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Bacillus subtilis multi-valent vector-based vaccine and application thereof

A Bacillus subtilis, carrier vaccine technology, applied in the direction of antibacterial drugs, bacterial antigen components, antibody medical components, etc., to achieve the effect of broad commercial development

Active Publication Date: 2014-02-05
马悦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Carrying out research and product development of fish vaccine with this strategy has not yet been reported at home and abroad, and the present invention proposes for the first time

Method used

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  • Bacillus subtilis multi-valent vector-based vaccine and application thereof
  • Bacillus subtilis multi-valent vector-based vaccine and application thereof
  • Bacillus subtilis multi-valent vector-based vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Preparation of Bacillus subtilis polyvalent carrier vaccine

[0037] 1. Culture medium preparation

[0038] (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.

[0039] (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.

[0040](3) LB medium: tryptone (Tryptone) 10g / L, yeast extract (Yeast extract) 5g / L, sodium chloride (NaCl) 10g / L, adjust the pH to 7.0.

[0041] 2. Recovery of the plate culture medium: the seeds of the recombinant Bacillus subtilis strain with the preservation number CCTCCNo: M 2012380 stored at -80°C were streaked and inoculated on the LB solid plate, and cultured at 37°C overnigh...

Embodiment 2

[0046] Example 2: Evaluation test of oral immune efficacy using turbot as experimental fish.

[0047] Preparation of pathogenic bacteria suspension: culture Vibrio anguillarum and Edwardsiella tarda in TSB (2% NaCl) liquid medium to OD600 of 0.6, culture Aeromonas hydrophila in TSB liquid medium to OD600 of 0.6, Then centrifuge at 5000 rpm for 10 minutes at room temperature, collect the precipitated cells, and suspend them with PBS buffer to a final concentration of 1×10 7 cfu / ml.

[0048] Breeding of experimental fish: The experimental fish (turboscus) purchased from Shandong was temporarily raised in a 200L aquarium, equipped with a flowing circulating water treatment and purification system, and the temperature of the breeding water was maintained at 18±2°C. unhealthy individuals. Healthy turbot with a body length of 12-15cm were randomly divided into 20L experimental aquariums, with 30 tails in each aquarium. The water in the experimental aquarium was changed twice a d...

Embodiment 3

[0054] Embodiment 3: Taking tilapia as the oral immune efficacy evaluation test of experimental fish

[0055] Preparation of pathogenic bacteria suspension: Brain heart infusion broth (BHI) medium (tryptone 10.0g / L, sodium chloride 5.0g / L, disodium hydrogen phosphate 2.5g / L, glucose 2.0g / L, bovine Heart extract (500 mL / L, pH 7.4) was used to culture Streptococcus agalactiae and Streptococcus iniae until OD600 was 0.6, and Aeromonas hydrophila was cultured in TSB medium until OD600 was 0.6, then centrifuged at room temperature at 5000rpm for 10 Minutes, the precipitated cells were collected and suspended in PBS buffer to a final concentration of 1×10 7 cfu / ml.

[0056] Breeding of experimental fish: The experimental fish (tilapia) purchased from Shandong were temporarily raised in a 200L aquarium, equipped with a flow circulation fresh water treatment and purification system, and the temperature of the breeding water was maintained at 24±2°C. unhealthy individuals. Select ...

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Abstract

The invention relates to a novel bacillus subtilis multi-valent vector-based vaccine and application thereof. The novel bacillus subtilis multi-valent vector-based vaccine comprises a bacillus subtilis recombination strain obtained by using a genetic engineering method, wherein the bacillus subtilis recombination strain is classified and named as bacillus subtilis HT5302 and is preserved in the China Center For Type Culture Collection (CCTCC) with a preservation number of CCTCC No:M2012380. The novel bacillus subtilis multi-valent vector-based vaccine is prepared by using the bacillus subtilis recombination strain as a core. A spore with a surface having no a streptococcus agalactiae 3-glyceraldehyde-phosphate dehydrogenase (GAPDH) protein can be generated through inducing the bacillus subtilis recombination strain, and secretes a fusion protein which formed by cell-penetrating peptide and vibrio anguillarum 3-GAPDH when germinating to form a nutrient cell. The novel bacillus subtilis multi-valent vector-based vaccine has a multi-valent protection function on bacterial diseases such as streptococcicosis, vibriosis, edwardsienosis and aeromonas hydrophila.

Description

technical field [0001] The invention belongs to the fields of microbiology, immunology and aquatic animal disease prevention and control, and in particular relates to a novel Bacillus subtilis used for preventing and treating fish diseases caused by Vibrio, Edwardsiella, Streptococcus and Aeromonas hydrophila Multivalent carrier vaccine and its application. Background technique [0002] The aquaculture industry has become one of the fastest growing food production industries of animal origin in the world. According to the 2010 statistics of the World Food and Agriculture Organization (FAO), since 2000, the growth of fish products has mainly come from aquaculture. With the development of scale, intensification and industrialization of the aquaculture industry, what will be displayed in front of us will be an aquaculture industry focusing on fish farming. [0003] However, with the rapid development of the aquaculture industry, the outbreak of various diseases followed. The...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/02A61K39/09A61K39/106A61P31/04
Inventor 不公告发明人
Owner 马悦
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