CN112481221A

CN112481221A - Edwardsiella tarda efficient lytic phage vB _ EtaM-IME523 and application thereof

Info

Publication number: CN112481221A
Application number: CN201910885029.5A
Authority: CN
Inventors: 林威; 李登峰; 孙智同; 童贻刚; 许丽华; 秦伟南
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2019-09-10
Filing date: 2019-09-10
Publication date: 2021-03-12

Abstract

The invention discloses an efficient virulent phage vB _ EtaM-IME523 of Edwardsiella tarda and application thereof, and relates to biological treatment of Edwardsiella tarda pollution and infection. The vB _ EtaM-IME523 is preserved in the common microorganism center of the microorganism culture preservation management committee, and the preservation number is CGMCC No. 18191. vB _ EtaM-IME523 has a head exhibiting an icosahedral approximately spherical shape, a diameter of about 95nm, a contractible tail, a length of about 125 nm; the vB _ EtaM-IME523 can form transparent plaques on the Edwardsiella tarda bacterial plate and can crack the Edwardsiella tarda to clarify bacterial liquid; vB _ EtaM-IME523 has obvious protective effect on animals. Use of vB EtaM-IME523 for specifically inhibiting, killing, tardive edwardsies.

Description

Edwardsiella tarda efficient lytic phage vB _ EtaM-IME523 and application thereof

Technical Field

The invention relates to a bacteriophage of pathogenic bacteria, in particular to an Edwardsiella tarda high-efficiency virulent bacteriophage vB _ EtaM-IME523 and application thereof.

Background

Antibiotics and chemical disinfectants have long been widely used for the prevention and control of diseases. However, antibiotics tend to induce bacterial resistance, forming "superbacteria" that seriously threaten human and animal health; antibiotics and chemical disinfectants have no specificity, destroy normal flora which we depend on, damage health and endanger ecological balance of the environment; antibiotics and chemical disinfectants remain in the cultured products, and after being ingested by human bodies, the antibiotics and the chemical disinfectants affect intestinal bacterial communities, inhibit immune systems and harm human health.

Phage (phase) is a virus that infects bacteria, fungi. Lytic phages are also known as virulent phages or virulent phage (virophage). The virulent phage specifically infects and cracks target bacteria, does not infect human, animals and plants, does not pollute normal microbial communities and the environment, has host dependence, dies along with host elimination, and does not remain in animals. The virulent phage has high safety, is the most potential antibiotic substitute, and has great potential and advantage in the research and development of antibacterial drugs.

Edwardsiella tarda (Edwardsiella tarda) is a representative bacterium of Enterobacteriaceae (Enterobacteriaceae) and Edwardsiella (Edwardsiella), is a gram-negative facultative anaerobe, is widely distributed, is a pathogenic bacterium of a human-animal-fish common pathogenic condition, can cause infection and morbidity of various mammals, fish, shrimps, crabs, soft-shelled turtles and the like, can cause diarrhea, septicemia, lumbar and epidural abscess and the like, and even can cause death. Feeding edwardsiella tarda contaminated water products can cause severe food-borne infections. In recent years, fish diseases caused by Edwardsiella tarda are frequently seen, and in aquaculture, the diseases caused by Edwardsiella tarda are frequently outbreaked and prevalent in many provinces of China, so that huge economic losses are caused to farmers. The development of the phage capable of efficiently cracking Edwardsiella tarda has important practical significance.

Disclosure of Invention

The invention aims to solve the technical problem of providing a novel phage capable of efficiently and quickly cracking Edwardsiella tarda and application thereof. The bacteriophage specifically infects and cracks Edwardsiella tarda, and has protective effect on animals.

