KR101040321B1 - Bio-marker for diagnosing Cardiovascular disease - Google Patents

Abstract

The present invention relates to a protein that can be used as a diagnostic marker for cardiovascular diseases using blood, and more particularly, to a biomarker protein whose cardiac disease is reduced or increased in blood of a cardiovascular disease patient than a normal blood and a cardiovascular disease using the same. It relates to a diagnostic kit. Moreover, the protein whose expression amount is increased or decreased is recognized as a value as a therapeutic agent for cardiovascular disease.

According to the present invention, cardiovascular diseases can be diagnosed from human blood to determine the hardness of the symptoms. In addition, monoclonal antibodies prepared on the basis of the biomarker protein of the present invention can be used in immunoassay kits (ELISA, antibody coated tube test, lateral-flow test, potable biosensor), as well as to diagnose and progress cardiovascular disease. Judgment can be used to develop new drugs for therapeutic purposes.

Cardiovascular diseases, biomarkers, antibodies, diagnostic kits

Description

Biomarker for Diagnosing Cardiovascular Disease {Bio-marker for diagnosing Cardiovascular disease}

The present invention relates to a protein that appears as a disease progresses in cardiovascular patients, and more particularly, to a biomarker protein that is expressed more or less in the blood of a cardiovascular disease patient and a diagnostic kit for early diagnosis of cardiovascular disease using the same. will be.

Cardiovascular diseases include heart disease and vascular disease. Specific diseases include heart failure, hypertensive heart disease, arrhythmia, valve disease, congenital heart disease, cardiomyopathy, pericardial disease, and other important heart diseases. Vascular diseases include stroke, peripheral vascular disease, aneurysm, and the like. Coronary artery disease, an important part of heart disease, is usually caused by blockage or narrowing of the coronary arteries that supply the heart with atherosclerosis. This includes myocardial infarction or angina pectoris. Coronary artery disease has increased rapidly in the last 30 years in Korea.

In general, myocardial infarction, stroke, angina pectoris, hypertension, heart failure, etc. can be referred to, myocardial infarction is a disease in which the heart muscle tissue dies because the blood vessels that supply blood to the heart muscle is blocked. Myocardial infarction means that the heart muscle dies from lack of oxygen. This lack of oxygen is often caused by narrowing of blood vessels (coronary arteries) that provide oxygen to the heart due to atherosclerosis or the formation of blood clots. When myocardial infarction occurs, the patient feels severe chest pain and difficulty breathing.

Stroke is a disorder of the arteries that supply blood to the brain, which leads to a sudden loss of brain function due to a lack of normal blood supply. It is caused by a temporary or persistent lack of blood supply or bleeding. As the brain is damaged, it causes abnormalities in the body's functions controlled by damaged areas of the brain, such as speech and motor disorders. Strokes can be classified into ischemic strokes (brain infarctions), which are caused by blockage of blood vessels in the brain, and blood circulation in certain areas, and hemorrhagic strokes caused by brain vessels bursting.

Angina is a type of coronary artery disease caused by a partial narrowing of the coronary artery that supplies blood to the heart because the heart muscle temporarily fails to receive enough blood and feels chest compressions or chest pain. It is divided into stable angina, unstable angina and heterogeneous angina (Prinz Metal Angina). Unlike myocardial infarction, where the heart's blood vessels are blocked and the heart muscle dies, the pain of angina is reduced within minutes when you rest or take angina. Angina is caused by insufficient blood supply to the heart muscle. The most common cause is atherosclerosis.

Hypertension is a high state of blood pressure without going down. Blood pressure can vary considerably depending on a number of factors, but hypertension refers to when it is always high. The reason for the treatment of hypertension is that untreated hypertension can lead to various complications caused by hypertension, such as coronary artery disease such as stroke, heart failure, angina pectoris, acute myocardial infarction, and kidney failure.

Heart failure is a condition in which the pumping of the heart is impaired and the required amount of blood is not released to other organs of the body. Many heart diseases can interfere with the heart's ability to contract and relax, causing heart failure.

Cardiovascular disease is diagnosed differently according to the classification, but in general, electrocardiography, computerized tomography (CT), nuclear magnetic resonance imaging (MRI), and a small amount of radioactive isotope are used to record the electrical rhythm of the heart. Positron emission tomography (PET) to obtain precise three-dimensional pictures of flow, heart or brain metabolism, blood flow tests to determine how narrow blood vessels are using ultrasound, and long, thin, flexible tubes (catheter) There is a coronary angiography and blood test method to find the narrowed part of the coronary artery by inserting it through an artery in the heart and moving it to the coronary artery of the heart and injecting a contrast agent or dye. Blood tests show that myocardial infarction or cardiovascular disease results in necrosis of the heart muscle, causing enzymes to flow from the dead cells into the blood. By measuring the amount of these enzymes in the blood, creatine kinase (CK-MB), AST, and LDH are elevated in cardiovascular disease. This increase in enzyme levels can be seen in other diseases, and thus is highly associated with other disease markers, making it difficult to determine the extent of direct cardiovascular disease.

Currently, biomarkers in the blood to determine the progress of cardiovascular disease are limited. Therefore, if a new diagnostic marker with high specificity and sensitivity is developed, as well as a monoclonal antibody capable of detecting it, it is possible to develop a kit that can predict the early diagnosis and progression of cardiovascular disease in advance.

Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior arts, and as a result, by using two-dimensional electrophoresis technology to identify biomarker proteins that decrease and increase in the blood of cardiovascular patients than in normal blood, The invention was completed.

Accordingly, the main object of the present invention is to provide a new biomarker or immunogenic fragment thereof for early diagnosis of cardiovascular disease.

Another object of the present invention to provide an antibody to the biomarker protein and a diagnostic kit using the same.

According to one aspect of the present invention, the present invention provides a biomarker for diagnosing cardiovascular diseases comprising a protein selected from the following group, wherein the expression in the blood of a cardiovascular disease patient is relatively lower than that of a normal person:

Hemopexin precusor having an amino acid sequence of SEQ ID NO: 1 and having a molecular weight of 51-53 KD and a pi of 5.6-7.6;

Heptoglobin having the amino acid sequence of SEQ ID NO: 2 and a molecular weight of 38-40 KD and a pi of 5.6-7.6;

Alpha-1-microglobulin having the amino acid sequence of SEQ ID NO: 3 and having a molecular weight of 39-41 KD and a pi of 4.9-6.9;

C-type lectin domain family 3 having the amino acid sequence of SEQ ID NO: 4 and having a molecular weight of 22-24 KD and a pi of 4.2-6.2;

Complex-forming glycoprotein HC having an amino acid sequence of SEQ ID NO: 5 and having a molecular weight of 20-22 KD and a pi of 4.8-6.8;

Heptoglobin precursors having an amino acid sequence of SEQ ID NO: 6 and having a molecular weight of 45-47 KD and a pi of 5.4-7.4;

Complement factor I light chain having an amino acid sequence of SEQ ID NO: 7 and having a molecular weight of 22-24 KD and a pi of 4.2-6.2;

Inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) having an amino acid sequence of SEQ ID NO: 8 and a molecular weight of 102-104 KD and a pi of 5.5-7.5 );

Pre-serum amyloid P component having an amino acid sequence of SEQ ID NO: 9 and having a molecular weight of 24-26 KD and a pi of 5.1-7.1;

Prialbumin having the amino acid sequence of SEQ ID NO: 10 and having a molecular weight of 15-17 KD and a pi of 4.5-6.5;

Complement factor H-related 1 having the amino acid sequence of SEQ ID NO: 11 and having a molecular weight of 38-40 KD and a pi of 6.8-8.8;

Heptoglobin-related protein precursors having an amino acid sequence of SEQ ID NO: 12 and having a molecular weight of 38-40 KD and a pi of 5.4-7.4;

Complement component C4B3 having an amino acid sequence of SEQ ID NO: 13 and having a molecular weight of 47-49 KD and a pi of 4.8-6.8; And

Pigment epithelial-differentiating factor having an amino acid sequence of SEQ ID NO: 14 and having a molecular weight of 45-47 KD and a pi of 4.8-6.8.

According to another aspect of the present invention, the present invention provides a biomarker for diagnosing cardiovascular disease, comprising a protein selected from the following group, the expression of which is increased in the blood of a cardiovascular disease patient rather than the blood of a normal person:

Retinol Binding Protein 4 having an amino acid sequence of SEQ ID NO: 15 and having a molecular weight of 22-24 KD and a pi of 4.8-6.8;

Proapo-A-I protein having an amino acid sequence of SEQ ID NO: 16 and having a molecular weight of 28-30 KD and a pi of 4.5-6.5;

Transthyretin having the amino acid sequence of SEQ ID NO: 17 and having a molecular weight of 15-17 KD and a pi of 4.5-6.5; And

Ceruloplasmin having an amino acid sequence of SEQ ID NO: 18 and having a molecular weight of 97-99 KD and a pi of 4.3-6.3.

In the present invention, the biomarker may be part of the full-length protein having the sequence number as long as it has the molecular weight and pi, in which case it has trypsin digested peptide sequences shown in red in the MS / MS results of FIGS. 3A-3R. The trypsin digested peptide refers to a peptide of a protein digested with trypsin for MALDI-TOF or MS / MS analysis. In addition, the molecular weight and pi are values determined on two-dimentional electrophoresis and include generally accepted experimental error ranges.

In the present invention, cardiovascular disease (CVD) refers to heart disease and vascular disease, and specific examples include heart failure, hypertensive heart disease, arrhythmia, valve disease, congenital heart disease, cardiomyopathy, pericardial disease, etc. Vascular diseases such as peripheral vascular diseases and aneurysms.

According to another aspect of the present invention, the present invention provides a cardiovascular disease diagnostic agent comprising an antibody that specifically binds to the protein or immunogenic fragment thereof.

In the present invention, the immunogenic fragment means a fragment of a biomarker protein having one or more epitopes that can be recognized by an antibody against the biomarker protein of the present invention.

In the cardiovascular disease diagnostic agent of the present invention, the antibody may be a polyclonal antibody, but is preferably a monoclonal antibody.

The polyclonal antibodies of the present invention can be prepared by injecting a biomarker protein or fragment thereof as an immunogen into an external host according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, rabbits. Immunogens, when injected intramuscularly, intraperitoneally or subcutaneously, are generally administered with an adjuvant to increase antigenicity. Blood is periodically collected from external hosts to collect serum showing improved titers and specificity for antigens or to separate and purify antibodies from them.

