Antimicrobial peptides or their salts, pharmaceutical compositions and their applications

Abstract

The present invention discloses an antimicrobial peptide or its salt, a pharmaceutical composition and application, belonging to the field of biotechnology. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 1, and it has a broad-spectrum antibacterial effect, and has a strong antibacterial effect on various Gram-positive bacteria such as Staphylococcus aureus, Enterococcus and Streptococcus, and various Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa, with strong antibacterial activity, a broad antibacterial spectrum and low toxicity. The present invention also provides derivatives and salts of the antimicrobial peptide, and the antimicrobial peptide, the derivatives and salts of the antimicrobial peptide are used in the research and development of various antimicrobial products or / antimicrobial drugs, and are used in the treatment of bacterial infectious diseases, with significant advantages and broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to an antimicrobial peptide or a salt thereof, a pharmaceutical composition and application thereof. Background Art

[0002] As the problem of bacterial resistance to current antibiotics becomes more and more serious, antimicrobial peptides have gradually come into people's attention as a new anti-infection drug, and some antimicrobial peptides have entered the clinical stage. As an alternative to antibiotic therapy, antimicrobial peptides have become people's hope for solving the problem of bacterial resistance. Antimicrobial peptides are a class of polypeptides with natural biological activity that have inhibitory effects on bacteria, viruses, and fungi. Antimicrobial peptides are widely present in animals and plants. They are the natural immune barrier of organisms (such as amphibians, mammals, insects, and plants) against pathogen invasion. They even exist in the microorganisms' own system and are called the "second defense system." Antimicrobial peptides have the characteristics of high antimicrobial activity, broad antimicrobial spectrum, many types, and low resistance. Therefore, they have broad application prospects in combating drug-resistant pathogen infections. Antimicrobial peptides can also be used in combination with existing drugs as a combination therapy to produce synergistic or even superimposed effects. In addition, antimicrobial peptides can also be used as immunomodulators to mobilize the body's immune response. The above characteristics determine that antimicrobial peptides have great potential for clinical application.

[0003] Although antimicrobial peptides have advantages over current drug classes, their clinical application still faces challenges. For example, the mass production cost of antimicrobial peptides is high; peptide drugs are easily hydrolyzed by proteases and have poor in vivo stability; they are highly cytotoxic and cause hemolysis of red blood cells. Therefore, it is necessary to synthesize new antimicrobial peptides through artificial design and develop stable, low-toxic, and highly effective broad-spectrum antimicrobial drugs to effectively solve the problem of drug resistance of pathogenic bacteria. Summary of the invention

[0004] The purpose of the present invention is to provide an antimicrobial peptide or its salt, its derivative, pharmaceutical composition and application.

[0005] According to one aspect of the present invention, an antimicrobial peptide is provided, the amino acid sequence of the antimicrobial peptide is:

[0006] a1) SEQ ID NO: 1; or

[0007] a2) an amino acid sequence in which one or more amino acid sequences are substituted and / or deleted and / or added to SEQ ID NO: 1 and has the same function as SEQ ID NO: 1; or

[0008] a3) an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology to SEQ ID NO: 1 and has the same function as SEQ ID NO: 1.

[0009] In some embodiments, the configuration of the antimicrobial peptide includes an L-type polypeptide, a D-type polypeptide, a Retro-inverso polypeptide or a cyclic peptide.

[0010] According to another aspect of the present invention, an antimicrobial peptide derivative is provided, which is a chemically modified product of the amino acid sequence of the above antimicrobial peptide, and the chemical modification includes acetylation, amination, methylation, phosphorylation, glycosylation, liposylation, ubiquitination, biotin labeling or fluorescent protein labeling.

[0011] According to another aspect of the present invention, a salt of an antimicrobial peptide is provided, including a metal salt of the above antimicrobial peptide, a salt formed by the above antimicrobial peptide and an inorganic acid or an organic acid, the metal including lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum, the inorganic acid including hydrochloric acid, sulfuric acid, boric acid or carbonic acid, and the organic acid including acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid or oxalic acid.

