Antibody for resisting GP2 protein as well as preparation method and application of antibody

Abstract

The invention provides an anti-porcine reproductive and respiratory syndrome virus antibody or an antigen-binding fragment thereof, a coding nucleic acid comprising the antibody or the antigen-binding fragment thereof, an expression vector, a cell, a composition, a method for preparing the antibody or the antigen-binding fragment thereof, and an application of the antibody or the antigen-binding fragment thereof. The anti-porcine reproductive and respiratory syndrome virus antibody disclosed by the invention can be used for diagnosing, preventing and treating porcine reproductive and respiratory syndrome virus infection.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more particularly to antibodies against porcine reproductive and respiratory syndrome virus and their applications. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV), characterized by abortion in sows and respiratory disorders in piglets, and can cause severe immunosuppression. This virus has spread widely in pig herds worldwide, causing enormous economic losses to the global pig industry. It has become one of the major diseases affecting large-scale pig farms globally and a significant challenge in global pig disease control.

[0003] PRRSV is an enveloped, single-stranded, positive-sense RNA virus belonging to the Arteriviridae family and the Arterivir genus. The PRRSV genome is approximately 15 kb in length and contains at least ten open reading frames (ORFs) and two untranslated regions (UTRs) at the 5' and 3' ends. ORF1, further divided into ORF1a and ORF1b, accounts for about 75% of the entire genome and encodes two large replicase precursor proteins, pp1a and pp1ab. ORF2-ORF7 are located at the 3' end of the PRRSV genome, accounting for about 25% of the remaining genome, and encode eight structural proteins: GP2, E, GP3, GP4, GP5, GP5a, M, and N proteins. GP2 and GP4 form a stable binary complex via intramolecular and intermolecular disulfide bonds. During the viral budding stage, GP3 interacts with the complex formed by GP2 and GP4 to form a stable ternary complex of GP2, GP3, and GP4, which is incorporated into progeny viral particles to facilitate interaction between the progeny virus and the receptor, establishing effective infection. Due to the crucial role of the GP234 complex in the viral infection process, it is an effective target for vaccine development.

[0004] Therefore, it is necessary to develop antibodies against the GP234 protein of porcine reproductive and respiratory syndrome virus (PRRSV) for the detection and / or prevention and treatment of PRRSV or the diseases it causes. Summary of the Invention

[0005] This disclosure provides an antibody against porcine reproductive and respiratory syndrome virus (PRRSV) or an antigen-binding fragment thereof, a composition including the antibody or the antigen-binding fragment thereof, a method for preparation and application.

[0006] According to one aspect of this disclosure, an antibody against porcine reproductive and respiratory syndrome virus (PRV) or an antigen-binding fragment thereof is provided, said antibody or antigen-binding fragment comprising: a1) The amino acid sequences CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region as shown in SEQ ID NO: 1; and the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region as shown in SEQ ID NO: 2; or a2) Compared with CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 described in a1), at least one CDR contains one or more amino acid substitutions, deletions or additions; The CDR is defined by IMGT, Kabat, Chothia, or the Contact numbering system.

[0007] In some embodiments, the antibody or its antigen-binding fragment comprises: b1) A heavy chain variable region comprising amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and a light chain variable region comprising amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 14, YAS, and SEQ ID NO: 15, respectively; wherein the CDRs are defined by the IMGT numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 15, respectively; wherein the CDRs are defined by the Kabat numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 15, respectively; wherein the CDRs are defined by the Chothia numbering system, or The heavy chain variable region comprises the amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and the light chain variable region comprises the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; wherein the CDRs are defined by the Contact numbering system, or b2) Heavy chain variable region; and, light chain variable region, wherein, compared with the heavy chain variable region and / or light chain variable region of b1), at least one CDR contains a substitution, deletion or addition of one or more amino acids.

[0008] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region, which may contain an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 75% sequence identity with it.

[0009] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region, which may contain an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 75% sequence identity with it.

[0010] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment further includes a framework region of the heavy chain variable region. In some embodiments, the light chain variable region of the antibody or its antigen-binding fragment further includes a framework region of the light chain variable region.

[0011] In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region of immunoglobulins derived from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese, or a mutant thereof; further, it includes the framework region of the heavy chain variable region of mouse immunoglobulins, or a mutant thereof.

[0012] In some embodiments, the light chain variable region of the anti-porcine reproductive and respiratory syndrome virus antibody or its antigen-binding fragment further includes the framework region of the light chain variable region.

[0013] In some embodiments, the framework region of the light chain variable region includes the framework region of the light chain variable region of immunoglobulins derived from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese, or a mutant thereof; further, it includes the framework region of the light chain variable region of mouse immunoglobulins, or a mutant thereof.

[0014] In some embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0015] In some embodiments, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

[0016] In some embodiments, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

[0017] In some embodiments, the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having at least 80% sequence identity with it.

[0018] In some embodiments, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins.

[0019] In some embodiments, the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence having at least 80% sequence identity with it.

[0020] In some embodiments, the antibody may be a monoclonal antibody.

[0021] In some embodiments, the antibody may be a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0022] In some embodiments, the antibody may be murine, porcine, porcine-derived, chimeric, humanized, or fully human.

[0023] In some embodiments, the antibody may be of type IgA, IgD, IgE, IgG, or IgM. In some specific embodiments, the antibody may be of type IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0024] In some embodiments, the antigen-binding fragment may be one known in the art. In specific embodiments, the antigen-binding fragment may include scFv, Fab, Fab', (Fab')2, Fv fragment, Fd, dsFv.

