CN114774371A - Vibrio phage and application thereof - Google Patents

Abstract

The invention provides a vibrio phage and application thereof, belonging to the field of marine biological engineering. The phage is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of: CCTCC NO: m2022200, deposit date: 04.03/2022, with a deposit address of: wuhan university in Wuhan City, China, zip code: 430072, classified and namedVibrio neocaledonicusphase vB _ Vne-09SG 01. The phage provided by the invention is used for the new Vibrio harveyivibrio neocaledonicusHas quick and lasting killing effect, and can obviously improve the food intake of the infected shellfish, thereby being applied to shellfish death caused by vibrio as a medicament and providing important value in aquaculture.

Description

Vibrio bacteriophage and application thereof

Technical Field

The invention relates to the field of marine bioengineering, in particular to a vibrio phage and application thereof.

Background

The aquaculture industry is one of the important agricultural activities in China, can provide high-quality food for China, contributes about 30% to total protein, and provides a large amount of raw materials for the development of industries such as medicine, chemical engineering, feed and the like.

In aquaculture activities, vibrio diseases are one of the important disasters of aquaculture. Vibrio is a gram-negative, short rod-shaped bacterium with polar flagellum, motion, no spore and capsule, wherein some Vibrio has certain bending and is arc-shaped or comma-shaped. Part of vibrios are important pathogenic bacteria of various marine organisms, and the caused vibriosis has wide epidemic area and high morbidity. At present, dozens of vibrios which have pathogenicity on aquaculture fishes and shrimps are found, mainly including vibrio parahaemolyticus, vibrio alginolyticus, vibrio anguillarum, vibrio harveyi and the like, and meanwhile, part of the vibrios have pathogenicity on mammals, particularly human beings. Vibriosis has become the leading road barrage of diseases in aquaculture industry, even to the extent of "talking about arc discoloration". Vibrio exists for a long time in aquaculture, and the Vibrio is controlled in a certain amount without treatment, but if the Vibrio is influenced by environment, aquatic weeds and harmful bacteria, the Vibrio can be stimulated to rapidly propagate, pollute water and damage cultured organisms.

Taking 2020 spring as an example, the prawn seedling field in China has a serious glass seedling phenomenon, the disease firstly presents an explosive trend in the south seedling field and gradually spreads in the first generation of coastal areas in the north, and the supply of the prawn seedlings in spring in China is seriously insufficient. Month 4-6, middleThe national institute of aquatic science and research on yellow sea aquatic products detects 10 units of aquaculture water and shrimp larvae pancreatic tissue in 5 nursery sites to find out new Vibrio carlidiensisvibrio neocaledonicus) The detection rate is highest, the pathogenicity is very strong as shown by indoor artificial infection, and the compound has obvious drug resistance to common antibiotics such as rifampicin, pipemidic acid, streptomycin, polymyxin, penicillin, compound sulfamethoxazole and the like.

The control of vibrio diseases is always the focus of aquatic product research. The emergence and prevalence of drug-resistant bacteria due to antibiotic abuse, and in particular the recent years the emergence of some "superbugs" that are insensitive to almost all antibiotics, resulting in the infection of humans and animals with such bacteria that is almost drug-free, and there is a strong pressing need for new anti-infective weapons to replace antibiotics. In this context, humans aim at phage therapy. Bacteriophage has received increasing attention as an emerging probiotic antibacterial agent because of its potential to replace antibiotics to treat disease. Many studies have shown that the phage has a good effect in treating vibriosis, for example, the survival rate of the group of young shrimps treated with the phage in the presence of vibrio harveyi is increased from 10% to 40% compared with that of the blank control group; stalin and other researches prove that the application of the phage in an aquaculture system can be used as an effective mode for preventing and treating the vibrio parahaemolyticus disease of the prawns; li and the like use a 'cocktail therapy' to mix 3 strains of bacteriophage in equal proportion and the effect of treating the vibrio splendidus is superior to that of a single strain; the bacteriophage researched by Sasikala and the like can crack 6 strains of vibrio alginolyticus and can effectively control the number of the vibrio alginolyticus. Moreover, the bacteriophage has strict host specificity, does not infect organisms of other species (including farmed organisms), and therefore does not die of organisms other than the infected object, and thus has specificity and safety.

Disclosure of Invention

One of the objects of the present invention is to provide a vibrio phage; the other purpose is to provide the application of the bacteriophage so as to make up the defects of the prior art.

In order to achieve the technical purpose, the invention adopts the technical scheme that:

the vibrio phage is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number as follows: CCTCC NO: m2022200, deposit date: 03/04/2022, with the preservation address: wuhan university in Wuhan City, China, zip code: 430072, classification nameVibrio neocaledonicus phage vB_Vne-09SG01。

The phage is a strain capable of specifically cracking Vibrio neocarlidinii (V) ((V))Vibrio neocaledonicus) The long-tail phage has a tail length of 141 +/-5 nm and a head of about 81 +/-2 nm of a regular icosahedron; the incubation period of the bacteriophage NF is about 40 min, and the lysis period is about 60 min; the phage can keep the activity relatively stable under the conditions of pH 5-9, 0-33.0 permillage and the temperature less than or equal to 32 ℃.

