Streptococcus suis lyase Ply2413 and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses streptococcus suis lyase Ply2413 and application thereof. Wherein the amino acid sequence of the lyase is as shown in SEQ ID NO: 1, or the lyase is a functional variant which has the same CHAP catalytic structural domain and SH3b binding structural domain as the lyase and retains broad-spectrum splitting activity. The lyase can be prepared from the coding gene of the lyase through a recombinant expression system. The preparation method comprises the following steps: cloning a gene for coding the lyase, constructing a recombinant expression vector, transforming host cells, carrying out induced expression, purifying to obtain target lyase, and further preparing various antibacterial products. The streptococcus suis lyase Ply2413 disclosed by the invention can be used for preventing and controlling streptococcus suis, staphylococcus aureus and streptococcus uberis infection, and has the advantages of wide lysis spectrum, multiple action targets and good environmental stability; in addition, the preparation method disclosed by the invention has the advantages of simplicity and convenience in operation, high expression efficiency, large-scale production and high technical repeatability.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more specifically, to a streptococcal lysin Ply2413 and its applications. Background Technology

[0002] Streptococcus suis is a zoonotic pathogen that seriously threatens the global swine industry and public health. Its diverse serotypes and geographically specific epidemiological characteristics, coupled with widespread mixed infections in the field, pose significant challenges to disease control. Traditional antibiotic treatment faces severe limitations due to the rapid evolution of bacterial resistance. Phage lyases, as novel antimicrobial agents, have emerged as important candidates as alternatives due to their low resistance rate and high bactericidal efficiency. These lyases exert their bactericidal effect by specifically hydrolyzing the peptidoglycan of bacterial cell walls, demonstrating promising application potential in the control of Streptococcus suis infections and offering a safer antimicrobial solution for the swine industry.

[0003] However, existing Streptococcus suis lysins generally suffer from core technological defects, namely, a narrow lysis spectrum and a single target. Most existing lysins are only effective against a few Streptococcus suis serotypes, failing to cover the many important serotypes prevalent in the field, resulting in significant blind spots in prevention and control. Furthermore, they only act against Streptococcus suis and have no lysis activity against pathogens commonly co-infecting with Streptococcus suis, such as Staphylococcus aureus and Streptococcus lactis, leading to the need for multiple antimicrobial products in combination when mixed infections occur, increasing prevention and control costs and operational complexity. Summary of the Invention

[0004] To address the issues of narrow lysis spectrum and single target of suicidal streptococcal lysins in existing technologies, this application provides a suicidal streptococcal lysin Ply2413 and its applications.

[0005] In a first aspect, this application provides a suicidal streptococcal lysin Ply2413, which adopts the following technical solution: A Streptococcus suis lysin Ply2413, said lysin being a recombinant protein having the amino acid sequence shown in SEQ ID NO:1.

[0006] By employing the above technical solution, a typical modular structure is formed using the recombinant Streptococcus suis lyase Ply2413 with a specific amino acid sequence SEQ ID NO:1. Its N-terminal CHAP catalytic domain hydrolyzes bacterial cell wall peptidoglycan, while its C-terminal SH3b cell wall binding domain specifically recognizes and anchors the Streptococcus suis cell wall. The two domains work synergistically to achieve precise binding and efficient destruction of the target bacterial cell wall, thereby achieving the lysis of multiple bacteria. This specific amino acid sequence endows the lyase with a clear target and highly efficient catalytic activity, enabling it to specifically target 11 serotypes of clinical Streptococcus suis isolates. It also lyses common co-infecting pathogens such as Staphylococcus aureus and Streptococcus lactis, providing a concrete and feasible material basis for bacterial control in complex infection scenarios. Furthermore, it lays the core material foundation for the subsequent large-scale preparation of this lyase through genetic engineering and the expansion of its applications in veterinary drugs, disinfectants, and feed additives.

[0007] Preferably, the lyase comprises a CHAP catalytic domain and an SH3b cell wall binding domain.

