CN114250262A - Method for screening novel coronavirus inhibitor - Google Patents

Method for screening novel coronavirus inhibitor Download PDF

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CN114250262A
CN114250262A CN202011005586.2A CN202011005586A CN114250262A CN 114250262 A CN114250262 A CN 114250262A CN 202011005586 A CN202011005586 A CN 202011005586A CN 114250262 A CN114250262 A CN 114250262A
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protein
inhibitor
rbd
hace2
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徐兆超
苗露
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Dalian Institute of Chemical Physics of CAS
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Abstract

The invention provides a method for screening a novel coronavirus inhibitor, belonging to the field of biological analysis and detection. The method realizes the screening of the inhibitor by collecting the changed fluorescent signals in the living cells through fluorescence imaging. The specific method is that firstly, a gene engineering method is used for fusing label proteins on a receptor binding protein (RBD) of a novel coronavirus and a receptor human angiotensin converting enzyme 2(hACE2), and a small molecular fluorescent probe can specifically label the two proteins. The hACE2 fusion protein is overexpressed in cells and is fluorescently labeled, and the fluorescently-labeled RBD protein can specifically recognize hACE2 protein in living cells and presents an overlapped fluorescent signal under a fluorescent microscope. When the inhibitor molecule is incubated with the fluorescence-labeled RBD protein by taking the fluorescence signal of hACE2 as a reference, the RBD fluorescence signal is weakened, so that a new coronavirus inhibitor is screened out, and the inhibition efficiency can be detected. The method has the characteristics of sensitivity and rapidness, and can screen the new coronavirus inhibitor in real time in living cells.

Description

Method for screening novel coronavirus inhibitor
Technical Field
The invention belongs to the field of biological analysis and detection, and particularly relates to a method for screening a novel coronavirus inhibitor.
Background
At present, no specific medicine for treating the new coronavirus exists, so that the medicine discovery and screening work becomes the focus of attention of part of researchers.
Coronavirus is composed of nucleocapsid protein wrapped single-stranded RNA, and COVID-19 infects cells through a mechanism similar to SARS-CoV, wherein the mechanism is that the spike S protein on the surface of the virus is combined with human cell membrane protein angiotensin converting enzyme 2(hACE2), and the coronavirus is induced into cells by protease and then releases RNA infection. The S protein of COVID-19 has nearly 80% homology with the S protein of SARS-CoV, and both consist of two subunits, S1 and S2, wherein the S2 subunit contains a hydrophobic unit for infecting cell membranes. Whereas the S1 subunit contains a receptor-binding domain (RBD) for recognizing the ACE2 protein of the host cell. The study showed that hACE2 bound to COVID-19 RBD-SD1 protein 34.6 nM and is bound to SARS-CoV RBD-SD1 protein (K)D325.8nM) that explains why codid-19 is more infectious.
Since the interaction of the RBD and hACE2 is the key point for the virus to infect the cells, the active central site of the RBD is an important target point for vaccine and drug development, and researchers think that screening out drugs targeting the active site of hACE2-RBD action and preventing the virus from binding hACE2 to infect the cells can be a rapid treatment scheme. Therefore, a rapid, efficient and sensitive screening method is essential. At present, a large number of researchers screen hACE2 or RBD inhibitors by using a computer simulation method, but the computer simulation method cannot reduce the real environment of protein, and has large error; a commercial screening kit for the new coronavirus RBD inhibitor is usually detected by an ELISA method in an in-vitro environment, and has the defects of ex-situ, large error, weak signal and the like; however, the method of infecting the hACE2 cells in situ by using the pseudovirus is long in time and cannot be used for flux screening.
The fluorescence detection technology is a common means for drug screening because of the outstanding advantages of in-situ detection, high screening quantity, low detection cost, mature detection instrument and the like. However, no fluorescence system is applied to screening of the hACE2-RBD interaction active site inhibitor at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to develop a novel coronavirus inhibitor screening method, which collects the changed fluorescent signals in living cells through fluorescence imaging to realize the screening of the inhibitors. The specific method is that firstly, a gene engineering method is used for fusing label proteins on the RBD of the novel coronavirus and a receptor hACE2 thereof respectively, so that the two proteins can be specifically marked by the small molecular fluorescent probe. Then the hACE2 fusion protein is overexpressed in cells and is fluorescently labeled, and the fluorescently-labeled RBD protein can specifically recognize hACE2 protein in living cells and presents an overlapped fluorescent signal under a fluorescent microscope. When the inhibitor molecule is incubated with the fluorescently labeled RBD protein by taking the fluorescence signal of hACE2 as a reference, the RBD fluorescence signal is weakened, so that the inhibitor molecule is screened out, and the inhibition efficiency can be detected. The method has the advantages of high sensitivity and high speed, and can screen new coronavirus inhibitor in living cells in real time
In order to achieve the above objects, the present invention provides a method for screening a novel coronavirus inhibitor, comprising the following steps:
(1) the tag protein-hACE 2 fusion protein plasmid was overexpressed in cells.
