KR20230060173A - Novel peptides that specifically bind to okadaic acid - Google Patents
Novel peptides that specifically bind to okadaic acid Download PDFInfo
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- KR20230060173A KR20230060173A KR1020210144582A KR20210144582A KR20230060173A KR 20230060173 A KR20230060173 A KR 20230060173A KR 1020210144582 A KR1020210144582 A KR 1020210144582A KR 20210144582 A KR20210144582 A KR 20210144582A KR 20230060173 A KR20230060173 A KR 20230060173A
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- peptide
- okadaic acid
- present
- detecting
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- 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
Abstract
본 발명은 박테리오파지 디스플레이 스크리닝을 이용하여 발굴한 신규한 펩타이드에 관한 것으로, 본 발명에 따르면 상기 펩타이드는 오카다산과 특이적으로 결합할 수 있어 오카다산을 정확하게 검출할 수 있음을 확인한 바, 이를 이용하여 다양한 대상, 예를 들어 식품 및 이의 원재료의 오카다산 보유 여부를 확인할 수 있어, 안전한 식품 공급에 기여할 수 있다.The present invention relates to a novel peptide discovered using bacteriophage display screening. According to the present invention, it was confirmed that the peptide can specifically bind to okadaic acid and thus accurately detect okadaic acid. It is possible to confirm whether an object, for example, food and its raw materials contain okadaic acid, thereby contributing to safe food supply.
Description
본 발명은 오카다산에 특이적으로 결합하는 신규한 펩타이드 및 이의 용도에 대한 것이다.The present invention relates to a novel peptide that specifically binds to okadaic acid and uses thereof.
현재 전세계적으로 발생 빈도가 증가하고 있는 적조현상(Harmful Algal Blooms(HABs), red tide)은 유독화, 조밀화되고 있어 수산자원, 해양환경, 및 공중보건에도 심각한 위협이 되고 있어 이에 대한 대책이 요구되고 있다. 최근 들어 우리나라 근해, 특히 남해안에서 주로 발생하는 적조현상은 대체로 유독성 편모조류에 의한 것으로, 우리나라 총 어업생산의 30 %를 차지하는 양식산업에 막대한 피해를 주고 있는 실정이다. 이에 따라 국내 연구소뿐만 아니라 대학 등에서도 적조방제를 위한 다각적인 노력을 기울이고 있다.Harmful Algal Blooms (HABs), red tides, which are currently increasing in frequency worldwide, are becoming toxic and dense, posing a serious threat to fisheries resources, the marine environment, and public health, and countermeasures are required. It is becoming. Recently, the red tide phenomenon, which mainly occurs in the coastal waters of Korea, especially in the southern coast, is caused by toxic flagellate algae, which is causing enormous damage to the aquaculture industry, which accounts for 30% of the total fishery production in Korea. Accordingly, not only domestic research institutes but also universities are making various efforts to control red tide.
적조에 의한 어패류의 피해로는, 식물 플랑크톤의 대발생으로 인해 해수 내의 용존산소가 감소되는 경우와, 식물 플랑크톤이 아가미 등에 모이면서 어패류의 사멸을 유도하는 경우가 일반적으로 알려져 있으나, 더욱 커다란 문제는 일부 유해 적조생물들에 의해 합성되어 분비 또는 세포내 함유되는 독성물질로 인한 피해라 할 수 있다. 이와 같이 유독 식물 플랑크톤에 의해 합성, 분비되는 독성물질은 크게 PSP(Paralytic Shellfish Poison), DSP(Diarrhetic Shellfish Poison), ASP(Amnestic Shellfish Poison) 등으로 분류되는데, 상기 DSP에 있어서, 오카다산(Okadaic acid)이 주된 독성 성분으로 주목받고 있다. 상기 오카다산에 감염되는 경우, 무기력증, 메스꺼움, 구토, 복부 통증 등이 발생할 수 있다.As damage to fish and shellfish caused by red tide, it is generally known that the dissolved oxygen in seawater is reduced due to the large occurrence of phytoplankton and the case that phytoplankton gathers on the gills and induces the death of fish and shellfish. It can be said to be damage caused by toxic substances synthesized and secreted by some harmful red algal organisms or contained in cells. Toxic substances synthesized and secreted by toxic phytoplankton are largely classified into PSP (Paralytic Shellfish Poison), DSP (Diarrhetic Shellfish Poison), ASP (Amnestic Shellfish Poison), etc. In the DSP, Okadaic acid ) is attracting attention as the main toxic component. In case of infection with Okadasan, lethargy, nausea, vomiting, abdominal pain, etc. may occur.
국내에서 환경 오염물질을 검출할 수 있는 바이오센서의 개발은 수질 모니터링, 토양독성 모니터링, 가스독성 모니터링 등을 위해 연구되고 있다. 특히 생물학적인 바이오센서로는 물벼룩, 해조류, 어류 등을 이용하는 방법과, 대장균, 바실러스, 슈도모나스, 살모넬라 등의 미생물에서 비롯된 유전자 재조합 미생물을 이용하는 방법 등이 주를 이루고 있다. 이중에서 물벼룩, 해조류, 어류 등을 이용하는 방법은 생물체의 유지, 보관 등의 문제로 인하여 일회용 탐지기능만을 제공할 수밖에 없다는 단점이 있다. 한편, 유전자 재조합 미생물을 이용하는 방법에 있어서는 현재까지 생물학적 빛(bioluminescence)과 녹색형광(green fluorescence)을 표지유전자로 사용하는 연구가 활발히 진행 중에 있다. 그리고, 최근에는 DNA 칩을 이용한 마이크로어레이(microarray) 기법도 적용되고 있다.In Korea, the development of biosensors capable of detecting environmental pollutants is being studied for water quality monitoring, soil toxicity monitoring, and gas toxicity monitoring. In particular, as biological biosensors, methods using Daphnia, algae, fish, etc. and methods using recombinant microorganisms derived from microorganisms such as Escherichia coli, Bacillus, Pseudomonas, and Salmonella are mainly used. Among them, the method using daphnia, algae, fish, etc. has a disadvantage in that it can only provide a one-time detection function due to problems such as maintenance and storage of living organisms. On the other hand, in the method using genetically recombinant microorganisms, studies using bioluminescence and green fluorescence as marker genes have been actively conducted. And, recently, a microarray technique using a DNA chip is also being applied.
그러나, 상기와 같은 바이오센서 관련 연구들은 환경호르몬, 내분비계 장애물질 등과 같은 유해 화학물질과 중금속 등에 국한되어 있을 뿐, 유독 식물 플랑크톤에 의해 합성, 분비되어 어패류 뿐만 아니라 인간에게까지 치명적인 영향을 미치는 독성물질에 대해서는 전혀 연구 개발이 이루어지지 않고 있는 실정이다.However, the biosensor-related studies as described above are limited to harmful chemicals and heavy metals such as endocrine disruptors, endocrine disruptors, etc., and are synthesized and secreted by toxic phytoplankton and have a fatal effect on fish and shellfish as well as humans. Research and development of the material is not being carried out at all.
해수 중의 유독 프랑크톤 수가 1 ml 당 200 개 이상일 경우에는 독소에 의한 피해가 예상되므로, 패독(貝毒) 발생을 대비한 광범위한 실태 조사와 함께 채취 시기의 지도 및 조기 수확 등 적절한 사전관리가 필요하게 된다. 그러나, 단순히 유독 프랑크톤의 수를 기준으로 하여 독소 발생을 예측하는 것은 매우 어려운 일이 될 수밖에 없다. 식품의약품안전청에서는 해수에 기준치 이상(80 ㎍/g 이상)의 독소성분이 검출될 경우 발생지역에서 생산되는 조개류에 대한 판매 금지 및 가공식품 원료 사용 금지를 취하고 있다. 그러나, 이처럼 독소 발생 지역을 발표하는 것은 정확한 검정법이 될 수 없으므로, 대상으로부터 독소를 정확하게 검출할 수 있는 기술의 개발이 필요하다.If the number of toxic plankton in seawater is more than 200 per 1 ml, damage by toxins is expected. Therefore, it is necessary to conduct an extensive fact-finding survey in preparation for the occurrence of shellfish poisoning, as well as proper prior management such as guidance on collection time and early harvest. do. However, it is bound to be very difficult to predict toxin generation simply based on the number of toxic plankton. The Korea Food and Drug Administration (KFDA) bans the sale of shellfish produced in the area and the use of raw materials for processed food if a toxin component is detected in seawater at a level higher than the standard (80 μg/g or more). However, it is necessary to develop a technology capable of accurately detecting the toxin from a target since it is not an accurate assay to announce the toxin-generating region.
본 발명자들은 종래 기술의 문제점을 해결하기 위하여, 오카다산을 신속하고 정확하게 검출할 수 있는 기술에 대해 연구하던 중, 오카다산에 특이적으로 결합하는 신규한 펩타이드를 발굴하고 상기 펩타이드가 오카다산만 특이적으로 검출할 수 있음을 확인하여 본 발명을 완성하였다.In order to solve the problems of the prior art, the inventors of the present invention discovered a novel peptide that specifically binds to Okadaic acid while studying a technology capable of quickly and accurately detecting Okadaic acid, and the peptide is specific to Okadaic acid. The present invention was completed by confirming that it could be detected by.
따라서 본 발명의 목적은 오카다산(Okadaic acid) 검출용 펩타이드를 제공하는 것이다.Accordingly, an object of the present invention is to provide a peptide for detecting okadaic acid.
본 발명의 다른 목적은 오카다산 검출용 조성물을 제공하는 것이다. Another object of the present invention is to provide a composition for detecting okadaic acid.
