KR102474263B1 - A combination vaccine based on virus like particle - Google Patents

A combination vaccine based on virus like particle Download PDF

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KR102474263B1
KR102474263B1 KR1020170109597A KR20170109597A KR102474263B1 KR 102474263 B1 KR102474263 B1 KR 102474263B1 KR 1020170109597 A KR1020170109597 A KR 1020170109597A KR 20170109597 A KR20170109597 A KR 20170109597A KR 102474263 B1 KR102474263 B1 KR 102474263B1
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오미경
최재방
이시내
김현일
박철세
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Abstract

본 발명은 PRRSV (Porcine reproductive and respiratory syndrome virus) 또는 구제역바이러스 (Foot-and-mouth disease virus) 의 병원성 에피토프가 삽입된 재조합 PCV2 (Porcine circovirus type 2) 의 ORF2, 이를 이용한 복합 백신 조성물 및 이를 이용한 감염 질환 예방 방법에 관한 것이다. 본 발명의 재조합 PCV2 ORF2 를 이용하는 경우, 곤충 또는 대장균 세포에서 바이러스 유사 입자를 효과적으로 생산할 수 있고, 이는 2종 이상의 돼지 바이러스에 대하여 효과적으로 면역 효과를 유도할 수 있으므로, PCV2 기반의 돼지 복합 백신으로 유용하게 사용될 수 있다. The present invention is a recombinant ORF2 of Porcine circovirus type 2 (PCV2) into which a pathogenic epitope of Porcine reproductive and respiratory syndrome virus (PRRSV) or foot-and-mouth disease virus is inserted, a composite vaccine composition using the same, and an infection using the same It is about disease prevention methods. When using the recombinant PCV2 ORF2 of the present invention, virus-like particles can be effectively produced in insect or Escherichia coli cells, which can effectively induce an immune effect against two or more porcine viruses, making it useful as a PCV2-based swine combination vaccine. can be used

Description

바이러스 유사입자 기반의 복합백신 {A combination vaccine based on virus like particle}A combination vaccine based on virus like particle}

본 발명은 PRRSV (Porcine reproductive and respiratory syndrome virus) 또는 구제역바이러스 (Foot-and-mouth virus) 의 병원성 에피토프가 삽입된 재조합 PCV2 (Porcine circovirus type 2) ORF2, 이를 이용한 복합 백신 조성물 및 이를 이용한 감염 질환 예방 방법에 관한 것이다. The present invention is a recombinant PCV2 (Porcine circovirus type 2) ORF2 into which a pathogenic epitope of PRRSV (Porcine reproductive and respiratory syndrome virus) or Foot-and-mouth virus is inserted, a composite vaccine composition using the same and prevention of infectious diseases using the same It's about how.

바이러스 유사입자(Virus like particle, VLP)는 바이러스와 유사한 구조를 갖는 게놈이 제거된 입자로 자가 조립 (self-assembly)되는 것이 가장 큰 특징이며 차세대 백신으로 그 가능성을 높이 평가 받고 있다. 게놈이 없기 때문에 복제와 감염이 불가능하고, 크기가 작아 세포 내 침투가 용이하며, virion과 유사한 형태로 강력한 체액성 및 세포성 면역을 유도하는 것이 특징이다. VLP기반으로 인체에서는 HBV, HPV, HEV와 influenza에 대한 백신이 허가되어 판매되고 있으며 가축에서는 PCV2에 대한 예방백신이 개발되어 적용되고 있다. Virus-like particles (VLPs) are characterized by self-assembly into particles from which the genome has been removed, and their potential as a next-generation vaccine is highly evaluated. Because it has no genome, replication and infection are impossible, and its small size makes it easy to penetrate cells. Based on VLP, vaccines against HBV, HPV, HEV and influenza are licensed and sold in humans, and preventive vaccines against PCV2 are developed and applied to livestock.

돼지 써코바이러스 2형(PCV2), 돼지생식기호흡기바이러스(PRRSV) 및 구제역 바이러스 (FMDV) 는 돼지에서 질병을 유발하는 대표적인 바이러스이다. Porcine circovirus type 2 (PCV2), porcine reproductive and respiratory virus (PRRSV) and foot-and-mouth disease virus (FMDV) are representative viruses that cause disease in pigs.

PCV2 (Porcine Circovirus type2)는 원형의 단일 가닥 DNA 게놈을 함유하는 작은(17 nm) 정이십면체 비외피형 바이러스로서 지금까지 알려진 가장 작은 동물 바이러스 중 하나이다. PCV2 게놈의 길이는 약 1768 bp이며, 이 바이러스는 PCV1(Porcine Circovirus type1)과 대략 80%의 서열 동일성을 공유한다. 하지만 일반적으로 비-병독성인 PCV1과는 대조적으로, PCV2에 감염된 돼지는 통상적으로 이유 후 전신성 소모성 증후군 (PMWS; Post-weaning Multisystemic Wasting Syndrome)이라고 하는 증상을 나타낸다. PMWS는 임상적으로 소모 (wasting), 피부의 창백함, 수척함, 호흡 곤란, 설사, 황달을 보이는 것이 특징이다. PCV2 감염 돼지를 부검하는 동안, 미시적 병변 및 육안 병변이 다수의 조직 및 기관에서 또한 나타나는데, 림프 기관이 가장 흔한 병변 부위이다. PCV2 핵산 또는 항원의 양과 미시적 림프 병변의 중증도 간에 강한 상관관계가 관찰되었다. PCV2에 감염된 돼지의 사망률은 80%에 이르며, PMWS 외에도, PCV2는 가성 광견병 (pseudorabies), 돼지 생식기호흡기 증후군 (PRRS: Porcine Reproductive and Respiratory Syndrome), 글래서병 (Glasser's disease), 연쇄구균성 수막염 (streptococcal meningitis), 살모넬라증 (salmonellosis), 이유후 대장균증 (postweaning colibacillosis), 식이성간증 (dietetic hepatosis) 및 화농성 기관지폐렴 (suppurative bronchopneumonia)을 포함하는 다수의 다른 감염을 동반한다. 따라서, PCV2의 감염을 예방할 수 있는 백신 치료제에 대한 필요성이 매우 크다.Porcine Circovirus type2 (PCV2) is a small (17 nm) icosahedral non-enveloped virus containing a circular single-stranded DNA genome and is one of the smallest animal viruses known so far. The length of the PCV2 genome is about 1768 bp, and the virus shares approximately 80% sequence identity with Porcine Circovirus type 1 (PCV1). However, in contrast to PCV1, which is generally non-virulent, pigs infected with PCV2 commonly display a condition called Post-weaning Multisystemic Wasting Syndrome (PMWS). PMWS is clinically characterized by wasting, pale skin, emaciation, dyspnoea, diarrhea, and jaundice. During necropsy of PCV2-infected pigs, microscopic and macroscopic lesions also appear in a number of tissues and organs, with lymphoid organs being the most common site of lesion. A strong correlation was observed between the amount of PCV2 nucleic acid or antigen and the severity of microscopic lymphatic lesions. PCV2-infected pigs have a mortality rate of up to 80%. In addition to PMWS, PCV2 also causes pseudorabies, Porcine Reproductive and Respiratory Syndrome (PRRS), Glasser's disease, and streptococcal meningitis. meningitis), salmonellosis, postweaning colibacillosis, dietetic hepatosis and suppurative bronchopneumonia. Therefore, there is a great need for a vaccine treatment capable of preventing PCV2 infection.

한편, PCV2의 OFR2 (Open Reading Frame 2) 유전자는 외피단백질 (capsid protein)을 암호화하는 것으로, 상기 ORF2에 의해 생성된 외피단백질은 바이러스 유사 입자(virus like particle)을 형성한다. 대략 30kDa의 분자량을 갖는 PCV2의 ORF2 단백질은 상기 ORF2 유전자를 포함하는 바이러스 벡터로 세포를 감염시켜, 이로부터 분리하는 방법을 통해 일반적으로 수득할 수 있다. 또한 발현된 ORF2 단백질은 자가 조립에 의해 자연적으로 바이러스 유사 입자 형태를 형성할 수 있으며 이를 통한 백신이 개발되어 양돈산업 분야에 적용되고 있다.On the other hand, the OFR2 (Open Reading Frame 2) gene of PCV2 encodes a capsid protein, and the capsid protein produced by ORF2 forms a virus like particle. The ORF2 protein of PCV2 having a molecular weight of approximately 30 kDa can be generally obtained by infecting cells with a viral vector containing the ORF2 gene and isolating the ORF2 gene. In addition, the expressed ORF2 protein can naturally form virus-like particles by self-assembly, and a vaccine has been developed and applied to the pig farming industry.

돼지생식기호흡기바이러스(Porcine reproductive and respiratory syndrome virus; PRRSV)는 아테리비리데 (Arteriviridae)에 속하는 단일가닥 양성 RNA 게놈을 함유하는 45~80 nm의 구형의 바이러스이다. PRRSV 게놈은 약 15 kb 로 8개의 ORF (open reading frame)을 가지며, ORF1a와 ORF1b는 유전물질을 복제하는 효소가 암호화된 비구조 단백질과, ORF2~7까지 각각 바이러스의 외형을 이루는 6개의 구조 단백질 (GP2, GP3, GP4, E, M, N)로 구성되어 있다. PRRSV는 번식장애와 호흡기 질환이 복합적으로 나타나는 질병으로 모돈의 유사산 및 분만 저하, 식욕결핍, 발열, 허약자돈, 폐청색증, 성장지연, 호흡곤란 등의 다양한 증상을 보이며, 다른 병원성 바이러스 및 세균과 상호작용으로 인하여 전세계 양돈산업에 경제적으로 엄청난 피해를 주고 있다. 이러한 PRRSV는 1980년대 후반에 유럽에서 처음으로 보고 되었으며 1994년에 북미에서 분리되면서 이를 각각 유럽형(Genotype 1)과 북미형(Genotype 2)으로 명명하였다. PRRSV 유럽형과 북미형은 50~60% 유전적 유사성을 보이며, 2006년 중국에서 Highly pathogenic PRRSV (HP-PRRSV)가 중국에서 최초로 보고되었다. PRRSV는 대식세포에 존재하는 바이러스 수용기인 CD163을 인식하여 감염되는 특이적이 바이러스로 심한 변이와 특이적인 면역반응으로 인해 기존 백신보다 현지에서 특이적으로 적용할 수 있는 새로운 백신이 요구되는 상황이다.Porcine reproductive and respiratory syndrome virus (PRRSV) is a 45-80 nm spherical virus containing a single-stranded positive RNA genome belonging to Arteriviridae. The PRRSV genome is about 15 kb and has eight open reading frames (ORFs). ORF1a and ORF1b are non-structural proteins encoding enzymes that replicate genetic material, and ORFs 2 to 7 are six structural proteins that make up the appearance of the virus, respectively. It consists of (GP2, GP3, GP4, E, M, N). PRRSV is a disease with a combination of reproductive disorders and respiratory diseases. It shows various symptoms such as lactation and parturition decline in sows, lack of appetite, fever, weak piglets, pulmonary cyanosis, growth retardation, and respiratory difficulties. The interaction is causing tremendous economic damage to the global pig farming industry. PRRSV was first reported in Europe in the late 1980s and was isolated in North America in 1994, and was named European (Genotype 1) and North American (Genotype 2), respectively. PRRSV European and North American types show 50-60% genetic similarity, and in 2006, Highly pathogenic PRRSV (HP-PRRSV) was first reported in China. PRRSV is a specific virus that infects by recognizing CD163, a viral receptor present in macrophages, and requires a new vaccine that can be applied more locally than existing vaccines due to severe mutation and specific immune response.

PRRSV 백신은 생독 백신 (Ingelvac PRRS MLV, Boehringer ingelheim)이 주를 이루고 있으며, 이로 인해 생독 백신에 의한 바이러스 전파가 문제가 되고 있고, 아시아 국가들의 분리주 들도 북미주와 가까운 것이 밝혀졌다. 이는 무분별한 백신남용으로 인해 돼지의 질병에 미치는 영향에 대해 시사하는 바가 크다고 할 수 있다. 이 문제를 개선하고자 구조단백질을 이용한 백신을 개발하고자 하였으나 현재까지 그 효과가 미비한 것으로 보고되었다. PRRSV 차세대 백신 개발을 위해 최근 미국에서 PRRSV의 5개 구조 단백질을 이용한 PRRSV VLP를 개발하였으며 그 가능성을 확인하였다. PRRSV vaccines are mainly live vaccines (Ingelvac PRRS MLV, Boehringer ingelheim), and this causes virus transmission by live vaccines to be a problem, and it has been found that isolates from Asian countries are also close to North America. This can be said to have great implications for the effects of indiscriminate vaccine abuse on pig diseases. In order to improve this problem, an attempt was made to develop a vaccine using structural proteins, but the effect has been reported to be insufficient so far. PRRSV VLP using five structural proteins of PRRSV was recently developed in the United States for the development of a PRRSV next-generation vaccine, and its potential was confirmed.

구제역 바이러스 (Foot-and-mouth virus; FMD)는 전염성이 매우 강하여 무리에서 한 마리가 감염되면 나머지 가축에게 모두 급속하게 감염된다. 또한 치사율이 50% 이상에 달하는 가축의 제 1종 바이러스성 법정전염병으로 특별한 치료법은 없고 감염 동물과 접촉한 다른 동물들을 살처분해야 하기 때문에 신속한 진단과 대처가 필요하다. Foot-and-mouth virus (FMD) is highly contagious, and if one animal in the herd is infected, it rapidly infects the rest of the livestock. In addition, it is a type 1 viral legal infectious disease of livestock with a mortality rate of more than 50%, and there is no special treatment, and since other animals in contact with the infected animal must be culled, rapid diagnosis and response are necessary.

구제역의 원인이 되는 바이러스는 피코나비리대과 (Picornaviridae family)의 아프소바이러스 (aphthovirus)에 속하는 바이러스이며, 바이러스 게놈은 약 8,000개의 핵산 염기로 이루어진 단일 RNA 가닥 (single-stranded RNA)으로 구성되어 있다. 상기 RNA는 초기에 단일 폴리펩타이드로 번역된 후 감염된 세포 내에서 바이러스에 의해 암 호화된 프로테아제 (viral protease)에 의해 연속적으로 절단되어 4개의 캡시드 단백질(VP1∼VP4) 및 비구조 폴리펩타이드 (2C, 3A, 3B, 3C 및 3D)를 생성한다. 이러한 구제역 바이러스의 혈청형은 7가지 타입 (A, O, C, Asia 1, SAT 1, SAT 2, SAT 3)이 존재하고 80여 가지의 아형(subtype)이 있다고 알려져 있다.The virus that causes foot-and-mouth disease is a virus belonging to the aphthovirus of the Picornaviridae family, and the viral genome consists of a single-stranded RNA consisting of about 8,000 nucleotide bases. . The RNA is initially translated into a single polypeptide and then sequentially cleaved by a viral protease in infected cells to form four capsid proteins (VP1 to VP4) and non-structural polypeptides (2C, 3A, 3B, 3C and 3D). It is known that there are 7 types (A, O, C, Asia 1, SAT 1, SAT 2, SAT 3) of serotypes of foot-and-mouth disease virus and about 80 subtypes.

구제역 백신으로 생독백신은 잘 사용되고 있지 않으며, 현재 사독백신이 전 세계적으로 널리 사용되고 있다. 국내에서도 2010년 11월 28일 구제역이 발생한 이후 독점적으로 공급되는 외국계 제약회사의 사독백신을 사용하고 있으나, 이 백신은 단가가 높아 축산업자들에게 적지 않은 부담이 되고 있다. 또한 사독백신은 일반적으로 면역원성이 떨어져 어주번트 (oil adjuvant)를 사용하는데, 이로 인해 백신을 접종한 이근부 이하 목근육에 비화농성병소가 발생되는 부작용이 나타난다. 그런데 이 부위는 식육으로서 가치가 특히 높은 부분이기 때문에 이로 인해 국내에서 연간 1천억원에 달하는 피해가 발생하고 있다. 국내에서 현재 사용되고 있는 사독백신의 접종 프로토콜은 모돈에게 6개월 간격으로 백신을 접종하고 자돈에게 모체이행항체가 소실된 후 1회 백신을 접종하도록 되어 있다. 그러나 기존 백신의 높은 단가 및 목근육에 생기는 부작용으로 인하여 구제역 백신 접종 기피현상이 발생하고 있어, 국내의 구제역 방어선이 허물어지고 있는 상황이다. 따라서 이러한 단점을 개선한 구제역 백신의 개발이 절실한 실정이다. As a foot-and-mouth disease vaccine, live poison vaccine is not used well, and dead poison vaccine is currently widely used around the world. In Korea, since the outbreak of foot-and-mouth disease on November 28, 2010, Zadok vaccine from foreign pharmaceutical companies, which has been supplied exclusively, is being used, but the vaccine is high in unit price, which is a considerable burden to livestock farmers. In addition, Zadok vaccine is generally less immunogenic and uses an adjuvant (oil adjuvant), which causes side effects of non-purulent lesions in the neck muscles below the vaccinated ear. However, since this part is a part with a particularly high value as meat, it causes damages of up to 100 billion won annually in Korea. The inoculation protocol of Zadok vaccine currently used in Korea is to inoculate sows at 6-month intervals and inoculate piglets once after the loss of maternal antibodies. However, due to the high unit price of existing vaccines and side effects occurring in the neck muscles, there is a phenomenon of avoiding foot-and-mouth disease vaccination, and the domestic defense line for foot-and-mouth disease is breaking down. Therefore, there is an urgent need to develop a foot-and-mouth disease vaccine that improves these disadvantages.

