KR20230124282A - Antibody for Detection Specific for Non-structural protein 1 of ZIKA virus and Use thereof - Google Patents
Antibody for Detection Specific for Non-structural protein 1 of ZIKA virus and Use thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
본 발명의 지카 바이러스의 비구조 단백질 1 (Non-structural protein 1, NS1)에 특이적인 항체에 관한 것이다. 보다 구체적으로는, 본 발명의 항체는 지카 바이러스의 NS1에 특이적으로 결합하는 능력을 가지고 다른 플라비바이러스에는 특이적이지 않으므로, 지카 바이러스의 진단에 매우 유용하다.The present invention relates to an antibody specific to non-structural protein 1 (NS1) of Zika virus. More specifically, the antibody of the present invention has the ability to specifically bind to NS1 of Zika virus and is not specific to other flaviviruses, so it is very useful for diagnosis of Zika virus.
Description
지카 바이러스 (ZIKA virus)의 비구조 단백질 1 (Non-structural protein 1, NS1)에 특이적으로 결합할 수 있는 진단용 항체 및 이의 용도에 관한 것이다.It relates to a diagnostic antibody capable of specifically binding to non-structural protein 1 (NS1) of ZIKA virus and its use.
지카 바이러스(Zika Virus)는 대표적인 모기매개(Mosquito-Borne) 바이러스 중의 하나다. 지카 바이러스는 단일 (+)가닥 RNA 바이러스(Single Positive-Stranded RNA Virus)로, 플라비비리데과(Flaviviridae Family)의 하위 속인 플라비바이러스속(Flavivirus Genus)에 해당한다. 플라비바이러스에는 지카 바이러스 외에도 뎅기 바이러스(Dengue Virus), 황열 바이러스(Yellow Fever Virus), 일본뇌염 바이러스(Japanese Encephalitis Virus), 세인트루이스 뇌염 바이러스(St. Louis Encephalitis Virus), 웨스트나일 바이러스(West Nile Virus) 등이 있다.Zika virus is one of the representative mosquito-borne (Mosquito-Borne) viruses. Zika virus is a single positive-stranded RNA virus and corresponds to the Flavivirus Genus, a subgenus of the Flaviviridae Family. In addition to Zika virus, Flaviviruses include Dengue Virus, Yellow Fever Virus, Japanese Encephalitis Virus, St. Louis Encephalitis Virus, and West Nile Virus. etc.
플라비바이러스는 모두 구형(Spherical)으로, 약 10,000 내지 11,000개의 염기(Base)로 구성된 공통의 구조를 가지고 있다. 플라비바이러스를 구성하는 단백질은 크게 구조단백질(Structural Protein)과 비구조 단백질(Non-Structural Protein)로 분류할 수 있다. 아직까지 모든 단백질의 기능이나 역할이 규명되지는 않았지만, 전구체 막 단백질, 외피 단백질, 비구조 단백질 1, 비구조 단백질 2A 및 2B의 일부, 비구조 단백질 4A 및 4B의 일부가 바이러스 외부에 노출되어 있는 것으로 파악되고 있다. 따라서 플라비바이러스에 해당하는 지카 바이러스를, 항체를 이용하여 검출하고자 할 경우 지카 바이러스의 외부에 노출되어 있어 항체와의 접근 및 결합(또는 반응)이 용이한 외피 단백질, 비구조 단백질 1, 비구조 단백질 2A 및 2B, 비구조 단백질 4A 및 4B 등이 유용한 바이오마커(타겟, 목표물질)라고 할 수 있다.Flaviviruses are all spherical and have a common structure consisting of about 10,000 to 11,000 bases. Proteins constituting flaviviruses can be largely classified into structural proteins and non-structural proteins. Although the functions or roles of all proteins have not yet been identified, precursor membrane proteins, envelope proteins, non-structural protein 1, parts of non-structural proteins 2A and 2B, and parts of non-structural proteins 4A and 4B are exposed to the outside of the virus. is understood to be Therefore, when detecting the Zika virus corresponding to the flavivirus using an antibody, the envelope protein, nonstructural protein 1, and nonstructural protein 1, which are exposed to the outside of the Zika virus and are easily accessible and bonded (or reacted) with the antibody Proteins 2A and 2B, non-structural proteins 4A and 4B, and the like are useful biomarkers (targets, targets).
최근 소아기 감염 및 지카 바이러스 감염과 관련된 잠재적인 소두증 및 기타 신경계 질환의 증가로 지카 바이러스 감염을 탐지하는 실험실 검사에 대한 수요가 증가하게 되었다. 이러한 내용에서, 지카 바이러스 감염과 다른 플라비바이러스의 감염을 구분하기 위해서는 항체의 높은 특이성이 필요하다. 그러나, 알려진 항-지카 항체는 전형적으로 다른 플라비바이러스에 대해 교차-반응성이어서, 지카 바이러스 감염을 다른 플라비바이러스의 감염과 구별하는데 유용하지 않다.The recent rise in childhood infections and latent microcephaly and other neurologic diseases associated with Zika virus infection has increased the demand for laboratory tests to detect Zika virus infection. In this context, high specificity of the antibody is required to distinguish Zika virus infection from infection with other flaviviruses. However, known anti-Zika antibodies are typically cross-reactive to other flaviviruses and are therefore not useful for differentiating Zika virus infections from those of other flaviviruses.
따라서, 다른 플라비바이러스에는 특이적이지 않으면서, 지카 바이러스에만 특이적인 항체 및 이를 이용한 진단 방법의 개발의 필요성이 요구되고 있다.Therefore, there is a need for the development of antibodies specific only to Zika virus and diagnostic methods using the same, while not being specific to other flaviviruses.
본 발명자들은 상기 문제점을 해결하고자 지카 바이러스의 비구조 단백질 1 (Non-structural protein 1, NS1)에 특이적인 결합 능력을 갖는 항체를 개발하였고, 이 항체가 지카 바이러스에 대하여 우수한 결합력을 가짐을 확인함으로써 본 발명을 완성하였다.In order to solve the above problems, the present inventors developed an antibody having specific binding ability to non-structural protein 1 (NS1) of Zika virus, and by confirming that this antibody has excellent binding ability to Zika virus completed the present invention.
일 양상은 지카 바이러스(ZIKV)의 비구조 단백질 1(Non-structural protein 1)에 특이적으로 결합하며, 중쇄 가변영역과 경쇄 가변영역을 포함하는, 지카 바이러스 특이적 항체 또는 이의 기능적 단편을 제공하는 것이다.One aspect is to provide a Zika virus-specific antibody or functional fragment thereof that specifically binds to non-structural protein 1 of Zika virus (ZIKV) and includes a heavy chain variable region and a light chain variable region will be.
다른 양상은 상기 항체 또는 이의 결합 단편을 암호화하는, 폴리뉴클레오티드를 제공하는 것이다.Another aspect is to provide a polynucleotide encoding the antibody or binding fragment thereof.
다른 양상은 상기 핵산 분자가 삽입된 발현 벡터를 제공하는 것이다.Another aspect is to provide an expression vector into which the nucleic acid molecule is inserted.
다른 양상은 상기 항체 또는 이의 결합 단편을 생산하는 하이브리도마 세포를 제공하는 것이다.Another aspect is to provide a hybridoma cell that produces the antibody or binding fragment thereof.
다른 양상은 지카 바이러스(ZIKV) 항원을 진단하는 제제를 포함하는, 지카 바이러스 진단용 키트를 제공하는 것이다.Another aspect is to provide a kit for diagnosing Zika virus, comprising an agent for diagnosing a Zika virus (ZIKV) antigen.
다른 양상은 상기 항체 또는 이의 결합 단편을 포함하는, 지카 바이러스 진단용 조성물을 제공하는 것이다.Another aspect is to provide a composition for diagnosing Zika virus comprising the antibody or binding fragment thereof.
1. 지카 바이러스(ZIKV)의 비구조 단백질 1(Non-structural protein 1)에 특이적으로 결합하며, 1. It binds specifically to non-structural protein 1 of Zika virus (ZIKV),
중쇄 가변영역과 경쇄 가변영역을 포함하는, 지카 바이러스 특이적 항체 또는 이의 결합 단편.A Zika virus-specific antibody or binding fragment thereof comprising a heavy chain variable region and a light chain variable region.
2. 위 1에 있어서, 상기 중쇄 가변영역은 서열번호 1의 아미노산 서열을 포함하는 CDR1-VH, 서열번호 2의 아미노산 서열을 포함하는 CDR2-VH 및 서열번호 3의 아미노산 서열을 포함하는 CDR3-VH를 포함하며,2. The method of 1 above, wherein the heavy chain variable region is CDR1-VH comprising the amino acid sequence of SEQ ID NO: 1, CDR2-VH comprising the amino acid sequence of SEQ ID NO: 2, and CDR3-VH comprising the amino acid sequence of SEQ ID NO: 3 Including,
상기 경쇄 가변영역은 서열번호 4의 아미노산 서열을 포함하는 CDR1-VK, 서열번호 5의 아미노산 서열을 포함하는 CDR2-VK 및 서열번호 6의 아미노산 서열을 포함하는 CDR3-VK를 포함하는 것을 특징으로 하는, 지카 바이러스 특이적 항체 또는 이의 결합 단편.The light chain variable region comprises CDR1-VK comprising the amino acid sequence of SEQ ID NO: 4, CDR2-VK comprising the amino acid sequence of SEQ ID NO: 5, and CDR3-VK comprising the amino acid sequence of SEQ ID NO: 6. , Zika virus-specific antibodies or binding fragments thereof.
