A sars-cov-2-n nanobody and derivative proteins and uses thereof

Abstract

This invention belongs to the field of immunology and relates to SARS-CoV-2-N nanobodies, their derived proteins, and applications. The single-domain antibody is composed of a heavy chain, which includes heavy chain CDR1, CDR2, and CDR3; the amino acid sequences of heavy chain CDR1, CDR2, and CDR3 are combinations of sequences (a) or (b) or sequences with high homology. This invention uses bioengineering technology to screen SARS-CoV-2-N nanobodies. These antibodies exhibit significant initial affinity and good binding activity upon prokaryotic expression, making them suitable for SARS-CoV-2 detection.

Description

Technical Field

[0001] This invention belongs to the field of immunology and relates to a SARS-Cov-2-N nanobody, its derived protein, and its applications. Background Technology

[0002] Coronaviruses are enveloped, asegmented, positive RNA viruses belonging to the family Coronaviridae and order Nidovirales. They are the largest known positive-sense RNA viruses, with a genome length of 26,000-32,000 bp. Mature coronaviruses have a diameter of 60-220 nm. They are named coronaviruses because they appear corona-like or crown-like under an electron microscope. Beaudette and Hudson first isolated coronaviruses from birds in 1937. In 1965, Tyr-Rell et al. inoculated nasal irrigation fluid from patients with the common cold into human embryonic tracheal cells, detecting viral replication, and identified human coronaviruses in 1968. Based on serological and genomic characteristics, the Coronavirus subfamily is now divided into four genera: α, β, γ, and δ. β-coronaviruses are further divided into four lineages: A, B, C, and D. Coronaviruses have been found in several avian hosts and various mammals, including camels, bats, masked palm civets, rats, pangolins, dogs, and cats. Coronaviruses are frequently discovered in new mammals and can cause respiratory, intestinal, liver, and nervous system infections in both animals and humans. The severity of illness varies among different animal species. Human colds caused by coronaviruses often occur in winter and early spring. Because the RNA polymerases of coronaviruses are prone to mutation, leading to high RNA recombination rates, coronaviruses not only exhibit various phenotypes and genotypes but also easily generate new variants that can adapt to new hosts and microclimates, thus triggering new human coronavirus infection outbreaks. Currently, the following seven coronaviruses are known to cause human infection: human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the 2019 novel coronavirus (2019-nCoV).

[0003] Since the emergence of the novel coronavirus (SARS-CoV-2, previously known as 2019-nCoV) in late 2019, the development of the epidemic has attracted global attention. On February 11, 2020, the World Health Organization officially named the virus causing COVID-19 SARS-CoV-2. Increasing evidence suggests a link between the 2019 novel coronavirus and other known bat coronaviruses, specifically the horseshoe bat (Phinolophusbat). It is currently the seventh known coronavirus capable of infecting humans, exhibiting high infectivity and high latency, and is clinically challenging to treat. SARS-CoV-2 belongs to the β-coronavirus genus, and its genetic material is positive-sense single-stranded RNA. The RNA genome contains 29,891 nucleotides and shares 82% homology with the SARS coronavirus (SARS-CoV) of 2003. Currently, most patients present with fever, fatigue, and dry cough, while a few patients also experience symptoms such as nasal congestion, runny nose, sore throat, and diarrhea. Severe patients often develop dyspnea and hypoxemia one or two weeks after the onset of illness. In severe cases, the condition can rapidly progress to acute respiratory distress syndrome, septic shock, or even uncorrectable metabolic acidosis and coagulation disorders.

[0004] Currently, research institutions both domestically and internationally have extensively conducted research on neutralizing antibodies against the novel coronavirus and antibodies against cytokine storms. Their research and development strategies mainly focus on the S protein, that is, using neutralizing antibodies targeting the S protein or ACE2 protein to bind to the S protein or ACE2 receptor on the surface of viral particles to block the binding of the S protein to ACE2, thereby blocking the virus from entering cells. However, the S protein is easily mutated by environmental stress, and the variants of the novel coronavirus that have appeared around the world are caused by mutations in the novel coronavirus S protein.

