Broad-spectrum antibacterial peptide and application thereof

Abstract

The invention discloses a broad-spectrum antibacterial peptide and application thereof. According to the invention, a broad-spectrum antibacterial peptide IYKILQLFHKRFKKGFFG is identified from a proteome of toads on the basis of an artificial intelligence screening method. The antibacterial peptide has six positive charges, the hydrophobicity of GRAVY in the sequence is-0.06, the hydrophobic moment is 0.522, and the antibacterial peptide shows the amphiphilic characteristic; and the sequence is short in length, easy to synthesize and low in cost. Experimental tests show that the antibacterial peptide can inhibit the growth of 11 different strains including 4 gram negative strains and 7 gram positive strains, and is a broad-spectrum antibacterial peptide. Meanwhile, the semi-maximum hemolysis concentration and the semi-maximum cytotoxicity concentration of the antibacterial peptide are both larger than 128 micrograms per milliliter, and the antibacterial peptide has good biological safety and can be applied to antibacterial drugs and other antibacterial products (such as disinfectants, detergents and feed additives).

Description

Technical Field

[0001] The invention relates to an antimicrobial peptide, in particular to a broad-spectrum antimicrobial peptide and application thereof in drug preparation, belonging to the field of biomedicine. Background Art

[0002] Drug-resistant bacterial infection is one of the most serious public health problems at present. According to statistics from the World Health Organization, 1.3 million people die from drug-resistant bacterial infections each year. Antimicrobial peptides are a class of short peptides composed of 10 to 50 amino acids. Their unique membrane-damaging antibacterial mechanism is not easy to induce bacterial resistance, so they are regarded as ideal antibiotic substitutes in the post-antibiotic era. Antimicrobial peptides can be isolated from natural organisms. In recent years, the development of artificial intelligence technology has also accelerated the process of finding new antimicrobial peptides. The number of antimicrobial peptides included in the DRAMP database has exceeded 30,000. However, due to differences in the mechanism of action, different antimicrobial peptides have different inhibitory abilities against different types of bacteria, and most antimicrobial peptides do not have broad-spectrum antibacterial abilities. Summary of the invention

[0003] The purpose of the present invention is to discover new membrane-permeable antimicrobial peptides with broad-spectrum bactericidal ability and low cytotoxicity from the proteome. To this end, based on the artificial intelligence screening method, a broad-spectrum antimicrobial peptide was identified from the proteome of toads.

[0004] The broad-spectrum antimicrobial peptide provided by the present invention comprises 18 amino acids, and its sequence is IYKILQLFHKRFKKGFFG.

[0005] The antimicrobial peptide has 6 positive charges, the GRAVY hydrophobicity in the sequence is -0.06, and the hydrophobic moment is 0.522, showing the characteristics of amphiphilicity ( Figure 1 ).

[0006] The antimicrobial peptide sequence is short in length and can be synthesized by chemical methods such as peptide solid phase synthesis. It is easy to synthesize and has low cost. Experimental tests have shown that this antimicrobial peptide can inhibit the growth of 11 different strains, including 4 Gram-negative strains and 7 Gram-positive strains. It is a broad-spectrum antimicrobial peptide. At the same time, the half-maximum hemolytic concentration and half-maximum cytotoxic concentration of this antimicrobial peptide are both greater than 128μg / mL, with good biosafety, and can be used in antimicrobial drugs and other antimicrobial products (such as disinfectants, detergents, and feed additives).

[0007] In summary, the antimicrobial peptides provided by the present invention have broad-spectrum bactericidal ability, low hemolytic activity and cytotoxicity, good biosafety, and exert their antibacterial effects by destroying cell membranes. They are not likely to cause bacterial resistance and are ideal targets for the development of antimicrobial drugs and antimicrobial products. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 It is a sequence wheel diagram of the antimicrobial peptide of the present invention.

[0009] Figure 2 This is the secondary structure test result of the antimicrobial peptide of the present invention. DETAILED DESCRIPTION

[0010] The present invention will be further described below through specific embodiments in conjunction with the accompanying drawings.

[0011] Example 1 Determination of Minimum Inhibitory Concentration of Antimicrobial Peptides

[0012] In this example, the minimum inhibitory concentration (MIC) of antimicrobial peptides was determined using the broth microdilution method. The selected strains included four Gram-negative strains (Acinetobacterbaumannii ATCC 17978, Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922, Escherichia coli DH5α) and seven Gram-positive strains (Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Micrococcus luteus CMCC(B)28001, Staphylococcus capitis ATCC27842, Staphylococcus hominis ATCC 27844). The bacteria were cultured at 37°C overnight and the bacterial solution was diluted to 5×10 5 cfu / mL, and then 99μL of bacterial suspension was added to each well of the 96-well plate. The antimicrobial peptide was dissolved in cation-adjusted Mueller-Hinton medium with an initial concentration of 1mg / mL, and then diluted in a two-fold gradient concentration ranging from 0.125μg / mL to 128μg / mL, and 1μL was added to each well. The 96-well plate was placed in a 37°C incubator and incubated for 20 hours. The lowest peptide concentration at which no bacterial growth was observed was observed and recorded, which was the MIC of the peptide, as shown in Table 1. After testing, the antimicrobial peptide can inhibit the growth of 11 different strains and has a broad-spectrum antibacterial ability.

