KR102195989B1 - Novel epitope of chikungunya virus and use thereof - Google Patents
Novel epitope of chikungunya virus and use thereof Download PDFInfo
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- KR102195989B1 KR102195989B1 KR1020180102973A KR20180102973A KR102195989B1 KR 102195989 B1 KR102195989 B1 KR 102195989B1 KR 1020180102973 A KR1020180102973 A KR 1020180102973A KR 20180102973 A KR20180102973 A KR 20180102973A KR 102195989 B1 KR102195989 B1 KR 102195989B1
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- chikungunya virus
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
본 발명은 치쿤군야 바이러스의 표준항원으로서 신규한 에피토프 및 이의 용도에 관한 것으로, 본 발명의 항원 단백질은 당화 부위를 포함하지 않고, 우수한 수용성을 갖는바 산업적인 수준의 항원 단백질 및 항체의 대량생산에 매우 적합하고, 중화항체를 유도한다는 점에서 우수한 특성을 갖는다. 또한, 항체에 의한 검출 민감성이 우수하여 치쿤군야 바이러스에 대한 감염 확인, 감염 예방 또는 감염 치료에 매우 효과적으로 활용될 수 있다. The present invention relates to a novel epitope and its use as a standard antigen of Chikungunya virus, and the antigenic protein of the present invention does not contain a glycosylation site and has excellent water solubility, so it is suitable for mass production of antigenic proteins and antibodies at an industrial level. It is very suitable and has excellent properties in that it induces neutralizing antibodies. In addition, since the detection sensitivity by the antibody is excellent, it can be very effectively used to confirm infection, prevent infection, or treat infection against Chikungunya virus.
Description
본 발명은 치쿤군야 바이러스의 표준항원으로서 신규한 에피토프 및 이를 이용한 치쿤군야 바이러스를 검출과 같은 새로운 에피토프의 용도에 관한 것이다. The present invention relates to a novel epitope as a standard antigen of chikungunya virus, and to the use of a novel epitope such as detecting chikungunya virus using the same.
치쿤군야 바이러스(Chikungunya virus, CHIKV)는 1952년 탄자니아의 마콘데 고원(Makonde Plateau)에서 처음 보고된 이래 동남아시아 지역에서 60-80년대 국지적인 유행을 보이다가, 2000년대에 들어와 케냐(2004), 코모도(2005), 인도(2005-2007), 프랑스(2006), 이탈리아(2007) 등 전 세계적으로 대규모 유행이 예상치 못하게 산발적으로 발생하고 있다. Chikungunya virus (CHIKV) was first reported in Tanzania's Makonde Plateau in 1952, and has been a local epidemic in Southeast Asia in the 60s and 80s, then entered Kenya (2004) and Komodo in the 2000s. (2005), India (2005-2007), France (2006), Italy (2007), and other large-scale outbreaks are unexpectedly and sporadic.
특히, 코모로 제도 인구의 70 % 이상 감염, 프랑스 발생 지역 인구의 30 % 감염 등과 같이 일부 지역에서는 감염자의 발생 정도의 규모가 상당히 컸고, 전세계적으로 매년 100만 명의 감염자가 발생하는 것으로 보고된 바 있고, 최근 들어 세계 여행의 증가로 인해 치쿤군야 바이러스 호발지역(인도, 아프리카, 인도양 섬 등)을 방문했던 여행자들이 지역 감염을 일으킨 사례가 2003년부터 발생하고 있다. In particular, in some regions, such as infection with more than 70% of the population of the Comoros Islands and 30% of the population in France, the incidence of infection was quite large, and it has been reported that 1 million infections occur annually worldwide. , In recent years, due to the increase in world travel, tourists who have visited the Chikungunya virus-prone regions (India, Africa, Indian Ocean islands, etc.) have caused local infections from 2003.
점차 심각해지는 지구 온난화로 인해 치쿤군야 바이러스 전염 매개체인 모기의 활동 반경이 점차 확대되고 있어 열대성의 기존의 치쿤군야 바이러스 호발지역을 넘어서 비열대성의 새로운 지역에도 치쿤군야 바이러스 감염의 위험성이 증가하고 있다. 전세계적으로 2007년부터 치쿤군야 바이러스 감염은 더 이상 열대성 질환으로 분류하지 않으며 우리나라에서도 2010년 12월에 처음으로 법정감염으로 지정되었다. 2013-2015년 사이 5건의 치쿤군야 바이러스의 국내 감염사례가 보고된 바 있으며, 대부분의 감염자들은 치쿤군야 바이러스 호발지역을 방문한 전력이 있는 것으로 나타났다. Due to the increasingly serious global warming, the radius of activity of mosquitoes, which is a carrier of chikungunya virus transmission, is gradually expanding.Therefore, the risk of chikungunya virus infection is increasing even in new non-tropical regions beyond the existing chikungunya virus-prone areas in tropical regions. Worldwide, since 2007, Chikungunya virus infection is no longer classified as a tropical disease, and Korea was also designated as a legal infection for the first time in December 2010. Between 2013 and 2015, 5 cases of infection with the chikungunya virus were reported in Korea, and most of the infected people had a history of visiting the Chikungunya virus-prone area.
치쿤군야 바이러스는 뎅기 바이러스와 지카 바이러스의 매개체로도 알려진 이집트 숲모기(Aedes aegypti)와 흰줄 숲모기(Aedes albopictus)에 의해 전파되는 모기 매개성 아보바이러스(Arbovirus)에 해당하는 것으로, 급성열성질환인 치쿤군야 열병을 일으키는 바이러스로 알려져 있다. 치쿤군야 바이러스가 감염되는 경우 10~15 % 는 무증상을 보이지만, 나머지 감염자는 1 ~ 12일의 잠복기를 거쳐 갑작스러운 고열, 간헐적 오한, 두통, 구역질, 구토, 극심한 관절통, 점상출혈 발진 및 구진발진 등의 증세를 보이며, 39~40 ℃에 이르는 고열이 수일 동안 지속되는 것으로 보고되어 있다. 특히, 관절통의 경우 감염자의 30~40 % 가 수년간 지속되는 만성질환으로 나타날 수 있을 뿐만 아니라, 신생아의 경우 뇌수막염으로까지 이어질 수 있는 질병으로 알려져 있다.Chikungunya virus is a mosquito-borne arbovirus transmitted by Egyptian forest mosquito (Aedes aegypti) and Aedes albopictus, also known as vectors of dengue virus and Zika virus, and is an acute febrile disease. It is known as a virus that causes Chikungunya fever. In the case of Chikungunya virus infection, 10-15% show no symptoms, but the rest of the infected suffer from sudden high fever, intermittent chills, headache, nausea, vomiting, severe joint pain, petechial rash and papule rash after an incubation period of 1 to 12 days. It has been reported to show symptoms of, and a high fever ranging from 39 to 40 ℃ persists for several days. In particular, in the case of joint pain, 30-40% of infected people can appear as a chronic disease that lasts for several years, and in newborns, it is known as a disease that can lead to meningitis.
치쿤군야 바이러스 감염이 1952년 최초로 보고된 이후부터 2000년대의 대유행을 거치면서 다양한 CHIKV의 변이주들이 임상환자에서 분리되었는데, 유전체 서열을 기준으로 작성한 계통학적 분류(phylogenic tree)에 따르면 치쿤군야 바이러스는 인도양(Indian ocean) 변이주, ESCA(East/Central/South Africa) 변이주, 아시아(Asian) 변이주, 서아프리카 계통(West Africa lineage) 변이주로 총 4 종류의 변이주가 존재하는 것으로 알려져 있다. 최근의 대유행에서 나타난 인도양(Indian ocean) 변이주는 이전의 서아프리카 계통과 비교할 때 E1 단백질 아미노산의 치환으로 인해 흰줄숲모기(Aedes albopictus)에 대한 감염력이 증가하는 한편, 아시아 변이주는 흰줄 숲모기 감염에 저항성을 나타내도록 변이된 것으로 밝혀졌다.From the first report of chikungunya virus infection in 1952 to the pandemic of the 2000s, various CHIKV mutants were isolated from clinical patients.According to the phylogenic tree based on the genome sequence, chikungunya virus (Indian ocean) mutant, ESCA (East/Central/South Africa) mutant, Asian mutant, and West Africa lineage mutant are known to exist in total of four types. The Indian ocean mutant strain, which appeared in the recent pandemic, increased infectivity against Aedes albopictus due to the substitution of the E1 protein amino acid compared to the previous West African strain, while the Asian mutant strain was more susceptible to infection by the Aedes albopictus. It was found to have been mutated to show resistance.
이러한 치쿤군야 바이러스 유행을 예방하기 위한 대비가 필요하다. 특히, 다양한 변이주에 대한 표준항원의 개발은 치쿤군야 바이러스의 예방, 진단 또는 치료에 매우 효과적일 것이다. 또한, 검출, 치료 등을 위해 대량생산이 가능한 시스템을 구축할 수 있다면, 치쿤군야 바이러스의 유행에 효과적으로 대비할 수 있을 것이다.It is necessary to prepare to prevent the chikungunya virus outbreak. In particular, the development of standard antigens for various mutant strains will be very effective in preventing, diagnosing, or treating chikungunya virus. In addition, if a system capable of mass production can be built for detection and treatment, it will be possible to effectively prepare for the outbreak of the chikungunya virus.
상기한 치쿤군야 바이러스 감염에 의한 문제를 신속히 해결하고, 의료 산업분야에서 효과적인 대비책을 마련하기 위한 목적으로, 본 발명은 다양한 변이주에 대한 항원 및 이에 의한 항원을 제공한다. 본 발명의 발명자는 본 발명의 신규 에피토프가 치쿤군야 바이러스에 대한 중화항체를 유도할 수 있고, 항체에 의하여 주요하게 인식되는 항원 부위임을 밝혔고, 당 수식 부위를 포함하지 않아 대장균 시스템을 이용에 유리하고 수용성을 가져 대량생산 시스템에 적합함을 확인하여 본 발명을 완성하였다. For the purpose of quickly solving the problem caused by the above-described chikungunya virus infection and to prepare an effective countermeasure in the medical industry, the present invention provides antigens for various mutant strains and antigens thereby. The inventors of the present invention revealed that the novel epitope of the present invention can induce a neutralizing antibody against the chikungunya virus, and is an antigenic site that is mainly recognized by the antibody. Since it does not contain a sugar modification site, it is advantageous to use the E. coli system. The present invention was completed by confirming that it has water solubility and is suitable for mass production systems.
따라서, 본 발명은 신규 에피토프 및 이를 이용한 다양한 치쿤군야 바이러스 변이주에 대한 예방 및/또는 치료 효과를 갖는 중화항체의 제공, 또는 신규 에피토프를 포함하는 재조합 단백질 또는 이의 항체를 이용한 백신, 진단키트, 감염 진단 등을 위한 용도를 제공하는 것을 그 목적으로 한다.Accordingly, the present invention provides a novel epitope and a neutralizing antibody having a prophylactic and/or therapeutic effect against various chikungunya virus mutants using the same, or a vaccine, diagnostic kit, and infection diagnosis using a recombinant protein or antibody thereof containing a novel epitope. Its purpose is to provide a use for, etc.
본 발명은 상기한 목적을 달성하기 위하여, 치쿤군야 바이러스의 신규한 에피토프를 제공한다.The present invention provides a novel epitope of chikungunya virus in order to achieve the above object.
이와 관련하여, 본 발명은 치쿤군야 바이러스에 대한 중화항체 유도능을 갖는 서열번호 1 또는 서열번호 2의 아미노산 서열로 표시되는 폴리펩티드를 제공한다.In this regard, the present invention provides a polypeptide represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 having the ability to induce neutralizing antibodies against Chikungunya virus.
본 발명은 또한 상기 폴리펩티드를 코딩하는 폴리뉴클레오티드를 제공한다.The present invention also provides a polynucleotide encoding the polypeptide.
본 발명은 또한 상기 폴리뉴클레오티드를 포함하는 재조합 발현 벡터를 제공한다.The present invention also provides a recombinant expression vector comprising the polynucleotide.
본 발명은 또한 상기 재조합 발현벡터를 포함하는 형질전환체를 제공한다.The present invention also provides a transformant comprising the recombinant expression vector.
본 발명은 또한 상기 형질전환체를 배양하는 것을 포함하는, 재조합 단백질 생산방법을 제공한다.The present invention also provides a method for producing a recombinant protein comprising culturing the transformant.
본 발명은 또한 서열번호 1 또는 서열번호 2의 아미노산 서열로 표시되는 에피토프에 특이적으로 결합하는 치쿤군야 바이러스 특이적 항체를 제공한다.The present invention also provides a chikungunya virus specific antibody that specifically binds to the epitope represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
본 발명은 또한 서열번호 1 또는 서열번호 2의 아미노산 서열로 표시되는 에피토프를 포함하는 치쿤군야 재조합 단백질을 인간을 제외한 동물에 주입하는 것을 포함하는, 치쿤군야 바이러스 특이적 항체를 생산하는 방법을 제공한다.The present invention also provides a method for producing a chikungunya virus-specific antibody, comprising injecting a chikungunya recombinant protein comprising an epitope represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 into an animal other than humans. .
본 발명은 또한 서열번호 1의 아미노산 서열로 표시되는 에피토프를 포함하는 재조합 단백질 또는 서열번호 1의 아미노산 서열로 표시되는 에피토프에 특이적으로 결합하는 항체를 포함하는 치쿤군야 바이러스 검출용 조성물을 제공한다.The present invention also provides a composition for detecting Chikungunya virus comprising a recombinant protein comprising an epitope represented by the amino acid sequence of SEQ ID NO: 1 or an antibody that specifically binds to the epitope represented by the amino acid sequence of SEQ ID NO: 1.
본 발명은 또한 서열번호 1의 아미노산 서열로 표시되는 에피토프를 포함하는 재조합 단백질 또는 서열번호 1의 아미노산 서열로 표시되는 에피토프에 특이적으로 결합하는 항체를 포함하는 치쿤군야 바이러스 검출용 키트를 제공한다.The present invention also provides a kit for detecting a chikungunya virus comprising a recombinant protein comprising an epitope represented by the amino acid sequence of SEQ ID NO: 1 or an antibody specifically binding to the epitope represented by the amino acid sequence of SEQ ID NO: 1.
본 발명은 또한 서열번호 1 또는 서열번호 2의 아미노산 서열로 표시되는 에피토프에 특이적으로 결합하는 항체와 시료를 접촉시키는 것을 포함하는, 시료에서 치쿤군야 바이러스를 검출하는 방법을 제공한다.The present invention also provides a method for detecting chikungunya virus in a sample, comprising contacting the sample with an antibody that specifically binds to the epitope represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
본 발명은 또한 서열번호 1 또는 서열번호 2의 아미노산 서열로 표시되는 에피토프에 특이적으로 결합하는 항체를 포함하는 치쿤군야 바이러스 감염 치료용 조성물을 제공한다.The present invention also provides a composition for treating chikungunya virus infection comprising an antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
본 발명은 또한 제1항의 폴리펩티드를 포함하는 치쿤군야 바이러스에 대한 백신 조성물을 제공한다.The present invention also provides a vaccine composition against chikungunya virus comprising the polypeptide of
본 발명의 치쿤군야 바이러스에 대한 신규한 에피토프는 다양한 치쿤군야 바이러스 변이주의 대하여 표준항원으로 이용될 수 있어, 다양한 변이주의 바이러스감염 여부 진단, 예방 또는 치료 효과를 제공할 수 있고, 수용성 특성을 가지며, 대장균 시스템에서 재조합 단백질 생산에 적합하므로, 본 발명의 에피토프를 포함하는 재조합 단백질을 이용한 백신 및 항체의 제조가 용이하다. 따라서, 치쿤군야 바이러스 검출 키트, 백신 및 항체 등과 같은 치쿤군야 바이러스의 예방 및 치료를 위한 산업분야에서 매우 효과적으로 활용될 수 있다.The novel epitope for the chikungunya virus of the present invention can be used as a standard antigen for various chikungunya virus mutants, so that it can diagnose, prevent or treat viral infection of various mutants, and has water-soluble properties, Since it is suitable for the production of recombinant proteins in the E. coli system, it is easy to prepare vaccines and antibodies using the recombinant protein containing the epitope of the present invention. Therefore, it can be very effectively utilized in the industrial field for the prevention and treatment of chikungunya virus such as chikungunya virus detection kit, vaccine and antibody.
도 1은 치쿤군야 바이러스의 변이주에 대한 유전적 관계도를 보여준다.
도 2는 본 발명의 일 실시예에 따라 알파바이러스에 대한 범용 중화항체(CHK-48, CHK-88, CHK-124, CHK-265, CHK-65, CHK-98, CHK-187)들의 항원 인지부위를 맵핑한 결과를 나타낸다: 치쿤군야 바이러스(CHKV-LR2006_CPY1), O'nyong'nyong 바이러스(ONNV-IBH10964), Se mliki Forest 바이러스(SFV-L10), Mayaro 바이러스(MAYV-MAYLC) 및 Ross river 바이러스(RRV-T48).
도 3a 및 3b는 치쿤군야 바이러스-단일 클론항체 복합체의 구조를 토대로 항원-항체 인터페이스의 표면 잔기 보존성을 구조생물학을 기반으로 분석한 결과이다.
도 4a, 4b 및 4c는 본 발명의 일 실싱예 따라 E2B-6xHis의 발현 테스트 결과를 나타내는 사진이다: 4a는 전기영동 SDS 젤 사진; 4b는 anti-6xHis 항체를 이용한 웨스턴블롯 결과; 4c는 E2B_6xHis_OVA의 발현테스트 결과, 도면에서 T은 전체(total) 레인, S는 상층액(supernatant) 레인, P는 펠렛(pellet) 레인을 의미한다.
도 5는 본 발명의 일 실시예 따라 설계한 E2 단백질 전체를 포함하는 항원 E2L-OVA와 E2Y-OVA의 구조단백질에서 위치 및 단백질의 특성을 보여준다.
도 6a 및 6b는 본 발명의 일 실시예 따라 E2L-OVA와 E2Y-OVA 단백질의 E.coli 시스템을 이용한 발현을 확인한 SDS-폴리아크릴아마이드 젤 전기영동 결과로, 6a는 E2L-OVA의 대장균 시스템에서 발현을 확인한 것이고, 도 6은 E2Y-OVA의 대장균 시스템에서 발현을 확인한 결과이다. 도면에서 T은 전체(total) 레인, S는 상층액(supernatant) 레인, P는 펠렛(pellet) 레인을 의미한다.
도 7a, 7b 및 7c는 본 발명의 일 실시예 따라 E2B 단백질의 정제 과정 및 결과를 나타내는 것으로, 7a는 친화도 크로마토그래피; 7b는 젤 여과 크로마토그래피(Gel filtration chromatography) 그리고 7c는 SCS-PAGE를 통해 확인한 최종 순도를 나타낸다.
도 8a 및 8b는 본 발명의 일 실시예 따라 E2B_-OVA의 정제 과정을 보여주는 것으로, 8a는 친화도 크로마토그래피; 8b는 젤 여과 크로마토그래피 결과를 나타낸다.
도 9a 및 9b는 본 발명의 일 실시예 따라 E2L-OVA의 정제 과정을 보여주는 것으로, 9a는 친화도 크로마토그래피; 9b는 젤 여과 크로마토그래피 결과를 나타낸다.
도 10은 본 발명의 일 실시예 따라 E2Y-OVA의 정제 과정을 보여주는 친화도 크로마토그래피 결과를 나타낸다.
도 11a, 11b, 11c 및 11d는 본 발명의 일 실시예 따른 E2B-6xHis-OVA, E2B-6xHis, E2Y-6xHis-OVA, E2L-6xHis-OVA 재조합 단백질의 대장균 시스템을 이용한 생산 결과를 나타낸다.
