Norovirus VP1 protein antibody and its application

Abstract

The present invention discloses an anti-norovirus VP1 protein antibody and its applications. The present invention discloses an anti-VP1 protein antibody, its fragment or fusion protein, which comprises a heavy chain variable region and a light chain variable region. The present invention also provides an isolated polynucleotide, a vector, a host cell and a pharmaceutical composition comprising the aforementioned anti-VP1 protein antibody, its fragment or fusion protein. The present invention also discloses a method for preparing the aforementioned anti-VP1 protein antibody, its fragment or fusion protein, a method for detecting the presence or amount of VP1 protein or norovirus in a sample. The present invention also provides the use in a corresponding medicament for treating, preventing or alleviating a disease or disorder responsive to the inhibition of VP1 protein, and the use in the production of a product for detecting VP1 protein or norovirus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an anti-norovirus VP1 protein antibody and its application. Background Art

[0002] Norovirus infections impose a substantial global disease burden, estimated to be associated with 20% of all diarrheal cases worldwide and more than 200,000 deaths annually. Norovirus is the leading cause of foodborne disease outbreaks in North America and the pathogen responsible for most hospital-acquired outbreaks among the elderly. Norovirus strains are also considered the leading cause of gastrointestinal illness in children globally.

[0003] Norovirus belongs to one of several genera within the family Caliciviridae. Noroviruses are classified based on the phylogenetic clustering of their VP1 amino acid sequences. To date, seven genogroups (GI to GVII) have been classified, with only GI, GII, and GIV known to infect humans. Among the 32 specific genotypes currently associated with human infections, GII.4 norovirus is responsible for most recent norovirus outbreaks. New strains of GII.4 emerge every two to three years, evolving through a process driven by mutations in epitopes that define regions of the hypervariable P2 domain of VP1. This process allows norovirus to evade the humoral immune response acquired from prior exposure to earlier strains.

[0004] Today, norovirus has become the main cause of non-bacterial acute diarrhea. Among all non-bacterial diarrhea outbreaks in the United States each year, 60% - 90% are caused by norovirus. Among children under 5 years old with diarrhea in China, the detection rate of norovirus is about 15%. However, so far, there is no specific drug on the market that can effectively treat norovirus infections. Therefore, studying rapid and accurate early detection methods for norovirus is of profound significance for the prevention and treatment of this virus. Summary of the Invention

[0005] To solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0006] The present invention provides an anti-VP1 protein antibody, a fragment or a fusion protein thereof, which comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises H-CDR1 with the amino acid sequence shown in SEQ ID NO: 1, H-CDR2 with the amino acid sequence shown in SEQ ID NO: 2, and H-CDR3 with the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof containing up to 3, 2 or 1 amino acid substitutions in the H-CDRs; and the VL comprises L-CDR1 with the amino acid sequence shown in SEQ ID NO: 4, L-CDR2 with the amino acid sequence shown as YAS, and L-CDR3 with the amino acid sequence shown in SEQ ID NO: 5, or a variant thereof containing up to 3, 2 or 1 amino acid substitutions in the L-CDRs.

[0007] Furthermore, the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region with the amino acid sequences shown in SEQ ID NOs: 4, YAS and SEQ ID NO: 5;

[0008] Furthermore, the heavy chain variable region comprises one or more heavy chain framework regions HFR1, HFR2, HFR3 and HFR4, and / or one or more light chain framework regions LFR1, LFR2, LFR3 and LFR4, wherein HFR1 comprises the amino acid sequence shown in SEQ ID NO: 6, or a homologous sequence having at least 85% sequence identity therewith,

[0009] HFR2 comprises the amino acid sequence shown in SEQ ID NO: 7, or a homologous sequence having at least 85% sequence identity therewith,

[0010] HFR3 comprises the amino acid sequence shown in SEQ ID NO: 8, or a homologous sequence having at least 85% sequence identity therewith,

[0011] HFR4 comprises the amino acid sequence shown in SEQ ID NO: 9, or a homologous sequence having at least 85% sequence identity therewith,

