Vibrio alginolyticus bacteriophage and application thereof

Abstract

The invention relates to the technical field of microorganisms, and provides a vibrio alginolyticus phage and application thereof in order to screen and accumulate a phage with a good lysis effect, the latin classification name of the vibrio alginolyticus phage is Vibrio alginolyticus phage, the vibrio alginolyticus phage is named as GYN01, the vibrio alginolyticus phage is preserved in the China General Microbiological Culture Collection Center, the preservation number is CGMCC No.46683, and the vibrio alginolyticus phage is named as GYN01. The invention relates to an icosahedron-shaped tail sheath, which has a typical tadpole-shaped composite symmetrical structure, comprises an icosahedron head part and a retractable tail sheath, a tail plate visible at the tail end of the tail part, a non-retractable tail part and a hollow tail tube visible in the center of the tail sheath, has good temperature stability and a wider acid-base tolerance range, and is sensitive to ultraviolet rays; the compound has good cracking capacity and killing effect on pathogenic vibrio alginolyticus, is good in safety and environment-friendly, and can be used for preventing and treating vibrio alginolyticus diseases in the aquaculture process.

Description

A Vibrio alginolyticus bacteriophage and its application Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Vibrio alginolyticus bacteriophage and its applications. Background Technology

[0002] Vibrio alginolyticus is an important pathogen in aquaculture, a conditionally pathogenic bacterium widely distributed in marine environments within the Vibrio family. It harms many important economically farmed species (such as fish, shrimp, crabs, and shellfish), exhibiting strong pathogenicity to aquaculture organisms and causing enormous losses to aquaculture industries worldwide. It poses a significant threat to the aquaculture environment and can also lead to gastroenteritis and other diseases in humans through seafood contamination. The pathogenic process of Vibrio alginolyticus generally involves five steps: adhesion, invasion, colonization, proliferation, and toxin production. The virulence varies considerably among different species, and its pathogenicity is related to the types and quantities of virulence factors it carries. Currently, Vibrio alginolyticus has developed multidrug resistance, and its control has become a global public health issue. Therefore, there is an urgent need to find alternative solutions such as bacteriophage therapy.

[0003] Bacteriophages are a class of viruses with extremely high specificity, capable of infecting microorganisms such as bacteria, fungi, algae, actinomycetes, or spirochetes. They are also known as specific viruses and natural enemies of bacteria. Bacteriophage control primarily utilizes the lytic action of virulent bacteriophages to kill pathogenic bacteria, thereby treating and preventing bacterial diseases in aquatic animals. A single bacteriophage typically infects and lyses only one or a specific type of host bacteria, without disrupting the structure of other bacterial communities within the aquatic product or in the aquatic environment, and without harming the aquatic animal's cells. After invading the host, virulent bacteriophages rapidly replicate and multiply exponentially until the host lyses, then infect and lyse other host bacteria, repeating this process repeatedly. This allows for the rapid and effective killing of pathogenic bacteria. Bacteriophage therapy also has advantages such as low resistance development, no drug residues, short development cycles, and low costs. Overall, using bacteriophages for control not only has a good killing effect on pathogenic bacteria in aquatic animals but is also environmentally friendly, and can completely replace antibiotics for the prevention and control of bacterial diseases and even drug-resistant pathogens. Macrophages are a class of tailed bacteriophages with genomes longer than 200 kbp. Due to limitations in isolation and sequencing technologies, macrophages are difficult to purify, culture, or obtain through metagenomic assembly. To date, the number of macrophage species obtained through metagenomic assembly is less than one thousand, and only about one hundred macrophage species have been isolated and cultured. Compared to ordinary bacteriophages, macrophage genomes contain more genes related to DNA replication and metabolism. The proteins encoded by these genes may be able to perform functions normally performed by bacterial proteins, thereby reducing the phage's dependence on the host organism and increasing its ability to lyse host bacteria. Therefore, utilizing macrophages for the control of pathogenic bacteria has significant application potential.

