KR102469657B1 - Method for screening biomarkers diagnosing cardiovascular diseases, biomarkers screened thereby and method for diagnosing cardiovascular diseases using the same - Google Patents

Method for screening biomarkers diagnosing cardiovascular diseases, biomarkers screened thereby and method for diagnosing cardiovascular diseases using the same Download PDF

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KR102469657B1
KR102469657B1 KR1020210191376A KR20210191376A KR102469657B1 KR 102469657 B1 KR102469657 B1 KR 102469657B1 KR 1020210191376 A KR1020210191376 A KR 1020210191376A KR 20210191376 A KR20210191376 A KR 20210191376A KR 102469657 B1 KR102469657 B1 KR 102469657B1
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강민정
유영숙
마흐무드 조예타
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한국과학기술연구원
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    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
    • G16B50/30—Data warehousing; Computing architectures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Abstract

본 발명은 심혈관 질환(cardiovascular disease) 진단용 바이오마커로서 14-3-3 단백질, 이의 선별방법, 이를 이용한 심혈관 질환 진단용 키트, 심혈관 질환 진단을 위한 정보제공방법, 이의 억제제를 포함하는 심혈관 질환의 예방 또는 치료용 약학적 조성물 및 심혈관 질환 치료제의 스크리닝 방법에 관한 것이다.The present invention is a 14-3-3 protein as a biomarker for diagnosing cardiovascular disease, a method for screening thereof, a kit for diagnosing cardiovascular disease using the same, a method for providing information for diagnosing cardiovascular disease, and preventing or preventing cardiovascular disease including inhibitors thereof. It relates to a pharmaceutical composition for treatment and a method for screening a therapeutic agent for cardiovascular disease.

Description

심혈관 질환 진단용 바이오마커의 선별방법, 이에 따라 선별된 바이오마커 및 상기 바이오마커를 이용한 심혈관 질환의 진단방법{Method for screening biomarkers diagnosing cardiovascular diseases, biomarkers screened thereby and method for diagnosing cardiovascular diseases using the same}Method for screening biomarkers diagnosing cardiovascular diseases, biomarkers screened thereby and method for diagnosing cardiovascular diseases using the same}

본 발명은 심혈관 질환(cardiovascular disease) 진단용 바이오마커로서 14-3-3 단백질, 이의 선별방법, 이를 이용한 심혈관 질환 진단용 키트, 심혈관 질환 진단을 위한 정보제공방법, 이의 억제제를 포함하는 심혈관 질환의 예방 또는 치료용 약학적 조성물 및 심혈관 질환 치료제의 스크리닝 방법에 관한 것이다.The present invention is a 14-3-3 protein as a biomarker for diagnosing cardiovascular disease, a method for screening thereof, a kit for diagnosing cardiovascular disease using the same, a method for providing information for diagnosing cardiovascular disease, and preventing or preventing cardiovascular disease including inhibitors thereof. It relates to a pharmaceutical composition for treatment and a method for screening a therapeutic agent for cardiovascular disease.

심혈관 질환(cardiovascular disease; CVD)은 뇌와 심장에 혈액을 공급하는 혈관에 이상이 생긴 질환을 포함하며, 협심증, 심근경색 및 뇌경색 등이 이에 해당한다. 보다 구체적으로, 심혈관 질환은 심장이나 혈관에 관련된 모든 질환을 포함하며, 예컨대, 관상동맥 심장질환, 울혈성 심부전, 말초 혈관 질환, 심장 마비, 뇌졸중 또는 일과성 허혈 발작, 신장 혈관 질환 등을 폭넓게 포함한다. 한국인에 있어서, 심장 질환은 암에 이어 사망원인 2위이며, 뇌혈관 질환으로 인한 사망률이 3위로 나타나고 있다. 나아가, 2015년 세계보건기구(WHO)가 발표한 전세계 사망원인 1위는 심혈관 질환이다. 이처럼 심혈관 건강은 생명과 밀접한 연관이 있으므로, 관리와 예방이 중요하다. 나아가, 이들 질환은 치료 가능한 질환이므로, 질환의 발병을 초기에 진단하고 치료하는 것은 중요하다.Cardiovascular disease (CVD) includes diseases in which there is an abnormality in blood vessels supplying blood to the brain and heart, and includes angina pectoris, myocardial infarction, and cerebral infarction. More specifically, cardiovascular disease includes all diseases related to the heart or blood vessels, such as coronary heart disease, congestive heart failure, peripheral vascular disease, heart attack, stroke or transient ischemic attack, renal vascular disease, and the like. . For Koreans, heart disease is the second leading cause of death after cancer, and cerebrovascular disease is the third leading cause of death. Furthermore, the number one cause of death worldwide announced by the World Health Organization (WHO) in 2015 is cardiovascular disease. As such, cardiovascular health is closely related to life, so management and prevention are important. Furthermore, since these diseases are treatable diseases, it is important to diagnose and treat the onset of the diseases at an early stage.

심혈관 질환을 진단하기 위해, 심전도 검사, 운동부하 검사, 심초음파 검사, 심장 핵의학검사, 관상동맥 촬영(CT), 및 심혈관조영술 등이 있다. 그러나 이들 진단 방법은 특별한 장치를 요구하거나, 조영제를 투여하기 위해 침습적인 시술이 선행되어야 하며, 비용이 높아, 쉽게 실시하기 어렵다.For diagnosing cardiovascular disease, there are electrocardiogram, exercise load test, echocardiography, nuclear heart medicine test, coronary angiography (CT), and cardiovascular angiography. However, these diagnostic methods require a special device or require an invasive procedure to administer a contrast agent, and are expensive, making it difficult to perform easily.

이에, 상대적으로 간편한 방법으로 심혈관 질환을 진단할 수 있는 바이오마커를 발굴하고자 하는 시도가 있다. 최근 시장보고에 의하면 종래에 사용되는 CK-MB, 심장 트로포닌(cardiac Troponin), 미오글로빈(myoglobin) 및 콜레스테롤과 함께 골수세포형과산화효소(myeloperoxidase), 뇌 나트륨이뇨 펩티드(brain natriuretic peptide; BNP), hs-CRP, mehocysteine, 지방산 결합 단백질(fatty acid binding protein; FABP), 뇨 알부민(urinary albumin), 아스파르테이트아미노전달효소(aspartate transaminase), LDH, HbA1c 등이 심장 질환 관련 바이오마커로 사용되고 있다. 심근경색을 포함, 심혈관 질환의 현장 진단 마커 시장은 빠른 성장을 보이는 분야로 응급실 뿐 아니라 병동에 이르기까지 폭넓게 이용되고 있다. 응급 및 현장 진단용 심근 경색 마커들은 각 마커 별로 혈액 내 나타나는 기간과 이에 따른 민감도와 특이도가 특징적으로 발현되는 것으로 조사되고 있다.Accordingly, there are attempts to discover biomarkers capable of diagnosing cardiovascular diseases in a relatively simple way. According to a recent market report, myeloperoxidase, brain natriuretic peptide (BNP), hs-CRP, mehocysteine, fatty acid binding protein (FABP), urinary albumin, aspartate transaminase, LDH, HbA1c, etc. are used as biomarkers related to heart disease. The point-of-care diagnostic marker market for cardiovascular diseases, including myocardial infarction, is a rapidly growing field and is widely used in emergency rooms as well as wards. Myocardial infarction markers for emergency and on-site diagnosis are being investigated for the characteristic expression of each marker's duration in the blood and its corresponding sensitivity and specificity.

바이오마커를 활용한 진단키트는 체외 진단형 키트 형태로 2007년 이래로 시장규모는 연간 3 내지 10%씩 꾸준히 증가하고 있고, 2019년 현재 약 71.7억 달러의 매출 규모에 달한다. 한편 진단키트 중 가장 큰 비중을 차지하는 것은 면역화학적 진단기기로 2017년을 기준으로 약 23.3억 달러의 시장 규모를 달성하였으며, 로슈가 가장 높은 점유율을 차지하고, 지멘스와 애보트가 그 뒤를 잇는다. 국내 체외 진단기기 시장규모는 약 4174억원(2014년 기준)으로 추정되며, 앞으로도 지속적인 성장이 예상된다. 이는 BT 산업에 큰 영향을 받고 있으며, 타 국가 대비 우위에 있는 IT 기술을 융합하여 경쟁력 있는 산업을 육성할 수 있을 것으로 예측되고 있다.Diagnostic kits using biomarkers are in the form of in vitro diagnostic kits. Since 2007, the market size has been steadily increasing by 3 to 10% per year, and as of 2019, the sales amount reached about 7.17 billion dollars. Meanwhile, immunochemical diagnostic devices account for the largest share among diagnostic kits, achieving a market size of about 2.33 billion dollars as of 2017, with Roche occupying the highest share, followed by Siemens and Abbott. The domestic in vitro diagnostic device market size is estimated to be approximately KRW 417.4 billion (as of 2014), and continuous growth is expected in the future. This is greatly influenced by the BT industry, and it is expected that competitive industries can be nurtured by converging IT technologies that are superior to other countries.

본 발명자들은 간단한 방법으로 심혈관 질환을 특이적으로 진단할 수 있는 체외진단 키트에 적용가능한 바이오마커를 발굴하기 위하여 예의 연구 노력한 결과, 심근비대 유도 세포에서 14-3-3 단백질, 특히 이의 베타, 입실론 및 제타 아형(isoform)의 발현이 정상 심근 세포는 물론 다른 조직 및/또는 다른 조직의 암세포들에 비해 현저하게 증가하는 것을 확인하고, 본 발명을 완성하였다.As a result of intensive research efforts to discover biomarkers applicable to in vitro diagnostic kits capable of specifically diagnosing cardiovascular diseases by a simple method, the inventors of the present invention found that 14-3-3 proteins, particularly beta and epsilon proteins, were found in myocardial hypertrophy-inducing cells. And it was confirmed that the expression of the zeta isoform was remarkably increased compared to normal myocardial cells as well as other tissues and/or cancer cells of other tissues, thereby completing the present invention.

본 발명의 제1양태는 14-3-3 단백질의 수준을 측정하는 제제를 포함하는, 심혈관 질환(cardiovascular disease) 진단용 조성물을 제공한다.A first aspect of the present invention provides a composition for diagnosing cardiovascular disease, including an agent for measuring the level of 14-3-3 protein.

본 발명의 용어 "진단"은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명의 목적상, 진단은 심혈관 질환의 발병 여부 또는 발병 가능성을 확인하는 것이다.As used herein, the term “diagnosis” refers to confirming the presence or character of a pathological condition. For purposes of the present invention, diagnosis is the determination of whether a cardiovascular disease has occurred or is likely to develop.

본 발명의 용어, "14-3-3 단백질"은 모든 진핵세포에서 발현되는 서열이 유지된 조절 분자(conserved regulatory molecules)의 패밀리이다. 14-3-3 단백질은 키나아제, 포스파타아제, 및 막관통수용체(transmembrane receptors)를 포함한 다수의(multitude) 기능적으로 다양한 신호전달 단백질에 대한 결합능을 갖는다. 14-3-3 리간드로서 200여종 이상의 신호전달 단백질이 보고되었다. 크로이츠펠트-야콥병(Creutzfeldt-Jakob disease) 환자의 뇌척수액(cerebrospinal fluid)에서 증가된 양의 14-3-3 단백질이 관찰된다. 또한, 이의 에타 아형(isoform)은 활액(synovial fluid)에 존재하는 류마티스성 관절염(rheumatoid arthritis)에 대한 바이오마커임이 보고된 바 있다. 그러나, 14-3-3 단백질 및 이의 아형들의 심혈관 질환의 상관관계에 대해서는 확인된 바 없다.As used herein, the term "14-3-3 protein" is a family of conserved regulatory molecules whose sequences are expressed in all eukaryotic cells. The 14-3-3 protein has the ability to bind to a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. More than 200 signaling proteins have been reported as 14-3-3 ligands. An increased amount of 14-3-3 protein is observed in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease. In addition, it has been reported that its eta isoform is a biomarker for rheumatoid arthritis present in synovial fluid. However, the correlation of 14-3-3 protein and its subtypes with cardiovascular disease has not been confirmed.

대부분의 포유류에서 7가지 구별되는 14-3-3 단백질을 부호화(encode)하는 7개 유전자가 존재한다: 인간에 대해, YWHAB(14-3-3 베타), YWHAE(14-3-3 입실론), YWHAG(14-3-3 감마), YWHAH(14-3-3 에타), YWHAQ(14-3-3 세타), 및 YWHAZ(14-3-3 제타). 진핵생물(eukaryotes)은, 다중 유전자가 발현되는 경우, 단일 14-3-3 유전자의 결실을 수용할 수 있으나, 모든 14-3-3의 결손(deletion)은 죽음을 야기한다.In most mammals, there are seven genes that encode seven distinct 14-3-3 proteins: in humans, YWHAB (14-3-3 beta), YWHAE (14-3-3 epsilon). , YWHAG (14-3-3 gamma), YWHAH (14-3-3 eta), YWHAQ (14-3-3 theta), and YWHAZ (14-3-3 zeta). Eukaryotes can accommodate deletion of a single 14-3-3 gene if multiple genes are expressed, but deletion of all 14-3-3 causes death.

14-3-3 단백질은 일반적으로 9 또는 10 알파 나선(helices)을 가지며, 이들의 아미노-말단 나선을 따라 동종(homo)- 및/또는 이종(hetero)-이합체(dimer) 상호작용을 형성하는 테트라트리코 펩티드 반복 수퍼패밀리(Tetratrico Peptide Repeat(TPR) superfamily)와 구조적으로 유사하다. 이들 단백질은, 2가 양이온 상호작용(divalent cation interaction), 인산화(phosphorylation)와 아세틸화(acetylation), 및 단백질분해 절단(proteolytic cleavage)을 포함한, 다수의 알려진 일반적인 개질 도메인(modification domains)을 포함한다.14-3-3 proteins usually have 9 or 10 alpha helices and form homo- and/or hetero-dimer interactions along their amino-terminal helices. It is structurally similar to the Tetratrico Peptide Repeat (TPR) superfamily. These proteins contain a number of known common modification domains, including divalent cation interactions, phosphorylation and acetylation, and proteolytic cleavage. .

14-3-3은 펩티드에 결합한다. 비-인산화된 리간드에 대한 결합이 보고되었음에도 불구하고, 인산화된 세린 또는 트레오닌 잔기를 포함하는 14-3-3 단백질에 대한 공통의 인식 모티프(common recognition motif)가 존재한다. 이러한 상호작용은, 자연 상태에서 양친매성(amphipathic)인, 이른바 결합 홈(groove) 또는 틈(cleft)을 따라 발생한다. 지금까지, 이들 단백질의 6개 클래스에 대한 결정 구조가 밝혀지고 공개적으로 기탁되었다.14-3-3 binds to the peptide. Although binding to non-phosphorylated ligands has been reported, there is a common recognition motif for 14-3-3 proteins containing phosphorylated serine or threonine residues. These interactions occur along so-called bonding grooves or clefts, which are amphipathic in nature. To date, crystal structures for six classes of these proteins have been identified and publicly deposited.

구체적으로, 본 발명의 심혈관 질환 진단용 조성물에 바이오마커로 사용되는 상기 14-3-3 단백질은 14-3-3 베타, 14-3-3 입실론, 14-3-3 감마, 14-3-3 에타, 14-3-3 세타 및 14-3-3 제타로 구성된 군으로부터 선택되는 하나 이상일 수 있다. 보다 구체적으로, 상기 14-3-3 단백질은 14-3-3 베타, 14-3-3 입실론, 및 14-3-3 제타로 구성된 군으로부터 선택되는 하나 이상일 수 있으나, 이에 제한되지 않는다.Specifically, the 14-3-3 protein used as a biomarker in the composition for diagnosing cardiovascular diseases of the present invention is 14-3-3 beta, 14-3-3 epsilon, 14-3-3 gamma, 14-3-3 It may be one or more selected from the group consisting of eta, 14-3-3 theta and 14-3-3 zeta. More specifically, the 14-3-3 protein may be one or more selected from the group consisting of 14-3-3 beta, 14-3-3 epsilon, and 14-3-3 zeta, but is not limited thereto.

상기 6종의 14-3-3 단백질 아형인 14-3-3 베타, 14-3-3 입실론, 14-3-3 감마, 14-3-3 에타, 14-3-3 세타 및 14-3-3 제타는 각각 서열번호 1 내지 6의 유전자에 의해 코딩될 수 있으며, 서열번호 7 내지 12의 아미노산 서열로 표현될 수 있다. 나아가, 이들 단백질은 서열번호 13 내지 18로 표시되는, 20개 내외(±3)의 아미노산 서열로 구성되는, 펩타이드 단편 및/또는 이의 이온에 의해 질량분석기법으로 동정될 수 있다.The six 14-3-3 protein subtypes 14-3-3 beta, 14-3-3 epsilon, 14-3-3 gamma, 14-3-3 eta, 14-3-3 theta and 14-3 -3 zeta may be encoded by the genes of SEQ ID NOs: 1 to 6, respectively, and may be represented by the amino acid sequences of SEQ ID NOs: 7 to 12. Furthermore, these proteins can be identified by mass spectrometry by means of peptide fragments and/or ions thereof, which are represented by SEQ ID NOs: 13 to 18 and consist of an amino acid sequence of around 20 (±3).

본 발명의 구체적인 실시예예서는 심근 비대 세포로부터 발현되는 일련의 단백질 중 정상 심근 세포에 비해 과발현되는 일련의 단백질을 확인하였다. 나아가, 인간 정상 폐 상피 세포주, 인간 폐암 세포주, 인간 정상 위 세포주, 인간 위암 세포주, 인간 정상 간 세포주, 및 인간 간암 세포주에서 발현되는 단백질의 종류 및 발현량과 비교하여 심근 비대 세포에서 특이적으로 발현량이 증가하는 단백질을 분리 및 동정하여, 14-3-3 단백질; 예컨대, 이의 6종 아형 14-3-3 베타, 14-3-3 입실론, 14-3-3 감마, 14-3-3 에타, 14-3-3 세타 및 14-3-3 제타;의 발현이 정상 심근 세포에 비해 심근 비대 세포에서 현저히 증가하는 것을 확인하였다. 특히 14-3-3 베타, 14-3-3 입실론, 및 14-3-3 제타의 3종 아형은 정상 심근 세포는 물론 다른 조직의 정상 세포주 및 암 세포주에 비해 심근 비대 세포에서 특이적으로 증가하는 것을 확인하였다. 이는 이들 단백질의 발현을 측정함으로써 심혈관 질환을 진단할 수 있음을 나타내는 것이다(도 6 및 7).In the specific examples of the present invention, among a series of proteins expressed from myocardial hypertrophic cells, a series of proteins overexpressed compared to normal myocardial cells were identified. Furthermore, compared to the types and expression levels of proteins expressed in human normal lung epithelial cell lines, human lung cancer cell lines, human normal gastric cell lines, human gastric cancer cell lines, human normal liver cell lines, and human liver cancer cell lines, the specific expression levels in myocardial hypertrophic cells By isolating and identifying increasing proteins, 14-3-3 proteins; For example, expression of its six subtypes 14-3-3 beta, 14-3-3 epsilon, 14-3-3 gamma, 14-3-3 eta, 14-3-3 theta and 14-3-3 zeta; It was confirmed that the myocardial hypertrophic cells significantly increased compared to normal myocardial cells. In particular, the three subtypes of 14-3-3 beta, 14-3-3 epsilon, and 14-3-3 zeta are specifically increased in myocardial hypertrophic cells compared to normal myocardial cells as well as normal cell lines and cancer cell lines in other tissues. confirmed that. This indicates that cardiovascular disease can be diagnosed by measuring the expression of these proteins (FIGS. 6 and 7).

본 발명의 조성물을 이용하여 진단 가능한 심혈관 질환은 심근 비대 세포에 의해 유발되는 질환일 수 있다. 예컨대, 상기 심혈관 질환은 안지오텐신 II(angiotensin II; Ang II) 또는 엔도텔린-1(endothelin-1; ET-1)에 의해 유발되는 질환으로서, 구체적으로는 심근비대증(비대성 심근병증, hypertrophic cardiomyopathy), 심근조직 괴사(심근 경색증, myocardial infarction), 고혈압(hypertension), 아테롬성 동맥경화증(atherosclerosis), 관동맥성 심장병(coronary heart disease), 재발협착증(restenosis), 심부전(heart failure), 전신성 고혈압(systemic hypertension), 폐동맥 고혈압(pulmonary hypertension), 관상동맥질환(coronary artery disease), 신부전증(renal failure), 만성 심부전증(chronic heart failure), 및 허혈(ischaemia)/재관류(reperfusion) 손상일 수 있으나, 이에 제한되지 않는다.Cardiovascular diseases that can be diagnosed using the composition of the present invention may be diseases caused by myocardial hypertrophic cells. For example, the cardiovascular disease is a disease caused by angiotensin II (angiotensin II; Ang II) or endothelin-1 (ET-1), specifically, myocardial hypertrophy (hypertrophic cardiomyopathy), Myocardial tissue necrosis (myocardial infarction), hypertension, atherosclerosis, coronary heart disease, restenosis, heart failure, systemic hypertension , pulmonary hypertension, coronary artery disease, renal failure, chronic heart failure, and ischaemia/reperfusion injury.

이때, 상기 14-3-3 단백질의 수준을 측정하는 제제는 이에 특이적으로 결합하는 단백질, 항체, 앱타머, 수용체 또는 리간드를 포함할 수 있다. 또는 상기 14-3-3 단백질을 코딩하는 유전자를 검출할 수 있는 제제일 수 있다. 구체적으로, 14-3-3 단백질의 수준을 측정하는 제제는 이에 특이적으로 결합하는 키나아제, 포스파타아제, 막관통 수용체 및/또는 다양한 14-3-3 리간드일 수 있으나, 이에 제한되지 않으며, 생체 시료 중 특이적으로 14-3-3 단백질 및/또는 이를 코딩하는 유전자를 정량할 수 있는 한, 해당 물질을 제한없이 사용할 수 있다. 상기 정량은 절대적인 양을 측정하는 것 뿐만 아니라 상대적인 양의 변화를 측정하는 것을 제한없이 포함할 수 있다.In this case, the agent for measuring the level of the 14-3-3 protein may include a protein, antibody, aptamer, receptor or ligand that specifically binds thereto. Alternatively, it may be an agent capable of detecting a gene encoding the 14-3-3 protein. Specifically, the agent for measuring the level of 14-3-3 protein may be a kinase, phosphatase, transmembrane receptor, and/or various 14-3-3 ligands that specifically bind thereto, but is not limited thereto, As long as the 14-3-3 protein and/or the gene encoding the 14-3-3 protein can be specifically quantified in a biological sample, the material may be used without limitation. The quantification may include, without limitation, measuring an absolute amount as well as measuring a change in a relative amount.

