KR20220106589A - 톡소포자충 단백질 및 인플루엔자 바이러스 단백질을 포함하는 바이러스 유사 입자 및 이를 이용한 진단 방법 - Google Patents
톡소포자충 단백질 및 인플루엔자 바이러스 단백질을 포함하는 바이러스 유사 입자 및 이를 이용한 진단 방법 Download PDFInfo
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Abstract
본 발명은 T. gondii 표면 항원 단백질이 포함된 VLP 및 인플루엔자 바이러스 M1 단백질을 포함하는 바이러스-유사 입자(VLPs)를 포함하는 톡소포자충 및 인플루엔자 바이러스 다중 진단용 조성물을 제공한다. 일 구체예로 상기 VLPs는 TgAMA1 VLP, TgROP4 VLP 및 TgROP18로 구성된 군으로부터 적어도 하나의 단백질을 포함한다. 상기 다중 진단용 조성물은 톡소포자충에 감염된 대상체 및 인플루엔자 바이러스에 감염된 대상체로부터 분리된 생물학적 시료와 높은 민감도와 특이성으로 반응할 수 있다. 따라서, 상기 다중 진단용 조성물은 톡소포자충증 및 인플루엔자 바이러스 다중 진단 키트에 활용될 수 있다.
Description
본 발명은 톡소포자충 단백질 및 인플루엔자 바이러스 단백질을 포함하는 바이러스-유사 입자를 포함하는 톡소포자충 및 인플루엔자 바이러스를 진단하기 위한 키트 및 이를 이용한 톡소포자충 또는 인플루엔자 바이러스 감염 여부를 진단하기 위한 방법에 관한 것이다.
톡소포자충(Toxoplasma gondii)은 세포 내 기생충으로서 인간 및 동물에 대한 톡소플라즈마증(Toxoplasmosis)을 야기하는 원인 원충이다(Astrid M. Tenter et al.). 톡소포자충은 크게 난포낭(oocyst, 오시스트), 영양형(tachyzoit, 타키조이트), 감염형(bradyzoit, 브래디조이트), 분열체(schizont, 시존트) 및 생식모체(gametocyte, 거미토사이트) 단계의 5가지 발육 단계를 거친다. 톡소포자충은 종숙주인 고양이 분변의 난포낭(oocyst)에 의해 오염된 물 또는 야채를 섭취함으로써 감염되거나, 중간숙주인 돼지, 양, 소 등의 육류에 씨스트(cyst)로 존재하는 톡소포자충을 섭식할 때 감염될 수 있다. 전 세계 인구의 약 3분의 1 이상이 톡소포자충에 감염된 것으로 추정되며, 국내에서도 그룹에 따라 2 내지 25%의 감염율을 나타내는 것으로 보고되고 있다.
특히, 톡소포자충이 산모를 감염시켰을 때 그 영양형(trophozoite)은 태반을 거쳐 태아를 감염시킬 수 있다. 톡소포자충의 감염에 의해 초기의 태아는 유산 또는 사산에 이를 수 있으며, 중후기의 태아는 정상적 분만에도 불구하고 시력 손상, 수두증, 정신박약 등의 선천적 기형이 야기될 수 있다. 또한, 건강한 개체를 감염시킨 톡소포자충은 면역계 세포, 망상내피계 세포 등을 파괴시켜 림프선염, 망막맥락막염, 뇌척수염 등의 질병을 유발할 수 있으며, 숙주의 면역부전시 뇌에서 증식된 씨스트가 활성화 되어 뇌수막염 또는 망막맥락막염을 일으킬 수 있다.
한편, 인플루엔자는 호흡기 바이러스로 인한 인간의 사망에 있어서 주요한 원인중 하나이다. 일반적인 증상은 특히 발열, 인후통, 숨가쁨 및 근육통을 포함한다. 독감 시즌의 인플루엔자 바이러스는 강한 전염성으로 인해 세계 인구의 10% ~ 20%를 감염시키고 사망자만 연간 250,000명 ~ 500,000명에 이른다. 이들 질병의 공통점은 다른 질병의 증상이 유사한 증상을 가지고 있다는 점이다. 따라서, 다른 질병과의 혼동을 줄이기 위해 라텍스 응집 테스트(Latex Agglutination Test), 항혈청진단법(enzyme-linked immunosorbent assay, enzyme-linked immunospecific assay, ELISA)과 같은 면역학적 방법, Nested PCR, real-time PCR과 같은 DNA 검출 방법 등 다양한 진단 방법이 사용되고 있다.
그러나, 종래의 진단 방법은 진단까지 신속성이 떨어지거나, 고가의 장비 사용으로 인한 낮은 경제성 또는 낮은 민감성의 문제가 있기 때문에, 신속성, 경제성 및 민감성이 높은 새로운 진단 방법이 필요한 상황이다.
Astrid M. Tenter et al., "Toxoplasma gondii: from animals to humans", Int J Parasitol. 2000 Nov; 30(12-13), 1217-1258
이에 본 발명자들은 톡소포자충 및 인플루엔자 바이러스를 동시에 진단할 수 있으면서도 신속성, 경제성 및 민감성이 높은 방법을 개발하기 위해 연구한 결과, 본 발명에 따른 VLP 형태 진단용 조성물을 포함한 진단 방법 및 진단 키트가 항원 용해물(lysate antigen) 및 항원 단백질에 비해 면역학적 특이성 및 민감성이 더 우수함을 확인함으로써 본 발명을 완성하였다.
상기 목적을 달성하기 위하여, 톡소포자충 유래의 표면 항원 단백질; 및 인플루엔자 바이러스 매트릭스 단백질 1을 포함하는 바이러스-유사 입자를 포함하는 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물을 제공한다.
본 발명의 다른 측면으로, 상기 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물을 포함하는 톡소포자충 및 인플루엔자 바이러스 다중 진단 키트를 제공한다.
본 발명의 다른 측면으로, 톡소포자충 유래의 표면 항원 단백질; 및 인플루엔자 바이러스 매트릭스 단백질 1을 포함하는 바이러스-유사 입자를 포함하는 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물에 개체의 혈청을 접촉시키는 단계; 및 바이러스-유사 입자에 결합하는 항체를 검출하는 단계;를 포함하는 톡소포자충 및/또는 인플루엔자 바이러스 감염 여부에 대한 정보를 제공하는 방법을 제공한다.
본 발명에 따른 톡소포자충 항원 단백질 및 인플루엔자 바이러스 M1 단백질이 포함된 바이러스-유사 입자를 포함하는 진단용 조성물은 높은 특이성 및 민감성으로 톡소포자충에 감염된 개체의 혈청에 존재하는 자가 항체와 반응할 수 있다. 뿐만 아니라, 인플루엔자 바이러스에 감염된 개체에 존재하는 자가 항체와 반응할 수 있다. 따라서, 상기 바이러스-유사 입자는 톡소포자충 진단 및/또는 인플루엔자 바이러스 진단에 유용하게 사용될 수 있다.
도 1a 내지 도 1c는 AMA1, ROP4 또는 ROP18 유전자를 포함한 rBV(재조합 배큘로 바이러스) 및 인플루엔자 M1 유전자를 포함한 rBV를 SF9 곤충 세포에 형질 감염시켜 AMA1, ROP4 또는 ROP18 VLPs(Virus-like particles)를 수득하는 과정을 도식화한 것이다.
도 2는 본 명세서의 일 실시예에서 마우스로부터 감염 혈청을 수득하는 In vivo 시험 과정을 시계열적으로 나타낸 것이다.
도 3은 본 명세서의 일 실시예에서 제작한 VLP를 이용하여 ELISA를 이용하여 진단하는 방법에 관한 모식도를 나타낸 것이다.
도 4는 Naive, P. berghei ANKA(말라리아 원충) 또는 T. gondii(톡소포자충) ME49로 감염된 마우스의 혈청을 각각 TLA(RH Tachyzoite lysate antigen), AMA1-M1-VLPs 및 AMA1 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 AMA1-M1-VLPs의 특이성을 확인한 것이다.
도 5는 T. gondii ME49로 감염된 마우스의 혈청을 각각 TLA, AMA1-M1-VLPs 및 AMA1 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 AMA1-M1-VLPs의 민감성을 확인한 것이다.
도 6은 T. gondii ME49로 감염된 인간 혈청을 각각 TLA, AMA1-M1-VLPs 및 AMA1 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 AMA1-M1-VLPs의 민감성을 확인한 것이다.
도 7은 Naive, P. berghei ANKA 또는 T. gondii ME49로 감염된 마우스 혈청을 각각 TLA, ROP4-M1-VLPs 및 ROP4 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP4-M1-VLPs의 특이성을 확인한 것이다.
도 8은 T. gondii ME49로 감염된 마우스 혈청을 각각 TLA, ROP4-M1-VLPs 및 ROP4 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP4-M1-VLPs의 민감성을 확인한 것이다.
도 9는 T. gondii ME49로 감염된 인간 혈청을 각각 TLA, ROP4-M1-VLPs 및 ROP4 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP4-M1-VLPs의 민감성을 확인한 것이다.
도 10은 Naive, P. berghei ANKA 또는 T. gondii ME49로 감염된 마우스 혈청을 각각 TLA, ROP18-M1-VLPs 및 ROP18 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP18-M1-VLPs의 특이성을 확인한 것이다.
도 11은 T. gondii ME49로 감염된 마우스 혈청을 각각 TLA, ROP18-M1-VLPs 및 ROP18 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP18-M1-VLPs의 민감성을 확인한 것이다.
도 12는 T. gondii ME49로 감염된 인간 혈청을 각각 TLA, ROP18-M1-VLPs 및 ROP18 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP18-M1-VLPs의 민감성을 확인한 것이다.
