Antibacterial peptide and application thereof

Abstract

The invention provides an antibacterial peptide and application thereof, and belongs to the technical field of biological medicine. Based on bioinformatics and an AI technology, a novel broad-spectrum antibacterial peptide is expected to be screened from tick (hard tick shoulder) genomes, and the antibacterial peptide has antibacterial activity on bacteria (including clinical drug-resistant strains) and fungi, does not have obvious cytotoxicity and hemolytic activity, and is an important candidate molecule of anti-infective drugs. In the embodiment of the invention, the minimum inhibitory concentration of the antibacterial peptide on gram-positive bacteria, gram-negative bacteria and fungi is verified, and the antibacterial peptide is proved to have obvious antibacterial activity on the gram-positive bacteria, the gram-negative bacteria and the fungi. The invention also verifies that the minimum inhibitory concentration of the antibacterial peptide to clinical drug-resistant bacteria is equivalent to the minimum inhibitory concentration of the antibacterial peptide to standard strains, and verifies that the antibacterial peptide can be used as a broad-spectrum antibacterial peptide and is named as SL-15.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an antimicrobial peptide and application thereof. Background Art

[0002] Antibiotic resistance refers to the increased resistance of bacteria and other microorganisms to antibiotics, which makes antibiotics ineffective in inhibiting or killing these microorganisms. At present, the problem of antibiotic resistance needs to be controlled urgently.

[0003] Antimicrobial peptides are a class of small molecule polypeptides that exist in nature. They are usually composed of 10 to 50 amino acids and are widely found in animals, plants and microorganisms. Antimicrobial peptides usually kill bacteria by binding to bacterial cell membranes, causing membrane rupture. They kill bacteria quickly, so they are not prone to drug resistance and are an ideal alternative to antibiotics.

[0004] At present, all the antimicrobial peptides that have become drugs are narrow-spectrum antimicrobial peptides, so there is an urgent need to develop broad-spectrum antimicrobial peptides. Summary of the invention

[0005] The object of the present invention is to provide an antimicrobial peptide and application thereof. The antimicrobial peptide of the present invention has broad-spectrum antimicrobial activity.

[0006] The present invention provides an antimicrobial peptide, wherein the amino acid sequence of the broad-spectrum antimicrobial peptide is shown in any one of the following:

[0007] (1) SEQ ID No. 1;

[0008] (2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1);

[0009] (3) An amino acid sequence obtained by converting one or more amino acids in the amino acid sequence described in (1) into D-amino acids, and having the same function as the amino acid sequence described in (1).

[0010] Preferably, the C-terminus of the antimicrobial peptide further includes an amidation modification.

[0011] The present invention also provides the use of the antimicrobial peptide described in the above scheme in the preparation of an antimicrobial agent.

[0012] Preferably, the antibacterial spectrum of the antibacterial agent includes bacteria and / or fungi; the bacteria include Gram-positive bacteria and / or Gram-negative bacteria.

[0013] Preferably, the antibacterial spectrum of the antibacterial agent also includes drug-resistant bacteria.

[0014] The present invention also provides an antimicrobial agent, comprising the antimicrobial peptide described in the above scheme.

[0015] The present invention also provides the use of the antimicrobial peptide or the antimicrobial agent described in the above scheme in the preparation of products with antibacterial and / or bactericidal properties.

[0016] The present invention also provides the use of the broad-spectrum antimicrobial peptide or the antimicrobial agent in the above scheme in the preparation of a drug for preventing and / or treating infection.

[0017] The present invention also provides a drug for preventing and / or treating infection, wherein the active ingredient comprises the antimicrobial peptide or the antimicrobial agent described in the above scheme.

[0018] Preferably, the infection includes bacterial infection and fungal infection; the bacteria include Gram-positive bacteria and / or Gram-negative bacteria; or, the infection includes drug-resistant bacterial infection.

