An ai-2 quencher and applications thereof

Abstract

This invention discloses an AI-2 quenching enzyme and its application. The AI-2 quenching enzyme is protein QQ-5, with the amino acid sequence shown in SEQ ID NO:1. Experiments have demonstrated that protein QQ-5 possesses AI-2 quenching enzyme activity, which can significantly improve the disease resistance of aquatic animals to Vibrio, manifested in effectively increasing the survival rate of aquatic animals infected with Vibrio and delaying the time to death. Therefore, protein QQ-5 plays an important role in the control of AI-2 family signaling-mediated pathogenic bacteria in aquatic animals. This invention has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an AI-2 quenching enzyme and its applications. Background Technology

[0002] Aquaculture is an important component of my country's agricultural economy. However, with the widespread adoption of intensive farming methods, acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio bacteria (such as Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio harveyi) is becoming increasingly frequent, causing significant economic losses to shrimp, fish, and other aquatic products. Currently, the main method for preventing and controlling AHPND is still the use of antibiotics, but the overuse and residues of antibiotics have led to a series of serious challenges, including the emergence of drug-resistant strains, water pollution, and food safety risks to aquatic products. Therefore, developing efficient, safe, and environmentally friendly antibiotic alternatives has become an urgent need for the sustainable development of aquaculture.

[0003] Among numerous alternative strategies, the "antiviral strategy" targeting the bacterial quorum sensing system shows great potential. Quorum sensing is an intercellular communication mechanism in which bacteria coordinate group behavior by secreting and sensing signaling molecules. The pathogenicity of Vibrio is closely related to its quorum sensing system, especially its type 2 quorum sensing system, in which the key signaling molecule is autoinducer-2 (AI-2). Since AI-2 is considered a universal signaling molecule for interspecies communication, targeting the AI-2 signaling pathway can effectively interfere with the pathogenic process of Vibrio without easily inducing strong selective pressure, thereby slowing down the development of drug resistance.

[0004] Existing technologies primarily employ interference strategies against AI-2, including synthesizing AI-2 analogues for signal antagonism and utilizing AI-2 quenching enzymes to degrade signal molecules. AI-2 quenching enzymes (such as AI-2 kinase LsrK) can directly and irreversibly degrade AI-2, fundamentally blocking signal transduction, and are considered a promising alternative. However, the types of natural AI-2 quenching enzymes screened from nature are currently limited, and their enzyme activity and stability are insufficient for practical applications. Furthermore, traditional enzyme screening methods are time-consuming, labor-intensive, and inefficient, severely hindering the development of such alternatives.

[0005] Therefore, there is an urgent need to discover novel AI-2 quenching enzymes with high activity and high stability to provide core materials and technical support for the green prevention and control of diseases caused by Vibrio. Summary of the Invention

[0006] The purpose of this invention is to provide a novel AI-2 quenching enzyme. The activity of the AI-2 quenching enzyme is to inhibit the formation of biofilms in Vibrio.

[0007] This invention first protects a novel AI-2 quenching enzyme—protein QQ-5. The protein QQ-5 can be a1), a2), or a3). a1) A protein whose amino acid sequence is shown in SEQ ID NO:1; a2) A protein that has 98% or more identity with a1) and has the same function, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of a1). a3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1) or a2).

[0008] To facilitate the purification of the protein shown in a1), a tag can be attached to the amino or carboxyl terminus of the protein shown in a1).

[0009] The protein shown in a2) above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0010] The protein shown in a2) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0011] The substitution of any of the amino acid residues mentioned above can be a conservative substitution of the amino acid residue.

[0012] The coding gene of the protein in a2) above can be obtained by deleting one or more amino acid residues from the codon of the DNA sequence encoding the protein, and / or by performing a missense mutation of one or more base pairs, and / or by attaching a tag coding sequence to its 5′ end and / or 3′ end.

[0013] The present invention also protects nucleic acid molecules encoding any of the proteins described above, QQ-5.

