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A hylarana guentheri secretory peptide, a gene thereof and applications of the secretory peptide in pharmacy

A technology for secreting peptides and marsh water frogs, applied in the field of biomedicine, can solve the problem of less research on skin active peptide substances, and achieve the effect of convenient artificial synthesis, wide antibacterial spectrum and inhibiting bacteria.

Active Publication Date: 2017-03-15
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The application of amphibian drugs has a long history in my country, but the research on its active ingredients and pharmacological properties mainly focuses on small organic molecules such as alkaloids, and there are not many studies on its skin active peptides

Method used

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  • A hylarana guentheri secretory peptide, a gene thereof and applications of the secretory peptide in pharmacy
  • A hylarana guentheri secretory peptide, a gene thereof and applications of the secretory peptide in pharmacy
  • A hylarana guentheri secretory peptide, a gene thereof and applications of the secretory peptide in pharmacy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1, the cloning of the secretory peptide gene of the marsh water frog:

[0030] 1. Extraction of total RNA from the skin of marsh water frog: the live marsh water frog was cleaned with water, put into liquid nitrogen and quick-frozen for 4h, got skin tissue, weighed, got 300mg skin tissue, added 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.

[0031] II. Purification of marsh water frog skin mRNA: the separation and purification of marsh water frog skin mRNA was carried out by the American PROMEG...

Embodiment 2

[0043] Example 2, preparation of secreted peptide from marsh water frog:

[0044] Ⅰ. The preparation method of the secreted peptide of the marsh water frog: according to the gene of the secreted peptide of the marsh water frog, the amino acid sequence of the mature living 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 polypeptide appears at 15 minutes, the concentration of solution B is 32-47%, and the detection wavelength is 220nm.

[0045] Ⅱ. The molecular weight is ...

Embodiment 3

[0048] Embodiment 3, the activity test of the secreted peptide of marsh water frog

[0049] Ⅰ. Determination of the ability to inhibit bacterial growth

[0050] The antibacterial activity was detected by the cup and saucer method, and the medium was ordinary agar medium. Inject 20m1 of heated and melted culture medium into the plate as the bottom layer, spread it evenly in the bottom of the plate, after solidification, take another appropriate amount of culture medium and heat it to melt, then add 5m1 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 test compound solution at a concentration of 0.1-0.3 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. The bacteriostatic zone above...

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Abstract

The invention relates to a hylarana guentheri secretory peptide, a gene thereof and applications of the secretory peptide in pharmacy. The secretory peptide is an annular polypeptide formed by 43 amino acids. The amino acid sequence of the secretory peptide is shown as SEQ ID NO.1, and cysteine at the number 37 site and cysteine at the number 43 site form an intramolecular disulfide bond. The gene sequence of the secretory peptide is shown as SEQ ID NO.4, wherein the number 133 to number 261 nucleotides encode a functional mature hylarana guentheri secretory peptide. A mature functional polypeptide amino acid sequence is induced from the gene of the hylarana guentheri secretory peptide. The synthesized hylarana guentheri secretory peptide has high anti-bacterial, anti-influenza virus and antioxidant functions. The hylarana guentheri secretory peptide has characteristics of a simple structure and convenient artificial synthesis, and can be applied in medicines treating pathogenic microorganism infectious diseases and diseases caused by radical oxidation, beautifying and skincare medicines and food additives.

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] The discovery of penicillin has enabled people to deal with related diseases caused by various pathogenic microbial infections, and various antibiotics derived from it have made great contributions to the protection of human health. However, due to the long-term use and abuse of these "traditional antibiotics", many bacteria have developed resistance to them. The emergence of multidrug-resistant superbugs has made the development of new antimicrobial drugs particularly urgent. Antimicrobial peptides are a class of active polypeptides with a molecular weight of less than 10,000, composed of 10-50 amino acids, and capable of killing or inhibiting the growth of microorganisms. The latest research shows that antimicrobial peptides not only have broad-spectrum antimicrob...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C12N15/12A61K38/17A61P31/04A61P31/10A61P31/12A61P39/06A61P1/04A61P1/18A61P25/16A23L29/00
CPCA23V2002/00A61K38/00C07K14/463A23V2250/55Y02A50/30
Inventor 徐学清曾白霜柴金为
Owner SOUTHERN MEDICAL UNIVERSITY
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