KR102307241B1 - Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same - Google Patents

Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same Download PDF

Info

Publication number
KR102307241B1
KR102307241B1 KR1020180155244A KR20180155244A KR102307241B1 KR 102307241 B1 KR102307241 B1 KR 102307241B1 KR 1020180155244 A KR1020180155244 A KR 1020180155244A KR 20180155244 A KR20180155244 A KR 20180155244A KR 102307241 B1 KR102307241 B1 KR 102307241B1
Authority
KR
South Korea
Prior art keywords
protein
fucosyllactose
phosphor
fret
fucosyl lactose
Prior art date
Application number
KR1020180155244A
Other languages
Korean (ko)
Other versions
KR20200068359A (en
Inventor
권대혁
김후연
Original Assignee
성균관대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 성균관대학교산학협력단 filed Critical 성균관대학교산학협력단
Priority to KR1020180155244A priority Critical patent/KR102307241B1/en
Publication of KR20200068359A publication Critical patent/KR20200068359A/en
Application granted granted Critical
Publication of KR102307241B1 publication Critical patent/KR102307241B1/en

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

본 발명은 푸코실락토오스 검출용 바이오센서 및 이를 이용하여 푸코실락토오스의 정량 분석 방법에 관한 것으로, 구체적으로 에너지 공여체와 에너지 수여체로 작용하는 형광 단백질들을 푸코실락토오스와 결합하는 단백질과 융합시킨 푸코실락토오스 바이오센서를 이용하여 세포 내부 또는 외부의 푸코실락토오스를 빠르게 정량하여 신규한 푸코실락토오스 발현 균주를 찾아낼 뿐만 아니라 모유와 같은 여러 다른 조건에서도 푸코실락토오스의 농도를 빠르게 정량할 수 있으므로 매우 유용한 기술이다. The present invention relates to a biosensor for detecting fucosyl lactose and a method for quantitative analysis of fucosyl lactose using the same. Using a lactose biosensor, it is very useful not only to quickly quantify fucosyllactose inside or outside the cell to find a new fucosyllactose-expressing strain, but also to quickly quantify the concentration of fucosyllactose in various other conditions, such as breast milk. it's technology

Description

푸코실락토오스 검출용 바이오센서 및 이를 이용하여 푸코실락토오스의 정량 분석 방법{Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same}Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same}

본 발명은 푸코실락토오스 검출용 바이오센서 및 이를 이용하여 푸코실락토오스의 정량 분석 방법에 관한 것으로, 구체적으로 eCFP-FBP-eYFP로 구성되는 세포 내부 또는 외부의 푸코실락토오스 정량을 위한 바이오센서에 관한 것으로서, 푸코실락토오스를 인식하는 푸코실락토오스 결합 단백질을 삽입함으로써 신호발생부인 형광단백질 사이의 거리 변화를 통해 FRET 변화(FRET Ratio)를 특징으로 하는 신규한 바이오센서에 관한 것이다.The present invention relates to a biosensor for detecting fucosyl lactose and a method for quantitative analysis of fucosyl lactose using the same, and specifically, to a biosensor for quantifying fucosyl lactose inside or outside a cell composed of eCFP-FBP-eYFP As such, it relates to a novel biosensor characterized by a change in FRET (FRET Ratio) through a change in the distance between a fluorescent protein, which is a signal generating part, by inserting a fucosyl lactose binding protein that recognizes fucosyl lactose.

해파리에서 유래한 GFP(green fluorescence protein)의 다양한 유전자 변이들인 CFP(cyan fluorescence protein)와 YFP(yellow fluorescence protein) 사이의 '형광 공명 에너지 전이 (fluorescence resonance energy transfer, FRET)' 현상을 이용한 바이오센서로 세포 내 대사물질들을 정량적으로 측정한 연구는 1997년 Tsien 그룹에서 '칼모듈린 (calmodulin)'과 이와 상호 작용하는 'M13 펩타이드 (M13 peptide)'에 BFP와 GFP를 연결시킨 '카멜레온 (cameleon)'으로 명명한 융합단백질로 세포내 칼슘 (calcium) 농도를 측정하면서부터 본격적으로 시작되었다. It is a biosensor using the phenomenon of 'fluorescence resonance energy transfer (FRET)' between CFP (cyan fluorescence protein) and YFP (yellow fluorescence protein), which are various genetic mutations of GFP (green fluorescence protein) derived from jellyfish. A study that quantitatively measured intracellular metabolites was conducted in 1997 by Tsien's group on 'cameleon' by linking BFP and GFP to 'calmodulin' and 'M13 peptide' that interacts with it. It started in earnest by measuring the intracellular calcium concentration with a fusion protein named .

또한, 1997년 Hellinga 그룹에서는 리간드의 결합에 의해 구조가 바뀌는 미생물의 '세포막간 단백질들(periplasmic-binding proteins, PBPs)'의 일종인 말토오스 결합 단백질 (maltose binding protein, MBP)에 형광 염료를 결합하여 인공적인 조건(in vitro)에서 높은 감지 신호를 갖는 바이오센서를 구축하였으며, 최근까지도 다양한 종류의 단백질들에 적용한 사례가 보고 되어 있다.Also, in 1997, the Hellinga group combined a fluorescent dye with maltose binding protein (MBP), a type of 'periplasmic-binding proteins (PBPs)' of microorganisms whose structure is changed by binding of ligands. A biosensor with a high detection signal has been constructed in artificial conditions (in vitro), and cases of application to various types of proteins have been reported until recently.

이러한 연구들을 바탕으로 2002년 Frommer 그룹에서는 MBP의 양 말단에 CFP 와 YFP를 결합시킨 융합 단백질을 살아있는 효모에서 안정적으로 발현시키고, CFP와 YFP의 형광 강도의 차이를 비교하는 방법으로 살아있는 효모 내에서 맥아당 농도의 변화를 정량적으로 측정한 결과를 발표하였다. 그 후 세포막간 단백질인 '라이보오스 결합 단백질 (ribose-binding protein)'과 '글루코스 결합 단백질 (glucose-binding protein)'로 동일 한 연구를 수행하여 동물세포 내에서 각각의 당을 정량적으로 분석한 연구 결과를 발표했다. 뿐만 아니라 최근에는 세포내에 존재하는 다양한 물질들을 감지할 수 있는 형태의 바이오센서들이 개발되어지고 있는바, Champman 등은 최근에 보툴리눔(C. botulinum)의 신경독소 활성 (neurotoxin activity)을 측정할 수 있는 형광 바이오센서를 개발하여 발표하였고, Tojyo 등은 inositol 1,4,5-trisphosphate를 측정할 수 있는 바이오센서의 개발로 단일세포 수준에서 형광을 관찰하는 방법으로 정량적으로 분석할 수 있는 방법을 발표한 바 있다.Based on these studies, Frommer's group in 2002 stably expressed a fusion protein combining CFP and YFP at both ends of MBP in live yeast, and compared the difference in fluorescence intensity between CFP and YFP. The results of quantitatively measuring the change in concentration were published. After that, the same study was conducted with the intercellular proteins 'ribose-binding protein' and 'glucose-binding protein' to quantitatively analyze each sugar in animal cells. published the research results. In addition, recently, biosensors of a type that can detect various substances present in cells have been developed, and Champman et al. Tojyo et al. developed a biosensor capable of measuring inositol 1,4,5-trisphosphate and announced a method for quantitative analysis by observing fluorescence at the single cell level. there is a bar

