SARS-CoV-2 BINDING PEPTIDE

Abstract

To provide a peptide that binds to N protein of SARS-CoV-2 and a method for using the same.SOLUTION: The present invention provides: a peptide binding to, with high reactivity, N protein of SARS-CoV-2, obtained using the cDNA display screening method, from a VHH antibody library with a construct including CDRs 1-3, consisting of a specific number of amino acids; a nucleic acid encoding the peptide; a method and a kit for detecting SARS-CoV-2; and a pharmaceutical including the peptide.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a peptide that binds to the nucleocapsid protein of SARS-CoV-2.

Background Art

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the genus Betacoronavirus, like SARS coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and is a SARS-related coronavirus that causes acute respiratory disease (COVID-19). Its occurrence was first confirmed near Wuhan City, Hubei Province, China in 2019, and has since caused a global pandemic of COVID-19.

[0003] The viral genome of SARS-CoV-2 is about 29,903 bases and is a single-stranded positive-strand RNA virus. The virus particles (virions) are about 50 to 200 nm in size. Similar to common coronaviruses, it is composed of four proteins known as spike protein, nucleocapsid protein, membrane protein, and envelope protein, and RNA. Among these, the nucleocapsid protein binds to RNA to form a nucleocapsid, and the spike protein, membrane protein, and envelope protein bound to lipids surround it to form an envelope (Non-Patent Documents 2 and 3).

[0004] The nucleocapsid protein of SARS-CoV-2 (hereinafter, N protein) is a protein that binds to the RNA genome of SARS-CoV-2. It has been reported to be involved in virus particle formation such as folding of the viral genome, assembly of virus proteins, and budding of the virus, and also has a function of suppressing the response of host cells (Non-Patent Document 4).

[0005] The virological diagnosis of COVID-19 primarily relies on the detection of SARS-CoV-2 genes using gene amplification (PCR). PCR testing is considered to be the most sensitive virus test currently available. Other detection methods include antigen tests, which detect fragments of proteins on the surface of the virus (e.g., spike proteins). Antigen test kits based on immunochromatography and ELISA are already commercially available. While generally less sensitive than PCR testing, they are superior in terms of speed and ease of use.

[0006] In viral testing, methods that eliminate false positives and false negatives are required. For false positives, a measurement principle with high specificity for the target molecule is crucial, while for false negatives, the detection sensitivity of the test method is important. Furthermore, even in bodily fluids (such as saliva and pharyngeal mucus) and sewage from SARS-CoV-2 infected individuals, some viral structures are present, albeit at low concentrations. Existing PCR and antigen tests may not be sufficient to detect these viruses. Therefore, while PCR and antigen tests for SARS-CoV-2 have become commonplace technologies, there is a strong need for the development of technologies that can detect SARS-CoV-2 more specifically and with higher sensitivity.

[0007] The N protein of SARS-CoV-2 has been reported to be one of the most numerous proteins in the virus (Non-Patent Literature 4). Because the number of N proteins is far greater than the spike proteins exposed on the surface of SARS-CoV-2, it is a preferred target molecule for SARS-CoV-2 antigen testing, particularly in terms of detection sensitivity. Furthermore, the N protein exists bound to the SARS-CoV-2 RNA genome as a nucleocapsid. Therefore, if a peptide that selectively and strongly binds to the N protein can be developed, for example, to enrich the SARS-CoV-2 N protein-RNA genome complex, it will be possible to detect SARS-CoV-2 with higher sensitivity even in PCR testing. Similarly, by combining the process of enriching the N protein with antigen testing, it will also be possible to increase the sensitivity of SARS-CoV-2 antigen testing. Furthermore, the N protein of SARS-CoV-2 is one of the proteins mainly expressed in infected cells and exhibits strong immunogenicity. ELISA methods for the N protein of SARS-CoV, which was prevalent from November 2002, have been reported to have high specificity, enabling the diagnosis of patients in the early stages of infection (Non-Patent Literature 5).

[0008] Conventional antibodies (such as IgG antibodies) bind to antigens using two variable regions: a heavy chain and a light chain. However, heavy chain antibodies found in the serum of camelid animals exhibit binding activity and antigen specificity using only one variable region. This variable region of the antibody is called VHH (Variable domain of Heavy chain of Heavy chain antibody), and it is considered to have the lowest molecular weight (about one-tenth that of conventional antibodies (such as IgG antibodies)) among immunoglobulin fragments that can bind to antigens (Non-patent Literature 6). Functional characteristics of VHH antibodies include: 1) having binding activity equal to or greater than that of conventional antibodies; 2) being able to penetrate regions inaccessible to conventional antibodies; 3) being highly resilient to the formation and regeneration of physiological structures; and 4) having a very simple structure because it is a single-chain peptide. In particular, due to reason 3), VHH antibodies are extremely stable against heat and pressure, and it is possible to mass-produce functional antibodies in prokaryotes (such as Escherichia coli, Bacillus subtilis, and Corynebacterium), which was previously difficult. Furthermore, for the reasons stated in 4), it is easy to modify its function through protein engineering and chemical modification, and it has the advantage of being easy to create antibody-drug conjugates (ADCs).

[0009] Thus, VHH antibodies have many advantages in terms of properties and production compared to conventional antibodies (such as IgG antibodies). Therefore, detection technology using VHH antibodies that target the SARS-CoV-2 N protein is expected to be more advantageous in terms of performance and cost than conventional technology using IgG antibodies. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020 Mar;579(7798):270-273. [Non-Patent Document 2] A new coronavirus associated with human respiratory disease in China. Nature. 2020 Mar;579(7798):265-269. [Non-Patent Document 3] The Coronavirus Nucleocapsid Is a Multifunctional Protein. Viruses. Volume 6, Issue 8, August 2014, Pages 2991-3018. [Non-Patent Document 4] The SARS-CoV nucleocapsid protein: A protein with multifarious activities. Infection, Genetics and Evolution. Volume 8, July 2008, Pages 397-405. [Non-Patent Document 5] Diagnosis of Severe Acute Respiratory Syndrome (SARS) by Detection of SARS Coronavirus Nucleocapsid Antibodies in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay. Journal of Clinical Microbiology. Volume 41, No. 12, December 2003, Pages 5781-5782. [Non-Patent Document 6] The Therapeutic Potential of Nanobodies. BioDrugs. Volume 34, November 2019, Pages 11-26. [Overview of the project] [Problems that the invention aims to solve]

[0011] This invention relates to providing a peptide that binds to the N protein of SARS-CoV-2 and a method for utilizing the same. [Means for solving the problem]

[0012] The inventors investigated how to obtain peptides that bind to the SARS-CoV-2 N protein. As a result, they succeeded in obtaining clones highly reactive to the SARS-CoV-2 N protein from a VHH antibody library containing CDR1-3 constructs with specific amino acid numbers, by screening using cDNA display.

[0013] In other words, the present invention relates to the following 1) to 5). 1) A peptide that binds to SARS-CoV-2, having one or more structural domains containing a CDR as shown in (a), (b), (c), (d), (e), (f), or (g) below. (a) CDR1 consisting of the amino acid sequence shown by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids are substituted with other amino acids, or an amino acid sequence in which 1 to 3 amino acids are deleted; CDR2 consisting of the amino acid sequence shown by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by PWGY (SEQ ID NO: 3), or an amino acid sequence in which 1 amino acid is substituted with another amino acid. (b) CDR1 consisting of the amino acid sequence indicated by GFTFSHYFMH (SEQ ID NO: 4) or an amino acid sequence in which one to three amino acids in that sequence are substituted with other amino acids, CDR2 consisting of the amino acid sequence indicated by TIGSSSTF (SEQ ID NO: 5) or an amino acid sequence in which one amino acid in that sequence is substituted with other amino acids, and CDR3 consisting of the amino acid sequence indicated by TLQYYTGLHLNIVGT (SEQ ID NO: 6) (c) CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7) or an amino acid sequence in which one to ten amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8) or an amino acid sequence in which one or two amino acids in said amino acid sequence are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids. (d) CDR1 consisting of the amino acid sequence indicated by GRAFSSYMMG (SEQ ID NO: 10) or an amino acid sequence in which one to five amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence indicated by AIGWSTY (SEQ ID NO: 11) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids; and CDR3 consisting of the amino acid sequence indicated by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence shown by GFTFSVYWMF (SEQ ID NO: 13) or an amino acid sequence in which one to four amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence shown by VINPDGSSTH (SEQ ID NO: 14) or an amino acid sequence in which one or two amino acids in said amino acid sequence are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by GISSTMDMDPWISLHY (SEQ ID NO: 15) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids. (f) CDR1 consisting of the amino acid sequence shown by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence shown by GIRWNNVSTY (SEQ ID NO: 17) or an amino acid sequence in which one amino acid is substituted with another amino acid, and CDR3 consisting of the amino acid sequence shown by LALSSDRLGLYAY (SEQ ID NO: 18). (g) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids are substituted with other amino acids, or an amino acid sequence in which 1 amino acid is inserted; CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids are substituted with other amino acids; and CDR3 consisting of the amino acid sequence indicated by SGT, or an amino acid sequence in which 1 amino acid is substituted with other amino acids. 2) A nucleic acid that codes for the peptide in 1). 3) A method for detecting SARS-CoV-2 in a sample, comprising the step of contacting a test sample with a peptide that binds to SARS-CoV-2, having one or more structural domains containing CDRs as shown in (a), (b), (c), (d), (e), (f), or (g) below. (a) CDR1 consisting of the amino acid sequence shown by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids are substituted with other amino acids, or an amino acid sequence in which 1 to 3 amino acids are deleted; CDR2 consisting of the amino acid sequence shown by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by PWGY (SEQ ID NO: 3), or an amino acid sequence in which 1 amino acid is substituted with another amino acid. (b) CDR1 consisting of the amino acid sequence indicated by GFTFSHYFMH (SEQ ID NO: 4) or an amino acid sequence in which one to three amino acids in that sequence are substituted with other amino acids, CDR2 consisting of the amino acid sequence indicated by TIGSSSTF (SEQ ID NO: 5) or an amino acid sequence in which one amino acid in that sequence is substituted with other amino acids, and CDR3 consisting of the amino acid sequence indicated by TLQYYTGLHLNIVGT (SEQ ID NO: 6) (c) CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7) or an amino acid sequence in which one to ten amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8) or an amino acid sequence in which one or two amino acids in said amino acid sequence are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids. (d) CDR1 consisting of the amino acid sequence indicated by GRAFSSYMMG (SEQ ID NO: 10) or an amino acid sequence in which one to five amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence indicated by AIGWSTY (SEQ ID NO: 11) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids; and CDR3 consisting of the amino acid sequence indicated by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence shown by GFTFSVYWMF (SEQ ID NO: 13) or an amino acid sequence in which one to four amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence shown by VINPDGSSTH (SEQ ID NO: 14) or an amino acid sequence in which one or two amino acids in said amino acid sequence are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by GISSTMDMDPWISLHY (SEQ ID NO: 15) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids. (f) CDR1 consisting of the amino acid sequence shown by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence shown by GIRWNNVSTY (SEQ ID NO: 17) or an amino acid sequence in which one amino acid is substituted with another amino acid, and CDR3 consisting of the amino acid sequence shown by LALSSDRLGLYAY (SEQ ID NO: 18). (g) CDR1 consisting of the amino acid sequence represented by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids in the amino acid sequence are substituted with other amino acids, or an amino acid sequence in which 1 amino acid is inserted, CDR2 consisting of the amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids in the amino acid sequence are substituted with other amino acids, and CDR3 consisting of the amino acid sequence represented by SGT, or an amino acid sequence in which 1 amino acid in the amino acid sequence is substituted with other amino acids 4) A SARS-CoV-2 detection kit containing the peptide of 1). 5) A medicament containing the peptide of 1).

