CN111718907A

CN111718907A - Novel Vibrio alginolyticus bacteriophage, composition thereof and application thereof

Info

Publication number: CN111718907A
Application number: CN202010621978.5A
Authority: CN
Inventors: 潘强; 任慧英; 孙虎芝; 闫艳新; 徐帆
Original assignee: Qingdao No Antibiotics Biotechnology Co ltd
Current assignee: Qingdao No Antibiotics Biotechnology Co ltd
Priority date: 2020-07-01
Filing date: 2020-07-01
Publication date: 2020-09-29
Anticipated expiration: 2040-07-01
Also published as: CN111718907B

Abstract

The invention discloses a novel vibrio alginolyticus bacteriophage, a composition and an application thereof, wherein the vibrio alginolyticus bacteriophage vB _ ValP _ PJ32 is preserved in China general microbiological culture Collection center (CGMCC No. 18860) in 11/04 in 2019, and the preservation number is CGMCC No. 18860. The novel vibrio alginolyticus bacteriophage has a wider cracking spectrum, has excellent acid-base tolerance capacity, can adapt to severer application environment, and is used for preventing and treating vibrio alginolyticus infection. The phage and the composition compounded by the phage can be used for preparing medicines for preventing and treating diseases caused by vibrio alginolyticus infection in aquaculture, prawn feed additives, an aquaculture farm environment disinfectant, a detection kit and a biological bacteriostatic agent for disinfecting fresh foods, can be used for each link of loss easily caused by vibrio alginolyticus infection in the aquaculture process, daily disinfection of the aquaculture market and the aquaculture environment and the like, and are beneficial to the healthy development of the aquaculture industry.

Description

Novel Vibrio alginolyticus bacteriophage, composition thereof and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a novel vibrio alginolyticus bacteriophage, a composition containing the bacteriophage and application of the bacteriophage.

Background

Vibrio alginolyticus (Vibrio algirlyticus) belongs to Vibrionaceae (Vibrionaceae), Vibrio (Vibrio), gram-negative brevibacterium, without capsule and spore, is halophilic and facultative anaerobic marine Vibrio, exists independently or is connected with the tail end to form a C shape or an S shape. Vibrio alginolyticus is widely present in seawater, river mouths and marine animals all over the world, and its amount is the top of Vibrio in the sea.

The vibrio alginolyticus is a conditional pathogenic bacterium, and can infect various marine culture fishes, shrimps and shellfishes through damaged skins and oral cavities of the fishes to cause the occurrence and the prevalence of digestive tract diseases such as fish ulcer disease, ascites disease, shrimp erythrosis or hemorrhage disease and the like. The suitable temperature for the propagation of the vibrio alginolyticus is 17-35 ℃, so that the large-area disease is easily caused in summer, and huge economic loss is brought to the breeding industry. Vibrio alginolyticus also causes food-borne diseases in humans, and eating food containing the bacteria may cause typical gastroenteritis reactions such as diarrhea, intestinal spasm, nausea, vomiting and fever, and the bacteria may also cause diseases such as otitis media, otitis externa, septicemia, surface wound infection and ulcer, cellulitis, necrotizing fasciitis, conjunctivitis, osteomyelitis, meningitis, and the like.

In recent years, vibrio alginolyticus has caused more and more loss in fishery. Because the separation rate of vibrio in marine bacteria in four sea areas in China is as high as 52.9%, the vibrio alginolyticus accounts for 62% of the common strains in the four sea areas. In addition, the Vibrio alginolyticus is easy to form a biofilm on the surfaces of foods and processing apparatuses during the growth process, and the bacteria in the biofilm have enhanced stress resistance to conventional preservatives and bacteriostats and are difficult to remove, thereby causing continuous pollution. At present, the prevention and treatment of vibrio alginolyticus are generally carried out by adopting a large amount of traditional antibiotics or disinfectants in fishery production. However, the literature data shows that the generation of the Vibrio alginolyticus drug-resistant strain is aggravated along with the overuse and abuse of drugs, and the food safety problems such as the residue of harmful drugs in aquatic products are also caused. For example, Vibrio alginolyticus has high tetracycline resistance up to 100.0% and ampicillin and carbenicillin resistance up to 97.0% and 93.9% in coastal sea areas of Shanghai, and some of the isolated strains have severe multi-drug resistance. Therefore, the traditional antibiotics or disinfectants are not suitable for the effective prevention and treatment of vibrio alginolyticus and bring environmental pollution.

