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Derived peptide IR2 of pig-derived antibacterial peptide as well as preparation method and application thereof

A technology of derivatized peptides and antimicrobial peptides, applied in the field of porcine-derived antimicrobial peptide IR2 and its preparation, can solve the problems of unsatisfactory antibacterial activity and high cytotoxicity of natural antimicrobial peptides

Active Publication Date: 2014-07-16
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with traditional antibiotics, the antibacterial activity of natural antimicrobial peptides is not ideal, and the cytotoxicity is high

Method used

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  • Derived peptide IR2 of pig-derived antibacterial peptide as well as preparation method and application thereof
  • Derived peptide IR2 of pig-derived antibacterial peptide as well as preparation method and application thereof
  • Derived peptide IR2 of pig-derived antibacterial peptide as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Design of Antimicrobial Peptides

[0018] The amino acid sequence of porcine antimicrobial peptide PG-1 is:

[0019] Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val Gly Arg-NH 2

[0020] 1 5 10 15 18

[0021] By intercepting the amino acid sequence CRRRFC (8-13 amino acids) of the β-turn position of PG-1, the charged arginine R and the hydrophobic amino acid I are selected to extend the sheets at both ends of the PG-1 turn position. By intercepting the amino acid sequence of the PG-1β-turn, and connecting the two ends of the turn with IR or RI respectively, the IR series peptides were designed: (IR) n H(RI) n -NH 2 , (n=1, 2; H is the β-turn amino acid sequence CRRRFC of PG-1), when n=1, the derived peptide is named IR1; when n=2, the derived peptide is named IR2. The amino acid sequences of the derived peptides are shown in Table 1.

[0022] Table 1 Amino Acid Sequence of Derived Peptides

[0023]

[0024] The charge numbers of IR1 and IR2 a...

Embodiment 2

[0026] Synthesis of Two Antimicrobial Peptides IR1 and IR2 by Solid Phase Chemical Synthesis

[0027] 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;

[0028] 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 evapor...

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 IR2 of a pig-derived antibacterial peptide as well as a preparation method and application thereof. The sequence of the derived peptide IR2 of the pig-derived antibacterial peptide is as shown in SEQ ID No.1. Antibacterial peptides IR1 and IR2 are obtained by using a fixed point amino acid fragment interception and binary amino acid sequence superimposing method for intercepting six amino acid fragments of a pig-derived PG-1 corner part and symmetrically and circularly arranging charged amino acid arginine and hydrophobic amino acid isoleucine serving as repeated binary sequence units at the two sides of the corner. The antibacterial activity of the antibacterial peptide IR2 is higher than that of the antibacterial peptide IR1. The therapeutic index of the antibacterial peptide IR2 reaches up to 43.2 and is 216 times of that of the pig-derived antibacterial peptide RG-1. By virtue of the method, the hemolytic activity of the antibacterial peptide is greatly reduced under the condition of increasing the antibacterial activity of the antibacterial peptide, the selectivity of the antibacterial peptide between bacterial cells and mammalian cells is increased, and the development potential of the antibacterial peptide serving as antibiotic substitutes is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a derivative peptide IR2 of a porcine antimicrobial peptide and a preparation method and application thereof. Background technique [0002] Antimicrobial peptides (AMPs) are a class of small molecular polypeptides that exist in the natural immune defense system of organisms, participate in the immune defense function of the body, and are the first barrier for the body to resist the invasion of pathogenic microorganisms. Since Boman et al first discovered a polypeptide substance with bactericidal effect in Hyalophora cecropia in 1972, and named it cecropia, people have successively isolated and identified polypeptides with antibacterial activity from various organisms. And collectively referred to as antimicrobial peptides. So far, the Antimicrobial Peptide Database (APD: the Antimicrobial Peptide Database) established by the University of Nebraska Medical Center in the U...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/04C07K1/16A61K38/10A61P31/04
Inventor 单安山董娜马清泉徐林
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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