KR20200122467A - Monoclonal Antibody specific for Canine Parvovirus and use thereof - Google Patents

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof which specifically binds to canine parvovirus, and a composition for diagnosing canine parvovirus infection comprising the antibody or antigen-binding fragment thereof. By using the antibody of the present invention, it is possible to quickly and accurately determine whether an object is infected with canine parvovirus.

Description

Monoclonal Antibody specific for Canine Parvovirus and Use thereof {Monoclonal Antibody specific for Canine Parvovirus and Use thereof}

The present invention relates to monoclonal antibodies specific for canine parvovirus and uses thereof.

Canine Parvovirus (CPV) infection is an infectious disease that has been reported worldwide since 1987. In Korea, it was confirmed that the first epidemic in Gyeonggi Province in the 1980s was confirmed, followed by national outbreaks. Clinically and pathologically, it can be largely divided into two types: heart type, which is mainly seen in young puppies aged 3-8 weeks, and enteritis type, which is seen in dogs after weaning. Severe vomiting, hemorrhagic diarrhea, dehydration, leukopenia are the main symptoms of enteritis, and mortality is high.

The clinical symptoms and pathologic findings are similar to Feline Panleukopenia (FPL), and the treatment for this is known as a popular therapy. Heart type is characterized by non-purulent myocarditis centered on the left ventricle, and generally progresses acutely, resulting in high mortality and poor prognosis.

Parvovirus is infected by mouth through diarrhea of an infected dog. In the case of acute type, a huge amount of virus is excreted through feces for 1-2 weeks after infection, and the excreted virus survives for several months in a normal living environment and remains pathogenic. do.

CPV is described as "CPV-2" to distinguish it from parvovirus canine minit virus (CMV or CPV-1). CPV-2 is generally thought to be a genetic variant of feline panleukopenia virus (FPV) or mink enteric virus (MEV), and parvo infects mink, fox, raccoon and other predators. It is a very close relationship with viruses in terms of genes and antigens. The open parvovirus capsid contains a single-stranded DNA genome consisting of approximately 5,200 bases with only two open reading frames (ORFs), although four or more proteins are encoded due to alternative mRNA splicing.

The capsid of parvovirus is composed of two types of virus proteins (VP) VP1 and VP2, of which VP2 is the main immunogenic parvovirus capsid protein. Genetic variants for the original canine parvovirus isolate were identified and named as canine parvovirus type 2a, another variant, type 2b, was named, and recently 2c was identified.

Vaccination is the best way to prevent canine parvovirus infection. An attenuated canine parvovirus vaccine can sustain immunity for a long time and is effective and ideal, but caution should be exercised as the effect of the vaccine may be halved under the influence of the maternal antibody.

Currently, the canine parvovirus vaccine, which is mainly used in Korea, is a live tissue-purifying vaccine and is marketed in the form of a combination of five vaccines such as canine distemper, canine infectious hepatitis, parainfluenza and leptospira.

The present inventors have made efforts to develop an accurate and simple method for diagnosing canine parvovirus infection that can improve and replace the problems of the currently used canine parvovirus infection diagnosis method. As a result, the present invention was completed by developing a monoclonal antibody capable of rapidly and accurately detecting the presence of canine parvovirus infection.

Accordingly, an object of the present invention is to provide an antibody or antigen-binding fragment thereof that specifically binds to canine parvovirus.

Another object of the present invention is to provide a recombinant vector comprising a polynucleotide encoding a light chain variable region and a polynucleotide encoding a heavy chain variable region of an antibody that specifically binds to canine parvovirus.

Another object of the present invention is to provide a host cell transformed with the recombinant vector.

Another object of the present invention is to provide a composition for diagnosing canine parvovirus infection.

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to Canine Parvovirus (CPV), and uses thereof.

Hereinafter, the present invention will be described in more detail.

Canine Parvovirus (CPV) is an intestinal pathogen that infects most dogs, especially puppies. Canine parvovirus infection is characterized by acute diarrhea, fever and leukopenia in dogs and puppies 4 to 5 weeks old, and cardiomyopathy in younger puppies. In unvaccinated dogs, the mortality rate from this disease is very high. Vaccines against canine parvovirus are available, but since canine parvovirus is a single-stranded DNA virus and has a very high mutagenicity, the virus exhibits a significant modification ability in terms of antigens, and therefore, immune protection is not achieved by vaccines.