[0014] The nucleic acid molecule encoding any of the aforementioned proteins QQ-5 may be a DNA molecule as shown in b1), b2), b3), or b4): b1) A DNA molecule with a coding region as shown in SEQ ID NO:2; b2) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO:2; b3) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in b1) or b2) and encodes any of the proteins described above, QQ-5; b4) A DNA molecule that hybridizes under stringent conditions with a DNA molecule defined by b1) or b2) and that encodes any of the proteins described above, QQ-5.

[0015] This invention also protects biological materials containing any of the nucleic acid molecules described above; said biological material may be at least one of the following: D1) An expression cassette containing the nucleic acid molecule; D2) A recombinant vector containing the nucleic acid molecule or a recombinant vector containing the expression cassette; D3) Recombinant microorganisms containing the nucleic acid molecule, recombinant microorganisms containing the expression cassette, or recombinant microorganisms containing the recombinant vector.

[0016] The expression cassette may include a promoter, a nucleic acid molecule encoding any of the proteins described above, QQ-5, and a terminator.

[0017] The recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding any of the proteins QQ-5 described above into an expression vector.

[0018] The recombinant vector can specifically be a recombinant plasmid obtained by inserting the DNA molecule shown in SEQ ID NO:2 into the multiple cloning site of the expression vector.

[0019] The expression vector may specifically be the vector pET-28a(+). The recombinant vector may specifically be the recombinant plasmid pET28a-QQ-5 mentioned in the examples.

[0020] The recombinant microorganism can be obtained by introducing any of the above-described recombinant vectors into the starting microorganism.

[0021] The starting microorganism can be yeast, bacteria, algae, or fungi. The bacteria can be Gram-positive or Gram-negative. The Gram-negative bacteria can be *Escherichia coli* (E. coli). E.coli The Escherichia coli (E. coli) E.coli ) can be Escherichia coli ( E.coli BL21 (DE3).

[0022] This invention also protects the application of any of the above-described proteins QQ-5, which may be at least one of the following c1)-c10): c1) acts as an AI-2 quenching enzyme; c2) Prepare products with AI-2 quenching enzyme activity; c3) Degradation of AI-2; c4) Prepare products for degrading AI-2; c5) Block AI-2 family signaling-mediated quorum sensing and / or virulence expression in aquatic pathogens; c6) Prepare products for blocking quorum sensing and / or virulence expression mediated by AI-2 family signaling in aquatic pathogens; c7) Enhance the resistance of aquatic animals to AI-2 family signal-mediated aquatic pathogens; c8) Prepare products that enhance the resistance of aquatic animals to AI-2 family signal-mediated pathogens; c9) Controlling diseases caused by AI-2 family signal-mediated pathogens in aquatic animals; c10) is used to prepare products for the prevention and control of diseases caused by AI-2 family signal-mediated pathogenic bacteria in aquatic animals.

[0023] This invention also protects the application of any of the above-described nucleic acid molecules or any of the above-described biological materials, which may be at least one of the following c2)-c10): c2) Prepare products with AI-2 quenching enzyme activity; c3) Degradation of AI-2; c4) Prepare products for degrading AI-2; c5) Block AI-2 family signaling-mediated quorum sensing and / or virulence expression in aquatic pathogens; c6) Prepare products for blocking quorum sensing and / or virulence expression mediated by AI-2 family signaling in aquatic pathogens; c7) Enhance the resistance of aquatic animals to AI-2 family signal-mediated aquatic pathogens; c8) Prepare products that enhance the resistance of aquatic animals to AI-2 family signal-mediated pathogens; c9) Controlling diseases caused by AI-2 family signal-mediated pathogens in aquatic animals; c10) is used to prepare products for the prevention and control of diseases caused by AI-2 family signal-mediated pathogenic bacteria in aquatic animals.

[0024] In any of the above applications, the product may be a drug or antibiotic feed.

[0025] In any of the above-described applications, the activity of the AI-2 quenching enzyme can manifest as inhibition of biofilm formation mediated by AI-2 family signaling in aquatic pathogens.

