Anti-Pseudomonas aeruginosa polypeptide, antibacterial polypeptide solution and application thereof

Abstract

The present invention relates to the field of medical technology, and specifically discloses an anti-Pseudomonas aeruginosa polypeptide, an antibacterial polypeptide solution, and applications thereof. The amino acid sequence of the polypeptide is shown in SEQ ID NO.1: Phe-Trp-Lys-Phe-Leu-Lys-Lys-Ile-Ala-Lys-Lys-Ala-Leu. The polypeptide can effectively kill Pseudomonas aeruginosa, has good stability, and has low toxicity. The polypeptide has the potential to be developed into an anti-Pseudomonas aeruginosa gel, cream, spray preparation, and drug coating for preventing and treating infectious diseases such as skin and wound infections caused by Pseudomonas aeruginosa.

Description

Technical Field

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

[0002] Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is a Gram-negative bacillus, a common opportunistic pathogen, and one of the main pathogens of hospital-acquired infections. Pseudomonas aeruginosa often causes postoperative wound infections, and can also cause abscesses, suppurative otitis media, keratitis, and other conditions. Pseudomonas aeruginosa is prone to developing resistance to conventional antibiotics, greatly increasing the difficulty of causing disease and treating it. In recent years, the resistance rate of Pseudomonas aeruginosa has increased year by year, posing an increasing challenge to traditional antibiotics. There is an urgent need for active pharmaceutical ingredients that can effectively kill and inhibit Pseudomonas aeruginosa while not easily leading to drug resistance.

[0003] Antimicrobial peptides possess excellent antibacterial activity, including strong activity against drug-resistant strains. They primarily kill bacteria through multiple modes of action, disrupting the bacterial cell membrane and simultaneously acting on intracellular targets, making it difficult for bacteria to develop drug resistance. Furthermore, antimicrobial peptides offer advantages such as high specificity and low toxicity, making them a hot topic in the medical field for the discovery of new antimicrobial drugs. Therefore, developing an antimicrobial peptide that is effective against Pseudomonas aeruginosa infection is of great significance to the medical field. Summary of the Invention

[0004] In order to prepare a new effective component of a drug that can resist Pseudomonas aeruginosa infection, the present invention provides an anti-Pseudomonas aeruginosa polypeptide, an antibacterial polypeptide solution and its application. The anti-Pseudomonas aeruginosa polypeptide provided by the present invention can effectively kill Pseudomonas aeruginosa, has good stability and low toxicity.

[0005] The present invention provides an anti-Pseudomonas aeruginosa polypeptide.

[0006] The amino acid sequence of the polypeptide is shown in SEQ ID NO. 1: Phe-Trp-Lys-Phe-Leu-Lys-Lys-Ile-Ala-Lys-Lys-Ala-Leu.

[0007] The present invention also provides an antibacterial polypeptide solution containing the anti-Pseudomonas aeruginosa polypeptide. The antibacterial polypeptide solution is prepared by mixing a solvent with the antibacterial polypeptide.

[0008] Furthermore, the solvent is any one of physiological saline, ultrapure water, phosphate buffer and acetic acid.

[0009] The present invention also provides a use of the anti-Pseudomonas aeruginosa polypeptide or the antibacterial polypeptide solution in the preparation of an antibacterial agent, wherein the antibacterial agent can inhibit the proliferation and growth of Pseudomonas aeruginosa, and the minimum inhibitory concentration of the antibacterial agent against Pseudomonas aeruginosa is 12.5 μg / mL.

[0010] Furthermore, the antibacterial agent is a preparation obtained by dissolving the anti-Pseudomonas aeruginosa polypeptide in 0.9% physiological saline to obtain a final concentration of ≥12.5 μg / mL.

[0011] The present invention also provides an application of the anti-Pseudomonas aeruginosa polypeptide or the antibacterial polypeptide solution in the preparation of a fungicide, wherein the fungicide is used to kill Pseudomonas aeruginosa. When the bactericidal concentration is 25 μg / mL, the fungicide has a Pseudomonas aeruginosa clearance rate of more than 95%.

[0012] The present invention also provides a use of the anti-Pseudomonas aeruginosa polypeptide or the antibacterial polypeptide solution in the preparation of a drug for preventing and / or treating skin wound infection, wherein the skin wound infection refers to infection caused by Pseudomonas aeruginosa.

[0013] Furthermore, the medicine also includes other pharmaceutically acceptable excipients.

[0014] Furthermore, the auxiliary material is any one or more combinations of chitosan, glycerol, triethanolamine, benzyl alcohol, fragrance, disodium edetate and purified water.

[0015] Furthermore, the drug is an antibacterial gel, cream, antibacterial spray or antibacterial drug coating.

