Rana grahami skin peptide, gene thereof and application in pharmacy

A technology of stinky frogs and frog skin, applied in the field of biomedicine, can solve the problems of limited research on skin active peptides, achieve the effects of inhibiting the growth of bacteria and fungi, wide antibacterial spectrum, and convenient artificial synthesis

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] 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Cloning of Peptide Gene of Fingerless Stink Frog:

[0038] I. Extraction of total RNA from the skin of the fingerless frog:

[0039]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,

[0040] 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.

[0041] 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.

[0042] 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 .

[0043] II. ...

Embodiment 2

[0119] Preparation of fingerless stinky frog frog skin peptide:

[0120] I, the preparation method of the stinky frog skin peptide of the fingerless disk: infer the amino acid sequence of the stinky frog skin peptide of the fingerless disk according to the gene encoding the stinky frog skin peptide of the fingerless disk, and synthesize its full sequence with an automatic polypeptide synthesizer. Reversed phase C by HPLC 18 Desalted and purified by column chromatography.

[0121] 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 1μA and the emission voltage is 25Kv.

[0122] III. The purity of the purified frog skin peptide was identified by high performance liquid chromatography (HPLC), the molecular weight was determined by fast atom bombardment mass spectrometry, the isoelectric...

Embodiment 3

[0125] The effect of anti-bacterial growth of the frog skin peptide of the fingerless stink frog:

[0126] 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, 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 0.1ml of the test compound solution with a concentration of 0.1-0.3mg / ml to the first steel cup, add the sample solution to the other steel cups by doubling dilution method, incubate at 37°C, and observe the size of the inhibition zone. The bacteriostatic zone above 10mm was regarded as the minimum inhibitory concentration (minimal inhibitory concent...

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Abstract

The invention discloses a non-finger frog skin 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 10.14, wherein the primary structure of peptide is Gly Leu Gly Gly Ala Lys Lys Asn Phe Ile Ile Ala Ala Asn Lys Thr Ala Pro Gln Ser Val Lys Lys Thr Phe Ser Cys Lys Leu Tyr Asn Gly; the gene of secretory peptide is composed of 731 nucleic acids, which codes 520-616th 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 a Rana Grahami frog skin peptide and its gene and application in pharmacy, belonging to the field of biomedicine. Background technique: [0002] Since the invention of antibiotics, humans have made great achievements in the control and treatment of microbial infection diseases. However, with the continuous use of "traditional antibiotics", many new problems continue to arise. At present, microbial resistance has become a major problem in the treatment of clinical microbial infection diseases, so that some pathogenic bacteria have no first-line drugs for clinical treatment. Vancomycin-resistant staphylococci, enterococci and other Gram-negative infectious diseases are currently clinical problems worldwide. Three types of bacteria that mainly cause meningitis have also emerged clinically with strong drug resistance. Penicillin-resistant , chloramphenicol, meningococci, pneumococci, and pneumococci resistant to neocephalosporin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46A61K38/17C12N15/12A61P31/00
Inventor 赖仞李建许李东升杨海龙
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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