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Multivalent carrier vaccine for crucian carp hemorrhagic disease and its application

A carrier vaccine and hemorrhagic disease technology, applied in the direction of antiviral agents, antibody medical ingredients, medical preparations containing active ingredients, etc., can solve the problems of crucian carp breeding industry impact, prevention and control difficulties, and vaccines are difficult to work, and achieve broad commercial development Prospects, effects of achieving high-efficiency immunization

Active Publication Date: 2016-05-18
马悦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since 2009, the virus has successively caused large-scale diseases in crucian carp breeding areas in many provinces and cities in my country, which has brought a huge impact on crucian carp breeding industry
In addition, Aeromonas hydrophila has also been isolated and purified from the diseased crucian carp, and back-staining experiments have proved that crucian carp has bleeding symptoms (Liu Yanan et al., 2012, Journal of Aquatic Ecology 05; Lu Wenhao et al., 2009, Journal of Guangdong Ocean University, Issue 01), the pathogen often occurs simultaneously with viral diseases in the crucian carp culture process, and the developed monovalent vaccines are often difficult to be effective, which brings difficulties to the prevention and treatment

Method used

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  • Multivalent carrier vaccine for crucian carp hemorrhagic disease and its application
  • Multivalent carrier vaccine for crucian carp hemorrhagic disease and its application

Examples

Experimental program
Comparison scheme
Effect test

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 O10ml, 1MNaOH1.5ml, adjust pH to 7.6, ddH 2 O was adjusted to 1000ml. After autoclaving, cool to 50°C, add sterilized 1MCa (NO 3 ) 2 , 0.01MMnCl 2 , 1mM FeSO 4 1ml each.

[0029] (2) PBS buffer: Na 2 HPO 4 2H 2 O2.74g / L, NaH 2 PO 4 ·H 2 0.63g / L, the composition was dissolved in 1000ml distilled water, and sterilized at 121°C for 20 minutes.

[0030] (3) LB medium: tryptone (Tryptone) 10g / L, yeast extract (Yeasextract) 5g / L, sodium chloride (NaCl) 10g / L, adjust the pH to 7.0.

[0031] 2. Recovery of the plate medium: take the seeds of the recombinant Bacillus subtilis strain with the preservation number CCTCCNo: M2013029 stored at -80°C and inoculate them on the LB solid plate by streaking and culturing overnight at 37...

Embodiment 2

[0036] Example 2: Evaluation of the immune effect of carp herpesvirus II (CyHV-2) on crucian carp immunized with multivalent carrier vaccine orally.

[0037] 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.

[0038] 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 (50mmol / LTris-HCl , pH7.6...

Embodiment 3

[0044] Example 3: Evaluation of the immune effect of crucian carp immunized orally with multivalent carrier vaccine against Aeromonas hydrophila.

[0045] 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.

[0046] 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 prec...

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Abstract

The invention relates to a biological control method for crucian carp haemorrhagic disease, which is characterized in that: immunize cultured crucian carp with a multivalent carrier vaccine; the multivalent carrier vaccine is a recombinant bacterial strain of Bacillus subtilis obtained by genetic engineering, and the classification name of the recombinant bacterial strain is Bacillus subtilis HT5304 (Bacillus? subtilis? HT5304), has been preserved in the China Center for Type Culture Collection, the preservation number is CCTCC? No: M2013029. The invention also provides the application of Bacillus subtilis HT5304 (Bacillus? subtilis? HT5304) in the prevention and treatment of crucian carp breeding diseases. The biological control method of the invention realizes multivalent immune prevention and treatment of crucian carp viral pathogens and bacterial pathogenic hemorrhagic disease by immunizing cultured crucian carp with multivalent carrier vaccine.

Description

technical field [0001] The invention relates to a multivalent carrier vaccine for crucian carp hemorrhagic disease immune prevention and treatment of crucian carp hemorrhagic disease and its application, belonging to the fields of microbiology, immunology and aquatic animal disease prevention and control. Background technique [0002] Crucian carp, belonging to Cyprinidae, is one of the main freshwater fish in my country. However, during the breeding process of crucian carp, not only is it threatened by virus infection, but it also faces the potential risk of bacterial infection in the environment. At present, the major disease risk in crucian carp breeding in my country mainly comes from crucian carp hemorrhagic disease caused by carp herpesvirus II (CyHV-2) and Aeromonas (mainly Aeromonas hydrophila). Healthy development has had devastating impacts and severe economic losses. [0003] Herpesviral haematopoietic necrosis (HVHN) is a highly pathogenic viral disease of cruci...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21A61K39/106A61K39/245A61K48/00A61P31/04A61P31/22
Inventor 不公告发明人
Owner 马悦
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