Specific Epitopes of the N Antigen of the Novel Coronavirus and Their Applications

Abstract

The present invention relates to specific epitopes of the N antigen of SARS-CoV-2 and their applications, and provides a polypeptide, a kit containing the polypeptide, and their applications in the preparation of products for the detection, diagnosis or drug screening of SARS-CoV-2. The present invention also provides a method for detecting SARS-CoV-2 and a method for drug screening. By using the polypeptide described in the present invention to design or screen antibodies, advantages such as low detection limit, high specificity and high sensitivity can be obtained.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to methods for detecting, diagnosing, and drug screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as the use of polypeptides or antibodies against polypeptides in the preparation of products for detecting, diagnosing, and drug screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Background Art

[0002] Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide pandemic. Antibodies are important materials for combating this pandemic disease. In addition, they also play an important role in helping people study the pathogenesis of COVID-19, conducting diagnostic tests, and developing therapeutic methods to neutralize the activity of SARS-CoV-2 virus. However, the amino acid sequences of epitopes that antibodies specifically recognize and bind to antigens are unknown for most SARS-CoV-2 antibodies.

[0003] SARS-CoV-2 has four main structural proteins: spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), and envelope protein (E protein). Among them, the S protein is abundant in coronaviruses. The receptor-binding domain (RBD) in the S protein contains a highly similar core structure and a highly variable receptor-binding motif (RBM), which is the main structure for recognizing angiotensin-converting enzyme 2 (ACE2) and plays a crucial role in antibody screening, vaccine preparation, and other processes. As a structural protein, the N antigen can also be used for detection or diagnosis.

[0004] At present, nucleic acid and serum antibody detection are still the main technologies for the diagnosis of COVID-19. For example, patent document CN112409462A discloses a detection kit for SARS-CoV-2 specific antigen and SARS-COV-2 immunoglobulin, which includes S protein and N protein as antigens for detection, and has a lower false positive and false negative rate. Patent document CN112485425A discloses a kit for detecting novel coronavirus N-S antigen in saliva and its application method, which is convenient to operate, has a short detection period, and is easy to interpret. Patent document CN111999496A discloses a kit for combined detection of SARS-CoV-2 antigen and antibody and its preparation method, which uses a kit for combined detection of antigen and antibody, and the kit contains a reaction plate coated with S antigen. However, the specific epitopes targeted by the antibodies are not disclosed in the above-mentioned prior arts, and the design of the antibodies only targets the full-length S protein or the full-length N protein, and their specificity and sensitivity are limited.

[0005] Therefore, the present application provides a polypeptide, which can be used for the design or screening of antibodies, achieving high specificity and sensitivity, and the specific recognition epitopes of the antibody pairs have not been reported yet. Summary of the Invention

[0006] In the first aspect of the present invention, there is provided an application of a polypeptide in the preparation of a product for the detection, diagnosis or drug screening of SARS-CoV-2, and the polypeptide comprises any one or a combination of two or more of the amino acids shown in SEQ ID NO: 1-15.

[0007] In the second aspect of the present invention, there is provided an application of a polypeptide in the detection of SARS-CoV-2, and the polypeptide comprises any one or a combination of two or more of the amino acids shown in SEQ ID NO: 1-15.

[0008] Preferably, the polypeptide comprises:

[0009] A) SEQ ID NO: 1, and SEQ ID NO: 2 and / or 3; or,

[0010] B) SEQ ID NO: 1, and any one or a combination of two or more of SEQ ID NO: 4-15.

[0011] In the third aspect of the present invention, there is provided a polypeptide, and the polypeptide comprises any one or a combination of two or more of the amino acids shown in SEQ ID NO: 1-15.

[0012] Preferably, the polypeptide comprises:

[0013] A) SEQ ID NO: 1, and SEQ ID NO: 2 and / or 3; or,

[0014] B) SEQ ID NO: 1, and any one or a combination of two or more of SEQ ID NOs: 4 - 15.

[0015] In the fourth aspect of the present invention, a kit for detecting or diagnosing SARS-CoV-2 is provided, and the kit contains the above-mentioned polypeptide.

