Anti-norovirus VP1 protein antibody and application thereof

Abstract

The invention discloses an anti-norovirus VP1 protein antibody and application thereof, discloses an anti-VP1 protein antibody, a fragment or a fusion protein thereof, comprising a heavy chain variable region and a light chain variable region, and also provides a separated polynucleotide, a carrier, a host cell and a pharmaceutical composition comprising the anti-VP1 protein antibody, the fragment or the fusion protein thereof, the invention also discloses a method for preparing the anti-VP1 protein antibody, the fragment or the fusion protein thereof, a method for detecting the existence or the amount of the VP1 protein or the norovirus in a sample, and an application of the anti-VP1 protein antibody, the fragment or the fusion protein in a corresponding medicament for treating, preventing or relieving diseases or symptoms responding to VP1 protein inhibition, and a method for preparing the anti-VP1 protein antibody, the fragment or the fusion protein and the method for detecting the existence or the amount of the VP1 protein or the norovirus in a sample. The invention also relates to application in production of a product for detecting the VP1 protein or the norovirus.

Description

Technical Field

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

[0002] Norovirus infection is responsible for a significant global disease burden, estimated to be associated with 20% of all diarrheal cases worldwide and more than 200,000 deaths each year. Norovirus is the leading cause of foodborne illness outbreaks in North America and is the etiology of the majority of hospital-acquired outbreaks among the elderly. Norovirus strains are also considered the leading cause of pediatric gastrointestinal illness worldwide.

[0003] Noroviruses include one of the multiple genera of the Caliciviridae family. Noroviruses are classified according to the phylogenetic clustering of their VP1 amino acid sequences. So far, seven genogroups (GI to GVII) have been classified, of which only GI, GII and GIV are known to infect humans. Of the 32 specific genotypes currently associated with human infection, GII.4 norovirus is the cause of most recent norovirus outbreaks. New strains of GII.4 appear every two to three years, evolving through a process driven by mutations in an epitope that determines the region of the hypervariable P2 domain of VP1. This process allows noroviruses to escape the humoral immune response previously obtained by exposure to earlier strains.

[0004] Nowadays, norovirus has become the main cause of non-bacterial acute diarrhea. Among all non-bacterial diarrhea outbreaks in the United States each year, 60% to 90% are caused by norovirus. In 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 infection. Therefore, the study of rapid and accurate early detection methods for norovirus has far-reaching significance for the prevention and treatment of the virus. Summary of the invention

[0005] In order 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, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an H-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and an H-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof comprising up to 3, 2 or 1 amino acid substitutions in the H-CDR; and the VL comprises an L-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, an L-CDR2 comprising the amino acid sequence shown in YAS, and an L-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, or a variant thereof comprising up to 3, 2 or 1 amino acid substitutions in the L-CDR.

[0007] Further, the heavy chain variable region comprises: H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region of 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 of the amino acid sequences shown in SEQ ID NOs: 4, YAS and SEQ ID NO: 5;

[0008] Further, 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,

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[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 thereto; 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.

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

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

[0027] Further, the anti-VP1 protein antibody, its fragment or fusion protein is a double-chain antibody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide bond-stabilized Fv fragment (dsFv), (dsFv)2, dual-specific dsFv (dsFv-dsFv'), disulfide bond-stabilized double-chain antibody (ds double-chain antibody), single-chain antibody molecule (scFv), scFv dimer (divalent double-chain antibody), multi-specific antibody, camelized single-domain antibody, nanobody, domain antibody or bivalent domain antibody.

[0028] Furthermore, the anti-VP1 protein antibody, its fragment or fusion protein is a mouse antibody, a monkey antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

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

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

[0031] Furthermore, the anti-VP1 protein antibody, fragment or fusion protein thereof also includes replacement or modification of one or more amino acid residues, but retains the binding specificity to the VP1 protein.

[0032] Furthermore, the anti-VP1 protein antibody, fragment or fusion protein thereof specifically binds to the VP1 protein; wherein the VP1 protein has an amino acid sequence as 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), which is a heavy chain constant region and / or a light chain constant region. In some embodiments, the heavy chain constant region is a heavy chain constant region of IgG, IgA, IgM, IgE or IgD or a variant thereof; the heavy chain constant region is preferably a heavy chain constant region of IgG or a variant thereof, such as a constant region of IgG1, IgG2, IgG3 or IgG4 or a variant thereof; the heavy chain constant region is more preferably a heavy chain constant region of IgG1 or a variant thereof; for example, a 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 portion comprises an agent for detection or separation, such as a clearance-improving agent, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent tag, a fluorescent tag, an enzyme-substrate tag, a DNA-alkylating agent, a local isomerase inhibitor, a microtubule-binding agent or other anti-Norovirus drug.

