Antimicrobial peptide and its application

Abstract

The present invention relates to the field of biomedicine, particularly to IPC C07K14, and more specifically to an antimicrobial peptide and its use. The present invention provides antimicrobial peptides 1 to 18 that can effectively inhibit the growth and reproduction of Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus, and have low hemolytic activity and good biosafety.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to IPC C07K14, and more specifically to an antimicrobial peptide and applications thereof. Background Art

[0002] Antimicrobial peptides are widely present in a variety of organisms and are small molecule polypeptides with biological activity. This type of active small molecule polypeptide generally has the characteristics of strong alkalinity, thermal stability and broad antibacterial spectrum, and can serve as an important line of defense against the invasion of pathogenic microorganisms.

[0003] Patent CN201910381420.1 discloses a method for isolating a plant antimicrobial peptide, a plant antimicrobial peptide, and its uses. The antimicrobial peptide, isolated from black sesame seeds, has inhibitory effects on Candida albicans and Haemophilus influenzae. However, it has not been shown to inhibit other bacteria.

[0004] Existing patent CN201811277061.7 discloses a method for purifying and preparing antimicrobial peptides. By optimizing the process steps and parameters, semipermeable membrane purification and reverse-phase chromatography purification are mutually reinforcing, resulting in the separation and purification of high-purity antimicrobial peptides. Existing technologies for separating antimicrobial peptides are well developed.

[0005] However, there are only a few types of antimicrobial peptides available, and some existing antimicrobial peptides have strong hemolytic properties, leaving room for improvement in their biosafety. This invention has developed a new antimicrobial peptide and its application, which primarily inhibits Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus, and has high biosafety. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the first aspect of the present invention provides an antimicrobial peptide, wherein the antimicrobial peptide is any one of antimicrobial peptide 1, antimicrobial peptide 2, antimicrobial peptide 3, antimicrobial peptide 4, antimicrobial peptide 5, antimicrobial peptide 6, antimicrobial peptide 7, antimicrobial peptide 8, antimicrobial peptide 9, antimicrobial peptide 10, antimicrobial peptide 11, antimicrobial peptide 12, antimicrobial peptide 13, antimicrobial peptide 14, antimicrobial peptide 15, antimicrobial peptide 16, antimicrobial peptide 17, and antimicrobial peptide 18, wherein the amino acid sequence of antimicrobial peptide 1 is shown in SEQ ID No: 1, the amino acid sequence of antimicrobial peptide 2 is shown in SEQ ID No: 2, the amino acid sequence of antimicrobial peptide 3 is shown in SEQ ID No: 3, the amino acid sequence of antimicrobial peptide 4 is shown in SEQ ID No: 4, the amino acid sequence of antimicrobial peptide 5 is shown in SEQ ID No: 5, the amino acid sequence of antimicrobial peptide 6 is shown in SEQ ID No: 6, the amino acid sequence of antimicrobial peptide 7 is shown in SEQ ID No: 7, and the amino acid sequence of antimicrobial peptide 8 is shown in SEQ ID No.: 8, the amino acid sequence of the antimicrobial peptide 9 is shown in SEQ ID No: 9, the amino acid sequence of the antimicrobial peptide 10 is shown in SEQ ID No: 10, the amino acid sequence of the antimicrobial peptide 11 is shown in SEQ ID No: 11, the amino acid sequence of the antimicrobial peptide 12 is shown in SEQ ID No: 12, the amino acid sequence of the antimicrobial peptide 13 is shown in SEQ ID No: 13, the amino acid sequence of the antimicrobial peptide 14 is shown in SEQ ID No: 14, the amino acid sequence of the antimicrobial peptide 15 is shown in SEQ ID No: 15, the amino acid sequence of the antimicrobial peptide 16 is shown in SEQ ID No: 16, the amino acid sequence of the antimicrobial peptide 17 is shown in SEQ ID No: 17, and the amino acid sequence of the antimicrobial peptide 18 is shown in SEQ ID No: 18.

[0007] The present invention generates antimicrobial peptides based on a machine learning method. A generative model is used to learn the feature vector representation of peptides from a large-scale peptide database, and then a machine model classifier is trained using the antimicrobial peptide database to screen out predicted sequences with antimicrobial function.

[0008] In a second aspect, the present invention provides a method for preparing the antimicrobial peptide, which can be artificially synthesized by genetic engineering methods, directly obtained from cells by separation and purification methods, or directly prepared by chemical synthesis.