도 12는 본 발명의 일 실시예에 따른 8종의 치쿤군야 바이러스 La reunion_05-115, India_KR51, DakArB16878, ugAg4155, SV0444, 070GaTv, ibH35 및 37997의 구조단백질의 다중서열배치(multiple sequence alignment) 결과를 나타낸다.
도 13은 본 발명의 일 실시예에 따라 생산된 치쿤군야 바이러스의 E2 구조단백질의 아미노산 서열과 실제 발현되는 E2B-6xHis 서열을 나타낸 것이고, B 도메인 부분은 빨간색으로 나타내었다.
도 14는 본 발명의 E2B 표준 항원에 대한 항체 제작을 위한 접종 일정을 나타낸 것이다.
도 15a 및 15b는 본 발명의 일 실시예에 따른 플라크감소 중화항체가 측정법의 유효성을 검증하기 위해 치쿤군야 바이러스 중화항체(chk-265, Absolute Antibody Ltd. Oxford, UK)를 이용한 중화항체가를 확인한 결과이다.
도 16은 본 발명의 일 실시예에 따른 E2B 표준 항원을 접종한 마우스의 혈장을 이용해서 본 발명의 일 실시예에서 마우스에 유도된 E2B에 대한 항체의 중화능을 확인한 결과이다.
도 17은 본 발명의 일 실시예에 따라 E2B, E2B-OVA, E2L-OVA, E2Y-OVA 4종의 치쿤군야 바이러스 항원을 주입한 마우스의 혈장 샘플을 이용한 항원-항체 반응성을 확인한 결과를 나타낸다.
도 18은 본 발명의 일 실시예에 따라 E2B, E2B-OVA, E2L-OVA, E2Y-OVA 4종의 치쿤군야 바이러스 항원에 대한 항체의 반응성을 확인한 결과이다.
도 19는 본 발명의 일 실시예에 따라 따라 E2B, E2B-OVA, E2L-OVA, E2Y-OVA 4 종의 치쿤군야 바이러스 항원을 주입한 마우스의 혈장 샘플에 포함된 항체에 대한 E2B 항원의 인지 가부를 ELISA로 확인한 결과이다.1 shows a genetic relationship diagram of the mutant strain of the chikungunya virus.
2 is an antigen recognition of universal neutralizing antibodies (CHK-48, CHK-88, CHK-124, CHK-265, CHK-65, CHK-98, CHK-187) against an alpha virus according to an embodiment of the present invention. The results of mapping the sites are shown: Chikungunya virus (CHKV-LR2006_CPY1), O'nyong'nyong virus (ONNV-IBH10964), Se mliki Forest virus (SFV-L10), Mayaro virus (MAYV-MAYLC) and Ross river virus. (RRV-T48).
3A and 3B are the results of analyzing the surface residue conservancy of the antigen-antibody interface based on the structure of the chikungunya virus-monoclonal antibody complex based on structural biology.
Figures 4a, 4b and 4c are photographs showing the results of the expression test of E2B-6xHis according to one example of the present invention: 4a is a photograph of an electrophoretic SDS gel; 4b is a Western blot result using anti-6xHis antibody; 4c is a result of the expression test of E2B_6xHis_OVA. In the figure, T denotes a total lane, S denotes a supernatant lane, and P denotes a pellet lane.
FIG. 5 shows the location and protein properties of the structural proteins of the antigens E2L-OVA and E2Y-OVA including the entire E2 protein designed according to an embodiment of the present invention.
6A and 6B are SDS-polyacrylamide gel electrophoresis results confirming the expression of E2L-OVA and E2Y-OVA proteins using the E.coli system according to an embodiment of the present invention, 6a is a result of E2L-OVA in the E. coli system. The expression was confirmed, and FIG. 6 is a result of confirming the expression in the E2Y-OVA E. coli system. In the figure, T denotes a total lane, S denotes a supernatant lane, and P denotes a pellet lane.
7a, 7b and 7c show the purification process and results of the E2B protein according to an embodiment of the present invention, 7a is affinity chromatography; 7b denotes gel filtration chromatography and 7c denotes the final purity confirmed through SCS-PAGE.
Figures 8a and 8b show the purification process of E2B_-OVA according to an embodiment of the present invention, 8a is affinity chromatography; 8b shows the results of gel filtration chromatography.
Figures 9a and 9b show the purification process of E2L-OVA according to an embodiment of the present invention, 9a is affinity chromatography; 9b shows the results of gel filtration chromatography.
10 shows the results of affinity chromatography showing the purification process of E2Y-OVA according to an embodiment of the present invention.
Figures 11a, 11b, 11c and 11d show the production results of E2B-6xHis-OVA, E2B-6xHis, E2Y-6xHis-OVA, E2L-6xHis-OVA recombinant proteins using the E. coli system according to an embodiment of the present invention.
FIG. 12 shows the results of multiple sequence alignment of structural proteins of 8 types of chikungunya viruses La reunion_05-115, India_KR51, DakArB16878, ugAg4155, SV0444, 070GaTv, ibH35 and 37997 according to an embodiment of the present invention. .
13 shows the amino acid sequence of the E2 structural protein of the chikungunya virus produced according to an embodiment of the present invention and the actually expressed E2B-6xHis sequence, and the B domain portion is shown in red.
14 shows the inoculation schedule for the production of antibodies against the E2B standard antigen of the present invention.
15A and 15B show the neutralizing antibody titer using a chikungunya virus neutralizing antibody (chk-265, Absolute Antibody Ltd. Oxford, UK) to verify the effectiveness of the plaque-reducing neutralizing antibody titer according to an embodiment of the present invention. It is the result.
16 is a result of confirming the neutralizing ability of an antibody against E2B induced in mice in an embodiment of the present invention using plasma of a mouse inoculated with an E2B standard antigen according to an embodiment of the present invention.
FIG. 17 shows the results of confirming antigen-antibody reactivity using plasma samples of mice injected with E2B, E2B-OVA, E2L-OVA, and E2Y-OVA four chikungunya virus antigens according to an embodiment of the present invention.
FIG. 18 is a result of confirming the reactivity of antibodies against four types of chikungunya virus antigens E2B, E2B-OVA, E2L-OVA, and E2Y-OVA according to an embodiment of the present invention.
FIG. 19 shows whether or not E2B antigens were recognized for antibodies contained in plasma samples of mice injected with E2B, E2B-OVA, E2L-OVA, and E2Y-OVA four kinds of chikungunya virus antigens according to an embodiment of the present invention. Is the result of confirming by ELISA.
본 발명의 발명자들은 치쿤군야 바이러스의 다양한 변이주에 적용 가능한 표준항원 및 이에 대한 항체의 제작을 위하여 노력한 결과, 중화능을 유발할 수 있는 최소 범위의 항원 부위로 E2 단백질의 B 도메인을 특정하고, 치쿤군야 바이러스의 진단 및 치료에 매우 중요한 역할을 하는 에피토프임를 포함하는 재조합 단백질을 대량 생산할 수 있는 기술을 확보하여, 본 발명을 완성하였다. The inventors of the present invention have made efforts to produce a standard antigen applicable to various mutant strains of chikungunya virus and antibodies thereto. As a result, the B domain of the E2 protein was specified as a minimum range of antigen sites capable of inducing neutralizing ability, The present invention has been completed by securing a technology capable of mass-producing a recombinant protein containing an epitope that plays a very important role in diagnosis and treatment of viruses.
본 발명은 신규하게 규명된 에피토프를 이용하여 다양한 변종에 대해서 동시에 검출, 예방 및 치료효과를 갖는 표준항원으로서 재조합단백질 및 이에 대한 항체, 상기 항체 또는 재조합 단백질을 포함하는 바이러스 검출용 조성물 또는 키트, 그리고 이를 이용한 바이러스 검출방법 또는 항체 제조방법을 제공할 수 있다. The present invention uses a newly identified epitope to simultaneously detect, prevent, and treat various strains as standard antigens, a recombinant protein and an antibody therefor, a composition or kit for virus detection comprising the antibody or recombinant protein, and A virus detection method or an antibody production method using this can be provided.
또한, 본 발명은 상기 재조합 단백질 생산을 위한 표준항원을 코딩하는 폴리뉴클레오티드, 이를 포함하는 재조합 벡터, 이를 포함하는 형질전환체 그리고, 상기 형질전환체를 이용한 치쿤군야 항원 재조합 단백질 생산방법을 제공할 수 있다. In addition, the present invention can provide a polynucleotide encoding a standard antigen for the production of the recombinant protein, a recombinant vector containing the same, a transformant containing the same, and a method for producing a recombinant Chikungunya antigen protein using the transformant. have.
이하에서, 본 발명의 에 대하여 상세히 설명한다.Hereinafter, the present invention will be described in detail.
다만, 본 발명은 다양한 변경을 가할 수 있고 여러 가지 형태를 가질 수 있는바, 이하에서 기술하는 특정 실시예 및 설명은 본 발명의 이해를 돕기 위한 것일 뿐, 본 발명을 특정한 개시 형태에 대해 한정하려는 것이 아니다. 본 발명의 범위는 본 발명의 사상 및 기술 범위에 포함되는 모든 변경, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다.However, since the present invention can be modified in various ways and has various forms, specific examples and descriptions described below are only to aid understanding of the present invention, and to limit the present invention to a specific disclosure form. It is not. It is to be understood that the scope of the present invention includes all changes, equivalents, and substitutes included in the spirit and scope of the present invention.
본 발명은 치쿤군야 바이러스의 신규한 에피토프에 관한 것이다.The present invention relates to a novel epitope of chikungunya virus.
치쿤군야 바이러스(Chikungunya virus, CHIKV)는 양성단일가닥 RNA(+ssRNA)를 게놈(genome)으로 갖는 토가바이러스과(Togaviridae)로 분류되는 알파바이러스(Alphavirus)이며 모기를 매개로 하는 아보바이러스(arbovirus)에 속한다. 바이러스 입자의 크기는 지름 60-70 nm 정도의 크기로 외피보유(enveloped) 바이러스이며, 바이러스 게놈은 11.6 kb 뉴클레오타이드의 양성단일가닥RNA(+ssRNA) 형태로 5'말단 캡(5'-cap)과 3'말단 폴리(A)꼬리(3'-poly(A)tail)의 구조를 가지며 4개의 비구조단백질(nsP1-nsP4)과 4개의 구조단백질(캡시드와 3종의 외피단백질)을 암호화하여 2종의 폴리단백질(polyprotein)을 생산하는 특성을 갖는다. 치쿤군야 바이러스의 구조단백질은 캡시드(C)와 외피단백질인 E1, E2, E3을 포함하고, E1과 E2의 이질이합체(heterodimer)가 숙주세포 부착에 있어 핵심적인 역할을 담당하는 것으로 알려져 있다. 구체적으로, E2 단백질은 숙주세포의 수용체와 결합하여 바이러스의 내포작용을 유도하며, E1 단백질은 낮은 pH 조건에서 숙주세포와의 융합 (fusion)에 관여한다(J Viol 2013 7:3774).Chikungunya virus (CHIKV) is an alphavirus classified as Togaviridae that has a positive single-stranded RNA (+ssRNA) as its genome, and is an arbovirus that mediates mosquitoes. Belongs to. The size of the virus particle is about 60-70 nm in diameter and is an enveloped virus, and the viral genome is in the form of a positive single-stranded RNA (+ssRNA) of 11.6 kb nucleotides with a 5'end cap (5'-cap). It has the structure of a 3'-poly(A)tail, and encodes 4 non-structural proteins (nsP1-nsP4) and 4 structural proteins (capsid and 3 types of envelope proteins) It has the property of producing polyprotein of. The structural proteins of the chikungunya virus include capsid (C) and envelope proteins E1, E2, and E3, and heterodimers of E1 and E2 are known to play a key role in host cell adhesion. Specifically, E2 protein binds to host cell receptors and induces viral inclusion, and E1 protein is involved in fusion with host cells under low pH conditions (J Viol 2013 7:3774).
치쿤군야 바이러스는 1248개의 아미노산으로 구성된 구조 단백질을 가지고 있는데, 이 단일 구조단백질에서 절단되어 캡시드(C)와 외피 당단백질인 E1, E2, E3를 형성하며, E1-E2 이형이합체(heterodimer)는 바이러스의 외피 바깥 부위에 노출되어 숙주세포 부착에 관여하고, 캡시드는 E1-E2 이형이합체의 짧은 외피 안쪽 도메인과 상호작용하며 바이러스 게놈을 안정적으로 바이러스 입자에 패키징시키는 역할을 한다. 이러한 치쿤군야 바이러스의 구조 단백질에서 E2 단백질이 항원 부위로 예상되었으나, 구체적으로 중화항체 유도능을 갖는 에피토프에 대해서는 아직까지 밝혀진 바 없다. 특히 치쿤군야 바이러스는 4종의 변이종에 대해서 복수 개의 종이 포함되어 있어, 치쿤군야 바이러스에 대해 특이적이면서도, 각 변이종에 범용적으로 사용될 수 있는 표준항원은 아직 개발되지 못하였다. 더욱이 E2 단백질은 다양한 당 수식 부위를 포함하고 있기 때문에, 대장균 시스템을 통해서 항원성 또는 항체 유도능이 유지되는 재조합 단백질을 생산하는데 어려움이 있었다. Chikungunya virus has a structural protein composed of 1248 amino acids, which is cleaved from this single structural protein to form the capsid (C) and envelope glycoproteins E1, E2, E3, and the E1-E2 heterodimer is a virus. It is exposed to the outer part of the outer coat of the cell and is involved in host cell adhesion, and the capsid interacts with the short inner coat domain of the E1-E2 heterodimer and serves to stably package the viral genome into the viral particle. E2 protein was expected to be an antigenic site in the structural protein of the chikungunya virus, but specifically, an epitope having neutralizing antibody-inducing ability has not yet been identified. In particular, since the chikungunya virus contains multiple species for the four mutant species, a standard antigen that is specific to the chikungunya virus and can be used universally for each mutant has not yet been developed. Moreover, since the E2 protein contains various sugar-modifying sites, it was difficult to produce a recombinant protein that maintains antigenicity or antibody-inducing ability through the E. coli system.
이러한 측면에서 본 발명에서 새롭게 밝힌 서열번호 1의 치쿤군야 바이러스의 신규한 에피토프는 바이러스가 인지할 수 있는 최소한의 중화항체 항원부위를 밝혔다는 점에서 특징이 있다. 특히, 서열번호 1의 항원 부위에는 당수식이 일어나지 않아, 대장균 시스템을 통한 재조합 단백질 대량 생산이 가능하며, 가용성(soluble) 단백질을 생산할 수 있다는 점에서 재조합 단백질, 항체, 키트 및 백신 등의 생산 측면에서 우수한 효과를 가지며, 특히, 감염질환에 대한 의료산업 분야의 신속한 대응을 위한 대량생산에 매우 유리하다.In this respect, the novel epitope of the chikungunya virus of SEQ ID NO: 1 newly identified in the present invention is characterized in that it reveals the least neutralizing antibody antigen site that the virus can recognize. In particular, in terms of production of recombinant proteins, antibodies, kits, vaccines, etc., in that the antigenic site of SEQ ID NO: 1 does not undergo sugar formula, mass production of recombinant proteins is possible through the E. coli system, and soluble proteins can be produced. It has an excellent effect, and is particularly advantageous for mass production for rapid response to infectious diseases in the medical industry.
또한, 본 발명의 일 실시예에서, 다양한 부위의 항원 단백질을 이용해서 유도된 항체를 이용해서 본 발명의 E2B 항원 단백질과의 항원-항체 반응성을 확인한 결과, 다양한 항원에 대한 항체가 매우 우수한 효율로 본 발명의 E2B 단백질을 인지하여 검출할 수 있음을 보였다. 이는, 본 발명의 E2B 항원 단백질은 치쿤군야 바이러스 항체의 매우 중요한 인식부위이면서, 잘 인식할 수 있는 부위로소, 치쿤군야 바이러스의 감염 진단 등에서 높은 검출 효율을 갖는다는 것을 의미한다. In addition, in one embodiment of the present invention, as a result of confirming the antigen-antibody reactivity with the E2B antigen protein of the present invention using antibodies derived using antigenic proteins of various sites, antibodies to various antigens are highly efficient. It was shown that the E2B protein of the present invention can be recognized and detected. This means that the E2B antigen protein of the present invention is a very important recognition site for the chikungunya virus antibody, as well as a well recognizable site, and has high detection efficiency in diagnosis of infection of the chikungunya virus.
이러한 측면에서 본 발명은 치쿤군야 바이러스에 대한 중화항체 유도능을 갖는 서열번호 1 또는 서열번호 2의 아미노산 서열로 표시되는 폴리펩티드 에 관한 것이다. 상기 폴리펩티드는 재조합 단백질일 수 있다. In this aspect, the present invention relates to a polypeptide represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 having the ability to induce neutralizing antibodies against chikungunya virus. The polypeptide may be a recombinant protein.
상기 재조합 단백질은 서열번호 1의 아미노산 서열의 일 말단에 히스티딘 표지(Histidine tag)를 더 포함할 수 있다. 이 경우, 재조합 단백질 생산에 있어, 단백질의 분리/정제가 용이한 장점이 있다. 바람직하게는 상기 히스티딘 표지를 2개 이상, 포함할 수 있다. 상기 히스티딘 표지는 본 발명의 항원 단백질을 발현하는 벡터시스템 제작 시 항원단백질 코딩하는 유전자 서열의 일 말단에 서열번호 15의 히스티딘 코딩하는 서열을 반복적으로 포함시켜, 항원단백질에 히스티딘 표지가 포함되는 형태로 발현될 수 있다. The recombinant protein may further include a histidine tag at one end of the amino acid sequence of SEQ ID NO: 1. In this case, in the production of a recombinant protein, there is an advantage of easy separation/purification of the protein. Preferably, two or more histidine labels may be included. The histidine label is a form in which the histidine label is included in the antigen protein by repeatedly including the histidine-coding sequence of SEQ ID NO: 15 at one end of the gene sequence encoding the antigen protein when constructing the vector system expressing the antigen protein of the present invention. Can be expressed.
본 발명의 서열번호 1 또는 서열번호 2의 재조합 단백질을 이용하면 치쿤군야 바이러스에서 변이주에 범용적으로 적용될 수 있는 항체를 제조할 수 있고, 특히, 중화능을 갖는 항체를 유도할 수 있다는 점에서 치쿤군야 바이러스의 치료 및 예방 측면에서 배우 매우 의미있다. By using the recombinant protein of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention, an antibody that can be universally applied to a mutant strain in Chikungunya virus can be prepared, and in particular, an antibody having neutralizing ability can be induced. The learning in terms of treatment and prevention of the military virus is very meaningful.
이러한 측면에서 본 발명은 서열번호 1 또는 서열번호의 아미노산 서열로 표시되는 에피토프 또는 상기 에피토프를 포함하는 재조합 단백질을 코딩하는 폴리뉴클레오티드; 상기 폴리뉴클레오티드를 포함하는 재조합 발현 벡터; 그리고 상기 재조합 발현벡터를 포함하는 형질전환체에 관한 것이다. In this aspect, the present invention provides a polynucleotide encoding an epitope represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: or a recombinant protein comprising the epitope; A recombinant expression vector comprising the polynucleotide; And it relates to a transformant comprising the recombinant expression vector.
단, 본 발명에서 상기 "폴리뉴클레오티드"란, 뉴클레오티드 단위체(monomer)가 공유결합에 의해 길게 사슬 모양으로 이어진 뉴클레오티드의 중합체(polymer)로 일정한 길이 이상의 DNA 또는 RNA 가닥으로서, 본 발명에 따른 재조합 단백질을 코딩하는 폴리뉴클레오티드를 의미한다. 본 발명의 상세한 설명 전체에서, 상기 폴리뉴클레오티드는 유전자와 상호교환적으로 사용된다. However, in the present invention, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide units are connected in a long chain by covalent bonds, and is a DNA or RNA strand of a certain length or longer, wherein the recombinant protein according to the present invention is used. It means an encoding polynucleotide. Throughout the detailed description of the invention, the polynucleotides are used interchangeably with genes.
