KR101231235B1 - Proteinic markers for diagnosing prostate cancer stem cells - Google Patents

Abstract

The present invention is a prostate cancer stem cell selection proteinaceous markers, prostate cancer stem cell selection composition comprising a substance for detecting a change in the proteinaceous markers, prostate cancer stem cell selection comprising the composition for selecting prostate cancer stem cells The present invention relates to a kit for detecting protein markers for selecting prostate cancer stem cells, and a method for screening prostate cancer stem cell inhibitors using the protein markers.

Description

Protein markers for diagnosing prostate cancer stem cells

The present invention relates to a proteinaceous marker for selecting prostate cancer stem cells, and more specifically, to a proteinaceous marker for selecting prostate cancer stem cells that are expressed more or less in prostate cancer stem cells than in normal prostate cancer cells. It relates to a composition for screening prostate cancer stem cells.

When the human prostate cancer cell line is separated into CD44 positive cells and CD44 negative cells by using CD44, which is known as a marker of prostate cancer stem cells, the present invention is to confirm the difference in protein expression between them, This study is about the development of a protein that can be used as a marker to select prostate cancer stem cells. In other words, the prostate gland can be identified through a protein study that can identify proteins expressing more specifically in CD44-positive cells and proteins expressing more in CD44-negative cells to identify characteristic differences between prostate and stem cells. It is an invention for the development of markers for the application of cancer stem cell-centered prostate cancer treatment.

The fact that cancer tissue consists of many different types of heterogeneous cells has been accepted as a fact in the academic world since the first report in 1977. The cancer stem cell hypothesis that there are some cancer cells that have the ability to form cancer among numerous cancer cells, and that they are produced by mutations of stem cells due to some environmental influence has already been proved through many studies in the academic community. There is a bar.

In the past studies, many researchers analyzed specific surface proteins of stem cells by labeling method, and found some stem cell markers in cancer tissues and compared some of the cells with excellent tumorigenicity by comparing tumor ability in selected cells. It was found and named cancer stem cells (Reya, T. et al., (2001) Stem cells, cancer, and cancer stem cells, Nature 414,105-111; Collins, A. et al) (2005) Prospective identification of tumorigenic prostate cancer stem cells, Cancer Res 65, 10946-10951.

The results to date suggest that cancer development may be due to excessive proliferation of stem cells by expression of cancer genes or accumulation of mutations. This cancer stem cell hypothesis may explain why anticancer drugs have not shown satisfactory therapeutic effects in cancer treatment. In other words, since cancer stem cells have the ability to recover cancer cells necrotic by at least the number of cancer cells, necrosis of cancer stem cells will have to be preceded for complete anti-cancer treatment. However, since the anticancer drugs do not distinguish between cancer cells and cancer stem cells, cancer stem cells, which are relatively resistant to drug absorption, may not be necroticed by anticancer drugs compared to general cancer cells. Therefore, this hypothesis could suggest a new concept of tumor research, the development of cancer stem cell specific anticancer agent.

However, studies on the isolation of cancer stem cells and their biological mechanisms are still in its infancy, and the markers of cancer stem cells to date are limited by the markers of known stem cells. This results in a problem that the cancer stem cells occupy a very small portion of the total cancer cells, but an excessive number of cells having a marker factor that is considered to be cancer stem cells in a particular cancer. Therefore, the establishment of cancer stem cell specific markers itself should be preceded by the development of cancer stem cell specific anticancer agents.

In addition, the most important thing in developing cancer stem cell-specific therapies is to analyze the biological characteristics of cancer stem cells in addition to separating cancer stem cells. If it is possible to isolate cancer stem cells from a large number of patients and characterize common cancer stem cells, we can develop specific therapies for cancer stem cells that are resistant to anticancer drugs and radiation therapy and dramatically improve the therapeutic effects of cancer patients. It is expected to be able to improve.

Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior arts, using two-dimensional electrophoresis to identify marker proteins that decrease and increase in comparison with normal cancer cells in cancer stem cells, and complete the present invention. It became.

Accordingly, the main object of the present invention is to apply prostate cancer stem cell selection protein for prostate cancer treatment centered on prostate cancer stem cells through protein research that can identify the characteristic differences between prostate cancer stem cells and general prostate cancer cells To provide sex markers.

Another object of the present invention is to provide a composition for selecting prostate cancer stem cells comprising a substance for detecting a change in the proteinaceous marker.

Another object of the present invention is to provide a prostate cancer stem cell selection kit comprising the composition for selecting prostate cancer stem cells.

Another object of the present invention is to provide a method for detecting the proteinaceous marker for selecting prostate cancer stem cells.

Another object of the present invention is to provide a method for screening a prostate cancer stem cell inhibitor using the proteinaceous marker.

According to one aspect of the invention, the invention provides a proteinaceous marker for prostate cancer stem cell selection, characterized in that one or more combinations selected from the group consisting of the following polypeptides:

KH-type splicing regulatory protein having an amino acid sequence of SEQ ID NO: 1 and having a molecular weight of 72-74 kD;

Isoform alpha-enolase of Alpha-enolase having the amino acid sequence of SEQ ID NO: 2 and having a molecular weight of 46-48 kD;

Annexin A5 having the amino acid sequence of SEQ ID NO: 3 and having a molecular weight of 35-37 kD;

60S acidic ribosomal protein P0 having the amino acid sequence of SEQ ID NO: 4 and having a molecular weight of 33-35 kD;

Osteoclast-stimulating factor 1 having an amino acid sequence of SEQ ID NO: 5 and a molecular weight of 23-25 kD;

Cofilin-1 having the amino acid sequence of SEQ ID NO: 6 and having a molecular weight of 18-20 kD;

Thioredoxin domain-containing protein 12 having the amino acid sequence of SEQ ID NO: 7 and having a molecular weight of 18-20 kD;

Prefoldin subunit 5 having the amino acid sequence of SEQ ID NO: 8 and having a molecular weight of 16-18 kD;

Hornerin having the amino acid sequence of SEQ ID NO: 9 and having a molecular weight of 281-283 kD;

COP9 signalosome complex subunit with amino acid sequence of SEQ ID NO: 10 and molecular weight of 22-24 kD 8.

FK506-binding protein 4 having the amino acid sequence of SEQ ID NO: 11 and having a molecular weight of 51-53 kD;

Crk-like protein having an amino acid sequence of SEQ ID NO: 12 and having a molecular weight of 33-35 kD;

Isoform 1 of Pyruvate dehydrogenase El component subunit beta, mitochondrial having an amino acid sequence of SEQ ID NO: 13 and having a molecular weight of 38-40 kD;

Thioredoxin-dependent peroxide reductase, mitochondrial having an amino acid sequence of SEQ ID NO: 14 and having a molecular weight of 27-29 kD;

Isoform 1 of Huntingtin-interacting protein K having the amino acid sequence of SEQ ID NO: 15 and having a molecular weight of 18-20 kD; And

Isoform Non-muscle of Myosin light polypeptide having an amino acid sequence of SEQ ID NO: 16 and a molecular weight of 16-18 kD. 6.

The present inventors analyzed the proteome of prostate cancer stem cells and prostate cancer cells to detect proteins whose expression increases or decreases in prostate cancer stem cells in comparison with prostate cancer cells, and can be used for the selection of prostate cancer stem cells. Markers were excavated.

As used herein, the term 'proteome' refers to the totality of all proteins that can be made from the genome, so that the pattern of the proteome always changes depending on the specific physiological or pathological condition in a cell or tissue. Being a dynamic concept. Proteomics is a generic term for methods and techniques for studying these proteomes. Cellular cells are studied by studying the properties of proteins, focusing on gene expression, post-translational modification, and binding to other proteins. It refers to the field of research that you want to understand holistically by linking my transformation process and network formation with the course of disease. As such, the proteome represents a physiological or pathological condition in a cell or tissue, and thus is most suitable as a method of finding a diagnostic marker that can be directly used for diagnosis of a disease. In addition, in the case of cancer stem cells, if expression of a specific gene is found to be involved in the proliferation and differentiation of cancer stem cells to promote cancer recurrence, the presence of the protein is confirmed and identified as a target protein for cancer stem cell inhibitor development. It can also be. The development of diagnostics and therapeutics using them has been progressing due to the superior sensitivity of Genomics and the easy amplification of genes, but changes in DNA or mRNA levels do not directly lead to changes in proteins that are actually active in cells. Theoretical problems remain in that it is not possible, and in reality, proteomics is the only research method for body fluids without genetic material. Currently, non-invasive approaches are used for diagnosis of body fluids such as plasma, serum, urine, cerebrospinal fluid, amniotic fluid, and secretion, and many researchers use proteomic methods to identify disease-specific proteins as diagnostic markers. I introduce it.

As used herein, the term "proteinaceous marker for prostate cancer stem cell selection" refers to a substance which is a protein as a major component among the substances which are the criteria for distinguishing prostate cancer stem cells from prostate cancer cells. In the present invention, the proteinaceous marker is characterized by a higher or lower amount in the early prostate cancer stem cells compared to the amount in the prostate cancer cells.

Since the proteome of prostate cancer stem cells is analyzed with the proteome of prostate cancer cells, the protein markers thus identified can be said to be specific for prostate cancer stem cells, and thus are useful for selecting prostate cancer stem cells. Can be used. Furthermore, given that the proteinaceous markers thus identified are prominent in prostate cancer stem cells, the physiological function of the proteinaceous markers may be directly related to the proliferation and differentiation of prostate cancer stem cells. Therefore, this protein marker may be useful as a target protein for studying the mechanism of the development or recurrence of prostate cancer or for the development of a prostate cancer therapeutic agent targeting prostate cancer stem cells.

In particular, since the proteomic markers for selecting prostate cancer stem cells of the present invention are found at the proteome level in actual prostate cancer stem cells, they are more clinically compared to the specific markers of prostate cancer stem cells found at conventional DNA or mRNA levels. These markers are highly useful markers and are very useful as targets for inhibitors of prostate cancer stem cells because they are limited to prostate cancer stem cells.

In the present invention, the proteinaceous marker may be part of the full-length protein having the sequence number as long as it has the molecular weight, in which case it has trypsin digested peptide sequences shown in red in the MS / MS results of FIGS. The trypsin digested peptide refers to a peptide of protein digested with trypsin for MALDI-TOF or MS / MS analysis as a method of proteome analysis. The molecular weight also includes a generally accepted experimental error range as a value identified on two-dimensional electrophoresis.

According to another aspect of the invention, the present invention is a prostate cancer stem cell selection proteinaceous marker, prostate cancer stem cells comprising a substance for detecting the presence, the amount, pattern or both of the presence of the proteinaceous marker It provides a composition for screening.

In the composition for screening prostate cancer stem cells of the present invention, the presence or absence of the proteinaceous marker for prostate cancer stem cell selection and the specific detection of the amount or pattern thereof are selected for prostate cancer stem cell selection proteinaceous markers in a biological sample. Means whether the presence and amount and pattern, for example, using the antibody that specifically binds to the proteinaceous marker for the prostate cancer stem cell selection to determine the presence and the amount or pattern Verification can be used. As used herein, the term “biological sample” refers to a cell capable of detecting the presence and amount or pattern of a proteinaceous marker for prostate cancer stem cell selection, or the expression and amount or expression pattern of a gene related to the proteinaceous marker. I mean organization. In addition, in the present specification, "antibody" refers to a protein that can specifically bind to epitopes of antigens, and is a concept including both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

In the present invention, the polyclonal antibody may be prepared by injecting an antigenic proteinaceous marker or a fragment having the epitope 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.

