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Method for high-flux screening of drugs used for pseudorabies viruses

A porcine pseudorabies virus, high-throughput technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of no drug prevention and treatment, achieve the effect of improving the hit rate and easy automation

Inactive Publication Date: 2013-07-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is still no report on the research on porcine pseudorabies virus medicine at home and abroad, and there is no available medicine for the prevention and treatment of the disease

Method used

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  • Method for high-flux screening of drugs used for pseudorabies viruses
  • Method for high-flux screening of drugs used for pseudorabies viruses
  • Method for high-flux screening of drugs used for pseudorabies viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] A method for high-throughput drug screening of porcine pseudorabies virus, the steps of which are:

[0036] (1) Determination of optimal cell seeding density and detection endpoint in high-throughput screening:

[0037] In order to find the optimal cell seeding density, 100 μL of 5000cells / well, 7500cells / well, 10000cells / well, 15000cells / well and 20000cells / well were seeded on a 96-well cell culture plate, and cultured continuously for 96 hours. Cell viability was detected by CellTiter glo every 24 h during the culture period. The results showed that the statistical results of the cell control group showed that the cell viability continued to increase within 72 hours; the cell viability began to decline from 72 hours to 96 hours, indicating that the cells had overgrown and died after being cultured for 96 hours (attached figure 1 ). The virus control group was cultivated under the same conditions for 96 hours, and tested by the same method every 24 hours. The test r...

Embodiment 2

[0045] BVDU as a drug candidate for porcine pseudorabies virus:

[0046] (1) The inhibitory effect of BVDU on wild strains of porcine pseudorabies virus:

[0047] Inoculate 5000 cells / well of IBRS-2 cells into a 96-well white bottom-permeable culture plate, and replace it with a maintenance solution after 12 hours. The maintenance solution contains 0.01 MOI of pseudorabies virus and BVDU with different concentrations (100 μM- 3.125 μM), set the cell control and virus control, continue to culture for 48 hours, add 100 μL Celltiter glo and measure the chemiluminescence value, calculate the inhibition rate of different concentrations of BVDU to pseudorabies virus, and find that when the BVDU concentration is lower than 3.125 μM, The inhibition rate was still greater than 67.2% (attached Figure 6 ), indicating that BVDU has a strong inhibitory effect on pseudorabies virus. The antiviral activity of the porcine pseudorabies virus attenuated vaccine strain TK- / gG- / gE- strain that...

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Abstract

The invention discloses a method for high-flux screening of drugs used for pseudorabies viruses. The method comprises the following steps of 1, inoculating a white optical-bottom 96-well cell plate with IBRS-2 cells, and carrying out culture in an incubator containing 5% of CO2 and having a temperature of 37 DEG C, 2, removing a growth solution, adding a maintenance media into the culture product, adding compounds needing to be screened into the culture product, orderly carrying out coubling dilution, carrying out uniform mixing, adding a pseudorabies virus Hubei A strain into each one of the wells of the white optical-bottom 96-well cell plate, and arranging a control group without the pseudorabies viruses and the compounds, and a control group with the pseudorabies viruses and without the compounds on the same white optical-bottom 96-well cell plate, wherein the compounds are dissolved in dimethylsulfoxide, 3, carrying out continue culture in the incubator containing 5% of CO2 and having a temperature of 37 DEG C, adding a Celltiterglo reagent into the culture, carrying out detection in a high-flux chemical luminescent detector, and calculating inhibition ratios of the compounds, and 4, utilizing the compound having a high inhibition rate as a potential drug for resisting pseudorabies viruses. The method is simple and fast, has a low cost, is convenient for high flux operation, can be used for screening aiming at the whole proliferation process of pseudorabies viruses in cells, and has a high success rate.

Description

technical field [0001] The invention relates to the technical field of animal infectious disease prevention and control and animal drug screening. More specifically, it relates to a high-throughput drug screening method for porcine pseudorabies virus. On the basis of drugs inhibiting the cytopathy caused by porcine pseudorabies virus, an in vitro drug screening method for porcine pseudorabies virus is established by measuring cell viability. Background technique [0002] Pseudorabies (PR) is an acute, contact and highly fatal infectious disease caused by pseudorabies virus (PRV) with fever, itching and encephalomyelitis as the main symptoms. The disease can cause abortion, stillbirth and mummification in pregnant sows. Boars show orchitis, testicular swelling and atrophy, and lose their seeding ability. Piglets show high fever, dyspnea, salivation, vomiting, diarrhea, loss of appetite, depression and tremors, etc. Symptoms followed by intermittent convulsions, coma, and dea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/02C12R1/93
Inventor 宋云峰周锐王翠陈焕春
Owner HUAZHONG AGRI UNIV
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