도 13은 TLA, AMA1-M1-VLPs, ROP4-M1-VLPs 및 ROP18-M1-VLPs가 각각 코팅된 면역플레이트에서 Naive 마우스 혈청, T. gondii ME49로 감염된 마우스 혈청, 인플루엔자 M1 VLPs 10 ㎍을 접종한 마우스의 혈청 및 NA(neuraminidase) VLPs 10 ㎍을 접종한 마우스의 혈청을 각각 접촉시킨 결과를 나타낸 것이다. 그 결과, AMA1-M1-VLPs, ROP4-M1-VLPs 및 ROP18-M1-VLPs로 톡소포자충증 및 인플루엔자 바이러스의 다중 진단이 가능함을 확인하였다.
도 2는 본 명세서의 일 실시예에서 마우스로부터 감염 혈청을 수득하는 In vivo 시험 과정을 시계열적으로 나타낸 것이다.
도 3은 본 명세서의 일 실시예에서 제작한 VLP를 이용하여 ELISA를 이용하여 진단하는 방법에 관한 모식도를 나타낸 것이다.
도 4는 Naive, P. berghei ANKA(말라리아 원충) 또는 T. gondii(톡소포자충) ME49로 감염된 마우스의 혈청을 각각 TLA(RH Tachyzoite lysate antigen), AMA1-M1-VLPs 및 AMA1 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 AMA1-M1-VLPs의 특이성을 확인한 것이다.
도 5는 T. gondii ME49로 감염된 마우스의 혈청을 각각 TLA, AMA1-M1-VLPs 및 AMA1 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 AMA1-M1-VLPs의 민감성을 확인한 것이다.
도 6은 T. gondii ME49로 감염된 인간 혈청을 각각 TLA, AMA1-M1-VLPs 및 AMA1 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 AMA1-M1-VLPs의 민감성을 확인한 것이다.
도 7은 Naive, P. berghei ANKA 또는 T. gondii ME49로 감염된 마우스 혈청을 각각 TLA, ROP4-M1-VLPs 및 ROP4 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP4-M1-VLPs의 특이성을 확인한 것이다.
도 8은 T. gondii ME49로 감염된 마우스 혈청을 각각 TLA, ROP4-M1-VLPs 및 ROP4 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP4-M1-VLPs의 민감성을 확인한 것이다.
도 9는 T. gondii ME49로 감염된 인간 혈청을 각각 TLA, ROP4-M1-VLPs 및 ROP4 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP4-M1-VLPs의 민감성을 확인한 것이다.
도 10은 Naive, P. berghei ANKA 또는 T. gondii ME49로 감염된 마우스 혈청을 각각 TLA, ROP18-M1-VLPs 및 ROP18 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP18-M1-VLPs의 특이성을 확인한 것이다.
도 11은 T. gondii ME49로 감염된 마우스 혈청을 각각 TLA, ROP18-M1-VLPs 및 ROP18 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP18-M1-VLPs의 민감성을 확인한 것이다.
도 12는 T. gondii ME49로 감염된 인간 혈청을 각각 TLA, ROP18-M1-VLPs 및 ROP18 단백질이 코팅된 면역플레이트에 접촉시킴으로써 감염 혈청에 대한 ROP18-M1-VLPs의 민감성을 확인한 것이다.
도 13은 TLA, AMA1-M1-VLPs, ROP4-M1-VLPs 및 ROP18-M1-VLPs가 각각 코팅된 면역플레이트에서 Naive 마우스 혈청, T. gondii ME49로 감염된 마우스 혈청, 인플루엔자 M1 VLPs 10 ㎍을 접종한 마우스의 혈청 및 NA(neuraminidase) VLPs 10 ㎍을 접종한 마우스의 혈청을 각각 접촉시킨 결과를 나타낸 것이다. 그 결과, AMA1-M1-VLPs, ROP4-M1-VLPs 및 ROP18-M1-VLPs로 톡소포자충증 및 인플루엔자 바이러스의 다중 진단이 가능함을 확인하였다.
본 발명의 일 측면은, 톡소포자충 유래의 표면 항원 단백질; 및 인플루엔자 바이러스 매트릭스 단백질 1을 포함하는 바이러스-유사 입자(Virus-like particle, VLP)를 포함하는 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물을 제공한다.
본 명세서에서 사용된 용어, "바이러스-유사 입자"(VLP) 또는 "바이러스-유사 입자들"(VLPs)은 바이러스성 단백질을 수반하거나 수반하지 않는 비감염성 바이러스성 아단위체를 의미한다. 예컨대, 상기 바이러스-유사 입자는 DNA 또는 RNA 게놈이 완전히 결여된 것일 수 있다. 일 구체예에서, 바이러스-유사 입자는 유전 공학적인 방법에 의해 제조될 수 있으며, 구조 단백질(core protein)로서 인플루엔자 바이러스 유래의 매트릭스 단백질(M1)을 포함한다. 본 발명의 바이러스-유사 입자는 별도의 원인 감염체, 즉 톡소포자충 없이도 유전 공학적인 방법에 의해 제조될 수 있다.
본 명세서에서 사용된 용어, "인플루엔자 바이러스 매트릭스 단백질 1"(M1)은 인플루엔자 바이러스의 구조 단백질로서, 인플루엔자 바이러스의 외피(envelop)인 지방층 안쪽에 코트(coat)를 형성하는 기질 단백질(matrix protein)을 의미한다. 상기 인플루엔자 바이러스는 A, B 및 C로 명명된 서브타입으로 구성된다. 상기 인플루엔자 바이러스는 코어와 바이러스 외피 간의 연결체로 작용하는 매트릭스 단백질(M1)의 층으로 둘러싸여 외형을 이루며, 상기 M1 단백질은 인플루엔자 바이러스-유사 입자의 개발에 있어서 구조 단백질로서 널리 사용될 수 있다. 본 명세서의 바이러스-유사 입자는 인플루엔자 바이러스 매트릭스 단백질 1(M1)을 구조 단백질로서 포함하며, 일 실시예로 상기 M1은 서열번호 1의 아미노산 서열을 가질 수 있다. 또한, 상기 M1은 서열번호 5의 핵산 서열에 의해 코딩될 수 있다.
상기 톡소포자충 유래의 표면 항원 단백질은 톡소포자충에 대한 면역 활성을 유도할 수 있으면 특별히 제한되지 않는다.
구체적으로, 본 명세서의 표면 항원 단백질은 톡소포자충에서 유래한 SAG1(Membrane-associated surface antigen), SAG2, GRA1(secreted dense-granule protein), GRA2, GRA4, GRA7, MIC1(Microneme protein), MIC2, MIC4, MIC6, MIC7, MIC8, MIC9, MIC10, MIC11, ROP1, ROP2, ROP3, ROP4, ROP5, ROP6, ROP7, ROP8, ROP9, ROP10, ROP11, ROP12, ROP13, ROP14, ROP15, ROP16, ROP17, ROP18, M2AP(MIC2 associated protein), AMA1(Plasmodium apical membrane antigen 1) 및 BAG1 등 다양한 종류의 단백질을 포함할 수 있고, 바람직하게는 AMA1, ROP4 및 ROP18 중 적어도 하나를 포함할 수 있다.
이때, 상기 AMA1은 서열번호 2의 아미노산 서열을 가질 수 있다. 또한, 상기 ROP4는 서열번호 3의 아미노산 서열을 가질 수 있다. 또한, 상기 ROP18은 서열번호 4의 아미노산 서열을 가질 수 있다.
또한, 상기 AMA1은 서열번호 6의 핵산 서열에 의해 코딩될 수 있고, ROP4는 서열번호 7의 핵산 서열에 의해 코딩될 수 있고, ROP18은 서열번호 8의 핵산 서열에 의해 코딩될 수 있다.
여기서 바이러스-유사 입자의 표면에 도입된 항원 단백질은 순수하게 분리된 재조합 단백질에 비해 높은 항원성을 가지므로 효과적인 중화항체를 형성할 수 있고, 이에 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물로 활용할 수 있다.
상기 바이러스-유사 입자에서 톡소포자충 유래의 표면 항원 단백질 및 인플루엔자 바이러스 매트릭스 단백질 1의 비율은 5:1 내지 1:5의 비율로 존재할 수 있다. 또한, 3:1, 1:1 또는 1:3의 비율로 존재할 수 있다. 바람직하게는, 상기 비율은 3:1일 수 있다.
상기 바이러스-유사 입자는 톡소포자충 유래의 표면 항원 단백질을 코딩하는 유전자를 포함하는 벡터 및 인플루엔자 바이러스 매트릭스 단백질 1을 코딩하는 유전자를 포함하는 벡터를 이용하여 제조된 것일 수 있다. 구체적으로, 상기 바이러스-유사 입자는 톡소포자충 유래의 표면 항원 단백질을 코딩하는 유전자를 포함하는 재조합 배큘로바이러스 및 인플루엔자 바이러스 매트릭스 단백질 1을 코딩하는 유전자를 포함하는 재조합 배큘로바이러스를 공동 감염시켜 제조된 것일 수 있다.
이때, 숙주세포는 곤충세포를 이용할 수 있다. 상기 곤충 세포는 유전자 발현을 위한 숙주 시스템으로서 개발되거나 시판되는 모든 세포가 사용될 수 있으며, 예컨대 스포도프테라 프루지페르다 곤충 세포(Spodoptera frugiperda) SF21, SF9, 트리코플루시아 니(Trichoplusia ni), 안티카르사 겜미탈리스(Anticarsa gemmitalis), 봄빅스 모리(Bombyx mori), 에스티그멘 아크레아(Estigmene acrea), 헬리오티스 비레쎈스(Heliothis virescens), 류카니아 세파라타(Leucania separata), 리만트리아 디스파(Lymantria dispar), 말라카소마 디스스트리아(Malacasoma disstria), 맘메스트라 브라씨카에(Mammestra brassicae), 만두카 섹스타(Manduca sexta), 플루텔라 질로스텔라(Plutella zylostella), 스토도프테라 엑시구아(Spodoptera exigua) 및 스포도프테라 리톨리스(Spodoptera littorlis)로 이루어진 군에서 하나 이상 선택될 수 있으나, 이에 한정되는 것은 아니다. 일 실시예에서 SF9 세포를 이용하였다.