The bacteriophage (bacteriophage or Phage) is a general name of microbial viruses such as infectious bacteria, fungi, actinomycetes or spirochetes, has small volume and simple structure, does not have a cell structure, can only live in a parasitic way, is widely distributed, and almost all places where bacteria exist have corresponding bacteriophage. The utilization of the phage preparation has a plurality of advantages in the treatment of bacterial aquaculture diseases: firstly, the bacteriophage has strong adaptability to bacterial drug resistance, although bacteria can also generate resistance to the bacteriophage, the bacteriophage has faster variation and replication speed and can adapt to variation of host organisms; secondly, the specificity of the phage is strong, only specific bacterial pathogenic bacteria of the phage are used as hosts, and normal flora cannot be destroyed; and thirdly, the phage has the advantages of no residue and no toxicity in use, the phage has host dependence, disappears along with the elimination of the host, and cannot remain in the animal body, and the degradation end products of the phage are amino acids and nucleotides, which have no influence on human and animals.

However, no effective Vibrio alginolyticus phage exists at present, which can be used for preventing and treating various diseases caused by Vibrio alginolyticus infection, so that the prior art needs to be further improved.

Disclosure of Invention

Aiming at the problems, the invention provides a novel Vibrio alginolyticus bacteriophage vB _ ValP _ PJ32, a bacteriophage composition and application thereof, wherein the bacteriophage and the composition are safe to use, and the problems of antibiotic residue caused by overuse of antibiotics and induction of drug-resistant Vibrio alginolyticus are avoided while infection caused by the Vibrio alginolyticus is solved.

The technical scheme of the invention is as follows:

in a first aspect, the invention provides a vibrio alginolyticus phage vB _ ValP _ PJ32, wherein the phage is separated from a sewage sample obtained from a city Yang seafood wholesale market, and the vibrio alginolyticus phage is preserved in China general microbiological culture Collection center (CGMCC) 11/04 in 2019 with the preservation number of CGMCC No. 18860.

The vibrio alginolyticus phage vB _ ValP _ PJ32 can form bright plaques with the diameter of 0.5mm on a double-layer agar culture medium, and the plaques are regular circles and have clear edges. Observed under an electron microscope: the head of the phage is of an icosahedron structure, the phage has a short but non-contractive tail, the diameter of the head is about 54nm, the length of the tail is about 15nm, the phage accords with the characteristics of the brachytail phage family, and the phage belongs to brachytail phage.

In a second aspect, the application also provides the application of the vibrio alginolyticus bacteriophage in preparing a medicament for preventing and treating diseases caused by vibrio alginolyticus infection in aquaculture. The term "prevention" is meant herein to include all actions that inhibit or delay the disease by administering the bacteriophage. The term "treatment" is meant herein to include all actions that would improve or ameliorate the disease by administration of the bacteriophage.

Preferably, the diseases infected by Vibrio alginolyticus include digestive tract diseases of mariculture products, such as fish ulcer disease, ascites disease, shrimp erythrosis or hemorrhagic disease.

In a third aspect, the present invention also provides a phage composition comprising the vibrio alginolyticus phage vB _ ValP _ PJ32 as described above. The phage composition can be compounded with other vibrio alginolyticus phages through vibrio alginolyticus vB _ ValP _ PJ32, and is used for preparing products for preventing and treating vibrio alginolyticus diseases of aquatic products. Other phage may be selected from other Vibrio alginolyticus phages or other pathogenic bacteriophages that cause digestive tract diseases in marine products.

Preferably, the above phage composition further comprises one or more of mutants of phage vB _ ValP _ PJ 32; the mutant has a homology of not less than 90% with the corresponding phage.

Since bacteriophages are very susceptible to mutations during replication, it is preferred that mutants of the aforementioned bacteriophages are also within the scope of the present application. The determination of homology can be suitably carried out by computer programs well known in the art, and the mutants of vB _ ValP _ PJ32 have at least 90% homology with the natural sequence of the phage; more preferably, the mutants are 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the native sequence of the respective phage. The sequence of vB _ ValP _ PJ32 can be determined by known methods from the biological materials deposited according to the invention. The mutants of the phage may be point, deletion or addition mutations, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bases may be changed relative to the original phage sequence. It is not necessary for the skilled person to inventively work to select a mutant with a similar trait from the phages provided according to the invention.

In a fourth aspect, the present invention also provides a phage pharmaceutical preparation, the active ingredient of which is mainly the vibrio alginolyticus phage or the phage composition. Preferably, the phage drug formulation further comprises phages to other specific pathogenic bacteria.

Optionally, the phage pharmaceutical preparation is in the form of oral administration dosage form, external application dosage form, such as solution, powder, gel, granule, lotion, ointment, patch or cream.

Optionally, the phage drug preparation further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the active ingredient being administered. In order to formulate the pharmaceutical composition as a liquid formulation, a pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered saline, albumin infusion solution, glucose solution, maltodextrin solution, glycerol and ethanol. They may be used alone or in any combination thereof. Other conventional additives, for example, antioxidants, buffers, bacteriostats, and the like, may be added if desired. The composition of the present invention may also be prepared into injections (e.g., aqueous solutions, suspensions, and emulsions), or pills, capsules, granules, or tablets, when further combined with diluents, dispersants, surfactants, binders, and/or lubricants.