본 발명의 제2양태는 정상 심근 세포 및 심근 비대 세포의 용해물 또는 이의 분비물에 포함된 일련의 단백질을 분리하는 제1단계; 분리된 단백질 중 정상 심근 세포에 비해 심근 비대 세포에서 보다 많이 발현되는 단백질을 선별하는 제2단계; 및 단백질체학 데이터베이스 검색 엔진(proteomics database search engine)을 이용하여 상기 제2단계로부터 선택된 단백질을 동정하는 제3단계를 포함하는, 심혈관 질환 진단용 바이오마커의 선별방법을 제공한다.A second aspect of the present invention is a first step of isolating a series of proteins included in lysates or secretions of normal myocardial cells and myocardial hypertrophic cells; A second step of selecting a protein that is more expressed in myocardial hypertrophic cells than in normal myocardial cells among the isolated proteins; and a third step of identifying the protein selected from the second step by using a proteomics database search engine.

제1양태에 사용된 14-3-3 단백질은 상기 본 발명의 바이오마터 선별방법을 통해 심혈관 질환 진단용 바이오마커로 동정되었다.The 14-3-3 protein used in the first aspect was identified as a biomarker for diagnosing cardiovascular disease through the biomarker screening method of the present invention.

상기 선별방법의 제1단계에 사용되는 심근 비대 세포는 정상 심근 세포에 안지오텐신 II 또는 엔도텔린-1을 처리하여 준비할 수 있으나, 이에 제한되지 않는다. 또는 심혈관 질환이 발병한 개체의 조직으로부터 분리된 세포 또는 상기 개체로부터 수집한 혈액이나 혈청, 또는 이로부터 분리한 엑소좀을 사용할 수 있다.Myocardial hypertrophic cells used in the first step of the screening method may be prepared by treating normal myocardial cells with angiotensin II or endothelin-1, but are not limited thereto. Alternatively, cells isolated from tissues of an individual suffering from cardiovascular disease, blood or serum collected from the individual, or exosomes isolated therefrom may be used.

예컨대, 상기 제1단계는 전기영동(electrophoresis), 웨스턴블롯(western blot) 또는 둘 모두에 의해 수행될 수 있다. 그러나, 이에 제한되는 것은 아니며, 당업계에 공지된 단백질 분리 방법을 제한없이 사용할 수 있다. 민감성을 높이기 위하여, 상기 제1단계에 따른 단백질 분리에 앞서 세포, 세포의 용해물, 및/또는 이의 분비물로부터 단백질을 추출하는 단계를 추가로 포함할 수 있으나, 이에 제한되지 않는다.For example, the first step may be performed by electrophoresis, western blot, or both. However, it is not limited thereto, and protein separation methods known in the art may be used without limitation. In order to increase sensitivity, a step of extracting proteins from cells, cell lysates, and/or their secretions may be further included prior to protein isolation according to the first step, but is not limited thereto.

나아가, 본 발명의 선별방법은 제2단계로부터 수득한 단백질을 액체 크로마토그래피-질량분석을 통해 분리분석하는 제2-1단계를 추가로 포함할 수 있으나, 이에 제한되지 않는다.Furthermore, the screening method of the present invention may further include a 2-1 step of separating and analyzing the protein obtained from the 2 step through liquid chromatography-mass spectrometry, but is not limited thereto.

예컨대, 상기 제2-1단계는 C18 트랩 나노컬럼을 구비한 나노 및 모세관 액체 크로마토그래피 컬럼과 연결된, 오비트랩(orbitrap) 질량 분석기와 선형 트랩 사극자(linear trap quadropole) 이온 트랩이 결합된 질량분석기를 이용하여 수행할 수 있으나, 이에 제한되지 않는다.For example, the 2-1 step is a mass spectrometer in which an orbitrap mass spectrometer and a linear trap quadropole ion trap are coupled to a nano and capillary liquid chromatography column having a C18 trap nanocolumn. It can be performed using, but is not limited thereto.

구체적으로, Nano-ESI(nEASY)-LTQ-Orbitrap Velos Pro장비를 이용하여 0.2% 포름산 수용액 및 0.2% 포름산 아세토니트릴 용액을 용매로 사용하여 비율을 조절하는 용매 구배에 의해, 직경 75 μm, 길이 2 cm의 C18 트랩 컬럼 및 이에 연결된 직경 75 μm, 길이 50 cm의 C18 분석 컬럼을 300 nL/min의 유속으로 통과시키고, 이어지는 오비트랩(orbitrap) 질량 분석기와 선형 트랩 사극자(linear trap quadropole) 이온 트랩이 결합된 질량분석기를 이용한 고해상도 질량분석을 통해 단백질을 분리 및 동정하였다. 질량분석시에는 배제시간(exclusion time) 180초, 반복 횟수(repeat count) 2, 반복 기간(repeat duration) 30초, 배제질량폭(exclusion mass width) 10 ppm 및 배제 크기 500의 변수를 사용하였으며, 단독으로 하전된 이온(singly charged ion)은 수집에서 제외하였다.Specifically, using a Nano-ESI (nEASY)-LTQ-Orbitrap Velos Pro equipment, 0.2% formic acid aqueous solution and 0.2% formic acid acetonitrile solution were used as solvents to adjust the ratio by a solvent gradient, with a diameter of 75 μm and a length of 2 cm C18 trap column and a C18 analytical column with a diameter of 75 μm and a length of 50 cm connected thereto at a flow rate of 300 nL/min, followed by an orbitrap mass spectrometer and a linear trap quadropole ion trap Proteins were separated and identified through high-resolution mass spectrometry using this coupled mass spectrometer. In mass spectrometry, variables of exclusion time of 180 seconds, repeat count of 2, repeat duration of 30 seconds, exclusion mass width of 10 ppm, and exclusion size of 500 were used, Singly charged ions were excluded from collection.

또한, 본 발명의 선별방법은 상기 제2-1단계에 앞서 제2단계로부터 수득한 단백질을 분해하는 제2-0단계를 추가로 포함할 수 있으나, 이에 제한되지 않는다. 아울러, 상기 제2-0단계는 당업계에 공지된 단백질 분해 방법을 제한없이 사용하여 수행할 수 있다.In addition, the screening method of the present invention may further include a step 2-0 of degrading the protein obtained from step 2 prior to step 2-1, but is not limited thereto. In addition, step 2-0 may be performed using a protein degradation method known in the art without limitation.

예컨대, 본 발명의 선별방법에 있어서, 상기 제3단계는 Mascot 또는 Sequest 엔진을 사용하여 수행될 수 있으나, 이에 제한되지 않는다. 현재 당업계에 사용되는 Mascow 프로그램 및 Sequest 프로그램 등이 있으나, 상기 목적을 달성할 수 있는 한, 특정 프로그램에 제한되지 않는다. 본 발명의 구체적인 실시예에서는, Sequest 프로그램을 이용하여 제3단계를 수행하였으나, 이에 제한되지 않는다. 이때, 전구체 이온 질량 오차(precursor ion mass tolerance) 10 ppm, 단편 이온 질량 오차(fragment ion mass tolerance) 0.6 Da, 최대 미절단(maximum missed cleavages) 2를 허용하는 조건 하에 수행할 수 있으나, 구체적인 수행 조건은 이에 제한되지 않는다.For example, in the screening method of the present invention, the third step may be performed using a Mascot or Sequest engine, but is not limited thereto. Currently, there are Mascow programs and Sequest programs used in the art, but they are not limited to specific programs as long as they can achieve the above object. In a specific embodiment of the present invention, the third step is performed using the Sequest program, but is not limited thereto. At this time, it can be performed under conditions allowing a precursor ion mass tolerance of 10 ppm, a fragment ion mass tolerance of 0.6 Da, and a maximum missed cleavages of 2, but specific performance conditions is not limited to this.

나아가, 본 발명의 선별방법은 상기 제3단계로부터 동정된 단백질들에 대해 가상 시뮬레이션 프로그램(in silico pathway analysis tool)을 이용하여 신호전달 경로를 분석하는 제4단계를 추가로 포함할 수 있다. 상기 제4단계를 통해, 선별된 단백질 후보군의 신호전달 경로 및 네트워크 등을 분석함으로써 해당 단백질의 심혈관 질환 진단용 바이오마커로서의 유용성을 검증할 수 있다.Furthermore, the screening method of the present invention may further include a fourth step of analyzing signal transduction pathways of the proteins identified in the third step using an in silico pathway analysis tool. Through the fourth step, the usefulness of the protein as a biomarker for diagnosing cardiovascular disease can be verified by analyzing the signaling pathway and network of the selected protein candidate group.

본 발명의 제3양태는 제1양태의 조성물을 포함하는, 심혈관 질환 진단용 키트를 제공한다.A third aspect of the present invention provides a kit for diagnosing cardiovascular disease comprising the composition of the first aspect.

본 발명의 키트는 검체 내의 14-3-3 단백질 또는 이를 코딩하는 유전자를 정성 및/또는 정량적으로 검출하기 위하여 사용될 수 있다.The kit of the present invention can be used to qualitatively and/or quantitatively detect the 14-3-3 protein or the gene encoding the 14-3-3 protein in a specimen.

예컨대, 상기 키트는 마이크로어레이, 앱타머 칩 키트, 엘라이자(ELISA, enzyme linked immunosorbent assay) 키트, RT-PCR(Reverse transcription polymerase chain reaction) 키트, DNA 칩 키트, ELISA(Enzymelinked immunosorbent assay) 키트, 블랏팅(blotting) 키트, 면역침전법 키트, 면역형광검사 키트, 단백질 칩 키트, MRM(Multiple reaction monitoring) 키트, 및 이들의 조합으로 이루어진 군으로부터 선택될 수 있으나, 제1양태의 조성물과 검체 중의 심혈관 질환 진단용 바이오마커인 14-3-3 단백질의 발현을 정성 및/또는 정량적으로 확인할 수 있는 한, 이에 제한되지 않는다.For example, the kit may include a microarray, an aptamer chip kit, an ELISA (enzyme linked immunosorbent assay) kit, a RT-PCR (Reverse transcription polymerase chain reaction) kit, a DNA chip kit, an ELISA (Enzymelinked immunosorbent assay) kit, a block It may be selected from the group consisting of a blotting kit, an immunoprecipitation kit, an immunofluorescence test kit, a protein chip kit, a multiple reaction monitoring (MRM) kit, and a combination thereof, but the composition of the first embodiment and the cardiovascular in the specimen As long as the expression of 14-3-3 protein, which is a biomarker for disease diagnosis, can be confirmed qualitatively and/or quantitatively, it is not limited thereto.

예컨대, 상기 키트는 RT-PCR(Reverse transcription polymerase chain reaction) 키트, DNA 칩 키트, ELISA(Enzymelinked immunosorbent assay) 키트, 단백질 칩 키트, 래피드(rapid) 키트 또는 MRM(Multiple reaction monitoring) 키트인 것일 수 있으나, 이에 제한되지 않는다.For example, the kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzymelinked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. , but not limited thereto.

본 발명의 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. RT-PCR 키트는, 상기 단백질들을 코딩하는 유전자에 대한 특이적인 각각의 프라이머 쌍 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드(dNTPs), Taq-폴리머라아제 및 역전사효소와 같은 효소, DNase, RNAse 억제제, DEPC-수(DEPC-water), 멸균수 등을 포함할 수 있다. 또한, 정량 대조구로 사용되는 유전자에 특이적인 프라이머 쌍을 포함할 수 있다.The kit of the present invention may be a kit containing essential elements required to perform RT-PCR. The RT-PCR kit consists of a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymer, in addition to each primer pair specific for the gene encoding the above proteins. enzymes such as enzymes and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. In addition, a primer pair specific to a gene used as a quantitative control may be included.

또한, 본 발명의 키트는 DNA 칩 분석법을 수행하기 위해 필요한 필수 요소를 포함할 수 있다. DNA 칩 분석용 키트는, 유전자 또는 그의 단편에 해당하는 cDNA가 프로브로 부착되어 있는 기판, 및 형광표식 프로브를 제작하기 위한 시약, 제제, 효소 등을 포함할 수 있다. 또한, 기판은 정량 대조구 유전자 또는 그의 단편에 해당하는 cDNA를 포함할 수 있다.In addition, the kit of the present invention may include essential elements necessary for performing the DNA chip assay. A DNA chip analysis kit may include a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, reagents, enzymes, and the like for producing a fluorescently labeled probe. In addition, the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof.

아울러, 본 발명의 키트는 상기 단백질들의 발현 수준을 측정하기 위한 단백질 칩 분석용 키트가 될 수 있는데, 상기 키트는 특별히 이에 제한되지 않으나, 항체의 면역학적 검출을 위하여 기재, 적당한 완충용액, 발색 효소 또는 형광물질로 표지된 2차 항체, 발색 기질 등을 포함할 수 있다. 상기 기재는 특별히 이에 제한되지 않으나 니트로셀룰로오스 막, 폴리비닐 수지로 합성된 96 웰 플레이트, 폴리스티렌 수지로 합성된 96 웰 플레이트 및 유리로 된 슬라이드글라스 등이 이용될 수 있고, 발색효소는 특별히 이에 제한되지 않으나 퍼옥시다아제(peroxidase), 알칼라인 포스파타아제(alkaline phosphatase)가 사용될 수 있으며, 형광물질은 특별히 이에 제한되지 않으나 FITC, RITC 등일 수 있고, 발색 기질액은 특별히 이에 제한되지 않으나 ABTS(2,2'-아지노-비스(3-에틸벤조티아졸린-6-설폰산)), OPD(o-페닐렌디아민) 또는 TMB(테트라메틸 벤지딘)일 수 있다.In addition, the kit of the present invention may be a protein chip analysis kit for measuring the expression level of the proteins. The kit is not particularly limited thereto, but a base material, an appropriate buffer solution, and a color-developing enzyme for immunological detection of antibodies. Alternatively, a secondary antibody labeled with a fluorescent material, a chromogenic substrate, and the like may be included. The substrate is not particularly limited thereto, but a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with polystyrene resin, and a slide glass made of glass may be used, and the chromogenic enzyme is not particularly limited thereto. However, peroxidase and alkaline phosphatase may be used, and the fluorescent material may be, but is not particularly limited to, FITC, RITC, etc., and the color-developing substrate solution is not particularly limited thereto, but ABTS (2,2' -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine) or TMB (tetramethyl benzidine).

본 발명의 구체적인 실시예에서는 질량분석기를 사용한 MRM 방법으로 14-3-3 단백질의 7종 아형을 동시에 정량분석함으로써 심혈관 질환 고위험군의 진단, 심근 비대 및 심근조직 괴사의 진단에 활용할 수 있음을 확인하였다.In a specific embodiment of the present invention, it was confirmed that 7 subtypes of 14-3-3 protein can be simultaneously quantitatively analyzed by the MRM method using mass spectrometry, which can be used for diagnosis of high-risk group of cardiovascular disease, myocardial hypertrophy, and myocardial tissue necrosis. .

본 발명의 제4양태는 심혈관 질환 발병이 의심되는 개체로부터 분리된 생체 시료를 제1양태의 조성물과 반응시켜 상기 시료 중의 14-3-3 단백질의 수준을 측정하는 제1단계; 및 상기 제1단계로부터 측정된 14-3-3 단백질의 수준이 정상 개체에 대해 측정된 14-3-3 단백질의 수준과 비교하여 상기 제1단계로부터 측정된 14-3-3 단백질의 수준이 더 높은 경우 개체에 심혈관 질환이 발병한 것으로 판단하는 제2단계를 포함하는, 심혈관 질환 진단을 위한 정보제공방법을 제공한다.A fourth aspect of the present invention is a first step of reacting a biological sample isolated from an individual suspected of having cardiovascular disease with the composition of the first aspect to measure the level of 14-3-3 protein in the sample; and comparing the level of 14-3-3 protein measured from the first step to the level of 14-3-3 protein measured for a normal subject, the level of 14-3-3 protein measured from the first step is Provided is an information providing method for diagnosing cardiovascular disease, including a second step of determining that the subject has a cardiovascular disease in the case of a higher level.

본 발명의 용어, "개체"는 심혈관 질환을 진단하고자 하는 대상을 의미한다. 이때, 상기 개체는 사람을 비롯하여, 개, 말, 소, 쥐, 염소, 토끼, 닭, 오리, 거위 등의 알츠하이머성 치매가 발병될 수 있는 동물이라면 제한 없이 포함될 수 있다.As used herein, the term "subject" refers to a subject for whom cardiovascular disease is to be diagnosed. In this case, the subject may be included without limitation as long as it is an animal that can develop Alzheimer's dementia, such as a human, dog, horse, cow, rat, goat, rabbit, chicken, duck, goose, and the like.

본 발명의 용어, "정상 개체"란 심혈관 질환이 발병한 것으로 진단되지 않은 개체를 의미하며, 정상 개체로부터 분리된 시료와 심혈관 질환 발병이 의심되는 개체로부터 분리된 시료에서 상기 유전자의 mRNA 또는 이로부터 발현되는 단백질의 발현 수준을 측정하고, 비교함으로써, 심혈관 질환 발병이 의심되는 개체의 심혈관 질환 발병 여부를 정확하게 예측할 수 있다.As used herein, the term "normal subject" refers to an individual who has not been diagnosed with cardiovascular disease, and in a sample isolated from a normal individual and a sample isolated from an individual suspected of having cardiovascular disease, the mRNA of the gene or from this By measuring and comparing the expression level of the expressed protein, it is possible to accurately predict whether a subject suspected of having a cardiovascular disease develops a cardiovascular disease.

상기 생체 시료는 세포, 세포 용해물, 세포 분비물 또는 혈액, 혈청 또는 소변으로부터 분리된 엑소좀을 포함할 수 있다.The biological sample may include cells, cell lysates, cell secretions, or exosomes isolated from blood, serum, or urine.

예컨대, mRNA 발현수준 측정은 생체 시료에서 바이오마커인 14-3-3 단백질을 코딩하는 유전자의 mRNA 존재 여부와 발현 정도를 확인하는 과정으로 mRNA의 양을 측정함으로써 알 수 있다. 이를 위한 분석 방법으로는 역전사효소 중합효소반응(RT-PCR), 경쟁적 역전사효소 중합효소반응(competitive RT-PCR), 실시간 역전사효소 중합효소반응(real time quantitative RT-PCR), RNase 보호 분석법(RNase protection method), 노던 블롯팅(Northern blotting), DNA칩 분석법(DNA chip technology assay) 등이 있다.For example, mRNA expression level measurement is a process of confirming the presence and expression level of mRNA of a gene encoding 14-3-3 protein, which is a biomarker, in a biological sample, and can be known by measuring the amount of mRNA. Analysis methods for this include reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time reverse transcriptase polymerase reaction (real time quantitative RT-PCR), RNase protection assay (RNase protection method), Northern blotting, and DNA chip technology assay.

예컨대, 단백질 발현수준 측정은 생체 시료에서 바이오마커인 14-3-3 단백질의 존재 여부와 발현 정도를 확인하는 과정으로, 상기 단백질에 대하여 특이적으로 결합하는 항체를 이용하여 단백질의 양을 확인할 수 있다. 이를 위한 분석 방법으로는 웨스턴 블랏(western blotting), ELISA(enzyme linked immunosorbent assay), 방사선면역분석(RIA: radioimmunoassay), 방사 면역 확산법(radial immunodiffusion), 오우크테로니 면역 확산법(Ouchterlony immunodiffusion), 로케트 면역전기영동(rocket immunoelectrophoresis), 면역조직화학염색법(immunohistochemical staining), 면역침전분석법(immunoprecipitation assay), 보체 고정 분석법(complement Fixation Assay), 면역형광법(immunofluorescence), 면역크로마토그래피법(immunochromatography), FACS 분석법(fluorescenceactivated cell sorter analysis) 및 단백질 칩 분석법(protein chip technology assay) 등이 있으나, 이에 제한되는 것은 아니다.For example, protein expression level measurement is a process of confirming the presence and expression level of 14-3-3 protein, which is a biomarker, in a biological sample, and the amount of protein can be confirmed using an antibody that specifically binds to the protein. have. Analysis methods for this include western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Outterlony immunodiffusion, rocket Rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, FACS assay (fluorescence activated cell sorter analysis) and protein chip technology assay, but are not limited thereto.

본 발명의 제5양태는 14-3-3 단백질 억제제를 유효성분으로 포함하는, 심혈관 질환의 예방 또는 치료용 약학적 조성물을 제공한다.A fifth aspect of the present invention provides a pharmaceutical composition for preventing or treating cardiovascular disease, comprising a 14-3-3 protein inhibitor as an active ingredient.

상기 14-3-3 단백질 억제제는 14-3-3 단백질의 활성을 저해하거나, 14-3-3 단백질을 코딩하는 유전자에 작용하여 14-3-3 단백질의 발현을 저해하는 물질일 수 있으나, 14-3-3 단백질의 활성을 저해하거나 발현을 감소시킴으로써 심혈관 질환을예방하거나 치료할 수 있는 한, 작용기전 및/또는 방식에 제한되지 않는다. 예컨대, 상기 억제제는 14-3-3 단백질에 특이적으로 결합하여 활성을 저해할 수 있는 단백질이나 소분자 화합물 또는, 14-3-3 단백질의 발현을 억제할 수 있는 siRNA(small interfering RNA 또는 silencing RNA) 또는 shRNA(short hairpin RNA 또는 small haripin RNA) 등 다양한 형태의 물질 또는 이들의 조합일 수 있으나, 이에 제한되지 않는다.The 14-3-3 protein inhibitor may be a substance that inhibits the activity of the 14-3-3 protein or inhibits the expression of the 14-3-3 protein by acting on a gene encoding the 14-3-3 protein. As long as cardiovascular disease can be prevented or treated by inhibiting the activity or reducing the expression of the 14-3-3 protein, the mechanism and/or mode of action are not limited. For example, the inhibitor may be a protein or small molecule compound capable of specifically binding to and inhibiting the activity of the 14-3-3 protein, or a siRNA (small interfering RNA or silencing RNA) capable of inhibiting the expression of the 14-3-3 protein. ) or shRNA (short hairpin RNA or small haripin RNA), or various types of substances or combinations thereof, but is not limited thereto.

본 발명의 용어 "개체"란, 상기 심혈관 질환이 발명하였거나 발병할 수 있는 인간을 포함한 원숭이, 소, 말, 양, 돼지, 닭, 칠면조, 메추라기, 고양이, 개, 마우스, 쥐, 토끼 또는 기니아 피그를 포함한 모든 동물을 의미하고, 본 발명의 약학적 조성물을 개체에게 투여함으로써 상기 질환을 효과적으로 예방 또는 치료할 수 있다. 본 발명의 약학적 조성물은 기존의 치료제와 병행하여 투여될 수 있다.The term "subject" of the present invention refers to monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs, including humans who have developed or may develop the above cardiovascular disease. It means all animals, including, and the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to a subject. The pharmaceutical composition of the present invention may be administered in parallel with existing therapeutic agents.

