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Chimeric antibacterial cell-permeable peptide T11N2 and application thereof

A T11N2, antimicrobial peptide technology, applied in the chimeric transmembrane antimicrobial peptide T11N2 and its application field, can solve the problems of restriction elimination, large cell stimulation, weak targeting, etc., and achieves low cost, relatively easy synthesis, and structural simple effect

Active Publication Date: 2018-03-23
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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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 antibacterial cell-permeable peptide T11N2 and application thereof
  • Chimeric antibacterial cell-permeable peptide T11N2 and application thereof
  • Chimeric antibacterial cell-permeable peptide T11N2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 Preparation of chimeric membrane-penetrating antimicrobial peptide

[0039] On the basis of highly active antimicrobial peptide N2 (SEQ ID NO:2), a cell-penetrating peptide Tat with 11 amino acids was connected to design a novel chimeric antimicrobial peptide T11N2, whose amino acid sequence is shown in SEQ ID NO:1 .

[0040] Antimicrobial peptide T11N2 can be prepared by Fmoc solid phase synthesis.

[0041] 1. Synthesis sequence: from the C-terminal to the N-terminal of the sequence, the steps are as follows:

[0042] 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 equivalent methanol , reacte...

Embodiment 2

[0055] Example 2 Cytotoxicity Experiment of Chimeric Membrane-penetrating Antimicrobial Peptides

[0056] 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).

[0057] 2. Prepare different concentrations of antimicrobial peptide T11N2 solutions (dissolved in 0.01M phosphate buffer), and set up three negative control wells at the same time, 37 ° C, 5% CO 2 Cultivate for 1-5h.

[0058] 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.

[0059] 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.

[0060] 5. At the same time, set th...

Embodiment 3

[0062] Example 3 Detection of Anti-Salmonella Activity

[0063] The minimum inhibitory concentration (MIC) of the antimicrobial peptide T11N2 against 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:

[0064] 1) Pick a single clone of the tested strain to MH medium, culture at 37°C, 250rpm, shake overnight;

[0065] 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;

[0066] 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;

[0067] 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...

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PUM

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Abstract

The invention discloses a novel chimeric antibacterial cell-permeable peptide T11N2. The amino acid sequence is as shown in SEQ ID NO:1. The novel chimeric antibacterial cell-permeable peptide T11N2 is a chimeric polypeptide formed by connecting a cell-permeable peptide Tat which has 11 amino acids on the basis of a high-activity antibacterial peptide N2. The antibacterial peptide T11N2 has a simple structure, low toxicity, safety, low cost and relatively easy synthesis, and is a novel chimeric antibacterial cell-permeable peptide capable of efficiently penetrating membranes and killing intracellular salmonella. The antibacterial peptide T11N2 disclosed by the invention can be applied to the fields of antibacterial drugs, food additives, cosmetics, feed additives and the like, and has a wide application value and market prospect.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a chimeric membrane-penetrating antimicrobial peptide T11N2 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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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A23L33/18A23K20/147A61K38/16A61P31/04A61P17/02A61K8/64A61Q19/00
CPCA23K20/147A23L33/18A61K8/64A61K38/00A61K2800/10A61Q19/00C07K14/00C07K2319/10Y02A50/30
Inventor 王建华王秀敏李占占毛若雨滕达郝娅王潇
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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