Antibacterial peptide FR and application thereof

Abstract

The invention relates to the technical field of agriculture, animal husbandry and veterinary medicine, in particular to a protease cracking resistant antibacterial peptide FR and application thereof. The invention provides a protease cracking resistant antibacterial peptide FR. The amino acid sequence of the antibacterial peptide FR is as shown in SEQ ID NO. 1. The specific implementation result shows that the protease cracking resistant antibacterial peptide FR provided by the invention has the activity of resisting gram-negative bacteria (escherichia coli, shigella flexneri, salmonella, campylobacter jejuni and the like), also has low hemolytic activity, can resist cracking of high-concentration protease, and has a very good application prospect. In conclusion, the antibacterial peptide FR provided by the invention has the advantages of gastrointestinal tract environment resistance, high salt resistance and protease lysis resistance, can realize the killing effect on gram-negative bacteria in the presence of enzymes and salt ions, has the characteristic of low hemolytic activity, and provides a new way for development of antibiotic substitutes.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural animal husbandry and veterinary medicine, and in particular to an antimicrobial peptide FR resistant to protease cleavage and application thereof. Background Art

[0002] In recent years, antimicrobial peptides have become a research hotspot in the field of veterinary antibiotic alternatives due to their strong antimicrobial activity, fast bactericidal rate, low resistance to drug resistance, no pollution and no residue. At present, there are thousands of antimicrobial peptides that have been extracted, newly synthesized or modified, but very few are put into actual production applications. Antimicrobial peptides are a type of natural active polypeptide, usually composed of 10-30 amino acid residues. Natural antimicrobial peptides are easily cleaved by digestive tract proteases into peptides without antimicrobial activity due to their lack of resistance to protease hydrolysis. Therefore, the antimicrobial peptides currently under development are mainly used to treat in vitro infections such as skin and eyes, which seriously restricts the application scenarios of antimicrobial peptides.

[0003] Antimicrobial peptides are small molecule polypeptides that are easily cleaved by various digestive enzymes in the gastrointestinal tract of animals, thereby losing their biological activity. At the same time, due to their unique antibacterial mechanism, they are easily affected by salt ions and serum in the body. In the past, chemical modification strategies such as D-amino acid substitution and fat modification were usually adopted to improve the anti-protease hydrolysis properties of antimicrobial peptides, but this would greatly increase the synthesis cost of antimicrobial peptides. Because these strategies all involve chemical modifications, protease-resistant antimicrobial peptides must be synthesized using chemical methods and cannot be obtained through bacterial expression systems, which greatly increases the synthesis cost of antimicrobial peptides. Therefore, in order to solve this dilemma, the present invention uses the reasonable arrangement of natural amino acids and the spatial interaction forces to maximize the salt ion stability and protease stability of the peptide. Summary of the invention

[0004] The purpose of the present invention is to provide an antimicrobial peptide FR resistant to protease cleavage and its application to solve the problems existing in the above-mentioned prior art. The antimicrobial peptide provided by the present invention has the advantages of resistance to gastrointestinal environment, high salt resistance and protease cleavage resistance, and can achieve the killing effect on Gram-negative bacteria in the presence of enzymes and salt ions.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an antimicrobial peptide FR (FR for short) resistant to protease cleavage. The amino acid sequence of the antimicrobial peptide FR is shown in SEQ ID NO.1.

[0007] As an additional solution, the present invention also provides a method for preparing the above antimicrobial peptide FR, comprising the following steps:

[0008] (FP)6 is used as a hydrophobic basic unit to provide hydrophobic force, (RP)6 is used as a positively charged basic unit, and an anti-protease hydrolysis turn structure is introduced in the middle of the peptide chain to make the hydrophobic unit and the positively charged amino acid symmetrically distributed, and the stability of the antimicrobial peptide is further improved through cation-π interaction; a peptide resin is obtained by solid phase chemical synthesis, and the obtained peptide resin is cut by TFA to obtain a polypeptide, and its amino acid sequence is shown in SEQ ID NO.1. After reversed-phase high performance liquid chromatography purification and mass spectrometry identification, the preparation of the polypeptide is completed; and then after antimicrobial activity assay containing multiple proteases and hemolytic activity assay, it is finally named antimicrobial peptide FR.

