Method for rapidly obtaining large amount of spore shells of nosema bombycis

A technology of microsporidia and microsporidia, which is applied in the diagnosis and identification of silkworm microsporidia and the biological field, can solve the problems of complicated operation, waste of time, cost, loss of alkali-soluble protein, etc., and achieve simple and fast operation, low equipment requirements, and high reliability. highly reproducible effect

Inactive Publication Date: 2013-11-20
SOUTHWEST UNIVERSITY
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Problems solved by technology

However, in the germination method, on the one hand, the germination process requires high spore vigor to ensure the germination rate, and sufficient spore shells can be obtained after purification, but during the storage of the spores, the viability of the spores drops sharply, resulting in a very low spore germination rate. Only about 10-20%, even no germination so that the spores cannot be obtained, and the spores after a germination treatment can no longer germinate, so the germination method has great non-repeatability in operation, resulting in time and cost. Waste; on the other hand, because the germination method needs to use alkaline conditions to germinate the spores in vitro due to the germination method to purify t

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  • Method for rapidly obtaining large amount of spore shells of nosema bombycis
  • Method for rapidly obtaining large amount of spore shells of nosema bombycis
  • Method for rapidly obtaining large amount of spore shells of nosema bombycis

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Embodiment 1

[0022] The preparation of silkworm microsporidia: get the 3rd instar of silkworm and add silkworms to feed on the silkworm microsporidia, collect tissues (comprising silk gland, midgut and silkworm epidermis) in the late stage of 5th instar, add 0.09g / L NaCl solution homogenate and grind, and The homogenate was filtered with 4 layers of gauze. After the filtrate was roughly extracted by differential centrifugation, the crude spores were filtered with absorbent cotton, and then subjected to Percoll gradient density centrifugation. Set different concentrations of Percoll solutions, respectively 30% (1.5mL), 45% (2mL), 60% (2mL), 75% (2mL), 90% (1.5mL), add the spore crude extract to the upper end of the gradient Percoll solution, use an ultra-fast refrigerated centrifuge at 4°C, centrifuge at 30000g for 40min, collect the bottom sediment For purified spores, store at 4°C.

[0023] Fragmentation method of the present invention: with 10 10 The purified spores were suspended in 40...

Embodiment 2

[0028] Two-dimensional electrophoresis sample preparation gets respectively 10 that embodiment 1 obtains 8 Add 100 μL of protein lysate (7mol / L urea, 2mol / L thiourea, 4% CHAPS, 100mmol / L DTT, 0.2%SDS) to each of the spore shells purified by the germination method and the spore shells purified by the scheme of the present invention, and vortex Extract for 6 hours, centrifuge at 12,000 r / min at 4°C for 15 minutes, take the supernatant as a protein extract, measure the protein concentration, and store in a -80°C refrigerator.

[0029] Using Ettan TM IPGphor TM The isoelectric focusing system (GE Company) was used to perform isoelectric focusing, and the hydrated loading buffer was fully mixed with the sample, and a total of 350 μL of the sample was loaded on a 18 cm IPG prefabricated gel strip (pH 3-10). The gradient step-up method is used: 50V hydration for 12 hours, 100V desalination for 1 hour, 200V desalination for 1 hour, 500V desalination for 1 hour, 1000V desalination fo...

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Abstract

The invention provides a method for rapidly obtaining large amount of spore shells of nosema bombycis. The method comprises the following steps of adding 10<9>-10<20> 400muL of nosema bombycis into a solution, mixing the nosema bombycis suspension with 212-300mu m of glass beads according to the proportion of 1mu L:1mg to obtain a mixture, putting the mixture into a micro burette mixer and oscillating for 8-12 hours, carrying out gradient density centrifugation on the spore suspension, and taking sediment. According to the method, the spore shell extracting and purification processes are simplified, the operation is simple and fast, the requirement on equipment is low, the repeatability is high, a large amount of spores can be rapidly broken in one day so as to obtain the spore shells, the yield of the spore shell can reach 70-90% or even 100%, and the acquisition of the spore shell is not affected by the weakened vitality of the spores.

Description

technical field [0001] The invention relates to the field of biological technology, and relates to the technical field of diagnosis and identification of the microsporidia silkworm. Background technique [0002] Microsporidia are a group of pathogens that obligately parasitize intracellularly in the form of spores, and have a wide range of hosts. Almost all animals, including protozoa and humans, can be infected with Microsporidia. Nosema bombycis is It was isolated and identified from diseased silkworms in 1857. It can infect silkworms and cause silkworm microparticle disease, which is a devastating disease in sericulture production. Therefore, the detection of silkworm microsporidia is very important in sericulture. In recent years, According to the different antigenicity of the interspecies sporozoite protein of microsporidia, some immunological detection methods have been gradually developed. The immunological detection method presents a certain reaction phenomenon th...

Claims

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Application Information

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IPC IPC(8): C12N1/10C12Q1/04
Inventor 周泽扬耿莉娜潘国庆党晓群李田龙梦娴
Owner SOUTHWEST UNIVERSITY
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