Studies on Vibrio Protease Inhibitors in the Serum of Grouper Epinephelus sp.

• Main Authors: Cheng-Hao Lee, 李正皓
• Other Authors: Lee, Kuo-Kau
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/74308655334453420872

碩士 === 國立海洋大學 === 水產養殖學系 === 89 === The identification, purification and characterization of Vibrio protease inhibitors in the serum of grouper Epinephelus sp. were studied. Protease activities in the extracellular products (ECP) of Vibrio alginolyticus S4y strain , V. harveyi 820514 strain and V. carchariae EmI82KL strain, previously isolated from surface lesions and/or head kidney of juvenile grouper, disease tiger prawn Penaeus monodon and the intestinal yellow fluid of diseased grouper exhibiting gastroenteritis, respectively, were inhibited by the presence of grouper serum. Therefor, the ECP of the three strains were used in the assay for protease inhibitor of fish serum. A protease inhibitor in grouper serum was purified by ammonium sulfate precipitation and using various columns (hydrophobic interaction chromatography column, RESOURCE Q column, Mono Q column and gel filtration column) on Fast Protein Liquid Chromatography system (Pharmacia). The molecular weight of the protease inhibitor was 55 kDa determined on SDS-PAGE (PhastSystem, Pharmacia) under reducing condition. The protease inhibitor was heat labile, inactivated at temperature above 60 0C and had a pI value of about 4~5. It was not an alpha 2 macroglobulin (not inactivated by methylamine), but was similar to an alpha 1 protease inhibitor. Only one precipitation arc was visualized in gel filtration fractions on crossed immunoelectrophoresis (CIE) gel using the rabbit antiserum to the grouper crude serum, thus further indicates that it is a purified component. However, more than three precipitation arcs were visualized in grouper crude serum on crossed immunoelectrophoresis (CIE) gel using the rabbit antiserum to the grouper serum protease inhibitor, thus indicates that there are more than three resemble protease inhibitors in grouper serum. After cross reaction of rabbit anti-grouper serum inhibitor antiserum with sera collecting from various fish species performed using CIE technique, this 55 kDa alpha 1 protease inhibitor could only be found in the sera of other groupers and related species.