[0025] In some embodiments, the antibody or its antigen-binding fragment specifically binds to the GP234 complex. In a preferred embodiment, the antibody or its antigen-binding fragment binds to the GP2 protein.

[0026] In some embodiments, the antibody or its antigen-binding fragment specifically binds to the GP234 complex of NADC30 and NADC34 strains. In a preferred embodiment, the antibody or its antigen-binding fragment binds to the GP2 protein of NADC30 and NADC34 strains.

[0027] According to another aspect of this disclosure, a nucleic acid molecule is provided that encodes an antibody or antigen-binding fragment thereof against porcine reproductive and respiratory syndrome virus as described in this disclosure.

[0028] According to another aspect of this disclosure, a carrier is provided that includes the nucleic acid molecules described in this disclosure.

[0029] In some embodiments, the vector includes prokaryotic expression vectors and eukaryotic expression vectors.

[0030] According to another aspect of this disclosure, a cell is provided comprising the anti-porcine reproductive and respiratory syndrome virus antibody or antigen-binding fragment thereof as described in this disclosure, the nucleic acid molecule as described in this disclosure, or the vector as described in this disclosure.

[0031] In some embodiments, the cells do not contain reproductive material.

[0032] According to another aspect of this disclosure, a conjugate is provided, comprising: an antibody against porcine reproductive and respiratory syndrome virus (PRRSV) as described in this disclosure or an antigen-binding fragment thereof; and a conjugation portion, wherein the conjugation portion is selected from detectable substances, small molecule drugs, and / or proteins.

[0033] In some embodiments, the protein may include, but is not limited to, cytokines, antibodies, antibody Fc fragments, antibody scFv fragments, enzymes, and viral capsid proteins.

[0034] In some embodiments, the detectable substance can be any substance conventionally used in the art, such as, but not limited to, fluorescent substances, luminescent markers, or radioactive substances. In a preferred embodiment, the detectable substance can be selected from any one of acridine esters, acridine sulfonamides, luminol, isoluminol, horseradish peroxidase, alkaline phosphatase, and isotopes.

[0035] According to another aspect of this disclosure, a method for preparing antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) or antigen-binding fragments thereof as described herein is provided.

[0036] In some embodiments, the method includes culturing the cells described in this disclosure and isolating the antibody under conditions that allow expression of the antibody or its antigen-binding fragment.

[0037] According to another aspect of this disclosure, a pharmaceutical composition is provided comprising: the antibody or antigen-binding fragment thereof described in this disclosure, the nucleic acid molecule, the carrier, the cell, or the conjugate.

[0038] In some embodiments, the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.

[0039] In some embodiments, the pharmaceutically acceptable carrier may be a carrier conventionally used in the art.

[0040] In some embodiments, the route of administration of the pharmaceutical composition may be parenteral, injection, oral, or topical. The pharmaceutical composition may be formulated into a form suitable for administration, such as a solid, semi-solid, or liquid form, and may be an aqueous solution, non-aqueous solution, or suspension, or in the form of powder, tablet, capsule, granules, injection, or infusion.

[0041] According to another aspect of this disclosure, the use of the antibody against porcine reproductive and respiratory syndrome virus (PRRSV) or its antigen-binding fragment thereof, the nucleic acid molecule, the vector, the cell, the conjugate, or the pharmaceutical composition described herein is provided, the use including one or more of the following: d1) Detect the presence or content of porcine reproductive and respiratory syndrome virus in the sample; d2) Diagnosis or prognostic assessment of diseases related to porcine reproductive and respiratory syndrome; d3) Prepare reagents for detecting the presence or content of porcine reproductive and respiratory syndrome virus in samples; d4) Prepare reagents for the diagnosis or prognostic assessment of porcine reproductive and respiratory syndrome.

[0042] In some embodiments, the sample includes whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions from the subject.

[0043] In some embodiments, a method for detecting porcine reproductive and respiratory syndrome virus (PRRSV) in a sample is provided, the method comprising the step of contacting the sample with an anti-PRRSV antibody or an antigen-binding fragment thereof as described in this disclosure.

[0044] In some embodiments, a method is provided for diagnosing or assessing the prognosis of porcine reproductive and respiratory syndrome (PRRS) using the anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibody or antigen-binding fragment thereof as described in this disclosure. In some embodiments, the method includes the step of contacting an anti-PRRSV antibody or antigen-binding fragment thereof as described in this disclosure with a sample from the subject.

[0045] In some implementations, the subject may include a pig.

[0046] According to another aspect of this disclosure, antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) or antigen-binding fragments thereof, nucleic acid molecules, vectors, cells, conjugates, or pharmaceutical compositions described herein are provided for the treatment and / or prevention of PRS.

[0047] In some embodiments, this disclosure provides the use of the nucleic acid molecules, the vectors, the cells, the conjugates, or the pharmaceutical compositions described herein in the preparation of medicaments for the treatment and / or prevention of porcine reproductive and respiratory syndrome.

[0048] In some embodiments, this disclosure provides a method for treating and / or preventing porcine reproductive and respiratory syndrome (PRRS), the method comprising: administering to a subject in need an effective amount of the anti-PRRS virus antibody or antigen-binding fragment thereof described in this disclosure, the nucleic acid molecule, the vector, the cell, the conjugate, or the pharmaceutical composition.