[0019] The present invention provides an antimicrobial peptide. Based on bioinformatics and AI technology, the present invention hopes to screen a new broad-spectrum antimicrobial peptide from the genome of ticks (Ixodes scapularis), which has antimicrobial activity against bacteria (including clinically resistant strains) and fungi, and has no obvious cytotoxicity and hemolytic activity, and is an important candidate molecule for anti-infective drugs. The minimum inhibitory concentration of the antimicrobial peptide against Gram-positive bacteria, Gram-negative bacteria and fungi was verified in the examples of the present invention, proving that the antimicrobial peptide has obvious antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and fungi. The present invention also verifies the minimum inhibitory concentration of the antimicrobial peptide against clinically resistant bacteria, which is equivalent to the minimum inhibitory concentration against standard strains, confirming that the antimicrobial peptide of the present invention can be used as a broad-spectrum antimicrobial peptide, named SL-15. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 is the structural diagram of the antimicrobial peptide SP-15;

[0022] Figure 2 The figure shows the results of the SL-15 cytotoxicity test on HaCaT cells;

[0023] Figure 3 This is the result of the hemolytic activity of SL-15 on mouse erythrocytes. DETAILED DESCRIPTION

[0024] The present invention provides an antimicrobial peptide, wherein the amino acid sequence of the broad-spectrum antimicrobial peptide is shown in any one of the following: (1) SEQ ID No. 1; (2) an amino acid sequence obtained by replacing, deleting or adding one or more amino acids in the amino acid sequence described in (1); (3) an amino acid sequence obtained by converting one or more amino acids in the amino acid sequence described in (1) into D-type amino acids, and maintaining the same function as the amino acid sequence described in (1).

[0025] The broad-spectrum antimicrobial peptide (SL-15) of the present invention is obtained by screening from the tick genome based on bioinformatics and AI technology. In the embodiment, it is screened from the genome of the Ixodes scapularis. It is verified by the embodiment that the broad-spectrum antimicrobial peptide SL-15 has antibacterial activity against bacteria (including clinical drug-resistant strains) and fungi, and has weak cytotoxicity and hemolytic activity, and is an important candidate molecule for anti-infective drugs.

[0026] In one embodiment of the present invention, the amino acid sequence of SL-15 may be as shown in SEQ ID No. 1: from N-terminus to C-terminus, SPLRWLSRVMKHRKR, or may be modified or mutated based on SEQ ID No. 1 as long as the corresponding antibacterial activity is retained.

[0027] In the specific implementation of the present invention, the C-terminus of the antimicrobial peptide also includes amidation modification, and the amino acid sequence is specifically: SPLRWLSRVMKHRKR-NH2, NH2 represents the amidation modification of the C-terminus.

[0028] In the present invention, the structure of the antimicrobial peptide is as follows Figure 1 As shown, the structure diagram can be obtained through https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py, where the blue amino acids are positively charged amino acids, among which the dark blue arginine and lysine and the light blue histidine are all basic amino acids, except that the isoelectric point of histidine is 7.59, which is weaker than the other two (arginine isoelectric point 10.8, lysine isoelectric point 9.74), the purple amino acids are polar uncharged amino acids, the green amino acids are special amino acids (no free amino groups), and the yellow amino acids are hydrophobic amino acids. In the present invention, in the antimicrobial peptide, the positively charged amino acids can form electrostatic adsorption with the negative charges on the cell membrane phospholipid molecules to bind to the membrane, and the hydrophobic amino acids can be inserted into the membrane, thereby destroying the cell membrane and completing the bactericidal effect.

[0029] The present invention does not specifically limit the preparation method of SL-12, which can be synthesized by a biological company, such as Hangzhou Gutuo Biotechnology Co., Ltd., which is commissioned to perform chemical synthesis in one embodiment, or obtained by other means such as expression.

[0030] The present invention also provides the use of the antimicrobial peptide described in the above scheme in the preparation of an antimicrobial agent.