The technical scheme adopted by the invention for solving the technical problems is as follows: a novel lytic bacteriophage which is obtained by separating and specifically infects Edwardsiella tarda and takes pathogenicity Edwardsiella tarda as a target is named as vB _ EtaM-IME523(vB _ EtaM is the abbreviation of Virus of bacteria, Edwards diella tarda, Myoviridae; IME523 is a strain number) according to the bacteriophage naming principle, and belongs to Myoviridae in the Myoviridae in classification. vB _ EtaM-IME523 is preserved in the general microbiological center of the culture Collection of microorganisms in 2019, 7 months and 10 days, the preservation number is CGMCC No.18191, and the preservation organization address is as follows: the microbial research institute of

western road

1, 3, national academy of sciences, north-south, morning-yang, Beijing, zip code: 100101. the phage vB _ EtaM-IME523 has a protective effect on animals, and the relative protection rate on zebra fish is 46.7%.

The biological characteristics of the phage were as follows: phage vB _ EtaM-IME523 has a head exhibiting an icosahedral approximately spherical shape, a diameter of about 95nm, a contractible tail, a length of about 125 nm; the phage vB _ EtaM-IME523 can form transparent plaques on the bacterium plate of the Edwardsiella tarda; the bacteriophage vB _ EtaM-IME523 can crack the Edwardsiella tarda and clarify the bacterial liquid; the host range of vB _ EtaM-IME523 is species specific.

The method for separating and purifying the phage vB _ EtaM-IME523 specifically comprises the following steps:

(1) activation and culture of Edwardsiella tarda

Edwardsiella tarda is derived from the institute of epidemiology, military medical academy of sciences. Edwardsiella tarda strains were streaked on LB solid medium plates containing 1.5% (W/V) agar and cultured overnight at 37 ℃ in an inverted state. Single colonies were picked from the plate, inoculated into a tube containing 5mL of LB liquid medium, and cultured on a shaker (37 ℃ C., 220rpm) for 12 hours. 1mL of the liquid culture medium was diluted 1: 100(V/V) from the test tube into a conical flask containing 100mL of LB liquid medium, and placed on a shaker (37 ℃ C.)220rpm) to the bacterial liquid OD₆₀₀And (5) the concentration is approximately equal to 0.5, and the logarithmic phase bacterial liquid is obtained.

(2) Enrichment of phages

Phage vB _ EtaM-IME523 was isolated from the fifth medical center wastewater of the general Hospital of the people's liberation force, Beijing, China (North laterude: 39.8622002; East longrude: 116.2957407). Filtering 5L of sewage by using neutral filter paper, centrifuging at 12000rpm for 5min, and collecting supernatant; filtering the supernatant with 0.22 μm filter, loading into 30kD dialysis bag, spreading PEG8000 around the dialysis bag, concentrating, and collecting the residual liquid in the dialysis bag after overnight; adding 200 μ L of the liquid into 5mL of Edwardsiella tarda cultured to logarithmic phase with LB liquid medium, culturing at 37 deg.C and 220rpm overnight; centrifuging the mixed culture solution at 12000rpm for 2min, collecting supernatant, and filtering bacteria with 0.22 μm filter to obtain filtrate as stock solution after phage enrichment.

Taking a conventional LB solid culture medium (containing 1.5% of agar) plate as a lower plate, adding 500 mu L of Edwardsiella tarda cultured to logarithmic phase into an upper semi-solid LB culture medium (containing 0.7% of agar) at 42-45 ℃, immediately mixing uniformly, pouring onto the lower plate, and after the upper plate is solidified. Dripping stock solution after phage enrichment into a corresponding double-layer flat plate, and dripping 2 mu L of stock solution into each zone; control zones were titrated with PBS as a control. After the drops were absorbed by the medium, the double-layered plate was inverted and incubated in a 37 ℃ incubator for 9 hours to observe and confirm the appearance of the phage loop.

