KR20210016850A - Monoclonal Antibody for Detecting Foot and Mouth Disease Virus, and Uses Thereof - Google Patents
Monoclonal Antibody for Detecting Foot and Mouth Disease Virus, and Uses Thereof Download PDFInfo
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- KR20210016850A KR20210016850A KR1020190095137A KR20190095137A KR20210016850A KR 20210016850 A KR20210016850 A KR 20210016850A KR 1020190095137 A KR1020190095137 A KR 1020190095137A KR 20190095137 A KR20190095137 A KR 20190095137A KR 20210016850 A KR20210016850 A KR 20210016850A
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- Prior art keywords
- foot
- seq
- mouth disease
- monoclonal antibody
- variable region
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Abstract
Description
본 발명은 구제역 바이러스의 혈청형 7종 중 O형에 특이적으로 결합 또는 모든 혈청형에 광범위하게 결합 가능한 소 유래 단일클론항체 및 이를 포함하는 구제역 바이러스 진단용 키트, 이를 이용한 구제역의 진단 방법, 구제역의 예방 또는 치료 방법에 관한 것이다. The present invention provides a bovine-derived monoclonal antibody capable of binding specifically to type O or broadly binding to all serotypes among 7 serotypes of foot-and-mouth disease virus, and a kit for diagnosis of foot-and-mouth disease virus including the same, a method for diagnosis of foot-and-mouth disease using the same, It relates to a method of prevention or treatment.
구제역은 소, 돼지, 염소, 사슴, 낙타 등 발굽이 2개인 우제류 동물에 대해 전염성이 높고 치사율이 5~55%인 바이러스성 질병으로서 국제수역사무국(Office International des Epizooties)에서 정한 가축 전염병 중 가장 위험한 A급(List A) 질병이며, 국내에서도 제1종 법정 전염병으로 분류된다. 구제역 바이러스(Foot and Mouth Disease Virus; FMDV)는 피코나바이러스과 애프도바이러스(Picornaviridae aphthovirus)에 속하는 RNA 바이러스로, 전세계적으로 항원 구조에 따른 7종의 혈청형 (O, A, Asia1, C, SAT1, SAT2, SAT3)과 70여 종의 아형 바이러스가 분포하고 있다. 구제역 바이러스에 감염된 동물은 혀, 점막 등 입과 발굽 주변에 수포 및 가피가 형성되고, 증세가 심해지면 수포가 터져 궤양으로 진전되어 죽게된다. Foot-and-mouth disease is a viral disease that is highly contagious and has a mortality rate of 5 to 55% for cows, pigs, goats, deer, camels, etc., and is the most dangerous among livestock infectious diseases determined by the Office International des Epizooties. It is a class A (List A) disease, and is classified as a first-class legal infectious disease in Korea. Foot and Mouth Disease Virus (FMDV) is an RNA virus belonging to Picornaviridae aphthovirus , and seven serotypes according to antigenic structure worldwide (O, A, Asia1, C, SAT1) , SAT2, SAT3) and more than 70 subtype viruses are distributed. In animals infected with foot-and-mouth disease virus, blisters and crusts form around the mouth and hooves such as tongue and mucous membranes, and when the symptoms worsen, blisters burst and develop into ulcers and die.
국내에서는 구제역이 1934년 처음 발생하여 2000년 이후 경기도, 충청도 등 전국 각지에서 꾸준히 발생하고 있고, 최근 2017년에 9건, 2018년에 2건이, 2019년 1월에 3건이 보고되었다. 구제역 발생시 질병의 확산을 억제하거나 예방하기 위해 구제역 감수성 개체에 백신을 접종하지만, 여전히 살처분이 최선의 확산 방지책으로 사용되고 있어 구제역 발생 농가의 막대한 재산 피해가 불가피하다.In Korea, foot-and-mouth disease first occurred in 1934, and since 2000, it has been steadily occurring in various places across the country, including Gyeonggi-do and Chungcheong-do, and recently, 9 cases in 2017, 2 cases in 2018, and 3 cases in January 2019. In the event of foot-and-mouth disease, vaccines are given to individuals susceptible to foot-and-mouth disease to inhibit 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.
현재 구제역 바이러스를 진단하는 방법으로는 바이러스 유전자를 대량으로 증폭시키는 중합효소 연쇄반응(polymerase chain reaction) 등의 유전자 검출 방법과 항원-항체 반응을 이용한 바이러스의 단백질 항원 검출 방법으로 나눌 수 있다. 단백질 항원 검출 방법은 유전자 검출 방법에 비해 검사 시간 및 비용이 적게 소요되며, 구제역 바이러스의 항원과 특이적으로 결합하는 항체를 이용하기 때문에 구제역 양성 판정과 동시에 혈청형까지 분석할 수 있는 장점이 있다. 그러나, 각 항원마다 구조가 뚜렷이 구분되어 해당 항원을 특이적으로 인식하는 항체가 없는 경우에는 구제역 확진 판정을 할 수 없다. 한편, 구제역 바이러스에 대한 항체는 구제역 예방 또는 치료 백신으로 사용할 수 있는데, 이때 백신 접종된 동물에 방어 면역이 형성되지만 혈청형 간의 상호 방어 면역이 이루어지지 않기 때문에 다른 혈청형의 바이러스가 침입하게 되면 방어하지 못하고 구제역이 발생하게 된다.Currently, methods for diagnosing foot-and-mouth disease viruses can be divided into a method of detecting a gene such as a polymerase chain reaction, which amplifies a viral gene in large quantities, and a method of detecting a protein antigen of a virus using an antigen-antibody reaction. The protein antigen detection method takes less time and cost compared to the gene detection method, and because it uses an antibody that specifically binds to the antigen of foot-and-mouth disease virus, it has the advantage of being able to analyze positive foot-and-mouth disease and even serotype at the same time. However, since each antigen has a distinct structure, if there is no antibody that specifically recognizes the antigen, it is not possible to confirm foot-and-mouth disease. On the other hand, antibodies against foot-and-mouth disease virus can be used as a vaccine for preventing or treating foot-and-mouth disease.At this time, protective immunity is formed in the vaccinated animal, but since mutual protective immunity between serotypes is not established, it is protected when viruses of other serotypes invade. Without doing so, foot-and-mouth disease occurs.
한국등록특허 제1960968호에는 A형 항원에 특이적으로 결합하는 단일클론항체가 개시되어 있고, 한국공개특허 제2019-0006154호에는 O형 항원에 특이적으로 결합하는 단일클론항체가 개시되어 있고, 일본공개특허 제2013-049645호에는 O형, A형 및 Asia 1형에 특이적으로 결합하지 않으면서 C형 항원에 특이적으로 결합하는 항체가 개시되어 있다. 이와 같이 개별 항원에 특이적으로 결합하는 항체는 꾸준히 개발되고 있으나, 구제역 바이러스의 검출 민감도가 높고 구제역 바이러스의 특정 단백질 항원 또는 모든 단백질 항원과 결합 가능하는 항체에 대해서는 여전히 연구 및 개발이 부족한 실정이다. Korean Patent No. 1960968 discloses a monoclonal antibody that specifically binds to type A antigen, and Korean Patent Publication No. 2019-0006154 discloses a monoclonal antibody that specifically binds to type O antigen, Japanese Patent Laid-Open No. 2013-049645 discloses an antibody that specifically binds to a type C antigen without specifically binding to type O, type A, and type 1 Asia. As described above, antibodies that specifically bind to individual antigens have been continuously developed, but there is still a lack of research and development for antibodies that have high detection sensitivity of foot-and-mouth disease virus and can bind to specific protein antigens or all protein antigens of foot-and-mouth disease virus.
본 발명의 일 양상은 구제역 바이러스 혈청형 O형에 특이적으로 결합하는 단일클론항체를 제공하는 것을 목적으로 한다.An object of the present invention is to provide a monoclonal antibody that specifically binds to foot-and-mouth disease virus serotype O.
본 발명의 다른 양상은 구제역 바이러스 혈청형 O, A, Asia1, C, SAT1, SAT2 및 SAT3으로 이루어진 군으로부터 선택되는 하나 이상의 혈청형에 특이적으로 결합하는 단일클론항체를 제공하는 것을 목적으로 한다.Another aspect of the present invention is to provide a monoclonal antibody that specifically binds to one or more serotypes selected from the group consisting of foot-and-mouth disease virus serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3.
본 발명의 또 다른 양상은 상기 단일클론항체를 포함하는 구제역 진단용 키트를 제공하는 것을 목적으로 한다.Another aspect of the present invention is to provide a kit for diagnosis of foot-and-mouth disease comprising the monoclonal antibody.
본 발명의 다른 양상은 상기 단일클론항체를 인간을 제외한 동물에게 투여하는 단계를 포함하는 구제역 바이러스 감염의 예방 또는 치료 방법을 제공하는 것을 목적으로 한다.Another aspect of the present invention is to provide a method for preventing or treating foot-and-mouth disease virus infection comprising administering the monoclonal antibody to animals other than humans.