In addition, the monoclonal antibodies can be prepared by the technology of generating 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 the 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 the ELISA method is used to select and propagate only hybridoma cells that produce the desired monoclonal antibody. The monoclonal antibody is isolated and purified from the culture.

Methods for measuring the presence, amount or pattern of the protein using the antibody include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, But are not limited to, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, complement fixation assays, FACS, and protein chips. no.

Through these analytical methods, it is possible to compare the amount of antigen-antibody complexes formed in biological samples of prostate cancer patients with the amount of antigen-antibody complexes formed in biological samples of prostate cancer stem cells, thereby prostate cancer of the present invention. The presence or absence and the amount or pattern of proteinaceous markers for stem cell selection can be determined, and ultimately, prostate cancer stem cells can be selected from tissues of prostate cancer patients.

Here, the term “antigen-antibody complex” means a combination of a proteinaceous marker for prostate cancer stem cell selection and an antibody specific thereto. The amount or pattern of formation of the antigen-antibody complex is usually a detection label (linked to a secondary antibody ( Measurement is possible by detecting the magnitude and pattern of the signal of the detection label. Such detection labels include, but are not limited to, enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, radioisotopes, and the like. When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase, luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate deca Carboxylase, β-latamase, and the like, but are not limited thereto. When fluorescent materials are used as detection labels, available fluorescent materials include fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, etc. However, this is not limiting. When a ligand is used as a detection label, available ligands include, but are not limited to, biotin derivatives. When a luminescent material is used as the detection label, available luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. When the microparticles are used as the detection label, the microparticles available include, but are not limited to, colloidal gold and colored latex. When redox molecules are used as detection labels, the available redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W (CN ) ₈ , [Os (bpy) ₃ ] ²⁺ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, etc., but are not limited to these. When radioisotopes are used as detection labels, the available radioisotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ^131. I or ¹⁸⁶ Re and the like, but is not limited thereto.

In the composition for screening prostate cancer stem cells of the present invention, the "substance that specifically detects the amount and pattern or the presence of proteinaceous markers for prostate cancer stem cell selection" is the presence and amount of the corresponding proteinaceous markers Or any substance that can be used specifically for the proteinaceous marker in the assay method for detecting the pattern, but is not necessarily limited to antibodies. Since the composition for screening the prostate cancer stem cell of the present invention has technical characteristics in detecting the difference between the presence and amount or pattern of the proteinaceous marker in prostate cancer stem cells and prostate cancer cells, Any substance that enables the presence and amount or pattern thereof may be used as "a substance which specifically detects the presence and amount or pattern of proteinaceous markers for selection of prostate cancer stem cells" and the object of the invention Technical effects can be achieved, and a person skilled in the art will be able to select and use a suitable material without particular difficulty according to specific embodiments with reference to average knowledge and well-known techniques in the art.

According to another aspect of the invention, the present invention provides a composition for screening prostate cancer stem cells comprising a substance for detecting the expression of the gene encoding the proteinaceous marker, and the amount of expression, expression pattern or both. .

In the composition for screening prostate cancer stem cells of the present invention, the presence and amount or pattern of the proteinaceous markers, in addition to using the method for detecting the protein itself, includes prostate cancer stem cells and prostate cancer cells. By detecting the expression of the gene encoding the proteinaceous marker in the sample or tissue and the expression amount or expression pattern thereof, a method of inferring the presence or absence and the amount or pattern of the proteinaceous marker may be used.

Here, whether the gene is expressed and the amount of expression or expression pattern detection can be generally performed by a process of confirming the presence and amount or pattern of mRNA generated by the transcription of the gene. Assays to determine the presence and amount or pattern of these mRNAs include RT-PCR, competitive RT-PCR, Real-time RT-PCR, and RNase protection assays. RNase protection assays, northern blots, DNA chips, and the like, but is not limited thereto.

Through these assay methods, the presence and amount or pattern of mRNA in a biological sample of a prostate cancer patient can be compared with the amount and pattern or presence of mRNA in prostate cancer stem cells, from which the prostate of the present invention It is possible to determine whether the gene encoding the proteinaceous marker for cancer stem cell selection and the difference in the expression amount or expression pattern, and ultimately to select prostate cancer stem cells in the tissue of the prostate cancer patient.

In the composition for selecting prostate cancer stem cells of the present invention, the kit for measuring the presence and amount or pattern of the mRNA by RT-PCR is specific for the mRNA for the proteinaceous marker for selecting prostate cancer stem cells of the present invention Primers. As used herein, a “primer” is a nucleic acid having a free 3 ′ hydroxyl group capable of complementarily binding to a template and allowing reverse transcriptase or DNA polymerase to initiate replication of the template. Refers to the sequence. A primer is a nucleotide having a sequence complementary to the nucleic acid sequence of a particular gene, and may be used in a length of about 7 bp to 50 bp, preferably about 10 bp to 30 bp. Other RT-PCR kits may include test tubes or other suitable containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC-water (DEPCwater), depending on the specific embodiment. , Sterile water and the like. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. The primer may also include additional other base sequences that do not change the basic properties of the primer that serve as the starting point for DNA synthesis. Primers can be chemically synthesized using well known methods, and such nucleic acid sequences can also be modified using many means known in the art.

In the composition for screening prostate cancer stem cells of the present invention, the "substance that specifically detects the expression of the gene encoding the proteinaceous marker for prostate cancer stem cell selection and its expression amount or expression pattern" is the corresponding proteinaceous marker. It may be any substance that can be used specifically for the proteinaceous marker in the expression method and the expression amount or expression pattern analysis of the gene encoding the, and is not necessarily limited to the primer for RT-PCR. The composition for screening prostate cancer stem cells of the present invention is the expression of a gene encoding a proteinaceous marker for prostate cancer stem cell selection and its expression amount or expression pattern in biological samples of prostate cancer patients and biological samples of prostate cancer stem cells. Since there is a technical characteristic in detecting differences, any substance that enables detection of such differences may be described as “the expression of the gene encoding the proteinaceous marker for prostate cancer stem cell selection and its expression amount or expression pattern. It can be used as a "specifically detectable substance" and the invention can achieve the desired technical effect, those skilled in the art will be able to select and use a suitable substance according to the specific embodiments with reference to the average knowledge in the art.

According to another aspect of the present invention, the present invention provides a kit for selecting prostate cancer stem cells comprising the composition for selecting prostate cancer stem cells.

Prostate cancer stem cell selection kit of the present invention, a substance that specifically detects the presence and amount or pattern of prostate cancer stem cell selection protein markers included in the composition for screening prostate cancer stem cells, or prostate In addition to the expression of the gene encoding the proteinaceous marker for cancer stem cell selection and the substance that specifically detects the expression amount or expression pattern, the presence or absence of the protein and the method of analyzing the amount or pattern or the expression of the gene and its expression amount or One or more other components, solutions or devices suitable for the method of analyzing the expression pattern may be further included. For example, if the diagnostic kit is a diagnostic kit for detecting the presence of the protein and its amount or pattern, the diagnostic kit may be, for example, a diagnostic kit including essential components necessary for performing an ELISA. Such ELISA kits include components capable of detecting bound antibodies, such as labeled secondary antibodies, chromopores, enzymes (eg, enzymes conjugated with antibodies) and substrates thereof, and quantitative control proteins. It may include an antibody specific to, and the like. On the other hand, when the diagnostic kit is a diagnostic kit for detecting the expression of the gene and its expression amount or expression pattern, the diagnostic kit may be a diagnostic kit containing the essential components necessary to perform RT-PCR, These RT-PCR kits can be used in addition to individual primers specific for the marker gene, depending on the specific embodiment, such as, for example, test tubes or other suitable containers, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases. Enzymes, DNAse, RNAse inhibitor DEPC-water (DEPCwater), sterile water, primer pairs specific for the gene used as a quantitative control, and the like. In addition, according to a specific embodiment, the prostate cancer stem cell selection kit may include a DNA chip or a protein chip.

According to another aspect of the present invention, the present invention provides a method for detecting a protein marker for selecting prostate cancer stem cells of the present invention in order to provide information necessary for selection of prostate cancer stem cells.

The method for detecting the prostate cancer stem cell selection proteinaceous marker, the prostate cancer stem cell selection composition of the present invention is treated to a biological sample collected from a patient with prostate cancer, the prostate cancer stem of the present invention from the treatment result It can be carried out by detecting the presence of the proteinaceous marker for cell selection and the difference in the amount or pattern thereof. In addition, the composition for screening prostate cancer stem cells of the present invention is treated to a biological sample collected from a patient with prostate cancer, and the results of the treatment indicate whether the gene encoding the proteinaceous marker for prostate cancer stem cell selection of the present invention and It can be carried out by detecting the difference in the expression amount or expression pattern.

According to another aspect of the present invention, the present invention provides a method of treating a protein and a cell expressing a protein marker, the method comprising the steps of treating the test compound and whether the test compound promotes or inhibits the physiological activity of the protein marker. It provides a method for screening a prostate cancer stem cell inhibitor comprising the step.

Since the proteinaceous marker for prostate cancer stem cell selection is prominent in the prostate cancer stem cells, the proteomic marker may be a protein directly involved in proliferation and differentiation of prostate cancer stem cells, and thus, the mechanism of prostate cancer recurrence It can be usefully used as a target protein for the development of inhibitors of prostate cancer stem cells. That is, since the proteinaceous marker for prostate cancer stem cell selection of the present invention solves an important premise of prostate cancer treatment, a method for screening a prostate cancer therapeutic agent using this proteinaceous marker also belongs to the scope of the present invention.

Samples containing test compounds for use in the method of screening the inhibitors include, but are not limited to, tissue extracts, expression products of gene libraries, synthetic compounds, synthetic peptides, natural compounds, and the like.

As described above, according to the present invention, a prostate cancer stem cell selection composition comprising a proteinaceous marker for selecting prostate cancer stem cells and a substance for detecting a change in the proteinaceous marker, and a composition for selecting prostate cancer stem cells. A prostate cancer stem cell selection kit, a method for detecting the prostate cancer stem cell selection proteinaceous marker, and a prostate cancer stem cell inhibitor screening method using the proteinaceous marker is provided.

FIG. 1 is a graph showing the separation of CD44 positive cells and CD44 negative cells using magnetic cell sorting, followed by flow cytometry.
Figure 2 is an image showing a protein expressing CD44 positive cells using two-dimensional electrophoresis (two-dimensional electrophoresis).
Figure 3 is an image showing a protein expressing CD44 negative cells using two-dimensional electrophoresis.
4A is a western blot image showing that Cofilin and Annexin A5 proteins have increased expression levels in CD44 positive cells.
Figure 4b is a graph comparing the expression level of Cofilin in CD44 positive cells and CD44 negative cells.
Figure 4c is a graph comparing the expression level of Annexin A5 in CD44 positive cells and CD44 negative cells.
5 to 20 show the results of identifying proteins expressed differently in prostate cancer stem cells and prostate cancer general cells using ESI-Q-TOF / MS / MS. Red in the figure represents the sequence of matched trypsin digesting peptides.