본 발명에 따른 재조합 단백질을 코딩하는 폴리뉴클레오티드는 코돈의 축퇴성(degeneracy), 또는 상기 단백질을 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여, 코딩영역으로부터 발현되는 재조합 단백질의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩영역에 다양한 변형이 이루어질 수 있고, 코딩영역을 제외한 부분에서도 유전자의 발현에 영향을 미치지 않는 범위 내에서 다양한 변형 또는 수식이 이루어질 수 있으며, 상기 변형 유전자 역시 본 발명의 보호범위 내에 포함된다.The polynucleotide encoding the recombinant protein according to the present invention does not change the amino acid sequence of the recombinant protein expressed from the coding region in consideration of the codon degeneracy or the preferred codon in the organism to express the protein. Various modifications may be made to the coding region within a range that does not affect the expression of the gene, and the modified gene may also be made within the protection scope of the present invention. Included.
본 발명의 또 다른 구현 예에서는 본 발명에 따른 상기 폴리뉴클레오티드를 포함하는 발현 벡터를 제공한다. 바람직하게는 상기 발현 벡터는 서열번호 2의 염기서열로 표시되는 유전자 절편을 포함하는 것일 수 있으나, 이에 제한되는 것은 아니다.Another embodiment of the present invention provides an expression vector comprising the polynucleotide according to the present invention. Preferably, the expression vector may include a gene fragment represented by the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.
본 발명에 있어서 상기 "발현 벡터"란, 숙주세포에 DNA를 도입하여 본 발명의 재조합 단백질을 미생물에서 발현시키기 위한 수단으로서, 상기 발현 벡터의 제작 시에는 상기 재조합 단백질을 생산하고자 하는 숙주세포의 종류에 따라 프로모터 (promoter), 종결자 (terminator), 인핸서 (inhancer) 등과 같은 발현 조절 서열 및 막 표적화 또는 분비를 위한 서열 등을 적절히 선택하고 목적에 따라 다양하게 조합할 수 있다.In the present invention, the "expression vector" refers to a means for expressing the recombinant protein of the present invention in a microorganism by introducing DNA into a host cell, and when the expression vector is produced, the type of host cell to be used to produce the recombinant protein Depending on the expression control sequences such as promoters, terminators, enhancers, etc., and sequences for membrane targeting or secretion, etc. may be appropriately selected and variously combined according to the purpose.
본 발명에서 발현 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오 파아지 벡터, 및 바이러스 벡터 등을 포함할 수 있다. 적합한 발현 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 발현 벡터의 프로모터는 구성적 또는 유도성일 수 있다. In the present invention, the expression vector may include a plasmid vector, a cozmid vector, a bacteriophage vector, and a viral vector. Suitable expression vectors include, in addition to expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals and enhancers, signal sequences or leader sequences for membrane targeting or secretion, and can be variously prepared according to the purpose. The promoter of the expression vector can be constitutive or inducible.
상기 발현 벡터는 벡터를 포함하는 숙주 세포를 선택하기 위한 선택 마커를 포함할 수 있고, 이는 예를 들어, 발현벡터는 엠피실린 저항 유전자, 테트라사이클린 저항 유전자, 가나마이신 저항 유전자, 클로람페니콜 저항 유전자 등 일 수 있으나, 이에 한정되는 것은 아니다. The expression vector may include a selection marker for selecting a host cell containing the vector, for example, the expression vector is an ampicillin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, etc. However, it is not limited thereto.
상기 재조합 발현 벡터는 당업계에서 통상적으로 이용되는 벡터를 이용할 수 있는데, 예를 들어 pGEM-T, pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19 또는 pET를 이용할 수 있으나, 이에 한정되는 것은 아니다. 본 발명에서 바람직한 발현벡터는 pET, 특히 pET21a 일 수 있다. The recombinant expression vector may use a vector commonly used in the art, for example, pGEM-T, pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19 or pET, but is not limited thereto. . A preferred expression vector in the present invention may be pET, particularly pET21a.
본 발명의 상기 형질전환체는 본 발명의 기술분야에서 통상적으로 이용할 수 있는 숙주세포를 모두 포함할 수 있고, 예를 들어 대장균(E- coli), 바실러스 서브틸리스, 바실러스 츄린겐시스와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움, 세라티아 마르세슨스 및 다양한 슈도모나스 종과 같은 장내균과 균주를 이용할 수 있다. 상기 대장균은 예를 들어, E. coli JM109, E. coli BL21(DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, E. coli Rosetta 또는 E. coli BL21 일 수 있다. The transformant of the present invention may include all host cells commonly used in the technical field of the present invention, for example, Bacillus such as E- coli , Bacillus subtilis, and Bacillus thuringensis. Genus strains, and enterobacteriaceae strains such as Salmonella typhimurium, Serratia marsessons, and various Pseudomonas species can be used. The E. coli is, for example, E. coli JM109, E. coli BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, E. coli Rosetta Or E. coli BL21.
본 발명의 바람직한 실시예에서는 대장균에 발현 벡터를 삽입하여, 유전자를 발현시켜 본 발명의 재조합 단백질을 생산하였다. 특히, 본 발명의 서열반호 1 또는 서열번호 2의 폴리펩티드는 당 수식 부위를 포함하지 않기 때문에 대장균 시스템을 통해서 매우 효과적으로 생산될 수 있다. 본 발명의 일 실시예에서는 대장균 시스템으로 생산된 재조합 단백질을 투여한 마우스 혈장에 포함된 항체가 중화능을 가짐을 실험적으로 확인했다.In a preferred embodiment of the present invention, an expression vector was inserted into E. coli to express the gene to produce the recombinant protein of the present invention. In particular, since the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention does not contain a sugar modification site, it can be produced very effectively through the E. coli system. In one embodiment of the present invention, it was experimentally confirmed that the antibody contained in mouse plasma to which the recombinant protein produced by the E. coli system was administered has neutralizing ability.
본 발명에서 발현은 당업계에 공지된 통상의 분자생물학 방법을 이용하여 실시할 수 있다. Expression in the present invention can be carried out using conventional molecular biology methods known in the art.
본 발명은 또한 상기 형질전환체를 배양하는 것을 포함하는, 재조합 단백질 생산방법에 관한 것이다. The present invention also relates to a method for producing a recombinant protein, comprising culturing the transformant.
본 발명의 실시예에서, 서열번호 1 또는 서열번호 2의 아미노산 서열로 표시되는 치쿤군야 바이러스의 신규 에피토프를 코딩하는 유전자를 증폭하기 위하여 상기 유전자의 5'-말단과 3'- 말단에 대응하는 중합효소 연쇄반응(polymerase chain reaction, PCR)용 프라이머를 고안하여 합성하였다. 유전자의 5'-말단에 대응하는 프라이머와 3'- 말단에 대응하는 프라이머에는 대장균 발현 벡터로 클로닝이 용이하도록 제한 효소 인지 부위가 존재하도록 설계하였다. E2 단백질의 유전자를 주형으로 하고, 상기와 같이 제조된 프라이머를 사용하여 PCR 반응을 수행하여 B 도메인 유전자를 증폭하였다. In an embodiment of the present invention, polymerization corresponding to the 5'-end and 3'-end of the gene to amplify a gene encoding a novel epitope of chikungunya virus represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 A primer for polymerase chain reaction (PCR) was designed and synthesized. The primers corresponding to the 5'-end of the gene and the primers corresponding to the 3'-end were designed to have a restriction enzyme recognition site to facilitate cloning into an E. coli expression vector. Using the E2 protein gene as a template, PCR reaction was performed using the primers prepared as described above to amplify the B domain gene.
당업계에서 통상적으로 이용되는 벡터, 예컨대 pET에 PCR로 증폭한 유전자를 삽입하고, 삽입 후 적합한 숙주 세포, 대장균에 형질전환시키고, 형질전환된 세포를 배양하여 본 발명의 E2B 단백질을 대량으로 발현시켰다. 단백질의 생성여부는 SDS-폴리아크릴아미드 겔 전기영동 또는 웨스턴 블롯팅 방법 등과 같은 통상적인 방법으로 확인하였다. The gene amplified by PCR was inserted into a vector commonly used in the art, such as pET, and then transformed into a suitable host cell, E. coli, and the transformed cell was cultured to express the E2B protein of the present invention in large quantities. . Whether the protein was produced was confirmed by conventional methods such as SDS-polyacrylamide gel electrophoresis or Western blotting.
일 실 시예에서, 본 발명의 서열번호 2의 에피토프의 일 말단에 6개의 히스티딘 표지가 결합된 단백질을 코딩하는 핵산서열을 포함하는 발현 벡터로 형질전환된 대장균을 엠피실린, 테트라사이클린, 카나마이신, 클로람페니콜 등 선별마커에 대응하는 항생제가 함유된 배지에서 진탕 배양하여 일정한 세포농도에 다다랐을 때, IPTG(isopropyl-β-D-galactopyranoside)를 첨가하여 E2B도메인 발현을 유도하였다. 상기 배양액을 원심분리하여 대장균 세포를 침전시킨 후, 대장균을 적당한 완충 용액에 현탁한 후 파쇄한다. 그 후 친화도 크로마토그래피와 사이즈 배제 크로마토그래피 과정을 통해 에피토프 단백질을 정제하여 순도를 높인 후 농축하여 원하는 농도의 항원을 생산하였다. In one embodiment, E. coli transformed with an expression vector containing a nucleic acid sequence encoding a protein to which six histidine labels are bound at one end of the epitope of SEQ ID NO: 2 of the present invention is used as empicillin, tetracycline, kanamycin, chloramphenicol. When the cell concentration reached a certain cell concentration by shaking culture in a medium containing antibiotics corresponding to such selection markers, isopropyl-β-D-galactopyranoside (IPTG) was added to induce E2B domain expression. After the culture solution is centrifuged to precipitate E. coli cells, E. coli is suspended in an appropriate buffer solution and then disrupted. Thereafter, the epitope protein was purified through affinity chromatography and size exclusion chromatography to increase purity, and then concentrated to produce an antigen of a desired concentration.
본 발명은 또한 서열번호 1 또는 서열번호 2의 아미노산 서열을 포함하는 치쿤군야 바이러스의 에피토프에 특이적으로 결합하는 항체와 상기 서열번호 1 또는 서열번호 2의 폴리펩티드를 포함하는 재조합 단백질을 인간을 제외한 동물에 주입하는 것을 포함하는 치쿤군야 바이러스 특이적 항체를 생산하는 방법을 제공한다. 본 발명의 서열번호 1 또는 서열번호 2의 폴리펩티드는 당 수식 부위를 포함하지 않아 대장균 시스템을 이용한 대량생산에 매우 유리하며, 이를 통해서 항체의 대량생산도 가능하게 한다는 점에서 우수한 효과를 갖는다. The present invention also provides an antibody specifically binding to an epitope of Chikungunya virus comprising the amino acid sequence of SEQ ID NO: 1 or 2 and a recombinant protein comprising the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 in animals other than humans. It provides a method of producing a chikungunya virus specific antibody comprising injecting into. The polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention does not contain a sugar modification site, so it is very advantageous for mass production using the E. coli system, and thus has an excellent effect in that it enables mass production of antibodies.
본 발명에서 항체는 특정 항원에 대하여 특이적으로 반응하는 단백질 분자를 의미하는 것으로, 특정 항원(하나의 항원 결정기)에 대해서 특이적으로 반응하는 단일클론항체 및 다중클론 항체를 모두 포함하는 의미이다. 또한, 본 발명의 항체는 바이러스의 감염능에는 전혀 변화를 주지 않는 비중화 항체와, 바이러스의 감염능을 억제, 즉 증식을 억제할 수 이는 중화항체를 모두 포함한다.In the present invention, an antibody refers to a protein molecule that specifically reacts to a specific antigen, and includes both monoclonal and polyclonal antibodies that specifically react to a specific antigen (one antigenic determinant). In addition, the antibody of the present invention includes both a non-neutralizing antibody that does not change the infectivity of the virus at all, and a neutralizing antibody capable of inhibiting the infectivity of the virus, ie, proliferation.
상기 서열번호 1의 에피토프에 의하여 유도된 항체는 바람직하게는 중화항체(neutralizing antibody) 일 수 있다. 본 발명의 일 실시예에서, 서열번호 2의 아미노산 일 말단에 6개의 히스티딘 표지가 결합된 E2B 재조합 단백질을 마우스에 접종하여, 마우스 혈액으로부터 치쿤군야 바이러스에 대한 항체가 잘 생성되었음을 확인하였다. 또한, 상기 항체의 중화능을 시험한 결과, 효과적으로 치쿤군야 바이러스의 감염가를 낮추는 중화능을 갖는 것을 확인하였다. The antibody induced by the epitope of SEQ ID NO: 1 may preferably be a neutralizing antibody. In an embodiment of the present invention, it was confirmed that an antibody against Chikungunya virus was well produced from mouse blood by inoculating mice with an E2B recombinant protein having six histidine labels bound to one end of the amino acid of SEQ ID NO: 2. In addition, as a result of testing the neutralizing ability of the antibody, it was confirmed that it has a neutralizing ability to effectively lower the infectious value of the chikungunya virus.
따라서, 본 발명의 서열번호 1 또는 2의 폴리펩티드는 치쿤군야 바이러스 감염에 대하여 예방 또는 치료 효과를 함께 제공할 수 있다는 점에서 의의를 갖는다. Therefore, the polypeptide of SEQ ID NO: 1 or 2 of the present invention is significant in that it can provide both a prophylactic or therapeutic effect against Chikungunya virus infection.
이러한 측면에서, 본 발명은 서열번호 1 또는 서열번호 2의 아미노산 서열로 표시되는 에피토프에 특이적으로 결합하는 항체를 포함하는 치쿤군야 바이러스 감염 치료용 조성물을 제공한다. 본 발명의 서열번호 1 또는 서열번호 2의 폴리펩티드는 당 수식 부위를 포함하지 않아 대장균 시스템을 이용한 대량생산에 매우 유리하며, 이를 통해서 항체의 대량생산도 가능하게 한다는 점에서 우수한 효과를 갖는다.In this aspect, the present invention provides a composition for treating chikungunya virus infection comprising an antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention does not contain a sugar modification site, so it is very advantageous for mass production using the E. coli system, and thus has an excellent effect in that it enables mass production of antibodies.
또한, 본 발명은 서열번호 1의 아미노산 서열로 표시되는 폴리펩티드를 포함하는 치쿤군야 바이러스에 대한 백신 조성물을 제공한다. 상기 백신 조성물은 치쿤군야 바이러스 감염 예방용 조성물로 표현될 수 있다. 본 발명의 서열번호 1 또는 서열번호 2의 폴리펩티드는 당 수식 부위를 포함하지 않아 대장균 시스템을 이용한 대량생산에 매우 유리하며, 이를 통해서 백신의 대량생산도 가능하게 한다는 점에서 우수한 효과를 갖는다.In addition, the present invention provides a vaccine composition against chikungunya virus comprising a polypeptide represented by the amino acid sequence of SEQ ID NO: 1. The vaccine composition may be expressed as a composition for preventing Chikungunya virus infection. The polypeptide of SEQ ID NO: 1 or 2 of the present invention does not contain a sugar modification site, so it is very advantageous for mass production using the E. coli system, and has an excellent effect in that it enables mass production of vaccines through this.
본 발명의 일 실시예에서, 서열번호 2의 아미노산 일 말단에 6개의 히스티딘 표지가 결합된 E2B 재조합 단백질을 마우스에 접종하여, 마우스 혈액으로부터 치쿤군야 바이러스에 대한 항체가 잘 생성되었음을 확인하였다. 또한, 상기 항체의 중화능을 시험한 결과, 효과적으로 치쿤군야 바이러스의 감염가를 낮추는 중화능을 갖는 것을 확인하였다.In an embodiment of the present invention, it was confirmed that an antibody against Chikungunya virus was well produced from mouse blood by inoculating mice with an E2B recombinant protein having six histidine labels bound to one end of the amino acid of SEQ ID NO: 2. In addition, as a result of testing the neutralizing ability of the antibody, it was confirmed that it has a neutralizing ability to effectively lower the infectious value of the chikungunya virus.
따라서, 본 발명의 서열번호 1 또는 서열번호 2의 폴리펩티드에 대한 항체를 이용해서 치쿤군야 바이러스 감염의 치료가 가능하며, 서열번호 1 또는 서열번호 2의 폴리펩티드를 이용해서 치쿤군야 바이러스에 대한 항체 생산의 유도가 가능하므로, 치쿤군야 바이러스 감염을 예방하기 위한 백신의 제조가 가능한다. Therefore, it is possible to treat chikungunya virus infection using an antibody against the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention, and the production of antibodies against the chikungunya virus using the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 Since induction is possible, it is possible to manufacture a vaccine to prevent infection with the chikungunya virus.
상기 치료용 조성물의 경우 약학적으로 허용되는 담체 외에, 약제학적으로 허용되는 부형제, 첨가제 등을 모두 포함할 수 있다. In the case of the therapeutic composition, in addition to a pharmaceutically acceptable carrier, all pharmaceutically acceptable excipients and additives may be included.
상기 백신 조성물의 경우 백신에 본 발명의 기술분야에서 통상적으로 이용되는 야쥬반트, 부형제 및 기타 약학적으로 허용되는 담체 또는 첨가제를 본 발명의 서열번호 1 또는 서열번호 2의 폴리펩티드의 효과에 영향을 주지 않는 범위에서 제한없이 포함할 수 있다.In the case of the vaccine composition, adjuvants, excipients, and other pharmaceutically acceptable carriers or additives commonly used in the technical field of the present invention do not affect the effect of the polypeptide of SEQ ID NO: 1 or 2 of the present invention. It can be included without limitation to the extent not limited.
또한, 상기 치료용 조성물 또는 백신 조성물의 유효성분의 농도, 투여량, 제제의 형태 등을 결정하기 위해, 본 발명의 기술분야에서 바이러스 감염 치료 제제, 항체를 포함하는 약학적 제제 또는 백신 제제의 제조 및 사용 시 적용될 수 있는 통상적인 구성 및 기술을 상기 치료용 조성물 또는 백신 조성물에 적용할 수 있다. In addition, in order to determine the concentration, dosage, form of the preparation, etc. of the active ingredient of the therapeutic composition or vaccine composition, preparation of a viral infection treatment preparation, a pharmaceutical preparation containing an antibody, or a vaccine preparation in the technical field of the present invention And conventional configurations and techniques that can be applied during use can be applied to the therapeutic composition or vaccine composition.
본 발명은 또한 서열번호 1의 아미노산 서열로 표시되는 에피토프에 특이적으로 결합하는 항체를 포함하는 치쿤군야 바이러스 검출용 조성물을 제공하는 것이다.The present invention also provides a composition for detecting chikungunya virus comprising an antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 1.
본 발명은 또한 서열번호 1의 아미노산 서열로 표시되는 에피토프에 특이적으로 결합하는 항체를 포함하는 치쿤군야 바이러스 검출용 키트를 제공하는 것이다.The present invention also provides a kit for detecting Chikungunya virus comprising an antibody that specifically binds to an epitope represented by the amino acid sequence of SEQ ID NO: 1.
본 발명에서 용어 "키트"란, 특정한 목적을 위해 필요한 조성물 및 부속품을 모아놓은 세트를 의미한다. 구체적으로 상기 키트에는 본 발명에 따른 치쿤군야 바이러스 검출용 조성물 뿐 아니라, 상기 항원-항체 복합체 형성 수준을 측정하는 분석에 사용되는 본 발명의 해당 분야에서 일반적으로 사용되는 도구, 시약 등이 포함될 수 있다. In the present invention, the term "kit" refers to a set of compositions and accessories necessary for a specific purpose. Specifically, the kit may include not only the composition for detecting the chikungunya virus according to the present invention, but also tools and reagents generally used in the relevant field of the present invention, which are used for an analysis measuring the level of formation of the antigen-antibody complex. .
