Method for screening blocking agent for blocking novel coronavirus infection and blocking agent

Abstract

The invention provides a method for screening a blocking agent for blocking novel coronavirus infection and the blocking agent. On one hand, the invention provides a method for screening a blocking agent for blocking novel coronavirus infection, and the method comprises the following steps: detecting the ability of a test sample to competitively bind to ACE2 with S protein and / or the ability of the test sample to block the binding of the S protein to ACE2 by adopting gel chromatography and / or flow cytometry; therefore, the blocking agent with the effect of blocking the novel coronavirus infection is screened out. The screening method provided by the invention is simple to operate, high in screening efficiency, low in screening cost and relatively high in practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of protein polypeptide technology, and specifically relates to a method and a blocker for screening a blocker for blocking novel coronavirus infection. Background Art

[0002] At present, the specific drug for the treatment of coronavirus has not been identified. The main prevention methods are to develop a novel coronavirus-specific vaccine (eight types, including RNA, protein, virus inactivation, and adenovirus), directly inhibit the virus proliferation mechanism, and block the binding between the coronavirus and its cell receptor ACE2. Due to the high variability of the novel coronavirus, it is very difficult to develop novel coronavirus-specific therapeutic drugs. So far, the novel coronavirus has mutated five generations in order to escape autoimmune attacks: alpha, beta, gamma, delta, and Omicron mutant viruses. At present, many scientific studies focus on the study of the novel coronavirus binding receptor hACE2 in human cells. Antagonists are used to block the receptor binding of the novel coronavirus and the cell receptor angiotensin converting enzyme 2 (ACE2; 805aa), so that the virus cannot enter the cell body, thereby inhibiting the proliferation and survival of the virus. Existing studies mainly focus on known ACE2 enzyme inhibitors, but since ACE2 also plays an important role in the human renin-angiotensin system, inhibiting ACE2 enzyme activity may cause ACE2 to affect blood pressure and cause side effects. Therefore, finding a new inhibitor that has no effect on ACE2 enzyme activity is the key to the development of specific drugs for the new coronavirus. Summary of the invention

[0003] The present invention provides a method for screening a blocking agent for blocking novel coronavirus infection and a blocking agent.

[0004] In one aspect, the present invention provides a method for screening a blocking agent for blocking novel coronavirus infection, the method comprising using gel chromatography and / or flow cytometry to detect the ability of a test article to competitively bind to ACE2 with S protein and / or the ability of a test article to block S protein from binding to ACE2, thereby screening out a blocking agent having the effect of blocking novel coronavirus infection;

[0005] The gel chromatography detection process includes:

[0006] Prepare experimental group samples for gel chromatography analysis; wherein the experimental group samples include: samples for detecting the ability of the test article to competitively bind to ACE2 with the S protein, and / or samples for detecting the ability of the test article to block the S protein from binding to ACE2; the preparation process of the sample for detecting the ability of the test article to competitively bind to ACE2 with the S protein includes mixing the test article and the S protein with ACE2 at the same time for a competitive binding reaction; the preparation process of the sample for detecting the ability of the test article to block the S protein from binding to ACE2 includes first mixing the test article with ACE2 for a binding reaction, and then adding the S protein for a reaction;

[0007] Gel chromatography was used to analyze the gel chromatography behavior of the experimental group samples at a wavelength of 280 nm;

[0008] The ability of the test article to competitively bind to ACE2 with the S protein and / or the ability of the test article to block the binding of the S protein to ACE2 is determined based on the peak area corresponding to ACE2 in the chromatogram;

[0009] The flow cytometry detection process includes: preparing FITC-S protein and flow cytometry screening;

[0010] Prepare experimental group samples for flow cytometry analysis and detection; wherein the experimental group samples include: samples for detecting the ability of the test article to competitively bind to ACE2 with the S protein, and / or samples for detecting the ability of the test article to block the S protein from binding to ACE2; the preparation process of the sample for detecting the ability of the test article to competitively bind to ACE2 with the S protein includes mixing the test article and the FITC-S protein with cells overexpressing ACE2 at the same time for a competitive binding reaction; the preparation process of the sample for detecting the ability of the test article to block the S protein from binding to ACE2 includes first mixing the test article with cells overexpressing ACE2 for a binding reaction, and then adding the FITC-S protein for a reaction;

[0011] Flow cytometry was used to analyze the fluorescence signal coverage of the experimental group samples, and the ability of the test article to competitively bind to ACE2 with the S protein and / or the ability of the test article to block the binding of the S protein to ACE2 was determined based on the fluorescence signal coverage.

[0012] The S protein described in the present invention is preferably a novel coronavirus spike protein trimer.

[0013] According to a specific embodiment of the present invention, the test article comprises a peptide;

[0014] Preferably, the test article comprises an oligopeptide;

[0015] More preferably, the test substance is a dipeptide, a tripeptide, a tetrapeptide and / or a pentapeptide; further preferably, it is a straight-chain dipeptide composed of unmodified amino acids.

[0016] The test sample of the above method of the present invention is not limited to peptides, and can be applied to any substance that can block the binding between ACE2 and S protein. The complex of ACE2 and S protein will show a specific gel chromatogram in gel chromatography analysis, and any substance that has a blocking effect on the binding between ACE2 and S protein will change this specific gel chromatogram.

