Restricted Replication West Nile Virus System Expressing Green Fluorescent Protein and Its Application

A green fluorescent protein, West Nile virus technology, applied in the direction of virus, application, viral peptide, etc., can solve the problems of hindering vaccine and drug research, and achieve the effect of high safety and simplicity

Active Publication Date: 2021-03-09
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

However, WNV is a zoonotic pathogen, and the live virus operation of vaccines and other related research needs to be carried out in high-level biosafety laboratories, which hinders related vaccine and drug research to a certain extent

Method used

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  • Restricted Replication West Nile Virus System Expressing Green Fluorescent Protein and Its Application
  • Restricted Replication West Nile Virus System Expressing Green Fluorescent Protein and Its Application
  • Restricted Replication West Nile Virus System Expressing Green Fluorescent Protein and Its Application

Examples

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Effect test

Embodiment 1

[0036] Example 1 Construction of Restricted Replication West Nile Virus System Expressing Green Fluorescent Protein

[0037] 1 Materials and methods

[0038] 1.1 Carriers, cells and main reagents

[0039] Expression vector plasmid pCAG-neo plasmid (Li Yenan et al., Chinese Journal of Preventive Veterinary Medicine, 2013, 35(3)::189-192; Hua RH, et al, BMC Biotechnology, 2014, 14:62) and pCAG-WNV plasmid (Guo Liping .Construction of a cell line stably expressing West Nile virus prM-E protein [D]. Chinese Academy of Agricultural Sciences, 2015) was constructed according to the method reported in the literature; BHK-21 cells were preserved by our laboratory, and W-92 cell line was constructed by our laboratory. DMEM, superior fetal bovine serum (FBS) and Opti-MEM were purchased from Gibco Company; X-treme HP transfection reagent was purchased from Roche Company; FITC-labeled goat anti-mouse IgG was purchased from Zhongshan Jinqiao Company; infrared fluorescent-labeled anti-mouse...

Embodiment 2

[0065] Example 2 Application of Restricted Replication West Nile Virus System Expressing Green Fluorescent Protein

[0066] 1 method

[0067] 1.1 Restricted replication virus system for WNV neutralizing antibody detection

[0068] Will 4×10 5 / mL BWNV-CME cells were inoculated in 24-well cell culture plates, 500 μL / well, cultured overnight at 37°C, 5% CO2, to make the cells grow into a single layer; inactivate the WNV positive serum and negative serum and start the gradient from 1:10 After dilution, mix it with an equal volume of △WNV, set up multiple wells, and place it in a 37°C, 5% CO2 incubator for 1 hour; then inoculate 100 μl of the △WNV virus antibody mixture on a monolayer of BWNV-CME cells, and incubate at 37°C, 5% CO2 After culturing for 6 hours, add 3% methylcellulose in DMEM, and observe the expression of green fluorescence after culturing for 3 days. Plaque counts were performed 6 days later. The neutralizing antibody titer of each serum to be tested was calcu...

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Abstract

The invention discloses a restricted replication West Nile virus system expressing green fluorescent protein and its application. The restricted replication West Nile virus system expressing green fluorescent protein of the present invention includes a West Nile virus restricted replicon plasmid and a recombinant cell line stably expressing West Nile virus C-prM-E protein. Restricted replication West Nile virus (△WNV) obtained using the system of the present invention, which has similar characteristics to WNV whole virus in BWNV-CME cell line, and can express GFP, has a visual effect, and more importantly , the virus can only replicate in this system, and cannot produce offspring viruses when it infects other cells, so it has extremely high safety and can be used for WNV neutralizing antibody detection and antiviral drug screening. The proposal of the present invention improves the safety and simplicity of WNV-related operations, does not depend on the high-level biosafety conditions of BSL-3, and provides a good technical means for WNV vaccine evaluation, antiviral drug screening and virus pathogenic mechanism research.

Description

technical field [0001] The invention relates to a virus replication system and application thereof, in particular to a limited replication West Nile virus system expressing green fluorescent protein and application thereof, and the invention belongs to the field of biotechnology. Background technique [0002] West Nile Virus (WNV) belongs to the Flaviviridae family and belongs to the Flavivirus genus. It is an important zoonotic virus that can cause severe neurological diseases in humans. The diameter of WNV virus particles is about 40nm-60nm, with an envelope, and the viral nucleocapsid in the membrane is icosahedral, with a diameter of about 30nm. The WNV genome is a single-stranded positive-sense RNA with a length of about 11kb. The genome consists of a non-coding region (UTR) with a length of 96nt at the 5' end and a length of 337-649nt at the 3' end, and a single open reading frame (ORF) about 10.3kb in length. ORF starts at position 97 at the 5' end and ends at posit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/40C12N15/85C12N5/10C12N7/01C07K14/435G01N33/569
CPCC07K14/005C07K14/43595C12N7/00C12N15/85C12N2770/24121C12N2770/24122G01N33/56983G01N2500/00
Inventor 华荣虹步志高
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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