Endolysin derived from staphylococcus warneri mild bacteriophage and application of endolysin

Abstract

The invention belongs to the technical field of biology, and particularly relates to endolysin derived from staphylococcus warneri mild bacteriophage and application of the endolysin. The endolysin has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence obtained after further optimization of the endolysin. The endolysin gene sequence is completely and successfully obtained on the basis of the staphylococcus warneri mild bacteriophage vBG3001, the endolysin protein (endolysin 65) is successfully expressed and purified, and the endolysin protein has an inhibition effect on the growth of a gram-positive bacterium bacillus velezensis and a gram-negative bacterium burkholderia. The staphylococcus warneri mild bacteriophage perforin gene and endolysin gene are co-expressed in escherichia coli, so that the staphylococcus warneri mild bacteriophage has relatively strong bactericidal activity on host escherichia coli. The effects provide a theoretical basis for prevention and treatment of gram-negative pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an endolysin derived from Staphylococcus warwick temperate phage and its applications. Background Technology

[0002] Coagulase is considered a major virulence factor in Staphylococcus aureus. Based on their ability to coagulate rabbit blood, bacteria are classified into coagulase-positive and coagulase-negative bacteria. Coagulase-negative bacteria (CNS) are generally non-pathogenic and are classified as minor pathogens, widely distributed in the natural environment and possessing richer genetic diversity. CNS are indispensable fermentation microorganisms in traditional fermented meat product production, exhibiting significant enzymatic characteristics such as proteases and lipases. *Staphylococcus warwickii* (… Staphylococcus warneri Staphylococcus warwick is a relatively common CNS (concentrated natural gas) used as a fermentation agent for meat products due to its highly efficient enzymatic properties. In addition, it can produce a series of antimicrobial peptides as bactericides. In industrial applications, Staphylococcus warwick not only has the potential to produce heat- and organic solvent-resistant lipases, but also plays an important role in the field of metal nanoparticles, which has received considerable attention in recent years.

[0003] Bacteriophages are a general term for viruses that specifically infect bacteria. Based on their different growth cycles during their interaction with the host, they can be divided into two categories: virulent phages and temperate phages. After infecting host bacteria, virulent phages immediately enter the lysis cycle. During this process, the phage genome is replicated extensively and packaged into progeny phage particles, which are released after programmed cell lysis of overloaded cells. Temperate phages, on the other hand, have lysogenic and lysis cycles. During the lysogenic cycle, the phage can integrate its genome into the host's chromosome. At this stage, the temperate phage is called a prophage, which remains in a latent or dormant state. The phage replicates along with the bacterial host chromosome, and this lysogenic state is maintained by inhibiting the phage's lysis genes. Under the stimulation of induced bacterial SOS responses (antibiotic treatment, oxidative stress, or DNA damage), prophages activate the lysis cycle pathway, lysing bacterial cells and releasing progeny phages, just like virulent phages. For most double-stranded DNA (dsDNA) bacteriophages, host lysis at the end of the replication cycle is primarily due to the coordinated action of two proteins. Holin is a protein that controls lysis time by permeating the host inner membrane to allow endolysin to diffuse, which then acquires and degrades cell wall peptidoglycan to induce lysis. Bacteriophages infecting Gram-negative bacteria also require a third protein to destabilize the outer membrane. Furthermore, some endolysins are independent of holin, interacting with general host secretion pathways via signaling.

[0004] With the overuse of antibiotics, problems such as the increasing number of multidrug-resistant bacteria, the inhibitory effects of some antibiotics on beneficial microorganisms, and the inability of some novel antibiotics to kill bacteria while only inhibiting the growth of pathogens, have emerged. Bacteriophages and their derivatives, possessing host specificity and not producing resistance, have attracted increasing attention. Therefore, the search for new antibacterial drugs is urgent, and phage-derived proteins offer a potential solution to this pressing issue. Perforin and endolysin are key genes in phage lysis of the host and have great application potential as phage derivatives. Unlike traditional broad-spectrum antibiotics, endolysin has many advantages, including high efficiency, non-destruction of non-target cells and animal cells, easy production and modification of enzyme proteins, and no impact on its antibacterial activity and efficacy by antibodies. Furthermore, the target specificity of endolysin gives it a broader antibacterial spectrum than phages. Research on endolysin in Gram-negative bacteria faces significant challenges, as the outer membrane prevents exogenous endolysin from contacting peptidoglycan; therefore, further development and utilization of endolysin are needed. This challenge has been addressed by: (1) identifying natural endonolysins capable of penetrating the outer membrane; (2) synergistic effects of endonolysins with outer membrane permeabilizers (such as EDTA, chloroform, cationic peptides, and antibacterial agents); (3) fusing endonolysins with a peptide; and (4) combining endonolysins with perforin. In the later stages of the phage lysis cycle, endonolysins are initially produced and accumulated in the cytoplasm, but they cannot penetrate the cytoplasmic membrane to function in the peptidoglycan layer. The synergistic effect of perforin is another important component of the phage binary lysis system. When perforin aggregates, it promotes inner membrane depolarization. Perforin dimers are the basic form of perforin functional assembly, which then polymerize into oligomers, forming pores in the cytoplasmic membrane, allowing endonolysins to be released into the periplasm. In the periplasm, endonolysins, acting as peptidoglycan hydrolases, begin to digest the peptidoglycan layer of the bacterial cell wall, ultimately leading to cell lysis due to the osmotic pressure difference between the cell and its surrounding environment. Therefore, studying the combined use of endosomalin and perforin has positive significance for the prevention and control of Gram-negative pathogens.