3. 위 1에 있어서, 상기 중쇄 가변영역은 서열번호 17의 아미노산 서열을 포함하는 CDR1-VH, 서열번호 18의 아미노산 서열을 포함하는 CDR2-VH 및 서열번호 19의 아미노산 서열을 포함하는 CDR3-VH를 포함하며,3. The method of 1 above, wherein the heavy chain variable region is CDR1-VH comprising the amino acid sequence of SEQ ID NO: 17, CDR2-VH comprising the amino acid sequence of SEQ ID NO: 18, and CDR3-VH comprising the amino acid sequence of SEQ ID NO: 19 Including,
상기 경쇄 가변영역은 서열번호 20의 아미노산 서열을 포함하는 CDR1-VK, 서열번호 21의 아미노산 서열을 포함하는 CDR2-VK 및 서열번호 22의 아미노산 서열을 포함하는 CDR3-VK를 포함하는 것을 특징으로 하는, 지카 바이러스 특이적 항체 또는 이의 결합 단편.The light chain variable region comprises CDR1-VK comprising the amino acid sequence of SEQ ID NO: 20, CDR2-VK comprising the amino acid sequence of SEQ ID NO: 21, and CDR3-VK comprising the amino acid sequence of SEQ ID NO: 22. , Zika virus-specific antibodies or binding fragments thereof.
4. 위 1 내지 3 중 어느 한 항에 있어서, 상기 항체 또는 그의 결합 단편은 scFv 절편, scFv-Fc 절편, Fab 절편, Fv 절편, 디아바디(diabody), 키메라 항체, 인간화 항체 또는 인간 항체인, 항체 또는 이의 결합 단편.4. The antibody or binding fragment thereof according to any one of 1 to 3 above, wherein the antibody or binding fragment thereof is a scFv fragment, a scFv-Fc fragment, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody, Antibodies or binding fragments thereof.
5. 위 4에 있어서, 상기 항체 또는 그의 결합 단편은 Fab 절편인, 항체 또는 이의 결합 단편.5. The antibody or binding fragment thereof according to 4 above, wherein the antibody or binding fragment thereof is a Fab fragment.
6. 위 1 내지 3 중 어느 한 항의 항체 또는 이의 결합 단편을 암호화하는, 폴리뉴클레오티드.6. A polynucleotide encoding the antibody or binding fragment thereof according to any one of 1 to 3 above.
7. 위 6에 있어서, 상기 폴리뉴클레오티드는 서열번호 7의 염기서열 및 서열번호 8의 염기서열을 포함하는 것인, 폴리뉴클레오티드.7. The polynucleotide according to 6 above, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 8.
8. 위 6에 있어서, 상기 폴리뉴클레오티드는 서열번호 23의 염기서열 및 서열번호 24의 염기서열을 포함하는 것인, 폴리뉴클레오티드.8. The polynucleotide according to 6 above, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 23 and the nucleotide sequence of SEQ ID NO: 24.
9. 위 6의 폴리뉴클레오티드가 삽입된 발현 벡터.9. An expression vector into which the polynucleotide of 6 above is inserted.
10. 위 9에 있어서, 상기 폴리뉴클레오티드는 서열번호 7의 염기서열 및 서열번호 8의 염기서열을 포함하는 것인, 발현 벡터.10. The expression vector according to 9 above, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 8.
11. 위 9에 있어서, 상기 폴리뉴클레오티드는 서열번호 23의 염기서열 및 서열번호 24의 염기서열을 포함하는 것인, 발현 벡터.11. The expression vector according to 9 above, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 23 and the nucleotide sequence of SEQ ID NO: 24.
12. 위 1 내지 3 중 어느 한 항의 항체 또는 이의 결합 단편을 생산하는 하이브리도마 세포.12. A hybridoma cell producing the antibody or binding fragment thereof of any one of 1 to 3 above.
13. 지카 바이러스(ZIKV) 항원을 진단하는 제제를 포함하는, 지카 바이러스 진단용 키트로서,13. A kit for diagnosing Zika virus (ZIKV), including an agent for diagnosing a Zika virus (ZIKV) antigen,
상기 바이러스 항원은 지카 바이러스의 비구조 단백질 1(Non-structural protein 1) 이고, The viral antigen is non-structural protein 1 of Zika virus,
상기 제제는 청구항 1 내지 3 중 어느 한 항의 항체 또는 이의 결합 단편인, 진단용 키트.The agent is an antibody or a binding fragment thereof according to any one of claims 1 to 3, a diagnostic kit.
14. 위 1 내지 3 중 어느 한 항의 항체 또는 이의 결합 단편을 포함하는, 지카 바이러스 진단용 조성물.14. A composition for diagnosis of Zika virus comprising the antibody or binding fragment thereof of any one of 1 to 3 above.
본 발명의 항체는 지카 바이러스의 비구조 단백질 1 (Non-structural protein 1, NS1)에 특이적인 결합 능력을 가지고, 다른 플라비바이러스에는 특이적이지 않으므로, 지카 바이러스 감염 환자 유래 생물학적 샘플 내 존재하는 지카 바이러스 항체가 효과적으로 결합할 수 있게 된다. 지카 바이러스 특이적 항원을 인지하는 항체의 효과적 결합은 진단시 예민성(민감성) 및 정확도를 획기적으로 높일 수 있다.Since the antibody of the present invention has a specific binding ability to non-structural protein 1 (NS1) of Zika virus and is not specific to other flaviviruses, Zika virus-infected patients are present in biological samples. Viral antibodies can bind effectively. Effective binding of antibodies that recognize Zika virus-specific antigens can dramatically increase sensitivity (sensitivity) and accuracy in diagnosis.
도 1은 지카 바이러스 NS1 단백질 (ZIKV NS1 protein)의 생산을 위한 구조체를 나타낸 도이다.
도 2는 지카 바이러스의 비구조 단백질 1 (Non-structural protein 1, NS1) 항원 단백질을 분리 및 정제한 것을 나타낸 도이다.
도 3은 지카 NS1_CTD에 대한 Fusion screening 결과를 나타낸 도이다.
도 4는 지카 NS1_CTD fusion 클론의 지카 NS1과 뎅기 NS1에 대한 ELISA 테스트 결과를 나타낸 도이다.
도 5는 지카 NS1_CTD 단클론 항체 정제 단백질의 SDS-PAGE 분석 결과를 나타낸 도이다.
도 6은 지카 바이러스 NS1 특이적 단일사슬항체의 지카 바이러스 NS1 항원 및 뎅기 바이러스 NS1 항원에 대한 결합 정도를 확인한 도이다.
도 7은 지카 바이러스 진단용 항체 페어를 포함하는 현장 검사용 신속진단키트를 나타낸 도이다. 도 7a는 지카 바이러스 특이적 항체의 농도에 따른 지카 바이러스 특이적 진단 결과를 나타낸 도이다. 도 7b는 지카 바이러스 특이적 항체의 농도가 변화하더라도 뎅기 바이러스에는 결합하지 않은 것을 보여주는 도이다.
도 8은 지카 바이러스 특이적 항체 중 4B1 항체의 VH 및 VL의 염기서열을 나타낸 도이다.
도 9는 지카 바이러스 특이적 항체 중 5D12 항체의 VH 및 VL의 염기서열을 나타낸 도이다.1 is a diagram showing a construct for production of Zika virus NS1 protein (ZIKV NS1 protein).
2 is a diagram showing the separation and purification of non-structural protein 1 (NS1) antigen protein of Zika virus.
3 is a diagram showing the results of fusion screening for Zika NS1_CTD.
4 is a diagram showing ELISA test results for Zika NS1 and Dengue NS1 of the Zika NS1_CTD fusion clone.
5 is a diagram showing the results of SDS-PAGE analysis of Zika NS1_CTD monoclonal antibody purified protein.
6 is a diagram confirming the degree of binding of Zika virus NS1-specific single chain antibodies to Zika virus NS1 antigen and dengue virus NS1 antigen.
7 is a diagram showing a rapid diagnostic kit for on-site testing including an antibody pair for diagnosing Zika virus. 7a is a diagram showing Zika virus-specific diagnosis results according to the concentration of Zika virus-specific antibodies. Figure 7b is a diagram showing that Zika virus-specific antibodies did not bind to dengue virus even when the concentration was changed.
8 is a diagram showing the nucleotide sequences of VH and VL of antibody 4B1 among Zika virus-specific antibodies.
9 is a diagram showing the nucleotide sequences of VH and VL of 5D12 antibody among Zika virus-specific antibodies.
본 발명자들은 지카 바이러스의 비구조 단백질 1 (Non-structural protein 1, NS1)에 특이적인 결합 능력을 가지고, 다른 바이러스에는 특이적으로 결합하지 않는 항체 또는 이의 결합 단편을 개발하였으며, 상기 항체 또는 이의 결합 단편이 지카 바이러스 진단, 예방 및 치료에 매우 유용하게 사용될 수 있음을 확인하였다.The present inventors have developed an antibody or binding fragment thereof that has a specific binding ability to non-structural protein 1 (NS1) of Zika virus and does not specifically bind to other viruses, and the antibody or binding fragment thereof It was confirmed that the fragment can be very useful for diagnosis, prevention and treatment of Zika virus.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 지카 바이러스(ZIKV)의 비구조 단백질 1(Non-structural protein 1, NS1)에 특이적으로 결합하는 항체를 제공한다.The present invention provides an antibody that specifically binds to non-structural protein 1 (NS1) of Zika virus (ZIKV).
본 발명의 항체는 지카 바이러스(ZIKV)의 비구조 단백질 1(Non-structural protein 1)에 우수한 결합능을 가진다. 일 구체예로, 본 발명의 항체는 지카 바이러스의 NS1에만 특이적으로 결합함으로서 우수한 지카 바이러스의 진단, 예방 및 치료 효과를 나타낼 수 있다.The antibody of the present invention has excellent binding ability to Non-structural protein 1 of Zika virus (ZIKV). In one embodiment, the antibody of the present invention can exhibit excellent diagnostic, preventive and therapeutic effects of Zika virus by specifically binding only to NS1 of Zika virus.