[0005] Studies have confirmed that screened N protein antibodies can activate the body's primary immune response to viral infection, similar to S protein antibodies, and are superior to S protein antibodies in the secondary immune response. Sequencing studies also show that N protein antibodies have a higher mutation frequency, enabling them to stimulate a stronger immune response in the host than S protein antibodies. The structure of the N protein is highly similar across different coronaviruses and different SARS-CoV-2 variants. Due to its conserved nature, the N protein has remained conserved throughout the evolution and mutation of the novel coronavirus. The fact that the N protein is encapsulated within the virus also protects it from environmental stresses that could lead to changes in the S protein. The nucleocapsid protein (N protein) of the novel coronavirus is the most expressed protein after SARS-CoV-2 infection. It is located in the core of the viral particle during viral assembly, existing in a form that binds to genomic RNA and interacts with RNA during viral RNA transcription and replication. However, the N protein is more conserved than other proteins such as S and M. The N protein can induce both humoral and cellular immune responses.

[0006] The SARS-CoV-2 viral N protein is 419 amino acids long and consists of an N-terminal domain (NTD), a C-terminal domain (CTD), and three random flexible regions (IDRs) connecting the two domains and their flanking sides. The NTD is a monomer, while the CTD forms a homodimer. Both the NTD and CTD contain relatively conserved positively charged regions for binding RNA. The N protein is a core component of SARS-CoV-2; it binds to the viral genomic RNA, packaging it into a ribonucleoprotein (RNP) complex. Besides assembly, the N protein also plays a crucial role in viral mRNA transcription and replication and participates in immune regulation.

[0007] Nanobodies are the newest and smallest antibody molecules developed by biomedical scientists by combining molecular biology techniques with the concepts of nanoparticle science, based on traditional antibodies. In 1993, Hamers-Cazterman et al. discovered a naturally occurring heavy chain antibody (HCAb) lacking the light chain in the serum of dromedary and Bactrian camels and South American camels. Cloning the variable region of the heavy chain antibody yielded a single-domain antibody consisting of only one heavy chain variable region, called a VHH antibody (variable domain of heavy chain of heavy-chain antibody). Its crystal structure is elliptical, with a diameter of 2.5 nm and a length of 4 nm. It is the smallest functional antigen-binding fragment and is also known as a nanobody. In 1993, Hamers et al. discovered that half of the antibodies in camel blood naturally lacked both the light chain and the constant region 1 (CH1) of the heavy chain. Cloning the variable region of the heavy chain of this antibody constructed a single-domain antibody (VHH) consisting of only one heavy chain variable region, which has now been renamed "nanobody." It is the smallest antigen-binding fragment with complete function, with a molecular weight of 15 kDa. The camel heavy chain antibody variable region nanobody (VHH) has a molecular weight of 15 kDa, much smaller than the Fab segment (60 kDa) and ordinary antibodies (150 kDa). The molecular structure of naturally occurring heavy chain antibodies in alpacas is as follows: two identical heavy chains are connected by interchain disulfide bonds formed in the hinge region. Each heavy chain molecule has a unique heavy chain variable region (VhH), a hinge region, and two constant regions, CH2 and CH3. In the IgG heavy chain molecule, the constant region CH1 is the site where it is linked to the light chain via interchain disulfide bonds. CH1 also exists in the genome of heavy chain antibodies, but it is cleaved during mRNA formation, resulting in camel heavy chain antibodies without light chains and the CH1 region.

[0008] Nanobodies are a rising star in the antibody field. Due to their small molecular weight, bivalent, trivalent, or bispecific antibodies can be obtained through simple molecular cloning techniques. Because of their small molecular size, nanobodies can achieve high yields in both prokaryotic expression systems (E. coli) and eukaryotic expression systems (CHO cells, 293 cells, etc.). The rapid development of nanobodies represents a powerful force with immense potential in antibody drug development, signifying an important direction for future antibody drug development.

