An antimicrobial polypeptide derived from Bacillus subtilis and its application

Abstract

This invention relates to an antimicrobial polypeptide extracted from Bacillus subtilis and its applications. Its amino acid sequence is shown in SEQ ID NO:1. The DNA molecule encoding the antimicrobial polypeptide extracted from Bacillus subtilis has a nucleotide sequence shown in SEQ ID NO:2. The antimicrobial polypeptide of this invention reduces the drug resistance of Staphylococcus aureus by inhibiting the formation of biofilms by Staphylococcus aureus and clearing existing biofilms. It can be used to prepare drugs for the treatment or prevention of diseases caused by Staphylococcus aureus, especially MRSA infection, and can also be used to prepare drugs that promote tissue repair and wound healing.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antimicrobial polypeptide derived from Bacillus subtilis, a method for the natural extraction and artificial synthesis of the antimicrobial polypeptide, and the medicinal uses of the antimicrobial polypeptide. Background Technology

[0002] Bacterial infection is an acute systemic or local infection caused by pathogenic or opportunistic pathogens invading the bloodstream or local environment, growing and multiplying, and producing toxins and other metabolic products. Pathogenic bacteria include methicillin-resistant Staphylococcus aureus (MRSA), hemolytic streptococci, Escherichia coli, and Pseudomonas aeruginosa.

[0003] Bacterial biofilms are sticky membranes formed by the excessive accumulation of bacteria, surrounded and encased by fibrin and other aggregates secreted by the bacteria themselves. The impact of bacterial biofilms on chronic wounds is mainly reflected in, but not limited to, the following aspects: 1. As a protective membrane for bacteria, bacterial biofilms can resist the killing power of external drugs, making wounds more severe, inducing cell death, affecting cell proliferation, and delaying wound healing. 2. When using antibiotics or other drugs to treat chronic wounds, the presence of bacterial biofilms in the wound indicates the development of drug resistance, affecting the host's immune function. This increases the difficulty of drug treatment, as bacterial biofilm formation is considered a major form of antibiotic and other drug resistance. 3. Bacterial biofilms can also damage proteins needed for wound healing, affecting cell remodeling and making wounds difficult to heal. 4. Some studies have reported that bacterial biofilms are a major factor promoting chronic inflammation.

[0004] If a bacterial biofilm is present in the wound, targeted measures must be taken. The following factors can serve as clinical indicators for the presence of a biofilm: 1. Failure of appropriate antibiotic treatment; 2. Poor response to appropriate antimicrobial treatment; 3. Recurrence of delayed healing after discontinuation of antibiotic treatment; 4. Failure to heal despite good wound management and individual support; 5. Increased exudate / humidity; 6. Low-level chronic inflammation; 7. Mild erythema; 8. Poor granulation tissue growth or excessively fragile granulation tissue proliferation; 9. Manifestations of secondary infection, with reinfection of chronic wounds being associated with the dissemination of biofilms.

[0005] Staphylococcus aureus is a major pathogenic bacterium in humans, ranking first among all Gram-positive cocci. Belonging to the genus Staphylococcus, it can cause a variety of serious infections. Staphylococcus aureus can develop drug-resistant mutations in the body, thus transforming from common Staphylococcus aureus into MRSA. The mechanism of MRSA resistance is the production of... mecAGenetic predisposition leads to resistance to multiple antimicrobial drugs, making clinical treatment difficult. The current lack of products to eradicate MRSA bacteria means that MRSA infections can only be treated with high-level antibiotics, such as vancomycin, or a combination of two antibiotics. This further leads to bacterial mutation, making the bacteria more resistant and giving rise to superbugs. Antibiotic production consistently lags behind bacterial mutation. There are two main reasons why MRSA is difficult to eradicate in the body: first, bacterial biofilms, which studies have shown increase the minimum inhibitory concentration (MIC) of sensitive antibiotics by 10-1000 times; second, antibiotic resistance resulting from bacterial mutation, which reduces the number of clinically effective antibiotics.