The monoclonal antibodies can be prepared by techniques for the generation of immortalized cell lines by fusion known to those skilled in the art (Koeher and Milstein (1975) Nature, 256: 495). The manufacturing method is briefly described as follows. First, 20 μg of pure protein is immunized to Balb / C mice or a peptide is synthesized and combined with bovine serum albumin to immunize mice. Thereafter, antigen-producing lymphocytes isolated from mice are fused with myeloma in humans or mice to produce immortalized hybridomas, and only hybridoma cells that produce the desired monoclonal antibody using ELISA method. After selection and propagation, monoclonal antibodies are isolated and purified from the culture.

According to another aspect of the present invention, the present invention provides a cardiovascular disease diagnostic kit comprising an antibody that specifically binds to the protein or immunogenic fragment thereof.

Diagnostic kits of the present invention are prepared by conventional methods known to those skilled in the art, and typically include lyophilized antibodies, buffers, stabilizers, inactive proteins and the like. The antibody can be labeled by radionuclides, fluorescors, enzymes, and the like.

The monoclonal antibodies of the present invention can be used in various immunoassay kits (ELISA, antibody coated tube test, lateral-flow test, potable biosensor), and various cardiovascular diseases through the development of antibodies showing higher specificity and sensitivity. It can also be used to develop protein chips with a detection spectrum.

According to another aspect of the present invention, the present invention provides a composition for treating and preventing cardiovascular disease, comprising as an active ingredient a protein selected from the following group which is decreased in blood of a cardiovascular disease patient than normal blood:

Hemopexin precusor having an amino acid sequence of SEQ ID NO: 1 and having a molecular weight of 51-53 KD and a pi of 5.6-7.6;

Heptoglobin having the amino acid sequence of SEQ ID NO: 2 and a molecular weight of 38-40 KD and a pi of 5.6-7.6;

Alpha-1-microglobulin having the amino acid sequence of SEQ ID NO: 3 and having a molecular weight of 39-41 KD and a pi of 4.9-6.9;

C-type lectin domain family 3 having the amino acid sequence of SEQ ID NO: 4 and having a molecular weight of 22-24 KD and a pi of 4.2-6.2;

Complex-forming glycoprotein HC having an amino acid sequence of SEQ ID NO: 5 and having a molecular weight of 20-22 KD and a pi of 4.8-6.8;

Heptoglobin precursors having an amino acid sequence of SEQ ID NO: 6 and having a molecular weight of 45-47 KD and a pi of 5.4-7.4;

Complement factor I light chain having an amino acid sequence of SEQ ID NO: 7 and having a molecular weight of 22-24 KD and a pi of 4.2-6.2;

Inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) having an amino acid sequence of SEQ ID NO: 8 and a molecular weight of 102-104 KD and a pi of 5.5-7.5 );

Pre-serum amyloid P component having an amino acid sequence of SEQ ID NO: 9 and having a molecular weight of 24-26 KD and a pi of 5.1-7.1;

Prialbumin having the amino acid sequence of SEQ ID NO: 10 and having a molecular weight of 15-17 KD and a pi of 4.5-6.5;

Complement factor H-related 1 having the amino acid sequence of SEQ ID NO: 11 and having a molecular weight of 38-40 KD and a pi of 6.8-8.8;

Heptoglobin-related protein precursors having an amino acid sequence of SEQ ID NO: 12 and having a molecular weight of 38-40 KD and a pi of 5.4-7.4;

Complement component C4B3 having an amino acid sequence of SEQ ID NO: 13 and having a molecular weight of 47-49 KD and a pi of 4.8-6.8; And

Pigment epithelial-differentiating factor having an amino acid sequence of SEQ ID NO: 14 and having a molecular weight of 45-47 KD and a pi of 4.8-6.8.

Since the biomarker proteins of the present invention are components lacking in the blood of a cardiovascular disease patient, supplementing them with the blood of the patient may exert an effect on the treatment and prevention of cardiovascular disease.

The pharmaceutical composition of the present invention may be mixed with a pharmaceutically acceptable carrier, excipient, diluent, etc. by a method known in the pharmaceutical art, prepared in the form of an injection, etc., and administered by intravenous injection.

The dosage of the pharmaceutical composition according to the present invention may be appropriately selected according to the age, sex, condition and symptoms of the disease of the patient, and preferably 0.01-1 mg of protein per day may be administered on an adult basis.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Sample preparation

At Korea University Guro Hospital, experiments were performed based on the fasting blood glucose level and vascular narrowing of cardiovascular disease. The number of patients with cardiovascular disease was 19 and the normal group for comparison was 30.

Serum obtained from the hospital is a column that removes albumin, transferrin, IgG, IgA, haptoglobin, antitrysin, etc. which are known to be the most protein in the blood and albumin, transferrin, IgG, Agilent Multiple Affinity Removal Column; Technologies, Wilmington, DE, USA). Each sample was diluted 5-fold with buffer A (buffer A [5185-5987], composition: phosphates and 0.02% sodium azide, pH 7.4 in water, Agilent Technologies) and placed in a 0.22 um spin filter and centrifuge (Union- 55R centrifuge (Hanil Science, Korea) was used for 1 hour at 4 ° C and 16,000 × g, leaving by-products at the top of the column and leaving only the solution with by-products removed at the bottom. The lower layer solution was injected into a 4.6 × 100 mm immunoaffinity column to give a flow-through fraction that was run at a flow rate of 0.5 ml per minute. Less protein was obtained from the fraction flowing out in about 3 minutes, and by about 13 minutes, six large proteins bound to the immunoaffinity column. All samples were treated in this manner, each sample using a molecular cut-off column (Molecular cut-off column; Amicon, Millipore Corp., USA) to remove salts and lipids present in HPLC fractions. It was. 5 times cold acetone was added to the concentrated sample and stored at -20 ° C for one day. The protein precipitated at -20 ° C was run for 10 minutes at 13,000 rpm at 4 ° C with a centrifuge (Union-55R centrifuge, Hanil Science, Korea) to remove excess lipids and leave only protein. These precipitates were dried in air, sample buffered and protein quantified and stored in a -70 ° C. freezer. The composition of the sample buffer is as follows: 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris-Cl, pH 8.5, 65 mM DTT, 1 mM EDTA, 1% IPG buffer.

Example 2: Two-Dimensional Electrophoresis

end. Isoelectroforcusing (IEF)

One-dimensional electrophoresis was performed with a protein amount of 60 µg of the sample treated with the sample buffer. Mix the rehydration buffer (8 Murea, 2% CHAPS, 13 mM DTT, 1% IPG buffer) to a total volume of 450 μl and place it in a one-dimensional electrophoretic 24 cm strip holder, in the pH 4-7 range. Drystrip (Amerahsm Pharmacia Biotech, Uppsala, Sweden) was mounted in a strip holder. The strip was soaked at 20 ° C. for 5 hours in rehydration buffer and then subjected to one-dimensional electrophoresis under the following conditions: 80V 5 hours, 200V 30 minutes, 500V 1 minute, 8000V 45 minutes, 8000V 142000 Vhrs, 17 hours 45 Min, total 146 KVhr. One-dimensional electrophoresis was performed with IPGphore (Amerahsm Pharmacia Biotech, Uppsala, Sweden).

I. Two-dimensional electrophoresis

(Sodoum Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE)

For two-dimensional electrophoresis of one-dimensional electrophoretic strips, place the one-dimensional electrophoretic strips in a cap tube and add the first equilibration solution (6 M urea, 50 mM Tris-cl (pH 8.8), 30% glycerol, 2% SDS). After reacting with 10 ml of 1% DTT for 15 minutes, discard and add 10 ml of secondary equilibration solution (6 M urea, 50 mM Tris-cl (pH 8.8), 30% glycerol, 2% SDS, 1.5% IAA). Reaction was carried out for 10 minutes. The reaction strip was mounted on a polyacylamide gel (25 × 30cm) made of 11-16% and sealed with 0.5% agarose. Two-dimensional electrophoresis was performed by adding electrophoresis buffer (24 mM Tris, 192 mM glycine, 0.1% SDS) to Ettan Dalt (Amerahsm Pharmacia Biotech, Uppsala, Sweden). The electrophoresis conditions are as follows: 70 V 1 hour, 180 V 2 hours, 360 V 2 hours, 430 V 3 hours 30 minutes.

All. Scanning

After two-dimensional electrophoresis, silver staining was performed to visualize the gel. The silver dyeing process is as follows. After fixing for 3 hours in a solution consisting of 50% methanol and 12% acetic acid, the solution was fixed and then washed three times with 50% ethanol for 20 minutes. This was again reacted with 0.2% sodium thiosulfate solution for 1 minute and then sensitized, and washed with distilled water (DW). After reacting for 20 minutes with 0.1% Silver nitrate, 0.075% formaldehyde (37%) solution, and then washed again with distilled water. This was again visualized by reacting with 6% Sodium carbonate, 0.075% formaldehyde (37%) solution, which had been chilled for about 7 minutes, and treated with 50% methanol and 12% acetic acid solution to stop the reaction.

la. Image Analysis

To analyze the gels tested, they were first scanned with ImageScanner (Amersham Pharmacia Bio-tech, Uppsala, Sweden). To analyze the differences in protein expression, the analysis was performed with ImageMaster (Amersham Pharmacia Biotech, Uppsala, Sweden), a program dedicated to two-dimensional electrophoresis analysis.

Example 3: Protein Identification Using Mass Spectrometer (ESI-Q-TOF / MS / MS Analysis)

In order to identify the spots found in the image analysis program, spots were cut out from the gel after two-dimensional electrophoresis. First, 100 μl of a destaining solution mixed with a 30 mM Potassium Ferricyanide and a 100 mM Sodium Thiosulfate solution in a 1: 1 ratio was removed to treat the gel pieces for 5 minutes. Then washed three times with 400 μl of water. Reaction with 200 mM ammonium bicarbonate and washing with water. Acetonitrile was added to dehydrate the gel until it turned white, and the gel was placed in a speed vacuum centrifuge to completely remove the solution from the gel. The dried gel pieces were incubated for 45 minutes on ice with 20 μl of 50 mM ammonium bicarbonate containing 0.2 μg of trypsin (Promega). After removing the reaction solution, 30 μl of 50 mM ammonium bicarbonate was added and reacted at 37 ° C. overnight. The peptide solution was desalted using a C18 nano column (home made).

