KR102563238B1 - Transgenic cloned porcine for animal models with increased susceptibility to severe acute respiratory syndrome coronavirus 2, and method for producing the same - Google Patents

Transgenic cloned porcine for animal models with increased susceptibility to severe acute respiratory syndrome coronavirus 2, and method for producing the same Download PDF

Info

Publication number
KR102563238B1
KR102563238B1 KR1020210058660A KR20210058660A KR102563238B1 KR 102563238 B1 KR102563238 B1 KR 102563238B1 KR 1020210058660 A KR1020210058660 A KR 1020210058660A KR 20210058660 A KR20210058660 A KR 20210058660A KR 102563238 B1 KR102563238 B1 KR 102563238B1
Authority
KR
South Korea
Prior art keywords
cov
sars
cell line
transgenic
recombinant vector
Prior art date
Application number
KR1020210058660A
Other languages
Korean (ko)
Other versions
KR20210136873A (en
Inventor
최기명
심주현
고나영
김형주
이용진
이정웅
김현일
Original Assignee
주식회사 옵티팜
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 옵티팜 filed Critical 주식회사 옵티팜
Publication of KR20210136873A publication Critical patent/KR20210136873A/en
Application granted granted Critical
Publication of KR102563238B1 publication Critical patent/KR102563238B1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Humanized animals, e.g. knockin
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2511/00—Cells for large scale production

Abstract

본 발명은 돼지에서 인간 안지오텐신 전환효소 2(Angiotensin-converting enzyme 2, ACE2)를 발현시키기 위한 재조합 벡터를 제조하고, 이를 이용하여 제조한 형질전환 세포주와 형질전환 돼지에 관한 것으로, 본 발명에 따르면 상기 형질전환 세포주 및 형질전환 돼지는 인간 ACE2를 발현하며, SARS-CoV-2에 대하여 높은 감수성을 보이는 바, 이를 SARS-CoV-2에 대한 연구 또는 상기 바이러스 감염 질환의 예방 또는 치료제 개발을 위한 비임상 동물모델로서 유용하게 활용할 수 있다.The present invention manufactures a recombinant vector for expressing human angiotensin-converting enzyme 2 (ACE2) in pigs, and relates to a transformed cell line prepared using the same and a transgenic pig. Transgenic cell lines and transgenic pigs express human ACE2 and show high sensitivity to SARS-CoV-2, which is a non-clinical study for SARS-CoV-2 or development of preventive or therapeutic agents for viral infectious diseases. It can be useful as an animal model.

Description

제2형 중증급성호흡기증후군 코로나바이러스에 대한 감수성이 증가된 동물모델용 형질전환 돼지 및 이의 제조방법{Transgenic cloned porcine for animal models with increased susceptibility to severe acute respiratory syndrome coronavirus 2, and method for producing the same}Transgenic cloned porcine for animal models with increased susceptibility to severe acute respiratory syndrome coronavirus 2, and method for producing the same}

본 발명은 제2형 중증급성호흡기증후군 코로나바이러스(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)에 대한 감수성이 증가되어, SARS-CoV-2 감염 질환의 비임상 평가를 위하여 사용될 수 있는 형질전환 돼지 및 이의 제조방법에 관한 것이다.The present invention is a trait that can be used for non-clinical evaluation of SARS-CoV-2 infectious disease by increasing susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) type 2 It relates to a converted pig and a method for producing the same.

코로나바이러스감염증-19(coronavirus disease 2019, COVID-19)는 2019년 중국 후베이성의 우한시에서 처음 발견된 바이러스인 SARS-CoV-2(Severe acute respiratory syndrome coronavirus 2)에 의한 것으로, 상기 SARS-CoV-2는 코로나바이러스(Coroviridae) 군에 속하며 사스 코로나바이러스(SARS-CoV, 중증급성호흡기증후군 코로나바이러스), 메르스 코로나바이러스(Middle East respiratory syndrome coronavirus, MERS-CoV, 중동호흡기증후군 코로나바이러스)와 유사한 바이러스로 알려져 있다. 아직까지 명확한 감염원과 감염경로는 확인되지 않았으나, 우한 지역의 박쥐와의 접촉을 통해 감염되었을 가능성이 높고 사람 간 밀접접촉에 의한 전파가 가능하다고 보고되었다.Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), a virus first discovered in Wuhan City, Hubei Province, China in 2019. belongs to the family of coronaviruses (Coroviridae) and is a virus similar to SARS-CoV (severe acute respiratory syndrome coronavirus) and Middle East respiratory syndrome coronavirus (MERS-CoV). It is known. Although the clear source of infection and route of infection have not yet been identified, it has been reported that it is likely to have been infected through contact with bats in Wuhan, and transmission through close contact between humans is possible.

그러나 현재까지 SARS-CoV-2 감염 질환에 대하여 확실한 치료 효과를 보이는 치료제가 개발되지 못하여 증상에 대한 치료를 위주로 하고 있다. 따라서 SARS-CoV-2 감염 질환의 예방 또는 치료제에 대한 연구가 활발히 수행되고 있다.However, until now, a therapeutic agent showing a clear therapeutic effect on SARS-CoV-2 infectious disease has not been developed, so treatment is mainly focused on symptoms. Therefore, research on preventive or therapeutic agents for SARS-CoV-2 infectious diseases is being actively conducted.

한편, SARS-CoV-2는 바깥쪽에 왕관처럼 튀어나온 돌기 단백질(Spike protein)을 가지고 있으며, 상기 돌기 단백질이 인간 안지오텐신 전환효소 2(Angiotensin-converting enzyme 2, ACE2) 유전자에 결합하고 세포의 내포 작용 (endocytosis)에 의해 감염을 일으키는 것으로 보고되었다(Daniel Wrapp et al., Science, 2020). 인간과 다른 종(species)에 있어서, ACE2 유전자간 비슷한 아미노산 서열을 갖고 있으나 코로나 바이러스의 돌기 단백질은 사람 ACE2 유전자에 특이적으로 결합한다. 따라서 동물 모델을 이용한 SARS-CoV-2 감염의 비임상 시험시 사람과 비슷한 감수성을 나타내기 위해서는 인간 ACE2가 삽입된 형질전환 모델을 사용하는 것을 권고하고 있다.On the other hand, SARS-CoV-2 has a spike protein protruding like a crown on the outside, and the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) gene and acts as a cell endocytosis It has been reported to cause infection by endocytosis (Daniel Wrapp et al., Science, 2020). In humans and other species, the ACE2 gene has a similar amino acid sequence, but the spike protein of the coronavirus binds specifically to the human ACE2 gene. Therefore, in non-clinical tests of SARS-CoV-2 infection using animal models, it is recommended to use a transgenic model in which human ACE2 is inserted to show sensitivity similar to that of humans.

따라서 인간 ACE2를 발현하는 마우스 동물모델이 개발된 바 있으나, 동물 질환 모델에 있어서, 동물 질환모델의 병리양상과 증상이 사람에서 관찰되는 것과 많은 차이를 보이고 있어 설치류 질환모델에서 나온 결과를 토대로 임상시험을 시행할 경우 많은 문제점이 있어왔다.Therefore, a mouse animal model expressing human ACE2 has been developed, but in animal disease models, the pathological aspects and symptoms of animal disease models are very different from those observed in humans, so clinical trials based on the results from rodent disease models There have been many problems with implementation.

그러므로, 인간과의 디멘존, 번식, 수명 및 행동양상의 확연한 차이로 인해 보다 인간과 가까운 종을 이용한 질환동물 모델에 대한 필요성이 제기되었다. 이에 있어 영장류의 경우 그 희소성 및 사육관리의 비용 및 어려움으로 인해 극히 제한적인 분야에서만 질환연구에 활용 가능한 제약이 있으므로, 이에 따라 비교적 저렴한 비용과 시설에서 보다 정확한 난치질환 연구를 할 수 있는 돼지를 새로운 모델 동물로서 바이오 메디컬 분야에 활용하고자 하는 요구가 증가되고 있다.Therefore, the need for a disease animal model using a species closer to humans has been raised due to clear differences in dimension, reproduction, lifespan, and behavioral patterns from humans. In this regard, in the case of primates, there are restrictions that can be used for disease research only in extremely limited fields due to their scarcity and cost and difficulty in breeding management. As a model animal, there is an increasing demand for use in the biomedical field.

돼지는 해부 생리학적으로 인간과 유사성이 인정되어 이미 각종 질환의 조직병리학적 기전과 치료를 위한 연구에 이용되고 있으며, 특히 오랫동안 경제 동물로 가치가 인정되어 다른 중/대 동물을 질환모델로 사용할 때보다 윤리적인 문제점을 피해갈 수 있으며, 안정적인 사육 시스템이 구축되어 있고, 임신 기간이 짧은 바 실험동물 모델 개발 시 유지 및 관리가 용이한 장점이 있다.Pigs have been recognized for their anatomical and physiological similarity to humans, and are already being used for research on histopathological mechanisms and treatment of various diseases. Ethical problems can be avoided, a stable breeding system is established, and the gestation period is short, so it has the advantage of easy maintenance and management when developing an experimental animal model.

따라서 SARS-CoV-2 감염 질환에 대한 효과적인 예방 또는 치료제의 개발을 위하여 돼지를 이용한 동물모델의 개발이 요구된다.Therefore, in order to develop an effective preventive or therapeutic agent for SARS-CoV-2 infectious disease, the development of an animal model using pigs is required.

본 발명자들은 제2형 중증급성호흡기증후군 코로나바이러스(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)에 대한 감수성을 증가시킨 동물모델의 개발을 위하여 연구하던 중, 인간 안지오텐신 전환효소 2(Angiotensin-converting enzyme 2, ACE2)를 발현시킨 형질전환 세포주 및 형질전환 돼지를 제조하고 상기 형질전환 세포주와 형질전환 돼지의 SARS-CoV-2에 대한 감수성이 야생형 대비 증가했음을 확인하여 본 발명을 완성하였다.The inventors of the present invention studied for the development of an animal model with increased susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human angiotensin converting enzyme 2 (Angiotensin-CoV-2). converting enzyme 2, ACE2) was prepared, and the present invention was completed by confirming that the susceptibility to SARS-CoV-2 of the transgenic cell line and the transgenic pig was increased compared to the wild type.

따라서 본 발명의 목적은 제2형 중증급성호흡기증후군 코로나바이러스(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터를 제공하는 것이다.Therefore, an object of the present invention is to provide a recombinant vector for producing an animal model with increased susceptibility to type 2 severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2).

본 발명의 또 다른 목적은 SARS-CoV-2에 대한 감수성이 증가된 형질전환 세포주를 제공하는 것이다. Another object of the present invention is to provide a transformed cell line with increased sensitivity to SARS-CoV-2.

본 발명의 또 다른 목적은 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지 생산을 위한 핵 이식란을 제공하는 것이다. Another object of the present invention is to provide nuclear transfer embryos for producing transgenic pigs for animal models with increased susceptibility to SARS-CoV-2.

본 발명의 또 다른 목적은 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지를 제공하는 것이다. Another object of the present invention is to provide a transgenic pig for animal models with increased susceptibility to SARS-CoV-2.

본 발명의 또 다른 목적은 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지의 제조방법을 제공하는 것이다. Another object of the present invention is to provide a method for producing transgenic pigs for animal models with increased susceptibility to SARS-CoV-2.

본 발명의 또 다른 목적은 SARS-CoV-2 감염 질환의 예방 또는 치료제의 스크리닝 방법을 제공하는 것이다.Another object of the present invention is to provide a screening method for preventing or treating SARS-CoV-2 infectious diseases.

상기 목적을 달성하기 위하여, 본 발명은 인간 안지오텐신 전환효소 2(Angiotensin-converting enzyme 2, ACE2) 유전자, 인핸서 및 프로모터를 포함하는, 제2형 중증급성호흡기증후군 코로나바이러스(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터를 제공한다.In order to achieve the above object, the present invention is a type 2 severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) provides a recombinant vector for constructing an animal model with increased susceptibility.

또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 재조합 벡터를 체세포에 형질전환시켜 제조한, SARS-CoV-2에 대한 감수성이 증가된 형질전환 세포주를 제공한다.In addition, in order to achieve the above another object, the present invention provides a transformed cell line with increased sensitivity to SARS-CoV-2 prepared by transforming the recombinant vector according to the present invention into somatic cells.

또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 형질전환 세포주를 탈핵시킨 돼지 수정란에 도입하여 핵치환된, SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지 생산을 위한 핵 이식란을 제공한다.In addition, in order to achieve the above another object, the present invention introduces the transformed cell line according to the present invention into enucleated pig fertilized eggs to produce nuclear-transferred, transgenic pigs for animal models with increased susceptibility to SARS-CoV-2. Provides nuclear transfer embryos for

또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 형질전환 세포주를 핵 이식하여 제조된 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지를 제공한다.In addition, in order to achieve the above another object, the present invention provides a transgenic pig for animal model with increased susceptibility to SARS-CoV-2 prepared by nuclear transfer of the transformed cell line according to the present invention.

또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 형질전환 세포주를 탈핵화된 난자에 이식하여 재구성된 난자를 제조하는 핵이식 단계; 및 상기 재구성된 난자를 대리모의 난관에 이식하는 단계;를 포함하는 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지의 제조방법을 제공한다.In order to achieve the above another object, the present invention provides a nuclear transfer step of preparing a reconstructed egg by transplanting the transformed cell line according to the present invention to a denuclearized egg; and transplanting the reconstituted oocyte into the fallopian tube of a surrogate mother.

또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 SARS-CoV-2에 대한 감수성이 증가된 형질전환 세포주 또는 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지를 이용한 SARS-CoV-2 감염 질환의 예방 또는 치료제의 스크리닝 방법을 제공한다.In addition, in order to achieve the above another object, the present invention provides a transgenic cell line with increased sensitivity to SARS-CoV-2 or a transgenic pig for animal model with increased sensitivity to SARS-CoV-2 according to the present invention. It provides a screening method for preventing or treating a SARS-CoV-2 infectious disease using the present invention.

본 발명은 인간 안지오텐신 전환효소 2(Angiotensin-converting enzyme 2, ACE2)를 발현시키기 위한 재조합 벡터, 이를 이용한 인간 ACE2를 발현하는 형질전환 세포주, 형질전환 돼지 생산을 위한 핵 이식란, 형질전환 돼지, 상기 형질전환 돼지의 제조 방법 및 이의 용도에 대한 것으로, 상기 인간 ACE2를 발현하는 형질전환 돼지는 SARS-CoV-2에 대하여 높은 감수성을 보이는 바, 이를 SARS-CoV-2에 대한 연구 또는 상기 바이러스 감염 질환의 예방 또는 치료제 개발을 위한 비임상 동물모델로서 유용하게 활용할 수 있다.The present invention provides a recombinant vector for expressing human angiotensin-converting enzyme 2 (ACE2), a transformed cell line expressing human ACE2 using the same, a nuclear transfer embryo for producing a transgenic pig, a transgenic pig, and the above trait Regarding a method for producing a transgenic pig and a use thereof, the transgenic pig expressing human ACE2 shows a high sensitivity to SARS-CoV-2, which can be used for research on SARS-CoV-2 or for viral infectious diseases. It can be usefully used as a non-clinical animal model for preventive or therapeutic development.

