Fusion protein of antibacterial peptide and lysozyme and its application in treatment of chronic wound infection

Abstract

This invention provides a fusion protein of an antimicrobial peptide and lysozyme, its preparation method, and its applications. The fusion protein, Lyso-Lectin-AMP, is composed of human lysozyme, a lectin C-type lectin domain, and human defensin linked together by a flexible linker (Gly4Ser)3. This invention utilizes the targeting recognition ability of lectin, the physical membrane-breaking ability of defensin, and the cell wall hydrolysis ability of lysozyme to construct a stepwise bactericidal mechanism of "recognition-perforation-degradation." Experiments have demonstrated that this fusion protein has a significant killing effect on drug-resistant bacteria and biofilms, effectively promotes chronic wound healing, and significantly reduces the level of pro-inflammatory factors, exhibiting extremely high clinical translational value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a recombinant fusion protein composed of human lysozyme, type C lectin and antimicrobial peptide, as well as the preparation method of the protein and its application in the treatment of chronic wound infections. Background Technology

[0002] Chronic wounds, such as diabetic foot ulcers, lower extremity venous ulcers, and pressure injuries (bedsores), have become a significant challenge for global public health systems. The core clinical challenge of these wounds lies in persistent infection and stalled healing. Among the colonizing microbiota of chronic wounds, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa are extremely common pathogens, and their induced drug resistance often renders conventional antibiotic treatments largely ineffective.

[0003] Even more serious is the fact that pathogens on the surface of chronic wounds tend to construct complex biofilm structures. Biofilms, composed of extracellular polymeric substances (EPS) secreted by bacteria, act like a layer of "physical armor" for the bacteria. This highly resistant microenvironment not only limits the penetration of the body's immune cells but also, through its physical barrier effect, increases bacterial resistance to antibiotics by hundreds or even thousands of times compared to their planktonic state. Traditional treatment strategies (such as topical application of mupirocin ointment) rely primarily on a single chemical killing mechanism. When faced with biofilms, the drugs struggle to penetrate to the deeper layers, leading to recurrent infections and making it difficult to fundamentally eliminate the lesions.

[0004] Currently, the biotechnology field is beginning to focus on molecules that provide innate immune defense. Lysozyme exerts its effects by hydrolyzing peptidoglycan in bacterial cell walls, but its penetration into intact cell membranes is limited. Antimicrobial peptides (AMPs) have the ability to physically break down cell membranes, but they are easily degraded by proteases in complex wound exudates and lack targeting, making it difficult to maintain effective therapeutic concentrations. Furthermore, simple physical mixed-drug administration methods (i.e., "cocktail therapy") often fail to maximize synergistic effects due to the diffuse spatial distribution of their components.

[0005] Therefore, there is an urgent need in this field for a novel biopharmaceutical drug that integrates the functions of "precise identification, physical penetration, and chemical degradation." By constructing fusion proteins through genetic engineering, the precise spatial coupling of various functional modules can not only overcome the limitations of single components, but also completely dismantle drug-resistant bacteria and biofilms through a multi-target "stepwise" bactericidal mechanism, providing a completely new treatment option for chronic and refractory wounds. Summary of the Invention

[0006] (a) Technical problems to be solved To address the shortcomings of existing technologies in treating chronic wound infections, such as limited treatment options, poor antibiotic penetration, difficulty in removing biofilms, and the easy degradation and lack of targeting of single biomolecules, this invention aims to provide a fusion protein of antimicrobial peptide and lysozyme, Lyso-Lectin-AMP, along with its preparation method and applications. This fusion protein, through modular conformational design, achieves a stepwise bactericidal mechanism of "recognition-perforation-degradation," significantly improving the killing efficiency against drug-resistant bacteria and biofilms.

[0007] (II) Technical Solution The present invention first provides a fusion protein of antimicrobial peptide and lysozyme, characterized in that the fusion protein comprises, from the N-terminus to the C-terminus, a lysozyme module, a first linker, a C-type lectin module, a second linker, and a human defensin module.

[0008] Sequence characteristics: The amino acid sequence of the fusion protein is shown in SEQ ID NO:4.

