KR102103857B1 - 메르스 코로나바이러스의 뉴클레오캡시드 단백질의 n-말단 도메인 단편 및 c-말단 도메인 단편을 포함한 nc 융합 단백질 및 이를 이용한 메르스 코로나바이러스 감염 진단용 키트 - Google Patents
메르스 코로나바이러스의 뉴클레오캡시드 단백질의 n-말단 도메인 단편 및 c-말단 도메인 단편을 포함한 nc 융합 단백질 및 이를 이용한 메르스 코로나바이러스 감염 진단용 키트 Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C—CHEMISTRY; METALLURGY
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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Abstract
본 발명은 메르스 코로나바이러스 뉴클레오캡시드 단백질의 N-말단 및 C-말단 도메인 단편을 포함하는 NC 융합 단백질 및 이를 이용한 메르스 코로나바이러스 감염 진단용 키트에 관한 것이다. 본 발명의 NC 융합 단백질은 대장균에서 뉴클레오캡시드 전장 단백질보다 훨씬 더 안정하게 대량 생산이 가능하다. 또한, 본 발명에 따른 서열번호 8로 표시되는 NC 융합 단백질을 항원으로 하였을 때, 메르스 코로나바이러스에 특이적인 항체를 효과적으로 생산할 수 있다. 또한, 상기 NC 융합 단백질은 메르스 코로나바이러스에 의해 유도되는 항체에 반응을 하므로, 상기 NC 융합 단백질은 메르스 코로나바이러스 진단 키트에 활용될 수 있다.
Description
본 발명은 메르스 코로나바이러스 뉴클레오캡시드 단백질(Nucleocapsid protein)의 N-말단 도메인 단편 및 C-말단 도메인 단편을 포함한 NC 융합 단백질 및 이를 이용한 메르스 코로나바이러스 감염 진단용 키트에 관한 것이다.
메르스(middle east respiratory syndrome, MERS)는 메르스 코로나바이러스 감염으로 인한 중증 급성 호흡기 질환이다. 메르스 코로나바이러스(MERS coronavirus, MERS-CoV)는 2012년 사우디아라비아에서 새로 발견된 신종 베타코로나 바이러스이다(Azhar EI, et al., 2014). MERS-CoV의 명칭은 2013년 5월 국제바이러스 분류 위원회에 의해 명명되었으며, 이전에는 신종 코로나 바이러스, SARS 유사 바이러스, 중동 사스, 또는 사우디 사스라고 불렸다. 이러한 MERS-CoV의 감염경로는 박쥐로부터 낙타와 사람에게 전염되는 것으로 알려져 있다. 또한, MERS-CoV는 사람 간의 전염이 가능하며 40%의 치사율을 보이고 있다.
MERS-CoV의 유전체는 비분절 형태이며 크기는 약 30 kb로 11개의 단백질을 암호화하고 있다. 상기 11개의 단백질은 2개의 복제 다단백질, 4개의 구조 단백질, 및 5개의 비구조적(non-structural) 단백질로 구성되어 있다.
현재, 사스 코로나바이러스의 연구를 기반으로, 영장류 모델에서 메르스 코로나바이러스 백신을 개발하고 있다. 개발중인 메르스 코로나 바이러스 백신은 재조합된 수용체 결합 도메인(RBD)을 기반으로 한 백신으로, 접종을 통해 체액성 및 세포성 면역반응을 증가시키는 것으로 나타났다. 그 외에도 다양한 메르스 바이러스 백신이 개발 중에 있다.
또한, MERS-CoV의 신속 진단을 위해 안티젠 래피드 사에서 제품이 개발되었다. 그러나, 안티젠 래피드 사 제품의 경우, 뉴클레오캡시드 단백질의 일부 펩타이드를 대장균에서 발현시킨 후 이를 정제함으로써 단일 클론 항체를 만들어 이용하고 있다. Antigen Rapid MERS-CoV Ag Test 제품은 현재 낙타의 메르스 진단을 위하여 사용이 승인되어 사우디아라비아와 아랍에미리트에서 사용 중이다.
높은 치사율을 보인 MERS는 감염시 호흡기뿐만 아니라 폐렴 또는 급성신부전 등의 합병증을 동반하기 때문에 빠른 진단을 통한 초기 치료가 매우 중요하다. 그러나 아직 인체용으로 승인된 MERS 신속 진단 키트가 없어, 개발이 필요한 실정이다.
Azhar EI, Hashem AM, El-Kafrawy SA, Sohrab SS, Aburizaiza AS, Farraj SA, Hassan AM, Al-Saeed MS, Jamjoom GA, Madani TA. 2014. Detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient. mBio 5(4):e01450-14. doi:10.1128/mBio.01450-14.
본 발명의 목적은 MERS-CoV 진단을 위한 항원을 제공하는 것이다.
본 발명의 다른 목적은 상기 항원을 포함한 MERS-CoV를 진단할 수 있는 MERS-CoV 감염 진단용 키트를 제공하는 것이다.
상기 목적을 달성하기 위하여, 본 발명은 MERS-CoV의 뉴클레오캡시드 단백질의 N-말단 도메인 단편 및 C-말단 도메인 단편을 포함하는 융합 단백질(이하, NC 융합 단백질)을 제공한다.
또한, 본 발명은 MERS-CoV의 뉴클레오캡시드 단백질의 NC 융합 단백질에 특이적으로 결합하는 단클론 항체를 제공한다.
또한, 본 발명은 MERS-CoV의 뉴클레오캡시드 단백질의 NC 융합 단백질을 코딩하는 뉴클레오티드를 포함하는 발현 벡터를 제공한다.
또한, 본 발명은 상기 발현 벡터로 형질감염된 숙주세포를 제공한다.
또한, 본 발명은 상기 형질감염된 숙주세포로부터 NC 융합 단백질을 수득하는 단계를 포함하는 NC 융합 단백질 제조방법을 제공한다.
또한, (a) MERS-CoV의 뉴클레오캡시드 단백질의 NC 융합 단백질을 항원으로서 실험동물에 주입하여 면역반응을 유도하는 단계; 및 (b) 상기 (a)단계의 실험동물로부터 항체를 회수하는 단계를 포함하는 MERS-CoV 항체 제조 방법을 제공한다.
상기 다른 목적을 달성하기 위하여, 본 발명은 MERS-CoV의 뉴클레오캡시드 단백질의 NC 융합 단백질을 포함하는 MERS-CoV 감염 진단용 키트를 제공한다.
또한, 본 발명은 상기 MERS-CoV 감염 진단용 키트에 진단 대상으로부터 분리한 시료를 첨가하는 단계를 포함하는 MERS-CoV 감염 정보제공 방법을 제공한다.
본 발명에 따른 서열번호 8로 표시되는 MERS-CoV의 뉴클레오캡시드 단백질의 NC 융합 단백질은 대장균에서 대량 생산이 가능하다. 또한, 개체 내에서 생성된 MERS-CoV의 항체에 특이적으로 결합하여 MERS-CoV 진단에 널리 활용될 수 있다.
도 1은 NC 융합 단백질을 발현 시킬 수 있는 대장균 발현 벡터 pET28a-MERS-CoV_NP_NC를 나타낸 것이다.
도 2a 내지 도 2c는 SDS-PAGE를 이용하여 N-말단 도메인(a), C-말단 도메인(b) 및 NC 융합 단백질 도메인(c)을 확인한 결과를 나타낸 것이다. 이때, Sup은 상층액, ppt는 펠렛, FT는 통과액(flow through), HS-W는 고염도 워시(high salt wash), 그리고 7%-W는 7% 이미다졸 워시(imidazole wash)를 의미한다.