Since the canine parvovirus virus can be mutated and mutated into a mutant with new antigenicity, it is necessary to develop a vaccine and diagnostic test that reflects the current canine parvovirus variant by examining the gene composition of the canine parvovirus variant.

One aspect of the present invention is a heavy chain variable region comprising at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4, SEQ ID NO: 5, and sequence It relates to an antibody or antigen-binding fragment thereof that specifically binds to canine parvovirus comprising a light chain variable region comprising at least one light chain CDR amino acid sequence selected from the group consisting of number 6.

The antibody or antigen-binding fragment thereof is CDR1 (complementarity determining region 1) consisting of the amino acid sequence represented by SEQ ID NO: 1, CDR2 (complementarity determining region 2) consisting of the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NO: 3. A heavy chain variable region comprising a CDR3 (complementarity determining region 3) consisting of the amino acid sequence shown; And

It may include a light chain variable region including CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6. .

In addition, the heavy chain variable region may be composed of the amino acid sequence of SEQ ID NO: 7, and the light chain variable region may be composed of the amino acid sequence of SEQ ID NO: 8.

Another aspect of the present invention is a heavy chain variable region comprising at least one heavy chain complementarity determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 14, SEQ ID NO: 15, and It relates to an antibody or antigen-binding fragment thereof that specifically binds to canine parvovirus comprising a light chain variable region comprising at least one light chain CDR amino acid sequence selected from the group consisting of SEQ ID NO: 16.

The antibody or antigen-binding fragment thereof is a heavy chain comprising CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 11, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 12, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 13 Variable region; And

It may include a light chain variable region including CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 14, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 15, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 16. .

In addition, the heavy chain variable region may be composed of the amino acid sequence of SEQ ID NO: 17, and the light chain variable region may be composed of the amino acid sequence of SEQ ID NO: 18.

As used herein, the term "antibody" refers to a specific antibody against canine parvovirus, which specifically binds to canine parvovirus, and includes antigen-binding fragments of antibody molecules as well as complete antibody forms.

A complete antibody has two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and subclasses It has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

The antibody may be a monoclonal antibody.

As used herein, the term "monoclonal antibody" means that the antibody molecule is prepared to consist of a single molecule. Monoclonal antibodies have specificity to react only to antigens having the same epitope, and also exhibit affinity only to specific epitopes.

As used herein, the term "heavy chain" refers to a full length comprising a variable region domain VH and three constant region domains CH1, CH2 and CH3 comprising an amino acid sequence having a sufficient variable region sequence to confer antigen specificity. It is interpreted to include both heavy chains and fragments thereof.

As used herein, the term "light chain" refers to both a full-length light chain including a variable region domain VL and a constant region domain CL comprising an amino acid sequence having a sufficient variable region sequence to confer antigen specificity and fragments thereof. It is interpreted to mean including.

As used herein, the term "complementarity determining region (CDR)" refers to an amino acid sequence of a hypervariable region of a heavy and light chain of an immunoglobulin. Each of the heavy and light chains may comprise three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs can provide key contact residues for the antibody to bind to an antigen or epitope.

As used herein, the term "antigen-binding fragment" refers to a fragment thereof for the entire structure of an immunoglobulin, and refers to a portion of a polypeptide including a portion to which an antigen can bind. For example, it may be F(ab') ₂ , Fab', Fab, Fv, or scFv, but is not limited thereto. Among the antigen-binding fragments, Fab has a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region of a heavy chain (C _H1 ), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing at least one cysteine residue at the C-terminus of the heavy chain C _H1 domain. F(ab') ₂ antibodies are produced by disulfide bonds between cysteine residues in the hinge region of Fab'. Fv is the smallest antibody fragment having only the heavy chain variable region and the light chain variable region. Recombination techniques for generating Fv fragments are well known in the art. The double-chain Fv (two-chain Fv) is a non-covalent bond where the heavy chain variable region and the light chain variable region are connected, and the single-chain Fv (single-chain Fv) is generally shared by the variable region of the heavy chain and the variable region of the single chain through a peptide linker. It is linked by a bond or is directly linked at the C-terminus to form a dimer-like structure such as a double-chain Fv. The antigen-binding fragment can be obtained using proteolytic enzymes (e.g., Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab') ₂ fragment can be obtained by digestion with pepsin), It can be produced through gene recombination technology.