(3) Purification culture of bacteriophage

100 μ L of phage-enriched stock confirmed to result in phage loop was diluted to 8 drops in PBS buffer gradient (10%^-1～10^-8) 100. mu.L of each dilution was incubated with 500. mu.L of each dilution to logarithmic phase (OD)₆₀₀About 0.5), standing and incubating for 10min, then respectively adding the mixed liquid into 5mL of upper layer semi-solid LB culture medium at 42-45 ℃, immediately mixing and containing a culture dish of the lower layer solid LB culture medium, and inverting the culture dish to culture for 9h at 37 ℃ after solidification; picking from double-layer flat plate with uniformly distributed plaques by using a cut sterile gun headThe plaques with the difference in morphology and size are respectively inoculated into 5mL of Edwardsiella tarda cultured to logarithmic phase, and are cultured overnight at 37 ℃ and 220 rpm; centrifuging the overnight culture solution at 12000rpm for 2min, collecting supernatant, and filtering with 0.22 μm filter; and (3) repeating the double-layer flat plaque test on the filtrate, obtaining plaques with uniform shape and size after the third generation, centrifuging the mixed culture solution of the third generation plaques and the logarithmic phase Edwardsiella tarda liquid at 12000rpm for 2min, filtering the supernatant by a 0.22-micrometer filter, and obtaining the filtrate which is the purified and cultured phage suspension.

(4) Amplification culture of bacteriophage

Taking Edwardsiella tarda monoclonal colonies into a liquid LB culture medium, and culturing at 37 ℃ and 220rpm until logarithmic phase; then inoculating 100 mu L of the bacterial liquid into 5mL of LB liquid culture medium, and inoculating 3 tubes in total, marking as 1, 2, 3, 37 ℃, and culturing at 220rpm until logarithmic phase; adding the bacterial liquid of the tube 1 into 1000mL of freshly prepared sterile LB liquid medium, culturing at 37 ℃ and 220rpm until logarithmic phase; inoculating 500 μ L of phage suspension purified and cultured in step (3) into tube 2, culturing at 37 deg.C and 220rpm for 5 h; tube 3 is a control group of tube 2, 500 mul LB culture medium is added, and the culture is carried out for 5h at 37 ℃ and 220rpm, which is used for judging the cracking degree of the bacterial liquid in tube 2; the culture solution in the tube 2 is subpackaged in a centrifuge tube, centrifuged at 12000rpm for 2min to collect supernatant, added into 1000mL of host bacteria solution cultured to logarithmic phase, and cultured at 37 ℃ and 220rpm overnight.

(5) Concentration and purification of phages

Centrifuging 1000mL of the phage lysate prepared in step (4) at 12000rpm for 2min, collecting the supernatant, and filtering with a 0.22 μm filter; 25mL of the filtrate was added to each 50mL ultracentrifuge tube (Beckman Co.), 8mL of 20% (w/v) sucrose solution was slowly added from the bottom using a syringe, 4mL of 50% (w/v) sucrose solution was slowly added from the bottom, and the mixture was centrifuged at 27500rpm/min for 3 hours at 4 ℃; sucking the phage in the centrifuge tube to precipitate into a dialysis bag of 100kD, and dialyzing for 12h by using SM buffer solution to remove sucrose; collecting dialysate, adding 20% and 50% sucrose into a new 50mL ultracentrifuge tube, centrifuging, collecting phage, precipitating into 100kD dialysis bag, dialyzing with SM buffer solution for 12 hr to remove sucrose, and repeating twice; collecting the dialysate of the last dialysis to an ultrafiltration tube of 30kD, centrifuging at 3500rpm for 10min, and collecting the upper layer liquid, i.e. the concentrated and purified bacteriophage.

The application of the lytic phage vB _ EtaM-IME523 is used for inhibiting the growth of Edwardsiella tarda and killing the Edwardsiella tarda.

Compared with the prior art, the invention has the advantages that: the invention discloses an Edwardsiella tarda lytic phage vB _ EtaM-IME523 and a separation method and application thereof, wherein the genome sequence of the vB _ EtaM-IME523 does not exist in an existing database and is a new unreported phage; the virus has high specificity, specifically infects and lyses Edwardsiella tarda, which is an important precondition for ensuring ecological safety; the large-scale culture and purification of the lytic virus vB _ EtaM-IME523 are easy, the operation is simple, the cost is low, and the environmental pollution is avoided; vB _ EtaM-IME523 has obvious protective effect on animals.

