Nanometer antibody targeting coronavirus N antigen and application thereof

Abstract

The invention provides a nano antibody targeting a coronavirus N antigen and application of the nano antibody. Wherein the nano antibody or the antigen binding fragment comprises a heavy chain variable region, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The nano antibody disclosed by the invention has specificity and broad-spectrum binding activity and can be specifically bound with SARS-CoV-2 and a mutant thereof, the binding capacity reaches picomole level, and the phenomena of missing detection and the like in clinical detection are avoided; the antigen binding epitope is a brand new epitope; the production cost is lower, and popularization and application are facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and specifically relates to a nano antibody targeting coronavirus N antigen and an application thereof. Background Art

[0002] Coronaviruses belong to the Coronaviridae family of the Nidovirales order. They are a type of virus with an envelope and linear single-stranded positive-strand RNA (ssRNA) genetic material. They are widely found in nature. So far, seven coronaviruses that infect humans have been found, namely HCoV-229E and HCoV-NL63 of the α genus; HCoV-OC43, SARS-CoV-1, HCoV-HKU1, MERS-CoV and SARS-CoV-2 of the β genus. All seven coronaviruses can cause respiratory diseases in humans. HKU1, NL63, OC43 and 229E mainly cause mild respiratory symptoms and are the second largest type of common influenza-related infection viruses; while SARS-CoV-1, MERS-CoV and SARS-CoV-2 can cause severe respiratory diseases (Corman et al., 2018). Compared with several other coronaviruses, the new coronavirus (SARS-CoV-2) is highly contagious and has a wide range of prevalence. It can be transmitted through close contact, droplet transmission, and even environmental transmission, and can survive for a long time in extreme environments. In addition, asymptomatic virus carriers further increase the risk of virus transmission. In view of these characteristics, it is particularly important to design a specific diagnostic method for the virus.

[0003] SARS-CoV-2 is round or oval in shape, with a diameter of about 80-120nM. It is a positive-stranded single-stranded RNA virus with an envelope structure, and its genome size is about 30Kb. Nucleic acid-based diagnostic testing, that is, detecting the genetic material of SARS-CoV-2, is currently the gold standard for SARS-CoV-2 testing, but this test requires specific locations, specifically trained technicians, equipment and instruments, etc., and requires a long operating time, and is usually used for large-scale screening.

[0004] Immunoassay-based methods, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, fluorescence immunoassay chromatography, up-conversion luminescence chromatography, immunoturbidimetry, and colloidal gold chromatography that does not require a matching detection device, etc. Immunoassay is a technology mainly developed based on the antigen-antibody reaction principle. It is particularly crucial to develop antibodies against SARS-CoV-2 antigens or reagents for detecting antibodies in patients infected with SARS-CoV-2. Antibody detection, that is, detecting the antibodies produced by the body against SARS-CoV-2 infection. This detection can often only detect the antibodies IgM or IgG produced in the body for a period of time after infection, and often has a certain lag; antigen detection, that is, the detection of the SARS-CoV-2 itself protein, is used for rapid diagnosis (or exclusion) of active coronavirus infection, also known as rapid detection. Nucleic acid detection is the basis for the diagnosis of SARS-CoV-2 infection. Antigen detection can be used as a supplementary means for the screening of specific populations, which is beneficial to improving the ability of early detection. Developing specific detection antibodies against SARS-CoV-2 antigens has a wide range of application spaces.

[0005] According to the structure of SARS-CoV-2, scientific researchers have developed corresponding antibodies. The related structural proteins of SARS-CoV-2 are envelope protein (E protein), membrane protein (membrane, M protein), spike protein (spike, S protein), and nucleocapsid protein (nucleocapsid, N), respectively. Among them, the S, E, and M proteins together constitute the outer shell of the virus, and the N protein binds to the viral RNA to form the viral nucleocapsid. The S protein is expressed on the surface of SARS-CoV-2 virus particles and is a homotrimer composed of S1 and S2 subunits through non-covalent bonds (Wrapp et al., 2020b). The virus particle binds to the host cell expressing ACE-2 through the receptor binding domain (RBD) of the surface spike protein, and then fuses with the host cell and invades and infects the host through the functional domain of the S2 subunit. By developing antibodies against spike or spike RBD, it can block the virus from infecting the host and play the role of neutralizing antibodies. However, due to the easy mutation of the spike protein, it is generally not used for the development of detection antibodies.

