KR100968838B1 - Composition for drug delivery comprising the polypeptide fragment of EGF-like domain repeat protein of stabilin-2 and use thereof - Google Patents

Abstract

The present invention relates to a polypeptide comprising a repeat fragment of the EGF-like domain of Stabilin-2, a composition for detecting phosphatidylserine comprising the same, and more particularly, the use of stabilin-2 This is achieved by the fact that a polypeptide that is sequentially linked to a second atypical EGF-like domain and at least three EGF-like domains in an EGF-like domain repeat protein is specifically bound to phosphatidylserine. A polypeptide, a composition for detecting phosphatidylserine comprising the polypeptide, and a use thereof. The second atypical EGF-like domain and three or more EGF-like domains are sequentially linked in an EGF-like domain repeat protein (EGFrp) of stabilin-2 of the present invention. The polypeptide may specifically bind to phosphatidylserine on the cell surface. Therefore, the present invention can be used in various ways, such as drug delivery to cells expressing phosphatidylserine on the cell surface, prevention, treatment or imaging of tumor diseases through delivery of drugs or markers to tumor cells.

Stabilin-2, EGFrp, Phosphatidylserine, Killing Cells, Tumor Disease

Description

Composition for drug delivery comprising the polypeptide fragment of EGF-like domain repeat protein of stabilin-2 and use according to the present invention.

The present invention relates to a composition for drug delivery comprising a repeat region fragment of the EGF-like domain of Stabilin-2 as an active ingredient, and more specifically, to EGF-like of Stabilin-2. The polypeptide is exploited by the fact that a polypeptide that is sequentially linked to a second atypical EGF-like domain and at least three EGF-like domains in an EGF-like domain repeat protein is specifically bound to phosphatidylserine It relates to a composition for drug delivery and its use.

Phosphatidylserine (PS) is an important marker for macrophages to recognize and eliminate apoptosis (Schlegel, RA et al., Cell Death and Differentiation, 2001, 8: 551-563; Lauber, K. et al., Mol Cell 2004, 14: 277-287 Henson, PM et al., Curr Biol 2001, 11: R795-805 Grimsley, C. et al., Trends Cell Biol. 2003, 13: 648-656). Normally, phosphatidylserine is present inside the cell membrane but is exposed to the outside of the cell membrane when the cell receives a death signal or when erythrocytes age (Fadeel, B. et al., Cell Mol Life Sci, 2003, 60: 2575-2585). Macrophages are recognized through cell surface receptors to cause phagocytosis (Fadok, VA et al., J immunol 1992, 148: 2207-2216; Fadok, VA et al., Nature 2000, 405: 85-90; Park , SY et al., Cell Death and Differentiation, in press).

In addition to killer cells, phosphatidylserine is also exposed to the outside of the cell membrane under various pathological conditions (Zwaal, R.F.A. et al., Cell.Mol.Life Sci 2005, 62: 971-988). Examples include Scott syndrome, antiphospholipid syndrome, sickle cell anemia, thalassemia, stomatocytosis, uremia, and kidney stones. kidney stone disease, diabetes, hyperglycemia, viral and microbial infections, malaria, pre-eclampsia, hyperbilirubinemia, neoplasia, etc. have. In particular, many tumor cells show increased expression of phosphatidylserine outside of the cell membrane (Utsugi, T. et al., Cancer Res. 1991, 15: 3062-3066; Rao, L. et al., Thromb Res. 1992, 67: 517-531; Sigimura, M. et al., Fibrinolysis. 1994, 5: 365-373; Ran, S. et al., Cancer Res. 2002, 62: 6132-6140; Woehlecke, H. et al. , Biochem J. 2003, 376: 489-495), which is more prominent in undifferentiated tumorigenic cells. Tumor tissue also releases small blood vessels that expose phosphatidylserine to the outside of the cell membrane (Ran, S. et al., Cancer Res. 2002, 62: 6132-6140; Zwaal, RFA et al., Blood. 1997, 89 : 1121-1132).

Moreover, phosphatidylserine is also observed in non-killing cells among various intracellular physiological processes. Examples include platelet activation, muscle cell fusion, syncytiotrophoblast formation, immunoglobulin-dependent stimulation of mast cells, and T cell migration. (Fadeel, B. et al., Cell Death Differ 2006, 13: 360-2 Ran, S. et al., Int J Radiat Oncol Biol Phys 2002, 54: 1479-84 Schlegel, RA et al., Cell Death Differ 2001, 8: 551-63.). In particular, blocking phosphatidylserine, which is expressed during the activation of platelets, may have a therapeutic effect by inhibiting the thrombosis process that may be formed by atherosclerosis (Cederholm, A. and Frosteg, J.). , Ann NY Acad Sci. 2007, 1108: 96-103 Cederholm, A. and Frosteg, J., Drug News Perspect. 2007 20 (5): 321-6). Therefore, these roles of phosphatidylserine have been suggested as phosphatidylserine as a target material for diagnosis, treatment, and treatment tracking, especially in various situations including neoplastic and inflammatory diseases.

In addition, inhibition of the recognition of phosphatidylserine has been reported to increase the cell's immunogenicity in irradiated lymphoma (Bondanza, A. et al., J Exp Med 2004, 200: 1157-65). Thus, proteins that can effectively bind phosphatidylserine may also be used to augment the effects of apoptotic cell-based vaccines by interfering with phosphatidylserine recognition and immunosuppressive clearance of killer cells. .

Therefore, the present inventors have studied to search for a novel protein or fragment thereof capable of targeting phosphatidylserine. As a result, the repeat region (EGFrp) of the EGF-like domain of stabilin-2 is expressed on the surface of dead cells. The present invention was completed by confirming that it can specifically bind to phosphatidylserine.

It is therefore an object of the present invention to provide a composition for drug delivery comprising the EGFrp fragment of Stabilin-2 which specifically binds to phosphatidylserine and its use.

In order to achieve the above object, the present invention provides a drug delivery composition comprising a polypeptide of the EGF-like domain repeat protein of the stabilin-2 (stabilin-2) as an active ingredient to provide.

In order to achieve the other object of the present invention, the present invention provides a pharmaceutical composition for the prevention and treatment of neoplastic diseases comprising the polypeptide and the anti-tumor disease agent coupled thereto as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention relates to a polypeptide in which a second atypical EGF-like domain and three or more EGF-like domains are sequentially linked in an EGF-like domain repeat protein of stabilin-2. In view of the specific binding to phosphatidylserine exposed to the cell surface, a second sequence of EGF-like domains and three or more EGF-like domains are sequentially combined to include a polypeptide of a new sequence linked thereto as an active ingredient. It provides a pharmaceutical delivery composition and a pharmaceutical composition for the prevention and treatment of tumor diseases comprising the polypeptide and an anti-tumor disease agent coupled thereto as an active ingredient.

