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Amolops hainanensis antimicrobial peptide Hainanenin-1, and gene, separation purification, chemical synthesis and application thereof

A technology of hainanenin-1, an antimicrobial peptide, applied in the field of biomedicine to achieve the effect of small molecular weight, simple structure and strong antimicrobial activity

Active Publication Date: 2013-06-26
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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 few reports on the research on its skin active peptides

Method used

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  • Amolops hainanensis antimicrobial peptide Hainanenin-1, and gene, separation purification, chemical synthesis and application thereof
  • Amolops hainanensis antimicrobial peptide Hainanenin-1, and gene, separation purification, chemical synthesis and application thereof
  • Amolops hainanensis antimicrobial peptide Hainanenin-1, and gene, separation purification, chemical synthesis and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Isolation and purification of Hainanenin-1: Adult Hainannensis frogs (A.hainanensis) (n=5) in good health were selected, and their backs were rinsed with ultrapure water. The skin secretions were collected by electric stimulation method, and the skin on the back of the frog was electrically stimulated with an electric shocker, the voltage was I0V, and the electric shock time was 3ms. After the stimulation, the skin secretions were washed with 0.9% normal saline into a clean container, and centrifuged at 12000rpm After 20 min, the supernatant was discarded, freeze-dried and stored. Store at -20°C for later use.

[0022]The first step Sephadex G-50 gel filtration chromatography: 10ml0.1M phosphate (Na 2 HPO 4 -NaH 2 PO 4 , pH 6.0) buffer was dissolved, centrifuged at 12000rpm for 10min, and the supernatant was loaded onto a balanced Sephadex G-50 gel exclusion chromatography column (1.6cm x 90cm, Amersham Bioscience), and eluted with the same buffer, The flow rate is...

Embodiment 2

[0026] Cloning and gene sequencing of antimicrobial peptide Hainanenin-1 precursor gene, including:

[0027] AxyPrepTM Multisource Total RNA Miniprep Kit was used to extract total RNA from the skin of Amolops hainanensis, and the Creator TM SMART TM The cDNA library construction kit was used to construct the skin cDNA library of Amolops hainanensis. Reverse Transcriptase reverse transcription synthesizes the first strand of cDNA, the primers are:

[0028] Forward SMARTer V Oligonucleotide: 5′-AAGCAGTGGTATCAACGCAGAGTACXXXXX-3′(X=undisclosed base in the proprietary SMARTer oligo sequence)

[0029] Reverse 3'In-Fusion SMARTer CDS Primer:

[0030] 5'-CGGGGTACGATGAGACACCATTTTTTTTTTTTTTTTTTTTTTVN-3' (N = A, C, G, or T; V = A, G, or C).

[0031] Use Advantage DNA Polymerase to synthesize the second strand, and the primer is: forward 5'-primer: 5'-AAGCAGTGGTATCAACGCAGAGT-3'. The reverse primer is also 3'In-Fusion SMARTer CDS Primer.

[0032] A specific forward primer (P1) and...

Embodiment 3

[0077] The chemical synthesis method of antimicrobial peptide Hainanenin-1:

[0078] 1. According to the deduced amino acid sequence of the mature peptide Hainanenin-1, its full sequence was synthesized with an automatic peptide synthesizer (Applied Biosystems), and desalted and purified by HPLC reverse-phase column chromatography.

[0079] 2. The molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

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Abstract

The invention relates to an Amolops hainanensis antimicrobial peptide Hainanenin-1, and a gene, separation purification, chemical synthesis and application thereof, belonging to the technical field of biomedicine. The Hainanenin-1 is a small peptide containing heptatomic ring at the C terminal, which is prepared by the following steps: electrically stimulating Amolops hainanensis, collecting the skin secretion, centrifuging, freeze-drying, carrying out Sephadex column chromatography and RP-HPLC (reversed phase-high performance liquid chromatography), and purifying. The molecular weight of the Hainanenin-1 is 2278.90, and the isoelectric point is 9.50. The primary structure is phenylalanine-alanine-leucine-glycine-alanine-valine-threonine-lysine-leucine-leucine-proline-serine-leucine-leucine-cysteine-methionine-isoleucine-threonine-arginine-lysine-cysteine. The precursor gene for coding the Hainanenin-1 is composed of 320 nucleotides, and the part for coding the mature peptide is 139th-201st nucleotides. When being used for preparing therapeutic drugs for pathogenic microbe infectious diseases, the Hainanenin-1 has the characteristics of broad-spectrum antimicrobial activity and low hemolysis.

Description

technical field [0001] The invention provides a broad-spectrum antimicrobial peptide Hainanenin-1 derived from Amolops hainanensis and its gene, separation and chemical synthesis methods and application in the field of biopharmaceuticals, belonging to the field of biomedicine technology. Background technique [0002] With the widespread and extensive use of antibiotics, the resistance of pathogenic microorganisms to antibiotics has gradually increased, which has become a difficult point in the treatment of infectious diseases. Microbial agents. Recent studies have found that peptide antibiotics have broad-spectrum antibacterial activity, and at the same time have incomparable advantages over "traditional antibiotics": such as rapid and broad-spectrum killing of microorganisms (including current clinical drug-resistant bacteria) at the minimum effective concentration; It also has inhibitory effect on fungi; it will not induce the production of drug-resistant strains; it is e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C07K1/36C07K1/20C07K1/16C12N15/12A61K38/17A61P31/04A61P31/10C12Q1/18
Inventor 于海宁王义鹏冯菲菲广慧娟何伟玉
Owner DALIAN UNIV OF TECH
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