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Enzymolysis-resisting antibacterial peptide I9H12 as well as preparation method and application thereof

An I9H12, anti-enzyme technology, applied in the preparation method of peptides, antibacterial drugs, antifungal agents, etc., can solve the problem of less antibacterial peptides

Active Publication Date: 2019-05-28
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, thousands of antimicrobial peptides have been extracted or newly designed, but few antimicrobial peptides have been put into practical use as antibiotic substitutes

Method used

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  • Enzymolysis-resisting antibacterial peptide I9H12 as well as preparation method and application thereof
  • Enzymolysis-resisting antibacterial peptide I9H12 as well as preparation method and application thereof
  • Enzymolysis-resisting antibacterial peptide I9H12 as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Antimicrobial Peptide Design:

[0013] The amino acid sequence of the antimicrobial peptide I9H12 is:

[0014]

[0015] Referring to the properties of amino acids, while avoiding the cleavage sites of major proteases in the body as much as possible, the hydrophobic amino acid isoleucine (Ile) and the positively charged amino acid histidine (His) were selected as the main amino acids to newly design the I9H12 antimicrobial peptide. The carboxyl terminus of the peptide is amidated to add a positive charge and increase the stability of the peptide. The sequences of the antimicrobial peptides are shown in Table 1.

[0016] Table 1 Main parameters of newly designed peptides

[0017]

[0018] I9H12 is a small peptide with 21 amino acids, the charge number is +13 at pH 6.0, and the hydrophobic amino acid ratio is 0.4286.

Embodiment 2

[0020] The above-mentioned antimicrobial peptides were synthesized using a peptide synthesizer. The method was solid-phase chemical synthesis, and the specific steps were:

[0021] 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;

[0022] 2. Add a cleavage reagent to the peptide resin obtained above, react at 20°C for 2 hours in the dark, filter; precipitate with TFA (trifluoroacetic acid) and wash, m...

specific Embodiment approach 3

[0025] Detect the antibacterial activity, hemolytic activity and protease hydrolysis ability of the designed and synthesized antimicrobial peptide in vitro;

[0026] 1. Determination of antibacterial activity: The minimum inhibitory concentration of several antibacterial peptides was determined by the micro broth dilution method. Bacterial single colonies were picked and cultured overnight in MHB medium, and transferred to new MHB to grow to mid-logarithmic phase. Then the above bacterial solution was centrifuged and resuspended in MHB adjusted to pH 6.0 to a final concentration of 1 × 10 5 CFUml -1 , and transferred to a 96-well plate, 50 μl per well, and the fungi were diluted in RPMI 1640 (pH=6.0) medium containing morpholine propanesulfonic acid (MOPS). 50 μl of BSA (pH=6.0) containing different concentrations of peptides were added to the above-mentioned 96-well plate, and the final peptide concentration in the 96-well plate ranged from 0.125 to 64 μM. After incubating...

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Abstract

The invention provides enzymolysis-resisting antibacterial peptide I9H12 as well as a preparation method and application thereof. A sequence of the antibacterial peptide I9H12 is shown as SEQ ID No. 1in a sequence table. The preparation method comprises the following steps: referring to the properties of amino acids; meanwhile, avoiding enzyme digestion sites of main proteinase in a body as muchas possible, and selecting hydrophobic amino acid isoleucine (Ile) and positive-charge amino acid histidine (His) as main amino acids to newly design the antibacterial peptide I9H12; carrying out amidation on a carboxyl terminal of the peptide to improve one positive charge and increase the stability of the peptide. The invention also provides the application of the antibacterial peptide to the preparation of targeting antibacterial medicines for treating gram-negative bacterium or fungus infectious diseases. According to the enzymolysis-resisting antibacterial peptide I9H12, the enzymolysis-resisting capability of the antibacterial peptide is effectively improved, and the application potential in actual production is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-enzymolysis antimicrobial peptide I9H12 and its preparation method and application. Background technique [0002] Antimicrobial peptides (AMPs) refer to a class of basic polypeptide substances with antibacterial activity induced in insects, which protect the body by inhibiting the growth of pathogens on the skin and mucosal surfaces of organisms. Many cells can release antimicrobial peptides, for example, epithelial cells, endothelial cells, leukocytes, platelets, etc. Antimicrobial peptides are usually composed of 12-50 amino acid residues, with a molecular weight of less than 10kDa. Most antimicrobial peptides contain positively charged amino acids, which are amphipathic and cationic. They are effective against a variety of bacteria, fungi, viruses, parasites and even cancers. Cells have inhibitory effect, and also have biological activities such as promoting w...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K1/04C07K1/06A61K38/16A61P31/10A61P31/04
Inventor 单安山来振衡邵长轩谭鹏朱永杰陈婷婷
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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