(2) Adding a fluorescent probe containing the specific substrate of the tag protein, incubating for 10-30min, and washing once;
(3) adding fluorescence-labeled RBD-tag protein fusion protein or mixture of inhibitor and fluorescence-labeled RBD-tag protein, and incubating for 30-120 min;
(4) fluorescence imaging, and qualitatively screening the inhibitor;
(5) analyzing the imaging data, and quantitatively detecting the inhibition rate of the inhibitor.
The tag protein-hACE 2 fusion protein in the step (1) is obtained by fusing SNAP, Halo, CLIP or PYP tag protein at the N terminal of hACE2 protein.
The fluorescent probe containing the label protein specific substrate in the step (2) is a membrane-impermeable probe.
The RBD-tag protein fusion protein in the step (3) is formed by fusing SNAP, Halo, CLIP or PYP tag protein at the C end or the N end of the RBD protein
The specific process for qualitatively screening the inhibitor through the fluorescence imaging result in the step (4) is that a cell over expressing hACE2 is determined by utilizing a fluorescence channel of tag protein-hACE 2, then the inhibition effect of the inhibitor is determined by observing the fluorescence intensity of the RBD fluorescence channel of the cell only added with the RBD-tag protein-dye, the inhibition effect is indicated by the weak fluorescence, and the inhibition effect is not indicated by the weak fluorescence intensity.
The specific process of the data analysis in the step (5) is to count the average fluorescence intensity of 10 to 100 cells over-expressing the tag protein-hACE 2
Figure BDA0002695728870000031
And
Figure BDA0002695728870000032
and average fluorescence intensity of the background of the imaged picture
Figure BDA0002695728870000033
And
Figure BDA0002695728870000034
Figure BDA0002695728870000035
Figure DA00026957288738844235
IF with RBD-tagged protein-dye only imaging pictures640/IF561IF imaged by inhibitor-added cells as 100% relative RBD activity640/IF561In comparison with this value, the inhibition ratio of the inhibitor was obtained.
The method for screening the novel coronavirus inhibitor is characterized by comprising the following steps: the selected inhibitor can inhibit the binding of hACE2 to S protein of novel coronavirus, and the inhibitor can be an inhibitor of hACE2, namely, the inhibitor interacts with hACE 2; or an inhibitor of the receptor binding portion of the S protein, RBD.
The invention has the advantages and beneficial effects that:
(1) the invention has the advantages that the in-situ imaging and screening in living cells are realized, and the screened inhibitor has higher applicability;
(2) the protein label is fused to the target protein by a genetic engineering method, and the protein label has the advantage that any small molecule fluorophore with a specific substrate can be selectively marked on the target protein for application according to different instruments or fluorescent channels.
(3) The fluorescence of hACE2 is taken as a reference, and the inhibitor can be qualitatively and quantitatively screened through the average fluorescence ratio of two channels;
(4) the screening method can simultaneously screen the hACE2 or RBD inhibitor molecules of the hACE2-RBD interaction site.
Drawings
FIG. 1 imaging of Hela cells labeled with various dyes of the various tagged proteins, hACE2
FIG. 2 is an SDS-PAGE electrophoresis of the fluorescent probe before and after interaction with RBD protein.
FIG. 3 is a confocal image of fluorescence from Hela cells transfected with SNAP-hACE 2.
FIG. 4 is a map showing the viability of RBD525 and RBD541 proteins measured by the fluorescence ratio method.
FIG. 5 is confocal imaging of fluorescence of SNAP-hACE2 transfected Hela cells after addition of mixed solution of wild-type protein and RBD.
FIG. 6 is confocal imaging of fluorescence of SNAP-hACE2 transfected Hela cells after addition of mixed solutions of different inhibitors and RBD.