본 발명에서 사용되는 용어 "결합 분자"는 키메라, 인간화 또는 인간 단일클론 항체와 같은 단일클론 항체를 포함하는 온전한(intact) 이뮤노글로블린(immunoglobulin), 또는 항원에 결합하는 이뮤노글로블린인 항원-결합 단편을 포함한다.As used herein, the term "binding molecule" refers to an antigen-binding antibody that is an intact immunoglobulin, including a monoclonal antibody, such as a chimeric, humanized or human monoclonal antibody, or an immunoglobulin that binds an antigen. contains a fragment
본 발명의 일 실시예에서, 상기 지카 바이러스의 NS1에 특이적으로 결합하는 결합 분자는 지카 바이러스의 NS1에 특이적으로 결합하는 항체, 펩타이드, 펩타이드 유사체, 압타머 및 화합물로 구성된 군에서 선택되는 어느 하나일 수 있다. 구체적으로, 상기 결합 분자는 항체 또는 이의 단편일 수 있다.In one embodiment of the present invention, the binding molecule that specifically binds to NS1 of Zika virus is any one selected from the group consisting of antibodies, peptides, peptide analogs, aptamers, and compounds that specifically bind to NS1 of Zika virus. can be one Specifically, the binding molecule may be an antibody or fragment thereof.
본 발명의 일 실시예에서, 상기 항체는 지카 바이러스의 비구조 단백질 1에 특이적으로 결합하며, 중쇄 가변영역과 경쇄 가변영역을 포함하는, 지카 바이러스 특이적 항체 또는 이의 결합 단편일 수 있다.In one embodiment of the present invention, the antibody may be a Zika virus-specific antibody or binding fragment thereof that specifically binds to nonstructural protein 1 of Zika virus and includes a heavy chain variable region and a light chain variable region.
본 발명에서, 상기 지카 바이러스(Zika Virus)는 대표적인 모기매개(Mosquito-Borne) 바이러스 중의 하나로서, 단일 (+)가닥 RNA 바이러스(Single Positive-Stranded RNA Virus)로, 플라비비리데과(Flaviviridae Family)의 하위 속인 플라비바이러스속(Flavivirus Genus)에 해당하는 바이러스를 의미한다.In the present invention, the Zika virus is one of the representative mosquito-borne (Mosquito-Borne) viruses, a single positive-stranded RNA virus, and belongs to the Flaviviridae family. It means a virus corresponding to the genus Flavivirus, which is a subgenus of .
상기 지카 바이러스는 구형(Spherical)의 구조로 크기는 40 내지 65 nm이며, 약 10,000 내지 11,000개의 염기(Base)로 구성되어 있다. 외피 단백질(Envelope Protein)과 정이십면체형의 캡시드 단백질(Icosahedral-Like Nucleocapsid Protein)은 대칭 형태일 수 있다. 지카 바이러스를 구성하는 단백질은 크게 구조단백질(Structural Protein)과 비구조 단백질(Non-Structural Protein)로 분류할 수 있으며, 구조단백질에는 캡시드 단백질(Capsid Protein), 전구체 막 단백질 (Precursor Membrane Protein), 막 단백질(Membrane Protein), 외피 단백질(Envelope Protein) 등이 있으며, 비구조 단백질에는 비구조 단백질 1(Non-Structural Protein 1, NS1), 비구조 단백질 2A(NS2A), 비구조 단백질 2B(NS2B), 비구조 단백질 3(NS3), 비구조 단백질 4A(NS4A), 비구조 단백질 4B(NS4B), 비구조 단백질 5(NS5) 등이 있다.The Zika virus has a spherical structure, has a size of 40 to 65 nm, and is composed of about 10,000 to 11,000 bases. The envelope protein and the icosahedral-like nucleocapsid protein may have symmetrical shapes. Proteins constituting Zika virus can be largely classified into structural proteins and non-structural proteins. Structural proteins include capsid protein, precursor membrane protein, and membrane protein. There are membrane protein and envelope protein. Non-structural protein includes non-structural protein 1 (NS1), non-structural protein 2A (NS2A), non-structural protein 2B (NS2B), There are nonstructural protein 3 (NS3), nonstructural protein 4A (NS4A), nonstructural protein 4B (NS4B), and nonstructural protein 5 (NS5).
본 발명에서 상기 비구조 단백질 1 (NS1)은 세포 내부의 이종이량체 (dimer)로서, 상기 이량체 3개가 모여서 육량체 (hexamer) 지단백질 입자로 세포외 공간으로 분비된다. NS1은 바이러스 복제에 필요하며, 면역 회피 및 발병에 관여하는 바이러스 감염의 주요 항원 표지자이다.In the present invention, the non-structural protein 1 (NS1) is a heterodimer inside the cell, and the three dimers are gathered and secreted into the extracellular space as hexamer lipoprotein particles. NS1 is required for viral replication and is a major antigenic marker of viral infection involved in immune evasion and pathogenesis.
본 발명에서 상기 지카 바이러스 특이적 항원 예를 들어, 비구조 단백질 1(NS1)를 인식하는 항체를 제조하고자 하였다. 이에, 상기 항원을 이용하여 지카 바이러스 비구조 단백질 1(NS1)에 대하여 우수한 결합 친화도를 나타내는 단일클론항체 2종 4B1 및 5D12를 선별하였다.In the present invention, an antibody recognizing the Zika virus-specific antigen, for example, non-structural protein 1 (NS1), was prepared. Accordingly, two types of monoclonal antibodies, 4B1 and 5D12, showing excellent binding affinity to Zika virus non-structural protein 1 (NS1) were selected using the antigen.
본 명세서에서 '항체'는 최대한 넓은 의미로 사용되며, 구체적으로 무손상(intact) 단일클론 항체, 다클론 항체, 2종 이상의 무손상 항체로부터 형성된 다중특이성 항체(예를 들어, 이중특이성 항체), 및 목적하는 생물학적 활성을 나타내는 항체 단편을 포함한다. 항체는 특이적인 항원을 인식하고 결합할 수 있는 면역계에 의하여 생성되는 단백질이다. 그 구조적인 면에서, 항체는 통상적으로 4개의 아미노산 쇄(2개의 중쇄 및 2개의 경쇄)로 이루어진 Y-형상의 단백질을 가진다. 각각의 항체는 주로 가변 영역 및 불변 영역의 2개의 영역을 가진다. Y의 팔의 말단 부분에 위치한 가변 영역은 표적 항원에 결합하고 상호작용한다. 상기 가변 영역은 특정 항원 상의 특이적 결합 부위를 인식하고 결합하는 상보성 결정 영역(CDR)을 포함한다. Y의 꼬리 부분에 위치한 불변 영역은 면역계에 의하여 인식되고 상호작용한다. 표적 항원은 일반적으로 다수의 항체 상의 CDR에 의하여 인식되는, 에피토프라고 하는 다수의 결합 부위를 가지고 있다. 상이한 에피토프에 특이적으로 결합하는 각각의 항체는 상이한 구조를 가진다. 그러므로 한 항원은 하나 이상의 상응하는 항체를 가질 수 있다.In the present specification, 'antibody' is used in the broadest sense, specifically intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from two or more intact antibodies (eg, bispecific antibodies), and antibody fragments that exhibit the desired biological activity. Antibodies are proteins produced by the immune system capable of recognizing and binding to specific antigens. In terms of their structure, antibodies usually have Y-shaped proteins consisting of four amino acid chains (two heavy chains and two light chains). Each antibody has primarily two regions, a variable region and a constant region. A variable region located at the distal portion of the arm of Y binds and interacts with the target antigen. The variable region includes a complementarity determining region (CDR) that recognizes and binds to a specific binding site on a specific antigen. The constant region located in the tail of Y is recognized and interacted with by the immune system. A target antigen usually has multiple binding sites, called epitopes, that are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, one antigen may have more than one corresponding antibody.
본 발명의 일 구체예에서, 상기 지카 바이러스 특이적 항체 또는 이의 결합 단편은 서열번호 1의 아미노산 서열을 포함하는 CDR1-VH, 서열번호 2의 아미노산 서열을 포함하는 CDR2-VH 및 서열번호 3의 아미노산 서열을 포함하는 CDR3-VH를 포함하는 중쇄 가변영역 및 서열번호 4의 아미노산 서열을 포함하는 CDR1-VK, 서열번호 5의 아미노산 서열을 포함하는 CDR2-VK 및 서열번호 6의 아미노산 서열을 포함하는 CDR3-VK를 포함하는 경쇄 가변영역을 포함할 수 있다.In one embodiment of the present invention, the Zika virus-specific antibody or binding fragment thereof is CDR1-VH comprising the amino acid sequence of SEQ ID NO: 1, CDR2-VH comprising the amino acid sequence of SEQ ID NO: 2, and amino acids of SEQ ID NO: 3 A heavy chain variable region comprising CDR3-VH comprising the sequence and CDR1-VK comprising the amino acid sequence of SEQ ID NO: 4, CDR2-VK comprising the amino acid sequence of SEQ ID NO: 5, and CDR3 comprising the amino acid sequence of SEQ ID NO: 6 -Can include a light chain variable region including VK.
본 발명의 일 구체예에서, 상기 지카 바이러스 특이적 항체 또는 이의 결합 단편은 서열번호 17의 아미노산 서열을 포함하는 CDR1-VH, 서열번호 18의 아미노산 서열을 포함하는 CDR2-VH 및 서열번호 19의 아미노산 서열을 포함하는 CDR3-VH를 포함하는 중쇄 가변영역 및 서열번호 20의 아미노산 서열을 포함하는 CDR1-VK, 서열번호 21의 아미노산 서열을 포함하는 CDR2-VK 및 서열번호 22의 아미노산 서열을 포함하는 CDR3-VK를 포함하는 경쇄 가변영역을 포함할 수 있다.In one embodiment of the present invention, the Zika virus-specific antibody or binding fragment thereof comprises CDR1-VH comprising the amino acid sequence of SEQ ID NO: 17, CDR2-VH comprising the amino acid sequence of SEQ ID NO: 18 and amino acids of SEQ ID NO: 19 A heavy chain variable region comprising CDR3-VH comprising the sequence and CDR1-VK comprising the amino acid sequence of SEQ ID NO: 20, CDR2-VK comprising the amino acid sequence of SEQ ID NO: 21, and CDR3 comprising the amino acid sequence of SEQ ID NO: 22 -Can include a light chain variable region including VK.
