KR20230066722A - Synagis-resistant respiratory syncytial virus detection method - Google Patents
Synagis-resistant respiratory syncytial virus detection method Download PDFInfo
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- KR20230066722A KR20230066722A KR1020210152000A KR20210152000A KR20230066722A KR 20230066722 A KR20230066722 A KR 20230066722A KR 1020210152000 A KR1020210152000 A KR 1020210152000A KR 20210152000 A KR20210152000 A KR 20210152000A KR 20230066722 A KR20230066722 A KR 20230066722A
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- synagis
- protein
- respiratory syncytial
- syncytial virus
- fluorescent protein
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- C—CHEMISTRY; METALLURGY
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Abstract
본 발명은 시나지스 내성 호흡기세포융합바이러스에 특이적으로 결합하는 항체 및 검출 보조용 단백질에 관한 것으로, 보다 상세하게는 상기 항체 및 검출 보조용 단백질을 이용한 시나지스 내성 호흡기세포융합바이러스 검출에 관한 것이다. 본 발명에 따른 항체 및 검출 보조용 단백질을 포함하는 시나지스 내성 호흡기세포융합바이러스 검출용 조성물은 시나지스 내성 호흡기세포융합바이러스가 존재할 경우 강한 형광 신호가 검출되는 것을 실험적으로 확인하였다. 이는 본 발명의 검출용 조성물이 시나지스 내성 호흡기세포융합바이러스를 민감하고 특이적으로 검출할 수 있음을 의미하는바, 시나지스 내성 호흡기세포융합바이러스 진단 및 관련 연구 분야에서 다양하게 활용될 수 있다.The present invention relates to an antibody that specifically binds to Synagis-resistant respiratory syncytial virus and an auxiliary protein for detection, and more particularly, to the detection of Synagis-resistant respiratory syncytial virus using the antibody and an auxiliary protein for detection. . It was experimentally confirmed that the composition for detecting Synagis-resistant respiratory syncytial virus, including the antibody and the protein for detection according to the present invention, detects a strong fluorescence signal when Synagis-resistant respiratory syncytial virus is present. This means that the composition for detection of the present invention can sensitively and specifically detect Synagis-resistant respiratory syncytial virus, and thus can be variously used in the diagnosis of Synagis-resistant respiratory syncytial virus and related research fields.
Description
본 발명은 시나지스 내성 호흡기세포융합바이러스에 특이적으로 결합하는 항체 및 검출 보조용 단백질에 관한 것으로, 보다 상세하게는 상기 항체 및 검출 보조용 단백질을 이용한 시나지스 내성 호흡기세포융합바이러스 검출에 관한 것이다.The present invention relates to an antibody that specifically binds to Synagis-resistant respiratory syncytial virus and an auxiliary protein for detection, and more particularly, to the detection of Synagis-resistant respiratory syncytial virus using the antibody and an auxiliary protein for detection. .
미숙아; 및 만성 폐 질환 또는 선천성 심장 질환이 있는 유아;는 중증 호흡기 세포융합 바이러스(respiratory syncytial virus, RSV) 감염 위험이 증가한다. Synagis(Palivizumab; MedImmune)은 RSV의 융합단백질(F-protein)에 특이적인 인간화 IgG1 단일클론항체(MAb)로 RSV에 대한 중화 및 융합억제 활성을 갖고 있다. 무작위 대조 시험에서 시나지스는 미숙아 또는 기관지폐 이형성증/미숙아 만성 폐 질환이 있는 소아 및 선천성 심장 질환이 있는 소아에서 RSV 질환으로 인한 입원을 ~50%까지 줄였다. 시나지스는 RSV 질병의 위험이 높은 어린이의 RSV로 인한 심각한 하기도 질환 예방에 사용하기 위해 1998년 FDA에 의해 허가되었다.premie; and infants with chronic lung disease or congenital heart disease; are at increased risk of severe respiratory syncytial virus (RSV) infection. Synagis (Palivizumab; MedImmune) is a humanized IgG1 monoclonal antibody (MAb) specific to the RSV fusion protein (F-protein) and has neutralizing and fusion inhibitory activities against RSV. In a randomized controlled trial, Synagis reduced hospitalizations for RSV disease by ~50% in children with prematurity or bronchopulmonary dysplasia/chronic lung disease of prematurity and children with congenital heart disease. Synagis was approved by the FDA in 1998 for use in the prevention of serious lower respiratory tract disease caused by RSV in children at high risk of RSV disease.
RSV와 같은 RNA 바이러스는 돌연변이 비율이 높은 것으로 알려져 있다. F-단백질의 A 영역에 있는 시나지스 결합 에피토프의 존재는 RSV의 중화와 관련이 있다. 이 에피토프는 실험실에서 선택된 팔리비주맙 내성 분리주에서 변경된 것을 확인하였다. 다른 연구에서 다른 항-RSV F-단백질 MAb(anti-RSV F-protein MAb)에 대한 시험관 내 돌연변이가 생성되는 것을 확인하였다. 이에, 시나지스 내성 RSV 검출에 대한 연구가 필요한 실정이다.RNA viruses such as RSV are known to have high mutation rates. The presence of synagis binding epitopes in the A region of the F-protein is associated with neutralization of RSV. This epitope was confirmed to be altered in palivizumab-resistant isolates selected in the laboratory. Other studies have confirmed the generation of in vitro mutations to other anti-RSV F-protein MAbs. Therefore, research on the detection of Synagis-resistant RSV is required.
이에 본 발명자들은 시나지스 내성 RSV 검출을 위한 항체 및 검출 보조용 단백질을 개발하고, 이의 우수한 시나지스 내성 RSV 검출능을 확인함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors have completed the present invention by developing an antibody for detecting Synagis-resistant RSV and an auxiliary protein for detection, and confirming its excellent ability to detect Synagis-resistant RSV.
따라서 본 발명의 목적은, 시나지스 내성 호흡기세포융합바이러스에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편을 제공하는 것이다.Accordingly, an object of the present invention is to provide an antibody or antigen-binding fragment thereof that specifically binds to Synagis-resistant respiratory syncytial virus.
본 발명의 다른 목적은, 시나지스 내성 호흡기세포융합바이러스 검출 보조용 단백질을 제공하는 것이다.Another object of the present invention is to provide a protein for assisting in the detection of Synagis-resistant respiratory syncytial virus.
본 발명의 또 다른 목적은, 상기 시나지스 내성 호흡기세포융합바이러스에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편; 및 시나지스 내성 호흡기세포융합바이러스 검출 보조용 단백질;을 포함하는 시나지스 내성 호흡기세포융합바이러스 검출용 조성물을 제공하는 것이다.Another object of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to the Synagis-resistant respiratory syncytial virus; To provide a composition for detecting Synagis-resistant respiratory syncytial virus, including; and a synagis-resistant respiratory syncytial virus detection auxiliary protein.
본 발명의 또 다른 목적은, 상기 시나지스 내성 호흡기세포융합바이러스 검출용 조성물과 시료를 반응시키는 단계;를 포함하는 시나지스 내성 호흡기세포융합바이러스 검출 방법을 제공하는 것이다.Another object of the present invention is to provide a method for detecting Synagis-resistant respiratory syncytial virus comprising the step of reacting the sample with the composition for detecting Synagis-resistant respiratory syncytial virus.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1로 표시되는 아미노산 서열로 구성되는 CDR1(complementarity determining region 1), 서열번호 2로 표시되는 아미노산 서열로 구성되는 CDR2(complementarity determining region 2) 및 서열번호 3으로 표시되는 아미노산 서열로 구성되는 CDR3(complementarity determining region 3)을 포함하는 중쇄 가변영역; 및 서열번호 5로 표시되는 아미노산 서열로 구성되는 CDR1, 서열번호 6로 표시되는 아미노산 서열로 구성되는 CDR2 및 서열번호 7으로 표시되는 아미노산 서열로 구성되는 CDR3을 포함하는 경쇄 가변영역을 포함하는, 시나지스 내성 호흡기세포융합바이러스(Respiratory syncytial virus, RSV)에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편을 제공한다.In order to achieve the above object, the present invention consists of CDR1 (complementarity determining region 1) composed of the amino acid sequence represented by SEQ ID NO: 1, CDR2 (complementarity determining region 2) composed of the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NO: a heavy chain variable region comprising a complementarity determining region 3 (CDR3) composed of the amino acid sequence represented by 3; and a light chain variable region comprising CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 5, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 7; Provided is an antibody or antigen-binding fragment thereof that specifically binds to Jis-resistant respiratory syncytial virus (Respiratory syncytial virus, RSV).
또한 본 발명은 N 말단에 위치한 제1 형광 단백질 단편; C 말단에 위치한 제2 형광 단백질 단편; 및 서열번호 11의 아미노산 서열로 표시되는 에피토프;를 포함하고, 상기 제1 및 제2 형광 단백질 단편은 각각 전장 형광단백질의 N 말단 및 C 말단 단편인, 시나지스 내성 호흡기세포융합바이러스(Respiratory syncytial virus, RSV) 검출 보조용 단백질을 제공한다.In addition, the present invention is a first fluorescent protein fragment located at the N-terminus; a second fluorescent protein fragment located at the C-terminus; And an epitope represented by the amino acid sequence of SEQ ID NO: 11; wherein the first and second fluorescent protein fragments are N-terminal and C-terminal fragments of full-length fluorescent protein, respectively, Synagis-resistant respiratory syncytial virus , RSV) to assist in detection.
