CN108727488B - Preparation and application of anti-norovirus GII.17 monoclonal antibody - Google Patents

Abstract

The invention provides preparation and application of an anti-norovirus GII.17 monoclonal antibody, and particularly relates to a monoclonal antibody aiming at norovirus GII.17, which is prepared by immunizing a mouse by using a recombinant expressed norovirus GII.17 virus-like particle and adopting a hybridoma technology. Through screening, a monoclonal antibody capable of binding GII.17 is obtained, and the monoclonal antibody not only can specifically recognize the GII.17 virus-like particles, but also has strong neutralizing activity.

Description

Preparation and application of anti-norovirus GII.17 monoclonal antibody

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to preparation and application of an anti-norovirus GII.17 monoclonal antibody.

Background

Norovirus (NoVs) is one of the major pathogens that causes sporadic cases and large outbreaks of acute gastroenteritis, and can infect people of all ages. Although symptoms caused by norovirus infection are generally mild and have a self-limiting course lasting for about 1-3 days, they can cause more severe symptoms and even death in children, the elderly, and people with immune insufficiency. Norovirus can be divided into 6 genotypes (G1-GVI) and multiple genotypes, based on the amino acid sequence of the VP1 capsid protein, but only GI, GII and GIV can infect humans. Infection with human norovirus is mainly caused by norovirus GII, while a large outbreak is caused by norovirus gii.4. However, recently, norovirus gii.17 strains have caused large outbreaks of acute gastroenteritis in different asian countries, and norovirus gii.17 was detected in south america, north america and europe. According to the sequence of VP1, the newly circulating gii.17 strain belongs to gii.17cluster C, which is prevalent in asia and rapidly spread around the world, and gii.17 may replace gii.4 as the main pathogen of norovirus causing acute gastroenteritis in the near future.

Norovirus is prevalent in developed and developing countries, causing serious economic loss to each country and posing great threat to the health of children and the elderly. To date, there are no prophylactic vaccines and specific therapeutic drugs on the market. Norovirus lacks simple cell culture models, nor small animal models, which presents a significant obstacle to the study of vaccines and antiviral drugs. The humanized mouse-derived monoclonal antibody is an effective method for developing a medicament for preventing and treating viral infection, and the monoclonal antibody can also be used for diagnosing viral infection. Monoclonal antibodies against GII.17 have not been reported so far.

Disclosure of Invention

The invention aims to provide preparation and application of an anti-norovirus GII.17 monoclonal antibody.

In a first aspect of the invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region having one or more of the following complementarity determining regions CDRs:

CDR1 shown in SEQ ID NO.1,

CDR2 shown in SEQ ID NO.2, and

CDR3 shown in SEQ ID NO. 3.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 4.

In a second aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region and a heavy chain constant region as described in the first aspect of the invention.

In another preferred embodiment, the heavy chain constant region is of human or murine origin.

In another preferred embodiment, the amino-selected sequence of the heavy chain of said antibody is shown in SEQ ID NO. 10.

In a third aspect of the present invention, there is provided a light chain variable region of an antibody, said light chain variable region having complementarity determining regions CDRs selected from the group consisting of:

CDR 1' shown in SEQ ID NO.5,

CDR 2' shown in SEQ ID NO.6, and

CDR 3' shown in SEQ ID NO. 7.

In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 8.

In a fourth aspect of the invention, there is provided a light chain of an antibody, said light chain having a light chain variable region and a light chain constant region as described in the third aspect of the invention.

In another preferred embodiment, the constant region of the light chain is of human or murine origin.

In a fifth aspect of the invention, there is provided an antibody having:

(1) a heavy chain variable region according to the first aspect of the invention; and/or

(2) A light chain variable region according to the third aspect of the invention.

In another preferred embodiment, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.

In another preferred embodiment, the antibody is an antibody specific against norovirus gii.17.

In another preferred embodiment, the antibody comprises: a single chain antibody (scFv), a diabody, a monoclonal antibody, a chimeric antibody (e.g., a human-murine chimeric antibody), a murine antibody, or a humanized antibody.

