KR102009267B1 - Recombinant foot-and-mouth disease virus expressing protective antigen of type SAT3 ZIM - Google Patents

Recombinant foot-and-mouth disease virus expressing protective antigen of type SAT3 ZIM Download PDF

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KR102009267B1
KR102009267B1 KR1020170153664A KR20170153664A KR102009267B1 KR 102009267 B1 KR102009267 B1 KR 102009267B1 KR 1020170153664 A KR1020170153664 A KR 1020170153664A KR 20170153664 A KR20170153664 A KR 20170153664A KR 102009267 B1 KR102009267 B1 KR 102009267B1
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박종현
이광녕
유수화
김병한
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    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32151—Methods of production or purification of viral material

Abstract

본 발명은 구제역 SAT 3형 I 지역형 ZIM 방어항원을 발현하는 재조합 구제역 바이러스 및 이의 제조 방법에 관한 것으로, 구제역 SAT3형 혈청형에 대해 보다 넓은 범위에서 방어효과를 극대화할 수 있다.The present invention relates to a recombinant foot-and-mouth virus that expresses foot-and-mouth disease SAT type 3 regional type ZIM protective antigen, and a method for preparing the foot-and-mouth disease SAT3 serotype.

Description

구제역 SAT 3형 ZIM의 방어항원이 발현되는 재조합 바이러스 {Recombinant foot-and-mouth disease virus expressing protective antigen of type SAT3 ZIM}Recombinant foot-and-mouth disease virus expressing protective antigen of type SAT3 ZIM}

본 발명은 구제역 SAT 3형 I 지역형 ZIM의 방어항원이 발현되는 재조합 바이러스, 그 제조방법 및 백신으로서의 용도에 관한 것이다.The present invention relates to a recombinant virus expressing the protective antigen of foot-and-mouth disease SAT type I regional type ZIM, a preparation method thereof, and a use as a vaccine.

구제역(Foot and mouth disease; FMD)은 발굽이 둘로 갈라진 동물에 감염되는 바이러스성 수포질병으로 빠른 복제와 빠른 전파력이 특징이다. 이 질병은 그 경제적 중요성으로 인하여 세계동물보건기구(OIE)에 의하여 매우 중요한 동물 질병으로 분류되어 있으며, 발생시 국가간 교역이 불가능하여 축산물의 교역에 있어 가장 중요한 요소로 작용하고 있다.Foot and mouth disease (FMD) is a viral bullous disease that infects animals with two hoofs and is characterized by rapid replication and rapid spread. This disease is classified as a very important animal disease by the World Animal Health Organization (OIE) due to its economic importance, and it is the most important factor in the trade of livestock products because it is impossible to trade between countries when it occurs.

상기 구제역 병원체는 단일가닥의 양극성 RNA 바이러스로 피코나비리데(Piconaviridae)과, 아프소바이러스(Aphthovirus)속에 속하며 7개의 다른 혈청형(A, O, C, Asia1, SAT1, SAT3, SAT3)으로 분류되고 있다.The foot-and-mouth disease pathogen is a single-stranded bipolar RNA virus belonging to the genus Piconaviridae and Aphthovirus and classified into seven different serotypes (A, O, C, Asia1, SAT1, SAT3, SAT3). It is becoming.

구제역 바이러스(Foot and mouth disease virus) SAT3형 중에서 다른 특성을 지닌 6개의 지역형이 존재한다. SAT3형은 I~Ⅵ의 6가지의 지역형이 있다. I (SEZ) 지역형은 남아프리카, 남 짐바브웨, Ⅱ지역형(WZ)은 나미비아, 보스와나, 서부 짐바브웨, Ⅲ지역형(NWZ)은 말라위, 북 짐바브웨, Ⅳ지역형은 잠비아, Ⅴ, Ⅵ 지역형은 우간다에서 주로 발생한다. 엄격하게 개발한다면 이러한 지역형 또는 그 아형에 대한 백신을 모두 개발하여 백신주로 사용하여야 한다. 그러나 모든 백신주 개발을 위해서는 야외주를 지속적으로 세포에 배양하여 적절한 증식력을 지녀야 하고, 이러한 방법은 시간이 많이 소요되며 결과가 좋으리라는 보장도 없다. 따라서 기존의 정보가 많고 이미 증명된 백신 바이러스를 이용하여 필요로 하는 백신주를 새로 신속하게 개발하여 사용할 수 있도록 적절한 시기에 제작 가능한 형태로 만들어져야 할 것이다.Foot and mouth disease virus There are six regional types of SAT3 with different characteristics. There are six regional types of SAT3 type I ~ VI. Region I (SEZ) is South Africa, South Zimbabwe, Region II (WZ) is Namibia, Boswana, Western Zimbabwe, Region III (NWZ) is Malawi, Northern Zimbabwe, Region IV is Zambia, Regions Ⅴ and VI. The older brother occurs mainly in Uganda. If rigorously developed, all vaccines against these regional or subtypes should be developed and used as vaccine strains. However, in order to develop all vaccine strains, it is necessary to continuously culture the field strains in cells to have a proper proliferation ability. This method is time-consuming and there is no guarantee that the result will be good. Therefore, it is necessary to make a form that can be produced in a timely manner so that new vaccines can be rapidly developed and used by using existing information and proven vaccine virus.

이에 본 발명자들은 상기 문제점을 해결하기 위하여, 이미 구제역 백신주로 알려져 있는, 병원성이 약화된 바이러스를 기초로 바이러스 유전자로 구성된 기본 벡터를 이용하여 SAT3 형 구제역 발생시 방어가 가능한 백신을 생산할 수 있는 새로운 재조합 바이러스를 제작하고자 지속적으로 연구 노력하였다. 특허문헌 0001은 본 발명자들의 선행연구 중 하나로 구제역 SAT 3의 I 지역형인 SA 바이러스 방어항원 단백질을 코딩하는 유전자가 삽입된 재조합 플라스미드를 개시하고 있지만, 유전적인 상동성은 13%의 큰 차이를 보여 이러한 백신 바이러스에 보강적인 역할을 수행할 수 있는 추가적인 백신주의 개발이 여전히 요구되고 있는 실정이다. 이에 본 발명자들은 방어가 필요한 새로운 구제역 재조합 바이러스로 병원성이 감소된 바이러스 형태로 방어효과가 극대화됨을 확인하고 본 발명을 완성하게 되었다.In order to solve the above problems, the present inventors use a basic vector composed of a viral gene based on a pathogenic weakened virus, which is already known as a foot-and-mouth vaccine vaccine, and a new recombinant virus that can produce a vaccine that can protect against SAT3-type foot-and-mouth disease. Efforts were continuously made to produce the Patent document 0001 discloses a recombinant plasmid in which a gene encoding SA virus protective antigen protein, which is the I region type of foot-and-mouth disease SAT 3, is inserted as one of the prior studies of the present inventors, but the genetic homology shows a large difference of 13%. There is still a need to develop additional vaccine strains that can play a protective role in viruses. Accordingly, the inventors have confirmed that the protective effect is maximized in the form of a virus having reduced pathogenicity as a new foot-and-mouth disease recombinant virus that requires defense and completed the present invention.

한국등록특허 제10-1602921호Korean Patent Registration No. 10-1602921

따라서, 본 발명의 목적은 구제역의 SAT 3형 바이러스 중 I 지역형의 방어항원 단백질을 코딩하는 재조합 유전자가 삽입된 재조합 플라스미드를 제공하는 것이다.Accordingly, it is an object of the present invention to provide a recombinant plasmid in which a recombinant gene encoding the region I protective antigen protein of the SAT type 3 virus of foot-and-mouth disease is inserted.

본 발명의 다른 목적은 상기 재조합 플라스미드를 이용하여 백신 종독주로 유용하게 사용될 수 있는 재조합 구제역 바이러스를 제공함에 있다.Another object of the present invention to provide a recombinant foot-and-mouth virus that can be usefully used as a vaccine strain using the recombinant plasmid.

본 발명의 또 다른 목적은 상기 재조합 바이러스의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing the recombinant virus.

본 발명의 또 다른 목적은 상기 재조합 구제역 바이러스를 유효성분으로 포함하는 구제역 예방용 백신 조성물을 제공하는 것이다.Still another object of the present invention is to provide a vaccine for foot-and-mouth disease prevention comprising the recombinant foot-and-mouth virus as an active ingredient.

본 발명의 또 다른 목적은 상기 백신 조성물을 이용하여 구제역을 예방하는 방법을 제공하는 것이다.Another object of the present invention to provide a method for preventing foot and mouth disease using the vaccine composition.

본 발명은 구제역 O형 Manisa 유전체의 P1 유전자 부위가 구제역 SAT3 ZIM의 P1 유전자로 치환되고, 상기 구제역 O형 Manisa 유전체의 3B 부위 중, 3B12 부위가 서열번호 2로 표시되는 3B3의 염기서열로 치환된 재조합 플라스미드를 제공한다.In the present invention, the P1 gene region of the foot-and-mouth disease O type Manisa genome is substituted with the P1 gene of the foot-and-mouth disease SAT3 ZIM, and in the 3B region of the foot-and-mouth disease O type Manisa genome, the 3B12 site is substituted with the nucleotide sequence of 3B3 represented by SEQ ID NO: 2. Provide a recombinant plasmid.

또한, 본 발명은 상기 재조합 플라스미드를 이용하여 제조한 재조합 구제역 바이러스를 제공한다.The present invention also provides a recombinant foot and mouth virus produced using the recombinant plasmid.

또한, 본 발명은 (a) 구제역 O형 Manisa의 유전자를 재조합 벡터에 삽입하는 단계; (b) 상기 (a)단계에서 얻은 재조합 벡터에서, 구제역 O형 Manisa의 3B12 부위를 치환하는 단계; (c) 상기 (b)단계에서 얻은 재조합 벡터에서, 상기 구제역 O형 Manisa의 P1 유전자 중 3C 142번째 아미노산 시스테인을 트레오닌(C142T)으로 치환하는 단계; (d) 상기 (c)단계에서 얻은 재조합 벡터에서, 상기 구제역 O형 Manisa의 P1 유전자를 SAT3 ZIM 유전자로 치환하는 단계를 포함하는 재조합 벡터의 제조방법을 제공한다.In addition, the present invention comprises the steps of (a) inserting a gene of foot-and-mouth disease O type Manisa into a recombinant vector; (b) replacing the 3B12 site of foot-and-mouth disease O type Manisa in the recombinant vector obtained in step (a); (c) in the recombinant vector obtained in step (b), replacing 3C 142 th amino acid cysteine in the P1 gene of the foot-and-mouth type O manisa with threonine (C142T); (d) In the recombinant vector obtained in the step (c), it provides a method for producing a recombinant vector comprising the step of replacing the P1 gene of the foot-and-mouth disease O type Manisa with SAT3 ZIM gene.

