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A kind of antifungal antibacterial peptide gl4w and its preparation method and application

An antimicrobial peptide and fungal technology, applied in the biological field, can solve problems such as broad antibacterial spectrum, secondary infection, and damage to the balance of the body's flora, and achieve high application value and high cell selectivity.

Active Publication Date: 2022-02-25
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although an existing antimicrobial peptide L4 (RLRLLRLR-NH2) has good antifungal (GM=8.00μM) and antibacterial (GM=4.76μM) antibacterial activities, but its antibacterial spectrum is relatively broad, and broad-spectrum antibacterial The use of drugs can disrupt the balance of the body's flora can cause secondary infections and more likely to develop drug resistance

Method used

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  • A kind of antifungal antibacterial peptide gl4w and its preparation method and application
  • A kind of antifungal antibacterial peptide gl4w and its preparation method and application
  • A kind of antifungal antibacterial peptide gl4w and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0016] Design of Antimicrobial Peptides

[0017] L4 (RLRLLRLR) designed from the α-helical peptide sequence template Rn(XRXXXRX)Rn (n=1,2; X represents L) is the active center, with glycine forming a cap structure at its N-terminus and adding color at its C-terminus A fungicidal antibacterial peptide was designed by using amino acid to form a tail anchor structure, named GL4W. The amino acid sequence of GL4W is shown in Table 1.

[0018] Table 1 Amino acid sequence of GL4W

[0019]

[0020] The charge number of GL4W is +5, and the average hydrophobic value is 0.610 respectively. The carboxyl terminus of GL4W was amidated to add a positive charge and increase the stability of the peptide.

Embodiment 2

[0022] GL4W was synthesized 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 Resin for chain protection.

[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 add 10 time...

Embodiment 3

[0028] 1. Determination of GL4W antibacterial activity: This experiment refers to the method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) to determine the minimum inhibitory concentration (Minimal inhibitory concentration, MIC) and the minimum bactericidal concentration ( Minimumbactericidal concentration, MBC), and make appropriate improvements according to the cationic characteristics of antimicrobial peptides, the specific steps are as follows:

[0029] (1) Bacteria preparation: Streak inoculation of strains frozen at -20°C on MHA solid medium, and culture overnight at 37°C. Then pick a single colony and inoculate it in MHB, culture at 220rpm, 37°C until the logarithmic growth phase, adjust its concentration to OD with MHB 600nm =0.1, and finally further diluted 1000 times with MHB to (0.5-1)×10 5 CFU / mL.

[0030] (2) Dilution of polypeptide: Add 95 μL of 0.2% BSA diluent to row A of the 96-well plate, and add 50 μL of 0.2% BSA diluent t...

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Abstract

The invention provides an antifungal antibacterial peptide GL4W and its preparation method and application. The sequence of the antibacterial peptide GL4W is shown in SEQ ID No.1. Taking the antimicrobial peptide L4 (RLRLLRLR) as the active center, a cap structure was formed with glycine at its N-terminus, and tryptophan was added at its C-terminus to form a tail anchor structure to design an antimicrobial peptide, which was named GL4W. Application of the antimicrobial peptide GL4W in the preparation of drugs for treating infectious diseases caused by fungi. The invention changes the antibacterial spectrum of antibacterial peptide L4, becomes a kind of antifungal antibacterial peptide, and improves the selectivity of antibacterial peptide to bacterial cells, which has the development potential of antifungal infection drug, and the therapeutic index of antibacterial peptide GL4W is as high as 39.61.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an antifungal antibacterial peptide GL4W and its preparation method and application. Background technique [0002] The global rate of fungal infections continues to rise, and fungal infections are placing an increasing clinical burden on healthcare systems worldwide, yet treatment options that can combat these diseases are still limited to four major classes of antifungal agents, namely polyenes, Triazoles, nikkomycins, and echinocandins. Since some effective drug targets in eukaryotic pathogens are often shared with human genes / gene products, the development of safe / non-toxic antifungal drugs is more challenging than that of antibacterial drugs. [0003] Antimicrobial peptides are a class of small molecular polypeptides used by animals to resist external microorganisms and eliminate mutant cells in the body. The quantity is small, fully meets the needs of safe producti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K1/06C07K1/04C07K1/20A61K38/08A61P31/10
CPCC07K7/06A61P31/10A61K38/00
Inventor 单安山宋静张珊珊王梓航
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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