Broad-spectrum antibacterial peptide, antibacterial agent and application

Abstract

The invention provides a broad-spectrum antibacterial peptide, an antibacterial agent and application, and belongs to the technical field of biological medicine. The invention provides a broad-spectrum antibacterial peptide. The broad-spectrum antibacterial peptide comprises an amino acid sequence as shown in SEQ ID No. 1. The minimum inhibitory concentration of the antibacterial peptide to gram-positive bacteria, gram-negative bacteria and fungi proves that the antibacterial peptide has obvious antibacterial activity to the gram-positive bacteria, the gram-negative bacteria and the fungi. The invention also verifies the minimum inhibitory concentration of the antibacterial peptide to clinical drug-resistant bacteria. The cytotoxicity of the SL-12 disclosed by the invention is relatively weak, the SL-12 only shows obvious cytotoxicity at 200 [mu] g / ml, and the minimum inhibitory concentration of the SL-12 is 6.25-12.5 [mu] g / ml; the SL-12 has no obvious hemolytic activity when the concentration is 100 [mu] g / ml or lower, and is high in safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a broad-spectrum antimicrobial peptide and an antimicrobial agent and their application. 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. According to the World Health Organization (WHO) and other public health agencies, if the problem of antibiotic resistance is not controlled, by 2050, antibiotic-resistant bacteria may become a major threat to global health, causing up to 10 million deaths each year.

[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] In the 1980s, Swedish scientist Boman and others discovered and named the first antimicrobial peptide, cecropin, in Cecropinus cecropinus. To date, more than 3,000 natural or synthetic antimicrobial peptides have been discovered and identified, but only 9 of them have become drugs. Unfortunately, all the druggable antimicrobial peptides are narrow-spectrum antimicrobial peptides, so the development of new broad-spectrum antimicrobial peptides is urgent. Summary of the invention

[0005] The present invention provides a broad-spectrum antimicrobial peptide and an antimicrobial agent and application thereof, which has antimicrobial 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.

[0006] The present invention provides a broad-spectrum antimicrobial peptide, wherein the broad-spectrum antimicrobial peptide comprises any of the following amino acid sequences: (1) SEQ ID No. 1;

[0007] (2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1);

[0008] (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).

[0009] In a specific embodiment of the present invention, the C-terminus of the broad-spectrum antimicrobial peptide also includes an amidation modification.

[0010] The present invention also provides the use of the broad-spectrum antimicrobial peptide in the preparation of an antimicrobial agent.

[0011] In a specific embodiment of the present invention, the antibacterial spectrum of the antibacterial agent includes Gram-positive bacteria, Gram-negative bacteria and fungi.

[0012] In a specific embodiment of the present invention, the antibacterial spectrum of the antibacterial agent also includes drug-resistant bacteria.

[0013] The present invention also provides an antibacterial agent, comprising the above broad-spectrum antibacterial peptide.

[0014] The present invention also provides the use of the broad-spectrum antimicrobial peptide or the antimicrobial agent in preparing products with antibacterial and / or bactericidal properties.

[0015] The present invention also provides the use of the broad-spectrum antimicrobial peptide or the antimicrobial agent in the preparation of a drug for preventing and / or treating infection.

[0016] The present invention also provides a drug for preventing and / or treating infection, wherein the active ingredient comprises the above broad-spectrum antimicrobial peptide or the above antimicrobial agent.

[0017] In a specific embodiment of the present invention, the infection includes infections caused by Gram-positive bacteria, Gram-negative bacteria and fungi, and also includes infections caused by drug-resistant bacteria.

[0018] Beneficial effects: The present invention is based on bioinformatics and AI technology and hopes to screen new antimicrobial peptides from the genome of ticks (Ixodes scapularis). The antimicrobial peptides have antimicrobial activity against bacteria (including clinically resistant strains) and fungi, and have weak cytotoxicity and hemolytic activity, and are important candidate molecules 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 for standard strains, proving that the antimicrobial peptide of the present invention can be used as a broad-spectrum antimicrobial peptide, named SL-12.

