Binding antibodies against sars-cov-2 virus n protein and uses thereof

Abstract

The application discloses a binding antibody against SARS-CoV-2 virus N protein and application thereof. The amino acid sequence of the heavy chain variable region of the monoclonal antibody N2E5 protected by the application is shown in SEQ ID NO. 1 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2 in the sequence listing; the amino acid sequence of the heavy chain variable region of the monoclonal antibody N2E5 is shown in SEQ ID NO. 5 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 6 in the sequence listing. The kinetic constants K D of N2E5 and N8C6 with the N protein are 1.42*10 ‑8 M and 1.31*10 ‑8 M respectively. The antibody has high affinity with the N protein of the novel coronavirus and can be widely used in a novel coronavirus antibody detection kit and clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to antibodies that bind to the N protein of SARS-CoV-2 virus and their applications. Background Technology

[0002] The novel coronavirus pneumonia (now known as COVID-19) is caused by the coronavirus SARS-CoV-2. COVID-19 patients often present with mild to moderate symptoms such as fever and cough. Severe cases can develop into acute respiratory distress syndrome, acute cardiac injury, multiple organ failure, and secondary infections. Even recovered patients may experience irreversible pulmonary fibrosis, and potential damage to the reproductive and hematopoietic systems.

[0003] SARS-CoV-2 belongs to the subgenus Sarbecovirus of the subfamily Orthocoronavirus within the family Coronaviridae. It is a different subgenus within the same genus as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The SARS-CoV-2 single-stranded RNA genome is approximately 29.9 kb in size and consists of 11 genes arranged in the order of 5′-replicaase (orf 1 / ab)-structural protein [spike protein (S)-enveloping protein (E)-membrane protein (M)-nucleocapsid protein (N)]-3′.

[0004] The envelope-anchored S protein attaches to the host during infection. The extracellular domain of the S protein has two subunits, S1 and S2. The S1 subunit binds to receptors on the host cell surface, and the S2 subunit mediates the fusion of the viral membrane and the host membrane. The M protein binds to the nucleocapsid and plays a role in envelope formation and assembly. The E protein is primarily responsible for viral assembly and release. The N protein is a nucleocapsid protein that binds to the viral RNA genome. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to identify the N protein of the novel coronavirus and / or how to obtain binding antibodies against the N protein of the novel coronavirus.

[0006] To address the aforementioned technical problems, this invention first provides an antibody against the novel coronavirus N protein. The antibody is A and / or B. A may be a monoclonal antibody N2E5 or its antigen-binding portion and / or a monoclonal antibody N8C6 or its antigen-binding portion. The monoclonal antibody N2E5 or its antigen-binding portion contains a component that may be named N2E5-V. HThe heavy chain variable region and its name is N2E5-V L The light chain variable region. The N2E5-V H and N2E5-V L Both consist of a cluster complement region and a framework region. The N2E5-V... H and the N2E5-V L The complementary regions of the determinant clusters can all be composed of CDR1, CDR2 and CDR3.

[0007] The N2E5-V H The amino acid sequence of CDR1 can be positions 26-33 of SEQ ID NO.1 in the sequence listing.

[0008] The N2E5-V H The amino acid sequence of CDR2 can be positions 51-57 of SEQ ID NO.1 in the sequence listing.

[0009] The N2E5-V H The amino acid sequence of CDR3 can be positions 96-110 of SEQ ID NO.1 in the sequence listing.

[0010] The N2E5-V L The amino acid sequence of CDR1 can be positions 26-31 of SEQ ID NO.2 in the sequence listing.

[0011] The N2E5-V L The amino acid sequence of CDR2 can be positions 49-51 of SEQ ID NO.2 in the sequence listing.

[0012] The N2E5-V L The amino acid sequence of CDR3 can be positions 88-98 of SEQ ID NO.2 in the sequence listing.

[0013] B may be a monoclonal antibody N8C6 or its antigen-binding moiety. The monoclonal antibody N8C6 or its antigen-binding moiety may contain a compound named N8C6-V. H The heavy chain variable region and its name are N8C6-V L The light chain variable region. The N8C6-V H and N8C6-V L Both consist of a cluster-determining complementary region and a framework region. The N8C6-V... H and the N8C6-V L The complementary regions of the determinant clusters are all composed of CDR1, CDR2 and CDR3.