The antibodies include monoclonal antibodies, bispecific antibodies, non-human antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-binding Fvs (sdFV) and anti- Idiotype (anti-Id) antibodies, and epitope-binding fragments of the antibodies are included, but are not limited thereto.

As used herein, the term "chimeric" means that the antibody or antigen-binding site includes sequences derived from two different species.

The antibody or antigen-binding fragment thereof that specifically binds to the canine parvovirus may include a variant of the amino acid sequence described in the attached SEQ ID NO: within a range that can specifically recognize the canine parvovirus.

For example, changes can be made to the amino acid sequence of an antibody to improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody. These amino acid variations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. For example, arginine, lysine and histidine are all positively charged residues, alanine, glycine and serine have similar sizes, and phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on the above considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine may be referred to as biologically functional equivalents.

On the other hand, amino acid exchange in proteins that do not change the activity of the molecule as a whole is known in the art. The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

If the mutation having the biologically equivalent activity is considered, the antibody or antigen-binding fragment thereof that specifically binds to the canine parvovirus may be interpreted as including a sequence showing substantial identity to the sequence described in SEQ ID NO: have.

The actual identity is at least 60% when the amino acid sequence of the above sequence number and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It may be a sequence showing homology of, at least 70%, at least 80%, or at least 90% of homology.

Alignment methods for comparison of sequences are known in the art. For example, through the NCBI Basic Local Alignment Search Tool (BLAST), sequence analysis programs such as blastp, blastx, tblastn and tblastx can be used on the Internet.

Another aspect of the present invention relates to a polynucleotide encoding a heavy chain variable region and/or a light chain variable region of an antibody that specifically binds to canine parvovirus.

According to one embodiment of the present invention, the polynucleotide encoding the heavy chain variable region may be a polynucleotide encoding the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 of an antibody that specifically binds to canine parvovirus. .

The polynucleotide encoding the heavy chain variable region may be composed of the nucleotide sequence of SEQ ID NO: 9.

According to an embodiment of the present invention, the polynucleotide encoding the light chain variable region may be a polynucleotide encoding the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8 of an antibody that specifically binds to canine parvovirus. .

The polynucleotide encoding the light chain variable region may consist of the nucleotide sequence of SEQ ID NO: 10.

According to another embodiment of the present invention, the polynucleotide encoding the heavy chain variable region may be a polynucleotide encoding the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17 of an antibody specifically binding to canine parvovirus. have.

The polynucleotide encoding the heavy chain variable region may consist of the nucleotide sequence of SEQ ID NO: 19.

According to another embodiment of the present invention, the polynucleotide encoding the light chain variable region may be a polynucleotide encoding the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 18 of an antibody that specifically binds to canine parvovirus. have.

The polynucleotide encoding the light chain variable region may be composed of the nucleotide sequence of SEQ ID NO: 20.

As used herein, the term "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides present in a single-stranded or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and unless specifically stated otherwise, includes analogs of natural polynucleotides.

The polynucleotide includes not only a nucleotide sequence encoding an amino acid sequence of a heavy chain variable region or a light chain variable region of an antibody specifically binding to the canine parvovirus, but also a sequence complementary to the sequence.

The complementary sequence includes not only a perfectly complementary sequence, but also a substantially complementary sequence, which under stringent conditions known in the art, for example, specifically binding to the canine parvovirus. It means a sequence capable of hybridizing with a nucleotide sequence of a nucleotide sequence encoding an amino acid sequence of a heavy chain variable region or a light chain variable region of an antibody.

In addition, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region and/or light chain variable region may be modified. Such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides.

The polynucleotide encoding the amino acid sequence of the heavy chain variable region and/or the light chain variable region of the antibody that specifically binds to the canine parvovirus is interpreted as including a nucleotide sequence showing substantial identity to the nucleotide sequence.

The actual identity is at least 80% homology when the nucleotide sequence and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art, It may be a sequence showing at least 90% homology or at least 95% homology.

Another aspect of the present invention relates to a recombinant vector comprising a polynucleotide encoding a heavy chain variable region and a polynucleotide encoding a light chain variable region of an antibody that specifically binds to canine parvovirus.