[0026] The aforementioned improvement in the resistance of aquatic animals to AI-2 family signal-mediated aquatic pathogens can manifest as an increase in the survival rate of aquatic animals infected with AI-2 family signal-mediated aquatic pathogens and / or a delay in the time of death of aquatic animals infected with AI-2 family signal-mediated aquatic pathogens.

[0027] In any of the above-described applications, the AI-2 family signal-mediated aquatic animal pathogens may be Vibrio.

[0028] The Vibrio species mentioned above may be Vibrio parahaemolyticus or Vibrio alginolyticus.

[0029] In any of the above-described applications, the disease caused by the AI-2 family signal-mediated aquatic pathogens can specifically be vibriosis.

[0030] This invention targets AI-2, a universal signaling molecule in the Vibrio quorum sensing system (a key pathway for Vibrio pathogenicity), as a precise target. By efficiently screening for AI-2 quenching enzymes that specifically degrade AI-2, it fundamentally blocks Vibrio quorum sensing and virulence expression. The AI-2 quenching enzyme is protein QQ-5, with the amino acid sequence shown in SEQ ID NO:1. Experiments have demonstrated that protein QQ-5 possesses AI-2 quenching enzyme activity, significantly improving the resistance of aquatic animals (such as Litopenaeus vannamei) to Vibrio, manifested in effectively increasing the survival rate and delaying the mortality time of infected aquatic animals. Therefore, protein QQ-5 plays an important role in the control of AI-2 family signaling-mediated pathogenic bacteria in aquatic animals. This invention provides a novel green biological agent that does not induce drug resistance and works by "decoding" rather than killing bacteria, offering a clear and feasible solution for the control of Vibrio-induced diseases as an alternative to antibiotics. This invention has significant application value.

[0031] Terminology Definition In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0032] The term "expression cassette" generally refers to a nucleic acid construct containing sufficient nucleic acid elements to express a target gene. A typical expression cassette includes a promoter, a multiple cloning site (MCS), and / or a terminator. Expression cassettes may also include the target gene, marker genes (such as TK, DHFR, CAT, and NEO genes), ribosome recognition and binding sites (SDs), transcription factor binding sites (TFBSs), enhancers, silencers, repressors, introns, poly(A) signal sequences, and / or mRNA splicing signal sequences. Elements within an expression cassette can be directly linked or indirectly linked through adapters.

[0033] The term "tag" includes, but is not limited to: GST (glutathione thioredoxin) tagged protein, Trx (thioredoxin) tagged protein, nitrogen utilization substrate A (NusA) tagged protein, His-tag protein, MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA (influenza hemagglutinin) tagged protein, Myc tagged protein, LacZ tagged protein, CBD (cellulose-binding domain) tagged protein, phage T7 protein kinase (T7PK) tagged protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein. Those skilled in the art know how to select appropriate tagged proteins according to the desired purpose. The use of tags does not alter the function of the target protein; its purpose is to separate, purify, detect, or trace it. Therefore, the tagged proteins applicable to this invention are not limited to a specific type. Tags can be separated from the target protein by chemical cleavage methods or enzymatic methods (such as introducing protease cleavage sites to remove the tag using TEV protease).

[0034] The term "vector" generally refers to a vector capable of delivering exogenous DNA or a target gene into host cells for amplification and / or expression. This vector can be a cloning vector or an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be amplified and / or expressed within the host cells. Those skilled in the art can select appropriate vectors based on the purpose of genetic engineering and the properties of the recipient cells. The vectors include, but are not limited to: plasmids, phages (such as λ phage or M13 phage), cosmids (i.e., Cosmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC), or Ti plasmid artificial chromosomes (TAC)), and viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, papillomaviruses (such as SV40), and herpesviruses (such as herpes simplex virus)). A vector may contain multiple elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication origin site.

