A fusion protein and its application in constructing a system for screening coronavirus 3cl protease inhibitors

A protease inhibitor and fusion protein technology, applied in the field of medical virology, can solve the problems of difficult to obtain efficient targeted inhibitory drugs, limited screening and validation of high-efficiency inhibitory drugs, etc., and achieves intuitive effect, low false positive rate, and screening mechanism. clear effect

Active Publication Date: 2022-04-01
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 Tropical Marine Sponge Axinella Corrugata, 2006; Junwei Zhou et al, Identification of Novel Proteolytically Inactive Mutations in Coronavirus 3C-like Protease Using a Combined Approach, 2019), cannot truly reproduce the working state of 3CL protease in cells, thus limiting the process of screening highly effective inhibitory drugs and further verifying the activity of the screened drugs, making it difficult to obtain highly effective targeted inhibitory drugs

Method used

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  • A fusion protein and its application in constructing a system for screening coronavirus 3cl protease inhibitors
  • A fusion protein and its application in constructing a system for screening coronavirus 3cl protease inhibitors
  • A fusion protein and its application in constructing a system for screening coronavirus 3cl protease inhibitors

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 its application in constructing a system for screening coronavirus 3CL protease inhibitors. The present invention firstly provides a fusion protein, which is formed by fusing the ubiquitinated protein at the N-terminus and the protein with reporter function at the C-terminus through the amino acid fragment of the 3CL protease cleavage site; the amino acid fragment of the 3CL protease cleavage site is 3CL Proteases can recognize and digest amino acid fragments. A system for screening coronavirus 3CL protease inhibitors was constructed by using the fusion protein, its coding gene, its vector or its expressing recombinant cells. The system is convenient, fast, easy to operate, and has good specificity and sensitivity. ;Using this system can conveniently and effectively screen coronavirus 3CL protease inhibitors. The screening mechanism is clear, the speed is fast, the effect is intuitive, and the versatility is strong, the repeatability is good, and the false positive rate is low. It has a good application prospect.

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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Patent Type & Authority Patents(China)
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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