In a sixth aspect of the present invention, there is provided a recombinant protein having:

(i) the sequence of a heavy chain variable region according to the first aspect of the invention, the sequence of a heavy chain according to the second aspect of the invention, the sequence of a light chain variable region according to the third aspect of the invention, the sequence of a light chain according to the fourth aspect of the invention, or the sequence of an antibody according to the fifth aspect of the invention;

(ii) polypeptide, protein drug sequences; and

(iii) optionally a tag sequence to facilitate expression and/or purification.

In another preferred embodiment, the polypeptide protein drug is a single chain antibody (scFv), a diabody, a monoclonal antibody, or a chimeric antibody.

In another preferred embodiment, the tag sequence is selected from the group consisting of: 6 × His tag, GGGS sequence, FLAG tag.

In another preferred embodiment, the recombinant protein comprises a bispecific antibody or a chimeric antibody.

In a seventh aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, an antibody according to the fifth aspect of the invention; or

(2) A recombinant protein according to the sixth aspect of the invention.

In another preferred embodiment, the polynucleotide has the sequence shown in SEQ ID No.13, 14, 15, 16, 17, 18, 11, or 9.

According to an eighth aspect of the invention, there is provided a vector comprising a polynucleotide according to the seventh aspect of the invention.

In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

According to a ninth aspect of the invention there is provided a genetically engineered host cell comprising a vector according to the eighth aspect of the invention or a genome into which has been integrated a polynucleotide according to the seventh aspect of the invention.

In a tenth aspect of the invention, there is provided an immunoconjugate comprising:

(a) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, an antibody according to the fifth aspect of the invention, or a recombinant protein according to the sixth aspect of the invention; and

(b) a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.

In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, liposomes, nanomagnetic particles, or any form of nanoparticles, and the like.

In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or an immunoconjugate according to the tenth aspect of the invention; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is in the form of injection.

In a twelfth aspect of the invention, there is provided a use of the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, or the immunoconjugate according to the tenth aspect of the invention for the preparation of a medicament, a reagent, a detection plate or a kit.

In another preferred embodiment, the reagent comprises a chip and immune microparticles coated with antibodies.

In a thirteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) culturing the host cell of the ninth aspect of the invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

FIG. 1 polyacrylamide gel electrophoresis analysis of purified anti-GII.17 mAb. The 5 purified antibodies were treated with a reducing agent-containing buffer, applied to a 12% polyacrylamide gel, electrophoresed, and stained with Coomassie Brilliant blue to reveal protein bands. M, protein molecular weight standards; 1, 1D3-2 monoclonal antibody; 2, 3a3-3 monoclonal antibody; 3, 2C1-1 monoclonal antibody; 4, 4B1-2 monoclonal antibody.

FIG. 2 enzyme-linked immunosorbent assay (Elisa) identifies the binding ability of the mAb to different antigens. 50ng of GII.7 (FIG. 2A), GII.4 (FIG. 2B) or GI.1 (FIG. 2C) virus-like particles were coated on each well of an Elisa plate, and each well was incubated with different concentrations of purified mAb at 37 ℃ for 2 hours, followed by incubation with HRP-labeled anti-mouse secondary antibody. Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody was used as an irrelevant control. Each point in the figure shows the mean and standard deviation of OD450nm determined for three replicate samples.

FIG. 3 Western blot analysis. After treatment, the virus-like particles were applied to a 12% polyacrylamide gel, electrophoresed, transferred to PVDF membrane, and hybridized with purified monoclonal antibodies. 1, gi.1 virus-like particle; 2, gii.3 virus-like particles; 3, gii.17 virus-like particles; control, monoclonal antibody against hepatitis B surface antigen (HBsAg).

FIG. 4 detection of GII.17 Virus-like particles by sandwich Elisa. 50ul1:2000 diluted rabbit anti-GII.17 serum was coated on each well of an Elisa plate, and different concentrations of GII.17 virus-like particles were added to each well and incubated at 37 ℃ for 2 hours, followed by 50ng of purified monoclonal antibody per well and finally incubated with HRP-labeled anti-mouse secondary antibody. Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody was used as an irrelevant control.