Custom-made chromatographic columns were used for peptide desalination and concentration prior to mass spectrometry. A column consisting of 100-300 nL of Poros reverse R2 material (20-30 um bead size, PerSeptive Biosystems) was packed in a tightly tightened GELoader tip (Eppendorp, hamburg, Germany). A 10 mL syringe passed the material through the column at pneumatic pressure. 30 μl of the peptide solution was diluted in 5% formic acid solution and transferred into the column. And 30 μl was washed with 5% formic acid solution. For MS / MS analysis, the peptide was eluted with 50% methnol / 49% H ₂ 0/1% formic acid in a borosilicate nanoelectrospray needle (Micromass, Manchester, UK).

MS / MS was performed with nano-ESI on a Q-TOF2 mass spectrometer (Micromass, Mancheser, UK). The source temperature was 80 ° C. 1 kV voltage on borosilicate nanoelectrospray needles (EconoTipTM, New Objective, USA) in an ion source mixed with nitrogen back-pressure of 0-5 psi to produce a stable flow rate (10-30 nL / min) Was applied. Its cone current is 40 volts. Quardrupole analyser was used to select precursor ions for segmentation in hexapole collision cells. Collision gas is argon (Ar) of 6-7 x 10-5 mbar and collision energy is 20-30V. Product ions were analyzed using a reflector, a micro channel plate detector, and a TOF analyzer fitted with a time-to-digital transducer. The data were analyzed using the Mass Lynx Widows NT PC system.

To identify proteins, all MS / MS spectra were examined for protein sequences in the NCBInr database using the MASCOT search program (www.matrixscience.com).

As described above, the amino acid sequence of the protein expressed differently during the progression of cardiovascular disease was confirmed using ESI-Q-TOF / MS / MS, and shown in FIGS. 3A to 3R.

Example 4: Western blotting

end. SDS-PAGE Electrophoresis

40 μg of serum protein of cardiovascular disease patients was subjected to 10% SDS-PAGE electrophoresis. 5-8 μl of serum sample (40 μg of protein) and 5 μl of SDS-PAGE loading buffer (60 mM Tris-Cl, 2% SDS, 25% Glycerol, 14.4 mM 2-Mercaptoethnol, 0.1% Bromophenol Blue, pH 6.8) Separation was carried out by mini gel (6 × 8 cm) by molecular weight (loading at 100 V on a separating gel). The composition of the running buffer used at this time is 0.025 M Tris-Cl, 0.192 M Glycine, 1% SDS (pH 8.3).

I. Blotting

After the SDS-PAGE, the gel protein was transferred to a NitroCellulose membrane using a semi-dry transfer kit (GE Healthcare) and carried out at 45 mA for 1 hour 30 minutes.

All. Antibody reaction

Retinol binding protein 4 (RBP4) was performed on the subject. Membrane was blocked with 5% w / v nonfat dry milk for 1 hour 30 minutes at room temperature. Retinol binding protein 4 was used as a primary antibody, and Retinol binding protein 4 is a polyclonal antibody. The antibody of Retinol binding protein 4 was reacted at 4 ° C. for 24 hours. The dilution concentration of the primary antibody was 1/2000. After washing with T-TBS (0.1%, Tris-buffered saline-tween 20), it was reacted with a secondary antibody at room temperature for 1 hour. Anti-rabbit was used as a secondary antibody and anti-rabbit dilution was 1 / 10,000. After the secondary antibody reaction was washed with T-TBS and visualized by ECL (Pierce Biotechnology, Inc., USA).

Example 5: Enzyme Linked Immunosorbent Assay (ELISA) Assay

Normal and cardiovascular disease patients, a total of 66 serum samples and 100 μl of Retino binding protein 4 standard are placed in each well. At this time, the protein concentration of the reference material was continuously diluted to 300, 150, 75, 37.5, 18.75, 9.4, 4.7 ng / ml. The lid was capped and allowed to react at room temperature for 1 hour to attach the antigen to the plate to which the polyclonal antibody was attached. After 1 hour, the plate was emptied, washed three times with a washing buffer, and 100 μl of the diluted secondary antibody anti-Retinol binding protein 4 solution was added to each well. The lid was then well covered and allowed to react for 1 hour at room temperature. After 1 hour, the plate was emptied and washed five times with the washing buffer, and 100 μl of Streptavidin-PAL solution was added to each well. The lid was then well covered and allowed to react for 1 hour at room temperature. After mixing for 20 minutes, the plate was emptied and washed five times with a washing buffer. 100 μl of substrate reaction solution pNPP was added to each well, and then the lid was capped and the color change was induced by reacting the absorbance with a standard concentration of 300 ng / ml at 1.5-2.0 units (about 20 minutes) at 37 ℃. 50 μl of the reaction stop solution was added to each well, and the absorbance was measured at 450 nm using an ELISA reader. The amount of Retinol binding protein 4 present in each serum sample was determined using the standard curve obtained by the concentration of the standard.

As a result of performing the two-dimensional electrophoresis experiment, proteins newly expressed or reduced or increased in the amount of the cardiovascular disease patients were identified.

1. Proteins Expressed in Serum of Cardiovascular Patients

Electrophoresis of the cardiovascular disease group resulted in 15 decreased proteins (# 3, # 15, # 18, # 28, # 84, # 125, # 132, # 153, # 162, # 196, # 228, # 232, # 259, # 300, # 3182) and four increasing proteins (# 66, # 69, # 74, # 104) were found (FIG. 1A). The reduced protein was reduced by about 45% or more at the level of 0.05 compared to the normal group, and the increased protein was increased by about 100% or more at the level of 0.05. In addition, ESI-Q-TOF was performed to identify proteins whose expression decreased or increased as cardiovascular disease progressed, as shown in Table 1 below.

TABLE 1 Protein Expressed in Serum of Cardiovascular Patients

2. Determination of the amino acid sequence of a protein

As described above, amino acid sequences of proteins expressed differently during the progression of cardiovascular disease were identified using ESI-Q-TOF / MS / MS to obtain the results of FIGS. 3A to 3R. Red in the figure represents matched trypsin digested peptide sequences. The amino acid sequences of these proteins were searched in the NCBInr database (http: //www.ncbi.nlm,nih.gov/) for hemopexin precusor (SEQ ID NO: 1), heptoglobin (SEQ ID NO: 2). ), Alpha-1-microglobulin (SEQ ID NO: 3), C-type lectin domain family 3 (SEQ ID NO: 4), complex-forming-glycoprotein H Seed (Complex-forming glycoprotein HC) (SEQ ID NO: 5), Heptoglobin precursor (SEQ ID NO: 6), Complement factor I light chain (SEQ ID NO: 7), inter-alpha- Inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) (SEQ ID NO: 8), Pre-serum amyloid P component (SEQ ID NO: 9), Prialbumin (SEQ ID NO: 10), Complement factor H-rela ted 1) (SEQ ID NO: 11), Heptoglobin-related protein precursor (SEQ ID NO: 12), Complement component C4B3 (SEQ ID NO: 13), pigment epithelium Pigment epithelial-differentiating factor (SEQ ID NO: 14), Retinol Binding Protein 4 (SEQ ID NO: 15), Proapo-AI protein (SEQ ID NO: 16) , Transthyretin (SEQ ID NO: 17), and Celluloplasmin (SEQ ID NO: 18).

Tables 2 and 3 below show trypsin digested peptide sequences of proteins expressing differently in patients with cardiovascular disease.

TABLE 2 Trypsin digested peptide sequence of reduced expression protein

TABLE 3 Trypsin digested peptide sequence of protein with increased expression

3. Western blotting and ELISA test results

Experimental results of Western blot of Retinol binding protein 4 (RBP4), a protein identified in the blood of cardiovascular disease patients, such as the protein whose expression was changed, are shown in FIG. 4A, and the relative strength thereof is shown in FIG. 4B. In addition, the results of Western blot of the protein Ceruloplasmin identified in the blood of the cardiovascular disease patient is shown in Figure 6a and the relative strength is shown in Figure 6b. As shown in the results, the amount of RBP4 and Ceruloplasmin was increased in cardiovascular patients than normal. This result was the same as the result of the second electrophoresis. 5 is a test result of ELISA of RBP4 with a total of 66 blood of normal and cardiovascular disease patients, and increased in cardiovascular disease patients as in the result of the second electrophoresis and Western blot. From this, RBP4 and Ceruloplasmin are considered to be very useful for the diagnosis of cardiovascular disease, and also show potential as a composition for treating and preventing cardiovascular disease.

As described above, 15 proteins with reduced expression and 4 proteins with increased expression in the cardiovascular disease obtained as a result of the present invention can be used as a biomarker for early diagnosis of cardiovascular disease. The biomarker protein of the present invention can be measured in the blood of a cardiovascular disease patient. In addition, monoclonal antibodies prepared on the basis of the biomarker protein of the present invention can be used in immunoassay kits for early diagnosis of cardiovascular diseases, as well as early development of various cardiovascular diseases through the development of antibodies showing higher specificity and sensitivity. It can also be used to develop protein chips with detection spectra for diagnosis.

1 is a 2-D electrophoresis image showing reduced and increased protein in blood of cardiovascular disease patients than in normal blood.

FIG. 2 is an enlarged image showing in detail the proteins shown in FIG. 1. FIG. Here, Healthy is normal, and CVD represents a cardiovascular disease patient.

3A-3R show hemopexin precursor, heptoglobin, alpha-1-microglobin, C-type lectin domain family 3, complex-forming-glycoprotein HC, heptoglobin precursor, complement element eye light chain, inter Alpha-Trypsin Inhibitor Family Heavy Chain-Related Proteins, Pre-Serum Amyloid P-Components, Prialbumin, Complement H-Related 1, Heptoglobin-Related Protein Precursors, Complement C4B3, Pigment Epithelium ESI-Q-TOF / MS / MS results of Retinol Adhesion Protein 4, Proapo-A-E Protein, Transthyretin, and Celluloplasmin.

Figure 4a is Western blot results of the protein identified in the blood of normal and cardiovascular patients Retinol binding protein 4.

4B is a result showing the relative strength of FIG. 4A.

Figure 5 shows the results of ELISA of Retinol binding protein 4 in the blood of 47 normal and 19 patients with cardiovascular disease.

Figure 6a is Western blot results of the protein Ceruloplasmin identified in the blood of normal and cardiovascular patients.

FIG. 6B is a result showing the relative strength of FIG. 6A.