본 발명의 또 다른 목적은 오카다산 검출용 키트를 제공하는 것이다. Another object of the present invention is to provide a kit for detecting okadaic acid.
본 발명의 또 다른 목적은 오카다산 검출용 바이오칩을 제공하는 것이다. Another object of the present invention is to provide a biochip for detecting okadaic acid.
본 발명의 또 다른 목적은 오카다산 검출 방법을 제공하는 것이다. Another object of the present invention is to provide a method for detecting okadaic acid.
본 발명의 또 다른 목적은 오카다산에 특이적으로 결합하는 펩타이드의 스크리닝 방법을 제공하는 것이다. Another object of the present invention is to provide a method for screening peptides that specifically bind to okadaic acid.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1 및 서열번호 2로 이루어진 군에서 선택된 어느 하나로 표시되는 아미노산 서열을 포함하는 오카다산(Okadaic acid) 검출용 펩타이드를 제공한다.In order to achieve the above object, the present invention provides a peptide for detecting okadaic acid comprising an amino acid sequence represented by any one selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
또한 상기 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 펩타이드를 포함하는 오카다산 검출용 조성물을 제공한다.In addition, in order to achieve the above other object, the present invention provides a composition for detecting okadaic acid comprising the peptide according to the present invention.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 펩타이드를 포함하는 오카다산 검출용 키트를 제공한다.In addition, in order to achieve the above another object, the present invention provides a kit for detecting okadaic acid comprising the peptide according to the present invention.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 펩타이드를 포함하는 오카다산 검출용 바이오칩을 제공한다.In addition, in order to achieve the above another object, the present invention provides a biochip for detecting okadaic acid comprising the peptide according to the present invention.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 펩타이드를 시료에 처리하는 단계; 및 상기 펩타이드와 시료의 반응을 검출하는 단계;를 포함하는, 오카다산 검출 방법을 제공한다.In addition, in order to achieve the above another object, the present invention comprises the steps of treating a sample with the peptide according to the present invention; And detecting the reaction between the peptide and the sample; it provides a method for detecting okadaic acid, including.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 오카다산과 운반체 단백질이 결합한 복합체를 제조하는 단계; 및 상기 복합체에 특이적으로 결합하는 펩타이드를 선별하는 단계;를 포함하는 오카다산에 특이적으로 결합하는 펩타이드의 스크리닝 방법을 제공한다.In addition, in order to achieve the above another object, the present invention comprises the steps of preparing a complex in which okadaic acid and carrier protein are bound; and selecting a peptide that specifically binds to the complex.
본 발명에 따른 신규한 오카다산 검출용 펩타이드는 오카다산에 대해 높은 정확도 및 민감도를 보여 오카다산을 정확하게 검출할 수 있는 바, 이를 이용하여 다양한 대상, 예를 들어 식품 및 이의 원재료의 오카다산 보유 여부를 확인할 수 있어, 안전한 식품 공급에 기여할 수 있다.The novel peptide for detecting okadaic acid according to the present invention shows high accuracy and sensitivity to okadaic acid and can accurately detect okadaic acid. can be identified, contributing to safe food supply.
도 1은 오카다산과 운반체 단백질인 소 혈청 알부민 (Bovine serum albumin, BSA)의 결합 여부를 SDS-PAGE로 확인한 결과이다.
도 2는 오카다산과 운반체 단백질인 소 혈청 알부민 (Bovine serum albumin, BSA)의 결합 여부를 MALDI-TOF 분석으로 확인한 결과이다.
도 3은 본 발명에 따라 선별된 박테리오파지 후보군의 오카다산에 대한 결합력을 확인한 결과이다.
도 4는 본 발명에 따라 선별된 펩타이드를 포함하는 박테리오파지 후보군의 오카다산에 대한 선택성을 확인한 결과이다.
도 5는 본 발명에 따라 선별된 펩타이드를 포함하는 박테리오파지 후보군의 파지 입자 농도에 따른 오카다산에 대한 결합력을 확인한 결과이다.
도 6은 본 발명에 따라 선별된 펩타이드를 포함하는 박테리오파지 후보군의 오카다산 농도에 따른 결합력을 확인한 결과이다.1 is a result of confirming the binding between Okadaic acid and bovine serum albumin (BSA), which is a carrier protein, by SDS-PAGE.
FIG. 2 is a result of confirming the binding between Okadaic acid and bovine serum albumin (BSA), which is a carrier protein, by MALDI-TOF analysis.
Figure 3 is a result of confirming the binding ability to Okadaic acid of the bacteriophage candidates selected according to the present invention.
Figure 4 is the result of confirming the selectivity for okadaic acid of the bacteriophage candidate group containing the selected peptide according to the present invention.
5 is a result of confirming the binding ability to okadaic acid according to the phage particle concentration of the bacteriophage candidate group including the peptide selected according to the present invention.
Figure 6 is a result of confirming the binding force according to the concentration of okadaic acid of the bacteriophage candidate group containing the peptide selected according to the present invention.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 오카다산을 특이적으로 검출할 수 있는 펩타이드에 대한 것으로, 하기 서열번호 1 및 서열번호 2로 이루어진 군에서 선택된 어느 하나로 표시되는 아미노산 서열을 포함하는 오카다산(Okadaic acid) 검출용 펩타이드를 제공한다.The present invention relates to a peptide capable of specifically detecting okadaic acid, and a peptide for detecting okadaic acid comprising an amino acid sequence represented by any one selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 below to provide.
HDKLGWLHWRHY(서열번호 1).HDKLGWLHWRHY (SEQ ID NO: 1).
WPWAHVHRGMSF(서열번호 2).WPWAHVHRGMSF (SEQ ID NO: 2).
상기 서열번호 1 및 서열번호 2로 이루어진 군에서 선택된 어느 하나로 표시되는, 본 발명에 따른 펩타이드의 아미노산 서열은 박테리오파지 무작위 펩타이드 라이브러리(phage display peptide library) Ph.D.-C7C(New England BioLab) 또는 Ph.D.-12(New England BioLab)를 이용하여 오카다산에 결합하는 서열을 찾아낸 것이다. 상기 Ph.D.-C7C 또는 Ph.D.-12는 각각 M13 박테리오파지의 게놈중에서 외피단백질(coat protein)의 일종인 pⅢ를 생산하는 유전자 말단에 7개의 무작위 아미노산이 서열 말단에 시스테인(cysteine) 잔기 간 이황화 결합(disulfide bond)에 의해 고리 모형을 갖는 펩타이드 또는, 12개의 무작위 아미노산 서열을 갖는 선형의 펩타이드가 발현되도록 인위적으로 유전자 서열을 삽입한 후, 대장균 (E.coli)에 감염시켜 얻은 수억 이상의 서로 다른 펩타이드를 발현한 재조합 M13 박테리오파지로 구성되어 있다.The amino acid sequence of the peptide according to the present invention, represented by any one selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, is a bacteriophage random peptide library (phage display peptide library) Ph.D.-C7C (New England BioLab) or Ph. A sequence binding to okadaic acid was found using .D.-12 (New England BioLab). The Ph.D.-C7C or Ph.D.-12 has 7 random amino acids at the end of a gene producing pIII, a kind of coat protein in the genome of M13 bacteriophage, respectively, and a cysteine residue at the end of the sequence. After artificially inserting a gene sequence so that a peptide having a ring model by a liver disulfide bond or a linear peptide having a random amino acid sequence of 12 is expressed, more than hundreds of millions obtained by infecting E.coli It consists of recombinant M13 bacteriophages expressing different peptides.
본 발명에서 사용된 상기 M13 박테리아파지는 특정 형질(strain)의 대장균만 감염되는 성질을 지니는 나노크기의 물질이며 돌연변이를 일으켜 인체에 감염될 가능성에 대해서는 현재까지 전혀 보고된 바 없다. 특히 2006년 FDA의 승인을 얻어 인스턴트식품의 세균 오염 방지용 첨가물로 활용되고 있으며, 항생제의 내성 문제는 해결할 수 있는 대체제뿐만 아니라 인체에 무해한 생체 진화형 나노물질로 각광을 받고 있다. 더불어 상기 박테리아파지를 이용하는 경우, 제조 공정이 간단하며 생산성과 안정성의 향상을 기대할 수 있고, 다양한 유전공학적, 화학적 변형을 활용할 수 있는 장점이 있다.The M13 bacteriophage used in the present invention is a nano-sized material having a property of infecting only a specific strain of E. coli, and there has been no report on the possibility of infecting humans by mutating. In particular, it has been approved by the FDA in 2006 and is used as an additive to prevent bacterial contamination of instant food, and is in the limelight as a bio-evolving nanomaterial that is harmless to the human body as well as an alternative that can solve the problem of antibiotic resistance. In addition, when using the bacteriophage, the manufacturing process is simple, productivity and stability can be expected to be improved, and various genetic engineering and chemical modifications can be utilized.
본 발명에 따른 펩타이드는 저분자 펩타이드로서 그 크기가 작아서 3차원적으로 안정화되어 있고 점막을 쉽게 통과하며 조직 깊은 곳에서도 분자 목표물을 인지할 수 있는 장점이 있다. 또한 본 발명에 따른 저분자 펩타이드는 대량 생산이 항체에 비해 상대적으로 간단하고, 독성이 거의 없다. 이외에도 본 발명에 따른 저분자 펩타이드는 표적 물질에 대한 결합력이 높은 장점이 있으며, 열/화학 처리시에도 변성이 일어나지 않는다. 또한 분자 크기가 작기 때문에 다른 단백질에 붙여서 융합 단백질로의 이용이 가능하다.The peptide according to the present invention is a low-molecular-weight peptide, and has the advantage of being three-dimensionally stable due to its small size, easily passing through mucous membranes, and being able to recognize molecular targets even in deep tissues. In addition, the small-molecular-weight peptide according to the present invention is relatively simple to mass-produce compared to antibodies, and has little toxicity. In addition, the low-molecular-weight peptide according to the present invention has the advantage of high binding force to the target substance, and does not undergo denaturation even during thermal/chemical treatment. In addition, because of its small molecular size, it can be attached to other proteins and used as a fusion protein.