FIG. 5 is the identification of the monoclonal antibody expressed by gene recombination. 50ng of GII.4 virus-like particles were coated on each well of an Elisa plate, and each well was incubated with different concentrations of purified monoclonal antibody at 37 ℃ for 2 hours, followed by incubation with HRP-labeled anti-mouse secondary antibody. The culture supernatant of cells not transfected with plasmid served as a blank control. The histogram in the figure shows the mean and standard deviation of OD450nm for three replicate sample determinations.

Detailed Description

The present inventors have made extensive and intensive studies to produce a monoclonal antibody capable of specifically recognizing GII.17 using GII.17 virus-like particles as an immunogen. Elisa and alternative neutralization experiments, etc., indicate that these antibodies can be used for sensitive detection and analysis of GII.17, and more importantly, some monoclonal antibodies also have strong neutralizing activity.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

Norovirus

Norovirus is one of the major pathogens responsible for acute gastroenteritis in children and adults. Epidemiological data have shown that norovirus gii.4 is the primary pathogen responsible for the epidemic of norovirus outbreaks, but recently norovirus gii.17 has caused a number of acute gastroenteritis outbreaks in countries throughout asia, and norovirus gii.17 was detected in south america, north america, and europe. This brings serious economic loss to each country and also poses great threat to the health of children and the old. To date, there are no prophylactic vaccines and therapeutic drugs against norovirus gii.17. The invention utilizes recombinant expressed norovirus GII.17 virus-like particles to immunize mice, and prepares the monoclonal antibody aiming at the norovirus GII.17 by a hybridoma technology. Through screening, four monoclonal antibodies capable of binding GII.17 are obtained and named as 1D3-2, 2C1-1, 3A3-3 and 4B1-2 respectively. The results of ELISA and Western blot experiments showed that 1D3-2, 2C1-1, 3A3-3 and 4B1-2 specifically recognized the GII.17 virus-like particle with minimal detection limits of 0.078ng, 0.156ng and 0.156ng, respectively. In addition, substitution neutralization experiments show that the monoclonal antibodies 1D3-2 and 3A3-3 have strong potential neutralization activity. In conclusion, these antibodies are not only tools for laboratory testing, but also reliable candidates for the production of therapeutic humanized mabs and useful reagents for the development of diagnostic methods.

Antibodies

As used herein, the term "antibody" or "immunoglobulin" is an isotetraglycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a number of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the antibodies or fragments thereof of the present invention. The invention also comprises a cell surface marker or antigen bound to the anti-norovirus gii.17 antibody or fragment thereof.

As used herein, the terms "heavy chain variable region" and "V_H"may be used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3, wherein

CDR1：GYTFSSYW，SEQ ID NO.1；

CDR2：ILPGNDNS，SEQ ID NO.2；

CDR3：ARSTWDKGYYYPLDY，SEQ ID NO.3。

In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 4:

QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKLRPGHGLEWIGEILPGNDNSNYNKKFKGKATFTADTSSNTAYIQLGSLTSEDSAVYYCARSTWDKGYYYPLDYWGQGTSVTV，SEQ ID NO.4。

in a preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.

As used herein, the terms "light chain variable region" and "V_L"may be used interchangeably.

In a preferred embodiment of the invention, the light chain variable region of the antibody comprises three complementarity determining regions CDR1 ', CDR2 ', and CDR3 ', wherein

CDR1’：SSINY，SEQ ID NO.5；

CDR2’：DTS，SEQ ID NO.6；

CDR3’：HQRSSSPWT，SEQ ID NO.7。

In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 8:

QIVLTQSPAIMSASPGEKVTLTCSASSSINYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYHCHQRSSSPWTFGGGTELEIK，SEQ ID NO.8。

in a preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.

In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and all refer to a polypeptide that specifically binds to norovirus gii.17, e.g. a protein or polypeptide having a heavy chain variable region and/or a light chain variable region as described above. They may or may not contain the initial methionine.

The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.

In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy chain variable region, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest, as at least some of them are involved in binding antigen. Thus, the invention includes molecules having the variable regions of the heavy and/or light chains of antibodies with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

The invention includes not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The antibody of the present invention refers to a polypeptide having norovirus gii.17 binding activity, including the CDR regions described above. The term also includes variants of the polypeptides comprising the above CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

In the present invention, "conservative variants of the antibody of the present invention" refers to the substitution of up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with qualitatively similar or analogous amino acids compared to the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to table I.