[0009] Preferably, the synthesis steps are as follows:

[0010] (1) The order of peptide synthesis is from C-terminus to N-terminus: 10 g of 2-Chlorotrityl Chloride Resin is placed in a reaction tube, 15 mL / g (milliliter / gram) DCM (dichloromethane) is added, and shaken for 30 minutes.

[0011] (2) First amino acid: Filter the solvent through a sand core, add a 3-fold molar excess of Fmoc-amino acid-OH amino acid, dissolve in DMF (dimethylformamide), then add a 10-fold molar excess of DIEA (N,N-diisopropylethylamine), shake for 90 minutes, and block with methanol.

[0012] (3) Deprotection: Remove DMF, add 15 ml / g 20% ​​piperidine DMF solution, wash for 5 minutes, remove the 20% piperidine DMF solution, add 15 ml / g 20% ​​piperidine DMF solution again, and wash for 15 minutes.

[0013] (4) Detection: Remove the piperidine solution, take 10-20 pellets of resin, wash them three times with ethanol, and add the detection reagent for detection. Heat at 105-110°C for 5 minutes. A positive reaction is indicated when the color changes to dark blue.

[0014] (5) Rinse the resin: rinse the resin twice with DMF (10 ml / g), DCM (10 ml / g), and DMF (10 ml / g) respectively.

[0015] (6) Condensation: Add a three-fold molar excess of the protected amino acid and a three-fold molar excess of HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), both dissolved in a small amount of DMF, and added to the reaction tube. Immediately add a ten-fold molar excess of DIEA and react for 30 minutes.

[0016] (7) Detection: Take 10 to 20 resin pellets, wash them three times with ethanol, add detection reagent for detection, heat at 105℃-110℃ for 5 minutes, and a colorless reaction indicates a negative reaction.

[0017] (8) Rinse the resin: rinse the resin twice with DMF (10 ml / g), DCM (10 ml / g), and DMF (10 ml / g) respectively.

[0018] (9) Repeat steps (3) to (6) to connect the amino acids in the sequence from right to left.

[0019] (10) Drain and wash the resin as follows: DMF (10 ml / g) twice, methanol (10 ml / g) twice, DMF (10 ml / g) twice, and DCM (10 ml / g) twice, drain for 10 min.

[0020] (11) Cleavage of the peptide from the resin: The cleavage solution used was composed of 95% TFA (trifluoroacetic acid), 1% water, 2% EDT (mercaptoethanol), and 2% TIS (triisopropylsilane); the cleavage time was 120 min.

[0021] (12) Blow-drying and washing: The lysate was blown dry with nitrogen, washed with ether six times, and then allowed to evaporate at room temperature.

[0022] (13) Analysis, purification and freeze drying: The crude polypeptide was purified by high performance liquid chromatography; the polypeptide solution was collected and placed in a freeze dryer for concentration, and freeze-dried to produce a white powder.

[0023] The third aspect of the present invention provides a use of the above antimicrobial peptide in the preparation of an antimicrobial agent.

[0024] Preferably, the bacteria acted by the antibacterial agent is any one of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, Candida albicans, and Bacillus subtilis; more preferably, it is any one of Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus.

[0025] Preferably, the minimum inhibitory concentration of the antimicrobial peptide against Propionibacterium acnes is 1 to 25 μg / mL.

[0026] Preferably, the minimum inhibitory concentration of the antimicrobial peptide against Staphylococcus epidermidis is 1 to 25 μg / mL.

[0027] Preferably, the minimum inhibitory concentration of the antimicrobial peptide against Staphylococcus aureus is 1 to 25 μg / mL.

[0028] Preferably, the minimum hemolytic concentration of the antimicrobial peptide is greater than or equal to 500 μg / mL.

[0029] Method for determining the inhibitory effect of antimicrobial peptides on Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus.

[0030] 1. Determination of the inhibitory effect of antimicrobial peptides on bacteria

[0031] (i) Preparation of antimicrobial peptides

[0032] Liquid culture screening: Use a dispenser or pipette to directly dispense 10 μL of an antimicrobial peptide dissolved in pure water to 1 mg / mL. Add the sample to a new 96-well plate in order from left to right to complete the positive and negative controls. Place the 96-well plate horizontally. Controls include pure water and bacteria at the same concentration as the antimicrobial peptide, a control antimicrobial peptide and bacteria, ciprofloxacin and bacteria, and sterile culture medium.