그러나 초기에 개발된 상기 바이오센서들은 매우 미약한 수준의 감지력을 보이는 바, 보다 정확한 측정수단으로의 활용을 위해서는 그 감지 능력을 높이는 요구가 계속되어지고 있으며, 최근에는 이러한 단점을 보완하고자 하는 시도가 있었으며, 그 중 하나로 Miyawaki 그룹에서는 'cameleon'의 EYFP 유전자를 순환치환(circular permutation)하는 방법으로 FRET 감지신호를 증가시킬 수 있다는 연구결과를 2001년부터 2004년까지 계속적으로 발표되고 있다.However, the initially developed biosensors show a very weak level of detection power, and there is a continuous demand to increase the detection ability for use as a more accurate measurement means. As one of them, the Miyawaki group has been continuously publishing research results from 2001 to 2004 that the FRET detection signal can be increased by a method of circular permutation of the EYFP gene of 'cameleon'.

그러나 유전자 조작의 어려움과 탐색 공정의 단순함이 다른 유사한 종류의 바이오센서에 적용하기 쉽지 않다는 점에서 보다 효율적인 방식의 접근이 요구된다. 따라서 세포 내에서 특정 물질의 농도를 정량적으로 측정할 수 있는 바이오센서는 바이오센서의 감지능도 높아야 하고, 감지능을 높이는 개발 과정도 보편적 유전자 조작 방법을 사용하여 다른 유사한 종류의 바이오센서에도 적용이 가능한 개발 방법을 제공하는 것이 필요하다.However, a more efficient approach is required in that it is not easy to apply to other similar types of biosensors due to the difficulty of genetic manipulation and the simplicity of the search process. Therefore, a biosensor that can quantitatively measure the concentration of a specific substance in a cell must have high detection capability, and the development process to increase the detection capability can also be applied to other similar types of biosensors using a universal genetic manipulation method. It is necessary to provide a possible development method.

또한, 푸코실락토오스 생산 균주의 개량을 위해서는 푸코실락토오스의 농도를 고속탐색(high-throughput)으로 측정하는 것이 매우 중요하나, 푸코실락토오스의 농도를 분석하기 위한 방법으로는 HPLC를 통한 당분석법이 유일한 방법이다. 이 방법은 1개의 시료당 분석 시간이 30분 이상 소요되므로 균주개발이나 발효연구에서 발생하는 다수의 시료를 분석하는데 많은 시간이 소요된다. 따라서 대량의 시료를 빠르게 분석할 수 있는 분석방법의 개발이 필수적이며 현재의 기술로는 고속탐색(high-throughput)이 불가능하다.In addition, it is very important to measure the concentration of fucosyl lactose by high-throughput in order to improve the fucosyl lactose-producing strain, but as a method for analyzing the concentration of fucosyl lactose, the glucose analysis method through HPLC is the only way Since this method takes more than 30 minutes of analysis time per sample, it takes a lot of time to analyze a large number of samples generated in strain development or fermentation research. Therefore, it is essential to develop an analysis method that can quickly analyze a large amount of sample, and high-throughput is impossible with current technology.

(0001) 한국 등록 특허 KR 10-1073989(0001) Korean registered patent KR 10-1073989

(0001) Marcus Fehr. et. al. (2002) PNAS 99: 9846-9851(0001) Marcus Fehr. et. al. (2002) PNAS 99: 9846-9851 (0002) Marcus. Fehr. et. al. (2004) Current Opinion in Plant Biology 7: 345-351(0002) Marcus. Fehr. et. al. (2004) Current Opinion in Plant Biology 7: 345-351

본 발명의 목적은 푸코실락토오스 검출능 및 농도 측정능이 개선된 바이오센서 및 농도 측정방법을 제공하는 것이다. It is an object of the present invention to provide a biosensor and a concentration measurement method having improved fucault room lactose detection ability and concentration measurement ability.

상기 목적을 해결하기 위하여, 본 발명은 공여 형광체 단백질(Fluorescence donor), 푸코실락토오스 결합 단백질(Fucosyllactose Binding Protein, FBP) 및 수용 형광체 단백질(Fluorescence acceptor)을 포함하는 푸코실락토오스(fucosyllactose) 검출용 FRET 센서를 제공한다. In order to solve the above object, the present invention provides FRET for detecting fucosyllactose including a donor fluorescent donor, a fucosyllactose binding protein (FBP), and a fluorescent acceptor. sensor is provided.

또한, 본 발명은 공여 형광체 단백질(Fluorescence donor), 푸코실락토오스 결합 단백질(Fucosyllactose Binding Protein, FBP) 및 수용 형광체 단백질(Fluorescence acceptor)을 포함하는 푸코실락토오스 검출용 FRET 센서를 푸코실락토오스를 포함하는 시료에 접촉시키는 단계; 및 In addition, the present invention provides a FRET sensor for detecting fucosyllactose comprising a donor fluorescent protein (Fluorescence donor), a fucosyllactose binding protein (FBP), and a fluorophore protein (Fluorescence acceptor) comprising fucosyllactose. contacting the sample; and

상기 공여 형광체 단백질과 수용 형광체 단백질의 방사광 비율의 변화를 측정하여 푸코실락토오스의 농도를 측정하는 방법을 제공한다. Provided is a method for measuring the concentration of fucosyllactose by measuring a change in the emission light ratio between the donor phosphor protein and the acceptor phosphor protein.

본 발명은 에너지 공여체와 에너지 수여체로 작용하는 형광 단백질들을 푸코실락토오스와 결합하는 단백질과 융합시킨 푸코실락토오스 바이오센서로서 세포 내부 또는 외부의 푸코실락토오스를 빠르게 정량하여 신규한 푸코실락토오스 발현 균주를 찾아낼 뿐만 아니라 모유와 같은 여러 다른 조건에서도 푸코실락토오스의 농도를 빠르게 정량할 수 있다.The present invention is a fucosyl lactose biosensor in which fluorescent proteins acting as an energy donor and energy acceptor are fused with a protein that binds to fucosyl lactose. In addition to the detection, it is possible to quickly quantify the concentration of fucosyllactose in several other conditions, such as breast milk.

