Antibiofilm peptides and uses thereof

Abstract

The application discloses an antibiofilm peptide and application thereof. The antibiofilm peptide P21 has an amino acid sequence as shown in SEQ ID NO. 1. The antibiofilm peptide P21 has the advantages of small molecular weight, simple artificial synthesis and activity of resisting biofilm formation of drug-resistant bacteria, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an anti-biofilm peptide and its application in inhibiting bacterial biofilms. Background Technology

[0002] With the shortage of new antibiotics, the emergence and spread of multidrug-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) pose a serious threat to public health and the food industry, representing a critical global problem. Biofilms are complex community structures formed by bacteria and their secreted extracellular polysaccharides, proteins, and eDNA, acting as natural barriers to protect bacteria from adverse environments and to resist attacks from drugs and the host's immune system. Persistent bacteria within biofilms exhibit significantly higher tolerance to drugs and host immunity compared to planktonic bacteria, and are a major cause of persistent infections in humans and animals, as well as contamination of medical devices and food. Furthermore, biofilms promote the transfer of resistance genes between internal strains, leading to the emergence of more drug-resistant strains. Therefore, biofilms are one of the main mechanisms underlying the hyperdrug resistance of pathogens such as MRSA at the population level, urgently requiring the development of effective biofilm control strategies. Summary of the Invention:

[0003] The present invention aims to provide an anti-biofilm peptide P21 and its application in inhibiting bacterial biofilms.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention proposes an anti-biomembrane peptide P21, which consists of 21 amino acid residues and has a molecular weight of 2306.55 Da. Its amino acid sequence is shown in SEQ ID NO.1, specifically: Val-Ala-Leu-Tyr-Tyr-Ala-Pro-Gly-Thr-Gly-Ile-Ala-Trp-Ala-Asp-Ala-Trp-Glu-Arg-Val-Pro.

[0006] The present invention further proposes a method for preparing the above-mentioned anti-biomembrane peptide P21, characterized in that the full sequence is synthesized using an automated peptide synthesizer according to the amino acid sequence shown in SEQ ID NO.1, and then purified by HPLC reverse-phase column chromatography for desalting.

[0007] Furthermore, the present invention proposes the application of the above-mentioned anti-biomembrane peptide P21 in antibacterial drugs.

[0008] Preferably, the bacteria is Staphylococcus aureus.

[0009] Further preferred, the bacteria are methicillin-resistant Staphylococcus aureus.

[0010] The anti-biofilm peptide P21 in this invention has the advantages of small molecular weight, simple artificial synthesis, and anti-biofilm formation activity of drug-resistant bacteria, and has broad application prospects. Attached Figure Description

[0011] Figure 1 Mass spectrometry results of anti-membrane peptide P21.

[0012] Figure 2 : The inhibitory activity of anti-biomembrane peptide P21 on biomembrane formation.

[0013] Figure 3 Synergistic antibacterial effect of anti-biomembrane peptide P21 and vancomycin. CK was the blank control group; P21 was the P21 treatment group; Van was the vancomycin treatment group; and Van+P21 was the P21 and vancomycin synergistic treatment group. Detailed Implementation

[0014] The following are specific implementation examples of the present invention. It should be noted that these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Modifications and substitutions to the details and form of the implementation schemes made within the scope and spirit of the present invention all fall within the protection scope of the present invention.

[0015] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0016] Example 1: Preparation of anti-membrane peptide P21

[0017] The anti-biomembrane peptide consists of 21 amino acid residues with a molecular weight of 2306.55 Da. Its amino acid sequence is shown in SEQ ID NO.1, specifically: Val-Ala-Leu-Tyr-Tyr-Ala-Pro-Gly-Thr-Gly-Ile-Ala-Trp-Ala-Asp-Ala-Trp-Glu-Arg-Val-Pro, and is named anti-biomembrane peptide P21. Based on the amino acid sequence, its full sequence was synthesized using an automated peptide synthesizer via solid-phase synthesis, and purified by HPLC reversed-phase column chromatography. Its purity was identified by high-performance liquid chromatography, its molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS), and its amino acid sequence was determined using an automated amino acid sequencer.

[0018] Depend on Figure 1 It can be seen that the synthesized polypeptide matches the predicted molecular weight.

[0019] Example 2: Anti-biomembrane activity of anti-biomembrane peptide P21

[0020] Methicillin-resistant Staphylococcus aureus (MRSA) ATCC43300 cultured overnight in LB medium was diluted 1:100 with fresh M63 medium (3 g / L KH₂PO₄, 7 g / L K₂HPO₄, 2 g / L (NH₄)₂SO₄, 1 mM MgSO₄, 0.2% glucose, 0.5% tyrosine, 0.4% arginine). The diluted bacterial culture was transferred to 100 μL per well of a 96-well plate. Different concentrations of P21 (0.018 mg / mL, 0.037 mg / mL, 0.075 mg / mL, 0.15 mg / mL, 0.3 mg / mL) were added to the experimental groups, while no P21 was added to the control group. Eight replicates were set up for each group, and the plates were incubated at 37°C for 48 h.

[0021] After culture, discard the culture medium and suspended cells. Gently wash the culture plate twice with sterile water and drain. Add 125 μL of 0.1% crystal violet staining solution to each well and incubate at room temperature for 10-15 min. Gently wash the culture plate 3-4 times with sterile water and air dry. Add 125 μL of 30% glacial acetic acid to each well and incubate at room temperature for 10-15 min. Measure the absorbance at 550 nm using a spectrophotometer, with 30% glacial acetic acid as a blank control.

[0022] The results are as follows Figure 2 As shown, at concentrations of 0.037–0.3 mg / mL, P21 can inhibit the formation of biofilms from methicillin-resistant Staphylococcus aureus ATCC43300.

[0023] Example 3: Synergistic antibacterial activity of anti-membrane peptide P21 with other antibacterial drugs

[0024] Methicillin-resistant Staphylococcus aureus (ATCC43300) cultured overnight in LB medium was inoculated into 0.8 mL of fresh LB medium at a 1% inoculation rate. The P21 treatment group received P21 at a final concentration of 0.2 mg / mL, the vancomycin treatment group received vancomycin at a final concentration of 1.5 mg / L, and the P21 and vancomycin co-treatment group received both P21 and vancomycin at a final concentration of 0.2 mg / mL and 1.5 mg / L, respectively. The control group received the corresponding volume of LB medium. Each group was divided into three replicates. After incubation at 37°C in a metal bath at 800 rpm for 15 h, the OD (exposure potential) was measured. 600 .

[0025] The results are as follows Figure 3 As shown, P21 has a synergistic antibacterial effect with vancomycin.

Claims

1. An anti-biomembrane peptide P21, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing the anti-membrane peptide P21 according to claim 1, characterized in that, The complete sequence of the peptide was synthesized using an automated peptide synthesizer based on the amino acid sequence shown in SEQ ID NO. 1, and then purified by desalting using HPLC reversed-phase column chromatography.

3. The use of the anti-biomembrane peptide P21 according to claim 1 in the preparation of antibacterial drugs, wherein the bacteria is methicillin-resistant Staphylococcus aureus.

Images