[0016] Preferably, the kit further contains a substrate that can enable the polypeptide to be distributed in an array. More preferably, the substrate includes but is not limited to glass, silicon wafer, ceramic, mica, metal, plastic, polymer film, etc., and is preferably glass, silicon wafer, and ceramic.

[0017] The polypeptide of the present invention can be an amino acid sequence or a combination of multiple amino acid sequences.

[0018] In the fifth aspect of the present invention, a method for drug screening or verifying drug function is provided, and the method includes contacting the above-mentioned polypeptide with a sample to be screened.

[0019] Preferably, the method includes:

[0020] Contacting the polypeptide containing SEQ ID NO: 1 with the sample to be screened, and then adding the polypeptide containing SEQ ID NO: 2 and / or 3; or, contacting the polypeptide containing SEQ ID NO: 1 with the sample to be screened, and then adding the polypeptide containing any one or a combination of two or more of SEQ ID NOs: 4 - 15.

[0021] In the sixth aspect of the present invention, a method for detecting SARS-CoV-2 is provided, which includes contacting a capture antibody with a sample to be detected, and then adding a detection antibody. The capture antibody recognizes the polypeptide shown in SEQ ID NO: 1, and the detection antibody recognizes any one or a combination of two or more of the polypeptides of SEQ ID NOs: 2 - 15.

[0022] Preferably, the detection method includes coating the capture antibody on a carrier, blocking; adding the sample to be detected, incubating; adding the detection antibody, incubating; adding a labeled antibody, incubating; and terminating the reaction.

[0023] In the seventh aspect of the present invention, there is provided the use of an antibody pair in the preparation of a product for detecting or diagnosing SARS-CoV-2, wherein the antibody pair comprises a capture antibody and a detection antibody, the capture antibody recognizes an epitope comprising the epitope shown in SEQ ID NO: 1, and the detection antibody recognizes an epitope comprising a) the epitopes shown in SEQ ID NO: 2 and SEQ ID NO: 3, and / or b) any one or a combination of two or more of the epitopes shown in SEQ ID NOs: 4-15.

[0024] In the eighth aspect of the present invention, there is provided the use of an antibody pair in detecting SARS-CoV-2, wherein the antibody pair comprises a capture antibody and a detection antibody, the capture antibody recognizes an epitope comprising the epitope shown in SEQ ID NO: 1, and the detection antibody recognizes an epitope comprising a) the epitopes shown in SEQ ID NO: 2 and SEQ ID NO: 3, and / or b) any one or a combination of two or more of the epitopes shown in SEQ ID NOs: 4-15.

[0025] Preferably, the antibody pair targets the orf1ab polyprotein, S glycoprotein, ORF3a protein, envelope protein, membrane glycoprotein, ORF6 protein, ORF7a protein, ORF8 protein, nucleocapsid phosphoprotein and / or ORF10 protein of SARS-CoV-2, as specifically shown in Table 1.

[0026] In a specific embodiment of the present invention, the antibody pair targets a specific epitope of the N protein (NP_828858.1) of SARS-CoV-2.

[0027] Table 1 Proteins encoded by SARS-CoV-2 coronavirus

[0028]

[0029]

[0030] Specifically, the epitopes included in the epitope recognized by the capture antibody and the epitope recognized by the detection antibody are shown in Table 2:

[0031] Table 2

[0032]

[0033]

[0034] Preferably, the product can be a kit, a chip, and the like.

[0035] In a ninth aspect of the present invention, there is provided the use of an antibody pair for recognizing an epitope or a polypeptide as a drug target or a therapeutic target in the preparation of a therapeutic drug for a disease caused by SARS-CoV-2.

[0036] In a tenth aspect of the present invention, there is provided an antibody pair, the antibody pair comprising a capture antibody and a detection antibody, the capture antibody recognizing an epitope comprising the epitope shown in SEQ ID NO: 1, and the detection antibody recognizing an epitope comprising a) the epitopes shown in SEQ ID NO: 2 and SEQ ID NO: 3, and / or b) any one or a combination of two or more of the epitopes shown in SEQ ID NOs: 4-15.

