Monoclonal antibody against c-terminal region of novel coronavirus nucleoprotein and application thereof

Abstract

The application discloses a monoclonal antibody against a C-terminal region of a novel coronavirus nucleoprotein and application thereof. In the monoclonal antibody, the amino acid sequences of light chain CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4; the amino acid sequences of heavy chain CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8. The monoclonal antibody against the C-terminal region of the novel coronavirus nucleoprotein has good specificity and affinity, and can be applied to preparation of a product for detecting the novel coronavirus and preparation of a product for treating a patient with the novel coronavirus.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a monoclonal antibody against the C-terminal region of the novel coronavirus nucleoprotein and its application. Background Technology

[0002] Novel coronavirus specific antigen detection targets antigens synthesized by the novel coronavirus itself, providing direct evidence of infection. The nucleoprotein and spike glycoprotein of the novel coronavirus possess strong antigenicity, serving as the primary antigens inducing host immune responses and the main targets for antigen detection. Due to the high conservation of the amino acid sequence of the novel coronavirus nucleoprotein, it has become a favorite among antigen detection reagent developers. Currently, novel coronavirus antigen detection is mainly based on the double-antibody sandwich principle. Developing antigen detection reagents requires paired antibodies with high affinity and high specificity; therefore, preparing antibodies with high affinity and specificity is crucial for the development of novel coronavirus antigen detection reagents.

[0003] Furthermore, targeted therapy refers to the process where therapeutic drugs, after entering the body, specifically bind to target organs, cells, or pathogens through specific guidance mechanisms, allowing them to exert their effects more directly and achieve targeted treatment. Utilizing targeted drug delivery can significantly increase drug concentration at the lesion site, improving efficacy while reducing toxic side effects. Targeted therapy mainly includes biological targeting and physicochemical targeting. Biological targeted therapy involves conjugating target molecules with drugs to deliver medication directly to specific targets. In recent years, the use of monoclonal antibodies with high affinity and specificity to conjugate therapeutic drugs has yielded targeted drugs with good therapeutic effects, becoming a hot topic and major area of ​​biological targeted drug delivery therapy. Monoclonal antibodies with high affinity and specificity are key factors in the success of this biological targeted therapy. Therefore, obtaining monoclonal antibodies with high affinity and specificity is particularly important for biological targeted therapy.

[0004] Currently, research on the treatment of novel coronavirus infection mainly focuses on oral drug therapy and neutralizing antibody therapy. There is a lack of research on biological targeted drug therapy using high-affinity and specific monoclonal antibodies targeting the novel coronavirus pathogen. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a monoclonal antibody against the C-terminal region of the novel coronavirus nucleoprotein prepared using a fused hybridoma cell line. The monoclonal antibody obtained through experiments has good specificity and affinity and can be used to detect the novel coronavirus nucleoprotein or to prepare products for treating novel coronavirus infection.

[0006] Therefore, one aspect of the present invention relates to a monoclonal antibody against the nucleoprotein of a novel coronavirus or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR1, CDR2, and CDR3, and the heavy chain variable region comprises CDR1, CDR2, and CDR3, wherein,

[0007] The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.2 or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO.2, or an amino acid sequence containing the above sequence.

[0008] The amino acid sequence of the light chain CDR2 is the sequence shown in SEQ ID NO.3 or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO.3, or an amino acid sequence containing the above sequence.

[0009] The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.4 or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO.4, or an amino acid sequence containing the above sequence.

[0010] The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.6 or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO.6, or an amino acid sequence containing the above sequence.

[0011] The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.7 or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO.7, or an amino acid sequence containing the above sequence.

[0012] The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.8, or an amino acid sequence having one or two conserved amino acid substitutions compared to the sequence shown in SEQ ID NO.8, or an amino acid sequence containing the above sequence.

[0013] In a further aspect, the present invention also relates to a monoclonal antibody or an antigen-binding fragment thereof, wherein the light chain variable region sequence is the sequence shown in SEQ ID NO.1 and the heavy chain amino acid sequence is the sequence shown in SEQ ID NO.5.

