KR20210136752A - Monoclonal Antibody for Detecting Serotype A of Foot and Mouth Disease Virus, and Uses Thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody derived from pigs capable of specifically binding to the serotype A of foot-and-mouth disease virus, to a kit for diagnosing foot-and-mouth disease virus comprising the same, to a method for diagnosing foot-and-mouth disease using the same, and to a method for preventing or treating foot-and-mouth disease. More specifically, the monoclonal antibody that specifically binds to the foot-and-mouth disease virus of the present invention can be used as an antibody for detecting foot-and-mouth disease virus by specifically binding to an antigen of the foot-and-mouth disease virus. Furthermore, the monoclonal antibody derived from pigs exhibits excellent neutralizing ability against the foot-and-mouth disease virus, so that it can be used as a vaccine for prevention and treatment of foot-and-mouth disease in an emergency without risks provoking rejection of an immune response in pigs.

Description

Monoclonal Antibody for Detecting Serotype A of Foot and Mouth Disease Virus, and Uses Thereof

The present invention relates to a pig-derived monoclonal antibody capable of specifically binding to serotype A of foot-and-mouth disease virus, a kit for diagnosing foot-and-mouth disease virus comprising the same, a method for diagnosing foot-and-mouth disease, and a method for preventing or treating foot-and-mouth disease using the same.

Foot-and-mouth disease is a viral disease with high contagiousness and a mortality rate of 5 to 55% for two-hoofed animals such as cattle, pigs, goats, deer, and camels. It is a Class A disease and is classified as a type 1 legal infectious disease in Korea. Foot and Mouth Disease Virus (FMDV) is an RNA virus belonging to the Picornaviridae aphthovirus , and has seven serotypes (O, A, Asia1, C, SAT1) according to the antigenic structure worldwide. , SAT2, SAT3) and about 70 subtype viruses are distributed. In animals infected with foot-and-mouth disease virus, blisters and crusts are formed around the mouth and hoofs, such as the tongue and mucous membranes, and when the symptoms worsen, the blisters burst and develop into ulcers and die.

In Korea, foot-and-mouth disease first occurred in 1934 and has been steadily occurring in various parts of the country including Gyeonggi-do and Chungcheong-do since 2000. Recently, 9 cases were reported in 2017, 2 cases in 2018, and 3 cases in January 2019. When foot-and-mouth disease occurs, vaccines are vaccinated to individuals susceptible to foot-and-mouth disease to suppress or prevent the spread of the disease, but killing is still used as the best way to prevent the spread of foot-and-mouth disease.

Currently, methods for diagnosing foot-and-mouth disease virus can be divided into gene detection methods such as polymerase chain reaction, which amplifies viral genes in large quantities, and virus protein antigen detection methods using antigen-antibody reaction. The protein antigen detection method requires less testing time and cost compared to the gene detection method, and since it uses an antibody that specifically binds to the antigen of the foot-and-mouth disease virus, it has the advantage of being able to analyze foot-and-mouth disease as well as the serotype at the same time. Foot-and-mouth disease virus is distinctly divided into 7 serotypes, and there is no mutual protection between the serotypes, so serotype analysis is absolutely necessary to select a vaccine for preventing the spread of foot-and-mouth disease.

Therefore, for accurate diagnosis and serotype analysis of foot-and-mouth disease infection, the development of antibodies with high detection sensitivity to individual antigens of foot-and-mouth disease virus should be more actively progressed.

An object of the present invention is to provide a monoclonal antibody that specifically binds to foot-and-mouth disease virus serotype A.

Another object of the present invention is to provide a polynucleotide encoding a heavy chain variable region or a light chain variable region of a monoclonal antibody that specifically binds to foot-and-mouth disease virus serotype A.

Another object of the present invention is to provide a recombinant vector comprising the polynucleotide and a host cell comprising the same.

Another object of the present invention is to provide a kit for diagnosing foot-and-mouth disease comprising the monoclonal antibody.

Another object of the present invention is to provide a method for preventing or treating foot-and-mouth disease comprising administering the monoclonal antibody to an animal other than a human.

One aspect of the present invention provides a monoclonal antibody that specifically binds to foot-and-mouth disease virus (FMDV) serotype A.