도 2d는 N-말단, C-말단, 및 NC 융합 단백질을 니켈-친화성 크로마토그래피로 정제하여 SDS-PAGE로 확인한 결과를 나타낸 것이다. 이때, 5 ug은 로딩한 각 단백질의 양을 의미한다.
도 3은 마우스를 이용하여 NC 융합 단백질의 면역 항체 형성 효과를 ELISA 테스트를 통해 확인한 결과를 나타낸 것이다. 이때, OD는 흡광도(optical density)를 의미한다.
도 4는 기존에 상용화 되어있는 MERS-CoV 진단 키트인 euroimmun사의 키트와 본 발명의 NC 융합 단백질을 이용한 키트의 검출 감도를 비교하여 나타낸 것이다.
도 5는 MERS-CoV, SARS-CoV, HCoV-229E 및 HCoV-OC43 단백질의 NC 융합 단백질 크로마토그래피로 정제하여 SDS-PAGE로 확인한 결과를 나타낸 것이다. 이때, 5 ug은 로딩한 각 단백질의 양을 의미한다.
도 6은 MERS-CoV, SARS-CoV, HCoV-229E 및 HCoV-OC43 단백질의 인간 CoV 항체와의 교차 반응성을 확인한 것이다.
도 2a 내지 도 2c는 SDS-PAGE를 이용하여 N-말단 도메인(a), C-말단 도메인(b) 및 NC 융합 단백질 도메인(c)을 확인한 결과를 나타낸 것이다. 이때, Sup은 상층액, ppt는 펠렛, FT는 통과액(flow through), HS-W는 고염도 워시(high salt wash), 그리고 7%-W는 7% 이미다졸 워시(imidazole wash)를 의미한다.
도 2d는 N-말단, C-말단, 및 NC 융합 단백질을 니켈-친화성 크로마토그래피로 정제하여 SDS-PAGE로 확인한 결과를 나타낸 것이다. 이때, 5 ug은 로딩한 각 단백질의 양을 의미한다.
도 3은 마우스를 이용하여 NC 융합 단백질의 면역 항체 형성 효과를 ELISA 테스트를 통해 확인한 결과를 나타낸 것이다. 이때, OD는 흡광도(optical density)를 의미한다.
도 4는 기존에 상용화 되어있는 MERS-CoV 진단 키트인 euroimmun사의 키트와 본 발명의 NC 융합 단백질을 이용한 키트의 검출 감도를 비교하여 나타낸 것이다.
도 5는 MERS-CoV, SARS-CoV, HCoV-229E 및 HCoV-OC43 단백질의 NC 융합 단백질 크로마토그래피로 정제하여 SDS-PAGE로 확인한 결과를 나타낸 것이다. 이때, 5 ug은 로딩한 각 단백질의 양을 의미한다.
도 6은 MERS-CoV, SARS-CoV, HCoV-229E 및 HCoV-OC43 단백질의 인간 CoV 항체와의 교차 반응성을 확인한 것이다.
본 발명은 일 측면으로, 메르스(middle east respiratory syndrome, MERS) 코로나바이러스의 뉴클레오캡시드 단백질의 N-말단 도메인 단편 및 C-말단 도메인 단편의 융합 단백질을 제공한다.
상기 메르스 코로나바이러스(이하, MERS-CoV 라고 한다)의 뉴클레오캡시드는 MERS 유전체가 암호화하는 총 11개의 단백질(2개의 복제 다단백질, 4개의 구조 단백질, 및 5개의 비구조적 단백질) 중 구조 단백질에 속한다. 이러한 뉴클레오캡시드는 MERS-CoV가 세포 밖에 있을 때 유전물질을 보호하고 새로운 숙주세포에 침투할 때 유전자가 그 세포 내로 유입되도록 도와주는 기능을 한다.
구체적으로, 상기 MERS-CoV의 뉴클레오캡시드 단백질의 N-말단 도메인은 서열번호 4의 아미노산 서열을 갖는 폴리펩타이드일 수 있다. 이때, 상기 단백질은 서열번호 3의 염기서열에 의해 코딩될 수 있다. 또한, 본 발명에서 상기 N-말단 도메인의 단편은 서열번호 4의 36번 위치의 아미노산부터 169번 위치의 아미노산 서열 중 1개 내지 134개, 10개 내지 120개, 30개 내지 100개, 또는 50개 내지 80개의 연속된 아미노산 서열일 수 있다.
상기 MERS-CoV의 뉴클레오캡시드 단백질의 C-말단 도메인은 서열번호 6의 아미노산 서열을 갖는 폴리펩타이드일 수 있다. 이때, 상기 단백질은 서열번호 5의 염기서열에 의해 코딩될 수 있다. 또한, 본 발명에서 상기 C-말단 도메인의 단편은 서열번호 6의 37번 위치의 아미노산부터 153번 위치의 아미노산 서열 중 1개 내지 117개, 10개 내지 100개, 20개 내지 80개, 또는 30개 내지 50개의 연속된 아미노산 서열일 수 있다.
상기 MERS-CoV의 뉴클레오캡시드 단백질의 NC 융합 단백질은 서열번호 8의 아미노산 서열을 가질 수 있다. 또한, 상기 NC 융합 단백질은 서열번호 7의 염기서열에 의해 코딩될 수 있다.
본 발명은 또 다른 측면으로, MERS-CoV의 뉴클레오캡시드 단백질의 N-말단 도메인 단편 및 C-말단 도메인 단편을 포함하는 융합 단백질에 특이적으로 결합하는 단클론 항체를 제공한다.
상기 용어 "항체"는 면역계 내에서 항원의 자극에 의하여 만들어지는 성분으로서 특정한 항원과 특이적으로 결합하여 림프와 혈액을 떠돌며 항원-항체반응을 일으키는 단백질이다. 항원-항체 반응은 각 항원에 대하여 높은 특이성을 갖는다. 이는 림프구의 B세포에서 항체가 만들어질 때 특정항원에 의해 생성된 항체는 원칙적으로 다른 항원과 반응하지 않는다. 이러한 높은 특이성은 면역, 알레르기, 각종 병, 및 감염 바이러스의 종류 및 유형의 결정 등의 검사에 사용된다.
이때, 상기 벡터는 pET28a일 수 있으며, 상기 형질전환체는 대장균 속(Escherichia) 미생물일 수 있다. 본 발명은 일 실시예에서는, 서열번호 4로 표시되는 MERS-CoV 뉴클레오캡시드 단백질의 N-말단 도메인의 아미노산 잔기 36-159를 pET28a 벡터의 NdeI과 BamHI 부위에 서브클론하고 대장균 균주를 이용하여 과발현하였다. 또한, 서열번호 6으로 표시되는 MERS-CoV 뉴클레오캡시드 단백질의 C-말단 도메인의 아미노산 잔기 37-153을 pET28a의 EcoRI과 XhoI 부위에 서브클론하고 대장균 균주를 이용하여 과발현하였다. 이후, 각각의 단백질을 대장균에 접종하고 배양하였다.
상기 용어 "항원"은 바이러스의 구성성분 중 면역기능을 일으킬 수 있는 성분으로서 바이러스가 발현하는 단백질이다. 본 발명의 일 실시예에서는, 서열번호 8의 아미노산 서열을 가지는 MERS-CoV 뉴클레오캡시드 단백질의 NC 융합 단백질 도메인이 개체에 투여되었을 때 면역 항체 형성을 유도하는 항원으로 작용하였다.
