Mouse monoclonal antibody for identifying unique specific B cell epitopes in the spike protein S on the surface of novel coronavirus and its application

Abstract

The present invention discloses a mouse monoclonal antibody that recognizes unique specific B-cell epitopes in the spike protein S on the surface of the novel coronavirus and its applications. The monoclonal antibody includes a heavy chain and a light chain. The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the heavy chain are SDFYIN, YISGNDHNNYTESFKS, and TSLDY, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the light chain are TASESISGSYLA, STTDLAS, and HQFHGSPWT, respectively. The mouse monoclonal antibody of the present invention is of the IgG2 subtype. Because it recognizes unique B-cell epitopes in the spike protein S of the novel coronavirus (SARS-CoV-2), it has strong specificity, high sensitivity, and good accuracy, and has been proven to be applicable to detections such as enzyme-linked immunosorbent assay (ELISA), western blot (WB), and immunofluorescence (IF). The mouse monoclonal antibody of the present invention can be used in the reagent kit of an automatic immunoanalyzer, and can also be applied to immunochromatographic test strips for detecting the spike protein S and virus particles on the surface of SARS-CoV-2, which is conducive to large-scale SARS-CoV-2 antigen detection and epidemiological investigations.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a mouse monoclonal antibody that recognizes unique specific B-cell epitopes in the spike protein S on the surface of the novel coronavirus (SARS-CoV-2) and its application. Background Art

[0002] Coronaviruses are a class of enveloped viruses with a linear single-stranded positive-sense RNA genome. Coronaviruses that can cause human diseases to date include HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and the novel coronavirus SARS-CoV-2 that is currently in a global pandemic. The spike protein (S) on the surface of coronaviruses is related to the infectivity of the virus, is the most important surface structural protein, and is also the main target for inducing antibodies including neutralizing antibodies in the host immune system after infection. The nucleocapsid protein (N) inside the coronavirus particle is also one of the main structural proteins of the virus, has high immunogenicity and is abundantly expressed during infection. Therefore, both the S protein and the N protein are targets for SARS-CoV-2 antigens and diagnosis.

[0003] At present, there are three categories of detection methods and their kits for the novel coronavirus SARS-CoV-2, which respectively target nucleic acids, antibodies, and antigen proteins. Nucleic acid detection uses real-time fluorescence RT-PCR as the detection method. Due to its characteristics such as early diagnosis, high sensitivity, and high specificity, it is regarded as the main means for the diagnosis of SARS-CoV-2 virus infection. However, nucleic acid detection has strict requirements for aspects such as the collection of specimen samples, sample preservation and transportation, and experimental conditions. Any oversight in any link may lead to deviations in the test results. Immunological detection kits that identify the antigen protein and its antibody of SARS-CoV-2 virus have the advantages of easy specimen collection, simple specimen processing and experimental operations, short testing time, and suitability for large-scale sample screening. However, IgM antibodies can only be produced 7 days after human infection with the virus or 3 days after the onset of the disease, while IgG antibodies usually appear after the human body enters the recovery period. Therefore, using antibodies as the target has limitations in the early detection of SARS-CoV-2 infection. The SARS-CoV-2 antigen detection kits approved and marketed at home and abroad use the colloidal gold method to detect its nucleocapsid protein N. Although it is fast and easy to use and can be used for the primary screening of a large number of people, it may also lead to false positives due to its high homology (29% to 91% identity and 42% to 94% similarity) with the nucleocapsid proteins N of 6 other coronaviruses. In addition, since the nucleocapsid protein N is located inside the virus particles and must be released by lysis, protease inhibitors must be added to the lysis solution to prevent its degradation, thus increasing the production and cost of the kit. Therefore, developing a corresponding specific antigen detection kit targeting the spike protein S located on the surface of the SARS-CoV-2 virus particles and using it in combination with qRT-PCR virus nucleic acid detection can provide strong support for the prevention and control plan of early detection, early isolation, and early treatment of the SARS-CoV-2 virus.

