KR20080028642A - Protein marker endorepellin lg3 fragment for breast cancer diagnosis and diagnosis kit for breast cancer using antibody against the same - Google Patents

Protein marker endorepellin lg3 fragment for breast cancer diagnosis and diagnosis kit for breast cancer using antibody against the same Download PDF

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KR20080028642A
KR20080028642A KR1020060094199A KR20060094199A KR20080028642A KR 20080028642 A KR20080028642 A KR 20080028642A KR 1020060094199 A KR1020060094199 A KR 1020060094199A KR 20060094199 A KR20060094199 A KR 20060094199A KR 20080028642 A KR20080028642 A KR 20080028642A
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KR100847274B1 (en
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이철주
장종욱
유명희
강운범
노동영
이종원
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한국과학기술연구원
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates

Abstract

A kit for diagnosing breast cancer comprising a marker endorepellin LG3 fragment for diagnosing the breast cancer and an antibody thereof is provided to show high sensitivity and accuracy of diagnosis and be used for an initial diagnosis of the breast cancer by conveniently diagnosing the breast cancer using blood of a patient. A protein marker endorepellin LG3 fragment for diagnosing breast cancer comprises an amino acid sequence of SEQ ID : NO. 2. A kit for diagnosing the breast cancer comprises an antibody specific to the protein marker endorepellin LG3 fragment. A method for detecting the protein marker endorepellin LG3 fragment comprises the steps of: (a) coating a fixed body with a test body and a protein of a control group; (b) adding the antibody specific to the protein marker endorepellin LG3 fragment to the fixed body to perform an antigen-antibody reaction; (c) detecting the antigen-antibody reaction product obtained from the step(b) using a secondary antibody conjugate and a chromogenic substrate solution; and (d) comparing results regarding the test body and the control group. Further, the antibody is multi clone antibody or single clone antibody.

Description

유방암 진단용 단백질 마커 엔도레펠린 ＬＧ3 절편 및 이에 대한 항체를 포함하는 유방암 진단키트{PROTEIN MARKER ENDOREPELLIN LG3 FRAGMENT FOR BREAST CANCER DIAGNOSIS AND DIAGNOSIS KIT FOR BREAST CANCER USING ANTIBODY AGAINST THE SAME}PROTEIN MARKER ENDOREPELLIN LG3 FRAGMENT FOR BREAST CANCER DIAGNOSIS AND DIAGNOSIS KIT FOR BREAST CANCER USING ANTIBODY AGAINST THE SAME}

도　1은 정상 세포주인 Hs578Bst와 유방암 세포주인 Hs578T의 배양액을 이차원 전기영동으로 분석하여 유방암 세포주에서 특이적으로 분비가 감소한 스팟을 도식화한 결과이고, 1 is a result of analyzing the culture medium of Hs578Bst, a normal cell line, and Hs578T, a breast cancer cell line, by two-dimensional electrophoresis, and plotting the spots with reduced specific secretion in breast cancer cell lines.

도　2는 상기 도 1에서 검출된 유방암 세포주에서 특이적으로 분비가 감소한 스팟을 트립신으로 처리하여 얻은 펩타이드들 중 하나인 서열번호: 3의 펩타이드에 대한 탬덤질량분석(tandem mass spectrometry) 스펙트럼 결과를 나타낸 것이고, Figure 2 is specific to the one of the peptides obtained by secreting the handle down spot by trypsin sequence number in the breast cancer cell line detected by the Figure 1: shows the taemdeom mass spectrometry (tandem mass spectrometry) spectrum results for the three peptides Will,

도 3은 각각 6명의 정상인 혈장과 6명의 유방암 환자의 혈장을 전기영동으로 분석한 후 항-엔도레펠린 LG3 절편 항체로 면역블럿팅을 수행한 결과이고, FIG. 3 shows the results of immunoblotting with anti-endorepelin LG3 fragment antibody after electrophoresis of the plasma of six normal and six breast cancer patients, respectively.

도 4는 상기 도 3의 면역블럿팅에 의해 측정된 항체의 농도를 그래프로 나타낸 것이다. Figure 4 shows the concentration of antibody as determined by immune blot of Figure 3 in a graph.

본 발명은 유방암을 진단할 수 있는 단백질 마커 엔도레펠린 LG3 절편 및 이의 항체를 포함하는 유방암 진단키트에 관한 것이다.The present invention relates to a breast cancer diagnostic kit comprising a protein marker endorepelin LG3 fragment and an antibody thereof for diagnosing breast cancer.

유방암의 원인에 대해 명확하게 밝혀진 것은 없지만, 여성 호르몬, 가족력, 과거력, 출산력, 식생활 습관 등의 다양한 인자들이 거론되고 있다. 2005년 통계청의 조사에 의하면, 한국 여성의 유방암 발생은 최근 급격히 증가하여 1998년 자궁경부암을 추월한 이래 2001년 발생한 한국 여성암 환자의 16.1%를 차지하면서 위암을 제치고 여성암 1위가 되었다. 특히, 2002년에는 2001년에 비해 유방암(11.1%)이 가장 급증한 암으로 나타나 저출산, 짧은 수유기간, 이른 초경, 늦은 폐경 등 생리적으로 왕성한 신체적 변화를 겪는 시기의 여성들에서는 여성호르몬의 자극을 받는 횟수의 급격한 증가로 인한 유선조직의 민감도 증가, 식생활의 서구화, 생활환경의 오염 등의 이유로 유방암 발생이 급격하게 증가하고 있다.Although the cause of breast cancer is not clear, various factors such as female hormones, family history, past history, fertility, and dietary habits have been discussed. According to a survey by the National Statistical Office in 2005, breast cancer in Korean women has recently increased rapidly, exceeding cervical cancer in 1998, accounting for 16.1% of Korean cancer patients in 2001, surpassing stomach cancer, becoming the number one female cancer. In particular, in 2002, breast cancer (11.1%) was the most rapid cancer compared to 2001, and female hormones were stimulated by females in physiologically active physical changes such as low birth, short lactation, early menarche and late menopause. The incidence of breast cancer is increasing rapidly due to the increased sensitivity of the mammary gland tissues due to the rapid increase in the number of people, the westernization of the diet, and the pollution of the living environment.

이러한 유방암의 발생빈도 및 유방암으로 인한 사망률의 증가는 현재의 서구화 실태로 보아 앞으로도 상당기간 지속될 것으로 예상된다. 유방암은 암세포의 성장으로 인한 주변 조직의 침범 또는 림프절 전이 등의 증상을 초래하는 것이 보통이지만, 대부분이 아무런 증상 없이도 자가검진으로 진단될 수 있다. 따라서, 유방암으로 인한 사망률을 줄이기 위해서는 유방암을 효과적으로 조기에 진단하는 것이 매우 중요하다(Tuli et al., Breast J. 12: 343-348, 2006).The increase in the incidence of breast cancer and mortality due to breast cancer is expected to continue for some time in view of the current westernization. Breast cancer usually causes symptoms such as invasion of surrounding tissues or lymph node metastasis due to the growth of cancer cells, but most of them can be diagnosed by self-examination without any symptoms. Therefore, it is very important to effectively and early diagnose breast cancer in order to reduce mortality from breast cancer (Tuli et al., Breast J. 12: 343-348, 2006).

유방암을 진단하기 위해서 여러 가지 방법이 복합적으로 사용되고 있는데, 현재까지는 유방암 환자의 70%가 자가진단에 의해서 내원하고 있다. 그러나, 이러한 자가진단 방법은 악성종양과 양성혹을 구분하는 것이 매우 어렵다는 단점이 있 다. 그 밖에, 유방암의 진단방법으로 X-선 유방촬영법, 초음파검사법, 세침흡입세포검사법, 자기공명촬영법 등이 있는데, 최종적으로는 조직검사를 통해 확인하는 것이 중요하다. X-선 유방촬영법은 X-선으로 유방을 찍어 검사하는 방법으로 혹이 양성인지 악성인지를 감별하는데 우수할 뿐만 아니라, 숨어 있는 혹을 발견하는 방법으로서 자가진단으로 혹이 만져지기 이전에 초기의 유방암을 진단하는데 가장 효과적인 방법이다. 그러나, 유방촬영법은 젊은 여성같이 유선이 많이 발달되어 있다거나 유방이 작고 섬유질이 많은 우리나라 여성에게서는 진단율이 떨어지는 단점이 있으며, 자주 찍으면 오히려 유방암이 유발될 수도 있다는 논란이 있다. 이러한 유방촬영법의 대안으로 초음파검사법이 사용되고 있는데, 초음파검사법은 물혹과 단단한 혹을 구별하는데 효과적이긴 하지만, 악성종양과 양성혹을 감별하는 능력은 떨어진다.Various methods are used to diagnose breast cancer. To date, 70% of breast cancer patients come by self-diagnosis. However, this self-diagnosis method has a disadvantage in that it is very difficult to distinguish between malignant tumors and benign nodules. In addition, X-ray mammography, ultrasonography, fine needle aspiration cytology, magnetic resonance imaging, etc. are diagnosed as breast cancer, and finally, it is important to confirm through histological examination. X-ray mammography is an excellent method for screening breasts with X-rays to distinguish whether the bumps are benign or malignant, and to detect hidden nodules before they are touched by self-diagnosis. It is the most effective way to diagnose breast cancer. However, mammography has a disadvantage in that a lot of mammary glands are developed like young women, or in Korean women with small breasts and a lot of fiber, the diagnosis rate is lowered, and frequent taking may cause breast cancer. Ultrasonography is used as an alternative to mammography, which is effective in distinguishing between water and hard nodules, but lacks the ability to differentiate between malignant tumors and benign nodules.

이러한 기존 진단방법의 단점을 보완하기 위한 방법으로 환자의 혈액에서 종양 표지자(marker)의 농도를 측정하여 유방암을 진단하려는 시도가 있었다(Clinton et al., Biomed Sci . Instrum. 39: 408-414, 2003; Rui et al., Proteomics. 3: 433-439, 2003; Soletormos et al., 48: 229-255, 2001). 그러나, 이러한 종양 표지자들의 진단적 또는 예후 인자로서의 가치가 연구되고는 있지만, 아직까지 제한적으로 사용되고 있을 뿐으로 공식적으로 권장되고 있는 유방암 표지자는 없는 실정이다.In order to make up for the shortcomings of the existing diagnostic methods, there have been attempts to diagnose breast cancer by measuring the concentration of tumor markers in the blood of patients (Clinton et al., Biomed) . Sci . Instrum . 39: 408-414, 2003; Rui et al., Proteomics . 3: 433-439, 2003; Soletormos et al., 48: 229-255, 2001). However, although the value of such tumor markers as a diagnostic or prognostic factor is being studied, there are no breast cancer markers that are officially recommended due to their limited use.

이에, 본 발명자들은 유방암 환자로부터 얻어진 생물학적 검체 시료 내에서 특이하게 변화하는 단백질을 발굴하고 이를 유방암의 조기 진단에 이용하는 방법을 개발하기 위하여 예의 연구 노력한 그 결과, 엔도레펠린 LG3 절편 단백질이 정상인에 비하여 유방암 환자의 혈액에서 특이적으로 그 양이 감소하여 존재한다는 사실을 발견하고, 이의 항체를 이용한 면역화학적 방법으로 유방암을 간편하게 진단할 수 있음을 확인함으로써 본 발명을 완성하였다.Therefore, the present inventors have made intensive studies to develop a method for detecting a specific protein in a biological sample obtained from a breast cancer patient and using it for early diagnosis of breast cancer. The present invention was completed by discovering that the amount of blood in a breast cancer patient was specifically reduced, and confirming that breast cancer could be easily diagnosed by an immunochemical method using an antibody thereof.

따라서, 본 발명의 목적은 혈액내 존재하는 엔도레펠린 LG3 절편을 이용하여 간편하게 유방암을 진단할 수 있는 방법을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a method for easily diagnosing breast cancer using an endrephelin LG3 fragment present in blood.

상기 목적을 달성하기 위하여, 본 발명은 서열번호: 2의 아미노산 서열을 갖는 유방암 진단용 엔도레펠린 LG3 단백질 마커를 제공한다.In order to achieve the above object, the present invention provides a endorepelin LG3 protein marker for diagnosing breast cancer having the amino acid sequence of SEQ ID NO: 2 .

또한, 본 발명은 상기 엔도레펠린 LG3 절편 단백질 마커를 선택적으로 인지하는 항체를 포함하는 유방암 진단키트를 제공한다.The present invention also provides a kit for diagnosing breast cancer comprising an antibody that selectively recognizes the endolephelin LG3 fragment protein marker.

아울러, 본 발명은 검체에서 항원-항체 결합반응을 통해 엔도레펠린 LG3 절편 단백질 마커를 검출하는 방법을 제공한다.In addition, the present invention provides a method for detecting an endorepelin LG3 fragment protein marker through an antigen-antibody binding reaction in a sample.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명의 일 측면에 의하면, 본 발명은 유방암 진단을 위한 단백질 마커로서 서열번호: 2의 아미노산 서열을 갖는 엔도레펠린 LG3 절편 단백질 마커를 제공한다.According to one aspect of the present invention, the present invention provides an endorepelin LG3 fragment protein marker having an amino acid sequence of SEQ ID NO: 2 as a protein marker for breast cancer diagnosis.

엔도레펠린 LG3 절편은 세포막에 존재하는 펠리칸(perlecan)이라는 분비단백질의 C-말단 LG 도메인이 BMP-1(bone morphogenic protein-1)이라는 단백질 분해 효소에 의해 절단되어 생긴 절편이다(Gonzalez et al., J. Biol . Chem . 280: 7080-7087, 2005). 최초에 펠리칸 단백질은 주변세포 표면의 수용체와의 상호작용을 통하여 세포의 부착(attachment), 분화(differentiation), 이동(migration) 등의 기능 조절에 관여하는 세포외 기질(extracellular matrix)의 일종으로 보고되었다. 펠리칸 단백질은 크기가 480 kDa이 되는 헤파란 설페이트 프로테오글리칸(heparan sulfate proteoglycan)으로 5개의 도메인으로 나뉘어져 있으며, 각각의 도메인은 다른 단백질과 결합하기에 용이하도록 작용한다. 펠리칸 단백질의 5개 도메인 중 다섯 번째 도메인이 엔도레펠린(endorepellin)이다. 최근 보고에 의하면, 이 절편을 재조합 단백질로 만든 후 혈관내피세포에 처리하면 혈관생성(angiogenesis)이 억제된다는 사실이 보고되었다(Mongiat et al., J. Biol . Chem . 278: 4238-4249, 2003; Bix et al., J. Cell Biol. 166: 97-109, 2004).Endolephelin LG3 fragments are fragments in which the C-terminal LG domain of the secretory protein called perlecan, which is present in the cell membrane, is cleaved by proteolytic enzymes called bone morphogenic protein-1 (Gonzalez et al. , J. Biol . Chem . 280: 7080-7087, 2005). Initially, the pelican protein is reported as a type of extracellular matrix that is involved in the regulation of functions such as attachment, differentiation and migration of cells through interaction with receptors on the surface of surrounding cells. It became. The pelican protein is heparan sulfate proteoglycan, which is 480 kDa in size, divided into five domains, each of which acts to facilitate binding to other proteins. The fifth of the five domains of the pelican protein is endorepellin. Recent reports have reported that treatment of vascular endothelial cells with this fragment from recombinant proteins inhibits angiogenesis (Mongiat et al., J. Biol . Chem . 278: 4238-4249, 2003). Bix et al., J. Cell Biol . 166: 97-109, 2004).

펠리칸 유전자에 대한 염기서열 및 아미노산 서열 정보는 사람, 생쥐, 닭 등에서 보고된 바 있고, 이들 중 사람의 펠리칸 단백질 유래 엔도레펠린 LG3 절편은 서열번호: 2로 기재되는 아미노산 서열을 갖는다. 서열번호: 2의 엔도레펠린 LG3 절편은 서열번호: 1의 아미노산 서열을 갖는 펠리칸 단백질의 4196번과 4197번째 아미노산 사이가 BMP-1에 의해 절단되어 생성된 것으로, 상기 펠리칸 단백질의 서열번호: 1의 아미노산 서열 중 4197번째부터 4391번째까지의 영역에 해당한다.Nucleotide sequence and amino acid sequence information for the pelican gene has been reported in humans, mice, chickens, etc. Among these, human pelican protein-derived endelin pelin LG3 fragment has an amino acid sequence set forth in SEQ ID NO: 2 . SEQ ID NO: endo Rappel of incorrect LG3 fragments are SEQ ID NO: to be between 4196 times and the 4197th amino acid of Pelican protein having an amino acid sequence of 1 is cut by the BMP-1 generation, sequence number of the pelican protein: 1 Corresponds to the 4197th to 4391th regions of the amino acid sequence of.

