Duttaphrynus melanostictus antimicrobial peptide as well as gene and application thereof

A technology of antimicrobial peptides and toads, applied in the field of biomedicine, can solve problems such as unknown sequence functions of protein and polypeptide components, and achieve the effect of convenient artificial synthesis, simple structure, and strong antibacterial activity

Active Publication Date: 2014-10-29
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sequence and specific function of the polypeptide components of the protein are still unknown

Method used

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  • Duttaphrynus melanostictus antimicrobial peptide as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: the cloning of bufo antibacterial peptide gene

[0034] 1. Extraction of total RNA from toad skin:

[0035] A, the living body black-orbited toad was cleaned with water, put into liquid nitrogen and quick-frozen for 4 hours, took skin tissue, weighed, got 300 mg skin tissue, added 10 ml total RNA extraction buffer (Trizol solution, product of Invitrogen Company of the United States), Homogenize in a 20 ml glass homogenizer for 10 minutes;

[0036] B. Add an equal volume of phenol / chloroform solution, oscillate and mix well, place at room temperature for 10 minutes, centrifuge at 12000rpm at 4°C for 10 minutes, and absorb the upper aqueous phase liquid;

[0037] 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 precipitate at the bottom of the tube is the total RNA of toad skin. .

[0038] 2. Synthesis o...

Embodiment 2

[0096] Embodiment 2: the application of antimicrobial peptide of Bufo melanogaster

[0097] One, the preparation method of the antibacterial peptide of Bufo melanogaster

[0098] The amino acid sequence of bufo bufo antimicrobial peptide was deduced according to the gene encoding antimicrobial peptide of bufo bufo. The whole sequence was synthesized by automatic peptide synthesizer, desalted and purified by HPLC reverse phase C18 column chromatography.

[0099] 2. Quality Evaluation and Molecular Weight Determination of Bufo Bufo Antimicrobial Peptides

[0100] The purity of the purified bufo 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, the amino acid sequence structure was determined by an automatic amino acid sequencer for confirmation.

[0101] 3. Detection of pharmacological ac...

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Abstract

The invention discloses a duttaphrynus melanostictus antimicrobial peptide and a gene thereof. The amino acid sequence of the duttaphrynus melanostictus antimicrobial peptide is shown as SEQ ID NO: 2, the nucleotide sequence of the gene for coding the duttaphrynus melanostictus antimicrobial peptide is shown as SEQ ID NO: 1, and the duttaphrynus melanostictus antimicrobial peptide obtained by a solid-phase polypeptide synthesis means has strong antimicrobial activity on multiple microbes including clinically isolated multi-drug tolerance pathogens, has a quick antimicrobial effect and does not have obvious hemolytic activity. The duttaphrynus melanostictus antimicrobial peptide has the characteristics of convenience in in-vitro production, strong antimicrobial effect, quickness, safety and the like, so that the duttaphrynus melanostictus antimicrobial peptide can be used as a novel antimicrobial medicament.

Description

technical field [0001] The invention provides acquisition, synthesis and application of a bufo bufo antimicrobial peptide gene, which belongs to the technical field of biomedicine. Background technique [0002] Black-orbited toad, Anura, Bufoidae, Bufo genus, is one of the main sources of traditional toad-like Chinese medicinal materials (toad venom, dried toad, toad skin and toad clothing). In recent years, a large number of studies have shown that toad venom has a wide spectrum of pharmacological activities, such as anti-tumor, cardiac strengthening, blood pressure boosting, stimulating breathing, antitussive and asthma-relieving, diuretic, stimulating smooth muscle, anti-platelet aggregation, anti-inflammation, enhancing immunity and local anesthetics, etc. effect. In the 1980s, toad venom was used clinically for the treatment of malignant tumors, various infectious diseases, chronic hepatitis, osteomyelitis, peripheral facial paralysis, and anesthesia for pain relief. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C12N15/12A61K38/17A61P31/04
CPCA61K38/00C07K14/463
Inventor 宋玉竹王梅孙鹥刘娃张阿梅韩芹芹张金阳
Owner KUNMING UNIV OF SCI & TECH
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