[0017] According to a specific embodiment of the present invention, the gel chromatography detection process further includes: comparing the peak area corresponding to ACE2 in the chromatogram of the experimental group sample with the peak area of ​​the control group to which the test article is not added, to determine the ability of the test article to competitively bind to ACE2 with the S protein and / or the ability of the test article to block the S protein from binding to ACE2;

[0018] The flow cytometry detection process also includes: comparing the fluorescence signal coverage of the experimental group samples with the fluorescence signal coverage of the control group without the test article, to determine the ability of the test article to competitively bind to ACE2 with the S protein and / or the ability of the test article to block the S protein from binding to ACE2;

[0019] Preferably, the screened blocking agent with the effect of blocking novel coronavirus infection has: the ability to competitively bind to ACE2 with the S protein, and the ability to block the S protein from binding to ACE2;

[0020] More preferably, gel chromatography is used to detect the ability of the test article to competitively bind to ACE2 with the S protein, and flow cytometry is used to detect the ability of the test article to block the binding of the S protein to ACE2.

[0021] According to a specific embodiment of the present invention, in the method of the present invention, the chromatographic column filler used in the gel chromatography is a hydrophilic gel chromatography filler suitable for protein analysis;

[0022] Preferably, the exclusion limit of the gel chromatography column is 5kd-2500kd;

[0023] Preferably, the chromatography column length of the gel chromatography column is ≥25 cm;

[0024] Preferably, the inner diameter of the gel chromatography column is ≤7.8 mm;

[0025] Preferably, the number of chromatography column plates of the gel chromatography column is ≥25,000.

[0026] According to a specific embodiment of the present invention, the hydrophilic gel chromatography filler suitable for protein analysis of the present invention is a porous substrate, and the substrate is one of a hydrophilic vinyl polymer, a hydrophilic modified silica gel, a dextran gel and an agarose gel; preferably a hydrophilic silica gel;

[0027] Preferably, the pore size of the hydrophilic gel chromatography filler suitable for protein analysis is:

[0028] Particle size: 3-5μm;

[0029] According to a specific embodiment of the present invention, preferably, in the method of the present invention, the chromatographic column used for gel chromatography is preferably TSK-G3000SWxl (7.8×300 mm) provided by TSK Corporation of Japan.

[0030] According to a specific embodiment of the present invention, in the method of the present invention, the mobile phase used for gel chromatography is 0.15-0.3M PB containing 0-5% IPA; the mobile phase pH is 6.5-7.5;

[0031] Preferably, the pH of the mobile phase is 6.8-7.2;

[0032] Preferably, the mobile phase is PB containing 1%-2% IPA;

[0033] Preferably, the mobile phase is 0.2-0.3 M PB.

[0034] According to a specific embodiment of the present invention, in the method of the present invention, the flow rate used for gel chromatography analysis is 0.2-1.0 ml / min; preferably, the flow rate used for gel chromatography is 0.35-1.0 ml / min; further preferably, the flow rate for gel chromatography analysis is 0.5 ml / min.

[0035] According to a specific embodiment of the present invention, in the method of the present invention, the mobile phase used for gel chromatography is 0.2M PB containing 2% IPA, and its pH is 7.2; the flow rate of gel chromatography analysis is 0.5 ml / min.

[0036] According to a specific embodiment of the present invention, in the experimental group of samples for gel chromatography analysis of the present invention: the solvent of the sample is the mobile phase and / or water used in gel chromatography;

[0037] Preferably, the molar ratio of S protein to ACE2 is 1:0.5-2; more preferably 1:1, 1.19:1, 1:2 or 2:1;

[0038] Preferably, the concentration of S protein is 0.5-2 μg / 10 μl; more preferably 1 μg / 10 μl;

[0039] Preferably, the concentration of the test article is 0.5-2 μg / 10 μl; more preferably 0.5-1 μg / 10 μl;

[0040] Preferably, during the preparation of the experimental group samples, the binding reaction conditions are room temperature reaction for 5-30 min; more preferably, 10-30 min.

[0041] The FITC-S protein described in the present invention refers to a FITC-labeled novel coronavirus spike protein, which is prepared by the following method:

[0042] FITC (fluorescein 5-isothiocyanate) acetonitrile solution and S protein / NaHCO3 solution (adjusted to pH 7-9) were mixed and reacted at room temperature for 2-4 hours, acetic acid was added to adjust pH to 6 to terminate the reaction, and FITC-S protein was separated and purified; wherein the concentration of FITC in the FITC acetonitrile solution was 0.5-1.5 mg / ml;

[0043] Preferably, in the FITC acetonitrile solution, the concentration of FITC is 1 mg / ml, the pH of the S protein / NaHCO3 solution is 8.5, and FITC and the S protein / NaHCO3 solution are reacted in a centrifuge tube at room temperature for 2 hours.

