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Method for inhibiting in-vitro replication and proliferation of pseudorabies virus by using inhibitor A2ti-1 and application

A pseudorabies virus, a2ti-1 technology, applied in the field of cell biology and virology, to achieve the effect of inhibiting replication and proliferation

Active Publication Date: 2022-04-05
河南省农业科学院动物免疫学重点实验室
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Given that there is currently no specific treatment for PRV, the widespread prevalence of pseudorabies virus poses a certain threat to practitioners in the pig industry and other people in close contact with it

Method used

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  • Method for inhibiting in-vitro replication and proliferation of pseudorabies virus by using inhibitor A2ti-1 and application
  • Method for inhibiting in-vitro replication and proliferation of pseudorabies virus by using inhibitor A2ti-1 and application
  • Method for inhibiting in-vitro replication and proliferation of pseudorabies virus by using inhibitor A2ti-1 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Detection of cytotoxicity of A2ti-1 on PK-15 cells

[0028] CellTiter 96 AQueous Single Solution Cell Proliferation Assay Kit was used to determine the cytotoxic effect of inhibitors on PK-15 cells.

[0029] (1) PK-15 cells were treated with 1×10 4 Cells / well were plated in a 96-well plate, and the next step could be performed after the cells grew to 70-80% abundance. A blank control was set, and at least three wells were replicated for each concentration.

[0030] (2) Dilution of inhibitors: Dilute A2ti-1 inhibitors with DMEM cell culture medium containing 10% (v / v) FBS (fetal bovine serum), and obtain the concentrations of A2ti-1 inhibitors in the medium as: 0 μM, 31.25μM, 62.5μM, 125μM, 250μM, 500μM.

[0031] (3) Incubation of inhibitors: Discard the old medium, and add 100 μL of inhibitors of corresponding concentration to each well. Place the 96-well plate in a cell culture incubator for 36-48 hours.

[0032] (4) Reading: After adding 20 μL of CellT...

Embodiment 2

[0035] Example 2. Immunofluorescence method to evaluate the inhibitory effect of A2ti-1 on PRV infection

[0036] (1) PK-15 cells were treated with 1×10 4Cells / well were plated in a 96-well plate, and the next step could be performed when the cells grew to about 80% abundance. Set up a blank control. At least three wells were replicated for each concentration.

[0037] (2) Incubate different concentrations of A2ti-1 (0 μM, 62.5 μM and 125 μM) with the PRV-GFP strain (MOI=0.1) at 37°C for 1 hour, rinse with PBS buffer for 3 times, and then add The DMEM cell maintenance solution (containing 2% (v / v) FBS) containing the corresponding concentration of inhibitors was further cultured for 24 hours.

[0038] (3) Discard the culture supernatant, rinse with PBS three times, then add 200 μL of 4% (wt%) paraformaldehyde to each well for fixation for 15 minutes, and then rinse with PBS three times.

[0039] (4) Add 100 μL of DAPI staining solution to each well, stain at room temperatu...

Embodiment 3

[0042] Example 3. A2ti-1 inhibits the dose-dependent evaluation of PRV replication and proliferation

[0043] (1) PK-15 cells were divided into 2.5×10 4 24-well cell culture plate was inoculated with each cell / well, 500 μL DDMEM cell culture medium (containing 10% FBS) was added to each well, and cultured in a cell culture incubator for 24 hours.

[0044] (2) Discard the medium, rinse with PBS for 3 times, and then use the PRV virus (PRV-HeNLH / 2017 strain) with a multiplicity of infection (MOI) of 0.01 and the corresponding concentration of inhibitor A2ti-1 (0μM , 62.5 μM and 125 μM) were added to the cell culture wells and incubated for 1 hour, 0 μM was the control group without inhibitors, and each group had three replicates; discard the medium after incubation, wash the cells with PBS three times, and then add The DMEM cell maintenance solution (containing 2% (v / v) FBS) containing the corresponding concentration of A2ti-1 was further incubated in the cell culture box for 2...

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Abstract

The invention relates to a method for inhibiting replication and proliferation of a pseudorabies virus in vitro by using an annexin A2 / S100A10 heterotetramer inhibitor A2ti-1 and application of the inhibitor A2ti-1. The inhibitor A2ti-1 can be used for developing related antiviral drugs for inhibiting the replication and proliferation of the pseudorabies virus in vitro. On PK-15 cells, it is verified that the DNA content, the virus titer and the expression level of structural protein after PRV infection are all remarkably reduced under the use concentration of A2ti-1 not lower than 62.5 [mu] M compared with a control group, and it is indicated that A2ti-1 can be used for inhibiting in-vitro replication and proliferation of the pseudorabies virus.

Description

technical field [0001] The invention relates to a method and application of annexin A2 / S100A10 heterotetramer (A2t) inhibitor A2ti-1 for inhibiting the replication and proliferation of pseudorabies virus in vitro, and belongs to the technical fields of cell biology and virology. Background technique [0002] Pseudorabies virus (PRV) belongs to the Herpesviridae family, the Alphaherpesvirinae subfamily Varicellovirus genus, the surface of the virus particles is enveloped, the genome is a double-stranded DNA virus, the size is about 150,000 bases, including 72 open reading frames (open reading frame, ORF), encoding 70 different proteins. PRV can infect a variety of animals, such as dogs, cats, sheep, cattle, foxes, minks, and rats, with a lethal rate of almost 100%. After pigs are infected with pseudorabies virus, the symptoms of pigs at different stages are also different. Sows mainly Manifested as reproductive disorders, such as abortion, stillbirth, mummies, weak piglets, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N33/569G01N21/76C07K14/005A61K38/16A61P31/22
CPCY02A50/30
Inventor 郭振华张改平翁茂洋宋佳姜瑶乔松林郭军庆
Owner 河南省农业科学院动物免疫学重点实验室
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