The heavy chain variable region may be a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7, and the light chain variable region may be a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

The polynucleotide encoding the heavy chain variable region may be composed of the nucleotide sequence of SEQ ID NO: 9, and the polynucleotide encoding the light chain variable region may be composed of the nucleotide sequence of SEQ ID NO: 10.

In addition, the heavy chain variable region may be a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17, and the light chain variable region may be a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 18.

The polynucleotide encoding the heavy chain variable region may be composed of the nucleotide sequence of SEQ ID NO: 19, and the polynucleotide encoding the light chain variable region may be composed of the nucleotide sequence of SEQ ID NO: 20.

In the present specification, the term "vector" means a means for expressing a gene of interest in a host cell. For example, plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retroviral vectors, and viral vectors such as adeno-associated viral vectors. Vectors that can be used as the recombinant vector are plasmids often used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series, and pUC19, etc.), phage (for example, λgt4λB, λ-Charon, λΔz1 and M13, etc.), or a virus (for example, SV40, etc.).

In the recombinant vector, the polynucleotide encoding the amino acid sequence of the heavy chain variable region and the light chain variable region may be operably linked to a promoter. The term “operatively linked” refers to a functional linkage between a nucleotide expression control sequence (eg, a promoter sequence) and another nucleotide sequence. Thus, thereby the regulatory sequence can regulate the transcription and/or translation of the other nucleotide sequence.

The recombinant vector can typically be constructed as a vector for cloning or as a vector for expression. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms. The recombinant vector can be constructed through various methods known in the art.

The recombinant vector can be constructed using prokaryotic or eukaryotic cells as a host. For example, the vector used is an expression vector, and when prokaryotic cells are used as a host, a strong promoter capable of progressing transcription (eg, p _L ^λ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), It is common to include a ribosome binding site and a transcription/translation termination sequence for initiation of translation. In the case of eukaryotic cells as a host, the origin of replication operating in eukaryotic cells included in the vector includes the f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adeno origin of replication, AAV origin of replication and BBV origin of replication, etc. It is not limited.

In addition, a promoter derived from the genome of mammalian cells (eg, metallotionine promoter) or a promoter derived from mammalian virus (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter) And the tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

Meanwhile, the vector capable of expressing the heavy and light chain variable regions of the antibody is a vector system in which the heavy chain variable region and the light chain variable region are simultaneously expressed in one vector, or the heavy chain variable region and the light chain variable region are each separate vector Any system expressed in is possible. In the latter case, the two vectors can be introduced into host cells through co-transformation and targeted transformation.

Another aspect of the present invention relates to a host cell transformed with the recombinant vector.

Any host cell known in the art may be used as a host cell capable of stably and continuously cloning or expressing the recombinant vector, and as prokaryotic cells, for example, E. coli JM109, E. coli BL21, E. strains of the genus Bacillus such as coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringensis, and Salmonella typhimurium, Serratia marsessons, and There are enterobacteriaceae and strains such as various Pseudomonas species, and when transforming into eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells, plant cells and animal cells, such as Sp2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines and the like can be used.

Transport of the polynucleotide or a recombinant vector containing the same into a host cell may be performed using a transport method well known in the art. For example, when the host cell is a prokaryotic cell, a CaCl ₂ method or an electroporation method may be used, and when the host cell is a eukaryotic cell, a microinjection method, a calcium phosphate precipitation method, an electroporation method, and a liposome -Mediated transfection method and gene bombardment may be used, but are not limited thereto.

When using microorganisms such as E. coli , the productivity is higher than that of animal cells, but it is not suitable for the production of intact Ig antibodies due to glycosylation problems, but antigen-binding fragments such as Fab and Fv Can be used in the production of

The method of selecting the transformed host cells can be easily carried out according to a method well known in the art using a phenotype expressed by a selection label. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

Another aspect of the present invention is the step of culturing the host cell; And recovering the antibody or antigen-binding fragment thereof from the cultured cells. It relates to a method of producing an antibody or antigen-binding fragment thereof that specifically binds to canine parvovirus.

The cells may be cultured in a medium, and a commercially available medium or the like may be used as a culture medium without any limitation.

Other known essential supplements may be included in suitable concentrations. Culture conditions, such as temperature, pH, etc., are already known for cells selected for expression, and can be appropriately selected by a person skilled in the art.

