Polypeptide with antibacterial and anti-inflammatory double activities or stereoisomer and application thereof

Abstract

The invention relates to the technical field of biomedical polypeptides, in particular to IPC C07K14, and more particularly relates to a polypeptide with antibacterial and anti-inflammatory double activities or a stereoisomer and application thereof. The invention provides a polypeptide with antibacterial and anti-inflammatory double activities, or a stereoisomer thereof. The polypeptide comprises a polypeptide 1; according to the synthesized polypeptide with the antibacterial and anti-inflammatory effects, the minimum inhibitory concentration range of escherichia coli is 50 mu g / mL, the minimum inhibitory concentration range of staphylococcus aureus is 50 mu g / mL, the minimum inhibitory concentration range of propionibacterium acnes is 8 mu g / mL, and the minimum inhibitory concentration range of candida albicans is 25 mu g / mL.

Description

[0001] This is a divisional application with the application date of July 7, 2023, application number CN202310833786.4, and invention name “A polypeptide with dual antibacterial and anti-inflammatory activities or its stereoisomers and applications”. Technical Field

[0002] The present invention relates to the technical field of biopharmaceutical polypeptides, in particular to IPC C07K14, and more specifically to a polypeptide having antibacterial and anti-inflammatory dual activities or a stereoisomer thereof and application thereof. Background Art

[0003] Antimicrobial peptides pass through the plasma membrane structure of bacteria, causing a large amount of cell contents to leak out, eventually leading to the death of pathogenic microorganisms. It is the host's natural defense mechanism 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 cause inflammation, such as infection and tissue damage. Persistent inflammation has become a key pathological basis for many diseases, including arthritis, inflammatory bowel disease, skin diseases, etc.

[0004] The existing patent CN116120422A discloses an antibacterial and anti-inflammatory dual-active polypeptide and its application. The dual-active polypeptide of the invention has good biocompatibility, can specifically inhibit the growth of periodontitis pathogens such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, inhibit the formation of bacterial biofilm, and has high biosafety and acellular biological toxicity, and can prevent bacteria from developing drug resistance. However, it cannot produce an inhibitory effect on bacteria such as Propionibacterium acnes, Staphylococcus aureus, Escherichia coli, and Candida albicans.

[0005] The existing patent CN116217674A discloses an antibacterial / anti-inflammatory polypeptide and its use. The polypeptide of the invention has a short amino acid sequence, a 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 trauma. However, its antibacterial and anti-inflammatory effects need to be improved. Summary of the invention

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

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

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

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

[0010] Preferably, the H at the C-terminus of the amino acid sequence shown in SEQ ID NOs: 1-6 in the amino acid sequence derivatives can be -NR2R3 or -OR2, wherein each R2 and R3 are 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, linoleyl or oleyl.

[0013] Preferably, the polypeptides 1 to 6 comprise an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NOs: 1-35.

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

[0015] In the present invention, polypeptides with dual antibacterial and anti-inflammatory activities are obtained through machine deep learning and experimental screening. The inventors obtained a large number of antibacterial polypeptides through machine deep learning. When studying antibacterial polypeptides, they unexpectedly found that some polypeptides also have anti-inflammatory effects. Further in-depth research has obtained polypeptides with broad-spectrum bactericidal and anti-inflammatory effects.

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

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

[0018] (1) The order of peptide synthesis is from C-terminus to N-terminus: Place 2-chlorotrityl chloride resin in a reaction tube, add dichloromethane (DCM), and shake.

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

[0020] (3) Deprotection: DMF was evaporated off by rotary evaporation, and the mixture was washed twice with piperidine-DMF solvent, and the solvent was removed.

[0021] (4) Detection: Draw piperidine solution, take a dozen resins, wash them three times with ethanol, add detection reagent for detection. Heat at 105-110℃ for 5 minutes. A positive reaction is indicated when the color changes to dark blue.

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

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

[0024] (7) Detection: Take a dozen 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.

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

[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, and DCM (10 mL / g) twice, drain for 10 min.

[0028] (11) Cutting the polypeptide from the resin: The cutting solution used is composed 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 is 120 minutes.

[0029] (12) Blow dry and wash: Blow dry the cutting liquid with nitrogen, wash it with ether six times, and then leave it to evaporate at room temperature.

[0030] (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.

[0031] The third aspect of the present invention provides a polypeptide having dual antibacterial and anti-inflammatory activities or the use of its stereoisomers for preparing cosmetically or pharmaceutically acceptable salts.