[0014] The N8C6-V HThe amino acid sequence of CDR1 can be positions 26-33 of SEQ ID NO.5 in the sequence listing;

[0015] The N8C6-V H The amino acid sequence of CDR2 can be positions 51-58 of SEQ ID NO.5 in the sequence listing;

[0016] The N8C6-V H The amino acid sequence of CDR3 can be positions 97-107 of SEQ ID NO.5 in the sequence listing;

[0017] The N8C6-V L The amino acid sequence of CDR1 can be positions 26-34 of SEQ ID NO.6 in the sequence listing;

[0018] The N8C6-V L The amino acid sequence of CDR2 can be positions 52-54 of SEQ ID NO.6 in the sequence listing;

[0019] The N8C6-V L The amino acid sequence of CDR3 can be positions 91-101 of SEQ ID NO.6 in the sequence listing.

[0020] In the aforementioned antibodies, the amino acid sequence of the heavy chain variable region of the monoclonal antibody N2E5 may be SEQ ID NO.1 in the sequence listing or have at least 80% identity with SEQ ID NO.1. The amino acid sequence of the light chain variable region of the monoclonal antibody N2E5 may be SEQ ID NO.2 in the sequence listing or have at least 80% identity with SEQ ID NO.2. Inconsistencies in the amino acid sequence may occur in the frame region (FR).

[0021] The amino acid sequence of the heavy chain variable region of the monoclonal antibody N8C6 may be SEQ ID NO. 5 in the sequence listing or have at least 80% identity with SEQ ID NO. 5. The amino acid sequence of the light chain variable region of the monoclonal antibody N8C6 may be SEQ ID NO. 6 in the sequence listing or have at least 80% identity with SEQ ID NO. 6. Inconsistencies in the amino acid sequence may occur in the frame region (FR).

[0022] The aforementioned at least 80% identity can be at least 80%, 85%, or 95% identity.

[0023] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0024] In this document, the at least 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0025] Variants of the antibodies described in this invention, exhibiting improved affinity and / or titer, can be obtained using methods known in the art and are included within the scope of this invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can also be used to improve translation efficiency in expression systems used to generate antibodies. Furthermore, polynucleotides comprising sequences whose antibody specificity or neutralizing activity is optimized by applying directed evolution to any nucleic acid sequence of this invention are also within the scope of this invention.

[0026] Of the antibodies mentioned above, the monoclonal antibody may be any of the following:

[0027] a) Single-chain antibodies;

[0028] b) A fusion antibody containing the single-chain antibody described in a);

[0029] c) Fab fragment;

[0030] d) Fv fragment;

[0031] The term "Fab fragment" refers to a heterodimer composed of a heavy chain Fd and a complete light chain linked by disulfide bonds, containing only one antigen-binding site. By linking the genes encoding the heavy chain Fd and the complete light chain and fusing them with a bacterial protein signal peptide gene, Fab antibodies (Fab fragments) can be secreted and expressed in *E. coli*, exhibiting complete stereofolding and intra- and inter-chain disulfide bonds. The heavy chain Fd refers to approximately half of the H chain portion of the Fab fragment (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region).

[0032] The term "Fv fragment" refers to a vector containing VH and VL genes that can be constructed separately, co-transfected into cells to express them separately, and then assembled into a functional Fv antibody; alternatively, a stop codon can be set between VH and VL in the vector to express two small protein fragments, which can then be bound together by non-covalent bonds to form an Fv antibody (Fv fragment).

[0033] The term "Fab′ fragment" contains a portion of a light chain and a heavy chain containing the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of two Fab′ fragments to form the F(ab′)2 molecule.

[0034] The term "F(ab′)2 segment" contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab′)2 segment consists of two Fab′ segments held together by the disulfide bond between the two heavy chains.

[0035] The term "single-chain antibody (ScFv)" refers to an antibody that expresses a single polypeptide chain by linking the light and heavy chain variable regions of a gene with an appropriate oligonucleotide linker. The polypeptide chain can spontaneously fold into its natural construct, maintaining the specificity and affinity of the Fv.

[0036] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody's binding affinity to the target. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target.