[0008] By employing the above-mentioned technical solution, the CHAP catalytic domain and SH3b cell wall-binding domain contained in the lyase work synergistically. The CHAP catalytic domain hydrolyzes bacterial cell wall peptidoglycan, while the SH3b cell wall-binding domain specifically recognizes and anchors the cell walls of *Streptococcus suis* and related co-infecting pathogens. This precise coordination between the two achieves a coherent process of recognition and hydrolysis, thereby achieving targeted destruction of the target bacterial cell wall. This domain combination is the core technological basis for the lyase's broad-spectrum cleavage activity, ensuring its targeted action against 11 serotypes of *Streptococcus suis*, as well as *Staphylococcus aureus* and *Streptococcus lactis*. It also provides a clear structural basis for the retention of core cleavage function in functional variants and active fragments, making the prediction and verification of protein function during genetic engineering more targeted and the implementation of the technical solution more feasible.

[0009] Preferably, the lysin can maintain its lysing activity under conditions of pH 4-9 and temperature 4°C to 45°C.

[0010] By adopting the above technical solution, and by defining the optimal pH range for maintaining the lysin's lytic activity as 4 to 9 and the temperature range as 4°C to 45°C, this technique ensures that the lysin's activity maintains a clear environmental adaptation range. Within this range, the CHAP catalytic domain and the SH3b cell wall binding domain can stably maintain their spatial conformation, thus continuously fulfilling their core functions of hydrolyzing peptidoglycan and recognizing and anchoring to the cell wall. This avoids structural denaturation and loss of activity due to environmental acid-base imbalances or temperature fluctuations. This limitation not only clearly defines the applicable environmental boundaries of the lysin but also leverages the structural stability advantage provided by its specific amino acid sequence. It can adapt to various practical scenarios, including mild high temperatures during feed processing, ambient or low temperatures during storage and transportation, and acidic environments for in vivo application. This provides a clear technical basis for the storage, transportation, and diverse applications of the lysin after large-scale production, making the implementation of the technical solution more feasible.

[0011] Preferably, the lysin is capable of specifically lysing 11 serotypes of Streptococcus suis, Staphylococcus aureus, and Streptococcus lactis.

[0012] By employing the above-mentioned technical solution, through the precise synergy between the CHAP catalytic domain of the lyase and the SH3b cell wall-binding domain, the SH3b cell wall-binding domain specifically recognizes and anchors to the cell wall characteristic sites of 11 serotypes of Streptococcus suis, Staphylococcus aureus, and Streptococcus lactis. The CHAP catalytic domain then hydrolyzes the peptidoglycan of the target bacterial cell wall, disrupting the cell wall's structural integrity, thereby achieving specific lysis of these bacteria. This technology directly relies on the specific structure and mechanism of action of the lyase, covering the major prevalent serotypes of Streptococcus suis and common co-infecting pathogens in clinical practice. It solves the problems of limited lysis spectrum and single target in existing technologies, providing a clear material basis for addressing the complex and variable serotypes and mixed infections in the field.

[0013] Preferably, the lyase is a functional variant or an active fragment; the functional variant contains the same CHAP catalytic domain and SH3b cell wall binding domain as SEQ ID NO:1, and retains broad-spectrum lytic activity against Streptococcus suis, Staphylococcus aureus, and Streptococcus lactis; the active fragment contains the CHAP catalytic domain and SH3b cell wall binding domain as in SEQ ID NO:1, and retains the broad-spectrum lytic activity.

[0014] By employing the above technical solution, the functional variant retains the CHAP catalytic domain and SH3b cell wall binding domain corresponding to SEQ ID NO:1 during amino acid sequence modification. The CHAP catalytic domain hydrolyzes bacterial cell wall peptidoglycan, while the SH3b cell wall binding domain specifically recognizes and anchors the cell walls of *Streptococcus suis*, *Staphylococcus aureus*, and *Streptococcus lactis*. Their synergistic effect supports the lytic action against the target bacteria. The active fragment, by including the aforementioned CHAP catalytic domain and SH3b cell wall binding domain, achieves a coherent recognition and hydrolysis process through the precise coordination of these two domains, thereby maintaining its lytic function.

[0015] Preferably, the amino acid sequence of the functional variant is obtained by conservative substitution, deletion, addition or insertion of one or more amino acids into the amino acid sequence shown in SEQ ID NO:1.