도 1은 푸코실락토오스 바이오센서의 구성 및 작동원리를 나타낸 모식도이다.
도 2는 푸코실락토오스 바이오센서의 구조를 나타낸 모식도이다.
도 3은 푸코실락토오스 결합 단백질(Fucosyllactose Binding Protein, FBP)로서 DC-SIGN을 이용한 바이오센서의 FRET 변화를 나타낸 그래프이다.
도 4는 푸코실락토오스 결합 단백질(Fucosyllactose Binding Protein, FBP)로서 BoGT6a를 이용한 바이오센서의 FRET 변화를 나타낸 그래프이다. 1 is a schematic diagram showing the configuration and operating principle of a fucosyl lactose biosensor.
Figure 2 is a schematic diagram showing the structure of the fucosyl lactose biosensor.
3 is a graph showing the FRET change of the biosensor using DC-SIGN as a fucosyllactose binding protein (FBP).
4 is a graph showing the FRET change of the biosensor using BoGT6a as a fucosyllactose binding protein (FBP).

다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

본 발명의 상세한 설명 등에서 사용되는 주요 용어의 정의는 다음과 같다.Definitions of key terms used in the detailed description of the present invention are as follows.

본 발명에서 "FRET"이란 서로 다른 발광 파장대의 두 형광물질 사이에서 발생하는 비방사성 (non-radiative) 에너지 전이현상으로, 여기(excitation)된 상태의 형광공여체의 여기 준위 에너지가 형광수용체로 전달되어 형광수용체로부터 발광 (emission)이 관찰되거나, 형광공여체의 형광감소(quenching)가 관찰되는 현상이다(Lakowicz,JR Principles of Fluorescence Spectroscopy, 2nd ed., New York:Plenum Press, 1999)In the present invention, "FRET" is a non-radiative energy transfer phenomenon that occurs between two fluorescent materials in different emission wavelength bands. It is a phenomenon in which emission from a fluorescent receptor or quenching of a fluorescent donor is observed (Lakowicz, JR Principles of Fluorescence Spectroscopy, 2nd ed., New York: Plenum Press, 1999).

본 발명에서 "형광공여체"란 FRET 현상에서 공여체로 작용하는 형광물질을 의미하고, "형광수여체"란 FRET 현상에서 수여체로 작용하는 형광물질을 의미한다.In the present invention, "fluorescence donor" means a fluorescent material that acts as a donor in the FRET phenomenon, and "fluorescence acceptor" refers to a fluorescent material that acts as an acceptor in the FRET phenomenon.

본 발명에서 "시료"란, 관심있는 푸코실락토오스를 함유하거나 함유하고 있는 것으로 추정되어 분석이 행해질 조성물을 의미하며, 세포, 물, 토양, 공기, 식품, 폐기물, 동식물 장내 및 동식물 조직 중 어느 하나 이상에서 수집된 것임을 특징으로 할 수 있으나, 이에 한정되는 것은 아니다. 이때, 상기 동식물은 인체를 포함한다.In the present invention, "sample" means a composition to be analyzed that contains or is presumed to contain fucosyllactose of interest, and any one of cells, water, soil, air, food, waste, animal and plant intestinal tract and animal and plant tissue. It may be characterized as being collected above, but is not limited thereto. In this case, the animals and plants include the human body.

본 발명은 공여 형광체 단백질(Fluorescence donor), 푸코실락토오스 결합 단백질(Fucosyllactose Binding Protein, FBP) 및 수용 형광체 단백질(Fluorescence acceptor)을 포함하는 푸코실락토오스(fucosyllactose) 검출용 FRET 센서에 관한 것이다. The present invention relates to a FRET sensor for detecting fucosyllactose comprising a donor phosphor protein (Fluorescence donor), a fucosyllactose binding protein (FBP) and a fluorescence acceptor protein (FBP).

본 발명의 일실시예에 있어서, 상기 형광 단백질은 녹색 형광체 단백질 (GFP), 변형된 녹색 형광체 단백질 (mGFP), 증강된 녹색 형광체, 단백질 (eGFP), 적색 형광체 단백질 (RFP), 변형된 적색 형광체 단백질 (mRFP), 증강된 적색 형광체 단백질(eRFP), 청색 형광체 단백질 (BFP), 변형된 청색 형광체 단백질 (mBFP), 증강된 청색 형광체 단백질 (eBFP), 황색 형광체 단백질 (YFP), 변형된 황색 형광체 단백질 (mYFP), 증강된 황색 형광체 단백질 (eYFP), 남색 형광체 단백질 (CFP), 변형된 남색 형광체 단백질 (mCFP), 증강된 남색 형광체 단백질 (eCFP), 및 이들의 조합들로 이루어진 군으로부터 선택되는 것을 포함하는 것일 수 있다.In one embodiment of the present invention, the fluorescent protein is a green phosphor protein (GFP), a modified green phosphor protein (mGFP), an enhanced green phosphor, a protein (eGFP), a red phosphor protein (RFP), a modified red phosphor protein (mRFP), enhanced red phosphor protein (eRFP), blue phosphor protein (BFP), modified blue phosphor protein (mBFP), enhanced blue phosphor protein (eBFP), yellow phosphor protein (YFP), modified yellow phosphor protein (mYFP), enhanced yellow phosphor protein (eYFP), indigo phosphor protein (CFP), modified indigo phosphor protein (mCFP), enhanced indigo phosphor protein (eCFP), and combinations thereof may include.

본 발명의 일실시예에 있어서, 상기 푸코실락토오스 결합 단백질은 DC-SIGN(Dendritic Cell Specific Intracellular adhesion molecule-3-grabbing Non integrin, CD209) 또는 BoGT6a(Bacteroides ovatus glycotransferase)인 것이며, 상기 DC-SIGN 단백질은 서열번호 7로 표기되고, 상기 BoGT6a 단백질은 서열번호 8로 표기되는 것이다. In one embodiment of the present invention, the fucosyllactose binding protein is DC-SIGN (Dendritic Cell Specific Intracellular adhesion molecule-3-grabbing Non integrin, CD209) or BoGT6a (Bacteroides ovatus glycotransferase), and the DC-SIGN protein is represented by SEQ ID NO: 7, and the BoGT6a protein is represented by SEQ ID NO: 8.

또한, 본 발명은 공여 형광체 단백질(Fluorescence donor), 푸코실락토오스 결합 단백질(Fucosyllactose Binding Protein, FBP) 및 수용 형광체 단백질(Fluorescence acceptor)을 포함하는 푸코실락토오스 검출용 FRET 센서를 푸코실락토오스를 포함하는 시료에 접촉시키는 단계; 및In addition, the present invention provides a FRET sensor for detecting fucosyllactose comprising a donor fluorescent protein (Fluorescence donor), a fucosyllactose binding protein (FBP), and a fluorophore protein (Fluorescence acceptor) comprising fucosyllactose. contacting the sample; and

상기 공여 형광체 단백질과 수용 형광체 단백질의 방사광 비율의 변화를 측정하여 푸코실락토오스의 농도를 측정하는 방법을 제공한다. Provided is a method for measuring the concentration of fucosyllactose by measuring a change in the emission light ratio between the donor phosphor protein and the acceptor phosphor protein.