도 1은 본 발명에 따른 인간 안지오텐신 전환효소 2(Angiotensin-converting enzyme 2, ACE2)를 발현시키기 위한 재조합 벡터의 모식도이다.
도 2는 본 발명에 따른 인간 ACE2 벡터를 돼지 암컷, 수컷의 귀 섬유아세포에 형질도입한 후 이의 형질도입 여부를 확인한 것으로, 도 2a는 각 세포의 GFP 단백질 발현을 확인한 것이고, 도 2b는 각 세포의 FLAG 단백질 발현을 확인한 것이며, 도 2c는 각 세포의 ACE2 단백질 발현을 확인한 것이다.
도 3은 본 발명에 따른 형질전환 세포주에서 인간 ACE2 발현 여부를 확인한 결과이다.
도 4는 본 발명에 따른 형질전환 세포주에서 인간 ACE2 발현을 확인한 것으로, 도 4a는 인간 ACE2 단백질의 발현을 확인한 것이고, 도 4b는 인간 ACE2 유전자에 태깅된 FLAG 단백질의 발현을 확인한 것이다(WT: 섬유아 세포주, TG: 형질전환 세포주).
도 5는 본 발명에 따른 형질전환 세포주 4 종의 SARS-CoV-2에 대한 감수성을 확인한 것이다(WT: 섬유아 세포주, TG: 형질전환 세포주).
도 6은 본 발명에 따른 형질전환 세포주를 공여세포로 체세포 핵 치환한 복제란(embryos)의 사진이다.
도 7은 상기 복제 배아를 이용하여 생산한 형질전환 복제돼지의 유전자 분석 결과이다(PC: 공여세포, NC: 야생형 유카탄 미니돼지, #1 내지 #7: 형질전환 복제돼지).
도 8은 야생형 돼지 및 본 발명에 따른 형질전환 복제돼지의 귀섬유아세포의 GFP 형광을 확인한 것이다.
도 9는 야생형 돼지 및 본 발명에 따른 형질전환 복제돼지의 염색체를 확인한 것으로, 도 9a 및 도 9b는 형질전환 복제돼지 2개체의 염색체 분석 결과이며, 도 9c는 야생형 돼지의 염색체 분석 결과이다.
도 10은 야생형 돼지 및 본 발명에 따른 형질전환 복제돼지 유래 귀섬유아세포에서 FLAG 태깅(taging) 단백질과 인간 ACE2 단백질의 발현을 확인한 결과이다(PC: 공여세포, NC: 야생형 돼지 유래 섬유아세포, TG1 및 TG2: 형질전환 복제돼지 유래 귀섬유아세포).
도 11은 야생형 돼지 및 본 발명에 따른 형질전환 복제돼지의 장기에서 인간 ACE2 단백질의 발현을 확인한 결과이다(PC: 공여세포, WT(야생형), TG(형질전환 복제돼지).
도 12는 야생형 돼지 및 본 발명에 따른 형질전환 복제돼지의 SARS-CoV-2 임상 증상을 비교한 것으로, 도 12a는 각 개체의 체중 증가율을 비교한 것이고, 도 12b는 각 개체의 체온을 비교한 것이다(Control: 야생형 돼지, ACE1 및 ACE2: 형질전환 복제돼지).
도 13은 야생형 돼지 및 본 발명에 따른 형질전환 복제돼지의 비강에서 바이러스의 배출을 확인한 결과로, 도 13a는 Egene RNA를 확인한 결과이고, 도 13b는 RdRP 유전자의 RNA를 확인한 결과이다(Control: 야생형 돼지, ACE1 및 ACE2: 형질전환 복제돼지, D1: 접종 1일후, D2: 접종 2일후, D3: 접종 3일후, D4: 접종 4일후, D5: 접종 5일 후).
도 14는 야생형 돼지 및 본 발명에 따른 형질전환 복제돼지의 폐 조직에서의 조직병리학적 증상을 비교한 결과이다(WT: 야생형 돼지, hACE2#1 및 hACE2#2: 형질전환 복제돼지).1 is a schematic diagram of a recombinant vector for expressing human angiotensin-converting enzyme 2 (ACE2) according to the present invention.
Figure 2 shows the transduction of the human ACE2 vector according to the present invention into female and male pig ear fibroblasts and then confirms whether or not the transduction was performed. It confirmed the FLAG protein expression, and FIG. 2c confirms the ACE2 protein expression of each cell.
3 is a result of confirming whether human ACE2 is expressed in the transformed cell line according to the present invention.
Figure 4 confirms the expression of human ACE2 in the transformed cell line according to the present invention, Figure 4a confirms the expression of human ACE2 protein, Figure 4b confirms the expression of the FLAG protein tagged to the human ACE2 gene (WT: fiber Blast cell line, TG: transformed cell line).
Figure 5 confirms the sensitivity to SARS-CoV-2 of four transformed cell lines according to the present invention (WT: fibroblast cell line, TG: transformed cell line).
6 is a photograph of cloned embryos in which the transformed cell line according to the present invention was replaced with a somatic cell nucleus by a donor cell.
7 shows the results of genetic analysis of transgenic cloned pigs produced using the cloned embryos (PC: donor cells, NC: wild-type Yucatan mini-pigs, #1 to #7: transgenic cloned pigs).
Figure 8 confirms the GFP fluorescence of otic fibroblasts of wild-type pigs and transgenic cloned pigs according to the present invention.
FIG. 9 shows the chromosomes of the wild-type pig and the transgenic cloned pig according to the present invention. FIGS. 9a and 9b show the chromosome analysis results of two transgenic cloned pigs, and FIG. 9c shows the chromosome analysis result of the wild-type pig.
10 is a result of confirming the expression of FLAG-tagged protein and human ACE2 protein in wild-type pig and otic fibroblasts derived from a transgenic cloned pig according to the present invention (PC: donor cells, NC: wild-type pig-derived fibroblasts, TG1 and TG2: otic fibroblasts derived from transgenic cloned pigs).
11 is a result of confirming the expression of human ACE2 protein in the organs of wild-type pigs and transgenic cloned pigs according to the present invention (PC: donor cells, WT (wild-type), TG (transgenic cloned pig)).
12 is a comparison of SARS-CoV-2 clinical symptoms of a wild-type pig and a transgenic cloned pig according to the present invention, FIG. 12a is a comparison of the weight gain rate of each individual, and FIG. 12b is a comparison of the body temperature of each individual (Control: wild-type pig, ACE1 and ACE2: transgenic cloned pig).
13 is a result of confirming the discharge of viruses from the nasal cavities of wild-type pigs and transgenic cloned pigs according to the present invention, FIG. 13a is the result of confirming the Egene RNA, and FIG. 13b is the result of confirming the RNA of the RdRP gene (Control: wild type). Pigs, ACE1 and ACE2: transgenic cloned pigs, D1: 1 day after inoculation, D2: 2 days after inoculation, D3: 3 days after inoculation, D4: 4 days after inoculation, D5: 5 days after inoculation).
14 is a comparison result of histopathological symptoms in lung tissues of wild-type pigs and transgenic cloned pigs according to the present invention (WT: wild-type pig, hACE2#1 and hACE2#2: transgenic cloned pig).

이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명은 인간 안지오텐신 전환효소 2(Angiotensin-converting enzyme 2, ACE2) 유전자, 인핸서 및 프로모터를 포함하는, 제2형 중증급성호흡기증후군 코로나바이러스(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터를 제공한다.The present invention is directed to Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including human angiotensin-converting enzyme 2 (ACE2) gene, enhancer and promoter. Provides a recombinant vector for producing animal models with increased susceptibility to

본 발명에 있어서, 상기 “인간 안지오텐신 전환효소 2(Angiotensin-converting enzyme 2, ACE2)”는 메탈로카복시펩티다제 (metallo-carboxypeptidase)의 하나로, 안지오텐신 전환효소와 상동하는 1형 막관통 단백질이며, 진핵생물과 세균에서 발견된다. 안지오텐신 전환효소 2는 체내 수분과 혈압을 조절하는 레닌-안지오텐신-알도스테론계(renin-angiotensin-aldosterone system, RAAS)에서 중요한 역할을 담당하며 심장, 폐, 콩팥, 혈관내피와 소화계통에 발현된다.In the present invention, the "human angiotensin-converting enzyme 2 (ACE2)" is a type 1 transmembrane protein homologous to angiotensin-converting enzyme, which is one of metallo-carboxypeptidases, It is found in eukaryotes and bacteria. Angiotensin converting enzyme 2 plays an important role in the renin-angiotensin-aldosterone system (RAAS), which regulates body water and blood pressure, and is expressed in the heart, lungs, kidneys, vascular endothelium, and digestive system.

본 발명에 있어서, 상기 인간 안지오텐신 전환효소 2(Human angiotensin converting enzyme 2, ACE2) 유전자는 SARS-CoV-2의 돌기 단백질(spike-protein)의 수용체 결합부위(Receptor Binding Domain, RBD)로 작용하는 것이다.In the present invention, the human angiotensin converting enzyme 2 (ACE2) gene is to act as a Receptor Binding Domain (RBD) of the spike-protein of SARS-CoV-2 .

본 발명에 있어서 상기 인간 안지오텐신 전환효소 2를 암호화하는 서열은 서열번호 1의 염기서열로 표시되는 것이 바람직하다. 또한 상기 SARS-CoV-2에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터는 서열번호 1의 염기서열로 표시되는 인간 안지오텐신 전환효소 2의 전체 또는 일부 서열을 포함할 수 있으며, 이의 기능적 동등물을 포함할 수 있다. 상기 “기능적 동등물”이란 염기의 결실(deletion), 치환(substitution) 또는 삽입(insertion)의 결과, 상기 서열번호 1의 염기서열과 적어도 70 % 이상, 바람직하게는 80 % 이상, 더욱 바람직하게는 90 % 이상, 더 더욱 바람직하게는 95 % 이상의 서열 상동성을 갖는 것으로, 서열번호 1의 염기서열로 표시되는 폴리뉴클레오티드와 실질적으로 동질의 생리활성을 나타내는 폴리뉴클레오티드를 말한다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.In the present invention, the sequence encoding the human angiotensin converting enzyme 2 is preferably represented by the nucleotide sequence of SEQ ID NO: 1. In addition, the recombinant vector for constructing an animal model with increased susceptibility to SARS-CoV-2 may include the entire or partial sequence of human angiotensin converting enzyme 2 represented by the nucleotide sequence of SEQ ID NO: 1, and a functional equivalent thereof can include The “functional equivalent” means at least 70%, preferably 80% or more, more preferably 80% or more of the nucleotide sequence of SEQ ID NO: 1 as a result of deletion, substitution or insertion of bases. It refers to a polynucleotide having a sequence homology of 90% or more, and more preferably 95% or more, and exhibiting substantially the same physiological activity as the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1. The "percentage of sequence homology" for polynucleotides is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. may include additions or deletions (i.e., gaps) compared to (not including).

본 발명에 있어서 상기 "벡터"는 적합한 숙주 내에서 목적 유전자를 발현시킬 수 있도록 적합한 조절 서열에 작동 가능하게 연결된 유전자의 염기서열을 포함하는 유전자 제작물을 의미하는 것으로, 상기 조절 서열은 전사를 개시할 수 있는 프로모터, 그러한 전사를 조절하기 위한 임의의 오퍼레이터 서열, 및 전사 및 해독의 종결을 조절하는 서열을 포함할 수 있다. 본 발명의 벡터는 세포 내에서 복제 가능한 것이면 특별히 한정되지 않고 당업계에 알려진 임의의 벡터를 이용할 수 있으며, 예컨대 플라스미드, 코즈미드, 파지 입자, 바이러스 벡터일 수 있다.In the present invention, the "vector" refers to a genetic construct comprising a nucleotide sequence of a gene operably linked to a suitable control sequence so as to express a target gene in a suitable host, and the control sequence is capable of initiating transcription. promoters capable of controlling transcription, optional operator sequences for regulating such transcription, and sequences regulating termination of transcription and translation. The vector of the present invention is not particularly limited as long as it can replicate in cells, and any vector known in the art may be used, for example, a plasmid, a cosmid, a phage particle, or a viral vector.

본 발명에 있어서 상기 "재조합 벡터"는, 발현시키고자 하는 목적 폴리펩타이드의 암호화 유전자가 작동 가능하게 연결될 경우, 적절한 숙주 세포에서 상기 목적 폴리펩타이드를 높은 효율로 발현시킬 수 있는 목적 폴리펩타이드의 발현 벡터로 사용될 수 있으며, 상기 재조합 벡터는 숙주 세포에서 발현 가능할 수 있다. 숙주 세포는 바람직하게는 진핵세포일 수 있으며, 숙주세포의 종류에 따라 프로모터(promoter), 종결자(terminator), 인핸서(enhancer) 등과 같은 발현 조절 서열, 막 표적화 또는 분비를 위한 서열 등을 적절히 선택하고 목적에 따라 다양하게 조합할 수 있다.In the present invention, the "recombinant vector" is an expression vector of a target polypeptide capable of expressing the target polypeptide with high efficiency in an appropriate host cell when the coding gene of the target polypeptide to be expressed is operably linked. It can be used as, and the recombinant vector can be expressed in a host cell. The host cell may preferably be a eukaryotic cell, and expression control sequences such as a promoter, terminator, and enhancer, sequences for membrane targeting or secretion, etc. are appropriately selected according to the type of host cell. and can be combined in various ways depending on the purpose.

본 발명에 있어서, 상기 “인핸서”는 DNA 주형의 구조적 변화를 유발하여 전사(transcription)가 더욱 활발하게 일어나도록 하는 부분이다. 상기 인핸서는 유전자마다 특유한 염기서열로 표시되며, 유전자 속의 어떤 위치에서도 전사를 촉진시킨다는 특징이 있다.In the present invention, the "enhancer" is a part that induces structural changes in the DNA template so that transcription occurs more actively. The enhancer is represented by a unique nucleotide sequence for each gene, and is characterized in that it promotes transcription at any position in the gene.

본 발명에 있어서, “프로모터”는 전사조절인자들이 결합하는 DNA 염기서열 부위를 의미하며, 목적 유전자의 과발현을 유도하기 위한 것이다. 상기 프로모터의 예로는 프리노브 박스(Pribnow box), 타타박스(TATA box) 등이 있다.In the present invention, "promoter" refers to a DNA base sequence region to which transcriptional regulators bind, and is intended to induce overexpression of a target gene. Examples of the promoter include a Pribnow box and a TATA box.

본 발명에 있어서, 상기 프로모터는 CAG 프로모터, CMV 프로모터, EF1α 프로모터, ICAM2 프로모터, SV40 프로모터, PGK1 프로모터, Ubc 프로모터 및 β-Act 프로모터로 이루어진 군에서 선택된 어느 하나의 외인성 프로모터 (exogenous promoter)일 수 있으나, 이에 제한되는 것은 아니다. 본 발명에 있어서 상기 인간 ACE2 유전자는 상기 외인성 프로모터를 이용한 무작위 삽입(random integration) 또는 넉인(knock-in) 방법으로 숙주에 도입될 수 있으나 이에 제한되는 것은 아니다.In the present invention, the promoter may be any one exogenous promoter selected from the group consisting of CAG promoter, CMV promoter, EF1α promoter, ICAM2 promoter, SV40 promoter, PGK1 promoter, Ubc promoter and β-Act promoter. , but is not limited thereto. In the present invention, the human ACE2 gene may be introduced into the host by random integration or knock-in using the exogenous promoter, but is not limited thereto.

본 발명에 있어서, 상기 재조합 벡터는 하기 도에 기재된 벡터맵을 갖는 것으로, 본 발명의 인간 ACE2를 발현시킬 수 있는 벡터의 구성이라면, 이에 제한되지 않는다.In the present invention, the recombinant vector has the vector map shown in the following figure, and is not limited thereto, as long as it is a vector capable of expressing human ACE2 of the present invention.

[도][do]

더불어 본 발명에 있어서, 상기 재조합 벡터는 서열번호 2의 염기서열로 표시되는 서열을 포함하는 것이 바람직하나, 이는 예시일 뿐 이에 본 발명이 제한되는 것은 아니다. 상기 서열번호 2는 태그 단백질(tag protein) 및 hACE2를 암호화하는 서열을 포함한다.In addition, in the present invention, the recombinant vector preferably includes the sequence represented by the nucleotide sequence of SEQ ID NO: 2, but this is only an example and the present invention is not limited thereto. SEQ ID NO: 2 includes a sequence encoding a tag protein and hACE2.

본 발명의 일실시예에 따르면, 서열번호 3의 염기서열로 표시되는, pCMV-5’FLAG 벡터 내에 서열번호 1의 인간 ACE2 유전자, CAGGS 프로모터 및 EGFP 형광 단백질을 포함하는 재조합 벡터를 사용하였다(도 1 참고).According to one embodiment of the present invention, a recombinant vector containing the human ACE2 gene of SEQ ID NO: 1, the CAGGS promoter, and the EGFP fluorescent protein in the pCMV-5'FLAG vector represented by the nucleotide sequence of SEQ ID NO: 3 was used (Fig. 1).

본 발명에 있어서, 상기 프로모터는 GGTA1(alpha 1,3- galactosyltransferase) 프로모터, ROSA26 프로모터 및 AAVS1(PPP1R12C locus) 프로모터로 이루어진 군에서 선택된 어느 하나의 내인성 프로모터(endogenous promoter)일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the promoter may be any one endogenous promoter selected from the group consisting of GGTA1 (alpha 1,3-galactosyltransferase) promoter, ROSA26 promoter, and AAVS1 (PPP1R12C locus) promoter, but is not limited thereto no.

상기 GGTA1은 돼지 고유의 유전자로 전 장기 및 혈액 등에서 잘 발현하는 유전자이며, 즉, 레프트 암(left arm)과 라이트 암(right arm) 사이의 GGTA1 단백질을 암호화하는 서열에 외래 유전자를 삽입하고 이를 GGTA1 유전자의 고유한 프로모터를 이용하여 발현될 수 있도록 하는 것이다. 상기 ROSA26이나 AAVS1은 외래 유전자를 삽입할 경우 유전자가 안전적, 안정적으로 잘 발현되는 좌위로, 외래 유전자가 삽입되어도 개체에 영향이 없다고 알려져 있어 넉-인 시스템(knock-in system)에서 주로 삽입되는 위치이다.The GGTA1 is a pig-specific gene that is well expressed in all organs and blood, that is, a foreign gene is inserted into the sequence encoding the GGTA1 protein between the left arm and the right arm, and this is GGTA1 It is to be expressed using the unique promoter of the gene. The ROSA26 or AAVS1 is a locus where the gene is expressed safely and stably when a foreign gene is inserted, and it is known that there is no effect on the individual even when the foreign gene is inserted, so it is mainly inserted in the knock-in system It is a location.

본 발명에 있어서, “레프트 암(left arm)” 및 “라이트 암(right arm)”은 상동 재조합(homologous recombination)이 일어나는 영역을 의미한다.In the present invention, “left arm” and “right arm” refer to a region where homologous recombination occurs.

본 발명에 있어서, “상동 재조합”은 상동성을 지닌 유전자 좌위에서 교환을 통하여 일어나는 유전자 재조합을 의미하며, 기능이 소실된 대립유전자를 지닌 형질전환 동물의 제조에 이용된다.In the present invention, “homologous recombination” refers to genetic recombination that occurs through exchange at homologous genetic loci, and is used to produce transgenic animals having functionally lost alleles.

본 발명에 있어서 상기 인간 ACE2 유전자는 상기 내인성 프로모터를 이용한 넉인 방법으로 숙주에 도입될 수 있으나 이에 제한되는 것은 아니다.In the present invention, the human ACE2 gene may be introduced into a host by a knock-in method using the endogenous promoter, but is not limited thereto.

본 발명에 있어서, 상기 재조합 벡터는 하기 도에 기재된 벡터맵을 갖는 것으로, 본 발명의 인간 ACE2를 발현시킬 수 있는 벡터의 구성이라면, 이에 제한되지 않는다.In the present invention, the recombinant vector has the vector map shown in the following figure, and is not limited thereto, as long as it is a vector capable of expressing human ACE2 of the present invention.

[도][do]

상기 도의 벡터맵은 내인성 프로모터를 포함하는 벡터로, 자체의 고유 프로모터를 사용하여 인간 ACE2를 발현하는 것을 특징으로 한다.The vector map of the above figure is a vector containing an endogenous promoter, and is characterized in that human ACE2 is expressed using its own promoter.

[도][do]

상기 도의 벡터맵은 넉인 시스템을 위한 외인성 프로모터를 포함하는 벡터를 나타낸다.The vector map in the above figure shows a vector containing an exogenous promoter for the knock-in system.