또한, 본 발명은 상기 융합 단백질을 코딩하는 뉴클레오티드를 포함하는 발현 벡터를 제공하며, 상기 발현 벡터로 형질감염된 숙주세포를 제공한다. 또한, 본 발명은 상기 형질감염된 숙주세포로부터 NC 융합 단백질을 수득하는 단계를 포함하는 NC 융합 단백질 제조방법을 제공한다.
본 발명의 일 실시예에서는, MERS-CoV 뉴클레오캡시드 단백질의 N-말단 및 C-말단 도메인을 코딩하는 뉴클레오티드를 pET28a 벡터에 서브클론하고, 이를 대장균에 접종하여 배양함으로써 NC 융합 단백질을 수득하였다.
본 발명은 또 다른 측면으로, (a) MERS-CoV의 뉴클레오캡시드 단백질의 N-말단 도메인 단편 및 C-말단 도메인 단편의 융합 단백질을 항원으로서 실험동물에 주입하여 면역반응을 유도하는 단계; 및 (b) 상기 (a)단계의 실험동물로부터 항체를 회수하는 단계를 포함하는 메르스 코로나바이러스 항체 제조 방법을 제공한다.
또한, 본 발명은 MERS-CoV의 뉴클레오캡시드 단백질의 NC 융합 단백질을 포함하는 MERS-CoV 감염 진단용 키트를 제공한다.
본 발명의 일 실시예에서는, 서열번호 8의 아미노산 서열을 가지는 MERS-CoV 뉴클레오캡시드 단백질의 NC 융합 단백질은 메르스 코로나바이러스에 대한 항체와 특이적으로 결합할 수 있어, MERS-CoV 감염 진단 키트에 적용될 수 있다. 항원-항체 반응을 유도하여 기존에 시판되고 있는 MERS-CoV 감염 진단 키트에 비해 낮은 농도의 항원으로도 효과적으로 MERS-CoV 감염 여부를 진단할 수 있었다. 특히, MERS-CoV에 감염된 개체에서 생성된 항체는 본 발명의 NC 융합 단백질과 결합할 수 있으므로, 개체가 MERS-CoV에 감염되었는지 여부를 용이하게 진단할 수 있다.
또한, 본 발명은 상기 MERS-CoV 감염 진단용 키트에 진단 대상에서 분리된 시료를 첨가하는 단계를 포함하는 메르스 코로나바이러스 감염 정보제공 방법을 제공한다.
상기 용어 "MERS-CoV 감염 진단용 키트"는 MERS-CoV 감염 및 이에 의한 질병을 신속하게 진단할 수 있는 키트이다. 본 발명의 일 실시예에서는, 환자로부터 추출한 검체에 항원이 코팅되어 있는 스트립을 담가 항원-항체 반응을 통한 발색반응을 확인할 수 있다. 이러한 키트를 이용한 검사들은 10분 내지 15분 이내에 검사 결과를 알 수 있으며, 전문가 또는 진단을 위한 장비가 필요하지 않아 누구나 쉽게 MERS-CoV 감염에 의한 질병을 진단할 수 있다는 장점이 있다.
상기 "메르스 코로나바이러스 감염에 의한 질병"은 호흡기 질환일 수 있으며, 바이러스 감염 후 2일 내지 14일의 잠복기를 거친 뒤 38℃ 이상의 고열, 기침 또는 호흡곤란 등의 심한 호흡기 증상이 나타날 수 있다. 설사 또는 변비 등의 소화기 증상을 보이는 경우도 있으며, 만성질환 또는 면역 저하자의 경우 폐렴 또는 급성신부전 등의 합병증이 동반되어 사망에 이를 수도 있다.
이하, 본 발명을 실시예 및 실험예에 의해 상세히 설명한다. 단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다.
실시예
1.
MERS
-
CoV
뉴클레오캡시드
N-말단 단편 및 C-말단 단편을 결합한 NC 융합 단백질의 제조
진단 키트 개발을 위한 항원으로 사용될 수 있는 MERS-CoV 뉴클레오캡시드 단백질의 서열을 정립하기 위하여, 뉴클레오캡시드 단백질의 구조를 분석하였다. 상기 구조 분석을 통해 안정한 형태의 2차 구조를 갖는 뉴클레오캡시드 단백질의 아미노산 서열을 결정하였다. 이때, 상기 MERS-CoV(Human betacoronavirus 2cEMC/2012)의 뉴클레오캡시드 단백질(413개 아미노산)의 합성은 바이오니아에서 수행되었으며, 이의 염기서열은 서열번호 1에 나타내었다.
상기 뉴클레오캡시드 단백질의 N-말단 및 C-말단 도메인 단편을 결합한 뉴클레오캡시드 융합 단백질을 제조하기 위하여, 서열번호 4로 표시되는 MERS-CoV 뉴클레오캡시드 단백질의 N-말단 도메인의 아미노산 잔기 36-159를 코딩하는 뉴클레오티드를 pET28a 벡터의 NdeI과 BamHI 부위에 서브클론하고 대장균(Escherichia coli BL21(DE3) pLysS Rosetta) 균주를 이용하여 N-말단의 36-159 부분을 코딩하는 뉴클레오티드를 과발현하였다. 뉴클레오캡시드 단백질의 또 다른 부위로서 서열번호 6으로 표시되는 C-말단 도메인의 아미노산 잔기 37-153을 코딩하는 뉴클레오티드를 pET28a의 EcoRI과 XhoI 부위에 서브클론하고 대장균(Escherichia coli BL21(DE3) pLysS Rosetta) 균주를 사용하여 C-말단의 37-153 부분을 코딩하는 뉴클레오티드를 과발현하였다. 또는, N-말단의 36-159를 코딩하는 뉴클레오티드 및 C-말단의 37-153 부분을 코딩하는 뉴클레오티드를 각각 NdeI/BamHI 및 EcoRI/XhoI를 pET28a 벡터에 도입하였다.
각각의 단백질을 코딩하는 벡터를 대장균에 접종하여 OD 600 nm에서 0.2 mM의 IPTG(isopropyl β-D-1-thiogalactopyranoside) 시약을 넣고 18시간 동안 291K에서 배양하였다. 상기에서 사용된 대장균 발현 벡터 pET28a-MERS-CoV_NP_NC를 도 1에 나타내었다.
His-태그(tag)된 N-말단, C-말단 및 NC 융합 단백질은 니켈-친화성 크로마토그래피로 일차 정제하였다. NC 융합 단백질의 경우, 2배 농도의 인산완충생리식염수(phosphate buffered saline, PBS)를 사용하여 HiPrep 16/60 Sephacryl S-200 HR 겔-여과 크로마토그래피로 한번 더 정제하였다. 상기 과정을 통해, 정제된 N-말단, C-말단 및 NC 융합 단백질 도메인을 얻었다. 그 결과를 도 2에 나타내었다.
도 2a 내지 도 2c에 나타난 바와 같이, N-말단 도메인(a), C-말단 도메인(b) 및 NC 융합 단백질 도메인(c)이 SDS-PAGE 상에서 확인되었다. 또한, 도 2d에 나타난 바와 같이, 상기 각 도메인은 크로마토그래피에 의하여 순도 95% 이상으로 정제되었다. 이때, NC 융합 단백질 도메인의 염기 서열을 서열번호 7에 나타내었고, 이에 의해 제조되는 276개의 아미노산 서열을 서열번호 8에 나타내었다.
실험예
1. NC 융합 단백질의 항체 생성 확인
상기 실시예 1에서 제조된 NC 융합 단백질이 효과적으로 항체 생성을 유도하는지 여부를 확인하기 위하여, 이의 항체 생성 효과를 확인하였다.