The phage (vB _ Vne-09SG 01) was at 10⁵ PFU/mL can achieve better sterilization effect, and more than or equal to 90 percent of vibrio can be killed within 6 hours; low temperature storage is recommended, but 54.4% of the phage survived even after 24h treatment at 32 ℃; the optimum pH value is 7-8, which accords with most mariculture conditions; has wide tolerance to salinity, is suitable for being applied under the condition of 26.4-33.0 per mill of salinity, has 52.5 percent of phage survival under the condition of 0 per mill of fresh water, and can be applied to culture environments from estuaries to seawater and the like.

A phage preparation comprises phage (vB _ Vne-09SG 01) and effective amount of total phage is 10 or more⁵ PFU/mL; or mixing the preparation with other agents having Vibrio inhibiting effect.

Wherein, in the preparation, the auxiliary materials are one or more of SM buffer solution, sterile seawater and the like; the SM buffer solution comprises 10 mM Tris-HCl, 100 mM NaCl and 10 mM MgSO₄，pH 7.8。

The method for separating the bacteriophage comprises the following steps:

mixing 0.22 μm seawater with Vibrio cultured to logarithmic phase, culturing for 7 days, and performing phage separation by double-layer plate method on days 1, 3, and 7. And selecting a single plaque, continuously purifying the phage for 5 times by adopting a double-layer plate method, and then enriching to obtain a phage suspension. Then, after gradual amplification culture, multiple centrifugation, PEG 8000 precipitation, cesium chloride density gradient centrifugation purification and staining, the phage is identified as a long-tail phage by adopting transmission electron microscope observation.

The phage (vB _ Vne-09SG 01) can be used for cracking Vibrio neocarviensis: (Vibrio paradisi)vibrio neocaledonicus) The use of (1); can be specifically applied to the prevention and treatment of marine shellfish against Vibrio neocarlidinii ((V))vibrio neocaledonicus) In the infection of (1), or in the application to an aqueous environment, to Vibrio neocarlidinii ((II))vibrio neocaledonicus) And (4) killing.

Further, the bacteriophage (vB _ Vne-09SG 01) and its preparation for Vibrio neocarlidinii (V.V.V.V.sub.09)vibrio neocaledonicus) The final concentration of the pesticide is more than or equal to 10⁵ PFU/mL。

The phage can be used in the preparation of medicaments for pathogenic vibrio, in particular to the preparation of medicaments for diseases caused by vibrio neocarlidinii.

The invention has the beneficial effects that:

(1) the bacteriophage is used for specifically killing Vibrio karlidonii (Vibrio karlidii) capable of causing shellfish death and food intake reductionvibrio neocaledonicus) And the other 2 new species of Vibrio neolyticus can be infected, so that the method has effective application value.

(2) The phage is separated from the seawater environment, the effect is lasting and stable, individual auxiliary reagents also belong to nontoxic and harmless chemicals, the influence on cultivated organisms and the environment is avoided, and the phage is nontoxic, harmless, safe and environment-friendly.

(3) The phage is used for the treatment of Vibrio neocarlidae: (vibrio neocaledonicus) Has quick and lasting killing effect, and can obviously improve the food intake of the infected shellfish, so the vibrio can be used as a medicine for shellfish death and the like caused by the vibrio.

Drawings

FIG. 1 is a schematic plaque drawing of the phage of the example (vB _ Vne-09SG 01).

FIG. 2 is a SEM image of phage (vB _ Vne-09SG 01) in the example.

FIG. 3 is a graph showing the bactericidal effect of the bacteriophage (vB _ Vne-09SG 01) at different concentrations in the examples.

FIG. 4 is a graph showing the tendency of the phage (vB _ Vne-09SG 01) in the examples to survive under different pH conditions.

FIG. 5 is a graph showing the trend of the survival of the phage (vB _ Vne-09SG 01) under different temperature conditions in the examples.

FIG. 6 is a graph showing the survival trend of phage (vB _ Vne-09SG 01) under different salinity conditions in the examples.

FIG. 7 is a graph showing the change of food intake of different treatments when the Vibrio is infected with Japanese scallop in the examples.

Detailed Description

The vibrio phage and its application are described in detail below with reference to the accompanying drawings and examples.

Example 1:

1. phage (vB _ Vne-09SG 01) isolation and purification

Taking diseased scallops as objects, adopting a TCBS culture medium to carry out streak purification, and then adopting a strain hemolysis plate to select suspected pathogenic bacteria with hemolytic activity; the pathogenic and Vibrio neocarlidae (V.neocarlidae) were identified by 16S sequencingvibrio neocaledonicus) The similarity was 100%. Under the condition of 12 ℃, the feeding rate of the comb scallops is reduced by 67 percent, the feeding rate of the bay scallops is reduced by 34 percent, the feeding rate of the chlamys farreri is reduced by 44 percent, and the fatality rate is between 5 and 20 percent.

Mixing 0.22 μm seawater with Vibrio cultured to logarithmic phase, culturing for 7 days, and performing phage separation by double-layer plate method on days 1, 3, and 7. Selecting a single plaque, continuously purifying the phage for 5 times by adopting a double-layer plate method, wherein a double-layer plate photo is shown in figure 1, after the phage is infected for 24 hours, the phage forms a plaque with the average size of 0.20-0.50cm (the average size is 0.75 +/-0.09 cm) on the double-layer plate, the plaque consists of a transparent ring in the middle and a fuzzy ring at the edge, and the edge is relatively regular; as the time of infection increases, plaques grow slowly until the entire plate is eroded.