본 발명의 벡터는 개체 또는 세포 내에서 복제 가능한 것이면 특별히 한정되지 않고 당업계에 알려진 임의의 벡터를 이용할 수 있으며, 본 발명에 사용될 수 있는 벡터는 바이러스 벡터(viral vector), 플라스미드 벡터(plasmid vector), 코스미드 벡터(cosmid vector), 포스미드 벡터(fosmid vector), 파아지 벡터(phage vector), BAC(bacterial artificial chromosome) 벡터, 박테리오파아지 벡터(bacteriopharge vector), PAC(P1-based artificial chromosome) 벡터 및 YAC(yeast artificial chromosome) 벡터로 이루어진 군에서 선택된 하나 이상일 수 있다.The vector of the present invention is not particularly limited as long as it can be replicated in an individual or cell, and any vector known in the art can be used, and the vector that can be used in the present invention is a viral vector, a plasmid vector , cosmid vector, fosmid vector, phage vector, BAC (bacterial artificial chromosome) vector, bacteriophage vector, PAC (P1-based artificial chromosome) vector, and It may be one or more selected from the group consisting of yeast artificial chromosome (YAC) vectors.

더불어 상기 바이러스 벡터로서 이에 제한되는 것은 아니나, 렌티바이러스, 아데노바이러스, 레트로바이러스, 폭스바이러스, 헤르페스 바이러스, 알파바이러스, 포미 바이러스 및 아데노-관련 바이러스로 이루어진 군에서 선택된 어느 하나를 사용한 감염(infection)을 통해 인간 ACE2 유전자를 도입할 수 있다.In addition, infection using any one selected from the group consisting of, but not limited to, lentivirus, adenovirus, retrovirus, poxvirus, herpes virus, alphavirus, pomivirus, and adeno-associated virus as the viral vector can introduce the human ACE2 gene.

본 발명은 SARS-CoV-2의 연구 또는 비임상 시험을 위한 동물모델의 제조를 위한 것으로, 상기 "동물"은 인간 이외의 임의의 포유류 동물을 의미한다. 상기 동물은 배아, 태아, 신생아 및 성인을 포함하는 모든 연령의 동물을 포함한다. 본 발명에서 사용하기 위한 동물들은, 예를 들어, 상업용 소스로부터 이용할 수 있다. 이런 동물들은 실험용 동물 또는 다른 동물, 토끼, 설치류(예를 들어, 생쥐, 쥐, 햄스터, 게르빌루스 및 기니피그), 소, 양, 돼지, 염소, 말, 개, 고양이, 새(예를 들어, 닭, 칠면조, 오리, 거위), 영장류(예를 들어, 침팬지, 원숭이, 붉은털원숭이)를 포함하나 이에 제한되는 것은 아니다. 해부 생리학적으로 인간과 유사성이 높은 돼지인 것이 가장 바람직하다.The present invention is for the preparation of an animal model for research or non-clinical testing of SARS-CoV-2, and the "animal" means any mammal other than human. The animals include animals of all ages including embryos, fetuses, neonates and adults. Animals for use in the present invention are available, for example, from commercial sources. These animals include laboratory animals or other animals, rabbits, rodents (eg mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, horses, dogs, cats, birds (eg chickens, turkeys, ducks, geese), primates (eg chimpanzees, monkeys, rhesus monkeys). It is most preferable that the pig is highly similar to human anatomy and physiology.

또한 본 발명은 본 발명에 따른 재조합 벡터를 체세포에 형질전환시켜 제조한, SARS-CoV-2에 대한 감수성이 증가된 형질전환 세포주를 제공한다.In addition, the present invention provides a transformed cell line with increased sensitivity to SARS-CoV-2 prepared by transforming the recombinant vector according to the present invention into somatic cells.

본 발명에 있어서 상기 "세포주"는 세포를 분리해서 순수 배양하여 계대배양해 나갈때 세포계의 각 개체를 말하며, 이때 세포주는 유전적 형질에 의해 다른 세포주와 구별될 수 있으며, 계대배양에도 원 세포의 형질이 유지되는 것을 말한다.In the present invention, the "cell line" refers to each individual of the cell line when cells are separated, pure cultured, and subcultured. At this time, the cell line can be distinguished from other cell lines by genetic characteristics, and the original cell's characteristics it says to keep

본 발명에 있어서 상기 세포주는 난모세포주, 섬유아세포주 또는 신장세포주일 수 있으며, 바람직하게는 섬유아세포주이다. 상기 세포주는 보다 구체적으로 태아 유래 세포주를 이용할 수 있으며, 동시에 1차(primary) 세포주를 이용할 수 있는바, 본 발명의 세포주는 1차 섬유아세포주 또는 1차 신장세포주를 이용하는 것이 보다 바람직하고, 1차 섬유아세포주를 이용하는 것이 가장 바람직하다.In the present invention, the cell line may be an oocyte cell line, a fibroblast cell line or a renal cell line, preferably a fibroblast cell line. The cell line may more specifically use a fetal-derived cell line, and at the same time, a primary cell line may be used, and it is more preferable to use a primary fibroblast cell line or a primary renal cell line for the cell line of the present invention. It is most preferable to use a tea fibroblast cell line.

본 발명에 있어서 상기 "형질전환"은 외부로부터 주어진 DNA에 의하여 생물의 유전적인 성질이 변하는 것으로, 형질변환, 형전환 또는 형변환 등이라고도 한다. 즉, "형질전환"이란 유전자를 숙주세포 내에 도입하여 숙주세포 내에서 발현시킬 수 있도록 하는 것을 의미한다.In the present invention, the "transformation" refers to a change in the genetic properties of an organism by DNA given from the outside, and is also referred to as transformation, transformation or transformation. That is, "transformation" means introducing a gene into a host cell so that it can be expressed in the host cell.

본 발명의 SARS-CoV-2에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터를 세포주에 도입하여 형질전환하는 방법은 당분야에 공지된 방법을 이용하여 숙주세포 내로 도입할 수 있다. 본 발명에 따른 재조합 벡터를 숙주세포내로 도입하는 방법으로는, 이에 한정되지는 않으나 뉴클레오펙션(nucleofection), 일시적인 형질감염(transient transfection), 미세주사, 형질도입(transduction), 세포융합, 칼슘 포스페이트 침전법, 리포좀 매개된 형질감염(liposem-mediated transfection), DEAE 덱스트란-매개된 형질감염(DEAE Dextran-mediated transfection), 폴리브렌-매개된 형질감염(polybrene-mediated transfection), PEG(polyethylenglycol)에 의한 침전법, 입자 총 충격법(particle gun bombardment), 초음파 처리(sonication), 전기 침공법(electroporation), 열 쇼크(heat shock) 방법일 수 있다. 본 발명의 일실시예에서는 양이온성 지질을 사용한 리포펙션(lipofection) 방법으로 형질전환하였다.A method for transforming a cell line by introducing the recombinant vector for preparing an animal model with increased sensitivity to SARS-CoV-2 of the present invention can be introduced into a host cell using a method known in the art. Methods for introducing the recombinant vector according to the present invention into host cells include, but are not limited to, nucleofection, transient transfection, microinjection, transduction, cell fusion, calcium phosphate Precipitation method, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, PEG (polyethylenglycol) It may be a precipitation method, a particle gun bombardment method, a sonication method, an electroporation method, or a heat shock method. In one embodiment of the present invention, it was transformed by a lipofection method using a cationic lipid.

본 발명에 있어서, 상기 형질전환 세포주는 한국세포주연구재단(KCLRF)에 2021년 05월 04일자로 기탁하였으며, 수탁번호 KCLRF-BP-00512를 부여받았다.In the present invention, the transformed cell line was deposited with the Korea Cell Line Research Foundation (KCLRF) on May 04, 2021, and was given accession number KCLRF-BP-00512.

상기 형질전환된 세포주는 본원 발명에 속하는 기술분야에 공지된 방법에 따라 증식 및 배양될 수 있다. 적절한 배지는 동물 세포 및 특히, 포유동물 세포의 배양을 위해 개발되거나, 또는 동물 세포 성장에 필요한 적절한 성분, 예컨대 동화성 탄소, 질소 및/또는 미량 영양소와 함께 실험실 내에서 제조될 수 있는 임의의 이용가능한 배지를 사용할 수 있다.The transformed cell line may be propagated and cultured according to methods known in the art pertaining to the present invention. Suitable media are either developed for the culture of animal cells and in particular mammalian cells, or any use that can be prepared in the laboratory together with suitable components necessary for animal cell growth, such as assimilable carbon, nitrogen and/or micronutrients. Any available medium can be used.

상기 배지는 동물 세포 성장에 적절한 임의의 기본 배지, 비제한적인 예로서, 일반적으로 배양에 이용되는 기본 배지로는 MEM(Minimal Essential Medium), DMEM(Dulbecco modified Eagle Medium), RPMI(Roswell ParkMemorial Institute Medium), K-SFM(Keratinocyte Serum Free Medium)이 있으며, 이 외에도 당해 업계에서 이용되는 배지라면 제한없이 사용할 수 있다. 바람직하게는, α-MEM 배지(GIBCO), K-SFM 배지, DMEM배지(Welgene), MCDB 131배지(Welgene), IMEM배지(GIBCO), DMEM/F12 배지, PCM 배지, M199/F12(mixture)(GIBCO) 및 MSC 확장배지(Chemicon)로 구성된 군에서 선택될 수 있다.The medium is any basal medium suitable for animal cell growth, including but not limited to, basal medium generally used for culture includes MEM (Minimal Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium) ), K-SFM (Keratinocyte Serum Free Medium), and other media used in the industry can be used without limitation. Preferably, α-MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM/F12 medium, PCM medium, M199/F12 (mixture) (GIBCO) and MSC expansion medium (Chemicon).

이러한 기본 배지에, 탄소, 질소 및 미량 영양소의 동화성 공급원, 비제한적인 예로서, 혈청 공급원, 성장인자, 아미노산, 항생제, 비타민, 환원제, 및/또는 당 공급원이 첨가될 수 있다. 본원 발명에 속하는 기술분야에서 통상의 지식을 가진 자가 다양한 조직 기원 유래 줄기세포에 가장 적합한 배지를 선택 또는 조합하여 공지의 방법으로 적절히 배양할 수 있음은 자명할 것이다.To this basal medium can be added anabolic sources of carbon, nitrogen and micronutrients, such as but not limited to serum sources, growth factors, amino acids, antibiotics, vitamins, reducing agents, and/or sugar sources. It will be apparent that those skilled in the art can properly culture by a known method by selecting or combining the most suitable medium for stem cells derived from various tissues.

또한, 이 분야의 통상의 지식에 기초하여 적합한 배양 환경, 시간, 온도 등의 조건을 조절하면서 배양할 수 있음은 자명하다.In addition, it is obvious that culture can be performed while adjusting conditions such as a suitable culture environment, time, and temperature based on conventional knowledge in the field.

더불어 본 발명은 본 발명에 따른 형질전환 세포주를 탈핵시킨 돼지 수정란에 도입하여 핵치환된, SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지 생산을 위한 핵 이식란을 제공한다.In addition, the present invention provides nuclear transfer embryos for producing transgenic pigs for animal models with increased susceptibility to SARS-CoV-2, which are nuclear-transferred by introducing the transformed cell line according to the present invention into enucleated pig fertilized eggs.

본 발명에 있어서, 상기 "핵 이식"은 탈핵된 난자에 다른 세포 또는 핵을 인공적으로 결합시켜 동일한 형질을 갖도록 하는 유전자 조작기술을 말한다. "핵 이식란"은 핵 공여 세포가 도입 또는 융합된 난자를 말하며, 상기 핵 이식란은 복제란을 의미하는 것일 수 있다.In the present invention, the "nuclear transfer" refers to a genetic engineering technique in which other cells or nuclei are artificially incorporated into enucleated eggs to have the same traits. "Nuclear transfer embryo" refers to an egg into which a nuclear donor cell has been introduced or fused, and the nuclear transfer embryo may refer to a cloned egg.

본 발명에 있어서, 상기 “탈핵된 난자”는 핵이 제거된 난자를 의미한다.In the present invention, the “enucleated egg” means an egg whose nucleus has been removed.

본 발명에 있어서, 상기 "복제"는 한 개체와 동일한 유전자 세트를 가진 새로운 개체를 만드는 유전자 조작기술로서 특히 본 발명에서는 돼지의 체세포, 배아 세포, 태아 유래 세포 및/또는 성체 유래 세포가 다른 세포의 핵 DNA 서열과 실질적으로 동일한 핵 DNA 서열을 갖는 것을 말한다. 본 발명은 핵 이식 기술을 이용하여 돼지를 복제하는 기술을 이용한다. 특히, 체세포 핵 이식 기술은 생식과정에서 일반적으로 이루어지는 감수분열 및 반수 염색체 보유 생식세포를 경유하지 않고도 자손을 탄생시킬 수 있는 기술로서 성체가 가진 배수체 보유 체세포를 핵이 제거된 난자에 이식하여 수정란을 생산하고 상기 수정란을 생체 내로 이식하여 새로운 개체를 발생시키는 방법이다.In the present invention, the "cloning" is a genetic engineering technique to create a new individual having the same set of genes as one individual, and in particular, in the present invention, pig somatic cells, embryonic cells, fetal-derived cells, and/or adult-derived cells are used to reproduce other cells. It refers to having a nuclear DNA sequence that is substantially identical to a nuclear DNA sequence. The present invention uses a technology for cloning pigs using nuclear transfer technology. In particular, somatic cell nuclear transfer technology is a technology that allows the birth of offspring without going through meiosis and reproductive cells containing hemichromosomes, which are common in the reproductive process. It is a method of generating a new organism by producing and transplanting the fertilized egg into a living body.

본 발명에 있어서, 상기 "핵 공여 세포"는 핵 수용체인 수핵 난자로 핵을 전달하는 세포 또는 세포의 핵을 말한다. "난자"는 바람직하게는 제2차 감수분열 중기까지 도달한 성숙난자를 말한다. 본 발명에서 상기 핵 공여 세포로는 돼지의 체세포 또는 줄기세포를 사용할 수 있다.In the present invention, the "nuclear donor cell" refers to a cell or a nucleus of a cell that transfers a nucleus to a nuclear acceptor, that is, a recipient oocyte. "Oocyte" preferably refers to a mature egg that has reached the second meiosis metaphase. In the present invention, porcine somatic cells or stem cells may be used as the nuclear donor cells.

상기 “체세포(somatic cell)”란, 다세포 생물을 구성하는 세포 중 생식 세포 이외의 세포이며, 어떤 목적으로 특화하여 그 이외의 세포는 되지 않는 분화된 세포와, 몇종류인가의 다른 기능을 갖는 세포로 분화되는 능력을 갖는 세포를 포함한다.The above “somatic cell” refers to a cell other than a germ cell among cells constituting a multicellular organism, a differentiated cell that is specialized for a certain purpose and does not become a cell other than that, and a cell having several types of different functions. It includes cells having the ability to differentiate into.

또한 본 발명은 본 발명에 따른 형질전환 세포주를 핵 이식하여 제조된 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지를 제공한다.In addition, the present invention provides a transgenic pig for animal model with increased sensitivity to SARS-CoV-2 prepared by nuclear transfer of the transformed cell line according to the present invention.

본 발명에 있어서 상기 돼지는 당업자가 적절히 선택하여 사용할 수 있는, 공지된 임의의 종류를 사용할 수 있다. 상기 돼지(학명 :Susscrofadomestica)는 수아강,진수하강,맹수유제구 측추상목구,우제목(소, 염소를 포함), 멧돼지과에 속하며,염색체 수는 2n=38이다. 본래 돼지는 육용 가축으로 육성된 잡식동물이나, 생리 해부학적 소견이 사람과 유사한 점과 근년 동물복지에 대한 높은 관심과 함께 실험동물로서도 크게 주목받고 있다. 돼지는 해부학적으로 장기의 크기가 인간과 비슷하여 이종 장기모델로서 적합할 뿐만 아니라 생리, 유전학적으로도 인간과 유사한 점이 아주 많다. 또한 높은 번식능력을 가지고 있으므로 생산 및 치료용 생물 신소재 개발에 있어서도 적합할 뿐만 아니라 독성 및 안전성 평가에 좋은 동물모델이라 할 수 있다.In the present invention, the pig may be any known type that can be appropriately selected and used by those skilled in the art. The pig (scientific name: Susscrofadomestica) belongs to the Sua class, Jinsu class, Bengsu Yujegu sidechusang neck, Cattle (including cows and goats), and Boar family, and the number of chromosomes is 2n = 38. Originally, pigs are omnivores raised as livestock for meat, but their physiological anatomical findings are similar to those of humans, and in recent years, they have attracted great attention as laboratory animals with high interest in animal welfare. Pigs are anatomically similar in organ size to humans, making them suitable as xenogeneic organ models, as well as physiologically and genetically similar to humans. In addition, since it has high reproductive ability, it is suitable for the development of new biological materials for production and treatment, and it can be said to be a good animal model for toxicity and safety evaluation.

본 발명에서는 성숙시의 최대 체중이 사람에 가까운 60-70kg의 미니돼지(대표예: 피트만,무어)를 사용하거나, 실험에 사용하기 더 쉬운 소형돈(성체중: 30-40kg) 괴팅겐계, 유카탄계 미니돼지, 마이크로 돼지 등을 사용할 수도 있다. 또는 체형이 작을 뿐 아니라 특성이 분명한 근교계 미니돼지(NIH계)를 사용할 수 있다. 본 발명의 일 실시예에서는 유카탄 미니돼지를 사용하였다.In the present invention, mini pigs (representative examples: Pittman, Moore) of 60-70 kg, which have a maximum weight at maturity close to humans, are used, or small pigs (adult weight: 30-40 kg) that are easier to use in experiments, Goettingen-type, Yucatan-based mini-pigs and micro-pigs may also be used. Alternatively, an inbred mini-pig (NIH system) with a small body size and clear characteristics may be used. In one embodiment of the present invention, Yucatan mini-pigs were used.

더불어 본 발명은 본 발명에 따른 형질전환 세포주를 탈핵화된 난자에 이식하여 재구성된 난자를 제조하는 핵이식 단계; 및 상기 재구성된 난자를 대리모의 난관에 이식하는 단계;를 포함하는 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지의 제조방법을 제공한다.In addition, the present invention provides a nuclear transfer step of preparing a reconstructed egg by transplanting the transformed cell line according to the present invention to a denuclearized egg; and transplanting the reconstituted oocyte into the fallopian tube of a surrogate mother.