[0012] According to the fourth aspect of the present invention, there is provided the use of the above-mentioned antimicrobial peptides, antimicrobial peptide derivatives, and antimicrobial peptide salts in the preparation of antibacterial products or / antibacterial drugs, wherein the bacteria are one or more of Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Streptococcus suis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, Salmonella, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

[0013] According to a fifth aspect of the present invention, there is provided a pharmaceutical composition comprising at least one of the above-mentioned antimicrobial peptides, antimicrobial peptide derivatives and antimicrobial peptide salts.

[0014] In some embodiments, the pharmaceutical composition further comprises other active ingredients for combating bacterial infection, the antibacterial activity of the other active ingredients for combating bacterial infection is complementary to the antibacterial activity of the above-mentioned antimicrobial peptides, antimicrobial peptide derivatives, and antimicrobial peptide salts, and the synergistic effect index is less than 0.8.

[0015] In some embodiments, the active ingredient against bacterial infection is directed against infections caused by the following bacteria: Streptococcus zooepidemicus, Streptococcus pneumoniae, Streptococcus milleri, Streptococcus constellatus, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus bovis, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Haemophilus paragallinarum, Moraxella catarrhalis, Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Escherichia coli, Enterobacter aerogenes, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Nocardia asteroides, Alcaligenes xylosoxidans, Salmonella typhimurium, Salmonella pullorum, Salmonella enteritidis, Salmonella gallinarum, Salmonella paratyphi, Salmonella hafnia Bacteria, Edwardsiella, Citrobacter cosseri, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Proteus morganii, Proteus raegi, Proteus anomalous, plague, Yersinia enteritidis, Yersinia pseudotuberculosis, Serratia marcescens, Brucella, phlegm Tatum, non-decarboxylated Leukocytosis, Gleminoella, Morganella morganii, Pasteurella multocida, Providencia, Pasteurella pneumophila, Pasteurella hemolytica, Pasteurella urina, Pasteurella anatipestifer, Lasix West ... , Clostridium botulinum, Clostridium perfringens, Clostridium tetani, intestinal Clostridium, Clostridium putrefaciens, Clostridium difficile, necrotic bacillus, Neisseria gonorrhoeae, Neisseria meningitidis, Vibrio alginolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholerae, Campylobacter fetus, Campylobacter sputum, Campylobacter feces, Campylobacter jejuni, Listeria monocytogenes, Spirillum, Bacteroides faecalis, Bacteroides fragilis, Bacteroides gibbsii, Bacteroides feces, Bacteroides pyogenes, Bacteroides polytrichoides, Bacteroides vulgaris, Bacteroides aeger, Bacteroides ovale, Bacteroides feces, Bacteroides thetaiotaomicron, Bacteroides monothecae, Citrobacter pyogenes, Citrobacter freundii, Citrobacter difficile, Citrobacter amalonicus, Corynebacterium diphtheriae, Corynebacterium pseudodiphtheriae, Corynebacterium conjunctivalis dry, Corynebacterium vaginalis, Corynebacterium acnes, ulcer Corynebacterium, Corynebacterium bovis, Bibacterium multiinum, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium ulcerans, Mycobacterium leprae, Treponema pallidum, Relapsing fever spirochetes, Borrelia burgdorferi, Rickettsia prowazekii, Rickettsia morozekii, Bartonella henselae, Orientia tsutsugamushi, Anaplasma phagocytophilum, Rickettsia burnetii, Bartonella quinquefasciatus, Spotted fever group Rickettsia, Louse-borne Rickettsia, Ornithophilus rhinotracheitis, Bordetella avium, Riemerella anatipestifer, Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia ei, Anaplasma phagocytophilum, Anaplasma thrombocytophilum, Leptospira, Borrelia burgdorferi, Brucella canis, Brucella abortus, Brucella malta, Brucella suis,Mycobacterium bovis, Mycobacterium voles, Mycobacterium voles-like bacteria, Actinomycetes viscolyticus, Actinomycetes odontolyticus, Nocardia asteroides, Nocardia brasiliensis, Nocardia caviae, Bartonella henselae, Acholerae leishmanii, Prevotella melaninogenica, Mycoplasma bovis, Mycoplasma bovis genitalium, Mycoplasma californica, Mycoplasma difficile.