본 발명에 따른 펩타이드는 당업계에 공지된 화학적 합성에 의해 제조될 수 있다. 대표적인 방법으로는 액체 또는 고체상 합성, 단편 응축, F-MOC(9-fluorenylmethoxycarbonyl) 또는 T-BOC(tert-butyloxycarbonyl) 화학법이 포함되나, 반드시 이에 제한되는 것은 아니다. 또한 본 발명의 펩타이드는 유전공학적 방법에 의해 제조될 수 있다. 우선, 통상적인 방법에 따라 상기 펩타이드를 코딩하는 DNA 서열을 작제한다. DNA 서열은 적절한 프라이머를 사용하여 PCR 증폭함으로써 작제할 수 있다. 다른 방법으로 당업계에 공지된 표준 방법에 의해, 예컨대, 자동 DNA 합성기(예: Biosearch 또는 Applied Biosystems사에서 판매하는 것)를 사용하여 DNA 서열을 합성할 수도 있다. 제작된 DNA 서열은 이 DNA 서열에 작동가능하게 연결되어(operatively linked) 그 DNA 서열의 발현을 조절하는 하나 또는 그 이상의 발현 조절 서열(expression control sequence)(예: 프로모터, 인핸서 등)을 포함하는 벡터에 삽입시키고, 이로부터 형성된 재조합 발현 벡터로 숙주세포를 형질전환시킨다. 생성된 형질전환체를 상기 DNA 서열이 발현되도록 적절한 배지 및 조건 하에서 배양하여, 배양물로부터 상기 DNA 서열에 의해 코딩된 실질적으로 순수한 펩타이드를 회수한다. 상기 회수는 이 기술분야에서 공지된 방법(예컨대, 크로마토그래피)을 이용하여 수행할 수 있다. 상기에서 '실질적으로 순수한 펩타이드'라 함은 본 발명에 따른 펩타이드가 숙주로부터 유래된 어떠한 다른 단백질도 실질적으로 포함하지 않는 것을 의미한다. 더불어 본 발명에 따른 펩타이드는 생체 내의 단백질 절단 효소들로부터 보호하고 안정성을 증가시키기 위해서 N 말단 또는 C 말단을 변형하거나 여러 유기단으로 보호한 형태일 수 있다. 즉, 상기 펩타이드의 C 말단은 안정성을 증가시키기 위해서 변형될 수 있는 형태라면, 특별한 제한은 없으나 바람직하게는 히드록시기(-OH) 또는 아미노기(-NH2)로 변형되는 것일 수 있다. 또한 상기 펩타이드의 N 말단은 안정성을 증가시키기 위해서 변형될 수 있는 형태라면, 특별한 제한은 없으나 바람직하게는 아세틸(Acetyl)기, 플루오레닐 메톡시카르보닐(Fmoc)기, 포르밀(Formyl)기, 팔미토일(Palmitoyl)기, 미리스틸(Myristyl)기, 스테아릴(Stearyl)기 및 폴리에틸렌글리콜(PEG)로 이루어진 군에서 선택되는 기로 변형되는 것일 수 있다.Peptides according to the present invention can be prepared by chemical synthesis known in the art. Representative methods include, but are not necessarily limited to, liquid or solid phase synthesis, fragment condensation, F-MOC (9-fluorenylmethoxycarbonyl) or T-BOC (tert-butyloxycarbonyl) chemistry. In addition, the peptides of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, such as using an automated DNA synthesizer (eg, sold by Biosearch or Applied Biosystems). The constructed DNA sequence is a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) operatively linked to the DNA sequence to control the expression of the DNA sequence. and the host cell is transformed with the recombinant expression vector formed therefrom. The resulting transformants are cultured under appropriate media and conditions to allow expression of the DNA sequence, and a substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery may be performed using a method known in the art (eg, chromatography). As used herein, 'substantially pure peptide' means that the peptide according to the present invention does not substantially contain any other proteins derived from the host. In addition, the peptide according to the present invention may be modified at the N-terminus or C-terminus or protected with various organic groups in order to protect from protein cleavage enzymes in vivo and increase stability. That is, the C terminus of the peptide is not particularly limited as long as it can be modified to increase stability, but may be preferably modified with a hydroxy group (-OH) or an amino group (-NH 2 ). In addition, if the N-terminus of the peptide is in a form that can be modified to increase stability, there is no particular limitation, but preferably an acetyl (Acetyl) group, a fluorenyl methoxycarbonyl (Fmoc) group, a formyl group , Palmitoyl group, myristyl group, stearyl group and may be modified with a group selected from the group consisting of polyethylene glycol (PEG).
또한 본 발명에 따른 펩타이드는 D형, L형, 펩토이드 모노머를 포함한 펩타이드 모방체 또는 비천연 아미노산 중의 어느 하나의 아미노산을 갖는 것을 특징으로 한다.In addition, the peptide according to the present invention is characterized in that it has any one amino acid among peptide mimics including D-type, L-type, and peptoid monomers, or non-natural amino acids.
또한 본 발명에 따른 펩타이드는 이중체(dimer), 삼중체(trimer) 또는 다중체(multimer)인 것을 특징으로 하며, N 말단 혹은 C 말단에 다양한 기능기를 포함할 수 있다.In addition, the peptide according to the present invention is characterized in that it is a dimer, a trimer, or a multimer, and may include various functional groups at the N-terminus or C-terminus.
상기 다양한 기능기의 예시로서, N 말단의 기능기는 자유아민(free amine), 아세틸화(acetylation), 바이오틴(biotin) 및 형광단(fluorophore)으로 이루어진 군에서 선택된 어느 하나일 수 있고, C 말단의 기능기는 유리산(free acid), 아미드화(amidation), 비오틴(biotin) 및 형광단(fluorophore)으로 이루어진 군에서 선택된 어느 하나일 수 있으나 이에 제한되는 것은 아니다. 더불어 본 발명에 따른 펩타이드는 검출 또는 동정을 위하여 표지될 수 있으며, 발색효소, 방사선 동위원소, 크로모포어(chromopore), 발광물질 및 형광물질로 이루어진 군에서 선택된 어느 하나로 표지될 수 있으나, 이에 제한되는 것은 아니다.As an example of the various functional groups, the N-terminal functional group may be any one selected from the group consisting of free amine, acetylation, biotin, and fluorophore, and the C-terminal The functional group may be any one selected from the group consisting of free acid, amidation, biotin, and fluorophore, but is not limited thereto. In addition, the peptide according to the present invention may be labeled for detection or identification, and may be labeled with any one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material, but is limited thereto it is not going to be
상기 발색효소는 예를 들어, 퍼옥시다제(peroxidase), 알칼라인 포스파타제(alkaline phosphatase)일 수 있고, 상기 방사성 동위원소는 예를 들어, 124I, 125I, 111In, 99mTc, 32P, 35S일 수 있으며, 상기 발광물질 또는 형광물질은 예를 들어, FITC, RITC, 로다민(rhodamine), 텍사스레드(Texas Red), 플로레신(fluorescein), 피코에리트린(phycoerythrin), 퀀텀닷(quantum dots) 등일 수 있다.The chromogenic enzyme may be, for example, peroxidase or alkaline phosphatase, and the radioactive isotope may be, for example, 124I, 125I, 111In, 99mTc, 32P, or 35S. The light emitting material or fluorescent material may be, for example, FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots, and the like.
유사하게, 상기 검출 가능한 표지는 항체 에피토프(epitope), 기질(substrate), 보조인자(cofactor), 저해제 또는 친화 리간드일 수 있다. 이러한 표지는 본 발명의 펩타이드를 합성하는 과정 중에 수행할 수도 있고, 이미 합성된 펩타이드에 추가로 수행될 수도 있다.Similarly, the detectable label may be an antibody epitope, substrate, cofactor, inhibitor or affinity ligand. This labeling may be performed during the process of synthesizing the peptide of the present invention, or may be additionally performed on already synthesized peptides.
만약 검출가능한 표지로 형광물질을 이용하는 경우에는 형광단층촬영(Fluorescence mediated tomography, FMT)으로 오카다산에 결합한 펩타이드의 형광 패턴을 관찰할 수 있고, 형광이 관찰되는 패턴에 따라, 오카다산을 검출할 수 있다.If a fluorescent substance is used as a detectable label, the fluorescence pattern of the peptide bound to okadaic acid can be observed by fluorescence mediated tomography (FMT), and okadaic acid can be detected according to the pattern in which fluorescence is observed there is.
본 발명의 오카다산 검출용 펩타이드 범위에 포함될 수 있는 생물학적 기능 균등물은 본 발명의 펩타이드와 균등한 생물학적 활성을 발휘하는 아미노산 서열의 변이를 포함하는데에 한정될 것이라는 것은 당업자에게 명확하다.It is clear to those skilled in the art that biological functional equivalents that can be included in the scope of the peptide for detecting okadaic acid of the present invention will be limited to those containing amino acid sequence mutations that exhibit the same biological activity as the peptide of the present invention.
