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A kind of antibacterial peptide and preparation method thereof

A technology of antibacterial peptides and amino acids, applied in the field of artificially synthesized new antibacterial peptides and their preparation

Inactive Publication Date: 2011-12-14
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At home and abroad, there are no reports and studies using arginine, isoleucine and phenylalanine as structural units to study the relationship between the structure and function of antimicrobial peptides

Method used

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  • A kind of antibacterial peptide and preparation method thereof
  • A kind of antibacterial peptide and preparation method thereof
  • A kind of antibacterial peptide and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0014] According to the mechanism of action of antimicrobial peptides and the characteristics of amino acid composition, the basic amino acid arginine (Arg) residues are used to provide positive charges to attract the negative charges on the surface of bacteria, and the aliphatic amino acids isoleucine (Ile) and The aromatic amino acid phenylalanine (Phe) has a hydrophobic interaction with phospholipids, thereby enabling antimicrobial peptides to damage bacterial cell membranes. According to this principle, there are five antimicrobial peptides in this embodiment, namely A-E in Table 1;

Embodiment 2

[0016] The above five peptides were synthesized using a peptide synthesizer, using solid-phase organic synthesis, and the synthesis direction was carried out one by one from the C-terminal to the N-terminal, and the Fmoc protection synthesis method was adopted. The specific steps were as follows:

[0017] Select the Wang resin that has been linked to the first amino acid at the C-terminal, that is, Fmoc-A(trt)-Wang (9-fluorenylmethoxycarboxy-trimethyl-A, where A is the first amino acid at the C-terminal), use two Soak in methylformamide (DMF) for about 15 minutes to remove impurities; remove the Fmoc protection on the resin with DMF containing 20% ​​piperidine, react for 20 minutes, and wash the resin until it is complete. The piperidine was washed away with DMF, and the remaining solid suspension was the deprotected A-Wang. The quality of A-Wang deprotection was checked with ptrintrione detection reagent.

[0018] Fmoc-B(trt)-OH (9-fluorenylmethoxycarboxy-trimethyl-B, B is t...

Embodiment 3

[0024] 1) Determination of antibacterial activity: Prepare the peptide into a certain 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 pc / 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 naked eye.

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Abstract

The invention relates to antibacterial peptides and a preparation method thereof. An amino acid sequence of the antibacterial peptide is IleTrpArgIlePheArgArgIlePhe. The method comprises the following steps of: 1) designing a group of antibacterial peptides consisting of isoleucine, arginine and phenylalanine which serve as basic units by utilizing the bacteriostatic mechanism of the antibacterial peptides and the characteristic of amino acid composition; 2) synthesizing a polypeptide crude product by using a polypeptide synthesizer in a solid-phase synthetic process; 3) purifying the synthesized polypeptide by reversed phase high performance liquid chromatography, and identifying by electrospray mass spectrometry; and 4) performing an in-vitro activity test, wherein a result of the activity test indicates that antibacterial peptides B containing 3 basic units have high bacteriostatic activity and weak hemolytic activity, a treatment index is the highest, and cell selectivity is optimum, so the antibacterial peptides have high application value.

Description

technical field [0001] The invention relates to a novel artificially synthesized antibacterial peptide and a preparation method thereof. Background technique [0002] Due to the problem of drug residue and bacterial resistance to antibiotics, it is urgent to find a green, pollution-free and non-bacterial resistance substitute. Antimicrobial peptides are small peptides with broad-spectrum antibacterial activity that widely exist in organisms. They are an important part of biological non-specific defense systems and can inhibit the growth of various bacteria or fungi, even tumor cells. Antimicrobial peptides have a net positive charge, so they physically interact with bacterial cell membranes; and then inhibit or kill bacteria through the interaction of hydrophobic residues with hydrophobic components in phospholipids. For microorganisms such as bacteria, it is difficult to change the membrane structure of their own phospholipid bilayers, so the probability of antimicrobial p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/06C07K1/04C07K1/20A61P31/04
Inventor 单安山马清泉李峰郑燕斌
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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