본 발명의 일 양상은 구제역 바이러스 혈청형 O형에 특이적으로 결합하는 단일클론항체를 제공한다.One aspect of the present invention provides a monoclonal antibody that specifically binds to foot-and-mouth disease virus serotype O.
본 발명의 다른 양상은 구제역 바이러스 혈청형 O, A, Asia1, C, SAT1, SAT2 및 SAT3으로 이루어진 군으로부터 선택되는 하나 이상의 혈청형에 특이적으로 결합하는 단일클론항체를 제공한다.Another aspect of the present invention provides a monoclonal antibody that specifically binds to one or more serotypes selected from the group consisting of foot-and-mouth disease virus serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3.
구제역 바이러스는 항원 구조에 따라 혈청형이 O, A, Asia1, C, SAT1, SAT2 및 SAT3으로 구분되며, 특히 국내에서는 주로 O형 구제역이 발생한다. 구제역 바이러스의 항원은 구조가 서로 뚜렷이 구분되기 때문에 하나의 혈청형에 대한 특이적인 항체를 사용하여 다른 혈청형을 검출하거나, 또는 면역을 형성시키는 것이 불가능하다. 즉, 구제역 진단시 O형 FMDV에 대한 항체를 포함하는 진단 키트를 이용할 경우, O형이 아닌 다른 혈청형의 구제역 감염 동물로부터 구제역 확진 판정을 할 수 없다. 또한, 구제역 의심 동물에 O형 FMDV 에 대한 백신(항체)을 접종할 경우, O형에 대한 방어 면역만 형성할 뿐 다른 혈청형에 대한 면역이 형성되지 않아 A형 바이러스가 침입하면 구제역이 발생하게 된다. 따라서, 본 발명에서는 국내에서 주로 발생되는 O형에 특이적으로 결합하거나, 또는 모든 혈청형에 광범위하게 결합하는 구제역 바이러스에 대한 단일클론항체를 개발하였다.Foot-and-mouth disease virus is classified into O, A, Asia1, C, SAT1, SAT2 and SAT3 according to the antigenic structure, and especially O-type foot-and-mouth disease occurs in Korea. Since the antigens of foot-and-mouth disease virus have distinct structures, it is impossible to detect another serotype or to form immunity using an antibody specific for one serotype. That is, when a diagnostic kit containing an antibody against FMDV type O is used for diagnosis of foot-and-mouth disease, foot-and-mouth disease diagnosis cannot be determined from an animal infected with foot-and-mouth disease other than type O. In addition, if an animal with suspected foot-and-mouth disease is inoculated with a vaccine (antibody) against type O FMDV, only protective immunity against type O is formed, and immunity against other serotypes is not formed, so that the invasion of type A virus causes foot and mouth disease. do. Therefore, in the present invention, a monoclonal antibody against foot-and-mouth disease virus that specifically binds to type O, or broadly binds to all serotypes, was developed.
본 명세서에서, "단일클론항체"는 하나의 항원 결정기(epotope)만을 인식하여 반응하는 특이성을 가지며, 단일 항체 형성세포로 형성되어 하나의 분자 구조로 이루어진 항체를 의미한다. 완전한 항체는 전체적으로 Y 모양을 하며, 2개의 긴 중쇄(heavy chain; H)과 2개의 짧은 경쇄(light chain; L)로 구성된다. 각 중쇄와 경쇄는 서로 황화결합으로 연결되며, 항원과 반응하는 부위인 가변영역(variable region; V)와 효과기능을 발현하는 부위인 불변영역(constant region; C)로 구분된다. 가변영역에는 항원과 특이적인 결합을 형성할 수 있도록 가변영역의 구조를 결정하며 항체결합세기를 조절하는 상보성 결정 부위(complementarity-determining region; CDR)가 존재한다. 일례로, O형 FMDV의 항체는 O형에 대해 특이적으로 결합하며, 완전한 형태의 항체뿐만 아니라 CDR을 포함하는 항체 분자의 항원 결합 단편(antigen binding fragment; Fab)일 수 있다.In the present specification, "monoclonal antibody" refers to an antibody that has a specificity to recognize and react to only one antigenic determinant (epotope), and is formed into a single antibody-forming cell to have a single molecular structure. Complete antibodies are entirely Y-shaped and consist of two long heavy chains (H) and two short light chains (L). Each heavy chain and light chain are linked to each other by a sulfiding bond, and are divided into a variable region (V), which is a site that reacts with an antigen, and a constant region (C), which is a site that expresses an effect function. In the variable region, there is a complementarity-determining region (CDR) that determines the structure of the variable region to form a specific binding with an antigen and controls the strength of antibody binding. For example, an antibody of type O FMDV specifically binds to type O, and may be an antigen binding fragment (Fab) of an antibody molecule including a CDR as well as a complete antibody.
본 발명의 다른 양상은 서열번호 7의 아미노산 서열을 포함하는 구제역 바이러스 혈청형 O형에 특이적으로 결합하는 단일클론항체의 중쇄 가변영역을 코딩하는 폴리뉴클레오티드 및 서열번호 8의 아미노산 서열을 포함하는 구제역 바이러스 혈청형 O형에 특이적으로 결합하는 단일클론항체의 경쇄 가변영역을 코딩하는 폴리뉴클레오티드를 제공한다.Another aspect of the present invention is a polynucleotide encoding a heavy chain variable region of a monoclonal antibody specifically binding to foot-and-mouth disease virus serotype O comprising the amino acid sequence of SEQ ID NO: 7 and foot-and-mouth disease comprising the amino acid sequence of SEQ ID NO: 8 A polynucleotide encoding the light chain variable region of a monoclonal antibody that specifically binds to viral serotype O is provided.
본 발명의 또 다른 양상은 상기 폴리뉴클레오티드를 포함하는 재조합 벡터 및 상기 재조합 벡터를 포함하는 숙주세포를 제공한다.Another aspect of the present invention provides a recombinant vector comprising the polynucleotide and a host cell comprising the recombinant vector.
본 발명의 다른 양상은 서열번호 15의 아미노산 서열을 포함하는 구제역 바이러스 혈청형 O, A, Asia1, C, SAT1, SAT2 및 SAT3으로 이루어진 군으로부터 선택되는 하나 이상의 혈청형에 특이적으로 결합하는 단일클론항체의 중쇄 가변영역을 코딩하는 폴리뉴클레오티드 및 서열번호 16의 아미노산 서열을 포함하는 구제역 바이러스 혈청형 O, A, Asia1, C, SAT1, SAT2 및 SAT3으로 이루어진 군으로부터 선택되는 하나 이상의 혈청형에 특이적으로 결합하는 단일클론항체의 경쇄 가변영역을 코딩하는 폴리뉴클레오티드를 제공한다.Another aspect of the present invention is a monoclonal that specifically binds to one or more serotypes selected from the group consisting of foot-and-mouth disease virus serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 comprising the amino acid sequence of SEQ ID NO: 15. Specific to one or more serotypes selected from the group consisting of foot-and-mouth disease virus serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 comprising the polynucleotide encoding the heavy chain variable region of the antibody and the amino acid sequence of SEQ ID NO: 16 A polynucleotide encoding the light chain variable region of a monoclonal antibody that binds to is provided.
본 발명의 또 다른 양상은 상기 폴리뉴클레오티드를 포함하는 재조합 벡터 및 상기 재조합 벡터를 포함하는 숙주세포를 제공한다.Another aspect of the present invention provides a recombinant vector comprising the polynucleotide and a host cell comprising the recombinant vector.
상기 단일클론항체는 당 업계에 공지된 방법을 사용하여 제조될 수 있다. 일례로, 소의 혈액에서 분리된 말초 혈액 림프구(peripheral blood lymphocyte)에서 총 RNA를 추출하여 cDNA를 합성하고, 그 cDNA와 소 항체의 중쇄 가변영역 및 경쇄 가변영역에 특이적인 프라이머를 이용하여 발현 벡터를 제조한 후 그 발현 벡터를 숙주세포에 형질전환시키고, 그 중에서 O형 FMDV에 특이적으로 결합 또는 모든 혈청형에 광범위하게 결합하는 항체만을 선택함으로써 FMDV 단일클론항체를 제조할 수 있다. The monoclonal antibody can be prepared using a method known in the art. For example, cDNA is synthesized by extracting total RNA from peripheral blood lymphocytes isolated from bovine blood, and an expression vector is prepared by using primers specific for the heavy and light chain variable regions of the cDNA and bovine antibodies. After preparation, the expression vector is transformed into host cells, and among them, only antibodies that specifically bind to O-type FMDV or broadly bind to all serotypes can be selected to prepare FMDV monoclonal antibodies.