본 발명자들은 유방암 환자의 혈액에서 특이적으로 그 양이 감소하여 유방암의 진단에 유용하게 사용될 수 있는 진단 마커를 선발하기 위하여, 유방암 세포주와 정상 세포주를 배양하여 얻은 배양액을 IEF(isoelectricfocusing)와 SDS 전기 영동법으로 등전점과 질량에 따라 분리한 후, 분리된 단백질을 실버 염색법(silver staining)으로 염색한 후 이들을 컴퓨터 프로그램으로 비교, 분석하고 정상세포의 배양액과 비교하여 유방암 세포의 배양액에서 특이적으로 분비가 감소한 단백질을 선별한다(도 1 참조). 이를 동정하기 위하여 선별된 단백질을 트립신으로 처리하여 다수의 펩티드 절편으로 분리한 다음, 각 절편의 질량을 측정하고, 컴퓨터 분석 프로그램을 사용하여 데이터베이스를 검색한 결과, 상기 선별된 단백질이 서열번호: 2의 아미노산 서열을 갖고 등전점이 5.6, 분자량이 26 kDa인 엔도레펠린 LG3 절편인 것을 확인한다.In order to select a diagnostic marker that can be usefully used for the diagnosis of breast cancer due to the specific decrease in the blood of breast cancer patients, the present inventors have used the IEF (isoelectricfocusing) and SDS electrochemical cultures obtained by culturing breast cancer cell lines and normal cell lines. After electrophoresis and separation according to isoelectric point and mass, the isolated proteins were stained by silver staining, and then compared and analyzed by a computer program, and compared with the culture of normal cells. The reduced protein is selected (see FIG. 1 ). By treating the selected protein with trypsin, separated by a plurality of peptide fragments in order to identify them, and then measuring the mass of each fragment and, as a result of using a computer analysis program searches the database, the selected protein is SEQ ID NO: 2 It confirms that it is an endorepelin LG3 fragment which has an amino acid sequence of 5.6, and has an isoelectric point of 5.6 and a molecular weight of 26 kDa.

본 발명에서 유방암 진단 마커로 선별된 엔도레펠린 LG3 절편 단백질은 지금까지 유방암의 발병 및 진행과 직접적으로 연관된다고 알려지지 않았기 때문에, 엔도레펠린 LG3 절편을 유방암의 진단 마커로 이용하는 것은 본 발명이 최초이다. 또한, 상기 단백질은 혈액에서 검출이 가능하기 때문에 생검(biopsy)을 통해서만 진단이 가능하던 기존의 단백질과는 달리 환자에게 불편을 초래하지 않으면서 간편한 방법으로 유방암을 진단하는데 활용될 수 있다.Endorphelin LG3 fragment protein selected as a breast cancer diagnostic marker in the present invention has not been known to be directly related to the onset and progression of breast cancer until now, the present invention is the first to use the endorephelin LG3 fragment as a diagnostic marker of breast cancer . In addition, since the protein can be detected in the blood, unlike the conventional protein, which can be diagnosed only through biopsy, the protein can be used to diagnose breast cancer in a convenient manner without causing inconvenience to the patient.

본 발명에서는 상기 엔도레펠린 LG3 절편이 유방암 환자의 혈액에서만 특이적으로 그 양이 감소함을 확인하고, 이를 선택적으로 인지하는 항체를 이용하여 정상인과 유방암 환자의 혈장 시료를 대상으로 한 면역화학 분석에서 유의한 결과를 얻어(도 3 및 4 참조), 상기 엔도레펠린 LG3 절편이 유방암의 진단 마커로 유용하게 사용될 수 있음을 확인한다.In the present invention, it was confirmed that the amount of the endorphelin LG3 fragment was specifically reduced only in the blood of breast cancer patients, and immunochemical analysis of plasma samples from normal and breast cancer patients using an antibody that selectively recognizes the endorepellin LG3 fragment. By obtaining a significant result in (see FIGS. 3 and 4 ), it is confirmed that the endolephelin LG3 fragment can be usefully used as a diagnostic marker of breast cancer.

본 발명의 일 실시예에 의하면, 피검자의 혈액 시료를 채취하고, 피검자의 혈액 시료에 함유된 상기 유방암 진단 마커인 엔도레펠린 LG3 절편을 이에 대한 항체를 이용하여 검출함으로써 유방암의 발병 여부를 확인할 수 있다.According to one embodiment of the present invention, by taking a blood sample of a subject and detecting the endorphelin LG3 fragment, the breast cancer diagnostic marker contained in the blood sample of the subject by using an antibody against it, it is possible to confirm the onset of breast cancer. have.

따라서, 본 발명의 엔도레펠린 LG3 절편 및 이를 선택적으로 인지하는 항체를 이용한 유방암의 발병 확인방법은 환자의 혈액을 이용하는 새로운 면역학적 진단도구로서 민감도가 우수할 뿐만 아니라 생검을 이용하지 않고 혈액을 대상으로 간편하게 분석할 수 있어, 유방암의 조기 진단에 유용하게 사용될 수 있다.Therefore, the method for confirming the onset of breast cancer using the endorepelin LG3 fragment of the present invention and an antibody that selectively recognizes the same is not only excellent in sensitivity but also sensitive to blood without using a biopsy as a new immunological diagnostic tool using the blood of the patient. As it can be easily analyzed, it can be usefully used for the early diagnosis of breast cancer.

본 발명의 일 실시예에 의하면, 피검자의 혈액 시료를 채취하고, 피검자의 혈액 시료에 함유된 상기 유방암 진단 마커인 엔도레펠린 LG3 절편을 이에 대한 항체를 이용하여 검출함으로써 유방암의 발병 여부를 확인할 수 있다.According to one embodiment of the present invention, by taking a blood sample of a subject and detecting the endorphelin LG3 fragment, the breast cancer diagnostic marker contained in the blood sample of the subject by using an antibody against it, it is possible to confirm the onset of breast cancer. have.

따라서, 본 발명의 엔도레펠린 LG3 절편 및 이를 선택적으로 인지하는 항체를 이용한 유방암의 발병 확인방법은 환자의 혈액을 이용하는 새로운 면역학적 진단도구로서 민감도가 우수할 뿐만 아니라 생검을 이용하지 않고 혈액을 대상으로 간편하게 분석할 수 있어, 유방암의 조기 진단에 유용하게 사용될 수 있다.Therefore, the method for confirming the onset of breast cancer using the endorepelin LG3 fragment of the present invention and an antibody that selectively recognizes the same is not only excellent in sensitivity but also sensitive to blood without using a biopsy as a new immunological diagnostic tool using the blood of the patient. As it can be easily analyzed, it can be usefully used for the early diagnosis of breast cancer.

이에, 본 발명의 다른 측면에 의하면, 본 발명은 유방암 진단 마커인 엔도레펠린 LG3 절편에 선택적으로 결합하는 항체를 포함하는 유방암 진단키트를 제공한다.Accordingly, according to another aspect of the present invention, the present invention provides a breast cancer diagnostic kit comprising an antibody that selectively binds to the endorephelin LG3 fragment which is a breast cancer diagnostic marker.

엔도레펠린 LG3 절편 마커 단백질에 선택적으로 결합하는 항체를 제조하기 위해서는 엔도레펠린 LG3 절편의 입수가 선행되어야 하며, 이는 서열번호: 2의 아미노산 서열을 이용하여 합성하거나 유전자 재조합을 이용하여 미생물에서 생산할 수 있고, 또는 혈액으로부터 직접 분리하여 준비할 수도 있다.In order to prepare an antibody that selectively binds to the endorephelin LG3 fragment marker protein, the acquisition of the endorephelin LG3 fragment must be preceded, which may be synthesized using the amino acid sequence of SEQ ID NO: 2 or produced in a microorganism using genetic recombination. It may be prepared directly or separated from the blood.

본 발명의 목적상, 상기 항체는 엔도레펠린 LG3 절편 마커 단백질뿐만 아니라 펠리칸 단백질을 선택적으로 인지하는 항체를 의미하며, 다클론 항체 및 단클론 항체를 모두 포함할 수 있으나, 항원과 보다 특이적으로 결합할 수 있는 단클론 항체가 바람직하다.For the purposes of the present invention, the antibody refers to an antibody that selectively recognizes a pelican protein as well as an endolephelin LG3 fragment marker protein, and may include both polyclonal and monoclonal antibodies, but more specifically binds to an antigen. Monoclonal antibodies are preferred.

다클론 항체는 당업자에 알려진 종래 방법에 따라 면역원(antigen)으로 엔도레펠린 LG3 절편 마커 단백질 또는 그의 단편을 외부 숙주에 주사함으로써 제조될 수 있다. 이러한 외부 숙주로는 마우스, 래트, 양, 토끼와 같은 포유동물을 예로 들 수 있으며, 면역원은 일반적으로 항원성을 증가시키기 위한 보조제(adjuvant)와 함께 근육내, 복강내 또는 피하주사 등의 방법으로 투여되어 외부 숙주를 면역화시킨다. 면역화된 외부 숙주로부터 정기적으로 혈청을 채취하여 향상된 역가 및 항원에 대한 특이성을 보이는 혈청을 수거하거나 이로부터 항체를 분리·정제하여 엔도레펠린 LG3 절편에 특이적인 다클론 항체를 제조할 수 있다.Polyclonal antibodies can be prepared by injecting an endorepellin LG3 fragment marker protein or fragment thereof into an external host with an immunogen according to conventional methods known to those skilled in the art. Examples of such external hosts include mammals such as mice, rats, sheep and rabbits, and immunogens are generally administered by intramuscular, intraperitoneal or subcutaneous injections with adjuvants to increase antigenicity. Is administered to immunize an external host. Serum from the immunized external host can be collected periodically to collect serum showing enhanced titers and specificity for antigens or to isolate and purify antibodies therefrom to produce polyclonal antibodies specific for the endorepelin LG3 fragment.

단클론 항체는 당업자에 알려진 융합(fusion)에 의한 불멸화된 세포주 생성방법(Koeher and Milstein, Nature 256: 495, 1975)을 이용하여 제조될 수 있다. 상기 방법을 간략히 설명하면, 먼저 순수한 엔도레펠린 LG3 절편 단백질 또는 그의 단편으로 쥐를 면역화시키거나, 이의 펩타이드를 합성하여 소혈청 알부민과 결합시켜 쥐에 면역화시킨다. 면역화된 쥐로부터 분리한 항원-생산 B 임파구를 인간 또는 마우스의 골수종세포와 융합하여 불멸화된 하이브리도마 세포를 생성한다. 이어, 효소면역분석법(enzyme-linked immunosorbent assay, ELISA)로 하이브리도마 세포의 단클론 항체의 생성 여부를 조사하여 양성 클론을 선발하고 이를 배양한 후 항체를 분리·정제하거나 쥐의 복강에 주입한 후 복수를 채취하여 엔도레펠린 LG3 절편에 특이적인 단클론 항체를 제조할 수 있다.Monoclonal antibodies can be prepared using methods of generating immortalized cell lines by fusion known to those skilled in the art (Koeher and Milstein, Nature 256: 495, 1975). Briefly, the method is first immunized with a pure endorefelin LG3 fragment protein or fragment thereof, or a peptide thereof is synthesized and combined with bovine serum albumin to immunize mice. Antigen-producing B lymphocytes isolated from immunized mice are fused with myeloma cells of human or mouse to produce immortalized hybridoma cells. Subsequently, the enzyme-linked immunosorbent assay (ELISA) was used to investigate the production of monoclonal antibodies in hybridoma cells, to select positive clones, to culture them, to isolate and purify the antibodies, or to inject into the abdominal cavity of mice. Ascites can be harvested to prepare monoclonal antibodies specific for the endorepelin LG3 fragment.

본 발명의 엔도레펠린 LG3 절편 단백질의 검출에 사용되는 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란 적어도 항원 결합능을 보유하고 있는 단편을 의미하며, Fab, F(ab'), F(ab')2 및 Fv 등이 있다.Antibodies used in the detection of the endorepellin LG3 fragment protein of the invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule means a fragment having at least antigen-binding ability, and includes Fab, F (ab '), F (ab') 2 and Fv.

본 발명의 유방암 진단키트에는 엔도레펠린 LG3 절편을 선택적으로 인지하는 항체뿐만 아니라 당분야에서 면역학적 분석에 일반적으로 사용되는 도구, 시약 등이 포함된다.The breast cancer diagnosis kit of the present invention includes not only an antibody that selectively recognizes an endrepelin LG3 fragment, but also tools, reagents, and the like, which are generally used for immunological analysis in the art.

본 발명의 일 실시예에 따르면, 상기 유방암 진단키트는 엔도레펠린 LG3 절편 단백질에 특이적인 항체; 기질과의 반응에 의해서 발색하는 표지체가 접합된 이차항체 접합체(conjugate); 상기 표지체와 발색 반응할 발색기질 용액; 세척액; 및 효소반응 정지용액을 포함할 수 있다.According to one embodiment of the invention, the breast cancer diagnostic kit is an antibody specific for the endorephelin LG3 fragment protein; Secondary antibody conjugates to which a label that is developed by reaction with a substrate is conjugated; A color substrate solution to be colored and reacted with the label; Washing liquid; And it may include a solution for stopping the enzyme reaction.

또한, 본 발명의 유방암 진단키트는 엔도레펠린 LG3 절편 표준 항원을 포함하는 양성 대조군과 상기 항원이 주입되지 않은 동물의 항혈청을 포함하는 음성 대조군을 추가적으로 포함할 수 있다.In addition, the breast cancer diagnostic kit of the present invention may further include a positive control comprising an endorephelin LG3 fragment standard antigen and a negative control comprising an antiserum of an animal not injected with the antigen.

본 발명의 유방암 진단키트는 항원-항체 결합반응을 통하여 항체 단백질에 대한 항원을 정량 또는 정성적으로 분석함으로써 유방암을 진단할 수 있으며, 상기 항원-항체 결합반응은 통상의 효소면역분석법(ELISA), 방사능면역분석 법(radioimmnoassay, RIA), 샌드위치 측정법(sandwich assay), 웨스턴 블롯팅, 면역침강법, 면역조직화학염색법(immnohistochemical staining), 형광면역법, 효소기질발색법, 항원-항체 응집법 등의 방법을 이용하여 측정할 수 있다. 예를 들어, 검체 및 대조군을 표면에 코팅시킨 96-웰 마이크로타이터 플레이트 등을 이용하여 재조합 단클론 항체 단백질과 반응하는 ELISA를 수행하도록 상기 진단키트를 제공할 수 있다.Breast cancer diagnostic kit of the present invention can diagnose breast cancer by quantitatively or qualitatively analyzing the antigen to the antibody protein through an antigen-antibody binding reaction, the antigen-antibody binding reaction is a conventional enzyme immunoassay (ELISA), Methods such as radioimmunoassay (RIA), sandwich assay, western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloration, antigen-antibody aggregation Can be measured. For example, the diagnostic kit may be provided to perform an ELISA that reacts with a recombinant monoclonal antibody protein using a 96-well microtiter plate or the like coated on a surface of a sample and a control.

항원-항체 결합반응을 위한 고정체로는 니트로셀룰로오즈 막, PVDF 막, 폴리비닐(polyvinyl) 수지 또는 폴리스티렌(polystyrene) 수지로 합성된 웰 플레이트(Well plate), 유리로 된 슬라이드 글래스 등이 사용될 수 있다.As a fixture for the antigen-antibody coupling reaction, a nitrocellulose membrane, a PVDF membrane, a well plate synthesized with a polyvinyl resin or a polystyrene resin, a glass slide glass, or the like may be used.

이차항체의 표지체는 발색반응을 하는 통상의 발색제가 바람직하며, HRP(horseradish peroxidase), 염기성 탈인산화효소(alkaline phosphatase), 콜로이드 골드(coloid gold), FITC(poly L-lysine-fluorescein isothiocyanate), RITC(rhodamine-B-isothiocyanate) 등의 형광물질(fluorescein) 및 색소(dye) 등의 표지체가 사용될 수 있다.As for the label of the secondary antibody, a conventional color developing agent having a color reaction is preferable, and horseradish peroxidase (HRP), basic alkaline phosphatase, colloid gold, poly L-lysine-fluorescein isothiocyanate (FITC), Labels such as fluorescents such as rhodamine-B-isothiocyanate (RITC) and dyes may be used.

발색을 유도하기 위한 발색기질은 발색반응을 하는 표지체에 따라 사용하는 것이 바람직하며, TMB(3,3',5,5'-tetramethyl bezidine), ABTS[2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD(o-phenylenediamine) 등을 사용할 수 있다. 이때, 발색기질은 완충용액(0.1 M NaAc, pH 5.5)에 용해된 상태로 제공되는 것이 더욱 바람직하다. TMB와 같은 발색기질은 이차항체 접합체의 표지체로 사용된 HRP에 의해 분해되어 발색 침적체를 생성하고, 이 발색 침적체의 침적 정도 를 육안으로 확인함으로써 엔도레펠린 LG3 절편 단백질 항원의 존재 유무를 검출한다.Color substrate to induce color development is preferably used according to the labeling reaction, TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3) -ethylbenzothiazoline-6-sulfonic acid), and OPD (o-phenylenediamine) can be used. At this time, the color substrate is more preferably provided in a dissolved state in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is decomposed by HRP used as a marker of the secondary antibody conjugate to generate a chromosome deposit, and the presence or absence of the endorepellin LG3 fragment protein antigen is detected by visually confirming the deposition degree of the chromosome deposit. do.

