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Derived peptide for chicken origin antibacterial peptide as well as preparation method and application thereof

An antibacterial peptide and derivatized peptide technology, applied in the field of chicken-derived antibacterial peptide derived peptides and their preparation, can solve the problem of anti-infection treatment falling into the crisis of drug-resistant bacteria, and achieve low cytotoxicity, high application value, and simple experimental technology. Effect

Inactive Publication Date: 2015-05-27
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the widespread use and abuse of antibacterial drugs, drug-resistant strains continue to increase, making anti-infection therapy fall into a crisis of drug-resistant bacteria

Method used

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  • Derived peptide for chicken origin antibacterial peptide as well as preparation method and application thereof
  • Derived peptide for chicken origin antibacterial peptide as well as preparation method and application thereof
  • Derived peptide for chicken origin antibacterial peptide as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Design of Antimicrobial Peptide MGW21

[0018] The 20 amino acids of the leader peptide of chicken-derived antibacterial peptide AvBD4 were intercepted, and the cysteine ​​at the 5th position of the leader peptide, and the phenylalanine at the 6th, 7th and 12th positions were replaced with the charged amino acid lysine respectively. , to obtain the antimicrobial peptide MGW21.

[0019] The charge number of the obtained antimicrobial peptide MGW21 is +7, the hydrophobic value is -0.01, and the molecular weight is 2331.06kDa. The carboxyl terminal of the antimicrobial peptide is amidated to add a positive charge to improve the stability of the antimicrobial peptide.

[0020] The amino acid sequence of the chicken source antibacterial peptide AvBD4 leader peptide is:

[0021] Met Lys Ile Leu Cys Phe Phe Ile Val Leu Leu Phe Val Ala Val His Gly Ala Val Gly

[0022] 15101520

[0023] The amino acid sequence of the obtained antimicrobial peptide MGW21 is:

[0024] Met Lys...

Embodiment 2

[0027] Synthesis of Antimicrobial Peptide MGW21

[0028] The above-mentioned antimicrobial peptide MGW21 was synthesized using a peptide synthesizer. The method was solid-phase chemical synthesis, and the specific steps were:

[0029] (1) The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus, 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.

[0030] (2) Add a cleavage reagent to the peptide resin obtained in (1), react at 20° C. for 2 hours in the dark,...

Embodiment 3

[0034] Determination of antimicrobial activity of antimicrobial peptides

[0035] (1) Determination of antibacterial activity: the minimum inhibitory concentration of several antimicrobial peptides was determined by the micro broth dilution 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 pcs / mL) in each well, respectively set a positive control (containing bacteria solution but not antimicrobial peptide) and a negative control (neither containing bacteria solution nor peptide), and incubated at 37°C for 20 hours, so that the bottom of the well was not visible to the naked eye. The one with turbidity is the minimum inhibitory concentration. The test results are shown in Table 1. It can be seen ...

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Abstract

The invention provides a derived peptide for a chicken origin antibacterial peptide as well as a preparation method and application thereof. The sequence of the derived peptide is shown as SEQ No.1. The preparation method comprises the following steps: intercepting 20 amino acids of a chicken origin AvBD4 leader peptide, replacing 5th cysteine and 6th, 7th and 12th phenylalanine by amino acid lysine with charges respectively, meanwhile, carrying out amidation on a carboxyl terminal of the antibacterial peptide, and increasing a positive charge to improve the stability of the antibacterial peptide; adopting a solid-phase chemical synthesis method to obtain crude MGW21; and purifying and identifying synthesized polypeptide by using reversed-phase high performance liquid chromatography and electrospray mass spectrometry. The invention provides the application of the antibacterial peptide in preparing a medicament for treating gram-positive bacteria or gram-negative bacteria infectious diseases. The antibacterial peptide is good in amphiphilic property; compared with an antibiotic and Melittin, the antibacterial peptide is high in antibacterial activity and low in cytotoxicity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a chicken-derived antimicrobial peptide derivative peptide and a preparation method and application thereof. Background technique [0002] Antimicrobial peptides (AMPs), also known as antimicrobial peptides or peptide antibiotics, are a class of small molecule polypeptides produced by specific bacterial genes and have broad-spectrum antibacterial properties. Antimicrobial peptides were first discovered from Hyatophora cecropia in the 1980s. Antimicrobial peptides usually consist of 12-50 amino acid residues, with a molecular weight of less than 10kDa. Most antimicrobial peptides contain positively charged amino acids and are amphipathic and cationic. The main biological function of antimicrobial peptides is that they can kill a variety of bacteria including Gram-negative bacteria and Gram-positive bacteria, and have the characteristics of broad antibacterial spectrum and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/465C07K1/20C07K1/04A61K38/17A61P31/04
CPCA61K38/00C07K14/465
Inventor 董娜吴迪毕重鹏
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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