[0016] By employing the above-described technical solution, a functional variant is constructed by conserved substitution, deletion, addition, or insertion of one or more amino acids in the amino acid sequence shown in SEQ ID NO:1. This technique selectively preserves the core sequence regions of the CHAP catalytic domain and the SH3b cell wall binding domain, modifying only non-critical functional regions, thereby maintaining the stability and functional integrity of the variant's core structure. These modification methods are all conventional techniques in molecular biology. Combined with the requirement that the functional variant maintains at least 80% sequence identity with the original sequence, this ensures that the variant retains its core function of specifically recognizing and lysing target bacteria. Furthermore, it provides a flexible technical path for optimizing enzyme stability, expression efficiency, and other characteristics according to practical application needs.

[0017] Secondly, this application provides a technical solution for isolating nucleic acid molecules, using the following approach: An isolated nucleic acid molecule encoding a Streptococcus suis lysin Ply2413, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0018] By employing the above-described technical solution, a nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:2 is used to precisely encode the Streptococcus suis lyase Ply2413 and its core domain. This nucleic acid molecule serves as the core template for gene cloning and expression, playing a role in the targeted transmission of the lyase's functional genetic information, thus providing a clear genetic basis for the recombinant preparation of the target protein. Based on this specific nucleotide sequence, specific primers can be designed for PCR amplification, constructing recombinant cloning and expression vectors, which can then be transformed into host cells such as Escherichia coli Trans5α or BL21(DE3) to achieve targeted expression of the lyase. Its sequence specificity ensures that the encoded lyase retains the complete amino acid sequences of the CHAP catalytic domain and the SH3b cell wall binding domain, providing genetic assurance for the lyase to perform its specific cleavage function.

[0019] Thirdly, this application provides a recombinant expression vector, which adopts the following technical solution: A recombinant expression vector comprising the aforementioned isolated nucleic acid molecule, the isolated nucleic acid molecule being operatively linked to an expression regulatory sequence; the recombinant expression vector is a recombinant plasmid constructed by inserting the nucleic acid molecule between the EcoRI and XhoI restriction sites of the prokaryotic expression vector pET-28a.

[0020] By employing the above-mentioned technical solution, a recombinant expression vector is constructed by directionally inserting the target isolated nucleic acid molecule between the EcoRI and XhoI restriction sites of the prokaryotic expression vector pET-28a, and operably linking this nucleic acid molecule with expression regulatory sequences such as promoters and terminators. The selection of specific restriction sites precisely mediates the insertion of the nucleic acid molecule into the vector, while the expression regulatory sequences guide the orderly transcription and translation of the nucleic acid molecule within the host cell, thereby enabling the recombinant expression vector to express the target lyase in a targeted and efficient manner. This construction method leverages the prokaryotic expression advantages of the pET-28a vector, combined with the specificity of the dual restriction sites to ensure correct insertion direction and sequence integrity, avoiding reverse linkage or multiple copy insertion. Simultaneously, the synergistic effect of the regulatory sequences and the nucleic acid molecule ensures the expression efficiency of the lyase gene, providing a structurally stable vector basis for subsequent transformation into *E. coli* Trans5α for cloning and identification, and transformation into BL21(DE3) for large-scale protein expression.

[0021] Fourthly, this application provides a host cell, employing the following technical solution: A host cell comprising the above-described recombinant expression vector, wherein the host cell is Escherichia coli, preferably Escherichia coli Trans5α or BL21(DE3).

[0022] By employing the above technical solution, a recombinant expression vector containing the target isolated nucleic acid molecule is introduced into host cells selected from Escherichia coli Trans5α, Escherichia coli BL21(DE3), yeast, or insect cells. Different host cells play a role in adapting to different technical steps. Escherichia coli Trans5α, with its high transformation efficiency and plasmid stability, plays a role in carrying the recombinant expression vector for cloning identification and ensuring the correctness of the vector sequence. Escherichia coli BL21(DE3), relying on the advantages of the prokaryotic expression system, plays a role in efficiently transcribing and translating the target gene and realizing the large-scale expression of lyase. Fifthly, this application provides an application of the suicidal streptococcal lysin Ply2413, employing the following technical solution: An application of a suicidal streptococcal lysin Ply2413, specifically for use in an antimicrobial product for the prevention and control of infections caused by Streptococcus suis, Staphylococcus aureus, or Streptococcus lactis, wherein the antimicrobial product is a veterinary drug, disinfectant, or feed additive.