이에 있어, 공지의 임의의 방법을 이용하여 인간 ACE2 유전자로 형질전환시킬 수 있으며 바람직하게는 체세포 핵이식 방법(somatic cell nuclear transfer, SCNT)을 이용할 수 있다. 즉, 인간 ACE2 유전자를 발현시킨 형질전환 세포주를 이용한 체세포핵이식(SCNT) 방식을 이용하여 본 발명의 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지를 생산할 수 있다.In this regard, human ACE2 gene can be transformed using any known method, and preferably, somatic cell nuclear transfer (SCNT) can be used. That is, it is possible to produce a transgenic pig for an animal model with increased sensitivity to SARS-CoV-2 of the present invention using a somatic cell nuclear transfer (SCNT) method using a transgenic cell line expressing the human ACE2 gene.

본 발명의 일실시예에 따르면, 본 발명에 따른 인간 ACE2를 발현하는 형질전환 세포주 또는 인간 ACE2를 발현하는 형질전환 돼지는 SARS-CoV-2에 감염되었을 때 야생형 대비 높은 감수성을 보였다.According to one embodiment of the present invention, the transgenic cell line expressing human ACE2 or the transgenic pig expressing human ACE2 according to the present invention showed higher sensitivity than wild type when infected with SARS-CoV-2.

따라서 본 발명에 따른 인간 ACE2를 발현하는 형질전환 세포주 또는 인간 ACE2를 발현하는 형질전환 돼지는 SARS-CoV-2에 대한 연구 또는 상기 바이러스 감염 질환의 예방 또는 치료제 개발을 위한 비임상 모델로서 유용하게 활용할 수 있다. Therefore, the transgenic cell line expressing human ACE2 or the transgenic pig expressing human ACE2 according to the present invention can be usefully used as a non-clinical model for research on SARS-CoV-2 or development of a preventive or therapeutic agent for the viral infectious disease can

그러므로 본 발명은 본 발명에 따른 SARS-CoV-2에 대한 감수성이 증가된 형질전환 세포주 또는 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지를 이용한 SARS-CoV-2 감염 질환의 예방 또는 치료제의 스크리닝 방법을 제공한다.Therefore, the present invention is a treatment for SARS-CoV-2 infectious disease using a transgenic cell line with increased susceptibility to SARS-CoV-2 or a transgenic pig for animal model with increased susceptibility to SARS-CoV-2 according to the present invention. A method for screening a prophylactic or therapeutic agent is provided.

보다 구체적으로 상기 방법은 하기 단계를 포함한다:More specifically, the method includes the following steps:

1) 본 발명에 따른 SARS-CoV-2에 대한 감수성이 증가된 형질전환 세포주 또는 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지에 SARS-CoV-2를 접종하는 단계;1) inoculating SARS-CoV-2 to a transgenic cell line with increased sensitivity to SARS-CoV-2 or a transgenic pig for an animal model with increased sensitivity to SARS-CoV-2 according to the present invention;

2) 상기 SARS-CoV-2가 접종된 형질전환 세포주 또는 형질전환 돼지에 SARS-CoV-2 감염 질환의 예방 또는 치료제 후보물질을 투여하는 단계; 및2) administering a candidate material for preventing or treating SARS-CoV-2 infectious disease to the transformed cell line or transgenic pig inoculated with SARS-CoV-2; and

3) 후보물질을 투여한 군과 후보물질을 투여하지 않은 대조군을 비교하여 SARS-CoV-2 감염 질환의 증상 또는 조직병리학적 지표 값을 유의미하게 변화시키는 후보물질을 선택하는 단계.3) A step of selecting a candidate substance that significantly changes symptoms or histopathological index values of SARS-CoV-2 infectious disease by comparing the group administered with the candidate substance and the control group not administered with the candidate substance.

상기 증상 또는 조직병리학적 지표 값은 SARS-CoV-2 감염시 나타나는 공지된 임상 증상 또는 조직병리학적 증상일 수 있다. 이에 제한되는 것은 아니나 상기 임상 증상은 발열, 호흡곤란, 두통, 인후통, 객혈, 오심, 기침, 객담, 설사, 후각 상실, 권태감, 식욕부진, 근육통 및 인지장애로 이루어진 군에서 선택된 어느 하나 이상일 수 있다.The symptoms or histopathological index values may be known clinical symptoms or histopathological symptoms that appear during SARS-CoV-2 infection. Although not limited thereto, the clinical symptoms may be any one or more selected from the group consisting of fever, dyspnea, headache, sore throat, hemoptysis, nausea, cough, expectoration, diarrhea, loss of smell, malaise, anorexia, muscle pain and cognitive impairment. .

더불어 상기 조직병리학적 지표 값은 염증성 사이토카인의 분비, 부종, 국소 출혈 및 염증세포 침윤 여부로 이루어진 군에서 선택된 어느 하나 이상일 수 있으나 이에 제한되는 것은 아니다.In addition, the histopathological index value may be one or more selected from the group consisting of inflammatory cytokine secretion, edema, local hemorrhage, and inflammatory cell infiltration, but is not limited thereto.

또한 상기 조직병리학적 지표 값은 SARS-CoV-2가 배출될 수 있는 조직 또는 SARS-CoV-2 감염에 의하여 영향을 받는 다양한 조직에서 평가될 수 있으며, 이에 제한되는 것은 아니나 폐, 간, 뇌, 비강, 두피, 눈, 신장, 심장 및 장으로 이루어진 군에서 선택된 어느 하나 이상의 조직에서 평가될 수 있다.In addition, the histopathological index value can be evaluated in tissues where SARS-CoV-2 can be excreted or various tissues affected by SARS-CoV-2 infection, but are not limited thereto, such as lung, liver, brain, It may be evaluated in any one or more tissues selected from the group consisting of nasal cavity, scalp, eye, kidney, heart and intestine.

더불어 본 발명은 본 발명에 따른 인간 ACE2를 발현하는 형질전환 세포주를 이용하여, 인간 ACE2 단백질을 생산하는 방법을 제공할 수 있다.In addition, the present invention can provide a method for producing human ACE2 protein using a transformed cell line expressing human ACE2 according to the present invention.

더불어 본 발명은 본 발명에 따른 인간 ACE2를 발현하는 형질전환 세포주를 배양하는 단계; 및 상기 배양된 인간 ACE2를 발현하는 형질전환 세포주 또는 상기 세포주 배양 배지로부터 발현된 인간 ACE2 단백질을 수득하는 단계를 포함하는 인간 ACE2 단백질 제조방법을 제공할 수 있다.In addition, the present invention comprises the steps of culturing a transformed cell line expressing human ACE2 according to the present invention; and obtaining human ACE2 protein expressed from the cultured human ACE2-expressing transformed cell line or the cell line culture medium.

본 발명에 있어서, 상기 "배양"은 세포를 적당히 인공적으로 조절한 환경조건에서 생육시키는 것을 의미한다. 상기 세포는 통상의 배지에서 생육 가능하다. 배지는 특정 세포를 배양하기 위하여 배양대상 즉 배양체가 되는 세포가 필요로 하는 영양물질을 포함하는 것으로 특수한 목적을 위한 물질이 추가로 첨가되어 혼합된 것일 수 있다. 상기 배지는 배양기 또는 배양액이라고도 하며, 천연배지, 합성배지 또는 선택배지를 모두 포함하는 개념이다. In the present invention, the "cultivation" means to grow the cells in an appropriately artificially controlled environmental conditions. The cells can grow in a normal medium. The medium contains nutrients required by the cells to be cultured, that is, cultured cells, in order to culture specific cells, and may be mixed with additional substances for special purposes. The medium is also referred to as an incubator or a culture medium, and is a concept that includes all of a natural medium, a synthetic medium, or a selective medium.

상술한 본 발명의 내용은 상호 모순되지 않는 한, 서로 동일하게 적용되며, 당해 기술분야의 통상의 기술자가 적절한 변경을 가해 실시하는 것 또한 본 발명의 범주에 포함된다.The contents of the present invention described above are equally applied to each other unless contradictory to each other, and implementation by adding appropriate changes by a person skilled in the art is also included in the scope of the present invention.

이하 본 발명을 실시예를 통해 상세하게 설명하나 본 발명의 범위가 하기 실시예로만 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail through examples, but the scope of the present invention is not limited only to the following examples.

실시예 1. 인간 안지오텐신 전환효소 2(Angiotensin-converting enzyme 2, ACE2) 발현을 위한 재조합 벡터 제조Example 1. Preparation of a recombinant vector for the expression of human angiotensin-converting enzyme 2 (ACE2)

인간 ACE2 유전자 발현을 위하여 염기서열을 분석한 후 human cDNA library를 주형가닥으로 증폭을 실시하였다. 보다 구체적으로, pCMV-5’FLAG vector에 제한효소를 이용하여 서열번호 1의 인간 ACE2 유전자와 CAGGS 프로모터에 의해 EGFP 형광 단백질이 발현될 수 있도록 클로닝을 진행하였다. 벡터 모식도는 도 1에 나타내었으며 완성된 벡터 서열 분석을 수행하여 유전자 삽입 여부를 확인하였다(솔젠트사에서 수행). 전체 벡터 서열은 서열번호 3에 나타내었다.After analyzing the nucleotide sequence for human ACE2 gene expression, the human cDNA library was amplified with the template strand. More specifically, cloning was performed using a restriction enzyme in the pCMV-5'FLAG vector so that EGFP fluorescent protein could be expressed by the human ACE2 gene of SEQ ID NO: 1 and the CAGGS promoter. A schematic diagram of the vector is shown in FIG. 1, and gene insertion was confirmed by performing sequence analysis of the completed vector (performed by Solgent). The full vector sequence is shown in SEQ ID NO:3.

실시예 2. ACE2를 발현하는 형질전환 세포주 구축Example 2. Construction of transformed cell lines expressing ACE2

상기 실시예 1을 통해 제조한 재조합 벡터를 야생형 암컷과 수컷 유카탄 미니돼지 귀 섬유아세포에 리포펙타민 3000(lipofectamine 3000(Invitrogen, CA, USA))을 이용하여 형질도입하였다. 보다 구체적으로, 건강한 야생형 유카탄 미니돼지의 귀를 70 % 에탄올로 닦은 후 이각기를 이용해 귀 조직을 수득했다. 1X anti-anti(항생제, Gibco, NY, USA)가 포함된 DPBS에 넣어서 실험실로 운반한 후 6-웰 멀티디쉬(6-well multidish)에 조직을 부착해 귀 조직 유래 섬유아세포(porcine ear fibroblast, PEF)를 15% 소태아혈청(fetal bovine serum(Hyclone, Utah, USA))과 1X 페니실린(penicilin)/스트렙토마이신(streptomycin)(Gibco, NY, USA)가 포함된 DMEM(Welgene, 한국) 배지에 분리배양하였다. 형질도입 전날 상기 세포 1 ×106개를 10cm 세포 배양 디쉬로 옮기고 다음날 리포펙타민 3000의 프로토콜에 따라 형질도입하였다. 도입된 세포는 FLAG 태깅(taging) 단백질을 이용해 FACS(Fluorescence activated cell sorter (FACS AriaIII, BDbioscience, CA, USA))로 선별하였고(sorting) 그 결과를 도 2a 내지 도 2c에 나타내었다. The recombinant vector prepared in Example 1 was transduced into wild-type female and male Yucatan minipig ear fibroblasts using lipofectamine 3000 (Invitrogen, CA, USA). More specifically, the ears of healthy wild-type Yucatan mini-pigs were cleaned with 70% ethanol, and then ear tissues were obtained using a decapsulator. After putting it in DPBS containing 1X anti-anti (antibiotic, Gibco, NY, USA) and transporting it to the laboratory, the tissue was attached to a 6-well multidish and porcine ear fibroblasts (porcine ear fibroblast, PEF) in DMEM (Welgene, Korea) medium containing 15% fetal bovine serum (Hyclone, Utah, USA) and 1X penicillin/streptomycin (Gibco, NY, USA). cultured separately. The day before transduction, 1 × 10 6 of the cells were transferred to a 10 cm cell culture dish, and transduced according to the protocol of Lipofectamine 3000 the next day. The introduced cells were sorted by FACS (Fluorescence activated cell sorter (FACS AriaIII, BDbioscience, CA, USA)) using FLAG-tagged protein, and the results are shown in FIGS. 2A to 2C.

그 결과 도 2a 내지 도 2와 같이, 형질도입한 세포에서 FLAG 단백질이 잘 발현되는 것을 확인하였고 인간 ACE2 단백질 또한 잘 발현되는 것을 확인하였다. 분리된 세포는 단일세포주 배양을 통해 증식시킨 후 단백질 발현 확인을 위해 FACS 분석을 실시하였다. 그 결과 도 3에 나타낸 바와 같이, 선별된 세포주 4종(#2, #27, #33, #37)에서 인간 ACE2 단백질이 잘 발현되는 것을 확인하였다. 더불어 추가적으로 단백질 발현 확인을 위해 웨스턴 블랏을 실시하였다. 보다 구체적으로 구축된 세포주를 단백질가수분해효소 억제제(proteinase inhibitor)가 포함된 RIPA buffer(Biosesang, 한국)를 세포에 처리한 후 얼음에 10 분간 인큐베이션하였다. 이를 음파처리(sonication)한 후, 10분간 다시 얼음에 인큐베이션하고 4℃에서 13000 rpm의 속도로 20 분간 원심분리하였다. 상등액을 새로운 튜브로 옮긴 뒤 10% 아크릴아미드(acrylamide) SDS-PAGE젤에 로딩하였고 니트로셀룰로오스 막(nitrocellulose membrane)에 단백질을 옮겼다. 5% 탈지유(skim milk)가 포함된 TBST(Tris Buffered Saline with Tween 20)에 1시간동안 교반하였다. 그 후 인간 ACE2 항체(Santacruz, CA, USA)와 FLAG(Siama, MO, USA) 항체를 각각 1:1000의 농도로 처리하고 4 ℃에서 하룻밤동안 반응시켰다. 이를 TBST 버퍼를 이용해 10 분간 3 회 세척한 뒤 5% 탈지유가 포함된 TBST에 1:1000의 농도로 각 항체에 맞는 HRP가 접합된 2 차 항체를 처리하고 상온에서 1 시간동안 반응시켰다. 그 후 TBST 버퍼로 10 분간 3 회 세척한 뒤 ECL(enhanced chemiluminescence) 용액을 이용해 발색하여 그 결과를 확인했다. 그 결과 도 4에 나타낸 바와 같이, 같은 사이즈(130 kDa)에서 ACE2 단백질(도 4a)과 인간 ACE2 유전자에 태깅된 FLAG 단백질(도 4b)의 밴드가 확인되었다. 따라서 본 결과를 기반으로 본 발명의 실시예에서 구축한 형질전환 세포주에서 인간 ACE2 단백질이 잘 발현되는 것을 확인하였다.As a result, as shown in FIGS. 2a to 2, it was confirmed that the FLAG protein was well expressed in the transduced cells, and that the human ACE2 protein was also well expressed. The isolated cells were proliferated through single cell line culture and FACS analysis was performed to confirm protein expression. As a result, as shown in FIG. 3, it was confirmed that the human ACE2 protein was well expressed in the four selected cell lines (#2, #27, #33, and #37). In addition, Western blotting was performed to additionally confirm protein expression. More specifically, the constructed cell line was treated with RIPA buffer (Biosesang, Korea) containing a proteinase inhibitor, and then incubated on ice for 10 minutes. After sonication, it was incubated on ice again for 10 minutes and centrifuged at 4° C. at 13000 rpm for 20 minutes. The supernatant was transferred to a new tube, loaded on a 10% acrylamide SDS-PAGE gel, and the protein was transferred to a nitrocellulose membrane. The mixture was stirred for 1 hour in TBST (Tris Buffered Saline with Tween 20) containing 5% skim milk. Thereafter, human ACE2 antibody (Santacruz, CA, USA) and FLAG (Siama, MO, USA) antibody were treated at a concentration of 1:1000, respectively, and reacted overnight at 4°C. After washing with TBST buffer three times for 10 minutes, the secondary antibody was treated with HRP-conjugated secondary antibodies at a concentration of 1:1000 in TBST containing 5% skim milk and reacted at room temperature for 1 hour. Then, after washing with TBST buffer three times for 10 minutes, the color was developed using an ECL (enhanced chemiluminescence) solution to confirm the result. As a result, as shown in FIG. 4, bands of the ACE2 protein (FIG. 4a) and the human ACE2 gene-tagged FLAG protein (FIG. 4b) of the same size (130 kDa) were identified. Therefore, based on this result, it was confirmed that the human ACE2 protein was well expressed in the transformed cell line constructed in Examples of the present invention.

실시예 3. 형질전환 세포주의 SARS-CoV-2에 대한 감수성 검증Example 3. Verification of susceptibility to SARS-CoV-2 of transformed cell lines

상기 실시예 2에서 구축된 형질전환 세포주 4 종과 야생형 세포주(섬유아세포주)를 SARS-CoV-2에 감염시킨 후 이에 대한 감수성을 확인하였다. 감염된 세포는 SARS-CoV-2의 ORF1b 유전자와 RdRp 유전자에 대해 실시간 역전사 중합효소 연쇄방법(Realtime RT-PCR)으로 증폭하여 바이러스 존재여부를 검사하였고 그 결과를 도 5에 나타내었다(Median에서 수행, 한국). After infecting the four transformed cell lines constructed in Example 2 and the wild-type cell line (fibroblast cell line) with SARS-CoV-2, their susceptibility was confirmed. Infected cells were amplified by real-time reverse transcription polymerase chain method (Realtime RT-PCR) for the ORF1b gene and RdRp gene of SARS-CoV-2 to examine the presence of the virus, and the results are shown in FIG. 5 (carried out by Median, korea).

그 결과, 야생형 대비 인간 ACE2 단백질이 발현되는 4 종의 형질전환 세포주에서 SARS-CoV-2에 대해 높은 감수성이 나타남을 확인하였다.As a result, it was confirmed that the 4 types of transformed cell lines expressing human ACE2 protein showed higher susceptibility to SARS-CoV-2 compared to the wild type.

상기 형질전환 세포주 TG#2(hACE2-TG라 명명), TG#27, TG#33 및 TG#37 중 TG#2(hACE2-TG)을 한국세포주연구재단에 KCLRF-BP-00512로 수탁하였다.Among the transformed cell lines TG#2 (named hACE2-TG), TG#27, TG#33 and TG#37, TG#2 (hACE2-TG) was entrusted to the Korea Cell Line Research Foundation as KCLRF-BP-00512.

