Monoclonal antibody for resisting norovirus and application thereof

Abstract

The invention discloses an anti-norovirus monoclonal antibody and application thereof, and discloses an anti-VP1 protein monoclonal antibody, complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region of the monoclonal antibody are as shown in SEQ ID NO: 1-3, complementarity determining regions LCDR1 and LCDR3 of a light chain variable region of the monoclonal antibody are as shown in SEQ ID NO: 4-5, and the amino acid sequence of the complementarity determining region LCDR2 of the light chain variable region is NAK. The anti-norovirus monoclonal antibody disclosed by the invention has strong affinity and high binding activity with norovirus type 1 VP1 protein, and is an excellent monoclonal antibody for detecting and treating norovirus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a monoclonal antibody against norovirus and an application thereof. Background Art

[0002] Norovirus (English: Norovirus, Winter Vomiting Bug, abbreviated: NV), also known as norovirus, Norwalk virus or pyomel virus, is a virus that causes non-bacterial acute gastroenteritis. The characteristic of norovirus is that it infects places with high population density and poor sanitary environment, such as cruise ships. Norovirus is mainly transmitted by the fecal-oral route, that is, food is contaminated by feces full of viruses. It may also be transmitted through droplets in places with high population density.

[0003] Norovirus is divided into genogroup I and genogroup II according to its genetic genes. Genogroup I includes Norovirus, Southampton virus, and Desert Shield virus; genogroup II includes Snow Mountain virus, Hawaii virus, and Toronto virus. The general symptoms include abdominal pain, diarrhea, vomiting, low-grade fever, and muscle aches all over the body. The symptoms usually disappear after 12 to 60 hours, but the excretion of the virus will continue for about 2 weeks. If the diarrhea is severe, dehydration and shock are likely to occur. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0005] The present invention provides a monoclonal antibody against VP1 protein, wherein the heavy chain variable region of the monoclonal antibody includes complementary determining regions HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 1-3, and the light chain variable region includes complementary determining regions LCDR1 and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 4-5, and the amino acid sequence of the complementary determining region LCDR2 of the light chain variable region is NAK.

[0006] Furthermore, the anti-VP1 protein is an anti-Norovirus type 1 VP1 protein.

[0007] Furthermore, the monoclonal antibody also includes framework regions FR1, FR2, FR3, FR4 of the heavy chain variable region and framework regions FR1, FR2, FR3, FR4 of the light chain variable region.

[0008] Furthermore, the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the monoclonal antibody have at least 70% identity, preferably at least 75% identity, preferably at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, and preferably at least 99% identity to the amino acid sequences of SEQ ID NOs: 6-9, respectively.

[0009] Furthermore, the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region of the monoclonal antibody are at least 70% identical to the amino acid sequences of SEQ ID NOs: 10-13, respectively, preferably at least 75% identical, preferably at least 80% identical, preferably at least 85% identical, preferably at least 90% identical, preferably at least 95% identical, preferably at least 96% identical, preferably at least 97% identical, preferably at least 98% identical, and preferably at least 99% identical.

[0010] Furthermore, the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NOs: 6-9, and the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region are shown in SEQ ID NOs: 10-13.

[0011] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody has at least 60% identity, preferably at least 65% identity, preferably at least 70% identity, preferably at least 75% identity, preferably at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity with the amino acid sequence of SEQ ID NO:14.

[0012] Furthermore, the amino acid sequence of the light chain variable region of the monoclonal antibody has at least 60% identity, preferably at least 65% identity, preferably at least 70% identity, preferably at least 75% identity, preferably at least 80% identity, preferably at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity with the amino acid sequence of SEQ ID NO:15.

[0013] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:15.

[0014] Furthermore, the CDR division of the monoclonal antibody can also be performed using the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0015] Furthermore, the CDR of the monoclonal antibody is divided based on the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 14 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 15 as the basic sequence.

[0016] The terms "sequence identity" and "identity" as used herein are synonymous and refer to the "percentage of sequence identity" or "percentage of identity" between two polynucleotides, i.e., the number of identical matching positions shared by the sequences in the comparison window, taking into account the additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position in which the same nucleotides are present in both the target sequence and the reference sequence. Since the gap is not a nucleotide, the gap present in the target sequence is not counted. Similarly, since the target sequence nucleotides are counted, and the nucleotides from the reference sequence are not counted, the gap present in the reference sequence is not counted. At least 60% sequence identity includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the total length of the sequence having a continuous segment with sequence identity. "Percentage of sequence identity" can be calculated by comparing the two best aligned sequences in the window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) appears in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0017] The term "antibody" as used herein is generally an immunoglobulin molecule consisting of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain may also include a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) in each heavy chain and light chain form antigen binding sites, respectively.