또한, 본 발명은 상기 재조합 구제역 바이러스를 유효성분으로 포함하는 구제역 예방용 백신 조성물을 제공한다.The present invention also provides a vaccine for foot-and-mouth disease prevention comprising the recombinant foot-and-mouth virus as an active ingredient.

또한, 본 발명은 상기 재조합 구제역 바이러스를 인간을 제외한 개체에 투여하는 단계;를 포함하는 구제역의 예방 방법을 제공한다.The present invention also provides a method for preventing foot-and-mouth disease, comprising administering the recombinant foot-and-mouth virus to a subject other than a human.

본 발명에 의한 재조합 구제역 바이러스는 구제역 O형 바이러스의 유전자 일부를 조작하여 동물에 병원성을 약화시킴으로써, 소독제 실험 또는 항바이러스 실험 등 실험실 내에서 다루기에 병원성 바이러스보다 더욱 안전하게 사용할 수 있는 장점을 지니고 있다.Recombinant foot-and-mouth virus according to the present invention has the advantage that can be used more safely than the pathogenic virus to handle in the laboratory, such as disinfectant experiments or antiviral experiments by manipulating the gene part of the foot-and-mouth virus O virus.

또한, 본 발명에 의한 재조합 구제역 바이러스를 유효성분으로 하여 백신을 제작함으로써, SAT3 형 I 지역형 바이러스까지 방어할 수 있는 효과가 있다.In addition, by producing a vaccine using the recombinant foot-and-mouth virus according to the present invention as an active ingredient, there is an effect that can protect up to SAT3 type I local type virus.

또한, 본 발명에 의한 재조합 구제역 바이러스는 야외주와의 감별진단도 가능하여 백신 제작에 효과적으로 이용될 수 있다.In addition, the recombinant foot-and-mouth virus according to the present invention can be differentially diagnosed with the outdoor strain and thus can be effectively used for vaccine production.

도 1는 구제역 SAT 3형 구제역 바이러스 I 지역형 ZIM 백신주의 방어 항원이 발현되는 재조합 구제역 바이러스의 게놈 모식도를 나타낸 것이다.
도 2은 구제역 O형 백신주 Manisa에 SAT3 ZIM (I 지역형)의 방어항원 단백질을 코딩하는 유전자 염기서열이 삽입된 본 발명의 재조합 플라스미드(pO-SAT3 ZIM)의 모식도를 나타낸 것이다.
도 3은 도 2의 재조합 플라스미드를 세포에 도입하여 새로 제작된 SAT2 ZIM 바이러스 입자를 전자현미경 사진을 통하여 확인한 것이다.
도 4는 야외 주(SAT3 ZIM-wild type)와 본 발명의 재조합 구제역 바이러스(SAT3 ZIM-R)를 포유 마우스에 접종한 후 폐사율을 측정하여 재조합 구제역 바이러스의 병원성이 감소된 것을 확인한 것이다.
도 5는 본 발명의 재조합 구제역 바이러스(SAT3 ZIM-R)가 감염된 세포에서 항원 단백질이 발현됨을 간이항원 키트로 확인한 것이다(Svanova 키트, UK; SP 밴드는 밴드형성, C는 실험대조, 375 ng[1/40 dose]까지 검출 가능하다).도 6은 마우스에서의 항원 dose 별로 7일 면역 후 (1.5~0.023 μg, 소 및 돼지 1/10~1/640 dose) 공격접종 실험 (SAT3 ZIM wild type)을 통해서 마우스의 생존율 및 체중감소를 조사한 결과이다. 이 결과에 따르면 0.023 μg (1/640 dose) 백신을 접종하여도 I 지역형 바이러스에 대해 완벽한 방어가 가능함을 확인하였다.
도 7은 소(n=5)에서 면역시험 (0주, 4주에 백신접종)을 통해 얻어진 혈청으로 면역된 항체에 대해 중화 항체수준을 조사한 결과이다.
도 8은 돼지(n=20)에서 면역시험 (0주, 4주에 백신접종)을 통해 얻어진 혈청으로 면역된 항체에 대해 중화항체수준을 조사한 결과이다.Figure 1 shows a genomic schematic of recombinant foot-and-mouth virus in which the protective antigen of the foot-and-mouth disease SAT type 3 foot-and-mouth virus I region type ZIM vaccine strain is expressed.
Figure 2 shows a schematic diagram of the recombinant plasmid (pO-SAT3 ZIM) of the present invention is inserted into the foot-and-mouth disease O vaccine strain Manisa nucleotide sequence encoding the protective antigen protein of SAT3 ZIM (I region type).
FIG. 3 shows the newly prepared SAT2 ZIM virus particles by introducing the recombinant plasmid of FIG. 2 into the cells through an electron micrograph.
Figure 4 confirms that the pathogenicity of recombinant foot-and-mouth disease virus was reduced by measuring mortality after inoculation of mammalian mice with SAT3 ZIM-wild type and the recombinant foot-and-mouth virus (SAT3 ZIM-R) of the present invention.
Figure 5 confirms that the antigenic protein is expressed in cells infected with the recombinant foot-and-mouth virus (SAT3 ZIM-R) of the present invention (Svanova kit, UK; SP band is band formation, C is experimental control, 375 ng [ 1/40 dose]. FIG. 6 shows challenge after 7-day immunization (1.5-0.023 μg, bovine and swine 1 / 10-1 / 640 dose) by antigen dose in mice (SAT3 ZIM wild type) ), The survival rate and weight loss of mice. According to these results, even when inoculated with 0.023 μg (1/640 dose) vaccine, it was confirmed that complete protection against I-type virus was possible.
Figure 7 shows the results of neutralizing antibody levels for antibodies immunized with serum obtained through the immunization test (vaccinates 0 and 4 weeks) in cattle (n = 5).
FIG. 8 shows the results of investigating neutralizing antibody levels of antibodies immunized with serum obtained through immunoassay (vaccination at 0 and 4 weeks) in pigs (n = 20).

본 발명은 구제역 O형 바이러스의 유전자를 포함하는, 서열번호 1의 염기서열을 갖는 재조합 플라스미드에 관한 것으로서, 구제역 O형 Manisa 유전체의 P1 유전자가 구제역 I 지역형 SAT3-ZIM의 P1 유전자로 치환되고, 상기 구제역 O형 Manisa 유전체의 3B 부위 중, 3B12 부위가 치환되고 3C 부위 중 142번째 아미노산인 시스테인이 트레오닌으로 치환된 것인, 구제역 O형 Manisa 유전체 염기서열을 포함하는 재조합 플라스미드에 관한 것이다.The present invention relates to a recombinant plasmid having a nucleotide sequence of SEQ ID NO: 1 comprising a gene of foot-and-mouth disease O virus, wherein the P1 gene of the foot-and-mouth disease O type Manisa genome is substituted with a P1 gene of foot-and-mouth disease I region type SAT3-ZIM, It relates to a recombinant plasmid comprising the foot-and-mouth type O manisa genome sequence, wherein the 3B12 site is substituted and the 142th amino acid cysteine in the 3C site is substituted with threonine.

본 발명에서 "P1 유전자"는 P1 단백질을 코딩하는 염기서열로, VP1, VP2, VP3 및 VP4 부위를 포함할 수 있으며, 본 발명에서는 상기 부위는 단백질을 코딩하는 염기서열, 단백질을 코딩하지 않은 염기서열 및 유전자를 포함하는 개념으로 사용된다.In the present invention, "P1 gene" is a base sequence encoding the P1 protein, may include VP1, VP2, VP3 and VP4 sites, in the present invention, the site is a base sequence encoding the protein, the base without the protein coding Used as a concept involving sequences and genes.

상기 구제역 O형 바이러스의 유전자는 구제역 O형 백신주 Manisa의 전체 유전자인 것을 특징으로 한다. 상기 전체 유전자 부위 중에서 야외주와의 감별진단 목적으로 3B1 부위 및 3B2 부위 일부를 치환시킬 수 있다. 상기 3B1 및 3B2의 치환부위는 서열번호 1의 염기서열로 표시되는 본 발명의 재조합 플라스미드 중 6501 내지 6644 bp 부위로서 서열번호 2의 염기서열로 표시되는 144bp의 서열인 것이 바람직하나 이에 제한되지 않는다.The gene of foot-and-mouth disease O virus is characterized in that the entire gene of foot-and-mouth disease O vaccine strain Manisa. Part of the 3B1 region and 3B2 region may be substituted for the purpose of differential diagnosis with the outdoor strain among the entire gene region. Substitution sites of the 3B1 and 3B2 are 6501 to 6644 bp of the recombinant plasmid of the present invention represented by the nucleotide sequence of SEQ ID NO: 1 The site is preferably a 144 bp sequence represented by the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

본 발명에서 "구제역 바이러스 SAT3형 I 지역형 SAT3 ZIM 주"는 SAT3 형 백신에서 널리 쓰이는 백신 바이러스로, SAT3 형 중 I 지역형에 대한 바이러스에 대한 방어가 가능한 것으로 알려져 있다.In the present invention, "foot and mouth virus SAT3 type I regional type SAT3 ZIM strain" is a vaccine virus widely used in the SAT3 type vaccine, and is known to be able to defend against the virus against I type among the SAT3 types.

본 발명의 실시예에 의하면, 상기 본 발명의 재조합 플라스미드는 상기 구제역 O형 Manisa 유전체 중 3C 부위의 142번째 아미노산 시스테인이 트레오닌으로 치환(C142T)된 것일 수 있다.According to an embodiment of the present invention, the recombinant plasmid of the present invention may be substituted with threonine at the 142th amino acid cysteine of the 3C region of the foot-and-mouth type O Manisa genome (C142T).

본 발명의 또 다른 실시 예에 의하면, 본 발명은 상기 재조합 플라스미드를 이용하여 제조한 재조합 구제역 바이러스에 관한 것이다.According to another embodiment of the present invention, the present invention relates to a recombinant foot-and-mouth virus produced using the recombinant plasmid.

본 발명의 재조합 바이러스는 구제역 SAT3 형 ZIM 주의 방어항원이 발현되는 것을 특징으로 한다.The recombinant virus of the present invention is characterized in that the protective antigen of foot-and-mouth disease SAT3 type ZIM strain is expressed.

본 발명의 다른 일 실시예에 의하면, 본 발명은According to another embodiment of the present invention, the present invention

(a) 구제역 O형 Manisa의 유전자를 재조합 벡터에 삽입하는 단계; (b) 상기 (a)단계에서 얻은 재조합 벡터에서, 구제역 O형 Manisa의 3B12 부위를 치환하는 단계; (c)상기 (b)단계에서 얻는 재조합 벡터에서, 상기 구제역 O형 Manisa의 3C 부위 중 142번째 아미노산 시스테인을 트레오닌으로 치환(C142T)하는 단계; 및 (d) 상기 (c)단계에서 얻은 재조합 벡터에서, 상기 구제역 O형 Manisa의 P1 유전자를 구제역 SAT3-ZIM의 P1 유전자로 치환하는 단계를 포함하는 재조합 벡터의 제조방법에 관한 것이다.(a) inserting a gene of foot-and-mouth type O Manisa into a recombinant vector; (b) replacing the 3B12 site of foot-and-mouth disease O type Manisa in the recombinant vector obtained in step (a); (c) in the recombinant vector obtained in step (b), substituting threonine for the 142th amino acid cysteine of the 3C region of the foot-and-mouth type O Manisa (C142T); And (d) replacing the P1 gene of foot-and-mouth disease O type Manisa with the P1 gene of foot-and-mouth disease SAT3-ZIM in the recombinant vector obtained in step (c).