본 발명의 일 실시예에서, 상기 항체 또는 그의 결합 단편은 scFv 절편, scFv-Fc 절편, Fab 절편, Fv 절편, 디아바디 (diabody), 키메라 항체, 인간화 항체 또는 인간 항체일 수 있으나, 이에 한정되지 않는다. 본 발명의 일 구체예는 지카 바이러스 비구조 단백질 1(NS1) 영역에 결합하는 Fab 절편을 제공한다.In one embodiment of the present invention, the antibody or binding fragment thereof may be a scFv fragment, scFv-Fc fragment, Fab fragment, Fv fragment, diabody, chimeric antibody, humanized antibody, or human antibody, but is not limited thereto. don't One embodiment of the present invention provides a Fab fragment that binds to the Zika virus non-structural protein 1 (NS1) region.
아울러 본 발명은 상기 결합 분자의 기능적 변이체를 포함한다. 결합 분자들은 변이체들이 지카 바이러스 비구조 단백질 1(NS1) 영역에 특이적으로 결합하기 위해 본 발명의 결합 분자와 경쟁할 수 있고, 지카 바이러스 비구조 단백질 1(NS1) 영역과 결합 능력을 보유한다면 본 발명의 결합 분자의 기능적 변이체로 간주된다. 기능적 변이체는 1차 구조적 서열이 실질적으로 유사한 유도체를 포함하지만, 이에 제한되는 것은 아니며, 예를 들면, 생체외(in vitro) 또는 생체내(in vivo) 변형, 화학약품 및/또는 생화학 약품을 포함하며, 이들은 본원 발명의 부모 단일클론 항체에서는 발견되지 않는다. 이와 같은 변형으로는 예를 들어 아세틸화, 아실화, 뉴클레오티드 또는 뉴클레오티드 유도체의 공유 결합, 지질 또는 지질 유도체의 공유 결합, 가교, 이황화 결합 형성, 글리코실화, 수산화, 메틸화, 산화, 페길화, 단백질 분해 및 인산화 등이 포함된다. 기능적 변이체는 선택적으로 부모 항체의 아미노산 서열과 비교하여 하나 이상의 아미노산의 치환, 삽입, 결실 또는 그들의 조합을 함유하는 아미노산 서열을 포함하는 항체일 수 있다. 더욱이 기능적 변이체는 아미노 말단 또는 카르복시 말단 중 하나 또는 모두에서 아미노산 서열의 절단체 (truncated form)를 포함할 수 있다. 본 발명의 기능적 변이체는 본 발명의 부모 항체와 비교하여 동일하거나 다르거나, 더 높거나 낮은 결합 친화력을 가질 수 있지만, 여전히 지카 바이러스의 NS1에 결합할 수 있다. 일 예로, 골격구조, 초가변(Hypervariable) 영역, 특히 경쇄 또는 중쇄의 상보성 결정 영역 (Complementarity-determining region, CDR)을 포함하나 이에 한정되지 않는 가변 영역의 아미노산 서열이 변형될 수 있다. 일반적으로 경쇄 또는 중쇄 영역은 3개의 CDR 영역을 포함하는, 3개의 초가변 영역 및 더욱 보존된 영역, 즉 골격 영역(FR)을 포함한다. 초가변 영역은 CDR로부터의 아미노산 잔기와 초가변 루프로부터의 아미노산 잔기를 포함한다. 본 발명의 범위에 속하는 기능적 변이체는 본 명세서의 부모 항체와 약 50%~99%, 약 60%~99%, 약 80%~99%, 약 90%~99%, 약 95%~99%, 또는 약 97%~99% 아미노산 서열 동질성을 가질 수 있다. 비교될 아미노산 서열을 최적으로 배열하고 유사하거나 또는 동일한 아미노산 잔기를 정의하기 위해 컴퓨터 알고리즘 중 당업자에게 알려진 Gap 또는 Bestfit를 사용할 수 있다. 기능적 변이체는 부모 항체 또는 그것의 일부를 PCR 방법, 올리고머 뉴클레오티드를 이용한 돌연변이 생성 및 부분 돌연변이 생성을 포함하는 공지의 일반 분자 생물학적 방법에 의해 변화시키거나 유기합성 방법으로 얻을 수 있으나 이에 제한되는 것은 아니다.In addition, the present invention includes functional variants of the binding molecule. Binding molecules can compete with binding molecules of the present invention for specific binding to the Zika virus non-structural protein 1 (NS1) region, and retain the ability to bind to the Zika virus non-structural protein 1 (NS1) region. It is considered a functional variant of the binding molecule of the invention. Functional variants include, but are not limited to, derivatives with substantially similar primary structural sequences, including, for example, in vitro or in vivo modifications, chemical and/or biochemical agents. and they are not found in the parental monoclonal antibodies of the present invention. Such modifications include, for example, acetylation, acylation, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, cross-linking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolysis, and phosphorylation. A functional variant may be an antibody comprising an amino acid sequence that optionally contains a substitution, insertion, deletion or combination of one or more amino acids compared to the amino acid sequence of a parent antibody. Moreover, functional variants may include truncated forms of amino acid sequences at either or both the amino terminus or the carboxy terminus. A functional variant of the invention may have the same, different, higher or lower binding affinity compared to the parental antibody of the invention, but still be able to bind NS1 of Zika virus. For example, the amino acid sequence of the variable region including, but not limited to, a backbone structure, a hypervariable region, particularly a complementarity-determining region (CDR) of a light chain or a heavy chain may be modified. Generally, a light or heavy chain region comprises three hypervariable regions, comprising the three CDR regions, and a more conserved region, the framework region (FR). Hypervariable regions include amino acid residues from CDRs and amino acid residues from hypervariable loops. Functional variants within the scope of the present invention are about 50% to 99%, about 60% to 99%, about 80% to 99%, about 90% to 99%, about 95% to 99%, or about 97%-99% amino acid sequence identity. Among computer algorithms known to those skilled in the art, Gap or Bestfit can be used to optimally align amino acid sequences to be compared and define similar or identical amino acid residues. Functional variants can be obtained by changing the parent antibody or part thereof by known general molecular biology methods, including PCR, oligomeric nucleotide mutagenesis and partial mutagenesis, or by organic synthesis, but are not limited thereto.
본 발명의 일 실시예에서, 상기 항체 또는 이의 결합 단편은 하이브리도마 세포에 의해 생산될 수 있다.In one embodiment of the present invention, the antibody or binding fragment thereof may be produced by hybridoma cells.
본 발명의 일 실시예에서는, 상기 지카 바이러스 특이적 항체를 조합하여 사용하는 경우, 지카 바이러스 진단 효과가 우수함을 확인하였다. 구체적으로 지카 바이러스 특이적 항체 중 2종 (4B1 및 5D12)을 페어로 사용하는 경우, 우수한 지카 바이러스 진단 효과를 나타낼 수 있다.In one embodiment of the present invention, it was confirmed that the Zika virus diagnostic effect was excellent when the Zika virus-specific antibodies were used in combination. Specifically, when two of the Zika virus-specific antibodies (4B1 and 5D12) are used as a pair, excellent Zika virus diagnostic effects can be exhibited.
또한, 본 발명은 상기 항체를 암호화하는 핵산 분자를 제공한다. 구체적으로, 상기 핵산 분자는 상기 항체 또는 그의 결합 단편을 암호화하는 폴리뉴클레오티드일 수 있다.In addition, the present invention provides a nucleic acid molecule encoding the antibody. Specifically, the nucleic acid molecule may be a polynucleotide encoding the antibody or binding fragment thereof.
본 발명의 핵산 분자는 본 발명에서 제공하는 항체의 아미노산 서열을 당업자에게 알려진 바와 같이 폴리뉴클레오티드 서열로 번역된 핵산 분자 모두를 포함한다. 그러므로 ORF(open reading frame)에 의한 다양한 폴리뉴클레오티드 서열이 제조될 수 있으며 이 또한 모두 본 발명의 핵산 분자에 포함된다.The nucleic acid molecules of the present invention include all nucleic acid molecules in which the amino acid sequence of the antibody provided in the present invention is translated into a polynucleotide sequence as known to those skilled in the art. Therefore, various polynucleotide sequences by ORF (open reading frame) can be prepared, and all of them are also included in the nucleic acid molecule of the present invention.
본 발명의 일 구체예에서, 상기 폴리뉴클레오티드는 서열번호 7의 염기서열 및 서열번호 8의 염기서열을 포함할 수 있다.In one embodiment of the present invention, the polynucleotide may include the nucleotide sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 8.
본 발명의 일 구체예에서, 상기 폴리뉴클레오티드는 서열번호 23의 염기서열 및 서열번호 24의 염기서열을 포함할 수 있다.In one embodiment of the present invention, the polynucleotide may include the base sequence of SEQ ID NO: 23 and the base sequence of SEQ ID NO: 24.
또한, 본 발명은 상기 핵산 분자가 삽입된 발현 벡터를 제공한다. 일 실시예에서, 상기 발현 벡터는 상기 폴리뉴클레오티드가 삽입된 발현 벡터일 수 있다.In addition, the present invention provides an expression vector into which the nucleic acid molecule is inserted. In one embodiment, the expression vector may be an expression vector into which the polynucleotide is inserted.
본 발명의 일 구체예에서, 상기 발현 벡터는 서열번호 7의 염기서열 및 서열번호 8의 염기서열을 포함할 수 있다.In one embodiment of the present invention, the expression vector may include the nucleotide sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 8.
