Antibacterial polypeptide, antibacterial polypeptide solution and application thereof

Abstract

The present invention relates to the field of medical applications, and specifically discloses an antimicrobial polypeptide, an antimicrobial polypeptide solution, and applications thereof. The amino acid sequence of the antimicrobial polypeptide is Lys-Leu-Trp-Lys-Phe-Leu-Lys-Lys-Ile-Leu. The antimicrobial polypeptide provided by the present invention can effectively kill the pathogenic bacteria Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans that cause skin or wound infections. At the same time, the polypeptide has good stability and low toxicity. The antimicrobial polypeptide has the potential to be developed into antimicrobial gels, creams, spray preparations, and drug coatings with anti-skin or wound infection efficacy, and is used to prevent and treat skin or wound infections caused by Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans.

Description

Technical Field

[0001] The present invention relates to the field of medical applications, and in particular to an antibacterial polypeptide, an antibacterial polypeptide solution and applications thereof. Background Art

[0002] Skin and wound infections are the most common health problems in daily life, in outpatient clinics, and in hospitals. Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans are the main pathogens causing skin and wound infections. In recent years, the incidence of skin and wound infections has been gradually increasing, especially with the emergence of infections associated with drug-resistant pathogens, resulting in significantly higher morbidity, mortality, length of hospital stay, and costs than infections associated with susceptible strains. However, the development of new antibiotics is relatively lagging, and pathogens are also facing the problem of rapid development of drug resistance.

[0003] Antimicrobial peptides are a class of polypeptide molecules with excellent antimicrobial activity. These molecules also have a good killing and inhibitory effect on drug-resistant strains. Compared with the specific action sites of traditional antibiotics, antimicrobial peptides mainly destroy the integrity and function of the cell membrane of pathogenic bacteria, while also interfering with the physiological and metabolic processes within the bacteria. This multiple and complex mechanism of action makes it difficult for pathogens to develop drug resistance. In addition, antimicrobial peptides also have good specificity, low toxicity, and immunomodulatory effects. Therefore, antimicrobial peptides are extremely attractive and have application prospects in the research and development and utilization of new antimicrobial drugs in the medical field. Therefore, there is an urgent need to obtain an antimicrobial peptide that can fight pathogenic bacteria that cause skin and wound infections. Summary of the Invention

[0004] The present invention provides an antimicrobial polypeptide, an antimicrobial polypeptide solution, and applications thereof. The antimicrobial polypeptide provided by the present invention can effectively kill pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans that cause skin or wound infections. At the same time, the polypeptide has good stability and low toxicity.

[0005] The present invention provides an antibacterial polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 1: Lys-Leu-Trp-Lys-Phe-Leu-Lys-Lys-Ile-Leu.

[0006] The present invention also provides an antimicrobial polypeptide solution containing the antimicrobial polypeptide. The antimicrobial polypeptide solution is prepared by mixing a solvent with the antimicrobial polypeptide.

[0007] Furthermore, the solvent is any one of physiological saline, ultrapure water, phosphate buffer and acetic acid.

[0008] The present invention also provides a use of the antibacterial polypeptide or the antibacterial polypeptide solution in the preparation of an antibacterial agent, wherein the antibacterial agent can inhibit Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans.

[0009] Furthermore, the minimum inhibitory concentration of the antibacterial agent against Staphylococcus aureus is 6.25 μg / mL, the minimum inhibitory concentration against Pseudomonas aeruginosa is 12.5 μg / mL, the minimum inhibitory concentration against Escherichia coli is 12.5 μg / mL, and the minimum inhibitory concentration against Candida albicans is 12.5 μg / mL.

[0010] The present invention also provides a use of the antimicrobial polypeptide or the antimicrobial polypeptide solution in preparing a fungicide, wherein the fungicide can kill Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans.

[0011] Furthermore, when the Staphylococcus aureus is completely killed, the concentration of the antimicrobial polypeptide in the bactericidal agent is ≥25 μg / mL;

[0012] When the Pseudomonas aeruginosa, Escherichia coli and Candida albicans are completely killed, the concentration of the antibacterial polypeptide in the bactericide is ≥50 μg / mL.

[0013] The present invention also provides a use of the antimicrobial polypeptide or the antimicrobial polypeptide solution in the preparation of a drug for treating skin or wound infections, wherein the pathogen causing the skin or wound infection is any one or a combination of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans.

[0014] Furthermore, the medicine also includes other pharmaceutically acceptable excipients.

[0015] Furthermore, the auxiliary materials include any one or more combinations of sodium alginate, chitosan, polyvinyl alcohol, glutamine, formaldehyde-modified dextran, adipic acid dihydrazide, Tween 60, lauramidopropyl betaine, gardenia glycoside and gardenia oil.

