A kind of vibrio anguillarum bivalent vaccine and its preparation and use method
A bivalent inactivated vaccine, Vibrio eel technology, applied in the directions of antibacterial drugs, pharmaceutical formulations, bacterial antigen components, etc. The effect of good safety and low production cost
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Embodiment 1
[0023] Example 1: Pathogenicity of Vibrio anguillaris to flounder (Paralichthys olivaceus)
[0024] 1) The median lethal dose (LD) of flounder infected by Vibrio anguillarum injection 50 )
[0025] Healthy test fish with a body weight of 10-12g are temporarily raised in a 1000L aquarium, equipped with a flowing circulating water treatment and purification system, and the temperature of the breeding water is maintained at 18-20°C. After 4 weeks of temporary breeding, they were randomly divided into 40L experimental aquariums, with 20 fish in each group, and continued to be temporarily raised for 2 weeks. In the test aquarium, the water was changed once a day in the morning and afternoon, the amount of water change was 1 / 2, and the water temperature was maintained at 18-20°C.
[0026] In the artificial infection test, live Vibrio anguillarum CGMCC No.6942 or CGMCC No.6944 cells were diluted with sterile PBS buffer solution to a serial concentration of 10 5 -10 9 Bacterial su...
Embodiment 2
[0036] Example 2: Pathogenicity of Vibrio anguillaris to turbot (Scophthalmus maximus)
[0037] 1) The median lethal dose (LD) of turbot infected by Vibrio anguillarum injection 50 )
[0038] Healthy test fish with a body weight of 9-12g are temporarily raised in a 1000L aquarium, equipped with a flowing circulating water treatment and purification system, and the temperature of the breeding water is maintained at 16-18°C. After 4 weeks of temporary raising, they were randomly divided into 30L experimental aquariums, with 10 fish in each aquarium, and continued to be temporarily raised for 2 weeks. In the test aquarium, the water was changed once a day in the morning and afternoon, the amount of water changed was 1 / 2, and the water temperature was maintained at 16-18°C.
[0039] In the artificial infection test, live Vibrio anguillarum CGMCC No.6942 or CGMCC No.6944 cells were diluted with sterile PBS buffer solution to a serial concentration of 10 5 -10 9 cfu / ml bacterial...
Embodiment 3
[0049] Embodiment 3: Preparation of Vibrio anguillarum inactivated vaccine
[0050] A. Take Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 and inoculate them into common broth medium containing 1.5% NaCl (w / v), respectively, and culture them in shake flasks at 25-30°C for 24-36 hours ; Add formaldehyde with a final concentration of 0.3-0.5% to the obtained cultures, and place them in shake flasks at 25-30° C. for 24-36 hours;
[0051] B. Centrifuge the cultures obtained above at a speed of 4000-5000rpm / min for 10-20min, respectively collect the precipitated bacteria, and then use PBS phosphate buffer solution to suspend the sediment respectively, and adjust the bacterial cell concentration to ~10 9 cells / ml, respectively Vibrio anguillarum strain CGMCC No.6942 inactivated vaccine and Vibrio anguillarum CGMCC No.6944 inactivated vaccine.
[0052] C. Mix the inactivated cells of Vibrio anguillarum CGMCC No.6942 and CGMCC No.6944 in a sterile phosphate-buffered saline soluti...
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