In a fifth aspect, the application also provides a prawn feed additive, which comprises the vibrio alginolyticus bacteriophage or the bacteriophage composition, and the prawn feed additive is fed after being mixed with the prawn feed to achieve the effect of preventing or treating vibrio alginolyticus disease⁹PFU/g。

Sixth aspect of the inventionIn one aspect, the present application also provides an environmental disinfectant comprising the effective ingredient mainly the Vibrio alginolyticus bacteriophage or the bacteriophage composition, preferably, the bacteriophage has a titer of 1 × 10⁹PFU/ml or more. The environmental disinfectant also contains other active ingredients for inhibiting or eliminating viruses and bacteria in the environment.

The environment disinfectant can be used for daily disinfection of aquaculture places, disinfection of empty ponds, and disinfection and purification of environments in aquaculture markets, can be used for replacing antibiotics or traditional disinfection products which cause resistance forbidding or burden on the environment, and the bacteriophage and the metabolite of the environment disinfectant can not cause damage to human bodies or other animals. The environment disinfectant can be used for comprehensively disinfecting culture environments, feeding appliances and the like by spraying and soaking. The liquid soaking, spraying forms include but are not limited to detergents, disinfectants, detergents, and the like.

In a seventh aspect, the present invention also provides a detection kit comprising a vibrio alginolyticus phage or phage composition as described above. The skilled in the art can use the above-mentioned Vibrio alginolyticus bacteriophage or the bacteriophage composition thereof to prepare a detection kit for detecting Vibrio alginolyticus that is specifically infected with the Vibrio alginolyticus bacteriophage or for preventing and controlling diseases caused by infection of Vibrio alginolyticus that is a host thereof, according to the present disclosure and the common general knowledge in the art.

In the ninth aspect, the invention also provides a biological bacteriostatic agent for disinfecting fresh aquatic products, and the effective component of the biological bacteriostatic agent is mainly the vibrio alginolyticus bacteriophage or the bacteriophage composition. The use method of the biological bacteriostatic agent comprises the following steps: the surface of the fresh product is soaked or sprayed for disinfection to inhibit the proliferation of the vibrio alginolyticus in the processing or fresh-keeping process of the product.

The invention has the following beneficial effects:

1. the invention provides a vibrio alginolyticus phage vB _ ValP _ PJ32 and a phage preparation thereof, wherein the phage preparation can kill pathogenic vibrio alginolyticus with antibiotic multi-drug resistance, has a cracking rate of 90.30 percent, has a broad-spectrum sterilization effect, and can be used for preventing and treating aquatic diseases caused by vibrio alginolyticus; has no toxic and side effects and high safety, and can not cause the appearance of a large amount of Vibrio alginolyticus with drug resistance. In addition, the bacteriophage has a better hydrophilic phase, and is easy to prepare into spraying liquid or injection to effectively kill the vibrio alginolyticus in the environment.

2. The bacteriophage is obtained from nature, is easy to carry out industrial production, and the medicament or disinfectant prepared from the bacteriophage not only can reduce the cost, but also has the advantage of environmental protection.

3. The phage has strong acid-base tolerance, the titer of the phage is basically unchanged after the phage acts for 3h in the environment of pH 4-11, the phage has certain tolerance in the environment of pH 3 and pH 12, and the phage can be applied to the sterilization of vibrio alginolyticus in a severe acid-base environment.

4. The vibrio alginolyticus bacteriophage and the bacteriophage composition thereof can be widely used in each link easily causing loss due to vibrio alginolyticus infection in the aquaculture process, daily disinfection of the aquaculture market and the aquaculture environment and the like, and are beneficial to the healthy development of the aquaculture industry.

Drawings

FIG. 1 is a plaque photograph of the bacteriophage vB _ ValP _ PJ 32;

FIG. 2 is an electron micrograph of the bacteriophage vB _ ValP _ PJ 32;

FIG. 3 shows the result of the thermostability detection of the phage vB _ ValP _ PJ 32;

FIG. 4 shows the results of pH stability test of the phage vB _ ValP _ PJ 32;

FIG. 5 is a one-step growth curve of phage vB _ ValP _ PJ 32.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In the present invention, the equipment and materials used are commercially available or commonly used in the art, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.

Experimental materials:

1. the test of the invention relates to a standard strain of vibrio alginolyticus: ATCC17749 is available from Shanghai Fuxiang Co., Ltd, and ATCC33787 is available from Shanghai Fenghui Biotechnology Co., Ltd.

2. The Vibrio alginolyticus phage vB _ ValP _ PJ32 is preserved in China general microbiological culture Collection center (CGMCC) No.18860 in 11 th and 04 th month in 2019, and is named as vB _ ValP _ PJ 32.