[0005] Currently, there is relatively little research on the mild bacteriophage of Staphylococcus wartii, and there are no reports on the inolysin of the mild bacteriophage of Staphylococcus wartii. Summary of the Invention

[0006] The first objective of this invention is to provide the mild phage in vivo lysin gene of Staphylococcus warwickii ( endolysin65 The protein sequence encoded by the protein was then further expressed and purified in E. coli.

[0007] A second objective of this invention is to provide a protein encoded by the mild bacteriophage in vivo lysin gene of Staphylococcus warwick that has antibacterial activity against Gram-positive and Gram-negative bacteria.

[0008] The third objective of this invention is to provide the co-expression and application of the endosynovitis gene and its perforin gene of Staphylococcus wartii mild phage in Escherichia coli. The co-expression of proteins encoded by the perforin gene and endosynovitis gene of Staphylococcus wartii mild phage described in this invention has strong bactericidal activity against the host Escherichia coli.

[0009] To achieve the objectives described in this invention, the following technical solutions are provided: The nucleotide sequence of an endolysin gene derived from Staphylococcus warwick temperate phage is shown in SEQ ID NO:1, or a further optimized nucleotide sequence thereof.

[0010] The optimized endosomalin has the nucleotide sequence shown in SEQ ID NO:2.

[0011] The amino acid sequence of the protein encoded by the endolysin of the nucleotide sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:3. The amino acid sequence of the protein encoded by the endolysin of the nucleotide sequence shown in SEQ ID NO:2 is shown in SEQ ID NO:3.

[0012] The primer pair for obtaining the nucleotide sequence endosomalin shown in SEQ ID NO:1 is: endolysin65- F CGGGATCCATGGCTACAGAGAATTGGAAAGG; endolysin65- R CGAGCTCTTATTTCCACTTAATGGAACCCC.

[0013] The primer pair for obtaining the optimized endosomal is: endolysin65- F GAATTCATGGCTACTGAAAACTGG endolysin65- R GCGGCCGCTTATTTCCAC The application of the endolysin derived from Staphylococcus warwick temperate phage, wherein the endolysin, or the use of a mixture of endolysin and perforin in the preparation of an antibacterial agent.

[0014] The use of the endolysin in the preparation of antibacterial agents that inhibit Gram-positive Bacillus belyssus and / or Gram-negative Burkholderia.

[0015] The use of the mixture of endosomalin and perforin in the preparation of an antibacterial agent that inhibits Gram-negative host bacteria.

[0016] The perforin is the nucleotide sequence shown in SEQ ID NO:4, or a further optimized nucleotide sequence shown in SEQ ID NO:5.

[0017] The amino acid sequence of the protein encoded by the perforin of the nucleotide sequence shown in SEQ ID NO:4 is shown in SEQ ID NO:6. The amino acid sequence of the protein encoded by the perforin of the nucleotide sequence shown in SEQ ID NO:5 is shown in SEQ ID NO:6.

[0018] The primer pair for obtaining perforin derived from Staphylococcus warwick temperate phage is: holin64-F GAGCTCGATGCAATTCCAGAAAAACAATACA; holin64-R CTGCAGCTATTTTAAATTATTTTCTTTTAAGTATT.

[0019] The primer pair used to obtain the optimized nucleotide sequence of perforin is: holin64F GAATTCATGCAGTTTCCAAAAGAACAACACC; holin64R GCGGCCGCTTACTTCAGGGTTGTTCTCCTTCAGATATT.

[0020] An antibacterial agent containing the endosomalin, or containing the endosomalin and perforin.

[0021] When the antibacterial agent contains endosomalin and perforin, the two are used in a 1:1 mass ratio.

[0022] A method for lysing cells in vivo involves introducing the endolysin and perforin into recipient cells, culturing them, and then lysing the recipient cells.

[0023] Specifically, the process involves amplifying the endolysin and perforin, digesting the amplification products and plasmids with restriction endonucleases, integrating them to obtain a recombinant plasmid, and then transforming the recombinant plasmid into recipient cells. Successfully transformed *E. coli* cells are then incubated with 50 μg / mL [a specific solution / container / etc.]. -1 Amp cultured in LB liquid medium until OD 600 The value was approximately 0.6, and the final concentration added was 0.5 mg·mL. -1 IPTG-induced overnight low-temperature induction leads to host cell lysis.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention successfully obtained the endolysin gene sequence based on the mild phage vB_G30_01 of *Staphylococcus wartii* and successfully expressed and purified the endolysin protein (endolysin65). This endolysin protein exhibits inhibitory effects on the growth of one Gram-positive bacterium, *Bacillus belyssiensis*, and one Gram-negative bacterium, *Burkholderia gravidarum*. Co-expression of the perforin gene and the endolysin gene from the mild phage of *Staphylococcus wartii* in *Escherichia coli* showed strong bactericidal activity against the host bacterium *Escherichia coli*. These results provide a theoretical basis for the prevention and control of Gram-negative pathogens. Attached Figure Description

[0025] Figure 1 This diagram illustrates the transcription of the endosomalin gene in Staphylococcus warwick temperate phage vB_G30_01 under mitomycin C (MMC) induction, as provided in this embodiment of the invention.

