Antibacterial peptide and application thereof

Abstract

The invention discloses an antibacterial peptide and application thereof. The amino acid sequence of the antibacterial peptide disclosed by the invention is KKKEHRLLHRVLFHRL. The minimum inhibitory concentration of the antibacterial peptide to staphylococcus aureus is 4.5 mu g / mL, and the minimum bactericidal concentration of the antibacterial peptide to staphylococcus aureus is 72.7 mu g / mL. The antibacterial peptide disclosed by the invention has remarkable antibacterial activity on staphylococcus aureus and is expected to be widely applied as a novel bacteriostatic agent for preventing and treating 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 application in the medical field has greatly saved countless lives. However, with the overuse of antibiotics, bacterial resistance has continued to increase, leading to the emergence of superbugs, which poses a severe challenge to the global medical system. Therefore, it is particularly important to find and develop new alternatives to antibiotics. Peptide antimicrobial drugs, especially antimicrobial peptides (AMPs), have become a hot topic of current research due to their high antimicrobial activity, low drug resistance and wide range of biological activities.

[0003] Staphylococcus aureus is an important foodborne zoonosis pathogen that can cause pneumonia, meningitis, bacteremia and other infectious diseases in humans and livestock, seriously affecting the healthy development of livestock and poultry farming and public health. Antibiotics play an important role in ensuring the healthy development of livestock and poultry farming and public health. However, antibiotics promote the generation and development of bacterial resistance, especially the emergence of super-resistant Staphylococcus aureus (MRSA), which poses a huge challenge to its infection inhibition. The development of new anti-Staphylococcus aureus infection inhibitors has become a "bottleneck" problem in the prevention and control of Staphylococcus aureus infection.

[0004] Antimicrobial peptides are a class of natural polypeptide substances with antimicrobial activity. Their molecular composition is usually less than 100 amino acids, with good thermal stability, no drug shielding, and a broad spectrum of biological activities against bacteria, fungi, viruses, etc. At present, more than 3,200 types of antimicrobial peptides have been discovered, and their sources are wide-ranging, including bacteriophages, bacteria, fungi, plants, and animals. Among them, plant-derived antimicrobial peptides have shown great application potential in the fields of medical treatment and food preservation due to their natural, safe, environmentally friendly, and non-resistant characteristics.

[0005] Although a variety of antimicrobial peptides have been discovered and applied, there is still a lack of antimicrobial peptides with high killing effect and high stability against Staphylococcus aureus. Therefore, the development of a new plant-derived antimicrobial peptide, especially an antimicrobial peptide against 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 control effect on Staphylococcus aureus, and provides an antimicrobial peptide and application thereof.

[0007] The technical solution of the present invention is 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 of the present invention 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 antimicrobial peptide (SEQ ID NO: 1).

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

[0014] More preferably, the concentration of the antimicrobial peptide in the antibacterial agent is 0.1-200 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; the antimicrobial peptide exhibits significant antibacterial activity against Staphylococcus aureus when made into an aqueous bactericide. When the concentration of the antimicrobial peptide reaches 4.5 μg / mL or more, it has a strong antibacterial effect on Staphylococcus aureus; when the concentration reaches 72.7 μg / mL or more, it exhibits a strong killing effect on Staphylococcus aureus. The antimicrobial peptide of the present invention has the advantages of environmental protection, 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 antibacterial effect: Through comparative experiments, the antimicrobial peptide described in 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 peptide of Example 2-4 of the present invention on Staphylococcus aureus, Escherichia coli and Listeria monocytogenes.

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

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

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

[0029] The specific implementation of the present invention is 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 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 raw materials and reagents: 2-CL resin; Fmoc-AA-OH; anhydrous DCM; DIEA; methanol; DCM; industrial 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 0.3g of 2-CL resin, put it into a vertical reactor (20×250mm, No. 1 sand core), add 5mL DCM and soak for 30min. Weigh 0.08mM α-amino (the first amino acid at the C-terminus) into a centrifuge tube, and add 5mL anhydrous DCM and 0.2mM DIEA to shake well. Add it to the reactor in the previous step, and react with nitrogen bubbling for 90min. After the reaction is completed, add 2mL methanol and 4mL DCM and react for 20min.