또한, 공동 감염시에 톡소포자충 유래의 표면 항원 단백질을 코딩하는 유전자를 포함하는 재조합 배큘로바이러스와 인플루엔자 바이러스 매트릭스 단백질 1을 코딩하는 유전자를 포함하는 재조합 배큘로바이러는 3:1로 감염시켜 바이러스-유사 입자를 제조할 수 있다.
특히, 상기 톡소포자충 유래의 표면 항원 단백질; 및 인플루엔자 바이러스 매트릭스 단백질 1을 포함하는 바이러스-유사 입자는 특이성 및 민감성이 매우 우수하다. 따라서, 톡소포자충 및/또는 인플루엔자 바이러스에 감염된 개체내에 생성된 자가 항체를 검출하는 데 사용될 수 있다.
예를 들어, 톡소포자충의 감염시에 개체내에 생성되는 항-AMA1 항체, 항-ROP4 항체 또는 항-ROP18 항체의 존재 여부를 판단하는데, 상기 VLPs가 활용될 수 있다. 뿐만 아니라, 인플루엔자 바이러스에 감염시에 개체내에 생성되는 항-M1 항체의 존재 여부를 판단하는데 활용될 수 있다.
더욱이, 상기 VLPs는 톡소포자충 및 인플루엔자 바이러스가 동시에 감염된 개체에서는 더욱 효율적으로 톡소포자충 및 인플루엔자 바이러스의 감염 여부를 진단할 수 있다.
본 발명의 또 다른 측면은, 톡소포자충 유래의 표면 항원 단백질; 및 인플루엔자 바이러스 매트릭스 단백질 1을 포함하는 바이러스-유사 입자를 포함하는 톡소포자충증 및 인플루엔자 바이러스 다중 진단 키트를 제공한다.
이때, 톡소포자충 유래의 표면 항원 단백질, 인플루엔자 바이러스 매트릭스 단백질 및 바이러스-유사 입자는 상술한 바와 같다. 일 구체예로 톡소포자충 AMA1 단백질을 발현하는 VLPs인 AMA1-M1-VLPs, 톡소포자충 ROP4 단백질을 발현하는 VLPs인 ROP4-M1-VLPs, 톡소포자충 ROP18 단백질을 발현하는 VLPs인 ROP18-M1-VLPs를 이용하여 키트를 제작할 수 있다. 이때, 상기 VLPs는 진단 키트의 검출부에 존재할 수 있다.
본 명세서에서 사용된 용어 "검출부"는 톡소포자충 표면 항원 단백질이 포함된 VLP 조성물이 고정화된 지지체를 포함하는 부분으로서, 톡소포자충에 대한 항체 및 인플루엔자 바이러스에 대한 항체 중 적어도 1개와 상기 VLP 조성물이 특이적으로 반응하는 장소를 의미한다.
상기 검출부의 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물은 디스펜싱 기구를 사용하여 검출부의 지지체(예컨대, 니트로 셀룰로오스 막)에 코팅하며, 해당 부분에 대상체로부터 분리된 생물학적 시료를 접촉시킴으로서 톡소포자충 및 인플루엔자 바이러스 다중 진단이 수행될 수 있다.
일 구체예에서, 상기 진단 키트의 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물은 대상체로부터 분리된 생물학적 시료의 IgG 항체, IgA 또는 IgM 항체의 존재 여부를 탐지하여 톡소포자충 및 인플루엔자 바이러스 감염여부를 개별적으로 또는 동시에 진단할 수 있으며, 그 과정에서 ELISA(Enzyme-linked immunosorbent assay), RIA(Radioimmnoassay), 샌드위치 측정법(Sandwich assay), 웨스턴 블롯(Western Blot), 면역블롯 분석(Immunoblot assay) 및 면역조직화학염색 방법(Immnohistochemical staining)으로 구성된 군으로부터 적어도 하나를 포함하여 수행될 수 있으나, 이에 한정되는 것은 아니다.
본 발명의 또 다른 측면은, 톡소포자충 유래의 표면 항원 단백질; 및 인플루엔자 바이러스 매트릭스 단백질 1을 포함하는 바이러스-유사 입자를 포함하는 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물에 대상체로부터 분리된 생물학적 시료를 접촉시키는 단계; 및 바이러스-유사 입자에 결합하는 항체를 검출하는 단계;를 포함하는 톡소포자충 및/또는 인플루엔자 바이러스 감염 여부에 대한 정보를 제공하는 방법을 제공한다.
이때, 톡소포자충 유래의 표면 항원 단백질, 인플루엔자 바이러스 매트릭스 단백질 및 바이러스-유사 입자는 상술한 바와 같다. 또한, 본 명세서에서 사용된 용어 "대상체로부터 분리된 생물학적 시료"는 바람직하게는 체외로 분비되는 체액으로서, 혈액, 혈청, 혈장, 소변, 눈물, 침, 젖 등을 포함하며, 보다 바람직하게는 개체부터 수득한 혈액일 수 있으나 이에 한정되지는 않는다. 이때, 개체는 개, 고양이, 또는 인간과 같은 포유동물일 수 있으며, 바람직하게 인간일 수 있다.
상기 항원-항체 반응물 또는 표지인자를 검출하는 방법은 그 반응물 또는 표지인자의 종류에 따라 당업자에게 공지된 방법을 이용할 수 있으며, 구체적으로는 육안, 검출장비, 검출시약 등을 이용할 수 있다. 상기 검출장비로는 콘포칼 레이저 스캐너(confocal laser scanner)을 이용할 수 있으며, 상기 검출시약은 검사하고자 하는 표지인자(analyte)의 존재를 육안 또는 기타 기구를 통해 외부에서 식별이 가능하도록 해 주는 소정의 표지 시약(labeled reagent), 보조적 특이결합부재 및/또는 신호발생 시스템의 구성 성분을 포함할 수 있다. 표지된 검출시약은 이미 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 잘 알려져 있다.
이러한 표지의 예는 촉매, 효소(예를 들어, 포스파타제, 퍼옥시다제 등으로서, 보다 구체적인 예로는 효소 기질과 결합하여 함께 사용되는 염기성 포스퍼타제와 양고추 퍼옥시다제 등), 효소기질(예를 들어, 니트로블루테트라졸리움, 3,5',5,5'-테트라니트로벤지딘, 4-메톡시-1-나프톨, 4-클로로-1-나프톨, 5-브로모-4-클로로-3-인돌일포스페이트, 화학발광 효소기질로는 디옥세탄, 및 유도체와 이들의 유사체), 형광화합물(예를 들어, 플로우레세인(fluorescein), 피코빌리프로틴(phycobiliprotein), 로다민(rhodamine) 등과 이들 유도체 및 유사체), 화학발광화합물, 금속졸(Metal sol), 염료졸(Dye sol), 미립자 라텍스(Particulate latex), 칼라인디케이터(Color Indicator), 리포좀에 포함된 색재료(Color matter), 탄소졸 및 셀레늄과 같은 비금속졸 등이 있으나, 이에 한정되지 않는다.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Ⅰ. 톡소포자충 항원 및 인플루엔자 항원을 포함한 VLP의 제조
제조예 1. 톡소포자충 항원을 발현하는 배큘로바이러스 제조
제조예 1.1. 톡소포자충 cDNA 수득
톡소포자충 RH로부터 AMA1, ROP4 및 ROP18을 코딩하는 RNA를 추출하기 위해, 톡소포자충 RH 샘플에서 RNA를 수득하였다. 이 후, 추출된 항원 RNA를 RT-PCR을 수행하여 톡소포자충 유래의 cDNA를 수득하였다. 그 후, 수득된 cDNA를 기반으로 dsDNA를 수득하였다.
제조예 1.2. 항원 cDNA 수득
목적 유전자에 대한 PCR을 수행하기 위해, AMA1, ROP4 및 ROP18를 증폭하기 위한 프라이머를 디자인 및 주문하였다. PCR용 효소 TaKaRa Ex TaqTM 0.25 ㎕, 10X Ex Taq 버퍼 5 ㎕, dNTP 혼합물 4 ㎕, 템플릿 1 ㎕, 정방향 프라이머 2 ㎕, 및 역방향 프라이머 2 ㎕의 혼합물에 총 부피가 50 ㎕가 되도록 증류수를 넣었다. 그 후, PCR을 98℃에서 10초, 55℃에서 30초, 72℃에서 1분 조건으로 30 ~ 40 사이클 수행하였다. 이후, 아가로오스 겔에 DNA 마커 및 로딩 염료(loading dye)(6x)를 희석한 PCR 증폭 산물을 겔에 로딩하였다. 그 후, UV 등을 이용하여 PCR 증폭 산물을 확인하였다. 그 후, 원하는 유전자가 위치한 겔에서 항원을 코딩하는 DNA를 추출하였다.