세척액은 인산염 완충용액, NaCl 및 트윈 20(Tween 20)을 포함하는 것이 바람직하며, 0.02 M 인산염 완충용액, 0.13 M NaCl, 및 0.05% 트윈 20으로 구성된 완충용액(PBST)이 더욱 바람직하다. 세척액은 항원-항체 결합반응 후 항원-항체 결합체에 이차항체를 반응시킨 다음 적당량을 고정체에 첨가하여 3 내지 6회 세척한다. 반응 정지용액은 황산용액(H2SO4)이 바람직하게 사용될 수 있다.The wash preferably comprises phosphate buffer, NaCl and Tween 20, more preferably a buffer consisting of 0.02 M phosphate buffer, 0.13 M NaCl, and 0.05% Tween 20 (PBST). The wash solution is washed 3 to 6 times by reacting the secondary antibody to the antigen-antibody conjugate after the antigen-antibody binding reaction, and then adding an appropriate amount to the fixture. As the reaction terminating solution, sulfuric acid solution (H 2 SO 4 ) may be preferably used.

아울러, 본 발명의 또 다른 측면은 검체에서 유방암 진단용 마커 단백질인 엔도레펠린 LG3 절편을 항원-항체 결합반응을 이용하여 검출하는 방법을 제공한다. In addition, another aspect of the present invention provides a method for detecting an endrepelin LG3 fragment, which is a marker protein for diagnosing breast cancer in a sample by using an antigen-antibody binding reaction.

상기 검출방법은 혈액내 단백질을 고정화시키거나 전기영동(SDS-PAGE)으로 분리시킨 후 니트로셀룰로즈 막으로 전이시키고 엔도레펠린 LG3 절편을 선택적으로 인지하는 항체와 접촉시켜 항원-항체 결합반응을 통해 엔도레펠린 LG3 절편 단백질의 존재를 간접적으로 확인하는 것을 포함한다. 상기 항원-항체 결합반응으로는 효소면역분석법(ELISA), 방사능면역분석법(RIA), 샌드위치 측정, 웨스턴 블롯팅, 면역침강법, 면역조직화학염색법, 형광면역법, 효소기질발색법, 항원-항체 응집법 등을 예로 들 수 있다. 검체로서는 혈청, 혈장 또는 혈액을 사용할 수 있고, 혈장이 가장 바람직하다.The detection method is an endo-antibody-binding reaction by immobilizing proteins in the blood or by electrophoresis (SDS-PAGE), transferring them to a nitrocellulose membrane, and contacting them with an antibody that selectively recognizes endorepellin LG3 fragments. Indirectly confirming the presence of the lepelin LG3 fragment protein. The antigen-antibody binding reaction includes enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich measurement, western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloration, and antigen-antibody aggregation. Etc. can be mentioned. Serum, plasma or blood may be used as the sample, with plasma being most preferred.

본 발명의 바람직한 실시예에 의하면, 상기 검출방법은,According to a preferred embodiment of the present invention, the detection method,

1) 검체 및 대조군의 단백질을 고정체에 코팅시키는 단계;1) coating the sample and the protein of the control to the fixture;

2) 상기 고정체에 엔도레펠린 LG3 절편에 특이적인 항체를 첨가하여 항원-항체 결합반응을 수행하는 단계;2) performing an antigen-antibody binding reaction by adding an antibody specific for an endrepelin LG3 fragment to the fixture;

3) 상기 항원-항체 결합반응을 통해 생성된 항원-항체 결합반응물을 이차항체 접합체(conjugate) 및 발색기질 용액을 이용하여 검출하는 단계; 및3) detecting the antigen-antibody binding reactant produced through the antigen-antibody binding reaction using a secondary antibody conjugate and a color substrate solution; And

4) 검체와 대조군에 대한 검출 결과를 비교하는 단계를 포함할 수 있다.4) comparing the detection results for the sample and the control.

상기 검출방법을 구체적으로 설명하면, 먼저 검체로부터 혈장단백질을 분자량에 따라 분리한다. 엔도레펠린 LG3 절편은 분자량이 26 kDa이므로 12% 폴리아크릴아마이드 겔을 이용하여 전기영동을 수행하고, 이로부터 분리된 단백질들을 니트로셀룰로즈 막과 같은 고정체로 전이시켜 고정한다. 이어, 고정된 단백질 항원에 엔도레펠린 LG3 절편에 특이적인 항체를 가하여 항원-항체 결합반응을 수행한다. 검체에 엔도레펠린 LG3 절편 단백질이 존재한다면, 상기 단백질이 고정된 막에 엔도레펠린 LG3 절편 특이적인 항체가 가해졌을 때 항원-항체 결합반응이 일어나게 된다. 엔도레펠린 LG3 절편과 이에 대한 항체의 결합 정도를 측정하기 위해서, 엔도레펠린 LG3 절편 항체에 친화성을 갖는 이차항체, 예컨대 항-인간 IgG-HRP와 결합시키는 단계를 수행하는데, 이차항체에 접합된 HRP(horseradish peroxidase)가 기질인 ECL(enhanced chemiluminescence)과 반응하여 발색반응을 일으키는지의 여부와 그 정도를 대조군과 비교함으로써 검체 시료내 유방암 진단 마커인 엔도레펠린 LG3 절편 단백질의 존재 여부를 검출하게 된다.Specifically, the plasma protein is separated from the sample according to the molecular weight. Since the endolephelin LG3 fragment has a molecular weight of 26 kDa, electrophoresis is performed using a 12% polyacrylamide gel, and the proteins separated therefrom are fixed by transferring to a fixture such as a nitrocellulose membrane. Subsequently, an antigen-antibody binding reaction is performed by adding an antibody specific to the endorephelin LG3 fragment to the immobilized protein antigen. If the sample contains an endorepellin LG3 fragment protein, an antigen-antibody binding reaction occurs when an endorphelin LG3 fragment-specific antibody is added to the membrane to which the protein is immobilized. In order to determine the degree of binding of the endorephelin LG3 fragment and the antibody to it, a step of binding to a secondary antibody having an affinity for the endorephelin LG3 fragment antibody, such as an anti-human IgG-HRP, is performed. Whether or not HRP (horseradish peroxidase) reacts with enhanced chemiluminescence (ECL) as a substrate and develops a color reaction, and compares it with a control group to detect the presence of endorephelin LG3 fragment protein, a breast cancer diagnostic marker in a sample. do.

한편, 엔도레펠린 LG3 절편에 특이적인 항체를 생물학적 마이크로칩(biological microchip) 상에 고정시킨 후 대상자로부터 분리된 검체 시료와 반 응시켜 상기 항체 단백질에 대한 항원을 검출할 수 있는 생물학적 마이크로칩 및 자동화된 미세배열 시스템(microarray system)을 이용하면, 한 번의 분석으로 대량의 시료를 분석할 수 있는 장점이 있다.On the other hand, the biological microchip and automation capable of detecting the antigen for the antibody protein by immobilizing the antibody specific for the endorephrine LG3 fragment on a biological microchip and reacting with a sample sample isolated from the subject Using a microarray system, there is an advantage that can analyze a large number of samples in one analysis.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, but the scope of the present invention is not limited by these examples.

실시예Example 1: 유방암 특이적 진단  1: Breast Cancer Specific Diagnosis 마커로서As a marker 엔도레펠린Endolephelin LG3LG3 절편 Intercept 의 동정Pity

본 발명자들은 정상인에 비하여 유방암 환자의 혈액 내에서 특이적인 양적 변화를 나타내는 단백질 마커를 선발하기 위하여, 유방암 세포주 Hs578T(ATCC 등재번호: HTB-126) 및 정상 세포주 Hs578Bst(ATCC 등재번호: HTB-125)를 배양하여 얻은 배양액에 들어 있는 단백질체를 IEF(isoelectricfocusing)와 SDS 전기영동법으로 등전점과 질량에 따라 분리하였다. 분리된 단백질을 실버 염색법(silver staining)으로 염색한 후 이들을 컴퓨터 프로그램으로 비교, 분석하고 정상세포의 배양액과 비교하여 유방암 세포의 배양액에서 특이적으로 분비가 감소한 단백질 스팟을 선별하였다(도 1). 이를 동정하기 위하여 선별된 단백질 스팟을 트립신으로 처리(100 ng)하여 다수의 펩티드 절편으로 분리한 다음, 각 절편의 질량을 탠덤질량분석기(Linear Iontrap quadrupole mass spectrometery, Thermofinnigan사)로 측정하고, 컴퓨터 분석 프로그램을 사용하여 데이터베이스를 검색하였다. 상기 과정에서 서열번호: 3 내지 7로 기재되는 아미노산 서열을 갖는 5개의 트립틱 펩티드(tryptic peptide)가 펠리칸 단백질의 C-말단 도메인, 특히 서열번호: 1의 펠리 칸 단백질의 4197번째부터 4391번째 영역에 해당하는 것으로 동정되었다. 이로부터 유방암 세포주에서 특이적으로 분비가 감소하는 것으로 검출된 스팟이 서열번 호: 2의 아미노산 서열을 갖고 등전점이 5.6, 분자량이 26 kDa인 엔도레펠린 LG3 절편인 것을 확인하였다.In order to select protein markers that exhibit specific quantitative changes in the blood of breast cancer patients compared to normal individuals, the present inventors have identified breast cancer cell lines Hs578T (ATCC accession no .: HTB-126) and normal cell lines Hs578Bst (ATCC accession no .: HTB-125). Protein bodies contained in the culture solution obtained by culturing were separated by isoelectric point and mass by IEF (isoelectricfocusing) and SDS electrophoresis. The isolated proteins were stained by silver staining, and then compared and analyzed by a computer program, and the protein spots that specifically reduced secretion in cultures of breast cancer cells were selected by comparison with cultures of normal cells ( FIG. 1 ). To identify this, the selected protein spot was treated with trypsin (100 ng), separated into a number of peptide fragments, and the mass of each fragment was measured by a tandem mass spectrometer (Linear Iontrap quadrupole mass spectrometery, Thermofinnigan). The database was searched using the program. In this process, five tryptic peptides having an amino acid sequence represented by SEQ ID NOs: 3 to 7 are located at the C-terminal domain of the pelican protein, particularly the 4197 to 4391 regions of the pelican protein of SEQ ID NO: 1 Was identified as From this, a spot is detected sequence number that is less secreted specifically in breast cancer cell lines reduced: has an amino acid sequence of 2 was found that the isoelectric point is 5.6, the endo Rappel lean the LG3 fragments having a molecular weight of 26 kDa.

도 2는 상기 동정 과정에서 트립신에 의해 절단된 펩티드들 중의 하나인 서열번호: 3의 트립틱 펩티드에 대한 탠덤질량분석 스펙트럼 결과를 나타낸 것으로, 이로부터 질량분석기에 의해 동정된 스팟에 해당하는 단백질이 엔도레펠린 LG3임을 확인할 수 있다. Figure 2 shows the tandem mass spectrometry results of the tryptic peptide of SEQ ID NO: 3 , one of the peptides cleaved by trypsin in the identification process, from which the protein corresponding to the spot identified by the mass spectrometer It can be confirmed that it is an endorelin LG3.

실시예Example 2:  2: 면역블롯팅을Immunoblotting 이용한  Used 혈액내In the blood 엔도레펠린Endolephelin LG3LG3 절편의 검출 Detection of intercept

유방암 환자 6명 및 정상인 6명으로부터 각각 혈장을 30 ㎕씩 취하고 혈청 단백질의 대부분을 차지하는 단백질들인 알부민, 면역글로불린, 트랜스페린, 항-트립신 등을 제거하기 위해서 MARS(multiple affinity removal system) 컬럼을 장착한 크로마토그래피를 수행하였다. 상기 컬럼에 결합하지 않는 단백질 구획을 자외선 흡광도 280 ㎚로 추적하여 모으고 컬럼에 결합한 혈액 단백질들을 분리하여 제거하였다. 이와 같이 혈액 단백질이 제거된 구획을 모아 3 kDa 이상의 크기를 갖는 단백질들만을 농축한 후 1% SDS가 포함된 완충용액에 섞은 후 12% SDS 폴리아크릴아마이드 겔을 이용하여 전기영동을 수행하였다. 분리된 혈청 단백질을 50 mM 트리스(Tris-HCl), 130 mM 글리신(glycine), 0.05% SDS 및 12.5% 메탄올(methanol)이 포함된 용액 내에서 니트로셀룰로즈 막으로 전이시키고 전이된 막을 5% 탈지분유가 함유된 TBST(Tris-buffered saline Tween 20)에서 2시간 동안 반응시켰다.30 μl of plasma was collected from 6 breast cancer patients and 6 normal patients, and a MARS (multiple affinity removal system) column was equipped to remove albumin, immunoglobulin, transferrin, anti-trypsin, etc. Chromatography was performed. Protein compartments that do not bind to the column were collected by tracking with an ultraviolet absorbance of 280 nm, and blood proteins bound to the column were separated and removed. Thus, the blood protein compartments were collected, concentrated only proteins having a size of 3 kDa or more, mixed in a buffer solution containing 1% SDS and electrophoresis was performed using a 12% SDS polyacrylamide gel. The separated serum proteins were transferred to nitrocellulose membranes in a solution containing 50 mM Tris-HCl, 130 mM glycine, 0.05% SDS and 12.5% methanol and the transferred membranes were 5% skim milk powder. The reaction was carried out in a TBST containing Tri-buffered saline Tween 20 for 2 hours.

이어, 상기 니트로셀룰로즈 막을 항-엔도레펠린 LG3 절편 항체(1:500, Abcam사, UK)와 2시간 동안 반응시키고 TBST로 3회 세척한 후 쥐의 면역글로불린과 결합하는 쥐 IgG-HRP(1:5000, Amersham Bioscience)와 반응시켰다. 그 뒤, 상기 니트로셀룰로즈 막을 ECL(enhanced chemiluminescence, Amersham Bioscience)과 반응시킨 후 X-ray 필름에 감광시켜 항-엔도레펠린 LG3 절편 항체의 농도를 측정하였다(도 3).Subsequently, the nitrocellulose membrane was reacted with an anti-endorephrine LG3 fragment antibody (1: 500, Abcam, UK) for 2 hours, washed three times with TBST, and then bound to mouse IgG-HRP (1). : 5000, Amersham Bioscience). Thereafter, the nitrocellulose membrane was reacted with enhanced chemiluminescence (ECL) and then subjected to photosensitive X-ray film to determine the concentration of anti-endorepelin LG3 fragment antibody ( FIG. 3 ).

도 4는 도 3의 면역블롯팅에 의해 측정된 항체의 농도를 그래프로 도시한 것으로, 유방암 환자의 경우 혈액 내에 엔도레펠린 LG3 절편 단백질이 정상인에 비하여 평균 2.3배 감소하는 경향을 나타냄을 알 수 있다. 상기 결과로부터 혈액 내에 엔도레펠린 LG3 절편을 선택적으로 인지하는 항체의 농도를 측정하고 이를 정상인과 비교하여 상대적으로 감소한 값을 나타내는 피시험자를 유방암 환자로 진단할 수 있음을 확인하였다. Figure 4 is a graph showing the concentration of the antibody measured by the immunoblotting of Figure 3 , it can be seen that the average tendency to decrease the endorephelin LG3 fragment protein in the blood 2.3 times compared to normal in the case of breast cancer patients have. From the above results, it was confirmed that breast cancer patients could be diagnosed as having a relatively decreased value by measuring the concentration of the antibody that selectively recognizes the endorephelin LG3 fragment in the blood and comparing it with a normal person.

상기에서 살펴본 바와 같이, 본 발명의 유방암 진단용 마커 단백질 및 이의 항체를 이용한 유방암 진단키트는 비교적 채취가 용이한 혈액을 검체로 하기 때문에 생검을 대상으로 하는 기존의 유방암 진단방법과는 달리 환자에게 부담을 주지않고 매우 간편하게 유방암을 진단할 수 있을 뿐만 아니라 진단의 정확도 및 민감도가 높아 유방암의 조기 진단에 유용하게 사용될 수 있다.As described above, the breast cancer diagnostic kit using the marker protein for diagnosing breast cancer of the present invention and an antibody thereof has a burden on a patient unlike a conventional breast cancer diagnosis method for biopsy because blood samples are relatively easy to collect. Not only can breast cancer be diagnosed very easily, but also the accuracy and sensitivity of the diagnosis can be useful for early diagnosis of breast cancer.

