Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Coronavirus protease activity measurement method based on fluorescence resonance energy transfer

A fluorescence resonance energy and coronavirus technology, which is applied in the field of enzyme activity detection, can solve the problem that the substrate cannot be cut, and achieve the effect of improving enzyme reaction efficiency, less workload, and low detection limit

Pending Publication Date: 2021-03-09
苏州新格诺康生物技术有限公司
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, substrates with disrupted sequence structure at the cleavage site may not be cleaved as efficiently as substrates containing intact recognition sequences

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Coronavirus protease activity measurement method based on fluorescence resonance energy transfer
  • Coronavirus protease activity measurement method based on fluorescence resonance energy transfer
  • Coronavirus protease activity measurement method based on fluorescence resonance energy transfer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] A coronavirus protease assay method based on fluorescence resonance energy transfer, specifically including the following:

[0024] (1) thawing papain-like samples on ice;

[0025] (2) Use a mixture of 20mM Tris-HCl, pH 7.3, 100mM NaCl and 1mM EDTA, and then add 1mM freshly prepared DTT as a solution to prepare papain-like sample solution with 2 times final concentration and 2 times final concentration FRET peptide solution;

[0026] (3) Add 25 μl of 2-fold final concentration of papain sample solution and 25 μl of 2-fold final concentration of FRET peptide solution to a black half-area 96-well plate to achieve a reaction volume of 50 μl;

[0027] (4) Mix on a shaker for 1 minute, seal the experimental well with a microplate sealing film, and incubate at 37°C for 60-90min;

[0028] (5) When the temperature of the orifice plate drops to ambient temperature, remove the sealing film of the orifice plate;

[0029] (6) Read the fluorescence intensity value when excitation...

Embodiment 2

[0032] A coronavirus protease assay method based on fluorescence resonance energy transfer, specifically including the following:

[0033] (1) Thaw the chymotrypsin sample on ice;

[0034] (2) Use 50mM HEPES, pH 7.5 solution and 1mM newly prepared DTT as solutions to prepare 2 times final concentration of chymotrypsin sample solution and 2 times final concentration of FRET peptide solution;

[0035] (3) Add 25 μl of 2-fold final concentration of chymotrypsin sample solution and 25 μl of 2-fold final concentration of FRET peptide solution to a black half-area 96-well plate to achieve a reaction volume of 50 μl;

[0036] (4) Mix on a shaker for 1 minute, seal the experimental well with a microplate sealing film, and incubate at 37°C for 60-90min;

[0037] (5) When the temperature of the orifice plate drops to ambient temperature, remove the sealing film of the orifice plate;

[0038] (6) Read the fluorescence intensity value when excitation wavelength / emission wavelength=340 / 4...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a coronavirus protease activity measurement method based on fluorescence resonance energy transfer. The coronavirus protease activity measurement method specifically comprises the following steps: unfreezing a coronavirus active protease sample on ice; preparing a coronavirus active protease sample solution and an FRET peptide solution by using a lysis buffer solution; adding a coronavirus active protease sample solution and an FRET peptide solution into a black half-region 96-well plate; performing mixing on a shaking table for 1 minute, sealing the experiment holes byusing a microporous plate sealing film, and performing incubating at 37 DEG C; after the temperature of the pore plate is reduced to the ambient temperature, removing the sealing film of the pore plate; reading a fluorescence intensity value when the excitation wavelength / emission wavelength is equal to 340 / 495nm on a luminoscope; and determining the concentration of the generated Edans labeled peptide fragment by using an Edans / Dabcyl standard curve, and calculating the enzyme specific activity by using a formula. The same experimental scheme provided by the invention can be used for detecting the activities of two virus proteases.

Description

technical field [0001] The invention relates to the technical field of enzyme activity detection, in particular to a coronavirus protease activity detection method based on fluorescence resonance energy transfer. Background technique [0002] The new coronavirus produces two proteases that cut the viral polyprotein, papain-like protease (PL pro ), and chymotrypsin (3CL pro ). The measurement of the activities of these two proteases currently lacks a general quantitative assay protocol, although separate assays have been reported for each, such as polyacrylamide gel electrophoresis or high-pressure liquid chromatography. However, these methods are based on the separation of cleaved peptide fragments, so they are quite cumbersome and non-quantitative, and are only suitable for endpoint analysis. It is not practical to apply to high-throughput screening of compound libraries. [0003] In recent years, assays of protease activity using fluorescent probes have also been descri...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6486G01N2021/6432
Inventor 姚晓晖谢志伟张延翀冯颖欣严俊
Owner 苏州新格诺康生物技术有限公司
Features
  • Generate Ideas
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com