Bacteriophage polysaccharide degrading enzyme, nucleic acid molecule, expression vector, recombinant cell, bactericidal composition and bactericide and application thereof

Abstract

The present invention belongs to the technical field of microbial control, and specifically relates to a bacteriophage polysaccharide degrading enzyme, a nucleic acid molecule, an expression vector, a recombinant cell, a bactericidal composition, a bactericide and an application thereof. The polysaccharide degrading enzyme Dep193 is a metabolite of bacteriophage VnaB1, and its amino acid sequence is shown in SEQ ID NO.1. The composition and the bactericide of the present invention have the effect of strongly lysing and killing atypical Vibrio, can effectively inhibit the production of resistant bacteria, and achieve a lasting and stable bactericidal effect. The composition has important application value in the prevention and control of atypical Vibrio infectious diseases in marine aquaculture animals.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial control, and specifically relates to a bacteriophage polysaccharide degrading enzyme, a nucleic acid molecule, an expression vector, a recombinant cell, a bactericidal composition, a bactericide and an application thereof. Background Art

[0002] In aquaculture, Vibrio diseases occur frequently and affect a wide range of areas, and are the "culprit" for economic losses in the aquaculture industry. Phage therapy is an effective method of using bacteriophages as a biological control method to treat bacterial diseases. Compared with antibiotics, bacteriophages have the advantages of strong lysis, high specificity, and environmental friendliness. At a time when bacterial resistance is becoming increasingly serious, they have shown unique antibacterial advantages and have gradually become the most promising antibiotic alternative in the fields of aquaculture, medicine, agriculture, and food.

[0003] However, when using bacteriophages to control Vibrio, the pathogens can easily develop resistance to phage infection, making the phages quickly "ineffective", thus limiting the widespread application of phages in aquaculture. Some studies have used phages in combination with probiotics, Chinese herbal medicines, disinfectants, etc. to control pathogens, but the results are not ideal. Phage-derived polysaccharide depolymerases can remove bacterial extracellular "barriers" by specifically degrading bacterial surface polysaccharides (such as capsular polysaccharides, lipopolysaccharides, extracellular polysaccharides, etc.), assisting phages in adsorbing and lysing bacteria. Utilizing this characteristic of polysaccharide degrading enzymes and combining them with phages can greatly improve the efficiency of pathogen removal. Atypical Vibrio is a common conditional pathogen. The outbreak of pathogens often causes large-scale deaths of abalone, causing serious economic losses to the aquaculture industry. However, there are currently no reports on polysaccharide degrading enzymes and phage compositions for removing atypical Vibrio. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a bacteriophage polysaccharide degrading enzyme, a nucleic acid molecule, an expression vector, a recombinant cell, a bactericidal composition, a bactericide and applications.

[0005] The present invention is achieved through the following technical solutions:

[0006] A bacteriophage polysaccharide degrading enzyme. The polysaccharide degrading enzyme Dep193 is a metabolite of bacteriophage VnaB1, and its amino acid sequence is shown in SEQ ID NO.1.

[0007] The bacteriophage VnaB1 is a phage of Vibrio natrii and can lyse atypical Vibrio. It has been sent to the China Center for Type Culture Collection, address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, and is classified and named Vibrio natriegensphage VnaB1, the deposit date is August 10, 2023, and the deposit number is: CCTCC NO: M20231425.

[0008] A nucleic acid molecule, which encodes the polysaccharide degrading enzyme Dep193.

[0009] The nucleic acid sequence of the nucleic acid molecule is shown as SEQ ID NO.2.

[0010] An expression vector contains the nucleic acid molecule.

[0011] A recombinant cell contains the polysaccharide degrading enzyme Dep193, the nucleic acid molecule or the expression vector.

[0012] An atypical Vibrio bactericidal composition contains the polysaccharide degrading enzyme Dep193 and / or bacteriophage VnaB1.

[0013] The composition comprises the polysaccharide degrading enzyme Dep193 and bacteriophage VnaB1, the final concentration of the polysaccharide degrading enzyme Dep193 is 10 μg / mL, and the final concentration of the bacteriophage is 10 6 PFU / mL.