[0031] In the specific implementation of the present invention, the antibacterial spectrum of the antibacterial agent includes bacteria and / or fungi; the bacteria include Gram-positive bacteria and / or Gram-negative bacteria.

[0032] In the specific implementation process of the present invention, the Gram-positive bacteria include Staphylococcus aureus (S.aureus); the Staphylococcus aureus includes Staphylococcus aureus ATCC 6538; the Gram-negative bacteria include one or more of Acinetobacter baumannii (A.baumannii), Pseudomonas aeruginosa (P.aeruginosa) and Escherichia coli (E.coli); the Acinetobacter baumannii includes Acinetobacter baumannii ATCC 19606; the Pseudomonas aeruginosa includes Pseudomonas aeruginosa ATCC 9027; the Escherichia coli includes Escherichia coli ATCC25922; the fungus includes Candida albicans (C.albicans); the Candida albicans includes Candida albicans ATCC 10231.

[0033] The antimicrobial peptide of the present invention has a broad-spectrum antimicrobial activity, and has obvious antimicrobial activity against Gram-positive bacteria (S. aureus), Gram-negative bacteria (A. baumannii, P. aeruginosa and E. coli) and fungi (C. albicans).

[0034] The minimum inhibitory concentration of the antimicrobial peptide of the present invention to clinical drug-resistant strains is equivalent to that of standard strains, and the antibacterial ability to some clinical drug-resistant strains of S. aureus is close to that of vancomycin.

[0035] In addition, the antimicrobial peptide of the present invention has no obvious cytotoxicity, even at 200 μg / ml, and the minimum inhibitory concentration of the antimicrobial peptide of the present invention is 6.25-25 μg / ml. The antimicrobial peptide of the present invention also has no obvious hemolytic activity. It can be seen that the antimicrobial peptide of the present invention is highly safe.

[0036] In the specific implementation of the present invention, the antibacterial spectrum of the antibacterial agent also includes drug-resistant bacteria. In the specific implementation of the present invention, the drug-resistant bacteria include clinical drug-resistant bacteria; the clinical drug-resistant bacteria include clinical drug-resistant bacteria of Staphylococcus aureus, clinical drug-resistant bacteria of Acinetobacter baumannii and clinical drug-resistant bacteria of Pseudomonas aeruginosa.

[0037] The present invention also provides an antimicrobial agent, comprising the antimicrobial peptide described in the above scheme.

[0038] In the specific implementation of the present invention, the antimicrobial peptide is the only active ingredient of the antimicrobial agent.

[0039] Under the conditions of the embodiment of the present invention, the minimum inhibitory concentration MIC of the S. aureus standard strain (ATCC 6538) was measured, and its minimum inhibitory concentration MIC was 6.25 μg / ml; the minimum inhibitory concentration MIC of the A. baumannii standard strain (ATCC 19606) was 25 μg / ml; the minimum inhibitory concentration MIC of the P. aeruginosa standard strain (ATCC 9027) was 12.5 μg / ml; the minimum inhibitory concentration MIC of the E. coli standard strain (ATCC25922) was 12.5 μg / ml; the minimum inhibitory concentration MIC of the C. albicans standard strain (ATCC 10231) was 12.5 μg / ml.

[0040] Under the conditions of the embodiment of the present invention, when the minimum inhibitory concentration MIC of clinical drug-resistant bacteria was detected, it was found that the MIC for MRSA-Z (clinical drug-resistant bacteria of S. aureus) was 6.25 μg / ml; the MIC for MRSA 11 (clinical drug-resistant bacteria of S. aureus) was 6.25 μg / ml; the MIC for MRSA22 (clinical drug-resistant bacteria of S. aureus) was 6.25 μg / ml; the MIC for CRAB (clinical drug-resistant bacteria of A. baumannii) was 25 μg / ml; and the MIC for CRPA (clinical drug-resistant bacteria of P. aeruginosa) was 25 μg / ml.