In conclusion, the invention provides a novel Edwardsiella tarda lytic phage vB _ EtaM-IME523 and a separation method and application thereof, and the phage can efficiently, rapidly and specifically infect and kill Edwardsiella tarda and has obvious protective effect on animals.

Drawings

FIG. 1 shows the plaque morphology of bacteriophage vB _ EtaM-IME523 on double-layer plates of Edwardsiella tarda

FIG. 2 shows control bacterial liquid of Edwardsiella tarda in the left test tube, and bacterial liquid in the right test tube is clarified by adding phage vB _ EtaM-IME523

FIG. 3 is a transmission electron micrograph of negatively stained phage vB _ EtaM-IME523

FIG. 4 shows zebrafish in the experimental group and the control group of animal protection, wherein the upper fish belongs to the experimental group added with the phage vB _ EtaM-IME 523; the fish in the lower part belong to the control group and present swelling due to edwardsiella tarda infection.

FIG. 5 is a line graph showing cumulative mortality of groups of zebrafish in animal protection experiments

Detailed Description

The invention is described in further detail below with reference to the accompanying examples

Example 1

Separation and purification of phage vB _ EtaM-IME523

(1) Activation and culture of Edwardsiella tarda

Edwardsiella tarda strains are streaked and inoculated on LB solid medium plates containing 1.5% (W/V) agar, and the plates are placed upside down at 37 ℃ for overnight culture. Single colonies were picked from the plate, inoculated into a tube containing 5mL of LB liquid medium, and cultured on a shaker (37 ℃ C., 220rpm) for 12 hours. Taking 1mL of liquid culture medium from the test tube, diluting 1: 100(V/V) to a conical flask containing 100mL of LB liquid culture medium, placing on a shaking table (37 ℃, 220rpm) for amplification culture until the OD of the bacterial liquid₆₀₀And (5) the concentration is approximately equal to 0.5, and the logarithmic phase bacterial liquid is obtained.

(2) Enrichment of phages

Taking a conventional LB solid culture medium (containing 1.5% of agar) plate as a lower plate, adding 500 mu L of Edwardsiella tarda cultured to logarithmic phase into an upper semi-solid LB culture medium (containing 0.7% of agar) at 42-45 ℃, immediately mixing uniformly, pouring onto the lower plate, and after the upper plate is solidified. Dripping stock solution after phage enrichment into a corresponding double-layer flat plate, and dripping 2 mu L of stock solution into each zone; the control was performed by dropping 0.01M PBS on the individual control area. After the drops were absorbed by the medium, the double-layered plate was inverted and incubated in a 37 ℃ incubator for 9 hours to observe and confirm the appearance of the phage loop.

(3) Purification culture of bacteriophage

100 μ L of the enriched stock, which was confirmed to be phage causing phage loops, was diluted to 8 titers (10) with a 0.01M PBS buffer gradient^-1～10^-8) 100. mu.L of each dilution was incubated with 500. mu.L of each dilution to logarithmic phase (OD)₆₀₀About 0.5), standing and incubating for 10min, then respectively adding the mixed liquid into 5mL of upper-layer semisolid LB culture medium at 42-45 ℃, immediately mixing and containing a culture dish of the lower-layer solid LB culture medium, and inverting the culture dish to culture for 9h in a 37 ℃ culture box after solidification; from a double-layer plate with evenly distributed plaques, the plaques with different shapes and sizes are picked by a cut aseptic gun head and respectively inoculated into 5mL of Edwardsiella tarda cultured to logarithmic phase, and are cultured overnight at 37 ℃ and 220 rpm; centrifuging the overnight culture solution at 12000rpm for 2min, collecting supernatant, and filtering with 0.22 μm filter; and (3) repeating the double-layer flat plaque test on the filtrate, obtaining plaques with uniform shape and size after the third generation, centrifuging the mixed culture solution of the third generation plaques and the logarithmic phase Edwardsiella tarda liquid at 12000rpm for 2min, filtering the supernatant by a 0.22-micrometer filter, and obtaining the filtrate which is the purified and cultured phage suspension.