이러한 아미노산 변이는 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. 아미노산 곁사슬 치환체의 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 라이신과 히스티딘은 모두 양전하를 띤 잔기이고; 알라닌, 글라이신과 세린은 유사한 크기를 가지며; 페닐알라닌, 트립토판과 타이로신은 유사한 모양을 갖는다는 것을 알 수 있다. 따라서 이러한 고려 사항에 기초하여, 아르기닌, 라이신과 히스티딘; 알라닌, 글라이신과 세린; 그리고 페닐알라닌, 트립토판과 타이로신은 생물학적으로 기능 균등물이라 할 수 있다.Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substituents revealed that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.
변이를 도입하는 데 있어서, 아미노산의 소수성 인덱스(hydropathic index)가 고려될 수 있다. 각각의 아미노산은 소수성과 전하에 따라 소수성 인덱스가 부여되어 있다: 아이소루이신 (+4.5); 발린 (+4.2); 루이신(+3.8); 페닐알라닌(+2.8); 시스테인/시스타인 (+2.5); 메티오닌 (+1.9); 알라닌 (+1.8); 글라이신 (-0.4); 트레오닌 (-0.7); 세린 (-0.8); 트립토판 (-0.9); 타이로신 (-1.3); 프롤린 (-1.6); 히스티딘 (-3.2); 글루타메이트 (-3.5); 글루타민 (-3.5); 아스파르테이트 (-3.5); 아스파라긴 (-3.5); 라이신 (-3.9); 및 아르기닌 (-4.5).In introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); Tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); Lysine (-3.9); and arginine (-4.5).
펩타이드의 상호적인 생물학적 기능(interactive biological function)을 부여하는 데 있어서 소수성 아미노산 인덱스는 매우 중요하다. 유사한 소수성 인덱스를 가지는 아미노산으로 치환하여야 유사한 생물학적 활성을 보유할 수 있다는 것은 공지된 사실이다. 소수성 인덱스를 참조하여 변이를 도입시키는 경우, 바람직하게는 ±2 이내, 보다 바람직하게는 ±1 이내, 보다 더 바람직하게는 ±0.5 이내의 소수성 인덱스 차이를 나타내는 아미노산 사이에 치환을 한다.The hydrophobic amino acid index is very important in conferring the interactive biological function of peptides. It is a known fact that amino acids having similar hydrophobicity indexes should be substituted to retain similar biological activities. When a mutation is introduced with reference to the hydrophobicity index, substitution is made between amino acids exhibiting a difference in hydrophobicity index, preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.
한편, 유사한 친수성 값(hydrophilicity value)을 가지는 아미노산 사이의 치환이 균등한 생물학적 활성을 갖는 펩타이드를 초래한다는 것도 잘 알려져 있다. 미국 특허 제4,554,101호에 개시된 바와 같이, 다음의 친수성 값이 각각의 아미노산 잔기에 부여되어 있다: 아르기닌 (+3.0); 라이신 (+3.0); 아스팔테이트(+3.0±1); 글루타메이트 (+3.0±1); 세린 (+0.3); 아스파라긴 (+0.2); 글루타민 (+0.2); 글라이신 (0); 쓰레오닌 (-0.4); 프롤린 (-0.5±1); 알라닌 (-0.5); 히스티딘 (-0.5); 시스테인 (-1.0); 메티오닌 (-1.3); 발린 (-1.5); 루이신 (-1.8); 아이소루이신 (-1.8); 타이로신 (-2.3); 페닐알라닌 (-2.5); 트립토판 (-3.4). On the other hand, it is also well known that substitution between amino acids having similar hydrophilicity values results in peptides having equivalent biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); Asphaltate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); Alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4).
친수성 값을 참조하여 변이를 도입시키는 경우, 바람직하게는 ±2 이내, 보다 바람직하게는 ±1 이내, 보다 더 바람직하게는 ±0.5 이내의 친수성 값 차이를 나타내는 아미노산 사이에 치환을 한다.When a mutation is introduced by referring to the hydrophilicity value, substitution is made between amino acids showing a difference in hydrophilicity value, preferably within ±2, more preferably within ±1, even more preferably within ±0.5.
분자의 활성을 전체적으로 변경시키지 않는 펩타이드에서의 아미노산 교환은 당해 분야에 공지되어 있다(H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly 간의 교환이다.Amino acid exchanges in peptides that do not entirely alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.
상술한 생물학적 균등 활성을 갖는 변이를 고려한다면, 본 발명의 오카다산 검출용 펩타이드는 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 본 발명의 펩타이드 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 80% 이상의 상동성, 보다 바람직하게는 90% 이상의 상동성을 나타내는 서열을 의미한다. 서열 비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있는 어떤 방법이라도 제한없이 사용할 수 있다. Considering the mutation having the above-described biological equivalent activity, the peptide for detecting okadaic acid of the present invention is interpreted to include a sequence showing substantial identity with the sequence described in the sequence listing. The above substantial identity is at least 80% or more when the peptide sequence of the present invention and any other sequence are aligned so as to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art. It means a sequence showing homology, more preferably 90% or more homology. As an alignment method for sequence comparison, any method known in the art may be used without limitation.
더불어 본 발명은 본 발명에 따른 펩타이드를 포함하는 오카다산 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting okadaic acid comprising the peptide according to the present invention.
또한 본 발명은 본 발명에 따른 펩타이드를 포함하는 오카다산 검출용 키트를 제공한다.In addition, the present invention provides a kit for detecting okadaic acid comprising the peptide according to the present invention.
본 발명의 오카다산 검출용 키트에는 오카다산 검출을 위한 펩타이드의 분석 방법에 적합한 하나 또는 그 이상의 다른 구성 성분 조성물 또는 장치가 포함될 수 있고, 펩타이드의 구조 또는 생리 활성을 안정하게 유지시키는 완충액 또는 반응액이 추가로 포함될 수 있다. 또한, 안정성을 유지하기 위해, 4℃로 유지된 상태로 제공될 수 있다. The kit for detecting okadaic acid of the present invention may include one or more other component compositions or devices suitable for the peptide analysis method for detecting okadaic acid, and a buffer or reaction solution that stably maintains the structure or physiological activity of the peptide This may additionally be included. In addition, in order to maintain stability, it may be provided in a state maintained at 4 ° C.
오카다산에 결합한 본 발명에 따른 펩타이드의 확인, 검출 및 정량을 용이하게 하기 위하여, 본 발명의 키트에 포함되는 펩타이드는 상술한 바와 같이 표지된 상태로 제공될 수 있다. 한편, 본 발명의 펩타이드가 표지되지 않은 채로 제공되는 경우에는, 본 발명의 키트는 본 발명의 펩타이드의 시험관 내 또는 생체 내에서 오카다산과의 결합 정도 또는 그 위치를 탐색하기 위한 성분을 추가로 포함할 수 있다. 상기 성분은 본 발명의 펩타이드의 표지를 위한 공지의 화합물이거나, 항원-항체반응을 통한 탐색을 위하여 본 발명의 펩타이드 또는 본 발명의 펩타이드와 결합하는 특정 수용체에 대한 항체 또는 이들에 대한 2차 항체 및 이의 검출을 위한 시약일 수 있다. 일 구체예로서 본 발명의 키트는 다양한 대상, 예를 들어 농산물 또는 수산물의 오카다산 보유 여부를 확인하기 위하여 사용될 수 있고, 오카다산을 보유한 물질의 섭취 여부를 확인하기 위하여 사용될 수 있다. 상기 확인은 종래의 면역분석(immunoassay) 방식 또는 프로토콜을 변형하여 실시될 수 있다. 예컨대, 종래 면역분석에서 항체 대신에, 본 발명의 펩타이드를 사용하고, 프로세스는 동일하게 하여 진단을 실시할 수 있다.In order to facilitate identification, detection and quantification of the peptides according to the present invention bound to okadaic acid, the peptides included in the kit of the present invention may be provided in a labeled state as described above. On the other hand, when the peptide of the present invention is provided unlabeled, the kit of the present invention may further include a component for examining the degree or position of the peptide of the present invention bound to okadaic acid in vitro or in vivo. can The component is a known compound for labeling the peptide of the present invention, or an antibody against a specific receptor binding to the peptide of the present invention or a peptide of the present invention for a search through an antigen-antibody reaction, or a secondary antibody against them, and It may be a reagent for its detection. As one embodiment, the kit of the present invention can be used to check whether various objects, for example, agricultural products or marine products contain okadaic acid, and can be used to check whether substances containing okadaic acid are ingested. The confirmation may be performed by modifying a conventional immunoassay method or protocol. For example, diagnosis can be performed by using the peptide of the present invention instead of an antibody in a conventional immunoassay and using the same process.
더불어 본 발명은 본 발명에 따른 펩타이드를 포함하는 오카다산 검출용 바이오칩을 제공한다.In addition, the present invention provides a biochip for detecting okadaic acid containing the peptide according to the present invention.
본 발명에 따른 펩타이드는 바이오칩으로 사용 가능한 다양한 칩 (금(gold), 은(silver), 자성 비드(magnetic bead), 실리카(silica), 그래핀(graphene), 탄소 나노튜브(carbon nanotube) 등) 상에 특이적으로 고정될 수 있다. 다만, 본 발명의 특징은 전술한 펩타이드의 특징과 관련되며 이와 같이 펩타이드를 바이오칩으로 제조하는 것은 공지기술에 따라 할 수 있는 부분이다. The peptide according to the present invention can be used as a biochip (gold, silver, magnetic bead, silica, graphene, carbon nanotube, etc.) It can be specifically immobilized on top. However, the characteristics of the present invention are related to the characteristics of the above-described peptides, and manufacturing of the peptides into a biochip can be performed according to known technologies.