[0004] Based on the specific characteristics of bacteriophages, it is necessary to continuously screen and accumulate bacteriophages that are effective against different pathogenic strains and have good lysis properties, and to establish and expand a bacteriophage library covering various pathogenic species and strains, so as to respond to different aquatic diseases in a timely manner and lay the foundation for developing new biological agents for the prevention and control of Vibrio alginolyticus bacteriophages. Summary of the Invention

[0005] To address the problems mentioned above in the background section, this invention aims to propose an alginolytic vibrio phage GYN01 and its applications. This alginolytic vibrio phage GYN01 has good lysis ability and killing effect, and can effectively prevent and control the pollution and infection of alginolytic vibrio during aquaculture. It is a phage strain that can be studied and applied on a large scale, which is beneficial to the sustainable development of aquaculture.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: The first aspect of the technical solution disclosed in the present invention is: a Vibrio alginolyticus phage, the Latin classification name of the Vibrio alginolyticus phage being named GYN01, which was deposited at the China General Microbiological Culture Collection Center on October 17, 2025, with the accession number CGMCC No. 46683.

[0007] The Vibrio alginolyticus phage of the present invention exhibits strong lytic activity against specific Vibrio alginolyticus, with large plaques, a lysis rate of approximately 83 PFU / cell in infected cells, short adsorption time, a wide acid-base tolerance range, good temperature tolerance, and extreme sensitivity to ultraviolet light. Observation of the morphology of the phage and the resulting plaques, combined with genome sequencing and comparison results, confirms it as a novel Vibrio alginolyticus schistolyticus phage.

[0008] Furthermore, transmission electron microscopy revealed that the *Vibrio alginolyticus* phage of the present invention possesses a typical tadpole-shaped complex symmetrical structure, including an icosahedral head and a retractable tail sheath. A tail plate is visible at the end of the tail. The head measures 120 nm (±5 nm) × 55 nm (±5 nm). The tail is non-retractable, with a diameter of approximately 10 nm (±1.5 nm) and a length of approximately 25 nm (±2 nm). A hollow tail tube is visible in the center of the tail sheath. Based on the latest virus classification system of the International Committee on Taxonomy of Viruses (ICTV) and its phylogenetic tree analysis, *Vibrio alginolyticus* phage GYN01 is identified as belonging to the genus *Schizotequatrovirus*, family Myoviridae, and the giant phage *jumbo phage*.

[0009] Secondly, this invention discloses the application of Vibrio alginolyticus bacteriophage in the prevention and control of Vibrio alginolyticus, wherein the Vibrio alginolyticus is Vibrio alginolyticus VAFJ201706 or Vibrio alginolyticus VAFJ202412.

[0010] Thirdly, the present invention also discloses the application of Vibrio alginolyticus bacteriophage in the preparation of biological agents for the prevention and treatment of Vibrio alginolyticus, wherein the Vibrio alginolyticus is Vibrio alginolyticus VAFJ201706 or Vibrio alginolyticus VAFJ202412.

[0011] Fourthly, the present invention discloses a biological agent for preventing and controlling Vibrio alginolyticus, the biological agent containing Vibrio alginolyticus bacteriophage GYN01.

[0012] Fifthly, the present invention also discloses a method for preparing a biological agent for preventing and treating Vibrio alginolyticus, comprising the following steps: (1) Vibrio alginolyticus is added to liquid culture medium 2116E and cultured with shaking at 37°C; (2) Phage solution is added and cultured on a shaker until the mixture becomes clear. The mixture is centrifuged at 4°C and the supernatant is filtered through a 0.22 μm filter membrane to obtain phage fluid; (3) RNase A and Dnase I enzymes are added to the phage fluid. After being placed at room temperature for 30 min, solid NaCl is added to a final concentration of 1 mol / L. After the NaCl is completely dissolved, the mixture is placed in an ice bath for 1 h; (4) After the ice bath, the volume of the supernatant is measured after centrifugation at 4°C. Solid polyethylene glycol is added and placed in an ice-water mixture overnight after the polyethylene glycol is completely dissolved. The mixture is centrifuged at 4°C the next day. The precipitate after centrifugation is washed with SM buffer and resuspended; (5) Phage particles are prepared by CsCl density gradient centrifugation to obtain a biological agent for Vibrio alginolyticus.

