KR20120066555A - Transformants expressing epitope of porcine epidemic diarrhea virus and vaccine compositions containing the same - Google Patents
Transformants expressing epitope of porcine epidemic diarrhea virus and vaccine compositions containing the same Download PDFInfo
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- KR20120066555A KR20120066555A KR1020100127957A KR20100127957A KR20120066555A KR 20120066555 A KR20120066555 A KR 20120066555A KR 1020100127957 A KR1020100127957 A KR 1020100127957A KR 20100127957 A KR20100127957 A KR 20100127957A KR 20120066555 A KR20120066555 A KR 20120066555A
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Abstract
Description
본 발명은 신규한 PEDV(porcine epidemic diarrhea virus) 에피토프 단백질, 이에 대한 항체, 상기 에피토프 단백질을 암호화하는 유전자를 포함하는 재조합 벡터, 상기 재조합 벡터로 형질전환된 형질전환체, 상기 형질전환체를 포함하는 PEDV 백신 조성물 및 이의 제조방법에 관한 것이다.
The present invention is a novel PEDV (porcine epidemic diarrhea virus) epitope protein, an antibody thereto, a recombinant vector comprising a gene encoding the epitope protein, a transformant transformed with the recombinant vector, comprising the transformant It relates to a PEDV vaccine composition and a method for preparing the same.
돼지 유행성 설사병(PED)은 1978년에 벨기에와 영국에서 처음 보고된 후 돼지를 생산하는 많은 국가(유럽, 일본, 중국, 한국을 포함한 아시아)에서 발생되었으며 유럽 및 아시아에서 큰 경제적 손실을 일으켰다(Pensaert 1978; Chasey 1978). 돼지 유행성 설사병을 일으키는 PEDV는 주로 소장 융모세포에 증식하여 융모 상피세포를 변성 또는 괴사시켜 융모의 위축과 탈락을 동반하고, 흡수장애를 초래하여 지속적인 수양성 설사를 일으킨다(Straw 2006). 돼지의 연령에 상관없이 장염을 일으키며 심한 설사와 탈수현상을 동반하고 심지어 자돈에서의 치사율은 80~90%에 이른다(Jinghui 2005). Swine epidemic diarrheal disease (PED) was first reported in Belgium and the United Kingdom in 1978 and has occurred in many pig-producing countries (Asia, including Europe, Japan, China and Korea) and has caused significant economic losses in Europe and Asia (Pensaert). 1978; Chasey 1978). PEDV, which causes swine epidemic diarrheal disease, mainly proliferates in small intestinal chollocytes, denatures or necrosis of chorionic epithelial cells, causes atrophy and excretion of villi, and causes malabsorption, resulting in sustained diarrhea (Straw 2006). Regardless of the age of pigs, enteritis is associated with severe diarrhea and dehydration, and even mortality in piglets ranges from 80 to 90% (Jinghui 2005).
국내에서의 돼지 유행성 설사병은 1992년에 처음으로 PED 바이러스가 분리된 이래 2년간 크게 유행한 바 있으며, TGEV(Transmissible Gastroenteritis virus)와 임상적으로 감별되지 않아 막대한 경제적 피해를 초래했다(Duarte 1994). 최근에는 PED의 단독발생보다는 TGE와 혼합감염이 증가하고 계절에 관계없이 발생하나 겨울철에 주로 빈번하게 발생한다.
Swine epidemic diarrhea in Korea has been in vogue for two years since the PED virus was first isolated in 1992, and it has not been clinically differentiated from TGEV (Transmissible Gastroenteritis virus), causing massive economic damage (Duarte 1994). In recent years, mixed infection with TGE is increased rather than PED alone, and occurs regardless of season, but occurs frequently in winter.
한편, 항생제는 미생물에 의해 생성되는 가용성 유기물질로서 다른 미생물의 성장을 억제, 파괴하는 물질이다. 원래 미생물에서만 생산되었으나 현재는 공업적으로 생산되는 화합물도 항생제작용을 나타낸다. 이러한 항생제는 병원균을 억제하기 위한 의학 및 수의학적 목적으로 개발되었으나 동물의 성장 촉진 및 사료효율 개선효과도 인정되어 사료첨가제로 사용되기도 한다(Sarmah 2006). Meanwhile, antibiotics are soluble organic substances produced by microorganisms and are substances that inhibit and destroy the growth of other microorganisms. Originally produced only by microorganisms, now industrially produced compounds also exhibit antibiotic activity. These antibiotics have been developed for medical and veterinary purposes to suppress pathogens, but they are also used as feed additives because they are recognized for promoting animal growth and improving feed efficiency (Sarmah 2006).
양돈 산업에 있어서 항생제의 사용은 성장률 개선 이외에도 어린 돼지의 폐사율을 감소시키는 효과와 위생상태가 불량한 환경에서 자돈 폐사율 감소 효과로 인해 사육밀도가 높은 농장일수록 사용 효과가 크게 나타난다. 그러나 가축에 대한 무분별한 항생제의 사용으로 항생제 잔류물, 세균의 내성 증가 및 동물에서 사람으로 세균의 내성 전이 등 인체에 미치는 안전문제가 제기됨에 따라 전세계적으로 가축에 대한 사료 첨가제용 항생제 사용이 금지되고 있는 추세이다. In the swine industry, the use of antibiotics increases the mortality rate of young pigs in addition to improving the growth rate, and in farms with higher densities due to the lower mortality rate in poor hygiene. However, the use of indiscriminate antibiotics on livestock raises safety concerns on humans such as antibiotic residues, increased bacterial tolerance, and the transfer of bacterial resistance from animals to humans. There is a trend.
최근 미국 FDA에서 가축 성장을 촉진하는 항생제에 한해 제한을 권고하고 인체에 영향을 줄 수 있는 강력한 항생제에 관해서는 금지 법안검토 중이다. 국내에서도 2011년 7월부터 사료에서의 항생제첨가가 전면 금지됨에 따라 인체에 무해하고 성장효율이 우수한 사료첨가제의 개발이 시급한 실정이다. The US FDA recently recommends restrictions on antibiotics that promote livestock growth and is reviewing bans on potent antibiotics that could affect the human body. In Korea, since antibiotics were completely banned from July 2011, it is urgent to develop feed additives that are harmless to humans and have excellent growth efficiency.
한편, 사람이나 가축의 질병을 예방하는 방법으로 생독백신, 사독백신, 재조합백신의 접종으로 이루어진다. 생독백신과 사독백신은 병원체를 약화시키거나 죽인 것으로 적절한 접종을 시행하지 못할 경우 부작용이 나타날 수 있다. 재조합백신은 유전적으로 조작된 박테리아, 효모 또는 포유류의 세포에서 병원균의 항원성을 갖는 유전자를 분리하여 발현시킨 것을 백신으로 사용하는 것이다. 대장균에서 생산된 재조합 백신의 경우 병원체 오염의 배제와 저렴한 대량 생산비용의 이점이 있으나 동식물 단백질의 경우 대장균과의 번역 후 변형체계의 차이로 인해 면역성이 저하되기도 하며, 재조합 대장균 백신의 경구 투여 시 장의 비우호적 조건에 의해 단백질이 파괴되기도 한다(Amanda and Charles 2000; Tacket 2005). 식물과 같은 고등생물의 단백질 발현 체계를 이용할 경우 면역성 저하 문제나 장의 비우호적 조건을 회피할 수 있다(Kong 2001; Youm 2007). On the other hand, as a way to prevent disease of humans or livestock consists of inoculation of live vaccine, dead poison vaccine, recombinant vaccine. Live and dead vaccines are attenuated or killed by pathogens and can cause side effects if proper inoculation is not given. Recombinant vaccine is a vaccine that isolates and expresses a gene having an antigenicity of a pathogen in cells of genetically engineered bacteria, yeasts or mammals. Recombinant vaccines produced by E. coli have the advantages of eliminating pathogen contamination and low cost of mass production. However, animal and plant proteins may be less immune due to differences in post-translational modifications with E. coli. Proteins are also destroyed by unfriendly conditions (Amanda and Charles 2000; Tacket 2005). Higher organism protein expression systems, such as plants, can be used to circumvent immunity deprivation problems and intestinal unfriendly conditions (Kong 2001; Youm 2007).
식물 경구백신은 투여 시 고통과 스트레스를 동반하지 않은 쉬운 접종과 생산비용 및 저온 운반에 대한 비용 발생하지 않는 장점으로 의료 활동이 어려운 지역에 손쉬운 공급이 가능하여 경제적으로나 의료적으로 큰 효과를 가져 올 수 있다(Lamphear 2004). 그러나 식물 백신은 유전자변형체(genetically modified organism)로서 박테리아, 바이러스 또는 동물에 있는 유전자를 삽입하여 새로운 종을 만들어내는 것에는 매우 유용하나 GMO를 섭취함으로 인한 인체에 대한 안전성과 GMO 재배로 인한 유전자오염으로 환경 위해성에 대한 문제가 제기되고 있다(Uzogara 2000; Conner 2003). GMO 재배로 인한 환경에 대한 위해성을 피하고자 세대진전을 할 수 없는 유전자 변형체 종을 만들거나 식물의 꽃가루가 발생할 수 없는 상태의 기내 배양된 조직을 이용하는 방법이 주된 추세이다(Wilkinson 2003).
Plant oral vaccines have the advantages of easy inoculation without the pain and stress of administration, and no cost for production and low temperature transportation, so they can be easily supplied to areas where medical activities are difficult. (Lamphear 2004). Plant vaccines, however, are genetically modified organisms that are very useful for creating new species by inserting genes from bacteria, viruses, or animals. Environmental risks are being raised (Uzogara 2000; Conner 2003). In order to avoid the environmental risks caused by GMO cultivation, the main trend is to create genetically modified species that cannot progress generation or to use in-flight cultures without plant pollen (Wilkinson 2003).
이러한 배경하에 본 발명자들은 PEDV의 새로운 에피토프 유전자를 개발하고, 상기 PEDV 에피토프 단백질을 발현하는 재조합 벡터를 제조하였으며, 상기 벡터가 형질전환된 형질전환체를 PEDV에 감염된 돼지에 투여 시 돼지의 설사증이 개선됨을 확인함으로써 본 발명을 완성하기에 이르렀다.
Against this background, we developed a new epitope gene of PEDV, prepared a recombinant vector expressing the PEDV epitope protein, and improved diarrhea in pigs when the transformed transformed vector was applied to pigs infected with PEDV. By confirming that the present invention was completed.
본 발명의 목적은 서열번호 1의 PEDV(porcine epidemic diarrhea virus) 에피토프 단백질을 제공하는 것이다.It is an object of the present invention to provide a porcine epidemic diarrhea virus (PEDV) epitope protein of SEQ ID NO: 1.
본 발명의 다른 목적은 상기 에피토프 단백질을 포함하는 항원을 제공하는 것이다. Another object of the present invention is to provide an antigen comprising the epitope protein.
본 발명의 또 다른 목적은 상기 에피토프 단백질을 암호화하는 유전자를 포함하는 재조합 벡터를 제공하는 것이다.It is another object of the present invention to provide a recombinant vector comprising a gene encoding the epitope protein.
본 발명의 또 다른 목적은 상기 재조합 벡터로 형질전환된 형질전환체를 제공하는 것이다.Still another object of the present invention is to provide a transformant transformed with the recombinant vector.
본 발명의 또 다른 목적은 상기 형질전환체, 상기 형질전환체의 단백질 추출물 또는 상기 형질전환체로부터 분리된 재조합 PEDV 에피토프 단백질을 포함하는 PEDV의 백신 조성물을 제공하는 것이다.Still another object of the present invention is to provide a vaccine composition of PEDV comprising the transformant, the protein extract of the transformant or recombinant PEDV epitope protein isolated from the transformant.
본 발명의 또 다른 목적은 상기 형질전환체, 상기 형질전환체의 단백질 추출물 또는 상기 형질전환체로부터 분리된 재조합 PEDV 에피토프 단백질을 포함하는 PEDV 감염의 예방 또는 치료용 사료 조성물 및 사료 첨가용 조성물을 제공하는 것이다.Still another object of the present invention is to provide a feed composition and feed composition for preventing or treating PEDV infection comprising the transformant, a protein extract of the transformant or a recombinant PEDV epitope protein isolated from the transformant. It is.
본 발명의 또 다른 목적은 (1) 서열번호 1의 PEDV 에피토프 단백질을 암호화하는 유전자를 발현하는 벡터를 제조하는 단계; (2) 상기 단계 (1)의 발현벡터를 아그로박테리아에 도입시키는 단계; 및 (3) 상기 단계 (2)의 아그로박테리아와 식물세포를 형질전환시키는 단계를 포함하는 PEDV 백신의 제조방법을 제공하는 것이다.Another object of the present invention is to prepare a vector expressing a gene encoding a PEDV epitope protein of SEQ ID NO: 1; (2) introducing the expression vector of step (1) into Agrobacteria; And (3) transforming the agrobacteria and plant cells of step (2).
본 발명의 또 다른 목적은 본 발명의 에피토프 단백질에 대한 항체를 제공하는 것이다.
Another object of the present invention is to provide an antibody against the epitope protein of the present invention.
상기 목적을 달성하기 위한 하나의 양태로서, 본 발명은 서열번호 1의 PEDV(porcine epidemic diarrhea virus) 에피토프 단백질에 관한 것이다.
As one aspect for achieving the above object, the present invention relates to a porcine epidemic diarrhea virus (PEDV) epitope protein of SEQ ID NO: 1.