[0032] Preferably, the cosmetically or pharmaceutically acceptable salt is a salt formed by polypeptides 1 to 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 to 6 and an inorganic acid or an organic acid; 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 the present invention, a polypeptide with dual antibacterial and anti-inflammatory activities is obtained through machine deep learning and experimental screening.

[0036] 2. In the present invention, the corresponding polypeptide can be synthesized in large quantities through solid phase synthesis, and the difficulty of product purification is greatly reduced.

[0037] 3. The present invention synthesizes 1 to 6 polypeptides with antibacterial and anti-inflammatory effects, which can inhibit the expression of IL-6, IL-1b, CCL-2, IL-1 factor and NO while inhibiting one or more of Escherichia coli, Staphylococcus aureus, Propionibacterium acnes and Candida albicans. The small inhibitory concentration is less than the hemolytic concentration and the maximum non-cytotoxic concentration, and can be used commercially.

[0038] 4. The polypeptide with antibacterial and anti-inflammatory effects synthesized in the present invention has a minimum inhibitory concentration that is lower than the hemolytic concentration and the maximum non-cytotoxic concentration, and has excellent anti-inflammatory effects and can be used commercially.

[0039] 5. The synthetic polypeptide with antibacterial and anti-inflammatory effects in the present invention has a minimum inhibitory concentration range of 50-100 μg / mL for Escherichia coli, a minimum inhibitory concentration range of 4-50 μg / mL for Staphylococcus aureus, a minimum inhibitory concentration range of 2-12.5 μg / mL for Propionibacterium acnes, and a minimum inhibitory concentration range of 25-50 μg / mL for Candida albicans, among which GLVSRLRRLVTPLL has the strongest anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a bar graph showing the effects of the polypeptides of Examples 1 to 6 and LPS, a control group, and dexamethasone on the gene expressions of IL-6, IL-1b, and CCL-2.

[0041] Figure 2 It is a bar graph showing the effects of the polypeptides of Examples 1 to 6 and LPS, a control group, and dexamethasone on IL-10-FR1 gene expression.

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

[0043] Example 1

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

[0045] The polypeptide 1 shown in SEQ ID NOs: 1 in this example is synthesized by the following synthesis steps:

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

[0047] (2) Connect the first amino acid: remove the solvent DCM through the sand core, add 3 times molar excess Fmoc-Lys-OH, add DMF (dimethylformamide) to dissolve, then add 6 times molar excess DIEA (N,N-diisopropylethylamine), shake for 90 minutes, and block with methanol.

[0048] (3) Deprotection: DMF was evaporated off by rotary evaporation, 15 mL / g of 20% (V / V) piperidine-DMF solvent was added, washed for 5 minutes, removed, and then 15 mL / g of 20% (V / V) piperidine-DMF solvent was added, and washed for 15 minutes.

[0049] (4) Detection: Draw piperidine solution, take 15 resins, wash them three times with ethanol, add detection reagent for detection. Heat at 105-110℃ for 5 minutes. A positive reaction is indicated when the color changes to dark blue.

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

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

[0052] (7) Detection: Take 15 resin beads, 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.

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

[0054] (9) Repeat steps (3) to (8) to sequentially connect the amino acids in the sequence 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, and DCM (10 mL / g) twice, drain for 10 min.

[0056] (11) Cutting the polypeptide from the resin: The cutting solution used is composed 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 is 120 minutes, and the cleavage solution is obtained.

[0057] (12) Blow-drying and washing: The lysate was blown dry with nitrogen, washed with ether six times, and then left to evaporate at room temperature.

[0058] (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 the small molecule peptide shown in SEQ ID No: 1.

[0059] The detection reagent is ninhydrin

[0060] Example 2

[0061] The specific implementation 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 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 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 peptides on Candida albicans

[0072] (i) Preparation of peptides

[0073] Liquid culture initial screening: Use a spray gun to directly take 10 μL of peptide dissolved in pure water to 100 μg / mL, and add Examples 1 to 6 to a new 96-well plate from left to right starting from the second grid, to complete the positive and negative controls for standby use. The 96-well plate is placed vertically, and the controls are pure water and bacteria at the same concentration as the antimicrobial peptide, control antimicrobial peptide (FK13) and bacteria, control antimicrobial peptide (HPA3NT3) and bacteria, and sterile culture medium. FK13 amino acid sequence: FPLTWLKWWKWKK; HPA3NT3 amino acid sequence: FKRLKKLFKKIWNWK.

