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Fejervarya multistriata antioxidative peptide and gene and application thereof

A technology of antioxidant peptides and uses, applied in the field of biomedicine

Inactive Publication Date: 2017-12-22
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is based on the above technical background, aiming at the shortage of natural antioxidants and the safety problems of artificially synthesized antioxidants, to provide a new antioxidant peptide and its gene and other antioxidants with antioxidant and immune regulation activities Application in the preparation of drugs and food additives with antioxidant effect

Method used

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  • Fejervarya multistriata antioxidative peptide and gene and application thereof
  • Fejervarya multistriata antioxidative peptide and gene and application thereof
  • Fejervarya multistriata antioxidative peptide and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1, Cloning of Antioxidant Peptide Gene of Frog Rana:

[0028] 1. Extraction of total RNA from the skin of Frog syringa: the living Frog syringa was cleaned with water, put into liquid nitrogen and quick-frozen for 4h, took skin tissue, weighed, got 300mg skin tissue, and added 10m1 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 it 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 in the air.

[0029] II. Purification of Frog skin mRNA: The mRNA of Frog skin was isolated and purified by the American PROMEGA company. mRNA Isolation Systems Kit. The...

Embodiment 2

[0041] Example 2, Preparation of Antioxidant Peptides of Frog Rana:

[0042] Ⅰ. The preparation method of the anti-oxidative peptide of Frog sylvestris: According to the gene of Frog sylvestris anti-oxidant peptide, the amino acid sequence of the mature live secreted 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 B liquid gradient is 15-37% within 22 minutes, the detection wavelength is 220nm, and the polypeptide appears at 15.662 minutes.

[0043] Ⅱ. The molecular weight is de...

Embodiment 3

[0046] Example 3, Experiment on the Activity of Antioxidant Peptides of Frog Frog

[0047] Ⅰ. Determination of antioxidant capacity

[0048] 1) Determination of DPPH free radical scavenging ability

[0049] 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.

[0050] Clearance (%) = [1-(A i -A j ) / A 0 】×100%

[0051] 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 o...

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Abstract

The invention relates to active polypeptide, a gene thereof and an application of active polypeptide to pharmacy. The fejervarya multistriata antioxidative peptide is a cyclopeptide comprises nine amino acids, has the molecular weight of 1122.62 Dalton and the isoelectric point of 8.01, and has the amino acid sequence of Arg Cys Phe Tyr Phe Gly Gln Cys Thr (RCFYFGQCT) (SEQ ID NO.1). Cysteines in the second and eighth places of the polypeptide form an intramolecular disulfide bond, and threonine in the ninth place is amidated. The gene sequence of the fejervarya multistriata antioxidative peptide comprises SEQ ID NO.4, and nucleotides in the 196-222<nd> are coded with functional mature fejervarya multistriata antioxidative peptide. The fejervarya multistriata antioxidative peptide is simple in structure and convenient to synthesize, and is applied to prepare a medicine and food additive having an antioxidative effect.

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 a 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 has been linked to various diseases such as Alzheimer's disease, Parkinson's disease, this pathology is caused by diabetes, complications caused by diabetes, rheumatoid arthritis and amyotrophic lateral sclerosis It is related to the induced neurodegeneration (Food Chemistry, 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 phy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/12A23L33/18A61K38/10A61P39/06A61P25/16A61P25/28A61P3/10A61P21/00A61P19/02A61P29/00A61K8/64A61Q19/08
CPCA23L33/18A23V2002/00A61K8/64A61K38/00A61Q19/08C07K7/08A23V2200/30A23V2200/324A23V2250/55
Inventor 吴继国蒋云霞张国霞郑钟黛西
Owner SOUTHERN MEDICAL UNIVERSITY
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