Example 1: isolation, purification and amplification of bacteriophages

Taking a sewage sample of urban male seafood wholesale market in Qingdao city, Shandong province, adding Vibrio alginolyticus logarithmic phase bacterial liquid J20, adding a proper amount of 2216E culture medium, and placing at 37 ℃ and 170rpm for shake cultivation for 16 h. Taking out 5ml of liquid, centrifuging at 11000rpm/min for 10min, filtering the supernatant with a 0.22 μm sterile microporous filter membrane to obtain phage stock solution, and storing at 4 deg.C.

Separating phage by double-layer plate method, diluting phage stock solution by 10 times, mixing 100 μ l of diluted solution with 200 μ l of corresponding Vibrio alginolyticus bacterial solution, incubating at 37 deg.C for 5min, mixing with upper layer agar (agar concentration is 0.8%), spreading on lower layer 2216E agar (agar concentration is 1.5%) plate, cooling, and culturing at 37 deg.C in incubator upside down overnight. The next day, spotting condition was observed, and single plaques were picked and purified repeatedly 3 times to obtain single phage.

And respectively picking single plaques, placing the plaques in 1ml of PBS solution, and leaching for 30min at 37 ℃ and 170rpm to obtain phage leachate. 100 mul of phage extract and 100 mul of Vibrio alginolyticus host bacteria log phase bacteria liquid are taken to be put in 5ml of liquid 2216E culture medium and are shake cultured in a shaking table at 37 ℃ and 170 rpm. And centrifuging at 11000rpm for 10min until the liquid becomes clear, taking the supernatant, and filtering by using a 0.22-micron bacterial filter to obtain a phage proliferation liquid.

The vibrio alginolyticus phage can form transparent plaques with the diameter of 0.5mm, the plaques are regular and round, the edges are clear, and the plaque forming picture is shown in figure 1. The solution isThe phage of the vibrio alginolyticus is proliferated by using host bacteria, can be shaken up after 3h, and has the titer of 8.76 × 10⁹PFU/ml。

EXAMPLE 2 Electron microscopy of phages

And dripping the separated phage suspension liquid on a copper net covered with a polyethylene formaldehyde film, dyeing for 5-10 min by using 2% phosphotungstic acid, and observing by using a Hitachi HT7700 type transmission electron microscope after drying.

Observed under a transmission electron microscope, the shape of the phage is shown in figure 2, the head of the phage is in an icosahedral structure, the phage has a short but non-contractive tail, the diameter of the head is about 54nm, and the length of the tail is about 15 nm; according to the classification method of the international committee for virus classification (ICTV), the phage morphology of the present application conforms to the characteristics of the brachyphagidae, belongs to the brachyphagidae, and is named as vB _ ValP _ PJ 32.

Example 3 genomic analysis of phages

Extracting the genome of pseudomonas aeruginosa bacteriophage vB _ ValP _ PJ32, and performing whole genome sequencing to obtain:

the whole genome length of phage vB _ ValP _ PJ32 is: 80293 bp; wherein, the gene sequence of tail protein related to the recognition of the phage host is shown in sequence 1 in the sequence table, the gene sequence of highly conserved terminal enzyme large subunit (DNA polymerase) is shown in sequence 2 in the sequence table, and the gene sequence of DNA polymerase (DNA polymerase) is shown in sequence 3 in the sequence table. The specific information of the above genes is shown in Table 1 below.

The phage genome was subjected to sequence similarity alignment analysis using the BLAST online tool (http:// BLAST. ncbi. nlm. nih. gov /). The phage with the highest homology is Vibrio phase Ares1, and the phage vB _ ValP _ PJ32 has the homology with the phage of only 76.22%. The above results indicate that the phage vB _ ValP _ PJ32 is a new Vibrio alginolyticus phage, and has a distant relationship with the existing closely related phage.

TABLE 1 Gene sequence information Table of phage vB _ ValP _ PJ32

Example 4 determination of phage lysis Profile

132 vibrio alginolyticus strains separated from farms and seafood markets stored in laboratories are selected, the 132 vibrio alginolyticus strains are separated from farms or seafood markets in various urban areas of the provinces of Hainan, Jiangsu, Shandong and the like, and single colonies of 2 vibrio alginolyticus standard strains (ATCC17749 and ATCC33787) are respectively inoculated into 5ml of 2216E liquid culture medium, and the logarithmic phase bacterial liquid is obtained by culturing in a shaking table. 100 mul of the bacterial liquid and 100 mul of the phage are mixed into the upper layer agar and evenly spread on the lower layer agar. And (5) after solidification and drying, culturing for 8-12 h in an incubator at 37 ℃, and observing and recording a spot forming result. Meanwhile, the drug resistance condition of 132 clinical isolates is determined by using a drug sensitive paper method, and the virulence genes of the 132 clinical isolates are identified by PCR.

