Anti-biofilm peptide and application thereof

Abstract

The invention relates to an anti-biofilm peptide and application thereof, and belongs to the technical field of antibacterial peptides. The invention relates to an anti-biofilm peptide VKLR. The amino acid sequence of the anti-biofilm peptide VKLR is as shown in SEQ ID NO: 1: LVKLRVKLRVKLR. The broad-spectrum antibacterial agent aims at bacteria including gram-negative bacteria and gram-positive bacteria. The anti-biofilm peptide VKLR disclosed by the invention has broad-spectrum antibacterial activity and has a remarkable antibacterial effect on various gram-negative bacteria and gram-positive bacteria such as escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and staphylococcus epidermidis, and infection caused by a pathogen biofilm seriously threatens human and animal health; in the breeding process, abuse of traditional antibiotics is likely to generate drug resistance risks, and people pay attention to novel antibacterial drug substitutes. According to the present invention, the 14-element library of the AMPs is obtained, the peptide VKLR shows the huge potential in the antibacterial effect aspect, and is the ideal candidate for treating bacterial infection, such that the research provides the new insight for the future design of the biofilm removing peptide, and the wide application of the biofilm removing peptide in the medicine and the animal husbandry is promoted.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide technology, specifically to an anti-biomembrane peptide and its applications. Background Technology

[0002] In recent decades, the frequent and irregular use of antibiotics in medicine, animal husbandry, and other fields has accelerated the mutation and evolution rate of pathogenic microorganisms, leading to their increasing drug resistance and the emergence of "superbugs." Currently, the overuse of antibiotics in animal husbandry is causing their effectiveness to decline rapidly, and the problem of drug resistance is becoming increasingly serious.

[0003] Besides antibiotic overuse, bacterial biofilms are another significant factor exacerbating drug resistance. Biofilms not only exhibit natural resistance to conventional antibacterial agents but also promote the transfer of drug resistance genes between bacteria, accelerating the development of bacterial resistance and leading to pathogens developing resistance to multiple antibiotics, posing a significant challenge to clinical treatment and disease control.

[0004] Antimicrobial peptides, as a class of small molecule polypeptides, possess broad-spectrum antibacterial properties and are less likely to induce bacterial resistance. Among them, antibiofilm peptides can penetrate and eliminate bacterial biofilms with low cytotoxicity. Therefore, designing antimicrobial peptides with biofilm penetration and elimination capabilities has become an important strategy for eliminating drug-resistant bacteria. Based on this, the present invention aims to provide a novel antibiofilm peptide to address the problem of poor efficacy of existing antimicrobial agents against drug-resistant bacteria and biofilms. Summary of the Invention

[0005] The purpose of this invention is to provide an anti-biofilm peptide that has good antibacterial and bactericidal effects against a variety of bacteria and can effectively inhibit and remove biofilms produced by bacteria; at the same time, it provides the application of the anti-biofilm peptide and products containing the anti-biofilm peptide.

[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0007] An anti-biomembrane peptide, VKLR, has the amino acid sequence SEQ ID NO: 1 as: LVKLRVKLRVKLR.

[0008] The application of the anti-biomembrane peptide VKLR in the preparation of broad-spectrum antibacterial agents, which target both Gram-negative and Gram-positive bacteria.

[0009] Preferably, the Gram-negative bacteria are selected from Escherichia coli and Pseudomonas aeruginosa.

[0010] Preferably, the Gram-positive bacteria are selected from Staphylococcus aureus and Staphylococcus epidermidis.

[0011] Preferably, the Escherichia coli includes E. coli 25922 and E. coli K88; the Pseudomonas aeruginosa includes P. aeruginosa 15442 and P. aeruginosa 27853.

[0012] Preferably, the Staphylococcus aureus includes S. aureus 25923 and S. aureus 6538; the Staphylococcus epidermidis includes S. epidermidis 43300 and S. epidermidis 49134.

[0013] A broad-spectrum antibacterial agent, the active ingredient of which is the aforementioned anti-biomembrane peptide VKLR.

[0014] Preferably, the broad-spectrum antibacterial agent is capable of killing bacteria in the presence of salt ions.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The anti-biomembrane peptide VKLR has broad-spectrum antibacterial activity and has significant antibacterial effects against a variety of Gram-negative and Gram-positive bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis, with a low minimum inhibitory concentration (1-4 μM).

[0017] (2) This peptide can effectively inhibit and remove bacterial biofilms, and has a clear inhibitory effect on the biofilms of Pseudomonas aeruginosa, Staphylococcus aureus, etc.

