A polypeptide with antibacterial and anti-inflammatory dual activity or a stereoisomer thereof and application

Abstract

The present application relates to the technical field of biological medicine polypeptide, especially IPC C07K14, more particularly to a polypeptide with antibacterial and anti-inflammatory activity or stereoisomer and application thereof.The present application provides a polypeptide with antibacterial and anti-inflammatory activity or stereoisomer, including polypeptide 3; the polypeptide with antibacterial and anti-inflammatory effect synthesized in the present application has a minimum inhibitory concentration range of 50 mu g / mL for escherichia coli, a minimum inhibitory concentration range of 4 mu g / mL for staphylococcus aureus, a minimum inhibitory concentration range of 2 mu g / mL for propionibacterium acnes, and a minimum inhibitory concentration range of 50 mu g / mL for candida albicans.

Description

[0001] This application is a divisional application filed on July 7, 2023, with application number CN202310833786.4 and invention title "A polypeptide or its stereoisomer with dual antibacterial and anti-inflammatory activities and its application". Technical Field

[0002] This invention relates to the field of biomedical peptide technology, particularly to IPC C07K14, and more specifically to a peptide or its stereoisomer with dual antibacterial and anti-inflammatory activities and its applications. Background Technology

[0003] Antimicrobial peptides penetrate the plasma membrane structure of harmful bacteria, causing a large outflow of cellular contents and ultimately leading to the death of pathogenic microorganisms. This is a natural defense mechanism of the host against exogenous pathogenic microorganisms. Inflammation is a defensive response of the body's tissues to damaging stimuli. Any factor that can cause tissue and cell damage can induce inflammation, such as infection and tissue injury. Persistent inflammation has become a key pathological basis for many diseases, including arthritis, inflammatory bowel disease, and skin diseases.

[0004] Existing patent CN116120422A discloses an antibacterial and anti-inflammatory dual-activity polypeptide and its applications. This invention's dual-activity polypeptide exhibits good biocompatibility, specifically inhibiting the growth of periodontitis pathogens such as *Porphyromonas gingivalis* and *Actinomyces actinomycetes*, suppressing bacterial biofilm formation, and possessing high biosafety and no cellular toxicity, while also preventing bacterial resistance. However, it does not show inhibitory effects against bacteria such as *Propionibacterium acnes*, *Staphylococcus aureus*, *Escherichia coli*, and *Candida albicans*.

[0005] Existing patent CN116217674A discloses an antibacterial / anti-inflammatory polypeptide and its uses. The polypeptide has a short amino acid sequence, simple structure, convenient synthesis, and high antibacterial / anti-inflammatory activity. It can be used for the prevention and treatment of diseases caused by microbial infections and has a good effect on the healing of skin wounds. However, its antibacterial and anti-inflammatory effects need to be improved. Summary of the Invention

[0006] To address the problems in the prior art, the first aspect of the present invention provides a polypeptide or its stereoisomer with dual antibacterial and anti-inflammatory activities, including any one of polypeptide 1, polypeptide 2, polypeptide 3, polypeptide 4, polypeptide 5, and polypeptide 6; wherein polypeptide 1 to 6 include any 6 of the amino acid sequences or their derivatives shown in SEQ ID NOs: 1-6.

[0007] Preferably, the H in the amino acid sequence of polypeptides 1-6 can be replaced by deuterium or tritium.

[0008] Preferably, the N-terminal H of the amino acid sequence shown in SEQ ID NOs: 1-6 in the derivative of the amino acid sequence can be replaced by CH3CH(OH)CO- or R1-CO-, wherein R1 is selected from: H, hydroxyl, amino, alkyl, alkenyl.

[0009] Preferably, the N-terminal H of the amino acid sequence shown in SEQ ID NOs: 1-6 in the derivative of the amino acid sequence can be replaced by CH3CH(OH)CO- or R1-CO-, wherein R1 is selected from: H, hydroxyl, amino, alkyl, alkenyl.

