CN112461802A - Coronavirus protease activity detection method based on fluorescence resonance energy transfer - Google Patents
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- 108091005804 Peptidases Proteins 0.000 title claims abstract description 27
- 239000004365 Protease Substances 0.000 title claims abstract description 27
- 238000002866 fluorescence resonance energy transfer Methods 0.000 title claims abstract description 25
- 230000000694 effects Effects 0.000 title claims abstract description 24
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 23
- 238000001514 detection method Methods 0.000 title abstract description 11
- 241000004176 Alphacoronavirus Species 0.000 title 1
- 241000711573 Coronaviridae Species 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 24
- 102000004190 Enzymes Human genes 0.000 claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 17
- 238000007789 sealing Methods 0.000 claims abstract description 15
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 11
- 239000011148 porous material Substances 0.000 claims abstract description 10
- 239000012488 sample solution Substances 0.000 claims abstract description 10
- 239000000523 sample Substances 0.000 claims abstract description 9
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 102000007079 Peptide Fragments Human genes 0.000 claims abstract description 6
- 108010033276 Peptide Fragments Proteins 0.000 claims abstract description 6
- 230000005284 excitation Effects 0.000 claims abstract description 6
- 238000012360 testing method Methods 0.000 claims abstract description 6
- 239000012139 lysis buffer Substances 0.000 claims abstract description 5
- 238000010257 thawing Methods 0.000 claims abstract description 5
- 239000012528 membrane Substances 0.000 claims abstract description 3
- 102100025566 Chymotrypsin-like protease CTRL-1 Human genes 0.000 claims description 12
- 101000856199 Homo sapiens Chymotrypsin-like protease CTRL-1 Proteins 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
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- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000003614 protease activity assay Methods 0.000 claims description 2
- 238000013268 sustained release Methods 0.000 claims description 2
- 239000012730 sustained-release form Substances 0.000 claims description 2
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- 108700022715 Viral Proteases Proteins 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
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- 238000000954 titration curve Methods 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 3
- NTQCWVJNMKBKFB-UHFFFAOYSA-N 2-(7-amino-2-oxochromen-4-yl)acetamide Chemical compound C1=C(N)C=CC2=C1OC(=O)C=C2CC(=O)N NTQCWVJNMKBKFB-UHFFFAOYSA-N 0.000 description 2
- JYOAXOMPIXKMKK-YUMQZZPRSA-N Leu-Gln Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C([O-])=O)CCC(N)=O JYOAXOMPIXKMKK-YUMQZZPRSA-N 0.000 description 2
- BTKUIVBNGBFTTP-WHFBIAKZSA-N Ser-Ala-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCC(O)=O BTKUIVBNGBFTTP-WHFBIAKZSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- 230000017854 proteolysis Effects 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 108010021648 semen liquefaction factor Proteins 0.000 description 1
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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Abstract
The invention provides a coronavirus protease activity detection method based on fluorescence resonance energy transfer, which specifically comprises the following steps: thawing a coronavirus active protease sample on ice; preparing a coronavirus active protease sample solution and a FRET peptide solution by using a lysis buffer solution; adding coronavirus active protease sample solution and FRET peptide solution into a black half-zone 96-well plate; mixing on a shaking table for 1 minute, sealing the test well with a microplate sealing membrane, and incubating at 37 ℃; removing the sealing film of the pore plate when the temperature of the pore plate is reduced to the ambient temperature; reading the fluorescence intensity value of 340/495nm at the excitation wavelength/emission wavelength on a fluorometer; the concentration of the produced Edans-labeled peptide fragment was determined using an Edans/Dabcyl standard curve, and the specific enzyme activity was calculated using the formula. The same experimental protocol provided by the invention can be used for detecting the activity of two viral proteases.