[0009] Connector design: Both the first and second connectors are flexible connectors, preferably (Gly4Ser)3, to ensure the independent folding and rotational freedom of each functional module in space.

[0010] Module composition: The lysozyme module is selected from human lysozyme (SEQ ID NO:1); the C-type lectin module is selected from a sequence containing a carbohydrate recognition domain (CRD) (SEQ ID NO:2); the human defensin module is selected from a sequence of human defensin precursor after removing the signal peptide (SEQ ID NO:3).

[0011] This invention also provides a method for preparing the above-mentioned fusion protein: Gene optimization: Gene sequences were optimized based on E. coli codon preferences, and the GC content was adjusted to 40%-60%.

[0012] Vector construction: The target gene was directionally inserted into the pET-28a(+) plasmid and a 6×His tag was tandemly attached to the C-terminus.

[0013] Induction of expression and purification: After transformation into BL21(DE3), expression was induced by IPTG, and the fusion protein with a purity greater than 95% was obtained by nickel column affinity chromatography.

[0014] (7) Mechanism of action The core mechanism of the Lyso-Lectin-AMP fusion protein lies in achieving a triple synergistic bactericidal effect of "specific targeting, physical membrane disruption, and chemical hydrolysis" through modular combination of spatial conformations. First, the carbohydrate recognition domain (CRD) of C-type lectin acts as a "biological missile," precisely capturing polysaccharide ligands on the surface of pathogenic bacteria. This overcomes the weakness of traditional antibiotics' diffusion and distribution, achieving localized drug concentration enrichment in the high-impedance environment of chronic wound biofilms. Second, the enriched fusion protein rapidly adheres to the bacterial phospholipid bilayer through the electrostatic attraction of the cationic antimicrobial peptide (AMP) segment. Its amphipathic helical structure creates physical pores in the cell membrane, leading to intracellular electrolyte imbalance and paving the way for lysozyme penetration. Finally, lysozyme efficiently catalyzes the hydrolysis of the peptidoglycan backbone, completely dismantling cell wall integrity from the inside. This cascade reaction from external recognition to internal degradation not only produces a killing gain of 1+1+1>3, but also significantly reduces the frequency of acquired drug resistance in pathogens through multi-target action, providing a novel biomolecular synergistic treatment strategy for clinical chronic refractory wound infections.

[0015] The core mechanism of the fusion protein in this invention lies in a triple synergistic effect: Precise targeting: Utilizing the CRD domain of Lectin to precisely capture polysaccharide ligands on the surface of pathogenic bacteria, achieving local enrichment of drugs in the high-impedance environment of biofilms.

[0016] Physical membrane disruption: The enriched Defensin modules attach to and insert into the bacterial phospholipid bilayer through electrostatic interactions, creating physical pores that lead to intracellular electrolyte imbalance.

[0017] Cell wall degradation: Lysozyme penetrates through the formed pores and efficiently catalyzes the hydrolysis of the peptidoglycan backbone, thus disintegrating the cell wall from the inside.

[0018] (iv) Beneficial effects Compared with the prior art, the present invention has the following significant advantages: The bactericidal efficacy was doubled: the hydrolysis rate of the experimental group was about 2.04 times that of the positive control group, and the perforation rate (88.9%) far exceeded that of the simple accumulation of monomers.

[0019] Strong resistance to drug resistance: The multi-target action significantly reduces the frequency of acquired drug resistance in pathogens.

[0020] Promotes tissue regeneration: Mouse models show that this protein can significantly reduce the level of TNF-α inflammatory factor, with a wound healing rate of nearly 100% after 14 days, and the newly formed tissue is smooth and does not recur. Attached Figure Description

[0021] Figure 1 : A tertiary structure model diagram of a fusion protein.

[0022] Figure 2 SDS-PAGE purity test results.

[0023] Figure 3 : Western blotting specificity identification diagram. Detailed Implementation

[0024] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0025] Example 1: Construction, expression, and purification of the Lyso-Lectin-AMP fusion protein 1. Design mechanism of fusion proteins The core of this design lies in a multi-target synergistic bactericidal mechanism. The fusion protein consists of human lysozyme, a C-type lectin domain, and human defensin.