[0010] Preferably, the C-terminal H of the amino acid sequence shown in SEQ ID NOs: 1-6 in the derivative of the amino acid sequence can be -NR2R3 or -OR2, wherein each R2 and R3 is independently selected from: H, hydroxyl, amino, alkyl or alkenyl.

[0011] Preferably, the alkyl group is selected from any one of methyl, ethyl, isopropyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl, 2-ethylhexyl, 2-methylbutyl, or 5-methylhexyl.

[0012] Preferably, the alkenyl group is selected from any one of vinyl, oleylene, or oleylene.

[0013] Preferably, the polypeptides 1 to 6 contain amino acid sequences that have at least 95% identity with the amino acid sequences shown in SEQ ID NOs: 1-35.

[0014] Preferably, the amino acid sequence of peptide 1 is as shown in SEQ ID No: 1, GIFKKITGKLFKWIK; the amino acid sequence of peptide 2 is as shown in SEQ ID No: 2, KRIVHRLLKKLHSLF; the amino acid sequence of peptide 3 is as shown in SEQ ID No: 3, KKWRKVLKLIKRLVG; the amino acid sequence of peptide 4 is as shown in SEQ ID No: 4, AKRIVKLIKNFFRKL; the amino acid sequence of peptide 5 is as shown in SEQ ID No: 5, LSKWLKKLGKLLAG; and the amino acid sequence of peptide 6 is as shown in SEQ ID No: 6, HFLGVVAKLVSKLF.

[0015] In this invention, peptides with dual antibacterial and anti-inflammatory activities were identified through machine deep learning and experimental screening. The inventors obtained a large number of antibacterial peptides through machine deep learning. While studying these antibacterial peptides, they unexpectedly discovered that some peptides also possessed anti-inflammatory effects. Further in-depth research yielded peptides with broad-spectrum bactericidal and anti-inflammatory effects.

[0016] A second aspect of the present invention provides a method for preparing a polypeptide or its stereoisomer with dual antibacterial and anti-inflammatory activities, wherein the preparation method is selected from any one of genetic engineering methods or chemical synthesis methods.

[0017] The specific steps of the chemical synthesis method (solid-phase synthesis) are as follows:

[0018] (1) The order of polypeptide synthesis is from C-terminus to N-terminus: 2-chlorotriphenylmethyl chloro resin is placed in a reaction tube, dichloromethane (DCM) is added, and the mixture is shaken.

[0019] (2) To add the first amino acid: Filter the solvent through a sand filter, add Fmoc-amino acid-OH, add dimethylformamide (DMF) to dissolve, then add N,N-diisopropylethylamine (DIEA) and shake. Seal with methanol.

[0020] (3) Deprotection: Remove DMF by rotary evaporation, wash twice with piperidine-DMF solvent to remove the solvent.

[0021] (4) Detection: Take a dozen or so resin grains from the piperidine solution, wash them three times with ethanol, and add the test reagent. Heat at 105-110℃ for 5 minutes. A positive reaction is indicated when the color turns dark blue.

[0022] (5) Rinse the resin: Rinse the resin twice in sequence with DMF (10mL / g), DCM (10mL / g), and DMF (10mL / g).

[0023] (6) Condensation: Add three times the excess of DMF to dissolve the next amino acid derivative (from right to left), dissolve three times the excess of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) in DMF in the reaction tube, and then immediately add 10 times the molar amount of DIEA. React for 30 minutes.

[0024] (7) Detection: Take a dozen resin grains, wash them three times with ethanol, add the test reagent, heat at 105℃-110℃ for 5 minutes, and the colorless result is a negative reaction.

[0025] (8) Rinse the resin: Rinse the resin twice in sequence with DMF (10mL / g), DCM (10mL / g), and DMF (10mL / g).

[0026] (9) Repeat steps (3) to (8) to connect the amino acids in the sequence from right to left.