[0037] In an eleventh aspect of the present invention, there is provided a kit for detecting or diagnosing SARS-CoV-2, the kit comprising the above-mentioned antibody pair for recognizing an epitope or a polypeptide or the above-mentioned antibody pair. Preferably, the kit further comprises a carrier, a washing solution, a blocking solution, a labeled antibody, a chromogenic solution, and / or a reaction termination solution.

[0038] More preferably, the carrier can be any container or plate capable of supporting the antigen-antibody reaction. More preferably, it is a 96-well plate.

[0039] More preferably, the washing solution can be PBST or a solution containing PBST.

[0040] More preferably, the blocking solution can be PBST and / or milk. More preferably, it is 0.2% PBST + 5% skim milk.

[0041] More preferably, the labeled antibody can be a horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody.

[0042] More preferably, the chromogenic solution can be 3’,3’,5’,5’,-tetramethylbenzidine (TMB).

[0043] More preferably, the reaction termination solution can be sulfuric acid, hydrochloric acid, or NaOH.

[0044] In a twelfth aspect of the present invention, there is provided a method for detecting SARS-CoV-2, comprising contacting the capture antibody with a sample to be detected and then adding the detection antibody.

[0045] Preferably, the detection method comprises coating the capture antibody on a carrier, blocking; adding the sample to be detected, incubating; adding the detection antibody, incubating; adding the labeled antibody, incubating; and terminating the reaction.

[0046] Further preferably, the carrier can be a 96-well plate.

[0047] Further preferably, the blocking is carried out by blocking with a blocking solution (preferably 0.2% PBST + 5% skim milk) at 30 - 40 °C (preferably 37 °C).

[0048] Further preferably, the labeled antibody can be a goat anti-rabbit IgG antibody labeled with horseradish peroxidase (HRP).

[0049] In a specific embodiment of the present invention, the sample to be detected can be any sample containing SARS-CoV-2 proteins (including S protein, N protein, M protein or E protein, preferably N protein). Specifically, it can be human or non-human animal blood, serum, cells, tissues or organs (such as nasopharyngeal swabs, deep sputum, bronchoalveolar lavage fluid, lung tissue biopsy specimens), or it can be in vitro human or non-human animal blood, serum, cells, tissues or organs (such as nasopharyngeal swabs, deep sputum, bronchoalveolar lavage fluid, lung tissue biopsy specimens), or it can also be any articles that can be attached by SARS-CoV-2, such as ice cream, meat, seafood, food packaging, staircase handrails, table tops, etc.

[0050] Based on the differences in the above samples to be detected, the detection method of SARS-CoV-2 described in this application can be for therapeutic purposes or non-therapeutic purposes.

[0051] Preferably, the incubation temperature is 30 - 40, more preferably 37 °C.

[0052] Preferably, the incubation time is 0.5 - 2 h, more preferably 1 h.

[0053] Preferably, the labeled antibody can be a goat anti-rabbit IgG antibody labeled with horseradish peroxidase (HRP).

[0054] In a specific embodiment of the present invention, the capture antibody is coated in a 96-well plate and incubated overnight at 4 °C. After washing, it is blocked in 0.2% PBST + 5% skim milk at 37 °C for 1 h; after washing with 0.2% PBST, the sample to be detected is added and incubated at 37 °C for 1 h; after washing, the detection antibody is added and incubated at 37 °C for 1 h; after washing with PBST, a goat anti-rabbit IgG antibody labeled with horseradish peroxidase (HRP) is added and incubated at 37 °C for 1 h; after washing with PBST, 3’,3’,5’,5’-tetramethylbenzidine (TMB) is added, and then the termination reaction solution is added.

[0055] Preferably, it further includes the step of detecting the absorbance at 450 nm.

[0056] The capture antibody or detection antibody described in the present invention can be in the conventional form of an antibody or an antigen-binding fragment. Of course, it can also be a single-chain antibody or a single-domain antibody. It can be a monoclonal antibody or a polyclonal antibody. It also includes fragments such as Fab, Fab’, Fab’-SH, Fv, scFv, (Fab’)2, single-domain antibody, diabody (dAb) or linear antibody.