[0006] Antimicrobial peptides are a class of basic polypeptides with antimicrobial activity induced in insects or bacteria. They consist of 20–60 amino acid residues with a molecular weight of approximately 2000–7000. Most are strongly alkaline, thermally stable, and possess broad-spectrum antimicrobial activity, exhibiting strong bactericidal effects, particularly against drug-resistant pathogens. Many natural antimicrobial peptides are positively charged amphiphilic peptide molecules, maintaining their activity even under conditions of high ionic strength and extreme acidity or alkalinity. Further research has revealed that some antimicrobial peptides also exhibit potent killing effects against certain fungi, protozoa, viruses, and cancer cells, and may even enhance immunity and accelerate wound healing. While the exact mechanism of action of antimicrobial peptides remains unclear, the cell membrane is a primary target. Based on their antibacterial, antifungal, antiviral, and antitumor effects, antimicrobial peptides are increasingly being applied in various fields, including medicine, agriculture, and food.

[0007] Therefore, studying the drug resistance and biofilm formation mechanisms of Staphylococcus aureus in infection and exploring prevention and control measures for Staphylococcus aureus is one of the key scientific issues that urgently need to be addressed in the treatment of Staphylococcus aureus infection. Preventing the formation of new biofilms and clearing existing biofilms have important scientific value and clinical significance. Summary of the Invention

[0008] The purpose of this invention is to provide an antimicrobial polypeptide extracted from Bacillus subtilis, the amino acid sequence of which is shown in SEQ ID NO:1. The antimicrobial polypeptide can inhibit the formation of biofilm by Staphylococcus aureus and remove existing biofilm, change the minimum inhibitory concentration of Staphylococcus aureus, and reduce the drug resistance of Staphylococcus aureus.

[0009] Another object of the present invention is to provide the application of the above-mentioned antimicrobial peptides in the preparation of antimicrobial preparations.

[0010] Preferably, the antibacterial agent is used to inhibit Staphylococcus aureus.

[0011] Preferably, the Staphylococcus aureus is MRSA.

[0012] Another object of the present invention is to provide the use of the above-mentioned antimicrobial peptides in the preparation of medicaments for the prevention and / or treatment of Staphylococcus aureus infections.

[0013] Preferably, the infection is a local infection, systemic infection, or toxin disease caused by Staphylococcus aureus.

[0014] Preferably, the Staphylococcus aureus is MRSA.

[0015] Another object of the present invention is to provide the use of the above-mentioned antimicrobial peptides in the preparation of medicaments that promote tissue repair and / or wound healing.

[0016] Another object of the present invention is to provide the application of the above-mentioned antimicrobial peptides in the preparation of antibiotic sensitizers.

[0017] Another object of the present invention is to provide a DNA molecule encoding the above-mentioned antimicrobial polypeptide having the nucleotide sequence shown in SEQ ID NO:2.

[0018] Another object of the present invention is to provide a recombinant vector containing the aforementioned DNA molecule.

[0019] Another objective of this invention is to provide a transformant, wherein the DNA molecule is fused with an expression vector to construct a recombinant expression vector; the recombinant expression vector is then transferred into a host cell to obtain the transformant.

[0020] Another object of the present invention is to provide a pharmaceutical preparation for treating Staphylococcus aureus infection, wherein the pharmaceutical preparation is composed of the above-mentioned antimicrobial peptides and pharmaceutically acceptable excipients.

[0021] Another object of the present invention is to provide a pharmaceutical preparation for treating wound infections and / or promoting tissue repair and healing, said pharmaceutical preparation being composed of the above-mentioned antimicrobial peptides and pharmaceutically acceptable excipients.

[0022] Preferably, the pharmaceutical preparation is a gel, spray, film, patch, ointment, liniment, coating, eye drops, nasal drops, mouthwash, or iontophoresis agent.

[0023] Another object of the present invention is to provide the application of the above-mentioned antimicrobial peptides in the preparation of feed or cosmetic additives.