본 발명에서 용어, "PEDV(porcine epidemic diarrhea virus)"는 코로나바이러스과(coronaviridae)에 속하며 단일 가닥 RNA를 게놈(genome)으로 가지며 길이는 약 28Kb이고, 3개의 주요 구성 단백질인 spike 단백질(180-220KDa)과 막단백질 또는 외피 단백질 (27-32KDa) 그리고 뉴클레오캡시드 단백질(55-58KDa)을 암호화 하고 있다(Shenyang 2007). 이는 같은 종인 SARS 코로나바이러스와 유사한 구조를 가지며 트립신이 첨가된 아프리카 녹색원숭이 신장세포(Vero cell)에서 배양된다(Kim 2003; Stadler 2003; 권 2009). As used herein, the term "porcine epidemic diarrhea virus" (PEDV) belongs to the family of coronaviridae ( coronaviridae ) and has a single stranded RNA in the genome (genome), the length of about 28Kb, three major constituent proteins spike protein (180-220KDa) ) And membrane protein or envelope protein (27-32 KDa) and nucleocapsid protein (55-58 KDa) (Shenyang 2007). It has a structure similar to that of the same species SARS coronavirus and is cultured in African green monkey kidney cells with trypsin added (Kim 2003; Stadler 2003; Vol. 2009).
본 발명에서 용어, "Spike 단백질"은 PEDV의 주요 구성 단백질로서, 목표 세포를 인식하고 바이러스와 세포막(cellular membrane)를 융합시키는 생물학적으로 중요한 기능을 가지고 있다(Chang 2002). Spike 단백질의 일부인 COE(CO-26K fragment equivalent) 유전자를 약독화 항원결정기(neutrali zation epitope)로 이용할 수 있다.As used herein, the term "Spike protein" is a major constituent protein of PEDV and has a biologically important function of recognizing target cells and fusing viruses and cellular membranes (Chang 2002). COE (CO-26K fragment equivalent) gene, which is part of the Spike protein, can be used as an attenuating epitope.
본 발명에서 용어, "에피토프"는 특정 항체에 의해 인식되는 항원결합부위의 아미노산 잔기 세트, 또는 T 세포에서는 T 세포 수용체 단백질 및/또는 주요 조직적합성 복합체 (Major Histocompatibility Complex, MHC) 수용체에 의해 인식되는 잔기이다. 에피토프는 항체, T 세포 수용체 또는 HLA 분자에 의해 인식되는 부위를 형성하는 분자로, 일차, 이차 및 삼차 펩티드 구조, 또는 전하를 의미한다. As used herein, the term "epitope" refers to a set of amino acid residues at an antigen binding site recognized by a particular antibody, or in T cells, recognized by a T cell receptor protein and / or a Major Histocompatibility Complex (MHC) receptor. Residue. Epitopes are molecules that form a site recognized by an antibody, T cell receptor, or HLA molecule and refer to primary, secondary, and tertiary peptide structures, or charges.
본 발명의 일 실시예에서는 PEDV spike 단백질의 일부이며 항원성이 있는 COE 유전자를 클로닝하기 위해서, 기존에 사용되는 Brl/27 strain이 아닌 KPEDV-9 strain에서 새로운 COE 유전자 즉, PEDV 에피토프를 얻었다. In one embodiment of the present invention, in order to clone a COE gene that is part of the PEDV spike protein and antigenic, a new COE gene, ie, PEDV epitope, was obtained from a KPEDV-9 strain instead of the Brl / 27 strain.
상기 본 발명의 새로운 PEDV 에피토프 단백질은 서열번호 1의 아미노산 서열을 포함하며, 서열번호 2의 염기서열을 갖는 것이 바람직하다.
The new PEDV epitope protein of the present invention comprises an amino acid sequence of SEQ ID NO: 1, preferably having a nucleotide sequence of SEQ ID NO: 2.
다른 양태로서 본 발명은 상기 서열번호 1의 에피토프 단백질을 포함하는 PEDV 항원에 관한 것이다. 상기 PEDV 항원은 서열번호 2의 염기서열을 가질 수 있으며, mRNA의 안정성을 위해 서열번호 2의 염기 중 148번째 염기가 구아닌, 151번째 염기가 시토신으로 변형된 것일 수 있다. 또한 spike 단백질의 C-말단에 존재하는 아미노산 서열의 항원성이 부분이 융합될 수도 있다. In another aspect, the present invention relates to a PEDV antigen comprising the epitope protein of SEQ ID NO: 1. The PEDV antigen may have a nucleotide sequence of SEQ ID NO: 2, 148 base of the base of SEQ ID NO: 2 guanine, 151 base may be modified to cytosine for the stability of the mRNA. In addition, the antigenic portion of the amino acid sequence present at the C-terminus of the spike protein may be fused.
본 발명의 일 실시예에서는 서열번호 1의 PEDV 에피토프를 COE로 표기하였으며, 서열번호 3의 아미노산 서열이 결합된 형태를 COEGY로 표기하였다.
In one embodiment of the present invention, the PEDV epitope of SEQ ID NO: 1 is denoted by COE, and the form in which the amino acid sequence of SEQ ID NO: 3 is bound is denoted by COEGY.
또 다른 하나의 양태로서, 본 발명은 서열번호 1의 PEDV 에피토프 단백질을 암호화하는 유전자를 포함하는 재조합 벡터에 관한 것이다.In another aspect, the present invention relates to a recombinant vector comprising a gene encoding a PEDV epitope protein of SEQ ID NO: 1.
본 발명에서 용어, "벡터"는 숙주 세포로 염기의 클로닝 및/또는 전이를 위한 임의의 매개물을 말한다. 벡터는 다른 DNA 단편이 결합하여 결합된 단편의 복제를 가져올 수 있는 복제단위 (replicon)일 수 있다. "복제단위"란 생체 내에서 DNA 복제의 자가 유닛으로서 기능하는, 즉, 스스로의 조절에 의해 복제가능한, 임의의 유전적 단위 (예를 들면, 플라스미드, 파지, 코스미드, 염색체, 바이러스)를 말한다. 용어 "벡터"는 시험관 내, 생체 외 또는 생체 내에서 숙주 세포로 염기를 도입하기 위한 바이러스 및 비 바이러스 매개물을 포함한다. 용어 "벡터"는 또한 미니구형 DNA를 포함할 수 있다. 예를 들면, 상기 벡터는 박테리아 DNA 서열을 갖지 않는 플라스미드일 수 있다. CpG 영역에서 풍부한 박테리아 DNA 서열의 제거는 전이유전자 발현 사일런싱을 감소시키고 플라스미드 DNA 벡터로부터 보다 지속적인 발현을 가져오기 위해 행해지고 있다. 용어 "벡터"는 또한 슬리핑 뷰티(Sleeping Beauty)같은 트랜스포존(Izsvak et al. J. MoI. Biol. 302:93-102 (2000)), 또는 인공 염색체를 포함할 수 있다. As used herein, the term "vector" refers to any medium for cloning and / or transferring bases to a host cell. The vector may be a replica, in which other DNA fragments can bind to result in replication of the bound fragment. A "replicating unit" refers to any genetic unit (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as a self-unit of DNA replication in vivo, i.e., is replicable by its own regulation. . The term "vector" includes viral and non-viral mediators for introducing bases into host cells in vitro, ex vivo or in vivo. The term "vector" may also include minispherical DNA. For example, the vector may be a plasmid that does not have a bacterial DNA sequence. Removal of bacterial DNA sequences enriched in the CpG region has been done to reduce transgene expression silencing and lead to more sustained expression from plasmid DNA vectors. The term “vector” may also include transposons (Szping, et al. J. MoI. Biol. 302: 93-102 (2000)), such as Sleeping Beauty, or artificial chromosomes.
본 발명에서는 PEDV 에피토프 단백질을 암호화하는 유전자 및 spike 단백질의 c-말단에 존재하는 단백질의 일부를 암호화하는 유전자가 작동가능하게 연결된 벡터일 수 있으며, 바람직하게 상기 PEDV 에피토프 단백질을 암호화하는 유전자는 서열번호 2로 표시되는 염기서열일 수 있고, 더욱 바람직하게 상기 유전자는 상기 서열번호 2의 염기 중 148번째 염기가 구아닌, 151번째 염기가 시토신으로 변형된 서열번호 3으로 표시되는 염기서열일 수 있다. 또한, 상기 spike 단백질의 c-말단에 존재하는 단백질의 일부를 암호화하는 유전자는 서열번호 4의 아미노산을 암호화하는 유전자일 수 있으며, 상기 유전자는 서열번호 5로 표시되는 염기서열일 수 있다.
In the present invention, the gene encoding the PEDV epitope protein and the gene encoding a portion of the protein present at the c-terminus of the spike protein may be a vector operably linked, preferably the gene encoding the PEDV epitope protein is SEQ ID NO: 2 may be a nucleotide sequence represented by 2, and more preferably, the gene may be a nucleotide sequence represented by SEQ ID NO: 3 in which the 148th base is guanine and the 151th base is cytosine among the bases of SEQ ID NO: 2. In addition, a gene encoding a part of the protein present at the c-terminus of the spike protein may be a gene encoding the amino acid of SEQ ID NO: 4, the gene may be a nucleotide sequence represented by SEQ ID NO: 5.
또 다른 하나의 양태로서, 본 발명은 상기 재조합 벡터로 형질전환된 형질전환체에 관한 것이다. 보다 구체적으로, 서열번호 1의 에피토프 단백질을 암호화하는 유전자가 포함된 재조합 벡터 또는 서열번호 1의 에피토프 단백질을 암호화하는 유전자에 서열번호 4의 아미노산을 암호화하는 유전자가 융합된 유전자를 포함하는 제조합 벡터일 수 있다. As another aspect, the present invention relates to a transformant transformed with the recombinant vector. More specifically, a recombinant vector comprising a gene encoding an epitope protein of SEQ ID NO: 1 or a production vector comprising a gene in which a gene encoding an amino acid of SEQ ID NO: 4 is fused to a gene encoding an epitope protein of SEQ ID NO: 1 Can be.
본 발명에서 용어, “형질전환”은 외부로부터 주어진 DNA에 의하여 생물의 유전적인 성질이 변하는 것으로, 즉 생물의 어떤 계통의 세포에서 추출된 핵산의 일종인 DNA를 다른 계통의 살아있는 세포의 주었을 때 DNA가 그 세포에 들어가서 유전형질이 변화하는 현상으로 형질변환 ?형전환 또는 형변환 등이라고도 한다. In the present invention, the term “transformation” refers to a change in the genetic properties of an organism by DNA given from the outside, that is, when DNA is a kind of nucleic acid extracted from a cell of a certain organism, the DNA of a living cell of another strain Is a phenomenon in which the genetic trait changes when the cell enters the cell, also called transformation, transformation, or transformation.
본 발명에서 용어, “형질전환체”는 형질전환으로 인해 생성된 형질전환식물 또는 형질전환동물을 의미하며, 유전자 재조합 기술을 이용하여 특정 유전자의 변형 또는 변이가 유발되어 생성된 유전자재조합체를 포함한다. 바람직하게 본 발명의 형질전환체는 식물세포 유래의 형질전환체일 수 있다.As used herein, the term “transformer” refers to a transformed plant or a transgenic animal produced by transformation, and includes a genetic recombinant produced by modification or mutation of a specific gene using genetic recombination technology. do. Preferably, the transformant of the present invention may be a transformant derived from plant cells.
본 발명에서 용어, “식물세포”는 식물체를 구성하는 기본이 되는 단위로, 식물 조직의 배양 세포, 배양 조직, 배양기관 또는 전체 식물일 수 있으며, 바람직하게 배양 세포, 배양 조직 또는 배양 기관일 수 있고, 더욱 바람직하게는 배양 세포의 가능한 모든 형태일 수 있으며, 가장 바람직하게는 당근 유래의 세포일 수 있다.As used herein, the term “plant cell” is a basic unit constituting a plant, and may be cultured cells, culture tissues, culture organs or whole plants of plant tissue, preferably culture cells, culture tissues, or culture organs. And more preferably all possible forms of cultured cells, most preferably cells derived from carrots.
본 발명에서 용어, “식물 조직”은 분화된 또는 미분화된 식물의 조직, 예를 들면 이에 한정되진 않으나, 뿌리, 줄기, 잎, 꽃가루, 종자, 암 조직 및 배양에 이용되는 다양한 형태의 세포들, 즉 단일 세포, 원형질체(protoplast), 싹 및 캘러스 조직을 포함한다. 식물 조직은 인 플란타(in planta)이거나 기관 배양, 조직 배양 또는 세포 배양 상태일 수 있다.As used herein, the term “plant tissue” refers to tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, Ie, single cells, protoplasts, shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture.
본 발명에서 용어, "재조합"은 세포가 이종의 핵산을 복제하거나, 상기 핵산을 발현하거나 또는 펩티드, 이종의 펩티드 또는 이종의 핵산에 의해 암호화된 단백질을 발현하는 세포를 지칭하는 것이다. 재조합 세포는 상기 세포의 천연 형태에서는 발견되지 않는 유전자 또는 유전자 절편을, 센스 또는 안티센스 형태 중 하나로 발현할 수 있다. 또한 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 상기 유전자는 변형된 것으로써 인위적인 수단에 의해 세포 내 재도입된 것이다. As used herein, the term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. Recombinant cells can also express genes found in natural cells, which are modified and reintroduced into cells by artificial means.
본 발명의 일 실시예에서는 PEDV 에피토프 단백질을 암호화하는 유전자 또는 상기 PEDV 에피토프 단백질을 암호화하는 유전자 및 spike 단백질의 일부 서열을 암호화하는 유전자가 항원을 작동가능하게 연결한 벡터를 제작하고, 상기 벡터를 형질전환한 형질전환체를 제작하였다. 본 발명의 형질전환체는 당업계에 공지된 형질전환에 이용될 수 있는 세포이면 제한없이 당업자에 의해 적절하게 선택되어 사용될 수 있으며, 본 발명의 형질전환체는 아그로박테리움, 대장균 또는 형질전환 식물일 수 있고, 바람직하게 상기 형질전환체는 식물세포일 수 있으며, 더욱 바람직하게는 담배, 애기장대, 감자, 상추, 무, 배추, 토마토, 콩, 쌀, 보리, 밀, 옥수수 및 당근으로 이루어진 군으로부터 선택되는 것인 형질전환체 일 수 있다.