[0013] Sixthly, the present invention also discloses a method for preventing and controlling harmful infections caused by Vibrio alginolyticus in vitro, which utilizes the lytic effect of Vibrio alginolyticus bacteriophage on Vibrio alginolyticus to kill Vibrio alginolyticus in vitro, wherein the Vibrio alginolyticus is Vibrio alginolyticus VAFJ201706 or Vibrio alginolyticus VAFJ202412.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The alginolyticus bacteriophage GYN01 of the present invention is a new lytic myotail giant bacteriophage, which has high specificity against the pathogenic Vibrio alginolyticus (Vibrio alginolyticus VAFJ201706 and / or Vibrio alginolyticus VAFJ202412), and can specifically kill and prevent the above two Vibrio alginolyticus diseases. At the same time, no lysogenic inhibitors or integrase genes were found in this bacteriophage, nor were antibiotic resistance genes or virulence genes detected. It can replace antibiotics as a safe and effective biological agent for the prevention and control of Vibrio alginolyticus in aquaculture.

[0015] 2. The alginolytic Vibrio phage GYN01 of the present invention maintains stable activity after treatment at 37~60℃ for 1h, and still has lytic activity after incubation at 37℃ for 1h within a pH range of 4~10, exhibiting good temperature stability and a wide acid-base tolerance range. At the same time, its titer decreases significantly after irradiation under ultraviolet light for 30min, and decreases by about 5 orders of magnitude after irradiation for 40min, proving that the alginolytic Vibrio phage GYN01 is extremely sensitive to ultraviolet light. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 shows the plaque morphology of Vibrio alginolyticus phage GYN01 of the present invention; Figure 2 shows an electron micrograph of Vibrio alginolyticus phage GYN01 of the present invention; Figure 3 shows the 134 functionally annotated genes in the genome of Vibrio alginolyticus phage GYN01 of the present invention obtained by alignment with the NCBI nr database; Figure 4 shows the thermostability test results of Vibrio alginolyticus phage GYN01 of the present invention; Figure 5 shows the acid-base tolerance test results of Vibrio alginolyticus phage GYN01 of the present invention; Figure 6 shows the ultraviolet sensitivity test results of Vibrio alginolyticus phage GYN01 of the present invention; Figure 7 shows the one-step growth curve test results of Vibrio alginolyticus phage GYN01 of the present invention; Figure 8 shows the in vitro antibacterial experiment results of Vibrio alginolyticus phage GYN01 of the present invention; Figure 9 shows the chloroform sensitivity test results of Vibrio alginolyticus phage GYN01 of the present invention; Figure 10 shows the phylogenetic tree analysis of Vibrio alginolyticus phage GYN01 of the present invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0019] Unless otherwise specified, the raw materials and equipment used in this invention can be purchased from the market or are commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0020] The Vibrio alginolyticus phage GYN01 of this invention has been deposited in accordance with regulations. The deposit information is as follows: Phage name: Vibrio alginolyticus phage GYN01; Depository institution: China General Microbiological Culture Collection Center (CGMCC); Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: October 17, 2025; Deposit number: CGMCC No. 46683; Postal code: 100101; Latin classification name: Vibrio alginolyticus phage.

[0021] Example 1: Isolation and purification of Vibrio alginolyticus phage GYN01: 1.1 Sample collection; The water samples of this invention were taken from seawater in the large yellow croaker farming area of ​​Ningde, Fujian, and brought back to the laboratory under refrigeration (stored in a refrigerator at 4°C).