상기 발현 벡터는 하나의 벡터에서 중쇄 가변영역 및 경쇄 가변영역이 동시에 발현되는 벡터 시스템이거나, 또는 각각의 벡터에서 중쇄 가변영역 및 경쇄 가변영역이 발현되는 벡터 시스템일 수 있다. 각각의 벡터를 이용하는 시스템인 경우에는 두 벡터를 동시 형질전환(co-transfomation) 또는 표적 형질전환(targeted transformation)하여 숙주세포에 도입시킬 수 있다.The expression vector may be a vector system in which the heavy chain variable region and the light chain variable region are simultaneously expressed in one vector, or a vector system in which the heavy chain variable region and the light chain variable region are expressed in each vector. In the case of a system using each vector, the two vectors can be simultaneously transformed (co-transfomation) or targeted transformation to be introduced into host cells.
상기 숙주세포는 발현 벡터를 안정화시켜 연속적으로 클로닝 또는 발현시킬 수 있는 세포로서 당 업계에 공지된 숙주세포를 제한 없이 사용할 수 있다. 일례로, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, 바실러스 속 균주, 효모(Saccharomyce cerevisiae), 곤충 세포, 식물 세포, Sp2/0, CHO(chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK 등의 동물 세포일 수 있다.The host cell is a cell that can be continuously cloned or expressed by stabilizing an expression vector, and host cells known in the art may 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 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, MDCK, etc. may be animal cells.
최종 얻어진 FMDV 단일클론항체는 IgG1, IgG2a, IgG2b, IgG3, IgA, IgM 타입일 수 있다.The finally obtained FMDV monoclonal antibody may be of IgG 1 , IgG 2a , IgG 2b , Ig G3 , IgA, IgM type.
본 발명의 일 구체예에 따르면, 상기 혈청형 O형에 특이적으로 결합하는 단일클론항체는 서열번호 1로 이루어진 중쇄 CDR1(complementarity determining region 1), 서열번호 2로 이루어진 중쇄 CDR2(complementarity determining region 2) 및 서열번호 3으로 이루어진 중쇄 CDR3(complementarity determining region 3)을 포함하는 중쇄 가변영역; 및According to one embodiment of the present invention, the monoclonal antibody specifically binding to the serotype O is a heavy chain CDR1 (complementarity determining region 1) consisting of SEQ ID NO: 1, and a heavy chain CDR2 (complementarity determining region 2 consisting of SEQ ID NO: 2). ) And a heavy chain variable region comprising a heavy chain CDR3 (complementarity determining region 3) consisting of SEQ ID NO: 3; And
서열번호 4로 이루어진 경쇄 CDR1(complementarity determining region 1), 서열번호 5로 이루어진 경쇄 CDR2(complementarity determining region 2) 및 서열번호 6으로 이루어진 경쇄 CDR3(complementarity determining region 3)을 포함하는 경쇄 가변영역을 포함할 수 있으며, 바람직하게는, 상기 단일클론항체는 서열번호 7의 아미노산 서열을 포함하는 중쇄 가변영역 및 서열번호 8의 아미노산 서열을 포함하는 경쇄 가변영역을 포함할 수 있다.A light chain variable region comprising a light chain CDR1 (complementarity determining region 1) consisting of SEQ ID NO: 4, a light chain CDR2 (complementarity determining region 2) consisting of SEQ ID NO: 5, and a light chain CDR3 (complementarity determining region 3) consisting of SEQ ID NO: 6 It may, and preferably, the monoclonal antibody may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.
본 발명의 다른 구체예에 따르면, 상기 혈청형 O, A, Asia1, C, SAT1, SAT2 및 SAT3으로 이루어진 군으로부터 선택되는 하나 이상의 혈청형에 결합하는 단일클론항체는 서열번호 9로 이루어진 중쇄 CDR1(complementarity determining region 1), 서열번호 10으로 이루어진 중쇄 CDR2(complementarity determining region 2) 및 서열번호 11로 이루어진 중쇄 CDR3(complementarity determining region 3)을 포함하는 중쇄 가변영역; 및According to another embodiment of the present invention, the monoclonal antibody binding to one or more serotypes selected from the group consisting of the serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 is a heavy chain CDR1 consisting of SEQ ID NO:9 ( a heavy chain variable region comprising a complementarity determining region 1), a heavy chain CDR2 (complementarity determining region 2) consisting of SEQ ID NO: 10, and a heavy chain CDR3 (complementarity determining region 3) consisting of SEQ ID NO: 11; And
서열번호 12로 이루어진 경쇄 CDR1(complementarity determining region 1), 서열번호 13으로 이루어진 경쇄 CDR2(complementarity determining region 2) 및 서열번호 14로 이루어진 경쇄 CDR3(complementarity determining region 3)을 포함하는 경쇄 가변영역을 포함할 수 있으며, 바람직하게는, 상기 단일클론항체는 서열번호 15의 아미노산 서열을 포함하는 중쇄 가변영역 및 서열번호 16의 아미노산 서열을 포함하는 경쇄 가변영역을 포함할 수 있다.A light chain variable region comprising a light chain CDR1 (complementarity determining region 1) consisting of SEQ ID NO: 12, a light chain CDR2 (complementarity determining region 2) consisting of SEQ ID NO: 13, and a light chain CDR3 (complementarity determining region 3) consisting of SEQ ID NO: 14 It may, and preferably, the monoclonal antibody may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.
본 명세서에서, "항체(antibody)"는 구제역 바이러스 혈청형 O형 또는 혈청형 O, A, Asia1, C, SAT1, SAT2 및 SAT3으로 이루어진 군으로부터 선택되는 하나 이상의 혈청형에 대한 특이 항체로서, 상기 혈청형에 대해 특이적으로 결합하며, 완전한 항체 형태뿐만 아니라 항체 분자의 항원 결합 단편을 포함하는 것으로 해석된다. 완전한 항체에 대한 설명은 상기 언급한 바와 같다.In the present specification, "antibody" is a specific antibody against one or more serotypes selected from the group consisting of foot-and-mouth disease virus serotype O or serotype O, A, Asia1, C, SAT1, SAT2 and SAT3, wherein It specifically binds to the serotype and is interpreted to include the antigen-binding fragment of the antibody molecule as well as the complete antibody form. The description of the complete antibody is as mentioned above.
본 명세서에서, "중쇄(heavy chain)"는 항원에 특이성을 부여하기 위해 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VH 및 3개의 불변영역 도메인 CH1, CH2 및 CH3를 포함하는 전장 중쇄 및 이의 단편을 모두 포함하는 의미로 해석되며, "경쇄(light chain)"는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VL 및 불변영역 도메인 CL을 포함하는 전장 경쇄 및 이의 단편을 모두 포함하는 의미로 해석된다.In the present specification, "heavy chain" refers to a full-length heavy chain 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 impart specificity to an antigen. And it is interpreted to include both fragments thereof, and "light chain" includes a variable region domain VL and a constant region domain CL comprising an amino acid sequence having a sufficient variable region sequence to impart specificity to an antigen. It is interpreted to include both full-length light chains and fragments thereof.
본 명세서에서, "CDR(complementarity determining region)"은 면역글로불린의 중쇄 및 경쇄의 고가변영역(hypervariable region)의 아미노산 서열을 의미한다. 중쇄 및 경쇄는 각각 3개의 CDR을 포함할 수 있다(CDRH1, CDRH2, CDRH3 및 CDRL1, CDRL2, CDRL3). 상기 CDR은 항체가 항원 또는 에피토프에 결합하는 데 있어서 주요한 접촉 잔기를 제공할 수 있다.In the present specification, "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region of the heavy and light chains 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.
본 명세서에서, "항원 결합 단편"은 면역글로불린 전체 구조에 대한 그의 단편으로, 항원이 결합할 수 있는 부분을 포함하는 폴리펩티드의 일부를 의미한다. 예를 들어, F(ab')2, Fab', Fab, Fv 또는 scFv일 수 있으나, 이에 한정하지 않는다. 상기 항원 결합 단편 중 Fab는 경쇄 및 중쇄의 가변영역과 경쇄의 불변영역 및 중쇄의 첫 번째 불변영역(CH1)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 C-말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역(hinge region)을 가진다는 점에서 Fab와 차이가 있다. F(ab')2 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv는 중쇄 가변부위 및 경쇄 가변부위만을 가지고 있는 최소의 항체조각으로 Fv 단편을 생성하는 재조합 기술은 당업계에 널리 공지되어 있다. 이중쇄 Fv(two-chain Fv)는 비공유 결합으로 중쇄 가변부위와 경쇄 가변부위가 연결되어 있고 단쇄 Fv(single-chain Fv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변영역과 단쇄의 가변영역이 공유 결합으로 연결되거나 또는 C-말단에서 바로 연결되어 있어서 이중쇄 Fv와 같이 다이머와 같은 구조를 이룰 수 있다. 상기 항원 결합 단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고 (예를 들어, 전체 항체를 파파인으로 제한 절단하면 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab')2 단편을 얻을 수 있다), 유전자 재조합 기술을 통하여 제작할 수 있다.In the present specification, "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 (CH1) of a heavy chain, 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 CH1 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 connected by a bond or is directly connected 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.