[0018] In some embodiments, the present invention provides an "anti-Norovirus type 1 VP1 protein antibody" that is a monoclonal antibody. A monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the antibody population are identical except for possible naturally occurring mutations that may be present in small amounts. The modifier "monoclonal" refers to the characteristic of an antibody obtained from a substantially homogeneous antibody population, and is not to be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in accordance with the present invention may be prepared by the hybridoma (murine or human) method first described by Kohler et al., Nature, 256:495 (1975), or may be prepared by recombinant DNA methods (see, e.g., US 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J Mol. Biol., 222:581-597 (1991). It should also be understood that certain embodiments of the present invention relate to compositions comprising more than one different monoclonal antibody that specifically binds to the VP1 protein, ie, a polyclonal antibody composition comprising a plurality of monoclonal antibodies with different epitope specificities.

[0019] The present invention provides a nucleic acid molecule, which encodes the monoclonal antibody mentioned above.

[0020] Furthermore, the nucleic acid molecules include DNA, cDNA, mRNA, and recombinant nucleic acid.

[0021] Furthermore, the nucleotide sequence of the nucleic acid molecule is codon optimized.

[0022] The term "nucleic acid" as used herein is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and DNA / RNA hybrids, and also including DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or thiophosphates) or modified bases. Therefore, nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0023] The present invention provides a vector, which comprises the nucleic acid molecule described above.

[0024] Furthermore, the vector includes retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus, lambda phage, M13 phage, and plasmid.

[0025] The term "vector" as used herein refers to the nucleic acid sequence being directly or indirectly connected to the regulatory elements on the vector when the above-mentioned isolated nucleic acid molecule is connected to the vector, as long as these regulatory elements can regulate the translation and expression of the nucleic acid molecule. Of course, these regulatory elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. In other words, the nucleic acid molecule is operably connected to the regulatory element. Commonly used vectors include prokaryotic expression vectors, eukaryotic expression vectors, or viral expression vectors, etc.

[0026] In some embodiments, the selection of a specific vector will depend on the target cell and the conditions being treated. Introduction can be by standard techniques such as infection, transfection, transduction or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE polydextrose, electroporation, protoplast fusion, liposome transfection, cell microinjection and viral vectors.

[0027] The present invention provides a cell, which comprises the above-mentioned monoclonal antibody, the above-mentioned nucleic acid molecule, and the above-mentioned vector.

[0028] Furthermore, the cells include eukaryotic cells and prokaryotic cells.

[0029] Furthermore, the prokaryotic cells include bacterial cells, Escherichia coli, and Streptomyces.

[0030] Furthermore, the eukaryotic cells include yeast cells, mammalian cells, and insect cells.

[0031] Furthermore, the mammalian cells include humans, monkeys, mice, rats, hamsters, goats, sheep, cattle, pigs, dogs, and cats.

[0032] Furthermore, the eukaryotic cells include human cells, mouse cells, rat cells, hamster cells, goat cells, sheep cells or any other cells.

[0033] Furthermore, the mammalian cells include A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, WI38, HeLa, 293 cells (which can express functional adenovirus E1), Saos, C2C12, L cells, HT1080, and HepG2.

[0034] Furthermore, the mammalian cells include fibroblasts, hepatocytes and myoblasts.

[0035] The present invention provides a conjugate, which comprises the above-mentioned monoclonal antibody and a directly or indirectly conjugated linker thereof.

[0036] Furthermore, the attachable substance includes a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0037] The term "detectable marker" as used herein refers to any part that generates a measurable signal by a change in the optical, electrical or other physical index of the molecular state coupled to the part. Such physical indexes include spectral, photochemical, biochemical, immunochemical, electromagnetic, radiochemical and chemical means, such as but not limited to fluorescence, chemiluminescence and chemiluminescence, etc. When used with respect to a labeled detection agent, a "direct marker" is a detectable marker attached to a detection agent by any means. When used with respect to a labeled detection agent, an "indirect marker" is a detectable marker that specifically binds to a detection agent. Therefore, an indirect marker includes the following parts: a specific binding partner of a detection agent. Biotin and avidin are examples of such parts that are used, for example, by contacting a biotinylated antibody with a labeled avidin to produce an indirect labeled antibody for application.

[0038] In some embodiments, the label can be selected from isotopic labels, isobaric labels, enzyme labels, colored labels, fluorescent labels, chromogenic labels, luminescent labels, radioactive labels, haptens, biotin, metal complexes, metals and colloidal gold, among which isotopic labels and isobaric labels are particularly preferred.