[0037] The term "nanobody (single-domain antibody)" refers to an antibody containing only the VH fragment, obtained by expressing the V region of the antibody heavy chain through genetic engineering. The ability of single-domain antibodies to bind to antigens and their stability are essentially the same as those of complete antibodies.

[0038] The term "bispecific antibody" refers to a high-yield, uniform, and pure bispecific antibody obtained by introducing two sets of light chain and heavy chain genes into myeloma cells and selecting appropriate antibody constant regions and Ig types. Alternatively, bispecific antibodies can also be obtained using chemical cross-linking techniques or hybridization-hybridoma techniques.

[0039] The term "minimum recognition unit (MRU)" refers to a single CDR structure containing only the variable region, with a molecular weight of only about 1% of that of a complete antibody, which can bind to the corresponding antigen.

[0040] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector, then transfected into host cells, and the transfected host cells are cultured under specific conditions to express the antibodies of the present invention.

[0041] As is well known to those skilled in the art, the antigen-binding fragment can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody to produce an antigen-binding fragment of the antibody.

[0042] To address the aforementioned technical problems, the present invention also provides nucleic acid molecules encoding the monoclonal antibody N2E5 or its antigen-binding portion described above, and / or the monoclonal antibody N8C6 or its antigen-binding portion described above.

[0043] The nucleic acid molecule may be a DNA molecule as described in c1), c2), or c3):

[0044] c1) The encoding DNA molecule of the monoclonal antibody, wherein the coding sequence of the heavy chain variable region of the monoclonal antibody may be a DNA molecule with the sequence shown in SEQ ID NO.3, and the sequence of the light chain variable region of the monoclonal antibody may be a DNA molecule with the sequence shown in SEQ ID NO.4.

[0045] c2) The encoding DNA molecule of the monoclonal antibody; the heavy chain variable region encoding sequence of the monoclonal antibody is a DNA molecule with the sequence shown in SEQ ID NO.7, and the light chain variable region encoding sequence of the monoclonal antibody is a DNA molecule with the sequence shown in SEQ ID NO.8.

[0046] c3) has more than 90% identity with the DNA molecule defined by c1) or c2) and encodes the DNA of the monoclonal antibody or its antigen-binding portion.

[0047] In this document, the at least 90% identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0048] To address the aforementioned technical problems, the present invention also provides biological materials. These biological materials may be expression cassettes containing the nucleic acid molecules described above, recombinant vectors, recombinant microorganisms, and / or recombinant animal cell lines.

[0049] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), and viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpesviruses (such as herpes simplex virus)). In one embodiment of the present invention, the vector may specifically be the pADSCFV-S vector or the pcDNA3.1(+) vector.

[0050] The microorganisms described in this article may be yeast, bacteria, or fungi. Among them, bacteria may be from the genera Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc.; yeast may be Pichia pastoris.

[0051] The cell line (host cell) refers to cells that can be used to introduce the vector, including but not limited to: eukaryotic cells (such as yeast cells, Aspergillus), animal cells (such as mammalian cells, insect cells), or prokaryotic cells. In one embodiment of the present invention, the cell line may specifically be HEK293-F cells.

[0052] The terms “cell” and “cell line” are used interchangeably, and all such names include their descendants.

[0053] The application of the antibodies described above in the detection of the N protein of the novel coronavirus also falls within the scope of protection of this invention.

[0054] To address the aforementioned technical problems, this invention also provides the application of the antibodies and / or biological materials described above in the preparation of products for detecting or diagnosing the novel coronavirus.

[0055] To address the aforementioned technical problems, this invention also provides the application of the antibodies and / or biomaterials described above in the preparation or development of products for the clinical treatment of novel coronavirus.

[0056] To address the aforementioned technical problems, this invention also provides the application of the antibodies and / or biological materials described above in the preparation or development of drugs for diseases caused by the novel coronavirus.

[0057] To address the aforementioned technical problems, this invention also provides the application of the antibodies and / or the biological materials described above in the preparation of products for detecting or diagnosing diseases caused by the novel coronavirus.

