Anti-porcine norovirus antibody

Abstract

To provide anti-porcine norovirus antibodies that exhibit specific binding activity to norovirus particles of a specific genotype.SOLUTION: Provided is an antibody that binds to porcine norovirus, which has one or more structural domains including a CDR represented by, for example, CDR1 having an amino acid sequence represented by GLTFSMYSMG (sequence number 1), a CDR2 having an amino acid sequence represented by SINWSGGSTY (sequence number 2), and a CDR3 having an amino acid sequence represented by DFSFLSRCKLGSSGYDY (sequence number 3).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an anti-porcine norovirus antibody that exhibits specific binding activity to norovirus particles of a specific genotype. [Background technology]

[0002] Norovirus is a non-enveloped, spherical virus with a diameter of about 30 to 40 nm belonging to the Caliciviridae family. Norovirus causes acute gastroenteritis symptoms such as vomiting and diarrhea in humans, but it has been reported that it is infectious to various hosts other than humans. The genome of norovirus is a single-stranded (+)RNA of about 7.6 kb, and in recent years, it has been classified into seven gene groups based on genome homology: gene group 1 (GI), gene group 2 (GII), gene group 3 (GIII), gene group 4 (GIV), gene group 5 (GV), gene group 6 (GVI), and gene group 7 (GVII). Of these, GI is infectious to humans, GII to humans and pigs, GIII to cattle, GIV to humans, cats, and dogs, GV to mice, and GVI and GVII to dogs (see Non-Patent Document 1).

[0003] There are multiple genotypes in each gene group of norovirus. In the case of norovirus, each genotype exhibits different antigenicity, but the same genotype is said to exhibit the same antigenicity. The norovirus genome mutates very quickly, and it has been reported that viruses of different genotypes recombine, and the emergence of noroviruses with new genotypes has also been confirmed. Therefore, it is very important to accurately classify noroviruses and identify their genotypes.

[0004] To date, human-infectious noroviruses (hereafter referred to as human noroviruses) have been reported to have GI norovirus genotypes GI.1 to GI.9, and GII norovirus genotypes GII.1 to GII.10, GII.12 to GII.17, and GII.20 to GII.22. Human noroviruses are one of the main causes of infectious gastroenteritis that occurs annually in Japan, mainly from autumn to winter. Human noroviruses can cause mass outbreaks when brought into public facilities such as schools, medical facilities, and food handling facilities, and can cause food poisoning if they contaminate food. Therefore, epidemiological information on human noroviruses is important, and in Japan, the Ministry of Health, Labor, and Welfare and the National Institute of Infectious Diseases are collecting epidemiological data, including norovirus gene groups and genotypes.

[0005] On the other hand, among the noroviruses classified as GII, the same as human noroviruses, GII.11, GII.18, and GII.19 have been reported to infect pigs (hereinafter referred to as porcine noroviruses). These porcine noroviruses are said to be closely related to GII noroviruses, which are highly prevalent among human noroviruses. To date, no human infection with porcine noroviruses has been reported. However, among GII noroviruses, porcine noroviruses with genotypes similar to highly prevalent human noroviruses have been reported (see Non-Patent Documents 2 and 3). Therefore, pigs are of concern as a public health problem not only as a source of norovirus infection for humans, but also as a reservoir for the occurrence of new recombinant noroviruses (see Non-Patent Document 4). Therefore, epidemiological surveys are being conducted on porcine noroviruses.

[0006] The ability to specifically detect porcine norovirus is also important for epidemiological investigations of porcine norovirus. In particular, because human norovirus and porcine norovirus form the same genetic group (GII), a method for distinguishing between genotypes is necessary to obtain epidemiological information.

[0007] Antibodies are used to detect proteins derived from viruses, for example antigen test methods that use immunochromatography or ELISA (Enzyme-linked immuno-sorbent assay) as their measurement principle. In the case of norovirus, each genotype exhibits different antigenicity, but the same genotype is said to exhibit the same antigenicity. In addition, the VP1 protein, which is the structural protein of norovirus, has regions where the amino acid sequence is conserved for each gene group. For this reason, antigen test kits for norovirus that use immunochromatography or ELISA as their measurement principle are also commercially available.

[0008] However, such commercially available kits use monoclonal or polyclonal IgG antibodies that bind broadly to the capsid proteins of GI and GII noroviruses, and thus do not allow for genotyping of GII noroviruses. Furthermore, no antibodies specific to porcine noroviruses GII.11, GII.18, or GII.19 have been published to date. Antibodies that exhibit excellent binding activity and binding specificity to porcine noroviruses are important for rapid and simple detection and identification of porcine noroviruses. However, for noroviruses, which undergo rapid genome mutation and have a large number of reported genotypes, it is difficult to develop antibodies that exhibit high specificity at the genotype level.

[0009] On the other hand, most of the antigen test kits available on the market use monoclonal or polyclonal IgG antibodies derived from mice, rabbits, etc. VHH (variable domain of heavy chain of heavy chain antibody) is a single domain antibody consisting only of the variable domain of the heavy chain antibody possessed by camelids. VHH shows the same binding activity as IgG antibodies, but has the following characteristics: (1) its molecular weight is about one-tenth that of IgG antibodies, and it is expected to find new epitopes that cannot be bound by conventional antibodies; (2) unlike IgG antibodies, it has a highly reversible protein structure and shows excellent resistance to heat and pressure; and (3) unlike IgG antibodies, it can be produced using microorganisms such as yeast and bacteria. Furthermore, (4) VHH has a very high affinity with in-vitro antibody selection techniques such as cDNA display and phage display, and can be developed in a shorter period of time than IgG antibodies obtained by immunization of mice, rabbits, etc. Therefore, VHHs are expected to be used as alternative antibodies to conventional immunoglobulins such as IgG and IgA antibodies that have been used in pharmaceuticals and diagnostic reagents.

[0010] The superiority of VHHs is also clear in the development of antigen test kits. In particular, the properties of VHHs are considered to be superior to IgG antibodies in the following ways: (1) VHHs can be densely immobilized on particles or nitrocellulose membranes, allowing more paratopes to be presented on the substrate, (2) VHHs have excellent protein stability, and products can be expected to have better storage stability, (3) VHHs can be mass-produced inexpensively using microorganisms, allowing for further reduction in manufacturing costs, and (4) VHHs do not have an Fc region that can cause nonspecific reactions in immunochromatography.

[0011] As described above, VHHs have many advantages in terms of properties and production compared to conventional antibodies (such as IgG antibodies). Therefore, detection technology using single domain antibodies targeting norovirus can be provided with advantages in terms of performance and cost compared to conventional technology using IgG antibodies. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Advances in laboratory methods for detection and typing of norovirus. J Clin Microbiol. 2015;53(2):373-81. [Non-Patent Document 2] Frequent Detection of Noroviruses and Sapoviruses in Swine and High Genetic Diversity of Porcine Sapovirus in Japan during Fiscal Year 2008. J Clin Microbiol. 2010;48(4):1215-1222. [Non-Patent Document 3] Prevalence of Porcine Noroviruses, Molecular haracterization of Emerging Porcine Sapoviruses from Finisher Swine in the United States, and Unified Classification Scheme for Sapoviruses. J Clin Microbiol. 2013;51(7):2344-2353. [Non-Patent Document 4] Toshiharu Morimitsu et al., "Survey of Norovirus in Pigs in Kochi Prefecture," Kochi Prefectural Health Research Report, 2014, Vol. 60, p. 25-27 Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention relates to providing an anti-porcine norovirus antibody that exhibits specific binding activity to norovirus particles of a specific genotype. [Means for solving the problem]

[0014] The inventors conducted studies to obtain single domain antibodies that exhibit specific binding activity to the virus-like particles (VLPs) of GII.11, GII.18, and GII.19 noroviruses, which have been reported as porcine noroviruses. As a result, they succeeded in obtaining clones with high binding specificity for the target molecule by screening a VHH antibody library containing CDRs 1 to 3 with specific numbers of amino acids in its construct using a cDNA display method.

[0015] That is, the present invention relates to the following 1) to 5). 1) An antibody that binds to a porcine norovirus, which has one or more structural domains containing CDRs shown in (a), (b), (c), (d) or (e) below. (a) CDR1 consisting of the amino acid sequence represented by GLTFSMYSMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by SINWSGGSTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DFSFLSRCKLGSSGYDY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GIVFSVYPMG (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GINSFHNTT (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by GAYRRLCPIEEYGMDF (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence represented by GRAFSSYMVG (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AIAWSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by GFPTLVALPYEYDY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence represented by GITFSNTAMT (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by SINKSGDEVA (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by PYFGS (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence represented by GSIFSFNAMA (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence represented by GITSGGRTS (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence represented by TRWATNSIAIRQVESYDY (SEQ ID NO: 15). 2) A nucleic acid encoding the antibody of 1). 3) A method for detecting porcine norovirus in a sample, comprising the step of contacting a test sample with an antibody that binds to norovirus and has one or more structural domains containing a CDR shown in (a), (b), (c), (d) or (e) below. (a) CDR1 consisting of the amino acid sequence represented by GLTFSMYSMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by SINWSGGSTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DFSFLSRCKLGSSGYDY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GIVFSVYPMG (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GINSFHNTT (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by GAYRRLCPIEEYGMDF (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence represented by GRAFSSYMVG (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AIAWSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by GFPTLVALPYEYDY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence represented by GITFSNTAMT (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by SINKSGDEVA (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by PYFGS (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence represented by GSIFSFNAMA (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence represented by GITSGGRTS (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence represented by TRWATNSIAIRQVESYDY (SEQ ID NO: 15). 4) A porcine norovirus detection kit containing the antibody of 1). 5) A preventive or therapeutic drug for porcine norovirus infection containing the antibody of 1). Effect of the Invention

[0016] According to the present invention, a single domain antibody having high reactivity with norovirus particles of GII.11, GII.18, and GII.19 can be provided. Since the antibody of the present invention exhibits excellent specificity with respect to porcine norovirus particles, the antibody can be used to specifically detect porcine norovirus, i.e., to rapidly detect porcine norovirus infection. In addition, by testing a norovirus-positive sample using the antibody and confirming that there is no reaction, it is possible to confirm that the norovirus contained in the sample is porcine norovirus. [Brief description of the drawings]

[0017] [Figure 1] Detection of porcine norovirus VLPs by SDS-PAGE. [Diagram 2] The frequency of occurrence of each VHH clone in the DNA library after selection. [Diagram 3] Library titer confirmation by phage ELISA. [Figure 4] Selection of VHHs by phage ELISA. [Diagram 5] Detection of His-tagged VHHs by SDS-PAGE. [Figure 6] Activity evaluation of anti-GII norovirus polyclonal antibodies by ELISA. [Figure 7] Criteria for determining coloration obtained with lateral flow assays. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The antibody of the present invention that binds to the norovirus (hereinafter referred to as the "antibody of the present invention") is an anti-porcine norovirus monoclonal antibody having one or more structural domains including CDR1 to 3 shown in (a), (b), (c), (d) or (e) below. (a) CDR1 consisting of the amino acid sequence represented by GLTFSMYSMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by SINWSGGSTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DFSFLSRCKLGSSGYDY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GIVFSVYPMG (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GINSFHNTT (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by GAYRRLCPIEEYGMDF (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence represented by GRAFSSYMVG (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AIAWSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by GFPTLVALPYEYDY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence represented by GITFSNTAMT (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by SINKSGDEVA (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by PYFGS (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence represented by GSIFSFNAMA (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence represented by GITSGGRTS (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence represented by TRWATNSIAIRQVESYDY (SEQ ID NO: 15).