[0026] Figure 2 This is an analysis diagram of the conserved domains of the endosomal protein provided in an embodiment of the present invention.

[0027] Figure 3 This is a predicted tertiary structure diagram of the endosomal protein provided in an embodiment of the present invention.

[0028] Figure 4 The electrophoresis diagram of the clonal endosin gene provided in the embodiments of the present invention, wherein M: 2000 Marker; 1-2: holin64 .

[0029] Figure 5 The image shows the purified electrophoresis result of the recombinant protein pET32a-endolysin65 provided in this embodiment of the invention, where M: Marker; 1: Sample eluent; 2-6: Washing Buffer eluted sequentially; 7-10: Elution Buffer eluted sequentially.

[0030] Figure 6 This is an SDS-PAGE electrophoresis image of the endosomal protein TCA precipitated by Pichia pastoris GS115 eukaryotic expression provided in an embodiment of the present invention, where M: Marker; 1-6: recombinant protein pPIC9K-endolysin65; 7: pPIC9K.

[0031] Figure 7 The diagram shows the antibacterial effect of recombinant protein pET32a-endolysin65 and its combined antibacterial effect with outer membrane permeability agent EDTA provided in the embodiments of the present invention, wherein A: Bacillus belye; B: Burkholderia; 1: CK; 2: pET32a-endolysin65; 3: EDTA; 4: EDTA+pET32a-endolysin65.

[0032] Figure 8 The diagram shows the inhibitory effect of endosomalin protein expressed by Pichia pastoris GS115 eukaryotically against Gram-positive Staphylococcus warwick G30, provided in the embodiments of the present invention, where 1-2: CK; 3-4: perforin protein.

[0033] Figure 9 The transformation and recombinant plasmid pETDuet provided in this embodiment of the invention TM -1- holin64 - endolys-in65 PCR electrophoresis image of Escherichia coli Rosetta-gami2 (DE3) colonies, where M: 2000 Marker; 1-2: endolysin65 3-4: holin64 .

[0034] Figure 10 The diagram shows the inhibitory effect of perforin protein expressed by Pichia pastoris GS115 eukaryotically on Gram-positive bacteria, as provided in the embodiments of the present invention. A: Staphylococcus aureus; B: Staphylococcus aureus ATCC25923; C: Bacillus cereus; D: Staphylococcus wartii G30. 1-2: CK; 3-4: perforin protein.

[0035] Figure 11 The diagram shows the inhibitory effect of perforin protein expressed by Pichia pastoris GS115 eukaryotically on Gram-negative bacteria, as provided in this embodiment of the invention. A: Escherichia coli ATCC25922; B: Salmonella; C: Burkholderia D4-24. 1-2: CK; 3-4: perforin protein.

[0036] Figure 12 The transformation plasmid pETDuet provided in the embodiments of the present invention TM -1. Recombinant plasmid pETDuet TM -1- holin64 and recombinant plasmid pETDuet TM -1- holin64 - endolysin65 Growth curve of Escherichia coli Rosetta-gami2 (DE3).

[0037] Figure 13 The transformation plasmid pETDuet provided in the embodiments of the present invention TM -1. Recombinant plasmid pETDuet TM -1- holin64 and recombinant plasmid pETDuet TM -1- holin64 - endolysin65Scanning electron microscopy (SEM) images of Escherichia coli Rosetta-gami2 (DE3) before and after IPTG induction; where A is the SEM image before IPTG induction and B is the SEM image after IPTG induction.

[0038] Figure 14 The plate inhibition diagram of Staphylococcus aureus by the combined use of perforin and endosomal protein expressed by Pichia pastoris GS115 eukaryotically provided in the embodiments of the present invention is shown in the figure, where A: endosomal protein; B: perforin protein; C: perforin protein + endosomal protein; 1-2: pPIC9K; 3-4: perforin or endosomal protein. Detailed Implementation

[0039] The following examples further illustrate this study, but the examples do not constitute any limitation on the invention.

[0040] This experiment obtained samples from Staphylococcus warwick temperate phage vB_G30_01 (Pu, F.; Zhang, N.; Pang, J.; Zeng, N.; Baloch, FB; Li, Z.; Li, B.). Deciphering the Genetic Architecture of Staphylococcus warneri Prophage vB_G30_01: A Comprehensive MolecularAnalysis. Viruses 2024, 16 (1631.) The candidate gene sequence of endosynovitis was obtained, its protein structure was predicted, and its function was successfully cloned and identified. The bactericidal activity of the encoded protein was investigated, providing theoretical support for the use of temperate phages to control Gram-negative pathogens. The protein encoded by the endosynovitis gene of this Staphylococcus warwickii temperate phage exhibited significant bactericidal activity against Escherichia coli.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional experimental methods already existing in the prior art.