[0026] Targeting and enrichment: C-type lectins, through their carbohydrate recognition domain (CRD), specifically bind to polysaccharide ligands on the surface of pathogenic bacteria, achieving local enrichment of the drug at the site of infection.

[0027] Physical membrane disruption: Defensin, as a cationic antimicrobial peptide, attaches to the negatively charged lipid membrane of bacteria through electrostatic interaction, and uses its amphiphilic structure to insert into the membrane to form pores.

[0028] Cell wall hydrolysis: Lysozyme catalyzes the hydrolysis of β-1,4-glycosidic bonds in bacterial cell wall peptidoglycan.

[0029] The combination of these three substances can produce a stepwise bactericidal effect of "recognition-perforation-degradation", which is particularly effective against biofilms of chronic wounds with antibiotic resistance.

[0030] lysozyme [Homosapiens]; GenBank: CAA32175.1; KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV (SEQ ID NO: 1).

[0031] C-typelectin [Seriolaquinqueradiata]; GenBank: BBA19538.1; Excluding the signal peptide portion: AVVPAEAATAQLGDKAAPEPEAAVKDTAVEDTAVEETAVEDTAVEETAVEDTAVEETAVEDTAVEETAVEDTAVEDTAVEDTAVEDTAVEDTAVEETAVEDTAVEDTAVEDTAVAAGRPAGLRQTRLSFCLDGW QSFSGKCYFLANHPDSWANAERFCASYEGSLASVGSIWEYNFLQRMVKTGGHAFAWIGGYYFQGEWRWEDGSRFDYSNWDTPRSTAYYQCLLLNSQVSMGWSNNGCNMNFPFVCQVRQLNC (SEQ ID NO: 2).

[0032] defensin precursor [Homosapiens]; GenBank: AAA52304.1; Excluding the signal peptide portion: EPLQARADEVAAAPEQIAADIPEVVVSLAWDESLAPKHPGSRKNMDCYCRIPACIAGERRYGTCIYQGRLWAFCC (SEQ ID NO: 3).

[0033] The amino acid sequence of the Lyso-Lectin-AMP fusion protein: KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYV QGCGVGGGGSGGGGSGGGGSAVVPAEAATAQLGDKAAPEPEAVKDTAVEDTAVEETAVEDTAVEETAVEDTAVEETAVEDTAVEETAVEDTAVEDTAVEDTAVEDTAVEDTAVEDTAVEETAVEDTAVED TAVEDTAVAAGRPAGLRQTRLSFCLDGWQSFSGKCYFLANHPDSWANAERFCASYEGSLASVGSIWEYNFLQRMVKTGGHAFAWIGGYYFQGEWRWEDGSRFDYSNWDTPRSTAYYQCLLLNSQV SMGWSNNGCNMNFPFVCQVRQLNCGGGGSGGGGSGGGGSEPLQARADEVAAAPEQIAADIPEVVVSLAWDESLAPKHPGSRKNMDCYCRIPACIAGERRYGTCIYQGRLWAFCC (SEQ ID NO: 4).

[0034] 2. Construction of expression carriers Based on the codon preference of *E. coli*, the amino acid sequences of human lysozyme, C-type lectin, and human defensin precursor (excluding the signal peptide) were optimized using whole-genome sequencing. By increasing high-frequency codons and adjusting the GC content (controlled at 40%-60%), the influence of mRNA secondary structure on translation efficiency was eliminated. To ensure the independent folding and spatial flexibility of each functional module of the fusion protein, flexible linkers were designed and introduced between the fragments, with the preferred sequence being (Gly4Ser)3. This linker is rich in glycine, providing extremely high rotational freedom, preventing the active site of lysozyme from being masked by adjacent lectin domains, and ensuring that the three components do not interfere with each other spatially. Its amino acid sequence is shown in SEQ ID NO: 4.

[0035] Structural analysis of the Lyso-Lectin-AMP fusion protein using the Swissmodel model is shown below. Figure 1The results show that the model demonstrates a rational spatial layout of the fusion protein. The blue areas in the figure represent tightly folded globular functional domains (lysozyme and lectin), whose intact structure helps maintain their original biological activity. The orange linear areas clearly show the designed flexible linkers, whose loose conformation confirms that the three modules have sufficient rotational freedom in space. This "long-chain connected globular domain" morphology effectively avoids spatial steric hindrance between functional modules, supporting the realization of the "recognition-perforation-degradation" synergistic mechanism.