[0009] Further preferably, the design principle of the antimicrobial peptide FR of the present invention is:

[0010] 1) Aromatic amino acids with benzene rings (phenylalanine) are selected as the hydrophobic core to provide hydrophobic force for the peptide chain, so that it can be embedded in the bacterial cell membrane, which is convenient for destroying the integrity of the bacterial cell membrane and ensuring low toxicity to mammalian red blood cells to the greatest extent; 2) Proline (Pro) is placed at the N-terminus of phenylalanine to prevent cleavage by pepsin; 3) Three flexible amino acids (glycine) that are resistant to protease hydrolysis are added to the end of the hydrophobic amino acid to act as the turn structure of the peptide chain, which facilitates the symmetrical distribution of subsequent hydrophobic amino acids and positively charged amino acids; 4) Positively charged amino acids (arginine) are used as the positive point core to ensure that the peptide chain has sufficient positive charge number, providing electrostatic interaction force between the antimicrobial peptide and the negatively charged components on the bacterial cell membrane. At the same time, a Pro is placed at the N-terminus of each Arg to prevent cleavage by chymosin; 5) Through the turn structure, the hydrophobic amino acids and positively charged amino acids are symmetrically distributed on one side, and the β-folding conformation of the antimicrobial peptide is further stabilized with the help of cation-π interaction force.

[0011] The present invention provides the use of the antimicrobial peptide FR in the preparation of an anti-Gram-negative bacteria agent.

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

[0013] The present invention provides the use of the antimicrobial peptide FR in the preparation of a preservative.

[0014] The present invention provides a preservative, which comprises the above-mentioned antimicrobial peptide FR.

[0015] The present invention provides the use of the antimicrobial peptide FR in the preparation of feed additives.

[0016] The present invention provides a feed additive, which comprises the above-mentioned antimicrobial peptide FR.

[0017] The present invention provides application of the above feed additive in preparing feed.

[0018] The present invention provides a feed, which comprises the above feed additive and a basic diet.

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

[0020] The present invention provides an antimicrobial peptide FR, the amino acid sequence of which is shown in SEQ ID NO.1. The present invention introduces proline P to reasonably avoid protease cleavage sites, optimizes the amino acid arrangement, and further improves the overall stability of the peptide chain by means of cation-π interaction forces, thereby maximally ensuring the peptide's ability to resist protease hydrolysis and salt ion and serum stability. The results of the specific implementation of the present invention show that the antimicrobial peptide FR provided by the present invention has anti-Gram-negative bacteria (Escherichia coli, Shigella flexneri, Salmonella and Campylobacter jejuni, etc.) activity, and at the same time has low hemolytic activity, can resist the cleavage of high-concentration proteases, and has very good application prospects. Therefore, the antimicrobial peptide FR provided by the present invention has the advantages of resistance to gastrointestinal environment, high salt resistance and resistance to protease cleavage, can achieve the killing effect on Gram-negative bacteria in the presence of enzymes and salt ions, and at the same time, the antimicrobial peptide has the characteristics of low hemolytic activity, which provides a new way for the development of antibiotic substitutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

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

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1 Synthesis of antimicrobial peptide FR (antimicrobial peptide FR resistant to protease hydrolysis) by solid phase chemical synthesis

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

[0032] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus by a peptide synthesizer, specifically: first, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) is connected to Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); according to this procedure, it is synthesized from the C-terminus to the N-terminus in sequence until the synthesis is completed, and a resin with side chain protection removed from the Fmoc group is obtained.

[0033] 2. Add a cleavage reagent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter; wash with precipitated TFA (trifluoroacetic acid), mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, and then add about 10 times the volume of pre-cooled anhydrous ether, precipitate at -20°C for 3 hours, precipitate a white powder, centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain a polypeptide, wherein the cleavage reagent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5.

[0034] 3. Use 0.2M sodium sulfate (adjusted to pH 7.5 with phosphoric acid) to balance the column for 30 min, dissolve the peptide in 90% acetonitrile aqueous solution, filter, and 18 Reverse phase atmospheric pressure column, gradient elution (eluent is methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70-70:30), flow rate is 1mL / min, detection wave is 220nm, main peak is collected, lyophilized; then reverse phase C 18 The column was further purified, eluent A was 0.1% TFA / water solution; eluent B was 0.1% TFA / acetonitrile solution, elution concentration was 25% B-40% B, elution time was 12 min, flow rate was 1 mL / min, and the main peak was collected and lyophilized as above.

[0035] 4. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray mass spectrometry. The molecular weight ( Figure 1 ) is basically consistent with the theoretical molecular weight, and it is named antimicrobial peptide FR. The purity of antimicrobial peptide FR is greater than 95% ( Figure 2 ).