[0057] "Detection" as described in the present invention is to determine whether a test sample contains severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or whether it contains autoantibodies.

[0058] "Diagnosis" as described in the present invention is to confirm whether an individual has coronavirus disease 2019 (COVID-19) due to infection with SARS-CoV-2 BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings, where:

[0060] Figure 1 : Antibody recognition epitopes of the SARS-CoV-2 N antigen antibody pair, where a is the #14 recognition epitope, b is the #12 recognition epitope, c is the #13 recognition epitope, Z-score = (x - μ) / σ, where x is the observed value, μ is the mean of this set of data, and σ is the standard deviation of this set of data.

[0061] Figure 2 : Results of detecting SARS-CoV-2 recombinant N protein by ELISA method, where a represents pair 1 and b represents pair 2.

[0062] Figure 3 : Results of detecting N protein in the culture medium of VERO cells infected with SARS-CoV-2 virus by ELISA method, where a represents pair 1 and b represents pair 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0063] The following specifically describes the specific embodiments provided by the present invention in combination with the embodiments.

[0064] Sources of antibodies, cells and reagents used in this application:

[0065] Antibody #12 was purchased from Sino Biological Inc., catalog number 40143-R001, antibody type MAb, host Rabbit;

[0066] Antibody #13 was purchased from Sino Biological Inc., catalog number 40143-RP01, antibody type PAb, host Rabbit;

[0067] Antibody #14, purchased from Sino Biological Inc., catalog number 40143-MM05, antibody type MAb, host Mouse;

[0068] Recombinant SARS-CoV-2 N protein, purchased from Sino Biological, Inc, catalog number 40588-V08B.

[0069] Example 1 Determination of antibody epitopes for detecting SARS-CoV-2

[0070] According to all the coding protein sequences of SARS-CoV-2 extracted from NCBI data recorded in CN202010215184.9 (application date: 2020-03-24), a SARS-CoV-2 virus proteome polypeptide chip was designed and prepared to achieve a panoramic scan of all SARS-CoV-2 virus antibodies in the blood of patients infected with the novel coronavirus pneumonia. All the inventive content thereof can be used as the content of the present invention.

[0071] The antibody epitopes that specifically react with the polypeptide chip are as Figure 1 shown.

[0072] Example 2 Detection of SARS-CoV-2 nucleocapsid protein by enzyme-linked immunosorbent assay

[0073] Experimental procedure: Antibody #14 was coated onto a 96-well plate (Corning, USA) and incubated overnight at 4°C. After washing, it was blocked in 0.2% PBST + 5% skim milk at 37°C for 1 h. After washing with 0.2% PBST, the diluted recombinant SARS-CoV-2 N protein or the inactivated VERO cell culture infected with SARS-CoV-2 IME-BJ01 strain for 2 - 3 days (reference: Esparza, Thomas J et al. “High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme.” Scientific reports vol. 10, 1 22370. 22 Dec. 2020.) was added to the 96-well plate and incubated at 37°C for 1 h. After washing, antibody #12 or #13 was added. After incubating at 37°C for 1 h, it was washed with PBST. Goat anti-rabbit IgG antibody labeled with horseradish peroxidase (HRP) (CoWin Biosciences, China) was added to the 96-well plate and incubated at 37°C for 1 h. After washing with PBST, 3’,3’,5’,5’,-tetramethylbenzidine (TMB) (CoWin Biosciences, China) was added, and then the stop solution for enzyme-linked immunosorbent assay (Solarbio, China) was added. After briefly shaking the plate, the absorbance at 450 nm was read.

[0074] Among them, the capture antibody is: #14; the detection antibodies are (#12, #13), representing two antibody pairs (pair 1: #14 and #12, pair 2: #14 and #13).

[0075] Statistical analysis: Statistical analysis was performed using GraphPad Prism software and Microsoft Excel.

