CN109180810B - Antibody specifically binding norovirus GI.1 genotype VP1 protein or VLP, and preparation method and application thereof - Google Patents

Abstract

The invention discloses an antibody specifically binding to norovirus GI.1 genotype VP1 protein and/or VLP, which comprises CDR1, CDR2 and CDR3 regions of a heavy chain variable region shown in SEQ ID NO.1-3 and/or CDR1, CDR2 and CDR3 regions of a light chain variable region shown in SEQ ID NO. 4-6. The antibody or antigen binding part of the norovirus GI.1 genotype VP1 protein and/or VLP provided by the invention has no cross reaction with the GII.4 genotype, has high specificity, has the characteristic of blocking the binding of the norovirus GI.1 genotype VP1 protein and/or VLP and HBGA, has high affinity, can be used for detecting the existence or the development of a kit for detecting the norovirus GI.1 genotype VP1 protein and/or VLP in a sample, and has good application prospect in treating patients and performing passive immunity on susceptible people.

Description

Antibody specifically binding norovirus GI.1 genotype VP1 protein or VLP, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an antibody specifically binding to a norovirus GI.1 genotype VP1 protein and/or VLP, and a preparation method and application thereof.

Background

Norovirus is an important food-borne virus on a global scale and can infect humans and animals causing acute gastroenteritis. Norovirus epidemic infection, whether in developed or developing countries, involves people of all ages and can cause fulminant epidemics, has become a non-negligible problem affecting the daily health of humans. In 1995, China reported a first case of norovirus infection, and over 20 years, many groups were erupted and spread norovirus infectious diarrhea in succession across the country, especially in semi-closed environments such as kindergarten, school, military, hospital, etc., causing certain social panic. Norovirus is mainly derived from marine products and is commonly found in shellfish such as oyster. Can be transmitted by food-derived route, water-derived route, and human-human direct contact (feces-mouth route or vomit aerosol); virions are highly infectious, allowing infection by about 18 to 100 virions, rapid spread of the virus over a small area, and frequent aggregate infections and outbreaks in semi-enclosed and populated locations. There are no specific anti-NoV drugs and prophylactic and therapeutic vaccines on the market today, and antibiotics have little effect on diarrhea caused by this viral infection.

The norovirus belongs to the family of human Caliciviridae, the genus norovirus, is a group of non-enveloped single-stranded RNA viruses, and the virus particles are in an icosahedral symmetric structure and have diameters of about 26-35 nm. The viral genome is approximately 7.7kb in length and comprises 3 Open Reading Frames (ORFs). ORF1 is about 5kb in length and encodes a polyprotein (proprotein) which, after proteolysis during viral replication, forms 6 nonstructural proteins necessary for viral replication, including N-terminal protein, NTPase (nucleoside phosphatase), p22 (3A-like protein), VPg (covalently bound to the viral genome), Pro (3C-like protease), RNA-dependent RNA polymerase, etc.; ORFs 2 and 3 encode structural proteins, ORF2 is about 1.8kb in length, encodes the main structural protein VP1 of 57kD, and VP1 protein can be packaged in vitro into VLPs; ORF3 is approximately 0.6kb in length and encodes the 22kD minor structural protein VP2, VP2, whose function may be involved in the packaging of the genome into virions.

The norovirus capsid consists of 90 VP1 dimers, 180 VP1 constituting an icosahedral symmetric structure of T ═ 3. The VP1 protein can be structurally divided into two adjacent regions, namely an S region and a P region, which are connected through a flexible hinge. Region S forms an inner shell, which forms the base of VP1 and is unable to bind to the receptor; the dimeric structure of the P domain, which protrudes from the inner shell, contains a receptor binding domain, determining the antigenicity of NoV. The P region can be further divided into two subregions, P1 and P2, the latter being located at the outermost layer of VP1, highly variable and currently considered as a key site for immune recognition and receptor binding. The norovirus has high diversity of genes and antigenicity, and can be divided into 7 genomes, G I to G VII, according to the difference of complete VP1 sequences, and each genome can be further divided into genotypes. More than 30 genotypes can infect human, mainly comprising GI and GIII genomes, and in human norovirus infection, GI.1 and GII.4 genotypes occupy the dominant position and account for 70-80% of the worldwide epidemic NoV.

Norovirus cannot be cultured in vitro and there is no suitable animal model, and therefore, there is no accurate method to evaluate the level of neutralizing antibodies in serum after norovirus vaccine immunization. Viral receptors are one of the accepted initial steps and key factors for viral infection of host cells. Receptors are critical for the host, and infectious pathogens often evolve selectively to adapt to different receptor phenotypes. Hutson et al first proposed a hypothesis in 2002 that Norwalk virus infection is linked to ABO blood group antigens. Since then, receptor localization of norovirus has been a research hotspot internationally, and several years of research have confirmed that human histo-blood group antigens (HBGAs) on intestinal mucosa are NoV virus receptors or synergists, which is a milestone in norovirus research. HBGAs are a highly polymorphic class of carbohydrate antigens on the surface of the epithelial cells of the mucosa of the erythrocytes, respiratory tract, urogenital tract and digestive tract, and are expressed as ABO, secretory and Lewis on the epithelial cells of the intestinal tract. The expression of HBGA on the cell surface is controlled by ABO blood type, and has diversity due to different connection modes between monomeric saccharides, including A, B, Htype 1, Htype2, Lewis y, Lewis x, and leb.

At present, the research on the mechanisms of norovirus infection, pathogenic mechanisms, virus-host interaction, target cell receptors and the like is relatively few, the development of the traditional attenuated vaccine and inactivated vaccine is limited, on the other hand, the development process of the vaccine is slow due to the complex and rapid variability of the norovirus genotype, so far, no related vaccine is on the market, and the subunit vaccine based on genetic engineering is the main research strategy of the norovirus vaccine. The recombinant NoV VLPs vaccine developed by the Kyowa (Takeda) company through the insect cell/baculovirus expression system has been developed for phase II clinical studies, and the oral norovirus vaccine developed by the Vaxart company in the United states has been developed for phase I clinical studies using a non-replicating adenovirus as a vector.