[0022] 1.2 Isolation and purification of bacteriophages; Water samples collected from aquaculture areas were sterilized by passing them through a 0.22 μm filter membrane; To enrich the target bacteriophages in the aquaculture water, 20 mL of sterilized water sample was filtered, 60 mL of 2116E medium was mixed with 1 mL of Vibrio alginolyticus bacterial solution, and the mixture was placed in a constant temperature shaker at 37℃ for 24 h. Centrifuge the mixture at 4200 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane to remove residual bacterial cells. Repeat this process at least three times. Isolate and detect bacteriophages using the double-layer plate method: Prepare 1.5% agar 2116E solid medium, autoclave it, and then place it at room temperature (approximately 40-60°C). Pour 10-15 mL into a petri dish, spread it evenly on the bottom, and let it solidify at room temperature for 30 min. Use this as the bottom layer medium. Mix 100 μL of the filtered supernatant with 100 μL of Vibrio alginolyticus and incubate at 28°C for 5 min. Add 5 μL of autoclaved and cooled agar to 55°C. In a 0.7% 2116E semi-solid nutrient agar medium, the agar was shaken to mix thoroughly and quickly poured onto a prepared bottom agar plate. The plate was rotated to distribute the agar evenly to form the top agar layer. After the agar solidified, the plate was incubated overnight at 37°C, inverted, and the presence of phage plaques was observed. The phage plaques were observed to be round and transparent spots with a diameter of approximately 1 ± 0.5 mm, as shown in Figure 1. The largest and most transparent phage plaque was picked and placed in 1 mL of SM buffer and incubated overnight at 4°C. The next day, the plaque was centrifuged at 4200 rpm for 10 min at 4°C. The supernatant was filtered through a 0.22 μm filter membrane. The filtrate was purified using the double-layer plate method, and the purification was repeated at least 3-5 times to obtain phage plaques of consistent size and morphology. This phage plaque was named GYN01.

[0023] Example 2: Amplification and enrichment of Vibrio alginolyticus phage GYN01: (1) 500 μL of Vibrio alginolyticus VAFJ201706 in the logarithmic phase was added to 500 mL of liquid culture medium 2116E and cultured with shaking at 37°C; (2) 50 mL of phage solution was added and cultured on a shaker for 18 hours until the mixture became clear. The mixture was centrifuged at 4200 rpm for 10 min at 4°C. The supernatant was filtered using a 0.22 μm filter membrane to obtain the phage solution; (3) RNase A and DNase I enzymes were added to the phage solution to a final concentration of 1 μg / mL. The mixture was placed at room temperature for 30 min. Solid NaCl was added to a final concentration of 1 mol / L. After the NaCl dissolved, the mixture was placed on ice for 1 h; (4) The mixture was centrifuged at 12000 rpm for 10 min at 4°C. After min, the volume of the supernatant was measured; solid polyethylene glycol (PEG8000) was added to a final concentration of 10% (w / v), and after complete dissolution, it was placed in an ice-water mixture overnight; the next day, it was centrifuged at 12000 rpm for 15 min at 4℃, and the precipitate after centrifugation was washed 3 times with SM buffer and resuspended in SM buffer; (5) the phage titer of the resuspended phage after expansion culture was determined by the double-layer plate method; (6) high-purity phage particles were prepared by CsCl density gradient centrifugation and the high-purity phage particles were observed by electron microscopy. Specifically, CsCl solutions of different densities (1.3 g / mL, 1.5 g / mL and 1.7 g / mL) were added to the ultra-bright centrifuge tubes in descending order of density. After each CsCl solution of a certain density was added, a mark was made with a marker, and then the phage solution was added and balanced. The error should be less than 0.001. The cells were centrifuged at 34200 rpm at 4°C for 3 h. After centrifugation, phage particles located near the 1.5 g / mL CsCl density band were collected.

[0024] Example 3: Morphological observation and identification of Vibrio alginolyticus phage GYN01: The phage was directly observed by transmission electron microscopy using uranyl acetate negative staining, as shown in Figure 2. Transmission electron microscopy showed that Vibrio alginolyticus phage GYN01 of the present invention has a typical tadpole-shaped complex symmetrical structure, including an icosahedral head and a retractable tail sheath. A tail plate is visible at the end of the tail. The head size is 120 nm (±5 nm) × 55 nm (±5 nm). The tail is not retractable, with a tail diameter of about 10 nm (±1.5 nm) and a length of about 25 nm (±2 nm). A hollow tail tube is visible in the center of the tail sheath.