(4) Amplification culture of bacteriophage

(5) Concentration and purification of phages

Centrifuging 1000mL of the phage lysate prepared in step (4) at 12000rpm for 2min, collecting the supernatant, and filtering with a 0.22 μm filter; 25mL of the filtrate was added to each 50mL ultracentrifuge tube (Beckman Co.), 8mL of 20% (w/v) sucrose solution was slowly added from the bottom using a syringe, 4mL of 50% (w/v) sucrose solution was slowly added from the bottom, and the mixture was centrifuged at 27500rpm/min for 3 hours at 4 ℃; sucking the phage in the centrifuge tube to precipitate into a dialysis bag of 100kD, and dialyzing for 12h by using SM buffer solution to remove sucrose; collecting dialysate, adding into a new ultracentrifuge tube with weight of 50mL, adding 20% and 50% sucrose from the bottom of the centrifuge tube, centrifuging, collecting phage layer, and performing liquid dialysis twice; collecting the dialysate of the last dialysis to an ultrafiltration tube of 30kD, centrifuging at 3500rpm for 10min, and collecting the upper layer liquid, i.e. the concentrated and purified bacteriophage.

The purified phage vB _ EtaM-IME523 is mixed with Edwardsiella tarda infection in the logarithmic growth phase to carry out a plaque experiment, so that transparent round plaques can be obtained, no halo is formed around the plaques, and the edges are clear and regular (figure 1). After the phage vB _ EtaM-IME523 was added to the Edwardsiella tarda bacterial solution, the bacterial lysate became clear (FIG. 2).

The LB culture medium comprises the following formula: 10g of tryptone, 5g of yeast extract and 10g of NaCl, diluting to 1L with distilled water, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20min under high pressure; the formulation of SM buffer was as follows: 5.8g of sodium chloride, 2.0g of magnesium sulfate, 50mL of 1mol/L Tris-HCl and 5mL of 2% gelatin, adding water to 1L, adjusting the pH value to 7.5, and autoclaving at 121 ℃ for 20 min; the 0.01M PBS formulation was as follows: NaCl 8g, KCl 0.2g, Na₂HPO₄·12H₂O 2.9g、KH₂PO₄0.2g, water is added to 1L, pH7.4, and autoclaving is carried out at 121 ℃ for 20 min.

The purified phage is preserved in China general microbiological culture Collection center with the preservation number: CGMCC No.18191, preservation date of 2019, 7 months and 10 days, preservation unit address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North Chen, zip code 100101.

Example 2

Morphological Observation of phage vB _ EtaM-IME523

The phage-bacteria culture lysate separated and purified in example 1 was centrifuged at a low speed (4 ℃, 12000g, 15min) to remove the precipitate, 1mL of the supernatant was centrifuged at a high speed (4 ℃, 58000g, 1h) to remove the supernatant, and the precipitate was suspended in 200. mu.L of 0.01M PBS to obtain a phage suspension. A drop of phage suspension was applied to a copper mesh using a pipette gun, left to stand for 10min and excess water was sucked off laterally with neutral filter paper. A drop of 3% uranyl acetate was placed on the copper mesh and after staining for 20s, the stain was sucked off laterally with neutral filter paper. Standing for 10min, air drying, and observing phage morphology with transmission electron microscope (Hitachi H-7650).

As a result, as shown in FIG. 2, the phage vB _ EtaM-IME523 has a head exhibiting an icosahedral approximately spherical structure, a diameter of about 95nm, and a tail length of about 125 nm.