[0035] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasmas, chlamydiae, spirochetes, algae, etc. For example, the bacteria mentioned could be from the genus *Escherichia* (…). Escherichia sp.(such as Escherichia coli), Erwinia spp. Erwinia sp. ), Agrobacterium ( Agrobacterium sp. (such as Agrobacterium tumefaciens), Flavobacterium spp. ( Flavobacterium sp. Alcaligenes ( ) Alcaligenes sp. ), Pseudomonas spp. Pseudomonas sp. ) and Bacillus spp. ( Bacillus sp. (e.g., Bacillus subtilis). The viruses may include rotavirus, baculovirus, retrovirus (e.g., lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (e.g., SV40), and herpesvirus (e.g., herpes simplex virus). The fungi may be derived from yeasts (e.g., Bacillus subtilis). Saccharomyces sp. (such as Saccharomyces cerevisiae, Saccharomyces methylbenzene, Pichia pastoris), Fusarium genus ( Fusarium sp. ), Rhizoctonia spp. ( Rhizoctonia sp. Verticillium ( Verticillium sp. ), Penicillium ( Penicillium sp. Aspergillus ( ) Aspergillus sp. ) and Cephalosporin ( Cephalosporium sp. The actinomycetes may originate from the genus Streptomyces (…). Streptomyces sp. (e.g., Streptomyces). The algae may originate from the phylum Cyanophyta (e.g., cyanobacteria), genus Fucus (e.g., fucus vesiculosus). Fucus sp. ), genus *Cyclocarya* ( Achnanthes sp. ), genus *Codonopsis* ( Amphiprora sp. ), genus Dipterocarpa ( Amphora sp. ), Fiber Algae ( Ankistrodesmus sp. ), genus Styracula ( Asteromonas sp. ) and the genus *Golden Color Algae* ( Boekelovia sp. )wait.

[0036] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by linking a foreign target gene to a vector in vitro. It can be constructed in any suitable way, as long as the constructed recombinant vector can carry the foreign target gene into the recipient cell and provide the foreign target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0037] The term "linkage" generally refers to the association of two or more molecules. Linkages can be covalent or non-covalent. The linkages described herein can be direct peptide bonds or linkages via linkers (connectors).

[0038] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is usually expressed as a percentage. The identity described herein can refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies having completely identical sequences have 100% identity. Those skilled in the art will recognize that the identity of an amino acid sequence or nucleotide sequence can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, the identity of an amino acid sequence can be calculated by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), and performing a search, thus obtaining the identity value (%). Alternatively, sequence analysis software such as CLC Main Workbench and MegAlign can be used. TM The determination can be performed, for example, using a computer program BLAST with default parameters, especially BLASTP or TBLASTN. The 90% or higher identity mentioned herein can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity.

[0039] The term "comprising" is not intended to be restrictive, but rather inclusive and implies the presence of other elements besides those listed, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "substantially composed of". In this document, the terms "comprising" and "including" are used interchangeably. Attached Figure Description

[0040] Figure 1 The structural diagram of the recombinant plasmid pET28a-QQ-5.

[0041] Figure 2 The results of 12% SDS-PAGE analysis of the purified QQ-5 protein solution.

[0042] Figure 3 The effect of QQ-5 protein on the growth and biofilm formation of Vibrio parahaemolyticus and Vibrio alginolyticus; where ** means p < 0.01, *** means p < 0.001, and **** means p < 0.0001.

[0043] Figure 4 QQ-5 protein enhances the resistance of Litopenaeus vannamei to Vibrio parahaemolyticus. Detailed Implementation

[0044] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0046] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0047] Example 1, Recombinant bacteria E.coli Obtaining BL21(DE3) / pET28a-QQ-5 1. The amino acid sequences of publicly available AI-2 quenchers were compared with the Uniprot database, and sequences with an amino acid sequence similarity of over 80% were selected. Then, the E2P2 tool was used to perform functional annotation on a metagenomic database containing millions of sequences, and EC numbers were selected. Subsequently, the Enzyme-GLM model was used to calculate the specificity of these candidate sequences against AI-2 substrates, and approximately 500 sequences with a score greater than 0.9 were selected. Next, the catalytic efficiency of these 500 sequences was predicted using the DLKcat and UniKP models, and the target sequences were finally selected.