[0027] (10) Drain and wash the resin as follows: DMF (10 mL / g) twice, methanol (10 mL / g) twice, DMF (10 mL / g) twice, DCM (10 mL / g) twice, and drain for 10 minutes.

[0028] (11) Cutting peptides from resin: The cutting solution used consisted of 95% TFA (V / V) (trifluoroacetic acid), 1% (V / V) water, 2% (V / V) EDT (mercaptoethanol), and 2% TIS (V / V) (triisopropylsilane); the cutting time was 120 minutes.

[0029] (12) Drying and washing: Dry the cutting fluid with nitrogen, wash it six times with ether, and then let it evaporate at room temperature.

[0030] (13) Analysis, purification and freeze drying: The crude peptide was purified by high performance liquid chromatography; the peptide solution was collected and concentrated in a freeze dryer and freeze-dried to produce a white powder.

[0031] The third aspect of this invention provides an application of a polypeptide or its stereoisomer with dual antibacterial and anti-inflammatory activities in the preparation of cosmetically or pharmaceutically acceptable salts.

[0032] Preferably, the cosmetically or pharmaceutically acceptable salt is a salt formed by polypeptides 1-6 and an organic base; the organic base includes any one of ethylamine, diethylamine, arginine, lysine, histidine, or piperazine.

[0033] Preferably, the cosmetically or pharmaceutically acceptable salt is a salt formed by polypeptides 1-6 and inorganic or organic acids; the organic acid is any one of acetic acid, citric acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, succinic acid, oxalic acid, and gluconic acid; the inorganic acid is hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

[0034] Beneficial effects:

[0035] 1. In this invention, a polypeptide with dual antibacterial and anti-inflammatory activities is obtained through machine deep learning and experimental screening.

[0036] 2. In this invention, the solid-phase synthesis method can synthesize a large number of corresponding peptides, and greatly reduces the difficulty of product purification.

[0037] 3. This invention synthesizes 1 to 6 polypeptides with antibacterial and anti-inflammatory effects. While inhibiting one or more of *Escherichia coli*, *Staphylococcus aureus*, *Propionibacterium acnes*, and *Candida albicans*, these polypeptides also inhibit the expression of IL-6, IL-1β, CCL-2, IL-1, and NO. The minimum inhibitory concentration (MIC) is lower than the hemolytic concentration and also lower than the maximum non-cytotoxic concentration (NCC), making them suitable for commercial application.

[0038] 4. The polypeptide synthesized in this invention has antibacterial and anti-inflammatory effects, with a minimum inhibitory concentration (MIC) lower than the hemolytic concentration and a maximum non-cytotoxic concentration (NCC), and exhibits excellent anti-inflammatory effects, making it suitable for commercial application.

[0039] 5. The synthesized polypeptides with antibacterial and anti-inflammatory effects in this invention have a minimum inhibitory concentration (MIC) range of 50–100 μg / mL against Escherichia coli, 4–50 μg / mL against Staphylococcus aureus, 2–12.5 μg / mL against Propionibacterium acnes, and 25–50 μg / mL against Candida albicans. Among them, GLVSRLRRLVTPLL has the strongest anti-inflammatory effect. Attached Figure Description

[0040] Figure 1 Bar chart showing the effects of peptides and LPS from Examples 1-6, control group, and dexamethasone on the expression of IL-6, IL-1b, and CCL-2 genes.

[0041] Figure 2 The bar chart shows the effects of the peptides and LPS from Examples 1-6, the control group, and dexamethasone on IL-10-FR1 gene expression.

[0042] Figure 3 The graph shows the inhibition of NO levels in each experimental group in the LPS-stimulated RAW264.7 cell model. Detailed Implementation

[0043] Example 1

[0044] This embodiment provides a polypeptide or its stereoisomer with dual antibacterial and anti-inflammatory activities, including polypeptide 1. The amino acid sequence of polypeptide 1 is shown in SEQ ID No: 1, GIFKKITGKLFKWIK.