본 발명의 일실시예에 있어서, 상기 푸코실락토오스 결합 단백질은 DC-SIGN(Dendritic Cell Specific Intracellular adhesion molecule-3-grabbing Non integrin, CD209) 또는 BoGT6a(Bacteroides ovatus glycotransferase)인 것이며, 상기 DC-SIGN 단백질은 서열번호 7로 표기되고, 상기 BoGT6a 단백질은 서열번호 8로 표기되는 것이다. In one embodiment of the present invention, the fucosyllactose binding protein is DC-SIGN (Dendritic Cell Specific Intracellular adhesion molecule-3-grabbing Non integrin, CD209) or BoGT6a (Bacteroides ovatus glycotransferase), and the DC-SIGN protein is represented by SEQ ID NO: 7, and the BoGT6a protein is represented by SEQ ID NO: 8.

본 발명의 일실시예에 있어서, 상기 공여 형광체 단백질과 수용 형광체 단백질의 방사광 비율은 하기 수학식 1에 개시된 바와 같이 계산하고 도 3 또는 도 4와 같이 공여 형광체 단백질과 수용 형광체 단백질의 방사광 비율(FRET Ratio)에 따라 푸코실락토오스의 농도를 측정할 수 있으므로 푸코실락토오스를 정량할 수 있다. FBP로서 DC-SIGN를 이용한 경우 푸코실락토오스의 농도가 10- 2uM에서 102uM까지 FRET Ratio가 변화되는 것을 측정할 수 있으며, FRET Ratio는 10- 2uM의 푸코실락토오스 농도에서 0.84의 측정값을 갖고 102uM 농도까지 푸코실락토오스의 양을 증가시켰을 때 FRET ratio가 0.78까지 감소하는 것을 확인할 수 있었다(도 3).In one embodiment of the present invention, the emission ratio of the donor phosphor protein and the recipient phosphor protein is calculated as described in Equation 1 below, and the emission ratio between the donor phosphor protein and the recipient phosphor protein (FRET) as shown in FIG. 3 or FIG. Ratio), so that the concentration of fucosyl lactose can be measured, so that fucosyl lactose can be quantified. When DC-SIGN is used as FBP, it can be measured that the FRET Ratio changes from 10 - 2 uM to 10 2 uM of fucosyllactose, and the FRET Ratio is 0.84 at a concentration of 10 - 2 uM fucosyllactose. When the amount of fucosyllactose was increased to a concentration of 10 2 uM with a value, it was confirmed that the FRET ratio decreased to 0.78 (FIG. 3).

또한, FBP로서 BoGT6a를 이용한 경우 푸코실락토오스의 농도가 10-2uM에서 102uM에서 FRET 변화되는 것을 측정할 수 있으며, FRET Ratio는 10- 2uM 농도에서 0.80의 측정값을 갖고 102uM 농도까지 푸코실락토오스의 양을 증가시켰을 때 0.91까지 증가하는 것을 확인할 수 있으므로(도 4). 이를 통해 표준곡선이 구할 수 있으며, 측정된 FRET Ratio을 이용하여 용액 내 푸코실락토오스의 농도를 구할 수 있다.In addition, when a BoGT6a as FBP can measure the concentration of the Foucault room lactose which FRET changes from 10 2 uM at 10 uM -2, FRET Ratio 10 - has a value of 0.80 measured in a concentration of 2 uM 10 uM 2 As it can be seen that the concentration increases to 0.91 when the amount of fucosyllactose is increased (FIG. 4). Through this, a standard curve can be obtained, and the concentration of fucosyllactose in the solution can be obtained using the measured FRET Ratio.

본 발명은 도 1에 나타낸 바와 같이, CFP-FBP-YFP(도 2 모식도 참고)로 구성되는 세포 내·외부의 푸코실락토오스 정량을 위한 바이오센서에 관한 것으로서, 푸코실락토오스 인식부위인 푸코실락토오스 결합 단백질(Fucosyllactose Binding Protein, FBP)에 푸코실락토오스가 결합하면 푸코실락토오스의 결합에 의해 FBP의 구조가 바뀌면서 양 말단에 결합한 CFP와 YFP의 거리(distance)와 배향(orientation)이 바뀌는 구조적 변화(conformational change)가 일어나게 되고, 이로 인해 발생하는 FRET의 정도 차이로 발생하는 형광을 비교 측정함에 있다. FRET이 발생하기 위한 조건으로는 에너지 공여체(donor)의 방사광(emission) 스펙트럼과 수여체(acceptor)의 흡수광(absorbtion) 스펙트럼이 서로 중첩되어야 하며, 공여체와 수여체의 전위쌍극자 상호배향(transition dipole orientation)이 평행이 되어야 유리하다. 상기의 바이오센서는 푸코실락토오스의 결합으로 FBP의 구조 변화를 유발하며, 결과적으로 FBP의 양 말단에 위치한 CFP와 YFP의 거리와 전위 쌍극자 상호배향을 변화시켜 FRET의 조건을 변화시킴으로 인해 발생하는 CFP와 YFP의 형광차이를 비교 분석하는 방법으로 세포 내·외부의 푸코실락토오스를 정량적으로 분석한다.As shown in FIG. 1, the present invention relates to a biosensor for quantifying fucosyl lactose inside and outside cells composed of CFP-FBP-YFP (refer to the schematic diagram of FIG. 2). When fucosyllactose is bound to the binding protein (Fucosyllactose Binding Protein, FBP), the structure of FBP is changed by the binding of fucosyllactose, and the distance and orientation of CFP and YFP bound to both ends are changed ( conformational change) occurs, and the fluorescence generated by the difference in the degree of FRET caused by this is compared and measured. As a condition for FRET to occur, the emission spectrum of the energy donor and the absorption spectrum of the acceptor must overlap each other, and the transition dipole of the donor and acceptor orientation) should be parallel. The biosensor induces a structural change of FBP due to the binding of fucosyllactose, and as a result, the distance between CFP and YFP located at both ends of FBP and the potential dipole mutual orientation are changed to change the conditions of FRET. By comparing and analyzing the difference in fluorescence between YFP and YFP, it quantitatively analyzes fucosyllactose inside and outside the cell.

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.