[0019] Porcine norovirus is a non-enveloped, spherical virus with a diameter of about 30 to 40 nm that belongs to the Caliciviridae family, and causes porcine norovirus infection. The antibody of the present invention is an antibody that binds to porcine norovirus (GII.11, GII.18, GII.19), and more specifically, an antibody that binds to the structural proteins of the porcine norovirus and complexes thereof. The complexes include virus-like particles (VLPs), VP1 protein, and complexes thereof.

[0020] The structural domain of the antibody of the present invention has three CDRs, namely, CDR1, CDR2, and CDR3. A CDR (Complementarity Determining Region) includes a sequence-variable antigen recognition site or a random sequence region, and is also called a hypervariable region. In the structural domain of the antibody of the present invention, the three CDRs are present in the order of CDR1, CDR2, and CDR3 from the N-terminus.

[0021] The binding ability to norovirus can be evaluated by a method known to those skilled in the art. Specifically, as shown in the Examples described below, the binding ability 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 interference.

[0022] The structural domain of the antibody of the present invention may have framework regions on both ends of CDR1, CDR2, and CDR3. The framework region is a region in the variable region of an antibody molecule excluding the complementarity determining regions, and refers to a highly conserved region. That is, one embodiment of the structural domain of the present invention includes a first framework region (FR1), CDR1, second framework region (FR2), CDR2, third framework region (FR3), CDR3 and fourth framework region (FR4) in this order. Examples of amino acid sequences of framework regions in structural domains include the amino acid sequences shown below or amino acid sequences having 80% or more identity to said amino acid sequences. FR1: AEVQLVESGGGLVQAGDSLRLSCVAS (SEQ ID NO: 16) AEVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 17) AQLQLVESGGGLVTAGGSLSLSCAAS (SEQ ID NO: 18) AEVQLVESGGGQVQPGGSLRLSCIVS (SEQ ID NO: 19) AEVQLVESGGGLVQAGGSLKLSCAAS (SEQ ID NO: 20) FR2: WFRQAPGKEREFVA (SEQ ID NO:21) WYRQAPGKQREWVA (SEQ ID NO:22) WVRRAPGKGLEWIS (SEQ ID NO: 23) WYRQVPGKQREFIA (SEQ ID NO: 24) FR3: YADSVKGRFTISRDNAKNTVYLQMNSLSPEDTAVYYCAT (SEQ ID NO: 25), YEESVKGRFTISRDNAKNAVYLQMNDLKPEDTAVYYCAA (SEQ ID NO:26), YDESVKGRFTISRDSAKNTVYLQMDDLKPEDTAVYYCAA (SEQ ID NO: 27), YADSVKGRFTISRDNAKNTLYLQMNSLKAEDTAVYYCHA (SEQ ID NO: 28), YADSVKGRFTISRDNAKNTVYLRMNSLRPEDSAVYYCAA (SEQ ID NO: 29), FR4: WGQGTQVTVSS (SEQ ID NO: 30) WGKGTLVTVSS (SEQ ID NO: 31)

[0023] Examples of framework regions consisting of amino acid sequences having 80% or more identity to the amino acid sequences shown in SEQ ID NOs: 16 to 31 include framework regions consisting of amino acid sequences having preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more identity.

[0024] Here, the identity of amino acid sequences refers to the percentage (%) of the number of positions at which identical amino acid residues exist in two amino acid sequences when the two sequences are aligned relative to the total number of amino acid residues. The identity of sequences can be calculated, for example, by performing an analysis using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI).

[0025] Among the antibodies of the present invention, an antibody having a structural domain (SEQ ID NO: 32) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 being the amino acid sequence shown in SEQ ID NO: 1, CDR2 being the amino acid sequence shown in SEQ ID NO: 2, CDR3 being the amino acid sequence shown in SEQ ID NO: 3, FR1 being the amino acid sequence shown in SEQ ID NO: 16, FR2 being the amino acid sequence shown in SEQ ID NO: 21, FR3 being the amino acid sequence shown in SEQ ID NO: 25, and FR4 being the amino acid sequence shown in SEQ ID NO: 30, and an antibody having a structural domain (SEQ ID NO: 32) in which CDR1 is shown in SEQ ID NO: 4 an antibody having a structural domain (SEQ ID NO: 33) 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, CDR3 is the amino acid sequence shown in SEQ ID NO: 9, FR1 is the amino acid sequence shown in SEQ ID NO: 10, FR2 is the amino acid sequence shown in SEQ ID NO: 11, FR3 is the amino acid sequence shown in SEQ ID NO: 12, FR4 is the amino acid sequence shown in SEQ ID NO: 13, and and an antibody having a structural domain (SEQ ID NO: 35) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 13, CDR2 is the amino acid sequence shown in SEQ ID NO: 14, CDR3 is the amino acid sequence shown in SEQ ID NO: 15, FR1 is the amino acid sequence shown in SEQ ID NO: 20, FR2 is the amino acid sequence shown in SEQ ID NO: 24, FR3 is the amino acid sequence shown in SEQ ID NO: 29, and FR4 is the amino acid sequence shown in SEQ ID NO: 30, respectively, in the Examples described below.The clones were obtained by screening using the cDNA display method and are highly reactive to norovirus structural proteins (SEQ ID NO: 32 is NoVHH200, SEQ ID NO: 33 is NoVHH201, SEQ ID NO: 34 is NoVHH191E, SEQ ID NO: 35 is NoVHH53, and SEQ ID NO: 36 is NoVHH54), and are suitable anti-porcine norovirus monoclonal antibodies.

[0026] The form of the antibody of the present invention is not limited as long as it has at least one of the above structural domains, and it may be a single domain antibody (also called a nanobody) or a multimer (e.g., a dimer) in which multiple single domain antibodies are linked. Multimers include multimers in which multiple structural domains of the present invention are linked, as well as multimers in which one or more of the structural domains are linked to one or more other structural domains that have different antigen specificity from the structural domains. A single domain antibody refers to an antibody that has the property of specifically binding to an antigen through a single variable region (antigen-binding domain). Single domain antibodies include antibodies whose variable region consists only of the variable region of a heavy chain (heavy chain single domain antibody) and antibodies whose variable region consists only of the variable region of a light chain (light chain single domain antibody). VHH, which is a heavy chain antibody identified in camelids (e.g., camel, llama, alpaca, etc.), and VNAR, which is a heavy chain antibody derived from cartilaginous fish (e.g., shark), are known as types of single domain antibodies, and VHH is preferred in the present invention. The antibody of the present invention may be modified to evade the immune system of a pig. Since the antibody modified to evade the immune system of a pig can be administered to a pig, it can be used as a livestock medicine.

[0027] The method for producing the antibody of the present invention is not particularly limited, and the antibody can be easily produced by known techniques in the art. For example, the antibody can be produced by combining solid-phase peptide synthesis and native chemical ligation (NCL) or by genetic engineering, but a preferred method is to design an artificial gene optimized for the expression of the antibody of interest in a host cell by subjecting a nucleic acid encoding the antibody of the present invention to a process such as codon optimization, incorporate the artificial gene into an appropriate vector, and introduce the vector into a host cell to produce the antibody as a recombinant antibody.

[0028] Examples of host cells used in the production of recombinant antibodies include Escherichia coli, Bacillus subtilis, mold, animal cells, plant cells, baculovirus / insect cells, and yeast cells. The expression vector for expressing the antibody can be a vector suitable for various host cells. Examples of expression vectors that can be used include vectors derived from Escherichia coli such as pBR322, pBR325, pUC12, and pUC13; vectors derived from Bacillus subtilis such as pUB110, pTP5, and pC194; shuttle vectors that can be used in common between Escherichia coli and Bacillus subtilis such as pHY300PLK; vectors derived from yeast such as pSH19 and pSH15; bacteriophages such as λ phage; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, and baculovirus; and vectors modified from these. These expression vectors have a replication origin, a selection marker, and a promoter suitable for each vector, and may have an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, etc. Furthermore, in order to facilitate purification of the expressed polypeptide, the expression vector may have inserted therein a base sequence for expressing a fused tag such as a FLAG tag, a His tag, an HA tag, or a GST tag.

[0029] When extracting the expressed antibody of the present invention from cultured bacteria or cells, after culturing, the bacteria or cultured cells are collected by a known method, suspended in an appropriate buffer, and the bacteria or cells are disrupted by ultrasonication, lysozyme and / or freeze-thawing, etc., and then a soluble extract is obtained by centrifugation or filtration. The target antibody can be obtained from the obtained 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 utilize mainly 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 reversed-phase high performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing.