Example 3

Protection experiment of phage vB _ EtaM-IME523 on animals

Healthy zebra fish with a body length of 3-4 cm is purchased as an experimental subject, and the zebra fish is divided into three groups, namely an experimental group, a control group and a blank group. The original water addition concentration of the experimental group and the control group is 5.6 multiplied by 10⁹CFU/mL Edwardsiella tarda to a final concentration of 1X 10⁸CFU/mL, three hours later, the original concentration of 2 x 10 was added to the water in the experimental group¹¹PFU/mL vB _ EtaM-IME523 at a final concentration of 1X 10⁸PFU/mL; the control group was replaced with an equal volume of SM buffer as the phage. The blank water body was not treated. The zebra fish is fed twice a day and observed and recorded. The experiment was carried out to day 8 by supplementing the test group and the control group with Edwardsiella tarda once more and the test group with phage once again in the same amount as the initial one.

The blank group of experimental fish was as usual. By the end of the experiment by day 12, the cumulative mortality of the control group was 100% and the cumulative mortality of the experimental group was 53.3% (table 1; fig. 5). The cumulative mortality rate of the experimental group is 46.7% less than that of the control group, namely the relative protection rate of the phage vB _ EtaM-IME523 on the zebra fish is 46.7%.

TABLE 1 survival number of Zebra fish in animal protection experiment with bacteriophage vB _ EtaM-IME523

The results show that the bacteriophage vB _ EtaM-IME523 has obvious protection effect on the zebra fish infected by Edwardsiella tarda.

Example 4

Host Range assay for phage vB _ EtaM-IME523

Edwardsiella tarda and other strains to be tested (see Table 2 for details) were cultured to logarithmic phase (OD)₆₀₀0.6). In this case, marine-origin bacteria (see right column in Table 2) were cultured in LB seawater, and terrestrial-origin bacteria (see left column in Table 2) were cultured in LB seawater. The logarithmic phase bacteria liquid and bacteriophage vB _ EtaM-IME523 suspension are mixed according to the volume of 100: 1 respectively to be used as an experimental group, a control group replaces the bacteriophage with corresponding culture medium, each group is provided with two parallel groups, and each group is cultured on a shaking table overnight. Wherein the culture conditions of marine origin bacteria are 29 ℃ and 180 rpm; the culture conditions of terrestrial bacteria were 37 ℃ and 220 rpm. After overnight incubation, the OD of each group was measured with a microplate reader₆₀₀And taking the average value of the parallel groups, and calculating the ratio of the average value of the control group to the average value of the experimental group. If the ratio is more than 1.2, the phage can infect the bacterium, and the result is positive; otherwise, the phage is considered to be incapable of infecting the bacterium and is a negative result; observing infection and lysis with naked eye and microscope to confirm OD₆₀₀And (4) judging the result. Results vB _ EtaM-IME523 has species specificity and strain specificity for host infection, and only infects and lyses pathogenic Edwardsiella tarda from terrestrial sources.

TABLE 2 host Range assay results for phage vB _ EtaM-IME523

"+" represents infection and "-" represents no infection

The LB seawater culture medium comprises the following formula: 10g of tryptone and 5g of yeast extract, adding filtered seawater to a constant volume of 1L, adjusting the pH value to 7.2, and carrying out autoclaving at 121 ℃ for 20 min.

The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

1. A virulent phage vB _ EtaM-IME523 of Edwardsiella tarda is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 7 months and 10 days, and the preservation number is CGMCC No. 18191.

2. A virulent phage vB _ EtaM-IME523 of edwardsiella tarda according to claim 1, having the following biological characteristics: the phage obtained by taking pathogenic Edwardsiella tarda as a target and being separated is named as vB _ EtaM-IME 523; vB _ EtaM-IME523 clarified pathogenic Edwardsiella tarda bacterial liquid to form transparent plaques on the bacterial plate.

3. A virulent phage vB _ EtaM-IME523 of edwardsiella tarda according to claim 1, having the following biological characteristics: presents an icosahedral approximately spherical head, 95nm in diameter, and a contractible tail, 125nm in length.