본 발명의 일 구체예에서, 상기 발현 벡터는 서열번호 23의 염기서열 및 서열번호 24의 염기서열을 포함할 수 있다.In one embodiment of the present invention, the expression vector may include the nucleotide sequence of SEQ ID NO: 23 and the nucleotide sequence of SEQ ID NO: 24.
상기 발현 벡터로는 셀트리온 고유의 발현 벡터인 MarEx 벡터(한국특허등록 제10-1076602호 참조) 및 상업적으로 널리 사용되는 pCDNA 벡터, F, R1, RP1, Col, pBR322, ToL, Ti 벡터; 코스미드; 람다, 람도이드(lambdoid), M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7 등의 파아지; 식물 바이러스로 이루어진 군으로부터 선택된 어느 하나에서 선택된 발현 벡터를 이용할 수 있으나 이에 한정되지 않으며, 당업자에게 발현 벡터로 알려진 모든 발현 벡터는 본 발명에 사용 가능하며, 발현 벡터를 선택할 때에는 목적으로 하는 숙주 세포의 성질에 따른다. 숙주세포로의 벡터 도입시 인산칼슘 트랜스펙션, 바이러스 감염, DEAE-덱스트란 조절 트랜스펙션, 리포펙타민 트랜스펙션 또는 전기천공법에 의해 수행될 수 있으나 이에 한정되지 않으며 당업자는 사용하는 발현 벡터 및 숙주 세포에 알맞은 도입 방법을 선택하여 이용할 수 있다. 예를 들어, 벡터는 하나 이상의 선별 마커를 함유하나 이에 한정되지 않으며, 선별 마커를 포함하지 않은 벡터도 이용하여 생산물 생산 여부에 따라 선별이 가능하다. 선별 마커의 선택은 목적하는 숙주 세포에 의해 선택되며, 이는 이미 당업자에게 알려진 방법을 이용하므로 본 발명은 이에 제한을 두지 않는다.As the expression vector, MarEx vector (refer to Korean Patent Registration No. 10-1076602), which is Celltrion's own expression vector, and commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors; cosmid; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, and T7; An expression vector selected from any one selected from the group consisting of plant viruses can be used, but is not limited thereto, and all expression vectors known to those skilled in the art as expression vectors can be used in the present invention, and when selecting an expression vector, the host cell of interest according to the nature When the vector is introduced into the host cell, it may be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection or electroporation, but is not limited thereto, and expression used by those skilled in the art An introduction method suitable for the vector and host cell can be selected and used. For example, vectors contain one or more selectable markers, but are not limited thereto, and vectors without selectable markers may also be used for selection depending on whether a product is produced. Selection of the selection marker is selected by the host cell of interest, and since this method is already known to those skilled in the art, the present invention is not limited thereto.
본 발명의 항체의 정제를 용이하게 하기 위하여 태그 서열을 발현 벡터 상에 삽입하여 융합시킬 수 있다.To facilitate purification of the antibody of the present invention, a tag sequence may be inserted and fused onto an expression vector.
상기 태그로는 헥사-히스티딘 태그, 헤마글루티닌 태그, myc 태그 또는 flag 태그를 포함하나 이에 한정되지 않으며 당업자에게 알려진 정제를 용이하게 하는 태그는 모두 본 발명에서 이용 가능하다.The tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag that facilitates purification known to those skilled in the art can be used in the present invention.
또한, 본 발명은 항체 또는 이의 결합 단편을 생산하는 하이브리도마 세포를 제공한다. 일 실시예에서, 상기 하이브리도마 세포는 상기 발현 벡터가 도입된 세포일 수 있다.In addition, the present invention provides a hybridoma cell producing the antibody or binding fragment thereof. In one embodiment, the hybridoma cells may be cells into which the expression vector is introduced.
본 발명의 용어 '하이브리도마 세포'란, 2개의 다른 종류의 세포를 인공적으로 융합시켜 만든 세포로, 폴리에틸렌글리콜(Polyethylene glycol, PEG) 등 세포융합을 일으키게 하는 물질이나 어떤 종의 바이러스를 사용하여 둘 이상의 동종 세포나 이종세포가 융합되어, 각기 다른 세포가 갖는 다른 기능을 하나의 세포 속에 통합시킨 세포 또는 세포주를 의미한다.The term 'hybridoma cell' of the present invention is a cell made by artificially fusing two different types of cells, using a substance that causes cell fusion, such as polyethylene glycol (PEG), or a certain type of virus. It refers to a cell or cell line in which two or more allogeneic cells or heterogeneous cells are fused to integrate different functions of different cells into one cell.
상기 하이브리도마 세포주는, '지카 바이러스 비구조 단백질 1'를 사용하여 마우스를 면역화시키는 단계; 상기 면역화시킨 마우스의 비장세포를 골수종 세포와 융합하여 배양하는 단계; 및 융합된 하이브리도마 세포주에서 '지카 바이러스 비구조 단백질 1' 단백질에 특이적인 항체 또는 이의 결합 단편을 생산하는 하이브리도마 세포주를 선별하는 단계를 통해 제조될 수 있다.In the hybridoma cell line, immunizing a mouse using 'Zika virus non-structural protein 1'; Fusing the spleen cells of the immunized mouse with myeloma cells and culturing them; and selecting a hybridoma cell line producing an antibody or binding fragment thereof specific to the 'Zika virus non-structural protein 1' protein in the fused hybridoma cell line.
또한, 본 발명의 '지카 바이러스 비구조 단백질 1'에 대해 특이적인 항체 또는 이의 결합 단편은 본 발명이 속하는 기술분야에 잘 알려져 있는 융합 방법(Fusion method)에 의해 제조될 수 있다(Khler G. & Milstein C., Nature (1975) Vol. 256, pp. 495-497., Galfre G. & Milstein C., Methods Enzymol. (1981) Vol. 73, pp. 3-46. 참조). 상기 항체 또는 이의 결합 단편을 분비하는 '하이브리도마 세포주'를 만들기 위해 융합되는 두 세포 집단 중 하나의 집단은 '지카 바이러스 비구조 단백질 1'를 주사한 마우스와 같은 면역학적으로 적합한 숙주동물의 세포를 이용하고, 나머지 하나의 집단으로는 암 또는 골수종 세포주를 이용할 수 있다. 이러한 두 집단의 세포들을 폴리에틸렌글리콜과 같은 본 발명이 속하는 기술 분야에 공지되어 있는 방법에 의해 융합시키고 나서 항체-생산 세포를 표준적인 조직 배양 방법에 의해 증식시킨다. 한계 희석법(Limited dilution technique)에 의한 서브클로닝에 의해 균일한 세포 집단을 수득하고 나서, '지카 바이러스 비구조 단백질 1'에 대해 특이적인 항체를 생산할 수 있는 하이브리도마 세포주를 효소면역흡착분석법(Enzyme-linked immunosorbent assay, ELISA) 또는 웨스턴블럿 분석법으로 선별하고, 표준 기술에 따라 시험관 내에서 또는 생체 내에서 대량으로 배양할 수 있다. 상기 생체내 대량배양은 하이브리도마 세포주를 마우스의 복강에 주사하여 고농도의 항체 생산을 유도한 후, 복수액(Ascites)에서 분리하는 것을 의미한다.In addition, the antibody or binding fragment thereof specific for 'Zika virus non-structural protein 1' of the present invention can be prepared by a fusion method well known in the art to which the present invention belongs (Khler G. & See Milstein C., Nature (1975) Vol. 256, pp. 495-497, Galfre G. & Milstein C., Methods Enzymol. (1981) Vol. One of the two cell populations fused to create a 'hybridoma cell line' secreting the antibody or binding fragment thereof is a cell of an immunologically compatible host animal such as a mouse injected with 'Zika virus non-structural protein 1'. , and a cancer or myeloma cell line may be used as the other group. These two populations of cells are fused by methods known in the art, such as polyethylene glycol, and then the antibody-producing cells are propagated by standard tissue culture methods. After obtaining a uniform cell population by subcloning by the limited dilution technique, a hybridoma cell line capable of producing an antibody specific to 'Zika virus non-structural protein 1' was determined by enzyme immunosorbent assay (Enzyme Immunosorbent Analysis). -linked immunosorbent assay (ELISA) or Western blot analysis, and can be cultured in vitro or in vivo in large quantities according to standard techniques. The in vivo mass culture means that the hybridoma cell line is injected into the peritoneal cavity of a mouse to induce high concentration of antibody production, and then separated from ascites fluid (Ascites).
상기 하이브리도마 세포주가 생산하는 단일클론항체는 정제하지 않고 사용하거나, 본 발명이 속하는 기술분야에 잘 알려져 있는 방법에 따라 고순도(예컨대, 90% 이상)로 정제하여 사용할 수 있다. 이러한 정제 기술로는, 예를 들어 겔 전기영동, 투석, 염 침전, 이온교환 크로마토그래피, 친화성 항원 컬럼 크로마토그래피 등의 방법을 이용하여 배양 배지 또는 복수액(Ascites)에서 분리할 수 있다.The monoclonal antibody produced by the hybridoma cell line may be used without purification or purified to high purity (eg, 90% or more) according to a method well known in the art to which the present invention belongs. As such a purification technique, for example, gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity antigen column chromatography, etc. may be used to separate from culture medium or ascites.
본 발명의 일 구체예에서, 상기 하이브리도마 세포는 서열번호 7의 염기서열 및 서열번호 8의 염기서열을 포함하는 발현 벡터가 도입된 세포일 수 있다.In one embodiment of the present invention, the hybridoma cell may be a cell into which an expression vector containing the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8 has been introduced.
본 발명의 일 구체예에서, 상기 하이브리도마 세포는 서열번호 23의 염기서열 및 서열번호 24의 염기서열을 포함하는 발현 벡터가 도입된 세포일 수 있다.In one embodiment of the present invention, the hybridoma cell may be a cell into which an expression vector containing the nucleotide sequence of SEQ ID NO: 23 and SEQ ID NO: 24 has been introduced.