[0018] (3) It can retain antibacterial activity under simulated in vivo salt environment conditions, making it suitable for in vivo application scenarios;

[0019] (4) It has low cytotoxicity (92.4% survival rate of human embryonic kidney cells 293T at 128 μM concentration), low hemolytic activity (98.7% survival rate of bovine erythrocytes at 128 μM concentration), good biocompatibility, and potential for clinical application.

[0020] (5) It can be widely used in the preparation of broad-spectrum antibacterial agents, preservatives, antibacterial drugs or compositions, and has broad application prospects.

[0021] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application 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 this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The mass spectrum of the anti-membrane peptide VKLR of this invention (molecular weight consistent with the theoretical value of 1621.11);

[0024] Figure 2 : Liquid chromatogram of the anti-biomembrane peptide VKLR of this invention (purity > 95%);

[0025] Figure 3 Results of hemolytic activity assay of the anti-membrane peptide VKLR (red blood cell survival rate at different concentrations) of this invention;

[0026] Figure 4 The cytotoxicity test results of the anti-membrane peptide VKLR of this invention (survival rate of 293T cells at different concentrations). Detailed Implementation

[0027] Specific embodiments of the invention will now be described in detail. Although the invention is described in conjunction with these specific embodiments, it should be understood that the invention is not intended to be limited to these specific embodiments. Rather, these embodiments are intended to cover alternative, modified, or equivalent embodiments that may be included within the scope of the invention as defined by the claims. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. The invention may be practiced without some or all of these specific details. In other instances, well-known processes have not been described in detail in order not to unnecessarily obscure the invention.

[0028] When used in conjunction with the terms "comprising," "method comprising," or similar language in this specification and appended claims, the singular forms "a," "some," and "the" include plural references unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] The present invention will now be described in further detail with reference to the full text.

[0030] Combined with appendix Figures 1-4 This invention provides an anti-biofilm peptide that exhibits good antibacterial and bactericidal effects against bacteria and can inhibit and eliminate bacterial biofilms. Another objective of this invention is to provide applications of this anti-biofilm peptide and products containing it.

[0031] An anti-biofilm peptide and its applications are disclosed. The amino acid sequence of the anti-biofilm peptide is shown in SEQ ID NO: 1 (LVKLRVKLRVKLR). This anti-biofilm peptide exhibits broad-spectrum antibacterial activity, showing significant inhibitory effects against both Gram-negative and Gram-positive bacteria, including *Escherichia coli*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, and *Staphylococcus epidermidis*. It effectively inhibits and eliminates biofilms produced by these bacteria, retains its antibacterial activity even under simulated in vivo saline conditions, exhibits low cytotoxicity, and demonstrates good biocompatibility. It shows broad application prospects in the preparation of broad-spectrum antibacterial agents, preservatives, antibacterial drugs, or compositions.

[0032] An anti-membrane peptide, VKLR, has the amino acid sequence SEQ ID NO: 1 as: LVKLRVKLRVKLR (L: leucine; V: valine; K: lysine; R: arginine).

[0033] The application of the anti-biomembrane peptide VKLR in the preparation of broad-spectrum antibacterial agents, which target both Gram-negative and Gram-positive bacteria.

[0034] The Gram-negative bacteria were selected from Escherichia coli and Pseudomonas aeruginosa.

[0035] The Gram-positive bacteria are selected from Staphylococcus aureus and Staphylococcus epidermidis.

[0036] The *Escherichia coli* includes *E. coli* 25922 and *E. coli* K88; the *Pseudomonas aeruginosa* includes...

[0037] P. aeruginosa15442, P. aeruginosa27853.

[0038] The Staphylococcus aureus includes S. aureus 25923 and S. aureus 6538; the Staphylococcus epidermidis includes S. epidermidis 43300 and S. epidermidis 49134.

[0039] A broad-spectrum antibacterial agent, the active ingredient of which is the aforementioned anti-biomembrane peptide VKLR.

[0040] The broad-spectrum antibacterial agent can kill bacteria in the presence of salt ions.

[0041] Example 1: Synthesis of the anti-biomembrane peptide VKLR.

[0042] The anti-biomembrane peptide VKLR (amino acid sequence shown in SEQ ID NO: 1) was synthesized using a solid-phase chemical synthesis method. The specific steps are as follows:

[0043] 1. The preparation of antimicrobial peptides proceeded sequentially from the C-terminus to the N-terminus using a peptide synthesizer. First, Fmoc-X (where X is the first amino acid at the C-terminus of each antimicrobial peptide) was inscribed into Wang resin, and then the Fmoc group was removed to obtain X-Wang resin. Next, Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxylic acid-trimethyl-Y, where Y is the second amino acid at the C-terminus of each antimicrobial peptide) was deprotected. This procedure was repeated sequentially from the C-terminus to the N-terminus until complete synthesis, yielding resin with the side chains protected by the removed Fmoc group.