[0014] The present invention also relates to the above-mentioned monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a single-chain antibody or a humanized antibody, which, because they retain the variable regions of the light and heavy chains, are able to recognize and bind to the C-terminal region of the novel coronavirus nucleoprotein.

[0015] Furthermore, the present invention relates to a nucleic acid molecule comprising a nucleic acid encoding the aforementioned antibody or its antigen-binding fragment, and an expression vector comprising the aforementioned nucleic acid molecule, the expression vector being capable of expressing the aforementioned antibody or its antigen-binding fragment. The present invention also relates to a recombinant comprising the aforementioned nucleic acid molecule or the aforementioned expression vector, which can produce the aforementioned antibody or its antigen-binding fragment.

[0016] On the other hand, this invention relates to a monoclonal antibody hybridoma cell line against the nucleoprotein of the novel coronavirus, wherein the monoclonal antibody hybridoma cell line secretes the aforementioned monoclonal antibody. Further, this invention relates to a monoclonal antibody hybridoma cell line against the nucleoprotein of the novel coronavirus, wherein the monoclonal antibody hybridoma cell line is mouse hybridoma cell line 5D85163, with accession number CGMCC No. 45130.

[0017] Furthermore, this invention relates to the use of the aforementioned monoclonal antibody or its antigen-binding fragment in the preparation of products for detecting or treating novel coronavirus infection. Further, this invention relates to a kit for detecting the novel coronavirus, the kit comprising the aforementioned monoclonal antibody or its antigen-binding fragment for recognizing and binding to the C-terminal region of the novel coronavirus nucleoprotein.

[0018] Instructions for the Preservation of Biological Materials

[0019] The monoclonal antibody hybridoma cell line of this invention, mouse hybridoma cell line 5D85163, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45130 and deposit date of March 10, 2022. The address of the China General Microbiological Culture Collection Center is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Attached Figure Description

[0020] Figure 1 This is an SDS-PAGE electrophoresis image showing the prokaryotic expression of the novel coronavirus nucleoprotein. The labels are as follows: M is the marker; 1 is the C-terminal region of the nucleoprotein expressed by the purified pET-NP-C recombinant plasmid; 2 is the C-terminal region of the nucleoprotein expressed by the purified pGEX-NP-C recombinant plasmid.

[0021] Figure 2This image shows the identification results of monoclonal antibody subclasses against the C-terminal region of the novel coronavirus nucleoprotein. The antibody subtype was identified as IgG1.

[0022] Figure 3 This image shows the results of an ELISA immunoassay of the nucleoprotein C-terminal region of the novel coronavirus nucleoprotein using a monoclonal antibody against the nucleoprotein. NP represents the full-length nucleoprotein of the novel coronavirus expressed in eukaryotes; TB represents the protein of Mycobacterium tuberculosis expressed in prokaryotes; EB represents the protein of Epstein-Barr virus expressed in prokaryotes; and B19 represents the protein of human parvovirus B19 expressed in prokaryotes. The results demonstrate that the monoclonal antibody against the novel coronavirus nucleoprotein can specifically recognize the nucleoprotein of the novel coronavirus. Detailed Implementation

[0023] The purpose of this invention is to provide a monoclonal antibody against the C-terminal region of the novel coronavirus nucleoprotein prepared using a fused hybridoma cell line.