[0048] The target sequence is the amino acid sequence of the QQ-5 protein.

[0049] The amino acid sequence of the QQ-5 protein is shown in SEQ ID NO:1.

[0050] 2. Based on the amino acid sequence of the QQ-5 protein, codon optimization was performed by Nanjing Genscript Biotech Co., Ltd., and the nucleotide sequence shown in SEQ ID NO:2 was synthesized. QQ-5 Gene. QQ-5 The gene encodes the QQ-5 protein.

[0051] 3. After completing step 2, Nanjing Genscript Biotech Co., Ltd. will replace the small DNA fragment between the restriction endonucleases XbaI and BlpⅠ in the vector pET-28a(+) with... QQ-5 Genes were extracted to obtain the recombinant plasmid pET28a-QQ-5.

[0052] The structural diagram of the recombinant plasmid pET28a-QQ-5 is as follows: Figure 1 As shown.

[0053] The recombinant plasmid pET28a-QQ-5 expresses the QQ-5 protein with the amino acid sequence shown in SEQ ID NO:1.

[0054] 4. After completing step 2, import the recombinant plasmid pET28a-QQ-5. E.coli BL21 (DE3) yielded recombinant Escherichia coli, which was named recombinant bacteria. E.coli BL21(DE3) / pET28a-QQ-5.

[0055] Example 2: Expression and purification of QQ-5 protein I. Expression and crude extraction of QQ-5 protein 1. Recombinant bacteria E.coli BL21(DE3) / pET28a-QQ-5 was inoculated into 1 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 200 rpm to obtain culture 1.

[0056] 2. After completing step 1, transfer 1 mL of culture medium 1 to 50 mL of LB liquid medium containing 50 μg / mL kanamycin, and incubate at 37°C with shaking at 200 rpm to obtain OD. 600nm 2. The culture solution reaches a pH of 0.6.

[0057] 3. After completing step 2, add IPTG to the culture medium 2 to achieve a concentration of 0.4 mM. Then, induce culture at 18°C ​​and 180 rpm for 16 hours with shaking. After induction, centrifuge at 4°C and 12000 rpm for 2 minutes and collect the bacterial pellet.

[0058] 4. After completing step 3, resuspend the bacterial cell precipitate in pre-cooled 0.01 mol / L PBS buffer (pH 7.4) and sonicate it under ice bath conditions (power 300W, working for 2 seconds, intermittent for 3 seconds, total time 15 minutes) to obtain the lysate.

[0059] 5. After completing step 4, take the broken liquid, centrifuge at 4°C and 12000 rpm for 10 minutes, and collect the supernatant. The supernatant is the crude enzyme solution.

[0060] II. Affinity chromatography purification Protein purification was performed using Ni-NTA affinity chromatography. The specific steps are as follows: 1. Equilibrate 1 mL of Ni NTA Beads 6FF (Smart-Lifesciences) with 10 column volumes of wash buffer (solute and concentration of 20 mM Tris-Cl, 150 mM NaCl and 20 mM imidazole, solvent of water, pH 8.0) to obtain an equilibrated column.

[0061] 2. After completing step 1, gently mix the crude enzyme solution collected in step 1, step 5, with the equilibrated column material at 4°C for 30 minutes (the purpose is to ensure that the QQ-5 protein with the His tag is fully bound to the filler material) to obtain the mixture.

[0062] 3. After completing step 2, pack the mixture into a chromatography column and elute sequentially with 10 column volumes of wash buffer and 5 column volumes of elution buffer (solute and concentration of 20 mM Tris-Cl, 200 mM NaCl and 250 mM imidazole, solvent of water, pH 8.0), and collect the eluent.

