Imperfect amphiphilic antimicrobial peptide PRW4-R and preparation method and application thereof

A PRW4-R, amphiphilic technology, used in cationic antimicrobial peptides, antibacterial drugs, chemical instruments and methods, etc., can solve the problems of abnormal cytotoxicity, poor biocompatibility, and poor bactericidal activity.

Inactive Publication Date: 2017-04-05
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, synthetic peptide drugs or materials often have poor biocompatibility, produce abnormal cytotoxicity, and have poor bactericidal activity

Method used

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  • Imperfect amphiphilic antimicrobial peptide PRW4-R and preparation method and application thereof
  • Imperfect amphiphilic antimicrobial peptide PRW4-R and preparation method and application thereof
  • Imperfect amphiphilic antimicrobial peptide PRW4-R and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Design of Antimicrobial Peptides

[0016] The perfect amphiphilic structure improves the bactericidal activity of antimicrobial peptides, but at the same time inevitably increases the cytotoxicity. Therefore, moderately disrupting the amphiphilicity of antimicrobial peptides may increase antibacterial activity while reducing cytotoxicity. The 16 N-terminal amino acid sequences of cationic antimicrobial peptide PMAP-36 containing 36 amino acids in porcine bone marrow were intercepted to obtain RI16 with a perfect amphipathic structure. According to the principle of α-helical protein folding, tryptophan (Trp) was used to replace the paired lysine (Lys) in the polar face of the amphipathic structure, and the imperfect amphipathic antimicrobial peptide PRW4 was designed; the sequence of PRW4 was All Lys are replaced by Arg to get PRW4-R. The amino acid sequence is shown in Table 1.

[0017] Table 1 Amino acid sequences of derived peptides

[0018]

[0019] The carbox...

Embodiment 2

[0021] Synthesis of Antimicrobial Peptides by Solid Phase Chemical Synthesis

[0022] 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;

[0023] 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 then a...

Embodiment 3

[0027] Determination of antimicrobial activity of antimicrobial peptides

[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 the naked eye....

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Abstract

The invention provides an imperfect amphiphilic antimicrobial peptide PRW4-R and a preparation method and application thereof. The sequence of the imperfect amphiphilic antimicrobial peptide PRW4-R is shown as SEQ ID No. 1. The preparation method comprises the following steps: cutting out 16 amino acid sequences at a PMAP-36N end to obtain RI16 with a perfect amphiphilic structure; based on an Alpha-helix protein folding principle, substituting Trp for paired Lys in the polar face of the amphiphilic structure to obtain imperfect amphiphilic antimicrobial peptide PRW4; and substituting Arg for all lysine in the sequence of the PRW4 to obtain the PRW4-R. The therapeutic index of the PRW4-R is up to 161.5; and the peptide PRW4-R shows high cell selectivity and has low hemolytic activity, so that the development potential of the peptide PRW4-R in becoming a substituent of antibiotics is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an imperfect amphiphilic antibacterial peptide PRW4-R and its preparation method and application. Background technique [0002] Antimicrobial peptides have received extensive attention as potential antimicrobial drugs or alternatives to antibiotics due to their broad-spectrum activity against Gram-negative and Gram-positive bacteria. Most antimicrobial peptides mainly kill bacteria through irreversible and high-strength membrane disruption, so this unique mechanism of action helps to reduce the occurrence of bacterial resistance. However, synthetic peptide drugs or materials often have poor biocompatibility, produce abnormal cytotoxicity, and have poor bactericidal activity. Currently, it is widely believed that amphiphilicity has a great influence on antibacterial activity and cytotoxicity. [0003] Amphiphilicity, mainly the spatial distribution of clusters of hydroph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47A61K38/17A61P31/04
CPCA61K38/00C07K14/4723
Inventor 单安山朱鑫王志华丑淑丽
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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