[0044] According to a specific embodiment of the present invention, the process of separating and purifying FITC-S protein includes: terminating the reaction and placing the FITC-S protein solution in a 5K ultrafiltration tube and centrifuging it in a centrifuge at 3000g centrifugal force for 15 minutes. After the centrifugation, the filtrate of the ultrafiltration tube is discarded, water is added to the ultrafiltration tube again, and centrifuged again to complete one washing. The washing process is repeated 6 times to obtain FITC-S protein without free FITC.

[0045] According to a specific embodiment of the present invention, in the experimental group samples used for flow cytometry analysis and detection: the cells are 293T cells in the logarithmic growth phase that overexpress hACE2 on the membrane;

[0046] Preferably, in the reaction system, the number of cells is 5.5×10 5 -2×10 6 , FITC-S protein 0.8μg / tube-4μg / tube, test article 1-5μg / tube;

[0047] Preferably, during the preparation of the experimental group samples, the binding reaction conditions are incubation at a temperature suitable for cell growth for half an hour to one hour.

[0048] According to a specific embodiment of the present invention, the method described in the present invention includes constructing 293T cells that overexpress hACE2 on the membrane, specifically, including the following steps: inserting mem-ACE2 (human) -3 × FLAG into the vector pcDNA3.1, transfecting into 293T cells, screening out 293T cells that highly express hACE2 by the G418 method, recovering the whole cell protein, performing mRNA and Western Blotting to detect the protein expression amount, confirming that the protein level hACE2 is highly expressed, and freezing the cells.

[0049] According to a specific embodiment of the present invention, the flow cytometry screening of the present invention includes: dividing the 293T cells overexpressing hACE2 on the membrane in the logarithmic growth phase into two groups, adding the FITC-S protein to the cells in group one, incubating, washing with PBS and centrifuging, setting the fluorescent label to 100%, resuspending, and serving as a positive control group; adding the test article to the cells in group two, incubating, washing with PBS, adding the FITC-S protein, incubating, washing with PBS and centrifuging, resuspending, and analyzing the fluorescence signal coverage by flow cytometry. Preferably, the incubation conditions are 4-36°C, 20-60min; more preferably 36°C, 30 minutes.

[0050] After the test article was added to Group 2, the binding of FITC-S protein and ACE2 was blocked. After washing with PBS, the FITC-S protein that failed to bind would be washed away, and the fluorescence coverage of the cells would be reduced, thereby analyzing the flow cytometric screening blocking efficiency of the test article.

[0051] The present invention preliminarily screens out drugs with blocking effects on novel coronavirus infection by observing the changes in gel chromatography behavior of a mixture of S protein and ACE2 under the action of different test samples. The gel chromatography method can also be used to screen other blocking drugs. The present invention also provides a flow cytometry screening method, which can further improve the accuracy of the blocker by combining gel chromatography screening with flow cytometry screening.

[0052] In another aspect, the present invention provides a blocking agent for blocking novel coronavirus infection, wherein the blocking agent comprises one or more dipeptides having an amino acid sequence of Asn-Phe, Cys-Asp, Ala-Thr, His-Phe and Cys-Gly.

[0053] The dipeptides of the present invention: Asn-Phe, Cys-Asp, Ala-Thr, His-Phe and Cys-Gly, analyzed according to the gel chromatography screening method of the present invention, have blocking activities on the binding of ACE2 and S protein of 29.3%, 4.1%, 16.7%, 10.8% and 13.8%, respectively.

[0054] The dipeptides of the present invention: Asn-Phe, Cys-Asp, Ala-Thr, His-Phe and Cys-Gly, analyzed according to the flow cytometric screening method of the present invention, have blocking activities on the binding of ACE2 and S protein of 43.25%, 23.84%, 34.69%, 19.27% ​​and 37.31%, respectively.

[0055] In another aspect, the present invention provides the use of the blocker in the preparation of a drug for blocking novel coronavirus infection and / or a preparation for blocking novel coronavirus binding to ACE2.

[0056] In another aspect, the present invention provides a pharmaceutical composition comprising the blocking agent.

[0057] In the present invention, the ability of the test sample to competitively bind to ACE2 with the S protein and / or the ability of the test sample to block the S protein from binding to ACE2 detected by gel chromatography is represented by the blocking activity Z1, which is calculated as follows:

[0058] Chromatographic blocking rate

[0059] S1: chromatographic peak area of ​​ACE2 after adding the test sample;

[0060] S2: chromatographic peak area of ​​ACE2 after the reaction of S protein and ACE2 (if any);

[0061] S3: Chromatographic peak area after ACE2 analysis alone.

[0062] In the present invention, the ability of the test article to competitively bind to ACE2 with the S protein and / or the ability of the test article to block the S protein from binding to ACE2 detected by flow cytometry is represented by blocking activity Z2, which is calculated as follows:

[0063] Cell blocking rate Z2 = C1-C2;

[0064] C1: fluorescence coverage after adding S-FITC to cells;

[0065] C2: Fluorescence coverage after adding the test article to the cells.

[0066] In the present invention, the "oligopeptide" refers to a peptide consisting of 2-20 amino acid residues, and each amino acid residue in the peptide may be modified or unmodified.

[0067] In the present invention, the term “blocking” refers to preventing the S protein from binding to the ACE2 active site or reducing the binding ability.