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

Example 1. Sample preparation

Human prostate cancer cell line DU145 was purchased from Korea Cell Line Bank and cultured using GIBCO's RPMI1640 medium. When the cells grew in the culture dish more than 80% was collected and put into a lysis buffer (lysis buffer) to keep the cold state and the cells were broken down by using a sonicator (sonicator). Five times the volume of acetone of the lysis buffer containing crushed cells was added so that there was no lump, and the mixture was cooled by shaking at -20 ° C for 150 minutes. The protein precipitated at -20 ° C was rotated 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. Isolation of CD44 +, CD44- Cells

Human prostate cancer cell line DU145 was cultured using RPMI1640 medium of GIBCO. When the cells grow in the culture dish more than 80% was collected and precipitated at 1400 g for 5 minutes in a centrifuge and the medium was removed. The phosphate buffered saline (PBS) pH 7.2, 0.5% bovine serum albumin (BSA) and 2 mM EDTA MACS buffer solution were diluted 1:20 with autoMACS Rinsing solution (Miltenyi Biotec, CA, USA). The cells were washed twice with this dilution solution, and then reacted with 30 μl of CD44 antibody at 4 ° C. for 15 minutes. The cells were washed twice with 1 ml of MACS buffer solution and reacted with 30 μl Anti-PE microbeads for 10 minutes at 4 ° C. Cells were washed with MAC buffer solution and LS column was installed in the magnetic field of MACS Separator. After the cells were suspended in 500 μl of MACS buffer solution, the loaded CD44 negative cells were slowly loaded into the column and received in the tube. After passing 3 ml of MACS buffer solution three more times, the column was separated from the Separator to load 5 ml of MACS buffer solution, and pressed with a piston to receive CD44 positive cells in the tube.

The isolated CD44 positive cells and CD44 negative cells were immediately confirmed in flow cytometry, and as shown in the graph of FIG. The first graph is DU145 cells without antibody reaction, corresponding to the negative control. CD44 positive cells appear in a second graph measured at the wavelength at which PE is absorbed, confirming that the isolation was successful at a high rate of 84.8%. In the third graph, CD44 negative cells were also successfully separated at a high rate of 84.07%.

Example 2 Two-Dimensional Electrophoresis

2-1. 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 M urea, 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, pH 4 Drystrip (Amerahsm Pharmacia Biotech, Uppsala, Sweden) in the -7 range was mounted in a strip holder. The strip was soaked in rehydration buffer for 5 hours at 20 ° C and subjected to one-dimensional electrophoresis under the following conditions: 200 V 30 minutes, 500 V 1 minute, 8,000 V 45 minutes, 8,000 V 72000 Vhrs, 11 30 minutes. 146 kVhr total. One-dimensional electrophoresis was performed with IPGphore (Amerahsm Pharmacia Biotech, Uppsala, Sweden).

2-2. Two-dimensional Electrophoresis (SDS-PAGE)

For two-dimensional electrophoresis of one-dimensional electrophoretic strips, place the one-dimensional electrophoretic strips in a cap tube and place the primary equilibration solution (6 M urea, 50 mM Tris-cl (pH 8.8), 30% glycerol). , 2% SDS, 1% DTT) in 10 ml for 15 minutes and discard, again equilibrate with secondary equilibration solution (6 M urea, 50 mM Tris-cl (pH 8.8), 30% glycerol, 2% SDS, 1.5% IAA) 10 ml was added and re-reacted for 10 minutes. The reaction strip was mounted on a polyacrylamide gel (polyacrylamide 25 × 30 cm) 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.

2-3. Staining

After two-dimensional electrophoresis, silver staining was performed to visualize the gel. The silver staining process is as follows. Fix by reacting with a solution of 50% methanol and 12% acetic acid for 12 hours, rinsing three times with 50% ethanol for 20 minutes, and sensitizing by reacting with 0.2% sodium thiosulfate solution for 1 minute. ), Distilled water (DW), 0.1% silver nitrate, 0.075% formaldehyde (37%) solution for 20 minutes to react (Improving), washed again with distilled water, The reaction was stopped by reacting with cold 6% sodium carbonate, 0.075% formaldehyde (37%) solution for about 7 minutes, and treated with 50% methanol and 12% acetic acid solution to stop the reaction.

2-4. Image Analysis

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

2 is an image analysis result showing a protein expressing CD44 positive cells, Figure 3 is an image analysis result showing a protein expressing CD44 negative cells. The differences in expression in these results were analyzed below.

Example 3. Mass Spectrometry (ESI-Q-TOF / MS / MS Analysis)

In order to identify the spots found with the image analysis program, the spots were cut out from the gel after two-dimensional electrophoresis. First, 100 μl of a destaining solution mixed with 30 mM Potassium Ferricyanide and 100 mM Sodium Thiosulfate solution in a 1: 1 ratio was applied to the gel pieces for 5 minutes to remove silver. It was then washed three times with 400 μl of water.

The reaction was again performed with 200 mM ammonium bicarbonate and washed with water. Acetonitrile was added and dehydrated until the gel turned white. 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 hydrocarbon containing 0.2 μg of trypsin (promega). After removing the reaction solution was added 30 μl of 50 mM ammonium hydrocarbon and reacted overnight at 37 ℃. 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 were 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 is 80 ° C. A voltage of 1 kV 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) Applied to. 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 sequences of proteins expressed differently in prostate cancer stem cells and prostate cancer general cells were confirmed using ESI-Q-TOF / MS / MS, and the results are shown in FIGS. 5 to 20.

Example 4. Western blotting

4-1. SDS-PAGE Electrophoresis

Human colon cancer cell line DU145 isolated from CD44 positive and CD44 negative cells was subjected to 10% SDS-PAGE electrophoresis using a magnetic cell sorting system. Mix 15 μl of serum sample (20 μg of protein) with 4 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 molecular weight (loading at 120 V on a separating gel) in a mini gel (6 × 8 cm). The composition of the running buffer used was 0.025 M Tris-Cl, 0.192 M Glycine, 1% SDS (pH 8.3).

4-2. Blotting

After the SDS-PAGE, the gel protein was transferred to a NitroCellulose membrane using a semi-dry transfer kit (Bio-rad). The gel was carried out for 1 hour 30 minutes at 50 mA per gel.

4-3. Antibody reaction

Cofilin and Annexin A5 were performed. The membrane was blocked with 5% w / v nonfat dry milk for 1 hour at room temperature. For the measurement of Cofilin and Annexin A5, monoclonal antibodies Cofilin and Annexin A5 were used as primary antibodies, and the antibodies of Cofilin and Annexin A5 were reacted at 4 ° C. for 24 hours. The dilution concentration of the primary antibody is 1/500. After washing with TBS / T (0.1%, Tris-buffered saline-tween 20) three times for 10 minutes, the reaction was performed for 1 hour at room temperature with a secondary antibody. Secondary antibody was used anti-goat, dilution concentration was 1 / 5,000 anti-goat. After the secondary antibody reaction was washed with TBS / T three times for 15 minutes, and then visualized by ECL (Pierce Biotechnology, Inc., USA).

As a result of performing the two-dimensional electrophoresis experiment as described above, proteins newly expressed in CD44 positive cells or decreased and increased in expression amount were identified in CD44 negative cells as follows.

1) Protein with increased or decreased expression in prostate cancer stem cells

Electrophoresis showed 10 increased proteins (# 227, # 1089, # 1265, # 1293, # 1373, # 1530, # 1540, # 1570, # 1618, # 1737) and 6 decreasing proteins (# 588, # 1083, # 1201, # 1483, # 1578, # 1723) could be found (FIGS. 2A-3B). The reduced expression of the protein was reduced by about 50% or more than that of CD44 negative cells, and the increased protein was increased by about 100% or more. As a result of performing ESI-Q-TOF, proteins with reduced and increased expression in CD44 positive cells were identified as shown in Table 2 below.

2) Determination of the amino acid sequence of the protein

As described above, proteins expressed differently from prostate cancer stem cells and prostate cancer general cells were identified using ESI-Q-TOF / MS / MS, thereby obtaining the results of FIGS. 5 to 20. Red in the figure represents the sequence of matched trypsin digesting peptides. The amino acid sequences of these proteins were searched in the NCBInr database (http://www.ncbi.nlm.nih.gov/) to search for KH-type splicing regulatory protein (SEQ ID NO: 1), Isoform alpha-enolase of Alpha-enolase (SEQ ID NO: 2), Annexin A5 (SEQ ID NO: 3), 60S acidic ribosomal protein P0 (SEQ ID NO: 4), Osteoclast-stimulating factor 1 (SEQ ID NO: 5), Cofilin-1 (SEQ ID NO: 6), Thioredoxin domain-containing protein 12 (SEQ ID NO: 2) Number 7), Prefoldin subunit 5 (SEQ ID NO: 8), Hornerin (SEQ ID NO: 9), COP9 signalosome complex subunit 8 (SEQ ID NO: 10), FK506-binding protein 4 (SEQ ID NO: 11), Crk-like protein (SEQ ID NO: 12) ), Isoform 1 of Pyruvate dehydrogenase E1 component subunit beta, mitochondrial (SEQ ID NO: 13), Thioredoxin-dependent peroxide reductase, mitochondrial (SEQ ID NO: 14), Isoform 1 of Huntingtin-interacting protein K (SEQ ID NO: 15), Isoform Non-muscle of Myosin light polypeptide 6 (SEQ ID NO: 16) was confirmed to have.

3) Western blotting test result

Among the proteins whose expression was changed, Western blot experiment results of Cofilin (spot No. 1265) and Annexin A5 (spot No. 1530), which are proteins of increased expression in prostate cancer stem cells, are shown in FIG. And FIG. 4C. As shown in the results, the amount of Cofilin and Annexin A5 was significantly increased in prostate cancer stem cells. These results were the same as the results of the second electrophoresis. These results suggest that Cofilin and Annexin A5 may be useful for the diagnosis of prostate cancer and the study of prostate cancer stem cells.