Foot-and-mouth disease virus is divided into serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 according to the antigenic structure. Since the antigens of foot-and-mouth disease virus have distinct structures, a specific antibody for one serotype is used. Therefore, it is impossible to detect other serotypes or to develop immunity. That is, when a diagnostic kit containing an antibody to FMDV type A is used for diagnosing foot-and-mouth disease, it is impossible to confirm the diagnosis of foot-and-mouth disease from animals infected with foot-and-mouth disease of a serotype other than type A. In addition, when an animal suspected of foot-and-mouth disease is inoculated with a vaccine (antibody) against type A FMDV, only protective immunity to type A is formed, but immunity to other serotypes is not formed, so viruses of serotypes other than type A invade This will cause foot-and-mouth disease.

As used herein, the term "monoclonal antibody" refers to an antibody having specificity to react by recognizing only one epitope, and is formed from a single antibody-forming cell and has a single molecular structure. A complete antibody is generally Y-shaped and consists of two long heavy chains (H) and two short light chains (L). Each heavy chain and light chain are connected to each other by a sulfide bond, and are divided into a variable region (V), a region that reacts with an antigen, and a constant region (C), a region that expresses an effector function. In the variable region, there is a complementarity-determining region (CDR) that determines the structure of the variable region so as to form specific binding with an antigen and controls antibody binding strength. For example, the antibody of type A FMDV specifically binds to type A, and may be an antigen binding fragment (Fab) of an antibody molecule including a CDR as well as a complete antibody.

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

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

As used herein, the term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure, and refers to a portion of a polypeptide including a portion capable of binding an antigen. 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 variable regions of light and heavy chains, constant regions of light chain and the first constant region (CH1) of heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab') ₂ antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and a recombinant technique for generating an Fv fragment is well known in the art. In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and in single-chain Fv (single-chain Fv), the heavy chain variable region and the single chain variable region are generally shared through a peptide linker. They are linked by a bond or are linked directly at the C-terminus to form a dimer-like structure like a double-stranded Fv. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, by restriction digestion of the whole antibody with papain, Fab can be obtained, and when digested with pepsin, a F(ab') ₂ fragment can be obtained), It can be produced through genetic recombination technology.

The monoclonal antibody against type A FMDV is derived from a pig, and may include some or all of the nucleotide sequence of an antibody formed from a pig infected with the foot-and-mouth disease virus. For example, a heavy chain variable region comprising a heavy chain CDR1 (complementarity determining region 1) consisting of SEQ ID NO: 2, a heavy chain complementarity determining region 2 (CDR2) consisting of SEQ ID NO: 3 and a heavy chain CDR3 (complementarity determining region 3) consisting of SEQ ID NO: 4 ; and a light chain variable region comprising a light chain CDR1 (complementarity determining region 1) of SEQ ID NO: 6, a light chain CDR2 (complementarity determining region 2) of SEQ ID NO: 7, and a light chain CDR3 (complementarity determining region 3) of SEQ ID NO: 8 may be doing Preferably, it may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5.

The monoclonal antibody may be prepared using methods known in the art. For example, total RNA is extracted from peripheral blood lymphocytes isolated from swine blood to synthesize cDNA, and the cDNA and an expression vector using primers specific for the heavy chain variable region and the light chain variable region of the porcine antibody After preparing the FMDV monoclonal antibody can be prepared by transforming the expression vector into a host cell, and selecting only an antibody that specifically binds to type A FMDV among them.

The finally obtained FMDV monoclonal antibody may be of IgG ₁ , IgG _2a , IgG _2b , Ig _G3 , IgA or IgM type.

Another aspect of the present invention is foot-and-mouth disease comprising a polynucleotide encoding the heavy chain variable region of a monoclonal antibody that specifically binds to foot-and-mouth disease virus serotype A comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 5 Provided is a polynucleotide encoding the light chain variable region of a monoclonal antibody that specifically binds to virus serotype A.

Another aspect of the present invention provides a recombinant vector comprising the polynucleotide and a host cell comprising the recombinant vector.

The recombinant vector may be a vector system in which a heavy chain variable region and a light chain variable region are simultaneously expressed in one vector, or a vector system in which a heavy chain variable region and a light chain variable region are expressed in each vector. In the case of a system using each vector, the two vectors may be introduced into a host cell by co-transformation or targeted transformation.

The host cell is a cell that can be continuously cloned or expressed by stabilizing the expression vector, and a host cell known in the art can be used without limitation. For example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus sp. strain, yeast ( Saccharomyce cerevisiae ), insect cells , plant cells, Sp2/0, CHO (chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, animal cells such as MDCK.

