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Restrictive replicated west nile virus system for expressing green fluorescent protein and application thereof

A technology of green fluorescent protein and West Nile virus, applied in applications, viruses, viral peptides, etc., can solve problems hindering vaccine and drug research, and achieve high safety and simplicity

Active Publication Date: 2017-05-31
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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  • Restrictive replicated west nile virus system for expressing green fluorescent protein and application thereof
  • Restrictive replicated west nile virus system for expressing green fluorescent protein and application thereof
  • Restrictive replicated west nile virus system for expressing green fluorescent protein and application thereof

Examples

Experimental program
Comparison scheme
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 restrictive replicated west nile virus system for expressing green fluorescent protein (GFP) and an application thereof. The restrictive replicated west nile virus system for expressing the green fluorescent protein, disclosed by the invention, comprises a west nile virus restrictive replicon plasmid and a recombinant cell line capable of stably expressing west nile virus C-prM-E protein. A restrictive replicated west nile virus (deltaWNV) obtained by the system is obtained, and the virus has properties similar with a WNV whole virus in a BWNV-CME cell line; the GFP can be expressed and the system has a visual effect; more importantly, the virus only can be replicated in the system; when other cells are infected, progeny viruses are not generated, so that the safety is extremely high; and the restrictive replicated west nile virus system can be used for detecting a WNV neutralizing antibody and screening an anti-virus medicine. According to the restrictive replicated west nile virus system, the safety and convenience of WNV related operation are improved, and the system does not depend on BSL-3 high-grade biological safety condition; and a good technical means is provided for WNV vaccine evaluation, anti-virus medicine screening and researches of a virus pathogenic mechanism.

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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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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