[0014] A non-typical Vibrio bactericide, comprising the polysaccharide degrading enzyme or the composition.

[0015] The preparation for eliminating atypical Vibrio also includes auxiliary materials acceptable in aquaculture.

[0016] An application of the atypical Vibrio fungicide, and application of the atypical Vibrio fungicide in controlling Vibrio infection in aquaculture.

[0017] Beneficial effects of the present invention:

[0018] The present invention provides a bacteriophage polysaccharide degrading enzyme Dep193, which has a degradation effect on bacterial capsular polysaccharides. The combination of polysaccharide degrading enzyme Dep193 and bacteriophage VnaB1 has a strong effect of lysing and killing atypical Vibrio, can effectively inhibit the generation of resistant bacteria, and achieve a long-lasting and stable bactericidal effect. The composition has important application value in the prevention and control of atypical Vibrio infectious diseases in marine aquaculture animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram showing the results of protein purification of the polysaccharide degrading enzyme Dep193 in Example 1 of the present invention.

[0020] Figure 2 This is a diagram showing the results of enzymatic activity verification of the polysaccharide degrading enzyme Dep193 in Example 2 of the present invention.

[0021] Figure 3 This is a diagram showing the results of polysaccharide degradation verification by the polysaccharide degrading enzyme Dep193 in Example 3 of the present invention.

[0022] Figure 4 This is a plate culture photograph of bacteriophage VnaB1 lysing atypical Vibrio in Example 4 of the present invention.

[0023] Figure 5 This is a diagram showing the antibacterial effect of the combination of polysaccharide degrading enzyme Dep193 and bacteriophage VnaB1 in Example 6 of the present invention.

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0026] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0027] As mentioned above, there are no reports on the synergistic application of atypical Vibrio phages and phage-derived polysaccharide-degrading enzymes, and the existing technology urgently needs to be improved.

[0028] In view of this, in a specific embodiment of the present invention, a bacteriophage polysaccharide degrading enzyme Dep193 is provided, which is composed of 603 amino acids, has a molecular weight of 65.2 kDa, and an isoelectric point of 4.62. The amino acid sequence of the bacteriophage polysaccharide degrading enzyme Dep193 is shown in SEQ ID NO.1.

[0029] MTYPATNSNEATKLIIDGGNQLHEIINEDALTTVTTESGEIPSVRKAITDTFLFQAPITWSEGSDETVFNQLRTFNNETYWAPTATLSTPIPMGATPVGDSNWKLAPIGSNKELIRQWDTEVEERLLGIGSSLYKGSNGEFVQDGDTVPI GTTHLTVQINGITEDVVMSPITSGVVSLLTETSATIGGTPIEFISMNMKPFKTLSDAVSSTNVYVGMSLDIKEYTLGHGGGAIWDVVLTSSVTPNGVDIIQCTGVPTLSLVLRINGAVNTMQIGASTDKTAAQNFSIINAFIDSCEGRLYG VHITIPLESEYGYIFTDATTYLQKTNPDSSCVCEDFTIGPNTNAAGLPTYGSQRRLWMFAEGLVEQNGENTGIGNTAGNSTLIHGLWNPGLVMNVNGENIDNRRVKIFFGALGENYWSIGQGNLTIPYNAGDPSEIDGSNEKRRRHWNIAG KTNDATGYQNKWVIRQDNGHQGWNIGEPNYTYHFTDNSNTVSTLTFMVENRESQSTQLVLKTGDEQVNILQDNSSGGRLLIKNTAGETILQIGADGEIFALKGVNSGNFTTSERNALTGISFGTMVFDSTLQKPVWFSSPSVWRDATGVVV (SEQ ID NO. 1).

[0030] The polysaccharide degrading enzyme Dep193 of the present invention is a prokaryotic expression and purification product of the 193rd open reading frame of bacteriophage VnaB1.