[0045] In this embodiment, polypeptide 1, as shown in SEQ ID NOs: 1, was synthesized using the following synthetic steps:

[0046] (1) The order of polypeptide synthesis is from C-terminus to N-terminus: 10g of 2-chlorotriphenylmethyl chloro resin was placed in a reaction tube, 15mL / g of DCM (dichloromethane) was added, and the mixture was shaken for 30 minutes.

[0047] (2) Inoculation of the first amino acid: Remove the solvent DCM by vacuum filtration through a sand core filter, add 3 molar excess of Fmoc-Lys-OH, add DMF (dimethylformamide) to dissolve, then add 6 molar excess of DIEA (N,N-diisopropylethylamine), and shake for 90 minutes. Block with methanol.

[0048] (3) Deprotection: Evaporate DMF by rotary evaporation, add 15 mL / g 20% ​​(V / V) piperidine-DMF solvent, wash for 5 minutes, remove the DMF, add 15 mL / g 20% ​​(V / V) piperidine-DMF solvent again, and wash for 15 minutes.

[0049] (4) Detection: Take 15 resin grains from the piperidine solution, wash them three times with ethanol, and add the test reagent. Heat at 105-110℃ for 5 minutes. A positive reaction is indicated when the color turns dark blue.

[0050] (5) Rinse the resin: Rinse the resin twice in sequence with DMF (10mL / g), DCM (10mL / g), and DMF (10mL / g).

[0051] (6) Condensation: Add three times the excess of DMF to dissolve the next amino acid derivative (from right to left), dissolve three times the excess of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) in DMF in the reaction tube, and then immediately add 10 times the molar amount of DIEA. React for 30 minutes.

[0052] (7) Detection: Take 15 resin grains, wash them three times with ethanol, add the test reagent, heat at 105℃-110℃ for 5 minutes, and the colorless reaction is a negative reaction.

[0053] (8) Rinse the resin: Rinse the resin twice in sequence with DMF (10mL / g), DCM (10mL / g), and DMF (10mL / g).

[0054] (9) Repeat steps (3) to (8) to connect the amino acids in sequence as shown in SEQ ID Nos: 1 from right to left.

[0055] (10) Drain and wash the resin as follows: DMF (10 mL / g) twice, methanol (10 mL / g) twice, DMF (10 mL / g) twice, DCM (10 mL / g) twice, and drain for 10 minutes.

[0056] (11) Cutting peptides from resin: The cutting solution used consisted of 95% (V / V) TFA (trifluoroacetic acid), 1% (V / V) water, 2% (V / V) EDT (mercaptoethanol), and 2% (V / V) TIS (triisopropylsilane); the cutting time was 120 minutes, and the resulting lysate was obtained.

[0057] (12) Drying and washing: Dry the lysate with nitrogen, wash it six times with ether, and then let it evaporate at room temperature.

[0058] (13) Analysis, purification and freeze drying: The crude peptide was purified by high performance liquid chromatography; the peptide solution was collected and concentrated in a freeze dryer and freeze-dried to obtain a white powder, thus obtaining the small molecule peptide shown in SEQ ID No: 1.

[0059] The detection reagent is ninhydrin.

[0060] Example 2

[0061] The specific implementation method of Example 2 is the same as that of Example 1, except that the amino acid sequence of the polypeptide 2 is as shown in SEQ ID No: 2, KRIVHRLLKKLHSLF.

[0062] Example 3

[0063] The specific implementation method of Example 3 is the same as that of Example 1, except that the amino acid sequence of the polypeptide 3 is as shown in SEQ ID No: 3, KKWRKVLKLIKRLVG.

[0064] Example 4

[0065] The specific implementation method of Example 4 is the same as that of Example 1, except that the amino acid sequence of the polypeptide 4 is as shown in SEQ ID No: 4, AKRIVKLIKNFFRKL.