또한, 본 발명은 지카 바이러스(ZIKV) 항원을 진단하는 제제를 포함하는, 지카 바이러스 진단용 키트를 제공한다. 일 구체적예에서, 상기 바이러스 항원은 지카 바이러스의 비구조 단백질 1(Non-structural protein 1) 이고, 상기 제제는 상기 항체 또는 이의 결합 단편인, 진단용 키트일 수 있다.In addition, the present invention provides a kit for diagnosing Zika virus, including an agent for diagnosing a Zika virus (ZIKV) antigen. In one specific embodiment, the viral antigen is non-structural protein 1 of Zika virus, and the agent may be the antibody or binding fragment thereof, a diagnostic kit.
본 발명의 일 구체예에서, 진단용 키트에 사용되는 본 발명의 항체는 진단 가능하게 표식될 수 있다. 생분자들을 표식시키는데 사용 가능한 다양한 방법들이 당업자에게 잘 알려져 있고, 본 발명의 범주 내에서 고려된다. 본 발명에서 사용될 수 있는 표식 종류의 예로는 효소, 방사성 동위원소, 콜로이드금속, 형광 화합물, 화학발광 화합물 및 생발광 화합물이 있다. 통상적으로 사용되는 표식들은 형광물질(가령, 플루레신, 로다민, 텍사스 레드 등), 효소(가령, 고추냉이 퍼옥시다아제, β-갈락토시다아제, 알칼리포스파타 아제), 방사성 동위원소(가령, 32P 또는 125I), 바이오틴, 디곡시게닌, 콜로이드 금속, 화학발광 또는 생발광 화합물(가령, 디옥세탄, 루미놀 또는 아크리디늄)을 포함한다. 효소 또는 바이오티닐기의 공유 결합법, 요오드화법, 인산화법, 바이오틴화법 등과 같은 표식 방법들이 당 분야에 잘 알려져 있다. 진단 방법들로는 오토라디오그래피, 형광 현미경, 직접 및 간접 효소반응 등이 있으며, 이에 제한되지는 않는다. 통상적으로 사용되는 진단 분석법으로는 방사성 동위원소 또는 비-방사성 동위원소 방법이 있다. 이들은 그 중에서도 웨스턴블롯팅, 오버레이-분석법, RIA(Radioimmuno Assay) 및 IRMA(ImmuneRadioimmunometric Assay), EIA(Enzyme Immuno Assay), ELISA(Enzyme Linked Immuno Sorbent Assay), FIA(Fluorescent Immuno Assay) 및 CLIA(Chemioluminescent Immune Assay)이 있다.In one embodiment of the present invention, the antibody of the present invention used in a diagnostic kit may be diagnostically labeled. A variety of methods available for labeling biomolecules are well known to those skilled in the art and are contemplated within the scope of the present invention. Examples of types of labels that can be used in the present invention include enzymes, radioactive isotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds. Commonly used labels include fluorescent substances (eg, flurescin, rhodamine, Texas red, etc.), enzymes (eg, horseradish peroxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes (eg, 32P or 125I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds such as dioxetane, luminol or acridinium. Labeling methods such as covalent bonding of enzymes or biotinyl groups, iodination, phosphorylation, and biotinylation are well known in the art. Diagnostic methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, and the like. Commonly used diagnostic assays include radioactive isotope or non-radioactive isotope methods. These include, among others, Western blotting, overlay-assay, Radioimmuno Assay (RIA) and ImmuneRadioimmunometric Assay (IRMA), Enzyme Immuno Assay (EIA), Enzyme Linked Immuno Sorbent Assay (ELISA), Fluorescent Immuno Assay (FIA) and Chemioluminescent Immunometric Assay (CLIA). Assay).
본 발명의 진단용 키트는 샘플과 상기 항체를 접촉시킨 후, 반응을 확인하여 지카 바이러스의 존재 여부를 진단하는 데 사용될 수 있다. 상기 샘플은 대상의 가래, 침, 혈액, 땀, 폐 세포, 폐 조직의 점액, 호흡기 조직 및 타액으로 이루어진 군으로부터 선택된 어느 하나일 수 있으나, 이에 한정되지 않으며 당업자에게 알려진 통상적인 방법으로 샘플 준비가 가능하다.The diagnostic kit of the present invention can be used to diagnose the presence or absence of Zika virus by confirming the reaction after contacting the sample with the antibody. The sample may be any one selected from the group consisting of sputum, saliva, blood, sweat, lung cells, mucus of lung tissue, respiratory tissue and saliva of the subject, but is not limited thereto, and the sample is prepared by a conventional method known to those skilled in the art possible.
본 발명의 일 실시예는, a) 상기 항체; 및 b) 용기를 포함하는 키트일 수 있다. 구체적으로, 상기 a)의 항체는 지카 바이러스의 비구조 단백질 1(Non-structural protein 1)에 특이적으로 결합하는 항체 또는 이의 결합 단편일 수 있다.One embodiment of the present invention, a) the antibody; and b) a kit comprising a container. Specifically, the antibody of a) may be an antibody or binding fragment thereof that specifically binds to non-structural protein 1 of Zika virus.
또한, 본 발명은 지카 바이러스 비구조 단백질(NS1) 영역에 결합하는 항체 또는 이의 결합 단편을 포함하는, 지카 바이러스 관련 질환의 진단용 키트를 제공한다.In addition, the present invention provides a kit for diagnosing a Zika virus-related disease, comprising an antibody or binding fragment thereof that binds to a Zika virus non-structural protein (NS1) region.
본 발명의 진단용 키트에 있어서, 키트 용기에는 고체 담체가 포함될 수 있다. 본 발명의 항체는 고체 담체에 부착될 수 있고, 이와 같은 고체 담체는 다공성 또는 비다공성, 평면 또는 비평면일 수 있다.In the diagnostic kit of the present invention, a solid carrier may be included in the kit container. An antibody of the present invention may be attached to a solid carrier, and such a solid carrier may be porous or non-porous, planar or non-planar.
또한, 본 발명은 상기 항체 또는 이의 결합 단편을 포함하는, 지카 바이러스 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing Zika virus comprising the antibody or binding fragment thereof.
또한, 본 발명의 다른 일 구체예로, 본 발명은 상기 진단용 키트를 이용하여 지카 바이러스를 진단하는 방법을 제공한다.In another embodiment of the present invention, the present invention provides a method for diagnosing Zika virus using the diagnostic kit.
또한, 본 발명의 다른 일 구체예로, 본 발명은 상기 진단용 키트를 이용하여 지카 바이러스 관련 질환을 진단하는 방법을 제공한다.In addition, as another embodiment of the present invention, the present invention provides a method for diagnosing a Zika virus-related disease using the diagnostic kit.
본원에 기재된 상기 각 특징들은 조합되어 사용될 수 있으며, 상기 각 특징들이 특허청구범위의 서로 다른 종속항에 기재된다는 사실은 이들이 조합되어 사용될 수 없음을 나타내는 것은 아니다.Each of the features described herein may be used in combination, and the fact that each of the features are recited in different dependent claims of the claims does not indicate that they cannot be used in combination.
이하 본 발명을 실시예 및 실험예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예 및 실험예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples and experimental examples.
실시예 및 실험예Examples and experimental examples
실험예 1. 지카 바이러스 NS1 재조합 항원 단백질 분리 정제Experimental Example 1. Zika Virus NS1 Recombinant Antigen Protein Isolation and Purification
지카 바이러스 NS1 단백질의 아미노산 서열 1-352 부위 (full-length)와 172-352 부위에 대하여 유전자 클로닝하였다 (도 1 참조).Genes were cloned for the amino acid sequences 1-352 (full-length) and 172-352 of the Zika virus NS1 protein (see FIG. 1).
E. coli 발현시스템을 이용하여 바이러스 상기 항원 재조합 172-352 부위 단백질 (지카 NS1_CTD) refolding 분리 정제 기술을 확립하여 10 mg 이상 항원 단백질을 고순도로 생산하여 마우스 면역 항체 개발에 사용하였다 (도 2 참조).An E. coli expression system was used to establish a refolding separation and purification technology for the recombinant 172-352 site protein (Zika NS1_CTD) of the virus above, to produce 10 mg or more antigen protein with high purity and use it for the development of mouse immune antibodies (see Fig. 2). .
실험예 2. 지카 바이러스 NS1 특이적 항체 클로닝Experimental Example 2. Zika virus NS1 specific antibody cloning
상기 분리된 항원 단백질을 이용하여 상기 항원 단백질과 결합 능력이 있는 지카 바이러스 특이적 단일사슬항체를 확인하였다 (도 3a 내지 3d 참조). 상기 항원 단백질과 반응(OD>0.5)하는 41개의 클론 (Clones)을 ELISA (ELISA 조건은 antigen coating: 100ng/well, 2nd Ab dilution: 1:10,000, substrate: TMB)를 통해 확인하였다 (붉은색으로 표기, H12 well은 positive control).Using the isolated antigen protein, a Zika virus-specific single chain antibody capable of binding to the antigen protein was identified (see FIGS. 3a to 3d). 41 clones reacting with the antigen protein (OD>0.5) were identified through ELISA (ELISA conditions: antigen coating: 100ng/well, 2nd Ab dilution: 1:10,000, substrate: TMB) (color in red) notation, H12 well is a positive control).
상기 41개의 positive clones을 24 well plate에 옮긴 후 ELISA (ELISA 조건은 antigen coating: 100ng/well, 2nd Ab dilution: 1:10,000, substrate: TMB)로 확인한 결과, 뎅기바이러스 NS1과는 교차반응이 없고, 지카 NS1 항원과 특이적으로 반응하는 22개 클론을 확인하였다 (도 4 참조). After transferring the 41 positive clones to a 24 well plate, ELISA (ELISA conditions: antigen coating: 100ng/well, 2nd Ab dilution: 1:10,000, substrate: TMB) was confirmed. As a result, there was no cross-reaction with dengue virus NS1, Twenty-two clones that specifically reacted with the Zika NS1 antigen were identified (see FIG. 4).
