Fish-derived high-temperature-resistant antibacterial peptide and application thereof

Abstract

The invention provides a polypeptide which is derived from zebra fish antibacterial peptide and is improved in heat resistance. The amino acid sequence of the polypeptide AR10 is WKRKRWRIWK, and the polypeptide AR10 has higher antibacterial activity than the unmodified polypeptide AR10, and has better high temperature resistance, salt resistance and serum resistance. The antibacterial peptide provided by the invention not only has a good antibacterial effect on common Gram-positive and Gram-negative pathogenic bacteria, but also has a good antibacterial effect on clinically common drug-resistant bacteria, and cell experiment results show that the screened antibacterial peptide has biological safety. Therefore, the antibacterial peptide provided by the invention has broad-spectrum antibacterial property and high biological safety, and can be used for developing antibacterial and bacteriostatic products related to human bodies.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a fish-derived high-temperature-resistant antibacterial peptide and application thereof. Background Art

[0002] Antimicrobial peptides (AMPs) are an important part of innate immunity, and together with complement, lysozyme, interferon, etc., they constitute the first line of defense of the body's immunity. When pathogens invade, antimicrobial peptides, as a small molecule peptide, can respond quickly and play an antibacterial role to resist the invasion of pathogens to the body. Antimicrobial peptides have a broad-spectrum killing effect on pathogens, such as bacteria, viruses, fungi, tumor cells and protozoa. In addition, some antimicrobial peptides can relieve inflammation, promote wound healing, reduce the chance of infection, and are not easy to develop drug resistance. Therefore, antimicrobial peptides have become a research hotspot in recent years.

[0003] The characteristics of antimicrobial peptides are that most of them are positively charged, the peptide chain is short (composed of 10 to 80 amino acid residues), the molecular weight is small (1.8-8kDa), the water solubility is good, and the isoelectric point is 8.9-10.7. The advantages of antimicrobial peptides due to their mechanism of action are: they act on the inherent structure of pathogens and thus have a broad-spectrum antimicrobial effect; they do not rely on a specific receptor and therefore are not likely to induce drug resistance in strains. Antimicrobial peptides themselves are small molecule proteins that can be naturally degraded in the body without the worry of drug residues. For clinical treatment, antimicrobial peptides can be used together with antibiotics to achieve the purpose of low concentration and good effect.

[0004] Most fish reproductive processes take place in the external environment, including in vitro fertilization and in vitro development of embryos after fertilization. There are various pathogenic microorganisms in the water bodies of the external environment where fish live. When the eggs leave the mother's body and enter the water body, various microorganisms will attach to the surface of the embryo, especially pathogenic bacteria, which will cause serious damage to the embryo. In addition, early fish embryos are basically unable to synthesize specific antibodies independently, and the development of the immune system and lymphatic organs is relatively late. How fish embryos survive and develop under harsh conditions is a research direction with great potential application value. There are many fish antimicrobial peptides and they may show different types of antimicrobial peptides in different tissues of the same fish; and the molecular structure and mechanism of action of these antimicrobial peptides show great differences between different types of fish. Analysis of the amino acid sequences of reported fish antimicrobial peptides shows that they have low homology, but there are some common characteristics in structure, such as rich in hydrophobic amino acids such as lysine and arginine, and the α-helical structure is easy to form inside the molecule. Therefore, finding new antimicrobial peptides from fish has become a hot topic of research.

[0005] In previous studies, the present inventors screened an antimicrobial peptide AP10 derived from zebrafish, but found that the antibacterial ability of the antimicrobial peptide AP10 was poor (Gong, Y.; Li, H.; Wu, F.; Zhang, X.; Zhou, Y.; Zhang, S. A shortpeptide derived from zebrafish AP-2 complex subunit mu-AAP2M1A 354-382 has antimicrobial activity against multi-drug resistant bacteria. Pept. Sci. 2022, e24258.). Summary of the invention

[0006] The inventors studied an antimicrobial peptide AP10 from zebrafish screened in previous studies and found that the antimicrobial peptide AP10 has antibacterial ability, but the effect is poor and it is not resistant to high temperatures. Therefore, the present invention modified the antimicrobial peptide AP10 to obtain an antimicrobial peptide AP10RW with higher antimicrobial activity, high temperature resistance, salt resistance, and serum resistance, thus contributing to the present invention.

[0007] Therefore, the present invention provides a polypeptide derived from zebrafish antimicrobial peptide that has been modified to improve its heat resistance, namely, fish-derived high-temperature-resistant antimicrobial peptide, whose amino acid sequence is WKRKRWRIWK.

[0008] Preferably, it is amidated at its C-terminus.

[0009] The present invention also provides application of the fish-derived high-temperature-resistant antimicrobial peptide in the preparation of antibacterial or antibacterial products.

