Polypeptide, preparation method and application thereof

A peptide chain and aspect technology, applied in the field of peptide preparation, can solve the problems of reversal and inability to cure the disease course, and achieve the effect of convenient operation and simple process

Active Publication Date: 2015-01-21
TIANJIN BIOFEED TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no specific drug for the treatment of this disease. For example, the treatment of asthma with inh

Method used

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  • Polypeptide, preparation method and application thereof
  • Polypeptide, preparation method and application thereof
  • Polypeptide, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The tissue extraction and preparation method (animal tissue extraction method) of the polypeptide of this embodiment includes the following steps:

[0036] 1) Extraction by acidified ethanol: take the skin of the northern leopard frog, mince it and digest it in acidified ethanol at 4°C for 12 hours, add 8 mL of acidified ethanol per gram of skin tissue (the acidified ethanol is made of ethanol with a concentration of 0.7mol / L Hydrochloric acid prepared according to the volume ratio of 3:1), centrifuged at 4°C and 3000g for 30min, and the supernatant was concentrated by rotation and freeze-dried to obtain a freeze-dried product;

[0037] 2) Separation by size exclusion chromatography: the lyophilized product obtained in step 1) was treated in acetic acid with a concentration of 2mol / L, and then loaded into a Sephadex G50 column (90×1.6cm) at a concentration of 2mol / L acetic acid balance, column flow rate 10mL / h, collect 60 components of the chromatographic peak, freeze-d...

Embodiment 2

[0041] The chemical synthesis preparation method (artificial synthesis method) of the polypeptide of this embodiment includes the following steps:

[0042] 1) Using Wang resin (0.6mmol / g) as the starting resin, Fmoc solid-phase synthesis method was used to couple each protected amino acid one by one. The condensing agent used in the coupling was 2-(1H-benzotrisazo L-1 -base)-1,1,3,3-tetramethyluronium tetrafluoroborate, synthesized to obtain a peptide chain resin with fully protected side chains;

[0043] 2) Cleave the peptide chain resin with fully protected side chains with a cleavage reagent, cleave the peptide chain from the resin and remove the side chain protection group to obtain a crude polypeptide; the cleavage reagent is trifluoroacetic acid, phenol, water, The mixture that triisopropylsilane is formulated according to the volume ratio is 88:5:5:2;

[0044] 3) Dissolve the crude peptide in a mixture of dimethyl sulfoxide and water at a volume ratio of 2:8 to prepare...

Embodiment 3

[0057] In this example, the polypeptide obtained in Example 1 was used for animal experiments.

[0058] Materials and Methods:

[0059] (1) Grouping of animals: Select 50 healthy female Wistar rats, weighing 120-140 g, and randomly divide them into 5 groups, namely control group, model group, treatment groups I, II and III, with 10 rats in each group, and raise them in single cages.

[0060] (2) Animal model of allergic rhinitis: on the first day, 1 mL of normal saline, 1.5 mg of ovalbumin, 2 mg of aluminum hydroxide sol and 1×10 10 Each inactivated Bacillus pertussis mixture was injected subcutaneously into rats at 0.2 mL each at 5 points. On the fifth day, the mixture was injected subcutaneously again except for pertussis. Rats were challenged from the 14th day: intranasally with 1% ovalbumin saline solution, 30 μL in each nostril, once a day, 10 times in total. Modeling was successful if the behavior (number of sneezing and nose scratching) and symptoms (nasal secretions...

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Abstract

The invention discloses polypeptide, a preparation method and an application thereof. An amino acid sequence of the polypeptide is shown as SEQID NO:1. The polypeptide provided by the invention is a bioactive polypeptide extracted from skins of northern leopard frogs, is leucine-valine-arginine-glycine-cysteine-tryptophan-threonine-lysine-serine-tyrosine-proline-proline-lysine-proline-cysteine-phenylalanine-valine-arginine polypeptide, can penetrate in unilamellar lipid vesicles with negative charges, inhibit formation of eosinophilic granulocyte and granulocyte-macrophage colonies and reduce activity of eosinophilic granulocyte for forming precursor cells and mast cells, and achieves the object of treating allergic rhinitis and asthma syndrome by combining active centers of tryptase to inhibit the activity of protease.

Description

technical field [0001] The invention belongs to the technical field of medicine, specifically relates to a polypeptide, and also relates to a preparation method and application of the polypeptide. Background technique [0002] Allergic rhinitis and asthma syndrome refers to the simultaneous occurrence of clinical or subclinical allergic symptoms of the upper and lower respiratory tract, and the two often coexist at the same time. The immunological and pathological changes of the upper and lower respiratory tracts of allergic rhinitis and asthma are allergic inflammation in nasal mucosa and bronchial mucosa respectively, that is, inflammatory cell exudation, eosinophils, mast / basophils increase. At present, there is no specific drug for the treatment of this disease. For example, the treatment of asthma such as application of inhaled corticosteroids or β-2 stimulants can only relieve symptoms, and cannot cure or reverse the course of the disease. [0003] Tryptase is an infl...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/16C07K1/06A61K38/10A61P11/06A61P11/02A61P37/08
CPCY02P20/55
Inventor 刘宁翟仙敦王帅宝张学智
Owner TIANJIN BIOFEED TECH CO LTD
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