또한 본 발명은 상기 시나지스 내성 호흡기세포융합바이러스에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편; 및 상기 시나지스 내성 호흡기세포융합바이러스 검출 보조용 단백질;을 포함하는 시나지스 내성 호흡기세포융합바이러스 검출용 조성물을 제공한다.In addition, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the Synagis-resistant respiratory syncytial virus; It provides a composition for detecting Synagis-resistant respiratory syncytial virus comprising; and a protein for assisting in the detection of the Synagis-resistant respiratory syncytial virus.
또한 본 발명은 상기 시나지스 내성 호흡기세포융합바이러스 검출용 조성물을 포함하는 시나지스 내성 호흡기세포융합바이러스 검출 키트를 제공한다.In addition, the present invention provides a Synagis-resistant respiratory syncytial virus detection kit comprising the composition for detecting the Synagis-resistant respiratory syncytial virus.
또한 본 발명은 상기 시나지스 내성 호흡기세포융합바이러스 검출용 조성물과 시료를 반응시키는 단계;를 포함하는 시나지스 내성 호흡기세포융합바이러스 검출 방법을 제공한다.In addition, the present invention provides a method for detecting Synagis-resistant respiratory syncytial virus comprising the step of reacting the sample with the composition for detecting Synagis-resistant respiratory syncytial virus.
본 발명에 따른 항체 및 검출 보조용 단백질을 포함하는 시나지스 내성 호흡기세포융합바이러스 검출용 조성물은 시나지스 내성 호흡기세포융합바이러스가 존재할 경우 강한 형광 신호가 검출되는 것을 실험적으로 확인하였다. 이는 본 발명의 검출용 조성물이 시나지스 내성 호흡기세포융합바이러스를 민감하고 특이적으로 검출할 수 있음을 의미하는바, 시나지스 내성 호흡기세포융합바이러스 진단 및 관련 연구 분야에서 다양하게 활용될 수 있다.It was experimentally confirmed that the composition for detecting Synagis-resistant respiratory syncytial virus, including the antibody and the protein for detection according to the present invention, detects a strong fluorescence signal when Synagis-resistant respiratory syncytial virus is present. This means that the composition for detection of the present invention can sensitively and specifically detect Synagis-resistant respiratory syncytial virus, and thus can be variously used in the diagnosis of Synagis-resistant respiratory syncytial virus and related research fields.
도 1은 본 발명에 따른 시나지스 내성 RSV에 특이적으로 결합하는 항체 1A12의 Kd값을 확인한 결과를 나타낸 도이다.
도 2a는 본 발명에 따른 단백질 11L의 3D 모델 및 작동메커니즘을 나타낸 도이다.
도 2b는 본 발명에 따른 단백질 11L의 Kd값을 확인한 결과를 나타낸 도이다.
도 2c는 본 발명에 따른 1A12 항체가 결합된 단백질 11L의 형광 값을 측정한 결과를 나타낸 도이다.
도 3은 본 발명에 따른 단백질 11L의 시나지스 내성 RSV S275F에 대한 형광 검출 분석 결과를 나타낸 도이다.
도 4 및 5는 웨스턴 블롯팅을 통해 바이러스 유사입자 생성 결과를 확인한 결과를 나타낸 도이다.
도 6은 본 발명에 따른 단백질 11L 및 항체 1A12를 이용한 시나지스 내성 RSV 검출 결과를 나타낸 도이다.
도 7은 본 발명에 따른 단백질 11L 및 항체 1A12를 이용하여 시료 내 시나지스 내성 RSV를 형광신호를 통해 확인한 결과를 나타낸 도이다.1 is a diagram showing the results of confirming the Kd value of antibody 1A12 specifically binding to Synagis-resistant RSV according to the present invention.
Figure 2a is a diagram showing a 3D model and operation mechanism of protein 11L according to the present invention.
Figure 2b is a diagram showing the results of confirming the Kd value of the protein 11L according to the present invention.
Figure 2c is a diagram showing the results of measuring the fluorescence value of protein 11L bound to the 1A12 antibody according to the present invention.
3 is a diagram showing the results of fluorescence detection analysis of protein 11L according to the present invention against Synagis-resistant RSV S275F.
4 and 5 are diagrams showing the results of confirming the virus-like particle production results through Western blotting.
6 is a diagram showing the results of detection of Synagis-resistant RSV using protein 11L and antibody 1A12 according to the present invention.
7 is a diagram showing the results of confirming Synagis-resistant RSV in a sample through fluorescence signals using protein 11L and antibody 1A12 according to the present invention.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 양태에 따르면, 본 발명은 서열번호 1로 표시되는 아미노산 서열로 구성되는 CDR1(complementarity determining region 1), 서열번호 2로 표시되는 아미노산 서열로 구성되는 CDR2(complementarity determining region 2) 및 서열번호 3으로 표시되는 아미노산 서열로 구성되는 CDR3(complementarity determining region 3)을 포함하는 중쇄 가변영역; 및 서열번호 5로 표시되는 아미노산 서열로 구성되는 CDR1, 서열번호 6로 표시되는 아미노산 서열로 구성되는 CDR2 및 서열번호 7으로 표시되는 아미노산 서열로 구성되는 CDR3을 포함하는 경쇄 가변영역을 포함하는, 시나지스 내성 호흡기세포융합바이러스(Respiratory syncytial virus, RSV)에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편을 제공한다.According to an aspect of the present invention, CDR1 (complementarity determining region 1) composed of the amino acid sequence represented by SEQ ID NO: 1, CDR2 (complementarity determining region 2) composed of the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NO: a heavy chain variable region comprising a complementarity determining region 3 (CDR3) composed of the amino acid sequence represented by 3; and a light chain variable region comprising CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 5, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 7; Provided is an antibody or antigen-binding fragment thereof that specifically binds to Jis-resistant respiratory syncytial virus (Respiratory syncytial virus, RSV).
본 발명에 있어서, 항체(antibody)는 전체 항체(whole) 형태일 뿐만 아니라 항체 분자의 기능적인 단편을 포함한다. 전체 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 구조이며 각각의 경쇄는 중쇄와 다이설파이드 결합으로 연결되어 있다. 항체 분자의 기능적인 단편이란 항원 결합 기능을 보유하고 있는 단편을 뜻하며 Fab, F(ab'), F(ab')2 및 Fv 등을 포함한다. 항체 단편 중 Fab는 경쇄 및 중쇄의 가변영역과 경쇄의 불변 영역 및 중쇄의 첫 번째 불변 영역(CH1)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 C 말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역(hinge region)을 가진다는 점에서 Fab와 차이가 있다. F(ab')2 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv는 중쇄 가변부위 및 경쇄 가변부위만을 가지고 있는 최소의 항체조각으로 Fv 단편을 생성하는 재조합 기술은 국제공개 특허 WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 및 WO88/09344에 개시되어 있다. 이중쇄 Fv(dsFv)는 디설파이드 결합으로 중쇄 가변부위와 경쇄 가변부위가 연결되어 있고 단쇄 Fv(scFv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변 영역과 경쇄의 가변 영역이 공유 결합으로 연결되어 있다. 이러한 항체 단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고(예를 들어, 전체 항체를 파파인으로 제한 절단하며 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab')2 단편을 얻을 수 있다) 바람직하게는 유전자 재조합 기술을 통하여 제작할 수 있다. 본 발명에서 항체는 바람직하게는 전체 항체 형태이다.In the present invention, antibodies include functional fragments of antibody molecules as well as whole antibodies. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. A functional fragment of an antibody molecule refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure having variable regions of light and heavy chains, constant regions of light chains, and a first constant region (CH1) of heavy chains, and has one antigen-binding site. Fab' is different from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. An F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv is a minimal antibody fragment having only the heavy chain variable region and the light chain variable region. Recombinant techniques for generating Fv fragments are disclosed in International Publication Patents WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO88/ 09344. In double-chain Fv (dsFv), the heavy chain variable region and light chain variable region are linked by a disulfide bond, and in single-chain Fv (scFv), the heavy chain variable region and light chain variable region are generally covalently linked through a peptide linker. Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), preferably can be produced through genetic recombination technology. Antibodies in the present invention are preferably in the form of whole antibodies.
본 발명의 구체예에서, 상기 중쇄 가변영역은 서열번호 4의 아미노산 서열로 표시되는 것이 바람직하다.In an embodiment of the present invention, the heavy chain variable region is preferably represented by the amino acid sequence of SEQ ID NO: 4.
본 발명의 구체예에서, 상기 경쇄 가변영역은 서열번호 8의 아미노산 서열로 표시되는 것이 바람직하다.In an embodiment of the present invention, the light chain variable region is preferably represented by the amino acid sequence of SEQ ID NO: 8.
