A method for purifying Enterocystoma hepatica of prawns

A technology of hepatic enterocystosis and prawns, which is applied in the field of microsporidia purification, can solve the problem of inability to obtain purified hepatic enterocystosis, achieve the effects of reducing the interference of microorganisms such as bacteria, high purity, and obvious separation effect

Active Publication Date: 2022-04-01
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] For the above-mentioned technical problems, the purpose of the present invention is to solve the problem that the method in the existing data cannot obtain the purified Enterococcus hepatica, and to provide a method for purifying Enterococcus hepatica of prawns. The purification method of the present invention is simple and convenient, and does not require Requires large equipment such as high-speed centrifuges for easy operation in routine laboratories

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: purification method of the present invention, comprises the steps:

[0045] One, the preparation of crude extract:

[0046] 1. Take multiple shrimps on ice or at room temperature (the number varies according to the degree of shrimp infection, 20 are selected in this example) of the hepatopancreas of shrimp infected with Enterocystis hepatica (note: avoid taking the intestine and stomach, in this example, the Litopenaeus vannamei), remove the membrane, and grind vigorously in a mortar with a grinding rod until there are no obvious tissue pieces.

[0047] 2. Dilute the grinding solution with sterile water to 35-40ml, filter it with a 70μm pore size filter and a 40μm cell filter, and place 50ml of the obtained filtrate containing Enteroplasma hepatica in a centrifuge tube.

[0048] 3. Set the centrifuge to 15°C for centrifugation, centrifuge at 1500rpm for 10min, discard the supernatant to get the precipitate. Add 30ml of sterile water to dissolve the precipi...

Embodiment 2

[0065] Embodiment 2: purification method of the present invention, comprises the steps:

[0066] One, the preparation of crude extract:

[0067] 1. Take multiple shrimps on ice or at room temperature (the number varies according to the degree of shrimp infection, 10 are selected in this example) of the hepatopancreas of shrimp infected with Enterocystis hepatica (note: avoid taking the intestine and stomach, the harvested in this example Litopenaeus vannamei), remove the membrane, and grind vigorously in a mortar with a grinding rod until there are no obvious tissue pieces.

[0068] 2. Dilute the grinding solution with 20ml of sterile water to 35ml, filter it with a 70μm pore size filter and a 40μm cell filter, and place 50ml of the obtained filtrate containing Enteroplasma hepatica in a centrifuge tube.

[0069] 3. Set the centrifuge to 15°C and centrifuge at 1600rpm for 10min, discard the supernatant to get the precipitate. Add 22ml of sterile water to dissolve the precipi...

Embodiment 3

[0086] Embodiment 3: purification method of the present invention, comprises the steps:

[0087] One, the preparation of crude extract:

[0088] 1. Take multiple shrimps on ice or at room temperature (the number varies depending on the degree of shrimp infection, 15 are selected in this example) of the hepatopancreas of shrimp infected with Enterocystis hepatica (note: avoid taking the intestine and stomach, in this example, the Litopenaeus vannamei), remove the membrane, and grind vigorously in a mortar with a grinding rod until there are no obvious tissue pieces.

[0089] 2. The grinding solution was diluted to 40 ml with 25 ml of sterile water, filtered with a 70 μm pore size and 40 μm cell filter, and 50 ml of the obtained filtrate containing Enteroplasma hepatica was placed in a centrifuge tube.

[0090] 3. Set the centrifuge to 15°C for centrifugation, centrifuge at 2000rpm for 15min, discard the supernatant to get the precipitate. Add 30ml of sterile water to dissolve...

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Abstract

A kind of method that is used for purifying prawn enterocystoma hepatica, the steps are: take a plurality of prawn hepatopancreas infected with enterococcus hepatica, remove the membrane and grind; dilute to 35-40ml with sterile water; alternately at 1500-2000rpm and 500-600rpm Centrifuge until no precipitation is observed with the naked eye; then centrifuge the supernatant without precipitation at 3000-3500rpm to obtain the precipitation, dissolve the precipitation in sterile water as the crude extract; use different concentrations of Percoll cell separation medium for gradient separation; Add 1ml-1.5ml of the crude extract to the top of the gradient layer; use a centrifuge to centrifuge at 15°C and 9000-10000rpm; wash to obtain a purified suspension of Enterocystis hepatica. The purified worm body of the invention has high purity, less impurities and simple operation.

Description

technical field [0001] The invention belongs to the technical field of microsporidia purification, in particular to a method for purifying Enterocystis hepatica of prawns. Background technique [0002] Enterocytozoon hepatopenaei (EHP) belongs to the family Microsporidiaceae and the genus Enterocystis, and is a highly infectious intracellular parasitic microsporidia. Monodon) was isolated from the epithelial cells of the hepatopancreas tubules, and in recent years, it has spread and prevailed in major shrimp farming countries such as Thailand, India, Vietnam, Indonesia and China, and mainly infects the economical species such as Penaeus vannamei and Penaeus monodon. Shrimp farming can cause extremely slow or stagnant growth of prawns, seriously affecting the production and economic value of prawns, and is one of the important diseases that endanger the global prawn farming industry. [0003] Since 2013, EHP has been detected in several coastal shrimp farming areas in my cou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/10C12R1/90
CPCC12N1/10
Inventor 姜宏波宁梓健陈启军
Owner SHENYANG AGRI UNIV
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