[0009] Nanobodies exhibit high affinity and good penetration ability for their target binding sites, making them easier to bind to receptors and penetrate into tissues with low blood vessels. Furthermore, given the low immunogenicity of nanobodies, repeated administration to mice did not induce any humoral or cellular immunity. Additionally, nanobodies can be used to construct various molecular structures, enabling molecular adjuvant therapy. Currently, there is a lack of SARS-CoV-2-N nanobodies targeting the N protein in existing technologies. Summary of the Invention

[0010] To overcome the above-mentioned defects, the purpose of this invention is to provide a SARS-CoV-2-N nanobody, its derived protein, and its applications. SARS-CoV-2-N nanobodies specifically targeting the N protein of the novel coronavirus SARS-CoV-2 are screened using bioengineering technology. These antibodies show significant initial affinity and exhibit good binding activity upon prokaryotic expression, enabling effective detection of the novel coronavirus.

[0011] In a first aspect, the present invention provides a SARS-Cov-2-N nanobody, wherein the single-domain antibody is composed of a heavy chain, the heavy chain including heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;

[0012] The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(5):

[0013] (1) CDR1 shown in SEQ ID NO:31, CDR2 shown in SEQ ID NO:32, and CDR3 shown in SEQ ID NO:38;

[0014] (2) CDR1 shown in SEQ ID NO:27, CDR2 shown in SEQ ID NO:35, and CDR3 shown in SEQ ID NO:36;

[0015] (3) CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:33, and CDR3 shown in SEQ ID NO:37;

[0016] (4) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:34, and CDR3 shown in SEQ ID NO:39;

[0017] (5) CDR1 shown in SEQ ID NO:29, CDR2 shown in SEQ ID NO:34, and CDR3 shown in SEQ ID NO:40.

[0018] That is, the heavy chain includes a complementarity-determining region (CDR); the CDR includes the amino acid sequences of heavy chain CDR1, CDR2, and CDR3. The above CDR sequences (1)-(5) correspond to SEQ ID NO. 1-5 in sequence. All of the above sequences can be replaced with sequences that have "at least 80% homology" or sequences that replace only one or a few amino acids; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0019] In a preferred embodiment, the sequence of the single-domain antibody further includes a frame region FR; the frame region FR includes the amino acid sequences of FR1, FR2, FR3 and FR4;

[0020] The frame region FR sequence of the single-domain antibody is one of the following (a)-(e);

[0021] (a) FR1 shown in SEQ ID NO:12, FR2 shown in SEQ ID NO:18, FR3 shown in SEQ ID NO:21, FR4 shown in SEQ ID NO:26, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR;

[0022] (b) FR1 shown in SEQ ID NO:15, FR2 shown in SEQ ID NO:19, FR3 shown in SEQ ID NO:24, FR4 shown in SEQ ID NO:26, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR;

[0023] (c) FR1 shown in SEQ ID NO:13, FR2 shown in SEQ ID NO:17, FR3 shown in SEQ ID NO:20, FR4 shown in SEQ ID NO:25, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR;

[0024] (d) FR1 shown in SEQ ID NO:11, FR2 shown in SEQ ID NO:16, FR3 shown in SEQ ID NO:23, FR4 shown in SEQ ID NO:26, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR;

[0025] (e) FR1 shown in SEQ ID NO:14, FR2 shown in SEQ ID NO:16, FR3 shown in SEQ ID NO:22, FR4 shown in SEQ ID NO:26, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR.

[0026] In one embodiment, the single-domain antibody against SARS-Cov-2-N has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with an amino acid sequence selected from SEQ ID NO: 1-5, and is capable of specifically binding to the N protein of the SARS-Cov-2 virus.

