Production Process Of Vibrio Anguillarum MVAV6203A-1 Multivalent Vaccine MVAV6203A-1 For Marine Flatfishes

Vibrio anguillarum and Aeromonas hydrophila are important fish pathogens infecting many commercial farmed fish species, causing great loss of profit in aquaculture around the world. Vaccination is now recognized as a viable strategy in the prophylaxis of infectious diseases for farmed fish. In our previous work, a recombinant vector vaccine, V. anguillarum MVAV6203A-1, was constructed to display the A. hydrophila originated antigen-GapA34 on the cell surface of the live attenuated V. anguillarum MVAV6203. The immune protection evaluation in turbot (Scophtalmus maximus) by injection demonstrated that this vaccine effectively protected turbot from the infection of both A. hydrophila and V. anguillarum and had a great potential value in commercial application.In this work, a new submerged culture medium were designed and optimized by using component swapping, Plackett-Burman design and response surface methodology. The optimal medium (g/L) (soluble amylum 10, NH4Cl 3, yeast extract 3.57, FeCl3·6H2O 0.11, trisodium citrate 0.22 and NaCl 34.6) was obtained. In the optimized medium, although the dry cell weight (DCW) obtained was 0.78 g/L which was almost the same as before optimization, the specific yields of antigen-Gap A34 expressed in whole cell and displayed on outer membrane were enhanced by 179% (736μg/gbiomass) and 44% (83μg/gbiomass) respectively. Furthermore, compared with the modified Luria-Bertani medium, the optimized medium significantly increased the specific yield of antigen-GapA34 displayed on outer membrane to 1.73 fold. In 5 L batch culture, the DCW of 1.71 g/L with the GapA34 specific yield of 1138μg/gbiomass was obtained. A suitable fed-batch system with dissolved oxygen as substrate feed indicator was developed. Using this strategy, the obtained DCW and specific yield of GapA34 were 5.98 g/L and 3384μg/gbiomass respectively. Then we performed scale-up fermentation in 30 L bioreactor with pH control. The DCW of 5.61 g/L with the GapA34 specific yield of 3283μg/gbiomass was attained. Besides the study on fermentation process, various groups of protective agents and their synergistic effects on V. anguillarum MVAV6203A-1 in the freeze drying were tested and evaluated. A favorable protective medium was established and the composition was determined as:galactose 46 g/L, trehalose 22.5 g/L and skimmed milk 101.5 g/L. In this protective medium, the cells obtained a viability of up to 77.3% after freeze drying.