| Summary: | 碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 94 === Abstract
The genome of grouper iridovirus consists 140 Kb and encodes 120 open reading frames. Three putative protein with molecular weight of 100 KDa, 60 KDa and 45 KDa were detected after Western immunoblot analysis of the antibodies produced from fish and mice against the grouper iridovirus. Five candidate genes including GIV001L、GIV010R、GIV021L、GIV042L and GIV045R were selected from the grouper iridovirus genome. One of the candidate genes, the GIV045R gene encodes the major capsid protein of iridovirus. These putative target genes were cloned and their encoded protein were expression from the E. coil expression system. We have already constructed GIV042L and GIV010R in pET20b(+) expression vector. We produced polyclonal antibodies from mice and New Zealand rabbit that were immunized with the recombinant proteins expressed from Rosetta DE3 pLysS. Results obtained from Western immunoblot analysis indicate that these antibodies could recognize the grouper iridovirus particles.
The protection efficiency of these subunit vaccines against grouper iridovirus were estimated by injecting these recombinant protein into grouper fry and subsequently challenged with grouper iridovirus. After intraperitoneal immunizing with these recombinant proteins as subunit vaccines for 14 days, the immunizing grouper fry were challenged with 109 TCID50 mL-1 grouper iridovirus. The highest mortality of the sample group were reached at 13-16 days after virus challenge and the cumulative mortality of 60 % was found at the end of 24 days. On the contrary, the cumulative mortality of negative control group was 100% after 14 days grouper iridovirus infection and positive control group was 20% after 24 days. These subunit vaccine protection against grouper iridovirus infection is significant with 40% relative percentage of survival.
In summary, several putative and effective subunit vaccine were developed successfully from the grouper iridovirus genome. Preliminary results indicted that significant protection effects of these subunit vaccines were observed.
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