[0049] The beneficial effects of this invention are: This invention provides an antibody against porcine reproductive and respiratory syndrome virus (PRRSV) or its antigen-binding fragment, which can specifically recognize and bind to PRRSV or its GP2 protein and has good affinity for it; it can be used to prepare products for the diagnosis, prevention and / or treatment of PRRSV infection or diseases caused by PRRSV, for detecting the presence or level of PRRSV or its GP2 protein in samples, or for screening drugs for the prevention and / or treatment of PRRSV infection or diseases caused by PRRSV. Attached Figure Description

[0050] Figure 1 The results of the GP2-2 antibody subtype assay are shown.

[0051] Figure 2 The results of SDS-PAGE electrophoresis identification of the genetically engineered antibody are shown.

[0052] Figure 3 The results of flow cytometry binding verification of the genetically engineered antibody are shown, where PE is phycoerythrin and FITC isothiocyanate fluorescein.

[0053] Figure 4 The affinity results for the genetically engineered antibody are shown.

[0054] Figure 5 The results of ELISA-level GP2-2 antibody binding to different strains are shown. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0056] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0057] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0058] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0059] The term "antibody" in this article encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single-chain molecules, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0060] The terms "antibody or antigen-binding fragment thereof" and "antibody" are used interchangeably herein, referring to an immunoglobulin capable of specifically binding to a target antigen via at least one of its antigen-binding domains. Antibodies can be classified into different types based on the amino acid sequence of their heavy chain constant regions. There are five major types of immunoglobulins: IgG, IgA, IgM, IgD, and IgE, with heavy chains of γ, α, μ, δ, and ε chains, respectively. Antibodies can also be further classified into subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The light chains of antibodies, based on the amino acid sequence of their constant domains, can be classified into two types: κ and λ.

[0061] The term "variable region" or "variable domain" used in this article refers to the domain of the antibody heavy or light chain in an antigen-binding molecule that participates in antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain is sufficient to confer antigen-binding specificity. The HVRs in each chain are tightly held together by the FR regions and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site. Constant regions do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0062] The term "hypervariant region" or "HVR" in this article refers to a region in the variable domain region of an antibody that is highly variable in sequence and / or forms a structurally defined loop ("hypervariant loop"). Typically, a natural tetrachain antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariant loop and / or from the "complementarity-determining region (CDR)," the amino acid residues from the "CDR" having the highest sequence variability and / or being involved in antigen recognition.

[0063] The term "framework" or "FR" in this article refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences typically appear in the VH (or VL) in the following sequence: FR-H1(L1)-FR-H2(L2)-FR-H3(L3)-FR4.

[0064] The term "substitution" or "replacement" of amino acids used in this article can refer to the substitution of conserved amino acids, in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine (L), arginine (R), histidine (H)), acidic side chains (e.g., aspartic acid (D), glutamic acid (E)), uncharged polar side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C)), nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), β-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)), and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, such substitution is considered conserved. In another respect, an amino acid chain can be conservatively replaced by a structurally similar amino acid chain that differs in the order and / or composition of its side chain family members.

[0065] The term "nucleic acid molecule" used in this article refers to one of the most important biological macromolecules in organisms. It is the material basis for carrying and transmitting genetic information and includes two major categories: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0066] The term "antibody fragment" or "antigen-binding fragment" used in this article refers to a portion of a complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments, linear antibodies, single-chain antibody molecules (e.g., scFv), single-domain antibodies (single-domain antibodies), etc.

[0067] After obtaining the DNA fragments encoding the VH and / or VL of the antibody, these DNA fragments can be further manipulated using recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding the VH and / or VL is operatively ligated to another DNA fragment encoding a different protein, such as the antibody constant region or a flexible linker. As used herein, the term "operatively ligated" means that two DNA fragments are joined together in such a way that the amino acid sequences encoded by both DNA fragments remain within the reading frame.

[0068] By operatively linking the DNA encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3), isolated DNA encoding the VH region can be converted into a full-length heavy chain gene. Similarly, by operatively linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene).

[0069] The terms "antigen-binding domain" or "antigen-binding site" used herein refer to the portion of an antibody or its antigen-binding fragment that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a region of an antibody that specifically binds to and is complementary to a portion or all of an antigen. In cases where the antigen molecule is large, the antibody may bind only to a specific portion of the antigen, called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In some embodiments, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0070] The term "antigenic determinant" used herein is interchangeable with "antigen" and "epitope" and refers to a site on a polypeptide molecule (e.g., a continuous amino acid sequence or a conformation composed of discontinuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Unless otherwise stated, the protein used as an antigen in this disclosure can be any naturally occurring protein of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), dogs, cats, etc.

[0071] The terms "vector" or "expression vector" and "expression construct" used herein are used interchangeably to describe a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vectors disclosed herein contain expression cassettes. Expression vectors can perform transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, the cellular transcription and / or translation mechanisms generate a ribonucleic acid molecule or protein encoded by the gene. The term "expression cassette" herein refers to a recombinant or synthetically produced polynucleotide having a set of nucleic acid elements that allow transcription of a specific nucleic acid in the target cell.

[0072] The term "pharmaceutical composition" herein refers to a mixture containing an antibody or antibody- or antigen-binding fragment thereof, biological material, or conjugate of the present disclosure, and other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0073] The term "treatment" herein refers to the administration of an oral or topical therapeutic agent, such as a composition comprising any antibody of the present disclosure or an antibody thereof or an antigen-binding fragment thereof, or a nucleic acid molecule encoding an antibody thereof or an antibody thereof or an antigen-binding fragment thereof, to a subject who has one or more diseases or symptoms, and the therapeutic agent has a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent.

[0074] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0075] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0076] Those skilled in the art will understand that the reference herein to having “at least 75% sequence identity” compared to a sequence is intended to include all sequences having more than 75% sequence identity with that sequence, such as sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity.

