Antimicrobial peptide and its application

Abstract

The present invention discloses an antimicrobial peptide and its use. The amino acid sequence of the antimicrobial peptide is KKKEHRLLHRVLFHRL. The antimicrobial peptide has a minimum inhibitory concentration of 4.5 μg / mL against Staphylococcus aureus and a minimum bactericidal concentration of 72.7 μg / mL. The antimicrobial peptide exhibits significant antimicrobial activity against Staphylococcus aureus and is expected to be widely used as a novel antimicrobial agent for the prevention and treatment of Staphylococcus aureus.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen prevention and treatment, and in particular to an antimicrobial peptide and application thereof. Background Art

[0002] Since the discovery of antibiotics, their use in healthcare has significantly saved countless lives. However, with the overuse of antibiotics, bacterial resistance has continued to grow, leading to the emergence of superbugs, which pose a serious challenge to the global healthcare system. Therefore, the search and development of new alternatives to antibiotics has become increasingly important. Peptide antimicrobial drugs, especially antimicrobial peptides (AMPs), have become a hot topic of research due to their high antimicrobial activity, low resistance, and broad biological activity.

[0003] Staphylococcus aureus is a major foodborne zoonosis that can cause infectious diseases such as pneumonia, meningitis, and bacteremia in humans and livestock, severely impacting the healthy development of the livestock and poultry industry and public health. Antibiotics play a vital role in safeguarding the healthy development of the livestock and poultry industry and public health. However, antibiotics promote the development and progression of bacterial resistance, particularly the emergence of super-resistant Staphylococcus aureus (MRSA), posing a significant challenge to infection control. The development of new anti-S. aureus inhibitors has become a bottleneck in the prevention and control of S. aureus infections.

[0004] Antimicrobial peptides are a class of naturally occurring polypeptides with antimicrobial activity. Their molecules typically consist of fewer than 100 amino acids, exhibit excellent thermal stability, lack drug shielding, and possess broad-spectrum antibacterial, antifungal, and antiviral activity. Currently, over 3,200 antimicrobial peptides have been discovered, originating from a wide range of sources, including bacteriophages, bacteria, fungi, plants, and animals. Plant-derived antimicrobial peptides, due to their natural, safe, environmentally friendly, and low resistance to drug resistance, hold great potential for application in healthcare and food preservation.

[0005] Although a variety of antimicrobial peptides have been discovered and applied, antimicrobial peptides with high efficacy and stability against Staphylococcus aureus are still scarce. Therefore, the development of a new plant-derived antimicrobial peptide, especially one targeting Staphylococcus aureus, has important practical significance and application value. Summary of the Invention

[0006] The present invention aims to solve the problem that the current antimicrobial peptide field has limited effect on the prevention and treatment of Staphylococcus aureus, and provides an antimicrobial peptide and its application.

[0007] The technical solutions of the present invention are as follows:

[0008] The present invention provides an antimicrobial peptide, the amino acid sequence of which is KKKEHRLLHRVLFHRL (SEQ ID NO: 1). The antimicrobial peptide has a highly effective killing effect on Staphylococcus aureus and exhibits good stability.

[0009] Preferably, the minimum inhibitory concentration of the antimicrobial peptide against Staphylococcus aureus is 4.5 μg / mL.

[0010] Preferably, the minimum bactericidal concentration of the antimicrobial peptide against Staphylococcus aureus is 72.7 μg / mL.

[0011] Preferably, the antimicrobial peptide is obtained by solid phase synthesis.

[0012] The present invention also provides an antibacterial agent comprising the above-mentioned antimicrobial peptide (SEQ ID NO: 1).

[0013] Preferably, the antibacterial agent further contains water.

[0014] More preferably, the concentration of the antimicrobial peptide in the antibacterial agent is 0.1 to 200 μg / mL.

[0015] More preferably, the concentration of the antimicrobial peptide in the antibacterial agent is 0.625-80 μg / mL.

[0016] More preferably, the concentration of the antimicrobial peptide in the antibacterial agent is 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0 or 80.0 μg / mL, preferably 5 μg / mL.

[0017] The present invention also provides a method for preparing the above-mentioned antibacterial agent, comprising the following steps:

[0018] The antimicrobial peptide is mixed with water and stirred.

[0019] The present invention also provides a use of the antimicrobial peptide or the antibacterial agent or the antibacterial agent obtained by the above preparation method in inhibiting the growth of Staphylococcus aureus or killing Staphylococcus aureus.

[0020] The present invention also provides a use of the antimicrobial peptide or the antibacterial agent or the antibacterial agent obtained by the above preparation method in the preparation of a Staphylococcus aureus infection inhibitor.

