Application of domestic silkworm cocoon antifungal protease inhibitor in fungus prevention and preparation method of domestic silkworm cocoon antifungal protease inhibitor

A protease inhibitor, antifungal technology, applied in the direction of protease inhibitor, peptide preparation method, antifungal agent, etc., to achieve the effects of high stability, high activity and low production cost

Active Publication Date: 2015-12-23
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Silk protein is considered to have antibacterial, anti-oxidant and cell proliferation-promoting properties, indicating that there may be other biologically active proteins in silk, but their components and properties have not been reported yet.

Method used

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  • Application of domestic silkworm cocoon antifungal protease inhibitor in fungus prevention and preparation method of domestic silkworm cocoon antifungal protease inhibitor
  • Application of domestic silkworm cocoon antifungal protease inhibitor in fungus prevention and preparation method of domestic silkworm cocoon antifungal protease inhibitor
  • Application of domestic silkworm cocoon antifungal protease inhibitor in fungus prevention and preparation method of domestic silkworm cocoon antifungal protease inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, the separation method of silkworm silkworm cocoon antifungal protease inhibitor

[0021] Divide silkworm cocoons into 6 layers, then cut them into small pieces, sterilize with ultraviolet light overnight, add appropriate amount of 100mM Tris-HCl (pH7.5) buffer solution, place at 37°C, and extract in a shaker at 220rpm for 30min; Then centrifuge at room temperature (18-25° C.) at 10,000 rpm for 15 minutes, take the supernatant, filter it with a 0.22 μm filter membrane, and store it at -20° C.

[0022] Using the BCA method, taking bovine serum albumin as a standard, measure the absorbance of the extracted protein at 595nm, and then calculate according to the standard curve to measure the concentration of cocoon layer 1 (innermost layer) to cocoon layer 6 (outermost layer). Sample concentrations were 0.79, 1.13, 1.09, 0.90, 0.83 and 0.97 mg / mL.

[0023] The protein extracted from the outermost cocoon was identified by mass spectrometry using Thermo Scientifi...

Embodiment 2

[0027] Embodiment 2, measure the inhibitory activity of the fungal protease K of silkworm silkworm cocoon antifungal protease inhibitor

[0028] The silkworm cocoon antifungal protease inhibitor is tested for the inhibitory activity of fungal proteinase K, and the specific steps are as follows:

[0029] (1) After the 1-6 layers of silkworm cocoon antifungal protease inhibitors (5 μg each) extracted in Example 1 were subjected to non-denaturing polyacrylamide gel electrophoresis, the gel was placed in a 0.07% proteinase K solution ( 100mM Tris-HCl, pH 7.5), 37°C, 60rpm shaker conditions, shake and incubate in the dark for 20min;

[0030] (2) Remove the proteinase K solution, wash the protein glue twice with MilliQ water, and let stand at room temperature for 15 minutes;

[0031] (3) Add mass fraction of 0.1% dianisidine staining solution (pH7.5, dissolved in 100mM Tris-HCl buffer) and 0.2% N-acetyl-D, L-phenylalanine-β-aphthylester matrix (formed by N , N'-dimethylformamide d...

Embodiment 3

[0034] Example 3, Determination of the inhibitory activity of silkworm cocoon antifungal protease inhibitors to spore germination of Beauveria bassiana

[0035] Determination of the inhibitory activity of silkworm cocoon antifungal protease inhibitors on spore germination of Beauveria bassiana, the specific method is as follows:

[0036] (1) Beauveria bassiana was inoculated on the PDA solid medium, and cultivated in a constant temperature incubator at 26°C for 14 days;

[0037] (2) Add Tween-80 with a mass fraction of 0.05% after autoclaving, scrape the spores, vortex to suspend the spores, and use sterilized cotton to filter the spore suspension twice to remove mycelium;

[0038] (3) Centrifuge the effluent at 4°C and 6000g for 10min, discard the supernatant, add MilliQ water to wash the spores, and centrifuge at 4°C and 6000g to discard the supernatant;

[0039] (4) Store the spores in sterilized MilliQ water, count them on a hemocytometer, and store them at 4°C for later ...

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Abstract

The invention discloses an application of a domestic silkworm cocoon antifungal protease inhibitor in fungus prevention and a separation method of the domestic silkworm cocoon antifungal protease inhibitor. The separation method comprises the following steps: layering and cutting a silkworm cocoon into pieces, then adding 100mm of Tris-HCL buffer solution with pH of 7.5, and extracting for 30 minutes under the condition that the temperature is 37 DEG C and the rotation speed is 220 r / pm, centrifuging for 15 minutes under the rotation speed of 10000rpm, filtering supernatant by utilizing a 0.22-micrometer filter membrane, to obtain the domestic silkworm cocoon antifungal protease inhibitor. The prepared protease inhibitor can inhibit the activity of fungal protease secreted by Tritirachium album limber, can inhibit the spore germination of silkworm pathogenic fungal beauveria bassiana and still can maintain the activity after being incubated for 10 minutes in boiled water. The prepared domestic silkworm cocoon antifungal protease inhibitor belongs to the natural, high-activity and high-stability antifungal protease and is non-toxic and harmless; and meanwhile, the silk used in the method is rich in resource, and a novel way is provided for diversified development of the silk.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of the silkworm cocoon antifungal protease inhibitor in the preparation of antifungal medicine, and also relates to a method for isolating the silkworm cocoon antifungal protease inhibitor and a product obtained by the method. Background technique [0002] my country is the largest country in silk production in the world. The annual output of silkworm cocoons is several hundred thousand tons, and the output value of silk industry is tens of billions. Silk products account for more than 70% of the world's total. However, due to the influence of the domestic silk weaving industry's backward technology and technology, my country's silk industry has low technical content and low added value. Therefore, it is of great significance to develop the application field of silk and improve the application value of silk by conducting in-depth research on silk and diggi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81A61K38/57A61P31/10C07K1/34
CPCA61K38/57C07K14/8121
Inventor 赵萍董照明郭晓朦张艳夏庆友
Owner SOUTHWEST UNIV
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