[0030] Since the antibodies of the present invention bind to the structural proteins of porcine norovirus, by contacting them with a test sample that contains or may contain porcine norovirus, it is possible to confirm the presence or absence of porcine norovirus in the sample. Specifically, detection of porcine norovirus using an antibody of the present invention comprises the steps of contacting the antibody of the present invention with a test sample to form a complex between the antibody of the present invention and the porcine norovirus in the test sample, and detecting the porcine norovirus in the complex. In addition, the antibody of the present invention can also be used as an antibody for detecting virus-specific antibodies in serum, by adding a portion of an antigen containing a structural protein of the porcine norovirus to anti-porcine norovirus antibodies (e.g., serum antibodies) contained in an immobilized test sample (serum) to allow binding, and confirming the presence of the porcine norovirus antigen in the bound state.

[0031] Test samples include, but are not limited to, biological samples (e.g., saliva, body fluids, blood, serum, urine, feces, tissues, cells, tissue or cell fragments, etc.), as well as solutions and solid surfaces that may be contaminated with porcine norovirus. From the viewpoint of binding to VLP, it is preferable to dissolve the virus in the test sample in a solution containing a surfactant, etc., and allow the virus to bind to the antibody in the solution. The antibody may or may not be immobilized on a solid phase. The step of detecting the porcine norovirus in the conjugate can be carried out, for example, by reacting the conjugate with an anti-porcine norovirus antibody that recognizes an epitope different from that of the antibody of the present invention in the conjugate. Alternatively, the porcine norovirus in the conjugate may be detected in a liquid phase by a homogeneous assay.

[0032] Furthermore, the antibody of the present invention can be a component of a kit for detecting porcine norovirus. The kit can be used as a diagnostic agent for porcine norovirus infection and as a tool for developing preventive or therapeutic agents for porcine norovirus infection. The detection kit can include, in addition to the anti-porcine norovirus antibody of the present invention, reagents and instruments necessary for detection, such as an antibody that recognizes the antibody of the present invention or an anti-porcine norovirus antibody that recognizes an epitope different from the antibody of the present invention, a solid phase support, a buffer solution, an enzyme reaction stopping solution, a microplate reader, etc. In the detection kit, the antibody of the present invention may be immobilized on a solid phase, such as beads, a membrane, the side or bottom surface of a reaction vessel, a plate-like substrate such as a slide glass, or a well substrate such as an immunoplate, to which the antibody of the present invention is directly or indirectly immobilized.

[0033] In such a preventive or therapeutic drug for porcine norovirus infection, the content of the antibody of the present invention in the composition can be appropriately adjusted. Such a medicine can be applied by administering an effective amount of the antibody of the present invention to a patient at intervals of about once to several times a week. In this case, suitable methods of administration include intravenous injection, drip infusion, etc.

[0034] In the present invention, the following aspects are further disclosed in relation to the above-mentioned embodiment. <1> An antibody that binds to a porcine norovirus, having one or more structural domains containing CDRs shown in (a), (b), (c), (d) or (e) below. (a) CDR1 consisting of the amino acid sequence shown in (SEQ ID NO:1), CDR2 consisting of the amino acid sequence shown in (SEQ ID NO:2), and CDR3 consisting of the amino acid sequence shown in (SEQ ID NO:3). (b) CDR1 consisting of the amino acid sequence shown in (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence shown in (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence shown in (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence shown in (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown in (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown in (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence shown in (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence shown in (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence shown in (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence shown in (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence shown in (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence shown in (SEQ ID NO: 15). <2> Binding to a porcine norovirus structural protein, preferably a viral structural protein consisting of porcine norovirus VP1; <1> antibodies. <3> A single domain antibody or a multimer thereof. <1> or <2> antibodies. <4> the single domain antibody is a VHH antibody, <3> antibodies. <5> A multimer of a single domain antibody is a multimer, preferably a dimer, in which multiple of the structural domains are linked together. <3> antibodies. <6> A multimer of a single domain antibody is a multimer in which one or more of the structural domains are linked to one or more structural domains having different antigen specificity from the structural domains. <3> antibodies.

[0035] <7> The structural domain has, in this order, a first framework region (FR1), a CDR1, a second framework region (FR2), a CDR2, a third framework region (FR3), a CDR3 and a fourth framework region (FR4); <1> ~ <6> Any of the antibodies. <8> FR1 to FR4 are composed of the following amino acid sequences: <7> antibodies. FR1: an amino acid sequence represented by SEQ ID NO: 16, 17, 18, 19, or 20, or an amino acid sequence having 80% or more identity thereto FR2: an amino acid sequence represented by SEQ ID NO: 21, 22, 23, or 24, or an amino acid sequence having 80% or more identity thereto FR3: an amino acid sequence represented by SEQ ID NO: 25, 26, 27, 28, or 29, or an amino acid sequence having 80% or more identity thereto FR4: an amino acid sequence shown in SEQ ID NO: 30 or 31, or an amino acid sequence having 80% or more identity thereto <9> a structural domain (SEQ ID NO: 32) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 is the amino acid sequence shown in SEQ ID NO: 1, CDR2 is the amino acid sequence shown in SEQ ID NO: 2, and CDR3 is the amino acid sequence shown in SEQ ID NO: 3, FR1 is the amino acid sequence shown in SEQ ID NO: 16, FR2 is the amino acid sequence shown in SEQ ID NO: 21, FR3 is the amino acid sequence shown in SEQ ID NO: 25, and FR4 is the amino acid sequence shown in SEQ ID NO: 30; <7> antibodies. <10> a structural domain (SEQ ID NO: 33) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, 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, FR1 is the amino acid sequence shown in SEQ ID NO: 17, FR2 is the amino acid sequence shown in SEQ ID NO: 22, FR3 is the amino acid sequence shown in SEQ ID NO: 26, and FR4 is the amino acid sequence shown in SEQ ID NO: 31; <7> antibodies. <11> a structural domain (SEQ ID NO: 34) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, 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, FR1 is the amino acid sequence shown in SEQ ID NO: 18, FR2 is the amino acid sequence shown in SEQ ID NO: 21, FR3 is the amino acid sequence shown in SEQ ID NO: 27, and FR4 is the amino acid sequence shown in SEQ ID NO: 30; <7> antibodies. <12> a structural domain (SEQ ID NO: 35) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, 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, FR1 is the amino acid sequence shown in SEQ ID NO: 19, FR2 is the amino acid sequence shown in SEQ ID NO: 23, FR3 is the amino acid sequence shown in SEQ ID NO: 28, and FR4 is the amino acid sequence shown in SEQ ID NO: 30; <7> antibodies. <13> a structural domain (SEQ ID NO: 36) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, 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, FR1 is the amino acid sequence shown in SEQ ID NO: 20, FR2 is the amino acid sequence shown in SEQ ID NO: 24, FR3 is the amino acid sequence shown in SEQ ID NO: 29, and FR4 is the amino acid sequence shown in SEQ ID NO: 30; <7> antibodies. <14> <1> ~ <13> A nucleic acid encoding any one of the antibodies. <15> A method for detecting porcine norovirus in a sample, comprising the step of contacting a test sample with an antibody that binds to norovirus and has one or more structural domains containing CDRs shown in (a), (b), (c), (d), (e), (f) or (g) below. (a) CDR1 consisting of the amino acid sequence shown in (SEQ ID NO:1), CDR2 consisting of the amino acid sequence shown in (SEQ ID NO:2), and CDR3 consisting of the amino acid sequence shown in (SEQ ID NO:3). (b) CDR1 consisting of the amino acid sequence shown in (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence shown in (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence shown in (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence shown in (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence shown in (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence shown in (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence shown in (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence shown in (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence shown in (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence shown in (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence shown in (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence shown in (SEQ ID NO: 15). <16> <1> ~ <15> A porcine norovirus detection kit comprising any one of the antibodies described above. <17> <1> ~ <15> A preventive or therapeutic drug for porcine norovirus infection, comprising any one of the antibodies. EXAMPLES

[0036] (Example 1) Preparation of virus-like hollow particles of norovirus 1-1. Cell lines, media and primers used The bacterial strains and cell lines used in this study are shown in Table 1. LB medium (polypeptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7-7.4, Wako Pure Chemical Industries, Ltd.) was used for culturing DH5α and DH10bac. Agar medium contained 15 g / L agarose (Wako Pure Chemical Industries, Ltd.). Cultivation was performed at 37°C, with shaking (200 rpm) as necessary. Antibiotics, etc. were added during transformation as necessary. Sf-900III SFM (Thermo Fisher Scientific) was used for culturing Sf9. 10 mL of 10000 U / mL Pencillin-streptomycin (Thermo Fisher Scientific) was added per 1 L of medium (hereinafter referred to as Sf900 medium). Express Five SFM (Thermo Fisher Scientific) was used for culturing H5. 10 mL of 10000 U / mL Pencillin-streptomycin (Thermo Fisher Scientific) and 100 mL of 200 mM L-Glutamine (Thermo Fisher Scientific) were added per 1 L of medium (hereinafter referred to as H5 medium). The culture temperature for both Sf9 and H5 was 27°C. Subculture of cells was performed in a stationary culture system using T flasks (Becton Dickinson). Amplification of baculovirus and VLP production were performed in a suspension culture system using Erlenmeyer flasks.

[0037] [Table 1]

[0038] 1-2. Artificial synthesis of genes The sequences of ORF2 and ORF3, which code for norovirus capsid proteins, were obtained from the public gene database NoroNet (https: / / www.rivm.nl / en / noronet) or NCBI (https: / / www.ncbi.nlm.nih.gov / ). The strain names, accession numbers, etc. of the obtained virus genotypes are listed in Table 2. For the genotypes for which sequences were available, the 4 bases upstream of ORF2 (GI type: GTAA, GII type: GTGA) were added to the 5' side of the ORF2 / ORF3 sequence, and the 3'-untranslated region (UTR, Table 3) of each genotype was added to the 3' side to design the target gene construct. The designed target construct was obtained by artificial gene synthesis. For GII.2 and GII.3, synthesis was requested to Genscript, and a pUC57 vector with the target construct inserted was obtained. For the other genotypes, synthesis was requested to FASMAC, and a pUCFa vector with the target construct inserted was obtained. For constructs other than GI.1, GII.2, GII.3, and GII.4, the sequences were optimized to correspond to the codon frequency of H5 cells for protein expression.