TABLE I

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the above-described sequences and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) denaturant is added during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ℃ and the like; or (3) hybridization only when the identity between two sequences is at least 90% or more, preferably 95% or more. And, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides shown in one of SEQ ID Nos. 32-37.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acid, protein, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or e.g., vectors) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.

Transformation of a host cell with recombinant DNA may be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, such as E.coli, competent cells, which are capable of DNA uptake, can be harvested after exponential growth phase and subsequently treated with CaCl₂Methods of treatment, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Therapeutic agents that may be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis reviews)24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer communication (Cancer letters)239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 5. viral particles (Peng et al, 2004, Gene therapy 11, 1234); 6. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 7. nano magnetic particles; 8. a prodrug activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, and the like.

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-described antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous, or topical administration.

The pharmaceutical composition of the present invention can be directly used for binding norovirus gii.17 molecules, and thus can be used for preventing and treating norovirus infection. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described antibody (or conjugate thereof) of the present invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

When using pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Labeled immunoglobulins

In a preferred embodiment of the invention, the antibody carries a detectable label. More preferably, the marker is selected from the group consisting of: a colloidal gold label, a colored label, or a fluorescent label.

The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the present invention, the monoclonal antibody against norovirus gii.17 is labeled with colloidal gold to obtain a colloidal gold-labeled monoclonal antibody.

The norovirus GII.17 monoclonal antibody has good specificity and high titer.

Method and sample

The present invention relates to a method for detecting norovirus gii.17 in a sample lysed in cells and/or tissue. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of norovirus gii.17 in the lysed sample. The sample used in the method of the present invention may be any sample comprising cells present in a cell preservation solution, as used in liquid based cytology.

Reagent kit

The present invention also provides a kit comprising the antibody (or fragment thereof) of the present invention or the assay plate of the present invention, and in a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, and the like.

The invention further designs a detection kit for detecting the norovirus GII.17 level, which comprises an antibody for identifying the norovirus GII.17, a lysis medium for dissolving a sample, and general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates and the like. The test kit may be an in vitro diagnostic device.

The main advantages of the invention are:

(1) provides an anti-norovirus GII.17 monoclonal antibody for the first time;

(2) the anti-norovirus GII.17 monoclonal antibody provided by the invention not only can sensitively and specifically recognize non-denatured norovirus GII.17-like particles, but also has the capacity of binding to denatured norovirus GII.17-like particles.

(3) The anti-norovirus GII.17 monoclonal antibody provided by the invention has strong potential neutralization activity.

(4) The anti-norovirus GII.17 monoclonal antibody provided by the invention can recognize denatured norovirus GI.1 virus-like particles and norovirus GII.4 virus-like particles.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Experimental procedures without specifying detailed conditions in the following examples are generally carried out under conventional conditions such as those described in molecular cloning laboratory Manual, Sambrook, et al, U.S.A. (Huang Pepper's et al, Beijing: scientific Press, 2002), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Materials and methods

1. Antigen preparation and mouse immunization

Virus-like particles were prepared by expressing norovirus GII.17VP1 using the Pichia expression system. Female Balb/c mice were immunized intraperitoneally for 6 weeks after 5ug of virus-like particles (50ul volume) mixed with an equal volume of aluminum adjuvant (500ug), once at each of 0, 2, and 4 weeks. At week 6, mouse sera were taken to test the titer of antibodies specific for gii.17 virus-like particles and the effect of inhibiting the binding of gii.17 virus-like particles to porcine gastric mucin III. At 7 weeks, one mouse with the highest specific antibody titer and the better effect of inhibiting the combination of GII.17 virus-like particles and porcine gastric mucin III was selected to strengthen 15ug of GII.17 virus-like particles through tail vein. After 3 days, spleen from mice was used to prepare hybridoma cells.