먼저, 정제된 NC 융합 단백질 도메인의 항원을 마우스(코아텍, 한국)에 주사하기 전에 전-면역 혈청(pre-immune serum)을 채취하여 이를 음성 대조군으로 사용하였다). 일차적 면역화(Primary immunize)를 위하여 항원 100 ug을 프로인트 어주번트(freund's adjuvant)와 혼합하여 4마리의 마우스에 각각 근육주사하고, 2주 후 프로인트 어주번트와 혼합하여 1차 부스팅(boosting)을 진행하였다. 2주 후, 1차 혈액을 채취하여 ELISA 테스트 및 2차 부스팅을 진행하였다. 2주 후, 각 마우스를 채혈하고 최종 혈청을 분리하여 최종 ELISA 테스트를 수행하였다. 이때, ELISA 테스트에서 항원은 웰(well) 당 100 ng을 사용하였다. 또한, 면역 혈청은 1XPBS로 1:100 내지 1:1,000,000 비율로 희석하였고, 2차 확인을 위하여 항-마우스 IgG-HRP(ABC5001)을 1:5,000 비율로 희석하여 사용하였다. ELISA reader를 이용하여 광학 밀도 405 nm에서 발색을 검출하여 최종적으로 면역 항체 생성 여부를 확인하였으며, 이를 도 3에 나타내었다.
도 3에 나타낸 바와 같이, NC 융합 단백질은 상기 실험에 사용한 4마리의 마우스에서 모두 면역 항체 형성 효과를 나타내었다. 이는 NC 융합 단백질 도메인이 개체 내에서 항원으로 작용하여 항체를 효과적으로 유도할 수 있다는 것을 의미한다. 이를 통해, MERS-CoV 감염을 진단하기 위한 항체를 제작하기 위해 NC 융합 단백질을 항원으로서 사용할 수 있음을 확인하였다.
실험예
2. NC 융합 단백질을 이용하여 개발한
MERS
-
CoV
진단 키트
및 시판 되고
있는
키트의
진단능
비교
상기 실시예 1에서 제조된 NC 융합 단백질을 이용하여 개발한 MERS-CoV 진단 키트의 우수성을 시판되고 있는 MERS-CoV 진단 키트와 비교하여 확인하였다.
400 ng의 NC 융합 단백질 항원을 코팅한 플레이트(plate)에 40배 희석한 인간 혈청을 이용하여 ELISA를 수행하였으며, 플레이트에 2차 항체 HRP conjugated goat anti-human IgG를 PBS와 1:100 (12 ug/ml)으로 희석하여 indirect ELISA을 수행하였다.
시판되고 있는 MERS-CoV 진단 키트로서 euroimmun사의 키트를 사용하였다. 상기 키트에 들어있는 항원이 코팅된 플레이트에 인간 혈청을 이용하여 제조사의 메뉴얼대로 ELISA를 수행하였으며, 제조사 프로토콜에 따라 키트에 들어있는 플레이트에 사람 혈청을 샘플 버퍼와 1:100으로 희석하여 ELISA를 수행하였다. 그 결과를 도 4에 나타내었다.
도 4에 나타난 바와 같이, NC 융합 단백질 항원을 코팅한 플레이트를 이용하여 제작된 MERS-CoV 진단 키트가 시판되는 MERS-CoV 진단 키트인 euroimmun사의 키트에 비해 4배 정도 더 높은 진단능을 나타냈다. 이는 NC 융합 단백질 항원이 코팅된 플레이트를 이용한 MERS-CoV 진단 키트가 MERS-CoV 감염 초기 또는 회복기에 이르러 체내 바이러스의 양이 적은 환자에게서도 효과적인 진단이 가능함을 의미한다.
실험예
3. 인간
CoV
NC 융합 단백질 항체의 교차반응성 확인
실험예
3.1. 인간
CoV
NC 융합 단백질의 제조
MERS-CoV, SARS-CoV, HCoV-229E 및 HCoV-OC43 뉴클레오캡시드 단백질을 대장균 발현 시스템을 통해 제조하였다. 이때, SARS-CoV 뉴클레오캡시드 단백질은 서열번호 9의 아미노산 서열을 갖는 폴리펩타이드일 수 있으며, 상기 단백질은 서열번호 10의 염기서열에 의해 코딩될 수 있다. 또한, HCoV-229E 뉴클레오캡시드 단백질은 서열번호 11의 아미노산 서열을 갖는 폴리펩타이드일 수 있으며, 상기 단백질은 서열번호 12의 염기서열에 의해 코딩될 수 있다. 또한, HCoV-OC43 뉴클레오캡시드 단백질은 서열번호 13의 아미노산 서열을 갖는 폴리펩타이드일 수 있으며, 상기 단백질은 서열번호 14의 염기서열에 의해 코딩될 수 있다. 상기 각 인간 CoV 뉴클레오캡시드 단백질의 전체 유전자를 pET28a 벡터의 NdeI 및 BamHI 부위에 서브클론한 후, 대장균(Escherichia coli BL21(DE3) pLysS Rosetta) 균주를 이용하여 각 인간 CoV 뉴클레오캡시드 단백질을 과발현 하였다. 이때, 상기 각각의 인간 CoV 뉴클레오캡시드 단백질을 과발현 및 정제하는 방법은 상기 실시예 1과 동일한 방법으로 수행하였다. 상기 과정을 통해, SARS-CoV, HCoV-229E 및 HCoV-OC43 뉴클레오캡시드 단백질의 정제된 NC 융합 단백질 도메인을 수득하였으며, 그 결과를 도 5에 나타내었다.
도 5에 나타난 바와 같이, MERS-CoV, SARS-CoV, HCoV-229E 및 HCoV-OC43 뉴클레오캡시드 단백질의 NC 융합 단백질 도메인은 크로마토그래피에 의하여 순도 95% 이상으로 정제되었다.
실험예
3.2. 인간
CoV
항체와의 교차반응성 측정
상기 실험예 3.1에서 수득한 MERS-CoV, SARS-CoV, HCoV-229E 및 HCoV-OC43 단백질의 NC 융합 단백질이 인간 CoV 항체와 교차반응성(cross activity)을 일으키는지 확인하였다. 구체적으로, 96 웰 플레이트 표면 코팅에 사용된 단클론 항체는 MERS-CoV 뉴클레오캡시드 단백질의 N-말단 특이 항체로, 100 ng/웰의 농도로 코팅하여 4℃, O/N 코팅 진행하였다. 코팅된 96 웰 플레이트는 PBST 버퍼로 1회 세척한 후, 2% BSA로 37℃에서 1시간 동안 블로킹(blocking) 진행하였다. 블로킹 진행 후, 정제된 MERS-CoV, SARS-CoV, HCoV-229E 및 HCoV-OC43 뉴클레오캡시드 재조합 단백질을 100 ng/웰 내지 0.8 ng/웰까지 2배 희석하여 37℃에서 2시간 동안 인큐베이션하였다. 이후, PBST로 5분씩 3회 세척한 후 HRP 컨쥬게이션된 MERS-CoV 뉴클레오캡시드 C-말단 특이 단클론 항체를 200 ng/웰의 농도로 처리하여 37℃에서 1시간 동안 인큐베이션하였다. 그 다음, PBST로 5분씩 5회 세척한 후, TMB 용액으로 발색 반응을 진행하였고, 그 결과를 도 6에 나타내었다.
도 6에 나타난 바와 같이, SARS-CoV, HCoV-229E 및 HCoV-OC43 뉴클레오캡시드 단백질의 NC 융합 단백질과 다르게, MERS-CoV 뉴클레오캡시드 NC 융합 단백질은, 낮은 단백질 농도에서도 인간 CoV 항체와 교차 반응이 잘 이루어졌다. 이는 상기 항체가 MERS-CoV를 진단하기 위한 항체로서 사용할 수 있음을 의미한다.