[0024] Another objective of this invention is to provide a natural extraction method for the aforementioned antimicrobial peptides. Specifically, a single Bacillus subtilis colony is cultured in a medium, centrifuged, and filtered through an ultrafiltration membrane to obtain a supernatant of the Bacillus subtilis colony. The pH of the supernatant is adjusted to 2 and then refrigerated at 4°C for 8 hours. The precipitate is discarded after centrifugation. The upper layer obtained after ultrafiltration of the acid-precipitated supernatant is mixed with different concentrations of ammonium sulfate. The precipitate with an ammonium sulfate concentration of 30-40% is collected to obtain a primary purified protein. Different peaks are collected in preparative liquid chromatography to obtain a secondary purified protein. The peptides are then collected after high-precision purification using ultra-high performance liquid chromatography-mass spectrometry (UHPLC), which is the antimicrobial peptide of this invention.

[0025] Another object of the present invention is to provide a chemical synthesis method for the above-mentioned antimicrobial peptides, wherein the antimicrobial peptides are chemically synthesized by a peptide solid-phase synthesis method, and a linear peptide as shown in SEQ ID NO:1 is synthesized according to its amino acid sequence.

[0026] Preferably, the chemical synthesis is the Fmoc solid-phase synthesis method.

[0027] Another object of the present invention is to provide a pharmaceutical combination product, wherein the combination product comprises the above-mentioned antimicrobial peptides and antibiotics.

[0028] The antimicrobial peptides provided by this invention can achieve the purpose of eradicating Staphylococcus aureus by inhibiting the formation of biofilms and eliminating existing biofilms, thereby changing the minimum inhibitory concentration of MRSA and making it easier to eradicate MRSA, which is difficult to eradicate.

[0029] The antimicrobial peptides provided by this invention can also alter bacterial resistance, making previously penicillin-resistant MRSA resensitive, thereby reducing the clinical use of antibiotics. Experiments have demonstrated that after treatment with the antimicrobial peptides, MRSA regained its sensitivity to penicillin and was no longer resistant. Furthermore, MRSA pretreated with the aforementioned antimicrobial peptides for 1 hour showed a reduction in sterilization time under penicillin treatment from 22.36±1.13 hours to 7.54±1.08 hours, and a reduction in gentamicin sterilization time from 3.5±0.18 hours to 0.99±0.13 hours.

[0030] The antimicrobial peptides provided by this invention resulted in a high survival rate and a low bone infection rate in a mouse model of MRSA infection. Significant differences were also observed in bacterial load between the tibia and femur in the mouse model. In a study conducted by the Department of Orthopedics at Sun Yat-sen Memorial Hospital of Sun Yat-sen University, the topical antimicrobial spray containing the antimicrobial peptides of this invention was more effective than antibiotics alone in treating 20 cases of wound infection. Clinical follow-up showed no recurrence, demonstrating significant efficacy. Attached Figure Description

[0031] Figure 1 This is a structural diagram of the antimicrobial polypeptide extracted from Bacillus subtilis according to the present invention.

[0032] Figure 2 yes Subtilisin The figure shows the verification results of significantly inhibiting biofilm formation and clearing existing biofilms.

[0033] Figure 3 yes Subtilisin The results of the validation of Staphylococcus aureus's sensitivity to penicillin and gentamicin have been added.

[0034] Figure 4 yes Subtilisin The figure shows the validation results of inhibiting Staphylococcus aureus colonization in a mouse model of implant-associated osteomyelitis.

[0035] Figure 5 This is a flowchart of the extraction process of the antibacterial polypeptide of the present invention. Detailed Implementation

[0036] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0037] This invention extracts an antimicrobial polypeptide from Bacillus subtilis (CMCC-B-63501), the amino acid sequence of which is shown in SEQ ID NO:1. The DNA molecule encoding the above-mentioned antimicrobial polypeptide has the nucleotide sequence shown in SEQ ID NO:2. This invention also provides a recombinant vector containing the aforementioned DNA molecule. This invention further provides a transformant, wherein the DNA molecule is fused with an expression vector to construct a recombinant expression vector; the recombinant expression vector is then transformed into a host cell to obtain a transformant.