These host cells can produce monoclonal antibodies through culture. Culture conditions can be adjusted according to the proliferation characteristics of the host cells. The culture may be performed in batchwise, semi-batchwise, continuous culture or fed-batch culture. The culture temperature may be 15° C. to 45° C., preferably 25° C. to 40° C., more preferably 32° C. to 37° C., and the pH of the medium is 5 to 9, preferably 6 to 8, more preferably It could be 6.8. The incubation time is preferably continued until the formation of the desired product reaches a maximum, and in order to show the optimal production efficiency of the host cells, the culture may be performed for 7 to 9 days. As such, the monoclonal antibody produced by culturing the host cell remains secreted into the medium, and can be purified using a purification method known in the art.

Another aspect of the present invention provides a kit for diagnosing foot-and-mouth disease comprising the monoclonal antibody and a method for diagnosing foot-and-mouth disease by using the same to rapidly and accurately diagnose foot-and-mouth disease.

The kit can be used to quantitatively measure whether foot-and-mouth disease virus is infected using the FMDV monoclonal antibody against type A, and includes an enzyme immunoassay using an antigen-antibody reaction, a radio immunoassay, A method such as fluorescence immunoassay may be used.

The diagnosis method can diagnose foot-and-mouth disease by directly using the FMDV monoclonal antibody or by using a diagnostic kit using the FMDV monoclonal antibody. Specifically, a biological sample isolated from an animal suspected of foot-and-mouth disease, such as a pig or cow, can be reacted with an FMDV monoclonal antibody to detect the antigen-antibody reaction. In this case, the biological sample may be blood, serum, plasma, urine, tears, saliva, milk, etc., and it is preferable to use a body fluid secreted outside the body for easy collection.

In addition, the serotype can be analyzed according to the FMDV monoclonal antibody used. When using an antibody (A-serotype) that specifically binds to type A FMDV or a kit including the same, type A foot-and-mouth disease can be diagnosed, but other serotypes other than type A foot-and-mouth disease cannot be diagnosed.

Another aspect of the present invention provides a method for preventing or treating foot-and-mouth disease, comprising administering the above-described monoclonal antibody to an animal other than a human.

The prevention or treatment method can form immunity to the foot-and-mouth disease virus by administering the FMDV monoclonal antibody to a pig that has already been infected with the foot-and-mouth disease virus or has a high possibility of infection. At this time, depending on the FMDV monoclonal antibody used, immunity to the corresponding serotype can be formed. When an antibody (A-serotype) that specifically binds to type A FMDV is administered, immunity to type A FMDV is formed, and thus a preventive or therapeutic effect against type A foot and mouth disease can be exhibited.

The monoclonal antibody that specifically binds to the foot-and-mouth disease virus according to the present invention can be used as an antibody for detecting foot-and-mouth disease virus by specifically binding to the antigen of the foot-and-mouth disease virus. Since there is no concern about the occurrence of immune rejection, it can be used as a vaccine for prevention and treatment of foot-and-mouth disease in an emergency.

1 is a graph confirming the binding specificity for various serotypes of FMDV of clones selected after panning as an experimental result according to an embodiment of the present invention.
Figure 2 is an experimental result according to an embodiment of the present invention, the monoclonal antibodies pigA4, pigA2, pigA25 expressed in the CHO cell line are purified and confirmed by SDS-PAGE.
3 is a graph confirming the binding affinity of monoclonal antibodies pigA4, pigA2, and pigA25 to type A FMDV as an experimental result according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. However, these descriptions are provided for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions.

Example 1. Preparation of FMDV monoclonal antibody

PBL was isolated by centrifugation from blood of pigs challenged with type A FMDV (Yeoncheonju). Total RNA was extracted from PBL using an RNA extraction kit (TRIzol, Invitrogen), and cDNA was synthesized from it using an RT-PCR kit (SuperScript IV First-Strand Synthesis System, Invitrogen).

From the synthesized cDNA, the VH, Vk, and Vλ genes were respectively amplified using a primer set specific to the porcine antibody variable region (see Table 1 below), and the heavy chain variable region gene (VH) and light chain variable region gene (Vk, Vλ) was linked in the form of scFv using the G4S linker sequence. The synthesized library scFv sequence was cut using restriction enzyme SfiI and ligated to pDR-D1 vector cut with the same SfiI using T4 Ligase. This ligate DNA was introduced into electrocompetent ER2738 E. coli using Electroporator (Bio-rad). Helper phage (VCSM13, Stratagene) was superinfected and cultured overnight in order to obtain a recombinant phage in which the antibody library was introduced from E. coli, finally present in the supernatant using PEG8000 (Sigma). The phage was concentrated.