[0010] Specifically, the antibacterial or bacteriostatic activity is directed against Gram-positive or Gram-negative bacteria.

[0011] More preferably, the antibacterial or antibacterial activity is directed against Escherichia coli or Staphylococcus aureus.

[0012] The present invention also provides an antibacterial or antibacterial product, which uses the fish-derived high-temperature-resistant antibacterial peptide as an active ingredient.

[0013] Specifically, it is food, medicine, feed or an additive thereof.

[0014] The antimicrobial peptide provided by the present invention has a good antimicrobial effect not only on common Gram-positive and Gram-negative pathogens, but also on common clinical drug-resistant bacteria, especially with a significant effect on high temperature resistance. The cell experiment results show that the screened antimicrobial peptide has biosafety. The antimicrobial peptide AP10RW provided by the present invention has a broad-spectrum antibacterial property and high biosafety, and can be used in the development of antibacterial and antibacterial products related to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Antibacterial activity of AP10RW against multidrug-resistant Escherichia coli 577 strain after heating at 55°C for 30 min.

[0016] Figure 2 Antibacterial activity of AP10RW against multidrug-resistant Staphylococcus aureus USA500 strain after heating at 55°C for 30 min.

[0017] Figure 3 Antibacterial activity of AP10RW against multidrug-resistant Escherichia coli 577 strain after heating at 85°C for 30 min.

[0018] Figure 4 Antibacterial activity of AP10RW against multidrug-resistant Staphylococcus aureus USA500 strain after heating at 85°C for 30 min.

[0019] Figure 5 Antibacterial activity of AP10RW against multidrug-resistant Escherichia coli 577 strain in normal saline.

[0020] Figure 6 Antibacterial activity of AP10RW against multidrug-resistant Staphylococcus aureus USA500 strain in normal saline.

[0021] Figure 7 The antibacterial activity of AP10RW against multidrug-resistant Escherichia coli 577 strain in 25% serum.

[0022] Figure 8 The antibacterial activity of AP10RW against multidrug-resistant Staphylococcus aureus USA500 strain in 25% serum. DETAILED DESCRIPTION

[0023] The present invention is described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0024] Example 1: Modification of antimicrobial peptides

[0025] Based on the analysis of the sequence of the antimicrobial peptide AP10 (WKIKRMAGMK), AP10 was modified by amino substitution, and the antimicrobial ability of each modified mutant was screened after high-temperature stimulation. Finally, the antimicrobial peptide mutant with no obvious change in antimicrobial ability after high-temperature treatment was selected.

[0026] The final screened antimicrobial peptide AP10RW is the antimicrobial peptide AP10 in which the hydrophobic amino acid Ile at the third position and the hydrophobic amino acid Ala at the seventh position are replaced with the hydrophilic positively charged amino acid Arg, the hydrophobic amino acid Met at the sixth position is replaced with the hydrophobic amino acid Trp with a larger side chain, and the Gly at the eighth position is replaced with the hydrophobic amino acid Ile. Its amino acid sequence from N-terminus to C-terminus is WKRKRWRIWK. During use, it is amidated at its C-terminus, and the sequence is Ac-WKRKRWRIWK-NH2. Amidation at the C-terminus is used to improve the stability of the polypeptide, enhance the biological activity and eliminate the influence of the terminal charge.

[0027] The peptide is synthesized by a commercial company and amidated at the C-terminus. The purity of the synthesized peptide is over 95% and can be used for various biological tests.

[0028] Example 2: Detection of the antibacterial effect of antimicrobial peptide AP10RW on representative bacteria Escherichia coli and Staphylococcus aureus

[0029] The synthesized antimicrobial peptide was dissolved in PBS to prepare a 1 mg / ml stock solution.

[0030] Bacterial culture and bacterial solution preparation: Gram-positive bacteria Staphylococcus aureus (S. aureus 25923) and Gram-negative bacteria Escherichia coli (E. coli 25922) were inoculated into sterilized LB medium and cultured at 37°C and 120 rpm for 16 hours. Then centrifuged at 6000g for 10 minutes at room temperature. Washed three times with PBS (pH 7.4), resuspended in PBS, and adjusted to a density of 1×10 5 Pieces / mL.

[0031] Microdilution assay: 30 μl of each bacterial suspension was mixed with 270 μl of fresh LB medium and 300 μl of antimicrobial peptide solution, with final peptide concentrations ranging from 5 to 300 μg / ml. A total of 600 μl of the mixture was added to 3 wells (200 μl / well) in a sterile 96-well microplate and incubated at 37°C for 9 hours. The blank control group was subjected to the experiment using PBS instead of the peptide solution. The absorbance (OD 600nm) was measured every hour at a wavelength of 600 nm using a microplate reader to determine the inhibitory effect of each well on bacterial growth at each time point. The minimum inhibitory concentration (MIC) is defined as the lowest concentration that completely inhibits the growth of the corresponding bacteria. The results showed that AP10RW had a good inhibitory effect on both Staphylococcus aureus and Escherichia coli, and the MIC value was more than 3 times lower than that of the parent peptide AP10.