[0016] In some embodiments, the dosage form of the pharmaceutical composition is any one of an aerosol, a spray, a powder, a pill, a tablet, a film, an ointment, a suppository, a paste, a solution, an injection, a mixture, a lotion, or a liniment.

[0017] According to a sixth aspect of the present invention, there is provided the use of the above-mentioned antimicrobial peptides, antimicrobial peptide derivatives, antimicrobial peptide salts or pharmaceutical compositions in the preparation of drugs for preventing or treating bacterial infectious diseases, wherein the bacterial infectious diseases are selected from one or more of acute bacterial upper respiratory tract infection, acute bacterial lower respiratory tract infection, urinary tract infection, bacterial prostatitis, acute infectious diarrhea, bacterial meningitis, brain abscess, bloodstream infection, infective endocarditis, abdominal infection, bone infection, joint infection, skin and soft tissue infection, oral infection, maxillofacial infection, eye infection, vaginal infection, gonorrhea, cervicitis, pelvic inflammatory disease, mycobacterial infection, diphtheria, whooping cough, scarlet fever, plague, anthrax, tetanus, gas gangrene, typhoid and paratyphoid fever and other Salmonella infections, brucellosis, leptospirosis, relapsing fever, Lyme disease, rickettsial disease, and neutropenia with fever.

[0018] Beneficial effects of the present invention: The antimicrobial peptide SAMP17 of the present invention has a broad-spectrum antibacterial effect, and has a strong antibacterial effect on various Gram-positive bacteria such as Staphylococcus aureus, Enterococcus and Streptococcus, and various Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa. It has strong antibacterial activity, a broad antibacterial spectrum, and low toxicity. It can be used in the research and development of various antibacterial drugs and for the treatment of bacterial infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the mass spectrum of the antimicrobial peptide SAMP17 in Example 1 of the present invention.

[0020] Figure 2 This is a liquid chromatogram of the antimicrobial peptide SAMP17 in Example 1 of the present invention.

[0021] Figure 3 This is a graph showing the in vitro bactericidal kinetics of the antimicrobial peptide SAMP17 in Example 3 of the present invention.

[0022] Figure 4 This is a diagram showing the drug resistance development of the antimicrobial peptide SAMP17 in Example 4 of the present invention.

[0023] Figure 5 middle Figure 5 a The survival rate of Hep-2 cells in Example 5 of the present invention when the antimicrobial peptide SAMP17 is added, Figure 5 b is the survival rate of Caco-2 cells when antimicrobial peptide SAMP17 is added. Figure 5 c is the hemolysis rate of red blood cells when the antimicrobial peptide SAMP17 was added.

[0024] Figure 6 middle Figure 6 a is a wound photograph in Example 6 of the present invention, Figure 6 b is the calculation result of wound area change on the 11th day. Figure 6 c is the calculation result of the bacterial load in the wound tissue on the 11th day.