[0012] LFR1 comprises the amino acid sequence shown in SEQ ID NO: 10, or a homologous sequence having at least 85% sequence identity therewith,

[0013] LFR2 comprises the amino acid sequence shown in SEQ ID NO: 11, or a homologous sequence having at least 85% sequence identity therewith,

[0014] LFR3 contains the amino acid sequence shown in SEQ ID NO: 12, or a homologous sequence having at least 85% sequence identity therewith, and

[0015] LFR4 contains the amino acid sequence shown in SEQ ID NO: 13, or a homologous sequence having at least 85% sequence identity therewith.

[0016] In some embodiments, the HFR1 contains the amino acid sequence shown in SEQ ID NO: 6, or a homologous sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith,

[0017] HFR2 contains the amino acid sequence shown in SEQ ID NO: 7, or a homologous sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith,

[0018] HFR3 contains the amino acid sequence shown in SEQ ID NO: 8, or a homologous sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith,

[0019] HFR4 contains the amino acid sequence shown in SEQ ID NO: 9, or a homologous sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith,

[0020] LFR1 contains the amino acid sequence shown in SEQ ID NO: 10, or a homologous sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith,

[0021] LFR2 contains the amino acid sequence shown in SEQ ID NO: 11, or a homologous sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith,

[0022] LFR3 comprises the amino acid sequence shown in SEQ ID NO: 12, or a homologous sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith, and

[0023] LFR4 comprises the amino acid sequence shown in SEQ ID NO: 13, or a homologous sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

[0024] Furthermore, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14, or a homologous sequence having at least 85% sequence identity therewith; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or a homologous sequence having at least 85% sequence identity therewith.

[0025] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14, or a homologous sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or a homologous sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

[0026] Furthermore, the anti-VP1 protein antibody, its fragment or fusion protein is a monoclonal antibody, bispecific antibody, multispecific antibody, recombinant antibody, chimeric antibody, labeled antibody, bivalent antibody, anti-idiotype antibody or fusion protein.

[0027] Furthermore, the anti-VP1 protein antibody, its fragment or fusion protein is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single-domain antibody, nanobody, domain antibody or bivalent domain antibody.

[0028] Further, the anti-VP1 protein antibody, its fragment or fusion protein is a murine antibody, a simian antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0029] Further, the anti-VP1 protein antibody, its fragment or fusion protein is humanized.

[0030] In some embodiments, the anti-VP1 protein antibody, its fragment or fusion protein is selected from at least one of monoclonal antibody, polyclonal antibody, neutralizing antibody, antagonistic antibody, non-trehalosylated antibody, monospecific antibody, multispecific antibody, nanobody, Fab fragment, Fab' fragment, F(ab')2 fragment, Fd fragment, Fv fragment, dAb fragment, isolated CDR region, and scFv.

[0031] Further, the anti-VP1 protein antibody, its fragment or fusion protein further comprises substitution or modification of one or more amino acid residues, but retains the binding specificity to the VP1 protein.

[0032] Further, the anti-VP1 protein antibody, its fragment or fusion protein specifically binds to the VP1 protein; wherein the VP1 protein has the amino acid sequence shown in SEQ ID NO: 16.

[0033] In some embodiments, the anti-VP1 protein antibody, its fragment or fusion protein further comprises a constant region (Fc region), and the constant region is a heavy chain constant region and / or a light chain constant region. In some embodiments, the heavy chain constant region is the heavy chain constant region of IgG, IgA, IgM, IgE or IgD or a variant thereof; the heavy chain constant region is preferably the heavy chain constant region of IgG or a variant thereof, such as the constant region of IgG1, IgG2, IgG3 or IgG4 or a variant thereof; the heavy chain constant region is more preferably the heavy chain constant region of IgG1 or a variant thereof; for example, the heavy chain constant region of human IgG1 or a variant thereof. In some embodiments, the Fc region binds to an Fcγ receptor, such as: FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa or FcγRIIIb.