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

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

[0076] (ii) Bacterial recovery and bacterial suspension preparation

[0077] Candida albicans was inoculated into LB medium and cultured at normal oxygen concentration (21%) for 48 hours. Escherichia coli clones were selected and cultured in LB medium at 37°C and normal oxygen concentration (21%) for 48 hours. The bacterial concentration was measured and diluted to 10 7 CFU / mL, set aside.

[0078] (iii) Liquid culture detection of peptide minimum inhibitory concentration

[0079] Prepare fresh bacterial suspension diluted to 10 5 CFU / mL, add 96-well plates pre-added with peptides, 90μL per well. Incubate at 37℃ with normal oxygen concentration for 24-48h. Measure the absorbance at 630nm with an enzyme reader, and observe the turbidity with the naked eye. The lowest concentration without turbidity is the minimum inhibitory concentration of the peptide under the gradient of 100μg / mL, 50μg / mL, 25μg / mL, 12.5μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, and 0.25μg / mL.

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

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

[0082] The inhibitory effects of the six polypeptides on Candida albicans are shown in Table 1. In the same operating system, the polypeptides in Examples 1 and 5 have a better inhibitory effect on Candida albicans, which is 25 μg / mL.

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

[0084] (i) Preparation of peptides

[0085] Liquid culture initial screening: Use a spray gun to directly take 10 μL of peptide dissolved in pure water to 100 μg / mL, and add the peptides in Examples 1 to 6 to a new 96-well plate from left to right starting from the second grid, to complete the positive and negative controls for later use. The 96-well plate is placed vertically, and the controls are pure water and bacteria at the same concentration as the peptide, control antimicrobial peptide (FK13) and bacteria, control antimicrobial peptide (HPA3NT3) and bacteria, ciprofloxacin and bacteria, and sterile culture medium. Prepare three groups.

[0086] A 96-well plate containing 10 μL of peptides at different concentrations: dilute to 15 concentrations, namely 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: If using a 96-well deep-well plate to culture bacteria, add 100 μL of sterile culture medium to all wells)

[0088] (ii) Bacterial recovery and bacterial suspension preparation

[0089] Take E. coli and inoculate it in LB medium, normal oxygen concentration (21%), and culture it for 48 hours. Select E. coli clones and culture them in LB medium at 37℃ and normal oxygen concentration (21%) for 48 hours, measure the bacterial concentration, and dilute it to 10 7 CFU / mL, set aside.

[0090] Take Staphylococcus aureus and inoculate it in LB medium, normal oxygen concentration (21%), and culture it for 48 hours. Select Staphylococcus aureus clones and culture them in LB medium at 37℃ and normal oxygen concentration (21%) for 48 hours, measure the bacterial concentration, and dilute it to 10 7 CFU / mL, set aside.

[0091] Take Propionibacterium acnes and inoculate it on anaerobic blood agar plate, place it in an anaerobic bag, and culture it anaerobically at 37℃ for 48h. Select 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 with culture medium. 7 CFU / mL, set aside.

[0092] (iii) Liquid culture detection of peptide minimum inhibitory concentration

[0093] Prepare fresh bacterial suspension diluted to 10 5CFU / mL, add to a 96-well plate pre-added with the peptide, 90μL per well. Culture at 37℃ normal oxygen concentration for 24-48h; (Propionibacterium acnes is cultured in an anaerobic bag at 37℃ for 24-48h). Measure the absorbance at 630nm with an ELISA reader, and observe the turbidity with the naked eye. The lowest concentration without turbidity is the minimum inhibitory concentration of the peptide at 100μg / mL, 50μg / mL, 25μg / mL, 12.5μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL.

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

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

[0096] The LB 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 plate was purchased from Henan Meikai Biotechnology Co., Ltd.

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

[0100] The inhibitory effects of the six polypeptides on Staphylococcus aureus are shown in Table 1. In the same operating system, the polypeptide in Example 3 has a better inhibitory effect on Staphylococcus aureus, which is 4 μg / mL.

[0101] The inhibitory effects of the six polypeptides on Propionibacterium acnes are shown in Table 1. In the same operating system, the polypeptide in Example 3 has a better inhibitory effect on Propionibacterium acnes, which is 2 μg / mL.

[0102] In Table 1, “ / ” indicates that the experiment of the corresponding bacterial species was not conducted. Table 4 is the same.