본 발명의 용어 "예방"이란 본 발명의 조성물의 투여로 심혈관 질환의 발생, 확산 및 재발을 억제시키거나 지연시키는 모든 행위를 의미하고, "치료"란 본 발명의 조성물의 투여로 상기 질환의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.The term "prevention" of the present invention refers to any action that inhibits or delays the occurrence, spread, and recurrence of cardiovascular disease by administration of the composition of the present invention, and "treatment" refers to symptoms of the disease by administration of the composition of the present invention. means any action that improves or changes favorably.

본 발명의 약학적 조성물은 14-3-3 단백질의 활성 및/또는 발현을 저해으로써 이와 관련 질환을 예방 또는 치료할 수 있다.The pharmaceutical composition of the present invention inhibits the activity and/or expression of 14-3-3 protein, thereby preventing or treating diseases related thereto.

바람직하게, 본 발명에 따른 약학적 조성물은 유효성분으로서 14-3-3 단백질 억제제를 조성물의 총중량을 기준으로 0.1 내지 75 중량%로, 보다 바람직하게는 1 내지 50 중량%로 함유할 수 있다.Preferably, the pharmaceutical composition according to the present invention may contain the 14-3-3 protein inhibitor as an active ingredient in an amount of 0.1 to 75% by weight, more preferably 1 to 50% by weight, based on the total weight of the composition.

본 발명의 조성물은 약학적으로 허용가능한 담체, 희석제 또는 부형제를 추가로 포함할 수 있으며, 각각의 사용 목적에 맞게 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁제, 에멀젼, 시럽, 에어로졸 등의 경구 제형, 멸균 주사 용액의 주사제 등 다양한 형태로 제형화하여 사용할 수 있으며, 경구 투여하거나 정맥내, 복강내, 피하, 직장, 국소 투여 등을 포함한 다양한 경로를 통해 투여될 수 있다. 이러한 조성물에 포함될 수 있는 적합한 담체, 부형제 또는 희석제의 예로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸셀룰로즈, 미정질셀룰로스, 폴리비닐피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 들 수 있다. 또한, 본 발명의 조성물은 충전제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.The composition of the present invention may further include a pharmaceutically acceptable carrier, diluent or excipient, and may be prepared according to a conventional method according to each purpose of use, such as powder, granule, tablet, capsule, suspension, emulsion, syrup, It can be formulated and used in various forms such as oral formulations such as aerosols and injections of sterile injection solutions, and can be administered through various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like. Examples of suitable carriers, excipients or diluents that may be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil; and the like. In addition, the composition of the present invention may further include fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like.

경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면 전분, 탄산칼슘, 수크로스, 락토즈, 젤라틴 등을 혼합하여 제형화한다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크와 같은 윤활제가 사용될 수 있다.Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the composition, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. Formulated by mixing. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

경구용 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 예시될 수 있으며, 흔히 사용되는 단순 희석제인 물, 액체 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Oral liquid preparations may include suspensions, solutions for internal use, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. can

비경구 투여를 위한 제제에는 멸균된 수용액제, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈61. 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. 한편, 주사제에는 용해제, 등장화제, 현탁화제, 유화제, 안정화제, 방부제 등과 같은 종래의 첨가제가 포함될 수 있다.Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. The suppositories are Witepsol, Macrogol, and Tween 61. Cacao fat, laurin fat, glycerogeratin and the like can be used. Meanwhile, conventional additives such as solubilizers, tonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives may be included in the injection.

이때, 본 발명의 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명의 용어 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.At this time, the composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dose level is the patient's health condition, Depending on the type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field can The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

예컨대, 투여 경로, 질병의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.For example, the dosage may increase or decrease depending on the route of administration, severity of disease, sex, weight, age, etc., so the dosage is not limited to the scope of the present invention in any way.

구체적으로, 본 발명의 조성물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 kg 당 1 내지 100 mg, 바람직하게는 5 내지 60 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나, 투여 경로, 질병의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the composition of the present invention may vary depending on the patient's age, sex, and weight, and is generally 1 to 100 mg, preferably 5 to 60 mg per kg of body weight, administered daily or every other day, or administered once a day. It can be divided into 3 doses. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

본 발명의 제6양태는 심혈관 질환 치료용 후보물질을 14-3-3 단백질을 과발현하는 세포에 처리하는 제1단계; 상기 심혈관 질환 치료용 후보물질을 처리 전과 후 14-3-3 단백질을 과발현하는 세포의 14-3-3 단백질 수준을 측정하여 비교하는 제2단계; 및 상기 심혈관 질환 치료용 후보물질 처리 후 세포에서 측정된 14-3-3 단백질 수준이 심혈관 질환 치료용 후보물질 처리 전 세포에 비해 감소된 경우 심혈관 질환 치료 효과를 갖는 것으로 판단하는 제3단계를 포함하는, 심혈관 질환 치료제의 스크리닝 방법을 제공한다.A sixth aspect of the present invention is a first step of treating cells overexpressing 14-3-3 protein with a candidate substance for treating cardiovascular disease; A second step of measuring and comparing 14-3-3 protein levels of cells overexpressing 14-3-3 protein before and after treatment with the candidate substance for treating cardiovascular disease; and a third step of judging that the 14-3-3 protein level measured in the cells after treatment with the candidate substance for treatment of cardiovascular disease is reduced compared to the cells before treatment with the candidate substance for treatment of cardiovascular disease, and determining that the cell has a therapeutic effect on cardiovascular disease. To provide a screening method for a therapeutic agent for cardiovascular disease.

예컨대, 상기 14-3-3 단백질은 14-3-3 베타, 14-3-3 입실론, 및 14-3-3 제타로 구성된 군으로부터 선택되는 하나 이상일 수 있다.For example, the 14-3-3 protein may be one or more selected from the group consisting of 14-3-3 beta, 14-3-3 epsilon, and 14-3-3 zeta.

예컨대, 상기 14-3-3 단백질을 과발현하는 세포는 정상 심근 세포에 안지오텐신 II, 엔도텔린-1 또는 둘 모두를 처리하여 준비한 심근 비대 세포일 수 있으나, 이에 제한되지 않는다.For example, cells overexpressing the 14-3-3 protein may be myocardial hypertrophic cells prepared by treating normal myocardial cells with angiotensin II, endothelin-1, or both, but are not limited thereto.

본 발명의 용어, "후보물질"은 임의의 물질(substance), 분자(molecule), 원소(element), 화합물(compound), 실재물(entity) 또는 이들의 조합을 포함한다. 예컨대, 이들로 한정되지는 않으나, 단백질, 폴리펩티드, 소 유기분자(small organic molecule), 다당류(polysaccharide), 폴리뉴클레오티드 등을 포함할 수 있다. 또한, 천연 산물(natural product), 합성 화합물 또는 화학 화합물 또는 2개 이상의 물질의 조합일 수도 있다. 달리 지시되지 않는 한, 제제, 물질 및 화합물은 호환성 있게(interchangeably) 사용할 수 있다. 예컨대, 상기 후보물질은 합성 또는 천연 화합물의 라이브러리를 포함하는 광범위하고 다양한 출처로부터 얻어질 수 있다.As used herein, the term “candidate” includes any substance, molecule, element, compound, entity, or combination thereof. Examples include, but are not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, a synthetic compound or a chemical compound or a combination of two or more substances. Unless otherwise indicated, agents, materials and compounds can be used interchangeably. For example, the candidates can be obtained from a wide variety of sources including libraries of synthetic or natural compounds.

본 발명은 심혈관 질환 진단을 위한 새로운 바이오마커로서 14-3-3 단백질을 발굴하였으며, 이는 정상 심근 세포에 비해 심근 비대 세포에서 현저히 발현이 증가될 뿐만 아니라 다른 조직 세포 및 이의 암세포에 비해서도 현저히 증가된 발현량을 나타내므로 심혈관 질환의 진단에 유용하게 사용될 수 있으며, 나아가 이를 이용한 심혈관 질환 예방 또는 치료 용도 및 심혈관 질환 치료제의 스크리닝에도 활용될 수 있다.The present invention discovered 14-3-3 protein as a new biomarker for diagnosing cardiovascular disease, and its expression was significantly increased in myocardial hypertrophic cells compared to normal myocardial cells, as well as significantly increased compared to other tissue cells and their cancer cells. Since it shows the expression level, it can be usefully used for diagnosing cardiovascular diseases, and furthermore, it can be used for preventing or treating cardiovascular diseases and screening cardiovascular disease therapeutics.

도 1은 정상 심장 세포주(T0445, control) 및 이에 심근비대증을 유도하는 것으로 알려진 엔도테린-1(endothelin-1, ET-1) 또는 안지오텐신 II(angiotensin II, Ang II)를 처리한 지 각각 4시간 및 24시간 후, (A) 정상 심장 세포와 심근비대증이 유도된 세포의 면역염색법 결과, 및 (B) 세포 면적의 평균값을 나타낸 도이다. ET-1 또는 Ang II 처리 후 4시간 만에 심장 세포의 비대증이 유도되었고, 해당 효과는 24시간까지 지속되었다.
도 2는 정상 세포로부터 심근비대증이 유도되는 과정의 각 시점에서 세포로부터의 분비물(secretome)에서, 심혈관질환 환자에서 발현이 증가하는 것으로 알려진 바이오마커인, 양성 나트륨이뇨 펩티드(benign natriuretic peptide; BNP) 및 심방 나트륨이뇨 펩티드(atrial natriuretic peptide; ANP)를 웨스턴 블롯팅 방법으로 분석한 결과를 나타낸 도이다. (A-1 및 A-2)는 ET-1을 처리 후 시간에 따른 마커의 발현의 변화를, (B-1 및 B-2)는 Ang II를 처리 후 시간에 따른 마커의 발현의 변화를 반(semi)-정량적으로 나타낸다. ET-1 및 Ang II 처리 후 15분 후로부터 BNP 및 ANP의 발현량이 모두 증가하기 시작하였으며 24시간까지 발현량의 증가와 감소를 2번 반복하였다.
도 3은 나노-액체 크로마토그래피-고분해능 질량분석기법을 활용하여 정상 심장 세포와 심근 비대 유도 세포에서 유래된 분비물을 비교·분석하고, 발현량에서 차이를 보이는 예컨대, 심근 비대 유도 세포에서 유의미하게 증가한, 단백질을 동정한 결과를 나타낸 도이다. 구체적으로, 세포를 분주하고 각각 2개 플레이트씩 ET-1 또는 Ang II를 처리하여 심근 비대를 유도하여, 정상 심장 세포주와 함께 분석하였다. 총 4개 플레이트의 심근 비대 세포 시료를 2개 플레이트의 정상 세포주와 비교하여 존재비(abundance ratio)＞1.4의 심근 비대 유도 세포에서 발현이 증가된 단백질 중, 공통된 것을 선별하여 32개의 단백질을 검출하였다.
도 4는 도 3에 개시한 본 발명의 일 실시예에 따라 스크리닝된 심근 비대 유도 세포에서 과발현된 32가지 단백질 중 가장 존재비가 높은 14-3-3 단백질, (A) 14-3-3 감마(gamma, m/z 745.72), (B) 14-3-3 입실론(epsilon, m/z 696.65) 및 (C) 14-3-3 세타(theta, m/z 715.66)의 펩타이드 MS/MS 서열분석(sequencing) 결과를 나타낸 도이다. 공지의 심혈관질환 바이오마커 후보(볼드로 표시)는 제외하였으며, 전하(charge)와 교차 상관 스코어(cross-correalation score; Xcorr) 값이 가장 높은 펩티드 단편을 선택하였다.
도 5는 도 3에 개시한 본 발명의 일 실시예에 따라 스크리닝된 심근 비대 유도 세포에서 과발현된 32가지 단백질 중 가장 존재비가 높은 14-3-3 단백질, (A) 14-3-3 에타(eta, m/z 720.66), (B) 14-3-3 제타(zeta, m/z 711.33) 및 (C) 14-3-3 베타(beta, m/z 796.43)의 펩타이드 MS/MS 서열분석(sequencing) 결과를 나타낸 도이다. 공지의 심혈관질환 바이오마커 후보(볼드로 표시)는 제외하였으며, 전하와 교차 상관 스코어(Xcorr) 값이 가장 높은 펩티드 단편을 선택하였다.
도 6은 다양한 조직 세포 시료의 분비물과 세포 용해물(cell lysate)에서 발굴된 14-3-3 단백질 바이오마커 후보군 펩타이드들의 MRM 정량분석 결과를 나타낸 도이다. 14-3-3 단백질의 발현을 폐 정상 세포(L132), 폐암 세포(A549), 간암 세포(Hep-3B), 간 정상 세포(HRG), 위 정상 세포(HFE-145), 위암 세포(MKN-1), 심장 정상 세포(T0445)와 각각 ET-1와 Ang II로 심근 비대를 유도한 세포에 대해 비교·분석하였다. (A) 내지 (C)는 각각 14-3-3 입실론, 14-3-3 제타, 및 14-3-3 에타의 발현량을 나타내며, 좌측의 회색 바는 세포 용해물에서, 우측의 검은색 바는 분비물에 대한 결과는 나타낸다. 14-3-3 입실론, 14-3-3 제타, 및 14-3-3 베타의 경우, 분비물에서 다른 조직의 세포보다 심근 비대 유도 심장 세포에서 현저하게 높은 양의 14-3-3 단백질 동형(isotype)이 발현되었다.
도 7은 다양한 조직 세포 시료의 분비물과 세포 용해물에서 발굴된 14-3-3 단백질 바이오마커 후보군 펩타이드들의 MRM 정량분석 결과를 나타낸 도이다. 14-3-3 단백질의 발현을 폐 정상 세포(L132), 폐암 세포(A549), 간암 세포(Hep-3B), 간 정상 세포(HRG), 위 정상 세포(HFE-145), 위암 세포(MKN-1), 심장 정상 세포(T0445)와 각각 ET-1와 Ang II로 심근 비대를 유도한 세포에 대해 비교·분석하였다. (A) 및 (B)는 각각 14-3-3 베타, 및 14-3-3 세타의 발현량을 나타내며, 좌측의 회색 바는 세포 용해물에서, 우측의 검은색 바는 분비물에 대한 결과는 나타낸다. 14-3-3 입실론, 14-3-3 제타, 및 14-3-3 베타의 경우, 분비물에서 다른 조직의 세포보다 심근 비대 유도 심장 세포에서 현저하게 높은 양의 14-3-3 단백질 동형이 발현되었다.
도 8은 나노-액체크로마토그래피-고분해능 질량분석기로 측정한 secretome 단백질 분석결과를 IPA 프로그램으로 인-실리코 경로(in silico pathway)를 분석한 결과를 나타낸 도이다. (A) ET-1 처리 후, 및 (B) Ang II 처리 후의 세포 모두에서, Hippo 신호전달(signaling)이 현저하게 저하되었고, 당분해(glycolysis), 당신생합성(gluconeogenesis), 액틴 세포골격 신호전달(actin cytoskeleton signaling), 및 14-3-3 매개 신호전달이 현저하게 증가하였다. 이들은 모두 세포의 크기 조절, 증식, 및 사멸(apotosis)과 관련된 신호전달과 매우 밀접한 관계를 갖는 네트워크에 해당하여, 세포의 크기가 비대해 지면서, 이를 억제하기 위한 또는 세포의 사멸을 막기 위한 신호전달 네트워크가 활성화 된 것으로 보인다.
도 9는 마이크로-액체 크로마토그래피-질량분석기를 이용하여, 정상 개체(healthy control; HC) 및 급성 심근경색 환자군(acute myocardial infarction; AMI)으로부터 채취한 혈액으로부터 측정한 혈청 내 14-3-3 베타, 14-3-3 입실론, 및 14-3-3 제타의 발현량을 비교 분석한 결과를 나타낸 도이다(각각 n=5). 정상 개체에 비해 급성 심근경색 환자군에서 상기 3종 아형 모두의 발현이 현저히 증가하여 7 내지 8배 높게 검출되었다.Figure 1 shows a normal heart cell line (T0445, control) and its treatment with endothelin-1 (ET-1) or angiotensin II (angiotensin II, Ang II) known to induce myocardial hypertrophy for 4 hours, respectively. and 24 hours later, (A) immunostaining results of normal heart cells and cells with myocardial hypertrophy induced, and (B) a diagram showing the average value of the cell area. Cardiac cell hypertrophy was induced 4 hours after treatment with ET-1 or Ang II, and the effect lasted up to 24 hours.
Figure 2 is a positive natriuretic peptide (BNP), a biomarker known to increase expression in patients with cardiovascular disease, in secretomes from cells at each time point of the process of inducing myocardial hypertrophy from normal cells. and atrial natriuretic peptide (ANP) analysis by Western blotting. (A-1 and A-2) show changes in marker expression over time after treatment with ET-1, and (B-1 and B-2) show changes in marker expression over time after treatment with Ang II. Shown semi-quantitatively. The expression levels of both BNP and ANP began to increase from 15 minutes after ET-1 and Ang II treatment, and the increase and decrease in expression levels were repeated twice until 24 hours.
Figure 3 compares and analyzes secretions derived from normal cardiac cells and myocardial hypertrophy-induced cells using nano-liquid chromatography-high-resolution mass spectrometry, and shows a difference in expression level, for example, significantly increased in myocardial hypertrophy-induced cells , It is a diagram showing the result of identifying the protein. Specifically, the cells were divided and treated with ET-1 or Ang II to induce myocardial hypertrophy, and analyzed together with normal cardiac cell lines. A total of four plates of myocardial hypertrophy cell samples were compared with normal cell lines of two plates, and among the proteins whose expression was increased in myocardial hypertrophy-induced cells with an abundance ratio > 1.4, common ones were selected and 32 proteins were detected.
4 is a 14-3-3 protein with the highest abundance among 32 proteins overexpressed in myocardial hypertrophy-inducing cells screened according to an embodiment of the present invention disclosed in FIG. 3, (A) 14-3-3 gamma ( gamma, m/z 745.72), (B) 14-3-3 epsilon (m/z 696.65) and (C) 14-3-3 theta (theta, m/z 715.66) MS/MS sequence analysis It is a diagram showing the result of sequencing. Known cardiovascular disease biomarker candidates (shown in bold) were excluded, and a peptide fragment with the highest charge and cross-correlation score (Xcorr) value was selected.
5 is a 14-3-3 protein with the highest abundance among 32 proteins overexpressed in myocardial hypertrophy-inducing cells screened according to one embodiment of the present invention disclosed in FIG. 3, (A) 14-3-3 eta ( eta, m/z 720.66), (B) 14-3-3 zeta (zeta, m/z 711.33) and (C) 14-3-3 beta (beta, m/z 796.43) Peptide MS/MS sequencing It is a diagram showing the result of sequencing. Known cardiovascular disease biomarker candidates (shown in bold) were excluded, and a peptide fragment having the highest charge and cross-correlation score (Xcorr) value was selected.
6 is a diagram showing the results of MRM quantitative analysis of 14-3-3 protein biomarker candidate peptides discovered from secretions and cell lysates of various tissue cell samples. 14-3-3 Protein expression was analyzed in lung normal cells (L132), lung cancer cells (A549), liver cancer cells (Hep-3B), liver normal cells (HRG), gastric normal cells (HFE-145), and gastric cancer cells (MKN). -1), cardiac normal cells (T0445) and cells induced myocardial hypertrophy with ET-1 and Ang II, respectively, were compared and analyzed. (A) to (C) represent the expression levels of 14-3-3 epsilon, 14-3-3 zeta, and 14-3-3 eta, respectively, with gray bars on the left in cell lysates and black bars on the right Bars represent results for secretions. For 14-3-3 epsilon, 14-3-3 zeta, and 14-3-3 beta, significantly higher amounts of 14-3-3 protein isoforms ( isotype) was expressed.
7 is a diagram showing the results of MRM quantitative analysis of 14-3-3 protein biomarker candidate peptides discovered from secretions and cell lysates of various tissue cell samples. 14-3-3 Protein expression was analyzed in lung normal cells (L132), lung cancer cells (A549), liver cancer cells (Hep-3B), liver normal cells (HRG), gastric normal cells (HFE-145), and gastric cancer cells (MKN). -1), cardiac normal cells (T0445) and cells induced myocardial hypertrophy with ET-1 and Ang II, respectively, were compared and analyzed. (A) and (B) show the expression levels of 14-3-3 beta and 14-3-3 theta, respectively. The gray bar on the left is for the cell lysate and the black bar on the right is for the secretion. indicate For 14-3-3 epsilon, 14-3-3 zeta, and 14-3-3 beta, significantly higher amounts of 14-3-3 protein isoforms were found in myocardial hypertrophy-induced cardiac cells than in cells of other tissues in the secretion. has been manifested
8 is a diagram showing the results of analyzing the in silico pathway of secretome protein analysis results measured by nano-liquid chromatography-high-resolution mass spectrometry using an IPA program. In both cells after (A) ET-1 treatment and (B) Ang II treatment, Hippo signaling was significantly reduced, and glycolysis, gluconeogenesis, and actin cytoskeletal signaling (actin cytoskeleton signaling) and 14-3-3-mediated signaling were markedly increased. All of these correspond to a network that is very closely related to signal transduction related to cell size control, proliferation, and apoptosis. The network appears to be active.
Figure 9 shows 14-3-3 beta in serum measured from blood collected from normal subjects (healthy control; HC) and acute myocardial infarction (AMI) patients using micro-liquid chromatography-mass spectrometry. , 14-3-3 epsilon, and 14-3-3 zeta expression levels are compared and analyzed (n=5 each). Compared to normal subjects, the expression of all three subtypes was significantly increased in the acute myocardial infarction patient group and was detected 7 to 8 times higher.

이하, 하기 실시예에 의하여 본 발명을 더욱 상세하게 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited only to these.

