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Alpha helical antibacterial peptide RL as well as preparation method thereof and application thereof

An antimicrobial peptide and α-helix technology, applied in the field of α-helical antibacterial peptide RL and its preparation, can solve the problems of high cytotoxicity, limited natural production, and poor antibacterial effect

Active Publication Date: 2017-10-20
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2. Antimicrobial peptides do not involve specific receptors
[0003] However, due to the small molecular weight of natural antibacterial peptides, the content in the body is very small, and separation and purification are difficult, so the natural production is very limited
Moreover, the therapeutic index of natural antimicrobial peptides (such as melittin and cecropin) is not high, and they are highly toxic to the body cells
Using traditional methods to extract natural antimicrobial peptides faces problems such as poor antibacterial effect or excessive cytotoxicity, which requires a new method to solve
There are more and more clinical reports that the extensive use of foreign homologous natural antimicrobial peptides or natural antimicrobial peptide derivatives will harm the body's own immune system, thus causing serious public health problems

Method used

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  • Alpha helical antibacterial peptide RL as well as preparation method thereof and application thereof
  • Alpha helical antibacterial peptide RL as well as preparation method thereof and application thereof
  • Alpha helical antibacterial peptide RL as well as preparation method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Design of Antimicrobial Peptides

[0018] The amino acid sequence of the antimicrobial peptide RL is:

[0019]

[0020] Based on the folding principle of imperfect amphipathic α-helical peptides, an imperfect amphipathic α-helical peptide template Ac-WXKYWXZZYKXWYK-NH with a turn unit was designed 2 , X is a positively charged amino acid, Y is a hydrophobic amino acid, ZZ is a corner unit, when X=R, Y=L, ZZ= D When PG, the antimicrobial peptide is named RL; when D When the PG turn is disrupted, the antimicrobial peptide is named RLα. The sequences of the antimicrobial peptides are shown in Table 1.

[0021] Table 1 Amino Acid Sequence of Derived Peptides

[0022]

[0023] The charges of RL and RLα are both +7, and the hydrophobic value is -0.469. The C-termini of the two peptides were amidated to increase a positive charge, and the N-termini were acetylated to increase the stability of the peptides. The method enables the two peptides to have high antibacter...

Embodiment 2

[0025] Synthesis of Two Antimicrobial Peptides RL and RLα by Solid Phase Chemical Synthesis

[0026] 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 chain protection resin;

[0027] 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 evapora...

Embodiment 3

[0031] Determination of antimicrobial activity of antimicrobial peptides

[0032] 1. Determination of antibacterial activity: The minimum inhibitory concentration of several antibacterial 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 per mL) in each well. Positive controls (containing bacterial fluid but not antimicrobial peptides) and negative controls (neither bacterial fluid nor peptides) were set up. Cultivate at a constant temperature of 37°C for 14-18h, measure the light absorption value at 492nm (OD492nm) with a microplate reader, and determine the minimum inhibitory concentration. The test results are shown in Table 2.

[0033] Ant...

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Abstract

The invention provides alpha helical antibacterial peptide RL as well as a preparation method thereof and application thereof. The sequence of antibacterial peptide RL is as shown in SEQ ID No.1. The preparation method comprises the following step: designing an imperfect amphipathic alpha helical peptide template WXKYWXZZYKXWYK-NH2 containing a turn unit on the basis of imperfect amphipathic alpha helical multi-peptide folding principle, wherein X is positive charge amino acid, Y is hydrophobic amino acid, Z is the turn unit, and the antibacterial peptide is named as RL when X is equal to R, Y is equal to L and ZZ is equal to <D>PG. The preparation method is simple in technology, and antibacterial and hemolytic activity detection is performed on the obtained antibacterial peptide and proves that RL has efficient inhibiting effect on seven bacteria of escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis, salmonella typhimurium, salmonella gallinarum and bacillus subtilis, and has very low hemolytic activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an α-helical antibacterial peptide RL and its preparation method and application. Background technique [0002] At present, the excessive use of antibiotics has led to drug resistance of pathogenic microorganisms, and even caused drug residues in livestock and poultry products, seriously affecting the health status, product quality and safety of livestock and poultry, thus threatening human health. Antimicrobial peptides (AMPs), also known as antimicrobial peptides or peptide antibiotics, are a type of small molecule polypeptides produced by specific genes of organisms that can resist external microorganisms and eliminate mutant cells in the body. The natural immune defense system of organisms important parts of. There are three main biological characteristics of antimicrobial peptides: 1. Broad-spectrum antibacterial properties. Most antimicrobial peptides can kill a v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/04C07K1/20A61K38/10A61P31/04
CPCA61K38/00C07K7/08Y02A50/30
Inventor 单安山邵长轩
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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