<110> Korea Institute of Science and Technology <120> PROTEIN MARKER ENDOREPELLIN LG3 FRAGMENT FOR BREAST CANCER DIAGNOSIS AND DIAGNOSIS KIT FOR BREAST CANCER USING ANTIBODY AGAINST THE SAME <130> KC062710 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 4391 <212> PRT <213> human perlecan protein <400> 1 Met Gly Trp Arg Ala Ala Gly Ala Leu Leu Leu Ala Leu Leu Leu His 1 5 10 15 Gly Arg Leu Leu Ala Val Thr His Gly Leu Arg Ala Tyr Asp Gly Leu 20 25 30 Ser Leu Pro Glu Asp Ile Glu Thr Val Thr Ala Ser Gln Met Arg Trp 35 40 45 Thr His Ser Tyr Leu Ser Asp Asp Glu Asp Met Leu Ala Asp Ser Ile 50 55 60 Ser Gly Asp Asp Leu Gly Ser Gly Asp Leu Gly Ser Gly Asp Phe Gln 65 70 75 80 Met Val Tyr Phe Arg Ala Leu Val Asn Phe Thr Arg Ser Ile Glu Tyr 85 90 95 Ser Pro Gln Leu Glu Asp Ala Gly Ser Arg Glu Phe Arg Glu Val Ser 100 105 110 Glu Ala Val Val Asp Thr Leu Glu Ser Glu Tyr Leu Lys Ile Pro Gly 115 120 125 Asp Gln Val Val Ser Val Val Phe Ile Lys Glu Leu Asp Gly Trp Val 130 135 140 Phe Val Glu Leu Asp Val Gly Ser Glu Gly Asn Ala Asp Gly Ala Gln 145 150 155 160 Ile Gln Glu Met Leu Leu Arg Val Ile Ser Ser Gly Ser Val Ala Ser 165 170 175 Tyr Val Thr Ser Pro Gln Gly Phe Gln Phe Arg Arg Leu Gly Thr Val 180 185 190 Pro Gln Phe Pro Arg Ala Cys Thr Glu Ala Glu Phe Ala Cys His Ser 195 200 205 Tyr Asn Glu Cys Val Ala Leu Glu Tyr Arg Cys Asp Arg Arg Pro Asp 210 215 220 Cys Arg Asp Met Ser Asp Glu Leu Asn Cys Glu Glu Pro Val Leu Gly 225 230 235 240 Ile Ser Pro Thr Phe Ser Leu Leu Val Glu Thr Thr Ser Leu Pro Pro 245 250 255 Arg Pro Glu Thr Thr Ile Met Arg Gln Pro Pro Val Thr His Ala Pro 260 265 270 Gln Pro Leu Leu Pro Gly Ser Val Arg Pro Leu Pro Cys Gly Pro Gln 275 280 285 Glu Ala Ala Cys Arg Asn Gly His Cys Ile Pro Arg Asp Tyr Leu Cys 290 295 300 Asp Gly Gln Glu Asp Cys Glu Asp Gly Ser Asp Glu Leu Asp Cys Gly 305 310 315 320 Pro Pro Pro Pro Cys Glu Pro Asn Glu Phe Pro Cys Gly Asn Gly His 325 330 335 Cys Ala Leu Lys Leu Trp Arg Cys Asp Gly Asp Phe Asp Cys Glu Asp 340 345 350 Arg Thr Asp Glu Ala Asn Cys Pro Thr Lys Arg Pro Glu Glu Val Cys 355 360 365 Gly Pro Thr Gln Phe Arg Cys Val Ser Thr Asn Met Cys Ile Pro Ala 370 375 380 Ser Phe His Cys Asp Glu Glu Ser Asp Cys Pro Asp Arg Ser Asp Glu 385 390 395 400 Phe Gly Cys Met Pro Pro Gln Val Val Thr Pro Pro Arg Glu Ser Ile 405 410 415 Gln Ala Ser Arg Gly Gln Thr Val Thr Phe Thr Cys Val Ala Ile Gly 420 425 430 Val Pro Thr Pro Ile Ile Asn Trp Arg Leu Asn Trp Gly His Ile Pro 435 440 445 Ser His Pro Arg Val Thr Val Thr Ser Glu Gly Gly Arg Gly Thr Leu 450 455 460 Ile Ile Arg Asp Val Lys Glu Ser Asp Gln Gly Ala Tyr Thr Cys Glu 465 470 475 480 Ala Met Asn Ala Arg Gly Met Val Phe Gly Ile Pro Asp Gly Val Leu 485 490 495 Glu Leu Val Pro Gln Arg Gly Pro Cys Pro Asp Gly His Phe Tyr Leu 500 505 510 Glu His Ser Ala Ala Cys Leu Pro Cys Phe Cys Phe Gly Ile Thr Ser 515 520 525 Val Cys Gln Ser Thr Arg Arg Phe Arg Asp Gln Ile Arg Leu Arg Phe 530 535 540 Asp Gln Pro Asp Asp Phe Lys Gly Val Asn Val Thr Met Pro Ala Gln 545 550 555 560 Pro Gly Thr Pro Pro Leu Ser Ser Thr Gln Leu Gln Ile Asp Pro Ser 565 570 575 Leu His Glu Phe Gln Leu Val Asp Leu Ser Arg Arg Phe Leu Val His 580 585 590 Asp Ser Phe Trp Ala Leu Pro Glu Gln Phe Leu Gly Asn Lys Val Asp 595 600 605 Ser Tyr Gly Gly Ser Leu Arg Tyr Asn Val Arg Tyr Glu Leu Ala Arg 610 615 620 Gly Met Leu Glu Pro Val Gln Arg Pro Asp Val Val Leu Met Gly Ala 625 630 635 640 Gly Tyr Arg Leu Leu Ser Arg Gly His Thr Pro Thr Gln Pro Gly Ala 645 650 655 Leu Asn Gln Arg Gln Val Gln Phe Ser Glu Glu His Trp Val His Glu 660 665 670 Ser Gly Arg Pro Val Gln Arg Ala Glu Leu Leu Gln Val Leu Gln Ser 675 680 685 Leu Glu Ala Val Leu Ile Gln Thr Val Tyr Asn Thr Lys Met Ala Ser 690 695 700 Val Gly Leu Ser Asp Ile Ala Met Asp Thr Thr Val Thr His Ala Thr 705 710 715 720 Ser His Gly Arg Ala His Ser Val Glu Glu Cys Arg Cys Pro Ile Gly 725 730 735 Tyr Ser Gly Leu Ser Cys Glu Ser Cys Asp Ala His Phe Thr Arg Val 740 745 750 Pro Gly Gly Pro Tyr Leu Gly Thr Cys Ser Gly Cys Asn Cys Asn Gly 755 760 765 His Ala Ser Ser Cys Asp Pro Val Tyr Gly His Cys Leu Asn Cys Gln 770 775 780 His Asn Thr Glu Gly Pro Gln Cys Asn Lys Cys Lys Ala Gly Phe Phe 785 790 795 800 Gly Asp Ala Met Lys Ala Thr Ala Thr Ser Cys Arg Pro Cys Pro Cys 805 810 815 Pro Tyr Ile Asp Ala Ser Arg Arg Phe Ser Asp Thr Cys Phe Leu Asp 820 825 830 Thr Asp Gly Gln Ala Thr Cys Asp Ala Cys Ala Pro Gly Tyr Thr Gly 835 840 845 Arg Arg Cys Glu Ser Cys Ala Pro Gly Tyr Glu Gly Asn Pro Ile Gln 850 855 860 Pro Gly Gly Lys Cys Arg Pro Val Asn Gln Glu Ile Val Arg Cys Asp 865 870 875 880 Glu Arg Gly Ser Met Gly Thr Ser Gly Glu Ala Cys Arg Cys Lys Asn 885 890 895 Asn Val Val Gly Arg Leu Cys Asn Glu Cys Ala Asp Gly Ser Phe His 900 905 910 Leu Ser Thr Arg Asn Pro Asp Gly Cys Leu Lys Cys Phe Cys Met Gly 915 920 925 Val Ser Arg His Cys Thr Ser Ser Ser Trp Ser Arg Ala Gln Leu His 930 935 940 Gly Ala Ser Glu Glu Pro Gly His Phe Ser Leu Thr Asn Ala Ala Ser 945 950 955 960 Thr His Thr Thr Asn Glu Gly Ile Phe Ser Pro Thr Pro Gly Glu Leu 965 970 975 Gly Phe Ser Ser Phe His Arg Leu Leu Ser Gly Pro Tyr Phe Trp Ser 980 985 990 Leu Pro Ser Arg Phe Leu Gly Asp Lys Val Thr Ser Tyr Gly Gly Glu 995 1000 1005 Leu Arg Phe Thr Val Thr Gln Arg Ser Gln Pro Gly Ser Thr Pro Leu 1010 1015 1020 His Gly Gln Pro Leu Val Val Leu Gln Gly Asn Asn Ile Ile Leu Glu 1025 1030 1035 1040 His His Val Ala Gln Glu Pro Ser Pro Gly Gln Pro Ser Thr Phe Ile 1045 1050 1055 Val Pro Phe Arg Glu Gln Ala Trp Gln Arg Pro Asp Gly Gln Pro Ala 1060 1065 1070 Thr Arg Glu His Leu Leu Met Ala Leu Ala Gly Ile Asp Thr Leu Leu 1075 1080 1085 Ile Arg Ala Ser Tyr Ala Gln Gln Pro Ala Glu Ser Arg Val Ser Gly 1090 1095 1100 Ile Ser Met Asp Val Ala Val Pro Glu Glu Thr Gly Gln Asp Pro Ala 1105 1110 1115 1120 Leu Glu Val Glu Gln Cys Ser Cys Pro Pro Gly Tyr Arg Gly Pro Ser 1125 1130 1135 Cys Gln Asp Cys Asp Thr Gly Tyr Thr Arg Thr Pro Ser Gly Leu Tyr 1140 1145 1150 Leu Gly Thr Cys Glu Arg Cys Ser Cys His Gly His Ser Glu Ala Cys 1155 1160 1165 Glu Pro Glu Thr Gly Ala Cys Gln Gly Cys Gln His His Thr Glu Gly 1170 1175 1180 Pro Arg Cys Glu Gln Cys Gln Pro Gly Tyr Tyr Gly Asp Ala Gln Arg 1185 1190 1195 1200 Gly Thr Pro Gln Asp Cys Gln Leu Cys Pro Cys Tyr Gly Asp Pro Ala 1205 1210 1215 Ala Gly Gln Ala Ala His Thr Cys Phe Leu Asp Thr Asp Gly His Pro 1220 1225 1230 Thr Cys Asp Ala Cys Ser Pro Gly His Ser Gly Arg His Cys Glu Arg 1235 1240 1245 Cys Ala Pro Gly Tyr Tyr Gly Asn Pro Ser Gln Gly Gln Pro Cys Gln 1250 1255 1260 Arg Asp Ser Gln Val Pro Gly Pro Ile Gly Cys Asn Cys Asp Pro Gln 1265 1270 1275 1280 Gly Ser Val Ser Ser Gln Cys Asp Ala Ala Gly Gln Cys Gln Cys Lys 1285 1290 1295 Ala Gln Val Glu Gly Leu Thr Cys Ser His Cys Arg Pro His His Phe 1300 1305 1310 His Leu Ser Ala Ser Asn Pro Asp Gly Cys Leu Pro Cys Phe Cys Met 1315 1320 1325 Gly Ile Thr Gln Gln Cys Ala Ser Ser Ala Tyr Thr Arg His Leu Ile 1330 1335 1340 Ser Thr His Phe Ala Pro Gly Asp Phe Gln Gly Phe Ala Leu Val Asn 1345 1350 1355 1360 Pro Gln Arg Asn Ser Arg Leu Thr Gly Glu Phe Thr Val Glu Pro Val 1365 1370 1375 Pro Glu Gly Ala Gln Leu Ser Phe Gly Asn Phe Ala Gln Leu Gly His 1380 1385 1390 Glu Ser Phe Tyr Trp Gln Leu Pro Glu Thr Tyr Gln Gly Asp Lys Val 1395 1400 1405 Ala Ala Tyr Gly Gly Lys Leu Arg Tyr Thr Leu Ser Tyr Thr Ala Gly 1410 1415 1420 Pro Gln Gly Ser Pro Leu Ser Asp Pro Asp Val Gln Ile Thr Gly Asn 1425 1430 1435 1440 Asn Ile Met Leu Val Ala Ser Gln Pro Ala Leu Gln Gly Pro Glu Arg 1445 1450 1455 Arg Ser Tyr Glu Ile Met Phe Arg Glu Glu Phe Trp Arg Arg Pro Asp 1460 1465 1470 Gly Gln Pro Ala Thr Arg Glu His Leu Leu Met Ala Leu Ala Asp Leu 1475 1480 1485 Asp Glu Leu Leu Ile Arg Ala Thr Phe Ser Ser Val Pro Leu Ala Ala 1490 1495 1500 Ser Ile Ser Ala Val Ser Leu Glu Val Ala Gln Pro Gly Pro Ser Asn 1505 1510 1515 1520 Arg Pro Arg Ala Leu Glu Val Glu Glu Cys Arg Cys Pro Pro Gly Tyr 1525 1530 1535 Ile Gly Leu Ser Cys Gln Asp Cys Ala Pro Gly Tyr Thr Arg Thr Gly 1540 1545 1550 Ser Gly Leu Tyr Leu Gly His Cys Glu Leu Cys Glu Cys Asn Gly His 1555 1560 1565 Ser Asp Leu Cys His Pro Glu Thr Gly Ala Cys Ser Gln Cys Gln His 1570 1575 1580 Asn Ala Ala Gly Glu Phe Cys Glu Leu Cys Ala Pro Gly Tyr Tyr Gly 1585 1590 1595 1600 Asp Ala Thr Ala Gly Thr Pro Glu Asp Cys Gln Pro Cys Ala Cys Pro 1605 1610 1615 Leu Thr Asn Pro Glu Asn Met Phe Ser Arg Thr Cys Glu Ser Leu Gly 1620 1625 1630 Ala Gly Gly Tyr Arg Cys Thr Ala Cys Glu Pro Gly Tyr Thr Gly Gln 1635 1640 1645 Tyr Cys Glu Gln Cys Gly Pro Gly Tyr Val Gly Asn Pro Ser Val Gln 1650 1655 1660 Gly Gly Gln Cys Leu Pro Glu Thr Asn Gln Ala Pro Leu Val Val Glu 1665 1670 1675 1680 Val His Pro Ala Arg Ser Ile Val Pro Gln Gly Gly Ser His Ser Leu 1685 1690 1695 Arg Cys Gln Val Ser Gly Ser Pro Pro His Tyr Phe Tyr Trp Ser Arg 1700 1705 1710 Glu Asp Gly Arg Pro Val Pro Ser Gly Thr Gln Gln Arg His Gln Gly 1715 1720 1725 Ser Glu Leu His Phe Pro Ser Val Gln Pro Ser Asp Ala Gly Val Tyr 1730 1735 1740 Ile Cys Thr Cys Arg Asn Leu His Gln Ser Asn Thr Ser Arg Ala Glu 1745 1750 1755 1760 Leu Leu Val Thr Glu Ala Pro Ser Lys Pro Ile Thr Val Thr Val Glu 1765 1770 1775 Glu Gln Arg Ser Gln Ser Val Arg Pro Gly Ala Asp Val Thr Phe Ile 1780 1785 1790 Cys Thr Ala Lys Ser Lys Ser Pro Ala Tyr Thr Leu Val Trp Thr Arg 1795 1800 1805 Leu His Asn Gly Lys Leu Pro Thr Arg Ala Met Asp Phe Asn Gly Ile 1810 1815 1820 Leu Thr Ile Arg Asn Val Gln Leu Ser Asp Ala Gly Thr Tyr Val Cys 1825 1830 1835 1840 Thr Gly Ser Asn Met Phe Ala Met Asp Gln Gly Thr Ala Thr Leu His 1845 1850 1855 Val Gln Ala Ser Gly Thr Leu Ser Ala Pro Val Val Ser Ile His Pro 1860 1865 1870 Pro Gln Leu Thr Val Gln Pro Gly Gln Leu Ala Glu Phe Arg Cys Ser 1875 1880 1885 Ala Thr Gly Ser Pro Thr Pro Thr Leu Glu Trp Thr Gly Gly Pro Gly 1890 1895 1900 Gly Gln Leu Pro Ala Lys Ala Gln Ile His Gly Gly Ile Leu Arg Leu 1905 1910 1915 1920 Pro Ala Val Glu Pro Thr Asp Gln Ala Gln Tyr Leu Cys Arg Ala His 1925 1930 1935 Ser Ser Ala Gly Gln Gln Val Ala Arg Ala Val Leu His Val His Gly 1940 1945 1950 Gly Gly Gly Pro Arg Val Gln Val Ser Pro Glu Arg Thr Gln Val His 1955 1960 1965 Ala Gly Arg Thr Val Arg Leu Tyr Cys Arg Ala Ala Gly Val Pro Ser 1970 1975 1980 Ala Thr Ile Thr Trp Arg Lys Glu Gly Gly Ser Leu Pro Pro Gln Ala 1985 1990 1995 2000 Arg Ser Glu Arg Thr Asp Ile Ala Thr Leu Leu Ile Pro Ala Ile Thr 2005 2010 2015 Thr Ala Asp Ala Gly Phe Tyr Leu Cys Val Ala Thr Ser Pro Ala Gly 2020 2025 2030 Thr Ala Gln Ala Arg Ile Gln Val Val Val Leu Ser Ala Ser Asp Ala 2035 2040 2045 Ser Pro Pro Pro Val Lys Ile Glu Ser Ser Ser Pro Ser Val Thr Glu 2050 2055 2060 Gly Gln Thr Leu Asp Leu Asn Cys Val Val Ala Gly Ser Ala His Ala 2065 2070 2075 2080 Gln Val Thr Trp Tyr Arg Arg Gly Gly Ser Leu Pro Pro His Thr Gln 2085 2090 2095 Val His Gly Ser Arg Leu Arg Leu Pro Gln Val Ser Pro Ala Asp Ser 2100 2105 2110 Gly Glu Tyr Val Cys Arg Val Glu Asn Gly Ser Gly Pro Lys Glu Ala 2115 2120 2125 Ser Ile Thr Val Ser Val Leu His Gly Thr His Ser Gly Pro Ser Tyr 2130 2135 2140 Thr Pro Val Pro Gly Ser Thr Arg Pro Ile Arg Ile Glu Pro Ser Ser 2145 2150 2155 2160 Ser His Val Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Pro 2165 2170 2175 Gly Gln Ala His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu 2180 2185 2190 Pro Ala Arg His Gln Thr His Gly Ser Leu Leu Arg Leu His Gln Val 2195 2200 2205 Thr Pro Ala Asp Ser Gly Glu Tyr Val Cys His Val Val Gly Thr Ser 2210 2215 2220 Gly Pro Leu Glu Ala Ser Val Leu Val Thr Ile Glu Ala Ser Val Ile 2225 2230 2235 2240 Pro Gly Pro Ile Pro Pro Val Arg Ile Glu Ser Ser Ser Ser Thr Val 2245 2250 2255 Ala Glu Gly Gln Thr Leu Asp Leu Ser Cys Val Val Ala Gly Gln Ala 2260 2265 2270 His Ala Gln Val Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ala Arg 2275 2280 2285 His Gln Val Arg Gly Ser Arg Leu Tyr Ile Phe Gln Ala Ser Pro Ala 2290 2295 2300 Asp Ala Gly Gln Tyr Val Cys Arg Ala Ser Asn Gly Met Glu Ala Ser 2305 2310 2315 2320 Ile Thr Val Thr Val Thr Gly Thr Gln Gly Ala Asn Leu Ala Tyr Pro 2325 2330 2335 Ala Gly Ser Thr Gln Pro Ile Arg Ile Glu Pro Ser Ser Ser Gln Val 2340 2345 2350 Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Pro Gly Gln Ser 2355 2360 2365 His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro Val Arg 2370 2375 2380 His Gln Thr His Gly Ser Leu Leu Arg Leu Tyr Gln Ala Ser Pro Ala 2385 2390 2395 2400 Asp Ser Gly Glu Tyr Val Cys Arg Val Leu Gly Ser Ser Val Pro Leu 2405 2410 2415 Glu Ala Ser Val Leu Val Thr Ile Glu Pro Ala Gly Ser Val Pro Ala 2420 2425 2430 Leu Gly Val Thr Pro Thr Val Arg Ile Glu Ser Ser Ser Ser Gln Val 2435 2440 2445 Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Leu Val Ala Gly Gln