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Beta hairpin antimicrobial peptide containing d-type proline and glycine turn angle and preparation method thereof

An antimicrobial peptide and proline technology, applied in the biological field, can solve the problems of long β-fold antibacterial peptide sequence, high synthesis cost, and difficult synthesis, and achieve the effects of low hemolytic activity, short sequence length, and simple preparation technology

Active Publication Date: 2021-12-14
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Based on the above deficiencies, the present invention provides a β-hairpin antibacterial peptide containing D-proline and glycine corners and a preparation method, which solves the problem of excessively long sequence, high toxicity, difficult synthesis, and difficult synthesis in the preparation process of β-fold antibacterial peptides. high cost

Method used

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  • Beta hairpin antimicrobial peptide containing d-type proline and glycine turn angle and preparation method thereof
  • Beta hairpin antimicrobial peptide containing d-type proline and glycine turn angle and preparation method thereof
  • Beta hairpin antimicrobial peptide containing d-type proline and glycine turn angle and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0013] Design of Antimicrobial Peptide RWFKFpGRWFKF-NH 2

[0014] The amino acid sequence of the antimicrobial peptide WKFpG is:

[0015] Arg Trp Phe Lys Phe D-Pro Gly Arg Trp Phe Lys Phe-NH 2

1 5 10 12

[0016] With the rigid D-Pro-Gly corner as the corner unit, two pairs of tryptophan and lysine are placed at the non-hydrogen bond site of the β-hairpin side chain, and the interaction is used to assist the D-Pro-Gly corner to form a hairpin structure , design antimicrobial peptide template XWYKYZZXWYKY-NH 2 , where X is a positively charged amino acid, Y is a hydrophobic amino acid, and ZZ is a β-turn unit. When X=R, Y=F, and ZZ=pG, the antimicrobial peptide is named WKFpG. The sequence of the antimicrobial peptide is shown in Table 1 as peptide WKFpG. A hairpin structure is formed with an Asn-Gly turn angle, and the sequence of the formed antimicrobial peptide is shown in Table 1, peptide WKFNG.

[0017] Table 1 Amino Acid Sequence of Derived Peptides

[0018]

...

Embodiment 2

[0022] Synthesis of WKFpG Antimicrobial Peptide by Solid Phase Chemical Synthesis

[0023] 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;

[0024] 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

[0027] Embodiment 3: the mensuration of antimicrobial peptide antibacterial activity

[0028] 1. Determination of antibacterial activity: the minimum inhibitory concentration of antimicrobial peptides was determined by micro broth dilution 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 18h, and use a microplate reader at 492nm (OD 492 ) to determine the minimum inhibitory concentration. The test was repeated 3 times with two parallels for each repetition. The test re...

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Abstract

The invention provides a β-hairpin antimicrobial peptide containing D-type proline and glycine corners and a preparation method thereof. The sequence of the antimicrobial peptide WKFpG of the present invention is shown in SEQ ID No.1. The present invention uses the rigid D-Pro-Gly corner as the corner unit, symmetrically places tryptophan and lysine at the non-hydrogen bond site of the β-hairpin side chain, and uses its interaction to assist the D-Pro-Gly corner to form a hairpin Structure, designed antimicrobial peptide template XWYKYZZXWYKY‑NH 2 . The application of the antibacterial peptide in the preparation of drugs for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria. The length of the amino acid sequence of this antibacterial peptide is only 12, which reveals the influence of rigid pG turn on the biological activity of β-hairpin antibacterial peptide, and has high inhibitory effect on various Gram-negative bacteria and positive bacteria, and has low hemolytic activity , the therapeutic index reaches 110.30, which is 1.5 times higher than that of the β-hairpin antimicrobial peptide with the same side chain containing NG turn, and has the potential to become a green and efficient antibiotic substitute.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a β-hairpin antibacterial peptide containing D-type proline and glycine corners and a preparation method thereof. Background technique [0002] Over the past few decades, the overuse and inappropriate use of antibiotics has contributed to the emergence of drug residues and bacterial resistance, which has caused serious global health problems and contributed to reduced economic output in animal production or other industries. Exploring novel therapeutic strategies has become a top priority. Antimicrobial peptides (AMPs) play an intrinsic role in almost all multicellular organisms as the first line of host defense, showing inhibitory activity against a variety of pathogens. Although multiple targets of AMPs in bacteria have been reported in recent years, there is no doubt that most AMPs release their hydrophobic domains after electrostatic interactions between their cation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K1/06C07K1/04A61K38/10A61P31/04
CPCC07K7/08A61P31/04A61K38/00
Inventor 单安山邵长轩菅俏
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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