Microhyla pulchra antioxidation peptide and gene thereof, and application of microhyla pulchra antioxidation peptide to pharmacy

A technology for antioxidant peptides and drugs, which is applied in the field of biomedicine to achieve significant red blood cell agglutination and antioxidant effects, convenient artificial synthesis and simple structure.

Active Publication Date: 2017-02-22
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] 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 its Application in the preparation of drugs and food additives with antioxidant effect

Method used

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  • Microhyla pulchra antioxidation peptide and gene thereof, and application of microhyla pulchra antioxidation peptide to pharmacy
  • Microhyla pulchra antioxidation peptide and gene thereof, and application of microhyla pulchra antioxidation peptide to pharmacy
  • Microhyla pulchra antioxidation peptide and gene thereof, and application of microhyla pulchra antioxidation peptide to pharmacy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, Cloning of Antioxidant Peptide Gene of Rana japonica:

[0036] 1. Extraction of total RNA from the skin of Frog japonica: the live Frog japonica was cleaned with water, put into liquid nitrogen and quick-frozen for 4h, got skin tissue, weighed, got 300mg skin tissue, 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 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.

[0037] 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 details are a...

Embodiment 2

[0049] Example 2, preparation of antioxidative peptides of Rana japonica:

[0050] Ⅰ. The preparation method of the anti-oxidative peptide of Rana japonica: according to the anti-oxidative peptide gene of Rana japonica, 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 30-50% within 20 minutes, the detection wavelength is 220nm, and the polypeptide appears at 12.962 minutes.

[0051] Ⅱ. The molecular weight i...

Embodiment 3

[0054] Example 3, Activity Experiment of Antioxidant Peptide of Frog Rana

[0055] Ⅰ. Determination of antioxidant capacity

[0056] 1) Determination of DPPH free radical scavenging ability

[0057] 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.

[0058] Clearance (%) = [1-(A i -A j ) / A 0 】*100%

[0059] 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 sample...

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Abstract

The invention relates to active polypeptide and a gene thereof, and application of the active polypeptide to pharmacy. Microhyla pulchra antioxidation peptide is cyclopeptide consisting of 12 pieces of amino acid, wherein the molecular weight is 1334.91 daltons; the isoelectric point is 8.002; the amino acid sequence is SEQ ID NO.1; and the third-position cysteine and the eleventh-position cysteine of the polypeptide form intramolecular disulfide bonds. The gene sequence of the microhyla pulchra antioxidation peptide consists of SEQ ID NO.4, and the gene encoded with the functional mature microhyla pulchra antioxidation peptide is the 187th to the 222nd position nucleotide. The gene of the microhyla pulchra antioxidation peptide deduces the mature functional polypeptide amino acid sequence, and the synthesized microhyla pulchra antioxidation peptide has strong erythrocyte agglutination and anti-oxidation functions.

Description

[0001] Technical field: [0002] The invention relates to the field of biomedicine, in particular to a protein obtained from animal tissue and its use in biopharmaceuticals. [0003] Background technique: [0004] 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/12A61K38/10A61K8/64A61K47/42A61P39/06A61P25/16A61P1/04A61P1/18A61Q19/08
CPCA61K8/64A61K38/00A61K47/42A61Q19/08C07K7/08
Inventor 徐学清陈新曾白霜张贝
Owner SOUTHERN MEDICAL UNIVERSITY
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