[0033] Liquid culture testing for minimum inhibitory concentration (MIC) of antimicrobial peptides: Dilute to four concentrations. Use a dispenser to pipette 10 μL of sterile water into rows B, C, and D of a new 96-well cell culture plate. Use a dispenser to pipette 10 μL of the peptide directly into rows A and B of a new 96-well cell culture plate. Mix row B thoroughly with a dispenser and transfer 10 μL to row C. Mix row C thoroughly and transfer 10 μL to row D. Mix row D thoroughly and discard 10 μL. Repeat for rows E, F, G, and H. Complete positive and negative controls (10 μL volume) and set aside.

[0034] (Additional information: If using a 96-well deep-well plate to culture bacteria, add 100 μL of sterile culture medium to all wells.)

[0035] (ii) Bacterial recovery and bacterial suspension preparation

[0036] Take Propionibacterium acnes and inoculate it on anaerobic blood agar plate, place it in anaerobic bag, and culture it anaerobically at 37℃ for 48h. Pick Propionibacterium acnes clones and culture them anaerobically in liquid anaerobic broth medium at 37℃ for 48h. Measure the bacterial concentration and dilute it to 10 7 CFU / mL, set aside.

[0037] (iii) Liquid culture detection of the minimum inhibitory concentration of antimicrobial peptides

[0038] Prepare fresh bacterial suspension diluted to 10 5 CFU / mL, add 90 μL per well of a 96-well plate pre-inoculated with antimicrobial peptide. Incubate anaerobically at 37°C in an anaerobic bag for 24-48 hours. Measure absorbance at 630 nm using a microplate reader and visually inspect for turbidity. The minimum inhibitory concentration (MIC) of the antimicrobial peptide at which turbidity does not occur is defined as the minimum inhibitory concentration (MIC) of 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL.

[0039] (iv) Repeat steps 3 and 4 twice, and decide whether to repeat the third time based on the repeatability of the experiment.

[0040] (v) Based on the results, further minimum inhibitory concentration (MIC) tests were performed, and the minimum concentration was adjusted to 1 μg / mL. 25 concentration gradients were performed to finally determine the MIC of the antimicrobial peptide.

[0041] (vi) All synthesized antimicrobial peptides were further tested for their inhibitory activity against Staphylococcus epidermidis and Staphylococcus aureus. The method was the same as above, but the culture medium was changed to LB medium and cultured under normal oxygen concentration (21%).

[0042] (2) Determination of hemolytic activity of antimicrobial peptides

[0043] Prepare 4 mL of 2% mouse erythrocytes (RBCs), rinse twice with 4 mL of PBS, centrifuge at 3000 rpm for 5 minutes, discard the supernatant, and resuspend the RBCs in 4 mL of PBS. Dissolve 8 mg of peptide in 2 mL of PBS to prepare a 4000 μg / mL peptide stock solution. Subsequently, dilute the peptide stock solution with PBS to 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, and 125 μg / mL. To each well of a 96-well plate, 100 μL of 2% mouse cells and 100 μL of peptide solution at various concentrations (4000 μg / mL, 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, and 125 μg / mL) were added. For negative and positive controls, 100 μL of PBS buffer and 100 μL of 0.1% (w / v) Triton X-100 were added, respectively. Three replicates were set up for each group and incubated at 37°C for 1 hour. The 96-well plate was removed and centrifuged at 3000 rpm for 5 minutes. 100 μL of the supernatant was transferred to a new 96-well plate and the absorbance was measured at 540 nm using a microplate reader. Hemolysis rate (%) = (absorbance sample - absorbance negative) / (absorbance positive - absorbance negative) * 100%.