본 발명에 따른 오카다산 검출용 바이오칩을 이용하여 단백질과 단백질 간의 반응, 단백질과 반응리간드 간의 반응, 목적리간드와 반응단백질 간의 반응 또는 목적리간드와 반응리간드간의 반응을 검출할 수 있다.A reaction between a protein and a protein, a reaction between a protein and a reactive ligand, a reaction between a target ligand and a reactive protein, or a reaction between a target ligand and a reactive ligand can be detected by using the biochip for detecting okadaic acid according to the present invention.
상기 반응리간드는 단백질이나 펩타이드와 반응하는 저분자량의 생체물질, 펩티드, 탄수화물, 앱타머, 지방산 또는 지질일 수 있으나, 이에 제한되는 것은 아니다. 더불어 상기 반응단백질은 효소 또는 항체일 수 있으나, 이에 제한되는 것은 아니다. 또한 상기 목적리간드는 단백질이나 펩타이드와 반응하는 저분자량의 화합물, 핵산, 펩티드, 탄수화물, 앱타머, 지방산 또는 지질일 수 있으나, 이에 제한되는 것은 아니다. 더불어 상기 반응 검출은 표지 혹은 비표지체를 이용하여 수행할 수 있다.The reactive ligand may be a low molecular weight biomaterial, peptide, carbohydrate, aptamer, fatty acid or lipid that reacts with a protein or peptide, but is not limited thereto. In addition, the reactive protein may be an enzyme or an antibody, but is not limited thereto. In addition, the target ligand may be a low molecular weight compound, nucleic acid, peptide, carbohydrate, aptamer, fatty acid or lipid that reacts with a protein or peptide, but is not limited thereto. In addition, the detection of the reaction may be performed using a labeled or non-labeled material.
또한, 본 발명은 본 발명에 따른 펩타이드를 시료에 처리하는 단계; 및 상기 펩타이드와 시료의 반응을 검출하는 단계;를 포함하는, 오카다산 검출 방법을 제공한다.In addition, the present invention comprises the steps of treating the sample with the peptide according to the present invention; And detecting the reaction between the peptide and the sample; it provides a method for detecting okadaic acid, including.
이를 통해 시료 내 오카다산의 존재 유무를 검출하거나, 오카다산의 양을 정량할 수 있고, 오카다산을 보유한 물질의 섭취 여부를 확인할 수 있다.Through this, the presence or absence of okadaic acid in the sample can be detected, the amount of okadaic acid can be quantified, and whether or not a substance containing okadaic acid can be ingested can be confirmed.
본 발명에 있어서 상기 "시료"는 가장 광범위한 의미로 사용된다. 한편으로는 생물학적 및 환경적 시료 양자 모두를 포함하는 의미이다. 더불어 시료는 합성 기원의 검체를 포함할 수 있다. 또한 한편으로 이는 검체 또는 배양물(예를 들어 미생물 배양물)을 포함할 수 있다. 상기 시료는 농산물 또는 수산물 유래 시료일 수 있다. 상기 수산물 시료에 있어서, 바람직하게는 어패류일 수 있다. 더불어 상기 생물학적 시료는 본 발명에 따른 펩타이드를 이용하여 오카다산을 보유한 물질의 섭취 여부를 확인할 때 사용될 수 있으며, 전혈(whole blood), 혈장(plasma), 혈청(serum), 객담(sputum), 눈물(tears), 점액(mucus), 세비액(nasal washes), 비강 흡인물(nasal aspirate), 호흡(breath), 소변(urine), 정액(semen), 침(saliva), 복강 세척액(peritoneal washings), 복수(ascites), 낭종액(cystic fluid), 뇌척수막 액(meningeal fluid), 양수(amniotic fluid), 백혈구(leukocytes), 말초혈액 단핵 세포(peripheral blood mononuclear cells), 백혈구 연층(buffy coat), 선액(glandular fluid), 췌장액(pancreatic fluid), 림프액(lymph fluid), 흉수(pleural fluid), 유두 흡인물(nipple aspirate), 기관지 흡인물(bronchial aspirate), 활액(synovial fluid), 관절 흡인물(joint aspirate), 기관 분비물(organ secretions), 세포(cell), 세포 추출물(cell extract) 및 뇌척수액(cerebrospinal fluid)으로 이루어진 군에서 선택된 어느 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the "sample" is used in the broadest sense. On the one hand, it is meant to include both biological and environmental samples. In addition, samples may include specimens of synthetic origin. Alternatively, it may include samples or cultures (eg microbial cultures). The sample may be an agricultural or aquatic product-derived sample. In the aquatic product sample, it may be preferably fish and shellfish. In addition, the biological sample can be used to determine whether or not a substance containing okadaic acid is ingested using the peptide according to the present invention, and whole blood, plasma, serum, sputum, tears Tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings , ascites, cystic fluid, meningeal fluid, amniotic fluid, leukocytes, peripheral blood mononuclear cells, buffy coat, glandular fluid (glandular fluid), pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate aspirate), organ secretions, cells, cell extracts, and cerebrospinal fluid, but may be at least one selected from the group consisting of, but is not limited thereto.
본 발명의 방법에 있어서, 상기 반응의 검출은 순환 전압 전류법(Cyclic Voltammetry), 사각파전압전류법(Square Wave Voltammetry), 전기화학 임피던스 분광법(Electrochemical impedance spectroscopy), 수정 진동자 마이크로밸런스(quartz crystal microbalance), 표면 플라스몬 공명(Surface plasmon resonance), LFA(Lateral flow assay)/VFA(Variable flip angle), 효소 결합 면역 분석법(Enzyme linked immunoassay), 형광 공명 에너지 전달(Fluorescence resonance energy transfer) 및 표면증강 라만 분광법(Surface-enhanced Raman spectroscopy)으로 이루어진 군에서 선택된 어느 하나 이상의 방법으로 수행될 수 있으나, 이에 제한되는 것은 아니다.In the method of the present invention, the detection of the reaction is carried out by cyclic voltammetry, square wave voltammetry, electrochemical impedance spectroscopy, quartz crystal microbalance ), Surface plasmon resonance, Lateral flow assay (LFA)/Variable flip angle (VFA), Enzyme linked immunoassay, Fluorescence resonance energy transfer, and Surface-enhanced Raman It may be performed by one or more methods selected from the group consisting of surface-enhanced Raman spectroscopy, but is not limited thereto.
또한 본 발명은 오카다산에 특이적으로 결합하는 펩타이드의 스크리닝 방법을 제공한다.In addition, the present invention provides a screening method for a peptide that specifically binds to okadaic acid.
보다 구체적으로, 상기 스크리닝 방법은 오카다산과 운반체 단백질이 결합한 복합체를 제조하는 단계; 및 상기 복합체에 특이적으로 결합하는 펩타이드를 선별하는 단계;를 포함할 수 있다.More specifically, the screening method comprises preparing a complex in which okadaic acid and a carrier protein are bound; and selecting a peptide that specifically binds to the complex.
본 발명의 스크리닝 방법에 있어서, 상기 복합체는 저분자 물질인 오카다산의 파지 디스플레이에 대한 효율성을 증대시키기 위한 것으로, 상기 운반체 단백질은 알부민(albumin) 또는 오발부민(ovalbumin)일 수 있다.In the screening method of the present invention, the complex is used to increase the efficiency of phage display of okadaic acid, which is a low-molecular substance, and the carrier protein may be albumin or ovalbumin.
더불어 본 발명의 스크리닝 방법에 있어서, 상기 복합체에 특이적으로 결합하는 펩타이드는 서열번호 1 및 서열번호 2로 이루어진 군에서 선택된 어느 하나로 표시되는 아미노산 서열을 포함할 수 있다.In addition, in the screening method of the present invention, the peptide specifically binding to the complex may include an amino acid sequence represented by any one selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
상술한 본 발명의 내용은 상호 모순되지 않는 한, 서로 동일하게 적용되며, 당해 기술분야의 통상의 기술자가 적절한 변경을 가해 실시하는 것 또한 본 발명의 범주에 포함된다.The contents of the present invention described above are equally applied to each other unless contradictory to each other, and implementation by adding appropriate changes by a person skilled in the art is also included in the scope of the present invention.
이하 본 발명을 실시예를 통해 상세하게 설명하나 본 발명의 범위가 하기 실시예로만 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail through examples, but the scope of the present invention is not limited only to the following examples.
실시예 1. 오카다산의 합텐(hapten) 및 오카다산 운반체 형성Example 1. Formation of Okadaic acid hapten and Okadaic acid transporter
오카다산은 저분자 물질이기 때문에 바로 플레이트에 고정화하여 파지 디스플레이를 수행한다면 효율성이 저하된다. 따라서 펩타이드의 효율적인 발굴을 위하여 오카다산을 운반체 단백질에 고정화시켰다. 이를 위하여 운반체 단백질로서 카르복실산(carboxylic acid)과 아민 작용기를 가진 알부민(albumin)을 이용하였다. Since okadaic acid is a low-molecular substance, efficiency is reduced if phage display is performed by immobilizing it directly on a plate. Therefore, for efficient discovery of peptides, okadaic acid was immobilized on the carrier protein. To this end, albumin having a carboxylic acid and an amine functional group was used as a carrier protein.
