Broad-spectrum neutralizing monoclonal antibodies against classical swine fever virus and their applications

Abstract

The present invention discloses a broad-spectrum neutralizing monoclonal antibody against classical swine fever virus and its application, belonging to the field of immunoglobulins. Specifically, an antibody or its antigen-binding fragment is disclosed, which comprises a heavy-chain variable region and a light-chain variable region; the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy-chain variable region sequentially comprise positions 9-15, 33-40, and 79-90 of SEQ ID No.1; the amino acid sequences of CDR1, CDR2, and CDR3 in the light-chain variable region sequentially comprise positions 21-31, 49-51, and 88-97 of SEQ ID No.3. The monoclonal antibody of the present invention can specifically react with all genotype strains of classical swine fever virus, and the established blocking ELISA method using it can detect classical swine fever virus E2 antibody. The monoclonal antibody of the present invention can be used for the identification of CSFV antigen and the development of serological antibody detection kits, providing antibody resources for the accurate diagnosis, prevention, control, and purification of CSFV.

Description

Technical Field

[0001] The present invention belongs to the field of immunoglobulins, and particularly relates to a broad-spectrum neutralizing monoclonal antibody against classical swine fever virus and its application. Background Art

[0002] Classical swine fever is a disease that seriously endangers the global pig industry. The World Organization for Animal Health (WOAH) lists it as a notifiable animal disease, and it belongs to Class II animal diseases in China. Domestic pigs and wild boars are susceptible animals to classical swine fever virus. Diseased pigs mainly show symptoms such as persistent high fever, dyspnea, extensive systemic hemorrhage, and reproductive disorders. Acute classical swine fever has a short incubation period and a high fatality rate. Diseased animals can die only 10 - 20 days after infection. Subacute and chronic classical swine fever can cause intermittent fever, retarded growth and development in diseased animals, and ultimately death. Persistently infected animals have no obvious clinical symptoms, but pregnant sows are prone to abortion and stillbirth, and they are also a relatively large source of infection.

[0003] Classical swine fever virus (CSFV) is the pathogen that causes classical swine fever. It is a single-stranded positive-sense RNA virus with an envelope. The virus particles are spherical with a diameter of about 40 - 60 nm, and the genome size is about 12.3 kb, containing a large open reading frame that encodes a polyprotein composed of 3,898 amino acid residues. Further cleavage by proteases generates 4 structural proteins (Core, E rns 、E1 and E2) and 8 non-structural proteins (N pro 、P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). Among them, the structural proteins E rns and E2, and the non-structural protein NS3 can stimulate the body to produce antibodies. The E2 protein is the main protective antigen of CSFV, and its size is about 51 - 58 kDa. The amino terminus of the E2 protein has four antigenic domains B, C, D, and A in sequence, which can be further divided into two antigenic units B / C and D / A, and it contains most of the antigenic epitopes.

[0004] In China, classical swine fever is currently prevalent in a multi-point sporadic manner, and the prevention and control strategy combines vaccination and culling. The lapinized attenuated vaccine strain C of classical swine fever can still provide good protection against classical swine fever virus. Therefore, clarifying the humoral immune protection mechanism of the vaccine and developing an antibody detection kit for scientifically evaluating the immune effect of the vaccine are very important for the prevention and control of classical swine fever. The prerequisite for carrying out these works is to obtain broad-spectrum neutralizing monoclonal antibodies that can react with different genotypes of classical swine fever virus. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a new broad-spectrum neutralizing monoclonal antibody against classical swine fever virus and its application.

[0006] To solve the above technical problem, in the first aspect, the present invention provides an antibody or its antigen-binding fragment, which comprises a heavy-chain variable region and a light-chain variable region;

[0007] The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy-chain variable region sequentially comprise positions 9-15, 33-40 and 79-90 of SEQ ID No.1;

[0008] The amino acid sequences of CDR1, CDR2 and CDR3 in the light-chain variable region sequentially comprise positions 21-31, 49-51 and 88-97 of SEQ ID No.3.

[0009] The above-mentioned antibody or its antigen-binding fragment comprises 4 heavy-chain variable region framework regions named HFR1, HFR2, HFR3 and HFR4, and 4 light-chain variable region framework regions named LFR1, LFR2, LFR3 and LFR4;

[0010] The amino acid sequence of the HFR1 comprises an amino acid sequence selected from the following:

[0011] A1) positions 1 to 8 of SEQ ID NO:1;

[0012] A2) a sequence having more than 75% identity with A1);

[0013] The amino acid sequence of the HFR2 comprises an amino acid sequence selected from the following:

[0014] A3) positions 16 to 32 of SEQ ID NO:1;

[0015] A4) a sequence having more than 75% identity with A3);

[0016] The amino acid sequence of the HFR3 comprises an amino acid sequence selected from the following:

[0017] A5) positions 41 to 78 of SEQ ID NO:1;

[0018] A6) a sequence having more than 75% identity with A5);

[0019] The amino acid sequence of the HFR4 comprises an amino acid sequence selected from the following:

[0020] A7) positions 91 to 101 of SEQ ID NO:1;

[0021] A8) A sequence having more than 75% identity with A7);

[0022] The amino acid sequence of said LFR1 comprises an amino acid sequence selected from the following:

[0023] A9) positions 1 to 20 of SEQ ID NO:3;

[0024] A10) A sequence having more than 75% identity with A9);

[0025] The amino acid sequence of said LFR2 comprises an amino acid sequence selected from the following:

[0026] A11) positions 32 to 48 of SEQ ID NO:3;

[0027] A12) A sequence having more than 75% identity with A11);

[0028] The amino acid sequence of said LFR3 comprises an amino acid sequence selected from the following:

[0029] A13) positions 52 to 87 of SEQ ID NO:3;

[0030] A14) A sequence having more than 75% identity with A13);

[0031] The amino acid sequence of said LFR4 comprises an amino acid sequence selected from the following:

[0032] A15) positions 98 to 106 of SEQ ID NO:3;

[0033] A16) A sequence having more than 75% identity with A15).

[0034] Among the antibodies or antigen-binding fragments thereof described above,

[0035] The heavy chain variable region is any of the following:

[0036] B1) The amino acid sequence of the heavy chain variable region shown comprises SEQ ID No.1;

[0037] B2) The amino acid sequence of the heavy chain variable region shown comprises an amino acid sequence having an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID No.1;

[0038] And / or, the light chain variable region is any of the following:

[0039] B3) The amino acid sequence of the light chain variable region shown comprises SEQ ID No.3;

[0040] The amino acid sequence of the light chain variable region shown in (B4) includes an amino acid sequence having an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80%, or more than 75% with SEQ ID No. 3.