[0006] The N protein is a phosphorylated protein composed of two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD). It participates in and regulates RNA transcription, replication, and protein translation. It is the structural protein with the most expressed copies during the virus replication process and is closely related to pathogenicity. Additionally, among different SARS-CoV-2 variants, unlike the S protein which is prone to mutation, the N protein, especially its two domains NTD and CTD, has very few mutations and relatively conserved structures. At the same time, when compared with the N proteins of other coronaviruses, the N protein of the novel coronavirus and its NTD and CTD domains maintain obvious specificity and can be identified and distinguished from other coronaviruses through specific binding detection. The N protein of SARS-CoV-2, with its characteristic pattern of being both conservative and specific, as well as its high-copy and abundant content, makes it a key target protein in research such as the clinical diagnosis of the novel coronavirus, epidemiological studies, and pathogenic infection mechanisms. Developing high-affinity antibodies against the N protein of SARS-CoV-2 is the key to the above immunological analysis-based detection methods.

[0007] Nanobody (Nb) is a novel antibody derived from the variable domain VHH of the heavy-chain antibody that is naturally lacking a light chain in camelids. Nb is composed of four conserved framework regions (FR) and three complementarity-determining regions (CDR). The number of amino acids in its CDR region is generally longer than that of the CDR3 of traditional human or murine antibodies, forming a convex loop structure (traditional antibodies usually have concave or flat antigen-binding sites), with a large number of CDR loops exposed in the solvent, which improves the specificity and affinity for antigen binding (Vanlandschoot et al., 2011b; Desmyter et al., 2001). Four hydrophilic residues in the FR2 region of Nb replace the four hydrophobic residues in the FR2 of traditional antibodies, resulting in higher solubility. The internal disulfide bonds make it more heat-resistant, acid- and alkali-resistant than traditional antibodies, with reduced polymerization, and still have biological activity after long-term placement in high-temperature environments or under strong denaturing conditions, showing higher in vitro stability (Vanlandschoot et al., 2011b; Muyldermans et al., 1994; Hamers-Casterman et al., 1993). Due to the natural lack of a light chain and its small molecular weight, it can be amplified and expressed in large quantities using prokaryotic and yeast systems, with relatively lower production costs, which is conducive to its popularization and application in infectious diseases such as pandemic viruses. Four hydrophilic residues in the FR2 region of Nb replace the four hydrophobic residues in the FR2 of traditional antibodies, having higher water solubility. The internal disulfide bonds make it more heat-resistant, acid- and alkali-resistant than traditional antibodies, with reduced polymerization, and still have biological activity after long-term placement in high-temperature environments or under strong denaturing conditions, showing higher in vitro stability (Vanlandschoot et al., 2011b; Muyldermans et al., 1994; Hamers-Casterman et al., 1993), which is more conducive to the development of antigen detection products that require room temperature conditions. Summary of the Invention

[0008] To solve the problem of the lack of reagents for detecting coronaviruses with high sensitivity and high specificity in the prior art, the present invention provides a nanobody targeting the N antigen of coronaviruses and its application. Specifically, the present invention uses the N proteins of wild-type and mutant strains of SARS-CoV-2 to cross-immunize alpacas, and detects and evaluates the antibody titers in alpaca sera. After the immunization, the peripheral blood of alpacas is collected, and plasma and peripheral blood lymphocytes (PBMCs) are isolated. The total RNA of PBMCs is extracted and reverse transcribed into cDNA, and then the alpaca nanobody (VHH) is amplified by multiplex PCR, and the nanobody phage library is constructed. Then, through the method of multi-antigen cross-selection, a SARS-CoV-2 N nanobody with high affinity and broad-spectrum binding activity is screened. The nanobody is subjected to multivalent modification and detection and evaluation of binding specificity, binding broad-spectrum, binding sensitivity, binding affinity, etc., and it is found that the antibody is an antibody against a new binding epitope of the SARS-CoV-2 N protein, and belongs to different binding epitopes from the currently commercially available N antibodies. At the same time, the antibody can specifically bind to the N antigen of SARS-CoV-2 and does not bind to the N antigens of other respiratory viruses such as influenza viruses, showing the binding specificity characteristics of the coronavirus N antigen. In addition, the nanobody can broadly recognize the N antigens of different novel coronavirus variants, and can avoid the phenomenon of "missed detection" in the detection of different SARS-CoV-2 variants. In summary, the nanobody of the present invention is a broad-spectrum novel coronavirus N antibody with high specificity, high sensitivity and high affinity, and has extremely high application value in the detection of SARS-CoV-2.

[0009] To solve the above technical problems, a first aspect of the present invention provides an antibody or antigen-binding fragment targeting the N antigen of coronaviruses, wherein it comprises a heavy chain variable region, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are respectively as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0010] In some preferred embodiments, the amino acid sequences of FR1, FR2, FR3, and FR4 of the framework region of the heavy chain variable region are respectively as shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

[0011] In some preferred embodiments, the antibody or antigen-binding fragment is selected from one or more of the following:

[0012] (1) IgG antibody, F(ab')2, F(ab)2, Fab, scFv, dSFv, nanobody, and heavy chain antibody;

[0013] (2) Monoclonal antibody or polyclonal antibody;

[0014] (3) A monospecific antibody, bispecific antibody or multispecific antibody.