[0039] The present invention provides a pharmaceutical composition, which comprises the aforementioned monoclonal antibody, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, and / or the aforementioned conjugate.

[0040] Further, the pharmaceutical composition is administered systemically, specifically via intranasal route, intravenous route, intraperitoneal route, intramuscular route, intradermal route, subcutaneous route, subdermal route, transdermal route, intracranial route, mucosal route, anal route, vaginal route, oral route, buccal route or they can be delivered by inhalation.

[0041] Furthermore, the pharmaceutical composition includes a pharmaceutically acceptable carrier or excipient.

[0042] In some embodiments, excipients include pharmaceutically acceptable solvents, dispersants, additives, plasticizers, etc. In general, these substances can be formulated in non-toxic, inert and pharmaceutically acceptable carrier media. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration. In other embodiments, the pharmaceutical composition is an aqueous pharmaceutical composition (e.g., an aqueous solution), or the nanobody and salt are provided in water without a buffer, or contain an aqueous buffer or other types of solvents (e.g., an organic solvent).

[0043] The present invention provides any of the following applications, including:

[0044] 1) Use of the aforementioned monoclonal antibody, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, and / or the aforementioned conjugate in the preparation of a product for detecting norovirus;

[0045] Furthermore, the norovirus includes norovirus type 1.

[0046] Furthermore, the norovirus includes norovirus type 1 VP1 protein.

[0047] Furthermore, the product includes a kit, a test paper, a nucleic acid membrane strip, a chip, a system, or a device.

[0048] 2) Use of the aforementioned monoclonal antibody, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, and / or the aforementioned conjugate in the preparation of a pharmaceutical composition for preventing or treating norovirus;

[0049] Furthermore, the norovirus includes norovirus type 1.

[0050] Furthermore, the norovirus includes norovirus type 1 VP1 protein.

[0051] The terms "treatment" or "prevention" as used herein refer to any type of intervention or treatment performed on an individual, or the administration of an active agent to a subject, with the intent to reverse, alleviate, ameliorate, inhibit, or slow or prevent the progression, occurrence, aggravation or recurrence of symptoms, complications, conditions or biochemical markers associated with a disease. Prevention refers to administration to a subject who does not have the disease in order to prevent the disease from occurring or to minimize its effects if it occurs.

[0052] 3) Use of the aforementioned monoclonal antibody, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, and / or the aforementioned conjugate in the preparation of a product for regulating the activity or level of Norovirus Type 1 VP1 protein.

[0053] Furthermore, the product includes a kit, a test paper, a nucleic acid membrane strip, a chip, a system, or a device.

[0054] The present invention provides any of the following methods, comprising:

[0055] 1) A method for producing the aforementioned monoclonal antibody, the method comprising: transforming the aforementioned nucleic acid molecule or the aforementioned vector into cells, culturing the cells, or directly using the aforementioned cells for culturing, and isolating and purifying the aforementioned monoclonal antibody from the cell culture fluid;

[0056] 2) A method for inhibiting the activity or level of Norovirus 1 VP1 protein in vitro for non-therapeutic purposes, the method comprising: applying the aforementioned monoclonal antibody, the aforementioned nucleic acid molecule, the aforementioned vector, and / or the aforementioned cell to cells or tissues containing Norovirus 1 in vitro;

[0057] 3) A method for detecting norovirus in a sample for non-diagnostic and non-therapeutic purposes, the method comprising contacting the sample with the monoclonal antibody and / or the conjugate described above, and detecting the formation of the complex or the change of the marker.

[0058] Advantages and beneficial effects of the present invention:

[0059] The present invention provides a monoclonal antibody against Norovirus Type 1 VP1 protein, which has strong affinity and high binding activity with Norovirus Type 1 VP1 protein and is an excellent monoclonal antibody for detecting and treating Norovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is the electrophoresis diagram of monoclonal antibody 1B6;

[0061] Figure 2 It is the HPLC chart of monoclonal antibody 1B6;

[0062] Figure 3 It is a graph of the binding activity of monoclonal antibody 1B6. DETAILED DESCRIPTION

[0063] Hereinafter, the present invention will be described in further detail with reference to the following examples. However, these examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0064] Example

[0065] 1. Recombinant expression of immunogens

[0066] Synthesize the Norovirus 1 VP1 protein sequence and construct it into the pEM5.1 vector; extract the transfection plasmid; 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 Norovirus 1 VP1 protein. The recombinant Norovirus 1 VP1 protein sequence comes from Uniprot, and the sequence information is as follows:

[0067] (SEQ ID NO:16)

[0068] 2. Immunity

[0069] Immunization subjects: mice, SP2 / 0 fusion, screening, subcloning, specific steps are: the first time with Freund's complete adjuvant, 100μg per mouse, intraperitoneal injection, total dose 0.5ml / mouse, the second immunization is carried out after 3 weeks; the second time with Freund's incomplete adjuvant, the dose is 50μg / 0.5ml / mouse, the third immunization is carried out after 2 weeks; prepare cell fusion 10 days after the third injection.