Description
Technical Field
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
The novel coronaviruses produce two proteases that cleave the viral polyprotein, papain-like (PL)pro) And chymotrypsin-like enzyme(3CLpro). The measurement of the activity of these two proteases currently lacks a universal quantitative detection scheme, although separate methods for their activation have been reported, such as polyacrylamide gel electrophoresis or high pressure liquid chromatography. However, these methods are based on the isolation of peptide fragments after cleavage and are therefore rather cumbersome and non-quantitative and only suitable for end-point analysis, which is not practical if applied to high-throughput screening of compound libraries.
In recent years, the use of fluorescent probes for protease activity measurements has also been described in the literature. Fluorophores, such as 7-amino-4-carbamoylmethylcoumarin (ACC) covalently attached to the terminus of a substrate polypeptide, can be used as reporter groups for proteolysis. However, recognition of these substrates by proteases may be affected because only one side contains the homologous sequence of the native substrate and the other side is replaced by a fluorophore at the site that would otherwise require cleavage. For example, 3CLproThere is a Leu-Gln ↓ Ser-Ala-Gly recognition sequence, where the arrow marks the cleavage site. Although the N-terminal Leu-Gln sequence plays a key role in binding specificity, the C-terminal Ser-Ala-Gly sequence may play an important role in binding affinity and cleavage efficiency. Thus, a substrate whose sequence structure at the cleavage site is disrupted may not be cleaved as efficiently as a substrate comprising the complete recognition sequence.
Disclosure of Invention
The present invention is to provide a coronavirus protease activity assay method based on fluorescence resonance energy transfer, so as to solve the problems in the background art.
The technical problem solved by the invention is realized by adopting the following technical scheme: a coronavirus protease activity detection method based on fluorescence resonance energy transfer specifically comprises the following steps:
(1) thawing a coronavirus active protease sample on ice;
(2) preparing a coronavirus active protease sample solution with 2 times of final concentration and a FRET peptide solution with 2 times of final concentration by using a lysis buffer solution;
(3) in a black half 96-well plate, 25. mu.l of a coronavirus active protease sample solution at 2-fold final concentration and 25. mu.l of a FRET peptide solution at 2-fold final concentration were added to reach a reaction volume of 50. mu.l;
(4) mixing on a shaking table for 1 minute, sealing the test well with a microplate sealing membrane, and incubating at 37 ℃;
(5) removing the sealing film of the pore plate when the temperature of the pore plate is reduced to the ambient temperature;
(6) reading the fluorescence intensity value of 340/495nm at the excitation wavelength/emission wavelength on a fluorometer;
(7) the concentration of the produced Edans-labeled peptide fragment was determined using an Edans/Dabcyl standard curve, and the specific enzyme activity was calculated using the formula.
Preferably, the coronavirus active protease samples include a papain-like sample and a chymotrypsin-like sample.
Preferably, the lysis buffer comprises a papain-like buffer solution and a chymotrypsin-like buffer solution, wherein the papain-like buffer solution is: a mixture of 20mM Tris-HCl, pH 7.3, 100mM NaCl and 1mM EDTA, with 1mM fresh DTT added prior to use; the chymotrypsin-like sustained release solution is prepared from the following components: 50mM HEPES, pH 7.5 solution, and 1mM fresh DTT added prior to use.
Preferably, the calculation formula of the specific activity of the enzyme in the content (7) is: enzyme Specific Activity (SA) (pmol/min/mg) ═ Edans (μ M) × reaction volume (μ l))/(reaction time (min) × enzyme amount (mg)).
Preferably, the incubation time in said content (4) is 60-90 min.
Compared with the prior art, the invention has the following advantages: completely quantifying; lower detection limits enable a wider enzyme activity analysis window; less effort and shorter experimental period; applications compatible with high throughput screening; the enzyme reaction efficiency can be improved by utilizing a natural substrate sequence; the same protocol can be used to test the activity of both viral proteases.