이를 위하여 소 혈청 알부민 (Bovine serum albumin, BSA), 인산완충 생리식염수(phosphate-buffered saline, PBS, pH 7.4), N,N’디싸이클로헥실카르보디이미드(N,N′-dicyclohexylcarbodiimide, DCC), N-히드록시 석신이미드(N-hydroxy succinimide, NHS), 쿠마시 브릴리언트 블루 R(Coomassie Brilliant Blue R)은 Sigma-Aldrich (St. Louis, MO, USA)에서 구매하였다. 또한 메탄올은 Merck (Darmstadt, Germany)에서 구매하였으며, 빙초산(Glacial acetic acid)은 덕산(안산, 한국)에서 구매하였다. 오카다산(Okadaic acid, OA)은 Fermentek (Jerusalem, Israel)에서 구매하였고, N,N-디메틸포름아마이드(N,N-dimethylformamide, DMF)는 Alfa Aesar(Ward Hill, MA, USA)에서 구매하였다. 모든 화합물질 및 용액들은 탈이온수(deionized (DI) water)를 이용하여 준비하였다. For this purpose, bovine serum albumin (BSA), phosphate-buffered saline (PBS, pH 7.4), N,N'-dicyclohexylcarbodiimide (DCC), N-hydroxy succinimide (NHS) and Coomassie Brilliant Blue R were purchased from Sigma-Aldrich (St. Louis, MO, USA). In addition, methanol was purchased from Merck (Darmstadt, Germany), and glacial acetic acid was purchased from Deoksan (Ansan, Korea). Okadaic acid (OA) was purchased from Fermentek (Jerusalem, Israel), and N,N-dimethylformamide (DMF) was purchased from Alfa Aesar (Ward Hill, MA, USA). All compounds and solutions were prepared using deionized (DI) water.
먼저 오카다산(0.1000mg, 0.124μmol)을 20μL의 DMF에 용해하고, 이에 NHS(0.0157mg, 0.137μmol)을 첨가하였다. 이를 10초간 혼합한 뒤 5분간 상온에서 반응시켰다. 이후 DCC(0.0282mg, 0.137μmol)을 첨가한 뒤 상온에서 3일간 반응시켰다. BSA(0.1836mg, 0.003 μmol)는 PBS(10mM, pH 7.4)에 용해시켰다. DCC 및 NHS로 활성화한 합텐을 BSA 용액에 투여한 뒤 상온에서 1일간 반응시켰다. 디사이클로헥실유레아(dicyclohexylurea) 및 미반응 물질을 제거하기 위해 아미콘 튜브(Amicon tube (3K, 1.5mL))로 여과하였다. 여과 후 수득한 오카다산-BSA는 SDS-PAGE(sodium dodecyl sulphate-polyacrylamide gel electrophoresis) 분석 및 MALDI-TOF(Matrix-assisted laser desorption/ionization) 분석으로 확인하였다. SDS-PAGE 분석을 위해 탈이온수를 사용하여 BSA 및 획득한 오카다산-BSA의 농도를 각각 100 μg/mL로 희석하였다. 상기 용액 5.5 μL와 ELPIS-BIOTECH(대전, 한국)사의 샘플 버퍼(Sample Buffer (Laemmli's buffer 5x)) 2.2 μL, 탈이온수 3.3 μL을 완전히 혼합한 후 100 ℃에서 5분간 가열해 전처리를 했다. 각 10 μL의 전처리된 BSA 및 오카다산-BSA 시료를 12 % SDS-PAGE 겔에서 전기영동을 통해 분리한 뒤, 단백질 착색 용액(0.1 % 쿠마시 브릴리언트 블루 R, 50 % 메탄올, 10 % 빙초산)을 사용해 1시간 동안 염색한 후 탈색 용액(40 % 메탄올, 10 % 빙초산)을 사용하여 시각화하였다. First, okadaic acid (0.1000 mg, 0.124 μmol) was dissolved in 20 μL of DMF, to which NHS (0.0157 mg, 0.137 μmol) was added. After mixing this for 10 seconds, it was reacted at room temperature for 5 minutes. Then DCC (0.0282mg, 0.137μmol) was added and reacted at room temperature for 3 days. BSA (0.1836 mg, 0.003 μmol) was dissolved in PBS (10 mM, pH 7.4). Haptens activated by DCC and NHS were administered to the BSA solution and reacted at room temperature for 1 day. It was filtered through an Amicon tube (3K, 1.5mL) to remove dicyclohexylurea and unreacted substances. Okadaic acid-BSA obtained after filtration was confirmed by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) analysis and MALDI-TOF (matrix-assisted laser desorption/ionization) analysis. For SDS-PAGE analysis, the concentrations of BSA and okadaic acid-BSA obtained were each diluted to 100 μg/mL using deionized water. 5.5 μL of the above solution, 2.2 μL of Sample Buffer (Laemmli's buffer 5x) from ELPIS-BIOTECH (Daejeon, Korea), and 3.3 μL of deionized water were thoroughly mixed, and pre-treatment was performed by heating at 100 ° C. for 5 minutes. Each 10 μL of pretreated BSA and Okadaic acid-BSA samples was separated by electrophoresis on a 12% SDS-PAGE gel, followed by protein staining solution (0.1% Coomassie Brilliant Blue R, 50% methanol, 10% glacial acetic acid). After staining for 1 hour, it was visualized using a decolorizing solution (40% methanol, 10% glacial acetic acid).
상기 MALDI-TOF 질량 스펙트럼은 MALDI-TOF voyager DE-STR (Applied Biosystems)에 기록하였다.The MALDI-TOF mass spectrum was recorded on a MALDI-TOF voyager DE-STR (Applied Biosystems).
그 결과 도 1에 나타낸 바와 같이, SDS-PAGE상 오카다산을 결합하기 전 BSA의 밴드(66.4kDa)보다 더 높은 위치(합텐이라 기재된 라인(Line))에서 밴드가 나타남을 확인하였다(최고점은 73.1kDa). 오카다산의 MW는 805.00Da이기 때문에, BSA에 결합된 합텐의 수는 약 8.4개인 것으로 추측되었다. 더불어 도 2와 같이, MALDI-TOF 분석 결과 또한 BSA 피크가 오카다산과 결합하여 합텐 형성 후 70000 (m/z) 부근으로 이동한 것을 통해 오카다산의 합텐화가 성공적으로 이루어졌음을 확인하였다.As a result, as shown in Figure 1, it was confirmed that a band appeared at a higher position (line described as hapten) than the BSA band (66.4 kDa) before binding okadaic acid on SDS-PAGE (the highest point was 73.1). kDa). Since the MW of Okadaic acid is 805.00 Da, the number of haptens bound to BSA was estimated to be about 8.4. In addition, as shown in FIG. 2, as a result of MALDI-TOF analysis, it was confirmed that haptenization of okadaic acid was successfully achieved, as the BSA peak moved to around 70000 (m/z) after combining with okadaic acid to form a hapten.
실시예 2. 오카다산에 특이적으로 결합하는 펩타이드의 발굴Example 2. Discovery of peptides specifically binding to okadaic acid
상기 실시예 1에서 제조한 오카다산 운반체(오카다산-BSA 복합체라 기재)와 박테리오파지 무작위 펩타이드 라이브러리(phage display peptide library)를 이용하여 오카다산에 특이적으로 결합하는 펩타이드를 발굴하였다. Peptides specifically binding to okadaic acid were discovered using the okadaic acid transporter prepared in Example 1 (described as an okadaic acid-BSA complex) and a bacteriophage random peptide library (phage display peptide library).
상기 박테리오파지 무작위 펩타이드 라이브러리(phage display peptide library)는 Ph.D.-C7C 및 Ph.D.-12(New England BioLab)를 사용했다. 이들은 각각 M13 박테리오파지의 게놈중에서 외피단백질(coat protein)의 일종인 pⅢ를 생산하는 유전자 말단에 7개의 무작위 아미노산이 서열 말단에 시스테인(cysteine) 잔기 간 이황화 결합(disulfide bond)에 의해 고리 모형을 갖는 펩타이드 또는, 12개의 무작위 아미노산 서열을 갖는 선형의 펩타이드가 발현되도록 인위적으로 유전자 서열을 삽입한 후, 대장균 (E.coli)에 감염시켜 얻은 수억 이상의 서로 다른 펩타이드를 발현한 재조합 M13 박테리오파지로 구성되어 있다.Ph.D.-C7C and Ph.D.-12 (New England BioLab) were used as the bacteriophage random peptide library (phage display peptide library). These are peptides having a ring model by disulfide bonds between cysteine residues at the end of a sequence of seven random amino acids at the end of a gene that produces pIII, a kind of coat protein in the genome of M13 bacteriophage. Alternatively, after artificially inserting a gene sequence so that a linear peptide having 12 random amino acid sequences is expressed, it is composed of recombinant M13 bacteriophage expressing hundreds of millions of different peptides obtained by infecting E.coli .