실시예 4. 형질전환 복제돼지 생산Example 4. Production of transgenic cloned pigs

4-1. 난모세포(oocyte)의 준비, 핵이식란 제작 및 대리모돈 이식 4-1. Preparation of oocytes, production of nuclear transfer embryos, and transplantation into surrogate mothers

상기 실시예 2에서 구축한 형질전환 세포주를 이용해 기존 공지된 방법(Theriogenology. 127 80-87, 2019 참고)을 약간 수정하여 핵이식란을 제작하였고, 이를 대리모돈에 이식하였다. 먼저 미성숙 암돼지의 난소를 수득한 후, 35 ℃로 유지된 0.9 % NaCl 용액에 넣어 실험실로 운반하였다. 10 mL 일회용 주사기에 고정된 18-게이지 바늘을 이용해 2-6 mm 지름의 미성숙 난포로부터 축적 난모세포 복합체(Cumulus-oocyte complexes, COCs)를 흡입하였다. 상기 COCs를 0.1 % 폴리비닐알코올(Polyvinyl alcohol), 3.05 mM D-글루코스(D-glucose), 0.91 mM 피부르산 나트륨(Sodium Pyruvate), 0.57 mM 시스테인(cysteine), 0.5 μg/ml 황체형성호르몬(luteinizing hormone, LH, (Sigma)), 0.5 μg/ml 여포자극호르몬(follicle stimulating hormone, FSH, (Sigma)), 10 ng/mL 표피생장인자(epithelial growth factor, EGF, (Sigma)), 75 μg/ml 페니실린 G 및 50 μg/ml 스트렙토마이신(streptomycin (Gibco))을 포함한 TCM-199(Gibco)로 3회 세척하였다. 약 50 내지 60 COCs를 미네랄 오일(Mineral oil)로 덮인 4-웰 멀티디쉬에 옮긴 후, 500 μL의 동일한 배지를 첨가하고, 5 %의 CO2 및 39 ℃의 조건에서 배양하였다. 배양된 COCs는 42 내지 44 시간 후에 0.1 % PVA 및 0.2 % 히알루로니다아제(hyaluronidase)를 포함하는 TL-HEPES에서 4분동안 강하게 볼텍싱(vortexing)하여 난구세포로부터 난모세포를 분리하였다. 0.3 % 소혈청알부민(bovine serum albumin, BSA, (Sigma)) 및 7.5 μg/ml 사이토칼라신 B(cytochalasin B)를 포함하는 TCM-199에서 미세 유리 피펫을 이용하여 제1 극체 및 인접 세포질을 흡입함으로써 난모세포에서 세포핵을 제거하였다. 체세포 핵치환 이식(Somatic cell nuclear transfer, SCNT)에 앞서 혈청 기아(serum starvation)을 위하여 상기 실시예 2에서 제조한 공여 세포를 0.5 % FBS를 포함하는 DMEM 배지에서 3 일동안 배양하였다. 공여 세포를 난모세포 막과 접촉하는 난모세포의 위란강(periviteline space)에 위치하였다. 접종된 난모세포를 0.3 M 만니톨(mannitol), 1.0 mM CaCl2, 0.1 mM MgCl2 및 0.5 mM HEPES로 이루어진 배지에서 1 mm 간격의 0.2 mm 직경의 두 백금 전극 사이에 배치하였다. 융합/활성화(Fusion/activation)는 30 μs(BTX, USA) 동안 1.1 kV/cm의 DC 펄스를 2 회 연속적으로 가하여 유도하였다. 그 후, 20 내지 30 개의 재구성된 배아(reconstrcted embryos)를 미네랄 오일이 덮인 4-웰 멀티디쉬에 옮기고, 500 mL의 0.4 % BSA를 보충한 NCSU(North Carolina State University)-23 배지를 첨가하였다. 배양 7 일 후 배아를 형광현미경하에 관찰한 결과 도 6과 같이, 핵이식란된 배아에서 GFP 형광이 잘 나타남을 확인하였다. 재구성된 배아를 승가 허용기(standing estrus)의 첫 날인 암돼지의 난관으로 외과적인 수술 방법을 이용해 이식하였다. 임신 상태는 초음파 스캐너(Mysono 201, Medison Co. LTD, 한국)로 확인하였다.Using the transformed cell line constructed in Example 2, a previously known method (refer to Theriogenology. 127 80-87, 2019) was slightly modified to prepare nuclear transfer embryos, which were transplanted into surrogate sows. First, ovaries of immature female pigs were obtained, and then put in a 0.9% NaCl solution maintained at 35° C. and transported to a laboratory. Cumulus-oocyte complexes (COCs) were aspirated from immature follicles with a diameter of 2-6 mm using an 18-gauge needle fixed to a 10 mL disposable syringe. The COCs were mixed with 0.1% polyvinyl alcohol, 3.05 mM D-glucose, 0.91 mM Sodium Pyruvate, 0.57 mM cysteine, and 0.5 μg/ml luteinizing hormone. hormone, LH, (Sigma)), 0.5 μg/ml follicle stimulating hormone (FSH, (Sigma)), 10 ng/mL epidermal growth factor (EGF, (Sigma)), 75 μg/ml Washed three times with TCM-199 (Gibco) containing ml penicillin G and 50 μg/ml streptomycin (Gibco). About 50 to 60 COCs were transferred to a 4-well multi-dish covered with mineral oil, 500 μL of the same medium was added, and cultured at 5% CO 2 and 39 °C. The cultured COCs were vortexed vigorously for 4 minutes in TL-HEPES containing 0.1% PVA and 0.2% hyaluronidase after 42 to 44 hours to separate oocytes from cumulus cells. Aspirate the first polar body and adjacent cytoplasm using a fine glass pipette in TCM-199 containing 0.3% bovine serum albumin (BSA, (Sigma)) and 7.5 μg/ml cytochalasin B By doing so, the cell nucleus was removed from the oocyte. Prior to somatic cell nuclear transfer (SCNT), the donor cells prepared in Example 2 were cultured for 3 days in DMEM medium containing 0.5% FBS for serum starvation. Donor cells were placed in the periviteline space of the oocyte in contact with the oocyte membrane. Inoculated oocytes were placed between two 0.2 mm diameter platinum electrodes spaced 1 mm apart in medium consisting of 0.3 M mannitol, 1.0 mM CaCl 2 , 0.1 mM MgCl 2 and 0.5 mM HEPES. Fusion/activation was induced by applying two consecutive DC pulses of 1.1 kV/cm for 30 μs (BTX, USA). Then, 20 to 30 reconstructed embryos were transferred to a 4-well multi-dish covered with mineral oil, and 500 mL of NCSU (North Carolina State University)-23 medium supplemented with 0.4% BSA was added. As a result of observing the embryos under a fluorescence microscope after 7 days of culture, as shown in FIG. 6, it was confirmed that GFP fluorescence was well displayed in the nuclear transfer embryos. The reconstructed embryos were surgically implanted into the fallopian tubes of sows on the first day of standing estrus. Pregnancy status was confirmed with an ultrasound scanner (Mysono 201, Medison Co. LTD, Korea).

4-2. 형질전환 복제돼지의 검증4-2. Verification of transgenic cloned pigs

상기 실시예 4-1을 통해 생산된 형질전환 복제돼지 7 두의 탯줄을 이용해 유전자 분석을 실시하였다. 생산된 자돈 유래 꼬리 조직을 수득한 후 Dneasy Blood & Tissue Kits(Qiagen, GmbH, Germany)을 이용해 프로토콜을 따라 게놈 DNA를 분리하였다. 분리된 게놈 DNA를 주형으로 정방향 프라이머 (5'-GTACATCTACGTATTAGTCATCGC-3' (서열번호 4)), 역방향 프라이머(5'-GTTCAACCGTTTGCTCTTGTCTTC-3' (서열번호 5)), Profi PCR premix(Bioneer, 한국)를 혼합하여 최초 변성 95 ℃에서 5 분 후 95 ℃에서 40 초, 60 ℃에서 40 초, 72 ℃에서 1 분의 조건으로 35회 반복, 마지막 72 ℃에서 7분동안 PCR(Applied Biosystems)을 수행하였다. 완료된 PCR 산물은 1 % 아가로오스(agarose) TAE 젤에 로딩한 뒤 밴드를 확인하였고 그 결과를 도 7에 나타내었다. 그 결과 형질전환 복재돼지 #1, #2, #3 및 #6 개체에서 인간 ACE2 발현 벡터가 잘 삽입된 것을 확인하였다. 더불어 선별된 개체 유래 귀조직 섬유아세포를 실시예 2에 기재한 바와 같이 수득한 후 형광현미경(Olympus, Japan)을 이용해 벡터내 삽입된 GFP 형광 단백질을 확인하였다. 그 결과 도 8과 같이, 인간 ACE2 발현 벡터가 삽입된 형질전환 돼지 유래 귀섬유아세포에서 GFP 형광이 잘 나타남을 확인하였다. 또한 개체 유래 귀섬유아세포를 이용해 karyotyping과 FISH (Fluorescence In-situ hybridization)를 통해 염색체 분석을 실시하여(Gendix, 한국) 그 결과를 도 9에 나타내었다. 그 결과, 정상적인 돼지의 염색체 (상동염색체 18쌍+성염색체)를 갖는 것을 확인하였고 인간 ACE2 특이 프로브를 합성한 후 분석한 결과 두 개체 모두 돼지 염색체 17번에 삽입된 것을 확인하였다(화살표 참고).Genetic analysis was performed using the umbilical cords of 7 transgenic cloned pigs produced in Example 4-1. After obtaining the produced piglet-derived tail tissue, genomic DNA was isolated using Dneasy Blood & Tissue Kits (Qiagen, GmbH, Germany) according to the protocol. Using the isolated genomic DNA as a template, forward primer (5'-GTACATCTACGTATTAGTCATCGC-3' (SEQ ID NO: 4)), reverse primer (5'-GTTCAACCGTTTGCTCTTGTCTTC-3' (SEQ ID NO: 5)), Profi PCR premix (Bioneer, Korea) After mixing, PCR (Applied Biosystems) was performed at 95 ° C for 5 minutes, 95 ° C for 40 seconds, 60 ° C for 40 seconds, 72 ° C for 1 minute, repeated 35 times, and finally at 72 ° C for 7 minutes. The completed PCR product was loaded on a 1% agarose TAE gel, and the band was confirmed, and the results are shown in FIG. 7 . As a result, it was confirmed that the human ACE2 expression vector was well inserted in the transgenic pigs #1, #2, #3, and #6. In addition, after obtaining otic tissue fibroblasts derived from the selected individual as described in Example 2, the GFP fluorescent protein inserted into the vector was confirmed using a fluorescence microscope (Olympus, Japan). As a result, as shown in FIG. 8, it was confirmed that GFP fluorescence was well displayed in the transgenic pig-derived otic fibroblasts into which the human ACE2 expression vector was inserted. In addition, chromosome analysis was performed through karyotyping and FISH (Fluorescence In-situ hybridization) using individual-derived otic fibroblasts (Gendix, Korea), and the results are shown in FIG. 9 . As a result, it was confirmed to have normal pig chromosomes (18 pairs of homologous chromosomes + sex chromosomes), and as a result of analysis after synthesizing a human ACE2-specific probe, it was confirmed that both individuals were inserted into pig chromosome 17 (see arrow).

실시예 5. 형질전환 복제돼지의 특성 분석Example 5. Analysis of characteristics of transgenic cloned pigs

5-1. 단백질 발현 분석5-1. Protein expression analysis

상기 실시예 4를 통해 생산된 인간 ACE2 발현 벡터가 삽입된 형질전환 복제돼지 유래 귀섬유아세포를 이용해 상기 실시예 2와 동일한 방법으로 웨스턴 블랏을 실시하였다. 그 결과 도 10과 같이, PC(Positive control, 공여세포)와 같은 사이즈에서 FLAG 태깅 단백질과 인간 ACE2 단백질 밴드가 잘 생성됨을 확인하였고, 도 11에 나타낸 바와 같이, 안락사시킨 형질전환 복제돼지 1두 유래 심장, 폐, 소장을 이용한 결과에서도 인간 ACE2 단백질 밴드가 잘 생성되는 것을 확인하였다. Western blotting was performed in the same manner as in Example 2 using the transgenic cloned pig-derived otic fibroblasts into which the human ACE2 expression vector produced in Example 4 was inserted. As a result, as shown in FIG. 10, it was confirmed that FLAG-tagged protein and human ACE2 protein bands were well produced in the same size as PC (Positive control, donor cells), and as shown in FIG. 11, derived from one euthanized transgenic cloned pig. It was also confirmed that the human ACE2 protein band was well produced in the results using the heart, lung, and small intestine.

5-2. SARS-CoV-2 접종시험5-2. SARS-CoV-2 inoculation test

상기 실시예 4를 통해 생산된 형질전환 복제돼지와 야생형 돼지(유카탄 미니돼지)를 이용해 SARS-CoV-2 공격접종 시험을 실시하였다(인수공통전염병연구소에서 수행). 개체에 비강으로 SARS-CoV-2를 접종한 후 매일 체중과 체온을 측정한 결과 도 12a와 같이 야생형 대비 인간 ACE2 발현 형질전환 복제돼지에서 체중 증가율이 감소하였고, 도 12b와 같이 체온은 증가한 것으로 나타났다. 이는 SARS-CoV-2 감염의 일반적인 임상 증상이며 특히 공격접종 후 유카탄 미니돼지는 정상 체온(39.2 ℃ ±0.5, sinclair)을 넘는 고열 증상을 나타냈고 무기력 증상 또한 확인되었다. 접종 3 일 이후 각 개체의 E gene, RdRP gene 유전자 분석(seegene사 키트 이용, 한국)을 수행한 결과, 도 13과 같이 야생형 대비 형질전환 복제돼지의 비강에서 바이러스 배출(viral shedding)이 확인되었다. 또한 접종 5 일 후 각 개체를 안락사한 후 조직에 대한 조직병리학적 검사를 실시하였다. 분석 결과 도 14에 나타낸 바와 같이, 야생형 대비 인간 ACE2 발현 형질전환 복제돼지의 폐 일부에서 폐포 내 부종성 변화를 동반한 국소 출혈이 확인되었고, 폐엽 중 절반 이상에서 경도의 혈관 및 세기관지주위 염증세포 침윤을 확인하였으며 주로 침윤된 세포는 단핵염증세포 계열로 확인되었다. A SARS-CoV-2 challenge inoculation test was conducted using the transgenic cloned pigs produced in Example 4 and wild-type pigs (Yucatan mini-pigs) (conducted by the Institute of Zoonotic Infectious Diseases). As a result of measuring body weight and body temperature every day after inoculating SARS-CoV-2 intranasally into the individual, as shown in FIG. 12a, the weight gain rate was reduced in the human ACE2-expressing transgenic cloned pig compared to the wild type, and the body temperature increased as shown in FIG. 12b. . This is a common clinical symptom of SARS-CoV-2 infection, and in particular, Yucatan mini-pigs showed symptoms of high fever exceeding normal body temperature (39.2 ℃ ± 0.5, sinclair) after challenge inoculation, and symptoms of lethargy were also confirmed. After 3 days of inoculation, genetic analysis of the E gene and RdRP gene of each individual (seegene kit, Korea) was performed. As a result, as shown in FIG. 13, viral shedding was confirmed in the nasal cavity of the transgenic cloned pig compared to the wild type. In addition, after 5 days of inoculation, each individual was euthanized, and histopathological examination of tissues was performed. As a result of the analysis, as shown in FIG. 14, local hemorrhage accompanied by edematous changes in the alveoli was confirmed in some of the lungs of the human ACE2-expressing transgenic pig compared to the wild type, and mild vascular and peribronchiolar inflammatory cell infiltration was observed in more than half of the lung lobes. It was confirmed, and the mainly infiltrated cells were identified as mononuclear inflammatory cells.

따라서 본 발명에 따른 인간 ACE2를 발현하는 형질전환 복제돼지는 SARS-CoV-2에 대하여 높은 감수성을 나타냄을 확인하였다.Therefore, it was confirmed that the transgenic cloned pig expressing human ACE2 according to the present invention exhibits high sensitivity to SARS-CoV-2.