[0034] Further, it is linked to one or more conjugate moieties.

[0035] Further, the conjugate moiety comprises a reagent for detection or separation, such as a clearance-improver, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent tag, a fluorescent tag, an enzyme-substrate tag, a DNA-alkylating agent, a topoisomerase inhibitor, a tubulin-binding agent, or other anti-norovirus drugs.

[0036] Further, the VP1 protein is a norovirus VP1 protein.

[0037] Further, the VP1 protein is a norovirus type 1 VP1 protein.

[0038] As a non-limiting example, the anti-VP1 protein antibody, its fragment or fusion protein has one or more of the following characteristics:

[0039] 1) It can bind to human VP1;

[0040] 2) It can bind to murine VP1;

[0041] 3) It can bind to simian VP1;

[0042] 4) It can bind to rabbit VP1;

[0043] 5) It competes with one or more VP1 ligands for binding to VP1;

[0044] 6) It inhibits and / or blocks the binding of VP1 to the ligand;

[0045] 7) It inhibits and / or blocks the intracellular signal transduction mediated by the binding of VP1 to the ligand;

[0046] 8) It enhances the immune response;

[0047] 9) It has at least one of antibody-dependent cell-mediated cytotoxic activity, complement-dependent cytotoxic activity, and antibody-mediated phagocytosis activity.

[0048] In some embodiments, the one or more VP1 ligands are selected from Escherichia coli cells, apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids, negatively charged phospholipids, phosphatidylserine, sulfatide, phosphatidylcholine, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides, and lipidated amyloid β peptides.

[0049] The present invention provides an isolated polynucleotide encoding the aforementioned anti-VP1 protein antibody, its fragment or fusion protein.

[0050] The present invention provides a vector comprising the aforementioned isolated polynucleotide.

[0051] The present invention provides a host cell comprising the aforementioned vector.

[0052] The present invention provides a pharmaceutical composition comprising:

[0053] The aforementioned anti-VP1 protein antibody, its fragment or fusion protein, or a polynucleotide encoding the aforementioned anti-VP1 protein antibody, its fragment or fusion protein; and

[0054] One or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

[0055] Further, the pharmaceutical composition further comprises other therapeutic agents.

[0056] Further, the other therapeutic agent is an agent for treating norovirus.

[0057] The present invention provides a method for expressing the aforementioned anti-VP1 protein antibody, its fragment or fusion protein, which comprises culturing the aforementioned host cell under conditions for expressing the aforementioned vector, and finally isolating the aforementioned anti-VP1 protein antibody, its fragment or fusion protein.

[0058] The present invention provides a method for detecting the presence or amount of VP1 protein or norovirus in a sample, which comprises contacting the sample with the aforementioned anti-VP1 protein antibody, its fragment or fusion protein, and determining the presence or amount of VP1 protein or norovirus in the sample.

[0059] Further, the method further comprises the step of determining whether the VP1 protein is expressed in the cells in the sample.

[0060] Further, the VP1 protein is norovirus VP1 protein.

[0061] Further, the norovirus is norovirus type 1.

[0062] The present invention provides the use of the aforementioned anti-VP1 protein antibody, its fragment or fusion protein, the aforementioned isolated polynucleotide, the aforementioned vector, and the aforementioned host cell in the production of a medicament for treating, preventing or alleviating a disease or disorder responsive to inhibition of VP1 protein.

[0063] Further, the VP1 protein is norovirus VP1 protein.

[0064] Further, the VP1 protein is norovirus type 1 VP1 protein.

[0065] The present invention provides the use of the aforementioned anti-VP1 protein antibody, its fragment or fusion protein, the aforementioned isolated polynucleotide, the aforementioned vector, and the aforementioned host cell in the production of a product for detecting VP1 protein or norovirus.

[0066] Further, the VP1 protein is norovirus VP1 protein.

[0067] Further, the norovirus is norovirus type 1.