[0039] [Table 2]

[0040] [Table 3]

[0041] 1-3. Insertion of NoV gene into pDEST8 vector Using the plasmid vector containing the artificially synthesized construct as a template, each NoV gene fragment was amplified by PCR using the primers in Table 4. In addition, a linear pDEST8 vector was obtained using the pDEST8 vector (Thermo Fisher Scientific) as a template and inverse PCR primers (SEQ ID NO: 87, SEQ ID NO: 88). KOD Plus Neo (TOYOBO) was used as the PCR enzyme. Each NoV gene fragment was inserted into the pDEST8 vector using In-Fusion (registered trademark) HD ​​Cloning Kit (TAKARA BIO). The NoV gene fragment, the linear pDEST8 vector, and the reaction reagents were mixed as shown in Table 5 and incubated at 50°C for 15 minutes to obtain an In-Fusion reaction solution.

[0042] [Table 4]

[0043] [Table 5]

[0044] 1-4. Transformation of DH5α 2.5 μL of the infusion reaction solution was added to 100 μL of DH5α competent cells melted on ice and mixed gently. After standing on ice for 5 minutes, the cells were incubated at 42 °C for 45 seconds and then left on ice for 2 minutes. 1 mL of LB medium was added, and the appropriately diluted solution was plated on LB agar medium containing 100 μg / mL ampicillin. After overnight culture at 37 °C, the grown colonies were picked up and cultured overnight in LB medium containing 100 μg / mL ampicillin. Plasmids were extracted from the culture medium using a QIAprep Spin Miniprep Kit (QIAGEN), and the pDEST8 vector into which the target construct was inserted was obtained.

[0045] 1-5. Construction of recombinant Bacmid (rBacmid) DH10Bac competent cells were thawed on ice, and 1 μL of pDEST8 vector solution (final concentration 100 ng) was added to 50 μL of competent cells. pDEST8 vector was introduced by heat shock in the same manner as in 1-4. 900 μL of SOC medium was added to the heat-shocked cells, and recovery culture was performed at 37 °C and 200 rpm for 4 hours. Appropriately diluted bacterial solution was plated on LB agar medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 40 μg / mL IPTG, and 100 μg / mL X-gal. Static culture was performed at 37 °C, and white colonies were picked up on LB agar medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline. Transformants containing the target sequence were selected by PCR. The transformant of interest was cultured in LB medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline, and then rBacmid was extracted from the culture medium using a QIAprep Spin Miniprep Kit.

[0046] 1-6. Introduction of rBacmid into insect cells To obtain recombinant baculovirus (rBV) carrying the target construct, rBacmid was introduced into Sf9 cells by transfection. 6Sf9 cells were seeded at 1000 cells / well and allowed to settle on the bottom of the plate. While the cells were settling, the transfection reagent was prepared as follows. 8 μL of Cellfectin Reagent (Thermo Fisher Scientific) was mixed with 100 μL of Grace's insect medium, unsupplemented (Thermo Fisher Scientific). 100 μL of rBacmid solution prepared at 25 μg / mL in Grace's insect medium, unsupplemented was added to this solution, and the mixture was gently mixed by pipetting and then incubated at room temperature for 15 minutes. The cells were confirmed to have settled under a microscope, and the medium was removed. Plating medium (1.5 mL Grace's insect medium Supplemented with 10% FBS, 8.5 mL Grace's insect medium Unsupplemented (Thermo Fisher Scientific)) was added at 2.5 mL / well. The prepared transfection reagent was added to the wells in its entirety and incubated at 27°C for 3 to 5 hours. The supernatant was removed and 2 mL of Sf900 medium was added. To prevent evaporation of the culture medium, the wells were wrapped in plastic wrap, protected from light with aluminum foil, and cultured at 27°C for 1 week. The collected culture medium was centrifuged at 8000 rpm, 10 min, and 4°C, and the supernatant was used as P0 rBV seed.

[0047] 1-7. Amplification of rBV seeds 4x10 in a 250mL Erlenmeyer flask (Nalgene) 5 30 mL of Sf9 cell solution prepared at 1000 cells / mL was added. 1 mL of P0 rBV seed solution was added and cultured under shaking conditions at 27°C and 125 rpm for 1 week. The culture was centrifuged at 8000 rpm for 10 min at 4°C, and the collected supernatant was used as P1 rBV seed. The infectious titer of the rBV seed was measured by plaque assay. The obtained rBV seed was dispensed in 1 mL aliquots and frozen and stored at -80°C. If the infectious titer of the rBV seed was insufficient, further amplification was performed using the same procedure as necessary.

[0048] 1-8.Plaque assay-1 2.4x10 in a 6-well plate 6 Sf9 was seeded at 100 cells / well. After leaving the plate at room temperature to allow the cells to settle, the supernatant was removed and 900 μL of fresh Sf900 medium was added. 100 μL of rBV seed solution diluted 10-fold with Sf900 medium was added to each well. The plate was protected from light with aluminum foil and incubated at 27°C for 1 hour with gentle stirring every 15 minutes. The supernatant was removed and 3 mL of Overlay1 medium prepared as shown in Table 6 was added to each well. After leaving the plate at room temperature until solidified, the plate was wrapped in plastic wrap and aluminum foil, turned upside down, and cultured at 27°C for 4 days.

[0049] [Table 6]

[0050] 1-9.Plaque assay-2 The reagents were mixed as shown in Table 7 to prepare Overlay 2 medium. 2 mL of Overlay 2 medium was layered on each well and allowed to stand at room temperature to solidify. The plate was wrapped in plastic wrap and aluminum foil, turned upside down, and allowed to stand at 27°C for 2 days. When plaques could be visually confirmed, white light was applied to the bottom of the plate, and the plaques in the wells with the appropriate dilution ratio were counted. The average number of plaques in two wells was multiplied by the dilution ratio, and then multiplied by 10 to obtain the infectious titer (pfu / mL) of the rBV seed.

[0051] [Table 7]

[0052] 1-10.Production of VLPs VLP production was carried out in a suspension culture system using a 250 mL Erlenmeyer flask. 6The H5 cell solution prepared in H5 medium to give 100 cells / mL and rBV seeds were added to a 250 mL Erlenmeyer flask, and VLP production was carried out in a culture volume of 40 mL. The rBV seeds were added to the culture medium so that the MOI (Multiplicity of Infection) was 0.01 or more. The flask was shielded from light with aluminum foil, and cultured with shaking at 27°C and 125 rpm for one week. Two days after the start of culture, protease inhibitor (Roche) was added. One tablet of protease inhibitor was dissolved in 1 mL of H5 medium, and 100 μL was added per 10 mL of medium.

[0053] Purification of VLPs The entire culture was centrifuged at 8000 rpm, 10 min, and 4°C to roughly remove the cells. The mixture was then centrifuged at 11000 rpm, 1 h, and 4°C, and the supernatant was collected in a new centrifuge tube. The supernatant was transferred to a centrifuge tube (Beckman Coulter) and ultracentrifuged at 32000 rpm, 2 h, and 4°C using an Optima XPN-100 (Beckman Coulter) and an SW32-Ti rotor to precipitate proteins including VLPs. The supernatant was discarded, 1 mL of fresh H5 medium was added, and the mixture was left to stand at 4°C for several hours to swell the pellet. The swollen pellet was well suspended by pipetting until no precipitate remained. 1.8 g of CsCl (Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed out per tube, dissolved in 2 mL of H5 medium, and mixed with the pellet suspension. The mixture was placed in a centrifuge tube (Beckman Coulter) and ultracentrifuged at 40,000 rpm, 20 h, and 4°C using an SW55-Ti rotor. After centrifugation, white light was applied from above the tube, and only the band portion derived from VLP was collected with a pipette. The collected solution was transferred to a centrifuge tube for pelleting. The solution was made up with 1xPBS (Thermo Fisher Scientific), and ultracentrifuged at 32000 rpm, 2 h, and 4°C to precipitate the VLPs. The supernatant was discarded, and fresh H5 medium was added and left to stand overnight at 4°C to swell the pellet. The swollen pellet was well suspended by pipetting to obtain a purified VLP solution.

[0054] Quantification of VLPs The VLP concentration was quantified by the Bradford method. BSA diluted 2-fold from 0.5 mg / mL to 0.067 mg / mL was used as the standard substance. 200 μL of staining solution (Bio-Rad) was added to 800 μL of ion-exchanged water, and 20 μL of appropriately diluted sample was added and mixed well. The mixture was left to stand at room temperature for 5 minutes, and the absorbance at 600 nm was measured using a spectrophotometer.

[0055] 1-13. SDS-PAGE analysis of VLPs The acquisition of VLPs was confirmed by detection of VP1 protein by SDS-PAGE. 6.5 μL of sample prepared so that 1 μg was electrophoresed was mixed with 2.5 μL of 4xLDS sample buffer (Thermo Fisher Scientific), and 1 μL of 10xSample Reducing Agent (Thermo Fisher Scientific), and heat-treated at 100°C for 5 minutes. Electrophoresis was performed using NuPAGE 10% Bis-Tris Protein Gels (Thermo Fisher Scientific) at a constant pressure of 200 V for 40 minutes. After electrophoresis, the gel was stained with GelCode Blue Safe Protein Stain (Thermo Fisher Scientific). After decolorization with tap water, the gel image was captured with a scanner (EPSON, GT-9300UF) (Figure 1). As a result, synthesis of the target norovirus VP1 protein was confirmed.

[0056] (Example 2) Selection of VHH by cDNA display method 2-1. Preparation of cDNA for VHH display Construction of a DNA library encoding the single variable domain of the heavy chain of a heavy-chain antibody (VHH) of a full-length alpaca-derived antibody Two types of alpaca-derived naive VHH library genes, S-hinge and L-hinge, provided by RePHAGEN, were used as templates and amplified by PCR using an S-hinge VHH-specific primer pair (sequence numbers 126 and 127) and an L-hinge VHH-specific primer pair (sequence numbers 126 and 128).The genes were then amplified by extension PCR using overlapping PCR primers (sequence numbers 129 and 130) to prepare DNA fragments consisting of a T7 promoter, an omega (ω) enhancer, a Kozak consensus sequence, a VHH gene, a His tag, and a linker hybridization region (Y tag), to create a full-length VHH-encoding DNA library.