종합적으로 본 발명은 인간 안지오텐신 전환효소 2(Angiotensin-converting enzyme 2, ACE2)를 발현시키기 위한 재조합 벡터를 제조하고, 이를 이용하여 형질전환 세포주와 형질전환 돼지를 제조한 것으로, 상기 형질전환 세포주 및 형질전환 돼지는 인간 ACE2를 발현하며, SARS-CoV-2에 대하여 높은 감수성을 보이는 바, 이를 SARS-CoV-2에 대한 연구 또는 상기 바이러스 감염 질환의 예방 또는 치료제 개발을 위한 비임상 동물모델로서 유용하게 활용할 수 있다.Overall, the present invention is to prepare a recombinant vector for expressing human angiotensin-converting enzyme 2 (ACE2), and prepare a transgenic cell line and a transgenic pig using the same. Transgenic pigs express human ACE2 and show high susceptibility to SARS-CoV-2, which is useful as a non-clinical animal model for research on SARS-CoV-2 or development of preventive or therapeutic agents for viral infectious diseases can be utilized

한국세포주연구재단Korea Cell Line Research Foundation KCLRF-BP-00512KCLRF-BP-00512 2021050420210504

<110> Optipharm.CO.,LTD <120> Transgenic cloned porcine for animal models with increased susceptibility to severe acute respiratory syndrome coronavirus 2, and method for producing the same <130> 1-88P-1 <150> KR 1020200053935 <151> 2020-05-06 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 2381 <212> DNA <213> Artificial Sequence <220> <223> Human angiotensin converting enzyme 2(ACE2) gene <400> 1 ggtggaggcg gtagccagtc caccattgag gaacaggcca agacattttt ggacaagttt 60 aaccacgaag ccgaagacct gttctatcaa agttcacttg cttcttggaa ttataacacc 120 aatattactg aagagaatgt ccaaaacatg aataatgctg gggacaaatg gtctgccttt 180 ttaaaggaac agtccacact tgcccaaatg tatccactac aagaaattca gaatctcaca 240 gtcaagcttc agctgcaggc tcttcagcaa aatgggtctt cagtgctctc agaagacaag 300 agcaaacggt tgaacacaat tctaaataca atgagcacca tctacagtac tggaaaagtt 360 tgtaacccag ataatccaca agaatgctta ttacttgaac caggtttgaa tgaaataatg 420 gcaaacagtt tagactacaa tgagaggctc tgggcttggg aaagctggag atctgaggtc 480 ggcaagcagc tgaggccatt atatgaagag tatgtggtct tgaaaaatga gatggcaaga 540 gcaaatcatt atgaggacta tggggattat tggagaggag actatgaagt aaatggggta 600 gatggctatg actacagccg cggccagttg attgaagatg tggaacatac ctttgaagag 660 attaaaccat tatatgaaca tcttcatgcc tatgtgaggg caaagttgat gaatgcctat 720 ccttcctata tcagtccaat tggatgcctc cctgctcatt tgcttggtga tatgtggggt 780 agattttgga caaatctgta ctctttgaca gttccctttg gacagaaacc aaacatagat 840 gttactgatg caatggtgga ccaggcctgg gatgcacaga gaatattcaa ggaggccgag 900 aagttctttg tatctgttgg tcttcctaat atgactcaag gattctggga aaattccatg 960 ctaacggacc caggaaatgt tcagaaagca gtctgccatc ccacagcttg ggacctgggg 1020 aagggcgact tcaggatcct tatgtgcaca aaggtgacaa tggacgactt cctgacagct 1080 catcatgaga tggggcatat ccagtatgat atggcatatg ctgcacaacc ttttctgcta 1140 agaaatggag ctaatgaagg attccatgaa gctgttgggg aaatcatgtc actttctgca 1200 gccacaccta agcatttaaa atccattggt cttctgtcac ccgattttca agaagacaat 1260 gaaacagaaa taaacttcct gctcaaacaa gcactcacga ttgttgggac tctgccattt 1320 acttacatgt tagagaagtg gaggtggatg gtctttaaag gggaaattcc caaagaccag 1380 tggatgaaaa agtggtggga gatgaagcga gagatagttg gggtggtgga acctgtgccc 1440 catgatgaaa catactgtga ccccgcatct ctgttccatg tttctaatga ttactcattc 1500 attcgatatt acacaaggac cctttaccaa ttccagtttc aagaagcact ttgtcaagca 1560 gctaaacatg aaggccctct gcacaaatgt gacatctcaa actctacaga agctggacag 1620 aaactgttca atatgctgag gcttggaaaa tcagaaccct ggaccctagc attggaaaat 1680 gttgtaggag caaagaacat gaatgtaagg ccactgctca actactttga gcccttattt 1740 acctggctga aagaccagaa caagaattct tttgtgggat ggagtaccga ctggagtcca 1800 tatgcagacc aaagcatcaa agtgaggata agcctaaaat cagctcttgg agataaagca 1860 tatgaatgga acgacaatga aatgtacctg ttccgatcat ctgttgcata tgctatgagg 1920 cagtactttt taaaagtaaa aaatcagatg attctttttg gggaggagga tgtgcgagtg 1980 gctaatttga aaccaagaat ctcctttaat ttctttgtca ctgcacctaa aaatgtgtct 2040 gatatcattc ctagaactga agttgaaaag gccatcagga tgtcccggag ccgtatcaat 2100 gatgctttcc gtctgaatga caacagccta gagtttctgg ggatacagcc aacacttgga 2160 cctcctaacc agccccctgt ttccatatgg ctgattgttt ttggagttgt gatgggagtg 2220 atagtggttg gcattgtcat cctgatcttc actgggatca gagatcggaa gaagaaaaat 2280 aaagcaagaa gtggagaaaa tccttatgcc tccatcgata ttagcaaagg agaaaataat 2340 ccaggattcc aaaacactga tgatgttcag acctcctttt a 2381 <210> 2 <211> 2405 <212> DNA <213> Artificial Sequence <220> <223> recombinant vector(Flag,hACE2) <400> 2 gattacaagg atgacgacga taagggtgga ggcggtagcc agtccaccat tgaggaacag 60 gccaagacat ttttggacaa gtttaaccac gaagccgaag acctgttcta tcaaagttca 120 cttgcttctt ggaattataa caccaatatt actgaagaga atgtccaaaa catgaataat 180 gctggggaca aatggtctgc ctttttaaag gaacagtcca cacttgccca aatgtatcca 240 ctacaagaaa ttcagaatct cacagtcaag cttcagctgc aggctcttca gcaaaatggg 300 tcttcagtgc tctcagaaga caagagcaaa cggttgaaca caattctaaa tacaatgagc 360 accatctaca gtactggaaa agtttgtaac ccagataatc cacaagaatg cttattactt 420 gaaccaggtt tgaatgaaat aatggcaaac agtttagact acaatgagag gctctgggct 480 tgggaaagct ggagatctga ggtcggcaag cagctgaggc cattatatga agagtatgtg 540 gtcttgaaaa atgagatggc aagagcaaat cattatgagg actatgggga ttattggaga 600 ggagactatg aagtaaatgg ggtagatggc tatgactaca gccgcggcca gttgattgaa 660 gatgtggaac atacctttga agagattaaa ccattatatg aacatcttca tgcctatgtg 720 agggcaaagt tgatgaatgc ctatccttcc tatatcagtc caattggatg cctccctgct 780 catttgcttg gtgatatgtg gggtagattt tggacaaatc tgtactcttt gacagttccc 840 tttggacaga aaccaaacat agatgttact gatgcaatgg tggaccaggc ctgggatgca 900 cagagaatat tcaaggaggc cgagaagttc tttgtatctg ttggtcttcc taatatgact 960 caaggattct gggaaaattc catgctaacg gacccaggaa atgttcagaa agcagtctgc 1020 catcccacag cttgggacct ggggaagggc gacttcagga tccttatgtg cacaaaggtg 1080 acaatggacg acttcctgac agctcatcat gagatggggc atatccagta tgatatggca 1140 tatgctgcac aaccttttct gctaagaaat ggagctaatg aaggattcca tgaagctgtt 1200 ggggaaatca tgtcactttc tgcagccaca cctaagcatt taaaatccat tggtcttctg 1260 tcacccgatt ttcaagaaga caatgaaaca gaaataaact tcctgctcaa acaagcactc 1320 acgattgttg ggactctgcc atttacttac atgttagaga agtggaggtg gatggtcttt 1380 aaaggggaaa ttcccaaaga ccagtggatg aaaaagtggt gggagatgaa gcgagagata 1440 gttggggtgg tggaacctgt gccccatgat gaaacatact gtgaccccgc atctctgttc 1500 catgtttcta atgattactc attcattcga tattacacaa ggacccttta ccaattccag 1560 tttcaagaag cactttgtca agcagctaaa catgaaggcc ctctgcacaa atgtgacatc 1620 tcaaactcta cagaagctgg acagaaactg ttcaatatgc tgaggcttgg aaaatcagaa 1680 ccctggaccc tagcattgga aaatgttgta ggagcaaaga acatgaatgt aaggccactg 1740 ctcaactact ttgagccctt atttacctgg ctgaaagacc agaacaagaa ttcttttgtg 1800 ggatggagta ccgactggag tccatatgca gaccaaagca tcaaagtgag gataagccta 1860 aaatcagctc ttggagataa agcatatgaa tggaacgaca atgaaatgta cctgttccga 1920 tcatctgttg catatgctat gaggcagtac tttttaaaag taaaaaatca gatgattctt 1980 tttggggagg aggatgtgcg agtggctaat ttgaaaccaa gaatctcctt taatttcttt 2040 gtcactgcac ctaaaaatgt gtctgatatc attcctagaa ctgaagttga aaaggccatc 2100 aggatgtccc ggagccgtat caatgatgct ttccgtctga atgacaacag cctagagttt 2160 ctggggatac agccaacact tggacctcct aaccagcccc ctgtttccat atggctgatt 2220 gtttttggag ttgtgatggg agtgatagtg gttggcattg tcatcctgat cttcactggg 2280 atcagagatc ggaagaagaa aaataaagca agaagtggag aaaatcctta tgcctccatc 2340 gatattagca aaggagaaaa taatccagga ttccaaaaca ctgatgatgt tcagacctcc 2400 tttta 2405 <210> 3 <211> 13856 <212> DNA <213> Artificial Sequence <220> <223> pCMV-5'FLAG vector <400> 3 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 60 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 120 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 180 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 240 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 300 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 360 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 420 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 480 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 540 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 600 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 660 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 720 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 780 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 840 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 900 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 960 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 1020 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 1080 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 1140 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 1200 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 1260 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 1320 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 1380 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 1440 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 1500 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 1560 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 1620 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 1680 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 1740 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 1800 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 1860 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 1920 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 1980 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 2040 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 2100 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt 2160 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg 2220 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg 2280 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta 2340 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg 2400 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa 2460 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct 2520 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct 2580 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 2640 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 2700 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata 2760 cgactcacta tagggcgaat tggagctcca ccgcgggctg gttctttccg cctcagaagc 2820 catagagccc accgcatccc cagcatgcct gctattgtct tcccaatcct cccccttgct 2880 gtcctgcccc accccacccc ccagaataga atgacaccta ctcagacaat gcgatgcaat 2940 ttcctcattt tattaggaaa ggacagtggg agtggcacct tccagggtca aggaaggcac 3000 gggggagggg caaacaacag atggctggca actagaaggc acagtcgagg ctgatcagcg 3060 agctctagga tctgcattcc accactgctc ccattcatca gttccatagg ttggaatcta 3120 aaatacacaa acaattagaa tcagtagttt aacacattat acacttaaaa attttatatt 3180 taccttagag ctttaaatct ctgtaggtag tttgtccaat tatgtcacac cacagaagta 3240 aggttccttc acaaagagat cgcctgacac gatttcctgc acaggcttga gccatatact 3300 catacatcgc atcttggcca cgttttccac gggtttcaaa attaatctca agttctacgc 3360 ttaacgcttt cgcctgttcc cagttattaa tatattcaac actagaactc ccctcagcga 3420 agggaaggct gagcactaca cgcgaagcac catcaccgaa ccttttgata aactcttccg 3480 ttccgacttg ctccatcaac ggttcagtga gacttaaacc taactctttc ttaatagttt 3540 cggcattatc cacttttagt gcgagaacct tcgtcaatcc tggatacgtc actttgacca 3600 cgcctccagc ttttccagag agcgggtttt cattatctac agagtatccc gcagcgtcgt 3660 atttattgtc ggtactataa aaccctttcc aatcatcgtc ataatttcct tgtgtaccag 3720 attttggctt ttgtatacct ttttgaatgg aatctacata accaggttta gtcccgtggt 3780 acgaagaaaa gttttccatc acaaaagatt tagaagaatc aacaacatca tcaggatcca 3840 tggcgaggac ctgcaggtcg aaaggcccgg agatgaggaa gaggagaaca gcgcggcaga 3900 cgtgcgcttt tgaagcgtgc agaatgccgg gcctccggag gaccttcggg cgcccgcccc 3960 gcccctgagc ccgcccctga gcccgccccc ggacccaccc cttcccagcc tctgagccca 4020 gaaagcgaag gagcaaagct gctattggcc gctgccccaa aggcctaccc gcttccattg 4080 ctcagcggtg ctgtccatct gcacgagact agtgagacgt gctacttcca tttgtcacgt 4140 cctgcacgac gcgagctgcg gggcgggggg gaacttcctg actaggggag gagtagaagg 4200 tggcgcgaag gggccaccaa agaacggagc cggttggcgc ctaccggtgg atgtggaatg 4260 tgtgcgaggc cagaggccac ttgtgtagcg ccaagtgccc agcggggctg ctaaagcgca 4320 tgctccagac tgccttgcgg ccgccgagta catttatatt ggctcatgtc caatatgacc 4380 gccatgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 4440 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 4500 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 4560 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 4620 agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg acggtaaatg 4680 gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt ggcagtacat 4740 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca ccaatgggcg 4800 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 4860 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaataacc ccgccccgtt 4920 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt 4980 gaaccgtcag atcctcactc tcttccgcat cgctgtctgc gagggccagc tgttgggctc 5040 gcggttgagg acaaactctt cgcggtcttt ccagtactct tggatcggaa acccgtcggc 5100 ctccgaacgg tactccgcca ccgagggacc tgagcgagtc cgcatcgacc ggatcggaaa 5160 acctctcgag aaaggcgtct aaccagtcac agtcgcaagg taggctgagc accgtggcgg 5220 gcggcagcgg gtggcggtcg gggttgtttc tggcggaggt gctgctgatg atgtaattaa 5280 agtaggcggt cttgagacgg cggatggtcg aggtgaggtg tggcaggctt gagatccagc 5340 tgttggggtg agtactccct ctcaaaagcg ggcattactt ctgcgctaag attgtcagtt 5400 tccaaaaacg aggaggattt gatattcacc tggcccgatc tggccataca cttgagtgac 5460 aatgacatcc actttgcctt tctctccaca ggtgtccact cccaggtcca agtttaaact 5520 ttaatacgac tcactatagg ggccgccacc aagcttggta ccatgccact gctgctcttg 5580 ctgcctctgc tttgggctgg agctctggct gattacaagg atgacgacga taagggtgga 5640 ggcggtagcc agtccaccat tgaggaacag gccaagacat ttttggacaa gtttaaccac 5700 gaagccgaag acctgttcta tcaaagttca cttgcttctt ggaattataa caccaatatt 5760 actgaagaga atgtccaaaa catgaataat gctggggaca aatggtctgc ctttttaaag 5820 gaacagtcca cacttgccca aatgtatcca ctacaagaaa ttcagaatct cacagtcaag 5880 cttcagctgc aggctcttca gcaaaatggg tcttcagtgc tctcagaaga caagagcaaa 5940 cggttgaaca caattctaaa tacaatgagc accatctaca gtactggaaa agtttgtaac 6000 ccagataatc cacaagaatg cttattactt gaaccaggtt tgaatgaaat aatggcaaac 6060 agtttagact acaatgagag gctctgggct tgggaaagct ggagatctga ggtcggcaag 6120 cagctgaggc cattatatga agagtatgtg gtcttgaaaa atgagatggc aagagcaaat 6180 cattatgagg actatgggga ttattggaga ggagactatg aagtaaatgg ggtagatggc 6240 tatgactaca gccgcggcca gttgattgaa gatgtggaac atacctttga agagattaaa 6300 ccattatatg aacatcttca tgcctatgtg agggcaaagt tgatgaatgc ctatccttcc 6360 tatatcagtc caattggatg cctccctgct catttgcttg gtgatatgtg gggtagattt 6420 tggacaaatc tgtactcttt gacagttccc tttggacaga aaccaaacat agatgttact 6480 gatgcaatgg tggaccaggc ctgggatgca cagagaatat tcaaggaggc cgagaagttc 6540 tttgtatctg ttggtcttcc taatatgact caaggattct gggaaaattc catgctaacg 6600 gacccaggaa atgttcagaa agcagtctgc catcccacag cttgggacct ggggaagggc 6660 gacttcagga tccttatgtg cacaaaggtg acaatggacg acttcctgac agctcatcat 6720 gagatggggc atatccagta tgatatggca tatgctgcac aaccttttct gctaagaaat 6780 ggagctaatg aaggattcca tgaagctgtt ggggaaatca tgtcactttc tgcagccaca 6840 cctaagcatt taaaatccat tggtcttctg tcacccgatt ttcaagaaga caatgaaaca 6900 gaaataaact tcctgctcaa acaagcactc acgattgttg ggactctgcc atttacttac 6960 atgttagaga agtggaggtg gatggtcttt aaaggggaaa ttcccaaaga ccagtggatg 7020 aaaaagtggt gggagatgaa gcgagagata gttggggtgg tggaacctgt gccccatgat 7080 gaaacatact gtgaccccgc atctctgttc catgtttcta atgattactc attcattcga 7140 tattacacaa ggacccttta ccaattccag tttcaagaag cactttgtca agcagctaaa 7200 catgaaggcc ctctgcacaa atgtgacatc tcaaactcta cagaagctgg acagaaactg 7260 ttcaatatgc tgaggcttgg aaaatcagaa ccctggaccc tagcattgga aaatgttgta 7320 ggagcaaaga acatgaatgt aaggccactg ctcaactact ttgagccctt atttacctgg 7380 ctgaaagacc agaacaagaa ttcttttgtg ggatggagta ccgactggag tccatatgca 7440 gaccaaagca tcaaagtgag gataagccta aaatcagctc ttggagataa agcatatgaa 7500 tggaacgaca atgaaatgta cctgttccga tcatctgttg catatgctat gaggcagtac 7560 tttttaaaag taaaaaatca gatgattctt tttggggagg aggatgtgcg agtggctaat 7620 ttgaaaccaa gaatctcctt taatttcttt gtcactgcac ctaaaaatgt gtctgatatc 7680 attcctagaa ctgaagttga aaaggccatc aggatgtccc ggagccgtat caatgatgct 7740 ttccgtctga atgacaacag cctagagttt ctggggatac agccaacact tggacctcct 7800 aaccagcccc ctgtttccat atggctgatt gtttttggag ttgtgatggg agtgatagtg 7860 gttggcattg tcatcctgat cttcactggg atcagagatc ggaagaagaa aaataaagca 7920 agaagtggag aaaatcctta tgcctccatc gatattagca aaggagaaaa taatccagga 7980 ttccaaaaca ctgatgatgt tcagacctcc ttttaatcta gagcggccgc cgaattcggg 8040 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 8100 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 8160 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 8220 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 8280 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac 8340 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 8400 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 8460 ttcgcaggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 8520 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 8580 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 8640 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 8700 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 8760 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag 8820 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc 8880 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag 8940 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 9000 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc 9060 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctctcgg 9120 gagcttgtat atccattttc ggatctgatc agcacgtgat gaaaaagcct gaactcaccg 9180 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc 9240 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc 9300 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg 9360 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga 9420 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac 9480 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta 9540 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc 9600 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg 9660 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact 9720 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca 9780 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg 9840 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct 9900 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc 9960 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt 10020 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac 10080 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata 10140 gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgtg ctacgagatt 10200 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgctg 10260 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt 10320 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 10380 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 10440 tctgtatacc gtcgacattg attattgact agttattaat agtaatcaat tacggggtca 10500 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 10560 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 10620 acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac 10680 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 10740 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 10800 tacatctacg tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt 10860 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt 10920 attttgtgca gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg 10980 gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc 11040 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg 11100 cgcggcgggc gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc 11160 gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc 11220 cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc 11280 tgcgtgaaag ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg 11340 ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct 11400 gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc 11460 ggccgggggc ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg 11520 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc 11580 cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc 11640 ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg 11700 cggggcgggg ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc 11760 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag 11820 agggcgcagg gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc 11880 gcaccccctc tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg 11940 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg 12000 ccgcaggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt 12060 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag 12120 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcatggtg 12180 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac 12240 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag 12300 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg 12360 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac 12420 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag 12480 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac 12540 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg 12600 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc 12660 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac 12720 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg 12780 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg 12840 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaagaattc 12900 actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct 12960 cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc 13020 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg 13080 aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag 13140 aatgagtatt tggtttagag tttggcaaca tatgccatat gctggctgcc atgaacaaag 13200 gtggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca 13260 tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc 13320 tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg 13380 actactccca gtcatagctg tccctcttct cttatgaaga tccctcgacc tgcagttggg 13440 gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg 13500 ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag 13560 cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc 13620 cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc 13680 acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc 13740 gcgatgggct gtggccaata gcggctgctc agcagggcgc gccggtacca gcttttgttc 13800 cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctg 13856 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ACE2 forward primer <400> 4 gtacatctac gtattagtca tcgc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ACE2 reverse primer <400> 5 gttcaaccgt ttgctcttgt cttc 24 <110> Optipharm.CO.,LTD <120> Transgenic cloned porcine for animal models with increased susceptibility to severe acute respiratory syndrome coronavirus 2, and method for producing the same <130> 1-88P-1 <150> KR 1020200053935 <151 > 2020-05-06 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 2381 <212> DNA <213> Artificial Sequence <220> <223> Human angiotensin converting enzyme 2(ACE2) gene <400> 1 ggtggaggcg gtagccagtc caccattgag gaacaggcca agacattttt ggacaagttt 60 aaccacgaag ccgaagacct gttctatcaa agttcacttg cttcttggaa ttataacacc 120 aatattactg aagagaatgt ccaaaacatg aataatgctg gggacaaatg gtctgcc ttt 180 ttaaaggaac agtccacact tgcccaaatg tatccactac aagaaattca gaatctcaca 240 gtcaagcttc agctgcaggc tcttcagcaa aatgggtctt cagtgctctc agaagacaag 300 agcaaacggt tgaacacaat tctaaataca atgagcacca tctacagtac tggaaaagtt 360 tgtaacccag ataatccaca agaatgctta ttacttgaac caggtttgaa tgaaataatg 420 gcaaacagtt tagactacaa tgagaggctc tgggcttggg aaagctggag atctgaggtc 480 ggcaagcagc tgaggccatt atatgaagag tatgtggtct tgaaaaatga gatggcaaga 540 gcaaatcatt atgaggacta tggggattat tggagaggag actatgaagt aaatggggta 600 gat ggctatg actacagccg cggccagttg attgaagatg tggaacatac ctttgaagag 660 attaaaccat tatatgaaca tcttcatgcc tatgtgaggg caaagttgat gaatgcctat 720 ccttcctata tcagtccaat tggatgcctc cctgctcatt tgcttggtga tatgtggggt 780 agattttgga caaatctgta ctctttgaca gttccctttg gacagaaacc aaacatagat 840 gttactgatg aag ggcgact tcaggatcct tatgtgcaca aaggtgacaa tggacgactt cctgacagct 1080 catcatgaga tggggcatat ccagtatgat atggcatatg ctgcacaacc ttttctgcta 1140 agaaatggag ctaatgaagg attccatgaa gctgttgggg aaatcatgtc actttctgca 120 0 gccacaccta agcatttaaa atccattggt cttctgtcac ccgattttca agaagacaat 1260 gaaacagaaa taaacttcct gctcaaacaa gcactcacga ttgttgggac tctgccattt 1320 acttacatgt tagagaagtg gaggtggatg gtctttaaag gggaaattcc caaagaccag 1380 tggatgaaaa agtggtggga gatgaagcga gagatagttg gggtggtgga acctgtgccc 1440 catgatgaaa cat actgtga ccccgcatct ctgttccatg tttctaatga ttactcattc 1500 attcgatatt acacaaggac cctttaccaa ttccagtttc aagaagcact ttgtcaagca 1560 gctaaacatg aaggccctct gcacaaatgt gacatctcaa actctacaga agctggacag 1620 aaactgtt ca atatgctgag gcttggaaaa tcagaaccct ggaccctagc attggaaaat 1680 gttgtaggag caaagaacat gaatgtaagg ccactgctca actactttga gcccttattt 1740 acctggctga aagaccagaa caagaattct tttgtgggat ggagtaccga ctggagtcca 1800 tatgcagacc aaagcatcaa agtgaggata agcctaaaat cagctcttgg agataaagca 1860 tatgaatgga acgacaatga aatgtacctg ttccgatcat ctgttgcata tgctatgagg 1920 cagtactttt taaaagtaaa aaatcagatg attctttttg gggaggagga tgtgcgagtg 1980 gctaatttga aaccaagaat ctcctttaat ttctttgtca ctgcacctaa aaatgtgtct 2040 gatatcattc ct agaactga agttgaaaag gccatcagga tgtcccggag ccgtatcaat 2100 gatgctttcc gtctgaatga caacagccta gagtttctgg ggatacagcc aacacttgga 2160 cctcctaacc agccccctgt ttccatatgg ctgattgttt ttggagttgt gatgggagtg 2220 atagtggttg gcattgtcat cctgatcttc actgggatca gagatcggaa gaagaaaaat 2280 aaagcaagaa gtggagaaaa tcctta tgcc tccatcgata ttagcaaagg agaaaataat 2340 ccaggattcc aaaacactga tgatgttcag acctcctttt a 2381 <210> 2 <211> 2405 <212> DNA <213> Artificial Sequence <220> <223> recombinant vector (Flag, hACE2) <400> 2 gattacaagg atgacgacga taagggtgga ggcggtagcc agtccaccat tgaggaacag 60 gccaagacat ttttggacaa gtttaaccac gaagccgaag acctgttcta tcaaagttca 120 cttgcttctt ggaattataa cacca atatt actgaagaga atgtccaaaa catgaataat 180 gctggggaca aatggtctgc ctttttaaag gaacagtcca cacttgccca aatgtatcca 240 ctacaagaaa ttcagaatct cacagtcaag cttcagctgc aggctcttca gcaaaatggg 300 tcttcagtgc tctcagaaga caagagcaaa cggttgaaca caattctaaa tacaatgagc 360 accatctaca gtactggaaa agtttgtaac ccagataatc cacaagaatg cttattactt 420 gaaccaggtt tgaatgaaat aatggcaaac agtttagact acaatgagag gctctgggct 480 tgggaaag ct at acctttga agagattaaa ccattatatg aacatcttca tgcctatgtg 720 agggcaaagt tgatgaatgc ctatccttcc tatatcagtc caattggatg cctccctgct 780 catttgcttg gtgatatgtg gggtagattt tggacaaatc tgtactcttt gacagttccc 840 tttggacaga aaccaaacat agatgttact gatgcaatgg tggaccaggc ctgggatgca 900 cagagaatat t a caatggacg acttcctgac agctcatcat gagatggggc atatccagta tgatatggca 1140 tatgctgcac aaccttttct gctaagaaat ggagctaatg aaggattcca tgaagctgtt 1200 ggggaaatca tgtcactttc tgcagccaca cctaagcatt taaaatccat tggtcttctg 1260 tcaccgatt ttcaagaaga caatgaaaca gaaataaact tcctgctcaa acaagcactc 1320 acgattgttg ggactctgcc atttacttac atgttagaga agtggaggtg gatggtcttt 1380 aaaggggaaa ttcccaaaga ccagtggatg aaaaagtggt gggagatgaa gcgagagata 1440 gttggggtgg tggaacctgt gccccatgat gaaacatact gtgaccccgc atctctgttc 1500 catgt ttcta atgattactc attcattcga tattacacaa ggacccttta ccaattccag 1560 tttcaagaag cactttgtca agcagctaaa catgaaggcc ctctgcacaa atgtgacatc 1620 tcaaactcta cagaagctgg acagaaactg ttcaatatgc tgaggcttgg aaaatcagaa 1680 ccctggaccc tagcattgga aaatgttgta ggagcaaaga acatgaatgt aaggccactg 1740 ctcaactact ttgagccctt atttacctgg ctga aagacc agaacaagaa ttcttttgg 1800 ggatggagta ccgactggag tccatatgca gaccaaagca tcaaagtgag gataagccta 1860 aaatcagctc ttggagataa agcatatgaa tggaacgaca atgaaatgta cctgttccga 1920 tcatctgttg catatgctat ga ggcagtac tttttaaaag taaaaaatca gatgattctt 1980 tttggggagg aggatgtgcg agtggctaat ttgaaaccaa gaatctcctt taatttcttt 2040 gtcactgcac ctaaaaatgt gtctgatatc attcctagaa ctgaagttga aaaggccatc 2100 aggatgtccc ggagccgtat caatgatgct ttccgtctga atgacaacag cctagagttt 2160 ctggggatac agccaacact tggacctcct aacca gcccc ctgtttccat atggctgatt 2220 gtttttggag ttgtgatggg agtgatagtg gttggcattg tcatcctgat cttcactggg 2280 atcagagatc ggaagaagaa aaataaagca agaagtggag aaaatcctta tgcctccatc 2340 gatattagca aaggagaaaa ta atccagga ttccaaaaca ctgatgatgt tcagacctcc 2400 tttta 2405 <210> 3 <211> 13856 <212> DNA <213> Artificial Sequence <220> <223> pCMV-5'FLAG vector <400> 3 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 60 aagcctgggg tgcctaatga gtgagctaac tcacattaat t gcgttgcgc tcactgcccg 120 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 180 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 240 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 300 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaa ag gccaggaacc 360 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 420 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 480 ttccccctgg aagctccctc gtgcgctctc ctgttcc gac cctgccgctt accggatacc 540 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 600 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 660 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 720 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 780 ct acagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 840 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 900 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgca gaa 960 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 1020 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 1080 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 1140 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 1200 ccatagtt gc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 1260 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 1320 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 1380 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 1440 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 1500 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 1560 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 1620 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 1680 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 1740 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 1800 tgctcat cat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 1860 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 1920 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 1980 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 2040 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 2100 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt 2160 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg 2220 caa aatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg 2280 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta 2340 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg 2400 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa 2460 gccggcgaac gtggcgagaa aggaaggga a gaaagcgaaa ggagcgggcg ctagggcgct 2520 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct 2580 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 2640 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 2700 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata 2760 cgactcacta tagggcgaat tggagctcca ccgcgggctg gttctttccg cctcagaagc 2820 catagagccc accgcatccc cagcatgcct gctattgtct tcccaatcct cccccttgct 2880 gtcctgcccc accccacccc ccagaataga atgacaccta ctcagacaat gcgat gcaat 2940 ttcctcattt tattaggaaa ggacagtggg agtggcacct tccagggtca aggaaggcac 3000 gggggagggg caaacaacag atggctggca actagaaggc acagtcgagg ctgatcagcg 3060 agctctagga tctgcattcc accactgctc ccattcatca gttccatagg ttggaatcta 3120 aaatacacaa acaattagaa tcagtagttt aacacattat acacttaaaa attttatatt 3180 taccttagag ctttaaatct ctgtaggtag tttgtccaat tatgtcacac cacagaagta 3240 aggttccttc acaaagagat cgcctgacac gatttcctgc acaggcttga gccatatact 3300 catacatcgc atcttggcca cgtttccac gggtttcaaa attaatctca agttctac gc 3360 ttaacgcttt cgcctgttcc cagttattaa tatattcaac actagaactc ccctcagcga 3420 agggaaggct gagcactaca cgcgaagcac catcaccgaa ccttttgata aactcttccg 3480 ttccgacttg ctccatcaac ggttcagtga gacttaaacc taactctttc tta atagttt 3540 cggcattatc cacttttagt gcgagaacct tcgtcaatcc tggatacgtc actttgacca 3600 cgcctccagc ttttccagag agcgggtttt cattatctac agagtatccc gcagcgtcgt 3660 atttattgtc ggtactataa aaccctttcc aatcatcgtc ataatttcct tgtgtaccag 3720 attttggctt ttgtatacct ttttgaatgg aatctacata accaggttta gtcccgtggt 3 780 acgaagaaaa gttttccatc acaaaagatt tagaagaatc aacaacatca tcaggatcca 3840 tggcgaggac ctgcaggtcg aaaggcccgg agatgaggaa gaggagaaca gcgcggcaga 3900 cgtgcgcttt tgaagcgtgc agaatgccgg gcctccggag gacctt cggg cgcccgcccc 3960 gcccctgagc ccgcccctga gcccgccccc ggacccaccc cttcccagcc tctgagccca 4020 gaaagcgaag gagcaaagct gctattggcc gctgccccaa aggcctaccc gcttccattg 4080 ctcagcggtg ctgtccatct gcacgagact agtgagaacgt gctacttcca tttgtcacgt 4140 cctgcacgac gcgagctgcg gggcgggggg gaacttcctg actaggggag gagtagaagg 4200 tggc gcgaag gggccaccaa agaacggagc cggttggcgc ctaccggtgg atgtggaatg 4260 tgtgcgaggc cagaggccac ttggttagcg ccaagtgccc agcggggctg ctaaagcgca 4320 tgctccagac tgccttgcgg ccgccgagta catttatatt ggctcatgtc ca atatgacc 4380 gccatgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 4440 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 4500 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 4560 aatagggact ttccattgac gtcaatgggt ggaggtattta cggtaaactg cccacttggc 4620 agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg acggtaaatg 4680 gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt ggcagtacat 4740 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca ccaatgggcg 4800 tggatagcgg tttg actcac ggggatttcc aagtctccac cccattgacg tcaatgggag 4860 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaataacc ccgccccgtt 4920 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt 4980 gaaccgtcag atcctcactc tcttccgcat cgctgtctgc gagggccagc tgttgggctc 5040 gcggttgagg acaaactctt c gcggtcttt ccagtactct tggatcggaa acccgtcggc 5100 ctccgaacgg tactccgcca ccgagggacc tgagcgagtc cgcatcgacc ggatcggaaa 5160 acctctcgag aaaggcgtct aaccagtcac agtcgcaagg taggctgagc accgtggcgg 5220 gcggca gcgg gtggcggtcg gggttgtttc tggcggaggt gctgctgatg atgtaattaa 5280 agtaggcggt cttgagacgg cggatggtcg aggtgaggtg tggcaggctt gagatccagc 5340 tgttggggtg agtactccct ctcaaaagcg ggcattactt ctgcgctaag attgtcagtt 5400 tccaaaaacg aggaggattt gatattcacc tggcccgatc tggccataca cttgagtgac 5460 aatgacatcc actttgcctt tctctccaca ggtgtccact cccaggtcca agtttaaact 5520 ttaatacgac tcactatagg ggccgccacc aagcttggta ccatgccact gctgctcttg 5580 ctgcctctgc tttgggctgg agctctggct gattacaagg atgacgacga taagggtgga 5640 ggcggtagcc agtccaccat tgag gaacag gccaagacat ttttggacaa gtttaaccac 5700 gaagccgaag acctgttcta tcaaagttca cttgcttctt ggaattataa caccaatatt 5760 actgaagaga atgtccaaaa catgaataat gctggggaca aatggtctgc ctttttaaag 5820 gaacagtcca cacttgccca aatgtatcca ctacaagaaa ttcagaatct cacagtcaag 5880 cttcagctgc aggctcttca gcaaaatggg tcttcagtgc tctcagaaga ca agagcaaa 5940 cggttgaaca caattctaaa tacaatgagc accatctaca gtactggaaa agtttgtaac 6000 ccagataatc cacaagaatg cttattactt gaaccaggtt tgaatgaaat aatggcaaac 6060 agtttagact acaatgagag gctctgggct tgggaaagct ggagatct ga ggtcggcaag 6120 cagctgaggc cattatatga agagtatgtg gtcttgaaaa atgagatggc aagagcaaat 6180 cattatgagg actatgggga tattggaga ggagactatg aagtaaatgg ggtagatggc 6240 tatgactaca gccgcggcca gttgattgaa gatgtggaac atacctttga agagattaaa 6300 ccattatatg aacatcttca tgcctatgtg agggcaaagt tgatgaatgc ctatcctt cc 6360 tatatcagtc caattggatg cctccctgct catttgcttg gtgatatgtg gggtagattt 6420 tggacaaatc tgtactcttt gacagttccc tttggacaga aaccaaacat agatgttact 6480 gatgcaatgg tggaccaggc ctgggatgca cagagaatat tcaaggagg c cgagaagttc 6540 tttgtatctg ttggtcttcc taatatgact caaggattct gggaaaattc catgctaacg 6600 gacccaggaa atgttcagaa agcagtctgc catcccacag cttgggacct ggggaagggc 6660 gacttcagga tccttatggg cacaaaggtg acaatggacg acttcctgac agctcatcat 6720 gagatggggc atatccagta tgatatggca tatgctgcac aaccttttct gctaagaaat 6780 gg agctaatg aaggattcca tgaagctgtt ggggaaatca tgtcactttc tgcagccaca 6840 cctaagcatt taaaatccat tggtcttctg tcaccgatt ttcaagaaga caatgaaaca 6900 gaaataaact tcctgctcaa acaagcactc acgattgttg ggactctgcc attact tac 6960 atgttagaga agtggaggtg gatggtcttt aaaggggaaa ttcccaaaga ccagtggatg 7020 aaaaagtggt gggagatgaa gcgagagata gttggggtgg tggaacctgt gcccccatgat 7080 gaaacatact gtgaccccgc atctctgttc catgtttcta atgattactc attcattcga 7140 tattacacaa ggacccttta ccaattccag tttcaagaag cactttgtca agcagctaaa 7200 catgaaggcc ctctg cacaa atgtgacatc tcaaactcta cagaagctgg acagaaactg 7260 ttcaatatgc tgaggcttgg aaaatcagaa ccctggaccc tagcattgga aaatgttgta 7320 ggagcaaaga acatgaatgt aaggccactg ctcaactact ttgagccctt atttacctgg 7380 ctgaaagacc aga acaagaa ttcttttgtg ggatggagta ccgactggag tccatatgca 7440 gaccaaagca tcaaagtgag gataagccta aaatcagctc ttggagataa agcatatgaa 7500 tggaacgaca atgaaatgta cctgttccga tcatctgttg catatgctat gaggcagtac 7560 tttttaaaag taaaaaatca gatgattctt tttggggagg aggatgtgcg agtggctaat 7620 ttgaaaccaa gaatctcctt taatttcttt gtcactgcac ctaaaaatgt gtctgatatc 7680 attcctagaa ctgaagttga aaaggccatc aggat gtccc ggagccgtat caatgatgct 7740 ttccgtctga atgacaacag cctagagttt ctggggatac agccaacact tggacctcct 7 800 aaccagcccc ctgtttccat atggctgatt gtttttggag ttgtgatggg agtgatagtg 7860 gttggcattg tcatcctgat cttcactggg atcagagatc ggaagaagaa aaataaagca 7920 agaagtggag aaaatcctta tgcctccatc gatattagca aaggagaaaa taatccagga 7980 ttccaaaaca ctgatgatgt tcagacctcc ttttaatcta gagcggccgc cgaattcggg 8040 cccgtttaaa cccgctgatc agcct cgact gtgccttcta gttgccagcc atctgttgtt 8100 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 8160 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 8220 gtggggca gg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 8280 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac 8340 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 8400 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 8460 ttcgcaggct ttcccc gtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 8520 gctttacggc acctcgaccc caaaaaactt gattagggg atggttcacg tagtgggcca 8580 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 8640 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattctt t tgatttataa 8700 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 8760 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag 8820 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc 8880 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag 8940 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 9000 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc 9060 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctctcgg 9 120 gagcttgtat atccattttc ggatctgatc agcacgtgat gaaaaagcct gaactcaccg 9180 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc 9240 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt agg agggcgt ggatatgtcc 9300 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg 9360 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga 9420 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac 9480 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta 9540 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc 9600 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg 9660 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact 9720 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca 9780 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg 9840 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct 9900 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc 9960 gcatt ggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt 10020 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac 10080 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaag ta ctcgccgata 10140 gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgtg ctacgagatt 10200 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgctg 10260 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt 10320 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 10380 catttttttc actgcat tct agttgtggtt tgtccaaact catcaatgta tcttatcatg 10440 tctgtatacc gtcgacattg attattgact agttattaat agtaatcaat tacggggtca 10500 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 10560 ggctgaccg c ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 10620 acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac 10680 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 10740 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 10800 tacatctacg tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt 10860 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt 10920 attttgtgca gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg 10980 gcgaggggcg gggcggggcg a ggcggagag gtgcggcggc agccaatcag agcggcgcgc 11040 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg 11100 cgcggcgggc gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc 11160 gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc 11220 cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc 11280 tgcgtgaaag ccttaaaggg ctccgggagg gccctttgg cgggggggag cggctcgggg 11340 ggtgcgtgcg tgtgtgtgg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct 11400 gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc 11460 ggccgggggc ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg 11520 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc 11580 cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc 11640 ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg 11700 cggggcgggg ccgcctcggg ccggggaggg ctcgggggg gggcgcggcg gccccggagc 11760 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag 11820 agggcgcagg gact tccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc 11880 gcaccccctc tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg 11940 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg 12000 ccgcaggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt 12060 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt t ttcctacag 12120 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcatggtg 12180 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac 12240 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag 12300 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg 12360 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac 12420 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag 12480 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac 1254 0 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg 12600 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc 12660 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac 1 2720 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg 12780 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg 12840 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaagaattc 12900 actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct 12960 cacaaatacc actg agatct ttttccctct gccaaaaatt atggggacat catgaagccc 13020 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg 13080 aattttttgt gtctctcact cggaaggaca tatgggaggg caatcattt aaaacatcag 1314 0 aatgagtatt tggtttagag tttggcaaca tatgccatat gctggctgcc atgaacaaag 13200 gtggctataa agaggtcatc agtatatgaa acagccccct gtcatagctg tccctcttct cttatgaaga tccctcgacc tgcagttggg 13440 gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg 13500 ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt cc gttcgcag 13560 cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc 13620 cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc 13680 acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc 13740 gcgatgggct gtggccaata gcggctgctc agcagggcgc gccggtacca gcttttgttc 13800 cctttagtga gggttaattt cgagcttggc g taatcatgg tcatagctgt ttcctg 13856 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ACE2 forward primer < 400> 4 gtacatctac gtattagtca tcgc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ACE2 reverse primer<400> 5 gttcaaccgt ttgctcttgt cttc 24