[0015] In a more preferred embodiment, the antibody or antigen-binding fragment is a nanobody, and its amino acid sequence is as shown in SEQ ID NO:8.

[0016] In a more preferred embodiment, the antibody or antigen-binding fragment is a heavy-chain antibody, including VHH and Fc, wherein the amino acid sequence of VHH is as shown in SEQ ID NO:8.

[0017] Preferably, the Fc is selected from human or rabbit IgG, IgA, IgD, IgE and IgM.

[0018] More preferably, the amino acid sequence of the heavy-chain antibody is as shown in SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

[0019] In some preferred embodiments, the heavy-chain antibody is a monovalent antibody, bivalent antibody or tetravalent antibody. For example, bivalent VHH1-FC, tetravalent VHH1-VHH1-FC or IgM antibody VHH1-IgM.

[0020] The second aspect of the present invention provides an isolated nucleic acid, wherein the nucleic acid encodes the antibody or antigen-binding fragment as described in the first aspect.

[0021] The third aspect of the present invention provides a recombinant expression vector, wherein the recombinant expression vector contains the nucleic acid as described in the second aspect. Preferably, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO:9.

[0022] The fourth aspect of the present invention provides a transformant, wherein the transformant contains the nucleic acid as described in the second aspect or the recombinant expression vector as described in the third aspect.

[0023] The fifth aspect of the present invention provides a method for preparing an antibody or antigen-binding fragment targeting coronavirus N antigen, and the method is to culture the transformant as described in the fourth aspect of the present invention under suitable conditions to obtain the antibody or antigen-binding fragment.

[0024] The sixth aspect of the present invention provides a kit, wherein the kit contains the antibody or antigen-binding fragment as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant expression vector as described in the third aspect and / or the transformant as described in the fourth aspect.

[0025] The seventh aspect of the present invention provides a method for detecting coronavirus for non-diagnostic purposes. In this method, an antibody or antigen-binding fragment as described in the first aspect is contacted with a sample, and based on the binding of the antibody or antigen-binding fragment to the sample, it is quantitatively or qualitatively determined whether the coronavirus is present in the sample. Application scenarios for non-diagnostic purposes include, for example, detecting coronavirus in the environment to decide whether disinfection is required, or detecting whether a sample in the laboratory is contaminated with coronavirus.

[0026] The eighth aspect of the present invention provides the application of an antibody or antigen-binding fragment as described in the first aspect, a nucleic acid as described in the second aspect, a recombinant expression vector as described in the third aspect, or a transformant as described in the fourth aspect in the preparation of a reagent for detecting coronavirus.

[0027] On the basis of conforming to common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0028] The present invention provides a method for discovering and prokaryotic preparation of nanobodies, and also provides a method for preparing multivalent nanobodies by modifying nanobodies. The present invention provides the above-mentioned biological activity characteristics of nanobodies, including data such as antibody binding sensitivity, specificity, broad spectrum, affinity dissociation equilibrium constant, epitope detection, application of colloidal gold and enzyme-linked immunosorbent assay, etc. The application of the nanobodies in the detection and diagnosis of SARS-CoV-2, epidemiological research, pathogenic mechanism research, etc. are all within the protection scope of the present invention. The nanobodies involved in the present invention, no matter in any form of genetic engineering modification, including but not limited to the following modifications: amino acid mutation, small molecule structure modification, humanization, bispecific nanobody modification, bivalent or multivalent nanobody modification, coupling with other structural proteins or chemical reagents, etc., are all within the protection scope of the present invention.

[0029] The reagents and raw materials used in the present invention are all commercially available.

[0030] The positive and progressive effects of the present invention are as follows:

[0031] (1) Through the method of multi-antigen cross-immunization and panning, the nanobodies targeting the N antigen of SARS-CoV-2 screened and obtained by the present invention have specific and broad-spectrum binding activities, and can specifically bind to SARS-CoV-2 and its mutants, avoiding phenomena such as missed detection in clinical tests.

[0032] (2) The novel coronavirus N nanobodies of the present invention can be highly expressed prokaryotically. Compared with the eukaryotic mammalian cell expression of traditional antibodies, the production cost is lower, which is more conducive to popularization and application in such infectious diseases.

[0033] (3) The binding ability of the novel coronavirus N nanobodies of the present invention to the novel coronavirus N antigen reaches the picomolar level, and the specificity is strong.

[0034] (4) The antigen-binding epitope of the novel coronavirus N nanobody of the present invention is a brand-new epitope, which is different from commercially available antibodies and has more application possibilities in SARS-CoV-2 antigen detection or other immunoassay-based detection methods. Description of the Drawings

[0035] Figure 1 SDS-PAGE electrophoresis was used to detect the purified nanobody. Among them, lanes 1-3: VHH1 class nanobodies; lane M: protein ladder.

[0036] Figure 2 Electrophoresis was used to detect the bivalent nanobody. Among them, the electrophoresis conditions were 140V, 50min.

