Rana nigromaculata antibacterial peptide and gene and application thereof

An antimicrobial peptide and black spot technology, which is applied in the field of biomedicine, can solve the problem of few antibacterial peptides in the black spotted frog, and achieve the effect of strong antibacterial activity, convenient artificial synthesis, and broad-spectrum antibacterial activity

Inactive Publication Date: 2011-11-02
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

Black-spotted frog is a kind of traditional Chinese medicinal material in my country. It is widely distributed in East Asia. Its wide distribution may be related to its strong ability to adapt to the environment. There are few reports on the antimicrobial peptide of black-spotted frog.

Method used

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  • Rana nigromaculata antibacterial peptide and gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 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, the skin tissue was taken, weighed, 300 mg of skin tissue was taken, and 10 ml of total RNA extraction buffer (Trizol solution, product of Invitrogen, USA) was added to 20 ml 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...

Embodiment 2

[0076] Example 2 Preparation and Pharmacological Experiments of Antimicrobial Peptides 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 antibacterial peptide of the black-spotted frog according to the gene encoding the antibacterial peptide of the black-spotted frog, synthesize the whole sequence with an automatic peptide synthesizer, and perform HPLC reversed-phase C18 column chromatography Desalting and purification.

[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), and finally determined by an automatic amino acid sequencer Amino acid sequence structure for confirmat...

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Abstract

The invention relates to a rana nigromaculata antibacterial peptide and a gene and application thereof, belonging to the technical field of medical technology. The rana nigromaculata antibacterial peptide is a single-chain polypeptide coded by a gene of a rana nigromaculata amphibious animal in China and has molecular weight of 1495.85D and isoelectric point of 9.0; and complete sequence of the rana nigromaculata antibacterial peptide is valine-isoleucine-proline-isoleucine-valine-serin-glycine-leucine-leucine-serin-serin-leucine-leucine-glycine-lysine. A coding gene of the rana nigromaculata antibacterial peptide is composed of 333 nucleotides, wherein nucleotides No 142-186 are used for coding a mature part. The artificially synthesized rana nigromaculata antibacterial peptide has strong antibacterial action and high safety, also has the advantages of simple sequence and convenience for synthesis and can be applied to a novel antiinfection medicine.

Description

technical field [0001] The invention provides a black-spotted frog ( Rana nigromaculata ) antimicrobial peptide and its gene and application belong to the field of biomedical technology. Background technique [0002] In Chinese traditional medicine and ethnic medicine, many amphibians are used as medicinal materials and widely used, such as the Chinese toad ( Bufo gargarizans ), Bombina grandis ( Bombina maxima ) and black-spotted frogs ( Rana nigromaculata )Wait. The skin and viscera of these amphibians have a wide range of pharmacological activities and clinical curative effects. It has been reported that the pharmacological activities include broad-spectrum antibacterial, antitumor, antiviral, analgesic, immune regulation, etc., which have the value of biomedical industry development. At present, there is also a major problem, that is, in traditional Chinese medicine and ethnic medicine, medicinal materials are processed and applied in a holistic manner, and the co...

Claims

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Application Information

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