또한 불활성화 바이러스 백신의 제작은 생물재해/생물보안 위험을 일으키는데, 인접 환경의 오염을 방지하기 위해 바이러스가 고-봉쇄 설비에서 제조되어야 하기 때문이다. 추가적으로, 불활성화 바이러스 제품의 무해성이 완전히 보장될 수 없다. 실제로, 유럽에서의 여러 최근의 FMDV 사례가 불완전하게 불활성화된 바이러스로 추적되었다. 이러한 잠재적인 생물재해/생물보안 위험은 적격인 백신 공급원의 수를 소수로 제한한다. 이와 같은 제한은 지역이 돌발에 효과적 및 즉각적으로 반응하는 능력을 방해할 수 있다. In addition, the production of inactivated virus vaccines poses a biohazard/biosecurity risk, as the virus must be manufactured in high-containment facilities to prevent contamination of the immediate environment. Additionally, the innocence of inactivated virus products cannot be fully guaranteed. Indeed, several recent cases of FMDV in Europe have been traced to incompletely inactivated virus. These potential biohazard/biosecurity risks limit the number of eligible vaccine sources to a small number. Such restrictions may hinder the ability of a region to respond effectively and promptly to an outbreak.

불활화된 백신의 가장 큰 단점은 바이러스 항원을 주기적으로 접종하는 것이 필요하며, 바이러스가 방출될 위험이 있다는 것이다. 이러한 단점을 보안하기 위해서 할 수 있는 것은 재조합 단백질을 이용한 안전한 백신을 생산하는 것이다. 현재 이용 가능한 구제역 바이러스 백신의 단점 및 한계를 해결하기 위하여 바이러스 유사입자인 VLP(Virus like particle)백신을 이용할 수 있다. 재조합 백신은 생성과정에서 전염성이 있는 바이러스를 필요로 하지 않기 때문에 바이러스가 방출될 위험이 없다. 재조합 단백질이나 펩타이드는 생산과정이 쉽고, 보관에 용이한 장점도 있다. 또한 구제역 바이러스의 RNA가 포함되지 않아 안정성이 높으면서도 방어능이 향상된 백신이며 오일 어주번트(oil adjuvant)를 필수적으로 사용하지 않아도 되므로 돼지 목근육 부위의 폐기로 인한 경제적 손실을 막을 수 있다.A major disadvantage of inactivated vaccines is that periodic vaccination with viral antigens is required and there is a risk of virus shedding. What can be done to secure these disadvantages is to produce safe vaccines using recombinant proteins. In order to solve the disadvantages and limitations of currently available foot-and-mouth disease virus vaccines, virus like particle (VLP) vaccines can be used. Recombinant vaccines do not require any infectious virus to be produced, so there is no risk of virus shedding. Recombinant proteins or peptides have advantages of easy production and easy storage. In addition, it is a vaccine with high stability and improved protective ability because it does not contain RNA of foot-and-mouth disease virus, and it does not require the use of oil adjuvant, so it can prevent economic loss due to the disposal of pig neck muscle parts.

FMDV 관련 대표적인 VLP 연구는 Asia 1형 VLP를 제작하기 위하여 구제역 바이러스의 구조 단백질인 P1과 3C유전자를 Dual vector에 삽입한 재조합 배큘로바이러스를 제작하고 Bac-to-Bac system을 이용하여 곤충세포에서 발현한 것이라 할 수 있다. 또한 FMDV 관련 VLP 연구는 전 세계적으로 많이 이루어지고 있다. 1970대 중반까지 구제역바이러스의 주요한 면역물질인 VP1을 이용하여 발현하였고, 이 단백질을 기초로 하여 재조합 단백질을 생산하였다. 대장균에서 발현된 VP1은 돼지, 소에서 중화항체를 유도할 수 있었다. 바이러스의 VP1의 G-H loop는 중화항체를 유도하기 위한 주요한 면역성 유발부위이므로 이 부위의 펩타이드를 이용한 백신개발 연구가 보고되고 있다. 하지만 소와 돼지에 G-H loop 합성 펩타이드를 접종하였을 때에는 높은 수준의 중화항체가 생성되었으나 질병으로부터 방어되지 않았다. 구제역의 특이 T 세포 에피토프만을 면역하였을 경우, 감수성 소에서 바이러스T 세포 에피토프 백신은 기대하는 만큼의 방어효과가 나타나지 않았다. A representative VLP study related to FMDV was to construct a recombinant baculovirus by inserting the P1 and 3C genes, structural proteins of foot-and-mouth disease virus, into a dual vector to produce Asia type 1 VLP, and expressed in insect cells using the Bac-to-Bac system. can be said to have been In addition, many VLP studies related to FMDV are being conducted around the world. Until the mid-1970s, foot-and-mouth disease virus was expressed using VP1, a major immune substance, and recombinant proteins were produced based on this protein. VP1 expressed in E. coli was able to induce neutralizing antibodies in pigs and cattle. Since the G-H loop of VP1 of the virus is a major immune-inducing site for inducing neutralizing antibodies, vaccine development studies using peptides from this site have been reported. However, when cattle and pigs were inoculated with the G-H loop synthesized peptide, high levels of neutralizing antibodies were produced, but were not protected from disease. When only the specific T-cell epitope of foot-and-mouth disease was immunized, the viral T-cell epitope vaccine did not show the expected protective effect in susceptible cows.

따라서 돼지에 감염되는 대표적인 바이러스인 PRRSV, PCV2, FMDV 를 모두 효과적으로 예방할 수 있는 새로운 백신에 대한 필요성 및 이를 제조하기 위한 방법에 대한 필요성이 있으며, 특히 상기 바이러스에 대하여 동시에 면역화할 수 있는 새로운 복합 백신에 대한 필요성이 있다. Therefore, there is a need for a new vaccine that can effectively prevent all of PRRSV, PCV2, and FMDV, which are representative viruses that infect pigs, and a method for preparing the same, and in particular, a new combination vaccine that can simultaneously immunize against the viruses there is a need for

본 발명자들은 PRRSV, PCV2, FMDV 를 모두 효과적으로 예방할 수 있는 새로운 백신에 대해 연구하던 중, 국내 유래 PCV2(Porcine circovirus type 2)의 ORF2 (Open Reading Frame 2) 유전자의 특정 부위에 선별된 병원성 바이러스의 에피토프를 삽입해도 3차 구조의 형태가 변형되지 않으며, 바이러스 유사 입자 (virus like particle, VLP)가 효과적으로 생산됨을 확인하고 본 발명을 완성하였다. While researching a new vaccine that can effectively prevent PRRSV, PCV2, and FMDV, the present inventors found an epitope of a pathogenic virus selected at a specific region of the ORF2 (Open Reading Frame 2) gene of domestically derived PCV2 (Porcine circovirus type 2) The present invention was completed by confirming that the shape of the tertiary structure was not modified even when inserted, and virus like particle (VLP) was effectively produced.

따라서 본 발명의 목적은 PCV2의 ORF2 부위에 PRRSV 또는 FMDV 의 병원성 에피토프가 삽입된 재조합 PCV2의 ORF2 및 이를 이용한 복합 백신 조성물을 제공하는 것이다. Accordingly, an object of the present invention is to provide ORF2 of recombinant PCV2 in which a pathogenic epitope of PRRSV or FMDV is inserted into the ORF2 region of PCV2 and a combination vaccine composition using the same.

상기 목적을 제공하기 위하여, 본 발명은 PRRSV (Porcine reproductive and respiratory syndrome virus) 또는 구제역 바이러스 (Foot-and-mouth disease virus) 의 병원성 에피토프가 삽입된 재조합 PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2)를 제공한다. In order to provide the above object, the present invention is a recombinant PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame) into which a pathogenic epitope of PRRSV (Porcine reproductive and respiratory syndrome virus) or foot-and-mouth disease virus 2) is provided.

또한 본 발명은 상기 재조합 PCV2 ORF2 가 도입된 세포 또는 대장균을 제공한다. In addition, the present invention provides a cell or E. coli into which the recombinant PCV2 ORF2 is introduced.

또한 본 발명은 상기 세포 또는 대장균에서 생산된 바이러스 유사 입자를 제공한다. In addition, the present invention provides virus-like particles produced from the cells or Escherichia coli.

또한 본 발명은 바이러스 유사 입자를 포함하는 복합 백신 조성물을 제공한다. The present invention also provides a combination vaccine composition comprising virus-like particles.

본 발명의 재조합 PCV2 ORF2 를 이용하는 경우, 곤충 또는 대장균 세포에서 바이러스 유사 입자를 효과적으로 생산할 수 있고, 이는 2종 이상의 돼지 바이러스에 대하여 효과적으로 면역 효과를 유도할 수 있으므로, PCV2 기반의 돼지 복합 백신으로 유용하게 사용될 수 있다. When using the recombinant PCV2 ORF2 of the present invention, virus-like particles can be effectively produced in insect or Escherichia coli cells, which can effectively induce an immune effect against two or more porcine viruses, making it useful as a PCV2-based swine combination vaccine. can be used

도 1은 PCV2 VLP 기반의 복합백신 생산 결과를 나타낸 도이다.
도 2는 코돈 최적화된 PCV2의 ORF2 유전자의 3차 구조를 분석해서 외부 에피토프의 삽입 가능 부위를 예측한 도이다.
도 3은 코돈 최적화된 PCV2의 ORF2 유전자 기반의 복합백신 발현용 재조합 발현벡터를 구축하는 과정의 모식도이다.
도 4는 곤충세포가 재조합 베큘로바이러스에 감염되었는지 여부를 확인하고 웨스턴 블랏을 통해 삽입 에피토프의 발현을 확인한 도이다.
도 5는 대장균에서 IPTG 유도에 따른 재조합 단백질의 발현을 정제하여 전기영동을 통해 확인한 결과를 나타낸 도이다.
도 6은 대장균에서 생산 정제한 바이러스 유사입자를 전자현미경(TEM)으로 확인한 도이다.
도 7은 재조합 바이러스 곤충세포 감염 뒤 에피토프 유전자의 발현을 확인한 결과를 나타낸 도이다.
도 8은 제작한 재조합바이러스의 곤충세포 감염뒤 에피토프 유전자의 발현 확인 (IFA) 결과를 나타낸 도이다. 1 is a diagram showing the production results of a PCV2 VLP-based combination vaccine.
FIG. 2 is a diagram illustrating a prediction of a site capable of inserting an external epitope by analyzing the tertiary structure of the ORF2 gene of codon-optimized PCV2.
3 is a schematic diagram of a process of constructing a recombinant expression vector for expressing a combination vaccine based on the ORF2 gene of codon-optimized PCV2.
Figure 4 is a diagram confirming whether insect cells are infected with recombinant baculovirus and confirming the expression of the inserted epitope through Western blotting.
5 is a diagram showing the result confirmed through electrophoresis by purifying the expression of a recombinant protein according to IPTG induction in E. coli.
6 is a diagram confirming virus-like particles produced and purified from Escherichia coli by electron microscopy (TEM).
7 is a diagram showing the results of confirming the expression of epitope genes after infection with recombinant virus insect cells.
8 is a diagram showing the result of confirming the expression of epitope genes (IFA) after infection of insect cells with the prepared recombinant virus.

본 발명은 PRRSV (Porcine reproductive and respiratory syndrome virus) 또는 구제역바이러스 (Foot-and-mouth disease virus) 의 병원성 에피토프가 삽입된 재조합 PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2) 를 제공한다. The present invention provides a recombinant Porcine circovirus type 2 (PCV2) Open Reading Frame 2 (ORF2) into which a pathogenic epitope of Porcine reproductive and respiratory syndrome virus (PRRSV) or Foot-and-mouth disease virus is inserted.

본 발명은 PCV2 ORF2의 3차 구조 예측에 의해 바이러스 유사입자 생성 시 구조에 영향을 주지 않는 부위를 선정하고 선정 부위에 PRRSV와 FMDV의 주요항원부위라고 알려진 에피토프를 선발하여 PCV2 바이러스 유사입자 표면에 표출될 수 있도록 융합 발현한 것을 특징으로 할 수 있다. 상기 재조합 PCV2의 ORF2 는 PCV2 (Porcine circovirus type 2) ORF2 아미노산 서열의 61 내지 62 번 아미노산 사이, 85 내지 86 번 아미노산 사이, 130 내지 131 번 아미노산 사이, 190 내지 191 번 아미노산 사이로 이루어진 군에서 선택된 1종 부위에 삽입되는 것일 수 있으며, 상기 위치는 PCV2의 ORF2 (NCBI 등록번호 FJ755686) 를 기준으로 설명될 수 있다. The present invention selects a site that does not affect the structure when generating virus-like particles by predicting the tertiary structure of PCV2 ORF2, selects an epitope known as the main antigenic region of PRRSV and FMDV at the selected site, and expresses it on the surface of PCV2 virus-like particles It can be characterized by fusion expression so that it can be. ORF2 of the recombinant PCV2 is one selected from the group consisting of between amino acids 61 and 62, between amino acids 85 and 86, between amino acids 130 and 131, and between amino acids 190 and 191 of the PCV2 (Porcine circovirus type 2) ORF2 amino acid sequence It may be inserted into the site, and the site can be described based on ORF2 of PCV2 (NCBI registration number FJ755686).

본 발명의 재조합 PCV2 (Porcine circovirus type 2) 의 ORF2 는 서열번호 18 내지 서열번호 30으로 이루어진 군에서 선택된 1종의 아미노산 서열인, 재조합 PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2) 일 수 있으며, 상기 아미노산 서열은 각각 서열번호 5 내지 서열번호 17 로 이루어진 군에서 선택된 염기서열에 의해 코딩되는 것일 수 있다. ORF2 of the recombinant PCV2 (Porcine circovirus type 2) of the present invention can be an amino acid sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 30, ORF2 (Open Reading Frame 2) of recombinant PCV2 (Porcine circovirus type 2) And, the amino acid sequence may be encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 17, respectively.

본 발명의 PCV2 의 ORF2 기반의 재조합 PCV2 ORF2 유전자는 국내 분리주의 ORF2 서열과 항원인식부위를 곤충 세포 및 대장균 시스템에 맞도록 코돈 최적화시킨 것이다. The ORF2-based recombinant PCV2 ORF2 gene of PCV2 of the present invention is a codon optimization of the ORF2 sequence and antigen recognition site of a domestic isolate to suit insect cells and E. coli systems.

본 발명에서 "코돈 최적화(codon optimization)"란 특정 유전자를 다른 개체에서 발현시키는 경우, 유전자가 동일한 아미노산 서열을 갖는 단백질을 발현할 수 있도록 유전자의 염기서열을 변환시키는 것을 말한다. In the present invention, "codon optimization" refers to converting the nucleotide sequence of a gene so that a protein having the same amino acid sequence can be expressed when a specific gene is expressed in another individual.

본 발명에서 용어 "벡터"는 세포 내로 전달하는 DNA 단편(들), 핵산 분자를 지칭할 때 사용된다. 벡터는 DNA를 복제시키고, 숙주세포에서 독립적으로 재생산될 수 있다. 벡터는 목적한 코딩 서열과 특정 숙주 생물에서 작동 가능하게 연결된 코딩 서열을 발현하는 데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. In the present invention, the term "vector" is used to refer to DNA fragment(s) and nucleic acid molecules that are delivered into cells. Vectors replicate DNA and can reproduce independently in host cells. A vector refers to a recombinant DNA molecule containing a coding sequence of interest and appropriate nucleic acid sequences necessary for the expression of an operably linked coding sequence in a particular host organism.

또한 본 발명은 재조합 PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2) 가 도입된 세포를 제공한다. In addition, the present invention provides a cell into which recombinant Porcine circovirus type 2 (PCV2) ORF2 (Open Reading Frame 2) is introduced.

본 발명에서 "세포"는 Sf9, Sf21, High five 세포 또는 Tn5 등의 일반적인 곤충 세포를 포함하며, 바람직하게는 Sf9 또는 High five 세포이나, 이에 제한되지 않는다. In the present invention, "cell" includes general insect cells such as Sf9, Sf21, High five cells or Tn5, preferably Sf9 or High five cells, but is not limited thereto.