[0044] 2. Cutting and purification: Add cutting reagent (TFA:water:TIS = 95:2.5:2.5, mass ratio) to peptide resin, react at 20℃ in the dark for 2 hours, and filter; wash the precipitate with TFA, combine the filtrate and washings, concentrate by rotary evaporation, add 10 times the volume of pre-cooled anhydrous diethyl ether, precipitate at -20℃ for 3 hours, a white powder will precipitate, centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous diethyl ether, and vacuum dry to obtain crude peptide.

[0045] 3. Purification: Equilibrate a C18 reverse-phase atmospheric pressure column for 30 min with 0.2 mol / L sodium sulfate solution (adjust pH to 7.5 with phosphoric acid). Dissolve the crude peptide in 90% acetonitrile aqueous solution and filter. Use gradient elution (methanol:sodium sulfate aqueous solution = 30:70 to 70:30, volume ratio) at a flow rate of 1 mL / min and a detection wavelength of 220 nm. Collect the main peak and freeze-dry it. Then, further purify it through a reverse-phase C18 column (elution buffer A: 0.1% TFA / aqueous solution; elution buffer B: 0.1% TFA / acetonitrile solution, elution gradient of 25% B to 40% B for 12 min at a flow rate of 1 mL / min). Collect the main peak and freeze-dry it to obtain pure peptide.

[0046] 4. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray ionization mass spectrometry. The mass spectrum is shown in the figure. Figure 1 The molecular weight shown is basically consistent with the theoretical molecular weight of 1621.11; the purity of the antimicrobial peptide is greater than 95%. Figure 2 The liquid chromatography column was a Kromasil C18-5 (4.6 × 250 mm, 220 nm, 10 μL), using a non-linear gradient of water / acetonitrile (containing 0.1% trifluoroacetic acid) at a flow rate of 1.0 mL / min.

[0047] Example 2: Determination of the antibacterial activity of the anti-biomembrane peptide VKLR.

[0048] The minimum inhibitory concentration (MIC) was determined using the microdilution method, and the steps are as follows:

[0049] 1. Add PBS buffer and 0.2% fetal bovine serum albumin as diluents to a 96-well plate and prepare a series of peptide solutions using serial dilution to make the solution volume in each well 50 μL.

[0050] 2. Then add 50 μL of the test bacterial solution (approximately 10 μL). 5 (CFU / mL) was added to each well, and the culture medium was MHB;

[0051] 3. Incubate at 37℃ for 18–24 h, then measure the optical density at 492 nm using a microplate reader to determine the minimum inhibitory concentration (MIC) of peptide VKLR against bacteria. A value less than 0.1 is considered to indicate bacterial inhibition. Perform two replicates for each test, repeating three times.

[0052] The results are shown in Table 1. The anti-biomembrane peptide VKLR showed significant antibacterial activity against a variety of bacteria, demonstrating broad-spectrum antibacterial properties.

[0053] Table 1. Antibacterial activity (μM) of the anti-biomembrane peptide VKLR against bacteria.

[0054] strain Minimum inhibitory concentration E. coli 25922 2 E.coliK88 2 P. aeruginosa15442 2 P.aeruginosa27853 4 S.aureus25923 2 S.aureus6538 1 S.epidermidis43300 4 S.epidermidis49134 2

[0055] Example 3: Determination of the biomembrane inhibitory activity of the anti-biomembrane peptide VKLR.

[0056] The minimum biofilm inhibitory concentration was determined using the microdilution method, and the steps are as follows:

[0057] 1. In a 96-well plate, use the test bacterial suspension (approximately 10 μL) 8 Prepare a series of gradient peptide solutions using the CFU / mL serial dilution method, so that the solution volume in each well is 100 μL.

[0058] 2. Incubate at 37℃ for 24 hours. Then, remove the culture medium and wash three times with PBS (pH=7.4).

[0059] 3. Use crystal violet for biofilm assay. Finally, add 200 μL of 75% ethanol to dissolve the crystal violet, and measure the absorbance at 595 nm using a microplate reader. Perform two replicates for each test, repeating three times.

[0060] The results are shown in Table 2. The anti-biofilm peptide VKLR has a significant inhibitory effect on bacterial biofilms.