[0036] Furthermore, the VP1 protein is Norovirus VP1 protein.

[0037] Furthermore, the VP1 protein is Norovirus type 1 VP1 protein.

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

[0039] 1) Able to bind to human VP1;

[0040] 2) able to bind to mouse VP1;

[0041] 3) able to bind to monkey VP1;

[0042] 4) able to bind to rabbit VP1;

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

[0044] 6) inhibiting and / or blocking the binding of VP1 to ligand;

[0045] 7) inhibiting and / or blocking intracellular signal transduction mediated by VP1 binding ligand;

[0046] 8) Enhance immune response;

[0047] 9) Having at least one of antibody-dependent cell-mediated cytotoxicity activity, complement-dependent cytotoxicity 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, sulfatides, phosphatidylcholine, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides, and lipidated amyloid-β peptide.

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

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

[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] Furthermore, the pharmaceutical composition also includes other therapeutic agents.

[0056] Furthermore, 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 the conditions of 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 anti-VP1 protein antibody, fragment thereof or fusion protein described above, and determining the presence or amount of VP1 protein or norovirus in the sample.

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

[0060] Furthermore, the VP1 protein is Norovirus VP1 protein.

[0061] Furthermore, 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 condition responsive to VP1 protein inhibition.

[0063] Furthermore, the VP1 protein is Norovirus VP1 protein.

[0064] Furthermore, 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 producing a product for detecting VP1 protein or norovirus.

[0066] Furthermore, the VP1 protein is Norovirus VP1 protein.

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

[0068] The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multi-specific antibody or bispecific antibody that binds to a specific antigen. A natural complete antibody comprises two heavy chains (H) and two light chains (L). Mammalian heavy chains are divided into α, δ, ε, γ and μ, each heavy chain consisting of a variable region (VH) and a first, second, third and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are divided into λ or κ, and each light chain consists of a variable region (VL) and a constant region. The antibody has a "Y" shape, wherein the stem of the Y consists of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y includes a variable region and a first constant region of a single heavy chain bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains cause 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, heavy chain CDRs, including HCDR1, HCDR2, HCDR3).The 5th edition of the invention can be described by Kabat, IMGT, Chothia or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, 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, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252): 877-83 (1989); Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th edition. 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 edition), Chapter 26, 481-514, (2015)) The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein are conventionally defined or identified. The three CDRs are inserted between flanking extensions called framework regions (FRs) (light chain FRs, including LFR1, LFR2, LFR3 and LFR4, heavy chain FRs, including HFR1, HFR2, HFR3 and HFR4), which are more highly conserved than CDRs and form a skeleton to support hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen-binding, but exhibit a variety of effector functions. Antibodies are divided into different types based on the amino acid sequence of the constant region of their heavy chains. The five major 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 major 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 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 to another sequence when optimally aligned.

[0070] An "isolated" material has been altered by man from its natural state. If an "isolated" composition or substance occurs in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated", but the same polynucleotide or polypeptide is "isolated" if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state. An "isolated nucleic acid sequence" refers to an isolated nucleic acid molecule sequence. In certain embodiments, an “isolated antibody or antigen-binding fragment thereof” refers to an antibody or antigen-binding fragment thereof that is 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% pure as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatography (e.g., ion exchange chromatography or reverse phase HPLC).

[0071] The term "vector" as used herein refers to a medium into which a genetic element can be operably inserted to cause the expression of the genetic element, thereby producing a protein, RNA or DNA encoded by the genetic element or replicating the genetic element. The vector can be used to transform, transduce or transfect a host cell to cause the expression of the genetic element it has in the host cell. Examples of vectors include plasmids, phasmids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages, such as lambda phages or M13 phages, and animal viruses. The vector can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements and reporter genes. In addition, the vector can contain a replication origin. The vector can also include materials that help it enter the cell, including (but not limited to) viral particles, liposomes or protein coatings. The vector can be an expression vector or a cloning vector.