상기 도구 또는 시약의 일 예로, 적합한 담체, 검출 가능한 신호를 생성할 수 있는 표지 물질, 발색단(chromophores), 용해제, 세정제, 완충제, 안정화제 등이 포함되나 이에 제한되지 않는다. Examples of the tool or reagent include, but are not limited to, a suitable carrier, a labeling substance capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, and the like.
표지 물질이 효소인 경우에는 효소 활성을 측정할 수 있는 기질 및 반응 정지제를 포함할 수 있다. 담체는 가용성 담체, 불용성 담체가 있고, 가용성 담체의 일 예로 당 분야에서 공지된 생리학적으로 허용되는 완충액, 예를 들어 PBS가 있고, 불용성 담체의 일 예로 폴리스틸렌, 폴리에틸렌, 폴리프로필렌, 폴리에스테르, 폴리아크릴로니트릴, 불소 수지, 가교 덱스트란, 폴리사카라이드, 라텍스에 금속을 도금한 자성 미립자와 같은 고분자, 기타 종이, 유리, 금속, 아가로오스 및 이들의 조합일 수 있다.When the labeling substance is an enzyme, a substrate capable of measuring enzyme activity and a reaction terminator may be included. The carrier includes a soluble carrier, an insoluble carrier, and an example of a soluble carrier includes a physiologically acceptable buffer solution known in the art, for example, PBS, and an example of an insoluble carrier includes polystyrene, polyethylene, polypropylene, polyester, poly It may be acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymers such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.
본 발명에서 제공하는 키트를 이용하는 경우, 예를 들어, 개체로부터 얻은 시료와 본 발명의 서열번호 1 또는 2의 서열을 포함하는 재조합 단백질을 접촉하여, 시료 내의 항체와 본 발명의 재조합 단백질의 항원-항체 복합체 형성 수준을 비교하여, 개체의 치쿤군야 바이러스 감염 여부를 진단할 수 있다. 또는, 이와 반대로, 본 발명의 개체로부터 얻은 시료와 본 발명의 서열번호 1 또는 2의 에피토프를 특이적으로 인식하는 항체를 접촉하여, 시료 내 치쿤군야 바이러스와 본 발명의 항체의 항원-항체 복합체 형성 수준을 비교하여, 개체의 치쿤군야 바이러스 감염 여부를 진단할 수 있다.In the case of using the kit provided in the present invention, for example, by contacting a sample obtained from an individual with a recombinant protein comprising the sequence of SEQ ID NO: 1 or 2 of the present invention, the antibody in the sample and the antigen of the recombinant protein of the present invention- By comparing the level of antibody complex formation, it is possible to diagnose whether an individual is infected with the Chikungunya virus. Alternatively, on the contrary, by contacting a sample obtained from an individual of the present invention with an antibody that specifically recognizes the epitope of SEQ ID NO: 1 or 2 of the present invention, the antigen-antibody complex of the chikungunya virus and the antibody of the present invention in the sample is formed. By comparing the levels, it is possible to diagnose whether an individual is infected with the Chikungunya virus.
본 발명에서 "개체"란, 치쿤군야 바이러스 감염의 여부가 확실하지 않거나 감염 여부가 의심되는 개체를 의미한다. In the present invention, the term "individual" refers to an individual who is not sure whether it is infected with Chikungunya virus or is suspected of being infected.
또한 본 발명에서 "시료"란 개체의 조직, 세포, 혈액, 혈청, 혈장, 타액, 체액 또는 객담을 포함하는 물질을 의미한다. 상기 시료는 바람직하게는 치쿤군야 바이러스 감염여부의 확인이 필요한 개체로부터 분리한 조직, 세포, 혈액, 혈청, 혈장, 타액, 체액 또는 객담일 수 있다. 일 예로, 혈액, 혈청 또는 혈장은 IgG 또는 IgM 항체를 포함하는 것 일 수 있으나, 이에 제한되는 것은 아니다. In addition, in the present invention, the term "sample" refers to a substance including tissue, cells, blood, serum, plasma, saliva, body fluids, or sputum of an individual. The sample may be preferably a tissue, cell, blood, serum, plasma, saliva, body fluid, or sputum isolated from an individual requiring confirmation of infection with the chikungunya virus. For example, blood, serum, or plasma may contain IgG or IgM antibodies, but are not limited thereto.
또한, 본 발명의 상기 키트는 단백질 칩 분석을 수행하기 위해 필요한 필수 요소를 포함하는 키트 또는 ELISA를 수행하기 위해 필요한 필수 요소를 포함하는 키트 일 수 있으나, 이에 제한되는 것은 아니다.In addition, the kit of the present invention may be a kit including essential elements necessary for performing protein chip analysis or a kit including essential elements necessary for performing ELISA, but is not limited thereto.
본 발명에 있어서, 상기 "단백질 칩 키트"란, 특정 단백질과 반응할 수 있는 항체 등을 단일 칩 상에 고밀도로 고정시킨 키트를 의미하며, 일 예로, 본 발명의 서열번호 1 또는 서열번호 2의 에피토프에 특이적으로 결합할 수 있는 항체가 고정된 것 일 수 있다. In the present invention, the "protein chip kit" means a kit in which an antibody capable of reacting with a specific protein is immobilized on a single chip at high density, and as an example, of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention An antibody capable of specifically binding an epitope may be immobilized.
본 발명에 따른 상기 항원-항체 복합체 형성 수준을 측정하기 위한 키트는 항체의 면역학적 검출을 위하여 기재, 완충용액, 발색효소 또는 형광물질로 표지된 2차 항체, 발색기질 등을 포함할 수 있다. 상기에서 기재로는 니트로셀룰로오스 막, 폴리비닐 수지로 합성된 96-웰 플레이트, 폴리스틸렌 수지로 합성된 96-웰 플레이트, 유리로 된 슬라이드 글라스 등이 이용될 수 있고, 발색효소로는 퍼옥시다아제(peroxidase), 알칼라인 포스파타아제(alkaline phosphatase) 등이 사용될 수 있다. 또한, 형광물질로는 FITC, RITC 등이 사용될 수 있고, 발색기질로는 ABTS(2,2'-아지노-비스-(3-에틸벤조티아졸린-6-설폰산)), OPD(o-페닐렌디아민), TMB(테트라메틸 벤지딘) 등이 사용될 수 있다. 또한, 상기 단백질의 항원-항체 복합체 형성 수준 측정은 본 발명의 기술분야에서 항체의 결합물을 계수하는 방법이 사용될 수 있다.The kit for measuring the level of formation of the antigen-antibody complex according to the present invention may include a substrate, a buffer solution, a secondary antibody labeled with a color developing enzyme or a fluorescent substance, a color developing substrate, and the like for immunological detection of the antibody. As the substrate in the above, a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc. may be used, and as a coloring enzyme, peroxidase (peroxidase) ), alkaline phosphatase, etc. may be used. In addition, FITC, RITC, etc. may be used as a fluorescent material, and ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenyl) as a color developing substrate Rendiamine), TMB (tetramethyl benzidine), and the like may be used. In addition, the measurement of the level of antigen-antibody complex formation of the protein may be performed by a method of counting antibody conjugates in the technical field of the present invention.
본 발명에 있어서, 상기 "ELISA 키트"란, 효소를 표식자로 하여 항원-항체 반응을 이용한 항원 또는 항체량 측정 방법을 일반적으로 효소면역분석법을 총칭하며, 본 발명의 목적상 상기 ELISA 키트는 본 발명에 따른 재조합 단백질을 포함한다. 또한, 상기 ELISA 키트는 상기 재조합 단백질에 결합된 목적하는 개체 내의 항체를 검출할 수 있는 시약, 예를 들면, 표지된 2차 항체, 발색단(chromophores), 항체와 결합(link)되어 있는 효소 및 그의 기질 또는 항체와 결합할 수 있는 다른 물질 등을 포함할 수 있다.In the present invention, the "ELISA kit" refers to a method for measuring the amount of antigen or antibody using an antigen-antibody reaction using an enzyme as a marker, and generally refers to an enzyme immunoassay method. For the purposes of the present invention, the ELISA kit is the present invention It includes a recombinant protein according to. In addition, the ELISA kit is a reagent capable of detecting an antibody in a target individual bound to the recombinant protein, for example, labeled secondary antibodies, chromophores, enzymes linked to the antibody, and Other substances capable of binding to a substrate or antibody may be included.
본 발명은 또한 서열번호 1 또는 서열번호 2의 아미노산 서열로 표시되는 에피토프에 특이적으로 결합하는 항체를 시료와 접촉시키는 것을 포함하는, 시료에서 치쿤군야 바이러스를 검출하는 방법에 관한 것이다. The present invention also relates to a method for detecting chikungunya virus in a sample comprising contacting the sample with an antibody that specifically binds to the epitope represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
또한, 본 발명은 상기 검출 결과를 가지고 치쿤군야 바이러스 감염 여부에 대한 정보를 제공할 수 있다는 점에서, 본 발명은 치쿤군야 바이러스에 대한 감염 여부에 대한 정보를 제공하는 방법에 관한 것이다.In addition, the present invention relates to a method of providing information on whether or not the chikungunya virus is infected with the detection result.
상기 치쿤군야 바이러스 검출 방법 또는 감염에 대한 정보 제공 방법은 개체로부터 얻은 시료와 본 발명의 서열번호 1 또는 서열번호 2의 아미노산 서열로 표시되는 에피토프를 포함하는 재조합 단백질; 또는 이에 특이적으로 결합하는 항체를 접촉시키는 것 이후에, 상기 시료에서 항원-항체 복합체 형성 수준을 측정하는 것을 더 포함할 수 있다.The method for detecting chikungunya virus or providing information on infection may include a sample obtained from an individual and a recombinant protein comprising an epitope represented by the amino acid sequence of SEQ ID NO: 1 or 2 of the present invention; Alternatively, after contacting the antibody that specifically binds thereto, measuring the level of antigen-antibody complex formation in the sample may be further included.
또한, 본 발명의 바이러스 검출방법 또는 감염에 대한 정보를 제공하는 방법은 정상 대조군의 시료에서의 항원-항체 복합체 형성수준과 상기에서 측정된 복합체 형성 수준을 비교하는 것을 더 포함할 수 있다. In addition, the virus detection method of the present invention or the method of providing information on infection may further include comparing the antigen-antibody complex formation level in a sample of a normal control group with the complex formation level measured above.
상기 복합체 형성 수준의 차이를 판단하여, 치쿤군야 바이러스 감염 여부를 예측할 수 있다. 바람직하게는, 상기 항원-항체 복합체 형성 수준이 치쿤군야 바이러스가 감염되지 않은 정상 대조군의 시료보다 높은 경우에 목적하는 개체가 치쿤군야 바이러스에 감염되었을 것으로 진단할 수 있으나, 이에 제한되는 것은 아니다.By determining the difference in the level of formation of the complex, it is possible to predict whether the chikungunya virus is infected. Preferably, when the level of formation of the antigen-antibody complex is higher than that of a sample of a normal control group that is not infected with the chikungunya virus, it can be diagnosed that the target individual is infected with the chikungunya virus, but is not limited thereto.
본 발명에서 상기 "항원-항체 복합체"란, 본 발명에 따른 항체와 개체로부터 얻어진 시료 내에 존재하는 이에 특이적인 항체의 결합물을 의미하고, 항원-항체 복합체의 형성 수준은 검출 라벨(detection label)의 시그널의 세기를 통해서 정량적으로 측정 가능하다.In the present invention, the "antigen-antibody complex" means a combination of the antibody according to the present invention and an antibody specific for it present in a sample obtained from an individual, and the level of formation of the antigen-antibody complex is a detection label. It can be quantitatively measured through the intensity of the signal of.
본 발명에서 상기 "항원-항체 복합체 형성 수준 측정"이란, 치쿤군야 바이러스를 검출 또는 감염 여부를 진단하기 위하여, 본 발명에 다른 항체와 결합하는 시료 내 존재하는 항원 단백질의 존재 또는 존재하는 양을 확인하는 것을 의미한다. In the present invention, the "antigen-antibody complex formation level measurement" means, in order to detect or diagnose Chikungunya virus, the presence or amount of antigenic protein present in a sample binding to another antibody according to the present invention Means to do.
상기 항원-항체 복합체 형성 수준을 측정하기 위한 분석 방법으로는 웨스턴 블럿, ELISA (enzyme linked i mMunosorbent assay), 방사선면역분석 (RIA: Radioi mMunoassay), 방사면역확산법 (radioi mMunodiffusion), 오우크테로니 (Ouchterlony) 면역 확산법, 로케트(rocket) 면역전기영동, 조직면역 염색, 면역침전 분석법 (I mMunoprecipitation assay), 보체고정분석법 (Complement Fixation Assay), FACS, 단백질 칩 (protein chip) 등이 있으나, 이에 제한되는 것은 아니다.As an analysis method for measuring the level of antigen-antibody complex formation, Western blot, ELISA (enzyme linked i mMunosorbent assay), radioimmunoassay (RIA: Radioi mMunoassay), radioi mMunodiffusion, and ouchteroni ( Ouchterlony) immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay (ImMunoprecipitation assay), complement fixation assay (Complement Fixation Assay), FACS, protein chip, etc., but are limited thereto. It is not.
본 발명에서 "검출 라벨"은 예를 들어, 효소, 형광물, 리간드, 발광물, 미소입자(microparticle), 레독스 분자 및 방사선 동위원소로 이루어진 그룹 중에서 선택할 수 있으며, 이에 제한되는 것은 아니다. 보다 구체적으로, 검출 라벨로서 이용 가능한 효소에는 β-글루쿠로니다제, β-D-글루코시다제, β-D-갈락토시다제, 우레아제, 퍼옥시다아제 또는 알칼라인 포스파타아제, 아세틸콜린에스테라제, 글루코즈 옥시다제, 헥소키나제와 GDPase, RNase, 글루코즈 옥시다제와 루시페라제, 포스포프럭토키나제, 포스포에놀피루베이트 카복실라제, 아스파르테이트 아미노트랜스페라제, 포스페놀피루베이트 데카복실라제, β-라타마제 등이 있으며, 이에 제한되지 않는다. 또한 상기 형광물로서 이용 가능한 것에는 플루오레신, 이소티오시아네이트, 로다민, 피코에리테린, 피코시아닌, 알로피코시아닌, o-프탈데히드, 플루오레스카민 등이 있으나, 이에 제한되지 않는다. In the present invention, the "detection label" may be selected from, for example, an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule, and a radioisotope, but is not limited thereto. More specifically, the enzymes usable as detection labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinestera. Agent, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phospholpyruvate decarboxyl Lase, β-latamase, and the like, but are not limited thereto. In addition, fluorescein, isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescarmine, and the like may be used as the fluorescent material, but are not limited thereto. .
또한, 상기 리간드에는 바이오틴 유도체 등이 있고, 이에 제한되지 않는다. In addition, the ligand includes a biotin derivative, but is not limited thereto.
또한, 상기 발광물에는 아크리디늄 에스테르, 루시페린, 루시퍼라아제 등이 있으며, 이에 제한되지 않는다. In addition, the light-emitting material includes acridinium ester, luciferin, luciferase, and the like, but is not limited thereto.
또한, 미소입자에는 콜로이드 금, 착색된 라텍스 등이 있으며, 이에 제한되지 않는다. 또한, 레독스 분자에는 페로센, 루테늄 착화합물, 바이올로젠, 퀴논, Ti 이온, Cs 이온, 디이미드, 1,4-벤조퀴논, 하이드로퀴논, K4W(CN)8, [Os(bpy)3]2+, [RU(bpy)3]2+, [MO(CN)8]4 - 등이 포함되며, 이에 제한되지 않는다.In addition, the microparticles include colloidal gold and colored latex, but are not limited thereto. In addition, the redox molecule includes ferrocene, ruthenium complex, biologene, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K 4 W(CN) 8 , [Os(bpy) 3 ] 2+ , [RU(bpy) 3 ] 2+ , [MO(CN) 8 ] 4 -, and the like are included, but are not limited thereto.
또한, 상기 방사선 동위원소에는 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re이 포함되나, 이에 제한되지 않는다.In addition, the radioisotope includes 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re, but Not limited.
이하, 본 발명을 제조예 및 실험예를 통해 상세히 설명한다. 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐 본 발명의 범위가 이들에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail through Preparation Examples and Experimental Examples. The following examples and experimental examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto.
실험예Experimental example 1. One. 치쿤군야Chikungunya 바이러스에서 새로운 New from virus 에피토프의Epitope 개발 Development
1.1. 1.1. 치쿤군야Chikungunya 바이러스의 Viral 타겟target 및 항원 선정 And antigen selection
중화항체 제작을 위해 Indian Ocean 변이종 중 La reunion_05-115(Indian Ocean strain, 도 1)를 선정하여 항원 선발에 사용하였다. 또한, X-선 결정학과 Cryo-EM 기법으로 규명된 총 9 종의 La reunion 변이주의 당단백질 구조를 표준 항원 선정을 위해 이용하였다. For the production of neutralizing antibodies, La reunion_05-115 (Indian Ocean strain, Fig. 1) among Indian Ocean variants was selected and used for antigen selection. In addition, the glycoprotein structures of a total of 9 La reunion mutants identified by X-ray crystallography and Cryo-EM techniques were used for selection of standard antigens.
치쿤군야 바이러스는 1248개의 아미노산으로 구성된 구조 단백질을 가지고 있는데, 이 구조 단백질이 잘려나가 캡시드와 당단백질인 E1, E2 및 E3를 형성하는데, E1, E2가 바이러스의 표면을 구성하므로, E1과 E2를 항원 후보 단백질로 선정하였다. 또한, 종래의 중화항체들의 항원인지 부위 맵핑(mapping)한 결과, 마우스 유래 중화항체와 인간 유래 중화항체의 중화효능이 특정 잔기의 치환에 민감하게 나타남을 확인하였고, 이는 중화항체의 항원인지를 제시하는 것으로 해석할 수 있다. 도 2에 나타낸 바와 같이, 항원 인지부위는 구조연구결과와 일치하게 주로 E2의 B도메인 메인 부위에 있음을 알 수 있었다. Chikungunya virus has a structural protein composed of 1248 amino acids, which is cut to form the capsid and glycoproteins E1, E2 and E3. Since E1 and E2 constitute the surface of the virus, E1 and E2 are formed. It was selected as an antigen candidate protein. In addition, as a result of mapping the antigen recognition sites of conventional neutralizing antibodies, it was confirmed that the neutralizing efficacy of the neutralizing antibodies derived from mice and the neutralizing antibodies derived from humans was sensitive to the substitution of specific residues, indicating whether the neutralizing antibodies were antigens. It can be interpreted as doing. As shown in Fig. 2, it was found that the antigen recognition site was mainly located at the main site of the B domain of E2, consistent with the results of the structural study.