It was found that monoclonal antibodies with neutralizing capacity essentially recognized conformational epitopes and that most monoclonal antibodies recognizing conformational epitopes specific for type have neutralizing capacity. Different neutralizing monoclonal antibodies can prevent virus infection from entering cells by blocking binding sites of viruses and cell receptors, and research on the binding epitopes of the monoclonal antibodies is beneficial to explaining key sites of virus infection entering cells. The determination of the activity of neutralizing antibodies induced by a vaccine is an important criterion for the detection of the immunoprotection of a prophylactic vaccine. The neutralizing active antibody has important application prospect in immunogenicity detection and content determination of norovirus vaccines.

Since no more effective specific antibody against the norovirus GI.1 genotype VP1 protein exists, it is necessary to find such an antibody and apply it to diagnosis and detection of norovirus GI.1 genotype infection.

Disclosure of Invention

The invention aims to solve the technical problem that a specific antibody for resisting norovirus GI.1 genotype VP1 protein is lack in the prior art, and provides an antibody specifically binding norovirus GI.1 genotype VP1 protein and/or VLP, and a preparation method and application thereof.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

an antibody which specifically binds to norovirus GI.1 genotype VP1 protein and/or VLP, comprising the CDR1, CDR2 and CDR3 regions of the heavy chain variable region shown in SEQ ID nos. 1-3 and/or the CDR1, CDR2 and CDR3 regions of the light chain variable region shown in SEQ ID nos. 4-6.

Another aspect of the invention provides an antibody that specifically binds to norovirus gi.1 genotype VP1 protein and/or VLP, comprising the heavy chain variable region amino acid sequence set forth in SEQ ID No.10 and/or the light chain variable region amino acid sequence set forth in SEQ ID No. 11.

Another aspect of the invention provides an antigen-binding portion that specifically binds norovirus gi.1 genotype VP1 protein and/or VLP, comprising the CDR1, CDR2 and CDR3 regions of the heavy chain variable region shown in SEQ ID nos. 1-3 and/or the CDR1, CDR2 and CDR3 regions of the light chain variable region shown in SEQ ID nos. 4-6;

wherein said antigen binding portion is selected from the group consisting of Fab, Fab ', F (ab')₂Fd, dAb, a complementarity determining region fragment, a single chain antibody, a humanized antibody, a chimeric antibody or a diabody.

The antigen-binding portion that specifically binds to norovirus gi.1 genotype VP1 protein and/or VLP can be obtained using methods well known to those skilled in the art, such as treatment with chemical reagents, or digestion with proteases, such as papain, pepsin, and the like.

In the present invention, sequences having 90% or more homology (preferably 95% or more) with the above-mentioned CDR amino acid sequences are also considered to be included in the scope of the present invention. Likewise, other variations of the above-described CDR amino acid sequences, such as deletion, insertion and/or substitution of one or more amino acids, addition of one or more amino acids at the C-terminus and/or N-terminus, and the like, are considered to be included within the scope of the present invention as long as they do not alter the biological function of the above-described CDR amino acid sequences.

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. Preferably, however, in one embodiment of the present invention, the antibody of the present invention is a monoclonal antibody.

Preferably, in one embodiment of the present invention, the antibody is a monoclonal antibody produced by a cell having a accession number of CGMCC NO. 15693. The cell is a hybridoma obtained by fusing immunized host spleen cells and myeloma cells, is preserved in China general microbiological culture Collection center (CGMCC), has a preservation number of CGMCC NO.15693, and has a preservation date of 2018, 04 months and 27 days.

The antibody provided by the invention is a type-specific antibody, can only be specifically combined with norovirus GI.1 genotype VP1 protein and/or VLP, and has no cross reaction to GII.4 genotype.

In the present invention, the antibody has a neutralizing activity, and is capable of blocking binding of VLPs to HBGA.

Preferably, in one embodiment of the invention, the antibody is a monovalent or bivalent antibody.

In the present invention, the monoclonal antibody can be prepared by a hybridoma preparation method as reported by Kohler et al, Nature 256:495 (1975). Mice or other suitable host animals are first immunized with the immunogen (with adjuvant added if necessary). In the invention, the immunogen is a virus-like particle (VLP) formed by automatically assembling norovirus GI.1 genotype VP1 protein which is generated by Hansenula polymorpha cells and obtained after chromatography purification and/or norovirus GI.1 genotype VP1 protein in vitro. The amino acid sequence of the antigen is shown in SEQ ID NO. 16.

The mode of injection of the immunogen or adjuvant is usually subcutaneous multi-site injection or intraperitoneal injection. Freund's adjuvant (Freund's complete adjuvant or Freund's incomplete adjuvant) or MPL-TDM, etc. can be used as the adjuvant. After an animal is immunized, lymphocytes that secrete antibodies that specifically bind the immunogen are produced in vivo. The lymphocytes of interest are harvested and fused with myeloma cells using a suitable fusing agent (e.g., PEG4000) to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996).

The hybridoma cells prepared as described above are inoculated into a suitable medium containing one or more substances capable of inhibiting the growth of unfused, maternal myeloma cells. For example, for parental myeloma cells that lack hypoxanthine guanine phosphotransferase (HGPRT or HPRT), the addition of hypoxanthine, aminopterin, and thymidine (HAT medium) to the culture medium will inhibit the growth of HGPRT-deficient cells.

Preferred myeloma cells should have high fusion rate, stable antibody secretion ability, sensitivity to HAT medium, and the like. Among them, THE myeloma cells are preferably derived from murine myelomas such as MOP-21 and MC-11 mouse tumor-derived strains (THE salt Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2/0 or X63-Ag8-653 Cell strain (American Type C. mu. culture Collection, Rockville, Md. USA). In addition, human Monoclonal antibodies can also be prepared using human myeloma and human murine allogeneic myeloma cell lines (Kozbor, J.Immunol., 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., New York, 1987).

