Diagnostic kit for bovine diarrhea-causing virus

Abstract

The present invention relates to a diagnostic kit for bovine viral diarrhea virus (BVDV), which enables rapid on-site identification and diagnosis of BVDV1 and BVDV2, which are genotypes of the BVD virus that are pathogens causing bovine diarrhea, within a short time of approximately 15 minutes.

Description

Bovine diarrhea-causing virus diagnostic kit

[0001] The present invention relates to a diagnostic kit for a virus causing small bowel diarrhea.

[0002] Bovine viral diarrhea (BVD) is a very serious viral disease caused by infection with the bovine viral diarrhea virus (BVDV) and causes high fever, diarrhea, runny nose, cough, leukopenia, thrombocytopenia, ulcers of the gastrointestinal mucosa, respiratory symptoms, bleeding, decreased milk yield, reproductive disorders (miscarriage and stillbirth), immunosuppression, and in severe cases, death. It causes enormous economic losses (at least 100 billion won per year) in cattle farms. In Korea, the infection rate of BVD varies depending on the farm, but it is known to have an incidence rate of 20-60% in general, and its incidence has been rapidly increasing recently.

[0003] The BVD virus infects cattle of all ages, has a high rate of transmission and morbidity, and its mortality rate continues to increase. Recent research has identified BVD virus infection as a major cause of abortion in pregnant cattle in Korea. Furthermore, BVD virus infection has been identified as a major cause of calf birth defects, including cerebellar hypoplasia, inability to stand, and ocular dysplasia.

[0004] According to data from the Ministry of Agriculture, Food and Rural Affairs, the economic loss to domestic livestock farms due to BVD is estimated at 60 to 100 billion won per year, and in the United States and Europe, it is known that losses of 1.5 to 2 billion dollars and 4.5 billion dollars per year, respectively, are caused by bovine diarrhea.

[0005] The bovine vulgaris disease (BVDV) virus that causes bovine diarrhea is known to have two genotypes (BVDV-1 and BVDV-2). BVDV-1 has 22 subtypes (1a-1u), and BVDV-2 has 4 subtypes (2a-2d). It was first reported in the 1990s in North America as an acute infectious disease characterized by diarrhea and gastrointestinal erosions, and since then, outbreaks have occurred in Europe, Japan, Korea, and South America, and it has been found to be accompanied by hemorrhagic syndrome (HS) such as thrombocytopenia, bloody diarrhea, epistaxis, petechial and ecchymotic hemorrhages, and a high mortality rate.

[0006] In addition, BVDV can be divided into two biotypes, NCP (non-cytopathic; non-cytopathic) and CP (cytopathic; cytopathic) types, depending on the presence or absence of cytopathic changes in cultured cells. Infection with the NCP type shows miscarriage, respiratory symptoms, and mostly persistent infection symptoms. On the other hand, the CP type is characterized by showing clinical symptoms of mucosal disease (MD), showing bleeding symptoms in the mouth and mucosa and eventually leading to death. Above all, it is known that the clinical symptoms become much more severe when superinfection with the antigenically similar NCP and CP types occurs.

[0007] Furthermore, the most widely used products for BVDV diagnosis in Korea are real-time PCR and BVDV Ag detection kits. While the commercially available real-time PCR kit (Lilif Co.) boasts high sensitivity, it is difficult to apply in the field and requires expensive equipment and expert skills. Furthermore, it suffers from significantly lower sensitivity compared to conventional PCR methods, posing numerous challenges for field application. Therefore, there is an urgent need to develop a field-ready kit that can provide more accurate results.

[0008] Accordingly, the inventors of the present invention completed the present invention by confirming that it is possible to rapidly diagnose BVDV1 and BVDV2, which are the main genotypes of BVDV (Bovine viral diarrhea virus), a major pathogen of bovine diarrhea, by using an antigen protein produced based on the genotype with the highest distribution in Korea.

[0009] Accordingly, the present invention aims to provide a diagnostic kit comprising a recombinant protein containing an antigen of bovine viral diarrhea virus (BVDV) and an antibody binding thereto.

[0010] To achieve the above object, the present invention provides a recombinant protein comprising at least one antigen of bovine viral diarrhea virus (BVDV) selected from the group consisting of BVDV nucleocapsid protein (BVDV-CP), BVDV1 envelope glycoprotein E2 (BVDV1-E), and BVDV2 envelope glycoprotein E2 (BVDV2-E).

[0011] In addition, the present invention provides an expression vector comprising a gene encoding the above recombinant protein.

[0012] In addition, the present invention provides a transformant transformed with the above expression vector.

[0013] In addition, the present invention provides a vaccine composition for bovine viral diarrhea virus (BVDV), comprising at least one selected from the group consisting of the above recombinant protein, the above expression vector, the above transformant, and a combination thereof.

[0014] In addition, the present invention provides an antibody or fragment thereof that specifically binds to the recombinant protein or a protein produced by the transformant.

[0015] In addition, the present invention provides a bovine viral diarrhea virus (BVDV) diagnostic kit comprising the antibody or a fragment thereof.