[0021] Beneficial effects of the present invention:

[0022] The antimicrobial peptide of the present invention has significant antimicrobial activity; when the antimicrobial peptide is made into an aqueous solution bactericide, it exhibits significant antibacterial activity against Staphylococcus aureus. When the concentration of the antimicrobial peptide reaches 4.5 μg / mL or above, it has a strong antibacterial effect on Staphylococcus aureus; when the concentration reaches 72.7 μg / mL or above, it exhibits a strong killing effect on Staphylococcus aureus. The antimicrobial peptide of the present invention has the advantages of being environmentally friendly, low toxicity and low drug resistance, and is expected to be widely used as a new antibacterial agent for the prevention and treatment of Staphylococcus aureus. Verification of antimicrobial effect: Through comparative experiments, the antimicrobial peptide of the present invention has a more efficient killing effect on Staphylococcus aureus than traditional antibiotics (such as kanamycin). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a high performance liquid chromatogram of the antimicrobial peptide of Example 1 of the present invention.

[0024] Figure 2 This is a mass spectrometry analysis diagram of the antimicrobial peptide in Example 1 of the present invention.

[0025] Figure 3 This is a graph showing the inhibitory effect of the antimicrobial peptides of Examples 2-4 of the present invention on Staphylococcus aureus, Escherichia coli and Listeria monocytogenes.

[0026] Figure 4 This is a bar graph showing the diameter of the inhibition zone of the antimicrobial peptide of Example 2 of the present invention against Staphylococcus aureus.

[0027] Figure 5 This is a graph showing the results of culturing Staphylococcus aureus with antimicrobial peptides at different concentrations according to Example 5 of the present invention.

[0028] Figure 6 This is a graph showing the results of culturing Staphylococcus aureus with antimicrobial peptides at different concentrations according to Example 6 of the present invention. DETAILED DESCRIPTION

[0029] The specific implementation of the present invention will be further described below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0030] Example 1

[0031] (1) Solid-phase synthesis of antimicrobial peptides

[0032] Experimental materials and reagents: 2-CL resin; Fmoc-AA-OH; anhydrous DCM; DIEA; methanol; DCM; technical-grade DMF; analytical-grade DMF; 20% (v / v) piperidine / DMF; detection reagent A (5 g ninhydrin dissolved in 100 mL anhydrous ethanol), detection reagent B (analytical-grade pyridine); HOBT; DIC.

[0033] Weigh 0.3 g of 2-CL resin into a vertical reactor (20 × 250 mm, No. 1 sand core) and soak in 5 mL of DCM for 30 minutes. Weigh 0.08 mM of α-amino (the first amino acid at the C-terminus) into a centrifuge tube and add 5 mL of anhydrous DCM and 0.2 mM DIEA to mix well. Add this to the reactor from the previous step and react with nitrogen bubbling for 90 minutes. Upon completion of the reaction, add 2 mL of methanol and 4 mL of DCM and react for 20 minutes.

[0034] After draining the liquid in the reactor with a circulating water vacuum pump, add industrial-grade DMF to the reactor using a wash bottle. The volume of the reagent is about 3 times the volume of the resin. The resin is completely soaked in the solution and washed for 30 seconds. Then, drain the liquid in the reactor with a circulating water vacuum pump. The draining time is about 30 seconds. Repeat this operation 4 times.

[0035] A 20% (v / v) piperidine / DMF solution was added to the reactor at a volume of approximately 3 times the resin volume, allowing the resin to be completely soaked in the solution. The reaction was then bubbling with nitrogen for 20 minutes. After the liquid in the reactor was drained with a circulating water vacuum pump, technical-grade DMF was added to the reactor with a wash bottle at a volume of approximately 3 times the resin volume, allowing the resin to be completely soaked in the solution. The reaction was then washed for 30 seconds. The liquid in the reactor was then drained with a circulating water vacuum pump for 30 seconds. This operation was repeated five times, with the technical-grade DMF being replaced with analytical-grade DMF during the fifth washing.

[0036] Pipette 10-20 resins from the reactor and place them at the bottom of the test tube. Take two drops of detection reagent A and B and drop them into the test tube to ensure full contact between the resin and the detection reagent. Then place the test tube in a 100°C constant temperature oven and heat for 2 minutes. Observe the color of the resin. If the resin shows color, it means that the Fmoc removal is successful.