본 발명에 있어서, 가변 영역은 항원과 특이적으로 결합하는 기능을 수행하면서 서열상의 많은 변이(variation)을 보이는 항체 분자의 부분을 의미하고, 변영역에는 CDR1, CDR2 및 CDR3가 존재한다. 상보성 결정 영역(complementarity determining regions: CDR)은 항원의 인식에 관여하는 고리모양의 부위로서 이 부위의 서열이 변함에 따라 항체의 항원에 대한 특이성이 결정되는 중요한 부위이다.In the present invention, the variable region refers to a portion of an antibody molecule that exhibits many variations in sequence while performing a function of specifically binding to an antigen, and CDR1, CDR2, and CDR3 are present in the variable region. Complementarity determining regions (CDRs) are ring-shaped regions involved in antigen recognition, and are important regions in which the specificity of an antibody for an antigen is determined according to changes in the sequence of this region.
본 발명의 범위에는 각 아미노산 서열로 표시되는 펩타이드의 기능적 동등물 및 그들의 염을 포함한다. 상기 기능적 동등물이란 아미노산의 부가, 치환 또는 결실의 결과, 본 발명의 각 아미노산 서열로 표시되는 펩타이드와 적어도 80％ 이상의, 바람직하게는 90％, 더욱 바람직하게는 95％이상의 서열 상동성(즉, 동일성)을 갖는 것으로 예를 들면, 80％, 81％, 82％, 83％, 84％, 85％, 86％, 87％, 88％, 89％, 90％, 91％, 92％, 93％, 94％, 95％, 96％, 97％, 98％, 99％, 100％의 서열 상동성을 갖는 것을 포함한다. 실질적으로 동질의 생리활성을 나타내는 펩타이드를 말한다. 본 명세서에서 서열 상동성 및 동질성은 본 발명의 각각의 아미노산 서열과 후보 서열을 정렬하고 갭(gaps)을 도입한 후 각 아미노산 서열에 대한 후보 서열의 아미노산 잔기의 백분율로서 정의된다. 필요한 경우, 최대 백분율 서열 동질성을 수득하기 위하여 서열 동질성의 부분으로서 보존적 치환은 고려하지 않는다. 각 아미노산 서열의 N-말단, C-말단 또는 내부 신장, 결손 또는 삽입은 서열 동질성 또는 상동성에 영향을 주는 서열로서 해석되지 않는다. 또한 상기 서열 동질성은 두개의 폴리펩타이드의 아미노산 서열의 유사한 부분을 비교하기 위해 사용되는 일반적인 표준 방법에 의해 결정할 수 있다. BLAST 또는 FASTA와 같은 컴퓨터 프로그램은 두개의 폴리펩타이드를 각 각의 아미노산이 최적으로 매칭(matching) 되도록 정렬한다(하나 또는 두 서열의 전장서열을 따라 또는 하나 또는 두 서열의 예측된 부분을 따라). 상기 프로그램은 디펄트 오프닝 패널티(default opening penalty) 및 디펄트 갭 페널티(default gap penalty)를 제공하며 컴퓨터 프로그램과 함께 연계되어 사용될 수 있는 PAM250(표준 스코링 매트릭스; Dayhoff et al., in Atlas of Protein Sequence and Structure, vol 5, supp. 3, 1978)와 같은 스코링 매트릭스를 제공한다. 예를 들어, 백분율 동질성은 다음과 같이 계산할 수 있다: 일치하는 서열(identical matches)의 총 수에 100을 곱한 다음 대응되는 스팬(machted span) 내의 보다 긴 서열의 길이와 두 서열을 정렬하기 위해 보다 긴 서열내로 도입된 갭(gaps)의 수의 합으로 나눈다.The scope of the present invention includes functional equivalents of the peptides represented by each amino acid sequence and their salts. The functional equivalent is at least 80% or more, preferably 90%, more preferably 95% or more sequence homology (i.e., identity), for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, including those with 100% sequence homology. It refers to a peptide that exhibits substantially the same physiological activity. Sequence homology and identity herein are defined as the percentage of amino acid residues in a candidate sequence for each amino acid sequence after aligning each amino acid sequence of the present invention with the candidate sequence and introducing gaps. If necessary, conservative substitutions are not considered as part of sequence identity in order to obtain the maximum percent sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions of each amino acid sequence are not to be construed as affecting sequence identity or homology. In addition, the sequence identity can be determined by standard methods commonly used to compare similar portions of the amino acid sequences of two polypeptides. A computer program such as BLAST or FASTA aligns two polypeptides so that each amino acid is optimally matched (along the full length of one or both sequences or along a predicted portion of one or both sequences). The program provides a default opening penalty and a default gap penalty and is PAM250 (Standard Scoring Matrix; Dayhoff et al., in Atlas of Protein) that can be used in conjunction with a computer program. Sequence and Structure,
본 발명의 기능적 동등물 범위에는 본 발명의 각 아미노산 서열로 표시되는 펩타이드의 기본골격을 유지하면서 펩타이드의 일부 화학 구조가 변형된 유도체가 포함된다. 예를 들어 펩타이드의 안정성, 저장성, 휘발성 또는 용해도 등을 변경시키기 위한 구조변경이 이에 포함된다.The range of functional equivalents of the present invention includes derivatives in which a part of the chemical structure of the peptide is modified while maintaining the basic skeleton of the peptide represented by each amino acid sequence of the present invention. For example, structural modification to change the stability, storage stability, volatility or solubility of the peptide is included in this.
본 발명의 구체예에서, 상기 시나지스 내성 호흡기세포융합바이러스는 N262D, K272N 및 S275F로 이루어진 군에서 선택된 1종 이상인 것이 바람직하며, 더 바람직하게는 S275F일 수 있고, 이에 본 발명의 범위가 제한되지 않는다.In an embodiment of the present invention, the Synagis-resistant respiratory syncytial virus is preferably one or more selected from the group consisting of N262D, K272N and S275F, more preferably S275F, and the scope of the present invention is not limited thereto. don't
본 발명의 다른 양태에서, 본 발명은 N 말단에 위치한 제1 형광 단백질 단편; C 말단에 위치한 제2 형광 단백질 단편; 및 서열번호 11의 아미노산 서열로 표시되는 에피토프;를 포함하고, 상기 제1 및 제2 형광 단백질 단편은 각각 전장 형광단백질의 N 말단 및 C 말단 단편인, 시나지스 내성 호흡기세포융합바이러스 검출 보조용 단백질을 제공한다.In another aspect of the invention, the invention provides a first fluorescent protein fragment located at the N-terminus; a second fluorescent protein fragment located at the C-terminus; And an epitope represented by the amino acid sequence of SEQ ID NO: 11; wherein the first and second fluorescent protein fragments are N-terminal and C-terminal fragments of full-length fluorescent protein, respectively. provides
본 발명의 구체예에서, 상기 형광 단백질은 YFP(yellow fluorescent protein), EYFP(enhanced yellow fluorescent protein), GFP(green fluorescent protein), EGFP(enhanced GFP), BFP(blue fluorescent protein), EBFP(enhanced BFP), CFP(cyan fluorescent protein) 또는 ECFP(enhanced CFP)인 것이 바람직하나, 이에 제한되지 않는다.In an embodiment of the present invention, the fluorescent protein is YFP (yellow fluorescent protein), EYFP (enhanced yellow fluorescent protein), GFP (green fluorescent protein), EGFP (enhanced GFP), BFP (blue fluorescent protein), EBFP (enhanced BFP) ), CFP (cyan fluorescent protein) or ECFP (enhanced CFP) is preferred, but is not limited thereto.
본 발명의 구체예에서, 상기 제1 형광 단백질 단편은 서열번호 9의 아미노산 서열로 표시되는 것이 바람직하다.In an embodiment of the present invention, the first fluorescent protein fragment is preferably represented by the amino acid sequence of SEQ ID NO: 9.
본 발명의 구체예에서, 상기 제2 형광 단백질 단편은 서열번호 10의 아미노산 서열로 표시되는 것이 바람직하다.In an embodiment of the present invention, the second fluorescent protein fragment is preferably represented by the amino acid sequence of SEQ ID NO: 10.
본 발명의 구체예에서, 상기 단백질은 링커를 더 포함하는 것이 바람직하고, 더 바람직하게는 상기 링커는 서열번호 13의 염기서열로 코딩되는 것이나, 이에 제한되지 않는다.In an embodiment of the present invention, the protein preferably further includes a linker, and more preferably, the linker is encoded by the nucleotide sequence of SEQ ID NO: 13, but is not limited thereto.
본 발명의 구체예에서, 상기 단백질은 서열번호 12의 염기서열로 암호화되는 것이 바람직하다.In an embodiment of the present invention, the protein is preferably encoded by the nucleotide sequence of SEQ ID NO: 12.
본 발명의 바람직한 구체예에서, 상기 단백질은 하기 구조식의 형태로 배열된 것이 바람직하다.In a preferred embodiment of the present invention, the protein is preferably arranged in the form of the following structural formula.
[구조식][constitutional formula]
제1 형광 단백질 단편-에피토프-링커-에피토프-제2 형광단백질 단편 First fluorescent protein fragment - epitope - linker - epitope - second fluorescent protein fragment
본 발명에 따른 검출 보조용 단백질은 N 말단 및 C 말단에 각각 제1 및 제2 형광단백질 단편이 결합되어 있다. 이에 따라, 상기 제1 및 제2 형광단백질 단편의 물리적 거리가 가까워질 경우 형광 신호를 검출할 수 있다.In the auxiliary detection protein according to the present invention, first and second fluorescent protein fragments are bound to the N-terminus and the C-terminus, respectively. Accordingly, a fluorescence signal can be detected when the physical distance between the first and second fluorescent protein fragments becomes close.