[0016] In addition, the present invention provides a method for diagnosing bovine viral diarrhea virus (BVDV) using the diagnostic kit.

[0017] Using the bovine diarrhea disease BVDV diagnostic kit of the present invention, the BVD virus genotypes BVDV1 and BVDV2, which are the pathogens causing bovine diarrhea, can be quickly differentiated and diagnosed on-site within a short time of about 15 minutes. In addition, the diagnostic kit of the present invention uses the blood, plasma, serum, etc. of the subject, so that livestock farmers can easily and quickly diagnose infection with the bovine diarrhea disease pathogen themselves without expensive samples, equipment, or specialized skills.

[0018] Figure 1 is a diagram showing the selection of the gene for the nucleocapsid protein (BVDV-CP) of the bovine diarrhea pathogen BVDV and the production and purification of the recombinant antigen protein: (A) Cloning of the BVDV-CP gene into an Escherichia coli expression vector. (B) Expression and isolation and purification of the BVDV-CP recombinant protein in E. coli.

[0019] Figure 2 is a diagram showing the synthesis of genes for envelope proteins (BVDV1-E2, BVDV2-E2) of bovine diarrhea pathogens BVDV1 and BVDV2, and the production and purification of recombinant antigen proteins: (A) Cloning of BVDV1-E2 and BVDV2-E2 genes into E. coli expression vectors. (B) Expression and purification of BVDV1-E2 recombinant protein in E. coli. (C) Isolation and purification of BVDV2-E2 recombinant protein.

[0020] Figure 3 is a diagram showing the selection of monoclonal antibody-producing cell lines and antibody pair selection against the antigen protein of a bovine diarrhea pathogen: (A) List of monoclonal antibody-producing cell lines against the bovine diarrhea pathogen BVDV-CP antigen protein. (B) List of monoclonal antibody-producing cell lines against the bovine diarrhea pathogen BVDV1-E2 antigen protein. (C) List of monoclonal antibody-producing cell lines against the bovine diarrhea pathogen BVDV2-E2 antigen protein.

[0021] Figure 4 shows the results of a specificity test of a diagnostic kit for bovine diarrhea virus BVDV.

[0022] Figure 5 shows the results of comparing the performance of diagnostic kits for bovine diarrhea virus: (A) Specific detection performance of the BVDV diagnostic kit of the present invention for BVDV1 and BVDV2 pathogens. (B) Detection performance of BVDV1 and BVDV2 of Company B's BVDV detection kit.

[0023] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of well-known technologies to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such detailed descriptions may unnecessarily obscure the gist of the present invention. Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of the user or operator, or the customs of the field to which the present invention pertains.

[0024] Therefore, definitions of these terms should be based on the overall content of this specification. Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather implies the inclusion of additional components, unless otherwise specifically stated.

[0025] Hereinafter, terms used in the present invention are explained.

[0026] The present invention provides a recombinant protein comprising at least one antigen of bovine viral diarrhea virus (BVDV) selected from the group consisting of BVDV nucleocapsid protein (BVDV-CP), BVDV1 envelope glycoprotein E2 (BVDV1-E), and BVDV2 envelope glycoprotein E2 (BVDV2-E).

[0027] The term "antigen" in the present invention refers to a substance that specifically induces an immune response when a substance invades the body, and a substance that reacts with an antibody is called an antigen. The property of an antigen to establish immunity is called immunogenicity, and the property of specifically reacting with an antibody is called reactogenicity. An antigen that possesses both properties is called a complete antigen. Generally, proteins or polysaccharides with large molecular weights belong to this category, and bacteria, rickettsiae, viruses, microorganisms, red blood cells, cells, and other substances with complex chemical structures are complete antigens. Among various antigens, those that are reactogenic but not immunogenic are called incomplete antigens or haptens. Haptens include complex haptens that react with antibodies to form a precipitate, and simple haptens that do not form a precipitate and whose presence is only detected when the antibody is consumed. The conditions for an antigen include being non-self, having a certain molecular weight, and being able to induce an immune response by immune cells when parenterally introduced into the body.

[0028] According to one embodiment of the present invention, the BVDV-CP may include an amino acid sequence of SEQ ID NO: 2, the BVDV1-E may include an amino acid sequence of SEQ ID NO: 4, and the BVDV2-E may include an amino acid sequence of SEQ ID NO: 6, but is not limited thereto.

[0029] The “bovine viral diarrhea virus (BVDV)” of the present invention is an RNA virus classified into the genus Pestivirus of the family Flaviviridae, and is a virus classified into cytopathic strains (NADL, Singer, T20 strains, etc.) and non-cytopathic strains (New York strain, etc.) depending on whether it causes a cytopathic effect in a cell line. Morphologically, it is a spherical virus with a diameter of 40 to 60 nm and has an envelope, and is transmitted by oral infection through feed contaminated with feces, placental infection through the placenta, respiratory and reproductive infections, etc. It is a virus that mainly infects ruminants, and when infected, causes ulcers of the digestive tract mucosa, diarrhea, respiratory lesions, etc., and in severe cases, leads to death.