2. Preparation and screening of hybridoma cell lines

After 3 days of tail vein boosting immunization, spleen cells of mice were taken and fused with myeloma cells SP2/0 by PEG1500 to prepare hybridoma cells. After 9 days, antibodies specifically secreting against gii.17 virus like particles were screened by enzyme linked immunosorbent assay. Briefly, GII.17 virus-like particles were coated in 96-well plates with 30ng per well, overnight at 4 ℃, blocked with PBST containing 5% skim milk, 50ul of hybridoma culture medium per well incubated at 37 ℃ for 2 hours, followed by HRP-labeled secondary antibody for 1 hour, and finally subjected to color development to read the absorbance at OD 450.

3. Ascites production and antibody purification

Female Balb/c mice were injected intraperitoneally with 500ul liquid paraffin two weeks later, 30 ten thousand hybridoma cells per mouse. After 7 days, ascites was collected with a 12-gauge needle, centrifuged at 10,000rpm for 10min, the upper layer of oil and the lower layer of precipitate were removed, and clarified ascites was subjected to antibody purification. The ascites fluid was purified using HiTrap HiTraptM Protein G affinity column (GE health care) according to the instructions to obtain the antibody.

4. Monoclonal antibody for enzyme-linked immunosorbent assay identification

Binding capacity of the mAbs was identified by coating 96-well Elisa plates with 50ng GI.1 or GII.4 or GII.17 virus-like particles per well overnight at 4 ℃. After blocking the Elisa plates by PBST containing 5% skim milk for 1 hour at 37 ℃, different concentrations (5ug/ml, 1ug/ml, 0.2ug/ml and 0.04ug/ml) were added to the monoclonal antibody at 50ul per well for 2 hours at 37 ℃, followed by incubation with HRP-labeled anti-mouse secondary antibody and reading of the absorbance OD 450.

5. Polyacrylamide gel electrophoresis and western blot analysis

Mixing the protein sample with SDS-PAG loading buffer solution, boiling for 10min, and separating the protein sample by 12% polyacrylamide gel. Protein bands were visualized by Coomassie blue staining or western blot analysis was performed by transferring the proteins onto PVDF membrane. The monoclonal antibody was diluted to a final concentration of 1ug/ml in PBST containing 1% skim milk. The murine anti-GII.17 polyclonal antibody 1:1000 was used diluted, followed by incubation with HPR-labeled murine secondary antibody and finally recorded with LAS-400 luminescence image analyzer.

6. Sandwich Elisa detection of GII.17 virus-like particles

96-well Elisa plates were coated with rabbit anti-GII.17 virus-like particle polyclonal antiserum at 1:2000 dilution (50 ul/well) overnight at 4 ℃, after blocking the Elisa plates with 5% skim milk-containing PBST for 2 hours at 37 ℃, virus-like particles were added to the Elisa plates, 40ng/50 ul/well started, 2 fold diluted to 12 concentrations, incubated for 2 hours at 37 ℃, followed by virus-like particle specific monoclonal antibody 50ng/50 ul/well incubated for 1 hour at 37 ℃, followed by incubation with HPR-labeled murine secondary antibody, and finally read the absorbance OD 450.

7. In vitro surrogate neutralization assay

96-well Elisa plates were coated with 10ug/ml PGMIII (50 ul/well) at room temperature and were blocked with 5% skim milk in PBST at 4 ℃ overnight for use. 8ug/ml of monoclonal antibody specific to GII.17 virus-like particles was diluted at 2-fold, incubated with equal volume of 0.5ug/ml of GII.17 virus-like particles at room temperature for 1 hour, and then applied to PGMIII-coated 96-well Elisa plates, incubated at room temperature for 1 hour, and then rabbit anti-GII.17VLP polyclonal antiserum 1:1000 diluted solution was added, incubated at 37 ℃ for 1 hour, and then incubated with HPR-labeled rabbit secondary antibody, and finally the absorbance OD450 was read.

