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
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Problems solved by technology

Thus, substrates with disrupted sequence structure at the cleavage site may not...
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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.

Application Domain

Fluorescence/phosphorescence

Technology Topic

EDANSExcitation wavelength +8

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(2)

Example Embodiment

[0022] Example 1
[0023] A coronavirus protease survival method based on fluorescence resonance energy transfer, including the following:
[0024] (1) Detylpaboba protease samples on ice;
[0025] (2) A mixture of 20 mM Tris-HCl, pH 7.3, 100 mM NaCl and 1 mM EDTA was added, and 1 mM was added to the solution to prepare a 2-final concentration of papaya protease sample liquid and 2 final concentration. FRET peptide solution;
[0026] (3) In the black half region 96-well plate, 25 μl of 2 times the final concentration of papaya protease sample solution and 25 μl of the final concentration of FRET peptide solution were added to 50 μl of reaction volume;
[0027] (4) In the shaker for 1 minute, the experimental holes were sealed with a microplate sealing film, and incubated at 37 ° C for 60-90 min;
[0028] (5) The temperature of the hole is lowered to the ambient temperature, remove the sealing film of the orifice plate;
[0029] (6) The fluorescence intensity value when the excitation wavelength / emission wavelength = 340/495 nm is read on the fluorescent device;
[0030] (7) Curve with edans / dabcyl standard image 3 As shown, it is determined that the concentration of the generated EDANS label peptide fragment is determined, and the modifier specific activity is calculated using formula, the calculation formula of the enzyme specific activity is: enzyme ratio activity (SA) (PMol / min / mg) = (EDANS (μM) * Reaction volume (μL)) / (reaction time (min) * enzyme (Mg)), the generated papaya protease titration curve figure 2 Indicated.

Example Embodiment

[0031] Example 2
[0032] A coronavirus protease survival method based on fluorescence resonance energy transfer, including the following:
[0033] (1) Thade the pancreatic curlin sample on ice;
[0034] (2) Use 50 mM hepes, pH 7.5 solutions and 1 mm new DTT as a solution to formulate 2 times the final concentration of glutamin sample liquid and 2 times the final concentration of FRET peptide solution;
[0035] (3) In a black half-region 96-well plate, 25 μl of 2 times the final concentration of glutamin sample solution and 25 μl of 2 times the final concentration of FRET peptide solution were added to 50 μl of reaction volume;
[0036] (4) In the shaker for 1 minute, the experimental holes were sealed with a microplate sealing film, and incubated at 37 ° C for 60-90 min;
[0037] (5) The temperature of the hole is lowered to the ambient temperature, remove the sealing film of the orifice plate;
[0038] (6) The fluorescence intensity value when the excitation wavelength / emission wavelength = 340/495 nm is read on the fluorescent device;
[0039] (7) Curve with edans / dabcyl standard image 3 As shown, it is determined that the concentration of the generated EDANS label peptide fragment is determined, and the modifier specific activity is calculated using formula, the calculation formula of the enzyme specific activity is: enzyme ratio activity (SA) (PMol / min / mg) = (EDANS (μM) * Reaction volume (μL)) / (reaction time (min) * enzyme (Mg)), the resulting generated class celase titration curve figure 1 Indicated.
[0040] The working principle of the present invention is that the enzyme activity of the two coronavirus proteases of papaya proteases and prolovirus can be detected by detecting the detection method based on fluorescence resonance energy transfer (FRET), and EDANS Fluorescence is quenched because it is close to the dabcyl quencher; when the substrate is cleaved into two separate segments, the fluorescence is recovered and monitored when excited wavelength / emission wavelength = 340 nm / 495 nm.

PUM

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