In an embodiment of the present invention, a vector encoding a gene encoding a PEDV epitope protein or a gene encoding the PEDV epitope protein and a gene encoding a partial sequence of a spike protein is operably linked to an antigen. Converted transformants were produced. The transformant of the present invention may be appropriately selected and used by those skilled in the art without limitation as long as the cell can be used for transformation known in the art, and the transformant of the present invention may be Agrobacterium, Escherichia coli or a transgenic plant. Preferably, the transformant may be a plant cell, more preferably a group consisting of tobacco, Arabidopsis, potato, lettuce, radish, Chinese cabbage, tomato, soybean, rice, barley, wheat, corn and carrot It may be a transformant is selected from.
또 다른 하나의 양태로서, 본 발명은 본 발명의 형질전환체, 상기 형질전환체의 단백질 추출물 또는 상기 형질전환체로부터 분리된 재조합 PEDV 에피토프 단백질을 포함하는 PEDV의 백신 조성물에 관한 것이다.As another aspect, the present invention relates to a vaccine composition of PEDV comprising a transformant of the present invention, a protein extract of the transformant or a recombinant PEDV epitope protein isolated from the transformant.
본 발명에서 용어, “형질전환체” 및 “PEDV 에피토프 단백질”은 상기에서 설명한 바와 같다. As used herein, the terms "transformer" and "PEDV epitope protein" are as described above.
본 발명에서 용어, “백신”은 생체에 면역을 주는 항원을 함유한 생물학적인 제제로서, 감염증의 예방을 위하여 사람이나 동물에 주사하거나 경구 투여함으로써 생체에 면역이 생기게 하는 면역원 또는 항원성 물질을 말한다. 생체 내 면역은 병원균의 감염 후에 생체 내 면역력이 자동으로 얻어지는 자동면역과 외부에서 주입한 백신에 의하여 얻어지는 수동 면역으로 크게 나누어진다. 자동면역이 면역에 관계하는 항체의 생성 기간이 길고 지속적인 면역력의 특징이 있는 반면, 백신에 의한 수동 면역은 감염증 치료에 즉시 작용하나 지속력이 떨어지는 단점이 있다.As used herein, the term “vaccine” refers to a biological agent containing an antigen that immunizes a living body, and refers to an immunogen or antigenic substance that immunizes the living body by injection or oral administration to a human or animal to prevent infection. . In vivo immunization is largely divided into autoimmunity, in which immunity is automatically obtained after infection by pathogens, and passive immunity obtained by an externally injected vaccine. While autoimmunity is characterized by a long period of generation of antibodies related to immunity and continuous immunity, passive immunization with vaccines acts immediately to treat infectious diseases, but has a disadvantage of poor sustainability.
본 발명에서 용어, “면역원” 또는 “항원성 물질”은 펩티드, 폴리펩티드, 상기 폴리펩티드를 발현하는 유산균, 단백질, 상기 단백질을 발현하는 유산균, 올리고뉴클레오티드, 폴리뉴클레오티드, 재조합 박테리아 및 재조합 바이러스로 구성된 군에서 선택된 어느 하나인 것을 특징으로 할 수 있다. 구체적인 예를 들면, 상기 항원 물질은 불활성화된 전체 또는 부분 세포 제제 형태, 또는 통상적인 단백질 정제, 유전 공학 기법 또는 화학 합성에 의해 수득되는 항원 분자 형태일 수 있다.As used herein, the term “immunogen” or “antigenic substance” refers to a peptide, polypeptide, lactic acid bacteria expressing the polypeptide, protein, lactic acid bacteria expressing the protein, oligonucleotide, polynucleotide, recombinant bacteria and recombinant virus in the group consisting of It may be characterized in that it is any one selected. In specific embodiments, the antigenic substance may be in the form of an inactivated whole or partial cell preparation, or in the form of an antigen molecule obtained by conventional protein purification, genetic engineering techniques or chemical synthesis.
바람직하게 본 발명의 항원성 물질은 돼지에서 유행성 설사병을 유발하는 PEDV일 수 있고, 더욱 바람직하게는 상기 PEDV 유래 COE 단백질 및 spike 단백질의 C-말단에 존재하는 유전자 서열이 융합된 형태일 수 있으며, 가장 바람직하게는 서열번호 1의 아미노산 서열 또는 서열번호 1의 아미노산 서열 및 서열번호 4의 아미노산 서열이 융합된 형태의 항원일 수 있다. Preferably, the antigenic material of the present invention may be PEDV causing pancreatic diarrhea in pigs, more preferably, may be a fused form of the gene sequence present at the C-terminus of the PEDV-derived COE protein and spike protein, Most preferably, the antigen may be a fused form of the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 4.
상기 항원의 함량은 백신 조성물 총 중량에 대해서 1 내지 10 중량%일 수 있으며, 바람직하게는 5% 중량이내 일 수 있다.
The content of the antigen may be 1 to 10% by weight relative to the total weight of the vaccine composition, preferably 5% by weight or less.
본 발명에 따른 백신 조성물은 안정제, 유화제, 수산화알루미늄, 인산알루미늄, pH 조정제, 계면활성제, 리포솜, 이스콤(iscom) 보조제, 합성 글리코펩티드, 증량제, 카복시폴리메틸렌, 세균 세포벽, 세균 세포벽의 유도체, 세균백신, 동물 폭스바이러스 단백질, 서브바이랄(subviral) 입자 보조제, 콜레라 독소, N, N-디옥타데실-N',N'-비스(2-하이드록시에틸)-프로판디아민, 모노포스포릴 지질 A, 디메틸디옥타데실-암모늄 브로마이드 및 이의 혼합물로 구성된 군에서 선택된 어느 하나 이상의 제 2 보조제를 추가로 함유하는 것을 특징으로 할 수 있다.
Vaccine compositions according to the invention are stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylenes, bacterial cell walls, derivatives of bacterial cell walls, Bacterial vaccine, animal poxvirus protein, subviral particle adjuvant, cholera toxin, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium bromide and mixtures thereof may be further contained in at least one second adjuvant.
또한, 본 발명의 백신 조성물은 수의학적으로 허용 가능한 담체를 포함할 수 있다. 본 발명에서 용어, "수의학적으로 허용 가능한 담체"란 임의의 및 모든 용매, 분산 매질, 코팅제, 항원보강제, 안정제, 희석제, 보존제, 항균제 및 항진균제, 등장성 작용제, 흡착지연제 등을 포함한다. 백신용 조성물에 포함될 수 있는 담체, 부형제, 희석제로는 락토즈, 덱스트로스, 슈크로스, 솔비톨, 만니톨, 자일리톨, 말티톨, 전분, 글리세린, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘포스페이트, 칼슘실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.In addition, the vaccine composition of the present invention may comprise a veterinary acceptable carrier. As used herein, the term "veterinarily acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvant, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorptive delay agents and the like. Carriers, excipients, and diluents that may be included in the composition for vaccines include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
또한, 본 발명의 백신용 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 제제화할 경우에는 보통 사용되는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제할 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 레시틴 유사 유화제에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 슈크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용할 수 있다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등을 사용할 수 있으며, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수용성제, 현탁제, 유제, 동결건조제제가 포함된다. 비수용성제제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.
In addition, the vaccine composition of the present invention can be used in the form of oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions, respectively, according to conventional methods. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used can be prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate and sucrose in the lecithin-like emulsifier. (sucrose), lactose (lactose), gelatin, etc. can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc may also be used. As a liquid preparation for oral administration, suspending agents, liquid solutions, emulsions, syrups, etc. may be used, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations. As the non-aqueous preparation and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used.
또한 본 발명은, 상기 백신용 조성물을 인간을 제외한 동물에 주입하여 항원에 대한 항체 생성률을 높이는 방법에 관한 것이다.The present invention also relates to a method of increasing the antibody production rate against the antigen by injecting the vaccine composition into animals other than humans.
본 발명에서 용어, "인간을 제외한 동물"은 포유류 또는 조류인 것을 특징으로 할 수 있고, 상기 "주입"은 피하주사, 근육내 주사, 피하내 주사, 복막내 주사, 비강투여, 구강투여, 경피투여 및 경구투여로 구성된 군에서 선택된 어느 하나의 방법으로 수행되는 것을 특징으로 할 수 있다. 바람직하게 본 발명의 백신은 경구용 백신일 수 있다.In the present invention, the term "animal except human" may be characterized as a mammal or a bird, the "injection" is subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, transdermal It may be characterized in that it is carried out by any one method selected from the group consisting of administration and oral administration. Preferably the vaccine of the present invention may be an oral vaccine.
또한, 본 발명에서 용어, "주사" 또는 "투여"는 투여대상의 나이, 성별, 체중 등에 따라 투여량이 달라질 수 있고, 투여경로, 질병의 정도, 성별, 체중, 나이 등에 따라서도 백신의 투여량이 본 발명의 백신용 면역증강제는 비경구, 점막(경구 및 비강 등) 및 경피성 경로에 의한 백신 투여시 함께 사용할 수 있다. In the present invention, the term "injection" or "administration" may vary depending on the age, sex, and weight of the subject to be administered, and the dose of the vaccine also depends on the route of administration, the degree of disease, sex, weight, age, and the like. The immunoadjuvant for the vaccine of the present invention can be used together when administering the vaccine by parenteral, mucosal (oral and nasal, etc.) and transdermal routes.
본 발명의 일 실시예에서는 본 발명의 항원으로 제조된 백신을 PEDV 감염된 돼지에 투여하고, 설사지수, 항체역가, 및 해부를 통한 PEDV 감염여부를 확인한 결과, 본 발명의 PEDV 항원을 포함하는 백신을 투여한 그룹에서 설사증이 완화되었으며, 항체 역가도 높았고, 해부를 통한 육안검사에서도 PEDV 감염 증상이 완화되는 것을 확인하였다. 이러한 효과는, 본 발명의 PEDV 백신의 투여 및 상기 항원으로 형질전환된 형질전환 당근을 동시에 투여한 경우, 더욱 강화되는 것을 확인할 수 있었다(도 16 내지 19).
In one embodiment of the present invention, a vaccine prepared with the antigen of the present invention is administered to a PEDV infected pig, and diarrhea index, antibody titer, and confirmed whether PEDV infection through dissection, the vaccine containing the PEDV antigen of the present invention Diarrhea was relieved, antibody titers were high, and PEDV infection was alleviated by dissection gross examination. This effect, when the administration of the PEDV vaccine of the present invention and the transformed carrot transformed with the antigen at the same time was confirmed to be further enhanced (Fig. 16 to 19).
또 다른 하나의 양태로서, 본 발명은 본 발명의 형질전환체, 상기 형질전환체의 단백질 추출물 또는 상기 형질전환체로부터 분리된 재조합 PEDV 에피토프 단백질을 포함하는 PEDV 감염의 예방 또는 치료용 사료 조성물에 관한 것이다. As another aspect, the present invention relates to a feed composition for the prevention or treatment of a PEDV infection comprising a transformant of the present invention, a protein extract of the transformant or a recombinant PEDV epitope protein isolated from the transformant. will be.
본 발명에서 용어, “예방”은 본 발명의 PEDV 에피토프 단백질을 발현하는 형질전환체 또는 상기 형질전환체를 유효성분으로 하는 조성물의 투여로 상기 PEDV 감염을 억제 또는 지연시키는 모든 행위를 말한다.As used herein, the term "prevention" refers to any action that inhibits or delays the PEDV infection by administration of a transformant expressing the PEDV epitope protein of the present invention or a composition containing the transformant as an active ingredient.
본 발명에서 용어, “치료”는 본 발명의 PEDV 에피토프 단백질을 발현하는 형질전환체 또는 상기 형질전환체를 유효성분으로 하는 조성물의 투여로 상기 PEDV의 감염 증상이 호전되거나 이롭게 되는 모든 행위를 말한다.In the present invention, the term "treatment" refers to any action that improves or benefits the symptoms of infection of the PEDV by administration of a transformant expressing the PEDV epitope protein of the present invention or a composition comprising the transformant as an active ingredient.
본 발명에서 사용한 사료 조성물은 본 발명에 따른 사료 첨가제를 유효성분으로 포함하는 한 당업계에 공지된 다양한 형태의 조성비로 당업자에 의해 적절하게 구성될 수 있으며, 바람직하게는 단백질 20%, 지방, 에테르 추출물 4.5%, 지방, 산 가수분해 5.4%, 조섬유질 4.7%, 회분 6%, 칼슘 0.80% 및 인산염 0.62%를 포함하는 사료 조성물일 수 있다.
The feed composition used in the present invention may be appropriately configured by those skilled in the art in various forms of composition known in the art as long as it includes the feed additive according to the present invention as an active ingredient, preferably 20% protein, fat, ether It may be a feed composition comprising 4.5% extract, 5.4% fat, acid hydrolysis, 4.7% crude fiber, 6% ash, 0.80% calcium and 0.62% phosphate.
또 다른 양태로서, 본 발명은 본 발명의 형질전환체, 상기 형질전환체의 단백질 추출물 또는 상기 형질전환체로부터 분리된 재조합 PEDV 에피토프 단백질을 포함하는 PEDV 감염의 예방 또는 치료용 사료 첨가용 조성물에 관한 것이다. In still another aspect, the present invention relates to a composition for preventing or treating PEDV infection comprising a transformant of the present invention, a protein extract of the transformant or a recombinant PEDV epitope protein isolated from the transformant. will be.
본 발명의 사료 첨가용 조성물은 당업계에 공지된 다양한 형태로 제조될 수 있으며, 바람직하게는 PEDV 항원으로 형질전환된 형질전환체를 포함하는 것을 특징으로 하는 것인 사료 첨가제일 수 있다. The feed composition of the present invention may be prepared in various forms known in the art, it may be a feed additive that is characterized in that it comprises a transformant transformed with a PEDV antigen.
본 발명에 따른 사료 첨가제는 개별적으로 사용될 수 있고 종래 공지된 사료 첨가제와 병용하여 사용될 수 있고 종래의 사료첨가제와 순차적 또는 동시에 사용될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.
