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Hylarana guentheri antibacterial peptide and application thereof

An antibacterial peptide, a technology of swamp water frog, applied in the field of biomedicine, can solve the problem of undiscovered antibacterial peptides of the swamp water frog skin

Inactive Publication Date: 2015-12-23
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that frogs need at least 20-30 kinds of antimicrobial peptides to form a good antibacterial barrier, and there are 9 reported antibacterial peptides of marshwater frogs, which belong to the brevinin, temporin and guentherin families, and there are still many marshwater frogs. Antimicrobial peptides found in water frog skin

Method used

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  • Hylarana guentheri antibacterial peptide and application thereof
  • Hylarana guentheri antibacterial peptide and application thereof
  • Hylarana guentheri antibacterial peptide and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0021] The separation and purification of the antimicrobial peptide of the present invention includes two steps of Sephadex G-50 gel filtration chromatography and reverse phase high performance liquid chromatography (RP-HPLC).

[0022] Extract the skin secretions of marsh water frogs: adult marsh water frogs were washed with deionized water, and a commercially available 9V DC battery was used to shock the skin of the post-auricular glands and the developed part of the back glands of marsh water frogs. Contains 0.05% ( V / V ) was washed with deionized water of trifluoroacetic acid and the secretion was collected, centrifuged at 12,000 rpm for 15 min, and the supernatant was freeze-dried and stored at -80°C.

[0023] SephadexG-50 Gel Filtration Chromatography: The lyophilized powder of the skin secretions of the marsh water frog was obtained according to the above method, dissolved in deionized water, and centrifuged at 12000 rpm for 15 min. After the supernatant was filtered w...

Embodiment 2

[0027] Antibacterial activity of fractions separated by gel filtration chromatography by agar spread method:

[0028] The strains of Escherichia coli (EscherichiacoliATCC8099) and Staphylococcus aureus (StaphylococcusaureusCMCC26003) were preserved in our laboratory. The specific operation is as follows: select a single colony and inoculate it in beef extract peptone liquid medium, shake it overnight (37°C, 130rpm), inoculate 200 μL of the overnight culture solution into 20 mL beef extract peptone liquid medium, and shake it for 4 hours . The cultured bacterial suspension was diluted 1000 times, and 100 μL was added to the surface of the plate medium, and spread evenly. On the surface of the plate culture medium coated with the good bacterial solution, evenly paste circular filter paper pieces with a diameter of 5 mm, and add 10 μL of the sample to be tested on each filter paper piece. Gentamicin was used as a positive control, and sterile water was used as a negative contro...

Embodiment 3

[0030] Utilize the liquid growth inhibition method to detect the inhibitory activity of the polypeptide of the present invention to bacteria:

[0031] The antimicrobial peptide used in this example was synthesized by Shanghai Jier Biochemical Co., Ltd. according to the sequence SEQIDNO.1 and the sequence SEQIDNO.2. The synthesized antimicrobial peptide maintained the original modification, wherein the C-terminal cysteine ​​of antimicrobial peptide A formed a disulfide bond. C-terminal amidation of antimicrobial peptide B. The purity of the two synthetic peptides was not less than 95%. The minimal inhibitory concentration (MIC) of the sample was determined by the 96-well plate method, and the specific operation was as follows:

[0032] Recover the strains, inoculate the slant and incubate overnight at 37°C, pick a single colony and inoculate it in ordinary LB medium, cultivate overnight at 37°C, dilute the bacterial solution to make the bacterial concentration 10 4 -10 5CFU / ...

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Abstract

The invention relates to sequences, a preparation method and application of two types of hylarana guentheri antibacterial peptides. The two types of antibacterial peptides are separated from skin secreta of hylarana guentheri, and antibacterial and hemolytic activities of the antibacterial peptides are detected; a secondary structure of the antibacterial peptides under a simulated film environment is researched. The two antibacterial peptides are obtained: the antibacterial peptide A with an amino acid sequence shown as SEQ ID NO.1; the antibacterial peptide B with an amino acid sequence shown as SEQ ID NO.2. The antibacterial peptide A provided by the invention has an inhibition effect on staphylococcus aureus, colon bacillus and bacillus subtilis; the antibacterial peptide B has a very good sterilization effect on the bacillus subtilis and the staphylococcus aureus. The two types of antibacterial peptides, provided by the invention, have the characteristics of novel structure, strong antibacterial activities and the like, and have an important value in the aspects of medicine development and application. The sequences, the preparation method and the application of two types of the hylarana guentheri antibacterial peptides have important meanings on researches of an antibacterial mechanism of the antibacterial peptides, and further improvement, and development and utilization of the antibacterial peptides.

Description

technical field [0001] The invention relates to two natural antibacterial peptides and their application in the field of pharmacy, belonging to the field of biomedicine. Background technique [0002] Antimicrobial peptides have important theoretical research and drug development application value due to their broad-spectrum antibacterial activity and special antibacterial mechanism. Amphibian skin can secrete a large number of antimicrobial peptides to resist the invasion of foreign microorganisms. Since researchers discovered the first amphibian antimicrobial peptide magainins from Xenopus laevis skin secretions, thousands of antimicrobial peptides have been secreted from amphibian skin These antimicrobial peptides are generally composed of 10-50 amino acids. According to the amino acid sequence of antimicrobial peptides, they are divided into more than 100 different families. The antimicrobial peptides of these different families are different in molecular weight, charge,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46A61K38/17A61P31/04
CPCA61K38/00A61K38/10C07K14/463Y02A50/30
Inventor 洪晶汪少芸俞世宏
Owner FUZHOU UNIV
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