KR20180074109A - Marker for predicting lymph node metastasis and method for predicting lymph node metastasis using same - Google Patents

Abstract

The present invention relates to a composition for diagnosing a metastatic gastric cancer, and a method to predict lymph node metastasis in an initial stage of gastric cancer using the method. According to the present invention, the composition comprises a probe, a primer set, or a nucleotide specifically bonded to a polynucleotide sequence coding one or more proteins selected from a group including apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3A (APOBEC3A), fragment of IgG, low affinity IIIb (FCGR3B), serpin family B member 5 (SERPINB5), neuromedin-U (NMU), or protein-arginine deiminase type-2 (PADI2), or an antibody, an antigen-binding fragment, or a polypeptide, which are specifically bonded to one or more proteins or each fragment thereof selected from a group including APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 or each fragment thereof.

Description

[0001] The present invention relates to a marker for predicting lymph node metastasis and a method for predicting lymph node metastasis using the same,

Compositions and kits for predicting lymph node metastasis in early stomach cancer comprising substances specifically binding to one or more proteins selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2, and prediction of lymph node metastasis in early gastric cancer using the composition .

Stomach cancer is the fifth most common cancer in the world and the second leading cause of death from cancer (Int J Cancer 2015; 136: E359-386). Local lymph node metastasis is known to be one of the most important prognostic factors in gastric cancer. Lymph node dissection is also important in the treatment of gastric cancer (J Clin Oncol 2012; 30: 3834-3840; Ann Surg 2012; J Surg 1995; 82: 346-351). Recently, our retrospective clinical study reassessed the importance of lymph node metastasis in mucosal stomach cancer (Oh SY et al., Ann Surg 2016).

On the other hand, the depth of tumor invasion in the TNM staging system is frequently associated with local lymph node metastasis (Edge S et al., New York, NY: Springer 2009). However, in rare cases, stomach cancer may occur when the T stage and N stage are conflicting (T stage is high and no lymph node metastasis or T stage is low, but lymph node metastasis is high). In such cases, independent biomarkers for lymph node metastasis are sought It can be a good target. In previous studies, DNA microarray technique has shown that overexpression of plasminogen activator inhibitor-1 (PAI-1) in gastric cancer is associated with advanced gastric cancer with high lymph node metastasis, There was a difficulty in verifying results with depth limitations (Cancer Res Treat 2015; 47: 718-726).

 With the development of next-generation sequencing (NGS), it has become possible to analyze the molecular characteristics of rare mutations in cancer genomic studies (Nat Rev Genet 2013; 14: 681-691). Therefore, the present study aims to find independent molecular biomarkers for lymph node metastasis by analyzing the NGS technique for gastric cancer with high T stage and no lymph node metastasis and gastric cancer with low T stage but extremely high lymph node metastasis.

An aspect is an antibody, an antigen-binding fragment, or a polypeptide that specifically binds to one or more proteins selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2, or each of the fragments, or the APOBEC3A, FCGR3B, SERPINB5, NMU And a probe, a primer set, or a nucleotide, which specifically binds to a polynucleotide sequence encoding at least one protein selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and PADI2.

Another aspect provides a metastatic gastric cancer diagnostic kit comprising the metastatic gastric cancer diagnostic composition and a reagent for detecting the metastatic gastric cancer diagnostic composition.

Another aspect is an antibody, peptide, protein, or combination thereof that specifically binds to one or more proteins selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2, or each fragment thereof, Contacting a polynucleotide sequence that specifically binds to at least one protein selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 with a probe, a primer, a nucleotide, or a combination thereof to form a complex; And measuring the expression level of at least one protein selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 from the complex, and a method for diagnosing metastatic gastric cancer.

One aspect is APOPEC3A (catalytic polypeptide-like 3A), FCGR3B (Fc fragment of IgG, low affinity IIIb, receptor), SERPINB5 (serpin family B member 5), NMU (neuromedin-U) and PADI2 An antigen-binding fragment or a polypeptide that specifically binds to one or more proteins selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: A primer set, or a nucleotide, which specifically binds to a polynucleotide sequence encoding at least one protein selected from the group consisting of SEQ ID NOs.

The APOBEC3A, FCGR3B, SERPINB5, NMU, or PADI2 proteins are human ( Homo sapiens ) or mouse ( Mus the same protein can be expressed in other mammals such as a mouse, a mouse, a mouse, a mouse, a mouse, a mouse, a mouse, a horse, a horse, (SEQ ID NO: 1) of NM_007365.2 or a codon which is expressed by PADI2 among them, and may contain the sequence of SEQ ID NO: 2. From the mRNA containing the amino acid sequence of NP_031391.2, RTI ID = 0.0 > 14 < / RTI > Even though the mRNA of SEQ ID NO: 1 or 2 and the protein of SEQ ID NO: 14 do not coincide with some polynucleotide sequence or amino acid sequence, a polynucleotide sequence or amino acid sequence having a biologically equivalent activity can be regarded as mRNA or PADI2 protein of PADI2 have. APOBEC3A has a polynucleotide sequence of SEQ ID NO: 3 (NM_145699.3), a nucleotide sequence of 171 to 770 in SEQ ID NO: 3, a polynucleotide sequence of SEQ ID NO: 4 (NM_001270406.1) and a polynucleotide sequence of 171 to 716 in SEQ ID NO: 4, FCGR3B has a sequence (NM_001244753.1), a polynucleotide sequence from 261 to 1070 in SEQ ID NO: 5, a polynucleotide sequence from SEQ ID NO: 6 (NM_000570.4), a polynucleotide sequence from 123 to 824 in SEQ ID NO: 6, a polynucleotide sequence from SEQ ID NO: 7 (NM_001271035.1) , A polynucleotide sequence from 261 to 1067 in SEQ ID NO: 7, a polynucleotide sequence from SEQ ID NO: 8 (NM_001271036.1), a polynucleotide sequence from 286 to 936 in SEQ ID NO: 8, a polynucleotide sequence from SEQ ID NO: 9 (NM_001271037.1) SERPINB5 is a polynucleotide sequence of SEQ ID NO: 10 (NM_002639.4), SEQ ID NO: 10 is a polynucleotide sequence of SEQ ID NOs: 143 to 1270, and NMU is a polynucleotide sequence of SEQ ID NO: 11 (NM_006681.3) (NM_001292045.1), a polynucleotide sequence from 107 to 583 in SEQ ID NO: 12, a polynucleotide sequence from SEQ ID NO: 13 (NM_001292046.1) in SEQ ID NO: 12, and a polynucleotide sequence from 107 to 107 in SEQ ID NO: (SEQ ID NO: 15), and FCGR3B is NP_001231682.1 (SEQ ID NO: 16), which are translated from the mRNA comprising any one of the polynucleotide sequences of SEQ ID NOs: , NP_000561.3 (SEQ ID NO: 17), NP_001257964.1 (SEQ ID NO: 18), NP_001257965.1 (SEQ ID NO: 19), and NP_001257966.1 (SEQ ID NO: 20), SERPINB5 is NP_002630.2 ) And NMU may be a polypeptide or protein comprising the amino acid sequence of any of NP_006672.1 (SEQ ID NO: 22), NP_001278974.1 (SEQ ID NO: 23), and NP_001278975.1 (SEQ ID NO: 24) Also, an amino acid sequence or polynucleotide sequence having biologically equivalent activity can be regarded as the mRNA of the protein or the protein even if it does not coincide with the amino acid sequence or the polynucleotide sequence of each of the above proteins.

The polynucleotide encoding each of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 may have 60% or more, such as 70% or more, 80% or more, 90% or more, 95% or more, 99% or more , ≪ / RTI > or 100% sequence identity. The polynucleotide encoding each of the APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 is selected from the group consisting of one or more nucleotides, two or more nucleotides, three or more nucleotides, four or more nucleotides , 5 or more nucleotides, 6 or more nucleotides, or 7 or more nucleotides may be polynucleotides having different sequences.

Wherein each of the proteins selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 has at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% Or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In addition, each of the proteins selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 has at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, More than 6 amino acids, or more than 7 amino acids.

Wherein the composition for diagnosing metastatic gastric cancer comprises an antibody, an antigen-binding fragment, or a polypeptide that specifically binds to each protein or each fragment thereof selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2, or the APOBEC3A, FCGR3B, A set of primers, or a nucleotide, which specifically binds to a polynucleotide sequence encoding each protein selected from the group consisting of SERPINB5, NMU, and PADI2.

The term " metastatic " refers to the spread of cancer from an organ or location in which it develops to other organs or other locations, and primarily refers to the transfer to a site (or organ) that is not directly connected. Some cancer cells can pass through the lymphatic or vascular walls and into other organs. When they are transferred to the lymph nodes through such processes, they are called 'lymph node metastasis'.

In one embodiment, the composition of the present invention may be for diagnosing the metastatic potential of gastric cancer or for predicting the probability of metastasis, and the metastasis may specifically refer to lymph node metastasis.

In another embodiment, the composition of the present invention may be for predicting the diagnostic or metastatic potential for early metastatic gastric cancer in gastric cancer. Metastatic cancer can be assessed on the basis of the TNM staging system. In the TNM stage, T refers to the tumor size in the original tissue, N refers to the nearby lymph nodes and M refers to the long distance. T is Tx (inability to measure), Tis (in situ carcinoma) (No tumor signal), and T1 to T4 according to size, where N is the number of tumor cells in the tumor, in the presence of Nx (not measurable), N0 (no local lymph node in the tumor), N1 (local lymph node metastasis, (Metastasis to local lymph nodes), N2 (degree of lymph node metastasis between N1 and N3), and N3 (distant lymph node metastasis or multiple local lymph node metastases), M is classified as M0 (no distant metastasis) and M1 ). ≪ / RTI >

In one embodiment, the composition of the present invention is capable of diagnosing metastatic or metastatic potential in stomach cancer corresponding to T1 to T2, based on the TNM staging system, among the stomach cancer. In addition, the initial stomach cancer may be diagnosed as metastatic or non-metastatic in gastric cancer corresponding to N0 to N2, N0 to N1, or N1 to N2 in N stage, preferably N stage to N stage.

In one embodiment, the composition of the present invention may further comprise reagents for isolating the nucleus from the sample to be diagnosed. Techniques for isolating nuclei from tissue samples or cell samples can be made according to methods well known to those of ordinary skill in the art.

In one embodiment, the PADI2 protein to be detected in the isolated nucleus nucleus is a polypeptide translated from a polynucleotide of truncated-PADI2 (SEQ ID NO: BC009701.1), wherein the polypeptide comprises an amino acid sequence from 1 to 437 of the sequence of SEQ ID NO: 14 It may contain the amino acid sequence of SEQ ID NO: 26. The polynucleotide may also comprise a polynucleotide sequence of SEQ ID NO: 25 or 65 to 1378 in SEQ ID NO:

According to the embodiment of the present invention, the accuracy of the diagnosis of metastatic gastric cancer using PADI2 protein as a biomarker can be further improved by measuring the expression levels of IL-1β, IL-6 and TNF-β. Thus, in one embodiment, the composition for diagnosing metastatic gastric cancer of the invention comprises an antibody, peptide, protein, or combination thereof that specifically binds to IL-1 [beta], IL-6, and TNF- A probe, a primer, a nucleotide or a combination thereof that specifically binds to a nucleotide sequence encoding the IL-1β, IL-6, and TNF-β protein.

The substance capable of measuring the expression level of a protein selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 may be an antibody, an antigen-binding fragment, a polypeptide, a protein or the like that specifically binds to the protein or each fragment thereof . ≪ / RTI >

The term "antibody" means a specific immunoglobulin directed against an antigenic site. The antibody refers to an antibody specifically binding to each of APOBEC3A, FCGR3B, SERPINB5, NMU or PADI2 which is a metastatic gastric cancer biomarker. APOBEC3A, FCGR3B, SERPINB5, NMU or PADI2 gene is cloned into an expression vector, The APOBEC3A, FCGR3B, SERPINB5, NMU or PADI2 protein to be coded can be obtained and the antibody can be prepared from the obtained protein according to a conventional method in the art. The forms of the antibodies include polyclonal or monoclonal antibodies, including all immunoglobulin antibodies. The antibody does not have the structure of a complete intact antibody with two light and two heavy chains as well as a complete form with two light and two heavy chains as well as two light and two heavy chains, But also functional fragments of antibody molecules which have specific antigen binding sites (binding domains) and which possess antigen binding function.

The substance capable of measuring the expression level of APOBEC3A, FCGR3B, SERPINB5, NMU or PADI2 may be a probe, a primer, a nucleotide sequence or a sequence which specifically binds to each polynucleotide sequence encoding APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 Or a combination thereof.

Since the nucleotide sequences of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 genes are known, a person skilled in the art can design primers or probes which specifically bind to the sequence of these genes based on the above sequence, according to a conventional method in the art .

The term "probe" means a nucleotide fragment such as RNA or DNA corresponding to a nucleotide of mRNA or the like and capable of specifically binding to a nucleotide sequence of a few nucleotides or several hundreds of nucleotides, and is labeled with a radioactive element. Presence, content (amount of expression) can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. According to one embodiment of the present invention, hybridization is performed using a probe complementary to the mRNA of PADI2 gene, which is a biomarker of metastatic gastric cancer, and the expression level of mRNA is measured through hybridization degree to predict the diagnosis and metastatic potential of metastatic gastric cancer have. Selection of suitable probes and hybridization conditions can be appropriately selected according to techniques known in the art.

The term "primer" refers to a nucleotide sequence having a short free 3 'hydroxyl group, which can form a base pair with a complementary template and which has a short nucleotide sequence serving as a starting point for template strand copying It says. Primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase / polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffer solutions and temperatures. According to one embodiment of the present invention, PCR amplification is carried out using a primer set of each of the markers APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 to measure the expression amount of a desired protein to determine whether it is a metastatic stomach cancer and the possibility of lymph node metastasis Can be predicted. The PCR conditions, and the length of the primer set, can be appropriately selected according to techniques known in the art.

The term "nucleotide" refers to a deoxyribonucleotide or ribonucleotide, and may include analogs of natural nucleotides and analogs in which the sugar or base moieties are modified, unless specifically stated otherwise.

The probe, the primer or the nucleotide can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. Such nucleotide sequences may also be modified through a variety of methods known in the art. Examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate Etc.) or charged conjugates (e.g., phosphorothioates, phosphorodithioates, etc.).

The probe, primer or nucleotide may be 10 to 100 nucleotides in length, 10 to 90 nt, 10 to 80 nt, 10 to 70 nt, 10 to 60 nt, 10 to 50 nt, 10 to 100 nt, 40 nt, 10 to 30 nt, 10 to 25 nt, 20 to 100 nt, 30 to 90 nt, 40 to 80 nt, 50 to 70 nt, 20 to 60 nt, 20 to 50 nt, 30 to 40 nt, 30 nt, or 20 to 25 nt.

The mRNA expression levels of the APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 proteins or their genes are specifically increased when the risk of lymph node metastasis is high in metastatic gastric cancer, especially early stomach cancer. Therefore, the metastatic gastric cancer composition according to the present invention is The expression levels of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 proteins or the expression levels of genes (mRNA, etc.) in gastric cancer cells (or compared with normal cells) are measured to diagnose metastatic gastric cancer and predict the possibility of metastasis to the lymph nodes .

Other aspects include the above compositions capable of measuring the expression levels of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 of the present invention and the expression levels of IL-1β, IL-6, and TNF-β and reagents for detecting the same The present invention provides a kit for diagnosing metastatic gastric cancer.

