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Rana grahami secretory peptide, gene thereof and application in pharmacy

A technology for secreting peptides and stinky frogs, applied in the field of biomedicine, can solve the problem of little research on skin active peptide substances, and achieve the effects of inhibiting the growth of bacteria and fungi, convenient artificial synthesis, 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] 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

[0027] Cloning of secretory peptide gene of the fingerless frog:

[0028] I. Extraction of total RNA from the skin of the fingerless frog:

[0029] A. The live fingerless frog is cleaned with water, put into liquid nitrogen and quick-frozen for 4 hours, get the skin tissue, weigh, get 300mg skin tissue, add 10ml total RNA extraction buffer (Trizol solution, U.S. GIBCOBRL company product), Homogenize for 30 minutes in a 20ml glass homogenizer.

[0030] 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.

[0031] 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 .

[0032] II. Purification of the skin mRN...

Embodiment 2

[0099] Preparation of fingerless frog secreted peptide:

[0100] I. The preparation method of the peptide secreted by the stinky frog: infer the amino acid sequence of the secreted peptide by the stinky frog according to the gene encoding the secreted peptide by the stinky frog, and synthesize its full sequence with an automatic polypeptide synthesizer. Reversed phase C by HPLC 18 Desalted and purified by column chromatography.

[0101] 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 bombardment particles, the current is 1μA and the emission voltage is 25Ky.

[0102] III. The purity of the purified secretory peptide of the stinky frog was identified by high performance liquid chromatography (HPLC), the molecular weight was determined by fast atom bombardment mass spectrometry, the isoelectric point was determined by isoe...

Embodiment 3

[0105] Inhibition of bacterial growth by peptides secreted by the stinkfrog:

[0106] 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 0.1 ml of the test compound solution with a concentration of 0.1-0.3 mg / ml to the first steel cup, add the sample solution to the other steel cups by doubling the 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 concentration, MIC). ...

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Abstract

The invention discloses a non-finger frog secretory peptide and gene and application in the drug in the biological medical domain, which codes a cyclic peptide with molecular weight at 1587.98 and isoelectric point at 9.7, wherein the primary structure of peptide is Phe Met Pro Ile Leu Ser Cys Ser Arg Phe Lys Arg Cys with disulfide bond in the seventh cysteine and thirteenth cysteine; the gene of secretory peptide is composed of 300 nucleic acids, which codes 138-177th 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 secretion peptide and its gene and its application in pharmacy, belonging to the field of biomedicine. Background technique: [0002] The discovery of penicillin enables people to deal with various 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. So people began to search for new antimicrobial drugs. Studies in recent years have found that polypeptide antibiotics not only have broad-spectrum antimicrobial activity, but also have incomparable advantages over "traditional antibiotics": for example, at very small concentrations, they can kill microorganisms (including Currently clinical drug-resistant strains); it also has inhibitory effect on fungi; it is difficult to ind...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10C12N15/12A61P31/00
Inventor 赖仞李建许李东升徐学清韩耀平
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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