Ala 2450 2455 2460 His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro Ala Arg 2465 2470 2475 2480 His Gln Val His Gly Ser Arg Leu Arg Leu Leu Gln Val Thr Pro Ala 2485 2490 2495 Asp Ser Gly Glu Tyr Val Cys Arg Val Val Gly Ser Ser Gly Thr Gln 2500 2505 2510 Glu Ala Ser Val Leu Val Thr Ile Gln Gln Arg Leu Ser Gly Ser His 2515 2520 2525 Ser Gln Gly Val Ala Tyr Pro Val Arg Ile Glu Ser Ser Ser Ala Ser 2530 2535 2540 Leu Ala Asn Gly His Thr Leu Asp Leu Asn Cys Leu Val Ala Ser Gln 2545 2550 2555 2560 Ala Pro His Thr Ile Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ser 2565 2570 2575 Arg His Gln Ile Val Gly Ser Arg Leu Arg Ile Pro Gln Val Thr Pro 2580 2585 2590 Ala Asp Ser Gly Glu Tyr Val Cys His Val Ser Asn Gly Ala Gly Ser 2595 2600 2605 Arg Glu Thr Ser Leu Ile Val Thr Ile Gln Gly Ser Gly Ser Ser His 2610 2615 2620 Val Pro Ser Val Ser Pro Pro Ile Arg Ile Glu Ser Ser Ser Pro Thr 2625 2630 2635 2640 Val Val Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Ala Arg Gln 2645 2650 2655 Pro Gln Ala Ile Ile Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ser 2660 2665 2670 Arg His Gln Thr His Gly Ser His Leu Arg Leu His Gln Met Ser Val 2675 2680 2685 Ala Asp Ser Gly Glu Tyr Val Cys Arg Ala Asn Asn Asn Ile Asp Ala 2690 2695 2700 Leu Glu Ala Ser Ile Val Ile Ser Val Ser Pro Ser Ala Gly Ser Pro 2705 2710 2715 2720 Ser Ala Pro Gly Ser Ser Met Pro Ile Arg Ile Glu Ser Ser Ser Ser 2725 2730 2735 His Val Ala Glu Gly Glu Thr Leu Asp Leu Asn Cys Val Val Pro Gly 2740 2745 2750 Gln Ala His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro 2755 2760 2765 Ser His His Gln Thr Arg Gly Ser Arg Leu Arg Leu His His Val Ser 2770 2775 2780 Pro Ala Asp Ser Gly Glu Tyr Val Cys Arg Val Met Gly Ser Ser Gly 2785 2790 2795 2800 Pro Leu Glu Ala Ser Val Leu Val Thr Ile Glu Ala Ser Gly Ser Ser 2805 2810 2815 Ala Val His Val Pro Ala Pro Gly Gly Ala Pro Pro Ile Arg Ile Glu 2820 2825 2830 Pro Ser Ser Ser Arg Val Ala Glu Gly Gln Thr Leu Asp Leu Lys Cys 2835 2840 2845 Val Val Pro Gly Gln Ala His Ala Gln Val Thr Trp His Lys Arg Gly 2850 2855 2860 Gly Asn Leu Pro Ala Arg His Gln Val His Gly Pro Leu Leu Arg Leu 2865 2870 2875 2880 Asn Gln Val Ser Pro Ala Asp Ser Gly Glu Tyr Ser Cys Gln Val Thr 2885 2890 2895 Gly Ser Ser Gly Thr Leu Glu Ala Ser Val Leu Val Thr Ile Glu Pro 2900 2905 2910 Ser Ser Pro Gly Pro Ile Pro Ala Pro Gly Leu Ala Gln Pro Ile Tyr 2915 2920 2925 Ile Glu Ala Ser Ser Ser His Val Thr Glu Gly Gln Thr Leu Asp Leu 2930 2935 2940 Asn Cys Val Val Pro Gly Gln Ala His Ala Gln Val Thr Trp Tyr Lys 2945 2950 2955 2960 Arg Gly Gly Ser Leu Pro Ala Arg His Gln Thr His Gly Ser Gln Leu 2965 2970 2975 Arg Leu His Leu Val Ser Pro Ala Asp Ser Gly Glu Tyr Val Cys Arg 2980 2985 2990 Ala Ala Ser Gly Pro Gly Pro Glu Gln Glu Ala Ser Phe Thr Val Thr 2995 3000 3005 Val Pro Pro Ser Glu Gly Ser Ser Tyr Arg Leu Arg Ser Pro Val Ile 3010 3015 3020 Ser Ile Asp Pro Pro Ser Ser Thr Val Gln Gln Gly Gln Asp Ala Ser 3025 3030 3035 3040 Phe Lys Cys Leu Ile His Asp Gly Ala Ala Pro Ile Ser Leu Glu Trp 3045 3050 3055 Lys Thr Arg Asn Gln Glu Leu Glu Asp Asn Val His Ile Ser Pro Asn 3060 3065 3070 Gly Ser Ile Ile Thr Ile Val Gly Thr Arg Pro Ser Asn His Gly Thr 3075 3080 3085 Tyr Arg Cys Val Ala Ser Asn Ala Tyr Gly Val Ala Gln Ser Val Val 3090 3095 3100 Asn Leu Ser Val His Gly Pro Pro Thr Val Ser Val Leu Pro Glu Gly 3105 3110 3115 3120 Pro Val Trp Val Lys Val Gly Lys Ala Val Thr Leu Glu Cys Val Ser 3125 3130 3135 Ala Gly Glu Pro Arg Ser Ser Ala Arg Trp Thr Arg Ile Ser Ser Thr 3140 3145 3150 Pro Ala Lys Leu Glu Gln Arg Thr Tyr Gly Leu Met Asp Ser His Ala 3155 3160 3165 Val Leu Gln Ile Ser Ser Ala Lys Pro Ser Asp Ala Gly Thr Tyr Val 3170 3175 3180 Cys Leu Ala Gln Asn Ala Leu Gly Thr Ala Gln Lys Gln Val Glu Val 3185 3190 3195 3200 Ile Val Asp Thr Gly Ala Met Ala Pro Gly Ala Pro Gln Val Gln Ala 3205 3210 3215 Glu Glu Ala Glu Leu Thr Val Glu Ala Gly His Thr Ala Thr Leu Arg 3220 3225 3230 Cys Ser Ala Thr Gly Ser Pro Ala Pro Thr Ile His Trp Ser Lys Leu 3235 3240 3245 Arg Ser Pro Leu Pro Trp Gln His Arg Leu Glu Gly Asp Thr Leu Ile 3250 3255 3260 Ile Pro Arg Val Ala Gln Gln Asp Ser Gly Gln Tyr Ile Cys Asn Ala 3265 3270 3275 3280 Thr Ser Pro Ala Gly His Ala Glu Ala Thr Ile Ile Leu His Val Glu 3285 3290 3295 Ser Pro Pro Tyr Ala Thr Thr Val Pro Glu His Ala Ser Val Gln Ala 3300 3305 3310 Gly Glu Thr Val Gln Leu Gln Cys Leu Ala His Gly Thr Pro Pro Leu 3315 3320 3325 Thr Phe Gln Trp Ser Arg Val Gly Ser Ser Leu Pro Gly Arg Ala Thr 3330 3335 3340 Ala Arg Asn Glu Leu Leu His Phe Glu Arg Ala Ala Pro Glu Asp Ser 3345 3350 3355 3360 Gly Arg Tyr Arg Cys Arg Val Thr Asn Lys Val Gly Ser Ala Glu Ala 3365 3370 3375 Phe Ala Gln Leu Leu Val Gln Gly Pro Pro Gly Ser Leu Pro Ala Thr 3380 3385 3390 Ser Ile Pro Ala Gly Ser Thr Pro Thr Val Gln Val Thr Pro Gln Leu 3395 3400 3405 Glu Thr Lys Ser Ile Gly Ala Ser Val Glu Phe His Cys Ala Val Pro 3410 3415 3420 Ser Asp Arg Gly Thr Gln Leu Arg Trp Phe Lys Glu Gly Gly Gln Leu 3425 3430 3435 3440 Pro Pro Gly His Ser Val Gln Asp Gly Val Leu Arg Ile Gln Asn Leu 3445 3450 3455 Asp Gln Ser Cys Gln Gly Thr Tyr Ile Cys Gln Ala His Gly Pro Trp 3460 3465 3470 Gly Lys Ala Gln Ala Ser Ala Gln Leu Val Ile Gln Ala Leu Pro Ser 3475 3480 3485 Val Leu Ile Asn Ile Arg Thr Ser Val Gln Thr Val Val Val Gly His 3490 3495 3500 Ala Val Glu Phe Glu Cys Leu Ala Leu Gly Asp Pro Lys Pro Gln Val 3505 3510 3515 3520 Thr Trp Ser Lys Val Gly Gly His Leu Arg Pro Gly Ile Val Gln Ser 3525 3530 3535 Gly Gly Val Val Arg Ile Ala His Val Glu Leu Ala Asp Ala Gly Gln 3540 3545 3550 Tyr Arg Cys Thr Ala Thr Asn Ala Ala Gly Thr Thr Gln Ser His Val 3555 3560 3565 Leu Leu Leu Val Gln Ala Leu Pro Gln Ile Ser Met Pro Gln Glu Val 3570 3575 3580 Arg Val Pro Ala Gly Ser Ala Ala Val Phe Pro Cys Ile Ala Ser Gly 3585 3590 3595 3600 Tyr Pro Thr Pro Asp Ile Ser Trp Ser Lys Leu Asp Gly Ser Leu Pro 3605 3610 3615 Pro Asp Ser Arg Leu Glu Asn Asn Met Leu Met Leu Pro Ser Val Arg 3620 3625 3630 Pro Gln Asp Ala Gly Thr Tyr Val Cys Thr Ala Thr Asn Arg Gln Gly 3635 3640 3645 Lys Val Lys Ala Phe Ala His Leu Gln Val Pro Glu Arg Val Val Pro 3650 3655 3660 Tyr Phe Thr Gln Thr Pro Tyr Ser Phe Leu Pro Leu Pro Thr Ile Lys 3665 3670 3675 3680 Asp Ala Tyr Arg Lys Phe Glu Ile Lys Ile Thr Phe Arg Pro Asp Ser 3685 3690 3695 Ala Asp Gly Met Leu Leu Tyr Asn Gly Gln Lys Arg Val Pro Gly Ser 3700 3705 3710 Pro Thr Asn Leu Ala Asn Arg Gln Pro Asp Phe Ile Ser Phe Gly Leu 3715 3720 3725 Val Gly Gly Arg Pro Glu Phe Arg Phe Asp Ala Gly Ser Gly Met Ala 3730 3735 3740 Thr Ile Arg His Pro Thr Pro Leu Ala Leu Gly His Phe His Thr Val 3745 3750 3755 3760 Thr Leu Leu Arg Ser Leu Thr Gln Gly Ser Leu Ile Val Gly Asp Leu 3765 3770 3775 Ala Pro Val Asn Gly Thr Ser Gln Gly Lys Phe Gln Gly Leu Asp Leu 3780 3785 3790 Asn Glu Glu Leu Tyr Leu Gly Gly Tyr Pro Asp Tyr Gly Ala Ile Pro 3795 3800 3805 Lys Ala Gly Leu Ser Ser Gly Phe Ile Gly Cys Val Arg Glu Leu Arg 3810 3815 3820 Ile Gln Gly Glu Glu Ile Val Phe His Asp Leu Asn Leu Thr Ala His 3825 3830 3835 3840 Gly Ile Ser His Cys Pro Thr Cys Arg Asp Arg Pro Cys Gln Asn Gly 3845 3850 3855 Gly Gln Cys His Asp Ser Glu Ser Ser Ser Tyr Val Cys Val Cys Pro 3860 3865 3870 Ala Gly Phe Thr Gly Ser Arg Cys Glu His Ser Gln Ala Leu His Cys 3875 3880 3885 His Pro Glu Ala Cys Gly Pro Asp Ala Thr Cys Val Asn Arg Pro Asp 3890 3895 3900 Gly Arg Gly Tyr Thr Cys Arg Cys His Leu Gly Arg Ser Gly Leu Arg 3905 3910 3915 3920 Cys Glu Glu Gly Val Thr Val Thr Thr Pro Ser Leu Ser Gly Ala Gly 3925 3930 3935 Ser Tyr Leu Ala Leu Pro Ala Leu Thr Asn Thr His His Glu Leu Arg 3940 3945 3950 Leu Asp Val Glu Phe Lys Pro Leu Ala Pro Asp Gly Val Leu Leu Phe 3955 3960 3965 Ser Gly Gly Lys Ser Gly Pro Val Glu Asp Phe Val Ser Leu Ala Met 3970 3975 3980 Val Gly Gly His Leu Glu Phe Arg Tyr Glu Leu Gly Ser Gly Leu Ala 3985 3990 3995 4000 Val Leu Arg Ser Ala Glu Pro Leu Ala Leu Gly Arg Trp His Arg Val 4005 4010 4015 Ser Ala Glu Arg Leu Asn Lys Asp Gly Ser Leu Arg Val Asn Gly Gly 4020 4025 4030 Arg Pro Val Leu Arg Ser Ser Pro Gly Lys Ser Gln Gly Leu Asn Leu 4035 4040 4045 His Thr Leu Leu Tyr Leu Gly Gly Val Glu Pro Ser Val Pro Leu Ser 4050 4055 4060 Pro Ala Thr Asn Met Ser Ala His Phe Arg Gly Cys Val Gly Glu Val 4065 4070 4075 4080 Ser Val Asn Gly Lys Arg Leu Asp Leu Thr Tyr Ser Phe Leu Gly Ser 4085 4090 4095 Gln Gly Ile Gly Gln Cys Tyr Asp Ser Ser Pro Cys Glu Arg Gln Pro 4100 4105 4110 Cys Gln His Gly Ala Thr Cys Met Pro Ala Gly Glu Tyr Glu Phe Gln 4115 4120 4125 Cys Leu Cys Arg Asp Gly Phe Lys Gly Asp Leu Cys Glu His Glu Glu 4130 4135 4140 Asn Pro Cys Gln Leu Arg Glu Pro Cys Leu His Gly Gly Thr Cys Gln 4145 4150 4155 4160 Gly Thr Arg Cys Leu Cys Leu Pro Gly Phe Ser Gly Pro Arg Cys Gln 4165 4170 4175 Gln Gly Ser Gly His Gly Ile Ala Glu Ser Asp Trp His Leu Glu Gly 4180 4185 4190 Ser Gly Gly Asn Asp Ala Pro Gly Gln Tyr Gly Ala Tyr Phe His Asp 4195 4200 4205 Asp Gly Phe Leu Ala Phe Pro Gly His Val Phe Ser Arg Ser Leu Pro 4210 4215 4220 Glu Val Pro Glu Thr Ile Glu Leu Glu Val Arg Thr Ser Thr Ala Ser 4225 4230 4235 4240 Gly Leu Leu Leu Trp Gln Gly Val Glu Val Gly Glu Ala Gly Gln Gly 4245 4250 4255 Lys Asp Phe Ile Ser Leu Gly Leu Gln Asp Gly His Leu Val Phe Arg 4260 4265 4270 Tyr Gln Leu Gly Ser Gly Glu Ala Arg Leu Val Ser Glu Asp Pro Ile 4275 4280 4285 Asn Asp Gly Glu Trp His Arg Val Thr Ala Leu Arg Glu Gly Arg Arg 4290 4295 4300 Gly Ser Ile Gln Val Asp Gly Glu Glu Leu Val Ser Gly Arg Ser Pro 4305 4310 4315 4320 Gly Pro Asn Val Ala Val Asn Ala Lys Gly Ser Val Tyr Ile Gly Gly 4325 4330 4335 Ala Pro Asp Val Ala Thr Leu Thr Gly Gly Arg Phe Ser Ser Gly Ile 4340 4345 4350 Thr Gly Cys Val Lys Asn Leu Val Leu His Ser Ala Arg Pro Gly Ala 4355 4360 4365 Pro Pro Pro Gln Pro Leu Asp Leu Gln His Arg Ala Gln Ala Gly Ala 4370 4375 4380 Asn Thr Arg Pro Cys Pro Ser 4385 4390 <210> 2 <211> 195 <212> PRT <213> human endorepellin LG3 fragment <400> 2 Asp Ala Pro Gly Gln Tyr Gly Ala Tyr Phe His Asp Asp Gly Phe Leu 1 5 10 15 Ala Phe Pro Gly His Val Phe Ser Arg Ser Leu Pro Glu Val Pro Glu 20 25 30 Thr Ile Glu Leu Glu Val Arg Thr Ser Thr Ala Ser Gly Leu Leu Leu 35 40 45 Trp Gln Gly Val Glu Val Gly Glu Ala Gly Gln Gly Lys Asp Phe Ile 50 55 60 Ser Leu Gly Leu Gln Asp Gly His Leu Val Phe Arg Tyr Gln Leu Gly 65 70 75 80 Ser Gly Glu Ala Arg Leu Val Ser Glu Asp Pro Ile Asn Asp Gly Glu 85 90 95 Trp His Arg Val Thr Ala Leu Arg Glu Gly Arg Arg Gly Ser Ile Gln 100 105 110 Val Asp Gly Glu Glu Leu Val Ser Gly Arg Ser Pro Gly Pro Asn Val 115 120 125 Ala Val Asn Ala Lys Gly Ser Val Tyr Ile Gly Gly Ala Pro Asp Val 130 135 140 Ala Thr Leu Thr Gly Gly Arg Phe Ser Ser Gly Ile Thr Gly Cys Val 145 150 155 160 Lys Asn Leu Val Leu His Ser Ala Arg Pro Gly Ala Pro Pro Pro Gln 165 170 175 Pro Leu Asp Leu Gln His Arg Ala Gln Ala Gly Ala Asn Thr Arg Pro 180 185 190 Cys Pro Ser 195 <210> 3 <211> 14 <212> PRT <213> tryptic peptide <400> 3 Ser Leu Pro Glu Val Pro Glu Thr Ile Glu Leu Glu Val Arg 1 5 10 <210> 4 <211> 14 <212> PRT <213> tryptic peptide <400> 4 Leu Val Ser Glu Asp Pro Ile Asn Asp Gly Glu Trp His Arg 1 5 10 <210> 5 <211> 14 <212> PRT <213> tryptic peptide <400> 5 Gly Ser Ile Gln Val Asp Gly Glu Glu Leu Val Ser Gly Arg 1 5 10 <210> 6 <211> 18 <212> PRT <213> tryptic peptide <400> 6 Gly Ser Val Tyr Ile Gly Gly Ala Pro Asp Val Ala Thr Leu Thr Gly 1 5 10 15 Gly Arg <210> 7 <211> 22 <212> PRT <213> tryptic peptide <400> 7 Asn Leu Val Leu His Ser Ala Arg Pro Gly Ala Pro Pro Pro Gln Pro 1 5 10 15 Leu Asp Leu Gln His Arg 20 <110> Korea Institute of Science and Technology <120> PROTEIN MARKER ENDOREPELLIN LG3 FRAGMENT FOR BREAST CANCER          DIAGNOSIS AND DIAGNOSIS KIT FOR BREAST CANCER USING ANTIBODY          AGAINST THE SAME <130> KC062710 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 4391 <212> PRT <213> human perlecan protein <400> 1 Met Gly Trp Arg Ala Ala Gly Ala Leu Leu Leu Ala Leu Leu Leu His   1 5 10 15 Gly Arg Leu Leu Ala Val Thr His Gly Leu Arg Ala Tyr Asp Gly Leu              20 25 30 Ser Leu Pro Glu Asp Ile Glu Thr Val Thr Ala Ser Gln Met Arg Trp          35 40 45 Thr His Ser Tyr Leu Ser Asp Asp Glu Asp Met Leu Ala Asp Ser Ile      50 55 60 Ser Gly Asp Asp Leu Gly Ser Gly Asp Leu Gly Ser Gly Asp Phe Gln  65 70 75 80 Met Val Tyr Phe Arg Ala Leu Val Asn Phe Thr Arg Ser Ile Glu Tyr                  85 90 95 Ser Pro Gln Leu Glu Asp Ala Gly Ser Arg Glu Phe Arg Glu Val Ser             100 105 110 Glu Ala Val Val Asp Thr Leu Glu Ser Glu Tyr Leu Lys Ile Pro Gly         115 120 125 Asp Gln Val Val Ser Val Val Phe Ile Lys Glu Leu Asp Gly Trp Val     130 135 140 Phe Val Glu Leu