[0004] Our previous work predicted and identified the unique specific immune-dominant linear B-cell epitopes contained in the spike protein S on the surface of SARS-CoV2 through bioinformatics program analysis and experimental verification, and established a polypeptide-ELISA method for detecting SARS-CoV2-specific antibodies, which can distinguish between antibodies against COVID-19 virus and antibodies against other coronaviruses (see the invention application with the publication number CN112213497A for details). Further analysis showed that the amino acid sequences of one of the above-mentioned unique B-cell epitopes in the spike proteins S of all currently known SARS-CoV2 epidemic strains, including the prototype strain and variant strains (from the earliest Alpha to the most recent Omicron), are completely identical, that is to say, the antibodies induced by this epitope S9 should be able to specifically recognize all SARS-CoV2 variant strains of the novel coronavirus. On this basis, the present invention developed and obtained a mouse monoclonal antibody targeting the unique epitope in the spike protein of SARS-CoV2, which can be used to specifically recognize SARS-CoV2 and its antigen / spike protein S, and laid a foundation for the further development of a sensitive and specific detection kit for SARS-CoV2 virus and its antigen / spike protein S. While expanding the antigen detection targets of SARS-CoV2, this antibody and its kit can distinguish SARS-CoV2 and its antigen / spike protein S from other coronaviruses and their antigen / spike protein S, reducing the false positive rate of antigen detection, which has important practical significance. Summary of the Invention

[0005] The object of the present invention is to provide a mouse monoclonal antibody that recognizes the unique specific B-cell epitope in the spike protein S on the surface of the novel coronavirus and its application in view of the deficiencies of the prior art.

[0006] The object of the present invention is achieved by the following technical solutions: A mouse monoclonal antibody that recognizes the unique specific B-cell epitope in the spike protein S on the surface of the novel coronavirus (SARS-CoV2), including a heavy chain and a light chain. The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the heavy chain are SDFYIN, YISGNDHNNYTESFKS, and TSLDY respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the light chain are TASESISGSYLA, STTDLAS, and HQFHGSPWT respectively.

[0007] Preferably, for the mouse monoclonal antibody, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.4.

[0008] Preferably, the mouse monoclonal antibody is a full-length antibody, F(ab) antibody or F(ab’)2 antibody.

[0009] The present invention also provides a gene encoding the mouse monoclonal antibody. Preferably, the gene sequence encoding the heavy chain variable region is as shown in SEQ ID NO.1; the gene sequence encoding the light chain variable region is as shown in SEQ ID NO.2.

[0010] The present invention also provides the application of the monoclonal antibody in the preparation of a kit for detecting SARS-CoV2. Preferably, in the above application, the detection methods include enzyme-linked immunosorbent assay (ELISA), Western blot (WB) and immunofluorescence (IF).

[0011] The present invention further provides a kit for detecting SARS-CoV2, including the monoclonal antibody. Preferably, the kit is an ELISA detection kit or an immunochromatographic diagnostic kit using an immunochromatographic test strip.

[0012] The mouse monoclonal antibody of the present invention is of IgG2 subtype. Since it recognizes a unique B cell epitope in the spike protein S of SARS-CoV2, it has strong specificity, high sensitivity and good accuracy. It can be used in the kit of an automatic immunoanalyzer, and can also be applied to the immunochromatographic test strip to detect the spike protein S and virus particles on the surface of SARS-CoV2, which is beneficial to large-scale SARS-CoV2 antigen detection and epidemiological investigation. Description of the Drawings

[0013] Figure 1 It is the SDS-PAGE electrophoresis pattern of the mouse monoclonal antibody in Example 1; M: Protein Marker.

[0014] Figure 2 It is the detection result of the titer of the mouse monoclonal antibody in Example 2.

[0015] Figure 3 It is the mouse monoclonal antibody in Example 3 used for enzyme-linked immunosorbent assay (ELISA) to detect the NTD region of the recombinant expressed SARS-CoV2 spike protein S.

[0016] Figure 4For the mouse monoclonal antibody in Example 4, it was used for protein immunoblot hybridization (WB) to detect the S1 subunit of the spike protein S of 7 recombinant-expressed coronaviruses; M: protein Marker, and 1-7 were successively the S1 subunits of the spike proteins S of 7 coronaviruses including SARS-CoV2, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-HKU1, HCoV-OC43, and HCoV-229E that were recombinantly expressed and purified. Detailed implementation mode

[0017] The following are the preferred implementation modes of the present invention. The embodiments are used to explain and illustrate the present invention, rather than limit the present invention. Within the spirit of the present invention and the protection scope of the claims, any modifications and changes made by those of ordinary skill in the art on the basis of the present invention fall within the protection scope of the present invention. For the experimental methods without specific conditions noted in the embodiments of the present invention, they are usually carried out under conventional conditions; unless otherwise specified, the reagents and consumables used are commercially available products.