[0041] The present invention also provides the use of the antimicrobial peptide or the antimicrobial agent described in the above scheme in the preparation of products with antibacterial and / or bactericidal properties.

[0042] The present invention also provides the use of the antimicrobial peptide or the antimicrobial agent in the above scheme in the preparation of a drug for preventing and / or treating infection.

[0043] The present invention also provides a drug for preventing and / or treating infection, wherein the active ingredient comprises the antimicrobial peptide or the antimicrobial agent described in the above scheme.

[0044] In the specific implementation of the present invention, the infection includes bacterial infection and fungal infection; the bacteria include Gram-positive bacteria and / or Gram-negative bacteria; or, the infection includes drug-resistant bacterial infection.

[0045] In the specific implementation of the present invention, the antimicrobial peptide or the antimicrobial agent is the only active ingredient of the drug.

[0046] The present invention does not specifically limit the dosage form of the drug, and it can be conventionally prepared using conventional technical means in the art with the aid of pharmaceutically acceptable excipients.

[0047] In the specific implementation of the present invention, the medicine contains an effective dose of the antimicrobial peptide; the effective dose is 6.25-25 μg / ml.

[0048] In order to further illustrate the present invention, an antimicrobial peptide and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] 1. The sequence of the antimicrobial peptide: SPLRWLSRVMKHRKR-NH2 (NH2 represents amidation modification of the C-terminus), named SP-15.

[0051] 2. Source of antimicrobial peptides: obtained from the genome of Ixodes scapularis through the prediction of antimicrobial peptides. Experimental method: Download high-quality Lxodes persulcatus genome data (GCA_013358835.2) from NCBI, and use TBtools to predict its full ORFs to obtain its protein / peptide / protein fragment sequence; after obtaining the sequence, use a special script to perform preliminary filtering and screening on the data, remove redundant and invalid sequences, and output candidate peptide sequences. The candidate peptide sequences are interactively verified by the deep learning antimicrobial peptide prediction model (iAMPCN-master) and the AI ​​learning antimicrobial peptide prediction model (Macrel), and the predicted antimicrobial peptide sequences are selected from them, and then the candidate antimicrobial peptide sequences are chemically synthesized and their specific antimicrobial activity is verified based on in vitro experiments.

[0052] 3. Synthesis of antimicrobial peptides: synthesized by Hangzhou Gutuo Biotechnology Co., Ltd.

[0053] Test Example 1 Minimum inhibitory concentration of the antimicrobial peptide of Example 1 against different standard strains

[0054] 1. Standard strains and culture medium

[0055] Bacteria: Staphylococcus aureus (S. aureus) ATCC 6538, Acinetobacter baumannii (A. baumannii) ATCC 19606, Pseudomonas aeruginosa (P. aeruginosa) ATCC 9027 and Escherichia coli (E. coli) ATCC 25922;

[0056] Fungi: Candida albicans (C.albicans) ATCC 10231; the above standard strains were purchased from Guangdong Microbiological Culture Collection Center;

[0057] Culture medium formula:

[0058] LB liquid medium: Tryptone: 10 g / L; Yeast extract: 5 g / L; Sodium chloride (NaCl): 10 g / L;

[0059] LB solid medium: Tryptone: 10 g / L; Yeast extract: 5 g / L; Sodium chloride (NaCl): 10 g / L; Agar powder: 15 g / L;

[0060] YM liquid medium: YM 21g / L;

[0061] YM solid medium: YM 21g / L; agar powder: 15g / L;

[0062] RPMI1640 medium: CORNING, 10-040-CV.