본 발명의 또 다른 양태에서, 본 발명은 상기 시나지스 내성 호흡기세포융합바이러스에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편; 및 상기 시나지스 내성 호흡기세포융합바이러스 검출 보조용 단백질;을 포함하는 시나지스 내성 호흡기세포융합바이러스 검출용 조성물을 제공한다. 또한 본 발명은 상기 조성물을 포함하는 시나지스 내성 호흡기세포융합바이러스 검출 키트를 제공한다.In another aspect of the present invention, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the Synagis-resistant respiratory syncytial virus; It provides a composition for detecting Synagis-resistant respiratory syncytial virus comprising; and a protein for assisting in the detection of the Synagis-resistant respiratory syncytial virus. In addition, the present invention provides a Synagis-resistant respiratory syncytial virus detection kit comprising the composition.
본 발명에 따른 시나지스 내성 호흡기세포융합바이러스 검출용 조성물은 시나지스 내성 호흡기세포융합바이러스에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편; 및 시나지스 내성 호흡기세포융합바이러스 검출 보조용 단백질;을 포함한다. 본 발명의 실시예에서, 상기 ‘항체 및 시나지스 내성 RSV의 Kd 값’은 ‘검출 보조용 단백질 및 시나지스 내성 RSV의 Kd 값’보다 약 15배 높은 것을 실험적으로 확인하였다. 이는 시료에 시나지스 내성 RSV가 존재할 경우 상기 항체는 검출 보조용 단백질이 아닌 시나지스 내성 RSV에 결합한다는 것을 의미한다. 이 때 본 발명의 검출 보조용 단백질의 N 말단 및 C 말단에 결합된 제1 및 제2 형광단백질 단편의 물리적 거리가 가까워지므로, 강한 형광신호가 검출된다(도 3a). 따라서 형광 신호의 검출 여부에 따라 시나지스 내성 RSV의 존재 여부를 확인할 수 있다.The composition for detecting Synagis-resistant respiratory syncytial virus according to the present invention comprises an antibody or antigen-binding fragment thereof that specifically binds to Synagis-resistant respiratory syncytial virus; and synagis-resistant respiratory syncytial virus detection aid proteins; In the examples of the present invention, it was experimentally confirmed that the 'Kd value of the antibody and Synagis-resistant RSV' was about 15 times higher than the 'Kd value of the protein for detection and Synagis-resistant RSV'. This means that when Synagis-resistant RSV is present in the sample, the antibody binds to Synagis-resistant RSV, not to the protein for detection. At this time, since the physical distance of the first and second fluorescent protein fragments bound to the N-terminus and C-terminus of the auxiliary detection protein of the present invention becomes closer, a strong fluorescence signal is detected (FIG. 3a). Therefore, the presence or absence of Synagis-resistant RSV can be confirmed according to whether a fluorescent signal is detected.
또한, 키트는 특정 반응에서 사용되는 시약의 최적량은, 본 명세서에 개시사항을 습득한 당업자에 의해서 용이하게 결정될 수 있다. 전형적으로, 본 발명의 키트는 앞서 언급된 구성성분들을 포함하는 별도의 포장 또는 컴파트먼트(compartment)로 제작된다. 또한 상기 키트는 사용 지침(instruction) 및 기타 검출에 필요한 도구 또는 장비를 더 포함할 수 있다.In addition, the optimal amount of reagents to be used in a particular reaction of the kit can be easily determined by a person skilled in the art having learned the disclosure herein. Typically, the kits of the present invention are manufactured in separate packages or compartments containing the aforementioned components. In addition, the kit may further include instructions for use and other tools or equipment necessary for detection.
본 발명의 또 다른 양태에서, 본 발명은 상기 시나지스 내성 호흡기세포융합바이러스 검출용 조성물과 시료를 반응시키는 단계;를 포함하는 시나지스 내성 호흡기세포융합바이러스 검출 방법을 제공한다.In another aspect of the present invention, the present invention provides a method for detecting Synagis-resistant respiratory syncytial virus comprising the step of reacting the sample with the composition for detecting Synagis-resistant respiratory syncytial virus.
본 발명의 구체예에서, 상기 시료는 호흡기세포융합바이러스 항원, 호흡기세포융합바이러스 배양액 또는 환자 유래 임상 시료 형태인 것이 바람직하나, 이에 제한되지 않는다. In an embodiment of the present invention, the sample is preferably in the form of respiratory syncytial virus antigen, respiratory syncytial virus culture medium, or patient-derived clinical sample, but is not limited thereto.
상기 환자 유래 임상 시료는 생물학적 체액, 예컨대, 비인두도말물, 혈청, 혈장, 유리체액, 림프액, 활액, 소포액, 정액, 양수액, 젖, 전혈, 뇨, 뇌-척수액, 타액, 객담, 눈물, 땀, 점액, 및 조직 배양 배지뿐만 아니라, 균질화된 조직 및 세포 추출물과 같은 조직 추출물을 포함한다. 바람직하게 상기 환자 유래 임상 시료는 비인두도말물일 수 있다.The patient-derived clinical sample is a biological fluid such as nasopharyngeal swab, serum, plasma, vitreous humor, lymph fluid, synovial fluid, vesicular fluid, semen, amniotic fluid, milk, whole blood, urine, brain-spinal fluid, saliva, sputum, tears , sweat, mucus, and tissue culture media, as well as tissue extracts such as homogenized tissue and cell extracts. Preferably, the patient-derived clinical sample may be a nasopharyngeal swab.
본 발명의 구체예에서, 상기 검출 방법은 반응물로부터 형광신호를 검출하는 단계를 더 포함할 수 있다.In an embodiment of the present invention, the detection method may further include detecting a fluorescence signal from a reactant.
중복되는 내용은 본 명세서의 복잡성을 고려하여 생략하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 호흡기세포융합바이러스에 특이적으로 결합하는 항체 개발Example 1. Development of antibodies that specifically bind to respiratory syncytial virus
호흡기세포융합바이러스(Respiratory syncytial virus, RSV)에 특이적으로 결합하는 항체(RSV Protein-1A12 Ab)를 개발하였다. 개발된 항체의 서열은 표 1에 나타내었다.An antibody (RSV Protein-1A12 Ab) that specifically binds to respiratory syncytial virus (RSV) was developed. The sequences of the developed antibodies are shown in Table 1.
상기 항체 1A12 Ab의 결합친화도 Kd값을 측정하였다. 구체적으로, S275F 및 WT 항원에 대한 항체의 결합 친화도를 측정하기 위해 100ng의 항원을 96-웰 플레이트에 코팅하고 4℃에서 12시간 동안 배양하였다. 배양 후 PBS에 2% 탈지유를 처리하여 블로킹하였다. 37°C에서 1시간 동안 항체(0-50 nM)를 웰 플레이트에 첨가하고 37°C에서 2시간 동안 배양하였다. 상업적으로 이용 가능한 항-RSV F 단백질 항체(Ab94968, Abcam, UK)도 항원의 항원성을 확인하기 위해 사용되었다. 플레이트를 PBST로 세척하고, 항-인간 IgG FC-HRP(Thermo Fisher Scientific Inc.)를 첨가하였다(DPBS에서 1:2,000 희석). 37℃에서 1시간 동안 배양한 후, 플레이트를 PBST로 세척하였다. 그 후 100 μL의 TMB 용액 및 50 μL의 H2SO4(1 M)를 첨가하였다. Multiscan FC를 사용하여 450 nm에서 흡광도를 측정하였으며, 그 결과는 도 1에 나타내었다.The binding affinity Kd value of the antibody 1A12 Ab was measured. Specifically, in order to measure the binding affinity of antibodies to the S275F and WT antigens, 100 ng of the antigen was coated on a 96-well plate and incubated at 4° C. for 12 hours. After culturing, blocking was performed by treating 2% skim milk in PBS. Antibodies (0-50 nM) were added to the well plate for 1 h at 37 °C and incubated for 2 h at 37 °C. A commercially available anti-RSV F protein antibody (Ab94968, Abcam, UK) was also used to confirm the antigenicity of the antigen. Plates were washed with PBST and anti-human IgG FC-HRP (Thermo Fisher Scientific Inc.) was added (diluted 1:2,000 in DPBS). After incubation at 37° C. for 1 hour, the plate was washed with PBST. Then 100 μL of TMB solution and 50 μL of H 2 SO 4 (1 M) were added. Absorbance was measured at 450 nm using Multiscan FC, and the results are shown in FIG. 1 .
도 1에 나타낸 바와 같이, 항체 1A12 Ab는 시나지스 내성 RSV인 S275F와 선택적으로 결합하며, 항원에 대한 Kd값은 2.7 x 10-9임을 확인하였다.As shown in FIG. 1 , it was confirmed that antibody 1A12 Ab selectively binds to Synagis-resistant RSV, S275F, and has a Kd value of 2.7 x 10 -9 for the antigen.
