Hoplobatrachus rugulosus proteinase inhibition peptide and its gene and application of inhibition peptide in pharmacy

A technology of protease inhibition and tiger frog, which is applied in the field of biomedicine, can solve the problems of little research on skin pharmacological active substances, achieve the effects of inhibiting pancreatic enzyme activity and anti-oxidation, strong activity, and convenient artificial synthesis

Active Publication Date: 2017-03-08
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few studies on its skin pharmacological active substances

Method used

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  • Hoplobatrachus rugulosus proteinase inhibition peptide and its gene and application of inhibition peptide in pharmacy
  • Hoplobatrachus rugulosus proteinase inhibition peptide and its gene and application of inhibition peptide in pharmacy
  • Hoplobatrachus rugulosus proteinase inhibition peptide and its gene and application of inhibition peptide in pharmacy

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, tiger frog protease inhibitory peptide gene cloning:

[0032] 1, tiger frog skin total RNA extraction: the living tiger frog is cleaned with water, put into liquid nitrogen and quick-frozen for 4h, get skin tissue, weigh, get 300mg skin tissue, add 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 tiger frog skin.

[0033] II. Purification of tiger frog skin mRNA: the separation and purification of tiger frog skin mRNA was carried ...

Embodiment 2

[0045] Embodiment 2, preparation of tiger frog protease inhibitory peptide:

[0046] Ⅰ. The preparation method of protease inhibitory peptide of tiger frog: deduce the amino acid sequence of functional mature multifunctional protease inhibitory peptide according to the gene of tiger frog protease inhibitory peptide, and synthesize the polypeptide with an automatic polypeptide synthesizer. Desalted and purified by HPLC reverse phase C18 column chromatography. 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. During purification, liquid A is 0.05% TFA+2% CH3CN, liquid B is 0.05% TFA+90% CH3CN, the concentration gradient of liquid B is 25-40% within 15 minutes, the detection wavelength is 220nm, and...

Embodiment 3

[0050] Embodiment 3, activity test of tiger frog protease inhibitory peptide

[0051] Ⅰ. Determination of antibacterial ability

[0052] The antibacterial activity was detected by the cup-and-saucer method, the common agar medium was used for bacterial culture, and the modified Sabousand medium was used for fungal culture. Inject 20ml of heated and melted medium into the plate as the bottom layer, spread it evenly in the bottom of the plate, after solidification, take another appropriate amount of medium and heat and melt, add 5ml of bacterial suspension to each plate, shake well, Spread it evenly on the bottom layer as a bacterial layer. After cooling, put 6 sterilized stainless steel cups evenly in the plate at equal distances. Add 0.1 ml of the sample solution to be tested at a concentration of 0.3 mg / ml to the first steel cup, add the sample solution to the remaining steel cups by doubling the dilution method, incubate at 37°C, and observe the size of the inhibition zone...

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Abstract

The invention relates to an active polypeptide and its gene and an application of the inhibition peptide in pharmacy. The hoplobatrachus rugulosus proteinase inhibition peptide is cyclopeptide composed of 33 amino acids, a molecular weight is 3768.33 dalton, the isoelectric point is 6.303, and an amino acid sequence is SEQ ID NO.1, and a third cysteine and an eighth cysteine of the polypeptide form intramolecular disulfide bond. A gene sequence of the hoplobatrachus rugulosus proteinase inhibition peptide is composed of SEQ ID NO.4, wherein, the matured hoplobatrachus rugulosus proteinase inhibition peptide is 379th-477th site nucleotide. The hoplobatrachus rugulosus proteinase inhibition peptide has the characteristics of simple structure, convenient artificial synthesis, wide antibacterial spectrum, and strong activity. and can be taken as a medicine for treating pathogenic microorganism infectious diseases and a medicine having functions of beauty treatment and skin caring.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a protein obtained from animal tissues. Background technique [0002] The most basic function of serine protease inhibitors (serine protease inhibitor, serpin) is to prevent proteolysis and regulate the hydrolysis balance of serine proteases. Through the regulation of serine proteases, serine protease inhibitors have important effects on the physiological and biochemical functions in organisms. For example, they are involved in blood coagulation, complement formation, fibrinolysis, protein folding, cell migration, cell differentiation, cell matrix remodeling, hormone formation and transport, intracellular proteolysis, blood pressure regulation, tumor suppression, and viral or parasitic pathogenicity. Formation and so on play an important role. Serine protease inhibitors regulate so many physiological processes that they have a wide range of clinical application values. For example, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C12N15/15A61K38/57A61K8/64A61P39/06A61P1/18A61P1/04A61P31/04A61P31/10A61Q19/00A61Q19/08
CPCA61K8/64A61K38/00A61Q19/00A61Q19/08C07K14/81Y02A50/30
Inventor 徐学清陈新曾白霜张贝
Owner SOUTHERN MEDICAL UNIVERSITY
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