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A kind of antimicrobial peptide rich in phenylalanine and its preparation method and application

A technology of phenylalanine and antibacterial peptide is applied in the field of phenylalanine-rich antibacterial peptide RFEK and its preparation, which can solve the problems of long sequence of natural antibacterial peptide, toxicity to eukaryotic cells, shortening of peptide chain length, etc. The effect of short sequence, high antibacterial, low toxicity, and high therapeutic index

Active Publication Date: 2022-02-08
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a phenylalanine-rich antibacterial peptide RFEK and its preparation method and application, shorten the length of the peptide chain, and solve the problems of long natural antibacterial peptide sequence, high synthesis cost and toxicity to eukaryotic cells

Method used

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  • A kind of antimicrobial peptide rich in phenylalanine and its preparation method and application
  • A kind of antimicrobial peptide rich in phenylalanine and its preparation method and application
  • A kind of antimicrobial peptide rich in phenylalanine and its preparation method and application

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Embodiment 1

[0012] Design and preparation of embodiment 1 antimicrobial peptide synthesis

[0013] Based on the de novo design of antimicrobial peptides and the structure-activity relationship, the dominant hydrophobic amino acid Phe and the charged amino acid Arg that stand out in the short peptide design are alternately arranged to ensure the hydrophobicity required for its antibacterial function, and Glu / Lys is used to replace Arg. When the peptide interacts with the cell membrane, the attraction ability between the peptides is used to increase its aggregation concentration, and to ensure a certain number of positive charges required for attraction to the bacterial cell membrane. The antimicrobial peptide RFEK is newly obtained, and its amino acid sequence is shown in Table 1.

[0014] Table 1 Amino acid sequence of antimicrobial peptide RFEK

[0015]

[0016] The charge number of the antibacterial peptide RFEK is +4, the hydrophobic value is 0.278, and the hydrophobic moment is 0.0...

Embodiment 2

[0018] Synthesis of RFEK Antimicrobial Peptides by Solid Phase Chemical Synthesis

[0019] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminal to the N-terminal, and is completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminal of each antimicrobial peptide) is inserted into Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9 -Fmoxy-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 until the synthesis is completed, and the side of the Fmoc group is removed chain protection resin;

[0020] 2. Add a cleavage reagent to the peptide resin obtained above, react for 2 hours at 20°C in the dark, filter; wash the precipitate with TFA (trifluoroacetic acid), mix the washing liquid with the above filtrate, concentrate with a rotary evaporator, and t...

Embodiment 3

[0024] 1. Bacteriostatic activity of antimicrobial peptides

[0025] The peptide was configured as a 2.56mM / L storage solution for future use. The minimum inhibitory concentrations of several antimicrobial peptides were determined by the broth microdilution method. Using 0.01% acetic acid (containing 0.2% BSA) as the diluent, a series of gradient antimicrobial peptide solutions were sequentially prepared using the double dilution method. Take 100 μL of the above solution and place it in a 96-well cell culture plate, then add an equal volume of the bacteria solution to be tested (~10 5 individual / mL) in each well. Positive controls (containing bacterial fluid but not antimicrobial peptides) and negative controls (neither bacterial fluid nor peptides) were set up. Incubate at a constant temperature of 37°C for 18 hours, and the minimum inhibitory concentration is the one where no turbidity is seen at the bottom of the well with the naked eye. The results are shown in Table 2...

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Abstract

The invention provides a phenylalanine-rich antimicrobial peptide RFEK as well as its preparation method and application. The sequence of the antimicrobial peptide is shown in SEQ ID No.1. Preparation method: Alternate arrangement of amino acids Phe and Arg is selected to ensure the hydrophobicity required for its antibacterial function, and Arg is replaced by Glu and Lys, so that when the peptide interacts with the cell membrane, the attraction ability between the peptides is used to increase its aggregation concentration. And to ensure a certain number of positive charges required to attract the bacterial cell membrane, the antibacterial peptide was designed. The antimicrobial peptide is used in the preparation of drugs for treating Gram-negative bacterial infectious diseases. The preparation method is simple in technology, and the antibacterial and hemolytic activities of the obtained antibacterial peptides are detected to have strong antibacterial activities against Gram-negative bacterial strains such as Escherichia coli, Salmonella typhimurium, and Salmonella pullorum, and no hemolysis occurs at 128 μM. Has a high therapeutic index.

Description

technical field [0001] This invention belongs to the field of agricultural animal husbandry and veterinary applications, and specifically relates to a phenylalanine-rich antimicrobial peptide RFEK and its preparation method and application Background technique [0002] In animal husbandry, due to the long-term use and abuse of traditional antibiotics, the problem of bacterial resistance and drug residues seriously affects the quality of animal products and human health, making it a problem that cannot be ignored. Therefore, there is an urgent need to develop new antibacterial drugs to solve this problem. Antimicrobial peptides are a class of bioactive small molecules in plants, animals, insects and other organisms, which play an important role in the innate defense mechanism. Compared with traditional antibiotics, antimicrobial peptides have broad-spectrum antibacterial activity and stable physical and chemical properties. Its unique mechanism of action is not easy to produ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K1/06C07K1/04A61K38/08A61P31/04
CPCC07K7/06A61P31/04A61K38/00Y02A50/30
Inventor 单安山陈婷婷杨城义吕银凤
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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