본 발명의 다른 일 실시예에 의하면, 본 발명은 상기 제조방법에 의해 제조된 재조합 벡터를 세포에 도입하여 증식시키는 단계;를 포함하는 재조합 구제역 바이러스의 제조방법에 관한 것이다.According to another embodiment of the present invention, the present invention relates to a method for producing a recombinant foot-and-mouth virus, comprising the steps of: introducing and propagating a recombinant vector prepared by the production method into a cell.

본 발명에서 용어 "벡터(vector)"는 적합한 숙주 내에서 DNA를 발현시킬 수 있는 적합한 조절 서열에 작동 가능하게 연결된 DNA 서열을 함유하는 DNA 제조물을 의미한다. 벡터는 플라스미드, 파지 입자 또는 간단하게 잠재적 게놈 삽입물일 수 있다. 적당한 숙주로 형질전환되면, 벡터는 숙주 게놈과 무관하게 복제하고 기능할 수 있거나, 또는 일부 경우에 게놈 그 자체에 통합될 수 있다. 플라스미드가 현재 벡터의 가장 통상적으로 사용되는 형태이므로, 본 발명의 명세서에서 "플라스미드(plasmid)" 및 "벡터(vector)"는 때로 상호 교환적으로 사용된다. 본 발명의 목적상, 플라스미드 벡터를 이용하는 것이 바람직하다. 이러한 목적에 사용될 수 있는 전형적인 플라스미드 벡터는 (a) 숙주세포당 수백 개의 플라스미드 벡터를 포함하도록 복제가 효율적으로 이루어지도록 하는 복제 개시점, (b) 플라스미드 벡터로 형질전환된 숙주세포가 선발될 수 있도록 선별 표지 및 (c) 외래 DNA 절편이 삽입될 수 있는 제한효소 절단부위를 포함하는 구조를 지니고 있다. 적절한 제한효소 절단부위가 존재하지 않을지라도, 통상의 방법에 따른 합성 올리고뉴클레오타이드 어댑터(oligonucleotide adaptor) 또는 링커(linker)를 사용하면 벡터와 외래 DNA를 용이하게 라이게이션(ligation)할 수 있다.As used herein, the term "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising a screening label and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

본 발명의 재조합 벡터 및 재조합 구제역 바이러스는 통상의 유전자조작법, 형질전환법에 의해 제조될 수 있으며, 적은 양으로 형성된 바이러스를 세포배양을 통한 연속 계대로 적절한 양의 바이러스를 수득할 수 있다.Recombinant vectors and recombinant foot-and-mouth virus of the present invention can be prepared by conventional genetic engineering, transformation method, it is possible to obtain an appropriate amount of virus in a continuous passage through the cell culture of the virus formed in a small amount.

본 발명의 세포는 개과 동물, 고양이과 동물, 멧돼지과 동물, 소과 동물, 사슴과 동물, 기린과 동물, 페커리과 동물, 낙타과 동물, 하마과 동물, 말과 동물, 맥과 동물, 코뿔소과 동물, 족제비과, 토끼과, 설치류 및 영장류의 세포로 이루어진 군에서 선택된 1종 이상의 세포에서 유래된 것일 수 있고, 바람직하게는 염소 혀 세포 (ZZR) 및 햄스터 신장 세포 (BHK-21), 흑염소 신장세포 (BGK), 돼지 신장세포 (IBRS-2) 및 소 신장세포 (LF-BK)로 이루어진 군에서 선택된 1종 이상을 사용할 수 있다.The cells of the present invention are canine, feline, boar, bovine, deer, animal, giraffe, animal, pelican, camel, hippopotamus, horse, animal, maca, rhinoceros, weasel, rabbit, It may be derived from one or more cells selected from the group consisting of cells of rodents and primates, preferably goat tongue cells (ZZR) and hamster kidney cells (BHK-21), black goat kidney cells (BGK), porcine kidney cells (IBRS-2) and small kidney cells (LF-BK) can be used one or more selected from the group consisting of.

본 발명의 다른 일 실시예에 의하면, 본 발명은 상기 재조합 구제역 바이러스를 유효성분으로 포함하는 구제역 예방용 백신 조성물에 관한 것이다.According to another embodiment of the present invention, the present invention relates to a vaccine for foot-and-mouth disease prevention comprising the recombinant foot-and-mouth virus as an active ingredient.

본 발명의 구제역 예방용 백신 조성물에 있어서, 상기 백신은 생백신, 약독화된 백신, 또는 사백신일 수 있다.In the vaccine composition for foot-and-mouth disease prevention of the present invention, the vaccine may be a live vaccine, an attenuated vaccine, or a dead vaccine.

본 발명의 백신 조성물은 재조합 구제역 바이러스과 함께 구제역 감염의 예방 효과가 우수한 공지의 유효성분을 1종 이상 함유할 수 있다.The vaccine composition of the present invention may contain one or more known active ingredients with excellent preventive effect against foot-and-mouth disease along with the recombinant foot-and-mouth virus.

본 발명의 백신 조성물은, 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약학적으로 허용가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로오스 용액, 수크로오스 용액, 글리세롤, 에탄올 및 이들 성분 중 1성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.The vaccine composition of the present invention may be prepared by further including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, sucrose solution, glycerol, ethanol and one or more of these components, as necessary, including antioxidants, buffers, Other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

본 발명의 백신 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 개체의 무게, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다.The vaccine composition of the present invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex, health of the individual. The range varies depending on the condition, diet, administration time, administration method, excretion rate and severity of the disease.

본 발명의 또 다른 일 실시예에 의하면, 본 발명은 상기 재조합 구제역 바이러스를 인간을 제외한 개체에 투여하는 단계;를 포함하는 구제역의 예방 방법에 관한 것이다.According to another embodiment of the present invention, the present invention relates to a method for preventing foot-and-mouth disease, comprising administering the recombinant foot-and-mouth virus to a subject other than human.

이하 본 발명의 내용을 실험예를 통하여 구체적으로 설명한다. 그러나 하기의 실험예는 본 발명을 보다 상세하게 설명하기 위한 것으로 본 발명의 권리범위가 이들에 의해 한정되는 것은 아니다.Hereinafter, the content of the present invention will be described in detail through experimental examples. However, the following experimental examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited thereto.

실시예 1. 구제역 야외주와 감별 진단을 위한 유전자 조작Example 1 Genetic Engineering for Differential Diagnosis of Foot-and-mouth Disease Outdoor Cells

구제역 O형 Manisa 바이러스 전체 유전자 (Genbank AY593823.1)를 PCR에 의하여 증폭하고 플라스미드(pBluescript SK I)에 O_Manisa 유전자를 삽입하여 재조합 플라스미드 pO-Manisa를 제조하였다. 그 후 제조된 pO-Manisa의 3B1 및 3B2 부위를 제거한 후 A형의 3B3 및 SAT형의 3B3 서열을 합성(144 bps, 서열번호 2)하여 삽입하는 작업을 통해 야외주와의 감별 진단을 위한 pO-Mansia-3B 플라스미드를 제작하였다.The foot-and-mouth disease O type Manisa virus whole gene (Genbank AY593823.1) was amplified by PCR, and the recombinant plasmid pO-Manisa was prepared by inserting the O_Manisa gene into the plasmid (pBluescript SK I). After removing the 3B1 and 3B2 sites of the prepared pO-Manisa and then inserting by inserting the AB 3B3 and SAT 3B3 sequence synthesis (144 bps, SEQ ID NO: 2) pO for differential diagnosis with the outdoor strain -Mansia-3B plasmid was made.

구체적으로 cDNA 합성시 일반적으로 사용되는 랜덤 프라이머를 이용하여 O-Manisa 바이러스에 대한 cDNA 작성후 전체 유전자가 클로닝된 것을 기초로 3A의 끝부분 유전자 및 3B3의 시작 부위 유전자 증폭이 가능한 특이 프라이머들(GenBank Accession No. AY593823.1를 기초로 5' 및 3' 유전자의 각 20 mers씩에 해당)를 작성하여 PCR을 실시하였다. PCR은 5X buffer (FINNZYMES, 10㎕), 10mM dNTPs (1㎕), Phusion enzyme (2U/㎕, 0.5㎕), 멸균증류수 (35.5㎕)의 용량으로 98℃에서 30초 후 98℃ 10초, 65℃ 30초, 72℃ 2분 30초간 25 싸이클, 최종 72℃ 10분으로 반응을 실시하였다. 상기에서 작성된 pO-Manisa와 PCR 반응에 의해 합성된 유전자와의 결찰반응 (TAKARA Long Ligation kit)을 수행하였으며, 전체 염기서열 분석을 통하여 적절히 클로닝된 것을 확인하였다. 하기 표 1은 O-Manisa의 3B1 및 3B2를 제거하고, A형 3B3 및 SAT형의 3B3 이 삽입된 아미노산 서열을 나타낸 것이다.Specifically, specific primers capable of amplifying the end gene of 3A and the start region of 3B3 based on cloning of the entire gene after cDNA preparation for O-Manisa virus using a random primer commonly used in cDNA synthesis (GenBank) PCR was carried out based on Accession No. AY593823.1, corresponding to 20 mers of each of the 5 'and 3' genes). PCR was performed at 98 ° C for 30 seconds at 98 ° C for 30 seconds at a dose of 5X buffer (FINNZYMES, 10µl), 10mM dNTPs (1µl), Phusion enzyme (2U / µl, 0.5µl), sterile distilled water (35.5µl). The reaction was carried out at 25 cycles of 30 ° C., 72 ° C. for 2 minutes 30 seconds, and final 72 ° C. for 10 minutes. The ligation reaction (TAKARA Long Ligation kit) with the pO-Manisa prepared by the PCR reaction was carried out, and it was confirmed that it was properly cloned through the entire sequence analysis. Table 1 below shows amino acid sequences in which 3B1 and 3B2 of O-Manisa were removed and 3B3 of type A 3B3 and SAT were inserted.

