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Antioxidant peptide sourced from limnonectes fragilis as well as gene and application thereof

A crispy-skinned frog and antioxidant peptide technology, which is applied in the field of biomedicine and achieves the effects of small molecular weight, low hemolytic activity and simple preparation method

Active Publication Date: 2015-06-17
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the research on antioxidant peptides derived from crispy big-headed frogs

Method used

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  • Antioxidant peptide sourced from limnonectes fragilis as well as gene and application thereof
  • Antioxidant peptide sourced from limnonectes fragilis as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Gene cloning of three kinds of crispy big-headed frog antioxidant peptides Fragilin-A1, Fragilin-B2, Odorranain-Q-Lf:

[0051] 1 Extraction of total RNA from skin of crispy frog skin:

[0052] ① Take 300 mg of skin tissue of crispy-skinned frog, put it into a mortar and add liquid nitrogen to grind it into powder, transfer it to an EP tube, add 1 ml of total RNA extraction buffer (Trizol, product of Invitrogen, USA), mix well, Then centrifuge at 12000 rpm for 10 min at 4°C.

[0053] ② Centrifuge to get the supernatant, add 0.2 ml chloroform solution, mix vigorously, let stand at room temperature for 10 minutes, then centrifuge at 4°C, 12000 rpm for 10 minutes, discard the precipitate.

[0054] ③ Add an equal volume of isopropanol to the supernatant, place at room temperature for 10 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, collect the precipitate, wash it once with 75% (V / V) ethanol, and dry it. The precipitate at the bottom of the tube is brittle. Tota...

Embodiment 2

[0106] Chemical synthesis of three antioxidant peptides Fragilin-A1, Fragilin-B2, and Odorranain-Q-Lf from the crispy frog:

[0107] 1. The chemical synthesis method of three kinds of crispy big-headed frog antioxidant peptides Fragilin-A1, Fragilin-B2, and Odorranain-Q-Lf: according to the amino acid sequence of the mature peptide derived from the gene, synthesize it with an automatic peptide synthesizer (433A, Applied Biosystems) The whole sequence was desalted by HPLC reverse phase column chromatography.

[0108] 2. The molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

[0109] 3. The purity of the three purified antioxidant peptides Fragilin-A1, Fragilin-B2, and Odorranain-Q-Lf of crispy-headed frogs was identified by high-performance liquid chromatography (HPLC), and the molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI -TOF), th...

Embodiment 3

[0114] Pharmacological experiments of three antioxidant peptides Fragilin-A1, Fragilin-B2, Odorranain-Q-Lf of crispy big-headed frog:

[0115] 1. Fragilin-A1, Fragilin-B2, Odorranain-Q-Lf antioxidant activity assay:

[0116] 1.1 DPPH radical scavenging activity (DPPH radical scavenging assay)

[0117] Weigh a certain amount of DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate, Sigma, USA), dissolve it in methanol, and make 6×10 -5 The solution of M is prepared and used immediately. Mix 48 μl DPPH solution with 2 μl sample (2 mg / ml) (the mass ratio of final sample to DPPH is 3:1), let stand in the dark at room temperature for 30 min, and measure the absorbance at 517 nm. In the blank control group, the sample to be tested was replaced by the sample dissolution medium. The experiment was done in triplicate, and methanol was used when the UV spectrophotometer was zeroed.

[0118] DPPH·clearance rate (%) = (AB-AA ) / A B×100 (AB: absorbance value of blank control group; AA: absorbanc...

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Abstract

The invention relates to antioxidant peptide sourced from limnonectes fragilis as well as a gene and an application thereof and belongs to the technical field of biomedicine. The primary structural sequences of three antioxidant peptides Fragilin-A1, Fragilin-B2 and Odorranain-Q-Lf which are sourced from the limnonectes fragilis, have the encoding genes obtained through a gene clone way, are obtained through a chemical synthesis method and respectively contain 15 amino acid residues, 12 amino acid residues and 21 amino acid residues; the molecular weight of each of the three antioxidant peptides is respectively 1720.95Da, 1464.66Da and 2597.18Da; an isoelectric point of each of the three antioxidant peptides is respectively 8.04, 8.75 and 9.53. The three antioxidant peptides are strong in antioxidant activity, small in molecular weight, simple in structure, low in hemolytic activity and simple in preparation method. When the antioxidant peptide is applied to antioxidant medicines, foods or cosmetic additives, the antioxidant effect of the medicines, the foods or the cosmetic additives can be improved, and the application prospect is promising.

Description

technical field [0001] The invention relates to an antioxidant peptide derived from a crispy-skinned frog and its gene and application, and belongs to the technical field of biomedicine. Background technique [0002] The free radicals produced by the body's oxidation reaction are atomic groups containing an unpaired electron, such as superoxide anion, hydroxyl radical, etc. Free radicals have strong oxidative properties. With age or under pathological conditions, if excessive free radicals cannot be removed in time, they will accumulate in cells and interact with biological macromolecules in the body, such as proteins, nucleic acids, and lipids. and other interactions to generate excessive oxides and peroxides, which will eventually affect the body's metabolism and cause irreversible damage to the body. Aging and many chronic diseases such as cancer, cardiovascular disease, emphysema, liver cirrhosis, arthritis, etc. are all related to the damage of free radicals. Although...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K14/46C12N15/12A61K38/10A61K38/17A61P39/06A23L1/305A61K8/64A61Q19/08A23L33/18
Inventor 于海宁王义鹏高久香
Owner DALIAN UNIV OF TECH
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