또한 본 발명은 재조합 PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2) 가 도입된 대장균을 제공한다. In addition, the present invention provides E. coli into which recombinant Porcine circovirus type 2 (PCV2) ORF2 (Open Reading Frame 2) is introduced.

또한 본 발명은 상기 세포 또는 대장균에서 생산된 바이러스 유사 입자를 제공한다. In addition, the present invention provides virus-like particles produced from the cells or Escherichia coli.

본 발명에서 "바이러스 유사 입자"는 바이러스 외피를 구성하도록 조립된 단백질 코트 또는 캡시드를 포함하는 것으로, 상기 바이러스 유사 입자는 바이러스의 외피 (capsid) 형태를 유지하여 살아있는 바이러스와 유사한 효과를 가진다. 또한 PCV2의 ORF2 단백질 단독 형태에 비해 면역원성이 증가된다.In the present invention, the "virus-like particle" includes a protein coat or capsid assembled to form a virus envelope, and the virus-like particle maintains the shape of a virus capsid and has an effect similar to that of a living virus. In addition, immunogenicity is increased compared to the ORF2 protein alone form of PCV2.

또한 본 발명은 상기 바이러스 유사 입자를 포함하는 복합 백신 조성물을 제공한다. In addition, the present invention provides a combination vaccine composition comprising the virus-like particles.

본 발명의 실시예에서는 코돈 최적화된 PCV2 ORF2 기반의 재조합 PCV2 ORF2 를 이용하여 제조된 바이러스 유사 입자를 이용하여 복합 백신 백신 조성물을 제조하였으며, 상기 복합 백신 조성물은 항체 생성능 및 바이러스 방어능이 우수한 바, PCV2와 PRRSV 또는 구제역 감염 질환의 예방에 유용하게 사용될 수 있다. In an embodiment of the present invention, a combination vaccine vaccine composition was prepared using virus-like particles prepared using recombinant PCV2 ORF2 based on codon-optimized PCV2 ORF2. and PRRSV or foot-and-mouth disease.

본 발명에서 "백신"은 항원 물질을 포함하는 수의학용 백신으로, PCV2와 PRRSV 또는 구제역에 대하여 특이적이고 능동 또는 수동의 면역성을 유도하기 위한 목적으로 투여된다. In the present invention, "vaccine" is a veterinary vaccine containing an antigenic material, which is specific for PCV2 and PRRSV or foot-and-mouth disease, and is administered for the purpose of inducing active or passive immunity.

본 발명의 "대상 가축 감염 질환"에는 이유후 전신성 소모성 증후군 (PMWS; Post-weaning Multisystemic Wasting Syndrome), 가성 광견병 (pseudorabies), 돼지 생식기 및 호흡기 증후군 (PRRS: Porcine Reproductive and Respiratory Syndrome), 글래서병 (Glasser's disease), 연쇄구균성 수막염 (streptococcal meningitis), 살모넬라증 (salmonellosis), 이유후 대장균증 (postweaning colibacillosis), 식이성 간증 (dietetic hepatosis) 및 화농성 기관지폐렴 (suppurative bronchopneumonia) 등이 포함될 수 있으나, 이에 제한되는 것은 아니다.The "target livestock infectious disease" of the present invention includes Post-weaning Multisystemic Wasting Syndrome (PMWS), pseudorabies, Porcine Reproductive and Respiratory Syndrome (PRRS), Glasser's disease ( Glasser's disease), streptococcal meningitis, salmonellosis, postweaning colibacillosis, dietetic hepatosis, and suppurative bronchopneumonia. It is not.

본 발명의 백신 조성물은 유효 성분인 바이러스 유사 입자 이외에도 백신 조성물을 구성하는 데 적절한 하나 이상의 면역 증강제 또는 부형제 또는 담체를 포함할 수 있다.The vaccine composition of the present invention may include one or more immune enhancers or excipients or carriers suitable for constituting the vaccine composition in addition to the active ingredient, virus-like particles.

본 발명에의 조성물에 포함될 수 있는 면역증강제는 주사한 동물의 면역 반응을 증대시키는 물질을 의미하는 것으로, 다수의 상이한 면역증강제가 기술 분야의 당업자에게 공지되어 있다. 상기 면역증강제는 프로인트 완전 및 불완전 면역 증강제, 비타민 E, 비이온성 차단 폴리머, 뮤라밀디펩티드, Quil A, 광유 및 무광물유 및 카보폴(Carbopol), 유중수형 유제 면역증강제등을 포함하며, 이에 제한되는 것은 아니다.An adjuvant that may be included in the composition of the present invention refers to a substance that enhances the immune response of an injected animal, and many different adjuvants are known to those skilled in the art. The immune enhancers include Freund's complete and incomplete immune enhancers, vitamin E, nonionic blocking polymers, muramyl dipeptide, Quil A, mineral oil and non-mineral oil and Carbopol, water-in-oil emulsion immune enhancers, etc., but are limited thereto it is not going to be

본 발명의 조성물에 포함될 수 있는 담체는 기술 분야의 당업자에게 공지되어 있으며, 단백질, 설탕 등을 포함하지만, 이로 제한되는 것은 아니다. 상기의 담체는 수용액 또는 비-수용액, 현탁액, 및 에멀전 일 수 있다. 비-수용액 담체의 예는 프로필렌 글리콜, 폴리에틸렌 글리콜, 식용유 예컨대 올리브 오일, 및 주사가능한 유기 에스테르 예컨대 에틸 올리에이트를 들 수 있다. 수용액 담체는 식염수 및 완충배지를 포함하는, 물, 알콜/수용액, 에멀전 또는 현탁액을 포함한다. 비경구 담체는 염화나트륨 용액, 링거 덱스트로스, 덱스트로스 및 염화나트륨, 유산처리 링거 또는 고정 오일을 포함한다. 정맥주사용 담체는 예컨대 링거 덱스트로스를 기본으로 하는 것과 같은 전해질 보충제, 액체 및 영양 보충제 등을 포함한다.Carriers that may be included in the composition of the present invention are known to those skilled in the art, and include, but are not limited to, proteins, sugars, and the like. The above carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Carriers for intravenous injection include electrolyte replenishers, liquid and nutritional supplements, and the like, such as those based on Ringer's dextrose.

본 발명의 백신 조성물은 방부제 및 기타 첨가제 예컨대 예를 들면 항미생물제제, 항산화제, 킬레이트제, 불활성 가스 등과 같은 것을 추가로 포함할 수 있다. 상기 방부제는 포르말린, 티메로살, 네오마이신, 폴리믹신 B 및 암포테리신 B 등을 포함한다. 본 발명의 백신 조성물은 하나 이상의 적절한 유화제, 예로서 스판(Span) 또는 트윈(Tween)을 포함할 수 있다. 또한 본 발명의 백신 조성물은 보호제를 포함할 수 있으며, 당업계에 공지된 보호제를 제한 없이 사용할 수 있고, 이는 락토오스(Lactose; LPGG) 또는 트레할로오스(Trehalose; TPGG)를 포함할 수 있으나, 이에 제한되는 것은 아니다.The vaccine composition of the present invention may further contain preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like. The preservatives include formalin, thimerosal, neomycin, polymyxin B and amphotericin B, and the like. The vaccine composition of the present invention may include one or more suitable emulsifiers, such as Span or Tween. In addition, the vaccine composition of the present invention may include a protecting agent, and a protecting agent known in the art may be used without limitation, which may include lactose (LPGG) or trehalose (TPGG), It is not limited thereto.

또한, 본 발명은 상기 백신 조성물을 가축에게 투여하는 단계를 포함하는 바이러스 감염 질환의 예방 방법을 제공한다. 본 발명에서 가축에 백신 조성물을 투여하는 방법은 일반적인 백신 투여 방법에 의할 수 있으며, 이에 제한되는 것은 아니지만 장내 또는 비경구 경로, 경구, 비강 내, 정맥 내, 근육 내, 피하, 피내 또는 다른 적절한 경로를 통한 투여를 수행할 수 있고, 바람직하게는 근육 내, 피내, 피하에 투여된다.In addition, the present invention provides a method for preventing viral infectious diseases comprising administering the vaccine composition to livestock. In the present invention, the method of administering the vaccine composition to livestock may be by a general vaccine administration method, but is not limited thereto, enteric or parenteral route, oral, intranasal, intravenous, intramuscular, subcutaneous, intradermal or other appropriate route. Administration can be performed via a route, and is preferably administered intramuscularly, intradermally, or subcutaneously.

본 발명에서 백신 접종의 대상인 가축은 돼지에 제한되지 않으며, 본 발명에서 돼지에게 백신 조성물 투여는 면역학적 유효량으로 투여할 수 있다. 상기 "면역학적 유효량"이란 PCV2와 구제역 또는 PRRS 관련된 질병의 예방 효과를 나타낼 수 있을 정도의 충분한 양과 부작용이나 심각한 또는 과도한 면역반응을 일으키지 않을 정도의 양을 의미하며, 정확한 투여 농도는 투여될 특정 면역원에 따라 달라지며, 예방 접종 대상의 연령, 체중, 건강, 성별, 개체의 약물에 대한 민감도, 투여 경로, 투여 방법 등 의학 분야에 잘 알려진 요소에 따라 당업자에 의해 용이하게 결정될 수 있으며, 1회 내지 수회 투여가능 하다.Livestock subject to vaccination in the present invention is not limited to pigs, and the vaccine composition may be administered to pigs in an immunologically effective amount in the present invention. The "immunologically effective amount" means an amount sufficient to exhibit a preventive effect of PCV2 and foot-and-mouth disease or PRRS-related diseases and an amount sufficient to not cause side effects or severe or excessive immune reactions, and the exact dosage concentration is the specific immunogen to be administered. Depending on the vaccination target, it can be easily determined by a person skilled in the art according to factors well-known in the medical field, such as age, weight, health, sex, sensitivity to drugs of the individual, administration route, and administration method. Multiple doses are possible.

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the content of the present invention is not limited by the examples.

실시예Example 1 - 코돈 최적화된 1 - codon optimized PCV2의PCV2's ORF2기반의ORF2-based 복합백신 유전자 제조 Complex vaccine gene manufacturing

곤충 세포에서 발현 효율을 높이기 위해 국내 분리주로부터 얻은 PCV2의 ORF2 유전자(NCBI 등록번호 FJ755686)의 염기서열의 3차 구조 분석을 수행하였으며 PCV2의 ORF2 유전자 부위에서 총 4군데의 에피토프 치환부위를 선정하였다. 에피토프 선정을 위한 3차원 구조 분석 및 선정된 부위를 도 2에 나타내었다. To increase expression efficiency in insect cells, tertiary structure analysis was performed on the nucleotide sequence of the ORF2 gene (NCBI registration number FJ755686) of PCV2 obtained from a domestic isolate, and a total of four epitope substitution sites were selected from the ORF2 gene region of PCV2. 3D structural analysis and selected sites for epitope selection are shown in FIG. 2 .

도 2에 나타낸 바와 같이, PCV2 ORF2의 아미노산 서열 번호 61~62, 85~86, 130~131, 190~191 부위를 에피토프 치환 부위로 선정하였다. 상기 부위는 PCV2 ORF2 구조단백질의 3차 구조 예측과 이전에 보고된 문헌을 참고하여 선정된 것으로, PCV2 바이러스 유사입자 형성에 영향이 없으며, PRRSV와 FMDV의 에피토프가 표면으로 돌출될 수 있는 부위를 선발하였다. 선발된 부위에 FMDV와 PRRSV의 에피토프 아미노산을 서열을 부가하여 PCV2와 이종의 바이러스의 에피토프가 융합발현 될 수 있는 유전자 서열을 제작하였다. 도입에 사용한 FMDV와 PRRSV의 주요 에피토프 대상 아미노산 서열을 하기 표 1에 나타내었다. As shown in FIG. 2, amino acid sequence numbers 61 to 62, 85 to 86, 130 to 131, and 190 to 191 of PCV2 ORF2 were selected as epitope substitution sites. The site was selected by referring to the prediction of the tertiary structure of the PCV2 ORF2 structural protein and previously reported literature, and it does not affect the formation of PCV2 virus-like particles, and selects a site where PRRSV and FMDV epitopes can protrude to the surface did By adding the amino acid sequences of the epitopes of FMDV and PRRSV to the selected regions, gene sequences capable of fusion expression of epitopes of PCV2 and heterogeneous viruses were prepared. Table 1 shows the amino acid sequences of major epitopes of FMDV and PRRSV used for introduction.

아미노산 서열amino acid sequence 서열번호sequence number FMDV epitope -1FMDV epitope -1 LPNVRGDLQVLAQKAARPLPLPNVRGDLQVLAQKAARPLP 서열번호 1SEQ ID NO: 1 FMDV epitope -2FMDV epitope -2 LPNVRGDLQVLAQKAARPLPRHKQKIVAPVKQSLLPNVRGDLQVLAQKAARPLPRHKQKIVAPVKQSL 서열번호 2SEQ ID NO: 2 PRRSV epitope -1PRRSV epitope -1 SHLQLIYNLSHLQLIYNL 서열번호 3SEQ ID NO: 3 PRRSV epitope -2PRRSV epitope-2 SNDSSSHLQLIYNLTLCEGSGSSLDDFCHGSSNDSSSHLQLIYNLTLCEGSGSSLDDFCHGS 서열번호 4SEQ ID NO: 4

상기 아미노산을 기반으로 발현에 필요한 유전자를 곤충세포와 대장균에 맞도록 코돈 최적화했다. 코돈 최적화는 원래의 염기서열을 곤충세포과 대장균에서 주로 사용되는 염기서열로 변경하는 방법으로 수행하였다. 본 발명의 코돈 최적화된 PCV2 ORF2기반의 복합백신 유전자가 곤충세포와 대장균에서 발현되는 경우 원래 단백질과 동일한 아미노산 서열을 갖는 단백질을 생산할 수 있다. Based on the above amino acids, genes required for expression were codon-optimized to suit insect cells and Escherichia coli. Codon optimization was performed by changing the original nucleotide sequence to a nucleotide sequence mainly used in insect cells and Escherichia coli. When the codon-optimized PCV2 ORF2-based combination vaccine gene of the present invention is expressed in insect cells and Escherichia coli, a protein having the same amino acid sequence as the original protein can be produced.

본 발명에서 재조합된 서열정보는 다음 표 2와 같이 표시하였다. Sequence information recombined in the present invention is shown in Table 2 below.