실시예Example 1: One: 푸코실락토오스Foucault room lactose 검출용 바이오센서의 제작 방법 Method of manufacturing biosensor for detection

도 2로 표시되는 His-eCFP-FBP-eYFP로 구성되는 융합 단백질을 발현시키기 위한 발현벡터를 구축하기 위해 하기 표 1에 기재된 DNA 염기서열을 갖는 프라이머를 사용하여 클로닝을 수행하였다.In order to construct an expression vector for expressing a fusion protein composed of His-eCFP-FBP-eYFP as shown in FIG. 2, cloning was performed using primers having the DNA sequences shown in Table 1 below.

구체적으로, eCFP와 eYFP가 들어있는 유전자는 Wolf Frommer에게 제공받은 FLIPRbs 발현벡터를 주형으로 하였고, 서열번호 1과 서열번호 2의 프라이머를 사용하여 PCR을 하였다. eCFP와 eYFP 사이에 삽입되는 푸코실락토오스 결합 단백질인 DC-SIGN과 BoGT6a의 아미노산 서열은 표 2에 기재된 서열과 같고, 이 서열을 대장균에서의 발현을 위해 DNA 염기서열 최적화 과정을 통해 Solid-phase oligonucleotide synthesis 법을 통해 DNA로 합성하였다. 합성된 DC-SIGN 염기서열은 서열번호 3과 서열번호 4의 프라이머를 이용해 PCR로 증폭시켰고, BoGT6a는 서열번호 5와 서열번호 6의 프라이머를 이용하여 PCR을 통해 증폭시켰다.Specifically, the genes containing eCFP and eYFP were performed using the FLIPRbs expression vector provided by Wolf Frommer as a template, and PCR was performed using the primers of SEQ ID NO: 1 and SEQ ID NO: 2. The amino acid sequences of DC-SIGN and BoGT6a, which are fucosyllactose-binding proteins inserted between eCFP and eYFP, are the same as those shown in Table 2, and this sequence is subjected to a DNA sequence optimization process for expression in E. coli to be a solid-phase oligonucleotide. It was synthesized into DNA through the synthesis method. The synthesized DC-SIGN base sequence was amplified by PCR using the primers of SEQ ID NO: 3 and SEQ ID NO: 4, and BoGT6a was amplified by PCR using the primers of SEQ ID NO: 5 and SEQ ID NO: 6.

주형인 서열번호 1과 서열번호 2로 증폭된 유전자와 서로 중첩되게 제작된 서열번호 3과 서열번호 4로 증폭된 DC-SIGN의 유전자를 SLIC(Sequence and Ligase Independent Cloning) 방법으로 클로닝 하였고 마찬가지로 서열번호 1과 서열번호 2로 증폭된 유전자와 서로 중첩되게 제작된 서열번호 5와 서열번호 6으로 증폭된 BoGT6a의 유전자를 SLIC 방법으로 클로닝하여 각각 eCFP와 eYFP 사이에 DC-SIGN 또는 BoGT6a가 융합된 DNA 염기서열을 얻을 수 있었다.The gene amplified with SEQ ID NO: 3 and SEQ ID NO: 4 prepared to overlap with the gene amplified by SEQ ID NO: 1 and SEQ ID NO: 2 as the template was cloned by SLIC (Sequence and Ligase Independent Cloning) method, and similarly, SEQ ID NO: The DNA base in which DC-SIGN or BoGT6a is fused between eCFP and eYFP by cloning the gene of SEQ ID NO: 5 and SEQ ID NO: 6 amplified by the SLIC method to overlap the gene amplified with SEQ ID NO: 1 and SEQ ID NO: 2, respectively. sequence could be obtained.

이름name 서열번호 SEQ ID NO: 서열order FLIPRbs
Forward primerFLIPRbs
forward primer 1One 5‘-GGCGCCGGTACCGGTGGAATG-3’ 5'-GGCGCCGGTACCGGTGGAATG-3' FLIPRbs
Reverse primerFLIPRbs
Reverse primer 22 5’-GCCTCCGGTACCACCCTTGTACAG-3’ 5’-GCCTCCGGTACCACCCTTGTACAG-3’ DC-SIGN
Forward primerDC-SIGN
forward primer 33 5‘-GGTGGTACCGGAGGCTGTCATCCATGCCCGTGGGAATGG-3’5'-GGTGGTACCGGAGGCTGTCATCCATGCCCGTGGGAATGG-3' DC-SIGN
Reverse primerDC-SIGN
Reverse primer 44 5‘-ACCGGTACCGGCGCCGCAGCTTGCCGCGGATTTTTTACAAATCCA-3’5'-ACCGGTACCGGCGCCGCAGCTTGCCGCGGATTTTTTACAAATCCA-3' BoGT6a
Forward primerBoGT6a
forward primer 55 5‘-GGTGGTACCGGAGGCATGCGGATCGGCATCCTGTATATCTGTACTGGG-3’5'-GGTGGTACCGGAGGCATGCGGATCGGCATCCTGTATATCTGTACTGGG-3' BoGT6a
Reverse primerBoGT6a
Reverse primer 66 5‘-ACCGGTACCGGCGCCGTTTTTACGTCTCAGCAACTCGTGTCCTCCGTACTG-3’5'-ACCGGTACCGGCGCCGTTTTTACGTCTCAGCAACTCGTGTCCTCCGTACTG-3'

이름name 서열번호SEQ ID NO: 서열order DC-SIGNDC-SIGN 77 CHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQNFLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNVGEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQNFLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNVGEEDCAEFSGNGWNDDKCNLAKFWICKKSAASC BoGT6aBoGT6a 88 MRIGILYICTGKYDIFWKDFYLSAERYFMQDQSFIIEYYVFTDSPKLYDEENNKHIHRIKQKNLGWPDNTLKRFHIFLRIKEQLERETDYLFFFNANLLFTSPIGKEILPPSDSNGLLGTMHPGFYNKPNSEFTYERRDASTAYIPEGEGRYYYAGGLSGGCTKAYLKLCTTICSWVDRDATNHIIPIWHDESLINKYFLDNPPAITLSPAYLYPEGWLLPFEPIILIRDKNKPQYGGHELLRRKNMRIGILYICTGKYDIFWKDFYLSAERYFMQDQSFIIEYYVFTDSPKLYDEENNKHIHRIKQKNLGWPDNTLKRFHIFLRIKEQLERETDYLFFFNANLLFTSPIGKEILPPSDSNGLLGTMHPGFYNKPNSEFTYERRDASTAYIPEGEGRYIIYYAGGLESSGGCYLINKPNSEFTYERRDASTAYIPEGEGRYIIYYAGGLESSGGCYLINKPNSEFTYERRDASTAYIPEGEGRYYYYYAGGLESSGGCYLINKPNGRR

실시예Example 2 : 바이오센서 단백질의 형광분석 2: Fluorescence analysis of biosensor protein

상기 실시예 1에서 제작된 eCFP-FBP-eYFP로 구성된 바이오센서 단백질의 형광 스펙트럼 분석은 형광분광장비인 Synergy H1(Biotek)을 사용하여 측정하였으며, CFP의 입사광(excitation)을 433nm로 하였을 때 측정되는 475nm의 방사광(emission)값과 FRET에 의해 여기(excited)된 YFP의 방사광인 528nm의 비율을 하기 수학식 1에 의해 계산하여 FRET 변화를 측정하였다. The fluorescence spectrum analysis of the biosensor protein composed of eCFP-FBP-eYFP prepared in Example 1 was measured using Synergy H1 (Biotek), which is a fluorescence spectrometer, and was measured when the incident light (excitation) of CFP was 433 nm. The FRET change was measured by calculating the ratio of the emission value of 475 nm and the emission light of YFP excited by FRET, 528 nm, by Equation 1 below.