본 발명의 다른 양상은 상기 단일클론항체를 포함하는 구제역 진단용 키트 및 이를 이용하여 구제역 바이러스를 신속하고 정확하게 진단할 수 있는 구제역의 진단 방법을 제공한다.Another aspect of the present invention provides a kit for diagnosis of foot-and-mouth disease, including the monoclonal antibody, and a method for diagnosing foot-and-mouth disease, which can quickly and accurately diagnose foot-and-mouth disease virus using the same.
상기 키트는 FMDV 단일클론항체를 사용하여 구제역 바이러스의 감염 여부를 정량적으로 측정하는데 사용될 수 있으며, 항원-항체 반응을 이용한 효소 면역분석법(enzyme immunoassay), 방사선 면역분석법(radio immunoassay), 형광 면역분석법(fluorescence immunoassay) 등의 방법을 이용할 수 있다. The kit can be used to quantitatively measure the infection of foot-and-mouth disease virus using FMDV monoclonal antibody, and enzyme immunoassay using an antigen-antibody reaction (enzyme immunoassay), radioimmunoassay, fluorescence immunoassay ( fluorescence immunoassay) or the like can be used.
상기 진단 방법은 FMDV 단일클론항체를 직접 사용하거나, 또는 FMDV 단일클론항체를 이용한 진단용 키트를 사용하여 구제역을 진단할 수 있다. 구체적으로, 소 또는 돼지 등의 구제역 의심 동물로부터 분리된 생물학적 시료를 FMDV 단일클론항체와 반응시켜 항원-항체 반응으로 검출할 수 있다. 이때, 생물학적 시료는 혈액, 혈청, 혈장, 소변, 눈물, 침, 젖 등일 수 있으며, 채취가 용이하도록 체외로 분비되는 체액을 이용하는 것이 바람직하다.The diagnostic method may diagnose foot-and-mouth disease using either an FMDV monoclonal antibody directly, or a diagnostic kit using an FMDV monoclonal antibody. Specifically, a biological sample isolated from an animal suspected of foot-and-mouth disease, such as a cow or a pig, may be reacted with an FMDV monoclonal antibody to detect an antigen-antibody reaction. At this time, 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 to facilitate collection.
이때, 사용되는 FMDV 단일클론항체에 따라 혈청형을 분석할 수 있다. O형 FMDV에 특이적으로 결합하는 항체 (O-serotype) 또는 이를 포함한 키트를 사용할 경우에는 O형 구제역을 진단할 수 있으나, O형이 아닌 다른 혈청형 구제역을 진단할 수 없다. 반면, 광범위 혈청형 FMDV에 결합하는 항체 (Pan-serotype) 또는 이를 포함하는 키트를 사용할 경우에는 O형, A형 등의 혈청형에 관계없이 모든 혈청형에 대한 구제역을 진단할 수 있다.At this time, the serotype can be analyzed according to the FMDV monoclonal antibody used. In the case of using an antibody that specifically binds to type O FMDV (O-serotype) or a kit containing the same, type O foot-and-mouth disease can be diagnosed, but serotype foot-and-mouth disease other than type O cannot be diagnosed. On the other hand, in the case of using an antibody (Pan-serotype) that binds to the broad serotype FMDV or a kit including the same, foot-and-mouth disease for all serotypes can be diagnosed regardless of serotypes such as O type and A type.
본 발명의 또 다른 양상은 전술한 단일클론항체를 인간을 제외한 동물에게 투여하는 단계를 포함하는 구제역의 예방 또는 치료 방법을 제공한다.Another aspect of the present invention provides a method for preventing or treating foot-and-mouth disease, comprising administering the aforementioned monoclonal antibody to animals other than humans.
상기 예방 또는 치료 방법은 이미 구제역 바이러스에 감염되었거나, 감염 가능성이 높은 우제류를 대상으로 FMDV 단일클론항체를 투여하여 구제역 바이러스에 대한 면역을 형성시킬 수 있다. 이때, 사용되는 FMDV 단일클론항체에 따라 해당 혈청형에 대한 면역을 형성시킬 수 있다. O형 FMDV에 특이적으로 결합하는 항체 (O-serotype)를 투여할 경우에는 O형 FMDV에 대해 면역이 형성되어 O형 구제역에 대한 예방 또는 치료 효과를 나타낼 수 있으나, O형이 아닌 다른 혈청형 구제역에 대해서는 예방 또는 치료 효과를 기대할 수 없다. 반면, 광범위 혈청형 FMDV에 결합하는 항체 (Pan-serotype)를 투여할 경우에는 O형, A형 등의 혈청형에 관계없이 모든 혈청형 구제역에 대해 면역이 형성되어, 구제역의 예방 또는 치료 효과를 기대할 수 있다.The prevention or treatment method is already infected with the foot-and-mouth disease virus, or by administering an FMDV monoclonal antibody to a right-handed animal with a high possibility of infection, thereby forming immunity against the foot-and-mouth disease virus. At this time, immunity against the serotype can be formed depending on the FMDV monoclonal antibody used. When an antibody that specifically binds to type O FMDV (O-serotype) is administered, immunity is formed against O type FMDV, which may show a preventive or therapeutic effect against O type foot-and-mouth disease, but other serotypes other than O type No preventive or therapeutic effect can be expected for foot-and-mouth disease. On the other hand, when an antibody (Pan-serotype) that binds to the broad serotype FMDV is administered, immunity is formed against all serotypes and foot-and-mouth disease irrespective of serotypes such as O-type and A-type, thereby preventing or treating foot-and-mouth disease. Can be expected.
본 발명에 따른 구제역 바이러스에 특이적으로 결합하는 단일클론항체는 구제역 바이러스의 항원과 특이적으로 결합하여 구제역 바이러스의 검출용 항체로 사용될 수 있으며, 구제역 바이러스에 대한 중화능이 우수하고 소에서 유래되어 소의 면역 거부 반응 발생에 대한 우려가 없으므로 긴급시 구제역 예방용 및 치료용 백신으로 사용될 수 있다.The monoclonal antibody specifically binding to foot-and-mouth disease virus according to the present invention specifically binds to the antigen of foot-and-mouth disease virus and can be used as an antibody for detection of foot-and-mouth disease virus, and has excellent neutralizing ability against foot-and-mouth disease virus and is derived from cattle. 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.
도 1은 본 발명의 일 구체예에 따른 실험 결과로, O형 FMDV (O manisa) 및 A형 FMDV (A22 Iraq)에 대해 FMDV 단일클론항체의 혈청형 특이성을 분석한 그래프이다.
도 2는 본 발명의 일 구체예에 따른 실험 결과로, FMDV 단일클론항체를 생산하는 Bv22 클론 및 Bv7 클론의 혈청형을 분석한 그래프이다.1 is an experimental result according to an embodiment of the present invention, a graph analyzing the serotype specificity of FMDV monoclonal antibodies against O type FMDV (O manisa) and A type FMDV (A22 Iraq).
2 is an experimental result according to an embodiment of the present invention, a graph analyzing the serotypes of Bv22 clones and Bv7 clones producing FMDV monoclonal antibodies.
이하, 첨부된 도면을 참조하며 본 발명에 따른 FMDV 단일클론항체를 보다 상세하게 설명한다. 그러나, 이러한 설명은 본 발명의 이해를 돕기 위하여 예시적으로 제시된 것일 뿐, 본 발명의 범위가 이러한 예시적인 설명에 의하여 제한되는 것은 아니다.Hereinafter, the FMDV monoclonal antibody according to the present invention will be described in more detail with reference to the accompanying drawings. However, these descriptions are provided by way of example only to aid understanding of the present invention, and the scope of the present invention is not limited by these exemplary descriptions.
실시예 1. FMDV 단일클론항체의 제조Example 1. Preparation of FMDV monoclonal antibody
국내 도축장에서 확보한 한우의 혈액으로부터 원심분리하여 PBL를 분리하였다. RNA 추출 키트 (TRIzol, Invitrogen)를 이용하여 PBL에서 total RNA를 추출한 후 이로부터 RT-PCR 키트 (SuperScript IV First-Strand Synthesis System, Invitrogen)를 이용하여 cDNA를 합성하였다. PBL was separated by centrifugation from the blood of Korean cattle obtained at domestic slaughterhouses. After total RNA was extracted from PBL using an RNA extraction kit (TRIzol, Invitrogen), cDNA was synthesized using an RT-PCR kit (SuperScript IV First-Strand Synthesis System, Invitrogen).