[0023] By adopting the above technical solutions, and using the suicidal streptococcal lysin Ply2413, which has a specific lysing function, as the core active ingredient, targeted antibacterial products such as veterinary drugs, disinfectants, or feed additives are prepared. These products, combined with specific forms and ingredients, are tailored to different prevention and control needs: veterinary drugs are available in dosage forms such as injections, powders, sprays, or ointments, combined with pharmaceutically acceptable carriers, to precisely target the bacteria infecting pigs through different administration methods, directly lysing pathogens and inhibiting infection progression; disinfectants are available in liquid, powder, or effervescent tablet forms, containing lysin and potentially compounded with organic acids or alcohols, suitable for different disinfection scenarios such as surface spraying and wiping in the breeding environment, enhancing bactericidal effects and eliminating residual pathogens in the environment; feed additives, with lysin as the core ingredient, are used in the daily feeding of pigs, building an antibacterial barrier through continuous intake, thus preventing bacterial infection in advance. The three types of products form a closed-loop prevention and control system from three dimensions: treatment, environmental disinfection, and daily prevention. They not only meet the usage needs of different breeding scenarios, but also fully leverage the broad-spectrum antibacterial advantages of lysin through the clear definition of specific dosage forms and ingredients. This enables full-process and multi-scenario prevention and control of Streptococcus suis, Staphylococcus aureus, and Streptococcus lactis infections, thus meeting the complex breeding and prevention and control needs in the field.

[0024] In summary, this application has the following beneficial effects: 1. Because this application uses recombinant Streptococcus suis lyase Ply2413 with the amino acid sequence shown in SEQ ID NO:1, this lyase can specifically lyse 11 serotypes of Streptococcus suis as well as Staphylococcus aureus and Streptococcus lactis, which are common co-infecting pathogens in clinical practice, it breaks through the limitations of existing lyases with narrow lysis spectrum and single target. It can deal with complex infection scenarios without the need for multiple drugs, and provides a more comprehensive technical solution for the prevention and control of streptococcal disease and mixed infections in suis.

[0025] 2. The preferred lyase in this application can maintain lysing activity under conditions of pH 4-9 and temperature 4℃ to 45℃. Compared with the lyases with limited stability in the prior art, its environmental adaptability is significantly improved. It can withstand a variety of harsh scenarios such as feed processing, storage and transportation and in vivo application, without the need for special and harsh storage or use conditions, providing technical support for industrial production and multi-scenario promotion and application.

[0026] 3. The lyase of this application comprises functional variants or active fragments. These functional variants are obtained by retaining the key CHAP catalytic domain and SH3b cell wall-binding domain of SEQ ID NO:1 while maintaining their broad-spectrum cleavage activity. Such variants can be obtained through conventional modifications such as conserved substitution, deletion, addition, or insertion of one or more amino acids. This enriches the technical implementation forms of the protein without altering its core function, and enhances the flexibility and applicability of the technical solution to different production or application scenarios.

[0027] 4. This application encodes the target lysin using a specific nucleotide sequence SEQ ID NO:2. The recombinant expression vector constructed is based on the insertion of this nucleic acid molecule into a specific restriction site of the prokaryotic expression vector pET-28a. The host cells selected are suitable strains such as Escherichia coli Trans5α and Escherichia coli BL21(DE3). This technical approach realizes the efficient cloning, targeted expression, and large-scale purification of the target lysin, ensuring the stable acquisition of high-purity active protein and laying a solid foundation for the industrial transformation and mass production of the technical solution.

