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Multi-stranded beta-hairpin short-peptide with tolerant protease and preparation method and application

A protease and hairpin technology, applied in the biological field, can solve the problems of high synthesis cost, narrow antibacterial spectrum, protease sensitivity, etc.

Inactive Publication Date: 2017-05-31
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, natural cationic antimicrobial peptides are not perfect. Most natural antimicrobial peptides have weak antibacterial activity, relatively narrow antibacterial spectrum, high synthesis cost, some antimicrobial peptides have certain toxicity to eukaryotes, and are highly effective against pathogenic microorganisms. Killing activity is often accompanied by hemolysis to eukaryotes and sensitivity to proteases

Method used

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  • Multi-stranded beta-hairpin short-peptide with tolerant protease and preparation method and application
  • Multi-stranded beta-hairpin short-peptide with tolerant protease and preparation method and application
  • Multi-stranded beta-hairpin short-peptide with tolerant protease and preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Design of Antimicrobial Peptides

[0017] According to the fact that the β-hairpin structure has better antibacterial activity and excellent effect of cell selectivity, and increasing the number is beneficial to improve the stability, this method gives full play to the structural advantages of the β-hairpin structure to obtain multi-strand β-hairpin structure antibacterial peptides, Among them, the common amino acids Trp and Arg are used to ensure the activity while further improving the stability, and changing the composition of the turn amino acids, and finally obtained a series of multi-strand β-hairpin antimicrobial peptides with a new structure: (WRXxRW)n-NH2, where n= 1, 2, 3, 4; PG is selected as the turning angle for illustration. When n=2, the antimicrobial peptide is named W2; when n=3, the antimicrobial peptide is named W3; its amino acid sequence is shown in Table 1.

[0018] Table 1 Amino Acid Sequence of Derived Peptides

[0019]

[0020] The charge nu...

Embodiment 2

[0022] Synthesis of Two Antimicrobial Peptides W2 and W3 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 evaporat...

Embodiment 3

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

[0028] 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 th...

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Abstract

The invention provides a multi-stranded beta-hairpin short-peptide with tolerant protease and a preparation method and an application. A sequence is as shown in a SEQ ID No.1. The short-peptide provided by the invention fully exerts the structural advantages of a beta-hairpin structure to obtain a multi-stranded beta-hairpin structural antibacterial peptide. The stability is further improved while the activity is ensured by using a common amino, and composition of a corner amino acid is changed, so that a series of multi-stranded beta-hairpin antibacterial peptides of brand new structures: (WRXxRW)n-NH2, wherein n is 1, 2, 3 and 4; PG is selected as a corner to describe; when n is equal to 2, the antibacterial peptide is named W2; and when n is equal to 3, the antibacterial peptide is named W3. The peptide verifies relatively high cell selectivity and salt ion tolerance, and moreover, based on the activity of the antibacterial peptide with structural stability, the obtained antibacterial peptide is also relatively short, and relatively strong tolerance of in vivo protease is also represented.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a protease-resistant multi-strand β-hairpin short peptide, a preparation method and application thereof. Background technique [0002] Since antibiotics were officially approved as additives in feed in 1950, they have not only effectively reduced the infection rate of diseases and improved the survival rate of piglets, but also showed obvious effects on promoting animal growth, increasing feed-to-meat ratio, and increasing reproduction rate, etc. good performance. However, since the discovery of vancomycin-resistant bacteria in Japan in 1996, the abuse of antibiotics has caused an increase in drug-resistant strains, and the resulting decline in the immunity of livestock and poultry and the frequent occurrence of imbalances in the intestinal flora of livestock and poultry. In recent years, the emergence of superbugs and a series of problems have made people pay more atten...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/06C07K1/04A61K38/10A61P31/04
CPCA61K38/00C07K7/08
Inventor 单安山丑淑丽王家俊王志华
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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