AHL quenching enzyme CP1 derived from actinobacillus succinogenes as well as preparation method and application of AHL quenching enzyme CP1

Abstract

The invention discloses an AHL quenching enzyme CP1 derived from actinobacillus succinogenes as well as a preparation method and application of the AHL quenching enzyme CP1. The invention belongs to the technical field of biology, and particularly relates to an AHL quenching enzyme CP1 derived from actinobacillus succinogenes as well as a preparation method and application of the AHL quenching enzyme CP1. The quenching enzyme CP1 disclosed by the invention can be applied as follows: 1, application of the quenching enzyme CP1 in preparation of an aquatic disease inhibitor; 2, application of the quenching enzyme CP1 in preparation of products for preventing or treating diseases caused by aeromonas veronii infection; 3, application of quenching enzyme CP1 in inhibition of pathogen biofilm formation; 4, application of quenching enzyme CP1 in inhibition of pathogenic bacteria hemolysis capability or extracellular protease activity; and 5, application of the quenching enzyme CP1 in aquaculture. The CP1 disclosed by the invention can be used as a novel aquatic feed additive, can prevent and control bacterial diseases in aquaculture by interfering a quorum sensing system of pathogenic bacteria, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an AHL quenching enzyme CP1 derived from Actinobacillus succinogenes, and a preparation method and application thereof. Background Art

[0002] With economic and social development and the continuous expansion of construction land, aquaculture waters are being severely squeezed. Consequently, the "low-input, high-output" model of intensive farming and factory-style farming is inevitable. Consequently, disease problems are becoming increasingly prominent. This is primarily due to the wide variety of pathogens, high incidence rates, rapid and widespread disease spread, high mortality rates after onset, and significant economic losses. Therefore, fish diseases have become a key constraint to the sustainable development of the aquaculture industry. Controlling aquatic animal diseases is imperative, and the use of feed control products has become a key approach. While traditional medications can help aquaculture operators recover some of their economic losses, they also raise issues with food hygiene and safety, chemical use, and residues.

[0003] Basic research on bacterial diseases has found that quorum sensing between bacteria is crucial for their physiological functions. For bacteria of the same species, the exchange of quorum sensing signal molecules can be used to regulate biological characteristics such as bacterial bioluminescence, biofilm formation, and secretion of extracellular polymers, as well as the immune response of the bacteria's own cells, thereby affecting their virulence and pathogenicity. For different species of bacteria, bacterial quorum sensing can affect the composition of bacterial communities. For infected hosts, bacterial quorum sensing signal molecules also affect the interaction between bacteria and eukaryotic hosts. For example, in mammalian models, it has been found that they can regulate the host's immune response and disease resistance. Therefore, interfering with the quorum sensing system between microbial cells, that is, the quorum quenching strategy, is a new green prevention and control approach to defend against pathogenic bacteria infections.

[0004] As a new method for regulating bacterial virulence, quorum quenching technology has a fundamentally different mechanism of action from traditional antibiotics. This technology significantly reduces the pathogenicity of pathogens by interfering with the bacterial quorum sensing system rather than inhibiting its growth and reproduction, and is less likely to induce bacterial resistance. Currently, the main approaches to achieving quorum quenching include competitive inhibition and signal molecule degradation strategies. Among them, the signal molecule degradation method based on quorum sensing quenching enzymes exhibits higher intervention efficiency due to its catalytic properties outside the cell.

[0005] N-acylhomoserine lactone (AHL) signaling molecules, commonly found in Gram-negative bacteria, are key regulators of the quorum sensing system of aquatic pathogens such as Aeromonas and Vibrio. Lactonase degradation of these signaling molecules has become an important research focus in aquatic disease prevention and control. Due to their excellent biosafety, AHL lactonase has multiple applications in aquaculture: as a functional feed additive, aquaculture water purifier, and a green alternative to antibiotics. Specific application strategies include: transferring the AHL lactonase gene into probiotic systems to create engineered strains that consistently express the enzyme, thereby achieving long-term inhibition of quorum sensing by pathogens in aquaculture environments; or directly treating aquaculture water with AHL lactonase to effectively remove AHL molecules from the environment and block the expression of bacterial virulence factors. Multiple studies have demonstrated that in typical aquaculture species such as zebrafish, tilapia, and shrimp, AHL lactonase not only significantly reduces the risk of pathogen infection but also enhances host immune defenses, demonstrating its feasibility as an alternative to antibiotics in aquaculture. Summary of the Invention

[0006] The main problem to be solved by the present invention is how to reduce the infection rate of pathogenic bacteria in aquatic animals and effectively prevent and control aquatic diseases.