[0057] 2-2. Preparation of cDNA display Each cDNA display was prepared according to the following steps 2-3 to 2-7. The buffers used are listed in Table 8.

[0058] [Table 8]

[0059] 2-3. Transcription The full-length VHH-encoding DNA library prepared in 2-1 was transcribed using the 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 for each selection was 0.1 to 1 μg from the second round onwards. The obtained transcription product was purified using RNAClean XP (Beckman Coulter) according to the attached manual. The concentration of the purified product was quantified using NanoPad DS-11FX (DeNovix).

[0060] 2-4. Ligation 20 pmol of purified mRNA and 20 pmol of puromycin linker, cnvK riboG linker (Epsilon Molecular Engineering), were added 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 temperature was then lowered to 70 °C at a rate of 0.1 °C / sec, and incubated at 70 °C for 1 minute. The temperature was then lowered to 25 °C at a rate of 0.1 °C / sec, and then lowered to 10 °C at a rate of 2 °C / sec, and the cnvK linker was hybridized to the 3' end of the mRNA. Then, a UVP CrossLinker (CL-3000), 365 nm, 100-115 V (Analytik Jena) was used to irradiate 365 nm UV at 4060 mJ / cm. 2 By irradiation, the cnvK linker and mRNA were photocrosslinked to obtain an mRNA-linker complex.

[0061] 2-5. Preparation of mRNA display 6 pmol of the mRNA-linker complex was incubated at 30°C for 30 minutes using a 50 μL-scale cell-free translation system (Rabbit reticulocyte Lysate (nuclease-treated), manufactured by Promega). Next, MgCL2 and KCL were added to final concentrations of 75 mM and 900 mM, respectively, and incubated at 37°C for 1 hour to display a peptide corresponding to the mRNA 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.

[0062] 2-6. Preparation of cDNA display 60 μL of Dynabeads Myone streptavidin C1 (manufactured by Thermo Fisher Scientific) was placed in a Protein Lobind Tube, washed with 200 μL of binding buffer, and the mRNA display prepared by the method 2-5 above was added and stirred at 25 ° C for 30 minutes. After washing with 200 μL of binding buffer, the reverse transcription reaction was carried out by incubating at 42 ° C for 30 minutes in a reaction solution having the composition shown in Table 7 to prepare an mRNA / cDNA - VHH conjugate. After washing with 200 μL of binding buffer, 39 μL of His tag binding / washing 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 conjugate (hereinafter referred to as cDNA display) from Dynabeads Myone streptavidin C1 (manufactured by Veritas).

[0063] [Table 9]

[0064] 2-7. 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 / washing buffer, after which the cDNA display prepared by methods 2-1 to 2-6 was added and stirred for 30 minutes at 25° C. After washing with 200 μL of His tag binding / washing buffer, 30 μL of His tag elution buffer was added and stirred at 37° C. for 15 minutes to elute the cDNA display.

[0065] 2-8. In vitro selection experiments Selection was performed using cDNA display prepared from the library obtained in 2-7. The synthesis scale of cDNA display used in each selection round is shown in Table 10.

[0066] [Table 10]

[0067] 2-9. Screening procedure for selection cycle 1 (R1) 100 μL of 10 μg / mL GII.11 VLP, GII.18 VLP, or GII.19 VLP prepared using PBS was added to each well of an F96 Cert.Maxisorp Nunc-Immuno Plate (manufactured by Thermo Fisher Scientific), and the plate was sealed and left to stand overnight at 4°C. After removing the norovirus VLP dispersion that was not adsorbed to the well, 200 μL of PBST (PBS containing 0.05% (v / v) Tween20) was added and immediately removed. This washing operation was performed three times. 200 μL of 5% skim milk solution (PBST solvent) was added and incubated at room temperature for 1 hour, and then the 5% skim milk solution was carefully removed using a pipette. 200 μL of HBST (20 mM HEPES, 500 mM NaCl, 0.05% Tween20, pH 7.2) was added and immediately removed. This washing operation was performed three times. 100 μL of the VHH-presenting cDNA display library prepared using HBST was added to each well containing immobilized VLPs and incubated at room temperature for 1 hour. After incubation, the unreacted VHH-presenting cDNA display library was carefully removed using a pipette. 200 μL of HBST was added, left to stand at room temperature for 5 minutes, and then removed. This washing procedure was performed six times. After washing, 100 μL of 100 mM Tris (hydroxymethyl) aminomethane (pH 11) adjusted with nuclease-free water was added to each well, carefully pipetted, and left to stand at 37 ° C for 10 minutes to elute the VHH-presenting cDNA display bound to the immobilized VLP. The eluate was subjected to PCR using cnvK NewYtag for poly A (sequence number 131) and T7 omeganew (sequence number 132) as primers. The reaction solution was prepared by mixing 25 μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa biosystems), 1.5 μL of 10 μM cnvK NewYtag for poly A primer, 1.5 μL of 10 μM T7 omeganew primer, 12.5 μL of VHH-presenting cDNA display, and 9.5 μL of nuclease free water per sample. PCR was performed at 95 ° C for 2 minutes, followed by 22 cycles of 98 ° C for 20 seconds, 68 ° C for 15 seconds, and 72 ° C for 20 seconds, and then 72 ° C for 5 minutes. Agencourt AMPure XP (Beckman Coulter) was used to purify the PCR product. The obtained purified DNA library (hereinafter referred to as R1 library) was subjected to R2 selection.

[0068] 2-10. Screening procedure for selection cycle 2 (R2) A cDNA display library was prepared from the R1 library by the same method as above, at the synthesis scale shown in Table 3. 100 μL of 1 μg / mL GII.11 type VLP, GII.18 type VLP, or GII.19 type VLP prepared with PBS was added to each well of an F96 Cert.Maxisorp Nunc-Immuno Plate (manufactured by Thermo Fisher Scientific), and after sealing, the plate was left to stand overnight at 4 ° C. After removing the norovirus VLP dispersion that was not adsorbed to the well, 200 μL of PBST was added and immediately removed. This washing operation was performed three times. 200 μL of 5% skim milk solution (PBST solvent) was added, and the plate was incubated at room temperature for 1 hour, after which the 5% skim milk solution was carefully removed using a pipette. 200 μL of HBST was added and immediately removed. This washing operation was performed three times. 100 μL of the VHH-presenting cDNA display library prepared using HBST was added to each well containing immobilized VLPs and incubated at room temperature for 1 hour. After incubation, the unreacted VHH-presenting cDNA display library was carefully removed using a pipette. 200 μL of HBST was added, left to stand at room temperature for 5 minutes, and then removed. This washing procedure was performed six times. After washing, 100 μL of 100 mM Tris(hydroxymethyl)aminomethane (pH 11) adjusted with nuclease-free water was added to each well, carefully pipetted, and left to stand at 37°C for 10 minutes to elute the VHH-presenting cDNA display bound to the immobilized VLP. The eluate was subjected to PCR using cnvK NewYtag for poly A (sequence number 131) and T7 omeganew (sequence number 132) as primers. The reaction solution was prepared by mixing 25μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa biosystems), 1.5μL of 10μM cnvK NewYtag for poly A primer, 1.5μL of 10μM T7 omeganew primer, 12.5μL of VHH-presenting cDNA display, and 9.5μL of nuclease free water per sample. PCR was performed at 95℃ for 2 minutes, followed by 24 cycles of 98℃ for 20 seconds, 68℃ for 15 seconds, and 72℃ for 20 seconds, and then 72℃ for 5 minutes. Agencourt AMPure XP (Beckman Coulter) was used to purify the PCR product. The obtained purified DNA library (hereinafter referred to as R2 library) was subjected to R3 selection.

[0069] 2-11. Screening procedure for selection cycles (R3 and R4) A cDNA display library was prepared from the R2 library using the same method as above, with the synthesis scale shown in Table 3. Screening was then carried out up to R4 according to the method in 2-10. The resulting purified DNA library (hereinafter referred to as the R4 library) was subjected to R5 selection.

[0070] 2-12. Screening procedure for selection cycle 5 (R5) A cDNA display library was prepared from the R4 library by the same method as above, at the synthesis scale shown in Table 3. 100 μL of 0.1 μg / mL GII.11 type VLP or GII.19 type VLP adjusted with PBS was added to each well of an F96 Cert.Maxisorp Nunc-Immuno™ Plate (manufactured by Thermo Fisher Scientific), and after sealing, the plate was left to stand overnight at 4°C. After removing the norovirus VLP dispersion that was not adsorbed to the well, 200 μL of PBST was added and immediately removed. This washing operation was performed three times. 200 μL of 5% skim milk solution (PBST solvent) was added, and the plate was incubated at room temperature for 1 hour, after which the 5% skim milk solution was carefully removed using a pipette. 200 μL of HBST was added and immediately removed. This washing operation was performed three times. 100 μL of the VHH-presenting cDNA display library prepared using HBST was added to each well containing immobilized VLPs and incubated at room temperature for 1 hour. After incubation, the unreacted VHH-presenting cDNA display library was carefully removed using a pipette. 200 μL of HBST was added, left to stand at room temperature for 5 minutes, and then removed. This washing procedure was performed six times. After washing, 100 μL of 100 mM Tris(hydroxymethyl)aminomethane (pH 11) adjusted with nuclease-free water was added to each well, carefully pipetted, and left to stand at 37°C for 10 minutes to elute the VHH-presenting cDNA display bound to the immobilized VLP. The eluate was subjected to PCR using cnvK NewYtag for poly A (sequence number 131) and T7 omeganew (sequence number 132) as primers. The reaction mixture was prepared by mixing 25μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa biosystems), 1.5μL of 10μM cnvK NewYtag for poly A primer, 1.5μL of 10μM T7 omeganew primer, 12.5μL of VHH-presenting cDNA display, and 9.5μL of nuclease free water per sample. PCR was performed at 95℃ for 2 minutes, followed by 24 cycles of 98℃ for 20 seconds, 68℃ for 15 seconds, and 72℃ for 20 seconds, and then 72℃ for 5 minutes. Agencourt AMPure XP (Beckman Coulter) was used to purify the PCR product. This resulted in the R5 library.