상기에서 AMA1의 유전자는 정방향 프라이머로 5'-AAAGAATTCACCATGGGGCTCGTGGGCGTACA-3'(서열번호 11) 및 역방향 프라이머로 5'-TTACTCGAGCTAGTAATCCCCCTCGACCA-3'(서열번호 12)을 이용하였고, ROP4의 유전자는 정방향 프라이머로 5'-AAAGCATGCACCATGGGGCACCCTACCTCTTT-3'(서열번호 13) 및 역방향 프라이머로 5'-TTAGGTACCTCACGTTTCCGGTGGTGGCAT-3'(서열번호 14)을 이용하였고, ROP18의 유전자는 정방향 프라이머로 5'-AAAGGATCCACCATGTTTTCGGTACAGCGGCC-3'(서열번호 15) 및 역방향 프라이머로 5'-TTATCTAGATTATTCTGTGTGGAGATGTTC-3'(서열번호 16)을 이용하였다.
제조예 1.3. 항원 유전자 증폭
실시예 1.2에서 추출한 톡소포자충 항원인 AMA1, ROP4 및 ROP18의 DNA를 TA 벡터에 적재시켰다. 그 후, 항원 유전자가 적재된 TA 벡터를 대장균 세포에 형질전환시킨 후 이를 배양하였다. 형질전환된 대장균 세포를 배양한 후, 이로부터 AMA1, ROP4 및 ROP18을 코딩하는 유전자를 수득하였다.
제조예 1.4. 항원 유전자가 적재된 pFast-Bac 플라스미드 제조
실시예 1.3에서 수득한 AMA1, ROP4 및 ROP18 항원 유전자를 pFast-Bac 벡터에 각각 적재시켰다. 항원 유전자가 적재된 pFast-Bac 벡터를 DH10B 세포에 형질전환시켰다. 형질전환된 세포를 배양하여 항원 유전자가 적재된 pFast-Bac 벡터를 다량 수득하였다.
제조예 1.5. 톡소포자충 항원을 발현하는 배큘로바이러스 제조
AMA1, ROP4 및 ROP18의 DNA가 적재된 pFast-Bac 벡터를 cellfectin II(Invitrogen,USA)를 사용하여 SF9 세포 내로 형질감염시켰다. Bac-to-Bac 발현 시스템(Invitrogen)을 통해 제조사의 매뉴얼에 따라 AMA1, ROP4 및 ROP18을 발현하는 재조합 배큘로바이러스(rBV)를 제조하였다.
또한, 인플루엔자 바이러스 M1을 발현하는 rBV를 상기와 동일한 방법으로 제조하였다. 이때, M1의 유전자는 정방향 프라이머 5'-AAAGGATCCACCATGAGTCTTCTAACCGAGGT-3'(서열번호 9) 및 역방향 프라이머 5'-TTACTCGAGTTACTCTAGCTCTATGTTGAC-3'(서열번호 10)을 이용하여 RT-PCR을 통해 증폭하였다.
실시예 1. 톡소포자충 항원 및 인플루엔자 항원을 포함한 VLP 제조: AMA1
AMA1 및 M1 단백질을 포함하는 제1 톡소포자충 VLP를 제조하기 위하여, AMA1 rBV 및 M1 rBV를 SF9 세포에 공동 감염시켰다. 이때 M1 rBV와 AMA1 항원 rBV의 비율은 1:3으로 하여 감염시켰다. 이 후, 감염시킨 SF9 세포를 배양하고, SF9 세포 사멸율이 30% ~ 40%가 되었을 때 하베스트를 수행하였다. 구체적으로 배양한 세포 및 배양액을 6,000 rpm으로 30분간 원심분리 하여 상청액을 모은 후에, 30,000 rpm, 4℃에서 30분간 원심분리하여 펠렛화하였다. VLPs는 수크로오스 구배를 통해 정제하였다. 또한, VLPs는 사용시까지 인산염-완충 식염수(PBS)에서 재현탁시켰다. 이렇게 AMA1 항원 및 M1 항원을 발현하는 VLP를 AMA1-M1-VLPs라 명명하였다. AMA1-M1-VLP의 제조예는 도 1a에 도시하였다.
실시예 2. 톡소포자충 항원 및 인플루엔자 항원을 포함한 VLP 제조: ROP4
또한, ROP4 및 M1 단백질을 포함하는 제2 톡소포자충 VLP를 제조하기 위하여, 상기와 같은 방법으로 수행하여 ROP4-M1-VLPs를 수득하였다(도 1b).
실시예 3. 톡소포자충 항원 및 인플루엔자 항원을 포함한 VLP 제조: ROP18
또한, ROP18 및 M1 단백질을 포함하는 제3 톡소포자충 VLP를 제조하기 위하여, 상기와 같은 방법으로 수행하여 ROP18-M1-VLPs를 수득하였다(도 1c).
Ⅱ. VLPs를 이용한 톡소포자충 및 인플루엔자 진단
제조예 2. 톡소포자충 및 말라리아 감염 마우스로부터 혈청 채취
cyst(ME49 strain)를 300 cyst/마리(hd)로 7주령의 female BALB/c 마우스에 경구 감염(oral infection)시켰으며, 안와정맥총에서 채혈 후 원심분리하는 방식으로 감염 혈청을 수득하였다. 상기 in-vivo 과정에 대한 예시적인 순서는 도 2에 나타내었다. 또한, 말라리아 원충(P. berghei ANKA) 0.5%(0.5 × 104)를 마우스에 감염시킨 후, 감염된 마우스로부터 혈청을 수거하였다.
실험예 1. AMA1-M1-VLPs와 IgG 항체 반응 시험
실험예 1.1. 마우스 혈청에서 AMA1-M1-VLPs와 IgG 항체 반응의 특이성 시험
실시예 1에서 수득한 AMA1-M1-VLPs, AMA1 단백질 및 TLA(RH Tachyzoite lysate antigen)를 각각 4 ㎍/㎖의 농도로 96웰 면역플레이트에 코팅하였다. 이후, 말라리아 원충(P. berghei ANKA)을 감염시킨 마우스 혈청 14개, T. gondii(톡소포자충) ME49 300 cysts를 감염시킨 마우스 혈청 14개를 100배 희석하여 ELISA를 진행하였다(도 3).
그 결과, 3가지의 코팅한 항원 중 Naive 및 말라리아 원충을 감염시킨 혈청과는 거의 반응하지 않으면서, AMA1 VLPs의 톡소포자충 특이 IgG 항체에 대해서만 높은 반응성을 나타내는 것을 통해, 마우스 혈청에서 AMA1 VLPs와 IgG 항체간의 특이성이 있음을 확인하였다(도 4).
실험예 1.2. 마우스 혈청에서 AMA1-M1-VLPs와 IgG 항체 반응의 민감성 시험
실시예 1에서 수득한 AMA1-M1-VLPs, AMA1 단백질 및 TLA(RH Tachyzoite lysate antigen)를 각각 4 ㎍/㎖의 농도로 96웰 면역플레이트에 코팅하였다. 이후, T. gondii ME49 300 cysts를 감염시킨 마우스 혈청 14개를 100배, 300배, 900배 희석하여 ELISA를 진행하였다(도 3).
그 결과, 3가지의 코팅한 항원 중에서 AMA1-M1-VLPs의 톡소포자충 특이 IgG 항체가 가장 높게 측정됨을 확인함과 동시에, 100배 희석한 농도에 대해서 가장 민감성이 높음을 확인하였다(도 5).
실험예 1.3. 인간 혈청에서 AMA1-M1-VLPs와 IgG 항체 반응의 민감성 시험
실시예 1에서 수득한 AMA1-M1-VLPs, AMA1 단백질 및 TLA(RH Tachyzoite lysate antigen)를 각각 4 ㎍/㎖의 농도로 96웰 면역플레이트에 코팅하였다. 이후, T. gondii가 감염된 환자의 혈청 10개를 100배, 300배, 900배 희석하여 ELISA를 진행하였다(도 3).
그 결과, 3가지의 코팅한 항원 중에서 AMA1-M1-VLPs의 톡소포자충 특이 IgG 항체가 가장 높게 측정됨을 확인함과 동시에, 100배 희석한 농도에 대해서 가장 민감성이 높음을 확인하였다(도 6).
실험예 2. ROP4-M1-VLPs와 IgG 항체 반응 시험
실험예 2.1. 마우스 혈청에서 ROP4-M1-VLPs와 IgG 항체 반응의 특이성 시험
실시예 2에서 수득한 ROP4-M1-VLPs, ROP4 단백질 및 TLA(RH Tachyzoite lysate antigen)를 각각 4 ㎍/㎖의 농도로 96웰 면역플레이트에 코팅하였다. 이후, 말라리아 원충(P. berghei ANKA)을 감염시킨 마우스 혈청 14개, T. gondii(톡소포자충) ME49 300 cysts를 감염시킨 마우스 혈청 14개를 100배 희석하여 ELISA를 진행하였다(도 3).
그 결과, 3가지의 코팅한 항원 중 Naive 및 말라리아 원충을 감염시킨 혈청과는 거의 반응하지 않으면서, ROP4 VLPs의 톡소포자충 특이 IgG 항체에 대해서만 높은 반응성을 나타내는 것을 통해, 마우스 혈청에서 ROP4 VLPs와 IgG 항체간의 특이성이 있음을 확인하였다(도 7).
실험예 2.2. 마우스 혈청에서 ROP4-M1-VLPs와 IgG 항체 반응의 민감성 시험
실시예 2에서 수득한 ROP4-M1-VLPs, ROP4 단백질 및 TLA(RH Tachyzoite lysate antigen)를 각각 4 ㎍/㎖의 농도로 96웰 면역플레이트에 코팅하였다. 이후, T. gondii ME49 300 cysts를 감염시킨 마우스 혈청 14개를 100배, 300배, 900배 희석하여 ELISA를 진행하였다(도 3).
그 결과, 3가지의 코팅한 항원 중에서 ROP4-M1-VLPs의 톡소포자충 특이 IgG 항체가 가장 높게 측정됨을 확인함과 동시에, 100배 희석한 농도에 대해서 가장 민감성이 높음을 확인하였다(도 8).
