion exchange column on the Fast Liquid Chromatography System. After purification, an analysis for the homogeneity and molecular weight of the protease inhibitor(s) were determined by using Native-PAGE、crossed immunoelectrophoresis (CIE) and SDS-PAGE. One protein band was observed on Native-PAGE and one precipitation arc was observed on CIE gel using fractions purified from parts A and B, respectively. The protein band on Native-PAGE exhibiting anti-protease avtivity was confirmed by zymogram. The molecular weight of the protease inhibitors was 185 kDa and 74 kDa, respectively.
More than 85% of the anti-protease activity in the two partially purified fractions was inhibited after incubation at 60℃ for 20 minutes prior to the assay indicating that the two protease inhibitors were heat-labile. The anti-protease activity of the two protease inhibitors was both apparently inhibited by 0.2M methylamine (incubated 37℃ for 30 mintues) together with pI values estimated between 4.15-4.55 and 3.75-4.55, respectively. Therefore, the two protease inhibitors in cobia serum were suggested to be α2-macroglobulin-like proteins.
| Identifer | oai:union.ndltd.org:TW/092NTOU5086018 |
| Date | January 2004 |
| Creators | Zhi-Jian Pan, 潘志建 |
| Contributors | Ping-Chung Liu, 劉秉忠 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 117 |
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