그 결과, FBP로서 DC-SIGN를 이용한 경우 푸코실락토오스의 농도가 10- 2uM에서 102uM까지 FRET Ratio가 변화되는 것을 측정할 수 있다(도 3). 이 FRET Ratio는 10-2uM의 푸코실락토오스 농도에서 0.84의 측정값을 갖고 102uM 농도까지 푸코실락토오스의 양을 증가시켰을 때 FRET ratio가 0.78까지 감소하는 것을 확인할 수 있었다.As a result, when DC-SIGN is used as the FBP, it can be measured that the FRET Ratio changes from 10 - 2 uM to 10 2 uM of fucosyllactose (FIG. 3). This FRET Ratio has a measured value of 0.84 at a fucosyllactose concentration of 10 -2 uM, and it was confirmed that the FRET ratio decreased to 0.78 when the amount of fucosyllactose was increased to a concentration of 10 2 uM.

또한, FBP로서 BoGT6a를 이용한 경우 푸코실락토오스의 농도가 10-2uM에서 102uM에서 FRET 변화되는 것을 측정할 수 있다(도 4). 이 FRET Ratio는 10- 2uM 농도에서 0.80의 측정값을 갖고 102uM 농도까지 푸코실락토오스의 양을 증가시켰을 때 0.91까지 증가하는 것을 확인할 수 있었다. 이를 통해 표준곡선이 구해진다면, 측정된 FRET Ratio을 이용하여 용액 내 푸코실락토오스의 농도를 정량할 수 있다.In addition, when BoGT6a is used as the FBP, it can be measured that the concentration of fucosyllactose is changed from 10 -2 uM to 10 2 uM in FRET (FIG. 4). This FRET Ratio has a measured value of 0.80 at a concentration of 10 - 2 uM, and it was confirmed that it increased to 0.91 when the amount of fucosyllactose was increased to a concentration of 10 2 uM. If a standard curve is obtained through this, the concentration of fucosyllactose in the solution can be quantified using the measured FRET Ratio.

[수학식 1][Equation 1]

Figure 112018121897876-pat00001
Figure 112018121897876-pat00001

FRET: YFP와 CFP의 방사광 비율FRET: Emission ratio of YFP and CFP

528nm: FRET에 의해 측정되어지는 YFP의 방사광 값528nm: Emission value of YFP measured by FRET

475nm: 입사광을 433nm로 하였을 때 측정되는 CFP의 방사광 값475nm: CFP emission value measured when the incident light is 433nm

