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Edwardsiella tarda attenuated strain and application thereof

A technology of Edwardsiella tarda and Edwardsiella tarda, applied in the direction of bacteria, antibacterial drugs, bacterial antigen components, etc., can solve the problems of unclear genetic background of attenuated strains, weakened bacterial virulence, and unclear mechanism, etc., to eliminate the spread of a large number of Danger of pathogens, ease of differentiation, effects of clear genetic background

Inactive Publication Date: 2010-05-12
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the U.S. patent has the method that utilizes rifampicin (rifampicin) culture medium to carry out multiple subcultures to Edwardsiella tarda to screen the attenuated strain as an attenuated live vaccine (United States Patent 7067122). The genetic background of the attenuated strain obtained by this method is unclear, and the bacteria The mechanism of virulence attenuation is not clear, and it is easy to cause virulence recovery during use. At the same time, it is difficult to monitor the environment of vaccine strains. Attenuated vaccines constructed using transposon or gene insertion inactivation technology carry antibiotics and other foreign gene fragments. It is easy to release antibiotics and foreign gene fragments to the environment during administration, and there is a potential danger of gene transfer or integration into other pathogens or other organisms. Therefore, it is difficult for these attenuated live vaccines to pass the approval regulations of biological product safety inspection and management agencies in various countries, so that Difficult to commercialize.

Method used

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  • Edwardsiella tarda attenuated strain and application thereof
  • Edwardsiella tarda attenuated strain and application thereof
  • Edwardsiella tarda attenuated strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Preparation of attenuated live vaccine

[0020] 1) TSB medium: tryptone 17.0g / L, soybean peptone 3.0g / L, NaCl 5.0g / L, K 2 HPO4 2.5g / L, glucose 2.5g / L, phenylalanine 20mg / L, tyrosine 20mg / L, tryptophan 20mg / L, pH 7.3.

[0021] Dissolve the above ingredients in 1000ml of distilled water, adjust the pH with 10% NaOH, sterilize at 121 lbs, 125°C for 15 minutes, cool down at room temperature for later use.

[0022] 2) TSA medium: tryptone 17.0g / L, soybean peptone 3.0g / L, NaCl 5.0g / L, K 2 HPO4 2.5g / L, glucose 2.5g / L, phenylalanine 20mg / L, tyrosine 20mg / L, tryptophan 20mg / L, agar powder 1.5g / L, pH 7.3.

[0023] Dissolve the above components in 1000ml of distilled water, adjust the pH with 10% NaOH, sterilize at 121 pounds, 125°C for 15 minutes, cool to 60°C to make a plate for use.

[0024] 3) PBS buffer: Na 2 HPO4 2H2O 2.74g / L, NaH 2 PO4·H2O 0.63g / L. Dissolve the above ingredients in 1000ml of distilled water, sterilize at 121 lbs, 125°C for 15 minutes, co...

Embodiment 2

[0029] Embodiment 2: Take silk bamboo fish (Trichogaster trichopterus Pallas) as the median lethal dose LD of experimental animals 50 Determination of

[0030] Healthy test fish with a body weight of 7-8g are temporarily raised in a 100L aquarium, equipped with a flowing circulating water treatment and purification system, and the temperature of the breeding water is maintained at 20-22°C. After 2 weeks of temporary raising, they were randomly divided into 10L experimental aquariums, with 10 fish in each aquarium, and continued to be temporarily raised for 2 weeks. The water in the test aquarium was changed once a day in the morning, the amount of water changed was 1 / 2, and the water temperature was maintained at 20-22°C.

[0031] During the artificial infection test, each group of experimental fish was injected with different dilution concentrations (10 5 ~10 9 cfu / mL) of the attenuated vaccine strain and the wild-type bacterial strain, each fish abdominal muscle was injec...

Embodiment 3

[0036] Embodiment 3: Take flounder (Paralichthys olivaceus) as the median lethal dose LD of experimental animals 50 Determination of

[0037] Healthy test fish with a body length of 8-10cm are temporarily kept 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 raising, they were randomly divided into 20L 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 change was 1 / 2, and the water temperature was maintained at 18-20°C.

[0038] During the artificial infection test, each group of experimental fish was injected with different dilution concentrations (10 5 -10 6 cfu / mL) of the attenuated vaccine strain and wild-type strain, intramuscular injection of 0.1 mL per fish, observed ...

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Abstract

The invention relates to an aquiculture animal disease prevention and control technology, in particular to an Edwardsiella tarda attenuated strain for preventing and controlling Edwardsiella tarda infection and application thereof. An Edwardsiella tarda mutant strain is MZLLSE40aroA which is preserved in CGMCC and has the preservation number: CGMCC No.2886 and the Edwardsiella tarda mutant strain is MZL LSE40 aroAesrB which is preserved in CGMCC and has a preservation number: CGMCCNo.2887. Bacterial cells of 106-109cfu / mL of Edwardsiella tarda strain MZL LSE40aroA or the Edwardsiella tarda strain MZL LSE40 aroAesrB and sterile phosphoric buffer salt solution are mixed as an attenuated live vaccine for preventing and controlling Edwardsiella tarda disease of culture fish. The strain has intraframe large-scale deletion of virulence related genes and nutrition metabolization related genes and non-recoverable virulence, does not contain a foreign gene segment and an antibiotics resistance marker and is safe for the environment and animals.

Description

technical field [0001] The invention relates to aquaculture animal disease prevention technology, in particular to an Edwardsiella tarda attenuated strain for preventing and treating Edwardsiella tarda infection and its application. technical background [0002] Edwardsiella tarda is a Gram-negative pathogen that infects a wide range of hosts and can infect a variety of lower and higher animals. In aquaculture animals, the bacterium is an important pathogenic bacterium of fish, amphibians and reptiles. The Edwardsiella disease caused by it is a common infectious disease in farmed fish, which is very harmful to the health of farmed animals and the aquaculture industry. In my country, pathogenic Edwardsiella has been isolated from various animals such as bullfrogs, soft-shelled turtles, eels, flounder, turbot, and red sea bream. [0003] Due to the use of various antibiotics for disease prevention and control in the production process, more and more drug-resistant strains of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A61K39/02A61P31/04C12R1/01
Inventor 莫照兰李杰茅云翔肖鹏杨佳银王波
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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