[0042] Example 1: Detection of endosomal gene transcription level in Staphylococcus warwick temperate phage vB_G30_01 after mitomycin C (MMC) treatment The genomic data of bacteriophage vB_G30_01 has been submitted to the NCBI database, accession number PP213047. The endosin gene sequence (GenBank: WP_046464358.1) obtained from the whole genome sequencing of bacteriophage vB_G30_01 is shown in SEQ ID NO:1 and named as follows. endolysin65 .

[0043] SEQ ID NO:1 Staphylococcus warwick temperate phage vB_G30_01 endolysin gene ( endolysin65 Nucleotide sequence (5'-3') SEQ ID NO:3 Amino acid sequence of endolysin 65 from Staphylococcus warwick temperate phage vB_G30_01 MATENWKGVKVRYQLLTKGTRRYGETMDGGKPQFIVAHDTGNINTTAQSNVTYYENTYNIPWNNVASAHIFVDDKECIICIPTTEKAWHVLYDAPTDNIWYNKDANDVAIGVEICYF SDRERSRKALDNGARVLAYLAEYWHIDYKTRMPGHQDIQADKQDPGNALEASGYGRNTSNLDKLVAKYYKQNVKVKATPVKVEKGSTSFTREEFVKWLKSTVGKQYDYDLYAAFQCVD YANVGWDKLFGHGLKGNGAKDIPFNAYNKDKFKNEATVYKNTPSFLAKPGDLVVWGEQMGDGWGHVAWVVEATLDYIVVLEQNWLGGGWTSGPINNGTGWETVTRRKHEYDTQMWFI RPKFSNKKAESKLLKKSKEKKKEKQITWNWKGRFTTNTTIKVRRSPSLKGSVVPSSDWLLSNQWVDFVSITKKDGYWWAKFKYPTNPSSGYFYCALCKITDKQERIKKEKYWGSIKWK 1. Total RNA extraction Staphylococcus warwick G30 was cultured in beef extract peptone liquid medium until OD. 600 The concentration was approximately 0.4, and then a final concentration of 1 μg·mL⁻¹ was added to the system. -1 Mitomycin C (MMC) induction was performed, and an equal volume of fresh beef extract peptone liquid medium was added to the control. Bacterial cell pellets were collected at 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h of culture time, and RNA was extracted using the SteadyPure Universal RNA Extraction Kit.

[0044] 2. Synthesis of reverse-transcribed cDNA RNA template solutions were prepared using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (Evo M-MLV Reverse Transcription Premix Kit, which includes gDNA removal reagent for qPCR), and the reverse transcription reaction was performed. 3. Primers for Real-Time Quantitative PCR Table 1 Primers used for qPCR

[0045] Using the Premix Pro Taq HS qPCR Kit (ROX Plus) SYBR® Green Pro Taq HS premixed qPCR kit (containing ROX), 16S rDNA was selected as the internal reference gene.

[0046] Experimental results show that ( Figure 1 Under MMC treatment, the endosomalin gene of Staphylococcus warwick temperate phage vB_G30_01 began to be transcribed, but the relative expression level was not high, indicating that the gene was involved in the phage lysis and growth process. The relative expression level of the endosomalin gene reached its peak 8 h after the addition of MMC, indicating that it is a late-transcribed gene.

[0047] Example 2: Bioinformatics Analysis of Endolysin Protein 1. Physicochemical property analysis of endosomal proteins The physicochemical properties of proteins were analyzed using the Protparam software (https: / / web.expasy.org / protparam / ), including the protein's amino acid composition, relative molecular mass, isoelectric point, and hydrophilicity / hydrophobicity.

[0048] The physicochemical properties of the protein were analyzed using ProtParam software. The results showed that the endosomal protein consists of 470 amino acids, with a relative molecular mass of 54163.30, an isoelectric point of 9.32, and is slightly alkaline. It contains 52 negatively charged amino acids and 71 positively charged amino acids. Its molecular formula is C1. 2461 H 3726 N 658 O 705 S 11 With a total of 7561 atoms, an instability index of 26.01, a lipid index of 64.91, and an average hydrophilicity of -0.718, this protein is classified as a stable hydrophobic protein.

[0049] 2. Prediction of conserved domains of endolysin proteins The conserved domains of the endosomal protein were analyzed using the NCBI (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) online software for conserved domains.

[0050] Based on the comparison of perforin and endolysin proteins using the NCBI conserved domain database, the experimental results show that ( Figure 2Endolysin possesses two conserved structural domains: a homology domain of peptidoglycan recognition protein (PGRP) and a CHAP domain. PGRP is a pattern recognition receptor that binds to and, under certain conditions, hydrolyzes peptidoglycan (PGN) in the bacterial cell wall. PGRP has been classified into three classes: short PGRP (PGRP-S), an extracellular protein of approximately 20 kDa; intermediate PGRP (PGRP-I), a transmembrane protein of 40-45 kDa; and long PGRP (PGRP-L), up to 90 kDa, which can be both intracellular and transmembrane proteins. This family includes zinc-dependent N-acetylmuracil-L-alanine amidase, which cleaves the amide bond between the N-acetylmuracil group and the L-amino acid in the bacterial cell wall. The CHAP domain corresponds to the amidase function. Many related proteins are involved in bacterial cell wall hydrolysis. Cloning and expression of the CHAP domain has revealed a highly specialized antibacterial spectrum and strong antibacterial activity against Staphylococcus aureus.

