Polypeptide, polypeptide solution and application for resisting pathogenic bacteria of skin and wound infection

Abstract

The application belongs to the field of medical application, and particularly relates to an anti-skin and wound infection pathogenic bacteria polypeptide, a polypeptide solution and application. The anti-skin and wound infection pathogenic bacteria polypeptide can effectively inhibit or kill Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, and the amino acid sequence of the anti-skin and wound infection pathogenic bacteria polypeptide is Phe-Leu-Lys-Lys-Ala-Ala-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Phe. Meanwhile, the polypeptide has good stability and very low toxicity. The polypeptide has the potential to be developed into an antibacterial gel, a spray preparation and a drug coating with the efficacy of resisting Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, and is used for preventing and treating skin and wound infections and other infectious diseases caused by Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical applications, specifically to a polypeptide, polypeptide solution, and its application against pathogenic bacteria causing skin and wound infections. Background Technology

[0002] Bacterial infections are a common type of disease, especially skin and wound infections. Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli are the main pathogens causing skin and wound infections. Although they can be effectively treated with traditional antibiotics, drug resistance is a common problem. The drug resistance of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli is currently very serious, severely affecting treatment outcomes and also causing a series of social problems.

[0003] Antimicrobial peptides are a class of bioactive peptide molecules that can effectively kill different types of pathogens, including drug-resistant strains. Furthermore, due to their multiple mechanisms of action, which differ from traditional antibiotics, antimicrobial peptides are less likely to induce bacterial resistance. Therefore, antimicrobial peptides are highly attractive and have promising development prospects, making them a hot topic in the pharmaceutical field for discovering novel antimicrobial drugs.

[0004] Therefore, antimicrobial peptides are highly attractive and promising candidates for the research and development of novel antimicrobial drugs in the pharmaceutical field. Currently, existing peptides capable of simultaneously combating multiple pathogens are relatively limited. Therefore, there is an urgent need to obtain an antimicrobial peptide that can combat pathogens causing skin and wound infections. Summary of the Invention

[0005] This invention provides a polypeptide, a polypeptide solution, and its application for inhibiting pathogenic bacteria causing skin and wound infections. The polypeptide provided by this invention can effectively inhibit or kill pathogenic bacteria that cause skin or wound infections, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. At the same time, the polypeptide has good stability and low toxicity.

[0006] This invention provides a polypeptide against pathogenic bacteria causing skin and wound infections, the amino acid sequence of which is shown in SEQ ID NO.1: Phe-Leu-Lys-Lys-Ala-Ala-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Phe;

[0007] The pathogens include Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.

[0008] This invention provides a novel antimicrobial peptide, enriching the sources of antimicrobial peptide products. It also lays the foundation for the clinical preparation of drugs for skin and wound infections.

[0009] The present invention also provides a polypeptide solution containing the aforementioned anti-skin and wound infection pathogenic polypeptide, wherein the polypeptide solution is obtained by dissolving the anti-skin and wound infection pathogenic polypeptide in a solvent.

[0010] Furthermore, the saline solution can be replaced with ultrapure water, phosphate buffer, or acetic acid.

[0011] Furthermore, the concentration of the polypeptide solution is 6.25-100 μg / mL.

[0012] The present invention also provides the application of the aforementioned anti-skin and wound infection pathogenic peptide or the aforementioned peptide solution in the preparation of an antibacterial agent, wherein the antibacterial agent is used to inhibit Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.

[0013] Furthermore, the minimum inhibitory concentration (MIC) of the polypeptide solution against Staphylococcus aureus is 6.25 μg / mL, against Pseudomonas aeruginosa is 12.5 μg / mL, and against Escherichia coli is 12.5 μg / mL.

[0014] The present invention also provides the application of the aforementioned anti-skin and wound infection pathogenic peptide or the aforementioned peptide solution in the preparation of a bactericide, wherein the bactericide is used to kill Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli;

[0015] The minimum concentration of the polypeptide solution to kill Staphylococcus aureus is 25 μg / mL, and the minimum concentration to kill Pseudomonas aeruginosa and Escherichia coli is 50 μg / mL.

[0016] The present invention also provides the application of the aforementioned anti-skin and wound infection pathogenic polypeptide or the aforementioned polypeptide solution in the preparation of a drug for treating skin or wound infections, wherein the pathogenic bacteria causing the skin or wound infection are any one or a combination of several of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.

[0017] Furthermore, the drug uses a polypeptide with the sequence shown in SEQ ID NO.1 as its sole active ingredient.

[0018] Furthermore, the drug also includes other pharmaceutically acceptable excipients.

[0019] Furthermore, the excipients include any one or more combinations of sodium alginate, chitosan, polyvinyl alcohol, glutamine, aldehyde-modified dextran, adipic acid dihydrazide, Tween 60, lauramide propyl betaine, geniposide, and gardenia oil.