[0071] 2-13. Next-generation sequencer analysis To confirm the degree of convergence of the R4 library or the R5 library in detail, sequence analysis was performed using a next-generation sequencer. The preparation of the sequence samples was performed with reference to the 2-step PCR Amplicon Library Preparation method provided by Illumina. First, Amplicon PCR was performed using the PCR products after each selection as templates and NGS Fw 1st PCR primer (SEQ ID NO: 133) and NGS Rv 1st PCR primer (SEQ ID NO: 134) as primers. The PCR conditions were 98°C for 1 minute, followed by 15 cycles of 98°C for 10 seconds, 62°C for 5 seconds, and 72°C for 35 seconds, and then 72°C for 1 minute. The obtained PCR products were purified according to the instructions for Agencourt AMPure XP (Beckman Coulter), and then index PCR was performed according to the instructions for Illumina. The resulting PCR product was purified according to the Agencourt AMPure XP instructions, and then its concentration was quantified using a NanoPad DS-11FX (DeNovix). Sequence analysis was then performed using MiSeq (Illumina) and MiSeq Reagent Nano kit v2 (500 cycles) according to the Illumina instructions.

[0072] 2-14. Sequencing data analysis The sequence data demultiplexed using the MiSeq Controller was subjected to analysis. The base sequence region encoding VHH was extracted from the sequence data and translated into amino acid sequence. The number of amino acid sequences that perfectly matched each amino acid sequence was then counted. As a result, NoVHH200 (SEQ ID NO: 32), NoVHH201 (SEQ ID NO: 33), NoVHH53 (SEQ ID NO: 35), and NoVHH54 (SEQ ID NO: 36) appeared frequently and were selected as antibodies with high affinity to the antigen.

[0073] In the amino acid sequence of NoVHH200 (sequence number 32), positions 1 to 26 are FR1 (sequence number 16), positions 27 to 36 are CDR1 (sequence number 1), positions 37 to 50 are FR2 (sequence number 21), positions 51 to 60 are CDR2 (sequence number 2), positions 61 to 99 are FR3 (sequence number 25), positions 100 to 116 are CDR3 (sequence number 3), and positions 117 to 127 are FR4 (sequence number 30).

[0074] In the amino acid sequence of NoVHH201 (sequence number 33), positions 1 to 26 are FR1 (sequence number 17), positions 27 to 36 are CDR1 (sequence number 4), positions 37 to 50 are FR2 (sequence number 22), positions 51 to 59 are CDR2 (sequence number 5), positions 60 to 98 are FR3 (sequence number 26), positions 99 to 114 are CDR3 (sequence number 6), and positions 115 to 125 are FR4 (sequence number 31).

[0075] In the amino acid sequence of NoVHH53 (sequence number 35), positions 1 to 26 are FR1 (sequence number 19), positions 27 to 36 are CDR1 (sequence number 10), positions 37 to 50 are FR2 (sequence number 23), positions 51 to 60 are CDR2 (sequence number 11), positions 61 to 99 are FR3 (sequence number 28), positions 100 to 104 are CDR3 (sequence number 12), and positions 105 to 115 are FR4 (sequence number 30).

[0076] In the amino acid sequence of NoVHH54 (sequence number 36), positions 1 to 26 are FR1 (sequence number 20), positions 27 to 36 are CDR1 (sequence number 13), positions 37 to 50 are FR2 (sequence number 24), positions 51 to 59 are CDR2 (sequence number 14), positions 60 to 98 are FR3 (sequence number 29), positions 99 to 116 are CDR3 (sequence number 15), and positions 109 to 127 are FR4 (sequence number 30).

[0077] Example 3: Selection of VHH by phage display method 3-1. In 2-10, the R2 library obtained by the selection using GII.18 VLP as the target molecule was used as a template, and PCR was performed using Alp_VHH_phagemid_F (SEQ ID NO: 135: CGATGGCCGCGGCCCATATGGCCATGGCTSAGKTGCAGCTCGTGGAGTC) and Alp_VHH_phagemid_R (SEQ ID NO: 136: CTAGGATCCTGCGGCCGCTCCGCCGTGATGATGATGATGATGGCTGCC) as primers at an annealing temperature of 55°C and an extension reaction of 60 seconds to add a restriction enzyme recognition sequence for ligation with the phagemid vector, and purified with AMPure XP (Beckman Coulter). FastDigest NotI (Thermo Fisher Scientific) was added to the phagemid and DNA library, and the mixture was reacted at 37°C for 1 hour, and then Fast Digest SfiI (Thermo Fisher Scientific) was added and the mixture was reacted at 50°C for 1 hour. The restriction enzyme-treated phagemid vector and DNA library were electrophoresed at 100V for 30 minutes on a 1% agarose gel containing 1x Gel green (Fujifilm Wako Pure Chemical Industries, Ltd.), and then extracted and purified using a Fast Gene Gel / PCR Extraction Kit (Nihon Genetics Co., Ltd.) according to the attached manual. The purified phagemid vector was dephosphorylated by adding FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific) and reacting at 37°C for 60 minutes, and then the enzyme was inactivated by reacting at 75°C for 5 minutes. The DNA library was mixed with the phagemid vector prepared above at a molar ratio of 5 to 10 times, Ligation high Ver.2 (Toyobo Co., Ltd.) was added, and the mixture was allowed to react overnight at 16°C. The obtained phagemid vector was concentrated by ethanol precipitation, and then transformed into E. coli TG-1 (competent cells for phage display, Lucigen Co., Ltd.) by electroporation, and cultured overnight at 30°C on a 2YTAG agar medium plate. All colonies were collected in 2YTAG liquid medium and cultured at 30°C until the OD600 reached 0.5 to 1. Then, helper phage was added in an amount 20 times that of E. coli to infect the E. coli with the helper phage, and the mixture was cultured overnight at 30°C in 2YT / Amp / Kan medium (containing 100 mg / mL ampicillin and 50 mg / mL kanamycin). The culture solution containing the cultivated E. coli was centrifuged at 4,000×g for 30 minutes at 4°C, and the supernatant was collected. A 20% PEG (polyethylene glycol) solution containing 2.5M NaCl was added to the supernatant, mixed by inversion, cooled on ice for 1 hour, and then centrifuged to remove the supernatant. The precipitate was dissolved in PBS (Phosphate Buffered Saline) containing 10% glycerol, and used as a phage library solution (hereinafter referred to as F-R0 library).

[0078] 3-2. Biopanning using ELISA plates GII.18 VLP was diluted to 10 μg / mL with PBS to prepare a GII.18 VLP immobilization solution. 100 μL of the GII.18 VLP immobilization solution was added to each well of a 96-well ELISA plate (Immuno Clear Standard Modules_C8_MaxiSorp:catsha-pu445101, Thermo Fisher Scientific) and immobilized overnight at 4 ° C. The same procedure was performed using PBS as a negative control. Each well was washed three times with PBS, and then 200 μL of 3% skim milk-containing PBS was added and allowed to stand at room temperature for 1 hour for blocking. Then, each well was washed three times with PBS. 50 μL of the F-R0 library prepared in 3-1 was mixed with 1 mL of 3% skim milk and 5% BSA-containing PBS, and 100 μL was added to each well and reacted at room temperature for 1 hour. The plate was washed four times with 200 μL of PBST, and then 200 μL of PBST was added and the plate was shaken for 5 minutes. This washing process was repeated twice. After washing, 100 μL of 100 mM trimethylamine solution was added to each well and collected. 100 μL of 100 mM trimethylamine solution was added to each well and left at room temperature for 10 minutes to elute the VHH-displaying phages bound to the GII.18 VLP. The eluate was immediately neutralized with 100 μL of 0.5 M Tris-HC1 buffer (pH 6.8). The eluate was mixed with 1200 μL of E. coli TG-1 and left at 30°C for 1 hour. 10 μL of the mixture was spread on 2YTAG agar medium (10 cm dish). The remaining mixture was centrifuged, and 100 μL of the precipitate (Escherichia coli TG-1) was plated on 2YTAG agar medium (15 cm dish). Each agar medium was cultured overnight at 30°C. All colonies that appeared on each agar medium were collected in 2YTAG liquid medium in a 500 mL Erlenmeyer flask and cultured at 30°C until OD600 reached 0.5-1. Then, helper phage was added in an amount 20 times that of E. coli to infect the E. coli with the helper phage, and cultured overnight at 30°C in 2YT / Amp / Kan medium. The culture solution containing the cultured E. coli was centrifuged at 4,000×g for 30 minutes at 4°C, and the supernatant was collected. A 20% PEG (polyethylene glycol) solution containing 2.5 M NaCl was added to the supernatant, and after mixing by inversion, the mixture was cooled on ice for 1 hour, and then centrifuged to remove the supernatant. The precipitate was dissolved by adding PBS containing 10% glycerol to create the F-R1 library, and biopanning was performed again in the same manner (the phage library obtained in this way is called the F-R2 library).

[0079] 3-3.Phage ELISA To confirm whether VHHs that bind to GII.18 VLPs were enriched in the library, phage ELISA was performed. GII.18 VLPs were diluted to 10 μg / mL with PBS to prepare a GII.18 VLP immobilization solution. 100 μL of the GII.18 VLP immobilization solution was added to each well of a 96-well ELISA plate and immobilized overnight at 4°C. Each well was washed three times with PBS, and then blocked by adding 200 μL of PBS containing 3% skim milk and leaving it to stand at room temperature for 1 hour. Then, each well was washed three times with PBS. 50 μL of the F-R0 library, F-R1 library, or F-R2 library was mixed with 50 μL of PBS containing 10% BSA, and the mixture was added to each well and reacted at room temperature for 1 hour. Then, each well was washed five times with 200 μL of PBST. Anti-M13-mAb-HRP (Sinobiologica) diluted 3000-fold with PBST was added to each well in an amount of 50 μL, and the mixture was allowed to react at room temperature for 1 hour. Each well was then washed six times with 200 μL of PBST. After washing, an OPD tablet (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 10 mL of 0.1 M NaH2PO4 aqueous solution, and 100 μL of the solution was added to each well and incubated for 5 minutes in the dark. After incubation, 100 μL of 1 M sulfuric acid was added, and the absorbance of each well was immediately measured at 490 nm using a Microplate Reader Infinite 200Pro M PLEX (Tecan). As a result, it was confirmed that the titers of the F-R1 library and the F-R2 library against GII.18 type VLPs were improved by biopanning.