[0024] Based on the inventors' analysis of the dominant antigenic epitopes of the novel coronavirus, the region from amino acids 230 to 419 of the C-terminal region of the novel coronavirus nucleoprotein was identified as containing the dominant antigenic epitopes. Therefore, this region was selected for expression and subsequent immunization of mice to screen for monoclonal antibody hybridoma cell lines. After screening, a mouse hybridoma cell line expressing a monoclonal antibody with high specificity and affinity was obtained and named 5D85163. On March 10, 2022, this cell line was deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 45130, dated March 10, 2022. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0025] The inventors sequenced the monoclonal antibody produced by this mouse hybridoma cell line. The light chain variable region sequence of the monoclonal antibody is 107 amino acids, and its sequence is as follows: DIQMNQSPSSLSASLGDRVTISCSAS WHO LNWYQQKPDGTVKLLIY Federal Tax Service SLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYC QQYSKLPYTFGGGTKLELK (SEQ ID NO. 1), where the underlined sequences are CDR1, CDR2, and CDR3, respectively. CDR1 is located at amino acids 27-32 (aa) and has the amino acid sequence QGISNY (SEQ ID NO. 2); CDR2 is located at amino acids 50-52 (aa) and has the amino acid sequence FTS (SEQ ID NO. 3); CDR3 is located at amino acids 89-97 (aa) and has the amino acid sequence QQYSKLPYT (SEQ ID NO. 4). The heavy chain variable region has 119 amino acids, and its sequence is as follows: EVQLQQSGPELVKPGASVKISCKTS GYTFTEIT MHWMKQNHGKSLEWIGD INPNNGST NYNQMYKGKATFTLDKSSTTAYMELRRSLTSDNSAVYYC WRONG WGQGTTLTVS (SEQ ID NO. 5), wherein the underlined sequences are CDR1, CDR2, and CDR3, respectively. CDR1 is located at amino acid range 26-33 (Aa), with the amino acid sequence GYTFTEYT (SEQ ID NO. 6); CDR2 is located at amino acid range 51-58 (Aa), with the amino acid sequence INPNGST (SEQ ID NO. 7); and CDR3 is located at amino acid range 97-109 (Aa), with the amino acid sequence ARGYYGYLYYFDY (SEQ ID NO. 8). The above monoclonal antibody was determined to have high specificity and affinity for the SARS-CoV-2 nucleoprotein.

[0026] As is well known, the CDR regions of the antibody heavy chain and light chain are important sequences for recognizing and binding to corresponding antigens, and the substitution of one or two conserved amino acids in the amino acid sequence generally does not change or slightly alters the properties of the protein. Therefore, monoclonal antibodies or their antigen-binding fragments obtained by substituting one or two conserved amino acids into light chain CDR1 and / or light chain CDR2 and / or light chain CDR3 and / or heavy chain CDR1 and / or heavy chain CDR2 and / or heavy chain CDR3 can still recognize and bind to the novel coronavirus nucleoprotein. Conserved amino acid substitution refers to the replacement of one amino acid in a protein with another chemically similar amino acid. Examples include substitutions between aromatic amino acids Phe, Trp, and Tyr; between aliphatic amino acids Ala, Gly, Leu, Ile, and Val; between polar amino acids Gln and Asn; between basic amino acids Lys, Arg, and His; between acidic amino acids Asp and Glu; and between hydroxy amino acids Ser and Thr.

[0027] Furthermore, as is well known in the art, in antibodies or their antigen-binding fragments, the backbone region is located outside each CDR in the light or heavy chain. Adding a few amino acids, such as one or two amino acids, between the CDR sequence and the backbone region sequence has minimal impact on the stereostructure of the antibody or its antigen-binding fragment, thus allowing it to still recognize and bind to the corresponding antigen. Therefore, the CDR1 sequence of the light chain of the monoclonal antibody or its antigen-binding fragment of the present invention can be any sequence containing the aforementioned sequence, in addition to being the sequence shown in SEQ ID NO.2 or a sequence with one or two conserved amino acid substitutions compared to it. Similarly, the CDR2 sequence of the monoclonal antibody or its antigen-binding fragment light chain of the present invention, in addition to being the sequence shown in SEQ ID NO.3 or having one or two conserved amino acid substitutions compared to it, can also be a sequence containing the above-mentioned sequence; the CDR3 sequence of the monoclonal antibody or its antigen-binding fragment light chain of the present invention, in addition to being the sequence shown in SEQ ID NO.4 or having one or two conserved amino acid substitutions compared to it, can also be a sequence containing the above-mentioned sequence; the CDR1 sequence of the monoclonal antibody or its antigen-binding fragment heavy chain of the present invention, in addition to being the sequence shown in SEQ ID NO.6 or having one or two conserved amino acid substitutions compared to it, can also be a sequence containing the above-mentioned sequence; the CDR2 sequence of the monoclonal antibody or its antigen-binding fragment heavy chain of the present invention, in addition to being the sequence shown in SEQ ID NO.7 or having one or two conserved amino acid substitutions compared to it, can also be a sequence containing the above-mentioned sequence; the CDR3 sequence of the monoclonal antibody or its antigen-binding fragment heavy chain of the present invention, in addition to being the sequence shown in SEQ ID NO.8 or having one or two conserved amino acid substitutions compared to it, can also be a sequence containing the above-mentioned sequence.