The metastatic gastric cancer diagnostic kit can diagnose metastatic gastric cancer and determine the possibility of metastasis to the lymph node by measuring the expression levels of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 proteins as metastatic gastric cancer diagnostic markers. Can be predicted. The kit for diagnosing metastatic gastric cancer may further comprise antibodies, antigens or antibodies that specifically bind to the APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 proteins, But not limited to, primers or probes, nucleotides, or combinations thereof that specifically bind to each gene encoding a binding fragment, polypeptide, or protein, APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 proteins, Solution or device suitable for the assay method of measuring the expression levels of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 protein, respectively.

According to one embodiment, the kit for the diagnosis of metastatic gastric cancer of the present invention is a kit for measuring the expression level of mRNA of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 protein, Lt; / RTI > kit. RT-PCR kits can be used to detect the presence of enzymes such as test tubes or other appropriate containers, reaction buffers, deoxyribonucleotides (dNTPs), Taq polymerase and reverse transcriptase, DNase, RNase Inhibitors, DEPC-water (dEPC-water), sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.

According to another embodiment, the metastatic gastric cancer diagnostic kit of the present invention comprises a substrate for immunological detection of antibodies, antigen-binding fragments, polypeptides, protein antibodies that specifically bind to APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 proteins, A suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass. The chromogenic enzyme may be peroxidase, alkaline phosphatase alkaline phosphatase may be used as the fluorescent material, FITC or RITC may be used as the fluorescent material, and the chromogenic substrate may be 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) Phenylenediamine (OPD), tetramethylbenzidine (TMB), etc. may be used.

According to another embodiment, the kit of the present invention may be a microarray for the diagnosis of metastatic gastric cancer capable of measuring the amount of protein or mRNA expression of a gene encoding the protein. The microarray for diagnosing metastatic gastric cancer can be easily produced by those skilled in the art according to methods known in the art using the markers of the present invention. According to one embodiment, the APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 proteins The mRNA of each gene to be encoded or a cDNA having a sequence corresponding to the fragment may be attached to the substrate as a probe.

Also, the kit of the present invention is as described above for IL-1?, IL-6, and TNF- ?, and may further comprise a reagent for isolating the nucleus from the sample to be diagnosed or a container containing the reagent.

It is to be understood that the terms or elements mentioned in the above kits, such as those mentioned in the description of the claimed compositions, are as mentioned in the description of the compositions claimed above.

Another aspect relates to an antibody, a peptide, a protein or a combination thereof specifically binding to one or more proteins or fragments thereof selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2, Contacting a probe, a primer, a nucleotide or a combination thereof that specifically binds to a nucleotide sequence encoding at least one protein selected from the group consisting of FCGR3B, SERPINB5, NMU, and PADI2 to form a complex; And measuring the expression level of each of at least one protein selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 from the complex, and a method for diagnosing metastatic gastric cancer.

The subject to be diagnosed may be a mammal. The mammal may be a human, a mouse, a rat, a cow, a goat, a pig, a horse, a sheep, a dog, a cat, or a combination thereof. The sample isolated from the subject may be a normal tissue and a lesion tissue derived from the stomach tissue of the subject. In addition, the sample may include a cell derived from the tissues or a peripheral extracellular fluid.

The term "lesion tissue" refers to a tissue showing signs of cancer. Normal tissue refers to a tissue showing no signs of cancer, and may be a tissue derived from stomach in the present invention, and more specifically, gastric mucosal tissue. In one embodiment, the normal tissue and the lesion tissue may be derived from the same individual.

In one embodiment, the method for diagnosing metastatic gastric cancer according to the present invention further comprises the step of comparing the expression levels of each of the measured APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 with the standard expression levels of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 May include. The standard expression amount refers to an average expression amount of a specific protein in an unspecified number of normal tissues, for example, an average value obtained from such as TCGA Big Data can be used.

In another embodiment, the method for diagnosing metastatic gastric cancer of the present invention comprises comparing the expression levels of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2, respectively, measured from normal tissue and lesion tissue derived from the stomach tissue of the subject It can be more inclusive. In this case, the expression level of each of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 may be advantageous over individual differences in the expression level in the normal state.

In one embodiment, the method of diagnosing metastatic gastric cancer of the present invention may further comprise isolating the nucleus from the sample in a sample isolated from the subject. Referring to FIG. 8C, the amount of PADI2 expressed in the cytoplasm as well as the nucleus of the cell is high, and among the PADI2, the -PADI2 cleaved is highly expressed in the nucleus and is highly involved in invasion (FIG. 9D). Therefore, the use of a diagnostic method that further includes the step of isolating the nucleus can be used to increase the accuracy in the diagnosis and metastatic prediction of metastatic gastric cancer by measuring the expression level of PADI2 in the nucleus.

In one embodiment, the method of diagnosing the metastatic gastric cancer of the present invention may be to diagnose that the stomach cancer of the individual has a high probability of metastasis when the expression levels of each of the compared APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 are remarkably high . In one embodiment, the amount of PADI2 expression in metastatic gastric cancer tissue may be three to ten times greater than the standard expression level, and the amount of PADI2 expression in the gastric cancer tissue obtained from the same individual relative to the amount of expression of PADI2 in normal tissue is three times 10 times or more.

In one embodiment, the method for diagnosing the metastatic gastric cancer of the present invention may be for an individual who has an early gastric cancer. The initial stomach cancer may correspond to T1 to T2 on the basis of the TNM stage system, and may include N stage to N0 to N2, N0 to N1, or N1 to N2, preferably N The weapon may be N0 to N1.

In one embodiment, the method for diagnosing metastatic gastric cancer of the present invention may further comprise measuring the expression level of IL-1β, IL-6, and TNF-β from a sample isolated from the subject. That is, an antibody, a peptide, a protein, or a combination thereof that specifically binds to one or more proteins selected from the group consisting of IL-1 ?, IL-6 and TNF-? Primer, nucleotide, or a combination thereof that specifically binds to a nucleotide sequence encoding at least one protein selected from the group consisting of TNF-1, TNF-6, and TNF-beta; And measuring the expression level of each of the at least one protein selected from the group consisting of IL-1β, IL-6, and TNF-β from the complex.

The expression level of each of the APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 proteins in the diagnostic method of the present invention can be determined by measuring the expression amount of the protein using an antibody, an antigen-binding fragment, a polypeptide or a protein that specifically binds to the protein Can be confirmed. Analysis methods include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis Immunoprecipitation Assay, Complement Fixation Assay, FACS, and Protein Chip are examples of immunosuppressive agents. Such an analytical method can be appropriately performed using techniques known to those skilled in the art.

The expression levels of mRNAs of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 can be measured using a probe, a primer or a nucleotide that specifically binds to the mRNA. Analysis methods include RT-PCR, RNase protection assay (RPA), Northern blotting, or DNA chip. Such an analytical method can be appropriately performed using techniques known to those skilled in the art.

It is to be understood that the terms or elements mentioned in the above methods, such as those mentioned in the description of the claimed compositions and kits, are as mentioned in the description of the compositions and kits claimed above.

An antigen-binding fragment or polypeptide that specifically binds to one or more proteins selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 according to an embodiment, or each fragment thereof, or a nucleotide sequence encoding the PADI2 protein , A primer set or a nucleotide-containing composition for diagnosing metastatic gastric cancer, it is possible to anticipate the possibility of lymph node metastasis early in the stomach cancer and to prevent the metastasis in advance.

According to another aspect of the present invention, there is provided a metastatic gastric cancer diagnostic kit comprising the composition for diagnosing metastatic gastric cancer and a reagent for detecting the metastatic gastric cancer diagnosis composition.

Peptides, proteins, or combinations thereof that specifically bind to one or more proteins selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2, or fragments thereof, to a sample isolated from an individual according to another aspect, or Contacting a probe, a primer, a nucleotide or a combination thereof that specifically binds to a nucleotide sequence encoding at least one protein selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 to form a complex; And measuring the expression level of at least one protein selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 from the complex, and predicting the possibility of lymph node metastasis in early stage of gastric cancer, Can be prevented.

FIG. 1 shows the heatmap of a gene that is increased more than 2-fold in T1 / 2N3-T (tumor) using RNA extracted from normal tissues and cancer tissues classified by each stage of gastric cancer. (The higher the red color, the higher the fold change)
Figure 2 shows the significance (P <0.05) in patients with T1 / 2N3 (0107, 0160, 0399, 0486, and 1027) compared to patients with TNM stage T4N0 (1031, 1101, 211, 220, and 241) (APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2) overexpressing the gene. (N: normal tissue, T: cancer tissue)
FIG. 3 shows mRNA expression levels of 26 pairs of normal tissues and gastric cancer tissues according to TNM stage. FIG. 3A shows the expression levels of PADI2 in normal tissues and gastric cancer tissues in 26 patients, FIG. 3B shows the expression levels of PADI2 in T4N0 patients FIG. 3C shows PADI2 expression levels in gastric cancer tissues in 21 T4N0 patients and 5 T1 / 2N3 patients, FIG. 3D shows 21 patients in T4N0 patients and T1 / 2N3 patients in T1 / 2N3 patients The expression level of PADI2 in gastric cancer tissue relative to normal tissue is shown.
FIG. 4 is a graph showing expression levels in normal tissues and gastric cancer tissues of five genes that are significantly overexpressed in patients with T1 / 2N3 as compared to patients with TNM stage T4N0, as calculated from Cufflinks v2.1.1.
FIG. 5 shows the results of analysis of PADI2 expression in cancer tissues and normal tissues in various carcinomas by the TCGA database.
FIG. 6 shows the results of analysis of the expression of the PADI2 family in cancer tissues and normal tissues in stomach cancer by the TCGA database.
FIG. 7 shows the results of analysis of PADI2 expression level according to each stage of TNM stage by TCGA database.
FIG. 8A shows the amount of PADI2 expression in each gastric cancer cell, FIG. 8B is a schematic representation of the full-length form and PADI2 (truncated form), and FIG. FIG. 8D shows the level of PADI2 expression in cytoplasm and nucleus at SNU0638 at the protein level. FIG.
FIG. 9A shows the degree of cell proliferation by siPADI2 in SNU-638, FIG. 9B shows the degree of cell proliferation according to total-PADI2 and truncated-PADI2 in PADI2 stable cells, and FIG. FIG. 9D shows the degree of cell invasion by total-PADI2 and truncated-PADI2 in PADI2 stable cells.
FIG. 10A shows the results of qRT-PCR in which PADI2 overexpresses SNU-638, and PADI2-induced inflammatory cytokine expression in AGS stable cells overexpressing full-length and truncated-PADI2. FIG. 10B shows the results of RT-PCR on expression levels of TNF-α, IL-1β, and IL-6 in PADI2 by overexpression of full-length PADI2 and truncated-PADI2 in AGS stable cells .
11 shows the information of the patient who provided the sample.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: predictive of lymph node metastasis As a biomarker Of PADI2  excavation

1-1. Collection of patient tissue samples and classification of staging

Patients were classified according to each stage to obtain biomarkers capable of predicting lymph node metastasis at early stage of gastric cancer, and tissue samples were obtained from the patients.

In order to obtain the specimen, in a patient who underwent gastrectomy with primary gastric adenocarcinoma at Seoul National University Hospital, the gastric cancer tissue of fresh condition immediately after gastrectomy and the normal gastric mucosal tissue of the same patient were partially taken, Lt; RTI ID = 0.0 &gt; 80 &lt; / RTI &gt; Each of the above samples was approved by IRB at the Seoul National University Hospital Stomach Cancer Research Institute and was stored with IRB No. H-0806-072-248 along with the consent form.

In order to screen samples, we compared the IRB No: H-0603-072-170 with the IRB-approved IRB No: H-0603-072-170 for retrospective review of the literature, In the AJCC / UICC Seventh Edition TNM staging definition, patients with advanced stage gastric carcinoma of stage 4 had no lymph node metastasis, and patients with stage T stage 1 or stage 2 and stage N stage of lymph node metastasis were 3 Respectively. After that, fresh frozen tissue of gastric cancer tissue and normal gastric mucosal tissue were thawed and the experiment was conducted.

Therefore, each stage is divided into T stage and N stage according to the TNM staging system, which can classify malignant tumors. Among the 26 patients, T stage (stage T4N0, n = 21), and patients with low T stage and high lymph node metastasis (T1N3 and T2N3, n = 5). Tissue samples for analysis were obtained by isolating normal tissues (N) and tumor tissues (T) from a total of 26 patients, and then analyzed for the expression levels of each candidate biomarker in the tissues. The information of each patient is shown in Fig.

1-2. candidate Biomarker  Generate library for expression analysis

Total RNA was extracted from tissues collected from patients, and mRNA was isolated from oligo (dT) from 2 μg of RNA. 100bp Paired-end sequencing was performed. After the patient samples were mixed, a final library was prepared using Illumina's TruSeq RNA Sample Prep Kit.

Specifically, the isolated mRNA was fragmented and synthesized as single-stranded cDNA through random hexamer priming. Secondary strands were synthesized using this as a template and double-strand cDNA was synthesized. End repair was performed to make the blunt-end, and A-tailing and Adapter ligation to attach the adapter. The cDNA library was then amplified using PCR (Polymerase Chain Reaction). 2100 BioAnalyzer was used to confirm the concentration and size of the final product. The resulting library was finally quantified using a KAPA library quantification kit and sequenced using Hiseq2500.

1-3. candidate Biomarker  Expression level analysis

Filtering was performed to remove low quality sequences from the decoded sequence. Specifically, the nucleotide sequence represented by N in the sequence information contained at least 10% of the entire sequence, or the nucleotide sequence having a base of less than Q20 of 40% or more was removed, and the lid having an average quality of Q20 or less was also removed. The entire filtering process was performed by an internally generated program. The filtered sequence was aligned to the species reference genomic sequence (hg19) using STAR v2.4.0b (Dobin et al, 2013).

Expression levels were calculated using Cufflinks v2.1.1 (Trapnell C. et al, 2010) and the calculated expression values were expressed as FPKM (fragments read per kilobase of exon per million fragments mapped). The ensemble 72 was used as a genetic information database, and the non-coding gene region was excluded from the expression measurement using the -mask option. To increase the accuracy of the expression measurement, the multi-read-correction and frag-bias-correct options were additionally used.

Expression values of all 52 samples obtained through Cufflinks were used to identify genes involved in lymph node metastasis. A significant gene was selected for the expression of T4N0-T in normal tissues and non-metastatic gastric carcinoma (T4N0-T), more than 2 times higher than T1 / 2N3-T in lymph node metastasis group and P value <0.01. Tauno M., 2015).

Figure 1 shows the selected 78 genes using Clustvis as a heatmap. (T4N0-N: T4N0-T: normal tissue of the sample without stage 4 and lymph node metastasis (N stage), T4N0-T: stage 4 and no lymph node metastasis Tumor tissues in the sample, T1 / 2N3-T: normal tissue of the sample with lymph node metastasis to stage 1 or 2 and stage 3 of N stage, and lymph node metastasis to T1 / 2N3-T: T stage 1 or 2, Lt; / RTI &gt; tumor tissue). In the above heatmap, the closer the red color is, the higher the expression amount. The closer the color is to the blue color, the lower the expression amount is, and the more red color, the higher the fold change. The results showed that PADI2 was expressed more than 2 times in T1 / 2N3-T lymph node metastasis compared to the total expression level of non-metastatic gastric carcinoma (T4N0-T), and the arrow in the heat map indicates PADI2.

FIG. 2 is a graph showing the relationship between the number of patients (N3) (represented by 0107, 0160, 0399, 0486, and 1027) and the 4th gastric cancer (T4) Five genes with significance were selected in patients (N0) (1031, 1101, 211, 220, and 241) in which no metastasis was observed. In comparison of normal tissues (N) and cancer tissues (T) by selecting APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2, all selected genes were almost consistently expressed in T1 / 2N3, among which PADI2 was the most significant And showed high expression in cancer tissues.