따라서, 상기한 분석 결과를 종합하여, 본 발명에서는 La reunion_05-115(Indian Ocean strain) 변이주에 대해 강력한 중화항체 에피토프를 가지고 있고 보존성이 높은 잔기 서열을 가지고 있는 E1, E2, E2_B를 후보 항원 부위로 선정하였다. 재조합 항원 정제의 용이성과 원활한 항체 형성을 위해 6xHis tag 등을 첨가하였고, 항원의 올바른 접힘을 위해 6xHis tag의 위치를 N말단, C말단에 첨가하는 2가지 방식을 이용하였다(표 1, 위치는 전체 구조단백질(NCBI accession number: CAJ90470)에 대한 아미노산의 위치로 기재).Therefore, in summarizing the above analysis results, in the present invention, E1, E2, E2_B, which has a strong neutralizing antibody epitope and highly conserved residue sequence against the La reunion_05-115 (Indian Ocean strain) mutant, are used as candidate antigen sites. Selected. 6xHis tag was added for ease of purification of the recombinant antigen and smooth antibody formation, and two methods were used in which the position of the 6xHis tag was added to the N-terminal and C-terminal for correct folding of the antigen (Table 1, the positions are all). Structural protein (NCBI accession number: CAJ90470) in the amino acid position)
실험예Experimental example 2. 항원 유전자의 증폭 2. Amplification of antigen gene
E2B, E2L 및 E2Y 단백질을 코딩하는 유전자를 표 2의 프라이머들을 사용하여 PCR(중합효소 연쇄반응)로 증폭하였다. E2B 항원에 대한 정방향 프라이머(서열번호 5)는 BamHI 제한효소 인지부위(음영부분, ggatcc)에 해당하는 염기서열을 포함하고, 역방향 프라이머(서열번호 6)는 제한효소 HindⅢ 인지부위(음영부분, aagctt)에 해당하는 염기서열을 포함하도록 설계하였다. E2Y 항원에 대한 정방향 프라이머(서열번호 7)는 BamHI 제한효소 인지부위(음영부분, ggatcc)에 해당하는 염기서열을 포함하고, 역방향 프라이머(서열번호 8)는 제한효소 HindⅢ 인지부위(음영부분, aagctt)에 해당하는 염기서열을 포함하도록 설계하였다. 또한, E2L 항원에 대한 정방향 프라이머(서열번호 9)는 BamHI 제한효소 인지부위(음영부분, ggatcc)에 해당하는 염기서열을 포함하고, 역방향 프라이머(서열번호 10)는 제한효소 HindⅢ 인지부위(음영부분, aagctt)에 해당하는 염기서열을 포함하도록 설계하였다.Genes encoding E2B, E2L and E2Y proteins were amplified by PCR (polymerase chain reaction) using the primers in Table 2. The forward primer (SEQ ID NO: 5) for the E2B antigen contains the nucleotide sequence corresponding to the BamHI restriction enzyme recognition site (shaded portion, ggatcc), and the reverse primer (SEQ ID NO: 6) is the restriction enzyme HindIII recognition site (shaded portion, aagctt). It was designed to include the nucleotide sequence corresponding to ). The forward primer (SEQ ID NO: 7) for the E2Y antigen contains the nucleotide sequence corresponding to the BamHI restriction enzyme recognition site (shaded portion, ggatcc), and the reverse primer (SEQ ID NO: 8) is the restriction enzyme HindIII recognition site (shaded portion, aagctt). It was designed to include the nucleotide sequence corresponding to ). In addition, the forward primer (SEQ ID NO: 9) for the E2L antigen contains the base sequence corresponding to the BamHI restriction enzyme recognition site (shaded portion, ggatcc), and the reverse primer (SEQ ID NO: 10) is the restriction enzyme HindIII recognition site (shaded portion). , aagctt) was designed to include the corresponding nucleotide sequence.
PCR 반응액은 cDNA(200 ng/ul) 1 ㎕ , 10 mM dNTP 10 ㎕ (최종농도: 0.4 mM), 10pM 프라이머 1 ㎕ (최종농도: 0.2 uM), KOD FX Neo DNA 중합효소 1 ㎕ (1.0 U/ ㎕ ), 25 ℃ PCR 완충용액에 11 ㎕ 의 증류수를 넣어 제조하였다. 상기 반응액을 95 ℃에서 10분; 94 ℃에서 1분 30초; 59 ℃에서 1분; 72 ℃에서 30초의 반응을 28회 반복하였다. 반응용액을 1 % 아가로즈 젤에서 전기영동법으로 분리하여 약 300 염기쌍의 E2B 유전자를 용출하여 얻었다. E2L 유전자와 E2Y 유전자도 동일한 방법으로 용출하여 얻었다. PCR reaction solution was cDNA (200 ng/ul) 1 μl, 10
이 용출액 16 ㎕ 에 10배의 제한효소 반응 완충용액 2 ㎕ 와 제한효소 BamHI과 HindⅢ를 1 ㎕ 씩 첨가하여 37 ℃에서 20분 동안 반응시켰다. 다음 이 반응용액을 1 % 아가로즈 젤에서 전기영동법으로 분리하여 원하는 DNA 절편(이하 E2B BamHI/HindⅢ; E2L BamHI/HindⅢ; 또는 E2Y BamHI/HindⅢ)을 추출한 후 50 ㎕ 의 증류수 용액에 용존시켰다. E2Y 및 E2L에 대해서도 E2B와 동일한 방법으로 각 유전자를 얻고, 50 ㎕ 의 증류수 용액에 용존시켜, 다음 실험에 사용하였다. To 16 µl of this eluate, 2 µl of a 10-fold restriction enzyme reaction buffer solution and 1 µl of restriction enzymes BamHI and HindIII were added each, and reacted at 37°C for 20 minutes. Next, the reaction solution was separated by electrophoresis on a 1% agarose gel, and the desired DNA fragment (hereinafter, E2B BamHI/HindIII; E2L BamHI/HindIII; or E2Y BamHI/HindIII) was extracted and dissolved in 50 µl of distilled water solution. For E2Y and E2L, each gene was obtained in the same manner as in E2B, dissolved in 50 µl of distilled water, and used in the next experiment.
실험예Experimental example 3. 항원 유전자를 포함하는 발현벡터 제조 3. Preparation of expression vector containing antigen gene
E2B 단백질 발현벡터를 제조하기 위해서 플라스미드 pET21a(Quiagen)를 제한효소 BamHI과 HindⅢ를 이용하여 완전히 절단하고 1 % 아가로즈 젤 상에서 분리하였다(이하 pET21a BamHI/HindⅢ). E2B BamHI/HindⅢ 6 ㎕ 를 상기 플라스미드 벡터 pET21a BamHI/HindⅢ 3 ㎕ 와 함께 반응튜브에 넣은 후 2 ㎕ 의 10배 연결반응용액(500 mM 트리스-염산, pH 7.8, 100 mM 염화마그네슘, 100 mM DTT, 10 mM ATP), 10U의 T4 DNA 리가제를 넣고 총 부피가 20 ㎕ 가 되도록 증류수를 가한 다음 상온에서 30분 동안 반응시켰다. In order to prepare an E2B protein expression vector, the plasmid pET21a (Quiagen) was completely digested with restriction enzymes BamHI and HindIII and isolated on a 1% agarose gel (hereinafter, pET21a BamHI/HindIII).
상기 반응용액을 대장균 DH5α 컴피턴트 세포에 첨가하여 형질전환 시킨 다음 50 ㎕ /ml 농도의 LB-엠피실린 배지에 평판하여 대장균 형질전환체를 선별하였다. 이들로부터 플라스미드를 추출하고 제한효소 및 염기서열 분석에 의해 각각 pET21a-E2B를 얻은 것을 확인하였다. 상기에서 얻은 재조합 플라스미드에 클로닝된 E2B 유전자의 염기서열 확인은 Sanger 등(Sanger, F., et al. (1977). PNAS U.S.A 74, 5463)에 개시된 방법에 따라 빅-다이 사이클 시퀀싱 시스템(Big-Dye Cycle Sequencing System, Applied Biosystems, U.S.A)을 이용하여 수행하였다.The reaction solution was added to E. coli DH5α competent cells for transformation, and then plated on 50 µl/ml LB-ampicillin medium to select E. coli transformants. From these, the plasmid was extracted, and it was confirmed that pET21a-E2B was obtained by restriction enzyme and nucleotide sequence analysis, respectively. Confirmation of the nucleotide sequence of the E2B gene cloned into the recombinant plasmid obtained above was performed according to the method disclosed in Sanger et al. (Sanger, F., et al. (1977). PNAS USA 74, 5463), a big-die cycle sequencing system (Big-). Dye Cycle Sequencing System, Applied Biosystems, USA).
또한, E2B 부위 외의, E2 단백질의 다른 부위의 항원성을 무시할 수 없기 때문에, E2 단백질 전체를 포함하는 컨스트럭션을 대장균 시스템을 이용해서 발현하기 위해, E2L과 E2Y의 발현 벡터를 제조하였다. 구체적인 항원의 종류 및 특성은 표 3에 나타낸 바와 같다(표 3, 위치는 전체 구조단백질(NCBI accession number: CAJ90470)에 대한 아미노산의 위치로 기재).In addition, since antigenicity of other sites of the E2 protein other than the E2B site cannot be ignored, expression vectors of E2L and E2Y were prepared in order to express a construct including the entire E2 protein using the E. coli system. Specific antigen types and properties are as shown in Table 3 (Table 3, positions are described as amino acid positions relative to the entire structural protein (NCBI accession number: CAJ90470)).
길이AA
Length
(aliquote)Aliquot
(aliquote)
1490(reduced)1740,
1490(reduced)
2980 (reduced)3230,
2980 (reduced)
52830 (reduced)53580,
52830 (reduced)
OVA 단백질의 일부분을 포함하게 디자인된 플라스미드 pET21a-OVA를 제한효소 BamHI과 HindⅢ를 이용하여 완전히 절단하고 1 % 아가로즈 젤 상에서 분리하였다(이하 pET21a-OVA BamHI/HindⅢ). E2Y, E2L을 BamHI/HindⅢ 6 ㎕ 를 상기 플라스미드 벡터 pET21a-OVA BamHI/HindⅢ 3 ㎕ 와 함께 반응튜브에 넣은 후 2 ㎕ 의 10배 연결반응용액(500 mM 트리스-염산, pH 7.8, 100 mM 염화마그네슘, 100 mM DTT, 10 mM ATP), 10U의 T4 DNA 리가제를 넣고 총 부피가 20 ㎕ 가 되도록 증류수를 가한 다음 상온에서 30분 동안 반응시켰다. The plasmid pET21a-OVA designed to contain a portion of the OVA protein was completely digested with restriction enzymes BamHI and HindIII and isolated on a 1% agarose gel (hereinafter, pET21a-OVA BamHI/HindIII). Put E2Y and E2L into the reaction tube with 6 µl of BamHI/HindⅢ and 3 µl of the plasmid vector pET21a-OVA BamHI/HindⅢ, and then add 2 µl of 10-fold reaction solution (500 mM Tris-HCl, pH 7.8, 100 mM magnesium chloride). , 100 mM DTT, 10 mM ATP), 10U of T4 DNA ligase was added, distilled water was added so that the total volume became 20 µl, and then reacted at room temperature for 30 minutes.
상기 반응용액을 대장균 DH5α 컴피턴트 세포에 첨가하여 형질전환 시킨 다음 50 ㎕ /ml 농도의 LB-엠피실린 배지에 평판하여 대장균 형질전환체를 선별하였다. 이들로부터 플라스미드를 추출하고 제한효소 및 염기서열 분석에 의해 각각 pET21a-E2Y-OVA, pET21a-E2L-OVA를 얻은 것을 확인하였다. 상기에서 얻은 재조합 플라스미드에 클로닝된 E2L, E2Y 유전자의 염기서열 확인은 Sanger 등(Sanger, F., et al. (1977). PNAS U.S.A 74, 5463)을 이용하여 확인하였다. E2B_OVA 발현벡터 pET21a-E2B-OVA도 상기한 방법에 따라 제조하였다. The reaction solution was added to E. coli DH5α competent cells for transformation, and then plated on 50 µl/ml LB-ampicillin medium to select E. coli transformants. Plasmids were extracted from these, and it was confirmed that pET21a-E2Y-OVA and pET21a-E2L-OVA were obtained by restriction enzyme and nucleotide sequence analysis, respectively. The nucleotide sequence confirmation of the E2L and E2Y genes cloned into the recombinant plasmid obtained above was confirmed using Sanger et al. (Sanger, F., et al. (1977). PNAS U.S.A 74, 5463). E2B_OVA expression vector pET21a-E2B-OVA was also prepared according to the above method.
실험예Experimental example 4. 4. 항원단백질의Antigenic protein 정제 refine
4-1. 대장균 배양(4-1. E. coli culture ( E2BE2B 발현) 및 파쇄 Expression) and disruption
상기 실험예 3에서 얻은 발현 벡터 pET21a-E2B를 발현 숙주인 대장균 Rosetta(DE3)pLysS(Novagen Inc.)에 형질 전환시켰다. 형질 전환된 대장균 균주를 100㎍/ml의 엠피실린이 함유된 루리아 배지(LB, 1 % 박토트립톤, 0.5 % 효모 추출물, 1 % 염화나트륨)에서 12시간 동안 진탕 배양한 후 이중 1 ml을 100 ml의 루리아 배지(100 ug/ml의 카나마이신 함유)에 옮겨 37 ℃에서 박테리아의 흡광도가 600nm에서 약 0.6 정도가 될 때 IPTG(isopropyl-β-D-galactopyranoside)를 최종농도가 1 mM이 되게 첨가하였다. The expression vector pET21a-E2B obtained in Experimental Example 3 was transformed into E. coli Rosetta (DE3) pLysS (Novagen Inc.), an expression host. The transformed E. coli strain was cultured with shaking for 12 hours in Luria medium (LB, 1% Bacttotripton, 0.5% yeast extract, 1% sodium chloride) containing 100 μg/ml of ampicillin, and then 1 ml of 100 ml In Luria medium (containing 100 ug/ml kanamycin), when the absorbance of bacteria at 37° C. was about 0.6 at 600 nm, isopropyl-β-D-galactopyranoside (IPTG) was added to a final concentration of 1 mM.
IPTG 첨가한 후 18시간 후에 5000rpm에서 10분간 원심분리하여 각각 세포 침전물을 수거하였다. 수거한 세포 침전물을 20 mM Tris pH 8.0, 100 mM 염화나트륨, 5 mM MgCl2 조성의 완충용액에 현탁시킨 다음, 얼음중탕에서 초음파 분쇄기(Sonic Dismembrator, Fisher, 미국)를 이용하여 세포를 파쇄하였다. 이 용액을 원심분리기로 13000rpm에서 1시간 원심 분리한 후 그 상층액을 분리하여, 펠릿을 얻었다. pET21a-E2B-OVA도 상기와 동일한 방법으로 대장균 Rosetta(DE3) pLysS(Novagen Inc.)에 형질 전환하여 배양하여, 상층액을 분리하여 펠릿을 얻었다. Eighteen hours after the addition of IPTG, the cells were centrifuged at 5000 rpm for 10 minutes to collect cell precipitates. The collected cell precipitate was suspended in a buffer solution having a composition of 20 mM Tris pH 8.0, 100 mM sodium chloride, and 5 mM MgCl 2 , and then the cells were disrupted in an ice bath using an ultrasonic grinder (Sonic Dismembrator, Fisher, USA). The solution was centrifuged at 13000 rpm for 1 hour with a centrifuge, and the supernatant was separated to obtain a pellet. pET21a-E2B-OVA was transformed and cultured in E. coli Rosetta (DE3) pLysS (Novagen Inc.) in the same manner as described above, and the supernatant was separated to obtain a pellet.
발현 벡터 pET21a-E2Y-OVA를 발현 숙주인 대장균 BL21, 그리고 발현 벡터 pET21a-E2L-OVA를 대장균 BL21에 각각 형질 전환시킨 것을 제외하곤, 상기 E2B와 동일하게 대장균을 배양하여, 상층액을 분리하여 펠릿을 얻었다. Except that the expression vector pET21a-E2Y-OVA was transformed into E. coli BL21 as an expression host, and the expression vector pET21a-E2L-OVA was transformed into E. coli BL21, respectively, the E. coli was cultured in the same manner as E2B, and the supernatant was separated and pelleted. Got it.
4-2. 파쇄된 대장균에서 단백질의 정제4-2. Purification of protein from crushed E. coli
상기 실험예 4-1의 결과물을 20 mM Tris pH 8.0, 100 mM 염화나트륨 조성의 완충용액으로 미리 평형된 니켈-니트릴로트리아세틱 액시드(Ni-NTA)칼럼(파마시아, 미국)에 결합시킨 후 0-0.5M 이미다졸 농도구배를 이용하여 용출한 후 SDS-PAGE(12 % SDS 겔)하여 E2B 단백질 부분만 모았다. After binding the resultant of Experimental Example 4-1 to a nickel-nitrilotriacetic acid (Ni-NTA) column (Pharmacia, USA) previously equilibrated with a buffer solution of 20 mM Tris pH 8.0 and 100 mM sodium chloride, 0 After elution using -0.5M imidazole concentration gradient, only the E2B protein portion was collected by SDS-PAGE (12% SDS gel).
그 결과를, 도 4a, 4b 및 4c에 나타내었다. 특히, 도 4c를 확인하면, 37 ℃에서 발현된 상층액 레인(supernatant lane: S)에서 E2B 단백질이 확인되었는바, E2B 단백질이 수용성을 가짐을 확인 할 수 있다. The results are shown in Figs. 4a, 4b and 4c. In particular, when checking FIG. 4C, the E2B protein was confirmed in a supernatant lane (S) expressed at 37° C., and it can be confirmed that the E2B protein has water solubleness.
또한, E2L-OVA, E2Y-OVA 단백질의 경우, 실험예 4-1에서 얻은 펠릿을 20 mM Tris pH 8.0, 500 mM NaCl, 6M Urea, 5 mM B-ME 조성의 변성(denaturation) 용액으로 녹인 후, 20 mM Tris pH 8.0, 500 mM NaCl, 8M Urea, 5 mM B-ME 조성으로 미리 평형된 니켈-니트릴로트리아세틱 액시드(Ni-NTA)칼럼(파마시아, 미국)에 결합시킨 후 0-0.5M 이미다졸 농도구배를 이용하여 용출한 후 SDS-PAGE하여 E2L-OVA, E2Y-OVA 단백질 부분만 모았다.In the case of E2L-OVA and E2Y-OVA proteins, the pellet obtained in Experimental Example 4-1 was dissolved in a denaturation solution of 20 mM Tris pH 8.0, 500 mM NaCl, 6M Urea, and 5 mM B-ME. , 20 mM Tris pH 8.0, 500 mM NaCl, 8M Urea, 0-0.5 after binding to a nickel-nitrilotriacetic acid (Ni-NTA) column (Pharmacia, USA) previously equilibrated with a composition of 5 mM B-ME After elution using M imidazole concentration gradient, only the E2L-OVA and E2Y-OVA proteins were collected by SDS-PAGE.
도 6a 및 도 6b에서 각 단백질의 발현을 확인하였으나, T 레인(total lane)에서 확인된 타겟 단백질의 양이 S 레인(상층액 레인)에서는 확인되지 않고, P 레인(펠릿 레인)에서만 확인되는 것을 통해, E2L 및 E2Y 단백질 모두 수용성이 떨어짐을 확인하였다. Although the expression of each protein was confirmed in FIGS. 6A and 6B, the amount of the target protein identified in the T lane (total lane) was not confirmed in the S lane (supernatant lane), but only in the P lane (pellet lane). Through this, it was confirmed that both the E2L and E2Y proteins had poor water solubility.
따라서, 상기에서 제조한 E2B, E2Y 및 E2L 발현 벡터 각각에 대해서 대장균 시스템에서 발현을 시도한 결과, E2Y 및 E2L 단백질은 당수식이 되는 부분을 포함하나, 대장균 발현 시스템의 경우 당수식이 불가능하고, E2Y 및 E2L 단백질의 낮은 수용성을 보여, E2Y 및 E2L 단백질은 중화항체 스크리닝 또는 진단 키트를 위한 대량생산을 위한 항원으로서 적합하지 에 적합하지 않다고 판단하였다. 그에 반해 도 4a, 4b 및 4c에 나타낸 바와 같이 수용성이 우수하고, 당수식 부위를 포함하지 않아, 대장균 발현 시스템에 의한 대량생산이 가능한 E2B 단백질이 항원으로 매우 적합하다고 판단하였다. Therefore, as a result of attempting expression in the E. coli system for each of the E2B, E2Y and E2L expression vectors prepared above, the E2Y and E2L proteins contain a part that becomes a sugar formula, but in the case of the E. Due to the low solubility of the protein, it was judged that the E2Y and E2L proteins were not suitable as antigens for mass production for neutralizing antibody screening or diagnostic kits. On the other hand, as shown in Figs. 4a, 4b and 4c, it was judged that the E2B protein, which was excellent in water solubility and did not contain a sugar control site, was able to be mass-produced by the E. coli expression system, as an antigen.
