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Polypeptide Cbf-14 resisting infection of drug-resistant bacteria and application thereof

A technology of anti-drug-resistant bacteria and antibacterial peptides, applied in the field of biomedicine, can solve the problems of poor proteasome stability, high toxicity of antimicrobial peptides, and high production costs of antimicrobial peptides

Active Publication Date: 2013-12-11
NANJING YINGHAIYUE BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] But at present, there are three bottlenecks in the development of antimicrobial peptides, namely: high production cost of antimicrobial peptides; high toxicity of antimicrobial peptides; poor stability of antimicrobial peptides to proteasomes

Method used

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  • Polypeptide Cbf-14 resisting infection of drug-resistant bacteria and application thereof
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  • Polypeptide Cbf-14 resisting infection of drug-resistant bacteria and application thereof

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Experimental program
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Effect test

Embodiment 1

[0021] Synthesis and Structure Confirmation of Antibacterial Peptide Cbf-14:

[0022] The polypeptide of the present invention is artificially synthesized by conventional solid-phase synthesis method. The specific experimental steps are as follows:

[0023] The polypeptide sequence of the prepared Cbf-14 is:

[0024] H-Arg-Leu-Leu-Arg-Lys-Phe-Phe-Arg-Lys-Leu-Lys-Lys-Ser-Val-OH

[0025] Synthesis of peptides: The synthesis of peptides is carried out one by one from the C-terminal to the N-terminal. Soak Fmoc-Val-Wang Resin in dichloromethane for 15 minutes, wait for the resin to swell, and remove the dichloromethane; add hexahydropyridine / DMF solution with a volume ratio of 1:4, use nitrogen agitation, and react twice for After 5 minutes and 15 minutes, the resin was washed 9 times with DMF after the reaction. Take a small amount of resin and add 2-3 drops of each color test agent ABC (A solution: ninhydrin / absolute ethanol solution; B solution: pyridine; C solution: phenol...

Embodiment 2

[0034] The drug effect of polypeptide Cbf-14 of the present invention on penicillin-resistant bacteria:

[0035] (1) Preparation of inoculum

[0036] Connect the bacteria used in the experiment from the glycerol tube to the slant of nutrient agar, cultivate overnight at 37°C, then pick a little to inoculate in 2ml nutrient broth medium, culture at 37°C for 8h, and dilute to 100% with sterile MH broth culture solution. 5 Bacterial suspension around CFU / ml.

[0037] (2) Drug configuration

[0038] Accurately weigh Cbf-14 and penicillin to prepare a drug solution with a concentration of 1024 μg / ml, filter it through a 0.22 μm membrane aseptically, aliquot it, and store it at -70°C for future use.

[0039] (3) Determination of minimum inhibitory concentration (MIC)

[0040]Use sterile MH broth to dilute the drug stock solution to 1ml solution with drug concentration of 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μg / ml. Take 1ml of the above bacterial liquid and add it to ...

Embodiment 3

[0060] The toxicity of the polypeptide antibacterial peptide Cbf-14 of the present invention includes the determination of hemolysis and the determination of toxicity to eukaryotic cells.

[0061] Hemolytic determination of the polypeptide antimicrobial peptide Cbf-14 of the present invention:

[0062] Fresh sheep whole blood was centrifuged at 3000 rpm for 10 min in a refrigerated centrifuge (4°C), and the obtained sheep red blood cells were washed three times with PBS and suspended at a specific gravity of 10% by volume. Mix the same volume of resuspended sheep red blood cells with the serially diluted drugs to make the final concentration of Cbf-14 to be 512, 256, 128, 64, 32, 16, 8, 4 and 2, 1 μg / ml, and set the PBS group as negative Control, 0.1% Triton X-100 group was used as positive control, and each gradient was set to 6 parallels. All mixtures were incubated in a 37°C incubator for 1 h. Then centrifuge at 3000rpm for 10min in a refrigerated centrifuge (4°C), take t...

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Abstract

The invention relates to the field of biomedicine and in particular relates to an antibacterial polypeptide. Part of active amino acids of the antibacterial polypeptide of Cathelicidin family is mutated based on the antibacterial polypeptide of Cathelicidin family, and then the antibacterial polypeptide with high inhibitory activity to penicilin-resistant bacterium is obtained by solid phase chemical synthesis. The result of pharmacodynamic tests shows that the antibacterial polypeptide provided by the invention has good in vitro antibacterial activity and is applicable to treating diseases about drug-resistant bacterial infection.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to an antibacterial polypeptide and its application. The antibacterial polypeptide of the invention has the application of resisting drug-resistant bacterial infection. Background technique [0002] With the extensive use of antibiotics, especially non-indication use, inappropriate selection of backup antibacterial drugs, overtreatment and frequent dressing changes, the rate of bacterial drug resistance is getting higher and higher, and the degree of drug resistance is becoming more and more serious. The existence of spontaneous drug-resistant mutations and the continuous effect of antibiotic selection pressure, as well as the environmental adaptability of pathogenic bacteria and the evolutionary drive of ecological changes in the human microenvironment, are the basis for the emergence of clinically drug-resistant and multidrug-resistant bacteria. The emergence and development o...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61P31/04
CPCY02A50/30
Inventor 周长林贾源宾田玉伟李冰李博王慧窦洁
Owner NANJING YINGHAIYUE BIOLOGICAL SCI & TECH
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