[0068] The present invention screens for test products that can specifically bind to the active site of ACE2, thereby preventing the S protein from binding to the active site of ACE2 or reducing the binding ability.

[0069] The present invention has the following beneficial effects:

[0070] The gel chromatography blocking screening method provided by the present invention is simple to operate, has high screening efficiency and low screening cost, greatly reduces the screening cost while maintaining accuracy, and has high practical application value.

[0071] The gel chromatography combined with flow cytometry screening of the present invention can significantly improve the accuracy of screening blockers: firstly perform initial screening by gel chromatography, and then perform secondary screening of blockers by flow cytometry. This method is simple to operate, efficient, and greatly reduces the screening cost. It can be used as a conventional high-throughput screening of blocking drugs, thereby screening out drugs that have the ability to block the binding of S protein and ACE2. This drug can be used as a new drug for preventing and / or treating novel coronavirus infection to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The principle of gel chromatography blocking screening is shown, where A is the chromatogram of the S protein trimer, B is the chromatogram of ACE2, C is the chromatogram of the S protein and ACE2 complex, and D is the chromatogram after adding the blocker to the S protein and ACE2 mixture.

[0073] Figure 2 This is the chromatogram of the gel chromatography screening blocking dipeptide in Example 1.

[0074] Figure 3 Five blocking dipeptides obtained from gel chromatography screening and their blocking rates are shown.

[0075] Figure 4 Schematic diagram of the structure of pcDNA3.1-mem-ACE2(human)-3×FLAG.

[0076] Figure 5 Schematic diagram of flow cytometry screening principle and some results.

[0077] Figure 6 The fluorescence coverage and blocking rate results of five blocking dipeptides screened by flow cytometry are shown.

[0078] Figure 7 The chromatograms of Example, Comparative Example 1 and Comparative Example 2 when gel chromatography was used to screen the blocking dipeptide Asn-Phe.

[0079] Figure 8 The chromatograms of Example, Comparative Example 1 and Comparative Example 2 when gel chromatography was used to screen the blocking dipeptide Ala-Thr. DETAILED DESCRIPTION

[0080] The new coronavirus belongs to the β subgenus of coronaviruses and mainly binds to host cells through the S protein. The S protein is a large trimeric transmembrane glycoprotein with 1,200 amino acids per monomer. Each monomeric S protein is divided into S1 and S2 functional units. The S1 functional unit recognizes the host cell receptor, and the S1 functional unit is divided into four domains: NTD, CTD1, CTD2, and CTD3. However, its S1 functional unit is prone to mutation. Studies have shown that the new coronavirus enters human cells mainly through the recognition and binding of the human cell receptor angiotensin 2 converting enzyme (ACE2; 805aa) by the trimeric S protein. The essence of the binding of the S protein to ACE2 is the binding between its epitope peptides. Screening for blocking peptides that can competitively bind to the S protein or ACE2 epitope peptides can block the infection pathway of the new coronavirus, exposing the new coronavirus in the patient's body to the immune system for a long time, and achieve the purpose of treatment.

[0081] The present invention is further described in detail below in conjunction with embodiments and comparative examples, but the protection scope of the present invention is not limited to these embodiments.

[0082] Example 1

[0083] This embodiment provides a method for screening a blocking agent for blocking novel coronavirus infection. The method comprises the following steps:

[0084] 1. Gel chromatography screening

[0085] (a) Sample preparation and packaging

[0086] Prepare and aliquot S protein solution: Take a bottle of 500μg / tube trimer S protein, add 833μl ddH2O to dissolve, prepare 600μg / ml S protein solution, and then aliquot into 5 portions with 1ml EP tubes, each portion is about 167μl, and freeze at -70℃ for later use. The S protein used is S protein trimer, the monomer molecular weight is about 138kd, and the S protein trimer molecular weight is about 414kd.

[0087] Prepare and aliquot ACE2 solution: Take a bottle of 200μg / tube ACE2, add 656μl ddH2O to dissolve, make 305μg / ml ACE2 solution, and then aliquot into 40 portions with 200μl EP tubes. Each portion is 16.4μl and frozen at -70℃ for later use. The molecular weight of the ACE2 used is about 87.2kd.

[0088] (b) Preparation of S protein and ACE2 complex

[0089] Take the S protein solution and ACE2 solution prepared in step (a) above, add the mobile phase (0.2M PB + 2% IPA, pH 7.0) to prepare a reaction system with a total volume of 10 μl; the S protein content in the reaction system is 1 μg / 10 μl, and the ACE2 content is 0.18 μg / 10 μl. The reaction is carried out at room temperature for 20 minutes to obtain a complex of S protein and ACE2.

[0090] (c) Gel chromatography analysis

[0091] Instrument conditions: Agilent 1260 high performance liquid chromatograph;

[0092] Detector: UVD ultraviolet detector;

[0093] Wavelength: 280nm;

[0094] Chromatographic column: TSK-G3000SWxl, 7.8×300mm (TSK, Japan);

[0095] The experimental conditions are:

[0096] Mobile phase: 0.2M PB + 2% IPA; mobile phase pH 7.0; flow rate: 0.5 ml / min; room temperature; injection volume: 10 μl.