8. Gene sequence amplification of monoclonal antibody and construction of expression vector

Firstly, extracting total RNA from cells of a hybridoma cell strain by using Trizol reagent, and then amplifying full-length genes of a heavy chain and a light chain according to the specification of a 5' RACE reagent kit. HindIII and EcoRI restriction sites are respectively introduced to the 5 'end and the 3' end of the heavy chain and the light chain by utilizing a PCR amplification method, all the amplified genes of the heavy chain and the light chain are respectively cloned into pGEM-T (Promage), positive clone sequencing is screened out, then the clone with correct sequence is subjected to double digestion by HindIII and EcoRI, a target fragment is purified by agarose gel electrophoresis, and the target fragment is connected with plasmid pcDNA3.1(Promage) subjected to the same digestion by using T4DNA ligase to construct eukaryotic expression vectors pcDNA3.1- (m3A3-3H) and pcDNA3.1- (m3A 3-3L).

9. Recombinant expression identification of monoclonal antibody gene

Co-transfecting pcDNA3.1- (m3A3-3H) and pcDNA3.1- (m3A3-3L) to 293T cells by using a liposome method, collecting culture supernatant for analysis after 72 hours, and determining the expression of antibodies in the culture supernatant by using ELISA: the plates were coated with GII.17 virus-like particles, blocked with 5% milk in PBST for 1 hour at 37 deg.C, incubated for 2 hours at 37 deg.C with various dilutions of the culture supernatant to be tested, then incubated with HRP-labeled anti-mouse IgG secondary antibody, and the absorbance OD450 was read.

Example 1 screening of hybridoma cells secreting GII.17-specific antibodies

Spleen cells from mice immunized with GII.17 virus-like particles were used to prepare hybridoma cells. Hybridoma cell supernatants are screened by an Elisa experiment, so that hybridoma cell strains capable of secreting the hybridoma cell strains with the ability to bind GII.17 virus-like particles are obtained. Finally, four monoclonal antibodies were screened, which were able to bind gii.17 virus-like particles. Subtype identification showed that the heavy chains of 1D3-2, 2C1-1, 3A3-3 and 4B1-2 were all IgG1 and the light chains were kappa.

TABLE 1 identification of hybridoma cell lines secreting monoclonal antibodies

The samples used for the analysis were all 50ul hybridoma culture cell supernatant.

*,+：OD450＞0.15；++：OD450＞0.3；+++：OD450＞0.5。

Example 2 specificity assay for anti-GII.17 mAbs

The purity and integrity of GII.4 mab purified from ascites fluid was first identified by SDS-PAGE. FIG. 1 shows that the heavy and light chains of the four mAbs are around 50KD and 25KD, respectively. Next, the reactivity of the mabs with different antigens, including gi.1 virus-like particles, gii.4 virus-like particles, and gii.17 virus-like particles, was tested by the Elisa method. FIG. 2 shows that 1D3-2, 2C1-1, 3A3-3 and 4B1-2 can specifically recognize GII.17 virus-like particles, but cannot recognize GI.1 virus-like particles and GII.4 virus-like particles. Finally, the binding of the mabs to gi.1, gii.4 and gii.17 was analyzed by Western blot, and fig. 3 shows that of the four antibodies: none of 1D3-2, 2C1-1 and 4B1-2 recognized denatured GI.1, GII.4 and GII.17 virus-like particles, while 3A3-3 recognized denatured GI.1, GII.4 and GII.17 virus-like particles.

Example 3 monoclonal antibody-based sandwich Elisa can specifically and sensitively detect GII.17 virus-like particles

The monoclonal antibodies were minimally detected for virus-like particles by sandwich Elisa assay (positive when OD450 > 0.15). FIG. 4 shows that 1D3-2, 2C1-1, 3A3-3 and 4B1-2 mabs all detected GII.17 virus-like particles with a specific sensitivity and the minimal detection limits were 0.078ng, 0.156ng and 0.156ng, respectively, suggesting that they may be useful in the diagnosis of GII.17 infection.

EXAMPLE 4 potential neutralizing Activity of monoclonal antibodies

Histoblood group antigens (HBGA) are carbohydrates expressed on mucosal tissues and red blood cells and are required receptors for norovirus infection. The binding inhibition assay of HBGA is widely used as an alternative neutralization assay for antibody-mediated norovirus. Porcine gastric mucin III contains HBGA and has been shown to be useful in alternative neutralization assays. The potential neutralizing activity of the four monoclonal antibodies 1D3-2, 2C1-1, 3A3-3 and 4B1-2 was tested by alternative neutralization tests. FIG. 5 shows that 1D3-2 and 3A3-3 have strong potential neutralizing activity against GII.17, and EC50, which inhibits binding of virus-like particles to PGMIII, is: 0.050ug/ml and 0.059 ug/ml.