실험예 2.3. 인간 혈청에서 ROP4-M1-VLPs와 IgG 항체 반응의 민감성 시험
실시예 2에서 수득한 ROP4-M1-VLPs, ROP4 단백질 및 TLA(RH Tachyzoite lysate antigen)를 각각 4 ㎍/㎖의 농도로 96웰 면역플레이트에 코팅하였다. 이후, T. gondii가 감염된 환자의 혈청 10개를 100배, 300배, 900배 희석하여 ELISA를 진행하였다(도 3).
그 결과, 3가지의 코팅한 항원 중에서 ROP4-M1-VLPs의 톡소포자충 특이 IgG 항체가 가장 높게 측정됨을 확인함과 동시에, 100배 희석한 농도에 대해서 가장 민감성이 높음을 확인하였다(도 9).
실험예 3. ROP18-M1-VLPs와 IgG 항체 반응 시험
실험예 3.1. 마우스 혈청에서 ROP18-M1-VLPs와 IgG 항체 반응의 특이성 시험
실시예 3에서 수득한 ROP18-M1-VLPs, ROP18 단백질 및 TLA(RH Tachyzoite lysate antigen)를 각각 4 ㎍/㎖의 농도로 96웰 면역플레이트에 코팅하였다. 이후, 말라리아 원충(P. berghei ANKA)을 감염시킨 마우스 혈청 14개, T. gondii(톡소포자충) ME49 300 cysts를 감염시킨 마우스 혈청 14개를 100배 희석하여 ELISA를 진행하였다(도 3).
그 결과, 3가지의 코팅한 항원 중 Naive 및 말라리아 원충을 감염시킨 혈청과는 거의 반응하지 않으면서, ROP18 VLPs의 톡소포자충 특이 IgG 항체에 대해서만 높은 반응성을 나타내는 것을 통해, 마우스 혈청에서 ROP18 VLPs와 IgG 항체간의 특이성이 있음을 확인하였다(도 10).
실험예 3.2. 마우스 혈청에서 ROP18-M1-VLPs와 IgG 항체 반응의 민감성 시험
실시예 3에서 수득한 ROP18-M1-VLPs, ROP18 단백질 및 TLA(RH Tachyzoite lysate antigen)를 각각 4 ㎍/㎖의 농도로 96웰 면역플레이트에 코팅하였다. 이후, T. gondii ME49 300 cysts를 감염시킨 마우스 혈청 14개를 100배, 300배, 900배 희석하여 ELISA를 진행하였다(도 3).
그 결과, 3가지의 코팅한 항원 중에서 ROP18-M1-VLPs의 톡소포자충 특이 IgG 항체가 가장 높게 측정됨을 확인함과 동시에, 100배 희석한 농도에 대해서 가장 민감성이 높음을 확인하였다(도 11).
실험예 3.3. 인간 혈청에서 ROP18-M1-VLPs와 IgG 항체 반응의 민감성 시험
실시예 3에서 수득한 ROP18-M1-VLPs, ROP18 단백질 및 TLA(RH Tachyzoite lysate antigen)를 각각 4 ㎍/㎖의 농도로 96웰 면역플레이트에 코팅하였다. 이후, T. gondii가 감염된 환자의 혈청 10개를 100배, 300배, 900배 희석하여 ELISA를 진행하였다(도 3).
그 결과, 3가지의 코팅한 항원 중에서 ROP18-M1-VLPs의 톡소포자충 특이 IgG 항체가 가장 높게 측정됨을 확인함과 동시에, 100배 희석한 농도에 대해서 가장 민감성이 높음을 확인하였다(도 12).
실험예 4. AMA1-M1-VLPs, ROP4-M1-VLPs, ROP18-M1-VLPs를 이용한 인플루엔자 진단
실시예 1 내지 실시예 3에서 수득한 AMA1-M1-VLPs, ROP4-M1-VLPs, ROP18-M1-VLPs 및 TLA(RH Tachyzoite lysate antigen)를 각각 4 ㎍/㎖의 농도로 96웰 면역플레이트에 코팅하였다.
이후, Naive 마우스 혈청, T. gondii ME49 300 cysts를 감염시킨 마우스 혈청, 인플루엔자 M1 VLPs 10 ㎍을 접종한 마우스의 혈청 및 NA(neuraminidase) VLPs 10 ㎍을 접종한 마우스의 혈청을 100배 희석하여 ELISA를 진행하였다.
여기서, NA VLPs는 인플루엔자 바이러스 A/PR/8/34(H1N1)에서 NA의 cDNA를 증폭시켜, T. gondii VLPs를 제조하는 방법과 동일한 방법으로 제조하였다.
그 결과, 상기 4가지의 항원이 코팅된 면역플레이트 중 AMA1-M1-VLPs, ROP4-M1-VLPs 및 ROP18-M1-VLPs가 코팅된 면역플레이트에서 492 ㎚로 OD값을 측정했을 때 유의미하게 높은 IgG 항체 반응이 나타났다. 뿐만 아니라, 동시에 인플루엔자 M1 VLPs 또는 NA VLPs 10 ㎍을 접종한 마우스의 혈청에 대해서도 0.2 이상의 값을 측정함으로써 상기 AMA1-M1-VLPs, ROP4-M1-VLPs 및 ROP18-M1-VLPs가 코팅된 면역플레이트로 톡소포자충증 및 인플루엔자 바이러스의 다중 진단이 가능함을 확인하였다(도 13).
결론적으로, AMA1-M1-VLPs, ROP4-M1-VLPs 및 ROP18-M1-VLPs는 톡소포자충에 감염된 개체의 혈청에 존재하는 항-AMA1 항체, 항-ROP4 항체 및/또는 항-ROP18 항체를 높은 민감도와 특이성으로 검출 가능할 뿐 아니라, 인플루엔자 바이러스에 감염된 개체의 혈청에 존재하는 항-M1 항체 또는 높은 민감도와 특이성으로 검출 가능함을 확인하였다. 따라서, 본원의 AMA1-M1-VLPs, ROP4-M1-VLPs 및 ROP18-M1-VLPs는 톡소포자충에 감염된 개체 및/또는 인플루엔자 바이러스에 감염된 개체를 동시에 진단하기 위한 조성물로 사용될 수 있다.
<110> health park co., ltd.
<120> VIRUS-LIKE PARTICLES COMPRISING TOXOPLASMA PROTEIN AND INFLUENZA
PROTEIN AND DIAGNOSTIC METHOD USING THE SAME
<130> SPD20-138
<160> 16
<170> KoPatentIn 3.0
<210> 1
<211> 252
<212> PRT
<213> Artificial Sequence
<220>
<223> Influenza virus matrix protein 1(M1) Amino acid sequence
<400> 1
Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro
1 5 10 15
Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe
20 25 30
Ala Gly Lys Asn Thr Asp Leu Glu Val Leu Met Glu Trp Leu Lys Thr
35 40 45
Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe
50 55 60
Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val
65 70 75 80
Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala
85 90 95
Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala
100 105 110
Lys Glu Ile Ser Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met
115 120 125
Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Val Ala Phe
130 135 140
Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg
145 150 155 160
Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu
165 170 175
Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met
180 185 190
Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln
195 200 205
Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser
210 215 220
Ser Ser Ala Gly Leu Lys Asn Asp Leu Leu Glu Asn Leu Gln Ala Tyr
225 230 235 240
Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys
245 250
<210> 2
<211> 541
<212> PRT
<213> Artificial Sequence
<220>
<223> TgAMA1 Amino acid sequence
<400> 2
Met Gly Leu Val Gly Val Gln Val Leu Leu Val Leu Val Ala Asp Cys
1 5 10 15
Thr Ile Phe Ala Ser Gly Leu Ser Ser Ser Thr Arg Ser Arg Glu Ser
20 25 30
Gln Thr Leu Ser Ala Ser Thr Ser Gly Asn Pro Phe Gln Ala Asn Val
35 40 45
Glu Met Lys Thr Phe Met Glu Arg Phe Asn Leu Thr His His His Gln
50 55 60
Ser Gly Ile Tyr Val Asp Leu Gly Gln Asp Lys Glu Val Asp Gly Thr
65 70 75 80
Leu Tyr Arg Glu Pro Ala Gly Leu Cys Pro Ile Trp Gly Lys His Ile
85 90 95
Glu Leu Gln Gln Pro Asp Arg Leu Pro Tyr Arg Asn Asn Phe Leu Glu
100 105 110
Asp Val Pro Thr Glu Lys Glu Tyr Lys Gln Ser Gly Asn Pro Leu Pro
115 120 125
Gly Gly Phe Asn Leu Asn Phe Val Thr Pro Ser Gly Gln Arg Ile Ser
130 135 140
Pro Phe Pro Met Glu Leu Leu Glu Lys Asn Ser Asn Ile Lys Ala Ser
145 150 155 160
Thr Asp Leu Gly Arg Cys Ala Glu Phe Ala Phe Lys Thr Val Ala Met
165 170 175
Asp Lys Asn Asn Lys Ala Thr Lys Tyr Arg Tyr Pro Phe Val Tyr Asp
180 185 190
Ser Lys Lys Arg Leu Cys His Ile Leu Tyr Val Ser Met Gln Leu Met
195 200 205
Glu Gly Lys Lys Tyr Cys Ser Val Lys Gly Glu Pro Pro Asp Leu Thr
210 215 220
Trp Tyr Cys Phe Lys Pro Arg Lys Ser Val Thr Glu Asn His His Leu
225 230 235 240
Ile Tyr Gly Ser Ala Tyr Val Gly Glu Asn Pro Asp Ala Phe Ile Ser
245 250 255
Lys Cys Pro Asn Gln Ala Leu Arg Gly Tyr Arg Phe Gly Val Trp Lys
260 265 270
Lys Gly Arg Cys Leu Asp Tyr Thr Glu Leu Thr Asp Thr Val Ile Glu
275 280 285
Arg Val Glu Ser Lys Ala Gln Cys Trp Val Lys Thr Phe Glu Asn Asp
290 295 300
Gly Val Ala Ser Asp Gln Pro His Thr Tyr Pro Leu Thr Ser Gln Ala
305 310 315 320
Ser Trp Asn Asp Trp Trp Pro Leu His Gln Ser Asp Gln Pro His Ser
325 330 335
Gly Gly Val Gly Arg Asn Tyr Gly Phe Tyr Tyr Val Asp Thr Thr Gly
340 345 350
Glu Gly Lys Cys Ala Leu Ser Asp Gln Val Pro Asp Cys Leu Val Ser
355 360 365