[0025] Figure 7 The scanning electron microscope image and the transmission electron microscope image of Staphylococcus aureus in Example 7 of the present invention are shown. DETAILED DESCRIPTION

[0026] The present invention is further described in detail through specific implementation cases, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art are all limited by the claims attached to this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0027] Example 1

[0028] An antimicrobial peptide SAM17, whose amino acid sequence is AWQAIARLIKGRGRKKYGRIIIIG (SEQ ID NO.1), is synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd. (product name: TG-LG-16255 SAMP17, batch number: TP-20067853, molecular weight: 2738.39), with a purity greater than 95%. Its mass spectrum is as follows Figure 1 As shown, the mass spectrometry detection conditions are: flow rate: 0.2 mL / min, running time: 1 min, mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid acetonitrile solution; the HPLC chromatogram is shown Figure 2As shown, liquid chromatography detection conditions: chromatographic column: 4.6*150mm, kromasil C18-5, mobile phase A: 0.1% trifluoroacetic acid in acetonitrile solution, mobile phase B: 0.1% trifluoroacetic acid aqueous solution, elution conditions: 0-0.01min mobile phase A 5%, mobile phase B 95%; 0.01-25min mobile phase A increased from 5% to 50%, mobile phase B decreased from 95% to 50%; 25-30min mobile phase A increased from 50% to 90%, mobile phase B decreased from 50% to 10%, flow rate: 1mL / min, wavelength: 214nm, injection volume: 20μL.

[0029] Depend on Figure 1 and Figure 2 It can be seen that the antimicrobial peptide SAMP17 of the present invention, whose sequence is shown in SEQ ID NO: 1, was successfully synthesized and separated and purified.

[0030] Example 2 MIC and MBC determination of antimicrobial peptide SAMP17

[0031] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the antimicrobial peptide SAMP17 obtained in Example 1 were determined. 600 The test bacterial solution of 0.1 was diluted with MH medium at 1:100 for later use. The concentration of the antimicrobial peptide SAMP17 was adjusted to 256 μg / mL, and 100 μL of bacterial solution was added to the 1st to 11th columns of the 96-well plate. 100 μL of antimicrobial peptide SAMP17 solution was added to the first column, and 100 μL of bacterial solution was drawn from the first well and diluted to the 10th well in turn. 100 μL of sterile MHB was added to the 12th well. Each test compound was repeated 3 wells, and the cells were cultured in a 37°C incubator for 24 h. The antimicrobial peptide concentration at which no bacteria grew was taken as the minimum inhibitory concentration. After the minimum inhibitory concentration (MIC) results were read, 0.1 mL of the mixed solution in all wells where no bacteria grew was added to the drug-free nutrient agar medium, and the cells were cultured in a 37°C incubator overnight. If the number of colonies grown by the test bacteria on the plate was ≤0.1% of the inoculated bacteria, the antimicrobial peptide concentration was considered to be the minimum bactericidal concentration (MBC). When MBC / MIC≤2, the antimicrobial peptide is considered to have bactericidal effect; when MBC / MIC≥4, the antimicrobial peptide is considered to have antibacterial effect. The specific test results are shown in Table 1.

[0032] Table 1 Antibacterial activity test table of antimicrobial peptide SAMP17

[0033]

[0034] As shown in Table 1, the antimicrobial peptide SAMP17 has good antibacterial and bactericidal activities against both Gram-positive and Gram-negative bacteria, showing a broad-spectrum antibacterial effect.

[0035] Example 3 In vitro bactericidal kinetics of antimicrobial peptide SAMP17

[0036] Draw a line on the frozen Staphylococcus aureus ATCC29213 plate and place it in a 37℃ incubator for culture. Pick a single colony and inoculate it in MH broth for overnight enrichment. Adjust the OD value with MH broth the next day. 600 The value is 0.1 for standby use. According to the MIC value (8µg / ml) of the test bacteria in Example 2, the antimicrobial peptide SAMP17 working solution with a concentration of 10×MIC, 20×MIC, 40×MIC, 80×MIC, 160×MIC, and 320×MIC was prepared. 0.6ml of bacterial solution and 0.6ml of antimicrobial peptide SAMP17 working solution were added to the 6ml MHB system to make the final concentration of the antimicrobial peptide SAMP17 to be tested 1×MIC, 2×MIC, 4×MIC, 8×MIC, 16×MIC, and 32×MIC, and placed in a 37°C shaking incubator for shaking culture. The bacterial solution without the addition of antimicrobial peptide SAMP17 was used as the control group, and the colony count was performed. 0.1 mL of the sample was taken from all tubes at 30 min, 1 h, 2 h, 3 h, and 5 h, and diluted 10-fold and 100-fold respectively, and then evenly spread on the surface of drug-free nutrient agar medium using a disposable spreader stick. All plates were placed in a 36°C incubator for overnight culture for 18 h. The colonies were counted the next day to draw a time-killing curve, see Figure 3 .