The feed additives according to the present invention can be used individually and in combination with conventionally known feed additives and can be used sequentially or simultaneously with conventional feed additives. And single or multiple administrations. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.
본 발명의 사료용 조성물을 적용할 수 있는 개체는 특별히 한정되지 않고, 어떠한 형태의 것이든 적용 가능하다. 예를 들면, 닭, 돼지, 원숭이, 개, 고양이, 토끼, 모르모트, 래트, 마우스, 소, 양, 염소 등과 같은 동물에 제한없이 적용가능하며, 본 발명의 일 실시예에 따르면 PEDV 감염 돼지의 사료 조성물로 첨가한 경우, 설사 지수가 완화되었으며, PEDV에 대한 항체 역가가 증가하였고, 해부를 통한 육안검사에서도 PEDV 감염 증세가 완화되는 것을 확인하였다.
The individual to which the composition for feed of the present invention can be applied is not particularly limited and may be applied in any form. For example, it is applicable without limitation to animals such as chickens, pigs, monkeys, dogs, cats, rabbits, morphotes, rats, mice, cows, sheep, goats, etc., according to one embodiment of the invention the feed of PEDV infected pigs When added as a composition, diarrhea index was alleviated, antibody titer to PEDV was increased, and visual inspection through anatomical analysis confirmed that the symptoms of PEDV infection were alleviated.
또 다른 양태로서, 본 발명은 PEDV 감염의 예방 또는 치료하는 방법에 관한 것이다. 구체적으로, 상기 PEDV 백신 조성물을 PEDV 감염의 예방 또는 치료가 필요한 개체에게 투여함으로써 PEDV 감염을 예방 또는 치료하는 방법에 관한 것이다. 또한, 상기 PEDV 감염의 예방 또는 치료용 사료 조성물 또는 사료 첨가용 조성물을 개체에 투여함으로써 PEDV 감염을 예방 또는 치료하는 방법을 포함한다.
In another aspect, the invention relates to a method of preventing or treating PEDV infection. Specifically, the present invention relates to a method for preventing or treating PEDV infection by administering the PEDV vaccine composition to a subject in need of prevention or treatment of PEDV infection. The present invention also includes a method for preventing or treating PEDV infection by administering to a subject a feed composition for preventing or treating PEDV infection or a composition for feed addition.
또 다른 하나의 양태로서, 본 발명은 (1) 서열번호 1의 PEDV 에피토프 단백질을 암호화하는 유전자를 발현하는 벡터를 제조하는 단계; (2) 상기 단계 (1)의 발현벡터를 아그로박테리아에 도입시키는 단계; 및 (3) 상기 단계 (2)의 아그로박테리아와 식물세포를 형질전환시키는 단계를 포함하는 PEDV 백신의 제조방법에 관한 것이다.In another embodiment, the present invention provides a method for preparing a vector comprising: (1) preparing a vector expressing a gene encoding a PEDV epitope protein of SEQ ID NO: 1; (2) introducing the expression vector of step (1) into Agrobacteria; And (3) transforming the agrobacteria and plant cells of step (2).
본 발명에서 용어 “PEDV” 및 “에피토프 단백질” 는 상기에서 설명한 바와 같다.In the present invention, the terms "PEDV" and "epitope protein" are as described above.
또한, 본 발명에서 용어, “작동가능하게 연결된”은 상기에서 설명한 바와 같다.In the present invention, the term “operably connected” is as described above.
상기 단계 (1)은 본 발명의 PEDV(porcine epidemic diarrhea virus) 에피토프 단백질을 암호화하는 유전자를 발현하는 벡터를 제조하는 단계에 관한 것이다. PEDV(porcine epidemic diarrhea virus) 에피토프 단백질을 암호화하는 유전자는 PEDV 항원성을 가진 서열을 포함하는 부위이면 제한없이 선택될 수 있으며, 바람직하게는 서열번호 2로 표시되는 염기서열일 수 있고, 더욱 바람직하게 상기 유전자는 서열번호 2의 염기 중 148번째 염기가 구아닌, 151번째 염기가 시토신으로 변형된 것인 서열번호 3으로 표시되는 염기서열일 수 있다. 또한, PEDV(porcine epidemic diarrhea virus) 에피토프 단백질을 암호화하는 유전자 및 spike 단백질의 C-말단의 단백질을 암호화하는 유전자 서열이 융합된 형태의 유전자를 발현하는 벡터일 수 있고, PEDV(porcine epidemic diarrhea virus) 에피토프 단백질을 암호화하는 유전자는 상기에서 설명한 바와 같다. spike 단백질의 C-말단의 단백질을 암호화하는 유전자는 항원성을 나타내는 서열이면 제한없이 포함될 수 이으며, 바람직하게 상기 유전자는 서열번호 5로 표시되는 염기서열일 수 있다.
The step (1) relates to a step of preparing a vector expressing a gene encoding a PPEV epicinemic epitope protein of the present invention. The gene encoding a PEDV epitope protein may be selected without limitation as long as the site includes a sequence having a PEDV antigenicity, preferably the nucleotide sequence represented by SEQ ID NO: 2, and more preferably. The gene may be a nucleotide sequence represented by SEQ ID NO: 3, wherein the 148th base is guanine and the 151th base is modified with cytosine. In addition, it may be a vector expressing a gene in a fused form of the gene encoding the epicinemic epidemic diarrhea virus (PEDV) epitope protein and the gene sequence encoding the protein at the C-terminal end of the spike protein, and may be a porcine epidemic diarrhea virus (PEDV). Genes encoding epitope proteins are as described above. Gene encoding the C-terminal protein of the spike protein may be included without limitation so long as it shows an antigenic sequence, preferably the gene may be a nucleotide sequence represented by SEQ ID NO: 5.
상기 단계 (2) 및 단계 (3)은 단계 (1)의 발현벡터를 아그로박테리아에 도입시키는 단계; 및 아그로박테리아와 식물세포를 형질전환시키는 단계를 포함하는 PEDV 백신 제조방법에 관한 것이다.Step (2) and step (3) comprises the steps of introducing the expression vector of step (1) into Agrobacteria; And it relates to a method for producing a PEDV vaccine comprising the step of transforming agrobacteria and plant cells.
본 발명의 발현벡터를 아그로박테리아에 형질도입시키는 방법은 당업계에 공지된 방법을 당업자에 의해 적절하게 변형되어 적용될 수 있으며, 바람직하게 본 발명에서는 아그로박테리아 튜머파시엔스 GV3101에 본 발명의 재조합 벡터를 freeze-thaw 방법을 이용하여 형질전환하였다. 또한, 상기 백신제조방법은 당업계에 공지된 백신의 제조방법에 따라, 본 발명의 항원 조성물을 적절한 중량 %로 첨가하여 제조할 수 있으며, 바람직하게는 백신 조성물 총 중량에 대해서 1 내지 10 중량%일 수 있으며, 바람직하게는 5% 중량이내 일 수 있다.
The method for transducing the expression vector of the present invention to Agrobacteria may be appropriately applied by those skilled in the art by methods known in the art. Preferably, in the present invention, the recombinant vector of the present invention may be used in Agrobacteria tumerfaciens GV3101. Transformation was performed using the freeze-thaw method. In addition, the vaccine manufacturing method can be prepared by adding the appropriate weight% of the antigenic composition of the present invention, according to the preparation of vaccines known in the art, preferably 1 to 10% by weight relative to the total weight of the vaccine composition It may be, preferably less than 5% by weight.
또 다른 하나의 양태로서, 본 발명은 본 발명의 에피토프 단백질에 대한 항체에 관한 것이다.In another aspect, the present invention relates to an antibody against the epitope protein of the present invention.
본 발명에서 용어, “항체”는 면역계 내에서 항원의 자극에 의하여 만들어지는 물질로서, 면역글로불린이라고도 하며, 특정한 항원과 특이적으로 결합하여 림프와 혈액을 떠돌며 항원항체반응을 일으킨다. 항체가 특이적 항원에 대한 특이성을 나타내는 반면에, 면역글로불린은 항체, 및 항원 특이성이 결여된 항체 유사 물질 모두를 포함한다. 후자의 폴리펩티드는 예컨대 림프계에서는 낮은 수준으로 생산되며 골수종에 의해서 증가된 수준으로 생산된다. 본 발명에서는 PEDV 에피토프 단백질의 일부를 포함하는 유전자 서열 또는 상기 유전자 서열 및 spike 단백질의 C-말단의 일부를 포함하는 유전자 서열이 융합된 형태의 항원에 대한 항체일 수 있으며, 바람직하게는 서열번호 2의 유전자 서열 또는 서열번호 2 또는 3이 서열번호 5의 유전자 서열과 융합된 형태의 항원에 대한 항체일 수 있다.
In the present invention, the term "antibody" is a substance produced by the stimulation of the antigen in the immune system, also called immunoglobulin, and specifically binds to a specific antigen, floating the lymph and blood, causing an antigen-antibody reaction. While antibodies exhibit specificity for specific antigens, immunoglobulins include both antibodies and antibody-like substances lacking antigen specificity. The latter polypeptide is produced at low levels, for example in the lymphatic system, and at elevated levels by myeloma. In the present invention, the antibody may be a gene sequence comprising a portion of the PEDV epitope protein or a gene in which the gene sequence and a gene sequence including a portion of the C-terminus of the spike protein are fused to each other, preferably SEQ ID NO: 2 The gene sequence of or SEQ ID NO: 2 or 3 may be an antibody against the antigen in the form fused with the gene sequence of SEQ ID NO: 5.
본 발명의 PEDV 에피토프 단백질이 형질전환된 식물 형질전환체는 경구 백신으로 사용하여 PEDV에 대하여 중화활성을 가지는 항체형성을 유도할 수 있어 PEDV 감염을 예방할 수 있다. 또한 아직 조직이 형성되기 전의 상태이기 때문에 도입된 외래유전자 발현에 의해 식물생장에 영향을 받을 수 있는 문제를 해결 할 수 있는 장점이 있다. 아울러 본 발명의 경구 백신은 동물성 바이러스 오염 가능성이 낮아 안전하며, 식물 형질전환체의 장기간 보관 및 이동이 가능하여 백신 제조가 용이할 뿐만 아니라 경구투여로 주사접종으로 인한 비용소모를 대폭 절감시킬 수 있다.
The plant transformant transformed with the PEDV epitope protein of the present invention can be used as an oral vaccine to induce antibody formation having neutralizing activity against PEDV, thereby preventing PEDV infection. In addition, there is an advantage that can solve the problem that can be affected by the plant growth by the introduction of foreign gene expression because it is still before the tissue is formed. In addition, the oral vaccine of the present invention is safe due to the possibility of animal virus contamination, and it is possible to store and transport plant transformants for a long time, thus facilitating vaccine production and greatly reducing the cost of injection due to oral administration. .
도 1은 PCR 반응으로 얻은 COE 항원 유전자(430bp) 산물을 전기영동으로 확인한 결과를 나타낸 것이다(A). 또한 Topo-vector에 PCR 산물이 삽입되었는지 여부를 나타낸 것이다(B).
도 2는 NCBI blast 검색을 통해 증폭된 COE 항원 유전자의 얼라이먼트(Alignmnet)를 나타낸 것이다.
도 3은 엑스파시(expasy)에 의한 PEDV 내 아미노 서열의 다중 얼라인먼트를 나타내는 도이다.
도 4는 위치-특이적 돌연변이 COE(mCOE) 항원 유전자의 1% 아가로스 겔 전기영동 및 pENTR/D/TOPO-COE 플라스미드 DNA에 의해 증폭된 후 GPRLQPY 아미노 서열(mCOEGY) 항원 유전자(469bp) 재조합체를 나타내는 도이다(A). BglⅡ 및 EcoRⅤ으로 절단된 2.7Kb 및 0.28Kb의 두 가지 산물의 Topo-벡터(게이트웨이) 내로 mCOE의 삽입을 나타내는 도이다(B). NotⅠ및 BglⅡ로 절단된 2.7Kb 및 0.32Kb의 두 가지 산물의 Topo-벡터(게이트웨이) 내로 mCOE의 삽입을 나타내는 도이다(C).
도 5는 항원(mCOE, mCOEGY)의 구조 및 이를 아그로박테리움 튜머파시엔스 GV3101에 형질전환시키는 과정을 모식도로 나타낸 것이다.
도 6은 아가로즈 겔 전기영동 결과를 나타낸 것이다. pK2GW7-mCOE 및 pK2GW7-mCOEGY의 밴드를 확인하였다.
도 7은 아그로박테리움 튜머파시엔스 GV3101 내 mCOE(430bp)(A) 및 mCOEGY(469bp)(B)유전자 콜로니를 PCR로 확인한 결과이다.
도 8은 대장균에서 mCOEGY 항원의 발현을 나타낸 것이다.
도 9는 대장균에서 mCOEGY 단백질이 발현됨을 웨스턴 블롯으로 확인한 것이다(A). 또한 mCOEGY 항혈청의 웨스턴 블롯 결과를 나타낸 것이다(B).
도 10은 형질전환된 당근 캘러스에서 게놈 DNA PCR로 항원 유전자를 확인한 것이다. mCOE 430bp, mCOEGY 469bp.
도 11은 mCOE, mCOEGY로 형질전환된 당근 캘러스 유전자의 RT-PCR 분석을 나타내는 결과이다.
도 12는 형질전환 당근 캘러스의 SDS-PAGE 및 웨스턴 블롯 분석을 나타낸 결과이다. SDS-PAGE; 15% 아크릴아믹 겔, 50ug 전체 단백질 로딩양, C, 형질전환되지 않은 당근 캘러스; M, 저분자 단백질 마커; 1~5, COE J 1 형질전환 캘러스. mCOEGY 다클론 항체를 이용한 형질전환 당근 캘러스의 웨스턴 분석 결과를 나타낸 것이다. C, 형질전환되지 않은 당근 캘러스; M, 전체 범위의 레인보우 마커(rainbow marker); 1~5, COE J 1 형질전환 캘러스.
도 13은 마우스 경구 면역 반응 실험에서 시간별 실험 내용을 나타낸 것이다.