[0077] "Affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Binding affinity is typically expressed as a dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constants (Koff and Kon, respectively). Therefore, equivalent affinity can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured using conventional methods known in the art, such as surface plasmon resonance (SPR). When used herein, the antibody molecule is "bound" to the antigen, a binding understood by those skilled in the art. In one embodiment, the antibody binds to the antigen with a dissociation constant (KD) of approximately 1 × 10⁻⁶. -5 M or lower, 1×10 -6 M or lower, or 1×10 -7 M or lower, 1×10 -8 M or lower, 1×10 -9M or lower, 1×10 -10 M or lower, 1×10 -11 M or lower.

[0078] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0079] Table 1. Amino acid sequences of VH and VL of GP2-2 antibody

[0080] Table 2. Amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of GP2-2 antibody

[0081] Table 3. Amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 of GP2-2 antibody

[0082] Example Example 1. Expression and purification of GP2, GP3 and GP4 proteins 1. Plasmid construction 1) Eukaryotic recombinant plasmid expressing GP2: The truncated GP2 variant encoding the NADC30 strain (NCBI accession number MH500776) (named GP2-209, with the secreted LYSP signal peptide (MEFGLSWVFLVALFRGVQC, SEQ ID NO: 21) added to the N-terminus of the truncated GP2 variant of the NADC30 strain (SQSPDGWWYFASDWFAPRYSVRALPFTLSNYRRSYEAYLSQCRVDIPSWGTKHPLGMFWHHKVSTLIDAMVSRRVYHTMEKAGQAAWKQVVSEATLSRISSLDVVAHFQHLAAIEAETCKYLASRLPMLHNLRMSGSNVTIKYNSTSGRVFAIFPTPDSRPKLHDFQQW, SEQ ID NO: 21) was modified. 22) The gene with a His tag added to the C end was inserted between SalI and EcoRI of the pCMV plasmid (brand: Youbao Biotechnology; name: pCMV-HA-C vector; catalog number: VT1065; the same below) to obtain a eukaryotic recombinant plasmid expressing GP2.

[0083] 2) Eukaryotic recombinant plasmid expressing GP3: The gene encoding the GP3 mutant of the NADC30 strain (named: GP3-M3; a Flag tag was added to the N-terminus of the GP3 mutant of the NADC30 strain (APSANSTYCFWFPLVRGNFSFELMVNYTVCPPCLTRQAAAQILEPGGSLWCRIGHDRCEESDHDELEFAVPPGLSVEGRLTSVYAWLAFLSFSYTAQFHPEIFGIGNVSEVYVDIQNQLICAVHDGLNATLPSHDNITAVYQSYYQHQVDGGNWFHLEAARPAFSSALVANVSAFLRRSPVSRVSVRVFQTLRPTPPAQQVPLSSKTSAVLGIATRPLRRFAKSLNVARR, SEQ ID NO: 23), and a secretory LYSP signal peptide was added to the N-terminus of the Flag tag) was inserted between Sal I and EcoR I of the pCMV plasmid to obtain the eukaryotic recombinant plasmid expressing GP3.

[0084] 3) Eukaryotic recombinant plasmid expressing GP4: The gene encoding GP4 of the NADC30 strain (with a Strep tag added to the N-terminus of GP4 (SQAFACKPCFSSSLSDIKTNTTSAASSVVLQNIGCLRHSNPSPAAFRKIPQCRAAIGTPVYITVTANVTDENYLHSSDLLMLSSCLFYASEMSEKGFKVIFGNVSGIVAVCVNFTSYVQHVREFTQRSLVVDHV, SEQ ID NO: 24) of the NADC30 strain, and a secretory LYSP signal peptide added to the N-terminus of the Strep tag) was inserted between Sal I and EcoR I of the pCMV plasmid to obtain the eukaryotic recombinant plasmid expressing GP4.

[0085] 2. Large-scale preparation of eukaryotic recombinant plasmids Take 1 μg of the GP2, GP3, and GP4 eukaryotic recombinant plasmids obtained in step 1 and mix them with 100 μL of Top10 competent cells (purchased from Qingke Biotechnology), and place on ice for 15 minutes. After heat shock at 42℃ for 90 seconds, place on ice for 5 minutes, add antibiotic-free liquid LB medium, and incubate at 37℃ and 220 rpm for 40 minutes. After incubation, centrifuge at 2000×g for 5 minutes, discard most of the supernatant, resuspend the competent cells in the remaining medium, and spread evenly on solid LB (Amp+) culture dishes. Incubate at 37℃ for 12–16 hours. After incubation, single colonies with good growth can be picked from the culture dishes for expansion culture, and plasmid extraction can be performed according to the instructions of the Tiangen Plasmid Large-Scale Extraction Kit.

[0086] 3) Expression and purification of the immunogen (GP234 complex) Mix the GP2, GP3, and GP4 eukaryotic recombinant plasmids obtained in step 2 with the transfection reagent PEI (purchased from Polyscience), and let stand at room temperature for 5-10 minutes after mixing. Take a sample with a density of 3×10⁻⁶. 6 1 L of 293F cells / mL was added dropwise to the prepared plasmid / PEI mixture while shaking, and the cells were cultured at 130 rpm for 4 days in a 37°C incubator with 5% CO2. After 4 days of culture, the cells were harvested, and the culture was centrifuged to collect the supernatant. Cell debris was removed by filtration through a 0.45 μM filter. The supernatant was then concentrated by ultrafiltration (10 kD) using a membrane encapsulation method, and diluted with buffer (1×PBS) for later use.

