| An extracellular protease produced by Vibrio anguillarum strain M3 was purified following the steps of ultrafiltration, gel filtration with Sephadex G100, ion-exchange chromatography with DEAE-cellulose A52, HPLC gel filtration. The purified protease, with an isoelectric point of about 5.1 and molecular weights of about 36.5KDa, was lethal for goldfish at LD50 value of 1.2ug protein/g body weight. It was inhibited by EDTA,EDGA and 1,10-phenanthroline, which proved to be family of metalloproease, and showed maximal activity at pH8.0 and 55℃. It was found that this protease could degrade several protein substrates, including azocasein, geltin, transferrin, BSA, elastin, fibrinogen, collagen and fish IgM. Partial N-terminal amino acid sequence of the protease of Vibrio anguillarum strain M3 was AGATGTGPGGAGLTG.In order to demonstrate the role of metalloprotease in the pathogenesis of Vibrio anguillarum, we designed primers and successfully cloned the metalloprotease gene. According to this gene, two primers were designed and a internal homologous fragment was cloned. The fragment was subcloned into a suicide vector pNQ705, and then was transformed into the conjugation donor strain E.coli S17-1. Insertion mutation in Vibrio anguillarum chromosomal gene are isolated by mating the recombinant into M3 strain. PCR and sequence were used to validate the correct mutation. The mutant was analyzed to keep the original outmembrane antigens with serology test, but was found to grow obviously more slowly than the wild strain. Moreover, the SDS-PAGE pattern showed that extracellular products of mutant weredramatically different from those of M3, and the typical 36kD electrophoresis band disappeared in the pattern. The protease activity was also founded to have a greatly drop. No hemolysis phenomenon was found when the mutant was cultured in blood plate. In conclusion, the construction of metalloprotease gene mutant will surely be beneficial to further study of the pathogenesis of Vibrio anguillarum.The growth, production of extracellular products and protease of Vibrio anguillarum strain M3 cultured on solid or liquid medium were studied. An experiment was designed with Response Surface Methodology to assay the effects of NaCl concentration, pH value and temperature on growth of bacteria and protease production, and two regression models were established with SAS statistical software to evaluate the data. Protease activity was lower on the brain heart infusion medium than on the peptone medium. Compared with the peptone medium, strain growth and ECP production were stimulated on the trypticase soy broth medium, but protease production remained the same level. The presence of NH4Cl or casamino acids significantly decreased protease production. Muscle homogenate of flounder fish was able to stimulate the bacterial growth, ECP and protease production at different levels. The quantity of bacteria and ECP arrived at a maximal value at 4％ peptone in the medium, and protease production was steady at 2% peptone. The bacterial growth and ECP production increased, whereas the protease production decreased at the presence of 1％ glucose, sucrose or glycerin.Monoclonal antibodies (Mabs) against V.anguillarum strain M3 were produced, and their isotype were also characterized. Among them, C1C5 is the only Mab which did not cross-react with other eleven non-V.anguillarum strains. The proteinase K digestion test showed that the epitopes recognized by C1C5, C6C3 and C6C32 Mabs contain a protein in. While, the periodate oxidation test showed that the epitopes recognized by Mabs except C1C5 were glycosylated. In addition, results of additivity test indicated that theepitopes recognized by C6C3 and C6C32 Mabs were similar, which were quite different from that of Mab C1C5.The metalloprotease purified from the extracellular products(ECP) of Vibrio amguillarum M3, was used as antigen to immunize Bab/c mouse. Three monoclonal antibodies(MAbs), P2B1, P4A3 and P4B1, against the purified metalloprotease have been obtaine...
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