[0025] Example 4: Genome Sequencing and Bioinformatics Analysis of Vibrio alginolyticus Phage GYN01: 4.1 Genome Sequencing of Vibrio alginolyticus Phage GYN01; First, DNA was extracted, and the specific steps are as follows: The phage genome was extracted using a viral nucleic acid extraction kit (Omega Bio-Tek EZNA® Viral DNA Kit). After nucleic acid extraction, the whole phage genome was sequenced using the DNBSEQ-T7 platform. To ensure the reliability of subsequent information analysis results, we used FASTP to filter and quality control the obtained raw sequencing data, cut the adapter, and remove low-quality reads and reads with a high proportion of N to obtain clean reads. The clean reads were then assembled using metaSPAdes software for de novo assembly. At the same time, different k-mer lengths were selected for testing and the best assembly results were obtained. Then, bwa software was used to align the clean reads to the assembled genome sequence for statistical coverage.

[0026] Sequencing results showed that the full-length genome of this bacteriophage was 246,912 bp (its nucleotide sequence is shown in SEQ ID NO. 1), with a GC content of 41.32%. After bwa alignment, a total of 400 open reading frames (ORFs) were predicted, of which 134 (33.5%) were functionally annotated by alignment with the NCBI nr database (as shown in Figure 3), including genes related to DNA replication, structural proteins, packaging proteins, metabolism, and host lysis. In addition, 27 tRNA genes were predicted in the genome, with an average gene length of 200 bp (its nucleotide sequence is shown in SEQ ID NO. 2~28).

[0027] 4.2 Genome alignment of Vibrio alginolyticus phage GYN01: Phage genome alignment was performed using NCBI's BLASTN online tool. The results showed that the phage with the highest homology was Vibrio phage ValKK3, with 98.58% homology in 98% of the coverage area. This indicates that GYN01 is a new Vibrio alginolyticus phage (see Table 1). Its nucleic acid is double-stranded linear DNA and it belongs to the order Tailed Phages.

[0028] Table 1. Top 5 bacteriophages with the highest similarity to GYN01: .

[0029] 4.3 Virulence and resistance gene detection of Vibrio alginolyticus phage GYN01: Virulence gene detection of the phage was performed in the Virulence Factor Database (VFDB). No lysogenic inhibitors or integrase genes were found. No virulence genes were found. Antibiotic resistance genes were also detected in the Comprehensive Antibiotic Resistance Database (CARD). This indicates that the phage will not enhance the host's virulence intensity or cause antibiotic resistance pollution in the surrounding water environment. It is safe and environmentally friendly.

[0030] Example 5: Biochemical stability determination of Vibrio alginolyticus phage GYN01: 5.1 Thermal stability test; Take 500 μL of phage suspension (10 8 The bacterial count (PFU / mL) was divided into multiple groups and incubated at 37°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 20 min, 40 min, and 60 min, respectively. The number of bacterial plaques was determined using the double agar plate method to obtain the results of the heat stability test.

[0031] As shown in Figure 4, the Vibrio alginolyticus phage GYN01 of the present invention remained active after treatment at 37~60℃ for 1 hour, but was completely inactivated after treatment at 70℃ for 40 min or 80℃ for 20 min. This indicates that the Vibrio alginolyticus phage GYN01 of the present invention has strong titer stability in the temperature range of 37~60℃. In actual aquaculture production, even in the hot summer, the Vibrio alginolyticus phage GYN01 can maintain its activity. The number of bacterial plaques was determined by the double agar plate method to obtain the pH stability test results.

[0032] 5.2 pH tolerance test; 100 μL of phage suspension (10 8 Transfer (PFU / mL) to 900 μL of 2116E medium at pH 2-13 and incubate at 37°C for 1 hour.

[0033] As shown in Figure 5, Vibrio alginolyticus phage GYN01 retained lytic activity after incubation at 37℃ for 1 h within a pH range of 4–10; however, its titer decreased significantly under conditions of pH < 4 or pH > 10. These results indicate that Vibrio alginolyticus phage GYN01 has a wide acid-base tolerance range, and is particularly suitable for environments with a pH range of 4–10.