E2B, E2B-OVA, E2L-OVA, E2Y-OVA 단백질 각각을 2 ml 가량 농축한 후, 20 mM Tris pH 8.0, 100 mM 염화나트륨 조성의 완충용액으로 평형된 젤 필트레이션 칼럼(Superdex 75)에 주입시킨 후 단백질의 분자량에 의해 분리하여 SDS-PAGE로 확인하고 각 단백질을 정제하였다.E2B, E2B-OVA, E2L-OVA, E2Y-OVA proteins were each concentrated by about 2 ml, and then injected into a gel filtration column (Superdex 75) equilibrated with a buffer solution of 20 mM Tris pH 8.0 and 100 mM sodium chloride. After separation by the molecular weight of the protein, it was confirmed by SDS-PAGE, and each protein was purified.
E2B-6xHis는 Ni 컬럼(column)을 이용한 친화도 크로마토그래피, 젤-여과크로마토그래피(Gel filtration chromatography) 과정을 거쳐서 0.35 mg/L의 수득률로 95 퍼센트 이상의 순도로 정제가 되었다(도 7a, 7b 및 7c).E2B-6xHis was purified to a purity of 95 percent or more with a yield of 0.35 mg/L through affinity chromatography using a Ni column and gel filtration chromatography (FIGS. 7a, 7b and 7c).
E2B-6xHis-OVA는 발현 테스트에서 비수용성(insoluble)으로 발현되어 우레아 버퍼(Urea buffer)로 변성(denaturation) 과정을 거친 뒤 친화도 크로마토 그래피로 정제하였다. 그 후, 재접힘 과정을 통해 변성제(denaturant)로 작용하는 우레아(Urea)의 농도를 줄여 올바른 접힘을 유도하고 젤-여과크로마토그래피를 통해 단량체로 유지되는 부분만을 농축시켜 정제를 완료하였다(도 8a 및 8b).E2B-6xHis-OVA was expressed as insoluble in the expression test, subjected to denaturation with urea buffer, and then purified by affinity chromatography. After that, through the refolding process, the concentration of urea acting as a denaturant was reduced to induce correct folding, and purification was completed by concentrating only the portion retained as a monomer through gel-filtration chromatography (FIG. 8A). And 8b).
E2L-OVA와 E2Y-OVA는 발현 테스트에서 비-수용성(insoluble)으로 발현되어 우레아 버퍼(Urea buffer)로 변성(denaturation) 과정을 거친 뒤 친화도 크로마토 그래피로 정제하였다. 그 후, 재접힘 과정을 진행하였지만, 수득률이 극히 낮아, 이후에는 재접힘 과정을 거치지 않고 우레아 버퍼(Urea buffer) 상태에서 항체 제작에 들어갔다(도 9a 및 9b: 2L-OVA의 친화도 크로마토그래피와 젤-여과크로마토그래피 정제. 도 10: E2Y-OVA의 친화도 크로마토그래피). E2L-OVA and E2Y-OVA were expressed as insoluble in the expression test, subjected to denaturation with urea buffer, and then purified by affinity chromatography. Thereafter, the refolding process was performed, but the yield was extremely low, and thereafter, the antibody production was started in the urea buffer state without going through the refolding process (Figs. 9A and 9B: affinity chromatography of 2L-OVA and Gel-filtration chromatography purification, Fig. 10: Affinity chromatography of E2Y-OVA).
상기한 항원 후보 단백질 E2B_OVA, E2B, E2Y_OVA 및 E2L_OVA 각각을 대장균(E-coli) 시스템을 통해서 생산하였다. 총 생산량을 SDS-PAGE를 이용해서 UV 280nm에서 흡수도를 이용하여 정량하였다(도 11a 내지 11d).Each of the antigen candidate proteins E2B_OVA, E2B, E2Y_OVA, and E2L_OVA described above was produced through the E-coli system. The total production was quantified using the absorbance at UV 280nm using SDS-PAGE (FIGS. 11A to 11D ).
실험예Experimental example 5. 표준 항원으로서 특성 검토 5. Characterization as a standard antigen
또한, 본 발명의 E2B 항원이 특정 치쿤군야 바이러스 변이주에 특이적인 것인지를 확인하기 위해, 변이주들의 당단백질 서열 비교를 통해서 변이주들 간의 당단백질 표면 잔기들의 보존성을 평가하였다. 유전체 서열을 기준으로 작성한 계통학적 분류(phylogenic tree)에 따르면 치쿤군야 바이러스는 Indian ocean, ESCA(East/Central/South Africa), Asian, West Africa lineage로 총 4 종류의 변이주가 존재하므로, 각 변이주에 해당하는 서열정보 2개씩을 NCBI Gene Bank(Gene Bank 등록번호(accession code) CAJ90470.1, ACM00915.1, ADG95881.1, ADG95934.1, ADG95887.1, ABY57313.1, ADG95885.1, 및 AAU43881.1)에서 확보하여 분석하였다(도 12).In addition, in order to confirm whether the E2B antigen of the present invention is specific to a specific chikungunya virus mutant, the conservation of glycoprotein surface residues between the mutants was evaluated through comparison of the glycoprotein sequences of the mutants. According to the phylogenic tree based on the genome sequence, chikungunya virus has a total of 4 types of mutants: Indian ocean, ESCA (East/Central/South Africa), Asian, and West Africa lineage. Each of the two corresponding sequence information is NCBI Gene Bank (Gene Bank accession code) CAJ90470.1, ACM00915.1, ADG95881.1, ADG95934.1, ADG95887.1, ABY57313.1, ADG95885.1, and AAU43881. It was obtained and analyzed in 1) (Fig. 12).
또한, 변이주와 무관한 범용적인 중화항체 형성과 올바른 항원 선정을 위해 치쿤군야 바이러스 외피 단백질-단일 클론항체 복합체의 구조를 토대로 항원-항체 인터페이스의 표면 잔기 보존성을 구조생물학을 기반으로 분석하였다. In addition, the surface residue conservancy of the antigen-antibody interface was analyzed based on structural biology based on the structure of the chikungunya virus envelope protein-monoclonal antibody complex for the formation of universal neutralizing antibodies independent of the mutant strain and for correct antigen selection.
그 결과, 도 3a 및 3b에 나타낸 바와 같이, E1 E2의 복합체 형성에 관여하는 인터페이스의 표면잔기들의 보존 정도가 높게 나타났으며, 이 사실은 E1-E2 이형 이합체(heterodimer)가 바이러스 입자 형성에 중요하다는 연구 결과와 일치한다는 것을 알 수 있다. 또한, 도 12에 나타낸 바와 같이, 본 발명의 항원부위에서 항체와 직접적으로 상호작용하는 표면 잔기들은 예외없이 모든 변이주들에 대하여 잘 보존되어 있음을 확인하였는바, 이는 본 발명의 항원이 변이주의 종류와는 무관한 범용성 항체를 형성할 수 있음을 의미한다. As a result, as shown in FIGS. 3A and 3B, the degree of preservation of the surface residues of the interface involved in the formation of the E1 E2 complex was high, and this fact indicates that the E1-E2 heterodimer is important for viral particle formation. It can be seen that it is consistent with the research results. In addition, as shown in Figure 12, it was confirmed that the surface residues that directly interact with the antibody in the antigenic site of the present invention are well preserved for all mutant strains without exception, which indicates that the antigen of the present invention is the kind of mutant strain. It means that it is possible to form universal antibodies that are independent of and.
따라서, 본 발명은 당 수식 부위를 포함하지 않아, 대장균 시스템을 통해서 대량생산이 가능하고, 우수한 수용성을 갖는 E2B 부위(서열번호 1)가 범용의 중화항체 에피토프로서 효과적일 것이라 판단하여, E2B 부위를 최종 항원 부위로 선정하였다. 이후, His 태그 서열을 더 포함하는 재조합 단백질을 이용하여 항체 생산, 중화항체 스크리닝 등을 수행하였다. Therefore, the present invention does not contain a sugar-modified site, so mass production is possible through the E. coli system, and it is determined that the E2B site (SEQ ID NO: 1) having excellent water solubility will be effective as a universal neutralizing antibody epitope, and the E2B site It was selected as the final antigenic site. Thereafter, antibody production, neutralizing antibody screening, and the like were performed using a recombinant protein further comprising a His tag sequence.
실험예Experimental example 6. 6. 치쿤군야Chikungunya 바이러스 항원 주입을 통한 항체 유도 Antibody induction through viral antigen injection
대장균에서 발현하고 정제한 재조합 E2B, E2B-OVA 단백질 50 ㎍ 을 각각 아쥬반트로 명반(alum)과 1:1 부피비로 섞고 상온에서 회전시키면서 흡착시킨 후 마우스의 복강 내에 주입하거나, 다른 아쥬반트로 TiterMax® Gold와 1:1 부피비로 섞고 현탁액으로 만들어 주사하였다. 이와 같은 항원을 도 14의 접종 일정에 따라, 약 7-14일 간격으로 총 3번 접종하였다. 50 ㎍ of recombinant E2B and E2B-OVA proteins expressed and purified in Escherichia coli were mixed with alum in a 1:1 volume ratio, respectively, and adsorbed while rotating at room temperature, and then injected into the intraperitoneal cavity of mice, or with other adjuvants, TiterMax ® Gold was mixed in a volume ratio of 1:1 and made into a suspension for injection. According to the inoculation schedule of FIG. 14, the antigen was inoculated three times at intervals of about 7-14 days.
또한, 대장균에서 발현하고 정제한 재조합 E2L-OVA, E2Y-OVA 단백질 50㎍ 을 아쥬반트로서 명반(alum)과 1:1 부피비로 섞고 상온에서 회전시키면서 흡착시킨 후 마우스의 복강 내에 주입하거나, TiterMax® Gold와 1:1 부피비로 섞고 현탁액으로 만들어 주사하였고 도 14의 접종 일정에 따라 약 10일 간격으로 총 3번 접종하였다.In addition, 50 μg of recombinant E2L-OVA and E2Y-OVA proteins expressed and purified in E. coli were mixed as an adjuvant in a 1:1 volume ratio with alum, adsorbed while rotating at room temperature, and injected into the intraperitoneal cavity of a mouse or TiterMax® Gold was mixed in a 1:1 volume ratio, made into a suspension, and injected, and inoculated three times at about 10 day intervals according to the inoculation schedule of FIG.
마우스로는 OVA 펩타이드에 특이적으로 반응하는 형질전환 T세포(transgenic T cell)를 갖는 DO11.10 마우스에서 림프절을 분리하여 단일세포로 만들어 정맥 내에 약 1.5 x 106 또는 2.0 x 106 세포를 주입한 것과 주입하지 않은 마우스를 사용하였다.As a mouse, lymph nodes were isolated from DO11.10 mice with transgenic T cells that specifically respond to OVA peptides, made into single cells, and injected about 1.5 x 10 6 or 2.0 x 10 6 cells into a vein. One and no injection mice were used.
3회 접종 후, 3일 뒤인 최초 접종일 기준 24일 후에 헤파린이 코팅된 모세관을 사용하여 안와 채혈(eye bleeding)을 통하여 혈액을 얻었다. 혈액은 2,000×g, 4 ℃에서 약 10분간 침강시키고 상층액 혈장을 얻었다. Blood was obtained through eye bleeding using a heparin-coated
실험예Experimental example 7. 7. 치쿤군야Chikungunya 바이러스 virus 중화항체가Neutralizing antibody 확인 Confirm
7-1. 7-1. 치쿤군야Chikungunya 바이러스 플라크감소 Reduced viral plaque 중화항체가Neutralizing antibody 측정법( Measurement method ( plaqueplaque reductionreduction neutralization neutralization testtest , , PRNTPRNT ) 구축) build
본 발명에서는 메틸셀룰로스(Methylcellulose)를 포함하는 오버레이(overlay) 배양액을 이용하여 Vero 세포배양시스템을 기반으로 하는 플라크감소 중화항체가 측정법(plaque reduction neutralization test, PRNT)을 구축하였다. 양성대조군으로는 상업적으로 판매되는 치쿤군야 바이러스 중화항체(chk-265, Absolute Antibody Ltd. Oxford, UK)을 사용하여 중화효능을 분석하였다. In the present invention, a plaque reduction neutralization test (PRNT) based on the Vero cell culture system was constructed using an overlay culture medium containing methylcellulose. As a positive control, a commercially available chikungunya virus neutralizing antibody (chk-265, Absolute Antibody Ltd. Oxford, UK) was used to analyze the neutralizing efficacy.
우선, 중화항체가 측정 24 시간 전에 12웰 플레이트에 Vero 세포를 웰(well) 당 1.2x105개 세포를 분주하고, 항체가 측정 당일에 각 웰(Well)에 세포가 70~90 % 정도로 도말되도록 세포를 준비하였다. 중화능 시험 희석용액으로 56 ℃에서 30분 동안 놓아두어 열 불활성화(heat inactivated) 처리시킨 5 % FBS가 첨가된 DMEM 배지를 준비하였다. 또한, 치쿤군야 바이러스 중화항체(chk-265)를 중화능 시험 희석용액으로 희석하여, 단계별로 희석된 항체 시료 희석용액을 준비하였다. 치쿤군야 바이러스 용액은 웰(well) 당 플라크의 개수가 100 개가 되도록 제3(triplicate) 샘플에 대해 준비였다(~100 PFU/ 25 ㎕ ). 0.2 % 크리스탈 바이올렛 용액은 크리스탈 바이올렛을 20 % 에탄올 수용액에 0.2 % (w/v)으로 녹인 후 0.2㎛ 필터로 여과하여 준비하였다. 오버레이 배지(overlay media)는 열 불활성화(heat inactivated) 처리시킨 5 % FBS가 첨가된 DMEM 배지에 1 % 메틸-셀룰로오즈(methyl-cellulose)가 첨가된 것을 사용하였다. First, 24 hours before the neutralizing antibody is measured, Vero cells are dispensed into a 12-well plate, and 1.2x10 5 cells per well are dispensed, and the antibody is plated at about 70-90% of the cells in each well on the day of measurement. The cells were prepared. A DMEM medium to which 5% FBS was added was prepared by placing it at 56° C. for 30 minutes as a neutralization test diluted solution. In addition, the chikungunya virus neutralizing antibody (chk-265) was diluted with a neutralizing ability test dilution solution to prepare a dilution solution of the antibody sample diluted step by step. Chikungunya virus solution was prepared for the third (triplicate) sample so that the number of plaques per well was 100 (~100 PFU/25 μl). A 0.2% crystal violet solution was prepared by dissolving crystal violet in 0.2% (w/v) in a 20% ethanol aqueous solution and filtering through a 0.2 μm filter. As the overlay media, 1% methyl-cellulose was added to the DMEM medium to which 5% FBS was added to the heat inactivated treatment.
희석된 항체시료와 치쿤군야 바이러스 용액을 1:1 부피비로 혼합하여 총 160 ㎕ 반응양으로 37 ℃에서 60분간 배양하고, 12 웰 플레이트에 도말된 Vero 세포의 배양액을 흡입·제거한 후, 웰(well) 당 중화능 시험 희석용액 200 ㎕ 와 치쿤군야 바이러스-항체 혼합 용액 50 ㎕ 씩을 3개의 well에 나누어 분주하였다. 상기 용액이 첨가된 플레이트는 37 ℃, 5 % CO2 조건에서 90분간 배양하고, 배양시간 동안 10-15 분 마다 세포가 마르지 않도록 플레이트를 앞뒤로 흔들어 주었다.The diluted antibody sample and the chikungunya virus solution were mixed in a 1:1 volume ratio and incubated for 60 minutes at 37°C with a total reaction volume of 160 μl, and the culture solution of Vero cells smeared on a 12 well plate was aspirated and removed. ) Each of 200 µl of the diluted solution for the neutralizing activity test and 50 µl of the Chikungunya virus-antibody mixed solution were divided into 3 wells and dispensed. The plate to which the solution was added was incubated for 90 minutes at 37° C. and 5% CO 2 conditions, and the plate was shaken back and forth so that the cells did not dry out every 10-15 minutes during the incubation time.
첨가한 혼합 용액을 흡입·제거하고 1 % 메틸-셀룰로오즈(methyl-cellulose)가 첨가된 오버레이 배지(overlay media)를 웰 당 2 ml씩 넣고, 해당 플레이트를 37 ℃, 5 % CO2 조건에서 4일간 배양하였다. 4일간의 배양으로 플라크가 형성된 Vero 세포 플레이트에서 오버레이 배지(Overlay media)를 흡입·제거하고, 0.2 % 크리스탈 바이올렛 용액을 웰 당 2 ml 첨가하여 약 6-8 시간 실온에서 세포를 염색하였다. 이후 염색약을 흐르는 물로 세척하고 건조시킨 후, 웰 당 관찰되는 플라크 개수를 측정하여 chk-265 중화항체 시험 용액의 중화항체가를 계산하였다. Aspirate and remove the added mixed solution, add 2 ml of overlay media to which 1% methyl-cellulose is added per well, and place the plate at 37°C and 5% CO 2 for 4 days. Cultured. Overlay media was aspirated and removed from the Vero cell plate in which plaques were formed by incubation for 4 days, and 2 ml of 0.2% crystal violet solution was added per well to stain the cells at room temperature for about 6-8 hours. Thereafter, the dye was washed with running water and dried, and then the number of plaques observed per well was measured to calculate the neutralizing antibody value of the chk-265 neutralizing antibody test solution.
도 15a 및 도 15b에 나타낸 바와 같이, 본 발명의 플라크감소 중화항체가 측정법(PRNT)을 이용한 chk-265 중화능 확인결과 EC50=198ng/ml, EC90=483ng/ml의 결과를 반복적으로 도출할 수 있었으며, 본 실험을 통해 구축된 Vero 세포배양 시스템 기반 중화항체가 측정법(PRNT)의 유효성을 확인할 수 있었다. As shown in Figures 15a and 15b, the result of confirming the neutralizing ability of chk-265 using the plaque-reducing neutralizing antibody of the present invention (PRNT), EC 50 =198 ng/ml, EC 90 =483 ng/ml Through this experiment, the Vero cell culture system-based neutralizing antibody was able to confirm the effectiveness of the measurement method (PRNT).
7-2. 치쿤군야 바이러스 항원 주입 시 중화항체 형성 확인7-2. Confirmation of formation of neutralizing antibodies upon injection of chikungunya virus antigen
따라서, 상기 중화항체가 측정법에 따라, 본 발명의 항체 제작시스템을 이용해 본 발명의 서열번호 2의 E2B 치쿤군야 바이러스 항원을 Alum이나 TiterMax® Gold를 어쥬번트로 사용하여 실험예 6에서 설명한 바와 같이 접종한 마우스 혈장을 확보하였다. 상기 실험예 7-1의 중화항체가 측정법(PRNT)을 따라, chk-265 중화항체 대신 상기 마우스 혈장(plasma)의 중화능을 1:5 희석배율로 고정하여 스크리닝을 수행하였다.Therefore, according to the method of measuring the neutralizing antibody, the E2B chikungunya virus antigen of SEQ ID NO: 2 of the present invention was inoculated as described in Experimental Example 6 using Alum or TiterMax® Gold as an adjuvant using the antibody production system of the present invention. One mouse plasma was obtained. According to the neutralizing antibody assay method (PRNT) of Experimental Example 7-1, screening was performed by fixing the neutralizing capacity of the mouse plasma at a 1:5 dilution ratio instead of the chk-265 neutralizing antibody.