[0097] The gel chromatography behaviors of S protein, ACE2, and the complex of S protein and ACE2 at 280 nm were analyzed and recorded by gel chromatography respectively; then 1 μl of the test sample (1 μg / μl, the solvent was the mobile phase) was added to the mixture of S protein and ACE2, the concentration of the test sample in the reaction system was 1 μg / 10 μl, mixed well, reacted at room temperature for 20 minutes, and its chromatographic behavior was analyzed by gel chromatography, and the change in ACE2 peak area after the addition of the test sample was calculated to determine the ability of the test sample to block the formation of a complex between S protein and ACE2, thereby calculating its blocking activity.

[0098] Figure 1 Figure 2 shows the principle of gel chromatography blocking screening and some of the screening results, where A is the chromatogram of the S protein trimer, B is the chromatogram of ACE2, C is the chromatogram of the S protein and ACE2 complex, and D is the chromatogram after adding the blocker to the mixture of S protein and ACE2.

[0099] It can be seen from the chromatogram that in the gel chromatography screening method provided by the present invention, S protein, ACE2, and the S protein and ACE2 complex have different retention times (S protein retention is 11.6min, ACE2 retention value is 14.2min; S / ACE2 complex retention value is 10.9min). When S protein and ACE2 form a complex, a 10.9min complex peak will appear. Under this condition, the gel chromatography separation is good and the peak shape is better.

[0100] After adding the test sample, if the test sample cannot block its binding, then the ACE2 peak area should not change significantly; if it can block the binding of S protein and ACE2, then the peak area of ​​the complex will decrease, and the corresponding ACE2 peak area will increase (the amount of ACE2 not bound to S protein will increase). Through the changes in the gel chromatography behavior of the S protein and ACE2 mixture under different test samples, drugs with blocking effects on novel coronavirus infection were preliminarily screened out.

[0101] Figure 2 Shown is the chromatogram when screening blocking dipeptides with gel chromatography.

[0102] Figure 3 The blocking rates of the five blocking dipeptides obtained by gel chromatography screening: Asn-Phe, Cys-Asp, Ala-Thr, His-Phe and Cys-Gly are shown. Figure 3 It can be seen that according to the gel chromatography screening method of the present invention, their blocking activities on the binding of ACE2 and S protein are 29.3%, 4.1%, 16.7%, 10.8% and 13.8%, respectively.

[0103] By analyzing the changes in the gel chromatography behavior of the mixture of S protein and ACE2 under the action of different test products, drugs with blocking effects on novel coronavirus infection were preliminarily screened out. These preliminarily screened blockers can also be further confirmed by the subsequent flow cytometry screening method to see whether they have the effect of blocking the binding of ACE2 and S protein.

[0104] 2. Flow cytometry screening

[0105] (1) Fluorescent labeling of S protein and preparation of FITC-S protein

[0106] Dissolve an appropriate amount of FITC in acetonitrile solution to prepare a 1 mg / ml FITC acetonitrile solution for later use.

[0107] 500 μg of S protein was dissolved in NaHCO3 solution and the pH was adjusted to 8.5 with 0.5 M NaOH for later use.

[0108] S protein fluorescent labeling: FITC acetonitrile solution and S protein / NaHCO3 solution were mixed in a centrifuge tube and reacted at room temperature for 2 hours. The pH of the reaction solution was then adjusted to 6 with AcOH to terminate the reaction. The system at this time was recorded as FITC-S protein solution.

[0109] Isolation and purification of FITC-S protein: After the reaction is completed, the FITC-S protein solution is placed in a 2 ml 5K ultrafiltration tube and centrifuged at 3000g for 15 minutes. After the centrifugation, the filtrate in the ultrafiltration tube is discarded, and 500μl of ultrapure water is added to the ultrafiltration tube again, and centrifuged again to complete one wash. Repeat the washing process 6 times to obtain FITC-S protein without free FITC.

[0110] (2) Construction of 293T cells overexpressing hACE2 on the membrane

[0111] Insert the nucleic acid fragment encoding mem-ACE2(human)-3×FLAG into the vector pcDNA3.1 to construct the overexpression vector pcDNA3.1-mem-ACE2(human)-3×FLAG. The vector structure diagram is shown in Figure 4 .

[0112] The overexpression vector pcDNA3.1-mem-ACE2(human)-3×FLAG was transfected into 293T cells, and 293T cells with high expression of hACE2 were screened by G418 method (G418 concentration was 150μg / ml). The total cell protein was recovered, and the protein expression was detected by mRNA and Western Blotting. After confirming that the protein level hACE2 was highly expressed, the cells were frozen for later use.

[0113] (3) Flow cytometry screening

[0114] Take the 293T cells overexpressing hACE2 on the membrane in the logarithmic growth phase and place them in a flow cytometry tube. Add 3-4 ml of PBS to wash the cells. Centrifuge at 1200 rpm for 6 min and discard the supernatant. 5 cells / branch management.

[0115] The experimental groups are as follows:

[0116] Group 1: Add FITC-S protein to the cells, 0.8 μg / tube, and incubate at 36°C for half an hour. Add PBS wash solution and centrifuge at 1200 rpm for 6 minutes to wash twice. Set the fluorescent label to 100% (positive control), and finally add 500 μl PBS to suspend and prepare for loading.