The experiment shows that: among the 132 clinical isolates, 128 drug-resistant strains were obtained, the drug resistance rate was 96.97%, 127 strains harboring virulence genes were obtained, and the virulence gene harboring rate was 96.21%. For 132 strains of Vibrio alginolyticus, the Vibrio alginolyticus phage vB _ ValP _ PJ32 can crack 119 clinical isolates and 2 Vibrio alginolyticus standard strains ATCC17749 and ATCC33787 with the cracking rate of 90.30%.

Since 119 strains of the vibrio alginolyticus bacteriophage vB _ ValP _ PJ32 which can be split clinically isolated strains have different types of drug resistance except J05, J13 and J40 which do not have drug resistance, the 116 strains of the vibrio alginolyticus bacteriophage vB _ ValP _ PJ32 can effectively split pathogenic host bacteria with different drug resistance.

TABLE 2 lysis of clinical strains by Vibrio alginolyticus phages

Note: +: cracking, and brightening plaques; -: it is not cracked.

Example 5 measurement of phage titer and MOI

Phage and host bacteria were propagated as described in example 1, phage and host bacteria titers were determined, and phage vB _ ValP _ PJ32 and its host bacteria were appropriately diluted. 100. mu.l of each of the phage and the host bacterium was added to 2216E liquid medium at a multiplicity of infection of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, and 0.0000001, respectively. The cells were cultured at 37 ℃ with shaking at 170rpm until the cells became clear, and the time for clearing the cells was recorded. After centrifugation at 11000rpm for 5min, the supernatant was removed and the titer of phage was determined by the double-layer plate method.

The titer of the phage of the vibrio alginolyticus proliferated in the embodiment 1 can reach 8.76 × 10⁹PFU/ml, Vibrio alginolyticus phage vB _ ValP _ PJ32 at MOI of 0.01, titer can reach 3.43 × 10¹⁰PFU/ml。

Example 6 measurement of phage thermostability

Respectively packaging 500 μ l phage proliferation solution into sterile EP tube, and respectively treating in water bath at 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C and 80 deg.C for 20min, 40min and 60 min. Each temperature was set to 2 replicates. Immediately after the reaction, the sample is placed in an ice bath for cooling, and the titer of the phage is measured by a double-layer plate method after dilution by 10 times. And drawing a phage thermal stability curve by taking the temperature as an abscissa and taking the logarithm value of the phage titer as an ordinate.

As shown in FIG. 3, the activity of the phage vB _ ValP _ PJ32 is basically kept unchanged after 1h of action at the temperature of 40 ℃, 50 ℃ and 60 ℃; the inactivation is complete after 20min at 70 deg.C and 80 deg.C. The practical application environment and the production process flow of the bacteriophage and the composition thereof are combined, which shows that the bacteriophage has better thermal stability.

Example 7 determination of phage pH stability

Adding 4.5ml of PBS solutions with different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13) into a sterile test tube, placing the test tube into a water bath kettle at 37 ℃, adding 500 mu l of phage proliferation solution into the test tube respectively after temperature balance, mixing the test tube and the phage proliferation solution uniformly, and performing water bath at 37 ℃ for 1h, 2h and 3 h. Phage titers were determined using the double-plate method at 1h, 2h and 3h, respectively, at different pH, and pH was neutralized to pH 7 with a neutralization solution before the assay. The results of the examples were plotted on the pH abscissa and the log of the titer of the phage on the ordinate to obtain a stability curve of the pH of the phage.

FIG. 4 is a pH stability curve of Vibrio alginolyticus phage vB _ ValP _ PJ 32. The experimental results of fig. 4 show that: the titer of the phage is basically unchanged after the phage acts for 3h in the environment with the pH value of 4-11, and the titer is reduced by 3 titers in the environment with the pH value of 3 and the pH value of 12. By integrating the practical application environment, the Vibrio alginolyticus bacteriophage vB _ ValP _ PJ32 has strong acid and alkali resistance, and particularly has outstanding acid resistance.

EXAMPLE 8 determination of one-step growth curves of bacteriophages

1ml of each of the growth medium of Vibrio alginolyticus phage vB _ ValP _ PJ32 and the logarithmic phase of the host bacterium was added at an MOI of 0.01, the mixture was thoroughly mixed and started to be timed, incubated at 37 ℃ for 5min, centrifuged at 13000g for 30s, the supernatant was aspirated as much as possible by a micropipette, washed 1 time with 1ml of 2216E liquid medium (centrifuged at 13000g for 30s), and the supernatant was discarded. Suspending and precipitating with preheated 2216E liquid culture medium (total volume is 5ml), mixing well, quickly placing in a shaker at 37 deg.C and shaking at 170rpm for culturing, taking out 150 μ l at 0 time and every 10min, centrifuging at 10000rpm for 1min, sucking 100 μ l of supernatant, diluting with physiological saline 10 times, measuring phage titer by double-layer plate method, repeating for 3 times, and averaging the results. And (3) drawing a one-step growth curve by taking the infection time as a horizontal coordinate and the logarithm value of the titer of the phage in the infection system as a vertical coordinate to obtain the incubation period and the outbreak period of the phage.