Stabilin-2 (Genbank Accession No. MN_017564) of the present invention was first reported through its function as a hyaluronan receptor in sinusoidal endothelial cells of rats (McCourt). , PA et al., Hepatology. 1999, 30: 1276-1286; Politz, O. et al., Biochem J. 2002, 362: 155-164; Zhou, B. et al., J Biol Chem. 2000, 275 (37733-37741), then involved in phagocytosis of modified LDL and glycation end products, and has been reported to have binding capacity to Gram (-) and Gram (+) bacteria (Adachi, H. et al., J Biol Chem. 2002, 277: 34264-34270; Tamura, Y. et al., J Biol Chem. 2003, 278: 12613-126). Furthermore, we have recently identified that Stabilin-2 is an important receptor for phagocytosis of dead cells and aged erythrocytes (Park, S.Y. et al., Cell Death Differ. 2008, 15: 192-201). The extracellular portion of Stabilin-2 consists of 7 FAS1 domains, 4 repeats of EGF-like domains (23 EGF-like domains) and 1 Link domain, among which repeats of EGF-like domains The region (EGF-like domain repeat protein) consists of five or six EGF-like domains. Although EGF-like domains are not known for their exact function, they exist as repeats in the extracellular portion of many membrane receptor proteins and are known to be involved in ligand-receptor interactions (Campbell, ID and Bork, P. Curr Opin Struc Biol). 1993, 3: 385-39), depending on its length and structure: (1) standard EGF-like domains, (2) calcium binding EGF-like domains, (3) laminin-type EGF-like domains, and (4) Can be classified as an atypical EGF-like domain.

The polypeptide in the present invention refers to a polypeptide sequentially linked to a second atypical EGF-like domain and three or more EGF-like domains of an EGF-like domain repeat protein (EGFrp) of Stabilin-2. . Peptide fragments of the present invention include all kinds of peptides, polypeptides, proteins, peptide replicas, compounds and biologics, and have the activity of specifically binding to phosphatidylserine expressed on the surface of cells such as tumor cells. Say that. The repeating site of the EGF-like domain of Stabilin-2 from which the polypeptide of the present invention is derived may be from a mammal and is preferably any one selected from the group consisting of human, rat and mouse. It may be derived from one.

The second atypical EGF-like domain of the polypeptide of the present invention, the EGF-like domain repeat protein (EGFrp) of Stabilin-2, is the repeat site of the known EGF-like domain of Stabilin-2. The second atypical EGF-like domain of may be, but is not limited to, having an amino acid sequence consisting of SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 96, or SEQ ID NO: 102.

In addition, the EGF-like domain constituting three or more EGF-like domains except the second atypical EGF-like domain in the polypeptide of the present invention may be an EGF-like domain of the repeat region of the known EGF-like domain of Stabilin-2. It may be, but not limited to, a standard EGF-like domain or an atypical EGF-like domain. The standard EGF-like domain may preferably have an amino acid sequence selected from the group consisting of SEQ ID NO: 85 to SEQ ID NO: 88, SEQ ID NO: 91 to SEQ ID NO: 94, SEQ ID NO: 97 to SEQ ID NO: 100, and SEQ ID NO: 103 to SEQ ID NO: 105. And the atypical EGF-like domain preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 101, and SEQ ID NO: 102 Can have

The EGF-like domain included in the polypeptide of the present invention may comprise 3 or more, preferably 3 to 10, more preferably 3 to 5 EGF-like domains, each EGF-like The domain may be composed of the same sequence repeated and combined, for example, only the amino acid sequence of SEQ ID NO: 96. In addition, the polypeptide of the present invention may be combined such that the second atypical EGF-like domain is located at the N-terminus, C-terminus or any site of the polypeptide of the present invention. Preferably, the polypeptide of the present invention may be a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 75 to SEQ ID NO: 82 and SEQ ID NO: 106 to 116.

In addition, the present invention provides a polynucleotide having a nucleotide sequence encoding the polypeptide of the present invention. Preferably, the polynucleotide of the present invention may have a nucleotide sequence selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 40 and SEQ ID NO: 64 to SEQ ID NO: 73.

In addition, the present invention provides a vector having a nucleotide sequence encoding a polypeptide of the present invention and a transformant transformed with the vector.

Vectors of the invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Vectors of the present invention may be conventional cloning vectors or expression vectors, which may be used for membrane targeting or secretion in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers (promoter). It includes a signal sequence or leader sequence and can be prepared in various ways depending on the purpose. The vector also includes a selection marker for selecting a host cell containing the vector and, in the case of a replicable vector, the origin of replication.

Transformation with the vector can be carried out by transformation techniques known to those skilled in the art. Preferably, microprojectile bombardment, electroporation, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, PEG-mediated fusion, microinjection (microinjection) and liposome-mediated methods can be used, and the transformant is Escherichia coli ), Bacillus Subtilis ( Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis), Staphylococcus (Staphylococcus), Agrobacterium Tome Pacific Enschede (Agrobacterium tumefaciens ), but is not limited thereto.

The inventors conducted various experiments to confirm the function of the EGFrp, and as a result, the EGFrp mediates the adhesion and phagocytosis of the cells by specifically recognizing phosphatidylserine expressed on the surface of aged and dead cells. Fibroblasts (L / Stab-2 cells) transformed with Stabilin-2 gene were identified. In addition, the present inventors confirmed that phagocytosis of aged and dead cells by macrophages was also inhibited by EGFrp protein in vitro and in vivo. In addition, it was confirmed that calcium is required for stabilin-2 mediated phagocytosis and recognition of phosphatidylserine by EGFrp. Furthermore, the present inventors have created four C-terminal and N-terminal deletion proteins of EGFrp so that EGFrp requires four or more EGF-like domains to recognize phosphatidylserine, and a second atypical EGF-like in EGFrp. It was confirmed that the domain is important for the recognition of calcium dependent phosphatidylserine. From this, the inventors have identified that EGFrp is a novel binding protein of phosphatidylserine. Therefore, it was found that EGFrp of Stabilin-2 can be used as a composition for detecting phosphatidylserine, and furthermore, together with a diagnostic or therapeutic tracking agent for recognizing phosphatidylserine in tumor tissue, or a separate tumor therapeutic agent. It can be seen that it can be used as a pharmaceutical composition for the prevention and treatment of tumor diseases.

More specifically, in an embodiment of the present invention, after dividing the extracellular portion of the Stabilin-2 protein into four subunits to prepare a recombinant protein corresponding thereto (see Example 1), Stabilin-2 having a phagocytic ability After pretreatment to L cells (L / Stab-2 cells), which are overexpressed mouse fibroblasts, the effects of these proteins on phagocytosis of aged cells of the cells were examined (see Example 2). As a result, it was confirmed that phagocytosis of aged red blood cells by L / Stab-2 was inhibited by recombinant proteins corresponding to four subunits (see FIGS. 2 and 3). Thus, it was confirmed that the motifs involved in cell phagocytosis exist in all four subunits. Furthermore, the present invention is to prepare a recombinant protein for the repeat region (EGFrp, E3) and FAS1 domain (F5) of the EGF-like domain constituting the third subunit to further identify the domain involved in cell phagocytosis (Example As a result of the experiment, pretreatment of the protein corresponding to the repeat region of the EGF-like domain was confirmed that the phagocytosis of the aged red blood cells and the dead cells was suppressed (see FIGS. 4 and 5). In addition, repeat regions of four EGF-like domains present in other subunits also inhibited phagocytosis of aged erythrocytes (see FIG. 6). Therefore, it was found that the EGF-like domain repeat protein (EGFrp) is responsible for the recognition of phosphatidylserine.