[0034] 5.3 Ultraviolet sensitivity test; Take 20 mL of Vibrio alginolyticus phage GYN01 stock solution (10 8PFU / mL was poured into a culture dish and irradiated at a specific intensity (20 W, 35 cm). 100 μL of sample was collected every 10 minutes for analysis over 60 minutes. Phage GYN01, which was not exposed to UV irradiation, served as a control. The number of phage plaques in the samples was determined using the double agar plate method to obtain pH stability test results.

[0035] As shown in Figure 6, Vibrio alginolyticus phage GYN01 is extremely sensitive to ultraviolet light. After continuous irradiation for 30 minutes, its titer decreased significantly; after irradiation for 40 minutes, its titer decreased by approximately five orders of magnitude. The results indicate that Vibrio alginolyticus phage GYN01 of this invention has poor stability under ultraviolet conditions.

[0036] Example 6: Detection of the host range of Vibrio alginolyticus phage GYN01: In this example, several common species of Vibrio aquatic animal pathogens were selected, including 5 species of Vibrio alginolyticus (VAFJ201706, isolated from diseased large yellow croaker; VAFJ202412, isolated from diseased sea bass; 2014V-1011, isolated from diseased Pacific white shrimp; 2014V-1012, isolated from diseased oyster; FA2, isolated from diseased oyster) and 2 species of Vibrio harveyi (S090801, isolated from diseased oyster; VJFJ201701, isolated from diseased large yellow croaker).

[0037] The above-mentioned aquatic animal pathogens were isolated and preserved in our laboratory. The specific procedures are as follows: Select the lesion site of the diseased aquatic animal sample and take 0.5g of tissue under aseptic conditions; add the tissue to 4.5mL of sterile protein buffer (BPW), homogenize thoroughly, and centrifuge at low speed; take 1mL of supernatant and incubate at 37℃ for 12h, then dilute the homogenate 1:1000 and streak it onto TCBS solid medium. Incubate at 37℃ for 24h. Vibrio alginolyticus forms large yellow (sucrose fermentation) colonies, and Vibrio harveyi forms green (non-sucrose fermentation) colonies; pick single colonies with typical morphology (such as yellow or green, smooth, and with regular edges on TCBS solid medium) from the plates and perform three streak purifications on 2216E agar plates to obtain pure cultures. The pure cultures are then identified by 16S sequencing.

[0038] Based on the pure cultures of various aquatic animal pathogens obtained above, the lysis spectrum of Vibrio alginolyticus phage GYN01 was detected using the spot method. 100 μL of the logarithmic growth phase suspension of the aforementioned pathogenic Vibrio bacteria was added to 5 mL of 0.65% (w / v) 2116E agar melted at 48℃, poured onto a plate containing 1% (w / v) sterile bottom agar, and allowed to solidify at room temperature. Then, 5 μL of purified phage suspension (approximately 10 μL of pure culture) was added to the plate. 8 Add PFU (2,5-dimethylformamide) to the surface of freshly inoculated bacteria, allow to air dry, incubate at 37°C overnight, and observe the formation of lysis zones.

[0039] The test results are shown in Table 2. The Vibrio alginolyticus phage GYN01 can lyse two types of Vibrio alginolyticus from fish, but has no lysing ability against other Vibrio alginolyticus and Vibrio harveyi, indicating that the phage has strong specificity.

[0040] Table 2. Lysis spectrum of Vibrio alginolyticus phage GYN01: .

[0041] Note: "+" indicates that a clearly visible plaque can be formed; "-" indicates that there is no change.

[0042] Example 7: Detection of the Optimal Multiple of Infection (MOI) of Vibrio alginolyticus phage GYN01: The multiple of infection (MOI) refers to the ratio of the number of bacteriophages to the number of host bacteria when the host bacteria are infected by the phage. The optimal MOI is the ratio at which the phage can maximally infect the host bacteria under the same conditions. According to MOI ratios of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100, 50 μL of proportionally diluted phage and 50 μL of host bacteria were added to 900 μL of SM Buffer. The mixture was incubated at 37°C in a shaker until the mixture became clear. It was then centrifuged at 12000 rpm for 3 min at 4°C. The supernatant was filtered through a 0.22 μm filter to obtain the phage solution. Simultaneously, phage without host bacteria, host bacteria without phage, and a blank control group were set up. The phage titer in the filtrate was determined using the double-layer plate method. The MOI with the highest phage titer is the optimal multiple of infection (MOI).