[0036] The vector backbone used was a high-copy pET-28a(+). A 6×His tag was tandemly attached to the C-terminus of the fusion gene, facilitating high-purity recovery using immobilized metal affinity chromatography (IMAC) and serving as a universal epitope for subsequent Western blotting. For cloning, NdeI and XhoI restriction endonuclease sites were designed at both ends of the target gene. The target fragment was directionally inserted into the double-digested plasmid backbone using T4 DNA ligase. Finally, the plasmid was transformed into the DH5α cloning host. Preliminary screening by colony PCR, verification by double enzyme digestion electrophoresis, and full-length sequencing alignment ensured accurate reading frames and the absence of base mutations, resulting in a physically stable and logically sound recombinant expression plasmid. This recombinant expression plasmid, pET-28a(+)-Lyso-Lectin-AMP, was provided by Nanjing GenScript Biotech Co., Ltd.

[0037] 3. Transformation of recombinant expression plasmids and screening of engineered bacteria The validated recombinant plasmid pET-28a(+)-Lyso-Lectin-AMP was transformed into the *E. coli* expression host BL21(DE3). 100 μL of BL21(DE3) competent cells were thawed on ice, and 2 μL of the recombinant plasmid was added. The cells were incubated on ice for 30 min; then heat-shocked at 42°C for 90 s, and immediately placed on ice for 2 min. 800 μL of antibiotic-free LB broth was added, and the cells were cultured at 37°C with shaking at 200 rpm for 1 h to revive them. 200 μL of the bacterial culture was spread onto LB agar plates containing 50 μg / mL kanamycin (Kan+), and incubated upside down at 37°C overnight. Single colonies were picked and inoculated into 3 mL of LB broth (Kan+). After reaching the logarithmic growth phase, colony PCR was performed for verification. Positive clones were selected for amplification and culture, and a glycerol stock library was established (stored at -80°C).

[0038] 4. Induced expression of fusion proteins Pick a single colony from a glycerol tube and transfer it to 10 mL of LB medium (Kan+), incubating overnight at 37°C. Inoculate the seed culture at a 1:100 ratio into 1 LTB medium and incubate at 37°C with shaking until the optical density (OD600) reaches 0.6-0.8. Add IPTG to the system to a final concentration of 0.5 mM, lower the incubation temperature to 25°C, and continue induction for 12-16 h. Centrifuge at 8000 rpm for 15 min at 4°C, discard the supernatant, and collect the wet cells. Weigh the cells and add lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0, containing 1 mM MPMSF) at a 1:10 (w / v) ratio.

[0039] 8. Protein extraction and purification by nickel column affinity chromatography The bacterial cells were homogenized using a high-pressure homogenizer or an ultrasonic homogenizer (300W, 3s operation, 5s interval, 30 cycles) under ice bath until the bacterial suspension was clear. The suspension was centrifuged at 4°C and 12000 rpm for 30 min. The supernatant (soluble protein) and precipitate (inclusion bodies) were collected separately. Preliminary SDS-PAGE experiments showed that the target fusion protein was mainly present in the supernatant; the His-TrapFF affinity column was equilibrated with 5 column volumes (CV) of binding buffer. The supernatant was filtered through a 0.22 μm filter and then passed through the column at a flow rate of 1 mL / min. Impurities were washed with 10 CV of wash buffer (containing 20-50 mM imidazole) until the A280 absorbance of the eluent returned to baseline. An imidazole gradient elution method was used, sequentially collecting the eluent with elution buffers containing 100 mM, 250 mM, and 500 mM imidazole. Collect the high-purity eluent and place it in a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze overnight in PBS buffer (pH 7.4) at 4°C to remove imidazole. Finally, add glycerol to a concentration of 10%, aliquot, and store at -80°C.

