Antibacterial peptide FR and application thereof

Abstract

The present application relates to the field of agricultural husbandry and veterinary technology, in particular to a protease-resistant antibacterial peptide FR and application thereof. The present application provides a protease-resistant antibacterial peptide FR, and the amino acid sequence of the antibacterial peptide FR is shown as SEQ ID NO. 1. The results of the specific implementation of the present application show that the protease-resistant antibacterial peptide FR provided by the present application has the activity of resisting gram-negative bacteria (Escherichia coli, Shigella flexneri, Salmonella and Campylobacter jejuni, etc.), has low hemolytic activity, can resist the cleavage of high-concentration protease, and has very good application prospect. In summary, the antibacterial peptide FR provided by the present application has the advantages of resisting gastrointestinal environment, resisting high salt and resisting protease cleavage, can realize the killing effect on gram-negative bacteria in the presence of enzymes and salt ions, and has the characteristics of low hemolytic activity, thereby providing a new way for the development of antibiotic substitutes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural animal husbandry, in particular to a protease-resistant antibacterial peptide FR and application thereof. BACKGROUND

[0002] In recent years, antibacterial peptides have become a research hotspot in the field of veterinary antibiotics due to their strong antibacterial activity, fast bactericidal rate, no drug resistance, no pollution and no residue. At present, there are thousands of antibacterial peptides extracted, newly synthesized or modified, but only a few of them are used in practical production and application. Antibacterial peptides are natural active peptides, usually composed of 10-30 amino acid residues. Natural antibacterial peptides are easily hydrolyzed by digestive tract proteases due to the lack of resistance to protease hydrolysis, and are easily hydrolyzed into peptide segments without antibacterial activity. Therefore, the current research and development of antibacterial peptides are mainly used for the treatment of skin, eye and other external infections, which seriously restricts the application scenarios of antibacterial peptides.

[0003] Antibacterial peptides are small molecular peptides that are easily hydrolyzed by various digestive enzymes in the gastrointestinal tract of animals, thereby losing biological activity. At the same time, due to its unique antibacterial mechanism, it is easily affected by salt ions and serum. In the past, the improvement of the anti-protease hydrolysis characteristics of antibacterial peptides usually adopts chemical modification strategies such as D-type amino acid substitution and fat modification, but it will greatly increase the synthesis cost of antibacterial peptides. Because these strategies involve chemical modification, the protease-resistant antibacterial peptide must be synthesized by chemical method, and cannot be obtained by bacterial expression system, which greatly increases the synthesis cost of antibacterial peptides. Therefore, in order to solve this dilemma, the present application maximizes the salt ion stability and protease stability of the peptide by means of the reasonable arrangement of natural amino acids and the spatial interaction force. SUMMARY

[0004] The purpose of the present application is to provide a protease-resistant antibacterial peptide FR and application thereof to solve the problems existing in the prior art. The antibacterial peptide provided by the present application has the advantages of resisting gastrointestinal environment, resisting high salt and resisting protease hydrolysis, and can realize the killing effect on gram-negative bacteria in the presence of enzymes and salt ions.

[0005] To achieve the above purpose, the present application provides the following scheme:

[0006] The present application provides a protease-resistant antibacterial peptide FR (hereinafter referred to as FR), and the amino acid sequence of the antibacterial peptide FR is shown in SEQ ID NO. 1.

[0007] As an additional scheme, the present application further provides a preparation method of the above antibacterial peptide FR, comprising the following steps:

[0008] The hydrophobic force is provided by taking (FP)6 as a hydrophobic basic unit, (RP)6 as a positive charge basic unit, and introducing an anti-protease hydrolysis corner structure in the middle of the peptide chain to symmetrically distribute the hydrophobic unit and the positive charge amino acid, and the stability of the antibacterial peptide is further improved through cation-pi interaction; the peptide resin is obtained through solid-phase chemical synthesis, the obtained peptide resin is cut through TFA to obtain a polypeptide, the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1, and the preparation of the polypeptide is completed after reverse phase high performance liquid chromatography purification and mass spectrometry identification; and then the antibacterial activity determination and hemolytic activity determination containing multiple proteases are carried out, and finally the antibacterial peptide FR is named.