[0103] Table 1

[0104]

[0105] 3. Determination of hemolytic concentration of polypeptide

[0106] Take 4 mL of 2% rabbit red blood cells, rinse twice with 4 mL PBS, centrifuge at 3000 rpm for 5 min, discard the supernatant, and resuspend the rabbit red blood cells in 4 mL PBS. Weigh 160 μg of the polypeptides in Examples 1 to 6, FK13, and HPA3NT3 and dissolve them in 1 mL PBS buffer to prepare the polypeptide mother solutions of Examples 1 to 6 and FK13 and HPA3NT3 with a concentration of 160 μg / mL. Then dilute the polypeptide mother solutions to 80 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, and 8 μg / mL with PBS buffer. In each well of a 96-well plate, 100 μL of 2% rabbit red blood cells and 100 μL of peptide solutions of different concentrations (80 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL) were added; 100 μL of PBS buffer and 100 μL of 0.1% (w / v) Triton X-100 were added to the negative control and positive control, respectively; 3 replicate wells were set up for each group and incubated at 37°C for 1 hour. The 96-well plate was taken out, centrifuged at 3000 rpm for 5 minutes, 100 μL of supernatant was transferred to a new 96-well plate, and the absorbance was measured at 540 nm on an ELISA reader. Hemolysis rate (%) = (absorbance sample - absorbance negative) / (absorbance positive - absorbance negative) * 100%.

[0107] The test results of the hemolytic concentrations 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 count; inoculation into 96-well plates: according to 5×10 4 The number of cells plated was calculated, and 100 μL of cell suspension was added to each well. The same sample can be repeated 3 times. Culture in a 37°C incubator: After the cells were inoculated, they were cultured for 4 hours and the polypeptides in Examples 10, 17, 22, 24, and 26 and FK13 and HPA3NT3 polypeptides were added; the concentrations of each polypeptide were 80 μg / mL, 32 μg / mL, and 8 μg / mL, respectively; culture in a 37°C incubator: culture for 60 hours; add 10 μL of CCK8; culture for 2 hours; measure the absorbance at 450 nm: dual wavelength was used for measurement, with a detection wavelength of 450-490 nm and a reference wavelength of 600-650 nm. The results of the maximum non-cytotoxic concentration determined are shown in Table 3.

[0113] Table 3

[0114]

[0115] As can be seen from Tables 1 to 3, the polypeptides of Examples 1 to 3, 5, and 6 can simultaneously inhibit Escherichia coli, Staphylococcus aureus, Propionibacterium acnes, and Candida albicans, and the polypeptide of Example 4 can simultaneously inhibit Staphylococcus aureus, Propionibacterium acnes, and Candida albicans; the minimum inhibitory concentration of Example 4 for Escherichia coli is 100 μg / mL, which is greater than the minimum hemolytic concentration and the maximum non-cytotoxic concentration, and cannot be used to inhibit Escherichia coli. In short, the polypeptides of Examples 1 to 6 have excellent spectrum antibacterial effects.

[0116] 5. Determination of anti-inflammatory effect

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

[0118] (1) Cell culture: RAW264.7 cells to be tested were inoculated in a culture dish. 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 continued culture, the same amount of 100 μg / mL of the polypeptides of Examples 1, 2, 4, 5, and 6, 10 μM of the polypeptide in Example 3, 20 μM of the polypeptide in Example 3, and dexamethasone were added respectively; after the culture was completed, when the cells were collected, they were gently washed 3 times with PBS to remove the interference of the culture medium on subsequent experiments.

[0119] (2) RNA extraction: Use an RNA extraction kit to extract total RNA from cells, and use a UV spectrophotometer to detect RNA concentration and purity. 260 / A 280 At 1.8-2.1, it indicates that the RNA sample is usable.

[0120] (3) cDNA synthesis: According to the instructions of the reverse transcription reagent, it is divided into two steps: one is to remove genomic DNA, and the other is to obtain cDNA through reverse transcription reaction. After this step is completed, the cDNA can be frozen at -80℃ for future use.

[0121] (4) qRT-PCR reaction: According to the primer sequences of inflammatory factors provided in the primerbank, the primers were synthesized, and then the cDNA was used as a template and the specific primers of the cellular inflammatory factors were used to perform qRT-PCR amplification reaction. After the reaction was completed, the amplification curve and melting curve were confirmed, and the standard curve was analyzed.

[0122] (5) Data analysis: The fluorescence signal was detected using a qRT-PCR instrument, and the Ct value was derived. β-actin was used as the internal reference gene. The expression levels of inflammatory factors in cells were calculated by the method. The results are recorded in Table 4. Figure 1 , Figure 2 .