제조예 1: 심근비대 유도 세포의 제조Preparation Example 1: Preparation of myocardial hypertrophy-inducing cells

인간 정상 심장 세포주 T0445를 60 mm 배양 접시에 60%까지 차도록(confluent) 배양한 후, 비대작용제로서 1 μM 안지오텐신 II(angiotensin II; Ang II) 및 200 nM 엔도텔린-1(endothelin-1; ET-1)(Sigma-Aldrich, Germany)으로 처리하였다. 면역 세포 화학 염색을 위하여 상기 배양한 세포를 유리 커버 스립에 분주(seeding)하고 비대작용제와 함께 각각 4시간 및 24시간 동안 인큐베이션하였다. 이후 4% 포름알데히드(Sigma, USA)로 20분 동안 처리하여 세포를 고정하고, 면역 형광 염색을 수행하였다. 비특이적 결합을 차단하기 위하여 항체희석완충액(antibody dilution buffer; Abdil, 0.1% Triton X-100을 함유하는 2% 소혈청알부민 용액)을 30분 동안 사용하였다. 이후, 세포를 Abdil 용액에 혼합된 항-유전성 알파-액티닌(Abcam, USA)과 배양하고, 4℃에서 밤새도록 유지하였다. 트리스완충식염수(tris buffered saline; TBS)를 사용하여 종합적으로 세척하고, Abdil 용액에 혼합된 2차 항체, Alexa Fluor 488 염소 항-마우스 IgG(H+L)(Thermo Fusher Scientific, USA)를 실온에서 2시간 동안 사용하였다. 핵을 Hoechst 33342(Thermo Fisher Scientific, USA)로 10분 동안 염색하고, 장착 용액을 이용하여 커버 슬립을 장착하고, 니콘 도립 현미경 Eclipse Ti-U(Carl Zeiss, Germany)를 사용하여 10× 및 20× 배율로 이미지를 획득하였다. 이미지 J 소프트웨어(Java-based image processing program, USA)를 사용하여 상기 획득한 이미지로부터 제곱 마이크로미터에서의 세포 면적을 산출하였다. 나아가, 평균 세포 표면적을 대조군에 대해 계산하고 각 세포 유형을 처리하였다. 하나의 유리 슬라이드로부터 3개의 영역을 계산하고, 총 3개의 유리 슬라이드를 측정하였다.Human normal heart cell line T0445 was cultured in a 60 mm culture dish to 60% confluent, and then 1 μM angiotensin II (Ang II) and 200 nM endothelin-1 (ET-1) were added as hypertrophic agents. 1) (Sigma-Aldrich, Germany). For immunocytochemical staining, the cultured cells were seeded on glass coverslips and incubated with hypertrophic agents for 4 hours and 24 hours, respectively. Thereafter, cells were fixed by treatment with 4% formaldehyde (Sigma, USA) for 20 minutes, and immunofluorescence staining was performed. In order to block non-specific binding, antibody dilution buffer (Abdil, 2% bovine serum albumin solution containing 0.1% Triton X-100) was used for 30 minutes. Then, the cells were incubated with anti-hereditary alpha-actinin (Abcam, USA) mixed in Abdil solution and kept overnight at 4°C. After comprehensive washing with tris buffered saline (TBS), the secondary antibody, Alexa Fluor 488 goat anti-mouse IgG (H+L) (Thermo Fusher Scientific, USA) mixed in Abdil solution was incubated at room temperature. Used for 2 hours. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, USA) for 10 min, mounted on cover slips using mounting solution, and imaged at 10× and 20× using a Nikon inverted microscope Eclipse Ti-U (Carl Zeiss, Germany). Images were acquired at magnification. The cell area in square micrometers was calculated from the acquired images using Image J software (Java-based image processing program, USA). Further, the average cell surface area was calculated relative to the control and treated each cell type. Three areas were calculated from one glass slide, and a total of three glass slides were measured.

실시예 1: 세포주 배양, 단백질 추출 및 정량Example 1: Cell line culture, protein extraction and quantification

심근 비대 세포에서 특이적으로 발현하는 마커를 발굴하기 위한 비교예로서 한국 세포주 은행(KCLB, Seoul)에서 얻은 인간 정상 폐 상피 세포주 L132 및 인간 폐암 세포주 A549, 인간 위암 세포주 MKN-1, 인간 정상 위 세포주 HFE-145, 및 인간 간암 세포주 Hep3B와 인간 정상 간 세포주 Hepa RG(Thermo Fisher Scientific, MA, USA) 및 불멸화된 인간 심장 세포주 T0445(Abcam, CA)를 사용하였다. 구체적으로, 상기 일련의 세포주를 10% 열-불활성화 FBS 및 1% 페니실린-스트렙토마이신(Gibco, USA) 함유 RPMI, DMEM, William's medium E, 및 Prigrow 배지에 정기적으로 배양하였다. 배양 플레이트는 가습 세포 인큐베이터에서 5% CO2로 37℃에서 유지되었다. 60 mm 플라스티 배양 접시(Corning Life Science, Acton, MA, USA)에서 컨플루언시가 80% 이상될 때까지(약 2×106 세포/cm2) 배양하였다. HFE-145, MKN-1, L132, A549, Hep3B, Hepa RG, T0445 및 ET-1, Ang II 유도 T0445 세포의 분비물(secretome)을 차가운 PBS를 사용하여 수집하고, 다시 PBS로 2회 세척한 후, TNN-EDTA 용해 완충액(Thermo Scientific, Germany), 프로테아제 및 포스파타제 억제제 칵테일(Roche Diagnostics GmbH, Germany)을 혼합하여 세포막을 분해하고 단백질을 수집하였다. 상층액을 수집하기 위해 시료를 4℃에서 15분 동안 10,000×g로 원심분리하였다. 마지막으로 바이신코닉산(bicinchoninic acid; BCA) 키트(Thermo Scientific, USA)를 사용하여 제조업체의 매뉴얼에 따라 수집한 상층액으로부터 단백질 농도를 측정하였다.As comparative examples for discovering markers specifically expressed in myocardial hypertrophic cells, human normal lung epithelial cell line L132 and human lung cancer cell line A549 obtained from Korea Cell Line Bank (KCLB, Seoul), human gastric cancer cell line MKN-1, and human normal gastric cell line HFE-145, and human liver cancer cell line Hep3B and human normal liver cell line Hepa RG (Thermo Fisher Scientific, MA, USA) and immortalized human cardiac cell line T0445 (Abcam, CA) were used. Specifically, the series of cell lines were regularly cultured in RPMI, DMEM, William's medium E, and Prigrow medium containing 10% heat-inactivated FBS and 1% penicillin-streptomycin (Gibco, USA). Culture plates were maintained at 37° C. with 5% CO 2 in a humidified cell incubator. It was cultured in a 60 mm plastic culture dish (Corning Life Science, Acton, MA, USA) until confluency reached 80% or more (about 2×10 6 cells/cm 2 ). The secretome of HFE-145, MKN-1, L132, A549, Hep3B, Hepa RG, T0445 and ET-1, Ang II-induced T0445 cells was collected using cold PBS, washed twice with PBS, and , TNN-EDTA lysis buffer (Thermo Scientific, Germany), and a protease and phosphatase inhibitor cocktail (Roche Diagnostics GmbH, Germany) were mixed to dissolve the cell membrane and collect proteins. Samples were centrifuged at 10,000×g for 15 minutes at 4° C. to collect the supernatant. Finally, the protein concentration was measured from the collected supernatant using a bicinchoninic acid (BCA) kit (Thermo Scientific, USA) according to the manufacturer's manual.

실시예 2: 분비물 준비Example 2: secretion preparation

세포 배양 및 세포 배지(cell media; CM) 수집의 첫 단계는 신뢰할 수 있는 분비물(secretome) 분석에 가장 중요하며, 분석할 시료의 품질에 큰 영향을 받는다. 배지를 수집하기 전에, 완전히 합류된 세포 플레이트 배지를 24시간 동안 무혈청 배지로 교체하였다. 불멸화된 인간 심근 세포에서 비대를 유도하기 위해, 1 μM Ang II 또는 200 nM ET-1을 상이한 시간 간격으로 첨가하였다. 소정의 시간 동안 배야한 후, CM을 수집하고, 4000 RCF로 4℃에서 10분 동안 원심분리하여 온전한 세포를 펠렛화한 후, 상층액을 수집하고 임의의 세포 잔해물을 제거하였다. 수집한 상층액을 새로운 튜브에 옮기고 동결 건조기(CHRIST ALPHA 1-2 LD plus, Sigma)로 동결 건조시켰다.The first step of cell culture and cell media (CM) collection is the most important for reliable secretome analysis and is greatly influenced by the quality of the sample to be analyzed. Before collecting media, fully confluent cell plate media was replaced with serum-free media for 24 hours. To induce hypertrophy in immortalized human cardiomyocytes, 1 μM Ang II or 200 nM ET-1 were added at different time intervals. After incubation for the specified amount of time, the CM was collected and centrifuged at 4000 RCF for 10 minutes at 4° C. to pellet intact cells, then the supernatant was collected and any cell debris removed. The collected supernatant was transferred to a new tube and freeze-dried using a freeze dryer (CHRISST ALPHA 1-2 LD plus, Sigma).

실시예 3: 웨스턴블롯Example 3: Western blot

각 시료로부터 30 μg의 CM을 1-D 12% SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)에 로딩하여 분리한 후, 니트로셀룰로오스 막(Pall Corporation, USA)에 옮겼다. 막을 차단하기 위해, 5% 탈지유를 함유한 PBS로 1시간 동안 처리한 후, 0.5% 트윈 20(TBST)을 포함하는 TBS로 세척하였다. 상기와 같이 처리한 니트로셀룰로오스 막을 1차 항체 항-ANP, 항-BNP(이상 Thermo Fisher Scientific, USA), 및 항-β-액틴(Cell Signaling, USA)과 함께 밤새도록 인큐베이션 하였다. 상기 1차 항체와 인큐베이션 한 막을 TBST로 4회 세척한 후, sheep HRP(horseradish peroxidase) 접합된 이차 항체(GeneTex, USA)를 첨가하여 실온에서 1시간 동안 인큐베이션 하였다. 이어서, 웨스턴블롯을 위한 내강 시약(Santa Cruz Biotechnology, USA)을 화학 발광 이미징 시스템인 Ez-Capture MG(ATTO, NY, USA)에 의한 밴드의 가시화에 사용하였다.30 μg of CM from each sample was separated by loading on 1-D 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and then transferred to a nitrocellulose membrane (Pall Corporation, USA). To block the membrane, it was treated with PBS containing 5% skim milk for 1 hour and then washed with TBS containing 0.5% Tween 20 (TBST). The nitrocellulose membrane treated as described above was incubated overnight with primary antibodies anti-ANP, anti-BNP (above Thermo Fisher Scientific, USA), and anti-β-actin (Cell Signaling, USA). After washing the membrane incubated with the primary antibody 4 times with TBST, sheep HRP (horseradish peroxidase) conjugated secondary antibody (GeneTex, USA) was added and incubated for 1 hour at room temperature. Then, the luminal reagent for western blotting (Santa Cruz Biotechnology, USA) was used for the visualization of bands by a chemiluminescent imaging system, Ez-Capture MG (ATTO, NY, USA).

실시예 4: 단백질 분해Example 4: Proteolysis

각 시료(200 μg)의 농도를 측정한 후, 속도 진공 건조기에서 3시간 동안 건조시켰다. 건조된 시료를 6.0 M 우레아(Sigma, USA)로 재구성하고, 200 mM 디티오트레이트톨(dithiothreitol; DTT, Sigma-Aldrich, Darmstadt, Germany)에서 37℃로 30분 동안 배양함으로써 환원시켰다. 이어서, 100 mM 인돌-3-아세트산(indole-3-acetic acid; IAA, Sigma-Aldrich, Darmstadt, Germany)에서 37℃로 30분 동안 알킬화시켰다. 상기 시료에 최종 프로테아제:단백질 비율 1:30(w/w)로 트립신을 보충하고, 혼합물을 37℃에서 20시간 동안 배양하였다. 이후 C18 스핀 컬럼(Pierce, Thermo Fisher Scientific, USA)을 이용하여 제조업자의 매뉴얼에 따라 시료를 세척하였다. 세척한 시료를 진공 동결 건조기로 건조시키고 0,1% 포름산(Thermo Scientific, Spain)을 포함하는 50% 메탄올로 재구성하였다.After measuring the concentration of each sample (200 μg), it was dried for 3 hours in a speed vacuum dryer. The dried sample was reconstituted with 6.0 M urea (Sigma, USA) and reduced by incubation in 200 mM dithiothreitol (DTT, Sigma-Aldrich, Darmstadt, Germany) at 37°C for 30 minutes. This was followed by alkylation in 100 mM indole-3-acetic acid (IAA, Sigma-Aldrich, Darmstadt, Germany) at 37° C. for 30 min. The sample was supplemented with trypsin at a final protease:protein ratio of 1:30 (w/w), and the mixture was incubated at 37°C for 20 hours. Then, the sample was washed using a C18 spin column (Pierce, Thermo Fisher Scientific, USA) according to the manufacturer's manual. The washed samples were dried in a vacuum freeze dryer and reconstituted with 50% methanol containing 0,1% formic acid (Thermo Scientific, Spain).

실시예 5: 나노-액체 크로마토그래피-고분해능 질량분석기에 의한 분석Example 5: Analysis by Nano-Liquid Chromatography-High Resolution Mass Spectrometry

상기 실시예 4에 따라 수득한 시료를 EASY-nLC1000 액체 크로마토그래피 시스템이 연결된 LTQ Orbitrap Velos Pro 질량분석기(Thermo Fischer Scientific, Sunnyvale, CA, USA)를 이용하여 분석하였다. 용매로는 0.1% 포름산이 함유된 증류수(0.1% formic acid in DI warer, 용매 A)와 0.1% 포름산이 함유된 아세토니트릴(0.1% formic acid in acetonitrile, 용매 B)을 준비하여 사용하였다. 펩티드를 함유하는 시료를 Acclaim PepMap 100 트랩 컬럼(C18, 75 μm×2 cm, nanoViper, 100 Å, 3 μm, Thermo Scientific)을 통해 로딩하고, 이어지는 Acclaim PepMap 이지-스프레이 분석 컬럼(C18, 75 μm×50 cm, nanoViper, 100 Å, 2 μm, Thermo Scientific)에 의해 펩티드를 분리하였다. 각 액체 크로마토그래피 탠덤 질량 분석(LC-MS/MS)을 위해, 시료를 마이크로 리터 픽업이 있는 프리 컬럼에서 최대 2 μL 로딩했다. 펩티드의 분리는 120분에 걸쳐 5 내지 40%의 구배로 이동상에서 용매 B를 사용하여 250 nL/분의 유속으로 달성되었다. 용출된 펩티드는 +2.1 kV의 분무 전압을 사용하여 Orbitrap Velos 질량 분석기로 연구하였다. 전체 스캔은 m/z 300에서 2000에서 60,000의 해상도로 획득하였다. MS/MS 스캔 방법의 10가지 데이터-의존적 획득에 의해 관찰된 Top N 모드는 35 eV의 정규 에너지로 충돌유도분리(collision-induced dissociation; CID) 에너지를 사용하여 적용하였다. 이전에 단편화된 전구체 이온의 동적 배제를 위하여, 신호 임계 값 1000, 배제질량폭(exclusion mass width) 10 ppm, 배제시간(exclusion time) 180초, 반복 지속 시간 30초, 반복 횟수 2 및 분리 폭 1 m/z의 변수를 사용하였다. 단일 하전된 이온(singly charged ion)은 수집에서 제외하였다.Samples obtained according to Example 4 were analyzed using an LTQ Orbitrap Velos Pro mass spectrometer (Thermo Fischer Scientific, Sunnyvale, CA, USA) connected to an EASY-nLC1000 liquid chromatography system. As a solvent, distilled water containing 0.1% formic acid in DI warer (solvent A) and acetonitrile containing 0.1% formic acid (0.1% formic acid in acetonitrile, solvent B) were prepared and used. Samples containing peptides were loaded through an Acclaim PepMap 100 trap column (C18, 75 μm × 2 cm, nanoViper, 100 Å, 3 μm, Thermo Scientific) followed by an Acclaim PepMap easy-spray analytical column (C18, 75 μm × 2 cm). 50 cm, nanoViper, 100 Å, 2 μm, Thermo Scientific). For each liquid chromatography tandem mass spectrometry (LC-MS/MS), samples were loaded up to 2 μL on a pre-column with a microliter pickup. Separation of the peptides was achieved at a flow rate of 250 nL/min using solvent B in the mobile phase with a gradient of 5 to 40% over 120 min. Eluted peptides were studied with an Orbitrap Velos mass spectrometer using a spray voltage of +2.1 kV. Full scans were acquired at resolutions of 2000 to 60,000 at m/z 300. The Top N mode observed by 10 data-dependent acquisitions of the MS/MS scan method was applied using collision-induced dissociation (CID) energy with a normal energy of 35 eV. For dynamic exclusion of previously fragmented precursor ions, signal threshold of 1000, exclusion mass width of 10 ppm, exclusion time of 180 seconds, repetition duration of 30 seconds, number of repetitions of 2 and separation width of 1 A variable of m/z was used. Single charged ions were excluded from collection.

실시예 6: Sequest를 이용한 단백질 동정Example 6: Protein identification using Sequest

Proteome Discoverer v.2.2 프로그램(Thermo Fisher Scientific, Boston, MA, USA)을 이용하여 상기 실시예 5를 통해 액체 크로마토그래피-고분해능 질량분석기로 검출된 피크로부터 단백질을 동정하였다. 전구체 이온 질량 오차(precursor ion mass tolerance)는 10 ppm, 단편 이온 질량 오차(fragment ion mass tolerance)는 0.6 Da, 최대 미절단(maximum missed cleavages)은 2를 허용하는 것으로, 가변적 개질(variable modification)은 산화(+15.995 Da)와 카바미도메틸(carbamidomethyl, +57.021 Da)을 사용하였다. 펩티드 매칭 기준은 SEQUEST HT 스코어 ＞1를 사용하였으며, 교차 상관 스코어(cross-correalation score; Xcorr) 기준은 +1 펩티드에 대해 ＞1.2, +2 펩티드에 대해 ＞2.2, +3 펩티드에 대해 ＞3.0이었다. 단백질 당 2개 이상의 특이적인 펩티드가 발견되었을 때, 단백질을 동정하였다. 서로 다른 그룹간의 통계적 처리는 카이제곱 검정법(chi-square test)을 사용하였다.Proteins were identified from peaks detected by liquid chromatography-high-resolution mass spectrometry in Example 5 using the Proteome Discoverer v.2.2 program (Thermo Fisher Scientific, Boston, MA, USA). Precursor ion mass tolerance is 10 ppm, fragment ion mass tolerance is 0.6 Da, maximum missed cleavages allow 2, variable modification is Oxidation (+15.995 Da) and carbamidomethyl (+57.021 Da) were used. A SEQUEST HT score >1 was used as the peptide matching criterion, and the cross-correlation score (Xcorr) criterion was >1.2 for +1 peptides, >2.2 for +2 peptides, and >3.0 for +3 peptides. . Proteins were identified when two or more specific peptides per protein were found. Statistical treatment between different groups was performed using a chi-square test.

실시예 7: 펩티드 바이오마커 후보군 정량Example 7: Quantification of peptide biomarker candidates

용액 내 소화 후 시료를 LTQ Orbitrap Velos pro와 연결된 UHPLC-ESI-MS/MS 시스템으로 분석하였다. 5 μL의 시료를 로딩하고, 4 μm Proteo Phenomenex(90 Å 기공, LC 컬럼 250×4.6 mm, Jupiter, USA)를 사용하여 분리하였다. 분리 구배는 용매 구배를 사용하여 30분 동안 0.3 mL/분으로 수행하였으며, 0.1% 포름산이 함유된 증류수(0.1% formic acid in DI warer, 용매 A)와 0.1% 포름산이 함유된 아세토니트릴(0.1% formic acid in acetonitrile, 용매 B)을 준비하여, 다음과 같이 용매 B의 선행 구배를 사용하였다: 0분 0%, 17분 100%, 21분 100%, 22분 0%, 30분 0%. 15000의 해상도에서 전체 MS 스캔을 진행하였으며, 300 내지 2000 m/z의 스캔 범위로 처리하였다. 고분해능 질량 분석기의 분무 전압은 +3.9 kV였으며, 이를 통해 용리된 펩티드를 검사하였다. 총 스캔 사건은 7이고 충돌 에너지는 25였다. 14-3-3 감마, 엡실론, 세타, 에타, 제타, 베타 및 액틴의 전구체 이온 및 이에 상응하는 일치 생성물 이온은 각각 m/z=745.7, 696.6, 715.6, 720.6, 711.3, 796.4 및 652.0와 765.3, 742.3, 871, 698.7, 742.2, 761.5 및 595.0이었다.After in-solution digestion, samples were analyzed on a UHPLC-ESI-MS/MS system coupled to an LTQ Orbitrap Velos pro. 5 μL of sample was loaded and separated using a 4 μm Proteo Phenomenex (90 Å pore, LC column 250×4.6 mm, Jupiter, USA). The separation gradient was performed at 0.3 mL/min for 30 minutes using a solvent gradient, distilled water containing 0.1% formic acid in DI warer (solvent A) and acetonitrile containing 0.1% formic acid (0.1% Formic acid in acetonitrile, solvent B) was prepared, and a pre-gradient of solvent B was used as follows: 0% at 0 min, 100% at 17 min, 100% at 21 min, 0% at 22 min, 0% at 30 min. A full MS scan was performed at a resolution of 15000, and a scan range of 300 to 2000 m/z was processed. The spray voltage of the high-resolution mass spectrometer was +3.9 kV, through which the eluted peptides were examined. The total scan event was 7 and the collision energy was 25. 14-3-3 The precursor ions of gamma, epsilon, theta, eta, zeta, beta and actin and their corresponding homogenous product ions are m/z=745.7, 696.6, 715.6, 720.6, 711.3, 796.4 and 652.0 and 765.3, respectively. 742.3, 871, 698.7, 742.2, 761.5 and 595.0.

실시예 8: 통계적 분석Example 8: Statistical Analysis

모든 데이터는 3회 결과에 대해 평균±표준편차(standard deviation; SD)로 제시되었으며, 웨스턴블롯 결과는 β-액틴 및 대조군과 비교하여 배수 변화로 보고하였다. 두 그룹의 차이는 스튜던트 테스트(Student's test, Origin 2017, USA)에 의해 결정되었으며, p-값이 ＜0.05인 그룹만을 보고하였다.All data were presented as mean±standard deviation (SD) for triplicate results, and Western blot results were reported as fold change compared to β-actin and control. The difference between the two groups was determined by Student's test (Student's test, Origin 2017, USA), and only groups with a p-value of <0.05 were reported.

