Biological control method of crucian hemorrhagic disease and application thereof
A technology of hemorrhagic disease and crucian carp, which is applied in the field of biological control of crucian carp hemorrhagic disease, can solve the problems of crucian carp breeding industry impact, prevention and control difficulties, vaccines are difficult to work, etc., and achieve the effect of high-efficiency immunization and broad commercial development prospects
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Embodiment 1
[0026] Embodiment 1: Preparation of crucian carp haemorrhagic disease polyvalent carrier vaccine
[0027] 1. Culture medium preparation
[0028] (1) DSM liquid medium: bacterial nutrient broth (Difco) 8g, 10% (w / v) KCl 10ml, 1.2% (w / v) MgSO 4 ·7H 2 O 10ml, 1M NaOH 1.5ml, adjust pH to 7.6, ddH 2 O was adjusted to 1000ml. After autoclaving, cool to 50°C and add sterilized 1M Ca(NO 3 ) 2 , 0.01MMnCl 2 , 1mM FeSO 4 1ml each.
[0029] (2) PBS buffer: Na 2 HPO 4 2H 2 O 2.74g / L, NaH 2 PO 4 ·H 2 O 0.63g / L, the ingredients were dissolved in 1000ml of distilled water, and sterilized at 121°C for 20 minutes.
[0030] (3) LB medium: tryptone (Tryptone) 10g / L, yeast extract (Yeast extract) 5g / L, sodium chloride (NaCl) 10g / L, adjust the pH to 7.0.
[0031] 2. Recovery of the plate medium: take the seed of the recombinant Bacillus subtilis strain preserved at -80°C with the preservation number CCTCCNo: M2013029 and inoculate it on the LB solid plate by streaking and inoculat...
Embodiment 2
[0037] Example 2: Evaluation of the immune effect of carp herpesvirus II (CyHV-2) on crucian carp immunized with multivalent carrier vaccine orally.
[0038] Breeding of experimental fish: The experimental fish (crucian carp) purchased from Jiangsu was temporarily raised in a 2000L aquarium after being tested for the absence of CyHV-2, equipped with a flow circulation fresh water treatment and purification system. After raising for 1 week, remove unhealthy individuals. Select healthy crucian carp with a weight of 350±50g and randomly group them into 200L experimental aquariums, 30 in each aquarium, change the water twice a day in the experimental aquarium, and change the water volume to 1 / 3, maintain the water temperature at 20±2°C, and continue to raise temporarily 2 weeks.
[0039] Preparation of CyHV-2 virus suspension: dissect diseased fish, select gills, kidney and spleen, cut them into pieces and grind them under liquid nitrogen, add TN buffer (50 mmol / L Tris -HCl, ...
Embodiment 3
[0046] Example 3: Evaluation of the immune effect of crucian carp immunized orally with multivalent carrier vaccine against Aeromonas hydrophila.
[0047] Breeding of experimental fish: The experimental fish (crucian carp) purchased from Jiangsu was temporarily raised in a 2000L aquarium after being tested for the absence of CyHV-2, equipped with a flow circulation fresh water treatment and purification system. After raising for 1 week, remove unhealthy individuals. Select healthy crucian carp with a weight of 350±50g and randomly group them into 200L experimental aquariums, 30 in each aquarium, change the water twice a day in the experimental aquarium, and change the water volume to 1 / 3, maintain the water temperature at 20±2°C, and continue to raise temporarily 2 weeks.
[0048] Preparation of pathogenic bacteria suspension: culture Aeromonas hydrophila to OD in LB liquid medium 600 was 0.6, then centrifuged at 5000rpm for 10 minutes at room temperature, collected the pr...
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