Furthermore, the present inventors examined whether the EGFrp protein and phosphatidylserine bind directly (see Example 2-2), and the EGFrp protein directly binds to phosphatidylserine and phospholipids other than phosphatidylserine, that is, phosphatidylcholine and phosphatidyl It can be seen that it does not bind to inositol and phosphatidylethanolamine (see FIGS. 7 and 8). Therefore, it was possible to identify that EGFrp protein is a novel protein that specifically binds directly to phosphatidylserine.

On the other hand, the recognition of phosphatidylserine for phagocytosis of senescent cells and natural killer cells is mainly performed by receptors present on the surface of macrophages, so that the extracellular portion of the Stabilin-2 protein is used to express only EGFrp on the surface of cells. Was prepared an expression vector for making a protein substituted with EGFrp (see Fig. 9) (see Example 3). Thereafter, fibroblasts were transformed to prepare a cell line (L / EGF3 cells) in which EGFrp was expressed on the cell surface (see FIG. 10). The cells were used to examine whether EGFrp on the cell surface binds to phosphatidylserine (see Example 4). As a result, EGFrp-expressing cells bound to phosphatidylserine liposomes (see FIG. 11) and adhered well to phosphatidylserine coated plates (see FIG. 12). In addition, EGFrp-expressing cells mediated the attachment of aged erythrocytes (see FIGS. 13 and 14), and adhesion of erythrocytes was specifically inhibited by stabilin-2 monoclonal antibody and phosphatidylserine liposomes (see FIG. 15). ).

In addition, in order to determine whether EGFrp inhibits phagocytosis by macrophages, human monocyte-derived macrophages were separated and tested for aging of red blood cells by EGFrp (see FIG. 16). Furthermore, in order to determine whether EGFrp protein can inhibit phagocytosis in vivo, an animal model inducing macrophages intraperitoneally of rats was used (see Example 5). As a result, EGFrp protein inhibited macrophage phagocytic phagocytosis in rat abdominal cavity (see FIGS. 17 and 18).

On the other hand, proteins that bind to phosphatidylserine in the cell are known to be dependent on calcium ions. The present inventors investigated the effect of calcium on phagocytosis of aged erythrocytes using cells expressing Stabilin-2 (L / Stab-2 cells) (see Example 6-1). As a result, almost minimal aging of red blood cells was observed in serum and calcium-free medium. However, as calcium was added, the phagocytosis of aged erythrocytes increased (see FIG. 19). Therefore, it was found that calcium is required for the stabilization process mediated by Stabilin-2. In addition, whether calcium was required for the recognition of phosphatidylserine by EGFrp was investigated by using the extinction effect of NBD fluorescence (see Example 6-2). As a result, EGFrp adhered well to the PS liposomes, which were inhibited by calcium chelators (see FIG. 20). Therefore, it was found that calcium ion is involved in the recognition process of phosphatidylserine by EGFrp.

We prepared C-terminal and N-terminal defective proteins (see FIG. 21) of the EGFrp protein to investigate motifs that recognize phosphatidylserine of dead cells (see Example 7). In experiments using the C-terminal deletion protein, it was found that four or more EGF-like domains are required for EGFrp to recognize phosphatidylserine (see FIG. 22), and atypical through experiments using the N-terminal deletion protein. It was found that the EGF-like domain is important (see FIG. 23). The results showed similar results in phagocytosis inhibition experiments using L / Stab-2 cells (see FIGS. 24 and 25). Therefore, it was found that EGFrp should be composed of four or more EGF-like domains including atypical EGF-like domains in order to recognize phosphatidylserine.

Furthermore, the inventors have prepared proteins in which each atypical EGF-like domain is deleted to investigate which atypical EGF-like domain is important among the two atypical EGF-like domains in the EGFrp. (See FIG. 26 and Example 7). The degree of adhesion of phosphatidylserine to the protein was examined using the extinction effect of NBD fluorescence, indicating that the second atypical EGF-like domain was important for the recognition of calcium-dependent phosphatidylserine (Fig. 27 and Fig. 27). 28). A second atypical EGF-like domain also plays an important role in phagocytosis of aged erythrocytes (see FIG. 29).

In conclusion, the second atypical EGF-like domain and three EGF-like domains of the EGFrp-like domain repeat region of Stabilin-2, particularly the Stabilin-2, are specific for phosphatidylserine. In addition, it can be seen that it plays an important role in recognition and phagocytosis of dead cells and aged red blood cells in vivo.

For reference, reference may be made to the above-mentioned nucleotide and protein operations (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al. ., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA (1990). )).

Thus, the present invention is directed to a second atypical EGF-like domain and at least three EGF-s in the EGF-like domain repeat protein of the polypeptide of the invention, ie, stabilin-2. Provided is a composition for detecting phosphatidylserine comprising a polypeptide including a polypeptide in which a pseudo domain is sequentially linked.

In addition, since the polypeptide of the present invention specifically binds to phosphatidylserine, the present invention

(a) a second atypical EGF-like domain and three or more EGF-like domains in the polypeptide of the invention, ie the EGF-like domain repeat protein of stabilin-2 Mixing the sequentially linked polypeptide with a sample;

(b) removing the unbound or nonspecifically bound polypeptide; And

(c) it provides a method for detecting phosphatidylserine comprising the step of confirming the binding and position of the polypeptide.

In order to facilitate identification, detection and quantification of binding of the polypeptide of the present invention, the polypeptide of the present invention may be provided in a labeled state. In other words, the detectable label may be provided in a link (eg, covalently bonded or crosslinked). The detectable label may be a colorase (e.g. peroxidase, alkaline phosphatase), radioisotope (e.g. ¹²⁵ I, ³² P, ³⁵ S), chromophore, luminescent or fluorescent material (e.g. FITC , RITC, Fluorescent Protein (GFP (Green Fluorescent Protein); EGFP (Enhanced Green Fluorescent Protein), RFP (Red Fluorescent Protein); DsRed (Discosoma sp. Red fluorescent protein); CFP (Cyan Fluorescent Protein), CGFP (Cyan Green Fluorescent) Protein) and YFP (Yellow Fluorescent Protein)).

Detection methods according to labels are well known in the art, but can be performed, for example, by the following method. If fluorescent material is used as a detectable label, immunofluorescence staining may be used. For example, a fluorescently labeled polypeptide of the present invention may be reacted with a sample to remove unbound or nonspecific binding products, and then fluorescence by peptides may be observed under a fluorescence microscope. In addition, in the case of using the enzyme as a detectable label, the absorbance may be measured by the color reaction of the substrate through the enzymatic reaction, and in the case of the radioactive substance, the radiation emission may be measured.

