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Pseudovirus of COVID-19 coronavirus as well as preparation method and application of pseudovirus

A technology of COVID-19 and coronavirus, applied in the field of pseudovirus of COVID-19 coronavirus and its preparation, can solve problems such as harsh experimental conditions, achieve the effect of reducing risk, convenient and rapid detection and analysis

Inactive Publication Date: 2021-02-19
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to study drugs that can inhibit the virus from entering cells, direct research on 2019-nCoV is quite dangerous and requires harsh experimental conditions

Method used

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  • Pseudovirus of COVID-19 coronavirus as well as preparation method and application of pseudovirus
  • Pseudovirus of COVID-19 coronavirus as well as preparation method and application of pseudovirus
  • Pseudovirus of COVID-19 coronavirus as well as preparation method and application of pseudovirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The construction of embodiment 1COVID-19S protein expression vector plasmid one and plasmid two

[0045] Query the SARS-CoV-2 S glycoprotein gene sequence MN908947.3 expressing membrane protein in NCBI:

[0046] Plasmid 1: remove the 19 amino acids at the C-terminus of S glycoprotein.

[0047] Plasmid 2: Connect the signal peptide (GSP) of the VSVG protein corresponding to amino acid 16aa before the sequence corresponding to amino acid 14-1213 of the extracellular region of the COVID-19S protein, followed by the transmembrane region and the intracellular region of the VSVG protein (G TMC ), after codon optimization, it was handed over to Beijing Synbiosis Technology Co., Ltd. for gene synthesis and connected into the eukaryotic cell expression vector pCMV3 through homologous recombination, such as figure 1 shown.

Embodiment 2

[0048] Preparation and concentration of embodiment 2 pseudovirus

[0049] Using polyetherimide, plasmid one and plasmid two containing SARS-CoV-2 S glycoprotein membrane protein and plasmid three containing defective other viral genomes carrying reporter gene, and lentiviral packaging plasmid (plasmid four) were passed through three The plasmid lentiviral packaging system was transfected into the packaging cell line HEK293T, and the cell supernatant was collected after 48-72 hours, and the viral supernatant was concentrated by Lenti-X Concentrator, and then the obtained pseudo-covid-19 coronavirus was extracted and purified. virus particles.

Embodiment 3

[0050] The identification of embodiment 3 pseudoviruses

[0051] Take 50 microliters of the concentrate containing the pseudovirus of COVID-19 coronavirus, infect known COVID-19 susceptible cells 293T-hACE2 and non-susceptible cells 293T in 96-well plate, infect 30,000 cells per well . Discard the medium after 12 hours of infection, replace it with fresh complete medium of DMEM, and continue to culture at 37°C for 48 hours, then detect the expression of EGFP on the cell surface by flow cytometry, and further identify the infection efficiency of the pseudovirus by measuring the expression of luciferase .

[0052] Such as figure 2 As shown, pseudoviral particles can well infect susceptible cells 293T-hACE2, and the infection efficiency of the optimized plasmid is significantly improved, which is 10 times higher than that of the unoptimized S protein vector. It shows that using the second plasmid to package the pseudovirus is more effective than using the plasmid one to med...

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Abstract

The invention discloses a pseudovirus of COVID-19 coronavirus as well as a preparation method and application of the pseudovirus, and belongs to the technical field of biological medicines. Accordingto the pseudovirus, COVID-19 membrane protein participates in packaging of a defective virus genome with a reporter gene, one or more sections of exogenous sequences for encoding bioactive substancescan be included under the condition that the reporter gene sequence is carried or not carried, the reporter gene is selected from enhanced green fluorescent protein (EGFP) or luciferase. the defectivevirus genome or a portion thereof are derived from VSV, HIV, SARS and other viruses, and one or more structural genes contained in the defective virus genome can be removed or regulated to be inactivated. The pseudovirus can reduce the risk of virus research to the maximum extent, and can also be used for screening antiviral drugs, determining the titer of a neutralizing antibody in the body of an infected person, searching for an epitope combined with the neutralizing antibody on a COVID-19 coronavirus surface antigen and evaluating the immune effect of vaccines.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a pseudovirus of COVID-19 coronavirus and a preparation method and application thereof. Background technique [0002] Novel Coronavirus Disease 2019 (COVID-19), referred to as "New Coronary Pneumonia", was named "2019 Coronavirus Disease" by the World Health Organization. Coronavirus (CoV) is a large family of viruses. So far, outbreaks of human infection are mainly caused by SARS-CoV, MERS-CoV and SARS-CoV-2. Among them, SARS-CoV-2 (also known as 2019-nCoV) has never been A new strain of coronavirus found in humans. This virus is the seventh coronavirus that can infect humans. The incubation period for human infection with 2019-nCoV is generally 1-14 days. Common signs after infection with 2019-nCoV include respiratory symptoms, fever, cough, shortness of breath, and dyspnea. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/50C12N15/867C12N15/65C12N15/52C12N7/00C12N5/10C12Q1/18G01N33/68G01N33/569G01N33/58
CPCC07K14/005C12N15/86C12N15/65C12N15/52C12N7/00C12N5/0686G01N33/6854G01N33/56983G01N33/581C12N2740/15043C12N2770/20021C12N2770/20022C12N2800/107C12N2510/00C12N2503/02G01N33/5044G01N2333/165G01N2469/10G01N2500/10
Inventor 仝娇吴成豪朱宸希来函宇冯春朝周大鹏
Owner TONGJI UNIV
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