[0028] Various antibody fragments capable of binding to the novel coronavirus nucleoprotein, i.e., antigen-binding fragments, such as, but not limited to, Fab, Fab', and F(ab')2, can also be prepared from the monoclonal antibody of the present invention using existing techniques in the art. The Fab fragment is the antigen-binding region in the antibody structure, composed of a complete light chain and a variable region VH and a constant region CH1 domain (Fd segment). Both the light and heavy chains have a constant region and a variable region, and disulfide bonds link the light and heavy chains. The antigen-binding fragment can be prepared as follows: for example, after enzymatic hydrolysis with papain, antibody IgG is degraded into two Fab fragments and one Fc fragment (crystalline fragment). Under the action of pepsin, antibody IgG is degraded into one F(ab')2 fragment and one pFc' fragment, and the F(ab')2 fragment is further reduced to form two Fab' fragments. The above-mentioned antigen-binding fragments can be used to prepare products for detecting the novel coronavirus nucleoprotein and therapeutic products for treating patients with novel coronavirus.

[0029] Single-chain antibodies (scFvs) can also be prepared from the monoclonal antibodies of the present invention using existing techniques in the art. A single-chain antibody is an antibody composed of a heavy chain variable region and a light chain variable region linked by a short peptide linker of several amino acids; it has only one chain and is a synthetically produced antibody. The length and amino acid composition of the short peptide linker are well known in the art, and usable short peptide linkers for the monoclonal antibodies of the present invention can be determined through simple, repeatable experiments. Single-chain antibodies can be expressed in, for example, *E. coli* using genetic engineering techniques. Due to their small molecular weight, strong penetrability, and weak antigenicity, single-chain antibodies can be applied to the detection of novel coronavirus nucleoproteins and to diagnostic and therapeutic products for patients with novel coronavirus.

[0030] By utilizing existing techniques in the art, the light chain constant region and heavy chain constant region of the monoclonal antibody of the present invention can be replaced with the amino acid sequence of a human antibody, thereby humanizing the mouse monoclonal antibody of the present invention to obtain a humanized antibody for antibody treatment of human patients with COVID-19, thereby reducing the immune side effects of the mouse antibody on the human body. Therefore, the humanized monoclonal antibody of the present invention can be applied to the detection of the nucleoprotein of the novel coronavirus, and to diagnostic and therapeutic products for patients with COVID-19.

[0031] Those skilled in the art can design and synthesize nucleic acid molecules encoding the variable region of the monoclonal antibody against the novel coronavirus nucleoprotein based on the aforementioned amino acid sequence. They can also insert the synthesized nucleic acid molecules into a nucleic acid vector to construct an expression vector capable of expressing the monoclonal antibody against the novel coronavirus nucleoprotein or its antigen-binding fragment. Furthermore, those skilled in the art can introduce the synthesized nucleic acid molecules or constructed expression vectors into organisms such as cells, bacteria, or yeast to obtain recombinant bodies, and express the antibodies or their antigen-binding fragments of the present invention through these recombinant bodies. The expressed antibodies or their antigen-binding fragments can bind to and recognize the novel coronavirus nucleoprotein. Therefore, the aforementioned nucleic acid molecules, expression vectors, and recombinant bodies are within the scope of protection of the claims of this invention. Moreover, the above-mentioned techniques are all well-known in the art and can be carried out by those skilled in the art without inventive effort.