Also, referring to FIG. 4, the expression levels of five genes that are significantly overexpressed in T1 / 2N3 patients compared to patients with TNM stage T4N0 were compared with those of normal tissues and gastric cancer tissues. As a result, APOBEC3A, FCGR3B, SERPINB5, NMU And PADI2 in T1 / 2N3 patients compared with those in T4N0 patients. In particular, T1 / 2N3 patients showed a significant difference when comparing normal tissues and gastric cancer tissues in each stage.

Example  2: lymph node metastasis As a biomarker Of PADI2 mRNA  analysis

2-1. mRNA  Extraction and cDNA synthesis

To investigate whether PADI2 could serve as a lymph node metastatic biomarker, qRT-PCR was performed on 26 pairs of normal mucosal tissues and gastric cancer tissues. First, RNA was extracted from each tissue and cDNA was synthesized.

Specifically, in order to extract total RNA, 26 pairs of normal mucosal tissues and gastric cancer tissues frozen in liquid nitrogen were taken into a 2 mm ³ flask, and the mixture was pulverized with a mortar and then 1 mL of Trizol reagent (Invitrogen, Carlsbad, CA) Mix until completely dissolved. After adding 200 μl of chloroform, the supernatant was mixed with 500 μl of isopropanol, and centrifuged again to confirm the precipitated RNA pellet. After washing the pellet with 1 mL of 75% ethanol (ethanol), the RNA pellet was dried by centrifugation again and distilled water without RNase was added. Absorbance was measured by electrophoresis and spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific, Inc., Waltham, Mass., USA) to measure RNA quality. For cDNA synthesis, 500 ng of RNA was used and performed according to manufacturer's protocol using TOP script cDNA synthesis kit (Enzynomics, Daejeon, Korea).

2-2. real time Polymerase chain reaction  (RT- PCR )

Real-time quantitative real-time PCR was performed using a relative quantification method using the StepOne Plus Real-Time PCR system (Applied Biosystems, Carlsbad, Calif.) To analyze the expression of PADI2 gene. This reaction was carried out by adding 10 μl of 2 × SYBR Green PCR Master Mix (Applied Biosystems, Monza, Italy), 2.5 pmole / μl of each primer and 1 μl of cDNA to 20 μl total. The PCR reaction conditions were 95 deg. C for 10 min, followed by 40 cycles of 95 deg. C for 15 sec, 60 deg. C for 15 sec, and 72 deg. C for 1 min. Gene expression was quantified using the calculated Ct value (threshold cycle), and PADI2 expression in each sample was corrected to GAPDH expression value. The primer sequences used are shown in Table 1.

Gene Forward primer (5 '- &gt; 3') Reverse primer (5 'to 3') GAPDH TCTCAGGCCTGGTCTCCAT AAGATGGGAGTCAGAGGGGAAT PADI2 CATTTCCTGGTATGACAACGA CTTCCTCTTGTGCTCTTGCT

As a result, as shown in FIG. 3, PADI2 mRNA was significantly higher in gastric cancer tissues than in normal mucosal tissues ( p = 0.047, FIG. 3A). Patients were divided into two groups (T4N0; n = 21 and T1 / 2N3; n = 5). PADI2 expression was not significantly different between the two groups ( p = 0.981, FIG. 3B), but expression of PADI2 in gastric cancer tissues was significantly higher in the T1 / 2N3 group than in the T4N0 group ( P <0.001, Fig. 3C). In addition, when PADI2 expression in gastric cancer tissue was calculated as a ratio of PADI2 expression in normal mucosal tissues in each patient, there was also a significant difference (about 4 to 5 times) between the two groups ( p = 0.022, Figure 3D). Therefore, the value measured by the amount of mRNA expression of PADI2 can be used as an index of overexpression of PADI2, and PADI2 can function as a biomarker in early gastric cancer with high lymph node metastasis.

Example  3: Lymph node metastasis by big data analysis As a biomarker Of PADI2  evaluation

3-1. The Cancer Genome Atlas ( TCGA ) Big Data  analysis

The Cancer Genome Atlas (TCGA) Big Data analysis was performed to see if PADI2 could serve as a lymph node metastasis biomarker. TCGA is the most important study of the human genome project, led by the National Institutes of Health, and is a study of genomes of large-scale cancer patients and released to researchers worldwide in the form of big data. The TCGA homepage (https://cancergenome.nih.gov) and the UCSC Cancer Browser homepage (https://genome-cancer.ucsc.edu) contain clinical information on more than 10,000 patients, as well as genetic mutations, methylation profiles profile, and all data from raw data to processed data can be downloaded for gene expression information. We downloaded all published data for RNA expression and analyzed RNA expression data in 8,981 cancer tissues and 686 normal tissues in a total of 31 carcinomas (see bottom of FIG. 5). Among them, 401 cancer tissues and 35 normal tissues were analyzed in gastric cancer. Cancer stage information of the relevant tissues was referred to the clinical information data which are disclosed together. Differences in gene expression between the two groups were analyzed using the Wilcoxon rank sum method. The differences in gene expression levels were analyzed by the ANOVA method and the differences between the groups were analyzed using the Tukey method. P values less than 0.05 were considered statistically significant.

As a result, as shown in FIG. 5, in the case of thyroid carcinoma (THCA), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), cervical cancer (CESC), and stomach adenocarcinoma This phenomenon was confirmed, but the expression of PADI2, especially in gastric cancer, was found to be significantly higher in cancer tissues than in normal tissues ( P <0.001). In addition, the expression of PADI1, PADI3, and PADI4, which are the same as the PADI2 gene, in gastric cancer tissues and stomach tissues (FIG. 6), PADI1 and PADI3 were significantly increased in cancer tissues than in normal tissues In contrast, the expression of PADI4 was decreased in cancer tissues.

In order to check whether the increase of expression of PADI2 in the TCGA database was different according to the stage of gastric cancer, correlation analysis was performed with clinical information (FIG. 7). As a result, it was confirmed that the expression of PADI2 was different according to T stage ( P <0.001). Especially, the difference between T1 and T2 was the most significant ( P = 0.007). ( P <0.001), but the difference was mainly due to the presence of cancer tissues ( P = 0.036) in normal tissue and N1 stage, and cancer tissues ( P = 0.011), but there was no statistically significant difference. Thus, by measuring the overexpression of PADI2 in gastric cancer, it is possible to predict the lymph node metastasis in the patients of T1 to T2 stage in the early stage of gastric cancer and at least in the TNM stage, and PADI2 is initially (at least N0 to N1) And function as a biomarker for lymph node metastasis, and thus can serve as a good biomarker in predicting lymph node metastasis.

Example  4. PADI2  Location of expression

4-1. Cell line and cell culture

In order to select cells for PADI2 expression site analysis, SNU-638 overexpressing PADI2 and SNU-719 and AGS with little expression of PADI2 were selected by analysis on the gene database as in Example 1-3 8A)

For further experiments, human stomach cancer cells SNU-638 and SNU-719 were purchased from Korean Cell Line Bank (Seoul, Korea) and AGS were purchased from the American Type Culture Collection (ATCC) (Virginia, USA). All stomach cancer cells were cultured in RPMI 1640 medium (WelGENE) supplemented with 10% fetal bovine serum (FBS, WelGENE) and 1% penicillin / streptomycin in a 5% CO ₂ humidified incubator at 37 ° C.

4-2. Reagents and Antibodies

PADI2 antibodies were purchased from Proteintech (12110-1-AP), and α-tubulin (b-7) and Lamin B (c-20) antibodies were purchased from Santa Cruz Technology. β-actin antibodies were purchased from Sigma-aldrich (A2228).

4-3. Cell nucleus and cytoplasmic fraction, and Western Blot

Cells were washed twice with cold PBS to separate the nucleus and cytoplasm from the cells, and the cells were disrupted with M-PER ^™ Mammalian Protein Extract Reagent (ThermoFisher Scientific, 78501) supplemented with phosphatase and protease inhibitor. The cell lysate was centrifuged at 13,000 rpm / min for 20 min at 4 &lt; 0 &gt; C for purification. The supernatant was collected and the concentration of total protein was measured by BCA protein quantitation report method. The same amount of cell protein was heated at 95 ° C for 5 minutes, separated by SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to PVDF (polyvinylidene difluoride) membrane. The membranes were blocked with Tris-buffered saline containing 0.05% (v / v) Tween 20 and 5% (w / v) skim milk for 1 hour and then incubated with appropriate antibodies. Lt; / RTI &gt; overnight. Primary antibody binding was visualized with Chemiluminscent Substrate (GE, Visual Protein Biotechnology Corporation, LF08-500) using Horseradish peroxidase-attached goat anti-mouse or anti-rabbit immunoglobin G secondary antibody (Merck Millipore).

As a result, as shown in FIG. 8C, PADI2 was measured in two sizes, and it was found that full-length form and truncated form existed. In addition, as shown in Fig. 8D, the cytoplasm appeared to have a full-length form and the nucleus had a truncated form.

Example  5. On PADI2  Assessment of cell viability and evaluation of cell invasion by

5-1. Production of stable cell line

Human PADI2 cDNA was amplified from cDNA of SNU-638 cell line by PCR and then introduced into the EcoRI and SalI restriction sites of pFLAG-CMV-6c vector to obtain Flag-cPADI2 (full-length) and Flag-nPADI2 -Form). &Lt; / RTI &gt;

To induce the overexpression of the PADI2 gene, the Flag-cPADI2 (full-length form) and Flag-nPADI2 (truncated form) were introduced into the XhoI and ClaI restriction sites of the Lpcx vector to construct a retrovirus plasmid.

In this plasmid, PADI2 was amplified by the amino acid sequence of SEQ ID NO: 14 (SEQ ID NO: 14) according to the nucleotide sequence information NM_007365.2 (full length-PADI2) and truncated -PADI2 (BC009701.1) PADI2 (cPADI2) and LPCX-truncated-PADI2 (nPADI2) plasmids, respectively, with the cut-PADI2 (1 to 435 of the amino acid sequence of SEQ ID NO: 14)

The primers used are as shown in Tables 2 and 3.

Flag-cPADI2 Flag-nPADI2 Forward Primer AAAGAATTCATGCTGCGCGAGCGGACCGT AAAGAATTCATGCTGCGCGAGCGGACCGT Reverse Primer AAAGTCGACTCAGGGCACCATGTGCCACC GATCGTCGACTGACAGAGGAAAGCTGCTCCCGAT

Lpcx-flag-cPADI2 Lpcx-flag-nPADI2 Forward Primer GATCCTCGAGATGGACTACAAAGACGATGACGAC GATCCTCGAGATGGACTACAAAGACGATGACGAC Reverse Primer GATCATCGATCTAGGGCACCATGTGCCACCACTT GATCATCGATCTACAGAGGAAAGCTGCTCCCGAT

As a virus production host cell, a GP2-293 cell line was used. GP2-293 cell line was inoculated into 100-mm culture dish 24 hours before transfection. 10 DNA DNA and 5 VS VSV-G were transfected with polyethylenimine (PEI). Forty-eight hours after the transfection, the virus soup was collected from the cell culture and purified using a 0.45-μm filter. 3-3.5 ml of the purified virus was treated with AGS (1x10 ⁵ cells / 100-mm culture dish) with 8 mg / ml polybrene, and this was repeated three times at 8 hour intervals. After infecting the virus, the cells were changed to a new medium. After 24 hours, cells infected with 2 μg / ml puromycin were selected.

8E and 8F, the amount of PADI2 expression in the whole cell, cytoplasm and cell nucleus was measured as in Example 4-3. As a result, in accordance with the results in Example 4, the whole-PADI2 was found to be cytoplasmic, The truncated -PADI2 was found to be mainly expressed in the nucleus.

On the other hand, when the stable cells were cultured and counted every two days for 6 days, the cell proliferation rate was significantly increased when -PADI2 was overexpressed as shown in FIG. 9B.

5-2. siRNA  Used PADI2  Assessment of cell proliferation rate upon inhibition of expression

SNU-638 cells according to Example 4-1 were prepared, and the expression of PADI2 was inhibited using siRNA, and the cell proliferation rate was evaluated.

Specifically, SNU-638 cells were inoculated at 60-80% density in the culture dish. The next day, the siRNA transfection reagent and siRNA were added to the Opti-MEM medium (Gibco, 31985070) at a 1: 3 ratio and incubated for 5 minutes. The mixture was placed in the cell culture medium and incubated for 1-3 days at 37 ° C. PADI2 siRNA was purchased from Thermo Fisher Scientific (4392420) and its transfection reagent was purchased from invitrogen (13778-075).

As a result, consistent with the results of Example 5-1, it can be seen that when PADI2 expression is inhibited as shown in FIG. 9A, the cell proliferation rate is decreased as compared with the control group.

5-3. PADI2  Cells with active inhibition Survival rate  evaluation

SNU-638 overexpressing PADI2 and SNU-719 with little expression of PADI2 were prepared according to Example 4-1, and then the PAD inhibitor Cl-amidine was purchased from Merck Millipore (506282-10MGCN) Treated at the indicated concentrations.

Specifically, the cytotoxicity of Cl-amidine, a PADI2 inhibitor, was measured by measuring the ability of mitochondria to reduce the yellow water-soluble substrate MTT tetrazolium to a purple, water-insoluble MTT formazan by dehydrogenase MTT analysis was performed. Each of the SNU-638 (high PADI2) and SNU-719 (low PADI2) cell lines was inoculated into a 96-well plate at 2 × 10 ⁵ cells and incubated with Cl-amidine at a predetermined concentration (shown in FIG. 9C) Lt; / RTI &gt; Then, 1/10 MTT solution was added to the cell culture and incubated for 4 hours. The culture medium was carefully removed from the cell culture, and 200 μl of DMSO was added to each well. After incubation for 30 minutes, the absorbance was measured using a microplate reader at 540 nm.

As a result, as shown in Fig. 9C, SNU-638 overexpressing PADI2 and SNU-719 almost not expressing PADI2 all had decreased cell viability by treatment with Cl-amidine. However, consistent with the results of Examples 5-1 and 5-2, it was shown that SNU-638 overexpressing PADI2 was more sensitive to PADI2 inhibitors than to SNU-719 which was not, resulting in decreased cell survival.

5-4. Cellular invasion analysis

For cell invasion analysis, a BioCoat Matrigel invasion chamber (Corning, 354578) was used. The PADI2-overexpressing stable cells of Example 5-1 were cultured in the absence of FBS serum for 24 hours, and then the cells were added to the upper part of the Traswell at 1 × 10 ⁵ cells. The lower part of the Transwell was supplemented with a medium containing serum, and the upper part where the cells were present was cultured in a serum-free medium. After 24 hours, the cells remaining in the chamber were wiped with a cotton swab. To stain the cells that migrated to the lower part of the Trasnwell, the cells were fixed with 70% ethanal for 10 minutes. Ethanol was removed and the cells were stained with 0.05% crystal violet. Thereafter, the number of migrated cells was observed under a microscope, and 4 images of photographic images were obtained at random and the number of migrated cells was measured.

As a result, as shown in FIG. 9D, when the invasion overexpresses PADI2, the rate of cell invasion increases sharply as compared with that of the control. In particular, the incidence of invasion increases markedly when the cut-PADI2 is overexpressed.

Therefore, the overexpression of PADI2 in gastric cancer cells increases cell proliferation rate and increases cell invasion. Therefore, PADI2 can be a good marker for predicting metastatic gastric cancer and predicting metastatic potential.

In particular, the overexpression of PADI2 overexpression or truncated -PADI2 in the nucleus can be a marker for further increasing the accuracy of predicting the metastatic potential for metastatic gastric cancer.

Example  6. Metastatic gastric cancer As a biomarker PADI2  When measuring Cofactor  excavation

6-1. Cell line and siRNA  process

AGS stable cells of Example 5-1 and SNU-638 treated with the siRNA of Example 5-2 were prepared.

