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Screening method of antimicrobial peptide and application of screening method

A screening method and antibacterial peptide technology, applied in the field of antibacterial peptides, can solve the problems of long antibacterial peptide peptide chain, high synthesis cost, low biological activity of natural antibacterial peptides, etc., and achieve small molecular weight, high bactericidal activity, cytotoxicity and hemolytic toxicity. low effect

Active Publication Date: 2020-01-31
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention provides a screening method for antimicrobial peptides and its application, which solves the problems of the existing screening methods for antimicrobial peptides, such as long peptide chains of the screened antibacterial peptides, high synthesis costs, and natural antimicrobial peptides isolated from nature. The problem of low biological activity

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  • Screening method of antimicrobial peptide and application of screening method
  • Screening method of antimicrobial peptide and application of screening method
  • Screening method of antimicrobial peptide and application of screening method

Examples

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Embodiment 1

[0043] Example 1 Phage Display Screening

[0044] This example uses a phage display peptide library of random 7 peptides to conduct a phage display screening experiment against Staphylococcus aureus. The principle of the screening experiment is shown in figure 1 , the specific operation is as follows:

[0045] (1) Inoculate 50 μl of Staphylococcus aureus glycerol into 50 ml of LB medium, shake and culture in a constant temperature shaker at 37°C and 250 rpm until the OD600 is about 0.8;

[0046] (2) Take a 1.5ml centrifuge tube, add 1ml of PBS buffer solution containing 5% BSA, block at room temperature for 30min, and discard the solution;

[0047] (3) Take 200 μl of bacterial solution into a 1.5ml centrifuge tube, centrifuge at 5000 rpm for 2 minutes, discard the culture medium, resuspend the bacterial pellet in 200 μl of PBS solution, and take 100 μl into a closed centrifuge tube;

[0048] (4) Take 10 μl of random 7-peptide phage (2×10 11 pfu) was added to the above-menti...

Embodiment 2

[0058] Embodiment 2 Phage monoclonal amplification, extraction and sequencing

[0059] In order to obtain the amino acid sequence of the screened phage, it is necessary to pick a single clone of the phage for amplification and extract single-stranded DNA, and then perform DNA sequencing. The specific steps are as follows:

[0060] (1) Inoculate E.coli ER2738 into 20ml LB medium, culture in a constant temperature incubator at 37°C and 250rpm until OD600 is about 0.8, take 1ml of the bacterial solution into sterile centrifuge tubes;

[0061] (2) Pick 10 clearly separated blue phage plaques from the counting plate after the fifth round of screening, inoculate them into the above-mentioned centrifuge tubes, numbered P1-P10, and culture them in a constant temperature culture shaker at 37°C and 250rpm 5h;

[0062] (3) According to the operation method in the instructions of the phage single-stranded DNA extraction kit, extract the single-stranded DNA of P1-P10;

[0063] (4) The ex...

Embodiment 4

[0071] Embodiment 4 antibacterial experiment

[0072] The antibacterial effect of the polypeptide was preliminarily determined by the disc diffusion method, and the specific operation was as follows:

[0073] (1) Streak and inoculate Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Bacillus cereus on LB agar plates respectively, and incubator for 37 Cultivate at ℃ for 16-18 hours, pick a single colony and inoculate them into 10ml LB liquid culture, culture at 37℃, 250rpm until OD600 is about 0.5;

[0074] (2) Dilute the bacterial solution by 1:1000 respectively, at this time the number of colonies in the bacterial solution is 10 5 -10 6 In the range of cfu / ml, take 100 μl of the diluted bacterial solution and add it to the LB agar plate and spread it evenly;

[0075] (3) After the bacterial solution is completely dry, place 6 pieces of paper (6 mm in diameter) on the plate, add negative ...

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Abstract

The invention relates to the technical field of antibacterial peptide, in particular to a screening method of the antimicrobial peptide and an application of the screening method, and discloses a screening method of the antimicrobial peptide. In accordance with specific pathogenic bacteria, the screening method can quickly screen the antibacterial peptide having excellent resistance. The polypeptide molecular weight in a bacteriophage random peptide library selected in the screening method is small, so that the screened antibacterial peptide is small in molecular weight, and easy to obtain bya chemosynthesis method, and the production cost of the antibacterial peptide is reduced. The antibacterial peptide screened by the screening method has high bactericidal activity for pathogenic bacteria, and the cytotoxicity and the hemolytic activity are low.

Description

technical field [0001] The invention relates to the technical field of antimicrobial peptides, in particular to a method for screening antimicrobial peptides and applications thereof. Background technique [0002] Since the successful industrialization of penicillin in 1938, the research and development of antibiotics has entered a period of rapid development. Antibiotics have treated countless patients with bacterial or fungal infections and made great contributions to human health. However, with the inappropriate use of antibiotics and the absence of antibiotic management, many bacteria are resistant to antibiotics, which makes antibiotic treatment ineffective. Bacterial drug resistance has seriously threatened human health. According to the data from the China Bacterial Resistance Detection Network, in the first half of 2018, the situation of domestic bacterial resistance to antibacterial drugs is still relatively severe, especially carbapenem-resistant Gram-negative bac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/04A61K38/08A61P31/04
CPCA61K38/00A61P31/04C07K1/047C07K7/06
Inventor 瞿祥猛李至军张桂涛
Owner SUN YAT SEN UNIV
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