[0063] III. Protein Concentration and Detection 1. Place the eluent collected in step 2, step 3, into a pretreated dialysis bag (molecular weight cut-off (MWCO) 8-14 kDa). Dialyze overnight at 4°C in 0.01 mol / L PBS buffer (pH 7.4), changing the dialysis buffer every 4 hours. After dialysis, concentrate the protein using PEG8000 at 4°C to obtain a purified QQ-5 protein solution.

[0064] 2. Perform 12% SDS-PAGE on the purified QQ-5 protein solution.

[0065] Test results are shown Figure 2 (M represents the protein marker, and QQ-5 represents the purified QQ-5 protein solution). The results showed that the purified QQ-5 protein solution exhibited a single, clear band between approximately 25-35 kDa, consistent with the molecular weight predicted based on the QQ-5 protein amino acid sequence (the predicted molecular weight of QQ-5 protein is 27.08 kDa). This indicates that the purified QQ-5 protein solution contains high-purity QQ-5 protein.

[0066] Example 3: Observation of the effect of QQ-5 protein on the growth and biofilm formation of Vibrio parahaemolyticus and Vibrio alginolyticus using crystal violet staining. 1. Activation and culture of bacterial strains Vibrio parahaemolyticus stored at -80℃ was streaked onto TSB solid plates containing 3% NaCl and incubated at 30℃ for 24 h. Single colonies were then picked and inoculated into TSB liquid medium containing 3% NaCl and incubated at 30℃ with shaking at 180 rpm for 16 h. Finally, the culture was diluted with TSB liquid medium containing 3% NaCl to obtain the OD. 600nm The concentration of Vibrio parahaemolyticus bacterial suspension was 0.05.

[0067] Vibrio alginolyticus stored at -80℃ was streaked onto TSB solid plates containing 3% NaCl and incubated at 30℃ for 24 h. Single colonies were then picked and inoculated into TSB liquid medium containing 3% NaCl and incubated at 30℃ with shaking at 180 rpm for 16 h. Finally, the culture was diluted with TSB liquid medium containing 3% NaCl to obtain the OD. 600nm The concentration of Vibrio alginolyticus bacterial solution was 0.05.

[0068] 2. Biofilm formation (1) Take a 96-well polystyrene plate and randomly divide it into three groups: experimental group (QQ-5 group), positive control group (LsrK group), and blank control group (PBS group), with 6 replicate wells in each group. Then, add 10 μL of purified QQ-5 protein solution prepared in Example 2 to each well of the experimental group, add 10 μL of AI-2 kinase LsrK solution with a concentration of 5 μg / mL to each well of the positive control group, and add 10 μL of 0.01 mol / L PBS buffer (pH 7.4) to each well of the blank control group. Then add 190 μL of the test bacterial solution (Vibrio parahaemolyticus or Vibrio alginolyticus prepared in step 1) to each well and mix well.

[0069] AI-2 kinase LsrK solution was prepared according to the method described in the following literature: Zhu J, Hixon MS, Globisch D, Kaufmann GF, Janda KD. Mechanistic insights into the LsrK kinase required for autoinducer-2 quorum sensing activation. J Am Chem Soc. 2013 May 29;135(21):7827-30. doi: 10.1021 / ja4024989. Epub 2013 May 16. PMID: 23672516; PMCID:PMC3736694.

[0070] (2) After completing step (1), the 96-well polystyrene plate is placed at 30°C and incubated for 48 h.

[0071] The absorbance of each well was measured at 600 nm using a microplate reader. The results are shown below. Figure 3 (OD600 is the absorbance value). The absorbance value detection showed that QQ-5 protein and AI-2 kinase LsrK had no effect on the growth of Vibrio parahaemolyticus and Vibrio alginolyticus.

[0072] 3. Staining and quantification of biological membranes (1) After completing step 2, carefully aspirate the supernatant from each well and gently wash the precipitate twice with sterile 0.01 mol / L PBS buffer (pH 7.4).