[0019] The cytotoxicity of SL-12 of the present invention is relatively weak, and only exhibits significant cytotoxicity at 200 μg / ml, while its minimum inhibitory concentration is 6.25-12.5 μg / ml; SL-12 has no significant hemolytic activity at a concentration of 100 μg / ml or lower, and has high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The molecular structure diagram of SL-12 of the present invention;

[0021] Figure 2 This is a graph showing the results of the SL-12 cytotoxicity test on HaCaT cells;

[0022] Figure 3The graph is the result of the hemolytic activity of SL-12 on mouse erythrocytes, in which PC is the positive control: 10% TritonX-100. DETAILED DESCRIPTION

[0023] The present invention provides a broad-spectrum antimicrobial peptide, wherein the broad-spectrum antimicrobial peptide comprises any of the following amino acid sequences: (1) SEQ ID No. 1;

[0024] (2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1);

[0025] (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).

[0026] The broad-spectrum antimicrobial peptide (SL-12) 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-12 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.

[0027] In one embodiment of the present invention, the amino acid sequence of SL-12 can be as shown in SEQ ID No.1: SLWRKWWRWGRY, or it can be modified or mutated on the basis of SEQ ID No.1, and the corresponding antibacterial activity can be retained. The modification of SL-12 described in the present invention, for example, can be amidated at the C-terminus, and the specific sequence is as follows: SLWRKWWRWGRY-NH2. The present invention does not specifically limit the preparation method of the SL-12, and it can be synthesized by a biological company, such as entrusting Hangzhou Gutuo Biotechnology Co., Ltd. to perform chemical synthesis in one embodiment, or it can be obtained by other means such as expression.

[0028] The SL-12 of the present invention has Figure 1 The structure shown can be obtained through https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py, wherein the blue amino acids are positively charged amino acids, which can form electrostatic adsorption with the negative charges on the phospholipid molecules on the cell membrane and bind to the membrane, and the yellow amino acids are hydrophobic amino acids, which can be inserted into the membrane, thereby destroying the cell membrane and completing the bactericidal effect.

[0029] The present invention also provides the use of the broad-spectrum antimicrobial peptide in the preparation of an antimicrobial agent.

[0030] In one embodiment of the present invention, the antibacterial agent has an antibacterial spectrum including Gram-positive bacteria, Gram-negative bacteria and fungi, and its antibacterial spectrum also includes drug-resistant bacteria. In one embodiment of the present invention, Staphylococcus aureus (S. aureus) is taken as an example to illustrate the antibacterial activity of SL-12 against Gram-positive bacteria; Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) are taken as examples to illustrate the antibacterial activity of SL-12 against Gram-negative bacteria; Candida albicans (C. albicans) is taken as an example to illustrate the antibacterial activity of SL-12 against fungi.

[0031] In one embodiment of the present invention, clinical drug-resistant bacteria S. aureus and A. baumannii were taken as examples to demonstrate that SL-12 also has significant antibacterial activity against clinical drug-resistant bacteria.

[0032] The present invention also provides an antibacterial agent, comprising the above broad-spectrum antibacterial peptide.

[0033] Under the conditions of the embodiment of the present invention, the minimum inhibitory concentration MIC of the S. aureus standard strain (ATCC6538) was measured, and its minimum inhibitory concentration MIC was 6.25 μg / ml; the minimum inhibitory concentration MIC of the A. baumannii standard strain (ATCC19606) was 6.25 μg / ml; the minimum inhibitory concentration MIC of the P. aeruginosa standard strain (ATCC9027) was 12.5 μg / ml; the minimum inhibitory concentration MIC of the E. coli standard strain (ATCC25922) was 25 μg / ml; and the minimum inhibitory concentration MIC of the C. albicans standard strain (ATCC 10231) was 12.5 μg / ml.

