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Bactrocerin, and preparation method and application thereof

A kind of fruit fly, antibacterial peptide technology, applied in biological field

Inactive Publication Date: 2017-07-25
四川科劲生物科技有限公司
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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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  • Bactrocerin, and preparation method and application thereof
  • Bactrocerin, and preparation method and application thereof
  • Bactrocerin, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: The preparation steps of Bacteralis dorsalis antimicrobial peptide are as follows:

[0034] (1) Bacteralis dorsalis pupae are induced by immunization with mixed bacterial solution;

[0035] 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; Coccus and Escherichia coli DH5α mixture.

[0036] (2) Bactrocera dorsalis pupal tissue homogenate to obtain a crude extract;

[0037] 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 on ice bath, 4 ℃, 10,000 × g centrifugation for 30 to 35 minutes, ...

Embodiment 2

[0042] Example 2: Identification of Purified Bactrocera dorsalis Antimicrobial Peptides

[0043] 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.

[0044] 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

[0045] Embodiment 3: Experiment of the inhibitory effect of Bacteralis dorsalis antimicrobial peptides on microorganisms

[0046] 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). The fungus was carried out on potato agar medium with 80 μL of fungal spores (104 spores / ml), and 20 μL of Bacteralis dorsalis antimicrobial peptides of different concentrations were added to each well, mixed evenly, and incubated overnight at 25°C ...

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Abstract

The invention discloses a bactrocerin, and a preparation method and an application thereof. The bactrocerin is a polypeptide separated from pupas of Bactrocera dorsalis, the molecular weight is 2.3 Dalton, the isoelectric point is 11.43, and the amino acid sequence of the bactrocerin is represented by SEQ ID NO:1. The invention also discloses the preparation method of the antimicrobial peptides of the bactrocerin. The method comprises the following steps: inducing the pupas of Bactrocera dorsalis with a mixed bacterial liquid, carrying out tissue homogenization to obtain a crude extract, and carrying out ion exchange chromatography, reversed phase fast protein liquid chromatography and reversed phase high pressure liquid chromatography purification on the crude extract to prepare the bactrocerin. The bactrocerin has an inhibition effect on Gram-positive bacteria, Gram-negative bacteria and fungi, has no hemolytic activity, can be used as a raw material for developing biotechnological products for preventing pathogenic organism infection of humans and animals, and also can be used in transgenic plants and transgenic animal breeding as an anti-disease gene through transgene 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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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08A61K38/10A61P31/04A61P31/10A01N63/02A01P1/00A01P3/00
CPCA01N63/10A61K38/00C07K7/08
Inventor 林丹
Owner 四川科劲生物科技有限公司
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