As shown in the one-step growth curve of Vibrio alginolyticus phage vB _ ValP _ PJ32 in FIG. 5, the titer is basically stable within 30min after the phage infects host bacteria, which indicates that the latency period of the phage is about 30 min; within 30-100min after the phage infects the host bacteria, the number of the phage is increased sharply, and the outbreak period of the visible phage is about 70 min; during the subsequent 30min, the phage population was essentially unchanged and entered stationary growth phase.

Example 9 prevention and protection test method of phage preparation against Penaeus vannamei infected with Vibrio alginolyticus: healthy penaeus vannamei boone weighing about 5g is subjected to fasting overnight. 3 groups (blank, test and control groups, respectively) were set up, 50 shrimps per group. Wherein, the blank control group is not treated; the test group is characterized in that phage and prawn feed are uniformly mixed according to the addition amount of 5% of the volume to mass ratio, and the mixture is fed at the dosage of 3% of the weight of prawns after being dried in the shade, wherein the content of the phage is 1 multiplied by 108 PFU/ml; the control group is fed with the prawn after the feed is soaked in 2216E culture medium according to the same dosage (5 percent of addition).

After continuously feeding for 3 days, all prawns are injected with vibrio alginolyticus at a dose of 5.3 × 10⁷CFU/only. The number of dead prawns in each group of 5d was recorded and the protection rate of the phage was calculated.

The result is achieved: the mortality rate of the challenge control group is 56 percent, the mortality rate of the test group is 12 percent, and the prevention and protection rate of the phage on the penaeus vannamei boone can reach 88 percent. The results of this example demonstrate that feeding bacteriophage vB _ ValP _ PJ32 is effective in preventing infection of penaeus vannamei by vibrio alginolyticus.

EXAMPLE 10 therapeutic protection test of phage preparation against Penaeus vannamei infected with Vibrio alginolyticus

The experimental method comprises setting 3 groups of healthy Penaeus vannamei Boone with weight of about 5g overnight in an empty stomach, leaving 50 shrimps in each group, and leaving blank control group untreated, and performing intramuscular injection of Vibrio alginolyticus at a depth of about 0.5-1 mm from the second and third internode side muscles of all the Penaeus vannamei Boone, wherein the dosage is 5.3 × 10⁷CFU/only. After 1h of toxin counteracting, the phage and the prawn feed are evenly mixed according to the addition amount of 5 percent of the volume to mass ratio, and the mixture is used for killing the virusesAfter drying, the shrimp was fed at a dose of 3% of the shrimp weight. The control group was fed with 2216E medium soaked with the feed at the same dosage. The number of dead prawns in each group of 5d was recorded and the protection rate of the phage was calculated.

The experimental results are as follows: the mortality rate of the challenge control group is 54 percent, the mortality rate of the test group is 18 percent, and the treatment protection rate of the phage on the penaeus vannamei boone can reach 82 percent. The penaeus vannamei infected with vibrio alginolyticus can be effectively treated by feeding the phage vB _ ValP _ PJ 32.

EXAMPLE 11 phage preparation Water body Disinfection test

The experimental method comprises adding 3L of sterile seawater into each barrel, placing 5L of Penaeus vannamei Boone into each barrel, water-bathing at 28 deg.C, introducing oxygen via small air pump, adding certain volume of Vibrio alginolyticus into the system, and adjusting the final concentration to 1 × 10⁶CFU/ml; after 1h of treatment, phage vB _ ValP _ PJ32 was added to each barrel of seawater to make the final multiplicity of infection 1:1 and 0.1:1, respectively, each group was set in parallel, and an equal volume of 2216E medium was added to the challenge group, the detailed grouping is shown in Table 3. And detecting the quantity of the vibrio alginolyticus in the water body by a coating plate method after 1h, 7h, 10h and 25h respectively.

TABLE 3 Water disinfection experiment grouping

TABLE 4 residual quantity of Vibrio alginolyticus CFU/ml in water disinfection test

The experimental results as shown in fig. 4 can be obtained: after 7 hours of action, the phage can obviously reduce the number of viable bacteria of vibrio alginolyticus in the culture water body, and can reduce 4 orders of magnitude at most. The results of this example demonstrate that the bacteriophage vB _ ValP _ PJ32 can be used as a water disinfectant (or modifying agent) to effectively kill Vibrio alginolyticus.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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<400>2