[0038] The antimicrobial peptides described in this invention reduce the drug resistance of Staphylococcus aureus by inhibiting the formation of biofilms and clearing existing biofilms.

[0039] The antimicrobial peptides described in this invention can be used to prepare drugs for treating or preventing diseases related to Staphylococcus aureus infections, especially MRSA, and to promote tissue repair and wound healing. The antimicrobial peptides provided by this invention are additives for preventing Staphylococcus aureus infection and promoting tissue repair, such as cosmetic additives or feed additives. They are effective in preventing Staphylococcus aureus infection and promoting tissue repair, with good results, excellent skin tissue repair, and no side effects. The antimicrobial peptides provided by this invention are prepared by natural extraction. Single Bacillus subtilis colonies are cultured in a medium, centrifuged, and filtered through an ultrafiltration membrane to obtain the supernatant of the Bacillus subtilis colonies. The supernatant after acid precipitation is then added to the upper layer of the ultrafiltration membrane with different concentrations of ammonium sulfate to purify the peptides. The antimicrobial peptides provided by this invention can also be chemically synthesized using a solid-phase peptide synthesis method.

[0040] In this invention, treatment refers to any process, action, application, therapy, etc., in which a subject receives medical assistance with the aim of directly or indirectly curing a disease, eradicating a pathogen, or improving the subject's condition. Treatment also refers to reducing morbidity, alleviating symptoms, eliminating recurrence, preventing recurrence, preventing onset of disease, reducing the risk of disease, improving symptoms, improving prognosis, or a combination thereof. Treatment may further encompass reducing the bacterial population, growth rate, or virulence in the subject, thereby controlling or reducing bacterial infection in the subject or bacterial contamination of organs, tissues, or the environment. Thus, a treatment that reduces morbidity can, for example, effectively inhibit the growth of at least one Staphylococcus aureus in a specific environment.

[0041] In this invention, a pharmaceutically acceptable carrier refers to any and all physiologically compatible solvents, additives, excipients, dispersion media, solubilizers, coating agents, preservatives, isotonic and absorption-retarding agents, surfactants, propellants, diluents, mediators, etc. The carrier must be acceptable in the sense that it is not harmful to the treated subject at the amount normally used in a pharmaceutically acceptable formulation. A pharmaceutically acceptable carrier is compatible with other components of the composition without rendering the composition unsuitable for its intended purpose. Furthermore, a pharmaceutically acceptable carrier is suitable for use without causing undue adverse side effects in the subject. Non-limiting examples of pharmaceutically acceptable carriers or excipients include any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, water and emulsions, such as oil / water emulsions and microemulsions.

[0042] The present invention will be further illustrated below with reference to the embodiments.

[0043] Example 1: Preparation of the antibacterial polypeptide of the present invention by natural extraction method ( Subtilisin The extraction process is as follows: Figure 5 As shown.

[0044] Prepare 50mL test tubes in a clean bench and add 30mL of TSB medium. Take the frozen Bacillus subtilis solution, thaw it quickly, and add 10μL of the bacterial solution to the test tube. Mix well, seal with sealing film, and place in a 37℃ bacterial incubator for 24 hours at 150 rpm. Stop incubation when the bacterial OD600 value reaches 0.4±0.05. Centrifuge all supernatant at 4000g for 10min, discard the bacterial cell precipitate, and keep the supernatant. Filter the supernatant through a 0.22μm filter plug and check the pH. Centrifuge the supernatant through a 10kDa ultrafiltration membrane at 3000g for 20min. Retain solutions with a pH greater than 10kDa and discard those with a pH less than 10kDa. Adjust the pH of the supernatant solution to 2, then refrigerate at 4℃ for 8 hours. Centrifuge at 15000g, discard the precipitate, and retain the supernatant. After mixing the supernatant with ammonium sulfate, the ammonium sulfate concentration was adjusted to 30%. After standing at room temperature for 30 minutes, the mixture was centrifuged at 15000g, retaining 30% ammonium sulfate precipitate, which was then redissolved in 5 mL and desalted using a solid-phase extraction column. Purification was performed using a preparative reversed-phase high-performance liquid chromatography (RP-HPLC) system (1260 Infinity II). Mobile phase: A (water) and B (pure methanol), flow rate: 3 mL / min, isocratic elution: 25% A + 75% B; column temperature: 25℃; injection volume: 1 mL; detection wavelength: 214 nm; total run time: 30 minutes. The antibacterial function of each peak was collected and detected, purifying the peaks exhibiting antibacterial effects. The collected active peak liquid was vacuum dried, and 0.1 mg was redissolved in 1 mL of water. Mobile phase: A (water) and B (pure methanol); flow rate: 300 nL / min; gradient elution: methanol concentration gradually increased from 5% to 100%. Column temperature: 25℃; injection volume: 2μL; full scan range: 350~1500 amu, dynamic exclusion for 6 seconds. Total run time: 120 minutes. The identified substance is considered the final product. Subtilisin Its amino acid sequence is shown in SEQ ID NO:1, and its structural diagram is shown in... Figure 2 As shown.