Taking the purified phage, performing gradual amplification culture, respectively adopting culture medium with volume of 50mL, 100mL and 1L, and measuring bacterial OD with spectrophotometer₆₀₀The value is determined to determine whether lysis occurred or not, the culture conditions are 28-30 ℃ and 160 rmp; when large-volume virus lysis is obtained, a virus supernatant is obtained by adopting a multi-time centrifugation mode (12000 rmp, 5 min); then adding PEG 8000 with final concentration of 10% (w/w) and NaCl with final concentration of 1% (w/w), precipitating at 4 deg.C for more than 12 hr, and centrifuging at low temperature and high speed (10000 g, 45min, 4 deg.C) to obtain precipitate; and (3) removing the supernatant after the centrifugation is finished, adding an appropriate volume of SM buffer solution and an appropriate volume of chloroform, and storing at 4 ℃ and-80 ℃ to obtain the phage storage solution.

Preparation of phage (vB _ Vne-09SG 01) preparation

Taking the obtained phage, detecting its titer by double-layer plate method, wherein the titer should be greater than 10⁹PFU/mL, diluted with SM buffer solution, and the titer of phage in the preparation should be more than or equal to 10⁶ PFU/mL。

In the actual operation process, one or more of sterile seawater and seawater enrichment culture medium can be added into the bacteriophage concentration, as long as the total content is ensured to be more than or equal to 10⁶ PFU/mL, can achieve the purpose of the invention.

Example 2: phage functional genome extraction sequencing

After the phage capsid protein is degraded by DNAse-protease K-phenol chloroform extraction, the phage genome is obtained. Sequencing was performed using the Illumina high throughput sequencing platform NovaSeq 6000.

The sequencing result is compared by NCBI and viptree, in 859 phage genomes in the database, the phage vB _ Vne-09SG01 genome is only relatively high in similarity with the phage Vibrio phase qdvp001, but the coverage of similar sequences is only 47%, and the coverage of similar sequences with other phage genomes is 12% or below, which indicates that the novelty of the phage vB _ Vne-09SG01 is high.

Gene function prediction is carried out by adopting GeneMark to obtain 244 genes, the prediction result of selecting 10 genes is shown in Table 1, and the specific sequence of Gene1-10 is shown in a sequence table.

TABLE 1 alignment of predicted function and similarity of functional genomic Gene1-10

In the above table, the homing endonuclease synthesized by Gene1 is capable of degrading the genome of the host upon viral infection to provide deoxynucleotides for synthesis of the phage genome. Gene 5 and Gene 8 encode proteins involved in phage base assembly, Gene 7 encodes proteins involved in phage tail assembly, and the 3 genes encode proteins that are part of phage capsid protein assembly. Other genomes in the table, such as Gene2, Gene3, Gene4, etc., are homologous to functionally unknown phage sequences in the NCBI database (similarity between 67.14-98.45%), and these functionally unknown genomes also reflect the strong novelty of the phage.

Example 3: phage property test

1. Phage electron microscopy

Vibrio karlidinii (V.) (Vibrio neocaledonicus) The phage is named as vB _ Vne-09SG01, and an electron micrograph of the phage is shown in figure 2, the phage has a polyhedral head and a longer tail, belongs to the family Longidae, and the tail is 141 +/-5 nm long and the head is about 81 +/-2 nm.

2. Sterilization range analysis of phage vB _ Vne-09SG01

Taking 28 strains of diseased abalone, diseased scallop (patinopecten yessoensis, chlamys farreri and bay scallop) and seawater in a diseased area, separating and purifying the strains by adopting a TCBS culture medium, respectively preparing bacterial lawn, dripping 10 mu L of phage solution on the bacterial lawn, culturing for 1-7d by taking the sample application of natural seawater as a contrast, and observing the formation condition of the plaque. As shown in Table 2, the phage vB _ Vne-09SG01 has infectivity on 3 kinds of Vibrio neolyticus including isolated hosts (2 isolated from diseased abalone and 1 isolated from scallop), indicating that the host range of the phage vB _ Vne-09SG01 is relatively wide, and the phage vB _ Vne-09SG01 can be used as a broad-spectrum phage preparation for marine shellfish.

TABLE 2 bacteriocidal spectra of phage preparation vB _ Vne-09SG01

Note: + represents the ability to form significant plaques; -represents failure to form significant plaques.

3. Laboratory bactericidal effect of phage preparation (vB _ Vne-09SG 01)

Different concentrations (10)⁴-10⁹pfu/mL) of phage vB _ Vne-09SG01 agent to Vibrio neocarlidiensis (Vibrio neocarlidiensis) in logarithmic phase of cultureVibrio neocaledonicus) In the culture solution, the ratio of the number of the virus to the number of the bacteria is 10:1, 1:10, 1:100, 1:1000 and 1:10000 respectively, and no virus is added into a Control group (Control). The bacterial concentration OD was determined every 30min₆₀₀The result shows that the cracking ratio of the 10:1 adding group is 32.68 percent at most and the cracking ratio of other experimental groups is 10.24 to 15.45 percent in 30 min; when the time is 360min, the bacterial lysis of all experimental groups reaches more than 89.51 percent; compared with other experimental groups, the ratio of the virus concentration is 1:10000 (the virus concentration is 10)⁴ PFU/mL) was significantly less effective than the other treatment groups, as shown in figure 3, phage preparation vB _ Vne-09SG01 at 10⁵ Better sterilization effect can be achieved by PFU/mL.