바이오패닝(biopanning)은 96 마이트로웰 폴리스티렌(polystyrene) 플레이트 (96-well polystyrene plate)를 이용하여 수행하였다(Corning (NY, USA)사에서 구입). 하기 실시예 1에서 제조한 오카다산-BSA를 NaHCO3(pH 9.4)에 희석하고 폴리스티렌 플레이트에 64 μg/mL(1 μmol)의 오카다산-BSA 100 μL를 넣고 밀봉한 후 4℃에서 밤새 반응시켰다. NaHCO3(pH 9.4) 10 mL에 BSA 0.5 mg을 넣어 블로킹(blocking) 용액을 제조하였고, 배양이 완료된 용액을 제거한 후 블로킹 용액 200 μL을 넣고 밀봉한 후, 4℃에서 1시간 동안 반응시켰다. 세척 용액으로 PBST(0.1 M PBS with Tween 0.1%, pH 7.4)를 제조하였고, 세척 용액 200 μL로 반응이 완료된 플레이트를 6회 반복 세척하였다. 무작위 서열의 펩타이드 리셉터가 디스플레이(display)된 M13 박테리오파지를 PBS(0.1 M, pH 7.4)에 1011 PFU/mL 농도로 희석한 후 세척이 끝난 플레이트에 100 μL 넣고 150 rpm으로 교반하면서 1시간 동안 반응시켰다. 반응이 끝난 후 세척 용액 200 μL로 10회 세척하였다. 이후 100 μL의 글리신(Glycine)/HCl(0.2 M, pH 2.2)을 넣고 150 rpm으로 교반하면서 10분 동안 배양하여 오카다산-BSA와 결합한 M13 박테리오파지를 용해시켰다. 배양이 끝난 후 플레이트의 상층액(용출된 M13 박테리오파지)을 마이크로튜브로 옮기고 M13 박테리오파지의 변성을 막기 위해 Tris-HCl(1 M, pH 9.1) 15 μL를 넣어 중화하였다. 용출된 M13 박테리오파지 용액은 4℃에서 보관하였다. 오카다산에 특이적인 M13 박테리오파지를 얻기 위해서 회차(round) 진행에 따라 세척 용액의 트윈-20(Tween-20) 농도를 0.1, 0.3, 0.5% 순으로 증가시켜 주었다. 또한 운반체 단백질인 BSA에 결합하는 펩타이드 리셉터 서열을 제거하기 위하여 2, 3, 4 회차에서 바이오패닝을 시작하기 전 M13 박테리오파지 라이브러리를 BSA 단백질과 반응시켜 음성 선택(Negative selection)을 수행하였다. Biopanning was performed using a 96-well polystyrene plate (purchased from Corning (NY, USA)). Okadaic acid-BSA prepared in Example 1 was diluted in NaHCO 3 (pH 9.4), and 100 μL of 64 μg/mL (1 μmol) of okadaic acid-BSA was added to a polystyrene plate, sealed, and reacted overnight at 4° C. . A blocking solution was prepared by adding 0.5 mg of BSA to 10 mL of NaHCO 3 (pH 9.4), and after removing the cultured solution, 200 μL of the blocking solution was added, sealed, and reacted at 4° C. for 1 hour. PBST (0.1 M PBS with Tween 0.1%, pH 7.4) was prepared as a washing solution, and the reaction-completed plate was repeatedly washed 6 times with 200 μL of the washing solution. After diluting the M13 bacteriophage displaying the peptide receptors of random sequence to a concentration of 10 11 PFU/mL in PBS (0.1 M, pH 7.4), 100 μL was added to the washed plate and reacted for 1 hour while stirring at 150 rpm. made it After the reaction was finished, it was washed 10 times with 200 μL of the washing solution. Thereafter, 100 μL of glycine/HCl (0.2 M, pH 2.2) was added and incubated for 10 minutes while stirring at 150 rpm to dissolve M13 bacteriophages bound to Okadasan-BSA. After the incubation, the supernatant (eluted M13 bacteriophage) from the plate was transferred to a microtube and neutralized by adding 15 μL of Tris-HCl (1 M, pH 9.1) to prevent denaturation of M13 bacteriophage. The eluted M13 bacteriophage solution was stored at 4°C. In order to obtain Okadasan-specific M13 bacteriophage, the concentration of Tween-20 in the washing solution was increased in the order of 0.1, 0.3, and 0.5% according to the rounds. In addition, in order to remove the peptide receptor sequence binding to the carrier protein BSA, negative selection was performed by reacting the M13 bacteriophage library with the BSA protein before starting biopanning in
상기의 과정을 Ph.D.-12와 Ph.D.-C7C 두 종류의 라이브러리에서 각각 4회씩 반복하였으며 각 회차에서 수득한 M13 박테리오파지의 농도를 하기 표 1에 기재하였다. 더불어 DNA 서열 분석을 통해 반복적으로 나타나는 펩타이드 리셉터 서열을 포함한 M13 박테리오파지를 선별해 하기 표 2에 나타내었다.The above process was repeated 4 times each in two types of libraries, Ph.D.-12 and Ph.D.-C7C, and the concentrations of M13 bacteriophage obtained in each round are shown in Table 1 below. In addition, through DNA sequence analysis, M13 bacteriophages containing repetitive peptide receptor sequences were selected and shown in Table 2 below.
(sample)(sample)
(Cyclic 7-mer)BSA-Okadasan
(Cyclic 7-mer)
(12-mer)BSA-Okadasan
(12-mer)
실시예 3. 박테리오파지 후보군의 오카다산에 대한 결합력 확인Example 3. Confirmation of binding ability to Okadaic acid of bacteriophage candidates
상기 실시예 1을 통해 선별된 5개의 박테리오파지 후보군의 오카다산에 대한 결합력을 ELISA(enzymed-linked immunosorbent assay)를 이용하여 확인하였다. The binding ability to Okadasan of the five bacteriophage candidates selected through Example 1 was confirmed using an enzyme-linked immunosorbent assay (ELISA).
박테리오파지 후보군의 농도를 동일하게 맞추고 실험에 이용하기 위해 박테리오파지 후보군의 증폭(Amplification) 과정을 수행하였다. 대장균주(ER 2738) 콜로니를 20 mL LB배지에 넣고 210 rpm으로 교반하면서 37℃에서 밤새 배양하였다. 상기 대장균 배양액을 흡광도(Optical density, OD)값이 0.03-0.05 사이가 되도록 LB배지에 희석하였다. 희석된 대장균 배양액 20 mL에 박테리오파지 후보군의 용출용액 10 μL를 접종하고 210 rpm으로 교반하면서 37℃에서 4시간 30분간 배양하였다. 박테리오파지 후보군 배양액을 12,000g 속도로 4℃에서 10분동안 원심분리한 후 상층액을 수득하였다. 박테리오파지 후보군을 회수하기 위해서 상층액에 20% PEG(polyethylen glycol)/2.5 mol NaCl 3.3 mL을 넣고 4℃에서 12시간 동안 보관하였다. 이 후 12,000g 속도로 4℃에서 15분 동안 원심분리하여 상층액을 제거하였다. 상층액을 제거한 후 남은 침전물(Pellet)을 PBS(0.1M, pH7.4) 1 mL에 녹인 후 4℃에서 14,000rpm 속도로 5분 동안 원심분리 하였다. 원심분리가 끝난 후 상층액을 수득해 이에 20% PEG/2.5 mol NaCl 166.7 μL을 넣고 1시간 동안 얼음에서 보관하였다. 이 후 4℃에서 14,000rpm 속도로 10분 동안 원심분리하고 상층액을 제거한 후 침전물을 PBS(0.1 M, pH7.4) 200 μL에 녹였다. 그 후 적정 (Titration)을 실시하고 하기 수학식1을 통해 파지의 농도(PFU/mL: Plaque forming units/mL)를 계산하였다.Amplification process of the bacteriophage candidate group was performed in order to set the same concentration of the bacteriophage candidate group and use it in the experiment. E. coli strain (ER 2738) colonies were put in 20 mL LB medium and cultured overnight at 37°C while stirring at 210 rpm. The E. coli culture was diluted in LB medium so that the optical density (OD) value was between 0.03 and 0.05. Inoculated with 10 μL of elution solution of bacteriophage candidates in 20 mL of the diluted E. coli culture medium, and incubated at 37° C. for 4 hours and 30 minutes while stirring at 210 rpm. The supernatant was obtained after centrifuging the bacteriophage candidate culture solution at 12,000 g at 4° C. for 10 minutes. In order to recover the bacteriophage candidates, 3.3 mL of 20% PEG (polyethylen glycol)/2.5 mol NaCl was added to the supernatant and stored at 4° C. for 12 hours. Thereafter, the supernatant was removed by centrifugation at 12,000 g at 4° C. for 15 minutes. After removing the supernatant, the remaining precipitate (Pellet) was dissolved in 1 mL of PBS (0.1M, pH7.4), and then centrifuged at 4° C. at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was obtained, and 166.7 μL of 20% PEG/2.5 mol NaCl was added thereto and stored on ice for 1 hour. After centrifugation at 4° C. at 14,000 rpm for 10 minutes, the supernatant was removed, and the precipitate was dissolved in 200 μL of PBS (0.1 M, pH7.4). Thereafter, titration was performed and the concentration of phage (PFU/mL: Plaque forming units/mL) was calculated through
[수학식 1][Equation 1]
파지 농도(PFU/mL) = Plaque수 × 희석배수 × mL로 환산한 접종량Phage concentration (PFU/mL) = number of plaques × dilution factor × inoculum converted to mL
상기를 통해 증폭된 박테리오파지 후보군들을 이용하여 ELISA를 수행하였다. 먼저 합성된 오카다산-BSA를 NaHCO3(pH 9.4)에 희석하였다. 16 μg/mL(0.25 μmol)의 오카다산-BSA 및 BSA 100 μL를 플레이트에 넣고 밀봉한 후 4℃에서 밤새 반응시켰다. 블로킹 용액은 NaHCO3(pH 9.4) 10 mL에 BSA 0.5 mg을 넣어 제조하였다. 용액을 모두 제거한 후 블로킹 용액 200 μL을 상기 플레이트에 넣고 밀봉한 후 4℃에서 1시간 동안 반응시켰다. 이 후 200 μL의 PBST(0.1 M PBS with Tween 0.1%, pH 7.4)로 반응이 완료된 플레이트를 6회 반복 세척하였다. 증폭된 박테리오파지 후보군 용액을 PBS(0.1 M, pH7.4)를 이용하여 1012 PFU/mL 농도로 희석하였다. 희석한 박테리오파지 후보군 용액 100 μL를 세척한 플레이트에 넣고 150 rpm으로 교반하면서 1시간 동안 반응시킨 후 세척 용액 200 μL로 6회 반복 세척하였다. 검출 용액은 상기 블로킹 용액 5 mL에 2 μL의 Anti-M13박테리오파지 항체-HRP (Sinobio, China)를 넣어 제조하였다. 검출 용액 200 μL를 파지가 고정화되어 있는 플레이트에 넣고 150 rpm으로 교반하면서 1시간 동안 반응시켰다. 반응이 끝난 후 세척 용액 200 μL로 6회 반복 세척하였다. ABTS(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 용액 7 mL에 H2O2 12 μL 혼합한 직후 플레이트에 상기 혼합 용액 200 μL를 넣고 405 nm에서 OD 값을 측정하였다. 그 결과 도 3과 같이, 상기 5개의 박테리오파지 후보군 중 WPWAHVHRGMSF 및 HDKLGWLHWRHY 서열의 후보군이 오카다산에 대해 높은 결합력을 보이는 것을 확인하였다.ELISA was performed using the bacteriophage candidates amplified through the above. First, the synthesized okadaic acid-BSA was diluted in NaHCO 3 (pH 9.4). 16 µg/mL (0.25 µmol) of okadaic acid-BSA and 100 µL of BSA were added to the plate, sealed, and allowed to react overnight at 4°C. A blocking solution was prepared by adding 0.5 mg of BSA to 10 mL of NaHCO 3 (pH 9.4). After removing all the solutions, 200 μL of the blocking solution was added to the plate, sealed, and reacted at 4° C. for 1 hour. Thereafter, the reaction-completed plate was repeatedly washed 6 times with 200 μL of PBST (0.1 M PBS with Tween 0.1%, pH 7.4). The amplified bacteriophage candidate solution was diluted to a concentration of 10 12 PFU/mL using PBS (0.1 M, pH7.4). 100 μL of the diluted bacteriophage candidate solution was added to the washed plate, reacted for 1 hour while stirring at 150 rpm, and then washed 6 times with 200 μL of the washing solution. The detection solution was prepared by adding 2 μL of Anti-M13 bacteriophage antibody-HRP (Sinobio, China) to 5 mL of the blocking solution. 200 μL of the detection solution was added to the plate on which the phages were immobilized and reacted for 1 hour while stirring at 150 rpm. After the reaction was over, the mixture was washed 6 times with 200 μL of the washing solution. Immediately after mixing 12 µL of H 2 O 2 with 7 mL of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) solution, 200 µL of the mixed solution was put on a plate and the OD value was measured at 405 nm. As a result, as shown in Figure 3, it was confirmed that among the five bacteriophage candidates, the candidate groups having the WPWAHVHRGMSF and HDKLGWLHWRHY sequences showed high binding ability to Okadasan.