[0037] like Figure 3 As shown in the figure, at a concentration of 0.5×MIC, the concentration of the antimicrobial peptide SAMP17 increased sharply, while at a concentration of 1×MIC, the antimicrobial peptide SAMP17 showed an inhibitory effect on the growth of Staphylococcus aureus. In contrast, at concentrations of 2×MIC and 4×MIC, the antimicrobial peptide SAMP17 was able to completely kill Staphylococcus aureus after contacting with Staphylococcus aureus for 30 min and 2 h, respectively, showing a rapid bactericidal effect.

[0038] Example 4 Evaluation of the drug resistance of antimicrobial peptide SAMP17 to Staphylococcus aureus

[0039] According to the minimum inhibitory concentration (MIC) results of Staphylococcus aureus in Example 2, 0.5×MIC of Staphylococcus aureus in a 96-well plate was inoculated into MH broth with a final concentration of 0.5×MIC, incubated at 37°C for 24 hours and used for the next MIC determination. Ciprofloxacin was used as a control. After incubation at 37°C for 24 hours, a new MIC value was read. The experiment lasted for 28 generations. The results are shown in Figure 4 .

[0040] Depend on Figure 4It can be seen that after 28 generations of culture at subinhibitory concentrations, the MIC value of the antimicrobial peptide SAMP17 increased by no more than 2 times, while the MIC value of ciprofloxacin increased by 64 times, indicating that the antimicrobial peptide SAMP17 can avoid inducing drug resistance in Staphylococcus aureus.

[0041] Example 5 Cytotoxicity and erythrocyte toxicity assays of antimicrobial peptide SAMP17

[0042] Hep-2 cells and Caco-2 cells frozen in liquid nitrogen were revived and inoculated in DMEM medium containing 10% fetal bovine serum and subcultured at 37°C and 5% CO2. A 10 cm plate that had grown to 90% or a dense monolayer was selected, the medium was discarded, and it was washed 1-2 times with PBS. Trypsin digestion solution was added to digest the adherent cells to round them, and DMEM medium was added to terminate the digestion. The adherent cells were washed off with a pipette and transferred to a 10 ml centrifuge tube. The supernatant was discarded at 1500 rpm for 4 min, and new DMEM medium was added to make a cell suspension. After counting with a cell counting plate, 100 μL of the cell suspension was aspirated and inoculated in a 96-well plate, with 5000 cells in each well, and placed in a 37°C incubator for overnight culture. Different final concentrations of the antimicrobial peptide SAMP17 solution were added to intervene in the cells for 48 h. The culture medium in the wells was discarded, 100 µL of DMEM culture medium without antibiotics was added, 10 µL of CCK8 solution was added, and the 96-well plate was placed in a 37°C incubator for 40 min-60 min. The absorbance at 450 nm was measured using an ELISA reader. The wells without antimicrobial peptide SAMP17 were used as the control group, and the wells without antimicrobial peptide SAMP17 and cells were used as the blank group. The cell survival rate was calculated based on the OD value.

[0043] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%,

[0044] Among them, As is the OD value of the experimental group, Ac is the OD value of the control group, and Ab is the OD value of the blank well.