NSP 부위 조작NSP site manipulation 아미노산 서열Amino acid sequence 아미노산 크기Amino acid size 참고Reference 3B12 deletion 3B12 deletion GPYAGPLERQKPLRVKTKLPQQEGPYAGPMDRQKPLKVRARAPVVKE(서열번호 3)GPYAGPLERQKPLRVKTKLPQQEGPYAGPMDRQKPLKVRARAPVVKE (SEQ ID NO: 3) 47 aa47 aa -- 3B33 addition3B33 addition GPYEGPVKKPVALKVKAKNLIVTEGPYEGPVKKPVALKVKAKAPIVTE(서열번호 4)GPYEGPVKKPVALKVKAKNLIVTEGPYEGPVKKPVALKVKAKAPIVTE (SEQ ID NO: 4) 48 aa48 aa A type and SAT type A type and SAT type

실시예 2. 병원성 감소 유전자 조작Example 2. Pathogenicity Reduction Genetic Engineering

병원성 감소된 구제역바이러스를 제작하기 위해 우선 감염성 플라스미드를 만들기 위한 시도가 수행되었다. 실시예 1에서 제조된 pO-Mansia-3B 플라스미드에서, 그 벡터를 NEW ENGLAND BIOLABSⓡ Inc.의 Gibson Assemblyⓡ Master Mix 를 이용하여 pO-Manisa-3B 유전자 중 3C 부위의 142번째 아미노산인 시스테인을 트레오닌으로 치환(C142T)되도록 조작하였다(서열번호 1의 염기서열에서 7140-7141 bp 위치의 tg를 ac로 치환).PCR을 이용하여 증폭한 뒤 자가결찰 (Self ligation)하는 방법을 이용하여 유전자가 교체 플라스미드를 확보하였다.Attempts were first made to make infectious plasmids to produce pathogenic reduced foot and mouth virus. Carried out at a pO-Mansia-3B plasmid prepared in Example 1, the vector NEW ENGLAND BIOLABS ⓡ Inc. 142 the amino acid cysteine in the Gibson Assembly ⓡ Master Mix parts of pO-3C Manisa-3B gene using a threonine It was manipulated to replace (C142T) (substituting tg at 7140-7141 bp position with ac in the nucleotide sequence of SEQ ID NO: 1) .Amplification using PCR followed by self ligation. Secured.

실시예 3. 재조합 플라스미드 pO-SAT3 ZIM의 제작(도 1 및 도 2)Example 3 Construction of Recombinant Plasmid pO-SAT3 ZIM (FIGS. 1 and 2)

상기 실시예 2에서 제조된 재조합 플라스미드(pO-Manisa 3B333 3Cmut) 중에서 방어 항원인 P1 단백질을 코딩하는 유전자를 구제역 SAT1 바이러스 BOT 지역형의 방어 항원인 P1 단백질을 코딩하는 유전자로 치환하여 재조합 유전자가 삽입된 플라스미드(pO-SAT3 ZIM)를 제조하였다. 상기 P1 단백질을 코딩하는 유전자는 하기와 같이 치환시켰다.In the recombinant plasmid prepared in Example 2 (pO-Manisa 3B333 3Cmut), a gene encoding the P1 protein as a protective antigen was replaced with a gene encoding the P1 protein as a protective antigen of foot-and-mouth disease SAT1 virus BOT region type to insert a recombinant gene. Plasmid (pO-SAT3 ZIM) was prepared. The gene encoding the P1 protein was substituted as follows.

(1) 먼저 Gibson Assembly®Cloning Kit(NEB)을 이용하여 SAT3 ZIM4/81의 wild type virus에서 RNA를 추출 하여 cDNA를 합성 한 후 플라스미드 (100ng/μl, 1μl), 서열번호 5의 정방향 프라이머 (5‘-AACAAAGGTCCAGAAAAGGCTCAAGGGAGCGG GACAGTCATCA-3’, 43mer), 서열번호 6의 역방향 프라이머 (5‘-TTTGAGCAGGTCAAAATTTAGAAGTGCACAGTTGTTTCTCGACG-3’, 44mer)를 이용하여 SAT3 ZIM4/81 바이러스의 P1 유전자를 PCR에 의하여 증폭시켰다. 이때, 상기 PCR은 5X buffer (FINNZYMES, 10μl), 10mM dNTPs (1μl), Phusion enzyme (2U/μl, 1μl), 멸균증류수 (35μl)의 용량으로 98℃ 30초 후 98℃ 10초, 72℃ 30초, 72℃ 2분 30초를 한 주기로 25주기로 PCR을 수행한 후, 최종 72℃에서 10분 동안 반응을 실시하였다.(1) First, RNA was extracted from wild type virus of SAT3 ZIM4 / 81 using Gibson Assembly® Cloning Kit (NEB) to synthesize cDNA, followed by plasmid (100ng / μl, 1μl), forward primer of SEQ ID NO: 5 (5 P1 gene of SAT3 ZIM4 / 81 virus was amplified using '-AACAAAGGTCCAGAAAAGGCTCAAGGGAGCGG GACAGTCATCA-3', 43mer), reverse primer of SEQ ID NO: 6 (5'-TTTGAGCAGGTCAAAATTTAGAAGTGCACAGTTGTTTCTCGACG-3 ', 44mer) by PCR. At this time, the PCR is a capacity of 5X buffer (FINNZYMES, 10μl), 10mM dNTPs (1μl), Phusion enzyme (2U / μl, 1μl), sterile distilled water (35μl) after 98 ℃ 30 seconds 98 ℃ 10 seconds, 72 ℃ 30 Second, PCR was carried out in 25 cycles of 2 minutes 30 seconds at 72 ℃, and then the reaction was carried out for 10 minutes at 72 ℃.

(2) 3B 및 3C 부위가 조작된 플라스미드(pO-Manisa 3B333 3Cmut)에서 P1을 제거하기 위한 PCR은 플라스미드 (100ng/μl,1μl), 서열번호 7의 정방향 프라이머인 V_Fwd(5‘-ACTTCTAAATTTTGACCTGC-3’, 20mer), 서열번호 8의 역방향 프라이머인 V_Rev(5‘-CTTGAGCCTTTTCTGGAC-3’, 18mer)를 사용하였다. 이때, 상기 PCR조건은 5X buffer (FINNZYMES, 10μl), 10mM dNTPs (1μl), Phusion enzyme (2U/μl, 1μl), 멸균증류수 (35μl)의 용량으로 98℃에서 30초 후 98℃에서 10초, 65℃ 30초, 72℃ 2분 30초를 한 주기로 25주기로 PCR을 수행한 후, 최종 72℃에서 10분 동안 반응을 실시하였다.(2) PCR to remove P1 from the plasmid engineered with the 3B and 3C sites (pO-Manisa 3B333 3Cmut) was performed using _ plasmid (100ng / μl, 1μl), V_Fwd (5'-ACTTCTAAATTTTGACCTGC-3), the forward primer of SEQ ID NO: 7 20mer), V_Rev (5'-CTTGAGCCTTTTCTGGAC-3 ', 18mer), a reverse primer of SEQ ID NO: 8, was used. At this time, the PCR conditions are 5X buffer (FINNZYMES, 10μl), 10mM dNTPs (1μl), Phusion enzyme (2U / μl, 1μl), sterile distilled water (35μl) 30 seconds at 98 ℃ 30 seconds at 98 ℃, PCR was performed in 25 cycles of 65 ° C. 30 seconds, 72 ° C. 2 minutes 30 seconds, and then the reaction was performed at 72 ° C. for 10 minutes.

(3) 상기 SAT3 ZIM4/81의 P1이 증폭된 PCR산물과 pO-Manisa 3B333 3Cmut에서 P1 부위가 제거된 증폭산물(200ng/μl,1μl)과 Gibson Assembly Master Mix(2X, 10μl), 멸균 증류수(8μl)용량으로 56℃에서 30분간 반응시켰다. 그 이후에 Gibson Assembly®Cloning Kit에 포함된 competent cell에 transformation하여 본 발명의 pO-SAT3-ZIM 플라스미드(전체서열을 서열번호 1에 나타냄)를 제조하였으며 최종적으로 염기서열 분석을 실시하여 pO-Manisa 3B333 3Cmut의 플라스 미드의 P1 부위가 SAT3 ZIM 4/81의 P1 부위로 교체됨을 확인하였다. 제작된 재조합 플라스미드 pO-SAT3-ZIM의 구조 및 모식도를 각각 도 1 및 도2에 나타내었다.(3) the PCR product amplified P1 of the SAT3 ZIM4 / 81 and the amplified product (200ng / μl, 1μl) from which the P1 site was removed from pO-Manisa 3B333 3Cmut, Gibson Assembly Master Mix (2X, 10μl), sterile distilled water ( 8 μl) was reacted at 56 ° C. for 30 minutes. Subsequently, pO-SAT3-ZIM plasmid (the entire sequence is shown in SEQ ID NO: 1) of the present invention was prepared by transforming into competent cells included in the Gibson Assembly® Cloning Kit. Finally, pO-Manisa 3B333 was subjected to sequencing. It was confirmed that the P1 region of the 3 Cmut plasmid was replaced with the P1 region of SAT3 ZIM 4/81. The structure and schematic diagram of the produced recombinant plasmid pO-SAT3-ZIM are shown in FIGS. 1 and 2, respectively.

실시예 4. 본 발명의 플라스미드를 이용한 재조합 구제역 바이러스의 제조(도 3)Example 4 Preparation of Recombinant Foot-and-mouth Virus Using the Plasmid of the Invention (FIG. 3)

재조합 구제역 바이러스의 회복은 확보된 상기 재조합 플라스미드(pO-SAT3 ZIM)를 제한효소로 반응시켜 유전자를 단일 조각편으로 작성하고, BHKT7-9 세포(T7 RNA polymerase가 발현되는 세포주)에 리포펙타민 형질전환 시약 (라이프 테크놀로지)을 이용하여 정제된 DNA를 형질도입하여 2 내지 3일 동안 배양 후, 형성된 재조합 바이러스를 확보하였다. 이 후, 확보된 바이러스를 ZZ-R (염소 태아 혀)세포 또는 BHK-21 (어린 햄스터 신장) 세포에서 연속계대를 통해 접종하여 바이러스를 증식시켰다. 이후 구제역 바이러스가 감염된 세포에서 재조합 바이러스가 재형성되는 것을 도 3의 전자현미경 사진을 통해 확인하였다.Recovery of the recombinant foot-and-mouth virus was performed by reacting the obtained recombinant plasmid (pO-SAT3 ZIM) with a restriction enzyme to prepare genes into single fragments and transforming lipofectamine into BHKT7-9 cells (cell line expressing T7 RNA polymerase). Purified DNA was introduced using reagents (Life Technologies) to incubate for 2 to 3 days and the resulting recombinant virus was obtained. Thereafter, the secured virus was inoculated via serial passage in ZZ-R (goat fetal tongue) cells or BHK-21 (young hamster kidney) cells to propagate the virus. Thereafter, it was confirmed through the electron micrograph of FIG. 3 that the recombinant virus was remodeled in the cells infected with the foot and mouth virus.