[0051] 3. Prediction of the higher-order structure of endosomal proteins The secondary structure of the endosomal protein was analyzed using the SOPMA tool (https: / / npsaprabi.ibcp.fr / cgibin / npsa_automat.pl?page=npsa_sopma.htmL). The tertiary structure of the endosomal protein was predicted using AlphaFold (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynb), and a three-dimensional structural diagram of the protein was drawn.

[0052] SOPMA analysis of the secondary structure content of perforin revealed that the endorhein contained 27.66% α-helices, 22.55% extended chains, 10.21% β-sheets, and 39.57% random coils. This indicates that the secondary structure of endorhein is primarily random coils, followed by α-helices and extended chains in similar proportions, with β-sheets being the least abundant.

[0053] The tertiary structure of endosomal proteins was predicted using AlphaFold2 software, and the prediction results were modeled to generate 3D visualizations. The output results show that ( Figure 3 The three-dimensional model of the endosomal protein shows that its tertiary structure is mainly an α-helix structure with fewer β-sheet structures, which is basically consistent with the results of the secondary structure prediction.

[0054] Example 3 Cloning and Construction of Endolysin Gene Expression System 1. Primer design and synthesis The specific primers designed using Primer Premier 5.0 software (synthesized by Shanghai Sangon Biotech Co., Ltd.) are shown in the table below: Table 2 PCR cloning primer sequences and restriction enzyme sites

[0055] 2. Extraction of genomic DNA from Staphylococcus warwick temperate phage vB_G30_01 Staphylococcus warwick G30 was cultured in 800 mL of beef extract peptone broth at 37 °C and 180 rpm until OD reached [value missing]. 600 The concentration was approximately 0.4, and then a final concentration of 1 μg·mL⁻¹ was added to the system. -1 Mitomycin C (MMC) was induced at 37 ℃ and 180 rpm for 12 h. After centrifugation at 12000 rpm for 20 min, the supernatant was filtered sequentially through 0.45 μm and 0.22 μm microporous membranes, and extracted with chloroform to obtain crude phage particle extract. A CsCl gradient solution was prepared and added to centrifuge tubes sequentially from high to low density. The crude phage extract was added on top, and the tubes were centrifuged at 100000 g for 3 h at 4 ℃. The pale blue phage concentrate was slowly aspirated using a syringe. Finally, the phage particle concentrate obtained from CsCl was removed by ultrafiltration using a 100 kDa ultrafiltration tube. Genomic DNA of Staphylococcus warwick temperate phage vB_G30_01 was extracted using the phenol-chloroform method. The extracted DNA was dissolved in sterile water and stored at -20 ℃.

[0056] 3. Gene cloning and construction of prokaryotic expression vectors Using the genomic DNA of Staphylococcus warwick temperate phage vB_G30_01 obtained above as a template, endolysin65- F, endolysin65- R are primers, used for PCR amplification respectively. Figure 4 ).

[0057] PCR reaction system: Table 3 PCR reaction system

[0058] PCR reaction procedure: Table 4 PCR reaction procedure

[0059] The PCR reaction solution was subjected to agarose gel electrophoresis, and the target band was recovered using the SanPrep column DNA gel recovery kit. The operation steps were followed according to the detailed steps of the kit. The recovered target band was then sequenced for verification.

[0060] Using restriction endonucleases Sac I and Bam HI was used to double-digest the known plasmid pET32a(+) and the target gene that had not undergone sequencing to construct the recombinant plasmid pET32a-. endolysin65 The recombinant plasmid pET32a- endolysin65 Transformed Escherichia coli Rosetta-gami2 (DE3).

[0061] 4. Codon optimization and construction of eukaryotic expression vectors The endosomalin gene was optimized for codon bias in Pichia pastoris. The optimized nucleotide sequence is shown in SEQ ID NO:2 below. The gene was synthesized according to the optimized nucleotide sequence and ligated into the pPIC9K vector to construct the recombinant plasmid pPIC9K- endolysin65 Transformed E. coli DH5α, positive clones were screened by colony PCR, and plasmid pPIC9K- was extracted. endolysin65 Sequencing verification.

[0062] SEQ ID NO:2 Before transformation of yeast, the constructed recombinant plasmid was linearized using the restriction endonuclease Sac I to integrate the target gene into the Pichia pastoris genome. The reaction was carried out at 37 °C for 30 min, followed by treatment at 80 °C for 10 min to terminate the reaction. The linearized plasmid was detected by agarose gel electrophoresis, and the gel block was recovered and purified. Pichia pastoris GS115 competent cells and recombinant expression vector transformation were performed using a classic yeast transformation kit. Specific steps were followed according to the kit instructions. The transformed Pichia pastoris were plated on histidine-deficient medium onto MD plates and incubated upside down at 28 °C for 3 days.