[0037] Figure 3 Specificity detection of the antibody was performed. Among them, PC refers to the positive control nanobody; NC is the buffer solution.

[0038] Figure 4 Broad-spectrum detection of the binding of the antibody to the novel coronavirus N antigen was performed. Among them, PC refers to the novel coronavirus N antibody verified in another patent; NC is the buffer solution.

[0039] Figure 5 Sensitivity detection of the binding of the antibody to the novel coronavirus N antigen was performed. Among them, PC is the novel coronavirus N antibody verified in another patent. Detailed Description of the Invention

[0040] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0041] Example 1: Multi-antigen Cross-immunization

[0042] Take 200 μg of wild-type SARS-CoV-2 N full-length antigen (Sino Biological) and mix it with an equal volume of Freund's complete adjuvant (Sigma), and perform the first immunization by multiple subcutaneous injections near the front limb under the neck of the alpaca. After that, immunize once every 2 weeks. For the second and third times, repeat the immunization with the wild-type SARS-CoV-2 N full-length antigen, and change the immunization adjuvant from Freund's complete adjuvant (Sigma) in the first time to Freund's incomplete adjuvant (Sigma); for the fourth time, immunize with two domains of the N antigen, NTD and CTD, and for the fifth time, immunize with the N antigen of different mutant strains (Sino Biological). 7 days after the end of immunization, collect 20-30 ml of peripheral blood from the alpaca's vein, and separate the plasma and PBMC samples for subsequent immune library construction.

[0043] Example 2: Plasma Titer Detection

[0044] Coat an ELISA plate with 100 ng of N antigen and incubate overnight at 4°C. Wash 3 times with PBST (0.05%). Add 200 μl of 2% BSA to each well and incubate at room temperature for 2 h. Wash 3 times with PBST. Dilute the pre-immunization and post-immunization plasma in Example 1 by 10 3 、10 4 、10 5 、10 6 and 10 7 times respectively, then add them to the corresponding ELISA plates and incubate at room temperature for 1 h. Wash 5 times with PBST. Add 100 μl of anti-alpaca H&L IgG HRP (2500-fold dilution, abcam) to each well and incubate in the dark at room temperature for 1 h. Wash 5 times with PBST. Add 100 μl of TMB chromogenic solution (abcam) and develop color for 10 min. Add an equal volume of TMB stop buffer (abcam) to terminate the color development and read the OD 450 value.

[0045] The ELISA test results are shown in (Table 1). The plasma titer after immunization increased significantly. At a plasma dilution of 106, the OD450 of the antibody detection after immunization was 3.38 times that before immunization, and the plasma titer of the novel coronavirus N antigen reached 10 6 , indicating that after immunization, the alpaca has obtained abundant antibodies against the novel coronavirus N antigen in its body.

[0046] Table 1: Determination of Alpaca Immune Plasma Potency

[0047]

[0048] Example 3: Obtaining Nanobodies

[0049] Take the blood samples of immunized alpacas and isolate lymphocytes from the peripheral blood of alpacas using lymphocyte separation medium (GE, 17-1440-02) and density gradient centrifugation. Extract RNA from the lymphocytes according to the instructions of Trizol reagent (INVITROGEN, 15596-018). Then take 20 μg of RNA and according to SuperScript TMIII First-Strand Synthesis SuperMix (Invitrogen) kit and process were used to synthesize cDNA. Then, the reverse transcription product cDNA was used as a template to build a library according to the phage library construction and screening process of Vincke C et al. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs (doi: 10.1007 / 978-1-61779-974-7_8. PMID: 22907350), and a library capacity of about 10 8 , a clone library with an abundance greater than 94%. The phage library was enriched with the novel coronavirus antigen N for 2-3 rounds of panning, and clones were selected from the affinity-enriched library for antigen affinity clone ELISA testing.

[0050] 100 ng of N antigen was coated on the ELISA plate as the experimental group. At the same time, an equal number of wells were left without antigen as the negative control. Incubate at 4°C overnight. From the plate selected in step 3 above (2), randomly select a single clone in 1 ml of culture medium, culture at 37°C until the logarithmic phase, and add 1 mM IPTG for induction overnight. The next day, collect the bacterial pellet by centrifugation, break it, centrifuge it at 5,000g for 15 minutes, and collect the supernatant. At the same time, add 2% BSA to the ELISA plate and block it at room temperature for 1 hour. Add the supernatant of the single clone to each well of the experimental group and the negative control group, incubate at room temperature for 2 hours. Wash 10 times with PBST, add anti HA-HRP label antibody (abcam), and incubate at room temperature for 1 hour. Wash 5 times with PBST, add substrate colorimetric agent, react for 10-20 minutes, add terminator, and read the absorbance value on the microplate reader. When the absorbance value is greater than 2.1 compared with the control well, it is determined to be a positive clone. In this example, a total of 1 nanoantibody (VHH1) with specificity and affinity for N antigen was screened.