실시예 2. 호흡기세포융합바이러스 검출 보조용 단백질 개발Example 2. Development of a protein for assisting detection of respiratory syncytial virus
1A12 Ab와 결합하는 에피토프를 포함하고, ssGFP의 N 말단 및 C 말단 단편이 링커로 연결된 단백질(11L)을 고안하였다. 고안된 단백질 11L은 하기 구조식과 같이 배열되며, 구체적인 서열은 표 2에 나타내었다.A protein (11L) containing the 1A12 Ab-binding epitope and the N-terminal and C-terminal fragments of ssGFP connected by a linker was designed. The designed protein 11L is arranged as shown in the following structural formula, and its specific sequence is shown in Table 2.
[구조식][constitutional formula]
제1 형광 단백질 단편-에피토프-링커-에피토프-제2 형광단백질 단편First fluorescent protein fragment - epitope - linker - epitope - second fluorescent protein fragment
CATATGCATCATCACCACCACCACATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCAACAATCGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTCTGACTTATGGTGTTCAATGCTTTTCCCGTTATCC GGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAAATACAAGACGCGTGCTGTAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTACTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAATACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAA TGGAATCAAAGCTAACTTCACTGTTCGCCACAACGTTGAAGATGGCTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGAACCATTACCTGTCGACACAAACTGTTCTTTCGAAAGATCCCAACGAAAAGACTAGTGGTGGAAATAGTGAACTGCTTAGTCTGATAGATGACATGCCTATTACAAATGATCAAAAAAAATTAATGTCGAATAAT GGTGGTAGTGGTGGATCAGGTGGTTCTGGTGGTAGCGGTGGATCTGGTGGGTCTGGTGCAGAAGCAGCAGCTAAAGAAGCAGCAGCAAAAGAAGCCGCAGCAAAAGAAGCAGCTGCTAAAGAAGCAGCAGCTAAAGAAGCAGCAGCAAAAGCAGGTTCAGGTGGTTCGGGTGGTAGCGGTGGCTCCGGTGGTAGCGGTGGTTCAGGAGCAGAAGCAGCAGCAAAAGAAGCTGCAGCAAAAGAA GCTGCAGCAAAAGAAGCTGCTGCAAAAGAAGCAGCAGCAAAAGAAGCTGCAGCAAAAGCAGGTTCAGGAGGTAGCGGTGGTAGTGGTGGTTCCGGTGGATCTGGTGGTAGTGGAGGTAACTCTGAATTATTATCTCTGATCGACGATATGCCGATTACCAACGATCAGAAAAAATTGATGTCCAACAATGGAGGTGGTAGCGGTGGTGGTTCGACTTCAGGATCCCGTGACCACATGGTCCT TCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAAGGAGAATTC GGC GGT TGG AGC CAT CCG CAG TTT GAA AAA TAA GCG GCC GC
고안된 단백질 11L은 1A12 항체의 유무에 따라 형광이 ON/OFF된다. 고안된 단백질(11L)의 3D 모델 및 작동메커니즘은 도 2a와 같다.The fluorescence of the designed protein 11L is turned ON/OFF depending on the presence or absence of the 1A12 antibody. The 3D model and operating mechanism of the designed protein (11L) are shown in FIG. 2a.
단백질 11L에 대한 항체의 결합 친화도를 측정하기 위해 96-웰 플레이트에 100 ng의 단백질 11L를 코팅하고 4°C에서 12시간 배양한 후 PBS에 녹인 2% 탈지유로 37°에서 1시간 처리하여 차단하였다. 다음으로, 1A12 항체(0-250 nM)를 웰 플레이트에 첨가하고 37°C에서 2시간 동안 배양하였다. 플레이트를 PBST로 세척하고, 항-인간 IgG FC-HRP를 첨가하였다(DPBS에서 1:2,000 희석). 37℃에서 1시간 동안 배양한 후, 플레이트를 PBST로 세척하였다. 그 후, 100μL의 TMB 용액과 50μL의 H2SO4(1 M)를 첨가하였다. Multiscan FC를 사용하여 450 nm에서 측정하였고, 그 결과는 도 2b에 나타내었다.To measure the binding affinity of the antibody to
도 2b에 나타낸 바와 같이, 단백질 11L은 1A12 Ab와 4.278X10-8 의 Kd값으로 친화도가 높은 것을 확인하였다. 앞서 S275F와 1A12 Ab의 Kd값은 2.697X10-9 이고, 11L과 1A12 Ab의 값은 4.278X10-8 로, 1A12 Ab가 11L보다 S275F와 affinity가 약 15배 더 높으므로, 이를 이용하여 시나지스 내성 RSV를 검출할 수 있다.As shown in FIG. 2B , it was confirmed that protein 11L has a high affinity with 1A12 Ab with a Kd value of 4.278X10 -8 . Previously, the Kd values of S275F and 1A12 Ab were 2.697X10 -9 , and the values of 11L and 1A12 Ab were 4.278X10 -8 . RSV can be detected.
단백질 11L의 형광 값과 1A12 항체가 결합된 단백질 11L의 형광 값을 측정하였으며, 그 결과는 도 2c에 나타내었다.The fluorescence value of protein 11L and the fluorescence value of protein 11L bound with 1A12 antibody were measured, and the results are shown in FIG. 2c.
도 2c에 나타낸 바와 같이, 단백질 11L과 1A12 항체가 결합되면 형광값이 작아지는 것을 확인하였다(excitation 475nm, emission 510nm).As shown in Figure 2c, it was confirmed that the fluorescence value decreased when the protein 11L and the 1A12 antibody were bound (excitation 475 nm, emission 510 nm).
실시예 3. 칩(chip) 테스트를 통한 단백질 11L의 시나지스 내성 RSV S275F에 대한 형광 검출 분석Example 3. Fluorescence detection assay for Synagis resistant RSV S275F of protein 11L through chip test
Cy5 결합 항체를 제조하기 위해 항체(50 nM) 90 μL를 Cy5 N-히드록시숙신이미드(N-hydroxysuccinimide, NHS) 에스테르(디메틸 설폭사이드 중 0.48 mg, Abcam) 150 μL와 혼합하고 25°C에서 45분 동안 유지하였다. 반응 후 완충액을 DPBS로 교환하였다. 유리 슬라이드에 1A12 항체(2.5 μM)를 고정화하기 위해 N-ethyl-N’-(3-(dimethylamino) propyl) carbodiimide 및 NHS(0.4 M, Sigma-Aldrich)와의 반응을 통해 3 μL의 단백질 11L을 알데히드 유리 슬라이드(Arrayit Corporation, CA, USA)에 5분 동안 코팅했다. DPBS로 글라스를 세척한 후, 3 μL의 Cy5-conjugated antibody(50 nM)를 슬라이드에 코팅하고 1시간 후 DPBS로 세척하였다. RSV 항원을 검출하기 위해 3μL의 단백질(5 μM, WT 및 S275F)을 1A12 항체가 코팅된 유리에 15분 동안 반응시키고 DPBS로 세척하였다. 형광 신호는 녹색 형광용 Alexa-488 필터와 적색 형광용 Cy5 필터가 있는 Axon GenePix 4200A(Molecular Devices, CA, USA)를 사용하여 검출하였다.To prepare Cy5 binding antibody, 90 μL of antibody (50 nM) was mixed with 150 μL of Cy5 N-hydroxysuccinimide (NHS) ester (0.48 mg in dimethyl sulfoxide, Abcam) and incubated at 25 °C. It was held for 45 minutes. After the reaction, the buffer was exchanged with DPBS. To immobilize the 1A12 antibody (2.5 µM) on a glass slide, 3 µL of protein 11L was mixed with aldehyde by reaction with N-ethyl-N'-(3-(dimethylamino) propyl) carbodiimide and NHS (0.4 M, Sigma-Aldrich). Glass slides (Arrayit Corporation, CA, USA) were coated for 5 minutes. After washing the glass with DPBS, 3 μL of Cy5-conjugated antibody (50 nM) was coated on the slide and washed with DPBS after 1 hour. To detect the RSV antigen, 3 μL of protein (5 μM, WT and S275F) was reacted on 1A12 antibody-coated glass for 15 minutes and washed with DPBS. Fluorescent signals were detected using an Axon GenePix 4200A (Molecular Devices, CA, USA) equipped with an Alexa-488 filter for green fluorescence and a Cy5 filter for red fluorescence.
단백질 11L-항체 시스템을 구성하기 위해 단백질 11L(50 nM)과 BSA(40 nM)를 혼합하고 25°C에서 1시간 동안 배양하였다. 다음으로, 항체(50 nM)를 첨가하고 25℃에서 10분 동안 인큐베이션하였다. S275F RSV 항원을 검출하기 위해 200 μL의 단백질을 단백질 11L-항체 시스템(200 μL)에 추가하였다. 형광 신호는 마이크로플레이트 리더(BioTek, VT, USA)를 사용하여 여기 파장 475 nm 및 방출 파장 510 nm에서 측정하였다. VLP(WT 및 S275F)의 검출을 위해 200 μL의 준비된 VLP(103 PFU/mL) 또는 200 μL의 VLP 스파이크 비인두 면봉 샘플(103 PFU/mL)을 단백질 11L-항체 시스템(200μL)에 첨가하였다. 형광 신호는 475 nm의 여기 파장 및 510 nm의 방출 파장에서 마이크로플레이트 리더를 사용하여 측정하였다.To construct the protein 11L-antibody system, protein 11L (50 nM) and BSA (40 nM) were mixed and incubated at 25 °C for 1 hour. Next, antibody (50 nM) was added and incubated for 10 minutes at 25°C. 200 μL of protein was added to the protein 11L-antibody system (200 μL) to detect the S275F RSV antigen. Fluorescent signals were measured at an excitation wavelength of 475 nm and an emission wavelength of 510 nm using a microplate reader (BioTek, VT, USA). Add 200 μL of prepared VLPs (10 3 PFU/mL) or 200 μL of VLP-spiked nasopharyngeal swab sample (10 3 PFU/mL) to protein 11L-antibody system (200 μL) for detection of VLPs (WT and S275F) did Fluorescent signals were measured using a microplate reader at an excitation wavelength of 475 nm and an emission wavelength of 510 nm.