도 16에 나타낸 바와 같이, 약 1000 PFU의 고정된 플라크(plaque) 수로 진행한 PRNT에서 총 3 마리 마우스의 혈청(Gold 아쥬반트와 함께 접종된 DO11.10(+) 마우스에서 E2B#3, E2B#4, E2B#5 실험군)에서 유효한 중화능을 확인하였다. 이를 통해, 본 발명의 E2B 항원은 중화항체를 유도하는 항원부위 임을 알 수 있다.As shown in FIG. 16, serum of a total of 3 mice (
이후 추가로 E2B, E2B-OVA, E2L-OVA, E2Y-OVA 4 종의 치쿤군야 바이러스 항원을 Alum이나 TiterMax® Gold를 어쥬번트로 사용하여 실험예 6에서 설명한 바와 같이 접종한 마우스의 혈장을 확보하였으며 약 100 PFU의 고정된 플라크(plaque) 수로 진행한 플라크감소 중화항체가 측정 실험에서 총 8마리 마우스의 혈장(E2B, E2B-OVA#1, E2B-OVA#2, E2B-OVA#3와 E2L-OVA, E2Y-OVA#1, E2Y-OVA#2, E2Y-OVA#3)에서 유효한 중화능을 확인하였다(도 17).Subsequently, plasma of the mice inoculated as described in Experimental Example 6 was obtained by using four chikungunya virus antigens of E2B, E2B-OVA, E2L-OVA, and E2Y-OVA as an adjuvant using Alum or TiterMax® Gold. Plaque reduction neutralizing antibodies proceeded with a fixed number of plaques of about 100 PFU were measured in plasma of a total of 8 mice (E2B, E2B-
이들 혈장의 항원-항체 반응성을 선별하기 위해 ELISA를 수행하였다. 사용한 항원인 E2B 및 E2B-OVA, E2Y-OVA, E2L-OVA를 각각 0.1㎍ 항원/100 ㎕ PBS로 희석하여, 96웰 이뮤노플레이트(96 well i mMunoplate)에 웰 당 0.1㎍씩 담은 후 4 ℃에서 14시간 이상 보관하였다. 항원이 담겨있던 웰을 180 ㎕ 의 0.05 % Tween-20를 포함하는 PBS로 4번 씻어낸 후, 100 ㎕ 의 1 % BSA를 포함하는 PBS(assay diluent)/well을 이용해 상온에서 1시간 배양하였다. 마우스에서 분리한 혈장(plasma)을 각각 1x10-3, 1x10-4, 1x10-5의 농도로 희석한 후 혈장(plasma)을 처리하고 상온에서 2시간 동안 배양하였다. 분리한 마우스 혈장(plasma)에 특이적인 HRP-컨쥬게이트 Goat-anti-mouse 항체를 각 1x10-4, 1x10-5, 1x10-6으로 희석하여 처리하고, 상온에서 30분 배양하였다. 이후 웰 당 100 ㎕ TMB 기질을 10분 처리하고, 파란색을 띄게 되면 50 ㎕ /well의 2N H2SO4를 처리한 뒤, 플레이트 리더기(plate reader)를 이용해 450nm와 560nm에서 흡광율을 측정하였다. 이때 chk-265 항체를 대조군으로 사용하였다. ELISA was performed to screen for antigen-antibody reactivity of these plasmas. Each of the used antigens E2B, E2B-OVA, E2Y-OVA, and E2L-OVA was diluted with 0.1 μg antigen/100 μl PBS, added 0.1 μg per well to a 96 well immunoplate (96 well i mMunoplate), and then 4°C Stored for more than 14 hours. The wells containing the antigen were washed 4 times with 180 µl of PBS containing 0.05% Tween-20, and then incubated for 1 hour at room temperature using 100 µl of 1% BSA-containing PBS (assay diluent)/well. Plasma isolated from mice was diluted to a concentration of 1x10 -3 , 1x10 -4 , and 1x10 -5 , respectively, and then plasma was treated and incubated at room temperature for 2 hours. HRP-conjugated Goat-anti-mouse antibodies specific to the isolated mouse plasma were diluted with each of 1x10 -4 , 1x10 -5 , and 1x10 -6 and treated, and incubated for 30 minutes at room temperature. Thereafter, 100 µl TMB substrate per well was treated for 10 minutes, and 50 µl/well of 2N H 2 SO 4 was treated when it became blue, and the absorbance was measured at 450 nm and 560 nm using a plate reader. At this time, the chk-265 antibody was used as a control.
도 18에 나타낸 바와 같이, 마우스에 주입했던 항원에 대한 항체의 반응성은 농도 의존적인 반응으로 모두 양성으로 보였다. 특히, 대조군으로 사용한 chk-265 항체는 E2B에 대해서는 높은 항원 반응성을 보였으나, E2Y나 E2L에 대해서는 항원-항체 반응성이 높지 않았다. 이는 E2B가 E2Y나 E2L과 비교해서 더 우수한 항원능을 가지며, 더 높은 민감성으로 검출될 수 있음을 의미한다.As shown in Fig. 18, the reactivity of the antibody to the antigen injected into the mouse was a concentration-dependent reaction, and all were positive. In particular, the chk-265 antibody used as a control showed high antigen reactivity against E2B, but did not exhibit high antigen-antibody reactivity against E2Y or E2L. This means that E2B has better antigenic activity compared to E2Y or E2L and can be detected with higher sensitivity.
이와 관련하여, 도 19에 나타낸 바와 같이 E2B, E2B-OVA, E2Y-OVA 및 E2L-OVA 항원을 주입했던 각각 마우스의 혈장에 포함된 항체는, 항원의 종류와 관계없이 모두 E2B 항원을 인지 할 수 있음을 E2B를 ELISA 항원으로 사용한 분석을 통해 확인하였는바, 본 발명의 E2B 항원이 중화항체를 유도할 수 있으면서도, 치쿤군야 바이러스 검출, 항체 생산 등에 있어서 매우 중요한 인식 부위로 작용되는 에피토프 임을 알 수 있다. In this regard, antibodies contained in the plasma of each mouse injected with the E2B, E2B-OVA, E2Y-OVA and E2L-OVA antigens as shown in FIG. 19 can all recognize the E2B antigen regardless of the type of antigen. It was confirmed through an analysis using E2B as an ELISA antigen that the E2B antigen of the present invention can induce a neutralizing antibody and is an epitope that acts as a very important recognition site in the detection of chikungunya virus and antibody production. .
SEQUENCE LISTING <110> KOREA UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> NOVEL EPITOPE OF CHIKUNGUNYA VIRUS AND USE THEREOF <130> P18U13C0185/DP-2018-0294 <160> 15 <170> PatentIn version 3.2 <210> 1 <211> 59 <212> PRT <213> Chikungunya virus <400> 1 Pro Asp Thr Pro Asp Arg Thr Leu Met Ser Gln Gln Ser Gly Asn Val 1 5 10 15 Lys Ile Thr Val Asn Gly Gln Thr Val Arg Tyr Lys Cys Asn Cys Gly 20 25 30 Gly Ser Asn Glu Gly Leu Thr Thr Thr Asp Lys Val Ile Asn Asn Cys 35 40 45 Lys Val Asp Gln Cys His Ala Ala Val Thr Asn 50 55 <210> 2 <211> 80 <212> PRT <213> Chikungunya virus <400> 2 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Pro Asp 1 5 10 15 Thr Pro Asp Arg Thr Leu Met Ser Gln Gln Ser Gly Asn Val Lys Ile 20 25 30 Thr Val Asn Gly Gln Thr Val Arg Tyr Lys Cys Asn Cys Gly Gly Ser 35 40 45 Asn Glu Gly Leu Thr Thr Thr Asp Lys Val Ile Asn Asn Cys Lys Val 50 55 60 Asp Gln Cys His Ala Ala Val Thr Asn Lys Leu Ala Ala Ala Leu Glu 65 70 75 80 <210> 3 <211> 177 <212> DNA <213> Chikungunya virus <400> 3 ccagacaccc ctgatcgcac attaatgtca caacagtccg gcaacgtaaa gatcacagtc 60 aatggccaga cggtgcggta caagtgtaat tgcggtggct caaatgaagg actaacaact 120 acagacaaag tgattaataa ctgcaaggtt gatcaatgtc atgccgcggt caccaat 177 <210> 4 <211> 240 <212> DNA <213> Chikungunya virus <400> 4 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccccagacac ccctgatcgc 60 acattaatgt cacaacagtc cggcaacgta aagatcacag tcaatggcca gacggtgcgg 120 tacaagtgta attgcggtgg ctcaaatgaa ggactaacaa ctacagacaa agtgattaat 180 aactgcaagg ttgatcaatg tcatgccgcg gtcaccaata agcttgcggc cgcactcgag 240 <210> 5 <211> 27 <212> DNA <213> Artificial <220> <223> E2B forward primer <400> 5 cgggatcccc agacacccct gatcgca 27 <210> 6 <211> 26 <212> DNA <213> Artificial <220> <223> E2B reverse primer <400> 6 cccaagctta ttggtgaccg cggcat 26 <210> 7 <211> 30 <212> DNA <213> Artificial <220> <223> E2Y forward primer <400> 7 cgggatcccc atacttagct cactgtcccg 30 <210> 8 <211> 31 <212> DNA <213> Artificial <220> <223> E2Y reverse primer <400> 8 ccaagcttat gagctgtacc ccactatgac t 31 <210> 9 <211> 31 <212> DNA <213> Artificial <220> <223> E2L forward primer <400> 9 cgggatccag caccaaggac aacttcaatg t 31 <210> 10 <211> 31 <212> DNA <213> Artificial <220> <223> E2L reverse primer <400> 10 ccaagcttat gagctgtacc ccactatgac t 31 <210> 11 <211> 1224 <212> DNA <213> Chikungunya virus <400> 11 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccccatactt agctcactgt 60 cccgactgtg gagaagggca ctcgtgccat agtcccgtag cactagaacg catcagaaat 120 gaagcgacag acgggacgct gaaaatccag gtctccttgc aaatcggaat aaagacggat 180 gacagccacg attggaccaa gctgcgttat atggacaacc acatgccagc agacgcagag 240 agggcggggc tatttgtaag aacatcagca ccgtgtacga ttactggaac aatgggacac 300 ttcatcctgg cccgatgtcc aaaaggggaa actctgacgg tgggattcac tgacagtagg 360 aagattagtc actcatgtac gcacccattt caccacgacc ctcctgtgat aggtcgggaa 420 aaattccatt cccgaccgca gcacggtaaa gagctacctt gcagcacgta cgtgcagagc 480 accgccgcaa ctaccgagga gatagaggta cacatgcccc cagacacccc tgatcgcaca 540 ttaatgtcac aacagtccgg caacgtaaag atcacagtca atggccagac ggtgcggtac 600 aagtgtaatt gcggtggctc aaatgaagga ctaacaacta cagacaaagt gattaataac 660 tgcaaggttg atcaatgtca tgccgcggtc accaatcaca aaaagtggca gtataactcc 720 cctctggtcc cgcgtaatgc tgaacttggg gaccgaaaag gaaaaattca catcccgttt 780 ccgctggcaa atgtaacatg cagggtgcct aaagcaagga accccaccgt gacgtacggg 840 aaaaaccaag tcatcatgct actgtatcct gaccacccaa cactcctgtc ctaccggaat 900 atgggagaag aaccaaacta tcaagaagag tgggtgatgc ataagaagga agtcgtgcta 960 accgtgccga ctgaagggct cgaggtcacg tggggcaaca acgagccgta taagtattgg 1020 ccgcagttat ctacaaacgg tacagcccat ggccacccgc atgagataat tctgtattat 1080 tatgagctgt accccactat gactaagctt gtcgagaagt actcagcaga gagcctgaag 1140 atatctcaag ctgtccatgc agcacatgca gaaatcaatg aagcaggcag agaggtggta 1200 gggtcagcag cggccgcact cgag 1224 <210> 12 <211> 408 <212> PRT <213> Chikungunya virus <400> 12 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Pro Tyr 1 5 10 15 Leu Ala His Cys Pro Asp Cys Gly Glu Gly His Ser Cys His Ser Pro 20 25 30 Val Ala Leu Glu Arg Ile Arg Asn Glu Ala Thr Asp Gly Thr Leu Lys 35 40 45 Ile Gln Val Ser Leu Gln Ile Gly Ile Lys Thr Asp Asp Ser His Asp 50 55 60 Trp Thr Lys Leu Arg Tyr Met Asp Asn His Met Pro Ala Asp Ala Glu 65 70 75 80 Arg Ala Gly Leu Phe Val Arg Thr Ser Ala Pro Cys Thr Ile Thr Gly 85 90 95 Thr Met Gly His Phe Ile Leu Ala Arg Cys Pro Lys Gly Glu Thr Leu 100 105 110 Thr Val Gly Phe Thr Asp Ser Arg Lys Ile Ser His Ser Cys Thr His 115 120 125 Pro Phe His His Asp Pro Pro Val Ile Gly Arg Glu Lys Phe His Ser 130 135 140 Arg Pro Gln His Gly Lys Glu Leu Pro Cys Ser Thr Tyr Val Gln Ser 145 150 155 160 Thr Ala Ala Thr Thr Glu Glu Ile Glu Val His Met Pro Pro Asp Thr 165 170 175 Pro Asp Arg Thr Leu Met Ser Gln Gln Ser Gly Asn Val Lys Ile Thr 180 185 190 Val Asn Gly Gln Thr Val Arg Tyr Lys Cys Asn Cys Gly Gly Ser Asn 195 200 205 Glu Gly Leu Thr Thr Thr Asp Lys Val Ile Asn Asn Cys Lys Val Asp 210 215 220 Gln Cys His Ala Ala Val Thr Asn His Lys Lys Trp Gln Tyr Asn Ser 225 230 235 240 Pro Leu Val Pro Arg Asn Ala Glu Leu Gly Asp Arg Lys Gly Lys Ile 245 250 255 His Ile Pro Phe Pro Leu Ala Asn Val Thr Cys Arg Val Pro Lys Ala 260 265 270 Arg Asn Pro Thr Val Thr Tyr Gly Lys Asn Gln Val Ile Met Leu Leu 275 280 285 Tyr Pro Asp His Pro Thr Leu Leu Ser Tyr Arg Asn Met Gly Glu Glu 290 295 300 Pro Asn Tyr Gln Glu Glu Trp Val Met His Lys Lys Glu Val Val Leu 305 310 315 320 Thr Val Pro Thr Glu Gly Leu Glu Val Thr Trp Gly Asn Asn Glu Pro 325 330 335 Tyr Lys Tyr Trp Pro Gln Leu Ser Thr Asn Gly Thr Ala His Gly His 340 345 350 Pro His Glu Ile Ile Leu Tyr Tyr Tyr Glu Leu Tyr Pro Thr Met Thr 355 360 365 Lys Leu Val Glu Lys Tyr Ser Ala Glu Ser Leu Lys Ile Ser Gln Ala 370 375 380 Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val 385 390 395 400 Gly Ser Ala Ala Ala Ala Leu Glu 405 <210> 13 <211> 1368 <212> DNA <213> Chikungunya virus <400> 13 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccagcaccaa ggacaacttc 60 aatgtctata aagccacaag accatactta gctcactgtc ccgactgtgg agaagggcac 120 tcgtgccata gtcccgtagc actagaacgc atcagaaatg aagcgacaga cgggacgctg 180 aaaatccagg tctccttgca aatcggaata aagacggatg acagccacga ttggaccaag 240 ctgcgttata tggacaacca catgccagca gacgcagaga gggcggggct atttgtaaga 300 acatcagcac cgtgtacgat tactggaaca atgggacact tcatcctggc ccgatgtcca 360 aaaggggaaa ctctgacggt gggattcact gacagtagga agattagtca ctcatgtacg 420 cacccatttc accacgaccc tcctgtgata ggtcgggaaa aattccattc ccgaccgcag 480 cacggtaaag agctaccttg cagcacgtac gtgcagagca ccgccgcaac taccgaggag 540 atagaggtac acatgccccc agacacccct gatcgcacat taatgtcaca acagtccggc 600 aacgtaaaga tcacagtcaa tggccagacg gtgcggtaca agtgtaattg cggtggctca 660 aatgaaggac taacaactac agacaaagtg attaataact gcaaggttga tcaatgtcat 720 gccgcggtca ccaatcacaa aaagtggcag tataactccc ctctggtccc gcgtaatgct 780 gaacttgggg accgaaaagg aaaaattcac atcccgtttc cgctggcaaa tgtaacatgc 840 agggtgccta aagcaaggaa ccccaccgtg acgtacggga aaaaccaagt catcatgcta 900 ctgtatcctg accacccaac actcctgtcc taccggaata tgggagaaga accaaactat 960 caagaagagt gggtgatgca taagaaggaa gtcgtgctaa ccgtgccgac tgaagggctc 1020 gaggtcacgt ggggcaacaa cgagccgtat aagtattggc cgcagttatc tacaaacggt 1080 acagcccatg gccacccgca tgagataatt ctgtattatt atgagctgta ccccactatg 1140 actaagcttg tcgagaagta ctcagcagag agcctgaaga tatctcaagc tgtccatgca 1200 gcacatgcag aaatcaatga agcaggcaga gaggtggtag ggtcagcaaa gcttgtcgag 1260 aagtactcag cagagagcct gaagatatct caagctgtcc atgcagcaca tgcagaaatc 1320 aatgaagcag gcagagaggt ggtagggtca gcagcggccg cactcgag 1368 <210> 14 <211> 456 <212> PRT <213> Chikungunya virus <400> 14 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Ser Thr 1 5 10 15 Lys Asp Asn Phe Asn Val Tyr Lys Ala Thr Arg Pro Tyr Leu Ala His 20 25 30 Cys Pro Asp Cys Gly Glu Gly His Ser Cys His Ser Pro Val Ala Leu 35 40 45 Glu Arg Ile Arg Asn Glu Ala Thr Asp Gly Thr Leu Lys Ile Gln Val 50 55 60 Ser Leu Gln Ile Gly Ile Lys Thr Asp Asp Ser His Asp Trp Thr Lys 65 70 75 80 Leu Arg Tyr Met Asp Asn His Met Pro Ala Asp Ala Glu Arg Ala Gly 85 90 95 Leu Phe Val Arg Thr Ser Ala Pro Cys Thr Ile Thr Gly Thr Met Gly 100 105 110 His Phe Ile Leu Ala Arg Cys Pro Lys Gly Glu Thr Leu Thr Val Gly 115 120 125 Phe Thr Asp Ser Arg Lys Ile Ser His Ser Cys Thr His Pro Phe His 130 135 140 His Asp Pro Pro Val Ile Gly Arg Glu Lys Phe His Ser Arg Pro Gln 145 150 155 160 His Gly Lys Glu Leu Pro Cys Ser Thr Tyr Val Gln Ser Thr Ala Ala 165 170 175 Thr Thr Glu Glu Ile Glu Val His Met Pro Pro Asp Thr Pro Asp Arg 180 185 190 Thr Leu Met Ser Gln Gln Ser Gly Asn Val Lys Ile Thr Val Asn Gly 195 200 205 Gln Thr Val Arg Tyr Lys Cys Asn Cys Gly Gly Ser Asn Glu Gly Leu 210 215 220 Thr Thr Thr Asp Lys Val Ile Asn Asn Cys Lys Val Asp Gln Cys His 225 230 235 240 Ala Ala Val Thr Asn His Lys Lys Trp Gln Tyr Asn Ser Pro Leu Val 245 250 255 Pro Arg Asn Ala Glu Leu Gly Asp Arg Lys Gly Lys Ile His Ile Pro 260 265 270 Phe Pro Leu Ala Asn Val Thr Cys Arg Val Pro Lys Ala Arg Asn Pro 275 280 285 Thr Val Thr Tyr Gly Lys Asn Gln Val Ile Met Leu Leu Tyr Pro Asp 290 295 300 His Pro Thr Leu Leu Ser Tyr Arg Asn Met Gly Glu Glu Pro Asn Tyr 305 310 315 320 Gln Glu Glu Trp Val Met His Lys Lys Glu Val Val Leu Thr Val Pro 325 330 335 Thr Glu Gly Leu Glu Val Thr Trp Gly Asn Asn Glu Pro Tyr Lys Tyr 340 345 350 Trp Pro Gln Leu Ser Thr Asn Gly Thr Ala His Gly His Pro His Glu 355 360 365 Ile Ile Leu Tyr Tyr Tyr Glu Leu Tyr Pro Thr Met Thr Lys Leu Val 370 375 380 Glu Lys Tyr Ser Ala Glu Ser Leu Lys Ile Ser Gln Ala Val His Ala 385 390 395 400 Ala His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val