Antifungal protein, method for preparing same and application thereof

An antifungal protein and seed technology, applied in the preparation method of peptides, botanical equipment and methods, applications, etc., can solve the problems of difficult amplification of the preparation system, retention, and inability to achieve mass production, and achieve high purification multiples and purification steps little effect

Inactive Publication Date: 2009-12-02
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing antifungal protein purification processes involve high-pressure liquid phase (HPLC). The disadvantage of this is that it is difficult to scale up the preparation system and cannot achieve mass production. The scale can only stay at the stage of micro-level preparation in the laboratory

Method used

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  • Antifungal protein, method for preparing same and application thereof
  • Antifungal protein, method for preparing same and application thereof
  • Antifungal protein, method for preparing same and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: Preparation of buckwheat seed antifungal protein

[0031] Naked oats seeds come from the naked oats production area of ​​Datong City, Shanxi Province. Take 100g of naked oats seeds harvested in the same year, dry them at low temperature, grind them in a seed grinder, pass through a 0.2mm aperture sieve, add 300ml of acetone, and store them at 4°C Stir skim for 3 hours. Suction filtration under reduced pressure, discard the acetone, collect the filter cake, add a small amount of ether to stir, vacuum suction filtration, discard the ether, collect the filter cake, dry at low temperature, and the obtained powder is oatmeal degreased powder.

[0032] Take 20g of oatmeal defatted powder, add 200ml of extraction buffer, the buffer formula is: 20mM Tris-HCl, pH 8.0. Stir and extract at 4°C for 6 hours, centrifuge at 12000rpm to discard the precipitate, add ammonium sulfate to the supernatant to 80% saturation, stir and salt out at 4°C for 6 hours, centrifuge at ...

Embodiment 2

[0038]Example 2 The effect of naked oat seed antifungal protein on the growth inhibition of Trichoderma viride and its dose-dependent experiment

[0039] Prepare the PDA medium, plant the test strain Trichoderma reesei in the center of the medium, cultivate it at 28°C until the diameter of the mold colony reaches 1-2cm, and place a sterilized Oxford cup on the periphery of the colony, 1cm away from the edge of the colony , the sample group (containing the target protein) and the control group (the buffer with the same conditions) were sterilized by filtration with a 0.22 μm microporous membrane, and 200 μl of the prepared protein sample or the same volume of buffer solution was added to the Oxford cup as a control, and covered with a ceramic Tile cover, pre-diffusion at 4°C for 24h, transfer the medium to a 28°C incubator and culture for 72h, then observe the growth of the colony.

[0040] Such as Figure 4 -A shows: set up four Oxford cups, among which 1 is the control group...

Embodiment 3

[0041] Example 3 The effect of oat seed antifungal protein on the growth inhibition of white rot fungus and its dose-dependent experiment

[0042] Prepare the PDA medium, plant the white rot fungus (Panus conchatus) of the test strain in the center of the medium, cultivate it at 28°C until the diameter of the mold colony reaches 1-2cm, and place a sterilized Oxford cup on the periphery of the colony, 1cm away from the edge of the colony , the sample group (containing the target protein) and the control group (the buffer with the same conditions) were sterilized by filtration with a 0.22 μm microporous membrane, and 200 μl of the prepared protein sample or the same volume of buffer solution was added to the Oxford cup as a control, and covered with a ceramic Tile cover, pre-diffusion at 4°C for 24h, transfer the medium to a 28°C incubator and culture for 72h, then observe the growth of the colony.

[0043] Such as Figure 4 -B shows: three Oxford cups are set up, among which 1...

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Abstract

The invention provides an antifungal protein, which is obtained by purifying a water soluble extract of hulless oat seed, wherein the apparent molecular weight of the antifungal protein is about 35kDa. The preparation method comprises the following steps: crushing and degreasing the hulless oat seed; extracting the hulless oat seed by buffer solution to obtain a coarse protein; and obtaining the antifungal protein by the two-step chromatographic separation which is the Resource S cation exchange and Superdex 75 gel exclusion chromatographic purification. The antifungal protein has obvious effect of inhibiting the growth of white rot fungus and trichoderma viride, and the minimum inhibition for the white rot fungus is 0.42mu g, and 0.21mu g for the thetrichoderma viride. In addition, the antifungal protein also has certain effect of inhibiting the growth of fusarium. The antifungal protein can be used as a food anticorrosion additive, a crop antifungal damage spraying agent and the like, and has high practical values in the aspects of food anticorrosion, crop prophylaxis and crop disease resistance.

Description

technical field [0001] The invention relates to a plant antifungal protein, in particular to a plant seed antifungal protein and its preparation method and application. Background technique [0002] Antifungal proteins and peptides (AFPs) in plant seeds are a protective mechanism acquired by seed plants during the long-term growth and evolution process, which enables seeds to have a certain ability to resist natural microbial erosion. The whole process of seed plant growth faces the threat of microorganisms. Among them, because fungi (especially plant pathogenic molds) have better reproductive ability, diffusion speed and environmental adaptability than bacteria, the main threat to plant seed storage comes from fungi. , similarly, usually plant seeds contain one or more antifungal proteins or antifungal peptides with a broad antibacterial spectrum. These natural plant antifungal peptides or antifungal proteins have a wide range of sources, significant antibacterial effects, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C07K1/18C07K1/16C07K1/14A23L3/3472A01N65/44A01P3/00
Inventor 王转花白承之李玉英
Owner SHANXI UNIV
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