| Summary: | 碩士 === 國立海洋大學 === 水產養殖學系 === 90 === This thesis investigates the effects of bacterial cells, extracellular products (ECP) and partially purified protease of Vibrio harveyi, originally isolated from diseased tiger shrimp (Penaeus monodon), on plasma coagulogen of shrimps in vitro and in vivo, in order to further understand the pathogenesis and pathophysiological phenomenon caused by the Vibrio species.
The ECP of V. harveyi was prepared and the toxic protease was partially purified. This partially-purified protease was a 22 kDa protein, determined by SDS-PAGE, exhibiting caseinolytic activity without boiling while it was a 38 kDa protein after boiling in sample buffer (contained 2-mercaptoethanol).
On SDS-PAGE, the molecular weights of coagulogen and hemocyanin of white shrimp (Litopenaeus vannamei) and tiger shrimp found similar. In addition, the migration, molecular weights and immune cross-reactivity of coagulogen of both shrimp species were also found were similar.
The effects of bacterial cells, ECP and partially purified protease of V. harveyi were investigated using crossed immunoelectrophoresis (CIE), SDS-PAGE and Western blotting. The results revealed that a fast- migrating phenomenon of plasma coagulogen in CIE gels could be observed after pre-incubation with partially purified protease. In addition to fast-migrating of plasma coagulogen, the hemocyanin was also affected after pre-incubation with the ECP. Plasma coagulogen was proven to be degraded into smaller molecular-weight proteins on Western blot, plasma coagulogen was not remarkably affected after pre-incubation with bacterial cells. However, the reduction of plasma protein content and coagulogen amount was determined when the shrimps were pre-injected with bacterial cells, ECP and partially purified protease, separately. As for the degree of reduction, the group pre-injected with ECP was the highest while that pre-injected partially purified protease was the second. The plasma coagulogen was degraded into 89 kDa and 43 kDa, and 80 kDa protein in white shrimp and tiger shrimp, respectively.
The present results revealed that plasma components and blood clotting mechanism of shrimp could be affected by bacterial cells, ECP and partially purified protease. In particular, the plasma coagulogen was degraded. This is the first report which demonstrates that plasma coagulogen of shrimp is degraded when the animal is infected with V. harveyi leading to occurrence of unclottabe hemolymph. Meanwhile, the toxic protease may play a major role in the pathogenesis of V. harveyi infection.
|
|---|