[0041] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind the same antigen to which the full-length antibody binds and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion", "antigen-binding domain". In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and such polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0042] The antibody or its antigen-binding fragment described above includes a heavy chain variable region and a light chain variable region, both of which are composed of a complementarity-determining region and a framework region; the complementarity-determining regions of the heavy chain variable region and the light chain variable region are each composed of their respective CDR1, CDR2, and CDR3;

[0043] The CDR in the present invention is the "complementarity-determining region", which is a region in the variable domain of an antibody that is highly variable in sequence and forms a structurally defined "hypervariable loop" and / or contains antigen contact residues "antigen contact points". The CDR is mainly responsible for binding to the antigen epitope. A variable region usually contains 3 CDR regions, which are CDR1, CDR2, and CDR3 in sequence from the N-terminus.

[0044] The framework region of the heavy chain variable region is composed of 4 heavy chain variable region framework regions named HFR1, HFR2, HFR3, and HFR4;

[0045] The framework region of the light chain variable region is composed of 4 light chain variable region framework regions named LFR1, LFR2, LFR3, and LFR4.

[0046] The alignment for determining the percent sequence identity can be achieved in a variety of known ways, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning the sequences can be determined, including the algorithm required to achieve maximal alignment over the entire length of the sequences being compared. However, for the purposes of this disclosure, the percent sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office, Washington, D.C. 20559, along with user documentation, and has U.S. Copyright Office Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0047] Specifically, the identity above 75% is above 80%. Specifically, the identity above 75% is above 85%. Specifically, the identity above 75% is above 90%. Specifically, the identity above 75% is above 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. More specifically, the identity above 75% can be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0048] The term "antibody" in the present invention is used in the broadest sense and includes polyclonal antibodies and monoclonal antibodies, including intact antibodies and their functional (antigen-binding) antibody fragments, including antigen-binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (sFv or scFv) and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses immunoglobulins that are genetically engineered and / or otherwise modified, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies, such as bispecific antibodies, trispecific antibodies, and tetravalent antibodies, tandem bis-scFv, tandem tris-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass its functional antibody fragments. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region.

[0049] In the above-mentioned antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment may further comprise a constant region.

[0050] In the antibody or its antigen-binding fragment, the constant region may be a heavy-chain constant region and a light-chain constant region.

[0051] In the antibody or its antigen-binding fragment, the heavy-chain constant region may be IgG, IgM, or IgA.

[0052] In the antibody or its antigen-binding fragment, the IgG may be IgG1, IgG2, IgG3, or IgG4.

[0053] In the antibody or its antigen-binding fragment, the light-chain constant region may be a lamda (λ) constant region or a Kappa (κ) constant region.

[0054] The antibody or its antigen-binding fragment may be murine, human, chimeric, or humanized.

[0055] As used herein, the term "monoclonal antibody" or "Antibody", unless otherwise specified, generally refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair consisting of one "light" (L) chain and one "heavy" (H) chain. In general, the heavy chain can be understood as the polypeptide chain with a larger molecular weight in the antibody, and the light chain is the polypeptide chain with a smaller molecular weight in the antibody. The light chain can be classified into κ and λ light chains. The heavy chain is usually classified into μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be further divided into regions with high variability (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site. The assignment of amino acids to each region or domain follows the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342: 878-883. In particular, the heavy chain can also contain more than three CDRs, such as 6, 9, or 12. For example, in the bispecific antibody of the present invention, the heavy chain can be the C-terminus of the heavy chain of an IgG antibody linked to the ScFv of another antibody, in which case the heavy chain contains 9 CDRs. The term "antibody" is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be antibodies of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0056] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present invention, amino acids are generally represented by the single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0057] The term "identity" refers to sequence similarity to a native nucleic acid sequence. Identity can be evaluated by eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0058] In a second aspect, the present invention provides a biological material related to the antibody or antigen-binding fragment thereof described in the first aspect, and the biological material is any one of C1) to C12):

[0059] C1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described in the first aspect;

[0060] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0061] C3) A recombinant vector containing the nucleic acid molecule described in C1);

[0062] C4) A recombinant vector containing the expression cassette described in C2);

[0063] C5) A recombinant microorganism containing the nucleic acid molecule described in C1);

[0064] C6) A recombinant microorganism containing the expression cassette described in C2);

[0065] C7) A recombinant microorganism containing the recombinant vector described in C3);

[0066] C8) A recombinant microorganism containing the recombinant vector described in C4);

[0067] C9) A recombinant cell containing the nucleic acid molecule described in C1);

[0068] C10) A recombinant cell containing the expression cassette described in C2);

[0069] C11) A recombinant cell containing the recombinant vector described in C3);

[0070] C12) A recombinant cell containing the recombinant vector described in C4).

[0071] Among the above-mentioned biological materials, C1) the nucleic acid molecule can be a DNA molecule or an RNA molecule. The DNA molecule can be genomic DNA or cDNA molecule. The nucleic acid molecule can be the genomic gene or cDNA gene of the protein.

[0072] Among the above biological materials, C2) the expression cassette refers to a DNA that can express the protein in a host cell. The expression cassette can also include a single-stranded or double-stranded nucleic acid molecule of all regulatory sequences necessary for expressing the nucleic acid molecule of the above protein. The regulatory sequences can direct the coding sequence to express the above protein in a suitable host cell under its compatible conditions. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequences should include a promoter and transcription and translation termination signals. To introduce specific restriction enzyme sites into the vector for ligating the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, the regulatory sequences with linkers can be provided. The regulatory sequence can be a suitable promoter sequence, i.e., a nucleic acid sequence recognizable by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences mediating protein expression. The promoter can be any nucleic acid sequence having transcriptional activity in the selected host cell, including mutated, truncated, and chimeric promoters, and can be derived from genes encoding extracellular or intracellular proteins homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of the protein and can direct the encoded protein into the cell secretion pathway. Any signal peptide coding region that can direct the expressed protein into the secretion pathway of the used host cell can be used in the present invention. It may also be necessary to add regulatory sequences that can regulate protein expression according to the growth of the host cell. Examples of regulatory sequences are those systems that can respond to chemical or physical stimulants (including in the presence of regulatory compounds) to turn on or off gene expression. Other examples of regulatory sequences are those that can amplify genes. In these examples, the nucleic acid sequence encoding the protein should be operably linked to the regulatory sequences.

[0073] Among the above biological materials, the recombinant vector described in C3) can be a cloning vector or an expression vector. As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as λ phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain a variety of elements for controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain an origin of replication site.