[0037] Condensation: Weigh 0.2 mM Fmoc-AA-OH (second from the C-terminus) and 0.2 mM HOBT into a centrifuge tube and fully dissolve them with 1 mL of DMF; then add 500 μL of DIC, mix for 1 min, add to the dried resin, and react with nitrogen bubbling for 1 h.

[0038] Resin detection: colorless resin indicates that the connection is complete. According to the above condensation steps, the antimicrobial peptide Lys-Lys-Lys-Glu-His-Arg-Leu-Leu-His-Arg-Val-Leu-Phe-His-Arg-Leu (SEQ ID NO: 1) of the present invention was synthesized one by one from the C-terminus to the N-terminus of the peptide chain until the peptide connection is completed.

[0039] (2) Purification and identification of antimicrobial peptides

[0040] The antimicrobial peptide was loaded onto pump A and equilibrated with 5% (v / v) acetonitrile water for 5 min.

[0041] Mobile phase: Phase A (aqueous phase): deionized water (containing 0.1% FA (v / v)); Phase B (organic phase): 5% (v / v) acetonitrile aqueous solution (containing 0.1% TFA (v / v)).

[0042] Flow rate: 10 mL / min; elution gradient: Phase B increased from 10% to 50% at a rate of 1.33% per minute. The organic phase acetonitrile in the eluent was removed by rotary evaporator, and the sample was transferred to a freeze-drying bottle and placed in a freeze dryer for 24 hours to obtain the pure product. The sample peaks of the collected samples were detected and analyzed according to the HPLC chart ( Figure 1 ) The purity of the analyzed sample was greater than 75%. Figure 2 Its mass spectrum.

[0043] Example 2

[0044] Inhibitory effect of antimicrobial peptides against Staphylococcus aureus.

[0045] (1) Sample preparation

[0046] Dissolve 1 mg of the antimicrobial peptide of SEQ ID No. 1 in 500 μL of deionized water. Stir continuously for 10 minutes, then top up the volume to 1000 μL with deionized water to obtain the antibacterial agent. Control sample: 1 mg / mL kanamycin. Blank sample: sterile deionized water.

[0047] (2) Preparation of bacterial solution

[0048] Staphylococcus aureus was cultured in 5 mL of NB medium at 37°C on a constant temperature shaker at 180 rpm / min for 16 h. The overnight cultured Staphylococcus aureus was diluted 1:1000 and cultured for 3-4 h at 37°C on a constant temperature shaker at 180 rpm / min. The bacterial solution concentration was adjusted to 1×10 8 CFU / mL (OD600=0.2~0.3).

[0049] (3) Antimicrobial peptide antibacterial activity detection

[0050] Take 1 mL of antibacterial agent (KJT), control sample (Kan) and blank sample (CK), and use the Kirby-Bauer method to determine the size of the inhibition zone. Add 100 μL of bacterial solution (adjust the concentration to 1×10 8 CFU / mL) and pour into a 90 mm Petri dish and allow to solidify. Sterilize the filter paper by cutting it into 6 mm diameter discs. Place the filter paper containing 10 μL of sample on the surface of the Petri dish and let it sit for 5 minutes. Incubate the Petri dish at 37°C for 24 hours and measure the size of the inhibition zone with a vernier caliper.

[0051] Test results: The inhibitory effect of antimicrobial peptides and kanamycin on Staphylococcus aureus at a concentration of 1 mg / mL is shown in the figure below. Figure 3 The diameter of the inhibition zone is Figure 4 The results showed that the antimicrobial peptide had a better inhibitory effect on Staphylococcus aureus than kanamycin.

[0052] Example 3

[0053] Inhibitory effect of antimicrobial peptides on Escherichia coli.

[0054] The difference between Example 3 and Example 2 is that:

[0055] In step (2), the bacterial solution was prepared by culturing E. coli in 5 mL of LB medium at 37°C on a constant temperature shaker at 180 rpm / min for 16 h. The overnight cultured E. coli was diluted at 1:1000 and then cultured at 37°C on a constant temperature shaker at 180 rpm / min for 3-4 h to adjust the bacterial solution concentration to 1×10 8 CFU / mL (OD600=0.2~0.3).

[0056] Test results: The inhibitory effect of antimicrobial peptides and kanamycin on Escherichia coli at a concentration of 1 mg / mL is shown in the figure below. Figure 3 The results showed that the antimicrobial peptides had no inhibitory effect on Escherichia coli.

[0057] Example 4

[0058] Inhibitory effect of antimicrobial peptides against Listeria monocytogenes.