Advantages of the Invention

[0014] According to the present invention, a highly reactive anti-SARS-CoV-2 N protein binding peptide can be provided. Since the peptide of the present invention can bind universally to the N protein of mutant strains, by using the peptide, detection of SARS-CoV-2 including mutant strains, that is, rapid detection of acute respiratory disease (COVID-19) which is a SARS-CoV-2 infection, becomes possible.

Brief Description of the Drawings

[0015] [Figure 1] Alignment diagram of the amino acid sequences of EN2, EN3 and EN4 obtained by screening and the amino acid sequences of each CDR variant. Amino acids with a gray background indicate mutated amino acids. [Figure 2] Alignment diagram of the amino acid sequences of EN5, EN6, EN11 and EN12 obtained by screening and the amino acid sequences of each CDR variant. Amino acids with a gray background indicate mutated amino acids. [Figure 3] Detection of His-tagged EN2, EN3, EN4, EN5, EN6, EN11 and EN12 by SDS-PAGE. [Figure 4]Evaluation of the binding specificity of coronavirus N proteins EN2, EN3, EN4, EN5, EN6, EN11, and EN12 by ELISA method. [Figure 5] Evaluation of the binding specificity of EN2, EN3, EN4, EN5, EN6, EN11, and EN12 to the N protein of SARS-CoV-2 variant coronaviruses using ELISA. [Figure 6] Evaluation of the binding specificity of EN2, EN3, EN4, EN5, EN6, EN11, and EN12 to the N protein of SARS-CoV-2 variant coronaviruses using ELISA. [Figure 7] Evaluation of the binding specificity of EN2, EN3, EN4, EN5, EN6, EN11, and EN12 to the N protein of SARS-CoV-2 omicron strain coronavirus by ELISA method. [Modes for carrying out the invention]

[0016] The peptide that binds to SARS-CoV-2 according to the present invention (hereinafter referred to as "the peptide of the present invention") is a peptide having one or more structural domains containing CDR1 to CDR3 as shown in (a), (b), (c), (d), (e), (f), or (g) below. (a) CDR1 consisting of the amino acid sequence shown by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids are substituted with other amino acids, or an amino acid sequence in which 1 to 3 amino acids are deleted; CDR2 consisting of the amino acid sequence shown by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by PWGY (SEQ ID NO: 3), or an amino acid sequence in which 1 amino acid is substituted with another amino acid. (b) CDR1 consisting of the amino acid sequence indicated by GFTFSHYFMH (SEQ ID NO: 4) or an amino acid sequence in which one to three amino acids in that sequence are substituted with other amino acids, CDR2 consisting of the amino acid sequence indicated by TIGSSSTF (SEQ ID NO: 5) or an amino acid sequence in which one amino acid in that sequence is substituted with other amino acids, and CDR3 consisting of the amino acid sequence indicated by TLQYYTGLHLNIVGT (SEQ ID NO: 6) (c) CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7) or an amino acid sequence in which one to ten amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8) or an amino acid sequence in which one or two amino acids in said amino acid sequence are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids. (d) CDR1 consisting of the amino acid sequence indicated by GRAFSSYMMG (SEQ ID NO: 10) or an amino acid sequence in which one to five amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence indicated by AIGWSTY (SEQ ID NO: 11) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids; and CDR3 consisting of the amino acid sequence indicated by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence shown by GFTFSVYWMF (SEQ ID NO: 13) or an amino acid sequence in which one to four amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence shown by VINPDGSSTH (SEQ ID NO: 14) or an amino acid sequence in which one or two amino acids in said amino acid sequence are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by GISSTMDMDPWISLHY (SEQ ID NO: 15) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids. (f) CDR1 consisting of the amino acid sequence shown by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence shown by GIRWNNVSTY (SEQ ID NO: 17) or an amino acid sequence in which one amino acid is replaced by another amino acid, and CDR3 consisting of the amino acid sequence shown by LALSSDRLGLYAY (SEQ ID NO: 18) (g) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids are substituted with other amino acids, or an amino acid sequence in which 1 amino acid is inserted; CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids are substituted with other amino acids; and CDR3 consisting of the amino acid sequence indicated by SGT, or an amino acid sequence in which 1 amino acid is substituted with other amino acids.

[0017] SARS-CoV-2 is a SARS-related coronavirus that causes acute respiratory illness (COVID-19), and its viral genome is a single-stranded positive-sense RNA virus with approximately 29,903 nucleotides. The peptide of the present invention is a peptide that binds to SARS-CoV-2, and more specifically, a peptide that binds to the N protein of SARS-CoV-2.

[0018] The peptides of the present invention include peptides that have the ability to potentially bind to epitopes, and typically include antibodies or antibody fragments. "Antibodies" include IgA, IgD, IgE, IgG, IgM, as well as heavy chain antibodies, single chain antibodies, ScFv, single-domain antibodies containing the CDR3 sequence of heavy chain antibodies (hereinafter sometimes referred to as "VHH" or "VHH antibody"), aptamers that can bind to foreign substances such as pathogens that have entered the body, and modified or altered versions thereof.

[0019] The peptide of the present invention includes at least three complementarity determining regions (CDRs) and a plurality of framework regions. CDRs are also called hypervariable regions and refer to regions that include sequence-variable antigen recognition sites or random sequence regions. The relative positions of the CDRs are CDR1, CDR2, and CDR3 from the N-terminus. The framework regions (FRs) refer to regions in the variable region of an antibody molecule that exclude the complementarity determining regions, i.e., highly conserved regions, and may be located on both the 5' and 3' sides of CDR1 to CDR3.

[0020] One aspect of the peptide of the present invention is that in the structural domain, CDR1 consists of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which one to seven amino acids are substituted with other amino acids, or an amino acid sequence in which one to three amino acids are deleted. CDR2 consists of the amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2) or an amino acid sequence in which one to six amino acids in that amino acid sequence are substituted with other amino acids. CDR3 consists of an amino acid sequence represented by PWGY (Sequence ID 3) or an amino acid sequence in which one amino acid in that sequence is substituted with another amino acid.

[0021] Another embodiment of the peptide of the present invention is that in the structural domain, CDR1 consists of the amino acid sequence represented by GFTFSHYFMH (SEQ ID NO: 4) or an amino acid sequence in which one to three amino acids in that amino acid sequence are substituted with other amino acids. CDR2 consists of the amino acid sequence shown in TIGSSSTF (Sequence ID 5) or an amino acid sequence in which one amino acid in that amino acid sequence is substituted with another amino acid. CDR3 consists of the amino acid sequence shown as TLQYYTGLHLNIVGT (SEQ ID NO: 6).

[0022] Another embodiment of the peptide of the present invention is that in the structural domain, CDR1 consists of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7) or an amino acid sequence in which one to ten amino acids in that amino acid sequence are substituted with other amino acids. CDR2 consists of the amino acid sequence represented by SITKSGDTN (Sequence ID 8) or an amino acid sequence in which one or two amino acids in that amino acid sequence are substituted with other amino acids. CDR3 consists of the amino acid sequence indicated by AVFRGITLYTY (SEQ ID NO: 9) or an amino acid sequence in which one amino acid in that sequence is substituted with another amino acid.

[0023] Another embodiment of the peptide of the present invention is that in the structural domain, CDR1 consists of the amino acid sequence shown by GRAFSSYMMG (SEQ ID NO: 10) or an amino acid sequence in which one to five amino acids in that amino acid sequence are substituted with other amino acids. CDR2 consists of the amino acid sequence shown by AIGWSTY (SEQ ID NO: 11) or an amino acid sequence in which one amino acid in that amino acid sequence is substituted with another amino acid. CDR3 consists of the amino acid sequence shown in NHRFTGRVY (SEQ ID NO: 12).

[0024] Another embodiment of the peptide of the present invention is that in the structural domain, CDR1 consists of the amino acid sequence represented by GFTFSVYWMF (SEQ ID NO: 13) or an amino acid sequence in which one to four amino acids in that amino acid sequence are substituted with other amino acids. CDR2 consists of the amino acid sequence indicated by VINPDGSSTH (SEQ ID NO: 14) or an amino acid sequence in which one or two amino acids in that amino acid sequence are substituted with other amino acids. CDR3 consists of the amino acid sequence represented by GISSTMDMDPWISLHY (SEQ ID NO: 15) or an amino acid sequence in which one amino acid in that amino acid sequence is substituted with another amino acid.

[0025] Another embodiment of the peptide of the present invention is that in the structural domain, CDR1 consists of the amino acid sequence shown by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consists of the amino acid sequence shown by GIRWNNVSTY (SEQ ID NO: 17) or an amino acid sequence in which one amino acid in that amino acid sequence is substituted with another amino acid. CDR3 consists of the amino acid sequence shown in LALSSDRLGLYAY (SEQ ID NO: 18).

[0026] Another embodiment of the peptide of the present invention is that in the structural domain, CDR1 consists of an amino acid sequence represented by GLTFRTYTMG (SEQ ID NO: 1), an amino acid sequence in which one to seven amino acids are substituted with other amino acids, or an amino acid sequence in which one amino acid is inserted. CDR2 consists of the amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2) or an amino acid sequence in which one to six amino acids in that amino acid sequence are substituted with other amino acids. CDR3 consists of an amino acid sequence represented by SGT or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with another amino acid.

[0027] A CDR consisting of an amino acid sequence in which some amino acids are substituted with other amino acids, an amino acid sequence in which some amino acids are deleted, or an amino acid sequence in which one amino acid is inserted, means a CDR in which a mutation has been introduced into the CDR consisting of said amino acid sequence. Furthermore, even if mutations have been introduced into CDR1, CDR2, and CDR3, as long as they have the ability to bind to SARS-CoV-2, they are included in the CDR of the peptide of the present invention.

[0028] Furthermore, while the type of amino acid to be substituted is not limited, examples of substitutions include those with similar polarity and size.