[0074] When preparing an expression vector, a nucleic acid molecule encoding the above-mentioned protein can be located in the vector so as to be operably linked to an appropriate expression regulatory sequence. The recombinant expression vector can be any vector that facilitates recombinant DNA manipulation and expresses a nucleic acid sequence (such as a plasmid or a virus). The choice of the vector usually depends on the compatibility of the vector with the host cell into which it is to be introduced. The vector can be a linear or closed circular plasmid. The vector can be an autonomously replicating vector (i.e., a complete structure existing outside the chromosome that can replicate independently of the chromosome), such as a plasmid, an episome, a minichromosome, or an artificial chromosome. The vector can contain any mechanism that ensures self-replication. Alternatively, the vector is a vector that can integrate into the chromosome and replicate together with the integrated chromosome when introduced into the host cell. The said vector contains one or more selectable markers that facilitate the selection of transformed cells. A selectable marker is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or confers prototrophy to auxotrophs, etc. Examples of bacterial selectable markers are the dal gene of Bacillus subtilis or Bacillus licheniformis, or resistance markers to antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. The vector contains elements that can stably integrate the vector into the host cell genome or ensure autonomous replication of the vector in the cell independently of the cell genome. In the case of autonomous replication, the vector can also contain an origin of replication that enables the vector to replicate autonomously in the target host cell. The origin of replication can carry a mutation that makes it temperature-sensitive in the host cell (see, for example, fEhrlich, 1978, Proceedings of the National Academy of Sciences of the United States of America 75: 1433). One or more copies of the nucleic acid molecule encoding the above-mentioned protein can be inserted into the host cell to increase the yield of the gene product. The increase in the copy number of the nucleic acid molecule can be achieved by inserting at least one additional copy of the nucleic acid molecule into the host cell genome, or by inserting an amplifiable selectable marker together with the nucleic acid molecule, and by culturing the cells in the presence of a suitable selection reagent to select cells containing the amplified copy of the selectable marker gene and thus containing an additional copy of the nucleic acid molecule. The operations for ligating the above-mentioned elements to construct the recombinant expression vector of the present invention are well-known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0075] Among the above-mentioned biomaterials, the recombinant microorganism in C5) can specifically be a bacterium, a yeast, an alga, or a fungus. The bacterium can be any one of the following:

[0076] 1) Prokaryotic microorganism;

[0077] 2) Gram-negative bacterium;

[0078] 3) Bacterium of the genus Escherichia;

[0079] 4) Escherichia coli.

[0080] Among the above biological materials, the recombinant cells described in C9)-C12) can be animal cells, and the above animal cell line can be a non-reproductive material. The animal cells can be in vitro mammalian cells. The mammals include humans or mice. The mammalian cells may not include animal germ cells, animal fertilized eggs, and animal embryonic stem cells, and can be somatic cells or cell lines, for example. The animal cells can be cell lines or somatic cells derived from mice.

[0081] In a third aspect, the present invention provides an antibody conjugate, comprising an antibody portion and a conjugate portion, wherein the antibody portion comprises the antibody or its antigen-binding fragment described in the first aspect.

[0082] In the above text, the antibody portion is the antibody or its antigen-binding fragment described in the first aspect.

[0083] The conjugate portion can be an enzyme label. Further, the enzyme label can specifically be HRP.

[0084] In a fourth aspect, the present invention provides the use of the antibody or its antigen-binding fragment described in the first aspect, or the biological material described in the second aspect, or the antibody conjugate described in the third aspect in at least one of the following;

[0085] D1) Preparing a product for detecting or assisting in detecting classical swine fever virus;

[0086] D2) Preparing a product for detecting or assisting in detecting diseases caused by classical swine fever virus infection;

[0087] D3) Preparing a product for screening or assisting in screening classical swine fever virus;

[0088] D4) Preparing a product for screening or assisting in screening diseases caused by classical swine fever virus infection;

[0089] D5) Preparing a product for detecting or assisting in detecting the E2 protein of classical swine fever virus;

[0090] D6) Preparing a product that binds to the E2 protein of classical swine fever virus.

[0091] In the above text, the classical swine fever virus includes epidemic strains of classical swine fever virus.

[0092] In a fifth aspect, the present invention provides a product, which comprises the antibody or its antigen-binding fragment described in the first aspect, or the biological material described in the second aspect, or the antibody conjugate described in the third aspect.

[0093] The product described above is a drug, a composition, a health product, a functional food, a food for special medical purposes, or other biological products.

[0094] The dosage form of the above-mentioned drug is injection, freeze-dried powder for injection, aerosol, large volume infusion, dropping pill, pill, powder, granule, tablet, capsule, oral liquid or emulsion.

[0095] The above product also includes a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier may be an excipient, a stabilizer, a suspending agent or a diluent, etc., which are well known to those skilled in the art.

[0096] In the above text, the pharmaceutically acceptable excipients, diluents or carriers include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly water-soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric-soluble carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Using these materials, various dosage forms can be prepared, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. They can be conventional preparations, sustained-release preparations, controlled-release preparations and various particulate drug delivery systems. To prepare the unit dosage form into tablets, various carriers well-known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, laminaran, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. To prepare the unit dosage form into pills, various carriers well-known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc. To prepare the unit dosage form into suppositories, various carriers well-known in the art can be widely used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. To prepare the unit dosage form into injectable preparations, such as solutions, emulsions, freeze-dried powder injections and suspensions, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, etc.In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, conventional solubilizers, buffers, pH regulators, etc. can also be added. In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation.

[0097] In a sixth aspect, the present invention also provides a method for detecting classical swine fever virus E2 protein, characterized in that: the method includes the step of contacting the antibody or its antigen-binding fragment described in the first aspect, or the biological material described in the second aspect, or the antibody conjugate described in the third aspect, or the product with a sample to be tested, so as to detect whether the sample to be tested contains classical swine fever virus E2 protein.

[0098] The above-mentioned product has at least one of the following functions:

[0099] E1) Detecting or assisting in detecting classical swine fever virus;

[0100] E2) Detecting or assisting in detecting diseases caused by classical swine fever virus infection;

[0101] E3) Screening or assisting in screening classical swine fever virus;

[0102] E4) Screening or assisting in screening diseases caused by classical swine fever virus infection;

[0103] E5) Detecting or assisting in detecting classical swine fever virus E2 protein;

[0104] E6) Binding to classical swine fever virus E2 protein.

[0105] The present invention provides a new monoclonal antibody TCH053, which can recognize classical swine fever virus of all genotypes. The method established based on this antibody can detect classical swine fever virus infection with high sensitivity, which is beneficial to the prevention and control of classical swine fever in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 It is the result of an indirect immunofluorescence experiment of monoclonal antibody TCH053 with different genotype strains of classical swine fever virus.

