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Cell penetrating antibacterial peptide and application thereof

A technology of antimicrobial peptides and cells, which is applied in the direction of application, antibacterial drugs, peptides, etc., can solve the problems of difficult intracellular bacteria to play a role, and achieve the effect of less drug resistance, low cytotoxicity, and broad-spectrum antibacterial activity

Active Publication Date: 2021-12-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to the problem of drug resistance, the use of antibiotics has certain limitations: the biological barrier of the cell membrane prevents antibiotics from entering the cell, making it difficult to act on intracellular bacteria

Method used

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  • Cell penetrating antibacterial peptide and application thereof
  • Cell penetrating antibacterial peptide and application thereof
  • Cell penetrating antibacterial peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 solid phase chemical synthesis method synthetic antimicrobial peptide

[0047] In this example, the antimicrobial peptide was synthesized by solid-phase chemical synthesis. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.3. The specific method is as follows:

[0048] 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 deprotection group, 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 Fmoc Group side chain protected resin;

[0049]2. Add a cleavage reagent to t...

Embodiment 2

[0053] The mensuration of the antibacterial activity of embodiment 2 peptide

[0054] Utilize the microdilution method to measure the antibacterial peptide (P3I7) that embodiment 1 prepares to the minimum inhibitory concentration of bacteria, specifically as follows:

[0055] Add 0.2% fetal bovine serum albumin containing 0.01% acetic acid as a diluent into a 96-well plate, and sequentially prepare a series of gradient antimicrobial peptide solutions using the doubling dilution method, so that the volume of the solution in each well is 50 μL. Then add 50 μL of the bacteria solution to be tested (~10 5 CFU / mL) in each well, the medium is MHB (pH=7.0). 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 18 hours, then measure the light absorption value at 492nm with a microplate reader, and use the value greater than 0.1 as the judg...

Embodiment 3

[0059] Intracellular bacteriostatic activity assay of embodiment 3 peptide

[0060] The intracellular bacteriostatic activity of the antimicrobial peptide prepared in Example 1 is determined, specifically as follows:

[0061] The S.aureus 6538 strain was grown in culture medium for 4 hours, centrifuged at 3000×g for 10 minutes, bacterial cells were collected, washed three times with PBS, and resuspended in culture medium without fetal bovine serum. Concentration is 10 5 The macrophages of each cell / well were spread evenly in a 96-well plate, and S.aureus6538 (10 7 CFU / well), co-cultivate at 37°C for 1 hour, aspirate the culture supernatant and wash with PBS, add complete medium containing 100 μg / mL gentamicin at 37°C for 2 hours to kill extracellular bacteria, and wash the cells with PBS . Afterwards, 100 μL of doubly diluted antimicrobial peptides and vancomycin (Vancomycin) were added to treat for 6 hours, and then incubated with 0.1% TritonX-100 for 15 minutes to lyse th...

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Abstract

The invention relates to the technical field of antibacterial peptides, in particular to a cell penetrating antibacterial peptide and application thereof. The antibacterial peptide provided by the invention is a chimeric peptide formed by a polypeptide with an amino acid sequence as shown in SEQ ID NO.1 and a polypeptide with an amino acid sequence as shown in SEQ ID NO.2. The antibacterial peptide has cell penetrating and antibacterial functions, has a broad-spectrum antibacterial effect, has good antibacterial activity on escherichia coli, staphylococcus aureus, salmonella and the like, can enter cells and has a strong inhibition effect on intracellular bacteria; and meanwhile, the antibacterial peptide disclosed by the invention has relatively low cytotoxicity, good biocompatibility and wide application prospect.

Description

technical field [0001] The invention relates to the technical field of antimicrobial peptides, in particular to a cell-penetrating antimicrobial peptide and its application. Background technique [0002] The frequent and irregular use of antibiotics has exacerbated the mutation and continuous evolution of pathogenic microorganisms, making them increasingly resistant to antibiotics, leading to the emergence of superbugs. Today, antibiotics are rapidly losing effectiveness in the livestock industry, and antibiotic resistance is a growing problem. In addition to the problem of drug resistance, the use of antibiotics has certain limitations: the biological barrier of the cell membrane prevents antibiotics from entering the cell, making it difficult to act on intracellular bacteria. [0003] Intracellular bacteria are a class of pathogenic microorganisms that can invade host cells and grow and reproduce in cells, mainly including Staphylococcus aureus, Salmonella, and Brucella. ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/17A61K47/64A61P31/04A23K20/147A23L33/18A23L33/13
CPCC07K14/47A61K38/1709A61K47/64A61P31/04A23K20/147A23L33/18A23L33/13C07K2319/10A23V2002/00A23V2200/30A23V2250/55Y02A50/30
Inventor 马曦唐琪谭鹏孙菲泽
Owner CHINA AGRI UNIV
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