Asp Val Gly Ser Glu Gly Asn Ala Asp Gly Ala Gln 145 150 155 160 Ile Gln Glu Met Leu Leu Arg Val Ile Ser Ser Gly Ser Val Ala Ser                 165 170 175 Tyr Val Thr Ser Pro Gln Gly Phe Gln Phe Arg Arg Leu Gly Thr Val             180 185 190 Pro Gln Phe Pro Arg Ala Cys Thr Glu Ala Glu Phe Ala Cys His Ser         195 200 205 Tyr Asn Glu Cys Val Ala Leu Glu Tyr Arg Cys Asp Arg Arg Pro Asp     210 215 220 Cys Arg Asp Met Ser Asp Glu Leu Asn Cys Glu Glu Pro Val Leu Gly 225 230 235 240 Ile Ser Pro Thr Phe Ser Leu Leu Val Glu Thr Thr Ser Leu Pro Pro                 245 250 255 Arg Pro Glu Thr Thr Ile Met Arg Gln Pro Pro Val Thr His Ala Pro             260 265 270 Gln Pro Leu Leu Pro Gly Ser Val Arg Pro Leu Pro Cys Gly Pro Gln         275 280 285 Glu Ala Ala Cys Arg Asn Gly His Cys Ile Pro Arg Asp Tyr Leu Cys     290 295 300 Asp Gly Gln Glu Asp Cys Glu Asp Gly Ser Asp Glu Leu Asp Cys Gly 305 310 315 320 Pro Pro Pro Pro Cys Glu Pro Asn Glu Phe Pro Cys Gly Asn Gly His                 325 330 335 Cys Ala Leu Lys Leu Trp Arg Cys Asp Gly Asp Phe Asp Cys Glu Asp             340 345 350 Arg Thr Asp Glu Ala Asn Cys Pro Thr Lys Arg Pro Glu Glu Val Cys         355 360 365 Gly Pro Thr Gln Phe Arg Cys Val Ser Thr Asn Met Cys Ile Pro Ala     370 375 380 Ser Phe His Cys Asp Glu Glu Ser Asp Cys Pro Asp Arg Ser Asp Glu 385 390 395 400 Phe Gly Cys Met Pro Pro Gln Val Val Thr Pro Pro Arg Glu Ser Ile                 405 410 415 Gln Ala Ser Arg Gly Gln Thr Val Thr Phe Thr Cys Val Ala Ile Gly             420 425 430 Val Pro Thr Pro Ile Ile Asn Trp Arg Leu Asn Trp Gly His Ile Pro         435 440 445 Ser His Pro Arg Val Thr Val Thr Ser Glu Gly Gly Arg Gly Thr Leu     450 455 460 Ile Ile Arg Asp Val Lys Glu Ser Asp Gln Gly Ala Tyr Thr Cys Glu 465 470 475 480 Ala Met Asn Ala Arg Gly Met Val Phe Gly Ile Pro Asp Gly Val Leu                 485 490 495 Glu Leu Val Pro Gln Arg Gly Pro Cys Pro Asp Gly His Phe Tyr Leu             500 505 510 Glu His Ser Ala Ala Cys Leu Pro Cys Phe Cys Phe Gly Ile Thr Ser         515 520 525 Val Cys Gln Ser Thr Arg Arg Phe Arg Asp Gln Ile Arg Leu Arg Phe     530 535 540 Asp Gln Pro Asp Asp Phe Lys Gly Val Asn Val Thr Met Pro Ala Gln 545 550 555 560 Pro Gly Thr Pro Pro Leu Ser Ser Thr Gln Leu Gln Ile Asp Pro Ser                 565 570 575 Leu His Glu Phe Gln Leu Val Asp Leu Ser Arg Arg Phe Leu Val His             580 585 590 Asp Ser Phe Trp Ala Leu Pro Glu Gln Phe Leu Gly Asn Lys Val Asp         595 600 605 Ser Tyr Gly Gly Ser Leu Arg Tyr Asn Val Arg Tyr Glu Leu Ala Arg     610 615 620 Gly Met Leu Glu Pro Val Gln Arg Pro Asp Val Val Leu Met Gly Ala 625 630 635 640 Gly Tyr Arg Leu Leu Ser Arg Gly His Thr Pro Thr Gln Pro Gly Ala                 645 650 655 Leu Asn Gln Arg Gln Val Gln Phe Ser Glu Glu His Trp Val His Glu             660 665 670 Ser Gly Arg Pro Val Gln Arg Ala Glu Leu Leu Gln Val Leu Gln Ser         675 680 685 Leu Glu Ala Val Leu Ile Gln Thr Val Tyr Asn Thr Lys Met Ala Ser     690 695 700 Val Gly Leu Ser Asp Ile Ala Met Asp Thr Thr Val Thr His Ala Thr 705 710 715 720 Ser His Gly Arg Ala His Ser Val Glu Glu Cys Arg Cys Pro Ile Gly                 725 730 735 Tyr Ser Gly Leu Ser Cys Glu Ser Cys Asp Ala His Phe Thr Arg Val             740 745 750 Pro Gly Gly Pro Tyr Leu Gly Thr Cys Ser Gly Cys Asn Cys Asn Gly         755 760 765 His Ala Ser Ser Cys Asp Pro Val Tyr Gly His Cys Leu Asn Cys Gln     770 775 780 His Asn Thr Glu Gly Pro Gln Cys Asn Lys Cys Lys Ala Gly Phe Phe 785 790 795 800 Gly Asp Ala Met Lys Ala Thr Ala Thr Ser Cys Arg Pro Cys Pro Cys                 805 810 815 Pro Tyr Ile Asp Ala Ser Arg Arg Phe Ser Asp Thr Cys Phe Leu Asp             820 825 830 Thr Asp Gly Gln Ala Thr Cys Asp Ala Cys Ala Pro Gly Tyr Thr Gly         835 840 845 Arg Arg Cys Glu Ser Cys Ala Pro Gly Tyr Glu Gly Asn Pro Ile Gln     850 855 860 Pro Gly Gly Lys Cys Arg Pro Val Asn Gln Glu Ile Val Arg Cys Asp 865 870 875 880 Glu Arg Gly Ser Met Gly Thr Ser Gly Glu Ala Cys Arg Cys Lys Asn                 885 890 895 Asn Val Val Gly Arg Leu Cys Asn Glu Cys Ala Asp Gly Ser Phe His             900 905 910 Leu Ser Thr Arg Asn Pro Asp Gly Cys Leu Lys Cys Phe Cys Met Gly         915 920 925 Val Ser Arg His Cys Thr Ser Ser Ser Trp Ser Arg Ala Gln Leu His     930 935 940 Gly Ala Ser Glu Glu Pro Gly His Phe Ser Leu Thr Asn Ala Ala Ser 945 950 955 960 Thr His Thr Thr Asn Glu Gly Ile Phe Ser Pro Thr Pro Gly Glu Leu                 965 970 975 Gly Phe Ser Ser Phe His Arg Leu Leu Ser Gly Pro Tyr Phe Trp Ser             980 985 990 Leu Pro Ser Arg Phe Leu Gly Asp Lys Val Thr Ser Tyr Gly Gly Glu         995 1000 1005 Leu Arg Phe Thr Val Thr Gln Arg Ser Gln Pro Gly Ser Thr Pro Leu    1010 1015 1020 His Gly Gln Pro Leu Val Val Leu Gln Gly Asn Asn Ile Leu Glu 1025 1030 1035 1040 His His Val Ala Gln Glu Pro Ser Pro Gly Gln Pro Ser Thr Phe Ile                1045 1050 1055 Val Pro Phe Arg Glu Gln Ala Trp Gln Arg Pro Asp Gly Gln Pro Ala            1060 1065 1070 Thr Arg Glu His Leu Leu Met Ala Leu Ala Gly Ile Asp Thr Leu Leu        1075 1080 1085 Ile Arg Ala Ser Tyr Ala Gln Gln Pro Ala Glu Ser Arg Val Ser Gly    1090 1095 1100 Ile Ser Met Asp Val Ala Val Pro Glu Glu Thr Gly Gln Asp Pro Ala 1105 1110 1115 1120 Leu Glu Val Glu Gln Cys Ser Cys Pro Pro Gly Tyr Arg Gly Pro Ser                1125 1130 1135 Cys Gln Asp Cys Asp Thr Gly Tyr Thr Arg Thr Pro Ser Gly Leu Tyr            1140 1145 1150 Leu Gly Thr Cys Glu Arg Cys Ser Cys His Gly His Ser Glu Ala Cys        1155 1160 1165 Glu Pro Glu Thr Gly Ala Cys Gln Gly Cys Gln His His Thr Glu Gly    1170 1175 1180 Pro Arg Cys Glu Gln Cys Gln Pro Gly Tyr Tyr Gly Asp Ala Gln Arg 1185 1190 1195 1200 Gly Thr Pro Gln Asp Cys Gln Leu Cys Pro Cys Tyr Gly Asp Pro Ala                1205 1210 1215 Ala Gly Gln Ala Ala His Thr Cys Phe Leu Asp Thr Asp Gly His Pro            1220 1225 1230 Thr Cys Asp Ala Cys Ser Pro Gly His Ser Gly Arg His Cys Glu Arg        1235 1240 1245 Cys Ala Pro Gly Tyr Tyr Gly Asn Pro Ser Gln Gly Gln Pro Cys Gln    1250 1255 1260 Arg Asp Ser Gln Val Pro Gly Pro Ile Gly Cys Asn Cys Asp Pro Gln 1265 1270 1275 1280 Gly Ser Val Ser Ser Gln Cys Asp Ala Ala Gly Gln Cys Gln Cys Lys                1285 1290 1295 Ala Gln Val Glu Gly Leu Thr Cys Ser His Cys Arg Pro His His Phe            1300 1305 1310 His Leu Ser Ala Ser Asn Pro Asp Gly Cys Leu Pro Cys Phe Cys Met        1315 1320 1325 Gly Ile Thr Gln Gln Cys Ala Ser Ser Ala Tyr Thr Arg His Leu Ile    1330 1335 1340 Ser Thr His Phe Ala Pro Gly Asp Phe Gln Gly Phe Ala Leu Val Asn 1345 1350 1355 1360 Pro Gln Arg Asn Ser Arg Leu Thr Gly Glu Phe Thr Val Glu Pro Val                1365 1370 1375 Pro Glu Gly Ala Gln Leu Ser Phe Gly Asn Phe Ala Gln Leu Gly His            1380 1385 1390 Glu Ser Phe Tyr Trp Gln Leu Pro Glu Thr Tyr Gln Gly Asp Lys Val        1395 1400 1405 Ala Ala Tyr Gly Gly Lys Leu Arg Tyr Thr Leu Ser Tyr Thr Ala Gly    1410 1415 1420 Pro Gln Gly Ser Pro Leu Ser Asp Pro Asp Val Gln Ile Thr Gly Asn 1425 1430 1435 1440 Asn Ile Met Leu Val Ala Ser Gln Pro Ala Leu Gln Gly Pro Glu Arg                1445 1450 1455 Arg Ser Tyr Glu Ile Met Phe Arg Glu Glu Phe Trp Arg Arg Pro Asp            1460 1465 1470 Gly Gln Pro Ala Thr Arg Glu His Leu Leu Met Ala Leu Ala Asp Leu        1475 1480 1485 Asp Glu Leu Leu Ile Arg Ala Thr Phe Ser Ser Val Pro Leu Ala Ala    1490 1495 1500 Ser Ile Ser Ala Val Ser Leu Glu Val Ala Gln Pro Gly Pro Ser Asn 1505 1510 1515 1520 Arg Pro Arg Ala Leu Glu Val Glu Glu Cys Arg Cys Pro Pro Gly Tyr                1525 1530 1535 Ile Gly Leu Ser Cys Gln Asp Cys Ala Pro Gly Tyr Thr Arg Thr Gly            1540 1545 1550 Ser Gly Leu Tyr Leu Gly His Cys Glu Leu Cys Glu Cys Asn Gly His        1555 1560 1565 Ser Asp Leu Cys His Pro Glu Thr Gly Ala Cys Ser Gln Cys Gln His    1570 1575 1580 Asn Ala Ala Gly Glu Phe Cys Glu Leu Cys Ala Pro Gly Tyr Tyr Gly 1585 1590 1595 1600 Asp Ala Thr Ala Gly Thr Pro Glu Asp Cys Gln Pro Cys Ala Cys Pro                1605 1610 1615 Leu Thr Asn Pro Glu Asn Met Phe Ser Arg Thr Cys Glu Ser Leu Gly            1620 1625 1630 Ala Gly Gly Tyr Arg Cys Thr Ala Cys Glu Pro Gly Tyr Thr Gly Gln        1635 1640 1645 Tyr Cys Glu Gln Cys Gly Pro Gly Tyr Val Gly Asn Pro Ser Val Gln    1650 1655 1660 Gly Gly Gln Cys Leu Pro Glu Thr Asn Gln Ala Pro Leu Val Val Glu 1665 1670 1675 1680 Val His Pro Ala Arg Ser Ile Val Pro Gln Gly Gly Ser His Ser Leu                1685 1690 1695 Arg Cys Gln Val Ser Gly Ser Pro Pro His Tyr Phe Tyr Trp Ser Arg            1700 1705 1710 Glu Asp Gly Arg Pro Val Pro Ser Gly Thr Gln Gln Arg His Gln Gly        1715 1720 1725 Ser Glu Leu His Phe Pro Ser Val Gln Pro Ser Asp Ala Gly Val Tyr    1730 1735 1740 Ile Cys Thr Cys Arg Asn Leu His Gln Ser Asn Thr Ser Arg Ala Glu 1745 1750 1755 1760 Leu Leu Val Thr Glu Ala Pro Ser Lys Pro Ile Thr Val Thr Val Glu                1765 1770 1775 Glu Gln Arg Ser Gln Ser Val Arg Pro Gly Ala Asp Val Thr Phe Ile            1780 1785 1790 Cys Thr Ala Lys Ser Lys Ser Pro Ala Tyr Thr Leu Val Trp Thr Arg        1795 1800 1805 Leu His Asn Gly Lys Leu Pro Thr Arg Ala Met Asp Phe Asn Gly Ile    1810 1815 1820 Leu Thr Ile Arg Asn Val Gln Leu Ser Asp Ala Gly Thr Tyr Val Cys 1825 1830 1835 1840 Thr Gly Ser Asn Met Phe Ala Met Asp Gln Gly Thr Ala Thr Leu His                1845 1850 1855 Val Gln Ala Ser Gly Thr Leu Ser Ala Pro Val Val Ser Ile His Pro            1860 1865 1870 Pro Gln Leu Thr Val Gln Pro Gly Gln Leu Ala Glu Phe Arg Cys Ser        1875 1880 1885 Ala Thr Gly Ser Pro Thr Pro Thr Leu Glu Trp Thr Gly Gly Pro Gly    1890 1895 1900 Gly Gln Leu Pro Ala Lys Ala Gln Ile His Gly Gly Ile Leu Arg Leu 1905 1910 1915 1920 Pro Ala Val Glu Pro Thr Asp Gln Ala Gln Tyr Leu Cys Arg Ala His                1925 1930 1935 Ser Ser Ala Gly Gln Gln Val Ala Arg Ala Val Leu His Val His Gly            1940 1945 1950 Gly Gly Gly Pro Arg Val Gln Val Ser Pro Glu Arg Thr Gln Val His        1955 1960 1965 Ala Gly Arg Thr Val Arg Leu Tyr Cys Arg Ala Ala Gly Val Pro Ser    1970 1975 1980 Ala Thr Ile Thr Trp Arg Lys Glu Gly Gly Ser Leu Pro Pro Gln Ala 1985 1990 1995 2000 Arg Ser Glu Arg Thr Asp Ile Ala Thr Leu Leu Ile Pro Ala Ile Thr                2005 2010 2015 Thr Ala Asp Ala Gly Phe Tyr Leu Cys Val Ala Thr Ser Pro Ala Gly            2020 2025 2030 Thr Ala Gln Ala Arg Ile Gln Val Val Val Leu Ser Ala Ser Asp Ala        2035 2040 2045 Ser Pro Pro Pro Val Lys Ile Glu Ser Ser Ser Pro Ser Val Thr Glu    2050 2055 2060 Gly Gln Thr Leu Asp Leu Asn Cys Val Val Ala Gly Ser Ala His Ala 2065 2070 2075 2080 Gln Val Thr Trp Tyr Arg Arg Gly Gly Ser Leu Pro Pro His Thr Gln                2085 2090 2095 Val His Gly Ser Arg Leu Arg Leu Pro Gln Val Ser Pro Ala Asp Ser            2100 2105 2110 Gly Glu Tyr Val Cys Arg Val Glu Asn Gly Ser Gly Pro Lys Glu Ala        2115 2120 2125 Ser Ile Thr Val Ser Val Leu His Gly Thr His Ser Gly Pro Ser Tyr    2130 2135 2140 Thr Pro Val Pro Gly Ser Thr Arg Pro Ile Arg Ile Glu Pro Ser Ser 2145 2150 2155 2160 Ser His Val Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Pro                2165 2170 2175 Gly Gln Ala His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu            2180 2185 2190 Pro Ala Arg His Gln Thr His Gly Ser Leu Leu Arg Leu His Gln Val        2195 2200 2205 Thr Pro Ala Asp Ser Gly Glu Tyr Val Cys His Val Val Gly Thr Ser    2210 2215 2220 Gly Pro Leu Glu Ala Ser Val Leu Val Thr Ile Glu Ala Ser Val Ile 2225 2230 2235 2240 Pro Gly Pro Ile Pro Pro Val Arg Ile Glu Ser Ser Ser Ser Thr Val                2245 2250 2255 Ala Glu Gly Gln Thr Leu Asp Leu Ser Cys Val Val Ala Gly Gln Ala            2260 2265 2270 His Ala Gln Val Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ala Arg        2275 2280 2285 His Gln Val Arg Gly Ser Arg Leu Tyr Ile Phe Gln Ala Ser Pro Ala    2290 2295 2300 Asp Ala Gly Gln Tyr Val Cys Arg Ala Ser Asn Gly Met Glu Ala Ser 2305 2310 2315 2320 Ile Thr Val Thr Val Thr Gly Thr Gln Gly Ala Asn Leu Ala Tyr Pro                2325 2330 2335 Ala Gly Ser Thr Gln Pro Ile Arg Ile Glu Pro Ser Ser Ser Gln Val            2340 2345 2350 Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Pro Gly Gln Ser        2355 2360 2365 His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro Val Arg    2370 2375 2380 His Gln Thr His Gly Ser Leu Leu Arg Leu Tyr Gln Ala Ser Pro Ala 2385 2390 2395 2400 Asp Ser Gly Glu Tyr Val Cys Arg Val Leu Gly Ser Ser Val Pro Leu                2405 2410 2415 Glu Ala Ser Val Leu Val Thr Ile Glu Pro Ala Gly Ser Val Pro Ala            2420 2425 2430 Leu Gly Val Thr Pro Thr Val Arg Ile Glu Ser Ser Ser Ser Gln Val        2435 