[0044] Beneficial effects

[0045] This invention uses a machine learning approach to screen for antimicrobial peptide sequences with antibacterial properties. Subsequently, various antimicrobial peptides were synthesized through chemical synthesis and genetic engineering, and tested for their antibacterial properties. Antimicrobial peptides 1-18 all demonstrated good bioactivity and high biosafety. Based on commercial applications, appropriate antimicrobial peptide combinations can be selected and formulated to develop potential broad-spectrum antimicrobial drugs. They can also be used to treat acne. DETAILED DESCRIPTION

[0046] The amino acid sequences of antimicrobial peptides 1 to 18 of the present invention are shown in Table 1:

[0047] Table 1

[0048] name Serial number Peptide sequence Antimicrobial peptide 1 SEQ ID No: 1 IRLLKKAQAILA Antimicrobial peptide 2 SEQ ID No: 2 IKIVLHFKR Antimicrobial peptide 3 SEQ ID No: 3 RVFPGFHRIHFKS Antimicrobial peptide 4 SEQ ID No: 4 VKRILKWAFK Antimicrobial peptide 5 SEQ ID No: 5 LIKAFKK Antimicrobial peptide 6 SEQ ID No: 6 RAAFKWR Antimicrobial peptide 7 SEQ ID No: 7 AFKKLIK Antimicrobial peptide 8 SEQ ID No: 8 LVRLKRALVRAVS Antimicrobial peptide 9 SEQ ID No: 9 VFRAPKK Antimicrobial peptide 10 SEQ ID No: 10 IWKRALKP Antimicrobial peptide 11 SEQ ID No: 11 PRFRRLHGFVFKK Antimicrobial peptide 12 SEQ ID No: 12 VKRALKILSALLA Antimicrobial peptide 13 SEQ ID No: 13 VIRKFHV Antimicrobial peptide 14 SEQ ID No: 14 LFGKLFKHFK Antimicrobial peptide 15 SEQ ID No: 15 KLASAARKLVGRAIK Antimicrobial peptide 16 SEQ ID No: 16 IAASVKRLLAKVL Antimicrobial peptide 17 SEQ ID No: 17 LKLKRLVKVLLTL Antimicrobial peptide 18 SEQ ID No: 18 IARLIKRAISLAR

[0049] The common single-letter and three-letter codes for the amino acids of the polypeptides of the present invention are as follows:

[0050] G (Gly) Glycine M (Met) Methionine A (Ala) Alanine S (Ser) Serine K (Lys) Lysine V (Val) Valine L (Leu) Leucine I (Ile) Isoleucine D (Asp) Aspartic Acid E (Glu) Glutamic Acid H (His) Histidine F (Phe) Phenylalanine P (Pro) Proline W (Trp) Tryptophan Q (Gln) Glutamine R (Arg) Arginine.

[0051] The antimicrobial peptide of the present invention is synthesized by the following synthesis steps:

[0052] (1) The order of peptide synthesis is from C-terminus to N-terminus: add 10g of 2-Chlorotrityl Chloride Resin into a reaction tube, add (15ml / g) DCM (dichloromethane), and shake for 30min.

[0053] (2) First amino acid: Filter the solvent through a sand core, add 3.5 times the molar volume of Fmoc-Ala-OH amino acid, dissolve in DMF (dimethylformamide), then add 10.5 times the molar volume of DIEA (N,N-diisopropylethylamine), shake for 90 minutes, and block with methanol.

[0054] (3) Deprotection: Remove DMF, add 15 ml / g 20% ​​piperidine DMF solution, wash for 5 minutes, remove the 20% piperidine DMF solution, add 15 ml / g 20% ​​piperidine DMF solution again, and wash for 15 minutes.

[0055] (4) Detection: Remove the piperidine solution and take 15 pellets of resin (2-chlorochlorochloride resin adsorbed with deprotected amino acids). Wash three times with ethanol and add ninhydrin for detection. Heat at 110°C for 5 minutes. A positive reaction is indicated when the color changes to dark blue.

[0056] (5) Rinse the resin: rinse the resin twice with DMF (10 ml / g), DCM (10 ml / g), and DMF (10 ml / g) respectively.

[0057] (6) Condensation: Add 3.5 times the moles of the next nitrogen-terminus protected amino acid and 3.5 times the moles of HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), both dissolved in 10 ml of DMF, and add to the reaction tube. Immediately add 11 times the moles of DIEA and react for 30 minutes.

[0058] (7) Detection: Take 15 resin pellets, wash them three times with ethanol, add ninhydrin for detection, heat at 110°C for 5 minutes, and a colorless reaction indicates a negative reaction.

[0059] (8) Rinse the resin: rinse the resin twice with DMF (10 ml / g), DCM (10 ml / g), and DMF (10 ml / g) respectively.

[0060] (9) Repeat steps (2) to (8) to sequentially connect the amino acids shown in SEQ ID No: 1 in Table 1 from right to left.