[0061] Table 2. Biofilm inhibitory activity of peptide VKLR against bacteria (μM)

[0062]

[0063]

[0064] Example 4: Determination of the hemolytic activity of the anti-membrane peptide VKLR.

[0065] 1. Take fresh bovine red blood cells and wash them with PBS (pH=7.4) until the supernatant is clear to prepare a 10% (v / v) red blood cell suspension;

[0066] 2. Prepare 10% (v / v) RBCs by resuspending them in PBS, and add serially diluted anti-biomembrane peptides to the suspension (1 mL);

[0067] 3. After incubation at 37℃ for 2 hours, the RBCs were centrifuged at 6000 rpm for 10 minutes, and the absorbance of the supernatant was measured at 570 nm.

[0068] Calculate the hemolysis rate: Hemolysis rate (%) = [(sample OD570 - negative control OD570) / (positive control OD570 - negative control OD570)] × 100% (negative control is PBS, positive control is deionized water).

[0069] The results are as follows Figure 3 As shown, when bovine erythrocytes were treated with a 128 μM concentration of the anti-membrane peptide VKLR, the cell survival rate was 98.7%, indicating that VKLR has low hemolytic activity in erythrocytes and has certain potential for clinical application.

[0070] Example 5: Cytotoxicity assay of the anti-membrane peptide VKLR.

[0071] The toxicity to human embryonic kidney cells 293T was determined using the MTT assay, and the steps are as follows:

[0072] 1. After thawing the human embryonic kidney cell line 293T cells that had been frozen in liquid nitrogen, the cells were inoculated into a culture medium containing 10% fetal bovine serum and passaged at 37°C and 5% CO2.

[0073] 2. Once the cells have grown to 80% coverage of the culture dish, add 2 mL of 0.25% trypsin to digest the cells;

[0074] 3. Adjust the cell concentration using culture medium to a final concentration of approximately 1×10⁻⁶. 5 Cells / well. After the cells have fully adhered, 50 μL of cell suspension was mixed with 50 μL of serially diluted anti-biomembrane peptide in a 96-well plate and incubated at 37°C and 5% CO2 for 6 h. Then, 25 μL of MTT (5 mg / mL) was added to each well and incubated for another 2 h.

[0075] 4. After incubation, discard the supernatant. Dissolve the crystals at the bottom of the wells in 150 μL of DMSO, and measure the absorbance at 570 nm using a microplate reader. Set up a positive control (containing cells but no anti-biomembrane peptides) and a negative control (containing only culture medium). The experiment was performed in duplicate, with three replicates.

[0076] Cell viability = [(OD570 of test wells - OD570 of negative control) / (OD570 of positive control - OD570 of negative control)] × 100%.

[0077] The results are as follows Figure 4 As shown, when human embryonic kidney cells 293T were treated with 128 μM of the anti-membrane peptide VKLR, the cell survival rate was 92.4%, indicating that VKLR has low cytotoxicity and has certain clinical application potential.

[0078] In summary, the anti-biofilm peptide VKLR provided by this invention has broad-spectrum antibacterial and anti-biofilm activities, good adaptability to salt environments, low toxicity, and broad application prospects.

[0079] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown throughout are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. An anti-membrane peptide VKLR, characterized in that, Its amino acid sequence SEQ ID NO:1 is: LVKLRVKLRVKLR.

2. The application of the anti-biomembrane peptide VKLR according to claim 1 in the preparation of a broad-spectrum antibacterial agent, characterized in that, The broad-spectrum antibacterial agent targets both Gram-negative and Gram-positive bacteria.

3. The application according to claim 2, characterized in that, The Gram-negative bacteria were selected from Escherichia coli and Pseudomonas aeruginosa.

4. The application according to claim 2, characterized in that, The Gram-positive bacteria are selected from Staphylococcus aureus and Staphylococcus epidermidis.

5. The application according to claim 3, characterized in that, The *Escherichia coli* includes *E. coli* 25922 and *E. coli* K88; the *Pseudomonas aeruginosa* includes *P. aeruginosa* 15442 and *P. aeruginosa* 27853.

6. The application according to claim 4, characterized in that, The Staphylococcus aureus includes S. aureus 25923 and S. aureus 6538; the Staphylococcus epidermidis includes S. epidermidis 43300 and S. epidermidis 49134.

7. A broad-spectrum antibacterial agent, characterized in that, Its active ingredient is the anti-biomembrane peptide VKLR as described in claim 1.

8. The broad-spectrum antibacterial agent according to claim 7, characterized in that, The broad-spectrum antibacterial agent can kill bacteria in the presence of salt ions.

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