[0058] The present invention screened and obtained two novel binding antibodies against the novel coronavirus nucleocapsid protein (N protein, SARS-CoV-2 N protein). The monoclonal antibodies N2E5 and N8C6 can recognize the novel coronavirus N protein. The single-chain antibodies include a heavy chain variable region and a light chain variable region. The N2E5 heavy chain variable region has three complementarity-determining regions (CDRs) of the amino acid sequence shown in SEQ ID NO. 1, positions 26-33, 51-57, and 96-110. The N2E5 light chain variable region has three CDRs of the amino acid sequence shown in SEQ ID NO. 2, positions 26-31, 49-51, and 88-98. The N8C6 heavy chain variable region has three complementarity-determining regions of the amino acid sequence shown in positions 26-33, 51-58, and 97-107 of SEQ ID NO. 5; the N8C6 light chain variable region has three complementarity-determining regions of the amino acid sequence shown in positions 26-34, 52-54, and 91-101 of SEQ ID NO. 6.

[0059] The beneficial effects of this invention are as follows:

[0060] The single-chain antibodies against the SARS-CoV-2 virus N protein provided by this invention can specifically recognize the SARS-CoV-2 virus N protein. The kinetic constant K between the N2E5 and N8C6 monoclonal antibodies and the N protein is... D They are 1.42×10 -8 M and 1.31×10 -8 M.

[0061] This antibody has a high affinity for the N protein of the novel coronavirus and can be widely used for COVID-19 detection, clinical treatment, and other purposes. Attached Figure Description

[0062] Figure 1 SDS-PAGE protein electrophoresis images of monoclonal antibodies N2E5 and N8C6. A shows the detection results for monoclonal antibody N2E5; B shows the detection results for monoclonal antibody N8C6. M is the protein marker, 1 is the cell culture supernatant, 2 is the flow through, 3 is the purified product 1, 4 is the purified product 2, 5 is the purified product 3, 6 is the purified product 4, and 7 is the purified product 5.

[0063] Figure 2Western blotting results for N2E5 and N8C6 antibodies. A shows the identification results for monoclonal antibody N2E5; B shows the identification results for monoclonal antibody N8C6. M represents protein markers; 1 shows 5 μg of SARS-CoV-2N protein loaded, 2 shows 10 μg of SARS-CoV-2N protein loaded, 3 shows 15 μg of SARS-CoV-2N protein loaded, and 4 shows 20 μg of SARS-CoV-2N protein loaded.

[0064] Figure 3 Affinity identification diagram of N2E5 antibody and N8C6 antibody with SARS-CoV-2 virus N protein. Detailed Implementation

[0065] Animal viruses: The public may obtain the biological material from the applicant in accordance with the relevant national biosafety regulations. The biological material is only for repeating the relevant experiments of this invention and may not be used for other purposes.

[0066] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0068] The sources of the reagents and carriers in the embodiments of the present invention are as follows:

[0069] The antibody heavy chain expression vector Igγ1 and antibody light chain expression vector Igλ were provided by the State Key Laboratory of Infectious Disease Prevention and Control, National Center for STD and AIDS Prevention and Control, Chinese Center for Disease Control and Prevention (Related literature: Hu Yuanyuan, Kang Jiaxing, et al. Isolation and functional identification of a group of monoclonal antibodies targeting HIV membrane proteins [J] Chinese Journal of Experimental and Clinical Virology, April 2021, Vol. 35, No. 2: 141-147; Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann H. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods. 2008 Jan 1; 329(1-2):112-24).

[0070] FreeStyle TM 293 Expression Medium: Gibco, Catalog No. 12338026.

[0071] 293F cells: Cells were provided by the State Key Laboratory of Infectious Disease Prevention and Control, National Center for STD and AIDS Prevention and Control, Chinese Center for Disease Control and Prevention (Related literature: Hu Yuanyuan, Kang Jiaxing, et al. Isolation and functional identification of a group of monoclonal antibodies targeting HIV membrane proteins [J] Chinese Journal of Experimental and Clinical Virology, April 2021, Vol. 35, No. 2: 141-147).

[0072] SARS-CoV-2 N protein (a mixture of His-tagged and untagged N protein): Shanghai Beyotime Biotechnology Co., Ltd., catalog number P2328-1mg.