[0061] (10) Drain and wash the resin as follows: DMF (10 ml / g) twice, methanol (10 ml / g) twice, DMF (10 ml / g) twice, and DCM (10 ml / g) twice, drain for 10 min.

[0062] (11) Cleavage of the peptide from the resin: The cleavage solution used was composed of 95% TFA (trifluoroacetic acid), 1% water, 2% EDT (mercaptoethanol), and 2% TIS (triisopropylsilane); the cleavage time was 120 min.

[0063] (12) Blow-drying and washing: The lysate was blown dry with nitrogen, washed with ether six times, and then allowed to evaporate at room temperature.

[0064] (13) Analysis, purification and freeze drying: The crude polypeptide was purified by high performance liquid chromatography; the polypeptide solution was collected and placed in a freeze dryer for concentration, and freeze-dried to obtain a white powder to obtain antimicrobial peptide 1.

[0065] (14) According to the peptide sequences in Table 1, the types and amounts of the added amino acids were adjusted by the methods (1) to (13) to prepare antimicrobial peptides 2 to 18, respectively. These peptides were used in the antimicrobial inhibition experiment.

[0066] Example 1

[0067] Method for determining the inhibitory effect of antimicrobial peptides on Propionibacterium acnes

[0068] (i) Preparation of antimicrobial peptides

[0069] Liquid culture screening: Use a dispenser to directly dispense 10 μL of an antimicrobial peptide dissolved in pure water to 1 mg / mL. Add 10 antimicrobial peptides sequentially, starting from the second grid on the left, to a new 96-well plate to complete the positive and negative controls. Place the 96-well plate horizontally. Controls include pure water and bacteria at the same concentration as the antimicrobial peptide, a control antimicrobial peptide and bacteria, ciprofloxacin and bacteria, and sterile culture medium.

[0070] Liquid culture testing for minimum inhibitory concentration (MIC) of antimicrobial peptides: Dilute to four concentrations. Use a dispenser to pipette 10 μL of sterile water into rows B, C, and D of a new 96-well cell culture plate. Use a dispenser to pipette 10 μL of the peptide directly into rows A and B of a new 96-well cell culture plate. Mix row B thoroughly with a dispenser and transfer 10 μL to row C. Mix row C thoroughly and transfer 10 μL to row D. Mix row D thoroughly and discard 10 μL. Repeat for rows E, F, G, and H. Complete positive and negative controls (10 μL volume) and set aside.

[0071] (Additional information: If using a 96-well deep-well plate to culture bacteria, add 100 μL of sterile culture medium to all wells.)

[0072] (ii) Bacterial recovery and bacterial suspension preparation

[0073] Take Propionibacterium acnes and inoculate it on anaerobic blood agar plate, place it in anaerobic bag, and culture it anaerobically at 37℃ for 48h. Pick Propionibacterium acnes clones and culture them anaerobically in liquid anaerobic broth medium at 37℃ for 48h. Measure the bacterial concentration and dilute it to 10 7 CFU / mL, set aside.

[0074] (iii) Liquid culture detection of the minimum inhibitory concentration of antimicrobial peptides

[0075] Prepare fresh bacterial suspension diluted to 10 5 CFU / mL, add 90 μL per well of a 96-well plate pre-inoculated with antimicrobial peptide. Incubate anaerobically at 37°C in an anaerobic bag for 24-48 hours. Measure absorbance at 630 nm using a microplate reader and visually inspect for turbidity. The minimum inhibitory concentration (MIC) of the antimicrobial peptide at which turbidity does not occur is defined as the minimum inhibitory concentration (MIC) of 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL.

[0076] (iv) Repeat steps 3 and 4 twice, and decide whether to repeat the third time based on the repeatability of the experiment.

[0077] (v) Based on the results, further minimum inhibitory concentration (MIC) tests were performed and the minimum concentration was adjusted to (1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, 24 μg / ml, 25 μg / ml). 25 concentration gradients were performed to finally determine the MIC of the antimicrobial peptide.

[0078] The anaerobic blood agar plate is a CDC anaerobic blood agar plate.

[0079] The CDC anaerobic blood agar plates were purchased from Henan Meikai Biotechnology Co., Ltd.

[0080] The inhibitory effects of 18 antimicrobial peptides on Propionibacterium acnes are shown in Table 2.

