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Rana grahami anti-microorganism polypeptide, gene thereof and application in pharmacy

An anti-microbial peptide, stinky frog technology, applied in the field of biomedicine, can solve the problem of not much research on skin active peptide substances, and achieve the effect of inhibiting the growth of bacteria and fungi, strong antibacterial activity, and broad antibacterial spectrum.

Inactive Publication Date: 2007-08-08
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] my country has a long history of the application of amphibian drugs, but the research on its active ingredients and pharmacological properties mainly focuses on small organic molecules such as alkaloids, while there are not many studies on its skin active peptides

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Cloning of Antimicrobial Polypeptide Gene of Stink Frog:

[0033] I. Extraction of total RNA from the skin of the fingerless frog:

[0034] A. The live fingerless stink frog was cleaned with water, put into liquid nitrogen for quick freezing for 4 hours, and the skin tissue was taken,

[0035] Weigh, take 300 mg of skin tissue, add 10 ml of total RNA extraction buffer (Trizol solution, product of GIBCOBRL, USA), and homogenize in a 20 ml glass homogenizer for 30 minutes.

[0036] B. Add an equal volume of phenol / chloroform solution, mix vigorously, leave at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, and discard the precipitate.

[0037] C. Add an equal volume of isopropanol to the supernatant, leave it 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 in the air. The sediment at the bottom of the tube is the total RNA of the frog skin .

[00...

Embodiment 2

[0107] Preparation of the antimicrobial polypeptide of the stinkfrog:

[0108] I. The preparation method of the antimicrobial polypeptide of the stinky frog: deduce the amino acid sequence of the antimicrobial polypeptide of the stinky frog according to the gene encoding the stinky frog antimicrobial polypeptide, and synthesize its full sequence with an automatic polypeptide synthesizer. Reversed phase C by HPLC 18 Column desalting and purification.

[0109] II. The molecular weight is determined by fast atom bombardment mass spectrometry (FAB-MS), with glycerol: m-nitrobenzyl alcohol: dimethyl sulfoxide (1: 1: 1, V: V: V, volume ratio) as substrate, Cs + As the bombardment particles, the current is 1 μA, and the emission voltage is 25Kv.

[0110] III. The purity of the purified antimicrobial peptides of the fingerless frog was identified by high performance liquid chromatography (HPLC) and mass spectrometry, the molecular weight was determined by fast atom bombardment mass...

Embodiment 3

[0113] Pharmacological experiment of antimicrobial peptides of stinkfrog:

[0114] 1. The antimicrobial polypeptide of the fingerless frog to inhibit the growth of bacteria

[0115] The antibacterial activity was detected by the cup and saucer method, and the medium was ordinary agar medium. Inject 20ml of heated and melted medium into the plate as the bottom layer, and spread it evenly on the bottom of the plate. After solidification, take another appropriate amount of medium and heat it to dissolve. Add 5ml of bacterial suspension to each plate, shake well to make it on the bottom layer Spread evenly on the top as a bacterial layer. After cooling, put 6 sterilized stainless steel cups into the plate at equal distances. Add 0.1ml of the test compound solution at a concentration of 0.3mg / ml to the first steel cup, add the sample solution to the remaining steel cups by doubling dilution method, incubate at 37°C, and measure the size of the inhibition zone after 24 hours. A b...

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Abstract

The invention discloses a non-finger microbe-proof peptide and gene and application in the drug in the biological medical domain, which codes a single-chained peptide with molecular weight at 3355.95 and isoelectric point at 11.47, wherein the primary structure of peptide is Gly Leu Phe Thr Leu Ile Lys Gly Ala Ala Lys Leu Ile Gly Lys Thr Val Pro Lys Lys Gln Ala Arg Leu Gly Met Asn Leu Trp Leu Val Lys Leu Pro Thr Asn Val Lys Thr (GLFTLIKGAAKLIGKTVPKKQARLGMNLWLVKLPTNVKT); the gene of secretory peptide is composed of 343 nucleic acids, which codes 123-240th nucleic acid; the peptide possesses obvious bacteria and fungi inhibiting action, which can be applied to make microbe infection disease drug.

Description

Technical field: [0001] The invention relates to an antimicrobial polypeptide of Rana grahami and its gene and its application in pharmacy, belonging to the field of biomedicine. Background technique: [0002] In traditional Chinese medicine and ethnic medicines, many amphibians, such as Chinese toad (Bufogargarizans), large-webbed bell toad (Bombina maxima), black-spotted frog (pelophylax nigromaculata), marsh frog (Hylarana guentheri) and marsh frog (Euphlyctis limnocharis) etc. are used as medicines. The skin and viscera of these amphibians have a wide range of pharmacological activities and clinical efficacy. Pharmacological activities have been reported to include broad-spectrum antimicrobial effects, antitumor effects, analgesia, local anesthesia, immune regulation, and cardiovascular system effects. In foreign countries, the search for specific pharmacologically active monomeric compounds in amphibian skin has become a hot spot in the invention of new drugs. Accord...

Claims

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Application Information

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IPC IPC(8): C07K14/46A61K38/17C12N15/12A61P31/00
Inventor 赖仞李建许王义鹏李东升
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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