[0045] After anesthetizing the ICR mice, the eyeballs were removed and blood was collected. The cells were centrifuged at 1500 rpm for 10 min, the supernatant was carefully aspirated and discarded, PBS was added and gently inverted to resuspend the red blood cells, and the cells were centrifuged at 1500 rpm for 10 min, the supernatant was aspirated and discarded, and PBS was added again to wash until the supernatant was completely clear. The red blood cells were aspirated and added to PBS to prepare a 2% red blood cell suspension for later use. 50 µL of red blood cell suspension was added to a 1.5 mL centrifuge tube, and then the antimicrobial peptide SAMP17 with a final concentration of 1-16×MIC was added. The tube was placed in a 37°C incubator and incubated for 1 h. After centrifugation at 1500 rpm for 10 min, 30 µL of supernatant was aspirated and added to a 96-well plate containing 70 µL PBS. The absorbance of the mixed solution was measured at 450 nm. The negative control was a red blood cell suspension not treated with the antimicrobial peptide SAMP17, and the positive control was a red blood cell suspension treated with 2% Triton. The experiment was repeated three times, and the results are shown in Tables 2 and Figure 5 , the hemolytic calculation formula is:

[0046] Hemolysis (%) = [(Abs sample -Abs PBS) / (Abs Triton - Abs PBS)] × 100%.

[0047] Table 2 Results of in vitro toxicity test of SAMP17 on mammalian cells

[0048]

[0049] From Table 2 and Figure 5 It can be seen that at a concentration of 64 µg / mL, the survival rates of Hep-2 and Caco-2 were 74.53% and 87.95%, respectively, indicating that there was no obvious cytotoxicity within the range of its inhibitory concentration. The hemolytic rate of the antimicrobial peptide SAMP17 was 0% at a concentration of 8 µg / mL. As the concentration increased, the hemolytic effect of the antimicrobial peptide SAMP17 also increased. However, at a concentration of 128 µg / mL, the hemolytic rate of the antimicrobial peptide SAMP17 was 9%, indicating that at a concentration far exceeding the MIC, the antimicrobial peptide SAMP17 was less toxic to red blood cells.

[0050] Example 6 Antimicrobial peptide SAMP17 treats Staphylococcus aureus skin infection in mice

[0051] The frozen methicillin-resistant Staphylococcus aureus YZSA-24 was inoculated into TSB broth, cultured overnight at 37°C, transferred to 10 mL preheated TSB at a ratio of 3%, and cultured in a constant temperature shaker until the logarithmic phase, and then adjusted to an OD value of about 0.5 with TSB broth for later use (the corresponding concentration is about 10 8CFU / mL). The antimicrobial peptide SAMP17 was prepared into a stock solution, which was diluted with PBS to prepare antimicrobial peptide SAMP solutions with final concentrations of 1%, 0.5%, and 0.1% (W / V), with mupirocin (0.1%) as an antibiotic control and PBS as a solvent control.

[0052] Thirty ICR mice were randomly divided into five groups. The mice were anesthetized with isoflurane, and the hair was shaved to expose the skin, which was disinfected with alcohol. The skin with a diameter of 10 mm was cut with sterile surgical scissors to create a circular wound for infection and treatment. 50 µL of Staphylococcus aureus solution was aspirated and added to the skin surface to initiate infection. 40 µL of antimicrobial peptide SAMP17 solution was aspirated and applied to the infected area 1h, 12h, and 24h after infection. The antibiotic control group and the solvent control group were given mupirocin solution and PBS at the same time. Wound images were taken on the 1st, 3rd, 5th, 7th, 9th, and 11th days. On the 11th day, the mice were killed and the wound size was measured. Then the wound skin was excised, cut into pieces, and placed in a 10 mL tube. PBS (0.25 g of skin tissue was added to 4 ml of PBS) was added and vortexed to mix. After 10-fold dilution, 100 µL was aspirated and applied to the TSA plate for viable bacterial count. The results are shown in Figure 6 .