Drawings
FIG. 1 is a chymotrypsin-like titration curve generated by the present invention;
FIG. 2 is a papain-like titration curve generated in accordance with the present invention;
FIG. 3 is a standard curve of Edans/Dabcyl in the present invention.
Detailed Description
In order to make the technical means, the creation features, the work flow and the using method of the present invention easily understand and understand the purpose and the efficacy, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A coronavirus protease activity detection method based on fluorescence resonance energy transfer specifically comprises the following steps:
(1) thawing the papain-like sample on ice;
(2) using a mixed solution of 20mM Tris-HCl, pH 7.3, 100mM NaCl and 1mM EDTA, and adding 1mM newly-prepared DTT as a solution to prepare a papain-like sample solution with 2 times of final concentration and a FRET peptide solution with 2 times of final concentration;
(3) adding 25 ul of papain-like sample solution with 2-fold final concentration and 25 ul of FRET peptide solution with 2-fold final concentration into a black half-zone 96-well plate to reach a reaction volume of 50 ul;
(4) mixing on a shaking table for 1 minute, sealing the test hole with a micropore plate sealing film, and incubating at 37 ℃ for 60-90 min;
(5) removing the sealing film of the pore plate when the temperature of the pore plate is reduced to the ambient temperature;
(6) reading the fluorescence intensity value of 340/495nm at the excitation wavelength/emission wavelength on a fluorometer;
(7) the concentration of the produced Edans-labeled peptide fragment was determined using an Edans/Dabcyl standard curve as shown in FIG. 3, and the specific activity of the enzyme was calculated using the formula: the specific enzyme activity (SA) (pmol/min/mg) ═ Edans (μ M) × reaction volume (μ l))/(reaction time (min) × enzyme amount (mg)) yielded a papain-like titration curve as shown in fig. 2.
Example 2
A coronavirus protease activity detection method based on fluorescence resonance energy transfer specifically comprises the following steps:
(1) thawing the chymotrypsin-like sample on ice;
(2) preparing a chymotrypsin-like sample solution at 2-fold final concentration and a FRET peptide solution at 2-fold final concentration using 50mM HEPES, pH 7.5 solution and 1mM fresh DTT as solutions;
(3) in a black half-zone 96-well plate, 25. mu.l of a chymotrypsin-like sample solution at 2-fold final concentration and 25. mu.l of a FRET peptide solution at 2-fold final concentration were added to reach a reaction volume of 50. mu.l;
(4) mixing on a shaking table for 1 minute, sealing the test hole with a micropore plate sealing film, and incubating at 37 ℃ for 60-90 min;
(5) removing the sealing film of the pore plate when the temperature of the pore plate is reduced to the ambient temperature;
(6) reading the fluorescence intensity value of 340/495nm at the excitation wavelength/emission wavelength on a fluorometer;
(7) the concentration of the produced Edans-labeled peptide fragment was determined using an Edans/Dabcyl standard curve as shown in FIG. 3, and the specific activity of the enzyme was calculated using the formula: the resulting chymotrypsin-like titration curves are shown in fig. 1, for enzyme Specific Activity (SA) (pmol/min/mg) ═ Edans (μ M) × reaction volume (μ l))/(reaction time (min) × enzyme amount (mg)).
The working principle of the invention is as follows: the enzymatic activity of both papain-like and chymotrypsin-like coronaviral proteases can be detected by Fluorescence Resonance Energy Transfer (FRET) -based detection methods, in which the fluorescence of Edans is quenched due to proximity to a Dabcyl quencher; when the substrate is cleaved into two separate fragments, fluorescence is recovered and can be monitored at an excitation/emission wavelength of 340/495 nm.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. A coronavirus protease activity assay method based on fluorescence resonance energy transfer is characterized in that: the concrete content of the method comprises the following contents,
(1) thawing a coronavirus active protease sample on ice;
(2) preparing a coronavirus active protease sample solution with 2 times of final concentration and a FRET peptide solution with 2 times of final concentration by using a lysis buffer solution;
(3) in a black half 96-well plate, 25. mu.l of a coronavirus active protease sample solution at 2-fold final concentration and 25. mu.l of a FRET peptide solution at 2-fold final concentration were added to reach a reaction volume of 50. mu.l;
(4) mixing on a shaking table for 1 minute, sealing the test well with a microplate sealing membrane, and incubating at 37 ℃;
(5) removing the sealing film of the pore plate when the temperature of the pore plate is reduced to the ambient temperature;
(6) reading the fluorescence intensity value of 340/495nm at the excitation wavelength/emission wavelength on a fluorometer;
(7) the concentration of the produced Edans-labeled peptide fragment was determined using an Edans/Dabcyl standard curve, and the specific enzyme activity was calculated using the formula.
