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Cloning, construction and application of new coronavirus SARS-CoV-2 subgenome replicon based on bacterial artificial chromosome

A genome and replicon technology, applied in applications, viruses, genetic engineering, etc., can solve problems such as screening efficiency obstacles

Pending Publication Date: 2022-03-25
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, while developing vaccines, it is still urgent to screen new, highly efficient, and highly specific drugs that can directly fight viruses. There are still major obstacles

Method used

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  • Cloning, construction and application of new coronavirus SARS-CoV-2 subgenome replicon based on bacterial artificial chromosome
  • Cloning, construction and application of new coronavirus SARS-CoV-2 subgenome replicon based on bacterial artificial chromosome
  • Cloning, construction and application of new coronavirus SARS-CoV-2 subgenome replicon based on bacterial artificial chromosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1: the construction of new coronavirus (SARS-CoV-2) subgenome clone

[0072] Using BAC ( figure 1 ) and in vitro connection method ( Figure 4 ) to construct the subgenomic DNA of the new coronavirus (SARS-CoV-2), and use the in vitro transcription kit (mMESSAGE mMACHINE, ambion) to transcribe in vitro to produce subgenomic RNA containing a reporter gene (such as sGluc), and through transfection, it can initiate The viral replication cycle results in the expression of a reporter gene, and the level of expression of the reporter gene indicates the level of viral replication. And there is no difference in the replication ability between the RNA produced by ligation in vitro and the RNA produced by the BAC plasmid ( Figure 4 ).

Embodiment 2

[0073] Embodiment 2: the level of replication of new coronavirus (SARS-CoV-2) subgenomic RNA in cells

[0074] The RNA produced by in vitro transcription is transfected into different cells using conventional RNA transfection reagents, such as Mirus mRNA transfection reagent, and by comparing the RNA polymerase-deficient mutant (SAA) control, it shows obvious virus replication signals ( figure 2 ).

Embodiment 3

[0075] Embodiment 3: Utilize the replicon system with reporter gene to carry out new coronavirus (SARS-CoV-2) drug evaluation

[0076] The cells were pretreated with different compounds, and then transfected with the subgenomic RNA of the new coronavirus (SARS-CoV-2) as described above, and the luciferase signal secreted in the supernatant was detected at different times after transfection to evaluate the antiviral activity of the drug. Such as image 3 As shown, 10nM Remdesivir, 100U / ml IFN-α, 1μM Daclatasvir, 10μM Sofosbuvir, 50μM 2CMC were treated for 4 hours, and then transfected with subgenomic RNA of SARS-CoV-2, only Remdesivir and interferon IFN -α exhibited significant antiviral activity.

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Abstract

The invention belongs to the technical field of biological medicines, and relates to cloning, construction and application of a new coronavirus SARS-CoV-2 subgenome replicon based on a bacterial artificial chromosome BAC. The invention provides an expression product cloned by a new coronavirus subgenome replicon, the expression product does not contain structural protein S, M or E of the new coronavirus, and the invention also comprises a coding sequence, a preparation method and application of the expression product. The invention relates to DNA (Deoxyribose Nucleic Acid) cloning of a new coronavirus (SARS-CoV-2) with a Gaussian luciferase reporter gene based on a bacterial artificial chromosome BAC (Bacterial Artificial Chromosome); four DNA clones respectively containing four new coronavirus gene segments and a reporter gene Gluc; the invention also discloses a preparation method of a new coronavirus (SARS-CoV-2) subgenome replicon by removing virus structural proteins S, M and E and adding a reporter gene. The system provided by the invention can be used for screening new coronavirus antiviral drugs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to the cloning, construction and application of a new coronavirus SARS-CoV-2 subgenome replicon based on bacterial artificial chromosome BAC. It specifically involves a new coronavirus (SARS-CoV-2) DNA clone with a Gaussia luciferase reporter gene based on a bacterial artificial chromosome BAC; it can be used for in vitro linking to generate viral subgenomic DNA, which contains four new coronavirus gene fragments and a reporter 4 DNA clones of the gene Gluc; and a construction strategy for constructing a new coronavirus (SARS-CoV-2) subgenome replicon by deleting the viral structural proteins S, M and E, and adding a reporter gene; the system of the present invention can be used for screening the new coronavirus Viral antiviral drugs. Background technique [0002] In view of the current lack of effective anti-new coronavirus (SARS-CoV-2) drugs, related vaccines have high hopes as...

Claims

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Application Information

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IPC IPC(8): C12N15/50C12N15/52C12N5/10C12Q1/26
CPCC12N7/00C12N15/52C12Q1/26C12N2770/20021
Inventor 易志刚袁正宏
Owner FUDAN UNIV
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