Development Of Live Attenuated Vaccine And Vector Vaccine For Vibrio Anguillarum

Live attenuated vaccine has become the best choice compared with others, because of its convenient administration, low dose required and inducing a strong immune reaction. Now most attenuated vaccines are developed by genetic engineering, although they have got a well attenuated effect, the ability of infecting host or multipling in the host is greatly reduced which will inevitably reduce their immune effect. To solve this problem, we planned to develop vaccines which had high infection ability and low pathogenicity. We used iron-regulated promoter to control phage lysis genes, and introduced this system into pathogens. This recombinant pathogen would grow normally in vitro, but lyse in vivo because of the iron-limiting environment. In this work, lysis genes from different bacteriophages under the control of a strict iron-regulated promoter Pviua were respectively inserted into plasmid pUTat to generate a serial of lysis plasmids. An efficient iron-regulated lysis system Pviua-13-19-15was obtained which could lyse Vibrio anguillarum in vivo effectively. This system was introduced into V. anguillarum wild type MVM425, and the recombinant vaccine425/pUTP22was evaluated in zebra fish(Danio rerio). In intraperitoneal injection trial, the LD50of425/pUTP22was3.47×104CFU/tail compared with1.45×103CFU/tail of wild strain. Zabra fish vaccinated with425/pUTP22had relative percent survival of87%. In immersion trial, the mortality of zebra fish(108CFU/ml by immersion) infected with425/pUTP22was0%compared with60%of wild strain, the mortality of zebra fish (109CFU/ml by immersion) was10%compared with70%of wild strain. And zebra fish vaccinated with425/pUTP22(108CFU/ml by immersion) had relative percent survival of87.7%, suggesting that V. anguillarum live attenuated vaccine based on iron-regulated lysis system had great potential in vaccine application.Bacterial vector vaccine has been widely studied, because it has many advantages, such as immunizing without adjuvant, enhancing immunogenicity of antigens, carrying different antigens and similar infection characteristics with pathogens. Many vector vaccines were constructed by using plasmids as expression vector. But this model has several problems, such as genetic instability, resistant genes existence and heavy burden to vaccine vector from too much antigen synthesis. In this study, we tested the Tn7transposition system in Escherichia coli, Vibrio anguillarum and Edwardsiella. tarda and eliminated the resistance selection marker inserted in the chromosome. Then, we used Tn7to insert a heterologous antigen gene gapA to the chromosome of V. anguillarum MVAV6203and then introduced the iron-regulated system into the recombinant strain to generate bacterial vector vaccine with the purpose of enhancing the genetic stability and bio-security for the vector vaccine.