[0070] Take feeder cells and press 10 5 / well, plate 10 on the day before fusion 5 / 100μl / well; Mouse immune spleen cells were taken and fused with the prepared myeloma cells using the fusion agent PEG, and then plated into a 96-cell culture plate that had been added with feeder cells, 100μl / well.

[0071] Positive wells were screened by ELISA detection method, and recombinant Norovirus type 1 VP1 protein was plated overnight; the plates were washed, blocked with skim milk powder, and incubated at 37°C for 1h; the plates were washed, 100μl of 96-well culture supernatant was added, and incubated at 37°C for 1h; the plates were washed, HRP-labeled goat anti-mouse secondary antibody was added, and incubated at 37°C for 30min; the plates were washed, color development solution was added, color development was performed for 10min, stop solution was added, and OD450 value was read; high expression cell lines were screened for subcloning.

[0072] 3. Sequence Retrieval

[0073] Cells were collected, RNA was extracted, reverse transcribed, primers were designed, PCR was performed, transformation was performed, clones were selected, and sequencing was performed. Finally, the Norovirus 1 VP1 protein antibody was obtained, clone number 1B6, and the specific sequence is shown in Table 1.

[0074] Table 1

[0075]

[0076]

[0077] 4. Antibody Expression

[0078] Monoclonal Antibody Expression and Purification:

[0079] (1) The screened sequences are chemically synthesized and cloned into a eukaryotic expression vector.

[0080] (2) Amplify and extract the plasmid.

[0081] (3) The antibody encoding plasmid was transiently transfected into mammalian HEK293 cells.

[0082] (4) Collect the supernatant and purify the monoclonal antibody using affinity chromatography.

[0083] HEK293 cells were transiently transfected with PEI, and the results showed that the expression level of antibody 1B6 was 249 mg / L.

[0084] 5. Physical and chemical properties

[0085] 5.1 Gel electrophoresis detection

[0086] A. Sample preparation

[0087] Take 20 μL of sample and mix it evenly with 5 μL of 5× reducing buffer, heat it at 95°C for 5 min, and cool it down; take 20 μL of sample and mix it evenly with 5 μL of 5× non-reducing buffer.

[0088] B. Electrophoresis

[0089] Prepare gel, add appropriate amount of electrophoresis buffer, add sample, and perform electrophoresis.

[0090] C. Dyeing and bleaching

[0091] After the electrophoresis is finished, take the gel and put it into an appropriate amount of Coomassie Brilliant Blue staining solution, and stain it at room temperature for 1 hour or longer; pour out the staining solution, add an appropriate amount of Coomassie Brilliant Blue staining decolorizing solution, and decolorize it at room temperature for 4-24 hours. After the decolorization is completed, soak it in ddH2O, refer to the marker protein, compare it 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.

[0092] 5.2 HPLC detection of monoclonal antibody purity

[0093] A. Mobile phase preparation

[0094] Add potassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate and potassium chloride to about 900 mL of purified water, stir to dissolve, dilute to 1 L, measure with a pH meter to determine that the pH is between 6.2±0.1. Filter through a 0.22 μm filter membrane and store at room temperature.

[0095] B. Sample Preparation

[0096] System suitability sample: The standard was diluted to 2 mg / mL with mobile phase;

[0097] Test sample: Dilute the sample to be tested to 2 mg / mL with mobile phase.

[0098] The detection was carried out under conventional chromatographic conditions.

[0099] The results are as follows Figure 1 and Figure 2 As shown, the results showed that the purity of antibody 1B6 was greater than 95% by electrophoresis and liquid chromatography detection.

[0100] 6. Binding activity

[0101] Binding activity assay of monoclonal antibodies:

[0102] A. Coating: Dilute the anti-Norovirus type 1 VP1 protein to 2 μg / ml with coating solution, mix well, add to 96-well coating plate, 100 μl / well, seal the plate, and incubate at 4°C overnight.

[0103] B. Wash the plate 3 times in a plate washer. No liquid should remain on the plate during the last wash. Pat the liquid on the surface of the plate dry with absorbent paper.