상기 ELISA 결과를 토대로 #4B1, #5D12, #5G3, #8D11, #9E3, #4D2, #7H8, #8H5, #9C10, #9E5 클론을 선택하여 총 10종 단클론 항체 클로닝(cloning)하여 ELISA로 확인한 후, 이를 액체질소에 보관하였다.Based on the above ELISA results, clones #4B1, #5D12, #5G3, #8D11, #9E3, #4D2, #7H8, #8H5, #9C10, and #9E5 were selected, and a total of 10 monoclonal antibodies were cloned by ELISA. After checking, it was stored in liquid nitrogen.
그 결과, 정제된 항체의 순도가 낮거나 뎅기 NS1과 교차반응을 보이는 항체를 제외한 총 8종 단일클론항체를 확인하였다 (표 1 및 2 참조).As a result, a total of 8 monoclonal antibodies were identified, excluding antibodies with low purity or cross-reactivity with Dengue NS1 (see Tables 1 and 2).
실험예 3. 지카 바이러스 NS1 특이적 항체 정제 및 결합력 확인Experimental Example 3. Zika Virus NS1 Specific Antibody Purification and Confirmation of Binding Ability
상기 클로닝된 지카 NS1_CTD 단일클론항체인 #9E3, #9C10, #8H5, #9E5, #4D2, #4B1, #5D12 및 #8D11의 마우스 복수 (mouse ascites)를 각 10 mL씩 Protein A column으로 하여, IgG를 정제하고 BCA법 (BGG 스탠다드 사용)으로 정량하였다. 상기 정제된 항체를 SDS-PAGE한 후 coomassie blue로 염색하여 순도를 확인하였다 (도 5 참조).10 mL each of mouse ascites of the cloned Zika NS1_CTD monoclonal antibodies #9E3, #9C10, #8H5, #9E5, #4D2, #4B1, #5D12, and #8D11 were used as Protein A columns, IgG was purified and quantified by BCA method (using BGG standard). The purified antibody was subjected to SDS-PAGE and then stained with coomassie blue to confirm purity (see FIG. 5).
상기 정제된 지카 특이적 단일클론항체 8종의 HEK293 cell 발현 항원에 대하여 결합력을 가지고 있음을 Western blot (WB), Dot blot (DB) 실험을 통해 확인하였다.It was confirmed through Western blot (WB) and Dot blot (DB) experiments that the purified 8 types of Zika-specific monoclonal antibodies had binding ability to the HEK293 cell-expressed antigen.
그 결과, 상기 지카 바이러스 특이적 단일사슬항체 8종 중 4종은 판매되는 항체와 유사한 결합력을 가지고 있었다. 또한, 상기 4종의 항체는 모두 뎅기 바이러스 NS1에는 특이적 반응을 보이지 않았으나, 지카 바이러스 NS1에는 특이적으로 결합하는 것을 확인하였다 (도 6 참조).As a result, 4 out of 8 Zika virus-specific single chain antibodies had similar binding ability to commercially available antibodies. In addition, all of the above four types of antibodies did not show a specific reaction to dengue virus NS1, but it was confirmed that they specifically bind to Zika virus NS1 (see FIG. 6).
이러한 결과는 지카 바이러스 특이적 진단에 상기 항체들을 이용할 수 있다는 것을 의미한다.These results indicate that the antibodies can be used for Zika virus-specific diagnosis.
실험예 3. 지카 바이러스 특이적 항체 서열 분석Experimental Example 3. Zika virus-specific antibody sequence analysis
상기 지카 바이러스 특이적 단일사슬항체 4종 중, 2종에 대한 서열을 분석하였다.Sequences were analyzed for two of the four Zika virus-specific single chain antibodies.
표 3은 상기 단일사슬항체 중 4B1에 대한 서열이다.Table 3 shows the sequence of 4B1 among the single chain antibodies.
표 4는 상기 단일사슬항체 중 5D12에 대한 서열이다.Table 4 shows the sequence of 5D12 among the single chain antibodies.
실험예 4. 지카 바이러스 진단용 키트 제작Experimental Example 4. Production of Zika virus diagnosis kit
상기 분석된 항체 2종 (4B1 및 5D12)를 포함하는 지카 바이러스 진단용 키트를 제조하였다.A kit for diagnosing Zika virus containing the two types of antibodies analyzed above (4B1 and 5D12) was prepared.
상기 제조된 진단용 키트에 지카 바이러스 샘플 및 뎅기 바이러스 샘플을 농도별로 처리하여, 특이적인 진단 반응이 일어나는지 확인하였다.Zika virus samples and dengue virus samples were treated for each concentration in the diagnostic kit prepared above to confirm whether a specific diagnostic reaction occurs.
그 결과, 지카 바이러스 샘플에 대해서는 진단 반응이 나타났으나 (도 7a 참조), 뎅기 바이러스 샘플에 대해서는 반응이 일어나지 않는 것을 확인할 수 있었다 (도 7b 참조).As a result, it was confirmed that a diagnostic response was shown for the Zika virus sample (see FIG. 7a), but no response occurred for the dengue virus sample (see FIG. 7b).
이러한 결과는 상기 항체 2종을 포함하는 지카 바이러스 진단용 키트를 이용하여, 신속하고 정확하게 지카 바이러스를 진단할 수 있음을 의미한다.These results mean that the Zika virus can be rapidly and accurately diagnosed using the Zika virus diagnosis kit containing the above two types of antibodies.
<110> KOREA BASIC SCIENCE INSTITUTE <120> Antibody for Detection Specific for Non-structural protein 1 of ZIKA virus and Use thereof <130> PD21-446 <160> 32 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 1_VH <400> 1 Gly Tyr Thr Phe Thr Asp Tyr Tyr 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 2_VH <400> 2 Ile Tyr Pro Gly Ser Gly Asn Ile 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 3_VH <400> 3 Ala Arg Pro Thr Val Tyr Phe Asp Tyr 1 5 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 1_VK <400> 4 Gln Ser Ile Gly Thr Ser 1 5 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 2_VK <400> 5 Tyr Ala Ser 1 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 3_VK <400> 6 Gln Gln Ser Asn Ser Trp Pro Leu Thr 1 5 <210> 7 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> ZIKV mAb VH <400> 7 cagatccagc tgcagcagtc tggacctggg gccaaggcac cactctctga gctggtgaag 60 cctggggctt cactgaggat atcctgcaag ccttctggct acaccttcac tgactactat 120 ataaactggg tgaagcagaa gcctggacag ggacttgagt ggattggatg gatttatcct 180 ggaagcggta atattaagta caatgagaag ttcaagggca aggccacatt gactgtagac 240 acatcctcca gcacagccta catgcagctc agcagcctga catctgagga cactgctgtc 300 tatttctgtg caagacctac ggtctatttt gactatacag tctcctca 348 <210> 8 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> ZIKV mAb VL <400> 8 gacatcttgc tgactcagtc tccagccatc ctgtctgtga gtccaggaga aagagtcagt 60 ttctcctgca gggccagtca gagcattggc acaagcatac actggtatca gcaaagaaca 120 aatggttctc caaggcttct cataaagtat gcttctgagt ctatctctgg gatcccttcc 180 aggtttagtg gcagtggatc agggacagat tttactctta gcatcaacag tgtggagtct 240 gaagatattg cagattatta ctgtcaacaa agtaatagct ggccactcac gttcggtgct 300 gggaccaagc tggagctgaa a 321 <210> 9 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR1_VH <400> 9 Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Leu Arg Ile Ser Cys Lys Pro Ser 20 25 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR2_VH <400> 10 Ile Asn Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Trp <210> 11 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR3_VH <400> 11 Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr 1 5 10 15 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp 20 25 30 Thr Ala Val Tyr Phe Cys 35 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR4_VH <400> 12 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1 5 10 <210> 13 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR1_VK <400> 13 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser 20 25 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR2_VK <400> 14 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 1 5 10 15 Lys <210> 15 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR3_VK <400> 15 Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu Asp Ile Ala 20 25 30 Asp Tyr Tyr Cys 35 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR4_VK <400> 16 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 1 5 10 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 1_VH <400> 17 Gly His Thr Phe Thr Arg Tyr Trp 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 2_VH <400> 18 Ile Phe Pro Ser Asp Ser Tyr Thr 1 5 <210> 19 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 3_VH <400> 19 Thr Ser Ser Ser Asn Tyr Tyr Tyr Gly Ser Ser Trp Phe Ala Tyr 1 5 10 15 <210> 20 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 1_VK <400> 20 Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 21 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 2_VK <400> 21 Trp Ala Ser 1 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb CDR 3_VK <400> 22 Ile Tyr Pro Gly Ser Gly Asn Ile 1 5 <210> 23 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> ZIKV mAb VH <400> 23 caggtccaac tgcagcagcc tggggctgag gtggtgaggc ctggggcttc agtgaagctg 60 tcctgcaagg cttctggcca caccttcacc aggtactgga taaactgggt gaagcagagg 120 cctggacaag gccttgagtg gatcggaaat atttttcctt ctgatagtta tactaactac 180 aatcaaaagt tcaaggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240 atgcagctca gcagcccgac atctgaggac tctgcggtct attactgtac aagctcctcc 300 aattattact acggtagtag ctggtttgct tactggggcc aagggactct ggtcactgtc 360 tctgca 366 <210> 24 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> ZIKV mAb VL <400> 24 gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60 atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 120 tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240 atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 300 cctcggacgt tcggtggagg caccaagctg gaaatcaaa 339 <210> 25 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR1_VH <400> 25 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser 20 25 <210> 26 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR2_VH <400> 26 Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Asn <210> 27 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR3_VH <400> 27 Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys 1 5 10 15 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp 20 25 30 Ser Ala Val Tyr Tyr Cys 35 <210> 28 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR4_VH <400> 28 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1 5 10 <210> 29 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR1_VK <400> 29 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser 20 25 <210> 30 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR2_VK <400> 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 31 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR3_VK <400> 31 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala 20 25 30 Val Tyr Tyr Cys 35 <210> 32 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> ZIKV mAb FR4_VK <400> 32 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <110> KOREA BASIC SCIENCE INSTITUTE <120> Antibody for Detection Specific for Non-structural protein 1 of ZIKA virus and use its <130> PD21-446 <160> 32 