[0028] 5. The lyase of this application can be used to prepare antibacterial products such as veterinary drugs, disinfectants or feed additives, covering multiple core aspects of infection treatment, environmental cleaning and disinfection and daily feeding prevention in the breeding process. It eliminates the need to develop multiple products separately for different prevention and control needs, simplifies the operation process of breeding prevention and control, reduces prevention and control costs, and provides an integrated and multi-dimensional antibacterial technology solution for the pig industry. Attached Figure Description

[0029] Figure 1 This is a block diagram of the overall technical roadmap provided in this application; Figure 2This is a schematic diagram of the modular structural domains of the Ply2413 protein provided in this application; Figure 3 This is a fragmentation pattern of the Ply2413 protein provided in this application; Figure 4 This is a graph showing the effect of different temperatures on the lysing activity of Streptococcus suis lysin Ply2413, as provided in this application. Figure 5 This is a graph showing the effect of different pH values ​​on the lytic activity of Streptococcus suis lysin Ply2413, as provided in this application. Detailed Implementation

[0030] The present application will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used below are conventional methods. Unless otherwise specified, the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art, which can be obtained by those skilled in the art through commercial channels or prepared according to literature methods.

[0031] Technical concept: Streptococcus suis infection poses a serious threat to the swine industry and public health. Traditional antibiotic treatment faces challenges due to drug resistance, while existing Streptococcus suis lysin technology has significant limitations. Existing lysins generally suffer from narrow lysis spectra and uneven serotype coverage, with most only effective against a few serotypes. For example, some lysins cover only nine or fewer serotypes, making it difficult to address the complex and variable serotypes and mixed infections encountered in the field. Furthermore, existing lysins have single targets, specifically targeting Streptococcus suis, lacking lysis activity against pathogens commonly co-infecting with it clinically, such as Staphylococcus aureus and Streptococcus lactis. This necessitates the use of multiple drugs in combination for mixed infection treatment, increasing the complexity and cost of medication. In addition, some existing lysins lack stability, with temperature tolerance limited to 4℃-37℃ and pH adaptation concentrated in the alkaline region, failing to meet the demands of demanding applications such as feed processing, high-temperature storage, and acidic in vivo environments. The core reason for this lies in the inherent deficiencies in the amino acid sequence design and synergistic action mechanisms of existing lysins, failing to achieve synergistic optimization of lysis specificity, broad spectrum, and environmental adaptability.

[0032] This technical solution systematically addresses the aforementioned technical challenges through precise molecular design and a complete technical chain. First, it identifies and provides for the first time a Streptococcus suis lysin Ply2413 with a specific amino acid sequence (SEQ ID NO:1), whose encoding gene sequence is SEQ ID NO:2. The protein's unique modular structure—the N-terminal CHAP catalytic domain and the C-terminal SH3b cell wall binding domain—achieves highly efficient synergy between hydrolysis and recognition-anchoring functions, laying the structural foundation for its broad-spectrum lysing activity. Second, through a standardized genetic engineering approach, including the targeted design of primers containing specific restriction sites, the construction of the pMD19-T cloning vector and the pET-28a expression vector, the use of *E. coli* Trans5α and BL21(DE3) as cloning and expression hosts respectively, and IPTG-induced expression followed by nickel column affinity chromatography purification, high-purity activity is ensured. Furthermore, this lyase has been functionally verified to possess broad-spectrum lysing capabilities covering 11 serotypes of Streptococcus suis, and it can also lyse Staphylococcus aureus and Streptococcus lactis across species, achieving multi-target effects with a single drug. Simultaneously, it maintains high activity within a pH range of 4-9 and a temperature range of 4℃-45℃, significantly broadening its application boundaries. Finally, through sequence substitution schemes such as functional variants, active fragments, and fusion proteins, gene substitution schemes such as degenerate nucleotides and hybrid nucleotides, and application substitution schemes involving multiple dosage forms and multiple combinations, a complete technical protection system is formed to ensure the practicality and scalability of the technical solution, comprehensively addressing the core deficiencies of existing technologies.

[0033] This specific embodiment details the preparation, functional verification, and application of the broad-spectrum Streptococcus suis lysin Ply2413. This scheme is based on the technical route disclosed in this invention and covers the entire process, including gene cloning, recombinant vector construction, protein expression and purification, functional detection, and practical application, ensuring that those skilled in the art can replicate it without any inventive effort. The overall technical route of this invention is as follows: Figure 1 As shown, the core technology involves obtaining lysins with specific amino acid sequences through genetic engineering, which are then used in industrial applications after characterization and verification.

[0034] I. Preparation of lyase Ply2413 1. Gene cloning and construction of recombinant expression vectors First, based on the nucleotide sequence shown in SEQ ID NO:2, specific upstream and downstream primers for amplifying the lyase gene were designed using SnapGene software, with EcoRI and XhoI restriction sites added to both ends of the primers, respectively. Using the bacterial genome as a template, the target gene was amplified by PCR using the designed primers. After amplification, the PCR product was recovered by gel extraction to obtain the purified target fragment.

[0035] The target fragment recovered from the gel was ligated to the pMD19-T cloning vector overnight at 16°C to construct the recombinant cloning plasmid pMD19T-Ply2413. This recombinant cloning plasmid was transformed into *E. coli* Trans5α competent cells for positive clone selection. The selected positive clones were sequenced for verification. After confirming the correct target gene sequence, the correct recombinant cloning plasmid pMD19T-Ply2413 was obtained. The correct positive pMD19T-Ply2413 plasmid and pET-28a vector were double-digested using QuickCut™ EcoRI and XhoRI enzymes at 37°C for 30 minutes. After identification of the digestion products by agarose gel electrophoresis, the digested target fragment and vector fragment were recovered and purified using a kit. The recovered target fragment and vector fragment were ligated using T4 DNA Ligase overnight at 16°C. The ligation product was transformed into Escherichia coli Trans5α competent cells. The constructed clone strain pET-28a-Ply2413 was identified by PCR and double enzyme digestion, respectively, confirming that the recombinant expression plasmid was constructed correctly and obtaining the recombinant expression plasmid pET-28a-Ply2413.

[0036] 2. Protein Expression and Purification The validated recombinant plasmid pET-28a-Ply2413 was transformed into the expression host bacterium *Escherichia coli* BL21(DE3). Single colonies were picked and inoculated into LB medium containing 25 μg / mL kanamycin and cultured until the logarithmic growth phase, at which point the OD600 value reached 0.6 to 0.8. Isopropyl-β-D-thiogalactoside was added to the medium to a final concentration of 0.5 mM, and expression was induced at 16°C for 14 hours.

[0037] After induction of expression, bacterial cells were collected by centrifugation and then disrupted by ultrasonication to obtain a supernatant containing recombinant Ply2413 protein. The supernatant was purified by nickel affinity chromatography to obtain high-purity recombinant Ply2413 protein for subsequent functional validation experiments.

[0038] II. Functional Verification of Lysis Enzyme Ply2413 1. Pyrolysis spectrum determination The lysis profile of Ply2413 was determined using liquid lysis kinetics. Purified Ply2413 protein was adjusted to a concentration of 50 μg / mL and co-incubated with 23 clinical isolates of Streptococcus suis from different serotypes, covering 11 serotypes. Changes in bacterial turbidity were monitored during incubation, and the results are shown below. Figure 3 As shown, the turbidity of all Streptococcus suis bacterial cultures decreased significantly after treatment with Ply2413, confirming its broad-spectrum lytic activity covering 11 serotypes.

[0039] Simultaneously, Ply2413 protein was co-incubated with Staphylococcus aureus and Streptococcus lactis, and changes in bacterial turbidity were monitored. The results showed that Ply2413 produced significant lytic effects on both Staphylococcus aureus and Streptococcus lactis, confirming its cross-species lytic ability.

[0040] 2. Stability testing Buffer solutions with different pH values, ranging from 4 to 9, were prepared. Ply2413 protein was treated in each pH buffer for a certain period of time, and its residual cleavage activity was measured. Simultaneously, Ply2413 protein was treated at different temperatures ranging from 4°C to 70°C for a certain period of time, and its residual cleavage activity was also measured.

[0041] The test results showed that Ply2413 maintained more than 90% of its lytic activity after treatment in buffer solutions with pH 4 to 9; it also maintained more than 90% of its lytic activity after treatment in a temperature range of 4°C to 45°C, demonstrating its superior environmental stability.

[0042] III. Application Examples of Lysase Ply2413 1. Preparation of veterinary drug formulations A therapeutically effective amount of purified Ply2413 protein is mixed with a pharmaceutically acceptable carrier, including sterile saline and stabilizers, to prepare an injectable formulation for the prevention or treatment of Streptococcus suis infection. Alternatively, Ply2413 protein can be mixed with suitable excipients to prepare other veterinary drug dosage forms such as powders, sprays, or ointments to meet the needs of different application scenarios.