In addition, the polypeptide of the present invention can specifically bind to cells exposing phosphatidylserine to the cell surface, and since the phosphatidylserine is exposed to the cell membrane in apoptotic cells, the present invention provides the polypeptide of the present invention as an active ingredient. It provides a composition for detecting apoptotic cells comprising. The detection of dead cells in the above is not limited thereto. For example, a method for detecting phosphatidylserine may be used, and as described above, identification, detection, and quantification of binding of the polypeptide of the present invention may be performed. To facilitate, the polypeptides of the invention can be provided in a labeled state. The detected result may be imaged according to a known imaging method according to the detection label.

In addition, the polypeptide of the present invention can specifically bind to a cell exposing phosphatidylserine to a cell surface, and thus can be used as an intelligent drug carrier for selectively delivering a drug to the cell. Thus, the second atypical EGF-like domain and three or more EGF-like domains in the polypeptide of the present invention, ie, the EGF-like domain repeat protein of stabilin-2, It provides a drug delivery composition comprising a sequentially linked polypeptide as an active ingredient.

As noted above, increased expression of phosphatidylserine outside of the cell membrane has been associated with melanoma and colorectal cancer cells (Utsugi, T. et al., Cancer Res. 1991, 15: 3062-3066), ovarian cancer cells (Rao, L. et al. et al., Thromb Res. 1992, 67: 517-531), gastric and liver cancer cells (Sugimura, M. et al., Blood Coagul. Fibrinolysis. 1994, 5: 365-373; Woehlecke, H. et al., Biochem J. 2003, 376: 489-495), and endothelial cells of cancer tissues (Ran, S. et al., Cancer Res. 2002, 62: 6132-6140), which are found in undifferentiated carcinogenic cells. more pronounced in undifferentiated tumorigenic cells. Tumor tissue also releases small blood vessels that expose phosphatidylserine to the outside of the cell membrane (Ran, S. et al., Cancer Res. 2002, 62: 6132-6140; Zwaal, RFA et al., Blood. 1997, 89 : 1121-1132).

Thus, the drug delivery composition may be specific for a neoplastic disease. Tumorous diseases are pathological symptoms caused by malignant tumors, but are not limited to, but are not limited to, colon cancer, lung cancer, stomach cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer , Endometrial cancer, chorionic cancer, ovarian cancer, breast cancer, thyroid cancer, brain cancer, head and neck cancer, malignant melanoma, skin cancer, liver cancer, leukemia, lymphoma, multiple myeloma, chronic myelogenous leukemia myelogenous leukemia, neuroblastoma, aplastic anemia.

When the polypeptide of the present invention contained in the composition for drug delivery of the present invention is used for treatment in connection with a conventional anti-tumor disease agent, the effect of the drug may be exerted because the agent is selectively delivered only to tumor cells by the polypeptide of the present invention. It can increase and at the same time significantly reduce side effects on normal tissues.

Anti-tumor disease agents that can be linked to the polypeptides of the invention can be used without limitation as long as they are used in conventional tumor therapy. For example, paclitaxel, doxorubicin, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, etoposide, teniposide , Bisantrene, homomoharringtonine, Gleevec (STI-571), cisplain, 5-fluouracil, 5-fluouracil, adriamycin, methotrexate, Busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, nitrosourea, and the like. The linking of the agent with the polypeptide of the present invention may be carried out through methods known in the art, such as covalent bonds, crosslinking, and the like. To this end, the peptide of the present invention may be chemically modified if necessary so long as its activity is not lost. The amount of the peptide of the present invention included in the composition of the present invention may vary depending on the type and amount of the anticancer agent bound.

On the other hand, the present invention provides a polypeptide of the present invention, that is, a second atypical EGF-like domain and three or more EGF-s in the EGF-like domain repeat protein of stabilin-2. Provided is a pharmaceutical composition for the prevention and treatment of neoplastic diseases as an active ingredient using the polypeptide and the anti-tumor disease agent coupled to the similar domain sequentially.

At this time, the anti-tumor disease agent, the binding method and the neoplastic disease in the pharmaceutical composition are as described above.

On the other hand, the pharmaceutical composition according to the present invention can be provided by formulating in a suitable form together with the pure form of the polypeptide or a pharmaceutically acceptable carrier. 'Pharmaceutically acceptable' refers to a nontoxic composition which, when administered to humans, is physiologically acceptable and typically does not cause allergic or similar reactions such as gastrointestinal disorders, dizziness and the like. Such carriers include all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.

On the other hand, the pharmaceutical composition according to the present invention can be formulated with a suitable carrier depending on the route of administration. The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but may be administered orally or parenterally. Parenteral routes of administration include, for example, several routes such as transdermal, nasal, abdominal, muscle, subcutaneous or intravenous.

In the case of oral administration of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention is prepared in powder, granule, tablet, pill, dragee, capsule, liquid, gel according to a method known in the art together with a suitable oral carrier. , Syrups, suspensions, wafers and the like. Examples of suitable carriers include sugars, including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol and starch, cellulose, starch including corn starch, wheat starch, rice starch and potato starch, and the like. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like, including methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate and the like may optionally be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, and a preservative.

In addition, when administered parenterally, the pharmaceutical compositions of the present invention may be formulated according to methods known in the art in the form of injections, transdermal and nasal inhalants together with suitable parenteral carriers. Such injections must be sterile and protected from contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, solvents or dispersion media comprising water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycols, etc.), mixtures thereof and / or vegetable oils Can be. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be further included. In addition, the injection may in most cases further comprise an isotonic agent such as sugar or sodium chloride.

In the case of transdermal administrations, ointments, creams, lotions, gels, external preparations, pasta preparations, linen preparations, air rolls and the like are included. As used herein, "transdermal administration" means that the pharmaceutical composition is topically administered to the skin such that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin. These formulations are formulated in Remington's , a commonly known formula in pharmaceutical chemistry. Pharmaceutical Science , 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania.

In the case of inhaled dosages, the compounds used according to the invention may be pressurized packs or by means of suitable propellants, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be delivered conveniently from the nebulizer in the form of an aerosol spray. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in inhalers or blowers can be formulated to contain a mixture of the compound and a suitable powder base such as lactose or starch.

Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

In addition, the pharmaceutical compositions according to the invention may contain one or more buffers (eg saline or PBS), carbohydrates (eg glucose, mannose, sucrose or dextran), stabilizers (sodium bisulfite, Sodium sulfite or ascorbic acid) antioxidants, bacteriostatic agents, chelating agents (eg EDTA or glutathione), adjuvants (eg aluminum hydroxide), suspending agents, thickening agents and / or preservatives (benzalkonium chloride , Methyl- or propyl-paraben and chlorobutanol).