형광 분석 결과는 도 3에 나타내었다.The fluorescence analysis results are shown in FIG. 3 .
도 3에 나타낸 바와 같이, RSV S275F을 반응시킨 칩은 1A12와 S275F 단백질의 결합으로 인해 ssGFP 형광 단백질의 녹색을 띄는 반면, WT을 반응시킨 칩은 단백질 11L와 1A12 antibody의 결합으로 인해 cy5(빨간색) 형광이 나타남을 확인하였다.As shown in Figure 3, the chip reacted with RSV S275F shows green color of ssGFP fluorescent protein due to the binding of 1A12 and S275F protein, whereas the chip reacted with WT shows cy5 (red) due to the binding of protein 11L and 1A12 antibody. It was confirmed that fluorescence appeared.
실시예 4. 단백질 11L 및 항체 1A12를 이용한 시나지스 내성 RSV 검출 Example 4. Detection of Synagis-resistant RSV using protein 11L and antibody 1A12
인플루엔자 바이러스 기질(M1) 단백질과 WT RSV 또는 S275F RSV F 단백질로 구성된 바이러스 유사 입자(VLP)를 생성하였다. 구체적으로, A/Puerto Rico/8/1934(H1N1) 게놈 RNA에서 파생된 M1 유전자를 범용 역방향 프라이머 및 게놈 특이적 프라이머를 사용하여 pVP-M 벡터에 개별적으로 클로닝하였다. 또한 WT 및 S275F RSV F 단백질은 프라이머 세트(정방향 : 5’-TATTCGTCTCAGGGAGCAAAAGCAGGACTTTAAAATGGAGCTGCTGATCCTG-3’, 역방향 : 5’-ATATCGTCTCGTATTAGTAGAAACAAGGAGTTTTTTGAACAGATCAGTGGTGGTGGTGGTGGTG-3’)에 의해 F 단편 내에 도입되었다. 공동 배양된 293T 및 Madin-Darby Canine Kidney(MDCK) 세포(웰당 0.5 x 106 세포)를 WT 또는 S275F RSV 및 M1 플라스미드로 형질감염시켰다. 형질감염 후 3-6일에 세포 상청액을 HEp-2 세포 단층으로 적정하여 바이러스 역가를 추정하였다. VLP를 QuixStand(GE Healthcare)로 농축하고 4°C에서 1시간 동안 14,000 ×g의 불연속 자당 구배를 통해 정제하였다. 30%에서 60% 사이의 VLP 밴드를 수집하고 4°C에서 40분 동안 14,000 xg에서 원심분리하였다. VLP는 6x-His Tag 모노클로날 항체(MA1-21315, Invitrogen, MA, USA)를 사용하여 RSV F 단백질을 프로브하고 항-M1 항체를 사용하여 M1 단백질을 검출하는 닷블럿을 수행하였다. VLP생성 결과는 도 4 및 5에 나타내었다.Virus like particles (VLPs) composed of influenza virus substrate (M1) protein and WT RSV or S275F RSV F protein were generated. Specifically, the M1 gene derived from A/Puerto Rico/8/1934 (H1N1) genomic RNA was individually cloned into the pVP-M vector using a universal reverse primer and a genome-specific primer. In addition, WT and S275F RSV F proteins were introduced into the F fragment by a set of primers (forward: 5'-TATTCGTCTCAGGGAGCAAAAGCAGGACTTTAAAATGGAGCTGCTGATCCTG-3', reverse: 5'-ATATCGTCTCGTATTAGTAGAAACAAGGAGTTTTTTGAACAGATCAGTGGTGGTGGTGGTGGTG-3'). Co-cultured 293T and Madin-Darby Canine Kidney (MDCK) cells (0.5 x 10 6 cells per well) were transfected with WT or S275F RSV and M1 plasmids. Viral titers were estimated by titrating cell supernatants with HEp-2 cell monolayers 3-6 days after transfection. VLPs were concentrated with QuixStand (GE Healthcare) and purified via a discontinuous sucrose gradient at 14,000 × g for 1 h at 4 °C. VLP bands between 30% and 60% were collected and centrifuged at 14,000 xg for 40 min at 4 °C. For VLP, a dot blot was performed to probe the RSV F protein using a 6x-His Tag monoclonal antibody (MA1-21315, Invitrogen, MA, USA) and to detect the M1 protein using an anti-M1 antibody. The VLP production results are shown in FIGS. 4 and 5 .
도 4에 나타낸 바와 같이, 인플루엔자 바이러스 기질(M1) 단백질과 WT RSV 또는 S275F RSV F 단백질로 구성된 바이러스 유사 입자(VLP)가 생성된 것을 확인하였다. 웨스턴 블랏 결과 42KDa에서는 WT RSV 와 S275F RSV F 단백질을 확인하였으며, 25KDa에서는 인플루엔자 바이러스 M1 단백질을 확인하였다. VLP의 양이 늘어남에 따라 M1과 F단백질의 양 또한 증가하는 것을 확인하였다.As shown in FIG. 4, it was confirmed that virus-like particles (VLPs) composed of influenza virus substrate (M1) protein and WT RSV or S275F RSV F protein were produced. As a result of Western blotting, WT RSV and S275F RSV F proteins were confirmed at 42 KDa, and influenza virus M1 protein was confirmed at 25 KDa. As the amount of VLP increased, it was confirmed that the amount of M1 and F protein also increased.
도 5에 나타낸 바와 같이, RSV 1A12항체로 VLP를 detection한 결과 RSV 내성 S275F VLP에서는 signal을 확인하였으며(도 5a), palivizumab으로 VLP를 detection한 결과 WT VLP에서만 signal을 확인하였다(도 5b).As shown in FIG. 5, as a result of detecting VLPs with RSV 1A12 antibody, signals were confirmed in RSV-resistant S275F VLPs (FIG. 5a), and as a result of VLP detection with palivizumab, signals were confirmed only in WT VLPs (FIG. 5b).
상기 결과는 인플루엔자 바이러스 기질(M1) 단백질과 WT RSV 또는 S275F RSV F 단백질로 구성된 바이러스 유사 입자(VLP)가 생성된 것을 의미한다.The above results indicate that virus-like particles (VLPs) composed of influenza virus substrate (M1) protein and WT RSV or S275F RSV F protein were generated.
제조된 WT RSV 또는 S275F RSV F 단백질로 구성된 바이러스 유사 입자를 단백질 11L 및 1A12 항체를 반응시킨 후 형광을 검출하였다. 항원 존재 여부 및 항원 농도에 따른 형광 세기를 확인한 결과는 도 6에 나타내었다.Virus-like particles composed of the prepared WT RSV or S275F RSV F protein were reacted with protein 11L and 1A12 antibody, and then fluorescence was detected. The results of confirming the fluorescence intensity according to the presence or absence of the antigen and the concentration of the antigen are shown in FIG. 6 .
도 6에 나타낸 바와 같이, S275F RSV F 단백질로 구성된 바이러스 유사 입자가 존재하는 경우 WT군에 비해 강한 형광 신호가 검출된 것을 확인하였다. 또한 S275F RSV F 단백질로 구성된 바이러스 유사 입자의 농도가 증가함에 따라 형광 신호의 세기도 증가하는 것을 확인하였다.As shown in FIG. 6, it was confirmed that a strong fluorescence signal was detected compared to the WT group in the presence of virus-like particles composed of the S275F RSV F protein. In addition, it was confirmed that the intensity of the fluorescence signal increased as the concentration of the virus-like particles composed of the S275F RSV F protein increased.