4. A virulent phage vB _ EtaM-IME523 of edwardsiella tarda according to claim 1, having the following biological characteristics: the sewage is separated from the fifth medical center of general hospital of people liberation force of China, Beijing, and is positioned as North latitude: 39.8622002, East longituude: 116.2957407, respectively; the preparation method comprises the following steps:

(1) activation and culture of Edwardsiella tarda

Taking Edwardsiella tarda strains to streak and inoculate on an LB solid medium plate containing 1.5 percent agar, and carrying out inverted culture at 37 ℃ for overnight; picking single colony from the plate, inoculating to a test tube filled with 5mL LB liquid culture medium, and culturing for 12 hours at 37 ℃ and 220rpm on a shaker; taking 1mL of culture solution from the test tube, diluting the culture solution into a conical flask filled with 100mL of LB liquid culture medium, placing the conical flask on a shaker at 37 ℃ and 220rpm, and culturing until the bacterial solution OD₆₀₀Approximately closing to 0.5 to obtain logarithmic phase bacterial liquid;

the LB medium used therein has the following formulation: 10g of tryptone, 5g of yeast extract and 10g of NaCl, diluting to 1L with distilled water, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20min under high pressure;

(2) enrichment of phages

Taking sewage of the fifth medical center of China general Hospital of the liberation force of people in Beijing, performing filtration treatment by using neutral filter paper, centrifuging at 12000rpm for 5min, and collecting supernatant; filtering the supernatant with 0.22 μm filter, loading into 30kD dialysis bag, spreading PEG8000 around the dialysis bag, concentrating, and collecting the residual liquid in the dialysis bag after overnight; adding 200 μ L of the liquid into 5mL of Edwardsiella tarda cultured in LB liquid medium to logarithmic phase, culturing at 37 deg.C and 220rpm overnight; centrifuging the mixed culture solution at 12000rpm for 2min, collecting supernatant, and filtering the supernatant with 0.22 μm filter to obtain filtrate as stock solution after phage enrichment;

taking a conventional LB solid culture medium flat plate containing 1.5% of agar as a lower flat plate, adding 500 mu L of Edwardsiella tarda cultured to logarithmic phase into a semisolid LB culture medium containing 0.7% of agar at 42-45 ℃, immediately mixing uniformly and pouring onto the lower flat plate, after the upper flat plate is solidified, dripping stock solution after enrichment of phage into a corresponding double-layer flat plate, and dripping 2 mu L of stock solution into each flat plate; control zones were titrated with 0.01M PBS as a control. After the dropping liquid is absorbed by the culture medium, the double-layer flat plate is inverted and placed in a constant-temperature incubator at 37 ℃ for culturing for 9 hours, and the appearance of the phage ring is observed and determined;

the formulation of 0.01M PBS was as follows: NaCl 8g, KCl 0.2g, Na₂HPO₄·12H₂O 2.9g、KH₂PO₄0.2g, adding water to 1L, pH7.4, and autoclaving at 121 deg.C for 20 min;

(3) purification culture of bacteriophage

100 μ L of the enriched stock solution of phage confirmed to cause phage loop was diluted to 8 dilutions (10) with a 0.01M PBS buffer gradient^-1～10^-8) Uniformly mixing 100 mu L of diluent and 500 mu L of Edwardsiella tarda cultured to logarithmic phase, standing and incubating for 10min, respectively adding the mixed liquid into 5mL of 42-45 ℃ upper-layer semisolid LB culture medium, immediately mixing uniformly, pouring into a culture dish containing a lower-layer solid LB culture medium, and after solidification, inverting the culture dish in a 37 ℃ culture box for culturing for 9 h; from a double-layer plate with evenly distributed plaques, picking the plaques by using a cut sterile gun head, respectively inoculating the plaques into 5mL of Edwardsiella tarda cultured to logarithmic phase, and culturing at 37 ℃ overnight at 220 rpm; centrifuging the overnight culture solution at 12000rpm for 2min, collecting supernatant, and filtering with 0.22 μm filter; and (3) repeating the double-layer flat plaque test on the filtrate, obtaining plaques with uniform shape and size after the third generation, centrifuging the mixed culture solution of the third generation plaques and the logarithmic phase Edwardsiella tarda liquid at 12000rpm for 2min, filtering the supernatant by a 0.22-micrometer filter, and obtaining the filtrate which is the purified and cultured phage suspension.