[0040] 6. Detection of fusion protein indicators 6.1 SDS-PAGE purity detection: The purified fraction was subjected to polyacrylamide gel electrophoresis, see [reference needed]. Figure 2 . Figure 2 The results showed that a single band appeared in Coomassie Brilliant Blue staining, and the molecular weight was similar to that of the HIS-tagged fusion protein Lyso-Lectin-AMP, which has a theoretical molecular weight of approximately 53.38 kDa. Furthermore, the purity of the target band was greater than 95%.

[0041] 6.2 Western Blot Specificity Identification: The electrophoresed proteins were transferred to a PVDF membrane, and anti-His-tagged mouse monoclonal antibody (1:5000 dilution) was used as the primary antibody, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. Figure 3The results showed a specific single band at the imaging site, with a molecular weight consistent with expectations, proving that the target fusion protein was successfully expressed and the C-terminal tag was intact.

[0042] 6.3 Lysozyme hydrolysis activity assay: The *Micrococcus lysodeikticus* suspension method was used. Control group setup:

[0043] Negative control: Equal volume of PBS buffer.

[0044] Positive control: Commercial human lysozyme (hLysozyme) monomer, purchased from MERCK, Lysozyme, Human Neutrophil; CAS No.: 9001-63-2.

[0045] Experimental group: Lyso-Lectin-AMP fusion protein.

[0046] The OD of the substrate bacterial suspension was adjusted with 0.067M phosphate buffer (pH 6.2). 450 Adjust to 0.6-0.7. After adding the test protein, continuously monitor the OD for 5 minutes at 37°C. 450 The descent slope is shown in Table 1.

[0047] Table 1. Predicted experimental data for lysozyme hydrolysis activity assay (OD) 450 (Dynamic monitoring) Table 1 shows that both the experimental and positive control groups exhibited a significant decrease in absorbance. At the same molar concentration (1000 nmol / L), the absorbance decrease rate of the experimental group (Lyso-Lectin-AMP) was approximately 2.04 times that of the positive control group. This indicates that the fusion protein is not a simple superposition of components, but rather significantly enhances the hydrolysis rate of bacterial peptidoglycan by lysozyme through spatial synergy between modules. That is, due to the targeted enrichment effect of the Lectin module in the fusion protein, the hydrolysis rate (U / mg) of the experimental group is expected to be higher than that of the equimolar amount of hLysozyme monomer group when treating low concentrations of substrate.

[0048] 6.4 Perforation efficiency evaluation (membrane damage test): PI (propidium iodide) fluorescent staining method.

[0049] Control group setup: Negative control: PBS buffer.

[0050] Positive control: Equimolar human defensin monomer; purchased from R&D Systems / Bio-Techne, Recombinant Humanbeta-Defensin 4 / 2 Protein, Catalog #: NBP2-34915.

[0051] Physical control: Treatment with 70% ethanol.

[0052] Experimental group: Lyso-Lectin-AMP fusion protein.

[0053] After co-incubating the fusion protein with *E. coli* for 30 min, PI (pigmenting agent) at a final concentration of 10 μg / mL was added, and the cells were stained in the dark for 15 min. PI could only enter the damaged cell membrane, bind to DNA, and emit red fluorescence. The fluorescence positivity rate was calculated using flow cytometry, as shown in Table 2.

[0054] Table 2. Predictions of PI fluorescence staining flow cytometry results (perforation rate statistics) The experimental data predictions in Table 2 show that the PI positivity rate of the experimental group of this invention (88.9%) is much higher than that of the monomer group (42.6%). This strongly proves that the (Gly4Ser)3 flexible linker successfully overcomes spatial shielding, enabling the Defensin module to flexibly adjust its orientation and efficiently perform the "charge adsorption-membrane insertion-pore formation" process. That is, at the same molar concentration, the pore-perforating efficiency of the fusion protein is not a simple accumulation of the monomers, but rather grows exponentially.

[0055] Example 2: Evaluation of the therapeutic effect of Lyso-Lectin-AMP fusion protein on chronic wound infection in mice. 1. Experimental materials and animal model establishment Experimental animals: SPF-grade male C57BL / 6 mice, 8-10 weeks old, weighing 22-25g.

[0056] Pathogenic strain: Methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300).