[0016] Furthermore, the drug is an antibacterial gel, cream, antibacterial spray or antibacterial drug coating.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The antimicrobial polypeptide described herein can effectively kill Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans, exhibits good stability, and has very low toxicity. This polypeptide has the potential to be developed into gels, creams, sprays, and drug coatings with antimicrobial properties against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans, for use in preventing and treating skin and wound infections caused by these bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 The hemolytic activity of the antibacterial polypeptide prepared in Example 1 at different concentrations. DETAILED DESCRIPTION

[0021] 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 by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0022] Example 1: Preparation of an antibacterial polypeptide.

[0023] The antibacterial polypeptide proposed in the present invention is artificially designed based on the characteristics of antimicrobial polypeptides, and its amino acid sequence is shown in SEQ ID NO.1. Based on the shown amino acid sequence, it was synthesized by solid-phase chemical synthesis by Jier Biochemical (Shanghai) Co., Ltd., and purified by reverse-phase high-performance liquid chromatography and identified by electrospray ionization mass spectrometry to obtain a pure polypeptide with a purity greater than 95%.

[0024] SEQ ID NO. 1: Lys-Leu-Trp-Lys-Phe-Leu-Lys-Lys-Ile-Leu.

[0025] Example 2: Study on the antibacterial activity of the antibacterial polypeptide obtained in Example 1.

[0026] 1. Experimental Methods

[0027] 1. Study on the antibacterial activity of antibacterial peptides

[0028] (1) Preparation of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli cultures

[0029] Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli pure cultures were inoculated into LB liquid medium at 37°C and 150 rpm and cultured to the logarithmic growth phase to obtain Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli liquids, respectively. The concentrations of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli liquids were then adjusted to 10 using LB liquid medium. 5 -10 6 cfu / mL.

[0030] The specific strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli are shown in Table 1 .

[0031] (2) Preparation of Candida albicans liquid

[0032] The pure culture of Candida albicans was inoculated into YPD liquid medium at 37 ° C and 150 rpm and cultured to the logarithmic growth phase to obtain Candida albicans liquid. The concentration of Candida albicans liquid was adjusted to 10 3 -10 4 cfu / mL.

[0033] The information of Candida albicans strains is shown in Table 1.

[0034] 80 μL of the bacterial suspension prepared above and 20 μL of antimicrobial peptide solutions of varying concentrations, diluted twofold with normal saline, were added to a 96-well sterile cell culture plate. 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 plates were incubated in a 37°C biochemical incubator for 24 hours. The lowest peptide concentration that resulted in sterile growth was defined as the minimum inhibitory concentration (MIC) of the peptide against the test bacteria.

[0035] 2. Experimental Results

[0036] 1. Antibacterial activity of antimicrobial peptides

[0037] The antibacterial activity of the antibacterial polypeptide described in SEQ ID NO. 1 is shown in Table 1. As can be seen from Table 1, the antibacterial polypeptide has inhibitory effects on Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans, wherein the minimum inhibitory concentration for Staphylococcus aureus is 6.25 μg / mL, the minimum inhibitory concentration for Pseudomonas aeruginosa is 12.5 μg / mL, the minimum inhibitory concentration for Escherichia coli is 12.5 μg / mL, and the minimum inhibitory concentration for Candida albicans is 12.5 μg / mL.

[0038] Table 1 Antibacterial activity of the antibacterial peptides obtained in Example 1

[0039] strain Minimum inhibitory concentration (μg / mL) Staphylococcus aureus ATCC 25923 6.25 Staphylococcus aureus ATCC 6538 6.25 Staphylococcus aureus AB 94004 6.25 Pseudomonas aeruginosa ATCC 9027 12.5 Pseudomonas aeruginosa ATCC 27853 12.5 Pseudomonas aeruginosa AB 93066 12.5 Escherichia coli ATCC 25922 12.5 Escherichia coli AB 94012 12.5 Candida albicans ATCC 10231 12.5 Candida albicans CMCC 98001 12.5 Candida albicans AY 93025 12.5

[0040] Example 3: Study on the bactericidal effect of the antimicrobial polypeptide obtained in Example 1.

[0041] 1. Experimental Methods

[0042] Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC9027, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231 were cultured according to the culture steps in Example 2 to obtain Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans liquids;

[0043] The concentrations of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were adjusted to 10 6 -10 7 cfu / mL; the concentration of Candida albicans solution was adjusted to 10 5 -10 6 cfu / mL.

[0044] Antimicrobial peptides were added to the diluted bacterial solution to a final concentration of 2 or 4 times the minimum inhibitory concentration of the strain. After mixing, the mixture was incubated in a 37°C constant temperature biochemical incubator for 15 minutes. Samples were then taken and diluted with physiological salt gradients, spread on solid culture plates, and incubated in a 37°C constant temperature biochemical incubator for 24 hours. The colonies were counted and the clearance rate was calculated.

[0045] 2. Experimental Results

[0046] The bactericidal effects of the peptides are shown in Table 2.