[0032] As described above, the antibody or its antigen-binding fragment of the present invention can recognize and bind to the novel coronavirus nucleoprotein, and therefore can be used to prepare a kit for detecting the novel coronavirus nucleoprotein. The kit can be any kit that utilizes the binding reaction between the antibody or its antigen-binding fragment of the present invention and the novel coronavirus nucleoprotein, such as, but not limited to, colloidal gold immunochromatography, fluorescence immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, and immunohistochemistry kits.

[0033] Because the monoclonal antibody or its antigen-binding fragment of the present invention can specifically bind to the SARS-CoV-2 nucleoprotein, the antibody or its antigen-binding fragment, or humanized antibody, can be used to prepare products for treating SARS-CoV-2 infection, such as pharmaceuticals. Treatment can be, for example, but not limited to, targeted therapy. In targeted therapy, the monoclonal antibody or its antigen-binding fragment, or humanized antibody of the present invention is conjugated to a SARS-CoV-2 therapeutic drug. The conjugated drug is then targeted to the SARS-CoV-2 virus via the antibody or its antigen-binding fragment, or humanized antibody of the present invention, thereby improving the therapeutic effect.

[0034] To explain in detail the technical content, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments.

[0035] Example 1: Preparation of antigens for immunization and screening

[0036] Based on the amino acid sequence of the novel coronavirus nucleoprotein published in Genebank, which is 419 amino acids in length, the amino acid sequence was analyzed using BIOSUN bioinformatics software. First, the full-length sequence was input. Then, using the B-cell epitope and signal peptide analysis functions, the distribution of antigenic epitopes was analyzed based on the strength of hydrophobicity and the presence of a signal peptide. Analysis revealed a strongly hydrophobic region at amino acids 220-230, and further analysis showed that amino acids 1-19 constituted the signal peptide sequence. Therefore, we determined that the 230-419aa region at the C-terminus of the novel coronavirus nucleoprotein was the dominant epitope antigenic region. Thus, in this embodiment, the 230-419aa dominant epitope antigenic region was selected as the immunogen.

[0037] The gene encoding the dominant epitope antigen of nucleoprotein 230-419aa was synthesized by Beijing Nuosai Biotechnology Co., Ltd. Using EcoRI and XhoI restriction enzyme sites, the gene encoding the dominant epitope antigen of 230-419aa was double-digested and inserted into pET-28a plasmid and pGEX-4T-1 plasmid, respectively, to obtain pET-NP-C recombinant plasmid and pGEX-NP-C recombinant plasmid.

[0038] The correctly sequenced recombinant expression plasmid was transformed into BL21 competent cells. Single colonies were picked and cultured in 3 mL of LB broth containing ampicillin sodium at 37°C with shaking overnight. The next day, the colonies were inoculated into 250 mL of fresh LB broth and cultured at 37°C and 160 rpm for 4 h to reach the logarithmic growth phase. 150 μl of 1 mol / L IPTG induction medium was added, and the cells were induced at 15°C for 12-14 h. The induced cells were collected by centrifugation at 4°C and 6000 rpm for 10 min. The cells were resuspended in 25 mmol / L Tris-HCl (pH 8.5) and sonicated on ice. The supernatant was collected by centrifugation at 4°C and 12000 rpm for 10 min. SDS-PAGE gel electrophoresis was performed, and the dominant epitopes 230-419aa at the C-terminus of the nucleoprotein were mainly expressed in soluble form in the supernatant.

[0039] The pET-NP-C recombinant plasmid expression antigen was purified using a Ni column, and the pGEX-NP-C recombinant plasmid expression antigen was purified using a GST column. Protein peaks were collected, and the purified products were analyzed by SDS-PAGE electrophoresis to verify the expression of the purified target protein. The results are as follows: Figure 1 As shown. The molecular weight and purity of the purified nucleoprotein 230-419aa dominant epitope antigen were analyzed using Gel-ProR Analyzer Version 3.0 software. The molecular weight of the nucleoprotein 230-419aa dominant epitope antigen expressed by the pET-NP-C recombinant plasmid was 25.30 kDa, consistent with the molecular weight shown in the electrophoresis image, and the purity was 97.65%, meeting the immunogen purity requirements. The molecular weight of the nucleoprotein 230-419aa dominant epitope antigen expressed by the pGEX-NP-C recombinant plasmid was 45.40 kDa, consistent with the molecular weight shown in the electrophoresis image, and the purity was 96.12%, making it suitable as a detection antigen.