[0030] The scope of amino acids according to the present invention includes proteins having amino acid sequences represented by SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 and functional equivalents of the proteins. The term "functional equivalent" refers to a protein having at least 70%, preferably 80%, more preferably 90%, and even more preferably 95% sequence homology with the amino acid sequence represented by the above SEQ ID NO as a result of addition, substitution or deletion of amino acids, and exhibiting substantially the same physiological activity as the protein represented by the above SEQ ID NO. The amino acids of the present invention include not only proteins having their native amino acid sequences but also amino acid sequence variants thereof, within the scope of the present invention.

[0031] Amino acid variants refer to proteins whose sequences differ from the native amino acid sequence by deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues, or a combination thereof. Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are well known in the art. Furthermore, tetrapeptides or their variants can be derived from natural sources, synthesized, or produced by genetic recombination methods based on DNA sequences.

[0032] The above amino acid mutations are based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0033]

[0034] In addition, the present invention provides an expression vector that expresses a gene encoding the above recombinant protein.

[0035] A recombinant expression vector can be provided by operably linking a gene encoding a protein that can be an antigen of the bovine diarrhea pathogen of the present invention to a regulatory sequence of an expression vector. In the present invention, the term "expression vector" may include a plasmid known in the art into which the DNA sequence, gene sequence or base sequence according to the present invention can be inserted or introduced, and all other vectors known to those skilled in the art, such as phage, virus such as SV40, CMV, baculovirus and adenovirus, transposon, IS element, phasmid, cosmid, and linear or circular DNA, and these vectors can autonomously replicate or chromosomally replicate in a host organism. The DNA sequence, gene sequence or base sequence according to the present invention can be operably linked to an expression regulatory sequence, and the operably linked gene sequence and expression regulatory sequence can be included in one expression vector that includes a selection marker and a replication origin together. The term "operably linked" as described above may refer to a gene and an expression control sequence that are linked in such a way that gene expression is enabled when an appropriate molecule is bound to the expression control sequence. An "expression control sequence" refers to a DNA sequence that controls the expression of an operably linked polynucleotide sequence in a particular host cell. Such control sequences include a promoter for initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that controls the termination of transcription and translation.

[0036]

[0037] In addition, the present invention provides a transformant transformed with the above expression vector.

[0038] In addition, the present invention can provide a host cell transformed with the expression vector, and the host cell can be a prokaryotic or eukaryotic cell, and a host cell with high DNA introduction efficiency and high expression efficiency of the introduced DNA is preferred. Examples of such host cells include Escherichia coli, fungi, and yeast, and in one embodiment of the present invention, Escherichia coli was used.

[0039] The method for transforming the host cell may use a conventional transformation method, such as cationic lipid-mediated transfection, electroporation, DEAE-dextran mediated transfection, or calcium phosphate transfection. In addition, another optimal transformation method may be introduced depending on the type of host cell and expression vector, etc., with reference to what is known in the art.

[0040] The transformed host cells can be cultured on a culture medium suitable for the production of recombinant proteins, and the recombinant proteins can be isolated and purified from the cultured cells using protein purification methods known in the art. For example, but not limited to, the host cells can be cultured in a small-scale or large-scale culture system in a laboratory or industrial device operated under suitable media and conditions that facilitate the expression and / or isolation of the proteins, and the culture is performed in a suitable nutrient medium containing carbon, a nitrogen source, and inorganic salts using known techniques. Such suitable media can be obtained commercially or prepared according to methods known in the literature.

[0041] Recombinant proteins produced in transformed host cells can be isolated using conventional protein isolation methods. For example, recombinant proteins can be isolated from cultures using methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Furthermore, proteins can be purified using known methods, including chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion), electrophoresis, differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.

[0042]

[0043] In addition, the present invention provides a vaccine composition for bovine viral diarrhea virus (BVDV), comprising at least one selected from the group consisting of the above recombinant protein, the above expression vector, the above transformant, and a combination thereof.

[0044]

[0045] In addition, the present invention provides an antibody or fragment thereof that specifically binds to the recombinant protein or a protein produced by the transformant.

[0046] The above antibody may be a monoclonal or polyclonal antibody, and may be, for example, but is not limited to, a monoclonal antibody.

[0047] The term 'antibody' as used herein refers to immunoglobulins and immunoglobulin fragments, including any fragment comprising at least a portion of the variable region of an immunoglobulin molecule that retains the specific binding affinity of a full-length immunoglobulin, whether natural or partially or fully synthetic, e.g., produced recombinantly. Accordingly, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). The antibodies may include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, bispecific antibodies, multispecific antibodies, human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, or antibody fragments. For example, the antibody may comprise a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a disulfide-linked Fv (dsFv), an Fd fragment, an Fd' fragment, a single-chain Fv (scFv), a single-chain Fab (scFab), a diabody, a nanobody, an anti-idiotypic (anti-Id) antibody, or an antigen-binding fragment of any of the above.

[0048] The term "monoclonal antibody" used in the present invention is a term known in the art and refers to a highly specific antibody directed against a single antigenic site. Unlike polyclonal antibodies, which typically include different antibodies directed against different epitopes (epitopes), monoclonal antibodies are directed against a single determinant on an antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays that utilize antigen-antibody binding, and, because they are synthesized through hybridoma culture, they have the additional advantage of being free from contamination by other immunoglobulins.

