Fusion protein and application thereof to construction of system for screening coronavirus 3CL protease inhibitor

A technology of fusion protein and protease, which is applied in the field of medical virology, can solve the problems of difficulty in obtaining highly effective targeted inhibitory drugs, restrict screening and verification of highly effective inhibitory drugs, and achieve intuitive effect, low false positive rate, and clear screening mechanism Effect

Active Publication Date: 2020-11-03
SUN YAT SEN UNIV
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Problems solved by technology

At present, the existing drug screening methods targeting 3CL protease are mostly based on computer analysis and the extracellular environment of the extracellular system (Yu-ChihLiu et al, Screening of drugs by FRET analysis identifies inhibitors of SARS-CoV 3CLprotease, 2005; Pamela Hamilletal, Development of a Red-Shifted Fluorescence-Based Assay for SARS-coronavirus 3CL Protease: Identification of a Novel Class of anti-SARS AgentsFrom the Tropi...

Method used

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  • Fusion protein and application thereof to construction of system for screening coronavirus 3CL protease inhibitor
  • Fusion protein and application thereof to construction of system for screening coronavirus 3CL protease inhibitor
  • Fusion protein and application thereof to construction of system for screening coronavirus 3CL protease inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1UB (4)-CScon-Fluc fusion protein and 3CL protease single gene double expression viral vector construction

[0069] In this example, the enzyme cloning method was used to achieve the directional insertion of the small nucleic acid fragment CScon corresponding to the amino acid sequence of the 3CL protease cleavage site into the constructed gene expressing the UB(4)-Fluc fusion protein to obtain UB(4)-CScon -Fluc gene; then use the seamless cloning technology to recombine the constructed UB(4)-CScon-Fluc gene with the single-gene double-expression virus vector to obtain a single-gene double-expression virus containing the UB(4)-CScon-Fluc gene Vector; finally, the gene encoding 3CL protease is recombined with the single-gene double-expression viral vector to obtain a single-gene double-expression viral vector containing both the UB(4)-CScon-Fluc gene and the 3CL protease gene. Specific steps are as follows:

[0070] 1. Cloning of UB(4)-CScon-Fluc gene

[007...

Embodiment 2

[0091] Example 2 Using mammalian cells to construct stable expression cell lines

[0092] The single-gene double-expression viral vector constructed in Example 1 containing both the UB(4)-CScon-Fluc gene and the 3CL protease gene was used for the construction of a stable expression cell line, and it was integrated into the host cell by virus transfection (Mammalian cell) chromosome, so that host cells can express UB(4)-CScon-Fluc fusion protein and 3CL protease for a long time, and construct a drug screening system. Specific steps are as follows:

[0093] 1. Virus packaging

[0094] Prepare HEK-293T cells in a good state, and plate the cells 24 hours before transfection (select a cell culture dish with a diameter of 6 cm according to the experimental requirements), take HEK-293T cells in the logarithmic growth phase for virus packaging, and control the cell density At about 80%, take high-quality plasmids (including virus packaging plasmids and viral vectors) that have remov...

Embodiment 3

[0102] Example 3 High-throughput screening of anti-coronavirus infection drugs

[0103] 1. Drug treatment

[0104] The cell line with stable and high expression of the target protein obtained in Example 2, which is in a good preparation state, is plated 21 hours before drug treatment (cell culture dishes are selected according to experimental requirements), and the obtained candidate drugs are used at appropriate concentrations according to drug solubility and other characteristics. Cells were treated for 12h.

[0105] 2. Luciferase detection

[0106] The original medium was discarded, and the remaining medium was gently washed away with PBS solution. According to the instructions of the dual luciferase reporter gene detection kit, the overall expression level of luciferase at the cellular level was detected using imatinib (Imatinib) As a positive control, data analysis and comparison with the negative control (cells not added with the candidate drug) are carried out to scre...

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Abstract

The invention discloses a fusion protein and an application thereof to construction of a system for screening a coronavirus 3CL protease inhibitor. The invention firstly provides the fusion protein. The fusion protein is formed by fusing a ubiquitination protein at an N end and a protein with a reporting function at a C end through a 3CL protease restriction enzyme cutting site amino acid fragment, wherein the 3CL protease restriction enzyme cutting site amino acid fragment is an amino acid fragment which can be recognized by 3CL protease and can be subjected to restriction enzyme cutting. A system for screening the coronavirus 3CL protease inhibitor is constructed by utilizing the fusion protein, an encoding gene of the fusion protein, a carrier containing the fusion protein or recombinant cells for expressing the fusion protein, and the system is convenient and rapid, is simple to operate and has good specificity and sensitivity; by utilizing the system, the coronavirus 3CL proteaseinhibitor can be conveniently and effectively screened, the screening mechanism is clear, the speed is high, the action effect is visual, the universality is high, the repeatability is good, the falsepositive rate is low, and the system has a good application prospect in screening of the coronavirus 3CL protease inhibitor.

Description

technical field [0001] The invention belongs to the technical field of medical virology. More specifically, it relates to a fusion protein and its application in constructing a system for screening coronavirus 3CL protease inhibitors. Background technique [0002] Coronaviruses are one of the main pathogens that infect the human respiratory system, causing severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19). At present, there is still a lack of understanding of the mechanism of coronavirus-induced disease and the molecular and cellular biology of the disease, and there is no specific antiviral drug for this type of disease. [0003] The establishment of a drug screening model for coronavirus is an important basis for the pathogenic mechanism of coronavirus disease and its drug research. The existing drug screening model for coronavirus is mainly based on the in vitro enzyme activity detection system of t...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/86C12N5/10C12Q1/6897C12Q1/37C12Q1/02
CPCC07K7/06C12N15/86C12N5/0603C12N5/0686C12Q1/6897C12Q1/37G01N33/5008C07K2319/50C07K2319/95C07K2319/60C07K2319/61C12N2800/107C12N2510/00C12N2503/02G01N2500/10Y02A50/30
Inventor 潘纪安彭小雪杜柳冰蒋依伶
Owner SUN YAT SEN UNIV
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