[0020] Furthermore, the drug is an antibacterial gel, cream, antibacterial spray, or antibacterial drug coating.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. The antimicrobial polypeptide of this invention can effectively kill Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, with clearance rates of 99.7%, 98.9%, and 99.2% for Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, respectively. The antimicrobial polypeptide exhibits good stability and very low toxicity. This polypeptide has the potential to be developed into gels, creams, sprays, and drug coatings with anti-Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli functions, for the prevention and treatment of skin infections or wound infections caused by Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The hemolysis rate of the antimicrobial peptides prepared in Example 1 at different concentrations. Detailed Implementation

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0026] Example 1: Preparation of an antimicrobial polypeptide.

[0027] The antimicrobial peptide proposed in this invention is derived from our artificial design based on the characteristics of antimicrobial peptides, and its amino acid sequence is shown in SEQ ID NO.1. According to the amino acid sequence shown, it was synthesized by Jier Biochemical (Shanghai) Co., Ltd. using solid-phase chemical synthesis method. After purification by reversed-phase high-performance liquid chromatography and identification by electrospray mass spectrometry, a peptide with a purity greater than 95% was obtained.

[0028] SEQ ID NO. 1: Phe-Leu-Lys-Lys-Ala-Ala-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Phe.

[0029] Example 2: Study on the antibacterial activity of the antimicrobial peptides obtained in Example 1.

[0030] I. Experimental Methods

[0031] 1. Study on the antibacterial activity of antimicrobial peptides

[0032] (1) Preparation of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli bacterial suspensions

[0033] Pure cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were inoculated into LB liquid medium and cultured at 37°C and 150 rpm until the logarithmic growth phase, respectively, to obtain Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli suspensions. The concentrations of these suspensions were then adjusted to 10⁻⁶ using LB liquid medium. 5 -10 6 cfu / mL.

[0034] Specific strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli are shown in Table 1.

[0035] 80 μL of the bacterial suspension prepared above and 20 μL of antimicrobial peptide solutions of different concentrations, serially diluted twice with physiological saline, were added to 96-well sterile cell culture plates. The final peptide concentrations were 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.13 μg / mL, and 1.56 μg / mL. The culture plates were incubated in a 37℃ biochemical incubator for 24 h. The lowest peptide concentration at which sterile growth was observed was the minimum inhibitory concentration (MIC) of the peptide against the test bacteria.

[0036] II. Experimental Results

[0037] 1. Antibacterial activity of antimicrobial peptides

[0038] The antimicrobial activity of the antimicrobial peptide described in SEQ ID NO.1 is shown in Table 1. As can be seen from Table 1, the antimicrobial peptide exhibits inhibitory effects against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Specifically, the minimum inhibitory concentration (MIC) against Staphylococcus aureus is 6.25 μg / mL, against Pseudomonas aeruginosa is 12.5 μg / mL, and against Escherichia coli is 12.5 μg / mL.

[0039] Table 1. Antimicrobial activity of the antimicrobial peptides obtained in Example 1

[0040]

[0041] Example 3: Study on the bactericidal effect of the antibacterial peptides obtained in Example 1.

[0042] I. Experimental Methods

[0043] Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC9027 and Escherichia coli ATCC 25922 were cultured according to the culture steps in Example 2 to obtain Staphylococcus aureus culture, Pseudomonas aeruginosa culture and Escherichia coli culture.

[0044] The concentrations of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli bacterial suspensions were adjusted to 10. 6 -10 7 cfu / mL.

[0045] Add antimicrobial peptides to the diluted bacterial solution to a final concentration four times the minimum inhibitory concentration (MIC) of the corresponding strain, mix well, and incubate at 37°C for 15 min. Then, take samples, serially dilute with physiological saline, spread them on solid culture plates, and incubate at 37°C for 24 h. Count the number of colonies and calculate the clearance rate.

[0046] II. Experimental Results

[0047] The bactericidal effects of the peptides are shown in Table 2.

[0048] Table 2. Bactericidal activity of peptides

[0049]

[0050] As shown in Table 2, after treatment with the antimicrobial peptide at 4 times the minimum inhibitory concentration for 15 min, the antimicrobial peptide could kill 99.7% of Staphylococcus aureus, 98.9% of Pseudomonas aeruginosa, and 99.2% of Escherichia coli in the reaction system, respectively. This indicates that the antimicrobial peptide has good killing activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, and its killing efficiency is rapid and efficient.

[0051] Example 4: Stability study of the antimicrobial peptides prepared in Example 1.

[0052] I. Experimental Methods

[0053] Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 9027, and Escherichia coli ATCC 25922 were the subjects of this study.

[0054] 1. Effects of cationic ions on the antibacterial activity of antimicrobial peptides

[0055] The effect of salt ion concentration on the activity of antimicrobial peptides: Bacteria cultured to the logarithmic growth phase were diluted to a cell concentration of 10⁻⁶ using sterile LB liquid medium. 5 -10 6CFU / mL. Then, 80 μL of the diluted bacterial solution and 20 μL of the polypeptide solution, serially diluted twofold with 0.9% physiological saline, were added to sterile 96-well cell culture plates, along with the corresponding final concentrations of salt ions. The culture plates were incubated at 37 ℃ in a biochemical incubator for 18–24 h, and the minimum inhibitory concentration (MIC) of the polypeptide against the bacteria was determined.