The culture medium in which the hybridoma cells are grown is used to detect the production of monoclonal antibodies directed against specific antigens. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined using the following methods: immunoprecipitation or in vitro binding assays, such as Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). For example, the affinity of a mAb can be determined using the Scatchard assay described by Munson et al, anal. biochem.107:220 (1980).

After determining the specificity, affinity and reactivity of the Antibodies produced by the hybridomas, the cell lines of interest can be identified by Goding, Monoclonal Antibodies: subcloning was performed by limiting dilution as described by Principles and Practice, pp.59-103, Academic Press, 1996. Suitable media may be DMEM or RPMI-1640 or the like. In addition, hybridoma cells can also be grown in animals as ascites tumors.

The monoclonal antibody secreted by the subcloned cells can be separated from cell culture fluid, ascites fluid or serum by using a traditional immunoglobulin purification method, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, and the monoclonal antibody can be obtained.

Preferably, in one embodiment of the present invention, the obtained monoclonal antibody is numbered 401.

Another aspect of the present invention provides a polynucleotide encoding the above heavy chain variable region amino acid sequence or light chain variable region amino acid sequence.

Preferably, in one embodiment of the present invention, the polynucleotide sequence encoding the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO.12, and the polynucleotide sequence encoding the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 13.

Another aspect of the present invention provides a vector comprising the polynucleotide as described above.

Another aspect of the invention provides a kit comprising an antibody or antigen-binding portion thereof of the invention that specifically binds norovirus gi.1 genotype VP1 protein and/or VLP.

Preferably, in one embodiment of the invention, the kit further comprises a second antibody that specifically binds to norovirus gi.1 genotype VP1 protein and/or VLP.

In the above kit, the second antibody may be any antibody known in the art against the gi.1 genotype VP1 protein and/or VLP, thereby detecting the sample using a double sandwich method.

More preferably, the second antibody comprises the CDR1, CDR2 and CDR3 regions of the heavy chain variable region shown in SEQ ID Nos. 7-9.

Preferably, in one embodiment of the present invention, the second antibody comprises the heavy chain variable region amino acid sequence shown as SEQ ID No. 14.

Preferably, in one embodiment of the invention, the second antibody comprises an amino acid sequence encoded by the heavy chain variable region nucleotide sequence set forth in SEQ ID No. 15.

More preferably, the second antibody is a monoclonal antibody produced by a cell having a collection number of CGMCC NO.15694, which is collected in the China general microbiological culture Collection center (CGMCC) with a collection date of 2018, 04 and 27 days.

Preferably, in one embodiment of the present invention, the monoclonal antibody of the obtained secondary antibody is numbered 101.

Preferably, in one embodiment of the invention, the second antibody is prepared using the same method as the antibody of the invention which specifically binds norovirus gi.1 genotype VP1 protein and/or VLP.

Another aspect of the invention is to provide the use of an antibody, antigen binding portion, polynucleotide, vector or kit as described above for detecting the presence or level of norovirus gi.1 genotype VP1 protein and/or VLP in a sample.

The invention has the beneficial effects that:

the antibody or antigen binding part specifically binding to the norovirus GI.1 genotype VP1 protein and/or VLP provided by the invention has no cross reaction with the GII.4 genotype, has high specificity, has the characteristic of blocking the binding of the norovirus GI.1 genotype VP1 protein and/or VLP and HBGA, and has high affinity. The kit can be used for detecting the existence or level of norovirus GI.1 genotype VP1 protein and/or VLP in a sample, and has good application prospect in treating patients and performing passive immunization on susceptible people.

Biological preservation information:

the preservation number is: CGMCC NO.15693

The preservation date is as follows: 04 month and 27 days 2018

The preservation unit: china general microbiological culture Collection center (CGMCC) for short, the address: microbial research institute of western road 1 institute No. 3 of China academy of sciences, Beijing, Chaoyang

The preservation number is: CGMCC NO.15694

The preservation date is as follows: 04 month and 27 days 2018

The preservation unit: china general microbiological culture Collection center (CGMCC) for short, the address: microbial research institute of western road 1 institute No. 3 of China academy of sciences, Beijing, Chaoyang

Drawings

FIG. 1 is the electron microscopic observation result of GI.1 genotype VP1-VLP of antigen norovirus prepared in example 1 of the present invention.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO.1-3 are the amino acid sequences of the heavy chain variable region CDR1-3 of an antibody or antigen binding portion of the invention;

SEQ ID Nos. 4 to 6 are amino acid sequences of CDR1-3 in the light chain variable region of the antibody or antigen-binding portion of the present invention;

SEQ ID Nos. 7 to 9 show the amino acid sequence of CDR1-3 in the heavy chain variable region of the second antibody of the present invention;

SEQ ID NO.10 is the amino acid sequence of the heavy chain variable region of the antibody of the present invention;

SEQ ID NO.11 is the amino acid sequence of the light chain variable region of the antibody of the present invention;

SEQ ID NO.12 is the heavy chain variable region nucleotide sequence of the antibody of the present invention;

SEQ ID NO.13 is the light chain variable region nucleotide sequence of the antibody of the present invention;

SEQ ID No.14 is the heavy chain variable region amino acid sequence of the second antibody of the present invention;

SEQ ID NO.15 is the heavy chain variable region nucleotide sequence of the second antibody of the present invention;

SEQ ID NO.16 is the amino acid sequence of the antigen norovirus GI.1 genotype VP1 protein.

Detailed Description

The invention discloses an antibody specifically binding to norovirus GI.1 genotype VP1 protein and/or VLP, and a preparation method and application thereof, and can be realized by appropriately modifying process parameters by referring to the content. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference, and that the techniques of the invention may be practiced and applied by those skilled in the art without departing from the spirit, scope and range of equivalents of the invention.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

The term "antibody", as used herein, refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are 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 also contains 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 3 domains (CH1, CH2, and CH 3). 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 region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

The inventionThe term "antigen-binding portion" as used herein, refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding fragment". See generally, Fundamental Immunology, Ch.7(Paul, W., ed., 2 nd edition, Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes₂Fd, Fv, etc.