[0063] Single colonies were picked from MD plates and spotted onto YPD solid plates containing different concentrations of G418 on small square grids. The final concentration of G418 was 0.5 mg / mL. -1 1 mg·mL -1 2 mg·mL -1 Incubate at 28°C upside down for 3-5 days. Take 2 mg / mL of the culture. -1 Colonies from G418 concentration plates were spot-inoculated onto MM plates (with methanol as the sole carbon source) with small squares drawn on them, and simultaneously spot-inoculated onto histidine-deficient MD plates with small squares drawn on them. The plates were incubated upside down at 28 °C for 3 days, and transformants that could grow on both MM and MD plates were screened.

[0064] Pick 3-6 single colonies and place them into separate 200 μL PCR tubes. Add 30 μL of sterile water and mix well. Transfer 10 μL to a new 200 μL PCR tube and react using the boiling water method at 85 °C for 5 min. Immediately cool the tube at -20 °C for 15 min to use as the template for PCR identification. The remaining 20 μL is used as the seed culture. Refer to section 2.3.6.3 for the PCR reaction system and conditions. PCR products were detected by gel electrophoresis and verified by sequencing.

[0065] Example 4: Expression, purification, and antibacterial analysis of the fusion protein 1. Expression and purification of the fusion protein pET32a-endolysin65 The plasmid pET32a(+) and recombinant plasmid pET32a- were successfully transformed. endolysin65 Escherichia coli Rosetta-gami2 (DE3) in 50 μg·mL -1 Amp cultured in LB liquid medium until OD 600 The value was 0.6, and the final concentration added was 0.5 mg·mL. -1IPTG was used for overnight induction at 18 °C. Protein expression was observed by electrophoresis; the protein was highly expressed in the supernatant, with a size of approximately 72 kDa. The successfully expressed recombinant protein pET32a-endolysin65 was purified and analyzed by protein electrophoresis. Figure 5 The purified protein was then concentrated.

[0066] 2. Expression and purification of the fusion protein pPIC9K-endolysin65 Integrating plasmid pPIC9K and recombinant plasmid pPIC9K- endolysin65 Pichia pastoris was inoculated into 100 mL of BMGY medium and cultured at 28 °C with shaking at 220 rpm for 24 h. Yeast cells were aseptically collected by centrifugation at 4 °C and 4,000 rpm for 5 min, and the cell pellet was resuspended in 100 mL of BMMY induction medium. The culture was continued at 28 °C and 220 rpm, with 1 mL of sample taken every 24 h and 0.5 mL of methanol added, until 96 h. The sample was then centrifuged at 4 °C and 8,000 rpm for 5 min, and the supernatant was used to precipitate proteins using trichloroacetic acid (TCA) for SDS-PAGE analysis of protein expression. The recombinant protein pPIC9K-endolysin65 showed a specific band between 55-72 kDa. Figure 6 The results indicate that the endosomal protein was successfully expressed in the Pichia pastoris eukaryotic system. The molecular weight of the recombinant endosomal protein was calculated to be 64 kDa (54.7 + 9.3) using ProtParam. It contains the target protein and an α-factor secretion signal tag, which is consistent with the results obtained by protein electrophoresis.

[0067] 3. Detection of antibacterial activity of recombinant protein The antibacterial spectrum of the purified and concentrated recombinant proteins pET32a-endolysin65 and pPIC9K-endolysin65 was detected. The antibacterial effect of the recombinant protein against five strains of Gram-positive bacteria and five strains of Gram-negative bacteria was detected by disk diffusion method. The five Gram-positive bacteria were one strain of Bacillus cereus, one strain of Bacillus belyssus, one strain of Staphylococcus warwick, one strain of Staphylococcus aureus, and one standard strain of Staphylococcus aureus ATCC25923. The five Gram-negative bacteria were one strain of Escherichia coli, one standard strain of Escherichia coli ATCC25922, one strain of Salmonella, one strain of Burkholderia, and one strain of Ralstonia solanacearum. Two recombinant endosomal proteins were sterilized by passing them through a 0.45 μm microporous membrane. 10 μL of the recombinant endosomal protein was spot-inoculated onto filter paper. The controls were the supernatant of Escherichia coli transformed with pET32a and the supernatant of Pichia pastoris GS115 transformed with empty pPIC9K. The cultures were inverted at 37 ℃ for 12 h to observe the antibacterial effect.

[0068] The experimental results showed that the recombinant protein pET32a-endolysin65 had an inhibitory effect on a Gram-positive Bacillus belyssus strain and a Gram-negative Burkholderia strain. Figure 7 The recombinant protein pPIC9K-endolysin65 showed an inhibitory effect on a strain of Staphylococcus warwick. Figure 8 ).

[0069] Example 5: Functional Identification of Endolysin and Perforin 1. Construction of prokaryotic co-expression vectors Perforin gene of Staphylococcus warwick temperate phage vB_G30_01 (GenBank: WP_404774739.1) holin64 The nucleotide sequence (5'-3') is shown in SEQ ID NO:4 below.