<110> Korea University Industrial and Academic Collaboration Foundation <120> Proteinic markers for diagnosing prostate cancer stem cells <160> 16 <170> Kopatentin 1.71 <210> 1 <211> 711 <212> PRT <213> Homo sapiens <400> 1 Met Ser Asp Tyr Ser Thr Gly Gly Pro Pro Pro Gly Pro Pro Pro Pro   1 5 10 15 Ala Gly Gly Gly Gly Gly Ala Gly Gly Ala Gly Gly Gly Pro Pro Pro              20 25 30 Gly Pro Pro Gly Ala Gly Asp Arg Gly Gly Gly Gly Pro Gly Gly Gly          35 40 45 Gly Pro Gly Gly Gly Ser Ala Gly Gly Pro Ser Gln Pro Pro Gly Gly      50 55 60 Gly Gly Pro Gly Ile Arg Lys Asp Ala Phe Ala Asp Ala Val Gln Arg  65 70 75 80 Ala Arg Gln Ile Ala Ala Lys Ile Gly Gly Asp Ala Ala Thr Thr Val                  85 90 95 Asn Asn Ser Thr Pro Asp Phe Gly Phe Gly Gly Gln Lys Arg Gln Leu             100 105 110 Glu Asp Gly Asp Gln Pro Glu Ser Lys Lys Leu Ala Ser Gln Gly Asp         115 120 125 Ser Ile Ser Ser Gln Leu Gly Pro Ile His Pro Pro Pro Arg Thr Ser     130 135 140 Met Thr Glu Glu Tyr Arg Val Pro Asp Gly Met Val Gly Leu Ile Ile 145 150 155 160 Gly Arg Gly Gly Glu Gln Ile Asn Lys Ile Gln Gln Asp Ser Gly Cys                 165 170 175 Lys Val Gln Ile Ser Pro Asp Ser Gly Gly Leu Pro Glu Arg Ser Val             180 185 190 Ser Leu Thr Gly Ala Pro Glu Ser Val Gln Lys Ala Lys Met Met Leu         195 200 205 Asp Asp Ile Val Ser Arg Gly Arg Gly Gly Pro Pro Gly Gln Phe His     210 215 220 Asp Asn Ala Asn Gly Gly Gln Asn Gly Thr Val Gln Glu Ile Met Ile 225 230 235 240 Pro Ala Gly Lys Ala Gly Leu Val Ile Gly Lys Gly Gly Glu Thr Ile                 245 250 255 Lys Gln Leu Gln Glu Arg Ala Gly Val Lys Met Ile Leu Ile Gln Asp             260 265 270 Gly Ser Gln Asn Thr Asn Val Asp Lys Pro Leu Arg Ile Ily Gly Asp         275 280 285 Pro Tyr Lys Val Gln Gln Ala Cys Glu Met Val Met Asp Ile Leu Arg     290 295 300 Glu Arg Asp Gln Gly Gly Phe Gly Asp Arg Asn Glu Tyr Gly Ser Arg 305 310 315 320 Ile Gly Gly Gly Ile Asp Val Pro Val Pro Arg His Ser Val Gly Val                 325 330 335 Val Ile Gly Arg Ser Gly Glu Met Ile Lys Lys Ile Gln Asn Asp Ala             340 345 350 Gly Val Arg Ile Gln Phe Lys Gln Asp Asp Gly Thr Gly Pro Glu Lys         355 360 365 Ile Ala His Ile Met Gly Pro Pro Asp Arg Cys Glu His Ala Ala Arg     370 375 380 Ile Ile Asn Asp Leu Leu Gln Ser Leu Arg Ser Gly Pro Pro Gly Pro 385 390 395 400 Pro Gly Gly Pro Gly Met Pro Pro Gly Gly Arg Gly Arg Gly Arg Gly                 405 410 415 Gln Gly Asn Trp Gly Pro Pro Gly Gly Glu Met Thr Phe Ser Ile Pro             420 425 430 Thr His Lys Cys Gly Leu Val Ile Gly Arg Gly Gly Glu Asn Val Lys         435 440 445 Ala Ile Asn Gln Gln Thr Gly Ala Phe Val Glu Ile Ser Arg Gln Leu     450 455 460 Pro Pro Asn Gly Asp Pro Asn Phe Lys Leu Phe Ile Ile Arg Gly Ser 465 470 475 480 Pro Gln Gln Ile Asp His Ala Lys Gln Leu Ile Glu Glu Lys Ile Glu                 485 490 495 Gly Pro Leu Cys Pro Val Gly Pro Gly Pro Gly Gly Pro Gly Pro Ala             500 505 510 Gly Pro Met Gly Pro Phe Asn Pro Gly Pro Phe Asn Gln Gly Pro Pro         515 520 525 Gly Ala Pro Pro His Ala Gly Gly Pro Pro Pro His Gln Tyr Pro Pro     530 535 540 Gln Gly Trp Gly Asn Thr Tyr Pro Gln Trp Gln Pro Pro Ala Pro His 545 550 555 560 Asp Pro Ser Lys Ala Ala Ala Ala Ala Ala Asp Pro Asn Ala Ala Trp                 565 570 575 Ala Ala Tyr Tyr Ser His Tyr Tyr Gln Gln Pro Pro Gly Pro Val Pro             580 585 590 Gly Pro Ala Pro Ala Pro Ala Ala Pro Pro Ala Gln Gly Glu Pro Pro         595 600 605 Gln Pro Pro Pro Thr Gly Gln Ser Asp Tyr Thr Lys Ala Trp Glu Glu     610 615 620 Tyr Tyr Lys Lys Ile Gly Gln Gln Pro Gln Gln Pro Gly Ala Pro Pro 625 630 635 640 Gln Gln Asp Tyr Thr Lys Ala Trp Glu Glu Tyr Tyr Lys Lys Gln Ala                 645 650 655 Gln Val Ala Thr Gly Gly Gly Pro Gly Ala Pro Pro Gly Ser Gln Pro             660 665 670 Asp Tyr Ser Ala Ala Trp Ala Glu Tyr Tyr Arg Gln Gln Ala Ala Tyr         675 680 685 Tyr Gly Gln Thr Pro Gly Pro Gly Gly Pro Gln Pro Pro Pro Thr Gln     690 695 700 Gln Gly Gln Gln Gln Ala Gln 705 710 <210> 2 <211> 434 <212> PRT <213> Homo sapiens <400> 2 Met Ser Ile Leu Lys Ile His Ala Arg Glu Ile Phe Asp Ser Arg Gly   1 5 10 15 Asn Pro Thr Val Glu Val Asp Leu Phe Thr Ser Lys Gly Leu Phe Arg              20 25 30 Ala Ala Val Pro Ser Gly Ala Ser Thr Gly Ile Tyr Glu Ala Leu Glu          35 40 45 Leu Arg Asp Asn Asp Lys Thr Arg Tyr Met Gly Lys Gly Val Ser Lys      50 55 60 Ala Val Glu His Ile Asn Lys Thr Ile Ala Pro Ala Leu Val Ser Lys  65 70 75 80 Lys Leu Asn Val Thr Glu Gln Glu Lys Ile Asp Lys Leu Met Ile Glu                  85 90 95 Met Asp Gly Thr Glu Asn Lys Ser Lys Phe Gly Ala Asn Ala Ile Leu             100 105 110 Gly Val Ser Leu Ala Val Cys Lys Ala Gly Ala Val Glu Lys Gly Val         115 120 125 Pro Leu Tyr Arg His Ile Ala Asp Leu Ala Gly Asn Ser Glu Val Ile     130 135 140 Leu Pro Val Pro Ala Phe Asn Val Ile Asn Gly Gly Ser His Ala Gly 145 150 155 160 Asn Lys Leu Ala Met Gln Glu Phe Met Ile Leu Pro Val Gly Ala Ala                 165 170 175 Asn Phe Arg Glu Ala Met Arg Ile Gly Ala Glu Val Tyr His Asn Leu             180 185 190 Lys Asn Val Ile Lys Glu Lys Tyr Gly Lys Asp Ala Thr Asn Val Gly         195 200 205 Asp Glu Gly Gly Phe Ala Pro Asn Ile Leu Glu Asn Lys Glu Gly Leu     210 215 220 Glu Leu Leu Lys Thr Ala Ile Gly Lys Ala Gly Tyr Thr Asp Lys Val 225 230 235 240 Val Ile Gly Met Asp Val Ala Ala Ser Glu Phe Phe Arg Ser Gly Lys                 245 250 255 Tyr Asp Leu Asp Phe Lys Ser Pro Asp Asp Pro Ser Arg Tyr Ile Ser             260 265 270 Pro Asp Gln Leu Ala Asp Leu Tyr Lys Ser Phe Ile Lys Asp Tyr Pro         275 280 285 Val Val Ser Ile Glu Asp Pro Phe Asp Gln Asp Asp Trp Gly Ala Trp     290 295 300 Gln Lys Phe Thr Ala Ser Ala Gly Ile Gln Val Val Gly Asp Asp Leu 305 310 315 320 Thr Val Thr Asn Pro Lys Arg Ile Ala Lys Ala Val Asn Glu Lys Ser                 325 330 335 Cys Asn Cys Leu Leu Leu Lys Val Asn Gln Ile Gly Ser Val Thr Glu             340 345 350 Ser Leu Gln Ala Cys Lys Leu Ala Gln Ala Asn Gly Trp Gly Val Met         355 360 365 Val Ser His Arg Ser Gly Glu Thr Glu Asp Thr Phe Ile Ala Asp Leu     370 375 380 Val Val Gly Leu Cys Thr Gly Gln Ile Lys Thr Gly Ala Pro Cys Arg 385 390 395 400 Ser Glu Arg Leu Ala Lys Tyr Asn Gln Leu Leu Arg Ile Glu Glu Glu                 405 410 415 Leu Gly Ser Lys Ala Lys Phe Ala Gly Arg Asn Phe Arg Asn Pro Leu             420 425 430 Ala lys         <210> 3 <211> 320 <212> PRT <213> Homo sapiens <400> 3 Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp   1 5 10 15 Glu Arg Ala Asp Ala Glu Thr Leu Arg Lys Ala Met Lys Gly Leu Gly              20 25 30 Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala          35 40 45 Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp      50 55 60 Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu  65 70 75 80 Ile Val Ala Leu Met Lys Pro Ser Arg Leu Tyr Asp Ala Tyr Glu Leu                  85 90 95 Lys His Ala Leu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr Glu             100 105 110 Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln Val         115 120 125 Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp     130 135 140 Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn 145 150 155 160 Arg Asp Pro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala                 165 170 175 Gln Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu             180 185 190 Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys         195 200 205 Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr     210 215 220 Ile Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val 225 230 235 240 Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu Tyr                 245 250 255 Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg Val             260 265 270 Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg Lys Glu Phe         275 280 285 Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile Lys Gly Asp Thr     290 295 300 Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp 305 310 315 320 <210> 4 <211> 254 <212> PRT <213> Homo sapiens <400> 4 Met Pro Arg Glu Asp Arg Ala Thr Trp Lys Ser Asn Tyr Phe Leu Lys   1 5 10 15 Ile Ile Gln Leu Leu Asp Asp Tyr Pro Lys Cys Phe Ile Val Gly Ala              20 25 30 Asp Asn Val Gly Ser Lys Gln Met Gln Gln Ile Arg Met Ser Leu Arg          35 40 45 Gly Lys Ala Val Val Leu Met Gly Lys Asn Thr Met Met Arg Lys Ala      50 55 60 Ile Arg Gly His Leu Glu Asn Asn Pro Ala Leu Glu Lys Leu Leu Pro  65 70 75 80 Leu Arg Gly Asn Val Gly Phe Val Phe Thr Lys Glu Asp Leu Thr Glu                  85 90 95 Ile Arg Asp Met Leu Leu Ala Asn Lys Val Pro Ala Ala Ala Arg Ala             100 105 110 Gly Ala Ile Ala Pro Cys Glu Val Thr Val Pro Ala Gln Asn Thr Gly         115 120 125 Leu Gly Pro Glu Lys Thr Ser Phe Phe Gln Ala Leu Gly Ile Thr Thr     130 135 140 Lys Ile Ser Arg Gly Thr Ile Glu Ile Leu Gly Val Arg Asn Val Ala 145 150 155 160 Ser Val Cys Leu Gln Ile Gly Tyr Pro Thr Val Ala Ser Val Pro His                 165 170 175 Ser Ile Ile Asn Gly Tyr Lys Arg Val Leu Ala Leu Ser Val Glu Thr             180 185 190 Asp Tyr Thr Phe Pro Leu Ala Glu Lys Val Lys Ala Phe Leu Ala Asp         195 200 205 Pro Ser Ala Phe Val Ala Ala Ala Pro Val Ala Ala Ala Thr Thr Ala     210 215 220 Ala Pro Ala Ala Ala Ala Ala Ala Pro Ala Lys Val Glu Ala Lys Glu Glu 225 230 235 240 Ser Glu Glu Ser Asp Glu Asp Met Gly Phe Gly Leu Phe Asp                 245 250 <210> 5 <211> 214 <212> PRT <213> Homo sapiens <400> 5 Met Ser Lys Pro Pro Pro Lys Pro Val Lys Pro Gly Gln Val Lys Val   1 5 10 15 Phe Arg Ala Leu Tyr Thr Phe Glu Pro Arg Thr Pro Asp Glu Leu Tyr              20 25 30 Phe Glu Glu Gly Asp Ile Ile Tyr Ile Thr Asp Met Ser Asp Thr Asn          35 40 45 Trp Trp Lys Gly Thr Ser Lys Gly Arg Thr Gly Leu Ile Pro Ser Asn      50 55 60 Tyr Val Ala Glu Gln Ala Glu Ser Ile Asp Asn Pro Leu His Glu Ala  65 70 75 80 Ala Lys Arg Gly Asn Leu Ser Trp Leu Arg Glu Cys Leu Asp Asn Arg                  85 90 95 Val Gly Val Asn Gly Leu Asp Lys Ala Gly Ser Thr Ala Leu Tyr Trp             100 105 110 Ala Cys His Gly Gly His Lys Asp Ile Val Glu Met Leu Phe Thr Gln         115 120 125 Pro Asn Ile Glu Leu Asn Gln Gln Asn Lys Leu Gly Asp Thr Ala Leu     130 135 140 His Ala Ala Ala Trp Lys Gly Tyr Ala Asp Ile Val Gln Leu Leu Leu 145 150 155 160 Ala Lys Gly Ala Arg Thr Asp Leu Arg Asn Ile Glu Lys Lys Leu Ala                 165 170 175 Phe Asp Met Ala Thr Asn Ala Ala Cys Ala Ser Leu Leu Lys Lys Lys             180 185 190 Gln Gly Thr Asp Ala Val Arg Thr Leu Ser Asn Ala Glu Asp Tyr Leu         195 200 205 Asp Asp Glu Asp Ser Asp     210 <210> 6 <211> 166 <212> PRT <213> Homo sapiens <400> 6 Met Ala Ser Gly Val Ala Val Ser Asp Gly Val Ile Lys Val Phe Asn   1 5 10 15 Asp Met Lys Val Arg Lys Ser Ser Thr Pro Glu Glu Val Lys Lys Arg              20 25 30 Lys Lys Ala Val Leu Phe Cys Leu Ser Glu Asp Lys Lys Asn Ile Ile          35 40 45 Leu Glu Glu Gly Lys Glu Ile Leu Val Gly Asp Val Gly Gln Thr Val      50 55 60 Asp Asp Pro Tyr Ala Thr Phe Val Lys Met Leu Pro Asp Lys Asp Cys  65 70 75 80 Arg Tyr Ala Leu Tyr Asp Ala Thr Tyr Glu Thr Lys Glu Ser Lys Lys                  85 90 95 Glu Asp Leu Val Phe Ile Phe Trp Ala Pro Glu Ser Ala Pro Leu Lys             100 105 110 Ser Lys Met Ile Tyr Ala Ser Ser Lys Asp Ala Ile Lys Lys Lys Leu         115 120 125 Thr Gly Ile Lys His Glu Leu Gln Ala Asn Cys Tyr Glu Glu Val Lys     130 135 140 Asp Arg Cys Thr Leu Ala Glu Lys Leu Gly Gly Ser Ala Val Ile Ser 145 150 155 160 Leu Glu Gly Lys Pro Leu                 165 <210> 7 <211> 172 <212> PRT <213> Homo sapiens <400> 7 Met Glu Thr Arg Pro Arg Leu Gly Ala Thr Cys Leu Leu Gly Phe Ser   1 5 10 15 Phe Leu Leu Leu Val Ile Ser Ser Asp Gly His Asn Gly Leu Gly Lys              20 25 30 Gly Phe Gly Asp His Ile His Trp Arg Thr Leu Glu Asp Gly Lys Lys          35 40 45 Glu Ala Ala Ala Ser Gly Leu Pro Leu Met Val Ile Ile His Lys Ser      50 55 60 Trp Cys Gly Ala Cys Lys Ala Leu Lys Pro Lys Phe Ala Glu Ser Thr  65 70 75 80 Glu Ile Ser Glu Leu Ser His Asn Phe Val Met Val Asn Leu Glu Asp                  85 90 95 Glu Glu Glu Pro Lys His Glu Asp Phe Ser Pro Asp Gly Gly Tyr Ile             100 105 110 Pro Arg Ile Leu Phe Leu Asp Pro Ser Gly Lys Val His Pro Glu Ile         115 120 125 Ile Asn Glu Asn Gly Asn Pro Ser Tyr Lys Tyr Phe Tyr Val Ser Ala     130 135 140 Glu Gln Val Val Gln Gly Met Lys Glu Ala Gln Glu Arg Leu Thr Gly 145 150 155 160 Asp Ala Phe Arg Lys Lys His Leu Glu Asp Glu Leu                 165 170 <210> 8 <211> 154 <212> PRT <213> Homo sapiens <400> 8 Met Ala Gln Ser Ile Asn Ile Thr Glu Leu Asn Leu Pro Gln Leu Glu   1 5 10 15 Met Leu Lys Asn Gln Leu Asp Gln Glu Val Glu Phe Leu Ser Thr Ser              20 25 30 Ile Ala Gln Leu Lys Val Val Gln Thr Lys Tyr Val Glu Ala Lys Asp          35 40 45 Cys Leu Asn Val Leu Asn Lys Ser Asn Glu Gly Lys Glu Leu Leu Val      50 55 60 Pro Leu Thr Ser Ser Met Tyr Val Pro Gly Lys Leu His Asp Val Glu  65 70 75 80 His Val Leu Ile Asp Val Gly Thr Gly Tyr Tyr Val Glu Lys Thr Ala                  85 90 95 Glu Asp Ala Lys Asp Phe Phe Lys Arg Lys Ile Asp Phe Leu Thr Lys             100 105 110 Gln Met Glu Lys Ile Gln Pro Ala Leu Gln Glu Lys His Ala Met Lys         115 120 125 Gln Ala Val Met Glu Met Met Ser Gln Lys Ile Gln Gln Leu Thr Ala     130 135 140 Leu Gly Ala Ala Gln Ala Thr Ala Lys Ala 145 150 <210> 9 <211> 2850 <212> PRT <213> Homo sapiens <400> 9 Met Pro Lys Leu Leu Gln Gly Val Ile Thr Val Ile Asp Val Phe Tyr   1 5 10 15 Gln Tyr Ala Thr Gln His Gly Glu Tyr Asp Thr Leu Asn Lys Ala Glu              20 25 30 Leu Lys Glu Leu Leu Glu Asn Glu Phe His Gln Ile Leu Lys Asn Pro          35 40 45 Asn Asp Pro Asp Thr Val Asp Ile Ile Leu Gln Ser Leu Asp Arg Asp      50 55 60 His Asn Lys Lys Val Asp Phe Thr Glu Tyr Leu Leu Met Ile Phe Lys  65 70 75 80 Leu Val Gln Ala Arg Asn Lys Ile Ile Gly Lys Asp Tyr Cys Gln Val                  85 90 95 Ser Gly Ser Lys Leu Arg Asp Asp Thr His Gln His Gln Glu Glu Gln             100 105 110 Glu Glu Thr Glu Lys Glu Glu Asn Lys Arg Gln Glu Ser Ser Phe Ser         115 120 125 His Ser Ser Trp Ser Ala Gly Glu Asn Asp Ser Tyr Ser Arg Asn Val     130 135 140 Arg Gly Ser Leu Lys Pro Gly Thr Glu Ser Ile Ser Arg Arg Leu Ser 145 150 155 160 Phe Gln Arg Asp Phe Ser Gly Gln His Asn Ser Tyr Ser Gly Gln Ser                 165 170 175 Ser Ser Tyr Gly Glu Gln Asn Ser Asp Ser His Gln Ser Ser Gly Arg             180 185 190 Gly Gln Cys Gly Ser Gly Ser Gly Gln Ser Pro Asn Tyr Gly Gln His         195 200 205 Gly Ser Gly Ser Gly Gln Ser Ser Ser Asn Asp Thr His Gly Ser Gly     210 215 220 Ser Gly Gln Ser Ser Gly Phe Ser Gln His Lys Ser Ser Ser Gly Gln 225 230 235 240 Ser Ser Gly Tyr Ser Gln His Gly Ser Gly Ser Gly His Ser Ser Gly                 245 250 255 Tyr Gly Gln His Gly Ser Arg Ser Gly Gln Ser Ser Arg Gly Glu Arg             260 265 270 His Arg Ser Ser Ser Gly Ser Ser Ser Ser Tyr Gly Gln His Gly Ser         275 280 285 Gly Ser Arg Gln Ser Leu Gly His Gly Arg Gln Gly Ser Gly Ser Arg     290 295 300 Gln Ser Pro Ser His Val Arg His Gly Ser Gly Ser Gly His Ser Ser 305 310 315 320 Ser His Gly Gln His Gly Ser Gly Ser Ser Tyr Ser Tyr Ser Arg Gly                 325 330 335 His Tyr Glu Ser Gly Ser Gly Gln Thr Ser Gly Phe Gly Gln His Glu             340 345 350 Ser Gly Ser Gly Gln Ser Ser Gly Tyr Ser Lys His Gly Ser Gly Ser         355 360 365 Gly His Ser Ser Ser Gln Gly Gln His Gly Ser Thr Ser Gly Gln Ala     370 375 380 Ser Ser Ser Gly Gln His Gly Ser Ser Ser Arg Gln Ser Ser Ser Tyr 385 390 395 400 Gly Gln His Glu Ser Ala Ser Arg His Ser Ser Gly Arg Gly Gln His                 405 410 415 Ser Ser Gly Ser Gly Gln Ser Pro Gly His Gly Gln Arg Gly Ser Gly             420 425 430 Ser Gly Gln Ser Pro Ser Ser Gly Gln His Gly Thr Gly Phe Gly Arg         435 440 445 Ser Ser Ser Ser Gly Pro Tyr Val Ser Gly Ser Gly Tyr Ser Ser Gly     450 455 460 Phe Gly His His Glu Ser Ser Ser Glu His Ser Ser Gly Tyr Thr Gln 465 470 475 480 His Gly Ser Gly Ser Gly His Ser Ser Gly His Gly Gln His Gly Ser                 485 490 495 Arg Ser Gly Gln Ser Ser Arg Gly Glu Arg Gln Gly Ser Ser Ala Gly             500 505 510 Ser