6-2. RNA extraction and qRT - PCR (quantitative Reverse Transcriptase PCR )

RNA was extracted from the cells of Example 6-1 according to the manufacturer's instructions. Total RNA was extracted using the easy-BLUE total RNA extreactio kit (Promega, 17061). 2 쨉 g of extracted RNA was prepared using oligo dT primer, yeast transcription enzyme (promega, M1705) and an enzyme buffer. PCR was performed using AccuPower ^™ PCR PreMix kit (Bioneer Co., Daejon, S. Korea) using a gene-specific primer pair to be used as a template. Then, the PCR amplification product was electrophoresed, and the band was analyzed to measure changes in the expression level of the target gene. The primer pairs used in the above qRT-PCR are shown in Table 4.

Target gene Forward Primer Reverse Primer PADI2 TGAGAGCCTCGTGCAAGAGAACC CGTGAGCCCCAAACTCCTTCTTG IL-6 GTAGCCGCCCCACACAGA CATGTCTCCTTTCTCAGGGCTG IL- 1β AATCTGTACCTGTCCTGCGTGTT TGGGTAATTTTTGGGATCTACACTCT TNF -α TCTTCTCGAACCCCGAGTGA CCTCTGATGGCACCACCAG 18S CCCAACTTCTTAGAGGGACA TAGTCAAGTTCGACCGTCTT

The results of qRT-PCR are shown in Fig. 10A. The expression levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 were increased by PADI2 overexpression.

6-3. PADI2  Inflammation due to overexpression Cytokine mRNA  Measurement of expression level

RT-PCR was performed to assess the expression level of inflammatory cytokines at the mRNA level as PADI2 was overexpressed.

Specifically, quantitative real-time PCR was performed using SYBR Premix Ex Taq II (TAKARA, RR820A) on ViiA ^TM 7 real-time PCR systems (Applied Biosystems). The previously prepared cDNA was diluted 1/5 and then mixed with a target gene-specific primer pair and 2x SYBR premix and incubated. At this time, mRNA levels of various genes were measured in triplicate and corrected with GAPDH RNA. The primer pairs used in the RT-PCR of this Example are shown in Table 5.

Target gene Forward Primer Reverse Primer cPADI2 ATGGAGAGGCCATCATGTTC TGTCAGTCCCAGCTCCTTCT nPADI2 ACTCTCCCCGAGATGGAAAC GAATAATTCCCCCAGCCACT IL-6 CTCTGGGAAATCGTGGAAAT CCAGTTTGGTAGCATCCATC IL- 1β GGACAAGCTGAGGAAGATGC TCTTTCAACACGCAGGACAG TNF -α TGCTTGTTCCTCAGCCTCTT TGAGGTACAGGCCCTCTGAT GAPDH CCATCTTCCAGGAGCGAGAT GTGTCAGGTACGGTAGTGAC

RT-PCR results are shown in Figs. 10B and 10C. The expression levels of TNF-α, IL-1β and IL-6, which are inflammatory cytokines, are increased according to overexpression of total-PADI2 and truncated-PADI2 as in Example 6-2 above. Therefore, TNF-α, IL-1β, and IL-6 may be good candidates for the co-factor when measuring PADI2 as a biomarker for predicting lymph node metastatic gastric cancer.