FIG. 7 is confocal fluorescence imaging of SNAP-hACE2 transfected Hela cells after addition of mixed solutions of different concentrations of neutralizing antibody and RBD.
FIG. 8 relative viability profiles of RBD protein after addition of different concentrations of neutralizing antibody.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
Construction of hACE2 and tag protein fusion expression vector
The full-length cDNA of hACE2 was subcloned into commercial pcDNA3.1 vector by conventional molecular cloning method, and then cDNA of SNAP, CLIP, Halo, PYP, TC tag protein was subcloned into N-terminal signal peptide of hACE2 (1-51 base of hACE2 is signal peptide), respectively, to obtain pCMV-SNAP-hACE2, pCMV-CLIP-hACE2, pCMV-Halo-hACE2, pCMV-PYP-hACE2, and pCMV-TC-hACE 2.
Example 2
Fluorescent labeling of hACE2 in living cells
Hela cells were transferred to 5 confocal imaging dishes and 24 hours later, 500ng of plasmid vectors pCMV-SNAP-hACE2, pCMV-CLIP-hACE2, pCMV-Halo-hACE2, pCMV-PYP-hACE2 and pCMV-TC-hACE2 were transferred to Hela cells using Lipofectamine 2000 reagent according to the instructions, and after 4 hours, the culture medium was changed to DMEM high-sugar culture medium containing 10% fetal bovine serum and 5% CO at 37 ℃2The cultivation was continued in the incubator for 48 hours. SNAP-561, Halo-488, PYP-488, CLIP-488 and TC-488 fluorescent dyes were dissolved in DMEM high-glucose medium to a final concentration of 1. mu.M, respectively. The cells were incubated with the probe solution for 30min, then washed 2 times with DMEM, and 1ml of MEM medium was added. Imaging was performed with a fluorescence confocal microscope under 100 x oil microscopy as shown in FIG. 1.
FIG. 1a is an image of Hela cells transfected with pCMV-SNAP-hACE2 plasmid and excited at 561nm,
FIG. 1b is an image of Hela cells transfected with pCMV-Halo-hACE2 plasmid and excited at 488nm,
FIG. 1c shows the imaging of Hela cells transfected with pCMV-CLIP-hACE2 plasmid and excited at 488nm,
FIG. 1d is an image of Hela cells transfected with pCMV-PYP-hACE2 plasmid and excited at 488nm,
FIG. 1e is an image of Hela cells transfected with pCMV-TC-hACE2 plasmid and excited at 488nm,
as can be seen from FIG. 1, the dyes were all labeled on the cell membrane, indicating that it is hACE2 protein that is overexpressed on the cell membrane.
Example 3
Construction of RBD525-Halo fusion expression vector, expression and purification of protein
The cDNA of the novel coronavirus RBD (333-525) is firstly subcloned into a commercial pcDNA3.1 vector by using a conventional molecular cloning method, and then the cDNA of the Halo tag protein with 6His at the C end is subcloned to the C end of the RBD to obtain pCMV-RBD-Halo-6 His.
The pCMV-RBD-Halo-6His was overexpressed by conventional eukaryotic HEK293T cells, and then the protein was purified by Ni-NTA column, which was eluted with 200mM imidazole salt in PBS buffer to give about 0.5mg of the target protein RBD525-Halo with a molecular weight of 57 kDa.
Example 4
Fluorescence labeling RBD525-Halo protein and purifying
RBD525-Halo protein was dissolved in PBS (20mM, pH 7.4) buffer to make a 1.1mg/mL stock solution. Halo640 fluorescent dye was dissolved in DMSO to make up a 2mM stock solution. And (3) taking 20 mu L of RBD525-Halo protein solution, adding 0.6 mu L of Halo640 fluorescent dye to ensure that the molar ratio of the protein to the probe is 1:3, and reacting for 2 hours at room temperature to obtain the RBD525-Halo-640 dye. The total volume of the protein solution was made up to 100. mu.L, then desalted by passing through Sephadex column G-25 using PBS (20mM, pH 7.4), and the eluted RBD525-Halo-640dye was concentrated by concentration column, and the purified RBD525-Halo-640dye protein was 0.17mg/mL by Coomassie blue staining and had a molar concentration of about 3. mu.M.