The antibody or antigen-binding fragment thereof may be recovered by removing impurities by, for example, centrifugation or ultrafiltration, and the resultant may be purified through, for example, affinity chromatography. Additional purification techniques can be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and the like.

Another aspect of the present invention relates to a composition for diagnosing canine parvovirus infection comprising the antibody or antigen-binding fragment thereof.

As described above, the antibody or antigen-binding fragment thereof of the present invention exhibits specific immunoreactivity to canine parvovirus and can easily detect canine parvovirus. Thus, when applied as an antibody of a virus diagnostic reagent, infection of canine parvovirus It can be a kit that can diagnose effectively.

The composition for diagnosing canine parvovirus infection of the present invention may further include additives that may be included in a conventional virus detection composition, such as an auxiliary substance for maintaining antibody activity and confirming antigen-antibody binding.

In the composition for diagnosing canine parvovirus infection of the present invention, the presence or absence of a virus is confirmed from a biological sample using an antibody such as a tool for collecting a sample of a test object or applying a diagnostic reagent, a reagent or device for confirming antigen-antibody binding. Tools, devices, or reagents that may be included in a conventional diagnostic composition may be further included.

Tools/reagents that may be included in the diagnostic composition include, but are not limited to, a suitable carrier, a labeling substance capable of generating a detectable signal, a solubilizing agent, a detergent, a buffering agent, and a stabilizer. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymers such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

Another aspect of the present invention relates to a method for providing information necessary for diagnosis of canine parvovirus infection, comprising the step of detecting an antigenic protein of canine parvovirus from a biological sample of a dog using the antibody or antigen-binding fragment thereof. will be.

The antigenic protein detection of the canine parvovirus may be performed through an antigen-antibody reaction.

The antigen-antibody reaction includes tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), western blotting, immunoprecipitation assay (Immunoprecipitation Assay), immunodiffusion assay (Immunodiffusion Assay), complement fixation assay. One or more methods selected from the group consisting of (Complement Fixation Assay), fluorescence-activated cell sorter (FACS), and protein chip analysis can be used, for example, carried out using enzyme immunoassay (ELISA). can do.

The biological sample may be dog tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, and may be, for example, dog serum.

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to canine parvovirus, and a composition for diagnosing canine parvovirus infection comprising the antibody or antigen-binding fragment thereof. By using the antibody of the present invention, it is possible to quickly and accurately determine whether or not canine parvovirus infection.

Figure 1a is a result of confirming the canine parvovirus infection by reacting and staining a parvovirus-infected cell with a positive control monoclonal antibody (abcam anti-parvovirus antibody, ab140431) against canine parvovirus.
1B is a result of confirming canine parvovirus infection by reacting and staining canine parvovirus-infected cells with a negative control antibody (negative mouse antibody).
1C is a result of confirming canine parvovirus infection by reacting and staining canine parvovirus-infected cells with canine parvovirus monoclonal antibody CPV-620 obtained in Example 1. FIG.
1D is a result of confirming canine parvovirus infection by reacting and staining canine parvovirus-infected cells with canine parvovirus monoclonal antibody CPV-330 obtained in Example 1. FIG.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example 1: CPV specific monoclonal antibody

1-1. Antibody production

Spleens of balb/c mice immunized with canine parvovirus (CPV 2a) were excised to obtain splenocytes, and then fused with myeloma cells (SP2/0AG14). CPV-330) hybridoma clones were obtained.

1-2. CPV infection confirmation

After infecting A72 cells with canine parvovirus, fixing the cells, immunizing mice with canine parvovirus (CPV 2a), reacting with two monoclonal antibodies obtained, and confirming after DAB staining, two kinds (CPV-620, CPV-330) was specifically confirmed to be infected with canine parvovirus through a monoclonal antibody (FIGS. 1C and 1D ).

Example 2. Hemagglutination Inhibition test: HI Test

2-1. method of inspection

2-1-1. Kaoline treatment

The supernatant of two monoclonal hybridomas (CPV-620, CPV-330) subjected to inactivation at 56° C. for 30 minutes was diluted 1:5 with Sorensen buffer. 0.1-0.4 ml of diluted pseudogum serum was added to the tube, and the same amount of Kaoline suspension was added, followed by mixing. It was incubated for 1 hour at 4°C while mixing 3-4 times during the culture. After centrifugation (1,000Хg, 15 minutes, 4°C), the supernatant was used in the experiment. Serum in this state is diluted 1:10.