[0029] In CDR1, preferred combinations of the position of the amino acid to be substituted or deleted and the type of amino acid to be substituted are, for example, in CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, substitution of the second leucine residue with a glycine residue, arginine residue, phenylalanine residue, asparagine residue, tryptophan residue, lysine residue, serine residue, or glutamine residue, substitution of the third threonine residue with a leucine residue, lysine residue, alanine residue, asparagine residue, aspartic acid residue, arginine residue, or serine residue or Substitution to an isoleucine residue, substitution or deletion of the 4th phenylalanine residue to a leucine or methionine residue, substitution or deletion of the 5th arginine residue to an aspartic acid, serine, valine, glycine, or glutamic acid residue, substitution or deletion of the 6th threonine residue to a tyrosine, serine, alanine, valine, aspartic acid, isoleucine, or glycine residue, substitution of the 7th tyrosine residue to a leucine, asparagine, valine, or alanine residue, 8th threonine Examples include substitution of a rheonine residue with a serine, aspartic acid, alanine, methionine, valine, or asparagine residue; substitution of the 9th methionine residue with a threonine, valine, isoleucine, or leucine residue; and substitution of the 10th glycine residue with an arginine or alanine residue, or insertion of a proline residue before the 1st glycine residue. In CDR1, which consists of the amino acid sequence shown in SEQ ID NO: 4, substitution of the 2nd phenylalanine residue with an arginine residue, and the 3rd th One or more substitutions are selected from the following: substitution of a rheonine residue with alanine, substitution of the 5th serine residue with an isoleucine residue, substitution of the 6th histidine residue with serine, substitution of the 7th tyrosine residue with an aspartic acid residue, substitution of the 8th phenylalanine residue with a tryptophan residue, and substitution of the 10th histidine residue with a proline or leucine residue. In CDR1, which consists of the amino acid sequence shown in SEQ ID NO: 7, substitution of the 1st arginine residue with glycine, substitution of the 2nd serine residue with a phenylalanine or arginine residue,One or more substitutions are selected from the following: substitution of the 3rd isoleucine residue with a methionine, lysine, or alanine residue; substitution of the 4th serine residue with a phenylalanine or methionine residue; substitution of the 5th arginine residue with a serine or aspartic acid residue; substitution of the 6th phenylalanine residue with an isoleucine, tyrosine, or serine residue; substitution of the 7th asparagine residue with a tyrosine or serine residue; substitution of the 8th valine residue with an alanine or methionine residue; substitution of the 9th tryptophan residue with a leucine, methionine, or isoleucine residue; and substitution of the 10th alanine residue with a glycine residue. In CDR1, which consists of the amino acid sequence shown in Sequence ID No. 10, one or more substitutions are selected from the following: substitution of the 2nd arginine residue with a leucine or threonine residue; substitution of the 3rd alanine residue with a threonine or arginine residue; substitution of the 4th phenylalanine residue with a serine residue; and substitution of the 5th se In the case of CDR1, which consists of the amino acid sequence shown in Sequence ID No. 13, one or more substitutions are selected from the following: substitution of a phosphorus residue with an arginine residue, substitution of the 6th serine residue with a threonine or valine residue, substitution of the 7th tyrosine residue with a serine residue, substitution of the 8th methionine residue with a threonine or leucine residue, substitution of the 9th methionine residue with an arginine residue, and substitution of the 10th glycine residue with a valine residue. These amino acid sequence mutations (including substitutions, insertions, and deletions) in CDR1 originate from the amino acid sequences of CDR1 obtained through a screening process described in the examples, which selects VHHs that exhibit binding activity to the SARS-CoV-2 N protein.

[0030] In CDR2, preferred combinations of the position of the substituted amino acid and the type of substituted amino acid include, for example, in a CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, one or more selected from: substitution of the 1st alanine residue with an aspartic acid residue, a threonine residue, a valine residue, or a glycine residue; substitution of the 3rd threonine residue with a serine residue; substitution of the 4th proline residue with a tryptophan residue; substitution of the 5th serine residue with a glycine residue or an aspartic acid residue; substitution of the 7th glycine residue with an alanine residue; substitution of the 8th asparagine residue with a serine residue; and substitution of the 10th alanine residue with a tyrosine residue or a glycine residue. In a CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, a preferred combination is substitution of the 6th serine residue with an isoleucine residue or a glycine residue. In the CDR2 consisting of the amino acid sequence shown, one or more substitutions are selected from the following: substitution of the second isoleucine residue with a methionine residue, substitution of the fourth lysine residue with a methionine or isoleucine residue, and substitution of the eighth threonine residue with an alanine residue. In the CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11, one substitution is selected from the fifth serine residue with a glycine residue. In the CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, one or more substitutions are selected from the following: substitution of the fifth aspartic acid residue with a valine residue, substitution of the seventh serine residue with an arginine residue, substitution of the eighth serine residue with an asparagine residue, and substitution of the ninth threonine residue with a proline residue. In the CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, one substitution is selected from the third arginine residue with a serine residue. These amino acid sequence mutations (including substitutions, insertions, and deletions) in CDR2 originate from the amino acid sequences of CDR2 of VHH obtained through a screening process described in the examples, which selects VHH that exhibits binding activity to the SARS-CoV-2 N protein.

[0031] In CDR3, preferred combinations of the position of the substituted amino acid and the type of the substituted amino acid include, for example, the substitution of the third tryptophan residue with an arginine residue in the CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; the substitution of the third phenylalanine residue with serine in the CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9; the substitution of the fifth serine residue with an alanine residue in the CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15; and the substitution of the third threonine residue with an asparagine residue in the CDR3 consisting of the amino acid sequence shown in SGT. These amino acid sequence mutations (including substitutions, insertions, and deletions) in CDR3 originate from the amino acid sequences of CDR3 of VHH obtained by a screening process described in the examples, which selects VHH that exhibits binding activity to the SARS-CoV-2 N protein.

[0032] The binding ability to SARS-CoV-2 can be evaluated by methods known to those skilled in the art. Specifically, as shown in the examples described later, it can be evaluated by determining the equilibrium dissociation constant KD by surface plasmon resonance. It can also be evaluated by methods such as ELISA, immunochromatography, isothermal titration calorimetry, and biolayer interferometry.

[0033] The structural domain of the peptide of the present invention may have framework regions at both ends of CDR1, CDR2, and CDR3. The framework region refers to the region of the peptide molecule's variable region excluding the complementarity-determining region, and is a highly conserved region. In other words, one embodiment of the present invention includes a structural domain having a first framework region (FR1), CDR1, a second framework region (FR2), CDR2, a third framework region (FR3), CDR3, and a fourth framework region (FR4) in this order. The amino acid sequences of the framework region in the structural domain include the amino acid sequences shown below or amino acid sequences that have 80% or more identity with said amino acid sequences. FR1: EVQLVESGGGLVQAGGSQKLSCTTS (Sequence ID 19) EVQLVESGGGLVQPGGSLRLSCAAS (Sequence ID 20) EVQLVESGGGSVQAGGSLRLSCAAS (Sequence ID 21) EVQLVESGGGLVTAGGSLSLSCAAS (Sequence ID 22) EVQLVESGGGLVQPGGSLRLSCAAS (Sequence ID 23) EVQLVESGGGLVQPGGSLRLTCVAS (Sequence ID 24) FR2: WFRQAPGKEREFVA(Sequence ID 25) WVRQVPGKGLEWVS (Sequence ID 26) WYRQAPGKQRELVA (Sequence ID 27) WVRQAPGKGLEAVS(Sequence ID 28) FR3: YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRH (Sequence ID 29), YADSVKGRFTISRDNSKSTVYLQMNNLTPEDTAVYYCVG (Sequence ID 30), YAESVKGRFTISRDSAKRTVYLQMNSLKPEDTAVYYCNA (Sequence ID 31), YDESVKGRFTISRDNAKSTVYLQMNSLKPEDTAVYYCYA (Sequence ID 32), YGNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCIA (Sequence ID 33), YGDSVKGRFTISRDHAKNTVYLQMNSLKPEDTAVYYCYA(Sequence ID 34) YADSVKGRFHISRDKTKNTVDLFIRSLKPEDSGVYYCRA (Sequence ID 35) FR4: WGQGTQVTVSS (Sequence ID 36) WGQGTQVTVTS (Sequence ID 37) RGQGTQVTVSS (Sequence ID 38) WGPGTQVTVSE (Sequence ID 39)

[0034] Framework regions consisting of amino acid sequences having 80% or more identity with the amino acid sequences shown in SEQ ID NOs. 19-39 are preferably framework regions consisting of amino acid sequences having 85% or more identity, more preferably 90% or more identity, more preferably 95% or more identity, more preferably 96% or more identity, more preferably 97% or more identity, more preferably 98% or more identity, and more preferably 99% or more identity.

[0035] Here, amino acid sequence identity refers to the percentage (%) of the total number of amino acid residues at the positions where two amino acid sequences are identical when aligned. Sequence identity can be calculated, for example, by performing analysis using BLAST (Basic Local Alignment Search Tool) from the National Center for Biotechnology Information (NCBI).

[0036] Among the peptides of the present invention, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, and CDR1 is the amino acid sequence shown in SEQ ID NO: 1, CDR2 is the amino acid sequence shown in SEQ ID NO: 2, CDR3 is the amino acid sequence shown in SEQ ID NO: 3, and FR1 is the amino acid sequence shown in SEQ ID NO: 19, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 29, and FR4 is the amino acid sequence shown in SEQ ID NO: 36, and the peptide has a structural domain (SEQ ID NO: 41) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 4, CDR2 is the amino acid sequence shown in SEQ ID NO: 5, and CDR3 is the amino acid sequence shown in SEQ ID NO: 6, and FR1 is the amino acid sequence shown in SEQ ID NO: 20, FR2 is the amino acid sequence shown in SEQ ID NO: 26, FR3 is the amino acid sequence shown in SEQ ID NO: 30, and FR4 is the amino acid sequence shown in SEQ ID NO: 37, and the peptide has a structural domain (SEQ ID NO: 41) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 7, CDR2 is the amino acid sequence shown in SEQ ID NO: 8, and CDR3 is the amino acid sequence shown in SEQ ID NO: 9. , a peptide having a structural domain (SEQ ID NO: 42) in which FR1 is the amino acid sequence shown in SEQ ID NO: 21, FR2 is the amino acid sequence shown in SEQ ID NO: 27, FR3 is the amino acid sequence shown in SEQ ID NO: 31, and FR4 is the amino acid sequence shown in SEQ ID NO: 38, and CDR1 is the amino acid sequence shown in SEQ ID NO: 10, CDR2 is the amino acid sequence shown in SEQ ID NO: 11, and CDR3 is the amino acid sequence shown in SEQ ID NO: 12, and FR1 is the amino acid sequence shown in SEQ ID NO: 22, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 32, and FR4 is the amino acid sequence shown in SEQ ID NO: 36, and CDR1 is the amino acid sequence shown in SEQ ID NO: 13, CDR2 is the amino acid sequence shown in SEQ ID NO: 14, and CDR3 is the amino acid sequence shown in SEQ ID NO: 15, and FR1 is the amino acid sequence shown in SEQ ID NO: 23, FR2 is the amino acid sequence shown in SEQ ID NO: 28, FR3 is the amino acid sequence shown in SEQ ID NO: 33, and FR4 is the amino acid sequence shown in SEQ ID NO: 36,and a peptide having a structural domain (SEQ ID NO: 45) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 16, CDR2 is the amino acid sequence shown in SEQ ID NO: 17, CDR3 is the amino acid sequence shown in SEQ ID NO: 18, FR1 is the amino acid sequence shown in SEQ ID NO: 24, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 34, and FR4 is the amino acid sequence shown in SEQ ID NO: 36, and CDR1 is the amino acid sequence shown in SEQ ID NO: 1, CDR2 is the amino acid sequence shown in SEQ ID NO: 2, CDR3 is the amino acid sequence shown in SGT, and FR1 is the amino acid sequence shown in SEQ ID NO: 19 The peptide having an acid sequence, a structural domain (SEQ ID NO: 46) where FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 35, and FR4 is the amino acid sequence shown in SEQ ID NO: 39, is a clone (SEQ ID NO: 40 is EN2, SEQ ID NO: 41 is EN3, SEQ ID NO: 42 is EN4, SEQ ID NO: 43 is EN5, SEQ ID NO: 44 is EN6, SEQ ID NO: 45 is EN11, and SEQ ID NO: 46 is EN12) obtained by screening using the cDNA display method in the examples described later, and is a suitable SARS-CoV-2 binding peptide.

[0037] The peptide of the present invention is not limited in form as long as it has at least one of the above-described structural domains, and may be a single-domain antibody (also called a nanobody) such as a VHH antibody, a single-chain antibody, a heavy-chain antibody, a multimer formed by linking a variable region fragment with a peptide linker, for example, a dimer, or even a multimer formed by linking a variable region fragment with one or more variable region fragments having different antigen specificities. Furthermore, the peptides of the present invention may be humanized. Since humanized peptides can be administered to humans, they can be applied as pharmaceuticals.