[0031] The Vibrio natriureticus phage VnaB1 has been sent to the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), with a preservation date of August 10, 2023 and a preservation number of: CCTCC NO: M20231425.

[0032] In another specific embodiment of the present invention, a nucleic acid molecule is provided, which encodes a polysaccharide degrading enzyme Dep193, has a length of 1812 bp, and a nucleic acid sequence as shown in SEQ ID NO.2.

[0033]

[0034] Another specific embodiment of the present invention provides an expression vector, which contains the nucleic acid molecule described above.

[0035] The expression vector of the present invention can express the nucleic acid molecule as the bacteriophage polysaccharide degrading enzyme Dep193, which can specifically degrade the surface polysaccharide of atypical Vibrio, and the efficacy of the degrading enzyme will not be described in detail hereinafter.

[0036] The term "expression vector" described in the present invention generally refers to a vector used in molecular biology and genetic engineering to insert foreign genes and express them in host cells. These vectors can be plasmids, bacteriophages, etc.

[0037] Another specific embodiment of the present invention provides a recombinant cell, which contains at least one of the following: the polysaccharide degrading enzyme Dep193, the nucleic acid molecule and the expression vector mentioned above.

[0038] The term "recombinant cell" of the present invention is generally a cell that has been genetically engineered to contain foreign genes or DNA sequences. These foreign genes or DNA sequences can be genes from the same species or other species, which are inserted into the genome of the host cell and can be expressed in the host cell.

[0039] Another specific embodiment of the present invention provides an atypical Vibrio fungicide, which contains the above-mentioned polysaccharide degrading enzyme Dep193 and bacteriophage VnaB1, the final concentration of the polysaccharide degrading enzyme Dep193 is 10 μg / mL, and the final concentration of the bacteriophage is 10 6 PFU / mL.

[0040] In another specific embodiment of the present invention, the fungicide further comprises an auxiliary material acceptable in aquaculture.

[0041] Another specific embodiment of the present invention provides the use of an atypical Vibrio fungicide, wherein the atypical Vibrio fungicide is used in controlling Vibrio infection in aquaculture.

[0042] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0043] In the following embodiments, the materials used are mainly as follows.

[0044] The formula of 2216E medium is: 1 g yeast powder, 5 g peptone, 0.1 g ferric orthophosphate, 1000 mL Chenhai water, pH 7.6.

[0045] The 2216E solid culture medium comprises: 1 g yeast powder, 5 g peptone, 0.1 g ferric orthophosphate, 1000 mL Chenhai water, pH 7.6, and 15 g agar.

[0046] The 2216E semi-solid culture medium comprises: 1 g yeast powder, 5 g peptone, 0.1 g ferric orthophosphate, 1000 mL Chenhai water, pH 7.6, and 5 g agar.

[0047] The LB medium is: 10 g tryptone, 5 g yeast powder, 10 g sodium chloride, and deionized water to 1000 mL, pH 7.0.

[0048] SM buffer: purchased from Sangon Biotechnology Co., Ltd.

[0049] PBS buffer: purchased from Solarbio.

[0050] pRSFDuet-1 vector: purchased from Novagen.

[0051] BL21(DE3) competent cells: purchased from Novozymes.

[0052] PHanta Max Super-Fidelity DNA Polymerase: purchased from Novozymes.

[0053] FastPure Gel DNA Extraction Mini Kit DNA gel recovery kit: purchased from Novozymes.

[0054] ClonExpress® II One Step Cloning Kit: purchased from Novozymes.

[0055] 8%-16% SurePAGE™ protein precast gel: purchased from GenScript Biotech.

[0056] Example 1: Construction and expression of recombinant protein

[0057] 1. PCR amplification of target gene

[0058] (1) Design primers for constructing a polysaccharide-degrading enzyme expression vector. The target gene is 1812 bp in length.

[0059] Upstream primer F (SEQ ID NO.3) is:

[0060] 5'- ACCATCATCACCACAGCCAGACTTACCCAGCAACTAATAG-3';

[0061] Downstream primer R (SEQ ID NO.4) is:

[0062] 5'- GCATTATGCGGCCGCAAGCTTTAAACTACTACACCAGTTG-3' .