(4) Amplification culture of bacteriophage

Taking Edwardsiella tarda monoclonal colonies into a liquid LB culture medium, and culturing at 37 ℃ and 220rpm until logarithmic phase; then inoculating 100 mu L of the bacterial liquid into 5mL of LB liquid culture medium, and inoculating 3 tubes in total, marking as 1, 2, 3, 37 ℃, and culturing at 220rpm until logarithmic phase; adding the bacterial liquid of the tube 1 into 1000mL of freshly prepared sterile LB liquid medium, culturing at 37 ℃ and 220rpm until logarithmic phase; inoculating 500 μ L of phage suspension purified and cultured in step (3) into tube 2, culturing at 37 deg.C and 220rpm for 5 h; tube 3 is a control group of tube 2, 500 mul LB culture medium is added, and the culture is carried out for 5h at 37 ℃ and 220rpm, which is used for judging the cracking degree of the bacterial liquid in tube 2; the culture solution in the tube 2 is subpackaged in a centrifuge tube, centrifuged at 12000rpm for 2min to collect supernatant, added into 1000mL of host bacteria solution cultured to logarithmic phase, and cultured overnight at 37 ℃ and 220 rpm;

(5) concentration and purification of phages

Centrifuging 1000mL of the phage lysate prepared in step (4) at 12000rpm for 2min, collecting the supernatant, and filtering with a 0.22 μm filter; adding 25mL of the filtrate into each 50mL Beckman ultracentrifuge tube, slowly adding 8mL of 20% sucrose solution from the bottom by using a syringe, slowly adding 4mL of 50% sucrose solution from the bottom, and centrifuging at 4 ℃ and 27500rpm/min for 3 h; sucking the phage precipitate in the centrifuge tube into a dialysis bag of 100kD, and dialyzing for 12h by using SM buffer solution to remove sucrose; collecting dialysate, adding 20% and 50% sucrose into a new 50mL ultracentrifuge tube, centrifuging, collecting phage, precipitating into 100kD dialysis bag, dialyzing with SM buffer solution for 12 hr to remove sucrose, and repeating twice; collecting the dialysate of the last dialysis to an ultrafiltration tube with 30kD, centrifuging at 3500rpm for 10min, and collecting the upper layer liquid, namely the concentrated and purified phage vB _ EtaM-IME 523;

the SM buffer solution is prepared according to the following formula: 5.8g of sodium chloride, 2.0g of magnesium sulfate, 50mL of 1mol/L Tris-HCl and 5mL of 2% gelatin, adding water to 1L, adjusting the pH value to 7.5, and autoclaving at 121 ℃ for 20 min.

5. The use of a virulent phage vB _ EtaM-IME523 against edwardsiella tarda according to claim 1, wherein the host has species and strain specificity, specifically infects and lyses highly pathogenic edwardsiella tarda.

6. The use of a virulent phage vB _ EtaM-IME523 against Edwardsiella tarda according to claim 5, characterized by strong lytic action against highly pathogenic Edwardsiella tarda, for inhibiting and killing highly pathogenic Edwardsiella tarda.

7. Use of a virulent phage vB _ EtaM-IME523 according to any one of claims 1 to 6, characterized by protective effect on animals, in particular portunus trituberculatus.

8. Use of a virulent phage vB EtaM-IME523 according to any one of claims 1-7 of edwardsiella tarda for the preparation of, but not limited to, biologicals for disease control and disinfection of food products, including aquatic products, production environments or production equipment.