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Method for screening acceptor blocker for controlling prawn virus infection

A receptor blocker and virus infection technology, applied in antiviral agents, biochemical equipment and methods, pharmaceutical formulations, etc., to speed up the screening cycle and reduce the dosage

Inactive Publication Date: 2005-06-22
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] When the present invention was completed, there were no other units or individuals to carry out research reports on shrimp virus receptor blockers

Method used

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  • Method for screening acceptor blocker for controlling prawn virus infection
  • Method for screening acceptor blocker for controlling prawn virus infection
  • Method for screening acceptor blocker for controlling prawn virus infection

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0036] 1. Example 1: Analysis of specific binding between BCMP and WSSV

[0037] 1.1 WSSV purification

[0038] 1.1.1 Collect shrimps with leukoplakia, weigh 5 g of cephalothorax without hepatopancreas, add 5 times the volume of PPB pre-cooled at 4°C (short for "Physiological Buffer for Prawns", containing: NaCl, 22.0 g / L; K 2 SO 4 , 1.1g / L; CaCl 2 ·H 2 O, 1.9g / L; MgSO 4 ·7H 2 O, 1.6g / L; histidine hydrochloride, 5.0g / L; pH 6.5), homogenize in an ice bath at 20,000r / min for 3-5 times, each time for 5s.

[0039] 1.1.2 Centrifuge at 4,500×g for 15 minutes at 4°C, add sucrose to the supernatant to a concentration of 30% (W / W), and centrifuge at 30,000×g for 2 hours at 4°C.

[0040] 1.1.3 The pellet was resuspended with 30% (W / W) sucrose, spread on a 33%-62% (W / W) sucrose gradient, centrifuged at 60,000×g at 4°C for 4 hours, and carefully collected visible virus bands with a pipette.

[0041] 1.1.4 Add 5 times the volume of PPB to the collected virus zone solution to dilute ...

Embodiment 2

[0070] 2. Example 2: Screening of polysaccharides that inhibit the binding of WSSV and BCMP

[0071] 2.1 Binding inhibition analysis of heparin sodium:

[0072] 2.1.1 Treatment of DIG-WSSV by heparin sodium:

[0073] - Dilution of heparin sodium: Dilute heparin sodium with an original concentration of 0.16 g / ml in dHO in an Eppendorf tube 2 O was diluted into 4 gradients of heparin sodium by 2-fold dilution. Concentrations are: 0.08g / ml, 0.04g / ml, 0.02g / ml, 0.01g / ml.

[0074] - Heparin treatment of DIG-WSSV: Add 300 μl of 0.124 μg / μl DIG-WSSV (diluted in MPBS) to four Eppendorf tubes, and mix 120 μl of the above heparin sodium concentration gradient with the DIG-WSSV in the tube. The final concentrations of heparin sodium in each tube were: 32 μg / μl, 16 μg / μl, 8 μg / μl and 4 μg / μl.

[0075] 2.1.2 Coating: After diluting 1.0 μg / μl BCMP with coating solution, coat 1.5 μg / 100 μl / well on a 96-well microtiter plate, overnight at 4°C.

[0076] 2.1.3 Plate washing: The next day, ...

Embodiment 3

[0108] 3. Example 3: Infection experiment to verify the anti-infection effect of heparin sodium on WSSV treatment

[0109] 3.1 Experimental shrimp and grouping

[0110] 3.1.1 Litopenaeus vannamei is provided by Qingdao Jiaonan Farm, 9-12cm in size, with an average of 14.8g / tail.

[0111] 3.1.2 After the experimental shrimps were inflated for 3 days and stabilized in the laboratory, the healthy and lively ones were selected and divided into two groups. The P group was the WSSV infection group, which was used as the infection control group. The H group was the heparin sodium experimental group, 12 fish / group.

[0112] 3.2 Virus seed treatment

[0113] 3.2.1 Take 20 g of cephalothorax (with hepatopancreas removed) diseased material of prawns infected by WSSV, add 2% NaCl, and homogenize in an ice bath.

[0114] 3.2.2 The homogenate was centrifuged at 4,500×g for 20 minutes at 4°C, and the sedimentation was repeated 3 times.

[0115] 3.3.3 The supernatants were combined, and th...

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Abstract

Disclosed is a method for screening acceptor blocker for controlling prawn virus infection, wherein the combined analytic technique for virus and prawn cell membrane protein is employed for the screening process of the virus receptor bonding blocking agent, and screening is achieved by comparing the influence of the screening article treatment to the combination of virus and prawn cell membrane protein, the three screened substances have blocking-up action to the combination of leukoplakia syndrome virus and prawn branchia cell membrane.

Description

technical field [0001] The invention belongs to screening and application technology of drugs for prevention and treatment of aquaculture animal diseases, can be applied to screening of drugs for prevention and treatment of prawn viruses, and is a receptor blocker for screening and controlling prawn virus infection. Background technique [0002] Viruses have had a serious impact on the shrimp farming industry, especially the white spot syndrome virus (WSSV) is the most important pathogen of the shrimp, which has seriously hindered the development of the farming industry. The study of this pathogen has become one of the hot spots in the field of marine biology, and the development of drugs for the prevention and treatment of prawn diseases has attracted widespread attention. [0003] At present, most of the technical routes for the development of shrimp virus prevention and control drugs are to borrow medicine or veterinary drug control technology. For example, ribavirin is u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/727A61K38/16A61P31/12C12Q1/18G01N33/68
Inventor 黄倢陆春玲梁艳宋晓玲解飞霞易志刚刘庆慧杨冰刘莉
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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