[0040] Example 2: Establishment of hybridoma cell lines

[0041] The purified pET-NP-C recombinant plasmid expressing the dominant epitope antigen of nuclear protein 230-419aa was used as an immunogen. Eight-week-old female BALB / c mice were injected dorsally and intraperitoneally with the dominant epitope antigen plus an equal amount of Freund's complete adjuvant (50 μg / mouse). The same dose of immunization was performed for the second and third time at the fourth and eighth weeks, respectively, using Freund's incomplete adjuvant. Spleen cells were harvested 3 days later for fusion.

[0042] SP20 myeloma cells were revived and cultured until they entered the logarithmic growth phase. Immunized BALB / c mice were used, and blood was collected from the eyes of the mice to provide serum for positive controls. The mice were then euthanized by cervical dislocation. The body surface was disinfected with 75% alcohol for 3-5 minutes, and the spleen was harvested to prepare a spleen cell suspension.

[0043] Splenocytes and myeloma cells were mixed at a 5:1 ratio in serum-free DMEM medium and centrifuged at 1500 rpm for 5 minutes. The supernatant was thoroughly aspirated, and the cells were gently dispersed by shaking the bottom of the centrifuge tube. 1 mL of pre-warmed 50% PEG fusion cells was added over 45-60 seconds, while gently shaking to mix. After addition, the mixture was allowed to stand for 90 seconds. Serum-free DMEM medium was then added to terminate the fusion (1 mL in the first minute, 2 mL in the second minute, and 8 mL in the third minute). The mixture was incubated at 37°C for 10 minutes, centrifuged at 1500 rpm for 5 minutes, and the pellet was resuspended in HAT medium. The pellet was aliquoted into 96-well cell culture plates containing feeder cells and cultured in a 37°C, 5% CO2 incubator. After 5 days of culture, the medium was changed once with HAT medium. On day 10, the medium was changed again with HAT medium. When the fusion cells covered 10%-50% of the bottom of the wells, positive clones were screened using an indirect ELISA method. The specific method is as follows: ① Dilute the pGEX-NP-C recombinant plasmid expressing the dominant epitope antigen of nuclear protein 230-419aa with carbonate coating buffer to a concentration of 2.5 μg / ml, coating 100 μl per well and incubating overnight at 4℃; wash the plate twice with washing buffer, 200 μl / well; add 110 μl / well blocking buffer and block at room temperature for 6 hours; wash the plate five times with washing buffer, 200 μl / well. ② Add 200 μl of sample diluent to each well, then add 10 μl of cell culture supernatant, incubate at room temperature for 30 min, and discard the supernatant. ③ Wash the plate with 1× washing buffer, 300 μl per well, repeating the washing 5 times. Invert the washed ELISA plate onto absorbent paper and tap it vigorously to remove excess washing buffer. ④ Add 100 μl / well of HRP-labeled anti-mouse IgG antibody and incubate at room temperature for 20 min. ⑤ Wash the plate, following the same procedure as step 3. ⑥ Add 100 μl of freshly prepared substrate solution to each well and incubate at room temperature in the dark for 10 minutes. ⑦ Add 50 μl of 2M H2SO4 stop solution to each well to terminate the reaction. ⑧ Set the microplate reader to a detection wavelength of 450 nm and measure the OD value of each well. Read the value within 10 minutes after termination.

[0044] A total of 12 positive clones were obtained. The clone with the highest detection value (OD450nm=3.437) was selected for subsequent experiments and named mouse hybridoma cell line 5D85163.

[0045] Example 3: Preparation of Monoclonal Antibodies and Isotype Analysis

[0046] 5D85163 hybridoma cells were cultured in 1640 medium containing 10% fetal bovine serum. Each male BALB / c mouse was intraperitoneally injected with 0.5 mL of liquid paraffin. After 10 days, cells were collected, resuspended in 10 mL of physiological saline, and injected intraperitoneally with 0.5 mL of saline (cell density approximately 1 × 10⁷ cells / mL). Ascites fluid was collected 2 weeks later.

