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D-amino acid antibacterial peptide PRW4-d and preparation method and application thereof

An antimicrobial peptide and amino acid technology, applied in the biological field, can solve the problems of weak antibacterial activity, high synthesis cost, protease sensitivity, etc., and achieve the effect of low hemolytic activity, simple preparation technology and high cell selectivity

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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  • D-amino acid antibacterial peptide PRW4-d and preparation method and application thereof
  • D-amino acid antibacterial peptide PRW4-d and preparation method and application thereof
  • D-amino acid antibacterial peptide PRW4-d and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Design of Antimicrobial Peptides

[0016] First, the 16 amino acid sequence of the N-terminal of the cationic antimicrobial peptide PMAP-36 containing 36 amino acids derived from porcine bone marrow was intercepted, named RI16, and the sequence after interception has a perfect amphipathic structure. Secondly, an imperfect amphiphilic antimicrobial peptide was designed based on the α-helical protein framework and folding principles to obtain PRW4.

[0017] Using the antimicrobial peptide PRW4 as a template, the amino acids cysteine ​​(Cys), aspartic acid (Asp), isoleucine (Ile), proline (Pro) and D-color with different helical tendencies were used respectively. Tryptophan in PRW4 is replaced by D-Trp. At the same time, in order to study the important role of arginine (Arg), all Lys in PRW4 were replaced by Arg. Wherein, the amino acid sequence of the antimicrobial peptide PRW4-d replaced with D-tryptophan (D-Trp) is shown in Table 1.

[0018] Table 1 Amino Acid Sequen...

Embodiment 2

[0022] Synthesis of Antimicrobial Peptides 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 Resin for chain protection.

[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 evaporator, and th...

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 D-amino acid antibacterial peptide PRW4-d and a preparation method and an application thereof. A sequence of the peptide is as shown in a sequence table SEQ ID No.1. The preparation method comprises the following steps: by taking antibacterial peptide PRW4 as a template, replacing tryptophan in the antibacterial peptide PRW4 by using an amino D-tryptophan with different spiral tendentiousness; replacing all lysine in the PRW4 by arginine; and obtaining peptide resin through a polypeptide synthesizer by virtue of a solid phase chemical synthesizing method and performing TFA cutting on the obtained peptide resin to obtain the poly peptide PRW4-d. The polypeptide PRW4-d has an obvious inhibiting effect on various cultures such as escherichia coli, salmonella typhimurium, staphylococcus aureus and bacillus subtilis, is very low in hemolytic activity, and shows relatively high cell selectivity. In a word, the PRW4-d is the antibacterial peptide which is relatively high in application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a D-type amino acid antibacterial peptide PRW4-d and its preparation method and application. Background technique [0002] The application of antibiotics as feed additives can be traced back to 1943, but in recent years studies have shown that while the application of antibiotics has brought great benefits to people, antibiotic-resistant bacteria have begun to appear in animals fed antibiotics, as well as in humans with diseases and among animals. The development of resistance to in-feed antibiotics is far more of a concern than industry interests. [0003] Antimicrobial Peptide (AMP), also known as Host Defense Peptide (HDP), is an important part of the body's endogenous immune system and the primary barrier for the host to resist the invasion of foreign pathogens. Antimicrobial peptides come from a wide range of sources, including animals, plants, and microorganisms, ...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K1/20C07K1/04A61K38/17A61P31/04
CPCA61K38/00C07K14/4723Y02A50/30
Inventor 单安山朱鑫丑淑丽
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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