[0038] The method for producing the peptide of the present invention is not particularly limited and can be easily produced by known techniques in the art. For example, it can be produced by combining peptide solid-phase synthesis and native chemical ligation (NCL) or by genetic engineering. However, a preferred method involves designing an artificial gene optimized for the expression of the target antibody in host cells by performing steps such as codon optimization on the nucleic acid encoding the peptide of the present invention, incorporating it into a suitable vector, introducing it into host cells, and producing it as a recombinant peptide.

[0039] Examples of host cells used in the production of recombinant peptides include Escherichia coli, Bacillus subtilis, fungi, animal cells, plant cells, baculovirus / insect cells, or yeast cells. Expression vectors suitable for various host cells can be used to express the peptides. Examples of expression vectors include E. coli-derived vectors such as pBR322, pBR325, pUC12, and pUC13; Bacillus subtilis-derived vectors such as pUB110, pTP5, and pC194; shuttle vectors that can be used in both E. coli and Bacillus subtilis, such as pHY300PLK; yeast-derived vectors such as pSH19 and pSH15; bacteriophages such as λ phage; viruses such as adenoviruses, adeno-associated viruses, lentiviruses, vaccinia viruses, and baculoviruses; and modified vectors thereof. These expression vectors have replication start sites, selection markers, and promoters suitable for each vector, and may optionally contain enhancers, transcription aggregation sequences (terminators), ribosome binding sites, and polyadenylation signals. Furthermore, the expression vectors may have nucleotide sequences inserted to facilitate the purification of the expressed polypeptides by fusing them with FLAG tags, His tags, HA tags, and GST tags.

[0040] When extracting the expressed peptide of the present invention from cultured bacterial cells or cultured cells, after culturing, the bacterial cells or cultured cells are collected by a known method, suspended in a suitable buffer, and then destroyed by sonication, lysozyme, and / or freeze-thaw cycles. A soluble extract is then obtained by centrifugation or filtration. The target peptide can then be obtained from the resulting extract by appropriately combining known separation and purification methods. Known separation and purification methods include methods that utilize solubility, such as salting out and solvent precipitation; methods that primarily utilize differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reverse-phase high-performance liquid chromatography; or methods that utilize differences in isoelectric point, such as isoelectric focusing electrophoresis.

[0041] Since the peptide of the present invention binds to the N protein of SARS-CoV-2, contacting it with a test sample that contains or may contain SARS-CoV-2 can confirm whether or not SARS-CoV-2 is present in the sample. Specifically, the detection of SARS-CoV-2 using the peptide of the present invention comprises the steps of: contacting the peptide of the present invention with a test sample to form a conjugate of the peptide of the present invention and SARS-CoV-2 in the test sample; and detecting the SARS-CoV-2 in the conjugate. Furthermore, the peptide of the present invention can also be used in the detection of virus-specific antibodies in serum by adding a portion of the antigen containing the SARS-CoV-2 N protein to the anti-SARS-CoV-2 antibody (e.g., serum antibody) contained in an immobilized test sample (serum) and binding it to it, thereby confirming the presence of the SARS-CoV-2 antigen in the conjugated state.

[0042] Test samples include biological samples such as tracheal swabs, nasal swabs, pharyngeal swabs, nasal lavage solutions, nasal aspirates, nasal discharge, saliva, sputum, blood, serum, urine, feces, tissues, cells, and tissue or cell lysates, as well as samples collected from solid surfaces that may have virus contamination, such as doorknobs and toilet bowls. From the viewpoint of binding to the N protein, it is preferable to dissolve the virus in the test sample in a solution containing a surfactant and then bind it to the peptide in the solution. The peptide may or may not be immobilized on a solid phase. The step of detecting SARS-CoV-2 in the above-mentioned conjugate can be carried out, for example, by reacting the above-mentioned conjugate with an anti-SARS-CoV-2 antibody that recognizes an epitope different from the peptide of the present invention in the conjugate. Alternatively, SARS-CoV-2 in the above-mentioned conjugate may be detected in the liquid phase by a homogeneous assay.

[0043] Furthermore, the peptide of the present invention can be a component of a SARS-CoV-2 detection kit. This kit can be used as a diagnostic agent for SARS-CoV-2 infection (COVID-19) and as a tool for the development of therapeutic agents for SARS-CoV-2 infection. The detection kit may include, in addition to the SARS-CoV-2 binding peptide of the present invention, reagents and equipment necessary for detection, such as an antibody that recognizes the peptide of the present invention or an N protein antibody that recognizes an epitope different from the peptide of the present invention, a solid support, a buffer, an enzyme reaction stop solution, a microplate reader, and the like. In the detection kit, the peptide of the present invention may be immobilized on a solid phase. Examples of solid phases include beads, membranes, the sides or bottom of a reaction vessel, plate-shaped substrates such as glass slides, and well substrates such as immunoplates, on which the peptide of the present invention is immobilized directly or indirectly.

[0044] Furthermore, it is conceivable that the peptide of the present invention, when introduced into cells by means of introducing a cell membrane-permeable peptide, binds to the N protein of SARS-CoV-2 and inhibits viral replication within cells. Therefore, the peptide of the present invention can also be administered to mammals and used as a pharmaceutical for the prevention or treatment of SARS-CoV-2 infection. Examples of mammals include humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, and pigs, but humans are preferred. When the peptide of the present invention is used as a pharmaceutical, it may be administered orally or parenterally, and can be used in combination with a well-known, pharmaceutically acceptable, and non-toxic carrier or diluent as appropriate. Parenteral administration typically involves injection, but administration by inhalation with a spray or the like is also possible.

[0045] In such pharmaceuticals, the content of the peptide of the present invention in the composition can be adjusted as appropriate. Furthermore, such pharmaceuticals can be administered to patients by administering an effective amount of the peptide of the present invention at intervals of one to several times per week. Preferred methods of administration in this case include intravenous injection and intravenous drip.

[0046] In relation to the embodiments described above, the following embodiments are further disclosed in the present invention. <1> A peptide that binds to SARS-CoV-2, having one or more structural domains containing a CDR as shown in (a), (b), (c), (d), (e), (f), or (g) below. (a) CDR1 consisting of the amino acid sequence shown by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids are substituted with other amino acids, or an amino acid sequence in which 1 to 3 amino acids are deleted; CDR2 consisting of the amino acid sequence shown by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by PWGY (SEQ ID NO: 3), or an amino acid sequence in which 1 amino acid is substituted with another amino acid. (b) CDR1 consisting of the amino acid sequence indicated by GFTFSHYFMH (SEQ ID NO: 4) or an amino acid sequence in which one to three amino acids in that sequence are substituted with other amino acids, CDR2 consisting of the amino acid sequence indicated by TIGSSSTF (SEQ ID NO: 5) or an amino acid sequence in which one amino acid in that sequence is substituted with other amino acids, and CDR3 consisting of the amino acid sequence indicated by TLQYYTGLHLNIVGT (SEQ ID NO: 6) (c) CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7) or an amino acid sequence in which one to ten amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8) or an amino acid sequence in which one or two amino acids in said amino acid sequence are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids. (d) CDR1 consisting of the amino acid sequence indicated by GRAFSSYMMG (SEQ ID NO: 10) or an amino acid sequence in which one to five amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence indicated by AIGWSTY (SEQ ID NO: 11) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids; and CDR3 consisting of the amino acid sequence indicated by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence shown by GFTFSVYWMF (SEQ ID NO: 13) or an amino acid sequence in which one to four amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence shown by VINPDGSSTH (SEQ ID NO: 14) or an amino acid sequence in which one or two amino acids in said amino acid sequence are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by GISSTMDMDPWISLHY (SEQ ID NO: 15) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids. (f) CDR1 consisting of the amino acid sequence shown by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence shown by GIRWNNVSTY (SEQ ID NO: 17) or an amino acid sequence in which one amino acid is replaced by another amino acid, and CDR3 consisting of the amino acid sequence shown by LALSSDRLGLYAY (SEQ ID NO: 18) (g) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids are substituted with other amino acids, or an amino acid sequence in which 1 amino acid is inserted; CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids are substituted with other amino acids; and CDR3 consisting of the amino acid sequence indicated by SGT, or an amino acid sequence in which 1 amino acid is substituted with other amino acids. <2> Binding to N-protein, <1> The peptide. <3> It is a VHH antibody, a heavy chain antibody, or a VHH antibody polymer. <1> or <2> The peptide. <4> The VHH antibody polymer is a polymer formed by linking multiple of the aforementioned structural domains. <3> The peptide. <5> The VHH antibody polymer is a dimer formed by linking two of the aforementioned structural domains. <3> The peptide. <6> The VHH antibody polymer is a polymer formed by linking one or more of the aforementioned structural domains with one or more structural domains that have different antigen specificity from the aforementioned structural domains. <3> The peptide.

