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Edwardsiella tarda high-efficiency lytic bacteriophage vB_EtaM-IME523 and application thereof

A bacteriophage, blunt technology, applied in the direction of bacteriophage, virus/phage, medical raw materials derived from virus/phage, etc., can solve the problems of farmers' economic loss, food-borne infection, etc., and achieve easy purification, specific infection, and obvious protection. effect of action

Pending Publication Date: 2021-03-12
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serious foodborne infection may result from ingestion of seafood contaminated with Edwardsiella tarda
In recent years, fish diseases caused by Edwardsiella tarda are not uncommon. In aquaculture, diseases caused by Edwardsiella tarda have exploded in many provinces across the country, causing huge economic losses to farmers.

Method used

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  • Edwardsiella tarda high-efficiency lytic bacteriophage vB_EtaM-IME523 and application thereof
  • Edwardsiella tarda high-efficiency lytic bacteriophage vB_EtaM-IME523 and application thereof
  • Edwardsiella tarda high-efficiency lytic bacteriophage vB_EtaM-IME523 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Isolation and Purification of Phage vB_EtaM-IME523

[0031] (1) Activation and cultivation of Edwardsiella tarda

[0032] Streak inoculation of Edwardsiella tarda strains on LB solid medium plates containing 1.5% (W / V) agar, and culture overnight at 37°C upside down. A single colony was picked from the plate, inoculated into a test tube containing 5 mL of LB liquid medium, and cultured on a shaker (37° C., 220 rpm) for 12 hours. Take 1 mL of liquid culture solution from the test tube and dilute it 1:100 (V / V) into an Erlenmeyer flask containing 100 mL of LB liquid medium, and place it on a shaker (37°C, 220 rpm) to expand the culture to the OD of the bacterial solution 600 ≈0.5, that is, the logarithmic phase bacterial liquid is obtained.

[0033] (2) Enrichment of phage

[0034] Phage vB_EtaM-IME523 was isolated from the sewage of the Fifth Medical Center of the Chinese People's Liberation Army General Hospital in Beijing (North latitude: 39.8622002; East longitude:...

Embodiment 2

[0046] Morphological Observation of Phage vB_EtaM-IME523

[0047] Take the phage-bacteria culture lysate isolated and purified in Example 1 and centrifuge at low speed (4°C, 12000g, 15min) to discard the precipitate, take 1mL of the supernatant and centrifuge at high speed (4°C, 58000g, 1h) to discard the supernatant, add 200 μL of Suspended in 0.01M PBS to obtain phage suspension. Use a pipette gun to take a drop of phage suspension onto the copper grid, let it stand for 10 minutes, and then use neutral filter paper to absorb excess water from the side. Drop a drop of 3% uranyl acetate on the copper grid, and after staining for 20 seconds, use neutral filter paper to absorb the staining agent from the side. After standing for 10min to dry, the morphology of phage was observed with a transmission electron microscope (Hitachi H-7650).

[0048] The result is as figure 2 As shown, the phage vB_EtaM-IME523 has a head with an icosahedral approximately spherical structure, a dia...

Embodiment 3

[0050] Animal protection experiment of bacteriophage vB_EtaM-IME523

[0051] Purchasing healthy zebrafish with a body length of 3-4 cm is used as the experimental object, and the zebrafish is divided into three groups, which are respectively the experimental group, the control group and the blank group. The original concentration of water added in the experimental group and the control group was 5.6×10 9 CFU / mL of Edwardsiella tarda to a final concentration of 1×10 8 CFU / mL, add the original concentration of 2×10 to the water body of the experimental group after three hours 11 PFU / mL of vB_EtaM-IME523 to a final concentration of 1×10 8 PFU / mL; the control group was replaced with the same volume of SM buffer as the phage. The water body of the blank group did not receive any treatment. Feed twice a day, observe and record the condition of zebrafish. On the 8th day of the experiment, Edwardsiella tarda was supplemented once again in the experimental group and the control gr...

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Abstract

The invention discloses an Edwardsiella tarda high-efficiency virulent bacteriophage vBEtaMIME523 and an application thereof, and relates to biological treatment of Edwardsiella tarda pollution and infection. The vB_EtaM-IME523 is preserved in the General Microbiological Culture Collection Center, and the preservation number of the vB_EtaM-IME523 is CGMCC No. 18191. The vB_EtaM-IME523 is providedwith an icosahedron approximately spherical head, the diameter of the icosahedron approximately spherical head is 95nm, the vB_EtaM-IME523 is provided with a shrinkable tail with the length being 125nm; the vB_EtaM-IME523 can form transparent plaque on a bacteria plate of the Edwardsiella tarda, and can be used for cracking the Edwardsiella tarda to clarify bacteria liquid; and the vB_EtaM-IME523has an obvious protection effect on animals. The application of the vB_EtaM-IME523 is used for specifically inhibiting and killing Edwardsiella tarda.

Description

technical field [0001] The invention relates to a phage of pathogenic bacteria, in particular to a highly efficient and virulent phage vB_EtaM-IME523 of Edwardsiella tarda and its application. Background technique [0002] To prevent and control diseases, antibiotics and chemical disinfectants have been used for a long time and widely. However, antibiotics easily induce bacterial resistance and form "super bacteria", which seriously threaten human and animal health; antibiotics and chemical disinfectants are not specific, destroy the normal flora we rely on, damage health and endanger the ecological balance of the environment ; Antibiotics and chemical disinfectants remain in farmed products. After ingestion by the human body, they will affect the intestinal bacterial flora, suppress the immune system, and endanger human health. [0003] Phages are viruses that infect bacteria and fungi. Lytic phages are also called virulent phages or virulent phages. The virulent phage s...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/02A61K35/76A61P31/04C12R1/92
CPCC12N7/00A61K35/76A61P31/04C12N2795/10121C12N2795/10151C12N2795/10132
Inventor 林威李登峰孙智同童贻刚许丽华秦伟南
Owner NINGBO UNIV
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