<110> Korea University Industry and Academy Cooperation Foundation <120> Bio-marker for diagnosing Cardiovascular disease <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 461 <212> PRT <213> Homo sapiens <400> 1 Ala Arg Val Leu Gly Ala Pro Val Ala Leu Gly Leu Trp Ser Leu Cys   1 5 10 15 Trp Ser Leu Ala Ile Ala Thr Pro Leu Pro Pro Thr Ser Ala His Gly              20 25 30 Asn Val Ala Glu Gly Glu Thr Lys Pro Asp Pro Asp Val Thr Glu Arg          35 40 45 Cys Ser Asp Gly Trp Ser Phe Asp Ala Thr Thr Leu Asp Asp Asn Gly      50 55 60 Thr Met Leu Phe Phe Lys Gly Glu Phe Val Trp Lys Ser His Lys Trp  65 70 75 80 Asp Arg Glu Leu Ile Ser Glu Arg Trp Lys Asn Phe Pro Ser Pro Val                  85 90 95 Asp Ala Ala Phe Arg Gln Gly His Asn Ser Val Phe Leu Ile Lys Gly             100 105 110 Asp Lys Val Trp Val Tyr Pro Pro Glu Lys Lys Glu Lys Gly Tyr Pro         115 120 125 Lys Leu Leu Gln Asp Glu Phe Pro Gly Ile Pro Ser Pro Leu Asp Ala     130 135 140 Ala Val Glu Cys His Arg Gly Glu Cys Gln Ala Glu Gly Val Leu Phe 145 150 155 160 Phe Gln Gly Asp Arg Glu Trp Phe Trp Asp Leu Ala Thr Gly Thr Met                 165 170 175 Lys Glu Arg Ser Trp Pro Ala Val Gly Asn Cys Ser Ser Ala Leu Arg             180 185 190 Trp Leu Gly Arg Tyr Tyr Cys Phe Gln Gly Asn Gln Phe Leu Arg Phe         195 200 205 Asp Pro Val Arg Gly Glu Val Pro Pro Arg Tyr Pro Arg Asp Val Arg     210 215 220 Asp Tyr Phe Met Pro Cys Pro Gly Arg Gly His Gly His Arg Asn Gly 225 230 235 240 Thr Gly His Gly Asn Ser Thr His His Gly Pro Glu Tyr Met Arg Cys                 245 250 255 Ser Pro His Leu Val Leu Ser Ala Leu Thr Ser Asp Asn His Gly Ala             260 265 270 Thr Tyr Ala Phe Ser Gly Thr His Tyr Trp Arg Leu Asp Thr Ser Arg         275 280 285 Asp Gly Trp His Ser Trp Pro Ile Ala His Gln Trp Pro Gln Gly Pro     290 295 300 Ser Ala Val Asp Ala Ala Phe Ser Trp Glu Glu Lys Leu Tyr Leu Val 305 310 315 320 Gln Gly Thr Gln Val Tyr Val Phe Leu Thr Lys Gly Gly Tyr Thr Leu                 325 330 335 Val Ser Gly Tyr Pro Lys Arg Leu Glu Lys Glu Val Gly Thr Pro His             340 345 350 Gly Ile Ile Leu Asp Ser Val Asp Ala Ala Phe Ile Cys Pro Gly Ser         355 360 365 Ser Arg Leu His Ile Met Ala Gly Arg Arg Leu Trp Trp Leu Asp Leu     370 375 380 Lys Ser Gly Ala Gln Ala Thr Trp Thr Glu Leu Pro Trp Pro His Glu 385 390 395 400 Lys Val Asp Gly Ala Leu Cys Met Glu Lys Ser Leu Gly Pro Asn Ser                 405 410 415 Cys Ser Ala Asn Gly Pro Gly Leu Tyr Leu Ile His Gly Pro Asn Leu             420 425 430 Tyr Cys Tyr Ser Asp Val Glu Lys Leu Asn Ala Ala Lys Ala Leu Pro         435 440 445 Gln Pro Gln Asn Val Thr Ser Leu Leu Gly Cys Thr His     450 455 460 <210> 2 <211> 345 <212> PRT <213> Homo sapiens <400> 2 Ala Leu Gly Ala Val Ile Ala Leu Leu Leu Trp Gly Gln Leu Phe Ala   1 5 10 15 Val Asp Ser Gly Asn Asp Val Thr Asp Ile Ala Asp Asp Gly Cys Pro              20 25 30 Lys Pro Pro Glu Ile Ala His Gly Tyr Val Glu His Ser Val Arg Tyr          35 40 45 Gln Cys Lys Asn Tyr Tyr Lys Leu Arg Thr Glu Gly Asp Gly Val Tyr      50 55 60 Thr Leu Asn Asp Lys Lys Gln Trp Ile Asn Lys Ala Val Gly Asp Lys  65 70 75 80 Leu Pro Glu Cys Glu Ala Val Cys Gly Lys Pro Lys Asn Pro Ala Asn                  85 90 95 Pro Val Gln Arg Ile Leu Gly Gly His Leu Asp Ala Ile Gly Ser Phe             100 105 110 Pro Trp Gln Ala Lys Met Val Ser Arg His Asn Leu Thr Thr Gly Ala         115 120 125 Thr Leu Ile Asn Glu Gln Trp Leu Leu Thr Thr Ala Lys Asn Leu Phe     130 135 140 Leu Asn His Ser Glu Asn Ala Thr Ala Lys Asp Ile Ala Pro Thr Leu 145 150 155 160 Thr Leu Tyr Val Gly Lys Lys Gln Leu Val Glu Ile Glu Lys Val Val                 165 170 175 Leu His Pro Asn Tyr Ser Gln Val Asp Ile Gly Leu Ile Lys Leu Lys             180 185 190 Gln Lys Val Ser Val Asn Glu Arg Val Met Pro Ile Cys Leu Pro Ser         195 200 205 Lys Asp Tyr Ala Glu Val Gly Arg Leu Gly Tyr Val Ser Gly Trp Gly     210 215 220 Arg Asn Ala Asn Phe Lys Phe Thr Asp His Leu Lys Tyr Val Met Leu 225 230 235 240 Pro Val Ala Asp Gln Asp Gln Cys Ile Arg His Tyr Glu Gly Ser Thr                 245 250 255 Val Pro Glu Lys Lys Thr Pro Lys Ser Pro Val Gly Val Gln Pro Ile             260 265 270 Leu Asn Lys His Thr Phe Cys Ala Gly Met Ser Lys Tyr Gln Glu Asp         275 280 285 Thr Cys Tyr Gly Asp Ala Gly Ser Ala Phe Ala Val His Asp Leu Glu     290 295 300 Glu Asp Thr Trp Tyr Ala Ala Gly Ile Leu Ser Phe Asp Lys Ser Cys 305 310 315 320 Ala Val Ala Glu Tyr Gly Val Tyr Val Lys Val Thr Ser Ile Gln Asp                 325 330 335 Trp Val Gln Lys Thr Ile Ala Glu Asn             340 345 <210> 3 <211> 352 <212> PRT <213> Homo sapiens <400> 3 Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala   1 5 10 15 Val Ser Ala Gly Pro Val Pro Thr Pro Pro Asp Asn Ile Gln Val Gln              20 25 30 Glu Asn Phe Asn Ile Ser Arg Ile Tyr Gly Lys Trp Tyr Asn Leu Ala          35 40 45 Ile Gly Ser Thr Cys Pro Trp Leu Lys Lys Ile Met Asp Arg Met Thr      50 55 60 Val Ser Thr Leu Val Leu Gly Glu Gly Ala Thr Glu Ala Glu Ile Ser  65 70 75 80 Met Thr Ser Thr Arg Trp Arg Lys Gly Val Cys Glu Glu Thr Ser Gly                  85 90 95 Ala Tyr Glu Lys Thr Asp Thr Asp Gly Lys Phe Leu Tyr His Lys Ser             100 105 110 Lys Trp Asn Ile Thr Met Glu Ser Tyr Val Val His Thr Asn Tyr Asp         115 120 125 Glu Tyr Ala Ile Phe Leu Thr Lys Lys Phe Ser Arg His His Gly Pro     130 135 140 Thr Ile Thr Ala Lys Leu Tyr Gly Arg Ala Pro Gln Leu Arg Glu Thr 145 150 155 160 Leu Leu Gln Asp Phe Arg Val Val Ala Gln Gly Val Gly Ile Pro Glu                 165 170 175 Asp Ser Ile Phe Thr Met Ala Asp Arg Gly Glu Cys Val Pro Gly Glu             180 185 190 Gln Glu Pro Glu Pro Ile Leu Ile Pro Arg Val Arg Arg Ala Val Leu         195 200 205 Pro Gln Glu Glu Glu Gly Ser Gly Gly Gly Gln Leu Val Thr Glu Val     210 215 220 Thr Lys Lys Glu Asp Ser Cys Gln Leu Gly Tyr Ser Ala Gly Pro Cys 225 230 235 240 Met Gly Met Thr Ser Arg Tyr Phe Tyr Asn Gly Thr Ser Met Ala Cys                 245 250 255 Glu Thr Phe Gln Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn Phe Val             260 265 270 Thr Glu Lys Glu Cys Leu Gln Thr Cys Arg Thr Val Ala Ala Cys Asn         275 280 285 Leu Pro Ile Val Arg Gly Pro Cys Arg Ala Phe Ile Gln Leu Trp Ala     290 295 300 Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys 305 310 315 320 Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys Glu Cys Arg Glu Tyr                 325 330 335 Cys Gly Val Pro Gly Asp Gly Asp Glu Glu Leu Leu Arg Phe Ser Asn             340 345 350 <210> 4 <211> 202 <212> PRT <213> Homo sapiens <400> 4 Met Glu Leu Trp Gly Ala Tyr Leu Leu Leu Cys Leu Phe Ser Leu Leu   1 5 10 15 Thr Gln Val Thr Thr Glu Pro Pro Thr Gln Lys Pro Lys Lys Ile Val              20 25 30 Asn Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu Glu Leu Lys          35 40 45 Ser Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu Lys Glu Gln      50 55 60 Gln Ala Leu Gln Thr Val Cys Leu Lys Gly Thr Lys Val His Met Lys  65 70 75 80 Cys Phe Leu Ala Phe Thr Gln Thr Lys Thr Phe His Glu Ala Ser Glu                  85 90 95 Asp Cys Ile Ser Arg Gly Thr Leu Ser Thr Pro Gln Thr Gly Ser             100 105 110 Glu Asn Asp Ala Leu Tyr Glu Tyr Leu Arg Gln Ser Val Gly Asn Glu         115 120 125 Ala Glu Ile Trp Leu Gly Leu Asn Asp Met Ala Ala Glu Gly Thr Trp     