[0104] C. Blocking: Add 5% milk powder (0.5 g milk powder dissolved in 10 mL DPBS), 300 μL / well, incubate at 37°C for 1 h, and wash the plate 3 times according to step B.

[0105] D. Dilute the antibody in a gradient manner, 100 μL / well, react at 37°C for 1 h, and wash the plate 3 times according to step B.

[0106] E. Add secondary antibody: dilute with DPBS at 1:2000, add to 96-well plate, 100 μL / well, react at 37℃ for 1 hour, and wash the plate 3 times according to step B.

[0107] F. Color development: Add TMB, 100 μL / well, and develop color for 10 min at room temperature in the dark.

[0108] G. Stop: Add 2N H2SO4, 100 μL / well.

[0109] H. Measure OD450 with an enzyme-labeled instrument within 10 minutes.

[0110] Coat VP1 protein, add antibody, add secondary antibody, develop color, read, and analyze data.

[0111] The results are as follows Figure 3 As shown, the results showed that the EC50 of antibody 1B6 was 0.00955 ng / mL, with excellent performance.

Claims

1. A monoclonal antibody against VP1 protein, wherein the heavy chain variable region of the monoclonal antibody includes complementary determining regions HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 1-3, and the light chain variable region includes complementary determining regions LCDR1 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 4-5, and the amino acid sequence of the complementary determining region LCDR2 of the light chain variable region is NAK.

2. The monoclonal antibody according to claim 1, wherein the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the monoclonal antibody are as shown in SEQ ID NOs: 6-9, and the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region are as shown in SEQ ID NOs: 10-13.

3. The monoclonal antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

15.

4. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 3; Preferably, the nucleic acid molecule comprises DNA, cDNA, mRNA, or recombinant nucleic acid.

5. A vector comprising the nucleic acid molecule according to claim 4; Preferably, the vector comprises a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a poxvirus, a baculovirus, a papillomavirus, a papovavirus, a lambda phage, an M13 phage, or a plasmid.

6. A cell, comprising the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, and the vector according to claim 5; Preferably, the cells include eukaryotic cells and prokaryotic cells; Preferably, the prokaryotic cells include bacterial cells, Escherichia coli, and Streptomyces; Preferably, the eukaryotic cells include yeast cells, mammalian cells, and insect cells; Preferably, the mammalian cells include humans, monkeys, mice, rats, hamsters, goats, sheep, cattle, pigs, dogs, and cats; Preferably, the eukaryotic cell comprises a human cell, a mouse cell, a rat cell, a hamster cell, a goat cell, a sheep cell or any other cell; Preferably, the mammalian cells include A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, WI38, HeLa, 293 cells, Saos, C2C12, L cells, HT1080, HepG2; Preferably, the mammalian cells include fibroblasts, hepatocytes and myoblasts.

7. A conjugate, comprising the monoclonal antibody according to any one of claims 1 to 3 and a directly or indirectly conjugated linker thereof; Preferably, the attachable species comprises a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

8. A pharmaceutical composition, comprising the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the cell according to claim 6, and / or the conjugate according to claim 7; Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient.

9. Any application as described in any of the following, include: 1) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the cell according to claim 6, and / or the conjugate according to claim 7 in the preparation of a product for detecting norovirus; Preferably, the norovirus comprises norovirus type 1; Preferably, the norovirus comprises norovirus type 1 VP1 protein; 2) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the cell according to claim 6, and / or the conjugate according to claim 7 in the preparation of a pharmaceutical composition for preventing or treating norovirus; Preferably, the norovirus comprises norovirus type 1; Preferably, the norovirus comprises norovirus type 1 VP1 protein; 3) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the cell according to claim 6, and / or the conjugate according to claim 7 in the preparation of a product for regulating the activity or level of Norovirus Type 1 VP1 protein.

10. Any of the following methods, include: 1) A method for producing the monoclonal antibody according to any one of claims 1 to 3, the method comprising: transforming the nucleic acid molecule according to claim 4 or the vector according to claim 5 into cells, culturing the cells, or directly using the cells according to claim 6 for culturing, and isolating and purifying the monoclonal antibody according to any one of claims 1 to 3 from the cell culture fluid; 2) A method for inhibiting the activity or level of Norovirus 1 VP1 protein for non-therapeutic purposes in vitro, the method comprising: applying the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, and / or the cell according to claim 6 to cells or tissues containing Norovirus 1 in vitro; 3) A method for detecting norovirus in a sample for non-diagnostic and non-therapeutic purposes, the method comprising contacting the sample with the monoclonal antibody according to any one of claims 1 to 3, and / or the conjugate according to claim 7, and detecting the formation of the complex or the change of the marker.

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