[0123] As can be seen from Table 4, the polypeptides in Example 1 and Example 3 at 20 μM can inhibit the expression of IL-6, IL-1b, and CCL-2 genes, and affect the expression of IL-10-FR1 gene; the polypeptides in Examples 2 and 4 can affect the expression of IL-10-FR1 inflammatory factor gene; the polypeptide in Example 3 can inhibit the expression of IL-1b and CCL-2 genes, and affect the expression of IL-10-FR1 gene; the polypeptides in Examples 5 and 6 can inhibit the expression of IL-6, IL-1b, and CCL-2 inflammatory factor genes. The data show that the anti-inflammatory possibility of polypeptide 1 in Example 1 is greater than polypeptide 3, polypeptide 5, and polypeptide 6.

[0124] Table 4

[0125]

[0126] 6. NO expression

[0127] Experimental equipment: cell culture box (cell room), microplate reader; shaker, centrifuge, pipette, 8 / 12 channel pipette (10ul / 20ul), -80 / -20 refrigerator

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

[0129] Cell culture medium (DMEM, FBS, PS), PBS, LPS, Dexamethasone; NO detection kit Nitric Oxide Assay Kit (beyotime.com). Consumables: pipettes, pipette tips, centrifuge tubes, test tubes, cell culture dishes

[0130] Experimental procedures: 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 macrophages RAW264.7 were placed in a 96-well cell culture plate in a 37°C 5% CO2 incubator and cultured for 24 hours (cell density was adjusted according to actual conditions). 20 μL PBS (negative control) or test peptide or Dexamethasone (100 uM, control) was added to the wells and cultured for 2 hours. 10 ng / mL lipopolysaccharide (LPS) was added and co-cultured with RAW264.7 for 24 hours (LPS concentration was adjusted according to actual conditions).

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

[0133] The tested polypeptides 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 It can be seen that the polypeptides in Examples 1 to 6 all have anti-inflammatory effects.

[0135] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A polypeptide having antibacterial and anti-inflammatory dual activities, or a stereoisomer thereof, characterized in that: It comprises polypeptide 1; the polypeptide 1 comprises any one of the amino acid sequences shown in SEQ ID NO: 1 or its derivatives.

2. The polypeptide having dual antibacterial and anti-inflammatory activities according to claim 1, or its stereoisomer, characterized in that: The H in the amino acid sequence of the polypeptide 1 may be replaced by deuterium or tritium.

3. The polypeptide having dual antibacterial and anti-inflammatory activities according to claim 1, or its stereoisomer, characterized in that: The H at the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 in the amino acid sequence derivative can be replaced by CH3CH(OH)CO- or R1-CO-, wherein R1 is selected from: H, hydroxyl, amino, alkyl, alkenyl.

4. The polypeptide having antibacterial and anti-inflammatory dual activities according to claim 1, or its stereoisomer, characterized in that: The H at the C-terminus of the amino acid sequence shown in SEQ ID NO: 1 in the amino acid sequence derivative can be -NR2R3 or -OR2, wherein each R2 and R3 are independently selected from: H, hydroxyl, amino, alkyl or alkenyl.

5. The polypeptide having dual antibacterial and anti-inflammatory activities according to claim 3 or 4, or its stereoisomer, characterized in that: The alkyl group is selected from any one of methyl, ethyl, isopropyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl, 2-ethylhexyl, 2-methylbutyl, and 5-methylhexyl.

6. The polypeptide having dual antibacterial and anti-inflammatory activities according to claim 3 or 4, or its stereoisomer, characterized in that: The alkenyl group is selected from any one of vinyl, linoleyl or oleyl.

7. The polypeptide having dual antibacterial and anti-inflammatory activities according to claim 1, or its stereoisomer, characterized in that: The polypeptide 1 comprises an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NOs: 1-6.

8. The polypeptide having dual antibacterial and anti-inflammatory activities according to claim 1, or its stereoisomer, characterized in that: The amino acid sequence of the polypeptide 1 is shown in SEQ ID No: 1, GIFKKITGKLFKWIK.

9. A method for preparing a polypeptide having dual antibacterial and anti-inflammatory activities according to any one of claims 1 to 8, or a stereoisomer thereof, characterized in that: The preparation method is selected from any one of a genetic engineering method or a chemical synthesis method.

10. A use of a polypeptide having dual antibacterial and anti-inflammatory activities according to any one of claims 1 to 8, or a stereoisomer thereof, characterized in that: Used in preparing cosmetically or pharmaceutically acceptable salts.

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