I and i+4 site-directed mutation of porcine tp peptide highly active derivative antibacterial peptide and its preparation method and application

A technology of site-directed mutagenesis and antibacterial peptides, which is applied in the preparation methods of peptides, chemical instruments and methods, antibacterial drugs, etc., to achieve the effects of good activity, efficient inhibition, and clear structural characteristics

Active Publication Date: 2022-06-14
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]Based on the above deficiencies, the present invention provides a porcine source TP peptide i and i+4 site-directed mutation highly active derivative antibacterial peptide TP(i+4)5 , to solve the problems of low activity, poor stability and high synthesis cost of natural pig-derived TP peptide

Method used

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  • I and i+4 site-directed mutation of porcine tp peptide highly active derivative antibacterial peptide and its preparation method and application
  • I and i+4 site-directed mutation of porcine tp peptide highly active derivative antibacterial peptide and its preparation method and application
  • I and i+4 site-directed mutation of porcine tp peptide highly active derivative antibacterial peptide and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Design of Antimicrobial Peptides

[0017] The amino acid sequence of the antimicrobial peptide TP is:

[0018]

[0019] The amino acid sequence of the antimicrobial peptide TP(i+4)1,2 is:

[0020]

[0021] The amino acid sequence of the antimicrobial peptide TP(i+4)5 is:

[0022]

[0023] Using the porcine TP peptide as a template, lysine was used to replace the hydrophilic surface amino acid of the porcine TP peptide at the hydrogen bond formation position (i, i+4), and the amino acids for different pairs were named 1, 2, 3, 4 The best activity is the pair of 1 and 2 that are replaced at the same time. The optimal peptide obtained by charge replacement is named TP(i+4)1, 2. On this basis, tryptophan is used to replace the hydrogen bond formation The amino acid at position (i, i+4) expands the hydrophobic surface of the peptide, and the different pairs of amino acids replaced by tryptophan are respectively named 5, 6, and 7. The activity is best replaced by t...

Embodiment 2

[0028] The above-mentioned antimicrobial peptides were synthesized using a peptide synthesizer. The method was solid-phase chemical synthesis, and the specific steps were:

[0029] 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;

[0030] 2. Add a cleavage reagent to the peptide resin obtained above, react in the dark at 20°C for 2 hours, filter; wash the precipitate with TFA (trifluoroacetic acid), m...

Embodiment 3

[0034] Determination of Biological Activity of Antimicrobial Peptides

[0035] Determination of antibacterial activity: The minimum inhibitory concentration of several antimicrobial peptides was determined by the micro broth dilution method. Bacterial single colonies were picked and cultured overnight in MHB medium, and transferred to new MHB to grow to mid-logarithmic phase, diluted to OD600=0.4 before use, and the bacterial solution was diluted 1000 times before use. An equal volume (50 μL) of bovine serum albumin solution (bovine serum albumin solution: BSA 0.2%; 0.01% acetic acid) was added to a 96-well plate. The system in the first row is 90 μL + 10 μL (antimicrobial peptides), the ratio is multiplied to the tenth column, and the final peptide concentration in the 96-well plate ranges from 1 to 128 μM / L. Then add an equal volume (50 μL) of bacterial solution to the 96-well plate. The plates were incubated at 37°C for 18-24 hours. The MIC value is the lowest concentrati...

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Abstract

The present invention provides an antimicrobial peptide of i and i+4 site-directed mutation highly active derivatives of porcine TP peptide and its preparation method and application. The sequence of the antibacterial peptide TP(i+4)5 is shown in SEQ ID No.1. Select the porcine TP peptide, replace the paired amino acids on the hydrophilic surface of the porcine TP peptide with lysine at the hydrogen bond formation position (i, i+4), and obtain a peptide with the best effect TP(i+4)1 , 2. On the basis of it, tryptophan is used to replace the amino acid at position (i, i+4) to increase the hydrophobic surface of the antimicrobial peptide, and obtain an antimicrobial peptide TP(i+4) with high activity, low toxicity and high stability )5, and has a lower hemolytic activity; and discloses the application of the antibacterial peptide TP(i+4)5 in the preparation of antibacterial drugs for the treatment of Gram-negative bacteria and / or Gram-positive bacterial infectious diseases. The antimicrobial peptide of the present invention has the development potential of becoming a substitute for high-efficiency antibiotics.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an antibacterial peptide of i and i+4 site-directed mutation highly active derivatives of porcine TP peptide and its preparation method and application. Background technique [0002] The overuse and inappropriate use of antibiotics promote the emergence of drug residues and bacterial resistance, and the increasing number of resistant bacteria is seriously threatening public health. At the same time, with the advent of the era of banning antibiotics in animal husbandry, scientists are doing more and more research on antibiotic replacement products that are highly efficient, non-toxic and not easy to make bacteria resistant to antibiotics. [0003] Antimicrobial peptides (AMPs) widely exist in various organisms such as insects, plants, and animals, and are an important part of the natural defense system against invading pathogens. They not only have direct activity against...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K1/04C07K1/06A61K38/10A61P31/04
CPCC07K7/08A61P31/04A61K38/00
Inventor 单安山李国雨袁晓洁
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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