[0043] As shown in Table 3, the difference in titer of Vibrio alginolyticus phage GYN01 is minimal when the MOI is 0.1 or 0.01, while the efficiency of phage infection of the host remains almost unchanged when the MOI is below 0.01. Therefore, the optimal MOI for Vibrio alginolyticus phage GYN01 is 0.01.

[0044] Table 3. Determination of the optimal multiple of infection (MOI) for phage GYN01: .

[0045] Example 8: Determination of the one-step growth curve of Vibrio alginolyticus phage GYN01: The phage solution and host bacterial suspension were mixed at the optimal MOI ratio (MOI = 0.01). After standing at 37°C for 15 min, the mixture was centrifuged at 12000 rpm for 10 min at 4°C to remove phage particles not adsorbed onto the host bacteria. The precipitate was then resuspended in 1 mL of 2116E liquid medium, and this step was repeated 3-4 times. 100 μL of the resuspended solution was added to 10 mL of fresh 2116E liquid medium and vortexed to mix. This time was recorded as T0=0. Every 10 min, 100 μL of culture medium was taken for titer determination using the double-layer plate method. The experiment was set up in 3 parallel groups, and the average value was recorded.

[0046] As shown in Figure 7, under the condition of MOI = 0.01, after Vibrio alginolyticus phage GYN01 infected logarithmic-phase host bacteria, its latency period was 0.4 h, the lysis phase peaked at 1 h, and entered the plateau phase at 3 h, with a lysis time of approximately 3.1 h. Based on the lysis yield, the lysis yield of Vibrio alginolyticus phage GYN01 was calculated to be 83 PFU / cell. The lysis yield characterizes the number of phages ultimately released by the host, and its calculation formula is as follows: Lysis yield = Phage titer at the end of lysis / Host bacterial concentration at the initial stage of infection.

[0047] Example 9: In vitro antibacterial experiment of Vibrio alginolyticus phage GYN01: The host bacteria were cultured in a shaker at 37°C until the logarithmic growth phase. The bacterial concentration was measured using a gradient method, and the bacterial solution was diluted to 1×10⁻⁶. 8 The bacterial suspension was centrifuged at 4200 rpm for 5 min at 4℃, the supernatant was discarded, and an equal volume of 2116E liquid medium was added to resuspend the suspension. 100 μL of the resuspended bacterial suspension was added to a sterile 96-well plate, followed by 100 μL of phage dilution (diluted at MOI=0.01), and the suspension was gently mixed by pipetting. 100 μL of chloramphenicol (50 mg / L) was added as a positive control, and 100 μL of 2116E liquid medium was added as a blank control. Each group was divided into three replicates. The sterile 96-well plate was placed on a microplate reader to measure the absorbance (OD600), and the OD600 reading was recorded every 1 h.

[0048] After adding GYN01 (MOI = 0.01) during the logarithmic growth phase of the host bacteria, its OD600 value slightly increased within 4 hours, then decreased significantly, and slightly rebounded after 8 hours, as shown in Figure 8. The *Vibrio alginolyticus* phage GYN01 exhibited an overall antibacterial effect comparable to chloramphenicol. This indicates that the *Vibrio alginolyticus* phage GYN01 of the present invention has good in vitro antibacterial activity and can be used to kill *Vibrio alginolyticus* in vitro, thus preventing harmful infections caused by *Vibrio alginolyticus* from outside the body.