[0080] 3-4.Monoclonal phage ELISA A monoclonal phage ELISA was performed to identify the amino acid sequence of VHH that binds to GII.18 type VLP in the F-R2 library. The F-R2 library obtained in 3-2 was mixed with E. coli TG-1 and left to stand at 30°C for 1 hour. The mixture was centrifuged, and the precipitate (E. coli TG-1) was dissolved in 2YTAG and plated on 2YTAG agar medium (15 cm dish). Sixteen colonies on the agar medium were randomly selected, and each phage display was prepared using the same method as in 3-1. Phage ELISA was performed using the same method as in 3-3. As a result, a clear response with an absorbance of 0.1 or more was obtained for 12 clones (Figure 4). For the clones that gave a response, phagemids were extracted from E. coli, and the base sequences corresponding to VHH were analyzed by Eurofins Genomics for DNA sequence analysis. As a result, it was confirmed that the VHHs displayed on phages #1, #2, #6, #8, #10, #11, #14 and #15 in Figure 4 consist of the same amino acid sequence, and NoVHH191E (sequence number 34) was identified as a clone that binds to GII.18 VLP.

[0081] In the amino acid sequence of NoVHH191E (sequence number 34), positions 1 to 26 are FR1 (sequence number 18), positions 27 to 36 are CDR1 (sequence number 7), positions 37 to 50 are FR2 (sequence number 21), positions 51 to 57 are CDR2 (sequence number 8), positions 58 to 96 are FR3 (sequence number 27), positions 97 to 110 are CDR3 (sequence number 9), and positions 111 to 121 are FR4 (sequence number 30).

[0082] Example 4: Production of VHHs by protease-deficient recombinant Bacillus subtilis 4-1. Artificial synthesis of genes The amino acid sequence of the synthesized VHH was added to the C-terminal side of the amino acid sequence of VHH of NoVHH200 (SEQ ID NO: 32), NoVHH201 (SEQ ID NO: 33), NoVHH191E (SEQ ID NO: 34), NoVHH53 (SEQ ID NO: 35) or NoVHH54 (SEQ ID NO: 36) via a linker sequence. The VHHs synthesized in this manner are called His-tagged NoVHH200 (SEQ ID NO: 137), His-tagged NoVHH201 (SEQ ID NO: 138), His-tagged NoVHH191E (SEQ ID NO: 139), His-tagged NoVHH53 (SEQ ID NO: 140), and His-tagged NoVHH54 (SEQ ID NO: 141). These VHHs shown in SEQ ID NOs: 139 to 143 are collectively referred to as His-tagged VHHs. The artificial synthetic genes for synthesizing these His-tagged VHHs were synthesized using Thermo Fisher Scientific's gene synthesis service, and were used in the experiments: an artificial synthetic gene for His-tagged NoVHH200 (sequence number 142), an artificial synthetic gene for His-tagged NoVHH201 (sequence number 143), an artificial synthetic gene for His-tagged NoVHH191E (sequence number 144), an artificial synthetic gene for His-tagged NoVHH53 (sequence number 145), and an artificial synthetic gene for His-tagged NoVHH54 (sequence number 146).

[0083] 4-2. Construction of plasmid for expressing His-tagged VHH The plasmid sequence was amplified by PCR using the recombinant plasmid pHY-S237 (JP Patent Publication 2014-158430) prepared based on pHY300PLK as a template, a primer set of 5'-GATCCCCGGGAATTCCTGTTATAAAAAAAGG-3' (SEQ ID NO: 147) and 5'-ATGATGTTAAGAAAGAAAACAAAGCAG-3' (SEQ ID NO: 148) and PrimeSTAR Max DNA polymerase (TaKaRa). The promoter DNA derived from the spoVG gene was amplified by PCR using the genome of the 168 strain as a template and a primer set of 5'-GAATTCCCGGGGATCTAAGAAAAGTGATTCTGGGAGAG-3' (SEQ ID NO: 149) and 5'-CTTTCTTAACATCATAGTAGTTCACCACCTTTTCCC-3' (SEQ ID NO: 150). The obtained promoter DNA was incorporated into a plasmid sequence using In-Fusion HD Cloning Kit (Takara) to construct a VHH expression plasmid linked to the spoVG promoter. The plasmid sequence was amplified by PCR using a primer set of 5'-TGCTGCAAGAGCTGCCGGAAATAAA-3' (SEQ ID NO: 151) and 5'-TCTATTAAACTAGTTATAGGG-3' (SEQ ID NO: 152) and PrimeSTAR Max DNA polymerase (TaKaRa). DNA containing an artificially synthesized gene was incorporated into the obtained PCR fragment using In-Fusion HD Cloning Kit (Takara) to construct a VHH expression plasmid containing each of the artificially synthesized VHH genes.

[0084] 4-3. Construction of recombinant Bacillus subtilis A strain lacking extracellular protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, aprX) was prepared from Bacillus subtilis 168 strain (hereinafter referred to as 168 strain) according to the method described in Japanese Patent No. 4485341. In addition, the sigF gene involved in sporulation was deleted from Bacillus subtilis strain Dpr9, which lacks all of the above nine extracellular protease genes, according to the method described in Japanese Patent No. 4336082. The obtained extracellular protease multiple deletion strain is referred to as Dpr9ΔsigF. Plasmid introduction into the Dpr9ΔsigF strain was performed by the protoplast method described below. Glycerol-stocked Bacillus subtilis was inoculated into 1 mL of LB liquid medium and cultured overnight at 30°C and 210 rpm with shaking. The next day, 10 μL of this culture was inoculated into a new 1 mL of LB liquid medium and cultured at 37°C and 210 rpm with shaking for about 2 hours. This culture was collected in a 1.5 mL tube and centrifuged at 1,2000 rpm for 5 minutes. The pellet from which the supernatant was removed 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-Aldrich) and incubated at 37°C for 1 hour. The mixture was then centrifuged at 3,500 rpm for 10 minutes, the supernatant was removed, and the pellet was suspended in 400 μL of SMMP. 33 μL of this suspension was mixed with various plasmids, and 100 μL of 40% PEG was added and vortexed. 350 μL of SMMP was added to this solution, mixed by inversion, and shaken at 30°C and 210 rpm for 1 hour. The entire amount was then applied to a DM3 agar medium plate and incubated at 30°C for 2 to 3 days.

[0085] 4-4.VHH production The recombinant Bacillus subtilis prepared in 4-3 was inoculated into 1 mL of LB medium containing 50 ppm tetracycline, and shaken at 30°C overnight to prepare a preculture solution. The preculture solution was inoculated at 1% into 20 mL of 2×L-mal medium placed in a pleated Erlenmeyer flask, and cultured at 30°C for 72 hours with shaking. At the end of the culture, 1 mL of the culture solution 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 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the kit protocol. The purified protein was dissolved in PBS containing 30 mM imidazole.

[0086] 4-5.Confirmation by SDS-PAGE The sample collected in 4-4 was mixed with an equal amount of Laemmli Sample Buffer (BIO-RAD) and then heated at 99°C for 5 minutes to prepare the sample. Mini-PROTEIN TGX Stain-Free (BIO-RAD) was used as the gel. 5 μL of sample was applied to each well and electrophoresed 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 a ChemiDoc MP Imaging System. The detected protein was quantified using a calibration curve created based on a lysozyme standard (Sigma-Aldrich). As a result, it was confirmed that the desired His-tagged VHH had been synthesized (Figure 5).

[0087] Example 5: Evaluation of binding specificity by ELISA method 100 μL of 10 μg / mL each norovirus VLP prepared using PBS was added to each well of an F96 Cert.Maxisorp Nunc-Immuno™ Plate (manufactured by Thermo Fisher Scientific), and after sealing, the plate was left to stand overnight at 4° C. After removing the norovirus VLP dispersion that had not been adsorbed to the well, 200 μL of PBST (PBS containing 0.05% (v / v) Tween 20) was added and immediately removed. This washing operation was performed three times. 200 μL of 5% skim milk solution (PBST solvent) was added, incubated at room temperature for 1 hour, and then the 5% skim milk solution was carefully removed using a pipette. 200 μL of PBST was added and immediately removed. This washing operation was performed three times. To each well where VLPs were immobilized, 100 μL of 2 μg / mL of each His-tagged VHH prepared using PBST was added, and after incubation at room temperature for 1 hour, unreacted VHH was carefully removed using a pipette. 200 μL of PBST was added and immediately removed. This washing procedure was performed three times. Anti-His-tag mAb-Biotin (Monoclonal, OGHis) (manufactured by MEDICAL & BIOLOGICAL LABORATORIES) was used. The primary antibody was diluted 1 / 5,000 with PBST. 100 μL of the primary antibody was added to each well, and incubated at room temperature for 1 hour. After carefully removing the primary antibody using a pipette, 200 μL of PBST was added and carefully removed using a pipette. This washing procedure was performed three times. Streptavidin HRP Conjugate (manufactured by Tokyo Chemical Industry Co., Ltd.) was used for detection of the primary antibody. Streptavidin HRP Conjugate was diluted 1 / 5,000 with PBST, and 100μL was added to each well and incubated at room temperature for 1 hour. After carefully removing the Streptavidin HRP Conjugate with a pipette, 200μL of PBST was added and carefully removed with a pipette. This washing procedure was repeated three times.The colorimetric substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of the colorimetric substrate was added to each well, incubated for 30 minutes in the dark, and the absorbance was immediately measured at 450 nm using a Microplate Reader Infinite M1000 PRO (TECAN). The signal / noise (S / N) ratio was calculated based on the obtained values.

[0088] The S / N ratio of 1 / 10 or more of the maximum S / N ratio obtained by ELISA using each VHH was judged to be positive (denoted by ◯), and the S / N ratio of less than 1 / 10 was judged to be negative (denoted by ×). The binding activity of His-tagged VHH to each genotype VLP is shown in Table 9. It was confirmed that NoVHH200 and NoVHH201 exhibited specific binding activity to GII.11 type VLP, NoVHH191E exhibited specific binding activity to GII.18 type VLP, NoVHH53 exhibited specific binding activity to GII.11 type VLP and GII.19 type VLP, and NoVHH54 exhibited specific binding activity to GII.19 type VLP. In other words, it was found that porcine norovirus can be specifically detected by using NoVHH200, NoVHH201, NoVHH191E, NoVHH53, and NoVHH54.

