AHL lactonase NJ1 from pathogenic bacillus as well as preparation method and application of AHL lactonase NJ1

Abstract

The invention discloses AHL lactonase NJ1 from pathogenic bacillus as well as a preparation method and application of the AHL lactonase NJ1. The invention belongs to the technical field of biology, and particularly relates to AHL lactonase NJ1 derived from pathogenic bacteria as well as a preparation method and application of the AHL lactonase NJ1. The invention provides application of quenching enzyme NJ1 in preparation of an aquatic disease inhibitor. The quenching enzyme NJ1 is applied to preparation of a product for preventing or treating diseases caused by aeromonas veronii infection; the quenching enzyme NJ1 is applied to inhibition of pathogenic bacteria biofilm formation; the application of the quenching enzyme NJ1 in inhibiting the hemolysis capability of pathogenic bacteria or the activity of extracellular protease; the quenching enzyme NJ1 is applied to aquaculture. The quenching enzyme NJ1 has the capability of efficiently degrading AHL, can keep the activity for a long time in an aquaculture environment, and has application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an AHL lactonase NJ1 derived from Xenorhabdus, a preparation method and an application thereof. Background Art

[0002] Aquaculture, a vital component of the global food supply, has experienced rapid growth in recent years. However, high-density aquaculture environments can easily lead to bacterial diseases in aquatic animals such as fish, shrimp, and shellfish, resulting in significant economic losses. Antibiotics are widely used in aquaculture to prevent and control aquatic animal diseases. However, the long-term, extensive use of antibiotics not only leads to increased bacterial resistance but also poses potential risks to human health through the food chain. Furthermore, antibiotic residues entering water bodies can affect the balance of ecosystems and disrupt environmental microbial communities. Therefore, the search for efficient, safe, and antibiotic-free methods for preventing and controlling aquatic diseases has become a research hotspot.

[0003] Many pathogenic bacteria in aquaculture, such as Aeromonas and Vibrio, rely on the quorum sensing (QS) system to regulate the expression of virulence factors and thus trigger host infection. This mechanism mediates information exchange between bacteria through signaling molecules such as N-acetylhomoserine lactones (AHLs), and can regulate key pathogenic processes such as biofilm formation, efflux pump expression, antibiotic resistance, and virulence factor synthesis. Studies have shown that the pathogenicity of aquatic pathogens such as Aeromonas veronii and Aeromonas hydrophila is closely related to the AHL-mediated QS system. Inactivation of their QS function can significantly weaken their pathogenicity, making interference with the QS pathway a highly promising anti-infection strategy. Currently, quorum quenching (QQ) technology, which blocks bacterial communication by degrading or inhibiting QS signaling molecules, has been widely used in aquatic disease prevention and control. Among them, AHL lactonase, which can specifically hydrolyze the AHL lactone ring and inactivate it, has attracted much attention due to its clear mechanism of action. The enzyme has been successfully isolated and identified from a variety of microorganisms such as bacteria and fungi.

[0004] AHL lactonase, due to its excellent biosafety and efficient signal molecule degradation capabilities, holds great promise for application in aquaculture. As a novel quorum sensing quencher, this enzyme can be applied in a variety of ways: as a feed additive, it effectively blocks the quorum sensing system of pathogens, reducing the expression of virulence factors and thereby minimizing the risk of infection in aquatic animals. As an antibiotic alternative, its unique mechanism of action prevents bacterial resistance, offering a green solution to the problem of antibiotic overuse. Furthermore, this enzyme can be combined with probiotics to construct engineered strains for the sustained removal of signal molecules from aquaculture environments, or used directly in water treatment to maintain a healthy aquaculture environment. These applications collectively demonstrate the significant value of AHL lactonase in green disease control systems for aquaculture. Summary of the Invention

[0005] 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.

[0006] In order to solve the above problems, the present invention provides the use of quenching enzyme NJ1 in the following: 1. Application of quenching enzyme NJ1 in the preparation of aquatic disease inhibitors.

[0007] 2. Application of quenching enzyme NJ1 in the preparation of products for preventing or treating diseases caused by Aeromonas viridifolia infection.