[0009] Further preferably, the design principle of the antibacterial peptide FR in the application is:

[0010] 1) selecting aromatic amino acid (phenylalanine) with a benzene ring as a hydrophobic core to provide hydrophobic force for the peptide chain, so as to embed the peptide chain in the bacterial cell membrane, facilitate the destruction of the integrity of the bacterial cell membrane and maximize the low toxicity to mammalian red blood cells; 2) placing proline (Pro) at the N-terminal of phenylalanine to prevent cleavage by pepsin; 3) adding three flexible amino acids (glycine) resistant to protease hydrolysis at the end of the hydrophobic amino acid to act as a corner structure of the peptide chain, facilitating the symmetric distribution of the subsequent hydrophobic amino acid and the positive charge amino acid; 4) taking the positive charge amino acid (arginine) as a positive point core to ensure that the peptide chain has sufficient positive charge number to provide electrostatic interaction force for the antibacterial peptide and the negatively charged components on the bacterial cell membrane, and placing a Pro at the N-terminal of each Arg to prevent cleavage by chymotrypsin; 5) through the corner structure, the hydrophobic amino acid and the positive charge amino acid are symmetrically distributed on one side, and the beta-sheet conformation of the antibacterial peptide is further stabilized by means of cation-pi interaction.

[0011] The application provides application of the antibacterial peptide FR in preparation of an antibacterial agent against gram-negative bacteria.

[0012] Preferably, the gram-negative bacteria include one or more of Escherichia coli, Shigella flexneri, Salmonella and Campylobacter jejuni.

[0013] The application provides application of the antibacterial peptide FR in preparation of a preservative.

[0014] The application provides a preservative, which comprises the antibacterial peptide FR.

[0015] The application provides application of the antibacterial peptide FR in preparation of a feed additive.

[0016] The application provides a feed additive, which comprises the antibacterial peptide FR.

[0017] The present application provides the use of the above-mentioned feed additive in the preparation of feed.

[0018] The present application provides a feed comprising the above-mentioned feed additive and a basal diet.

[0019] The present application discloses the following technical effects:

[0020] The present application provides an antibacterial peptide FR, and an amino acid sequence of the antibacterial peptide FR is shown as SEQ ID NO. 1. The present application reasonably avoids protease cleavage sites by introducing proline P, optimizes the arrangement of amino acids, and further improves the overall stability of the peptide chain by means of cation-pi interaction force, so as to maximize the anti-protease hydrolysis ability of the peptide and the salt ion and serum stability. The specific implementation results of the present application show that the antibacterial peptide FR provided by the present application has activity against gram-negative bacteria (Escherichia coli, Shigella flexneri, Salmonella and Campylobacter jejuni, etc.), and has low hemolytic activity, can resist the cleavage of high-concentration protease, and has very good application prospect. Therefore, the antibacterial peptide FR provided by the present application has the advantages of resisting gastrointestinal environment, resisting high salt and resisting protease cleavage, and can realize the killing effect on gram-negative bacteria in the presence of enzymes and salt ions, and the antibacterial peptide has the characteristics of low hemolytic activity, which provides a new way for the development of antibiotic substitutes. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0022] Figure 1 is a mass spectrum of the antibacterial peptide FR;

[0023] Figure 2 is a liquid chromatogram of the antibacterial peptide FR;

[0024] Figure 3 is a hemolysis rate diagram of the antibacterial peptide FR. DETAILED DESCRIPTION

[0025] Now, various exemplary embodiments of the present application will be described in detail, and the detailed description should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0026] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentrations, solvent amounts, and the like, it is to be understood that each intervening value, to the upper and lower limits of the ranges is also specifically included within the scope of the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, as is also expressly stated above. These smaller ranges are thus each specifically included within the scope of the present application.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited.

[0028] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in scope by the described embodiments.

[0029] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0030] Example 1: Synthesis of antibacterial peptide FR (antiprotease hydrolysis resistant antibacterial peptide FR) by solid phase chemistry

[0031] The amino acid sequence of antibacterial peptide FR is: PFPFPFPFPFPFPGGGRPRPRPRPRPRP (SEQ ID NO. 1), and the synthesis method is as follows:

[0032] 1. The preparation of antibacterial peptide is carried out from C-terminal to N-terminal one by one by polypeptide synthesizer. Specifically, Fmoc-X (X is the first amino acid at the C-terminal of each antibacterial peptide) is first connected to Wang resin, and then X-Wang resin is obtained after removing the Fmoc group. Then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-trimethyl-Y, Y is the second amino acid at the C-terminal of each antibacterial peptide) is connected. This procedure is followed to synthesize from the C-terminal to the N-terminal in turn until the synthesis is completed, and the resin with side chain protection after removing the Fmoc group is obtained.

