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AMP (antimicrobial peptide) WW based on peptide miniaturization strategy as well as preparation method and application of AMP WW

A technology of antimicrobial peptides and strategies, applied in the biological field, can solve the problems of immune system dysfunction, rejection, and immunogenicity.

Active Publication Date: 2017-03-22
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in fact, there are still two problems that cannot be ignored in the application of antimicrobial peptides: on the one hand, the modified antimicrobial peptides still retain traces of the parent peptide, and are prone to immunogenic reactions. If they are used for a long time, they may inhibit the animal body itself. The secretion of antimicrobial peptides causes the dysfunction of the immune system; on the other hand, for the newly designed antimicrobial peptides, which have nothing to do with the fed livestock and poultry animals, theoretically there will be rejection reactions, which are prone to allergic reactions, which is not conducive to the health and improvement of livestock and poultry. Production performance

Method used

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  • AMP (antimicrobial peptide) WW based on peptide miniaturization strategy as well as preparation method and application of AMP WW
  • AMP (antimicrobial peptide) WW based on peptide miniaturization strategy as well as preparation method and application of AMP WW
  • AMP (antimicrobial peptide) WW based on peptide miniaturization strategy as well as preparation method and application of AMP WW

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Design of Antimicrobial Peptides

[0019] Based on the dermaseptin family secreted by amphibian frog skin, the micropeptide strategy is adopted. The dermaseptin family is composed of 55 peptides, which are retrieved from the APD database, and their antibacterial activity is not less than 100 micromolar; Some parameters of the dermaseptin family were designed, and at the same time, the necessary design to improve the activity was added to WW, such as the introduction of Trp at both ends, symmetrical structure, etc. Although WW is derived from this family, it has the same characteristics as the 55 parent peptides. Compared with the 55 parent peptides, there are obvious differences, and then compared with the 55 parent peptides, there is no comparability. In order to illustrate its activity, we used the common antimicrobial peptide melittin as a control, which can show that the design of WW is successful.

[0020] The key physical parameters are retained as shown in Table...

Embodiment 2

[0027] Synthesis of WW Antimicrobial Peptide by Solid Phase Chemical Synthesis

[0028] 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;

[0029] 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 evaporator, and then...

Embodiment 3

[0032] Embodiment 3: the mensuration of antimicrobial peptide antibacterial activity

[0033] 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 AMP (antimicrobial peptide) WW based on a peptide miniaturization strategy as well as a preparation method and an application of the AMP WW. The sequence of the AMP WW is shown as SEQ ID NO.1 in the sequence table. By adopting the peptide miniaturization theory and a bioactivity improving strategy and taking dermaseptin-family peptides secreted by amphibian frog skin as a template, the sequence containing key physical parameters of mother peptides and having the length shortened to 13 amino acids is obtained. The bacteriostatic activity of WW is slightly lower than that of melittin, while the therapeutic index is as high as 69.8 and is 698 times that of the melittin. With adoption of the method, risks of immunogenicity and heterogenous repellency are avoided theoretically, the selectivity of the AMP between bacterial cells and mammalian cells is improved, and the development potential of the AMP for becoming an antibiotic substitute is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an antimicrobial peptide WW based on a miniature peptide strategy and its preparation method and application. Background technique [0002] The abuse of antibiotics has led to serious consequences such as the imbalance of intestinal microecology, the emergence of drug-resistant bacteria, and antibiotic residues in animal products, which has brought double risks to human health and environmental protection. Various countries have implemented extremely strict limit standards for antibiotic residues in animal products, and even banned them. This has brought huge risks to the production of livestock products in our country. For example, under the "green trade barriers", frequent customs clearance processing has caused huge economic losses. Therefore, it is urgent to find new green, safe and efficient alternatives to antibiotics. [0003] Antimicrobial peptides (AMPs) are ub...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C07K1/20C07K1/04A61K38/17A61P31/04
CPCA61K38/00C07K14/463
Inventor 单安山李伟忠谭婷婷徐蔚
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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