[0007] In order to solve the above problems, the present invention provides the use of quenching enzyme CP1 in the following:

[0008] 1. Application of quenching enzyme CP1 in the preparation of aquatic disease inhibitors.

[0009] 2. Use of quenching enzyme CP1 in the preparation of products for preventing or treating diseases caused by Aeromonas vernix infection.

[0010] 3. Application of quenching enzyme CP1 in inhibiting pathogen biofilm formation.

[0011] 4. Application of quenching enzyme CP1 in inhibiting the hemolytic ability of pathogens or the activity of extracellular proteases.

[0012] 5. Application of quenching enzyme CP1 in aquaculture.

[0013] In the above application, the amino acid sequence of the quenching enzyme is any of the following proteins:

[0014] a1) a protein having the amino acid sequence of SEQ ID No: 3;

[0015] a2) a protein having the same function as the amino acid sequence of SEQ ID No: 3 after one or more amino acid residues are substituted and / or deleted and / or added;

[0016] a3) a protein having an amino acid sequence of at least 75% identity with any of the amino acids specified in a1) or (a2) and having the same function;

[0017] a4) A fusion protein obtained by ligating a tag to the end of the protein defined in any one of a1) to (a3).

[0018] In order to facilitate purification or detection of the protein in a1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No: 3 in the sequence listing.

[0019] The tag protein includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0020] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0021] Furthermore, the coding sequence of the protein described in a1) is a DNA molecule as shown in any one of the following:

[0022] d1) the nucleotide sequence is a DNA molecule shown in SEQ ID No: 2;

[0023] d2) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in d1) and encodes the protein described above;

[0024] d3) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in d1) and encodes the protein described above.

[0025] As used herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of amino acid sequences or nucleotide sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, all filters to OFF, BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The value (%) of identity can then be obtained.

[0026] Herein, the greater than 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0027] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0028] In the above application, the protein is derived from Actinobacillus succinogenes.

[0029] Herein, the aquatic diseases are derived from Aeromonas veronii and / or Aeromonas hydrophila.

[0030] Herein, the pathogenic bacteria may be Aeromonas veronii and / or Aeromonas hydrophila.

[0031] The present invention also provides the use of a biological material containing the aforementioned DNA molecule in the preparation of an aquatic disease inhibitor, wherein the biological material may be any of the following:

[0032] B1) an expression cassette containing the DNA molecule described above;

[0033] B2) a recombinant vector containing the aforementioned DNA molecule;

[0034] B3) a recombinant vector containing the expression cassette described in B2);

[0035] B4) A recombinant microorganism containing the recombinant vector described in B3).

[0036] The vectors described herein are well known to those skilled in the art, including but not limited to plasmids, phages (such as lambda phage or M13 filamentous phage), cosmids (i.e., cosmids), Ti plasmids, or viral vectors. Specifically, the vector may be pET28a.

[0037] Furthermore, the recombinant vector may be the recombinant expression vector pET28a-CP1. The structure of the recombinant expression vector pET28a-CP1 is described as follows: A DNA fragment having the sequence SEQ ID No: 2 is inserted between the XbaI and BlpI restriction sites of the starting vector pET28a, while maintaining the remaining sequences of the pET28a vector unchanged. The pET28a-CP1 vector can express the recombinant CP1 protein, and the nucleotide sequence of the recombinant vector is SEQ ID No: 1.

[0038] The recombinant microorganism may be E. coli BL21 / pET28a-CP1.

