Portunus trituberculatus reovirus receptor, screening method and application thereof

A technology for the portunus trituratus and reovirus, which is applied in the field of biology, can solve the problems such as the undisclosed portunus trituratus reovirus and the like, and achieves the effect of expanding the scope

Active Publication Date: 2015-05-13
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no relevant research reports on the reovirus receptor of portunus trituberculatus and its blocking inhibitor at home and abroad.

Method used

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  • Portunus trituberculatus reovirus receptor, screening method and application thereof
  • Portunus trituberculatus reovirus receptor, screening method and application thereof
  • Portunus trituberculatus reovirus receptor, screening method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0026] Screening of Reovirus Receptor in Portunus trituberculatus by Western Blot

[0027] 1. Purification of Portunus trituberculatus reovirus (SCRV)

[0028]Take the portunus trituberculatus that died from the infection of the virus, dissect the internal organs on ice, weigh them, and add them to the pre-ice-bathed 1mM phenylmethylsulfonyl fluoride (PMSF) TNE 2 Homogenize in buffer solution, centrifuge the homogenate at 4°C, 8000g for 20min, and use TNE containing 1mM PMSF for precipitation 2 After resuspending the homogenate in the buffer, centrifuge again at 4°C, 8000g for 20min, combine the supernatant obtained from the two centrifugations, centrifuge the supernatant at 4°C, 6000g for 10min, take the supernatant and centrifuge at 4°C, 38000g for 1.5h , with pre-cooled TM 2 The loose part of the upper layer of the precipitate was washed with the buffer solution, and the solid white precipitate part of the lower layer was washed with pre-cooled TM containing 1mM PMSF 2 ...

specific Embodiment 2

[0039] Preparation of Receptor Antibody of Portunus trituberculatus Reovirus (SCRV)

[0040] Homogenate the above portunus trituberculatus reovirus receptor protein from 8% SDS-PAGE gel, suspend with 0.9% sodium chloride, mix the receptor protein and complete Freund's adjuvant in equal volumes, and fully emulsify Afterwards, the mice were injected subcutaneously for basic immunization, and the immunization dose for each mouse was 10-20 μg / mL. After 15 days, the portunus trituberculatus reovirus receptor protein and Freund's incomplete adjuvant were mixed in equal volumes, fully After emulsification, the mice were injected subcutaneously for booster immunization. The immunization dose for each mouse was 8-15 μg / mL. The booster immunization was repeated 3 times with an interval of 7-10 days. After the last immunization, the mice were given Pick up the eyeball to take blood, put the blood at room temperature for 1 hour, transfer it to 4°C and let it stand for 3-5 hours, gently fi...

specific Embodiment 3

[0041] Verification of the Binding of Portunus trituberculatus Reovirus (SCRV) to SCRV Receptor by Co-immunoprecipitation Technique

[0042] Add 800ul of Portunus trituberculatus gill membrane protein extract and 30ul of Portunus trituberculatus reovirus antigen freshly prepared in Specific Example 1 into a centrifuge tube, shake slowly at 4°C for 8 hours, then add 10ul of mouse anti-receptor protein antibody, Anti-turtle systemic septicemia spheroid virus yolk antibody was used as a negative control, and incubated overnight at 4°C. Take 60ul of protein A / G agarose (Santa Cruz Biotechnology Company, sc-2003) and mix thoroughly by inverting, then add to a centrifuge tube and shake slowly at 4°C for 3h, add 1ml of high-salt NETN buffer, at 4°C, 3000rpm Centrifuge, take the precipitate and repeat the centrifugation operation 4 times for washing, finally add 1ml of NETN buffer, centrifuge at 4°C, 3000rpm, take the precipitate and add 30ul protein loading buffer, cook in boiling wa...

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Abstract

The invention discloses a portunus trituberculatus reovirus receptor, a screening method and application thereof. The portunus trituberculatus reovirus receptor is characterized by being microtubulin. The screening method comprises the following steps: anatomizing healthy trituberculatus on ice, taking branchiae and adding the branchiae to a membrane protein extracting liquid, homogenizing, and performing resuspension on sediments obtained through centrifugation by the membrane protein extracting liquid so as to obtain a branchia membrane protein extracting liquid of portunus trituberculatus; performing separation on the membrane protein by using 8% SDS-PAGE and electrotransferring the membrane protein to a PVDF (polyvinylidene fluoride) membrane, performing overnight incubation by low-fat milk under 4 DEG C, washing the incubated membrane for three times, incubating the washed membrane for an hour by SCRV virus under room temperature, washing the incubated membrane for three times again, incubating the washed membrane by chicken antibody SCRV under room temperature for two hours, washing the incubated membrane for three times, incubating the washed membrane by a goat-anti-chicken second antibody in HRP coupling under room temperature for an hour, washing the incubated membrane for four times, performing color development by OPD so as to obtain a protein band, namely the SCRV receptor. The portunus trituberculatus reovirus receptor has the advantages that a receptor blocking agent has inhibitory activity to SCRV infection cells, and a medicine for preventing and treating SCRV infection can be developed.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to a novel portunus trituberculatus reovirus (SCRV) receptor and the prevention and treatment of SCRV virus infection by using the receptor protein antibody. Background technique [0002] Swimming crab (Portunus trituberculatus) is an important marine economic animal. It has delicious meat and rich nutrition, and is favored by consumers. It is also an important foreign exchange earning species. Portunus trituberculatus is currently the largest marine economic crab in the world. In recent years, the epidemic caused by Portunus reovirus often occurs in autumn and winter, causing a large number of Portunus to die, which brings important economic losses to the aquaculture industry. The strain of the portunus trituberculatus reovirus is R1015 strain, the preservation number is CGMCC No.5379, and it was preserved in the General Microorganism Center of China Committee for Microorganism ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705G01N33/68C07K16/28C07K16/06A61K39/42A61P31/14
Inventor 方静李登峰刘联国徐然章礼萍
Owner NINGBO UNIV
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