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Black frame toad antioxidant peptide and its gene and its application in pharmaceuticals

A toad and black frame technology, applied in the field of biomedicine, can solve the problems of insufficient research on skin active peptides

Active Publication Date: 2020-07-10
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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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  • Black frame toad antioxidant peptide and its gene and its application in pharmaceuticals
  • Black frame toad antioxidant peptide and its gene and its application in pharmaceuticals
  • Black frame toad antioxidant peptide and its gene and its application in pharmaceuticals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1, black box toad antioxidant peptide gene cloning:

[0032] 1. Extraction of total RNA from black-framed toad skin: the living black-framed toad was cleaned with water, put into liquid nitrogen for quick freezing for 4h, got skin tissue, weighed, got 300mg skin tissue, added 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 black-framed toad skin.

[0033] II. Purification of mRNA from black-framed toad skin: the isolation and purificat...

Embodiment 2

[0045] Example 2, the preparation of black frame toad antioxidant peptide:

[0046] Ⅰ. The preparation method of black-frame toad antioxidant peptide: According to the gene of black-frame toad antioxidant peptide, the amino acid sequence of the mature active secreted peptide encoding the function is deduced, and then the peptide is synthesized by an automatic polypeptide synthesizer. The formation of the disulfide bond adopts the air oxidation method, specifically dissolving the polypeptide in a flask at 0.1 mg / 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 concentration gradient of solution B is 20-40% in 15 minutes, the detection wavelength is 220nm, and the polypeptide appears at 11.670 minutes.

[0047] Ⅱ. The molecular weight is ...

Embodiment 3

[0050] Example 3, the activity experiment of black box toad antioxidant peptide

[0051] Ⅰ. Determination of antioxidant capacity

[0052] 1) Determination of DPPH free radical scavenging ability

[0053] DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical scavenging rate assay was used to study antioxidant peptides. Prepare a DPPH ethanol solution with a concentration of 1×10-5 mol / L, and store it in the dark. Add 2ml, 0.1mM DPPH absolute ethanol solution into a clean test tube containing 2ml of different enzymatic hydrolysis samples, and mix well. After standing at room temperature for 30 minutes, measure the absorbance at 517nm, the smaller the absorbance value, the stronger the ability to scavenge free radicals.

[0054] Clearance (%)={1-(A i -A j ) / A 0}*100%

[0055] In the formula, A 0 2ml, 0.1mM DPPH absolute ethanol solution + 2ml sample reagent, blank control, A i 2ml, 0.1mM DPPH absolute ethanol solution + 2ml sample, A j 2ml of absolute ethanol + 2ml of sam...

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Abstract

The invention relates to a bufo melanostictus antioxidative peptide and its gene and an application in pharmacy. The bufo melanostictus antioxidative peptide is cyclic peptide consisting of 33 amino acids, the molecular weight is 3678.14 Dalton, and the isoelectric point is 10.33, and the amino acid sequence is shown as SEQ ID NO.1; the third cysteine and the eighth cysteine of the polypeptide are formed to be an intramolecular disulfide bond. The gene sequence of the bufo melanostictus antioxidative peptide consists of SEQ ID NO.4, wherein the matured bufo melanostictus antioxidative peptide encoded with functions is the 394-492 bit of nucleotide. The matured functional polypeptide amino acid sequence is deduced by gene of bufo melanostictus antioxidative peptide; the compounded bufo melanostictus antioxidative peptide has very strong agglutinin, serine protease inhibitor and antioxygenation.

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] Oxidation of biomolecules is an oxygen free radical mediated process that can have many adverse effects on food and biological systems. In aerobic organs, free radicals related to various diseases such as arteriosclerosis and cancer are inevitably produced along with the process of oxygen metabolism. In addition, oxidative stress is considered to be related to neurodegeneration caused by various diseases such as Alzheimer's disease, Parkinson's disease, diabetes, rheumatoid arthritis and amyotrophic lateral sclerosis (FoodChemistry, 2008, 107, 1485–1493). In food, the oxidation of nutrients will produce peroxides, which will not only affect the nutritional value of food, cause food quality to decline, and even lead to physical diseases of the ingestor (Journal of Food Science, 199...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46C12N15/12A61K38/17A61K8/64A61K47/42A61P1/04A61P1/18A61P25/16A61P39/06A61Q19/08
CPCA61K8/64A61K38/00A61K47/42A61Q19/08C07K14/463
Inventor 徐学清曾白霜张贝
Owner SOUTHERN MEDICAL UNIVERSITY
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