실시예 8: 인간 혈장 시료의 분석Example 8: Analysis of human plasma samples

건강한 정상 개체 및 급성심근경색 환자로부터 채취한 혈장 시료를 이용하여 고려대 의과대학병원에서 기관의 생명윤리심의위원회(Institutional Review Board KUGH12118-005)로부터 승인을 받아 분석을 진행하였다. 구체적으로, 각각 5개의 시료에 대해 마이크로-액체 크로마토그래피-질량분석기를 사용하여 단백질 단편을 정량적으로 분석하였다. MRM 총 스캔 사건은 4이고 충돌 에너지는 25였다. 이때, 14-3-3 입실론, 제타, 베타 및 액틴의 전구체 이온과 이에 상응하는 일치 생성물 이온은 각각 m/z=696.6, 711.3, 796.4 및 652.0와 742.3, 742.2, 761.5 및 595.0이었다.Plasma samples collected from normal healthy individuals and patients with acute myocardial infarction were used for analysis at Korea University Medical School after receiving approval from the Institutional Review Board KUGH12118-005. Specifically, protein fragments were quantitatively analyzed for each of the five samples using a micro-liquid chromatography-mass spectrometer. The MRM total scan event was 4 and the collision energy was 25. At this time, the precursor ions of 14-3-3 epsilon, zeta, beta, and actin and the corresponding product ions were m/z = 696.6, 711.3, 796.4, and 652.0, and 742.3, 742.2, 761.5, and 595.0, respectively.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Method for screening biomarkers diagnosing cardiovascular diseases, biomarkers screened thereby and method for diagnosing cardiovascular diseases using the same <130> KPA200227-KR-D1 <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 3020 <212> DNA <213> Artificial Sequence <220> <223> 14-3-3 beta <400> 1 ggaagtggag ctaccgccac cgccgccgcc gattccggag ccggggtagt cgccgccgcc 60 gccgccgctg cagccactgc aggcaccgct gccgccgcct gagtagtggg cttaggaagg 120 aagaggtcat ctcgctcgga gcttcgctcg gaagggtctt tgttccctgc agccctccca 180 cgggaatgac aatggataaa agtgagctgg tacagaaagc caaactcgct gagcaggctg 240 agcgatatga tgatatggct gcagccatga aggcagtcac agaacagggg catgaactct 300 ccaacgaaga gagaaatctg ctctctgttg cctacaagaa tgtggtaggc gcccgccgct 360 cttcctggcg tgtcatctcc agcattgagc agaaaacaga gaggaatgag aagaagcagc 420 agatgggcaa agagtaccgt gagaagatag aggcagaact gcaggacatc tgcaatgatg 480 ttctggagct gttggacaaa tatcttattc ccaatgctac acaaccagaa agtaaggtgt 540 tctacttgaa aatgaaagga gattatttta ggtatctttc tgaagtggca tctggagaca 600 acaaacaaac cactgtgtcg aactcccagc aggcttacca ggaagcattt gaaattagta 660 agaaagaaat gcagcctaca cacccaattc gtcttggtct ggcactaaat ttctcagtct 720 tttactatga gattctaaac tctcctgaaa aggcctgtag cctggcaaaa acggcatttg 780 atgaagcaat tgctgaattg gatacgctga atgaagagtc ttataaagac agcactctga 840 tcatgcagtt acttagggac aatctcactc tgtggacatc ggaaaaccag ggagacgaag 900 gagacgctgg ggagggagag aactaatgtt tctcgtgctt tgtgatctgt tcagtgtcac 960 tctgtaccct caacatatat cccttgtgcg ataaaaaaaa aaaaaaaaaa aaaaagagaa 1020 tcgtacgtcg actttcgatt tttcacagcc tcagcctagg aaaaatggtt catgggataa 1080 acagctggta tttgtatcta aaactcagat tggtcacata aatgccacgg cattccgaag 1140 ttttgatttt gattaacatt gacaggatta ctgtgtgttt aattttttaa aaactgaaca 1200 ctgtgattat ggggttttgt aatttagcag aactcttact ggtagaaaaa atagacctga 1260 attatgtgta actttttgga aggtttaatc tgatatcaaa ataatcattg aaatacaatt 1320 ccattgtaaa gttgtacaga aagttataga gattatattg tgatgctgga acttggagtg 1380 agacacacat catttggcat ttgagttgaa tggtaattca cagtaatgct gccgttgttc 1440 gggacttaaa gacacttgac ctgtttgggc tgttgccact taaaagttca tgaccacaaa 1500 tgtccacagt gtcttcctct gaggaaactc gaatcctgaa atggaaattc tttgtggcag 1560 ataactggct tatgacacct tgaaaagttc aagtgctcat ataacacacc acactgaacc 1620 ccctttccta cagcaatatg ttcactatgt taccaatttg caacttgtgc ttcaatagtg 1680 gaatctactt tcattgttaa cactgagcta aagaaaaaaa gccgtgtgtt ttatgaatga 1740 ccttatctgt ttcctggata atacctttaa gaataatgtc ctgagtcagg cgtggtggtg 1800 cgtgcatcta gtcccaacta tttgggaggc tgaggcagga ggatcgcttg agcccaggag 1860 tttaaagctg cagtgccctg tggttgcacc tgtgaataac tgcactccag cctgggcaac 1920 atagcgagac ctcatctcca aaaaagaaaa caaaaaacaa aaaaaggaat gatgttctgt 1980 agagatggcc tttcacttga ggagtactca gttttcaggt tcttcctagc tcggggcttt 2040 taaattttga aatctaaaca ttctttccca ccatcctttt tgactgttga ccttggtttt 2100 ctcttctaag tttctgtccc tctgcttcct tacttttttt cctttttgaa ttctatcttt 2160 atctgtcttt tgttcacttt ttaatgctat atatgggcag gggtgagaga cattactgag 2220 caccttggtg agcaagcctg gctttaaaga ttggagaaga gcttctggca ccagaaccct 2280 gtcttcctcc agttctcaac acggtgttgc tcttcagtca taccggaatc tgaatcaaaa 2340 aagtattttt aaatatccat gatttctccc tgtattgagg ctagccctga tcatgctttt 2400 tgtgcctgtc accaggtctc ccaagtgcac tcatccaggt cagtgctcag atgtgtttaa 2460 ggagacccta tattcaggga agttgcgtga acactgcagt ggggagaatt gagaatagtc 2520 aggcctatca gtctcacaga atcacccctc tacctttgat attccactta gctgtagagt 2580 ccatctgttt gtccatctgc tgaaatgaga aaagaaaaat ttatgcactg atttaaaaca 2640 aaccaaaaaa aaagaaaaaa acaaaaaaaa aaatccctcc tttctagctg aacaaaaatg 2700 tgcagttaat acttggcgct tgaaaatgca gtagtgaatg tggaaccaag cctgtctgta 2760 tatctggtag ctcttttctt gctttgtttt ttcttaccag tattctgcct aacgtttgct 2820 tctgtgatgg ttatattgcc tagcaagcac acccgtggtt gtgaaaatag tatagcaaaa 2880 aagaaaaatc cccggttatt gatgtactag atttgtgtat gtcttttaaa cagttctagt 2940 ttcaccttac acagaataat caggaaaagt gtaaaaattc aaaagtgaaa taaaaatttt 3000 atcagttagt tgcctgtgaa 3020 <210> 2 <211> 2052 <212> DNA <213> Artificial Sequence <220> <223> 14-3-3 epsilon <400> 2 ggattgaggc gccgccattt ttgctgcccg gacgcggagc gagaggctga gagagtcgga 60 gacactatcc gcttccatcc gtcgcgcaga ccctgccgga gccgctgccg ctatggatga 120 tcgagaggat ctggtgtacc aggcgaagct ggccgagcag gctgagcgat acgacgaaat 180 ggtggagtca atgaagaaag tagcagggat ggatgtggag ctgacagttg aagaaagaaa 240 cctcctatct gttgcatata agaatgtgat tggagctaga agagcctcct ggagaataat 300 cagcagcatt gaacagaaag aagaaaacaa gggaggagaa gacaagctaa aaatgattcg 360 ggaatatcgg caaatggttg agactgagct aaagttaatc tgttgtgaca ttctggatgt 420 actggacaaa cacctcattc cagcagctaa cactggcgag tccaaggttt tctattataa 480 aatgaaaggg gactaccaca ggtatctggc agaatttgcc acaggaaacg acaggaagga 540 ggctgcggag aacagcctag tggcttataa agctgctagt gatattgcaa tgacagaact 600 tccaccaacg catcctattc gcttaggtct tgctctcaat ttttccgtat tctactacga 660 aattcttaat tcccctgacc gtgcctgcag gttggcaaaa gcagcttttg atgatgcaat 720 tgcagaactg gatacgctga gtgaagaaag ctataaggac tctacactta tcatgcagtt 780 gttacgtgat aatctgacac tatggacttc agacatgcag ggtgacggtg aagagcagaa 840 taaagaagcg ctgcaggacg tggaagacga aaatcagtga gacataagcc aacaagagaa 900 accatctctg accaccccct cctccccatc ccaccctttg gaaactcccc attgtcactg 960 agaaccacca aatctgactt ttacatttgg tctcagaatt taggttcctg ccctgttggt 1020 tttttttttt tttttttaaa cagttttcaa aagttcttaa aggcaagagt gaatttctgt 1080 ggattttact ggtcccagct tttaggttct ttaagacact aacaggacta catagaggct 1140 ttttcagcat tactgtgtcg tctccgtgcc agatgtggca agatcaccat tagcaaatgg 1200 aaattacatt tgaaagccat tagacttata ggtgatgcaa gcatctaaga gagaggttaa 1260 tcacactata gaggcataag tggtatcagt tttcattttt ctaattgttt aaactgtgtt 1320 ttataccagt gtttgcaagt aattgggtgt tagcttgaga tggttaaagg tggtttgggg 1380 agggacttcg ttgtaatggt tttgctgtaa aaaatgtttc caactccgct gaaatgttgc 1440 tgaaaagcat ggtgctggta acagttcaac aatccgtggc tgctcattct tgcctacttt 1500 actctcccac tgaagcaggt tagcgttgaa ggtggtatgg aaaagcctgc atgcctgttc 1560 aattcttttg tttcttctcc ttccccctcc ccctacctcc ttcccctcac tcctcccctc 1620 cttcgctcgc tcaacctctt ttgttcagta tgtgtaactt gaagctaatt tgtactactg 1680 gatatctgac tggagccaca gatacagaat ctgtattgtt cttactgaaa cacagcatgg 1740 aattaacatt aaacttaaat aaaacaaacc taaattaaaa atgccaaata tcgcgcctcc 1800 atcctttata cttctaaaat aaaatctcta gtccaaacac actgacagta ttatgtttgc 1860 ctgaatgtga aacattcctc cttttttctt ccataaataa tttgtttgct tcccaaagat 1920 gtatgttttg gaaaaaaatt gcctgtagtc tggttgtgaa gaacactgag tcagttgatg 1980 gtgtgtcttg tcctccagac caggcagcaa catggtattt attttaatca ttaaagtaca 2040 acatgagtag ta 2052 <210> 3 <211> 3705 <212> DNA <213> Artificial Sequence <220> <223> 14-3-3 gamma <400> 3 gcagccgcct cgcgcccggt cccgcggtcg cagctccagc cgcctcctcc gcgcagccgc 60 cgcctcagct gctcgctctg tgggtcggtc ctctccggca cttgggctcc agtcgcgccc 120 tccaagccct tcaggccgcc ccagtgtcct cctccttctc cggccagacc cagccccgcg 180 aagatggtgg accgcgagca actggtgcag aaagcccggc tggccgagca ggcggagcgc 240 tacgacgaca tggccgcggc catgaagaac gtgacagagc tgaatgagcc actgtcgaat 300 gaggaacgaa accttctgtc tgtggcctac aagaacgttg tgggggcacg ccgctcttcc 360 tggagggtca tcagtagcat tgagcagaag acatctgcag acggcaatga gaagaagatt 420 gagatggtcc gtgcgtaccg ggagaagata gagaaggagt tggaggctgt gtgccaggat 480 gtgctgagcc tgctggataa ctacctgatc aagaattgca gcgagaccca gtacgagagc 540 aaagtgttct acctgaagat gaaaggggac tactaccgct acctggctga agtggccacc 600 ggagagaaaa gggcgacggt ggtggagtcc tccgagaagg cctacagcga agcccacgag 660 atcagcaaag agcacatgca gcccacccac cccatccgat taggcctggc tcttaactac 720 tccgtcttct actatgagat ccagaacgcc ccagagcaag cgtgccactt ggccaagacc 780 gcgttcgacg acgccatcgc cgagcttgac accctcaacg aggactccta caaggactcc 840 acgctcatca tgcagctcct ccgcgacaac ctcacgctct ggacgagcga ccagcaggac 900 gacgatggcg gcgaaggcaa caattaaggc cccaggggaa ctggcagcgc acgcggatgc 960 tactactgca gtctttattt ttttcccatg agttgggggt cgggtggggg agggaaaggg 1020 agggatgacc ttcccaggga gaaacccacg acctgtcctg tctttgatcg cctctttgac 1080 atttttgcca aaataccact agtggaaagt caggctagct gtgctggtat tggaatagca 1140 gcctcacact ggcgtctgga ctgttctgta gattcatgca agtggagctg tctgtctcta 1200 atttaactta ttgctagata atagggtttt cagatgaaaa gaaaacttaa agaggaatgg 1260 ccctcattca gtaagttctg tggttccagt aaggattttt atgtacatac gctctcgtct 1320 ctcgttttgg gtactttcta tctcatctgt ctcggctctg catgttttcc agggtgtagc 1380 ctacagacat ggaacagtgt aaatcccaga ctgacagact tagaacctga ggtctcattc 1440 atccttatgg tttaggcctt gccagttttc cgaagtctct gattagttga cagtattaac 1500 actaaattgc agtttacagt atttctacat tacagccata tgtaacatca agccatcgat 1560 tgtgtacttt tcctttgcta gttgtttggg ctttaacatc cttattcagc cttatccagg 1620 ttggttttgc tgttgatcgg tctcctaggc taaatgagaa tgaaagcgac ttcaggtcag 1680 gtggctgtgg gatttttttt ttttggtcct tctttcctct taacgtaaat ccaccaccaa 1740 aattattaat cctcttgaga gaaacgtgaa acgccacaaa aatagagaaa attcaggtct 1800 gtatgtcatg gatcgtgttg gtattttcag agaacatccc gcttctgaag ctgctgcagc 1860 tccctcctca gggatcacac tgccgtcacc cactctgcac tggggcgttt cctactgcgc 1920 ctcgtgctgg cggacgcagc tgggtgcaga agctgtgggg tcggagaggc gtttggagaa 1980 ggtctgtggt gcagtgtgtg aaaattcagg tgctagaagc ctactggtag aaaaacccaa 2040 aaggaagagc tatatcctta accattctgt ccaatttcgg gagccttgtc agtgtgtcag 2100 tttttcctcc ccgaagacac tccttcccca agtaattgta ggaagataaa aaaactgtta 2160 ccagataaca aacactgaac tcctatttga ccagaacttt ttcctctcga gatagttttt 2220 tctttttaat gaaaaaagca taggaattgg agattggctt gtctcacgca gccagtgcac 2280 atttggaatt gacggaaaca acgttgctat ttccacccat ttgttttcgg cagccttaag 2340 gccctcattc tcatttcggg tgaatctgtc tatctgtgaa cgtggcccgc atgtgcattc 2400 ttttttttat atatataaag tcagtgacga ggaactcccg agacgtgtaa tgacaccaca 2460 cttgttttct ttgtttcttt gttttattta ggcaagaaga ggtgtgagta attgaggaaa 2520 aactgacaga tgcttttgct aataccaaaa ttgagcttac aattaggaac tgagtatgtg 2580 taacaggata caggtgacag tgaagataga agaaccacga tgaccacaga ctcaatgtgc 2640 tctgtaacat cgcacagttt acccagcatg actttcctta ggaggccccc tcctcacgct 2700 agagtaaaag tcccagttaa gtgaagccta ccagaagaac tagtagaaga agctttgccg 2760 cttttgtgcc tctcacaggc gcctaaagtc attgccatgg gaggaagacg atttgggggg 2820 ggaggggggg gggggcaggg taggtggggc tttccctaat ttatcttcat gtccagtgag 2880 cagtgttgcg tttttccttg tagcatttgg aaatgattta ctggaattac aaaacctatt 2940 tttcctttaa atttcagctt tggctctggc tgctttttag aataatgcaa gataaaaatc 3000 acacctgagg gctgaaaacg gagagggaat gggagacttg atatttaagc agcttgaatg 3060 gtttttcttt tctttatttt taaagaaatg cacttgccta tgatactgtc tctccagtga 3120 aatgattact cctccattac tctattgata caatattgtg catgctagtg ttgtatttct 3180 atacagtagc ttgaaattga ttaacttata ctgtaggtgt tatgtattcc tatgacaaaa 3240 aaaattaagt cttcaaattt tttaaaggtt tttttttttt aatttaattt ttccttttgg 3300 gggtaaagtt tgctctacca aatagtgatt gtaacaaatt gatctgtttt ggatgttgct 3360 atagtgacat gcagttatat attttgtttt taaaaggggg ggagcaaaag aaacaccagt 3420 gttagcttaa tcttaatgtc tggtgtttgt catggtgaaa ttataactat tacagtgttg 3480 gagaacaaca aatatgttct ctgaatgagc ctttgtgctt tttgtcatgt tatgcagtga 3540 actattttta aggtctaatc agtgattatt tttccagctc cgtgtttctc taaggaatta 3600 tttcacacac ggaccatctt tagcagtttc ctcagtgatg gaatatcatg aatgtgagtc 3660 attatgtagc tgtcgtacat tgagcaaata aacttacaga tctga 3705 <210> 4 <211> 1751 <212> DNA <213> Artificial Sequence <220> <223> 14-3-3 eta <400> 4 acagcgcgcg gggcgagcca gcgagagggc gcgagcggcg gcgctgcctg cagcctgcag 60 cctgcagcct ccggccggcc ggcgagccag tgcgcgtgcg cggcggcggc ctccgcagcg 120 accggggagc ggactgaccg gcgggagggc tagcgagcca gcggtgtgag gcgcgaggcg 180 aggccgagcc gcgagcgaca tgggggaccg ggagcagctg ctgcagcggg cgcggctggc 240 cgagcaggcg gagcgctacg acgacatggc ctccgctatg aaggcggtga cagagctgaa 300 tgaacctctc tccaatgaag atcgaaatct cctctctgtg gcctacaaga atgtggttgg 360 tgccaggcga tcttcctgga gggtcattag cagcattgag cagaaaacca tggctgatgg 420 aaacgaaaag aaattggaga aagttaaagc ttaccgggag aagattgaga aggagctgga 480 gacagtttgc aatgatgtcc tgtctctgct tgacaagttc ctgatcaaga actgcaatga 540 tttccagtat gagagcaagg tgttttacct gaaaatgaag ggtgattact accgctactt 600 agcagaggtc gcttctgggg agaagaaaaa cagtgtggtc gaagcttctg aagctgccta 660 caaggaagcc tttgaaatca gcaaagagca gatgcaaccc acgcatccca tccggctggg 720 cctggccctc aacttctccg tgttctacta tgagatccag aatgcacctg agcaagcctg 780 cctcttagcc aaacaagcct tcgatgatgc catagctgag ctggacacac taaacgagga 840 ttcctataag gactccacgc tgatcatgca gttgctgcga gacaacctca ccctctggac 900 gagcgaccag caggatgaag aagcaggaga aggcaactga agatccttca ggtcccctgg 960 cccttccttc acccaccacc cccatcatca ccgattcttc cttgccacaa tcactaaata 1020 tctagtgcta aacctatctg tattggcagc acagctactc agatctgcac tcctgtctct 1080 tgggaagcag tttcagataa atcatgggca ttgctggact gatggttgct ttgagcccac 1140 aggagctccc tttttgaatt gtgtggagaa gtgtgttctg atgaggcatt ttactatgcc 1200 tgttgatcta tgggaaatct aggcgaaagt aatggggaag attagaaaga attagccaac 1260 caggctacag ttgatattta aaagatccat ttaaaacaag ctgatagtgt ttcgttaagc 1320 agtacatctt gtgcatgcaa aaatgaattc acccctccca cctctttctt caattaatgg 1380 aaaactgtta agggaagctg atacagagag acaacttgct cctttccatc agctttataa 1440 taaactgttt aacgtgaggt ttcagtagct ccttggtttt gcctctttaa attatgacgt 1500 gcacaaacct tcttttcaat gcaatgcatc tgaaagtttt gatacttgta actttttttt 1560 ttttttggtt gcaattgttt aagaatcatg gatttatttt ttgtaactct ttggctattg 1620 tccttgtgta tcctgacagc gccatgtgtg tcagcccatg tcaatcaaga tgggtgatta 1680 tgaaatgcca gacttctaaa ataaatgttt tggaattcaa tgggtaaata aatgctgctt 1740 tggggatatt a 1751 <210> 5 <211> 2196 <212> DNA <213> Artificial Sequence <220> <223> 14-3-3 theta <400> 5 aaagccaaaa gcagatcaaa gtggtgggac tcgcgtcgcg gccgcggaga cgtgaagctc 60 tcgaggctcc tcccgctgcg ggtcggcgct cgccctcgct ctcctcgccc tccgccccgg 120 ccccggcccc gcgcccgcca tggagaagac tgagctgatc cagaaggcca agctggccga 180 gcaggccgag cgctacgacg acatggccac ctgcatgaag gcagtgaccg agcagggcgc 240 cgagctgtcc aacgaggagc gcaacctgct ctccgtggcc tacaagaacg tggtcggggg 300 ccgcaggtcc gcctggaggg tcatctctag catcgagcag aagaccgaca cctccgacaa 360 gaagttgcag ctgattaagg actatcggga gaaagtggag tccgagctga gatccatctg 420 caccacggtg ctggaattgt tggataaata tttaatagcc aatgcaacta atccagagag 480 taaggtcttc tatctgaaaa tgaagggtga ttacttccgg taccttgctg aagttgcgtg 540 tggtgatgat cgaaaacaaa cgatagataa ttcccaagga gcttaccaag aggcatttga 600 tataagcaag aaagagatgc aacccacaca cccaatccgc ctggggcttg ctcttaactt 660 ttctgtattt tactatgaga ttcttaataa cccagagctt gcctgcacgc tggctaaaac 720 ggcttttgat gaggccattg ctgaacttga tacactgaat gaagactcat acaaagacag 780 caccctcatc atgcagttgc ttagagacaa cctaacactt tggacatcag acagtgcagg 840 agaagaatgt gatgcggcag aaggggctga aaactaaatc catacagggt gtcatccttc 900 tttccttcaa gaaacctttt tacacatctc cattccttat tccacttgga tttcctatag 960 caaagaaacc cattcatgtg tatggaatca actgtttata gtcttttcac actgcagctt 1020 tgggaaaact tcattccttg atttgtgttt gtcttggcct tcctggtgtg cagtactgct 1080 gtagaaaagt attaatagct tcatttcata taaacataag taactcccaa acacttatgt 1140 agaggactaa aaatgtatct ggtatttaag taatctgaac cagttctgca agtgactgtg 1200 ttttgtatta ctgtgaaaat aagaaaatgt agttaattac aatttaaaga gtattccaca 1260 taacttctta atttctacat tccctccctt actcttcggg ggtttccttt cagtaagcaa 1320 cttttccatg ctcttaatgt attccttttt agtaggaatc cggaagtatt agattgaatg 1380 gaaaagcact tgccatctct gtctaggggt cacaaattga aatggctcct gtatcacata 1440 cggaggtctt gtgtatctgt ggcaacaggg agtttcctta ttcactcttt atttgctgct 1500 gtttaagttg ccaacctccc ctcccaataa aaattcactt acacctcctg cctttgtagt 1560 tctggtattc actttactat gtgatagaag tagcatgttg ctgccagaat acaagcattg 1620 cttttggcaa attaaagtgc atgtcatttc ttaatacact agaaagggga aataaattaa 1680 agtacacaag tccaagtcta aaactttagt acttttccat gcagatttgt gcacatgtga 1740 gagggtgtcc agtttgtcta gtgattgtta tttagagagt tggaccacta ttgtgtgttg 1800 ctaatcattg actgtagtcc caaaaaagcc ttgtgaaaat gttatgccct atgtaacagc 1860 agagtaacat aaaataaaag tacattttat aaaccattta ctatggcttt gtaacaattg 1920 catacccata ttttaaggga caggtgaatt tactactttc taaagtttat tgatacttcc 1980 cttttatgta aaatgtagta gtgataccta tatttccaca ttgtgcattg tgacacactt 2040 gtctagggat gcctggaagt gtataaaatt ggactgcatt tcttagagtg ttttactata 2100 gatcagtctc atgggccatc tcttcctcag atgtaaatga tatctggtta agtgttatat 2160 ggaataaagt ggacatttta aaactagcaa agttaa 2196 <210> 6 <211> 5085 <212> DNA <213> Artificial Sequence <220> <223> 14-3-3 zeta <400> 6 gacacagatc cgccatgaca aaggaggaga gtcggggact tgagccgtgg ctccgacttg 60 ggcggagcct ggaggggggt ggttgcgata cgcggaccgg agaatttgca ctttaaagtc 120 cgggtctgcc cgttttcgtt tcacagtaac cgacgtctca agtcagaaca tccagtcatg 180 gataaaaatg agctggttca gaaggccaaa ctggccgagc aggctgagcg atatgatgac 240 atggcagcct gcatgaagtc tgtaactgag caaggagctg aattatccaa tgaggagagg 300 aatcttctct cagttgctta taaaaatgtt gtaggagccc gtaggtcatc ttggagggtc 360 gtctcaagta ttgaacaaaa gacggaaggt gctgagaaaa aacagcagat ggctcgagaa 420 tacagagaga aaattgagac ggagctaaga gatatctgca atgatgtact gtctcttttg 480 gaaaagttct tgatccccaa tgcttcacaa gcagagagca aagtcttcta tttgaaaatg 540 aaaggagatt actaccgtta cttggctgag gttgccgctg gtgatgacaa gaaagggatt 600 gtcgatcagt cacaacaagc ataccaagaa gcttttgaaa tcagcaaaaa ggaaatgcaa 660 ccaacacatc ctatcagact gggtctggcc cttaacttct ctgtgttcta ttatgagatt 720 ctgaactccc cagagaaagc ctgctctctt gcaaagacag cttttgatga agccattgct 780 gaacttgata cattaagtga agagtcatac aaagacagca cgctaataat gcaattactg 840 agagacaact tgacattgtg gacatcggat acccaaggag acgaagctga agcaggagaa 900 ggaggggaaa attaaccggc cttccaactt ttgtctgcct cattctaaaa tttacacagt 960 agaccatttg tcatccatgc tgtcccacaa atagtttttt gtttacgatt tatgacaggt 1020 ttatgttact tctatttgaa tttctatatt tcccatgtgg tttttatgtt taatattagg 1080 ggagtagagc cagttaacat ttagggagtt atctgttttc atcttgaggt ggccaatatg 1140 gggatgtgga atttttatac aagttataag tgtttggcat agtacttttg gtacattgtg 1200 gcttcaaaag ggccagtgta aaactgcttc catgtctaag caaagaaaac tgcctacata 1260 ctggtttgtc ctggcgggga ataaaaggga tcattggttc cagtcacagg tgtagtaatt 1320 gtgggtactt taaggtttgg agcacttaca aggctgtggt agaatcatac cccatggata 1380 ccacatatta aaccatgtat atctgtggaa tactcaatgt gtacaccttt gactacagct 1440 gcagaagtgt tcctttagac aaagttgtga cccattttac tctggataag ggcagaaacg 1500 gttcacattc cattatttgt aaagttacct gctgttagct ttcattattt ttgctacact 1560 cattttattt gtatttaaat gttttaggca acctaagaac aaatgtaaaa gtaaagatgc 1620 aggaaaaatg aattgcttgg tattcattac ttcatgtata tcaagcacag cagtaaaaca 1680 aaaacccatg tatttaactt ttttttagga tttttgcttt tgtgattttt ttttttttga 1740 tacttgccta acatgcatgt gctgtaaaaa tagttaacag ggaaataact tgagatgatg 1800 gctagctttg tttaatgtct tatgaaattt tcatgaacaa tccaagcata attgttaaga 1860 acacgtgtat taaattcatg taagtggaat aaaagtttta tgaatggact tttcaactac 1920 tttctctaca gcttttcatg taaattagtc ttggttctga aacttctcta aaggaaattg 1980 tacatttttt gaaatttatt ccttattccc tcttggcagc taatgggctc ttaccaagtt 2040 taaacacaaa atttatcata acaaaaatac tactaatata actactgttt ccatgtccca 2100 tgatcccctc tcttcctccc caccctgaaa aaaatgagtt cctatttttt ctgggagagg 2160 gggggattga ttagaaaaaa atgtagtgtg ttccatttaa aattttggca tatggcattt 2220 tctaacttag gaagccacaa tgttcttggc ccatcatgac attgggtagc attaactgta 2280 agttttgtgc ttccaaatca ctttttggtt tttaagaatt tcttgatact cttatagcct 2340 gccttcaatt ttgatccttt attctttcta tttgtcaggt gcacaagatt accttcctgt 2400 tttagccttc tgtcttgtca ccaaccattc ttacttggtg gccatgtact tggaaaaagg 2460 ccgcatgatc tttctggctc cactcagtgt ctaaggcacc ctgcttcctt tgcttgcatc 2520 ccacagacta tttccctcat cctatttact gcagcaaatc tctccttagt tgatgagact 2580 gtgtttatct ccctttaaaa ccctacctat cctgaatggt ctgtcattgt ctgcctttaa 2640 aatccttcct ctttcttcct cctctattct ctaaataatg atggggctaa gttataccca 2700 aagctcactt tacaaaatat ttcctcagta ctttgcagaa aacaccaaac aaaaatgcca 2760 ttttaaaaaa ggtgtatttt ttcttttaga atgtaagctc ctcaagagca gggacaatgt 2820 tttctgtatg ttctattgtg cctagtacac tgtaaatgct caataaatat tgatgatggg 2880 aggcagtgag tcttgatgat aagggtgaga aactgaaatc ccaaacactg ttttgttgct 2940 tgttttatta tgacctcaga ttaaattggg aaatattggc ccttttgaat aattgtccca 3000 aatattacat tcaaataaaa gtgcaatgga gaccttgggg tctttatttc caggagaaaa 3060 gataccttct gcatctaggg caacaatcct tttccctcgc agctctttct tttacctcac 3120 ttactgattt ctcctgaccc ccaagccaag gaggtaaaat aattatatat atatatatat 3180 atatatatat atatatataa agtaattata ttttatagct agactgaaga ggtgggaatt 3240 