[0089] [Table 11]

[0090] (Example 6) Preparation of anti-GII type norovirus polyclonal antibody 6-1. Preparation of anti-GII norovirus-binding polyclonal antibodies Anti-GII norovirus-binding rabbit polyclonal antibodies were prepared using a mixture of GII norovirus genotype VLPs (GII.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 17, 18, 19, 21, 22) as antigens. The antigens were sent to Eurofins Genomics, Inc., and whole blood antiserum was obtained. Contaminants were removed from 50 μL of antiserum using a Melon Gel IgG purification kit (manufactured by Thermo Fisher Scientific), and 500 μL of a solution containing IgG antibodies was obtained. The operation was performed according to the protocol attached to the kit. The antibody concentration of the purified solution was quantified by the Bradford method using BSA as a standard substance, as in 1-12. The antibody obtained in this manner is called anti-GII pAb.

[0091] 6-2. Evaluation of binding activity to norovirus VLPs 100 μL of 25 μg / mL norovirus VLP solution prepared using PBS was added to each well of F96 Cert.Maxisorp Nunc-Immuno Plate (manufactured by Thermo Fisher Scientific), and after sealing, the plate was left to stand overnight at 4 ° C. After removing the solution in the well, 200 μL of Blocking Reagent for ELISA-Chemically Defined- (manufactured by Cosmo Bio) was added, the top surface of the plate was sealed, and the plate was left to stand at room temperature for 2 hours. After removing the solution in the well, 250 μL of PBST (PBS containing 0.05% (v / v) Tween 20) was added and immediately removed. This washing operation was performed three times. 100 μL of anti-GII pAb prepared to 2 μg / mL using Blocking Reagent for ELISA-Chemically Defined- was added to the well and incubated at room temperature for 50 minutes. After removing the solution in the well, 250 μL of PBST was added and immediately removed. This washing operation was performed four times. 100μL of secondary antibody (Anti-rabbit IgG, HRP-linked Antibody, Cell Signaling Technology) diluted 1 / 1000 using Blocking Reagent for ELISA-Chemically Defined- was added to the wells and incubated at room temperature for 50 minutes. After removing the solution in the wells, 250μL of PBST was added and immediately removed. This washing procedure was performed four times. TMB ELISA Substrate Highest Sensitivity (Abcam) was used as the color-developing substrate. 100μL of color-developing substrate was added to each well and incubated for 25 minutes in the dark. 100μL of 0.5M sulfuric acid (Fujifilm Wako Pure Chemical) was added to stop the reaction. The absorbance at 450nm was measured using a microplate reader (SpectraMax190, Molecular Devices) (Figure 6). As a result, it was confirmed that anti-GII pAb exhibits a wide range of binding activity to GII norovirus VLPs.

[0092] Example 7 Detection of Norovirus VLPs by Lateral Flow Immunoassay 7-1. Sensitization of labeled antibodies to pigment particles Colored cellulose particles NanoAct (chemically bonded type, manufactured by Asahi Kasei Corporation) were used as the dye particles. Sensitization with the detection antibody was performed according to the attached protocol. 60 μL of NanoAct was dispensed into a 15 mL centrifuge tube, 540 μL of 100 mM MES (pH 6), 7.5 μL of 4 wt% EDC (N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide), and 15 μL of 4 wt% NHS were added thereto, and the tube was left to stand at room temperature for 15 minutes to perform esterification of the carboxyl group. The tube was centrifuged at 13,000 × g for 20 minutes to remove the supernatant, and 600 μL of 100 mM MES (pH 6) was added and sonicated. 60 μg of His-tagged NoVHH53 or His-tagged NoVHH54 was added as the detection antibody, followed by vortexing and incubation at 37 ° C for 120 minutes. Next, 7.2 mL of blocking buffer (1 wt% Casein, 100 mM Boric Acid, pH 8.5) was added, followed by vortexing and incubation at 37°C for 60 minutes. The mixture was centrifuged at 13,000×g for 20 minutes to remove the supernatant, and 600 μL of 100 mM MES (pH 6) was added and sonicated. The mixture was centrifuged again at 13,000×g for 15 minutes to remove the supernatant, and 1.3 mL of storage solution (33 mM Boric Acid, 0.2 wt% Casein, 15 wt% Sucrose, pH 9.2) was added, followed by sonication, and stored at 4°C until use (hereinafter referred to as sensitized particles).

[0093] 7-2. Assembling the Half Strip FF120HP Plus or FF80 HP Plus (Cytiva) cut to 5 mm x 40 mm was used as the nitrocellulose membrane. CM5 (Cytiva) cut to 5 mm x 20 mm was used as the absorbent pad. The nitrocellulose membrane and absorbent pad were laminated together with a 10 mm overlap and adhered with a backing sheet (GL-57888, Lohmann) to assemble a half strip.

[0094] 7-3. Immobilization of capture antibodies onto nitrocellulose membrane The capture antibody used was His-tagged NoVHH53 or His-tagged NoVHH54. EZ-Link was used as the biotinylation reagent. TM Biotin was added to the His-tagged VHH using NHS-LC-LC-Biotin (Thermo Fisher Scientific). 1 mg / mL of His-tagged VHH was prepared using PBS. 10 mM of biotinylation reagent was added to the VHH solution so that the number of molecules was 20 times that of VHH, and the solution was left to stand overnight at 4°C. Then, Zeba TM Unreacted biotinylation reagent was removed by replacing with PBS using a Spin Desalting Column, 7K MWCO, 0.5mL (Thermo Fisher Scientific). Biotinylated VHH prepared to 1mg / mL using PBS and streptavidin (SA) prepared to 20mg / mL (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed (biotinylated VHH (μL): SA (μL) = 25:1) and left to stand at room temperature for 5 minutes. This allowed the formation of a VHH-SA complex. 1μL of the VHH-SA complex was spotted with a pipette at a location 2cm from the bottom end of the strip assembled in 6-2. Anti-GII pAb prepared to 1mg / mL was used as a control. Then, immobilization was performed by drying at 37℃ for 30 minutes. In the following, the site where the capture antibody was immobilized is referred to as the test spot.

[0095] 7-4. Evaluation using dipstick-type lateral flow assay The developing solution used was borate buffer (100 mM boric acid, 1% casein, 150 mM NaCl, pH 8.2). Using the developing solution, 0.5 to 5000 ng / mL GII.19 type VLP dispersion was prepared (hereinafter referred to as positive sample). For conditions not containing GII.19 type VLP, borate buffer was used as the developing solution (hereinafter referred to as negative sample). 100 μL of positive or negative sample and 20 μL of sensitized particles prepared in 6-1 were added to one well of a 96-well plate and mixed well by pipetting. Then, the half strip prepared in 6-3 was immersed by standing it up against the well and left to stand until the developing solution had run out. Then, the presence or absence of coloring in the test spot was judged visually. The criteria for judging the presence or absence of coloring are as shown in FIG. 7. The results of the lateral flow assay are shown in Table 9. As a result, we found that GII.19 VLPs could be detected with high sensitivity by lateral flow assay using NoVHH53 and NoVHH54, indicating that the antibodies can be used in lateral flow assays.

[0096] [Table 12]

Claims

1. An antibody that binds to porcine norovirus, having one or more structural domains containing CDRs shown in (a), (b), (c), (d) or (e) below. (a) CDR1 consisting of the amino acid sequence represented by GLTFSMYSMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by SINWSGGSTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DFSFLSRCKLGSSGYDY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence represented by GIVFSVYPMG (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GINSFHNTT (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by GAYRRLCPIEEYGMDF (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence represented by GRAFSSYMVG (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AIAWSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by GFPTLVALPYEYDY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence represented by GITFSNTAMT (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by SINKSGDEVA (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by PYFGS (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence represented by GSIFSFNAMA (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence represented by GITSGGRTS (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence represented by TRWATNSIAIRQVESYDY (SEQ ID NO: 15).

2. The antibody of claim 1, which binds to a porcine norovirus structural protein.

3. The antibody of claim 1, which is a single domain antibody or a multimer thereof.

4. The antibody of claim 3, wherein the single domain antibody is a VHH antibody.

5. The antibody according to claim 3, wherein the single domain antibody multimer is a multimer in which multiple structural domains are linked together.

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

7. A nucleic acid encoding the antibody according to any one of claims 1 to 6.

8. A method for detecting porcine norovirus in a sample, comprising the step of contacting a test sample with an antibody that binds to norovirus and has one or more structural domains containing CDRs shown in (a), (b), (c), (d), or (e) below. (a) CDR1 consisting of the amino acid sequence represented by GLTFSMYSMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by SINWSGGSTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DFSFLSRCKLGSSGYDY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence represented by GIVFSVYPMG (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GINSFHNTT (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by GAYRRLCPIEEYGMDF (SEQ ID NO: 6). (c) CDR1 consisting of the amino acid sequence represented by GRAFSSYMVG (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AIAWSTY (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by GFPTLVALPYEYDY (SEQ ID NO: 9). (d) CDR1 consisting of the amino acid sequence represented by GITFSNTAMT (SEQ ID NO: 10), CDR2 consisting of the amino acid sequence represented by SINKSGDEVA (SEQ ID NO: 11), and CDR3 consisting of the amino acid sequence represented by PYFGS (SEQ ID NO: 12). (e) CDR1 consisting of the amino acid sequence represented by GSIFSFNAMA (SEQ ID NO: 13), CDR2 consisting of the amino acid sequence represented by GITSGGRTS (SEQ ID NO: 14), and CDR3 consisting of the amino acid sequence represented by TRWATNSIAIRQVESYDY (SEQ ID NO: 15).

9. A porcine norovirus detection kit comprising the antibody according to any one of claims 1 to 6.

10. A preventive or therapeutic agent for porcine norovirus infection, comprising the antibody according to any one of claims 1 to 6.

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