[0063] (2) PCR amplification

[0064] The PCR system was: 25 μL PHanta Max Super-Fidelity DNA Polymerase, 2 μL upstream primer F, 2 μL downstream primer R, 2 μL bacteriophage VnaB1 template, and 19 μL sterile ultrapure water.

[0065] The PCR amplification program was 34 cycles of pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 60 s, and complete extension for 5 min.

[0066] (3) Rubber recycling

[0067] Use FastPure Gel DNA Extraction Mini Kit DNA gel recovery kit to recover the target band according to the instructions to obtain the target fragment DNA.

[0068] 2. Construction of recombinant plasmid

[0069] (1) First, linearize the pRSFDuet-1 vector and digest it with restriction endonucleases BamHI and HindIII at 37°C for 30 min.

[0070] (2) The target fragment DNA recovered from the amplified gel and the product after enzyme digestion were connected using ClonExpress® II OneStep Cloning Kit to obtain the pRSFDuet-1-Dep recombinant plasmid.

[0071] (3) The successfully sequenced recombinant plasmid pRSFDuet-1-Dep193 was transformed into BL21 (DE3) competent cells to obtain recombinant cells.

[0072] 3. Expression of recombinant cells

[0073] The recombinant cells were inoculated into 1 L of LB medium (final concentration of kanamycin 50 mg / L) at a ratio of 1:1000 (v / v) and shaken at 37°C until OD600 reached 0.8. Isopropylthiogalactoside (IPTG) with a final concentration of 0.5 mM was added and induced at 16°C for 16 hours. After the induction, all the bacterial liquid was centrifuged at 5000 rpm and 4°C for 15 minutes, the supernatant was discarded and the bacterial liquid was resuspended with 15 mL of PBS solution (pH 7.0).

[0074] 4. Protein Purification

[0075] The collected bacterial liquid was placed in an ultrasonic wall breaking machine at 180W, ultrasonic for 3s and 4s on, for 40 minutes. The broken bacterial liquid was centrifuged at 15000 rpm and 4°C for 30 minutes. After centrifugation, the supernatant was quickly placed in a 4°C chromatography cabinet for protein purification.

[0076] The protein was purified using an AKTA pure protein purifier and a Ni-His affinity column. The elution was performed using 40, 60, 100, and 300 mM imidazole eluents (the buffer was a PBS solution at pH 7.0). The effluents of different gradients were collected and the 100-300 mM effluent was concentrated using a 30 kDa protein concentrator. The protein was verified to be correct using an 8% - 16% SDS-PAGE protein electrophoresis precast gel. The SDS-PAGE results showed that there was a specific band between 55-75 kDa, with a size of approximately 65 kDa (see Figure 1 ), which is consistent with the theoretical molecular weight of the recombinant protein Dep193.

[0077] Example 2 Verification of the activity of polysaccharide degrading enzyme Dep193

[0078] The dot-blot assay was used to determine the lysis of the polysaccharide-degrading enzyme Dep193 against atypical Vibrio spp. Figure 2 ), 1 mL of atypical Vibrio broth in the logarithmic phase was mixed with 2216E semi-solid medium and poured into 2216E solid medium. After solidification, 2 μL of the protein obtained in Example 1 above diluted with PBS at different concentrations was dripped into the medium. At the same time, a control without protein was used. After drying, it was placed in a 28°C incubator for culture. The lowest concentration of polysaccharide degrading enzyme Dep193 was 1.6 ng, which could show an obvious translucent circle on the plate (see Figure 3 ).