실시예 5. 제작 백신의 동물에서의 면역원성(도 4 내지 도 8)Example 5 Immunogenicity in Animals of Constructed Vaccines (FIGS. 4-8)

상술한 바와 같이 제작된 바이러스를 이용하여 동물실험을 위해서 정제한 구제역바이러스 항원 (15 μg/dose) 0.5ml을 오일 아쥬반트 ISA206이 포함된 아쥬반트 0.5ml를 이용하여 혼합하여 이중오일로 작성하였다. 이렇게 제작된 백신을 구제역 항체가 없는 마우스(C57BL/6)에 0.1mL, 2-3개월령 돼지, 6개월령 이상 소에는 1mL을 접종하여 마우스에서는 각각 7 일간 면역후 공격접종하여 체중변화, 생존율을 관찰하였으며(도 6), 돼지 및 소에서는 0주, 4주에 백신접종하고 각 시기별 항체 측정을 위해 채혈하였고, 그 SAT3형 항체 측정을 위해서 백신주에 대한 중화시험(OIE 메뉴얼 표준시험법 기준)을 실시하여 항체 수준을 결정하였다(도 7 및 도 8). 0.5 ml of the foot-and-mouth virus virus (15 μg / dose) purified for animal experiments using the virus prepared as described above was mixed with 0.5 ml of the adjuvant containing the oil adjuvant ISA206 to prepare a double oil. The vaccine thus prepared was inoculated with 0.1 mL, 2-3 months-old pigs, and 1 mL of cattle 6 months-old or older in mice without foot-and-mouth antibody (C57BL / 6). (Fig. 6), pigs and cows were vaccinated at 0 and 4 weeks, and blood was collected for antibody measurement at each time, and neutralization test (based on OIE manual standard test method) for vaccine strain was used to measure the SAT3 antibody. Was performed to determine antibody levels (FIGS. 7 and 8).

상술한 바와 같이 본 발명의 바람직한 실시예 및 시험예를 참조하여 설명하였지만 본 발명의 기술 분야에서 통상의 지식을 가진 통상의 기술자라면 하기의 특허청구범위에 기재된 본 발명의 사상 및 영역으로부터 벗어나지 않는 범위 내에서 본 발명을 다양하게 수정 및 변경시킬 수 있음을 이해할 수 있을 것이다.Although described with reference to the preferred embodiments and test examples of the present invention as described above, those skilled in the art without departing from the spirit and scope of the present invention described in the claims below It will be understood that various modifications and variations can be made in the present invention.

구제역 SAT3 혈청형의 광범위 방어항원 발현능을 보유하는 본 발명에 의한 재조합 구제역 바이러스를 이용한 구제역 백신을 제공함으로써, 구제역의 치료 및/또는 예방에 기여할 수 있다.By providing a foot-and-mouth vaccine using the recombinant foot-and-mouth virus according to the present invention, which has a broad protective antigen expression ability of foot-and-mouth disease SAT3 serotype, it can contribute to treatment and / or prevention of foot-and-mouth disease.