Ser Ser Ser Tyr Gly Gln His Gly Ser Gly Ser Arg Gln Ser Leu         515 520 525 Gly His Ser Arg His Gly Ser Gly Ser Gly Gln Ser Pro Ser Pro Ser     530 535 540 Arg Gly Arg His Glu Ser Gly Ser Arg Gln Ser Ser Ser Tyr Gly Pro 545 550 555 560 His Gly Tyr Gly Ser Gly Arg Ser Ser Ser Arg Gly Pro Tyr Glu Ser                 565 570 575 Gly Ser Gly His Ser Ser Gly Leu Gly His Gln Glu Ser Arg Ser Gly             580 585 590 Gln Ser Ser Gly Tyr Gly Gln His Gly Ser Ser Ser Gly His Ser Ser         595 600 605 Thr His Gly Gln His Gly Ser Thr Ser Gly Gln Ser Ser Ser Cys Gly     610 615 620 Gln His Gly Ala Thr Ser Gly Gln Ser Ser Ser His Gly Gln His Gly 625 630 635 640 Ser Gly Ser Ser Gln Ser Ser Arg Tyr Gly Gln Gln Gly Ser Gly Ser                 645 650 655 Gly Gln Ser Pro Ser Arg Gly Arg His Gly Ser Asp Phe Gly His Ser             660 665 670 Ser Ser Tyr Gly Gln His Gly Ser Gly Ser Gly Trp Ser Ser Ser Asn         675 680 685 Gly Pro His Gly Ser Val Ser Gly Gln Ser Ser Gly Phe Gly His Lys     690 695 700 Ser Gly Ser Gly Gln Ser Ser Gly Tyr Ser Gln His Gly Ser Gly Ser 705 710 715 720 Ser His Ser Ser Gly Tyr Arg Lys His Gly Ser Arg Ser Gly Gln Ser                 725 730 735 Ser Arg Ser Glu Gln His Gly Ser Ser Ser Gly Leu Ser Ser Ser Tyr             740 745 750 Gly Gln His Gly Ser Gly Ser His Gln Ser Ser Gly His Gly Arg Gln         755 760 765 Gly Ser Gly Ser Gly His Ser Pro Ser Arg Val Arg His Gly Ser Ser     770 775 780 Ser Gly His Ser Ser Ser His Gly Gln His Gly Ser Gly Thr Ser Cys 785 790 795 800 Ser Ser Ser Cys Gly His Tyr Glu Ser Gly Ser Gly Gln Ala Ser Gly                 805 810 815 Phe Gly Gln His Glu Ser Gly Ser Gly Gln Gly Tyr Ser Gln His Gly             820 825 830 Ser Ala Ser Gly His Phe Ser Ser Gln Gly Arg His Gly Ser Thr Ser         835 840 845 Gly Gln Ser Ser Ser Ser Gly Gln His Asp Ser Ser Ser Gly Gln Ser     850 855 860 Ser Ser Tyr Gly Gln His Glu Ser Ala Ser His His Ala Ser Gly Arg 865 870 875 880 Gly Arg His Gly Ser Gly Ser Gly Gln Ser Pro Gly His Gly Gln Arg                 885 890 895 Gly Ser Gly Ser Gly Gln Ser Pro Ser Tyr Gly Arg His Gly Ser Gly             900 905 910 Ser Gly Arg Ser Ser Ser Ser Gly Arg His Gly Ser Gly Ser Gly Gln         915 920 925 Ser Ser Gly Phe Gly His Lys Ser Ser Gly Gln Ser Ser Gly Tyr     930 935 940 Thr Gln His Gly Ser Gly Ser Gly His Ser Ser Ser Tyr Glu Gln His 945 950 955 960 Gly Ser Arg Ser Gly Gln Ser Ser Arg Ser Glu Gln His Gly Ser Ser                 965 970 975 Ser Gly Ser Ser Ser Ser Tyr Gly Gln His Gly Ser Gly Ser Arg Gln             980 985 990 Ser Leu Gly His Gly Gln His Gly Ser Gly Ser Gly Gln Ser Pro Ser         995 1000 1005 Pro Ser Arg Gly Arg His Gly Ser Gly Ser Gly Gln Ser Ser Ser Tyr    1010 1015 1020 Gly Pro Tyr Arg Ser Gly Ser Gly Trp Ser Ser Ser Arg Gly Pro Tyr 1025 1030 1035 1040 Glu Ser Gly Ser Gly His Ser Ser Gly Leu Gly His Arg Glu Ser Arg                1045 1050 1055 Ser Gly Gln Ser Ser Gly Tyr Gly Gln His Gly Ser Ser Ser Gly His            1060 1065 1070 Ser Ser Thr His Gly Gln His Gly Ser Thr Ser Gly Gln Ser Ser Ser        1075 1080 1085 Cys Gly Gln His Gly Ala Ser Ser Gly Gln Ser Ser Ser His Gly Gln    1090 1095 1100 His Gly Ser Gly Ser Ser Gln Ser Ser Gly Tyr Gly Arg Gln Gly Ser 1105 1110 1115 1120 Gly Ser Gly Gln Ser Pro Gly His Gly Gln Arg Gly Ser Gly Ser Arg                1125 1130 1135 Gln Ser Pro Ser Tyr Gly Arg His Gly Ser Gly Ser Gly Arg Ser Ser            1140 1145 1150 Ser Ser Gly Gln His Gly Ser Gly Leu Gly Glu Ser Ser Gly Phe Gly        1155 1160 1165 His His Glu Ser Ser Ser Gly Gln Ser Ser Ser Tyr Ser Gln His Gly    1170 1175 1180 Ser Gly Ser Gly His Ser Ser Gly Tyr Gly Gln His Gly Ser Arg Ser 1185 1190 1195 1200 Gly Gln Ser Ser Arg Gly Glu Arg His Gly Ser Ser Ser Gly Ser Ser                1205 1210 1215 Ser His Tyr Gly Gln His Gly Ser Gly Ser Arg Gln Ser Ser Gly His            1220 1225 1230 Gly Arg Gln Gly Ser Gly Ser Gly His Ser Pro Ser Arg Gly Arg His        1235 1240 1245 Gly Ser Gly Leu Gly His Ser Ser Ser His Gly Gln His Gly Ser Gly    1250 1255 1260 Ser Gly Arg Ser Ser Ser Arg Gly Pro Tyr Glu Ser Arg Ser Gly His 1265 1270 1275 1280 Ser Ser Val Phe Gly Gln His Glu Ser Gly Ser Gly His Ser Ser Ala                1285 1290 1295 Tyr Ser Gln His Gly Ser Gly Ser Gly His Phe Cys Ser Gln Gly Gln            1300 1305 1310 His Gly Ser Thr Ser Gly Gln Ser Ser Thr Phe Asp Gln Glu Gly Ser        1315 1320 1325 Ser Thr Gly Gln Ser Ser Ser Tyr Gly His Arg Gly Ser Gly Ser Ser    1330 1335 1340 Gln Ser Ser Gly Tyr Gly Arg His Gly Ala Gly Ser Gly Gln Ser Pro 1345 1350 1355 1360 Ser Arg Gly Arg His Gly Ser Gly Ser Gly His Ser Ser Ser Tyr Gly                1365 1370 1375 Gln His Gly Ser Gly Ser Gly Trp Ser Ser Ser Ser Gly Arg His Gly            1380 1385 1390 Ser Gly Ser Gly Gln Ser Ser Gly Phe Gly His His Glu Ser Ser Ser        1395 1400 1405 Trp Gln Ser Ser Gly Cys Thr Gln His Gly Ser Gly Ser Gly His Ser    1410 1415 1420 Ser Ser Tyr Glu Gln His Gly Ser Arg Ser Gly Gln Ser Ser Arg Gly 1425 1430 1435 1440 Glu Arg His Gly Ser Ser Ser Gly Ser Ser Ser Ser Tyr Gly Gln His                1445 1450 1455 Gly Ser Gly Ser Arg Gln Ser Leu Gly His Gly Gln His Gly Ser Gly            1460 1465 1470 Ser Gly Gln Ser Pro Ser Pro Ser Arg Gly Arg His Gly Ser Gly Ser        1475 1480 1485 Gly Gln Ser Ser Ser Tyr Ser Pro Tyr Gly Ser Gly Ser Gly Trp Ser    1490 1495 1500 Ser Ser Arg Gly Pro Tyr Glu Ser Gly Ser Ser His Ser Ser Gly Leu 1505 1510 1515 1520 Gly His Arg Glu Ser Arg Ser Gly Gln Ser Ser Gly Tyr Gly Gln His                1525 1530 1535 Gly Ser Ser Ser Gly His Ser Ser Thr His Gly Gln His Gly Ser Thr            1540 1545 1550 Ser Gly Gln Ser Ser Ser Cys Gly Gln His Gly Ala Ser Ser Gly Gln        1555 1560 1565 Ser Ser Ser His Gly Gln His Gly Ser Gly Ser Ser Gln Ser Ser Gly    1570 1575 1580 Tyr Gly Arg Gln Gly Ser Gly Ser Gly Gln Ser Pro Gly His Gly Gln 1585 1590 1595 1600 Arg Gly Ser Gly Ser Arg Gln Ser Pro Ser Tyr Gly Arg His Gly Ser                1605 1610 1615 Gly Ser Gly Arg Ser Ser Ser Ser Gly Gln His Gly Ser Gly Leu Gly            1620 1625 1630 Glu Ser Ser Gly Phe Gly His His Glu Ser Ser Ser Gly Gln Ser Ser        1635 1640 1645 Ser Tyr Ser Gln His Gly Ser Gly Ser Gly His Ser Ser Gly Tyr Gly    1650 1655 1660 Gln His Gly Ser Arg Ser Gly Gln Ser Ser Arg Gly Glu Arg His Gly 1665 1670 1675 1680 Ser Ser Ser Arg Ser Ser Ser Arg Tyr Gly Gln His Gly Ser Gly Ser                1685 1690 1695 Arg Gln Ser Ser Gly His Gly Arg Gln Gly Ser Gly Ser Gly Gln Ser            1700 1705 1710 Pro Ser Arg Gly Arg His Gly Ser Gly Leu Gly His Ser Ser Ser His        1715 1720 1725 Gly Gln His Gly Ser Gly Ser Gly Arg Ser Ser Ser Arg Gly Pro Tyr    1730 1735 1740 Glu Ser Arg Ser Gly His Ser Ser Val Phe Gly Gln His Glu Ser Gly 1745 1750 1755 1760 Ser Gly His Ser Ser Ala Tyr Ser Gln His Gly Ser Gly Ser Gly His                1765 1770 1775 Phe Cys Ser Gln Gly Gln His Gly Ser Thr Ser Gly Gln Ser Ser Thr            1780 1785 1790 Phe Asp Gln Glu Gly Ser Ser Thr Gly Gln Ser Ser Ser His Gly Gln        1795 1800 1805 His Gly Ser Gly Ser Ser Gln Ser Ser Ser Tyr Gly Gln Gln Gly Ser    1810 1815 1820 Gly Ser Gly Gln Ser Pro Ser Arg Gly Arg His Gly Ser Gly Ser Gly 1825 1830 1835 1840 His Ser Ser Ser Tyr Gly Gln His Gly Ser Gly Ser Gly Trp Ser Ser                1845 1850 1855 Ser Ser Gly Arg His Gly Ser Gly Ser Gly Gln Ser Ser Gly Phe Gly            1860 1865 1870 His His Glu Ser Ser Ser Trp Gln Ser Ser Gly Tyr Thr Gln His Gly        1875 1880 1885 Ser Gly Ser Gly His Ser Ser Ser Tyr Glu Gln His Gly Ser Arg Ser    1890 1895 1900 Gly Gln Ser Ser Arg Gly Glu Gln His Gly Ser Ser Ser Gly Ser Ser 1905 1910 1915 1920 Ser Ser Tyr Gly Gln His Gly Ser Gly Ser Arg Gln Ser Leu Gly His                1925 1930 1935 Gly Gln His Gly Ser Gly Ser Gly Gln Ser Pro Ser Pro Ser Arg Gly            1940 1945 1950 Arg His Gly Ser Gly Ser Gly Gln Ser Ser Ser Tyr Gly Pro Tyr Gly        1955 1960 1965 Ser Gly Ser Gly Trp Ser Ser Ser Arg Gly Pro Tyr Glu Ser Gly Ser    1970 1975 1980 Gly His Ser Ser Gly Leu Gly His Arg Glu Ser Arg Ser Gly