[0070] SEQ ID NO:4 ATGCAATTCCAGAAAAACAATACACTCACAAAGCTAACCTTTAAGGTTGGTTTTTTATTTTACTCAAAAGGAGATAATCAAATGACTTCAGATAAATTAAAACAATATATTGGCTTATTTGGTGGTATGTTAGGGGCTTTATACCTTGCATTAAAAGCAAGTGGAA TCGAAGTTCCTTTTTTAATGCCCGATAAATTAGACGCATGGCAAAACTTCGCTACGTCAATAGTACCTTTTGTAATTGCGATATATGGCGTCTATAAAAACACATATATTATTCACTCGCATTCAAAAGCGCAAGAAGAATACTTAAAAGAAAATAATTTAAAATAG SEQ ID NO:6 MQFQKNNTLTKLTFKVGFLFYSKGDNQMTSDKLKQYIGLFGGMLGALYLALKASGIEVPFLMPDKLDAWQNFATSIVPFVIAIYGVYKNTYIIHSHSKAQEEYLKENNLK Using restriction endonucleases Sac I and Pst I; and restriction endonucleases Nde I and Kpn I respectively applied plasmid pETDuet TM The target gene, which was free of mutations after sequencing, was double-digested with enzymes to construct the recombinant plasmid pETDuet.TM -1- holin64 - endolysin65 The recombinant plasmid pETDuet TM -1- holin64 - endolysin65 Transformed Escherichia coli Rosetta-gami2 (DE3) Figure 9 ).

[0071] 2. Perforin codon optimization and functional detection of eukaryotic expressed proteins The perforin gene derived from the temperate bacteriophage vB_G30_01 of Staphylococcus warwick ( holin64 The nucleotide sequence (5'-3') of Pichia pastoris after codon preference optimization is shown in SEQ ID NO:4 below. The construction of the eukaryotic expression vector and transformation of Pichia pastoris GS115 were performed according to Example 3. The method for detecting the antibacterial activity of perforin protein and the backup strain were the same as in Example 4.

[0072] SEQ ID NO:5 ATGCAGTCCAAAAGAACAACACCTTGACTAAGCTGACTTTCAAGGTTGGTTTCTTGTTCTACTCCAAGGGTGATAATCAGATGACTTCTGATAAGCTGAAGCAGTACATCGGTTTGTTTGGTGGTATGTTGGGTGCTTTGTACTTGGCTTTGAAGGCTTCTGGTA TTGAAGTCCATTCTTGATGCCTGATAAGTTGGATGCTTGGCAAAACTTCGCTACTTCTATTGTTCCTTTCGTCATCGCTATCTACGGTGTTTACAAAAACACCTACATCATCCACTCCCATTCTAAGGCTCAAGAAGAATATCTGAAGGAGAACAACCTGAAGTAA The experimental results showed that perforin protein had an inhibitory effect on five Gram-positive bacteria, including Staphylococcus aureus, Staphylococcus aureus ATCC25923, Bacillus cereus, and Staphylococcus warwickii, with the inhibitory effect on Staphylococcus aureus being particularly significant. Figure 10 It showed inhibitory effects against five strains of Gram-negative bacteria, including Escherichia coli ATCC25922, Salmonella, Burkholderia, and Ralstonia solanacearum. Figure 11 ).

[0073] 3. Growth curve of E. coli transformed with recombinant plasmid Successfully recombinant plasmid pETDuet TM -1- holin64and pETDuet TM -1- holin64 - endolysin65 Transformation plasmid pETDuet TM Escherichia coli Rosetta-gami2 (DE3) at -1 in 50 μg·mL -1 Escherichia coli Rosetta-gami2 (DE3) was cultured in LB liquid medium and the growth curve was measured at 600 nm wavelength every 1 h.

[0074] The research results indicate that ( Figure 12 Transformation into recombinant plasmid pETDuet TM -1- holin64 and recombinant plasmid pETDuet TM -1- holin64 - endolysin65 The growth rate of Escherichia coli Rosetta-gami2 (DE3) is lower than that of pETDuet. TM -1 control. Recombinant plasmid pETDuet TM -1- holin64 - endolysin65 The growth rate of Escherichia coli Rosetta-gami2 (DE3) was the lowest. Compared with the single expression of perforin, which inhibited the growth of E. coli, the combined expression of perforin and endosomal protein accelerated the in vivo lysis rate of E. coli and synergistically inhibited the growth of E. coli, thus enhancing the inhibitory ability.

[0075] 4. Scanning electron microscopy observation of E. coli transformed with recombinant plasmid before and after induction The plasmid pETDuet was successfully transformed. TM -1. Recombinant plasmid pETDuet TM -1- holin64 and recombinant plasmid pETDuet TM -1- holin64 - endolysin65 Rosetta-gami2 (DE3) of various Escherichia coli were respectively in a solution containing 50 μg·mL -1 Amp cultured in LB liquid medium until OD 600 The value was 0.6, and the final concentration added was 0.5 mg·mL. -1 IPTG was used for overnight induction at 18 °C. The bacterial cells were centrifuged, and the precipitate was thoroughly dispersed and suspended in glutaraldehyde fixative. After fixation at room temperature in the dark for 30 min, the cells were observed under a microscope.