[0073] Example 1: Discovery of Antibodies

[0074] By infecting COVID-19 patient B cells with EB virus, and through subcloning screening, amplification of the variable regions of the antibody light and heavy chains, construction of expression vectors, antibody expression and purification, and identification of antibody affinity, two humanized IgG antibodies that can specifically recognize the SARS-CoV-2 virus nucleocapsid protein (N protein, SARS-CoV-2 N protein) were obtained and named monoclonal antibody N2E5 and monoclonal antibody N8C6, respectively.

[0075] Monoclonal antibody N2E5: includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region of monoclonal antibody N2E5 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region of monoclonal antibody N2E5 is shown in SEQ ID NO.2. The monoclonal antibody N2E5 heavy chain variable region has three complementarity-determining regions (CDRs): heavy chain CDR1 (amino acid sequence of SEQ ID NO. 1, positions 26-33), heavy chain CDR2 (amino acid sequence of SEQ ID NO. 1, positions 51-57), and heavy chain CDR3 (amino acid sequence of SEQ ID NO. 1, positions 96-110); the monoclonal antibody N2E5 light chain variable region has three complementarity-determining regions (CDRs): light chain CDR1 (amino acid sequence of SEQ ID NO. 2, positions 26-31), light chain CDR2 (amino acid sequence of SEQ ID NO. 2, positions 49-51), and light chain CDR3 (amino acid sequence of SEQ ID NO. 2, positions 88-98).

[0076] Monoclonal antibody N8C6: includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region of monoclonal antibody N8C6 is shown in SEQ ID NO.5; the amino acid sequence of the light chain variable region of monoclonal antibody N8C6 is shown in SEQ ID NO.6. The heavy chain variable region of monoclonal antibody N8C6 has three complementarity-determining regions (CDRs): heavy chain CDR1 (amino acid sequence of SEQ ID NO. 5, positions 26-33), heavy chain CDR2 (amino acid sequence of SEQ ID NO. 5, positions 51-58), and heavy chain CDR3 (amino acid sequence of SEQ ID NO. 5, positions 97-107). The light chain variable region of monoclonal antibody N2E5 also has three complementarity-determining regions (CDRs): light chain CDR1 (amino acid sequence of SEQ ID NO. 6, positions 26-34), light chain CDR2 (amino acid sequence of SEQ ID NO. 6, positions 52-54), and light chain CDR3 (amino acid sequence of SEQ ID NO. 6, positions 91-101) (Table 1).

[0077] Table 1. Amino acid sequences of the CDR region of monoclonal antibodies N2E5 and N8C6

[0078]

[0079] Note: The naming system for the CDRs mentioned above is Kabat.

[0080] Example 2: Preparation of Antibody

[0081] 1. The monoclonal antibody N2E5 heavy chain variable region DNA molecules (nucleotide sequence of SEQ ID NO.3 in the sequence listing), monoclonal antibody N8C6 heavy chain variable region DNA molecules (nucleotide sequence of SEQ ID NO.4 in the sequence listing), monoclonal antibody N2E5 light chain variable region DNA molecules (nucleotide sequence of SEQ ID NO.7 in the sequence listing), and monoclonal antibody N8C6 light chain variable region DNA molecules (nucleotide sequence of SEQ ID NO.8 in the sequence listing) were respectively ligated into T-cloning vectors to obtain four recombinant T-vectors containing different variable region DNA molecules. The antibody variable regions were amplified using the four recombinant T-vectors as templates and with specific primers containing restriction enzyme sites (Table 2). In Table 2, primer pair N2E5H5′AgeI / N2E5H3′SalI was used to amplify the variable region of the heavy chain of monoclonal antibody N2E5, primer pair N2E5L5′AgeI / N2E5L3′XhoI was used to amplify the variable region of the light chain of monoclonal antibody N2E5, primer pair N8C6H5′AgeI / N8C6H3′SalI was used to amplify the variable region of the heavy chain of monoclonal antibody N8C6, and primer pair N8C6L5′AgeI / N8C6L3′XhoI was used to amplify the variable region of the light chain of monoclonal antibody N8C6. The PCR reaction system is shown in Table 3, and the reaction procedure is shown in Table 4.

[0082] Table 2. Primers for antibody variable region amplification with enzyme cleavage sites

[0083]

[0084] Note: Underlined areas indicate enzyme cleavage sites.

