Porcine thrombin carbon terminal derived antibacterial peptide and preparation method and application thereof

A pig thrombin, antimicrobial peptide technology, applied in biochemical equipment and methods, antibacterial drugs, enzymes and other directions, can solve the problems of drug residues, environmental pollution, drug-resistant strains, etc., to achieve low synthesis cost, good safety, The effect of broad-spectrum antibacterial activity

Active Publication Date: 2015-04-08
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] The application of traditional antibiotics in feed veterinary drugs has improved the production performance of animals and reduced the incidence of diseases; but at the same time, it has also led to problems such as drug residues, drug-resistant strains, and environmental pollution. Therefore, it is necessary to discover new antibacterial agents

Method used

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  • Porcine thrombin carbon terminal derived antibacterial peptide and preparation method and application thereof
  • Porcine thrombin carbon terminal derived antibacterial peptide and preparation method and application thereof
  • Porcine thrombin carbon terminal derived antibacterial peptide and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0020] Design of Antimicrobial Peptides Derived from Porcine Thrombin Carbon-Terminal

[0021] The complete amino acid sequence of porcine thrombin was obtained through NCBI, starting from the first amino acid at the carbon-terminal of the enzyme, and by truncation, five derived peptides ranging in length from 11 to 19 amino acids were obtained, PT11, PT13, PT15, PT17, PT19. As shown in Table 1.

[0022] Table 1 Amino acid sequences and molecular weights of antimicrobial peptides derived from porcine thrombin

[0023]

Embodiment 2

[0025] Synthesis of Antimicrobial Peptides Derived from Porcine Thrombin Carbon-Terminal

[0026] The five polypeptides shown in Table 1 were synthesized using a peptide synthesizer, using solid-phase organic synthesis, using the Fmoc protection synthesis method, and the synthesis direction was carried out one by one from the C-terminal to the N-terminal. The specific steps are as follows:

[0027] (1) Select the Wang resin that has been connected to the first amino acid at the C-terminal, that is, Fmoc-A(trt)-Wang (9-fluorenylmethoxy-trimethyl-A, where A is the first amino acid at the C-terminal) , use dimethylformamide (DMF) to soak for about 15 minutes to remove impurities; use DMF containing 20% ​​piperidine to remove the Fmoc protection on the resin, react for 20 minutes, and wash the resin until it is complete. The piperidine was washed away with DMF, and the remaining solid suspension was the deprotected A-Wang. The quality of A-Wang deprotection was checked with ptrin...

Embodiment 3

[0032] Determination of Antibacterial Activity of Porcine Thrombin Carbon-terminal Derivatized Antibacterial Peptides

[0033] The minimum inhibitory concentration (MIC) method recommended by the American Clinical Laboratory Standardization Institute (CLSI) was adopted, and corresponding improvements were made for the cationic characteristics of antimicrobial peptides. 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 50 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 antimicrobial peptides) were set up. Incubate at a constant temperature of 37°C for 20 hours, and the minimum inhibitory concentration...

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Abstract

The invention discloses a porcine thrombin carbon terminal derived antibacterial peptide and a preparation method and application thereof. The amino acid sequence of the antibacterial peptide is shown in SEQ No.1. The preparation method comprises the following steps: analyzing the amino acid composition characteristics of porcine thrombin, discovering amino acid residues enriched in hydrophobicity and positive charges at the carbon terminal, thereby obtaining derived peptides of different lengths in a mode of intercepting the carbon terminal; synthesizing a crude peptide by virtue of a solid-phase synthesis method by using a peptide synthesizer; and finally, purifying the crude peptide by using high performance liquid chromatography, and identifying by using electron spray mass spectrometry. The bacteriostatic activity of the derived antibacterial peptide is measured by virtue of the minimal inhibitory concentration and hemolysis test, and the peptide PT17 with high activity is screened. The porcine thrombin carbon terminal derived antibacterial peptide and the preparation method and application thereof disclosed by the invention have the advantages that the peptide is derived from the carbon terminal of the porcine thrombin, belongs to natural derivatives, is high in safety and has broad spectrum antibacterial activity and low toxicity; and moreover, the peptide chain is short, the preparation method and technology are mature, and the synthesizing cost is low.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a porcine thrombin carbon-terminal-derived antibacterial peptide and a preparation method and application thereof. Background technique [0002] The application of traditional antibiotics in feed veterinary drugs has improved the production performance of animals and reduced the incidence of diseases; but it has also led to problems such as drug residues, drug-resistant strains, and environmental pollution. Therefore, it is necessary to discover new antibacterial agents. As important substitutes, probiotics, Chinese herbal medicines, and antimicrobial peptides have attracted widespread attention. Among them, antimicrobial peptides are an important part of the body's immunity, because of their unique membrane-mediated antibacterial mechanism, it is not easy for bacteria to develop drug resistance. , Safety and non-toxicity and other characteristics are favored. Antimicrob...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/74A61K38/48A61P31/04
CPCA61K38/00C12N9/6429C12Y304/21005
Inventor 马清泉毕重朋单安山吕希婷
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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