Novel coronavirus SARS-CoV-2 replicon as well as construction method and application thereof

A coronavirus and construction method technology, applied in the fields of virology and molecular biology, can solve the problem of no SARS-CoV-2 replicon research report, etc., and achieve the effect of wide coverage, strong applicability, and reduction of biosafety risks

Inactive Publication Date: 2021-02-02
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are currently no reports of SARS-CoV-2 replicons with reporter genes

Method used

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  • Novel coronavirus SARS-CoV-2 replicon as well as construction method and application thereof
  • Novel coronavirus SARS-CoV-2 replicon as well as construction method and application thereof
  • Novel coronavirus SARS-CoV-2 replicon as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0208] To detect the expression of tGFP reporter gene and SARS-CoV-2NP protein in the novel coronavirus SARS-CoV-2 replicon of the present invention, the specific method is:

[0209] Step 1: 293T cells were inoculated into 12-well plates at a seeding density of 4×10 5 cells / well;

[0210] Step 2: 12 hours after inoculation, transfect with Lipofectamine 3000 transfection reagent, and transfect 2 μg of the above-mentioned SARS-CoV-2-GFP replicon plasmid per well;

[0211] Step 3: After 36 hours of transfection, observe the expression of tGFP under a fluorescent microscope, and take pictures for preservation; collect the cells into an Ep tube, and perform Western Blot to detect the expression of tGFP-Bla gene and the expression of SARS-CoV-2NP protein;

[0212] Step 4: Use cell lysate to lyse the cell pellet in the Ep tube, 60 μL for each sample, with a volume ratio of 1:100, add protease inhibitors, and lyse on ice for 30 minutes;

[0213] Step 5: Centrifuge at 12000g at 4°C f...

Embodiment 2

[0227] Detect the inhibitory effect of Remdesivir (English name Remdesivir) and E64-D (Chinese name Alostatin) on the novel coronavirus SARS-CoV-2-GFP replicon of the present invention, the specific method is:

[0228] Step 1: Remdesivir was purchased from selleck, and prepared as a 10mM remdesivir stock solution in DMSO for use; E64-D was purchased from selleck, and prepared as a 40mg / ml E64-D stock solution in DMSO for use;

[0229] Step 2: 293T cells were inoculated into 12-well plates at a seeding density of 4×10 5 cells / well;

[0230] Step 3: Before transfection, 0 μM, 5 μM, 10 μM and 20 μM plus remdesivir stock solutions were added to the cell culture supernatant in the remdesivir group;

[0231] Step 4: 12 hours after inoculation, transfect with Lipofectamine 3000 transfection reagent, and transfect 2 μg of the above-mentioned SARS-CoV-2-GFP replicon plasmid per well;

[0232] Step 5: 12 hours after transfection, add E64-D stock solution to the cell culture supernatan...

experiment example 3

[0235] The half-maximal effect concentration (concentration for 50% of maximal effect, EC 50 ), the specific method is:

[0236] Step 1: Remdesivir was diluted by 3 times, and 10 concentration gradients were prepared with complete medium (0 μM, 0.14 μM, 0.43 μM, 1.37 μM, 4.12 μM, 12.35 μM, 37.03 μM, 111.11 μM μM, 333.33μM and 1000μM), each concentration of remdesivir was added to a 96-well plate at 50 μL / well, and three parallel wells were set for each concentration;

[0237] Step 2: Digest and resuspend 293T cells at a seeding density of 4×10 4 Cells / well / 50 μL was calculated, and the number of cells to be transfected was taken from the resuspended 293T cells;

[0238] Step 3: Transfect the above SARS-CoV-2-GFP replicon with 200ng DNA / well;

[0239] Step 4: In the transfection system in step 3, add 50 μL / well to the 96-well plate containing different concentrations of remdesivir in step 1;

[0240] Step 5: After transfection for 36 hours, observe the expression of tGFP un...

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Abstract

The invention discloses a novel coronavirus SARS-CoV-2 replicon as well as a construction method and application thereof, and belongs to the technical field of virology and molecular biology. The novel coronavirus SARS-CoV-2 replicon is an SARS-CoV-2-GFP replicon with a single reporter gene or an SARS-CoV-2-GFP-Luc replicon with double reporter genes. The invention also discloses the constructionmethod and application of the novel coronavirus SARS-CoV-2 replicon. According to the novel coronavirus SARS-CoV-2 replicon disclosed by the invention, infectious virus particles are not generated, sothat the biological safety risk in the process of screening antiviral drugs by utilizing the infectious virus particles is effectively reduced. Besides, a reporter gene carried by the replicon can allow high-throughput screening to be performed, so that the screening efficiency of anti-novel coronavirus drugs is greatly improved.

Description

technical field [0001] The invention relates to a novel coronavirus SARS-CoV-2 replicon, its construction method and application, and belongs to the technical fields of virology and molecular biology. Background technique [0002] Through laboratory high-throughput deep sequencing technology and epidemiological etiological investigation, the pathogen causing the pneumonia was identified as a new type of coronavirus. On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) issued a statement, officially naming the new coronavirus (2019-nCoV) as "SARS-CoV-2", namely Severe Acute Respiratory Syndrome Coronavirus 2 (Severe Acute Respiratory Syndrome Coronavirus 2). Acute Respiratory Syndrome Coronavirus 2). As of August 2020, the World Health Organization has received reports of more than 20 million infections and more than 760,000 deaths worldwide. Since the outbreak of the novel coronavirus, scientists from all over the world have begun to unite and de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/50C12N15/65C12N15/81
CPCC07K14/005C12N15/65C12N15/81C12N2770/20022C12N2770/20051
Inventor 赵振东王蓓张重阳黄鹤
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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