[0045] Example 2 Subtilisin Verification experiment on the disruption of MRSA biofilm.

[0046] In order to evaluate Subtilisin The effect on MRSA biofilm formation was investigated by mixing equal amounts of MRSA with PBS, Subtilisin Co-culture with gentamicin and penicillin. Performed at different time points. Control Group, Subtilisin Group, Penicillin Group, Gentamicin The biofilms were stained with crystal violet, and the amount of biofilm formation was observed. Figure 2 As shown in B.

[0047] The absorbance of the solution was measured after dissolving the biofilm at the 24th hour time point. The results are as follows: Figure 2 As shown in Figures A and C, the results show Subtilisin It significantly inhibited biofilm formation.

[0048] To evaluate the removal of existing biofilms, bacteria were allowed to grow freely for 24 hours before being added to the treatment process, allowing biofilm to form before the bacteria were introduced. Subtilisin The results are as follows Figure 2 As shown in Figure D. After 8 hours, the biofilm clearance results were observed by fluorescent staining, as shown in Figure D. Figure 2 As shown in Figure E, the biofilm has been removed.

[0049] Figure 2 A in the middle is Subtilisin Effect on the growth of suspended MRSA: MRSA (5×10 7 CFU) contains Subtilisin The samples were grown in TSB medium containing gentamicin (0.75 μg / mL) or penicillin (32 μg / mL). Samples were taken at specified time points for OD600 evaluation. Data showed that... mean± SE (n = 4 biologically independent samples at each time point); Figure 2 In Figure B, the biofilm of MRSA was stained with crystal violet after growing in four different culture media for 2, 4, 6, 8, 12, or 24 hours. Figure 2 In the middle C, after co-culturing for 24 hours, the biofilm was dissolved and the OD600 value was measured. The data is shown as mean ± SE (n = 4 biologically independent samples). Figure 2 D is Subtilisin A schematic diagram illustrating the removal of mature biofilms; Figure 2 E is a biofilm that has matured for 24 hours and contains PBS. Subtilisin The cells were co-cultured in TSB medium containing gentamicin (0.75 μg / mL) or penicillin (32 μg / mL) for 8 hours. MRSA within the biofilm was examined using SYTO9 / PI, followed by analysis under a fluorescence microscope. SYTO9 stained all present MRSA green, while PI stained dead MRSA red. Scale bar: 100 μm.

[0050] The above results prove that: Subtilisin It can significantly inhibit biofilm formation and remove existing biofilms; Example 3 Subtilisin Experimental verification of altered MRSA resistance.

[0051] (1) Subtilisin The MIC value of MRSA was lowered, making the bacteria easier to kill with antibiotics.