염기서열base sequence 아미노산 서열amino acid sequence 61~ 62 위치에 삽입 - 56FMB (FMDV) Insert at positions 61-62 - 56FMB (FMDV) 서열번호 5 SEQ ID NO: 5 서열번호 18SEQ ID NO: 18 61~62 위치에 삽입 - 56FMBT(FMDV)Insert at positions 61-62 - 56FMBT (FMDV) 서열번호 6SEQ ID NO: 6 서열번호 19SEQ ID NO: 19 85~86 위치에 삽입 - 80FMB (FMDV)Insert at positions 85-86 - 80FMB (FMDV) 서열번호 7 SEQ ID NO: 7 서열번호 20SEQ ID NO: 20 85~86 위치에 삽입 - 80FMBT (FMDV)Insert at positions 85-86 - 80FMBT (FMDV) 서열번호 8 SEQ ID NO: 8 서열번호 21 SEQ ID NO: 21 130~131 위치에 삽입 - 125FMB (FMDV)Insert at positions 130-131 - 125FMB (FMDV) 서열번호 9 SEQ ID NO: 9 서열번호 22SEQ ID NO: 22 130~131 위치에 삽입 - 125FMBT (FMDV)Insert at position 130~131 - 125FMBT (FMDV) 서열번호 10 SEQ ID NO: 10 서열번호 23 SEQ ID NO: 23 190~191 위치에 삽입 - 185FMB (FMDV)Insert at positions 190-191 - 185FMB (FMDV) 서열번호 11SEQ ID NO: 11 서열번호 24 SEQ ID NO: 24 61~62 위치에 삽입 - 56PRGP5 (PRRSV)Insert at positions 61-62 - 56PRGP5 (PRRSV) 서열번호 12SEQ ID NO: 12 서열번호 25 SEQ ID NO: 25 61~62 위치에 삽입 - 56PRGP5M (PRRSV)Insert at positions 61-62 - 56PRGP5M (PRRSV) 서열번호 13SEQ ID NO: 13 서열번호 26 SEQ ID NO: 26 85~86 위치에 삽입 - 80PRGP5 (PRRSV)Insert at positions 85-86 - 80PRGP5 (PRRSV) 서열번호 14SEQ ID NO: 14 서열번호 27 SEQ ID NO: 27 85~86 위치에 삽입 - 80PRGP5M (PRRSV)Insert at positions 85-86 - 80PRGP5M (PRRSV) 서열번호 15SEQ ID NO: 15 서열번호 28 SEQ ID NO: 28 130~131 위치에 삽입 - 125PRGP5 (PRRSV)Insert at positions 130-131 - 125PRGP5 (PRRSV) 서열번호 16SEQ ID NO: 16 서열번호 29 SEQ ID NO: 29 130~131 위치에 삽입 - 125PRGP5 (PRRSV)Insert at positions 130-131 - 125PRGP5 (PRRSV) 서열번호 17SEQ ID NO: 17 서열번호 30 SEQ ID NO: 30

실시예Example 2 - 코돈 최적화된 2 - codon optimized PCV2의PCV2's ORF2기반의ORF2-based 복합백신 항원의 곤충세포 발현을 위한 재조합 벡터의 구축 Construction of recombinant vectors for expression of complex vaccine antigens in insect cells

상기 실시예 1에서 얻은 코돈 최적화된 PCV2 ORF2기반의 복합 백신 서열을 포함하는 재조합 벡터를 제조하였으며 이의 제작 과정을 도 3에 간략하게 나타내었다. 발현 벡터의 백본으로 pFastBacTMDual과 pGEX4T1벡터를 이용하여 코돈 최적화된 서열에 도입하여 클로닝하였다. 상기와 같은 방법으로 제조한 재조합 발현벡터의 발현을 확인하기 위해서, 정방향 프라이머 (GCGGCCGCATGACGTATCCAAGGAGGCAT) 및 역방향 프라이머 (GGATCCTCAGGGTTTAAGTGGGGGGTC)를 사용하여, PCR을 수행한 후, 전기영동을 통해 확인하였다. 구체적으로 상기 실시예 1에서 얻은 코돈 최적화된 FMDV-epitope-PCV2-ORF2의 복합백신 서열을 포함하는 DNA 단편을 pFastBac™Dual (ThermoFisher SCIENTIFIC Cat. No. 10359-016) 벡터에 삽입하여 전이벡터 pFastBac™Dual-56FMBT를 제작하였다. 이 벡터에서 FMDV-epitope-PCV2-ORF2 단백질 코딩 DNA 단편은 폴리헤드린 프로모터의 다운스트림에 위치한다. 제작한 전이벡터의 DNA를 MAX Efficiency®DH10Bac™(ThermoFisher SCIENTIFIC Cat. No. 10361012) 대장균에 제조사의 제공 방법에 따라 형질전환하였으며, 이를 통해 FMDV-epitope-PCV2-ORF2 유전자가 삽입된 Bacmid를 제작하였다. 상기 방법으로 제작한 Bacmid를 100㎕의 Sf-900™III SFM (ThermoFisher SCIENTIFIC Cat. No. 12658019) 배지에 혼합하고, 셀펙틴 II (Cellfectin II, Invitrogen) 5㎕를 100㎕의 Sf-900™III SFM배지에 혼합한 후 각각을 15분 동안 27 ℃에서 방치하였다. 그 후 두 혼합액을 섞어주고 다시 15분 동안 27 ℃에서 방치하였다. 밤나방과 세포주, Sf9 (Spodoptera frugiperda 9) (ThermoFisher SCIENTIFIC Cat. No. B82501)를 25 ㎟ 배양 플라스크당 1.0ⅹ 106개의 세포로 분주하여, 위 혼합액을 배양액에 접종하고, 더 배양하여 세포변성을 확인 후 재조합바이러스 rAc-PCV2-56FMBT를 제작하였다.A recombinant vector containing the codon-optimized PCV2 ORF2-based composite vaccine sequence obtained in Example 1 was prepared, and the construction process is briefly shown in FIG. 3 . As the backbone of the expression vector, pFastBac TM Dual and pGEX4T1 vectors were introduced into codon-optimized sequences and cloned. In order to confirm the expression of the recombinant expression vector prepared as described above, PCR was performed using a forward primer (GCGGCCGCATGACGTATCCAAGGAGGCAT) and a reverse primer (GGATCCTCAGGGTTTAAGTGGGGGGTC), followed by electrophoresis. Specifically, the DNA fragment containing the codon-optimized FMDV-epitope-PCV2-ORF2 composite vaccine sequence obtained in Example 1 was inserted into the pFastBac™ Dual (ThermoFisher SCIENTIFIC Cat. No. 10359-016) vector to create the transfer vector pFastBac™ A Dual-56FMBT was manufactured. In this vector, a DNA fragment encoding the FMDV-epitope-PCV2-ORF2 protein is located downstream of the polyhedrin promoter. The DNA of the prepared transfer vector was transformed into MAX Efficiency®DH10Bac™ (ThermoFisher SCIENTIFIC Cat. No. 10361012) E. coli according to the method provided by the manufacturer, and through this, a Bacmid into which the FMDV-epitope-PCV2-ORF2 gene was inserted was prepared. . Bacmid produced by the above method was mixed with 100 μl of Sf-900™III SFM (ThermoFisher SCIENTIFIC Cat. No. 12658019) medium, and 5 μl of Cellfectin II (Invitrogen) was added to 100 μl of Sf-900™III. After mixing in SFM medium, each was left at 27 °C for 15 minutes. After that, the two mixed solutions were mixed and allowed to stand at 27 ° C. for another 15 minutes. Night moth and cell line, Sf9 ( Spodoptera frugiperda 9) (ThermoFisher SCIENTIFIC Cat. No. B82501) was divided into 1.0ⅹ 10 6 cells per 25 ㎟ culture flask, the above mixture was inoculated into the culture medium, further cultured to confirm cell denaturation, and then the recombinant virus rAc-PCV2- 56FMBT was fabricated.

이와 같은 방법으로 제작된 재조합바이러스는 용균반점법 (Plaque assay)으로 순수하게 분리하였다. 순수 분리된 바이러스는 T-75 플라스크(Corning Inc.)에 재접종하여 증식시킴으로써 대량으로 바이러스를 수득할 수 있었다. 수득한 바이러스들은 BacPAK™ qPCR Titration Kit (Clontech)를 이용하여 정량 후 사용하였다. 재조합 벡터의 정상적인 발현을 SDS-PAGE 및 웨스턴 블랏으로 확인하였으며, 감염된 세포의 형태를 비감염된 대조군 Sf9 세포와 비교하고 그 결과를 도 4에 나타내었다. The recombinant virus prepared in this way was purely isolated by a plaque assay. Purely isolated viruses were re-inoculated into T-75 flasks (Corning Inc.) and propagated to obtain viruses in large quantities. The obtained viruses were quantified using the BacPAK™ qPCR Titration Kit (Clontech) and then used. Normal expression of the recombinant vector was confirmed by SDS-PAGE and Western blotting, and the morphology of infected cells was compared with that of uninfected control Sf9 cells, and the results are shown in FIG. 4 .

도 4에 나타낸 바와 같이, 재조합 벡터로 형질전환된 Sf9 세포가 수득됨을 확인하였으며, 재조합 벡터가 모두 정상적으로 발현됨을 확인하여, 이후 실시예에 사용하였다. As shown in Figure 4, it was confirmed that Sf9 cells transformed with the recombinant vector were obtained, and it was confirmed that all of the recombinant vectors were normally expressed, and used in the following examples.

실시예Example 3 - 코돈 최적화된 3 - codon optimized PCV2의PCV2's ORF2기반의ORF2-based 복합백신 항원의 대장균 발현을 위한 발현 벡터의 구축 Construction of expression vectors for E. coli expression of complex vaccine antigens

상기 실시예1에서 얻은 코돈 최적화된 PRRSV-epitope-PCV2-ORF2의 복합백신 서열을 포함하는 DNA 단편을 pGEX4T1 벡터에 삽입하여 전이벡터 pGEX4T1-PRGP5를 제작하였다. 구체적으로 PCV2에 PRRS 에피토프가 포함된 유전자를 정방향 프라이머 5'CCGGATCCATGACGTATCCAAGGCGCCGTTAC3' 와 역방향 프라이머 5' GGCTCGAGTTACGGCTTAAG CGGCGGATCTTT3' 을 이용하여 PCR로 증폭시킨 후, pGEX4T1 벡터의 BamH I/Xho I 위치에 삽입시켰다. 이 벡터에서 PRRSV-epitope-PCV2-ORF2 단백질 코딩 DNA 단편은 tac 프로모터의 다운스트림에 위치한다. The transfer vector pGEX4T1-PRGP5 was constructed by inserting the DNA fragment containing the codon-optimized PRRSV-epitope-PCV2-ORF2 composite vaccine sequence obtained in Example 1 into the pGEX4T1 vector. Specifically, the gene containing the PRRS epitope in PCV2 was amplified by PCR using a forward primer 5'CCGGATCCATGACGTATCCAAGGCGCCGTTAC3' and a reverse primer 5'GGCTCGAGTTACGGCTTAAG CGGCGGATCTTT3', and then inserted into the BamH I/Xho I position of the pGEX4T1 vector. In this vector, a DNA fragment encoding the PRRSV-epitope-PCV2-ORF2 protein is located downstream of the tac promoter.

실시예Example 4 - 4 - PCV2의PCV2's ORF2기반의ORF2-based 복합백신 항원의 곤충세포 발현 Insect cell expression of complex vaccine antigens

상기 실시예 2에서 제조한 재조합 발현벡터를 이용하여 코돈 최적화 된 PCV2 ORF2기반의 복합백신 유전자를 발현하는 재조합 베큘로바이러스에 감염된 곤충세포를 제조하였다. 구체적으로, Sf9 세포주를 60mm 디쉬에 1x106 세포로 넣어주고, Sf-900III SFM (Gibco-BRL, USA) 3ml을 첨가하여 27℃에서 최소 1시간 이상 배양함으로써 세포를 디쉬에 부착시켰다. 상기 실시예 2에서 제조한 재조합 벡터와 BD BaculoGold DNA를 Sf-900III SFM 100μl 에 혼합한 용액 A, 및 CELLFECTIN Reagent(Gibco-BRL, USA) 와 Sf-900III SFM 100μl를 혼합한 용액 B를 27℃에서 각 각 15-30분간 반응시켰다. 반응 후, 용액 A, B를 혼합한 후, 27℃에서 15-30분간 반응시켰으며, 1x106 세포로 분주된 Sf9 세포주에 접종하여 재조합 베큘로바이러스를 곤충세포에 감염시켰다.Using the recombinant expression vector prepared in Example 2, insect cells infected with recombinant baculovirus expressing a codon-optimized PCV2 ORF2-based composite vaccine gene were prepared. Specifically, the Sf9 cell line was put into 1x10 6 cells in a 60mm dish, and 3ml of Sf-900III SFM (Gibco-BRL, USA) was added and cultured at 27° C. for at least 1 hour to attach the cells to the dish. Solution A in which the recombinant vector prepared in Example 2 and BD BaculoGold DNA were mixed in 100 μl of Sf-900III SFM and solution B in which CELLFECTIN Reagent (Gibco-BRL, USA) and Sf-900III SFM were mixed in 100 μl were prepared at 27 ° C. Reacted for 15-30 minutes each. After the reaction, solutions A and B were mixed, reacted at 27° C. for 15-30 minutes, and inoculated into Sf9 cell line divided into 1x10 6 cells to infect insect cells with recombinant baculovirus.

상기 방법으로 제조한 재조합 베큘로바이러스를 접종하여 감염된 곤충세포 및 배양액으로부터 DNA를 추출하여 전기 영동으로 형질감염 여부를 확인하였다. 또한 세포 형태, 세포 크기 등의 변화를 통하여 재조합 베큘로바이러스에 감염된 세포와 그렇지 않은 세포를 비교하였다. 그 결과 곤충세포가 재조합 베큘로바이러스에 감염되었음을 확인하였다.The recombinant baculovirus prepared in the above manner was inoculated and DNA was extracted from infected insect cells and the culture medium, and transfection was confirmed by electrophoresis. In addition, cells infected with recombinant baculovirus were compared with cells that were not through changes in cell shape, cell size, etc. As a result, it was confirmed that the insect cells were infected with the recombinant baculovirus.

실시예Example 5 - 코돈 최적화된 5 - codon optimized PCV2의PCV2's ORF2기반의ORF2-based 복합백신 유전자의 대장균 발현 E. coli expression of complex vaccine genes

상기 실시예 3에서 제작한 전이벡터의DNA는 Rosetta™2(DE3) (Novagen Cat. No.71397-4) 대장균에 제조사의 제공 방법에 따라 형질전환을 수행하였다. 얻어진 콜로니는 순수배양을 할 수 있도록 적절한 항생제가 함유된 LB배지를 이용하여 37℃에서 전 배양을 진행하였고, 본 배양은 OD 600nm 값이 1.0될 때까지 배양한 후 단백질 발현을 유도하기 위하여 0.3 mM IPTG(Isopropyl-β-D-thio-galactoside)를 첨가하여 18 ℃에서overnight 배양하였다. 배양된 균을 침전시키기 위하여 저속으로 원심분리를 수행하였고, 침전된 균은 PBS(phosphate-buffered saline, pH7.4) 를 이용하여 풀어준 후 초음파분쇄를 실시하였다. 분해된 균은 원심분리를 통하여 상층액과 침전물을 분리한 후, SDS-PAGE 및 Western blotting을 통하여 단백질 발현을 확인하였다.The DNA of the transfer vector prepared in Example 3 was transformed into Rosetta™2(DE3) (Novagen Cat. No.71397-4) E. coli according to the method provided by the manufacturer. The obtained colony was pre-cultured at 37 ° C using LB medium containing appropriate antibiotics for pure culture, and this culture was cultured until the OD 600 nm value was 1.0, and then 0.3 mM to induce protein expression. IPTG (Isopropyl-β-D-thio-galactoside) was added and incubated overnight at 18 °C. Centrifugation was performed at low speed to precipitate the cultured bacteria, and the precipitated bacteria were released using PBS (phosphate-buffered saline, pH 7.4) and then ultrasonicated. After separating the supernatant and the precipitate through centrifugation, protein expression was confirmed through SDS-PAGE and Western blotting.

단백질 발현이 확인된 상층액은 GSTrap™ HP, 5 x 1 ml Columns (GE Healthcare, Cat. No.17-5281-01) 을 통과시킨 후 10mM Glutathione (reduced) 가 포함된 완충액을 이용하여 정제를 진행하였다. 다음으로 18℃에서 TEV protease를 처리하여 표지단백질인 GST를 제거하였다. GST제거와 동시에 단백질 침전이 발생하였고, 침전물은 원심분리를 통하여 상층액과 분리하였다. 이 침전을 20mM CAPS, 150mM NaCl, 4mM 2-mercaptoethanol, pH 11 buffer로 해리시켰으며, 이를 통해 최종적으로 순수한 목적단백질을 수득하였다. The supernatant for which protein expression was confirmed was passed through GSTrap™ HP, 5 x 1 ml Columns (GE Healthcare, Cat. No.17-5281-01), and then purified using a buffer containing 10 mM Glutathione (reduced) did Next, the TEV protease was treated at 18°C to remove the labeled protein, GST. Protein precipitation occurred simultaneously with GST removal, and the precipitate was separated from the supernatant through centrifugation. This precipitate was dissociated with 20mM CAPS, 150mM NaCl, 4mM 2-mercaptoethanol, pH 11 buffer, and through this, a pure target protein was finally obtained.

실시예Example 6 - 재조합 바이러스 유사 입자의 제조 6 - Preparation of recombinant virus-like particles

상기 실시예 4에서 제조한 재조합 베큘로바이러스를 곤충세포에 접종하여 이로부터 바이러스 유사 입자(VLP)를 얻었다. 보다 구체적으로, Sf9 세포주를 T-75 플라스크에 5x106 개의 세포주로 넣어주고, Sf-900III SFM (Gibco-BRL, USA) 10ml을 첨가하여 27℃에서 최소 1시간 이상 배양함으로써 세포를 디쉬에 부착시켰다. 그 후 세포에 재조합 베큘로바이러스를 접종하였고, 세포 및 배양액을 수거하여 이로부터 바이러스 유사 입자를 얻었다. 상기 방법으로 얻어진 바이러스 유사 입자를 전자현미경(TEM)으로 확인하였다. 그 결과 700nm 크기의 PCV2 바이러스 유사 입자 클러스터는 이질염색질(heterochromatin)과 구별되도록 생성되었음을 확인하였다.The recombinant baculovirus prepared in Example 4 was inoculated into insect cells to obtain virus-like particles (VLPs). More specifically, the Sf9 cell line was put into a T-75 flask as 5x10 6 cell lines, and 10 ml of Sf-900III SFM (Gibco-BRL, USA) was added and cultured at 27 ° C for at least 1 hour to attach the cells to the dish. . Thereafter, the cells were inoculated with the recombinant baculovirus, and the cells and the culture medium were harvested to obtain virus-like particles. The virus-like particles obtained by the above method were confirmed by electron microscopy (TEM). As a result, it was confirmed that PCV2 virus-like particle clusters having a size of 700 nm were generated to be distinguished from heterochromatin.