[0068] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen. A native intact antibody comprises two heavy chains (H) and two light chains (L). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and each heavy chain consists of a variable region (VH) and first, second, third, and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are classified as λ or κ, and each light chain consists of a variable region (VL) and a constant region. An antibody has a "Y" shape, wherein the stem of the Y consists of the second and third constant regions of the two heavy chains joined together by disulfide bonds. Each arm of the Y includes the variable region and the first constant region of a single heavy chain that binds to the variable region and the constant region of a single light chain. The variable regions of the light and heavy chains result in antigen binding. The variable regions in both chains typically contain three hypervariable loops called complementarity determining regions (CDRs) (light chain CDRs, including LCDR1, LCDR2, and LCDR3, and heavy chain CDRs, including HCDR1, HCDR2, and HCDR3).The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3):651-63(1985); Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83(1989); Kabat E.A. et al., Sequences of Proteins of immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77(2003); Marie-Paule Lefranc et al., Immunome Research, 1(3),(2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Chapter 26, 481-514, (2015)) conventions. The three CDRs are inserted between flanking extensions called framework regions (FRs) (light chain FRs, including LFR1, LFR2, LFR3, and LFR4, and heavy chain FRs, including HFR1, HFR2, HFR3, and HFR4), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen-binding but exhibit various effector functions. Based on the amino acid sequences of their heavy chain constant regions, antibodies are divided into different types. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the main antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0069] As used herein, the term "homologous" refers to a nucleic acid sequence (or its complementary strand) or an amino acid sequence that, when optimally aligned, has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence.

[0070] A "separated" substance has been changed from its natural state by a human. If a "separated" composition or substance occurs naturally, it has been changed or removed from its original environment or both. For example, a polynucleotide or polypeptide that occurs naturally in a living animal is not "separated", but the same polynucleotide or polypeptide is "separated" if it has been sufficiently separated from the coexisting materials in its natural state to exist in a substantially pure state. A "separated nucleic acid sequence" refers to a sequence of a separated nucleic acid molecule. In certain embodiments, a "separated antibody or its antigen-binding fragment" refers to an antibody or its antigen-binding fragment having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoresis (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatography (such as ion-exchange chromatography or reverse-phase HPLC).

[0071] As used herein, the term "vector" refers to a medium into which a genetic element can be operably inserted to cause expression of the genetic element, thereby producing a protein, RNA, or DNA encoded by the genetic element or replicating the genetic element. A vector can be used to transform, transduce, or transfect a host cell to cause expression of the genetic element it contains within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, the vector can contain an origin of replication. The vector can also include materials that assist its entry into cells, which include (but are not limited to) virus particles, liposomes, or protein coatings. A vector can be an expression vector or a cloning vector.

[0072] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced.

[0073] As used herein, the term "biological sample" or "sample" refers to a biological composition containing (e.g.) cells and / or other molecular entities that are identified and / or discriminated based on physical, biochemical, chemical, and / or physiological characteristics, which is obtained from or sourced from a subject of interest. Biological samples include (but are not limited to) cells, tissues, organs, and / or biological fluids of a subject obtained by any method known to those skilled in the art. In some embodiments, the biological sample is a fluid sample. In some embodiments, the fluid sample is whole blood, plasma, serum, mucus (including nasal mucus and sputum), peritoneal fluid, pleural fluid, thoracic fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, abdominal fluid, ascites, or pericardial fluid. In some embodiments, the biological sample is tissue or cells obtained from the heart, liver, spleen, lung, kidney, skin, or blood vessels of a subject.