The Sorensen buffer (pH 6.8) is Solution A (M/15 KH ₂ PO ₄ ) and Na ₂ HPO ₄ (9.47 g) prepared by mixing KH ₂ PO ₄ (9.078 g), NaCl (9.0 g) and 1 L of distilled water. ), NaCl (9.0 g) and 1 L of distilled water were mixed to prepare Solution B (M/15 Na ₂ HPO ₄ ) in a ratio of 508:492.

To the Kaoline suspension, 25 g of commercially available acid-washed kaoline was added to 90 ml of PBS, mixed, and allowed to settle for 24 hours to precipitate and discard the supernatant, and then PBS was added to make it 100 ml, followed by pH with 1N NaOH. Was prepared by adjusting to 7.2.

2-1-2. Adsorption of non-specific agglutinin using red blood cells

A 50% porcine red blood cell suspension was prepared, and 0.1 ml of a 50% red blood cell suspension was added to 1 ml of the supernatant of a monoclonal hybridoma treated with Kaoline. After adsorbing at 4° C. for 1 hour and centrifugation (1,000 Хg, 10 minutes), the supernatant was used in the experiment.

2-1-3. Readout

The monoclonal hybridoma supernatant was diluted twice with Sorensen buffer, and 50 μl of VP2 recombinant antigen or CPV (8 HA unit/50 μl) was added. After performing back titration of the antigen, the plates were gently mixed and incubated for 1 hour at room temperature. 50 μl of the red blood cell suspension was added, then gently mixed and incubated at 4° C. for 1-2 hours, followed by reading.

As a result of carrying out the HI test using porcine red blood cells with the supernatant of two monoclonal hybridomas (CPV-620, CPV-330), the HI response was confirmed against canine parvovirus. The HI titer of the CPV-620 monoclonal hybridoma supernatant was 2 ⁴ and the HI titer of the CPV-330 monoclonal hybridoma supernatant was 2 ⁵ .