atgacgagta aagcagagaa accgaaaaag aaagagacgg caaaggatcg ccgtcttaaa 60

gcggcacgcc gtagaaaaga ggttatacca ccgatagacc caaacaaagg caagcccgct 120

aaggacctag acaagctaga aaagatatta gaggaagaac taggggcctt ccctgaaaag 180

caacgcgcgc cgctagtgca ttacatgaaa cagtatctag ccggggaagt taaactagat 240

atttcggact accacacaat cccggtagac ccgagaacct tcttattaga cgacttttat 300

ttaggtctaa aagatgatgt ctacccggct ctaatagatg agttttgcga aatgaacagc 360

gggaagtata tcgagatcgt tttaacgggc ggtatcggta caggtaagac gacgctagcc 420

gtttggaata ccgcttatca actttatcta ttatcgtgcc ttaaagaccc gcacaagcta 480

tttaagctag ataagagctc agagatatta atagttttcc agagtataac ggcggaccta 540

gcacgtaagc ttaactataa ccgatttaag gcacttatcg atcaatcacc gtattttcaa 600

acgcgtttcc cttatgaccg cagggtcgaa accgaactta aataccctaa ccggatctat 660

atccgcccgg tatccggtga agataccgcg gctatcggtc agaacgtgat cggcggcatc 720

atagacgaaa ttaactatat ggccgtaacc gaaaactcta aaaaagagga aggcggcggg 780

acctacgacc aagcagcaaa ggtctataac tcaatagctc gacgccgtaa atctcgattt 840

atgactaagg gtcagatcgc cgggttattg tgcctagtgt cctcgcgtaa gtacccgggt 900

cagtttacag accggaagga agaagaagcc aagaaagaca aaactatcta tatctatgat 960

aaaacgatct ggcagattaa acccaagggg acctatggca acgcccgttt taatgtgttt 1020

atcggggacg ttaagcgtaa gccgcgtata cttgatgaca acgaggaccc gcgcaagact 1080

tacaagccaa aggatcaaca cctaatcaga tccatccctg tagagtttaa aggcgagttt 1140

caaaccgata tggttaacgc gctgcgtgag gtagcgggcg tggctacgct tgcctcacac 1200

ccgtttattt tgaataccga cgcgatagac ccatgcttta cagagaaaac caaaagcatt 1260

ttcaaagagt ctatttgcaa ctttgatgat aagaaactac acgtacttaa gaagaatata 1320

tttgcgccgg atattccgcg ttttattcac ttggacttat catctacagg agataaaacg 1380

ggctttgcta tgggatgtat cccgaggttt acgacaatgg accgcggcga cgttaaagag 1440

gttcaccccg tatttagaat agacggtgca ttagctattg aggccccgtt gggtggggag 1500

atcctatact ggcggattcg aaagctaatc tatgctttgc gggatatggg ttacaatatc 1560

aaatggatca cattcgatac ataccaaagt actgacagta agcaaatact agcgcaaaac 1620

ggctttgtaa ccggggttca atccgttgat acttctacgc taccttatga gcttttgaag 1680

tccgctattt acgacgggcg actagtggca ccaccacacg acctattaca attggagtta 1740

gcacgattag agaaagacac taaaaacgat aaaatagacc acccgcccgg cttttctaaa 1800

gacgtttccg acgcggtagc cggggtatgt tatgggctta tgatgcgccg tgaaatatgg 1860

gttagccacg gtattaaccc tgataagatc gtaaacgcag atatagaagc catgaaacga 1920

agtgagaaga aactagaaga tgctaacaaa cgcattaacc gatctagctc aggagacggt 1980

tataacatag aggactaa 1998

<210>3

<211>2358

<212>DNA

<213> DNA polymerase of phage vB _ ValP _ PJ32 (DNA polymerase I of vB _ ValP _ PJ32)