[0066] Example 5

[0067] The specific implementation of Example 5 is the same as that of Example 1, except that the amino acid sequence of the polypeptide 5 is as shown in SEQ ID No: 5, LSKWLKKLGKLLAG.

[0068] Example 6

[0069] The specific implementation of Example 6 is the same as that of Example 1, except that the amino acid sequence of the polypeptide 6 is as shown in SEQ ID No: 6, HFLGVVAKLVSKLF.

[0070] Performance testing

[0071] 1. Determination of the inhibitory effect of polypeptides on Candida albicans

[0072] (i) Preparation of peptides

[0073] Initial screening using liquid culture: Dissolve 10 μL of the peptide in pure water using a multi-channel pipette to a concentration of 100 μg / mL. Add the peptides sequentially from left to right, starting from the second well, to new 96-well plates (Examples 1-6). Complete the positive and negative controls. The 96-well plates are placed vertically. The controls are pure water and bacteria at the same concentration as the antimicrobial peptide, the control antimicrobial peptide (FK13) and bacteria, and the control antimicrobial peptide (HPA3NT3) and bacteria. Sterile culture medium is used. FK13 amino acid sequence: FPLTWLKWWKWKK; HPA3NT3 amino acid sequence: FKRLKKLFKKIWNWK.

[0074] 96-well plates containing different concentrations of peptides: dilute to four concentrations: 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL, for later use.

[0075] (Additional note: If culturing bacteria in a 96-well deep plate, add 100 μL of sterile culture medium to all wells.)

[0076] (ii) Bacterial resuscitation and preparation of bacterial suspension

[0077] Candida albicans was inoculated into LB medium at normal oxygen concentration (21%) and cultured for 48 h. Escherichia coli clones were selected and cultured in LB medium at 37°C with normal oxygen concentration (21%) for 48 h. Bacterial concentration was measured and diluted to 10⁻⁶ with the medium. 7 CFU / mL, ready for use.

[0078] (iii) Detection of minimum inhibitory concentration of peptides by liquid culture

[0079] The prepared fresh bacterial suspension was diluted to 10. 5 Add 90 μL of CFU / mL to each well of a 96-well plate pre-contained with the peptide. Incubate at 37°C with normal oxygen for 24-48 h. Measure the absorbance at 630 nm using a microplate reader and visually observe the degree of turbidity. The lowest concentration at which no turbidity appears is the minimum inhibitory concentration (MIC) of the peptide at a gradient of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL.

[0080] (iv) Repeat steps 3 and 4 twice, and decide whether to do it a third time based on the reproducibility of the experiment.

[0081] The LB culture medium was purchased from Zhongke Ruitai (Beijing) Biotechnology Co., Ltd.

[0082] The inhibitory effects of the six peptides on Candida albicans are shown in Table 1. In the same operating system, the peptides in Examples 1 and 5 showed better inhibitory effects on Candida albicans, with a concentration of 25 μg / mL.

[0083] 2. Determination of the inhibitory effects of polypeptides on Escherichia coli, Staphylococcus aureus, and Propionibacterium acnes

[0084] (i) Preparation of peptides

[0085] Initial screening using liquid culture: Dissolve 10 μL of the peptide in pure water using a multi-channel pipette to prepare a 100 μg / mL peptide concentration. Add the peptides from Examples 1-6 sequentially from left to right, starting from the second well, into a new 96-well plate to complete the positive and negative controls. The 96-well plates are placed vertically. The controls are pure water and bacteria at the same concentration as the peptide, the control antimicrobial peptide (FK13) and bacteria, the control antimicrobial peptide (HPA3NT3) and bacteria, ciprofloxacin and bacteria, and sterile culture medium. Prepare three sets.

[0086] 96-well plates containing 10 μL of peptides at different concentrations: dilute to 15 concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL, for later use.