Wherein, the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F (ab')₂By fragment "is meant an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region. The term "Fd fragment" means an antibody fragment consisting of the VH and CH1 domains; the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.

Herein, when the term "antibody" is referred to, it includes not only intact antibodies, but also antigen-binding fragments of antibodies, unless the context clearly indicates otherwise.

The term "monoclonal antibody" as used herein refers to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using hybridoma technology first reported by Kohler et al (Nature,256:495,1975), but can also be obtained using recombinant DNA technology (see, e.g., U.S. P4, 816, 567).

The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, such as between an antibody and the antigen against which it is directed.

The term "neutralizing antibody" as used herein refers to an antibody or antibody fragment that is capable of clearly or significantly reducing the virulence of a target virus or pseudovirus.

The term "epitope" as used herein refers to a linear epitope or a conformational epitope on an antigen at a site to which an immunoglobulin or antibody specifically binds.

"monoclonal antibody" and "monoclonal antibody" as used herein have the same meaning and are used interchangeably.

In the present invention, amino acids are generally represented by the single and three letter abbreviations known in the art, such as: alanine can be represented by A or Ala.

In the present invention, the term "adjuvant" refers to a non-specific immunopotentiator which, when mixed with an antigen, can enhance the immune response of the body to the antigen or change the type of immune response, and includes, but is not limited to, aluminum adjuvants such as aluminum hydroxide, Freund's complete adjuvant, Freund's incomplete adjuvant, etc.

In the invention: the term "norovirus VLPs" refers to virus-like particles assembled from in vitro expressed norovirus VP1 protein, such as norovirus gi.1VP 1-VLPs refer to virus-like particles assembled from in vitro expressed norovirus gi.1VP1 protein.

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

The main instruments and reagents used in this embodiment:

1. main reagent, kit and consumable

Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA) and PEG4000 were purchased from Sigma.

DMEM medium (containing 2% HAT) and Fetal Bovine Serum (FBS) were purchased from HyClone, USA.

HRP-conjugated goat anti-mouse IgG Fc was purchased from Jackson Immun, usa.

HRP substrate A and B were purchased from Beijing DuPont Biotechnology Ltd.

Protein G affinity column materials were purchased from general electric (china) medical group.

Sepharose CL-4B gel filtration was from general electric (China) medical group.

10% dimethyl sulfoxide protective solution: contains 10% of dimethyl sulfoxide, 20% of inactivated fetal calf serum and 70% of RPMI-1640 liquid.

20% FCS-1640 medium: contains 100U/ml of penicillin and 100 mu g/ml of streptomycin.

HBGA is available from GlycoTech.

The other reagents are all domestic analytical pure products.

2. Main instrument

CO₂Incubators were purchased from SANYO corporation.

Biosafety cabinets were purchased from Baker corporation.

Inverted fluorescence microscope was purchased from Olympus corporation.

Flow cytometry was purchased from BD.

The microplate reader was purchased from berle life medicine products (shanghai) ltd.

IMMAGE 800 is available from Beckmann Kulter, Inc., USA.

The UV spectrophotometer was purchased from Leiberttaceae, Inc.

3. Laboratory animals and cell lines

The experimental animal is an SPF-grade Balb/c female mouse, is 8-12 weeks old and has the weight of about 28g, and is purchased from Beijing Wintonlifa experimental animals technology Limited company and is bred under the barrier condition.

SP 2/0 cells were purchased from biotechnology limited, tokyo banko.

Example 1 preparation and selection of hybridoma cells

1. Preparation of immunogens

The immunogen used for preparing the monoclonal antibody of the norovirus GI.1VP1 protein is norovirus GI.1VP1-VLP protein, the amino acid shown as SEQ ID NO.16 is expressed by Hansenula polymorpha cells through in vitro recombination, the amino acid can be automatically assembled into VLP, and the virus-like particles shown in the figure 1 can be observed by a transmission electron microscope, have the diameter of between 20 and 40nm and are spherical. Norovirus GI.1VP1-VLP protein was mixed with equal volume of Freund's complete adjuvant (CFA) or Freund's incomplete adjuvant (IFA), and fully emulsified by sonication to prepare the corresponding immunogen.

2. Animal immunization

On day 0, the immunogen mixed with CFA is injected into mice at multiple points through subcutaneous back injection, 3 mice are immunized for 0.2mg times/mouse, 4 times of immunization are carried out, the interval of each time is 1 week, the serum of the mice is collected after 3 times of immunization, the antibody titer is detected by the indirect ELISA method shown below, the antibody titer of the serum of the mice is more than 1 ten thousand times, and the antibody titer can be used for cell fusion.

3. Indirect ELISA method

GI.1VP1 protein coated 96-well plates at 100 ng/well. Adding 100 μ l of PBS solution containing 5% skimmed milk powder into Negative Control (NC) wells, and standing overnight at 4 ℃; discarding the liquid in the hole, washing the plate with PBS for 3 times, adding PBS solution containing 5% skimmed milk powder, sealing at 200 μ l/hole for 1 hr at room temperature; discarding the liquid in the wells, washing the plate for 1 time by PBST, adding primary antibody (tail blood is diluted according to the proportion of 1: 10000; hybridoma cell culture supernatant is diluted according to the proportion of 1:10 and the gradient of 2 times; mouse ascites is diluted according to the gradient of 3 times from the proportion of 1: 10000) and incubating for 1 hour at room temperature; discarding the liquid in the hole, washing the plate for 3 times by PBST, adding goat anti-mouse IgG Fc coupled with HRP (diluted in a ratio of 1: 10000), and incubating for 1 hour at room temperature; washing the PBST for 3 times, adding the substrate A and the substrate B, reacting at 50 mu l/hole in a dark place at room temperature for 15 min; the reaction was stopped by adding 1mol/L sulfuric acid, and the A450/630 value was measured by a microplate reader, using A450/630 (positive) >2 times A450/630(NC) as a standard.