단백질 11L-항체 시스템을 구성하기 위해 단백질 11L(50 nM)과 BSA(40 nM)를 혼합하고 25°C에서 1시간 동안 배양하였다. 다음으로, 항체(50 nM)를 첨가하고 25℃에서 10분 동안 배양하였다. S275F RSV 항원을 검출하기 위해 200 μL의 단백질을 단백질 11L-항체 시스템(200 μL)에 첨가하였다. 형광 신호는 475 nm의 여기 파장과 510 nm의 방출 파장에서 마이크로플레이트 리더(BioTek, VT, USA)를 사용하여 측정하였다. VLP(WT 및 S275F)의 검출을 위해 200 μL의 준비된 VLP(103 PFU/mL) 또는 200 μL의 VLP 스파이크 비인두 면봉 샘플(103 PFU/mL)을 단백질 11L-항체 시스템(200 μL)에 추가하였다. 형광 신호는 475 nm의 여기 파장 및 510 nm의 방출 파장에서 마이크로플레이트 리더를 사용하여 측정하였다. 형광신호를 측정한 결과는 도 7에 나타내었다. To construct the protein 11L-antibody system, protein 11L (50 nM) and BSA (40 nM) were mixed and incubated at 25 °C for 1 hour. Next, antibody (50 nM) was added and incubated for 10 minutes at 25°C. 200 μL of protein was added to the protein 11L-antibody system (200 μL) to detect the S275F RSV antigen. Fluorescent signals were measured using a microplate reader (BioTek, VT, USA) at an excitation wavelength of 475 nm and an emission wavelength of 510 nm. For the detection of VLPs (WT and S275F), 200 μL of prepared VLPs (10 3 PFU/mL) or 200 μL of VLP-spiked nasopharyngeal swab sample (10 3 PFU/mL) was added to protein 11L-antibody system (200 μL). added. Fluorescent signals were measured using a microplate reader at an excitation wavelength of 475 nm and an emission wavelength of 510 nm. The results of measuring the fluorescence signal are shown in FIG. 7 .
도 7에 나타낸 바와 같이, 단백질 11L과 1A12 항체를 이용한 항체 시스템은 시나지스 내성 RSV를 형광 검출하였으며, 시나지스 내성 RSV와 비인두도말물(nasopharyngeal swab sample)을 혼합하였을 경우 또한 형광 검출이 가능하였다.As shown in FIG. 7, the antibody system using protein 11L and 1A12 antibody detected fluorescence of Synagis-resistant RSV, and fluorescence detection was also possible when Synagis-resistant RSV and nasopharyngeal swab samples were mixed. .
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다. In the above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
<110> Korea Research Institute of Bioscience and Biotechnology <120> Synagis-resistant respiratory syncytial virus detection method <130> 1-113 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> RSV Protein-1A12 Ab heavy chain CDR1 <400> 1 Phe Thr Ser Ser Trp Met Ser 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> RSV Protein-1A12 Ab heavy chain CDR2 <400> 2 Ser Glu Ile Asp Gly Tyr Asn Gly Ser 1 5 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RSV Protein-1A12 Ab heavy chain CDR3 <400> 3 Thr Val Pro Trp Trp Gly 1 5 <210> 4 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> RSV Protein-1A12 Ab heavy chain <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Ser 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asp Gly Tyr Asn Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Val Pro Trp Trp Gly Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 5 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RSV Protein-1A12 Ab light chain CDR1 <400> 5 Asn Ile Ser Asn Tyr Leu 1 5 <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RSV Protein-1A12 Ab light chain CDR2 <400> 6 Leu Ala Ser Ser Leu Glu 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RSV Protein-1A12 Ab light chain CDR3 <400> 7 Ala Asn Ser Pro Pro Arg 1 5 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> RSV Protein-1A12 Ab light chain <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Pro Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 9 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> fluorescent protein fragment 1 <400> 9 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Ile Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Ser Phe Lys Asp Asp Gly Lys Tyr Lys Thr Arg Ala Val Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Thr 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Ala Asn Phe Thr Val Arg His Asn Val Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Thr Val Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys 210 <210> 10 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> fluorescent protein fragment 2 <400> 10 Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr 1 5 10 15 His Gly Met Asp Glu Leu Tyr Lys 20 <210> 11 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> epitope <400> 11 Asn Ser Glu Leu Leu Ser Leu Ile Asp Asp Met Pro Ile Thr Asn Asp 1 5 10 15 Gln Lys Lys Leu Met Ser Asn Asn 20 <210> 12 <211> 1340 <212> DNA <213> Artificial Sequence <220> <223> 11L <400> 12 catatgcatc atcaccacca ccacatgagc aaaggagaag aacttttcac tggagttgtc 60 ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt ccgtggagag 120 ggtgaaggtg atgcaacaat cggaaaactt acccttaaat ttatttgcac tactggaaaa 180 ctacctgttc catggccaac acttgtcact actctgactt atggtgttca atgcttttcc 240 cgttatccgg atcacatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat 300 gtacaggaac gcactatatc tttcaaagat gacgggaaat acaagacgcg tgctgtagtc 360 aagtttgaag gtgataccct tgttaatcgt atcgagttaa aaggtactga ttttaaagaa 420 gatggaaaca ttctcggaca caaactcgaa tacaacttta actcacacaa tgtatacatc 480 acggcagaca aacaaaagaa tggaatcaaa gctaacttca ctgttcgcca caacgttgaa 540 gatggctccg ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct 600 gtccttttac cagacaacca ttacctgtcg acacaaactg ttctttcgaa agatcccaac 660 gaaaagacta gtggtggaaa tagtgaactg cttagtctga tagatgacat gcctattaca 720 aatgatcaaa aaaaattaat gtcgaataat ggtggtagtg gtggatcagg tggttctggt 780 ggtagcggtg gatctggtgg gtctggtgca gaagcagcag ctaaagaagc agcagcaaaa 840 gaagccgcag caaaagaagc agctgctaaa gaagcagcag ctaaagaagc agcagcaaaa 900 gcaggttcag gtggttcggg tggtagcggt ggctccggtg gtagcggtgg ttcaggagca 960 gaagcagcag caaaagaagc tgcagcaaaa gaagctgcag caaaagaagc tgctgcaaaa 1020 gaagcagcag caaaagaagc tgcagcaaaa gcaggttcag gaggtagcgg tggtagtggt 1080 ggttccggtg gatctggtgg tagtggaggt aactctgaat tattatctct gatcgacgat 1140 atgccgatta ccaacgatca gaaaaaattg atgtccaaca atggaggtgg tagcggtggt 1200 ggttcgactt caggatcccg tgaccacatg gtccttcttg agtttgtaac tgctgctggg 1260 attacacatg gcatggatga gctctacaaa ggagaattcg gcggttggag ccatccgcag 1320 tttgaaaaat aagcggccgc 1340 <210> 13 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> linker <400> 13 ggtggctctg gtggtagcgg cggcagcggt ggtagcggcg gtagcggtgg cagcggcgcg 60 gaagctgcag cgaaagaagc tgcagcgaaa gaagctgcag cgaaagaagc tgcagcgaaa 120 gaagctgcag cgaaagaagc tgcagcgaaa gccggctctg gtggtagcgg cggcagcggt 180 ggtagcggcg gtagcggtgg cagcggcgcg gaagctgcag cgaaagaagc tgcagcgaaa 240 gaagctgcag cgaaagaagc tgcagcgaaa gaagctgcag cgaaagaagc tgcagcgaaa 300 gccggctctg gtggtagcgg cggcagcggt ggtagcggcg gtagcggtgg cagcggcggt 360 360 <110> Korea Research Institute of Bioscience and Biotechnology <120> Synagis-resistant respiratory syncytial virus detection method <130> 1-113 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> PRT <213> artificial sequence <220> <223> RSV Protein-1A12 Ab heavy chain CDR1 <400> 1 Phe Thr Ser Ser Trp Met Ser 1 5 <210> 2 <211> 9 <212> PRT <213> artificial sequence <220> <223> RSV Protein-1A12 Ab heavy chain CDR2 <400> 2 Ser Glu Ile Asp Gly Tyr Asn Gly Ser 1 5 <210> 3 <211> 6 <212> PRT <213> artificial sequence <220> <223> RSV Protein-1A12 Ab heavy chain CDR3 <400> 3 Thr Val Pro Trp Trp Gly 1 5 <210> 4 <211> 118 <212> PRT <213> artificial sequence <220> <223> RSV Protein-1A12 Ab heavy chain <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Ser 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asp Gly Tyr Asn Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Val Pro Trp Trp Gly Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 5 <211> 6 <212> PRT <213> artificial sequence <220> <223> RSV Protein-1A12 Ab light chain CDR1 <400> 5 Asn Ile Ser Asn Tyr Leu 1 5 <210> 6 <211> 6 <212> PRT <213> artificial sequence <220> <223> RSV Protein-1A12 Ab light chain CDR2 <400> 6 Leu Ala Ser Ser Leu Glu 1 5 <210> 7 <211> 6 <212> PRT <213> artificial sequence <220> <223> RSV Protein-1A12 Ab light chain CDR3 <400> 7 Ala Asn Ser Pro Pro Arg 1 5 <210> 8 <211> 107 <212> PRT <213> artificial sequence <220> <223> RSV Protein-1A12 Ab light chain <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Pro Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 9 <211> 214 <212> PRT <213> artificial sequence <220> <223> fluorescent protein fragment 