[0027] In another preferred embodiment, the SARS-Cov-2-N nanobody has at least 95% sequence homology with the amino acid sequences selected from SEQ ID NO: 1-5 and is capable of specifically binding to the N protein of SARS-Cov-2.

[0028] A second aspect of the present invention is to provide nanobodies against SARS-CoV-2-N, said nanobodies being as shown in SEQ ID NO. 1-5, or said single-domain antibody having at least 95% sequence homology with the amino acid sequences of SEQ ID NO. 1-5.

[0029] In one embodiment, the nucleic acid molecule encoding the SARS-Cov-2-N nanobody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with nucleotide sequences selected from SEQ ID NO: 6-10, and the SARS-Cov-2-N nanobody encoded therein is capable of specifically binding to the N protein of SARS-Cov-2.

[0030] Preferably, the coding sequences of the nanobody are as shown in SEQ ID NO.6-10, or have at least 95% sequence homology with SEQ ID NO.6-10.

[0031] A third aspect of the present invention is to provide nucleotide molecules encoding the aforementioned SARS-Cov-2-N nanoantibodies, the nucleotide sequences of which are shown in SEQ ID NO: 6-10, or have at least 95% sequence homology with SEQ ID NO: 6-10.

[0032] A fourth aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding the aforementioned nanobody or the aforementioned nucleotide molecule.

[0033] A fifth aspect of the present invention is to provide a host cell that can express the aforementioned SARS-Cov-2-N nanobody, or an expression vector comprising the aforementioned nanobody.

[0034] The present invention also provides a method for generating SARS-Cov-2-N nanobodies, comprising the steps of: (a) culturing the aforementioned host cells under conditions suitable for nanobodies to obtain SARS-Cov-2-N nanobodies; (b) isolating or recovering the SARS-Cov-2-N nanobodies from the culture; and (c) optionally purifying and / or modifying the SARS-Cov-2-N nanobodies obtained in step (b).

[0035] The present invention also provides the use of the aforementioned SARS-Cov-2-N nanobody for the preparation of reagents, detection plates or kits; wherein the reagents, detection plates or kits are used to detect the presence and / or content of SARS-Cov-2 virus in a sample.

[0036] The single-domain antibody described is VHH, which contains only the antibody heavy chain and not the antibody light chain. In this article, single-domain antibody refers to nanobody.

[0037] Compared to existing technologies, this invention uses bioengineering technology to screen SARS-CoV-2-N nanobodies. These antibodies exhibit significant initial affinity and demonstrate good binding activity upon prokaryotic expression. These nanobodies offer the following advantages:

[0038] (1) The expression system of these nanobodies is flexible. They can be expressed in prokaryotic systems as well as in eukaryotic systems of yeast cells or mammalian cells. Moreover, the expression cost in prokaryotic systems is low, which can reduce the production cost in the later stage.

[0039] (2) Since nanobodies are single-domain antibodies, it is easier to modify them into multiple combinations. Multivalent and multispecific antibodies can be obtained by simply tandem through genetic engineering.

[0040] (3) As reported in many literatures, nanobodies have a wider affinity range. Before affinity maturation, their affinity range can be from nM to pM, providing multiple options for antibodies for different purposes in the later stage.

[0041] (4) It can be used to detect SARS-CoV-2 with high accuracy. Attached Figure Description

[0042] Figure 1 SDS-PAGE analysis of human recombinant SARS-CoV-2-N protein;

[0043] Figure 2 VHH sequence insertion rate analysis;

[0044] Figure 3Enrichment of libraries targeting SARS-CoV-2-N;

[0045] Figure 4 SDS-PAGE of partial prokaryotic expression antibody targeting SARS-CoV-2-N;

[0046] Figure 5 Antigen-binding activity of SARS-CoV-2-N target antibody. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0048] Single-domain antibodies (sdAbs, also referred to as nanobodies or VHHs by the developer Ablynx) are well known to those skilled in the art. A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Therefore, a single-domain antibody contains a single complementarity-determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies include antibodies containing only the heavy chain (which naturally does not contain a light chain), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0049] Single-domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species such as camels, dromedaries, llamas, and guanacos. Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. Single-domain antibodies may contain only variable domains of the immunoglobulin chain, which have CDR1, CDR2, and CDR3, as well as a frame region.