[0087] The Ni chromatography column was equilibrated with equilibration buffer (1×PBS buffer). The supernatant was loaded onto the Ni chromatography column, and 5-10 column volumes of washing buffer (1×PBS buffer, 20 mM imidazole) were added. After baseline equilibration, the target protein was eluted with elution buffer (1×PBS buffer, 500 mM imidazole). The eluted protein solution was filtered through a 0.22 μM filter membrane for sterilization to obtain the purified GP234 complex. The protein concentration was determined using NanoDrop.

[0088] Example 2. Screening of monoclonal antibodies 1. Mouse immunization Prepare 6-8 week old BALB / c mice. On days 1 and 30 of the experiment, immunize the mice with the GP234 complex purified in Example 1 via intraperitoneal injection. The immunization dose for each dose is 20 µg of GP234 complex, as detailed below: For the first immunization, Freund's complete adjuvant was used as the adjuvant and CpG as the enhancer: 240 µg of the GP234 complex was dissolved in 600 µL of PBS buffer to obtain the GP234 complex solution; the enhancer CpG 4 OD was dissolved in 600 µL of PBS buffer to obtain the CpG solution; the GP234 complex solution, CpG solution and 1200 µL of Freund's complete adjuvant were mixed to obtain a 2.4 mL emulsion system, which could be used to immunize 12 mice; The second immunization used Freund's incomplete adjuvant as an adjuvant and CpG as an enhancer: 240 µg of the GP234 complex was dissolved in 600 µL of PBS buffer to obtain the GP234 complex solution; the enhancer CpG 4 OD was dissolved in 600 µL of PBS buffer to obtain the CpG solution; the GP234 complex solution, CpG solution and 1200 µL of Freund's incomplete adjuvant were mixed to obtain a 2.4 mL emulsion system, which could be used to immunize 12 mice. Two weeks after the second immunization, blood was collected from the orbital sinus to obtain the corresponding immune serum.

[0089] 2. Fusion and screening of hybridoma cells Feeder cells (100 μL / well) were prepared by rinsing the peritoneal cavity of unimmunized mice of the same strain with 0.34 M sucrose solution, collecting the cells, resuspending them in HAT medium, and seeding them into 96-well plates. Spleen cells (spleen cells from mice immunized twice in step 1 were ground in DNase-containing 1640 medium, filtered through a 70 μm filter, treated with ACK erythrocyte lysis buffer for 2 minutes, and then resuspended) were also prepared. Spleen cells were mixed with Sp2 / 0 mouse myeloma cells in logarithmic growth phase at a ratio of 1:1 to 1:3, centrifuged at 1500 rpm for 10 minutes, discarded the supernatant, and then added dropwise with 1 mL of preheated 50% PEG solution in a 37°C water bath. This was followed by slow addition of 40 mL of preheated 1640 medium, centrifuged again, discarded the supernatant, resuspended in HAT medium, and replenished to approximately 90 mL. The mixture was then added dropwise to 96-well plates containing feeder cells (2 drops per well). Four days after fusion, half of the medium (HAT medium) was replaced. After 7-10 days, when the hybridoma cell clusters grew to a certain size, the culture supernatant was aspirated as the test antibody. The titer was tested using serum from immunized mice as a positive control and serum from unimmunized mice as a negative control to screen for positive wells.

[0090] Subcloning was performed on positive wells using a limiting dilution method: For the first subcloning, HT medium was used. After counting, 100-150 cells were seeded into 9.5 mL of medium, mixed, and added to a plate containing feeder cells. After approximately 5 days, cell counts were observed. Once the cell clusters had grown, positive clones were screened again. Subcloning was then repeated 2-3 times using 1640 complete medium until a stable antibody-secreting monoclonal hybridoma cell line was obtained. Finally, the positive hybridoma cells were expanded (in 24-well plates and T25 flasks sequentially until 80% confluence), centrifuged, resuspended in 3 mL of cell cryopreservation medium, and stored at -80°C for 24 hours after programmed cooling. They were then transferred to liquid nitrogen for long-term storage, thus obtaining hybridoma cells secreting the target monoclonal antibody.

[0091] Example 3. Determination of antibody subtypes and acquisition of variable regions 1. Antibody subtype determination The test antibody (GP2-2, obtained from hybridoma cells secreted in Example 2) was diluted to 5 μg / mL using 1×PBS and added to an ELISA plate for overnight coating at 4°C. After coating, the plate was washed three times with PBS the following day to ensure the removal of unbound antibodies. Blocking buffer was added, and the plate was blocked at room temperature for 1 hour to prevent non-specific binding. After blocking, biotin-labeled IgG1, IgG2a, IgG2b, and IgG3 antibodies were added at a concentration of 10 μg / mL, and the plate was incubated at room temperature for 2 hours. After incubation, the plate was washed five times with PBST to remove unbound labeled antibodies. HRP-labeled streptavidin was then added, and the plate was incubated at room temperature for 1 hour. Finally, TMB chromogenic substrate was added, and after sufficient color development, the OD450 was detected using a microplate reader to determine the antibody subtype. Results are shown below. Figure 1 The GP2-2 antibody is the IgG2b subtype.

[0092] 2. Obtaining the antibody variable region sequence Based on the subtype identification results, appropriate light chain and heavy chain primers were selected for PCR amplification. The amplified fragments were separated by 1.5% agarose gel electrophoresis, and the light and heavy chain amplification products were recovered separately. DNA was recovered using an agarose gel DNA purification and recovery kit, and the purified products were sent to the company for sequencing. The downstream primers of the PCR amplification system were used for sequencing.

