Bactrocerin, and preparation method and application thereof
A kind of fruit fly, antibacterial peptide technology, applied in biological field
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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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