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Anti-enzymolysis branched antibacterial peptide Pal-CRKP as well as preparation method and application thereof

An anti-enzymolysis, branched technology, applied in the biological field, can solve the problems of high sensitivity, short half-life, etc., and achieve the effects of high-efficiency inhibition, improved stability, and low hemolytic toxicity

Active Publication Date: 2022-07-29
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although antimicrobial peptides have many advantages such as high antibacterial activity, low toxicity and unique bactericidal mechanism, most antimicrobial peptides have many problems to be solved. So far, there are several reasons that limit the clinical application of AMPs. The high sensitivity of source and microbial source proteases, followed by the toxicity due to the high concentration required to inhibit bacteria, and finally the short half-life in the body due to the complex environment of body fluids, improving these problems has become the key to the application of antimicrobial peptides

Method used

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  • Anti-enzymolysis branched antibacterial peptide Pal-CRKP as well as preparation method and application thereof
  • Anti-enzymolysis branched antibacterial peptide Pal-CRKP as well as preparation method and application thereof
  • Anti-enzymolysis branched antibacterial peptide Pal-CRKP as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Design of branched antimicrobial peptides:

[0021] The amino acid sequence of the branched antimicrobial peptide Pal-CRKP is:

[0022] C 16 -Gly Gly Gly Lys (Cys Arg Lys Pro Cys Arg Lys Pro)Cys Arg Lys ProCys Arg Lys Pro

[0023] This design takes advantage of the characteristics of the lysine side chain, uses Lysine Lys as the site for linking n-hexadecanoic acid and anti-enzymatic hydrolysis sequences, uses the carboxyl group of Lysine Lys and the -NH on R group 2 Both link two anti-digestion sequences: Cys Arg Lys ProCys Arg Lys Pro, further use the flexible amino acid linker GGG to link n-hexadecanoic acid and lysine Lys, forming a branch with two anti-digestion sequence branches Antibacterial peptides and anti-enzymolysis sequences are based on the principle of reasonably avoiding the restriction site, avoiding restriction by trypsin, chymotrypsin and other proteases, and placing cysteine ​​Cys and lysine Lys on the N of arginine Arg respectively. end and C-ter...

Embodiment 2

[0030] The above-mentioned dendritic antibacterial peptide is synthesized using a peptide synthesizer, the method is a solid-phase chemical synthesis method, and the specific steps are:

[0031] Step 1. The preparation of the polypeptide backbone is carried out one by one from the C-terminus to the N-terminus, and is completed by a polypeptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) was inserted into Wang resin, and then the Fmoc group was removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9 -Fluorenemethoxycarboxy-trimethyl-Y, Y is the second amino acid at the C-terminus of each antibacterial peptide); follow this procedure to synthesize from the C-terminus to the N-terminus until the synthesis is completed, and obtain the side with the Fmoc group removed. Chain-protected resins;

[0032] Step 2. Use hydrazine hydrate to remove the Dde protecting group on the side chain of Fmoc-Lys(Dde)-OH, and repeat step 1 to ...

Embodiment 3

[0037] The antibacterial activity and hemolytic activity of the prepared anti-enzymatic branched antimicrobial peptide Pal-CRKP were detected in vitro;

[0038] 1. Determination of antibacterial activity: to measure the minimum inhibitory concentration (MIC) of the peptide is to inoculate the bacteria in Mueller-Hinton broth (MHB) at 37°C overnight and then transfer to a new medium until the logarithmic growth phase. Dilute the bacterial culture to 1×10 5 CFU / ml, 50 μl of BSA (pH=6.0) containing different concentrations of peptides were added to the above 96-well plates, and the final peptide concentrations in the 96-well plates ranged from 0.125 to 64 μM. Peptide solution and bacterial culture solution were mixed in equal volume 1:1 in a 96-well plate, cultured for 18 hours, and the optical density (OD) of the mixture was measured at 492 nm with a microplate reader (Tecan GENios F129004, Austria) to determine the minimum inhibitory concentration. , and the test results are ...

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Abstract

The invention provides an anti-enzymolysis branched antibacterial peptide Pal-CRKP as well as a preparation method and application of the anti-enzymolysis branched antibacterial peptide Pal-CRKP. The amino acid sequence of the branched antibacterial peptide Pal-CRKP is as follows: Pal-GGGK (CRKPCRKP) CRKPCRKP, and Pal is n-hexadecanoic acid. According to the preparation method, by utilizing the characteristics of a lysine side chain, n-hexadecanoic acid and an anti-enzymolysis peptide sequence unit are combined by utilizing lysine to form a branched antibacterial macromolecule, and the branched antibacterial peptide with high antibacterial activity, low toxicity and high stability is designed. The anti-enzymolysis branched antibacterial peptide Pal-CRKP not only shows high-efficiency bacteriostatic activity on gram-negative bacteria, but also has a high-efficiency inhibition effect on gram-positive bacteria, meanwhile, the anti-enzymolysis branched antibacterial peptide Pal-CRKP has relatively low hemolytic toxicity, and the degradation degree of high-concentration protease on the branched antibacterial peptide Pal-CRKP is very weak, so that the anti-enzymolysis branched antibacterial peptide Pal-CRKP can be used for preparing the anti-enzymolysis branched antibacterial peptide Pal-CRKP. The anti-enzymolysis branched antibacterial peptide disclosed by the invention has relatively high development potential of replacing antibiotics.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-enzymolysis branched antibacterial peptide Pal-CRKP and a preparation method and application thereof. Background technique [0002] The moment antibacterial drugs are used, bacteria begin to evolve multiple drug resistance pathways to fight back. The emergence of drug-resistant bacteria is a major threat, jeopardizing the efficacy of potent antibiotics and leading to an increased risk of death. Therefore, new approaches are urgently needed. against these resistant strains. Antimicrobial peptides (AMPs) are low molecular weight proteins, and in the past four decades of antimicrobial peptide research, it has been found that these molecules have a variety of biological activities, including antibacterial, antifungal, antiviral, antiparasitic, anticancer and immune regulation. AMPs initially bind to lipoteichoic acid on Gram-positive bacterial membranes or lipopolys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/36C07K1/30C07K1/20C07K1/06C07K1/04A61K38/10A61P31/04
CPCC07K7/08A61P31/04A61K38/00Y02A50/30
Inventor 单安山陈虹羽李博文来振衡
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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