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Antibacterial peptide containing two leucine repeating units as well as preparation method and application

A leucine repeating and repeating unit technology, applied in the biological field, can solve problems such as drug residues, and achieve the effects of low synthesis cost, mature preparation method and technology

Inactive Publication Date: 2014-05-14
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, problems such as drug-resistant strains and drug residues have also emerged, which have been plagued by human beings.

Method used

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  • Antibacterial peptide containing two leucine repeating units as well as preparation method and application
  • Antibacterial peptide containing two leucine repeating units as well as preparation method and application
  • Antibacterial peptide containing two leucine repeating units as well as preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: the design of leucine repeat unit antimicrobial peptide sequence

[0017] The known motif of a leucine repeat unit is LxxLxLxxNxL, where L is leucine and x is any amino acid. The amino acid composition of antimicrobial peptides is mainly characterized by hydrophobicity and net positive charge. Therefore, we enriched the motif with hydrophobic and positively charged residues, resulting in the amino acid sequence LeuArgArgLeuTrpLeuArgAlaAsnArgLeu of the leucine repeat unit. At the same time, this repeating unit is connected in series to increase the length of its peptide chain and the number of net charges to obtain two antimicrobial peptide sequences. As shown in Table 1.

[0018] Amino acid sequence and molecular weight of table 1 antimicrobial peptide

[0019]

Embodiment 2

[0020] Embodiment 2: the synthesis of leucine repeat unit antimicrobial peptide sequence

[0021] The two peptides shown in Table 1 were synthesized using a peptide synthesizer, using solid-phase organic synthesis, using the Fmoc protection synthesis method, and the synthesis direction was carried out one by one from the C-terminal to the N-terminal. The specific steps are as follows:

[0022] (1) Select the Wang resin that has been connected to the first amino acid at the C-terminal, that is, Fmoc-A(trt)-Wang (9-fluorenylmethoxy-trimethyl-A, where A is the first amino acid at the C-terminal) , use dimethylformamide (DMF) to soak for about 15 minutes to remove impurities; use DMF containing 20% ​​piperidine to remove the Fmoc protection on the resin, 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 ptrintrion...

Embodiment 3

[0026] Embodiment 3: the mensuration of antimicrobial peptide antibacterial activity

[0027] The method for determining the minimum inhibitory concentration (MIC) recommended by the American Clinical Laboratory Standards Institute (CLSI) was adopted, and corresponding improvements were made for the cationic characteristics of antimicrobial peptides. 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 50 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 antimicrobial 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 turbid...

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Abstract

The invention provides an antibacterial peptide containing two leucine repeating units as well as a preparation method and an application. The amino acid sequence of the antibacterial peptide containing two leucine repeating units is shown by SEQ No.1. The preparation method comprises the following steps: obtaining the amino acid sequences of the leucine repeating units according to the sequence motifs of the leucine repeating units and the amino acid composition characteristics of the antibacterial peptide, and connecting the repeating units in series to increase the length of peptide chain and the number of net charges; synthesizing a polypeptide crude product by a solid-phase synthesis method through using a polypeptide synthesizer; and finally, purifying the synthesized polypeptide with high performance liquid chromatography, and identifying with electrospray ionization mass spectrometry. The antibacterial peptide provided by the invention has the beneficial effects that (1) the polypeptide is designed based on the leucine repeating units widely available in organism and has broad-spectrum antibacterial activity; and (2) the polypeptide consists of 22 amino acids and has relatively short peptide chain; and the preparation method and technology are mature, and the synthesis cost is low.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an antibacterial peptide containing two leucine repeating units, a preparation method and application. Background technique [0002] The use of antibiotics improves human health and animal productivity. However, problems such as drug-resistant strains and drug residues have also emerged, which have been plagued by human beings. Due to the problems of drug residues and bacterial resistance, there is an urgent need to discover new antibacterial agents. As an important immune component of the human body, antimicrobial peptides have great potential for development and are a hot spot for researchers. It is generally believed that antimicrobial peptides destroy the cell membrane of bacteria through physical adsorption and other effects, thereby killing bacteria, so the probability of bacteria developing drug resistance is small. Antimicrobial peptides are generally composed o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K1/04C07K1/20A61K38/16A61P31/04
CPCC07K14/00A61K38/00
Inventor 马清泉单安山董娜吕银凤焦文静
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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