[0079] Example 3 Verification of the polysaccharide degradation function of polysaccharide-degrading enzyme Dep193

[0080] Extraction of atypical Vibrio Vibrio atypicus LMG 24781 (T) surface polysaccharide, take 1 L of atypical Vibrio in the logarithmic phase and add 10,000 × gAfter centrifugation for 5 minutes, the precipitate was resuspended in PBS solution, and an equal volume of saturated phenol aqueous solution was added. After thorough vortex mixing, the mixture was incubated at 65°C for 20 minutes, and then extracted three times with chloroform to remove cell debris. After the extraction, the aqueous phase was freeze-dried overnight to obtain crude polysaccharides. The freeze-dried sample was dissolved with PBS, and the polysaccharide content was measured using the phenol-sulfuric acid method. The principle of this method is: polysaccharides and oligosaccharides are hydrolyzed under the action of sulfuric acid to form monosaccharides, and monosaccharides are rapidly dehydrated under the action of concentrated sulfuric acid to form aldehyde derivatives. These aldehyde derivatives then undergo condensation reaction with phenol to form orange-yellow colored compounds. The absorbance OD was determined by colorimetry. 490 Take 50 μL of polysaccharide solution and 50 μL of 5% phenol aqueous solution, add 250 μL of sulfuric acid to the mixture, react for 20 minutes and measure the OD 490 The standard curve was prepared using glucose concentrations of 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, and 0.1 mg / mL. The absorbance OD was measured. 490 To make a standard curve.

[0081] Different experimental groups were set up to verify the degradation function of polysaccharide degrading enzyme Dep193 on EPS. Experimental groups were set up: polysaccharide + polysaccharide degrading enzyme Dep193 and three different control groups: polysaccharide + PBS, polysaccharide + inactivated polysaccharide degrading enzyme Dep193 (the enzyme was boiled at 100°C for 10 minutes, and the protein was inactivated), and PBS + polysaccharide degrading enzyme Dep193. The specific addition amount of polysaccharide / PBS and polysaccharide degrading enzyme / PBS in the above groups was as follows: 900 μL of EPS with a concentration of 1 mg / mL was mixed with 10 μL of polysaccharide degrading enzyme Dep193 with a concentration of 10 μg / mL, and samples were taken at 0 h and 1 h, respectively. The reducing sugar content was detected by 3,5-dinitrosalicylic acid (DNS) colorimetric method. The principle of this method is: under alkaline conditions, dinitrosalicylic acid (DNS) and reducing sugar undergo redox reaction to generate 3-amino-5-nitrosalicylic acid. The product is brown-red under boiling conditions, and the color depth is proportional to the reducing sugar content within a certain concentration range. The reducing sugar content is determined by colorimetry. The specific operation is: add 50 μL sample and 100 μL DNS and boil for 5 minutes. Use an enzyme marker to measure the reducing sugar content at OD 540 The experiment was performed in triplicate and the results are shown in Figure 4 , OD of the control group before and after degradation 540 There was no obvious change, and reducing sugar was detected in EPS under the action of Dep193 after 1 h, indicating that the surface polysaccharides of atypical Vibrio were degraded by the polysaccharide-degrading enzyme Dep193.

[0082] Example 4 Screening of atypical Vibrio phages

[0083] Use SM buffer to dilute the above phage VnaB1 in different gradients, take 1 mL of the bacterial solution of atypical Vibrio in the logarithmic growth period and 1 mL of the filtrate of different dilution gradients, mix them, add 5 mL of semi-solid culture medium melted and pre-cooled to 48°C after adsorption on a shaker at 28°C for 30 minutes, pour them on a 2216E solid plate after mixing, and place them in a 28°C incubator for overnight culture after the upper agar solidifies. If plaques appear, it is a phage infected with atypical Vibrio. The morphology of the phage plaques formed by phage VnaB1 on the atypical Vibrio plate is: there is a circle of translucent halo around the clear lysis center (see Figure 1 ).

[0084] Example 5 Combination and sterilization range of polysaccharide degrading enzyme Dep193 and bacteriophage VnaB1 composition

[0085] In bacteriophage VnaB1 (content 10 6 PFU / mL) was added with polysaccharide degrading enzyme Dep193 at a final concentration of 10 μg / mL as a bactericidal composition.

[0086] 1 mL of bacterial culture solution in the logarithmic growth phase (Table 1) was taken and mixed with 5 mL of semi-solid culture medium, and then poured onto a solid plate. 5 μL of the above combination was spotted on a double-layer plate and placed in a 28°C incubator for overnight culture to observe whether plaques appeared.