A small number of RBD525-Halo and RBD525-Halo-640dye were run on SDS-PAGE, and the Coomassie brilliant blue stained images and UV-excited image were shown in FIG. 2. The left panel of FIG. 2 is Coomassie Brilliant blue staining and the right panel is UV-excited fluorescence imaging with lanes 3 and 4 being RBD525-Halo and RBD525-Halo-640dye, respectively. The molecular weight of the protein RBD525-Halo is about 57kDa, which is between 44.3kDa and 66.4kDa of standard protein, and the fluorescently-labeled RBD525-Halo-640dye shows red fluorescence under the excitation of ultraviolet light.
Example 5
Construction of RBD541-Halo fusion expression vector, expression and purification of protein
The cDNA of the novel coronavirus RBD (319-541) is firstly subcloned into a commercial pcDNA3.1 vector by using a conventional molecular cloning method, and then the cDNA of the Halo tag protein with 6His at the C end is subcloned into the C end of the RBD to obtain pCMV-RBD-Halo-6 His.
The pCMV-RBD-Halo-6His was overexpressed by conventional eukaryotic HEK293T cells, and then the protein was purified by Ni-NTA column, which was eluted with 200mM imidazole salt in PBS buffer to give about 0.5mg of the target protein RBD541-Halo having a molecular weight of 59.6 kDa.
Example 6
Fluorescence labeling RBD541-Halo protein and purifying
RBD541-Halo protein was dissolved in PBS (20mM, pH 7.4) buffer to make a mother liquor of 0.55 mg/mL. Halo640 fluorescent dye was dissolved in DMSO to make up a 2mM stock solution. And (3) taking 20 mu L of RBD541-Halo protein solution, adding 0.6 mu L of Halo640 fluorescent dye to ensure that the molar ratio of the protein to the probe is 1:3, and reacting for 2 hours at room temperature to obtain RBD541-Halo-640 dye. Then, the buffer solution was passed through Sephadex column G-25 in PBS (20mM, pH 7.4) to remove salts, and the eluted RBD541-Halo-640dye was concentrated in a concentration column, and the concentration of the purified RBD541-Halo-640dye protein was 0.18 mg/mL and the molar concentration was about 3.1. mu.M as determined by Coomassie blue staining.
A small number of RBD541-Halo and RBD541-Halo-640dye were run on SDS-PAGE, and Coomassie brilliant blue stained images and UV-excited imaging are shown in FIG. 2. The left panel of FIG. 2 is Coomassie Brilliant blue staining and the right panel is UV-excited fluorescence imaging with lanes 1 and 2 being RBD541-Halo and RBD541-Halo-640dye, respectively. The molecular weight of the protein RBD541-Halo is about 59.6kDa and is between 44.3kDa and 66.4kDa of standard protein, and the fluorescently-labeled RBD541-Halo-640dye shows red fluorescence under the excitation of ultraviolet light.
Example 7
The interaction of fluorescently labeled RBD525-Halo with fluorescently labeled SNAP-hACE2 was imaged.
Hela cells were transferred to confocal imaging dishes and after 24 hours were transiently transferred to PCMV-SNAP-hACE2 plasmid with Lipo2000 at 37 ℃ with 5% CO2The culture was carried out in an incubator for 48 hours. Dissolving SNAP-561 fluorescent dye in DMEM for high-sugar cultureIn this case, the final concentration was 0.2. mu.M. The cells were incubated with the probe solution for 15min, then washed once with DMEM, 1mL of DMMEM medium was added, RBD525-Halo-640dye was added to a final concentration of 20nM, and at the same time, the nuclear dye Hoechst 33342 was added to a final concentration of 0.5. mu.M, 5% CO at 37 deg.C2Incubate in incubator for 10 min. Images were taken with a fluorescence confocal microscope under 100 x oil immersion as shown in figures 3 a-d.
FIG. 3a is an image of Hela nuclei under 405nm excitation, showing fluorescence from Hoechst 33342 dye; FIG. 3b is an image of Hela cells under 561nm excitation showing fluorescence of SNAP-561 dye, SNAP-561 being labeled on SNAP-hACE2 protein overexpressed in the cell membrane, thus 561 channel shows fluorescence labeled SNAP-hACE2 protein; FIG. 3c is an image of Hela cells under 640nm excitation, showing the fluorescence of RBD525-Halo-640 dye; FIG. 3d is a superimposed image of FIGS. 3a-c, showing that the fluorescence of FIGS. 3b and 3c overlap well, demonstrating the interaction of hACE2 protein with RBD protein.