[0117] Group 2: Add the test substance to the cells, 2 μg / tube, incubate at 36℃ for half an hour, add PBS washing solution and centrifuge at 1200rpm for 6 minutes to wash twice, add FITC-S protein, 0.8 μg / tube, incubate at 36℃ for half an hour. Add PBS washing solution and centrifuge at 1200rpm for 6 minutes, repeat the washing twice. Finally, add 500 μl PBS to suspend and prepare for the machine. Use flow cytometer to analyze the fluorescence signal coverage and positive control ratio.

[0118] Figure 5 The flow cytometry screening principle is shown. After the test article is added to Group 2, the binding of FITC-S protein and ACE2 is blocked. After washing with PBS, the FITC-S protein that fails to bind will be washed away, and the fluorescence coverage of the cells will decrease, thereby calculating the flow cytometry screening blocking efficiency of the test article.

[0119] Figure 6 The fluorescence coverage and blocking rate results of five blocking dipeptides screened by flow cytometry are shown. Figure 6 It can be seen that, according to the flow cytometric screening method of the present invention, the blocking activities of Asn-Phe, Cys-Asp, Ala-Thr, His-Phe and Cys-Gly on the binding of ACE2 and S protein are 43.25%, 23.84%, 34.69%, 19.27% ​​and 37.31%, respectively.

[0120] Comparative Example 1

[0121] This comparative example provides a gel chromatography screening method for measuring the blocking activity of the five dipeptides obtained in Example 1. The method comprises the following steps:

[0122] (a) Sample preparation and packaging

[0123] Prepare and aliquot S protein solution: Take a bottle of 500μg / tube trimer S protein, add 833μl ddH2O to dissolve, prepare 600μg / ml S protein solution, and then aliquot into 5 portions with 1ml EP tubes, 167μl each, and freeze at -70℃ for later use. The S protein used is S protein trimer, the molecular weight of the monomer is about 138kd, and the molecular weight of the S protein trimer is about 414kd.

[0124] Prepare and aliquot ACE2 solution: Take a bottle of 200μg / tube ACE2, add 656μl ddH2O to dissolve, make 305μg / ml ACE2 solution, and then aliquot into 40 portions with 200μl EP tubes. Each portion is 16.4μl and frozen at -70℃ for later use. The molecular weight of the ACE2 used is about 87.2kd.

[0125] (b) Preparation of S protein and ACE2 complex

[0126] Take the S protein solution and ACE2 solution prepared in the above step (a) respectively, add the mobile phase (0.2M PB, pH 6.2), and prepare a reaction system with a total volume of 10μl; the S protein content in the reaction system is: 1μg / 10μl, and the ACE2 content is: 0.36μg / 10μl. The reaction is carried out at room temperature for 5 minutes to obtain the S protein and ACE2 complex.

[0127] (c) Gel chromatography analysis

[0128] Instrument conditions:

[0129] Agilent 1260 high performance liquid chromatograph;

[0130] Detector: UVD-2 ultraviolet detector;

[0131] Wavelength: 280nm;

[0132] Chromatographic column: TSKgel G3000SWxl, 4.6×300 mm (TSK, Japan);

[0133] The experimental conditions are:

[0134] Mobile phase: 0.2M PB; mobile phase pH 6.2; flow rate: 0.3ml / min; room temperature; injection volume: 10μl.

[0135] The gel chromatography behaviors of S protein, ACE2, and the complex of S protein and ACE2 at 280 nm were analyzed and recorded by gel chromatography respectively; then 1 μl of the test sample (1 μg / μl, the solvent was the mobile phase) was added to the mixture of S protein and ACE2, the concentration of the test sample in the reaction system was 1 μg / 10 μl, mixed well, reacted at room temperature for 20 minutes, and its chromatographic behavior was analyzed by gel chromatography, and the change in ACE2 peak area after the addition of the test sample was calculated to determine the ability of the test sample to block the formation of a complex between S protein and ACE2, thereby calculating its blocking activity.

[0136] By changing the gel chromatography behavior of the S protein and ACE2 mixture under different test products, drugs with blocking effects on novel coronavirus infection were preliminarily screened out. The screening principle is the same as in Example 1.

[0137] The test products used in this comparative example are Asn-Phe, Cys-Asp, Ala-Thr, His-Phe and Cys-Gly. According to the gel chromatography screening method described in this comparative example, the blocking activities of Asn-Phe, Cys-Asp, Ala-Thr, His-Phe and Cys-Gly on the binding of ACE2 and S protein are 8.6%, 2.2%, 7.7%, 3.2% and 4.1%, respectively. Among them, the chromatograms of Asn-Phe and Ala-Thr screened by gel chromatography in this comparative example are shown in Figure 7 The Chinese Library and Figure 8 The middle picture of .

[0138] The experimental results show that under the chromatographic conditions, the separation between the ACE2 peak and the S protein peak after adding the test sample is insufficient, resulting in a relatively small calculated ACE2 peak area, and thus a low calculated blocking activity. There are three reasons for the above phenomenon: on the one hand, it may be caused by the use of too high a chromatographic flow rate; on the other hand, it may be due to the pH condition being close to the isoelectric point of ACE2, which affects its solubility; on the third hand, it may be due to the fact that the amount of ACE2 added is too large, and ACE2 has not reacted completely.

