β hairpin antimicrobial peptide containing asparagine and glycine turn angle and preparation method thereof

An asparagine and antibacterial peptide technology, applied in the biological field, can solve the problems of high synthesis cost and high concentration hemolysis of β-hairpin antibacterial peptide, and achieve the effects of low synthesis cost, short sequence length and stable structure.

Active Publication Date: 2022-02-08
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 antimicrobial peptide containing asparagine and glycine turns and a preparation method thereof, which solves the problem of high synthesis cost and high-concentration hemolysis of β-hairpin antimicrobial peptide

Method used

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  • β hairpin antimicrobial peptide containing asparagine and glycine turn angle and preparation method thereof
  • β hairpin antimicrobial peptide containing asparagine and glycine turn angle and preparation method thereof
  • β hairpin antimicrobial peptide containing asparagine and glycine turn angle and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Antimicrobial Peptide Design RWFRFNGRWFRF-NH 2

[0015] The amino acid sequence of the antimicrobial peptide WRFNG is:

[0016] Arg Trp Phe Arg Phe Asn Gly Arg Trp Phe Arg Phe-NH 2

1 5 10 12

[0017] Taking the Asn-Gly corner as the corner unit, the interaction of tryptophan and arginine forms the mutual attraction of two β-side chains, and designs the antimicrobial peptide template XWYRYZZXWYRY-NH according to the arrangement principle of the β hairpin structure 2 , where X is a positively charged amino acid, Y is a hydrophobic amino acid, and ZZ is a β-turn angle. When X=R, Y=F, and ZZ=NG, the antimicrobial peptide is named WRFNG. The sequence of the antimicrobial peptide is shown in Table 1, peptide WRFNG. The sequence of the antimicrobial peptide formed with the Pro-Gly turn as the turn unit is shown in Table 1 peptide WRFPG. The sequence of the antimicrobial peptide formed with the D-Pro-Gly turn as the turn unit is shown in Table 1 as peptide WRFpG.

[001...

Embodiment 2

[0023] Synthesis of WRFNG Antimicrobial Peptides by Solid Phase Chemical Synthesis

[0024] 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;

[0025] 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 ...

Embodiment 3

[0028] Embodiment 3: the mensuration of antimicrobial peptide antibacterial activity

[0029] 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 6 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, measure the light absorbance at 492nm (OD 492nm) with a microplate reader, and determine the minimum inhibitory concentration. The test was repeated 3 times, with two parallels...

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Abstract

The invention provides a β-hairpin antibacterial peptide containing asparagine and glycine turn angle and a preparation method. The sequence of the antimicrobial peptide WRFNG of the present invention is shown in SEQ ID No.1. The preparation method of the present invention uses the Asn-Gly corner as the corner unit, forms the mutual attraction of two β-side chains with the interaction of Trp and Arg, and designs the antibacterial peptide template XWYRYZZXWYRY-NH according to the arrangement principle of the β-hairpin structure 2 . X=R, Y=F, ZZ=NG. 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 therapeutic index of the antimicrobial peptide of the present invention reaches 138.30, which is 1.58 times higher than WRFPG with Pro-Gly corner, and 3.55 times higher than WRFpG with D-Pro-Gly corner, and has the development 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 asparagine and glycine turn angles and a preparation method thereof. Background technique [0002] Antimicrobial peptides (AMPs) found in the host immune system have attracted widespread attention due to their excellent antibacterial properties and unique mechanisms of action. Especially in the past few decades, the overuse and inappropriate use of antibiotics has made the problem of drug residues and bacterial resistance more serious. These peptides are broad-spectrum and have strong activity against microorganisms, viruses, parasites and even tumor cells. Furthermore, unlike conventional antibiotics that inhibit specific metabolic pathways, most antimicrobial peptides exert their bactericidal effects through irreversible disruption of bacterial membranes, leading to leakage of vital cellular contents and ultimately cell deat...

Claims

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Application Information

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