[0039] The present invention uses genome mining technology to identify the CP1 gene with potential AHL degradation activity from succinic acid-producing Actinobacillus, and successfully achieves heterologous expression in an Escherichia coli expression system to obtain a highly active recombinant enzyme. Experiments have confirmed that the recombinant CP1 lactonase can significantly inhibit the quorum sensing regulatory phenotype of Aeromonas veronii, including the expression of virulence factors such as biofilm formation ability, hemolytic activity and extracellular protease secretion. Based on its efficient AHL signal molecule degradation characteristics, the AHL lactonase CP1 provided by the present invention can be used as a new aquatic feed additive to prevent and control bacterial diseases in aquaculture by interfering with the quorum sensing system of pathogenic bacteria, and has broad prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the plasmid map of the expression vector pET28a-CP1.

[0041] Figure 2 The SDS-PAGE analysis results of the recombinant protein CP1 are shown in Figure 1. 1 represents the whole-cell protein after induction of the recombinant bacteria; 2 represents the precipitated protein after disruption of the recombinant bacteria; 3 represents the supernatant protein after disruption of the recombinant bacteria; M: protein marker.

[0042] Figure 3 are the enzymatic properties of the quenching enzyme CP1, where A is the optimum temperature, B is the optimum pH, and C is the temperature stability.

[0043] Figure 4This figure shows the effect of quenching enzyme CP1 on the growth and biofilm formation of Aeromonas viridis.

[0044] Figure 5 The results of the effect of quenching enzyme CP1 on the hemolytic ability and extracellular protease activity of Aeromonas vermiformis are shown in Figure 1. A represents the hemolytic ability and B represents the extracellular protease activity. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0046] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0047] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0048] The pET28a in the following examples was purchased from Beijing Zoman Biotechnology: ZK159.

[0049] Hm091 in the following examples has been deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection under the registration number CGMCC No. 22536 and is classified as Aeromonas veronii. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0050] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0051] Example 1. Construction of E. coli expression vector pET28a-CP1 and preparation of recombinant strains

[0052] Based on the AHL lactonase sequences reported in the literature, multiple sequence alignment analysis was performed using the UniProt database, and the enzyme activity parameters (Kcat / Km) were predicted using the UniKP tool. Based on the prediction results, the CP1 gene sequence with potential high catalytic activity was screened out, and Nanjing GenScript Biotechnology Co., Ltd. was commissioned to complete the optimization design and chemical synthesis of the gene. The synthesized CP1 gene fragment was directionally cloned into the pET28a expression vector through the XbaI and BlpI double restriction sites, and the recombinant expression vector pET28a-CP1 was successfully constructed (its structural schematic is shown in Figure 1 ).

[0053] The structure of the pET28a-CP1 vector is described as follows: The recombinant vector is created by inserting the DNA fragment (SEQ ID No: 2) between the XbaI and BlpI restriction sites of the starting vector pET28a, while maintaining the remaining sequences of the pET28a vector unchanged. The pET28a-CP1 vector expresses the CP1 protein, and the nucleotide sequence of the recombinant vector is SEQ ID No: 1.

[0054] Subsequently, the recombinant vector pET28a-CP1 was introduced into Escherichia coli BL21 (DE3) competent cells (Company: Nanjing Novozymes Biotech Co., Ltd., Catalog No.: C504-02) using the heat shock transformation method. After resistance screening and sequencing verification, the recombinant engineered strain E. coli BL21 / pET28a-CP1 was obtained.

[0055] Example 2: Induced expression and purification of recombinant protein CP1

[0056] 1. Inducible expression of recombinant protein

[0057] The recombinant strain E. coli BL21 / pET-28a-CP1 was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 200 rpm. 1% (v / v) of the overnight culture was transferred to 50 mL of fresh LB medium (containing the same concentration of antibiotics) and cultured at 37°C until the OD 600 When the p-value reached 0.6, IPTG was added to a final concentration of 0.4 mM, and expression was induced at 18°C ​​for 16 h. After induction, the cells were harvested by centrifugation at 4°C and 12,000 × g for 2 min, resuspended in pre-chilled 0.1 mol / L PBS buffer (pH 7.4), and disrupted by ultrasonication on ice (power 300 W, 2 s operation, 3 s interval, total time 15 min), followed by centrifugation at 4°C and 12,000 × g for 10 min. The supernatant was collected and used as the crude enzyme solution.