[0081] Example 2

[0082] Determination of the inhibitory effect of antimicrobial peptides on Staphylococcus epidermidis

[0083] (i) Preparation of antimicrobial peptides

[0084] Liquid culture screening: Use a dispenser to directly dispense 10 μL of an antimicrobial peptide dissolved in pure water to 1 mg / mL. Add 10 antimicrobial peptides sequentially, starting from the second grid on the left, to a new 96-well plate to complete the positive and negative controls. Place the 96-well plate horizontally. Controls include pure water and bacteria at the same concentration as the antimicrobial peptide, a control antimicrobial peptide and bacteria, ciprofloxacin and bacteria, and sterile culture medium.

[0085] Liquid culture testing for minimum inhibitory concentration (MIC) of antimicrobial peptides: Dilute to four concentrations. Use a dispenser to pipette 10 μL of sterile water into rows B, C, and D of a new 96-well cell culture plate. Use a dispenser to pipette 10 μL of the peptide directly into rows A and B of a new 96-well cell culture plate. Mix row B thoroughly with a dispenser and transfer 10 μL to row C. Mix row C thoroughly and transfer 10 μL to row D. Mix row D thoroughly and discard 10 μL. Repeat for rows E, F, G, and H. Complete positive and negative controls (10 μL volume) and set aside.

[0086] (Additional information: If using a 96-well deep-well plate to culture bacteria, add 100 μL of sterile culture medium to all wells.)

[0087] (ii) Bacterial recovery and bacterial suspension preparation

[0088] Take Staphylococcus epidermidis and inoculate it in LB medium, normal oxygen concentration (21%), and culture for 48 hours. Pick Staphylococcus epidermidis clones and culture them in LB medium at 37℃ normal oxygen concentration (21%) for 48 hours, measure the bacterial concentration, and dilute it to 10 7 CFU / mL, set aside.

[0089] (iii) Liquid culture detection of the minimum inhibitory concentration of antimicrobial peptides

[0090] Prepare fresh bacterial suspension diluted to 10 5 CFU / mL, add 90 μL per well of a 96-well plate pre-added with antimicrobial peptide. Incubate at 37°C with normal oxygen concentration for 24-48 hours. Measure absorbance at 630 nm using a microplate reader and visually inspect for turbidity. The minimum inhibitory concentration (MIC) of the antimicrobial peptide at which turbidity does not occur is defined as the minimum inhibitory concentration (MIC) of 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL.

[0091] (iv) Repeat steps 3 and 4 twice, and decide whether to repeat the third time based on the repeatability of the experiment.

[0092] (v) Based on the results, further minimum inhibitory concentration (MIC) tests were performed and the minimum concentration was adjusted to (1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, 24 μg / ml, 25 μg / ml). 25 concentration gradients were performed to finally determine the MIC of the antimicrobial peptide.

[0093] The LB culture medium was purchased from Zhongke Ruitai (Beijing) Biotechnology Co., Ltd.

[0094] The inhibitory effects of 18 antimicrobial peptides on Staphylococcus epidermidis are shown in Table 2.

[0095] Example 3

[0096] Determination of the inhibitory effect of antimicrobial peptides on Staphylococcus aureus

[0097] (i) Preparation of antimicrobial peptides

[0098] Liquid culture screening: Use a dispenser to directly dispense 10 μL of an antimicrobial peptide dissolved in pure water to 1 mg / mL. Add 10 antimicrobial peptides sequentially, starting from the second grid on the left, to a new 96-well plate to complete the positive and negative controls. Place the 96-well plate horizontally. Controls include pure water and bacteria at the same concentration as the antimicrobial peptide, a control antimicrobial peptide and bacteria, ciprofloxacin and bacteria, and sterile culture medium.

[0099] Liquid culture testing for minimum inhibitory concentration (MIC) of antimicrobial peptides: Dilute to four concentrations. Use a dispenser to pipette 10 μL of sterile water into rows B, C, and D of a new 96-well cell culture plate. Use a dispenser to pipette 10 μL of the peptide directly into rows A and B of a new 96-well cell culture plate. Mix row B thoroughly with a dispenser and transfer 10 μL to row C. Mix row C thoroughly and transfer 10 μL to row D. Mix row D thoroughly and discard 10 μL. Repeat for rows E, F, G, and H. Complete positive and negative controls (10 μL volume) and set aside.