[0107] Figure 2 It is the result of a Western blot experiment of monoclonal antibody TCH053 with different genotype strains of classical swine fever virus.

[0108] Figure 3 It is the result of an experiment to verify the neutralization ability of monoclonal antibody TCH053 by IFA.

[0109] Figure 4 It is the verification result of the purification effect of monoclonal antibody TCH053.

[0110] Figure 5Identification results of the reaction between monoclonal antibody TCH053 and classical swine fever virus E2 protein. Detailed implementation manners

[0111] The present invention will be further described in detail below in combination with specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0112] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0113] Unless otherwise specified, the quantitative tests in the following embodiments are all set with three repeated experiments, and the results are averaged.

[0114] The baculovirus expression vector pFastBac 1 in the following embodiments is described in the following literature: Shijiang Mi. Identification of monoclonal antibodies that differentiate between field isolates and vaccine strains of classical swine fever virus and analysis of their antigenic epitopes [D]. Jilin University, 2022.

[0115] The CSFV epidemic strains of each genotype (SM, CN021, CN069, GD53, CN049, CN057, CN063, CN068, CN002, HuB2) and the classical swine fever lapinized vaccine strain C (HCLV) isolated and preserved in the laboratory in the following embodiments are described in the following literature: Shijiang Mi, Lihua Wang, Hongwei Li, et al. Characterization of monoclonal antibodies that specifically differentiate field isolates from vaccine strains of classical swine fever virus. Frontiers in Immunology, 2022, 13, 930631. The public can obtain the biological material from the applicant (Jilin University) in accordance with the relevant regulations of national biosafety. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0116] The classical swine fever virus epidemic strains AH1 and GD23 in the following embodiments are recorded in the following literature: Gong W, Li J, Wang Z, et al. Commercial E2 subunit vaccine provides full protection to pigs against lethal challenge with 4 strains of classical swine fever virus genotype 2. Vet Microbiol. 2019;237:108403. The public can obtain the biological material from the applicant (Jilin University) in accordance with the relevant regulations of national biosafety. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0117] The protein tag antibody anti-His Tag mAb in the following embodiments was purchased from Solarbio, with the product number K200060M.

[0118] Example 1. Preparation and identification of monoclonal antibody against classical swine fever virus E2 protein

[0119] The immunogen in this example is the E2 protein of the classical swine fever virus (CSFV) vaccine strain LPC (CSFV-LPC). In this example, the insect baculovirus expression system was used to prepare the E2 protein of CSFV-LPC. The steps are as follows: The E2 protein coding gene of CSFV-LPC (the 1171-2259th positions of the sequence shown in GenBank accession number: AY526732) was cloned into the insect baculovirus expression vector pFastBac 1 after adding a His tag (the His tag nucleotide sequence is 5’-CATCATCACCATCACCAT-3’, SEQ ID No.5) at the 3' end to obtain a recombinant expression vector. The recombinant expression vector was transformed into DH10Bac competent cells to obtain a recombinant bacmid containing the target gene fragment. The recombinant bacmid was transfected into Sf9 cells for protein expression. The CSFV-LPC E2 protein was collected by immobilized metal ion affinity chromatography and purified to obtain the immunogen, denoted as CSFV-LPC E2 purified protein.

[0120] 1. Preparation and identification of hybridoma cell lines and monoclonal antibodies

[0121] (1) Mouse immunization

[0122] SPF-grade female BALB / c mice aged 6 - 8 weeks were immunized. The purified CSFV-LPC E2 protein was emulsified with an equal volume of 206 adjuvant (Seppic), and the mice were immunized by subcutaneous multi-point injection with 50 μg of the emulsified protein per mouse. Immunization was performed once every two weeks for a total of 3 times. One week after the third immunization, the mice were boosted by intraperitoneal injection of 100 μg of the purified CSFV-LPC E2 protein without adjuvant and continued to be raised for 3 days.

[0123] (2)Cell fusion

[0124] The mice were sacrificed by cervical dislocation, and the spleens were removed. The spleens were gently crushed in a nylon sieve, and the washing liquid was collected after rinsing with sterile PBS. After removing red blood cells with red blood cell lysate, the splenocytes were counted. SP2 / 0 cells and splenocytes were mixed at a ratio of 1:5 and centrifuged at 1200 r / min for 3 min to discard the supernatant. The precipitate was washed twice with 10 mL of electrofusion solution (BTX), resuspended in 9 mL of electrofusion solution, and the cells were transferred to an electrofusion instrument for fusion. The fusion parameters were set at 800 V, 40 μs, and 1 time. The fused cells were activated with 5% FBS Advanced RPMI 1640 medium (purchased from Thermo Fisher Scientific, catalog number 12633012) for 10 h and then transferred to a semi-solid medium (purchased from Bioron, catalog number BTYA0607) for culture for 10 days.

[0125] (3)Screening of hybridoma cells

[0126] Monoclonal cell clusters in the semi-solid medium in step (2) were picked into Advanced RPMI 1640 medium containing HAT (purchased from Thermo Fisher, catalog number 21060017). After culturing for 3 days, antibody-positive cell wells were screened by indirect immunofluorescence assay. After subcloning, a hybridoma cell line (named hybridoma cell TCH053) was identified, and the monoclonal antibody secreted by it was named monoclonal antibody TCH053.

[0127] (4)Identification of antibody type of monoclonal antibody

[0128] The Ig class and light chain type of the monoclonal antibody were identified by indirect ELISA method. The undiluted hybridoma culture supernatant was used as the primary antibody. The specific preparation method of the hybridoma cell culture supernatant was as follows: The hybridoma cells were cultured in a cell culture flask. When the cell density was greater than 90%, the culture supernatant was collected for use. The secondary antibodies against different types of antibodies used in the experiment were all from the enzyme-labeled secondary antibody kit for mouse monoclonal antibody Ig subclass identification (purchased from Bioron, catalog number BF16002X). The results showed that the antibody type of monoclonal antibody TCH053 was IgG2a, κ light chain.

[0129] (5)Identification of the reaction type between monoclonal antibody and virus

[0130] A. Reaction of monoclonal antibody with virus

[0131] The monoclonal antibody TCH053 was subjected to indirect immunofluorescence assay (IFA) with CSFV epidemic strains of various genotypes (SM, CN021, CN069, GD53, CN049, CN057, CN063, CN068, CN002, HuB2) isolated and preserved in our laboratory, as well as the lapinized attenuated classical swine fever vaccine strain C (HCLV). The steps were as follows:

[0132] ① Cell inoculation with virus: Simultaneously inoculate the CSFV cell strain at 100 TCID 50 / well into the PK-15 cells added to a 96-well plate, and culture in an incubator at 37 °C and 5% CO2 for 72 h.

