A stable self-assembling antimicrobial peptide RW7, its preparation method and applications

Abstract

A stable self-assembling antimicrobial peptide RW7, its preparation method and application, which relate to the field of biotechnology. The self-assembling antimicrobial peptide RW7 of the present invention is Arg-Trp-Trp-Arg-Trp-Arg-Trp-NH2. It is used for preparing drugs for treating infectious diseases caused by Gram-negative bacteria and Gram-positive bacteria. The stable self-assembling antimicrobial peptide RW7 of the present invention only contains 7 amino acid sequences and does not require complex chemical modification; the antimicrobial peptide RW7 provided by the present invention has excellent inhibitory effects on aquatic pathogenic bacteria. It also has excellent inhibitory effects on common Gram-negative bacteria and Gram-positive bacteria. It has strong inhibitory effects on Escherichia coli, Aeromonas hydrophila, etc., has almost no hemolysis on red blood cells, and has strong stability under physiological salt ion conditions. It has the potential to become an alternative to antibiotics.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a stable self-assembled antibacterial peptide RW7, a preparation method thereof, and an application thereof. Background Art

[0002] In recent years, high-density intensive farming and water eutrophication have increased the susceptibility of fish to pathogenic bacteria. After fish are infected with aquatic pathogenic bacteria, it can cause explosive diseases of large-scale cultured fish and cause huge economic losses. At present, the treatment plan for aquatic animal bacteria in China mainly relies on antibiotics. However, the long-term irregular use of antibiotics will cause the enhancement of bacterial drug resistance and even lead to the outbreak of super bacteria. At the same time, with the increase in the use of antibiotics, the immune system of aquatic animals is likely to be damaged, and ultimately it cannot resist the spread of diseases, resulting in a large number of fish deaths. More seriously, drug residues may also seriously endanger the safety and health of humans through aquatic products. Therefore, it is extremely urgent to find new, green, safe, and alternative antibiotics.

[0003] As a part of the self-immunity of animals and plants, antibacterial peptides have the functions of wide distribution, broad-spectrum antibacterial activity, and immune enhancement. Due to their antibacterial mechanism different from that of traditional antibiotics, the use of antibacterial peptides is not likely to lead to the generation of drug-resistant strains. Therefore, antibacterial peptides are considered to be the most potential and promising antibiotic alternatives. However, at present, very few antibacterial peptides are applied to aquatic animals. The main reasons restricting their application are as follows: First, the potential cytotoxicity of antibacterial peptides. The positive charge and amphiphilic structure that endow antibacterial peptides with antibacterial activity often cause cytotoxicity of antibacterial peptides to mammalian cells and produce toxic and side effects on animals. Second, the instability of natural antibacterial peptides under physiological conditions. Although many antibacterial peptides have shown effective antibacterial activity in vitro, the huge difference between their antibacterial effects in vivo and in vitro is still the main problem hindering the application of antibacterial peptides. Generally speaking, natural antibacterial peptides are extremely sensitive to proteases, plasma, salts, pH, etc. and are easily decomposed in vivo. This is because antibacterial peptides mainly utilize the electrostatic interaction with the bacterial cell membrane to fold into a higher-order conformation, and the ionic strength in the solution will affect the stability of the higher-order structure of antibacterial peptides. Third, the synthesis cost of antibacterial peptides is relatively high. The peptide chains of natural antibacterial peptides are often relatively long, and the longer the peptide chain, the higher the synthesis cost. Due to the above limitations and deficiencies of natural antibacterial peptides, it is necessary to design new antibacterial peptides that are economical, efficient, stable, and safe.

[0004] CN104292301A, the invention title "A Small Molecule Synthetic Antibacterial Peptide and Its Preparation Method and Application" discloses that the cationic antibacterial peptide sequence is: SQ1: R-W-W-R-F-NH2 (Arg-Arg-Trp-Trp-Arg-NH2), and the preparation method is solid-phase chemical synthesis. The small molecule synthetic antibacterial peptide of the present invention has broad-spectrum killing activity against Gram-positive bacteria and Gram-negative bacteria, and stronger bactericidal activity than natural antibacterial peptides; it has a simple structure, is convenient for artificial synthesis, and does not require any modification and connection; moreover, it has low hemolytic toxicity and no toxic effect on animal and plant cells, and has important value in the development and application of antibacterial drugs.

[0005] CN106916205B, the invention title "An Antibacterial Hexapeptide" discloses the sequence: Arg-Arg-Trp-Trp-Arg-Trp. It is applied to the preparation of drugs for treating or preventing bacterial infections. The present invention also provides an antibacterial hexapeptide derivative, and the sequence of this antibacterial hexapeptide derivative is: Arg-Arg-Trp-Trp-Arg-Trp-β-phenethylamine. It is applied to the preparation of drugs for treating or preventing bacterial infections. The bacterium is drug-resistant Acinetobacter baumannii. The artificially designed and synthesized antibacterial hexapeptide and its phenethylamine modification provided by the present invention can be conveniently obtained by solid-phase synthesis. The synthetic antibacterial peptide and its derivative have broad-spectrum killing activity against Gram-positive bacteria and Gram-negative bacteria. In particular, its derivative shows stronger antibacterial activity against drug-resistant Acinetobacter baumannii, has extremely low hemolytic toxicity, and can be applied to drugs for treating or preventing diseases caused by drug-resistant Acinetobacter baumannii.