[0051] The amino acid sequence of VHH1:

[0052] QVQLQESGGGLVQPGGSLRLSCX1ASGSIFSIQNMGWYRQAPEX2QRELVATITX3GGSTHYADSVKGRFX4ISRDNAKSTVYLQMNSLKPEDTAVYYCNAETX5X6GDGX7GYVFEDWGX8GTQVTVSS(SEQ ID NO:8)

[0053] Among them, the framework area:

[0054] The sequence of FR1 is QVQLQESGGGLVQPGGSLRLSCX1AS (SEQ ID NO:4); where X1 is V / A;

[0055] The sequence of FR2 is MGWYRQAPEX2QRELVAT (SEQ ID NO:5); where X2 is E / K;

[0056] The sequence of FR3 is HYADSVKGRFX4ISRDNAKSTVYLQMNSLKPEDTAVYYC (SEQ ID NO:6); where X4 is A / T;

[0057] The sequence of FR4 is WGX8GTQVTVSS (SEQ ID NO:7); where X8 is Q / H.

[0058] Complementary determining region:

[0059] The sequence of CDR1 is GSIFSIQN (SEQ ID NO:1);

[0060] The sequence of CDR2 is ITX3GGST (SEQ ID NO:2); where X3 is T / S;

[0061] The sequence of CDR3 is NAETX5X6GDGX7GYVFED (SEQ ID NO:3); where X5 is S / P; X6 is V / A; X7 is R / S.

[0062] In this article, the numerical numbering after the amino acid residue X has no specific meaning, but is only used to distinguish X at different positions. In the sequence listing, the same expression will be presented, that is, all are X.

[0063] The nucleotide sequence encoding VHH1 is:

[0064] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGYAGCCTCTGGAAGCATCTTCAGTATCCAAAACATGGGCTGGTACCGCCAGGCTCCAGAGRAGCAGCGCGAGTTGGTCGCAACTATTACTWCTGGTGGTAGCACACACTATGCAGACTCCGTGAAGGGCCGATTCRCCATCTCCAGAGACAACGCCAAGAGCACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGCGGAAACTYCGGYAGGGGACGGTMGCGGCTACGTTTTTGAGGACTGGGGCCASGGGACCCAGGTCACCGTCTCCTCA(SEQ IDNO:9)

[0065] Wherein, Y: T / C; R: G / A; W: A / T; R: G / A; S: G / C; M: C / A.

[0066] Example 4: Induced expression and purification of nanobody

[0067] (1) Induced expression of nanobody.

[0068] Separate monoclonal nanobodies from Example 3 were selected and inoculated into 10 ml of ampicillin-containing medium, and cultured overnight at 37 °C and 220 rpm. The next day, 2 ml of the overnight culture was inoculated into 200 ml of ampicillin-containing medium, and cultured at 37 °C and 220 rpm until the logarithmic phase (OD600 was 0.6 - 0.8), and IPTG was added for overnight induction of nanobody expression. The next day, the cell pellet was collected, the cells were lysed by the hypotonic method, and the supernatant was collected by high-speed centrifugation for subsequent protein purification.

[0069] (2) Purification of nanobody.

[0070] The purified nanobody was obtained by affinity purification with His-nickel filler (abbreviated as Ni filler, BioRad). The Ni filler was packed into a column, first washed with ultrapure water, and then washed with the equilibration buffer PBS; the above-mentioned lysed supernatant was added to the purification column at a flow rate of 1 ml / min; an appropriate volume of PBS was used to wash away the miscellaneous proteins until the OD280 was below 0.0001; then the target protein was eluted with 10 times the volume of the elution buffer (150 mM imidazole). For the purified target protein, an equal volume was taken and detected by 12% SDS-PAGE for the expression and purification of the nanobody ( Figure 1). As can be seen from the figure, the size of the nano antibody band is about 15KD, the purity is >95%, the yield is 5mg / L, and the expression is successful.

[0071] Example 5: SPR analysis of the binding affinity of nanobodies to the novel coronavirus antigen N

[0072] Take the CM5 chip, couple an appropriate amount of SARS-CoV-2N antigen, and then dilute the nanobody protein solution in Example 4 with HBS-P buffer to a suitable concentration gradient. In this case, it is from about 0.375 μg / ml (25nM) as the starting concentration, and then dilute 6 concentration gradients by 2 times. Each injection is 120s, dissociation is 180s, flow rate is 30 / min, and then regenerated with 10mMPH2.0 Gly-HCL, and the cycle is repeated until all concentration gradient injections are completed. After the program is run, the BiacoreT200 (GE) instrument comes with an analysis program for fitting analysis to obtain the nanobody affinity constant results (Table 2). The results showed that in this case, the affinity binding relay constant (KD value for short) of the nanoantibody with the new coronavirus N antigen was at the 10-8M level, and the antibody affinity was at the nanomolar level. The dissociation coefficient of the nanoantibody was 4.69E-03, and the dissociation was relatively fast. The antibody affinity can be improved and the dissociation can be reduced through structural modification, that is, transformation into a bivalent, tetravalent or IgM structure.