[0133] ② Cell fixation: Discard the cell culture supernatant, add 200 μL of PBS to each well of the 96-well plate and wash 3 times, then add 80% cold acetone (50 μL / well) stored at -20 °C, and fix in a -20 °C refrigerator for 1 h.

[0134] ③ Incubation with primary antibody: Discard the cold acetone fixing solution, add 200 μL of PBS to each well and wash 3 times, then add 100 μL of hybridoma cell culture supernatant (monoclonal antibody TCH053) to each well, and incubate at 37 °C for 1 h.

[0135] ④ Incubation with secondary antibody: Discard the primary antibody incubation solution, add 200 μL of PBS to each well and wash 3 times. Dilute the Alexa Fluor 488 fluorescent secondary antibody (Thermo Fisher Scientific, catalog number: A21202) 1:500 with PBS, and simultaneously add 0.01% Evans blue and 5% FBS. After thorough mixing, add 100 μL to each well of the cell plate and incubate at 37 °C for 1 h.

[0136] ⑤ Fluorescence observation: Discard the secondary antibody incubation solution, add 200 μL of PBS to each well and wash 3 times, and observe the reaction of serum antibody with infected cells under a fluorescence microscope.

[0137] The results were as Figure 1 shown that the monoclonal antibody TCH053 reacted with all strains and showed obvious fluorescence.

[0138] B. Reaction of monoclonal antibody with virus protein

[0139] The E2 proteins of different genotypes of CSFV are expressed through the insect baculovirus expression system, including the E2 proteins of the following 18 CSFV strains: LPC, HCLV, SM, Brescia (1.2), CSF0650 (1.3), CSF1056 (1.4), CN021(2.1a), CN069 (2.1b), GD53 (2.1c), CN049 (2.1g), CN057 (2.1h), CN063 (2.1i), CN068(2.1j), CN002 (2.2), HuB2 (2.3), CSF0410 (3.1), JJ9811 (3.2), 94.4 / IL / 94 / TWN(3.4). The preparation method of the E2 proteins of the above vaccine strains and strains refers to the preparation method of the CSFV-LPC E2 purified protein described above. Among them, the coding genes of each E2 protein cloned into the insect baculovirus expression vector pFastBac 1 are as follows:

[0140] The nucleotide sequence of the coding gene of the E2 protein of the classical swine fever virus (CSFV) vaccine strain LPC: positions 1171-2259 of GenBank Accession No. AY526732 (Update Date 26-JUL-2016).

[0141] The nucleotide sequence of the coding gene of the E2 protein of the classical swine fever lapinized attenuated vaccine strain C (HCLV): positions 2345-3433 of GenBank Accession No. Z46258.1 (Update Date 25-NOV-2005).

[0142] The nucleotide sequence of the coding gene of the SM E2 protein: positions 2345-3433 of GenBank Accession No. AY775178.2 (Update Date 27-NOV-2007).

[0143] The nucleotide sequence of the coding gene of the Brescia (1.2) E2 protein: positions 2333-3421 of GenBank Accession No. AY578687.1 (Update Date 31-MAR-2005).

[0144] The nucleotide sequence of the coding gene of the CSF0650 (1.3) E2 protein: positions 2147-3235 of GenBank Accession No. JX028200.1 (Update Date 19-AUG-2012).

[0145] Nucleotide sequence of the coding gene of CSF1056 (1.4) E2 protein: positions 2147 - 3235 of GenBank Accession No. JX028202.1 (Update Date 19 - AUG - 2012).

[0146] The nucleotide sequence of the coding gene of CN021 (2.1a) E2 protein is as shown in SEQ ID No. 6.

[0147] The nucleotide sequence of the coding gene of CN069 (2.1b) E2 protein is as shown in SEQ ID No. 7.

[0148] Nucleotide sequence of the coding gene of GD53 (2.1c) E2 protein: positions 2344 - 3432 of GenBank Accession No. KP343640.1 (Update Date 01 - MAY - 2016).

[0149] Nucleotide sequence of the coding gene of CN049 (2.1g) E2 protein: positions 2347 - 3435 of GenBank Accession No. KU504339.1 (Update Date 28 - JUN - 2016).

[0150] The nucleotide sequence of the coding gene of CN057 (2.1h) E2 protein is as shown in SEQ ID No. 8.

[0151] Nucleotide sequence of the coding gene of CN063 (2.1i) E2 protein: positions 2345 - 3433 of GenBank Accession No. KY132096.1 (Update Date 01 - FEB - 2017).

[0152] The nucleotide sequence of the coding gene of CN068 (2.1j) E2 protein is as shown in SEQ ID No. 9.

[0153] The nucleotide sequence of the coding gene of CN002 (2.2) E2 protein is as shown in SEQ ID No. 10.

[0154] The nucleotide sequence of the coding gene of HuB2 (2.3) E2 protein is as shown in SEQ ID No. 11.

[0155] Nucleotide sequence of the coding gene of E2 protein of CSF0410 (3.1): positions 2146 - 3234 of GenBank Accession No. JQ411575.1 (Update Date 09 - MAR - 2016).

[0156] Nucleotide sequence of the coding gene of E2 protein of JJ9811 (3.2): positions 2346 - 3434 of GenBank Accession No. KF669877.1 (Update Date 20 - DEC - 2012).

[0157] Nucleotide sequence of the coding gene of E2 protein of 94.4 / IL / 94 / TWN(3.4): positions 2344 - 3432 of GenBank Accession No. AY646427.1 (Update Date 29 - OCT - 2007).

[0158] Perform Western blot experiments on monoclonal antibody TCH053 and different genotypes of CSFV E2 proteins expressed by the insect baculovirus expression system, using the protein tag antibody anti - His Tag mAb as a control. The steps are as follows:

[0159] ① Protein treatment: Add each E2 protein to 4×loading buffer in proportion and boil for 10 min.

[0160] ② Protein electrophoresis: Load the treated protein (10 μL per lane) onto a 10% SDS - PAGE gel and perform protein electrophoresis. The program is 55 V - 50 min, 110 V - 80 min.

[0161] ③ Membrane transfer: Use the semi - dry membrane transfer method to transfer the protein after electrophoresis to the NC membrane. The program is 23 V - 25 min.

[0162] ④ Blocking: Add 5 g of skim milk powder to 100 mL of PBS, shake well to mix as the blocking solution. Add 5 mL of the blocking solution into a self - sealing bag and place it on a shaker to block the NC membrane at room temperature for 1 h.

[0163] ⑤ Incubate with primary antibody: Dilute the hybridoma cell culture supernatant (monoclonal antibody TCH053) 1:100 with the blocking solution, and dilute the Anti - His Tag antibody (Solarbio, K200060M) 1:3000 as a positive control. Incubate overnight at 4℃ on a shaker.