[0087] Table 1 Bactericidal range of the combination of polysaccharide degrading enzyme Dep193 and bacteriophage VnaB1

[0088]

[0089] Note: + means there is an inhibitory effect, - means there is no inhibitory effect.

[0090] Example 6 Evaluation of the bactericidal effect of the combination of polysaccharide degrading enzyme Dep193 and bacteriophage VnaB1

[0091] Add the stored bacteriophage VnaB1 at a ratio of 1% to 50 ml of logarithmic phase atypical Vibrio Vibrio atypicus LMG 24781 (T) bacterial solution was placed in a shaking incubator at 28°C for overnight culture to obtain phage lysate, and then the phage lysate was filtered through a 0.22 μm filter membrane to obtain VnaB1 phage suspension.

[0092] Three experimental groups were set up. In the first experimental group, atypical Vibrio in the logarithmic phase was isolated. Vibrio atypicus LMG 24781 (T) bacterial suspension and VnaB1 phage suspension (content 10 6PFU / mL), the second experimental group added polysaccharide degrading enzyme Dep193 with a final concentration of 10 μg / mL on the basis of the first group, and the control group only cultured the atypical Vibrio culture alone without any phage and polysaccharide degrading enzyme Dep193, made up the volume with PBS, and placed it in a 28 ℃ shaker for shaking culture. Starting from 0 h (mixing is 0), samples were taken every 1 hour to measure the OD600 absorbance of the culture solution, and the growth curves of atypical Vibrio in different experimental groups were drawn. Figure 5 It can be seen that compared with the control group, the antibacterial effect of the two experimental groups took effect quickly, and the OD600 value could be controlled below 0.2 within 4 hours, and the effect could last for 17 hours. However, the experimental group with only phage rebounded at the 20th hour, while the experimental group with phage plus polysaccharide degrading enzyme Dep193 could still continuously control the OD600 value at around 0.2. At the 42nd hour of experimental monitoring, the final inhibition rates were 11.8% and 86.7%, respectively, indicating that the combination can inhibit the growth of resistant bacteria and show better bactericidal effect than using phage alone.

[0093] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A bacteriophage polysaccharide degrading enzyme Dep193, characterized in that: The polysaccharide degrading enzyme Dep193 is a metabolite of bacteriophage VnaB1, and its amino acid sequence is shown in SEQ ID NO.1; The bacteriophage VnaB1 is a phage of Vibrio natriuresis ( Vibrio natriegens phage) VnaB1, which can also lyse atypical Vibrio, has been sent to the China Center for Type Culture Collection, address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, with a preservation date of August 10, 2023, and a preservation number of CCTCC M 20231425.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the polysaccharide degrading enzyme Dep193 as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid sequence of the nucleic acid molecule is shown as SEQ ID NO.

2.

4. An expression vector, characterized in that: Containing the nucleic acid molecule according to claim 2.

5. A recombinant cell, characterized in that Contains the polysaccharide degrading enzyme Dep193 according to claim 1, the nucleic acid molecule according to claim 2 or the expression vector according to claim 4.

6. An atypical Vibrio bactericidal composition, characterized in that: Contains the polysaccharide degrading enzyme Dep193 according to claim 1 and bacteriophage VnaB1.

7. The atypical Vibrio bactericidal composition according to claim 6, characterized in that: The composition is the polysaccharide degrading enzyme Dep193 and bacteriophage VnaB1 according to claim 1, the final concentration of the polysaccharide degrading enzyme Dep193 is 10 μg / mL, and the final concentration of the bacteriophage is 10 6 PFU / mL.

8. An atypical Vibrio bactericide, characterized in that: The bactericide contains the polysaccharide degrading enzyme Dep193 according to claim 1 or the composition according to claim 6.

9. Use of the atypical Vibrio bactericide according to claim 8, characterized in that: The atypical Vibrio bactericide is used in preparing a preparation for controlling aquaculture Vibrio infection.

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