130 135 140 Val Asp Met Thr Gly Ala Arg Ile Ala Tyr Lys Asn Trp Glu Thr Glu 145 150 155 160 Ile Thr Ala Gln Pro Asp Gly Gly Lys Thr Glu Asn Cys Ala Val Leu                 165 170 175 Ser Gly Ala Ala Asn Gly Lys Trp Phe Asp Lys Arg Cys Arg Asp Gln             180 185 190 Leu Pro Tyr Ile Cys Gln Phe Gly Ile Val         195 200 <210> 5 <211> 181 <212> PRT <213> Homo sapiens <400> 5 Gly Pro Val Pro Thr Pro Pro Asp Asn Ile Gln Val Gln Glu Asn Phe   1 5 10 15 Asx Ile Ser Arg Ile Tyr Gly Lys Trp Tyr Asn Leu Ala Ile Gly Ser              20 25 30 Thr Cys Pro Leu Lys Ile Met Asp Arg Met Thr Val Ser Thr Leu Val          35 40 45 Leu Gly Glu Gly Ala Thr Glu Ala Glu Ile Ser Met Thr Ser Thr Arg      50 55 60 Trp Arg Lys Gly Val Cys Glu Glu Thr Ser Gly Ala Tyr Glu Lys Thr  65 70 75 80 Asp Thr Asp Gly Lys Phe Leu Tyr His Lys Ser Lys Trp Asx Ile Thr                  85 90 95 Met Glu Ser Tyr Val Val His Thr Asn Tyr Asp Glu Tyr Ala Ile Phe             100 105 110 Leu Thr Lys Lys Phe Ser Arg His His Gly Pro Thr Ile Thr Ala Lys         115 120 125 Leu Tyr Gly Arg Ala Pro Gln Leu Arg Glu Thr Leu Leu Gln Asp Phe     130 135 140 Arg Val Val Ala Gln Gly Val Gly Ile Pro Glu Asp Ser Ile Phe Thr 145 150 155 160 Met Ala Asp Arg Gly Glu Cys Val Pro Gly Glu Gln Glu Pro Glu Pro                 165 170 175 Ile Leu Ile Pro Arg             180 <210> 6 <211> 406 <212> PRT <213> Homo sapiens <400> 6 Met Ser Ala Leu Gly Ala Val Ile Ala Leu Leu Leu Trp Gly Gln Leu   1 5 10 15 Phe Ala Val Asp Ser Gly Asn Asp Val Thr Asp Ile Ala Asp Asp Gly              20 25 30 Cys Pro Lys Pro Pro Glu Ile Ala His Gly Tyr Val Glu His Ser Val          35 40 45 Arg Tyr Gln Cys Lys Asn Tyr Tyr Lys Leu Arg Thr Glu Gly Asp Gly      50 55 60 Val Tyr Thr Leu Asn Asn Lys Lys Gln Trp Ile Asn Lys Ala Val Gly  65 70 75 80 Asp Lys Leu Pro Glu Cys Glu Ala Asp Asp Gly Cys Pro Lys Pro Pro                  85 90 95 Glu Ile Ala His Gly Tyr Val Glu His Ser Val Arg Tyr Gln Cys Lys             100 105 110 Asn Tyr Tyr Lys Leu Arg Thr Glu Gly Asp Gly Val Tyr Thr Leu Asn         115 120 125 Asn Glu Lys Gln Trp Ile Asn Lys Ala Val Gly Asp Lys Leu Pro Glu     130 135 140 Cys Glu Ala Val Cys Gly Lys Pro Lys Asn Pro Ala Asn Pro Val Gln 145 150 155 160 Arg Ile Leu Gly Gly His Leu Asp Ala Lys Gly Ser Phe Pro Trp Gln                 165 170 175 Ala Lys Met Val Ser His His Asn Leu Thr Thr Gly Ala Thr Leu Ile             180 185 190 Asn Glu Gln Trp Leu Leu Thr Thr Ala Lys Asn Leu Phe Leu Asn His         195 200 205 Ser Glu Asn Ala Thr Ala Lys Asp Ile Ala Pro Thr Leu Thr Leu Tyr     210 215 220 Val Gly Lys Lys Gln Leu Val Glu Ile Glu Lys Val Val Leu His Pro 225 230 235 240 Asn Tyr Ser Gln Val Asp Ile Gly Leu Ile Lys Leu Lys Gln Lys Val                 245 250 255 Ser Val Asn Glu Arg Val Met Pro Ile Cys Leu Pro Ser Lys Asp Tyr             260 265 270 Ala Glu Val Gly Arg Val Gly Tyr Val Ser Gly Trp Gly Arg Asn Ala         275 280 285 Asn Phe Lys Phe Thr Asp His Leu Lys Tyr Val Met Leu Pro Val Ala     290 295 300 Asp Gln Asp Gln Cys Ile Arg His Tyr Glu Gly Ser Thr Val Pro Glu 305 310 315 320 Lys Lys Thr Pro Lys Ser Pro Val Gly Val Gln Pro Ile Leu Asn Glu                 325 330 335 His Thr Phe Cys Ala Gly Met Ser Lys Tyr Gln Glu Asp Thr Cys Tyr             340 345 350 Gly Asp Ala Gly Ser Ala Phe Ala Val His Asp Leu Glu Glu Asp Thr         355 360 365 Trp Tyr Ala Thr Gly Ile Leu Ser Phe Asp Lys Ser Cys Ala Val Ala     370 375 380 Glu Tyr Gly Val Tyr Val Lys Val Thr Ser Ile Gln Asp Trp Val Gln 385 390 395 400 Lys Thr Ile Ala Glu Asn                 405 <210> 7 <211> 200 <212> PRT <213> Homo sapiens <400> 7 Ile Val Gly Gly Lys Arg Ala Gln Leu Gly Asp Leu Pro Trp Gln Val   1 5 10 15 Ala Ile Lys Asp Ala Ser Gly Ile Thr Cys Gly Gly Ile Tyr Ile Gly              20 25 30 Ile Ile Phe His Glu Asn Tyr Asn Ala Gly Glu Thr Tyr Gln Asn Asp          35 40 45 Ile Ala Leu Ile Met Lys Lys Asp Gly Asn Lys Lys Asp Cys Glu Leu      50 55 60 Pro Arg Ser Ile Pro Ala Cys Val Pro Trp Ser Pro Tyr Leu Phe Gln  65 70 75 80 Pro Asn Asp Thr Cys Ile Val Ser Gly Trp Gly Arg Glu Lys Asp Asn                  85 90 95 Glu Arg Val Phe Ser Leu Gln Trp Gly Glu Val Lys Leu Ile Ser Asn             100 105 110 Cys Ser Lys Phe Tyr Gly Asn Arg Phe Tyr Glu Lys Glu Met Glu Cys         115 120 125 Ala Gly Thr Tyr Asp Gly Ser Ile Asp Ala Cys Lys Gly Asp Ser Gly     130 135 140 Gly Pro Leu Val Cys Met Asp Ala Asn Asn Val Thr Tyr Val Trp Gly 145 150 155 160 Val Val Ser Trp Gly Glu Asn Cys Gly Lys Pro Glu Phe Pro Gly Val                 165 170 175 Tyr Thr Lys Val Ala Asn Tyr Phe Asp Trp Ile Ser Tyr His Val Gly             180 185 190 Arg Pro Phe Ile Ser Gln Tyr Asn         195 200 <210> 8 <211> 930 <212> PRT <213> Homo sapiens <400> 8 Met Lys Pro Pro Arg Pro Val Arg Thr Cys Ser Lys Val Leu Val Leu   1 5 10 15 Leu Ser Leu Leu Ala Ile His Gln Thr Thr Thr Ala Glu Lys Asn Gly              20 25 30 Ile Asp Ile Tyr Ser Leu Thr Val Asp Ser Arg Val Ser Ser Arg Phe          35 40 45 Ala His Thr Val Val Thr Ser Arg Val Val Asn Arg Ala Asn Thr Val      50 55 60 Gln Glu Ala Thr Phe Gln Met Glu Leu Pro Lys Lys Ala Phe Ile Thr  65 70 75 80 Asn Phe Ser Met Asn Ile Asp Gly Met Thr Tyr Pro Gly Ile Ile Lys                  85 90 95 Glu Lys Ala Glu Ala Gln Ala Gln Tyr Ser Ala Ala Val Ala Lys Gly             100 105 110 Lys Asn Ala Gly Leu Val Lys Ala Thr Gly Arg Asn Met Glu Gln Phe         115 120 125 Gln Val Ser Val Ser Val Ala Pro Asn Ala Lys Ile Thr Phe Glu Leu     130 135 140 Val Tyr Glu Glu Leu Leu Lys Arg Arg Leu Gly Val Tyr Glu Leu Leu 145 150 155 160 Leu Lys Val Arg Pro Gln Gln Leu Val Lys His Leu Gln Met Asp Ile                 165 170 175 His Ile Phe Glu Pro Gln Gly Ile Ser Phe Leu Glu Thr Glu Ser Thr             180 185 190 Phe Met Thr Asn Gln Leu Val Asp Ala Leu Thr Thr Trp Gln Asn Lys         195 200 205 Thr Lys Ala His Ile Arg Phe Lys Pro Thr Leu Ser Gln Gln Gln Lys     210 215 220 Ser Pro Glu Gln Gln Glu Thr Val Leu Asp Gly Asn Leu Ile Arg 225 230 235 240 Tyr Asp Val Asp Arg Ala Ile Ser Gly Gly Ser Ile Gln Ile Glu Asn                 245 250 255 Gly Tyr Phe Val His Tyr Phe Ala Pro Glu Gly Leu Thr Thr Met Pro             260 265 270 Lys Asn Val Val Phe Val Ile Asp Lys Ser Gly Ser Met Ser Gly Arg         275 280 285 Lys Ile Gln Gln Thr Arg Glu Ala Leu Ile Lys Ile Leu Asp Asp Leu     290 295 300 Ser Pro Arg Asp Gln Phe Asn Leu Ile Val Phe Ser Thr Glu Ala Thr 305 310 315 320 Gln Trp Arg Pro Ser Leu Val Pro Ala Ser Ala Glu Asn Val Asn Lys                 325 330 335 Ala Arg Ser Phe Ala Ala Gly Ile Gln Ala Leu Gly Gly Thr Asn Ile             340 345 350 Asn Asp Ala Met Leu Met Ala Val Gln Leu Leu Asp Ser Ser Asn Gln         355 360 365 Glu Glu Arg Leu Pro Glu Gly Ser Val Ser Leu Ile Ile Leu Leu Thr     370 375 380 Asp Gly Asp Pro Thr Val Gly Glu Thr Asn Pro Arg Ser Ile Gln Asn 385 390 395 400 Asn Val Arg Glu Ala Val Ser Gly Arg Tyr Ser Leu Phe Cys Leu Gly                 405 410 415 Phe Gly Phe Asp Val Ser Tyr Ala Phe Leu Glu Lys Leu Ala Leu Asp             420 425 430 Asn Gly Gly Leu Ala Arg Arg Ile His Glu Asp Ser Asp Ser Ala Leu         435 440 445 Gln Leu Gln Asp Phe Tyr Gln Glu Val Ala Asn Pro Leu Leu Thr Ala     450 455 460 Val Thr Phe Glu Tyr Pro Ser Asn Ala Val Glu Glu Val Thr Gln Asn 465 470 475 480 Asn Phe Arg Leu Leu Phe