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No mark gene deletion deoxidated mutant strain of wild Manhu bacteria and its use

A markerless gene, Vibrio eel technology, applied in the methods of using microorganisms, medical preparations containing active ingredients, bacteria, etc., can solve the problem that the virulence of wild Vibrio eel strains weakens the colonization ability and hinders the synthesis of aromatic amino acids. Synthesis and other problems, to achieve obvious multi-titer immune protection, high-efficiency immune protection, and significant immune effects

Inactive Publication Date: 2005-07-27
SHANGHAI HAOSI MARINE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] Deletion of the above target genes can hinder the synthesis of aromatic amino acids, folic acid and virulence factor siderophore anguibactin of the wild strain of Vibrio anguillarum, which leads to the greatly weakened virulence of the wild strain of Vibrio anguillarum and its ability to survive in the natural environment and in fish. The ability to colonize in the host to achieve the purpose of attenuation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Preparation of live attenuated vaccine

[0028] Composition of culture medium and seawater saline:

[0029] 1) LB slant medium: soybean peptone (Difco) 10g / L, yeast extract (Merck) 5g / L, NaCl 25g / L, agar 18g / L, pH7.5;

[0030] 2) Seed medium: soybean peptone (Difco) 10g / L, yeast extract (Merck) 5g / L, NaCl 25g / L, phenylalanine 20mg / L, tyrosine 20mg / L, tryptophan 20mg / L L, p-hydroxybenzoic acid 20mg / L, p-aminobenzoic acid 20mg / L, pH7.5;

[0031] 3) Fermentation medium: soybean peptone (Difco) 10g / L, yeast extract (Merck) 5g / L, NaCl 25g / L, ferric ammonium citrate 0.2-0.5mmol, phenylalanine 20mg / L, tyrosine 20mg / L, tryptophan 20mg / L, p-hydroxybenzoic acid 20mg / L, p-aminobenzoic acid 20mg / L, pH6.8;

[0032] 4) Seawater saline: NaCl 20, KCl 0.7, MgCl 2 ·6H 2 O 4.8, NaHCO 3 0.11, MgSO 4 ·7H 2 O3.5, CaCl 2 2H 2 O 1.6, g / L, pH7.2, filter sterilized.

[0033] Vaccine preparation: get 1 inoculation loop and preserve the seed of the attenuated vaccine stra...

Embodiment 2

[0033] Vaccine preparation: get 1 inoculation loop and preserve the seed of the attenuated vaccine strain on the LB slant medium, inoculate it into a 500ml shaker flask equipped with 100ml liquid LB seed medium, and cultivate it with shaking at 28°C (rotating speed 200 rpm). After 12 hours, take 5ml of vigorously growing bacterial solution (about O.D=4.0) and inoculate it into 100ml of fresh fermentation medium, and incubate at 28°C for 12 hours. Wash 3 times with sterile seawater saline, harvest the cells by centrifugation (2000×g, 15 minutes, 15°C), and dilute with sterile seawater saline to a certain concentration suspension (10 6 -10 9 CFU / ml), stored at 15°C for future use. Embodiment 2: Take flounder (Paralichthys olivaceus) as the median lethal dose LD of experimental animals 50 determination

[0034] The fish used in the test were first placed in the SPF (Specific Pathogen Free) laboratory to adapt to breeding for 1 week to eliminate abnormal individuals. Before th...

Embodiment 3

[0045] Embodiment 3: take grouper as the median lethal dose LD of experimental animals 50 determination

[0046] The fish used in the test should be placed in the SPF (Specific Pathogen Free) laboratory to adapt to breeding for 1 week to eliminate abnormal individuals. Before the infection test, the SPF test fish were stocked in a 20L infection test tank in the Challenge Lab, and continued to be fed for 1 week, with 10 fish (12-15cm) in each tank. The test tank maintains a flowing water flow (300-400mL / min, sterile old sea water), the water temperature is 28°C, and the fluctuation is 2°C.

[0047] The test fish were randomly divided into groups, and each group was tested in parallel with 3 tanks. In the infection test, each group of test fish was dosed with a certain concentration gradient (10 2 ~10 8 CFU / tail) of Vibrio alginolyticus wild strain and attenuated vaccine strain were artificially infected by intramuscular injection. Record the number of deaths per day under ...

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Abstract

A bacterin for fish farming is used to treat vibriosis with reduced toxicity. The vaccine is mainly formed by no marker gene deletion strains with toxicity reduced. It is effective for cross immunization of Rongzao vibrios. The vaccine made from it can effectively be used to prevent testing fishes from diseases caused by vibriosis.

Description

technical field [0001] The present invention is the prevention and control technology of aquaculture animal disease, relates to aquaculture fish with bacterial multipotency attenuated live vaccine, specifically the Vibiro anguillarum (Vibiro anguillarum) multipotent for preventing and treating Vibrio anguillarum and Vibrio alginolyticus vibriosis Potency attenuated live vaccine. Background technique [0002] Aquaculture is becoming increasingly important in view of the growing world population and the depletion of natural fisheries. It is estimated that by 2010, my country's marine fish farming output will reach 500,000 tons in order to meet people's demand for marine fish products. Due to the limited water and soil resources, it is difficult to continue to develop by increasing the production area by increasing the breeding area. Therefore, large-scale, intensive, and high-density farming models have gradually become the mainstream of the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/106C12N1/20C12N1/21
CPCA61K39/107A61K2039/522A61K2039/552C12N1/36A61P31/04A61P37/00
Inventor 马悦张元兴赵东玲王蓬勃
Owner SHANGHAI HAOSI MARINE BIOTECHNOLOGY CO LTD
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