Example 8
Interaction of fluorescently-labeled RBD-Halo protein with wild-type hACE2
Hela cells are transferred to a confocal imaging dish, and are transiently transferred into PCMV-hACE2-SV-EGFP plasmid by using Lipo2000 reagent after 24 hours at 37 ℃ with 5 percent CO2The culture was carried out in an incubator for 48 hours. The medium was changed to 1ml of MEM, RBD525-Halo-640dye was added to a final concentration of 20nM, and the nuclear dye Hoechst 33342 was added to a final concentration of 0.5. mu.M with 5% CO at 37 ℃ to obtain a mixture2Incubate in incubator for 10 min. Imaging was performed with a fluorescence confocal microscope under 100 x oil microscopy.
FIG. 3e is an image of Hela nuclei under 405nm excitation, showing fluorescence from Hoechst 33342 dye; FIG. 3f is the image of Hela cell excited at 488nm, which shows the fluorescence of green fluorescent protein EGFP, EGFP is a reporter gene in PCMV-hACE2-SV-EGFP plasmid, and human ACE2 protein which is wild type is over-expressed on the cell membrane of the cell as long as there is EGFP-expressed cell; FIG. 3g is an image of Hela cells under 640nm excitation, showing the fluorescence of RBD525-Halo-640 dye; FIG. 3h is the superimposed image of FIGS. 3e-h, showing that RBD525-Halo-640dye is marked on the cell membrane of the cell expressing EGFP protein, demonstrating that RBD525-Halo protein can interact with wild type hACE2 protein.
Example 9
Competitive binding of wild-type RBD and RBD-Halo fusion proteins to hACE2
Hela cells were transferred to confocal imaging dishes and after 24 hours were transiently transferred to PCMV-SNAP-hACE2 plasmid with Lipo2000 at 37 ℃ with 5% CO2The culture was carried out in an incubator for 48 hours. SNAP-561 fluorescent dye was dissolved in DMEM high-glucose medium to a final concentration of 0.2. mu.M. Incubating the cells with the probe solution for 15min, washing with DMEM, adding 1ml MEM medium, adding RBD525-Halo-640dye to a final concentration of 20nM, adding 50nM wild type RBD protein, 5% CO at 37 deg.C2Incubate in incubator for 30 min. Finally adding cell nucleus dye Hoechst 33342 with the final concentration of 0.5 mu M. After 10min, images were taken under a 100-fold oil microscope with a fluorescence confocal microscope, as shown in FIGS. 2 i-l.
FIG. 3i is an image of Hela nuclei under 405nm excitation, showing fluorescence from Hoechst 33342 dye; FIG. 3j is an image of Hela cells under 561nm excitation, showing a fluorescently labeled SNAP-hACE2 protein; FIG. 3k is an image of Hela cells under 640nm excitation, showing fluorescence of RBD525-Halo-640 dye; fig. 3l is an image superimposed on fig. 3 i-l. Wherein FIG. 3k shows only weak fluorescence, because the wild-type RBD protein binds to hACE2 protein more strongly than to hACE2 protein, and thus the wild-type RBD protein can inhibit the binding of RBD525-Halo protein to hACE2 protein. Meanwhile, the detection method can detect the inhibitor of the interaction of RBD525-Halo and hACE 2.
Example 10
RBD525 and RBD541 protein activity detection
Hela cells were transferred to three 8-pore chamber confocal imaging dishes, and after 24 hours, the cells were transiently transferred to PCMV-SNAP-hACE2 plasmid with Lipo2000 reagent, respectively, and 5% CO was added at 37 ℃2The culture was carried out in an incubator for 48 hours. SNAP-561 fluorescent dye was dissolved in DMEM high-glucose medium to a final concentration of 0.2. mu.M. Cells were incubated with the probe solution for 15min and then washed once with DMEM, 0.1mLD per wellMEM medium, RBD525-Halo-640dye was added to a final concentration of 1000nM, 600nM, 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 1.917nM, respectively. Simultaneously, RBD541-Halo-640dye was added to each of the other wells to a final concentration of 800nM, 600nM, 400nM, 200nM, 100nM, 50nM, 25nM, 6.25nM, 1.56nM, respectively. 5% CO at 37 ℃2Incubate in incubator for 60 min. Imaging with a fluorescence confocal microscope under a 10-fold lens to obtain fluorescence images under excitation of 561nm and 640nm, and respectively counting the average fluorescence intensity of different channels of 50 cells
Figure BDA0002695728870000101
And
Figure DA00026957288738919347
and average fluorescence intensity of the background of the imaged picture
Figure BDA0002695728870000103
And
Figure BDA0002695728870000104
Figure BDA0002695728870000105
protein concentration as abscissa and IF640/IF561As ordinate, fig. 4 was obtained.