[0008] 3. Application of quenching enzyme NJ1 in inhibiting pathogen biofilm formation.

[0009] 4. Application of quenching enzyme NJ1 in inhibiting the hemolytic ability of pathogens or the activity of extracellular proteases.

[0010] 5. Application of quenching enzyme NJ1 in aquaculture.

[0011] In the above application, the amino acid sequence of the quenching enzyme is any of the following proteins: a1) a protein having the amino acid sequence of SEQ ID No: 1; a2) a protein having the same function as the amino acid sequence of SEQ ID No: 1 after 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).

[0012] 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: 1 in the sequence listing.

[0013] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) 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.

[0014] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0015] Furthermore, the coding sequence of the protein a1) is a DNA molecule shown in any one 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 described above; d3) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in d1) and encodes the protein described above.

[0016] As used herein, identity refers to amino acid sequence or nucleotide sequence identity. Amino acid sequence or nucleotide sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of amino acid or nucleotide sequences can be calculated by searching using 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 identity value (%) can then be obtained.

[0017] Herein, the 80% or greater 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.

[0018] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0019] In the above application, the protein is derived from Xenorhabdus spp. ( Xenorhabdus kozodoii. ).

[0020] Herein, the aquatic diseases are caused by Aeromonas vernix ( Aeromonas veronii ) and / or Aeromonas hydrophila ( Aeromonas hydrophila ).

[0021] Herein, the pathogen may be Aeromonas viridis ( Aeromonas veronii ) and / or Aeromonas hydrophila ( Aeromonas hydrophila ).

[0022] 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: B1) an expression cassette containing the DNA molecule described above; B2) a recombinant vector containing the aforementioned DNA molecule; B3) a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the recombinant vector described in B3).

[0023] 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.

[0024] Furthermore, the recombinant vector may be the recombinant expression vector pET28a-NJ1. The structure of the recombinant expression vector pET28a-NJ1 is described as follows: The recombinant vector is obtained by inserting a DNA fragment with the sequence SEQ ID No: 2 between the XbaI and BlpI restriction sites of the starting vector pET28a, while maintaining the other sequences of the pET28a vector unchanged. The pET28a-NJ1 vector can express the NJ1 protein, whose amino acid sequence is SEQ ID No: 1.

[0025] The recombinant microorganism may be E. coli BL21 / pET28a-NJ1.

[0026] This patent provides an AHL lactonase derived from Xenorhabdus, its preparation method, and application, aiming to develop an efficient, stable AHL lactonase suitable for aquaculture. The enzyme has the ability to efficiently degrade AHLs and maintain activity for extended periods in aquaculture environments. The patented enzyme exhibits excellent thermal stability and pH adaptability, making it widely applicable in diverse aquaculture environments. It provides a safe and efficient solution for aquatic disease prevention and control, and is expected to become an effective alternative to antibiotics, providing technical support for green and sustainable aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the plasmid map of the expression vector pET28a-NJ1.

[0028] Figure 2 The SDS-PAGE analysis results of the recombinant protein NJ1 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.

[0029] Figure 3 The enzymatic properties of the quenching enzyme NJ1 are shown in Figure 2. A is the optimum temperature, B is the optimum pH, and C is the temperature stability.

[0030] Figure 4 This figure shows the effect of quenching enzyme NJ1 on the growth and biofilm formation of Aeromonas viridis.

[0031] Figure 5 The figure shows the effect of quenching enzyme NJ1 on the hemolytic ability and extracellular protease activity of Aeromonas vermiformis. A represents the hemolytic ability and B represents the extracellular protease activity. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0035] Hm091 in the following examples has been deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection, with the registration number of CGMCC No. 22536, and is classified as Aeromonas vermiformis ( Aeromonas veronii Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0036] The pET28a in the following examples was purchased from Beijing Zoman Biotechnology Company with the product number ZK159.

[0037] 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.