4. Temperature, salinity and pH stability of phage preparation (vB _ Vne-09SG 01)

To determine the pH stability of the phage, phage liquid with known concentration was mixed with culture media (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0) with different pH values at a volume ratio of 1:9, mixed well, placed in the dark at 4 ℃ for 24h, and then counted by the double-layer plate method. The results show (as shown in FIGS. 4, 5, 6) that phage (vB _ Vne-09SG 01) exhibited a tendency to reverse bell shape over the pH range of the experiment, stored best at pH 7 and pH 7.5, leaving 46.4% of active phage at pH 10. In an actual culture environment, the pH value is basically between 7 and 8, and the phage preparation can maintain high activity.

To determine the temperature stability of the phages, known concentrations of phage liquid were treated at different temperatures (4-32 ℃) for 24h in the dark and then the phages were counted by the double-layer plate method. The results show that the phage concentration decreased significantly with increasing temperature. It was demonstrated that phage (vB _ Vne-09SG 01) was best stored at low temperature, but 54.4% of phage survived even after 24h treatment at 32 ℃.

In order to determine the salinity stability of the phage, phage liquid with known concentration is mixed with culture medium (0-33 ‰) with different salinity values according to the volume ratio of 1:19, and after mixing, the mixture is placed in a dark place at 4 ℃ for 24h, and then the phage is counted by a double-layer plate method. The result shows that the phage preparation vB _ Vne-09SG01 is stored optimally under the condition of salinity of 29.7 per thousand, the residual active phage is 87.4 percent and 90.0 percent respectively under the conditions of salinity of 26.4 and 33.0 per thousand, and 52.5 percent of phage survive even under the condition of 0 per thousand fresh water. The tolerance of the phage (vB _ Vne-09SG 01) to salinity is wide, and the phage can be applied to culture environments from river mouths to seawater and the like.

Example 3: experiment of specific application

Application example of the phage preparation (vB _ Vne-09SG 01) to Japanese scallop infected at Vibrio

To explore the therapeutic effect of phage preparation vB _ Vne-09SG01, Vibrio neocarlidinii (Vibrio neocarlidii) was simulated under laboratory conditionsVibrio neocaledonicus) Infection of Patinopecten yessoensis. The experiment was set up with 5 treatment groups, the treatments are given in table 3 below. Wherein the final concentration of phage (vB _ Vne-09SG 01) was 10 per addition⁶ PFU/mL, Vibrio of 10⁶ cells/mL, antibiotic (florfenicol, lethal to Vibrio carlidiensis proved by advanced experiments) 0.01 g/L. Selected Japanese scallopIs scallop of 3 years old, the average height of the shell is 74.66 +/-4.94 mm, and the total wet weight is 51.92 +/-10.92 g; the experimental temperature is 12 ℃; the culture water body of each group is 4L; replacing all the culture water at about 9:00 a.m. every day, and adding phage, antibiotics or vibrios after replacement; and measuring the food intake in the afternoon, wherein the fed microalgae is the seawater chlorella solution.

TABLE 3 example experimental setup for the application experiment of the phage preparation vB _ Vne-09SG01

When active vibrio is added at 3d, the food intake of patinopecten yessoensis of the antibiotic addition group and the vibrio addition group is obviously reduced, the reduction ratio is 43.20 percent and 47.37 percent respectively, and the phage (advanced) addition group is basically equal to the control group. At 3-12d, the food intake of the phage-added group is basically consistent with that of the control group, and the food intake of the phage-added group is slightly lower than that of the control group but is obviously higher than that of the other treatment groups; the antibiotic-added group had a higher feed rate at 3-7 d than the Vibrio direct-added group and a lower feed rate at 8-12d than the Vibrio direct-added group, and the results are shown in FIG. 7.

The experimental result shows that the food intake of the Japanese scallops is obviously reduced when the new Vibrio carinii is infected; the damage caused by vibrio infection can be relieved or counteracted by adding the phage or adding the phage in advance; antibiotic addition only slightly ameliorated vibrio infestation, much less effective than phage (vB _ Vne-09SG 01).

The phage (vB _ Vne-09SG 01) obtained by the invention is also suitable for treating and preventing shellfish death caused by other Vibrio neocarlidinii, and can be used as an immune preparation for a long time in the shellfish culture process.

Sequence listing

<110> China oceanic university

<120> Vibrio bacteriophage and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

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<213> phage (vB _ Vne-09SG 01) (Vibrio neolyticus (vB _ Vne-09SG 01))