As shown in FIG. 4, IF increased with the increase in the concentration of RBD protein640/IF561The ratio of (A) and (B) is gradually increased, and after a certain concentration is reached, the ratio is kept unchanged, which indicates that the interaction of the RBD and hACE reaches saturation. After fitting the two scattergrams, the EC50 value for the effect of RBD525-Halo on SNAP-hACE2 was calculated to be 93 nM, while the EC50 value for the effect of RBD541-Halo on SNAP-hACE2 was calculated to be 59nM, indicating that RBD541-Halo is stronger than RBD525-Halo on SNAP-hACE 2.
Example 11
Detection of wild-type protein as an inhibitor of RBD or hACE2, respectively
Hela cells were transferred to 4 confocal imaging dishes and 24 hours later, the HeLa cells were assayed using Lipo2000The agent was transiently transferred into the PCMV-SNAP-hACE2 plasmid at 37 ℃ with 5% CO2The culture was carried out in an incubator for 48 hours. SNAP-561 fluorescent dye was dissolved in DMEM high-glucose medium to a final concentration of 0.2. mu.M. The cells were incubated with the probe solution for 30min, washed once, and 1mL DMEM medium was added, then RBD525-Halo-640dye was added to a final concentration of 20nM to 4 imaging dishes, and 80nM of wild type RBD protein, 40nM of wild type hACE2 protein, and 50nM of neocoronavirus S protein were added to 3 dishes. Here, wild-type RBD protein and S protein as inhibitors of hACE2 and wild-type hACE2 protein as an inhibitor of RBD, 5% CO at 37 deg.C2Incubate in incubator for 60 min. Imaging was performed with a fluorescence confocal microscope under a 10-fold microscope, as shown in FIG. 5.
FIG. 5 shows the first behavior of 640 nM-stimulated RBD525-Halo protein imaging channel and the second behavior of 561 nM-stimulated SNAP-ACE2 protein imaging channel, wherein only 20nM RBD525-Halo-640dye is added in FIG. 5 a; FIG. 5b shows that the red channel has weak fluorescence when 20nM RBD525-Halo-640dye and 80nM wild type RBD protein are added, indicating that it has binding inhibition effect; FIG. 5c shows that the red channel of 20nM RBD525-Halo-640dye and 40nM wild type hACE2 protein has weak cellular fluorescence, indicating its binding inhibition effect; FIG. 5d shows that the red channel imaged by 20nM RBD525-Halo-640dye and 50nM new coronavirus S protein is attenuated compared to FIG. 5a, indicating that it inhibits binding of SARS-CoV-2 RBD to hACE 2. The method constructed by the invention is proved to be capable of detecting the inhibitor of hACE2 or RBD for inhibiting the interaction of hACE 2-RBD.
Example 12
Detection of known inhibitor molecules using RBD541-Halo-640dye
Hela cells were transferred to 4 confocal imaging dishes and after 24 hours were transiently transferred to PCMV-SNAP-hACE2 plasmid with Lipo2000 reagent at 37 ℃ with 5% CO2The culture was carried out in an incubator for 48 hours. SNAP-561 fluorescent dye was dissolved in DMEM high-glucose medium to a final concentration of 0.2. mu.M. The cells were incubated with the probe solution for 15min, washed once, 1mL DMEM medium was added, then 4 imaging dishes were added with RBD525-Halo-640dye to a final concentration of 20nM, and 3 dishes were used with RBD525-Halo-640dyeAdding commercial RBD protein neutralizing antibody 80nM, commercial RBD protein antibody 80nM, and reported small molecule inhibitor 1mM capable of inhibiting SARS-CoV invading cells, 5% CO at 37 deg.C2Incubate in incubator for 60 min. Imaging was performed with a fluorescence confocal microscope under a 10-fold microscope, as shown in FIG. 6.