[0016] Furthermore, when the drug is an antibacterial gel, its component excipients are at least one of chitosan, glycerol, triethanolamine, polyethylene glycol, polyvinyl alcohol, polyacrylic acid and polymethacrylic acid;

[0017] When the drug is a cream, its component excipients are at least one of monostearate glyceryl, white petrolatum, liquid paraffin and glycerin;

[0018] When the drug is an antibacterial spray, its component excipient is at least one of water and nitrogen;

[0019] When the drug is an antibacterial drug coating, its component excipients are at least one of polyvinyl alcohol, polyethylene glycol, hyaluronic acid, calcium alginate and gelatin.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The anti-Pseudomonas aeruginosa polypeptide provided by the present invention can effectively kill Pseudomonas aeruginosa, with a clearance rate of up to 99.7% or more. It has good stability and low toxicity. The polypeptide has the potential to be developed into anti-Pseudomonas aeruginosa gels, creams, sprays, and drug coatings for preventing and treating infectious diseases such as skin and wound infections caused by Pseudomonas aeruginosa. It can also be used to prepare anti-infective drugs and serve as an active ingredient in the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 This is the hemolytic activity of the antibacterial polypeptide prepared in Example 1. DETAILED DESCRIPTION

[0024] 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.

[0025] Example 1: Obtaining an anti-Pseudomonas aeruginosa polypeptide (hereinafter referred to as antibacterial polypeptide or polypeptide).

[0026] The anti-Pseudomonas aeruginosa polypeptide provided by 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%.

[0027] SEQ ID NO. 1: Phe-Trp-Lys-Phe-Leu-Lys-Lys-Ile-Ala-Lys-Lys-Ala-Leu.

[0028] Example 2: Antibacterial activity of anti-Pseudomonas aeruginosa polypeptides.

[0029] Pseudomonas aeruginosa was cultured in sterile LB liquid medium until the logarithmic growth phase, and then the cultured bacterial solution was diluted to a bacterial concentration of 10 5 -10 6 cfu / mL. Dissolve the polypeptide in 0.9% saline to prepare polypeptide storage solution, and dilute the polypeptide storage solution with 0.9% saline in a two-fold gradient. 5 -10 6 80 μL of bacterial suspension (100 cfu / mL) and 20 μL of the diluted peptide solution were added to a sterile 96-well 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, respectively. Three replicates were performed for each peptide concentration. The plates were incubated in a 37°C incubator for 24 hours. The minimum inhibitory concentration (MIC) of the peptide against the test bacteria was determined as the lowest peptide concentration at which no bacterial growth was observed. Information on P. aeruginosa strains is provided in Table 1.

[0030] Table 1 Antibacterial activity of the anti-Pseudomonas aeruginosa polypeptide provided in Example 1

[0031] strain Minimum inhibitory concentration (μg / mL) Pseudomonas aeruginosa ATCC9027 12.5 Pseudomonas aeruginosa ATCC27853 12.5 Pseudomonas aeruginosa PAO1 12.5 Pseudomonas aeruginosa AB93066 12.5

[0032] The antibacterial activity of the anti-Pseudomonas aeruginosa polypeptide is shown in Table 1. As can be seen from Table 1, the polypeptide has good antibacterial activity against the four tested strains of Pseudomonas aeruginosa, and the minimum inhibitory concentration is 12.5 μg / mL.

[0033] Example 3: Bactericidal effect of the anti-Pseudomonas aeruginosa polypeptide obtained in Example 1.

[0034] Use sterile LB liquid medium to dilute Pseudomonas aeruginosa ATCC9027 cultured to the logarithmic growth phase to a bacterial concentration of 10 6 -10 7 cfu / mL. Antimicrobial peptides were then added to final concentrations of 12.5 μg / mL, 25 μg / mL, and 50 μg / mL. After incubation in a 37°C incubator for 15 minutes, samples were collected and serially diluted before being spread on LB plates. The samples were incubated in a 37°C incubator for 24 hours. Colonies were counted and the clearance rate was calculated. The results are reported in Table 2.

[0035] Clearance rate = (1-number of colonies in the polypeptide-treated group / initial number of colonies) × 100%

[0036] Table 2 Bactericidal activity of anti-Pseudomonas aeruginosa polypeptides provided in Example 1

[0037] Peptide concentration (μg / mL) Clearance rate (%) 12.5 61.2 25 95.1 50 99.7

[0038] The bactericidal effect of the anti-Pseudomonas aeruginosa polypeptide of the present invention is shown in Table 2. As can be seen from Table 2, after treatment with 12.5 μg / mL of the polypeptide for 15 minutes, 61.2% of Pseudomonas aeruginosa were killed, after treatment with 25 μg / mL of the polypeptide for 15 minutes, 95.1% of Pseudomonas aeruginosa were killed, and after treatment with 50 μg / mL of the polypeptide for 15 minutes, 99.7% of Pseudomonas aeruginosa were killed. This shows that the polypeptide has excellent bactericidal activity against Pseudomonas aeruginosa and can kill Pseudomonas aeruginosa quickly and efficiently.

[0039] Example 4: Stability study of the anti-Pseudomonas aeruginosa polypeptide obtained in Example 1.

