Broad-spectrum antibacterial peptide and application thereof

Abstract

The application discloses a broad-spectrum antibacterial peptide and application thereof. Based on an artificial intelligence screening method, a broad-spectrum antibacterial peptide IYKILQLFHKRFKKGFFG is identified from a protein group of a toad. The antibacterial peptide has six positive charges, a GRAVY hydrophobicity of-0.06 in a sequence, a hydrophobicity matrix of 0.522, and shows the characteristics of amphiphilicity; and the sequence length is short, easy to synthesize, and low in cost. Experimental tests show that the antibacterial peptide can inhibit the growth of 11 different strains including four gram-negative bacterial strains and seven gram-positive bacterial strains, and is a broad-spectrum antibacterial peptide. Meanwhile, the half maximum hemolysis concentration and the half maximum cytotoxicity concentration of the antibacterial peptide are greater than 128 mu g / mL, and the antibacterial peptide has good biological safety and can be applied to antibacterial drugs and other antibacterial products (such as disinfectants and detergents).

Description

TECHNICAL FIELD

[0001] The present application relates to an antibacterial peptide, in particular to a broad-spectrum antibacterial peptide and its application in the preparation of drugs, and belongs to the field of biological medicine. BACKGROUND

[0002] Drug-resistant bacterial infection is one of the most serious public health problems at present, and according to the statistics of WHO, 1.3 million people die of drug-resistant bacterial infection every year. Antibacterial peptides are a kind of short peptides composed of 10 to 50 amino acids, and their unique membrane damage antibacterial mechanism is not easy to induce drug resistance of bacteria, so they are regarded as the ideal antibiotic substitutes in the post-antibiotic era. Antibacterial peptides can be isolated from organisms in nature, and in recent years, the development of artificial intelligence technology has also accelerated the process of finding new antibacterial peptides. The number of antibacterial peptides included in the DRAMP database has reached more than 30,000. However, due to the difference in mechanism, different antibacterial peptides have different inhibitory abilities on different bacterial species, and most of the antibacterial peptides do not have broad-spectrum antibacterial ability. SUMMARY

[0003] The purpose of the present application is to mine a new antibacterial peptide with broad-spectrum bactericidal ability and low cytotoxicity from the proteome, and for this purpose, a broad-spectrum antibacterial peptide is identified from the proteome of the toad based on the artificial intelligence screening method.

[0004] The broad-spectrum antibacterial peptide provided by the present application contains 18 amino acids, and the sequence is IYKILQLFHKRFKKGFFG.

[0005] The antibacterial peptide has 6 positive charges, the GRAVY hydrophobicity in the sequence is -0.06, the hydrophobicity square is 0.522, and the characteristics of amphiphilicity are shown. Figure 1 ).

[0006] The antibacterial peptide sequence is short, and can be synthesized by chemical methods such as polypeptide solid-phase synthesis, is easy to synthesize, and has low cost. Through experimental test, the antibacterial peptide can inhibit the growth of 11 different strains including 4 strains of gram-negative bacteria and 7 strains of gram-positive bacteria, and is a broad-spectrum antibacterial peptide. At the same time, the half maximum hemolytic concentration and the half maximum cytotoxicity concentration of the antibacterial peptide are both greater than 128 μg / mL, and the antibacterial peptide has good biological safety, and can be applied to antibacterial drugs and other antibacterial products (such as disinfectants and detergents).

[0007] In summary, the antibacterial peptide provided by the present application has broad-spectrum bactericidal ability, low hemolytic activity and cytotoxicity, good biological safety, and the antibacterial peptide exerts antibacterial effect by damaging the cell membrane, and has the characteristics of not easily leading to drug resistance of bacteria, and is an ideal antibacterial drug and antibacterial product development object. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1Sequence wheel map of the antibacterial peptide of the present application.

[0009] Figure 2 Secondary structure test result of the antibacterial peptide of the present application. DETAILED DESCRIPTION

[0010] The present application will be further described in conjunction with the accompanying drawings and specific embodiments.

[0011] Example 1 Determination of minimal inhibitory concentration of the antibacterial peptide

[0012] In this example, the minimal inhibitory concentration (MIC) of the antibacterial peptide was determined by micro-broth dilution method. The selected strains included four Gram-negative strains (Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 4352, and Pseudomonas aeruginosa ATCC 27853) and seven Gram-positive strains (Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Micrococcus luteus CMCC(B) 28001, Staphylococcus capitis ATCC 27842, Staphylococcus hominis ATCC 27844, and Staphylococcus epidermidis ATCC 35984). Acinetobacter baumannii Acinetobacter baumannii ATCC 17978、 Escherichia coli ATCC 19606, Escherichia coli Escherichia coli Staphylococcus aureus ATCC 25922、 Staphylococcus aureus DH5α) and seven Gram-positive strains (Staphylococcus aureus Bacillus subtilis ATCC 25923、 Enterococcus faecalis ATCC 6538, Bacillus subtilis Micrococcus luteus ATCC 6633, Enterococcus faecalis Staphylococcus capitis ATCC 29212, Micrococcus luteus Staphylococcus hominis CMCC(B) 28001, Staphylococcus capitis Figure 2 ATCC 27842, Staphylococcus hominis ​ ATCC 27844). The bacteria were incubated overnight at 37°C, and the bacterial suspension was diluted to 5 x 10 5 cfu / mL, and 99 μL of the bacterial suspension was added to each well of a 96-well plate. The antibacterial peptide was dissolved in cation-adjusted Mueller-Hinton broth at an initial concentration of 1 mg / mL, and then diluted by two-fold gradient, ranging from 0.125 μg / mL to 128 μg / mL, and 1 μL was added to each well. The 96-well plate was incubated in a 37°C incubator for 20 hours. The lowest peptide concentration at which no bacterial growth was observed was recorded as the MIC of the peptide, as shown in Table 1. The antibacterial peptide was tested to inhibit the growth of 11 different strains, and had a broad-spectrum antibacterial ability.

