Separating and enriching method of nosema bombycis sporoplasm

A technology for separation and enrichment of microsporidia, which is applied in the field of microbial separation to achieve the effect of convenient operation

Inactive Publication Date: 2019-05-03
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is still no report on the method for the separation and purification of the spore plasma of No. spp.

Method used

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  • Separating and enriching method of nosema bombycis sporoplasm
  • Separating and enriching method of nosema bombycis sporoplasm

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Embodiment

[0029] A method for separating and enriching silkworm microsporidia spore protoplasm, the specific steps are as follows:

[0030] 1. Centrifuge the spores treated with 0.1M KOH at 3000rmp / min for 5min, discard the supernatant;

[0031] 2. Add 1mL TC100 insect culture medium to suspend the spore pellet, and place it on a rotary shaker for 10 minutes at room temperature;

[0032] 3. Take 5uL of the spore sample suspended in the culture medium, stain the cell nucleus with the live cell dye Hochest, and then use the laser confocal fluorescence microscope to observe the sporoplasm ejected from the polar tube, and count the germination rate of the spore;

[0033] 4. When the spore germination rate reaches 60%, use isotonic stable percoll solution (purchased from GE Healthcare company) to prepare percoll with 30% and 60% density to prevent the sporoplasm from being destroyed in centrifugation, and spore samples after germination Add to two density gradient percoll solutions, use a h...

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Abstract

The invention relates to a separating and enriching method of nosema bombycis sporoplasm. The separating and enriching method comprises the following specific steps of (1) treating nosema bombycis spores with a 0.1M KOH aqueous solution, performing centrifugation, removing supernatant, adding TC100 insect culture medium suspension spore precipitate, and performing incubation to obtain culture medium suspension spore samples; (2) absorbing the spore samples obtained in the step (1), performing cell nucleus dyeing, observing the situation of sporoplasm ejected from a polar tube, and counting thesprouting rate of the spores; (3) when the sprouting rate of the spores reaches 60%, adding the sprouted spore samples into a percoll solution having two density gradients of 30% and 60%, and performing density gradient centrifugal separation to obtain sporoplasm; and (4) collecting the percoll solution at 30% density layer of an upper layer, and performing cleaning and centrifugation. The separating and enriching method disclosed by the invention is convenient to operate, besides, the sporoplasm can be efficiently separated and enriched, and the separating and enriching method has far-reaching significance on deep researching of the infestation mechanism and the life history of the nosema bombycis.

Description

technical field [0001] The invention belongs to the technical field of microorganism separation, and relates to a method for separating and enriching the spore protoplasm of the silkworm microsporidia. Background technique [0002] Nosema bombycis is the pathogen of silkworm microsporidia. It is a single-celled eukaryotic organism that parasitizes in specialized cells. It can vertically spread through eggs and cause microsporidia. It is very harmful to sericulture production. Statutory quarantine objects in sericulture production. Microsporidia spores have a thick outer wall, and cannot be cultured in vitro or genetically manipulated, which greatly limits the research on the gene function of Microsporidia. At present, its life cycle, infection process, pathogenic mechanism and other important characteristics are still unclear. [0003] The life cycle of microsporidia mainly includes three stages, infection period, fission proliferation period, and spore proliferation perio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/10C12N1/02C12R1/90
Inventor 李田何强周泽扬
Owner SOUTHWEST UNIVERSITY
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