[0085] Table 3. Enzyme restriction sites introduced into the PCR reaction system

[0086]

[0087] Table 4. PCR reaction procedure for introducing enzyme restriction sites

[0088]

[0089] All PCR products were identified by electrophoresis, purified by gel extraction, and their concentrations were measured.

[0090] Enzyme digestion: The purified PCR product, antibody expression vector Igγ1, and antibody light chain expression vector Igλ were used to establish the systems shown in Tables 5 and 6 on an ultra-low temperature ice box.

[0091] Table 5 Enzyme digestion reaction system

[0092]

[0093] Table 6. Enzyme digestion reaction procedure

[0094]

[0095] After detecting the enzyme digestion efficiency by agarose gel electrophoresis, the target fragment was incubated with T4 DNA ligase at 37°C for 3 hours to ligate it into the antibody expression vector. The ligation system is shown in Table 7.

[0096] Table 7. Connection Reaction Procedure

[0097]

[0098] Transformation: The ligation product was transformed into competent DH 5α Escherichia coli cells, and single colonies were expanded and preserved. Plasmids were extracted from the bacterial culture and identified by enzyme digestion. The identified plasmids were sent to the company for sequencing identification, yielding successfully constructed recombinant heavy chain expression vectors Igγ1-N2E5 and Igγ1-N8C6, and recombinant light chain expression vectors Igλ-N2E5 and Igλ-N8C6.

[0099] 2. Construction of recombinant cells

[0100] The recombinant heavy chain expression vector (Igγ1-N2E5) and the recombinant light chain expression vector (Igλ-N2E5) of the monoclonal antibody N2E5 were co-transfected into FreeStyle. TM 293F cells cultured in 293 expression medium yielded recombinant cells 293F / γ1HC-N2E5 expressing the monoclonal antibody N2E5.

[0101] The recombinant heavy chain expression vector (Igγ1-N8C6) and recombinant light chain expression vector (Igλ-N8C6) of the monoclonal antibody N8C6 were co-transfected into FreeStyle. TM 293F cells cultured in 293 expression medium yielded recombinant cells 293F / γ1HC-N8C6 expressing the monoclonal antibody N8C6.

[0102] The co-transfection steps are as follows:

[0103] Cell preparation: Use 28 mL of fresh, preheated serum-free 293... TM Expression medium diluted 3×10 7 Add one 293F cell to a 125 mL sterile cell shake flask.

[0104] Preparation of lipid-DNA complexes: In Dilute 30 μg of plasmid in I, maintaining a 1:1 ratio of antibody to recombinant light and heavy chain expression vector concentrations. Dilute 60 μL of 293 fetin in Opti-MEM serum-free medium. TMTransfection reagent, bringing the total serum culture medium volume to 1 mL, incubate at room temperature for 5 min. Add the diluted plasmid to the dilution reagent, mix gently, and incubate at room temperature for 20–30 min to allow the DNA-reagent complex to form.

[0105] Add 2 mL of the above complex to each cell suspension flask. Culture the cells in a cell culture incubator at 125 rpm, 37°C, and 5% CO2.

[0106] 3. Antibody preparation

[0107] 3.1 Collecting Supernatant

[0108] After culturing the recombinant cells obtained in step 2 for 48 hours, cultures expressing monoclonal antibody N2E5 (named 293FN2E5 culture) and monoclonal antibody N8C6 (293F / N8C6 culture) were obtained. The cultures were transferred to centrifuge tubes, centrifuged at 400g for 10 min, and the supernatant was collected and filtered through a 0.22μm filter.

[0109] 3.2 Antibody purification

[0110] Using the Thermo Fisher Scientific Protein A / G Antibody Purification Kit (NO. 89980), the supernatant obtained by filtration was purified according to the instructions to obtain purified products of monoclonal antibody N2E5 and monoclonal antibody N8C6.

[0111] The purification steps are as follows:

[0112] Dilute the filtered supernatant with an equal volume of binding buffer. Loosen the top cap of the rotating column and pull open the bottom seal. Place the column into a 15 mL collection tube, centrifuge at 400 g for 1 min, and discard the stock solution. Add 2 mL of binding buffer to equilibrate the column. Centrifuge at 1000 g for 1 min, and discard the filtrate. Repeat this step once. Add the diluted sample to the column and collect the flow, naming it "FlowThrough".