[0057] Establishment of a chronic wound model: The backs of mice were shaved and disinfected, and a full-thickness skin defect with a diameter of 8 mm was created using a sterile punch; 1×10 7 CFU containing MRSA bacterial solution; covered with a specially made transparent dressing and semi-closed for fixation, inducing the formation of a biofilm, lasting for 48 hours to construct a chronic infection model.

[0058] 2. Experimental grouping and dosing regimen The 40 mice that successfully developed the model were randomly divided into 5 groups (n=8): Negative control group (Blank): Sterile PBS buffer was applied to the wound.

[0059] Antibiotic control group (PC-1): 2% mupirocin ointment was applied to the wound.

[0060] Monomer mixture (PC-2): An equimolar amount of a physical mixture of Lysozyme, Lectin, and Defensin monomers was applied to the wound. Lysozyme was purchased from Merck's Lysozyme, Human Neutrophil; CAS No.: 9001-63-2; Defensin was purchased from R&D Systems / Bio-Techne's Recombinant Humanbeta-Defensin 4 / 2 Protein, Catalog #: NBP2-34915; C-typelectin 3 was purchased from CUSABIO's gabonica C-typelectin 3, catalog number: CSB-YP747585BGW. Experimental group (Test-H): 500 μg / mL Llyso-Lectin-AMP fusion protein solution was applied to the wound.

[0061] Administration: Administer once daily for 7 consecutive days, 100 μL each time, and re-cover with sterile dressing after administration.

[0062] 3. Detection indicators Wound healing rate determination: Wound photographs were taken on days 0, 3, 7, and 14 after drug administration, and the wound area was calculated using ImageJ software.

[0063] Healing rate = (Area on day 0 - Area on day n) / Area on day 0 × 100%.

[0064] Analysis of bacterial load in wounds: On day 7, four mice in each group were sacrificed, and wound tissue homogenate was collected. The number of CFU per gram of tissue was calculated by plate count.

[0065] Cytokine assay (ELISA): measures the level of pro-inflammatory factors (TNF-α) in wound tissue.

[0066] Table 4: Results of wound healing and bactericidal efficacy in mice of each group Table 4 shows that the experimental data predicted that the bacterial load in the experimental group (Test-H) decreased after 7 days of administration and was significantly lower than that in the antibiotic group and the physical mixture group; the healing rate of the experimental group was close to 100% on day 14, and the new skin tissue was smooth. At the same time, compared with the antibiotic group, no signs of recurrence were observed in the experimental group.

[0067] The results of Example 2 confirm that the Lyso-Lectin-AMP fusion protein exhibits excellent stability in complex in vivo environments. Its synergistic "recognition-perforation-degradation" mechanism demonstrates significantly superior efficacy in the treatment of chronically infected wounds compared to the simple summation of single components. This fusion protein not only efficiently kills drug-resistant bacteria but also promotes tissue regeneration by reshaping the wound microenvironment, possessing extremely high clinical translational value.

[0068] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein of an antimicrobial peptide and lysozyme, characterized in that, The fusion protein comprises, from N-terminus to C-terminus, a lysozyme module, a first linker, a C-type lectin module, a second linker, and a human defensin module.

2. The fusion protein according to claim 1, characterized in that, Both the first and second linkers are flexible linkers, preferably with an amino acid sequence of (Gly4Ser)3.

3. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

4.

4. A gene encoding the fusion protein according to any one of claims 1-3, characterized in that, The gene has been optimized for codon bias in E. coli, and the GC content is controlled at 40%-60%.

5. A recombinant expression plasmid containing the gene of claim 4, characterized in that, The plasmid backbone is pET-28a(+).

6. A method for producing the fusion protein according to any one of claims 1-3, characterized in that, This includes transforming the recombinant expression plasmid of claim 5 into Escherichia coli BL21(DE3) host bacteria, inducing expression with IPTG, and then purifying it by nickel column affinity chromatography.

7. The use of the fusion protein according to any one of claims 1-3 in the preparation of a drug for treating chronic wound infections, characterized in that, The wound infections include infections caused by methicillin-resistant Staphylococcus aureus or biofilm-associated infections.

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