FIG. 6 shows the first behavior of 640 nM-excited imaging channel of RBD541-Halo protein and the second behavior of 561 nM-excited imaging channel of SNAP-ACE2 protein, wherein only 20nM of RBD541-Halo-640dye is added in FIG. 6 a; FIG. 6b shows the weak cellular fluorescence of the red channel with the addition of 20nM RBD541-Halo-640dye and 80nM of commercial RBD protein neutralizing antibody, indicating the binding inhibition; FIG. 6c shows the addition of 20nM RBD541-Halo-640dye and 80nM commercial antibody to RBD protein whose recognition site is not the active center of RBD protein, and the red channel cell fluorescence is strong as seen from the image, comparable to FIG. 6a, indicating that it does not inhibit binding; FIG. 6d is a graph showing the inhibition of SARS-CoV invading cells by the addition of 20nM RBD541-Halo-640dye and a reported small molecule inhibitor that exhibits reduced cellular fluorescence of the red channel as compared to FIG. 6a, indicating the inhibition of SARS-CoV-2 RBD binding to hACE 2.
In addition, we counted the mean fluorescence intensity of 20-80 cells separately
Figure BDA0002695728870000121
And
Figure BDA0002695728870000122
and average fluorescence intensity of the background of the imaged picture
Figure BDA0002695728870000123
And
Figure BDA0002695728870000124
Figure BDA0002695728870000125
at the IF of FIG. 6a640/ IF561As 100% relative RBD Activity (RA), other componentsComparing with fig. 6a, the obtained 80nM commercial neutralizing antibody can inhibit 99.8% of RBD activity with highest inhibition efficiency, while the small molecule inhibitor has lower activity and only inhibits 28% of RBD activity.
Example 13
Quantitative determination of RBD and hACE2 inhibition by neutralizing antibody
Hela cells were transferred to 1 8-cavity confocal imaging dish, and after 24 hours, the cells were transiently transferred to PCMV-SNAP-hACE2 plasmid with Lipo2000 reagent at 37 ℃ with 5% CO2The culture was carried out in an incubator for 48 hours. SNAP-561 fluorescent dye was dissolved in DMEM high-glucose medium to a final concentration of 0.2. mu.M. The cells were incubated with the probe solution for 15min, washed once, and added with 0.1mL of DMEM medium, then RBD541-Halo-640dye was added to the imaging dish to a final concentration of 25nM, while commercial RBD protein neutralizing antibodies 40nM, 20nM, 10nM, 5nM, 2.5nM, 1.25nM, 0.625nM, 0nM were added to the dish at 37 deg.C with 5% CO2Incubate in incubator for 60 min. Imaging was performed with a fluorescence confocal microscope under a 10-fold microscope, as shown in FIG. 7.
In FIG. 7, the RBD541-Halo protein imaging channel excited by the actions 1 and 3 at 640nm, the SNAP-hACE2 imaging channel excited by the actions 2 and 4 at 561nm, and the cells with the hACE2 channel showing stronger fluorescence all over-express SNAP-hACE2, and the corresponding RBD channel has stronger fluorescence, so that the interaction of the RBD protein and hACE2 is shown, and the interaction of the RBD and hACE2 is shown to be inhibited. FIGS. 7a-h show that the addition of neutralizing antibody at concentrations ranging from high to low, respectively, shows that the binding of RBD to hACE2 is almost completely inhibited when 40nM neutralizing antibody is added, while the inhibition efficiency gradually decreases with decreasing concentration of neutralizing antibody added, indicating the concentration dependence of the inhibition, which allows quantitative detection of the inhibition efficiency of the inhibitor.
Example 14
Quantitative determination of RBD and hACE2 inhibition by neutralizing antibody
Hela cells were transferred to 2 8-cavity confocal imaging dishes, and after 24 hours, the cells were transiently transferred to PCMV-SNAP-hACE2 plasmid with Lipo2000 reagent at 37 ℃ with 5% CO2The culture was carried out in an incubator for 48 hours. Dissolving SNAP-561 fluorescent dye in DMEM for high-sugar cultureIn this case, the final concentration was 0.2. mu.M. The cells were incubated with the probe solution for 15min, washed once, and added with 0.1mL of DMEM medium, then RBD541-Halo-640dye was added to the imaging dish to a final concentration of 25nM, while commercial RBD protein neutralizing antibodies 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 2.5nM, 1.25nM, 0.625nM, 0.3125nM, 0nM were added to the dish, 5% CO at 37 deg.C2Incubate in incubator for 60 min. Imaging with a fluorescence confocal microscope under a 10-fold microscope, and then counting the average fluorescence intensity of 60 cells
Figure BDA0002695728870000141
And
Figure BDA0002695728870000142
and average fluorescence intensity of the background of the imaged picture
Figure BDA0002695728870000143
And
Figure BDA0002695728870000144
Figure BDA0002695728870000145
by IF of RBD image only640/IF561As a reference, the IF of the other images was set to 100%640/IF561The ratio was compared with the reference to obtain FIG. 8.