[0063] 2. Bacterial activation, culture and determination of minimum inhibitory concentration (MIC) in standard strains

[0064] The above bacteria were inoculated into LB solid culture medium for activation. The activation conditions were as follows: inverted culture in a 37°C constant temperature incubator for 18 to 24 hours. Then pick a single colony on the LB solid culture medium and add it to the newly prepared and high-temperature sterilized LB liquid culture medium. Culture it in a constant temperature shaker at 180rpm and 37°C until the logarithmic growth phase of the bacteria. Take 2ml of the logarithmic growth phase bacterial solution and centrifuge it at 3500rpm for 5 minutes at room temperature. Discard the supernatant and resuspend the bacterial solution with 0.9% saline. Repeat the washing of the fresh bacterial solution 3 times and finally resuspend the bacterial solution with an appropriate amount of saline. Use a pipette to take 200μl of the bacterial solution and add it to a sterile 96-well plate. Use an enzyme reader to detect its light absorbance at 600nm and calculate it according to 1OD600=1×10 9 CFU / ml was used to calculate the bacterial concentration. The bacterial concentration was diluted to 2×10 5 CFU / ml. The drug was then diluted using a two-fold gradient dilution method. 100 μl of saline was added to a sterile 96-well plate in advance. 200 μl of the antimicrobial peptide of Example 1 (200 μg / ml) was added to the first well. 100 μl was pipetted and added to the second well, mixed and diluted in sequence. 100 μl was discarded from the last well. Finally, 100 μl of a 2×10 5 CFU / ml bacterial solution was pipetted and mixed, and then placed in a 37°C constant temperature incubator for 16 to 24 hours. The light absorption value of the bacterial solution at 600nm was detected by an ELISA instrument, and the wells where no bacterial growth was detected were taken as the minimum inhibitory concentration, i.e., the MIC value.

[0065] 3. Fungal activation, culture and minimum inhibitory concentration (MIC) determination in standard strains

[0066] Candida albicans was inoculated onto YM solid culture medium for activation. The activation conditions were as follows: inverted culture in a 37°C constant temperature incubator for 18 to 24 hours. Then, a single colony was picked from the YM solid culture medium and added to the newly prepared and high-temperature sterilized YM liquid culture medium. The culture was cultured in a constant temperature shaker at 180rpm and 37°C until the fungus was in the logarithmic growth phase. Take 2ml of the logarithmic growth phase bacterial solution and centrifuge at 3500rpm for 5 minutes at room temperature. Discard the supernatant and resuspend the bacterial solution with 0.9% saline. Repeat the washing of the fresh bacterial solution 3 times and finally resuspend the bacterial solution with an appropriate amount of saline. Use a pipette to take 200μl of the bacterial solution and add it to a sterile 96-well plate. The microplate reader detects its light absorbance at 600nm and calculates the value according to 1OD600=1×10 9 CFU / ml was used to calculate the bacterial concentration. The bacterial concentration was diluted to 2×10 5 CFU / ml. The drug was then diluted using a two-fold gradient dilution method. 100 μl of saline was added to a sterile 96-well plate in advance. 200 μl of the antimicrobial peptide of Example 1 (200 μg / ml) was added to the first well. 100 μl was pipetted and added to the second well, mixed and diluted in sequence. 100 μl was discarded from the last well. Finally, 100 μl of a 2×10 5 CFU / ml bacterial solution was pipetted and mixed, and then placed in a 37°C constant temperature incubator for 16 to 24 hours. The light absorption value of the bacterial solution at 600nm was detected by an ELISA instrument, and the wells where no bacterial growth was detected were taken as the minimum inhibitory concentration, i.e., the MIC value.

[0067] 4. Minimum inhibitory concentration of antimicrobial peptides against different standard strains. See Table 1 for the results.

[0068] Table 1 Minimum inhibitory concentration of antimicrobial peptides against different standard strains

[0069] Bacteria strain MIC (μg / ml) S. aureus (ATCC6538) 6.25 A.baumannii (ATCC19606) 25 P.aeruginosa (ATCC9027) 12.5 E.coli (ATCC25922) 12.5 C.albicans (ATCC10231) 12.5

[0070] The results showed that SP-15 has a broad-spectrum antibacterial activity, and has obvious antibacterial activity against Gram-positive bacteria (S. aureus), Gram-negative bacteria (A. baumannii, P. aeruginosa and E. coli) and fungi (C. albicans).