[0076] The experimental results show that ( Figure 13 Compared with the empty vector control, the expression of recombinant protein pETDuet TME. coli Rosetta-gami2 (DE3) with -1-holin64-endolysin65 exhibited an aggregated state without induction. This contrasts with cells expressing the recombinant protein pETDuet. TM Compared to uninduced E. coli Rosetta-gami2 (DE3) with -1-holin64-endolysin65, IPTG-induced E. coli Rosetta-gami2 (DE3) exhibited cell fragmentation and filamentous dissemination of cell contents, connecting with other cells. The recombinant protein pETDuet was expressed. TM -1-holin64-expressing E. coli Rosetta-gami2 (DE3) cells aggregated in large numbers after IPTG induction, indicating that perforin expression causes cells to release a substance that binds them together. Meanwhile, the expression of the recombinant protein pETDuet... TM In E. coli Rosetta-gami2 (DE3) cells containing -1-holin64-endolysin65, IPTG induction resulted in cell fragmentation and the leakage of cellular contents, which then connected to other cells in a filamentous manner. This is likely due to the function of perforin and endolysin, leading to the leakage of cellular contents. Compared to perforin expression alone, the combined expression of endolysin and perforin significantly increased cell fragmentation and leakage, indicating a synergistic effect.

[0077] 5. Combined functional detection of perforin and endosomal proteins The combined function of perforin and endosomal proteins expressed in the eukaryotic strain Pichia pastoris GS115 was investigated using a 1:1 ratio, with perforin and endosomal proteins as controls. Gram-positive Staphylococcus aureus was used as a backup strain.

[0078] Experimental results show that ( Figure 14 Eukaryotic endosomal protein alone has no antibacterial effect against Staphylococcus aureus, while eukaryotic perforin alone has an antibacterial effect against Staphylococcus aureus. However, compared with perforin protein alone, the combined use of perforin and endosomal protein in a 1:1 ratio enhances the inhibitory effect on Staphylococcus aureus, and the inhibition zone is significantly larger.

[0079] In summary, recombinant endosomal protein exhibits inhibitory effects against some Gram-positive and Gram-negative bacteria; co-expression of perforin and endosomal genes significantly inhibits and lyses the growth of its expression host, *Escherichia coli* Rosetta-gami2 (DE3); and the combined use of eukaryotically expressed endosomal perforin and endosomal protein in a 1:1 ratio significantly enhances the inhibitory effect against *Staphylococcus aureus*. These three points provide a theoretical basis for the prevention and control of pathogens, especially for the prevention and control of Gram-negative pathogens.

Claims

1. An endolysin derived from Staphylococcus warwick temperate phage, characterized in that: Endolysin as shown in SEQ ID NO:1, or a further optimized nucleotide sequence thereof.

2. The endolysin derived from Staphylococcus warwick temperate phage according to claim 1, characterized in that: The optimized endosomalin has the nucleotide sequence shown in SEQ ID NO:

2.

3. The endolysin derived from Staphylococcus warwick temperate phage according to claim 1 or 2, characterized in that: The amino acid sequence of the protein encoded by the content element of the nucleotide sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:3; The amino acid sequence of the protein encoded by the nucleotide sequence shown in SEQ ID NO:2 is shown in SEQ ID NO:

3.

4. The endolysin derived from Staphylococcus warwick temperate phage according to claim 1, characterized in that: The primer pair for obtaining the nucleotide sequence endosomalin shown in SEQ ID NO:1 is: endolysin65- F CGGGATCCATGGCTACAGAGAATTGGAAAGG; endolysin65- R CGAGCTCTTATTTCACTTAATGGAACCCC; The primer pair used to obtain the optimized endosomalin is: endolysin65 F GAATTCATGGCTACTGAAAACTGG; endolysin65 R GCGGCCGCTTATTTCCAC。 5. The application of the endolysin derived from Staphylococcus warwick temperate phage as described in claim 1, characterized in that: The use of the endosomalin, or the mixture of endosomalin and perforin, in the preparation of antibacterial agents.

6. The application of the endolysin derived from Staphylococcus warwick temperate phage according to claim 5, characterized in that: The use of the endolysin in the preparation of antibacterial agents that inhibit Gram-positive Bacillus belyssus and / or Gram-negative Burkholderia.

7. The application of the endolysin derived from Staphylococcus warwick temperate phage according to claim 5, characterized in that: The use of the mixture of endosomalin and perforin in the preparation of an antibacterial agent that inhibits Gram-negative host bacteria.

8. The application of the endolysin derived from Staphylococcus warwick temperate phage according to claim 5 or 7, characterized in that: The perforin is the nucleotide sequence shown in SEQ ID NO:4, or a further optimized nucleotide sequence shown in SEQ ID NO:

5.

9. An antibacterial agent, characterized in that: The antibacterial agent contains the endosomalin as described in claim 1, or contains the endosomalin as described in claim 1 and perforin.

10. The antibacterial agent according to claim 9, characterized in that: When the antibacterial agent contains endosomalin and perforin, the two are used in a 1:1 mass ratio.

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