In addition, the pharmaceutical compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

Pharmaceutical compositions formulated in such a manner can be administered in a effective amount via several routes, including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, an 'effective amount' refers to an amount of a compound or extract that, when administered to a patient, enables the tracking of a diagnostic or therapeutic effect. The dosage of the pharmaceutical composition according to the present invention may be appropriately selected according to the route of administration, the subject to be administered, the target disease and its severity, age, sex weight, individual difference, and disease state. Preferably, the pharmaceutical composition comprising the polypeptide of the present invention may vary in the amount of the active ingredient depending on the extent of the disease, but usually an effective dose of 10 μg to 10 mg per single dose based on an adult May be repeated several times a day.

In addition, a second atypical EGF-like domain and three or more EGF-like domains of the polypeptide of the present invention, ie, the EGF-like domain repeat protein of stabilin-2, Sequentially linked polypeptides specifically bind to phosphatidylserine and thus may be useful for imaging the onset of neoplastic disease. Accordingly, the present invention provides a composition for imaging tumor disease comprising the polypeptide as an active ingredient. The polypeptide may be provided in a labeled state to facilitate identification, detection, and quantification of binding, as described above.

In addition, phosphatidylserine is exposed to the outside of the cell membrane in a variety of pathological conditions in addition to the tumor disease (Zwaal, R.F.A. et al., Cell.Mol.Life Sci. 2005, 62: 971-988). Examples include Scott syndrome, antiphospholipid syndrome, sickle cell anemia, thalathemia, stomatocytosis, uremia, and kidney stones ( kidney stone disease, diabetes, hyperglycemia, viral and microbial infections, malaria, pre-eclampsia, hyperbilirubinemia, neoplasia, etc. have.

Thus, the present invention is directed to a second atypical EGF-like domain and at least three EGF-s in the EGF-like domain repeat protein of the polypeptide of the invention, ie, stabilin-2. Scott syndrome, antiphospholipid syndrome, sickle cell anemia, thalathemia, stomatocytosis, comprising polypeptides with similar domains sequentially linked as active ingredients (stomatocytosis), uremia, kidney stone disease, diabetes mellitus, hyperglycemia, viral infections and microbial infections, malaria, pre-eclampsia, gobilirubin A diagnostic composition for a disease selected from the group consisting of hyperbilirubinemia and neoplasia can be provided.

On the other hand, the sequences (SEQ ID NO: 33 to SEQ ID NO: 116) relating to the polypeptide and polynucleotide in Stabilin-2 described in the present invention are as described in Table 1 below. In addition, the fragments of the present invention are named according to the positions of the EGFrp and EGF-like domains, for example, fragment E1-14 of the present invention is the first EGF-like domain of the first EGFrp in Stabilin-2. Refers to a fragment consisting of an amino acid sequence from the fourth EGF-like domain, wherein fragment E3-25 of the present invention is from the second EGF-like domain of the third EGFrp in Stabilin-2 to the fifth EGF-like domain. It means a fragment consisting of the amino acid sequence. In the present invention, the order of subunits (Stab-U1, etc.), EGFrp, and EGF-like domains are all ordered according to their position at the N terminus.