[0047] <7> In CDR1, which consists of the amino acid sequence shown in SEQ ID NO: 1, any amino acid substitution or deletion is as follows: substitution of the second leucine residue with a glycine residue, arginine residue, phenylalanine residue, asparagine residue, tryptophan residue, lysine residue, serine residue, or glutamine residue; substitution of the third threonine residue with a leucine residue, lysine residue, alanine residue, asparagine residue, aspartic acid residue, arginine residue, serine residue, or isoleucine residue; or substitution of the fourth phenylalanine residue with a leucine residue or methionine residue. or deletion, substitution or deletion of the 5th arginine residue to an aspartic acid residue, serine residue, valine residue, glycine residue or glutamic acid residue, substitution or deletion of the 6th threonine residue to a tyrosine residue, serine residue, alanine residue, valine residue, aspartic acid residue, isoleucine residue or glycine residue, substitution of the 7th tyrosine residue to a leucine residue, asparagine residue, valine residue or alanine residue, or substitution of the 8th threonine residue to a serine residue, aspartic acid residue, alanine residue, methionine residue or valine residue or One or more amino acid substitutions in CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, an asparagine substitution, a threonine or valine or isoleucine or leucine substitution of the 9th methionine residue, and a arginine or alanine substitution of the 10th glycine residue or an insertion of a proline residue before the 1st glycine residue, or a substitution of the 2nd phenylalanine residue to an arginine residue, a 3rd threonine residue to an alanine, a 5th serine residue to an isoleucine residue, and a 6th phenylalanine One or more amino acid substitutions selected from the following: substitution of a sutidine residue with serine, substitution of the 7th tyrosine residue with an aspartic acid residue, substitution of the 8th phenylalanine residue with a tryptophan residue, and substitution of the 10th histidine residue with a proline or leucine residue, or the amino acid substitutions in CDR1 consisting of the amino acid sequence shown in SEQ ID NO: substitution of the 1st arginine residue with glycine, substitution of the 2nd serine residue with a phenylalanine or arginine residue, substitution of the 3rd isoleucine residue with a methionine, lysine or alanine residue,One or more amino acid substitutions in CDR1 consisting of the amino acid sequence shown in Sequence ID No. 10 are selected from the following: substitution of the 4th serine residue with a phenylalanine or methionine residue, substitution of the 5th arginine residue with a serine or aspartic acid residue, substitution of the 6th phenylalanine residue with an isoleucine, tyrosine, or serine residue, substitution of the 7th asparagine residue with a tyrosine or serine residue, substitution of the 8th valine residue with an alanine or methionine residue, substitution of the 9th tryptophan residue with a leucine, methionine, or isoleucine residue, and substitution of the 10th alanine residue with a glycine residue, or the amino acid substitution in CDR1 consisting of the amino acid sequence shown in Sequence ID No. 10 is a substitution of the 2nd arginine residue with a leucine or threonine residue, substitution of the 3rd alanine residue with a threonine or arginine residue, substitution of the 4th phenylalanine residue with a serine residue, substitution of the 5th serine residue with an arginine residue, and the 6th One or more amino acid substitutions in CDR1 consisting of the amino acid sequence shown in Sequence ID No. 13 are selected from the following: substitution of the serine residue of the first position with a threonine or valine residue, substitution of the 7th tyrosine residue with a serine residue, substitution of the 8th methionine residue with a threonine or leucine residue, substitution of the 9th methionine residue with an arginine residue, and substitution of the 10th glycine residue with a valine residue, or one or more amino acid substitutions in CDR1 consisting of the amino acid sequence shown in Sequence ID No. 13 are selected from the following: substitution of the 2nd phenylalanine residue with a glycine residue, substitution of the 3rd threonine residue with a proline or isoleucine residue, substitution of the 4th phenylalanine residue with a leucine residue, substitution of the 5th serine residue with an arginine residue, substitution of the 6th valine residue with a serine or alanine residue, substitution of the 7th tyrosine residue with an aspartic acid residue, substitution of the 9th methionine residue with an arginine residue, and substitution of the 10th phenylalanine residue with a tyrosine residue. <1> ~ <6> One of the following peptides. <8> The amino acid substitutions in the CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2 are one or more selected from the following: substitution of the 1st alanine residue with an aspartic acid residue, a threonine residue, a valine residue, or a glycine residue; substitution of the 3rd threonine residue with a serine residue; substitution of the 4th proline residue with a tryptophan residue; substitution of the 5th serine residue with a glycine residue or an aspartic acid residue; substitution of the 7th glycine residue with an alanine residue; substitution of the 8th asparagine residue with a serine residue; and substitution of the 10th alanine residue with a tyrosine residue or a glycine residue; or the amino acid substitutions in the CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5 are substitution of the 6th serine residue with an isoleucine residue or a glycine residue; or the amino acid substitutions in the CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 8 are One or more amino acid substitutions are selected from the following: substitution of the second isoleucine residue with a methionine residue, substitution of the fourth lysine residue with a methionine or isoleucine residue, and substitution of the eighth threonine residue with an alanine residue; or the amino acid substitution in the CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11 is a substitution of the fifth serine residue with a glycine residue; or the amino acid substitution in the CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14 is one or more selected from the following: substitution of the fifth aspartic acid residue with a valine residue, substitution of the seventh serine residue with an arginine residue, substitution of the eighth serine residue with an asparagine residue, and substitution of the ninth threonine residue with a proline residue; or the amino acid substitution in the CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17 is a substitution of the third arginine residue with a serine residue. <1> ~ <6> One of the following peptides. <9> The amino acid substitution in the CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3 is a substitution of the third tryptophan residue with an arginine residue, or the amino acid substitution in the CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9 is a substitution of the third phenylalanine residue with serine, or the amino acid substitution in the CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15 is a substitution of the fifth serine residue with an alanine residue, or the amino acid substitution in the CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 19 is a substitution of the third threonine residue with an asparagine residue. <1> ~ <6> One of the following peptides. <10> The structural domain has the following regions in this order: 1st framework region (FR1), CDR1, 2nd framework region (FR2), CDR2, 3rd framework region (FR3), CDR3, and 4th framework region (FR4). <1> ~ <9> One of the following peptides. <11> FR1~FR4 consists of the following amino acid sequence: <10> The peptide. FR1: The amino acid sequence shown in SEQ ID NOs. 19, 20, 21, 22, 23, or 24, or an amino acid sequence having 80% or more identity with said amino acid sequence. FR2: The amino acid sequence shown in Sequence ID No. 25, 26, 27, or 28, or an amino acid sequence having 80% or more identity with said amino acid sequence. FR3: The amino acid sequence shown in SEQ ID NOs. 29, 30, 31, 32, 33, 34, or 35, or an amino acid sequence having 80% or more identity with said amino acid sequence. FR4: The amino acid sequence shown in SEQ ID NOs. 36, 37, 38, or 39, or an amino acid sequence having 80% or more identity with said amino acid sequence. <12> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 1, CDR2 being the amino acid sequence shown in SEQ ID NO: 2, and CDR3 being the amino acid sequence shown in SEQ ID NO: 3. It also has a structural domain (SEQ ID NO: 40) where FR1 is the amino acid sequence shown in SEQ ID NO: 19, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 29, and FR4 is the amino acid sequence shown in SEQ ID NO: 36. <10> The peptide. <13> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 4, CDR2 being the amino acid sequence shown in SEQ ID NO: 5, and CDR3 being the amino acid sequence shown in SEQ ID NO: 6. It has a structural domain (SEQ ID NO: 41) where FR1 is the amino acid sequence shown in SEQ ID NO: 20, FR2 is the amino acid sequence shown in SEQ ID NO: 26, FR3 is the amino acid sequence shown in SEQ ID NO: 30, and FR4 is the amino acid sequence shown in SEQ ID NO: 37. <10> The peptide. <14> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 7, CDR2 being the amino acid sequence shown in SEQ ID NO: 8, and CDR3 being the amino acid sequence shown in SEQ ID NO: 9. It has a structural domain (SEQ ID NO: 42) where FR1 is the amino acid sequence shown in SEQ ID NO: 21, FR2 is the amino acid sequence shown in SEQ ID NO: 27, FR3 is the amino acid sequence shown in SEQ ID NO: 31, and FR4 is the amino acid sequence shown in SEQ ID NO: 38. <10> The peptide. <15> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 10, CDR2 being the amino acid sequence shown in SEQ ID NO: 11, CDR3 being the amino acid sequence shown in SEQ ID NO: 12, and FR1 being the amino acid sequence shown in SEQ ID NO: 22, FR2 being the amino acid sequence shown in SEQ ID NO: 25, FR3 being the amino acid sequence shown in SEQ ID NO: 32, and FR4 being the amino acid sequence shown in SEQ ID NO: 36, forming a structural domain (SEQ ID NO: 43). <10> The peptide. <16> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 13, CDR2 being the amino acid sequence shown in SEQ ID NO: 14, and CDR3 being the amino acid sequence shown in SEQ ID NO: 15. It also has a structural domain (SEQ ID NO: 44) where FR1 is the amino acid sequence shown in SEQ ID NO: 23, FR2 is the amino acid sequence shown in SEQ ID NO: 28, FR3 is the amino acid sequence shown in SEQ ID NO: 33, and FR4 is the amino acid sequence shown in SEQ ID NO: 36. <10> The peptide. <17> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 16, CDR2 being the amino acid sequence shown in SEQ ID NO: 17, and CDR3 being the amino acid sequence shown in SEQ ID NO: 18. It has a structural domain (SEQ ID NO: 45) where FR1 is the amino acid sequence shown in SEQ ID NO: 24, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 34, and FR4 is the amino acid sequence shown in SEQ ID NO: 36. <10> The peptide. <18> FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, with CDR1 being the amino acid sequence shown in SEQ ID NO: 1, CDR2 being the amino acid sequence shown in SEQ ID NO: 2, and CDR3 being the amino acid sequence shown in SGT. It has a structural domain (SEQ ID NO: 46) where FR1 is the amino acid sequence shown in SEQ ID NO: 19, FR2 is the amino acid sequence shown in SEQ ID NO: 25, FR3 is the amino acid sequence shown in SEQ ID NO: 35, and FR4 is the amino acid sequence shown in SEQ ID NO: 39. <10> The peptide. <19> <1> A nucleic acid that codes for peptides. <20> A method for detecting SARS-CoV-2 in a sample, comprising the step of contacting a test sample with a peptide that binds to SARS-CoV-2, having one or more structural domains containing CDRs as shown in (a), (b), (c), (d), (e), (f), or (g) below. (a) CDR1 consisting of the amino acid sequence shown by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids are substituted with other amino acids, or an amino acid sequence in which 1 to 3 amino acids are deleted; CDR2 consisting of the amino acid sequence shown by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by PWGY (SEQ ID NO: 3), or an amino acid sequence in which 1 amino acid is substituted with another amino acid. (b) CDR1 consisting of the amino acid sequence indicated by GFTFSHYFMH (SEQ ID NO: 4) or an amino acid sequence in which one to three amino acids in that sequence are substituted with other amino acids, CDR2 consisting of the amino acid sequence indicated by TIGSSSTF (SEQ ID NO: 5) or an amino acid sequence in which one amino acid in that sequence is substituted with other amino acids, and CDR3 consisting of the amino acid sequence indicated by TLQYYTGLHLNIVGT (SEQ ID NO: 6) (c) CDR1 consisting of the amino acid sequence shown by RSISRFNVWA (SEQ ID NO: 7) or an amino acid sequence in which one to ten amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence shown by SITKSGDTN (SEQ ID NO: 8) or an amino acid sequence in which one or two amino acids in said amino acid sequence are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by AVFRGITLYTY (SEQ ID NO: 9) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids. (d) CDR1 consisting of the amino acid sequence indicated by GRAFSSYMMG (SEQ ID NO: 10) or an amino acid sequence in which one to five amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence indicated by AIGWSTY (SEQ ID NO: 11) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids; and CDR3 consisting of the amino acid sequence indicated by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence shown by GFTFSVYWMF (SEQ ID NO: 13) or an amino acid sequence in which one to four amino acids in said amino acid sequence are substituted with other amino acids; CDR2 consisting of the amino acid sequence shown by VINPDGSSTH (SEQ ID NO: 14) or an amino acid sequence in which one or two amino acids in said amino acid sequence are substituted with other amino acids; and CDR3 consisting of the amino acid sequence shown by GISSTMDMDPWISLHY (SEQ ID NO: 15) or an amino acid sequence in which one amino acid in said amino acid sequence is substituted with other amino acids. (f) CDR1 consisting of the amino acid sequence shown by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence shown by GIRWNNVSTY (SEQ ID NO: 17) or an amino acid sequence in which one amino acid is replaced by another amino acid, and CDR3 consisting of the amino acid sequence shown by LALSSDRLGLYAY (SEQ ID NO: 18) (g) CDR1 consisting of the amino acid sequence indicated by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids are substituted with other amino acids, or an amino acid sequence in which 1 amino acid is inserted; CDR2 consisting of the amino acid sequence indicated by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids are substituted with other amino acids; and CDR3 consisting of the amino acid sequence indicated by SGT, or an amino acid sequence in which 1 amino acid is substituted with other amino acids. <21> <1> ~ <18> A SARS-CoV-2 detection kit containing one of the following peptides. <22> <1> ~ <18> A pharmaceutical product containing any of the following peptides. <23> For the prevention or treatment of SARS-CoV-2 infection <1> ~ <18> A pharmaceutical product containing any of the following peptides. [Examples]

[0048] (Example 1) cDNA preparation for VHH-presented cDNA display adjustment Construction of a DNA library encoding the single variable domain of the heavy chain of a heavy-chain antibody (VHH) from full-length alpaca-derived antibodies. Using two alpaca-derived naive VHH library genes, S-hinge and L-hinge, provided by RePHAGEN, as templates, the S-hinge VHH-specific primer pair (SEQ ID NOs. 47, 48) and L-hinge VHH-specific primer pair (SEQ ID NOs. 47, 49) were amplified by PCR. Then, to prepare a DNA fragment consisting of a T7 promoter, omega (ω) enhancer, Kozak consensus sequence, VHH gene, His tag, and linker hybridization region (Y tag), the fragments were extended by extension PCR using overlap PCR primers (SEQ ID NOs. 50, 51) to create a full-length VHH-coding DNA library.

[0049] (Example 2) Fabrication of a cDNA display Each cDNA display was prepared according to the following steps (1) to (5). The buffers used are listed in Table 1.

