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A kind of Escherichia coli targeting antimicrobial peptide ki-qk and its preparation method and application

A KI-QK, Escherichia coli technology, applied in the biological field, achieves high application value, simple experimental technology, and strong targeting effect

Active Publication Date: 2021-12-14
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The normal flora on the surface of the human mucosa plays a key role in nutrient absorption and protection of the body against foreign microorganisms, and the current antimicrobial peptides have a broad-spectrum bactericidal effect, which will non-selectively affect the beneficial bacteria and pathogenic bacteria in the flora on the mucosal surface. Bacteria have bactericidal effect

Method used

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  • A kind of Escherichia coli targeting antimicrobial peptide ki-qk and its preparation method and application
  • A kind of Escherichia coli targeting antimicrobial peptide ki-qk and its preparation method and application
  • A kind of Escherichia coli targeting antimicrobial peptide ki-qk and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Design of Antimicrobial Peptides

[0016] In this example, we used a known peptide QK (CN 103590116 A) with strong affinity for Escherichia coli screened by phage display technology, and connected it with an α-helical peptide KI with antibacterial activity of Escherichia coli , so as to design a new peptide KI-QK with strong targeting antibacterial activity against Escherichia coli. The QK antimicrobial peptide was connected to the C-terminus of KI of the original peptide, and then three glycines were added between KI and QK as a linker, and the above antimicrobial peptide was synthesized by solid-phase synthesis using a peptide synthesizer. The amino acid sequence of the antimicrobial peptide is:

[0017]

[0018] The sequences of the antimicrobial peptides are shown in Table 1.

[0019] Amino acid sequence of table 1 peptide

[0020]

[0021]

[0022] The charge number of KI-QK is +10, and the hydrophobic value is 0.244. The two peptide chains are connect...

Embodiment 2

[0024] Synthesis of KI-QK by Solid Phase Chemical Synthesis

[0025] 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;

[0026] 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 add 10 times Abou...

Embodiment 3

[0029] Embodiment 3: the mensuration of antimicrobial peptide biological activity

[0030] 1. Determination of antibacterial activity: The minimum inhibitory concentration of several antibacterial peptides was determined by the micro broth dilution 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 doubling 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 6 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. Cultivate at a constant temperature of 37°C for 24-25h, measure the light absorption value at 492nm (OD492nm) with a microplate reader, and determine the minimum inhibitory concentration. The test results are shown in Tabl...

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Abstract

The present invention provides a method for designing Escherichia coli targeting antimicrobial peptide KI-QK, the sequence of which is shown in SEQ ID No.1 in the sequence table. A peptide KI with an α-helical structure is used, and peptide QK is connected to its C-terminus. At the same time, three flexible amino acids glycine are used as linkers between KI and QK, and the peptide is named KI-QK. In the biological activity test, we found that KI‑QK has a very strong antibacterial activity against Escherichia coli, and its geometric mean minimum inhibitory concentration against Escherichia coli is 3.667, which is 4.2 times higher than that of the original peptide KI. The index is 69.812, which is 4.7 times higher than that of the original peptide. In summary, KI‑QK is a typical targeting antimicrobial peptide, which has strong targeting effect on Escherichia coli and has high application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an Escherichia coli targeting antimicrobial peptide KI-QK, a preparation method and application thereof. Background technique [0002] At present, the excessive use of antibiotics has led to drug resistance of pathogenic microorganisms, and even caused drug residues in livestock and poultry products, seriously affecting the health status, product quality and safety of livestock and poultry, thus threatening human health. Antimicrobial peptides (AMPs), an important part of the natural immune defense system of organisms, constitute the first line of defense of human immunity. One of the biological characteristics of antimicrobial peptides is their broad-spectrum antibacterial properties. Most antimicrobial peptides can kill a variety of bacteria, including Gram-negative and Gram-positive bacteria. However, bacteria are classified into beneficial bacteria and pathogenic ba...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C07K1/20C07K1/04A61K38/16A61P31/04
CPCY02A50/30
Inventor 单安山谭鹏邵长轩来振衡朱永杰
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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