[0034] 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 12.5 μg / ml; and the MIC for CRPA (clinical drug-resistant bacteria of P. aeruginosa) was 6.25 μg / ml.

[0035] In summary, the examples of the present invention demonstrate the broad-spectrum antibacterial activity of the antimicrobial peptide and its antibacterial activity against drug-resistant bacteria, and the antimicrobial peptide is useful for preparing antimicrobial agents.

[0036] The present invention also provides the use of the broad-spectrum antimicrobial peptide or the antimicrobial agent in preparing products with antibacterial and / or bactericidal properties.

[0037] The present invention also provides the use of the broad-spectrum antimicrobial peptide or the antimicrobial agent in the preparation of a drug for preventing and / or treating infection.

[0038] The examples of the present invention also confirmed that the broad-spectrum antimicrobial peptide SL-12 has weak cytotoxicity, and only exhibits obvious cytotoxicity at a concentration of 200 μg / ml or above, and its minimum inhibitory concentration is 6.25-12.5 μg / ml; SL-12 has no obvious hemolytic activity at a concentration of 100 μg / ml or lower, has high safety, and has the prospect of preparing drugs for preventing and / or treating infections.

[0039] The present invention also provides a drug for preventing and / or treating infection, wherein the active ingredient comprises the above broad-spectrum antimicrobial peptide or the above antimicrobial agent.

[0040] 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.

[0041] In a specific embodiment of the present invention, the infection includes infections caused by Gram-positive bacteria, Gram-negative bacteria and fungal infections, and also includes infections caused by drug-resistant bacteria.

[0042] In order to further illustrate the present invention, a broad-spectrum antimicrobial peptide, antimicrobial agent and application provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1 Obtaining SL-12

[0044] Prediction of antimicrobial peptides from the genome of Ixodes scapularis

[0045] Download high-quality Lxodes persulcatus genome data (GCA_013358835.2) from NCBI, and use TBtools to predict the 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.

[0046] The antimicrobial peptide SL-12 sequence obtained by screening: SLWRKWWRWGRY-NH2 (NH2 represents amidation modification of the C-terminus), was commissioned to Hangzhou Gutuo Biotechnology Co., Ltd. for synthesis.

[0047] Example 2 Verification of SL-12 antimicrobial peptide activity

[0048] 1. Verify the minimum inhibitory concentration (MIC) of SL-12 against different standard strains (purchased from Guangdong Microbial Culture Collection Center)

[0049] 1. Method for determining the minimum inhibitory concentration MIC:

[0050] Activate the bacterial strains preserved in the laboratory, inoculate them onto solid culture media (LB medium for bacteria, YM medium for fungi), and invert and culture them in a 37°C constant temperature incubator for 18 to 24 hours. Then pick a single colony on the solid culture medium and add it to a newly prepared and high-temperature sterilized liquid culture medium, and culture it in a constant temperature shaker at 180rpm and 37°C until the logarithmic growth phase. Take 2ml of the logarithmic growth phase bacterial solution and centrifuge it at 3500rpm for 5 minutes at room temperature. Discard the supernatant, resuspend the bacterial solution with 0.9% saline, wash the fresh bacterial solution repeatedly 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 1OD 600 =1×10 9 CFU / ml was used to calculate the bacterial concentration.

[0051] The bacterial solution was diluted to 2×10 using RPMI1640 medium (containing 10% fetal bovine serum for bacterial culture) or 1 / 4YM+3 / 4RPMI1640 medium (containing 10% fetal bovine serum for fungal culture). 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 sample to be tested (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.

[0052] 2. Culture medium formula:

[0053] LB liquid medium: Tryptone 10 g / L, yeast extract 5 g / L and sodium chloride (NaCl) 10 g / L;

[0054] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L and agar powder 15 g / L;

[0055] YM liquid medium: YM 21g / L;

[0056] YM solid medium: YM 21g / L and agar powder 15g / L;

[0057] RPMI1640 medium: CORNING and 10-040-CV.