[0013] Table 1. Minimal inhibitory concentration of the antibacterial peptide against 11 strains

[0014]

[0015] Example 2 Determination of hemolytic activity of the antibacterial peptide

[0016] This example tests the hemolytic activity of the antibacterial peptide on rat red blood cells. Fresh rat blood was centrifuged at 500 g for 5 minutes, the red blood cell pellet was retained and resuspended in PBS buffer (pH 7.4). The red blood cell solution was diluted and added to a 96-well plate at a density of 1 x 10 8 cells per well. The antibacterial peptide was dissolved in PBS and added to the plate in a two-fold dilution gradient, with a final concentration ranging from 0.125 μg / mL to 128 μg / mL. Triton X-100 at 10 mg / mL was used as a positive control to induce 100% hemolysis, and PBS was used as a blank control. After 1 hour of incubation, the plate was centrifuged at 500 g for 5 minutes. Subsequently, the absorbance (OD) of the supernatant was measured at 450 nm. The percentage of hemolysis was calculated as follows: (OD 450, peptide - OD 450, PBS ) / (OD 450, positive - OD 450, PBS ), where OD 450, peptide represents the absorbance at 450 nm of the current concentration of antibacterial peptide treated wells, OD 450, PBS represents the absorbance at 450 nm of the PBS solution treated wells, and OD 450, positive represents the absorbance at 450 nm of the positive control wells. Each concentration was tested in triplicate to obtain the Half Maximal Hemolysis Concentration (HC 50 ) and the Minimum Hemolytic Concentration (MHC) value that caused 10% hemolysis. The antibacterial peptide tested had an HC 50 > 128 μg / mL and an MHC > 128 μg / mL, indicating good biosafety.

[0017] Example 3 Cytotoxicity Assay of the Antibacterial Peptide

[0018] The cytotoxicity of the antimicrobial peptides on HEK293T was evaluated using Alamar Blue method. HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 environment. Staurosporine was used as a positive control for cell toxicity. The antimicrobial peptides were dissolved in PBS buffer at pH 7.4, with a final concentration ranging from 1 μg / mL to 128 μg / mL in a two-fold gradient. After 72 hours of co-treatment of cells with antimicrobial peptides, 10 μL of Alamar Blue reagent was added to each well. Subsequently, the 96-well plate was incubated for 4 hours in the dark, and the absorbance (OD) was measured at a wavelength of 570 nm. The formula for calculating the cell growth inhibition rate is: (OD 570, peptide - OD 570, PBS ) / (OD 570, staurosporine - OD 570, PBS ), where OD 570, peptide represents the absorbance at 570 nm of the current concentration of antimicrobial peptide-treated wells, OD 570, PBS represents the absorbance at 570 nm of the PBS solution-treated wells, and OD 570, staurosporine represents the absorbance at 570 nm of the staurosporine-treated wells. Each concentration was measured in triplicate. The Graphpad Prism software was used to fit the Half Maximal Cytotoxic Concentration (CC 50 ) value. The CC 50 of this antimicrobial peptide was >128 μg / mL.

[0019] Example 4 Determination of the secondary structure of the antimicrobial peptide

[0020] The secondary structure of the antimicrobial peptide was determined using a Chirascan V100 circular dichroism spectrometer and a 1-millimeter path-length quartz cuvette, with a buffer of 20 mM Tris, 200 mM KCl, pH 7.4, and a final concentration of 15 μM of the antimicrobial peptide. The phospholipid membrane components used to simulate the bacterial cell membrane were set at DOPE:DOPG=3:1, with a final concentration of 450 μM. The signal was read at a speed of 1 nm / s in the wavelength range of 200 nm to 260 nm. ​ The distribution of the Mean residue ellipticity of the antimicrobial peptide with wavelength is shown in Figure 4, with each curve being the average of three spectra, and the data was smoothed in the Graphpad Prism 10 software. The proportion of secondary structures of the antimicrobial peptide was statistically analyzed using the K2D algorithm on Dichroweb, as shown in Table 2. Compared with the test results in the solution environment, the proportion of α-helix of the antimicrobial peptide in the phospholipid membrane environment was significantly increased.

[0021] Table 2. The proportion of secondary structure components of antibacterial peptides

[0022]

Claims

1. A broad-spectrum antibacterial peptide, characterized in that, The amino acid sequence of the broad-spectrum antibacterial peptide is as follows: IYKILQLFHKRFKKGFFG.

2. Use of the broad-spectrum antibacterial peptide of claim 1 in the preparation of an antibacterial drug, wherein the bacteria against which the antibacterial drug is directed are one or more of the following: Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus capitis, Staphylococcus hominis, Acinetobacter baumannii, Escherichia coli.

3. Use according to claim 2, wherein the compound is ###0002### The effective concentration of the broad-spectrum antibacterial peptide in the antibacterial drug is greater than or equal to 4 μg / mL.

4. An antibacterial agent, characterized by, The antibacterial drug comprises the broad-spectrum antibacterial peptide of claim 1.

5. Use of the broad-spectrum antibacterial peptide of claim 1 in the preparation of an antibacterial product, wherein the bacteria against which the antibacterial product is directed are one or more of the following: Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus capitis, Staphylococcus hominis, Acinetobacter baumannii, Escherichia coli.

6. Use according to claim 5, wherein The antibacterial product is a disinfectant or a detergent.

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