[0093] The heavy chain primers are as follows: VH-F: SARGTNMAGCTGSAGSAGTC (SEQ ID NO: 25), IgG2A-R: CTTGACCAGGCATCCTAGAGTCA (SEQ ID NO: 26), IgA-R: GATGGTGGGATTTCTCGCAGACTC (SEQ ID NO: 27), IgE-R: TAAGGGGTAGAGCTGAGGGTTCCTG (SEQ ID NO: 28), IgM-R:GACATTTGGGAAGGACTGACTCTC (SEQ ID NO: 29), IgG1-R: ATAGACAGATGGGGGTGTCGTTTTGGC (SEQ ID NO: 30), IgG2B-R: AGGGGCCAGTGGATAGACTGATGG (SEQ ID NO: 31), IgG3-R: AGGGACCAAGGGATAGACAGATGG (SEQ ID NO: 32); The light chain primers are as follows: VL-F: GAYATTGTGMTSACCMCARWCTMCA (SEQ ID NO: 33), Igκ-R: GGATACAGTTGGTGCAGCATC (SEQ ID NO: 34), Igλ-R:GACATTCAGCTGACCCAGTCTCCA (SEQ ID NO: 35).

[0094] Next, the sequencing results were compared with the mouse genome in the NCBI database to determine the signal peptide sequences of the light and heavy chains. Based on this result, new primers were designed to amplify the entire CDS, and the DNA was purified again using an agarose gel DNA purification and recovery kit. The recovered product was sent to the company for sequencing. Sequencing was performed using the upstream and downstream primers of the PCR amplification system, and the full-length sequences of the variable regions of the light and heavy chains were finally determined. The amino acid sequence of the heavy chain variable region of antibody GP2-2 is shown in SEQ ID No: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 2.

[0095] The nucleotide sequence of the variable region of the antibody heavy chain is as follows: GTGGAGTCTGGGGGAGGCTTAGTGATGCTTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATTACATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTTGGTCGCATCCATTAATCGTAATGGTGGTAGTACCTACTATCCAGAC ACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCTTGTATTTCTGTGCAAGACCGGCCTACTATAGGTACGACTTTGACTACTGGGGCCAAGGCACCGCTCTCACAGTCTCCTCA (SEQ ID NO: 36) The nucleotide sequence of the variable region of the antibody light chain is as follows: ACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGTAGGGCCAGCCCAAGTGTCAGTACATCTAGTTATCGTTATATACACTGGTACCAACAGAAACCAGGACAGCCACCCAAAATCCACATCAAGTATGCATCCAACCTA GAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTGTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGATGATAATGCAACATATTACTGTCAGCACAGTTGGGAGATTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO: 37) 3. Expression and purification of genetically engineered antibodies Construction of recombinant expression vectors: DNA encoding the full-length heavy and light chains (wherein, the amino acid sequences of the variable regions of the heavy and light chains are shown in Table 1, the amino acid sequence of the constant region of the heavy chain is SEQ ID NO: 38, and the amino acid sequence of the constant region of the light chain is SEQ ID NO: 39) were inserted into the EcoRI and NotI restriction sites of the PTT3 vector, respectively. After sequencing verification, the heavy and light chain expression plasmids were obtained.

[0096] Heavy chain constant region amino acid sequence (derived from mouse mIgG2a): AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO: 38) The amino acid sequence of the light chain constant region (derived from mouse mIgκ): RADAAPTVSIFPPSSEEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO: 39) The plasmid / PEI transfection complex was then prepared, and subsequently added dropwise to 1 L of cell suspension. The cells were incubated at 37°C and 8% CO2 with shaking at 130 rpm for 5 days. The cell supernatant was aseptically collected, and cells were removed by centrifugation. The supernatant was then filtered using a 0.45 μM filter. The sample was concentrated using a 50 kDa membrane, and the antibody was transferred to PBS. Next, the antibody was purified using Protein A affinity chromatography. The Protein A column was equilibrated with Binding Buffer (PBS 20 mM sodium phosphate, pH 7.0), and the sample was filtered twice through the column, collecting the eluent. During washing, the column was washed with PBS and eluted with Elution Buffer (0.1 M Mglycine, pH 2.5). The antibody in the eluent was collected and neutralized with Neutralization Buffer (1 M Tris-HCl, pH 9.0), then concentrated and the eluent was transferred.

[0097] Antibody concentration was performed using a 30 kDa concentration column. Depending on the concentration effect, the supernatant was removed and the medium was repeatedly changed to obtain the final genetically engineered antibody solution. Antibody purity and concentration were assessed using SDS-PAGE, and the results are as follows: Figure 2 As shown, the sizes of the heavy and light chains of the antibody are consistent with the expected results, verifying the successful expression and purification of the antibody.

[0098] 4. Streaming cytometry verification GS linker and Flag tag (GSDYKDDDDK, SEQ ID NO: 40) were added to the C-terminus of GP2, GP3 and GP4 proteins respectively to form GP2-Flag, GP3-Flag and GP4-Flag.

[0099] GP2-Flag amino acid sequence: MKWGLCRAFLTKSANSLWMLSRSSWFPLLISSYFWPFCSASQSPDGWWYFASDWFAPRYSVRALPFTLSNYRRSYEAYLSQCRVDIPSWGTKHPLGMFWHHKVSTLIDAMVSRRVYHTMEKAGQAAWKQVVSEATLSRISSLDVVAHFQHLAAIE AETCKYLASRLPMLHNLRMSGSNVTIKYNSTSGRVFAIFPTPDSRPKLHDFQQWLIVVHSSIFSSVAASCTLFVVLWLRVPILRTVFGFHWLGATFLSSSWGSDYKDDDDK, SEQ ID NO: 41 (the underlined part is the signal peptide).

