To study the immune protection of fish vaccinated by the live attenuated Vibrio alginolyticus vaccine,we constructed an in-frame deletion mutant of kdpD/kdpE (kdpDE) genes using overlap PCR and homologous recombination technology,to investigate the effects of this mutant on the biological characteristics.We then compared the differences of the biological characteristics and virulence between the gene deletion mutant and wild strain.Furthermore,the immunoprotection of the vaccine was analyzed by challenge test.The mutant ΔkdpDE showed no differences of ECPase activity and growth from the wild-type strain.But the decreases in swarming ability and biofilm formation were found.Virulence of the mutant declined by 78.5 times in zebrafish model.No bacteria survived in zebrafish 7 days later after injection vaccination.Furthermore,the mutant was used as an antigen to immunize zebrafish.To estimate immunoprotection and immune routes of the live attenuated vaccine,the zebrafish were challenged by V.alginolyticus and V.harveyi at 28 days after immersed and injected vaccination.The relative percentage survival(RPS) value of injection group was 83.3%,and the RPS value of immersion group was 66.7%.The results showed that the ΔkdpDE of V.alginolyticus has a high immunogenicity.In addition,the vaccination led to cross-protection from V.harveyi and the RPS values of injection group and immersion group were 65.5% and 55.2% respectively.These results indicate that the kdpDE mutant is an effective vaccine candidate against V.alginolyticus infection.