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atgcaattag tacaagaatg taattggaca gtagaccaaa gaaacttttc aaatcagacc 60

tttaagacac ctaaaggtgg cgttttggag ataaaacaag tatataaagg taattgtggt 120

gttgcacatt ttggattgga atgtagtatc tgctcaaatg atgaagagtt gtttccagaa 180

ggtagtatca tttcagttaa gaaatcctta ctaaaagggc aagttccttg tggttgctct 240

tttaatccta agtggtccga ggaacagaat aaagtaagag tacgtcggtt atgtgaagat 300

aaaggctata tttttcaagg ttggagcgga gaatacaaag gaatccatac ctacctaaaa 360

ttatataatg gaagaacggg taatttttgg gaatcaacaa ctatcaataa cctgttatta 420

ggtcatggtg accctgcaga ggggacagag atagtcatat ctaagagact cctaccagat 480

agatatcaca tagataagtt tataaaagca ggtttcacag aagattataa gttctggaga 540

agcgacaggt tagggaagga taatcgtaaa aggtattggt attatacatg ccctgtctgc 600

tcacaagatg agtatgcaca aaatggtttg tgtaatggta ttttcgaatc ttatgagaac 660

tcattagcga aagggttcta cgcatgtcgg tgcggaagaa attttgacta taataaagaa 720

caatctgaac taagaataaa acttgtatgt gaaagagaag gtttaacttt taattcttgg 780

gagaaggatt atagaggagt agattctaaa tttatctggc attgctctca taaccataaa 840

aacagtacaa gtatctctaa ttttatgcat ggggctagat gtaggaagtg taaggattta 900

aaatcaccta ttaatggtta ttatagtaag agaaaagatg aggatgatat tttatatatt 960

ctgaatttca acggtgagta tattaaggtt ggtagatctt ttgatatgga gaaaaggttt 1020

aaaggtaata gaggattaat tgcaatgtct ggatgtaaaa gggaaaatat agaaatactt 1080

cacttgttta aaggtaaaca tgaagatgtt tatagagaag aacaatggat tcatgaagag 1140

ctagaaaaca gaggttttca atttaaagat gttacatgga gtacagagtt attcagtcta 1200

gattgctatg acactttaaa atacttgtta aaagagacac cgctaaaaga gatcgacctc 1260

agtttactgt ga 1272

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atgatagtgt ctacttacat atcggaatca tctaatccta atggatccaa actgtatatg 60

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gataaaagta ttattaataa taatattgtc catcagaacc cgattgtgaa tataacttgc 180

tttacgggtt ctaatcctat taaaagtttc gatgattctt tagtggggta tgaggactta 240

gatcagaggg taagcaatac gcatcaggtc ttgcttaaat ggtggcaaaa taaaacacag 300

ctctacatct ttaatgagtt catcactttt gataagtacg ttatcacatc ttatcaaccg 360

aaacaatatg aaactaccag tagtatgcaa tttgaactta ccatggagta tttcaggcct 420

gtatcctatg aaaggggtac tttaataact ttcatggatg cttctaaaac aactgactct 480

actgctaaat ctaatcaaag tgacagctct tcaaaggaaa acataagtaa tttatcacag 540

aaagaaaaag tattaaaaac ttacggagag cagtttggta atcttttaaa cttaattgat 600

aaggaggaga caagtggcaa ttag 624

<210> 3

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<213> phage (vB _ Vne-09SG 01) (Vibrio neolyticus (vB _ Vne-09SG 01))

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gtggcaatta gtttaatccc tcttgagcag aatccttacc gatcatacag ggttactctt 60

gacggtgaat catatgatgt tgttgtgcaa tacaatcagc gattaatcaa ccaaacttcc 120

acaaatccta atttccttcc tgaaagtgca gatagtttta caatttctat tgcattgaca 180

gggcgtgatc caattttaaa aactgcattg aaaactaacc gtgacatcct tggtccgtat 240

aaataccgtg aaggatgccc tcaaggaaat ctcatcctac gagatattgc agcagatggt 300

aatcttgttg acggtaagtt gtatgcacca gaacgtgtaa gttatgaagg aataggatcg 360

agatttctac tcttatatga aagcgaggga ta 392

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<213> phage (vB _ Vne-09SG 01) (Vibrio neocaledonicus (vB _ Vne-09SG 01))

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atgagaacag aaatactata tcgattcaca attggtaaac ctttaaagct tgaaaataac 60

ttctttgttc catataaaaa tgaatcattt ccaacaatct ccctcaacga ttactttgac 120

acagaagatc cgtttaacag ttatattttt acacaacatc aaattcaatt caatgtgaac 180

atggattcta gttctaaaat aaactctagt tatgtcacac tgtataacgt tgatgaagaa 240

gtgattaact ttgtcacaac caatcataat aacaatcttg tgtgtatttt agaagctggt 300

gacaatgagc aaggattgaa agaaattttc aaaggtacta tcactgctgc acaaaagatt 360

gatgatacac aagatacaca actcaagatg aatttagcgg atggtgcagt gaatgcaaag 420

aatgctaaaa ctatccgtac ataccctcgt gggacttcat atgaaactat tctacgtgat 480

atgaacaaag atatgaagtt accaatctca agttttgcag gggtggaagg taggttatta 540

aaccctgtta cttttgcagg aagtacacat caaattatgg agaagctatc tgatactctt 600

ggtgtgtctt attccattca gaacggtgtg acaagtatag taccttaccg tagttataaa 660

aaggtagaag tatctattat tacaccaaca tcaggtctta ttggtaatat taagaaaggt 720

gtggatgata gtaagagtgg tcagaaatct gcaagtatgg attcaaatag tatccaattc 780

atgtgtttgc tagatggtgc tttgaaacct aatgaaacag tttatgtgga tgatggcgat 840

attgttggac catataaaat aactagtatt aagttttctg gtgattatga aggtaatgat 900

tggacgtgta gtgttaaggc agcgaaagtt gaaggggtgt tggaataa 948

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<213> phage (vB _ Vne-09SG 01) (Vibrio neolyticus (vB _ Vne-09SG 01))