[0050] [Table 1]

[0051] (1) Transcription The full-length VHH-coding DNA library prepared in Example 1 was transcribed using a T7 RiboMAX Express Large Scale RNA Production System (Promega) according to the attached manual. The amount of DNA used was 6.6 μg. The amount of DNA used for each subsequent selection was 0.1 to 1 μg from the 2nd round onward. The obtained transcripts were purified using RNAClean XP (Beckman Coulter) according to the attached manual. The purified products were quantified by concentration using NanoPad DS-11FX (DeNovix).

[0052] (2) Ligation Purified 20 pmol mRNA and a 20 pmol puromycin linker, cnvK riboG linker (Epsilon Molecular Engineering), were mixed with NaCl (final concentration 0.2 M) and Tris-HCl (pH 7.5, final concentration 0.05 M) and incubated at 90°C for 1 minute. The mixture was then cooled to 70°C at a rate of 0.1°C / second and incubated at 70°C for 1 minute. Next, the mixture was cooled to 25°C at a rate of 0.1°C / second, and then to 10°C at a rate of 2°C / second to hybridize the cnvK linker to the 3' end of the mRNA. Subsequently, using a UVP CrossLinker (CL-3000), 365nm, 100-115V (Analytik Jena), 365nm UV was applied at 4060 mJ / cm². 2 Irradiation was performed to photocrosslink the cnvK linker and mRNA, obtaining an mRNA-linker complex.

[0053] (3) Preparation of mRNA display Six pmol of mRNA-linker complexes were incubated at 30°C for 30 minutes using a 50 μL cell-free translation system (Rabbit reticulocyte Lysate (nuclease-treated), Promega). MgCL2 and KCL were then added to final concentrations of 75 mM and 900 mM, respectively, and incubated at 37°C for 1 hour to display the mRNA-corresponding peptide on the puromycin on the mRNA-linker. Next, EDTA (pH 8.0) was added to a final concentration of 70 mM, and incubated at 4°C for 5 minutes to prepare the mRNA display.

[0054] (4) Preparation of cDNA display 60 μL of Dynabeads Myone streptavidin C1 (Thermo Fisher Scientific) was placed in a Protein Lobind Tube, washed with 200 μL of binding buffer, and then the mRNA display prepared by the methods of (1)-(4) above was added and stirred at 25°C for 30 minutes. After washing with 200 μL of binding buffer, a reverse transcription reaction was carried out by incubation at 42°C for 30 minutes in a reaction mixture with the composition shown in Table 2 to prepare mRNA / cDNA-VHH conjugates. After washing with 200 μL of binding buffer, 39 μL of His tag binding / wash buffer and 1 μL of 1,000 U / μL RNase T1 were added and stirred at 37°C for 15 minutes to elute the mRNA / cDNA-VHH conjugates (cDNA display) from Dynabeads Myone streptavidin C1.

[0055] [Table 2]

[0056] (5) Purification of cDNA display 30 μL of His Mag Sepharose Ni Beads (GE Health Care) were placed in an Eppendorf tube and washed with 200 μL of His tag binding / wash buffer. Then, the cDNA display prepared by the methods described in (1)-(5) above was added and the mixture was stirred at 25°C for 30 minutes. After washing with 200 μL of His tag binding / wash buffer, the cDNA display was eluted by adding 30 μL of His tag elution buffer and stirring at 37°C for 15 minutes.

[0057] (Example 3) Selection of VHH (1) In-vitro selection experiment Selection was performed using cDNA displays prepared from the libraries obtained in Example 2. The synthesis scale of the VHH-presenting cDNA display libraries used in each selection round is shown in Table 3.

[0058] [Table 3]

[0059] (1-1) Screening procedure for Selection Cycle 1 (R (Round) 1) SARS-CoV2 (2019-nCoV) Nucleocapsid-His Recombinant Protein, Biotinylated (SinoBiological) (hereinafter referred to as biotinylated N protein) was added to Dynabeads Myone streptavidin C1 and mixed by inversion for 30 minutes at 4°C to prepare SARS-CoV2 Nucleocapsid-coated beads. The cDNA display prepared in the same manner as in Example 2 on the synthesis scale shown in Table 3 was diluted to 200 μL with PBS-T containing 5 mg / mL heparin sodium, and 100 μL was added to the beads cohesive with 50 pmol of biotinylated N protein and mixed by inversion for 30 minutes at 4°C. The remaining 100 μL of cDNA display solution was mixed with 50 pmol of biotinylated N protein and mixed by inversion at 4°C for 30 minutes. The mixture was then added to Dynabeads Myone streptavidin C1 and mixed by inversion at 4°C for 30 minutes to immobilize the biotinylated N protein containing the cDNA display-N protein complex onto the beads. All beads were then combined and washed four times with PBS-T. The bound cDNA display was then collected and purified using AMpure XP (Beckman Coulter) according to the provided instructions. The purified solution was subjected to PCR using cnvK NewYtag for poly A (SEQ ID NO: 52) and T7 omeganew (SEQ ID NO: 53) as primers, under conditions of annealing temperature 65°C and extension reaction time 10 seconds using PrimeSTAR Max (Takara Bio). The PCR products were then analyzed by 4% denatured PAGE. The PCR products were purified using AMpure XP according to the accompanying manual, and the resulting purified R1 library was subjected to the second round of selection.

[0060] (1-2) Screening procedure for selection cycle 2 (R2) A cDNA display library was prepared from the R1 library using the same method as described above, according to the synthesis scale shown in Table 3. This library was diluted with 50 μL of PBS-T containing 5 mg / mL heparin sodium, added to 20 μL of Dynabeads Myone streptavidin T1 (Thermo Fisher Scientific), and mixed by inversion at 4°C for 30 minutes. The supernatant was mixed with 10 pmol of biotinylated N protein and mixed by inversion at room temperature for 60 minutes. The mixture was added to 20 μL of Dynabeads Myone streptavidin T1 and mixed by inversion at 4°C for 30 minutes to immobilize the biotinylated N protein containing the cDNA display-N protein complex onto beads. After washing the beads four times with 200 μL of PBS-T, the bound cDNA display was collected and purified using AMpure XP (Beckman Coulter) according to the accompanying manual. The purified solution was subjected to a PCR reaction under the same conditions as in Example 3 (1-1), and then purified using AMpure XP according to the attached manual to obtain the R2 library.

[0061] (1-3) Screening procedure for the selection cycle (R3) A cDNA display library was prepared from the R2 library using the same method as described above, according to the synthesis scale shown in Table 3. This library was diluted with 50 μL of PBS-T containing 5 mg / mL of heparin sodium, and added to 20 μL of Dynabeads Myone streptavidin C1 (Thermo Fisher Scientific). The mixture was inverted and mixed at 4°C for 30 minutes. Half of the supernatant was mixed with 10 pmol of biotinylated N protein and inverted and mixed at room temperature for 60 minutes. The mixture was added to 20 μL of Dynabeads Myone streptavidin T1, and the biotinylated N protein containing the cDNA display-N protein complex was immobilized onto beads by inverted and mixed at 4°C for 30 minutes. The beads were washed three times with 200 μL of PBS-T, then washed again by inverted and mixed with 20 μL of PBST at 25°C for 30 minutes. Subsequently, the bound cDNA display was recovered and purified using AMpure XP (Beckman Coulter) according to the attached manual. The purified solution was subjected to a PCR reaction under the same conditions as in Example 3 (1-1), and purified using AMpure XP according to the attached manual to obtain the R3 library.

[0062] (1-4) Screening procedure for Selection Cycle 4 (R4) A cDNA display library was prepared from the R3 library using the same method as described above, according to the synthesis scale shown in Table 3. This library was diluted in 50 μL of PBS-T containing 5 mg / mL heparin sodium, added to 20 μL of Dynabeads Myone streptavidin C1 (Thermo Fisher Scientific), and mixed by inversion at 4°C for 30 minutes. Half of the supernatant was mixed with 5 pmol of biotinylated N protein to form a Positine solution, and the remaining half was left as a Negative solution. Both solutions were mixed by inversion at room temperature for 30 minutes. Each mixture was added to 20 μL of Dynabeads Myone streptavidin T1, and the biotinylated N protein containing the cDNA display-N protein complex was immobilized onto beads by inversion at 4°C for 30 minutes. Each bead was washed three times with 200 μL of PBST containing 1 μM biotin, then 20 μL of PBST containing 1 μM biotin and 12.5 pmol biotinylated N protein was added and washed by inversion mixing at 25°C for 5 minutes. Subsequently, 20 μL of PBST containing 12.5 pmol biotinylated N protein was added and competitive elution was performed by inversion mixing at 25°C for 60 minutes. The bound cDNA display was then collected, and each solution was purified using AMpure XP (Beckman Coulter) according to the attached manual. Each purified solution was subjected to PCR reaction under the same conditions as in Example 3 (1-1), and purified using AMpure XP according to the attached manual to obtain the R4 library.

[0063] (2) Next-generation sequencer analysis To verify the convergence of the DNA library through in vitro selection cycles in detail, sequencing analysis was performed using a next-generation sequencer (NGS). The preparation of the sequencing samples followed the 2-step PCR Amplicon Library Preparation method provided by Illumina. First, using the PCR products after each selection as templates, Amplicon PCR was performed using NGS Fw 1st PCR primer (SEQ ID NO: 54) and NGS Rv 1st PCR primer (SEQ ID NO: 55) as primers. The PCR conditions were 98°C for 1 minute, followed by 98°C for 10 seconds, 62°C for 5 seconds, 72°C for 35 seconds (15 cycles), and then 72°C for 1 minute. After purifying the obtained PCR products according to the instructions for the Agentcourt AMPure XP, Index PCR was performed according to the instructions for Illumina. The obtained PCR products were purified according to the instructions for Agencourt AMPure XP, then their concentrations were quantified using NanoPad DS-11FX (DeNovix), and sequence analysis was performed using MiSeq (Illumina) and MiSeq Reagent Nano kit v2 (500 cycles) according to the instructions for Illumina.

[0064] The obtained DNA sequences were translated into VHH amino acid sequences and ranked by frequency of occurrence. As a result, Table 4 was selected as representative VHHs with high affinity to the N protein. Furthermore, analysis of the library after the R4 selection process revealed that VHHs containing amino acid sequence mutations (including substitutions, insertions, and deletions) were selected in the amino acid sequences of CDR1, CDR2, and CDR3 of EN2, EN3, EN4, EN5, EN6, EN11, and EN12 (these VHHs are called CDR variants). SEQ ID NOs. 77-98 were selected as CDR variants of EN2 (SEQ ID NO: 40), SEQ ID NOs. 99-106 as CDR variants of EN3 (SEQ ID NO: 41), SEQ ID NOs. 107-119 as CDR variants of EN4 (SEQ ID NO: 42), SEQ ID NOs. 120-126 as CDR variants of EN5 (SEQ ID NO: 43), SEQ ID NOs. 127-134 as CDR variants of EN6 (SEQ ID NO: 44), SEQ ID NOs. 135 as CDR variant of EN11 (SEQ ID NO: 45), and SEQ ID NOs. 136-150 as CDR variants of EN12 (SEQ ID NO: 46) (Figures 1 and 2). Since these CDR variants are VHHs selected through the selection process, they are considered highly likely to exhibit binding activity to the SARS-CoV-2 N protein.