[0059] The difference between Example 4 and Example 2 is that:

[0060] In step (2), the bacterial solution was prepared by culturing Listeria monocytogenes in 5 mL of LB medium at 37°C on a constant temperature shaker at 180 rpm / min for 16 h. The overnight cultured Listeria monocytogenes was diluted at 1:1000 and then cultured at 37°C on a constant temperature shaker at 180 rpm / min for 3-4 h to adjust the bacterial solution concentration to 1×10 8CFU / mL (OD600=0.2~0.3).

[0061] Test results: The inhibitory effect of antimicrobial peptides and kanamycin on Listeria monocytogenes at a concentration of 1 mg / mL is shown in the figure below. Figure 3 The results showed that the antimicrobial peptides had no inhibitory effect on Listeria monocytogenes.

[0062] Example 5

[0063] Minimum inhibitory concentration of antimicrobial peptides against Staphylococcus aureus.

[0064] (1) Sample preparation: The antimicrobial peptides were diluted with sterile deionized water to prepare a series of double concentration gradient antimicrobial peptide solutions using the serial dilution method. The solutions were then added to test tubes containing 10 mL of NB medium to achieve final concentrations of 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 μg / mL.

[0065] (2) Preparation of bacterial solution: Staphylococcus aureus was cultured in 5 mL of NB medium at 37°C on a constant temperature shaker at 180 rpm / min for 16 h. The overnight cultured Staphylococcus aureus was diluted 1:1000 and cultured for 3-4 h at 37°C on a constant temperature shaker at 180 rpm / min. The bacterial solution concentration was adjusted to 1×10 8 CFU / mL (OD600=0.2~0.3).

[0066] (3) Minimum inhibitory concentration (MIC) determination: 1 mL of NB culture medium containing antimicrobial peptides at final concentrations of 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 μg / mL was added to 100 μL of 1×10 8 Mix the Staphylococcus aureus solution at a concentration of 100 CFU / mL thoroughly and incubate it in a shaker at 37°C and 220 rpm / min for 24 hours. The minimum inhibitory concentration (MIC) is the minimum inhibitory concentration (MIC) at which the solution remains turbid.

[0067] Test results: The results of different concentrations of antimicrobial peptides and Staphylococcus aureus culture are shown in the figure below. Figure 5 The results showed that the minimum inhibitory concentration of the antimicrobial peptide against Staphylococcus aureus was 4.5 μg / mL.

[0068] Example 6

[0069] Minimum bactericidal concentration of antimicrobial peptides against Staphylococcus aureus.

[0070] The difference between Example 6 and Example 5 is that:

[0071] In step (3), the minimum bactericidal concentration (MBC) was determined by taking 1 mL of NB medium with a final concentration of antimicrobial peptide of 5.0, 10.0, 20.0, 40.0, and 80.0 μg / mL, and adding 100 μL of 1×10 8 Mix thoroughly with a Staphylococcus aureus culture solution containing 100 CFU / mL. Incubate at 37°C at 220 rpm / min on a shaker for 24 hours. Spread 20 μL of the remaining turbid culture solution evenly onto a NA culture dish. Incubate at 37°C for 48 hours. The minimum bactericidal concentration (MCC) is the lowest concentration at which no bacteria grow.

[0072] Test results: The results of different concentrations of antimicrobial peptides and Staphylococcus aureus culture are shown in the figure below. Figure 6 The results showed that the minimum bactericidal concentration of antimicrobial peptides against Staphylococcus aureus was 72.7 μg / mL.

[0073] In summary, the antimicrobial peptide of the present invention has a highly effective killing effect only on Staphylococcus aureus but has no antibacterial effect on Escherichia coli and Listeria monocytogenes.

[0074] The above embodiments are only preferred embodiments of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide, characterized in that Its amino acid sequence is KKKEHRLLHRVLFHRL.

2. A bacteriostatic agent, characterized in that The antimicrobial peptide according to claim 1.

3. The antibacterial agent according to claim 2, characterized in that The bacteriostatic agent also contains water.

4. The antibacterial agent according to claim 3, characterized in that The concentration of the antimicrobial peptide in the antibacterial agent is 0.1-200 μg / mL.

5. The antibacterial agent according to claim 4, characterized in that The concentration of the antimicrobial peptide in the antibacterial agent is 0.625-80 μg / mL.

6. Use of the antimicrobial peptide according to claim 1 or the antibacterial agent according to any one of claims 2 to 5 for inhibiting the growth of Staphylococcus aureus or killing Staphylococcus aureus, wherein the use is for purposes other than disease diagnosis or treatment.

7. Use of the antimicrobial peptide according to claim 1 or the antibacterial agent according to any one of claims 2 to 5 in the preparation of an inhibitor of Staphylococcus aureus infection.

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