[0058] The results are shown in Table 1. SL-12 has a broad-spectrum antibacterial activity, and has significant antibacterial activity against Gram-positive bacteria (S. aureus), Gram-negative bacteria (A. baumannii, P. aeruginosa and E. coli) and fungi (C. albicans).

[0059] Table 1 Antibacterial activity of SL-12 against standard strains

[0060] strain MIC (μg / ml) S. aureus (ATCC6538) 6.25 A.baumannii (ATCC19606) 6.25 P.aeruginosa (ATCC9027) 12.5 E.coli (ATCC25922) 25 C.albicans (ATCC10231) 12.5

[0061] 3. MIC determination of SL-12 against clinical drug-resistant bacteria

[0062] Clinical drug-resistant strains were isolated from the First Affiliated Hospital of Kunming Medical University, and the MIC determination method was the same as above. Among them, clinical drug-resistant bacteria MRSA-Z, MRSA11 and MRSA22 of S. aureus have been disclosed in the literature (Identification and Characterization of RK22, a Novel Antimicrobial Peptide from Hirudinaria manillensis against Methicillin Resistant Staphylococcus aureus); CRAB (clinical drug-resistant bacteria of A. baumannii) and CRPA (clinical drug-resistant bacteria of P. aeruginosa) have been disclosed in the literature (The antimicrobial peptide ZY4 combats multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii infection).

[0063] The results are shown in Table 2. The minimum inhibitory concentration of SL-12 against clinical drug-resistant strains is comparable to that of standard strains. The MICs of clinical drug vancomycin against MRSA-Z, MRSA11, and MRSA22 are 1.56μg / ml, 3.125μg / ml, and 0.39μg / ml, respectively; the antibacterial ability of SL-12 against some MRSA is close to that of vancomycin.

[0064] Table 2 Antibacterial activity of SL-12 against clinical drug-resistant bacteria

[0065] 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) 12.5 CRPA (clinical drug-resistant strain of P. aeruginosa) 6.25

[0066] 4. SL-12 Cytotoxicity Assay

[0067] 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 in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Then transfer to a cell culture bottle and culture in a 5% carbon dioxide, 37℃ constant temperature cell culture incubator.

[0068] 2) CCK8 detection of sample cytotoxicity

[0069] 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 37℃ 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 37℃, 5% CO 2 Incubate in a constant temperature incubator for 24 hours, add 10 μl of CCK8 to each well, continue to incubate in the incubator for about 2 to 4 hours, and measure the absorbance at 450 nm with a multifunctional microplate reader.

[0070] The results are as follows Figure 2As shown, the cytotoxicity of SL-12 was weak, and it only showed significant antibacterial activity at 200 μg / ml, while its minimum inhibitory concentration was 6.25-12.5 μg / ml.

[0071] 5. SL-12 hemolytic activity assay

[0072] 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 in a 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 3-5 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 Triton X-100 (10%) at 540nm of the microplate reader was 0.7-0.8. 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% TritonX-100.

[0073] The results are as follows Figure 3 As shown, SL-12 had no obvious hemolytic activity at concentrations of 100 μg / ml and below, and its minimum inhibitory concentration was 6.25-12.5 μg / ml.

[0074] 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. A broad-spectrum antimicrobial peptide, characterized in that: The broad-spectrum antimicrobial peptide comprises any of the following amino acid sequences: (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 broad-spectrum antimicrobial peptide according to claim 1, characterized in that: The C-terminus of the broad-spectrum antimicrobial peptide also includes amidation modification.

3. Use of the broad-spectrum 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 antibacterial spectrum of the antibacterial agent includes Gram-positive bacteria, Gram-negative bacteria and fungi.

5. The use 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 It comprises the broad-spectrum antimicrobial peptide described in claim 1 or 2.

7. 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 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 broad-spectrum 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 infections include those caused by Gram-positive bacteria, Gram-negative bacteria and fungi, and also include infections caused by drug-resistant bacteria.

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