[0072] The term "host cell" as used herein 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 cells and / or other molecular entities that are identified and / or differentiated based on, for example, physical, biochemical, chemical, and / or physiological characteristics, obtained or derived 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, pleural fluid, saliva, urine, joint fluid, cerebrospinal fluid (CSF), thoracentesis fluid, peritoneal 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] The term "norovirus" as used herein refers to a non-enveloped virus strain of the genus Norovirus of the family Caliciviridae characterized by having single-stranded positive-sense RNA. The length of the norovirus genome is 7,654 nucleotides. ORF1 encodes a nonstructural polyprotein that is cleaved into 6 proteins (including RNA-dependent RNA polymerase) by a viral 3C-like protease. ORF2 and ORF3 encode the major (VP1) and minor (VP2) capsid proteins, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 It is the electrophoresis diagram of monoclonal antibody 2G11;

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

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

[0078] Example 1

[0079] 1. Recombinant expression of immunogens

[0080] The Norovirus 1 VP1 protein sequence was synthesized and constructed into the pEM5.1 vector; the transfection plasmid was extracted; the cells were transfected into HEK293 cells and cultured for 7 days; the supernatant was harvested, purified by Ni column, and the recombinant Norovirus 1 VP1 protein was obtained after concentration and replacement of the buffer.

[0081] The recombinant norovirus 1 VP1 protein sequence was obtained from Uniprot.

[0082] The sequence information is as follows:

[0083] (SEQ ID NO:16)

[0084] 2. Immunity

[0085] Immunization of mice, SP2 / 0 fusion, screening, subcloning, specific steps:

[0086] The first immunization was performed with Freund's complete adjuvant, 100 μg per mouse, intraperitoneally, with a total dose of 0.5 ml / mouse. The second immunization was performed 3 weeks later. The second immunization was performed with Freund's incomplete adjuvant, with a dose of 50 μg / 0.5 ml / mouse. The third immunization was performed 2 weeks later. Cell fusion was prepared 10 days after the third injection.

[0087] Take feeder cells and press 10 5 / well, plate 10 one 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.

[0088] 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.

[0089] 3. Sequence Retrieval

[0090] Collect cells, extract RNA, reverse transcribe, design primers, PCR, transform, pick clones, send for sequencing, Norovirus type 1 VP1 protein antibody, targeting VP1 protein.

[0091] Clone No. 2G11,

[0092] The specific sequences are shown in Table 1.

[0093] Table 1

[0094]

[0095]

[0096] 4. Antibody Expression

[0097] Monoclonal Antibody Expression and Purification:

[0098] (1) The screened sequences are chemically synthesized and cloned into a eukaryotic expression vector.

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

[0100] (3) The antibody encoding plasmid was transiently transfected into mammalian HEK293 cells.

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

[0102] HEK293 cells were transiently transfected with PEI, and the results showed that the expression level of antibody 2G11 was 249 mg / L.

[0103] 5. Physical and chemical properties

[0104] 5.1 Gel electrophoresis detection

[0105] A. Sample preparation

[0106] 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.

[0107] B. Electrophoresis

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

[0109] C. Dyeing and bleaching

[0110] 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.

[0111] 5.2 HPLC detection of monoclonal antibody purity

[0112] A. Mobile phase preparation

[0113] 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.

[0114] B. Sample Preparation

[0115] System suitability sample: The standard was diluted to 2 mg / mL with mobile phase;

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

[0117] The detection was carried out under conventional chromatographic conditions.

[0118] The results are as follows Figure 1 and Figure 2 As shown, the results showed that the purity of the monoclonal antibody 2G11 was greater than 95% by electrophoresis and liquid phase detection.

[0119] 6. Binding activity

[0120] Binding activity assay of monoclonal antibodies:

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

[0122] B. Wash the plate three 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.

[0123] 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.

[0124] 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.

[0125] 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.

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

[0127] G. Stop: Add 2N H2SO4, 100 μL / well.

[0128] H. Measure OD450 with an enzyme-labeled instrument within 10 minutes.

[0129] Coat VP1 protein, add antibody, add secondary antibody, develop color, read, and analyze data.

[0130] The results are as follows Figure 3 As shown, the results showed that the EC50 of monoclonal antibody 2G11 was 0.586 ng / mL.