tttttgttgt tttttttttt tttggagaca agagtcttgc tctgtcaccc aggcttgagt 3300 gcagtggtgc gatctcggct gagtgcaacc tccgcctgct gggttcaagc gattctcctg 3360 cctcagcctc ccaagtagct gggattacag gtgcccgcca ccacacctgg ctaatttttg 3420 tgtttttagt agagacaggg tttcaccatg ttgtccaggc tggtcttgaa ttcccgacct 3480 caggcaatcc gcccatctca acctcccaaa gtgctgggat tacaggcata agccaccgcg 3540 cccagtagag atgtgatatt attcaagtaa ataaaacctg aaacttggca catttgccta 3600 tttaatgtgc atcagtcatc tgaactaggt tgatcctgga ataatgaaaa cacttcacta 3660 gtattaacta atgttttcta catagtagtt cccaatagat taaggaaaat ttccaacctg 3720 agattctttg ttttggaatt tgaataatgt gtttgtaact attccttgat tgtcccaaat 3780 ccaggcttta cgtctgcttt tgtcaatgta acatcctagt gccaggaaat tggaaaatcc 3840 aatgactttt tgtgtgtaaa gtactggaat ttttttgttc gtttgttttt aagacggagt 3900 gtcaactctc gcccaggctg gggtgcagtg gcctgatctc ggctcactgc aaccttctcc 3960 tcccaggctg aagtgattct cctgcctcag cctcgcaagt agctgggact gcaggcatgt 4020 gccaccactc ctggctaatt atttgtattt ttagtagaga gagggtttca ccgtgttagc 4080 catgatggtc tcattctcct gaccttgtga tccgcccacc tcggcctccc aaagtgctgg 4140 gattacaggc gtgagccact gtgcccaccc tggtatgttt tttttaaatt ggccacttaa 4200 gagttgtcct tcagaaagtg gagcagcagt gtgattagta aaaacgatgg gctttcctgc 4260 agctctggct tatggtcttt gtttttccag ttaacagctg ttacttcatg ctgttatagg 4320 gggtgggggg agaaagagaa agaatatgtc tagtggctta attttggggc tcaatttgtt 4380 tattatgttt ttcctgcaaa agagtcacca gtcttgtcat ggctcaatgc atattttaaa 4440 atgagtcaaa gccaatttgg aagctgctat cttaggttat ttgtagtcag gcttcaaatg 4500 gagagtgaaa ctttgacttt gaaaactggt gctctatgct caatgatggt cttacacatt 4560 cctctaggga aaggtcaaga aataaatttg gcttgattgt attctctcat ttaccatata 4620 ggaaatacta tggtagaact gaaaatatgt acaatagtaa agtggtggct gagactgggc 4680 acttggaaaa tagacttgga ttcttttcta agtgtgaaac taattaaata cttttatgtg 4740 actggtagta gtcacagtct ccttgattga ttctagttcc ttttcaaagt ggagagacct 4800 gaatatagat tgtaacagga tgtccaacct tgccagacaa gcaggcatat atgtctctgg 4860 gatgcagaag cagttacaag ttaatatatt taagcataat gttagccaat cctaggtgat 4920 accagagatg tgaatatttg atcatatttg agttcattcc ttgaaagtcc aaacctaact 4980 ggtcttaatt gattacttct tagtattccg aattttagaa tttaaaaccc tatgaatttt 5040 cagtttgtgc ttacattttc taacattgga tgtttgcttt ggcca 5085 <210> 7 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 beta <400> 7 Met Thr Met Asp Lys Ser Glu Leu Val Gln Lys Ala Lys Leu Ala Glu 1 5 10 15 Gln Ala Glu Arg Tyr Asp Asp Met Ala Ala Ala Met Lys Ala Val Thr 20 25 30 Glu Gln Gly His Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val 35 40 45 Ala Tyr Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Ile 50 55 60 Ser Ser Ile Glu Gln Lys Thr Glu Arg Asn Glu Lys Lys Gln Gln Met 65 70 75 80 Gly Lys Glu Tyr Arg Glu Lys Ile Glu Ala Glu Leu Gln Asp Ile Cys 85 90 95 Asn Asp Val Leu Glu Leu Leu Asp Lys Tyr Leu Ile Pro Asn Ala Thr 100 105 110 Gln Pro Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Phe 115 120 125 Arg Tyr Leu Ser Glu Val Ala Ser Gly Asp Asn Lys Gln Thr Thr Val 130 135 140 Ser Asn Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile Ser Lys Lys 145 150 155 160 Glu Met Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe 165 170 175 Ser Val Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Glu Lys Ala Cys Ser 180 185 190 Leu Ala Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu 195 200 205 Asn Glu Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg 210 215 220 Asp Asn Leu Thr Leu Trp Thr Ser Glu Asn Gln Gly Asp Glu Gly Asp 225 230 235 240 Ala Gly Glu Gly Glu Asn 245 <210> 8 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 epsilon <400> 8 Met Asp Asp Arg Glu Asp Leu Val Tyr Gln Ala Lys Leu Ala Glu Gln 1 5 10 15 Ala Glu Arg Tyr Asp Glu Met Val Glu Ser Met Lys Lys Val Ala Gly 20 25 30 Met Asp Val Glu Leu Thr Val Glu Glu Arg Asn Leu Leu Ser Val Ala 35 40 45 Tyr Lys Asn Val Ile Gly Ala Arg Arg Ala Ser Trp Arg Ile Ile Ser 50 55 60 Ser Ile Glu Gln Lys Glu Glu Asn Lys Gly Gly Glu Asp Lys Leu Lys 65 70 75 80 Met Ile Arg Glu Tyr Arg Gln Met Val Glu Thr Glu Leu Lys Leu Ile 85 90 95 Cys Cys Asp Ile Leu Asp Val Leu Asp Lys His Leu Ile Pro Ala Ala 100 105 110 Asn Thr Gly Glu Ser Lys Val Phe Tyr Tyr Lys Met Lys Gly Asp Tyr 115 120 125 His Arg Tyr Leu Ala Glu Phe Ala Thr Gly Asn Asp Arg Lys Glu Ala 130 135 140 Ala Glu Asn Ser Leu Val Ala Tyr Lys Ala Ala Ser Asp Ile Ala Met 145 150 155 160 Thr Glu Leu Pro Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn 165 170 175 Phe Ser Val Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Asp Arg Ala Cys 180 185 190 Arg Leu Ala Lys Ala Ala Phe Asp Asp Ala Ile Ala Glu Leu Asp Thr 195 200 205 Leu Ser Glu Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu 210 215 220 Arg Asp Asn Leu Thr Leu Trp Thr Ser Asp Met Gln Gly Asp Gly Glu 225 230 235 240 Glu Gln Asn Lys Glu Ala Leu Gln Asp Val Glu Asp Glu Asn Gln 245 250 255 <210> 9 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 gamma <400> 9 Met Val Asp Arg Glu Gln Leu Val Gln Lys Ala Arg Leu Ala Glu Gln 1 5 10 15 Ala Glu Arg Tyr Asp Asp Met Ala Ala Ala Met Lys Asn Val Thr Glu 20 25 30 Leu Asn Glu Pro Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val Ala 35 40 45 Tyr Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Ile Ser 50 55 60 Ser Ile Glu Gln Lys Thr Ser Ala Asp Gly Asn Glu Lys Lys Ile Glu 65 70 75 80 Met Val Arg Ala Tyr Arg Glu Lys Ile Glu Lys Glu Leu Glu Ala Val 85 90 95 Cys Gln Asp Val Leu Ser Leu Leu Asp Asn Tyr Leu Ile Lys Asn Cys 100 105 110 Ser Glu Thr Gln Tyr Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly 115 120 125 Asp Tyr Tyr Arg Tyr Leu Ala Glu Val Ala Thr Gly Glu Lys Arg Ala 130 135 140 Thr Val Val Glu Ser Ser Glu Lys Ala Tyr Ser Glu Ala His Glu Ile 145 150 155 160 Ser Lys Glu His Met Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala 165 170 175 Leu Asn Tyr Ser Val Phe Tyr Tyr Glu Ile Gln Asn Ala Pro Glu Gln 180 185 190 Ala Cys His Leu Ala Lys Thr Ala Phe Asp Asp Ala Ile Ala Glu Leu 195 200 205 Asp Thr Leu Asn Glu Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln 210 215 220 Leu Leu Arg Asp Asn Leu Thr Leu Trp Thr Ser Asp Gln Gln Asp Asp 225 230 235 240 Asp Gly Gly Glu Gly Asn Asn 245 <210> 10 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 eta <400> 10 Met Gly Asp Arg Glu Gln Leu Leu Gln Arg Ala Arg Leu Ala Glu Gln 1 5 10 15 Ala Glu Arg Tyr Asp Asp Met Ala Ser Ala Met Lys Ala Val Thr Glu 20 25 30 Leu Asn Glu Pro Leu Ser Asn Glu Asp Arg Asn Leu Leu Ser Val Ala 35 40 45 Tyr Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Ile Ser 50 55 60 Ser Ile Glu Gln Lys Thr Met Ala Asp Gly Asn Glu Lys Lys Leu Glu 65 70 75 80 Lys Val Lys Ala Tyr Arg Glu Lys Ile Glu Lys Glu Leu Glu Thr Val 85 90 95 Cys Asn Asp Val Leu Ser Leu Leu Asp Lys Phe Leu Ile Lys Asn Cys 100 105 110 Asn Asp Phe Gln Tyr Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly 115 120 125 Asp Tyr Tyr Arg Tyr Leu Ala Glu Val Ala Ser Gly Glu Lys Lys Asn 130 135 140 Ser Val Val Glu Ala Ser Glu Ala Ala Tyr Lys Glu Ala Phe Glu Ile 145 150 155 160 Ser Lys Glu Gln Met Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala 165 170 175 Leu Asn Phe Ser Val Phe Tyr Tyr Glu Ile Gln Asn Ala Pro Glu Gln 180 185 190 Ala Cys Leu Leu Ala Lys Gln Ala Phe Asp Asp Ala Ile Ala Glu Leu 195 200 205 Asp Thr Leu Asn Glu Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln 210 215 220 Leu Leu Arg Asp Asn Leu Thr Leu Trp Thr Ser Asp Gln Gln Asp Glu 225 230 235 240 Glu Ala Gly Glu Gly Asn 245 <210> 11 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 theta <400> 11 Met Glu Lys Thr Glu Leu Ile Gln Lys Ala Lys Leu Ala Glu Gln Ala 1 5 10 15 Glu Arg Tyr Asp Asp Met Ala Thr Cys Met Lys Ala Val Thr Glu Gln 20 25 30 Gly Ala Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr 35 40 45 Lys Asn Val Val Gly Gly Arg Arg Ser Ala Trp Arg Val Ile Ser Ser 50 55 60 Ile Glu Gln Lys Thr Asp Thr Ser Asp Lys Lys Leu Gln Leu Ile Lys 65 70 75 80 Asp Tyr Arg Glu Lys Val Glu Ser Glu Leu Arg Ser Ile Cys Thr Thr 85 90 95 Val Leu Glu Leu Leu Asp Lys Tyr Leu Ile Ala Asn Ala Thr Asn Pro 100 105 110 Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Phe Arg Tyr 115 120 125 Leu Ala Glu Val Ala Cys Gly Asp Asp Arg Lys Gln Thr Ile Asp Asn 130 135 140 Ser Gln Gly Ala Tyr Gln Glu Ala Phe Asp Ile Ser Lys Lys Glu Met 145 150 155 160 Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe Ser Val 165 170 175 Phe Tyr Tyr Glu Ile Leu Asn Asn Pro Glu Leu Ala Cys Thr Leu Ala 180 185 190 Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Asn Glu 195 200 205 Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg Asp Asn 210 215 220 Leu Thr Leu Trp Thr Ser Asp Ser Ala Gly Glu Glu Cys Asp Ala Ala 225 230 235 240 Glu Gly Ala Glu Asn 245 <210> 12 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 zeta <400> 12 Met Asp Lys Asn Glu Leu Val Gln Lys Ala Lys Leu Ala Glu Gln Ala 1 5 10 15 Glu Arg Tyr Asp Asp Met Ala Ala Cys Met Lys Ser Val Thr Glu Gln 20 25 30 Gly Ala Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr 35 40 45 Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Val Ser Ser 50 55 60 Ile Glu Gln Lys Thr Glu Gly Ala Glu Lys Lys Gln Gln Met Ala Arg 65 70 75 80 Glu Tyr Arg Glu Lys Ile Glu Thr Glu Leu Arg Asp Ile Cys Asn Asp 85 90 95 Val Leu Ser Leu Leu Glu Lys Phe Leu Ile Pro Asn Ala Ser Gln Ala 100 105 110 Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Tyr Arg Tyr 115 120 125 Leu Ala Glu Val Ala Ala Gly Asp Asp Lys Lys Gly Ile Val Asp Gln 130 135 140 Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile Ser Lys Lys Glu Met 145 150 155 160 Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe Ser Val 165 170 175 Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Glu Lys Ala Cys Ser Leu Ala 180 185 190 Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu 195 200 205 Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg Asp Asn 210 215 220 Leu Thr Leu Trp Thr Ser Asp Thr Gln Gly Asp Glu Ala Glu Ala Gly 225 230 235 240 Glu Gly Gly Glu Asn 245 <210> 13 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 beta fragment <400> 13 Glu Lys Ile Glu Ala Glu Leu Gln Asp Ile Cys Asn Asp Val Leu Glu 1 5 10 15 Leu Leu Asp Lys 20 <210> 14 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 epsilon fragment <400> 14 Ala Ala Phe Asp Asp Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu Glu 1 5 10 15 Ser Tyr Lys <210> 15 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 gamma fragment <400> 15 Glu Leu Glu Ala Val Cys Gln Asp Val Leu Ser Leu Leu Asp Asn Tyr 1 5 10 15 Leu Ile Lys <210> 16 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 eta fragment <400> 16 Gln Ala Phe Asp Asp Ala Ile Ala Glu Leu Asp Thr Leu Asn Glu Asp 1 5 10 15 Ser Tyr Lys <210> 17 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 theta fragment <400> 17 Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Asn Glu Asp 1 5 10 15 Ser Tyr Lys <210> 18 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 zeta fragment <400> 18 Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu Glu 1 5 10 15 Ser Tyr Lys <110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Method for screening biomarkers diagnosing cardiovascular diseases, biomarkers screened thereby and method for diagnosing cardiovascular diseases using the same <130> KPA200227-KR-D1 <160> 18 <170> KoPatentIn 3.0 < 210> 1 <211> 3020 <212> DNA <213> Artificial Sequence <220> <223> 14-3-3 beta <400> 1 ggaagtggag ctaccgccac cgccgccgcc gattccggag ccggggtagt cgccgccgcc 60 gccgccgctg cagccactgc aggcaccgct gccgccgcct gagtagtggg cttaggaagg 120 aagaggtcat ctcgctcgga gcttcgctcg gaagggtctt tgttccctgc agccctccca 180 cgggaatgac aatggataaa agtgagctgg tacagaaagc caaactcgct gagcaggctg 240 agcgatatga tgatatggct gcagccatga aggcagtcac agaacagggg catgaactct 300 ccaacgaaga gagaaatctg ctctctgttg cctacaagaa tgtggtaggc gcccgccgct 360 cttcctggcg tgtcatctcc agcattgagc agaaaacaga gaggaatgag aagaagcagc 420 agatgggcaa agagtaccgt gagaagatag aggcagaact gcaggacatc tgcaatgatg 480 ttctggagct gttggacaaa tatcttattc ccaatgctac acaaccagaa agtaaggtgt 540 tctacttgaa aatgaa agga gattatttta ggtatctttc tgaagtggca tctggagaca 600 acaaacaaac cactgtgtcg aactcccagc aggcttacca ggaagcattt gaaattagta 660 agaaagaaat gcagcctaca cacccaattc gtcttggtct ggcactaaat ttctcagtct 720 tttactatga gattctaaac tctcctgaaa aggcctgtag cctggcaaaa acggcatttg 780 atgaagcaat tgctgaattg gatacgctga atgaagagtc ttataaagac agcactctga 840 tcatgcagtt acttagggac aatctcactc tgtggacatc ggaaaaccag ggagacgaag 900 gagacgctgg ggagggagag aactaatgtt tctcgtgctt tgtgatctgt tcagtgtcac 960 tctgtaccct caacatatat cccttgtgcg ataaaaaaaa aaaaaaaaaa aaaaagagaa 1020 tcgtacgtcg actttcgatt tttcacagcc tcagcctagg aaaaatggtt catgggataa 1080 acagctggta tttgtatcta aaactcagat tggtcacata aatgccacgg cattccgaag 1140 ttttgatttt gattaacatt gacaggatta ctgtgtgttt aattttttaa aaactgaaca 1200 ctgtgattat ggggttttgt aatttagcag aactcttact ggtagaaaaa atagacctga 1260 attatgtgta actttttgga aggtttaatc tgatatcaaa ataatcattg aaatacaatt 1320 ccattgtaaa gttgtacaga aagttataga gattatattg tgatgctgga acttggagtg 1380 agacacacat catttggcat ttgagttg aa tggtaattca cagtaatgct gccgttgttc 1440 gggacttaaa gacacttgac ctgtttgggc tgttgccact taaaagttca tgaccacaaa 1500 tgtccacagt gtcttcctct gaggaaactc gaatcctgaa atggaaattc tttgtggcag 1560 ataactggct tatgacacct tgaaaagttc aagtgctcat ataacacacc acactgaacc 1620 ccctttccta cagcaatatg ttcactatgt taccaatttg caacttgtgc ttcaatagtg 1680 gaatctactt tcattgttaa cactgagcta aagaaaaaaa gccgtgtgtt ttatgaatga 1740 ccttatctgt ttcctggata atacctttaa gaataatgtc ctgagtcagg cgtggtggtg 1800 cgtgcatcta gtcccaacta tttgggaggc tgaggcagga ggatcgcttg agcccaggag 1860 tttaaagctg cagtgccctg tggttgcacc tgtgaataac tgcactccag cctgggcaac 1920 atagcgagac ctcatctcca aaaaagaaaa caaaaaacaa aaaaaggaat gatgttctgt 1980 agagatggcc tttcacttga ggagtactca gttttcaggt tcttcctagc tcggggcttt 2040 taaattttga aatctaaaca ttctttccca ccatcctttt tgactgttga ccttggtttt 2100 ctcttctaag tttctgtccc tctgcttcct tacttttttt cctttttgaa ttctatcttt 2160 atctgtcttt tgttcacttt ttaatgctat atatgggcag gggtgagaga cattactgag 2220 caccttggtg agcaagcctg gctttaaaga ttg gagaaga gcttctggca ccagaaccct 2280 gtcttcctcc agttctcaac acggtgttgc tcttcagtca taccggaatc tgaatcaaaa 2340 aagtattttt aaatatccat gatttctccc tgtattgagg ctagccctga tcatgctttt 2400 tgtgcctgtc accaggtctc ccaagtgcac tcatccaggt cagtgctcag atgtgtttaa 2460 ggagacccta tattcaggga agttgcgtga acactgcagt ggggagaatt gagaatagtc 2520 aggcctatca gtctcacaga atcacccctc tacctttgat attccactta gctgtagagt 2580 ccatctgttt gtccatctgc tgaaatgaga aaagaaaaat ttatgcactg atttaaaaca 2640 aaccaaaaaa aaagaaaaaa acaaaaaaaa aaatccctcc tttctagctg aacaaaaatg 2700 tgcagttaat acttggcgct tgaaaatgca gtagtgaatg tggaaccaag cctgtctgta 2760 tatctggtag ctcttttctt gctttgtttt ttcttaccag tattctgcct aacgtttgct 2820 tctgtgatgg ttatattgcc tagcaagcac acccgtggtt gtgaaaatag tatagcaaaa 2880 aagaaaaatc cccggttatt gatgtactag atttgtgtat gtcttttaaa cagttctagt 2940 ttcaccttac acagaataat caggaaaagt gtaaaaattc aaaagtgaaa taaaaatttt 3000 atcagttagt tgcctgtgaa 3020 <210> 2 <211> 2052 < 212> DNA <213> Artificial Sequence <220> <223> 14-3-3 epsilon <400 > 2 ggattgaggc gccgccattt ttgctgcccg gacgcggagc gagaggctga gagagtcgga 60 gacactatcc gcttccatcc gtcgcgcaga ccctgccgga gccgctgccg ctatggatga 120 tcgagaggat ctggtgtacc aggcgaagct ggccgagcag gctgagcgat acgacgaaat 180 ggtggagtca atgaagaaag tagcagggat ggatgtggag ctgacagttg aagaaagaaa 240 cctcctatct gttgcatata agaatgtgat tggagctaga agagcctcct ggagaataat 300 cagcagcatt gaacagaaag aagaaaacaa gggaggagaa gacaagctaa aaatgattcg 360 ggaatatcgg caaatggttg agactgagct aaagttaatc tgttgtgaca ttctggatgt 420 actggacaaa cacctcattc cagcagctaa cactggcgag tccaaggttt tctattataa 480 aatgaaaggg gactaccaca ggtatctggc agaatttgcc acaggaaacg acaggaagga 540 ggctgcggag aacagcctag tggcttataa agctgctagt gatattgcaa tgacagaact 600 tccaccaacg catcctattc gcttaggtct tgctctcaat ttttccgtat tctactacga 660 aattcttaat tcccctgacc gtgcctgcag gttggcaaaa gcagcttttg atgatgcaat 720 tgcagaactg gatacgctga gtgaagaaag ctataaggac tctacactta tcatgcagtt 780 gttacgtgat aatctgacac tatggacttc agacatgcag ggtgacggtg aagagcagaa 840 taaagaagcg ctgcag gacg tggaagacga aaatcagtga gacataagcc aacaagagaa 900 accatctctg accaccccct cctccccatc ccaccctttg gaaactcccc attgtcactg 960 agaaccacca aatctgactt ttacatttgg tctcagaatt taggttcctg ccctgttggt 1020 tttttttttt tttttttaaa cagttttcaa aagttcttaa aggcaagagt gaatttctgt 1080 ggattttact ggtcccagct tttaggttct ttaagacact aacaggacta catagaggct 1140 ttttcagcat tactgtgtcg tctccgtgcc agatgtggca agatcaccat tagcaaatgg 1200 aaattacatt tgaaagccat tagacttata ggtgatgcaa gcatctaaga gagaggttaa 1260 tcacactata gaggcataag tggtatcagt tttcattttt ctaattgttt aaactgtgtt 1320 ttataccagt gtttgcaagt aattgggtgt tagcttgaga tggttaaagg tggtttgggg 1380 agggacttcg ttgtaatggt tttgctgtaa aaaatgtttc caactccgct gaaatgttgc 1440 tgaaaagcat ggtgctggta acagttcaac aatccgtggc tgctcattct tgcctacttt 1500 actctcccac tgaagcaggt tagcgttgaa ggtggtatgg aaaagcctgc atgcctgttc 1560 aattcttttg tttcttctcc ttccccctcc ccctacctcc ttcccctcac tcctcccctc 1620 cttcgctcgc tcaacctctt ttgttcagta tgtgtaactt gaagctaatt tgtactactg 1680 gatatctgac tggagccaca gat acagaat ctgtattgtt cttactgaaa cacagcatgg 1740 aattaacatt aaacttaaat aaaacaaacc taaattaaaa atgccaaata tcgcgcctcc 1800 atcctttata cttctaaaat aaaatctcta gtccaaacac actgacagta ttatgtttgc 1860 ctgaatgtga aacattcctc cttttttctt ccataaataa tttgtttgct tcccaaagat 1920 gtatgttttg gaaaaaaatt gcctgtagtc tggttgtgaa gaacactgag tcagttgatg 1980 gtgtgtcttg tcctccagac caggcagcaa catggtattt attttaatca ttaaagtaca 2040 acatgagtag ta 2052 <210> 3 <211> 3705 <212> DNA <213> Artificial Sequence <220> <223> 14-3-3 gamma <400> 3 gcagccgcct cgcgcccggt cccgcggtcg cagctccagc cgcctcctcc gcgcagccgc 60 cgcctcagct gctcgctctg tgggtcggtc ctctccggca cttgggctcc agtcgcgccc 120 tccaagccct tcaggccgcc ccagtgtcct cctccttctc cggccagacc cagccccgcg 180 aagatggtgg accgcgagca actggtgcag aaagcccggc tggccgagca ggcggagcgc 240 tacgacgaca tggccgcggc catgaagaac gtgacagagc tgaatgagcc actgtcgaat 300 gaggaacgaa accttctgtc tgtggcctac aagaacgttg tgggggcacg ccgctcttcc 360 tggagggtca tcagtagcat tgagcagaag acatctgcag acggcaatga gaagaagatt 420 gag atggtcc gtgcgtaccg ggagaagata gagaaggagt tggaggctgt gtgccaggat 480 gtgctgagcc tgctggataa ctacctgatc aagaattgca gcgagaccca gtacgagagc 540 aaagtgttct acctgaagat gaaaggggac tactaccgct acctggctga agtggccacc 600 ggagagaaaa gggcgacggt ggtggagtcc tccgagaagg cctacagcga agcccacgag 660 atcagcaaag agcacatgca gcccacccac cccatccgat taggcctggc tcttaactac 720 tccgtcttct actatgagat ccagaacgcc ccagagcaag cgtgccactt ggccaagacc 780 gcgttcgacg acgccatcgc cgagcttgac accctcaacg aggactccta caaggactcc 840 acgctcatca tgcagctcct ccgcgacaac ctcacgctct ggacgagcga ccagcaggac 900 gacgatggcg gcgaaggcaa caattaaggc cccaggggaa ctggcagcgc acgcggatgc 960 tactactgca gtctttattt ttttcccatg agttgggggt cgggtggggg agggaaaggg 1020 agggatgacc ttcccaggga gaaacccacg acctgtcctg tctttgatcg cctctttgac 1080 atttttgcca aaataccact agtggaaagt caggctagct gtgctggtat tggaatagca 1140 gcctcacact ggcgtctgga ctgttctgta gattcatgca agtggagctg tctgtctcta 1200 atttaactta ttgctagata atagggtttt cagatgaaaa gaaaacttaa agaggaatgg 1260 ccctcattca gtaagtt ctg tggttccagt aaggattttt atgtacatac gctctcgtct 1320 ctcgttttgg gtactttcta tctcatctgt ctcggctctg catgttttcc agggtgtagc 1380 ctacagacat ggaacagtgt aaatcccaga ctgacagact tagaacctga ggtctcattc 1440 atccttatgg tttaggcctt gccagttttc cgaagtctct gattagttga cagtattaac 1500 actaaattgc agtttacagt atttctacat tacagccata tgtaacatca agccatcgat 1560 tgtgtacttt tcctttgcta gttgtttggg ctttaacatc cttattcagc cttatccagg 1620 ttggttttgc tgttgatcgg tctcctaggc taaatgagaa tgaaagcgac ttcaggtcag 1680 gtggctgtgg gatttttttt ttttggtcct tctttcctct taacgtaaat ccaccaccaa 1740 aattattaat cctcttgaga gaaacgtgaa acgccacaaa aatagagaaa attcaggtct 1800 gtatgtcatg gatcgtgttg gtattttcag agaacatccc gcttctgaag ctgctgcagc 1860 tccctcctca gggatcacac tgccgtcacc cactctgcac tggggcgttt cctactgcgc 1920 ctcgtgctgg cggacgcagc tgggtgcaga agctgtgggg tcggagaggc gtttggagaa 1980 ggtctgtggt gcagtgtgtg aaaattcagg tgctagaagc ctactggtag aaaaacccaa 2040 aaggaagagc tatatcctta accattctgt ccaatttcgg gagccttgtc agtgtgtcag 2100 tttttcctcc ccgaagacac tc cttcccca agtaattgta ggaagataaa aaaactgtta 2160 ccagataaca aacactgaac tcctatttga ccagaacttt ttcctctcga gatagttttt 2220 tctttttaat gaaaaaagca taggaattgg agattggctt gtctcacgca gccagtgcac 2280 atttggaatt gacggaaaca acgttgctat ttccacccat ttgttttcgg cagccttaag 2340 gccctcattc tcatttcggg tgaatctgtc tatctgtgaa cgtggcccgc atgtgcattc 2400 ttttttttat atatataaag tcagtgacga ggaactcccg agacgtgtaa tgacaccaca 2460 cttgttttct ttgtttcttt gttttattta ggcaagaaga ggtgtgagta attgaggaaa 2520 aactgacaga tgcttttgct aataccaaaa ttgagcttac aattaggaac tgagtatgtg 2580 taacaggata caggtgacag tgaagataga agaaccacga tgaccacaga ctcaatgtgc 2640 tctgtaacat cgcacagttt acccagcatg actttcctta ggaggccccc tcctcacgct 2700 agagtaaaag tcccagttaa gtgaagccta ccagaagaac tagtagaaga agctttgccg 2760 cttttgtgcc tctcacaggc gcctaaagtc attgccatgg