SEQ ID NO: Name / Content of Sequence 33 Sequence of the First Subunit of Human Stabilin-2 (Stab-U1) 34 Nucleotide sequence of the second subunit of human Stabilin-2 (Stab-U2) 35 Nucleotide sequence of the third subunit of human Stabilin-2 (Stab-U3) 36 Nucleotide sequence of the fourth subunit of human Stabilin-2 (Stab-U4) 37 Nucleotide Sequences of the First EGFrp of Human Stabilin-2 38 Nucleotide Sequences of the Second EGFrp of Human Stabilin-2 39 Nucleotide Sequences of the Third EGFrp of Human Stabilin-2 40 Nucleotide Sequences of the Fourth EGFrp of Human Stabilin-2 41 Sequences of the First EGF-Like Domain in the First EGFrp of Human Stabilin-2 42 Nucleotide Sequences of the Second EGF-Like Domain in the First EGFrp of Human Stabilin-2 43 Sequences of the Third EGF-Like Domain in the First EGFrp of Human Stabilin-2 44 Nucleotide Sequences of the Fourth EGF-Like Domain in the First EGFrp of Human Stabilin-2 45 Nucleotide Sequences of the Fifth EGF-Like Domain in the First EGFrp of Human Stabilin-2 46 Sequences of the Sixth EGF-Like Domain in the First EGFrp of Human Stabilin-2 47 Nucleotide Sequences of the First EGF-Like Domain in the Second EGFrp of Human Stabilin-2 48 Nucleotide Sequences of the Second EGF-Like Domain in the Second EGFrp of Human Stabilin-2 49 Nucleotide Sequences of the Third EGF-Like Domain in the Second EGFrp of Human Stabilin-2 50 Nucleotide Sequences of the Fourth EGF-Like Domain in the Second EGFrp of Human Stabilin-2 51 Nucleotide Sequences of the Fifth EGF-Like Domain in the Second EGFrp of Human Stabilin-2 52 Nucleotide Sequences of the Sixth EGF-Like Domain in the Second EGFrp of Human Stabilin-2 53 Nucleotide Sequences of the First EGF-Like Domain in the Third EGFrp of Human Stabilin-2 54 Nucleotide sequence of the second EGF-like domain within the third EGFrp of human Stabilin-2 55 Nucleotide Sequences of the Third EGF-Like Domain in the Third EGFrp of Human Stabilin-2 56 Nucleotide Sequences of the Fourth EGF-Like Domain in the Third EGFrp of Human Stabilin-2 57 Nucleotide Sequences of the Fifth EGF-Like Domain in the Third EGFrp of Human Stabilin-2 58 Nucleotide Sequences of the Sixth EGF-Like Domain in the Third EGFrp of Human Stabilin-2 59 Nucleotide Sequences of the First EGF-Like Domain in the Fourth EGFrp of Human Stabilin-2 60 Nucleotide sequence of the second EGF-like domain within the fourth EGFrp of human Stabilin-2 61 Nucleotide sequence of the third EGF-like domain within the fourth EGFrp of human Stabilin-2 62 Nucleotide Sequences of the Fourth EGF-Like Domain in the Fourth EGFrp of Human Stabilin-2 63 Nucleotide Sequences of the Fifth EGF-Like Domain in the Fourth EGFrp of Human Stabilin-2 64 Nucleotide Sequences of the E1-14 Fragment of the Invention in Human Stabilin-2 65 Nucleotide Sequences of the E1-15 Fragment of the Invention in Human Stabilin-2 66 Nucleotide Sequences of the E1-25 Fragment of the Invention in Human Stabilin-2 67 Nucleotide Sequences of the E2-14 Fragment of the Invention in Human Stabilin-2 68 Nucleotide Sequences of the E2-15 Fragment of the Invention in Human Stabilin-2 69 Nucleotide Sequences of the E2-25 Fragment of the Invention in Human Stabilin-2 70 Nucleotide Sequences of the E3-14 Fragment of the Invention in Human Stabilin-2 71 Nucleotide Sequences of the E3-15 Fragment of the Invention in Human Stabilin-2 72 Nucleotide Sequences of the E3-25 Fragment of the Invention in Human Stabilin-2 73 Nucleotide Sequences of the E4-14 Fragment of the Invention in Human Stabilin-2 74 Nucleotide Sequences of the E4-25 Fragment of the Invention in Human Stabilin-2 75 Amino Acid Sequence of First Subunit of Human Stabilin-2 (Stab-U1) 76 Amino Acid Sequence of Second Subunit of Human Stabilin-2 (Stab-U1) 77 Amino Acid Sequence of Third Subunit of Human Stabilin-2 (Stab-U1) 78 Amino acid sequence of the fourth subunit of human stabilin-2 (Stab-U1) 79 Amino Acid Sequences of First EGFrp of Human Stabilin-2 80 Amino Acid Sequences of Second EGFrp of Human Stabilin-2 81 Amino Acid Sequences of the Third EGFrp of Human Stabilin-2 82 Amino Acid Sequences of the Fourth EGFrp of Human Stabilin-2 83 Amino Acid Sequences of the First EGF-Like Domain in the First EGFrp of Human Stabilin-2 84 Amino acid sequence of the second EGF-like domain in the first EGFrp of human Stabilin-2 85 Amino Acid Sequences of the Third EGF-Like Domain in the First EGFrp of Human Stabilin-2 86 Amino acid sequence of the fourth EGF-like domain within the first EGFrp of human Stabilin-2 87 Amino Acid Sequences of the Fifth EGF-Like Domain in the First EGFrp of Human Stabilin-2 88 Amino acid sequence of the sixth EGF-like domain in the first EGFrp of human Stabilin-2 89 Amino Acid Sequences of the First EGF-Like Domain in the Second EGFrp of Human Stabilin-2 90 Amino acid sequence of the second EGF-like domain within the second EGFrp of human Stabilin-2 91 Amino Acid Sequences of the Third EGF-Like Domain in the Second EGFrp of Human Stabilin-2 92 Amino acid sequence of the fourth EGF-like domain within the second EGFrp of human Stabilin-2 93 Amino Acid Sequence of the Fifth EGF-Like Domain in the Second EGFrp of Human Stabilin-2 94 Amino Acid Sequences of the Sixth EGF-Like Domain in the Second EGFrp of Human Stabilin-2 95 Amino Acid Sequences of the First EGF-Like Domain in the Third EGFrp of Human Stabilin-2 96 Amino acid sequence of the second EGF-like domain within the third EGFrp of human Stabilin-2 97 Amino acid sequence of the third EGF-like domain within the third EGFrp of human Stabilin-2 98 Amino acid sequence of the fourth EGF-like domain within the third EGFrp of human Stabilin-2 99 Amino Acid Sequences of the Fifth EGF-Like Domain in the Third EGFrp of Human Stabilin-2 100 Amino Acid Sequences of the Sixth EGF-Like Domain in the Third EGFrp of Human Stabilin-2 101 Amino Acid Sequences of the First EGF-Like Domain in the Fourth EGFrp of Human Stabilin-2 102 Amino acid sequence of the second EGF-like domain within the fourth EGFrp of human Stabilin-2 103 Amino acid sequence of the third EGF-like domain in the fourth EGFrp of human Stabilin-2 104 Amino acid sequence of the fourth EGF-like domain within the fourth EGFrp of human Stabilin-2 105 Amino Acid Sequences of the Fifth EGF-Like Domain in the Fourth EGFrp of Human Stabilin-2 106 Amino Acid Sequences of the E1-14 Fragment of the Invention in Human Stabilin-2 107 Amino Acid Sequences of the E1-15 Fragments of the Invention in Human Stabilin-2 108 Amino Acid Sequences of the E1-25 Fragment of the Invention in Human Stabilin-2 109 Amino Acid Sequences of the E2-14 Fragment of the Invention in Human Stabilin-2 110 Amino Acid Sequences of the E2-15 Fragment of the Invention in Human Stabilin-2 111 Amino Acid Sequences of the E2-25 Fragment of the Invention in Human Stabilin-2 112 Amino Acid Sequences of the E3-14 Fragment of the Invention in Human Stabilin-2 113 Amino Acid Sequences of the E3-15 Fragment of the Invention in Human Stabilin-2 114 Amino Acid Sequences of the E3-25 Fragment of the Invention in Human Stabilin-2 115 Amino Acid Sequences of the E4-14 Fragment of the Invention in Human Stabilin-2 116 Amino Acid Sequences of the E4-25 Fragment of the Invention in Human Stabilin-2

As described above, the second atypical EGF-like domain and three or more EGF-like domains in the EGF-like domain repeat protein of stabilin-2 of the present invention. Sequentially linked polypeptides can specifically bind phosphatidylserine on the cell surface. Therefore, the present invention can be used in various ways, such as drug delivery to cells expressing phosphatidylserine on the cell surface, prevention, treatment or imaging of tumor diseases through delivery of drugs or markers to tumor cells.

Below. The present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1>

Stabilin -2( stabilin -2) Preparation of Recombinant Protein

<1-1> Preparation of Expression Vector

The extracellular portion of Stabilin-2 was divided into four subunits, each subunit comprising one EGF-like domain repeat protein (EGFrp, EGF-like domain repeat protein) and two FAS-1 domains. (See FIG. 1).

First, to prepare a recombinant expression vector comprising the subunits (Stab-U1, Stab-U2, Stab-U3 and Stab-U4), based on the human Stabilin-2 cDNA sequence (Genbank Accession No. NM_017564) Four pairs of primers (SEQ ID NO: 1 to SEQ ID NO: 8) were designed (see Table 2). Using each primer pair designed above, the polymerase was expressed using an expression vector (Park, SY et al., Cell Death Differ. 2008, 15: 192-201) containing the cDNA of human Stabilin-2 as a template. Polymerase chain reaction (PCR) was performed. Specifically, the PCR reaction using Pfu DNA polymerase (Promega) for 2 minutes at 95 ℃, then 1 minute at 94 ℃, 30 seconds at 55 ℃, 2 minutes at 72 ℃ 35 It was performed repeatedly. As a result, each cDNA corresponding to four subunits was obtained. Obtained above Stab-U1, Stab-U2, and Stab-U3 in the amplification products were digested with restriction enzymes BamHI and XhoI (TaKaRa) and Stab-U4 with restriction enzymes BamHI and EcoRI, respectively, followed by the same restriction of the pET43.1 (Novagen) vector. Recombinant vectors 'pET-Stab-U1', 'pET-Stab-U2', 'pET-Stab-U3', and 'pET-Stab-U4' are inserted by inserting the T4 ligase (Invitrogen) into the enzyme site. Were prepared respectively. The pET43.1 (Novagen) vector contains a coding sequence for the Nus tag at the N-terminus to increase the solubility of the recombinant protein.