[0049] Typically, immunoglobulins have heavy chains and light chains, each of which comprises a constant region and a variable region (also known as a "domain"). The variable regions of the light and heavy chains comprise three variable regions called "complementarity determining regions" (hereinafter referred to as "CDRs") and four "framework regions." The CDRs are primarily responsible for binding to epitopes on an antigen. The CDRs of each chain are typically named CDR1, CDR2, and CDR3, starting from the N-terminus, and are also identified by the chain on which the particular CDR is located.

[0050] The term "variable" as used herein refers to the significant differences in sequence of specific regions between antibodies, which determine the binding specificity of each antibody for a specific antigen. The variability of antibodies is not uniformly distributed throughout the antibody's variable domain, but is concentrated in the CDRs. The heavy and light chains of a monoclonal antibody each have three CDRs, and these regions recognize antigens of bovine diarrheal disease pathogens, thereby forming an antigen-antibody complex. These CDRs have a unique sequence for each monoclonal antibody, and some or all of these six CDRs can interact to allow a single monoclonal antibody to recognize a specific epitope.

[0051]

[0052] In addition, the present invention provides a method for producing a bovine viral diarrhea virus (BVDV) specific monoclonal antibody, comprising the steps of: administering the recombinant protein to a non-human subject; obtaining cells from the subject; and purifying antibodies from the obtained cells.

[0053] Methods for producing antibodies using the above proteins can be easily produced by those skilled in the art using known techniques. For example, polyclonal antibodies can be produced by methods well known in the art, such as injecting a recombinant antigen into an animal and collecting blood from the animal to obtain serum containing antibodies. Such polyclonal antibodies can be produced from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cows, dogs, and mice. Monoclonal antibodies can be produced using a hybridoma method well known in the art or can be produced using phage antibody library technology. In addition, the antibodies according to the present invention may include not only complete forms having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules. Functional fragments of antibody molecules refer to fragments that possess at least an antigen-binding function, such as Fab, F(ab'), F(ab') 2, and Fv.

[0054] In one embodiment of the present invention, the antibody is preferably a monoclonal antibody, but is not limited thereto, and in order to produce a monoclonal antibody, the immunized cells and myeloma cells, etc. are fused using a known method, and then individual clones that produce the desired antibody can be obtained by processing using a limiting dilution method (Current Protocols in Immunology).

[0055] In one embodiment of the present invention, the method for producing an antibody against the bovine diarrhea pathogen antigen protein may include the steps of administering the host cell described above or the recombinant protein (antigen) of the bovine diarrhea pathogen expressed therefrom to a mouse to immunize it; obtaining lymph node cells from the popliteal fossa or lymph nodes of the immunized mouse; fusing them with myeloma cells; and culturing the fused cells to isolate a monoclonal antibody.

[0056] In one embodiment of the present invention, immunization of the mouse can be performed according to a conventional method. For example, but not limited to, the cell as an antigen or the bovine diarrhea pathogen protein expressed therefrom is first administered intraperitoneally, intravenously, or subcutaneously, and then 14 to 21 days later, a second administration is performed, followed by another administration of the antigen, so that the total dosage is 5 to 20 μg per mouse. As conventional adjuvants, complete Freund's adjuvant and incomplete adjuvant antigen can be appropriately used as needed. Spleen cells of the mouse can be obtained 3 to 4 days after the final administration of the immunizing antigen and used as immune cells.

[0057]

[0058] In addition, the present invention provides a bovine viral diarrhea virus (BVDV) diagnostic kit comprising the above antibody or a fragment thereof.

[0059] There is no limitation on the type of the above diagnostic or detection kit. In the case of a kit for detecting bovine viral diarrhea virus by enzyme immunoassay, it may include an antibody immobilized on a solid phase, an antibody conjugated to an enzyme, a sample dilution solution, an enzyme substrate solution, a washing solution, and an antigen standard solution. For example, but not limited to, the enzyme may be horseradish peroxidase, and the enzyme substrate solution may be an ortho-phenylenediamine (OPD) solution. In addition, the sample dilution solution may be a dilution solution of human or livestock feces.

[0060] There is no limitation on the form of the above kit, and it may be in the form of, for example, a strip, a cassette, a stick, etc.

[0061] The term "detection" in this specification refers to the detection of antigen-antibody complexes, using various markers. Specific examples include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, radioisotopes, metal nanoparticles, lanthanide elements, Raman reporters, electrochemical tags, and magnetic particles. However, the present invention is not limited to these. Preferably, the antigen-antibody complex can be detected using an enzyme-linked immunosorbent assay (ELISA). Enzyme-linked immunosorbent assay (ELISA) includes various ELISA methods, such as direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize the antigen attached to the solid support, direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of antibodies and antigen attached to the solid support, and indirect sandwich ELISA using a labeled secondary antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in a complex of antibodies and antigen attached to the solid support. The monoclonal antibodies may have a detection label, and if they do not have a detection label, these monoclonal antibodies can be captured and identified by treating them with another antibody that has a detection label.