[0033] 2. In the above obtained peptide resin, a cleavage reagent is added, and the mixture is reacted at 20°C for 2 hours in the dark, and filtered; the precipitate is washed with TFA (trifluoroacetic acid), and the wash liquid is mixed with the above filtrate, concentrated on a rotary evaporator, and then about 10 times the volume of pre-cooled anhydrous ether is added, and the mixture is precipitated at -20°C for 3 hours to obtain a white powder, which is centrifuged at 2500g for 10 minutes, and the precipitate is collected and washed with anhydrous ether, and dried in a vacuum to obtain the polypeptide, wherein the cleavage reagent is a mixture of TFA, water and TIS (triisopropylchlorosilane) in a mass ratio of 95:2.5:2.5.

[0034] 3. The column is equilibrated with 0.2M sodium sulfate (phosphoric acid is added to adjust pH to 7.5) for 30 minutes, and the polypeptide is dissolved in 90% acetonitrile aqueous solution, filtered, and the filtrate is loaded onto the column. 18 The column is equilibrated with 0.2M sodium sulfate (phosphoric acid is added to adjust pH to 7.5) for 30 minutes, and the polypeptide is dissolved in 90% acetonitrile aqueous solution, filtered, and the filtrate is loaded onto the column. 18 The column is equilibrated with 0.2M sodium sulfate (phosphoric acid is added to adjust pH to 7.5) for 30 minutes, and the polypeptide is dissolved in 90% acetonitrile aqueous solution, filtered, and the filtrate is loaded onto the column.

[0035] 4. Identification of the antibacterial peptide: the above obtained antibacterial peptide is analyzed by electrospray mass spectrometry, and the molecular weight (M+H+) shown in the mass spectrum is basically consistent with the theoretical molecular weight, and the antibacterial peptide is named as antibacterial peptide FR, and the purity of the antibacterial peptide FR is greater than 95%. Figure 1 ) with the theoretical molecular weight, and the antibacterial peptide is named as antibacterial peptide FR, and the purity of the antibacterial peptide FR is greater than 95%. Figure 2

[0036] Example 2 Determination of the antibacterial activity of the antibacterial peptide FR subjected to protease hydrolysis

[0037] The minimum inhibitory concentration of the antibacterial peptide FR is determined by microdilution method, and the specific steps are as follows:

[0038] A 96-well plate is added with 0.01% acetic acid and 0.2% fetal bovine serum albumin as diluent, and a series of gradient antibacterial peptide solutions are prepared by using a two-fold dilution method. 100 μL of the above solution is placed in a 96-well cell culture plate, and then an equal volume of bacteria solution to be tested (about 10 5 CFU / mL) is added to each well, and a positive control (containing bacteria solution but not containing antibacterial peptide) and a negative control (neither containing bacteria solution nor containing antibacterial peptide) are set. The plate is incubated at 37°C for 14-18 hours, and the optical absorption value is determined at 492 nm (OD 492nm ) by using a microplate reader to determine the minimum inhibitory concentration. A value less than 0.1 is considered to be inhibited. Each test is repeated twice in parallel, and the results are shown in Table 1. ​

[0039] Table 1 Antimicrobial activity of peptide FR against bacteria (μM)

[0040] Strains Minimum inhibitory concentration (μM) E. coli 25922 16 E. coli 8739 8 E. coli 35218 8 E. coli 35218 32 E. coli 35218 16 E. coli 35218 16 E. coli 35218 8

[0041] As shown in Table 1, the antimicrobial peptide FR has good antimicrobial activity against E. coli (E. coli 25922, E. coli 8739 and E. coli 35218), pathogenic E. coli (EPEC 24189), Shigella flexneri (Sh. flexneri 12022), Salmonella (Salmonella 14028) and Campylobacter jejuni (C. jejuni 700819) and other bacteria.