<110> Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same <130> PN1811-489 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FLIPRbs Forward primer <400> 1 ggcgccggta ccggtggaat g 21 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FLIPRbs Reverse primer <400> 2 gcctccggta ccacccttgt acag 24 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> DC-SIGN Forward primer <400> 3 ggtggtaccg gaggctgtca tccatgcccg tgggaatgg 39 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> DC-SIGN Reverse primer <400> 4 accggtaccg gcgccgcagc ttgccgcgga ttttttacaa atcca 45 <210> 5 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> BoGT6a Forward primer <400> 5 ggtggtaccg gaggcatgcg gatcggcatc ctgtatatct gtactggg 48 <210> 6 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> BoGT6a Reverse primer <400> 6 accggtaccg gcgccgtttt tacgtctcag caactcgtgt cctccgtact g 51 <210> 7 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> DC-SIGN <400> 7 Cys His Pro Cys Pro Trp Glu Trp Thr Phe Phe Gln Gly Asn Cys Tyr 1 5 10 15 Phe Met Ser Asn Ser Gln Arg Asn Trp His Asp Ser Ile Thr Ala Cys 20 25 30 Lys Glu Val Gly Ala Gln Leu Val Val Ile Lys Ser Ala Glu Glu Gln 35 40 45 Asn Phe Leu Gln Leu Gln Ser Ser Arg Ser Asn Arg Phe Thr Trp Met 50 55 60 Gly Leu Ser Asp Leu Asn Gln Glu Gly Thr Trp Gln Trp Val Asp Gly 65 70 75 80 Ser Pro Leu Leu Pro Ser Phe Lys Gln Tyr Trp Asn Arg Gly Glu Pro 85 90 95 Asn Asn Val Gly Glu Glu Asp Cys Ala Glu Phe Ser Gly Asn Gly Trp 100 105 110 Asn Asp Asp Lys Cys Asn Leu Ala Lys Phe Trp Ile Cys Lys Lys Ser 115 120 125 Ala Ala Ser Cys 130 <210> 8 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> BoGT6a <400> 8 Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Asp Ile Phe 1 5 10 15 Trp Lys Asp Phe Tyr Leu Ser Ala Glu Arg Tyr Phe Met Gln Asp Gln 20 25 30 Ser Phe Ile Ile Glu Tyr Tyr Val Phe Thr Asp Ser Pro Lys Leu Tyr 35 40 45 Asp Glu Glu Asn Asn Lys His Ile His Arg Ile Lys Gln Lys Asn Leu 50 55 60 Gly Trp Pro Asp Asn Thr Leu Lys Arg Phe His Ile Phe Leu Arg Ile 65 70 75 80 Lys Glu Gln Leu Glu Arg Glu Thr Asp Tyr Leu Phe Phe Phe Asn Ala 85 90 95 Asn Leu Leu Phe Thr Ser Pro Ile Gly Lys Glu Ile Leu Pro Pro Ser 100 105 110 Asp Ser Asn Gly Leu Leu Gly Thr Met His Pro Gly Phe Tyr Asn Lys 115 120 125 Pro Asn Ser Glu Phe Thr Tyr Glu Arg Arg Asp Ala Ser Thr Ala Tyr 130 135 140 Ile Pro Glu Gly Glu Gly Arg Tyr Tyr Tyr Ala Gly Gly Leu Ser Gly 145 150 155 160 Gly Cys Thr Lys Ala Tyr Leu Lys Leu Cys Thr Thr Ile Cys Ser Trp 165 170 175 Val Asp Arg Asp Ala Thr Asn His Ile Ile Pro Ile Trp His Asp Glu 180 185 190 Ser Leu Ile Asn Lys Tyr Phe Leu Asp Asn Pro Pro Ala Ile Thr Leu 195 200 205 Ser Pro Ala Tyr Leu Tyr Pro Glu Gly Trp Leu Leu Pro Phe Glu Pro 210 215 220 Ile Ile Leu Ile Arg Asp Lys Asn Lys Pro Gln Tyr Gly Gly His Glu 225 230 235 240 Leu Leu Arg Arg Lys Asn 245 <110> Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same <130> PN1811-489 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FLIPRbs Forward primer <400> 1 ggcgccggta ccggtggaat g 21 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FLIPRbs Reverse primer <400> 2 gcctccggta ccacccttgt acag 24 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> DC-SIGN Forward primer <400> 3 ggtggtaccg gaggctgtca tccatgcccg tgggaatgg 39 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> DC-SIGN Reverse primer <400> 4 accggtaccg gcgccgcagc ttgccgcgga ttttttacaa atcca 45 <210> 5 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> BoGT6a Forward primer <400> 5 ggtggtaccg gaggcatgcg gatcggcatc ctgtatatct gtactggg 48 <210> 6 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> BoGT6a Reverse primer <400> 6 accggtaccg gcgccgtttt tacgtctcag caactcgtgt cctccgtact g 51 <210> 7 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> DC-SIGN <400> 7 Cys His Pro Cys Pro Trp Glu Trp Thr Phe Phe Gln Gly Asn Cys Tyr 1 5 10 15 Phe Met Ser Asn Ser Gln Arg Asn Trp His Asp Ser Ile Thr Ala Cys 20 25 30 Lys Glu Val Gly Ala Gln Leu Val Val Ile Lys Ser Ala Glu Glu Gln 35 40 45 Asn Phe Leu Gln Leu Gln Ser Ser Arg Ser Asn Arg Phe Thr Trp Met 50 55 60 Gly Leu Ser Asp Leu Asn Gln Glu Gly Thr Trp Gln Trp Val Asp Gly 65 70 75 80 Ser Pro Leu Leu Pro Ser Phe Lys Gln Tyr Trp Asn Arg Gly Glu Pro 85 90 95 Asn Asn Val Gly Glu Glu Asp Cys Ala Glu Phe Ser Gly Asn Gly Trp 100 105 110 Asn Asp Asp Lys Cys Asn Leu Ala Lys Phe Trp Ile Cys Lys Lys Ser 115 120 125 Ala Ala Ser Cys 130 <210> 8 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> BoGT6a <400> 8 Met Arg Ile Gly Ile Leu Tyr Ile Cys Thr Gly Lys Tyr Asp Ile Phe 1 5 10 15 Trp Lys Asp Phe Tyr Leu Ser Ala Glu Arg Tyr Phe Met Gln Asp Gln 20 25 30 Ser Phe Ile Ile Glu Tyr Tyr Val Phe Thr Asp Ser Pro Lys Leu Tyr 35 40 45 Asp Glu Glu Asn Asn Lys His Ile His Arg Ile Lys Gln Lys Asn Leu 50 55 60 Gly Trp Pro Asp Asn Thr Leu Lys Arg Phe His Ile Phe Leu Arg Ile 65 70 75 80 Lys Glu Gln Leu Glu Arg Glu Thr Asp Tyr Leu Phe Phe Phe Phe Asn Ala 85 90 95 Asn Leu Leu Phe Thr Ser Pro Ile Gly Lys Glu Ile Leu Pro Pro Ser 100 105 110 Asp Ser Asn Gly Leu Leu Gly Thr Met His Pro Gly Phe Tyr Asn Lys 115 120 125 Pro Asn Ser Glu Phe Thr Tyr Glu Arg Arg Asp Ala Ser Thr Ala Tyr 130 135 140 Ile Pro Glu Gly Glu Gly Arg Tyr Tyr Tyr Ala Gly Gly Leu Ser Gly 145 150 155 160 Gly Cys Thr Lys Ala Tyr Leu Lys Leu Cys Thr Thr Ile Cys Ser Trp 165 170 175 Val Asp Arg Asp Ala Thr Asn His Ile Ile Pro Ile Trp His Asp Glu 180 185 190 Ser Leu Ile Asn Lys Tyr Phe Leu Asp Asn Pro Pro Ala Ile Thr Leu 195 200 205 Ser Pro Ala Tyr Leu Tyr Pro Glu Gly Trp Leu Leu Pro Phe Glu Pro 210 215 220 Ile Ile Leu Ile Arg Asp Lys Asn Lys Pro Gln Tyr Gly Gly His Glu 225 230 235 240 Leu Leu Arg Arg Lys Asn 245

Claims (7)