Lys Gly Ser Glu Met Val Val Ala Gly Lys                 485 490 495 Leu Gln Asp Arg Gly Pro Asp Val Leu Thr Ala Thr Val Ser Gly Lys             500 505 510 Leu Pro Thr Gln Asn Ile Thr Phe Gln Thr Glu Ser Ser Val Ala Glu         515 520 525 Gln Glu Ala Glu Phe Gln Ser Pro Lys Tyr Ile Phe His Asn Phe Met     530 535 540 Glu Arg Leu Trp Ala Tyr Leu Thr Ile Gln Gln Leu Leu Glu Gln Thr 545 550 555 560 Val Ser Ala Ser Asp Ala Asp Gln Gln Ala Leu Arg Asn Gln Ala Leu                 565 570 575 Asn Leu Ser Leu Ala Tyr Ser Phe Val Thr Pro Leu Thr Ser Met Val             580 585 590 Val Thr Lys Pro Asp Asp Gln Glu Gln Ser Gln Val Ala Glu Lys Pro         595 600 605 Met Glu Gly Glu Ser Arg Asn Arg Asn Val His Ser Gly Ser Thr Phe     610 615 620 Phe Lys Tyr Tyr Leu Gln Gly Ala Lys Ile Pro Lys Pro Glu Ala Ser 625 630 635 640 Phe Ser Pro Arg Arg Gly Trp Asn Arg Gln Ala Gly Ala Ala Gly Ser                 645 650 655 Arg Met Asn Phe Arg Pro Gly Val Leu Ser Ser Arg Gln Leu Gly Leu             660 665 670 Pro Gly Pro Pro Asp Val Pro Asp His Ala Ala Tyr His Pro Phe Arg         675 680 685 Arg Leu Ala Ile Leu Pro Ala Ser Ala Pro Pro Ala Thr Ser Asn Pro     690 695 700 Asp Pro Ala Val Ser Arg Val Met Asn Met Lys Ile Glu Glu Thr Thr 705 710 715 720 Met Thr Thr Gln Thr Pro Ala Pro Ile Gln Ala Pro Ser Ala Ile Leu                 725 730 735 Pro Leu Pro Gly Gln Ser Val Glu Arg Leu Cys Val Asp Pro Arg His             740 745 750 Arg Gln Gly Pro Val Asn Leu Leu Ser Asp Pro Glu Gln Gly Val Glu         755 760 765 Val Thr Gly Gln Tyr Glu Arg Glu Lys Ala Gly Phe Ser Trp Ile Glu     770 775 780 Val Thr Phe Lys Asn Pro Leu Val Trp Val His Ala Ser Pro Glu His 785 790 795 800 Val Val Val Thr Arg Asn Arg Arg Ser Ser Ala Tyr Lys Trp Lys Glu                 805 810 815 Thr Leu Phe Ser Val Met Pro Gly Leu Lys Met Thr Met Asp Lys Thr             820 825 830 Gly Leu Leu Leu Leu Ser Asp Pro Asp Lys Val Thr Ile Gly Leu Leu         835 840 845 Phe Trp Asp Gly Arg Gly Glu Gly Leu Arg Leu Leu Leu Arg Asp Thr     850 855 860 Asp Arg Phe Ser Ser His Val Gly Gly Thr Leu Gly Gln Phe Tyr Gln 865 870 875 880 Glu Val Leu Trp Gly Ser Pro Ala Ala Ser Asp Asp Gly Arg Arg Thr                 885 890 895 Leu Arg Val Gln Gly Asn Asp His Ser Ala Thr Arg Glu Arg Arg Leu             900 905 910 Asp Tyr Gln Glu Gly Pro Pro Gly Val Glu Ile Ser Cys Trp Ser Val         915 920 925 Glu leu     930 <210> 9 <211> 223 <212> PRT <213> Homo sapiens <400> 9 Met Asn Lys Pro Leu Leu Trp Ile Ser Val Leu Thr Ser Leu Leu Glu   1 5 10 15 Ala Phe Ala His Thr Asp Leu Ser Gly Lys Val Phe Val Phe Pro Arg              20 25 30 Glu Ser Val Thr Asp His Val Asn Leu Ile Thr Pro Leu Glu Lys Pro          35 40 45 Leu Gln Asn Phe Thr Leu Cys Phe Arg Ala Tyr Ser Asp Leu Ser Arg      50 55 60 Ala Tyr Ser Leu Phe Ser Tyr Asn Thr Gln Gly Arg Asp Asn Glu Leu  65 70 75 80 Leu Val Tyr Lys Glu Arg Val Gly Glu Tyr Ser Leu Tyr Ile Gly Arg                  85 90 95 His Lys Val Thr Pro Lys Val Ile Glu Lys Phe Pro Ala Pro Val His             100 105 110 Ile Cys Val Ser Trp Glu Ser Ser Ser Gly Ile Ala Glu Phe Trp Ile         115 120 125 Asn Gly Thr Pro Leu Val Lys Lys Gly Leu Arg Gln Gly Tyr Phe Val     130 135 140 Glu Ala Gln Pro Lys Ile Val Leu Gly Gln Glu Gln Asp Ser Tyr Gly 145 150 155 160 Gly Lys Phe Asp Arg Ser Gln Ser Phe Val Gly Glu Ile Gly Asp Leu                 165 170 175 Tyr Met Trp Asp Ser Val Leu Pro Pro Glu Asn Ile Leu Ser Ala Tyr             180 185 190 Gln Gly Thr Pro Leu Pro Ala Asn Ile Leu Asp Trp Gln Ala Leu Asn         195 200 205 Tyr Glu Ile Arg Gly Tyr Val Ile Ile Lys Pro Leu Val Trp Val     210 215 220 <210> 10 <211> 147 <212> PRT <213> Homo sapiens <400> 10 Met Ala Ser His Arg Leu Leu Leu Leu Cys Leu Ala Gly Leu Val Phe   1 5 10 15 Val Ser Glu Ala Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys Pro Leu              20 25 30 Met Val Lys Val Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val          35 40 45 Ala Met His Val Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro Phe      50 55 60 Ala Ser Gly Lys Thr Ser Glu Ser Gly Glu Leu His Gly Leu Thr Thr  65 70 75 80 Glu Glu Glu Phe Val Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys                  85 90 95 Ser Tyr Trp Lys Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu             100 105 110 Val Val Phe Thr Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala         115 120 125 Ala Leu Leu Ser Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn     130 135 140 Pro Lys Glu 145 <210> 11 <211> 330 <212> PRT <213> Homo sapiens <400> 11 Met Trp Leu Leu Val Ser Val Ile Leu Ile Ser Arg Ile Ser Ser Val   1 5 10 15 Gly Gly Glu Ala Thr Phe Cys Asp Phe Pro Lys Ile Asn His Gly Ile              20 25 30 Leu Tyr Asp Glu Glu Lys Tyr Lys Pro Phe Ser Gln Val Pro Thr Gly          35 40 45 Glu Val Phe Tyr Tyr Ser Cys Glu Tyr Asn Phe Val Ser Pro Ser Lys      50 55 60 Ser Phe Trp Thr Arg Ile Thr Cys Thr Glu Glu Gly Trp Ser Pro Thr  65 70 75 80 Pro Lys Cys Leu Arg Leu Cys Phe Phe Pro Phe Val Glu Asn Gly His                  85 90 95 Ser Glu Ser Ser Gly Gln Thr His Leu Glu Gly Asp Thr Val Gln Ile             100 105 110 Ile Cys Asn Thr Gly Tyr Arg Leu Gln Asn Asn Glu Asn Asn Ile Ser         115 120 125 Cys Val Glu Arg Gly Trp Ser Thr Pro Pro Lys Cys Arg Ser Thr Asp     130 135 140 Thr Ser Cys Val Asn Pro Pro Thr Val Gln Asn Ala Tyr Ile Val Ser 145 150 155 160 Arg Gln Met Ser Lys Tyr Pro Ser Gly Glu Arg Val Arg Tyr Gln Cys                 165 170 175 Arg Ser Pro Tyr Glu Met Phe Gly Asp Glu Glu Val Met Cys Leu Asn             180 185 190 Gly Asn Trp Thr Glu Pro Pro Gln Cys Lys Asp Ser Thr Gly Lys Cys         195 200 205 Gly Pro Pro Pro Pro Ile Asp Asn Gly Asp Ile Thr Ser Phe Pro Leu     210 215 220 Ser Val Tyr Ala Pro Ala Ser Ser Val Glu Tyr Gln Cys Gln Asn Leu 225 230 235 240 Tyr Gln Leu Glu Gly Asn Lys Arg Ile Thr Cys Arg Asn Gly Gln Trp                 245 250 255 Ser Glu Pro Pro Lys Cys Leu His Pro Cys Val Ile Ser Arg Glu Ile             260 265 270 Met Glu Asn Tyr Asn Ile Ala Leu Arg Trp Thr Ala Lys Gln Lys Leu         275 280 285 Tyr Leu Arg Thr Gly Glu Ser Ala Glu Phe Val Cys Lys Arg Gly Tyr     290 295 300 Arg Leu Ser Ser Arg Ser His Thr Leu Arg Thr Thr Cys Trp Asp Gly 305 310 315 320 Lys Leu Glu Tyr Pro Thr Cys Ala Lys Arg                 325 330 <210> 12 <211> 385 <212> PRT <213> Homo sapiens <400> 12 Met His Val Cys Val Cys Val Cys Val Cys Val Tyr Met Pro Val Cys   1 5 10 15 Val Asp Ala Cys Met Cys Cys Glu Ala Gly Arg Pro Ala Phe Arg Ser              20 25 30 Phe Leu Phe Ser Leu Cys Ser Asp Leu Gly Ala Val Ile Ser Leu Leu          35 40 45 Leu Trp Gly Arg Gln Leu Phe Ala Leu Tyr Ser Gly Asn Asp Val Thr      50 55 60 Asp Ile Ser Asp Asp Arg Phe Pro Lys Pro Pro Glu Ile Ala Asn Gly  65 70 75 80 Tyr Val Glu His Leu Phe Arg Tyr Gln Cys Lys Asn Tyr Tyr Arg Leu                  85 90 95 Arg Thr Glu Gly Asp Gly Val Tyr Thr Leu Asn Asp Lys Lys Gln Trp             100 105 110 Ile Asn Lys Ala Val Gly Asp Lys Leu Pro Glu Cys Glu Ala Val Cys         115 120 125 Gly Lys Pro Lys Asn Pro Ala Asn Pro Val Gln Arg Ile Leu Gly Gly     130 135 140 His Leu Asp Ala Lys Gly Ser Phe Pro Trp Gln Ala Lys Met Val Ser 145 150 155 160 His His Asn Leu Thr Thr Gly Ala Thr Leu Ile Asn Glu Gln Trp Leu                 