<110> Korea Research Institute of Bioscience and Biotechnology
<120> NC FUSION PROTEIN COMPRISING N-TERMINUS DOMAIN FRAGMENTS AND
C-TERMINUS DOMAIN FRAGMENTS OF MERS-COV NUCLEOCAPSID PROTEIN AND
KIT FOR DIAGNOSING INFECTION OF MERS-COV USING THE SAME
<130> FPD/201809-0026
<150> KR 10-2017-0124394
<151> 2017-09-26
<160> 14
<170> KoPatentIn 3.0
<210> 1
<211> 1242
<212> DNA
<213> Artificial Sequence
<220>
<223> MERS-CoV nucleocapsid protein
<400> 1
atggcatccc ctgctgcacc tcgtgctgtt tcctttgccg ataacaatga tataacaaat 60
acaaacctat ctcgaggtag aggacgtaat ccaaaaccac gagctgcacc aaataacact 120
gtctcttggt acactgggct tacccaacac gggaaagtcc ctcttacctt tccacctggg 180
cagggtgtac ctcttaatgc caattctacc cctgcgcaaa atgctgggta ttggcggaga 240
caggacagaa aaattaatac cgggaatgga attaagcaac tggctcccag gtggtacttc 300
tactacactg gaactggacc cgaagcagca ctcccattcc gggctgttaa ggatggcatc 360
gtttgggtcc atgaagatgg cgccactgat gctccttcaa cttttgggac gcggaaccct 420
aacaatgatt cagctattgt tacacaattc gcgcccggta ctaagcttcc taaaaacttc 480
cacattgagg ggactggagg caatagtcaa tcatcttcaa gagcctctag cttaagcaga 540
aactcttcca gatctagttc acaaggttca agatcaggaa actctacccg cggcacttct 600
ccaggtccat ctggaatcgg agcagtagga ggtgatctac tttaccttga tcttctgaac 660
agactacaag cccttgagtc tggcaaagta aagcaatcgc agccaaaagt aatcactaag 720
aaagatgctg ctgctgctaa aaataagatg cgccacaagc gcacttccac caaaagtttc 780
aacatggtgc aagcttttgg tcttcgcgga ccaggagacc tccagggaaa ctttggtgat 840
cttcaattga ataaactcgg cactgaggac ccacgttggc cccaaattgc tgagcttgct 900
cctacagcca gtgcttttat gggtatgtcg caatttaaac ttacccatca gaacaatgat 960
gatcatggca accctgtgta cttccttcgg tacagtggag ccattaaact tgacccaaag 1020
aatcccaact acaataagtg gttggagctt cttgagcaaa atattgatgc ctacaaaacc 1080
ttccctaaga aggaaaagaa acaaaaggca ccaaaagaag aatcaacaga ccaaatgtct 1140
gaacctccaa aggagcagcg tgtgcaaggt agcatcactc agcgcactcg cacccgtcca 1200
agtgttcagc ctggtccaat gattgatgtt aacactgatt ag 1242
<210> 2
<211> 413
<212> PRT
<213> Artificial Sequence
<220>
<223> MERS-CoV nucleocapsid protein
<400> 2
Met Ala Ser Pro Ala Ala Pro Arg Ala Val Ser Phe Ala Asp Asn Asn
1 5 10 15
Asp Ile Thr Asn Thr Asn Leu Ser Arg Gly Arg Gly Arg Asn Pro Lys
20 25 30
Pro Arg Ala Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly Leu Thr
35 40 45
Gln His Gly Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly Val Pro
50 55 60
Leu Asn Ala Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp Arg Arg
65 70 75 80
Gln Asp Arg Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu Ala Pro
85 90 95
Arg Trp Tyr Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala Leu Pro
100 105 110
Phe Arg Ala Val Lys Asp Gly Ile Val Trp Val His Glu Asp Gly Ala
115 120 125
Thr Asp Ala Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn Asp Ser
130 135 140
Ala Ile Val Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys Asn Phe
145 150 155 160
His Ile Glu Gly Thr Gly Gly Asn Ser Gln Ser Ser Ser Arg Ala Ser
165 170 175
Ser Leu Ser Arg Asn Ser Ser Arg Ser Ser Ser Gln Gly Ser Arg Ser
180 185 190
Gly Asn Ser Thr Arg Gly Thr Ser Pro Gly Pro Ser Gly Ile Gly Ala
195 200 205
Val Gly Gly Asp Leu Leu Tyr Leu Asp Leu Leu Asn Arg Leu Gln Ala
210 215 220
Leu Glu Ser Gly Lys Val Lys Gln Ser Gln Pro Lys Val Ile Thr Lys
225 230 235 240
Lys Asp Ala Ala Ala Ala Lys Asn Lys Met Arg His Lys Arg Thr Ser
245 250 255
Thr Lys Ser Phe Asn Met Val Gln Ala Phe Gly Leu Arg Gly Pro Gly
260 265 270
Asp Leu Gln Gly Asn Phe Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr
275 280 285
Glu Asp Pro Arg Trp Pro Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser
290 295 300
Ala Phe Met Gly Met Ser Gln Phe Lys Leu Thr His Gln Asn Asn Asp
305 310 315 320
Asp His Gly Asn Pro Val Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys
325 330 335
Leu Asp Pro Lys Asn Pro Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu
340 345 350
Gln Asn Ile Asp Ala Tyr Lys Thr Phe Pro Lys Lys Glu Lys Lys Gln
355 360 365
Lys Ala Pro Lys Glu Glu Ser Thr Asp Gln Met Ser Glu Pro Pro Lys
370 375 380
Glu Gln Arg Val Gln Gly Ser Ile Thr Gln Arg Thr Arg Thr Arg Pro
385 390 395 400
Ser Val Gln Pro Gly Pro Met Ile Asp Val Asn Thr Asp
405 410
<210> 3
<211> 546
<212> DNA
<213> Artificial Sequence
<220>
<223> N-terminus of MERS-CoV nucleocapsid protein
<400> 3
atgggcagca gccatcatca ttatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcaccaa ataacactgt ctcttggtac actgggctta cccaacacgg gaaagtccct 120
cttacctttc cacctgggca gggtgtacct cttaatgcca attccacccc tgcgcaaaat 180
gctgggtatt ggcggagaca ggacagaaaa attaataccg ggaatggaat taagcaactg 240
gctcccaggt ggtacttcta ctacactgga actggacccg aagcagcact cccattccgg 300
gctgttaagg atggcatcgt ttgggtccat gaagatggcg ccactgatgc tccttcaact 360
tttgggacgc ggaaccctaa caatgattca gctattgtta cacaattcgc tcccggtact 420
aagcttccta aaaacttcca cattgagggg actggaggca atagtggatc cgaattcgag 480
ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc 540
tgctaa 546
<210> 4
<211> 181
<212> PRT
<213> Artificial Sequence
<220>
<223> N-terminus of MERS-CoV nucleocapsid protein
<400> 4
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly
20 25 30
Leu Thr Gln His Gly Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly
35 40 45
Val Pro Leu Asn Ala Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp
50 55 60
Arg Arg Gln Asp Arg Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu
65 70 75 80
Ala Pro Arg Trp Tyr Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala
85 90 95
Leu Pro Phe Arg Ala Val Lys Asp Gly Ile Val Trp Val His Glu Asp