[0074] As used herein, the term "norovirus" refers to an unenveloped viral strain of the genus Norovirus in the family Caliciviridae, which is characterized by having a single-stranded positive-sense RNA. The length of the norovirus genome is 7,654 nucleotides. ORF1 encodes a non-structural polyprotein that is cleaved by the viral 3C-like protease into 6 proteins (including an RNA-dependent RNA polymerase). ORF2 and ORF3 encode the major (VP1) and minor (VP2) capsid proteins, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 is the electrophoretogram of monoclonal antibody 2G11;

[0076] Figure 2 is the HPLC chromatogram of monoclonal antibody 2G11;

[0077] Figure 3 is the active binding diagram of monoclonal antibody 2G11. DETAILED DESCRIPTION OF THE INVENTION

[0078] Example 1

[0079] 1. Recombinant expression of immunogen

[0080] Synthesize the VP1 protein sequence of norovirus type 1, construct it into the pEM5.1 vector; extract the plasmid for transfection; transfect it into HEK293 cells and culture the cells for 7 days; harvest the supernatant, purify it with a Ni column, and concentrate and replace the buffer to obtain the recombinant VP1 protein of norovirus type 1.

[0081] The recombinant VP1 protein sequence of norovirus type 1 is from Uniprot,

[0082] The sequence information is as follows:

[0083] MMMASKDATSSVDGASGAGQLVPEVNASDPLAMDPVAGSSTAVATAGQVNPIDPWIINNFVQAPQGEFTISPNNTPGDVLFDLSLGPHLNPFLLHLSQMYNGWVGNMRVRIMLAGNAFTAGKIIVSCIPPGFGSHNLTIAQATLFPHVIADVRTLDPIEVPLEDVRNVLFHNNDRNQQTMRLVCMLYTPLRTGGGTGDSFVVAGRVMTCPSPDFNFLFLVPPTVEQKTRPFTLPNLPLSSLSNSRAPLPISSMGISPDNVQSVQFQNGRCTLDGRLVGTTPVSLSHVAKIRGTSNGTVINLTELDGTPFHPFEGPAPIGFPDLGGCDWHINMTQFGHSSQTQYDVDTTPDTFVPHLGSIQANGIGSGNYVGVLSWISPPSHPSGSQVDLWKIPNYGSSITEATHLAPSVYPPGFGEVLVFFMSKMPGPGAYNLPCLLPQEYISHLASEQAPTVGEAALLHYVDPDTGRNLGEFKAYPDGFLTCVPNGASSGPQQLPINGVFVFVSWVSRFYQLKPVGTASSARGRLGLRR(SEQ ID NO:16)

[0084] 2. Immunity

[0085] Immunize mice, fuse with SP2 / 0, screen, and subclone. The specific steps are as follows:

[0086] For the first immunization, use Freund's complete adjuvant, 100 μg per mouse, intraperitoneal injection, total dose 0.5 ml / mouse. The second immunization is carried out after a 3-week interval; from the second immunization, use Freund's incomplete adjuvant, dose 50 μg / 0.5 ml / mouse, and the third immunization is carried out after a 2-week interval; prepare cell fusion 10 days after the third injection.

[0087] Take feeder cells, which can be used at 10 5 / well, plate 10 5 cells / 100 μl / well one day before fusion; take mouse immune spleen cells and the prepared myeloma cells and fuse them with the fusogen PEG, and plate them into a 96-well cell culture plate that has already been added with feeder cells, 100 μl / well.

[0088] Positive wells were screened by ELISA. Recombinant norovirus type 1 VP1 protein was plated overnight; the plate was washed, blocked with skim milk powder at 37 °C for 1 h; the plate was washed, 100 μL of the supernatant of the 96-well culture medium was added, and incubated at 37 °C for 1 h; the plate was washed, HRP-labeled goat anti-mouse secondary antibody was added, and incubated at 37 °C for 30 min; the plate was washed, chromogenic solution was added, developed for 10 min, stop solution was added, and the OD450 value was read; high-expression cell lines were screened for subcloning.

[0089] 3. Sequence retrieval

[0090] Cells were collected, RNA was extracted, reverse transcription was performed, primers were designed, PCR was carried out, transformation was done, colonies were picked, sent for sequencing, and the norovirus type 1 VP1 protein antibody targeted the VP1 protein.

[0091] Clone number 2G11,

[0092] The specific sequence is shown in Table 1.

[0093] Table 1

[0094]

[0095]

[0096] 4. Antibody expression

[0097] Expression and purification of monoclonal antibodies:

[0098] (1) Chemically synthesize the screened sequence and clone it into a eukaryotic expression vector.