[0050] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% homology.

[0051] In this invention, sequences with high homology to the CDR1-3 sequences disclosed in this invention can also be used to obtain SARS-Cov-2-N nanobodies. In some embodiments, sequences with "at least 80% homology", or "at least 85% homology", "at least 90% homology", "at least 95% homology", or "at least 98% homology" with the sequences in (1)-(5) can achieve the purpose of the invention (i.e., derived proteins).

[0052] In some embodiments, the inventive objective can also be achieved by replacing only one or a few amino acids compared to the sequences in (1)-(5), for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art may consider so-called “conserved” amino acid substitutions, in which case the substitution will preferably be a conserved amino acid substitution, which can generally be described as an amino acid residue being replaced by another amino acid residue having a similar chemical structure, and the substitution having little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. The particularly preferred conserved amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. Furthermore, those skilled in the art will understand that the inventiveness of the single-domain antibody lies in the CDR1-3 regions, while the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0053] The preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0054] This patent involves preparing a target protein and a truncated form of the target protein using genetic engineering technology. The obtained antigen protein is then used to immunize Bactrian camels in Alashan, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. Through genetic engineering, the variable region coding sequence of the camel-derived antibody is recombined into a phage display vector. Specific antibodies against the antigen protein are screened using phage display technology, and their ability to bind to the antigen is further tested.

[0055] The above technical solution will now be broken down and explained in detail, and described with specific embodiments:

[0056] Example 1: Preparation of human recombinant SARS-CoV-2-N protein:

[0057] The human recombinant SARS-CoV-2-N protein used in this patent was expressed and purified by the company itself. The specific design scheme of the expression vector for the SARS-CoV-2-N protein is as follows:

[0058] (1) The coding sequence of SARS-Cov-2-N protein was obtained by searching in NCBI. Its nucleotide accession number is NC_045512.2, the amino acid sequence generated by this sequence is accession number YP_009724397, and the Uniprot ID is P0DTC9.

[0059] (2) Using sequence-specific primers, the nucleotide sequence encoding amino acids 1 to 419 of the SARS-Cov-2-N protein was cloned into the vector pcDNA3.4 using restriction endonucleases XbaI and AgeI.

[0060] (3) The constructed vector was subjected to Sanger sequencing. After comparing with the original sequence and confirming that there were no errors, the recombinant plasmid was extracted in batches, endotoxin was removed, and it was transfected into suspension 293F for expression and purification of the target protein (SARS-CoV-2-N protein). The purified human recombinant SARS-CoV-2-N protein was analyzed by SDS-PAGE as follows: Figure 1 As shown. By Figure 1 It can be seen that the purified protein has a purity of up to 90%, which meets the immune requirements of animals.

[0061] Example 2: Construction of a single-domain antibody library for the SARS-CoV-2-N protein

[0062] 1 mg of the purified human recombinant SARS-CoV-2-N protein obtained in step 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alashan, Inner Mongolia. The camel was immunized once a week for a total of 7 weeks. Except for the first immunization, the remaining six immunizations were performed by mixing 1 mg of SARS-CoV-2-N protein with an equal volume of Freund's incomplete adjuvant. This immunization process was intended to concentrate the stimulation of the camel to produce antibodies against SARS-CoV-2-N protein.

[0063] After animal immunization, 150 mL of peripheral blood lymphocytes were collected from camels, and RNA was extracted from the cells. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified using nested PCR with the cDNA as a template.