더불어 상기와 동일한 ELISA 방법으로 비어있는 플레이트와 BSA 16 μg/mL (0.25 μmol) 농도가 고정화된 플레이트를 대조군으로 이용하여 박테리오파지 후보군의 오카다산에 대한 선택성을 확인하였다. 그 결과 도 4에 나타낸 바와 같이, WPWAHVHRGMSF 및 HDKLGWLHWRHY 서열의 후보군이 대조군에 비해 높은 결합력을 나타내었으며, 특히 HDKLGWLHWRHY 서열의 후보군이 대조군들에 비해 오카다산에 대해 2배 이상의 더 높은 결합력을 나타내는 것을 확인하였다.In addition, the selectivity of the bacteriophage candidates for Okadaic acid was confirmed by using an empty plate and a plate to which 16 μg/mL (0.25 μmol) of BSA was immobilized as a control by the same ELISA method as described above. As a result, as shown in FIG. 4, the candidate group of WPWAHVHRGMSF and HDKLGWLHWRHY sequences showed higher binding ability than the control group, and in particular, the candidate group of HDKLGWLHWRHY sequence showed more than two times higher binding ability to Okadasan compared to the control group. .
또한 상기와 동일한 ELISA 방법으로 오카다산-BSA 16 μg/mL (0.25 μmol) 농도가 고정화된 플레이트에 HDKLGWLHWRHY 서열의 후보군을 농도별 (1×1010-1×1012 PFU/mL)로 처리하여 그 결합력을 비교하였다. 그 결과 도 5와 같이, 박테리오파지 후보군의 농도가 증가함에 따라서 오카다산에 대한 결합력이 증가함을 확인하였다.In addition, by the same ELISA method as above, the candidate group of the HDKLGWLHWRHY sequence was treated by concentration (1 × 10 10 -1 × 10 12 PFU / mL) on a plate in which 16 μg / mL (0.25 μmol) of Okadaic acid-BSA was fixed, and The bonding strength was compared. As a result, as shown in Figure 5, it was confirmed that the binding force to Okadaic acid increased as the concentration of the bacteriophage candidate group increased.
더불어 상기와 동일한 ELISA 방법으로 오카다산 농도에 따른 결합력을 확인하였다(오카다산-BSA 농도: 0-64 μg/mL, 0-1 μmol). 그 결과 도 6에 나타낸 바와 같이, 오카다산의 농도가 증가함에 비례하여 결합력도 증가하는 경향을 확인하였다(도 6A). 또한, 검출한계(Limit of detection, LOD)는 16 μg/mL (0.25μmol)인 것으로 확인하였다(도 6B).In addition, the binding force according to the concentration of okadaic acid was confirmed by the same ELISA method as above (concentration of okadaic acid-BSA: 0-64 μg/mL, 0-1 μmol). As a result, as shown in FIG. 6, it was confirmed that the bonding strength also increased in proportion to the increase in the concentration of okadaic acid (FIG. 6A). In addition, the limit of detection (LOD) was confirmed to be 16 μg/mL (0.25 μmol) (FIG. 6B).
종합적으로, 박테리오파지 디스플레이 스크리닝을 이용하여 발굴한 본 발명의 펩타이드는 오카다산과 특이적으로 결합할 수 있어 오카다산을 정확하게 검출할 수 있으므로 이의 검출 또는 정량을 위해 활용할 수 있음을 확인하였다.Overall, it was confirmed that the peptide of the present invention discovered using bacteriophage display screening can specifically bind to okadaic acid and thus accurately detect okadaic acid, so that it can be used for its detection or quantification.
<110> CHUNG ANG University industry Academic Cooperation Foundation
<120> Novel peptides that specifically bind to okadaic acid
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His Asp Lys Leu Gly Trp Leu His Trp Arg His Tyr
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Trp Pro Trp Ala His Val His Arg Gly Met Ser Phe
1 5 10
<110> CHUNG ANG University industry Academic Cooperation Foundation
<120> Novel peptides that specifically bind to okadaic acid
<130> 1-95P
<160> 2
<170> KoPatentIn 3.0
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<212> PRT
<213> artificial sequence
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His Asp Lys Leu Gly Trp Leu His Trp Arg His
Claims (11)
A peptide for detecting Okadaic acid comprising an amino acid sequence represented by any one selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
상기 펩타이드는 이중체(dimer), 삼중체(trimer) 또는 다중체(multimer)인 것을 특징으로 하는, 펩타이드.
According to claim 1,
The peptide is characterized in that the peptide is a dimer, a trimer or a multimer.
상기 펩타이드는 발색효소, 방사선 동위원소, 크로모포어(chromopore), 발광물질 및 형광물질로 이루어진 군에서 선택된 어느 하나로 표지되는 것을 특징으로 하는, 펩타이드.
According to claim 1,
Characterized in that the peptide is labeled with any one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material.
A composition for detecting okadaic acid comprising the peptide according to any one of claims 1 to 3.
A kit for detecting okadaic acid comprising the peptide according to any one of claims 1 to 3.
A biochip for detecting okadaic acid comprising the peptide according to any one of claims 1 to 3.
Processing a sample with the peptide according to any one of claims 1 to 3; And detecting the reaction between the peptide and the sample; containing, okadaic acid detection method.
상기 반응의 검출은 순환 전압 전류법(Cyclic Voltammetry), 사각파전압전류법(Square Wave Voltammetry), 전기화학 임피던스 분광법(Electrochemical impedance spectroscopy), 수정 진동자 마이크로밸런스(quartz crystal microbalance), 표면 플라스몬 공명(Surface plasmon resonance), LFA(Lateral flow assay)/VFA(Variable flip angle), 효소 결합 면역 분석법(Enzyme linked immunoassay), 형광 공명 에너지 전달(Fluorescence resonance energy transfer) 및 표면증강 라만 분광법(Surface-enhanced Raman spectroscopy)으로 이루어진 군에서 선택된 어느 하나 이상의 방법으로 수행되는 것을 특징으로 하는, 방법.
According to claim 7,
Detection of the reaction is cyclic voltammetry, square wave voltammetry, electrochemical impedance spectroscopy, quartz crystal microbalance, surface plasmon resonance ( Surface plasmon resonance (LFA), Lateral flow assay (LFA)/Variable flip angle (VFA), Enzyme linked immunoassay, Fluorescence resonance energy transfer, and Surface-enhanced Raman spectroscopy ) Characterized in that carried out by any one or more methods selected from the group consisting of, a method.
preparing a complex in which okadaic acid and carrier protein are bound; and selecting a peptide that specifically binds to the complex.
상기 운반체 단백질은 알부민(albumin) 또는 오발부민(ovalbumin)인 것을 특징으로 하는, 스크리닝 방법.
According to claim 9,
Characterized in that the carrier protein is albumin (albumin) or ovalbumin (ovalbumin), screening method.
상기 복합체에 특이적으로 결합하는 펩타이드는 서열번호 1 및 서열번호 2로 이루어진 군에서 선택된 어느 하나로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는, 스크리닝 방법.According to claim 9,
The peptide specifically binding to the complex is characterized in that it comprises an amino acid sequence represented by any one selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, screening method.
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