Method for purifying enterocytozoon hepatopenaei of prawns

A technology for 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, good technical repeatability, and simple purification methods

Active Publication Date: 2019-04-16
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 shrimps infected with Enterocystis hepatica (note: avoid taking the intestines and stomachs, 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.

[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...

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 method for purifying enterocytozoon hepatopenaei of prawns comprises the steps of taking hepatopancreas of a plurality of prawns infected with enterocytozoon hepatopenaei, removing membranes by grinding; diluting them to 35-40 ml with sterile water; conducting alternative centrifugation at 1500-2000 rpm and at 500-600 rpm till no precipitate is observed by the naked eyes; centrifuging the supernatant without precipitate at the high speed of 3000-3500 rpm, and taking and dissolving a precipitate in sterile water to serve as a crude extracting solution; adopting different concentrations of Percoll cell separation solutions for gradient separation; adding 1-1.5 ml of crude extracting solution at the topmost of the liquid level with set gradient; using a centrifugal machine to carry out centrifugation under the conditions of 15 DEG C and 9000-10000 rpm; and obtaining a purified enterocytozoon hepatopenaei suspension by washing. The purified enterocytozoon hepatopenaei has high purity andless impurities, and the operation is simple.

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 Applications(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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