[0073] (2) After completing step (1), add 200 μL of 0.5% crystal violet staining solution to each well and stain at room temperature in the dark for 20 min. After discarding the staining solution, wash twice with sterile 0.01 mol / L PBS buffer (pH 7.4) and air dry at room temperature.

[0074] (3) After completing step (2), add 250 μL of 30% (v / v) acetic acid aqueous solution to each well and decolorize for 20 min.

[0075] (4) After completing step (3), use a microplate reader to measure the absorbance of each well at a wavelength of 595 nm. Calculate the biofilm inhibition rate based on the absorbance values; the calculation formula is as follows: Biomembrane inhibition rate = [1 - (experimental group OD)] 595nm - Blank control group OD 595nm ) / (Positive control group OD 595nm - Blank control group OD 595nm )]×100%.

[0076] 4. Results and Analysis Test results are shown Figure 3 (A represents Vibrio parahaemolyticus, and B represents Vibrio alginolyticus; OD595 is the absorbance value measured in step 3). The results showed that, compared with the PBS group, the QQ-5 group significantly inhibited biofilm formation in both Vibrio species. The QQ-5 protein inhibited biofilm formation in Vibrio parahaemolyticus by 40.6% and in Vibrio alginolyticus by 31.1%, significantly exceeding the inhibitory effect of the positive control group (AI-2 kinase LsrK inhibited biofilm formation in Vibrio parahaemolyticus by 18.3% and in Vibrio alginolyticus by 21.3%).

[0077] The above results indicate that the QQ-5 protein inhibits Vibrio biofilm formation not by inhibiting bacterial growth, but by specifically interfering with the biofilm formation process regulated by the quorum sensing system.

[0078] The above results indicate that the QQ-5 protein has AI-2 quenching enzyme activity, meaning that the QQ-5 protein is an AI-2 quenching enzyme.

[0079] Example 4: QQ-5 protein enhances the resistance of Litopenaeus vannamei to Vibrio parahaemolyticus. 1. Preparation of mixed feed Add 7.5 mg of QQ-5 protein to every 1 kg of basic feed to obtain mixed feed 1.

[0080] Add 15mg of QQ-5 protein to every 1kg of basic feed to obtain mixed feed 2.

[0081] Add 30mg of QQ-5 protein to every 1kg of basic feed to obtain mixed feed 3.

[0082] Each kilogram of basic feed consists of 200g flour, 150g soybean meal, 120g peanut meal, 250g domestic fish meal, 140g chicken meal, 30g shrimp shell powder, 30g squid paste, 16g bentonite, 3g lysine, 1g DL methionine, 15g calcium dihydrogen phosphate, 25g soybean oil, 10g phospholipid oil, and 10g predigested premix.

[0083] 2. Four-week-old Litopenaeus vannamei shrimp were fed with basal feed, mixed feed 1, mixed feed 2, and mixed feed 3 for 5 weeks respectively. Afterwards, they were treated with Vibrio parahaemolyticus (disclosed in the following literature: W. Zhou, Y. Xie, M. Xie, ...). et al. The effect of dietary supplementation of medium-chain fatty acids products on gut and hepatopancreas health, and disease resistance in white shrimp ( Litopenaeus vannamei (Aquacult. Rep., 29(2023), Article 101481) Invitation (invitation dose was 4.92 × 10⁻⁶) 6 (CFU / ml), survival rate was calculated 4 days after infection.

[0084] Statistical results are shown below Figure 4(Control was the basal diet, 7.5 mg / kg was mixed diet 1, 15 mg / kg was mixed diet 2, and 30 mg / kg was mixed diet 3). The results showed that feeding Litopenaeus vannamei with mixed diet 3 (i.e., adding 30 mg of QQ-5 protein per kilogram of basal diet) significantly improved the survival rate after challenge with the virus; feeding Litopenaeus vannamei with mixed diet 2 (i.e., adding 15 mg of QQ-5 protein per kilogram of basal diet) or mixed diet 1 (i.e., adding 7.5 mg of QQ-5 protein per kilogram of basal diet) delayed the mortality time after challenge with the virus.