[0043] 2. Preparation of disinfectants Ply2413 protein is dissolved in buffer solution to prepare a disinfectant solution of effective concentration for disinfection of surfaces in aquaculture environments. This disinfectant can be formulated as a liquid, powder, or effervescent tablet, and can also be compounded with organic acids or alcohols to enhance the disinfection effect. It is suitable for spraying or wiping disinfection of surfaces in aquaculture environments.

[0044] IV. Alternative Implementation Plan In addition to the preferred embodiments described above, those skilled in the art can achieve the purpose of this invention through the following alternative solutions, all of which fall within the protection scope of this invention.

[0045] 1. Alternatives to protein sequences Functional variants: Proteins that retain the integrity and key functions of the CHAP catalytic domain and SH3b cell wall binding domain in SEQ ID NO:1, and maintain broad-spectrum cleavage activity against Streptococcus suis, Staphylococcus aureus, and Streptococcus lactis. These variants include sequences obtained by conserved substitution, deletion, addition, or insertion of one or more amino acids into SEQ ID NO:1.

[0046] Active fragment: A polypeptide fragment containing the CHAP catalytic domain and SH3b cell wall binding domain of SEQ ID NO:1, and retaining the broad-spectrum cleavage activity. The fragment can be obtained by deleting certain non-essential N-terminal, C-terminal, or cyclic regions of SEQ ID NO:1.

[0047] Fusion protein: A fusion protein formed by fusing the amino acid sequence shown in SEQ ID NO:1 with an additional protein sequence, wherein the additional protein sequence includes a tag protein for purification, a chaperone protein for enhancing stability, a guide peptide for targeting specific tissues, etc., and is an alternative to the present invention as long as the core cleavage activity is not substantially changed.

[0048] 2. Alternative solutions for coding genes and expression systems Degenerate nucleotide sequences: The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1 is not limited to SEQ ID NO:2, but also includes all nucleotide sequences that are degenerate from SEQ ID NO:2. Due to the degeneracy of the genetic code, such sequences may be different but encode the exact same amino acid sequence.

[0049] Hybrid nucleotide sequence: A nucleotide sequence that can hybridize with the complementary sequence of SEQ ID NO:2 under stringent conditions and encodes a lyase having the above-described cleavage activity of the present invention.

[0050] Alternatives to the expression system: The expression vector is not limited to pET-28a. Commonly used prokaryotic expression vectors in the field, such as the pET series and pCold series, and eukaryotic expression vectors, such as yeast expression vectors and insect cell expression vectors, can all be used to achieve the purpose of this invention. The host bacteria is not limited to Escherichia coli BL21(DE3). Other suitable expression hosts, such as other Escherichia coli strains, Pichia pastoris, and insect cells, are all alternative solutions.

[0051] 3. Alternative solutions for application formats Alternative formulations: In addition to injections, pharmaceutical compositions can be prepared into any suitable veterinary drug dosage form, such as oral preparations, powders, sprays, and ointments; disinfectants can be prepared into liquid, powder, or effervescent tablet forms and can be compounded with other safe disinfectant ingredients for synergistic effects.

[0052] Combination scheme: The lyase of the present invention can be used alone or in combination with other antibacterial lyases, antimicrobial peptides or low-dose antibiotics to further broaden the antibacterial spectrum or prevent the development of drug resistance.