[0018] Example 1: Preparation of a mouse monoclonal antibody targeting a unique specific B cell epitope in the spike protein S on the surface of the novel coronavirus (SARS-CoV2)

[0019] According to the unique specific B cell epitope in the spike protein S on the surface of the novel coronavirus (SARS-CoV2) determined in our previous work, one of them was selected to synthesize a polypeptide (LHRSYLTPGDSSSGWTAG) artificially. The C-terminus was coupled to the carrier protein KLH, and Balb / c mice were immunized intraperitoneally several times. Spleen cells with expected immune responses were fused with SP2 / 0 mouse myeloma cells. A total of 20 hybridoma cell master clones were obtained through culture, and 2 ml of cell culture supernatant was prepared for each. First, the above polypeptide was used as a capture antigen and coated on a 96-well enzyme-linked immunosorbent assay (ELISA) plate, and the polypeptide-ELISA method was used to preliminarily screen the positive clones secreting specific antibodies; then, the recombinantly expressed SARS-CoV NTD protein was used as a capture antigen for ELISA verification, and 5 hybridoma cells with relatively high positive values were determined; then, protein immunoblot hybridization (WB) was used to exclude cross-reactivity with the S1 subunits of the spike proteins S of 6 human coronaviruses other than SARS-CoV2. Finally, a hybridoma cell line 6H8 that specifically recognizes only the S1 of SARS-CoV2 was selected. A large amount of antibody was prepared through ascites, and the purity of the antibody was identified by SDS-PAGE electrophoresis after purification by Protein A chromatography. The results showed that two clear target bands appeared at 55 kDa and 25 kDa, indicating that a high-purity mouse monoclonal antibody targeting and specifically recognizing the unique specific B cell epitope in the spike protein S of the novel coronavirus (SARS-CoV2) was successfully prepared ( Figure 1 )

[0020] Example 2: Detection of the titer of mouse monoclonal antibody by polypeptide-ELISA

[0021] The artificially synthesized polypeptide LHRSYLTPGDSSSGWTAG was coated on a 96-well ELISA plate at a concentration of 1 μg / ml and 100 μl / well, and incubated overnight at 4°C. After washing the plate with PBST, 1% BSA was added at 200 μl / well and blocked at 37°C for 1 h. After washing the plate three times with PBST, the mouse monoclonal antibody 6H8 in Example 1 at a serially diluted ratio (starting concentration 1 mg / ml), namely 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, was added at 100 μl / well and incubated at 37°C for 1 h. After washing the plate five times with PBST, a goat anti-mouse antibody labeled with horseradish peroxidase (HRP) at an appropriate dilution was added and incubated at 37°C for 1 h. After washing the plate five times with PBST, the TMB chromogenic substrate was added at 100 μl / well for a reaction for 15 min, and 50 μl of 2 mol / L sulfuric acid termination solution was added to terminate the reaction. The OD values of each well were read by an ELISA reader at 450 nm. The results showed that the titer of the mouse monoclonal antibody 6H8 was greater than 512000 (the concentration used was 1.95 ng / ml)( Figure 2 ), with high sensitivity. Example 3: Use of mouse monoclonal antibody for indirect enzyme-linked immunosorbent assay (ELISA) to detect the NTD region of recombinant expressed SARS-CoV2 spike protein S

[0022] The recombinant expressed and purified SARS-CoV2 NTD protein was used as the capture antigen and coated on a 96-well ELISA plate at a concentration of 5 μg / ml and 100 μl / well, and incubated overnight at 4°C. After washing the plate with PBST, 1% BSA was added at 200 μl / well and blocked at 37°C for 1 h. After washing the plate three times with PBST, the mouse monoclonal antibody 6H8 at an appropriate dilution was added as the primary antibody and incubated at 37°C for 1 h. After washing the plate five times with PBST, a goat anti-mouse antibody labeled with horseradish peroxidase (HRP) at an appropriate dilution was added and incubated at 37°C for 1 h. After washing the plate five times with PBST, the TMB chromogenic substrate was added at 100 μl / well for a reaction for 15 min, and 50 μl of 2 mol / L sulfuric acid termination solution was added to terminate the reaction. The OD values of each well were read by an ELISA reader at 450 nm. The results indicated that the mouse monoclonal antibody 6H8 could be used for indirect enzyme-linked immunosorbent assay (ELISA) to specifically detect the NTD region of recombinant expressed SARS-CoV2 spike protein S( Figure 3 ).