[0087] (Additional note: If culturing bacteria in a 96-well deep plate, add 100 μL of sterile culture medium to all wells.)

[0088] (ii) Bacterial resuscitation and preparation of bacterial suspension

[0089] Escherichia coli was inoculated into LB medium at normal oxygen concentration (21%) and cultured for 48 h. E. coli clones were then picked and cultured in LB medium at 37°C with normal oxygen concentration (21%) for 48 h. Bacterial concentration was measured, and the culture was diluted to 10⁻⁶ with the appropriate amount of medium. 7 CFU / mL, ready for use.

[0090] Staphylococcus aureus was inoculated into LB medium at normal oxygen concentration (21%) and cultured for 48 h. Staphylococcus aureus clones were then picked and cultured in LB medium at 37°C with normal oxygen concentration (21%) for 48 h. Bacterial concentration was measured and diluted to 10⁻⁶ with the medium. 7 CFU / mL, ready for use.

[0091] Propionibacterium acnes was inoculated onto anaerobic blood agar plates, placed in an anaerobic bag, and incubated anaerobically at 37°C for 48 hours. Propionibacterium acnes clones were then picked and cultured anaerobically in liquid anaerobic broth at 37°C for 48 hours. Bacterial concentration was measured, and the culture was diluted to a 10⁻⁶ concentration with the broth. 7 CFU / mL, ready for use.

[0092] (iii) Detection of minimum inhibitory concentration of peptides by liquid culture

[0093] The prepared fresh bacterial suspension was diluted to 10. 5Add 90 μL of CFU / mL to each well of a 96-well plate pre-laced with the peptide. Incubate at 37°C with normal oxygen for 24-48 h (for Propionibacterium acnes, incubate anaerobically at 37°C for 24-48 h in an anaerobic bag). Measure the absorbance at 630 nm using a microplate reader and visually observe the degree of turbidity. The lowest concentration at which no turbidity appears is the minimum inhibitory concentration (MIC) of the peptide at the following concentrations: 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL.

[0094] (iv) Repeat steps 3 and 4 twice, and decide whether to do it a third time based on the reproducibility of the experiment.

[0095] The LB culture medium was purchased from Zhongke Ruitai (Beijing) Biotechnology Co., Ltd.

[0096] The LB culture medium was purchased from Zhongke Ruitai (Beijing) Biotechnology Co., Ltd.

[0097] The anaerobic blood agar plate is a CDC anaerobic blood agar plate.

[0098] The CDC anaerobic blood agar plates were purchased from Henan Meikai Biotechnology Co., Ltd.

[0099] The inhibitory effects of the six peptides on Escherichia coli, Staphylococcus aureus, and Propionibacterium acnes are shown in Table 1. In the same operating system, the peptides in Examples 1, 2, 3, and 4 all showed an inhibitory effect of 50 μg / mL on Escherichia coli.

[0100] The inhibitory effects of the six peptides on Staphylococcus aureus are shown in Table 1. In the same operating system, the peptide in Example 3 showed the best inhibitory effect on Staphylococcus aureus, at 4 μg / mL.

[0101] The inhibitory effects of the six peptides on Propionibacterium acnes are shown in Table 1. In the same operating system, the peptide in Example 3 showed the best inhibitory effect on Propionibacterium acnes, at 2 μg / mL.

[0102] In Table 1, the " / " indicates that no experiment was conducted with the corresponding bacterial species. Table 4 is the same.