실시예Example 7 - 코돈 최적화된 7 - codon optimized PCV2PCV2 ORF2ORF2 기반의 복합백신 유전자를 이용한 곤충세포에서 In insect cells using a combination vaccine gene based on PCV2PCV2 ORF2ORF2 단백질의 of protein 발현능Expression ability 확인 Confirm

상기 실시예 1에서 제조한 PCV2의 ORF2 유전자 이용시 곤충세포에서의 ORF2 단백질 발현양을 웨스턴 블롯팅, SDS-PAGE 및 ELISA 방법으로 확인하였다. 구체적으로, SDS-PAGE를 위해 시료를 10% SDS-폴리아크릴아마이드 겔에서 80V의 전압으로 전기영동을 수행하였다. 전기영동 후, 겔은 Coomassie brilliant blue solution로 염색하고 Destaining solution으로 탈색하여 관찰하였다. 웨스턴 블롯팅은, SDS-PAGE 겔을 nitrocellulose membrane에 300mA로 2-3시간 동안 트랜스퍼하였고, 차단을 위하여 TBS-T 완충액에 녹인 5% 스킴 밀크를 1시간 처리하였다. 1차 항체로 메디안디노스틱사의 PCV2 단클론항체 (Anti-PCV2, Cat.No. 9051, 1:500)를 5% 스킴 밀크를 녹인 TBS-T 완충액에 희석하여 1시간 처리한 후, 15분씩 3번 세척하였다. 2차 항체 역시 5% 스킴 밀크를 녹인 TBS-T 완충액에 희석하여 1시간 동안 처리하였고, 15분씩 3번 세척하였다. 최종적으로 membrane에 DAB (Sigma)를 처리하여 특이 밴드를 관찰하였다. 또한, ELISA는 Synbiotics사의 Ag detection ELISA 키트를 이용하였으며, 각 시료를 PBS로 10-1~10-4의 농도가 되게 10진 희석하여 웰당 100μl씩 1시간 처리한 후, PBS-T 완충액으로 3번 세척하였다. 여기에 2차 항체를 PBS에 희석하여 1시간 동안 처리하였고, PBS-T 완충액으로 3번 세척한 후, TMB 용액으로 발색정도를 관찰하였다. When using the ORF2 gene of PCV2 prepared in Example 1, the expression level of ORF2 protein in insect cells was confirmed by Western blotting, SDS-PAGE and ELISA methods. Specifically, for SDS-PAGE, samples were subjected to electrophoresis on a 10% SDS-polyacrylamide gel at a voltage of 80V. After electrophoresis, the gel was observed by staining with Coomassie brilliant blue solution and destaining with Destaining solution. For Western blotting, the SDS-PAGE gel was transferred to a nitrocellulose membrane at 300 mA for 2-3 hours, and 5% skim milk dissolved in TBS-T buffer was treated for 1 hour for blocking. As a primary antibody, Mediandinostic's PCV2 monoclonal antibody (Anti-PCV2, Cat.No. 9051, 1:500) was diluted in TBS-T buffer in which 5% skim milk was dissolved and treated for 1 hour, followed by 3 times of 15 minutes each. Washed. The secondary antibody was also diluted in TBS-T buffer in which 5% skim milk was dissolved, treated for 1 hour, and washed three times for 15 minutes each. Finally, the membrane was treated with DAB (Sigma) to observe specific bands. In addition, ELISA used Ag detection ELISA kit from Synbiotics, and each sample was diluted in decimal to a concentration of 10 -1 to 10 -4 with PBS, treated with 100 μl per well for 1 hour, and then washed three times with PBS-T buffer. Washed. Here, the secondary antibody was diluted in PBS and treated for 1 hour, washed three times with PBS-T buffer, and the degree of color development was observed with TMB solution.

그 결과 대장균 발현 정제 시료 및 곤충세포 발현 배양액에서 PCV2의 ORF2 기반의 복합백신 항원의 발현을 확인할 수 있었다.As a result, it was confirmed that the expression of the ORF2-based composite vaccine antigen of PCV2 was confirmed in the purified E. coli expression sample and the insect cell expression culture medium.

실시예 8 - PCV2 ORF2 기반의 백신 조성물의 제조 및 항체 생성능 확인Example 8 - Preparation of vaccine composition based on PCV2 ORF2 and confirmation of antibody generating ability

항원의 안정성 확인과 함께 항체 형성여부를 확인하기 위해서 모델 동물인 기니픽 (guinea pig)을 이용한 동물 실험으로 항체 생성 여부를 확인하였다. 동물 실험은 상업용 백신인 CircoFLEXTM, 구제역백신, Ingelvac MLV 백신을 대조군으로 하여 2종류 부형제에 대한 테스트를 진행하였다. 이때 사용한 부형제는 0.1% Carbopol (부형제 A) 또는 10% Rehydrogel이 포함된 30% Al(OH)3 (부형제 B)이다. 면역을 위하여, 3개체의 기니픽에 코돈 최적화된 PCV2 ORF2기반의 발현단백질을 부형제와 1:1 (v/v)로 희석하여 1ml씩 주사하였다. 주사는 3주 간격으로 2회 주사하였으며 주사 뒤 1, 2주일 뒤에 혈청을 분리하여 시료로 사용하였다. 항원에 대한 특이 항체의 생성여부 및 혈액내 항체가 를 분석하기 위해서 Synbiotics사의 항체가 측정용 ELISA 키트(SERELISA® PCV2 Ab Mono Blocking)를 사용하였다. 이때, 흡광도 값이 낮을수록(0.4 이하) PCV2에 대한 방어 항체가 대량 생산된 것으로 측정하였다. In order to confirm the stability of the antigen and the formation of antibodies, the formation of antibodies was confirmed by animal experiments using a model animal, guinea pig. Animal experiments were conducted using commercial vaccines, CircoFLEX TM , foot-and-mouth disease vaccine, and Ingelvac MLV vaccine as controls, and tests were conducted on two types of excipients. The excipients used at this time were 30% Al(OH) 3 with 0.1% Carbopol (Excipient A) or 10% Rehydrogel. (Excipient B). For immunization, the codon-optimized PCV2 ORF2-based expression protein was diluted 1:1 (v/v) with the vehicle and injected 1 ml each into three guinea pigs. Injections were injected twice at 3-week intervals, and 1 or 2 weeks after injection, serum was separated and used as a sample. An ELISA kit (SERELISA® PCV2 Ab Mono Blocking) for antibody titer measurement from Synbiotics was used to analyze the production of specific antibodies to the antigen and the antibody titer in blood. At this time, it was determined that the lower the absorbance value (0.4 or less), the larger amount of protective antibody against PCV2 was produced.

실시예Example 9 - SPF miniature pig에서 9 - SPF in miniature pigs PCV2PCV2 ORF2ORF2 기반 백신 조성물의 항체 Antibodies of Based Vaccine Compositions 생성능ability to produce 확인 Confirm

돼지에서 상기 실시예8에서 제조한 본 발명의 백신 조성물의 항체 생성능 확인을 위하여, PCV2/FMDV/PRRSV 항체가가 음성인 SPF miniature pig 3두에 본 발명의 백신 조성물을 접종한 뒤 1주 간격으로 혈액을 채혈하고 Synbiotics 사의 PCV2 Ab ELISA, Prionics사의 FMDV Ab ELISA 키트를 이용해 항체가를 측정하였다. 대조군으로는 CircoFLEXTM, 구제역백신, Ingelvac MLV백신을 사용하였고, 그 결과를 확인하였다. In order to confirm the antibody-producing ability of the vaccine composition of the present invention prepared in Example 8 in pigs, 3 SPF miniature pigs with negative PCV2/FMDV/PRRSV antibody titers were inoculated with the vaccine composition of the present invention at 1-week intervals. Blood was collected and antibody titers were measured using Synbiotics' PCV2 Ab ELISA and Prionics' FMDV Ab ELISA kits. As a control group, CircoFLEX TM , foot-and-mouth disease vaccine, and Ingelvac MLV vaccine were used, and the results were confirmed.

실시예Example 10 - 백신 투여에 의한 바이러스 10 - Virus by vaccine administration 방어능defensive ability 확인 Confirm

본 발명의 백신 투여에 의한 바이러스 방어능을 확인하기 위해서, 사양 농가의 사양 시험을 진행했다. 구체적으로 본 발명의 재조합 PCV2 ORF2 기반의 복합 항원을 주사한 10두, 양성 대조군으로서 상업용 백신 (CircoFLEXTM, 구제역백신, Ingelvac MLV)을 주사한 10두 및 음성 대조군으로서 백신 주사를 하지 않은 자돈 10두를 이용한 시험을 수행하였다. 백신은 1회 접종하였고 안정성 확인을 위해 접종량의 10배까지 접종을 진행하였다. 백신 접종 후 매주 채혈과 임상증상을 비교하였으며 그 결과를 확인하였다. In order to confirm the virus protection ability by administration of the vaccine of the present invention, a specification test of breeding farms was conducted. Specifically, 10 pigs injected with the recombinant PCV2 ORF2-based composite antigen of the present invention, 10 pigs injected with a commercial vaccine (CircoFLEX TM , foot-and-mouth disease vaccine, Ingelvac MLV) as a positive control group, and 10 unvaccinated piglets as a negative control group A test using was performed. The vaccine was inoculated once and inoculated up to 10 times the inoculated amount to confirm stability. After vaccination, weekly blood sampling and clinical symptoms were compared, and the results were confirmed.