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

Claims

1. An anti-VP1 protein antibody, a fragment or a fusion protein thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an H-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and an H-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof comprising up to 3, 2 or 1 amino acid substitutions in the H-CDR; and the VL comprises an L-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, an L-CDR2 comprising the amino acid sequence shown in YAS, and an L-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5, or a variant thereof comprising up to 3, 2 or 1 amino acid substitutions in the L-CDR.

2. The anti-VP1 protein antibody, fragment or fusion protein thereof according to claim 1, wherein the heavy chain variable region comprises: H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region of 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 of the amino acid sequences shown in SEQ ID NOs: 4, YAS and SEQ ID NO: 5; Preferably, 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-VP1 protein antibody, fragment or fusion protein thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14, or a homologous sequence thereof having at least 85% sequence identity; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or a homologous sequence thereof having at least 85% sequence identity; Preferably, the anti-VP1 protein antibody, its fragment or fusion protein is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein; Preferably, 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, dual-specific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (dsdiabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single domain antibody, nanobody, domain antibody or bivalent domain antibody; Preferably, the anti-VP1 protein antibody, fragment or fusion protein thereof is humanized; Preferably, the anti-VP1 protein antibody, fragment or fusion protein thereof further comprises replacement or modification of one or more amino acid residues, but retains the binding specificity to the VP1 protein; Preferably, the anti-VP1 protein antibody, fragment or fusion protein thereof specifically binds to the VP1 protein; wherein the VP1 protein has an amino acid sequence as shown in SEQ ID NO:

16.

4. The anti-VP1 protein antibody, fragment or fusion protein thereof according to claim 1, which is linked to one or more conjugate moieties; Preferably, the conjugate moiety comprises an agent for detection or separation, such as a clearance-improving agent, 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 drug; Preferably, the VP1 protein is norovirus VP1 protein; Preferably, the VP1 protein is Norovirus type 1 VP1 protein.

5. An isolated polynucleotide encoding the anti-VP1 protein antibody, fragment thereof or fusion protein according to any one of claims 1 to 4. A vector comprising the isolated polynucleotide according to claim 5.

7. A host cell comprising the vector according to claim 6.

8. A pharmaceutical composition comprising: The anti-VP1 protein antibody, fragment or fusion protein thereof according to any one of claims 1 to 4, or a polynucleotide encoding the anti-VP1 protein antibody, fragment or fusion protein thereof according to any one of claims 1 to 4; and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients; Preferably, the pharmaceutical composition further comprises other therapeutic agents; Preferably, the other therapeutic agent is an agent for treating Norovirus.

9. Any of the following methods, comprising: 1) A method for expressing the anti-VP1 protein antibody, fragment thereof or fusion protein according to any one of claims 1 to 4, comprising culturing the host cell according to claim 7 under the conditions of expressing the vector according to claim 6, and finally isolating the anti-VP1 protein antibody, fragment thereof or fusion protein according to any one of claims 1 to 4; 2) A method for detecting the presence or amount of VP1 protein or norovirus in a sample, comprising contacting the sample with the anti-VP1 protein antibody, fragment thereof or fusion protein according to any one of claims 1 to 4, and determining the presence or amount of VP1 protein or norovirus in the sample; Preferably, the method further comprises the step of determining whether VP1 protein is expressed in cells in the sample; Preferably, the VP1 protein is norovirus VP1 protein; Preferably, the norovirus is norovirus type 1.

10. Any of the following purposes, including: 1) Use of the anti-VP1 protein antibody, fragment or fusion protein thereof according to any one of claims 1 to 4, the isolated polynucleotide according to claim 5, the vector according to claim 6, or the host cell according to claim 7 in the production of a medicament for treating, preventing or alleviating a disease or condition responsive to VP1 protein inhibition; Preferably, the VP1 protein is norovirus VP1 protein; Preferably, the VP1 protein is Norovirus type 1 VP1 protein; 2) Use of the anti-VP1 protein antibody, fragment or fusion protein thereof according to any one of claims 1 to 4, the isolated polynucleotide according to claim 5, the vector according to claim 6, or the host cell according to claim 7 in producing a product for detecting VP1 protein or norovirus; Preferably, the VP1 protein is norovirus VP1 protein; Preferably, the norovirus is norovirus type 1.

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