[0100] GP3-Flag amino acid sequence: MVNSCALLHIFLCCSFLHFICCAM APSANSTYCFWFPLVRGNFSFELMVNYTVCPPCLTRQAAAQILEPGGSLWCRIGHDRCEESDHDELEFAVPPGLSVEGRLTSVYAWLAFLSFSYTAQFHPEIFGIGNVSEVYVDIQNQLICA VHDGLNATLPSHDNITAVYQSYYQHQVDGGNWFHLEWLRPFFSSWLVLNVSWFLRRSPVSRVSVRVFQTLRPTPPAQQVPLSSKTSAVLGIATRPLRRFAKSLNVARRGSDYKDDDDK, SEQ ID NO: 42 (the underlined part is the signal peptide).

[0101] GP4-Flag amino acid sequence: MAASFLFLLVGFERFMV SQAFACKPCFSSSLSDIKTNTTSAASSVVLQNIGCLRHSNPSPAAFRKIPQCRAAIGTPVYITVTANVTDENYLHSSDLLMLSSCLFYASEMSEKGFKVIFGNVSGIVAVCVNFTSYVQHVREFTQRSLVVDHVRLLHFMTPETMRWATVLACLFAILLAIGSDYKDDDDK, SEQ ID NO: 43 (the underlined part is the signal peptide).

[0102] The nucleotide sequences encoding GP2-Flag, GP3-Flag, and GP4-Flag were inserted between NheI and NotI in the pCDH-CMV-MCS-EF1-GFP+Puro vector (Yobot Biotech, catalog number VT8070), respectively, to obtain the GP2-Flag, GP3-Flag, and GP4-Flag plasmids. These plasmids were transfected into 293T cells, and cells were collected 24 hours later for flow cytometry analysis. First, cells were trypsinized to disperse them into a single-cell suspension. Digestion was terminated with PBS buffer containing 5% FBS, and the supernatant was removed by centrifugation, collecting the cell pellet. Then, 1 mL of fixative was added to fix the cells, and they were incubated at room temperature in the dark for 30 minutes before centrifugation to remove the fixative. Next, the cells were permeabilized twice with 1 mL of 1X permeabilization buffer, and the supernatant was removed by centrifugation after each treatment. Cells were then blocked with PBS buffer containing 5% FBS for 30 minutes. After blocking, the blocking solution was removed by centrifugation, and the cells were washed once with 1X permeabilization buffer. Cells were resuspended in 250 µL of 1X permeabilization buffer, and primary antibodies (the genetically engineered antibody (GP2-2 mAb) obtained in step 3) and Flag antibody (FlagmAb) diluted 1:1000 were added. Cells were incubated at 4°C in the dark for 60 minutes. After primary antibody incubation, cells were washed twice with 1X permeabilization buffer, and then an appropriate amount of secondary antibody (PE Goat anti-mouse IgG, Biolegend, 405307) was added. Cells were incubated at 4°C in the dark for 30 minutes. After secondary antibody incubation, cells were washed twice with 1X permeabilization buffer. Finally, cells were resuspended in PBS buffer containing 1% FBS and analyzed by flow cytometry. The results are shown below. Figure 3 As shown, the GP2-2 antibody specifically binds to the GP2 protein.

[0103] Example 4. Antibody Affinity Verification The antibody obtained in Example 3 was immobilized on the surface of a CM5 chip using an amine coupling method, with a target immobilization amount of 50-200 RU. Next, using HBS-EP+ as the run buffer, a series of concentration gradients (6.25 to 200 nM) of the GP234 complex antigen obtained in Example 1 were prepared. On the SPR instrument, the antigens were injected sequentially in ascending order of concentration, with each cycle including brief antigen binding, buffer dissociation, and chip regeneration using glycine (pH 1.5). Finally, after subtracting blank and reference signals using the instrument software, the binding and dissociation curves were globally fitted using a "1:1 binding model" to directly obtain the affinity constant KD value, which was 1.43 × 10⁻⁶. -8 ,like Figure 4 As shown.

[0104] Example 5. Binding of antibodies to different strains GP234 antigens for the NADC34 strain (NCBI accession number OP716076) and the HP strain (NCBI accession number EF112445) were prepared using the same method as in Example 1. The GP234 antigen for the NADC30 strain was obtained from Example 1. The GP234 antigens of the three different strains were diluted to 1 μg / mL with PBS buffer and coated onto ELISA plates. 100 μL of antigen solution was added to each well, and coating was performed overnight at 4°C. The next day, the plates were washed four times with 200 μL of washing buffer (PBS solution containing 0.05% Tween-20) per well. Next, the plates were blocked with 200 μL of blocking buffer (PBST solution containing 0.5% BSA) per well, and incubated at room temperature for 1 hour. After blocking, the plates were washed four times with washing buffer (200 μL per well). Then, the serum to be tested (immune serum obtained in Example 2) was diluted with blocking buffer at a dilution ratio of 1:10000. Add 100 μL of diluted serum to each well, and 100 μL of blocking buffer to each blank control well. Incubate on a shaker at room temperature for 1 hour. Wash the plate 4 times with washing buffer, 200 μL per well. Then add secondary antibody (HRP-labeled goat anti-mouse IgG, Promega, W4021), diluted with blocking buffer at a ratio of 1:10000. Add 100 μL of secondary antibody and incubate on a shaker at room temperature for 1 hour. After incubation, wash the plate 5 times with washing buffer, 200 μL per well. Add 100 μL of substrate TMB and operate in the dark. After about 5 minutes of substrate reaction, when the liquid in the wells turns dark blue, add 50 μL of stop buffer (2N H2SO4) to stop the reaction and measure the OD value at 450 nm. Results are as follows. Figure 5 As shown, the GP2-2 antibody can recognize the GP234 antigen of NADC30 and NADC34 strains, exhibiting a certain broad spectrum of activity.