[0053] Depend on Figure 6 It can be seen that by local administration to skin wounds infected with Staphylococcus aureus, the antimicrobial peptide SAMP17 can prevent wound suppuration and promote wound recovery compared with the solvent control. As the concentration of the antimicrobial peptide SAMP17 increases, the speed of wound recovery accelerates. On the 11th day, the area of ​​the wounds in the treatment group was significantly lower than that in the solvent control group. The results of tissue bacterial load showed that compared with the solvent control group, the antimicrobial peptide SAMP17 can significantly reduce the bacterial load of skin tissue, indicating that the antimicrobial peptide SAMP17 has a positive therapeutic effect on skin infections caused by Staphylococcus aureus.

[0054] Example 7 Membrane action mechanism of antimicrobial peptide SAMP17

[0055] The frozen Staphylococcus aureus ATCC29213 was inoculated into 5 mL LB broth and cultured in a 37°C shaker at 180 rpm until the logarithmic growth phase. The antimicrobial peptide SAMP17 was added at a final concentration of 2×MIC. After incubation at 37°C for 2 h, the cells were centrifuged at 4000 rpm for 8 min, the supernatant was discarded, and the cells were washed 3 times with PBS. The centrifuged cells were resuspended in PBS and fixed with 2% glutaraldehyde. The untreated control samples were treated in the same way. After standing at 4°C overnight, the samples were sent to the testing company for electron microscopy observation. The results are shown in Figure 7 .

[0056] Depend on Figure 7It can be seen that the scanning electron microscope showed that the control group of Staphylococcus aureus not treated with the antimicrobial peptide SAMP17 was round, smooth and intact, while the Staphylococcus aureus treated with the 2×MIC antimicrobial peptide SAMP17 had a rough and defective surface, and individual bacterial fragments were visible. Through the transmission electron microscope, it can be observed that the bacteria not treated with the antimicrobial peptide SAMP17 have a complete structure, clear cell membrane boundaries, and uniform cytoplasm. The treated bacteria are obviously broken and the structure is obviously destroyed.

[0057] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

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

1.

2. An antimicrobial peptide according to claim 1, characterized in that: The configuration of the antimicrobial peptide includes an L-type polypeptide or a D-type polypeptide.

3. An antimicrobial peptide derivative, characterized in that: The derivative is a chemically modified product of the amino acid sequence of the antimicrobial peptide according to claim 1, and the chemical modification includes acetylation, amination, methylation, phosphorylation, glycosylation, liposylation, ubiquitination, biotin labeling or fluorescent protein labeling.

4. A salt of an antimicrobial peptide, characterized in that The invention relates to a metal salt of the antimicrobial peptide according to claim 1, or a salt of the antimicrobial peptide according to claim 1 and an inorganic acid or an organic acid, wherein the metal comprises lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum, the inorganic acid comprises hydrochloric acid, sulfuric acid, boric acid or carbonic acid, and the organic acid comprises acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid or oxalic acid.

5. Use of the antimicrobial peptide according to any one of claims 1 to 2, the antimicrobial peptide derivative according to claim 3, or the salt of the antimicrobial peptide according to claim 4 in the preparation of an antibacterial product or / and antibacterial drug, wherein the bacteria is one or more of Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Streptococcus suis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, Salmonella, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

6. A pharmaceutical composition, characterized in that The invention comprises at least one of the antimicrobial peptide according to any one of claims 1 to 2, the antimicrobial peptide derivative according to claim 3, and the salt of the antimicrobial peptide according to claim 4.

7. The pharmaceutical composition according to claim 6, characterized in that The dosage form of the pharmaceutical composition is any one of aerosol, spray, powder, pill, tablet, film, ointment, suppository, paste, solution, injection, mixture, lotion or liniment.

8. Use of the antimicrobial peptide according to any one of claims 1 to 2, the antimicrobial peptide derivative according to claim 3, the antimicrobial peptide salt according to claim 4, or the pharmaceutical composition according to any one of claims 6 to 7 in the preparation of a medicament for preventing or treating Staphylococcus aureus infection.

Images