[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 immersed in the solution. Wash 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] Add 20% (v / v) piperidine / DMF solution to the reactor, the reagent volume is about 3 times the resin volume, the resin is completely soaked in the solution, and the nitrogen bubbling reaction is carried out for 20 minutes. After the liquid in the reactor is drained with a circulating water vacuum pump, industrial-grade DMF is added to the reactor with a washing bottle, the reagent volume is about 3 times the resin volume, the resin is completely soaked in the solution, and washed for 30 seconds, and then the liquid in the reactor is drained with a circulating water vacuum pump, and drained for 30 seconds. Repeat this operation 5 times, and replace the industrial-grade DMF with analytical-grade DMF during the 5th washing process.

[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 allow the resin and the detection reagent to fully contact. Then put the test tube into a 100℃ 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 in a centrifuge tube and fully dissolve them with 1 mL DMF; then add 500 μL 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 is 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 increases from 10% to 50% at a rate of 1.33% per minute. Use a rotary evaporator to remove the organic phase acetonitrile in the eluent, transfer the sample to a freeze-drying bottle, place it in a freeze dryer and freeze-dry for 24 hours to obtain a pure product, detect the sample peaks of the collected samples, and according to the high performance liquid chromatogram ( 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 on Staphylococcus aureus.

[0045] (1) Sample preparation

[0046] Take 1 mg of the antimicrobial peptide of SEQ ID No: 1 and dissolve it in 500 μL of deionized water, stir it continuously for 10 min, and add deionized water to 1000 μL to obtain an antibacterial agent. Control sample: kanamycin with a concentration of 1 mg / mL. 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 at 1:1000 and cultured for 3-4 h at 37°C on a constant temperature shaker at 180 rpm / min. The concentration of the bacterial solution 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 (the concentration is adjusted to 1×10 8 CFU / mL), pour into a culture dish (90mm) and wait for it to solidify. Break the filter paper into a 6mm diameter disc and sterilize it. Place the filter paper containing 10μL sample on the surface of the culture dish and let it stand for 5min. Incubate the culture dish at 37℃ for 24h 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 show that the inhibitory effect of antimicrobial peptides on Staphylococcus aureus is better than that of 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 E. coli cultured overnight 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 show that antimicrobial peptides have no inhibitory effect on Escherichia coli.

[0057] Example 4

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

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

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

[0066] (3) Minimum inhibitory concentration (MIC) determination: Take 1 mL of NB 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, and add 100 μL of 1×10 8 CFU / mL of Staphylococcus aureus solution was mixed thoroughly and incubated in a shaker at 37°C and 220 rpm / min for 24 hours. The minimum concentration that did not turn turbid was the minimum inhibitory concentration.

[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 show that the minimum inhibitory concentration of the antimicrobial peptide against Staphylococcus aureus is 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, or 80.0 μg / mL, adding 100 μL of 1×10 8 CFU / mL of Staphylococcus aureus liquid, mix thoroughly. Place in a shaker at 37℃ and 220rpm / min for 24h. Take 20μL of the liquid that has not turned turbid and spread it evenly on a NA culture dish. After culturing at 37℃ for 48h, the lowest concentration without bacterial growth is the minimum bactericidal concentration.

[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 show that the minimum bactericidal concentration of antimicrobial peptides against Staphylococcus aureus is 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 implementation modes 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. The antimicrobial peptide according to claim 1, characterized in that The minimum inhibitory concentration of the antimicrobial peptide to Staphylococcus aureus is 4.5 μg / mL.

3. The antimicrobial peptide according to claim 1, characterized in that The minimum bactericidal concentration of the antimicrobial peptide against Staphylococcus aureus is 72.7 μg / mL.

4. The antimicrobial peptide according to claim 1, characterized in that The antimicrobial peptide is obtained by solid phase synthesis.

5. An antibacterial agent, characterized in that The invention comprises the antimicrobial peptide according to any one of claims 1 to 4.

6. The antibacterial agent according to claim 5, characterized in that The bacteriostatic agent also contains water.

7. The antibacterial agent according to claim 6, characterized in that The concentration of the antimicrobial peptide in the antibacterial agent is 0.1-200 μg / mL.

8. The antibacterial agent according to claim 7, characterized in that The concentration of the antimicrobial peptide in the antibacterial agent is 0.625-80 μg / mL.

9. Use of the antimicrobial peptide according to any one of claims 1 to 4 or the antibacterial agent according to any one of claims 5 to 8 in inhibiting the growth of Staphylococcus aureus or killing Staphylococcus aureus.

10. Use of the antimicrobial peptide according to any one of claims 1 to 4 or the antibacterial agent according to any one of claims 5 to 8 in the preparation of an inhibitor of Staphylococcus aureus infection.

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