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Chimeric cell-penetrating antibacterial peptide B6N2 and application thereof

A B6N2, antibacterial peptide technology, applied in the field of biomedicine, can solve the problems of restriction elimination, large cell stimulation, weak targeting, etc., and achieves the effects of low cost, relatively easy synthesis and simple structure

Inactive Publication Date: 2018-01-23
北京生泰云科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above methods have advantages, they also have disadvantages, such as great stimulation to cells, low introduction rate and weak targeting, etc.
Antibacterial peptide N2 can penetrate the outer membrane of Escherichia coli, bind to DNA by intercalation, and affect DNA replication, but the cell membrane of Salmonella treated with N2 is intact, and the cell membrane penetration rate is only 5.84% after 2 hours of treatment (Antibacterial and detoxifying activity of NZ17074analogues with multi-layers of selective antimicrobial actions against Escherichia coli andSalmonella enteritidis.Scientific reports,2017,7(1):3392.), which largely limits its elimination of intracellular escaped Salmonella

Method used

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  • Chimeric cell-penetrating antibacterial peptide B6N2 and application thereof
  • Chimeric cell-penetrating antibacterial peptide B6N2 and application thereof
  • Chimeric cell-penetrating antibacterial peptide B6N2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Preparation of chimeric membrane-penetrating antimicrobial peptide

[0040] On the basis of highly active antimicrobial peptide N2 (SEQ ID NO:2), a cell-penetrating peptide bLFcin6 with 6 amino acids was connected to design a new chimeric membrane-penetrating antimicrobial peptide B6N2, whose amino acid sequence is shown in SEQ ID NO:1 .

[0041] Antimicrobial peptide B6N2 can be prepared by Fmoc solid phase synthesis.

[0042] 1. Synthesis sequence: from the C-terminal to the N-terminal of the sequence, the steps are as follows:

[0043] 1) Weigh n equivalents of resin into the reactor, add DCM to swell for half an hour, then remove the DCM, add 2n equivalents of the first amino acid (Fmoc-Asn(Trt)-OH) in the sequence, add 2n equivalents of DIEA, Appropriate amount of DMF, DCM (appropriate amount means that the resin can fully expand), DIEA (diisopropylethylamine), DMF (N,N-dimethylformamide), DCM, blowing nitrogen for 60min, and then adding 5n equivalen...

Embodiment 2

[0056] Example 2 Cytotoxicity Experiment of Chimeric Membrane-penetrating Antimicrobial Peptides

[0057] 1. Collect logarithmic phase RAW264.7 cells (mouse peritoneal macrophage cell line), adjust the concentration of cell suspension, add 7×10 3 cells, 5% CO 2 , and incubated at 37°C until the cell monolayer covered the bottom of the well (96-well flat bottom plate).

[0058] 2. Prepare different concentrations of antibacterial peptide B6N2 solutions (dissolved in 0.01M phosphate buffer), and set up three negative control wells at the same time, at 37 ° C, 5% CO 2 Cultivate for 1-5h.

[0059] 3. Add 20ul of MTT solution to each well, continue to cultivate for 4 hours, stop the culture, and carefully suck off the culture medium in the well.

[0060] 4. Add 150ul dimethyl sulfoxide to each well, shake on a shaker at low speed for 10min. The absorbance of each well was measured at OD490nm in an enzyme-linked immunosorbent assay instrument.

[0061] 5. At the same time, set ...

Embodiment 3

[0063] Example 3 Detection of Anti-Salmonella Activity

[0064] The minimum inhibitory concentration (MIC) of antimicrobial peptide B6N2 to Salmonella was established with reference to Tian et al. Micro broth dilution method, slightly modified according to the specific situation, the specific operation is as follows:

[0065] 1) Pick a single clone of the tested strain to MH medium, culture at 37°C, 250rpm, shake overnight;

[0066] 2) Serially dilute the antibacterial drugs into 1.5mL sterile centrifuge tubes according to a 2-fold gradient, and the concentrations are 10 times the final concentrations;

[0067] 3) Transfer the tested strains to MH liquid medium at 37°C with 1% inoculum amount, and shake at 250rpm to 0.5 McFarland standard turbidity;

[0068] 4) Dilute 1000 times of the test bacteria culture solution (the final bacterium concentration is about 10 5 CFU / mL), transferred to a sterile cell culture plate, each well containing 90 μL of diluted bacterial solution;...

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Abstract

The invention discloses a novel chimeric cell-penetrating antibacterial peptide B6N2. The amino acid sequence is as shown in SEQ ID NO:1, the chimeric cell-penetrating antibacterial peptide is a chimeric polypeptide, and cell-penetrating peptides bLFcin6 with six amino acids are connected based on high-activity antibacterial peptide N2 to form the chimeric polypeptide. The antibacterial peptide B6N2 is simple in structure, low in toxicity and cost, safe and relatively easy to synthesize and can efficiently penetrate cells and kill intracellular salmonellae. The antibacterial peptide B6N2 is applied to the fields such as antibacterial drugs, food additives, cosmetics and feed additives and has wide application values and market prospect.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a chimeric membrane-penetrating antimicrobial peptide B6N2 and its application. Background technique [0002] The toxic side effects of traditional antibiotics and the emergence of drug-resistant strains have prompted people to look for new antimicrobial agents. Natural antimicrobial peptides are mostly short peptides composed of 13-45 amino acid residues, which have broad-spectrum antibacterial, immune regulation, tumor suppression and other biological functions and unique mechanism of action, and are currently one of the hot spots in the research of antibiotic substitutes. However, due to their excessive molecular weight and their own hydrophilic properties, their application in vivo is limited to a certain extent. In addition, in animal husbandry, diseases caused by staphylococcus and salmonella, such as mastitis, arthritis, typhoid fever, enteric fever, gastroenteritis and sepsis,...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K38/17A61K8/64A61K47/64A61P31/04A61P17/02A61Q17/00A23L33/18A23K20/147
CPCY02A50/30
Inventor 王建华李占占王秀敏滕达毛若雨郝娅王潇
Owner 北京生泰云科技有限公司
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