도 14는 마우스 혈청의 mCOEGY 항체의 ELISA 측정값을 나타내는 결과이다. (A) IgG. (B) IgA. 공통적인 윗첨자를 제외한 열의 평균은 유의적인 차이를 나타낸다(*P<0.05).
도 15는 PEDV 형질전환 당근 백신의 돼지 경구 면역 반응 실험을 하기 위한 시간별 실험 내용을 나타낸 것이다.
도 16은 PEDV 또는 PEDV 및 mCOEJ1 당근 캘러스를 경구투여한 후 시간 경과에 따른 설사지수를 나타낸 것이다. 이는 형질전환된 당근으로 사육한 모돈에서 태어난 자돈의 보호면역반응을 나타내는 결과이다. NV-C; 기본 식이 및 PEDV 공격접종, V-C; mCOEJ1 당근 캘러스 및 PEDV 공격접종. 공통적인 윗첨자를 제외한 열의 평균은 유의적인 차이를 나타낸다(*P<0.1, **P<0.05).
도 17은 자돈 혈청 IgG에서 mCOEGY 항체의 ELISA 측정값을 나타내는 결과이다. 공통적인 윗첨자를 제외한 열의 평균은 유의적인 차이를 나타낸다(*P<0.05).
도 18은 자돈 혈청 IgA에서 mCOEGY 항체의 ELISA 측정값을 나타내는 결과이다. 공통적인 윗첨자를 제외한 열의 평균은 유의적인 차이를 나타낸다(*P<0.05).
도 19는 PDE 생벡신의 근육주사 및 형질전환된 당근 캘러스 mCOEJ1를 경구투여한 그룹에서 소장의 상태를 나타내는 결과이다.Figure 1 shows the results confirmed by electrophoresis of the product COE antigen gene (430bp) obtained by the PCR reaction (A). It also shows whether the PCR product is inserted into the topo-vector (B).
Figure 2 shows the alignment (Alignmnet) of the COE antigen gene amplified by NCBI blast search.
FIG. 3 shows multiple alignments of amino sequences in PEDV by expasy.
Figure 4 shows GPRLQPY amino sequence (mCOEGY) antigen gene (469 bp) recombinant after amplification by 1% agarose gel electrophoresis of the site-specific mutant COE (mCOE) antigen gene and pENTR / D / TOPO-COE plasmid DNA. (A) which shows. Figure B shows the insertion of mCOE into the Topo-vector (gateway) of two products of 2.7 Kb and 0.28 Kb digested with BglII and EcoRV (B). Figure (C) shows the insertion of mCOE into the Topo-vector (gateway) of two products of 2.7 Kb and 0.32 Kb cleaved with NotI and BglII.
Figure 5 shows the structure of the antigen (mCOE, mCOEGY) and a process for transforming it into Agrobacterium tumer faciens GV3101.
Figure 6 shows agarose gel electrophoresis results. The bands of pK2GW7-mCOE and pK2GW7-mCOEGY were identified.
Figure 7 shows the results of PCR confirmed the mCOE (430bp) (A) and mCOEGY (469bp) (B) gene colonies in Agrobacterium tumer faciens GV3101.
8 shows the expression of mCOEGY antigen in E. coli.
Figure 9 confirms the expression of mCOEGY protein in Escherichia coli by Western blot (A). It also shows the Western blot results of mCOEGY antiserum (B).
10 confirms the antigenic gene by genomic DNA PCR in the transformed carrot callus. mCOE 430 bp,
11 is a result showing the RT-PCR analysis of carrot callus gene transformed with mCOE, mCOEGY.
12 shows the results of SDS-PAGE and Western blot analysis of transformed carrot callus. SDS-PAGE; 15% acrylamide gel, 50 ug total protein loading, C, untransformed carrot callus; M, low molecular weight protein markers; 1-5,
Figure 13 shows the hourly experimental content in the mouse oral immune response experiments.
14 shows results of ELISA measurements of mCOEGY antibodies in mouse serum. (A) IgG. (B) IgA. The mean of the columns except common superscripts shows a significant difference (* P <0.05).
Figure 15 shows the contents of the hourly experiments for porcine oral immune response experiments of PEDV transgenic carrot vaccine.
Figure 16 shows the diarrhea index over time after oral administration of PEDV or PEDV and mCOEJ1 carrot callus. This is a result of the protective immune response of piglets born from sows raised with transformed carrots. NV-C; Basic dietary and PEDV challenge, VC; mCOEJ1 carrot callus and PEDV challenge. The mean of the columns except common superscripts shows a significant difference (* P <0.1, ** P <0.05).
17 shows the results of ELISA measurements of mCOEGY antibodies in piglet serum IgG. The mean of the columns except common superscripts shows a significant difference (* P <0.05).
18 shows the results of ELISA measurements of mCOEGY antibodies in piglet serum IgA. The mean of the columns except common superscripts shows a significant difference (* P <0.05).
19 is a result showing the state of the small intestine in the group injected with oral administration of intramuscular injection of transformed carrot callus mCOEJ1 and PDE Saint Bexin.
이하, 하기 실시예를 통하여 본 발명을 더욱 상세하게 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.
Hereinafter, the present invention will be described in more detail with reference to the following examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.
실시예 1: PEDV 단일 항원유전자의 클로닝Example 1 Cloning of PEDV Single Antigen
1-1) Virus RNA 추출 및 cDNA 합성1-1) Virus RNA Extraction and cDNA Synthesis
PEDV의 생독백신(KPEDV-9, 고려비앤피 사)에서 QIAamp MinEluteVirus Spin Kit(Qiagen 사)를 이용하여 RNA를 추출하였다. 추출한 1㎍ RNA 및 50 pmol Anchored-oligo(dT)18 프라이머 2㎕와 멸균 증류수를 혼합하여 총 13㎕의 용액을 만든 후 65℃에서 10분간 반응시켰다. 그런 후, 5X 역전사 효소 반응 완충액 4㎕, 40U 단백질 분해효소 억제제 0.5㎕, 10mM dNTP(Deoxynucleotide) mix 2㎕, 20U 역전사 효소 0.5㎕(Transcriptor First Strand cDNA Synthesis Kit, Roche 사)을 더하여 50℃에서 1시간, 85℃에서 5분간 PCR을 이용하여 cDNA를 합성하였다.
RNA was extracted from the live vaccine of PEDV (KPEDV-9, Korea B & P) using QIAamp MinEluteVirus Spin Kit (Qiagen). 2 μl of the extracted 1 μg RNA and 50 pmol Anchored-oligo (dT) 18 primer and sterile distilled water were mixed to make a total of 13 μl of solution, followed by reaction at 65 ° C. for 10 minutes. Then, add 4 µl of 5X reverse transcriptase reaction buffer, 0.5 µl of 40U protease inhibitor, 2 µl of 10 mM dNTP (Deoxynucleotide) mix, and 0.5 µl of 20U reverse transcriptase (Transcriptor First Strand cDNA Synthesis Kit, Roche). CDNA was synthesized using PCR for 5 minutes at 85 ° C.
1-2) 프라이머 합성1-2) Primer Synthesis
돼지 유행성 설사병 바이러스(PEDV) spike 단백질의 일부이며 항원성이 있다고 알려진 COE(CO-26K fragment equivalent, 15KDa)유전자와 spike 단백질의 c-terminal에 존재하는 아미노산서열(GPRLQPY)을 항원으로 선택하고 NCBI (National Center for Biotechnology Information, www.ncbi.nlm.gov/) 의 gene bank 검색을 토대로 프라이머(서열번호 6 내지 13)를 작성하였다(표 1). Part of the swine epidemic diarrheal virus (PEDV) spike protein, known as antigenic, is the COE (CO-26K fragment equivalent, 15KDa) gene and the amino acid sequence (GPRLQPY) present in the c-terminal of the spike protein. Primers (SEQ ID NOS: 6-13) were prepared based on gene bank searches of the National Center for Biotechnology Information, www.ncbi.nlm.gov/) (Table 1).
1-3) PCR 클로닝1-3) PCR Cloning
PEDV의 cDNA에서 COE(CO 26K equivalent) 유전자를 얻기 위하여 다음과 같은 조건에서 PCR을 수행하였다; 1ug PEDV cDNA, 10X pfu 반응 완충액 5㎕, 10mM dNTP 1㎕, 100pmol PEDs 5' 프라이머(서열번호 6) 1㎕, 100pmol PEDs 3' 프라이머(서열번호 7) 1㎕, 2.5U pfu 폴리머라제 0.5㎕(solgent 사); 95℃ 2분, 95℃ 20초, 60℃ 40초, 72℃ 2분, 72℃ 5분, 35cycle. PCR was performed under the following conditions to obtain a COE (CO 26K equivalent) gene in the cDNA of PEDV; 1 ug PEDV cDNA, 5 μl of 10 × pfu reaction buffer, 1 μl of 10 mM dNTP, 1 μl of 100 pmol
얻어진 PCR 산물은 1% TAE 아가로스젤을 이용한 전기영동을 통해 확인하였다(도 1A). 얻어진 밴드는 pENTRTM/D-TOPO(invitrogen 사)에 삽입하여 TOP10에 형질전환하였다. 플라스미드 DNA를 분리하고, Bgl II와 EcoR I 제한효소를 이용하여 3개의 콜로니에서 예상된 252bp와 2752bp크기의 밴드를 확인하였다(도 1B). 확인된 3개의 플라스미드 DNA를 염기서열 분석 의뢰하여 KPEDV-9 균주의 COE 유전자의 염기서열 정보를 확인하였다. KPEDV-9 COE 유전자의 염기서열 정보를 토대로 NCBI BLAST를 한 결과 다른 PEDV의 바이러스주들과 95% 이상의 상동성을 가짐을 알 수 있었다. 또한 KPEDV-9 COE 유전자와 다른 바이러스주의 spike 단백질 지역들의 아미노산서열을 alignment 비교분석하여 변이 지역(variable region)을 확인하였다(도 2 및 3).The obtained PCR product was confirmed by electrophoresis using 1% TAE agarose gel (Fig. 1A). The obtained band was inserted into pENTR ™ / D-TOPO (invitrogen) and transformed into TOP10. Plasmid DNA was isolated and identified the bands of 252bp and 2752bp expected in three colonies using Bgl II and EcoR I restriction enzymes (FIG. 1B). Three plasmid DNAs were confirmed by sequencing and confirmed nucleotide sequence information of the COE gene of the KPEDV-9 strain. NCBI BLAST based on the sequence information of KPEDV-9 COE gene showed more than 95% homology with other PEDV virus lines. In addition, the alignment region of the KPEDV-9 COE gene and the spike protein regions of other virus strains was compared and identified for the variable region (FIGS. 2 and 3).
또한 COE 유전자의 mRNA를 불안정하게 하는 ATTTA 염기서열을 당근 코돈 선호도에 맞춰 GTTCA로 위치-특이적 돌연변이하기 위하여 PCR을 다음과 같은 조건으로 수행하였다; 20ng pENTR/D /TOPO-COE, 10X fu 반응 완충액 5㎕, 10mM dNTP 1㎕, 100pmol PEDs 5' 프라이머(서열번호 6) 1㎕, 100pmol PEDs(mut) 5' 프라이머(서열번호 8) 1㎕, 100pmol PEDs(mut) 3' 프라이머(서열번호 7) 1㎕, 100pmol PEDs 3' 프라이머(서열번호 9) 1㎕ 및 2.5U pfu 폴리머라제 0.5㎕; 95℃ 2분, 95℃ 20초, 58℃ 40초, 72℃ 2분, 72℃ 5분, 35cycle. In addition, PCR was performed under the following conditions in order to position-specific mutation of the ATTTA sequence destabilizing mRNA of the COE gene to GTTCA according to the carrot codon preference; 20 ng pENTR / D / TOPO-COE, 5 μl 10X fu reaction buffer, 1
mCOE(돌연변이된 COE) PCR 산물은 1% TAE 아가로스젤에 전기영동한 후 HiYieldTMGel/PCR DNA 추출 키트(RBC 사)를 이용하여 DNA를 얻었고, 이를 pENTR/D/TOPO (invitrogen사)에 라이게이션 후 Not I과 Bgl II 제한효소로 절단하여 전기영동으로 확인하였다. 그런 후 코스모진텍에 염기서열분석을 의뢰하여 돌연변이된 염기서열을 alignment (http://www.expasy.ch/)를 통해 확인하였다.The mCOE (mutated COE) PCR product was electrophoresed on 1% TAE agarose gel and DNA was obtained using HiYield TM Gel / PCR DNA extraction kit (RBC), which was then transferred to pENTR / D / TOPO (invitrogen). After ligation, digestion with Not I and Bgl II restriction enzymes was confirmed by electrophoresis. Subsequently, the sequencing of the sequencing was requested to Cosmojintech to confirm the mutated nucleotide sequence through alignment (http://www.expasy.ch/).