[0064] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments were ligated to the vector. The ligated fragments were electroporated into competent TG1 cells to construct a phage display library of the SARS-Cov-2-N protein, and the library size was determined to be approximately 1 × 10⁻⁶. 9 Simultaneously, the correct insertion rate of the target fragment in the library was detected by colony PCR identification, and the results are as follows: Figure 2 As shown.

[0065] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 28 clones were able to amplify a band of 1110 bp (predicted size), while 2 clones amplified an incorrect band. Therefore, the correct insertion rate was 28 ÷ 30 × 100% ≈ 93.3%.

[0066] Example 3: Screening for single-domain antibodies against SARS-CoV-2-N protein

[0067] Take 200 μL of the recombinant TG1 cells from step 2 and culture them in 2×TY medium. During this period, add 40 μL of helper phage VCSM13 to infect the TG1 cells and culture them overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and collect the amplified phage by centrifugation.

[0068] 500 μg of SARS-CoV-2-N protein diluted in 100 mM pH 8.3 NaHCO3 was coupled onto an ELISA plate and incubated overnight at 4°C, with a negative control well included. The next day, 200 μL of 3% skim milk was added, and the plate was blocked at room temperature for 2 hours. After blocking, 100 μL of the amplified phage library (approximately 2 × 10⁻⁶) was added. 11 (1 phage particle), incubate at room temperature for 1 hour; after 1 hour, wash 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0069] Phages specifically binding to the SARS-CoV-2-N protein were dissociated using trypsin at a final concentration of 25 mg / mL and then used to infect *E. coli* TG1 cells in logarithmic growth phase. The cells were cultured at 37°C for 1 hour, and the resulting phages were collected for the next round of screening. This screening process was repeated once to gradually enrich the cells. When the enrichment factor reached 10-fold or more, the enrichment effect was as follows: Figure 3 As shown.

[0070] Figure 3 In this context, P / N = the number of monoclonal bacteria grown from phages eluted from positive wells in the biopanning process after infecting TG1 bacteria / the number of monoclonal bacteria grown from phages eluted from negative wells after infecting TG1 bacteria. This parameter gradually increases after enrichment occurs. I / E = the total number of phages added to positive wells in each round of the biopanning process / the total number of phages eluted from positive wells in each round of the biopanning process. This parameter gradually approaches 1 after enrichment occurs.

[0071] Example 4: Enzyme-linked immunosorbent assay (ELISA) screening for specific positive clones against SARS-CoV-2-N

[0072] According to the screening method in Example 3 above, single-domain antibodies against SARS-Cov-2-N protein were screened for three rounds. The phage enrichment factor against SARS-Cov-2-N protein reached more than 10. After screening, 384 single colonies were selected from the positive clones and inoculated into 96-well plates of 2×TY medium containing 100 μg / mL ampicillin. A blank control was set up. After incubation at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and incubated overnight at 28°C.

[0073] Crude antibodies were obtained using the osmotic burst method. SARS-CoV-2-N recombinant protein was released into 100 mM NaHCO3 (pH 8.3), and 100 μg of protein was coated overnight at 4°C in an ELISA plate. 100 μL of the obtained crude antibody extract was transferred to an ELISA plate containing the antigen and incubated at room temperature for 1 h. Unbound antibodies were washed away with PBST, and 100 μL of Mouse Anti-HAtag Antibody (HRP) (mouse anti-HA horseradish peroxidase labeled antibody, ThermoFisher) diluted 1:2000 was added. The plate was incubated at room temperature for 1 h. Unbound antibodies were washed away with PBST, and horseradish peroxidase chromogenic solution was added. The reaction was carried out at 37°C for 15 min, and then stop solution was added. The absorbance was read at 450 nm using an ELISA reader.