[0038] Example 1. Preparation of recombinant protein NJ1 1. Construction and transformation of expression vector pET28a-NJ1 The AHL lactonase sequence reported in the literature was used for multiple sequence alignment with the Uniprot database, and then the enzyme activity (Kcat / Km) was predicted using UniKP. The gene sequence predicted to be active was artificially synthesized and inserted into the E. coli expression vector pET28a between the XbaI and BlpⅠ restriction sites. The structure of the obtained recombinant expression vector pET28a-NJ1 is shown in the figure. Figure 1 As shown, gene synthesis and vector construction were completed by Nanjing GenScript Biotechnology Co., Ltd.

[0039] The structure of the recombinant expression vector pET28a-NJ1 is described as follows: The DNA fragment with the sequence SEQ ID No: 2 is inserted between the XbaI and BlpI restriction sites of the starting vector pET28a, while the other sequences of the pET28a vector remain unchanged. The pET28a-NJ1 vector expresses the NJ1 protein, whose amino acid sequence is SEQ ID No: 1.

[0040] The recombinant expression vector pET28a-NJ1 was transformed into Escherichia coli BL21 by heat shock transformation to obtain the recombinant bacteria. E. coli BL21 / pET28a-NJ1.

[0041] 2. Expression and purification of recombinant protein NJ1 1) Recombinant bacteria E. coliPreparation of BL21 / pET28a-NJ1 cell culture medium The recombinant E. coli BL21 / pET-28a-FadDD1 was inoculated into LB culture medium (containing kanamycin at a final concentration of 50 μg / mL) and cultured at 37°C overnight with shaking. The overnight culture was inoculated into 50 mL LB culture medium at a ratio of 1% and cultured until the OD 600 = 0.6, then add IPTG to a final concentration of 0.4 mM and induce at 18°C ​​for 16 h to obtain a recombinant bacterial cell culture. The resulting culture was centrifuged at 12,000 rpm for 2 min to collect the cell pellet. The cell pellet was washed with 0.1 mol / L PBS buffer ( p H 7.4) and resuspended, ultrasonically disrupted, and centrifuged at 12,000 rpm for 10 min (4°C), and the supernatant was collected.

[0042] 2) Purification of NJ1 recombinant protein Add 1 mL of washing buffer (20 mM Tris-Cl, 150 mM NaCl, 20 mM imidazole, p The activated Ni NTA Beads 6FF (Smart-Lifesciences, Catalog No. SA005100) were washed with 5% HCl (pH = 8.0) and allowed to bind at 4°C for 30 min. The column with the filter membrane was placed at 4°C and equilibrated with 3 column volumes of wash buffer. The supernatant and bead mixture was added, and the protein-bound beads were washed with 2 column volumes of wash buffer. Finally, the beads were eluted with elution buffer (20 mM Tris-Cl, 200 mM NaCl, 250 mM imidazole, p The protein was eluted with PBS (pH=8.0) and the eluate was collected. The collected eluate was dialyzed in 0.01M PBS, p The quenched enzyme was dialyzed overnight at 4°C (pH = 7.4) and then concentrated using PEG8000. The concentrate was collected to obtain the recombinant protein NJ1, also known as quenched enzyme NJ1. The amino acid sequence of the recombinant protein NJ1 is SEQ ID No: 3.

[0043] Protein analysis was performed by 12% polyacrylamide gel electrophoresis. The results showed that the size of the quenching enzyme NJ1 protein was approximately 30.2 kDa ( Figure 2 ), and the size was consistent with the amino acid sequence prediction.

[0044] Example 2: Determination of Enzyme Activity and Enzymatic Properties of Quenching Enzyme NJ1 1. Determination of quenching enzyme NJ1 activity To prepare the standard curve, a 10 mg / mL solution of 3-oxo-C8-HSL was diluted to 10, 20, 30, 40, 50, and 60 μg / mL in 0.01 M PBS buffer (pH 7.4) to a final volume of 200 μL. The solution was mixed using a vortex mixer and incubated at 37°C for 15 min. Then, 300 μL of methanol was added, and the solution was filtered through a 0.22 μm filter before analysis. Chromatographic conditions were an aqueous phase (0.3 mL of triethylamine in 800 mL of ultrapure water, pH 7.4) to organic phase (acetonitrile) ratio of 0.64:0.36, a flow rate of 1 mL / min, a column temperature of 30°C, a detection wavelength of 201 nm, and an injection volume of 20 μL.