100 105 110
Gly Ala Thr Asp Ala Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn
115 120 125
Asp Ser Ala Ile Val Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys
130 135 140
Asn Phe His Ile Glu Gly Thr Gly Gly Asn Ser Gly Ser Glu Phe Glu
145 150 155 160
Leu Arg Arg Gln Ala Cys Gly Arg Thr Arg Ala Pro Pro Pro Pro Pro
165 170 175
Leu Arg Ser Gly Cys
180
<210> 5
<211> 462
<212> DNA
<213> Artificial Sequence
<220>
<223> C-terminus of MERS-CoV nucleocapsid protein
<400> 5
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccag 60
atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcgc taaaaataag 120
atgcgccaca agcgcacttc caccaaaagt ttcaacatgg tgcaagcttt tggtcttcgc 180
ggaccaggag acctccaggg aaactttggt gatcttcaat tgaataaact cggcactgag 240
gacccacgtt ggccccaaat tgctgagctt gctcctacag ccagtgcttt tatgggtatg 300
tcgcaattta aacttaccca tcagaacaat gatgatcatg gcaaccctgt gtacttcctt 360
cggtacagtg gagccattaa acttgaccca aagaatccca actacaataa gtggttggag 420
cttcttgagc aaaatattga tgcctacaaa accttccctt ga 462
<210> 6
<211> 153
<212> PRT
<213> Artificial Sequence
<220>
<223> C-terminus of MERS-CoV nucleocapsid protein
<400> 6
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser Gln Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Glu Phe Ala Lys Asn Lys Met Arg His Lys Arg Thr Ser Thr
35 40 45
Lys Ser Phe Asn Met Val Gln Ala Phe Gly Leu Arg Gly Pro Gly Asp
50 55 60
Leu Gln Gly Asn Phe Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr Glu
65 70 75 80
Asp Pro Arg Trp Pro Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser Ala
85 90 95
Phe Met Gly Met Ser Gln Phe Lys Leu Thr His Gln Asn Asn Asp Asp
100 105 110
His Gly Asn Pro Val Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys Leu
115 120 125
Asp Pro Lys Asn Pro Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu Gln
130 135 140
Asn Ile Asp Ala Tyr Lys Thr Phe Pro
145 150
<210> 7
<211> 831
<212> DNA
<213> Artificial Sequence
<220>
<223> NC fusion protein of recombination MERS-CoV nucleocapsid protein
<400> 7
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggcaccaa ataacactgt ctcttggtac actgggctta cccaacacgg gaaagtccct 120
cttacctttc cacctgggca gggtgtacct cttaatgcca attccacccc tgcgcaaaat 180
gctgggtatt ggcggagaca ggacagaaaa attaataccg ggaatggaat taagcaactg 240
gctcccaggt ggtacttcta ctacactgga actggacccg aagcagcact cccattccgg 300
gctgttaagg atggcatcgt ttgggtccat gaagatggcg ccactgatgc tccttcaact 360
tttgggacgc ggaaccctaa caatgattca gctattgtta cacaattcgc tcccggtact 420
aagcttccta aaaacttcca cattgagggg actggaggca atagtggatc cgaattcgct 480
aaaaataaga tgcgccacaa gcgcacttcc accaaaagtt tcaacatggt gcaagctttt 540
ggtcttcgcg gaccaggaga cctccaggga aactttggtg atcttcaatt gaataaactc 600
ggcactgagg acccacgttg gccccaaatt gctgagcttg ctcctacagc cagtgctttt 660
atgggtatgt cgcaatttaa acttacccat cagaacaatg atgatcatgg caaccctgtg 720
tacttccttc ggtacagtgg agccattaaa cttgacccaa agaatcccaa ctacaataag 780
tggttggagc ttcttgagca aaatattgat gcctacaaaa ccttcccttg a 831
<210> 8
<211> 276
<212> PRT
<213> Artificial Sequence
<220>
<223> NC fusion protein of recombination MERS-CoV nucleocapsid protein
<400> 8
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly
20 25 30
Leu Thr Gln His Gly Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly
35 40 45
Val Pro Leu Asn Ala Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp
50 55 60
Arg Arg Gln Asp Arg Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu
65 70 75 80
Ala Pro Arg Trp Tyr Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala
85 90 95
Leu Pro Phe Arg Ala Val Lys Asp Gly Ile Val Trp Val His Glu Asp
100 105 110
Gly Ala Thr Asp Ala Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn
115 120 125
Asp Ser Ala Ile Val Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys
130 135 140
Asn Phe His Ile Glu Gly Thr Gly Gly Asn Ser Gly Ser Glu Phe Ala
145 150 155 160
Lys Asn Lys Met Arg His Lys Arg Thr Ser Thr Lys Ser Phe Asn Met
165 170 175
Val Gln Ala Phe Gly Leu Arg Gly Pro Gly Asp Leu Gln Gly Asn Phe
180 185 190
Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr Glu Asp Pro Arg Trp Pro
195 200 205
Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser Ala Phe Met Gly Met Ser
210 215 220
Gln Phe Lys Leu Thr His Gln Asn Asn Asp Asp His Gly Asn Pro Val
225 230 235 240
Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys Leu Asp Pro Lys Asn Pro
245 250 255
Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu Gln Asn Ile Asp Ala Tyr
260 265 270
Lys Thr Phe Pro
275
<210> 9
<211> 422
<212> PRT
<213> Artificial Sequence
<220>
<223> SARS-CoV nucleocapsid protein
<400> 9
Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile
1 5 10 15
Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly
20 25 30
Arg Asn Gly Ala Arg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn
35 40 45
Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Glu
50 55 60
Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Gly
65 70 75 80
Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Val Arg
85 90 95
Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr
100 105 110
Tyr Leu Gly Thr Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys
115 120 125
Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys
130 135 140
Asp His Ile Gly Thr Arg Asn Pro Asn Asn Asn Ala Ala Thr Val Leu
145 150 155 160
Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly
165 170 175
Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg
180 185 190
Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro
195 200 205
Ala Arg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu
210 215 220
Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Val Ser Gly Lys Gly Gln
225 230 235 240
Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser
245 250 255
Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn Val Thr
260 265 270
Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly
275 280 285
Asp Gln Asp Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln
290 295 300
Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg
305 310 315 320
Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr His Gly
325 330 335
Ala Ile Lys Leu Asp Asp Lys Asp Pro Gln Phe Lys Asp Asn Val Ile
340 345 350
Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu
355 360 365
Pro Lys Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro
370 375 380
Gln Arg Gln Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp
385 390 395 400
Met Asp Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser
405 410 415
Ala Asp Ser Thr Gln Ala
420
<210> 10
<211> 1269
<212> DNA
<213> Artificial Sequence
<220>
<223> SARS-CoV nucleocapsid protein
<400> 10
atgtctgata atggacccca atcaaaccaa cgtagtgccc cccgcattac atttggtgga 60
cccacagatt caactgacaa taaccagaat ggaggacgca atggggcaag gccaaaacag 120
cgccgacccc aaggtttacc caataatact gcgtcttggt tcacagctct cactcagcat 180
ggcaaggagg aacttagatt ccctcgaggc cagggcgttc caatcaacac caatagtggt 240
ccagatgacc aaattggcta ctaccgaaga gctacccgac gagttcgtgg tggtgacggc 300
aaaatgaaag agctcagccc cagatggtac ttctattacc taggaactgg cccagaagct 360
tcacttccct acggcgctaa caaagaaggc atcgtatggg ttgcaactga gggagccttg 420
aatacaccca aagaccacat tggcacccgc aatcctaata acaatgctgc caccgtgcta 480
caacttcctc aaggaacaac attgccaaaa ggcttctacg cagagggaag cagaggcggc 540
agtcaagcct cttctcgctc ctcatcacgt agtcgcggta attcaagaaa ttcaactcct 600
ggcagcagta ggggaaattc tcctgctcga atggctagcg gaggtggtga aactgccctc 660
gcgctattgc tgctagacag attgaaccag cttgagagca aagtttctgg taaaggccaa 720
caacaacaag gccaaactgt cactaagaaa tctgctgctg aggcatctaa aaagcctcgc 780
caaaaacgta ctgccacaaa acagtacaac gtcactcaag catttgggag acgtggtcca 840
gaacaaaccc aaggaaattt cggggaccaa gacctaatca gacaaggaac tgattacaaa 900
cattggccgc aaattgcaca atttgctcca agtgcctctg cattctttgg aatgtcacgc 960
attggcatgg aagtcacacc ttcgggaaca tggctgactt atcatggagc cattaaattg 1020
gatgacaaag atccacaatt caaagacaac gtcatactgc tgaacaagca cattgacgca 1080
tacaaaacat tcccaccaac agagcctaaa aaggacaaaa agaaaaagac tgatgaagct 1140
cagcctttgc cgcagagaca aaagaagcag cccactgtga ctcttcttcc tgcggctgac 1200
atggatgatt tctccagaca acttcaaaat tccatgagtg gagcttctgc tgattcaact 1260
caggcataa 1269
<210> 11
<211> 389
<212> PRT
<213> Artificial Sequence
<220>
<223> HCoV-229E nucleocapsid protein
<400> 11
Met Ala Thr Val Lys Trp Ala Asp Ala Ser Glu Pro Gln Arg Gly Arg
1 5 10 15
Gln Gly Arg Ile Pro Tyr Ser Leu Tyr Ser Pro Leu Leu Val Asp Ser
20 25 30
Glu Gln Pro Trp Lys Val Ile Pro Arg Asn Leu Val Pro Val Asn Lys
35 40 45
Lys Asp Lys Asn Lys Leu Ile Gly Tyr Trp Asn Val Gln Lys Arg Phe
50 55 60
Arg Thr Arg Lys Gly Lys Arg Val Asp Leu Ser Pro Lys Leu His Phe
65 70 75 80
Tyr Tyr Leu Gly Thr Gly Pro His Lys Asp Ala Lys Phe Arg Glu Arg
85 90 95
Val Glu Gly Val Val Trp Val Ala Val Asp Gly Ala Lys Thr Glu Pro
100 105 110
Thr Gly Tyr Gly Val Arg Arg Lys Asn Ser Glu Pro Glu Ile Pro His
115 120 125
Phe Asn Gln Lys Leu Pro Asn Gly Val Thr Val Ala Glu Glu Pro Asp
130 135 140
Ser Arg Ala Pro Ser Arg Ser Gln Ser Arg Ser Gln Ser Arg Ser Arg
145 150 155 160
Val Glu Ser Lys Ser Gln Ser Arg Asn Pro Ser Ser Asp Arg Asn His
165 170 175
Asn Ser Gln Asp Asp Ile Met Lys Ala Val Ala Ala Ala Leu Lys Ser
180 185 190
Leu Gly Phe Asp Lys Pro Gln Glu Lys Asp Lys Lys Ser Ala Lys Thr
195 200 205
Gly Thr Pro Lys Pro Ser Arg Asn Gln Ser Pro Ala Ser Ser Gln Ser
210 215 220
Ala Ala Lys Ile Leu Ala Arg Ala Gln Ser Ser Glu Thr Lys Glu Gln
225 230 235 240
Lys His Glu Met Gln Lys Pro Arg Trp Lys Arg Gln Pro Asn Asp Asp
245 250 255
Val Thr Ser Asn Val Thr Gln Cys Phe Gly Pro Arg Asp Leu Asp His
260 265 270
Asn Phe Gly Ser Ala Gly Val Val Ala Asn Gly Val Lys Ala Lys Gly
275 280 285
Tyr Pro Gln Phe Ala Glu Leu Val Pro Ser Thr Ala Ala Met Leu Phe
290 295 300
Asp Ser His Ile Val Ser Lys Glu Ser Gly Asn Thr Val Val Leu Thr
305 310 315 320
Phe Thr Thr Arg Val Thr Val Pro Lys Asp His Pro His Leu Gly Lys
325 330 335
Phe Leu Glu Glu Leu Asn Ala Phe Thr Arg Glu Met Gln Gln Gln Pro
340 345 350
Leu Leu Asn Pro Ser Ala Leu Glu Phe Asn Pro Ser Gln Thr Ser Pro
355 360 365
Ala Thr Val Glu Pro Val Arg Asp Glu Val Ser Ile Glu Thr Asp Ile
370 375 380
Ile Asp Glu Val Asn
385
<210> 12
<211> 1170
<212> DNA
<213> Artificial Sequence
<220>
<223> HCoV-229E nucleocapsid protein
<400> 12
atggctacag tcaaatgggc tgatgcatct gaaccacaac gtggtcgtca gggtagaata 60
ccttactctc tttatagccc tttgcttgtt gatagtgaac aaccttggaa ggtgatacct 120
cgtaatttgg tacccgtcaa caagaaagac aaaaataagc ttataggcta ttggaatgtt 180
caaaaacgtt tcagaactag aaagggcaaa cgggtggatt tgtcacccaa gttacatttc 240
tattatcttg gcacaggacc tcataaagat gcaaaattta gagagcgtgt tgaaggtgtt 300
gtctgggttg ctgttgatgg tgctaaaact gaacctacag gttacggtgt taggcgcaag 360
aattcagaac cagagatacc acacttcaat caaaagctcc caaatggtgt tactgttgct 420
gaagaacctg actcccgtgc tccttcccgt tctcagtcaa ggtctcagag tcgcagtcgt 480
gttgaatcca aatctcaatc tcgtaatcct tcaagtgaca gaaaccataa cagtcaggat 540
gacatcatga aggcagtcgc tgcggctctt aaatctttag gttttgacaa gcctcaggaa 600