[0099] (2) Amplify the plasmid and extract the plasmid.

[0100] (3) Transiently transfect the plasmid encoding the antibody into mammalian cells HEK293.

[0101] (4) Collect the supernatant and purify the monoclonal antibody by affinity chromatography.

[0102] Transient transfection was carried out using HEK293 cells and transfection was done using PEI. The results showed that the expression level of antibody 2G11 was 249 mg / L.

[0103] 5. Physicochemical properties

[0104] 5.1 Gel electrophoresis detection

[0105] A. Sample preparation

[0106] Take 20 μL of the sample and mix it evenly with 5 μL of 5× reducing buffer, heat at 95 °C for 5 min, and cool; take 20 μL of the sample and mix it evenly with 5 μL of 5× non-reducing buffer.

[0107] B, Electrophoresis

[0108] Prepare the gel, add an appropriate amount of electrophoresis buffer, load the sample, and perform electrophoresis.

[0109] C, Staining and Decolorization

[0110] After electrophoresis, take out the gel and put it into an appropriate amount of Coomassie Brilliant Blue staining solution, stain at room temperature for 1 h or longer; pour out the staining solution, add an appropriate amount of Coomassie Brilliant Blue staining decolorization solution, and decolorize at room temperature for 4 - 24 h. After completion of decolorization, soak it in ddH2O, refer to the Marker protein, compare with the unstained gel, cut out the gel of the required protein component, and collect it. Then separate the protein to be purified from the gel.

[0111] 5.2 HPLC Detection of the Purity of Monoclonal Antibody

[0112] A, Preparation of Mobile Phase

[0113] Add dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and potassium chloride to about 900 mL of purified water, stir to dissolve, make up the volume to 1 L, measure with a pH meter, and ensure that its pH is between 6.2 ± 0.1. Filter with a 0.22 μm filter membrane and store at room temperature.

[0114] B, Sample Preparation

[0115] System suitability sample: Dilute the standard product to 2 mg / mL with the mobile phase;

[0116] Test sample: Dilute the sample to be tested to 2 mg / mL with the mobile phase.

[0117] Perform detection under conventional chromatographic conditions.

[0118] The results are as Figure 1 and Figure 2 shown. The results show that the electrophoresis pattern and the purity detected by liquid phase of the monoclonal antibody 2G11 are both greater than 95%.

[0119] 6, Binding Activity

[0120] Detection of the binding activity of monoclonal antibody:

[0121] A, Coating: Dilute Norovirus type 1 VP1 protein to 2 μg / ml with the coating solution, mix well, add to a 96-well coating plate, 100 μl / well, seal with a film and incubate overnight at 4°C.

[0122] B, Wash the plate 3 times with a plate washer. There should be no liquid remaining on the plate for the last wash. Pat dry the liquid on the surface of the plate with absorbent paper.

[0123] C, Sealing: Add 5% milk powder (0.5 g milk powder dissolved in 10 mL DPBS), 300 μL per well, incubate at 37 °C for 1 h, and wash the plate 3 times according to step B.

[0124] D, Gradiently dilute the antibody, 100 μL per well, react at 37 °C for 1 h, and wash the plate 3 times according to step B.

[0125] E, Add secondary antibody: Dilute with DPBS at 1:2000, add to a 96-well plate, 100 μL per well, react at 37 °C for 1 h, and wash the plate 3 times according to step B.

[0126] F, Color development: Add TMB, 100 μL per well, develop color in the dark at room temperature for 10 min.

[0127] G, Termination: Add 2N H2SO4, 100 μL per well.

[0128] H, Measure OD450 with an enzyme-linked immunosorbent assay reader within 10 min.

[0129] Coat with VP1 protein, add antibody, add secondary antibody, develop color, read the value, and perform data analysis.

[0130] The results are as Figure 3 shown. The results show that the EC50 of the monoclonal antibody 2G11 is 0.586 ng / mL.