Claims (17)

서열번호 1의 염기서열로 표시되는 인간 안지오텐신 전환효소 2(Angiotensin-converting enzyme 2, ACE2) 유전자, 인핸서 및 프로모터를 포함하는, 제2형 중증급성호흡기증후군 코로나바이러스(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터.
Severe acute respiratory syndrome coronavirus 2 (SARS), including human angiotensin-converting enzyme 2 (ACE2) gene, enhancer and promoter represented by the nucleotide sequence of SEQ ID NO: 1 -CoV-2) recombinant vector for animal model production with increased susceptibility.
 제 1항에 있어서,
상기 동물모델은 돼지인 것을 특징으로 하는, SARS-CoV-2에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터.
According to claim 1,
The animal model is a recombinant vector for producing an animal model with increased susceptibility to SARS-CoV-2, characterized in that pig.
 제 1항에 있어서,
상기 프로모터는 CAG 프로모터, CMV 프로모터, EF1α 프로모터, ICAM2 프로모터, SV40 프로모터, PGK1 프로모터, Ubc 프로모터 및 β-Act 프로모터로 이루어진 군에서 선택된 어느 하나의 외인성 프로모터 (exogenous promoter)인 것을 특징으로 하는, SARS-CoV-2에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터.
According to claim 1,
SARS- A recombinant vector for producing animal models with increased susceptibility to CoV-2.
 제 1항에 있어서,
상기 재조합 벡터는 하기 벡터맵으로 표시되는 것을 특징으로 하는, SARS-CoV-2에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터.

According to claim 1,
The recombinant vector is a recombinant vector for producing an animal model with increased susceptibility to SARS-CoV-2, characterized in that represented by the vector map below.

제 1항에 있어서,
상기 재조합 벡터는 서열번호 2의 염기서열로 표시되는 서열을 포함하는 것을 특징으로 하는, SARS-CoV-2에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터.
According to claim 1,
The recombinant vector is a recombinant vector for producing animal models with increased susceptibility to SARS-CoV-2, characterized in that it comprises a sequence represented by the nucleotide sequence of SEQ ID NO: 2.
 제 1항에 있어서,
상기 프로모터는 GGTA1(alpha 1,3- galactosyltransferase) 프로모터, ROSA26 프로모터 및 AAVS1(PPP1R12C locus) 프로모터로 이루어진 군에서 선택된 어느 하나의 내인성 프로모터(endogenous promoter)인 것을 특징으로 하는, SARS-CoV-2에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터.
According to claim 1,
The promoter is any one endogenous promoter selected from the group consisting of GGTA1 (alpha 1,3-galactosyltransferase) promoter, ROSA26 promoter and AAVS1 (PPP1R12C locus) promoter. For SARS-CoV-2 A recombinant vector for producing animal models with increased sensitivity.
 제 1항에 있어서,
상기 재조합 벡터는 하기 벡터맵으로 표시되는 것을 특징으로 하는, SARS-CoV-2에 대한 감수성이 증가된 동물모델 제작용 재조합 벡터.

According to claim 1,
The recombinant vector is a recombinant vector for producing an animal model with increased susceptibility to SARS-CoV-2, characterized in that represented by the vector map below.

제 1항의 재조합 벡터를 체세포에 형질전환시켜 제조한, SARS-CoV-2에 대한 감수성이 증가된 형질전환 세포주.
A transformed cell line with increased sensitivity to SARS-CoV-2 prepared by transforming the recombinant vector of claim 1 into somatic cells.
 제 8항에 있어서,
상기 체세포는 돼지의 섬유아세포인 것을 특징으로 하는, SARS-CoV-2에 대한 감수성이 증가된 형질전환 세포주.
According to claim 8,
Characterized in that the somatic cells are porcine fibroblasts, a transformed cell line with increased sensitivity to SARS-CoV-2.
 제 8항에 있어서,
상기 형질전환 세포주는 수탁번호 KCLRF-BP-00512인 것을 특징으로 하는, SARS-CoV-2에 대한 감수성이 증가된 형질전환 세포주.
According to claim 8,
The transformed cell line is a transformed cell line with increased sensitivity to SARS-CoV-2, characterized in that accession number KCLRF-BP-00512.
 제 8항의 형질전환 세포주를 탈핵시킨 돼지 수정란에 도입하여 핵치환된, SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지 생산을 위한 핵 이식란.
A nuclear transfer embryo for the production of transgenic pigs for animal models with increased susceptibility to SARS-CoV-2, which is nuclear-transferred by introducing the transformed cell line of claim 8 into enucleated pig fertilized eggs.
 제 8항의 형질전환 세포주를 핵 이식하여 제조된 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지.
Transgenic pigs for animal models with increased susceptibility to SARS-CoV-2 prepared by nuclear transfer of the transformed cell line of claim 8.
 제 8항의 형질전환 세포주를 탈핵화된 난자에 이식하여 재구성된 난자를 제조하는 핵이식 단계; 및
상기 재구성된 난자를 대리모의 난관에 이식하는 단계;를 포함하는 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지의 제조방법.
a nuclear transfer step of transplanting the transformed cell line of claim 8 into enucleated eggs to produce reconstituted eggs; and
A method for producing a transgenic pig for an animal model with increased susceptibility to SARS-CoV-2, comprising the step of transplanting the reconstructed oocyte into the fallopian tube of a surrogate mother.
 제 8항의 SARS-CoV-2에 대한 감수성이 증가된 형질전환 세포주 또는 상기 형질전환 세포주를 핵 이식하여 제조된 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지를 이용한 SARS-CoV-2 감염 질환의 예방 또는 치료제의 스크리닝 방법.
SARS-CoV using the transformed cell line with increased susceptibility to SARS-CoV-2 of claim 8 or a transgenic pig for animal model with increased susceptibility to SARS-CoV-2 prepared by nuclear transfer of the transformed cell line -2 A screening method for preventive or therapeutic agents for infectious diseases.
 제 14항에 있어서,
상기 방법은 하기 단계를 포함하는 것을 특징으로 하는, SARS-CoV-2 감염 질환의 예방 또는 치료제의 스크리닝 방법:
1) 제 8항의 SARS-CoV-2에 대한 감수성이 증가된 형질전환 세포주 또는 상기 형질전환 세포주를 핵 이식하여 제조된 SARS-CoV-2에 대한 감수성이 증가된 동물모델용 형질전환 돼지에 SARS-CoV-2를 접종하는 단계;
2) 상기 형질전환 세포주 또는 형질전환 돼지에 SARS-CoV-2 감염 질환의 예방 또는 치료제 후보물질을 투여하는 단계; 및
3) 후보물질을 투여한 군과 후보물질을 투여하지 않은 대조군을 비교하여 SARS-CoV-2 감염 질환의 증상 또는 조직병리학적 지표 값을 유의미하게 변화시키는 후보물질을 선택하는 단계.
According to claim 14,
The method for screening for a preventive or therapeutic agent for SARS-CoV-2 infectious disease, characterized in that it comprises the following steps:
1) Transgenic pigs with increased sensitivity to SARS-CoV-2 prepared by nuclear transfer of the transformed cell line with increased sensitivity to SARS-CoV-2 of claim 8 or SARS-CoV-2 for animal model inoculating CoV-2;
2) administering a candidate substance for preventing or treating SARS-CoV-2 infectious disease to the transgenic cell line or transgenic pig; and
3) A step of selecting a candidate substance that significantly changes symptoms or histopathological index values of SARS-CoV-2 infectious disease by comparing the group administered with the candidate substance and the control group not administered with the candidate substance.
 제 15항에 있어서,
상기 증상은 발열, 호흡곤란, 두통, 인후통, 객혈, 오심, 기침, 객담, 설사, 후각 상실, 권태감, 식욕부진, 근육통 및 인지장애로 이루어진 군에서 선택된 어느 하나 이상인 것을 특징으로 하는, SARS-CoV-2 감염 질환의 예방 또는 치료제의 스크리닝 방법.
According to claim 15,
The symptoms are characterized in that at least one selected from the group consisting of fever, dyspnea, headache, sore throat, hemoptysis, nausea, cough, sputum, diarrhea, loss of smell, malaise, anorexia, muscle pain and cognitive impairment, SARS-CoV -2 A screening method for preventive or therapeutic agents for infectious diseases.
 제 15항에 있어서,
상기 조직병리학적 지표 값은 염증성 사이토카인의 분비, 부종, 국소 출혈 및 염증세포 침윤 여부로 이루어진 군에서 선택된 어느 하나 이상인 것을 특징으로 하는, SARS-CoV-2 감염 질환의 예방 또는 치료제의 스크리닝 방법.According to claim 15,
The histopathological index value is characterized in that at least one selected from the group consisting of inflammatory cytokine secretion, edema, local hemorrhage, and inflammatory cell infiltration.
KR1020210058660A 2020-05-06 2021-05-06 Transgenic cloned porcine for animal models with increased susceptibility to severe acute respiratory syndrome coronavirus 2, and method for producing the same KR102563238B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020200053935 2020-05-06
KR20200053935 2020-05-06

Publications (2)

Publication Number Publication Date
KR20210136873A KR20210136873A (en) 2021-11-17
KR102563238B1 true KR102563238B1 (en) 2023-08-04

Family

ID=78468153

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020210058660A KR102563238B1 (en) 2020-05-06 2021-05-06 Transgenic cloned porcine for animal models with increased susceptibility to severe acute respiratory syndrome coronavirus 2, and method for producing the same

Country Status (2)

Country Link
KR (1) KR102563238B1 (en)
WO (1) WO2021225384A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090083865A1 (en) 2006-01-11 2009-03-26 Chan Teh-Sheng Transgenic Mouse Lines Expressing Human Ace2 and Uses Thereof
WO2022232348A1 (en) 2021-04-28 2022-11-03 Academia Sinica Angiotensin-converting enzyme ii (ace2) transgenic animal and uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101890978B1 (en) * 2014-06-17 2018-08-24 서울대학교산학협력단 Transgenic cloned porcine Models for alzheimer's disease and the Use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090083865A1 (en) 2006-01-11 2009-03-26 Chan Teh-Sheng Transgenic Mouse Lines Expressing Human Ace2 and Uses Thereof
WO2022232348A1 (en) 2021-04-28 2022-11-03 Academia Sinica Angiotensin-converting enzyme ii (ace2) transgenic animal and uses thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GREY, F., `Generation of human ACE2-expressing pig as a model of Covid-19, In: Webinar: Edinhurgh`s response to the Covid-19 pandemic, pp.1-8(2020.04.28.)
Nature vol.583, pp.830-833(2020.05.07., https://doi.org/10.1038/s41586-020-2312-y)
Research Results in Pharmacology, vol.6(2), pp.1-7(2020.05.04.)

Also Published As

Publication number Publication date
KR20210136873A (en) 2021-11-17
WO2021225384A1 (en) 2021-11-11

Similar Documents

Publication Publication Date Title
CN110022906A (en) Anti- BCMA CAR T cell composition
US6824770B1 (en) Adenovirus gene expression system
CN110656090B (en) Expression plasmid, cell strain for packaging capacity-increased second-generation adenovirus and application of cell strain
WO1996040911A1 (en) Modified steroid hormones for gene therapy and methods for their use
CN113559281A (en) Regenerating functional neurons for treatment of disease and injury in the nervous system
US6759236B1 (en) Methods to enhance and confine expression of genes
CN112458058B (en) TRAF6 over-expression DC cell, DC cell vaccine, construction method and application
US11820998B2 (en) Porcine Thy1 gene promoter specifically expressed in neurons
CN111272998A (en) Method for simultaneously detecting central demyelinating autoantibodies AQP4, MOG and MBP
CN108707626B (en) Preparation method of monoclonal cell line capable of detecting pyrogen
KR102563238B1 (en) Transgenic cloned porcine for animal models with increased susceptibility to severe acute respiratory syndrome coronavirus 2, and method for producing the same
CN109097392A (en) A kind of Her2-CAR-T system constituting method based on PiggyBac carrier
KR102280546B1 (en) A method for converting a nucleic acid sequence of a cell, which specifically converts a nucleic acid base of a targeted DNA using a cell endogenous DNA modifying enzyme, and a molecular complex using the same
US20040154046A1 (en) Gfp expression vector localized in mitochondria
US7265262B2 (en) Telomerizing nuclear donor cells and improving the efficiency on nuclear transfer
CN112522205B (en) Cell line for over-expressing angiotensin converting enzyme 2 as well as preparation method and application thereof
CN109957551B (en) Recombinant vaccinia virus expressing human beta-defensin 2 and application thereof
CN112852759A (en) Recombinant rabies virus of chimeric canine parvovirus VP2 gene and application thereof
US6693226B1 (en) Transgenic mice expressing human p25
CN111349653B (en) Preparation method of human alpha-lactalbumin gene site-directed integration transgenic dairy cow
CN114085868A (en) Targeting vector, recombinant Huh7 cell line, construction method and application
CN114014924B (en) Method for improving homologous recombination efficiency in gene editing process through BRCA1 and BARD1 proteins
KR20200135852A (en) Method for producing genetically modified lymphocytes
KR20130048562A (en) MIS18α KNOCKOUT MOUSE MODEL AND PRODUCING METHOD THEREOF
CN113025651B (en) Novel application of drug screening cell model, triciribine and structural analogue of targeted HBV core promoter

Legal Events

Date Code Title Description
E701 Decision to grant or registration of patent right
GRNT Written decision to grant