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Separated aeromonas salmonicida phage, and composition and application thereof

A technology for killing Aeromonas salmonids and bacteriophages, which is applied in the fields of biotechnology and microbiology and can solve problems such as affecting the quality and safety of aquatic products

Pending Publication Date: 2021-10-22
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the large-scale use of antibiotics will be accompanied by the emergence of multi-drug resistant bacteria, which affects the quality and safety of aquatic products

Method used

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  • Separated aeromonas salmonicida phage, and composition and application thereof
  • Separated aeromonas salmonicida phage, and composition and application thereof
  • Separated aeromonas salmonicida phage, and composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1, Isolation and identification of Aeromonas salmonicida AS01

[0115] In order to obtain organisms that can effectively kill fish pathogens, the inventors have carried out many years of research and large-scale screening. The present inventor isolated a strain of Aeromonas salmonicida from diseased turbot fish in a turbot farm in Liaoning, and named it AS01. Using the Aeromonas salmonicida AS01 as a host, the inventors isolated an Aeromonas salmonicida phage from the environment and named it vB_AsM_ZHF.

[0116] The present inventor samples the liver and spleen tissue from the diseased fish, draws four-section lines on the TSA solid medium, and cultures them upside down at 28°C for 24 to 48 hours, then picks the colonies grown on the plate for Gram staining , after observation under the microscope, select the colonies of Gram-negative brevibacteria on the plate to streak again, repeat the above steps 3 times until colonies with uniform size and shape are forme...

Embodiment 2

[0138] Example 2. Isolation and identification of Aeromonas salmonicida phage vB_AsM_ZHF

[0139] (1) Sample pretreatment

[0140] Sampling the wastewater from the turbot breeding farm in Liaoning, filtered the collected water samples with 8 layers of gauze, then centrifuged the filtrate at 8000×g for 5 min at 4°C, and centrifuged it with a microporous filter membrane with a pore size of 0.22 μm. The supernatant was filtered and stored at 4°C for future use.

[0141] (2) Phage sample enrichment culture

[0142]Take 40ml of the filtrate, 10ml of 5×TSB medium and 1ml of the culture solution of Aeromonas salmonicida AS01 in logarithmic growth phase, add it to a 250ml shake flask, and culture overnight at 100rpm at 28°C. Take 1ml of the culture solution, centrifuge at 8000×g for 5min, and take the supernatant after centrifugation to filter and sterilize with a microporous filter membrane with a pore size of 0.22 μm.

[0143] (3) Phage isolation

[0144] Use SM buffer to dilute...

Embodiment 3

[0153] Embodiment 3, the morphological observation of bacteriophage

[0154] Phage samples were pretreated with phosphotungstic acid negative staining method. Take 20 μL of the bacteriophage culture liquid that was filtered through a 0.22 μm filter membrane and pour it on the copper grid, and after 10 min, absorb the excess liquid with absorbent paper. Let it stand at room temperature for 2 minutes, add 20 μL of 2% phosphotungstic acid staining dropwise on the copper grid, and immediately absorb the excess phosphotungstic acid solution with absorbent paper after staining for 30 seconds. Let stand at room temperature for 5 min, and observe the morphology of phage with a transmission electron microscope.

[0155] Electron microscope image of Aeromonas salmonicida phage vB_AsM_ZHF figure 2 . according to figure 2 As shown, the head of phage vB_AsM_ZHF is an icosahedron, and the tail is composed of a hollow structure and an outer sheath. The phage head is 95nm long, 91nm wi...

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Abstract

The invention provides a separated aeromonas salmonicida phage as well as a composition and application thereof. The inventor obtains the bacteriophage capable of efficiently killing aeromonas salmonicida through screening, and the bacteriophage has strong infection ability and strong splitting effect and is short in incubation period in a bacterial host. The bacteriophage disclosed by the invention can be applied to the fields of agriculture, medicine, environment and the like to resist bacterial hosts of the bacteriophage, and has a good development prospect as an antibiotic substitute.

Description

technical field [0001] The invention belongs to the field of biotechnology and microbiology, and more specifically, the invention relates to an isolated Aeromonas salmonicida phage, its composition and application. Background technique [0002] In recent years, with the rapid development of the aquaculture industry, the scale of aquaculture has continued to expand. With the continuous promotion of intensive and large-scale breeding methods, the prevention and treatment of aquatic animal diseases is becoming more and more important. Effective prevention and control can promote the development of aquaculture. Bacterial disease is an important aquatic disease. Effective control of this disease can promote the orderly operation of the aquaculture industry and avoid economic losses caused by large-scale diseases. [0003] Aeromonas salmonicida (Aeromonas salmonicida) is a Gram-negative short bacillus belonging to the psychrophilic adynamic Aeromonas, widely distributed in aquati...

Claims

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Application Information

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IPC IPC(8): C12N7/00A23K10/18A23K50/80A23L33/135A61K35/76A61P31/04A01N63/40A01P1/00
CPCC12N7/00A23L33/135A23K10/18A23K50/80A61K35/76A61P31/04A01N63/40C12N2795/10121A23V2002/00A23V2200/30
Inventor 刘琴张元兴王启要徐蓁禾刘晓红邵帅王蓬勃
Owner EAST CHINA UNIV OF SCI & TECH
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