博士 / 國立海洋大學 / 水產養殖學系 / 87 / This thesis investigated the characteristics of protease and pathogenesis of Vibrio alginolyticus strain Swy in kuruma prawn.
An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy was purified using the AKTA purifier system with hydrophobic interaction chromatography, anion exchange and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl methylsulphonyl fluoride (PMSF), antipain and shows maximal activity at pH 8 to 11(HPA used), having a pI of 4.3 and a molecular weight of 33 kDa. The purified protease was lethal for kuruma prawn at an LD50 of 0.29 g protein/g body weight. The addition of PMSF completely inhibited the lethal toxicity of the purified protease, indicating that this serine protease was a lethal toxin produced by the bacterium.
Four chromogenic substrates for characterizing serine protease of strain Swy were evaluated. The protease activity of bacterial extracellular products (ECP), or the 33 kDa purified protease, was completely inhibited by EDTA, EGTA using water-soluble substrates (azoalbumin and azocasein). Since chelating agents and 1,10-phenanthroline are commonly employed as inhibitors to identify metalloprotease, the two water-soluble substrates may not be appropriate for this purpose, except for using 1,10-phenanthroline as an inhibitor.
Virulence of ECPs and the lethality attributes of serine proteases secreted by five pathogenic V. alginolyticus strains from various sources in kuruma prawn were studied. The ECPs of organisms originally isolated from diseased kuruma prawn or small abalone Haliotis diversicolor supertexta was more virulent (LD50 value of 0.48 or 0.41 g protein/g body weight) than those from diseased tiger prawn P. monodon, yellowfin porgy Acanthopagrus latus or horse mackerel (LD50 value of 0.98-1.17 g protein/g body weight). All the ECPs manifested strong, weak and no activities against gelatin, sheep erythrocytes and chitin, respectively. The higher inhibition of serine protease activity resulted in lower mortality rate of the ECP injected into the prawns suggesting that the protease is the lethal factor secreted by V. alginolyticus. The toxicity of the respective ECPs to kuruma prawns was only partially neutralized by rabbit antiserum to the formalinized Swy ECP and/or rabbit antiserum to the formalinized Swy serine protease.
The haemolymph withdrawn from the moribund prawns injected with the ECP or toxic protease was unable to clot. The coagulogen was fast migrated in CIE following in vitro toxic protease and ECP treatment, but the amount of all components were reduced only in ECP treatment except coagulogen. After these treatment the plasma colour change from blue to pink was recorded. The amount of coagulogen was reduced in CIE following in vivo bacteria and ECP treatment, however that of toxic protease treatment was not remarkable.
| Identifer | oai:union.ndltd.org:TW/087NTOU0086029 |
| Date | January 1999 |
| Creators | Ferng-Ruey Chen, 陳逢叡 |
| Contributors | Kuo-Kau Lee, 李國誥 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 92 |
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