[0052] For evaluation SubtilisinWhether the drug susceptibility characteristics of MRSA can be altered was determined using the E-Test method with PBS and... Subtilisin The minimum inhibitory concentration (MIC) after 1 hour of pretreatment. Figure 3 As shown in B and 3C, via Subtilisin Pretreatment of MRSA altered the MICs of both penicillin and gentamicin. The effects of penicillin on PBS-treated MRSA (Control) and... Subtilisin The MIC of MRSA treated with gentamicin decreased from 32 μg / mL to 12 μg / mL. (Gentamicin effect on PBS-treated MRSA (Control) and...) Subtilisin The MIC of the treated MRSA decreased from 0.75 μg / mL to 0.31 ± 0.062 μg / mL.

[0053] (2) Subtilisin prolongs the antibacterial time of different antibiotics.

[0054] In bacterial proliferation experiments, penicillin (0.5×MIC) failed to inhibit the proliferation of MRSA pretreated with PBS, and after 8 hours of incubation, it began to grow rapidly in TSB. Subtilisin Pretreated MRSA was significantly inhibited by penicillin (0.5×MIC) for 14 hours, as... Figure 3 As shown in D. Therefore, MRSA passes through Subtilisin Pretreatment makes them sensitive to previously resistant penicillin.

[0055] (3) Subtilisin reduced the MDK of different antibiotics. 99 This improves sterilization efficiency.

[0056] MDK 99 The calculation represents the time required to kill 99% of MRSA. Subtilisin 99% of MRSA bacteria pretreated for 1 hour were killed within 24 hours. Figure 3 As shown in E, but not through Subtilisin The treated MRSA failed to kill bacteria after 24 hours. Subtilisin Pretreated MRSA is more sensitive to gentamicin than Control High strain, 99% of MRSA Subtilisin Killed within 4 hours after pretreatment, such as Figure 3 As shown in F. Subtilisin MDK for preprocessed MRSA 99 The value was significantly lower than that of the control group. Subtilisin Post-treatment penicillin MDK 90 The value decreased from 22.36±1.13 to 7.54±1.08. Subtilisin Gentamicin MDK after treatment 99The value decreased from 3.5±0.18 to 0.99±0.13, such as Figure 3 As shown in G.

[0057] The test results of Example 3 are as follows Figure 3 As shown, where Figure 3 In section A, the MRSA detection of penicillin and gentamicin is indicated by the E-test. The bottom of this ellipse intersects the test strip (see black arrow). Figure 3 Tables B and C show the quantitative analysis results of penicillin's effect on Staphylococcus aureus treated with PBS (Control) and Subtilisin The MICs for gentamicin against treated Staphylococcus aureus were 32 μg / mL and 12 μg / mL, respectively. Subtilisin The MICs for the treated Staphylococcus aureus were 0.75 μg / mL and 0.31 ± 0.062 μg / mL, respectively. Figure 3 D represents Staphylococcus aureus after passing through PBS or Subtilisin Pretreatment for 1 hour significantly prolonged the time of bacterial growth inhibition in TSB medium containing penicillin or gentamicin. Figure 3 In the middle, E and F represent the effects of penicillin at 4×MIC (E) and gentamicin at 2×MIC (F) on the efficacy of penicillin after treatment. Subtilisin Time-dependent killing analysis of pretreated MRSA showed that the time required to kill MRSA was significantly shortened. Figure 3 G in the middle is MRSA after Subtilisin After treatment, the MDK99 value in the presence of penicillin (4×MIC) or gentamicin (2×MIC) was significantly shortened, indicating that the killing efficiency was increased.

[0058] The above results prove that: Subtilisin It increases the susceptibility of Staphylococcus aureus to penicillin and gentamicin.

[0059] Example 4: Detection of withered plants Subtilisin Protective effect against MRSA infection in vivo.