The library phages in which the enriched scFv was surface-expressed were reacted for 30 minutes each in an immunotube coated with Fetal bovine serum (FBS) to remove phages of non-specific binding, and the remaining phages were transferred to an immunotube coated with FMDV antigen A22 Iraq or A zabaykalsky 2 reacted for an hour. Unbound phages were removed through repeated PBST washing 5 times, and finally bound phages were eluted using 0.1M Glycin HCl (pH 2.7). The phages neutralized with 1M Tris-HCl (pH 8.0) were infected with E. coli to amplify the phages again, and then the next round of panning was performed. As the panning cycle progressed, the number of washings was increased so that only phages with specific and strong binding were selectively amplified.

After the third panning, 96 clones were randomly selected and cultured in a 96 deep well plate (Bioneer), and then recombinant phage for each clone was obtained using a helper phage. The FMDV antigen and the FBS-coated microtiter plate were bound to the wells, and the binding ability of individual recombinant phage clones was confirmed using an anti-M13-Ab-HRP antibody. Antibody clones that specifically bind only to FMDV without binding to the control (FBS) were identified, and antibody clones with unique sequences were selected through sequence analysis.

primer base sequence (5'> 3') SEQ ID NO: VH-Forward PVH1-2 GCGGCCCAGC CGGCCATGGC Cgaggtgaag ctggtggagt g 9 PVH1m GCGGCCCAGC CGGCCATGGC CgaggWgaag ctggtggagt c 10 PVH1S6 GCGGCCCAGC CGGCCATGGC Ccaggagaag ctggtggagt c 11 PVH1 GCGGCCCAGC CGGCCATGGC Cgaggagaag ctggtggagt ctg 12 VH-Reverse PJH12R cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg agacggtgac cagg 13 PJH3R cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg agacggtgac ctcg 14 PJH4R cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacccgagg cgtcgtagac tagg 15 PJH5R cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg acacgacgac ttcaac 16 Vk-Forward PVk1 ggatctggcg gcggcggctc ggccatccag ctgacccagt c 17 PVk2-1 ggatctggcg gcggcggctc ggccatggtg ttgacccaga g 18 PVk2-2 ggatctggcg gcggcggctc ggccatYgtg ctgacccaga c 19 PVk3 ggatctggcg gcggcggctc ggaaattgtg ctgacccagt c 20 PVk5 ggatctggcg gcggcggctc ggaaacaaca gtcactcaat c 21 Vk-Reverse PJk12R CTGCTCGAGG CCTCCCGGGC CtttgagYtc cagcttggtY c 22 PJk3R CTGCTCGAGG CCTCCCGGGC Ctttgggctc cactttggtc c 23 PJk4R CTGCTCGAGG CCTCCCGGGC Ctttgatttc cagcttggtc c 24 PJk5R CTGCTCGAGG CCTCCCGGGC Cttcaatctc cacggatgtc c 25 Vλ-Forward PVL1/5 ggatctggcg gcggcggctc gcaggctgtg ctgacKcagc 26 PVL2 ggatctggcg gcggcggctc gcagtctgcc ctgactcagc 27 PVL3-1 ggatctggcg gcggcggctc gtcctgtgag ctgactcagc 28 PVL3-2 ggatctggcg gcggcggctc gtcctatgag gtgactcagc 29 PVL3-3 ggatctggcg gcggcggctc gtcctatgag ctgacccagc 30 PVL3-4 ggatctggcg gcggcggctc gtcttctMag ctgactcagc 31 PVL7-1 ggatctggcg gcggcggctc gtcccagatg gtgactcagg 32 PVL7-2 ggatctggcg gcggcggctc gccaagctgt gtgactcagg 33 PVL8 ggatctggcg gcggcggctc gtctcagact gtgatccagg 34 Vλ - Reverse PJL23R CTGCTCGAGG CCTCCCGGGC Cgaggacggt cagatgggtc c 35 PJL4R CTGCTCGAGG CCTCCCGGGC Cgaggacact tagacgggtc c 36 scFv-Forward GACGACGACG ACGACGCGGC CCAGCCGGCC ATGGCC 37 scFv-Reverse GACGACGACG ACGACCTGCT CGAGGCCTCC CGGGCC 38

Example 2. FMDV serotype specificity analysis of monoclonal antibodies

In order to confirm the FMDV serotype specificity of the selected monoclonal antibodies, FMDV O (O manisa), A (A22 Iraq or A zabaykalsky), SAT1, SAT2, SAT3 serotypes and the control group (BSA) were selected in Example 1 The binding capacity of the monoclonal antibody was analyzed.