[0058] 2. Purification of recombinant protein (1) Protein purification by nickel ion affinity chromatography: 1 mL Ni-NTABeads 6FF (Smart-Lifesciences, catalog number SA005100) was equilibrated with rinsing buffer (20 mM Tris-Cl, 150 mM NaCl, 20 mM imidazole, pH 8.0) for 3 column volumes.

[0059] (2) Combine the crude enzyme solution with the equilibrated nickel column packing at 4°C with gentle shaking for 30 min; (3) Transfer the mixture to a pre-cooled chromatography column and wash away unbound proteins with 2 column volumes of wash buffer; (4) Elute the target protein with elution buffer (20 mM Tris-Cl, 200 mM NaCl, 250 mM imidazole, pH 8.0) and collect the elution peak fractions;

[0060] (5) The eluate was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed against 0.01 M PBS (pH 7.4) at 4°C overnight. The dialyzed sample was collected to obtain the purified recombinant protein CP1 (AHL lactonase). The amino acid sequence of the recombinant protein CP1 is SEQ ID No: 3.

[0061] 3. Protein purity and molecular weight analysis

[0062] Analysis by 12% SDS-PAGE electrophoresis showed that ( Figure 2 ), CP1 protein showed a single band at approximately 37.5 kDa, which was consistent with the theoretical molecular weight calculated based on the amino acid sequence, indicating that a high-purity recombinant protein was successfully obtained.

[0063] Example 3: Analysis of Enzymatic Properties of Recombinant AHL Lactonase CP1

[0064] 1. Enzyme activity assay

[0065] A quantitative standard curve was established using high-performance liquid chromatography (HPLC): a 3-oxo-C8-HSL standard (10 mg / mL) was serially diluted in 0.01 M PBS buffer (pH 7.4) to a concentration series of 10-60 μg / mL (final volume 200 μL). After incubation at 37°C for 15 min, the reaction was terminated by the addition of 300 μL of methanol. The sample was filtered through a 0.22 μm filter and analyzed using the following chromatographic conditions: mobile phase: aqueous phase (0.3 mL triethylamine + 800 mL ultrapure water, pH 7.4): acetonitrile (64:36 v / v), flow rate: 1 mL / min, column temperature: 30°C, detection wavelength: 201 nm, and injection volume: 20 μL.

[0066] The reaction system (200 μL) consisted of 50 μL of enzyme solution (appropriately diluted) plus 50 μL of 3-oxo-C8-HSL (500 μg / mL) in 100 μL of PBS buffer (0.01 M, pH 7.4). The reaction was incubated at 37°C for 15 min, followed by the addition of 300 μL of methanol to terminate the reaction. A control group was assayed using the post-termination enzyme addition method, and the residual substrate concentration was determined using the standard curve method.

[0067] The enzyme activity was calculated using the formula: X = (ΔC × 0.5 × n) / (0.05 × M × t) × 1000, where ΔC represents the change in substrate content, 0.5 represents the volume of the reaction solution, n represents the dilution factor of the sample, 0.05 represents the volume of the enzyme solution added to the total reaction system, M represents the molar mass of 3-oxo-C8-HSL, t represents the reaction time of the enzyme, and 1000 represents the conversion factor.

[0068] 2. Characterization of Enzymatic Properties

[0069] Optimal pH: CP1 protein was added to a system containing 0.01 M PBS buffer (pH 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0) and 50 μg / mL 3-oxo-C8-HSL. The reaction was incubated at 37°C for 15 min. Methanol was added to terminate the reaction and the substrate concentration was determined. The maximum enzyme activity was defined as 100%, and the ratio of the enzyme activity at each pH to the maximum enzyme activity was used as the relative activity.

