Antibacterial peptide and application thereof

A technology of antimicrobial peptide and antibacterial concentration, which is applied in the field of antimicrobial peptide and its application, can solve the problems of lack of long-term toxicological evaluation data, high cost, and few antimicrobial peptides, and achieve reduced hemolysis rate and cytotoxicity, good structural properties, The effect of reducing cytotoxicity

Active Publication Date: 2020-11-17
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the yield of natural antimicrobial peptides is low, the cycle is long, and the cost of extraction is high. Artificial extraction cannot meet the needs of large-scale production and promotion. relatively mature
The antibacterial effect of antimicrobial peptide itself is the key to the research. At present, most domestic research is still in the preclinical stage, and there are relatively few antimicrobial peptides as medicines. The main difficulties include high production costs, lack of systematic long-term toxicological evaluation data and antibacterial Peptides are easily degraded by proteases and other shortcomings

Method used

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  • Antibacterial peptide and application thereof
  • Antibacterial peptide and application thereof
  • Antibacterial peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Indolicidin is an antimicrobial peptide isolated from bovine neutrophils, composed of 13 amino acids, rich in tryptophan, and has an obvious extended structure. Indolicidin has a broad antibacterial spectrum and has antibacterial activity against a variety of Gram-positive bacteria, Gram-negative bacteria and fungi. The rational molecular design provided by the present invention includes increasing the number of positively charged amino acids, increasing the ratio of hydrophobic amino acids, increasing the ratio of α-helices, and properly introducing unnatural amino acids (such as ornithine) and D-type amino acids to the antimicrobial peptide Indolicidin. Design, as follows:

[0028] Using the method of de novo replacement, the eight amino acids of alanine, proline, leucine, lysine, arginine, glycine, valine, and histidine are replaced sequentially from the first amino acid, and then Combine and replace in pairs, construct an optimized peptide library, and input the re...

Embodiment 2

[0041] Embodiment 2 antibacterial activity

[0042] Antimicrobial peptides were synthesized by Gill Biochemical (Shanghai) Co., Ltd.

[0043]Transfer the plate-activated bacteria to LB liquid medium, place on a shaker at 37°C for 10-12 hours, and then centrifuge at 5000r / min for 5 minutes in a refrigerated centrifuge. Discard the supernatant and wash with 0.85% sterile saline. Bacteria once, centrifuged at the same speed (5000r / min) for 5min to discard the supernatant, and the precipitate was diluted to 1×10 with LB liquid medium. 8 CFU / mL, as the bacterial solution to be tested.

[0044] ①Determination of minimum inhibitory concentration (MIC) in liquid medium

[0045] Add the bacterial solution cultured to the logarithmic growth phase (Staphylococcus aureus, Vibrio parahaemolyticus, Salmonella or Aspergillus flavus) to the 96-well plate, and put each antimicrobial peptide in a gradient concentration (μg / mL) in each well. Add 50 μL each, and make negative control group and...

Embodiment 3

[0061] Example 3 Detection of hemolytic activity

[0062] Blood from healthy chickens was anticoagulated with heparin, centrifuged at 1000×g for 10 min, and the precipitate was collected, washed three times with PBS buffer, and red blood cells were resuspended and counted and diluted to 1%. After doubling the antimicrobial peptides, add 50 μL per well into a 96-well plate; add diluted red blood cells (50 μL per well) to the 96-well plate and mix with the antimicrobial peptides, incubate at 37°C for 1 hour, then proceed at 4°C The supernatant was collected by centrifugation at 1000×g, and the absorbance was measured at OD 570nm. Add 50 μL red blood cell suspension and 50 μL 0.2% TritonX-100 as a positive control for 100% hemolysis, and add 50 μL red blood cell suspension and 50 μL phosphate buffer as a negative control.

[0063] The calculation formula of hemolysis rate is: hemolysis rate=(A T -A 0 ) / (A C -A 0 )×100%.

[0064] In the formula: A T is the absorbance value ...

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Abstract

The invention provides an antibacterial peptide and application thereof, and belongs to the technical field of biology. The antibacterial peptide is obtained through rational molecular design on the basis of antibacterial peptide Indolicidin separated from bovine neutrophils; and the antibacterial peptide is antibacterial peptide Indo-1 with the amino acid sequence shown as SEQ ID NO. 1 or antibacterial peptide Indo-2 with the amino acid sequence shown as SEQ ID NO. 2. The antibacterial peptide provided by the invention has good structural characteristics; the antibacterial effect on staphylococcus aureus, salmonella and aspergillus flavus can be obviously improved; and meanwhile, the hemolysis rate and the cytotoxicity of chicken red blood cells can be effectively reduced; the antibacterial peptide can be effectively used in animal-related production activities.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an antibacterial peptide and its application. Background technique [0002] With the extensive use of antibiotics, the resistance of microorganisms to antibiotics is becoming more and more serious, and many pathogenic bacteria have produced drug-resistant strains ("super bacteria"). According to data, China invested more than 160,000 tons of antibiotics in 2013, of which veterinary antibiotics accounted for more than 50%. Some domestic farmers blindly improve breeding efficiency and rely too much on the use of antibiotics in the breeding process, which not only greatly reduces the effect of drugs, but even increases the pathogenicity of pathogenic bacteria. [0003] Antimicrobial peptides have broad-spectrum and high-efficiency antibacterial properties and cell selectivity. They have a unique membrane destruction or intracellular bactericidal mechanism, and this mechanis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08A61P31/04A61P31/10A23K20/147
CPCC07K7/08A61P31/04A61P31/10A23K20/147
Inventor 孔青秦冲
Owner OCEAN UNIV OF CHINA
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