First, for precise antibody binding capacity analysis, scFv sequences of antibody clones were cloned into animal cell expression vector pDR-OriP-Fc1, expressed in scFv-Fc form in 293E cells, and purified using protein G affinity column (GE Healthcare). . 2.5 ng of scFv-Fc antibody was reacted with each antigen-coated microtiter well, and bound scFv-Fc was measured using anti-human IgG (Fc specific)-HRP (Jackson ImmunoResearch Laboratories). The results are shown in FIG. 1 .

1, clones pigA4, pigA2, and pigA25 that are specific only to the A-type FMDV antigen and exhibit high binding ability and have different sequence information were finally selected.

Example 3. FMDV monoclonal antibody production and purification using CHO cell line

To produce monoclonal antibodies from the three clones selected in Example 2, the scFv sequence of each clone was transformed into a pCEP vector to construct a recombinant vector. This was transformed into a CHO cell line and cultured for one day at 37°C and pH 6.8 in a fed-batch method, and then glucose was additionally added to promote expression and cell growth. Then, it was cultured for 7 days under conditions of 32 °C and pH 6.8. The protein expressed in the form of scFv-Fc was purified using protein G affinity column (GE Healthcare).

The monoclonal antibodies pigA4, pigA2, and pigA25 were obtained with a high yield as shown in FIG.

Example 4. Avidity analysis of FMDV monoclonal antibody

The binding capacity of the monoclonal antibody purified in Example 3 to type A FMDV was comparatively analyzed.

After coating the FMDV type A protein on a 96-well ELISA plate, ^{each monoclonal antibody diluted from 10 3} pM to 1/4 concentration was added to allow the protein and antibody to bind to each other. After three washes using PBST, an HRP-conjugated secondary antibody was added and reacted. Thereafter, after four washing processes using PBST, signal detection was performed with an absorbance meter through the reaction of HRP with a substrate. The results are shown in FIG. 3 and Table 2 below.

monoclonal antibody binding force (pM) pigA4 17.4 pigA2 8.8 pigA25 10.9

As shown in FIG. 3 and Table 2, the FMDV monoclonal antibodies pigA4, pigA2, and pigA25 were confirmed to exhibit high binding affinity at a level of 8.8 to 17.4 pM.

Example 5. Competition ELISA measurement

The existing FMDV diagnostic kit (eg PrioCHECK® FMDV Type A Antibody ELISA Kit) is a competitive ELISA method, in which a mouse-derived FMDV-specific monoclonal antibody competes with the FMDV antibody present in serum isolated from an animal, and FMDV coated on an ELISA plate. Detects inability to bind antigen.

Among the monoclonal antibodies prepared in Example 3, pigA4 and pigA25 having excellent binding strength were used to test whether they can be applied to the FMDV diagnostic kit. First, 9 types of bovine sera or 2 types of porcine sera were respectively bound to an ELISA plate coated with FMDV antigen, and then one washing process was performed. Thereafter, the HRP-binding secondary antibody complex for signal detection was added and reacted, and the HRP signal was detected using an absorbance meter after four washing steps. The results are shown in Table 3 below.

PI value (%) pigA4 pigA25 control bovine serum 1 98.3 97.2 82 bovine serum 2 98.2 96.1 75.9 bovine serum 3 96.8 93.6 74.3 bovine serum 4 97.8 97.8 73.4 bovine serum 5 97.8 74.1 69.2 bovine serum 6 98.0 95.3 78.9 bovine serum 7 96.5 95.4 77.6 bovine serum 8 92.7 86.5 74.2 bovine serum 9 97.9 92.4 72.5 Pig Serum 1 89.0 94.0 66.0 Pig Serum 2 72.0 97.0 57.0

As shown in Table 3 above, as a result of using the FMDV monoclonal antibodies pigA4 and pigA25 instead of the FMDV-specific monoclonal antibody derived from the mouse in the diagnostic kit, it was confirmed to have significantly improved result values compared to the existing kit (control group). In particular, it was confirmed that pigA4 exhibited excellent PI values for bovine serum. These results suggest that pigA4 can be used as a new type A FMDV monoclonal antibody for diagnosing foot-and-mouth disease type A antibody.