2. The method of claim 1, wherein the coronavirus protease assay is based on fluorescence resonance energy transfer, and the assay comprises: the coronavirus active protease samples include a papain-like sample and a chymotrypsin-like sample.
3. The fluorescence resonance energy transfer-based coronavirus protease assay of claim 2, wherein: the lysis buffer comprises a papain-like slow-release solution and a chymotrypsin-like slow-release solution, wherein the papain-like slow-release solution is as follows: a mixture of 20mM Tris-HCl, pH 7.3, 100mM NaCl and 1mM EDTA, with 1mM fresh DTT added prior to use; the chymotrypsin-like sustained release solution is prepared from the following components: 50mM HEPES, pH 7.5 solution, and 1mM fresh DTT added prior to use.
4. The method of claim 1, wherein the coronavirus protease assay is based on fluorescence resonance energy transfer, and the assay comprises: the calculation formula of the specific activity of the enzyme in the above-mentioned content (7) is: enzyme Specific Activity (SA) (pmol/min/mg) ═ Edans (μ M) × reaction volume (μ l))/(reaction time (min) × enzyme amount (mg)).
5. The method of claim 1, wherein the coronavirus protease assay is based on fluorescence resonance energy transfer, and the assay comprises: the incubation time in the content (4) is 60-90 min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113189074A (en) * | 2021-05-11 | 2021-07-30 | 深圳技术大学 | Ultrahigh-sensitivity protease digital detection method |
| US11603552B2 (en) | 2020-07-20 | 2023-03-14 | Mesa Photonics, LLC | Method for pathogen identification |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1690691A (en) * | 2004-04-23 | 2005-11-02 | 中国科学院上海药物研究所 | Process for activity determination of SARS coronavirus 3CL protease and inhibitor screening |
| CN1829736A (en) * | 2003-04-10 | 2006-09-06 | 希龙公司 | The severe acute respiratory syndrome coronavirus |
| CN111544442A (en) * | 2020-06-02 | 2020-08-18 | 华北制药集团新药研究开发有限责任公司 | New use of rutin as coronavirus broad-spectrum inhibitor |
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- 2020-09-27 CN CN202011029010.XA patent/CN112461802A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1829736A (en) * | 2003-04-10 | 2006-09-06 | 希龙公司 | The severe acute respiratory syndrome coronavirus |
| CN1690691A (en) * | 2004-04-23 | 2005-11-02 | 中国科学院上海药物研究所 | Process for activity determination of SARS coronavirus 3CL protease and inhibitor screening |
| CN111544442A (en) * | 2020-06-02 | 2020-08-18 | 华北制药集团新药研究开发有限责任公司 | New use of rutin as coronavirus broad-spectrum inhibitor |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11603552B2 (en) | 2020-07-20 | 2023-03-14 | Mesa Photonics, LLC | Method for pathogen identification |
| US11851698B2 (en) | 2020-07-20 | 2023-12-26 | Mesa Photonics, LLC | Method for pathogen identification |
| CN113189074A (en) * | 2021-05-11 | 2021-07-30 | 深圳技术大学 | Ultrahigh-sensitivity protease digital detection method |
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