<400>3

atgctttatt tgatggttgg cgaacgcact acgctacatg acctaaaacg agttgtagcg 60

ccagtagtaa aagcgcaagg cataccgcac cggatcatga gatggaccga gggcgaaccg 120

acgctaggac agggcgacgt attattctca atgggaaaag cggcctttga tcacttaacg 180

cgcctaggcg tataccctaa aaatcgaacg gtaactagct ttagagaaaa gccgcagtta 240

cgcggccaag ggcatgtact atgttcttat gacgctgcta taggacgcgt taagccagag 300

catgaagcat ttttgcagat tgatatcatg caagcgtgta gaatgcacca aaccgggacc 360

attaaaccgc agttaggatc ctaccgttgg gttgacgact tcagcgaaat gatcgaggaa 420

atagagcaga cgtataccga gacgggtaaa cccgtccgcg tagcgggaga cttggaaacc 480

gtaggctttg attatgtcgc taaagataag tttatcgtat ccgcgcaatt ctcgcataag 540

gaaggcatgg ccgatttagt gcgattctac agccctgaaa agcagcctaa gccgggggac 600

ttgttacacg atcaaataac ttggctactt aacagcgaaa aagttatcct atgtggggct 660

aactttaagt acgatcttaa ctggataaaa tacaagtggg ggatcgagtg taccaacttt 720

aaactagaca ctacgctagt aggctcactt ctaaacgaaa accgcagtaa tagcctaaat 780

acccacgcga agctattcac gcacctaggc ggctacgatg atgatttaaa tactacgttt 840

gataagggtc gcatggactt gatccctaac gagaacttat taccgtatgc ggggggcgat 900

acagacgcaa ccttaagagt tagaaacgtt ttcgcccgtg aactacagca agaccgttta 960

ttaactaact tctatgttaa tctacttcac ccggctagcc gagcctttga gaaaatggag 1020

caaaacgggg ttaaggttaa cgccgagaaa ttagaaaagt tgggcgaaga actagaacaa 1080

gagaagaacc aatatctggc ggaaatgttc gcaatgatgc caagaccgct agccctgaaa 1140

taccgcggcg aaacccgtat taaatcggct atcgttaaag attatctttt tggcgcgcgt 1200

ggcctaggtc taaagcccgt aatgttcagc gagaagaatc acgaaccgct aacaaacaaa 1260

agccacttga ataactttga cgacccggaa aagtacccgg aggcacacaa atttattcaa 1320

ttggttaaca agtggaacca agcggataag gcacagggga cctattgccg tggattcctt 1380

aagcaccgcc gggaggatgg atatttccac ccttcctata tgttgtttaa gggtgccttt 1440

gaaaacgcta gcggtacaga cgacagcggg accgttaccg ggcgactagc agctaaagac 1500

ccggcgatac aaaccttacc taagaaaacg agcctagcga aaagattgcg tgcatgttac 1560

gagccgccgg aaggttatag cattgttgag atagacttct cacagggtga gttaagggtt 1620

tgtgcggtta tggctaatga acccgctatg aaacaagcgt acaacgaagg cttagacttg 1680

cacgttatct caggcggcgc gacggcgggc ctaacctacg agcaaatgat cgagttagag 1740

aaaaccgacg cggcgcttta taaaaagtac cgtaactatg gtaaaccgca gaacttcggc 1800

cttttgtatg gtatgagttg gggaggcttc caaacgtatg ccaaggacca atacgggatc 1860

ttactatcgg accgtgaagc acaaaacatg cgctttaact tctttgatac atacgcccgc 1920

ttaccggatt ggcacgaaga aacaaaagag tttgctagac agtacggtta cattagatca 1980

cctttaggcc gtgtgcgaca cttaccactg attaattcac gggataattt cctacgaagt 2040

caggccgagc gccaagcggt taacagtggc gtgcagtcca cactatcgga cctacttatt 2100

tacggtatgg cccgcttcag agcacaatac ggcgaccctg acgaagttag atttatggca 2160

atgatccacg atgcgctagt gagctacatc aaaacggacc aattaagtta ttggattcca 2220

attttgcaag ctataatgca agacaccgaa attttatata aagtgtttgg ttgggaggtt 2280

gacatcccat ttatagcaga cgcggaacat tcagaaacta actttgcaga aatgcgagaa 2340

tacaaagagg cagcttaa 2358

Claims

1. The novel vibrio alginolyticus phage is named as vB _ ValP _ PJ32, and the preservation number is CGMCC No. 18860.

2. Use of a Vibrio alginolyticus bacteriophage of claim 1 in the manufacture of a medicament for preventing and treating a disease caused by Vibrio alginolyticus infection in aquaculture.

3. A phage composition comprising the vibrio alginolyticus phage vB ValP PJ32 of claim 1.

4. The phage composition of claim 4, further comprising one or more of mutants of phage vB _ ValP _ PJ 32; the mutant has a homology of not less than 90% with the corresponding phage.

5. A bacteriophage pharmaceutical preparation comprising as an active ingredient the Vibrio alginolyticus bacteriophage or the bacteriophage composition according to any one of claims 1 to 5; preferably, the phage drug formulation further comprises phages to other specific pathogenic bacteria.

6. A phage pharmaceutical preparation according to claim 5, wherein said pharmaceutical preparation is in the form of an oral, topical or parenteral dosage form.

7. The phage drug formulation of claim 5, further comprising a pharmaceutically acceptable carrier.

8. A prawn feed additive comprising Vibrio alginolyticus phage or phage composition according to any one of claims 1 to 4, preferably wherein the concentration of each phage in the feed is at least 1 × 10⁹PFU/g。

9. An environmental disinfectant, characterized in that the effective component comprises the vibrio alginolyticus phage or the phage composition of any one of claims 1-4, and other active components for inhibiting or eliminating viruses and bacteria in the environment, preferably the phage is used at a concentration of 1 × 10⁹PFU/ml or more.

10. A test kit comprising a Vibrio alginolyticus bacteriophage or bacteriophage composition according to any one of claims 1 to 4.

11. A biological bacteriostatic agent for disinfecting fresh food, which comprises the Vibrio alginolyticus bacteriophage or the bacteriophage composition according to any one of claims 1 to 4; the use method of the biological bacteriostatic agent comprises the following steps: the surface of the fresh food is soaked or sprayed for disinfection to inhibit the proliferation of the vibrio alginolyticus in the processing or fresh-keeping process of the product.