The amino acid sequence of the monoclonal antibody pigA4 is shown in Table 4 below.

pigA4 amino acid sequence VH (SEQ ID NO: 1) EEKLVESGGGWVWPGGSLRLSCVGSGFTFS SYEIS WVRQAPGKGLEWLA GIGSSGSNTYYVDSVKG RFTISRDNSQNTAYLQMNSLRTEDTAHYYCAR GGSRYGSSLMDL WGPGVEVVVSS VL (SEQ ID NO: 5) ELTQPSSESVALGNTAKITC SGDLLDEKYTQ WYQQKPGQAPLLLIY RDSERPS GIPERFSGSSSGKTATLTITGAQAEDEADYYC QSADSSDDAI FGGGTRLSVL

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Monoclonal Antibody for Detecting Serotype A of Foot and Mouth Disease Virus, and Uses Thereof <130> PN200054 <160> 38 <170> KoPatentIn 3.0 <210> 1 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> variable region of heavy chain <400> 1 Glu Glu Lys Leu Val Glu Ser Gly Gly Gly Trp Val Trp Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Glu Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ala Gly Ile Gly Ser Ser Gly Ser Asn Thr Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala His Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ser Arg Tyr Gly Ser Ser Leu Met Asp Leu Trp Gly 100 105 110 Pro Gly Val Glu Val Val Val Ser Ser 115 120 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> variable region of heavy chain - CDR1 <400> 2 Ser Tyr Glu Ile Ser 1 5 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> variable region of heavy chain - CDR2 <400> 3 Gly Ile Gly Ser Ser Gly Ser Asn Thr Tyr Tyr Val Asp Ser Val Lys 1 5 10 15 Gly <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> variable region of heavy chain - CDR3 <400> 4 Gly Gly Ser Arg Tyr Gly Ser Ser Leu Met Asp Leu 1 5 10 <210> 5 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> variable region of light chain <400> 5 Glu Leu Thr Gln Pro Ser Ser Glu Ser Val Ala Leu Gly Asn Thr Ala 1 5 10 15 Lys Ile Thr Cys Ser Gly Asp Leu Leu Asp Glu Lys Tyr Thr Gln Trp 20 25 30 Tyr Gln Gln Lys Pro Gly Gln Ala Pro Leu Leu Leu Ile Tyr Arg Asp 35 40 45 Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Ser Ser Ser 50 55 60 Gly Lys Thr Ala Thr Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu 65 70 75 80 Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Asp Asp Ala Ile Phe 85 90 95 Gly Gly Gly Thr Arg Leu Ser Val Leu 100 105 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> variable region of light chain - CDR1 <400> 6 Ser Gly Asp Leu Leu Asp Glu Lys Tyr Thr Gln 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> variable region of light chain - CDR2 <400> 7 Arg Asp Ser Glu Arg Pro Ser 1 5 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> variable region of light chain - CDR3 <400> 8 Gln Ser Ala Asp Ser Ser Asp Asp Ala Ile 1 5 10 <210> 9 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PVH1-2 <400> 9 gcggcccagc cggccatggc cgaggtgaag ctggtggagt g 41 <210> 10 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PVH1m <400> 10 gcggcccagc cggccatggc cgaggwgaag ctggtggagt c 41 <210> 11 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PVH1S6 <400> 11 gcggcccagc cggccatggc ccaggagaag ctggtggagt c 41 <210> 12 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> PVH1 <400> 12 gcggcccagc cggccatggc cgaggagaag ctggtggagt ctg 43 <210> 13 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> PJH12R <400> 13 cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg agacggtgac 60 cagg 64 <210> 14 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> PJH3R <400> 14 cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg agacggtgac 60 ctcg 64 <210> 15 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> PJH4R <400> 15 cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacccgagg cgtcgtagac 60 tag 64 <210> 16 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> PJH5R <400> 16 cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg acacgacgac 60 ttcaac 66 <210> 17 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PVk1 <400> 17 ggatctggcg gcggcggctc ggccatccag ctgacccagt c 41 <210> 18 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PVk2-1 <400> 18 ggatctggcg gcggcggctc ggccatggtg ttgacccaga g 41 <210> 19 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PVk2-2 <400> 19 ggatctggcg gcggcggctc ggccatygtg ctgacccaga c 41 <210> 20 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PVk3 <400> 20 ggatctggcg gcggcggctc ggaaattgtg ctgacccagt c 41 <210> 21 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PVk5 <400> 21 ggatctggcg gcggcggctc ggaaacaaca gtcactcaat c 41 <210> 22 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PJk12R <400> 22 ctgctcgagg cctcccgggc ctttgagytc cagcttggty c 41 <210> 23 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PJk3R <400> 23 ctgctcgagg cctcccgggc ctttgggctc cactttggtc c 41 <210> 24 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PJk4R <400> 24 ctgctcgagg cctcccgggc ctttgatttc cagcttggtc c 41 <210> 25 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PJk5R <400> 25 ctgctcgagg cctcccgggc cttcaatctc cacggatgtc c 41 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PVL1/5 <400> 26 ggatctggcg gcggcggctc gcaggctgtg ctgackcagc 40 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PVL2 <400> 27 ggatctggcg gcggcggctc gcagtctgcc ctgactcagc 40 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PVL3-1 <400> 28 ggatctggcg gcggcggctc gtcctgtgag ctgactcagc 40 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PVL3-2 <400> 29 ggatctggcg gcggcggctc gtcctatgag gtgactcagc 40 <210> 30 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PVL3-3 <400> 30 ggatctggcg gcggcggctc gtcctatgag ctgacccagc 40 <210> 31 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PVL3-4 <400> 31 ggatctggcg gcggcggctc gtcttctmag ctgactcagc 40 <210> 32 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PVL7-1 <400> 32 ggatctggcg gcggcggctc gtcccagatg gtgactcagg 40 <210> 33 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PVL7-2 <400> 33 ggatctggcg gcggcggctc gccaagctgt gtgactcagg 40 <210> 34 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PVL8 <400> 34 ggatctggcg gcggcggctc gtctcagact gtgatccagg 40 <210> 35 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PJL23R <400> 35 ctgctcgagg cctcccgggc cgaggacggt cagatgggtc c 41 <210> 36 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PJL4R <400> 36 ctgctcgagg cctcccgggc cgaggacact tagacgggtc c 41 <210> 37 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> scFv-Forward <400> 37 gacgacgacg acgacgcggc ccagccggcc atggcc 36 <210> 38 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> scFv-Reverse <400> 38 gacgacgacg acgacctgct cgaggcctcc cgggcc 36

