Papaya-like protease inhibitor screening kit and application thereof

A technology for inhibitor screening and protease, which is applied in the determination/inspection of microorganisms, measurement devices, fluorescence/phosphorescence, etc., and can solve problems such as inaccurate detection, wrong screening of inhibitors, and missing screening.

Active Publication Date: 2022-01-14
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using this method to detect covid-19 in 2020 is inaccurate and may lead to wrong and missed screening of inhibitors

Method used

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  • Papaya-like protease inhibitor screening kit and application thereof
  • Papaya-like protease inhibitor screening kit and application thereof
  • Papaya-like protease inhibitor screening kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The specific implementation process is:

[0031] (1) The stock solutions of SARS-Cov2 papain-like protease and substrate peptide were stored in a -80°C refrigerator;

[0032] (2) Melt the SARS-Cov2 papain-like protease in a cryopreservation plate (-4 to 4°C) at room temperature, take 1uL and dilute it in 98uL borate borax buffer (PH=7.4), and add it to the detection plate;

[0033] (3) Add 1uL of substrate peptides (concentrations: 0.1mM, 0.25mM, 0.5mM, 1mM, 2.5mM, 5mM) to the solution obtained in the above step (3), incubate at 37 degrees Celsius with a fluorescent microplate reader, and Use a fluorescent microplate reader to monitor the excitation at 342nm and the fluorescence emission value at 496nm, detect while incubating, and collect a little every 1 minute;

[0034] The fluorescence intensity of the substrate peptide slowly increases with time under enzyme metabolism as follows figure 1 .

Embodiment 2

[0036] The specific implementation process is as follows:

[0037] (1) The stock solutions of SARS-Cov2 papain-like protease and substrate peptide were stored in a -80°C refrigerator;

[0038] (2) Slowly melt the SARS-Cov2 papain-like protease on ice at room temperature, take 1uL in 98uL of borate borax buffer solution (PH=7.4), and add it to the detection plate;

[0039] (3) Add 1uL of the substrate peptide solution (stock solution concentration is 0.1mM, 0.25mM, 0.5mM, 1mM, 2.5mM, 5mM) into the above buffer system solution, incubate for 30min, detect while incubating, use fluorescent enzyme labeling The instrument monitors the 342nm excitation, collects the fluorescence emission value at 496nm, and collects a point every 1 minute; the result is expressed as the relationship between the substrate conversion efficiency and the substrate concentration, that is, the kinetic properties of the enzyme, such as figure 2 As shown, this result shows that this substrate peptide is hy...

Embodiment 3

[0042] The specific implementation process is as follows:

[0043] Both the stock solution of SARS-Cov2 papain-like protease and substrate peptide were stored in a -80°C refrigerator;

[0044] 1) Slowly melt SARS-Cov2 papain-like protease on ice at room temperature, take 1uL in 98uL of borate borax buffer solution (PH=7.4), and add it to a 384-well Corning assay plate;

[0045] 2) Add 1 uL of the inhibitor stock solution (the stock solution concentration is 0.05mM, 0.1mM, 0.25mM, 0.5mM, 1mM, 2.5mM) into the above buffer system solution, and incubate for 10min; the fluorescence intensity of the solution system without inhibitor as comparison;

[0046] 3) Add 1uL of the substrate peptide solution (concentration of the stock solution is 0.5mM) into the buffer system solution in the above step 2), incubate for 30min, detect while incubating, and use a fluorescent microplate reader to monitor the excitation at 342nm and the fluorescence at 496nm Emission value, one point every 1 ...

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Abstract

The invention relates to an SARS-Cov-2 papaya-like protease inhibitor screening kit and a screening method thereof. The invention provides a screening method of an inhibitor capable of being used for SARS-Cov-2 papaya-like protease. The kit comprises the following components: SARS-Cov-2 papaya-like protease, a substrate peptide Dabcyl-FTLKGGAPTKVT-E(Edans), and a boric acid and borax buffer solution. According to the substrate peptide, fluorophore quenching is relieved under the action of the SARS-Cov-2 papaya-like protease, activity detection of the SARS-Cov-2 papaya-like protease is achieved through the difference of fluorescence properties before and after the reaction, and the method can be used for rapid screening of high-flux inhibitors which are simple, convenient and good in specificity.

Description

technical field [0001] The invention relates to the field of drug screening, in particular to a method for screening inhibitors of SARS-Cov-2 papain-like protease. Background technique [0002] At present, no clinically approved vaccines and drugs against COVID-19 have been found, and technical breakthroughs in research on COVID-19 and the development of drugs and vaccines are still imminent. [0003] SARS-Cov-2 papain-like protease is the covid-19 proteolytic enzyme of the global pandemic in 2019-2020. It plays an important role in the virus replication process and is therefore an important target for antiviral drug research. Screening inhibitors of this enzyme is of great help in finding specific drugs for this virus. [0004] Most of the papain-like protease detection methods currently used use Z-RLRGG-AMC / RLRGG-AMC as the substrate. This method follows the papain-like protease detection method of SARS in 2003 and MERS in 2013. Using this method to detect covid-19 in 20...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/37G01N21/64
CPCC12Q1/37G01N21/6428Y02A50/30
Inventor 韩克利贾燕
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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