Gln Ser 1985 1990 1995 2000 Ser Gly Tyr Gly Gln His Gly Ser Ser Ser Gly His Ser Ser Thr His                2005 2010 2015 Gly Gln His Gly Ser Ala Ser Gly Gln Ser Ser Ser Cys Gly Gln His            2020 2025 2030 Gly Ala Ser Ser Gly Gln Ser Ser Ser His Gly Gln His Gly Ser Gly        2035 2040 2045 Ser Ser Gln Ser Ser Gly Tyr Gly Arg Gln Gly Ser Gly Ser Gly Gln    2050 2055 2060 Ser Pro Gly His Gly Gln Arg Gly Ser Gly Ser Arg Gln Ser Pro Ser 2065 2070 2075 2080 Tyr Gly Arg His Gly Ser Gly Ser Gly Arg Ser Ser Ser Ser Gly Gln                2085 2090 2095 His Gly Pro Gly Leu Gly Glu Ser Ser Gly Phe Gly His His Glu Ser            2100 2105 2110 Ser Ser Gly Gln Ser Ser Ser Tyr Ser Gln His Gly Ser Gly Ser Gly        2115 2120 2125 His Ser Ser Gly Tyr Gly Gln His Gly Ser Arg Ser Gly Gln Ser Ser    2130 2135 2140 Arg Gly Glu Arg His Gly Ser Ser Ser Gly Ser Ser Ser Arg Tyr Gly 2145 2150 2155 2160 Gln His Gly Ser Gly Ser Arg Gln Ser Ser Gly His Gly Arg Gln Gly                2165 2170 2175 Ser Gly Ser Gly His Ser Pro Ser Arg Gly Arg His Gly Ser Gly Ser            2180 2185 2190 Gly His Ser Ser Ser His Gly Gln His Gly Ser Gly Ser Gly Arg Ser        2195 2200 2205 Ser Ser Arg Gly Pro Tyr Glu Ser Arg Ser Gly His Ser Ser Val Phe    2210 2215 2220 Gly Gln His Glu Ser Gly Ser Gly His Ser Ser Ala Tyr Ser Gln His 2225 2230 2235 2240 Gly Ser Gly Ser Gly His Phe Cys Ser Gln Gly Gln His Gly Ser Thr                2245 2250 2255 Ser Gly Gln Ser Ser Thr Phe Asp Gln Glu Gly Ser Ser Thr Gly Gln            2260 2265 2270 Ser Ser Ser His Gly Gln His Gly Ser Gly Ser Ser Gln Ser Ser Ser        2275 2280 2285 Tyr Gly Gln Gln Gly Ser Gly Ser Gly Gln Ser Pro Ser Arg Gly Arg    2290 2295 2300 His Gly Ser Gly Ser Gly His Ser Ser Ser Tyr Gly Gln His Gly Ser 2305 2310 2315 2320 Gly Ser Gly Trp Ser Ser Ser Ser Gly Arg His Gly Ser Gly Ser Gly                2325 2330 2335 Gln Ser Ser Gly Phe Gly His His Glu Ser Ser Ser Trp Gln Ser Ser            2340 2345 2350 Gly Tyr Thr Gln His Gly Ser Gly Ser Gly His Ser Ser Ser Tyr Glu        2355 2360 2365 Gln His Gly Ser Arg Ser Gly Gln Ser Ser Arg Gly Glu Arg His Gly    2370 2375 2380 Ser Ser Ser Gly Ser Ser Ser Ser Tyr Gly Gln His Gly Ser Gly Ser 2385 2390 2395 2400 Arg Gln Ser Leu Gly His Gly Gln His Gly Ser Gly Ser Gly Gln Ser                2405 2410 2415 Pro Ser Pro Ser Arg Gly Arg His Gly Ser Gly Ser Gly Gln Ser Ser            2420 2425 2430 Ser Tyr Ser Pro Tyr Gly Ser Gly Ser Gly Trp Ser Ser Ser Arg Gly        2435 2440 2445 Pro Tyr Glu Ser Gly Ser Gly His Ser Ser Gly Leu Gly His Arg Glu    2450 2455 2460 Ser Arg Ser Gly Gln Ser Ser Gly Tyr Gly Gln His Gly Ser Ser Ser 2465 2470 2475 2480 Gly His Ser Ser Thr His Gly Gln His Gly Ser Thr Ser Gly Gln Ser                2485 2490 2495 Ser Ser Cys Gly Gln His Gly Ala Ser Ser Gly Gln Ser Ser Ser His            2500 2505 2510 Gly Gln His Gly Ser Gly Ser Ser Gln Ser Ser Gly Tyr Gly Arg Gln        2515 2520 2525 Gly Ser Gly Ser Gly Gln Ser Pro Gly His Gly Gln Arg Gly Ser Gly    2530 2535 2540 Ser Arg Gln Ser Pro Ser Tyr Gly Arg His Gly Ser Gly Ser Gly Arg 2545 2550 2555 2560 Ser Ser Ser Ser Gly Gln His Gly Ser Gly Leu Gly Glu Ser Ser Gly                2565 2570 2575 Phe Gly His His Glu Ser Ser Ser Gly Gln Ser Ser Ser Tyr Ser Gln            2580 2585 2590 His Gly Ser Gly Ser Gly His Ser Ser Gly Tyr Gly Gln His Gly Ser        2595 2600 2605 Arg Ser Gly Gln Ser Ser Arg Gly Glu Arg His Gly Ser Ser Ser Gly    2610 2615 2620 Ser Ser Ser His Tyr Gly Gln His Gly Ser Gly Ser Arg Gln Ser Ser 2625 2630 2635 2640 Gly His Gly Arg Gln Gly Ser Gly Ser Gly Gln Ser Pro Ser Arg Gly                2645 2650 2655 Arg His Gly Ser Gly Leu Gly His Ser Ser Ser His Gly Gln His Gly            2660 2665 2670 Ser Gly Ser Gly Arg Ser Ser Ser Arg Gly Pro Tyr Glu Ser Arg Leu        2675 2680 2685 Gly His Ser Ser Val Phe Gly Gln His Glu Ser Gly Ser Gly His Ser    2690 2695 2700 Ser Ala Tyr Ser Gln His Gly Ser Gly Ser Gly His Phe Cys Ser Gln 2705 2710 2715 2720 Gly Gln His Gly Ser Thr Ser Gly Gln Ser Ser Thr Phe Asp Gln Glu                2725 2730 2735 Gly Ser Ser Thr Gly Gln Ser Ser Ser Tyr Gly His Arg Gly Ser Gly            2740 2745 2750 Ser Ser Gln Ser Ser Gly Tyr Gly Arg His Gly Ala Gly Ser Gly Gln        2755 2760 2765 Ser Leu Ser His Gly Arg His Gly Ser Gly Ser Gly Gln Ser Ser Ser    2770 2775 2780 Tyr Gly Gln His Gly Ser Gly Ser Gly Gln Ser Ser Gly Tyr Ser Gln 2785 2790 2795 2800 His Gly Ser Gly Ser Gly Gln Asp Gly Tyr Ser Tyr Cys Lys Gly Gly                2805 2810 2815 Ser Asn His Asp Gly Gly Ser Ser Gly Ser Tyr Phe Leu Ser Phe Pro            2820 2825 2830 Ser Ser Thr Ser Pro Tyr Glu Tyr Val Gln Glu Gln Arg Cys Tyr Phe        2835 2840 2845 Tyr gln    2850 <210> 10 <211> 209 <212> PRT <213> Homo sapiens <400> 10 Met Pro Val Ala Val Met Ala Glu Ser Ala Phe Ser Phe Lys Lys Leu   1 5 10 15 Leu Asp Gln Cys Glu Asn Gln Glu Leu Glu Ala Pro Gly Gly Ile Ala              20 25 30 Thr Pro Pro Val Tyr Gly Gln Leu Leu Ala Leu Tyr Leu Leu His Asn          35 40 45 Asp Met Asn Asn Ala Arg Tyr Leu Trp Lys Arg Ile Pro Pro Ala Ile      50 55 60 Lys Ser Ala Asn Ser Glu Leu Gly Gly Ile Trp Ser Val Gly Gln Arg  65 70 75 80 Ile Trp Gln Arg Asp Phe Pro Gly Ile Tyr Thr Thr Ile Asn Ala His                  85 90 95 Gln Trp Ser Glu Thr Val Gln Pro Ile Met Glu Ala Leu Arg Asp Ala             100 105 110 Thr Arg Arg Arg Ala Phe Ala Leu Val Ser Gln Ala Tyr Thr Ser Ile         115 120 125 Ile Ala Asp Asp Phe Ala Ala Phe Val Gly Leu Pro Val Glu Glu Ala     130 135 140 Val Lys Gly Ile Leu Glu Gln Gly Trp Gln Ala Asp Ser Thr Thr Arg 145 150 155 160 Met Val Leu Pro Arg Lys Pro Val Ala Gly Ala Leu Asp Val Ser Phe                 165 170 175 Asn Lys Phe Ile Pro Leu Ser Glu Pro Ala Pro Val Pro Pro Ile Pro             180 185 190 Asn Glu Gln Gln Leu Ala Arg Leu Thr Asp Tyr Val Ala Phe Leu Glu         195 200 205 Asn     <210> 11 <211> 459 <212> PRT <213> Homo sapiens <400> 11 Met Thr Ala Glu Glu Met Lys Ala Thr Glu Ser Gly Ala Gln Ser Ala   1 5 10 15 Pro Leu Pro Met Glu Gly Val Asp Ile Ser Pro Lys Gln Asp Glu Gly              20 25 30 Val Leu Lys Val Ile Lys Arg Glu Gly Thr Gly Thr Glu Met Pro Met          35 40 45 Ile Gly Asp Arg Val Phe Val His Tyr Thr Gly Trp Leu Leu Asp Gly      50 55 60 Thr Lys Phe Asp Ser Ser Leu Asp Arg Lys Asp Lys Phe Ser Phe Asp  65 70 75 80 Leu Gly Lys Gly Glu Val Ile Lys Ala Trp Asp Ile Ala Ile Ala Thr                  85 90 95 Met Lys Val Gly Glu Val Cys His Ile Thr Cys Lys Pro Glu Tyr Ala             100 105 110 Tyr Gly Ser Ala Gly Ser Pro Pro Lys Ile Pro Pro Asn Ala Thr Leu         115 120 125 Val Phe Glu Val Glu Leu Phe Glu Phe Lys Gly Glu Asp Leu Thr Glu     130 135 140 Glu Glu Asp Gly Gly Ile Ile Arg Arg Ile Gln Thr Arg Gly Glu Gly 145 150 155 160 Tyr Ala Lys Pro Asn Glu Gly Ala Ile Val Glu Val Ala Leu Glu Gly                 165 170 175 Tyr Tyr Lys Asp Lys Leu Phe Asp Gln Arg Glu Leu Arg Phe Glu Ile             180 185 190 Gly Glu Gly Glu Asn Leu Asp Leu Pro Tyr Gly Leu Glu Arg Ala Ile         195 200 205 Gln Arg Met Glu Lys Gly Glu His Ser Ile Val Tyr Leu Lys Pro Ser     210 215 220 Tyr Ala Phe Gly Ser Val Gly Lys Glu Lys Phe Gln Ile Pro Pro Asn 225 230 235 240 Ala Glu Leu Lys Tyr Glu Leu His Leu Lys Ser Phe Glu Lys Ala Lys                 245 250 255 Glu Ser Trp Glu Met Asn Ser Glu Glu Lys Leu Glu Gln Ser Thr Ile             260 265 270 Val Lys Glu Arg Gly Thr Val Tyr Phe Lys Glu Gly Lys Tyr Lys Gln         275 280 285 Ala Leu Leu Gln Tyr Lys Lys Ile Val Ser Trp Leu Glu Tyr Glu Ser     290 295 300 Ser Phe Ser Asn Glu Glu Ala Gln Lys Ala Gln Ala Leu Arg Leu Ala 305 310 315 320 Ser His Leu Asn Leu Ala Met Cys His Leu Lys Leu Gln Ala Phe Ser                 325 330 335 Ala Ala Ile Glu Ser Cys Asn Lys Ala Leu Glu Leu Asp Ser Asn Asn             340 345 350 Glu Lys Gly Leu Phe Arg Arg Gly Glu Ala His Leu Ala Val Asn Asp         355 360 365 Phe Glu Leu Ala Arg Ala Asp Phe Gln Lys Val Leu Gln Leu Tyr Pro     370 375 380 Asn Asn Lys Ala Ala Lys Thr Gln Leu Ala Val Cys Gln Gln Arg Ile 385 390 395 400 Arg Arg Gln Leu