| Summary: | 碩士 === 國立臺灣海洋大學 === 水產養殖學系 === 95 === The strdy investigated whether phospholipase C ( PLC ) in the major extracellular lethal factor of Photobacterium damselae subsp. piscicida. Using tests for lethality inhibition and immuno-protection of PLC in cobia : on the hand to observe the protect ion efficacy in cobia, and on the other hand, to confirm PLC as a major toxin of Photobacterium damselae subsp. piscicida.
Ammonium sulfate was used to precipitate ECP of Photobaterium damselae subsp. piscicida (strain 9205), then purified by using anion exchange chromatography-HP column and the band exhibited PLC activity was cut to gain purified enzyme. The PLC was a single band (27 kDa) visualized on Native-PAGE and SDS-PAGE. The PLC could digest egg yolk. The phospholipase was around 1102.6 unit/mg. The purity of PLC was further confirmed by N-terminal amino acids of the purified PLC was sequenced with a similarity of 13% comparing to a previous phospholipase of Photobacterium damselae subsp. piscicida.
In lethality inhibition tests, PLC was inhibited by 20mM EDTA、20mM SDS and 5mM CuCl2 in vitro. No cobia was killed after challenge with ECP mixed with 20mM SDS and 5mM CuCl2. These results showed that, the PLC was a major toxin of Photobacterium damselae subsp. piscicida.
The results revealed that both active and passive immunization tests with ECP and purified PLC all exhibited protective effect in cobia. In active immunization test, the PLC group manifested better than ECP. However, in passive imunization test, rabbit antisera from both ECP and purified PLC all exhibited good protection in cobia.
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