In addition, to prepare a recombinant expression vector comprising a repeating site (EGFrp; E1, E2, E3 and E4) of the EGF-like domain present in each subunit of Stabilin-2, a human Stabilin-2 cDNA sequence was prepared. Four pairs of primers (SEQ ID NOs: 1, 3, 5, 7 and SEQ ID NOs: 9 to 12) were designed on the basis (see Table 2). Using the primer pairs designed above, the same polymerase chain reaction (PCR) was carried out using the expression vector including the Stabilin-2 cDNA as a template. The resulting amplification product was digested with BamHI and XhoI (TaKaRa), and then inserted into the same restriction enzyme site of pET43.1 (Novagen) vector using T4 ligase (Invitrogen) to recombinant vector 'pET-E1. ',' pET-E2 ',' pET-E3 ', and' pET-E4 'were prepared.

Meanwhile, in order to prepare a recombinant expression vector comprising the FAS-1 domain (F5) present in the third subunit of Stabilin-2 (Stab-U3), a pair of pairs based on the human Stabilin-2 cDNA sequence Primers (SEQ ID NO: 13 to SEQ ID NO: 14) were designed (see Table 2). Using the primer pairs designed above, the same polymerase chain reaction (PCR) was carried out using the expression vector including the Stabilin-2 cDNA as a template. Specifically, the PCR reaction was performed by using Pfu DNA polymerase (Promega) for 2 minutes at 95 ° C., followed by 1 minute at 94 ° C., 30 seconds at 55 ° C., and 1 minute at 72 ° C. for 35 times. Was performed. The resulting amplification product was digested with BamHI and XhoI (TaKaRa), and then inserted into the same restriction enzyme site of pET43.1 (Novagen) vector using T4 ligase (Invitrogen) to recombinant vector'pET-F5 'Was prepared.

Primers used to prepare expression vectors with stabilin-2 primer order SEQ ID NO: Stab-U1 sense 5’-AAAAGGATCCGTAGGGGTTCGAGATTG-3 ’ One Stab-U1 antisense 5’-AAAACTCGAGTCAGGGAGGAATGAGAAC-3 ’ 2 Stab-U2 sense 5’-AAAAGGATCCAACTCTGAGCCCACAG-3 ’ 3 Stab-U2 antisense 5’-AAAACTCGAGTCACTGAATTTCCAG-3 ’ 4 Stab-U3 sense 5’-AAAAGGATCCGAGAAGAGGAGATGC-3 ’ 5 Stab-U3 Antisense 5’-AAAACTCGAGTCAATCAATCAGCAGAC-3 ’ 6 Stab-U4 sense 5’-AAAAGGATCCAGGTGGAGTAAACCAAAG-3 ’ 7 Stab-U4 Antisense 5’-AAAACTCGAGTCAGGGTGCTTTTAAAGGC-3 ’ 8 E1 antisense 5'-AAAAACTCGAGTCAATAACACGTTAAGCC-3 ' 9 E2 antisense 5’-AAAAACTCGAGTCAATAGCACAGAAAGCC-3 ’ 10 E3 antisense 5’-AAAAACTCGAGTCAGCGGCAGGTAAATCC-3 ’ 11 E4 antisense 5'-AAAAACTCGAGTCACTCACAGTTCAGCCC-3 ' 12 F5 sense 5’-AAAAGGATCCACTGTTTTTGCACC-3 ’ 13 F5 antisense 5’-AAAACTCGAGTCATTTGGGAGATAGCAA-3 ’ 14

<1-2> Stabilin -2( stabilin -2) Preparation of Recombinant Protein

E. coli BL21 (DE3) was transformed using the respective expression vectors prepared in Example 1-1. The transformed Escherichia coli was cultured in LB medium containing 50 µg / ml kanamycin, and then, when the absorbance of the culture medium was 0.5 to 0.6 at 600 nm to induce the expression of recombinant protein, 1 mM IPTG (isopropyl) was used. -D-(-)-thiogalactopyranoside) was added and further incubated at 37 ° C for 4 hours.

The recombinant protein expressed after the culture was purified according to a known method (Kim, J.-E. et al., J. Cell . Biochem ., 77: 169-187, 2000). Specifically, after centrifuging the culture of the transformed E. coli to obtain the cells, lysis buffer (50mM Tris-HCl (pH 8.0), 100mM NaCl, 1mM EDTA, 1% Triton X-100, 1mM PMSF, 0.5mM Resuspended in DTT). The cell suspension was pulverized by ultrasonication and centrifuged to purify the recombinant protein using Ni-NTA resin (resin, Qiagen). The recombinant protein was eluted in 200mM imidazole solution, and the recombinant protein was identified by SDS-PAGE (result not shown).

As a result, a recombinant protein named 'Stab-U1', 'Stab-U2', 'Stab-U3' and 'Stab-U4' each containing the subunit of Stabilin-2 was prepared. Recombinant proteins named 'E1', 'E2', 'E3', and 'E4', each containing only the repetition site (EGFrp) of the EGF-like domain present in each subunit of, were prepared. A recombinant protein named 'F5' containing only the FAS-1 domain (F5) present in the third subunit (Stab-U3) was prepared.

<Example 2>

Identification of the recognition site of phosphatidylserine (PS) in stabilin-2

<2-1> Phagocytosis inhibitory effect by recombinant protein containing subunit of Stabilin-2

The recombinant protein (Stab-U1, Stab-U2, Stab-U3, and Stab-U4) prepared using Stabilin-2 in <Example 1-2> has L / Stab having phagocytic activity Whether or not the inhibition of senescence of aged red blood cells by -2 cells (Park, SY et al., Cell Death Differ. 2008, 15: 192-201) was investigated (Park, SY et al., Cell Death Differ. 2008) , 15: 192-201).

Specifically, first, the normal red blood cells collected from normal adults were diluted with phosphate buffered solution (PBS, phosphate buffered saline) to 20% hematocrit, and then aged cells were prepared by culturing at 37 ° C. for 4 days. Recombinant proteins (Stab-U1, Stab-U2, Stab-U3 and Stab-U4) prepared in 1-2> were preincubated with hematocrit 1% of the aged red blood cells for 1 hour at 37 ° C. At this time, as a control, Nus protein produced by the pET43.1 vector was pre-cultured with the aged red blood cells as described above. After the preculture, L / Stab-2 cells were added and further incubated at 37 ° C. for 1 hour. After completion of the culture, red blood cells not bound to L / Stab-2 cells were removed, and the cells were washed five times with phosphate buffer solution, and then treated with water for 10 seconds to dissolve all bound red blood cells. After lysing, the L / Stab-2 cells were stained using a Diff quick staining kit (IMEM Ins., San Marcos, Calif., USA) according to the manufacturer's instructions. Posted After counting the number of cells redeemed erythrocytes in five arbitrarily selected places, the number of cells engulfed the red blood cells among the total L / Stab-2 cells as a percentage to calculate the detection index (%) and the results are shown in Figure 2 Shown in

As a result, as shown in Figures 2 and 3, when the L / Statab-2 cells pre-cultured with the recombinant protein consisting of the four subunits of the Stabilin-2, it can be seen that the phagocytosis of aged erythrocytes is significantly inhibited In contrast, the pre-culture with the control group Nus protein did not significantly affect the phagocytosis of aged erythrocytes, as did the pre-culture. From the above results, it was confirmed that the motifs involved in phagocytosis by interacting with phosphatidylserine of aged red blood cells are present in all four subunits.