[0105] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody against porcine reproductive and respiratory syndrome virus (PRRSV) or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment comprises: a1) The amino acid sequences CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region as shown in SEQ ID NO: 1; and the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region as shown in SEQ ID NO: 2; or a2) Compared with CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 described in a1), at least one CDR contains one or more amino acid substitutions, deletions or additions; The CDR is defined by IMGT, Kabat, Chothia, or the Contact numbering system.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises: b1) A heavy chain variable region comprising amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively; and a light chain variable region comprising amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 14, YAS, and SEQ ID NO: 15, respectively; wherein the CDRs are defined by the IMGT numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 15, respectively; wherein the CDRs are defined by the Kabat numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 8, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 15, respectively; wherein the CDRs are defined by the Chothia numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; wherein the CDRs are defined by the Contact numbering system, or b2) Heavy chain variable region; and, light chain variable region, wherein, compared with the heavy chain variable region and / or light chain variable region of b1), at least one CDR contains a substitution, deletion or addition of one or more amino acids; Preferably, the heavy chain variable region of the antibody or its antigen-binding fragment further includes the framework region of the heavy chain variable region; Preferably, the light chain variable region of the antibody or its antigen-binding fragment further includes the framework region of the light chain variable region.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The frame region of the heavy chain variable region includes the frame region of the heavy chain variable region of immunoglobulins derived from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese, or a mutant thereof; further including the frame region of the heavy chain variable region of immunoglobulins derived from mice, or a mutant thereof. Preferably, the framework region of the light chain variable region includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; further, it includes the framework region of the light chain variable region or a mutant thereof derived from mouse immunoglobulins. Preferably, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 75% sequence identity with it; and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 75% sequence identity with it.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese. Preferably, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese. Preferably, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins; Preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having at least 80% sequence identity with it; Preferably, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence having at least 80% sequence identity with it; Preferably, the antibody is a monoclonal antibody. Preferably, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody. Preferably, the antibody is a murine, porcine, porcine-derived, chimeric, humanized, or fully human antibody. Preferably, the antibody is of type IgA, IgD, IgE, IgG, or IgM; more preferably, the antibody is of type IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. Preferably, the antigen-binding fragment includes scFv, Fab, Fab', (Fab')2, Fv fragment, Fd, or dsFv. Preferably, the antibody or its antigen-binding fragment specifically binds to the GP234 complex; more preferably, the antibody or its antigen-binding fragment binds to the GP2 protein. Preferably, the antibody or its antigen-binding fragment specifically binds to the GP234 complex of NADC30 and NADC34 strains; more preferably, the antibody or its antigen-binding fragment binds to the GP2 protein of NADC30 and NADC34 strains.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment as described in claims 1-4.

6. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 5.

7. A cell, characterized in that, The cell comprises the antibody or antigen-binding fragment thereof as described in claims 1-4, the nucleic acid molecule as described in claim 5, or the vector as described in claim 6. Preferably, the cells do not contain reproductive material.

8. A coupling, characterized in that, The conjugate comprises: the antibody or antigen-binding fragment thereof as described in claims 1-4; and a conjugation portion, wherein the conjugation portion is selected from detectable substances, small molecule drugs, and / or proteins. Preferably, the protein includes cytokines, antibodies, antibody Fc fragments, antibody scFv fragments, enzymes, and viral capsid proteins. Preferably, the detectable substance is a fluorescent substance, a luminescent marker, or a radioactive substance; more preferably, the detectable substance is selected from any one of acridine ester, acridine sulfonamide, luminol, isoluminol, horseradish peroxidase, alkaline phosphatase, and isotopes.

9. A method for preparing antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) or antigen-binding fragments thereof, characterized in that, The method includes culturing the cells of claim 7 and isolating the antibody under conditions that allow expression of the antibody or its antigen-binding fragment.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: the antibody or antigen-binding fragment thereof as described in claims 1-4; the nucleic acid molecule as described in claim 5; the carrier as described in claim 6; the cell as described in claim 7; or the conjugate as described in claim 8; and a pharmaceutically acceptable carrier. Preferably, the route of administration of the pharmaceutical composition is parenteral, injection, oral, or topical. Preferably, the pharmaceutical composition is in solid, semi-solid, or liquid form.

11. The use of the antibody or antigen-binding fragment thereof according to claims 1-4, the nucleic acid molecule according to claim 5, the carrier according to claim 6, the cell according to claim 7, the conjugate according to claim 8, or the pharmaceutical composition according to claim 10, wherein the use comprises one or more of the following: d1) Detect the presence or content of porcine reproductive and respiratory syndrome virus in the sample; d2) Diagnosis or prognostic assessment of diseases related to porcine reproductive and respiratory syndrome; d3) Prepare reagents for detecting the presence or content of porcine reproductive and respiratory syndrome virus in samples; d4) Prepare reagents for the diagnosis or prognostic assessment of porcine reproductive and respiratory syndrome.

12. The application according to claim 11, characterized in that, The samples include whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions from the subject.

Images