[0071] Test Example 2 Minimum inhibitory concentration of the antimicrobial peptide of Example 1 against different clinical strains

[0072] MRSA-Z (clinically resistant to S. aureus), MRSA 11 (clinically resistant to S. aureus), MRSA22 (clinically resistant to S. aureus), CRAB (clinically resistant to A. baumannii) and CRPA (clinically resistant to P. aeruginosa) were clinically isolated from the First Affiliated Hospital of Kunming Medical University. Among them, MRSA-Z, MRSA 11 and MRSA22 were disclosed in published articles, see [Identification and Characterization of RK22, a Novel Antimicrobial Peptide from Hirudinaria manillensis against Methicillin-Resistant Staphylococcus aureus]; CRAB and CRPA were disclosed in published articles, see [The antimicrobial peptide ZY4 combats multidrug-resistant Pseudomonas aeruginosaand Acinetobacter baumannii infection].

[0073] The MIC determination method was the same as in Experimental Example 1.

[0074] The minimum inhibitory concentrations of the antimicrobial peptides of Example 1 against different clinical strains are shown in Table 2.

[0075] Table 2 Minimum inhibitory concentration of the antimicrobial peptides of Example 1 against different clinical strains

[0076] Bacteria strain MIC (μg / ml) MRSA-Z (clinical drug-resistant bacteria of S. aureus) 6.25 MRSA11 (clinical drug-resistant bacteria of S. aureus) 6.25 MRSA22 (clinical drug-resistant bacteria of S. aureus) 6.25 CRAB (clinical drug-resistant bacteria of A. baumannii) 25 CRPA (clinical drug-resistant strain of P. aeruginosa) 25

[0077] The results showed that the minimum inhibitory concentration of SP-15 against clinical drug-resistant strains was equivalent to that of standard strains.

[0078] Comparative Test Example 1 Minimum Inhibitory Concentration of Vancomycin against Different Clinical Strains in Test Example 2

[0079] The MIC determination method was the same as that in Experimental Example 1.

[0080] The experiments showed that the MICs of the clinical drug vancomycin against MRSA-Z, MRSA 11 and MRSA22 were 1.56 μg / ml, 3.125 μg / ml and 0.39 μg / ml, respectively. It can be seen that the antibacterial ability of the antimicrobial peptide SP-15 of the present invention against some MRSA is close to that of vancomycin.

[0081] Test Example 3 Cytotoxicity of the Antimicrobial Peptide of Example 1

[0082] The determination method is as follows:

[0083] 1) Take out the human immortalized keratinocytes (HaCaT cells) frozen in the liquid nitrogen tank in the laboratory and put them into a preheated 37℃ water bath for rapid thawing. Centrifuge at 1500rpm for 5min at room temperature, then discard the supernatant on a sterile clean bench and resuspend them in DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Then transfer them to a cell culture bottle and culture them in a 5% carbon dioxide, 37℃ constant temperature cell culture incubator.

[0084] 2) CCK8 detection of sample cytotoxicity

[0085] After 1 to 3 days of culture, when the cells are observed to cover the bottom of the culture flask under a microscope, discard the culture medium and wash once with PBS. Add 1 ml of trypsin to the cell culture flask and gently shake the culture flask. When the cells are observed to fall off the culture flask, use fresh DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin to terminate the digestion and mix with a pipette. Count the cell suspension with a cell counter and adjust its concentration to about 1×10 5 / ml. Add the diluted cell suspension to a sterile 96-well plate, 100μl per well, and add 100μl of PBS buffer to the outermost well of the sterile 96-well plate to maintain the humidity in the plate. Culture in a 37C constant temperature cell culture incubator overnight to allow the cells to adhere to the wall. After the cells adhere to the wall, use a pipette to remove the culture medium and add 200μl of the sample to be tested diluted with DMEM culture medium at different concentrations. The concentration gradient is set to 200, 100, 50, 25, 12.5, 0μg / ml, and 4 replicates are set for each concentration. Place in a constant temperature incubator at 37℃ and 5% CO2 for 24h, and add 10μl CCK8 to each well. Continue to incubate in the incubator for about 2 to 4h, and use a multifunctional microplate reader to detect the absorbance at 450nm.