[0139] Comparative Example 2

[0140] This comparative example provides a gel chromatography screening method for measuring the blocking activity of the five dipeptides obtained in Example 1. The method comprises the following steps:

[0141] (a) Sample preparation and packaging

[0142] Prepare and aliquot S protein solution: Take a bottle of 500μg / tube trimer S protein, add 833μl ddH2O to dissolve, prepare 600μg / ml S protein solution, and then aliquot into 5 portions with 1ml EP tubes, 167μl each, and freeze at -70℃ for later use. The S protein used is S protein trimer, the monomer molecular weight is about 138kd, and the S protein trimer molecular weight is about 414kd.

[0143] Prepare and aliquot ACE2 solution: Take a bottle of 200μg / tube ACE2, add 656μl ddH2O to dissolve, make 305μg / ml ACE2 solution, and then aliquot into 40 portions with 200μl EP tubes. Each portion is 16.4μl and frozen at -70℃ for later use. The molecular weight of the ACE2 used is about 87.2kd.

[0144] (b) Preparation of S protein and ACE2 complex

[0145] Take the S protein solution and ACE2 prepared in step (a) respectively, add the mobile phase (0.1M PB + 2% IPA, pH 6.7), and prepare a reaction system with a total volume of 10 μl; the S protein content in the reaction system is: 2 μg / 10 μl, and the ACE2 content is: 0.18 μg / 10 μl. The reaction is carried out at room temperature for 30 minutes to obtain the S protein and ACE2 complex.

[0146] (c) Gel chromatography analysis

[0147] Instrument conditions:

[0148] Agilent 1260 high performance liquid chromatograph;

[0149] Detector: UVD-2 ultraviolet detector;

[0150] Wavelength: 280nm;

[0151] Column: TSK-UltraSW Aggregate, 7.8×300mm;

[0152] The experimental conditions are:

[0153] Mobile phase: 0.1M PB + 2% IPA; mobile phase pH 6.7; flow rate: 0.7 ml / min; room temperature; injection volume: 10 μl.

[0154] The gel chromatography behaviors of S protein, ACE2, and the complex of S protein and ACE2 at 280 nm were analyzed and recorded by gel chromatography respectively; then 1 μl of the test sample (1 μg / μl, the solvent was the mobile phase) was added to the mixture of S protein and ACE2, the concentration of the test sample in the reaction system was 1 μg / 10 μl, mixed well, reacted at room temperature for 20 minutes, and its chromatographic behavior was analyzed by gel chromatography, and the change in ACE2 peak area before and after the addition of the test sample was calculated to determine the ability of the test sample to block the formation of a complex between S protein and ACE2, thereby calculating its blocking activity.

[0155] By changing the gel chromatography behavior of the S protein and -ACE2 mixture under the action of different test products, drugs with blocking effects on novel coronavirus infection were preliminarily screened out. The screening principle is the same as in Example 1.

[0156] The test products used in this comparative example are Asn-Phe, Cys-Asp, Ala-Thr, His-Phe and Cys-Gly. According to the gel chromatography screening method described in this comparative example, the blocking activities of Asn-Phe, Cys-Asp, Ala-Thr, His-Phe and Cys-Gly on the binding of ACE2 and S protein are 6.3%, 1.1%, 8.3%, 2.9% and 3.1%, respectively. Among them, the chromatograms of Asn-Phe and Ala-Thr screened by gel chromatography in this comparative example are shown in Figure 7 The following figure and Figure 8 The picture below.

[0157] The experimental results show that under the chromatographic conditions, the separation between the ACE2 peak and the S protein peak after adding the test sample is insufficient, resulting in a relatively small calculated ACE2 peak area, and thus a low calculated blocking activity. Analysis shows that the reason for the above phenomenon may be that the low PB concentration in the mobile phase causes nonspecific adsorption, resulting in a low calculated ACE2 peak area.