[0040] 1. Detect the effect of salt ion concentration on the activity of antibacterial peptides under different physiological conditions

[0041] Na used + The final concentration of (NaCl) was 150 mM, and the Ca 2+ The final concentration of (CaCl2) was 1 mM, and the K + The final concentration of KCl was 4 mM, and the Mg 2+ The final concentration of (MgCl2) was 2 mM.

[0042] The specific detection method is as follows: Pseudomonas aeruginosa ATCC9027 cultured to the logarithmic growth phase was diluted to a bacterial concentration of 10 5 -10 6 cfu / mL. Then take the concentration obtained above as 10 5 -10 6 80 μL of bacterial solution containing 100 cfu / mL and 20 μL of peptide stock solution diluted twofold with 0.9% physiological saline were added to a sterile 96-well cell culture plate. Simultaneously, a single salt ion at the required final concentration was added to achieve peptide final concentrations of 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, respectively. Three replicates were set for each peptide concentration. The plates were incubated in a 37°C biochemical incubator for 24 hours, and the minimum inhibitory concentration of the peptide against the bacteria was determined.

[0043] 2. Heat-treat the peptide storage solution at 60°C for 24 hours to detect the effect of heat treatment on the activity of the antibacterial peptide.

[0044] For the specific detection method, please refer to the detection steps in 1 above. The results are recorded in Table 3.

[0045] Table 3 Stability of the anti-Pseudomonas aeruginosa polypeptide obtained in Example 1

[0046]

[0047] The stability of the anti-Pseudomonas aeruginosa peptide is shown in Table 3. As can be seen from Table 3, the minimum inhibitory concentration (MIC) of the antimicrobial peptide against Pseudomonas aeruginosa in the systems containing 150 mM NaCl, 1 mM CaCl2, 4 mM KCl, or 2 mM MgCl2 was 12.5 μg / mL, unchanged from the final activity value. Furthermore, after heat treatment at 60°C, the MIC of the peptide against Pseudomonas aeruginosa was 12.5 μg / mL, unchanged from the final activity value. This demonstrates the excellent stability of the peptide.

[0048] Example 5: Hemolytic activity of anti-Pseudomonas aeruginosa polypeptides

[0049] 2 mL of fresh anticoagulated blood was collected from a healthy individual and centrifuged at 3000 rpm / min for 5 minutes. The red blood cells were collected and washed three times with 0.9% saline. The red blood cells were resuspended in 0.9% saline to a final concentration of 2% (v / v). 100 μL of the above red blood cell resuspension and 100 μL of the peptide stock solution diluted twofold with 0.9% saline were added to a sterile 96-well cell culture plate, respectively, to achieve final peptide concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. Three replicates were set for each peptide concentration. 0.1% Tritonx-100 was used as a complete hemolysis control, and normal saline was used as an insoluble hemolysis control. The cells were incubated in a 37°C biochemical incubator for 1 hour, followed by centrifugation at 3000 rpm / min for 10 minutes. The supernatant was transferred to a new sterile 96-well cell culture plate, and the absorbance at 570 nm was measured with a microplate reader to calculate the peptide hemolysis rate.

[0050] Hemolytic activity of anti-Pseudomonas aeruginosa peptides Figure 1 As shown, the hemolytic rate of the polypeptide at a concentration of 12.5 μg / mL was 0.2%, at a concentration of 25 μg / mL was 1.2%, and at a concentration of 50 μg / mL was 7.4%, indicating that its toxic side effects were very low.

[0051] 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.

[0052] 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 anti-Pseudomonas aeruginosa polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO. 1: Phe-Trp-Lys-Phe-Leu-Lys-Lys-Ile-Ala-Lys-Lys-Ala-Leu.

2. An antimicrobial polypeptide solution containing the anti-Pseudomonas aeruginosa 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 anti-Pseudomonas aeruginosa polypeptide according to claim 1 or the antibacterial polypeptide solution according to any one of claims 2 to 3 in the preparation of an antibacterial agent, characterized in that: The bacteriostatic agent can inhibit the proliferation and growth of Pseudomonas aeruginosa, and the minimum inhibitory concentration of the bacteriostatic agent on Pseudomonas aeruginosa is 12.5 μg / mL.

5. The use according to claim 4, characterized in that The antibacterial agent is a preparation in which the anti-Pseudomonas aeruginosa polypeptide is dissolved in 0.9% physiological saline to obtain a final concentration of ≥12.5 μg / mL.

6. Use of the anti-Pseudomonas aeruginosa 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 is used for killing Pseudomonas aeruginosa. When the bactericidal concentration is 25 μg / mL, the bactericide has a Pseudomonas aeruginosa elimination rate of more than 95%.

7. Use of the anti-Pseudomonas aeruginosa 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 preventing and / or treating skin wound infection, characterized in that: The skin wound infection refers to the infection caused by Pseudomonas aeruginosa.

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 auxiliary material is any one or more combinations of chitosan, glycerol, triethanolamine, benzyl alcohol, fragrance, disodium edetate and purified water.

10. 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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