In fig. 8, the relative activity value gradually decreased with the decrease of the concentration of the added neutralizing antibody, and the IC50 value of the neutralizing antibody was 7.6nM after the fitting, which demonstrates that the method of the present invention can quantitatively detect the inhibition efficiency of the inhibitor.

Claims (10)

1. A novel screening method of coronavirus inhibitors is characterized in that the method collects the changed fluorescent signals in living cells through fluorescence imaging to realize screening of the inhibitors.
2. The method for screening a novel coronavirus inhibitor according to claim 1, wherein the method comprises the steps of fusing a tag protein to a receptor binding protein (RBD) of the novel coronavirus and a receptor human angiotensin converting enzyme 2(hACE2) thereof, and specifically labeling the RBD and hACE2 with a small-molecule fluorescent probe, and screening the novel coronavirus inhibitor by reducing the RBD fluorescent signal with the fluorescent signal of hACE2 as a reference.
3. The method for screening a novel coronavirus inhibitor according to claim 1, which comprises the steps of:
(1) overexpresses the tag protein-hACE 2 fusion protein plasmid in cells;
(2) adding a fluorescent probe containing the specific substrate of the tagged protein;
(3) adding fluorescence-labeled RBD-tag protein fusion protein or mixture of inhibitor and fluorescence-labeled RBD-tag protein, and incubating for 30-120 min;
(4) fluorescence imaging, and qualitatively screening the inhibitor;
(5) analyzing the imaging data, and quantitatively detecting the inhibition rate of the inhibitor.
4. The method for screening a novel coronavirus inhibitor according to claim 3, wherein: the tag protein-hACE 2 fusion protein in the step (1) is specifically as follows: the SNAP, Halo, CLIP, or PYP tag proteins were fused to the N-terminus of hACE2 protein, respectively.
5. The method for screening a novel coronavirus inhibitor according to claim 3, wherein: the fluorescent probe containing the label protein specific substrate in the step (2) is a membrane-impermeable probe.
6. The method of claim 3, wherein the screening for a novel coronavirus inhibitor comprises: the RBD-tag protein fusion protein in the step (3) is formed by fusing SNAP, Halo, CLIP or PYP tag protein at the C end or the N end of the RBD protein.
7. The method of claim 3, wherein the screening for a novel coronavirus inhibitor comprises: the specific process for qualitatively screening the inhibitor through the fluorescence imaging result in the step (4) comprises the following steps: the cell over expressing hACE2 is determined by using the fluorescence channel of the tag protein-hACE 2, and then the inhibition effect of the inhibitor is determined by observing the fluorescence intensity of the inhibitor-added cell by taking the fluorescence intensity of the RBD fluorescence channel of the RBD-tag protein-dye-added cell as a standard.
8. The method of claim 3, wherein the screening for a novel coronavirus inhibitor comprises: the specific process of the data analysis in step (5) is to count the average fluorescence intensity of 10-100 cells over-expressing the tag protein-hACE 2
Figure FDA0002695728860000021
And
Figure FDA0002695728860000022
and average fluorescence intensity of the background of the imaged picture
Figure FDA0002695728860000023
And
Figure FDA0002695728860000024
Figure FDA0002695728860000025
IF with RBD-tagged protein-dye only imaging pictures640/IF561IF imaged by inhibitor-added cells as 100% relative RBD activity640/IF561The inhibition rate of the inhibitor was obtained by comparison with this value.
9. A novel coronavirus inhibitor obtained by the screening method according to any one of claims 1 to 8, wherein the novel coronavirus inhibitor is a hACE2 inhibitor or an RBD inhibitor.
10. Use of a novel coronavirus inhibitor according to claim 9 in the manufacture of a medicament for the treatment of novel coronavirus pneumonia.
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