[0113] Wash the column with 15 mL of binding buffer, place the column in a clean 15 mL collection tube, centrifuge at 1000 g for 1 min, and discard the filtrate. Add 100 μL of neutralization buffer to five 1.5 mL Eppendorf tubes, add 1 mL of elution buffer to the column, and centrifuge at 1000 g for 1 min. Transfer the filtrate to an Eppendorf tube containing neutralization buffer, and save the collected solution as "Purified Product-1". Repeat this step four times to obtain five gradients of purified products, and store at 4 °C.

[0114] 3.3 Detection of purified products

[0115] The purified products were detected using the Human Total IgG Content ELISA Kit (NO. BDEL-0254-96T) from Beijing Bio-Long Immunoassay Technology Co., Ltd. The human total IgG concentrations of purified products 1–5 of monoclonal antibody N2E5 were 6608.04 ng / mL, 6222.81 ng / mL, 4632.42 ng / mL, 4594.73 ng / mL, and 3421.90 ng / mL, respectively. The human total IgG concentrations of purified products 1–5 of monoclonal antibody N8C6 were 6695.55 ng / mL, 6731.94 ng / mL, 5577.87 ng / mL, 5547.51 ng / mL, and 3991.43 ng / mL, respectively.

[0116] 3.4 Antibody Purity Analysis

[0117] The culture supernatant obtained in step 3.1, the "flow through" produced during purification in step 3.2, and the purified antibody product were subjected to SDS-PAGE protein electrophoresis. The results are as follows: Figure 1 As shown, the culture supernatant (293F / N2E5 culture and 293F / N8C6 culture supernatant) Figure 1 Lane 1 of the middle A and Figure 1 (as shown in lane 1 of B) and "Flow Through" ( Figure 1 Lane 2 of Middle A and Figure 1 As shown in lane 2 of the middle B, there are bands of extraneous proteins, while the antibody purification product only shows the target band at 55 kDa (antibody heavy chain size) and 25 kDa (antibody light chain size).

[0118] 3.5 Western Blot Detection

[0119] SARS-CoV-2N protein (approximately 47 kDa, including both His-tagged and His-untagged SARS-CoV-2N proteins) was loaded in gradients of 5 μg, 10 μg, 15 μg, and 20 μg, followed by SDS-PAGE protein electrophoresis. The protein was then transferred to a PVDF membrane and incubated with monoclonal antibody N2E5 or monoclonal antibody N8C6, purified in step 3.2, at a 1:1000 dilution. Western blot results are shown below. Figure 2 As shown, monoclonal antibodies N2E5 and N8C6 can bind to the SARS-CoV-2N protein, exhibiting two specific bands at approximately 47 kDa: one for the His-tagged SARS-CoV-2N protein and one for the untagged SARS-CoV-2N protein.

[0120] Example 3: Detection of antibody affinity for SARS-CoV-2 viral N protein

[0121] Biolayer interferometry (BLI) was used: the affinity of the purified monoclonal antibody N2E5 and monoclonal antibody N8C6 obtained in Example 2 was detected using a Gator biolayer interferometry instrument.

[0122] Test results as follows Figure 3 As shown. The kinetic constant K of monoclonal antibodies N2E5 and N8C6 with the N protein. D They are 1.42×10 -8 M and 1.31×10 -8 M indicates that the two monoclonal antibodies have a high affinity.

[0123] In summary, the monoclonal antibodies N2E5 and N8C6 obtained by screening in this invention can specifically recognize and bind to the N protein of SARS-CoV-2 virus, and have high affinity for the N protein. They can be used to prepare COVID-19 test kits and develop products for the clinical treatment of the novel coronavirus.

