Expression In Pichia Pastoris And Epitope Screening Of Antigen Protein FBA From Edwardsiella Tarda

Edwardsiella tar da is one of the most important pathogens of marine fish. As an intracellular pathogen, the effect to control E. tarda with antibiotic is limited. The previous study in our laboratory on pathogenic mechanism and developing new vaccine of E. tarda has found that fructose diphosphate aldolase (FBA), a housekeeping enzyme of E. tarda, has good immune protection on zebrafish and turbot, which is a potential subunit vaccine candidate of Edwardsiellosis.E. coli expression system is surfered from generation of endotoxin. In order to avoid problem of endotoxin and realize the large-scale preparation of FBA, Pichia pastoris was used to express FBA, which can be applied in new vaccine development. In the present work, recombinant P. pastoris GS115-pPIC9K::fba and GS115-pGAPZαA::fba were constructed. In shake flask, GS115-pPIC9K::fba was detected to express FBA with methanol inducing. After 5 L fermentor culture of recombinant P. pastoris, the yield of FBA in the fermental supernatant can reach 1.06 g/L. The recombinant protein was purified through Ni-chelating affinity chromatography. We evaluated the immune effect of purified recombinant FBA, FBA expressed by recombinant P. pastoris can effectively protect zebrafish and turbot from infection of E. tarda. The same to FBA expressed by recombinant E. coli, FBA expressed by recombinant P. pastoris also has high reactivity with turbot antiserum. FBA expressed by recombinant P. pastoris remain good immunogenicity, which lay a solid foundation for the large-scale preparation of FBA and the development of new vaccine.Homology of FBA is high in a variety of bacteria. Epitope screening of FBA will provide new target for the vaccine design against polymicrobial pathogens in aquaculture. The preliminary screening of antigen epitope of FBA was carried on. Firstly, epitope of FBA was predicted with "BepiPred 1.0 b Server", an epitope prediction website. Secondly, eight recombinant plasmids were constructed, which had different regions of FBA. After purification, their relative percent of survivals on zebrafish and reactivity with antisera of turbot were analyzed.The results showed that the key epitope is most likely to be in 181～240 aa of FBA.