[0065] [Table 4]

[0066] (Example 4) Production of VHH by protease-deficient recombinant Bacillus subtilis (1) Artificial synthesis of genes The amino acid sequences of the synthesized VHH were modified by adding a His tag sequence (SEQ ID NO: 56) via a linker sequence to the C-terminus of each VHH amino acid sequence listed in Table 4. The resulting synthesized VHHs are called His-tagged EN2 (SEQ ID NO: 57), His-tagged EN3 (SEQ ID NO: 58), His-tagged EN4 (SEQ ID NO: 59), His-tagged EN5 (SEQ ID NO: 60), His-tagged EN6 (SEQ ID NO: 61), His-tagged EN11 (SEQ ID NO: 62), and His-tagged EN12 (SEQ ID NO: 63). These VHHs represented by SEQ ID NOs. 56 to 63 are collectively referred to as His-tagged VHHs. To synthesize these His-tagged VHH cells, we synthesized the following artificial synthesis genes: His-tagged EN2 artificial synthesis gene (SEQ ID NO: 64), His-tagged EN3 artificial synthesis gene (SEQ ID NO: 65), His-tagged EN4 artificial synthesis gene (SEQ ID NO: 66), His-tagged EN5 artificial synthesis gene (SEQ ID NO: 67), His-tagged EN6 artificial synthesis gene (SEQ ID NO: 68), His-tagged EN11 artificial synthesis gene (SEQ ID NO: 69), and His-tagged EN12 artificial synthesis gene (SEQ ID NO: 70), and used them in experiments.

[0067] (2) Construction of a plasmid for VHH expression Recombinant plasmid pHY-S237 (JP 2014-158430 A), prepared based on pHY300PLK, was used as a template, and the plasmid sequence was amplified by PCR using the primer sets 5'-GATCCCCGGGAATTCCTGTTATAAAAAAAGG-3' (SEQ ID NO: 71) and 5'-ATGATGTTAAGAAAGAAAACAAAGCAG-3' (SEQ ID NO: 72) and PrimeSTAR Max DNA polymerase (TaKaRa). Promoter DNA derived from the spoVG gene was amplified by PCR using the primer sets 5'-GAATTCCCGGGGATCTAAGAAAAGTGATTCTGGGAGAG-3' (SEQ ID NO: 73) and 5'-CTTTCTTAACATCATAGTAGTTCACCACCTTTTCCC-3' (SEQ ID NO: 74) with the genomes of 168 strains as templates. The obtained promoter DNA was incorporated into the plasmid sequence using the In-Fusion HD Cloning Kit (Takara), and a VHH expression plasmid linked to the spoVG promoter was constructed. Plasmid sequences were amplified by PCR using the primer sets 5'-TGCTGCAAGAGCTGCCGGAAATAAA-3' (SEQ ID NO: 75) and 5'-TCTATTAAACTAGTTATAGGG-3' (SEQ ID NO: 76) and PrimeSTAR Max DNA polymerase (TaKaRa). DNA containing the synthetically synthesized gene was incorporated into the resulting PCR fragment using the In-Fusion HD Cloning Kit (Takara) to construct VHH expression plasmids containing each of the synthetically synthesized VHH genes.

[0068] (3) Preparation of recombinant Bacillus subtilis From 168 strains of Bacillus subtilis (hereinafter referred to as "168 strains"), knockout strains of extracellular protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, aprX) were prepared according to the method described in Japanese Patent No. 4485341. Furthermore, according to the method described in Japanese Patent No. 4336082, the sigF gene, which is involved in spore formation, was deleted from Bacillus subtilis strain Dpr9, which lacked all nine of the above extracellular protease genes. The resulting extracellular protease multiple knockout strain will be described as Dpr9ΔsigF. Plasmid introduction into the Dpr9ΔsigF strain was performed using the protoplast method described below. Bacillus subtilis was inoculated into 1 mL of LB liquid medium from a glycerol stock and incubated overnight at 30°C and 210 rpm with shaking. The following day, 10 μL of this culture was inoculated into a fresh 1 mL of LB liquid medium and incubated at 37°C and 210 rpm with shaking for approximately 2 hours. This culture was collected in a 1.5 mL tube, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed. The resulting pellet was suspended in 500 μL of SMMP (0.5 M sucrose, 20 mM disodium maleate, 20 mM magnesium chloride hexahydrate, 35% (w / v) Antibiotic medium 3 (Difco)) containing 4 mg / mL of Lysozyme (SIGMA), and incubated at 37°C for 1 hour. Next, the pellet was centrifuged at 3,500 rpm for 10 minutes, and the supernatant was removed. The pellet was then suspended in 400 μL of SMMP. 33 μL of this suspension was mixed with the various plasmids, and 100 μL of 40% PEG was added and vortexed. 350 μL of SMMP was added to this mixture and mixed by inversion. After shaking at 30°C and 210 rpm for 1 hour, the entire volume was spread onto a DM3 agar plate and incubated at 30°C for 2-3 days.

[0069] (4) VHH production The recombinant Bacillus subtilis prepared in (3) was inoculated into LB medium containing 1 mL of 50 ppm tetracycline and shaken back and forth overnight at 30°C to prepare the preculture medium. The preculture medium was inoculated at 1% into 20 mL of 2 × L-mal medium in a ribbed Erlenmeyer flask and cultured with shaking at 30°C for 72 hours. At the end of the culture, 1 mL of the culture medium was centrifuged in a microtube at 4°C, 15,000 rpm for 5 minutes, and the supernatant was collected. His-tagged VHH was purified using Ni-NTA agarose beads (Fujifilm Wako Pure Chemical Industries) according to the kit protocol. The purified protein was dissolved in PBS containing 30 mM imidazole.

[0070] (5) Verification by SDS-PAGE The sample collected in (4) was mixed in equal volumes with Laemmli Sample Buffer (BIO-RAD), and then heat-treated at 99°C for 5 minutes to prepare the sample. Mini-PROTEIN TGX Stain-Free (BIO-RAD) gel was used. 5 μL of sample was applied to each well and electrophoresis was performed at 210 V for 25 minutes. Precision Plus protein Unstained standard (BIO-RAD) was used as the molecular weight marker. Protein bands were detected using the ChemiDoc MP Imaging System. The detected proteins were quantified using a calibration curve created based on a lysozyme standard (Sigma-Aldrich). As a result, it was confirmed that the target His-tagged VHH was synthesized (Figure 3).

[0071] (Example 5) Evaluation of binding specificity by ELISA method Each well of the F96 Cert. Maxisorp Nunc-Immuno™ Plate (Thermo Fisher Scientific) contains 50 μL of 10 μg / mL SARS-CoV-2 (2019-nCoV) Nucleocapsid-His Recombinant Protein (His-tagged N protein), Human coronavirus (HCoV-229E) Nucleoprotein / NP Protein (His Tag) (His-tagged N protein), Human coronavirus (HCoV-NL63) Nucleoprotein / NP Protein (His Tag) (His-tagged N protein), Human coronavirus (HCoV-HKU1) Nucleoprotein / NP Protein (His Tag) (His-tagged N protein), Human coronavirus (HCoV-OC43) Nucleoprotein / NP Protein (His Tag) (His-tagged N protein) (all from SinoBiological), SARS Nucleocapsid protein, His Tag, MERS nucleocapsid protein, and His Tag (His-tagged N protein) (both from AcroBioSystems) were added, sealed, and allowed to stand overnight at 4°C. After removing the N protein dispersion that did not adsorb to the wells, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 200 μL of Pierce® Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific) was added and incubated at room temperature for 1 hour. After removing the blocking buffer, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 50 μL of 1 μg / mL EN2, EN3, EN4, EN5, EN6, EN11, or EN12 (all His-tagged) / PBST (PBS containing 0.05% (v / v) Tween20) was added to each well on which N protein was immobilized, and the mixture was incubated at room temperature for 1 hour.After removing the VHH antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. Peroxidase-AffiniPure Goat Anti-Alpaca IgG (H+L) (min X Bov, Hu, Ms, Rb, Rat Sr Prot) (Jackson Immuno Research Laboratories) was used as the detection antibody. The detection antibody was diluted to 1 / 10,000 with PBST (PBS containing 0.05% (v / v) Tween20). 50 μL of the detection antibody was added to each well and incubated at room temperature for 1 hour. After removing the detection antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. The chromogenic substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of the chromogenic substrate was added to each well, incubated under light shielding for 30 minutes, and then the absorbance at 450 nm was immediately measured using a Microplate Reader Infinite M1000 PRO (TECAN) (Figure 4). As a result, it was confirmed that all of the His-tagged VHH used in this example bound most strongly to the N protein of SARS-CoV-2.

[0072] (Example 6) Evaluation of binding activity to mutant N protein by ELISA method Add 50 μL of 10 μg / mL SARS-CoV-2 (2019-nCoV) Nucleocapsid-His Recombinant Protein (His-tagged N protein), SARS-CoV-2 (2019-nCoV) Nucleocapsid (R203K, G204R)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (P13L)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (S194L)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (I292T)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (D3L, R203K, G204R, S235F)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (D3L, S235F)-His Recombinant Protein, SARS-CoV-2 (2019-nCoV) Nucleocapsid (T205I) Protein (His Tag), SARS-CoV-2 (2019-nCoV) Nucleocapsid (P80R) Protein (His Tag), SARS-CoV-2 (2019-nCoV) Nucleocapsid (D377Y) Protein (His Tag), SARS-CoV-2 Nucleocapsid (A12G, T205I) Protein (His Tag), SARS-CoV-2 Nucleocapsid (R203M, D377Y) Protein (His Tag), SARS-CoV-2 Nucleocapsid (A119S, R203K, G204R, M234I) Protein (His Tag) (all from SinoBiological) to each well of a F96 Cert.Maxisorp Nunc-Immuno™ Plate (Thermo Fisher Scientific). After sealing, leave it standing at 4 °C overnight. After removing the N protein dispersion that did not adsorb to the wells, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 200 μL of Pierce® Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific) was added and incubated at room temperature for 1 hour. After removing the blocking buffer, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 50 μL of 1 μg / mL EN2, EN3, EN4, EN5, EN6, EN11, or EN12 (all His-tagged) / PBST (PBS containing 0.05% (v / v) Tween20) was added to each well with immobilized N protein and incubated at room temperature for 1 hour. After removing the VHH antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. Peroxidase-AffiniPure Goat Anti-Alpaca IgG (H+L) (min X Bov, Hu, Ms, Rb, Rat Sr Prot) (Jackson Immuno Research Laboratories) was used as the detection antibody. The detection antibody was diluted to 1 / 10,000 with PBST (PBS containing 0.05% (v / v) Tween20). 50 μL of the detection antibody was added to each well and incubated at room temperature for 1 hour. After removing the detection antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. The chromogenic substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of the chromogenic substrate was added to each well, incubated under light shielding for 30 minutes, and immediately afterward, the absorbance at 450 nm was measured using a Microplate Reader Infinite M1000 PRO (TECAN) (Figures 5 and 6). As a result, it was confirmed that all His-tagged VHH molecules used in this example universally bind to the N protein of SARS-CoV-2 mutant strains.