[0073] Table 2. Binding affinity constants of novel coronavirus N antigen nanoantibodies

[0074]

[0075] Example 6: Monovalent Nanobody Multivalent Modification, Eukaryotic Expression and Purification

[0076] The affinities of the nanoantibodies detected in Example 5 are all at the nanomolar level, and they are relatively easy to dissociate. Further structural modifications can be performed to reduce dissociation and enhance the ability of the antibody to bind to the new coronavirus N antigen.

[0077] The present invention fuses the nano antibody with rabbit IgG Fc to transform it into a bivalent and tetravalent structure; at the same time, the nano antibody is fused with human IgM to transform it into an IgM structure antibody. (The amino acid sequence is shown in the notes: bivalent VHH1-FC, tetravalent VHH1-VHH1-FC, IgM antibody VHH1-IgM), and the synthesized gene sequence is cloned by seamless PCR technology (for specific operations, refer to Hieff plus One Step Cloning Kit Instruction Manual) Subclone it into pCDNA3.1(+). Transfect the recombinant plasmid into HEK293F cells for expression. Dilute the recombinant expression plasmid with PBS and add the PEI (polyethylenimine) solution required for transformation. After mixing, add it to the HEK293F cell suspension, place it in an incubator, and culture at 37°C, 8% CO2, relative humidity ≥ 80% with 150 rpm. After culturing for 5 - 6 days, collect the transient expression culture supernatant. The supernatant is used for subsequent protein purification, and the modified bivalent, tetravalent, and IgM-structured nanobodies are purified by protein A affinity chromatography or molecular sieve separation respectively. After detection by reducing and non-reducing electrophoresis ( Figure 2 ). The results show that the band size of the purified bivalent nanobody is approximately 76 KD (under non-reducing conditions), and the single-chain size is approximately 38 KD under reducing conditions, which is consistent with the theoretical size, the purity is greater than 90%, and the transient transfection expression yield is at the level of 500 mg / L, with extremely high yield.

[0078] Among them, the amino acid sequences of various antibodies are as follows:

[0079] VHH1-FC:

[0080] QVQLQESGGGLVQPGGSLRLSCX1ASGSIFSIQNMGWYRQAPEX2QRELVA TITX3GGSTHYADSVKGRFX4ISRDNAKSTVYLQMNSLKPEDTAVYYCNAE TX5X6GDGX7GYVFEDWGX8GTQVTVSSAPSTCSKPTCPPPELLGGPSVFIF PPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:10)

[0081] VHH1-VHH1-FC:

[0082] QVQLQESGGGLVQPGGSLRLSCX1ASGSIFSIQNMGWYRQAPEX2QRELVA TITX3GGSTHYADSVKGRFX4ISRDNAKSTVYLQMNSLKPEDTAVYYCNAE TX5X6GDGX7GYVFEDWGX8GTQVTVSSGSQVQLQESGGGLVQPGGSLRL SCX1ASGSIFSIQNMGWYRQAPEX2QRELVATITX3GGSTHYADSVKGRFX4ISRDNAKSTVYLQMNSLKPEDTAVYYCNAETX5X6GDGX7GYVFEDWGX8GTQVTVSSAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:11)

[0083] VHH1-IgM:

[0084] QVQLQESGGGLVQPGGSLRLSCX1ASGSIFSIQNMGWYRQAPEX2QRELVA TITX3GGSTHYADSVKGRFX4ISRDNAKSTVYLQMNSLKPEDTAVYYCNAE TX5X6GDGX7GYVFEDWGX8GTQVTVSSIAELPPKVSVFVPPRDGFFGNPR KSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY(SEQ ID NO:12)

[0085] Example 7: SPR method for determining the affinity constant of multivalent nanobodies against the N antigen of SARS-CoV-2

[0086] Take a CM5 chip, couple the SARS-CoV-2 N antigen, and then dilute the bivalent antibody in Example 6 with HBS-P buffer to appropriate concentration gradients. In this example, the bivalent, tetravalent, and IgM antibodies start from approximately 20, 10, and 2.5 nM respectively, and are diluted in a 2-fold gradient successively for 6 concentration gradients. Each injection lasts for 120 s, dissociation lasts for 180 s, the flow rate is 30 / min, and then it is regenerated with 10 mM PH2.0 Gly-HCL, repeating the cycle until all concentration gradients are injected. After the program runs to completion, use the built-in analysis program of the Biacore T200 (GE) instrument for fitting analysis to obtain the results of the nanobody affinity constant (Table 3). The results show that in this example, after the VHH1 nanobody is transformed into bivalent, tetravalent, and IgM antibodies, the affinities are increased by more than 500, 700, and 20,000 times respectively. Especially for VHH1-IgM, the affinity of the antibody against the N antigen of SARS-CoV-2 is increased to the pM level (4.72E-12).