[0085] This demonstrates that QQ-5 protein can significantly enhance the resistance of Litopenaeus vannamei to Vibrio parahaemolyticus.

[0086] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Protein QQ-5, which may be a1), a2), or a3). a1) A protein whose amino acid sequence is shown in SEQ ID NO:1; a2) A protein that has 98% or more identity with a1) and has the same function, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of a1). a3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1) or a2).

2. A nucleic acid molecule encoding the protein QQ-5 of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule as shown in b1), b2), b3), or b4) below: b1) A DNA molecule with a coding region as shown in SEQ ID NO:2; b2) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO:2; b3) has 75% or more identity with the nucleotide sequence defined in b1) or b2) and encodes a DNA molecule of the protein QQ-5 of claim 1; b4) Hybridizes under stringent conditions with a DNA molecule defined in b1) or b2) and encoding a DNA molecule of the protein QQ-5 as claimed in claim 1.

4. A biomaterial containing the nucleic acid molecule of claim 2 or 3; said biomaterial is at least one of the following: D1) An expression cassette containing the nucleic acid molecule; D2) A recombinant vector containing the nucleic acid molecule or a recombinant vector containing the expression cassette; D3) Recombinant microorganisms containing the nucleic acid molecule, recombinant microorganisms containing the expression cassette, or recombinant microorganisms containing the recombinant vector.

5. The biomaterial according to claim 4, characterized in that: The recombinant vector is a recombinant plasmid obtained by inserting the DNA molecule shown in SEQ ID NO:2 into the multiple cloning site of the expression vector.

6. The biomaterial according to claim 4, characterized in that: The recombinant microorganism is the recombinant microorganism obtained by introducing the recombinant vector of claim 5 into the starting microorganism.

7. The application of the protein QQ-5 according to claim 1 is at least one of the following c1)-c10): c1) acts as an AI-2 quenching enzyme; c2) Prepare products with AI-2 quenching enzyme activity; c3) Degradation of AI-2; c4) Prepare products for degrading AI-2; c5) Block AI-2 family signaling-mediated quorum sensing and / or virulence expression in aquatic pathogens; c6) Prepare products for blocking quorum sensing and / or virulence expression mediated by AI-2 family signaling in aquatic pathogens; c7) Enhance the resistance of aquatic animals to AI-2 family signal-mediated aquatic pathogens; c8) Prepare products that enhance the resistance of aquatic animals to AI-2 family signal-mediated pathogens; c9) Controlling diseases caused by AI-2 family signal-mediated pathogens in aquatic animals; c10) is used to prepare products for the prevention and control of diseases caused by AI-2 family signal-mediated pathogenic bacteria in aquatic animals.

8. The use of a nucleic acid molecule as described in claim 2 or 3 or a biological material as described in any one of claims 4 to 6, comprising at least one of the following c2)-c10): c2) Prepare products with AI-2 quenching enzyme activity; c3) Degradation of AI-2; c4) Prepare products for degrading AI-2; c5) Block AI-2 family signaling-mediated quorum sensing and / or virulence expression in aquatic pathogens; c6) Prepare products for blocking quorum sensing and / or virulence expression mediated by AI-2 family signaling in aquatic pathogens; c7) Enhance the resistance of aquatic animals to AI-2 family signal-mediated aquatic pathogens; c8) Prepare products that enhance the resistance of aquatic animals to AI-2 family signal-mediated pathogens; c9) Controlling diseases caused by AI-2 family signal-mediated pathogens in aquatic animals; c10) is used to prepare products for the prevention and control of diseases caused by AI-2 family signal-mediated pathogenic bacteria in aquatic animals.

9. The application according to claim 7 or 8, characterized in that: The activity of the AI-2 quenching enzyme is manifested in inhibiting the formation of biofilms in aquatic pathogens mediated by AI-2 family signaling.

10. The application according to any one of claims 7 to 9, characterized in that: The AI-2 family of signal-mediated pathogenic bacteria in aquatic animals is Vibrio.

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