[0036] Example 2 Determination of the antimicrobial activity of protease-resistant antimicrobial peptide FR

[0037] The minimum inhibitory concentration of antimicrobial peptide FR was determined by microdilution method. The specific steps are as follows:

[0038] A diluent containing 0.01% acetic acid and 0.2% fetal bovine serum albumin was added to a 96-well plate, and a series of gradient antimicrobial peptide solutions were prepared in sequence using the two-fold dilution method. 100 μL of the above solution was placed in a 96-well cell culture plate, and then an equal volume of the test bacterial solution (about 10 5 CFU / mL) in each well, and set up a positive control (containing bacterial solution but not antimicrobial peptide) and a negative control (containing neither bacterial solution nor antimicrobial peptide). Incubate at 37°C for 14-18h, and read the sample at 492nm (OD 492nm ) to determine the minimum inhibitory concentration. A value less than 0.1 was considered to be bacterial inhibition. Each test was repeated three times with two parallels. The results are shown in Table 1.

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

[0040] strain Minimum inhibitory concentration (μM) Escherichia coli 25922 (E.coli25922) 16 Escherichia coli 8739 (E.coli8739) 8 Escherichia coli 35218 (E.coli35218) 8 Pathogenic Escherichia coli 24189 (EPEC24189) 32 Shigella flexmeri 12022 (Sh.flexmeri12022) 16 Salmonella 14028 16 Campylobacter jejuni 700819 (C.Jeuni700819) 8

[0041] The results in Table 1 show that the antimicrobial peptide FR has good antibacterial activity against bacteria such as Escherichia coli (E. coli 25922, E. coli 8739 and E. coli 35218), pathogenic Escherichia coli (EPEC24189), Shigella flexmeri (Sh.flexmeri 12022), Salmonella (Salmonella14028) and Campylobacter jejuni (C.Jeuni 700819).

[0042] Example 3 Protease stability determination of antimicrobial peptide FR

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

[0044] Table 2 Protease stability of antimicrobial peptide FR

[0045] Protease Minimum inhibitory concentration (μM) Comparison 8 8mg / mL pepsin 8 8mg / mL trypsin 8 8mg / mL chymotrypsin 8

[0046] As can be seen from Table 2, the antimicrobial peptide FR can resist the cleavage of 8 mg / mL pepsin and trypsin, and 8 mg / mL chymotrypsin, and still maintains its original antimicrobial activity after 8 hours of incubation.

[0047] Example 4 Determination of Salt Ion Stability of Antimicrobial Peptide FR

[0048] The antibacterial 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 MgCl2, and 4 μM FeCl3, and the specific operation method was the same as that in Example 3. The control group was the minimum inhibitory concentration determination result without salt ions, and the results are shown in Table 3.

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

[0050] Salt ions Minimum inhibitory concentration (μM) Comparison 8 150mM NaCl 24 4.5 mM KCl 8 <![CDATA[6μMNH4Cl]]> 8 <![CDATA[8μMZnCl2]]> 8 <![CDATA[1mMMgCl2]]> 16 <![CDATA[4μMFeCl3]]> 8

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

[0052] Example 5 Determination of hemolytic activity of antimicrobial peptide FR

[0053] Purchase 1% porcine red blood cells, collect the red blood cells by centrifugation at 3000rpm for 10min; wash with PBS solution for 3 times, and then resuspend with 10mL PBS solution; take 50μL of red blood cell suspension and 50μL of antimicrobial peptide solution of different concentrations (4, 8, 16, 32, 64 and 128μM), mix evenly, and incubate at a constant temperature of 37℃ in an incubator for 1h; then centrifuge at 4℃ and 3000rpm for 10min; take out the supernatant and measure the light absorption value at 570nm with an enzyme marker. 50μL of red blood cells plus 50μL of PBS solution is used as a negative control, and 50μL of red blood cells plus 50μL of 0.1% Tritonx-100 is used as a positive control. The minimum hemolytic concentration is the antimicrobial peptide concentration when the antimicrobial peptide causes a 10% hemolysis rate. The test results are shown in Figure 3 .pass Figure 3 It can be seen that the antimicrobial peptide FR did not show hemolytic activity within the detection range, causing 1% hemolysis of erythrocytes at a concentration of 128 μM and failing to cause 10% hemolysis of erythrocytes, indicating that the antimicrobial peptide FR with low hemolytic activity has the potential to be developed as an antibiotic alternative.

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

[0055] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

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. Use of the antimicrobial peptide FR according to claim 1 in the preparation of an anti-Gram-negative bacterial agent.

3. The use according to claim 2, characterized in that: The Gram-negative bacteria include one or more of Escherichia coli, Shigella flexneri, Salmonella and Campylobacter jejuni.

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

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

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

7. Use of the antimicrobial peptide FR according to claim 1 in the preparation of feed additives.

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

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

10. A feed, characterized in that: The feed comprises the feed additive according to claim 8 and a basic diet.

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