[0103] Table 1

[0104]

[0105] 3. Determination of polypeptide hemolytic concentration

[0106] Take 4 mL of 2% rabbit red blood cells, wash twice with 4 mL of PBS, centrifuge at 3000 rpm for 5 min, discard the supernatant, and resuspend the rabbit red blood cells in 4 mL of PBS. Weigh 160 μg of the peptides from Examples 1-6, FK13, and HPA3NT3, and dissolve them in 1 mL of PBS buffer to prepare peptide stock solutions of Examples 1-6 and FK13 and HPA3NT3 at a concentration of 160 μg / mL. Then dilute the peptide stock solutions with PBS buffer to 80 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, and 8 μg / mL. Add 100 μL of 2% rabbit red blood cells and 100 μL of peptide solutions at different concentrations (80 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL) to each well of a 96-well plate. Add 100 μL of PBS buffer and 100 μL of 0.1% (w / v) Triton X-100 to each negative and positive control, respectively. Set up three replicates per group and incubate at 37°C for 1 h. Remove the 96-well plate, centrifuge at 3000 rpm for 5 min, and transfer 100 μL of the supernatant to a new 96-well plate. Measure the absorbance at 540 nm using a microplate reader. Hemolysis rate (%) = (Absorbance of sample - Absorbance of negative) / (Absorbance of positive - Absorbance of negative) * 100%.

[0107] The test results of hemolysis concentration of the six peptides are shown in Table 2.

[0108] Table 2

[0109]

[0110]

[0111] 4. Cytotoxicity assay

[0112] Preparation of cell suspension: Hacat (human immortalized epidermal cells) cell counting; seeding into 96-well plates: according to 5 × 10⁻⁶ cells / well. 4 The number of cells seeded was determined, and 100 μL of cell suspension was added to each well. Three replicates were performed for the same sample. Cells were cultured at 37°C for 4 hours after seeding. Then, peptides from Examples 10, 17, 22, 24, and 26, as well as FK13 and HPA3NT3 peptides, were added; the concentrations of each peptide were 80 μg / mL, 32 μg / mL, and 8 μg / mL, respectively. Cells were cultured at 37°C for 60 hours. Then, 10 μL of CCK8 was added, and the cells were cultured for another 2 hours. Absorbance was measured at 450 nm using a dual-wavelength method: the detection wavelength was 450-490 nm, and the reference wavelength was 600-650 nm. The results of the maximum non-cytotoxic concentration are shown in Table 3.

[0113] Table 3

[0114]

[0115] As shown in Tables 1-3, the peptides in Examples 1-3, 5, and 6 can simultaneously inhibit *Escherichia coli*, *Staphylococcus aureus*, *Propionibacterium acnes*, and *Candida albicans*. The peptide in Example 4 can also simultaneously inhibit *Staphylococcus aureus*, *Propionibacterium acnes*, and *Candida albicans*. The minimum inhibitory concentration (MIC) of Example 4 against *Escherichia coli* is 100 μg / mL, which is greater than both the minimum hemolytic concentration and the maximum non-cytotoxic concentration, therefore it cannot be used to inhibit *Escherichia coli*. In conclusion, the peptides in Examples 1-6 exhibit excellent broad-spectrum antibacterial effects.

[0116] 5. Anti-inflammatory effect determination

[0117] 1) IL-6, IL-1b, CCL-2, and IL-10-FR1 content testing

[0118] (1) Cell culture: RAW264.7 cells to be tested were seeded in culture dishes. After culture, LPS was not added to the control group, and LPS (lipopolysaccharide) was added to the remaining culture medium to construct an inflammation model. After further culture, the same amount of 100 μg / mL peptides from Examples 1, 2, 4, 5, and 6, 10 μM peptides from Example 3, 20 μM peptides from Example 3, and dexamethasone were added respectively. After culture, when collecting the cells, they were gently washed three times with PBS to remove the interference of the culture medium on subsequent experiments.

[0119] (2) RNA extraction: Total RNA was extracted from cells using an RNA extraction kit, and the RNA concentration and purity were detected using a UV spectrophotometer. 260 / A 280 A value between 1.8 and 2.1 indicates that the RNA sample is usable.

[0120] (3) cDNA synthesis: According to the reverse transcription reagent instructions, it is divided into two steps: first, removing genomic DNA, and second, obtaining cDNA through reverse transcription. After this step is completed, the cDNA can be frozen at -80℃ for later use.