<110> Optipharm.CO.,LTD <120> A combination vaccine based on virus like particle <130> OTIP1-50p <160> 30 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> FMDV epitope -1 <400> 1 Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala 1 5 10 15 Arg Pro Leu Pro 20 <210> 2 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> FMDV epitope -2 <400> 2 Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala 1 5 10 15 Arg Pro Leu Pro Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln 20 25 30 Ser Leu <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> PRRSV epitope -1 <400> 3 Ser His Leu Gln Leu Ile Tyr Asn Leu 1 5 <210> 4 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> PRRSV epitope -2 <400> 4 Ser Asn Asp Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Leu 1 5 10 15 Cys Glu Gly Ser Gly Ser Ser Leu Asp Asp Phe Cys His Gly Ser 20 25 30 <210> 5 <211> 762 <212> DNA <213> Artificial Sequence <220> <223> 56FMB (FMDV) <400> 5 atgacgtacc caaggaggcg ttaccggcga cgtagacatc gccctcgcag ccaccttggt 60 cagatactcc gccgtagacc atggctcgtc cacccccgcc accgtcacag gtggaggagg 120 aaaaatggta tcttcaacac ccgcctgtca agaacgtttg gatatactat caagcgaacc 180 acactgccta atgtgcgtgg cgatctccaa gtgctggctc agaaggccgc gcgtccattg 240 cctgtcaaga cgccatcctg ggctgtggac atgatgagat tcaatattaa cgacttcctt 300 ccccctggag ggggctcaaa cccccgttct gtgccgtttg agtactacag aatcagaaag 360 gtcaaggttg aattctggcc atgctctccg atcacccagg gtgatcgggg agtgggctcc 420 agtgctgtta tcctagacga caactttgta actaaggcca cagcactgac atatgacccc 480 tatgtaaact actccagcag acataccata acccaaccct tctcgtacca tagcaggtac 540 ttcacaccta aacctgtctt agattccact attgattact ttcaaccaaa caacaaaaga 600 aaccagctgt ggttgagatt gcagactgct ggaaatgtgg accacgtagg cctcggtact 660 gcattcgaga acagtattta cgatcaggaa tacaatatcc gtgtcaccat gtatgttcaa 720 ttcagagagt tcaacttaaa ggacccgcct ttgaaaccct aa 762 <210> 6 <211> 804 <212> DNA <213> Artificial Sequence <220> <223> 56FMBT(FMDV) <400> 6 atgacgtatc caaggaggcg ttaccgtcgg agaagacacc gccctcgtag ccatcttggt 60 caaatcttgc gccgccgccc atggctcgtc cacccccgtc atcgtcaccg ctggagacgt 120 aaaaacggta tcttcaacac ccgactctcc cgcaccttcg gatacactat caagcgaacc 180 acactgccta acgtgagagg cgacctccaa gtgctggctc agaaggcagc taggccgctg 240 cctagacaca agcaaaagat tgtggcacct gtaaagcagt cattagtcaa aactcccagc 300 tgggctgtgg acatgatgag attcaatatt aacgactttt tgcccccagg cgggggttca 360 aatccacgtt ctgtgccctt cgagtattac agaatcagga aggttaaggt tgaattctgg 420 ccctgcagcc ctatcacgca aggagatagg ggagtgggca gtagtgcggt tattctggat 480 gataactttg taacaaaggc cacagccttg acctacgatc cttatgtgaa ttactcttcg 540 cgccatacca taactcagcc tttctcctac cactcgcgct acttcacgcc gaaaccagtc 600 ttagactcca ctattgacta cttccaacca aataataaaa gaaaccagtt gtggctgaga 660 ctacaaacag ctggaaacgt tgatcacgta ggtctcggca ctgcctttga aaactctata 720 tatgaccagg agtacaacat ccgtgtcacc atgtacgtcc agttccgaga gtttaacctt 780 aaggacccgc cactgaaacc ctaa 804 <210> 7 <211> 762 <212> DNA <213> Artificial Sequence <220> <223> 80FMBT (FMDV) <400> 7 atgacgtatc caaggcgtag ataccgtaga agaagacacc gtccccgcag ccaccttggt 60 cagatcctca ggcgccgacc ctggttggtc catccgaggc accgtcaccg gtggaggagg 120 aaaaatggca tcttcaacac gcgactgagc cgcacattcg gctacactat taagcgtacc 180 acagtaaaga cgccttcgtg ggcggttgac atgatgcgtt tcaacattaa cgactttctt 240 cccccgggag ggggtctgcc caacgtgaga ggcgatttac aagtgctggc tcaaaaggca 300 gctaggcctc tgccttcaaa tccccgctct gtgccattcg agtactacag aatcagaaaa 360 gttaaggttg agttctggcc ttgctcgccg atcacacagg gtgatcgcgg agtcggctca 420 agtgccgtta ttctagacga taacttcgtc acaaaggcca cagccctcac ctatgaccca 480 tatgtgaact actcctcccg ccataccata acccaaccat tctcatacca ctctcgatac 540 tttaccccta agccagtctt ggattccact atcgattact tccagccaaa caacaagaga 600 aatcagctgt ggttgagact ccaaactgct ggaaatgtag accatgtggg actcggtact 660 gcattcgaaa acagtatata cgaccaggaa tacaacatcc gtgtaactat gtatgtgcag 720 ttcagagagt ttaatttgaa agacccacct ttaaaaccct aa 762 <210> 8 <211> 804 <212> DNA <213> Artificial Sequence <220> <223> 80FMB (FMDV) <400> 8 atgacgtacc caaggaggcg ttaccggaga cgtcgtcaca ggccacgcag ccatcttggc 60 cagatcctgc gcagaaggcc ttggctcgtc cacccccgtc atcgtcaccg ctggagacgt 120 aaaaacggta tcttcaacac cagactcagc cgcactttcg gatacactat taagcgaacg 180 acagtcaaaa cgccatcctg ggccgtggac atgatgagat tcaatattaa cgactttctt 240 cccccaggag ggggcttacc caatgtgcga ggtgatctcc aagttctggc ccagaaggca 300 gcgaggcctc tgcctcgtca caagcagaaa attgtggctc ctgtaaagca gtctttatca 360 aaccccaggt ctgtgccctt tgaatactat agaatacgaa aagttaaggt tgaattctgg 420 ccatgctccc cgatcaccca gggtgacagg ggagttggtt cgtctgctgt tattctggat 480 gacaactttg tcacaaaggc aacagccctc acctatgacc cgtatgtgaa ctactcgtcc 540 cgccacacaa tcacccagcc tttctcatac cactcaagat acttcacccc taagcctgtg 600 ttggatagta ctatcgatta cttccaacca aacaataaga gaaaccaact gtggttgcgc 660 ttgcaaactg ctggaaacgt agatcatgtc ggcttgggca ctgctttcga gaacagtata 720 tacgaccaag agtacaatat ccgtgtaacc atgtatgtcc agttcagaga gttcaatcta 780 aaagacccgc ctctgaagcc ctaa 804 <210> 9 <211> 762 <212> DNA <213> Artificial Sequence <220> <223> 125FMB (FMDV) <400> 9 atgacgtatc caaggaggag gtatcgtaga agaagacacc gcccgcgcag ccatcttggc 60 cagatactcc gtagacgccc ctggctcgtc cacccacgcc accgtcaccg ttggcgtagg 120 aaaaatggta tcttcaacac acgcctgtcg cgaaccttcg gttacactat caaacgaacc 180 acagtcaaga cgccttcttg ggccgtggac atgatgagat tcaatattaa tgactttctt 240 cccccaggag ggggctcaaa cccacgctct gtgcccttcg aatactaccg tataagaaag 300 gttaaggttg agttctggcc ttgcagcccg atcacacaag gtgacagagg agtgggctcc 360 agtgctgtca ttctagatga taacttcgtg ctgccaaacg tacggggtga cctccaggtg 420 ctggcacaaa aggcagctag gccgctgcct acgaaagcca ccgccttgac ctacgacccc 480 tacgtcaatt actcgtctcg ccataccatc actcagccct tctcctacca ttcccgctac 540 tttaccccca agcctgtcct cgattcaact attgattact tccaacctaa caacaagaga 600 aaccagctgt ggttgagatt acaaactgct ggaaacgttg accacgtagg attgggcact 660 gcgttcgaga acagtatcta cgatcaggaa tataacatca gagtaacaat gtatgtgcag 720 tttagagagt tcaatttgaa agacccacct ttaaagccat aa 762 <210> 10 <211> 804 <212> DNA <213> Artificial Sequence <220> <223> 125FMBT (FMDV) <400> 10 atgacgtatc caagaaggcg ttacagacgg agaagacacc gcccacgcag ccatcttggt 60 caaattctgc gcagacgccc gtggctggtt catccccgcc atcggcatcg ctggagacga 120 aaaaatggca tcttcaacac ccgtctctcg cgcacattcg gatatactat caaacgtacc 180 acagtcaaaa caccctcatg ggcagtcgac atgatgagat tcaatattaa tgactttctt 240 ccacctgggg ggggctcaaa tccgcgctct gtcccgtttg aatattatag aatacggaag 300 gttaaagttg agttctggcc ctgtagtccg atcacccagg gtgatcgggg agtaggctct 360 agtgctgtta ttctggatga taactttgta ctgcccaacg tgagaggtga tttgcaagtg 420 ctggcccaga aagcagcgag gccactgcct cgtcacaaac agaaaatagt ggcaccggtt 480 aagcagtcct taactaaggc cacagcctta acgtatgatc cgtatgtgaa ctactccagc 540 agacatacca ttacgcagcc gttcagctat cactctcgct actttacccc taaacctgtc 600 ctggattcaa cgattgatta ttttcagccg aacaacaaaa gaaatcagtt atggttgcga 660 ctacaaactg ctggtaatgt agatcatgtg ggcctcggta ctgcgtttga gaatagtatt 720 tacgaccagg aatacaatat ccgtgttacc atgtatgttc aatttcgtga atttaattta 780 aaggaccctc cacttaaacc ttaa 804 <210> 11 <211> 762 <212> DNA <213> Artificial Sequence <220> <223> 185FMB (FMDV) <400> 11 atgacgtacc cacgcaggag gtatcggcga agacgccata gaccccgcag ccacttagga 60 cagatcctcc gccgccgccc atggctcgtc cacccccgtc accgtcaccg ttggagaagg 120 aagaatggca tcttcaacac ccgcttgtcc agaacattcg gatatactat caagcgaacc 180 acagtcaaaa cgccatcatg ggcagtggac atgatgagat ttaatatcaa tgacttcctt 240 cctcctggag ggggctcgaa ccctcgttct gtccccttcg agtactaccg tattagaaag 300 gttaaggttg agttctggcc gtgctccccc atcacccaag gtgatagggg tgtgggttct 360 agtgctgtga ttctcgatga caattttgtc acaaaggcca cagccttaac ctatgaccct 420 tatgtaaact acagctcgcg tcatacgata acccagccgt tctcatacca ctcccgttac 480 tttactccca agcctgtcct ggattctact attgattact tccaaccaaa caacaaaaga 540 aaccagctgt ggttgcgtct acaaactgcg ctgcctaacg tgagaggcga tttgcaagtg 600 ctggctcaga aggcagctag gccactgcca ggaaacgttg accatgtagg tctcggcact 660 gctttcgaga acagtatata cgaccaggaa tacaacatca gagtaaccat gtacgtgcag 720 ttcagagaat tcaatctgaa agacccccct cttaaaccgt aa 762 <210> 12 <211> 729 <212> DNA <213> Artificial Sequence <220> <223> 56PRGP5 (PRRSV) <400> 12 atgacgtatc caaggcgccg ttaccgccgt agaagacatc gccctcgtag ccatcttggc 60 cagattctcc gccgtcgtcc ctggttagtg cacccgcgtc accgtcatcg gtggagaagg 120 aaaaatggca tttttaacac ccgcctgtcg cgaacgtttg gatatactat caagcgaacc 180 acttcccatc tgcaactgat atacaacttg gtcaaaacgc caagttgggc agtggatatg 240 atgcggttca acattaatga ctttttaccg ccaggagggg gttcaaaccc ccgctctgtg 300 ccgtttgaat attaccgaat ccgcaaagtc aaagtggaat tctggccctg ttcccctatt 360 acacagggtg accggggtgt cggctctagt gcagttattc tagatgataa ttttgtaaca 420 aaggccacgg cccttaccta tgatccttat gttaattaca gcagccgcca tacaatcacc 480 cagcctttct cgtaccactc acgctatttt accccgaaac ctgtgctgga ttcaactatt 540 gattattttc aaccaaacaa taaacgtaat cagctgtggt tgcggctgca aactgctggg 600 aatgtagatc atgttggctt aggtaccgcg ttcgaaaata gtatatatga ccaggagtat 660 aacatccgtg taacaatgta tgttcagttc agagagttta atctgaaaga tccgccgctt 720 aagccgtaa 729 <210> 13 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> 56PRGP5M (PRRSV) <400> 13 atgacgtatc caaggcgccg ttaccggaga agaagacacc gcccgcgctc tcatttgggt 60 cagatcctcc gccggaggcc ttggctggtc catccccgcc accgtcaccg ctggagacgc 120 aaaaatggca tattcaatac ccgtctgtcg cgcacgttcg gatatactat taaacgaacc 180 acaagtaatg acagcagctc acatttacag ctgatttata acttgacgct ctgtgagggt 240 agcgggagct cgttagatga tttctgccac ggttcagtca aaacaccatc ttgggcggtg 300 gacatgatgc gttttaatat caatgatttt cttccgccag gagggggctc aaatcctcgc 360 tctgtgcctt ttgaatatta tagaataaga aaggttaagg ttgaattctg gccttgctct 420 ccgatcacac agggtgaccg aggtgtgggc tccagtgccg tgattctgga tgataacttt 480 gtgacgaagg caacagccct gacgtatgat ccgtatgtta attattcctc acgtcatacc 540 attacccaac ccttctccta ccatagccgg tactttaccc ccaaacctgt cctggatagt 600 accattgact attttcagcc aaacaacaaa cgtaatcaac tgtggttaag actacagacc 660 gctggaaacg tagatcatgt tgggttaggc actgcatttg aaaatagtat ttatgaccag 720 gagtacaaca tccgtgtaac tatgtacgta caatttcggg aatttaatct gaaagatccg 780 ccgcttaagc cgtaa 795 <210> 14 <211> 729 <212> DNA <213> Artificial Sequence <220> <223> 80PRGP5 (PRRSV) <400> 14 atgacgtatc cacgtcgccg ttatcgccgg cgcagacatc ggccgcgttc acatctgggc 60 caaatactca ggcgccgtcc gtggttagtc catcctcgtc atcgtcaccg ctggagacgt 120 aaaaatggga tctttaacac ccgcctgagc cgtactttcg gttacacgat caagcgaacc 180 acagtcaaaa cgcccagttg ggcggtggat atgatgagat ttaatattaa tgattttctg 240 ccgccaggag gtggctctca tctacaactg atttataacc tctcaaaccc tcgctctgtg 300 ccgtttgaat actatcgaat tcggaaagtt aaagtggaat tttggccgtg ttccccgatc 360 acacagggtg atcggggtgt gggcagcagt gcagttattc tggatgacaa ctttgttaca 420 aaggccacag cccttaccta tgatccttat gtaaattatt ccagccggca tactataacc 480 cagcccttct cgtatcactc acgctacttt acccccaaac cggtcttaga ttccactatt 540 gattatttcc agccaaacaa caagagaaat cagctgtggt tgagattaca aactgctgga 600 aatgttgacc acgttggcct ggggacggct tttgagaata gtatatatga ccaggagtac 660 aatatccgtg taaccatgta cgtacagttc cgagaattca atcttaaaga tccgccattg 720 aaaccttaa 729 <210> 15 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> 80PRGP5M (PRRSV) <400> 15 atgacgtatc caaggcgccg ttaccggaga cgaagacatc ggcctcgcag ccatcttggc 60 cagatcttac gccgccgtcc ctggctggtt catcctcgcc accggcatcg ctggcgcagg 120 aaaaatggca ttttcaatac ccgcctgtcc cgtacctttg gatatactat caaacgaacc 180 acagtcaaaa cgccatcttg ggcagtagac atgatgcgtt ttaatattaa tgatttttta 240 cctccaggtg ggggaagtaa cgatagctca tcccatctac aactgattta taatttgacg 300 ctgtgcgaag gcagcgggtc aagcttagac gacttttgcc acggctcctc aaacccccgt 360 tctgtgccgt ttgaatacta tcgtataaga aaggttaagg tggaattctg gccttgttcc 420 ccgatcactc agggtgaccg tggagtgggt tcttcggctg ttattctgga tgataatttt 480 gttacaaaag ccacggcctt aacctatgat ccgtatgtaa attacagttc ccgccatacc 540 attacacagc cattctcgta tcactctcgc tattttaccc ccaaacctgt cctcgattca 600 actattgatt atttccaacc aaataacaaa agaaaccagc tgtggttgcg gttgcaaacg 660 gcgggtaatg tcgaccacgt gggtctcggc actgcattcg aaaacagtat atacgatcag 720 gagtacaata tccgtgtaac aatgtatgtt cagtttcgtg agtttaatct gaaagatccg 780 ccgcttaagc cgtaa 795 <210> 16 <211> 729 <212> DNA <213> Artificial Sequence <220> <223> 125PRGP5 (PRRSV) <400> 16 atgacgtatc caaggcgccg ttaccgccgc cgtagacacc gcccccgtag ccatcttgga 60 cagatattac gccggcgtcc gtggttagtc cacccacggc atcgtcatcg gtggagaagg 120 aagaatggca tcttcaatac gcgacttagc cgcaccttcg ggtacactat taaacgaaca 180 acagtcaaaa cgccgtcctg ggcggtggac atgatgcgtt ttaatattaa cgatttttta 240 ccgccaggtg ggggctctaa cccgcgctct gtgccctttg aatattatag aattagaaaa 300 gttaaggttg aattctggcc gtgtagtccg atcacccagg gtgatagggg agtgggttca 360 agtgcagtta ttctagatga caactttgtt agtcatctgc agctgatcta taatttgaca 420 aaggcaacag ccctcacgta tgatccatac gtaaattact cctcacgcca tactataacc 480 cagcccttct cgtatcactc tcggtacttt accccgaaac ctgttctgga tagcacaatt 540 gattattttc aaccaaacaa caaacggaat cagctgtggt tgagactgca aactgctggt 600 aacgtggacc acgtgggcct gggtactgcg ttcgagaatt caatttatga tcaagaatat 660 aatatccgcg tcaccatgta tgtacagttt cgtgagttta atcttaaaga ccctccactg 720 aaaccttaa 729 <210> 17 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> 125PRGP5 (PRRSV) <400> 17 atgacgtatc caaggcgccg ttaccggaga cgccgtcatc gtccgcgcag ccatcttggc 60 caaattctaa gacgccgccc ttggttagta cacccgcgcc atcgtcaccg gtggagacgt 120 aaaaatggta tcttcaacac ccggctcagt agaacctttg gttatacgat caagcgaacc 180 acagtgaaaa cgccatcatg ggcggtggac atgatgaggt ttaatattaa tgatttcctg 240 cccccgggtg ggggctcaaa ccctcgtagt gttccgtttg aatattatcg tattagaaag 300 gtcaaggttg agttttggcc atgttccccg atcacccaag gtgatcgagg ggtgggctcc 360 agtgcagtta ttttagatga taattttgta agcaatgact ctagctccca tctgcagctg 420 atttacaact tgacactgtg cgagggctca ggcagctcgt tagatgattt ttgccatggc 480 tccacaaaag ccacagccct gacctatgat ccctatgtga actattcttc tcgccatacg 540 ataactcagc cgttctcata ccactcgcgc tatttcaccc cgaaaccggt cctcgattct 600 actatagact attttcagcc aaataataaa agaaatcagc tgtggttgcg gttgcaaact 660 gctggtaatg tagaccacgt tggactgggc actgcatttg aaaacagtat ttacgatcag 720 gaatataaca tccgtgttac aatgtacgtc cagtttagag aattcaatct taaagacccc 780 cctcttaaac cttaa 795 <210> 18 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> 56FMB (FMDV) <400> 18 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Leu Pro Asn 50 55 60 Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu 65 70 75 80 Pro Val Lys Thr Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile 85 90 95 Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro 100 105 110 Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys 115 120 125 Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile 130 135 140 Leu Asp Asp Asn Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro 145 150 155 160 Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr 165 170 175 His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp 180 185 190 Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln 195 200 205 Thr Ala Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn 210 215 220 Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln 225 230 235 240 Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 245 250 <210> 19 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> 56FMBT(FMDV) <400> 19 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Leu Pro Asn 50 55 60 Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu 65 70 75 80 Pro Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Ser Leu Val 85 90 95 Lys Thr Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp 100 105 110 Phe Leu Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu 115 120 125 Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro 130 135 140 Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp 145 150 155 160 Asp Asn Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val 165 170 175 Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser 180 185 190 Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe 195 200 205 Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala 210 215 220 Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile 225 230 235 240 Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg 245 250 255 Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 260 265 <210> 20 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> 80FMB (FMDV) <400> 20 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu 85 90 95 Ala Gln Lys Ala Ala Arg Pro Leu Pro Ser Asn Pro Arg Ser Val Pro 100 105 110 Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys 115 120 125 Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile 130 135 140 Leu Asp Asp Asn Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro 145 150 155 160 Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr 165 170 175 His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp 180 185 190 Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln 195 200 205 Thr Ala Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn 210 215 220 Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln 225 230 235 240 Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 245 250 <210> 21 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> 80FMBT (FMDV) <400> 21 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu 85 90 95 Ala Gln Lys Ala Ala Arg Pro Leu Pro Arg His Lys Gln Lys Ile Val 100 105 110 Ala Pro Val Lys Gln Ser Leu Ser Asn Pro Arg Ser Val Pro Phe Glu 115 120 125 Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro 130 135 140 Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp 145 150 155 160 Asp Asn Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val 165 170 175 Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser 180 185 190 Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe 195 200 205 Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala 210 215 220 Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile 225 230 235 240 Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg 245 250 255 Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 260 265 <210> 22 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> 125FMB (FMDV) <400> 22 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys 130 135 140 Ala Ala Arg Pro Leu Pro Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro 145 150 155 160 Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr 165 170 175 His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp 180 185 190 Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln 195 200 205 Thr Ala Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn 210 215 220 Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln 225 230 235 240 Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 245 250 <210> 23 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> 125FMBT (FMDV) <400> 23 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys 130 135 140 Ala Ala Arg Pro Leu Pro Arg His Lys Gln Lys Ile Val Ala Pro Val 145 150 155 160 Lys Gln Ser Leu Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val 165 170 175 Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser 180 185 190 Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe 195 200 205 Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala 210 215 220 Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile 225 230 235 240 Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg 245 250 255 Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 260 265 <210> 24 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> 185FMB (FMDV) <400> 24 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr 130 135 140 Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr 145 150 155 160 Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro 165 170 175 Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala Leu Pro 180 185 190 Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro 195 200 205 Leu Pro Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn 210 215 220 Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln 225 230 235 240 Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 245 250 <210> 25 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> 56PRGP5 (PRRSV) <400> 25 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Ser His Leu 50 55 60 Gln Leu Ile Tyr Asn Leu Val Lys Thr Pro Ser Trp Ala Val Asp Met 65 70 75 80 Met Arg Phe Asn Ile Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser Asn 85 90 95 Pro Arg Ser Val Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val 100 105 110 Glu Phe Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly 115 120 125 Ser Ser Ala Val Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Thr Ala 130 135 140 Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr 145 150 155 160 Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu 165 170 175 Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu 180 185 190 Trp Leu Arg Leu Gln Thr Ala Gly Asn Val Asp His Val Gly Leu Gly 195 200 205 Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val 210 215 220 Thr Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu 225 230 235 240 Lys Pro <210> 26 <211> 264 <212> PRT <213> Artificial Sequence <220> <223> 56PRGP5M (PRRSV) <400> 26 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Ser Asn Asp 50 55 60 Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys Glu Gly 65 70 75 80 Ser Gly Ser Ser Leu Asp Asp Phe Cys His Gly Ser Val Lys Thr Pro 85 90 95 Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu Pro 100 105 110 Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr Arg 115 120 125 Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr Gln 130 135 140 Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn Phe 145 150 155 160 Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser 165 170 175 Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe 180 185 190 Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn 195 200 205 Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala Gly Asn Val 210 215 220 Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln 225 230 235 240 Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe Asn 245 250 255 Leu Lys Asp Pro Pro Leu Lys Pro 260 <210> 27 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> 80PRGP5 (PRRSV) <400> 27 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser His Leu Gln Leu Ile Tyr Asn Leu Ser Asn 85 90 95 Pro Arg Ser Val Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val 100 105 110 Glu Phe Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly 115 120 125 Ser Ser Ala Val Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Thr Ala 130 135 140 Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr 145 150 155 160 Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu 165 170 175 Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu 180 185 190 Trp Leu Arg Leu Gln Thr Ala Gly Asn Val Asp His Val Gly Leu Gly 195 200 205 Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val 210 215 220 Thr Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu 225 230 235 240 Lys Pro <210> 28 <211> 264 <212> PRT <213> Artificial Sequence <220> <223> 80PRGP5M (PRRSV) <400> 28 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Asp Ser Ser Ser His Leu Gln Leu Ile 85 90 95 Tyr Asn Leu Thr Leu Cys Glu Gly Ser Gly Ser Ser Leu Asp Asp Phe 100 105 110 Cys His Gly Ser Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr Arg 115 120 125 Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr Gln 130 135 140 Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn Phe 145 150 155 160 Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser 165 170 175 Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe 180 185 190 Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn 195 200 205 Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala Gly Asn Val 210 215 220 Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln 225 230 235 240 Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe Asn 245 250 255 Leu Lys Asp Pro Pro Leu Lys Pro 260 <210> 29 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> 125PRGP5 (PRRSV) <400> 29 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Lys Ala Thr Ala 130 135 140 Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr 145 150 155 160 Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu 165 170 175 Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu 180 185 190 Trp Leu Arg Leu Gln Thr Ala Gly Asn Val Asp His Val Gly Leu Gly 195 200 205 Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val 210 215 220 Thr Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu 225 230 235 240 Lys Pro <210> 30 <211> 265 <212> PRT <213> Artificial Sequence <220> <223> 125PRGP5 (PRRSV) <400> 30 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Ser Asn Asp Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu 130 135 140 Thr Leu Cys Glu Gly Ser Gly Ser Ser Leu Asp Asp Phe Cys His Gly 145 150 155 160 Ser Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser 165 170 175 Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe 180 185 190 Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn 195 200 205 Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala Gly Asn Val 210 215 220 Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln 225 230 235 240 Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe Asn 245 250 255 Leu Lys Asp Pro Pro Leu Lys Pro *** 260 265 <110> Optipharm.CO.,LTD <120> A combination vaccine based on virus like particle <130> OTIP1-50p <160> 30 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> FMDV epitope -1 <400> 1 Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala 1 5 10 15 Arg Pro Leu Pro 20 <210> 2 <211> 34 < 212> PRT <213> Artificial Sequence <220> <223> FMDV epitope -2 <400> 2 Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala 1 5 10 15 Arg Pro Leu Pro Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln 20 25 30 Ser Leu <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> PRRSV epitope -1 <400> 3 Ser His Leu Gln Leu Ile Tyr Asn Leu 1 5 <210> 4 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> PRRSV epitope -2 <400> 4 Ser Asn Asp Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Leu 1 5 10 15 Cys Glu Gly Ser Gly Ser Ser Leu Asp Asp Phe Cys His Gly Ser 20 25 30 <210> 5 <211> 762 <212> DNA < 213> Artificial Sequence <220> <223> 56FMB (FMDV) <400> 5 atgacgtacc caaggaggcg ttaccggcga cgtagacatc gccctcgcag ccaccttggt 60 cagatactcc gccgtagacc atggctcgtc cacccccgcc accgtcacag gtggaggagg 120 aaaaatggta tcttcaacac ccgcctgtca agaacgtttg gatatactat caagcgaacc 180 acactgccta atgtgcgtgg cgatctccaa gtgctggctc agaaggccgc gcgtccattg 240 cctgtcaaga cgccatcctg ggctgtggac atgatgagat tcaatattaa cgacttcctt 300 ccccctggag ggggctcaaa cccccgttct gtgccgtttg agtactacag aatcagaaag 360 gtcaaggttg aattctggcc atgctctccg atcacccagg gtgatcgggg agtgggctcc 420 agtgctgtta tcctagacga caactttgta actaaggcca cagcactgac atatgacccc 480 tatgtaaact actccagcag acataccata acccaaccct tctcgtacca tagcaggtac 540 ttcacaccta aacctgtctt agattccact attgattact ttcaaccaaa caacaaaaga 600 aaccagctgt ggttgagatt gcagactgct ggaaatgtgg accacgtagg cctcggtact 660 gcattcgaga acagtattta cgatcaggaa tacaatatcc gtgtcaccat gtatgttcaa 720 ttcagagagt tcaacttaaa ggacccgcct ttgaaaccct aa 762 <210> 6 <211> 804 <212> DNA <213> Artificial Sequence e <220> <223> 56FMBT(FMDV) <400> 6 atgacgtatc caaggaggcg ttaccgtcgg agaagacacc gccctcgtag ccatcttggt 60 caaatcttgc gccgccgccc atggctcgtc cacccccgtc atcgtcaccg ctggagacgt 120 aaaaacggta tcttcaacac ccgactctcc cgcaccttcg gatacactat caagcgaacc 180 acactgccta acgtgagagg cgacctccaa gtgctggctc agaaggcagc taggccgctg 240 cctagacaca agcaaaagat tgtggcacct gtaaagcagt cattagtcaa aactcccagc 300 tgggctgtgg acatgatgag attcaatatt aacgactttt tgcccccagg cgggggttca 360 aatccacgtt ctgtgccctt cgagtattac agaatcagga aggttaaggt tgaattctgg 420 ccctgcagcc ctatcacgca aggagatagg ggagtgggca gtagtgcggt tattctggat 480 gataactttg taacaaaggc cacagccttg acctacgatc cttatgtgaa ttactcttcg 540 cgccatacca taactcagcc tttctcctac cactcgcgct acttcacgcc gaaaccagtc 600 ttagactcca ctattgacta cttccaacca aataataaaa gaaaccagtt gtggctgaga 660 ctacaaacag ctggaaacgt tgatcacgta ggtctcggca ctgcctttga aaactctata 720 tatgaccagg agtacaacat ccgtgtcacc atgtacgtcc agttccgaga gtttaacctt 780 aaggacccgc cactgaaacc ctaa 804 <210> 7 <211> 762 <212> D NA <213> Artificial Sequence <220> <223> 80FMBT (FMDV) <400> 7 atgacgtatc caaggcgtag ataccgtaga agaagacacc gtccccgcag ccaccttggt 60 cagatcctca ggcgccgacc ctggttggtc catccgaggc accgtcaccg gtggaggagg 120 aaaaatggca tcttcaacac gcgactgagc cgcacattcg gctacactat taagcgtacc 180 acagtaaaga cgccttcgtg ggcggttgac atgatgcgtt tcaacattaa cgactttctt 240 cccccgggag ggggtctgcc caacgtgaga ggcgatttac aagtgctggc tcaaaaggca 300 gctaggcctc tgccttcaaa tccccgctct gtgccattcg agtactacag aatcagaaaa 360 gttaaggttg agttctggcc ttgctcgccg atcacacagg gtgatcgcgg agtcggctca 420 agtgccgtta ttctagacga taacttcgtc acaaaggcca cagccctcac ctatgaccca 480 tatgtgaact actcctcccg ccataccata acccaaccat tctcatacca ctctcgatac 540 tttaccccta agccagtctt ggattccact atcgattact tccagccaaa caacaagaga 600 aatcagctgt ggttgagact ccaaactgct ggaaatgtag accatgtggg actcggtact 660 gcattcgaaa acagtatata cgaccaggaa tacaacatcc gtgtaactat gtatgtgcag 720 ttcagagagt ttaatttgaa agacccacct ttaaaaccct aa 762 <210> 8 <211> 804 <212> DNA <213> Artificial Se quence <220> <223> 80FMB (FMDV) <400> 8 atgacgtacc caaggaggcg ttaccggaga cgtcgtcaca ggccacgcag ccatcttggc 60 cagatcctgc gcagaaggcc ttggctcgtc cacccccgtc atcgtcaccg ctggagacgt 120 aaaaacggta tcttcaacac cagactcagc cgcactttcg gatacactat taagcgaacg 180 acagtcaaaa cgccatcctg ggccgtggac atgatgagat tcaatattaa cgactttctt 240 cccccaggag ggggcttacc caatgtgcga ggtgatctcc aagttctggc ccagaaggca 300 gcgaggcctc tgcctcgtca caagcagaaa attgtggctc ctgtaaagca gtctttatca 360 aaccccaggt ctgtgccctt tgaatactat agaatacgaa aagttaaggt tgaattctgg 420 ccatgctccc cgatcaccca gggtgacagg ggagttggtt cgtctgctgt tattctggat 480 gacaactttg tcacaaaggc aacagccctc acctatgacc cgtatgtgaa ctactcgtcc 540 cgccacacaa tcacccagcc tttctcatac cactcaagat acttcacccc taagcctgtg 600 ttggatagta ctatcgatta cttccaacca aacaataaga gaaaccaact gtggttgcgc 660 ttgcaaactg ctggaaacgt agatcatgtc ggcttgggca ctgctttcga gaacagtata 720 tacgaccaag agtacaatat ccgtgtaacc atgtatgtcc agttcagaga gttcaatcta 780 aaagacccgc ctctgaagcc ctaa 804 <210> 9 <211> 762 <2 12> DNA <213> Artificial Sequence <220> <223> 125FMB (FMDV) <400> 9 atgacgtatc caaggaggag gtatcgtaga agaagacacc gcccgcgcag ccatcttggc 60 cagatactcc gtagacgccc ctggctcgtc cacccacgcc accgtcaccg ttggcgtagg 120 aaaaatggta tcttcaacac acgcctgtcg cgaaccttcg gttacactat caaacgaacc 180 acagtcaaga cgccttcttg ggccgtggac atgatgagat tcaatattaa tgactttctt 240 cccccaggag ggggctcaaa cccacgctct gtgcccttcg aatactaccg tataagaaag 300 gttaaggttg agttctggcc ttgcagcccg atcacacaag gtgacagagg agtgggctcc 360 agtgctgtca ttctagatga taacttcgtg ctgccaaacg tacggggtga cctccaggtg 420 ctggcacaaa aggcagctag gccgctgcct acgaaagcca ccgccttgac ctacgacccc 480 tacgtcaatt actcgtctcg ccataccatc actcagccct tctcctacca ttcccgctac 540 tttaccccca agcctgtcct cgattcaact attgattact tccaacctaa caacaagaga 600 aaccagctgt ggttgagatt acaaactgct ggaaacgttg accacgtagg attgggcact 660 gcgttcgaga acagtatcta cgatcaggaa tataacatca gagtaacaat gtatgtgcag 720 tttagagagt tcaatttgaa agacccacct ttaaagccat aa 762 <210> 10 <211> 804 <212> DNA <213> Artific ial Sequence <220> <223> 125FMBT (FMDV) <400> 10 atgacgtatc caagaaggcg ttacagacgg agaagacacc gcccacgcag ccatcttggt 60 caaattctgc gcagacgccc gtggctggtt catccccgcc atcggcatcg ctggagacga 120 aaaaatggca tcttcaacac ccgtctctcg cgcacattcg gatatactat caaacgtacc 180 acagtcaaaa caccctcatg ggcagtcgac atgatgagat tcaatattaa tgactttctt 240 ccacctgggg ggggctcaaa tccgcgctct gtcccgtttg aatattatag aatacggaag 300 gttaaagttg agttctggcc ctgtagtccg atcacccagg gtgatcgggg agtaggctct 360 agtgctgtta ttctggatga taactttgta ctgcccaacg tgagaggtga tttgcaagtg 420 ctggcccaga aagcagcgag gccactgcct cgtcacaaac agaaaatagt ggcaccggtt 480 aagcagtcct taactaaggc cacagcctta acgtatgatc cgtatgtgaa ctactccagc 540 agacatacca ttacgcagcc gttcagctat cactctcgct actttacccc taaacctgtc 600 ctggattcaa cgattgatta ttttcagccg aacaacaaaa gaaatcagtt atggttgcga 660 ctacaaactg ctggtaatgt agatcatgtg ggcctcggta ctgcgtttga gaatagtatt 720 tacgaccagg aatacaatat ccgtgttacc atgtatgttc aatttcgtga atttaattta 780 aaggaccctc cacttaaacc ttaa 804 <210> 11 <2 11> 762 <212> DNA <213> Artificial Sequence <220> <223> 185FMB (FMDV) <400> 11 atgacgtacc cacgcaggag gtatcggcga agacgccata gaccccgcag ccacttagga 60 cagatcctcc gccgccgccc atggctcgtc cacccccgtc accgtcaccg ttggagaagg 120 aagaatggca tcttcaacac ccgcttgtcc agaacattcg gatatactat caagcgaacc 180 acagtcaaaa cgccatcatg ggcagtggac atgatgagat ttaatatcaa tgacttcctt 240 cctcctggag ggggctcgaa ccctcgttct gtccccttcg agtactaccg tattagaaag 300 gttaaggttg agttctggcc gtgctccccc atcacccaag gtgatagggg tgtgggttct 360 agtgctgtga ttctcgatga caattttgtc acaaaggcca cagccttaac ctatgaccct 420 tatgtaaact acagctcgcg tcatacgata acccagccgt tctcatacca ctcccgttac 480 tttactccca agcctgtcct ggattctact attgattact tccaaccaaa caacaaaaga 540 aaccagctgt ggttgcgtct acaaactgcg ctgcctaacg tgagaggcga tttgcaagtg 600 ctggctcaga aggcagctag gccactgcca ggaaacgttg accatgtagg tctcggcact 660 gctttcgaga acagtatata cgaccaggaa tacaacatca gagtaaccat gtacgtgcag 720 ttcagagaat tcaatctgaa agacccccct cttaaaccgt aa 762 <210> 12 <211> 729 <212> DNA <2 13> Artificial Sequence <220> <223> 56PRGP5 (PRRSV) <400> 12 atgacgtatc caaggcgccg ttaccgccgt agaagacatc gccctcgtag ccatcttggc 60 cagattctcc gccgtcgtcc ctggttagtg cacccgcgtc accgtcatcg gtggagaagg 120 aaaaatggca tttttaacac ccgcctgtcg cgaacgtttg gatatactat caagcgaacc 180 acttcccatc tgcaactgat atacaacttg gtcaaaacgc caagttgggc agtggatatg 240 atgcggttca acattaatga ctttttaccg ccaggagggg gttcaaaccc ccgctctgtg 300 ccgtttgaat attaccgaat ccgcaaagtc aaagtggaat tctggccctg ttcccctatt 360 acacagggtg accggggtgt cggctctagt gcagttattc tagatgataa ttttgtaaca 420 aaggccacgg cccttaccta tgatccttat gttaattaca gcagccgcca tacaatcacc 480 cagcctttct cgtaccactc acgctatttt accccgaaac ctgtgctgga ttcaactatt 540 gattattttc aaccaaacaa taaacgtaat cagctgtggt tgcggctgca aactgctggg 600 aatgtagatc atgttggctt aggtaccgcg ttcgaaaata gtatatatga ccaggagtat 660 aacatccgtg taacaatgta tgttcagttc agagagttta atctgaaaga tccgccgctt 720 aagccgtaa 729 <210> 13 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> 56PRGP5M (PRRSV) <4 00> 13 atgacgtatc caaggcgccg ttaccggaga agaagacacc gcccgcgctc tcatttgggt 60 cagatcctcc gccggaggcc ttggctggtc catccccgcc accgtcaccg ctggagacgc 120 aaaaatggca tattcaatac ccgtctgtcg cgcacgttcg gatatactat taaacgaacc 180 acaagtaatg acagcagctc acatttacag ctgatttata acttgacgct ctgtgagggt 240 agcgggagct cgttagatga tttctgccac ggttcagtca aaacaccatc ttgggcggtg 300 gacatgatgc gttttaatat caatgatttt cttccgccag gagggggctc aaatcctcgc 360 tctgtgcctt ttgaatatta tagaataaga aaggttaagg ttgaattctg gccttgctct 420 ccgatcacac agggtgaccg aggtgtgggc tccagtgccg tgattctgga tgataacttt 480 gtgacgaagg caacagccct gacgtatgat ccgtatgtta attattcctc acgtcatacc 540 attacccaac ccttctccta ccatagccgg tactttaccc ccaaacctgt cctggatagt 600 accattgact attttcagcc aaacaacaaa cgtaatcaac tgtggttaag actacagacc 660 gctggaaacg tagatcatgt tgggttaggc actgcatttg aaaatagtat ttatgaccag 720 gagtacaaca tccgtgtaac tatgtacgta caatttcggg aatttaatct gaaagatccg 780 ccgcttaagc cgtaa 795 <210> 14 <211> 729 <212> DNA <213> Artificial Sequence <220> <2 23> 80PRGP5 (PRRSV) <400> 14 atgacgtatc cacgtcgccg ttatcgccgg cgcagacatc ggccgcgttc acatctgggc 60 caaatactca ggcgccgtcc gtggttagtc catcctcgtc atcgtcaccg ctggagacgt 120 aaaaatggga tctttaacac ccgcctgagc cgtactttcg gttacacgat caagcgaacc 180 acagtcaaaa cgcccagttg ggcggtggat atgatgagat ttaatattaa tgattttctg 240 ccgccaggag gtggctctca tctacaactg atttataacc tctcaaaccc tcgctctgtg 300 ccgtttgaat actatcgaat tcggaaagtt aaagtggaat tttggccgtg ttccccgatc 360 acacagggtg atcggggtgt gggcagcagt gcagttattc tggatgacaa ctttgttaca 420 aaggccacag cccttaccta tgatccttat gtaaattatt ccagccggca tactataacc 480 cagcccttct cgtatcactc acgctacttt acccccaaac cggtcttaga ttccactatt 540 gattatttcc agccaaacaa caagagaaat cagctgtggt tgagattaca aactgctgga 600 aatgttgacc acgttggcct ggggacggct tttgagaata gtatatatga ccaggagtac 660 aatatccgtg taaccatgta cgtacagttc cgagaattca atcttaaaga tccgccattg 720 aaaccttaa 729 <210> 15 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> 80PRGP5M (PRRSV) <400> 15 atgacgtatc caaggcgccg tta ccggaga cgaagacatc ggcctcgcag ccatcttggc 60 cagatcttac gccgccgtcc ctggctggtt catcctcgcc accggcatcg ctggcgcagg 120 aaaaatggca ttttcaatac ccgcctgtcc cgtacctttg gatatactat caaacgaacc 180 acagtcaaaa cgccatcttg ggcagtagac atgatgcgtt ttaatattaa tgatttttta 240 cctccaggtg ggggaagtaa cgatagctca tcccatctac aactgattta taatttgacg 300 ctgtgcgaag gcagcgggtc aagcttagac gacttttgcc acggctcctc aaacccccgt 360 tctgtgccgt ttgaatacta tcgtataaga aaggttaagg tggaattctg gccttgttcc 420 ccgatcactc agggtgaccg tggagtgggt tcttcggctg ttattctgga tgataatttt 480 gttacaaaag ccacggcctt aacctatgat ccgtatgtaa attacagttc ccgccatacc 540 attacacagc cattctcgta tcactctcgc tattttaccc ccaaacctgt cctcgattca 600 actattgatt atttccaacc aaataacaaa agaaaccagc tgtggttgcg gttgcaaacg 660 gcgggtaatg tcgaccacgt gggtctcggc actgcattcg aaaacagtat atacgatcag 720 gagtacaata tccgtgtaac aatgtatgtt cagtttcgtg agtttaatct gaaagatccg 780 ccgcttaagc cgtaa 795 <210> 16 <211> 729 <212> DNA <213> Artificial Sequence <220> <223> 125PRGP5 (PRRSV) <400> 16 at gacgtatc caaggcgccg ttaccgccgc cgtagacacc gcccccgtag ccatcttgga 60 cagatattac gccggcgtcc gtggttagtc cacccacggc atcgtcatcg gtggagaagg 120 aagaatggca tcttcaatac gcgacttagc cgcaccttcg ggtacactat taaacgaaca 180 acagtcaaaa cgccgtcctg ggcggtggac atgatgcgtt ttaatattaa cgatttttta 240 ccgccaggtg ggggctctaa cccgcgctct gtgccctttg aatattatag aattagaaaa 300 gttaaggttg aattctggcc gtgtagtccg atcacccagg gtgatagggg agtgggttca 360 agtgcagtta ttctagatga caactttgtt agtcatctgc agctgatcta taatttgaca 420 aaggcaacag ccctcacgta tgatccatac gtaaattact cctcacgcca tactataacc 480 cagcccttct cgtatcactc tcggtacttt accccgaaac ctgttctgga tagcacaatt 540 gattattttc aaccaaacaa caaacggaat cagctgtggt tgagactgca aactgctggt 600 aacgtggacc acgtgggcct gggtactgcg ttcgagaatt caatttatga tcaagaatat 660 aatatccgcg tcaccatgta tgtacagttt cgtgagttta atcttaaaga ccctccactg 720 aaaccttaa 729 <210> 17 <211> 795 <212> DNA <213 > Artificial Sequence <220> <223> 125PRGP5 (PRRSV) <400> 17 atgacgtatc caaggcgccg ttaccggaga cgccgtcatc gtccgcgcag c catcttggc 60 caaattctaa gacgccgccc ttggttagta cacccgcgcc atcgtcaccg gtggagacgt 120 aaaaatggta tcttcaacac ccggctcagt agaacctttg gttatacgat caagcgaacc 180 acagtgaaaa cgccatcatg ggcggtggac atgatgaggt ttaatattaa tgatttcctg 240 cccccgggtg ggggctcaaa ccctcgtagt gttccgtttg aatattatcg tattagaaag 300 gtcaaggttg agttttggcc atgttccccg atcacccaag gtgatcgagg ggtgggctcc 360 agtgcagtta ttttagatga taattttgta agcaatgact ctagctccca tctgcagctg 420 atttacaact tgacactgtg cgagggctca ggcagctcgt tagatgattt ttgccatggc 480 tccacaaaag ccacagccct gacctatgat ccctatgtga actattcttc tcgccatacg 540 ataactcagc cgttctcata ccactcgcgc tatttcaccc cgaaaccggt cctcgattct 600 actatagact attttcagcc aaataataaa agaaatcagc tgtggttgcg gttgcaaact 660 gctggtaatg tagaccacgt tggactgggc actgcatttg aaaacagtat ttacgatcag 720 gaatataaca tccgtgttac aatgtacgtc cagtttagag aattcaatct taaagacccc 780 cctcttaaac cttaa 795 <210> 18 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> 56FMB (FMDV) <400> 18 Met Thr Tyr Pro Arg Arg Arg Tyr Arg A rg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Leu Pro Asn 50 55 60 Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu 65 70 75 80 Pro Val Lys Thr Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile 85 90 95 Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro 100 105 110 Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys 115 120 125 Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile 130 135 140 Leu Asp Asp Asn Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro 145 150 155 160 Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr 165 170 175 His Ser Arg Tyr Phe T hr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp 180 185 190 Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln 195 200 205 Thr Ala Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn 210 215 220 Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln 225 230 235 240 Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 245 250 <210> 19 <211> 267 <212> PRT < 213> Artificial Sequence <220> <223> 56FMBT(FMDV) <400> 19 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Leu Pro Asn 50 55 60 Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu 65 70 75 80 Pro Arg His Lys Gln L ys Ile Val Ala Pro Val Lys Gln Ser Leu Val 85 90 95 Lys Thr Pro Ser Trp Ala Val Asp Met Met Met Arg Phe Asn Ile Asn Asp 100 105 110 Phe Leu Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu 115 120 125 Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro 130 135 140 Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp 145 150 155 160 Asp Asn Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val 165 170 175 Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser 180 185 190 Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe 195 200 205 Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala 210 215 220 Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile 225 230 235 240 Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg 245 250 255 Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 260 265 <210> 20 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> 80FMB (FMDV) <400> 20 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu 85 90 95 Ala Gln Lys Ala Ala Arg Pro Leu Pro Ser Asn Pro Arg Ser Val Pro 100 105 110 Phe Glu Tyr Tyr Arg Ile Arg Val Lys Val Lys Val Glu Phe Trp Pro Cys 115 120 125 Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Ser Ser Ala Val Ile 130 135 140 Leu Asp Asp Asn Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro 145 150 155 160 Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr 165 170 175 His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp 180 185 190 Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln 195 200 205 Thr Ala Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn 210 215 220 Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln 225 230 235 240 Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 245 250 <210> 21 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> 80FMBT (FMDV) <400> 21 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 6 0 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu 85 90 95 Ala Gln Lys Ala Ala Arg Pro Leu Pro Arg His Lys Gln Lys Ile Val 100 105 110 Ala Pro Val Lys Gln Ser Leu Ser Asn Pro Arg Ser Val Pro Phe Glu 115 120 125 Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro 130 135 140 Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp 145 150 155 160 Asp Asn Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val 165 170 175 Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser 180 185 190 Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe 195 200 205 Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala 210 215 220 Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile 225 230 235 240 Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg 245 250 255 Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 260 265 <210> 22 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> 125FMB (FMDV) <400> 22 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys 130 135 140 Ala Ala Arg Pro Leu Pro Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro 145 150 155 160 Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr 165 170 175 His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp 180 185 190 Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln 195 200 205 Thr Ala Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn 210 215 220 Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln 225 230 235 240 Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 245 250 <210> 23 <211> 267 <212> PR T <213> Artificial Sequence <220> <223> 125FMBT (FMDV) <400> 23 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Leu Pro Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys 130 135 140 Ala Ala Arg Pro Leu Pro Arg His Lys Gln Lys Ile Val Ala Pro Val 145 150 155 160 Lys Gln Ser Leu Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val 165 170 175 Asn Tyr Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser 180 185 190 Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe 195 200 205 Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala 210 215 220 Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile 225 230 235 240 Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg 245 250 255 Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 260 265 <210> 24 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> 185FMB (FMDV) <400 > 24 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn G ly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr 130 135 140 Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr 145 150 155 160 Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro 165 170 175 Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala Leu Pro 180 185 190 Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro 195 200 205 Leu Pro Gly Asn Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn 210 215 220 Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln 225 230 235 240 Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro 245 250 <210> 25 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> 56PRGP5 (PRRSV) <400> 25 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Ser His Leu 50 55 60 Gln Leu Ile Tyr Asn Leu Val Lys Thr Pro Ser Trp Ala Val Asp Met 65 70 75 80 Met Arg Phe Asn Ile Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser Asn 85 90 95 Pro Arg Ser Val Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val 100 105 110 Gl u Phe Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly 115 120 125 Ser Ser Ala Val Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Thr Ala 130 135 140 Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr 145 150 155 160 Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu 165 170 175 Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu 180 185 190 Trp Leu Arg Leu Gln Thr Ala Gly Asn Val Asp His Val Gly Leu Gly 195 200 205 Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val 210 215 220 Thr Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu 225 230 235 240 Lys Pro <210> 26 <211> 264 <212> PRT <213> Artificial Sequence <220> <223> 56PRGP5M (PRRSV) <400> 26 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Ser Asn Asp 50 55 60 Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys Glu Gly 65 70 75 80 Ser Gly Ser Ser Leu Asp Asp Phe Cys His Gly Ser Val Lys Thr Pro 85 90 95 Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu Pro 100 105 110 Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr Arg 115 120 125 Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr Gln 130 135 140 Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn Phe 145 150 155 160 Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser 165 170 175 Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe 180 185 190 Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn 195 200 205 Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala Gly Asn Val 210 215 220 Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln 225 230 235 240 Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe Asn 245 250 255 Leu Lys Asp Pro Pro Leu Lys Pro 260 <210> 27 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> 80PRGP5 (PRRSV) <400> 27 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser His Leu Gln Leu Ile Tyr Asn Leu Ser Asn 85 90 95 Pro Arg Ser Val Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val 100 105 110 Glu Phe Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly 115 120 125 Ser Ser Ala Val Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Thr Lys Ala Thr Ala 130 135 140 Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Thr 145 150 155 160 Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu 165 170 175 Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu 180 185 190 Trp Leu Arg Leu Gln Thr Ala Gly Asn Val Asp His Val Gly Leu Gly 195 200 205 Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg V al 210 215 220 Thr Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu 225 230 235 240 Lys Pro <210> 28 <211> 264 <212> PRT <213> Artificial Sequence <220> <223> 80PRGP5M (PRRSV) <400> 28 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Asp Ser Ser Ser His Leu Gln Leu Ile 85 90 95 Tyr Asn Leu Thr Leu Cys Glu Gly Ser Gly Ser Ser Leu Asp Asp Phe 100 105 110 Cys His Gly Ser Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr Arg 115 120 125 Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr Gln 130 135 140 Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn Phe 145 150 155 160 Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser 165 170 175 Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe 180 185 190 Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn 195 200 205 Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala Gly Asn Val 210 215 220 Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln 225 230 235 240 Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe Asn 245 250 255 Leu Lys Asp Pro Pro Leu Lys Pro 260 <210> 29 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> 125PRGP5 (PRRSV) <400> 29 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asp Asn 115 120 125 Phe Val Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Lys Ala Thr Ala 130 135 140 Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Ser Arg His Thr Ile Thr 145 150 155 160 Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu 165 170 175 Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu 180 185 190 Trp Leu Arg Leu Gln Thr Ala Gly Asn Val Asp His Val Gly Leu Gly 195 200 205 Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg Val 210 215 220 Thr Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu 225 230 235 240 Lys Pro <210> 30 <211> 265 <212> PRT <213> Artificial Sequence <220> <223> 125PRGP5 (PRRSV ) <400> 30 Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg 1 5 10 15 Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 30 Arg His Arg His Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg 35 40 45 Leu Ser Arg Thr Phe Gly Tyr Thr Ile Lys Arg Thr Thr Val Lys Thr 50 55 60 Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu 65 70 75 80 Pro Pro Gly Gly Gly Ser Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr 85 90 95 Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr 100 105 110 Gln Gly Asp Arg Gly Val Gly Ser Ser Ala Val Ile Leu Asp Asp Asn 115 120 125 Phe Val Ser Asn Asp Ser Ser Ser His Leu Gln Leu Ile Tyr Asn Leu 13 0 135 140 Thr Leu Cys Glu Gly Ser Gly Ser Ser Leu Asp Asp Phe Cys His Gly 145 150 155 160 Ser Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser 165 170 175 Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe 180 185 190 Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn 195 200 205 Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Ala Gly Asn Val 210 215 220 Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln 225 230 235 240 Glu Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe Asn 245 250 255 Leu Lys Asp Pro Pro Leu Lys Pro *** 260 265