[0056] 2. Effect of temperature on the antibacterial activity of peptides

[0057] Effect of heat treatment on antimicrobial peptide activity: Bacteria cultured to the logarithmic growth phase were diluted to a cell concentration of 10⁻⁶ using sterile LB liquid medium. 5 -10 6 CFU / mL. Then, 80 μL of the diluted bacterial solution and 20 μL of the heat-treated peptide solution, serially diluted twofold with 0.9% physiological saline, were added to sterile 96-well cell culture plates. The culture plates were incubated in a 37°C biochemical incubator for 18–24 h, and the minimum inhibitory concentration (MIC) of the peptide against the bacteria was determined.

[0058] II. Experimental Results

[0059] The antibacterial stability of the peptides is shown in Table 3.

[0060] Table 3 Antibacterial stability of peptides

[0061]

[0062] As shown in Table 3, the minimum inhibitory concentrations (MICs) of the antimicrobial peptide against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli remained unchanged compared to the original activity values ​​when the system contained 150 mM NaCl, 1 mM CaCl2, 4 mM KCl, or 2 mM MgCl2, respectively. Furthermore, the MICs against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli remained unchanged after heat treatment at 60 °C. This indicates that the peptide exhibits good stability.

[0063] Example 5: Study on the hemolytic activity of the antimicrobial peptides prepared in Example 1.

[0064] I. Experimental Methods

[0065] 1. Study on the hemolytic activity of antimicrobial peptides

[0066] (1) The antimicrobial peptide was serially diluted twice with 0.9% physiological saline to obtain antimicrobial peptide solutions with concentrations of 50 μg / mL, 100 μg / mL and 200 μg / mL.

[0067] (2) Fresh anticoagulated blood from healthy individuals was centrifuged at 3000 rpm for 5 min to collect red blood cells. The collected red blood cells were washed three times with physiological saline and resuspended to a 2% (V / V) red blood cell suspension. 100 μL of the red blood cell suspension and 100 μL of an antimicrobial peptide solution serially diluted twice with 0.9% physiological saline were added to sterile 96-well cell culture plates, respectively, so that the final peptide concentrations were 25 μg / mL, 50 μg / mL and 100 μg / mL, respectively, and then incubated at 37 ℃ for 1 h. 0.1% Tritonx-100 was used as a complete hemolysis control and 0.9% physiological saline was used as a non-hemolysis control. After incubation, the 96-well cell culture plates were centrifuged at 3000 rpm for 10 min. Equal volumes of supernatant were transferred to new 96-well cell culture plates, and the absorbance of the supernatant at 570 nm was detected using an ELISA reader to calculate the hemolysis rate of the peptide.

[0068] II. Experimental Results

[0069] The hemolytic activity of antimicrobial peptides, such as Figure 1 As shown, the hemolysis rate of this peptide at a concentration of 200 μg / mL was only 1.9%. This indicates that the cytotoxicity of this peptide is very low.

[0070] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0071] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A polypeptide for combating pathogenic bacteria causing skin and wound infections, characterized in that, The amino acid sequence of the peptide against pathogenic bacteria causing skin and wound infections is shown in SEQ ID NO.1: Phe-Leu-Lys-Lys-Ala-Ala-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Phe; The pathogens include Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.

2. A polypeptide solution containing the polypeptide against pathogenic bacteria causing skin and wound infections as described in claim 1, characterized in that, The polypeptide solution is obtained by dissolving the polypeptide that is effective against pathogenic bacteria causing skin and wound infections in a solvent.

3. The polypeptide solution according to claim 2, characterized in that, The solvent is physiological saline.

4. The use of the polypeptide of claim 1, which is effective against pathogenic bacteria causing skin and wound infections, or the polypeptide solution of any one of claims 2-3, in the preparation of an antibacterial agent, characterized in that, The antibacterial agent is used to inhibit Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.

5. The use of the polypeptide of claim 1, which is effective against pathogenic bacteria causing skin and wound infections, or the polypeptide solution of any one of claims 2-3, in the preparation of a bactericide, characterized in that, The bactericide is used to kill Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli; The minimum concentration of the polypeptide solution to kill Staphylococcus aureus is 25 μg / mL, and the minimum concentration to kill Pseudomonas aeruginosa and Escherichia coli is 50 μg / mL.

6. The use of the polypeptide of claim 1, which is effective against pathogenic bacteria causing skin and wound infections, or the polypeptide solution of any one of claims 2-3, in the preparation of a medicament for treating skin or wound infections, characterized in that, The pathogens causing the skin or wound infection are any one or a combination of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.

7. The application according to claim 6, characterized in that, The drug uses a polypeptide with the sequence shown in SEQ ID NO.1 as its sole active ingredient.

8. The application according to claim 7, characterized in that, The drug also includes other pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The excipients include any one or more combinations of sodium alginate, chitosan, polyvinyl alcohol, glutamine, aldehyde-modified dextran, adipic acid dihydrazide, Tween 60, lauramide propyl betaine, geniposide, and gardenia oil.

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