[0062]

[0063] In addition, the present invention provides a method for diagnosing bovine viral diarrhea virus (BVDV) using the diagnostic kit.

[0064] The above diagnostic method may include, but is not limited to, a step of isolating a biological sample from an individual; a step of processing the separated sample with the above kit; and a step of testing the reactivity of the kit.

[0065] The term “biological sample” as used herein includes, but is not limited to, feces, tissues, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), urine, cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems. By reacting these biological samples with or without manipulation with the kit of the present invention, the presence of bovine diarrhea pathogen proteins and the presence or absence of bovine diarrhea can be confirmed.

[0066] Assay systems for use in the detection method and diagnostic kit of the present invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatographic test strips, radial fractionation immunoassay devices, and flow-through devices.

[0067]

[0068] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0069]

[0070] <Example 1> Cloning of antigen protein genes of bovine diarrhea pathogen

[0071] To effectively diagnose the pathogen causing bovine diarrhea, we conducted various literature reviews and selected the optimal target protein for each pathogen based on the results. Among the diarrheal pathogens, the BVDV nucleocapsid protein (BVDV-CP) was selected primarily for the diagnosis of BVDV. In addition, for the specific selective diagnosis of BVDV1 and BVDV2, we compared and analyzed the genomes of approximately 40 BDVD1 and BVDV2 using previously reported database analyses, and selected the region with the lowest homology and thus highest variability. The genetic information of these target proteins was obtained by analyzing the genetic database (NCBI, MD, USA) to obtain the genetic information of the pathogen viruses. Based on the obtained genetic information, the gene base sequence for cloning the recombinant antigen protein gene of the pathogen target protein was designed.

[0072] In order to produce BVDV-CP protein and BVDV1-E2 protein, and recombinant protein of BVDV1-E2 protein, amino acid information of BVDV1-CP, BVDV1-E2, and BVDV2-E2 proteins (UEE94259, MW054940, AF145967) was obtained from NCBI, and then the gene sequence was newly designed by optimizing the amino acid codon usage to be suitable for the production of recombinant proteins. Based on this optimized genetic information, genes for production of each BVDV recombinant protein were selected through gene synthesis, and then inserted into an E. coli expression vector to select genes for production of BVDV recombinant proteins (SEQ ID NOs: 1, 3, and 5).

[0073]

[0074] <Example 2> Production and purification of pathogen recombinant antigen proteins

[0075] In Example 1, the genes of BVDV1-CP (SEQ ID NO: 1), BVDV1-E2 (SEQ ID NO: 3), and BVDV2-E2 (SEQ ID NO: 5) selected were inserted into a protein production vector of E. coli through restriction enzyme treatment and ligation reaction. All recombinant proteins were designed to have a 6X His moiety linked to the C-terminus of all recombinant proteins considering the characteristics and purification of the proteins. In addition, a Thioredoxi tag sequence was inserted into the N-terminal region of the BVDV-CP recombinant protein for effective expression and selection of recombinant proteins. The selected BVDV1-CP gene was treated with EcoRI and XhoI restriction enzymes, and then inserted into the pET32a expression vector treated with the same restriction enzymes, EcoRI and XhoI, through T4 ligase (NEB co.) (Fig. 1).

[0076] Meanwhile, the BVDV1-E2 and BVDV2-E2 genes were isolated by treatment with Nde I and Xho I restriction enzymes, and then inserted into pET21b treated with the same restriction enzymes using T4 ligase. Based on the pET21b vector, a vector plasmid was constructed to stably express the protein by linking 6XHis to the C-terminus of the recombinant protein, effectively expressing and purifying the BVDV-E protein (Fig. 2).

[0077] The plasmid DNAs of pET-BVDV-CP, pET-BVDV1-E2, and pET-BVDV2-E2, expression vectors containing recombinant genes of bovine diarrhea pathogens, were transformed into protein-expressing Escherichia coli BL21 using the heat shock method. The transformed Escherichia coli were plated on LB solid medium treated with 100 ug / ml of antibiotics, and single colonies transformed with the plasmids were selected.

[0078] A single colony of an E. coli strain transformed with the bovine diarrhea pathogen genome was inoculated into LB liquid medium treated with the appropriate antibiotic. The cultures were grown overnight at 37°C with shaking, and then treated with 0.5 mM IPTG to induce expression of the recombinant proteins. The protein expression patterns were analyzed by SDS-PAGE analysis. Inoculation conditions, IPTG concentrations, incubation times, and temperatures were optimized for each recombinant protein, and the optimal recombinant protein expression conditions were established. The recombinant proteins BVDV-CP, BVDV1-E2, and BVDV2-E2 expressed under the optimal conditions were finally purified through affinity chromatography using NiKel and provided as antigen proteins.

[0079] For the purification of each recombinant protein, the E. coli cells were cultured under optimal conditions to induce the expression of the recombinant protein, and then centrifuged to recover the cells. The E. coli pellets were resuspended in 1X PBS (pH 7.4), and then the E. coli cells were disrupted using an ultrasonicator to elute the recombinant proteins. Only the solutions with eluted recombinant proteins were recovered through centrifugation. These solutions were injected into a Nikel ion resin column pre-equilibrated with 1X PBS (pH 7.4) to allow the recombinant proteins to bind to Nikel. After washing once with the same 1X PBS (pH 7.4) solution and three times with 1X PBS (pH 7.4) solution containing 30 mM Imidazole, the recombinant proteins were eluted using 1X PBS (pH 7.4) solution containing 250 mM Imidazole. The eluted solution was analyzed by SDS-PAGE to confirm the presence of recombinant proteins, and then dialyzed against 1X PBS (pH 7.4) to remove imidazole in the solution. Each finally purified recombinant protein was concentrated and quantified using Vivaspin Tubo 15 (Sartorius Co.) and then provided as an antigen for antibody protein production.

[0080] The BVDV-CP recombinant protein produced a recombinant protein of approximately 10 kDa with 105 amino acids (SEQ ID NO: 2), but included a thioredoxin tag, a specific linker, and additional bases for stable expression for stable expression to ensure stable expression and purification of the recombinant protein. As a result, the BVDV-CP recombinant protein produced a Trx-BVDV-CP recombinant protein with a total size of 33 kDa, including a 19 kDa thioredoxin tag (Fig. 1A). As shown in Fig. 1, it was confirmed that a 33 kDa protein was overexpressed in the lysate of E. coli cells expressing BVDV-CP, and it was shown that the BCV-NP recombinant protein was well purified through affinity chromatography using a Nikel resin column (Fig. 1B).

[0081] Meanwhile, the approximately 22 kDa BVDV1-E2 (SEQ ID NO: 4) and BVDV2-E2 (SEQ ID NO: 6) recombinant proteins consisting of 202 amino acids were produced by linking a 6XHis fragment to the C-terminus to produce a approximately 23 kDa recombinant protein for improved purification efficiency. As shown in Figures 2B and 2C, the two His-linked BVDV-E2 recombinant proteins were highly expressed in the form of inclusion bodies around 23 kDa. These proteins were eluted using an 8 M urea (pH 8.0) solution and then purified through a Nikel resin column equilibrated with the same 8 M urea (pH 8.0) solution. These purified recombinant proteins were dialyzed sequentially against 6 M urea (pH 8.0), 4 M urea (pH 8.0), 2 M urea (pH 8.0), and 1 M urea (pH 8.0) solutions to refold the proteins, and then concentrated and quantified for use as antigen proteins. As shown in Figures 2B and 2C, both BVDV1-E2 and BVDV2-E2 recombinant proteins were well purified to a size of approximately 23 kDa and used for antibody protein production.

[0082]

[0083] <Example 3> Production of antibody proteins for diagnosis of bovine diarrhea pathogens

[0084] For the production of monoclonal antibodies for the diagnosis of bovine diarrhea pathogens, the recombinant proteins expressed and purified above, such as BVDV-CP, BVDV1-E2, and BVDV2-E2, were used as antigen proteins.

[0085] To produce antibodies, the above antigen proteins were diluted to a concentration of 1 mg / ml and mixed with Freund's complete adjuvant (FCA, Sigma) to prepare an emulsion, which was then injected into the foot pad of BALB / c mice to induce immunization. The immunity was increased by administering an additional three times at two-week intervals with an emulsion containing the antigen proteins and Freund's incomplete adjuvant (FIA, Sigma).

[0086] Meanwhile, after the final completion of immunization for monoclonal antibody production, the scapular and popliteal lymph nodes were extracted from the immunized mice, and lymph node cells were recovered. The recovered lymph node cells were mixed with the SP2 / 0 myeloma cell line, which is widely used in monoclonal antibody development, and PEG 1500 (Invitrogen) to induce cell fusion. The fused cell solution was suspended in a medium containing HAT (hypoxanthine, aminopterin, thymidine), dispensed into 96-well plates (SPL), and cultured in a CO2 incubator.

[0087] To select monoclonal antibody-producing cell lines from the fused cell lines, fusion cells were screened using an enzyme-linked immunosorbent assay (ELISA). Each recombinant antigen protein was diluted in 50 mM sodium bicarbonate buffer (pH 9.6) to a concentration of 1 μg / ml, and 50 μl was dispensed into a 96-well immune plate (SPL) and incubated at 4°C for 16 hours. Each plate was washed three times with PBS-T (1X PBS-0.05% Tween-20) solution, and 100 μl of the fused cell line culture medium was added to each well and incubated for 1 hour at 37°C. After washing three times with the same PBS-T solution as above, 50 μl of a diluted anti-mouse IgG antibody (Fitzerald) conjugated with HRP (horseradish peroxidase) was added to each well. After reacting at 37℃ for 1 hour, the mixture was washed three times in the same manner as above, and TMB solution, an HRP substrate solution, was added. After adding TMB solution, the mixture was reacted at room temperature for 5 to 10 minutes, and then 0.5 M H2SO4 solution was added to complete the reaction. The ELISA reaction results were analyzed by measuring the absorbance at a wavelength of 250 nm using an ELISA reader. Cell lines that produce monoclonal antibodies show relatively strong absorbance values ​​in the ELISA analysis. Primary cell lines that produce monoclonal antibodies were selected through these ELISA analyses, and secondary ELISA analyses were performed for each antigen concentration, and finally, cell lines that produce monoclonal antibodies against each pathogen were selected.

[0088] As can be seen in Figure 3 below, 48 cell lines were finally selected as single antibody cell lines for BVDV-CP, 46 cell lines for BVDV1-E2, and 12 cell lines for BVDV1-E2, resulting in the final selection of 106 monoclonal cell lines.

[0089] The monoclonal antibody-producing cell lines selected above were sequentially cultured in T-25, T-75, and T-175 flasks to proliferate the cultured cell lines, and then the antibody-producing cell lines and the culture medium were separated and recovered. The recovered culture medium was concentrated to concentrate the protein through amonium sulfate precipitation, and the antibody protein was purified through affinity chromatography using Protein A / G-conjugated cellulose beads and eluted with elution buffer (Glycine buffer, pH 3.0). Each eluted antibody protein was neutralized by adding neutralization buffer (Tris-HCL, pH 9.0), dialyzed against 1X PBS buffer, quantified, and stored. Meanwhile, the recovered cell lines were cryopreserved in liquid nitrogen, and some were injected back into the peritoneal cavity of mice to produce monoclonal antibody-producing ascites, and each monoclonal antibody was purified from these ascites according to the same procedure as the antibody purification method described above.

[0090]

[0091] <Example 4> Selection of antibody pairs for diagnosis of bovine diarrhea pathogens

[0092] We performed a screening of optimal antibody pairs for the development of a multi-diagnostic kit for bovine diarrhea pathogens. Using purified monoclonal antibody proteins produced for each pathogen, the optimal antibody pairs for each pathogen antigen protein were selected through a half-test based on a lateral flow immunoassay liquid test. Goat anti-mouse IgG was dispensed onto the control line of a nitrocellolose membrane (Nupore, 10 μm), and each antibody was dispensed onto the test line. The membrane was then dried overnight. Adsorbent pads were attached to the dried nitrocelolose membrane and cut to 4 mm lengths to produce strips for pair testing. After injecting 5 μl of each antibody conjugated to gold nanoparticles into a 96-well plate, each recombinant antigen protein diluted to a concentration of 100 ng / ml was injected. The strips for the pair test were placed in each test well to initiate the reaction. After 10 minutes of reaction, the detection signal intensities of the C and T lines on the nitrocellulose membrane were compared, and antibody pairs with high T line generation intensity were first selected. Through the first antibody pair selection, 23 antibody pairs were selected, including A3E1, which reacts with BVDV-CP, BDVD1-E2, and BVDV2-E2, as shown in Fig. 6B below.

[0093] For polymer pads manufactured in a dry state, signal intensity may decrease or other non-specific problems may arise compared to liquid-based half-tests. Therefore, to find the optimal antibody pair for each pathogen, a secondary antibody pair selection using a full strip was performed centered on each pathogen-antibody pair selected through the first selection. First, goat anti-mouse IgG and the selected antibody pair were dispensed onto the C and T lines of a nitrocellulose membrane as in the half-test method, and then dried. Another antibody pair conjugated to gold nanoparticles was dispensed onto the gold nanoparticle polymer pad and then dried. The polymer pad was attached to the dried nitrocellulose membrane, and an absorption pad and a sample supply pad were attached, and then cut to 0.6 mm to produce a full strip. Each antigen protein was diluted in assay buffer (1X PBS, 0.1% triton X100) prepared at high and low concentrations, dispensed onto each Strip, reacted for 10 minutes, and the results were compared and analyzed with the results of the assay buffer, which was a negative control. Through secondary antibody pair selection, the optimal antibody pair with strong reactivity and no nonspecific reactivity was selected, and as shown in Figure 3C, three pairs of antibodies, A3E1, Y5A5, 6F12, A5H6, 14H3, and 4B33, were selected as the optimal antibody pairs for diagnosing BVDV infection (Figure 3C).

[0094]

[0095] <Example 5> Production of an immune strip for diagnosing bovine diarrhea pathogens

[0096] Prior to developing a multi-diagnostic kit for bovine diarrhea pathogens, optimal gold nanoparticle polymer formation conditions were established for each antibody pair selected above. First, using 40 nm gold nanoparticles and 10 mM PB, the color change and aggregation of the antibody and gold nanoparticle polymer were analyzed to establish optimal polymer conditions for each antibody.

[0097] In addition, to establish the optimal analytical solution conditions for effectively diagnosing novel pathogens in specimens such as feces, changes in salt concentration, pH conditions, and surfactant conditions of the analytical solution were analyzed. As a result, a phosphate solution (1X PBS, 0.1% Trinton-X100, pH 7.4) containing neutral pH conditions and low concentrations of surfactant was selected as the optimal analytical solution.

[0098] Meanwhile, a diagnostic strip capable of simultaneously diagnosing BVDV1 and BVDV2, the serotypes of the bovine diarrhea virus pathogen, was manufactured. In addition, as in the above-mentioned diagnostic kit, goat anti-mouse IgG and selected capture antibody pairs were dispensed onto the C and T lines of a nitrocellulose membrane for each BVDV serotype, respectively, and then dried to produce a detection membrane. According to the above-mentioned conditions, a gold nanoparticle polymer of an antibody pair conjugated to gold nanoparticles was dispensed onto a polymer pad, and then dried to produce a polymer pad. The polymer pad was attached to the dried nitrocellulose membrane, and an absorbent pad and a sample supply pad were attached, and then cut to 0.6 mm to complete the specimen pad. By inserting each of these pathogen pads into each of the above-mentioned quadruple strips and assembling the strips, the manufacture of a diagnostic kit capable of simultaneously diagnosing BVDV1 and BVDV2, the two serotypes of the bovine diarrhea virus pathogen, was completed.

[0099]

[0100] <Example 6> Test method for multiple test kits for bovine diarrhea pathogens

[0101] The testing method involves collecting feces with a cotton swab for sampling, placing it in a sample bottle containing a specimen analysis solution, and swirling the swab to extract the feces. After that, the sample bottle is left standing for about 1 minute until large particles settle, and then 3 drops of the supernatant are dropped into the drip holes of 4 strips. The results are read after waiting for 10 minutes. The test results are read as negative if only one band appears at the control line (C), and positive if two bands form at the control line location (C) and the test line location (T). As shown in Figure 4 below, the pathogen causing bovine diarrhea was identified by confirming the type of strip that was found to be positive.

[0102]

[0103] Example 7. Specificity and performance test of a diagnostic kit for bovine diarrhea pathogens.

[0104] To test the sensitivity and specificity of diagnosing bovine diarrhea pathogens, lateral flow assay kits for each pathogen were developed using the optimal antibody pairs selected above, and nonspecific reactions and cross-reactivity using each recombinant antigen protein and other proteins were analyzed. In Test 1, for BVDV2 diagnosis, an LFA kit was developed using A3E1 or 6F12 as a capture antibody and Y5A5 or A5H6 as a gold nano-polymerized antibody, and in Test 2, for BVDV1 diagnosis, an LFA kit was developed using 14H3 as a capture antibody and 4B33 as a gold nano-polymerized antibody. The detection performance for each antigen protein was analyzed using each diagnostic kit. As shown in Figure 4, the specificity of the developed BVDV diagnostic kit was investigated using Canine Corona Virus (CCV), Bovine Corona Virus (BCoV), Bovine Rota Virus (BRV), and BVDV1 Virus extracts and BVDV2 virus extracts as antigens. As a result, there was no reaction to other antigen virus extracts and reactivity was shown only to BVDV1 and BVDV2. Meanwhile, in the case of BVDV1 virus extract, it reacted to both test 1 and test 2, but BVDV2 virus extract only reacted to test 1 for BVDV2 detection (Fig. 4, Fig. 5A). Meanwhile, in order to compare the performance of the developed BVDV diagnostic kit, the same reactivity was compared and analyzed using the BVDV diagnostic kit developed by Company A. In the case of the diagnostic kit of the present invention, both BVDV1 and BVDV2 were distinguished and diagnosed, whereas the product of the other company showed reactivity only to BVDV1 and could not detect BVDV2 (Fig. 5B). In summary, when these results are combined, it seems that the diagnostic kit using these antibody proteins can conveniently and effectively quickly distinguish and diagnose BVDV1 and BVDV2, which are novel coronaviruses.

Claims

1. A recombinant protein comprising at least one antigen of bovine viral diarrhea virus (BVDV) selected from the group consisting of BVDV nucleocapsid protein (BVDV-CP), BVDV1 envelope glycoprotein E2 (BVDV1-E), and BVDV2 envelope glycoprotein E2 (BVDV2-E).

2. In paragraph 1, The above BVDV-CP is a recombinant protein comprising the amino acid sequence of sequence number 2.

3. In paragraph 1, The above BVDV1-E is a recombinant protein comprising the amino acid sequence of sequence number 4.

4. In paragraph 1, The above BVDV2-E is a recombinant protein comprising the amino acid sequence of sequence number 6.

5. An expression vector comprising a gene encoding the recombinant protein of paragraph 1.

6. A transformant transformed with the expression vector of Article 5.

7. A vaccine composition against bovine viral diarrhea virus (BVDV), comprising at least one selected from the group consisting of the recombinant protein of claim 1, the expression vector of claim 5, the transformant of claim 6, and a combination thereof.

8. An antibody or fragment thereof that specifically binds to a recombinant protein of paragraph 1 or a protein produced by a transformant of paragraph 6.

9. An antibody or a fragment thereof comprising a recombinant protein as an antigen, wherein the antibody is a monoclonal or polyclonal antibody.

10. A diagnostic kit for bovine viral diarrhea virus (BVDV), comprising the antibody of Article 8 or a fragment thereof.

11. A diagnostic kit according to claim 10, wherein the kit distinguishes between BVDV1 and BVDV2.

12. A method for diagnosing bovine viral diarrhea virus (BVDV) using the diagnostic kit of Article 10.

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