[0164] ⑥ Incubate the secondary antibody: Wash the membrane 3 times with PBS, add the Alexa Fluor 680-labeled fluorescent secondary antibody (Thermo Fisher, catalog number: A10038) diluted 1:5000 with PBS, and incubate at room temperature in the dark on a shaker for 1 h.

[0165] ⑦ Scan the NC membrane: Wash the membrane 3 times with PBS, place it in a dual-color infrared laser imaging system for scanning, and save the pictures.

[0166] The results are as Figure 2 shown, the monoclonal antibody TCH053 reacts with the E2 proteins of all strains.

[0167] 2. Preparation of monoclonal antibody ascites and identification of neutralization ability from hybridoma cell lines

[0168] Female Balb / C mice at 6 - 8 weeks of age were intraperitoneally injected with 300 μL of adjuvant specific for mouse ascites. After 12 days, 1 × 10 6 hybridoma cells TCH053 prepared as described in 1 above were intraperitoneally injected. When the abdominal circumference of the mice was significantly enlarged, ascites was aspirated using a syringe, centrifuged at 12000 r / min for 10 min, and the supernatant was collected and stored to obtain the monoclonal antibody TCH053 prepared from ascites.

[0169] The ability of the monoclonal antibody TCH053 prepared from ascites to neutralize the CSFV vaccine strain LPC strain and the epidemic strain JL23 of genotype 2.1b was identified by indirect immunofluorescence assay. The LPC and JL23 strains used are described in the following literature: Shijiang Mi. Identification of monoclonal antibodies for differentiating epidemic strains and vaccine strains of classical swine fever virus and broad-spectrum monoclonal antibodies and analysis of their antigenic epitopes [D]. Jilin University, 2022.

[0170] The identification method is as follows: CSFV strains of different genotypes were diluted to 2000 TCID 50 / mL with MEM respectively. 50 μL of the virus dilution was pipetted into a 96-well plate. The monoclonal antibody TCH053 prepared from ascites was serially diluted 4-fold starting from 1:100 with MEM, and 50 μL of each concentration gradient was added to the corresponding virus solution. A control group of MEM without antibody (without adding monoclonal antibody) was set up and incubated at 37°C for 1 h. 100 μL of PK-15 cells passaged at 1:4 were added to the virus / antibody neutralization solution, and cultured at 37°C and 5% CO2 for 3 days. IFA (the method is the same as steps ② - ⑤ in the reaction of monoclonal antibody and virus in 1) was used to identify whether the broad-spectrum monoclonal antibody against pestivirus can neutralize CSFV.

[0171] The results are as Figure 3As shown, when a higher concentration of purified monoclonal antibody was added, there was no obvious green fluorescence in the cells infected with the CSFV strain. As the antibody dilution factor increased continuously, green fluorescence appeared in the cells infected with different strains, indicating that the monoclonal antibody TCH053 has good neutralizing ability.

[0172] Example 2: Sequencing, genetic engineering preparation and identification of monoclonal antibody TCH053

[0173] 1. Sequencing of monoclonal antibody TCH053

[0174] The nucleic acid of the hybridoma cell TCH053 prepared in Example 1 was extracted using a nucleic acid extraction kit (Magen, R4410 - 250), reverse transcribed using M-MLV reverse transcriptase (TaKaRa, 2641B) to obtain hybridoma cell cDNA, and the variable regions of the heavy and light chains were amplified by PCR. The amplification primers for the heavy chain variable region were F: 5’-CAGGTTCAGCTGCAGCAGTC-3’; R: 5’- TGAGGAGACGGTGACTGAG-3’, and the amplification primers for the light chain variable region were F: 5’-GATGTTGTGATGACCCAA-3’; R: 5’-ACTGAGGCACCTCCAGATG-3’. The amplification products were sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing, and the sequences obtained were as follows:

[0175] The amino acid sequence of the heavy chain variable region of monoclonal antibody TCH053 is shown in SEQ ID No.1, and the nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID No.2; the amino acid sequence of the light chain variable region of monoclonal antibody TCH053 is shown in SEQ ID No.3, and the nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID No.4.

[0176] Among them:

[0177] The amino acid sequence of CDR1 of the heavy chain variable region is shown in positions 9 - 15 of SEQ ID No.1;

[0178] The amino acid sequence of CDR2 of the heavy chain variable region is shown in positions 33 - 40 of SEQ ID No.1;

[0179] The amino acid sequence of CDR3 of the heavy chain variable region is shown in positions 79 - 90 of SEQ ID No.1;

[0180] The amino acid sequence of CDR1 of the light chain variable region is shown in positions 21 - 31 of SEQ ID No.3;

[0181] The amino acid sequence of CDR2 of the light chain variable region is shown in positions 49 - 51 of SEQ ID No.3;

[0182] The amino acid sequence of CDR3 of the light chain variable region is shown as positions 88-97 of SEQ ID No.3;

[0183] The above CDRs are determined by the Kabat coding system.

[0184] The above nucleotide sequence can be artificially synthesized.

[0185] 2. Preparation of monoclonal antibody TCH053 by genetic engineering method

[0186] 1) Construction of recombinant expression plasmid

[0187] To express monoclonal antibody TCH053, a heavy chain expression vector and a light chain expression vector were prepared respectively: The nucleotide sequence of the heavy chain gene of monoclonal antibody TCH053 consists of the nucleotide sequence of the heavy chain variable region coding gene (SEQ ID No.2) and the mouse-IgG2a template sequence (heavy chain constant region sequence, SEQ ID No.12) in sequence, and the last base of SEQ ID No.2 is connected to the first base of SEQ ID No.12. The nucleotide sequence of the light chain gene of monoclonal antibody TCH053 consists of the nucleotide sequence of the light chain variable region coding gene (SEQ ID No.4) and the mouse-kappa template sequence (light chain constant region sequence, SEQ ID No.13) in sequence, and the last base of SEQ ID No.4 is connected to the first base of SEQ ID No.13. The heavy chain gene and the light chain gene of monoclonal antibody TCH053 were respectively cloned into the Xba I and Eco RV cleavage sites of the vector pcDNA3.4 (HonorGene, product number HG-VPH1386) to obtain the heavy chain expression vector and the light chain expression vector.

[0188] 2) Expression of antibody

[0189] a) Inoculate freshly digested 293T cells into a 175 cm 2 culture flask, add 35 mL of DMEM medium containing 8% FBS (purchased from Corning, product number 10-013-CVRC) and culture the cell density to 90%, with a total of 4 bottles cultured.