gaggaagacg atttgggggg 2820 ggaggggggg gggggcaggg taggtggggc tttccctaat ttatcttcat gtccagtgag 2880 cagtgttgcg tttttccttg tagcatttgg aaatgattta ctggaattac aaaacctatt 2940 tttcctttaa atttcagctt tggctctg gc tgctttttag aataatgcaa gataaaaatc 3000 acacctgagg gctgaaaacg gagagggaat gggagacttg atatttaagc agcttgaatg 3060 gtttttcttt tctttatttt taaagaaatg cacttgccta tgatactgtc tctccagtga 3120 aatgattact cctccattac tctattgata caatattgtg catgctagtg ttgtatttct 3180 atacagtagc ttgaaattga ttaacttata ctgtaggtgt tatgtattcc tatgacaaaa 3240 aaaattaagt cttcaaattt tttaaaggtt tttttttttt aatttaattt ttccttttgg 3300 gggtaaagtt tgctctacca aatagtgatt gtaacaaatt gatctgtttt ggatgttgct 3360 atagtgacat gcagttatat attttgtttt taaaaggggg ggagcaaaag aaacaccagt 3420 gttagcttaa tcttaatgtc tggtgtttgt catggtgaaa ttataactat tacagtgttg 3480 gagaacaaca aatatgttct ctgaatgagc ctttgtgctt tttgtcatgt tatgcagtga 3540 actattttta aggtctaatc agtgattatt tttccagctc cgtgtttctc taaggaatta 3600 tttcacacac ggaccatctt tagcagtttc ctcagtgatg gaatatcatg aatgtgagtc 3660 attatgtagc tgtcgtacat tgagcaaata aacttacaga tctga 3705 <210> 4 <211> 1751 <212> DNA <213> Artificial Sequence <220> <223> 14-3-3 eta <400> 4 acagcgcgcg gggcgagcca gcgagagggc gcga gcggcg gcgctgcctg cagcctgcag 60 cctgcagcct ccggccggcc ggcgagccag tgcgcgtgcg cggcggcggc ctccgcagcg 120 accggggagc ggactgaccg gcgggagggc tagcgagcca gcggtgtgag gcgcgaggcg 180 aggccgagcc gcgagcgaca tgggggaccg ggagcagctg ctgcagcggg cgcggctggc 240 cgagcaggcg gagcgctacg acgacatggc ctccgctatg aaggcggtga cagagctgaa 300 tgaacctctc tccaatgaag atcgaaatct cctctctgtg gcctacaaga atgtggttgg 360 tgccaggcga tcttcctgga gggtcattag cagcattgag cagaaaacca tggctgatgg 420 aaacgaaaag aaattggaga aagttaaagc ttaccgggag aagattgaga aggagctgga 480 gacagtttgc aatgatgtcc tgtctctgct tgacaagttc ctgatcaaga actgcaatga 540 tttccagtat gagagcaagg tgttttacct gaaaatgaag ggtgattact accgctactt 600 agcagaggtc gcttctgggg agaagaaaaa cagtgtggtc gaagcttctg aagctgccta 660 caaggaagcc tttgaaatca gcaaagagca gatgcaaccc acgcatccca tccggctggg 720 cctggccctc aacttctccg tgttctacta tgagatccag aatgcacctg agcaagcctg 780 cctcttagcc aaacaagcct tcgatgatgc catagctgag ctggacacac taaacgagga 840 ttcctataag gactccacgc tgatcatgca gttgctgcga gacaacctca ccc tctggac 900 gagcgaccag caggatgaag aagcaggaga aggcaactga agatccttca ggtcccctgg 960 cccttccttc acccaccacc cccatcatca ccgattcttc cttgccacaa tcactaaata 1020 tctagtgcta aacctatctg tattggcagc acagctactc agatctgcac tcctgtctct 1080 tgggaagcag tttcagataa atcatgggca ttgctggact gatggttgct ttgagcccac 1140 aggagctccc tttttgaatt gtgtggagaa gtgtgttctg atgaggcatt ttactatgcc 1200 tgttgatcta tgggaaatct aggcgaaagt aatggggaag attagaaaga attagccaac 1260 caggctacag ttgatattta aaagatccat ttaaaacaag ctgatagtgt ttcgttaagc 1320 agtacatctt gtgcatgcaa aaatgaattc acccctccca cctctttctt caattaatgg 1380 aaaactgtta agggaagctg atacagagag acaacttgct cctttccatc agctttataa 1440 taaactgttt aacgtgaggt ttcagtagct ccttggtttt gcctctttaa attatgacgt 1500 gcacaaacct tcttttcaat gcaatgcatc tgaaagtttt gatacttgta actttttttt 1560 ttttttggtt gcaattgttt aagaatcatg gatttatttt ttgtaactct ttggctattg 1620 tccttgtgta tcctgacagc gccatgtgtg tcagcccatg tcaatcaaga tgggtgatta 1680 tgaaatgcca gacttctaaa ataaatgttt tggaattcaa tgggtaaata aatgctgctt 1740 tggggatatt a 1751 <210> 5 <211> 2196 <212> DNA <213> Artificial Sequence <220> <223> 14-3-3 theta <400> 5 aaagccaaaa gcagatcaaa gtggtgggac tcgcgtcgcg gccgcggaga cgtgaagctc 60 tcgaggctcc tcccgctgcg ggtcggcgct cgccctcgct ctcctcgccc tccgccccgg 120 ccccggcccc gcgcccgcca tggagaagac tgagctgatc cagaaggcca agctggccga 180 gcaggccgag cgctacgacg acatggccac ctgcatgaag gcagtgaccg agcagggcgc 240 cgagctgtcc aacgaggagc gcaacctgct ctccgtggcc tacaagaacg tggtcggggg 300 ccgcaggtcc gcctggaggg tcatctctag catcgagcag aagaccgaca cctccgacaa 360 gaagttgcag ctgattaagg actatcggga gaaagtggag tccgagctga gatccatctg 420 caccacggtg ctggaattgt tggataaata tttaatagcc aatgcaacta atccagagag 480 taaggtcttc tatctgaaaa tgaagggtga ttacttccgg taccttgctg aagttgcgtg 540 tggtgatgat cgaaaacaaa cgatagataa ttcccaagga gcttaccaag aggcatttga 600 tataagcaag aaagagatgc aacccacaca cccaatccgc ctggggcttg ctcttaactt 660 ttctgtattt tactatgaga ttcttaataa cccagagctt gcctgcacgc tggctaaaac 720 ggcttttgat gaggccattg ctgaacttga tacactgaat gaagac tcat acaaagacag 780 caccctcatc atgcagttgc ttagagacaa cctaacactt tggacatcag acagtgcagg 840 agaagaatgt gatgcggcag aaggggctga aaactaaatc catacagggt gtcatccttc 900 tttccttcaa gaaacctttt tacacatctc cattccttat tccacttgga tttcctatag 960 caaagaaacc cattcatgtg tatggaatca actgtttata gtcttttcac actgcagctt 1020 tgggaaaact tcattccttg atttgtgttt gtcttggcct tcctggtgtg cagtactgct 1080 gtagaaaagt attaatagct tcatttcata taaacataag taactcccaa acacttatgt 1140 agaggactaa aaatgtatct ggtatttaag taatctgaac cagttctgca agtgactgtg 1200 ttttgtatta ctgtgaaaat aagaaaatgt agttaattac aatttaaaga gtattccaca 1260 taacttctta atttctacat tccctccctt actcttcggg ggtttccttt cagtaagcaa 1320 cttttccatg ctcttaatgt attccttttt agtaggaatc cggaagtatt agattgaatg 1380 gaaaagcact tgccatctct gtctaggggt cacaaattga aatggctcct gtatcacata 1440 cggaggtctt gtgtatctgt ggcaacaggg agtttcctta ttcactcttt atttgctgct 1500 gtttaagttg ccaacctccc ctcccaataa aaattcactt acacctcctg cctttgtagt 1560 tctggtattc actttactat gtgatagaag tagcatgttg ctgccagaat acaag cattg 1620 cttttggcaa attaaagtgc atgtcatttc ttaatacact agaaagggga aataaattaa 1680 agtacacaag tccaagtcta aaactttagt acttttccat gcagatttgt gcacatgtga 1740 gagggtgtcc agtttgtcta gtgattgtta tttagagagt tggaccacta ttgtgtgttg 1800 ctaatcattg actgtagtcc caaaaaagcc ttgtgaaaat gttatgccct atgtaacagc 1860 agagtaacat aaaataaaag tacattttat aaaccattta ctatggcttt gtaacaattg 1920 catacccata ttttaaggga caggtgaatt tactactttc taaagtttat tgatacttcc 1980 cttttatgta aaatgtagta gtgataccta tatttccaca ttgtgcattg tgacacactt 2040 gtctagggat gcctggaagt gtataaaatt ggactgcatt tcttagagtg ttttactata 2100 gatcagtctc atgggccatc tcttcctcag atgtaaatga tatctggtta agtgttatat 2160 ggaataaagt ggacatttta aaactagcaa agttaa 2196 <210> 6 <211> 5085 <212> DNA <213> Artificial Sequence <220> <223> 14-3-3 zeta < 400> 6 gacacagatc cgccatgaca aaggaggaga gtcggggact tgagccgtgg ctccgacttg 60 ggcggagcct ggaggggggt ggttgcgata cgcggaccgg agaatttgca ctttaaagtc 120 cgggtctgcc cgttttcgtt tcacagtaac cgacgtctca agtcagtgcat18 g agctggttca gaaggccaaa ctggccgagc aggctgagcg atatgatgac 240 atggcagcct gcatgaagtc tgtaactgag caaggagctg aattatccaa tgaggagagg 300 aatcttctct cagttgctta taaaaatgtt gtaggagccc gtaggtcatc ttggagggtc 360 gtctcaagta ttgaacaaaa gacggaaggt gctgagaaaa aacagcagat ggctcgagaa 420 tacagagaga aaattgagac ggagctaaga gatatctgca atgatgtact gtctcttttg 480 gaaaagttct tgatccccaa tgcttcacaa gcagagagca aagtcttcta tttgaaaatg 540 aaaggagatt actaccgtta cttggctgag gttgccgctg gtgatgacaa gaaagggatt 600 gtcgatcagt cacaacaagc ataccaagaa gcttttgaaa tcagcaaaaa ggaaatgcaa 660 ccaacacatc ctatcagact gggtctggcc cttaacttct ctgtgttcta ttatgagatt 720 ctgaactccc cagagaaagc ctgctctctt gcaaagacag cttttgatga agccattgct 780 gaacttgata cattaagtga agagtcatac aaagacagca cgctaataat gcaattactg 840 agagacaact tgacattgtg gacatcggat acccaaggag acgaagctga agcaggagaa 900 ggaggggaaa attaaccggc cttccaactt ttgtctgcct cattctaaaa tttacacagt 960 agaccatttg tcatccatgc tgtcccacaa atagtttttt gtttacgatt tatgacaggt 1020 ttatgttact tctatttgaa tttctatatt tcccatgtgg tttttatgtt taatattagg 1080 ggagtagagc cagttaacat ttagggagtt atctgttttc atcttgaggt ggccaatatg 1140 gggatgtgga atttttatac aagttataag tgtttggcat agtacttttg gtacattgtg 1200 gcttcaaaag ggccagtgta aaactgcttc catgtctaag caaagaaaac tgcctacata 1260 ctggtttgtc ctgg cgggga ataaaaggga tcattggttc cagtcacagg tgtagtaatt 1320 gtgggtactt taaggtttgg agcacttaca aggctgtggt agaatcatac cccatggata 1380 ccacatatta aaccatgtat atctgtggaa tactcaatgt gtacaccttt gactacagct 1440 gcagaagtgt tcctttagac aaagttgtga cccattttac tctggataag ggcagaaacg 1500 gttcacattc cattatttgt aaagttacct gctgttagct ttcattattt ttgctacact 1560 cattttattt gtatttaaat gttttaggca acctaagaac aaatgtaaaa gtaaagatgc 1620 aggaaaaatg aattgcttgg tattcattac ttcatgtata tcaagcacag cagtaaaaca 1680 aaaacccatg tatttaactt ttttttagga tttttgcttt tgtgattttt ttttttttga 1740 tacttgccta acatgcatgt gctgtaaaaa tagttaacag ggaaataact tgagatgatg 1800 gctagctttg tttaatgtct tatgaaattt tcatgaacaa tccaagcata attgttaaga 1860 acacgtgtat taaattcatg taagtggaat aaaagtttta tgaatggact tttcaactac 1920 tttctctaca gcttttcatg taaattagtc ttggttctga aacttctcta aaggaaattg 1980 tacatttttt gaaatttatt ccttattccc tcttggcagc taatgggctc ttaccaagtt 2040 taaacacaaa atttatcata acaaaaatac tactaatata actactgttt ccatgtccca 2100 tgatcccctc tcttcctccc caccctgaaa aaaatgagtt cctatttttt ctgggagagg 2160 gggggattga ttagaaaaaa atgtagtgtg ttccatttaa aattttggca tatggcattt 2220 tctaacttag gaagccacaa tgttcttggc ccatcatgac attgggtagc attaactgta 2280 agttttgtgc ttccaaatca ctttttggtt tttaagaatt tcttgatact cttatagcct 2340 gccttcaatt ttgatccttt attctttcta tttgtcaggt gcacaagatt accttcctgt 2400 tttagccttc tgtcttgtca ccaaccattc ttacttggtg gccatgtact tggaaaaagg 2460 ccgcatgatc tttctggctc cactcagtgt ctaaggcacc ctgcttcctt tgcttgcatc 2520 ccacagacta tttccctcat cctatttact gcagcaaatc tctccttagt tgatgagact 2580 gtgtttatct ccctttaaaa ccctacctat cctgaatggt ctgtcattgt ctgcctttaa 2640 aatccttcct ctttcttcct cctctattct ctaaataatg atggggctaa gttataccca 2700 aagctcactt tacaaaatat ttcctcagta ctttgcagaa aacaccaaac aaaaatgcca 2760 ttttaaaaaa ggtgtatttt ttcttttaga atgtaagctc ctcaagagca gggacaatgt 2820 tttctgtatg ttctattgtg cctagtacac tgtaaatgct caataaatat tgatgatggg 2880 aggcagtgag tcttgatgat aagggtgaga aactgaaatc ccaaacactg ttttgttgct 2940 tgttttatta tgacctcaga ttaaa ttggg aaatattggc ccttttgaat aattgtccca 3000 aatattacat tcaaataaaa gtgcaatgga gaccttgggg tctttatttc caggagaaaa 3060 gataccttct gcatctaggg caacaatcct tttccctcgc agctctttct tttacctcac 3120 ttactgattt ctcctgaccc ccaagccaag gaggtaaaat aattatatat atatatatat 3180 atatatatat atatatataa agtaattata ttttatagct agactgaaga ggtgggaatt 3240 tttttgttgt tttttttttt tttggagaca agagtcttgc tctgtcaccc aggcttgagt 3300 gcagtggtgc gatctcggct gagtgcaacc tccgcctgct gggttcaagc gattctcctg 3360 cctcagcctc ccaagtagct gggattacag gtgcccgcca ccacacctgg ctaatttttg 3420 tgtttttagt agagacaggg tttcaccatg ttgtccaggc tggtcttgaa ttcccgacct 3480 caggcaatcc gcccatctca acctcccaaa gtgctgggat tacaggcata agccaccgcg 3540 cccagtagag atgtgatatt attcaagtaa ataaaacctg aaacttggca catttgccta 3600 tttaatgtgc atcagtcatc tgaactaggt tgatcctgga ataatgaaaa cacttcacta 3660 gtattaacta atgttttcta catagtagtt cccaatagat taaggaaaat ttccaacctg 3720 agattctttg ttttggaatt tgaataatgt gtttgtaact attccttgat tgtcccaaat 3780 ccaggcttta cgtctgcttt tgtcaatgta acatcctagt gccaggaaat tggaaaatcc 3840 aatgactttt tgtgtgtaaa gtactggaat ttttttgttc gtttgttttt aagacggagt 3900 gtcaactctc gcccaggctg gggtgcagtg gcctgatctc ggctcactgc aaccttctcc 3960 tcccaggctg aagtgattct cctgcctcag cctcgcaagt agctgggact gcaggcatgt 4020 gccaccactc ctggctaatt atttgtattt ttagtagaga gagggtttca ccgtgttagc 4080 catgatggtc tcattctcct gaccttgtga tccgcccacc tcggcctccc aaagtgctgg 4140 gattacaggc gtgagccact gtgcccaccc tggtatgttt tttttaaatt ggccacttaa 4200 gagttgtcct tcagaaagtg gagcagcagt gtgattagta aaaacgatgg gctttcctgc 4260 agctctggct tatggtcttt gtttttccag ttaacagctg ttacttcatg ctgttatagg 4320 gggtgggggg agaaagagaa agaatatgtc tagtggctta attttggggc tcaatttgtt 4380 tattatgttt ttcctgcaaa agagtcacca gtcttgtcat ggctcaatgc atattttaaa 4440 atgagtcaaa gccaatttgg aagctgctat cttaggttat ttgtagtcag gcttcaaatg 4500 gagagtgaaa ctttgacttt gaaaactggt gctctatgct caatgatggt cttacacatt 4560 cctctaggga aaggtcaaga aataaatttg gcttgattgt attctctcat ttaccatata 4620 ggaaatacta tggtagaact gaaaatatgt acaata gtaa agtggtggct gagactgggc 4680 acttggaaaa tagacttgga ttcttttcta agtgtgaaac taattaaata cttttatgtg 4740 actggtagta gtcacagtct ccttgattga ttctagttcc ttttcaaagt ggagagacct 4800 gaatatagat tgtaacagga tgtccaacct tgccagacaa gcaggcatat atgtctctgg 4860 gatgcagaag cagttacaag ttaatatatt taagcataat gttagccaat cctaggtgat 4920 accagagatg tgaatatttg atcatatttg agttcattcc ttgaaagtcc aaacctaact 4980 ggtcttaatt gattacttct tagtattccg aattttagaa tttaaaaccc tatgaatttt 5040 cagtttgtgc ttacattttc taacattgga tgtttgcttt ggcca 5085 <210> 7 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 beta <400> 7 Met Thr Met Asp Lys Ser Glu Leu Val Gln Lys Ala Lys Leu Ala Glu 1 5 10 15 Gln Ala Glu Arg Tyr Asp Asp Met Ala Ala Ala Met Lys Ala Val Thr 20 25 30 Glu Gln Gly His Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val 35 40 45 Ala Tyr Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Ile 50 55 60 Ser Ser Ile Glu Gln Lys Thr Glu Arg Asn Glu Lys Lys Gln Gln Met 65 70 75 80 Gly Lys Glu Tyr Ar g Glu Lys Ile Glu Ala Glu Leu Gln Asp Ile Cys 85 90 95 Asn Asp Val Leu Glu Leu Leu Asp Lys Tyr Leu Ile Pro Asn Ala Thr 100 105 110 Gln Pro Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Phe 115 120 125 Arg Tyr Leu Ser Glu Val Ala Ser Gly Asp Asn Lys Gln Thr Thr Val 130 135 140 Ser Asn Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile Ser Lys Lys 145 150 155 160 Glu Met Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe 165 170 175 Ser Val Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Glu Lys Ala Cys Ser 180 185 190 Leu Ala Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu 195 200 205 Asn Glu Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg 210 215 220 Asp Asn Leu Thr Leu Trp Thr Ser Glu Asn Gln Gly Asp Glu Gly A sp 225 230 235 240 Ala Gly Glu Gly Glu Asn 245 <210> 8 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 epsilon <400> 8 Met Asp Asp Arg Glu Asp Leu Val Tyr Gln Ala Lys Leu Ala Glu Gln 1 5 10 15 Ala Glu Arg Tyr Asp Glu Met Val Glu Ser Met Lys Lys Val Ala Gly 20 25 30 Met Asp Val Glu Leu Thr Val Glu Glu Arg Asn Leu Leu Ser Val Ala 35 40 45 Tyr Lys Asn Val Ile Gly Ala Arg Arg Ala Ser Trp Arg Ile Ile Ser 50 55 60 Ser Ile Glu Gln Lys Glu Glu Asn Lys Gly Gly Glu Asp Lys Leu Lys 65 70 75 80 Met Ile Arg Glu Tyr Arg Gln Met Val Glu Thr Glu Leu Lys Leu Ile 85 90 95 Cys Cys Asp Ile Leu Asp Val Leu Asp Lys His Leu Ile Pro Ala Ala 100 105 110 Asn Thr Gly Glu Ser Lys Val Phe Tyr Tyr Lys Met Lys Gly Asp Tyr 115 120 125 His Arg Tyr Leu Ala Glu Phe Ala Thr Gly Asn Asp Arg Lys Glu Ala 130 135 140 Ala Glu Asn Ser Leu Val Ala Tyr Lys Ala Ala Ser Asp Ile Ala Met 145 150 155 160 Thr Glu Leu Pro Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn 165 170 175 Phe Ser Val Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Asp Arg Ala Cys 180 185 190 Arg Leu Ala Lys Ala Ala Phe Asp Asp Ala Ile Ala Glu Leu Asp Thr 195 200 205 Leu Ser Glu Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu 210 215 220 Arg Asp Asn Leu Thr Leu Trp Thr Ser Asp Met Gln Gly Asp Gly Glu 225 230 235 240 Glu Gln Asn Lys Glu Ala Leu Gln Asp Val Glu Asp Glu Asn Gln 245 250 255 <210> 9 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> 14- 3-3 gamma <400> 9 Met Val Asp Arg Glu Gln Leu Val Gln Lys Ala Arg Leu Ala Glu Gln 1 5 10 15 Ala Glu Arg Tyr Asp Asp Met Ala Ala Ala Met Lys Asn Val Thr Glu 20 25 30 Leu Asn Glu Pro Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val Ala 35 40 45 Tyr Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Ile Ser 50 55 60 Ser Ile Glu Gln Lys Thr Ser Ala Asp Gly Asn Glu Lys Lys Ile Glu 65 70 75 80 Met Val Arg Ala Tyr Arg Glu Lys Ile Glu Lys Glu Leu Glu Ala Val 85 90 95 Cys Gln Asp Val Leu Ser Leu Leu Asp Asn Tyr Leu Ile Lys Asn Cys 100 105 110 Ser Glu Thr Gln Tyr Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly 115 120 125 Asp Tyr Tyr Arg Tyr Leu Ala Glu Val Ala Thr Gly Glu Lys Arg Ala 130 135 140 Thr Val Val Glu Ser Glu Lys Ala Tyr Ser Glu Ala His Glu Ile 145 150 155 160 Ser Lys Glu His Met Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala 165 170 175 Leu Asn Tyr Ser Val Phe Tyr Tyr Glu Ile Gln Asn Ala Pro Glu Gln 180 185 190 Ala Cys His Leu Ala Lys Thr Ala P he Asp Asp Ala Ile Ala Glu Leu 195 200 205 Asp Thr Leu Asn Glu Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln 210 215 220 Leu Leu Arg Asp Asn Leu Thr Leu Trp Thr Ser Asp Gln Gln Asp Asp 225 230 235 240 Asp Gly Gly Glu Gly Asn Asn 245 <210> 10 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 eta <400> 10 Met Gly Asp Arg Glu Gln Leu Leu Gln Arg Ala Arg Leu Ala Glu Gln 1 5 10 15 Ala Glu Arg Tyr Asp Asp Met Ala Ser Ala Met Lys Ala Val Thr Glu 20 25 30 Leu Asn Glu Pro Leu Ser Asn Glu Asp Arg Asn Leu Leu Ser Val Ala 35 40 45 Tyr Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Ile Ser 50 55 60 Ser Ile Glu Gln Lys Thr Met Ala Asp Gly Asn Glu Lys Lys Leu Glu 65 70 75 80 Lys Val Lys Ala Tyr Arg Glu Lys Ile Glu Lys Glu Leu Glu Thr Val 85 90 95 Cys Asn Asp Val Leu Ser Leu Leu Asp Lys Phe Leu Ile Lys Asn Cys 100 105 110 Asn As p Phe Gln Tyr Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly 115 120 125 Asp Tyr Tyr Arg Tyr Leu Ala Glu Val Ala Ser Gly Glu Lys Lys Asn 130 135 140 Ser Val Val Glu Ala Ser Glu Ala Ala Tyr Lys Glu Ala Phe Glu Ile 145 150 155 160 Ser Lys Glu Gln Met Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala 165 170 175 Leu Asn Phe Ser Val Phe Tyr Tyr Glu Ile Gln Asn Ala Pro Glu Gln 180 185 190 Ala Cys Leu Leu Ala Lys Gln Ala Phe Asp Asp Ala Ile Ala Glu Leu 195 200 205 Asp Thr Leu Asn Glu Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln 210 215 220 Leu Leu Arg Asp Asn Leu Thr Leu Trp Thr Ser Asp Gln Gln Asp Glu 225 230 235 240 Glu Ala Gly Glu Gly Asn 245 <210> 11 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 theta <400> 11 Met Glu Lys Thr Glu Leu Ile Gln Lys Ala Lys Leu Ala Glu Gln Ala 1 5 10 15 Glu Arg Tyr Asp Asp Met Ala Thr Cys Met Lys Ala Val Thr Glu Gln 20 25 30 Gly Ala Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr 35 40 45 Lys Asn Val Val Gly Gly Arg Arg Ser Ala Trp Arg Val Ile Ser Ser 50 55 60 Ile Glu Gln Lys Thr Asp Thr Ser Asp Lys Lys Leu Gln Leu Ile Lys 65 70 75 80 Asp Tyr Arg Glu Lys Val Glu Ser Glu Leu Arg Ser Ile Cys Thr Thr Thr 85 90 95 Val Leu Glu Leu Leu Asp Lys Tyr Leu Ile Ala Asn Ala Thr Asn Pro 100 105 110 Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Phe Arg Tyr 115 120 125 Leu Ala Glu Val Ala Cys Gly Asp Asp Arg Lys Gln Thr Ile Asp Asn 130 135 140 Ser Gln Gly Ala Tyr Gln Glu Ala Phe Asp Ile Ser Lys Lys Glu Met 145 150 155 160 Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe Ser Val 165 170 175 Phe Tyr Tyr Glu Ile Leu Asn Asn Pro Glu Leu Ala Cys Thr Leu Ala 180 185 190 Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Asn Glu 195 200 205 Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg Asp Asn 210 215 220 Leu Thr Leu Trp Thr Ser Asp Ser Ala Gly Glu Glu Cys Asp Ala Ala 225 230 235 240 Glu Gly Ala Glu Asn 245 <210> 12 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 zeta <400> 12 Met Asp Lys Asn Glu Leu Val Gln Lys Ala Lys Leu Ala Glu Gln Ala 1 5 10 15 Glu Arg Tyr Asp Asp Met Ala Ala Cys Met Lys Ser Val Thr Glu Gln 20 25 30 Gly Ala Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr 35 40 45 Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Val Ser Ser 50 55 60 Ile Glu Gln Lys Thr Glu Gly Ala Glu Lys Lys Gln Gln Met Ala Arg 65 70 75 80 Glu Tyr Arg Glu Lys Il e Glu Thr Glu Leu Arg Asp Ile Cys Asn Asp 85 90 95 Val Leu Ser Leu Leu Glu Lys Phe Leu Ile Pro Asn Ala Ser Gln Ala 100 105 110 Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Tyr Arg Tyr 115 120 125 Leu Ala Glu Val Ala Ala Gly Asp Asp Lys Lys Gly Ile Val Asp Gln 130 135 140 Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile Ser Lys Lys Glu Met 145 150 155 160 Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe Ser Val 165 170 175 Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Glu Lys Ala Cys Ser Leu Ala 180 185 190 Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu 195 200 205 Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg Asp Asn 210 215 220 Leu Thr Leu Trp Thr Ser Asp Thr Gln Gly Asp Glu Ala Glu Ala Gly 2 25 230 235 240 Glu Gly Gly Glu Asn 245 <210> 13 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 beta fragment <400> 13 Glu Lys Ile Glu Ala Glu Leu Gln Asp Ile Cys Asn Asp Val Leu Glu 1 5 10 15 Leu Leu Asp Lys 20 <210> 14 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 epsilon fragment < 400> 14 Ala Ala Phe Asp Asp Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu Glu 1 5 10 15 Ser Tyr Lys <210> 15 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> 14 -3-3 gamma fragment <400> 15 Glu Leu Glu Ala Val Cys Gln Asp Val Leu Ser Leu Leu Asp Asn Tyr 1 5 10 15 Leu Ile Lys <210> 16 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 eta fragment <400> 16 Gln Ala Phe Asp Asp Ala Ile Ala Glu Leu Asp Thr Leu Asn Glu Asp 1 5 10 15 Ser Tyr Lys <210> 17 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 theta fragment <400> 17 Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Asn Glu Asp 1 5 10 15 Ser Tyr Lys <210> 18 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> 14-3-3 zeta fragment <400> 18 Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu Glu 1 5 10 15 Ser Tyr Lys