4. Preparation and screening of hybridoma cells

The mouse with the highest antibody titer was taken for fusion. 3 days before the fusion, the cells were fused by boosting with the immunogen mixed with IFA at a concentration of 0.08 ml/cell and spleen cells were isolated from the spleen. Firstly, separating single splenocytes, fusing the splenocytes with mouse myeloma SP 2/0 cells by PEG4000(500g/L) according to the proportion of 4:1, and culturing the cells in a 96-well plate at the constant temperature of 37 ℃ for 14 days by using a DMEM medium containing 20% FBS and 2% HAT; collecting supernatant, screening positive clone cells, further performing expanded culture by using a DMEM medium containing 20% FBS, collecting supernatant, performing re-screening, performing expanded culture on the re-screened positive clone cells, collecting supernatant, performing cryopreservation and resuscitation to obtain positive clones, and collecting supernatant. The culture supernatant was detected by indirect ELISA and positive cell lines were selected. The results are shown in Table 1. 42 positive cell lines were selected from 400 cell lines, and 14 cell lines in total, in which antibodies against GI.1VP1 protein were stably secreted, were selected from the positive cell lines, and NC was a negative control.

TABLE 1 ELISA test results of cell strain supernatants stably secreting anti-GI.1 VP1 protein antibody

5. Specific antibody detection of hybridoma cell secretory supernatant

The coating sources were GI.1VP1 protein and GII.4 VP1 protein, 100 ng/well, respectively, using an indirect ELISA method. The antibody secreted from the cell supernatant of the 14 hybridoma cells was detected, and the results showed that the antibody secreted from the cell supernatant of the 14 hybridoma cells reacted only positively with the gi.1VP1 protein (as shown in table 1), and both the results of the detection of the gi.1VP1 protein were negative, and the 15 hybridoma cells were identified as type-specific antibodies.

6. Detection of neutralizing antibody Activity (HBGA-VLP blocking assay)

The neutralizing activity of the antibody and its antibody titer were tested by Synthetic HBGA-VLPs blocking assay.

The specific method comprises the following steps:

1) HBGA is added into an avidin coated microporous plate, 2.5 mu g/ml and 100 mu l of HBGA is added into each hole, the avidin coated microporous plate is incubated at 25 ℃ for 1 hour, then the solution in the plate is discarded, and the plate is washed for 3 times by phosphate buffer for later use.

2) Diluting the serum or antibody to be detected by a dilution plate in a 2-fold gradient, fully mixing the diluted serum or antibody with norovirus GI.1VP1-VLP solution (3.2 mu g/ml) with the same volume, simultaneously establishing a quality control serum control, a positive control (serum-free maximum binding VLPs holes) and a blank control (diluent holes), incubating for 1 hour at 37 ℃,

3) pipette 100 μ l of VLP and serum, antibody or diluent mixture slowly into the blocked plate, incubate at 4 ℃ for 2 hours, wash the plate with phosphate buffer 3 times.

4) HRP-labeled gi.1VP1 rabbit polyclonal antibody was diluted to working concentration (1: 8000) the cells were added to a microplate in an amount of 100. mu.l/well, incubated at 4 ℃ for 1 hour, and washed 3 times with phosphate buffer.

5) Color development: and sequentially adding 50 mul of color developing agent A and 50 mul of color developing agent B into each hole, uniformly mixing, adding 50 mul of stop solution into each hole to stop reaction at room temperature for 5 minutes, and performing double-wavelength reading (A450nm/630nm) in an enzyme-linked immunosorbent assay.

6) And (4) calculating a result: with BT₅₀The reaction neutralizes antibody titers. BT (BT)₅₀I.e., the highest dilution of serum that was able to block 50% of norovirus gi.1VP 1-VLPs from binding to HBGA receptors.

TABLE 2 detection of neutralizing antibody Activity of hybridoma cell secretion supernatants

7. Detection of neutralizing antibody Activity (PGM-VLP blocking assay)

A96-well microplate was coated with 5. mu.g/ml of porcine gastric mucin III (PGM) (100. mu.l/well) of Yuanmu biosciences, Shanghai, Ltd. (100. mu.l/well) at room temperature (25 ℃ C.), and blocked with 5% skim milk-containing PBST at 4 ℃ overnight for use. GI.1 VLP specificmonoclonal antibody 10 mu g/ml is started, diluted by 16 gradients in 2-fold proportion, incubated with equal volume of 0.5 mu g/ml GI.1 VLP for 1 hour at 37 ℃, added to a 96-well ELISA plate coated with PGM, incubated for 1 hour at room temperature (25 ℃), added with HRP-labeled rabbit anti-GI.1 virus-like particle polyclonal antibody (1:1000 dilution), incubated for 1 hour at room temperature (25 ℃), and finally read the absorbance OD 450.

8. Ascites preparation, antibody purification and antibody titer detection

8 hybridoma cells with high neutralizing activity are selected to prepare ascites, each positive clone cell is inoculated to 2 mice, the ascites is collected after 10 to 14 days, and ELISA detection is carried out on antibodies in the ascites, and the results are shown in Table 3. Then, ascites is purified by utilizing a Protein A affinity column to obtain a purified antibody, an A280 value is measured by an ultraviolet spectrophotometer, and the concentration of the antibody is calculated.

TABLE 3 mouse ascites antibody ELISA test results

9. Subculturing and preserving hybridoma cells

After the hybridoma cells are continuously cultured and subcultured in a DMEM medium containing 10% fetal calf serum until 10 passages, the 4G2-D1-B10(101) and 4H4-E6(401) can stably secrete monoclonal antibodies against GI.1VP1 protein, and the growth of the monoclonal antibodies is good.

