Citrus fruit fly antibacterial peptide, preparation and use thereof

A technology of Bactrocera dorsalis and antimicrobial peptides, applied in the fields of food science and cosmetics technology, biomedicine, Bacteralis dorsalis antimicrobial peptides and their preparation, field of agronomy

Inactive Publication Date: 2008-12-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The related technical research on the antibacterial active substances of Bactrocera dorsalis is of great significance, and no related technical reports have been published yet.

Method used

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  • Citrus fruit fly antibacterial peptide, preparation and use thereof
  • Citrus fruit fly antibacterial peptide, preparation and use thereof
  • Citrus fruit fly antibacterial peptide, preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: The preparation steps of Bacteralis dorsalis antimicrobial peptide are as follows:

[0035] (1) Bacteralis dorsalis pupae are induced by immunization with mixed bacterial solution;

[0036] Use No. 1 insect needle dipped in the mixed bacterial solution resuspended in physiological saline to acupuncture the middle part of Bacteralis dorsalis pupae; the physiological saline contains 150mM NaCl and 5mM KCl; the mixed bacterial solution is OD 600 A mixture of Staphylococcus aureus and Escherichia coli DH5α at 0.3.

[0037] (2) Bactrocera dorsalis pupal tissue homogenate to obtain a crude extract;

[0038] A certain amount of induced Bactrocera dorsalis pupae was weighed, and a corresponding volume of extraction buffer was added in a ratio of 1:4 to 1:6 by weight to volume. The extraction buffer was 50 mmol / L ammonium acetate and 1.5 μmol / L (final Concentration) protease inhibitor Aprotinin and 20μmol / L (final concentration) phenylthiourea, thoroughly ground ...

Embodiment 2

[0043] Example 2: Identification of Purified Bactrocera dorsalis Antimicrobial Peptides

[0044] The molecular weight of Bacteralis dorsalis antimicrobial peptide was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, and the isoelectric point was calculated by software analysis. The amino acid sequence structure of the antibacterial peptide was determined by automatic Edman degradation method protein sequencer.

[0045] The Bacteralis dorsalis antimicrobial peptide prepared by the examples is a polypeptide that we isolated from Bacteralis dorsalis pupa for the first time. Its molecular weight is 2.3 Daltons, and its isoelectric point is 11.43. The complete sequence of the polypeptide is: valine- Glycine-Lysine-Threonine-Tryptophan-Isoleucine-Lysine-Jeline-Isoleucine-Arginine-Glycine-Isoleucine-Glycine-Lysine-Serine - Lysine - Isoleucine - Lysine - Tryptophan - Glutamine.

Embodiment 3

[0046] Embodiment 3: Experiment of the inhibitory effect of Bacteralis dorsalis antimicrobial peptides on microorganisms

[0047] The purified Bactrocera dorsalis antimicrobial peptide was diluted with 1 / 2 times of sterilized distilled water. Take 90 μL of bacterial LB culture medium in the logarithmic growth phase, add 10 μL of Bacteralis dorsalis antimicrobial peptides at different concentrations, mix well, incubate overnight at 30°C for 24 hours on a 96-well plate, and measure each concentration at 600 nm with a microplate reader. The absorbance of the tube. The lowest concentration of antimicrobial peptides with no significant change in absorbance is the minimum inhibitory concentration (Minimal Inhibitory concentration, MIC). Fungi used 80 μL of fungal spores (10 4 Spores / ml) were carried out on potato agar medium, and 20 μL of Bacteralis antimicrobial peptides of different concentrations were added to each well, mixed evenly, incubated overnight at 25°C for 24 hours on...

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Abstract

The invention discloses a citrus fruit fly antibacterial peptide, which is a polypeptide obtained from separation of a citrus fruit fly pupa, with a molecular weight of 2.3 daltons and an isoelectric point of 11.43, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO: 1. The invention simultaneously discloses a method for preparing the citrus fruit fly antibacterial peptide, comprising the following steps that: the citrus fruit fly pupa is induced by adoption of a mixed bacteria solution; a crude extract is obtained through tissue homogenate; and the citrus fruit fly antibacterial peptide is obtained after purification through ion exchange chromatography, reversed fast protein liquid phase chromatography and reversed high pressure liquid phase chromatography of the crude extract. The citrus fruit fly antibacterial peptide provided by the invention has inhibition on gram positive bacteria, gram negative bacteria and fungi, has no hemolytic activity, can be taken as a raw material for developing human and poultry biotechnical products with pathogenic microorganism infection resistance, and can be taken as a disease-resistant gene to be used for breeding transgenic plants and transgenic animals by means of gene transformation and other biotechnologies.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Bactrocera dorsalis antibacterial peptide and a preparation method thereof, as well as applications in the technical fields of biomedicine, agronomy, food science and cosmetics. Background technique [0002] Since the discovery of antibiotics such as penicillin, a revolutionary leap has taken place in the prevention and treatment of pathogenic bacterial infectious diseases. At the same time, it was also realized that pathogenic microbes would eventually develop resistance to antibiotics such as penicillin, but it was surprising how quickly and broadly resistance developed. With the widespread use and abuse of antibacterial drugs, in less than 50 years, bacteria that were easy to kill began to have drug-resistant strains and multi-drug resistant strains. [0003] In order to deal with drug-resistant bacterial infections, people are constantly researching and developing ...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/14C07K1/18C07K1/20A61K38/10A61P31/00A01H1/00A01K67/00
Inventor 党向利温硕洋易辉玉杨婉莹邓小娟曹阳
Owner SOUTH CHINA AGRI UNIV
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