Claims (6)

정상 심근 세포 및 심근 비대 세포의 용해물 또는 이의 분비물에 포함된 일련의 단백질을 분리하는 제1단계;
분리된 단백질 중 정상 심근 세포에 비해 심근 비대 세포에서 보다 많이 발현되는 단백질을 선별하는 제2단계;
상기 제2단계로부터 수득한 단백질을 분해 후 액체 크로마토그래피-질량분석을 통해 분리분석하는 제3단계; 및
단백질체학 데이터베이스 검색 엔진(proteomics database search engine)을 이용하여 상기 제2단계로부터 선택된 단백질을 동정하는 제4단계를 포함하는,
심근 비대 세포에 의해 유발되는 심혈관 질환 진단용 바이오마커의 선별방법으로서,
상기 심근 비대 세포는 정상 심근 세포에 안지오텐신 II 및 엔도텔린-1을 처리하여 준비되는 것이 특징인, 심근 비대 세포에 의해 유발되는 심혈관 질환 진단용 바이오마커의 선별방법으로서,
상기 바이오마커는 14-3-3 단백질 또는 이의 아형인 것인, 심혈관 질환 진단용 바이오마커의 선별방법.
A first step of isolating a series of proteins included in the lysates of normal myocardial cells and myocardial hypertrophic cells or secretions thereof;
A second step of selecting a protein that is more expressed in myocardial hypertrophic cells than in normal myocardial cells among the isolated proteins;
A third step of separating and analyzing the protein obtained from the second step through liquid chromatography-mass spectrometry after digestion; and
A fourth step of identifying the protein selected from the second step using a proteomics database search engine,
As a method for screening biomarkers for diagnosing cardiovascular diseases caused by myocardial hypertrophic cells,
The myocardial hypertrophic cells are prepared by treating normal myocardial cells with angiotensin II and endothelin-1, as a method for screening biomarkers for diagnosing cardiovascular diseases caused by myocardial hypertrophic cells,
The biomarker is a 14-3-3 protein or a subtype thereof, a method for screening a biomarker for diagnosing cardiovascular disease.
 삭제delete 제1항에 있어서,
상기 제1단계는 전기영동(electrophoresis), 웨스턴블롯(western blot) 또는 둘 모두에 의해 수행되는 것인, 심혈관 질환 진단용 바이오마커의 선별방법.
According to claim 1,
Wherein the first step is performed by electrophoresis, western blot, or both, a method for screening a biomarker for diagnosing cardiovascular disease.
 삭제delete 삭제delete 제1항에 있어서,
상기 제3단계는 Mascot 또는 Sequest 엔진을 사용하여 수행되는 것인, 심혈관 질환 진단용 바이오마커의 선별방법.
According to claim 1,
Wherein the third step is performed using a Mascot or Sequest engine, a method for screening biomarkers for diagnosing cardiovascular diseases.
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Giulio Agnetti et al., 'Proteomic profiling of endothelin-1-stimulated hypertrophic cardiomyocytes reveals the increase of four different desmin species and a-B-crystallin', Biochim Biophys Acta., 200*
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