[0087] Table 3. Binding affinity constants of bivalent nanobodies against the N antigen of SARS-CoV-2

[0088]

[0089] Example 8: Identifying the binding epitope of an antibody using the SPR method

[0090] Take the CM5 chip coupled with the novel coronavirus N antigen in Example 5, and respectively inject and bind for 240 s at a flow rate of 10 μl / min with commercially available antibodies diluted with HBS-P buffer at approximately 3 μg / ml: AB0046-1 (BioBAY), AB0046-2 (BioBAY), COVID19-PS-Mab15 (Fitzgerald Industries International), and COVID19-PS-Mab17 (Fitzgerald Industries International) to fully saturate the commercially available antibodies with the N antigen on the chip and occupy the epitopes where the antibody itself binds to the N antigen; then inject for the second time for 90 s at a flow rate of 30 μl / min to confirm that the antibody is fully saturated. Next, inject for the third time in sequence, add 1.5 μg / ml of VHH1-FC, inject for 90 s at a flow rate of 30 μl / min, and dissociate for 60 s at a flow rate of 30 μl / min. Finally, regenerate with 10 mM PH2.0 Gly-HCL and repeat the cycle until the injection of the four commercially available antibodies is completed in sequence. After the program runs to completion, use the built-in analysis program of the Biacore T200 (GE) instrument to perform reading analysis. As shown in the results of Table 4, the binding value of VHH1-FC to the N antigen on the chip is 71.86 RU. When the N antigen on the chip is nearly saturated and bound by the commercially available antibody, that is, after the N antigen on the chip is occupied by the commercially available antibody at its binding epitope, the bindings of VHH1-FC to it are 72.6, 44.06, 70.35, and 71.93 RU respectively, and it is basically not affected by the commercially available antibody that has been saturated and bound, indicating that VHH1-FC and the four commercially available antibodies measured in this example are all different epitopes, and the epitope similarities with the four antibodies are: AB0046-1 (1.02%), AB0046-2 (38.68%), COVID19-PS-Mab15 (2.1%), and COVID19-PS-Mab17 (0.1%), indicating that the antibody in the present invention is a novel epitope antibody.

[0091] Binding rate = Binding value of the N antigen complex bound to the commercially available antibody / Binding value only to the N antigen on the chip; Epitope similarity with VHH1-FC = |1 - Binding rate|

[0092] Table 4. Detection of binding epitope

[0093]

[0094] Example 9: Specificity detection

[0095] Coat 50 ng of the novel coronavirus N antigen (Sino Biological) and other viral N antigens, which are N antigens of different subtypes of influenza A and B (Sino Biological) in this example, in an ELISA plate, 50 μl per well; wash 3 times with PBST, add 2% BSA for blocking, and incubate at room temperature for 2 hours. Wash 3 times with PBST, add 0.4 μg / ml of the three antibodies in Example 7, 50 μl per well, repeat for 2 wells, and incubate at room temperature for 1 hour. Wash 5 times with PBST, add 50 μl of anti-rabbit IgG FC HRP (Sino Biological) diluted 5000-fold, and incubate at room temperature in the dark for 1 hour. Wash 5 times with PBST, add 50 μl of TMB solution for color development in the dark for 10 minutes, and then add 50 μl of TMB stop buffer (abcam) to terminate color development. Finally, place the ELISA plate in a microplate reader (BioTek) for reading values. Figure 3 The results showed that, like the positive control nanobody, the novel coronavirus N antibody in this example specifically binds only to the novel coronavirus N antigen and does not bind to N antigens of multiple different subtypes of influenza, with high binding specificity.

[0096] Among them, the positive control nanobody is also a nanobody targeting the coronavirus N antigen, and its amino acid sequence is shown as follows:

[0097] QVQLQESGGGLVQPGGSLRLSCAASGFTADYYTIGWFRQAPGKGREGVSCISGSG AGGETVYADSVKGRFTISRDSAKNTVYLQMSSLKPDDTAVYTCAASFRPNYMCYPGG GPDDYGYWGQGTQVTVSS (SEQ ID NO: 13)