또한 mCOE 유전자에 GPRLQPY (GGTCCTAGACTTCAACCTTAC; 서열번호 5)을 재조합하기 위하여 다음과 같은 조건에서 PCR 하였다; 20ng pENTR/D/TOPO-mCOE, 10X fu 반응 완충액 5㎕, 10mM dNTP 1㎕, 100pmol COE(fus) 5' 프라이머(서열번호 10) 1㎕, 100pmol COE(fus) 3' 프라이머(서열번호 11) 1㎕, 100pmol COE(c-ter) 5' 프라이머(서열번호 12) 1㎕, 100pmol COE(c-ter) 3' 프라이머(서열번호 13) 1㎕ 및 2.5U pfu 폴리머라제 0.5㎕; 95 2분, 95℃ 20초, 58℃ 40초, 72℃ 2분, 72℃ 5분, 35cycle. In addition, PCR was performed under the following conditions to recombine GPRLQPY (GGTCCTAGACTTCAACCTTAC; SEQ ID NO: 5) to the mCOE gene; 20ng pENTR / D / TOPO-mCOE, 5μl 10X fu reaction buffer, 1μl 10mM dNTP, 100pmol COE (fus) 5 'primer (SEQ ID NO: 10) 1μl, 100pmol COE (fus) 3' primer (SEQ ID NO: 11) 1 μl, 1
mCOEGY(돌연변이된 COE GPRLQPY 아미노산 염기서열) PCR 산물은 1% TAE 아가로스젤에 전기영동한 후 HiYieldTMGel/PCR DNA Extraction Kit(RBC 사)를 이용하여 DNA를 얻었고 이를 pENTR/D/TOPO (invitrogen사)에 ligation 후 Bgl II와 EcoR V 제한효소로 절단하여 전기영동으로 확인하였다(도 4A). 그런 후 코스모진텍에 염기서열 분석의뢰하여 GPRLQPY 아미노산 서열(서열번호 4)이 재조합된 것을 alignment (http://www.expasy.ch/)를 통해 확인하였다.The mCOEGY (mutated COE GPRLQPY amino acid sequence) PCR product was subjected to electrophoresis on 1% TAE agarose gel, and then obtained DNA using HiYield TM Gel / PCR DNA Extraction Kit (RBC), which was then converted into pENTR / D / TOPO (invitrogen). G) After ligation, it was confirmed by electrophoresis by cutting with Bgl II and EcoR V restriction enzymes (FIG. 4A). Subsequently, a sequencing request was made to Cosmojintech to confirm that the GPRLQPY amino acid sequence (SEQ ID NO: 4) was recombined through alignment (http://www.expasy.ch/).
아가로스 젤에서 분리한 mCOE와 mCOEGY 유전자를 pENTR/D/TOPO 벡터에 삽입하여 TOP10에 형질전환하고 항생제 배지에서 선발된 콜로니에서 플라스미드 DNA를 추출하였고 pENTR/D/TOPO-mCOE는 BglII와 EcoRV 제한효소 처리 후 예상된 289bp와 2717bp 밴드를 확인하였고 염기서열 분석하여 9번을 최종선발하였다(도 4B).MCOE and mCOEGY genes isolated from agarose gels were inserted into pENTR / D / TOPO vector, transformed to TOP10, plasmid DNA extracted from colonies selected from antibiotic medium, and pENTR / D / TOPO-mCOE were BglII and EcoRV restriction enzymes. After the treatment, the expected 289bp and 2717bp bands were identified and 9 was finally selected by sequencing (FIG. 4B).
pENTR/D/TOPO-mCOEGY는 NotI과 BglII 제한 효소를 처리하여 320bp와 2725bp의 예상된 밴드를 확인하였고 염기서열 분석결과 3번의 콜로니가 최종 선발되었다(도 4C).
pENTR / D / TOPO-mCOEGY was treated with NotI and BglII restriction enzymes to confirm the expected bands of 320bp and 2725bp, and 3 colonies were finally selected by sequencing analysis (FIG. 4C).
실시예 2: 식물형질전환용 벡터 구축 및 Example 2: vector construction for plant transformation and AgrobacteriaAgrobacteria 형질전환 Transformation
클로닝된 mCOE와 mCOEGY 유전자를 식물 형질전환용 벡터인 pK2GW7.0에 삽입한 후 TOP10 세포(invitrogen 사)에 형질전환 하였다. 스펙티노마이신 100㎍/㎕ 항생제가 포함된 LB배지에서 선발한 콜로니로 클로닝 여부를 확인하였으며 이를 Freeze-Thaw 방법(안ㆍ남 1999)을 통하여 아그로박테리움 튜머파시엔스(Agrobacterium tumefaciens) GV3101에 형질전환하였다(도 5). Cloned mCOE and mCOEGY genes were inserted into pK2GW7.0, a plant transformation vector, and transformed into TOP10 cells (invitrogen). Cloning was confirmed by colonies selected from LB medium containing 100 μg / μl antibiotics of spectinomycin and transformed into Agrobacterium tumefaciens GV3101 by Freeze-Thaw method (Ahn, South 1999). (FIG. 5) .
pK2GW7-mCOE와 pK2GW7-mCOEGY를 Bgl II와 Hind III 제한효소를 처리하여 각각 407bp와 446bp의 예상된 밴드를 확인하였다(도 6). 제한 효소 처리에 의해 유전자 삽입이 확인된 pK2GW7 플라스미드 DNA를 아그로박테리움 튜머파시엔스(Agrobacterium tumefaciens) GV3101에 형질전환하고 항생제 배지에서 선발된 콜로니를 주형으로 PCR을 이용하여 유전자를 증폭하고, 전기영동을 통해 확인하였다. pK2GW7-mCOE는 430bp, pK2GW7-mCOEGY는 469bp의 colony를 50배 희석한 주형을 이용하여 PCR로 삽입을 확인하였다(도 7A 및 B).
pK2GW7-mCOE and pK2GW7-mCOEGY were treated with Bgl II and Hind III restriction enzymes to confirm the expected bands of 407bp and 446bp, respectively (Figure 6). PK2GW7 plasmid DNA whose gene insertion was confirmed by restriction enzyme treatment was transformed into Agrobacterium tumefaciens GV3101, and colonies selected from antibiotic medium were amplified by PCR using PCR as a template, and electrophoresis was performed. It was confirmed through. pK2GW7-mCOE was 430bp, pK2GW7-mCOEGY was confirmed by PCR using a template diluted 50-fold colony of 469bp (Fig. 7A and B).
실시예 3: Example 3: AgrobacteriaAgrobacteria 를 이용한 당근 형질전환Transformation with Carrot
당근 형질전환을 위해 조춘 5촌(J종묘, H종묘), 춘홍 5촌(S종묘) 품종을 사용하였다. 당근 종자는 70% EtOH 로 3번 씻고 4% 유한락스로 30분간 소독한 후 멸균수를 이용하여 5~6번 씻어 준비하였다. 멸균 소독한 당근 종자를 0.8% Agar 배지에 치상하여 25℃ 암조건에서 7일간 발아 시킨 배축을 0.3~0.5㎝ 간격으로 자른 후 MS+배지 (4.4g/l MS 염 w/비타민 (Duchefa 사), 0.1mg/l 2iP, 1mg/l 2,4-D, 3% 수크로스)에서 1~2일간 진탕배양 하였다. 2일간 M9배지 (6g/l Na2HPO4, 3g/l KH2PO4, 0.5g/l NaCl, 1g/l NH4Cl, 0.5g/l MgSO4, 2g/l 글루코스, 0.015g/l CaCl2)에 항생제 (100㎍/㎕ 스펙티노마이신)를 첨가한 배지에서 키운 아그로박테리아(Agrobacteria)를 MS+배지에 재현탁하여 2일간 배양한 당근 배축과 암조건에서 5~10분간 공동배양한 후 식물절편을 멸균된 종인에 옮겨 공기 건조(air dry)한 후 액체 MS+배지에서 하루 현탁배양(25, 100rpm, 암조건)하여 카베니실린 400㎍/㎕와 카나마이신 100㎍/㎕이 포함된 고체 MS+배지에 옮긴 후 캘러스를 유도하였다. For the carrot transformation,
형질전환된 아그로박테리아 GV3101을 이용하여 2품종 당근의 절편과 공동배양한 후, 당근 절편에 남아있는 아그로박테리아를 제거하기 위하여 1차 선발배지에서 캘러스를 유도하고 2차 선발배지에서 총 87을 계통화 하였다(mCOE 30 계통, mCOEGY 57계통). 당근 품종별 형질전환 효율을 분석한 결과 조춘 품종의 형질전환이 높았다(표 2).After coculture with slices of two varieties of carrots using transformed Agrobacterium GV3101, the callus was induced in the primary selection medium and the total of 87 in the secondary selection medium to remove the Agrobacteria remaining in the carrot slices.
실시예 4: PEDV 항원단백질(mCOEGY) 생산 및 항체 생산Example 4: PEDV Antigen Protein (mCOEGY) Production and Antibody Production
4-1) 항원단백질 발현 벡터 구축4-1) Construction of Antigen Protein Expression Vector
항원유전자인 mCOEGY를 리보솜 결합 부위(RBS)가 있는 게이트웨이 시스템(Gateway system)을 이용할 수 있는 pENTRTM/SD/D-TOPO(invitrogen 사)에 클로닝하고 LR clonase II(invitrogen사)를 사용하여 E. coli 발현 벡터인 GatewayDEST14TMvector에 삽입하고 이를 BL21-AlTM(invitrogen 사)에 형질전환 하고 제한효소로 확인하였다(도 8A). 확인된 콜로니에서 플라스미드 DNA를 추출하여 데스티네이션 벡터(destination vector)인 pDEST14 벡터에 LR 반응시켰다. 그런 후 TOP10과 BL21에 형질전환하여 항생제에서 선발된 콜로니에서 플라스미드를 추출하였고, 제한효소를 이용하여 클로닝을 확인하였다(도 8B 및 C).
The antigenic gene, mCOEGY, was cloned into pENTR ™ / SD / D-TOPO (invitrogen) using a gateway system with a ribosomal binding site (RBS) and LR clonase II (invitrogen) using E. coli . The coli expression vector, GatewayDEST14 ™, was inserted into the vector and transformed into BL21-Al ™ (Invitrogen) and confirmed by restriction enzymes (FIG. 8A). Plasmid DNA was extracted from the identified colonies and subjected to LR reaction with pDEST14 vector, which is a destination vector. Then, plasmids were extracted from colonies selected from antibiotics by transforming to TOP10 and BL21, and cloning was confirmed using restriction enzymes (FIGS. 8B and C).
4-2) 대장균 항원 발현 및 분리4-2) E. Coli Antigen Expression and Isolation
pDEST14-mCOEGY가 있는 BL21의 단일 콜로니를 항생제(100㎍/㎕ 암피실린)가 있는 LB 5ml에 접종하여 밤새 키운 후 50ml LB배지에 1/25비율로 재접종하여 OD600nm=~0.4 될 때까지 키워 20% L-아라비노스를 최종 농도가 0.2% 되게 처리하고 4시간동안 키웠다. 4시간동안 키운 대장균을 5000rpm으로 원심분리하여 침전물만을 수확하여 40ml 의 2X 샘플 완충액을 넣고 10분간 끓인 후 SDS-PAGE로 확인하였다.Single colonies of BL21 with pDEST14-mCOEGY were inoculated in 5 ml of LB with antibiotics (100 μg / μl ampicillin) and grown overnight, then reinoculated in 50 ml LB medium at 1/25 ratio until OD600nm = ~ 0.4 20% L-arabinose was treated to a final concentration of 0.2% and grown for 4 hours. E. coli grown for 4 hours was centrifuged at 5000rpm to harvest only the precipitate, 40ml of 2X sample buffer, boiled for 10 minutes and confirmed by SDS-PAGE.
대량 생산한 단백질은 SDS-PAGE를 통하여 크기별로 분리한 후 16.6KDa 밴드를 막자사발을 이용하여 곱게 분쇄한 후 확산 버퍼(diffusion buffer)(50mM Tris-Cl pH8.0, 0.1% SDS)에 넣어서 4℃에서 2일간 진탕하여 젤로부터 단백질이 나오게 하였다. 4℃, 5000rpm, 30분간 원심분리하여 상층액만을 회수하였고 4배의 아세톤을 넣어 -20℃에서 3시간 이상 침전시킨 후 다시 원심분리하여 침전물을 PBS(Phosphate-Buffer Saline) 완충액에 녹였다. 얻어진 단백질은 BCA방법(Bicinchoninic Acid Protein Assay Kit, Sigma사)에 의하여 정량하였다. 그 결과과 16.6KDa의 mCOEGY 단백질 발현을 확인할 수 있었다(도 9A).
Mass-produced proteins were separated by size through SDS-PAGE, and finely crushed 16.6KDa bands using a mortar and pestle, and then placed in diffusion buffer (50mM Tris-Cl pH8.0, 0.1% SDS). The protein was released from the gel by shaking for 2 days at < RTI ID = 0.0 > The supernatant was recovered by centrifugation at 4 ° C., 5000 rpm for 30 minutes, and 4 times of acetone was added to precipitate at −20 ° C. for at least 3 hours, followed by centrifugation to dissolve the precipitate in PBS buffer. The obtained protein was quantified by BCA method (Bicinchoninic Acid Protein Assay Kit, Sigma). As a result, it was possible to confirm the expression of mCOEGY protein of 16.6 KDa (FIG. 9A).
4-3) 4-3) PolyclonalPolyclonal 항체 생산 Antibody production
SDS-PAGE 겔로부터 순수 분리한 70㎍ mCOEGY 단백질과 Freud`s adjuvant와 1:1 부피로 섞은 뒤에 complete Freud`s adjuvant 1회 토끼에 근육주사와 피하주사를 시행하며 2회 incomplete Freud`s adjuvant를 동량으로 섞어 주사하였다. 2주 간격으로 3회 시행하며 접종하기 전에 미리 혈액을 채취하였다. 3회의 접종이 끝난 후 토끼를 마취하여 심장 채혈하였고 얻어진 혈액은 상온에 하루 둔 후 혈청만을 분리하고 동결건조하여 -70℃에 보관하였다. 70 μg mCOEGY protein isolated from SDS-PAGE gel was mixed 1: 1 with Freud`s adjuvant, followed by intramuscular injection and subcutaneous injection in rabbit once complete Freud`s adjuvant, followed by incomplete Freud`s adjuvant twice. The same amount was mixed and injected. Three times every two weeks, blood was collected before inoculation. Rabbits were anesthetized after cardiac inoculation three times, and the blood was collected at room temperature for one day, and then serum was separated and lyophilized and stored at -70 ° C.
접종 전 토끼의 항혈청과 접종 후 토끼의 항혈청을 이용하여 웨스턴 블롯 수행한 결과 접종 후 토끼의 항혈청에서만 16.6KDa크기에서 순수분리된 mCOEGY의 10ug, 1ug 농도에서 예상된 mCOEGY 단백질이 검출되었고 접종 전에 없었던 항체가 형성되었음을 확인하였다(도 9B).