합성된 cDNA로부터 소 항체 가변영역에 특이적인 프라이머 세트 (하기 표 1 참조)를 이용하여 VH, Vk, Vλ 유전자를 각각 증폭하였고, 중쇄 가변영역 유전자 (VH)와 경쇄 가변영역 유전자 (Vk, Vλ)를 G4S linker 서열을 이용하여 scFv 형태로 연결하였다. 합성된 library scFv 서열은 제한효소 SfiI를 이용하여 자르고 동일한 SfiI로 잘라진 pDR-D1 벡터에 T4 Ligase를 이용하여 연결하였다. 이 ligate DNA를 electrocompetent ER2738 E. coli에 Electroporator (Bio-rad)를 이용하여 도입하였다. 항체 라이브러리가 도입된 대장균으로부터 항체가 디스플레이된 재조합 파지(phage)를 얻기 위해 Helper phage (VCSM13, Stratagene)를 superinfection시키고 밤새 배양하였고, 최종적으로 PEG8000 (Sigma)을 이용하여 상등액에 존재하는 파지를 농축하였다. From the synthesized cDNA, VH, Vk, and Vλ genes were amplified, respectively, using a primer set specific to the small antibody variable region (see Table 1 below), and heavy chain variable region genes (VH) and light chain variable region genes (Vk, Vλ) Was linked in the form of scFv using the G4S linker sequence. The synthesized library scFv sequence was cut using the restriction enzyme SfiI and ligated to the pDR-D1 vector cut with the same SfiI using T4 Ligase. This ligate DNA was introduced into electrocompetent ER2738 E. coli using an Electroporator (Bio-rad). Helper phage (VCSM13, Stratagene) was superinfected and cultured overnight to obtain a recombinant phage displaying an antibody from E. coli into which the antibody library was introduced, and finally, the phage present in the supernatant was concentrated using PEG8000 (Sigma). .
농축된 scFv가 표면발현된 라이브러리 파지를 BSA가 코팅된 immunotube에서 30분간 반응시켜서 비특이적 결합의 파지를 제거하고, 남은 파지를 FMDV 항원이 코팅된 immunotube에서 2시간 동안 반응시켰다. 5회의 반복적인 PBST 세척을 통해 결합하지 않은 파지를 제거하고, 최종적으로 결합하고 있는 파지를 0.1M Glycin HCl (pH 2.7)을 이용하여 용출하였다. 1M Tris-Cl (pH 8.0)으로 중화시킨 파지를 대장균에 감염시켜 다시 파지를 증폭한 후, 다음 회차의 패닝을 진행하였다. 패닝 회차가 진행될수록 세척 횟수를 증가시켜 특이적이고 강한 결합의 파지만 선택적으로 증폭되도록 하였다. The concentrated scFv surface-expressed library phage was reacted for 30 minutes in a BSA-coated immunotube to remove non-specific binding phage, and the remaining phage was reacted in an FMDV antigen-coated immunotube for 2 hours. Unbound phage was removed through repeated washing of PBST 5 times, and finally bound phage was eluted with 0.1M Glycin HCl (pH 2.7). The phage neutralized with 1M Tris-Cl (pH 8.0) was infected with E. coli to amplify the phage again, followed by panning of the next round. As the panning cycle proceeded, the number of washing was increased so that only specific and strong bound phages were selectively amplified.
4차 패닝 후 96개의 클론을 무작위로 선정하여 96well deep well plate (Bioneer)에서 배양한 후 helper phage를 이용하여 각 클론에 대한 재조합 파지를 얻었다. 이를 FMDV 항원과 BSA가 코팅된 microtiter plate의 well에 결합시키고, 개별 재조합 파지 클론들의 결합능을 anti-M13-HRP 항체를 이용하여 확인하였다. 대조군 (BSA)에 결합하지 않고 FMDV에만 특이적으로 결합하는 항체 클론들을 확인하였고, 서열 분석을 통해 Unique한 서열의 항체 클론들을 선별하였다. 선별된 항체 (O-serotype: Bv 22, Pan-serotype: Bv7)의 서열은 하기 표 2와 같다.After the fourth panning, 96 clones were randomly selected, cultured in a 96 well deep well plate (Bioneer), and recombinant phage for each clone was obtained using helper phage. This was bound to a well of a microtiter plate coated with FMDV antigen and BSA, and the binding capacity of individual recombinant phage clones was confirmed using an anti-M13-HRP antibody. Antibody clones that specifically bind only to FMDV without binding to the control (BSA) were identified, and antibody clones with a unique sequence were selected through sequence analysis. The sequence of the selected antibody (O-serotype: Bv 22, Pan-serotype: Bv7) is shown in Table 2 below.
(서열번호 17)BVH1
(SEQ ID NO: 17)
(서열번호 18)BVH2
(SEQ ID NO: 18)
(서열번호 19)BVH3
(SEQ ID NO: 19)
(서열번호 20)BJH1R
(SEQ ID NO: 20)
(서열번호 21)BJH2R
(SEQ ID NO: 21)
(서열번호 22)BJH3R
(SEQ ID NO: 22)
(서열번호 23)BVk1
(SEQ ID NO: 23)
(서열번호 24)BVk2
(SEQ ID NO: 24)
(서열번호 25)BJk1R
(SEQ ID NO: 25)
(서열번호 26)BVL1
(SEQ ID NO: 26)
(서열번호 27)BVL2
(SEQ ID NO: 27)
(서열번호 28)BVL3
(SEQ ID NO: 28)
(서열번호 29)BVL4
(SEQ ID NO: 29)
(서열번호 30)BVL5
(SEQ ID NO: 30)
(서열번호 31)BVL6
(SEQ ID NO: 31)
(서열번호 32)BVL7
(SEQ ID NO: 32)
(서열번호 33)BJL1R
(SEQ ID NO: 33)
(서열번호 34)BJL2R
(SEQ ID NO: 34)
(서열번호 35)scFv-F
(SEQ ID NO: 35)
(서열번호 36)scFv-R
(SEQ ID NO: 36)
(서열번호 37)VH
(SEQ ID NO: 37)
(서열번호 38)VL
(SEQ ID NO: 38)
(서열번호 39)VH
(SEQ ID NO: 39)
(서열번호 40)VL
(SEQ ID NO: 40)
실험예 1. 단일클론항체가 결합하는 FMDV 혈청형 분석Experimental Example 1. FMDV serotype analysis to which monoclonal antibody binds
선별된 단일클론항체의 FMDV 혈청형 특이성을 확인하기 위해, O형 FMDV 항원 (O manisa) 및 A형 FMDV 항원 (A22 Iraq)에 대하여 실시예 1에서 선별된 단일클론항체의 결합능을 분석하였다.In order to confirm the FMDV serotype specificity of the selected monoclonal antibodies, the binding ability of the monoclonal antibodies selected in Example 1 to the O type FMDV antigen (O manisa) and A type FMDV antigen (A22 Iraq) was analyzed.
먼저, 정밀한 항체 결합능 분석을 위해 항체 클론들의 scFv 서열을 동물세포 발현벡터 pDR-OriP-Fc1에 클로닝하여 293E 세포에서 scFv-Fc형태로 발현하고, protein G affinity column (GE Healthcare)을 이용하여 정제하였다. scFv-Fc항체 2.5 ng 를 각각 O형, A형 항원이 코팅된 microtiter well에 반응시키고 결합된 scFv-Fc를 anti-human IgG (Fc specific)-HRP (Jackson ImmunoResearch Laboratories)를 이용하여 측정하였다. 그 결과를 도 1에 나타내었다. First, for precise antibody binding ability analysis, the scFv sequences of antibody clones were cloned into the animal cell expression vector pDR-OriP-Fc1, expressed in the form of scFv-Fc in 293E cells, and purified using a protein G affinity column (GE Healthcare). . 2.5 ng of the scFv-Fc antibody was reacted to microtiter wells coated with O and A type antigens, respectively, and the bound scFv-Fc was measured using anti-human IgG (Fc specific)-HRP (Jackson ImmunoResearch Laboratories). The results are shown in FIG. 1.
도 1에서 보는 바와 같이, O형 FMDV 항원에만 특이적으로 결합하는 클론과 O형 및 A형 FMDV 항원 모두에 결합하는 클론들이 존재함을 확인하였다.As shown in Figure 1, it was confirmed that there are clones that specifically bind only to type O FMDV antigen and clones that bind to both type O and type A FMDV antigens.
또한, FMDV O형, A형 이외에 Asia1형, SAT1형, SAT2형, SAT3형, C형에 대해서도 실시예 1에서 선별된 단일클론항체의 결합능을 분석하였다. 그 결과를 도 2에 나타내었다.In addition, the binding capacity of the monoclonal antibodies selected in Example 1 was also analyzed for the FMDV O type and A type as well as Asia 1 type, SAT 1 type, SAT 2 type, SAT 3 type, and C type. The results are shown in FIG. 2.