&Lt; 110 > AICT (ADVANCED INSTITUTES OF CONVERGENCE TECHNOLOGY) <120> Marker for predicting lymph node metastasis and method for          predicting lymph node metastasis using same <130> PN116685 <160> 26 <170> Kopatentin 2.0 <210> 1 <211> 4363 <212> DNA <213> Homo sapiens <400> 1 gcaggctgct ggagaaggcg cacctgctgc aggtgctccc ggccgccccg gaccagcgag 60 cgcgggcact gcggcgggga ggatgctgcg cgagcggacc gtgcggctgc agtacgggag 120 ccgcgtggag gcggtgtacg tgctgggcac ctacctctgg accgatgtct acagcgcggc 180 cccagccggg gcccaaacct tcagcctgaa gcactcggaa cacgtgtggg tggaggtggt 240 gcgtgatggg gaggctgagg aggtggccac caatggcaag cagcgctggc ttctctcgcc 300 cagcaccacc ctgcgggtca ccatgagcca ggcgagcacc gaggccagca gtgacaaggt 360 caccgtcaac tactatgacg aggaagggag cattcccatc gaccaggcgg ggctcttcct 420 cacagccatt gagatctccc tggatgtgga cgcagaccgg gatggtgtgg tggagaagaa 480 caacccaaag aaggcatcct ggacctgggg ccccgagggc cagggggcca tcctgctggt 540 gaactgtgac cgagagacac cctggttgcc caaggaggac tgccgtgatg agaaggtcta 600 cagcaaggaa gatctcaagg acatgtccca gatgatcctg cggaccaaag gccccgaccg 660 cctccccgcc ggatacgaga tagttctgta catttccatg tcagactcag acaaagtggg 720 cgtgttctac gtggagaacc cgttcttcgg ccaacgctat atccacatcc tgggccggcg 780 gaagctctac catgtggtca agtacacggg tggctccgcg gagctgctgt tcttcgtgga 840 aggcctctgt ttccccgacg agggcttctc aggcctggtc tccatccatg tcagcctgct 900 ggagtacatg gcccaggaca ttcccctgac tcccatcttc acggacaccg tgatattccg 960 gattgctccg tggatcatga cccccaacat cctgcctccc gtgtcggtgt ttgtgtgctg 1020 catgaaggat aattacctgt tcctgaaaga ggtgaagaac cttgtggaga aaaccaactg 1080 tgagctgaag gtctgcttcc agtacctaaa ccgaggcgat cgctggatcc aggatgaaat 1140 tgagtttggc tacatcgagg ccccccataa aggcttcccc gtggtgctgg actctccccg 1200 agatggaaac ctaaaggact tccctgtgaa ggagctcctg ggcccagatt ttggctacgt 1260 gcccgggag cccctctttg agtctgtcac cagccttgac tcatttggaa acctggaggt 1320 cagtccccca gtgaccgtga acggcaagac atacccgctt ggccgcatcc tcatcgggag 1380 cagctttcct ctgtctggtg gtcggaggat gaccaaggtg gtgcgtgact tcctgaaggc 1440 ccagcaggtg caggcgcccg tggagctcta ctcagactgg ctgactgtgg gccacgtgga 1500 tgagttcatg tcctttgtcc ccatccccgg cacaaagaaa ttcctgctac tcatggccag 1560 cacctcggcc tgctacaagc tcttccgaga gaagcagaag gacggccatg gagaggccat 1620 catgttcaaa ggcttgggtg ggatgagcag caagcgaatc accatcaaca agattctgtc 1680 caacgagagc cttgtgcagg agaacctgta cttccagcgc tgcctagact ggaaccgtga 1740 catcctcaag aaggagctgg gactgacaga gcaggacatc attgacctgc ccgctctgtt 1800 caagatggac gaggaccacc gtgccagagc cttcttccca aacatggtga acatgatcgt 1860 gctggacaag gacctgggca tccccaagcc attcgggcca caggttgagg aggaatgctg 1920 cctggagatg cacgtgcgtg gcctcctgga gcccctgggc ctcgaatgca ccttcatcga 1980 cgacatttct gcctaccaca aatttctggg ggaagtccac tgtggcacca acgtccgcag 2040 gaagcccttc accttcaagt ggtggcacat ggtgccctga cctgccaggg gccctggcgt 2100 ttgcctcctt cgcttagttc tccagaccct ccctcacacg cccagagcct tctgctgaca 2160 tggactggac agccccgctg ggagaccttt gggacgtggg gtggaatttg gggtatctgt 2220 gccttgccct ccctgagagg ggcctcagtg tcctctgaag ccatccccag tgagcctcga 2280 ctctgtccct gctgaaaata gctgggccag tgtctctgta gccctgacat aaggaacaga 2340 acacaacaaa acacagcaaa ccatgtgccc aaactgctcc ccaaagaatt ttgagtctct 2400 aatctgacac tgaatgaggg gagaagggaa ggagattctg ggattgccag ttcttccagc 2460 agccatgctc tgaaaatcaa ggtagaatcc atggaaaggg accccaggac cccgggaccc 2520 tagacgtatc ttgaactgcc atcgtcattt caaatacatc tccctcaggg tttccaggtg 2580 gccaccccca attattcatt ccttaccaac ctctcaaatc ctcttggctt tctctctgca 2640 gtgtggacac tgttggctag tcctccccac tccctgaggg tccagtaagt tagcttagaa 2700 ccttcctgga aacatttcat ctgagcaggt ttccccacgt gtgggatgct ccttttgcct 2760 catctgtctc agggatgcag gctcccccgc atgcatgggg atttctcccc agaccagcat 2820 acttgtgacc tgagagttca atgcgtaaag atgcccctgg tcagccatat ccatcttctc 2880 ttgcctggtc cttgattctc tggccgctcc ctgaccttcc tccttccact gccttgactt 2940 tcttcctttt tattcctggt gccatctgtc caggcagcta gacaagaact tgttcgccag 3000 cagccagatt caggccttcc caggggcata ataagtgacc agcccctcct ctccggacat 3060 cagatccaac acataaggac cctggcctac cctccagccc aacagccagt tctgggtcag 3120 ctgccaactt aggggtggtt tgattatccc attgaaattc accagtgcct ttgccaaaga 3180 ccctctcatt tggacatacc cagattcatt ccctggctcc aactgaaaag actcagtttc 3240 aatcgttaaa agttccttta gggccagaag aataaatgaa ttataatccc attttgaaga 3300 accgatttat aaccaatgaa aaggttataa tgtaatttat attcttggag gaacaagatt 3360 ttcatttggg attatttcct tcaaccattc aacaaacatt tgttgtatgc cactaagcgc 3420 caggcacggc gttgggctct gcaaacacag tggttagtag cagtctggac ctggtcccta 3480 ctggcatgga acccatcact ccccaacatg caaagcccac atttaaaggc cagcctctgc 3540 cccttcagtg atgcgctctt tagaaatgcc agtccactat attcagaaat ccgcagggca 3600 caaaacttcc agcaagtcac tgttgtggtg aaatgggcag tgggggtggg gggtcttctt 3660 taaacaggcc cccttcccat ctacctagcc agtacccatc caatgagtcc ccagagcctc 3720 cagaagctgt tgtctcctct ctggggacag cagctcctgc ctttggaggc caaagcccca 3780 gatctctcca gccccagagc tgaaaacacc aagtgcctat ttgagggtgt ctgtctggag 3840 acttagagtt tgtcatgtgt gtgtgtgtgt ttggttaatg tgggtttatg ggttttcttt 3900 cttttttttc tttttttttt tagtctacat tagggggaag tgagcgcctc ccatgtgcag 3960 acagtgtgtc tttatagatt tttctaaggc tttccccaat gatgtcggta atttctgatg 4020 tttctgaagt tcccaggact cacacacccg ttcccatctc acttgcccac ccagtgtgac 4080 aaccctcggt gtggatatac ccccgtggac tcatggctct tccccacccc cactttctat 4140 aaatgtaggc ctagaatacg cttctctgtt gcaaaactca gctaagttcc tgcttccacc 4200 ttgatgttga aatatcttat gtaagagggc aggggatgtc gtgaagatgg caagaagaac 4260 acagtttcaa atttctggaa aagagcctgt ggtggagatc taaagatgtt tagggaagag 4320 ctcgactaaa gaacaatgaa ataaatggtc caaggggaag tca 4363 <210> 2 <211> 1998 <212> DNA <213> Homo sapiens <400> 2 atgctgcgcg agcggaccgt gcggctgcag tacgggagcc gcgtggaggc ggtgtacgtg 60 ctgggcacct acctctggac cgatgtctac agcgcggccc cagccggggc ccaaaccttc 120 agcctgaagc actcggaaca cgtgtgggtg gaggtggtgc gtgatgggga ggctgaggag 180 gtggccacca atggcaagca gcgctggctt ctctcgccca gcaccaccct gcgggtcacc 240 atgagccagg cgagcaccga ggccagcagt gacaaggtca ccgtcaacta ctatgacgag 300 gaagggagca ttcccatcga ccaggcgggg ctcttcctca cagccattga gatctccctg 360 gatgtggacg cagaccggga tggtgtggtg gagaagaaca acccaaagaa ggcatcctgg 420 acctggggcc ccgagggcca gggggccatc ctgctggtga actgtgaccg agagacaccc 480 tggttgccca aggaggactg ccgtgatgag aaggtctaca gcaaggaaga tctcaaggac 540 atgtcccaga tgatcctgcg gaccaaaggc cccgaccgcc tccccgccgg atacgagata 600 gttctgtaca tttccatgtc agactcagac aaagtgggcg tgttctacgt ggagaacccg 660 ttcttcggcc aacgctatat ccacatcctg ggccggcgga agctctacca tgtggtcaag 720 tacacgggtg gctccgcgga gctgctgttc ttcgtggaag gcctctgttt ccccgacgag 780 ggcttctcag gcctggtctc catccatgtc agcctgctgg agtacatggc ccaggacatt 840 cccctgactc ccatcttcac ggacaccgtg atattccgga ttgctccgtg gatcatgacc 900 cccaacatcc tgcctcccgt gtcggtgttt gtgtgctgca tgaaggataa ttacctgttc 960 ctgaaagagg tgaagaacct tgtggagaaa accaactgtg agctgaaggt ctgcttccag 1020 tacctaaacc gaggcgatcg ctggatccag gatgaaattg agtttggcta catcgaggcc 1080 ccccataaag gcttccccgt ggtgctggac tctccccgag atggaaacct aaaggacttc 1140 cctgtgaagg agctcctggg cccagatttt ggctacgtga cccgggagcc cctctttgag 1200 tctgtcacca gccttgactc atttggaaac ctggaggtca gtcccccagt gaccgtgaac 1260 gccaagacat acccgcttgg ccgcatcctc atcgggagca gctttcctct gtctggtggt 1320 cggaggatga ccaaggtggt gcgtgacttc ctgaaggccc agcaggtgca ggcgcccgtg 1380 gagctctact cagactggct gactgtgggc cacgtggatg agttcatgtc ctttgtcccc 1440 atccccggca caaagaaatt cctgctactc atggccagca cctcggcctg ctacaagctc 1500 ttccgagaga agcagaagga cggccatgga gaggccatca tgttcaaagg cttgggtggg 1560 atgagcagca agcgaatcac catcaacaag attctgtcca acgagagcct tgtgcaggag 1620 aacctgtact tccagcgctg cctagactgg aaccgtgaca tcctcaagaa ggagctggga 1680 ctgacagagc aggacatcat tgacctgccc gctctgttca agatggacga ggaccaccgt 1740 gccagagcct tcttcccaaa catggtgaac atgatcgtgc tggacaagga cctgggcatc 1800 cccaagccat tcgggccaca ggttgaggag gaatgctgcc tggagatgca cgtgcgtggc 1860 ctcctggagc ccctgggcct cgaatgcacc ttcatcgacg acatttctgc ctaccacaaa 1920 tttctggggg aagtccactg tggcaccaac gtccgcagga agcccttcac cttcaagtgg 1980 tggcacatgg tgccctga 1998 <210> 3 <211> 1444 <212> DNA <213> Homo sapiens <400> 3 ggagaagggg tggggcaggg tatcgctgac tcagcagctt ccaggttgct ctgatgatat 60 attaaggctc ctgaatccta agagaatgtt ggtgaagatc ttaacaccac gccttgagca 120 agtcgcaaga gcgggaggac acagaccagg aaccgagaag ggacaagcac atggaagcca 180 gcccagcatc cgggcccaga cacttgatgg atccacacat attcacttcc aactttaaca 240 atggcattgg aaggcataag acctacctgt gctacgaagt ggagcgcctg gacaatggca 300 cctcggtcaa gatggaccag cacaggggct ttctacacaa ccaggctaag aatcttctct 360 gtggctttta cggccgccat gcggagctgc gcttcttgga cctggttcct tctttgcagt 420 tggacccggc ccagatctac agggtcactt ggttcatctc ctggagcccc tgcttctcct 480 ggggctgtgc cggggaagtg cgtgcgttcc ttcaggagaa cacacacgtg agactgcgta 540 tcttcgctgc ccgcatctat gattacgacc ccctatataa ggaggcactg caaatgctgc 600 gggatgctgg ggcccaagtc tccatcatga cctacgatga atttaagcac tgctgggaca 660 cctttgtgga ccaccaggga tgtcccttcc agccctggga tggactagat gagcacagcc 720 aagccctgag tgggaggctg cgggccattc tccagaatca gggaaactga aggatgggcc 780 tcagtctcta aggaaggcag agacctgggt tgagcagcag aataaaagat cttcttccaa 840 gaaatgcaaa cagaccgttc accaccatct ccagctgctc acagacgcca gcaaagcagt 900 atgctcccga tcaagtagat ttttaaaaaa tcagagtggg ccgggcgcgg tggctcacgc 960 ctgtaatccc agcactttgg aggccaaggc gggtggatca cgaggtcagg agatcgagac 1020 catcctggct aacacggtga aaccctgtct ctactaaaaa tacaaaaaat tagccaggcg 1080 tggtggcggg cgcctgtagt cccagctact ctggaggctg aggcaggaga gtagcgtgaa 1140 cccgggaggc agagcttgcg gtgagccgag attgcgctac tgcactccag cctgggcgac 1200 agtaccagac tccatctcaa aaaaaaaaaa accagactga attaatttta actgaaaatt 1260 tctcttatgt tccaagtaca caatagtaag attatgctca atattctcag aataattttc 1320 aatgtattaa tgaaatgaaa tgataatttg gcttcatatc tagactaaca caaaattaag 1380 aatcttccat aattgctttt gctcagtaac tgtgtcatga attgcaagag tttccacaaa 1440 cact 1444 <210> 4 <211> 1390 <212> DNA <213> Homo sapiens <400> 4 ggagaagggg tggggcaggg tatcgctgac tcagcagctt ccaggttgct ctgatgatat 60 attaaggctc ctgaatccta agagaatgtt ggtgaagatc ttaacaccac gccttgagca 120 agtcgcaaga gcgggaggac acagaccagg aaccgagaag ggacaagcac atggaagcca 180 gcccagcatc cgggcccagg cataagacct acctgtgcta cgaagtggag cgcctggaca 240 atggcacctc ggtcaagatg gaccagcaca ggggctttct acacaaccag gctaagaatc 300 ttctctgtgg cttttacggc cgccatgcgg agctgcgctt cttggacctg gttccttctt 360 tgcagttgga cccggcccag atctacaggg tcacttggtt catctcctgg agcccctgct 420 tctcctgggg ctgtgccggg gaagtgcgtg cgttccttca ggagaacaca cacgtgagac 480 tgcgtatctt cgctgcccgc atctatgatt acgaccccct atataaggag gcactgcaaa 540 tgctgcggga tgctggggcc caagtctcca tcatgaccta cgatgaattt aagcactgct 600 gggacacctt tgtggaccac cagggatgtc ccttccagcc ctgggatgga ctagatgagc 660 acagccaagc cctgagtggg aggctgcggg ccattctcca gaatcaggga aactgaagga 720 tgggcctcag tctctaagga aggcagagac ctgggttgag cagcagaata aaagatcttc 780 ttccaagaaa tgcaaacaga ccgttcacca ccatctccag ctgctcacag acgccagcaa 840 agcagtatgc tcccgatcaa gtagattttt aaaaaatcag agtgggccgg gcgcggtggc 900 tcacgcctgt aatcccagca ctttggaggc caaggcgggt ggatcacgag gtcaggagat 960 cgagaccatc ctggctaaca cggtgaaacc ctgtctctac taaaaataca aaaaattagc 1020 caggcgtggt ggcgggcgcc tgtagtccca gctactctgg aggctgaggc aggagagtag 1080 cgtgaacccg ggaggcagag cttgcggtga gccgagattg cgctactgca ctccagcctg 1140 ggcgacagta ccagactcca tctcaaaaaa aaaaaaacca gactgaatta attttaactg 1200 aaaatttctc ttatgttcca agtacacaat agtaagatta tgctcaatat tctcagaata 1260 attttcaatg tattaatgaa atgaaatgat aatttggctt catatctaga ctaacacaaa 1320 attaagaatc ttccataatt gcttttgctc agtaactgtg tcatgaattg caagagtttc 1380 cacaaacact 1390 <210> 5 <211> 2394 <212> DNA <213> Homo sapiens <400> 5 attcttggtg ctgggtggat ctaaatccag gagatggggg caagcatcct gggaaagctg 60 agggcacact ctggcagatt ctgtgtgtgt cctcagatgc tcagccgcag acctttggga 120 gagtaaaggg ggcacaccca cccaccttgc ctccaggctc tttccttcct attcctgttc 180 tatggtgggg ctccattgcg agacttcaga ttgagaaatc agatgaagtt tcaagaaaag 240 gaaactggca ggtgacagag atgggtggag ggactgggga aaggctgttt actccctcct 300 gtctagtcgg cttggtccct ttagggctcc ggatatcttt ggtgacttgt ccactccagt 360 gtggcatcat gtggcagctg ctcctcccaa ctgctctgct acttctagtt tcagctggca 420 tgcggactga agatctccca aaggctgtgg tgttcctgga gcctcaatgg tacagcgtgc 480 ttgagaagga cagtgtgact ctgaagtgcc agggagccta ctcccctgag gacaattcca 540 cacagtggtt tcacaatgag aacctcatct caagccaggc ctcgagctac ttcattgacg 600 ctgccacagt caacgacagt ggagagtaca ggtgccagac aaacctctcc accctcagtg 660 acccggtgca gctagaagtc catatcggct ggctgttgct ccaggcccct cggtgggtgt 720 tcaaggagga agaccctatt cacctgaggt gtcacagctg gaagaacact gctctgcata 780 aggtcacata tttacagaat ggcaaagaca ggaagtattt tcatcataat tctgacttcc 840 acattccaaa agccacactc aaagatagcg gctcctactt ctgcaggggg cttgttggga 900 gtaaaaatgt gtcttcagag actgtgaaca tcaccatcac tcaaggtttg gcagtgtcaa 960 ccatctcatc attctctcca cctgggtacc aagtctcttt ctgcttggtg atggtactcc 1020 tttttgcagt ggacacagga ctatatttct ctgtgaagac aaacatttga agctcaacaa 1080 gagactggaa ggaccataaa cttaaatgga gaaaggaccc tcaagacaaa tgacccccat 1140 cccatggggg taataagagc agtggcagca gcatctctga acatttctct ggatttgcaa 1200 ccccatcatc ctcaggcctc tctacaagca gcaggaaaca tagaactcag agccagatcc 1260 tttatccaac tctcgatttt tccttggtct ccagtggaag ggaaaagccc atgatcttca 1320 agcagggaag ccccagtgag tagctgcatt cctagaaatt gaagtttcag agctacacaa 1380 acactttttc tgtcccaacc attccctcac agcaaagcaa caatacaggc tagggatggt 1440 aatcctttaa acatacaaaa attgctcgta ttataaatta cccagtttag agggaaaaaa 1500 agaaaataat tattcctaaa caaatggata agtagaatta atggttgagg caggacccta 1560 cagagtgtgg gaactgctgg ggatctagag aattcagtgg gaccaatgaa agcatggctg 1620 agaaatagca gggtagtcca ggatagtcta agggaggtgt tcccatctga gcccagagat 1680 aagggtgtct tcctagaaca ttagccgtag tggaattaac aggaaatcat gagggtgacg 1740 tagaattgag tcttccaggg gactctatca gaactggacc atttccaagt atataacgat 1800 gagtcctcta atgctaggag tagaaaatgg tcctaggaag gggactgagg attggggtgg 1860 gggtggggtg gaaaagaaag tacagaacaa accctgtgtc actgtctcaa gttaagctaa 1920 gtgaacagaa ctatctcagc atcagaatga gaaagcctga gaagaaagaa ccaaccagaa 1980 gcacacagga aggaaagcgc aggaggtgaa aatgctttct tggccggggt agtaagaatt 2040 agaggttaat gcagggactg taaaaccacc ttttctgctt caatgtctag ttcctgtata 2100 gctttgttca ttgcatttat taaacaaatg ttgtataacc aatactaaat gtactactga 2160 gcttcactga gttacgctgt gaaactttca aatccttctt catgtcagtt ccaatgaggt 2220 ggggatggag aagacaattg ttgcttatga aagaaagctt tagctgtctc tgttttgtaa 2280 gctttcagtg caacatttct tggttccaat aaagcatttt acaagatctt gcatgctact 2340 cttagataga agatggcaaa accatggtaa taaaatatga atgataaaaa aaaa 2394 <210> 6 <211> 2148 <212> DNA <213> Homo sapiens <400> 6 aggctgacag accagcccag atccagtggc ccggaggggc ctgagctaaa tccgcaggac 60 ctgggtaaca cgaggaaggg ctccggatat ctttggtgac ttgtccactc cagtgtggca 120 tcatgtggca gctgctcctc ccaactgctc tgctacttct agtttcagct ggcatgcgga 180 ctgaagatct cccaaaggct gtggtgttcc tggagcctca atggtacagc gtgcttgaga 240 aggacagtgt gactctgaag tgccagggag cctactcccc tgaggacaat tccacacagt 300 ggtttcacaa tgagaacctc atctcaagcc aggcctcgag ctacttcatt gacgctgcca 360 cagtcaacga cagtggagag tacaggtgcc agacaaacct ctccaccctc agtgacccgg 420 tgcagctaga agtccatatc ggctggctgt tgctccaggc ccctcggtgg gtgttcaagg 480 aggaagaccc tattcacctg aggtgtcaca gctggaagaa cactgctctg cataaggtca 540 catatttaca gaatggcaaa gacaggaagt attttcatca taattctgac ttccacattc 600 caaaagccac actcaaagat agcggctcct acttctgcag ggggcttgtt gggagtaaaa 660 atgtgtcttc agagactgtg aacatcacca tcactcaagg tttggcagtg tcaaccatct 720 catcattctc tccacctggg taccaagtct ctttctgctt ggtgatggta ctcctttttg 780 cagtggacac aggactatat ttctctgtga agacaaacat ttgaagctca acaagagact 840 ggaaggacca taaacttaaa tggagaaagg accctcaaga caaatgaccc ccatcccatg 900 ggggtaataa gagcagtggc agcagcatct ctgaacattt ctctggattt gcaaccccat 960 catcctcagg cctctctaca agcagcagga aacatagaac tcagagccag atcctttatc 1020 caactctcga tttttccttg gtctccagtg gaagggaaaa gcccatgatc ttcaagcagg 1080 gaagccccag tgagtagctg cattcctaga aattgaagtt tcagagctac acaaacactt 1140 tttctgtccc aaccattccc tcacagcaaa gcaacaatac aggctaggga tggtaatcct 1200 ttaaacatac aaaaattgct cgtattataa attacccagt ttagagggaa aaaaagaaaa 1260 taattattcc taaacaaatg gataagtaga attaatggtt gaggcaggac cctacagagt 1320 gtgggaactg ctggggatct agagaattca gtgggaccaa tgaaagcatg gctgagaaat 1380 agcagggtag tccaggatag tctaagggag gtgttcccat ctgagcccag agataagggt 1440 gtcttcctag aacattagcc gtagtggaat taacaggaaa tcatgagggt gacgtagaat 1500 tgagtcttcc aggggactct atcagaactg gaccatttcc aagtatataa cgatgagtcc 1560 tctaatgcta ggagtagaaa atggtcctag gaaggggact gaggattggg gtgggggtgg 1620 ggtggaaaag aaagtacaga acaaaccctg tgtcactgtc tcaagttaag ctaagtgaac 1680 agaactatct cagcatcaga atgagaaagc ctgagaagaa agaaccaacc agaagcacac 1740 aggaaggaaa gcgcaggagg tgaaaatgct ttcttggccg gggtagtaag aattagaggt 1800 taatgcaggg actgtaaaac caccttttct gcttcaatgt ctagttcctg tatagctttg 1860 ttcattgcat ttattaaaca aatgttgtat aaccaatact aaatgtacta ctgagcttca 1920 ctgagttacg ctgtgaaact ttcaaatcct tcttcatgtc agttccaatg aggtggggat 1980 ggagaagaca attgttgctt atgaaagaaa gctttagctg tctctgtttt gtaagctttc 2040 agtgcaacat ttcttggttc caataaagca ttttacaaga tcttgcatgc tactcttaga 2100 tagaagatgg caaaaccatg gtaataaaat atgaatgata aaaaaaaa 2148 <210> 7 <211> 