1 <400> 9 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Ile Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Ser Phe Lys Asp Asp Gly Lys Tyr Lys Thr Arg Ala Val Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Thr 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Ala Asn Phe Thr Val Arg His Asn Val Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Thr Val Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys 210 <210> 10 <211> 24 <212> PRT <213> artificial sequence <220> <223> fluorescent protein fragment 2 <400> 10 Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr 1 5 10 15 His Gly Met Asp Glu Leu Tyr Lys 20 <210> 11 <211> 24 <212> PRT <213> artificial sequence <220> <223> epitope <400> 11 Asn Ser Glu Leu Leu Ser Leu Ile Asp Asp Met Pro Ile Thr Asn Asp 1 5 10 15 Gln Lys Lys Leu Met Ser Asn Asn 20 <210> 12 <211> 1340 <212> DNA <213> artificial sequence <220> <223> 11L <400> 12 catatgcatc atcaccacca ccacatgagc aaaggagaag aacttttcac tggagttgtc 60 ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt ccgtggagag 120 ggtgaaggtg atgcaacaat cggaaaactt acccttaaat ttatttgcac tactggaaaa 180 ctacctgttc catggccaac acttgtcact actctgactt atggtgttca atgcttttcc 240 cgttatccgg atcacatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat 300 gtacaggaac gcactatatc tttcaaagat gacgggaaat acaagacgcg tgctgtagtc 360 aagtttgaag gtgataccct tgttaatcgt atcgagttaa aaggtactga ttttaaagaa 420 gatggaaaca ttctcggaca caaactcgaa tacaacttta actcacacaa tgtatacatc 480 acggcagaca aacaaaagaa tggaatcaaa gctaacttca ctgttcgcca caacgttgaa 540 gatggctccg ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct 600 gtccttttac cagacaacca ttacctgtcg acacaaactg ttctttcgaa agatcccaac 660 gaaaagacta gtggtggaaa tagtgaactg cttagtctga tagatgacat gcctattaca 720 aatgatcaaa aaaaattaat gtcgaataat ggtggtagtg gtggatcagg tggttctggt 780 ggtagcggtg gatctggtgg gtctggtgca gaagcagcag ctaaagaagc agcagcaaaa 840 gaagccgcag caaaagaagc agctgctaaa gaagcagcag ctaaagaagc agcagcaaaa 900 gcaggttcag gtggttcggg tggtagcggt ggctccggtg gtagcggtgg ttcaggagca 960 gaagcagcag caaaagaagc tgcagcaaaa gaagctgcag caaaagaagc tgctgcaaaa 1020 gaagcagcag caaaagaagc tgcagcaaaa gcaggttcag gaggtagcgg tggtagtggt 1080 ggttccggtg gatctggtgg tagtggaggt aactctgaat tattatctct gatcgacgat 1140 atgccgatta ccaacgatca gaaaaaattg atgtccaaca atggaggtgg tagcggtggt 1200 ggttcgactt caggatcccg tgaccacatg gtccttcttg agtttgtaac tgctgctggg 1260 attacacatg gcatggatga gctctacaaa ggagaattcg gcggttggag ccatccgcag 1320 tttgaaaaat aagcggccgc 1340 <210> 13 <211> 360 <212> DNA <213> artificial sequence <220> <223> linker <400> 13 ggtggctctg gtggtagcgg cggcagcggt ggtagcggcg gtagcggtgg cagcggcgcg 60 gaagctgcag cgaaagaagc tgcagcgaaa gaagctgcag cgaaagaagc tgcagcgaaa 120 gaagctgcag cgaaagaagc tgcagcgaaa gccggctctg gtggtagcgg cggcagcggt 180 ggtagcggcg gtagcggtgg cagcggcgcg gaagctgcag cgaaagaagc tgcagcgaaa 240 gaagctgcag cgaaagaagc tgcagcgaaa gaagctgcag cgaaagaagc tgcagcgaaa 300 gccggctctg gtggtagcgg cggcagcggt ggtagcggcg gtagcggtgg cagcggcggt 360 360
Claims (14)
서열번호 5로 표시되는 아미노산 서열로 구성되는 CDR1, 서열번호 6로 표시되는 아미노산 서열로 구성되는 CDR2 및 서열번호 7으로 표시되는 아미노산 서열로 구성되는 CDR3을 포함하는 경쇄 가변영역을 포함하는,
시나지스 내성 호흡기세포융합바이러스(Respiratory syncytial virus, RSV)에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편.CDR1 (complementarity determining region 1) composed of the amino acid sequence represented by SEQ ID NO: 1, CDR2 (complementarity determining region 2) composed of the amino acid sequence represented by SEQ ID NO: 2, and CDR3 composed of the amino acid sequence represented by SEQ ID NO: 3 (complementarity determining region 3) including a heavy chain variable region; and
CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 5, CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 7, comprising a light chain variable region comprising,
An antibody or antigen-binding fragment thereof that specifically binds to Synagis-resistant respiratory syncytial virus (RSV).
상기 중쇄 가변영역은 서열번호 4의 아미노산 서열로 표시되는 것인, 항체 또는 그의 항원 결합 단편.According to claim 1,
The heavy chain variable region is represented by the amino acid sequence of SEQ ID NO: 4, an antibody or antigen-binding fragment thereof.
상기 경쇄 가변영역은 서열번호 8의 아미노산 서열로 표시되는 것인, 항체 또는 그의 항원 결합 단편.According to claim 1,
The light chain variable region is represented by the amino acid sequence of SEQ ID NO: 8, an antibody or antigen-binding fragment thereof.
상기 시나지스 내성 호흡기세포융합바이러스는 N262D, K272N 및 S275F로 이루어진 군에서 선택된 1종 이상인, 항체 또는 그의 항원 결합 단편.According to claim 1,
The Synagis-resistant respiratory syncytial virus is at least one selected from the group consisting of N262D, K272N and S275F, an antibody or antigen-binding fragment thereof.
C 말단에 위치한 제2 형광 단백질 단편; 및
서열번호 11의 아미노산 서열로 표시되는 에피토프;를 포함하고,
상기 제1 및 제2 형광 단백질 단편은 각각 전장 형광단백질의 N 말단 및 C 말단 단편인, 시나지스 내성 호흡기세포융합바이러스(Respiratory syncytial virus, RSV) 검출 보조용 단백질.a first fluorescent protein fragment located at the N-terminus;
a second fluorescent protein fragment located at the C-terminus; and
An epitope represented by the amino acid sequence of SEQ ID NO: 11;
The first and second fluorescent protein fragments are N-terminal and C-terminal fragments of a full-length fluorescent protein, respectively, Synagis-resistant respiratory syncytial virus (RSV) detection aid protein.
상기 형광 단백질은 YFP(yellow fluorescent protein), EYFP(enhanced yellow fluorescent protein), GFP(green fluorescent protein), EGFP(enhanced GFP), BFP(blue fluorescent protein), EBFP(enhanced BFP), CFP(cyan fluorescent protein) 또는 ECFP(enhanced CFP)인, 단백질.According to claim 5,
The fluorescent protein is YFP (yellow fluorescent protein), EYFP (enhanced yellow fluorescent protein), GFP (green fluorescent protein), EGFP (enhanced GFP), BFP (blue fluorescent protein), EBFP (enhanced BFP), CFP (cyan fluorescent protein) ) or enhanced CFP (ECFP).
상기 제1 형광 단백질 단편은 서열번호 9의 아미노산 서열로 표시되는 것인, 단백질.According to claim 5,
The protein, wherein the first fluorescent protein fragment is represented by the amino acid sequence of SEQ ID NO: 9.
상기 제2 형광 단백질 단편은 서열번호 10의 아미노산 서열로 표시되는 것인, 단백질.According to claim 5,
The protein, wherein the second fluorescent protein fragment is represented by the amino acid sequence of SEQ ID NO: 10.
상기 단백질은 링커를 더 포함하는 것인, 단백질.According to claim 5,
Wherein the protein further comprises a linker.
상기 단백질은 하기 구조식의 형태로 배열된 것인, 단백질.
[구조식]
제1 형광 단백질 단편-에피토프-링커-에피토프-제2 형광단백질 단편 According to claim 9,
Wherein the protein is arranged in the form of the following structural formula, the protein.
[constitutional formula]
First fluorescent protein fragment - epitope - linker - epitope - second fluorescent protein fragment
제5항의 시나지스 내성 호흡기세포융합바이러스 검출 보조용 단백질;을 포함하는 시나지스 내성 호흡기세포융합바이러스 검출용 조성물.An antibody or antigen-binding fragment thereof that specifically binds to the Synagis-resistant respiratory syncytial virus of claim 1; and
A composition for detecting Synagis-resistant respiratory syncytial virus, comprising a protein for assisting in the detection of Synagis-resistant respiratory syncytial virus of claim 5.
상기 시료는 호흡기세포융합바이러스 항원, 호흡기세포융합바이러스 배양액 또는 환자 유래 임상 시료 형태인, 방법.According to claim 13,
Wherein the sample is in the form of respiratory syncytial virus antigen, respiratory syncytial virus culture medium, or patient-derived clinical sample.
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| KR1020210152000A KR20230066722A (en) | 2021-11-08 | 2021-11-08 | Synagis-resistant respiratory syncytial virus detection method |
| PCT/KR2022/016442 WO2023080527A1 (en) | 2021-11-08 | 2022-10-26 | Method for detection of synagis-resistant respiratory syncytial virus |
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| TW200636064A (en) * | 2004-10-28 | 2006-10-16 | Centocor Inc | Anti-respiratory syncytial virus antibodies, antigens and uses thereof |
| WO2010087927A2 (en) * | 2009-02-02 | 2010-08-05 | Medimmune, Llc | Antibodies against and methods for producing vaccines for respiratory syncytial virus |
| US20160039883A1 (en) * | 2013-03-15 | 2016-02-11 | Medimmune, Llc | Palivizumab epitope-based virus-like particles |
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