<110> REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Monoclonal Antibody specific for Canine Parvovirus and use thereof <130> PN190028 <160> 20 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 <400> 1 Gly Phe Ser Leu Ser Thr Ser Gly Met Gly 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 <400> 2 Ile Trp Trp Asp Asp Asp Lys 1 5 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 <400> 3 Ala Arg Ile Ala Glu Val Tyr Tyr Tyr Gly His Tyr Tyr Ala Met Asp 1 5 10 15 Tyr <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 <400> 4 Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 <400> 5 Phe Ala Ser One <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 <400> 6 Gln Gln His Tyr Ser Ile Pro Trp Thr 1 5 <210> 7 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> VH AA SEQ <400> 7 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ile Ala Glu Val Tyr Tyr Tyr Gly His Tyr Tyr Ala Met 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 125 <210> 8 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> VL AA SEQ <400> 8 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly 1 5 10 15 Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln 85 90 95 His Tyr Ser Ile Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 9 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> VH DNA SEQ <400> 9 caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60 acttgttctt tctctgggtt ttcactgagc acttctggta tgggtgtagg ctggattcgt 120 cagccttcag ggaagggtct ggagtggctg gcacacattt ggtgggatga tgacaagcgc 180 tataacccag ccctgaagag ccgactgaca atctccaagg atacctccag caaccaggta 240 ttcctcaaga tcgccagtgt ggacactgca gatactgcca catactactg tgctcgaata 300 gctgaggttt attactacgg tcattactat gctatggact actggggtca aggaacctca 360 gtcaccgtct cctca 375 <210> 10 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> VL DNA SEQ <400> 10 gacattgtga tgacccagtc tccatcctcc ctggctatgt cagtaggaca gaaggtcact 60 atgagctgca agtccagtca gagcctttta aatagtagca atcaaaagaa ctatttggcc 120 tggtaccagc agaaaccagg acagtctcct aaacttctgg tatactttgc atccactagg 180 gaatctgggg tccctgatcg cttcataggc agtggatctg ggacagattt cactcttacc 240 atcagcagtg tgcaggctga agacctggca gattacttct gtcagcaaca ttatagtatt 300 ccgtggacgt tcggtggagg caccaagctg gaaatcaaa 339 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 <400> 11 Gly Asp Ser Ile Thr Ser Gly Tyr 1 5 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 <400> 12 Ile Ser Tyr Ser Gly Asn Thr 1 5 <210> 13 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 <400> 13 Ala Arg Arg Pro Tyr Tyr Tyr Gly Ser Ser Tyr Phe Asp Tyr 1 5 10 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 <400> 14 Asp His Ile Asn Asn Trp 1 5 <210> 15 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 <400> 15 Gly Ala Thr One <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 <400> 16 Gln Gln Phe Trp Ser Thr Pro Pro Thr 1 5 <210> 17 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH AA SEQ <400> 17 Gln Val Gln Leu Glu Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Arg Phe Pro Gly Asn Lys Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Arg Pro Tyr Tyr Tyr Gly Ser Ser Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 18 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL AA SEQ <400> 18 Asp Ile Val Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly 1 5 10 15 Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Phe Trp Ser Thr Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg 100 105 <210> 19 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VH DNA SEQ <400> 19 caggtgcagc tggaggagtc aggacctagc ctcgtgaaac cttctcagac tctgtccctc 60 acctgttctg tcactggcga ctccatcacc agtggttact ggaactggat ccggagattc 120 ccagggaaca aacttgagta catggggtac ataagctaca gtggtaacac ttactacaat 180 ccatctctca aaagtcgaat ctccatcact cgagacacat ccaagaacca gtactacctg 240 cagttgaatt ctgtgactac tgaggacaca gccacatatt actgtgcaag acggccctat 300 tactacggta gtagctactt tgactactgg ggccaaggca ccactctcac agtctcctca 360 360 <210> 20 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> VL DNA SEQ <400> 20 gacattgtga tgacccagtc ttcatcctac ttgtctgtat ctctaggagg cagagtcacc 60 attacttgca aggcaagtga ccacattaat aattggttag cctggtatca gcagaaacca 120 ggaaatgctc ctaggctctt aatatctggt gcaaccagtt tggaaactgg ggttccttca 180 agattcagtg gcagtggatc tggaaaggat tacactctca gcattaccag tcttcagact 240 gaagatgttg ctacttatta ctgtcaacag ttttggagta ctcctccgac gttcggtgga 300 ggcaccaagc tggaaatcag a 321

Claims (12)

CDR1 (complementarity determining region 1) consisting of the amino acid sequence represented by SEQ ID NO: 1, CDR2 (complementarity determining region 2) consisting of the amino acid sequence represented by SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3 a heavy chain variable region including (complementarity determining region 3); And
Canine parvovirus comprising a light chain variable region comprising CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 An antibody or antigen-binding fragment thereof that specifically binds to (Canine Parvovirus: CPV).
The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 7, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 8.
CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 11 CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) consisting of the amino acid sequence represented by SEQ ID NO: 12, and the amino acid sequence represented by SEQ ID NO: 13 A heavy chain variable region comprising a complementarity determining region 3 (CDR3); And
Canine parvovirus comprising a light chain variable region comprising CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 14, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 15, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 16 An antibody or antigen-binding fragment thereof that specifically binds to (Canine Parvovirus: CPV).
The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 17, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 18.
A recombinant vector comprising a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 and a polynucleotide encoding a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.
The recombinant vector according to claim 5, wherein the polynucleotide encoding the heavy chain variable region consists of the nucleotide sequence of SEQ ID NO: 9, and the polynucleotide encoding the light chain variable region consists of the nucleotide sequence of SEQ ID NO: 10.
A recombinant vector comprising a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17 and a polynucleotide encoding a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 18.
The recombinant vector according to claim 7, wherein the polynucleotide encoding the heavy chain variable region consists of the nucleotide sequence of SEQ ID NO: 19, and the polynucleotide encoding the light chain variable region consists of the nucleotide sequence of SEQ ID NO: 20.
A host cell transformed with the recombinant vector of claim 5.
A host cell transformed with the recombinant vector of claim 7.
A composition for diagnosing canine parvovirus infection comprising the antibody of claim 1 or an antigen-binding fragment thereof.
A composition for diagnosing canine parvovirus infection, comprising the antibody of claim 3 or an antigen-binding fragment thereof.

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