[0047] Antibodies were purified using the Thermo Melon Gel Monoclonal IgG Purification Kit. The purified antibodies were aliquoted and stored at -20°C.

[0048] Antibody subtype identification was performed using the Pierce Papid Isotyping Kit-Mouse. First, the antibody was diluted to 100 ng / mL with sample dilution buffer. Then, 150 μl of the diluted antibody was added to each well, and the results were observed and recorded after 5-10 minutes. The results showed that this monoclonal antibody was mouse IgG1 subtype. Figure 2 As shown.

[0049] Example 4: Determination of the variable region sequence of a monoclonal antibody

[0050] Mouse hybridoma cell line 5D85163 was cultured, and total RNA was extracted from hybridoma cells using the Trizol method. cDNA was reverse transcribed using the Thermo Fisher High Capacity cDNA Rever Transcription Kit. Based on the mouse monoclonal antibody primer sequences in "Recombinant Antibodies" (Science Press, edited by Shen Beifen, published in 2005), heavy and light chain primers for the antibody were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd. PCR amplification was performed (amplification program: 95℃ preheating for 1 min, 30 cycles (95℃ 30 s, 58℃ 30 s, 72℃ 45 s), and a final extension at 72℃ for 5 min). The resulting fragments were ligated into the PMD18-T vector and expressed in E. coli HB109. Positive clones were selected for sequencing. The sequenced data were compared and analyzed against the CDR region sequence of the mouse-derived monoclonal antibody using the BLAST website (https: / / www.ncbi.nlm.nih.gov / igblast / ).

[0051] Sequence analysis revealed that the variable region of the light chain consists of 107 amino acids, with the following sequence: DIQMNQSPSSLSASLGDRVTISCSAS WHO LNWYQQKPDGTVKLLIY Federal Tax Service SLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYC QQYSKLPYTFGGGTKLELK (SEQ ID NO.1), wherein the underlined sequences are CDR1, CDR2 and CDR3 in sequence. CDR1 is located at 27-32 aa and has the amino acid sequence QGISNY (SEQ ID NO.2); CDR2 is located at 50-52 aa and has the amino acid sequence FTS (SEQ ID NO.3); CDR3 is located at 89-97 aa and has the amino acid sequence QQYSKLPYT (SEQ ID NO.4).

[0052] The heavy chain variable region has 119 amino acids, and its sequence is as follows: EVQLQQSGPELVKPGASVKISCKTS GYTFTEIT MHWMKQNHGKSLEWIGD INPNNGST NYNQMYKGKATFTLDKSSTTAYMELRRSLTSDNSAVYYC ARG YYGYLYFDY WGQGTTLTVS (SEQ ID NO.5), wherein the underlined sequences are CDR1, CDR2 and CDR3 in sequence. CDR1 is located at 26-33 aa and has the amino acid sequence GYTFTEYT (SEQ ID NO.6); CDR2 is located at 51-58 aa and has the amino acid sequence INPNNGST (SEQ ID NO.7); CDR3 is located at 97-109 aa and has the amino acid sequence ARGYYGYLYYFDY (SEQ ID NO.8).

[0053] Example 5: Immunodetection of novel coronavirus nucleoprotein by monoclonal antibodies

[0054] The immunodetection performance of the prepared monoclonal antibody against the SARS-CoV-2 nucleoprotein was detected using an indirect enzyme-linked immunosorbent assay (ELISA). To examine the specificity and affinity of the monoclonal antibody, the following proteins were simultaneously tested: SARS-CoV-2 NP protein (catalog number HDS-5056), Mycobacterium tuberculosis TB protein (catalog number HDS-A5040), EB virus protein (catalog number HDS-M1001), and human parvovirus B19 protein (catalog number HDS-M1009), all purchased from Qingdao Henderson Biotechnology Co., Ltd.