[0060] Establishment of a mouse model of plant-associated osteomyelitis: After weighing, mice were anesthetized via intraperitoneal injection. A 5mm incision was made on the medial side of the tibial plateau. A single cortical opening was created through the medial cortical region of the tibia using a 29-gauge steel needle. A 0.3mm diameter, 8mm long stainless steel needle was then inserted into the medullary cavity and the opening was closed. After 7 days of feeding, subsequent experiments were conducted. 100μL of PBS containing Staphylococcus aureus (5×10⁻⁶) was injected via the tail vein. 7 A CFU / mL, 100 μL injection model was established to induce MRSA infection in mice. Mice were injected with PBS once daily from the time of MRSA challenge. Control (group) or Subtilisin,like Figure 4 As shown in A. And Control Compared to the group, Subtilisin Treatment improved the survival rate of mice challenged with MRSA, such as Figure 4 As shown in B. In the surviving... Control In the group of mice, the infection rate increased between day 3 and day 14 post-infection. In contrast, Subtilisin The infection rate of surviving mice in the treatment group was significantly lower than that in the treatment group. Control The group, and the infection rate remained unchanged from day 3 to day 14 post-infection, such as Figure 4 As shown in C. Bacterial quantity statistics, Control The mice in the group had higher bacterial loads on days 3 and 14 post-infection, while Subtilisin The treatment did indeed significantly reduce the amount of bacteria in the tibia and implant, such as Figure 4 As shown in D and E. Subtilisin It can reduce biofilm formation and protect bone tissue in mouse animal models. In a mouse osteomyelitis model, Subtilisin It increased the number of mice with high survival rates and reduced the rate of bone infection in mice. Subtilisin The dosage is: 0.1 mg, 100 uL, IV.

[0061] The test results of Example 4 are as follows Figure 4 As shown, where Figure 4 A in the middle is Subtilisin A schematic diagram of a treatment model for implant-related osteomyelitis. Staphylococcus aureus is administered daily after intravenous injection via tail vein. Subtilisin or PBS ( Control Mice were treated for 3 or 14 days. Figure 4 B is the result of using PBS ( Control )and Subtilisin Survival curves of mice with osteomyelitis after treatment. Figure 4 C represents the administration of PBS on days 3 and 14 post-infection. Control )and Subtilisin Infection rate in treated surviving osteomyelitis mice. Figure 4 D and E represent bacterial counts detected from the implanted tibia (D) and implant (E) on days 3 and 14 post-infection, respectively, N = 12 / group, * P < 0.05, ** P < 0.01, *** P < 0.001, Mann-Whitney U test.

[0062] The above results prove that: Subtilisin It inhibited Staphylococcus aureus colonization in vivo and alleviated inflammation caused by pathogenic infection in a mouse model of implant-associated osteomyelitis.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antimicrobial polypeptide derived from Bacillus subtilis, characterized in that: The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:1, and the antimicrobial peptide is used to inhibit Staphylococcus aureus.

2. A DNA molecule encoding the Bacillus subtilis-derived antimicrobial polypeptide as described in claim 1, characterized in that: The nucleotide sequence of the DNA molecule is shown in SEQ ID NO:

2.

3. A recombinant vector, characterized in that: The recombinant vector contains the DNA molecule as described in claim 2.

4. A transformant, characterized in that: The recombinant vector as described in claim 3 is transferred into host cells to obtain a transformant.

5. A pharmaceutical preparation for treating Staphylococcus aureus infection, characterized in that: The pharmaceutical formulation comprises the Bacillus subtilis-derived antimicrobial peptide as described in claim 1 and pharmaceutically acceptable excipients.

6. A pharmaceutical preparation for treating wound infection and / or promoting tissue repair and healing, characterized in that: The pharmaceutical formulation comprises the Bacillus subtilis-derived antimicrobial peptide as described in claim 1 and pharmaceutically acceptable excipients.

7. The pharmaceutical preparation according to any one of claims 5 to 6, characterized in that: The pharmaceutical preparations mentioned are gels, sprays, films, patches, ointments, liniments, coatings, eye drops, nasal drops, mouthwashes, or iontophoresis agents.

8. A pharmaceutical combination product, characterized in that: The combined product comprises an antibiotic and an antimicrobial peptide derived from Bacillus subtilis as described in claim 1.

9. The method for preparing the Bacillus subtilis-derived antimicrobial polypeptide as described in claim 1, characterized in that: The antibacterial polypeptide is chemically synthesized using a polypeptide solid-phase synthesis method, and a linear peptide is synthesized according to the amino acid sequence shown in SEQ ID NO:1.

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