After obtaining hybridoma cells producing the desired monoclonal antibody, a portion of the hybridoma cells are preserved, otherwise, during serial passages, mutations or chromosomal shifts may occur such that the intrinsic properties are lost or antibody-producing properties are lost; in addition, in the long-term culture process, pollution and destruction cannot occur inevitably, so that the cell strain is necessary to be preserved. The preservation method comprises the following steps: the old culture medium in the cell culture flask was removed, and 10% FCS-1640 solution was added to suspend the cells. Centrifuging at 1000rpm/min for 10min, and removing supernatant. The cell sediment is made into suspension by using 10% dimethyl sulfoxide protective solution, so that the suspension is 1.0 multiplied by 10⁷Cells/ml. Sampling, staining with trypan blue, and counting the number of living cells to be more than 95%. Finally, the cells were dispensed into ampoules, 0.5-1.0 ml per vial, using sterile syringes, and the ampoules were sealed by fusion. Standing at 4 deg.C for 2 hr, transferring into refrigerator at-70 deg.C for 15 hr, and freezing in liquid nitrogen.

Example 24 identification of H4-E6(401) antibodies

1. Obtaining antibodies

Adult BALB/c mice were selected and intraperitoneally inoculated with 0.5ml of pristane per mouse. The 16 th generation of 4H4-E6(401) hybridoma cells were intraperitoneally inoculated 7-10 days later, at1X 10/mouse⁶-2×10⁶And (4) respectively. After 5 days, when the abdomen is obviously enlarged and the hand touches the abdomen, the skin is tense, and the ascites is collected by using a 9-gauge needle.

2. Purification of antibodies

Ascites was centrifuged at 13000rpm for 30 minutes, the cell fraction and other precipitates were removed, and the supernatant was collected. The final purification by Protein A affinity chromatography gave the monoclonal antibody 4H4-E6(401) of GI.1VP1 Protein at a concentration of 5mg/ml or more.

3. Antibody purity detection

The purified antibody was subjected to 12% SDS-PAGE, and the purity was found to be 95% or more.

4. Determination of antibody light chain and heavy chain variable region gene sequence

Extracting mRNA of 4H4-E6(401) hybridoma cells, performing reverse transcription to cDNA, performing high fidelity PCR amplification by using variable region universal primers, inserting PCR product fragments into a T vector for DNA sequence determination, and performing DNA sequence determination on the variable region gene sequences of 4H4-E6 (401): the heavy chain is shown as SEQ ID NO.12, and the light chain is shown as SEQ ID NO. 13. The obtained nucleotide sequence is translated into an amino acid sequence. 4H4-E6 (401): the heavy chain is shown as SEQ ID NO.10, and the light chain is shown as SEQ ID NO. 11.

5. Detection of antibody type specificity

As shown by the specific antibody detection results in the supernatant secreted by the hybridoma cells 5 in example 1,

the 4H4-E6(401) antibody only reacts positively to GI.1VP1 protein, and the detection result is negative to GI.1VP1 protein, thus proving that the 4H4-E6(401) antibody is a type-specific monoclonal antibody.

6. Identification of neutralizing Activity of antibodies (HBGA-VLP blocking assay)

As shown by the result of the detection of the neutralizing activity of the antibody 6 in example 1, the 4H4-E6(401) antibody was a monoclonal antibody having a neutralizing activity. The purified 4H4-E6(401) antibody was diluted to 25. mu.g/ml, and then serially diluted in 2-fold gradient, and the antibody neutralization activity assay was performed according to 6. neutralization antibody activity assay method in example 1, BT 4H4-E6(401) antibody bound to GI.1 and HBGA₅₀The corresponding antibody concentration was 3.125. mu.g/ml.

7. Detection of neutralizing antibody Activity (PGM-VLP blocking assay)

As shown by the result of the detection of the neutralizing activity of the antibody of 7 in example 1, the 4H4-E6(401) antibody was a monoclonal antibody having a neutralizing activity. The purified 4H4-E6(401) antibody was diluted to 10. mu.g/ml, and then serially diluted in 2-fold gradient, and the antibody neutralization activity assay was carried out according to 7. neutralization antibody activity assay method in example 1, wherein the antibody concentration of 4H4-E6(401) antibody against GI.1 and PGM-bound BT50 was 0.3125. mu.g/ml.

Example 34 identification of the G2-D1-B10(101) antibody

1. Obtaining antibodies

The same procedure as in example 2-1 was conducted, except that 4G2-D1-B10(101) hybridoma cells were seeded.

2. Purification of antibodies

The same procedures as in example 2-1 were carried out to finally obtain monoclonal antibody 4G2-D1-B10(101) of GI.1VP1 protein at a concentration of 5mg/ml or more.

3. Detection of antibody purity

The same procedure as in example 2-1 was carried out, and as a result, the purity was found to be 95% or more.

4. Determination of gene sequence of antibody heavy chain variable region

Extracting mRNA of 4G2-D1-B10(101) hybridoma cells, performing reverse transcription to form cDNA, performing high-fidelity PCR amplification by using variable region universal primers, inserting PCR product fragments into a T vector for DNA sequence determination, and determining the gene sequence of the variable region of 4G2-D1-B10 (101): the heavy chain is shown as SEQ ID NO. 15. The obtained nucleotide sequence is translated into an amino acid sequence. 4G2-D1-B10 (101): the heavy chain is shown as SEQ ID NO. 14.

5. Detection of antibody type specificity

As shown in example 1, 5. the detection result of the specific antibody in the supernatant secreted by the hybridoma cell shows that the 4G2-D1-B10(101) antibody only reacts positively to the GI.1VP1 protein, and the detection result of the GII.4 VP1 protein is negative, thus proving that the 4G2-D1-B10(101) antibody is the type-specific monoclonal antibody.

6. Identification of neutralizing Activity of antibodies

As shown by the result of the detection of the neutralizing activity of the antibody of 7 in example 1, the 4G2-D1-B10(101) antibody was a monoclonal antibody having a neutralizing activity. The purified 4G2-D1-B10(101) antibody was diluted to 25. mu.g/ml, followed by serial dilutions of 2-fold gradient according toExample 1 neutralization antibody Activity test method 6 neutralization antibody Activity test, 4G2-D1-B10(101) antibody to GI.1 and HBGA-bound BT₅₀The corresponding antibody concentration was 3.125. mu.g/ml.