Asp Ser Ala Ala Val Ser Tyr Thr Ala Ala Gly Ser Leu Ser Glu Glu
370 375 380
Thr Pro Asn Phe Ile Ile Pro Ser Asn Pro Ser Val Thr Pro Pro Thr
385 390 395 400
Pro Glu Thr Ala Leu Gln Cys Thr Ala Asp Lys Phe Pro Asp Ser Phe
405 410 415
Gly Ala Cys Asp Val Gln Ala Cys Lys Arg Gln Lys Thr Ser Cys Val
420 425 430
Gly Gly Gln Ile Gln Ser Thr Ser Val Asp Cys Thr Ala Asp Glu Gln
435 440 445
Asn Glu Cys Gly Ser Asn Thr Ala Leu Ile Ala Gly Leu Ala Val Gly
450 455 460
Gly Val Leu Leu Leu Ala Leu Leu Gly Gly Gly Cys Tyr Phe Ala Lys
465 470 475 480
Arg Leu Asp Arg Asn Lys Gly Val Gln Ala Ala His His Glu His Glu
485 490 495
Phe Gln Ser Asp Arg Gly Ala Arg Lys Lys Arg Pro Ser Asp Leu Met
500 505 510
Gln Glu Ala Glu Pro Ser Phe Trp Asp Glu Ala Glu Glu Asn Ile Glu
515 520 525
Gln Asp Gly Glu Thr His Val Met Val Glu Gly Asp Tyr
530 535 540
<210> 3
<211> 578
<212> PRT
<213> Artificial Sequence
<220>
<223> TgROP4 Amino acid sequence
<400> 3
Met Gly His Pro Thr Ser Phe Gly Gln Pro Ser Cys Leu Val Trp Leu
1 5 10 15
Ala Ala Ala Phe Leu Val Leu Gly Leu Cys Leu Val Gln Gln Gly Ala
20 25 30
Gly Arg Gln Arg Pro His Gln Trp Lys Ser Ser Glu Ala Ala Leu Ser
35 40 45
Val Ser Pro Ala Gly Asp Ile Val Asp Lys Tyr Ser Arg Asp Ser Thr
50 55 60
Glu Gly Glu Asn Thr Val Ser Glu Gly Glu Ala Glu Gly Ser Arg Gly
65 70 75 80
Gly Ser Trp Leu Glu Gln Glu Gly Val Glu Leu Arg Ser Pro Ser Gln
85 90 95
Asp Ser Gln Thr Gly Thr Ser Thr Ala Ser Pro Thr Gly Phe Arg Arg
100 105 110
Leu Leu Arg Arg Leu Arg Phe Trp Arg Arg Gly Ser Thr Arg Gly Ser
115 120 125
Asp Asp Ala Ala Glu Val Ser Arg Arg Thr Arg Val Pro Leu His Thr
130 135 140
Arg Leu Leu Gln His Leu Arg Arg Val Ala Arg Ile Ile Arg His Gly
145 150 155 160
Val Ser Ala Ala Ala Gly Arg Leu Phe Gly Arg Val Arg Gln Val Glu
165 170 175
Ala Glu Arg Pro Gln Pro Val Phe Thr Glu Gly Asp Pro Pro Asp Leu
180 185 190
Glu Thr Asn Ser Leu Tyr Tyr Arg Asp Lys Val Pro Gly Gln Gly Ile
195 200 205
Ile Gln Glu Ile Leu Arg Gln Lys Pro Gly Ile Ala His His Pro Glu
210 215 220
Ser Phe Ser Val Val Ala Ala Asp Glu Arg Val Ser Arg Thr Leu Trp
225 230 235 240
Ala Glu Gly Gly Val Val Arg Val Ala Ser Glu Leu Gly Gln Pro Gly
245 250 255
Arg Val Leu Val Arg Gly Arg Arg Ile Gly Leu Phe Arg Pro Gly Met
260 265 270
Gln Phe Glu Ala Thr Asp Gln Ala Thr Gly Glu Pro Met Thr Ala Leu
275 280 285
Val Gly His Thr Val Leu Glu Ala Thr Ala Arg Asp Val Asp Ser Met
290 295 300
Arg Asn Glu Gly Leu Ala Val Gly Leu Phe Gln Lys Val Lys Asn Pro
305 310 315 320
Tyr Leu Ala Asn Arg Tyr Leu Arg Phe Leu Ala Pro Phe Asp Leu Val
325 330 335
Thr Ile Pro Gly Lys Pro Leu Val Gln Lys Ala Lys Ser Arg Asn Glu
340 345 350
Val Gly Trp Val Lys Asn Leu Leu Phe Leu Leu Pro Pro Thr His Val
355 360 365
Asp Met Glu Thr Phe Val Asp Glu Ile Gly Arg Phe Pro Gln Glu Asp
370 375 380
Arg Pro Leu Ala Asp Ala Ala Arg Leu Tyr Leu Thr Val Gln Ala Val
385 390 395 400
Arg Leu Val Ala His Leu Gln Asp Glu Gly Val Val His Gly Lys Ile
405 410 415
Met Pro Asp Ser Phe Cys Leu Lys Arg Glu Gly Gly Leu Tyr Leu Arg
420 425 430
Asp Phe Gly Ser Leu Val Arg Ala Gly Ala Lys Val Val Val Pro Ala
435 440 445
Glu Tyr Asp Glu Tyr Thr Pro Pro Glu Gly Arg Ala Ala Ala Arg Ser
450 455 460
Arg Phe Gly Ser Gly Ala Thr Thr Met Thr Tyr Ala Phe Asp Ala Trp
465 470 475 480
Thr Leu Gly Ser Val Ile Phe Leu Ile Trp Cys Ser Arg Ala Pro Asp
485 490 495
Thr Lys Ser Gly Tyr Glu Tyr Ser Val Glu Phe Phe Phe Ser Arg Cys
500 505 510
Arg Arg Val Pro Glu Asn Val Lys Leu Leu Val Tyr Lys Leu Ile Asn
515 520 525
Pro Ser Val Glu Ala Arg Leu Leu Ala Leu Gln Ala Ile Glu Thr Pro
530 535 540
Glu Tyr Arg Glu Met Glu Glu Gln Leu Ser Ala Ala Ser Arg Leu Tyr
545 550 555 560
Ser Gly Asp Gly Thr Leu Thr Gly Gly Asp Asp Asp Met Pro Pro Leu
565 570 575
Glu Thr
<210> 4
<211> 554
<212> PRT
<213> Artificial Sequence
<220>
<223> TgROP18 Amino acid sequence
<400> 4
Met Phe Ser Val Gln Arg Pro Pro Leu Thr Arg Thr Val Val Arg Met
1 5 10 15
Gly Leu Ala Thr Leu Leu Pro Lys Thr Ala Cys Leu Ala Gly Leu Asn
20 25 30
Val Ala Leu Val Phe Leu Leu Phe Gln Val Gln Asp Gly Thr Gly Ile
35 40 45
Thr Leu Gly Pro Ser Lys Leu Asp Ser Lys Pro Thr Ser Leu Asp Ser
50 55 60
Gln Gln His Val Ala Asp Lys Arg Trp Leu Ala Thr Val Gly His Tyr
65 70 75 80
Lys His Leu Ala Gly Ala Thr Glu Ser Thr Arg Asp Val Ser Leu Leu
85 90 95
Glu Glu Arg Ala Gln His Arg Val Asn Ala Gln Glu Thr Asn Gln Arg
100 105 110
Arg Thr Ile Phe Gln Arg Leu Leu Asn Leu Leu Arg Arg Arg Glu Arg
115 120 125
Asp Gly Glu Val Ser Gly Ser Ala Ala Asp Ser Ser Ser Arg Pro Arg
130 135 140
Leu Ser Val Arg Gln Arg Leu Ala Gln Leu Trp Arg Arg Ala Lys Ser
145 150 155 160
Leu Phe Lys Arg Gly Ile Arg Arg Tyr Phe Pro Gln Gly Arg Asn Arg
165 170 175
Gln Arg Ser Leu Arg Ala Gln Arg Arg Arg Ser Glu Leu Val Phe Glu
180 185 190
Lys Ala Asp Ser Gly Cys Val Ile Gly Lys Arg Ile Leu Ala His Met
195 200 205
Gln Glu Gln Ile Gly Gln Pro Gln Ala Leu Glu Asn Ser Glu Arg Leu
210 215 220
Asp Arg Ile Leu Thr Val Ala Ala Trp Pro Pro Asp Val Pro Lys Arg
225 230 235 240
Phe Val Ser Val Thr Thr Gly Glu Thr Arg Thr Leu Val Arg Gly Ala
245 250 255
Pro Leu Gly Ser Gly Gly Phe Ala Thr Val Tyr Glu Ala Thr Asp Val
260 265 270
Glu Thr Asn Glu Glu Leu Ala Val Lys Val Phe Met Ser Glu Lys Glu
275 280 285
Pro Thr Asp Glu Thr Met Leu Asp Leu Gln Arg Glu Ser Ser Cys Tyr
290 295 300
Arg Asn Phe Ser Leu Ala Lys Thr Ala Lys Asp Ala Gln Glu Ser Cys
305 310 315 320
Arg Phe Met Val Pro Ser Asp Val Val Met Leu Glu Gly Gln Pro Ala
325 330 335
Ser Thr Glu Val Val Ile Gly Leu Thr Thr Arg Trp Val Pro Asn Tyr
340 345 350
Phe Leu Leu Met Met Arg Ala Glu Ala Asp Met Ser Lys Val Ile Ser
355 360 365
Trp Val Phe Gly Asp Ala Ser Val Asn Lys Ser Glu Phe Gly Leu Val
370 375 380
Val Arg Met Tyr Leu Ser Ser Gln Ala Ile Lys Leu Val Ala Asn Val
385 390 395 400
Gln Ala Gln Gly Ile Val His Thr Asp Ile Lys Pro Ala Asn Phe Leu
405 410 415
Leu Leu Lys Asp Gly Arg Leu Phe Leu Gly Asp Phe Gly Thr Tyr Arg
420 425 430
Ile Asn Asn Ser Val Gly Arg Ala Ile Gly Thr Pro Gly Tyr Glu Pro
435 440 445
Pro Glu Arg Pro Phe Gln Ala Thr Gly Ile Thr Tyr Thr Phe Pro Thr
