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A tail-anchored α-helix antimicrobial peptide gw4a and preparation method and application thereof

An antibacterial peptide and α-helix technology, applied in the field of α-helix antibacterial peptide GW4A and its preparation, can solve the problems of inactivated clinical application, poor stability, unsatisfactory antibacterial activity, etc.

Active Publication Date: 2021-03-16
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many antimicrobial peptides have poor antibacterial activity and poor stability in serum environment, which leads to their rapid inactivation in biological fluids and limits their further clinical application
Antimicrobial peptide W4 is a broad-spectrum synthetic peptide rich in tryptophan, but its antibacterial activity is not ideal

Method used

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  • A tail-anchored α-helix antimicrobial peptide gw4a and preparation method and application thereof
  • A tail-anchored α-helix antimicrobial peptide gw4a and preparation method and application thereof
  • A tail-anchored α-helix antimicrobial peptide gw4a and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Design of Antimicrobial Peptides

[0018] Taking W4 as the active center, the sequence of peptide W4 is: RWRWWWRWR, anchored with different hydrophobic amino acids (Ala, Trp, Val, Ile, Phe, Leu) at its tail, and designing a cap structure with glycine at the N-terminus. A series of antimicrobial peptides; when glycine is added to the N-terminal and alanine is anchored at the tail, it is named GW4A. The amino acid sequences of GW4A and W4A are shown in Table 1 when only glycine is anchored at the tail end.

[0019] Table 1 Amino acid sequences of GW4A and W4A

[0020] peptide amino acid sequence Molecular weight (Dalton) wxya Gly Arg Trp Arg Trp Trp Trp Arg Trp Arg Ala-NH2 1700.99 W4A Arg Trp Arg Trp Trp Trp Arg Trp Arg Ala-NH2 1643.94

[0021] The charge numbers of GW4A and W4A are +5, respectively, and the average hydrophobicity values ​​are 0.684 and 0.752, respectively. The carboxyl termini of both peptides, GW4A and W4A, were amid...

Embodiment 2

[0023] Synthesis of Two Antimicrobial Peptides GW4A and W4A 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 Resin for chain protection.

[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 e...

Embodiment 3

[0028] Embodiment 3: Determination of biological activity of antimicrobial peptides

[0029] 1. Determination of antibacterial activity: Prepare the peptide as a storage solution for 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 20 hours, and the minimum inhibitory concentration is the one where no turbidity is seen at the bottom of the well with the...

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Abstract

The invention discloses alpha spiral antibacterial peptide GW4A with an anchored tail end and a preparation method and application. The sequence of the antibacterial peptide GW4A is as shown in a sequence table SEQ ID No.1. According to the alpha spiral antibacterial peptide GW4A having an anchored tail end, peptide W4 is used as an active center, the tail end of the peptide W4 is anchored with alanine, a hat structure is formed at an N end with glycine, and the antibacterial peptide GW4A is designed. The therapeutic index of the GW4A is as high as 137.69. The antibacterial peptide GW4A has obvious restraining effects on 19 kinds of Gram-negative bacteria and gram-positive bacteria strains, such as escherichia coli, salmonella typhimurium, salmonella gallinarum, staphylococcus aureus, , and has very low hemolytic activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a tail-end-anchored α-helical antibacterial peptide GW4A, a preparation method and application thereof. Background technique [0002] Antibiotics have been used to protect humans and animals for more than 90 years, since penicillin was first discovered in 1928. However, in recent years, with the emergence of a large number of antibiotic-resistant strains, the effectiveness of antibiotics in the treatment of common infections has declined rapidly, especially the development of new generation antibiotics has not been fully supplemented. Therefore, the development of new antibacterial drugs is imminent. [0003] Antimicrobial peptides are a class of small molecular polypeptides used by animals to resist external microorganisms and eliminate mutant cells in the body. The quantity is small, which fully meets the needs of animal product safety, is suitable for use in the feed...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K1/20C07K1/04A61K38/08A61P31/04
CPCA61K38/00A61P31/04C07K7/06Y02A50/30
Inventor 单安山王家俊宋静展娜于伟康
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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