[0100] (Additional information: If using a 96-well deep-well plate to culture bacteria, add 100 μL of sterile culture medium to all wells.)

[0101] (ii) Bacterial recovery and bacterial suspension preparation

[0102] Take Staphylococcus aureus and inoculate it in LB medium, normal oxygen concentration (21%), and culture it for 48 hours. Pick Staphylococcus aureus clones and culture them in LB medium at 37℃ normal oxygen concentration (21%) for 48 hours, measure the bacterial concentration, and dilute it to 10 7 CFU / mL, set aside.

[0103] (iii) Liquid culture detection of the minimum inhibitory concentration of antimicrobial peptides

[0104] Prepare fresh bacterial suspension diluted to 10 5 CFU / mL, add 90 μL per well of a 96-well plate pre-added with antimicrobial peptide. Incubate at 37°C in normal oxygen for 24-48 hours. Measure absorbance at 630 nm using a microplate reader and visually inspect for turbidity. The minimum inhibitory concentration (MIC) of the antimicrobial peptide at which turbidity does not occur is defined as the minimum inhibitory concentration (MIC) of 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL.

[0105] (iv) Repeat steps 3 and 4 twice, and decide whether to repeat the third time based on the repeatability of the experiment.

[0106] (v) Based on the results, further minimum inhibitory concentration (MIC) tests were performed and the minimum concentration was adjusted to (1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, 24 μg / ml, 25 μg / ml). 25 concentration gradients were performed to finally determine the MIC of the antimicrobial peptide.

[0107] The LB culture medium was purchased from Zhongke Ruitai (Beijing) Biotechnology Co., Ltd.

[0108] The inhibitory effects of 18 antimicrobial peptides on Staphylococcus aureus are shown in Table 2.

[0109] Example 4

[0110] Determination of Hemolytic Activity of Antimicrobial Peptides

[0111] Prepare 4 mL of 2% mouse erythrocytes (RBCs), rinse twice with 4 mL of PBS, centrifuge at 3000 rpm for 5 minutes, discard the supernatant, and resuspend the RBCs in 4 mL of PBS. Dissolve 8 mg of peptide in 2 mL of PBS to prepare a 4000 μg / mL peptide stock solution. Subsequently, dilute the peptide stock solution with PBS to 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, and 125 μg / mL. To each well of a 96-well plate, 100 μL of 2% mouse cells and 100 μL of peptide solution at various concentrations (4000 μg / mL, 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, and 125 μg / mL) were added. For negative and positive controls, 100 μL of PBS buffer and 100 μL of 0.1% (w / v) Triton X-100 were added, respectively. Three replicates were set up for each group and incubated at 37°C for 1 hour. The 96-well plate was removed and centrifuged at 3000 rpm for 5 minutes. 100 μL of the supernatant was transferred to a new 96-well plate and the absorbance was measured at 540 nm using a microplate reader. Hemolysis rate (%) = (absorbance sample - absorbance negative) / (absorbance positive - absorbance negative) * 100%.

[0112] The test results of the hemolytic activity of 18 antimicrobial peptides are shown in Table 2.

[0113] Table 2

[0114]

[0115]

Claims

1. An antimicrobial peptide, characterized in that The antimicrobial peptide is antimicrobial peptide 1, and the amino acid sequence of the antimicrobial peptide 1 is shown in SEQ ID No:

1.

2. A method for preparing an antimicrobial peptide according to claim 1, characterized in that: The antimicrobial peptide is artificially synthesized through genetic engineering methods, and is directly obtained from cells through separation and purification methods or directly prepared through chemical synthesis.

3. A use of an antimicrobial peptide according to claim 1, characterized in that: The antimicrobial peptide is used in the preparation of an antimicrobial agent, and the antimicrobial agent acts on Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus aureus.

4. The use of an antimicrobial peptide according to claim 3, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide to Propionibacterium acnes is 10 μg / mL.

5. The use of an antimicrobial peptide according to claim 3, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide against Staphylococcus epidermidis is 18 μg / mL.

6. The use of an antimicrobial peptide according to claim 3, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide against Staphylococcus aureus is 13 μg / mL.

7. The use of an antimicrobial peptide according to claim 3, characterized in that: The minimum hemolytic concentration of the antimicrobial peptide 1 is greater than 2000 μg / mL.

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