450 455 460
Asp Ala Trp Gln Leu Gly Ile Thr Leu Tyr Cys Ile Trp Cys Lys Glu
465 470 475 480
Arg Pro Thr Pro Ala Asp Gly Ile Trp Asp Tyr Leu His Phe Ala Asp
485 490 495
Cys Pro Ser Thr Pro Glu Leu Val Gln Asp Leu Ile Arg Ser Leu Leu
500 505 510
Asn Arg Asp Pro Gln Lys Arg Met Leu Pro Leu Gln Ala Leu Glu Thr
515 520 525
Ala Ala Phe Lys Glu Met Asp Ser Val Val Lys Gly Ala Ala Gln Asn
530 535 540
Phe Glu Gln Gln Glu His Leu His Thr Glu
545 550
<210> 5
<211> 1027
<212> DNA
<213> Artificial Sequence
<220>
<223> Influenza virus matrix protein 1(M1) DNA sequence
<400> 5
agcgaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtacgtact 60
ctctatcatc ccgtcaggcc ccctcaaagc cgagatcgca cagagacttg aagatgtctt 120
tgcagggaag aacaccgatc ttgaggttct catggaatgg ctaaagacaa gaccaatcct 180
gtcacctctg actaagggga ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg 240
aggactgcag cgtagacgct ttgtccaaaa tgcccttaat gggaacgggg atccaaataa 300
catggacaaa gcagttaaac tgtataggaa gctcaagagg gagataacat tccatggggc 360
caaagaaatc tcactcagtt attctgctgg tgcacttgcc agttgtatgg gcctcatata 420
caacaggatg ggggctgtga ccactgaagt ggcatttggc ctggtatgtg caacctgtga 480
acagattgct gactcccagc atcggtctca taggcaaatg gtgacaacaa ccaatccact 540
aatcagacat gagaacagaa tggttttagc cagcactaca gctaaggcta tggagcaaat 600
ggctggatcg agtgagcaag cagcagaggc catggaggtt gctagtcagg ctagacaaat 660
ggtgcaagcg atgagaacca ttggaactca tcctagctcc agtgctggtc tgaaaaatga 720
tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc aacggttcaa 780
gtgatcctct cactattgcc gcaaatatca ttgggatctt gcacttgaca ttgtggattc 840
ttgatcgtct ttttttcaaa tgcatttacc gtcgctttaa atacggactg aaaggagggc 900
cttctacgga aggagtgcca aagtctatga gggaagaata tcgaaaggaa cagcagagtg 960
ctgtggatgc tgacgatggt cattttgtca gcatagagct ggagtaaaaa actaccttgt 1020
ttctact 1027
<210> 6
<211> 1626
<212> DNA
<213> Artificial Sequence
<220>
<223> TgAMA1 DNA sequence
<400> 6
atggggctcg tgggcgtaca agttttgctg gttcttgtgg cggattgcac catattcgca 60
tcgggactca gctcaagcac aaggtctcgc gagtcgcaga cgctgagtgc tagcacgtcg 120
gggaatccct ttcaggcaaa tgtagagatg aaaaccttca tggaaagatt caacctaact 180
catcatcatc agtctggtat ttacgtcgac cttggacaag acaaggaagt tgatggcaca 240
ttataccggg agcctgcggg gttgtgtccc atttggggaa agcacatcga actccagcag 300
ccggaccggc ttccgtaccg taacaacttc ttggaagatg ttccgactga aaaagaatac 360
aaacagtcag ggaatccttt gcccggaggc ttcaacttga atttcgtgac gcctagcggg 420
cagcgaattt caccatttcc gatggaactt cttgaaaaaa atagcaacat caaggcgagt 480
acggatcttg ggaggtgcgc cgagtttgcc tttaagacgg tcgctatgga taaaaacaat 540
aaggcgacga agtaccgtta cccatttgtt tatgactcca agaagcgact gtgccacatc 600
ctctacgtat cgatgcagct gatggagggt aaaaagtact gttcagtcaa gggcgaacct 660
ccagatctca catggtattg cttcaagccc cgaaagagtg ttacggagaa tcatcatctc 720
atctacggat cggcctatgt tggagagaac ccagatgcgt tcatcagtaa atgcccaaat 780
caagctcttc gcgggtacag gttcggtgtt tggaagaaag gccgttgcct cgactacact 840
gaattgaccg acactgtgat agaacgtgtt gagtcaaagg cacagtgctg ggtgaaaacc 900
tttgaaaacg acggggtcgc gagtgaccaa ccccatacgt atccactgac gtcgcaagca 960
tcatggaacg attggtggcc tctccaccag agtgaccaac ctcactcagg tggcgttggg 1020
cgtaattacg gtttctacta cgtggacacg actggagagg gcaagtgtgc actctctgac 1080
caggtacccg actgcctggt gtcggattct gccgccgtgt cgtatacagc agcggggagt 1140
ttgtctgaag agacgccgaa tttcataatt ccgtcaaatc cctctgttac tccgccaacg 1200
cccgagacgg cacttcagtg cacggccgac aagttccccg actctttcgg tgcctgcgac 1260
gttcaagcct gtaaaagaca gaagacgtcc tgcgttggcg gacagattca aagtactagc 1320
gtcgactgca ccgcggacga acaaaatgaa tgtggctcta acactgcgtt gatcgctgga 1380
ctcgccgtag gaggggttct gctgttggct cttctaggag gaggctgcta cttcgcgaag 1440
aggttggaca gaaacaaagg cgtccaggcg gctcatcatg aacatgagtt tcagtcagac 1500
agaggtgctc gaaaaaagag gccaagcgat ctcatgcaag aggctgaacc gtcgttttgg 1560
gatgaggcag aggagaacat tgaacaagat ggggaaacac atgttatggt cgagggggat 1620
tactag 1626
<210> 7
<211> 2039
<212> DNA
<213> Artificial Sequence
<220>
<223> TgROP4 DNA sequence
<400> 7
tccggcatgg aaaaaatgcg tctagctaac cgcgggcccc aatgtcgcac ggccttggtc 60
aagggacggc gcgacctgca gagacgggaa tccgagccaa gcgctgagtg tccttgcttc 120
tggctgagcc gccatcctgc ggcaccgtcc atgactgcgc acagcatccc gggagtatgg 180
tgattgtgtc agtcttattt cttttgaagg cacttcagat gtgtacttca gaatgttgtt 240
acgataattg cggatgcagc tgccacctct gaacggttga tttggttgtg agtcctccca 300
acatggggca ccctacctct ttcggacagc cgtcgtgtct tgtctggcta gctgcggcat 360
tccttgttct ggggctttgc ctcgtccagc aaggcgctgg cagacagcgg cctcaccagt 420
ggaagagctc ggaagccgct ttgagtgtca gtccggcagg agacatcgtg gacaagtatt 480
ctcgtgattc cactgaggga gaaaacactg tctcagaggg ggaagctgag ggcagtcggg 540
ggggttcatg gctggagcag gagggggttg aattaaggtc gccttcacag gacagccaga 600
caggaacctc gaccgcgtcc cccacaggct tcagaaggtt actcaggcgt ttgcgttttt 660
ggcgtcgtgg gagtacacgc ggatccgatg atgcagcaga agtttccagg aggactcggg 720
ttcctctgca tacccgtctg cttcagcatt tgcggcgagt agcgagaatc atccgacacg 780
gggtttcagc ggccgctggc agattgttcg ggcgagttcg gcaagttgag gcggaacgtc 840
cacaacccgt tttcactgaa ggtgatcctc cggatctcga gacgaattcc ttgtattatc 900
gcgacaaggt tccaggacag gggataatcc aggagatcct caggcagaag ccgggaatcg 960
cacaccaccc cgagtcattt agtgtggttg ctgctgacga gagagtatca cggactctgt 1020
gggctgaggg cggggttgtg agagttgctt cagaattggg ccaacctggg agagtgctcg 1080
tgagggggag acgaatcggt ttgtttcgcc caggaatgca gtttgaagca acagaccagg 1140
cgacaggaga accgatgact gcgcttgttg gccatactgt gcttgaggcc acggcaagag 1200
acgtagattc gatgcgcaac gaaggattag ctgttgggtt gtttcaaaag gtgaagaacc 1260
catatctagc taacaggtat ctaagatttc tggccccgtt cgatttggtg acgatcccgg 1320
ggaagccatt agttcagaaa gctaagtcac gtaatgaggt tggctgggtc aaaaatctgt 1380
tgtttctgct tcctccaact catgtcgata tggaaacgtt tgtcgacgag attggtcgat 1440
ttccacaaga ggaccgcccc ctagctgatg ccgctcgatt gtatcttact gttcaggcgg 1500
tgaggttggt agcgcacctc caagacgaag gagtggtgca cgggaagata atgcctgatt 1560
cgttttgttt gaagcgtgag gggggcctgt acttgcgtga ctttggctct ctggtgagag 1620
cgggcgcaaa agtggtggtg cccgcggaat atgatgagta tacgccgcca gaaggtcgtg 1680
cggccgcccg aagtcgtttc ggctctgggg cgaccacgat gacgtacgca ttcgacgcct 1740