[0190] b) Dilute 200 μg of the heavy chain expression vector and the light chain expression vector obtained in 1) above (100 μg each) and 200 μL of the QuickShuttle-293 cell-specific transfection reagent (Biolong, KX0110044) into 1 mL of physiological saline respectively.

[0191] c) Combine the two solutions in step b) above and mix well. This is the complex required for one bottle of cell transfection.

[0192] d) Add the above complex directly to the cell culture medium in step a), and pipette and mix well.

[0193] e) Transfer the cell plate to an incubator at 37ºC / 5% CO2 for culture. After 3 days, take the supernatant for purification.

[0194] 3) Purification of antibody:

[0195] a) Preparation of buffer: Add Na2HPO4·12H2O to sterile ddH2O to make its final concentration 0.2 M, and shake well.

[0196] b) Preparation of pre-elution buffer: Add 0.1 M citric acid to the buffer to make the volume ratio of citric acid 20%.

[0197] c) Preparation of elution buffer: Add 0.1 M citric acid to the buffer to make the volume ratio of citric acid 60%.

[0198] d) Sample treatment: Take 30 mL of the cell expression supernatant in step 2), add the buffer prepared in step a) according to a volume ratio of 1:1, filter through a 0.22 μm pore size filter, and prepare for loading onto the column.

[0199] e) Equilibrate the column: Use a constant flow pump to slowly pass 10 mL of buffer through a Protein A / G 4FF pre-packed chromatography column (Sangon Biotech, C600983) at a flow rate of 1 mL / min.

[0200] f) Loading: Use a constant flow pump to slowly pass the solution in step d) through the Protein A / G column at a flow rate of 1 mL / min.

[0201] g) Washing: Use a constant flow pump to slowly pass 10 mL of washing buffer through the Protein A / G column at a flow rate of 1 mL / min.

[0202] h) Pre-elution: Use a constant flow pump to slowly pass 10 mL of pre-elution buffer through the Protein A / G column at a flow rate of 1 mL / min.

[0203] i) Elution: Use a constant flow pump to slowly pass 15 mL of elution buffer through the Protein A / G column at a flow rate of 1 mL / min. Aliquot the eluted product into 1.5 mL centrifuge tubes to obtain the purified monoclonal antibody TCH053 solution.

[0204] The concentration of the purified monoclonal antibody TCH053 solution is 0.8 mg / ml.

[0205] 4) Verification of antibody expression:

[0206] Take the purified monoclonal antibody TCH053 solution obtained in step 3) above, and load it onto a non-reducing Buffer without DTT (lane 1) and a reducing Buffer containing DTT (lane 2) in proportion for Western blot experiment.

[0207] The results are as Figure 4 shown. There is an obvious single band at a position greater than 180 kDa in lane 1. After the monoclonal antibody TCH053 is treated with the reducing Buffer, as shown in lane 2, there are two obvious bands of light chain and heavy chain at about 25 kDa and 50 kDa, indicating that the antibody is well purified.

[0208] 3. Identification of antibodies prepared by genetic engineering methods

[0209] Perform Western blot experiment on the purified monoclonal antibody TCH053 solution obtained in step 2 above and different genotypes of CSFV E2 proteins (E2 protein of classical swine fever lapinized attenuated vaccine strain C (HCLV) and CN069 (2.1b) E2 protein) expressed by the insect baculovirus expression system to verify the antibody activity. Use the purified monoclonal antibody TCH053 solution diluted 1:1000 with 5% skim milk as the primary antibody, and the remaining steps are the same as those in step (5) of Example 1 for the identification of the reaction between the monoclonal antibody and the viral protein.

[0210] The results are as Figure 5 shown. The monoclonal antibody TCH053 reacts with both HCLV and CN069 E2 proteins.

[0211] Example 3. Preparation and identification of conjugated antibodies

[0212] 1. Conjugated antibody

[0213] Use an HRP conjugation kit (Sangon Biotech, D601047) to conjugate HRP to the monoclonal antibody TCH053 prepared in Example 2 to prepare the HRP-TCH053 conjugated antibody. The specific operation steps are as follows:

[0214] (1) Mix 500 μL of HRP solution with 200 μL of HRP activation buffer evenly, place it on a shaker and shake slowly, and react at room temperature for 30 min to obtain the HRP conjugation buffer.

[0215] (2) Add 200 μL of the HRP conjugation buffer and let it stand at room temperature for 30 min.

[0216] (3) Add 1 mL of the purified monoclonal antibody TCH053 solution prepared in Example 2 to the reaction system in step (2), and transfer it to a dialysis bag, and dialyze it at room temperature in 2 L of dialysis solution for 2 h.

[0217] (4) Add 100 μL of reducing agent to the dialysis product, let it stand at room temperature for 2 h, stir it every 30 min, aliquot and store the conjugate product, denoted as the conjugated antibody.

[0218] 2. Identification of the conjugated antibody

[0219] Identify the antibody conjugation effect using the direct ELISA method. The specific steps are as follows:

[0220] (1) Protein coating: Dilute the CSFV E2 protein (i.e., the CSFV-LPC E2 purified protein in Example 1) with ELISA coating buffer (Solarbio, catalog number C1050) at a concentration of 1 μg / mL. Coat 100 μL per well in a 96-well ELISA plate, coat overnight at 4°C, and discard the coating buffer after coating.

[0221] (2) Blocking: Block with 5% skim milk powder, 100 μL per well, block at 37°C for 1 h.

[0222] (3) Antibody incubation: Dilute the conjugated antibody prepared in step 1 above with PBS, perform two-fold serial dilution starting from 1:2000 and dilute to 1:128000, incubate at 37°C for 1 h, and use PBS as the negative control (NC).

[0223] (4) Wash the plate 3 times with PBST, 300 μL per well.

[0224] (5) Add 50 μL of TMB chromogenic solution to each well, incubate at room temperature in the dark for 10 min.

[0225] (6) Add 50 μL of stop solution to each well to terminate the reaction, and measure the OD 450 value

[0226] The results are shown in Table 1 below. When the conjugated antibody is diluted 128,000 times, it is still significantly positive, indicating that the antibody conjugation effect is good.

[0227]

[0228] Example 4. Application of monoclonal antibody TCH053 in the detection of classical swine fever virus

[0229] In this example, an ELISA method for detecting classical swine fever virus using monoclonal antibody TCH053 was exemplarily established. The specific operation method is as follows:

[0230] (1) Coat 32 ng of CSFV E2 protein (i.e., the CSFV-LPC E2 purified protein in Example 1) per well in the ELISA plate. After coating, discard the coating buffer (Solarbio, catalog number C1050), add 5% skim milk for blocking, and after blocking, dry at room temperature and then store in a vacuum-sealed manner.