<110> Animal and plant quaratine agency <120> Recombinant foot-and-mouth disease virus expressing protective antigen of type SAT3 ZIM <130> 17-10922 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 11114 <212> DNA <213> Artificial Sequence <220> <223> Full DNA sequence of pO-SAT3 ZIM <400> 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc atatgtaata cgactcacta tagggttgaa agggggcgct 660 agggtctcac ccctagcatg ccaacgacag ctcctacgtc gcactccaca ctaacgtttg 720 tgtgcgcgcg ggaaccgatg gacttttgtt cacccaccta cagttggact cacggcaccg 780 cgtggccatt ttagctgggt tgtgcggacg aacactgctt gcgcatctcg cgtgaccggt 840 tagtactctt accactatcc gcctacttgg tcgttagcgc tgtcctgggc actcttgttg 900 ggggctgttc aacgctctac ggtctcccct gcgtaacaga ctacggtgtt ggggccgctt 960 cgtgcgagcc gatcgcttgg tgtgcctcgg ctgtcgcccg aagcccgcct ttcacccccc 1020 cccccccccc ctaggtttta ccgtcgttcc cgacgttaat ggggaaacaa ccacaagctt 1080 aacaccgtct tgcccgacgt aaaagggctg caaccaaaaa gcttgtgccg cctttcccgg 1140 cgttaatggg aggtaaccac aagacaaacc ttcacccgga agtaaaacgg caacttcaca 1200 cagttttgcc cgttttcgtg agaaatgggc cgtcaacgca cgaaacgcgc cgtcgcttga 1260 ggaggacttg tacaaacacg atctatgcag gtttccacaa ctgacacaaa ccgtgcaact 1320 tgaaaccccg cctggtcttt ccaggtctag aggggcgaca ttttgtactg tgcttgactc 1380 cacgctcggt ccactagcga gtgttagtag tagcactgtt gcttcgtagc ggagcatgat 1440 ggccgtggga gcttcccctt ggtaacaagg acccacgggg ccaaaagcca cgtcctaccg 1500 gacccatcat gtgtgcaaac ccagcacggc aactttactg cgaaaaccac tttaaggtga 1560 cactgatact ggtactcaat cactggtgac aggctaagga tgcccttcag gtaccccgag 1620 gtaacacgcg acactcggga tctgagaagg ggactggggc ttctttaaaa gtgcccagtt 1680 taaaaagctt ctatgcctga ataggcgacc ggaggccggc gccttttcac tgttttacta 1740 ctgttttcat gaatacaact gactgtttca ccgccctgtt acacgctctc agagagatca 1800 aaacactgtt tcttttacgg acacaaggaa agatggaatt cacactttac aacggtgaga 1860 agaaaacctt ctactccaga cccaacaacc acgacaactg ctggcttaac accattctcc 1920 agttgttcag gtatgttgat gagcctttct ttgactgggt ctacgactcg cctgaaaacc 1980 tcactcttga ggcaatcaaa cagttggaag agacaaccgg tcttgagctg cacgagggtg 2040 gaccacccgc tctcgtcatc tggaacatca aacacttgct tcacaccgga atcggcactg 2100 cctcacgccc tagcgaggtg tgtatggtgg acggaacgga catgtgttta gctgattttc 2160 atgctggcat tttcctgaaa ggacaggaac atgctgtgtt cgcctgtgtc acctccaacg 2220 ggtggtacgc gattgatgac gaggactttt acccttggac accggacccg tccgacgttc 2280 tggtgtttgt cccgtacgat caagaaccgc ttaacggaga gtggaaaaca aaggtccaga 2340 aaaggctcaa gggagcaggc caatcctccc ccgccacggg gtctcaaaat caatcaggca 2400 atactggtag catcattaac aactactaca tgcaacagta ccagaactcc atggacaccc 2460 agcttggcga caacgccatc tcgggtggat cgaatgaagg tagcactgac accacttcca 2520 cccacaccaa caacacacag aacaacgact ggttctcaaa gttggcgcag tctgccattt 2580 cgggactctt tggagctctg ttggcggaca agaagacaga ggagacaact cttctggagg 2640 atcgcatcct caccacgcgc cacaacacaa ccacgtccac cacacagagt tctgtcggtg 2700 tcacatacgg ttacgcgtca gctcaccgtt ttctgcctgg gcccaacacc agtggactcg 2760 agacacgcgt tgaacaggcg gagagattct tcaaggagaa actcttcact tggaccgcga 2820 gccaagagta cgcacacgtg catctactgg aactacccgt ggaccacaaa ggcatctacg 2880 gtgccatgct ggacagccac gcatacgtgc gaaatggctg ggacgtgcag gtttccgcaa 2940 ccagcacaca gttcaatggt ggcactctcc tcgtcgccat ggtccccgag ctgaagactc 3000 tggacaagcg tgacgtgtca caactcacgc ttttccccca ccagttcatc aacccacgca 3060 ccaacaccac cgcacacatc gtggtaccgt acgtgggtgt caacagacac gaccaggcaa 3120 agatgcacaa ggcatggaca ctcgtggtcg cggtgcttgc gccgctcacc acgtcaagca 3180 tgggacagga caacgtagag gtgtatgcga acatcgcacc caccaatgtg tacgttgctg 3240 gagagaagcc aacaaaacaa ggcatcatcc ccgtggcctg caacgacggt tacggtggat 3300 tccagaatac tgacccaaag accgcggacc caatctacgg tctcgtgtcc aacccgccac 3360 gcacagcgtt ccccggcagg ttcaccaacc tgctggatgt cgccgaagcg tgcccaacct 3420 tcctggactt cgacggcaca ccctacgtca agaccaggca caacagcggg agcaaaatac 3480 tgacacacat tgatttggct tttggacata agagtttcaa gaacacctac ctagcaggac 3540 tcgcccagta ctacgcccag tacagcgggt ctctgaatct gcacttcatg tacaccggtc 3600 caacgcagtc taaagcacgc tttatggttg cgtacattcc accagggacc aaccctgtac 3660 ctgacactcc tgaggctgca gcacactgct accactcaga gtgggacacc ggactgaact 3720 ccaagttcac gtttacggtt ccctatatgt cagctgcaga ctttgcatac acgtactgtg 3780 atgagcctga acaggcttca gcacaggggt gggtgacact ctaccaaatt acagacacgc 3840 acgaccccga ttcggcggtg cttgtctcgg tcagtgctgg cgctgacctt gaatttcggc 3900 tcccaatcaa ccctgcaacc cagacaacca gctcaggtga aggtggtgat gttgtgacga 3960 ccgacgtcac gacacacggt ggaaccgtgg accaaccacg ccgccaacac accaacgtgg 4020 agtttctgct ggacagattc acacacattg gtgagatcac cgcctccaag acaattgacc 4080 tcctggacac gaaggaacac acgttggtgg gcgcaatcct gcgttctgcc acgtattact 4140 tctgtgatct agaggttgcc gtcctgggca caagccagtg ggtggcgtgg gttcccaacg 4200 gatgcccaca caccgaccgc gtggaggaca acccagtcgt tcacgcgaag aacggtgtca 4260 cccgcttcgc tctgccgtac acagcaccac accaggttct cgctaccgtg tacaacggca 4320 actgcaaata ttccaacacc caacacgtta cgccacgccg tggtgacatg gccgcgttag 4380 cacaacgtgt tgcgaatgaa accacaagat gcacgcccac aactttcaac ttcgggagat 4440 tgttgtgtga cactggttcg gtttactacc ggatgaagag agctgagctg tactgcccac 4500 ggcccctcat ggtcaggtac acgcacacca ctgaccggta caaaatcaaa ctggttgcac 4560 ctgacaaaca acttctaaat tttgacctgc tcaaattggc gggagatgtg gagtccaacc 4620 ctgggccctt cttcttctcc gacgtcaggt caaatttctc aaaactggta gaaaccatca 4680 atcagatgca ggaggacatg tcaacaaaac acgggcctga ctttaaccgg ttggtgtccg 4740 catttgagga attggccact ggagtgaagg ctatcagggc cggtctcgac gaggccaaac 4800 cctggtacaa actcatcaag ctcctgagcc gcttgtcgtg catggccgct gtagcagcac 4860 ggtcaaagga cccagtcctt gtggccatca tgctggctga caccggtctt gagattctgg 4920 acagcacctt tgtcgtgaag aagatctccg actcgctctc cagtctcttt cacgtgccgg 4980 cccccgtctt cagtttcgga gccccgattc tgttggccgg gttggtcaaa gtcgcctcga 5040 gtttcttccg gtccacaccc gaagaccttg agagagcaga aaaacagctc aaagcacgtg 5100 acattaacga catattcgcc attctcaaga acggcgagtg gctggtcaag ctgatccttg 5160 ccatccgcga ctggatcaaa gcgtggatcg cctcagaaga aaagtttgtc accatgacgg 5220 acttggtgcc tggtatcctt gaaaagcagc gggatctcaa cgacccgagt aagtacaagg 5280 aagccaagga gtggctcgac aacgcgcgcc aggcgtgttt gaagagcggg aacgttcaca 5340 ttgccaattt gtgcaaagtg gtcgccccgg cacccagcaa gtcgagaccc gaacccgtgg 5400 tcgtttgcct ccgcggcaaa tccggccagg ggaagagttt ccttgcgaac gtgctcgcgc 5460 aagcaatctc cacccacttc accggcagaa ctgattcggt ttggtactgc ccgcctgacc 5520 ctgaccactt cgacggttac aaccagcaga ccgttgtcgt gatggacgat ttgggccaga 5580 accccgatgg caaggacttc aagtacttcg cccagatggt ttcgaccacg gggttcatcc 5640 cgcccatggc ctcgcttgag gacaaaggca agcctttcaa cagcaaagtc atcattgcta 5700 ccaccaacct gtactcgggt ttcaccccga gaacaatggt gtgtcctgac gcgctgaacc 5760 ggaggttcca ctttgacatc gacgtgagtg ccaaggacgg gtacaaagtt aacaacaaat 5820 tggacataat caaagctctt gaagacaccc acaccaaccc agtggcgatg ttccaatacg 5880 actgtgccct tctaaacggt atggcagttg aaatgaagag aatgcaacag gatatgttca 5940 agcctcaacc acccctccag aacgtgtacc aactcgttca cgaggtgatt gaacgggtcg 6000 agctccacga gaaggtgtcg agccacccga ttttcaaaca gatatcaatt ccttcccaaa 6060 agtctgtgtt gtacttcctc attgagaaag gccaacacga agcagcaatt gaattctttg 6120 agggaatggt gcatgactcc atcaaggaag agctccggcc cctcatccaa cagacctcat 6180 ttgtgaaacg cgcttttaag cgcctgaagg aaaactttga gactgttgcc ctgtgtttga 6240 ctcttttggc aaacatagtg atcatgatcc gcgagactcg caagagacaa cagatggtgg 6300 acgatgcagt gaatgactac attgagaagg caaacatcac cacagatgac aagactcttg 6360 acgaggcgga aaagaaccct ctagagacca gcggtgccag cactattggt ttcagagaga 6420 gaactctccc ggggcacaag gcgagcgatg acgtgagctc cgagcccgcc aaacccgtgg 6480 aggaccgacc acaagctgaa ggaccttacg agggaccggt gaagaagcct gtcgctttga 6540 aagtgaaagc taagaacttg attgtcactg aggggccata tgaaggacca gtgaagaaac 6600 ctgtcgcttt gaaagtgaaa gcaaaagccc cgattgtcac tgaaggaccc tacgagggac 6660 cggtgaagaa gcctgtcgct ttgaaagtga aagccaagaa cttgattgtc actgagagtg 6720 gtgccccacc gaccgacttg cagaagatgg tcatgggcaa cactaagcct gttgagctca 6780 tcctcgacgg gaagacggta gccatctgct gtgctaccgg agtgtttggc actgcctacc 6840 tcgtacctcg tcacctcttc gcggagaagt acgacaagat aatgttggac ggtagagcca 6900 tgacagacag tgactacaga gtgtttgagt ttgagattaa agtaaaagga caggacatgc 6960 tctcagacgc tgcactcatg gtgcttcacc gtgggaaccg cgtgagagac atcacgaaac 7020 attttcgtga cacagcaaga atgaagaaag gcacccccgt tgtcggtgtg atcaacaacg 7080 ccgacgttgg gagactgatt ttctctggag aggcccttac ctacaaagac attgtagtga 7140 ccatggatgg agacaccatg ccgggcctgt ttgcctacag agccgccacc aaggctggtt 7200 actgcggggg agccgttctc gccaaggacg gagccgacac attcatcgtt ggcacccact 7260 ccgcaggtgg taacggagtt ggatactgct cgtgcgtgtc caggtccatg ctcctgaaaa 7320 tgaaggcaca cattgaccct gaaccacacc acgaggggtt gattgttgat accagagatg 7380 tggaagagcg cgtgcatgtc atgcgtaaaa ccaagcttgc acccaccgtg gcacacggtg 7440 tgtttaaccc tgaatttggt cccgctgcct tgtccaacaa ggacccgcgg ctgaacgaag 7500 gggttgtcct cgatgaagtc atcttctcca aacacaaggg agacacgaaa atgtctgagg 7560 aggacaaagc gctgttccgc cgctgcgctg ccgactacgc gtcgcacttg cacagcgtgc 7620 tggggacggc aaatgcccca ttgagcatct atgaggccat caaaggcgtc gacgggctcg 7680 atgccatgga gccggacacc gcgcccggcc tcccctgggc cctccagggg aaacgccgtg 7740 gtgcgttgat tgacttcgag aacggcacgg tcggacccga agtcgaggct gccctaaagc 7800 tcatggagaa aagagagtac aaatttgctt gccagacctt cctgaaagac gagattcgtc 7860 cgatggaaaa agtacgtgct ggcaagactc gcattgtcga cgttttgccc gtggaacaca 7920 ttctttacac caggatgatg attggcagat tctgtgctca aatgcacaca aacaatggac 7980 cgcagattgg ctcagcggtc ggttgcaatc ctgatgttga ttggcaaaga tttggcacac 8040 attttgctca gtacagaaac gtgtgggatg tggactattc ggcctttgat gctaaccact 8100 gcagtgacgc aatgaacatc atgtttgagg aggtatttcg cacagacttc ggtttccacc 8160 caaatgctga gtggattctg aagactcttg tgaacacgga gcacgcctat gagaacaaac 8220 gtatcactgt tgagggcggg atgccgtctg gctgttccgc gacaagcatc atcaacacaa 8280 ttttgaacaa catttatgtg ctctacgctc ttcgtagaca ctatgaggga gttgagctgg 8340 acacctacac catgatctcc tacggagatg acatcgtggt tgcaagtgac tacgatctgg 8400 attttgaggc tctcaaaccc cacttcaaat ctcttggtca aaccatcact ccagctgaca 8460 aaagcgacaa aggttttgtt cttggtcact ccattaccga tgtcactttc ctcaaaagac 8520 acttccacat ggactatgga actgggtttt acaaacctgt gatggcctca aagaccctcg 8580 aggccattct ctcctttgca cgccgtggga ccatacagga gaagttgatc tccgtggcag 8640 gactcgccgt ccactcagga cctgacgagt accggcgtct ctttgagccc ttccagggtc 8700 tcttcgagat tccaagctac agatcacttt acctgcgttg ggtgaacgcc gtgtgcggtg 8760 acgcataatc cctcagatgt cacaattggc agaaagactc tgaggcgagc gacgccgtag 8820 gagtgaaaag cccgaaaggg cttttcccgc ttcctattcc aaaaaaaaaa aaaaaaaact 8880 agttctagag cggccgccac cgcggtggag ctccagcttt tgttcccttt agtgagggtt 8940 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 9000 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 9060 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 9120 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 9180 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 9240 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 9300 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 9360 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 9420 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 9480 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 9540 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 9600 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 9660 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 9720 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 9780 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 9840 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 9900 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 9960 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 10020 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 10080 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 10140 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 10200 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 10260 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 10320 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 10380 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 10440 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 10500 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 10560 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 10620 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 10680 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 10740 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 10800 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 10860 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 10920 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 10980 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 11040 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 11100 ccgaaaagtg ccac 11114 <210> 2 <211> 144 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of substitution region in 3B1 and 3B2 of O-Manisa <400> 2 ggaccttacg agggaccggt gaagaagcct gtcgctttga aagtgaaagc taagaacttg 60 attgtcactg aggggccata tgaaggacca gtgaagaaac ctgtcgcttt gaaagtgaaa 120 gcaaaagccc cgattgtcac tgaa 144 <210> 3 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of deletion region in 3B1 and 3B2 of O-Manisa <400> 3 Gly Pro Tyr Ala Gly Pro Leu Glu Arg Gln Lys Pro Leu Arg Val Lys 1 5 10 15 Thr Lys Leu Pro Gln Gln Glu Gly Pro Tyr Ala Gly Pro Met Asp Arg 20 25 30 Gln Lys Pro Leu Lys Val Arg Ala Arg Ala Pro Val Val Lys Glu 35 40 45 <210> 4 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of substitution region in 3B1 and 3B2 of O-Manisa <400> 4 Gly Pro Tyr Glu Gly Pro Val Lys Lys Pro Val Ala Leu Lys Val Lys 1 5 10 15 Ala Lys Asn Leu Ile Val Thr Glu Gly Pro Tyr Glu Gly Pro Val Lys 20 25 30 Lys Pro Val Ala Leu Lys Val Lys Ala Lys Ala Pro Ile Val Thr Glu 35 40 45 <210> 5 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for P1 of SAT3 ZIM virus <400> 5 aacaaaggtc cagaaaaggc tcaagggagc gggacagtca tca 43 <210> 6 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for P1 of SAT3 ZIM virus <400> 6 tttgagcagg tcaaaattta gaagtgcaca gttgtttctc gacg 44 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for P1-deleted-O-manisa virus <400> 7 acttctaaat tttgacctgc 20 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for P1-deleted-O-manisa virus <400> 8 cttgagcctt ttctggac 18 <110> Animal and plant quaratine