2440 2445 Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Leu Val Ala Gly Gln Ala    2450 2455 2460 His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro Ala Arg 2465 2470 2475 2480 His Gln Val His Gly Ser Arg Leu Arg Leu Leu Gln Val Thr Pro Ala                2485 2490 2495 Asp Ser Gly Glu Tyr Val Cys Arg Val Val Gly Ser Ser Gly Thr Gln            2500 2505 2510 Glu Ala Ser Val Leu Val Thr Ile Gln Gln Arg Leu Ser Gly Ser His        2515 2520 2525 Ser Gln Gly Val Ala Tyr Pro Val Arg Ile Glu Ser Ser Ser Ala Ser    2530 2535 2540 Leu Ala Asn Gly His Thr Leu Asp Leu Asn Cys Leu Val Ala Ser Gln 2545 2550 2555 2560 Ala Pro His Thr Ile Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ser                2565 2570 2575 Arg His Gln Ile Val Gly Ser Arg Leu Arg Ile Pro Gln Val Thr Pro            2580 2585 2590 Ala Asp Ser Gly Glu Tyr Val Cys His Val Ser Asn Gly Ala Gly Ser        2595 2600 2605 Arg Glu Thr Ser Leu Ile Val Thr Ile Gln Gly Ser Gly Ser Ser His    2610 2615 2620 Val Pro Ser Val Ser Pro Pro Ile Arg Ile Glu Ser Ser Ser Pro Thr 2625 2630 2635 2640 Val Val Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Ala Arg Gln                2645 2650 2655 Pro Gln Ala Ile Ile Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ser            2660 2665 2670 Arg His Gln Thr His Gly Ser His Leu Arg Leu His Gln Met Ser Val        2675 2680 2685 Ala Asp Ser Gly Glu Tyr Val Cys Arg Ala Asn Asn Asn Ile Asp Ala    2690 2695 2700 Leu Glu Ala Ser Ile Val Ile Ser Val Ser Pro Ser Ala Gly Ser Pro 2705 2710 2715 2720 Ser Ala Pro Gly Ser Ser Met Pro Ile Arg Ile Glu Ser Ser Ser Ser                2725 2730 2735 His Val Ala Glu Gly Glu Thr Leu Asp Leu Asn Cys Val Val Pro Gly            2740 2745 2750 Gln Ala His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro        2755 2760 2765 Ser His His Gln Thr Arg Gly Ser Arg Leu Arg Leu His His Val Ser    2770 2775 2780 Pro Ala Asp Ser Gly Glu Tyr Val Cys Arg Val Met Gly Ser Ser Gly 2785 2790 2795 2800 Pro Leu Glu Ala Ser Val Leu Val Thr Ile Glu Ala Ser Gly Ser Ser                2805 2810 2815 Ala Val His Val Pro Ala Pro Gly Gly Ala Pro Pro Ile Arg Ile Glu            2820 2825 2830 Pro Ser Ser Ser Arg Val Ala Glu Gly Gln Thr Leu Asp Leu Lys Cys        2835 2840 2845 Val Val Pro Gly Gln Ala His Ala Gln Val Thr Trp His Lys Arg Gly    2850 2855 2860 Gly Asn Leu Pro Ala Arg His Gln Val His Gly Pro Leu Leu Arg Leu 2865 2870 2875 2880 Asn Gln Val Ser Pro Ala Asp Ser Gly Glu Tyr Ser Cys Gln Val Thr                2885 2890 2895 Gly Ser Ser Gly Thr Leu Glu Ala Ser Val Leu Val Thr Ile Glu Pro            2900 2905 2910 Ser Ser Pro Gly Pro Ile Pro Ala Pro Gly Leu Ala Gln Pro Ile Tyr        2915 2920 2925 Ile Glu Ala Ser Ser Ser His Val Thr Glu Gly Gln Thr Leu Asp Leu    2930 2935 2940 Asn Cys Val Val Pro Gly Gln Ala His Ala Gln Val Thr Trp Tyr Lys 2945 2950 2955 2960 Arg Gly Gly Ser Leu Pro Ala Arg His Gln Thr His Gly Ser Gln Leu                2965 2970 2975 Arg Leu His Leu Val Ser Pro Ala Asp Ser Gly Glu Tyr Val Cys Arg            2980 2985 2990 Ala Ala Ser Gly Pro Gly Pro Glu Gln Glu Ala Ser Phe Thr Val Thr        2995 3000 3005 Val Pro Pro Ser Glu Gly Ser Ser Tyr Arg Leu Arg Ser Pro Val Ile    3010 3015 3020 Ser Ile Asp Pro Pro Ser Ser Thr Val Gln Gln Gly Gln Asp Ala Ser 3025 3030 3035 3040 Phe Lys Cys Leu Ile His Asp Gly Ala Ala Pro Ile Ser Leu Glu Trp                3045 3050 3055 Lys Thr Arg Asn Gln Glu Leu Glu Asp Asn Val His Ile Ser Pro Asn            3060 3065 3070 Gly Ser Ile Ile Thr Ile Val Gly Thr Arg Pro Ser Asn His Gly Thr        3075 3080 3085 Tyr Arg Cys Val Ala Ser Asn Ala Tyr Gly Val Ala Gln Ser Val Val    3090 3095 3100 Asn Leu Ser Val His Gly Pro Pro Thr Val Ser Val Leu Pro Glu Gly 3105 3110 3115 3120 Pro Val Trp Val Lys Val Gly Lys Ala Val Thr Leu Glu Cys Val Ser                3125 3130 3135 Ala Gly Glu Pro Arg Ser Ser Ala Arg Trp Thr Arg Ile Ser Ser Thr            3140 3145 3150 Pro Ala Lys Leu Glu Gln Arg Thr Tyr Gly Leu Met Asp Ser His Ala        3155 3160 3165 Val Leu Gln Ile Ser Ser Ala Lys Pro Ser Asp Ala Gly Thr Tyr Val    3170 3175 3180 Cys Leu Ala Gln Asn Ala Leu Gly Thr Ala Gln Lys Gln Val Glu Val 3185 3190 3195 3200 Ile Val Asp Thr Gly Ala Met Ala Pro Gly Ala Pro Gln Val Gln Ala                3205 3210 3215 Glu Glu Ala Glu Leu Thr Val Glu Ala Gly His Thr Ala Thr Leu Arg            3220 3225 3230 Cys Ser Ala Thr Gly Ser Pro Ala Pro Thr Ile His Trp Ser Lys Leu        3235 3240 3245 Arg Ser Pro Leu Pro Trp Gln His Arg Leu Glu Gly Asp Thr Leu Ile    3250 3255 3260 Ile Pro Arg Val Ala Gln Gln Asp Ser Gly Gln Tyr Ile Cys Asn Ala 3265 3270 3275 3280 Thr Ser Pro Ala Gly His Ala Glu Ala Thr Ile Ile Leu His Val Glu                3285 3290 3295 Ser Pro Pro Tyr Ala Thr Thr Val Pro Glu His Ala Ser Val Gln Ala            3300 3305 3310 Gly Glu Thr Val Gln Leu Gln Cys Leu Ala His Gly Thr Pro Pro Leu        3315 3320 3325 Thr Phe Gln Trp Ser Arg Val Gly Ser Ser Leu Pro Gly Arg Ala Thr    3330 3335 3340 Ala Arg Asn Glu Leu Leu His Phe Glu Arg Ala Ala Pro Glu Asp Ser 3345 3350 3355 3360 Gly Arg Tyr Arg Cys Arg Val Thr Asn Lys Val Gly Ser Ala Glu Ala                3365 3370 3375 Phe Ala Gln Leu Leu Val Gln Gly Pro Pro Gly Ser Leu Pro Ala Thr            3380 3385 3390 Ser Ile Pro Ala Gly Ser Thr Pro Thr Val Gln Val Thr Pro Gln Leu        3395 3400 3405 Glu Thr Lys Ser Ile Gly Ala Ser Val Glu Phe His Cys Ala Val Pro    3410 3415 3420 Ser Asp Arg Gly Thr Gln Leu Arg Trp Phe Lys Glu Gly Gly Gln Leu 3425 3430 3435 3440 Pro Pro Gly His Ser Val Gln Asp Gly Val Leu Arg Ile Gln Asn Leu                3445 3450 3455 Asp Gln Ser Cys Gln Gly Thr Tyr Ile Cys Gln Ala His Gly Pro Trp            3460 3465 3470 Gly Lys Ala Gln Ala Ser Ala Gln Leu Val Ile Gln Ala Leu Pro Ser        3475 3480 3485 Val Leu Ile Asn Ile Arg Thr Ser Val Gln Thr Val Val Val Gly His    3490 3495 3500 Ala Val Glu Phe Glu Cys Leu Ala Leu Gly Asp Pro Lys Pro Gln Val 3505 3510 3515 3520 Thr Trp Ser Lys Val Gly Gly His Leu Arg Pro Gly Ile Val Gln Ser                3525 3530 3535 Gly Gly Val Val Arg Ile Ala His Val Glu Leu Ala Asp Ala Gly Gln            3540 3545 3550 Tyr Arg Cys Thr Ala Thr Asn Ala Ala Gly Thr Thr Gln Ser His Val        3555 3560 3565 Leu Leu Leu Val Gln Ala Leu Pro Gln Ile Ser Met Pro Gln Glu Val    3570 3575 3580 Arg Val Pro Ala Gly Ser Ala Ala Val Phe Pro Cys Ile Ala Ser Gly 3585 3590 3595 3600 Tyr Pro Thr Pro Asp Ile Ser Trp Ser Lys Leu Asp Gly Ser Leu Pro                3605 3610 3615 Pro Asp Ser Arg Leu Glu Asn Asn Met Leu Met Leu Pro Ser Val Arg            3620 3625 3630 Pro Gln Asp Ala Gly Thr Tyr Val Cys Thr Ala Thr Asn Arg Gln Gly        3635 3640 3645 Lys Val Lys Ala Phe Ala His Leu Gln Val Pro Glu Arg Val Val Pro    3650 3655 3660 Tyr Phe Thr Gln Thr Pro Tyr Ser Phe Leu Pro Leu Pro Thr Ile Lys 3665 3670 3675 3680 Asp Ala Tyr Arg Lys Phe Glu Ile Lys Ile Thr Phe Arg Pro Asp Ser                3685 3690 3695 Ala Asp Gly Met Leu Leu Tyr Asn Gly Gln Lys Arg Val Pro Gly Ser            3700 3705 3710 Pro Thr Asn Leu Ala Asn Arg Gln Pro Asp Phe Ile Ser Phe Gly Leu        3715 3720 3725 Val Gly Gly Arg Pro Glu Phe Arg Phe Asp Ala Gly Ser Gly Met Ala    3730 3735 3740 Thr Ile Arg His Pro Thr Pro Leu Ala Leu Gly His Phe His Thr Val 3745 3750 3755 3760 Thr Leu Leu Arg Ser Leu Thr Gln Gly Ser Leu Ile Val Gly Asp Leu                3765 3770 3775 Ala Pro Val Asn Gly Thr Ser Gln Gly Lys Phe Gln Gly Leu Asp Leu            3780 3785 3790 Asn Glu Glu Leu Tyr Leu Gly Gly Tyr Pro Asp Tyr Gly Ala Ile Pro        3795 3800 3805 Lys Ala Gly Leu Ser Ser Gly Phe Ile Gly Cys Val Arg Glu Leu Arg    3810 3815 3820 Ile Gln Gly Glu Glu Ile Val Phe His Asp Leu Asn Leu Thr Ala His 3825 3830 3835 3840 Gly Ile Ser His Cys Pro Thr Cys Arg Asp Arg Pro Cys Gln Asn Gly                3845 3850 3855 Gly Gln Cys His Asp Ser Glu Ser Ser Ser Tyr Val Cys Val Cys Pro            3860 3865 3870 Ala Gly Phe Thr Gly Ser Arg Cys Glu His Ser Gln Ala Leu His Cys        3875 3880 3885 His Pro Glu Ala Cys Gly Pro Asp Ala Thr Cys Val Asn Arg Pro Asp    3890 3895 3900 Gly Arg Gly Tyr Thr Cys Arg Cys His Leu Gly Arg Ser Gly Leu Arg 3905 3910 3915 3920 Cys Glu Glu Gly Val Thr Val Thr Thr Pro Ser Leu Ser Gly Ala Gly                3925 3930 3935 Ser Tyr Leu Ala Leu Pro Ala Leu Thr Asn Thr His His Glu Leu Arg            3940 3945 3950 Leu Asp Val Glu Phe Lys Pro Leu Ala Pro Asp Gly Val Leu Leu Phe        3955 3960 3965 Ser Gly Gly Lys Ser Gly Pro Val Glu Asp Phe Val Ser Leu Ala Met    3970 3975 3980 Val Gly Gly His Leu Glu Phe Arg Tyr Glu Leu Gly Ser Gly Leu Ala 3985 3990 3995 4000 Val Leu Arg Ser Ala Glu Pro Leu Ala Leu Gly Arg Trp His Arg Val                4005 4010 4015 Ser Ala Glu Arg Leu Asn Lys Asp Gly Ser Leu Arg Val Asn Gly Gly            4020 4025 4030 Arg Pro Val Leu Arg Ser Ser Pro Gly Lys Ser Gln Gly Leu Asn Leu        4035 4040 4045 His Thr Leu Leu Tyr Leu Gly Gly Val Glu Pro Ser Val Pro Leu Ser    4050 4055 4060 Pro Ala Thr Asn Met Ser Ala His Phe Arg Gly Cys Val Gly Glu Val 4065 4070 4075 4080 Ser Val Asn Gly Lys Arg Leu Asp Leu Thr Tyr Ser Phe Leu Gly Ser                4085 4090 4095 Gln Gly Ile Gly Gln Cys Tyr Asp Ser Ser Pro Cys Glu Arg Gln Pro            4100 4105 4110 Cys Gln His Gly Ala Thr Cys Met Pro Ala Gly Glu Tyr Glu Phe Gln        4115 4120 4125 Cys Leu Cys Arg Asp Gly Phe Lys Gly Asp Leu Cys Glu His Glu Glu    4130 4135 4140 Asn Pro Cys Gln Leu Arg Glu Pro Cys Leu His Gly Gly Thr Cys Gln 4145 4150 4155 4160 Gly Thr Arg Cys Leu Cys Leu Pro Gly Phe Ser Gly Pro Arg Cys Gln                4165 4170 4175 Gln Gly Ser Gly His Gly Ile Ala Glu Ser Asp Trp His Leu Glu Gly            4180 4185 4190 Ser Gly Gly Asn Asp Ala Pro Gly Gln Tyr Gly Ala Tyr Phe His Asp        4195 4200 4205 Asp Gly Phe Leu Ala Phe Pro Gly His Val Phe Ser Arg Ser Leu Pro    4210 4215 4220 Glu Val Pro Glu Thr Ile Glu Leu Glu Val Arg Thr Ser Thr Ala Ser 4225 4230 4235 4240 Gly Leu Leu Leu Trp Gln Gly Val Glu Val Gly Glu Ala Gly Gln Gly                4245 4250 4255 Lys Asp Phe Ile Ser Leu Gly Leu Gln Asp Gly His Leu Val Phe Arg            4260 4265 4270 Tyr Gln Leu Gly Ser Gly Glu Ala Arg Leu Val Ser Glu Asp Pro Ile        4275 4280 4285 Asn Asp Gly Glu Trp His Arg Val Thr Ala Leu Arg Glu Gly Arg Arg    4290 4295 4300 Gly Ser Ile Gln Val Asp Gly Glu Glu Leu Val Ser Gly Arg Ser Pro 4305 4310 4315 4320 Gly Pro Asn Val Ala Val Asn Ala Lys Gly Ser Val Tyr Ile Gly Gly                4325 4330 4335 Ala Pro Asp Val Ala Thr Leu Thr Gly Gly Arg Phe Ser Ser Gly Ile            4340 4345 4350 Thr Gly Cys Val Lys Asn Leu Val Leu His Ser Ala Arg Pro Gly Ala        4355 4360 4365 Pro Pro Pro Gln Pro Leu Asp Leu Gln His Arg Ala Gln Ala Gly Ala    4370 4375 4380 Asn Thr Arg Pro Cys Pro Ser 4385 4390 <210> 2 <211> 195 <212> PRT <213> human endorepellin LG3 fragment <400> 2 Asp Ala Pro Gly Gln Tyr Gly Ala Tyr Phe His Asp Asp Gly Phe Leu   1 5 10 15 Ala Phe Pro Gly His Val Phe Ser Arg Ser Leu Pro Glu Val Pro Glu              20 25 30 Thr Ile Glu Leu Glu Val Arg Thr Ser Thr Ala Ser Gly Leu Leu Leu          35 40 45 Trp Gln Gly Val Glu Val Gly Glu Ala Gly Gln Gly Lys Asp Phe Ile      50 55 60 Ser Leu Gly Leu Gln Asp Gly His Leu Val Phe Arg Tyr Gln Leu Gly  65 70 75 80 Ser Gly Glu Ala Arg Leu Val Ser Glu Asp Pro Ile Asn Asp Gly Glu                  85 90 95 Trp His Arg Val Thr Ala Leu Arg Glu Gly Arg Arg Gly Ser Ile Gln             100 105 110 Val Asp Gly Glu Glu Leu Val Ser Gly Arg Ser Pro Gly Pro Asn Val         115 120 125 Ala Val Asn Ala Lys Gly Ser Val Tyr Ile Gly Gly Ala Pro Asp Val     130 135 140 Ala Thr Leu Thr Gly Gly Arg Phe Ser Ser Gly Ile Thr Gly Cys Val 145 150 155 160 Lys Asn Leu Val Leu His Ser Ala Arg Pro Gly Ala Pro Pro Pro Gln                 165 170 175 Pro Leu Asp Leu Gln His Arg Ala Gln Ala Gly Ala Asn Thr Arg Pro             180 185 190 Cys pro ser         195 <210> 3 <211> 14 <212> PRT <213> tryptic peptide <400> 3 Ser Leu Pro Glu Val Pro Glu Thr Ile Glu Leu Glu Val Arg   1 5 10 <210> 4 <211> 14 <212> PRT <213> tryptic peptide <400> 4 Leu Val Ser Glu Asp Pro Ile Asn Asp Gly Glu Trp His Arg   1 5 10 <210> 5 <211> 14 <212> PRT <213> tryptic peptide <400> 5 Gly Ser Ile Gln Val Asp Gly Glu Glu Leu Val Ser Gly Arg   1 5 10 <210> 6 <211> 18 <212> PRT <213> tryptic peptide <400> 6 Gly Ser Val Tyr Ile Gly Gly Ala Pro Asp Val Ala Thr Leu Thr Gly   1 5 10 15 Gly arg         <210> 7 <211> 22 <212> PRT <213> tryptic peptide <400> 7 Asn Leu Val Leu His Ser Ala Arg Pro Gly Ala Pro Pro Pro Gln Pro   1 5 10 15 Leu Asp Leu Gln His Arg              20  