[0073] (Example 7) Measurement of binding activity by biolayer interferometry The concentrations of various His-tagged VHHs were quantified using the BCA method with the Pierce BCA Protein Assay Kit (Thermo Fisher), with bovine serum albumin (Fujifilm Wako Pure Chemical Industries, Ltd.) as the standard protein. The binding activity of various His-tagged VHHs to biotinylated N protein immobilized on a Streptavidin (SA) Biosensor chip (Sartorius) was measured using Octet® RED384 (Sartorius). Each measurement was performed by adding 60 μL of each measurement solution to a 384-well plate. Before measurement, the tip of the SA Biosensor chip was hydrated by immersing it in 200 μL of PBST for 10 minutes. 1) Baseline step: Measurement in PBST for 30 seconds 2) Association step: Measurement for 120 seconds with various His-tagged VHH solutions of 250 nM, 125 nM, 62.6 nM, 31.3 nM, 15.6 nM, and 0 nM, prepared by diluting with PBST. 3) Dissociation step: Measurement in PBS-T for 240 seconds 4) Regeneration step: Measurement is performed by repeating the process three times: 5 seconds in Glycin-HCl (pH 2.2) followed by 5 seconds in PBS-T. The adsorption of various His-tagged VHHs to the SA Biosensor chip was measured in steps 1) to 4) above. Subsequently, the biotinylated N protein was immobilized on the SA Biosensor chip by measuring with PBS-T containing biotinylated N protein for 600 seconds until a response of approximately 6.5 nm was obtained. Steps 1) to 4) above were performed using the SA Biosensor chip with the biotinylated N protein immobilized. Using Octet software version (1.2.1.5) (Molecular Devices), measurements of various His-tagged VHHs at a concentration of 0 nM and the results before immobilization of biotinylated N protein were taken as double references, and binding and dissociation rates were calculated by performing global fitting (Table 5).

[0074] [Table 5]

[0075] (Example 8) Evaluation of binding activity to Omicron strain N protein by ELISA method 50 μL of 10 μg / mL SARS-CoV-2 (2019-nCoV) Nucleocapsid-His Recombinant Protein (His-tagged N protein) and SARS-CoV-2 B.1.529 (Omicron) Nucleocapsid Protein (His Tag) (both SinoBiological) were added to each well of an F96 Cert. Maxisorp Nunc-Immuno® Plate (Thermo Fisher Scientific). After sealing, the plates were incubated overnight at 4°C. After removing any N protein dispersion that did not adsorb to the wells, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 200 μL of Pierce® Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific) was added and incubated at room temperature for 1 hour. After removing the blocking buffer, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. 50 μL of 1 μg / mL EN2, EN3, EN4, EN5, EN6, EN11, or EN12 (all His-tagged) / PBST (PBS containing 0.05% (v / v) Tween20) was added to each well immobilized with N protein, and incubated at room temperature for 1 hour. After removing the VHH antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing procedure was repeated three times. The detection antibody used was Peroxidase-AffiniPure Goat Anti-Alpaca IgG(H+L)(min X Bov,Hu,Ms,Rb,Rat Sr Prot)(Jackson Immuno Research Laboratories) (1.0 mg / mL). The detection antibody was diluted to 1 / 5,000 with PBST (PBS containing 0.05% (v / v) Tween20). 50 μL of the detection antibody was added to each well and incubated at room temperature for 1 hour. After removing the detection antibody, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed.This cleaning procedure was performed three times. The chromogenic substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of the chromogenic substrate was added to each well, incubated for 30 minutes under light shielding, and the absorbance at 450 nm was immediately measured using a Microplate Reader Infinite M1000 PRO (TECAN) (Figure 7). As a result, it was confirmed that all of the His-tagged VHH used in this example also bind to the N protein of the SARS-CoV-2 Omicron strain.

Claims

1. A peptide that binds to SARS-CoV-2, having one or more structural domains containing CDRs represented by (a), (b), (c), (d), (e), (f) or (g) below. (a) a CDR1 consisting of an amino acid sequence represented by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids in the amino acid sequence have been replaced with other amino acids, or an amino acid sequence in which 1 to 3 amino acids have been deleted; a CDR2 consisting of an amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids in the amino acid sequence have been replaced with other amino acids; and a CDR3 consisting of an amino acid sequence represented by PWGY (SEQ ID NO: 3), or an amino acid sequence in which 1 amino acid in the amino acid sequence has been replaced with another amino acid. (b) CDR1 consisting of an amino acid sequence represented by GFTFSHYFMH (SEQ ID NO: 4) or an amino acid sequence in which one to three amino acids in the amino acid sequence have been replaced with other amino acids, CDR2 consisting of an amino acid sequence represented by TIGSSSTF (SEQ ID NO: 5) or an amino acid sequence in which one amino acid in the amino acid sequence has been replaced with another amino acid, and CDR3 consisting of an amino acid sequence represented by TLQYYTGLHLNIVGT (SEQ ID NO: 6). (c) CDR1 consisting of an amino acid sequence represented by RSISRFNVWA (SEQ ID NO: 7) or an amino acid sequence in which 1 to 10 amino acids in the amino acid sequence have been replaced with other amino acids, CDR2 consisting of an amino acid sequence represented by SITKSGDTN (SEQ ID NO: 8) or an amino acid sequence in which 1 or 2 amino acids in the amino acid sequence have been replaced with other amino acids, and CDR3 consisting of an amino acid sequence represented by AVFRGITLYTY (SEQ ID NO: 9) or an amino acid sequence in which 1 amino acid in the amino acid sequence has been replaced with another amino acid. (d) CDR1 consisting of an amino acid sequence represented by GRAFSSYMMG (SEQ ID NO: 10) or an amino acid sequence in which 1 to 5 amino acids in the amino acid sequence have been replaced with other amino acids, CDR2 consisting of an amino acid sequence represented by AIGWSTY (SEQ ID NO: 11) or an amino acid sequence in which 1 amino acid in the amino acid sequence has been replaced with other amino acid, and CDR3 consisting of an amino acid sequence represented by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of an amino acid sequence represented by GFTFSVYWMF (SEQ ID NO: 13) or an amino acid sequence in which one to four amino acids in the amino acid sequence have been replaced with other amino acids, CDR2 consisting of an amino acid sequence represented by VINPDGSSTH (SEQ ID NO: 14) or an amino acid sequence in which one or two amino acids in the amino acid sequence have been replaced with other amino acids, and CDR3 consisting of an amino acid sequence represented by GISSTMDMDPWISLHY (SEQ ID NO: 15) or an amino acid sequence in which one amino acid in the amino acid sequence has been replaced with another amino acid. (f) CDR1 consisting of the amino acid sequence represented by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence represented by GIRWNNVSTY (SEQ ID NO: 17) or the amino acid sequence in which one amino acid in the amino acid sequence is substituted with another amino acid, and CDR3 consisting of the amino acid sequence represented by LALSSDRLGLYAY (SEQ ID NO: 18). (g) CDR1 consisting of an amino acid sequence represented by GLTFRTYTMG (SEQ ID NO: 1) or an amino acid sequence in which 1 to 7 amino acids in the amino acid sequence have been replaced with other amino acids or an amino acid sequence in which one amino acid has been inserted; CDR2 consisting of an amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2) or an amino acid sequence in which 1 to 6 amino acids in the amino acid sequence have been replaced with other amino acids; and CDR3 consisting of an amino acid sequence represented by SGT or an amino acid sequence in which one amino acid in the amino acid sequence has been replaced with another amino acid.

2. The peptide of claim 1, which binds to the N-protein.

3. The peptide according to claim 1 or 2, which is a VHH antibody, a heavy chain antibody or a VHH antibody multimer.

4. The peptide according to claim 3 , wherein the VHH antibody multimer is a multimer in which multiple structural domains are linked together.

5. The peptide according to claim 3, wherein the VHH antibody multimer is a multimer in which one or more of the structural domains are linked to one or more structural domains having antigen specificity different from that of the structural domains.

6. A nucleic acid encoding the peptide according to claim 1 or 2.

7. A method for detecting SARS-CoV-2 in a sample, comprising the step of contacting a test sample with a peptide that binds to SARS-CoV-2, the peptide having one or more structural domains containing CDRs represented by (a), (b), (c), (d), (e), (f) or (g) below. (a) a CDR1 consisting of an amino acid sequence represented by GLTFRTYTMG (SEQ ID NO: 1), or an amino acid sequence in which 1 to 7 amino acids in the amino acid sequence have been replaced with other amino acids, or an amino acid sequence in which 1 to 3 amino acids have been deleted; a CDR2 consisting of an amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2), or an amino acid sequence in which 1 to 6 amino acids in the amino acid sequence have been replaced with other amino acids; and a CDR3 consisting of an amino acid sequence represented by PWGY (SEQ ID NO: 3), or an amino acid sequence in which 1 amino acid in the amino acid sequence has been replaced with another amino acid. (b) CDR1 consisting of an amino acid sequence represented by GFTFSHYFMH (SEQ ID NO: 4) or an amino acid sequence in which one to three amino acids in the amino acid sequence have been replaced with other amino acids, CDR2 consisting of an amino acid sequence represented by TIGSSSTF (SEQ ID NO: 5) or an amino acid sequence in which one amino acid in the amino acid sequence has been replaced with another amino acid, and CDR3 consisting of an amino acid sequence represented by TLQYYTGLHLNIVGT (SEQ ID NO: 6). (c) CDR1 consisting of an amino acid sequence represented by RSISRFNVWA (SEQ ID NO: 7) or an amino acid sequence in which 1 to 10 amino acids in the amino acid sequence have been replaced with other amino acids, CDR2 consisting of an amino acid sequence represented by SITKSGDTN (SEQ ID NO: 8) or an amino acid sequence in which 1 or 2 amino acids in the amino acid sequence have been replaced with other amino acids, and CDR3 consisting of an amino acid sequence represented by AVFRGITLYTY (SEQ ID NO: 9) or an amino acid sequence in which 1 amino acid in the amino acid sequence has been replaced with another amino acid. (d) CDR1 consisting of an amino acid sequence represented by GRAFSSYMMG (SEQ ID NO: 10) or an amino acid sequence in which 1 to 5 amino acids in the amino acid sequence have been replaced with other amino acids, CDR2 consisting of an amino acid sequence represented by AIGWSTY (SEQ ID NO: 11) or an amino acid sequence in which 1 amino acid in the amino acid sequence has been replaced with other amino acid, and CDR3 consisting of an amino acid sequence represented by NHRFTGRVY (SEQ ID NO: 12). (e) CDR1 consisting of an amino acid sequence represented by GFTFSVYWMF (SEQ ID NO: 13) or an amino acid sequence in which one to four amino acids in the amino acid sequence have been replaced with other amino acids, CDR2 consisting of an amino acid sequence represented by VINPDGSSTH (SEQ ID NO: 14) or an amino acid sequence in which one or two amino acids in the amino acid sequence have been replaced with other amino acids, and CDR3 consisting of an amino acid sequence represented by GISSTMDMDPWISLHY (SEQ ID NO: 15) or an amino acid sequence in which one amino acid in the amino acid sequence has been replaced with another amino acid. (f) CDR1 consisting of the amino acid sequence represented by ARTIHRIEMG (SEQ ID NO: 16), CDR2 consisting of the amino acid sequence represented by GIRWNNVSTY (SEQ ID NO: 17) or the amino acid sequence in which one amino acid in the amino acid sequence is substituted with another amino acid, and CDR3 consisting of the amino acid sequence represented by LALSSDRLGLYAY (SEQ ID NO: 18). (g) CDR1 consisting of an amino acid sequence represented by GLTFRTYTMG (SEQ ID NO: 1) or an amino acid sequence in which 1 to 7 amino acids in the amino acid sequence have been replaced with other amino acids or an amino acid sequence in which one amino acid has been inserted; CDR2 consisting of an amino acid sequence represented by AITPSGGNTA (SEQ ID NO: 2) or an amino acid sequence in which 1 to 6 amino acids in the amino acid sequence have been replaced with other amino acids; and CDR3 consisting of an amino acid sequence represented by SGT or an amino acid sequence in which one amino acid in the amino acid sequence has been replaced with another amino acid.

8. A SARS-CoV-2 detection kit comprising the peptide according to claim 1 or 2.

9. A medicine comprising the peptide according to claim 1 or 2.

10. The pharmaceutical composition according to claim 9, for preventing or treating SARS-CoV-2 infection.

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