[0131] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An anti-Norovirus VP1 protein monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an H-CDR1 of the amino acid sequence shown in SEQ ID NO: 1, an H-CDR2 of the amino acid sequence shown in SEQ ID NO: 2, and an H-CDR3 of the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region comprises an L-CDR1 of the amino acid sequence shown in SEQ ID NO: 4, an L-CDR2 of the amino acid sequence shown in YAS, and an L-CDR3 of the amino acid sequence shown in SEQ ID NO:

5.

2. The anti-Norovirus VP1 protein monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises one or more heavy chain framework regions HFR1, HFR2, HFR3 and HFR4, and / or one or more light chain framework regions LFR1, LFR2, LFR3 and LFR4, wherein HFR1 comprises the amino acid sequence shown in SEQ ID NO: 6, or a homologous sequence having at least 85% sequence identity thereto, HFR2 comprises the amino acid sequence shown in SEQ ID NO: 7, or a homologous sequence thereof having at least 85% sequence identity, HFR3 comprises the amino acid sequence shown in SEQ ID NO: 8, or a homologous sequence thereof having at least 85% sequence identity, HFR4 comprises the amino acid sequence shown in SEQ ID NO: 9, or a homologous sequence thereof having at least 85% sequence identity, LFR1 comprises the amino acid sequence shown in SEQ ID NO: 10, or a homologous sequence thereof having at least 85% sequence identity, LFR2 comprises the amino acid sequence shown in SEQ ID NO: 11, or a homologous sequence thereof having at least 85% sequence identity, LFR3 comprises the amino acid sequence shown in SEQ ID NO: 12, or a homologous sequence thereof having at least 85% sequence identity, and LFR4 comprises the amino acid sequence shown in SEQ ID NO: 13, or a homologous sequence thereof having at least 85% sequence identity.

3. The anti-Norovirus VP1 protein monoclonal antibody or its antigen-binding fragment as described in claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14, or a homologous sequence having at least 85% sequence identity thereto; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or a homologous sequence having at least 85% sequence identity thereto.

4. The anti-Norovirus VP1 protein monoclonal antibody or its antigen-binding fragment according to claim 1, wherein the anti-Norovirus VP1 protein monoclonal antibody or its antigen-binding fragment is humanized.

5. The anti-Norovirus VP1 protein monoclonal antibody or its antigen-binding fragment as described in claim 1, wherein the anti-Norovirus VP1 protein monoclonal antibody or its antigen-binding fragment specifically binds to the Norovirus VP1 protein; wherein the Norovirus VP1 protein has an amino acid sequence as shown in SEQ ID NO:

16.

6. The anti-Norovirus VP1 protein monoclonal antibody or its antigen-binding fragment as described in claim 1, which is connected to one or more conjugate parts; the conjugate part contains a reagent for detection, specifically a radioactive isotope, a lanthanide, a fluorescent label, or an enzyme-substrate label.

7. The anti-Norovirus VP1 protein monoclonal antibody or its antigen-binding fragment as described in claim 1, which is connected to one or more conjugate parts; the conjugate part contains a reagent for detection, specifically a luminescent label.

8. The anti-Norovirus VP1 protein monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the Norovirus VP1 protein is Norovirus type 1 VP1 protein.

9. An isolated polynucleotide encoding the anti-Norovirus VP1 protein monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A vector comprising the isolated polynucleotide of claim 9. A host cell comprising the vector according to claim 10.

12. A method for expressing the anti-Norovirus VP1 protein monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-8, comprising culturing the host cell as described in claim 11 under the conditions of expressing the vector as described in claim 10, and finally isolating the anti-Norovirus VP1 protein monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-8.

13. The method of claim 12, wherein the norovirus is norovirus type 1.

14. Use of the anti-Norovirus VP1 protein monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 8, the isolated polynucleotide according to claim 9, the vector according to claim 10, and the host cell according to claim 11 in the production of a product for detecting Norovirus VP1 protein.

15. The use according to claim 14, wherein the norovirus is norovirus type 1.

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