도 2에서 보는 바와 같이, Bv22 클론은 O형 FMDV에 특이적으로 결합 (O-serotype)하며, Bv7 클론은 광범위 혈청형 FMDV 결합성을 갖는 것 (Pan-serotype)으로 확인되었다. As shown in Figure 2, the Bv22 clone specifically binds to type O FMDV (O-serotype), and the Bv7 clone was confirmed to have broad serotype FMDV binding (Pan-serotype).
실험예 2. Competition ELISA 측정Experimental Example 2. Competition ELISA measurement
기존 FMDV 진단 키트 (예: PrioCHECK?? FMDV Type O Antibody ELISA Kit)는 competitive ELISA 방식으로, 마우스 유래의 FMDV 특이 단일클론항체가 동물에서 분리된 혈청에 존재하는 FMDV 항체와 경쟁하여 ELISA 플레이트에 코팅된 FMDV 항원과 결합하지 못하는 것을 탐지한다.Existing FMDV diagnostic kits (e.g., PrioCHECK?? FMDV Type O Antibody ELISA Kit) are competitive ELISA methods, where FMDV-specific monoclonal antibodies derived from mice compete with FMDV antibodies present in serum isolated from animals and coated on ELISA plates. Detect failure to bind with FMDV antigen.
실시예 1에서 제조된 Bv22 클론의 단일클론항체를 이용하여 FMDV 진단 키트에 적용 가능한지를 테스트하였고, 그 결과를 하기 표 3에 나타내었다.Using the monoclonal antibody of the Bv22 clone prepared in Example 1, it was tested whether it is applicable to the FMDV diagnostic kit, and the results are shown in Table 3 below.
상기 표 3과 같이, 진단 키트 내 마우스 유래의 FMDV 특이 단일클론항체 대신 실시예 1의 FMDV 단일클론항체를 사용한 결과, 기존 키트 (대조군)와 유사한 값을 갖는 것으로 확인되었다. 또한, 소뿐만 아니라 돼지 혈청에 대해서도 기존 키트에 비해 개선된 결과를 나타내는 것으로 확인되었다.As shown in Table 3, as a result of using the FMDV monoclonal antibody of Example 1 instead of the mouse-derived FMDV-specific monoclonal antibody in the diagnostic kit, it was confirmed to have a value similar to that of the existing kit (control). In addition, it was confirmed that not only cattle but also pig serum showed improved results compared to the existing kit.
<110> Korea Research Institute of Bioscience and Biotechnology <120> Monoclonal Antibody for Detecting Foot and Mouth Disease Virus, and Uses Thereof <130> PN190098 <160> 40 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> O-serotype heavy chain CDR1 <400> 1 Asn Tyr Ala Val Asn 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> O-serotype heavy chain CDR2 <400> 2 Gly Ile Ser Arg Ser Gly Asn Lys Gly Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> O-serotype heavy chain CDR3 <400> 3 Cys Ser His Glu Tyr Ala Asn Tyr Ala Cys Tyr Asp Phe Glu Asp Glu 1 5 10 15 Ser Tyr Phe Asp Ala 20 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> O-serotype light chain CDR1 <400> 4 Ser Gly Ser Ser Gly Asn Val Gly Asn Gly Tyr Val Ser 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> O-serotype light chain CDR2 <400> 5 Gly Asp Thr Ser Arg Ala Ser 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> O-serotype light chain CDR3 <400> 6 Ala Ala His Asp Ser Ser Ile Asn Asn Gly Val 1 5 10 <210> 7 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> O-serotype heavy chain <400> 7 Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Val Asn Asn Tyr 20 25 30 Ala Val Asn Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Gly Ile Ser Arg Ser Gly Asn Lys Gly Tyr Asn Pro Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Ser Ile Asn Asn Val Ser Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Lys Cys Ser His Glu Tyr Ala Asn Tyr Ala Cys Tyr Asp Phe Glu Asp 100 105 110 Glu Ser Tyr Phe Asp Ala Trp Gly Gln Gly Leu Arg Val Thr Val Ser 115 120 125 Ser <210> 8 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> O-serotype light chain <400> 8 Gln Pro Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15 Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Gly Asn Val Gly Asn Gly 20 25 30 Tyr Val Ser Trp Tyr Gln Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu 35 40 45 Ile Tyr Gly Asp Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Asn Ser Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ala His Asp Ser Ser Ile 85 90 95 Asn Asn Gly Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu 100 105 110 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype heavy chain CDR1 <400> 9 Thr Tyr Gly Val Asp 1 5 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype heavy chain CDR2 <400> 10 Gly Ile Ser Thr Asn Gly Val Pro Asp Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> 11 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype heavy chain CDR3 <400> 11 Asn Met Gly Asp Met Gly Ser Cys Tyr Ala Trp Ala Asn Gly Tyr Val 1 5 10 15 Asp Ala <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype light chain CDR1 <400> 12 Ser Gly Ser Arg Ser Asn Ile Gly Ser Tyr Gly Val Gly 1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype light chain CDR2 <400> 13 Ser Ala Thr Ser Arg Ala Ser 1 5 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype light chain CDR3 <400> 14 Ala Ala Tyr Asp Ser Ser Ser Asn Ala Val 1 5 10 <210> 15 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype heavy chain <400> 15 Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Arg Thr Tyr 20 25 30 Gly Val Asp Trp Val Arg Gln Ala Pro Gly Lys Ala Pro Glu Cys Leu 35 40 45 Gly Gly Ile Ser Thr Asn Gly Val Pro Asp Tyr Asn Pro Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Ser Leu Ser Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Lys Asn Met Gly Asp Met Gly Ser Cys Tyr Ala Trp Ala Asn Gly Tyr 100 105 110 Val Asp Ala Trp Gly Pro Gly Leu Gln Val Thr Val Ser Ser 115 120 125 <210> 16 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype light chain <400> 16 Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15 Arg Val Ser Ile Thr Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Tyr 20 25 30 Gly Val Gly Trp Tyr Gln Gln Val Pro Gly Ser Gly Pro Arg Thr Leu 35 40 45 Ile Tyr Ser Ala Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ala Tyr Asp Ser Ser Ser 85 90 95 Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu 100 105 <210> 17 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BVH1 primer <400> 17 gcggcccagc cggccatggc ccaggtgcag ctgcgggagt c 41 <210> 18 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BVH2 primer <400> 18 gcggcccagc cggccatggc ccaggtgcag ctgcaggagt c 41 <210> 19 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BVH3 primer <400> 19 gcggcccagc cggccatggc caaggtgcag ctgcaggagt c 41 <210> 20 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> BJH1R primer <400> 20 cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg agacggtgac 60 cagg 64 <210> 21 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> BJH2R primer <400> 21 cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg agacggtgac 60 ctcg 64 <210> 22 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> BJH3R primer <400> 22 cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg agacggtgac 60 cctg 64 <210> 23 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BVk1 primer <400> 23 ggatctggcg gcggcggctc ggatgttgtg ctgacccaga c 41 <210> 24 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BVk2 primer <400> 24 ggatctggcg gcggcggctc ggacatccag gtgacccagt c 41 <210> 25 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> BJk1R primer <400> 25 ctgctcgagg cctcccgggc ctttgatctc taccttggtt cc 42 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL1 primer <400> 26 ggatctggcg gcggcggctc gcaggctgtg ctgactcagc 40 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL2 primer <400> 27 ggatctggcg gcggcggctc gcaggatgtg ctgactcagc 40 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL3 primer <400> 28 ggatctggcg gcggcggctc gcagtctggc ctgactcagc 40 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL4 primer <400> 29 ggatctggcg gcggcggctc gtcttctcag ctgactcagc 40 <210> 30 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> BVL5 primer <400> 30 ggatctggcg gcggcggctc gtcctatgaa ctgacccag 39 <210> 31 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL6 primer <400> 31 ggatctggcg gcggcggctc gcagcctgtg ctgactcagc 40 <210> 32 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL7 primer <400> 32 ggatctggcg gcggcggctc gcagactgtg atccaggaac 40 <210> 33 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BJL1R primer <400> 33 ctgctcgagg cctcccgggc ccaggacggt cactctggtc c 41 <210> 34 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BJL2R primer <400> 34 ctgctcgagg cctcccgggc ccaggacggt cagtgtggtc c 41 <210> 35 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> scFv-F primer <400> 35 gacgacgacg acgacgcggc ccagccggcc atggcc 36 <210> 36 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> scFv-R primer <400> 36 gacgacgacg acgacctgct cgaggcctcc cgggcc 36 <210> 37 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> O-serotype VH <400> 37 caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60 acctgcacgg tctcgggatt ctcagtgaac aactatgctg taaactgggt ccgccaggct 120 ccagggaagg ctctggagtg gcttgggggt ataagcagga gtggaaacaa aggctataac 180 ccagccctga aatcccggct cagcatcacc aaggataatt ccaagagcca agtctctctg 240 tcaattaaca acgtatcacc tgaggacacg gccacatact attgtgcgaa gtgtagtcac 300 gagtatgcta attatgcgtg ctatgatttt gaagatgagt cctacttcga tgcctggggc 360 caaggacttc gggtcaccgt ctcctca 387 <210> 38 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> O-serotype VL <400> 38 cagcctgtgc tgactcagcc atcatccgtg tccgggtccc tgggccagag ggtctccatc 60 acctgctctg gaagcagcgg caatgttgga aatggatatg tgagctggta ccaactgatc 120 ccaggatcgg cccccagaac cctcatctat ggtgacacca gtcgagcctc gggggtcccc 180 gaccgattct ccggctccag gtctgggaac acagccacgc tgaccatcaa ctcgctccag 240 gccgaggacg aggcggatta tttctgtgca gctcatgaca gtagtatcaa taatggtgtt 300 ttcggcagcg ggaccacact gaccgtcctg 330 <210> 39 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Pan-serotype VH <400> 39 caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60 acctgcacgg tctctggatt ctcactgaga acgtatggtg tggactgggt ccgccaggct 120 ccagggaagg