[0124] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An antibody against the novel coronavirus N protein, characterized in that: The antibody is A, or a combination of A and B, wherein A is a monoclonal antibody N2E5 or an antigen-binding portion thereof; the monoclonal antibody N2E5 or the antigen-binding portion thereof comprises a heavy chain variable region designated as N2E5-VH and a light chain variable region designated as N2E5-VL; both the N2E5-VH and the N2E5-VL comprise a CDR1, a CDR2 and a CDR3; the CDR1 of the N2E5-VH has an amino acid sequence of 26-33 of SEQ ID NO. 1 in the sequence listing; the CDR2 of the N2E5-VH has an amino acid sequence of 51-57 of SEQ ID NO. 1 in the sequence listing; the CDR3 of the N2E5-VH has an amino acid sequence of 96-110 of SEQ ID NO. 1 in the sequence listing; the CDR1 of the N2E5-VL has an amino acid sequence of 26-31 of SEQ ID NO. 2 in the sequence listing; the CDR2 of the N2E5-VL has an amino acid sequence of 49-51 of SEQ ID NO. 2 in the sequence listing; the CDR3 of the N2E5-VL has an amino acid sequence of 88-98 of SEQ ID NO. 2 in the sequence listing; the B is a monoclonal antibody N8C6 or an antigen-binding portion thereof; the monoclonal antibody N8C6 or the antigen-binding portion thereof comprises a heavy chain variable region designated as N8C6-VH and a light chain variable region designated as N8C6-VL; both the N8C6-VH and the N8C6-VL comprise a CDR1, a CDR2 and a CDR3; the CDR1 of the N8C6-VH has an amino acid sequence of 26-33 of SEQ ID NO. 5 in the sequence listing; the CDR2 of the N8C6-VH has an amino acid sequence of 51-58 of SEQ ID NO. 5 in the sequence listing; the CDR3 of the N8C6-VH has an amino acid sequence of 97-107 of SEQ ID NO. 5 in the sequence listing; the CDR1 of the N8C6-VL has an amino acid sequence of 26-34 of SEQ ID NO. 6 in the sequence listing; the CDR2 of the N8C6-VL has an amino acid sequence of 52-54 of SEQ ID NO. 6 in the sequence listing; the CDR3 of the N8C6-VL has an amino acid sequence of 91-101 of SEQ ID NO. 6 in the sequence listing.

2. The antibody of claim 1, wherein: the heavy chain variable region of the monoclonal antibody N2E5 has an amino acid sequence of SEQ ID NO. 1 in the sequence listing, and the light chain variable region of the monoclonal antibody N2E5 has an amino acid sequence of SEQ ID NO. 2 in the sequence listing. The amino acid sequence of the heavy chain variable region of the monoclonal antibody N8C6 is SEQ ID NO. 5 in the sequence listing, and the amino acid sequence of the light chain variable region of the monoclonal antibody N8C6 is SEQ ID NO. 6 in the sequence listing.

3. The antibody of claim 1 or 2, characterized in that: The monoclonal antibody is any one of the following: a) a single-chain antibody; b) a fusion antibody containing the single-chain antibody of a); c) a Fab fragment; d) a Fv fragment.

4. A nucleic acid molecule encoding the monoclonal antibody of claim 1 or 2, The DNA molecule encoding the monoclonal antibody N2E5 has a heavy chain variable region coding sequence as shown in SEQ ID NO. 3 and a light chain variable region coding sequence as shown in SEQ ID NO. 4; the DNA molecule encoding the monoclonal antibody N8C6 has a heavy chain variable region coding sequence as shown in SEQ ID NO. 7 and a light chain variable region coding sequence as shown in SEQ ID NO.

8.

5. A biomaterial characterized by: The biological material is an expression cassette, a recombinant vector, a recombinant microorganism and / or a recombinant animal cell line containing the nucleic acid molecule of claim 4.

6. The antibody of any one of claims 1-3 for use in detecting the novel coronavirus N protein.

7. Use of the antibody of any one of claims 1-3 and / or the biological material of claim 5 in the preparation of a product for detecting or diagnosing the novel coronavirus.

8. Use of the antibody of any one of claims 1-3 and / or the biological material of claim 5 in the preparation of a product for the clinical treatment of the novel coronavirus.

9. Use of the antibody of any one of claims 1-3 and / or the biological material of claim 5 in the preparation of a drug for the disease caused by the novel coronavirus.

10. Use of the antibody of any one of claims 1-3 and / or the biological material of claim 5 in the preparation of a product for detecting or diagnosing the disease caused by the novel coronavirus.

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