Western blot was performed using antiserum of rabbit and antiserum of rabbit before inoculation. Antibodies were detected at 10ug, 1ug concentration of mCOEGY purified at 16.6KDa size only in antiserum of rabbit after inoculation. It was confirmed that was formed (Fig. 9B).
실시예Example 5: 5: PEDVPEDV 항원 유전자 형질전환체 당근 분석 Antigen Gene Transformant Carrot Analysis
5-1) 당근 5-1) Carrot 캘러스Callus genomicgenomic DNADNA 추출 extraction
항생제 저항성을 통해 선발된 캘러스 0.5cm2 조직을 1.5㎖ 튜브에 넣고 플라스틱 페슬(pestle)을 이용하여 상온에서 분쇄하였다. DNA 추출 완충액(0.1M Tris-HCl. pH7.5, 0.5M NaCl, 50mM EDTA, 0.5% SDS)를 이용하여 게놈 DNA를 추출하였다(Kang et al., 2004). Callus selected with antibiotic resistance 0.5 cm 2 The tissue was placed in a 1.5 ml tube and ground at room temperature using a plastic pestle. Genomic DNA was extracted using DNA extraction buffer (0.1M Tris-HCl. PH7.5, 0.5M NaCl, 50mM EDTA, 0.5% SDS) (Kang et al., 2004).
각 유전자에 해당하는 프라이머를 사용하여 PCR을 수행하였다. 그 결과 총 53개의 세포주에서 중 38개의 세포주에서 유전자가 삽입되었음을 확인하였다(도 10).
PCR was performed using primers corresponding to each gene. As a result, it was confirmed that genes were inserted in 38 of the 53 cell lines (FIG. 10).
5-2) 형질전환 당근의 5-2) Transgenic Carrots RTRT -- PCRPCR 분석 analysis
게놈 DNA PCR 분석을 통해 선발된 당근 형질전환체에서 RNA를 추출하고 cDNA를 합성하였고 RT-PCR을 수행하여 전사체 수준에서의 분석을 하였다. 그 결과 총 15개의 세포주에서 11개의 세포주가 전사체 수준에서도 발현이 됨을 확인하였다(도 11).
RNA was extracted from carrot transformants selected through genomic DNA PCR analysis, cDNA was synthesized, and RT-PCR was performed for analysis at the transcript level. As a result, it was confirmed that 11 cell lines were expressed at the transcript level in a total of 15 cell lines (FIG. 11).
5-3) 식물 단백질 추출5-3) Plant Protein Extraction
동결건조시킨 조직 50mg에 500㎕ PBST(100mM NaCl, 10mM Na2HPO4, 3mM KH2PO4 pH7.2, 0.05% tween-20(v/v), 단백질 분해효소 억제제 칵테일(Roche 사)을 넣고 30초 간격으로 votexing과 얼음에 두는 과정을 5회 반복하고 4℃, 14,000rpm에서 10분간 원심분리하고 상층액만 회수하여 -20℃에 보관하였다(Rosales-Mendoza 2010).
To 50 mg of lyophilized tissue, 500 μl PBST (100 mM NaCl, 10 mM Na 2 HPO 4 , 3 mM KH 2 PO 4 pH7.2, 0.05% tween-20 (v / v), Protease Inhibitor Cocktail (Roche)) The procedure of votexing and placing on ice was repeated five times at 30 second intervals, centrifuged at 4 ° C and 14,000 rpm for 10 minutes, and only the supernatant was collected and stored at -20 ° C (Rosales-Mendoza 2010).
5-4) 5-4) 웨스턴Weston 블롯분석Blot Analysis
추출한 총 단백질을 15% SDS-PAGE를 이용하여 전기영동한 후 트랜스퍼 완충액(15% 메탄올)을 이용하여 Trans-Blot cell (bio-rad 사) 400mA에서 2시간 동안 PVDF 막(millipore 사)에 블롯팅하였다. 그런 후 막을 TBST (100mM Tris-Cl pH8.0, 150mM NaCl, 0.05% Tween 20)에 10분간 씻어 준 후 TBST에 5% 무지방 우유(nonfat dry milk)가 포함된 용액으로 1시간 블로킹하였다. 1차 항체는 제작된 mCOEGY 다클론 항체를 1:1,000의 비율로 TBST에 5% 무지방 우유(nonfat dry milk)가 들어간 용액에 희석하여 1시간 반응하였고 TBST로 15분간 1회, 5분 3회 씻어 주었다. 2차 항체는 항-토끼 IgG (GE healthcare 사)를 1:10,000의 비율로 TBST 용액에 희석하여 1시간 동안 반응하고 TBST로 15분간 1회, 5분 3회 씻어 주었다. 반응이 끝난 막은 ECL plus kit(GE healthcare 사)를 이용하여 실온 암실에서 x-ray 필름을 이용하여 검출하였다. The total protein extracted was electrophoresed using 15% SDS-PAGE and blotted on PVDF membrane (millipore) for 2 hours at 400 mA using Trans-Blot cell (bio-rad) using transfer buffer (15% methanol). It was. The membrane was then washed for 10 minutes in TBST (100 mM Tris-Cl pH8.0, 150 mM NaCl, 0.05% Tween 20) and blocked for 1 hour with a solution containing 5% nonfat dry milk in TBST. The primary antibody was reacted for 1 hour by diluting the prepared mCOEGY polyclonal antibody in a solution containing 5% nonfat dry milk in TBST at a ratio of 1: 1,000. Washed. The secondary antibody was diluted anti-rabbit IgG (GE healthcare) at a ratio of 1: 10,000 in TBST solution for 1 hour and washed once with TBST for 15 minutes and 3 times for 5 minutes. After the reaction, the membrane was detected using an X-ray film in a dark room at room temperature using an ECL plus kit (GE healthcare company).
그 결과 mCOE 형질전환체와 대조구에서 차이를 보이는 16.6KDa으로 예상된 크기의 밴드를 확인 할 수 있었다(도 12).
As a result, it was confirmed that the band of the expected size of 16.6KDa showing a difference in the mCOE transformant and the control (Fig. 12).
실시예Example 6: 6: 쥐에서의Rat 사료첨가제용Feed additives 백신 당근의 임상시험 Clinical Trials of Vaccine Carrots
6-1) 공시재료 및 처리6-1) Test Materials and Processing
8주령된 ICR 마우스(오리엔트 바이오 사)로 그룹당 8마리를 사용하였다. 대조구로는 일반사료와 비형질전환 당근을 먹이고 처리구는 일반사료와 mCOEJ 1, mCOEJ 13, mCOEGYJ 5계통의 당근 형질전환체 동결건조하여 곱게 간 후 증류수와 혼합하여 사용하였다. 총 4번의 경구투여로 마리당 80mg의 시료를 먹였으며 경투 투여 2일전과 경구 투여 4일 후에 혈청을 얻어냈다(도 13).
Eight weeks old ICR mice (Orient Bio Co.) used 8 mice per group. The control group was fed general feed and non-transformed carrots, and the treated group was lyophilized by normal feed and
6-2) 시험사료 및 사양관리6-2) Test feed and specification management
실험용 마우스 사료는 단백질, 지방, 조 섬유소 등이 포함되어 있고 감마멸균된 PICO 5035(오리엔트 바이오 사)를 사용하였다(표 3). 물과 사료는 자유채식으로 하였고 백신 당근 캘러스는 1회 경구 투여 시 파우더 상태의 20mg 시료와 물을 혼합하여 주사기를 이용하여 쥐에 먹이고 그 이후는 물과 사료를 자유 채식하였다. The experimental mouse feed contained protein, fat, crude fiber, and the like, and gamma sterilized PICO 5035 (Orien Bio) was used (Table 3). Water and feed were free vegan. Vaccine carrot callus was mixed with a 20 mg powder of powder and water in a single oral dose and fed to rats using a syringe.
6-3) 6-3) ELISAELISA 에 의한 항체 규명Identification of antibodies by
강원대학교 수의과학대에서 분양받은 살아있는 PEDV를 항원으로 이용하여 탄산염 코팅된 완충액(0.1M NaHCO3, 0.1M Na2CO3 pH 9.6)에 3,000배 희석하고 ELISA 플레이트 각 웰에 코팅 버퍼로 희석된 항원 100ul씩 분주하였다. 그런 다음 4℃에 하룻 동안 반응시키고, 세척 완충액(PBS + 0.1% Tween 20)을 각 웰에 200ul씩 분주한 다음 3회 세척하였다. 웰에 차단 완충액(2% BSA(Bovine serum albumin) in PBS) 200ul씩을 분주한 다음 37℃, 1시간 정치하였다. 세척 완충액을 각 웰에 200ul씩 분주한 다음 3회 세척한 후 혼성화된 항-소 IgA 퍼옥시다아제, 혼성화된 항-소 IgG 퍼옥시다아제를 PBS에 1:8000 비율로 희석하여 각 웰에 100ul씩 분주한 후 37℃, 1시간 반응시켰다. 세척 완충액을 각 웰에 200ul씩 분주한 다음 4회 세척하였다. 기질(OPD)을 각 웰에 100ul씩 분주한 다음 실온에서 정확히 10분간 반응시킨 다음 정지 용액 50ul를 가하여 반응을 중지시키고 ELISA reader로 흡광도 450nm에서 측정하였다.100 ul of antigen diluted in carbonate coated buffer (0.1 M NaHCO 3 , 0.1 M Na 2 CO 3 pH 9.6) and coated buffer in each well of the ELISA plate using the live PEDV distributed by Kangwon National University Each was busy. Then, the reaction was carried out at 4 ° C. for one day, and 200 μl of washing buffer (PBS + 0.1% Tween 20) was added to each well, followed by washing three times. The wells were dispensed with 200ul of blocking buffer (2% BSA (Bovine serum albumin) in PBS) and then allowed to stand at 37 ° C for 1 hour. 200 ul of wash buffer was dispensed into each well, followed by three washes, followed by diluting the hybridized anti-bovine IgA peroxidase and the hybridized anti-bovine IgG peroxidase in PBS at a 1: 8000 ratio. After reacting at 37 ° C. for 1 hour. Wash buffer was dispensed into each well of 200 ul and then washed four times. The substrate (OPD) was dispensed into each well for 100ul and then reacted for exactly 10 minutes at room temperature. Then, 50ul of stop solution was added to stop the reaction, and the absorbance was measured at 450nm with ELISA reader.
그 결과 일반사료와 mCOE J1과 mCOEJ13간의 IgG와 IgA에서 유의적으로 높은 차이를 보였으나 대조구(일반사료)를 제외한 각 처리구간의 IgG와 IgA의 유의적인 차이는 없었다(도 14).
As a result, there was a significant difference in IgG and IgA between normal feed and mCOE J1 and mCOEJ13, but there was no significant difference between IgG and IgA in each treatment except control (normal feed) (Fig. 14).
실시예Example 7: 7: 돼지에서의Pig 사료첨가제용Feed additives 백신 당근의 격리시험 Containment test of vaccine carrot
7-1) 공시재료 및 처리7-1) Materials and Processing
PEDV 형질전환 당근백신의 돼지 경구면역 최적조건 규명하기 위한 격리실험으로 강원대 수의과대학 동물실험실에서 3일령의 포유자돈을 한 그룹당 5마리씩 준비하였다. 3일령 포유자돈을 4군(NV-NC그룹: 대조구, V-NC그룹: 당근백신, NV-C그룹: PEDV 공격접종, V-C그룹: 당근백신, PEDV 공격접종)으로 나눈 후 3일령과 4일령에 당근백신 5g/두을 투여하고 5일령째에 PEDV(수의과학검역원) 1.5 X 105 TCID50/ml을 공격접종 하였다(도 15). 그리고 시험기간 동안에는 대용유(주)제일제당사료, 돈돈밀크)를 제조사의 권장사항에 따라 급여하였고 음수는 자유롭게 섭취하도록 하였다. 그리고 돈방주위는 정기적으로 소독하였다.
As a quarantine experiment to determine the optimal conditions for oral immunity of pigs from PEDV-transformed carrot vaccine, five piglets of three-day-old piglets were prepared in the animal laboratory of the College of Veterinary Medicine, Kangwon National University. 3 days old and 3 days old piglets divided into 4 groups (NV-NC group: control, V-NC group: carrot vaccine, NV-C group: PEDV challenge, VC group: carrot vaccine, PEDV challenge) Carrot vaccine was administered to 5g / two and challenged with 1.5 x 10 5 TCID 50 / ml PEDV (Veterinary Quarantine Service) at 5 days of age (Fig. 15). During the test period, Daeyong Oil Co., Ltd. (JeongJedang Feed, Dondon Milk) was provided according to the manufacturer's recommendation, and drinking water was freely consumed. And the pig area was regularly disinfected.
7-2) 7-2) 돼지에서의Pig PEDVPEDV 감염 검사 Infection Test
공격접종한 후 1일부터 7일간 NV-C그룹과 V-C 그룹의 설사지수를 측정하였다. 설사 지수는 선행연구를 통하여 알려진 방법으로 측정하였다(Normal; 0, mild;1, moderate; 2, and sever diarrhea; 3)(Sherman DM 1983). 공격접종한 후 10일 후에 자돈을 강원대 수의과학대학에서 해부하여 소화기관의 상태를 검사하여 PEDV의 감염 증상을 확인하였다.
Diarrhea index of NV-C group and VC group was measured from 1 to 7 days after challenge. Diarrhea index was measured by a known method through previous studies (Normal; 0, mild; 1, moderate; 2, and sever diarrhea; 3) (Sherman DM 1983). Ten days after challenge, piglets were dissected at the College of Veterinary Science, Kangwon National University to examine the condition of the digestive organs to identify the symptoms of PEDV infection.
7-3) 설사지수7-3) Diarrhea Index
당근 백신 mCOEJ1과 PEDV 공격접종한 V-C그룹에서 PEDV만 공격접종한 NV-C그룹의 자돈 5마리에서 설사지수를 측정하고 one-way ANOVA 분석을 한 결과 공격접종 2일 후부터 V-C 그룹에서 낮은 설사지수를 보였다(도 16).