cgccggagtg tcttggtgga ataagtacta atggagtccc agactataac 180 ccagccctaa aatcccggct cagcatcacc aaggacaact ccaagaatca agtgtctctg 240 tcactgagca gcctgacaac tgaggacacg gccacatatt actgtgcgaa gaatatgggt 300 gatatgggta gctgttatgc ttgggcaaat ggttacgtcg atgcctgggg cccaggactc 360 caggtcaccg tctcctca 378 <210> 40 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> Pan-serotype VL <400> 40 caggctgtgc tgactcagcc gtcctccgtg tccggctccc tgggccagag ggtctccatc 60 acctgctctg gaagcagaag caacatcggt agttatggcg tgggctggta ccagcaggtc 120 ccaggatcgg gccccagaac cctcatctat agtgcgacca gtcgagcctc tggggtcccc 180 gaccgattct ccggctccag gtctgggaac acagctacgc tgaccatcag ctcgctccag 240 gccgaggacg aggcggatta tttctgtgca gcttatgaca gcagtagcaa tgctgttttc 300 ggcagcggga ccacactgac cgtcctg 327 <110> Korea Research Institute of Bioscience and Biotechnology <120> Monoclonal Antibody for Detecting Foot and Mouth Disease Virus, and Uses Thereof <130> PN190098 <160> 40 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> O-serotype heavy chain CDR1 <400> 1 Asn Tyr Ala Val Asn 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> O-serotype heavy chain CDR2 <400> 2 Gly Ile Ser Arg Ser Gly Asn Lys Gly Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> O-serotype heavy chain CDR3 <400> 3 Cys Ser His Glu Tyr Ala Asn Tyr Ala Cys Tyr Asp Phe Glu Asp Glu 1 5 10 15 Ser Tyr Phe Asp Ala 20 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> O-serotype light chain CDR1 <400> 4 Ser Gly Ser Ser Gly Asn Val Gly Asn Gly Tyr Val Ser 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> O-serotype light chain CDR2 <400> 5 Gly Asp Thr Ser Arg Ala Ser 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> O-serotype light chain CDR3 <400> 6 Ala Ala His Asp Ser Ser Ile Asn Asn Gly Val 1 5 10 <210> 7 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> O-serotype heavy chain <400> 7 Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Val Asn Asn Tyr 20 25 30 Ala Val Asn Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Gly Ile Ser Arg Ser Gly Asn Lys Gly Tyr Asn Pro Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Ser Ile Asn Asn Val Ser Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Lys Cys Ser His Glu Tyr Ala Asn Tyr Ala Cys Tyr Asp Phe Glu Asp 100 105 110 Glu Ser Tyr Phe Asp Ala Trp Gly Gln Gly Leu Arg Val Thr Val Ser 115 120 125 Ser <210> 8 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> O-serotype light chain <400> 8 Gln Pro Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15 Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Gly Asn Val Gly Asn Gly 20 25 30 Tyr Val Ser Trp Tyr Gln Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu 35 40 45 Ile Tyr Gly Asp Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Asn Ser Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ala His Asp Ser Ser Ile 85 90 95 Asn Asn Gly Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu 100 105 110 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype heavy chain CDR1 <400> 9 Thr Tyr Gly Val Asp 1 5 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype heavy chain CDR2 <400> 10 Gly Ile Ser Thr Asn Gly Val Pro Asp Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> 11 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype heavy chain CDR3 <400> 11 Asn Met Gly Asp Met Gly Ser Cys Tyr Ala Trp Ala Asn Gly Tyr Val 1 5 10 15 Asp Ala <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype light chain CDR1 <400> 12 Ser Gly Ser Arg Ser Asn Ile Gly Ser Tyr Gly Val Gly 1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype light chain CDR2 <400> 13 Ser Ala Thr Ser Arg Ala Ser 1 5 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype light chain CDR3 <400> 14 Ala Ala Tyr Asp Ser Ser Ser Asn Ala Val 1 5 10 <210> 15 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype heavy chain <400> 15 Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Arg Thr Tyr 20 25 30 Gly Val Asp Trp Val Arg Gln Ala Pro Gly Lys Ala Pro Glu Cys Leu 35 40 45 Gly Gly Ile Ser Thr Asn Gly Val Pro Asp Tyr Asn Pro Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Ser Leu Ser Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Lys Asn Met Gly Asp Met Gly Ser Cys Tyr Ala Trp Ala Asn Gly Tyr 100 105 110 Val Asp Ala Trp Gly Pro Gly Leu Gln Val Thr Val Ser Ser 115 120 125 <210> 16 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Pan-serotype light chain <400> 16 Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln 1 5 10 15 Arg Val Ser Ile Thr Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Tyr 20 25 30 Gly Val Gly Trp Tyr Gln Gln Val Pro Gly Ser Gly Pro Arg Thr Leu 35 40 45 Ile Tyr Ser Ala Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ala Tyr Asp Ser Ser Ser 85 90 95 Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu 100 105 <210> 17 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BVH1 primer <400> 17 gcggcccagc cggccatggc ccaggtgcag ctgcgggagt c 41 <210> 18 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BVH2 primer <400> 18 gcggcccagc cggccatggc ccaggtgcag ctgcaggagt c 41 <210> 19 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BVH3 primer <400> 19 gcggcccagc cggccatggc caaggtgcag ctgcaggagt c 41 <210> 20 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> BJH1R primer <400> 20 cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg agacggtgac 60 cagg 64 <210> 21 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> BJH2R primer <400> 21 cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg agacggtgac 60 ctcg 64 <210> 22 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> BJH3R primer <400> 22 cgagccgccg ccgccagatc cacctccacc tgaacctcct ccacctgagg agacggtgac 60 cctg 64 <210> 23 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BVk1 primer <400> 23 ggatctggcg gcggcggctc ggatgttgtg ctgacccaga c 41 <210> 24 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BVk2 primer <400> 24 ggatctggcg gcggcggctc ggacatccag gtgacccagt c 41 <210> 25 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> BJk1R primer <400> 25 ctgctcgagg cctcccgggc ctttgatctc taccttggtt cc 42 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL1 primer <400> 26 ggatctggcg gcggcggctc gcaggctgtg ctgactcagc 40 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL2 primer <400> 27 ggatctggcg gcggcggctc gcaggatgtg ctgactcagc 40 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL3 primer <400> 28 ggatctggcg gcggcggctc gcagtctggc ctgactcagc 40 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL4 primer <400> 29 ggatctggcg gcggcggctc gtcttctcag ctgactcagc 40 <210> 30 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> BVL5 primer <400> 30 ggatctggcg gcggcggctc gtcctatgaa ctgacccag 39 <210> 31 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL6 primer <400> 31 ggatctggcg gcggcggctc gcagcctgtg ctgactcagc 40 <210> 32 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BVL7 primer <400> 32 ggatctggcg gcggcggctc gcagactgtg atccaggaac 40 <210> 33 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BJL1R primer <400> 33 ctgctcgagg cctcccgggc ccaggacggt cactctggtc c 41 <210> 34 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BJL2R primer <400> 34 ctgctcgagg cctcccgggc ccaggacggt cagtgtggtc c 41 <210> 35 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> scFv-F primer <400> 35 gacgacgacg acgacgcggc ccagccggcc atggcc 36 <210> 36 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> scFv-R primer <400> 36 gacgacgacg acgacctgct cgaggcctcc cgggcc 36 <210> 37 <211> 387 <212> DNA <213> Artificial Sequence <220> <223> O-serotype VH <400> 37 caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60 acctgcacgg tctcgggatt ctcagtgaac aactatgctg taaactgggt ccgccaggct 120 ccagggaagg ctctggagtg gcttgggggt ataagcagga gtggaaacaa aggctataac 180 ccagccctga aatcccggct cagcatcacc aaggataatt ccaagagcca agtctctctg 240 tcaattaaca acgtatcacc tgaggacacg gccacatact attgtgcgaa gtgtagtcac 300 gagtatgcta attatgcgtg ctatgatttt gaagatgagt cctacttcga tgcctggggc 360 caaggacttc gggtcaccgt ctcctca 387 <210> 38 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> O-serotype VL <400> 38 cagcctgtgc tgactcagcc atcatccgtg tccgggtccc tgggccagag ggtctccatc 60 acctgctctg gaagcagcgg caatgttgga aatggatatg tgagctggta ccaactgatc 120 ccaggatcgg cccccagaac cctcatctat ggtgacacca gtcgagcctc gggggtcccc 180 gaccgattct ccggctccag gtctgggaac acagccacgc tgaccatcaa ctcgctccag 240 gccgaggacg aggcggatta tttctgtgca gctcatgaca gtagtatcaa taatggtgtt 300 ttcggcagcg ggaccacact gaccgtcctg 330 <210> 39 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Pan-serotype VH <400> 39 caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60 acctgcacgg tctctggatt ctcactgaga acgtatggtg tggactgggt ccgccaggct 120 ccagggaagg cgccggagtg tcttggtgga ataagtacta atggagtccc agactataac 180 ccagccctaa aatcccggct cagcatcacc aaggacaact ccaagaatca agtgtctctg 240 tcactgagca gcctgacaac tgaggacacg gccacatatt actgtgcgaa gaatatgggt 300 gatatgggta gctgttatgc ttgggcaaat ggttacgtcg atgcctgggg cccaggactc 360 caggtcaccg tctcctca 378 <210> 40 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> Pan-serotype VL <400> 40 caggctgtgc tgactcagcc gtcctccgtg tccggctccc tgggccagag ggtctccatc 60 acctgctctg gaagcagaag caacatcggt agttatggcg tgggctggta ccagcaggtc 120 ccaggatcgg gccccagaac cctcatctat agtgcgacca gtcgagcctc tggggtcccc 180 gaccgattct ccggctccag gtctgggaac acagctacgc tgaccatcag ctcgctccag 240 gccgaggacg aggcggatta tttctgtgca gcttatgaca gcagtagcaa tgctgttttc 300 ggcagcggga ccacactgac cgtcctg 327
Claims (17)
A monoclonal antibody that specifically binds to foot-and-mouth disease virus serotype O.