Gly Ser Ala 405 410 415 Lys Leu Val Glu Lys Tyr Ser Ala Glu Ser Leu Lys Ile Ser Gln Ala 420 425 430 Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val 435 440 445 Gly Ser Ala Ala Ala Ala Leu Glu 450 455 <210> 15 <211> 3 <212> DNA <213> Artificial <220> <223> his tag sequence <400> 15 cac 3 SEQUENCE LISTING <110> KOREA UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> NOVEL EPITOPE OF CHIKUNGUNYA VIRUS AND USE THEREOF <130> P18U13C0185/DP-2018-0294 <160> 15 <170> PatentIn version 3.2 <210> 1 <211> 59 <212> PRT <213> Chikungunya virus <400> 1 Pro Asp Thr Pro Asp Arg Thr Leu Met Ser Gln Gln Ser Gly Asn Val 1 5 10 15 Lys Ile Thr Val Asn Gly Gln Thr Val Arg Tyr Lys Cys Asn Cys Gly 20 25 30 Gly Ser Asn Glu Gly Leu Thr Thr Thr Asp Lys Val Ile Asn Asn Cys 35 40 45 Lys Val Asp Gln Cys His Ala Ala Val Thr Asn 50 55 <210> 2 <211> 80 <212> PRT <213> Chikungunya virus <400> 2 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Pro Asp 1 5 10 15 Thr Pro Asp Arg Thr Leu Met Ser Gln Gln Ser Gly Asn Val Lys Ile 20 25 30 Thr Val Asn Gly Gln Thr Val Arg Tyr Lys Cys Asn Cys Gly Gly Ser 35 40 45 Asn Glu Gly Leu Thr Thr Thr Asp Lys Val Ile Asn Asn Cys Lys Val 50 55 60 Asp Gln Cys His Ala Ala Val Thr Asn Lys Leu Ala Ala Ala Leu Glu 65 70 75 80 <210> 3 <211> 177 <212> DNA <213> Chikungunya virus <400> 3 ccagacaccc ctgatcgcac attaatgtca caacagtccg gcaacgtaaa gatcacagtc 60 aatggccaga cggtgcggta caagtgtaat tgcggtggct caaatgaagg actaacaact 120 acagacaaag tgattaataa ctgcaaggtt gatcaatgtc atgccgcggt caccaat 177 <210> 4 <211> 240 <212> DNA <213> Chikungunya virus <400> 4 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccccagacac ccctgatcgc 60 acattaatgt cacaacagtc cggcaacgta aagatcacag tcaatggcca gacggtgcgg 120 tacaagtgta attgcggtgg ctcaaatgaa ggactaacaa ctacagacaa agtgattaat 180 aactgcaagg ttgatcaatg tcatgccgcg gtcaccaata agcttgcggc cgcactcgag 240 <210> 5 <211> 27 <212> DNA <213> Artificial <220> <223> E2B forward primer <400> 5 cgggatcccc agacacccct gatcgca 27 <210> 6 <211> 26 <212> DNA <213> Artificial <220> <223> E2B reverse primer <400> 6 cccaagctta ttggtgaccg cggcat 26 <210> 7 <211> 30 <212> DNA <213> Artificial <220> <223> E2Y forward primer <400> 7 cgggatcccc atacttagct cactgtcccg 30 <210> 8 <211> 31 <212> DNA <213> Artificial <220> <223> E2Y reverse primer <400> 8 ccaagcttat gagctgtacc ccactatgac t 31 <210> 9 <211> 31 <212> DNA <213> Artificial <220> <223> E2L forward primer <400> 9 cgggatccag caccaaggac aacttcaatg t 31 <210> 10 <211> 31 <212> DNA <213> Artificial <220> <223> E2L reverse primer <400> 10 ccaagcttat gagctgtacc ccactatgac t 31 <210> 11 <211> 1224 <212> DNA <213> Chikungunya virus <400> 11 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccccatactt agctcactgt 60 cccgactgtg gagaagggca ctcgtgccat agtcccgtag cactagaacg catcagaaat 120 gaagcgacag acgggacgct gaaaatccag gtctccttgc aaatcggaat aaagacggat 180 gacagccacg attggaccaa gctgcgttat atggacaacc acatgccagc agacgcagag 240 agggcggggc tatttgtaag aacatcagca ccgtgtacga ttactggaac aatgggacac 300 ttcatcctgg cccgatgtcc aaaaggggaa actctgacgg tgggattcac tgacagtagg 360 aagattagtc actcatgtac gcacccattt caccacgacc ctcctgtgat aggtcgggaa 420 aaattccatt cccgaccgca gcacggtaaa gagctacctt gcagcacgta cgtgcagagc 480 accgccgcaa ctaccgagga gatagaggta cacatgcccc cagacacccc tgatcgcaca 540 ttaatgtcac aacagtccgg caacgtaaag atcacagtca atggccagac ggtgcggtac 600 aagtgtaatt gcggtggctc aaatgaagga ctaacaacta cagacaaagt gattaataac 660 tgcaaggttg atcaatgtca tgccgcggtc accaatcaca aaaagtggca gtataactcc 720 cctctggtcc cgcgtaatgc tgaacttggg gaccgaaaag gaaaaattca catcccgttt 780 ccgctggcaa atgtaacatg cagggtgcct aaagcaagga accccaccgt gacgtacggg 840 aaaaaccaag tcatcatgct actgtatcct gaccacccaa cactcctgtc ctaccggaat 900 atgggagaag aaccaaacta tcaagaagag tgggtgatgc ataagaagga agtcgtgcta 960 accgtgccga ctgaagggct cgaggtcacg tggggcaaca acgagccgta taagtattgg 1020 ccgcagttat ctacaaacgg tacagcccat ggccacccgc atgagataat tctgtattat 1080 tatgagctgt accccactat gactaagctt gtcgagaagt actcagcaga gagcctgaag 1140 atatctcaag ctgtccatgc agcacatgca gaaatcaatg aagcaggcag agaggtggta 1200 gggtcagcag cggccgcact cgag 1224 <210> 12 <211> 408 <212> PRT <213> Chikungunya virus <400> 12 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Pro Tyr 1 5 10 15 Leu Ala His Cys Pro Asp Cys Gly Glu Gly His Ser Cys His Ser Pro 20 25 30 Val Ala Leu Glu Arg Ile Arg Asn Glu Ala Thr Asp Gly Thr Leu Lys 35 40 45 Ile Gln Val Ser Leu Gln Ile Gly Ile Lys Thr Asp Asp Ser His Asp 50 55 60 Trp Thr Lys Leu Arg Tyr Met Asp Asn His Met Pro Ala Asp Ala Glu 65 70 75 80 Arg Ala Gly Leu Phe Val Arg Thr Ser Ala Pro Cys Thr Ile Thr Gly 85 90 95 Thr Met Gly His Phe Ile Leu Ala Arg Cys Pro Lys Gly Glu Thr Leu 100 105 110 Thr Val Gly Phe Thr Asp Ser Arg Lys Ile Ser His Ser Cys Thr His 115 120 125 Pro Phe His His Asp Pro Pro Val Ile Gly Arg Glu Lys Phe His Ser 130 135 140 Arg Pro Gln His Gly Lys Glu Leu Pro Cys Ser Thr Tyr Val Gln Ser 145 150 155 160 Thr Ala Ala Thr Thr Glu Glu Ile Glu Val His Met Pro Pro Asp Thr 165 170 175 Pro Asp Arg Thr Leu Met Ser Gln Gln Ser Gly Asn Val Lys Ile Thr 180 185 190 Val Asn Gly Gln Thr Val Arg Tyr Lys Cys Asn Cys Gly Gly Ser Asn 195 200 205 Glu Gly Leu Thr Thr Thr Asp Lys Val Ile Asn Asn Cys Lys Val Asp 210 215 220 Gln Cys His Ala Ala Val Thr Asn His Lys Lys Trp Gln Tyr Asn Ser 225 230 235 240 Pro Leu Val Pro Arg Asn Ala Glu Leu Gly Asp Arg Lys Gly Lys Ile 245 250 255 His Ile Pro Phe Pro Leu Ala Asn Val Thr Cys Arg Val Pro Lys Ala 260 265 270 Arg Asn Pro Thr Val Thr Tyr Gly Lys Asn Gln Val Ile Met Leu Leu 275 280 285 Tyr Pro Asp His Pro Thr Leu Leu Ser Tyr Arg Asn Met Gly Glu Glu 290 295 300 Pro Asn Tyr Gln Glu Glu Trp Val Met His Lys Lys Glu Val Val Leu 305 310 315 320 Thr Val Pro Thr Glu Gly Leu Glu Val Thr Trp Gly Asn Asn Glu Pro 325 330 335 Tyr Lys Tyr Trp Pro Gln Leu Ser Thr Asn Gly Thr Ala His Gly His 340 345 350 Pro His Glu Ile Ile Leu Tyr Tyr Tyr Glu Leu Tyr Pro Thr Met Thr 355 360 365 Lys Leu Val Glu Lys Tyr Ser Ala Glu Ser Leu Lys Ile Ser Gln Ala 370 375 380 Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val 385 390 395 400 Gly Ser Ala Ala Ala Ala Leu Glu 405 <210> 13 <211> 1368 <212> DNA <213> Chikungunya virus <400> 13 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccagcaccaa ggacaacttc 60 aatgtctata aagccacaag accatactta gctcactgtc ccgactgtgg agaagggcac 120 tcgtgccata gtcccgtagc actagaacgc atcagaaatg aagcgacaga cgggacgctg 180 aaaatccagg tctccttgca aatcggaata aagacggatg acagccacga ttggaccaag 240 ctgcgttata tggacaacca catgccagca gacgcagaga gggcggggct atttgtaaga 300 acatcagcac cgtgtacgat tactggaaca atgggacact tcatcctggc ccgatgtcca 360 aaaggggaaa ctctgacggt gggattcact gacagtagga agattagtca ctcatgtacg 420 cacccatttc accacgaccc tcctgtgata ggtcgggaaa aattccattc ccgaccgcag 480 cacggtaaag agctaccttg cagcacgtac gtgcagagca ccgccgcaac taccgaggag 540 atagaggtac acatgccccc agacacccct gatcgcacat taatgtcaca acagtccggc 600 aacgtaaaga tcacagtcaa tggccagacg gtgcggtaca agtgtaattg cggtggctca 660 aatgaaggac taacaactac agacaaagtg attaataact gcaaggttga tcaatgtcat 720 gccgcggtca ccaatcacaa aaagtggcag tataactccc ctctggtccc gcgtaatgct 780 gaacttgggg accgaaaagg aaaaattcac atcccgtttc cgctggcaaa tgtaacatgc 840 agggtgccta aagcaaggaa ccccaccgtg acgtacggga aaaaccaagt catcatgcta 900 ctgtatcctg accacccaac actcctgtcc taccggaata tgggagaaga accaaactat 960 caagaagagt gggtgatgca taagaaggaa gtcgtgctaa ccgtgccgac tgaagggctc 1020 gaggtcacgt ggggcaacaa cgagccgtat aagtattggc cgcagttatc tacaaacggt 1080 acagcccatg gccacccgca tgagataatt ctgtattatt atgagctgta ccccactatg 1140 actaagcttg tcgagaagta ctcagcagag agcctgaaga tatctcaagc tgtccatgca 1200 gcacatgcag aaatcaatga agcaggcaga gaggtggtag ggtcagcaaa gcttgtcgag 1260 aagtactcag cagagagcct gaagatatct caagctgtcc atgcagcaca tgcagaaatc 1320 aatgaagcag gcagagaggt ggtagggtca gcagcggccg cactcgag 1368 <210> 14 <211> 456 <212> PRT <213> Chikungunya virus <400> 14 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Ser Thr 1 5 10 15 Lys Asp Asn Phe Asn Val Tyr Lys Ala Thr Arg Pro Tyr Leu Ala His 20 25 30 Cys Pro Asp Cys Gly Glu Gly His Ser Cys His Ser Pro Val Ala Leu 35 40 45 Glu Arg Ile Arg Asn Glu Ala Thr Asp Gly Thr Leu Lys Ile Gln Val 50 55 60 Ser Leu Gln Ile Gly Ile Lys Thr Asp Asp Ser His Asp Trp Thr Lys 65 70 75 80 Leu Arg Tyr Met Asp Asn His Met Pro Ala Asp Ala Glu Arg Ala Gly 85 90 95 Leu Phe Val Arg Thr Ser Ala Pro Cys Thr Ile Thr Gly Thr Met Gly 100 105 110 His Phe Ile Leu Ala Arg Cys Pro Lys Gly Glu Thr Leu Thr Val Gly 115 120 125 Phe Thr Asp Ser Arg Lys Ile Ser His Ser Cys Thr His Pro Phe His 130 135 140 His Asp Pro Pro Val Ile Gly Arg Glu Lys Phe His Ser Arg Pro Gln 145 150 155 160 His Gly Lys Glu Leu Pro Cys Ser Thr Tyr Val Gln Ser Thr Ala Ala 165 170 175 Thr Thr Glu Glu Ile Glu Val His Met Pro Pro Asp Thr Pro Asp Arg 180 185 190 Thr Leu Met Ser Gln Gln Ser Gly Asn Val Lys Ile Thr Val Asn Gly 195 200 205 Gln Thr Val Arg Tyr Lys Cys Asn Cys Gly Gly Ser Asn Glu Gly Leu 210 215 220 Thr Thr Thr Asp Lys Val Ile Asn Asn Cys Lys Val Asp Gln Cys His 225 230 235 240 Ala Ala Val Thr Asn His Lys Lys Trp Gln Tyr Asn Ser Pro Leu Val 245 250 255 Pro Arg Asn Ala Glu Leu Gly Asp Arg Lys Gly Lys Ile His Ile Pro 260 265 270 Phe Pro Leu Ala Asn Val Thr Cys Arg Val Pro Lys Ala Arg Asn Pro 275 280 285 Thr Val Thr Tyr Gly Lys Asn Gln Val Ile Met Leu Leu Tyr Pro Asp 290 295 300 His Pro Thr Leu Leu Ser Tyr Arg Asn Met Gly Glu Glu Pro Asn Tyr 305 310 315 320 Gln Glu Glu Trp Val Met His Lys Lys Glu Val Val Leu Thr Val Pro 325 330 335 Thr Glu Gly Leu Glu Val Thr Trp Gly Asn Asn Glu Pro Tyr Lys Tyr 340 345 350 Trp Pro Gln Leu Ser Thr Asn Gly Thr Ala His Gly His Pro His Glu 355 360 365 Ile Ile Leu Tyr Tyr Tyr Glu Leu Tyr Pro Thr Met Thr Lys Leu Val 370 375 380 Glu Lys Tyr Ser Ala Glu Ser Leu Lys Ile Ser Gln Ala Val His Ala 385 390 395 400 Ala His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val Gly Ser Ala 405 410 415 Lys Leu Val Glu Lys Tyr Ser Ala Glu Ser Leu Lys Ile Ser Gln Ala 420 425 430 Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val 435 440 445 Gly Ser Ala Ala Ala Ala Leu Glu 450 455 <210> 15 <211> 3 <212> DNA <213> Artificial <220> <223> his tag sequence <400> 15 cac 3
Claims (15)
상기 배양물을 항생제를 포함하는 다른 배지에 옮기고, 이에 IPTG(isopropyl-β-D-galactopyranoside)를 첨가하여 형질전환된 단백질에서 재조합 단백질 발현을 유도하는 것;
상기 배양물을 원심분리하여 대장균 세포를 침전하는 것;
상기 침전된 대장균을 Tris, 염화나트륨 및 염화 마그네슘을 포함하는 완충용액에 현탁하고, 파쇄하는 것;
상기 파쇄물을 원심분리한 후, Tris 및 염화나트륨을 포함하는 완충용액으로 평형된 니켈-니트릴로트리아세틱 액시드(Ni-NTA)칼럼에 결합시킨 후 0-0.5M 이미다졸 농도구배를 이용하여 히스티딘 태그(His 태그)와 서열번호 1 또는 서열번호 2의 아미노산 서열의 재조합 단백질을 수득하는 것을 포함하는, 재조합 단백질 생산방법. E. coli transformed with a recombinant expression vector containing a polynucleotide encoding a polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 amino acid sequence and histidine tag (His tag) sequence is used as a medium containing antibiotics, baktotripton, yeast extract and sodium chloride To cultivate in;
Transferring the culture to another medium containing antibiotics, and adding isopropyl-β-D-galactopyranoside (IPTG) thereto to induce expression of the recombinant protein in the transformed protein;
Centrifuging the culture to precipitate E. coli cells;
Suspending and crushing the precipitated E. coli in a buffer solution containing Tris, sodium chloride and magnesium chloride;
After centrifuging the lysate, the mixture was bound to a nickel-nitrilotriacetic acid (Ni-NTA) column equilibrated with a buffer solution containing Tris and sodium chloride, and a histidine tag using a 0-0.5M imidazole concentration gradient (His tag) and a method for producing a recombinant protein comprising obtaining a recombinant protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
제1항에서 수득한 서열번호 1 또는 서열번호 2의 아미노산 서열의 재조합 단백질을 Tris 및 염화나트륨을 포함하는 완충용액으로 평형된 젤 필트레이션 칼럼에 주입시킨 후 단백질의 분자량에 의해 분리하여 정제하는 것을 더 포함하는 재조합 단백질 생산방법.The method of claim 1,
After injecting the recombinant protein of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 obtained in claim 1 into a gel filtration column equilibrated with a buffer solution containing Tris and sodium chloride, separation and purification by the molecular weight of the protein are further performed. Method for producing a recombinant protein comprising.
상기 방법은 서열번호 1 또는 서열번호 2 아미노산 서열의 폴리펩티드를 코딩하는 폴리뉴클레오티드를 포함하는 재조합 발현 벡터로 형질전환된 대장균을 엠피실린가 함유되고, 박토트립톤, 효모추출물 및 염화나트륨을 포함하는 루리아 배지(LB)에서 12시간 동안 진탕 배양하는 것이고,
상기 진탕 배양물을 카나마이신을 포함하는 다른 루리아 배지에 옮기고, 37 ℃에서 박테리아의 흡광도가 600nm에서 0.6이 될 때, IPTG(isopropyl-β-D-galactopyranoside)를 최종농도가 1 mM이 첨가하여 형질전환된 단백질에서 재조합 단백질 발현을 유도하는 것인, 재조합 단백질 생산방법.The method of claim 1,
The above method comprises E. coli transformed with a recombinant expression vector containing a polynucleotide encoding a polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 amino acid sequence, containing empicillin, and Luria medium containing baktotripton, yeast extract and sodium chloride ( LB) for 12 hours shaking culture,
Transfer the shaking culture to another Luria medium containing kanamycin, and when the absorbance of the bacteria is 0.6 at 600 nm at 37°C, isopropyl-β-D-galactopyranoside (IPTG) is added to a final concentration of 1 mM for transformation. To induce the expression of the recombinant protein in the protein, recombinant protein production method.
상기 IPTG 첨가한 후 단백질 발현은 18시간 동안 유도하고, 5000rpm에서 10분간 원심분리하는 것인, 재조합단백질 생산방법. The method of claim 1,
After the IPTG is added, protein expression is induced for 18 hours and centrifuged at 5000 rpm for 10 minutes.
완충용액은 Tris는 20 mM, 염화나트륨은 100 mM 또는 염화마그네슘은 5 mM으로 포함하는 것인, 재조합 단백질 생산방법.The method according to any one of claims 1 to 4,
The buffer solution is 20 mM of Tris, 100 mM of sodium chloride, or 5 mM of magnesium chloride.
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