agency <120> Recombinant foot-and-mouth disease virus expressing protective          antigen of type SAT3 ZIM <130> 17-10922 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 11114 <212> DNA <213> Artificial Sequence <220> <223> Full DNA sequence of pO-SAT3 ZIM <400> 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc atatgtaata cgactcacta tagggttgaa agggggcgct 660 agggtctcac ccctagcatg ccaacgacag ctcctacgtc gcactccaca ctaacgtttg 720 tgtgcgcgcg ggaaccgatg gacttttgtt cacccaccta cagttggact cacggcaccg 780 cgtggccatt ttagctgggt tgtgcggacg aacactgctt gcgcatctcg cgtgaccggt 840 tagtactctt accactatcc gcctacttgg tcgttagcgc tgtcctgggc actcttgttg 900 ggggctgttc aacgctctac ggtctcccct gcgtaacaga ctacggtgtt ggggccgctt 960 cgtgcgagcc gatcgcttgg tgtgcctcgg ctgtcgcccg aagcccgcct ttcacccccc 1020 cccccccccc ctaggtttta ccgtcgttcc cgacgttaat ggggaaacaa ccacaagctt 1080 aacaccgtct tgcccgacgt aaaagggctg caaccaaaaa gcttgtgccg cctttcccgg 1140 cgttaatggg aggtaaccac aagacaaacc ttcacccgga agtaaaacgg caacttcaca 1200 cagttttgcc cgttttcgtg agaaatgggc cgtcaacgca cgaaacgcgc cgtcgcttga 1260 ggaggacttg tacaaacacg atctatgcag gtttccacaa ctgacacaaa ccgtgcaact 1320 tgaaaccccg cctggtcttt ccaggtctag aggggcgaca ttttgtactg tgcttgactc 1380 cacgctcggt ccactagcga gtgttagtag tagcactgtt gcttcgtagc ggagcatgat 1440 ggccgtggga gcttcccctt ggtaacaagg acccacgggg ccaaaagcca cgtcctaccg 1500 gacccatcat gtgtgcaaac ccagcacggc aactttactg cgaaaaccac tttaaggtga 1560 cactgatact ggtactcaat cactggtgac aggctaagga tgcccttcag gtaccccgag 1620 gtaacacgcg acactcggga tctgagaagg ggactggggc ttctttaaaa gtgcccagtt 1680 taaaaagctt ctatgcctga ataggcgacc ggaggccggc gccttttcac tgttttacta 1740 ctgttttcat gaatacaact gactgtttca ccgccctgtt acacgctctc agagagatca 1800 aaacactgtt tcttttacgg acacaaggaa agatggaatt cacactttac aacggtgaga 1860 agaaaacctt ctactccaga cccaacaacc acgacaactg ctggcttaac accattctcc 1920 agttgttcag gtatgttgat gagcctttct ttgactgggt ctacgactcg cctgaaaacc 1980 tcactcttga ggcaatcaaa cagttggaag agacaaccgg tcttgagctg cacgagggtg 2040 gaccacccgc tctcgtcatc tggaacatca aacacttgct tcacaccgga atcggcactg 2100 cctcacgccc tagcgaggtg tgtatggtgg acggaacgga catgtgttta gctgattttc 2160 atgctggcat tttcctgaaa ggacaggaac atgctgtgtt cgcctgtgtc acctccaacg 2220 ggtggtacgc gattgatgac gaggactttt acccttggac accggacccg tccgacgttc 2280 tggtgtttgt cccgtacgat caagaaccgc ttaacggaga gtggaaaaca aaggtccaga 2340 aaaggctcaa gggagcaggc caatcctccc ccgccacggg gtctcaaaat caatcaggca 2400 atactggtag catcattaac aactactaca tgcaacagta ccagaactcc atggacaccc 2460 agcttggcga caacgccatc tcgggtggat cgaatgaagg tagcactgac accacttcca 2520 cccacaccaa caacacacag aacaacgact ggttctcaaa gttggcgcag tctgccattt 2580 cgggactctt tggagctctg ttggcggaca agaagacaga ggagacaact cttctggagg 2640 atcgcatcct caccacgcgc cacaacacaa ccacgtccac cacacagagt tctgtcggtg 2700 tcacatacgg ttacgcgtca gctcaccgtt ttctgcctgg gcccaacacc agtggactcg 2760 agacacgcgt tgaacaggcg gagagattct tcaaggagaa actcttcact tggaccgcga 2820 gccaagagta cgcacacgtg catctactgg aactacccgt ggaccacaaa ggcatctacg 2880 gtgccatgct ggacagccac gcatacgtgc gaaatggctg ggacgtgcag gtttccgcaa 2940 ccagcacaca gttcaatggt ggcactctcc tcgtcgccat ggtccccgag ctgaagactc 3000 tggacaagcg tgacgtgtca caactcacgc ttttccccca ccagttcatc aacccacgca 3060 ccaacaccac cgcacacatc gtggtaccgt acgtgggtgt caacagacac gaccaggcaa 3120 agatgcacaa ggcatggaca ctcgtggtcg cggtgcttgc gccgctcacc acgtcaagca 3180 tgggacagga caacgtagag gtgtatgcga acatcgcacc caccaatgtg tacgttgctg 3240 gagagaagcc aacaaaacaa ggcatcatcc ccgtggcctg caacgacggt tacggtggat 3300 tccagaatac tgacccaaag accgcggacc caatctacgg tctcgtgtcc aacccgccac 3360 gcacagcgtt ccccggcagg ttcaccaacc tgctggatgt cgccgaagcg tgcccaacct 3420 tcctggactt cgacggcaca ccctacgtca agaccaggca caacagcggg agcaaaatac 3480 tgacacacat tgatttggct tttggacata agagtttcaa gaacacctac ctagcaggac 3540 tcgcccagta ctacgcccag tacagcgggt ctctgaatct gcacttcatg tacaccggtc 3600 caacgcagtc taaagcacgc tttatggttg cgtacattcc accagggacc aaccctgtac 3660 ctgacactcc tgaggctgca gcacactgct accactcaga gtgggacacc ggactgaact 3720 ccaagttcac gtttacggtt ccctatatgt cagctgcaga ctttgcatac acgtactgtg 3780 atgagcctga acaggcttca gcacaggggt gggtgacact ctaccaaatt acagacacgc 3840 acgaccccga ttcggcggtg cttgtctcgg tcagtgctgg cgctgacctt gaatttcggc 3900 tcccaatcaa ccctgcaacc cagacaacca gctcaggtga aggtggtgat gttgtgacga 3960 ccgacgtcac gacacacggt ggaaccgtgg accaaccacg ccgccaacac accaacgtgg 4020 agtttctgct ggacagattc acacacattg gtgagatcac cgcctccaag acaattgacc 4080 tcctggacac gaaggaacac acgttggtgg gcgcaatcct gcgttctgcc acgtattact 4140 tctgtgatct agaggttgcc gtcctgggca caagccagtg ggtggcgtgg gttcccaacg 4200 gatgcccaca caccgaccgc gtggaggaca acccagtcgt tcacgcgaag aacggtgtca 4260 cccgcttcgc tctgccgtac acagcaccac accaggttct cgctaccgtg tacaacggca 4320 actgcaaata ttccaacacc caacacgtta cgccacgccg tggtgacatg gccgcgttag 4380 cacaacgtgt tgcgaatgaa accacaagat gcacgcccac aactttcaac ttcgggagat 4440 tgttgtgtga cactggttcg gtttactacc ggatgaagag agctgagctg tactgcccac 4500 ggcccctcat ggtcaggtac acgcacacca ctgaccggta caaaatcaaa ctggttgcac 4560 ctgacaaaca acttctaaat tttgacctgc tcaaattggc gggagatgtg gagtccaacc 4620 ctgggccctt cttcttctcc gacgtcaggt caaatttctc aaaactggta gaaaccatca 4680 atcagatgca ggaggacatg tcaacaaaac acgggcctga ctttaaccgg ttggtgtccg 4740 catttgagga attggccact ggagtgaagg ctatcagggc cggtctcgac gaggccaaac 4800 cctggtacaa actcatcaag ctcctgagcc gcttgtcgtg catggccgct gtagcagcac 4860 ggtcaaagga cccagtcctt gtggccatca tgctggctga caccggtctt gagattctgg 4920 acagcacctt tgtcgtgaag aagatctccg actcgctctc cagtctcttt cacgtgccgg 4980 cccccgtctt cagtttcgga gccccgattc tgttggccgg gttggtcaaa gtcgcctcga 5040 gtttcttccg gtccacaccc gaagaccttg agagagcaga aaaacagctc aaagcacgtg 5100 acattaacga catattcgcc attctcaaga acggcgagtg gctggtcaag ctgatccttg 5160 ccatccgcga ctggatcaaa gcgtggatcg cctcagaaga aaagtttgtc accatgacgg 5220 acttggtgcc tggtatcctt gaaaagcagc gggatctcaa cgacccgagt aagtacaagg 5280 aagccaagga gtggctcgac aacgcgcgcc aggcgtgttt gaagagcggg aacgttcaca 5340 ttgccaattt gtgcaaagtg gtcgccccgg cacccagcaa gtcgagaccc gaacccgtgg 5400 tcgtttgcct ccgcggcaaa tccggccagg ggaagagttt ccttgcgaac gtgctcgcgc 5460 aagcaatctc cacccacttc accggcagaa ctgattcggt ttggtactgc ccgcctgacc 5520 ctgaccactt cgacggttac aaccagcaga ccgttgtcgt gatggacgat ttgggccaga 5580 accccgatgg caaggacttc aagtacttcg cccagatggt ttcgaccacg gggttcatcc 5640 cgcccatggc ctcgcttgag gacaaaggca agcctttcaa cagcaaagtc atcattgcta 5700 ccaccaacct gtactcgggt ttcaccccga gaacaatggt gtgtcctgac gcgctgaacc 5760 ggaggttcca ctttgacatc gacgtgagtg ccaaggacgg gtacaaagtt aacaacaaat 5820 tggacataat caaagctctt gaagacaccc acaccaaccc agtggcgatg ttccaatacg 5880 actgtgccct tctaaacggt atggcagttg aaatgaagag aatgcaacag gatatgttca 5940 agcctcaacc acccctccag aacgtgtacc aactcgttca cgaggtgatt gaacgggtcg 6000 agctccacga gaaggtgtcg agccacccga ttttcaaaca gatatcaatt ccttcccaaa 6060 agtctgtgtt gtacttcctc attgagaaag gccaacacga agcagcaatt gaattctttg 6120 agggaatggt gcatgactcc atcaaggaag agctccggcc cctcatccaa cagacctcat 6180 ttgtgaaacg cgcttttaag cgcctgaagg aaaactttga gactgttgcc ctgtgtttga 6240 ctcttttggc aaacatagtg atcatgatcc gcgagactcg caagagacaa cagatggtgg 6300 acgatgcagt gaatgactac attgagaagg caaacatcac cacagatgac aagactcttg 6360 acgaggcgga aaagaaccct ctagagacca gcggtgccag cactattggt ttcagagaga 6420 gaactctccc ggggcacaag gcgagcgatg acgtgagctc cgagcccgcc aaacccgtgg 6480 aggaccgacc acaagctgaa ggaccttacg agggaccggt gaagaagcct gtcgctttga 6540 aagtgaaagc taagaacttg attgtcactg aggggccata tgaaggacca gtgaagaaac 6600 ctgtcgcttt gaaagtgaaa gcaaaagccc cgattgtcac tgaaggaccc tacgagggac 6660 cggtgaagaa gcctgtcgct ttgaaagtga aagccaagaa cttgattgtc actgagagtg 6720 gtgccccacc gaccgacttg cagaagatgg tcatgggcaa cactaagcct gttgagctca 6780 tcctcgacgg gaagacggta gccatctgct gtgctaccgg agtgtttggc actgcctacc 6840 tcgtacctcg tcacctcttc gcggagaagt acgacaagat aatgttggac ggtagagcca 6900 tgacagacag tgactacaga gtgtttgagt ttgagattaa agtaaaagga caggacatgc 6960 tctcagacgc tgcactcatg gtgcttcacc gtgggaaccg cgtgagagac atcacgaaac 7020 attttcgtga cacagcaaga atgaagaaag gcacccccgt tgtcggtgtg atcaacaacg 7080 ccgacgttgg gagactgatt ttctctggag aggcccttac ctacaaagac attgtagtga 7140 ccatggatgg agacaccatg ccgggcctgt ttgcctacag agccgccacc aaggctggtt 7200 actgcggggg agccgttctc gccaaggacg gagccgacac attcatcgtt ggcacccact 7260 ccgcaggtgg taacggagtt ggatactgct cgtgcgtgtc caggtccatg ctcctgaaaa 7320 tgaaggcaca cattgaccct gaaccacacc acgaggggtt gattgttgat accagagatg 7380 tggaagagcg cgtgcatgtc atgcgtaaaa ccaagcttgc acccaccgtg gcacacggtg 7440 tgtttaaccc tgaatttggt cccgctgcct tgtccaacaa ggacccgcgg ctgaacgaag 7500 gggttgtcct cgatgaagtc atcttctcca aacacaaggg agacacgaaa atgtctgagg 7560 aggacaaagc gctgttccgc cgctgcgctg ccgactacgc gtcgcacttg cacagcgtgc 7620 tggggacggc aaatgcccca ttgagcatct atgaggccat caaaggcgtc gacgggctcg 7680 atgccatgga gccggacacc gcgcccggcc tcccctgggc cctccagggg aaacgccgtg 7740 gtgcgttgat tgacttcgag aacggcacgg tcggacccga agtcgaggct gccctaaagc 7800 tcatggagaa aagagagtac aaatttgctt gccagacctt cctgaaagac gagattcgtc 7860 cgatggaaaa agtacgtgct ggcaagactc gcattgtcga cgttttgccc gtggaacaca 7920 ttctttacac caggatgatg attggcagat tctgtgctca aatgcacaca aacaatggac 7980 cgcagattgg ctcagcggtc ggttgcaatc ctgatgttga ttggcaaaga tttggcacac 8040 attttgctca gtacagaaac gtgtgggatg tggactattc ggcctttgat gctaaccact 8100 gcagtgacgc aatgaacatc atgtttgagg aggtatttcg cacagacttc ggtttccacc 8160 caaatgctga gtggattctg aagactcttg tgaacacgga gcacgcctat gagaacaaac 8220 gtatcactgt tgagggcggg atgccgtctg gctgttccgc gacaagcatc atcaacacaa 8280 ttttgaacaa catttatgtg ctctacgctc ttcgtagaca ctatgaggga gttgagctgg 8340 acacctacac catgatctcc tacggagatg acatcgtggt tgcaagtgac tacgatctgg 8400 attttgaggc tctcaaaccc cacttcaaat ctcttggtca aaccatcact ccagctgaca 8460 aaagcgacaa aggttttgtt cttggtcact ccattaccga tgtcactttc ctcaaaagac 8520 acttccacat ggactatgga actgggtttt acaaacctgt gatggcctca aagaccctcg 8580 aggccattct ctcctttgca cgccgtggga ccatacagga gaagttgatc tccgtggcag 8640 gactcgccgt ccactcagga cctgacgagt accggcgtct ctttgagccc ttccagggtc 8700 tcttcgagat tccaagctac agatcacttt acctgcgttg ggtgaacgcc gtgtgcggtg 8760 acgcataatc cctcagatgt cacaattggc agaaagactc tgaggcgagc gacgccgtag 8820 gagtgaaaag cccgaaaggg cttttcccgc ttcctattcc aaaaaaaaaa aaaaaaaact 8880 agttctagag cggccgccac cgcggtggag ctccagcttt tgttcccttt agtgagggtt 8940 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 9000 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 9060 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 9120 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 9180 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 9240 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 9300 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 9360 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 9420 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 9480 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 9540 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 9600 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 9660 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 9720 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 9780 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 9840 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 9900 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 9960 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 10020 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 10080 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 10140 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 10200 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 10260 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 10320 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 10380 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 10440 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 10500 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 10560 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 10620 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 10680 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 10740 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 10800 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 10860 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 10920 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 10980 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 11040 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 11100 ccgaaaagtg ccac 11114 <210> 2 <211> 144 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of substitution region in 3B1 and 3B2 of O-Manisa <400> 2 ggaccttacg agggaccggt gaagaagcct gtcgctttga aagtgaaagc taagaacttg 60 attgtcactg aggggccata tgaaggacca gtgaagaaac ctgtcgcttt gaaagtgaaa 120 gcaaaagccc cgattgtcac tgaa 144 <210> 3 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of deletion region in 3B1 and 3B2 of O-Manisa <400> 3 Gly Pro Tyr Ala Gly Pro Leu Glu Arg Gln Lys Pro Leu Arg Val Lys   1 5 10 15 Thr Lys Leu Pro Gln Gln Glu Gly Pro Tyr Ala Gly Pro Met Asp Arg              20 25 30 Gln Lys Pro Leu Lys Val Arg Ala Arg Ala Pro Val Val Lys Glu          35 40 45 <210> 4 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of substitution region in 3B1 and 3B2 of          O-manisa <400> 4 Gly Pro Tyr Glu Gly Pro Val Lys Lys Pro Val Ala Leu Lys Val Lys   1 5 10 15 Ala Lys Asn Leu Ile Val Thr Glu Gly Pro Tyr Glu Gly Pro Val Lys              20 25 30 Lys Pro Val Ala Leu Lys Val Lys Ala Lys Ala Pro Ile Val Thr Glu          35 40 45 <210> 5 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for P1 of SAT3 ZIM virus <400> 5 aacaaaggtc cagaaaaggc tcaagggagc gggacagtca tca 43 <210> 6 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for P1 of SAT3 ZIM virus <400> 6 tttgagcagg tcaaaattta gaagtgcaca gttgtttctc gacg 44 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for P1-deleted-O-manisa virus <400> 7 acttctaaat tttgacctgc 20 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for P1-deleted-O-manisa virus <400> 8 cttgagcctt ttctggac 18