[0074] When the OD value of the sample well is more than 5 times that of the control well, it is determined to be a positive clone well. The bacteria in the positive clone well are transferred to LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0075] Gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while clones with different sequences were considered different clones. Finally, single-domain antibodies specifically targeting the SARS-CoV-2-N protein were obtained (nanobodies 1B2, 1C3, 1C11, 1E4, and 2B9, corresponding to SEQ ID NO. 1-5 respectively, and single-domain antibodies with sequences not shown 1A10, 1A6, 1B5, 1B7, 1C1, 1D7, 1F4, 1F7, 1H4, 2A2, 2C12, 2C3, 2D2, 2E2, 2F11, 1C4, 1C7, 1D2, and 1D5).

[0076] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, constituting the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system to ultimately obtain the single-domain antibody protein.

[0077] The CDR, FR, and amino acid sequences of the five single-domain antibodies are shown in Tables 1, 2, and 3, respectively.

[0078] Table 1. CDR sequences of five single-domain antibodies

[0079] Actual clone number CDR1 SEQ ID 1C3 GFSFDTSY SEQ ID NO:27 1C11 GGTFSRNC SEQ ID NO:28 2B9 GYIFRHYY SEQ ID NO:29 1E4 GYITRHYY SEQ ID NO:30 1B2 GYTYTSGC SEQ ID NO:31

[0080]

[0081] Actual clone number CDR3 SEQ ID 1C3 AADGISRCTVVRGVLRRHGY SEQ ID NO:36 1C11 AADLNRRWGGPY SEQ ID NO:37 1B2 AAGRTPYELASGGKN SEQ ID NO:38 1E4 AASTIPGAYDTPWLSRRQYNF SEQ ID NO:39 2B9 AASTVPGAYGTWWLSRRQYNY SEQ ID NO:40

[0082] Table 2. FR sequences of five single-domain antibodies

[0083] Actual clone number FR1 SEQ ID 1E4 ESGGGPVQAGGSLRLSCSVP SEQ ID NO:11 1B2 ESGGGSVQAGGSLKLSCVAF SEQ ID NO:12 1C11 ESGGGSVQAGGSLRLSCAAS SEQ ID NO:13 2B9 ESGGGSVQAGGSLRLSCEVP SEQ ID NO:14 1C3 ESGGGSVQAGGSLRLSCTAS SEQ ID NO:15

[0084]

[0085] Actual clone number FR3 SEQ ID 1C11 AYANSVKGRFTISKDDDKNTVYLQMDSLKPEDTAMYYC SEQ ID NO:20 1B2 AYANSVKGRFTISQDNAKNTVYLQMNSLKAEDSAMYYC SEQ ID NO:21 2B9 SYSESVKGRFTISKDNAKNILYLQMNSLKPEDTDMYYC SEQ ID NO:22 1E4 TYSDSVKGRFIISKDNVKNILYLQMNNLKPEDTDMYYC SEQ ID NO:23 1C3 YYVDSVKGRFTVSRDNAKNAVYLEMNNLKPDDTATYYC SEQ ID NO:24

[0086]

[0087] Table 3. Amino acid sequences of five single-domain antibodies

[0088]

[0089] Example 5: Purification and expression of single-domain antibody against SARS-Cov-2-N protein in host Escherichia coli.

[0090] The plasmids (pMECS-VHH) of different clones obtained from the sequencing analysis in Example 4 were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated overnight at 37°C. Single colonies were selected and inoculated into 5 mL of LB medium containing penicillin and incubated overnight on a shaker at 37°C.

[0091] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium and incubate at 37°C in a shaker. When the OD600nm value reaches 0.6-0.9, add 1 MIPTG and incubate overnight at 28°C in a shaker. Centrifuge to collect E. coli and obtain crude antibody extract using the osmotic rupture method.

[0092] The antibody was purified by nickel column affinity chromatography. The purified single-domain antibody, such as... Figure 4 As shown, it includes VHH1 to 20. Figure 4 VHH4, 9, 15, 17, and 20 correspond to single-domain antibodies 1B2, 1C3, 1C11, 1E4, and 2B9, respectively. The sequences of the remaining single-domain antibodies are not shown (as they are single-domain antibodies whose technical effects are not good enough or do not need to be protected in this application).