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 1_VH <400> 1 Gly Tyr Thr Phe Thr Asp Tyr Tyr 1 5 <210> 2 <211> 8 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 2_VH <400> 2 Ile Tyr Pro Gly Ser Gly Asn Ile 1 5 <210> 3 <211> 9 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 3_VH <400> 3 Ala Arg Pro Thr Val Tyr Phe Asp Tyr 1 5 <210> 4 <211> 6 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 1_VK <400> 4 Gln Ser Ile Gly Thr Ser 1 5 <210> 5 <211> 3 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 2_VK <400> 5 Tyr Ala Ser One <210> 6 <211> 9 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 3_VK <400> 6 Gln Gln Ser Asn Ser Trp Pro Leu Thr 1 5 <210> 7 <211> 348 <212> DNA <213> artificial sequence <220> <223> ZIKV mAb VH <400> 7 cagatccagc tgcagcagtc tggacctggg gccaaggcac cactctctga gctggtgaag 60 cctggggctt cactgaggat atcctgcaag ccttctggct acaccttcac tgactactat 120 ataaactggg tgaagcagaa gcctggacag ggacttgagt ggattggatg gatttacct 180 ggaagcggta atattaagta caatgagaag ttcaagggca aggccacatt gactgtagac 240 acatcctcca gcacagccta catgcagctc agcagcctga catctgagga cactgctgtc 300 tatttctgtg caagacctac ggtctatttt gactatacag tctcctca 348 <210> 8 <211> 321 <212> DNA <213> artificial sequence <220> <223> ZIKV mAb VL <400> 8 gacatcttgc tgactcagtc tccagccatc ctgtctgtga gtccaggaga aagagtcagt 60 ttctcctgca gggccagtca gagcattggc acaagcatac actggtatca gcaaagaaca 120 aatggttctc caaggcttct cataaagtat gcttctgagt ctatctctgg gatcccttcc 180 aggtttagtg gcagtggatc agggacagat tttactctta gcatcaacag tgtggagtct 240 gaagatattg cagattatta ctgtcaacaa agtaatagct ggccactcac gttcggtgct 300 gggaccaagc tggagctgaa a 321 <210> 9 <211> 25 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR1_VH <400> 9 Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Leu Arg Ile Ser Cys Lys Pro Ser 20 25 <210> 10 <211> 17 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR2_VH <400> 10 Ile Asn Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Trp <210> 11 <211> 38 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR3_VH <400> 11 Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr 1 5 10 15 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp 20 25 30 Thr Ala Val Tyr Phe Cys 35 <210> 12 <211> 11 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR4_VH <400> 12 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1 5 10 <210> 13 <211> 26 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR1_VK <400> 13 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser 20 25 <210> 14 <211> 17 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR2_VK <400> 14 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 1 5 10 15 Lys <210> 15 <211> 36 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR3_VK <400> 15 Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu Asp Ile Ala 20 25 30 Asp Tyr Tyr Cys 35 <210> 16 <211> 10 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR4_VK <400> 16 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 1 5 10 <210> 17 <211> 8 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 1_VH <400> 17 Gly His Thr Phe Thr Arg Tyr Trp 1 5 <210> 18 <211> 8 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 2_VH <400> 18 Ile Phe Pro Ser Asp Ser Tyr Thr 1 5 <210> 19 <211> 15 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 3_VH <400> 19 Thr Ser Ser Ser Asn Tyr Tyr Tyr Gly Ser Ser Trp Phe Ala Tyr 1 5 10 15 <210> 20 <211> 12 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 1_VK <400> 20 Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 21 <211> 3 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 2_VK <400> 21 Trp Ala Ser One <210> 22 <211> 8 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb CDR 3_VK <400> 22 Ile Tyr Pro Gly Ser Gly Asn Ile 1 5 <210> 23 <211> 366 <212> DNA <213> artificial sequence <220> <223> ZIKV mAb VH <400> 23 caggtccaac tgcagcagcc tggggctgag gtggtgaggc ctggggcttc agtgaagctg 60 tcctgcaagg cttctggcca caccttcacc aggtactgga taaactgggt gaagcagagg 120 cctggacaag gccttgagtg gatcggaaat atttttcctt ctgatagtta tactaactac 180 aatcaaaagt tcaaggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240 atgcagctca gcagcccgac atctgaggac tctgcggtct attactgtac aagctcctcc 300 aattattact acggtagtag ctggtttgct tactggggcc aagggactct ggtcactgtc 360 tctgca 366 <210> 24 <211> 339 <212> DNA <213> artificial sequence <220> <223> ZIKV mAb VL <400> 24 gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60 atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 120 tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240 atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 300 cctcggacgt tcggtggagg caccaagctg gaaatcaaa 339 <210> 25 <211> 25 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR1_VH <400> 25 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser 20 25 <210> 26 <211> 17 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR2_VH <400> 26 Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Asn <210> 27 <211> 38 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR3_VH <400> 27 Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys 1 5 10 15 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp 20 25 30 Ser Ala Val Tyr Tyr Cys 35 <210> 28 <211> 11 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR4_VH <400> 28 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1 5 10 <210> 29 <211> 26 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR1_VK <400> 29 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser 20 25 <210> 30 <211> 17 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR2_VK <400> 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 31 <211> 36 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR3_VK <400> 31 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala 20 25 30 Val Tyr Tyr Cys 35 <210> 32 <211> 10 <212> PRT <213> artificial sequence <220> <223> ZIKV mAb FR4_VK <400> 32 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10
Claims (14)
중쇄 가변영역과 경쇄 가변영역을 포함하는, 지카 바이러스 특이적 항체 또는 이의 결합 단편.
It binds specifically to non-structural protein 1 of Zika virus (ZIKV),
A Zika virus-specific antibody or binding fragment thereof comprising a heavy chain variable region and a light chain variable region.
상기 경쇄 가변영역은 서열번호 4의 아미노산 서열을 포함하는 CDR1-VK, 서열번호 5의 아미노산 서열을 포함하는 CDR2-VK 및 서열번호 6의 아미노산 서열을 포함하는 CDR3-VK를 포함하는 것을 특징으로 하는, 지카 바이러스 특이적 항체 또는 이의 결합 단편.
The method according to claim 1, wherein the heavy chain variable region comprises CDR1-VH comprising the amino acid sequence of SEQ ID NO: 1, CDR2-VH comprising the amino acid sequence of SEQ ID NO: 2, and CDR3-VH comprising the amino acid sequence of SEQ ID NO: 3 and
The light chain variable region comprises CDR1-VK comprising the amino acid sequence of SEQ ID NO: 4, CDR2-VK comprising the amino acid sequence of SEQ ID NO: 5, and CDR3-VK comprising the amino acid sequence of SEQ ID NO: 6. , Zika virus-specific antibodies or binding fragments thereof.
상기 경쇄 가변영역은 서열번호 20의 아미노산 서열을 포함하는 CDR1-VK, 서열번호 21의 아미노산 서열을 포함하는 CDR2-VK 및 서열번호 22의 아미노산 서열을 포함하는 CDR3-VK를 포함하는 것을 특징으로 하는, 지카 바이러스 특이적 항체 또는 이의 결합 단편.
The method according to claim 1, wherein the heavy chain variable region comprises CDR1-VH comprising the amino acid sequence of SEQ ID NO: 17, CDR2-VH comprising the amino acid sequence of SEQ ID NO: 18, and CDR3-VH comprising the amino acid sequence of SEQ ID NO: 19 and
The light chain variable region comprises CDR1-VK comprising the amino acid sequence of SEQ ID NO: 20, CDR2-VK comprising the amino acid sequence of SEQ ID NO: 21, and CDR3-VK comprising the amino acid sequence of SEQ ID NO: 22. , Zika virus-specific antibodies or binding fragments thereof.
The antibody or antibody according to any one of claims 1 to 3, wherein the antibody or binding fragment thereof is a scFv fragment, scFv-Fc fragment, Fab fragment, Fv fragment, diabody, chimeric antibody, humanized antibody or human antibody. binding fragments thereof.
The antibody or binding fragment thereof according to claim 4, wherein the antibody or binding fragment thereof is a Fab fragment.
A polynucleotide encoding the antibody or binding fragment thereof of any one of claims 1 to 3.
The polynucleotide according to claim 6, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 8.
The polynucleotide according to claim 6, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 23 and the nucleotide sequence of SEQ ID NO: 24.
An expression vector into which the polynucleotide of claim 6 is inserted.
The expression vector according to claim 9, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 8.
The expression vector according to claim 9, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 23 and the nucleotide sequence of SEQ ID NO: 24.
A hybridoma cell producing the antibody or binding fragment thereof of any one of claims 1 to 3.
상기 바이러스 항원은 지카 바이러스의 비구조 단백질 1(Non-structural protein 1) 이고,
상기 제제는 청구항 1 내지 3 중 어느 한 항의 항체 또는 이의 결합 단편인, 진단용 키트.
A kit for diagnosing Zika virus, comprising an agent for diagnosing a Zika virus (ZIKV) antigen,
The viral antigen is non-structural protein 1 of Zika virus,
The agent is an antibody or a binding fragment thereof according to any one of claims 1 to 3, a diagnostic kit.
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| KR1020220021393A KR20230124282A (en) | 2022-02-18 | 2022-02-18 | Antibody for Detection Specific for Non-structural protein 1 of ZIKA virus and Use thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| KR1020220021393A KR20230124282A (en) | 2022-02-18 | 2022-02-18 | Antibody for Detection Specific for Non-structural protein 1 of ZIKA virus and Use thereof |
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