A stable plasmid system for heterologous antigen expression in attenuated Vibrio anguillarum.

To stably synthesize heterologous protein in an attenuated Vibrio anguillarum strain (MVAV6203) for potential multivalent live vaccine application, plasmids with different replicons were used to construct protein expression systems in this work. The gfp fragment under control of a strict low-iron-regulated promoter P(viua) was inserted into seven plasmids with varied replicons derived from pAT153, pACYC184, pBBR1, pEC, pMW118, pRK2, and pSC101, to generate seven corresponding plasmids. Our results revealed that the plasmid pUTat with the replicon from pAT153 was retained by 100% of the host cells and mediated stable expression of heterologous protein in antibiotic-free medium within 250 generations. Further analyses in animal model (zebrafish larvae) demonstrated that the constructed plasmid pUTat was well retained by bacteria and continuously expressed GFP in vivo in zebrafish. The gapA40 gene, encoding Glyceraldehyde-3-phosphate dehydrogenase from the fish pathogen Edwardsiella tarda, was introduced into the pUTat-based protein expression system, and transformed into V. anguillarum MVAV6203. The resultant recombinant vector vaccine 6203/pUTatgap was evaluated in turbot (Scophtalmus maximus). After 30 days post vaccination, the fish showed an increased survival ratio by 80% and 67% under the challenge of wild V. anguillarum and E. tarda, respectively. Our results suggested that the pUTat-based antigen expression system had great potential with its efficiency and stability in the design of bacterial vector vaccine.