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Antimicrobial peptide Hainanenin-5 of Amolops hainanensis, gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis, and application of gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis

A technology of hainanenin-5, 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: 2014-04-16
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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  • Antimicrobial peptide Hainanenin-5 of Amolops hainanensis, gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis, and application of gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis
  • Antimicrobial peptide Hainanenin-5 of Amolops hainanensis, gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis, and application of gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis
  • Antimicrobial peptide Hainanenin-5 of Amolops hainanensis, gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis, and application of gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024]Isolation and purification of Hainanenin-5: 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.

[0025] The first step Sephadex G-50 gel filtration chromatography: 10ml0.1M phosphate (Na 2 HPO 4 -NaH 2 PO 4 , pH 6.0) buffer solution, 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 solution, The flow rat...

Embodiment 2

[0029] Cloning and gene sequencing of antimicrobial peptide Hainanenin-5 precursor gene, including:

[0030] 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:

[0031] Forward SMARTer V Oligonucleotide: 5′-AAGCAGTGGTATCAACGCAGAGTACXXXXX-3′(X=undisclosed base in the proprietary SMARTer oligo sequence)

[0032] Reverse 3'In-Fusion SMARTer CDS Primer:

[0033] 5'-CGGGGTACGATGAGACACCATTTTTTTTTTTTTTTTTTTTTTVN-3' (N = A, C, G, or T; V = A, G, or C).

[0034] 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.

[0035] A specific forward primer (P1) and...

Embodiment 3

[0081] The chemical synthesis method of antimicrobial peptide Hainanenin-5:

[0082] 1. According to the deduced amino acid sequence of the mature peptide Hainanenin-5, its full sequence was synthesized with an automatic peptide synthesizer (Applied Biosystems), and desalted and purified by HPLC reverse-phase column chromatography.

[0083] 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 discloses novel antimicrobial peptide Hainanenin-5 of Amolops hainanensis, a gene of the antimicrobial peptide Hainanenin-5 of Amolops hainanensis, a separation method and a chemical synthesis method for the antimicrobial peptide Hainanenin-5 of Amolops hainanensis and application of the gene of the antimicrobial peptide Hainanenin-5 of Amolops hainanensis, and belongs to the technical field of biomedicine. The Hainanenin-5 is small peptide which is obtained by the steps of electrically stimulating the Amolops hainanensis so as to collect a skin exudate, centrifuging, freeze-drying, performing gel filtration column chromatography and reverse phase-high performance liquid chromatography (RP-HPLC) and purifying, and has a C terminal containing a seven-membered ring; the molecular weight is 2,337.91; and the isoelectric point is 10.11. A primary structure of the Hainanenin-5 is Phe<1>a<2>Leu<3>Gly<4>Ala<5>Val<6>Thr<7>Lys<8>Arg<9>Leu<10>Pro<11>Ser<12>Leu<13>Phe<14>Cys<15>Leu<16>Ile<17>Thr<18>Arg<19>Lys<20>Cys<21>. A gene for encoding a Hainanenin-5 precursor consists of 321 nucleotides, wherein a mature peptide part is encoded by 139th to 201st nucleotides. The Hainanenin-5 has low molecular weight and a simple structure, only contains one disulfide bond, and can be conveniently prepared through chemical synthesis and genetic engineering. The Hainanenin-5 is used for preparing a therapeutic medicine for pathogenic microorganism infectious diseases, has broad-spectrum antimicrobial activity, has the activities on gram-positive bacteria, gram-negative bacteria and fungi (including clinically separated medicine-resistant strains), and also has the advantages of low hemolysis.

Description

technical field [0001] The invention provides a broad-spectrum antimicrobial peptide Hainanenin-5 derived from Amolops hainanensis and its gene, separation and chemical synthesis method and application, belonging to the technical field of biomedicine. 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 effective for both local infection and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K1/20C07K1/16C12N15/12A61K38/17A61K48/00A61P31/00
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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