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atgtcaggag aaggtttaaa agtaacacca aagcaaccac cttatcgtgg aggacttaac 60

tatgctatgg atcacgctat agaagacaaa cttcgttttg ctagaactat ggtaccagct 120

caagtattaa aagttgatta tgataaaagt ttagttaatg taaaaccact aatccgtttc 180

cgctttgata tgactacaga agatgaattt ggtatcgagg aaatccttga agtaccactt 240

attttctcct ctgcaaagcg tggtaccgct aaaatgacat ttcctgtacg agagggcgat 300

gtgggactgc ttctatgctc agaccgtcat acagagagtt tcttagctag tgatggtatt 360

tctgttgtgg atagtggaag tttctctaca ttgggtacag atggttatat caactatatc 420

ggtttcatac cagaaatatt tacaagtgct ataggtgaga gctttgatgc taatgatgtt 480

gtattaaccc acggcagtag tgaaacacga cataagcaag atggaacaat aatttctaaa 540

aacaattcat ccacctgtac gcaatcacct tctggcgagg tttcattggt taatggtaat 600

ggttatataa aacttttagc tgacgggagt gtagatatca atggttttgt tattcaagta 660

aatggagcag caagttcacc agttagtgtt caagcaccta ctattactgc aacgacttca 720

cttactgtca acggtaaaga gatgaatgag cacaaccatg atgcaggaac atatgaagca 780

ggtggtgatc cagtttcagg tacatcagga gatccagttt aa 822

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<212> DNA

<213> phage (vB _ Vne-09SG 01) (Vibrio neolyticus (vB _ Vne-09SG 01))

<400> 6

atggcaaaat ttgatatgtt tattgatgca actggtgtag atgatttaga tttttctaat 60

ggtatagatt tccgttggac agagacaata ttagaatcac taacacaacg tttgcaatta 120

aggtatgagg tttggacagg acagtggggt tataatctac agtttggcac accataccgt 180

gagttaatgc agcaaggttt aaataagcaa cagcttgatg cagagtttat ccgtattgca 240

cttcaagagg aagatgtaac ttctgtcaag attattaact cggttttaaa taacgtaacc 300

cgtcgttatg aaatacaaag tttagaagtt tacacagatg gtgggttatt agaaatacct 360

atttccaacc cttacacaaa aacgaataat tacccagaac cagttgaaat ttcagatttt 420

acattctgtc agaaaacaga ccaagagatt gaagattaca ataaattata tgagtttatt 480

aacttcacgg ggttgccaga gtttggtgac agtacatggt ggaatacgtg gggaggtaac 540

gatccttact ttagtccaga attgattact gcaattaata atatctacgg tcatgttaat 600

ttcgacggcc tacctgattc cggtgatagc tactggtctg gcgactgggg tggaacagat 660

ccaatttaa 669

<210> 7

<211> 2778

<212> DNA

<213> phage (vB _ Vne-09SG 01) (Vibrio neolyticus (vB _ Vne-09SG 01))