In the VC group challenged with carrot vaccine mCOEJ1, the diarrhea index was measured in five piglets of the NV-C group who were only vaccinated with PEDV and one-way ANOVA analysis showed that the diarrhea index was lower in the VC group from 2 days after challenge. (FIG. 16).
7-4) 7-4) ELISAELISA 항체 검사 Antibody Test
ELISA를 통하여 NV-C그룹과 V-C그룹이 NV-NC그룹과 V-NC그룹보다 공격접종 후 4일째 IgG와 IgA에서 모두 유의적으로 높은 항체반응을 보였다. 공격접종 후 7일차에는 NV-C그룹에서만 IgG와 IgA에서 높은 항체가를 보였다. 공격접종 후 7일차에서는 V-C그룹에서는 NV-NC그룹과 V-NC그룹과 비숫한 항체가를 보였고 NV-C그룹만 IgG와 IgA의 항체가가 다른 그룹들보다 높았다(도 17 및 18).
By ELISA, NV-C and VC groups showed significantly higher antibody responses in both IgG and IgA at 4 days after challenge than NV-NC and V-NC groups. On the 7th day after challenge, the antibody titers of IgG and IgA were high in the NV-C group only. On the 7th day after challenge, the VC group showed a higher antibody titer than the NV-NC group and the V-NC group, and only the NV-C group had higher antibody titers of IgG and IgA (Figs. 17 and 18).
7-5) 해부를 통한 7-5) Through dissection PEDVPEDV 감염 육안검사 Infection Visual Inspection
공격접종 후 10일 후에 자돈을 해부하여 소화기관을 검사한 결과 NV-C 그룹에서 PEDV의 감염증상 중 하나인 소장에서 가스가 차있음을 발견할 수 있었고 아직 PEDV가 계속 진행되고 있는 것을 확인하였고 mCOEJ1을 경구투여한 V-C그룹은 NV-NC그룹과 비교했을 때 차이가 없는 것으로 보아 PEDV 감염이 완화된 것으로 보였다(도 19)After 10 days of challenge, piglets were dissected to examine the digestive tract, and the NV-C group found gas in the small intestine, one of the symptoms of PEDV infection, and confirmed that PEDV was still ongoing. MCOEJ1 VC group administered orally did not show any difference compared to NV-NC group, and PEDV infection appeared to be alleviated (FIG. 19).
<110> Industry-Academic Cooperation Foundation, Dankook University <120> Trnasformants expressing epitope of porcine epidemic diarrhea virus and vaccine composition containning the same <130> PA100814KR <160> 13 <170> KopatentIn 1.71 <210> 1 <211> 142 <212> PRT <213> porcine epidemic diarrhea virus <400> 1 Thr Met Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val Asn Ile 1 5 10 15 Thr Val Ser Ala Ala Phe Gly Gly His Ser Gly Ala Asn Leu Ile Ala 20 25 30 Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val Asp Thr Arg 35 40 45 Gln Phe Thr Ile Thr Leu Phe Tyr Asn Val Thr Asn Ser Tyr Gly Tyr 50 55 60 Val Ser Lys Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln Ser Val 65 70 75 80 Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser Leu Leu 85 90 95 Ala Gly Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu Phe Gly Ser 100 105 110 Gly Val Lys Phe Thr Ser Leu Tyr Phe Gln Phe Thr Lys Gly Glu Leu 115 120 125 Ile Thr Gly Thr Pro Lys Pro Leu Glu Gly Ile Thr Asp Val 130 135 140 <210> 2 <211> 430 <212> DNA <213> porcine epidemic diarrhea virus <220> <221> gene <222> (1)..(430) <223> PEDV epitope gene sequence <400> 2 caccatggtt actttgccat cattcaatga tcattctttt gttaatatta ctgtctctgc 60 ggcttttggt ggtcatagtg gtgccaacct cattgcatct gacactacta tcaatgggtt 120 tagttctttc tgtgttgaca ctagacaatt taccattaca ctgttttata acgttacaaa 180 cagttatggt tatgtgtcta agtcacagga tagtaattgc cctttcacct tgcaatctgt 240 taatgattac ctgtctttta gcaaattttg tgtttcaacc agccttttgg ctggtgcttg 300 taccatagat ctttttggtt accctgagtt cggtagtggt gttaagttta cgtcccttta 360 ttttcaattc acaaagggtg agttgattac tggcacgcct aaaccacttg aaggtatcac 420 agacgtttaa 430 <210> 3 <211> 430 <212> DNA <213> Artificial Sequence <220> <223> mutant of COE gene <400> 3 caccatggtt actttgccat cattcaatga tcattctttt gttaatatta ctgtctctgc 60 ggcttttggt ggtcatagtg gtgccaacct cattgcatct gacactacta tcaatgggtt 120 tagttctttc tgtgttgaca ctagacagtt caccattaca ctgttttata acgttacaaa 180 cagttatggt tatgtgtcta agtcacagga tagtaattgc cctttcacct tgcaatctgt 240 taatgattac ctgtctttta gcaaattttg tgtttcaacc agccttttgg ctggtgcttg 300 taccatagat ctttttggtt accctgagtt cggtagtggt gttaagttta cgtcccttta 360 ttttcaattc acaaagggtg agttgattac tggcacgcct aaaccacttg aaggtatcac 420 agacgtttaa 430 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> C-terminal region of spike protein <400> 4 Gly Pro Arg Leu Gln Pro Tyr 1 5 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C-terminal region of spike gene <400> 5 ggtcctagac ttcaacctta c 21 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PEDs <400> 6 caccatggtt actttgccat cattt 25 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PEDs <400> 7 ttaaacgtct gtgatacctt caagtgg 27 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PEDs(mut) <400> 8 tgtgttgaca ctagacagtt caccattaca ctgttt 36 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PEDs(mut) <400> 9 aaacagtgta atggtgaact gtctagtgtc aacaca 36 <210> 10 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for COE(fus) <400> 10 caccatggtt actttgccat cattcaatga tcattcttt 39 <210> 11 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for COE(fus) <400> 11 aagtctagga cccctacaac aacctgaaaa aacgtctgt 39 <210> 12 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for COE(c-ter) <400> 12 acagacgttt tttcaggttg ttgtaggggt cctagactt 39 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for COE(c-ter) <400> 13 ttagtaaggt tgaagtctag gacccctaca acaacc 36 <110> Industry-Academic Cooperation Foundation, Dankook University <120> Trnasformants expressing epitope of porcine epidemic diarrhea virus and vaccine composition containning the same <130> PA100814KR <160> 13 <170> Kopatentin 1.71 <210> 1 <211> 142 <212> PRT <213> porcine epidemic diarrhea virus <400> 1 Thr Met Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val Asn Ile 1 5 10 15 Thr Val Ser Ala Ala Phe Gly Gly His Ser Gly Ala Asn Leu Ile Ala 20 25 30 Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val Asp Thr Arg 35 40 45 Gln Phe Thr Ile Thr Leu Phe Tyr Asn Val Thr Asn Ser Tyr Gly Tyr 50 55 60 Val Ser Lys Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln Ser Val 65 70 75 80 Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser Leu Leu 85 90 95 Ala Gly Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu Phe Gly Ser 100 105 110 Gly Val Lys Phe Thr Ser Leu Tyr Phe Gln Phe Thr Lys Gly Glu Leu 115 120 125 Ile Thr Gly Thr Pro Lys Pro Leu Glu Gly Ile Thr Asp Val 130 135 140 <210> 2 <211> 430 <212> DNA <213> porcine epidemic diarrhea virus <220> <221> gene (222) (1) .. (430) P223 epitope gene sequence <400> 2 caccatggtt actttgccat cattcaatga tcattctttt gttaatatta ctgtctctgc 60 ggcttttggt ggtcatagtg gtgccaacct cattgcatct gacactacta tcaatgggtt 120 tagttctttc tgtgttgaca ctagacaatt taccattaca ctgttttata acgttacaaa 180 cagttatggt tatgtgtcta agtcacagga tagtaattgc cctttcacct tgcaatctgt 240 taatgattac ctgtctttta gcaaattttg tgtttcaacc agccttttgg ctggtgcttg 300 taccatagat ctttttggtt accctgagtt cggtagtggt gttaagttta cgtcccttta 360 ttttcaattc acaaagggtg agttgattac tggcacgcct aaaccacttg aaggtatcac 420 agacgtttaa 430 <210> 3 <211> 430 <212> DNA <213> Artificial Sequence <220> <223> mutant of COE gene <400> 3 caccatggtt actttgccat cattcaatga tcattctttt gttaatatta ctgtctctgc 60 ggcttttggt ggtcatagtg gtgccaacct cattgcatct gacactacta tcaatgggtt 120 tagttctttc tgtgttgaca ctagacagtt caccattaca ctgttttata acgttacaaa 180 cagttatggt tatgtgtcta agtcacagga tagtaattgc cctttcacct tgcaatctgt 240 taatgattac ctgtctttta gcaaattttg tgtttcaacc agccttttgg ctggtgcttg 300 taccatagat ctttttggtt accctgagtt cggtagtggt gttaagttta cgtcccttta 360 ttttcaattc acaaagggtg agttgattac tggcacgcct aaaccacttg aaggtatcac 420 agacgtttaa 430 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> C-terminal region of spike protein <400> 4 Gly Pro Arg Leu Gln Pro Tyr 1 5 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C-terminal region of spike gene <400> 5 ggtcctagac ttcaacctta c 21 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PEDs <400> 6 caccatggtt actttgccat cattt 25 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PEDs <400> 7 ttaaacgtct gtgatacctt caagtgg 27 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PEDs (mut) <400> 8 tgtgttgaca ctagacagtt caccattaca ctgttt 36 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> Reverse primer for PEDs (mut) <400> 9 aaacagtgta atggtgaact gtctagtgtc aacaca 36 <210> 10 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for COE (fus) <400> 10 caccatggtt actttgccat cattcaatga tcattcttt 39 <210> 11 <211> 39 <212> DNA <213> Artificial Sequence <220> Reverse primer for COE (fus) <400> 11 aagtctagga cccctacaac aacctgaaaa aacgtctgt 39 <210> 12 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for COE (c-ter) <400> 12 acagacgttt tttcaggttg ttgtaggggt cctagactt 39 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for COE (c-ter) <400> 13 ttagtaaggt tgaagtctag gacccctaca acaacc 36
Claims (15)
Porcine epidemic diarrhea virus (PEDV) epitope protein of SEQ ID NO: 1.
A PEDV antigen comprising the epitope of claim 1.
A recombinant vector comprising a gene encoding the epitope protein of claim 1.
The recombinant vector according to claim 3, wherein the gene has a nucleotide sequence represented by SEQ ID NO: 2.
The recombinant vector according to claim 3, wherein the gene has a nucleotide sequence represented by SEQ ID NO: 3.
The recombinant vector of claim 3, further comprising a gene encoding the amino acid of SEQ ID NO: 4. 5.
The recombinant vector according to claim 6, wherein the gene has a nucleotide sequence represented by SEQ ID NO: 5.
A transformant transformed with the recombinant vector of any one of claims 3 to 7.
The transformant of claim 8, wherein the transformant is Agrobacterium, Escherichia coli or a transgenic plant.
The tobacco plant of claim 9, wherein the plant cell is tobacco. A transformant selected from the group consisting of Arabidopsis, potato, lettuce, radish, cabbage, tomato, soybean, rice, barley, wheat, corn, and carrot.
A PEDV vaccine composition comprising the transformant of any one of claims 8 to 10, a protein extract of the transformant or a recombinant PEDV epitope protein isolated from the transformant.
A feed composition for preventing or treating PEDV infection comprising the transformant of any one of claims 8 to 10, a protein extract of the transformant or a recombinant PEDV epitope protein isolated from the transformant.
11. A composition for preventing or treating PEDV infection comprising a transformant of any one of claims 8 to 10, a protein extract of the transformant or a recombinant PEDV epitope protein isolated from the transformant.
(2) 상기 단계 (1)의 발현벡터를 아그로박테리아에 도입시키는 단계; 및
(3) 상기 단계 (2)의 아그로박테리아와 식물세포를 형질전환시키는 단계를 포함하는 PEDV 백신의 제조방법.
(1) preparing a vector expressing a gene encoding the PEDV epitope protein of claim 1;
(2) introducing the expression vector of step (1) into Agrobacteria; And
(3) A method for producing a PEDV vaccine comprising the step of transforming the agrobacteria and plant cells of step (2).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9950061B2 (en) | 2014-04-03 | 2018-04-24 | Boehringer Ingelheim Vetmedica, Inc. | Porcine epidemic diarrhea virus vaccine |
| CN109438573A (en) * | 2015-12-25 | 2019-03-08 | 中国农业科学院兰州兽医研究所 | Porcine epidemic diarrhea virus M protein-specific heavy chain antibody |
| KR20200121447A (en) | 2019-04-16 | 2020-10-26 | 주식회사 바이오앱 | Vaccine composition for preventing porcine epidemic diarrhea and manufacturing method thereof |
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2010
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9950061B2 (en) | 2014-04-03 | 2018-04-24 | Boehringer Ingelheim Vetmedica, Inc. | Porcine epidemic diarrhea virus vaccine |
| US10702601B2 (en) | 2014-04-03 | 2020-07-07 | Boehringer Ingelheim Animal Health USA Inc. | Porcine epidemic diarrhea virus vaccine |
| CN109438573A (en) * | 2015-12-25 | 2019-03-08 | 中国农业科学院兰州兽医研究所 | Porcine epidemic diarrhea virus M protein-specific heavy chain antibody |
| CN109438573B (en) * | 2015-12-25 | 2020-11-10 | 中国农业科学院兰州兽医研究所 | Porcine epidemic diarrhea virus M protein specific heavy chain antibody |
| KR20200121447A (en) | 2019-04-16 | 2020-10-26 | 주식회사 바이오앱 | Vaccine composition for preventing porcine epidemic diarrhea and manufacturing method thereof |
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