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Anionic antibacterial peptide AcHy-a2 in bee venom as well as preparation method and application of anionic antibacterial peptide AcHy-a2

A technology of achy-a2 and antimicrobial peptides, applied in the field of biomedicine, can solve the problems of limiting the utilization of antibacterial properties, and achieve the effects of less drug resistance, simple structure, and less toxic and side effects

Active Publication Date: 2015-08-26
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reports on antimicrobial peptides in bee venom are basically about melittin, because most of the existing melittin has hemolytic effect, thus limiting the utilization of its antibacterial properties

Method used

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  • Anionic antibacterial peptide AcHy-a2 in bee venom as well as preparation method and application of anionic antibacterial peptide AcHy-a2
  • Anionic antibacterial peptide AcHy-a2 in bee venom as well as preparation method and application of anionic antibacterial peptide AcHy-a2
  • Anionic antibacterial peptide AcHy-a2 in bee venom as well as preparation method and application of anionic antibacterial peptide AcHy-a2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Preparation of honeybee venom polypeptide

[0040] 1. Honeybee Venom Extraction and Reductive Alkylation

[0041] The abdomen of 10 bees of the genus Apiscerana (Apiscerana) was dissected to extract the venom glands, and the venom was lysed with 8M urea (containing protease inhibitors). The protein concentration in the venom was determined to be 7.87mg / ml by Bradford method. The total protein amount was taken as 1mg, and DTT with a final concentration of 10mM was added, and reacted at 56°C for 1h. After reduction and cooling to room temperature, IAM with a final concentration of 55mM was added and reacted in a dark room at room temperature for 45min.

[0042] 2. Enrichment of venom peptides

[0043] The honeybee polypeptides treated above were used to enrich the polypeptides in the honeybee venom with a Strata-X C18 column. Strata-X C18 enrichment operation was performed according to standard procedures: 1) Add 1ml of methanol to activate the column; 2) ...

Embodiment 2

[0044] Embodiment 2 Identification of anionic antimicrobial peptide AcHy-a2 of the present invention

[0045] 1. Identification of honey bee polypeptide sequence

[0046] The enriched polypeptide obtained in Example 1 was lyophilized and reconstituted with 15 μl of 0.1% formic acid, and then analyzed by nanoLC-MS / MS.

[0047] 2. NanoLC-MS / MS analysis

[0048] The liquid-mass spectrometer adopts the nano HPLC chromatographic system of Shimadzu, Japan and the Q-Exactive mass spectrometer system of Thermo Fisher Scientific. The peptide liquid reconstituted in the previous step was separated by the built-in 12cm long, 75μm inner diameter, and Ultimate capillary analysis column filled with Welch Materials brand XB-C18 column material with a particle size of 3μm and a pore size of 120A, with a flow rate of 300nl / min. The detection injection volume is 12 μl, and the elution gradient is such that the B solution concentration rises uniformly from 5% to 30% over 40 minutes. The mass ...

Embodiment 3

[0053] Example 3 Bacteriostatic activity detection of bee venom anionic antimicrobial peptide AcHy-a2 of the present invention

[0054] The bee venom anionic antibacterial peptide AcHy-a2 synthesized in Example 2 was weighed, and the bactericidal activity of the antimicrobial peptide AcHy-a2 was detected by the microdilution method, and the broad-spectrum semi-synthetic antibiotic Amp and the aminoglycoside antibiotic Kana were used as positive controls , to evaluate the antibacterial activity of the antimicrobial peptide AcHy-a2 of the present invention.

[0055] The test steps are as follows: strain recovery, the standard strain Staphylococcus aureus [CMCC(B)26003-5a20] was inoculated in the nutrient broth medium, cultivated in a biochemical incubator at 37°C for 24 hours, and then purified by streaking on LB solid medium After culturing, take an inoculation loop and inoculate in nutrient broth medium, incubate at a constant temperature of 37°C for 6 hours, count with a bact...

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Abstract

The invention relates to anionic antibacterial peptide AcHy-a2 in bee venom. Anionic antibacterial peptide AcHy-a2 has an amino acid sequence as shown in SEQ ID NO:1 or is derived polypeptide having an inhibition effect on gram positive bacteria and formed by enabling the amino acid sequence as shown in SEQ ID NO:1 to be subjected to substitution, addition or deletion of one or more amino acids. The invention further relates to polynucleotide coding peptide, a construction body containing polynucleotide, an expression vector, a transformation cell as well as a preparation method and a medical application of peptide. Anionic antibacterial peptide AcHy-a2 in the bee venom can be extracted from natural animal resources, belongs to antibacterial peptides, has the advantages of high activity, small toxic or side effects, low possibility of generating drug resistance, no drug residues in bodies and the like in comparison with conventional antibacterial drugs, and can well solve a problem of increasingly enhanced drug resistance of the bacteria to the conventional antibacterial drugs due to simple structure and convenient artificial synthesis, thereby having a relatively good clinical application prospect.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to a new mee venom anionic antimicrobial peptide AcHy-a2, a polynucleotide encoding the peptide, a construct containing the polynucleotide, an expression vector and a host cell, the peptide Natural extraction and artificial synthesis methods, and the medical use of the peptide. Background technique [0002] Staphylococcus aureus is an important human pathogenic bacteria belonging to the genus Staphylococcus, which can cause a variety of serious infections. The Ministry of Health of the People's Republic of China formulated the "List of Pathogenic Microorganisms Infected Between Humans" and recorded the degree of harm of the bacteria as the third category (China Laboratory Medicine Network, 2010, 8). This bacterium is the most common pathogenic bacteria in human suppurative infection, which can cause local suppurative infection, pneumonia, pseudomembranous enteritis, per...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/14C12N15/12A61K38/10A61P31/04
Inventor 童婷林志龙闻博冯强
Owner SHENZHEN HUADA GENE INST
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