ggacactggg ttcggttata tttttaattt ggtgctctcg agctcccgat acaaaatcgg 1800
ggtacgaata cagtgtcgaa ttcttttttt cgaggtgccg gcgggtgcca gagaacgtga 1860
aacttctggt ttacaagttg attaaccctt ctgtcgaggc ccgcctgctc gccttgcaag 1920
cgatagagac tccggaatac agggagatgg aagaacagct gtccgctgct tcgcgcctgt 1980
actcaggtga tgggacgttg acagggggag acgatgacat gccaccactg gaaacgtga 2039
<210> 8
<211> 1665
<212> DNA
<213> Artificial Sequence
<220>
<223> TgROP18 DNA sequence
<400> 8
atgttttcgg tacagcggcc acctcttacg cgtaccgtcg tccgaatggg tttagcgact 60
cttctcccga agacagcctg tcttgcgggg ttaaatgtag cgcttgtctt cctgctcttc 120
caagtccagg atgggaccgg aatcacactt ggtccttcaa aactcgactc caaaccgaca 180
agtttggatt cgcaacagca cgttgctgac aagcggtggc ttgctacagt tggccactac 240
aaacatttag caggagcgac agaaagcact cgagacgttt cattgctgga ggaaagggct 300
caacaccggg taaatgcgca agaaacaaac caacggcgca cgatttttca gaggcttctg 360
aatctcttga gacggagaga aagagatggt gaagtctcgg gttccgcagc tgatagctcc 420
tcgagacccc gtctgtccgt acgacagagg cttgctcaac tttggcgtag agcgaaatcg 480
ttattcaaac gcggaatccg gaggtacttt cctcaagggc gtaaccgaca gcgaagtttg 540
cgggcacaaa gacggcgatc tgaattggtt tttgagaagg cggattctgg atgcgtcatc 600
ggcaaacgca tcctggcgca catgcaagaa caaatcgggc agcctcaagc gctagaaaat 660
agtgaacgac tggatagaat tctgactgtc gccgcctggc ctccggacgt tccaaaaaga 720
tttgtttctg tgactaccgg tgaaacccgg acgctggtga gaggtgcacc ccttggctct 780
ggtggattcg ccactgtata tgaggctaca gacgtggaga cgaatgaaga gttggctgtt 840
aaggttttca tgtcagaaaa ggagcccacc gatgagacta tgcttgactt gcagagggag 900
tcgtcctgct acaggaactt tagtctagcc aagacggcga aggatgccca ggaaagctgt 960
agattcatgg ttcctagtga tgttgtgatg ttagagggac agccagcatc cacagaggtc 1020
gtgattggtt tgacgactcg gtgggtacca aactattttc ttctcatgat gcgggcagaa 1080
gcggacatga gcaaagtcat ttcatgggta tttggagatg cgtctgtcaa taaaagtgaa 1140
tttggcctgg tcgttcgaat gtacctatcc agtcaggcaa tcaaactagt ggccaatgtt 1200
caagctcagg gaattgtgca tacggatatc aaaccggcga atttcctcct cttgaaagac 1260
ggtcgcctgt ttctcggcga cttcggaacg tatagaatca ataattcggt tggacgcgcg 1320
ataggtactc ccggttacga gcctccggag cgaccgtttc aggctacagg catcacctat 1380
acattcccca ctgacgcgtg gcaactcggt ataactttgt actgcatctg gtgcaaggaa 1440
cgtccaactc cggccgacgg catctgggac tacttacact tcgcagattg tccttccacg 1500
cctgagctgg ttcaagacct catccgaagc ctcttgaatc gagatcctca gaaacggatg 1560
ctcccgctac aagccttgga gacagcagcg tttaaagaga tggattcagt agtaaaaggc 1620
gccgcgcaaa acttcgaaca gcaggaacat ctccacacag aataa 1665
<210> 9
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> M1 primer F
<400> 9
aaaggatcca ccatgagtct tctaaccgag gt 32
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> M1 primer R
<400> 10
ttactcgagt tactctagct ctatgttgac 30
<210> 11
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> AMA1 primer F
<400> 11
aaagaattca ccatggggct cgtgggcgta ca 32
<210> 12
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> AMA1 primer R
<400> 12
ttactcgagc tagtaatccc cctcgacca 29
<210> 13
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> ROP4 primer F
<400> 13
aaagcatgca ccatggggca ccctacctct tt 32
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> ROP4 primer R
<400> 14
ttaggtacct cacgtttccg gtggtggcat 30
<210> 15
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> ROP18 primer F
<400> 15
aaaggatcca ccatgttttc ggtacagcgg cc 32
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> ROP18 primer R
<400> 16
ttatctagat tattctgtgt ggagatgttc 30
Claims (12)
- 톡소포자충 유래의 표면 항원 단백질; 및 인플루엔자 바이러스 매트릭스 단백질 1을 포함하는 바이러스-유사 입자를 포함하는 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물.
- 제1항에 있어서,
상기 톡소포자충 유래의 표면 항원 단백질은 AMA1, ROP4, ROP18로 구성된 군으로부터 선택된 어느 하나인 것인, 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물. - 제1항에 있어서,
상기 M1은 서열번호 1의 아미노산 서열을 가지는 것인, 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물. - 제2항에 있어서,
상기 AMA1은 서열번호 2의 아미노산 서열을 가지며,
상기 ROP4는 서열번호 3의 아미노산 서열을 가지며,
상기 ROP18은 서열번호 4의 아미노산 서열을 가지는 것인, 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물. - 제1항에 있어서,
바이러스-유사 입자에서 톡소포자충 유래의 표면 항원 단백질 및 인플루엔자 바이러스 매트릭스 단백질 1의 비율은 3:1인 것인, 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물. - 제1항에 있어서,
상기 바이러스-유사 입자는 톡소포자충 유래의 표면 항원 단백질을 코딩하는 유전자를 포함하는 재조합 배큘로바이러스 및 인플루엔자 바이러스 매트릭스 단백질 1을 코딩하는 유전자를 포함하는 재조합 배큘로바이러스를 공동 감염시켜 제조된 것인, 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물. - 제5항에 있어서,
공동 감염시에 톡소포자충 유래의 표면 항원 단백질을 코딩하는 유전자를 포함하는 재조합 배큘로바이러스와 인플루엔자 바이러스 매트릭스 단백질 1을 코딩하는 유전자를 포함하는 재조합 배큘로바이러는 3:1로 감염시키는 것을 특징으로 하는, 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물. - 제1항에 있어서,
톡소포자충 및/또는 인플루엔자 바이러스의 감염된 개체의 자가 항체의 수준을 진단하는 것을 특징으로 하는, 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물. - 제1항의 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물을 포함하는 톡소포자충 및 인플루엔자 바이러스 다중 진단 키트.
- 제9항에 있어서,
상기 진단 키트는 ELISA(Enzyme-linked immunosorbent assay), RIA(Radioimmnoassay), 샌드위치 측정법(Sandwich assay), 웨스턴 블롯(Western Blot), 면역블롯 분석(Immunoblot assay) 및 면역조직화학염색 방법(Immnohistochemical staining)으로 구성된 군으로부터 선택되는 어느 하나인 것인, 톡소포자충 및 인플루엔자 바이러스 다중 진단 키트. - 톡소포자충 유래의 표면 항원 단백질; 및 인플루엔자 바이러스 매트릭스 단백질 1을 포함하는 바이러스-유사 입자를 포함하는 톡소포자충증 및 인플루엔자 바이러스 다중 진단용 조성물에 대상체로부터 분리된 생물학적 시료를 접촉시키는 단계; 및
바이러스-유사 입자에 결합하는 항체를 검출하는 단계;를 포함하는 톡소포자충 및/또는 인플루엔자 바이러스 감염 여부에 대한 정보를 제공하는 방법. - 제12항에 있어서,
상기 톡소포자충 유래의 표면 항원 단백질은 AMA1, ROP4, ROP18로 구성된 군으로부터 선택된 어느 하나인 것인, 톡소포자충 및/또는 인플루엔자 바이러스 감염 여부에 대한 정보를 제공하는 방법.
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JP2023544409A JP2024509043A (ja) | 2021-01-22 | 2021-12-06 | トキソプラズマタンパク質およびインフルエンザウイルスタンパク質を含むウイルス様粒子およびこれを用いた診断方法 |
PCT/KR2021/018383 WO2022158705A1 (ko) | 2021-01-22 | 2021-12-06 | 톡소포자충 단백질 및 인플루엔자 바이러스 단백질을 포함하는 바이러스 유사 입자 및 이를 이용한 진단 방법 |
US18/262,327 US20240085420A1 (en) | 2021-01-22 | 2021-12-06 | Virus-like particle comprising toxoplasma gondii protein and influenza virus protein, and diagnostic method using same |
EP21921464.0A EP4283296A1 (en) | 2021-01-22 | 2021-12-06 | Virus-like particle comprising toxoplasma gondii protein and influenza virus protein, and diagnostic method using same |
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