Example 5 Gene sequence analysis of monoclonal antibody

The sequences of the heavy chain and the light chain of the cloned monoclonal antibody 3A3-3 are as follows (wherein,single underlinePart is a signal peptide sequence, and the italic part is a variable region sequence,dashed underlineAs constant region sequence):

3A3-3 monoclonal antibody heavy chain nucleotide sequence:

3A3-3 monoclonal antibody heavy chain amino acid sequence:

3A3-3 monoclonal antibody light chain nucleotide sequence:

3A3-3 monoclonal antibody light chain amino acid sequence:

further analysis using the in-line tool IgBLAST (https:// www.ncbi.nlm.nih.gov/IgBLAST /), under conditions of Mouse for query sequence, 3A3-3 single-antibody heavy chain variable region and light chain variable region sequences, 3A3-3 single-antibody heavy chain variable region amino acids are as follows (underlined as heavy chain CDR regions):

QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKLRPGHGLEWIGEILPGNDNSNYNKKFKGKATFTADTSSNTAYIQLGSLTSEDSAVYYCARSTWDKGYYYPLDYWGQGTSVTV(SEQ ID NO.4)

the heavy chain variable region described above belongs to the subgroup IGHV 1.

The amino acids in the light chain variable region of the 3A3-3 monoclonal antibody are as follows (the underlined parts are the CDR regions of the heavy chain):

QIVLTQSPAIMSASPGEKVTLTCSASSSINYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYHCHQRSSSPWTFGGGTELEIK(SEQ ID NO.8)

the light chain variable region described above belongs to subgroup IGKV 4.

The amino acid sequences and nucleotide sequences of the respective CDR regions are summarized in Table 2.

TABLE 2

EXAMPLE 6 recombinant expression and characterization of monoclonal antibody genes

To determine whether the cloned gene of 3A3-3 monoclonal antibody was correct, the coding sequences for the heavy and light chains were inserted into pcDNA3.1, respectively, expression vectors pcDNA3.1- (m3A3-3H) and pcDNA3.1- (m3A3-3L) were constructed, and 293T cells were co-transfected, and the cell supernatant was examined for the presence of antibodies specifically binding to GII.17 virus-like particles by ELISA. FIG. 5 shows that the cell supernatants expressing 3A3-3 mAb sequence showed high binding signals and only slight decrease in OD450 values at 8-fold dilution; whereas the supernatant of control cells not transfected with the relevant plasmid did not bind signal when undiluted. The results indicate that the amplified and expressed sequence is indeed the gene of 3A3-3 monoclonal antibody.

Discussion of the related Art

The initial objective of this study was to obtain monoclonal antibodies that specifically bind to gii.17 virions for further use in the development of kits for the diagnosis of norovirus disease, but unexpectedly two monoclonal antibodies 1D3-2 and 3A3-3 were obtained that have potent potential neutralizing activity and that will be humanized for use as therapeutic monoclonal antibody drugs. The monoclonal antibodies 1D3-2, 2C1-1, 3A3-3 and 4B1-2 have the minimal detection limits of 0.078ng, 0.156ng and 0.156ng on the GII.17 virus-like particles through sandwich ELISA, which provides a favorable theoretical basis for developing the monoclonal antibodies into norovirus detection kits. The resulting monoclonal antibodies 1D3-2 and 3A3-3 against gii.17 had EC50 for the interaction of gii.17 virus-like particles with PGMIII as: 0.050ug/ml and 0.059 ug/ml.

In this study, the feasibility of using monoclonal antibodies to detect GII.17 was demonstrated. Elisa experiments show that the monoclonal antibodies can effectively detect the GII.17 virus-like particles.