7. Detection of neutralizing antibody Activity (PGM-VLP blocking assay)

As shown by the result of the detection of the neutralizing activity of the antibody of 7 in example 1, the 4G2-D1-B10(101) antibody was a monoclonal antibody having a neutralizing activity. The purified 4G2-D1-B10(101) antibody was diluted to 10. mu.g/ml, and then serially diluted in 2-fold gradient, and the neutralizing activity of the antibody was measured according to the method for measuring the activity of neutralizing antibody described in example 1, wherein the concentration of the antibody against GI.1-PGM-bound BT50 by 4G2-D1-B10(101) antibody was 0.3125. mu.g/ml.

Example 4 GI.1VP1 antigen detection kit

The monoclonal antibodies 4H4-E6(401) and 4G2-D1-B10(101) obtained in example 1 were used to develop GI.1VP1 double antibody sandwich ELISA kits for detecting GI.1VP1 antigen level.

1. Establishment of double-antibody sandwich ELISA method

A. The principle of the kit is as follows:

both 4H4-E6(401) and 4G2-D1-B10(101) are monoclonal antibodies specific to the GI.1VP1 type and having neutralizing activity. The method comprises the steps of taking 4H4-E6(401) as a coating antibody, taking 4G2-D1-B10(101) as an enzyme-labeled antibody, establishing a double-antibody sandwich ELISA detection kit, adding a sample solution to be detected or a standard solution into an enzyme-labeled plate which is pre-coated with the 4H4-E6(401) antibody, adding 4G2-D1-B10(101) enzyme-labeled secondary antibody, developing with a developing solution, enabling the absorption value of a sample to be in positive correlation with the content of GI.1VP1 protein in the sample in a linear range, and comparing with a standard curve to obtain the content of GI.1VP1 protein in the sample. Meanwhile, the concentration range of the GI.1VP1 protein in the sample can be roughly judged by comparing the color of the enzyme label plate with the color of the GI.1VP1 protein standard solution with a series of concentrations according to the color depth of the enzyme label plate.

B. The kit comprises the following components:

(1) elisa plate coated with coating antibody: the coating antibody is a 4H4-E6(401) type specific monoclonal antibody, and is secreted and generated by a hybridoma cell strain clone 4H4-E6(401) with the preservation number of CGMCC NO. 15693. The coated antibody is diluted to 3 mu g/ml by using a coating buffer solution, coated on a 96-well enzyme label plate, added with 100 mu l per well, and incubated for 8 hours at the temperature of 4 ℃. The coating solution was discarded, washed 3 times with washing solution and patted dry. Then, 100. mu.l of blocking solution was added to the microplate and incubated at 37 ℃ for 2 hours. Pouring out the liquid in the hole, drying, and storing in an aluminum film vacuum seal mode. Wherein the used coating buffer solution is a carbonate buffer solution with the pH value of 9.5 and 0.1 mol/L; the blocking solution used was a barbiturate sodium-hydrochloric acid buffer solution containing bovine serum albumin at a final concentration of 8% (mass percentage) in the blocking solution, having a pH of 8.6 and 0.1 mol/L.

(2) Enzyme-labeled antibody: the labeled enzyme is horseradish peroxidase, the labeling method is a sodium periodate method, the enzyme-labeled antibody is a 4G2-D1-B10(101) type specific neutralizing monoclonal antibody, the monoclonal antibody is secreted and generated by a hybridoma cell strain clone 4G2-D1-B10(101) with the preservation number of CGMCC NO.15694, and the working concentration of the enzyme-labeled antibody is 0.1 mu G/ml.

(3) Standard solution: GI.1VP1 protein standard solution, 7 bottles, with concentrations of 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.0625. mu.g/ml, 0.03125. mu.g/ml and 0.015625. mu.g/ml, respectively. The prepared standard solution is DMSO with a final concentration of 5-10% (mass percentage content) in the solution for preparing the standard, and phosphate buffer solution with a pH of 6.5-6.7 and a concentration of 0.1-0.2 mol/L.

(4) Substrate color developing solution: the color developing solution A is hydrogen peroxide, and the color developing solution B is o-phenylenediamine.

(5) The stop solution is 1-2mol/L sulfuric acid solution.

(6) Concentrating the washing solution: a phosphate buffer solution with a pH value of 8.5, a final concentration of 0.05% (mass percent) sodium azide in the concentrated washing solution, a final concentration of 2.0% (mass percent) tween-20 in the concentrated washing solution and 0.3 mol/L; 40 ml/bottle, 1 bottle.

(7) Concentrating the compound solution: contains DMSO with a final concentration of 5-10% (mass percentage content) in a double solution, and phosphate buffer solution with pH of 6.5-6.7 and 0.1-0.2 mol/L; 200 ml/bottle, 1 bottle.

(8) Kit instructions.

2. Kit specificity verification

The GI.1VP1 and GII.4 VP1 proteins are prepared into a sample of 50 mu g/ml, and the sample is diluted to a diluent of 2 mu g/ml, 1 mu g/ml, 0.5 mu g/ml, 0.25 mu g/ml, 0.125 mu g/ml, 0.0625 mu g/ml and 0.03125 mu g/ml respectively, and the results of ELISA detection of the GI.1VP1 and GII.4 VP1 proteins by using the GI.1VP1 antigen detection kit are shown in Table 4, and the GI.1VP1 antigen detection kit can detect GI.1VP1 positively and GII.4 VP1 proteins negatively, and the kit specificity is good.

TABLE 4 kit specificity verification test results

Note: negative control (sample dilution) OD450 < 0.05

3. Kit sensitivity verification

The lowest antigen concentration detected by the kit is the key for determining the sensitivity of the kit. The GI.1VP1 protein was prepared as a 50. mu.g/ml sample, diluted to 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.0625. mu.g/ml, 0.03125. mu.g/ml and 0.015625. mu.g/ml, respectively. And (3) repeatedly measuring the diluted sample by using the kit for 6 times, taking the average value of the lower detection limit of the 6 standard curves, and finally determining the sensitivity of the kit to be 7.81 ng/ml.