2391 <212> DNA <213> Homo sapiens <400> 7 attcttggtg ctgggtggat ctaaatccag gagatggggg caagcatcct gggaaagctg 60 agggcacact ctggcagatt ctgtgtgtgt cctcagatgc tcagccgcag acctttggga 120 gagtaaaggg ggcacaccca cccaccttgc ctccaggctc tttccttcct attcctgttc 180 tatggtgggg ctccattgcg agacttcaga ttgagaaatc agatgaagtt tcaagaaaag 240 gaaactggca ggtgacagag atgggtggag ggactgggga aaggctgttt actccctcct 300 gtctagtcgg cttggtccct ttagggctcc ggatatcttt ggtgacttgt ccactccagt 360 gtggcatcat gtggcagctg ctcctcccaa ctgctctgct acttctagtt tcagctggca 420 tgcggactga tctcccaaag gctgtggtgt tcctggagcc tcaatggtac agcgtgcttg 480 agaaggacag tgtgactctg aagtgccagg gagcctactc ccctgaggac aattccacac 540 agtggtttca caatgagaac ctcatctcaa gccaggcctc gagctacttc attgacgctg 600 ccacagtcaa cgacagtgga gagtacaggt gccagacaaa cctctccacc ctcagtgacc 660 cggtgcagct agaagtccat atcggctggc tgttgctcca ggcccctcgg tgggtgttca 720 aggaggaaga ccctattcac ctgaggtgtc acagctggaa gaacactgct ctgcataagg 780 tcacatattt acagaatggc aaagacagga agtattttca tcataattct gacttccaca 840 ttccaaaagc cacactcaaa gatagcggct cctacttctg cagggggctt gttgggagta 900 aaaatgtgtc ttcagagact gtgaacatca ccatcactca aggtttggca gtgtcaacca 960 tctcatcatt ctctccacct gggtaccaag tctctttctg cttggtgatg gtactccttt 1020 ttgcagtgga cacaggacta tatttctctg tgaagacaaa catttgaagc tcaacaagag 1080 actggaagga ccataaactt aaatggagaa aggaccctca agacaaatga cccccatccc 1140 atgggggtaa taagagcagt ggcagcagca tctctgaaca tttctctgga tttgcaaccc 1200 catcatcctc aggcctctct acaagcagca ggaaacatag aactcagagc cagatccttt 1260 atccaactct cgatttttcc ttggtctcca gtggaaggga aaagcccatg atcttcaagc 1320 agggaagccc cagtgagtag ctgcattcct agaaattgaa gtttcagagc tacacaaaca 1380 ctttttctgt cccaaccatt ccctcacagc aaagcaacaa tacaggctag ggatggtaat 1440 cctttaaaca tacaaaaatt gctcgtatta taaattaccc agtttagagg gaaaaaaaga 1500 aaataattat tcctaaacaa atggataagt agaattaatg gttgaggcag gaccctacag 1560 agtgtgggaa ctgctgggga tctagagaat tcagtgggac caatgaaagc atggctgaga 1620 aatagcaggg tagtccagga tagtctaagg gaggtgttcc catctgagcc cagagataag 1680 ggtgtcttcc tagaacatta gccgtagtgg aattaacagg aaatcatgag ggtgacgtag 1740 aattgagtct tccaggggac tctatcagaa ctggaccatt tccaagtata taacgatgag 1800 tcctctaatg ctaggagtag aaaatggtcc taggaagggg actgaggatt ggggtggggg 1860 tggggtggaa aagaaagtac agaacaaacc ctgtgtcact gtctcaagtt aagctaagtg 1920 aacagaacta tctcagcatc agaatgagaa agcctgagaa gaaagaacca accagaagca 1980 cagagaagg aaagcgcagg aggtgaaaat gctttcttgg ccggggtagt aagaattaga 2040 ggttaatgca gggactgtaa aaccaccttt tctgcttcaa tgtctagttc ctgtatagct 2100 ttgttcattg catttattaa acaaatgttg tataaccaat actaaatgta ctactgagct 2160 tcactgagtt acgctgtgaa actttcaaat ccttcttcat gtcagttcca atgaggtggg 2220 gatggagaag acaattgttg cttatgaaag aaagctttag ctgtctctgt tttgtaagct 2280 ttcagtgcaa catttcttgg ttccaataaa gcattttaca agatcttgca tgctactctt 2340 agatagaaga tggcaaaacc atggtaataa aatatgaatg ataaaaaaaa a 2391 <210> 8 <211> 2260 <212> DNA <213> Homo sapiens <400> 8 aggatctccc tggttgaggg agaagtttga gatgccttgg gttcatcaga cacccctttt 60 caggctacga atgagactcc cacaaaggga tgggacccct caccacatct atagctgtgg 120 attgagctac caggacaagc caagatgggg ctagaaatga ggagaatgct ggttccaatt 180 gggtcatagt catgagtgag gccagtcact tcacccctct gggtcccaga atcactatgt 240 ggaaccaaag agcttcgact agatggtccc taggtttcag ctggcatgcg gactgaagat 300 ctcccaaagg ctgtggtgtt cctggagcct caatggtaca gcgtgcttga gaaggacagt 360 gtgactctga agtgccaggg agcctactcc cctgaggaca attccacaca gtggtttcac 420 aatgagaacc tcatctcaag ccaggcctcg agctacttca ttgacgctgc cacagtcaac 480 gacagtggag agtacaggtg ccagacaaac ctctccaccc tcagtgaccc ggtgcagcta 540 gaagtccata tcggctggct gttgctccag gcccctcggt gggtgttcaa ggaggaagac 600 cctattcacc tgaggtgtca cagctggaag aacactgctc tgcataaggt cacatattta 660 cagaatggca aagacaggaa gtattttcat cataattctg acttccacat tccaaaagcc 720 acactcaaag atagcggctc ctacttctgc agggggcttg ttgggagtaa aaatgtgtct 780 tcagagactg tgaacatcac catcactcaa ggtttggcag tgtcaaccat ctcatcattc 840 tctccacctg ggtaccaagt ctctttctgc ttggtgatgg tactcctttt tgcagtggac 900 acaggactat atttctctgt gaagacaaac atttgaagct caacaagaga ctggaaggac 960 cataaactta aatggagaaa ggaccctcaa gacaaatgac ccccatccca tgggggtaat 1020 aagagcagtg gcagcagcat ctctgaacat ttctctggat ttgcaacccc atcatcctca 1080 ggcctctcta caagcagcag gaaacataga actcagagcc agatccttta tccaactctc 1140 gatttttcct tggtctccag tggaagggaa aagcccatga tcttcaagca gggaagcccc 1200 agtgagtagc tgcattccta gaaattgaag tttcagagct acacaaacac tttttctgtc 1260 ccaaccattc cctcacagca aagcaacaat acaggctagg gatggtaatc ctttaaacat 1320 acaaaaattg ctcgtattat aaattaccca gtttagaggg aaaaaaagaa aataattatt 1380 cctaaacaaa tggataagta gaattaatgg ttgaggcagg accctacaga gtgtgggaac 1440 tgctggggat ctagagaatt cagtgggacc aatgaaagca tggctgagaa atagcagggt 1500 agtccaggat agtctaaggg aggtgttccc atctgagccc agagataagg gtgtcttcct 1560 agaacattag ccgtagtgga attaacagga aatcatgagg gtgacgtaga attgagtctt 1620 ccaggggact ctatcagaac tggaccattt ccaagtatat aacgatgagt cctctaatgc 1680 taggagtaga aaatggtcct aggaagggga ctgaggattg gggtgggggt ggggtggaaa 1740 agaaagtaca gaacaaaccc tgtgtcactg tctcaagtta agctaagtga acagaactat 1800 ctcagcatca gaatgagaaa gcctgagaag aaagaaccaa ccagaagcac acaggaagga 1860 aagcgcagga ggtgaaaatg ctttcttggc cggggtagta agaattagag gttaatgcag 1920 gt; atttattaaa caaatgttgt ataaccaata ctaaatgtac tactgagctt cactgagtta 2040 cgctgtgaaa ctttcaaatc cttcttcatg tcagttccaa tgaggtgggg atggagaaga 2100 caattgttgc ttatgaaaga aagctttagc tgtctctgtt ttgtaagctt tcagtgcaac 2160 atttcttggt tccaataaag cattttacaa gatcttgcat gctactctta gatagaagat 2220 ggcaaaacca tggtaataaa atatgaatga taaaaaaaaa 2260 <210> 9 <211> 2002 <212> DNA <213> Homo sapiens <400> 9 aggatctccc tggttgaggg agaagtttga gatgccttgg gttcatcaga cacccctttt 60 caggctacga atgagactcc cacaaaggga tgggacccct caccacatct atagctgtgg 120 attgagctac caggacaagc caagatgggg ctagaaatga ggagaatgct ggttccaatt 180 gggtcatagt catgagtgag gccagtcact tcacccctct gggtcccaga atcactatgt 240 ggaaccaaag agcttcgact agatggtccc taggtttcag ctggcatgcg gactgaagat 300 ctcccaaagg ctgtggtgtt cctggagcct caatggtaca gcgtgcttga gaaggacagt 360 gtgactctga agtgccaggg agcctactcc cctgaggaca attccacaca gtggtttcac 420 aatgagaacc tcatctcaag ccaggcctcg agctacttca ttgacgctgc cacagtcaac 480 gacagtggag agtacaggtg ccagacaaac ctctccaccc tcagtgaccc ggtgcagcta 540 gaagtccata tcggtttggc agtgtcaacc atctcatcat tctctccacc tgggtaccaa 600 gtctctttct gcttggtgat ggtactcctt tttgcagtgg acacaggact atatttctct 660 gtgaagacaa acatttgaag ctcaacaaga gactggaagg accataaact taaatggaga 720 aaggaccctc aagacaaatg acccccatcc catgggggta ataagagcag tggcagcagc 780 atctctgaac atttctctgg atttgcaacc ccatcatcct caggcctctc tacaagcagc 840 aggaaacata gaactcagag ccagatcctt tatccaactc tcgatttttc cttggtctcc 900 agtggaaggg aaaagcccat gatcttcaag cagggaagcc ccagtgagta gctgcattcc 960 tagaaattga agtttcagag ctacacaaac actttttctg tcccaaccat tccctcacag 1020 caaagcaaca atacaggcta gggatggtaa tcctttaaac atacaaaaat tgctcgtatt 1080 ataaattacc cagtttagag ggaaaaaaag aaaataatta ttcctaaaca aatggataag 1140 tagaattaat ggttgaggca ggaccctaca gagtgtggga actgctgggg atctagagaa 1200 ttcagtggga ccaatgaaag catggctgag aaatagcagg gtagtccagg atagtctaag 1260 ggaggtgttc ccatctgagc ccagagataa gggtgtcttc ctagaacatt agccgtagtg 1320 gaattaacag gaaatcatga gggtgacgta gaattgagtc ttccagggga ctctatcaga 1380 actggaccat ttccaagtat ataacgatga gtcctctaat gctaggagta gaaaatggtc 1440 ctaggaaggg gactgaggat tggggtgggg gtggggtgga aaagaaagta cagaacaaac 1500 cctgtgtcac tgtctcaagt taagctaagt gaacagaact atctcagcat cagaatgaga 1560 aagcctgaga agaaagaacc aaccagaagc acacakgaag gaaagcgcag gaggtgaaaa 1620 tgctttcttg gccggggtag taagaattag aggttaatgc agggactgta aaaccacctt 1680 ttctgcttca atgtctagtt cctgtatagc tttgttcatt gcatttatta aacaaatgtt 1740 gtataaccaa tactaaatgt actactgagc ttcactgagt tacgctgtga aactttcaaa 1800 tccttcttca tgtcagttcc aatgaggtgg ggatggagaa gacaattgtt gcttatgaaa 1860 gaaagcttta gctgtctctg ttttgtaagc tttcagtgca acatttcttg gttccaataa 1920 agcattttac aagatcttgc atgctactct tagatagaag atggcaaaac catggtaata 1980 aaatatgaat gataaaaaaa aa 2002 <210> 10 <211> 2633 <212> DNA <213> Homo sapiens <400> 10 agtgggcgtg gcggtgctgc ccaggtgagc caccgctgct tctgcccaga cacggtcgcc 60 tccacatcca ggtctttgtg ctcctcgctt gcctgttcct tttccacgca ttttccagga 120 taactgtgac tccaggcccg caatggatgc cctgcaacta gcaaattcgg cttttgccgt 180 tgatctgttc aaacaactat gtgaaaagga gccactgggc aatgtcctct tctctccaat 240 ctgtctctcc acctctctgt cacttgctca agtgggtgct aaaggtgaca ctgcaaatga 300 aattggacagta gttcttcatt ttgaaaatgt caaagatgta ccctttggat ttcaaacagt 360 aacatcggat gtaaacaaac ttagttcctt ttactcactg aaactaatca agcggctcta 420 cgtagacaaa tctctgaatc tttctacaga gttcatcagc tctacgaaga gaccgtatgc 480 aaaggaattg gaaactgttg acttcaaaga taaattggaa gaaacgaaag gtcagatcaa 540 caactcaatt aaggatctca cagatggcca ctttgagaac attttagctg acaacagtgt 600 gaacgaccag accaaaatcc ttgtggttaa tgctgcctac tttgttggca agtggatgaa 660 gaaattttct gaatcagaaa caaaagaatg tcctttcaga gtcaacaaga cagacaccaa 720 accagtgcag atgatgaaca tggaggccac gttctgtatg ggaaacattg acagtatcaa 780 ttgtaagatc atagagcttc cttttcaaaa taagcatctc agcatgttca tcctactacc 840 caaggatgtg gaggatgagt ccacaggctt ggagaagatt gaaaaacaac tcaactcaga 900 gtcactgtca cagtggacta atcccagcac catggccaat gccaaggtca aactctccat 960 tccaaaattt aaggtggaaa agatgattga tcccaaggct tgtctggaaa atctagggct 1020 gaaacatatc ttcagtgaag acacatctga tttctctgga atgtcagaga ccaagggagt 1080 ggccctatca aatgttatcc acaaagtgtg cttagaaata actgaagatg gtggggattc 1140 catagaggtg ccaggagcac ggatcctgca gcacaaggat gaattgaatg ctgaccatcc 1200 ctttatttac atcatcaggc acaacaaaac tcgaaacatc attttctttg gcaaattctg 1260 ttctccttaa gtggcatagc ccatgttaag tcctccctga cttttctgtg gatgccgatt 1320 tctgtaaact ctgcatccag agattcattt tctagataca ataaattgct aatgttgctg 1380 gatcaggaag ccgccagtac ttgtcatatg tagccttcac acagatagac cttttttttt 1440 tttccaattc tatcttttgt ttcctttttt cccataagac aatgacatac gcttttaatg 1500 aaaaggaatc acgttagagg aaaaatattt attcattatt tgtcaaattg tccggggtag 1560 ttggcagaaa tacagtcttc cacaaagaaa attcctataa ggaagatttg gaagctcttc 1620 ttcccagcac tatgctttcc ttctttggga tagagaatgt tccagacatt ctcgcttccc 1680 tgaaagactg aagaaagtgt agtgcatggg acccacgaaa ctgccctggc tccagtgaaa 1740 cttgggcaca tgctcaggct actataggtc cagaagtcct tatgttaagc cctggcaggc 1800 aggtgtttat taaaattctg aattttgggg attttcaaaa gataatattt tacatacact 1860 gtatgttata gaacttcatg gatcagatct ggggcagcac cctataaatc aacaccttaa 1920 tatgctgcaa caaaatgtag aatattcaga caaaatggat acataaagac taagtagccc 1980 ataaggggtc aaaatttgct gccaaatgcg tatgccacca acttacaaaa acacttcgtt 2040 cgcagagctt ttcagattgt ggaatgttgg ataaggaatt atagacctct agtagctgaa 2100 atgcaagacc ccaagaggaa gttcagatct taatataaat tcactttcat ttttgatagc 2160 tgtcccatct ggtcatttgg ttggcactag actggtggca ggggcttcta gctgacttgc 2220 acagggattc tcacaatagc cgatatcaga atttgtgttg aaggaacttg tctcttcatc 2280 taatatgata gcgggaaaag gagaggaaac tactgccttt agaaaatata agtaaagtga 2340 ttaaagtgct cacgttacct tgacacatag tttttcagtc tatgggttta gttactttag 2400 atggcaagca tgtaacttat attaatagta atttgtaaag ttggttggat aagctatccg 2460 tgttgcaggt tcatggatta cttctctata aaaaatatgt atttaccaaa aattttgtga 2520 cattccttct cccatctctt ccttgacctg cattgtaaat aggttcttct tgttctgaga 2580 ttcaatattg aatttttcct atgctattga caataaaata ttattgaact aca 2633 <210> 11 <211> 834 <212> DNA <213> Homo sapiens <400> 11 agtcctgtgt ccgggccccg aggcacagcc agggcaccag gtggagcacc agctacgcgt 60 ggcgcagcgc agcgtcccta gcaccgagcc tcccgcagcc gccgagatgc tgcgaacaga 120 gagctgccgc cccaggtcgc ccgccggaca ggtggccgcg gcgtccccgc tcctgctgct 180 gctgctgctg ctcgcctggt gcgcgggcgc ctgccgaggt gctccaatat tacctcaagg 240 attacagcct gaacaacagc tacagttgtg gaatgagata gatgatactt gttcgtcttt 300 tctgtccatt gattctcagc ctcaggcatc caacgcactg gaggagcttt gctttatgat 360 tatgggaatg ctaccaaagc ctcaggaaca agatgaaaaa gataatacta aaaggttctt 420 atttcattat tcgaagacac agaagttggg caagtcaaat gttgtgtcgt cagttgtgca 480 tccgttgctg cagctcgttc ctcacctgca tgagagaaga atgaagagat tcagagtgga 540 cgaagaattc caaagtccct ttgcaagtca aagtcgagga tattttttat tcaggccacg 600 gaatggaaga aggtcagcag ggttcattta aaatggatgc cagctaattt tccacagagc 660 aatgctatgg aatacaaaat gtactgacat tttgttttct tctgaaaaaa atccttgcta 720 aatgtactct gttgaaaatc cctgtgttgt caatgttctc agttgtaaca atgttgtaaa 780 tgttcaattt gttgaaaatt aaaaaatcta aaaatattaa aaaaaaaaaaaaaa 834 <210> 12 <211> 786 <212> DNA <213> Homo sapiens <400> 12 agtcctgtgt ccgggccccg aggcacagcc agggcaccag gtggagcacc agctacgcgt 60 ggcgcagcgc agcgtcccta gcaccgagcc tcccgcagcc gccgagatgc tgcgaacaga 120 gagctgccgc cccaggtcgc ccgccggaca ggtggccgcg gcgtccccgc tcctgctgct 180 gctgctgctg ctcgcctggt gcgcgggcgc ctgccgaggt gctccaatat tacctcaagg 240 attacagcct gaacaacagc tacagttgtg gaatgaggca tccaacgcac tggaggagct 300 ttgctttatg attatgggaa tgctaccaaa gcctcaggaa caagatgaaa aagataatac 360 taaaaggttc ttatttcatt attcgaagac acagaagttg ggcaagtcaa atgttgtgtc 420 gtcagttgtg catccgttgc tgcagctcgt tcctcacctg catgagagaa gaatgaagag 480 attcagagtg gacgaagaat tccaaagtcc ctttgcaagt caaagtcgag gatatttttt 540 attcaggcca cggaatggaa gaaggtcagc agggttcatt taaaatggat gccagctaat 600 tttccacaga gcaatgctat ggaatacaaa atgtactgac attttgtttt cttctgaaaa 660 aaatccttgc taaatgtact ctgttgaaaa tccctgtgtt gtcaatgttc tcagttgtaa 720 caatgttgta aatgttcaat ttgttgaaaa ttaaaaaatc taaaaatatt aaaaaaaaaa 780 aaaaaa 786 <210> 13 <211> 759 <212> DNA <213> Homo sapiens <400> 13 agtcctgtgt ccgggccccg aggcacagcc agggcaccag gtggagcacc agctacgcgt 60 ggcgcagcgc agcgtcccta gcaccgagcc tcccgcagcc gccgagatgc tgcgaacaga 120 gagctgccgc cccaggtcgc ccgccggaca ggtggccgcg gcgtccccgc tcctgctgct 180 gctgctgctg ctcgcctggt gcgcgggcgc ctgccgaggt gctccaatat tacctcaagg 240 attacagcct gaacaacagc tacagttgtg gaatgagata gatgatactt gttcgtcttt 300 tctgtccatt gattctcagc ctcaggcatc caacgcactg gaggagcttt gctttatgat 360 tatgggaatg ctaccaaagc ctcaggaaca agatgaaaaa gataatacta aaaggttctt 420 atttcattat tcgaagacac agaagttggg caagtcaaat gttgtggaag aattccaaag 480 tccctttgca agtcaaagtc gaggatattt tttattcagg ccacggaatg gaagaaggtc 540 agcagggttc atttaaaatg gatgccagct aattttccac agagcaatgc tatggaatac 600 aaaatgtact gacattttgt tttcttctga aaaaaatcct tgctaaatgt actctgttga 660 aaatccctgt gttgtcaatg ttctcagttg taacaatgtt gtaaatgttc aatttgttga 720 aaattaaaaa atctaaaaat attaaaaaaa aaaaaaaaa 759 <210> 14 <211> 665 <212> PRT <213> Homo sapiens <400> 14 Met Leu Arg Glu Arg Thr Val Arg Leu Gln Tyr Gly Ser Arg Val Glu   1 5 10 15 Ala Val Tyr Val Leu Gly Thr Tyr Leu Trp Thr Asp Val Tyr Ser Ala              20 25 30 Ala Pro Ala Gly Ala Gln Thr Phe Ser Leu Lys His Ser Glu His Val          35 40 45 Trp Val Glu Val Val Arg Asp Gly Glu Ala Glu Glu Val Ala Thr Asn      50 55 60 Gly Lys Gln Arg Trp Leu Leu Ser Ser Thr Thr Leu Arg Val Thr  65 70 75 80 Met Ser Gln Ala Ser Thr Glu Ala Ser Ser Asp Lys Val Thr Val Asn                  85 90 95 Tyr Tyr Asp Glu Glu Gly Ser Ile Pro Ile Asp Gln Ala Gly Leu Phe             100 105 110 Leu Thr Ala Ile Glu Ile Ser Leu Asp Val Asp Ala Asp Arg Asp Gly         115 120 125 Val Val Glu Lys Asn Asn Pro Lys Lys Ala Ser Trp Thr Trp Gly Pro     130 135 140 Glu Gly Gln Gly Ala Ile Leu Leu Val Asn Cys Asp Arg Glu Thr Pro 145 150 155 160 Trp Leu Pro Lys Glu Asp Cys Arg Asp Glu Lys Val Tyr Ser Lys Glu                 165 170 175 Asp Leu Lys Asp Met Ser Gln Met Ile Leu Arg Thr Lys Gly Pro Asp             180 185 190 Arg Leu Pro Ala Gly Tyr Glu Ile Val Leu Tyr Ile Ser Ser Ser Asp         195 200 205 Ser Asp Lys Val Gly Val Phe Tyr Val Glu Asn Pro Phe Phe Gly Gln     210 215 220 Arg Tyr Ile His Ile Leu Gly Arg Arg Lys Leu Tyr His Val Val Lys 225 230 235 240 Tyr Thr Gly Gly Ser Ala Glu Leu Leu Phe Phe Val Glu Gly Leu Cys                 245 250 255 Phe Pro Asp Glu Gly Phe Ser Gly Leu Val Ser Ile His Val Ser Leu             260 265 270 Leu Glu Tyr Met Ala Gln Asp Ile Pro Leu Thr Pro Ile Phe Thr Asp         275 280 285 Thr Val Ile Phe Arg Ile Ala Pro Trp Ile Met Thr Pro Asn Ile Leu     290 295 300 Pro Pro Val Ser Val Phe Val Cys Cys Met Lys Asp Asn Tyr Leu Phe 305 310 315 320 Leu Lys Glu Val Lys Asn Leu Val Glu Lys Thr Asn Cys Glu Leu Lys                 325 330 335 Val Cys Phe Gln Tyr Leu Asn Arg Gly Asp Arg Trp Ile Gln Asp Glu             340 345 350 Ile Glu Phe Gly Tyr Ile Glu Ala Pro His Lys Gly Phe Pro Val Val         355 360 365 Leu Asp Ser Pro Arg Asp Gly Asn Leu Lys Asp Phe Pro Val Lys Glu     370 375 380 Leu Leu Gly Pro Asp Phe Gly Tyr Val Thr Arg Glu Pro Leu Phe Glu 385 390 395 400 Ser Val Thr Ser Leu Asp Ser Phe Gly Asn Leu Glu Val Ser Pro Pro                 405 410 415 Val Thr Val Asn Gly Lys Thr Tyr Pro Leu Gly Arg Ile Leu Ile Gly             420 425 430 Ser Ser Phe Pro Leu Ser Gly Gly Arg Arg Met Thr Lys Val Val Arg         435 440 445 Asp Phe Leu Lys Ala Gln Gln Val Gln Ala Pro Val Glu Leu Tyr Ser     450 455 460 Asp Trp Leu Thr Val Gly His Val Asp Glu Phe Met Ser Phe Val Pro 465 470 475 480 Ile Pro Gly Thr Lys Lys Phe Leu Leu Leu Met Ala Ser Thr Ser Ala                 485 490 495 Cys Tyr Lys Leu Phe Arg Glu Lys Gln Lys Asp Gly His Gly