[0045] Activity Assay: Pipette 50 µL of a 10 mg / mL 3-oxo-C8-HSL solution and dilute to 500 µg / mL by adding 950 µL of 0.01 M PBS buffer (pH 7.4). Then, pipette 50 µL of the appropriately diluted enzyme solution into a 1.5 mL centrifuge tube. Add 50 µL of the 500 µg / mL 3-oxo-C8-HSL solution and 100 µL of 0.01 M PBS buffer (pH 7.4). Incubate at 37°C in a water bath for 15 min. Terminate the reaction by adding 30 µL of methanol to each sample tube. For the control tube, add 50 µL of enzyme solution after termination. Determine substrate concentration using the standard curve assay.

[0046] The enzyme activity was calculated as follows: X = (ΔC × 0.5 × n) / (0.05 × M × t) × 1000; Where ΔC is the change in substrate content, 0.5 is the volume of the reaction solution, n is the dilution factor of the sample, 0.05 is the volume of the enzyme solution added to the total reaction system, M is the molar mass of 3-oxo-C8-HSL, t is the reaction time of the enzyme, and 1000 is the conversion factor.

[0047] 2. Determination of enzyme properties Optimal pH: NJ1 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 solution. The reaction was incubated at 37°C for 15 minutes. 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.

[0048] Optimum Temperature: NJ1 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.

[0049] Temperature Stability: NJ1 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 min. Protein samples were then removed and used as test solutions. Substrate concentration was determined in a system containing 50 μg / mL 3-oxo-C8-HSL solution in 0.01M PBS buffer (pH 7.4) at 37°C. 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.

[0050] The enzymatic properties of the quenching enzyme NJ1 showed that its optimum temperature was 30°C and its optimum pH was 9, and its activity was close to that at pH 7. The results of the temperature stability test showed that the quenching enzyme NJ1 was greatly affected by temperature and had enzymatic activity only at 30°C ( Figure 3 AC).

[0051] Example 3: Crystal violet staining to investigate the effect of quenching enzyme NJ1 on Aeromonas vernix biofilm 1. Construction of Aeromonas viridis biofilm model Aeromonas virens Hm091 (CGMCC No. 22536) stored at -80°C was taken out and streaked onto LB solid plates. The plates were incubated at 37°C for 24 h. The next day, a single colony was picked with an inoculation loop and inoculated into LB liquid medium. The plates were incubated at 37°C overnight and the OD was measured. 600 Dilute the Hm091 bacterial suspension to OD 600 =0.05.

[0052] Add 20 μL of NJ1 at different concentrations to a 96-well polypropylene plate, set up 6 enzyme activity gradients (105.47 U / mL, 210.94 U / mL, 421.88 U / mL, 843.75 U / mL, 1687.5 U / mL, 3375 U / mL), set up 3 replicates, add 20 μL of PBS buffer to the control group, add 180 μL of the mixed bacterial solution to each well, and incubate at 37°C for 24 hours. Determine OD 600 Values ​​were used to record the effect of NJ1 on the growth of Aeromonas vickers.

[0053] 2. Crystal violet staining The supernatant was aspirated and discarded, followed by two washes with sterile PBS buffer. Crystal violet was diluted to a 0.5% staining solution with sterile distilled water. 250 μL of 0.5% crystal violet dye was added to each well. The cells were stained for 20 minutes in the dark. The stain was aspirated, and the cells were washed three times with sterile distilled water and air-dried. 250 μL of 30% acetic acid solution was added to each well and decolorized for 20 minutes to fully dissolve the crystal violet. The absorbance of each well was read at a wavelength of 595 nm to obtain quantitative biofilm results.