[0098] Example 10: Broad-spectrum detection

[0099] Coat 50 ng of different mutant novel coronavirus N antigens (Sino Biological) in an ELISA plate, 50 μl per well; wash 3 times with PBST, add 2% BSA for blocking, and incubate at room temperature for 2 hours. Wash 3 times with PBST, add 0.4 μg / ml of the three antibodies in Example 7, 50 μl per well, repeat for 2 wells, and incubate at room temperature for 1 hour. Wash 5 times with PBST, add 50 μl of anti-rabbit IgG FC HRP (Sino Biological) diluted 5000-fold, and incubate at room temperature in the dark for 1 hour. Wash 5 times with PBST, add 50 μl of TMB solution for color development in the dark for 10 minutes, and then add 50 μl of TMB stop buffer (abcam) to terminate color development. Finally, place the ELISA plate in a microplate reader (BioTek) for reading values. Figure 4The results showed that the novel coronavirus N antibody in this example binds to the N antigens of the wild type, the Alpha novel coronavirus mutant strain (B.1.1.7) in the UK, the Beta novel coronavirus mutant strain (B.1.351) in South Africa, the Gamma novel coronavirus mutant strain (P.1) in Brazil, the Delta novel coronavirus mutant strain (B.1.617.2) in India, and the omicron novel coronavirus mutant strain (B.1.1.529) in South Africa. It binds to the N antigens of a wide range of different subtypes of novel coronavirus mutant strains, and is suitable for the detection of continuously mutating novel coronavirus strains.

[0100] Example 11: Sensitivity detection

[0101] Coat three kinds of antibodies in Example 7 on an ELISA plate, 50 μl per well; wash 3 times with PBST, add 2% BSA for blocking, incubate at room temperature for 2 hours, and wash 3 times with PBST. Dilute the novel coronavirus N antigen 2-fold starting from 3000 pg / ml for 6 gradients, add the diluted novel coronavirus N antigen to the ELISA plate wells, 50 μl per well, repeat 3 wells for each dilution concentration, and incubate at room temperature for 1 hour. Wash 5 times with PBST, add 50 μl of anti-His HRP (abcam) diluted 3000-fold, and incubate at room temperature in the dark for 1 hour. Wash 5 times with PBST, add 50 μl of TMB solution for color development in the dark for 10 minutes, and then add 50 μl of TMB stop buffer (abcam) to terminate the color development. Finally, place the ELISA plate in a microplate reader (BioTek) for reading.

[0102] Figure 5 The results showed that the 3 structural novel coronavirus N antibodies in this example showed a linear binding to the novel coronavirus N antigen between 93.75 - 3000 pg / ml, detected the novel coronavirus N antigen at the pg level, and had extremely high detection sensitivity, which could be applied to the enzyme-linked immunosorbent assay of the novel coronavirus N antigen.

Claims

1. An antibody or antigen-binding fragment targeting the coronavirus N antigen, characterized in that, It includes a heavy chain variable region, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that, The amino acid sequences of FR1, FR2, FR3, and FR4 of the framework region of the heavy chain variable region are shown as SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 respectively.

3. The antibody or antigen-binding fragment according to claim 1 or 2, characterized in that, It is selected from one or more of the following: (1) IgG antibody, F(ab')2, F(ab)2, Fab, scFv, dSFv, nanobody, and heavy chain antibody; (2) Monoclonal antibody or polyclonal antibody; (3) Monospecific antibody, bispecific antibody, or multispecific antibody.

4. The antibody or antigen-binding fragment according to claim 3, characterized in that, It is a nanobody, and its amino acid sequence is shown as SEQ ID NO:

8.

5. The antibody or antigen-binding fragment according to claim 3, characterized in that, It is a heavy chain antibody, including VHH and Fc, wherein the amino acid sequence of VHH is shown as SEQ ID NO:8; Preferably, the Fc is selected from human or rabbit IgG, IgA, IgD, IgE, and IgM; More preferably, the amino acid sequence of the heavy chain antibody is shown as SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:

12.

6. The antibody or antigen-binding fragment according to claim 5, characterized in that, The heavy chain antibody is a monovalent antibody or a bivalent antibody.

7. An isolated nucleic acid, characterized in that, The nucleic acid encodes the antibody or antigen-binding fragment according to any one of claims 1 to 6; preferably, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO:

9.

8. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid according to claim 7.

9. A transformant, characterized in that, The transformant contains the nucleic acid according to claim 7 or the recombinant expression vector according to claim 8.

10. A method for preparing an antibody or antigen-binding fragment targeting the coronavirus N antigen, characterized in that, Culturing the transformant according to claim 9 under suitable conditions gives the antibody or antigen-binding fragment.

11. A kit, characterized in that, The kit contains the antibody or antigen-binding fragment according to any one of claims 1 to 6, the nucleic acid according to claim 7, the recombinant expression vector according to claim 8, and / or the transformant according to claim 9.

12. A method for detecting coronavirus for non-diagnostic purposes, characterized in that, Contacting the sample with the antibody or antigen-binding fragment according to any one of claims 1 to 6, and quantitatively or qualitatively determining whether the coronavirus is present in the sample according to the binding situation between the antibody or antigen-binding fragment and the sample.

13. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 6, a nucleic acid according to claim 7, a recombinant expression vector according to claim 8 or a transformant according to claim 9 in the preparation of a reagent for detecting coronavirus.

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