[0093] Example 6: Determination of the binding dose-response curve of a specific single-domain antibody against SARS-CoV-2-N protein

[0094] (1) Coat 50 μL of 1 μg / mL SARS-COV-2-N and incubate overnight at 4℃.

[0095] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 1 hour.

[0096] (3) Dilute VHH to 2 μg / mL, and then perform a 5-fold serial dilution of the antibody to a total of 8 concentration gradients. Here, VHH refers to the 1B2, 1C3, 1C11, 1E4, and 2B9 nanobodies prepared in Example 5, as well as the remaining nanobodies with sequences not shown.

[0097] (4) Wash the plate; add 50 μL of nanobody diluted in step (3), double replicates, and incubate at 37°C for 1 h.

[0098] (5) Wash the plate; add 50 μL of mouse anti-HA tag HRP secondary antibody and incubate at 37°C for 30 min.

[0099] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought back to room temperature, and react at room temperature in the dark for 15 min.

[0100] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0101] (8) Plot the curve and calculate EC50, as follows: Figure 5 As shown in Table 4, the five SARS-COV-2-N nanoantibodies of the present invention exhibit excellent binding efficacy and specificity against the N protein of the novel coronavirus.

[0102] Table 4 EC50 values ​​of various nanobodies

[0103]

[0104] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A SARS-CoV-2-N nanobody, characterized in that: The nanobody is composed of heavy chains, including heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The amino acid sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are as follows (a) or (b): (a) CDR1 shown in SEQ ID NO:29, CDR2 shown in SEQ ID NO:34, and CDR3 shown in SEQ ID NO:40; (b) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:34, and CDR3 shown in SEQ ID NO:

39.

2. The SARS-CoV-2-N nanobody according to claim 1, characterized in that: The SARS-Cov-2-N nanobody has at least 95% sequence homology with an amino acid sequence selected from SEQ ID NO: 5 or 4, and is capable of specifically binding to the N protein of the SARS-Cov-2 virus.

3. The SARS-CoV-2-N nanobody according to claim 1, characterized in that: The amino acid sequence of the framework region FR of the nanobody is as follows: FR1 shown in SEQ ID NO:14, FR2 shown in SEQ ID NO:16, FR3 shown in SEQ ID NO:22, FR4 shown in SEQ ID NO:26, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR; or, FR1 shown in SEQ ID NO:11, FR2 shown in SEQ ID NO:16, FR3 shown in SEQ ID NO:23, FR4 shown in SEQ ID NO:26, or variants thereof, wherein the variants contain substitutions of up to 3 amino acids in the FR.

4. A SARS-CoV-2-N nanobody, characterized in that: The amino acid sequence of the nanobody is shown in SEQ ID NO. 5 or 4.

5. A SARS-CoV-2-N nanobody according to claim 4, characterized in that: The coding sequence of the nanobody is shown in SEQ ID NO.10 or 9.

6. A polynucleotide molecule encoding the SARS-CoV-2-N nanobody according to any one of claims 1-5, characterized in that: Its nucleotide sequence is shown in SEQ ID NO: 10 or 9.

7. An expression carrier, characterized in that, It comprises a polynucleotide molecule encoding the nanobody of any one of claims 1-5 or the polynucleotide molecule of claim 6.

8. A host cell, characterized in that, It can express the SARS-Cov-2-N nanobody as described in any one of claims 1-5, or the expression vector as described in claim 7.

9. The use of the SARS-CoV-2-N nanobody according to any one of claims 1-5, characterized in that, Used to prepare reagents, detection plates, or kits; wherein the reagents, detection plates, or kits are used to detect the presence and / or content of SARS-CoV-2 in samples.

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