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A kind of enzymolysis-resistant α-helical antimicrobial peptide bound by full carbon and hydrogen side chains, its preparation method and application

An antimicrobial peptide and anti-enzymolysis technology, applied in the direction of peptide preparation methods, antibacterial drugs, chemical instruments and methods, etc., to achieve high application potential, stable secondary structure, and low degree of degradation

Active Publication Date: 2022-06-14
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]Although natural antimicrobial peptides show a high degree of specificity in their biological actions, their practical application is limited by problems such as long sequences, high toxicity, and easy hydrolysis by proteases Severe limitations. Endogenous proteases, such as trypsin and chymotrypsin, can easily degrade AMPs into short peptides or even single amino acid molecules. carboxy-terminus of amino acids, therefore, although thousands of natural antimicrobial peptides have been developed, few have been put into use as antibiotic substitutes

Method used

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  • A kind of enzymolysis-resistant α-helical antimicrobial peptide bound by full carbon and hydrogen side chains, its preparation method and application
  • A kind of enzymolysis-resistant α-helical antimicrobial peptide bound by full carbon and hydrogen side chains, its preparation method and application
  • A kind of enzymolysis-resistant α-helical antimicrobial peptide bound by full carbon and hydrogen side chains, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Design of Antimicrobial Peptides

[0015] The amino acid sequence of the antimicrobial peptide stLRL is from N-terminal to C-terminal:

[0016] Leu Xaa Leu Arg Leu Xaa Arg - NH 2

1 5 7

[0018] Wherein, Xaa is (S)-2-(4-pentenyl)alanine;

[0019] Referring to the properties of amino acids and the structural properties of "stitching", in order to avoid the enzymatic cleavage sites of trypsin and chymotrypsin, firstly, Arg was selected as a cationic amino acid to provide a positive charge, and secondly, the hydrophobic amino acid Leu was selected to enhance the hydrophobic depth. Leu is placed in the position (a position) to eliminate the cleavage of the chymotrypsin site, Arg and Leu are placed in the "staple" structure to improve antibacterial activity, and Arg is placed in the seventh position (g position) to provide positive charges, which can hinder pancreatic Cleavage by proteases, carboxy-terminal amidation for increased stability. The structural formula of th...

Embodiment 2

[0022] Example 2

[0023] Synthesis of Antimicrobial Peptide stLRL 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 the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorene Methoxycarboxy-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); as above, it is synthesized from the C-terminus to the N-terminus, until the synthesis is completed, and the resin obtained by removing the side chain protection of the Fmoc group ;

[0025] 2. Add a cleavage reagent to the peptide resin obtained above, react at 20°C for 2 hours in the dark, filter; precipitate with TFA (trifluoroacetic acid) and wash, mix the washing liquid with the above filtrate, concentrate wi...

Embodiment 3

[0029] Example 3

[0030] Detection of antimicrobial peptide antibacterial activity and hemolytic activity in vitro:

[0031] 1. Determination of antibacterial activity: the minimum inhibitory concentration of antimicrobial peptides was determined by microdilution method. Add different concentrations of peptides to 0.2% BSA dilution (containing 0.01% acetic acid) in a 96-well plate, and then add an equal volume of the final concentration of 1 × 10 5 CFUmL -1 of bacterial suspensions in 96-well plates with final peptide concentrations ranging from 0.25 to 128 μM. Incubate at 37° C. for 18-22 hours, measure the light absorption value at 492 nm (OD=492 nm) with a microplate reader, and determine the minimum inhibitory concentration. The test results are shown in Table 2.

[0032] 2. Determination of hemolytic activity: collect 1mL of human fresh blood into a sodium heparin anticoagulant tube, centrifuge at 1000g for 5min to collect red blood cells, wash with PBS for 3 times a...

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Abstract

The invention provides an anti-enzymolysis α-helical antibacterial peptide bound by full carbon and hydrogen side chains, a preparation method and application thereof. The antimicrobial peptide sequence is shown in SEQ ID No.1. Preparation method: Based on the α-helical polypeptide folding principle, design a polypeptide sequence containing seven amino acid residues, select (S)-2-(4-pentenyl) alanine to be placed at the second position (b position ) and the 6th position (f position) to form a full carbon hydrogen binding side chain "book" structure, located in the "book" structure and its adjacent positions can hinder the hydrolysis of chymotrypsin. The sequence length of this antimicrobial peptide is only 7 amino acids, and the implantation of full carbon-hydrogen binding side chains has been invented to affect the biological activity of linear α-helical antibacterial peptides and the ability to resist protease hydrolysis. It is effective against a variety of Gram-negative bacteria and positive It has a strong inhibitory effect on erythrocytes, has extremely low hemolytic toxicity to red blood cells, and has a better effect on resistance to protease hydrolysis. It is expected to become an efficient substitute for traditional antibiotics.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-enzymolysis α-helical antibacterial peptide bound by full carbon and hydrogen side chains, a preparation method and application thereof. Background technique [0002] In recent years, the emergence of multidrug-resistant bacteria has seriously threatened public health, which has made conventional antibiotics increasingly ineffective, so it is imperative to find new antibacterial strategies. Antimicrobial peptides (AMPs) are a subclass of natural polypeptides expressed by different species and are the first line of defense of the host's natural immune system. Most cationic antimicrobial peptides mainly kill bacteria through membrane lysis. This unique mechanism of action and its multiple biological functions make AMPs the most potential competitor to replace traditional antibiotics. [0003] Although natural antimicrobial peptides show a high degree of specificity...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K1/04A61K38/08A61P31/04
CPCC07K7/06A61P31/04A61K38/00
Inventor 单安山菅俏陈虹羽
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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