| The scale of aquaculture of Anhui province has been expanded constantly in recent years. Unfortunately, it leads to the outbreak and prevalence of bacteria diseases consequently. The Vibriosis caused by Vibrio mimicus is extremely common in aquatic animals and threatens the further development of the crab aquiculture seriously. Strengthenning the research of its biology characteristics and pathogensis will do a great help to offer some kinds of effective prevention and cure measure and revitalize the aquaculture. Vibrio mimicus HX-4 strain isolated from diseased Eriocheir sinensis was identified a pathogene through pathogenic test. In order to provide the scientific basis for clarifying the pathogeny and producing toxinic vaccine, the biological characteristics of the exotoxin produced by Vibrio mimicus were systematically studied on its pathogenesis, optimal conditions of exotoxin expression, extraction method, molecular weight, enzyme activities, stability and cytotoxicity by bacteriological and molecular biological methods.Firstly, pathogenicity, toxicity and enterotoxicity of the exotoxin were studied by animal experiment, toxicity test and rabbit skin blue spot test. The results showed the extracted exotoxin and live bacterium(3.8X 108 CFU/mL) were pathogenic to mice and Eriocheir sinensis, their lethal ratioes were all 100%, but their lethal time was different, the former was from 2 h to 10 h and the latter was from 24 h to 36 h. The median lethal dose (LD50) of the exotoxin to Eriocheir sinensis amounted to 1.40 g. It was possessed of heat-labile enterotoxicity. These suggested that the exotoxin produced by HX-4 strain played an important role in its pathogenesis.Secondly, cultural conditions on the exotoxin expression of Vibrio mimicus HX-4 strain were optimized in vitro. The results demonstrated that the types of media, NaCl percentage, cultural temperature and cultural time, initial pH of the media and soluble oxygen all affected the exotoxin expression. The optimal conditions were initial pH 8.0, 0.5% NaCl-BHI and at 28癈 for 36 h in shake culture or 48 h in stationary and aerobic culture.Thirdly, the extraction method of the exotoxin was established by solid ammonium sulfate precipitation and chromatography technique. Among four saturated ammonium sulfate (30%, 50%, 70% and 85%), the hemolytic and proteolytic activities of culture supernatant precipitated with 50% saturatedammonium sulfate were the strongest, but the protein percentage was more. Protein percentage was the highest and the exotoxin activities were the strongest when 0.05 mol/L Tris-HCl buffer (pH 7.8) was applied to DEAE-Sephadex A50 column equilibrated with the same buffer and the exotoxin was eluted with 0-0.6 mol/L NaCl and 0.05mol/L Tris-HCl buffer (pH 7.8). According to above optimal extraction conditions, the crude preparation precipitated with 50% saturated ammonium sulfate was applied to DEAE-Sephadex A50 column and then partial purified exotoxin to Sephadex G-100 column equilibrated and eluted with 0.3 mol/L NaCl and 0.05 mol/L Tris-HCl buffer (pH 7.8), so the output of the extracted exotoxin was 0.2 mg/100 mL cultural fluid.Furtherly, the exotoxin molecular weight and enzyme activities were estimated by SDS-PAGE electrophoresis and indicator gel technique, at the same time the conditions of indicator gel technique were optimized. After SDS-PAGE electrophoresis with about 3 jaL exotoxin, at first 2.5%Tritox-100 solution treated the gel to remove SDS, then the gel was transfered to 0.8% agarose plate containing 0.5% casein, 0.4% gelatin or 1% erythrocyte of rabbit respectively to digest for 1 h. At last the gel was stained with Coomassie brilliant blue R-250 and destained. So coloureless and bright band could be observed under blue (caseinase and gelatinase) or dark red (hemolysin) background. The results demonstrated that the exotoxin had three kinds of different protein whose molecular weights were 154 KD, 143 KD and 117 KD respectively. All kinds of the protein were possessed of caseinase activity, gelatin...
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