Glu Ala             500 505 510 Ile Met Phe Lys Gly Leu Gly Gly Met Ser Ser Lys Arg Ile Thr Ile         515 520 525 Asn Lys Ile Leu Ser Asn Glu Ser Leu Val Gln Glu Asn Leu Tyr Phe     530 535 540 Gln Arg Cys Leu Asp Trp Asn Arg Asp Ile Leu Lys Lys Glu Leu Gly 545 550 555 560 Leu Thr Glu Gln Asp Ile Ile Asp Leu Pro Ala Leu Phe Lys Met Asp                 565 570 575 Glu Asp His Arg Ala Arg Ala Phe Phe Pro Asn Met Val Asn Met Ile             580 585 590 Val Leu Asp Lys Asp Leu Gly Ile Pro Lys Pro Phe Gly Pro Gln Val         595 600 605 Glu Glu Cys Cys Leu Glu Met His Val Arg Gly Leu Leu Glu Pro     610 615 620 Leu Gly Leu Glu Cys Thr Phe Ile Asp Asp Ile Ser Ala Tyr His Lys 625 630 635 640 Phe Leu Gly Glu Val His Cys Gly Thr Asn Val Arg Arg Lys Pro Phe                 645 650 655 Thr Phe Lys Trp Trp His Met Val Pro             660 665 <210> 15 <211> 199 <212> PRT <213> Homo sapiens <400> 15 Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His   1 5 10 15 Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr              20 25 30 Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met          35 40 45 Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys      50 55 60 Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro  65 70 75 80 Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile                  85 90 95 Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala             100 105 110 Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg         115 120 125 Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg     130 135 140 Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His 145 150 155 160 Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp                 165 170 175 Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala             180 185 190 Ile Leu Gln Asn Gln Gly Asn         195 <210> 16 <211> 269 <212> PRT <213> Homo sapiens <400> 16 Met Gly Gly Gly Thr Gly Glu Arg Leu Phe Thr Pro Ser Cys Leu Val   1 5 10 15 Gly Leu Val Pro Leu Gly Leu Arg Ile Ser Leu Val Thr Cys Pro Leu              20 25 30 Gln Cys Gly Ile Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu          35 40 45 Leu Val Ser Ala Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val      50 55 60 Phe Leu Glu Pro Gln Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr  65 70 75 80 Leu Lys Cys Gln Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp                  85 90 95 Phe His Asn Glu Asn Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile             100 105 110 Asp Ala Ala Thr Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn         115 120 125 Leu Ser Thr Leu Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp     130 135 140 Leu Leu Leu Gln Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile 145 150 155 160 His Leu Arg Cys His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr                 165 170 175 Tyr Leu Gln Asn Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp             180 185 190 Phe His Ile Pro Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys         195 200 205 Arg Gly Leu Val Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile     210 215 220 Thr Ile Thr Gln Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro 225 230 235 240 Pro Gly Tyr Gln Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala                 245 250 255 Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile             260 265 <210> 17 <211> 233 <212> PRT <213> Homo sapiens <400> 17 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala   1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro              20 25 30 Gln Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln          35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu      50 55 60 Asn Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr  65 70 75 80 Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu                  85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln             100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys         115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn     130 135 140 Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val                 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln             180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln         195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly     210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile 225 230 <210> 18 <211> 268 <212> PRT <213> Homo sapiens <400> 18 Met Gly Gly Gly Thr Gly Glu Arg Leu Phe Thr Pro Ser Cys Leu Val   1 5 10 15 Gly Leu Val Pro Leu Gly Leu Arg Ile Ser Leu Val Thr Cys Pro Leu              20 25 30 Gln Cys Gly Ile Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu          35 40 45 Leu Val Ser Ala Gly Met Arg Thr Asp Leu Pro Lys Ala Val Val Phe      50 55 60 Leu Glu Pro Gln Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu  65 70 75 80 Lys Cys Gln Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe                  85 90 95 His Asn Glu Asn Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp             100 105 110 Ala Ala Thr Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu         115 120 125 Ser Thr Leu Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu     130 135 140 Leu Leu Gln Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His 145 150 155 160 Leu Arg Cys His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr                 165 170 175 Leu Gln Asn Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe             180 185 190 His Ile Pro Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg         195 200 205 Gly Leu Val Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr     210 215 220 Ile Thr Gln Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro 225 230 235 240 Gly Tyr Gln Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val                 245 250 255 Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile             260 265 <210> 19 <211> 216 <212> PRT <213> Homo sapiens <400> 19 Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln   1 5 10 15 Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly              20 25 30 Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Asn          35 40 45 Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val      50 55 60 Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser  65 70 75 80 Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln Ala                  85 90 95 Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys His             100 105 110 Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn Gly         115 120 125 Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro Lys     130 135 140 Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val Gly 145 150 155 160 Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln Gly                 165 170 175 Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Gly Tyr Gln Val             180 185 190 Ser Phe Cys Leu Val Met Le Leu Le Phe Ala Val Asp Thr Gly Leu         195 200 205 Tyr Phe Ser Val Lys Thr Asn Ile     210 215 <210> 20 <211> 130 <212> PRT <213> Homo sapiens <400> 20 Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln   1 5 10 15 Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly              20 25 30 Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Asn          35 40 45 Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val      50 55 60 Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser  65 70 75 80 Asp Pro Val Gln Leu Glu Val His Ile Gly Leu Ala Val Ser Thr Ile                  85 90 95 Ser Ser Phe Ser Pro Pro Gly Tyr Gln Val Ser Phe Cys Leu Val Met             100 105 110 Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr         115 120 125 Asn Ile     130 <210> 21 <211> 375 <212> PRT <213> Homo sapiens <400> 21 Met Asp Ala Leu Gln Leu Ala Asn Ser Ala Phe Ala Val Asp Leu Phe   1 5 10 15 Lys Gln Leu Cys Glu Lys Glu Pro Leu Gly Asn Val Leu Phe Ser Pro              20 25 30 Ile Cys Leu Ser Thr Ser Leu Ser Leu Ala Gln Val Gly Ala Lys Gly          35 40 45 Asp Thr Ala Asn Glu Ile Gly Gln Val Leu His Phe Glu Asn Val Lys      50 55 60 Asp Val Pro Phe Gly Phe Gln Thr Val Thr Ser Asp Val Asn Lys Leu  65 70 75 80 Ser Ser Phe Tyr Ser Leu Lys Leu Ile Lys Arg Leu Tyr Val Asp Lys                  85 90 95 Ser Leu Asn Leu Ser Thr Glu Phe Ile Ser Ser Thr Lys Arg Pro Tyr             100 105 110 Ala Lys Glu Leu Glu Thr Val Asp Phe Lys Asp Lys Leu Glu Glu Thr         115 120 125 Lys Gly Gln Ile Asn Asn Ser Ile Lys Asp Leu Thr Asp Gly His Phe     130 135 140 Glu Asn Ile Leu Ala Asp Asn Ser Val Asn Asp Gln Thr Lys Ile Leu 145 150 155 160 Val Val Asn Ala Ala Tyr Phe Val Gly Lys Trp Met Lys Lys Phe Ser                 165 170 175 Glu Ser Glu Thr Lys Glu Cys Pro Phe Arg Val Asn Lys Thr Asp Thr             180 185 190 Lys Pro Val Gln Met Met Asn Met Glu Ala Thr Phe Cys Met Gly Asn         195 200 205 Ile Asp Ser Ile Asn Cys Lys Ile Ile Glu Leu Pro Phe Gln Asn Lys     210 215 220 His Leu Ser Met Phe Ile Leu Leu Pro Lys Asp Val Glu Asp Glu Ser 225 230 235 240 Thr Gly Leu Glu Lys Ile Glu Lys Gln Leu Asn Ser Glu Ser Leu Ser                 245 250 255 Gln Trp Thr Asn Pro Ser Thr Met Ala Asn Ala Lys Val Lys Leu Ser             260 265 270 Ile Pro Lys Phe Lys Val Glu Lys Met Ile Asp Pro Lys Ala Cys Leu         275 280 285 Glu Asn Leu Gly Leu Lys His Ile Phe Ser Glu Asp Thr Ser Asp Phe     290 295 300 Ser Gly Met Ser Glu Thr Lys Gly Val Ala Leu Ser Asn Val Ile His 305 310 315 320 Lys Val Cys Leu Glu Ile Thr Glu Asp Gly Gly Asp Ser Ile Glu Val                 325 330 335 Pro Gly Ala Arg Ile Leu Gln His Lys Asp Glu Leu Asn Ala Asp His             340 345 350 Pro Phe Ile Tyr Ile Ile Arg His Asn Lys Thr Arg Asn Ile Ile Phe         355 360 365 Phe Gly Lys Phe Cys Ser Pro     370 375 <210> 22 <211> 174 <212> PRT <213> Homo sapiens <400> 22 Met Leu Arg Thr Glu Ser Cys Arg Pro Arg Ser Ser Ala Gly Gln Val   1 5 10 15 Ala Ala Ala Ser Leu Leu Leu Leu Leu Leu Leu              20 25 30 Ala Gly Ala Cys Arg Gly Ala Pro Ile Leu Pro Gln Gly Leu Gln Pro          35 40 45 Glu Gln Gln Leu Gln Leu Trp Asn Glu Ile Asp Asp Thr Cys Ser Ser      50 55 60 Phe Leu Ser Ile Asp Ser Gln Pro Gln Ala Ser Asn Ala Leu Glu Glu  65 70 75 80 Leu Cys Phe Met Ile Met Gly Met Leu Pro Lys Pro Gln Glu Gln Asp                  85 90 95 Glu Lys Asp Asn Thr Lys Arg Phe Leu Phe His Tyr Ser Lys Thr Gln             100 105 110 Lys Leu Gly Lys Ser Asn Val Val Ser Ser Val Val His Pro Leu Leu         115 120 125 Gln Leu Val Pro His Leu His Glu Arg Arg Met Lys Arg Phe Arg Val     130 135 140 Asp Glu Glu Phe Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe 145 150 155 160 Leu Phe Arg Pro Arg Asn Gly Arg Arg Ser Ala Gly Phe Ile                 165 170 <210> 23 <211> 158 <212> PRT <213> Homo sapiens <400> 23 Met Leu Arg Thr Glu Ser Cys Arg Pro Arg Ser Ser Ala Gly Gln Val   1 5 10 15 Ala Ala Ala Ser Leu Leu Leu Leu Leu Leu Leu              20 25 30 Ala Gly Ala Cys Arg Gly Ala Pro Ile Leu Pro Gln Gly Leu Gln Pro          35 40 45 Glu Gln Gln Leu Gln Leu Trp Asn Glu Ala Ser Asn Ala Leu Glu Glu      50 55 60 Leu Cys Phe Met Ile Met Gly Met Leu Pro Lys Pro Gln Glu Gln Asp  65 70 75 80 Glu Lys Asp Asn Thr Lys Arg Phe Leu Phe His Tyr Ser Lys Thr Gln                  85 90 95 Lys Leu Gly Lys Ser Asn Val Val Ser Ser Val Val His Pro Leu Leu             100 105 110 Gln Leu Val Pro His Leu His Glu Arg Arg Met Lys Arg Phe Arg Val         115 120 125 Asp Glu Glu Phe Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe     130 135 140 Leu Phe Arg Pro Arg Asn Gly Arg Arg Ser Ala Gly Phe Ile 145 150 155 <210> 24 <211> 149 <212> PRT <213> Homo sapiens <400> 24 Met Leu Arg Thr Glu Ser Cys Arg Pro Arg Ser Ser Ala Gly Gln Val   1 5 10 15 Ala Ala Ala Ser Leu Leu Leu Leu Leu Leu Leu              20 25 30 Ala Gly Ala Cys Arg Gly Ala Pro Ile Leu Pro Gln Gly Leu Gln Pro          35 40 45 Glu Gln Gln Leu Gln Leu Trp Asn Glu Ile Asp Asp Thr Cys Ser Ser      50 55 60 Phe Leu Ser Ile Asp Ser Gln Pro Gln Ala Ser Asn Ala Leu Glu Glu  65 70 75 80 Leu Cys Phe Met Ile Met Gly Met Leu Pro Lys Pro Gln Glu Gln Asp                  85 90 95 Glu Lys Asp Asn Thr Lys Arg Phe Leu Phe His Tyr Ser Lys Thr Gln             100 105 110 Lys Leu Gly Lys Ser Asn Val Val Glu Glu Phe Gln Ser Pro Phe Ala         115 120 125 Ser Gln Ser Arg Gly Tyr Phe Leu Phe Arg Pro Arg Asn Gly Arg Arg     130 135 140 Ser Ala Gly Phe Ile 145 <210> 25 <211> 1626 <212> DNA <213> Homo sapiens <400> 25 cgcacctgct gcaggtgctc ccggccgccc cggaccagcg agcgcgggca ctgcggcggg 60 gaggatgctg cgcgagcgga ccgtgcggct gcagtacggg agccgcgtgg aggcggtgta 120 cgtgctgggc acctacctct ggaccgatgt ctacagcgcg gccccagccg gggcccaaac 180 cttcagcctg aagcactcgg aacacgtgtg ggtggaggtg gtgcgtgatg gggaggctga 240 ggaggtggcc accaatggca agcagcgctg gcttctctcg cccagcacca ccctgcgggt 300 caccatgagc caggcgagca ccgaggccag cagtgacaag gtcaccgtca actactatga 360 cgaggaaggg agcattccca tcgaccaggc ggggctcttc ctcacagcca ttgagatctc 420 cctggatgtg gacgcagacc gggatggtgt ggtggagaag aacaacccaa agaaggcatc 480 ctggacctgg ggccccgagg gccagggggc catcctgctg gtgaactgtg accgagagac 540 accctggttg cccaaggagg actgccgtga tgagaaggtc tacagcaagg aagatctcaa 600 ggacatgtcc cagatgatcc tgcggaccaa aggccccgac cgcctccccg ccggatacga 660 gatagttctg tacatttcca tgtcagactc agacaaagtg ggcgtgttct acgtggagaa 720 cccgttcttc ggccaacgct atatccacat cctgggccgg cggaagctct accatgtggt 780 caagtacacg ggtggctccg cggagctgct gttcttcgtg gaaggcctct gtttccccga 840 cgagggcttc tcaggcctgg tctccatcca tgtcagcctg ctggagtaca tggcccagga 900 cattcccctg actcccatct tcacggacac cgtgatattc cggattgctc cgtggatcat 960 gacccccaac atcctgcctc ccgtgtcggt gtttgtgtgc tgcatgaagg ataattacct 1020 gttcctgaaa gaggtgaaga accttgtgga gaaaaccaac tgtgagctga aggtctgctt 1080 ccagtaccta aaccgaggcg atcgctggat ccaggatgaa attgagtttg gctacatcga 1140 ggccccccat aaaggcttcc ccgtggtgct ggactctccc cgagatggaa acctaaagga 1200 cttccctgtg aaggagctcc tgggcccaga ttttggctac gtgacccggg agcccctctt 1260 tgagtctgtc accagccttg actcatttgg aaacctggag gtcagtcccc cagtgaccgt 1320 gaatggcaag acatacccgc ttggccgcat cctcatcggg agcagctttc ctctgtaaga 1380 gaagccaggc tggggctagg ggctgtggga gtggggaagt cactgtttct cttttggggt 1440 ggcctgggtt gctcacacat ggagcaagtg gctgggggaa ttattccctc ccacgacttc 1500 ctgttaagag gccgacactc ttggaaagaa aatcaagcta gcctttatat ctttgtgtga 1560 tttaggatct aatatataac taaataaaca actcttttcc ccaagtgaaa aaaaaaaaaa 1620 aaaaaa 1626 <210> 26 <211> 437 <212> PRT <213> Homo sapiens <400> 26 Met Leu Arg Glu Arg Thr Val Arg Leu Gln Tyr Gly Ser Arg Val Glu   1 5 10 15 Ala Val Tyr Val Leu Gly Thr Tyr Leu Trp Thr Asp Val Tyr Ser Ala              20 25 30 Ala Pro Ala Gly Ala Gln Thr Phe Ser Leu Lys His Ser Glu His Val          35 40 45 Trp Val Glu Val Val Arg Asp Gly Glu Ala Glu Glu Val Ala Thr Asn      50 55 60 Gly Lys Gln Arg Trp Leu Leu Ser Ser Thr Thr Leu Arg Val Thr  65 70 75 80 Met Ser Gln Ala Ser Thr Glu Ala Ser Ser Asp Lys Val Thr Val Asn                  85 90 95 Tyr Tyr Asp Glu Glu Gly Ser Ile Pro Ile Asp Gln Ala Gly Leu Phe             100 105 110 Leu Thr Ala Ile Glu Ile Ser Leu Asp Val Asp Ala Asp Arg Asp Gly         115 120 125 Val Val Glu Lys Asn Asn Pro Lys Lys Ala Ser Trp Thr Trp Gly Pro     130 135 140 Glu Gly Gln Gly Ala Ile Leu Leu Val Asn Cys Asp Arg Glu Thr Pro 145 150 155 160 Trp Leu Pro Lys Glu Asp Cys Arg Asp Glu Lys Val Tyr Ser Lys Glu                 165 170 175 Asp Leu Lys Asp Met Ser Gln Met Ile Leu Arg Thr Lys Gly Pro Asp             180 185 190 Arg Leu Pro Ala Gly Tyr Glu Ile Val Leu Tyr Ile Ser Ser Ser Asp         195 200 205 Ser Asp Lys Val Gly Val Phe Tyr Val Glu Asn Pro Phe Phe Gly Gln     210 215 220 Arg Tyr Ile His Ile Leu Gly Arg Arg Lys Leu Tyr His Val Val Lys 225 230 235 240 Tyr Thr Gly Gly Ser Ala Glu Leu Leu Phe Phe Val Glu Gly Leu Cys                 245 250 255 Phe Pro Asp Glu Gly Phe Ser Gly Leu Val Ser Ile His Val Ser Leu             260 265 270 Leu Glu Tyr Met Ala Gln Asp Ile Pro Leu Thr Pro Ile Phe Thr Asp         275 280 285 Thr Val Ile Phe Arg Ile Ala Pro Trp Ile Met Thr Pro Asn Ile Leu     290 295 300 Pro Pro Val Ser Val Phe Val Cys Cys Met Lys Asp Asn Tyr Leu Phe 305 310 315 320 Leu Lys Glu Val Lys Asn Leu Val Glu Lys Thr Asn Cys Glu Leu Lys                 325 330 335 Val Cys Phe Gln Tyr Leu Asn Arg Gly Asp Arg Trp Ile Gln Asp Glu             340 345 350 Ile Glu Phe Gly Tyr Ile Glu Ala Pro His Lys Gly Phe Pro Val Val         355 360 365 Leu Asp Ser Pro Arg Asp Gly Asn Leu Lys Asp Phe Pro Val Lys Glu     370 375 380 Leu Leu Gly Pro Asp Phe Gly Tyr Val Thr Arg Glu Pro Leu Phe Glu 385 390 395 400 Ser Val Thr Ser Leu Asp Ser Phe Gly Asn Leu Glu Val Ser Pro Pro                 405 410 415 Val Thr Val Asn Gly Lys Thr Tyr Pro Leu Gly Arg Ile Leu Ile Gly             420 425 430 Ser Ser Phe Pro Leu         435