[0086] Results Figure 2 , indicating that SP-15 still had no obvious cytotoxicity even at 200μg / ml, and its minimum inhibitory concentration was 6.25-25μg / ml.

[0087] Test Example 4 Hemolytic activity of the antimicrobial peptide of Example 1

[0088] Experimental method: The collected fresh mouse blood was placed in an anticoagulant tube containing sodium citrate and mixed by inversion. The collected blood was mixed with sterilized Ash solution at a volume ratio of 1:1, centrifuged at 1500rpm for 5min, and the supernatant was discarded. Then the red blood cells were repeatedly washed with physiological saline for 3 to 5 times until the supernatant no longer appeared red after centrifugation. The red blood cells were resuspended in an appropriate amount of physiological saline, and the red blood cell concentration was adjusted so that the absorbance value of 100μl red blood cell suspension plus 100μl TritonX-100 (10%) at 540nm of the microplate reader was 0.7 to 0.8. Then the sample to be tested was prepared into 100μl of different gradient concentrations and added to a 96-well plate (each concentration was set to 3 replicates), and then 100μl of red blood cell suspension was added and incubated in a 37℃ constant temperature incubator for 30min, followed by centrifugation at 1500rpm for 5min, and the supernatant was aspirated and the light absorption value at 540nm was detected by a microplate reader. The concentrations of the samples to be tested were 0, 25, 50, 100, and 200 μg / ml, and the positive drug was 10% by volume of TritonX-100.

[0089] Results Figure 3 , PC was used as positive control. The results showed that SP-15 had no significant hemolytic activity.

[0090] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antimicrobial peptide, characterized in that: The amino acid sequence of the broad-spectrum antimicrobial peptide is shown in any one of the following: (1) SEQ ID No. 1; (2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1); (3) An amino acid sequence obtained by converting one or more amino acids in the amino acid sequence described in (1) into D-amino acids, and having the same function as the amino acid sequence described in (1).

2. The antimicrobial peptide according to claim 1, characterized in that: The C-terminus of the antimicrobial peptide also includes amidation modification.

3. Use of the antimicrobial peptide according to claim 1 or 2 in the preparation of an antimicrobial agent.

4. The use according to claim 3, characterized in that: The antimicrobial spectrum of the antimicrobial agent includes bacteria and / or fungi; the bacteria include Gram-positive bacteria and / or Gram-negative bacteria.

5. The application according to claim 3, characterized in that: The antibacterial spectrum of the antibacterial agent also includes drug-resistant bacteria.

6. An antibacterial agent, characterized in that Comprising the antimicrobial peptide according to claim 1 or 2.

7. Use of the antimicrobial peptide according to claim 1 or 2 or the antimicrobial agent according to claim 6 in the preparation of products with antibacterial and / or bactericidal properties.

8. Use of the broad-spectrum antimicrobial peptide according to claim 1 or 2 or the antimicrobial agent according to claim 6 in the preparation of a medicament for preventing and / or treating infection.

9. A drug for preventing and / or treating infection, characterized in that: The active ingredient comprises the antimicrobial peptide according to claim 1 or 2 or the antimicrobial agent according to claim 6.

10. The drug according to claim 9, characterized in that: The infection includes bacterial infection and fungal infection; the bacteria include Gram-positive bacteria and / or Gram-negative bacteria; or, the infection includes drug-resistant bacterial infection.

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