Rana rugulosa wiegmann antibacterial peptide, as well as gene and application thereof

An antimicrobial peptide and tiger frog technology, applied in the field of biomedicine, can solve the problems of insufficient research on skin active peptides

Inactive Publication Date: 2017-12-26
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The application of amphibian drugs has a long history in my country, but the research on its active ingredients and pharmacological properties mainly focuses on small organic molecules such as alkaloids, and there are not many studies on its skin active peptides

Method used

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  • Rana rugulosa wiegmann antibacterial peptide, as well as gene and application thereof
  • Rana rugulosa wiegmann antibacterial peptide, as well as gene and application thereof
  • Rana rugulosa wiegmann antibacterial peptide, as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, tiger frog antimicrobial peptide gene cloning:

[0028] 1, tiger frog skin total RNA extraction: the living tiger frog is cleaned with water, put into liquid nitrogen and quick-frozen for 4h, get skin tissue, weigh, get 300mg skin tissue, add 10m total RNA extraction buffer (Trizol solution, the U.S. GIBCOBRL company product), homogenized in 20m1 glass homogenizer for 30min. Add an equal volume of phenol / chloroform solution, mix vigorously, place at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, and discard the precipitate. Add an equal volume of isopropanol to the supernatant, place at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, wash the precipitate once with 75% ethanol, and dry it. The precipitate at the bottom of the tube is the total RNA of tiger frog skin.

[0029] II. Purification of tiger frog skin mRNA: the separation and purification of tiger frog skin mRNA was carried out by...

Embodiment 2

[0041] Embodiment 2, the preparation of tiger frog antimicrobial peptide:

[0042] Ⅰ. The preparation method of the antibacterial peptide of tiger frog: according to the gene of tiger frog antibacterial peptide, the amino acid sequence of the mature active antimicrobial peptide encoded by the function is deduced, and then the polypeptide is synthesized by an automatic polypeptide synthesizer. The formation of disulfide bond adopts the air oxidation method, specifically dissolving the polypeptide in the flask according to 0.1mg / ml in 0.1% acetic acid solution, titrating with ammonium hydroxide to pH 7.8, and then stirring overnight at room temperature. Desalted and purified by HPLC reverse phase C18 column chromatography. Liquid A is 0.05% TFA+2% CH during purification 3 CN, liquid B is 0.05% TFA+90% CH 3 CN, the gradient of peptide elution is that the concentration of solution B is 25-47% within 20 minutes, the detection wavelength is 220nm, and the peptide appears at 11.908...

Embodiment 3

[0046] Embodiment 3, activity test of tiger frog antimicrobial peptide

[0047] Ⅰ. Determination of the ability to inhibit bacterial growth

[0048]The antibacterial activity was detected by the cup and saucer method, and the medium was ordinary agar medium. Inject 20m1 of heated and melted culture medium into the plate as the bottom layer, spread it evenly in the bottom of the plate, after solidification, take another appropriate amount of culture medium and heat it to melt, then add 5m1 of bacterial suspension to each plate, shake well, Spread it evenly on the bottom layer as a bacterial layer. After cooling, put 6 sterilized stainless steel cups evenly in the plate at equal distances. Add 0.1 ml of the test compound solution at a concentration of 0.1-0.3 mg / ml to the first steel cup, add the sample solution to the remaining steel cups by doubling the dilution method, incubate at 37°C, and observe the size of the inhibition zone. The bacteriostatic zone above 10mm was reg...

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Abstract

The invention relates to a rana rugulosa wiegmann antibacterial peptide, as well as gene and application in pharmacy. The rana rugulosa wiegmann antibacterial peptide is a ring-type polypeptide consisting of 12 amino acids, and has an amino acid sequence as shown in SEQ ID NO.1, wherein the 3nd cysteine and the 11th cysteine are complemented to form intramolecular disulfide bond, and the 12th tyrosine carries amidating modification. The gene sequence of the rana rugulosa wiegmann antibacterial peptide consists of SEQ ID NO.4, wherein a mature rana rugulosa wiegmann antibacterial peptide with coding function is nucleotide from 196 to 231. The mature functional polypeptide amino acid sequence is deduced by genes of the rana rugulosa wiegmann antibacterial peptide, and the synthesized rana rugulosa wiegmann antibacterial peptide has strong antibacterial and antioxidative functions. The rana rugulosa wiegmann antibacterial peptide has the characteristics of simple structure and convenience in artificial synthesis, can be applied to preparation of therapeutic medicines, beautifying and skin-protecting medicines and food additives for pathogenic microorganism infectious diseases and free radical oxidation.

Description

Technical field: [0001] The invention relates to the field of biomedicine, in particular to a protein obtained from animal tissue and its use in biopharmaceuticals. Background technique: [0002] The discovery of penicillin has enabled people to deal with related diseases caused by various pathogenic microbial infections, and various antibiotics derived from it have made great contributions to the protection of human health. However, due to the long-term use and abuse of these "traditional antibiotics", many bacteria have developed resistance to them. The emergence of multidrug-resistant superbugs has made the development of new antimicrobial drugs particularly urgent. Antimicrobial peptides are a class of active polypeptides with a molecular weight of less than 10,000, composed of 10-50 amino acids, and capable of killing or inhibiting the growth of microorganisms. The latest research shows that antimicrobial peptides not only have broad-spectrum antimicrobial activity, b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/12A23L33/18A23L3/3463A61K38/10A61P31/04A61P31/10A61P31/12A61P17/16A61P17/18A61P39/06A61P25/16A61P25/28A61P3/10A61P29/00A61P19/02A61P25/00A61P21/00A61K8/64A61Q19/08
CPCA23L3/3463A23V2002/00A61K8/64A61K38/00A61Q19/08A23L33/18C07K7/08A23V2200/318A23V2200/10A23V2200/328A23V2200/322Y02A50/30
Inventor 吴继国张国霞蒋云霞郑钟黛西
Owner SOUTHERN MEDICAL UNIVERSITY
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