[0054] Effects of quenching enzyme NJ1 on the growth and biofilm of Aeromonas verticillioides Figure 4 Shown: By detecting OD 600 It was found that when quenching enzyme NJ1 with different enzyme activities was added to the culture medium of Aeromonas vickers Hm091 and co-cultured for 24 hours, the addition of quenching enzyme NJ1 did not affect the growth of Aeromonas vickers, further indicating that quenching enzyme NJ1 had no obvious inhibitory effect on the growth of Aeromonas vickers; at the same time, with the increase of the amount of quenching enzyme NJ1 added, the inhibitory ability on the biofilm formation of Aeromonas vickers gradually increased.

[0055] Example 4: Effects of Quenching Enzyme NJ1 on the Hemolytic and Extracellular Protease Capacities of Aeromonas vernix Aeromonas vermiformis Hm091 was inoculated into fresh NB medium (10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride) containing quenched enzyme NJ1 at 300 U / mL and 1000 U / mL, respectively. The mixture was then incubated at 30°C and 180 rpm for 20 hours. The supernatant was collected after centrifugation and assayed for hemolytic and extracellular protease activity.

[0056] 1. Hemolytic ability determination Trypsin (20 μg / mL) was added to the supernatant to activate aerolysin. The aerolysin in the bacterial supernatant was activated at room temperature for 10 min and used for hemolysis test.

[0057] The hemolysis assay reaction system is as follows: 890 μL of hemolysis buffer (20 mmol / L Tris, 150 mmol / L NaCl, pH 7.2), 10 μL of activated supernatant, and 5 × 10⁶ defibrinated sheep erythrocytes (100 μL). The reaction system is thoroughly mixed and incubated in a 37°C incubator for 20 minutes. After the reaction is complete, centrifuge at 12,000 rpm for 1 minute, and the supernatant is collected for determination of OD. 543The absorbance value was 0.1%. The test group containing only hemolysis buffer and red blood cells served as the negative control group, and the red blood cells treated with 0.1% Triton X-100 served as the positive control group. Hemolytic activity was calculated as follows: Hemolysis rate (%) = (sample D543 nm - negative D543 nm) / (positive D543 nm - negative D543 nm) × 100%.

[0058] The results of hemolysis test are as follows: adding 300U / mL and 1000U / mL of quenching enzyme NJ1 significantly inhibited the hemolytic ability of Hm091 ( Figure 5 Middle A).

[0059] 2. Extracellular protease activity assay The reaction mixture contained 250 μL of supernatant and 250 μL of 2% azocasein solution. After 3 hours of reaction at 30°C, 1.2 mL of 10% trichloroacetic acid was added and the mixture was centrifuged at 6000 g for 10 minutes. Then, 1.2 mL of supernatant was mixed with 1.0 mL of 1 mol / L NaOH, and the absorbance was measured at 440 nm. Extracellular proteolytic activity was calculated as follows: proteolytic activity = (sample absorbance - negative control absorbance) / 0.01.

[0060] The results of the extracellular protease test are as follows: adding 300U / mL and 1000U / mL of quenching enzyme can significantly inhibit the extracellular protease activity of Hm091. The inhibition rate of 300U / mL of quenching enzyme NJ1 on extracellular protease activity reaches 79%, and the inhibition rate is as high as 94% when adding 1000U / mL of quenching enzyme NJ1. Figure 5 Middle B).

[0061] 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 quenching enzyme NJ1 in the preparation of aquatic disease inhibitors.

2. Application of quenching enzyme NJ1 in the preparation of products for preventing or treating diseases caused by Aeromonas vernix infection.

3. Application of quenching enzyme NJ1 in inhibiting pathogen biofilm formation.

4. Application of quenching enzyme NJ1 in inhibiting the hemolytic ability of pathogens or the activity of extracellular proteases.

5. Application of quenching enzyme NJ1 in aquaculture.

6. The use according to any one of claims 1 to 5, characterized in that: The amino acid sequence of the quenching enzyme is any of the following proteins: a1) a protein having the amino acid sequence of SEQ ID No: 1; a2) a protein having the same function as the amino acid sequence of SEQ ID No: 1 after 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. The use according to claim 1, characterized in that The aquatic disease originates from Aeromonas vernix ( Aeromonas veronii ) and / or Aeromonas hydrophila ( Aeromonas hydrophila ).

9. 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).

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