[0047] Table 2 Bactericidal activity of polypeptides

[0048]

[0049] As can be seen from Table 2, after being treated with the corresponding 2-fold minimum inhibitory concentration of antimicrobial peptide for 15 minutes, the antimicrobial peptide can kill 99.3% of Staphylococcus aureus, 99.5% of Pseudomonas aeruginosa, 99.9% of Escherichia coli and 91.5% of Candida albicans in the reaction system respectively;

[0050] After 15 minutes of treatment with the corresponding antimicrobial peptide at 4 times the minimum inhibitory concentration, the antimicrobial peptide completely eliminated Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans in the reaction system. This demonstrates that the antimicrobial peptide has excellent killing activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, with rapid and efficient killing efficiency.

[0051] Example 4: Stability study of the antibacterial polypeptide prepared in Example 1.

[0052] 1. Experimental Methods

[0053] Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231 were used as research objects in this example.

[0054] 1. Effect of cations on the antibacterial activity of antimicrobial peptides

[0055] The antibacterial activity test method refers to Example 2, and the test system contains Na + (NaCl), 4 mM K + (KCl), 1 mM Ca 2+ (CaCl2), 2mM Mg 2+ (MgCl2), compared with the original minimum inhibitory concentration.

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

[0057] Activity detection was performed with reference to Example 2. Before detection, the antimicrobial polypeptide was heat-treated at 60° C. for 24 h, and the difference with the original minimum inhibitory concentration was compared.

[0058] 2. Experimental Results

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

[0060] Table 3 Antibacterial stability of polypeptides

[0061]

[0062] As can be seen from Table 3, the system contains 150mM Na + (NaCl), or 4 mM K + (KCl), or 1 mM Ca 2 + (CaCl2), or 2 mM Mg 2+(MgCl2), the minimum inhibitory concentration of the peptide against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans remained unchanged compared to the initial activity values. Furthermore, after heat treatment at 60°C, the minimum inhibitory concentration of the peptide against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans remained unchanged compared to the initial activity values. This demonstrates that the peptide has good antibacterial stability.

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

[0064] 1. Experimental Methods

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

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

[0067] (2) Anticoagulated blood from healthy individuals was centrifuged at 3000 rpm / min for 5 min to collect red blood cells. The collected red blood cells were washed three times with physiological saline and resuspended into a 2% (V / V) red blood cell suspension. 100 μL of red blood cell suspension and 100 μL of antibacterial peptide solution were added to 96-well cell culture plates, with the final concentrations of the peptides being 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively), and then incubated in a 37°C incubator for 1 hour. After incubation, the 96-well cell culture plates were centrifuged at 3000 rpm / min for 10 minutes. Equal volumes of supernatant were taken to new 96-well cell culture plates, and the absorbance of the supernatant at 570 nm was detected using an enzyme-linked microplate reader to calculate the hemolysis rate of the peptide.

[0068] 2. Experimental Results

[0069] Hemolytic activity of peptides Figure 1 As shown in the results, the hemolytic rate of the polypeptide at a concentration of 25 μg / mL was 1.4%, at a concentration of 50 μg / mL was 5.2%, and at a concentration of 100 μg / mL was 16.7%. This indicates that the polypeptide has very low toxicity at concentrations with effective antibacterial activity.

[0070] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0071] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. An antimicrobial polypeptide, characterized in that The amino acid sequence of the antibacterial polypeptide is shown in SEQ ID NO. 1: Lys-Leu-Trp-Lys-Phe-Leu-Lys-Lys-Ile-Leu.

2. An antimicrobial polypeptide solution containing the antimicrobial polypeptide according to claim 1, characterized in that: The antibacterial polypeptide solution is prepared by mixing a solvent and the antibacterial polypeptide.

3. The antimicrobial polypeptide solution according to claim 2, characterized in that: The solvent is any one of physiological saline, ultrapure water, phosphate buffer and acetic acid.

4. Use of the antimicrobial polypeptide according to claim 1 or the antimicrobial polypeptide solution according to any one of claims 2 to 3 in the preparation of an antibacterial agent, characterized in that: The antibacterial agent can inhibit Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans.

5. The use according to claim 4, characterized in that The minimum inhibitory concentration of the antibacterial agent against Staphylococcus aureus is 6.25 μg / mL, the minimum inhibitory concentration against Pseudomonas aeruginosa is 12.5 μg / mL, the minimum inhibitory concentration against Escherichia coli is 12.5 μg / mL, and the minimum inhibitory concentration against Candida albicans is 12.5 μg / mL.

6. Use of the antimicrobial polypeptide according to claim 1 or the antimicrobial polypeptide solution according to any one of claims 2 to 3 in the preparation of a bactericide, characterized in that: The bactericide can kill Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans.

7. Use of the antimicrobial polypeptide according to claim 1 or the antimicrobial polypeptide solution according to any one of claims 2 to 3 in the preparation of a drug 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, Escherichia coli and Candida albicans.

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

9. The use according to claim 8, characterized in that The medicine is an antibacterial gel, cream, antibacterial spray or antibacterial medicine coating.

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