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Anti-enzymolysis antibacterial peptide II4II and preparation method and application thereof

An antibacterial peptide, anti-enzymatic hydrolysis technology, applied in the direction of antibacterial drugs, chemical instruments and methods, medical preparations containing active ingredients, etc. The effect of enzymatic hydrolysis ability and strong anti-enzymatic hydrolysis ability

Active Publication Date: 2020-07-28
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Based on the above deficiencies, the object of the present invention is to provide an anti-enzymolysis antimicrobial peptide II4II and its preparation method and application, which solves the problem that it is easily degraded by protease to reduce or even lose its biological activity.

Method used

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  • Anti-enzymolysis antibacterial peptide II4II and preparation method and application thereof
  • Anti-enzymolysis antibacterial peptide II4II and preparation method and application thereof
  • Anti-enzymolysis antibacterial peptide II4II and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Design of Antimicrobial Peptides Against Enzyme

[0017] (1) Arg with high cationicity is selected as the cationic amino acid to provide the necessary net positive charge. In order to avoid the cleavage of Arg by trypsin, Cys and Lys were placed on the N-terminal side and C-terminal side of Arg, respectively, to block the cleavage of Arg by trypsin. At the same time, Pro was placed on the C-terminal side of Lys to block the cleavage of Lys by trypsin.

[0018] (2) In order to avoid cleavage by chymotrypsin and pepsin, Ile with a longer aliphatic side chain was selected as the hydrophobic amino acid and placed on the C-terminal side of Pro and the N-terminal side of Cys to provide the necessary hydrophobicity.

[0019] (3) Place two Ile respectively at the N-terminus and C-terminus of the sequence unit to enhance the hydrophobicity to further improve the activity.

[0020] The anti-enzymolytic antimicrobial peptide template is thus designed as: XX(XCRKPX)nXX-NH 2 , wh...

Embodiment 2

[0027] Synthesis of Antimicrobial Peptides by Solid Phase Chemical Synthesis

[0028] 1. The synthesis of antimicrobial peptides is carried out one by one from the C-terminal to the N-terminal 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; precipitate TFA (trifluoroacetic acid) and wash, then mix the washing liquid with the above filtrate, concentrate with a rotary evaporator, and then add About 10 times t...

Embodiment 3

[0033] Determination of Biological Activity of Antimicrobial Peptides

[0034] 1. Determination of antibacterial activity: The minimum inhibitory concentration (MIC) of the peptide was determined by the standard micro-broth dilution method. Dilute bacteria in log phase to 10 5 CFU / ml. Add 50 μl of different concentrations of peptides (the final concentration of the peptide is 1-128 μM) and an equal volume of bacterial suspension to each well of a 96-well plate, and set up a negative control (medium only) and a positive control (bacteria and medium) at the same time , and then place the 96-well plate in a constant temperature incubator at 37°C for 18-20 hours. Using a microplate reader at 492nm (OD 492 ) to determine the minimum inhibitory concentration. Three independent replicates were performed, with two parallels for each replicate. The results are shown in Table 2.

[0035] Bacteriostatic activity (μM) of table 2 antimicrobial peptides

[0036]

[0037]

[003...

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Abstract

The invention belongs to the technical field of biology and provides an anti-enzymolysis antibacterial peptide II4II and a preparation method and application thereof. The amino acid sequence of the antibacterial peptide II4II is shown in SEQ ID No. 1. The preparation method comprises the following steps of reasonably arranging amino acids in the sequence based on specific restriction enzyme cutting sites of trypsin, chymotrypsin and pepsin and basic characteristics of the antibacterial peptide, and selecting Ile as a hydrophobic amino acid to obtain the antibacterial peptide II4II. The invention further provides application of the antibacterial peptide II4II in preparation of medicines for treating gastrointestinal tract infection diseases caused by gram-negative bacteria or / and gram-positive bacteria. The antibacterial peptide II4II has a relatively good inhibition effect on multiple bacteria, is relatively low in hemolytic activity, has a treatment index of 80.63, has relatively goodanti-enzymolysis property, is kept unchanged in antibacterial activity after being treated by trypsin, chymotrypsin or pepsin, and has application potential as an antibiotic substitute.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-enzymolysis antimicrobial peptide II4II, a preparation method and application thereof. Background technique [0002] In the past 90 years, antibiotics have been widely used in medicine, food, animal husbandry and other industries. However, the abuse of antibiotics has accelerated the development of drug resistance, posing a great threat to global public health. Therefore, finding alternatives to antibiotics has become a top priority. Antimicrobial peptides (AMPs) widely exist in natural organisms and have various biological activities such as antibacterial, antifungal, and anti-inflammatory, and are an important part of the host immune system. The unique non-specific membrane destruction mechanism of AMPs makes it difficult for bacteria to develop drug resistance, therefore, AMPs are considered to be the most promising alternatives to antibiotics. [0003] Howe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00A61K38/16A61P31/04
CPCC07K14/00A61P31/04A61K38/00
Inventor 单安山朱永杰邵长轩
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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