Rana nigromaculata antimicrobial peptide as well as gene and application thereof

An anti-microbial peptide, black spot technology, applied in the field of biomedicine, can solve the problems of unreasonable, serious antibiotics, tolerance, etc., and achieve the effects of simple structure, strong antibacterial activity, and broad-spectrum antibacterial activity

Inactive Publication Date: 2011-11-23
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But on the other hand, the widespread use of antibiotics, especially some irrational use, has also led to the increasingly serious phenomenon of antibiotic resistance

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Cloning of Antimicrobial Peptide Gene of Black-spotted Frog

[0021] 1. Extraction of total RNA from skin of black-spotted frog:

[0022] A. The living black-spotted frog was cleaned with water, put into liquid nitrogen and quick-frozen for 4 hours, got skin tissue, weighed, got 300mg skin tissue, added 10ml total RNA extraction buffer (Trizol solution, U.S. Invitrogen company product), in 20ml Homogenize in a glass homogenizer for 10 minutes.

[0023] B. Add an equal volume of phenol / chloroform solution, vortex and mix well, place at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, and absorb the upper aqueous phase liquid.

[0024] C. Add 1 / 2 volume of isopropanol to the supernatant, leave it at room temperature for 10 minutes, centrifuge at 7500g for 10 minutes at 4°C, wash the precipitate once with 75% ethanol, and dry it. The sediment at the bottom of the tube is the skin of the black-spotted frog total RNA.

[0025] 2. ...

Embodiment 2

[0076] Example 2 Preparation and Pharmacological Experiment of the Antimicrobial Peptide of Black-spotted Rana

[0077] 1. Preparation of antimicrobial peptides from black-spotted frog:

[0078] (1) Preparation method of the antimicrobial peptide of the black-spotted frog: deduce the amino acid sequence of the anti-microbial peptide of the black-spotted frog according to the gene encoding the anti-microbial peptide of the black-spotted frog, and synthesize the whole sequence with an automatic peptide synthesizer. Desalted and purified by HPLC reverse phase C18 column chromatography.

[0079] (2) The purity of the purified black-spotted frog antimicrobial peptide was identified by high-performance liquid chromatography (HPLC), and its precise molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Finally, an automatic amino acid sequencer was used Determine the amino acid sequence structure for confirmati...

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Abstract

The invention relates to a rana nigromaculata antimicrobial peptide as well as a gene and application thereof, and belongs to the technical field of biomedicine. The rana nigromaculata antimicrobial peptide is a gene coded single-chain polypeptide separated from an amphibian rana nigromaculata of China, wherein the molecular weight is 1,647.02 Daltons, and the isoelectric point is 11.05; and the complete sequence of the rana nigromaculata antimicrobial peptide is phenylalanine-isoleucine-proline-leucine-valine-serine-glycine-leucine-phenylalanine-serine-arginine-leucine-leucine-glycine-lysine. The coded gene of the rana nigromaculata antimicrobial peptide consists of 316 nucleotides, wherein the mature coded part of the coded gene contains 142nd-186th nucleotides. The artificially synthesized rana nigromaculata antimicrobial peptide has a strong antibacterial effect, has the advantages of high safety, simple sequence and convenience in synthesis, and can be used as a novel anti-infective agent for application.

Description

technical field [0001] The invention relates to a black-spotted frog antimicrobial peptide and its gene and application, belonging to the technical field of biomedicine. Background technique [0002] The discovery of antibiotics is one of the most important discoveries in the history of medicine, and its clinical application has greatly reduced the mortality rate of infectious diseases. But on the other hand, the widespread use of antibiotics, especially some irrational use, has also led to the increasingly serious phenomenon of antibiotic resistance. According to the statistics of the World Health Organization, after antibiotics are put into clinical application on a large scale, tolerance will generally appear within 1-5 years. In order to monitor the phenomenon of antibiotic resistance, Europe and Asia have successively established intercontinental antimicrobial resistance monitoring agencies, and my country has also established a corresponding testing agency - the Natio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/12A61K38/10A61P31/00
CPCY02A50/30
Inventor 宋玉竹孟庆雄余旭亚李涛
Owner KUNMING UNIV OF SCI & TECH
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