aaagacaaaa agtcagcgaa aacgggtacc cctaagcctt ctcgtaatca gagtcctgct 660
tcttctcaat ctgctgccaa gattcttgct cgtgctcaga gttctgaaac aaaagaacaa 720
aagcatgaaa tgcaaaagcc acggtggaaa agacagccta acgatgatgt gacatctaat 780
gtcacacaat gttttggccc cagagacctt gaccacaact ttggaagtgc aggtgttgtg 840
gccaatggtg ttaaagctaa aggctatcca caatttgctg agcttgtgcc gtctacagct 900
gctatgcttt ttgatagtca cattgtttcc aaagagtcag gcaacactgt ggtcttgact 960
ttcaccacta gagtgactgt gcccaaagac catccacact tgggtaagtt tcttgaggaa 1020
ttaaatgcat tcactagaga aatgcaacaa cagcctcttc ttaaccctag tgcactagaa 1080
ttcaacccat cccaaacttc acctgcaact gttgaaccag tgcgtgatga agtttctatt 1140
gaaactgaca taattgatga agtcaactaa 1170
<210> 13
<211> 448
<212> PRT
<213> Artificial Sequence
<220>
<223> HCoV-OC43 nucleocapsid protein
<400> 13
Met Ser Phe Thr Pro Gly Lys Gln Ser Ser Ser Arg Ala Ser Ser Gly
1 5 10 15
Asn Arg Ser Gly Asn Gly Ile Leu Lys Trp Ala Asp Gln Ser Asp Gln
20 25 30
Phe Arg Asn Phe Gln Thr Arg Gly Arg Arg Ala Gln Pro Lys Gln Thr
35 40 45
Ala Thr Ser Gln Gln Pro Ser Gly Gly Asn Val Val Pro His Tyr Ser
50 55 60
Trp Phe Ser Gly Ile Thr Gln Phe Gln Lys Gly Lys Glu Phe Glu Phe
65 70 75 80
Ala Glu Gly Gln Gly Val Pro Ile Ala Pro Gly Val Pro Ala Thr Glu
85 90 95
Ala Lys Gly Tyr Trp Tyr Arg His Asn Arg Arg Ser Phe Lys Thr Ala
100 105 110
Asp Gly Asn Gln Arg Gln Leu Leu Pro Arg Trp Tyr Phe Tyr Tyr Leu
115 120 125
Gly Thr Gly Pro His Ala Lys Asp Gln Tyr Gly Thr Asp Ile Asn Gly
130 135 140
Val Tyr Trp Val Ala Ser Asn Gln Ala Asp Val Asn Thr Pro Ala Asp
145 150 155 160
Ile Val Asp Arg Asp Pro Ser Ser Asp Glu Ala Ile Pro Thr Arg Phe
165 170 175
Pro Pro Gly Thr Val Leu Pro Gln Gly Tyr Tyr Ile Glu Gly Ser Gly
180 185 190
Arg Ser Ala Pro Asn Ser Arg Ser Thr Ser Arg Thr Ser Ser Arg Ala
195 200 205
Ser Ser Ala Gly Ser Arg Ser Arg Ala Asn Ser Gly Asn Arg Thr Pro
210 215 220
Thr Ser Gly Val Thr Pro Asp Met Ala Asp Gln Ile Ala Ser Leu Val
225 230 235 240
Leu Ala Lys Leu Gly Lys Asp Ala Thr Lys Pro Gln Gln Val Thr Lys
245 250 255
His Thr Ala Lys Glu Val Arg Gln Lys Ile Leu Asn Lys Pro Arg Gln
260 265 270
Lys Arg Ser Pro Asn Lys Gln Cys Thr Val Gln Gln Cys Phe Gly Lys
275 280 285
Arg Gly Pro Asn Gln Asn Phe Gly Gly Gly Glu Met Leu Lys Leu Gly
290 295 300
Thr Ser Asp Pro Gln Phe Pro Ile Leu Ala Glu Leu Ala Pro Thr Ala
305 310 315 320
Gly Ala Phe Phe Phe Gly Ser Lys Leu Glu Leu Ala Lys Val Gln Asn
325 330 335
Leu Ser Gly Asn Pro Asp Glu Pro Gln Lys Asp Val Tyr Glu Leu Arg
340 345 350
Tyr Asn Gly Ala Ile Arg Phe Asp Ser Thr Leu Ser Gly Phe Glu Thr
355 360 365
Ile Met Lys Val Leu Ser Glu Asn Leu Asn Ala Tyr Gln Gln Gln Asp
370 375 380
Gly Met Met Asn Met Ser Pro Lys Pro Gln Arg Gln Arg Gly His Lys
385 390 395 400
Asn Gly Gln Gly Glu Asn Asp Asn Ile Ser Val Ala Val Pro Lys Ser
405 410 415
Arg Val Gln Gln Asn Lys Ser Ile Glu Leu Thr Ala Glu Asp Ile Ser
420 425 430
Leu Leu Lys Lys Met Asp Glu Pro Phe Thr Glu Asp Thr Ser Glu Ile
435 440 445
<210> 14
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> HCoV-OC43 nucleocapsid protein
<400> 14
atgtctttta ctcctggtaa gcaatccagt agtagagcgt cctctggaaa tcggtctggt 60
aatggcatcc tcaagtgggc cgatcagtcc gaccagttta gaaattttca aaccaggggt 120
agaagagctc aacccaagca aactgctact tctcagcaac catcaggagg gaatgttgta 180
ccccactatt cttggttctc tggaattact cagtttcaaa agggaaagga gtttgagttt 240
gcagaaggac aaggtgtgcc tattgcacca ggagtcccag ctactgaagc taaggggtac 300
tggtacagac acaacagacg ttcttttaaa acagccgatg gcaaccagcg tcaactgctg 360
ccacgatggt atttttacta tctgggaacc ggaccgcatg ctaaagacca gtatggcacc 420
gatattaacg gagtctactg ggtcgctagc aaccaggctg atgtcaatac cccggctgac 480
attgtcgatc gggacccaag tagcgatgag gctattccga ctaggtttcc gcctggcacg 540
gtactccctc agggttacta tattgaaggc tcaggaaggt ctgctcctaa ttccagatct 600
acttcgcgca catccagcag agcctctagt gcaggatcgc gtagtagagc caattctggc 660
aatagaaccc ctacctctgg tgtaacacct gacatggctg atcaaattgc tagtcttgtt 720
ctggcaaaac ttggcaagga tgccaccaaa cctcagcaag taactaagca tactgccaaa 780
gaagtcagac agaaaatttt gaataaaccc cgccagaaga ggagccccaa taaacaatgc 840
actgttcagc agtgttttgg taagagaggc cctaatcaga attttggtgg tggagaaatg 900
ttaaaacttg gaactagtga cccacagttc cccattcttg cagaactcgc acccacagct 960
ggtgcgtttt tctttggatc aaaattagag ttggccaaag tgcagaattt atctgggaat 1020
cctgatgagc ctcagaagga tgtttatgaa ttgcgctata acggcgcaat taggtttgac 1080
agtacacttt caggctttga gaccataatg aaggtgctga gtgagaattt gaatgcctat 1140
caacaacaag atggtatgat gaatatgagt ccaaaaccac agcgtcagcg tggtcataag 1200
aatggacaag gagaaaatga taatataagt gttgcagtgc ccaaaagccg cgtgcagcaa 1260
aataagagta tagagttgac tgcagaggac atcagccttc ttaagaagat ggatgagccc 1320
tttactgaag acacctcaga aatataa 1347
Claims (14)
- 메르스 코로나바이러스의 뉴클레오캡시드 단백질의 N-말단 도메인 단편 및 C-말단 도메인 단편을 포함하는 NC 융합 단백질로서, 상기 NC 융합 단백질은 서열번호 8의 아미노산 서열로 이루어지는 것을 특징으로 하는 NC 융합 단백질.
- 삭제
- 삭제
- 삭제
- 삭제
- 삭제
- 제1항에 있어서,
상기 NC 융합 단백질은 서열번호 7의 염기서열에 의해 코딩되는 것을 특징으로 하는, NC 융합 단백질. - 제1항에 따른 NC 융합 단백질에 특이적으로 결합하는 단클론 항체.
- 제1항의 NC 융합 단백질을 코딩하는 뉴클레오티드를 포함하는 발현 벡터.
- 제9항의 발현 벡터로 형질감염된 숙주세포.
- 제10항의 형질감염된 숙주세포로부터 NC 융합 단백질을 수득하는 단계를 포함하는 NC 융합 단백질 제조방법.
- (a) 제1항 또는 제7항에 따른 융합 단백질을 항원으로서 실험동물에 주입하여 면역반응을 유도하는 단계; 및
(b) 상기 (a)단계의 실험동물로부터 항체를 회수하는 단계를 포함하는 메르스 코로나바이러스 항체 제조 방법. - 제1항에 따른 NC 융합 단백질을 포함하는 메르스 코로나바이러스 감염 진단용 키트.
- 제13항에 따른 진단용 키트에 진단 대상으로부터 분리한 시료를 첨가하는 단계를 포함하는 메르스 코로나바이러스 감염 정보제공 방법.
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KR20230008867A (ko) * | 2020-05-12 | 2023-01-16 | 그레펙스 인코포레이티드 | 백신 항원으로서의 코로나바이러스 스파이크 단백질의 조작, 이의 설계 및 용도 |
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