[0006] The antibacterial peptides of the above patents have no obvious inhibitory effect on aquatic pathogenic bacteria. Therefore, they are not suitable as inhibitors of fish pathogenic bacteria. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides an antibacterial peptide RW7 that is economical, efficient, stable and safe and its application.

[0008] A highly efficient and stable self-assembled antibacterial peptide RW7 of the present invention, the amino acid sequence of the antibacterial peptide RW7 is as follows: Arg-Trp-Trp-Arg-Trp-Arg-Trp-NH2.

[0009] Method for preparing a highly efficient and stable self-assembled antibacterial peptide RW7 of the present invention:

[0010] (1) Design a short peptide sequence containing 7 amino acids, using XYYXYXY-NH2 as the simplest sequence model, where X is a positively charged basic amino acid, and Y is a hydrophobic amino acid with different R group structures;

[0011] (2) The above-mentioned polypeptide was synthesized by solid-phase chemical synthesis method. After being identified by mass spectrometry, the preparation of the polypeptide was completed;

[0012] Furthermore, the self-assembling antimicrobial peptide RW7 constructs a hydrophobic core with tryptophan (Trp), isoleucine (Ile), leucine (Leu) and phenylalanine (Phe), and provides positive charges with arginine (Arg), lysine (Lys) and histidine (His).

[0013] Furthermore, the C-terminus of the peptide chain of the self-assembling antimicrobial peptide RW7 is amidated with -NH2.

[0014] Application of a highly efficient and stable self-assembling antimicrobial peptide RW7 of the present invention, the application of the self-assembling antimicrobial peptide RW7 in the preparation of drugs for treating infectious diseases caused by Gram-negative bacteria and Gram-positive bacteria.

[0015] Furthermore, the application of the self-assembling antimicrobial peptide RW7 in the preparation of drugs for treating infectious diseases caused by aquatic pathogenic bacteria.

[0016] Furthermore, the Gram-negative bacteria and Gram-positive bacteria include several of Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila, Staphylococcus aureus, Bacillus subtilis, Aeromonas sobria, Aeromonas veronii, Aeromonas salmonicida.

[0017] The present invention has the following beneficial effects:

[0018] The highly efficient and stable self-assembling antimicrobial peptide RW7 of the present invention only contains 7 amino acid sequences and does not require complex chemical modification; the antimicrobial peptide RW7 provided by the present invention has excellent inhibitory effects on aquatic pathogenic bacteria, and also has excellent inhibitory effects on common Gram-negative bacteria and Gram-positive bacteria. It has strong inhibitory effects on Escherichia coli, Aeromonas hydrophila, etc., has almost no hemolysis on red blood cells, and has strong stability under physiological salt ion conditions. It has the potential to become an alternative to antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Reverse-phase high performance liquid chromatography chart of antimicrobial peptide RW7;

[0020] Figure 2 Mass spectrometry chart of antimicrobial peptide RW7. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] In order to make the purposes, technical solutions and advantages of the embodiments of the present invention clearer and more understandable, the spirit of the content disclosed by the present invention will be described in detail below. After any person skilled in the art in the technical field understands the embodiments of the content of the present invention, the techniques taught by the content of the present invention can be changed and modified, which does not deviate from the spirit and scope of the content of the present invention.

[0022] Schematic embodiments of the present invention and their descriptions are used to explain the present invention, but not to limit the present invention.

[0023] Example 1

[0024] Design and synthesis of antimicrobial peptides

[0025] Based on the "cationic amphiphilic" principle, a short peptide sequence containing only 7 amino acids was de novo designed, with XYYXYXY-NH2 (XY7) as the simplest sequence model. Among them, X is a positively charged basic amino acid, and Y is a hydrophobic amino acid with different R-group structures; Tryptophan (Trp), Isoleucine (Ile), Leucine (Leu), and Phenylalanine (Phe) were selected to construct the hydrophobic core, and Arginine (Arg), Lysine (Lys), and Histidine (His) with different isoelectric points were selected to provide positive charges. The C-terminus of the peptide chain was amidated with -NH2 to further increase stability. The above-mentioned polypeptide was synthesized by solid-phase chemical synthesis, and the synthesized polypeptide was purified and identified by reverse-phase high-performance liquid chromatography, and the polypeptide purity > 95%. The amino acid sequence of antimicrobial peptide RW7 is as follows:

[0026] Arg-Trp-Trp-Arg-Trp-Arg-Trp-NH2

[0027] The structural formula of antimicrobial peptide RW7 is as follows:

[0028]

[0029] Example 2

[0030] Determination of biological activity of antimicrobial peptides

[0031] (1) Determination of antibacterial activity: The minimum inhibitory concentration of the antimicrobial peptide was determined by the standard broth microdilution method. The bacteria were incubated until the logarithmic growth phase and diluted to 10 5 CFU / mL. 50 μL of antimicrobial peptides with different concentrations (the final concentration of the peptide was 1 - 128 μmol / L) and an equal volume of the diluted bacterial suspension were added to each well of a 96-well plate and incubated at 37 °C for 18 - 24 hours. Among them, the culture medium containing bacteria was used as the positive control, and the uninoculated culture medium was used as the negative control. The absorbance was measured at OD = 492 nm with an enzyme-linked immunosorbent assay reader to determine the minimum inhibitory concentration. Three independent replicate experiments were carried out, with 2 parallels for each replicate. The results are shown in Table 1.

[0032] Table 1 Minimum inhibitory concentration of antimicrobial peptides (μmol / L)

[0033]

[0034] As can be seen from Table 1, the antimicrobial peptide RW7 exhibits good antibacterial activity against common pathogenic bacteria.

[0035] (2) Determination of hemolytic activity: Collect fresh and healthy fish red blood cell suspension and store it in a heparin sodium anticoagulant tube. Centrifuge the collected fresh blood at 1000 g for 10 minutes, collect the red blood cells, wash them and resuspend them in PBS buffer (pH = 7.4). Mix 50 μL of antimicrobial peptides at different concentrations (the final concentration of the peptide is 1 - 512 μmol / L) with an equal volume of red blood cell suspension and place them in each well of a 96-well plate. After incubation at 37 °C for a certain number of hours, centrifuge at 1000 g for a certain number of minutes, transfer 50 μL of the supernatant to a new 96-well plate, and measure the absorbance value at OD = 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader to monitor the amount of hemoglobin released. Use red blood cells dissolved in 0.1% Triton X-100 and PBS buffer as positive and negative controls respectively. The hemolysis rate calculation formula is as follows:

[0036] Hemolysis rate (%) = (OD measured value - OD negative control) / (OD positive control - OD negative control) × 100%

[0037] Define the concentration of the peptide corresponding to 10% hemolytic activity as the minimum hemolytic concentration. The results are shown in Table 2.

[0038] Table 2 Hemolytic activity of antimicrobial peptides (μmol / L)

[0039]

[0040] As can be seen from Table 2, the antimicrobial peptide did not exhibit hemolytic activity within the detection range.

[0041] Example 3

[0042] Determination of the physiological stability of antimicrobial peptides

[0043] To detect the stability of the polypeptide under physiological conditions, Escherichia coli ATCC 25922 was used as a Gram-negative bacterial model. In the salt ion stability experiment, salt powder was dissolved in 0.2% polypeptide dilution solution to evaluate the change in the minimum inhibitory concentration of the polypeptide. The specific method is as described in Example 2. The final concentrations of salt ions are as follows: NaCl, 150 mmol / L; KCl, 4.5 mmol / L; NH4Cl, 6 mmol / L; CaCl2, 2 mmol / L; ZnCl2, 8 mmol / L; MgCl2, 1 mmol / L; and FeCl3, 4 mmol / L. The results are shown in Table 3.

[0044] Table 3 Minimum inhibitory concentration of antimicrobial peptide RW7 against Escherichia coli ATCC 25922 under physiological conditions (μmol / L)

[0045]

[0046] As can be seen from Table 3, the antibacterial activity of antibacterial peptide RW7 is less affected by physiological conditions, and the change in its minimum inhibitory concentration is within 4-fold. Especially under the conditions of CaCl2 and ZnCl2, the minimum inhibitory concentration of antibacterial peptide RW7 decreases, indicating that antibacterial peptide RW7 has a strong resistance to salt ions.

Claims

1. A stable self-assembling antimicrobial peptide RW7, characterized in that The amino acid sequence of the antimicrobial peptide RW7 is as follows: Arg-Trp-Trp-Arg-Trp-Arg-Trp-NH2.

2. A method for preparing a stable self-assembled antibacterial peptide RW7 according to claim 1, characterized in that The method is carried out as follows: (1) Design a short peptide sequence containing 7 amino acids, using XYYXYXY-NH2 as the simplest sequence model, where X is a positively charged basic amino acid and Y is a hydrophobic amino acid with different R-group structures; (2) Synthesize the short peptide sequence containing 7 amino acids by solid-phase chemical synthesis method. After mass spectrometry identification, the preparation of the polypeptide is completed.

3. Application of a stable self-assembling antimicrobial peptide RW7 according to claim 1, characterized in that The application of the self-assembled antimicrobial peptide RW7 in the preparation of drugs for treating infectious diseases caused by Gram-negative bacteria and Gram-positive bacteria; the Gram-negative bacteria and Gram-positive bacteria are Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila, Staphylococcus aureus, Bacillus subtilis, Aeromonas sobria, Aeromonas veronii or Aeromonas salmonicida.

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