[0023] Example 4: Mouse monoclonal antibody used for protein immunoblotting (WB) to detect the S1 subunit of the spike protein S of 7 recombinant-expressed coronaviruses

[0024] Take 1 μg each of the S1 subunits of the spike proteins S of 7 human coronaviruses, namely SARS-CoV2, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-HKU1, HCoV-OC43, and HCoV-229E, which are recombinantly expressed and purified, and perform SDS-PAGE separation. After electrotransfer to a PVDF membrane, block it with 5% BSA for 2 h. Then, use mouse monoclonal antibody 6H8 as the primary antibody and HRP-labeled goat anti-mouse antibody as the secondary antibody for protein immunoblotting. The results show that this monoclonal antibody can specifically recognize the S1 subunit of the SARS-COV2 spike protein S, and there is no cross-reaction with the S1 subunits of the spike proteins S of the other 6 coronaviruses ( Figure 4 ).

[0025] Example 5: Mouse monoclonal antibody used for cell immunofluorescence (IF) detection

[0026] Dilute and mix the recombinant plasmid pCDNA3.1-HnCoV-S encoding the full-length spike protein S of SARS-CoV2 and the control plasmid pCDNA3.1 with Lip2000 using OPTI-MEM. After standing at room temperature for 20 min, add the mixture to HEK293 cells cultured on coverslips in a 6-well plate. Gently shake and mix, and then place it in an incubator at 37°C and 5% CO2 for 48 h. Then discard the culture medium, rinse the cell coverslips 3 times with PBS at 37°C, add pre-warmed 4% paraformaldehyde at 37°C to fix for 15 min, and rinse 3 times with PBS; incubate at room temperature with 0.2% TritonX-100 (diluted with PBS) for 15 min, and rinse 3 times with PBS; block the cell coverslips with PBS containing 10% goat serum for 30 min; add mouse monoclonal antibody 6H8 as the primary antibody for incubation at 4°C for 14 h, rinse 3 times with PBS, and let it stand for 5 min each time; use Alexa Flour 647-labeled goat anti-mouse IgG antibody as the secondary antibody, dilute it 1:500 in PBS containing 1% BSA, incubate at room temperature for 2 h, rinse 3 times with PBS, and let it stand for 5 min each time; fix the coverslips on the glass slides with anti-fluorescence mounting medium; observe under a confocal microscope. The results show that cells transfected with the recombinant plasmid exhibit specific fluorescence signals, while blank and cells transfected with the control plasmid do not show specific fluorescence signals, indicating that mouse monoclonal antibody 6H8 can be used for cell immunofluorescence (IF) detection and specifically recognize the SARS-COV2 spike protein S expressed in cells.

[0027] Example 6: Sequencing and analysis of the gene encoding the mouse monoclonal antibody

[0028] Extract the RNA of hybridoma cells producing mouse monoclonal antibody 6H8 and reverse transcribe it into cDNA. Design 2 pairs of degenerate primers according to the constant regions of the heavy and light chains of the antibody, and perform PCR amplification respectively. Recover the target fragments and determine the nucleotide sequences. After analysis and comparison, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.1, and the gene sequence encoding the heavy chain variable region is shown as SEQ ID NO.3; the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2, and the gene sequence encoding the light chain variable region is shown as SEQ ID NO.4.

Claims

1. A murine monoclonal antibody that identifies unique specific B-cell epitopes in the spike protein S on the surface of the novel coronavirus, including a heavy chain and a light chain, characterized in that, The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the heavy chain are SDFYIN, YISGNDHNNYTESFKS, and TSLDY respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the light chain are TASESISGSYLA, STTDLAS, and HQFHGSPWT respectively; according to the unique specific B cell epitope in the spike protein S on the surface of the novel coronavirus, one of them, the synthetic polypeptide LHRSYLTPGDSSSGWTAG, is selected as the antigen.

2. The mouse monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.

4.

3. The murine monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is a full-length antibody, F(ab) antibody or F(ab’)2 antibody.

4. A gene encoding the monoclonal antibody according to any one of claims 1 to 3.

5. The gene according to claim 4, characterized in that, The gene sequence encoding the heavy chain variable region is as shown in SEQ ID NO.1; the gene sequence encoding the light chain variable region is as shown in SEQ ID NO.

2.

6. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a kit for detecting the novel coronavirus SARS-CoV2.

Images