In summary, the present data show that the monoclonal antibody screened by the present invention not only can be used as a useful laboratory detection tool, but also can be used as an effective material for developing a diagnostic method and a reliable candidate for humanized anti-norovirus therapeutic monoclonal antibody.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Pasteur institute of Chinese academy of sciences

<120> preparation and application of anti-norovirus GII.17 monoclonal antibody

<130> P2017-0090

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<170> PatentIn version 3.5

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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

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Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr

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Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser

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Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn

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Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser

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Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr

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Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu

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Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

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Met Ser Ala Ser Pro Gly Glu Lys Val Thr Leu Thr Cys Ser Ala Ser

35 40 45

Ser Ser Ile Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Thr Ser

50 55 60

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

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Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr His Cys His Gln Arg

100 105 110

Ser Ser Ser Pro Trp Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys

115 120 125

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

130 135 140

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

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Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

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Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

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Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

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Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

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Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

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<212> DNA

<213> Artificial sequence

<400> 17

gacacatcc 9

<210> 18

<211> 27

<212> DNA

<213> Artificial sequence

<400> 18

catcagcgga gtagttcccc ctggacg 27

Claims (18)

1. An antibody having a heavy chain and a light chain, said heavy chain having a heavy chain variable region and said light chain having a light chain variable region, wherein said heavy chain variable region has the following complementarity determining regions CDRs:

CDR1 shown in SEQ ID NO.1,

CDR2 shown in SEQ ID NO.2, and

a CDR3 shown in SEQ ID NO. 3;

and, the light chain variable region has a complementarity determining region CDR selected from the group consisting of:

CDR 1' shown in SEQ ID NO.5,

CDR 2' shown in SEQ ID NO.6, and

CDR 3' shown in SEQ ID NO. 7.

2. The antibody of claim 1, wherein said heavy chain variable region has the amino acid sequence set forth in SEQ ID No. 4.

3. The antibody of claim 1, wherein said heavy chain further comprises a heavy chain constant region.

4. The antibody of claim 1, wherein the amino acid sequence of the heavy chain of said antibody is set forth in SEQ ID No. 10.

5. The antibody of claim 1, wherein said light chain variable region has the amino acid sequence set forth in SEQ ID No. 8.

6. The antibody of claim 1, wherein said light chain further comprises a light chain constant region.

7. The antibody of claim 1, wherein said antibody has:

(1) a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 4; and

(2) the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

8. The antibody of claim 1, wherein said antibody has: a heavy chain with an amino acid sequence shown as SEQ ID NO. 10; and a light chain having an amino acid sequence shown in SEQ ID NO. 12.

9. A recombinant protein, said recombinant protein having:

(i) the sequence of the antibody of any one of claims 1-8;

(ii) polypeptide, protein drug sequences; and

(iii) optionally a tag sequence to facilitate expression and/or purification.

10. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) the antibody of any one of claims 1-8; or

(2) The recombinant protein of claim 9.

11. The polynucleotide of claim 10, wherein the polynucleotide has the sequence shown in SEQ ID No.13, 14, 15, 16, 17, 18, 11, or 9.

12. A vector comprising the polynucleotide of claim 10.

13. A genetically engineered host cell comprising the vector or genome of claim 12 having the polynucleotide of claim 10 integrated therein.

14. An immunoconjugate, comprising:

(a) the antibody of any one of claims 1-8, or the recombinant protein of claim 9; and

(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

15. The immunoconjugate of claim 14, wherein the conjugate is selected from the group consisting of: a fluorescent or luminescent label, a radiolabel, a magnetic resonance imaging or computed tomography contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle/nanorod, a liposome, or a nanomagnetic particle.

16. A pharmaceutical composition, comprising:

(i) the antibody of any one of claims 1-8, or the recombinant protein of claim 9, or the immunoconjugate of claim 14; and

(ii) a pharmaceutically acceptable carrier.

17. Use of the antibody of any one of claims 1 to 8, or the recombinant protein of claim 9, or the immunoconjugate of claim 14, for the preparation of a medicament for the treatment of norovirus infection, or for the preparation of a reagent, assay plate or kit for the detection of norovirus.

18. A method of producing a recombinant polypeptide, the method comprising:

(a) culturing the host cell of claim 13 under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being the antibody of any one of claims 1-8 or the recombinant protein of claim 9.

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