165 170 175 Leu Thr Thr Ala Lys Asn Leu Phe Leu Asn His Ser Glu Asn Ala Thr             180 185 190 Ala Lys Asp Ile Ala Pro Thr Leu Thr Leu Tyr Val Gly Lys Lys Gln         195 200 205 Leu Val Glu Ile Glu Lys Val Val Leu His Pro Asn Tyr His Gln Val     210 215 220 Asp Ile Gly Leu Ile Lys Leu Lys Gln Lys Val Leu Val Asn Glu Arg 225 230 235 240 Val Met Pro Ile Cys Leu Pro Ser Lys Asn Tyr Ala Glu Val Gly Arg                 245 250 255 Val Gly Tyr Val Ser Gly Trp Gly Gln Ser Asp Asn Phe Lys Leu Thr             260 265 270 Asp His Leu Lys Tyr Val Met Leu Pro Val Ala Asp Gln Tyr Asp Cys         275 280 285 Ile Thr His Tyr Glu Gly Ser Thr Cys Pro Lys Trp Lys Ala Pro Lys     290 295 300 Ser Pro Val Gly Val Gln Pro Ile Leu Asn Glu His Thr Phe Cys Val 305 310 315 320 Gly Met Ser Lys Tyr Gln Glu Asp Thr Cys Tyr Gly Asp Ala Gly Ser                 325 330 335 Ala Phe Ala Val His Asp Leu Glu Glu Asp Thr Trp Tyr Ala Ala Gly             340 345 350 Ile Leu Ser Phe Asp Lys Ser Cys Ala Val Ala Glu Tyr Gly Val Tyr         355 360 365 Val Lys Val Thr Ser Ile Gln His Trp Val Gln Lys Thr Ile Ala Glu     370 375 380 Asn 385 <210> 13 <211> 428 <212> PRT <213> Homo sapiens <400> 13 Gly Gln Tyr Ala Ser Pro Thr Ala Lys Arg Cys Cys Gln Asp Gly Val   1 5 10 15 Thr Arg Leu Pro Met Met Ser Ser Cys Glu Gln Arg Ala Ala Arg Val              20 25 30 Gln Gln Pro Asp Cys Arg Glu Pro Phe Leu Ser Cys Cys Gln Phe Ala          35 40 45 Glu Ser Leu Arg Lys Lys Ser Arg Asp Lys Gly Gln Ala Gly Leu Gln      50 55 60 Arg Ala Leu Glu Ile Leu Gln Glu Glu Asp Leu Ile Asp Glu Asp Asp  65 70 75 80 Ile Pro Val Arg Ser Phe Phe Pro Glu Asn Trp Leu Trp Arg Val Glu                  85 90 95 Thr Val Asp Arg Phe Gln Ile Leu Thr Leu Trp Leu Pro Asp Ser Leu             100 105 110 Thr Thr Trp Glu Ile His Gly Leu Ser Leu Ser Lys Thr Lys Gly Leu         115 120 125 Cys Val Ala Thr Pro Val Gln Leu Arg Val Phe Arg Glu Phe His Leu     130 135 140 His Leu Arg Leu Pro Met Ser Val Arg Arg Phe Glu Gln Leu Glu Leu 145 150 155 160 Arg Pro Val Leu Tyr Asn Tyr Leu Asp Lys Asn Leu Thr Val Ser Val                 165 170 175 His Val Ser Pro Val Glu Gly Leu Cys Leu Ala Gly Gly Gly Gly Leu             180 185 190 Ala Gln Gln Val Leu Val Pro Ala Gly Ser Ala Arg Pro Val Ala Phe         195 200 205 Ser Val Val Pro Thr Ala Ala Ala Ala Val Ser Leu Lys Val Val Ala     210 215 220 Arg Gly Ser Phe Glu Phe Pro Val Gly Asp Ala Val Ser Lys Val Leu 225 230 235 240 Gln Ile Glu Lys Glu Gly Ala Ile His Arg Glu Glu Leu Val Tyr Glu                 245 250 255 Leu Asn Pro Leu Asp His Arg Gly Arg Thr Leu Glu Ile Pro Gly Asn             260 265 270 Ser Asp Pro Asn Met Ile Pro Asp Gly Asp Phe Asn Ser Tyr Val Arg         275 280 285 Val Thr Ala Ser Asp Pro Leu Asp Thr Leu Gly Ser Glu Gly Ala Leu     290 295 300 Ser Pro Gly Gly Val Ala Ser Leu Leu Arg Leu Pro Arg Gly Cys Gly 305 310 315 320 Glu Gln Thr Met Ile Tyr Leu Ala Pro Thr Leu Ala Ala Ser Arg Tyr                 325 330 335 Leu Asp Lys Thr Glu Gln Trp Ser Thr Leu Pro Pro Glu Thr Lys Asp             340 345 350 His Ala Val Asp Leu Ile Gln Lys Gly Tyr Met Arg Ile Gln Gln Phe         355 360 365 Arg Lys Ala Asp Gly Ser Tyr Ala Ala Trp Leu Ser Arg Gly Ser Ser     370 375 380 Thr Trp Leu Thr Ala Phe Val Leu Lys Val Leu Ser Leu Ala Gln Glu 385 390 395 400 Gln Val Gly Gly Ser Pro Glu Lys Leu Gln Glu Thr Ser Asn Trp Leu                 405 410 415 Leu Ser Gln Gln Gln Ala Asp Gly Ser Phe Gln Asp             420 425 <210> 14 <211> 418 <212> PRT <213> Homo sapiens <400> 14 Met Gln Ala Leu Val Leu Leu Leu Cys Ile Gly Ala Leu Leu Gly His   1 5 10 15 Ser Ser Cys Gln Asn Pro Ala Ser Pro Pro Glu Glu Gly Ser Pro Asp              20 25 30 Pro Asp Ser Thr Gly Ala Leu Val Glu Glu Glu Asp Pro Phe Phe Lys          35 40 45 Val Pro Val Asn Lys Leu Ala Ala Ala Val Ser Asn Phe Gly Tyr Asp      50 55 60 Leu Tyr Arg Val Arg Ser Ser Met Ser Pro Thr Thr Asn Val Leu Leu  65 70 75 80 Ser Pro Leu Ser Val Ala Thr Ala Leu Ser Ala Leu Ser Leu Gly Ala                  85 90 95 Asp Glu Arg Thr Glu Ser Ile Ile His Arg Ala Leu Tyr Tyr Asp Leu             100 105 110 Ile Ser Ser Pro Asp Ile His Gly Thr Tyr Lys Glu Leu Leu Asp Thr         115 120 125 Val Thr Ala Pro Gln Lys Asn Leu Lys Ser Ala Ser Arg Ile Val Phe     130 135 140 Glu Lys Lys Leu Arg Ile Lys Ser Ser Phe Val Ala Pro Leu Glu Lys 145 150 155 160 Ser Tyr Gly Thr Arg Pro Arg Val Leu Thr Gly Asn Pro Arg Leu Asp                 165 170 175 Leu Gln Glu Ile Asn Asn Trp Val Gln Ala Gln Met Lys Gly Lys Leu             180 185 190 Ala Arg Ser Thr Lys Glu Ile Pro Asp Glu Ile Ser Ile Leu Leu Leu         195 200 205 Gly Val Ala His Phe Lys Gly Gln Trp Val Thr Lys Phe Asp Ser Arg     210 215 220 Lys Thr Ser Leu Glu Asp Phe Tyr Leu Asp Glu Glu Arg Thr Val Arg 225 230 235 240 Val Pro Met Met Ser Asp Pro Lys Ala Val Leu Arg Tyr Gly Leu Asp                 245 250 255 Ser Asp Leu Ser Cys Lys Ile Ala Gln Leu Pro Leu Thr Gly Ser Met             260 265 270 Ser Ile Ile Phe Phe Leu Pro Leu Lys Val Thr Gln Asn Leu Thr Leu         275 280 285 Ile Glu Glu Ser Leu Thr Ser Glu Phe Ile His Asp Ile Asp Arg Glu     290 295 300 Leu Lys Thr Val Gln Ala Val Leu Thr Val Pro Lys Leu Lys Leu Ser 305 310 315 320 Tyr Glu Gly Glu Val Thr Lys Ser Leu Gln Glu Met Lys Leu Gln Ser                 325 330 335 Leu Phe Asp Ser Pro Asp Phe Ser Lys Ile Thr Gly Lys Pro Ile Lys             340 345 350 Leu Thr Gln Val Glu His Arg Ala Gly Phe Glu Trp Asn Glu Asp Gly         355 360 365 Ala Gly Thr Thr Pro Ser Pro Gly Leu Gln Pro Ala His Leu Thr Phe     370 375 380 Pro Leu Asp Tyr His Leu Asn Gln Pro Phe Ile Phe Val Leu Arg Asp 385 390 395 400 Thr Asp Thr Gly Ala Leu Leu Phe Ile Gly Lys Ile Leu Asp Pro Arg                 405 410 415 Gly pro         <210> 15 <211> 199 <212> PRT <213> Homo sapiens <400> 15 Met Asn Tyr Ser Lys Ile Pro Ala Gln Val Asp Leu Arg Arg Gln Ala   1 5 10 15 Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn Phe Asp              20 25 30 Lys Ala Arg Phe Ser Gly Thr Trp Tyr Ala Met Ala Lys Lys Asp Pro          35 40 45 Glu Gly Leu Phe Leu Gln Asp Asn Ile Val Ala Glu Phe Ser Val Asp      50 55 60 Glu Thr Gly Gln Met Ser Ala Thr Ala Lys Gly Arg Val Arg Leu Leu  65 70 75 80 Asn Asn Trp Asp Val Cys Ala Asp Met Val Gly Thr Phe Thr Asp Thr                  85 90 95 Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val Ala Ser Phe             100 105 110 Leu Gln Lys Gly Asn Asp Asp His Trp Ile Val Asp Thr Asp Tyr Asp         115 120 125 Thr Tyr Ala Val Gln Tyr Ser Cys Arg Leu Leu Asn Leu Asp Gly Thr     130 135 140 Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn Gly Leu 145 150 155 160 Pro Pro Glu Ala Gln Lys Ile Val Arg Gln Arg Gln Glu Glu Leu Cys                 165 170 175 Leu Ala Arg Gln Tyr Arg Leu Ile Val His Asn Gly Tyr Cys Asp Gly             180 185 190 Arg Ser Glu Arg Asn Leu Leu         195 <210> 16 <211> 249 <212> PRT <213> Homo sapiens <400> 16 Arg His Phe Trp Gln Gln Asp Glu Pro Pro Gln Ser Pro Trp Asp Arg   1 5 10 15 Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu Lys Asp Ser Gly              20 25 30 Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys Gln Leu          35 40 45 Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr Phe Ser      50 55 60 Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp Asp Asn  65 70 75 80 Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser Lys Asp Leu                  85 90 95 Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys             100 105 110 Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu         115 120 125 Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu His Glu Leu Gln     130 135 140 Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala 145 150 155 160 His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp Glu Leu                 165 170 175 Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly             180 185 190 Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu Ser Thr         195 200 205 Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu     210 215 220 Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu 225 230 235 240 Glu Tyr Thr Lys Lys Leu Asn Thr Gln                 245 <210> 17 <211> 147 <212> PRT <213> Homo sapiens <400> 17 Met Ala Ser His Arg Leu Leu Leu Leu Cys Leu Ala Gly Leu Val Phe   1 5 10 15 Val Ser Glu Ala Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys Pro Leu              20 25 30 Met Val Lys Val Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val          35 40 45 Ala Val His Val Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro Phe      50 55 60 Ala Ser Gly Lys Thr Ser Glu Ser Gly Glu Leu His Gly Leu Thr Thr  65 70 75 80 Glu Glu Glu Phe Val Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys                  85 90 95 Ser Tyr Trp Lys Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu             100 105 110 Val Val Phe Thr Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala         115 120 125 Ala Leu Leu Ser Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn     130 135 140 Pro Lys Glu 145 <210> 18 <211> 852 <212> PRT <213> Homo sapiens <400> 18 Val Val Met Phe Ser Val Val Asp Glu Asn Phe Ser Trp Tyr Leu Glu   1 5 10 15 Asp Asn Ile Lys Thr Tyr Cys Ser Glu Pro Glu Lys Val Asp Lys Asp              20 25 30 Asn Glu Asp Phe Gln Glu Ser Asn Arg Met Tyr Ser Val Asn Gly Tyr          35 40 45 Thr Phe Gly Ser Leu Pro Gly Leu Ser Met Cys Ala Glu Asp Arg Val      50 55 60 Lys Trp Tyr Leu Phe Gly Met Gly Asn Glu Val Asp Val His Ala Ala  65 70 75 80 Phe Phe His Gly Gln Ala Leu Thr Asn Lys Asn Tyr Arg Ile Asp Thr                  85 90 95 Ile Asn Leu Phe Pro Ala Thr Leu Phe Asp Ala Tyr Met Val Ala Gln             100 105 110 Asn Pro Gly Glu Trp Met Leu Ser Cys Gln Asn Leu Asn His Leu Lys         115 120 125 Ala Gly Leu Gln Ala Phe Phe Gln Val Gln Glu Cys Asn Lys Ser Ser     130 135 140 Ser Lys Asp Asn Ile Arg Gly Lys His Val Arg His Tyr Tyr Ile Ala 145 150 155 160 Ala Glu Glu Ile Ile Trp Asn Tyr Ala Pro Ser Gly Ile Asp Ile Phe                 165 170 175 Thr Lys Glu Asn Leu Thr Ala Pro Gly Ser Asp Ser Ala Val Phe Phe             180 185 190 Glu Gln Gly Thr Thr Arg Ile Gly Gly Ser Tyr Lys Lys Leu Val Tyr         195 200 205 Arg Glu Tyr Thr Asp Ala Ser Phe Thr Asn Arg Lys Glu Arg Gly Pro     210 215 220 Glu Glu Glu His Leu Gly Ile Leu Gly Pro Val Ile Trp Ala Glu Val 225 230 235 240 Gly Asp Thr Ile Arg Val Thr Phe His Asn Lys Gly Ala Tyr Pro Leu                 245 250 255 Ser Ile Glu Pro Ile Gly Val Arg Phe Asn Lys Asn Asn Glu Gly Thr             260 265 270 Tyr Tyr Ser Pro Asn Tyr Asn Pro Gln Ser Arg Ser Val Pro Pro Ser         275 280 285 Ala Ser His Val Ala Pro Thr Glu Thr Phe Thr Tyr Glu Trp Thr Val     290 295 300 Pro Lys Glu Val Gly Pro Thr Asn Ala Asp Pro Val Cys Leu Ala Lys 305 310 315 320 Met Tyr Tyr Ser Ala Val Asp Pro Thr Lys Asp Ile Phe Thr Gly Leu                 325 330 335 Ile Gly Pro Met Lys Ile Cys Lys Lys Gly Ser Leu His Ala Asn Gly             340 345 350 Arg Gln Lys Asp Val Asp Lys Glu Phe Tyr Leu Phe Pro Thr Val Phe         355 360 365 Asp Glu Asn Glu Ser Leu Leu Leu Glu Asp Asn Ile Arg Met Phe Thr     370 375 380 Thr Ala Pro Asp Gln Val Asp Lys Glu Asp Glu Asp Phe Gln Glu Ser 385 390 395 400 Asn Lys Met His Ser Met Asn Gly Phe Met Tyr Gly Asn Gln Pro Gly                 405 410 415 Leu Thr Met Cys Lys Gly Asp Ser Val Val Trp Tyr Leu Phe Ser Ala             420 425 430 Gly Asn Glu Ala Asp Val His Gly Ile Tyr Phe Ser Gly Asn Thr Tyr         435 440 445 Leu Trp Arg Gly Glu Arg Arg Asp Thr Ala Asn Leu Phe Pro Gln Thr     450 455 460 Ser Leu Thr Leu His Met Trp Pro Asp Thr Glu Gly Thr Phe Asn Val 465 470 475 480 Glu Cys Leu Thr Thr Asp His Tyr Thr Gly Gly Met Lys Gln Lys Tyr                 485 490 495 Thr Val Asn Gln Cys Arg Arg Gln Ser Glu Asp Ser Thr Phe Tyr Leu             500 505 510 Gly Glu Arg Thr Tyr Tyr Ile Ala Ala Val Glu Val Glu Trp Asp Tyr         515 520 525 Ser Pro Gln Arg Glu Trp Glu Lys Glu Leu His His Leu Gln Glu Gln     530 535 540 Asn Val Ser Asn Ala Phe Leu Asp Lys Gly Glu Phe Tyr Ile Gly Ser 545 550 555 560 Lys Tyr Lys Lys Val Val Tyr Arg Gln Tyr Thr Asp Ser Thr Phe Arg                 565 570 575 Val Pro Val Glu Arg Lys Ala Glu Glu Glu His Leu Gly Ile Leu Gly             580 585 590 Pro Gln Leu His Ala Asp Val Gly Asp Lys Val Lys Ile Ile Phe Lys         595 600 605 Asn Met Ala Thr Arg Pro Tyr Ser Ile His Ala His Gly Val Gln Thr     610 615 620 Glu Ser Ser Thr Val Thr Pro Thr Leu Pro Gly Glu Thr Leu Thr Tyr 625 630 635 640 Val Trp Lys Ile Pro Glu Arg Ser Gly Ala Gly Thr Glu Asp Ser Ala                 645 650 655 Cys Ile Pro Trp Ala Tyr Tyr Ser Thr Val Asp Gln Val Lys Asp Leu             660 665 670 Tyr Ser Gly Leu Ile Gly Pro Leu Ile Val Cys Arg Arg Pro Tyr Leu         675 680 685 Lys Val Phe Asn Pro Arg Arg Lys Leu Glu Phe Ala Leu Leu Phe Leu     690 695 700 Val Phe Asp Glu Asn Glu Ser Trp Tyr Leu Asp Asp Asn Ile Lys Thr 705 710 715 720 Tyr Ser Asp His Pro Glu Lys Val Asn Lys Asp Asp Glu Glu Phe Ile                 725 730 735 Glu Ser Asn Lys Met His Ala Ile Asn Gly Arg Met Phe Gly Asn Leu             740 745 750 Gln Gly Leu Thr Met His Val Gly Asp Glu Val Asn Trp Tyr Leu Met         755 760 765 Gly Met Gly Asn Glu Ile Asp Leu His Thr Val His Phe His Gly His     770 775 780 Ser Phe Gln Tyr Lys His Arg Gly Val Tyr Ser Ser Asp Val Phe Asp 785 790 795 800 Ile Phe Pro Gly Thr Tyr Gln Thr Leu Glu Met Phe Pro Arg Thr Pro                 805 810 815 Gly Ile Trp Leu Leu His Cys His Val Thr Asp His Ile His Ala Gly             820 825 830 Met Glu Thr Thr Tyr Thr Val Leu Gln Asn Glu Gly Glu Tyr Pro Asp         835 840 845 Thr Lys Ser Gly     850  

Claims (6)

delete Biomarker composition for diagnosing non-diabetic cardiovascular disease comprising the following protein is relatively increased in the blood of patients with non-diabetic cardiovascular disease than the blood of normal people: Retinol Binding Protein 4 having an amino acid sequence of SEQ ID NO: 15 and a molecular weight of 22-24 KD and a pi of 4.8-6.8. A nondiabetic cardiovascular disease diagnostic agent comprising an antibody that specifically binds to the protein of claim 2 or an immunogenic fragment thereof. 4. The nondiabetic cardiovascular disease diagnostic agent according to claim 3, wherein the antibody is a monoclonal antibody. Non-diabetic cardiovascular disease diagnostic kit comprising an antibody that specifically binds to the protein of claim 2 or an immunogenic fragment thereof. delete

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