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β-hairpin antimicrobial peptide with cross-chain interaction between tryptophan and arginine and preparation method thereof

A technology of tryptophan-arginine cross-chain and arginine cross-chain, which is applied in the field of bioengineering, can solve the problems of shortening the length of polypeptide sequences, too long sequences of antimicrobial peptides, and high synthesis costs, achieving short sequence lengths and high application The effect of value and convenience of synthesis technology

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 with cross-chain interaction between tryptophan and arginine and its preparation method, which utilizes the positive charge formation of the aromatic side chain of tryptophan and arginine The cation-π effect across the chain in the polypeptide molecule replaces the disulfide bond formed by cysteine, shortens the length of the polypeptide sequence, provides sufficient positive charge and hydrophobicity to form the amphiphilic structure of the β-hairpin, and solves the problem of β -The problem of long hairpin antimicrobial peptide sequence, difficult synthesis and high synthesis cost

Method used

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  • β-hairpin antimicrobial peptide with cross-chain interaction between tryptophan and arginine and preparation method thereof
  • β-hairpin antimicrobial peptide with cross-chain interaction between tryptophan and arginine and preparation method thereof
  • β-hairpin antimicrobial peptide with cross-chain interaction between tryptophan and arginine and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0014] Design of Antimicrobial Peptides

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

[0016] Arg Trp Leu Arg Leu Pro Gly Arg Trp Leu Arg Leu-NH 2

1 5 10 12

[0017] Based on the arrangement of β-hairpin amphiphilic peptides, the template XWYRYPGXWYRY-NH was designed through the cross-chain interaction of tryptophan and arginine as the force to assist the PG corner unit to form the β-hairpin structure 2 , X is a positively charged amino acid, Y is a hydrophobic amino acid, when X=R, Y=L, the antimicrobial peptide is named WRLPG. The sequences of the antimicrobial peptides are shown in Table 1.

[0018] Table 1 Amino Acid Sequence of Derived Peptides

[0019]

[0020] The sequence length of WRLPG is 12, and the corner unit is a PG unit. The C-terminus of the peptide is amidated to increase a positive charge, and the charge number is +5. This method enables the polypeptide to form a stable β-hairpin structure ( figure 1 ), has high antibacte...

Embodiment 2

[0022] Synthesis of WRLPG 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 several antibacterial peptides was determined by the micro broth dilution method. Bacteria were incubated overnight at 37 °C with constant shaking at 220 rpm and then transferred to fresh MHB until log phase of growth. 50 μL of bacterial culture (~10 6 CFU / mL) was added to 50 μL of BSA containing different concentrations of antimicrobial peptides, and incubated at 37°C for ~18h. Observe and measure the optical density at 492nm at the end of the 18h incubation, and the antimicrobial peptide concentration at which no microbial growth is observed visually and spectrophotometrically is MIC. Broth containing microbial cells was used as a negative control and uninoculated broth was used as a positive control. The test was repeated 3 times, with two parallels for each repetition.

[0029] The test results...

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Abstract

The invention provides a β-hairpin antimicrobial peptide with cross-chain interaction between tryptophan and arginine and a preparation method thereof. Antimicrobial peptide WRLPG, the sequence of which is shown in SEQ ID No.1. The preparation method is based on the arrangement of β-hairpin amphiphilic peptides, and an antimicrobial peptide template XWYRYPGXWYRY-NH containing tryptophan-arginine cross-chain interaction and PG corner unit is designed 2 , X=R, Y=L. 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. This antimicrobial peptide forms a stable β-hairpin structure without the disulfide bond formed by cysteine ​​pairs. The amino acid sequence length is only 12, and it has good biosafety on the basis of maintaining high antibacterial activity. Sex, the therapeutic index reached 90.78. It has a high development potential to replace antibiotics.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a beta hairpin antibacterial peptide with cross-chain interaction between tryptophan and arginine and a preparation method thereof. Background technique [0002] With the increasing resistance of microorganisms to traditional antibiotics, the need for new antimicrobial infection drugs is becoming more and more urgent. Antimicrobial peptides (AMPs), widely found in various organisms from insects to plants to animals, are an important component of innate immunity and display broad-spectrum antimicrobial activity, including Gram-negative and Gram-positive Bacteria, viruses and fungi. Unlike conventional antibiotics that inhibit specific biosynthetic pathways such as cell wall or protein synthesis, most amphipathic AMPs exert their activity by physically disrupting the lipid bilayer of microbial membranes and inducing leakage of cytoplasmic components, resulting i...

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 单安山邵长轩来振衡
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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