[0013] Table 1. Minimum inhibitory concentrations of antimicrobial peptides against 11 strains

[0014]

[0015] Example 2 Determination of hemolytic activity of antimicrobial peptides

[0016] This example tests the hemolytic activity of antimicrobial peptides on rat erythrocytes. Fresh rat blood was centrifuged at 500 g for 5 minutes, and the erythrocyte pellet was retained and resuspended in PBS buffer (pH 7.4). The erythrocyte solution was diluted and 1×10 8 The cells were added to a 96-well plate at a density of 10 cells. The antimicrobial peptide was dissolved in PBS and added to the plate in a two-fold gradient dilution, with the final concentration ranging from 0.125 μg / mL to 128 μg / mL. 10 mg / mL Triton X-100 was used as a positive control to induce 100% hemolysis, while PBS was used as a blank control. After incubation for 1 hour, centrifugation was performed at 500 g for 5 minutes. Subsequently, the absorbance (OD) of the supernatant was measured at a wavelength of 450 nm. The formula for calculating the percentage of hemolysis is: (OD 450,peptide -OD 450,PBS ) / (OD 450,positive -OD 450,PBS ), where OD 450,peptide Represents the absorbance at 450nm of the well treated with the current concentration of antimicrobial peptide, OD 450,PBS Represents the absorbance of the wells treated with PBS solution at 450nm, OD 450,positive Represents the absorbance of the positive control well at 450nm. Each concentration was measured three times to obtain the half maximum hemolysis concentration (HC 50 ) and the minimum hemolytic concentration (MHC) value that causes 10% hemolysis. 50 >128μg / mL, MHC>128μg / mL, with good biosafety.

[0017] Example 3 Cytotoxicity assay of antimicrobial peptides

[0018] The cytotoxicity of antimicrobial peptides to HEK293T cells was evaluated using the Alamar Blue method. HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37°C and 5% CO2. Staurosporine was used as a positive control for cell toxicity. The antimicrobial peptides were dissolved in PBS buffer at pH 7.4, and the final concentration range was a two-fold gradient concentration from 1μg / mL to 128μg / mL. After the cells were co-treated with the antimicrobial peptides for 72 hours, 10μL of Alamar Blue reagent was added to each well. Subsequently, the 96-well plate was incubated in the dark for 4 hours, and the absorbance (OD) was measured at a wavelength of 570nm. 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 of the antimicrobial peptide treated well at 570 nm, OD 570,PBS Represents the absorbance at 570 nm of the wells treated with PBS solution only, OD 570,staurosporine The absorbance of the wells treated with staurosporine at 570 nm was represented. Each concentration was measured in triplicate. The half-maximal cytotoxic concentration (CC) was fitted using Graphpad Prism software. 50 ) value. The CC 50 >128 μg / mL.

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

[0020] The secondary structure of the antimicrobial peptide was determined using a Chirascan V100 circular dichroism spectrometer and a quartz cuvette with a 1 mm optical path. The buffer in the system was 20 mM Tris, 200 mM KCl, pH 7.4, and the final concentration of the antimicrobial peptide was 15 μM. The phospholipid membrane component used to simulate the bacterial cell membrane was set to 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. Figure 2 The distribution of the mean residue ellipticity of antimicrobial peptides with wavelength is shown in Figure 2. Each curve is the average of three spectra, and the data are smoothed in Graphpad Prism 10. The secondary structure proportion of antimicrobial peptides using the K2D algorithm on Dichroweb is shown in Table 2. Compared with the test results in the solution environment, the proportion of antimicrobial peptide α helices in the phospholipid membrane environment is significantly increased.

[0021] Table 2. Proportion of secondary structure components of antimicrobial peptides

[0022]

Claims

1. A broad-spectrum antimicrobial peptide, characterized in that: The broad-spectrum antimicrobial peptide has the following amino acid sequence: IYKILQLFHKRFKKGFFG.

2. Use of the broad-spectrum antimicrobial peptide according to claim 1 in the preparation of antimicrobial drugs.

3. The use according to claim 2, characterized in that The bacteria targeted by the antibacterial drug include Gram-positive bacteria and Gram-negative bacteria.

4. The use according to claim 3, characterized in that The bacteria targeted by the antibacterial drug are one or more of the following bacteria: Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus capitis, Staphylococcus hominis, Acinetobacter baumannii, and Escherichia coli.

5. The use according to claim 2, characterized in that The effective concentration of the broad-spectrum antimicrobial peptide in the antimicrobial drug is greater than or equal to 4 μg / mL.

6. An antibacterial drug, characterized in that: The invention comprises the broad-spectrum antimicrobial peptide according to claim 1.

7. Use of the broad-spectrum antimicrobial peptide according to claim 1 in the preparation of antimicrobial products.

8. The use according to claim 7, characterized in that The antimicrobial product is a disinfectant, a detergent or a feed additive.

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