[0076] Experimental results: When detecting the recombinant SARS-CoV-2 N protein by ELISA, the detection limits of pair 1 and pair 2 were 1.425 ng / mL and 1.03 ng / mL ( Figure 2 a and Figure 2 b); when detecting the N protein in the VERO cell culture fluid infected with SARS-CoV-2 virus by ELISA, the limits of detection (LOD) were 1.119×10 5 PFU / mL (pair 1, Figure 3 a) and 1.615×10 4 PFU / mL (pair 2, Figure 3b). That is, the ELISA method based on antibody pairs (#14 and #12, #14 and #13) has great potential in the clinical detection of SARS-CoV-2.

[0077] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Sequence Listing <110> Institute of Life Omics, Academy of Military Medical Sciences, Chinese Academy of Military Sciences <120> Specific Epitopes of N Antigen of New Coronavirus and Their Applications <130> 1 <160> 15 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <400> 1 Gly Gly Asp Gly Lys 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <400> 2 Arg Arg Gly Pro Glu 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <400> 3 Pro Ala Ala Asp Leu 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <400> 4 Thr Phe Gly Gly Pro 1 5 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <400> 5 Tyr Tyr Arg Arg Ala 1 5 <210> 6 <211> 25 <212> PRT <213> Artificial Sequence <400> 6 Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu 1 5 10 15 Pro Tyr Gly Ala Asn Lys Asp Gly Ile 20 25 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <400> 7 Ile Gly Thr Arg Asn 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <400> 8 Thr Leu Pro Lys Gly 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <400> 9 Ser Pro Ala Arg Met 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <400> 10 Arg Leu Asn Gln Leu 1 5 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <400> 11 Arg Arg Gly Pro Glu 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <400> 12 Thr Asp Tyr Lys His 1 5 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <400> 13 Pro Ser Gly Thr Trp 1 5 <210> 14 <211> 5 <212> PRT <213> Artificial Sequence <400> 14 Ala Asp Glu Thr Gln 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <400> 15 Pro Ala Ala Asp Leu 1 5

Claims

1. Use of a polypeptide and an antibody pair in the preparation of a product for detecting the N protein of SARS-CoV-2, characterized in that, the polypeptide is: SEQ ID NO: 1, and a combination of SEQ ID NOs: 4-15; the product is a kit, the antibody pair includes a capture antibody and a detection antibody, the capture antibody is purchased from Beijing Sino Biological Inc., product number 40143-MM05, recognizing the epitope SEQ ID NO: 1, and the detection antibody is purchased from Beijing Sino Biological Inc., product number 40143-RP01, recognizing the epitope which is a combination of SEQ ID NOs: 4-15.

2. A method for detecting the N protein of SARS-CoV-2 for non-diagnostic purposes, characterized in that, it includes coating the capture antibody on a carrier and blocking; adding the sample to be detected and incubating; adding the detection antibody and incubating; adding the labeled antibody and incubating; terminating the reaction; the capture antibody is purchased from Beijing Sino Biological Inc., product number 40143-MM05, recognizing the polypeptide shown in SEQ ID NO: 1, and the detection antibody is purchased from Beijing Sino Biological Inc., product number 40143-RP01, recognizing the polypeptide of SEQ ID NOs: 4-15.

3. The detection method according to claim 2, characterized in that, the incubation temperature is 30-40°C.

4. The detection method according to claim 2, characterized in that, the incubation time is 0.5-2 h.

5. The detection method according to claim 2, characterized in that, the labeled antibody is a horseradish peroxidase-labeled goat anti-rabbit IgG antibody.

6. The detection method according to claim 2, characterized in that, the detection method includes coating the capture antibody in a 96-well plate, overnight at 4°C, washing and then blocking at 37°C in 0.2% PBST + 5% skim milk for 1 h; after washing with 0.2% PBST, adding the sample to be detected and incubating at 37°C for 1 h; after washing, adding the detection antibody and incubating at 37°C for 1 h; after washing with PBST, adding the horseradish peroxidase-labeled goat anti-rabbit IgG antibody and incubating at 37°C for 1 h; after washing with PBST, adding 3’,3’,5’,5’,-tetramethylbenzidine, and then adding the reaction termination solution.

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