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Screening system for anti-Singapore grouper iridovirus (SGIV) drug

A screening method and drug technology, applied in biochemical equipment and methods, biomaterial analysis, microbial measurement/inspection, etc., can solve problems such as complex process and complex analysis, and achieve simple screening methods and optimized cell activity culture conditions Effect

Inactive Publication Date: 2018-03-13
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have problems such as complicated process and complex analysis.

Method used

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  • Screening system for anti-Singapore grouper iridovirus (SGIV) drug
  • Screening system for anti-Singapore grouper iridovirus (SGIV) drug
  • Screening system for anti-Singapore grouper iridovirus (SGIV) drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Quantification of CPE caused by SGIV infection, determination of multiplicity of infection and infection time

[0031] ZEB cells were plated on 96-well plates and infected with SGIV at different multiplicity of infection (0.15, 0.3 and 0.7). After 24, 48 and 72 hours of infection, the CPE induced by SGIV infection was detected with the CellTiter-Glo Luminescent Cell Viability kit. The results showed that after 24, 48 and 72 h, the fluorescence signal (equivalent to cell viability) of ZEB cells at MOIs of 0.3 and 0.7 was significantly reduced compared with cells not infected with virus. However, the cell viability of cells with a multiplicity of infection of 0.15 did not decrease significantly at 24 and 48 hours after infection ( figure 1 ). The results demonstrate that SGIV infection-induced CPE can be quantified.

[0032] Determination of virus infection time and multiplicity of infection (MOI): ZEB cells were spread to 96-well plates at a density of 5000 / ...

Embodiment 2

[0034] Example 2 Optimization of CPE-based cell culture conditions

[0035] Determination of serum concentration: ZEB cells were transferred to a 96-well plate at a density of 5000 / well, and cultured with DMEM medium containing different serum concentrations (2%, 5%, 10%). Cell viability was tested with CellTiter-Glo kit 24, 48, and 72 hours after passage. The experiment was repeated three times, and the obtained experimental data was the average of 8 parallel sample data.

[0036] Cell culture medium with high FBS content may cause excessive cell proliferation and reduce cell viability. The optimal concentration of FBS can be determined by detecting the effect of FBS concentration on the activity of ZEB cells. Analysis showed that ZEB cells were inoculated into DMEM medium containing 5% and 10% FBS, and the fluorescent signals at 48h and 72h after inoculation were stronger than those at 24h after inoculation. However, when ZEB cells were cultured with DMEM medium containin...

Embodiment 3

[0039] Example 3 Addition of screened drugs and screening of drugs by cell activity

[0040]Drugs to be screened were: ribavirin, arbidol hydrochloride and hydroxytetradecanoic acid purchased from Sigma (St.Louis, MO). Hypocretin A and harringtonine were purchased from Abcam (Cambridge, UK). Harringtonine was dissolved in DMSO to a concentration of 9.4×10 3 μM. Hypocretin A was dissolved in DMSO at a concentration of 1.83 × 10 3 μM. Hydroxytetradecanoic acid (20.4×10 3 μM), Arbidol hydrochloride (1×10 3 μM), ribavirin (40.95×10 3 μM) were dissolved in DMSO and stored at -20°C.

[0041] The ZEB cells were spread to 96 wells at a density of 5000 / well, and pre-treated with different concentrations of ribavirin, arbidol hydrochloride, hydroxytetradecanoic acid, hypocretin A or harringtonine at 28°C. Incubate for 24h. In the presence of the above compounds, SGIV (MOI=0.3) was added to the pretreated and untreated cells, and after incubation at 28°C for 48h, the fluorescenc...

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Abstract

The invention provides a cell screening system for anti-SGIV drug screening based on cell activity analysis, comprising: serum concentration of 5%, when DMSO needs to be added as a solvent, the DMSO concentration is not higher than 1%, the multiplicity of infection of the virus is 0.3, and the virus The infection time is 48h. The invention also provides an optimization method of a cell screening system for screening anti-SGIV drugs and a drug screening method. The advantage of the method of the present invention is that the cytopathic effect (CPE) caused by quantitative SGIV infection can be quantified for the first time, the cell screening system for anti-SGIV drug screening is optimized, and a simple screening method is established using this system, using ddPCR for The detection of screening results is accurate and fast.

Description

technical field [0001] The present invention relates to a kind of drug screening method, especially a kind of anti-Singapore grouper iridescent virus (SGIV) drug screening method based on cell activity analysis. Background technique [0002] Singapore grouper iridescent virus (SGIV), belonging to the Ranavirus genus of the family Iridoviridae, is one of the main pathogens causing huge economic losses in Asian grouper aquaculture. Although SGIV infection of grouper has caused severe economic losses to farmers, currently available treatments for SGIV are very limited. Currently, two SGIV vaccines have been proven to be effective in reducing the risk of SGIV infection in groupers, including formalin inactivated vaccine and β-propiolactone inactivated vaccine. However, vaccinating fish, especially juvenile fish, is time-consuming and cumbersome, making these vaccines difficult to popularize and commercialize. Therefore, there is an urgent need to develop other effective anti-S...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12Q1/6851
CPCG01N33/5008C12Q1/6851G01N2333/4603C12Q2531/113
Inventor 易梅生贾坤同
Owner SUN YAT SEN UNIV
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