[0070] The results showed that the enzyme had an optimum pH of 9.0 and maintained high activity in the pH range of 6.0-9.0, with a wide pH adaptability range ( Figure 3 Middle B).

[0071] Optimum Temperature: CP1 protein was added to a system containing 0.01 M PBS buffer and 50 μg / mL 3-oxo-C8-HSL solution. The reaction was incubated at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 15 min. The reaction was terminated and the substrate concentration was determined. The maximum enzyme activity was defined as 100%, and the ratio of the enzyme activity at each temperature to the maximum enzyme activity was used as the relative activity.

[0072] The results showed that the optimum temperature for CP1 was 30°C, and it maintained high activity (>80% of maximum activity) in the range of 20-50°C, and had a wide temperature adaptability range ( Figure 3 Middle A).

[0073] Temperature Stability: CP1 protein was incubated with 0.01M PBS buffer (pH 7.4) at 30°C, 40°C, 50°C, 60°C, and 70°C for 60 minutes. Protein samples were then removed and used as test solutions. Substrate concentration was determined in a 37°C, pH 7.4, 0.01M PBS buffer solution containing 50 μg / mL 3-oxo-C8-HSL. The maximum enzyme activity was defined as 100%, and the ratio of the enzyme activity at each treatment time to the maximum enzyme activity was used as the relative activity.

[0074] The results showed that CP1 was very stable at 30℃-60℃ and had good enzyme activity ( Figure 3 Middle C).

[0075] Example 4: Inhibitory effect of recombinant AHL lactonase CP1 on Aeromonas vernix biofilm formation

[0076] 1. Strain culture and biofilm model construction

[0077] (1) Aeromonas welchii Hm091 (CGMCC No. 22536) stored at -80°C was streaked onto LB solid plates;

[0078] (2) After culturing at 37°C for 24 h, a single colony was picked and inoculated into LB liquid medium and cultured at 37°C overnight;

[0079] (3) Use fresh LB medium to adjust the bacterial solution to OD 600 =0.05 as working concentration;

[0080] 2. Establishing the experimental system using 96-well polystyrene plates

[0081] Experimental groups: Add 20 μL of CP1 solution with varying enzyme activity concentrations (105.47-3375 U / mL, 6 gradients). Control group: Add an equal volume of PBS buffer (pH 7.4). Add 180 μL of bacterial solution to each well, for a final volume of 200 μL. Set up three replicate wells for each concentration and incubate at 37°C for 24 hours.

[0082] 3. Detection indicators

[0083] (1) Bacterial growth assay: Directly measure the OD of the culture medium 600 value.

[0084] (2) Biofilm Quantitative Analysis: Discard the supernatant and gently wash twice with PBS. Add 250 μL of 0.5% crystal violet stain and stain for 20 min in the dark. Wash three times with deionized water and dry at room temperature. Decolorize with 250 μL of 30% acetic acid for 20 min and measure the absorbance at 595 nm.

[0085] The experimental results are as follows:

[0086] (1) Effect on bacterial growth: After 24 h of culture, the OD of each experimental group 600 The values ​​were not significantly different from those in the control group (p>0.05), indicating that CP1 did not affect the normal growth of Aeromonas vernix within the test concentration range (105.47-3375U / mL). Figure 4 ).

[0087] (2) Inhibitory effect on biofilm formation: Quantitative results of crystal violet staining showed that ( Figure 4 ), CP1 inhibited biofilm formation in a dose-dependent manner, and showed an inhibitory effect at the lowest tested concentration (105.47 U / mL), indicating that recombinant AHL lactonase CP1 specifically interfered with the phenotype regulated by the quorum sensing system by degrading AHL signaling molecules.

[0088] Example 5: Inhibitory effect of recombinant AHL lactonase CP1 on virulence factors of Aeromonas vernix

[0089] 1. Strain processing and sample preparation

[0090] Aeromonas vermiformis Hm091 was inoculated into fresh NB medium containing CP1 (10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L NaCl). Two treatments were set up: 300 U / mL and 1000 U / mL CP1. Cultures were shaken at 30°C and 180 rpm for 20 hours, and the supernatant was collected by centrifugation at 4°C and 10,000 × g for 10 minutes.