[0032] Table 1: Minimum inhibitory concentration (MIC) (μg / ml) of antimicrobial peptide AP10RW against Gram-positive and Gram-negative bacteria

[0033]

[0034] Example 3: Antimicrobial effects of antimicrobial peptides on other Gram-positive and Gram-negative bacteria

[0035] Bacterial culture and bacterial solution preparation: Gram-positive bacteria Micrococcus luteus (M.luteus 49732), Bacillus subtilis (B.subtilis6633) and Gram-negative bacteria Vibrio anguillarum (V.Anguillarum43308) were inoculated into sterilized LB medium and cultured at 37°C and 120 rpm for 16 hours. Then centrifuged at 6000g for 10 minutes at room temperature. Washed three times with PBS (pH 7.4), resuspended in PBS, and adjusted to a density of 1×10 5 Pieces / mL.

[0036] Microdilution assay: 30 μl of each bacterial suspension was mixed with 270 μl of fresh LB medium and 300 μl of antimicrobial peptide solution, with final peptide concentrations ranging from 5 to 300 μg / ml. A total of 600 μl of the mixture was added to 3 wells (200 μl / well) in a sterile 96-well microplate and incubated at 37°C for 9 hours. The blank control group was subjected to the experiment using PBS instead of the peptide solution. The absorbance (OD 600nm) was measured every hour at a wavelength of 600 nanometers using a microplate reader to determine the inhibitory effect of each well on bacterial growth at each time point. The minimum inhibitory concentration (MIC) is defined as the lowest concentration that completely inhibits the growth of the corresponding bacteria. The results are shown in Table 2. AP10RW has a good inhibitory effect on Micrococcus luteus, Bacillus subtilis, and Vibrio anguillarum.

[0037] Table 2: Minimum inhibitory concentration of antimicrobial peptide AP10RW against Micrococcus luteus, Bacillus subtilis, and Vibrio anguillarum

[0038] strains AP10RW minimum inhibitory concentration MICs (μg / ml) Micrococcus luteus (M.luteus 49732) 25 Bacillus subtilis (B.subtilis6633) 75 Vibrio anguillarum (V. Anguillarum 43308) 50

[0039] Example 3: Antimicrobial effect of antimicrobial peptides against multidrug-resistant bacteria

[0040] Bacterial culture and bacterial solution preparation: Escherichia coli 577 strain (E.coli 577), Staphylococcus aureus USA500 strain (S.aureus USA500) and Klebsiella pneumoniae 2182 strain (K.pneumoniae 2182) were inoculated into sterilized LB medium and cultured at 37°C and 120 rpm for 16 hours. Then centrifuged at 6000g for 10 minutes at room temperature. Washed three times with PBS (pH 7.4), resuspended in PBS, and adjusted to a density of 1×10 5 Pieces / mL.

[0041] Microdilution assay: 30 μl of each bacterial suspension was mixed with 270 μl of LB fresh medium and 300 μl of antimicrobial peptide solution, and the final peptide concentrations were 5 to 300 μg / ml. A total of 600 μl of the mixture was added to 3 wells (200 μl / well) in a sterile 96-well microplate and incubated at 37°C for 9 hours. The blank control group was tested using PBS instead of the peptide solution. The absorbance (OD600nm) was measured at a wavelength of 600 nanometers every hour using a microplate reader to determine the inhibitory effect of each well on bacterial growth at each time point. The minimum inhibitory concentration (MIC) is defined as the lowest concentration that completely inhibits the growth of the corresponding bacteria. As shown in Table 3, AP10RW has a good inhibitory effect on multidrug-resistant bacteria Escherichia coli 577, Staphylococcus aureus USA500, and Klebsiella pneumoniae 2182.

[0042] Table 3: Minimum inhibitory concentration MIC (μg / ml) of antimicrobial peptide AP10RW against drug-resistant bacteria

[0043]

[0044]

[0045] Example 4: Antibacterial activity of antimicrobial peptide AP10RW under different conditions

[0046] 1. Antibacterial activity under high temperature conditions

[0047] AP10RW was heated at 55°C and 85°C for 30 min, cooled to room temperature, and 30 μl of Escherichia coli 577 or Staphylococcus aureus USA500 bacterial suspension (1×10 5 cells / ml) were mixed with 270 μl LB fresh medium and 300 μl AP10RW solution that was heated and then cooled to room temperature, with final concentrations of 1×MIC (40 ug / ml), 1.5×MIC (60 ug / ml) and 2×MIC (80 ug / ml), respectively. Then, the antibacterial activity against multidrug-resistant Escherichia coli 577 strain and Staphylococcus aureus USA500 strain was tested according to the above method. Figures 1 to 4 The results shown indicate that AP10RW is highly stable after being heated to 55°C and 85°C for 30 minutes.