<400> 7

atgagtactt ttccagcaac aaatagtaac gaagctgttg agttggttat cgatggaggt 60

aatcaattac accagataat caacgaagat gccactacag agattcaaac agaatcggga 120

ccaatcccat cagtacgtaa agcattggca gatacttttc tgtttcaaga cccaatccct 180

tggcaaaacg gtagtgatga gactgctttt aatcaactac gtacttttga taacaacgtt 240

tattgggcac cgacagcaac gctgtcaacc cccgtcccta tggcagctac ccctatcggg 300

gatagcaatt ggaaattagc tcctacaagc aatagtcctt atgtacaaca acaatctcat 360

acgggtgata gtcaacaatc acaggggttg atctggccca tcgacagcaa tgagttatta 420

ccacttggta cccaatcatc agttattgat gttataggca ttacgcgatt gcgtgtcgat 480

actggcgttt tatcggttgg tgaagaattg cgattatgga agaaaagtgg cgatttttca 540

gggacagtcc agacaatcac aaatatctat gcaaataatt taggcggtta tgatgttgta 600

acaagcgctc aaacatatga atttgtaaca aaagatatcg aaaagcttag aatggataga 660

aatcctatcg gttttggtgc tgttgtatat actgcggggg atacttcatt caactcaagt 720

ccggtagact ctttaccgtc attcatcgca gcaattcaag atagtaattc accccttcag 780

ttaccagaag gaaggtttgc gtggtatgga gacggtatag atcttaatga ttataattgt 840

agtggaatga tcgggaaagg atatgatttt tgggaaaccg ttttccagaa tgagaacaat 900

ctaaaaaagg atgacaaagg tacccatatc tacttggttg gtactggtaa taaaatacat 960

tccattaaca atctgggttg cccatctatt gggtcagtta cggacgcggg tataacttac 1020

agcttaactg attttacact agcagactca atagacgaca caccagcaac accaaaacct 1080

tttagctgcg gtttaaaata tgggaaaaac catattgtta aggggctaag gattttaccg 1140

tatcatgcag gtattactgg gtacaataac cctgtatcca ccacattatc tgatgattgg 1200

gatgtatgcc tatggggggc ttctgctgcg caatcagagg tcgatgtaca ggcagtaggt 1260

catgccagaa ttgcagctca cctccttaca gaaaactcag ggtacgggga atatggtgat 1320

tgtgagcgta tggatattaa aacttatgct caaggtcaac gtgggttagt aattcggaat 1380

acagagcaaa cgaaagtttt aagttttacg gcatcaagct ttacgatacc acacacacca 1440

tccatgacaa ttacagcatc aactaaagta caagcgattg gtaccactac aaacaaattc 1500

tttactgtta ccagcacctc attcagtggt ggagaagtta cgattaacgt ttcagaattg 1560

gttgatggga acccctcagc attaagaagt gggtttacag ggactgggtg ggctaagaca 1620

gtaattcgtg attctagatt tgagaccttg cagcatatta gcggtgagaa atctacaacc 1680

tttggtttgg aaacatctat tgcgcgagag gtgtcgggat tcccattgcg cgggatcacg 1740

tttgataact caaagtcaca aactactatt gatgatggta atacatactg gggagactgt 1800

attgatattc ggaacattgt aggtcaatat gaaggtggta caaaaatagc agttgagttg 1860

aatgatgcta ggtttactgc tccatcggga tacacaggga atctaagaga ggttgataca 1920

gtaacaggat cagttaatga ggtaccattc agccctcgcg atgcatatga ttcacaccga 1980

caatttccta ctcaatttac atcaggacag tttatcatta aaccgtggcg caatgaatta 2040

gtgcagatgc aggatgtgac tggagcgatc cgttatgaat acggtgctag tggtgtagat 2100

tcattcttga aaactggtgc taatttcagt ctaaagaacg attatgcaac atcaataatg 2160

gacgcattcg gtgggtccac taacgttatt ttcgggaata atgttacagc taatggggat 2220

ttaatctcag taaatgatac aagaccctca gcagcagggg gtagttcatg cgggacagca 2280

tcacatccgt ggtctgaggt ttttgcactg gttactgcta tcaacccttc cacgcgcgat 2340

attaaacagg atatcgcacc attccctgcc accctgcttg atgcttggga agccaatgtg 2400

aattggtgtc aattcaagat taaggcttct gttgctgaaa agggtgatga tgctagaatt 2460

catggcggtg ttatttttga agaaattgaa gctgcgttta atgaggctgg aatggaccca 2520

tatcgctatg ggctgttgtg cattgaatca tgggaagaca tctatgatga tgttcctgcg 2580

attacagaac tcaagagaga gacagtcttt atcgagaatg ataaaggtga acttgagcct 2640

gagataatcg agcgggttgt agaaatagaa ccagccaaac gagtattagt taaagaggca 2700

gggacgatga aagctgtgcg atatacagaa gcccaggcta ttgagaatgc catgatcaga 2760

agaaaactgg gattatag 2778

<210> 8

<211> 1479

<212> DNA

<213> phage (vB _ Vne-09SG 01) (Vibrio neolyticus (vB _ Vne-09SG 01))

<400> 8

atggcgagtg gatttattaa cggtattttt caaagagata ccttagcaga tattatcctc 60

agtatcgaaa atgatgtcag ggtgacatac aacaacccta aattcagtat agaagataat 120

gagaatatcg gtcaattatt aaaaatgatt gctggacgag agaataatat ctggcagaca 180

attgaacaag tttataattc atggaataga aatggtgctg aagggttatt cttagatgag 240

atattcgctt tgtcaggggt atttcgtgaa aaagccactt caggtagtgg tgatgcagtt 300

gttgaaacta attcatcagc tattgacagt acaagcgtga gtatcggtac aatctttagt 360

ggagagaatg gtggtcagta tgccgcaact tctacacaat ttgtaagtag ccgtgtaaca 420

gcgtaccgtg tagtgggaag caatatttca cttgctactt ataatttgtc aattgaagat 480

ttaagctcag gacaaatctt ttctcaaagt ttcactcttt cctcaagcac acctacagcc 540

cgtttaaatt tcctgaatag tctcaaaaca ttccttgaat cagtgaatcc atcagaaaca 600

aatattcaat tagacacaga taatctaatt ttatattggg gttttgatga agcttacgag 660

ctaaaaggtt tagagaaaac agttaagttt ctttctacac caagtttagg gaatcgttac 720

tcacttatag aatgtgttaa tactcaaaca ggttttaatc cattaggtgt tggtggtatt 780

gcaagtatca gtattcctcc attgggttat gttagcgtaa caaacttaag tgagttttca 840

agtggtacag atgtagaaac agatgcagcg tttgttgaaa gagctagaag tgaagttgat 900

agtccaagaa gtgctacaag atcagcaatt attgcaggtt tgttagctaa tgtaagtggc 960

atagagaaga tcaaatttaa taaaactgtg gatgtaggta ttgtaacagt gacacccatt 1020

attattggtg gagagattgc agatattgca caagaacttt atcgtacaca acctataaac 1080

aatgtttact ctggtgatat tcaatataca gttgatacag aagatgaaga tcaagaagtt 1140

ataagcttct ctcgtggtgt tcaacaacaa ctaagtgtcc gtgttagata taaaactact 1200

aataatacag agttgtctac gtccgagaaa agtaccgcga caagcaatct attagatgta 1260

tcagaaagct ggcaactggg tgataagata tttaatttct cactgatgtc tgctgttagt 1320

tctgcagtta gttacgggag attcaatcaa ctgattgtag aagttaagaa attagaagag 1380

ccagactcag cttattctaa tgcagattac caagcgggta atacagaact accagatttg 1440

atctctaaca atattacttt tgtacaggag attaactga 1479

<210> 9

<211> 636

<212> DNA

<213> phage (vB _ Vne-09SG 01) (Vibrio neocaledonicus (vB _ Vne-09SG 01))

