| Summary: | 碩士 === 大仁科技大學 === 生物科技研究所 === 95 === Photobacterium damselae subspecies piscicida ( PDP ) is a Gram-negative, short rod and halophilic bacterium, which is one of the major pathogens in marine cage cultured cobia ( Rachycentron canadum ) in Taiwan. Cobia was suffered from pseudotuberculosis ( or pasteurellosis ) caused by PDP resulting in a huge economic lose in marine aquaculture. The goal of this study is to develop a recombinant vaccine against PDP by using PCR ( polymerase chain reaction ) cloning technique. The target gene AIP56 was amplified by using PCR and inserted into the vector, pUC18, and then transformed into BL21 (DE-3) E. coli. The expressed recombinant protein could be obtained after doing a process of expression. The purified recombinant protein ,AIP56, showed a mass of MW 56 KDa by using ion exchanger column when SDS-PAGE was employed for the assay. Moreover, the purified rAIP56 can be strongly reactive to the ECP ( extracellular product ) of PDP by using Western blotting assay indicating AIP56 really having antigenicity against the ECP of PDP. However, two kinds of recombinant vaccines, which one is for injecting use and the other is for dipping, were prepared for the vaccination in cobia. Two groups for each kind of vaccine and 20 fishes ( each fish 50g in BW ) for each group were used for the immunization program. The vaccine for injecting ( i.p. ) was given 3 times with an interval of a week as the same that were done with the dipping vaccine. One week after the last immunization, 2.5×106 CFU living PDP bacteria/0.1ml/(i.p.) were given to each tested fish. The results showed 75% ( 15/20 ) immunoprotection and reveled 66% relative surviving rate in the injecting vaccine group. No protection showed in the dipping use vaccine. Further study should be needed.
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