Claims (9)

구제역 O형 Manisa 유전체의 P1 유전자 부위가 구제역 SAT3 ZIM의 P1 유전자로 치환되고, 상기 구제역 O형 Manisa 유전체의 3B 부위 중, 3B1 및 3B2 부위가 서열번호 2의 서열로 치환되었으며, 상기 구제역 O형 Manisa 유전체의 3C 부위 유전자 중 142번째 아미노산 시스테인이 트레오닌으로 치환(C142T)된 재조합 플라스미드에 있어서,
상기 재조합 플라스미드는 구제역 O형 Manisa 유전체 염기서열을 포함하는 것으로 서열번호 1(pO-SAT3 ZIM)의 염기서열로 이루어진 것을 특징으로 하는 재조합 플라스미드The P1 gene region of the foot-and-mouth disease O type Manisa genome was substituted with the P1 gene of the foot-and-mouth disease SAT3 ZIM, and among the 3B regions of the foot-and-mouth disease O type Manisa genome, the 3B1 and 3B2 sites were substituted with the sequence of SEQ ID NO: 2, and the foot-and-mouth type O manisa In a recombinant plasmid in which the 142th amino acid cysteine of the genome 3C region gene is substituted with threonine (C142T),
The recombinant plasmid is a recombinant plasmid comprising the nucleotide sequence of SEQ ID NO: 1 (pO-SAT3 ZIM) that includes foot-and-mouth type O Manisa genome sequence 제1항의 재조합 플라스미드를 이용하여 제조한 재조합 구제역 바이러스.Recombinant foot-and-mouth virus produced using the recombinant plasmid of claim 1. 제2항에 있어서, 상기 재조합 바이러스는 구제역 SAT3 I 지역형인, SAT3 ZIM 주의 방어항원이 발현되는 것인, 재조합 구제역 바이러스.The recombinant foot-and-mouth virus according to claim 2, wherein the recombinant virus is expressed in the defensive antigen of the SAT3 ZIM strain of foot-and-mouth disease SAT3 I region type. (a) 구제역 O형 Manisa의 유전자를 재조합 벡터에 삽입하는 단계;
(b) 상기 (a)단계에서 얻은 재조합 벡터에서, 구제역 O형 Manisa의 3B1 및 3B2 부위를 서열번호 2의 서열로 치환하는 단계;
(c) 상기 (b)단계에서 얻은 재조합 벡터에서, 상기 구제역 O형 Manisa의 3C 유전자 중 142번째 아미노산 시스테인을 트레오닌으로 치환(C142T)하는 단계; 및
(d) 상기 (c)단계에서 얻은 재조합 벡터에서, 상기 구제역 O형 Manisa의 P1 유전자 부위를 구제역 SAT3-ZIM의 P1 유전자로 치환하는 단계를 포함하는 재조합 벡터의 제조방법.(a) inserting a gene of foot-and-mouth type O Manisa into a recombinant vector;
(b) replacing the 3B1 and 3B2 sites of foot-and-mouth disease O type Manisa with the sequence of SEQ ID NO: 2 in the recombinant vector obtained in step (a);
(c) in the recombinant vector obtained in step (b), substituting threonine for the 142th amino acid cysteine of the 3C gene of the foot-and-mouth type O manisa (C142T); And
(d) in the recombinant vector obtained in step (c), the method of producing a recombinant vector comprising the step of replacing the P1 gene region of foot-and-mouth disease O type Manisa with the P1 gene of foot-and-mouth disease SAT3-ZIM. 제4항에서 제조된 재조합 벡터를 세포에 도입하여 증식시키는 단계;를 포함하는 재조합 구제역 바이러스의 제조방법.A method for producing a recombinant foot-and-mouth virus, comprising the steps of: introducing and propagating the recombinant vector prepared in claim 4 into a cell. 제5항에 있어서, 상기 세포는 염소 태아 혀 세포 및 햄스터 신장 세포 중에서 선택된 어느 하나 이상인 것을 특징으로 하는 재조합 구제역 바이러스의 제조방법.The method of claim 5, wherein the cells are any one or more selected from goat fetal tongue cells and hamster kidney cells. 제3항의 재조합 구제역 바이러스를 유효성분으로 포함하는 구제역 예방용 백신 조성물.A vaccine for foot-and-mouth disease prevention comprising the recombinant foot-and-mouth virus of claim 3 as an active ingredient. 제7항에 있어서, 상기 백신은 생백신, 약독화된 백신, 또는 사백신인 것인, 구제역 예방용 백신 조성물.The vaccine composition for preventing foot-and-mouth disease according to claim 7, wherein the vaccine is a live vaccine, an attenuated vaccine, or a dead vaccine. 제3항의 재조합 구제역 바이러스를 인간을 제외한 개체에 투여하는 단계;를 포함하는 구제역의 예방 방법.A method for preventing foot-and-mouth disease, comprising administering the recombinant foot-and-mouth virus of claim 3 to a subject other than a human.
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