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A proline-rich antimicrobial peptide pyr-2 targeting gram-negative bacteria and its preparation method and application

A Gram-negative bacteria, proline-rich technology, applied in the preparation method of peptides, chemical instruments and methods, medical preparations containing active ingredients, etc., can solve the complex synthesis process, high synthesis cost, peptide hemolytic activity Advanced problems, to achieve the effect of high application value, good biocompatibility, and simple technical route

Active Publication Date: 2022-04-19
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The natural antimicrobial peptide Pyrrhocoricin is a proline-rich antimicrobial peptide that exists in insects. It targets bacterial ribosomes and only has antibacterial activity against Gram-negative bacteria. However, the peptide has a high hemolytic activity and the synthesis process Complexity leads to high synthesis cost, and the high difficulty of synthesis makes it have great limitations in production and application

Method used

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  • A proline-rich antimicrobial peptide pyr-2 targeting gram-negative bacteria and its preparation method and application
  • A proline-rich antimicrobial peptide pyr-2 targeting gram-negative bacteria and its preparation method and application
  • A proline-rich antimicrobial peptide pyr-2 targeting gram-negative bacteria and its preparation method and application

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Effect test

Embodiment 1

[0018] The natural antimicrobial peptide Pyrrhocoricin derived from insects is replaced by amino acids, and the hydrophilic amino acids at the 4th and 5th positions in the sequence are replaced by proline, and the number of prolines is increased to increase the hydrophobicity of the antimicrobial peptide; Amino acid replaces threonine at position 11 to increase the number of positive charges of the antimicrobial peptide. The sequence and physical and chemical parameters of Pyr-2 are shown in Table 1.

[0019] Amino acid sequence and physical and chemical parameters of table 1 peptide

[0020]

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 chain protection resin;

[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 then a...

Embodiment 3

[0028] Determination of antimicrobial activity of antimicrobial peptides

[0029] 1. Determination of minimum inhibitory concentration: The minimum inhibitory concentration (MIC) of the peptide was determined by the standard micro-broth dilution method. Bacteria were incubated overnight at 37 °C with constant shaking at 220 rpm, then transferred to a new MHB until the log phase of growth, and the bacteria in the log phase were diluted to 10 5 CFU / ml. Add 50 μL of different concentrations of peptides (the final concentration of the peptide is 1-128 μM) and 50 μL of bacterial suspension to each well of the 96-well plate. The negative control is the MHB medium without adding bacteria, and the positive control is the MHB medium adding bacteria. The 96-well plate is placed in a constant temperature incubator at 37°C for 18-20 hours. The concentration of antimicrobial peptide at which no microbial growth is observed by naked eye and spectrophotometry is MIC. Three independent rep...

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Abstract

The present invention provides a proline-rich antimicrobial peptide Pyr-2 targeting Gram-negative bacteria and its preparation method and application. The sequence of the antibacterial peptide is shown in SEQ ID No.1. The 2-10th amino acid in the natural antimicrobial peptide Pyrrhocoricin is used as the core sequence, and the glycine and serine at the 4th and 5th positions in the sequence are replaced by proline respectively to increase the number of proline and improve hydrophobicity; use arginine The acid replaces the threonine at the 11th position, which increases the number of positive charges of the polypeptide, thereby improving the antibacterial activity of the polypeptide. Application of the antimicrobial peptide Pyr‑2 in the preparation of drugs for treating infectious diseases caused by Gram-negative bacteria. The antimicrobial peptide Pyr-2 of the present invention has almost no hemolysis on red blood cells and has good biocompatibility. To sum up, the Pyr-2 of the present invention has the potential to become an antibacterial drug specifically against Gram-negative bacterial infection, and has high application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a proline-rich antimicrobial peptide Pyr-2 targeting Gram-negative bacteria, a preparation method and application thereof. Background technique [0002] In the past few decades, the abuse of antibiotics has led to the continuous strengthening of bacterial resistance, and the number of resistant bacteria has also increased. In order to prevent the spread and infection of these bacteria, it has become a priority to develop new drugs that can replace antibiotics. It is a problem that people urgently need to solve in recent years. Antimicrobial peptides (AMPs) are a class of polypeptide substances with broad-spectrum antibacterial properties, and are important components of the innate immune system; they exist in a wide range and are found in animals and plants. Compared with antibiotics, antimicrobial peptides have the advantages of non-toxic side effects (only a small part...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K1/04C07K1/20C07K1/14A61K38/10A61P31/04
CPCC07K14/43563A61P31/04A61K38/00Y02A50/30
Inventor 吕银凤谭美姝张丽聪马清泉陈婷婷单安山杨城义
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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