[0055] Each protein was diluted to a concentration of 2.5 μg / mL using carbonate coating buffer, and 100 μl was coated per well, incubated overnight at 4°C. The plate was washed twice with washing buffer (200 μl per well). 100 μl of blocking buffer was added to each well, and the plate was blocked at room temperature for 6 hours. The plate was then washed five times with washing buffer (200 μl per well). The monoclonal antibody against the C-terminal region of the novel coronavirus nucleoprotein was diluted with antibody diluent to concentrations of 200, 100, 50, 25, 12.5, 6.25, 1.0, and 0. 5. Add 0.25, 0.125, and 0.0625 ng / mL of antibody to each well at a rate of 100 μl, and set up a blank well (100 μl of antibody dilution); incubate at 37°C for 30 min; wash the plate 5 times with washing buffer, 200 μl per well; incubate with HRP-labeled goat anti-mouse secondary antibody at 37°C for 20 min; wash the plate 5 times with washing buffer, 200 μl per well; add freshly prepared substrate solution, 100 μl per well, and incubate at 37°C for 10 min; stop the reaction by adding 50 μl of 2M H2SO4 to each well. Measure the absorbance of each well using a microplate reader at a wavelength of 450 nm. The results are as follows: Figure 3 As shown. Figure 3 The results showed that the prepared monoclonal antibody specifically recognizes the novel coronavirus nucleoprotein but not other proteins, indicating that the prepared monoclonal antibody has very high specificity. Furthermore, the lowest effective concentration of the prepared monoclonal antibody was 0.0625 ng / mL, indicating that the prepared monoclonal antibody has a very high affinity for the novel coronavirus nucleoprotein. sequence list <110> Zhejiang Orient Gene Biotech Co., Ltd. <120> A monoclonal antibody against the C-terminal region of the novel coronavirus nucleoprotein and its application <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 1 Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 2 <211> 6 <212> PRT <213> Mouse (Mus musculus) <400> 2 Gln Gly Ile Ser Asn Tyr 1 5 <210> 3 <211> 3 <212> PRT <213> Mouse (Mus musculus) <400> 3 Phe Thr Ser 1 <210> 4 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 4 Gln Gln Tyr Ser Lys Leu Pro Tyr Thr 1 5 <210> 5 <211> 119 <212> PRT <213> Mouse (Mus musculus) <400> 5 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Met His Trp Met Lys Gln Asn His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly Ser Thr Asn Tyr Asn Gln Met Tyr 50 55 60 Lys Gly Lys Ala Thr Phe Thr Leu Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Asp Asn Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Gly Tyr Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser 115 <210> 6 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 6 Gly Tyr Thr Phe Thr Glu Tyr Thr 1 5 <210> 7 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 7 Ile Asn Pro Asn Asn Gly Ser Thr 1 5 <210> 8 <211> 13 <212> PRT <213> Mouse (Mus musculus) <400> 8 Ala Arg Gly Tyr Tyr Gly Tyr Leu Tyr Tyr Phe Asp Tyr 1 5 10

Claims

1. A monoclonal antibody against the nucleoprotein of a novel coronavirus or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR1, CDR2, and CDR3, and the heavy chain variable region comprises CDR1, CDR2, and CDR3, characterized in that, The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO. 2; The amino acid sequence of the light chain CDR2 is the sequence shown in SEQ ID NO. 3; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO. 4; The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO. 6; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO. 7; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.

8.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region sequence is the sequence shown in SEQ ID NO. 1, and the heavy chain variable region sequence is the sequence shown in SEQ ID NO.

5.

3. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or antigen-binding fragment is a Fab fragment, Fab' fragment, F(ab')2 fragment, or a single-chain antibody.

4. A nucleic acid molecule, characterized in that, The nucleic acid is a nucleic acid encoding the antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4.

6. A recombinant, characterized in that, The recombinant comprises the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. A monoclonal antibody hybridoma cell line against novel coronavirus nucleoprotein, characterized in that, The monoclonal antibody hybridoma cell line is mouse hybridoma cell line 5D85163, with accession number CGMCC No. 45130.

8. The use of the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3 in the preparation of products for detecting the novel coronavirus.

9. A kit for detecting the novel coronavirus, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

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