4. Kit precision verification

A. Precision verification-in-plate differences

The gi.1VP1 protein was diluted to 1 μ g/ml, 0.5 μ g/ml, 0.25 μ g/ml, 0.125 μ g/ml and 0.0625 μ g/ml for a total of 5 different antigen concentrations, each antigen concentration being in 5 duplicate wells (n ═ 6) with the sample dilutions as negative controls. The OD variation Coefficient (CV) between wells of the same concentration in the microplate was calculated, and the results are shown in table 5, in which the CV (%) values in the plate were less than 15%, indicating that the precision in the kit was good.

TABLE 5 in-board Difference verification results for reagent kit

B. Precision verification-inter-plate difference

The gi.1VP1 protein was diluted to 1 μ g/ml, 0.5 μ g/ml, 0.25 μ g/ml, 0.125 μ g/ml and 0.0625 μ g/ml for a total of 5 different antigen concentrations, each antigen concentration being in 5 duplicate wells (n ═ 6) with the sample dilutions as negative controls. The OD value variation Coefficient (CV) of each well of the same concentration between different elisa plates was calculated, and the results are shown in table 6, wherein the CV (%) between the plates was less than 15%, indicating that the precision between the elisa plates was good.

TABLE 6 verification results of inter-plate differences of kits

5. Kit accuracy verification

Diluting GI.1VP1 protein to 50ng/ml, 60ng/ml and 70ng/ml, adding the diluted solution as a standard solution into a test solution (240 mu g/ml), making a standard curve according to the content of VP1 protein corresponding to the absorbance value of the test solution, calculating the concentration of GI.1VP1 protein in the test solution before and after standard addition, calculating the sample recovery rate, and analyzing the accuracy of the kit. The results are shown in Table 7, and show that the recovery rate of the spiked standard (95% CI) is between 71.21 and 125.73%, and the recovery rate of the kit is good.

Table 7 kit accuracy verification results

6. Kit repeatability verification

The GI.1VP1 protein was diluted to 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml, 0.125. mu.g/ml, 0.0625. mu.g/ml, 0.03125. mu.g/ml and 0.015625. mu.g/ml7 concentrations, taking the sample diluent as a negative control, making a standard curve according to the absorbance value of the sample corresponding to the GI.1VP1 protein content, detecting each type for 6 times, and obtaining the linear correlation coefficient R of the standard curve²The results are shown in table 8, and the correlation coefficient R2 is not lower than 0.99, which indicates that the detection method has good linear correlation and repeatability.

TABLE 8 kit repeatability verification results

7. Shelf life test of kit

The storage condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard) and the actual measurement value of the GI.1VP1 protein of the kit are within the normal range after 6 months of measurement. Considering that abnormal storage conditions occur in the transportation and use processes, the kit is placed for 6 days under the condition of being stored at 37 ℃ for accelerated aging experiments, and the results show that all indexes of the kit completely meet the requirements. And in consideration of the occurrence of the freezing condition of the kit, the kit is frozen for 5 days in a refrigerator at the temperature of-20 ℃, and the determination result also shows that all indexes of the kit are completely normal. From the above results, it was found that the kit could be stored at 2-8 ℃ for at least 6 months.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> national institute of Biotechnology, Biotechnology limited; limited liability company of Beijing institute of biological products

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Claims (12)

1. An antibody which specifically binds to norovirus gi.1 genotype VP1 protein or VLP, comprising the CDR1, CDR2 and CDR3 regions of the heavy chain variable region shown in SEQ ID nos. 1-3, and the CDR1, CDR2 and CDR3 regions of the light chain variable region shown in SEQ ID nos. 4-6.

2. An antibody specifically binding to norovirus gi.1 genotype VP1 protein or VLP, comprising a heavy chain variable region amino acid sequence as set forth in SEQ ID No.10 and a light chain variable region amino acid sequence as set forth in SEQ ID No. 11.

3. An antigen-binding portion which specifically binds norovirus gi.1 genotype VP1 protein or VLP, comprising the CDR1, CDR2 and CDR3 regions of the heavy chain variable region shown in SEQ ID nos. 1-3, and the CDR1, CDR2 and CDR3 regions of the light chain variable region shown in SEQ ID nos. 4-6;

wherein said antigen binding portion is selected from the group consisting of Fab, Fab ', F (ab')₂、Fd、dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody or diabody.

4. The antibody according to claim 1 or 2, wherein the antibody is a monoclonal antibody produced by a cell having a accession number of CGMCC No. 15693.

5. The antibody of claim 1 or 2, wherein the antibody is a monoclonal antibody having neutralizing activity.

6. A polynucleotide encoding the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of claim 2.

7. The polynucleotide of claim 6, wherein the polynucleotide sequence encoding the amino acid sequence of the heavy chain variable region is depicted in SEQ ID No.12 and the polynucleotide sequence encoding the amino acid sequence of the light chain variable region is depicted in SEQ ID No. 13.

8. A vector comprising the polynucleotide of claim 6 or 7.

9. A kit comprising the antibody of any one of claims 1-2 or the antigen-binding portion of claim 3.

10. The kit of claim 9, further comprising a second antibody that specifically binds to norovirus gi.1 genotype VP1 protein or VLP.

11. The kit according to claim 10, wherein the second antibody is a monoclonal antibody produced by a cell having a accession number of CGMCC No. 15694.

12. Use of the antibody of claim 1, 2, 4 or 5, the antigen-binding portion of claim 3, the polynucleotide of claim 6 or 7, the vector of claim 8 or the kit of any one of claims 9 to 11 for detecting the presence or level of norovirus gi.1 genotype VP1 protein or VLP in a sample; the use is for non-disease diagnostic purposes.

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