Claims

1. A method for screening a blocking agent for blocking novel coronavirus infection, the method comprising: Gel chromatography and / or flow cytometry are used to detect the ability of the test article to competitively bind to ACE2 with the S protein and / or the ability of the test article to block the S protein from binding to ACE2, so as to screen out blockers that have the effect of blocking the infection of the new coronavirus; The gel chromatography detection process includes: Prepare experimental group samples for gel chromatography analysis; wherein the experimental group samples include: samples for detecting the ability of the test article to competitively bind to ACE2 with the S protein, and / or samples for detecting the ability of the test article to block the S protein from binding to ACE2; the preparation process of the sample for detecting the ability of the test article to competitively bind to ACE2 with the S protein includes mixing the test article and the S protein with ACE2 at the same time for a competitive binding reaction; the preparation process of the sample for detecting the ability of the test article to block the S protein from binding to ACE2 includes first mixing the test article with ACE2 for a binding reaction, and then adding the S protein for a reaction; The gel chromatography behavior of the experimental group samples at a wavelength of 280 nm was analyzed by gel chromatography, and the ability of the test sample to competitively bind to ACE2 with the S protein and / or the ability of the test sample to block the S protein from binding to ACE2 was determined based on the peak area corresponding to ACE2 in the chromatogram; The flow cytometry detection process includes: preparing FITC-S protein and flow cytometry screening; Prepare experimental group samples for flow cytometry analysis and detection; wherein the experimental group samples include: samples for detecting the ability of the test article to competitively bind to ACE2 with the S protein, and / or samples for detecting the ability of the test article to block the S protein from binding to ACE2; the preparation process of the sample for detecting the ability of the test article to competitively bind to ACE2 with the S protein includes mixing the test article and the FITC-S protein with cells overexpressing ACE2 at the same time for a competitive binding reaction; the preparation process of the sample for detecting the ability of the test article to block the S protein from binding to ACE2 includes first mixing the test article with cells overexpressing ACE2 for a binding reaction, and then adding the FITC-S protein for a reaction; Flow cytometry was used to analyze the fluorescence signal coverage of the experimental group samples, and the ability of the test article to competitively bind to ACE2 with the S protein and / or the ability of the test article to block the binding of the S protein to ACE2 was determined based on the fluorescence signal coverage.

2. The method according to claim 1, wherein: The test article includes a peptide; Preferably, the test article comprises an oligopeptide; More preferably, the test substance is a dipeptide, a tripeptide, a tetrapeptide and / or a pentapeptide; further preferably, it is a straight-chain dipeptide composed of unmodified amino acids.

3. The method according to claim 1, wherein: The gel chromatography detection process also includes: comparing the peak area corresponding to ACE2 in the chromatogram of the experimental group sample with the peak area of ​​the control group without the test article, to determine the ability of the test article to competitively bind to ACE2 with the S protein and / or the ability of the test article to block the S protein from binding to ACE2; The flow cytometry detection process also includes: comparing the fluorescence signal coverage of the experimental group samples with the fluorescence signal coverage of the control group without the test article, to determine the ability of the test article to competitively bind to ACE2 with the S protein and / or the ability of the test article to block the S protein from binding to ACE2; Preferably, the screened blocking agent with the effect of blocking novel coronavirus infection has: the ability to competitively bind to ACE2 with the S protein, and the ability to block the S protein from binding to ACE2; More preferably, gel chromatography is used to detect the ability of the test article to competitively bind to ACE2 with the S protein, and flow cytometry is used to detect the ability of the test article to block the binding of the S protein to ACE2.

4. The method according to claim 1, wherein: The chromatographic column filler used in the gel chromatography is a hydrophilic gel chromatography filler suitable for protein analysis; Preferably, the exclusion limit of the gel chromatography column is 5kd-2500kd; Preferably, the chromatography column length of the gel chromatography column is ≥25 cm; Preferably, the inner diameter of the gel chromatography column is ≤7.8 mm; Preferably, the number of chromatography column plates of the gel chromatography column is ≥25,000.

5. The method according to claim 1, wherein: The mobile phase used for gel chromatography is 0.15-0.3M PB containing 0-5% IPA; the mobile phase pH is 6.5-7.5; Preferably, the pH of the mobile phase is 6.8-7.2; Preferably, the mobile phase is PB containing 1%-2% IPA; Preferably, the mobile phase is 0.2-0.3M PB; Preferably, the flow rate used in the gel chromatography is 0.35-1.0 ml / min; More preferably, the mobile phase used for gel chromatography is 0.2M PB containing 2% IPA, and its pH is 7.0; the flow rate of gel chromatography analysis is 0.5 ml / min.

6. The method according to claim 1, wherein: In the experimental group of samples used for gel chromatography analysis: the solvent of the sample is the mobile phase used for gel chromatography and / or water; Preferably, the molar ratio of S protein to ACE2 is 1:0.5-2; more preferably 1:1, 1.19:1, 1:2 or 2:1; Preferably, the concentration of S protein is 0.5-2 μg / 10 μl; more preferably 1 μg / 10 μl; Preferably, the concentration of the test article is 0.5-2 μg / 10 μl; more preferably 0.5-1 μg / 10 μl; Preferably, during the preparation of the experimental group samples, the binding reaction conditions are room temperature reaction for 10-30 minutes.

7. The method according to claim 1, wherein: In the experimental group samples used for flow cytometry analysis: the cells are 293T cells in the logarithmic growth phase that overexpress hACE2 on the membrane; Preferably, in the reaction system, the number of cells is 5.5×10 5 -2×10 6 , FITC-S protein 0.8μg / tube-4μg / tube, test article 1-5μg / tube; Preferably, during the preparation of the experimental group samples, the binding reaction conditions are incubation at a temperature suitable for cell growth for half an hour to one hour.

8. A blocking agent for blocking infection by the new coronavirus, the blocking agent comprising one or more dipeptides having an amino acid sequence of Asn-Phe, Cys-Asp, Ala-Thr, His-Phe and Cys-Gly.

9. Use of the blocking agent according to claim 8 in the preparation of a drug for blocking infection with the new coronavirus and / or a preparation for blocking the binding of the new coronavirus to ACE2.

10. A pharmaceutical composition comprising the blocking agent according to claim 8.

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