[0042] Example 3 Determination of protease stability of antimicrobial peptide FR

[0043] Different concentrations of proteases were mixed with 2560 μM of antimicrobial peptide FR at 37°C for 8 h, and then the minimum inhibitory concentration of the peptide against E. coli 8739 was determined by the microdilution method described in Example 2. The control group was the minimum inhibitory concentration test result without proteases. The results are shown in Table 2.

[0044] Table 2 Protease stability of antimicrobial peptide FR

[0045] E. coli 35218 E. coli 35218 E. coli 35218 8 E. coli 35218 8 E. coli 35218 8 E. coli 35218 8

[0046] As shown in Table 2, the antimicrobial peptide FR can resist the cleavage of 8 mg / mL pepsin and trypsin, and 8 mg / mL chymotrypsin, and still retains the original antimicrobial activity after 8 h of incubation.

[0047] Example 4 Determination of salt ion stability of antimicrobial peptide FR

[0048] The antimicrobial activity of peptide FR was determined in the presence of 150 mM NaCl, 4.5 mM KCl, 6 μM NH4Cl, 8 μM ZnCl2, 1 mM MgCl2and 4 μM FeCl3. The specific operation method is the same as that of Example 3. The control group was the minimum inhibitory concentration determination result without salt ions. The results are shown in Table 3.

[0049] Table 3 Salt ion stability of antimicrobial peptide FR

[0050] E. coli 35218 E. coli 35218 E. coli 35218 8 E. coli 35218 24 E. coli 35218 8 6 μM NH4CI 8 8 μM ZnCl2 8 1 mM MgCl2 16 4 μM FeCl3 8

[0051] As shown in Table 3, under physiological salt concentration, the antimicrobial peptide FR basically retains the original antimicrobial activity, and only in the presence of physiological concentrations of NaCl and MgCl2, the activity decreases, which indicates that the antimicrobial peptide FR has strong salt stability.

[0052] Example 5 Hemolytic activity assay of antibacterial peptide FR

[0053] Purchased 1% porcine red blood cells, centrifuged at 3000 rpm for 10 min, collected the red blood cells; washed with PBS solution for 3 times, resuspended with 10 mL PBS solution; took 50 μL red blood cell suspension and mixed with 50 μL antibacterial peptide solution of different concentrations (4, 8, 16, 32, 64 and 128 μM) uniformly, incubated in a 37℃ incubator for 1 h; then centrifuged at 4℃, 3000 rpm for 10 min; took the supernatant and determined the optical absorption value at 570 nm by microplate reader. 50 μL red blood cells plus 50 μL PBS solution was used as negative control, 50 μL red blood cells plus 50 μL 0.1% Triton x-100 was used as positive control. The minimum hemolytic concentration was the antibacterial peptide concentration when 10% hemolysis rate was caused by the antibacterial peptide, and the detection results were shown in Table 1. E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. coli 35218 E. It can be seen that the antibacterial peptide FR did not show hemolytic activity in the detection range, caused 1% red blood cell hemolysis at 128 μM concentration, and failed to cause 10% red blood cell hemolysis, indicating that the low hemolytic activity antibacterial peptide FR has the potential to be developed as an antibiotic substitute.

[0054] In summary, the antibacterial peptide FR composed of natural amino acids has broad-spectrum antibacterial activity, strong salt stability and protease stability, and wide clinical application prospect.

[0055] The above-described examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.​

Claims

1. An antimicrobial peptide FR, characterized in that The amino acid sequence of the antimicrobial peptide FR is shown in SEQ ID NO.

1.

2. The use of the antimicrobial peptide FR according to claim 1 in the preparation of an anti-Gram-negative bacterial agent, characterized in that: The Gram-negative bacteria are one or more of Escherichia coli, Shigella flexneri, Salmonella and Campylobacter jejuni.

3. An anti-Gram-negative antibacterial agent, characterized in that: The antimicrobial agent comprises the antimicrobial peptide FR according to claim 1.

4. Use of the antimicrobial peptide FR according to claim 1 in the preparation of a preservative.

5. A preservative, characterized in that The preservative comprises the antimicrobial peptide FR according to claim 1.

6. Use of the antimicrobial peptide FR according to claim 1 in the preparation of a feed additive.

7. A feed additive, characterized in that The feed additive comprises the antimicrobial peptide FR according to claim 1.

8. Use of the feed additive according to claim 7 in preparing feed.

9. A feed, characterized in that The feed comprises the feed additive according to claim 7 and a basic diet.

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