Claims (11)

A monoclonal antibody that specifically binds to foot and mouth disease virus serotype A.
The method according to claim 1,
The monoclonal antibody is a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5.
The method according to claim 1,
The monoclonal antibody is a heavy chain comprising a heavy chain CDR1 (complementarity determining region 1) consisting of SEQ ID NO: 2, a heavy chain complementarity determining region 2 (CDR2) consisting of SEQ ID NO: 3, and a heavy chain CDR3 (complementarity determining region 3) consisting of SEQ ID NO: 4 variable region; and
A light chain variable region comprising a light chain CDR1 (complementarity determining region 1) of SEQ ID NO: 6, a light chain CDR2 (complementarity determining region 2) of SEQ ID NO: 7 and a light chain CDR3 (complementarity determining region 3) of SEQ ID NO: 8 monoclonal antibody.
A polynucleotide encoding the heavy chain variable region of a monoclonal antibody that specifically binds to foot-and-mouth disease virus serotype A comprising the amino acid sequence of SEQ ID NO: 1.
A polynucleotide encoding the light chain variable region of a monoclonal antibody specifically binding to foot-and-mouth disease virus serotype A comprising the amino acid sequence of SEQ ID NO: 5.
A recombinant vector comprising the polynucleotide of claim 4 or 5 .
A host cell comprising the recombinant vector of claim 6.
A kit for diagnosing foot-and-mouth disease comprising the monoclonal antibody of claim 1.
9. The method of claim 8,
The kit is a kit for diagnosing foot-and-mouth disease in cattle or swine foot-and-mouth disease.
A method for preventing or treating foot-and-mouth disease, comprising administering the monoclonal antibody of claim 1 to an animal other than a human.
11. The method of claim 10,
The method for preventing or treating foot-and-mouth disease wherein the animal is a pig.

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