[0091] 2. Hemolytic activity detection

[0092] (1) Supernatant pretreatment: Add trypsin (20 μg / mL) and activate at room temperature for 10 min.

[0093] (2) Reaction system (1 mL): 890 μL hemolysis buffer (20 mM Tris, 150 mM NaCl, pH 7.2) + 10 μL activated supernatant + 100 μL defibrinated sheep red blood cells (5×10^6 cells).

[0094] (3) After incubation at 37°C for 20 min, centrifuge at 12,000 × g for 1 min and measure the OD value of the supernatant. 543 value.

[0095] (4) Control settings: The negative control was buffer + red blood cells, and the positive control was 0.1% Triton X-100 treatment.

[0096] (5) Hemolysis rate calculation formula: Hemolysis rate (%) = (sample D543 nm - negative D543 nm) / (positive D543 nm - negative D543 nm) × 100%.

[0097] 3. Extracellular protease activity detection

[0098] The reaction system (500 μL) consisted of 250 μL of supernatant and 250 μL of 2% azocasein. After incubation at 30°C for 3 h, 1.2 mL of 10% trichloroacetic acid was added to terminate the reaction. The mixture was then centrifuged at 6,000 × g for 10 min. 1.2 mL of the supernatant was mixed with 1.0 mL of 1 mol / L NaOH and the OD of the sample was measured. 440 Calculation of protease activity: Proteolytic activity = (sample OD 440 -Negative control OD 440 ) / 0.01.

[0099] The experimental results are as follows: Recombinant AHL lactonase CP1 can significantly inhibit the ability of Aeromonas viridis to hemolyze and produce extracellular proteases. In the concentration range of 300-1000 U / mL, the inhibition rate of hemolytic activity is 30.3-34.2% ( Figure 5 A), the inhibition rate of extracellular proteases reached 76.5-77.2% ( Figure 5 The inhibitory effect showed a significant dose-response relationship, further confirming that CP1 regulates virulence gene expression by interfering with the quorum sensing system.

[0100] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Application of recombinant quenching enzyme CP1 in the preparation of aquatic disease inhibitors.

2. Application of recombinant quenching enzyme CP1 in the preparation of products for preventing or treating diseases caused by Aeromonas vernix infection.

3. Application of recombinant quenching enzyme CP1 in inhibiting pathogen biofilm formation.

4. Application of recombinant quenching enzyme CP1 in inhibiting the hemolytic ability of pathogens or the activity of extracellular proteases.

5. Application of recombinant quenching enzyme CP1 in inhibiting virulence factors of Aeromonas vermiformis.

6. The use according to any one of claims 1 to 5, characterized in that: The amino acid sequence of the recombinant quenching enzyme is any of the following proteins: a1) a protein having the amino acid sequence of SEQ ID No: 3; a2) a protein having the same function as the amino acid sequence of SEQ ID No: 3, wherein one or more amino acid residues are substituted and / or deleted and / or added; a3) a protein with an amino acid sequence of at least 75% identity to any of the ones specified in a1) or (a2) and having the same function; a4) A fusion protein obtained by ligating a tag to the end of any of the proteins defined in a1) to (a3).

7. The use according to claim 6, characterized in that a1) The coding sequence of the protein is a DNA molecule shown in any of the following: d1) The nucleotide sequence is a DNA molecule shown in SEQ ID No: 2; d2) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in d1) and encodes the protein of claim 6; d3) A DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in d1) and encodes the protein of claim 6.

8. Use of a biological material containing the DNA molecule according to claim 7 in the preparation of an aquatic disease inhibitor, characterized in that: The biological material is any one of the following: B1) an expression cassette containing the DNA molecule according to claim 7; B2) a recombinant vector containing the DNA molecule according to claim 7; B3) a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the recombinant vector described in B3).

9. The use according to claim 1, characterized in that The aquatic disease originates from Aeromonas vermiformis ( Aeromonas veronii ) and / or Aeromonas hydrophila ( Aeromonas hydrophila ).

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