Claims (8)

PRRSV (Porcine reproductive and respiratory syndrome virus) 또는 구제역 바이러스 (Foot-and-mouth virus)의 에피토프가 PCV2 (Porcine circovirus type 2) ORF2 아미노산 서열(NCBI 등록번호 FJ755686)의 61 내지 62 번 아미노산 사이, 85 내지 86 번 아미노산 사이, 130 내지 131 번 아미노산 사이, 190 내지 191 번 아미노산 사이로 이루어진 군에서 선택된 1종 부위에 삽입되고,
상기 PRRSV 에피토프는 서열번호 3 또는 4의 아미노산 서열로 표시되고,
상기 구제역 바이러스 에피토프는 서열번호 1 또는 2의 아미노산 서열로 표시되는,
재조합 PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2).
The epitope of Porcine reproductive and respiratory syndrome virus (PRRSV) or Foot-and-mouth virus is between amino acids 61 and 62 of the PCV2 (Porcine circovirus type 2) ORF2 amino acid sequence (NCBI registration number FJ755686), between 85 and 86 It is inserted at one site selected from the group consisting of between amino acids No. 130 and No. 131, between amino acids No. 190 and No. 191,
The PRRSV epitope is represented by the amino acid sequence of SEQ ID NO: 3 or 4,
The foot-and-mouth disease virus epitope is represented by the amino acid sequence of SEQ ID NO: 1 or 2,
Recombinant PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2).
 삭제delete 제1항에 있어서, 상기 재조합 PCV2 ORF2는 서열번호 18 내지 서열번호 30으로 이루어진 군에서 선택된 1종의 아미노산 서열인, 재조합 PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2).
According to claim 1, wherein the recombinant PCV2 ORF2 is one amino acid sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 30, recombinant PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2).
 제1항의 재조합 PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2) 가 도입된 세포.
A cell into which the recombinant PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2) of claim 1 is introduced.
 제1항의 재조합 PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2) 가 도입된 대장균.
Escherichia coli into which the recombinant PCV2 (Porcine circovirus type 2) ORF2 (Open Reading Frame 2) of claim 1 has been introduced.
 제4항의 세포 또는 제5항의 대장균에서 생산된 바이러스 유사 입자.
Virus-like particles produced from the cells of claim 4 or E. coli of claim 5.
 삭제delete 삭제delete
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