[0049] Example 10: Sensitivity determination of Vibrio alginolyticus phage GYN01 to chloroform: The drop method was used for verification. The specific steps are as follows: 1 mL of phage fluid was added to 30 μL and 300 μL of chloroform solution respectively, mixed well, and allowed to stand at room temperature for 30 min. The upper layer solution after separation was taken. The host bacteria were cultured to the logarithmic phase. 100 μL of bacterial solution was mixed with 5 mL of 2116E semi-solid medium containing 0.65% agar and poured onto a 2116E agar plate. After standing at room temperature for 5 min, 10 μL of the above upper layer solution was dropped onto the solidified plate. After the dropped liquid dried, the plate was placed in a 37°C constant temperature incubator for incubation. The formation of phage plaques was observed the next day.

[0050] The results of the chloroform sensitivity test of Vibrio alginolyticus phage GYN01 are shown in Figure 9. The phage still showed obvious phage plaques after chloroform treatment, indicating that Vibrio alginolyticus phage GYN01 of the present invention is not sensitive to chloroform, which means that its capsid does not contain lipid substances.

[0051] Example 11: Phylogenetic Tree Construction: A phylogenetic tree was constructed using MEGA 6.0 software based on the amino acid sequence of the large subunit of the terminase, as shown in Figure 10. The neighbor-joining method was used, and 1000 guided replications were performed. The results showed that the *Vibrio alginolyticus* phage GYN01 of this invention is most genetically closely related to *Vibrio phage phi-pp2*, representing different species of macrophages within the genus *Schizotequatrovirus*. BLAST sequence alignment revealed that the genomic sequences of *Vibrio alginolyticus* phage GYN01 and *Vibrio phage phi-pp2* shared 87.47% homology within an 84% coverage region.

[0052] The detailed description of the above embodiments is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. Based on the embodiments of the present invention, they are only used to illustrate the technical solutions of the invention and not to limit it. Other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention, as long as they do not depart from the spirit and scope of the technical solutions of the present invention, should be covered within the scope of the claims of the present invention.

Claims

1. A Vibrio alginolyticus bacteriophage, characterized in that, The Latin taxonomic name of the Vibrio alginolyticus phage is Vibrio alginolyticus phage, named GYN01. It was deposited on October 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46683.

2. The application of Vibrio alginolyticus phage as described in claim 1 in the prevention and control of Vibrio alginolyticus.

3. The application of the Vibrio alginolyticus phage as described in claim 1 in the preparation of biological agents for the prevention and control of Vibrio alginolyticus.

4. The application as described in claim 2 or 3, characterized in that, The Vibrio alginolyticus mentioned is either Vibrio alginolyticus VAFJ201706 or Vibrio alginolyticus VAFJ202412.

5. A biological agent for preventing and controlling Vibrio alginolyticus, characterized in that, This biological agent contains the Vibrio alginolyticus phage as described in claim 1.

6. The method for preparing a biological agent for preventing and controlling Vibrio alginolyticus as described in claim 5, characterized in that, The steps include: (1) Add Vibrio alginolyticus to liquid culture medium 2116E and culture with shaking at 37°C; (2) Continue to add phage solution and culture on a shaker until the mixture becomes clear. Centrifuge the mixture at 4°C and filter the supernatant with a 0.22 μm filter membrane to obtain phage solution; (3) Add RNase A and Dnase I enzymes to the phage solution, place at room temperature for 30 min, add solid NaCl to the final concentration of 1 mol / L, and incubate on ice for 1 h after the NaCl is completely dissolved; (4) After the ice bath, centrifuge at 4°C and measure the volume of the supernatant. Add solid polyethylene glycol and incubate in an ice-water mixture overnight after the polyethylene glycol is completely dissolved. Centrifuge at 4°C the next day and wash the precipitate with SM buffer and resuspend the precipitate; (5) Prepare phage particles by CsCl density gradient centrifugation to obtain the biological agent of Vibrio alginolyticus.

7. A method for in vitro prevention and treatment of harmful infections caused by Vibrio alginolyticus, characterized in that, Vibrio alginolyticus bacteriophage as described in claim 1 is used to kill Vibrio alginolyticus in vitro through lysis. The Vibrio alginolyticus is Vibrio alginolyticus VAFJ201706 or Vibrio alginolyticus VAFJ202412.

Images