Drug screening system based on recombinant Zika virus expressing green fluorescent protein and application thereof

A technology of green fluorescent protein and Zika virus, which is applied in the direction of virus, virus/bacteriophage, positive-sense single-stranded RNA virus, etc., can solve the problems of low replication ability and instability of Zika virus, reduce the pressure of virus replication, and easily Observing, replicating the effects of reduced ability

Active Publication Date: 2019-09-03
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a system for anti-Zika virus drug screening, to overcome the low replication ability and instability of the reporter Zika virus. defect

Method used

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  • Drug screening system based on recombinant Zika virus expressing green fluorescent protein and application thereof
  • Drug screening system based on recombinant Zika virus expressing green fluorescent protein and application thereof
  • Drug screening system based on recombinant Zika virus expressing green fluorescent protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of a cell line stably expressing DC-SIGNR

[0048] 1.1 Experimental materials

[0049] 1.1.1 Main materials and reagents

[0050] BHK-21 cells; DMEM, fetal bovine serum, and Opti-MEM were purchased from Gibco; the mixture of trypsin and penicillin was purchased from Gibco; polylysine was purchased from Sigma; TritoX-100 was purchased from Sigma Infrared fluorescence-labeled anti-rabbit IgG antibody was purchased from KPL Company; FITC-labeled goat anti-mouse IgG was purchased from Zhongshan Jinqiao Company; QIAGEN plasmid extraction kit was purchased from QIAGEN Company; TransIT-293Transfection Reagent was purchased from Mirus Company.

[0051] 1.1.2 Experimental Instruments

[0052] PCR instrument (Takara); nucleic acid electrophoresis instrument (Beijing Liuyi Instrument); nucleic acid gel imaging system (Bio-Rad); pure water instrument (Millipore); optical microscope (Olympus); inverted fluorescence microscope (Life); electronic balance ( Be...

Embodiment 2

[0070] Example 2: Construction of reporter Zika virus ZIKV-GFP expressing green fluorescent protein

[0071] 2.1 Experimental materials

[0072] 2.1.1 Main materials and reagents

[0073] HEK-293T, Vero, and BHK-21 cells were preserved by our laboratory; DMSO was purchased from Sigma; DMEM, fetal bovine serum, and Opti-MEM were purchased from Gibco; trypsin (TRYPSIN 0.25% EDTA) and penicillin-streptomycin mixed Polylysine was purchased from Sigma Company; Hoechst was purchased from Pierce Company; Triton X-100 was purchased from Sigma Company; infrared fluorescent-labeled anti-rabbit IgG antibody was purchased from KPL Company; FITC-labeled goat anti-small Mouse IgG was purchased from Zhongshan Jinqiao Company; QIAGEN Plasmid Extraction Kit was purchased from QIAGEN Company; TransIT-293Transfection Reagent was purchased from Mirus Company; Chloroquine (Chloroquin) and 6-Azauridine (6-Azauridine) were purchased from Sigma Company; ZIKV E protein Antibodies were purchased from G...

Embodiment 3

[0102] Example 3 reporter virus system is used for antiviral drug screening

[0103] 3.1 Antiviral drug inhibition experiment

[0104] Drugs: Drugs Ribavirin (Rib) and 6-Azauridine (6-Az) were dissolved in DMSO, chloroquine (Chloroquine, CQ) was dissolved in PBS, and the final concentration of DMSO in the culture medium was 1%. The %DMSO effect was used as a negative control. Drugs were diluted with DMSO and DMEM medium containing 2% fetal bovine serum (FBS) to different concentrations for use.

[0105] Drug treatment and virus infection: BHK-DR cells (3×10 4 cells / well) inoculated in black 96-well plate with 2% v / v FBS 100ul medium, when the cell density reached 90%, acted with the drug diluted in 50ul DMEM medium containing 2% v / v FBS for 1h, after 1h Add 50ul ZIKV-GFP virus (10 6 TCID 50 / ml) infection at 37°C, 5% CO 2 Cultivate in the incubator for 48h.

[0106] Statistical analysis of the high-content system: After the cells were treated with drugs and infected wit...

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Abstract

The invention discloses a drug screening system based on a recombinant Zika virus expressing a green fluorescent protein and an application thereof. The drug screening system is composed of the recombinant Zika virus (reported zika virus) expressing the green fluorescent protein (GFP) and a BHK-21 cell line expressing DC-SIGNR. The reported Zika virus expresses the green fluorescent protein, has strong fluorescent signal, has uniform coloration in cytoplasm, and is easy to observe and prone to high throughput screening analysis. The BHK-21 cell line stably expressing DC-SIGNR increases the sensitivity to the infection of the reported Zika virus, increases the efficiency of virus replication, reduces the pressure of virus replication, and improves the stability of recombinant virus. The drug screening system can directly observe and analyze the viral replication condition before and after drug action in the state of living cells, and does not need to fix and immunostain cells, so that the whole operation process is simpler and faster, the operation steps of live viruses are reduced, and the safety of the whole operation process is improved.

Description

technical field [0001] The present invention relates to an antiviral drug screening system and its application, in particular to a drug screening system based on recombinant Zika virus expressing green fluorescent protein and its application. The invention belongs to the field of biotechnology. Background technique [0002] Zika virus (ZIKV) is a zoonotic pathogen mainly transmitted by mosquitoes, and it is related to dengue virus (DENV), West Nile virus (WNV), Japan B Japanese encephalitis virus (JEV) and yellow fever virus (YFV) belong to the Flavivirus genus of the family Flaviviridae. In 1947, researchers such as Dick isolated it from rhesus monkeys in the Zika Forest of Uganda. ZIKV can cause Zika fever after infecting the host. Most infected people have no obvious symptoms or mild symptoms, including conjunctivitis, rash, fever, joint and muscle pain and other symptoms. The course of the disease is about 2-7 days. However, if ZIKV infection occurs during pregnancy, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12N7/01
CPCC12Q1/04C07K14/43595C12N7/00C12N2770/24121Y02A50/30
Inventor 步志高华荣虹
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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