Ala Arg Glu Lys Lys Leu Tyr Ala Asn Met Phe Glu                 405 410 415 Arg Leu Ala Glu Glu Glu Asn Lys Ala Lys Ala Glu Ala Ser Ser Gly             420 425 430 Asp His Pro Thr Asp Thr Glu Met Lys Glu Glu Gln Lys Ser Asn Thr         435 440 445 Ala Gly Ser Gln Ser Gln Val Glu Thr Glu Ala     450 455 <210> 12 <211> 303 <212> PRT <213> Homo sapiens <400> 12 Met Ser Ser Ala Arg Phe Asp Ser Ser Asp Arg Ser Ala Trp Tyr Met   1 5 10 15 Gly Pro Val Ser Arg Gln Glu Ala Gln Thr Arg Leu Gln Gly Gln Arg              20 25 30 His Gly Met Phe Leu Val Arg Asp Ser Ser Thr Cys Pro Gly Asp Tyr          35 40 45 Val Leu Ser Val Ser Glu Asn Ser Arg Val Ser His Tyr Ile Ile Asn      50 55 60 Ser Leu Pro Asn Arg Arg Phe Lys Ile Gly Asp Gln Glu Phe Asp His  65 70 75 80 Leu Pro Ala Leu Leu Glu Phe Tyr Lys Ile His Tyr Leu Asp Thr Thr                  85 90 95 Thr Leu Ile Glu Pro Ala Pro Arg Tyr Pro Ser Pro Pro Met Gly Ser             100 105 110 Val Ser Ala Pro Asn Leu Pro Thr Ala Glu Asp Asn Leu Glu Tyr Val         115 120 125 Arg Thr Leu Tyr Asp Phe Pro Gly Asn Asp Ala Glu Asp Leu Pro Phe     130 135 140 Lys Lys Gly Glu Ile Leu Val Ile Ile Glu Lys Pro Glu Glu Gln Trp 145 150 155 160 Trp Ser Ala Arg Asn Lys Asp Gly Arg Val Gly Met Ile Pro Val Pro                 165 170 175 Tyr Val Glu Lys Leu Val Arg Ser Ser Pro His Gly Lys His Gly Asn             180 185 190 Arg Asn Ser Asn Ser Tyr Gly Ile Pro Glu Pro Ala His Ala Tyr Ala         195 200 205 Gln Pro Gln Thr Thr Thr Pro Leu Pro Ala Val Ser Gly Ser Pro Gly     210 215 220 Ala Ala Ile Thr Pro Leu Pro Ser Thr Gln Asn Gly Pro Val Phe Ala 225 230 235 240 Lys Ala Ile Gln Lys Arg Val Pro Cys Ala Tyr Asp Lys Thr Ala Leu                 245 250 255 Ala Leu Glu Val Gly Asp Ile Val Lys Val Thr Arg Met Asn Ile Asn             260 265 270 Gly Gln Trp Glu Gly Glu Val Asn Gly Arg Lys Gly Leu Phe Pro Phe         275 280 285 Thr His Val Lys Ile Phe Asp Pro Gln Asn Pro Asp Glu Asn Glu     290 295 300 <210> 13 <211> 359 <212> PRT <213> Homo sapiens <400> 13 Met Ala Ala Val Ser Gly Leu Val Arg Arg Pro Leu Arg Glu Val Ser   1 5 10 15 Gly Leu Leu Lys Arg Arg Phe His Trp Thr Ala Pro Ala Ala Val Gln              20 25 30 Val Thr Val Arg Asp Ala Ile Asn Gln Gly Met Asp Glu Glu Leu Glu          35 40 45 Arg Asp Glu Lys Val Phe Leu Leu Gly Glu Glu Val Ala Gln Tyr Asp      50 55 60 Gly Ala Tyr Lys Val Ser Arg Gly Leu Trp Lys Lys Tyr Gly Asp Lys  65 70 75 80 Arg Ile Ile Asp Thr Pro Ile Ser Glu Met Gly Phe Ala Gly Ile Ala                  85 90 95 Val Gly Ala Ala Met Ala Gly Leu Arg Pro Ile Cys Glu Phe Met Thr             100 105 110 Phe Asn Phe Ser Met Gln Ala Ile Asp Gln Val Ile Asn Ser Ala Ala         115 120 125 Lys Thr Tyr Tyr Met Ser Gly Gly Leu Gln Pro Val Pro Ile Val Phe     130 135 140 Arg Gly Pro Asn Gly Ala Ser Ala Gly Val Ala Ala Gln His Ser Gln 145 150 155 160 Cys Phe Ala Ala Trp Tyr Gly His Cys Pro Gly Leu Lys Val Val Ser                 165 170 175 Pro Trp Asn Ser Glu Asp Ala Lys Gly Leu Ile Lys Ser Ala Ile Arg             180 185 190 Asp Asn Asn Pro Val Val Val Leu Glu Asn Glu Leu Met Tyr Gly Val         195 200 205 Pro Phe Glu Phe Pro Pro Glu Ala Gln Ser Lys Asp Phe Leu Ile Pro     210 215 220 Ile Gly Lys Ala Lys Ile Glu Arg Gln Gly Thr His Ile Thr Val Val 225 230 235 240 Ser His Ser Arg Pro Val Gly His Cys Leu Glu Ala Ala Ala Val Leu                 245 250 255 Ser Lys Glu Gly Val Glu Cys Glu Val Ile Asn Met Arg Thr Ile Arg             260 265 270 Pro Met Asp Met Glu Thr Ile Glu Ala Ser Val Met Lys Thr Asn His         275 280 285 Leu Val Thr Val Glu Gly Gly Trp Pro Gln Phe Gly Val Gly Ala Glu     290 295 300 Ile Cys Ala Arg Ile Met Glu Gly Pro Ala Phe Asn Phe Leu Asp Ala 305 310 315 320 Pro Ala Val Arg Val Thr Gly Ala Asp Val Pro Met Pro Tyr Ala Lys                 325 330 335 Ile Leu Glu Asp Asn Ser Ile Pro Gln Val Lys Asp Ile Ile Phe Ala             340 345 350 Ile Lys Lys Thr Leu Asn Ile         355 <210> 14 <211> 238 <212> PRT <213> Homo sapiens <400> 14 Met Ala Ala Ala Val Gly Arg Leu Leu Arg Ala Ser Val Ala Arg His   1 5 10 15 Val Ser Ala Ile Pro Trp Gly Ile Ser Ala Thr Ala Ala Leu Arg Pro              20 25 30 Ala Ala Cys Gly Arg Thr Ser Leu Thr Asn Leu Leu Cys Ser Gly Ser          35 40 45 Ser Gln Ala Pro Tyr Phe Lys Gly Thr Ala Val Val Asn Gly Glu Phe      50 55 60 Lys Asp Leu Ser Leu Asp Asp Phe Lys Gly Lys Tyr Leu Val Leu Phe  65 70 75 80 Phe Tyr Pro Leu Asp Phe Thr Phe Val Cys Pro Thr Glu Ile Val Ala                  85 90 95 Phe Ser Asp Lys Ala Asn Glu Phe His Asp Val Asn Cys Glu Val Val             100 105 110 Ala Val Ser Val Asp Ser His Phe Ser His Leu Ala Trp Ile Asn Thr         115 120 125 Pro Arg Lys Asn Gly Gly Leu Gly His Met Asn Ile Ala Leu Leu Ser     130 135 140 Asp Leu Thr Lys Gln Ile Ser Arg Asp Tyr Gly Val Leu Leu Glu Gly 145 150 155 160 Ser Gly Leu Ala Leu Arg Gly Leu Phe Ile Ile Asp Pro Asn Gly Val                 165 170 175 Ile Lys His Leu Ser Val Asn Asp Leu Pro Val Gly Arg Ser Val Glu             180 185 190 Glu Thr Leu Arg Leu Val Lys Ala Phe Gln Tyr Val Glu Thr His Gly         195 200 205 Glu Val Cys Pro Ala Asn Trp Thr Pro Asp Ser Pro Thr Ile Lys Pro     210 215 220 Ser Pro Ala Ala Ser Lys Glu Tyr Phe Gln Lys Val Asn Gln 225 230 235 <210> 15 <211> 129 <212> PRT <213> Homo sapiens <400> 15 Met Arg Arg Arg Gly Glu Ile Asp Met Ala Thr Glu Gly Asp Val Glu   1 5 10 15 Leu Glu Leu Glu Thr Glu Thr Ser Gly Pro Glu Arg Pro Pro Glu Lys              20 25 30 Pro Arg Lys His Asp Ser Gly Ala Ala Asp Leu Glu Arg Val Thr Asp          35 40 45 Tyr Ala Glu Glu Lys Glu Ile Gln Ser Ser Asn Leu Glu Thr Ala Met      50 55 60 Ser Val Ile Gly Asp Arg Arg Ser Arg Glu Gln Lys Ala Lys Gln Glu  65 70 75 80 Arg Glu Lys Glu Leu Ala Lys Val Thr Ile Lys Lys Glu Asp Leu Glu                  85 90 95 Leu Ile Met Thr Glu Met Glu Ile Ser Arg Ala Ala Ala Glu Arg Ser             100 105 110 Leu Arg Glu His Met Gly Asn Val Val Glu Ala Leu Ile Ala Leu Thr         115 120 125 Asn     <210> 16 <211> 151 <212> PRT <213> Homo sapiens <400> 16 Met Cys Asp Phe Thr Glu Asp Gln Thr Ala Glu Phe Lys Glu Ala Phe   1 5 10 15 Gln Leu Phe Asp Arg Thr Gly Asp Gly Lys Ile Leu Tyr Ser Gln Cys              20 25 30 Gly Asp Val Met Arg Ala Leu Gly Gln Asn Pro Thr Asn Ala Glu Val          35 40 45 Leu Lys Val Leu Gly Asn Pro Lys Ser Asp Glu Met Asn Val Lys Val      50 55 60 Leu Asp Phe Glu His Phe Leu Pro Met Leu Gln Thr Val Ala Lys Asn  65 70 75 80 Lys Asp Gln Gly Thr Tyr Glu Asp Tyr Val Glu Gly Leu Arg Val Phe                  85 90 95 Asp Lys Glu Gly Asn Gly Thr Val Met Gly Ala Glu Ile Arg His Val             100 105 110 Leu Val Thr Leu Gly Glu Lys Met Thr Glu Glu Glu Val Glu Met Leu         115 120 125 Val Ala Gly His Glu Asp Ser Asn Gly Cys Ile Asn Tyr Glu Ala Phe     130 135 140 Val Arg His Ile Leu Ser Gly 145 150

Claims (8)

Prostate cancer stem, characterized in that Cofilin-1 having an amino acid sequence of SEQ ID NO: 6 and a molecular weight of 18-20 kD, the expression of which is increased in prostate cancer stem cells rather than normal prostate cancer cells Proteinaceous marker composition for cell selection.
Prostate cancer stem cell selection composition comprising an antibody that specifically recognizes the proteinaceous marker of claim 1.
delete Prostate cancer stem cell selection composition comprising a primer for RT-PCR for detecting the mRNA of the gene encoding the proteinaceous marker of claim 1.
delete Prostate cancer stem cell selection kit comprising a composition for selecting prostate cancer stem cells of any one of claims 2 and 4.
A method for detecting the proteinaceous marker of claim 1 in order to provide information necessary for the selection of prostate cancer stem cells.
A prostate cancer stem cell inhibitor comprising the step of treating a test compound in a cell or animal expressing the proteinaceous marker of claim 1 and identifying whether the test compound promotes or inhibits the physiological activity of the proteinaceous marker. Screening method.

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