Claims (20)

An antigen-binding fragment, or a polypeptide that specifically binds to one or more proteins selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2, or each of the fragments thereof, or the APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 A primer set, or a nucleotide, which specifically binds to a polynucleotide sequence encoding at least one protein selected from the group consisting of SEQ ID NO: The diagnostic composition for metastatic gastric cancer according to claim 1, wherein the protein is PADI2. The diagnostic composition according to claim 1, wherein the gastric cancer is an early gastric cancer. 4. The composition for diagnosing metastatic gastric cancer according to claim 3, wherein said early gastric cancer corresponds to T1 to T2 on the basis of TNM staging system. The polynucleotide according to claim 1, wherein the PADI2 protein comprises the sequence of SEQ ID NO: 14, the polynucleotide sequence encoding the PADI2 protein comprises the sequence of SEQ ID NO: 1 or 2,
Wherein the APOBEC3A protein comprises the sequence of SEQ ID NO: 15, the polynucleotide sequence encoding the APOBEC3A protein is SEQ ID NO: 3, SEQ ID NO: 3 is the sequence of 171 to 770, SEQ ID NO: 4 and SEQ ID NO: 4 is 171 to 716 Or &lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; sequence,
Wherein the FCGR3B protein comprises the sequence of any one of SEQ ID NOS: 16 to 20, the polynucleotide sequence encoding the FCGR3B protein is SEQ ID NO: 5, the sequence of 261 to 1070 of SEQ ID NO: 5, the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 7, SEQ ID NO: 26, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 8 to 286 to 936, SEQ ID NO: 9 and SEQ ID NO: 9 to 286 to 678 Or &lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt;
Wherein the SERPINB5 protein comprises the sequence of SEQ ID NO: 21 and the polynucleotide sequence encoding the SERPINB5 protein comprises the sequence of SEQ ID NO: 10 or SEQ ID NO:
Wherein the NMU protein comprises a sequence selected from the group consisting of SEQ ID NOs: 22 to 24, wherein the polynucleotide sequence encoding the NMU protein is SEQ ID NO: 11, SEQ ID NO: 11: 107 to 631, SEQ ID NO: 12, SEQ ID NO: 12 to 107 to 583, SEQ ID NO: 13, and SEQ ID NO: 13 to 107 to 556, respectively. The diagnostic composition for metastatic gastric cancer according to claim 1, further comprising a reagent for isolating the nucleus from the sample to be diagnosed. [Claim 6] The PADI2 protein of claim 6, wherein the PADI2 protein comprises the amino acid sequence of SEQ ID NO: 14, wherein the polynucleotide sequence encoding the PADI2 protein contained in the isolated nucleotide is SEQ ID NO: 25 or SEQ ID NO: 25 to 65 to 1378 of SEQ ID NO: &lt; RTI ID = 0.0 &gt; 25. &lt; / RTI &gt; The method of claim 1, wherein the antibody, peptide, protein, or combination thereof specifically binds to one or more proteins selected from the group consisting of IL-1 ?, IL-6, and TNF-? A primer, a nucleotide, or a combination thereof, which specifically binds to a nucleotide sequence encoding at least one protein selected from the group consisting of IL-1β, IL-6, and TNF-β. A kit for the diagnosis of metastatic gastric cancer comprising the composition of claim 1 and a reagent for detecting the same. A peptide, a protein, or a combination thereof, which specifically binds to one or more proteins selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2, or a fragment thereof, to the sample separated from the subject, or the APOBEC3A, FCGR3B , SERPINB5, NMU, and PADI2 to form a complex by contacting a probe, a primer, a nucleotide, or a combination thereof that specifically binds to a polynucleotide sequence encoding at least one protein selected from the group consisting of: And
And measuring the expression level of each of at least one protein selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 from the complex. [Claim 10] The method according to claim 10, wherein the expression level of each of the at least one protein selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 is at least one selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU and PADI2 And comparing each of the standard outbreaks with a standard outbreak rate. 11. The method of claim 10, wherein the sample isolated from the subject is normal tissue and lesion tissue derived from the stomach tissue of the subject. 13. The method of claim 12, further comprising comparing the amount of expression of at least one protein selected from the group consisting of APOBEC3A, FCGR3B, SERPINB5, NMU, and PADI2 measured from each of normal tissue and lesion tissue derived from the stomach tissue of the subject How to diagnose metastatic gastric cancer. 11. The method of claim 10, further comprising isolating a cell nucleus from the sample in a sample separated from the subject. [Claim 12] The method according to claim 12, wherein, in the case of PADI2 protein expression amount, the gastric cancer of the individual is diagnosed to have a high probability of metastasis when the compared expression level of PADI2 is three times or more.  11. The method of claim 10, wherein the subject is afflicted with an early gastric cancer.  17. The method according to claim 16, wherein the initial stomach cancer corresponds to T1 to T2 on a TNM stage system basis. The method of claim 6, wherein the antibody, peptide, protein, or a combination thereof specifically binds to one or more proteins selected from the group consisting of IL-1 ?, IL-6 and TNF-? Contacting a probe, a primer, a nucleotide or a combination thereof that specifically binds to a nucleotide sequence encoding at least one protein selected from the group consisting of IL-1β, IL-6, and TNF-β to form a complex; And
And measuring the expression level of each of the at least one protein selected from the group consisting of IL-1?, IL-6, and TNF-? From the complex. 11. The method of claim 10, wherein the expression level is determined by one or more methods selected from RT-PCR, RNase protection assay (RPA), Northern blotting, and DNA chip. The method according to claim 10, wherein the expression level is measured by Western blotting, ELISA, radioimmunoassay, radial immunodiffusion, Oucheronian immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, Or a protein chip.

Images