상기 단일클론항체는 서열번호 1로 이루어진 중쇄 CDR1(complementarity determining region 1), 서열번호 2로 이루어진 중쇄 CDR2(complementarity determining region 2) 및 서열번호 3으로 이루어진 중쇄 CDR3(complementarity determining region 3)을 포함하는 중쇄 가변영역; 및
서열번호 4로 이루어진 경쇄 CDR1(complementarity determining region 1), 서열번호 5로 이루어진 경쇄 CDR2(complementarity determining region 2) 및 서열번호 6으로 이루어진 경쇄 CDR3(complementarity determining region 3)을 포함하는 경쇄 가변영역을 포함하는 것인 단일클론항체.
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: 1, a heavy chain CDR2 (complementarity determining region 2) consisting of SEQ ID NO: 2, and a heavy chain CDR3 (complementarity determining region 3) consisting of SEQ ID NO: 3 Variable region; And
Comprising a light chain variable region comprising a light chain CDR1 (complementarity determining region 1) consisting of SEQ ID NO: 4, a light chain CDR2 (complementarity determining region 2) consisting of SEQ ID NO: 5, and a light chain CDR3 (complementarity determining region 3) consisting of SEQ ID NO: 6 Will monoclonal antibody.
상기 단일클론항체는 서열번호 7의 아미노산 서열을 포함하는 중쇄 가변영역 및 서열번호 8의 아미노산 서열을 포함하는 경쇄 가변영역을 포함하는 것인 단일클론항체.
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: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.
A polynucleotide encoding a heavy chain variable region of a monoclonal antibody specifically binding to foot-and-mouth disease virus serotype O comprising the amino acid sequence of SEQ ID NO: 7.
A polynucleotide encoding the light chain variable region of a monoclonal antibody that specifically binds to foot-and-mouth disease virus serotype O comprising the amino acid sequence of SEQ ID NO: 8.
A recombinant vector comprising the polynucleotide of claim 4 or 5.
A host cell comprising the recombinant vector of claim 6.
A monoclonal antibody that specifically binds to one or more serotypes selected from the group consisting of foot-and-mouth disease virus serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3.
상기 단일클론항체는 서열번호 9로 이루어진 중쇄 CDR1(complementarity determining region 1), 서열번호 10으로 이루어진 중쇄 CDR2(complementarity determining region 2) 및 서열번호 11로 이루어진 중쇄 CDR3(complementarity determining region 3)을 포함하는 중쇄 가변영역; 및
서열번호 12로 이루어진 경쇄 CDR1(complementarity determining region 1), 서열번호 13으로 이루어진 경쇄 CDR2(complementarity determining region 2) 및 서열번호 14로 이루어진 경쇄 CDR3(complementarity determining region 3)을 포함하는 경쇄 가변영역을 포함하는 것인 단일클론항체.
The method of claim 8,
The monoclonal antibody is a heavy chain comprising a heavy chain CDR1 (complementarity determining region 1) consisting of SEQ ID NO: 9, a heavy chain CDR2 (complementarity determining region 2) consisting of SEQ ID NO: 10, and a heavy chain CDR3 (complementarity determining region 3) consisting of SEQ ID NO: 11 Variable region; And
Comprising a light chain variable region comprising a light chain CDR1 (complementarity determining region 1) consisting of SEQ ID NO: 12, a light chain CDR2 (complementarity determining region 2) consisting of SEQ ID NO: 13, and a light chain CDR3 (complementarity determining region 3) consisting of SEQ ID NO: 14 Will monoclonal antibody.
상기 단일클론항체는 서열번호 15의 아미노산 서열을 포함하는 중쇄 가변영역 및 서열번호 16의 아미노산 서열을 포함하는 경쇄 가변영역을 포함하는 것인 단일클론항체.
The method of claim 8,
The monoclonal antibody is a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.
The heavy chain variable region of a monoclonal antibody specifically binding to one or more serotypes selected from the group consisting of foot-and-mouth disease virus serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 comprising the amino acid sequence of SEQ ID NO: 15 Encoding polynucleotide.
The light chain variable region of a monoclonal antibody specifically binding to one or more serotypes selected from the group consisting of foot-and-mouth disease virus serotypes O, A, Asia1, C, SAT1, SAT2 and SAT3 comprising the amino acid sequence of SEQ ID NO: 16 Encoding polynucleotide.
A recombinant vector comprising the polynucleotide of claim 11 or 12.
A host cell comprising the recombinant vector of claim 13.
Foot-and-mouth disease diagnosis kit comprising the monoclonal antibody of claim 1 or 8.
상기 키트는 소 구제역 또는 돼지 구제역을 진단하는 것인 구제역 진단용 키트.
The method of claim 15,
The kit is a kit for diagnosis of foot-and-mouth disease in bovine foot-and-mouth disease or porcine foot-and-mouth disease.
A method for preventing or treating foot-and-mouth disease, comprising administering the monoclonal antibody of claim 1 or 8 to animals other than humans.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118344474A (en) * | 2024-05-28 | 2024-07-16 | 中国农业科学院兰州兽医研究所(中国动物卫生与流行病学中心兰州分中心) | Foot-and-mouth disease virus type-O, type-A and Asia1 broad-spectrum neutralization swine monoclonal antibody and application thereof |
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| JPH08320324A (en) * | 1995-05-24 | 1996-12-03 | Norin Suisansyo Kachiku Eisei Shikenjo | Peptide for diganosis of foot-and-mouth disease and antigen, for diagnosis of foot-and-mouth disease, containing said peptide |
| KR20100001169A (en) * | 2008-06-26 | 2010-01-06 | 주식회사 제노바이오텍 | Hybridoma cell lines, monoclonal antibodies produced from the hybridoma cell lines, foot-and-mouth disease virus(fmdv) detection reagents, fmdv detection kits and detection method for fmdv neutralizing antibodies |
| KR20120132227A (en) * | 2011-05-27 | 2012-12-05 | 차의과학대학교 산학협력단 | Monoclonal antibody for detecting multiple type Foot and Mouth Disease Virus and method for detecting Foot and Mouth Disease Virus using the same |
| KR20190006154A (en) * | 2017-07-07 | 2019-01-17 | 주식회사 메디안디노스틱 | Monoclonal Antibodies for detecting Foot and Mouth Disease Virus serotype O and using the same |
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| CN118344474A (en) * | 2024-05-28 | 2024-07-16 | 中国农业科学院兰州兽医研究所(中国动物卫生与流行病学中心兰州分中心) | Foot-and-mouth disease virus type-O, type-A and Asia1 broad-spectrum neutralization swine monoclonal antibody and application thereof |
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