[0053] SEQ ID NO: 1 MTTVNEALNNVRAQVGSGISVGNGECYALASWYERMISPDATVGLGAGVGWVSGAIGDTISAKNIGSSYNWQANGWTVSTSGPFKAGQIVTLGATPGNPYGHVVIVEAVDGDRLTILEQNYG GKRYPVRNYYSATSYRQQVVHHITPPGTITQSAPNLAGSRSYRETGTMTVTVDALNVRRAPNTSGEIVAVYKRGESFDYDTVIIDVNGYVWVSYIGGSGKRNYVATGATKDGKRFGNAWGTFK SEQ ID NO: 2 ATGACAACAGTAAATGAAGCCTTAAATAACGTCCGTGCCCAAGTCGGGTCTGGCATATCAGTAGGTAACGGCGAATGCTACGCTTTGGCTAGTTGGTACGAGCGCATGATTAGTCCGGATGCAACTGTCGGACTTGGCGCTGGTGTGGGCTGGGTCAGCGGTGCAATCGGCGATACAATCTCT GCCAAAAACATCGGCTCATCATACAACTGGCAAGCTAACGGCTGGACAGTTTCCACATCTGGTCCATTTAAAGCAGGTCAGATTGTGACGCTTTGGGGCAACACCAGGAAACCCTTACGGACATGTGGTAATCGTCGAAGCAGTGGACGGCGATAGATTGACTATTTTGGAGCAAAACTACGGCG GGAAACGTTATCCCGTCCGTAATTACAGCGCTACAAGCTATCGTCAACAAGTCGTGCACCACATCACACCGCCTGGAACGATTACACAGTCAGCACCCAACCTTGCAGGCTCTCGTTCCTATCGCGAGACGGGCACTATGACTGTCACGGTCGATGCTCTCAATGTTCGCAGGGCGCCAAA TACTTCAGGCGAGATTGTAGCAGTATACAAGCGTGGTGAATCATTTGACTATGATACTGTCATCATCGATGTCAATGGCTATGTCTGGGTGTCTTACATAGGCGGCAGCGGCAAACGTAACTACGTTGCGACGGGCGCTACCAAAGACGGTAAGCGTTTCGGCAATGCTTGGGGTACATTTAAA This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A suicidal streptococcal lysin Ply2413, characterized in that: The lyase is a recombinant protein with the amino acid sequence shown in SEQ ID NO:

1.

2. The *Streptococcus suis* lysin Ply2413 according to claim 1, characterized in that: The lyase contains a CHAP catalytic domain and an SH3b cell wall binding domain.

3. The *Streptococcus suis* lysin Ply2413 according to claim 1, characterized in that: The lysin can maintain its lysing activity under conditions of pH 4-9 and temperature 4°C to 45°C.

4. The *Streptococcus suis* lysin Ply2413 according to claim 1, characterized in that: The lyase can specifically lyse 11 serotypes of Streptococcus suis, Staphylococcus aureus, and Streptococcus lactis.

5. The *Streptococcus suis* lysin Ply2413 according to claim 1, characterized in that: The lyase is a functional variant or an active fragment; wherein the functional variant contains the same CHAP catalytic domain and SH3b cell wall binding domain as SEQ ID NO:1, and retains broad-spectrum lytic activity against Streptococcus suis, Staphylococcus aureus, and Streptococcus lactis; the active fragment contains the CHAP catalytic domain and SH3b cell wall binding domain as in SEQ ID NO:1, and retains the broad-spectrum lytic activity.

6. The *Streptococcus suis* lysin Ply2413 according to claim 5, characterized in that: The amino acid sequence of the functional variant is obtained by conservative substitution, deletion, addition or insertion of one or more amino acids into the amino acid sequence shown in SEQ ID NO:

1.

7. A method for isolating nucleic acid molecules, characterized in that, The isolated nucleic acid molecule is used to encode a Streptococcus suis lysin Ply2413 as described in any one of claims 1-6, and its nucleotide sequence is shown in SEQ ID NO:

2.

8. A recombinant expression vector, characterized in that, The recombinant expression vector comprises an isolated nucleic acid molecule as described in claim 7, wherein the isolated nucleic acid molecule is operatively linked to an expression regulatory sequence; the recombinant expression vector is a recombinant plasmid constructed by inserting the nucleic acid molecule between the EcoRI and XhoI restriction sites of the prokaryotic expression vector pET-28a.

9. A host cell, characterized in that, The host cell comprises a recombinant expression vector as described in claim 8, wherein the host cell is selected from Escherichia coli.

10. The application of a Streptococcus suis lysin Ply2413, characterized in that, The Streptococcus suis lysin Ply2413 according to any one of claims 1-6 is used as an antimicrobial product for the prevention and control of Streptococcus suis, Staphylococcus aureus or Streptococcus lactis infections, wherein the antimicrobial product is a veterinary drug, disinfectant or feed additive.

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