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Swine derived hybrid antimicrobial peptide MDP-2 and preparation method and application thereof

A technology of MDP-2 and hybrid antimicrobial peptides, which is applied in the direction of hybrid peptides, antibacterial drugs, fusion peptides, etc., can solve the problems of limited application, antibacterial activity and bacterial targeting ability

Active Publication Date: 2019-09-27
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the antibacterial activity and bacterial targeting ability of these two antimicrobial peptides are not strong, and they also have certain problems such as cytotoxicity, which limits their application in actual production.

Method used

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  • Swine derived hybrid antimicrobial peptide MDP-2 and preparation method and application thereof
  • Swine derived hybrid antimicrobial peptide MDP-2 and preparation method and application thereof
  • Swine derived hybrid antimicrobial peptide MDP-2 and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Example 1: Design of pig-derived hybrid antimicrobial peptides

[0018] The complete amino acid sequences of porcine antimicrobial peptides PMAP-23 and PMAP-36 were obtained through NCBI. According to the amino acid composition and arrangement characteristics, PMAP-23 and PMAP-36 were truncated and amino acid replaced to obtain 4 peptide derivatives; The lipopolysaccharide-binding sequence FSKGKYKCV of D-like differentiation protein-2 was connected to each other to obtain four derived peptides MDP-1, MDP-2, MDP-3, MDP-4 ranging in length from 20 to 24 amino acids. As shown in Table 1.

[0019] Table 1 Amino acid sequence and molecular weight of pig-derived hybrid antimicrobial peptides

[0020]

Embodiment 2

[0021] Example 2: Synthesis of Design of Porcine-derived Hybrid Antimicrobial Peptides

[0022] The 4 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:

[0023] (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 ptrintrion...

Embodiment 3

[0027] Example 3: Antibacterial Activity Determination of Porcine-derived Hybrid Antimicrobial Peptides

[0028] Using the method for determining the minimum inhibitory concentration (MIC) recommended by the American Clinical Laboratory Standardization Institute (CLSI), and aiming at the cationic characteristics of antimicrobial peptides, 0.01% acetic acid (containing 0.2% BSA) was used as the polypeptide diluent. A series of gradient antimicrobial peptide solutions were sequentially prepared by the double dilution method. Specific steps are as follows:

[0029] (1) Preparation of bacteria: take the bacteria to be tested frozen at -20°C, streak inoculate them in MH(A) medium and incubate them. A single colony was picked, inoculated in 10 mL of MH(B) medium, and cultured overnight at 37°C and 200 rpm. Then inoculate the overnight bacteria in fresh culture medium and culture for 1-2 hours until the bacteria are in the logarithmic growth phase, making their OD 600 =0.4, adjust...

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Abstract

The invention provides a swine derived hybrid antimicrobial peptide MDP-2 and a preparation method and application thereof. The sequence of the peptide is as shown in a sequence table SEQ ID No.2. The preparation method comprises the following steps: carrying out truncation and residue replacement on a natural antimicrobial peptide according to the composition characteristic and distribution of amino acids of the antimicrobial peptide PMAP-23 or PMAP-36 to obtain a swine derived peptide; connecting the derived peptide to a bounded sequence of lipopolysaccharide of myeloid differentiation protein-2 to further enhance the bacteria targeting ability of the derived peptide and finally obtaining four derived hybrid peptides, the lengths of amino acids of which are 20-24; then synthesizing a polypeptide; carrying out purification and identification; and finally, testing the bacteriostatic activity and the cytotoxicity of the derived antimicrobial peptide through the minimal inhibitory concentration and a hemolysis test to screen the polypeptide with relatively ideal activity. The swine derived hybrid antimicrobial peptide has the beneficial effects that the polypeptide is a natural derivative which is relatively good in safety, has broad-spectrum antibacterial activity and low toxicity, and is sophisticated in preparation method and preparation technology and low in synthetic cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a pig-derived hybrid antimicrobial peptide MDP-2, a preparation method and application thereof. Background technique [0002] Since the invention of antibiotics in the last century, they have played an important role in animal breeding and production, playing an important role in preventing and treating livestock diseases and ensuring the health of livestock and poultry. However, with the continuous application of antibiotics, many problems have gradually emerged; especially the irregular use and abuse lead to the emergence of superbugs, and drug-resistant bacteria have caused panic in the animal husbandry industry. Residues are not up to standard, affecting consumer health and food safety. Following the end of the last century, countries such as Sweden began to gradually ban some antibiotics, and now many countries have introduced policies to restrict the use of antibio...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K38/17A61P31/04
CPCA61K38/00A61P31/04C07K14/47C07K2319/00
Inventor 马清泉孟庆维李锋陈志辉单安山朱佳良
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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