[0121] (4) qRT-PCR reaction: Primers were synthesized based on the primer sequences of inflammatory factors provided in PrimerBank. Then, using cDNA as a template, qRT-PCR amplification was performed using primers specific to inflammatory factors. After the reaction was completed, the amplification curve and melting curve were confirmed, and the standard curve was analyzed.

[0122] (5) Data Analysis: Fluorescence signals were detected using a qRT-PCR instrument, and Ct values ​​were derived. β-actin was used as an internal reference gene. The expression levels of cellular inflammatory factors were calculated using a method described above. The results are recorded in Table 4. Figure 1 , Figure 2 .

[0123] As shown in Table 4, the peptides in Examples 1 and 20 μM of Example 3 can inhibit the expression of IL-6, IL-1b, and CCL-2 genes and affect the expression of IL-10-FR1 gene; the peptides in Examples 2 and 4 can affect the expression of the IL-10-FR1 inflammatory cytokine gene; the peptide in Example 3 can inhibit the expression of IL-1b and CCL-2 genes and affect the expression of IL-10-FR1 gene; the peptides in Examples 5 and 6 can inhibit the expression of IL-6, IL-1b, and CCL-2 inflammatory cytokine genes. This data indicates that peptide 1 in Example 1 has a greater anti-inflammatory potential than peptides 3, 5, and 6.

[0124] Table 4

[0125]

[0126] 6. NO expression status

[0127] Experimental equipment: Cell culture incubator (cell culture room), microplate reader; shaker, centrifuge, pipette, 8 / 12 channel multipipe (10ul / 20ul), -80 / -20°C freezer.

[0128] Experimental reagents: Mouse macrophages RAW264.7 (mouse mononuclear macrophages) (C7505);

[0129] Cell culture media (DMEM, FBS, PS), PBS, LPS, Dexamethasone; Nitric Oxide Assay Kit (beyotime.com). Consumables: pipettes, pipette tips, centrifuge tubes, test tubes, cell culture dishes.

[0130] Experimental procedure: Mouse macrophages RAW264.7 were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P / S) at 37°C and 5% CO2.

[0131] 1×10 4 Mouse-derived RAW264.7 macrophages were cultured in 96-well cell culture plates at 37°C in a 5% CO2 incubator for 24 hours (cell density adjusted according to actual conditions). 20 μL of PBS (negative control), test peptide, or dexamethasone (100 μM, control) was added to each well, and the cells were cultured for 2 hours. Then, 10 ng / mL of lipopolysaccharide (LPS) was added and co-cultured with RAW264.7 cells for 24 hours (LPS concentration adjusted according to actual conditions).

[0132] Collect the supernatant, detect the NO content in the supernatant using a kit, and measure the OD using a microplate reader. 540 .

[0133] The tested peptides were: AMP5 (100 μg / mL, Example 1), AMP9 (100 μg / mL, Example 2), AMP25 (100 μg / mL, Example 4), AMP33 (100 μg / mL, Example 5), AMP38 (100 μg / mL, Example 6), AMP12-20 μM (20 μM, Example 3), and AMP12-10 μM (10 μM, Example 3).

[0134] from Figure 3 As can be seen from the examples, the polypeptides in Examples 1 to 6 all have anti-inflammatory effects.

[0135] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A polypeptide with dual antibacterial and anti-inflammatory activities, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO: 3: KKWRKVLKLIKRLVG.

2. A method for preparing a polypeptide with dual antibacterial and anti-inflammatory activities according to claim 1, characterized in that, The preparation method is selected from either genetic engineering methods or chemical synthesis methods.

3. The application of the polypeptide with dual antibacterial and anti-inflammatory activities according to claim 1, characterized in that, It is used in the preparation of cosmetic products or as a medicine for antibacterial and anti-inflammatory purposes.

Images