[0231] (2) Add 50 μL of PBS to each well of the coated plate, and then add the serum sample to be tested to the corresponding well, and incubate at 37 °C for 1 h.

[0232] The above serum samples to be tested are 261 porcine sera (including 191 sera from multiparous sows, 25 sera from gilts, 21 sera from suckling piglets, 14 sera from weaned piglets from a classical swine fever affected farm, and 10 sera from sows).

[0233] (3) Discard the liquid in each well, wash the wells of the plate with 300 μL of PBST for 5 times in total, and pat dry the washing liquid in the wells after the last washing.

[0234] (4) Add 100 μL of HRP-TCH053 conjugate antibody diluted 1:10000 with PBS to each well, and incubate at 37 °C for 1 h.

[0235] (5) Repeat step (3).

[0236] (6) Add 50 μL of TMB chromogenic solution (Beyotime, catalog number P0206) to each well, and incubate at room temperature in the dark for 10 min.

[0237] (7) Add 50 μL of stop solution (Sangon Biotech, catalog number E661006) to each well to terminate the reaction, and measure the OD 450 value with an ELISA reader.

[0238] Select the IDEXX CSFV antibody detection kit (purchased from IDEXX Company, catalog number 99-43220, hereinafter referred to as the IDEXX kit) as a control (comparison).

[0239] The results are shown in Table 2 and Table 3. Among the 261 porcine sera, there are 252 serum samples that are both positive detected by the method of the present invention and the IDEXX kit, and 7 samples that are both negative. There are 2 samples that are negative detected by the method of the present invention and positive detected by the IDEXX kit, and 0 samples that are positive detected by the method of the present invention and negative detected by the IDEXX kit. The overall coincidence rate of the two kits is 99.2%.

[0240]

[0241]

[0242] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art. The application of some basic features can be made within the scope of the appended claims below.

Claims

1. An antibody or an antigen-binding fragment thereof targeting the E2 protein of classical swine fever virus, which comprises a heavy chain variable region and a light chain variable region; The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are the 9th to 15th, 33rd to 40th, and 79th to 90th positions of SEQ ID No.1 in sequence; The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are the 21st to 31st, 49th to 51st, and 88th to 97th positions of SEQ ID No.3 in sequence.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The antibody or its antigen-binding fragment comprises 4 heavy chain variable region framework regions named HFR1, HFR2, HFR3, and HFR4, and 4 light chain variable region framework regions named LFR1, LFR2, LFR3, and LFR4; The amino acid sequence of the HFR1 comprises an amino acid sequence selected from the following: A1) The 1st to 8th positions of SEQ ID NO:1; A2) A sequence having more than 75% identity with A1); The amino acid sequence of the HFR2 comprises an amino acid sequence selected from the following: A3) The 16th to 32nd positions of SEQ ID NO:1; A4) A sequence having more than 75% identity with A3); The amino acid sequence of the HFR3 comprises an amino acid sequence selected from the following: A5) The 41st to 78th positions of SEQ ID NO:1; A6) A sequence having more than 75% identity with A5); The amino acid sequence of the HFR4 comprises an amino acid sequence selected from the following: A7) The 91st to 101st positions of SEQ ID NO:1; A8) A sequence having more than 75% identity with A7); The amino acid sequence of the LFR1 comprises an amino acid sequence selected from the following: A9) The 1st to 20th positions of SEQ ID NO:3; A10) A sequence having more than 75% identity with A9); The amino acid sequence of the LFR2 comprises an amino acid sequence selected from the following: A11) The 32nd to 48th positions of SEQ ID NO:3; A12) A sequence having more than 75% identity with A11); The amino acid sequence of the LFR3 comprises an amino acid sequence selected from the following: A13) The 52nd to 87th positions of SEQ ID NO:3; A14) A sequence having more than 75% identity with A13); The amino acid sequence of the LFR4 comprises an amino acid sequence selected from the following: A15) The 98th to 106th positions of SEQ ID NO:3; A16) A sequence having more than 75% identity with A15).

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that: The heavy chain variable region is any one of the following: B1) The amino acid sequence of the shown heavy chain variable region comprises SEQ ID No.1; B2) The amino acid sequence of the shown heavy chain variable region comprises an amino acid sequence having more than 90% identity with SEQ ID No.1; And / or, the light chain variable region is any one of the following: The amino acid sequence of the light chain variable region shown in B3) includes SEQ ID No. 3; The amino acid sequence of the light chain variable region shown in B4) includes an amino acid sequence having more than 90% identity with SEQ ID No.

3.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein: The amino acid sequence of the heavy chain variable region shown in B2) includes an amino acid sequence having more than 95% identity with SEQ ID No. 1; and / or, The amino acid sequence of the light chain variable region shown in B4) includes an amino acid sequence having more than 95% identity with SEQ ID No.

3.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein: The amino acid sequence of the heavy chain variable region shown in B2) includes an amino acid sequence having more than 99% identity with SEQ ID No. 1; and / or, The amino acid sequence of the light chain variable region shown in B4) includes an amino acid sequence having more than 99% identity with SEQ ID No.

3.

6. A biological material related to the antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the biological material is any one of C1) to C12): C1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-5; C2) An expression cassette containing the nucleic acid molecule of C1); C3) A recombinant vector containing the nucleic acid molecule of C1); C4) A recombinant vector containing the expression cassette of C2); C5) A recombinant microorganism containing the nucleic acid molecule of C1); C6) A recombinant microorganism containing the expression cassette of C2); C7) A recombinant microorganism containing the recombinant vector of C3); C8) A recombinant microorganism containing the recombinant vector of C4); C9) A recombinant cell containing the nucleic acid molecule of C1); C10) A recombinant cell containing the expression cassette of C2); C11) A recombinant cell containing the recombinant vector of C3); C12) A recombinant cell containing the recombinant vector of C4).

7. An antibody conjugate, comprising an antibody moiety and a conjugate moiety, characterized in that: The antibody portion comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-5; the conjugate portion is HRP.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-5, or the biological material according to claim 6, or the antibody conjugate according to claim 7 in at least one of the following: D1) Preparing a product for detecting classical swine fever virus; D2) Preparing a product for detecting a disease caused by classical swine fever virus infection; D3) Preparing a product for detecting classical swine fever virus E2 protein; D4) Preparing a product for binding to classical swine fever virus E2 protein.

9. A product comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-5, or the biological material according to claim 6, or the antibody conjugate according to claim 7.

10. The product according to claim 9, wherein: The product has at least one of the following functions: E1) Detecting classical swine fever virus; E2) Detecting a disease caused by classical swine fever virus infection; E3) Detecting classical swine fever virus E2 protein; E4) Binding to classical swine fever virus E2 protein.

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