<400> 9

atggctttac ctgattccaa tagactcaaa ttcaatgtta cattcacaca agatggttta 60

agtgaattac cctataaaca aactcaacga gaaaactttt ataaacttgc tgaactgatt 120

gttaaccgtt taaacaatat ccaaaaccaa gctgtatctt tagcttactc tagagtttta 180

gatttagcag aaggggatat gttggatgta attgcttctc actattttat agaacgtgaa 240

gggaaggatg atgaaggttt acgttcagct ataaaacttc atgctttacg acaaaacaca 300

gaaccaacac gtccagaaat agttagtatt ttaaacatat tgacagataa tggttttgtt 360

aagatttaca aaggtttaaa taattatgtg gaagttgtta tcagtgtaga ttgtctatcg 420

atacaatcac tttctgacca actacaagat ttatttccag tcaacactaa tttaaaggta 480

gcttctgttc cagtagggtc aaaacctttt ggtgttggta gtattcattc tacaccttca 540

gataagattg gacctttagg tagtatccat gattctatta tagatagacc gaacgtagca 600

tcagtgacag ttattaatga tgagagagat ttataa 636

<210> 10

<211> 768

<212> DNA

<213> phage (vB _ Vne-09SG 01) (Vibrio neolyticus (vB _ Vne-09SG 01))

<400> 10

atggcaacac aaccaacaaa ccctatcatc aggatagcgg agaatgatgt aaatttacca 60

agcacaggtc agccgaataa gaatgaacct agttcgactt tacagtctac aggttatgat 120

aatgatcaag ttgtaacagc agaagaactt aattatatct ttgataactt cgctgagtgg 180

ttagattacc tagtattgga agatagtgac actaatacta gaatagataa tttggaatcg 240

agaactatta caggtgggaa tggtttaaca ggtggtgggg atttaagtga aaacagatct 300

atagctttag gtacaccttc ctcattagat ggttcaacaa ctaactctgt tacgacaggt 360

tcacacacac acagtttaaa tatggcgagc caaccggaag ctgaaactgg tacagataac 420

actaaacctg tgacatcttt gagagtacac caagccgtac cttttacatt cacaccatct 480

tcaattagtg gttcgacaga atcgacaacc ctacctaacg gtttaataat taaatggggg 540

gagacacctt ctataggaga tggaggggat actatagtaa ccttcccaac tccctttccc 600

aacgcatgtt tccatgtagg tactgaaatc ttaagaaacg tatcaggtga tggtccaaac 660

gcatacggta caggtgttac taataaaacg gccagtagtg taactatctc ttttaatggt 720

acaggttccg tatcttccgt gataggttgg tgggcaatag gctactaa 768

Claims (8)

1. A vibrio phage, wherein the phage is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of: CCTCC NO: m2022200, deposit date: 04.03/2022, with a deposit address of: wuhan university in Wuhan City, China, zip code: 430072, classification nameVibrio neocaledonicus phage vB_Vne-09SG01。

2. The bacteriophage of claim 1, wherein the bacteriophage is a long tail bacteriophage capable of specifically cleaving vibrio neocardoidis, the tail being 141 ± 5 nm long and the head being a regular icosahedron of 81 ± 2 nm; the incubation period of the bacteriophage NF is about 40 min, and the lysis period is 60 min; the phage can keep the stability of activity under the conditions of pH 5-9, salinity 0-33.0 ‰, and temperature not higher than 32 deg.C.

3. A phage preparation comprising the phage of claim 1, wherein the total phage effective amount is greater than or equal to 105 PFU/mL.

4. A phage preparation according to claim 3 wherein the adjuvant is one or more of SM buffer and sterile seawater.

5. The method for separating phage according to claim 1, comprising the steps of: mixing 0.22 μm seawater with Vibrio neocarviensis cultured to logarithmic phase, culturing for 7 days, and performing phage separation by double-layer plate method in 1 st, 3 rd and 7 th days; selecting a single plaque, continuously purifying the phage by a double-layer plate method for 5 times, and then enriching to obtain a phage suspension; then, after gradual amplification culture, multiple centrifugation, PEG 8000 precipitation, cesium chloride density gradient centrifugation purification and dyeing, the phage is identified as long-tail phage by adopting transmission electron microscope observation.

6. Use of the bacteriophage of claim 1 for lysing Vibrio neocardoyiniVibrio neocaledonicusThe use of (1); the application specifically comprises the following steps: application to prevention and treatment of marine shellfish to treat Vibrio neocarlidiniiVibrio neocaledonicusOr to the aquatic environment, to Vibrio neocarlidiniiVibrio neocaledonicusAnd (4) killing.

7. Use according to claim 6, wherein the bacteriophage is directed to Vibrio neocarlidiniiVibrio neocaledonicusThe final concentration for killing is more than or equal to 105 PFU/mL.

8. Use according to claim 6, characterized in that the use of said bacteriophage for the preparation of a medicament of Vibrio infestans, in particular Vibrio neocarlidiniiVibrio neocaledonicusIn the preparation of medicaments for treating the caused diseases.

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