공여 형광체 단백질(Fluorescence donor), 푸코실락토오스 결합 단백질(Fucosyllactose Binding Protein, FBP) 및 수용 형광체 단백질(Fluorescence acceptor)을 포함하는 푸코실락토오스(fucosyllactose) 검출용 FRET 센서에 있어서,
상기 푸코실락토오스 결합 단백질은 DC-SIGN(Dendritic Cell Specific Intracellular adhesion molecule-3-grabbing Non integrin, CD209)이고, 상기 센서는 푸코실락토오스가 푸코실락토오스 결합 단백질에 결합시 FRET 시그널이 감소되는 것인, 푸코실락토오스 검출용 FRET 센서.In the FRET sensor for detecting fucosyllactose comprising a donor fluorescent protein (Fluorescence donor), a fucosyllactose binding protein (FBP) and a fluorescent acceptor protein (Fluorescence acceptor),
The fucosyl lactose binding protein is DC-SIGN (Dendritic Cell Specific Intracellular adhesion molecule-3-grabbing Non integrin, CD209), and the sensor is that the FRET signal is reduced when fucosyl lactose binds to the fucosyl lactose binding protein. , FRET sensor for detection of fucosyllactose. 제 1 항에 있어서,
상기 형광 단백질은 녹색 형광체 단백질 (GFP), 변형된 녹색 형광체 단백질 (mGFP), 증강된 녹색 형광체, 단백질 (eGFP), 적색 형광체 단백질 (RFP), 변형된 적색 형광체 단백질 (mRFP), 증강된 적색 형광체 단백질(eRFP), 청색 형광체 단백질 (BFP), 변형된 청색 형광체 단백질 (mBFP), 증강된 청색 형광체 단백질 (eBFP), 황색 형광체 단백질 (YFP), 변형된 황색 형광체 단백질 (mYFP), 증강된 황색 형광체 단백질 (eYFP), 남색 형광체 단백질 (CFP), 변형된 남색 형광체 단백질 (mCFP), 증강된 남색 형광체 단백질 (eCFP), 및 이들의 조합들로 이루어진 군으로부터 선택되는 것을 포함하는 것인, 푸코실락토오스 검출용 FRET 센서.The method of claim 1,
The fluorescent protein is green phosphor protein (GFP), modified green phosphor protein (mGFP), enhanced green phosphor, protein (eGFP), red phosphor protein (RFP), modified red phosphor protein (mRFP), enhanced red phosphor protein (eRFP), blue phosphor protein (BFP), modified blue phosphor protein (mBFP), enhanced blue phosphor protein (eBFP), yellow phosphor protein (YFP), modified yellow phosphor protein (mYFP), enhanced yellow phosphor fucosyllactose, comprising one selected from the group consisting of protein (eYFP), indigo phosphor protein (CFP), modified indigo phosphor protein (mCFP), enhanced indigo phosphor protein (eCFP), and combinations thereof. FRET sensor for detection. 삭제delete 제 1항에 있어서, 상기 DC-SIGN 단백질은 서열번호 7로 표기되는 것인 푸코실락토오스 검출용 FRET 센서. The FRET sensor for detecting fucosyllactose according to claim 1, wherein the DC-SIGN protein is represented by SEQ ID NO: 7. 공여 형광체 단백질(Fluorescence donor), 푸코실락토오스 결합 단백질(Fucosyllactose Binding Protein, FBP) 및 수용 형광체 단백질(Fluorescence acceptor)을 포함하는 푸코실락토오스 검출용 FRET 센서를 푸코실락토오스를 포함하는 시료에 접촉시키는 단계; 및
상기 공여 형광체 단백질과 수용 형광체 단백질의 방사광 비율(FRET Ratio)의 변화를 측정하여 푸코실락토오스의 농도를 측정하는 방법에 있어서,
상기 푸코실락토오스 결합 단백질은 DC-SIGN(Dendritic Cell Specific Intracellular adhesion molecule-3-grabbing Non integrin, CD209)이고, 상기 센서는 푸코실락토오스가 푸코실락토오스 결합 단백질에 결합시 FRET 시그널이 감소되는 것인, 푸코실락토오스의 농도를 측정하는 방법.Contacting a FRET sensor for fucosyllactose detection comprising a donor fluorophore protein (Fluorescence donor), a fucosyllactose binding protein (FBP) and a fluorescence acceptor to a sample containing fucosyllactose ; and
In the method of measuring the concentration of fucosyllactose by measuring the change in the FRET ratio of the donor fluorescent protein and the receiving fluorescent protein,
The fucosyl lactose binding protein is DC-SIGN (Dendritic Cell Specific Intracellular adhesion molecule-3-grabbing Non integrin, CD209), and the sensor is that the FRET signal is reduced when fucosyl lactose binds to the fucosyl lactose binding protein. , a method for measuring the concentration of fucosyllactose. 삭제delete 제 5항에 있어서, 상기 DC-SIGN 단백질은 서열번호 7로 표기되는 것인, 푸코실락토오스의 농도를 측정하는 방법.The method of claim 5, wherein the DC-SIGN protein is represented by SEQ ID NO: 7, a method for measuring the concentration of fucosyllactose.
KR1020180155244A 2018-12-05 2018-12-05 Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same KR102307241B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020180155244A KR102307241B1 (en) 2018-12-05 2018-12-05 Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020180155244A KR102307241B1 (en) 2018-12-05 2018-12-05 Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same

Publications (2)

Publication Number Publication Date
KR20200068359A KR20200068359A (en) 2020-06-15
KR102307241B1 true KR102307241B1 (en) 2021-09-29

Family

ID=71081888

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020180155244A KR102307241B1 (en) 2018-12-05 2018-12-05 Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same

Country Status (1)

Country Link
KR (1) KR102307241B1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006044611A2 (en) 2004-10-14 2006-04-27 Carnegie Institution Of Washington Neurotransmitter sensors and methods of using the same
KR101073989B1 (en) * 2009-04-27 2011-10-17 한국생명공학연구원 Method for Detecting Ligands Using Biosensor Based on Fluorescence Resonance Energy Transfer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006044611A2 (en) 2004-10-14 2006-04-27 Carnegie Institution Of Washington Neurotransmitter sensors and methods of using the same
KR101073989B1 (en) * 2009-04-27 2011-10-17 한국생명공학연구원 Method for Detecting Ligands Using Biosensor Based on Fluorescence Resonance Energy Transfer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Biochem J., vol.473, pp.1343-1353 (2016) 1부.*

Also Published As

Publication number Publication date
KR20200068359A (en) 2020-06-15

Similar Documents

Publication Publication Date Title
Kaur et al. Förster resonance energy transfer (FRET) and applications thereof
Bermejo et al. Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors
Markwardt et al. An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching
De et al. Noninvasive imaging of protein‐protein interactions from live cells and living subjects using bioluminescence resonance energy transfer
De et al. An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects
Goedhart et al. Quantitative co-expression of proteins at the single cell level–application to a multimeric FRET sensor
US9606121B2 (en) Nicotinamide adenine dinucleotide gene encoding fluorescent probe, preparation method therefor and application thereof
Day Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy
Veiksina et al. Fluorescence anisotropy assay for pharmacological characterization of ligand binding dynamics to melanocortin 4 receptors
KR100739529B1 (en) Maltose Biosensor for quantitative measurement of maltose in the cell
Dimri et al. Use of BRET to study protein–protein interactions in vitro and in vivo
Ahmad et al. Live cell monitoring of glycine betaine by FRET-based genetically encoded nanosensor
US20030096243A1 (en) Methods and reagents for live-cell gene expression quantification
Chen et al. A tandem near-infrared fluorescence complementation system with enhanced fluorescence for imaging protein–protein interactions in vivo
Deng et al. Design strategies for fluorescent proteins/mimics and their applications in biosensing and bioimaging
US20090203035A1 (en) Fluorescent proteins with increased photostability
Chen et al. Chemical labeling of intracellular proteins via affinity conjugation and strain-promoted cycloadditions in live cells
Dunsing et al. Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection
Kannaiah et al. Methods for studying RNA localization in bacteria
Petazzi et al. Fluorescence microscopy methods for the study of protein oligomerization
KR102307241B1 (en) Biosensor for detecting fucosyllactose and detecting method of fucosyllactose using the same
Bauer et al. Visualizing RNA granule transport and translation in living neurons
Chen et al. The smallest near-infrared fluorescence complementation system for imaging protein–protein and RNA–protein interactions
KR101435605B1 (en) METHOD FOR QUANTITATIVE ANALYSIS OF REAL TIME INTERACTIONS BETWEEN HIF-1 ALPHA AND p300 PROTEINS
Meiresonne et al. Detection of protein interactions in the cytoplasm and periplasm of Escherichia coli by Förster resonance energy transfer

Legal Events

Date Code Title Description
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant