Short-chain antibacterial peptide vklr, preparation method and application thereof

Abstract

The application discloses a short-chain antibacterial peptide VKLR and a preparation method and application thereof, and belongs to the technical field of agricultural livestock and veterinary application. The amino acid sequence of the short-chain antibacterial peptide VKLR is shown as SEQ ID No. 1. The VKLR is a short-chain antibacterial peptide with excellent antibacterial activity and is composed of natural amino acids. The antibacterial peptide has a significant bacteriostatic effect and does not show cytotoxicity in an in-vitro cytotoxicity experiment, and has extremely high safety. The preparation process of the antibacterial peptide is mature, the acquisition channel is convenient, and the antibacterial peptide can be produced through an engineering bacteria expression system.

Description

Technical Field

[0001] The present invention relates to the field of agricultural, animal husbandry and veterinary application technology, and in particular to a short-chain antimicrobial peptide VKLR and a preparation method and application thereof. Background Art

[0002] Antibiotics, one of the most important medical discoveries of the 20th century, have saved countless lives and made significant contributions to the prevention and treatment of infectious diseases. Approximately 70% of antimicrobial drugs currently used in human medicine were obtained during this golden age of antibiotic discovery, with the majority isolated from actinomycetes and fungi. However, since the 1970s, it has become increasingly difficult to isolate promising antibiotics from microbial metabolites using pure microbial culture methods under laboratory conditions. Currently, antimicrobial drug research and development focuses on modifying or optimizing combinations of known compounds. Amidst the increasing challenges of antibiotic discovery, bacterial resistance to antibiotics has been increasing annually. Antibiotic resistance can enable bacteria to tolerate clinically effective concentrations of antibiotics, rendering them ineffective. It is predicted that by 2050, the number of deaths caused by multidrug resistance will reach 10 million, surpassing cancer as a leading cause of death worldwide. Therefore, controlling the development of drug resistance is urgent.

[0003] Antimicrobial peptides, as naturally derived biomaterials, have recently become popular candidates for antimicrobial agents, particularly as alternatives to veterinary antibiotics, due to their unique mechanisms for disrupting pathogen biofilms. However, naturally occurring antimicrobial peptides suffer from a range of issues, including weak bioactivity and high toxicity, which severely limit and hinder their translational applications. Therefore, the development of antimicrobial peptides with high bioactivity and low toxicity is of great significance. Summary of the Invention

[0004] The present invention aims to provide a short-chain antimicrobial peptide (VKLR), its preparation method, and its use, to address the aforementioned problems of the prior art. The present invention provides an antimicrobial peptide (VKLR) with high antimicrobial activity. Comprising only 13 amino acids, it offers low synthesis costs and excellent broad-spectrum antimicrobial activity. This antimicrobial peptide not only exhibits significant antimicrobial effects but also exhibits no cytotoxicity in in vitro cytotoxicity experiments, demonstrating its exceptional safety.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] Technical solution 1: A highly active short-chain antimicrobial peptide VKLR, whose amino acid sequence is shown in SEQ ID NO.1, and its amino acid sequence is as follows: LVKLRVKLRVKLR.

[0007] The present invention screened the peptides using the following technical scheme: 1) collecting all antimicrobial peptide sequences with antimicrobial function from the antimicrobial peptide database (https: / / aps.unmc.edu / AP / ); 2) statistically analyzing the amino acid ratio, secondary structure, sequence length, positive charge number, hydrophobicity, and adjacent amino acid richness of all sequences; 3) rationally designing and synthesizing a batch of antimicrobial peptides based on the above parameters; and 4) screening an antimicrobial peptide VKLR with excellent antimicrobial activity and minimal cytotoxicity based on in vitro antimicrobial activity, cytotoxicity, and hemolytic activity.

[0008] Technical Solution 2: The use of the highly active short-chain antimicrobial peptide VKLR in any of the following items, including:

[0009] (1) Used to prepare broad-spectrum antibacterial agents;

[0010] (2) Used in the preparation of preservatives;

[0011] (3) Used for preparing antibacterial drugs or antibacterial compositions;

[0012] (4) Used for preparing medical dressings.

[0013] Furthermore, the antibacterial activity includes anti-Gram-negative bacteria and / or anti-Gram-positive bacteria.

[0014] Furthermore, the antibacterial agent includes anti-Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and / or Staphylococcus epidermidis.

[0015] Technical solution three: A broad-spectrum antibacterial agent, which contains the highly active short-chain antibacterial peptide VKLR.

[0016] Technical solution 4: A preservative, comprising the highly active short-chain antimicrobial peptide VKLR.

[0017] Technical Solution 5: An antibacterial drug or antibacterial composition, which contains the highly active short-chain antibacterial peptide VKLR.

[0018] Technical Solution 6: A medical dressing comprising the highly active short-chain antimicrobial peptide VKLR.

[0019] Technical Solution 7: Application of the highly active short-chain antimicrobial peptide VKLR in the preparation of medical imaging reagents, daily chemical cleaning products and / or medical devices with antimicrobial effects.

[0020] The present invention discloses the following technical effects:

[0021] Based on an antimicrobial peptide database, this invention synthesized a highly active short-chain antimicrobial peptide, VKLR, through database filtering technology and computer-aided design. By optimizing the amino acid arrangement, it maintains high antimicrobial activity while minimizing its hemolytic activity. Results from the examples show that the antimicrobial peptide of this invention not only exhibits significant antibacterial effects but also exhibits no cytotoxicity in in vitro cytotoxicity experiments, demonstrating its extremely high safety profile. The antimicrobial peptide of this invention has a mature preparation process, is readily available, and can be produced through fermentation using an engineered bacterial expression system. 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. 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 is the mass spectrum of the antimicrobial peptide VKLR;

[0024] Figure 2 is the liquid chromatogram of the antimicrobial peptide VKLR;

[0025] Figure 3 This is the bactericidal kinetic curve of the antimicrobial peptide VKLR against Pseudomonas aeruginosa;

[0026] Figure 4 This is the bactericidal kinetic curve of the antimicrobial peptide VKLR against Staphylococcus aureus;

[0027] Figure 5 This is a bar graph showing the effect of the antimicrobial peptide VKLR on bacterial outer membrane permeability;

[0028] Figure 6 The bar graph shows the affinity of the antimicrobial peptide VKLR to LPS / LTA;

[0029] Figure 7 This is the result of the antimicrobial peptide VKLR depolarizing the inner membrane of Pseudomonas aeruginosa;

[0030] Figure 8 This is the result of the antimicrobial peptide VKLR depolarizing the inner membrane of Staphylococcus aureus;

[0031] Figure 9 This is the result of the antimicrobial peptide VKLR on the hemolysis rate of red blood cells. DETAILED DESCRIPTION

[0032] The following detailed description of various example embodiments of the application will not be considered limiting of the application, but rather as a description of certain aspects, features and embodiments of the application.

[0033] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value by each intervening value, as well as any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these intervening values can independently be included or excluded in the range, and each smaller range that falls within the ambit of the recited ranges is also encompassed. These smaller ranges are not necessarily provided in this disclosure unless otherwise specified.

[0034] Unless defined otherwise, 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 application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0035] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples presented herein are illustrative only and should not be considered limiting.

[0036] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0037] Example 1 Design of antibacterial peptide

[0038] (1) Collect all antibacterial peptide sequences with antibacterial function in the antibacterial peptide database (https: / / aps.unmc.edu / AP / ); (2) Statistics of the proportion of amino acids, secondary structure, sequence length, positive charge number, hydrophobicity and adjacent amino acid richness of all sequences; (3) According to the above parameters, a batch of antibacterial peptides are designed and synthesized; (4) According to the in vitro antibacterial activity, cytotoxicity, hemolytic activity, an antibacterial peptide VKLR with excellent antibacterial activity and the lowest cytotoxicity is screened out. Figure 1 Figure 1 is the mass spectrum of antibacterial peptide VKLR; Figure 2 Figure 2 is the liquid chromatogram of antibacterial peptide VKLR. The sequence and physicochemical parameters of antibacterial peptide VKLR are shown in Table 1.

[0039] Table 1

[0040] peptides sequence Theoretical molecular weight Actual molecular weight Charge number hydrophobicity hydrophobic moment VKLR LVKLRVKLRVKLR (SEQ ID NO. 1) 1621.14 1621.11 +6 0.343 0.322

[0041] Example 2 Synthesis of antimicrobial peptide VKLR by solid phase chemical synthesis

[0042] The synthesis method is as follows:

[0043] The polypeptides of the present invention are all prepared by solid phase synthesis:

[0044] (1) The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus using a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) is connected to the Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); according to this procedure, the synthesis is carried out from the C-terminus to the N-terminus until the synthesis is completed, and the side chain protection resin with the Fmoc group removed is obtained;

[0045] (2) Add a cleavage reagent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter; wash with TFA (trifluoroacetic acid) to precipitate, mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, and then add about 10 times the volume of pre-cooled anhydrous ether. Precipitate at -20°C for 3 hours to precipitate a white powder. Centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain a polypeptide, wherein the cleavage reagent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0046] (3) Use 0.2M sodium sulfate (adjusted to pH 7.5 with phosphoric acid) to balance the column for 30 minutes, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, and use C18 reverse phase atmospheric pressure column, gradient elution (eluent is methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), flow rate of 1 mL / min, detection wave of 220 nm, collect the main peak, and freeze-dry; then use reverse phase C18 column for further purification, eluent A is 0.1% TFA / water solution; eluent B is 0.1% TFA / acetonitrile solution, elution concentration is 25%B to 40%B, elution time is 12 minutes, flow rate of 1 mL / min, collect the main peak as above, and freeze-dry;

[0047] (4) Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray mass spectrometry. The molecular weight ( Figure 1 ) is basically consistent with the theoretical molecular weight, and the purity of the antimicrobial peptide is greater than 95%.

[0048] Example 3 Determination of the antimicrobial activity of protease-resistant antimicrobial peptide VKLR

[0049] The minimum inhibitory concentration (μg / mL) of the antimicrobial peptide VKLR was determined using the microdilution method. A diluent containing 0.01% acetic acid and 0.2% fetal bovine serum albumin was added to a 96-well plate, and a series of antimicrobial peptide solutions were prepared using a two-fold dilution method. 100 μL of the above solution was placed in a 96-well cell culture plate, and then an equal volume of the test bacteria solution (~10 5 CFU / mL) in each well. Set up positive control (containing bacterial solution but not antimicrobial peptide) and negative control (containing neither bacterial solution nor antimicrobial peptide) respectively. Incubate at 37℃ for 14-18h and read the sample at 492nm (OD 492nm ) was measured to determine the minimum inhibitory concentration (MIC). A value less than 0.1 was considered to be inhibitory. Each test was repeated three times in duplicate. The antibacterial activity of the peptide VKLR against bacteria is detailed in Table 2.

[0050] Table 2

[0051] strains Minimum inhibitory concentration Escherichia coli 25922 2 Escherichia coli K88 2 Pseudomonas aeruginosa 15442 4 Pseudomonas aeruginosa 27853 4 Staphylococcus aureus 25923 2 Staphylococcus aureus 6538 2 Staphylococcus aureus 43300 4 Staphylococcus epidermidis 49134 2

[0052] From the results in Table 2, it can be seen that the antimicrobial peptide VKLR has a low minimum inhibitory concentration against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis, showing excellent broad-spectrum antibacterial activity.

[0053] Example 4 Bactericidal kinetics determination of antimicrobial peptide VKLR

[0054] Pseudomonas aeruginosa ATCC 15442 in the logarithmic growth phase was diluted to (1×10 5 )CFU mL -1 , and in PBS (10 × 10 -3 M, pH = 7.4) and the concentration was 4×10 6 , 8×10 6 and 16×10 6 Similarly, Staphylococcus aureus ATCC 6538 cells in the logarithmic growth phase were diluted to (1×10 6 )CFU mL -1 and with a concentration of 2×10 6 , 4×10 6 and 8×10 6 M of the antimicrobial membrane peptide in PBS (10 × 10 -3M, pH = 7.4). Samples were taken at different time intervals (0, 3, 5, 15, 30, 60 and 120 minutes) and diluted to the appropriate multiple. The diluted bacterial suspension was spread on Mueller-Hinton agar (MHA) plates. After the plates were incubated in a 37°C incubator for 24 hours, the bacterial colonies were counted. Each test had two parallel items and was repeated three times independently.

[0055] Depend on Figure 3 It can be seen that the antimicrobial peptide VKLR can kill 99.9% of Pseudomonas aeruginosa within 15 minutes at the bactericidal concentration. Figure 4 It can be seen that at the bactericidal concentration, the antimicrobial peptide VKLR takes 120 minutes to kill 99.9% of Staphylococcus aureus. This is because Gram-positive bacteria have a dense peptidoglycan layer, which to some extent hinders the antimicrobial peptide VKLR from breaking down the membrane. In summary, the antimicrobial peptide VKLR has a stronger antibacterial effect on Gram-negative bacteria than on Gram-positive bacteria.

[0056] Example 5 Analysis of the Permeability of the Antimicrobial Peptide VKLR to the Outer Membrane of Pseudomonas aeruginosa

[0057] The outer membrane permeability of Pseudomonas aeruginosa ATCC15442 induced by antimicrobial membrane peptides was measured using the fluorescent probe N-phenyl-1-naphthylamine (NPN). -3 Dilute the logarithmic phase Pseudomonas aeruginosa ATCC 15442 with 4-M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer until the OD at 600 nm is 0.2. Then, add the final concentration of 10×10 -6 M fluorescent dye NPN was added to the Pseudomonas aeruginosa bacterial suspension. Next, 100 μL of bacterial suspension was mixed with 100 μL of peptide (final concentration range: 2×10 -6 -64×10 -6 M) were mixed into a 96-well plate. Fluorescence values ​​were measured using a microplate reader at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. 10 μg mL -1 The polymyxin B-treated group was used as a positive control, and the bacterial suspension without peptide was used as a negative control for initial fluorescence. Each test was performed in two parallel experiments and repeated three times independently.

[0058] Depend on Figure 5 It can be seen that the antimicrobial peptide VKLR has a dose-dependent effect on the outer membrane permeability of Pseudomonas aeruginosa. -6 M, it can destroy about 40% of the bacterial outer membrane,

[0059] Example 6 Antimicrobial Peptide VKLR Binding Affinity Test for LPS / LTA

[0060] The ability of the antimicrobial peptide VKLR to bind to LPS of Pseudomonas aeruginosa and LTA of Staphylococcus aureus was determined using the fluorescent probe BODIPY-TR cadaverine (BC). -6 M BC and the final concentration was 50 × 10 -6 M of LPS or LTA were incubated at 37°C in the dark for 4 hours. Next, the peptide VKLR solution was serially diluted in a 96-well plate to produce 1×10 -6 Up to 64×10 -6 The concentration of LPS or LTA was 100 M. Subsequently, the LPS or LTA probe was added to an equal volume of the peptide solution and incubated for 1 hour. Finally, the fluorescence intensity was measured using a microplate reader at an excitation wavelength of 580 nm and an emission wavelength of 620 nm. Each test was repeated three times in duplicate.

[0061] Depend on Figure 6 VKLR binds to both P. aeruginosa LPS and S. aureus LTA in a dose-dependent manner, with stronger binding to LPS. Binding kinetic analysis revealed that VKLR exhibits a stronger binding ability to LPS, which contributes to its more rapid bactericidal activity against P. aeruginosa.

[0062] Example 7 Effect of the Antimicrobial Peptide VKLR on the Degree of Bacterial Inner Membrane Depolarization

[0063] The lipophilic membrane probe 3,3'-dipropylthiodicarbocyanine iodide (DiSC3-5) was used to measure the changes in the plasma membrane potential of Pseudomonas aeruginosa ATCC15442 and Staphylococcus aureus ATCC6538 induced by the peptide VKLR. Logarithmically growing bacteria were resuspended in 5×10 -3 M HEPES buffer (pH 7.4, containing 40×10 -3 M glucose and 200×10 -3 M KCl) and adjusted the OD to 0.1 at 600 nm. Immediately thereafter, 0.8×10 -6 M DiSC3-5 and incubated in the dark for 90 min. Meanwhile, peptide VKLR solution was serially diluted in a 96-well plate to generate a concentration range of 8 × 10 -6 to 16×10 -6 M. Next, the peptide solution was mixed with the bacterial suspension (1:1, v / v), and the fluorescence changes at an excitation wavelength of 620 nm and an emission wavelength of 670 nm were immediately detected using a microplate reader.

[0064] Fluorescent probe DiSC3-5 was used as a tracer dye to assess changes in membrane potential. This environmentally sensitive cationic dye diffuses and accumulates in the phospholipid bilayer, leading to dye self-quenching. When VKLR changes the plasma membrane potential or structure, the fluorescence intensity increases. Figure 7 and Figure 8 It can be seen that the antibiotic membrane peptide VKLR causes cytoplasmic membrane depolarization in a dose-dependent manner. At the same time, Pseudomonas aeruginosa reaches its peak faster under VKLR than under Staphylococcus aureus, and its fluorescence reaches its peak at 120S.

[0065] Example 8 Determination of Hemolytic Activity of Antimicrobial Peptides

[0066] Purchase 1% porcine red blood cells, centrifuge at 3000rpm for 10 minutes, collect the red blood cells; wash with PBS solution 3 times, and then resuspend with 10mL PBS solution; take 50μL of red blood cell suspension and mix evenly with 50μL of antimicrobial peptide solution of different concentrations, incubate at a constant temperature of 37℃ in an incubator for 1h; then centrifuge at 4℃, 3000rpm for 10min; take out the supernatant and use a microplate reader to measure the light absorption value at 570nm. Among them, 50μL of red blood cells plus 50μL of PBS solution is used as a negative control, and 50μL of red blood cells plus 50μL of 0.1% Tritonx-100 is used as a positive control. The minimum hemolytic concentration is the antimicrobial peptide concentration when the antimicrobial peptide causes a hemolysis rate of 10%. The test results are shown in Figure 9 .pass Figure 9 It can be seen that the antimicrobial peptide VKLR did not show hemolytic activity within the detection range, caused 1% hemolysis of red blood cells at a concentration of 128 μM, and failed to cause 10% hemolysis of red blood cells, indicating that the low hemolytic activity antimicrobial peptide VKLR has the potential to be developed as an antibiotic alternative.

[0067] In summary, the peptide VKLR composed of natural amino acids has excellent broad-spectrum antibacterial activity, strong salt stability and protease stability, and has broad prospects for clinical application.

[0068] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A short-chain antimicrobial peptide VKLR, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. Use of the short-chain antimicrobial peptide VKLR according to claim 1 in any of the following items, characterized in that: include: (1) Used in the preparation of preservatives; (2) Used for preparing medical dressings.

3. Use of the short-chain antimicrobial peptide VKLR according to claim 1 in the preparation of a broad-spectrum antimicrobial agent, characterized in that: The antibacterial agent is anti-Escherichia coli, anti-Pseudomonas aeruginosa, anti-Staphylococcus aureus and / or anti-Staphylococcus epidermidis.

4. Use of the short-chain antimicrobial peptide VKLR according to claim 1 in the preparation of an antimicrobial composition, characterized in that: The antibacterial agent is anti-Escherichia coli, anti-Pseudomonas aeruginosa, anti-Staphylococcus aureus and / or anti-Staphylococcus epidermidis.

5. A broad-spectrum antibacterial agent, characterized in that: The broad-spectrum antibacterial agent comprises the short-chain antibacterial peptide VKLR according to claim 1; The antibacterial agent is anti-Escherichia coli, anti-Pseudomonas aeruginosa, anti-Staphylococcus aureus and / or anti-Staphylococcus epidermidis.

6. A preservative, characterized in that The preservative comprises the short-chain antimicrobial peptide VKLR according to claim 1.

7. An antibacterial composition, characterized in that The antibacterial composition comprises the short-chain antibacterial peptide VKLR according to claim 1; The antibacterial agent is anti-Escherichia coli, anti-Pseudomonas aeruginosa, anti-Staphylococcus aureus and / or anti-Staphylococcus epidermidis.

8. A medical dressing, characterized in that: The medical dressing comprises the short-chain antimicrobial peptide VKLR according to claim 1.

9. Use of the short-chain antimicrobial peptide VKLR according to claim 1 in the preparation of medical devices with antimicrobial effects, characterized in that: The antibacterial agent is anti-Escherichia coli, anti-Pseudomonas aeruginosa, anti-Staphylococcus aureus and / or anti-Staphylococcus epidermidis.

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