Derived peptide W8 based on amphibious frog-source antibacterial peptides and preparation method and application of derived peptide W8

An antimicrobial peptide and amphibian technology, applied in the field of derivative peptides based on amphibian frog-derived antimicrobial peptides, can solve the problems of weak antibacterial activity, narrow antibacterial spectrum, and protease sensitivity, and achieve low hemolytic activity and simple experimental techniques.

Active Publication Date: 2019-04-02
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, natural cationic antimicrobial peptides are not perfect. Most natural antimicrobial peptides have weak antibacterial activity, relatively narrow antibacterial spectrum, high synthesis cost, some antim

Method used

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  • Derived peptide W8 based on amphibious frog-source antibacterial peptides and preparation method and application of derived peptide W8
  • Derived peptide W8 based on amphibious frog-source antibacterial peptides and preparation method and application of derived peptide W8
  • Derived peptide W8 based on amphibious frog-source antibacterial peptides and preparation method and application of derived peptide W8

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1: the design of antibacterial peptide

[0014] Based on the sequence of the naturally occurring amphibian frog-derived antimicrobial peptide Kunitzin-RE (AAKIILNPKFRCKAAFC), the -CKAAFC-sequence was truncated, and the 3, 7, and 9 amino acids were replaced with the positively charged amino acid - arginine. Antimicrobial peptide P8 (AARIILRPRFR): Based on the template, a mutant peptide was designed for proline co-amino acid Trp at position 8. The amino acid sequence of the peptide is shown as W8 in Table 1.

[0015] Table 1 Amino Acid Sequence of Derived Peptides

[0016]

[0017] The charges of P8 and W8 are both +4, the hydrophobic values ​​are 0.399 and 0.538, and the hydrophobic moments are 0.211 and 0.275, respectively. The carboxyl termini of the two peptides P8 and W8 were amidated to increase a positive charge and increase the stability of the peptides. In the case of improving the bactericidal activity of the W8 antimicrobial peptide, the hemolyt...

Embodiment 2

[0018] Embodiment 2: Synthetic W8 antimicrobial peptide by solid-phase chemical synthesis

[0019] 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;

[0020] 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 evaporato...

Embodiment 3

[0031] Embodiment 3: the mensuration of antimicrobial peptide antibacterial activity

[0032] 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 th...

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Abstract

The invention provides a derived peptide W8 based on amphibious frog-source antibacterial peptides and a preparation method and application of the derived peptide W8. The sequence of the derived peptide W8 is shown as SEQID No.1. The preparation method comprises the following steps of designing an antibacterial peptide P8 according to the amphibious frog-source antibacterial peptides, wherein thesequence of the designed antibacterial peptide P8 is showed as AARIILRPRFR, based on the sequence, enabling No. 8 proline to be assisted with an amino acid Trp, and designing the derived peptide W8 having the sequence shown as AARIILRWRFR. The invention provides an application of the derived peptide W8 to preparation of medicines for treating diseases infected with Gram-negative bacteria, gram-positive bacteria and fungi. The bacterium-restraining activity of the antibacterial peptide W8 is obviously higher than that of Kunitzin-RE, besides, the antibacterial peptide W8 has remarkable broad spectrum bacterium-restraining activity which is not possessed by the Kunitzin-RE, and the selectivity of the antibacterial peptide W8 between bacterium cells and lactation animal cells is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a derivative peptide W8 based on amphibian frog-derived antimicrobial peptides, a preparation method and application thereof. Background technique [0002] The application of antibiotics as feed additives can be traced back to 1943. Americans first discovered that some antibiotic fermentation residues can promote the growth of pigs. In 1949, Americans Cunha and Stokstad conducted a systematic comparative experiment with penicillin in animals for the first time, and the results confirmed that the application of antibiotics had better disease prevention and growth-promoting effects on animals. In 1950, the U.S. Food and Drug Administration officially approved the application of penicillin and other antibiotics in feed. Because it can promote the growth of animals, increase the rate of weight gain, improve the reproductive performance of animals, increase the survival rate, ...

Claims

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Application Information

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IPC IPC(8): C07K14/46C07K1/04A61K38/17A61P31/04A61P31/10
CPCA61P31/04A61P31/10C07K14/463A61K38/00
Inventor 单安山杨占一何诗琪王家俊
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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