[0048] 2. Antibacterial activity under physiological salt conditions

[0049] Although AMPs have strong antibacterial activity in vitro, they partially or completely lose this activity in saline. To test the effect of salt on antibacterial activity, 30 μl of Escherichia coli 577 or Staphylococcus aureus USA500 bacterial suspension (1×10 5cells / ml) were mixed with 270 μl LB fresh medium and 300 μl AP10RW solution dissolved in 0.9% NaCl (154 mM) saline to a final concentration of 1×MIC (40 ug / ml), 1.5×MIC (60 ug / ml) and 2×MIC (80 ug / ml), and then the antibacterial activity was tested according to the above method. The results are shown in Figure 5 and Figure 6 As shown, the results showed that in normal saline, the antibacterial activity remained almost unchanged in the presence of high concentrations of sodium ions. AP10RW with the original MIC value (40ug / ml) could completely inhibit the growth of bacteria.

[0050] 3. Antibacterial activity in the presence of serum

[0051] Complex serum components are considered to be the main reason for the reduction of antibacterial activity of AMPs. Fresh human serum was diluted with PBS to prepare a 25% (v / v) human serum solution. 30 μl of Escherichia coli 577 or Staphylococcus aureus USA500 bacterial suspension (1×10 8 cells / ml) were mixed with 270 μl of LB fresh medium and 300 μl of AP10RW solution dissolved in 25% human serum, with the final concentrations being 1×MIC (40 ug / ml), 1.5×MIC (60 ug / ml) and 2×MIC (80 ug / ml), respectively. At the same time, 25% human serum was treated in the same way as a blank control. The results are shown in Figure 7 and Figure 8 As shown, it is shown that in the presence of 25% serum, the antibacterial activity of the peptide has no significant change. 1×MIC (40ug / ml) of AP10RW can completely inhibit the growth of bacteria (this result shows to a certain extent that the peptide can be used to sterilize bleeding wounds).

[0052] Example 5: Cytotoxicity and hemolysis of AP10RW

[0053] 1. Cytotoxicity of AP10RW

[0054] Using RAW264.7 mouse macrophage cell line as experimental material, the survival rate of cells under the action of antimicrobial peptide AP10RW was determined by MTT colorimetry. The experimental results are shown in Table 4. The data showed that within the concentration range of 200 μg / ml, the antimicrobial peptide AP10RW had no obvious toxic and damaging effects on RAW264.7 mouse macrophages, indicating that the antimicrobial peptide AP10RW has no significant toxicity to normal mammalian tissue cells.

[0055] Table 4: Survival rate of RAW264.7 cells in the presence of AP10RW

[0056] AP10RW concentration (μg / ml) Survival rate of RAW264.7 cells 0 100 50 127±5 100 147±1 200 115±4

[0057] 2. Study on the hemolytic activity of antimicrobial peptides

[0058] Human red blood cells were used as experimental materials to detect the hemolytic effect of antimicrobial peptide AP10RW on mammalian red blood cells. The experimental results showed that antimicrobial peptide AP10RW did not have significant hemolytic effect at a concentration of up to 200μg / ml.

[0059] In summary, the antimicrobial peptide AP10RW provided by the present invention has a broad-spectrum antibacterial activity and high biological safety, and has the potential to be used in the development of antibacterial and antibacterial products related to the human body.

Claims

1. A fish-derived high temperature resistant antimicrobial peptide, characterized in that: Its amino acid sequence is WKRKRWRIWK.

2. The fish-derived high temperature resistant antimicrobial peptide according to claim 1, characterized in that: It is amidated at its C-terminus.

3. Use of the fish-derived high-temperature-resistant antimicrobial peptide as described in claim 1 or 2 in the preparation of antibacterial or antibacterial products.

4. The use according to claim 3, characterized in that The antibacterial or bacteriostatic activity is directed against Gram-positive or Gram-negative bacteria.

5. The use according to claim 4, characterized in that The antibacterial or antibacterial agent is directed against Escherichia coli or Staphylococcus aureus.

6. The use according to claim 5, characterized in that The product is food, medicine, feed or an additive thereof.

7. An antibacterial or antibacterial product, characterized in that: The fish-derived high-temperature-resistant antimicrobial peptide as claimed in claim 1 or 2 is used as an active ingredient.

8. The antibacterial or antibacterial product according to claim 7, characterized in that: It is food, medicine, feed or its additive.

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