Claims (13)

서열번호: 2의 아미노산 서열을 갖는 유방암 진단용 엔도레펠린 LG3 절편 단백질 마커. An endrepelin LG3 fragment protein marker for diagnosing breast cancer having the amino acid sequence of SEQ ID NO: 2 . 제 1항의 엔도레펠린 LG3 절편 단백질 마커에 특이적인 항체를 포함하는 유방암 진단키트.Claim 1 breast cancer diagnostic kit comprising an antibody specific for the endorphelin LG3 fragment protein marker. 제 2항에 있어서,The method of claim 2, 상기 항체가 다클론 항체 또는 단클론 항체인 것을 특징으로 하는 유방암 진단키트.Breast cancer diagnostic kit, characterized in that the antibody is a polyclonal antibody or a monoclonal antibody. 제 2항에 있어서,The method of claim 2, 기질과의 반응에 의하여 발색하는 표지체가 접합된 이차항체 접합체; 상기 표지체와 발색 반응할 발색기질 용액; 세척액; 및 효소반응 정지액을 추가로 포함하는 것을 특징으로 하는 유방암 진단키트.Secondary antibody conjugates to which a label that is developed by reaction with a substrate is conjugated; A color substrate solution to be colored and reacted with the label; Washing liquid; And a breast cancer diagnostic kit further comprises an enzyme reaction stopper. 제 4항에 있어서,The method of claim 4, wherein 이차항체 접합체의 표지체가 HRP(horseradish peroxidase), 염기성 탈인산화효소(alkaline phosphatase), 콜로이드 골드(coloid gold), 형광물질(fluorescein) 및 색소(dye)로 구성된 군으로부터 선택되는 것을 특징으로 하는 진단키트.Diagnostic kit characterized in that the label of the secondary antibody conjugate is selected from the group consisting of horseradish peroxidase (HRP), basic alkaline phosphatase, colloid gold, fluorescent (fluorescein) and dye (dye) . 제 4항에 있어서,The method of claim 4, wherein 발색기질이 TMB(3,3',5,5'-tetramethyl bezidine), ABTS[2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] 및 OPD(o-phenylenediamine)로 구성된 군으로부터 선택되는 것을 특징으로 하는 진단키트.Group consisting of TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD (o-phenylenediamine) The diagnostic kit, characterized in that selected from. 검체로부터 항원-항체 결합반응을 이용하여 엔도레펠린 LG3 절편 단백질 마커를 검출하는 방법.A method for detecting an endorepelin LG3 fragment protein marker using an antigen-antibody binding reaction from a sample. 제 7항에 있어서,The method of claim 7, wherein 1) 검체 및 대조군의 단백질을 고정체에 코팅시키는 단계;1) coating the sample and the protein of the control to the fixture; 2) 상기 고정체에 엔도레펠린 LG3 절편에 특이적인 항체를 첨가하여 항원-항체 결합반응을 수행하는 단계;2) performing an antigen-antibody binding reaction by adding an antibody specific for an endrepelin LG3 fragment to the fixture; 3) 상기 항원-항체 결합반응을 통해 생성된 항원-항체 결합반응물을 이차항체 접합체(conjugate) 및 발색기질 용액을 이용하여 검출하는 단계; 및3) detecting the antigen-antibody binding reactant produced through the antigen-antibody binding reaction using a secondary antibody conjugate and a color substrate solution; And 4) 검체와 대조군에 대한 검출 결과를 비교하는 단계를 포함하는 것을 특징으로 하는 방법.4) comparing the detection results for the sample and the control. 제 8항에 있어서,The method of claim 8, 단계 1)의 검체가 혈청, 혈장 또는 혈액인 것을 특징으로 하는 방법.And the sample of step 1) is serum, plasma or blood. 제 8항에 있어서,The method of claim 8, 단계 1)의 고정체가 니트로셀룰로오즈 막, PVDF 막, 폴리비닐(polyvinyl) 또는 폴리스티렌(polystyrene) 수지로 합성된 웰 플레이트 및 유리로 된 슬라이드 글래스로 구성된 군으로부터 선택되는 것을 특징으로 하는 방법.The fixture of step 1) is selected from the group consisting of nitrocellulose membrane, PVDF membrane, well plate synthesized from polyvinyl or polystyrene resin and slide glass made of glass. 제 8항에 있어서,The method of claim 8, 단계 2)의 상기 항원-항체 결합반응이 효소면역분석법(enzyme-linked immunosorbent assay, ELISA), 방사능면역분석법(radioimmnoassay, RIA), 샌드위치 측정법(sandwich assay), 웨스턴 블롯팅, 면역침강법, 면역조직화학염색법(immnohistochemical staining), 형광면역법, 효소기질발색법, 항원-항체 응집법으로 구성된 군으로부터 선택되는 것을 특징으로 하는 방법.The antigen-antibody binding reaction of step 2) is performed by enzyme-linked immunosorbent assay (ELISA), radioimmuno assay (RIA), sandwich assay, western blotting, immunoprecipitation method, immune tissue Immunohistochemical staining (Immnohistochemical staining), fluorescence immunoassay, enzyme substrate coloration, antigen-antibody aggregation method characterized in that it is selected from the group consisting of. 제 8항에 있어서,The method of claim 8, 단계 3)의 이차항체 접합체의 표지체가 HRP, 염기성 탈인산화효소, 콜로이드 골드, 형광물질 및 색소로 구성된 군으로부터 선택되는 것을 특징으로 하는 방법.The label of the secondary antibody conjugate of step 3) is selected from the group consisting of HRP, basic dephosphatase, colloidal gold, fluorescent substance and pigment. 제 8항에 있어서,The method of claim 8, 단계 3)의 발색기질이 TMB, ABTS 및 OPD로 구성된 군으로부터 선택되는 것을 특징으로 하는 방법.The chromogenic substrate of step 3) is selected from the group consisting of TMB, ABTS and OPD.
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WO2010015659A1 (en) * 2008-08-07 2010-02-11 Proteomika, S.L. Cancer markers and methods for their detection
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WO2010015659A1 (en) * 2008-08-07 2010-02-11 Proteomika, S.L. Cancer markers and methods for their detection
US20120003180A1 (en) * 2010-07-01 2012-01-05 The Texas A&M University System Perlecan domain v protects, repairs and restores ischemic brain stroke injury and motor function
US9072713B2 (en) * 2010-07-01 2015-07-07 The Texas A&M University System Perlecan domain V protects, repairs and restores ischemic brain stroke injury and motor function

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