<2-2> Inhibition of phagocytosis by recombinant protein comprising repeat region (EGFrp) of EGF-like domain of Stabilin-2

In order to more specifically identify the motif interacting with phosphatidylserine in Stabilin-2 as in <Example 2-1>, the recombinant prepared using Stabilin-2 in <Example 1-2> It was further confirmed whether the recombinant protein (Stab-U3) and the deleted recombinant protein (E3, F5) for the third subunit of the protein had phagocytic activity.

Specifically, each recombinant protein (Stab-U3, E3, F5) is pre-incubated with the dead cells at concentrations of 0.1, 1, and 10 µg, respectively, and then the recombinant protein is separated from the dead cells by L / Stab-2 cells. It was confirmed whether or not to inhibit the eating. At this time, the dead cells were cultured in RPM-I1640 medium (containing 10% fetal bovine serum). Human leukemia Jurkat T cells (Korea Cell Line Bank) were added to a medium to which 100ng / mg of anti-Fas antibody (CH-11) was added. Refers to cells prepared by inducing death by incubating for 6 hours by inoculation (Oka, K. et al., Proc Natl Acad Sci USA , 95: 9535-40, 1998), to stain L / Stab-2 cells Annexin-V Apoptosis Detection Kit (Santa Cruz) was used, and the experimental method was performed in the same manner as in <Example 2-1>, and the results are shown in FIGS. 4 and 5. Indicated.

As a result, as shown in Figures 4 and 5, it can be seen that Stab-U3 and E3 protein containing the repeat site (EGFrp) of the EGF-like domain inhibits the phagocytosis of the dead cells in a concentration-dependent manner. From the above results, it was confirmed that a motif involved in phagocytosis by interacting with phosphatidylserine of apoptotic cells is present at the repeating site (EGFrp) of the EGF-like domain.

On the other hand, in order to confirm whether the repetition site (EGFrp) of the EGF-like domain in another subunit exhibits the same effect, the recombinant example (E1, E2 and E4) prepared in Example 1-2 may be used in the above <Example 2-1> the same experiment was carried out and the results are shown in FIG.

As a result, as shown in FIG. 6, it was found that all of the recombinant proteins (E1, E2, E3 and E4) including only the repetition site (EGFrp) of the EGF-like domain all concentration-dependently inhibit the aging of aged red blood cells. Could. From the above results, it was confirmed that the motif involved in phagocytosis by interacting with phosphatidylserine of aged erythrocytes or apoptosis cells is present at the repeating site (EGFrp) of the EGF-like domain. In other words, it was found that phagocytosis of aged erythrocytes and apoptotic cells was competitively inhibited by the repetition site (EGFrp) of the EGF-like domain of the recombinant protein, and this result was EGF-like in the extracellular portion of Stabilin-2. It was found that repeat sites in the domain mediate the recognition of aged red blood cells and dead cells.

<2-3> Confirmation of Direct Binding of Phosphatidylserine with Recombinant Protein Containing Repeating Site (EGFrp) of EGF-Like Domain of Stabilin-2

In order to confirm whether the recombinant protein prepared in Example 1-2 directly binds to phosphatidylserine, phosphatidylserine is dissolved in methanol to make a concentration of 3 µg / ml, and then added to an ELISA plate, followed by air. It was left at room temperature until methanol evaporated. Then, in order to inhibit nonspecific binding, 2% bovine serum albumin (BSA) was added to each well and incubated at room temperature for 1 hour, followed by Stab-U3, E3, and F5 prepared in Example 1-2. And, as a control, Nus protein was added and further incubated for 1 hour at room temperature. After the incubation, the unbound protein was removed with a phosphate buffer solution, and the protein bound to phosphatidylserine in the well was left for 1 hour by the addition of an anti-His antibody with peroxidase, followed by hydrogen peroxide solution. And by adding a coloring reagent containing o-phenylenediamine (o-phenylenediamine) was confirmed the binding degree of the recombinant protein prepared in <Example 1-2> and phosphatidylserine and the results are shown in FIG.

As a result, as shown in FIG. 7, Stab-U3 and E3 proteins containing the repeating site (EGFrp) of the EGF-like domain, as in the experimental results of <Example 2-2>, directly with phosphatidylserine Could be combined.

On the other hand, to determine whether the repeat site (EGFrp) of the EGF-like domain present in Stabilin-2 selectively binds directly to phosphatidylserine, phosphatidylserine (PS), phosphatidylcholine (PC), After coating phosphatidylinositol (PI) and phosphatidyl ethanolamine (PE), the same experiment as above was performed by adding the E3 protein prepared in Example 1-2, and the results are shown in FIG. 8.

As a result, as shown in Figure 8, it was found that the repeat site (EGFrp) of the EGF-like domain selectively binds directly to phosphatidylserine.

<Example 3>

Cloning of cDNA of Recombinant Proteins Containing Only Repeat Sites of the EGF-Like Domain of Stabilin-2

<3-1> Preparation of Expression Vector

To determine if the repeat site (EGFrp) of the EGF-like domain actually recognizes phosphatidylserine that is present on the surface of apoptotic cells at the cell surface, first the extracellular portion of Stabilin-2 is referred to as the repeat site of the third EGF-like domain ( An expression vector was prepared to express a protein (see FIG. 9) substituted with EGFrp).

First, in order to obtain cDNA corresponding to the signal peptide region of Stabilin-2, an expression vector containing Stabilin-2 cDNA was used as a template, and the primers (SEQ ID NO: 15 and SEQ ID NO: 16) shown in Table 3 below were used. A polymerase chain reaction (PCR) was performed. Specifically, the PCR reaction was performed by using Pfu DNA polymerase (Promega) for 2 minutes at 95 ° C. and then repeating the reaction 35 times for 1 minute at 94 ° C., 30 seconds at 55 ° C., and 1 minute at 72 ° C. It was. The product amplified by the PCR was digested with NheI and XhoI (TaKaRa) restriction enzymes, and then inserted into the same restriction enzyme site of the pcDNA3.1 / Myc-His (Invitrogen) vector using T4 ligase (Invitrogen). Recombinant vector 'pcDNA-S' was prepared.

On the other hand, in order to obtain cDNA containing the transmembrane and intracellular site of Stabilin-2 as a template, the expression vector of Stabilin-2 was used as a template, and the primers shown in Table 3 (SEQ ID NO: 17 and SEQ ID NO: 18) were used. The same polymerase chain reaction (PCR) was performed. The product amplified by the PCR was digested with BamHI and HindIII (TaKaRa) restriction enzymes, and then inserted into the restriction enzyme site of the pcDNA-S vector using a T4 ligase (Invitrogen) to recombinant vector'pcDNA-S. -TMC 'was prepared.

In addition, in order to obtain a cDNA corresponding to the repeat site (EGFrp) of the EGF-like domain of Stabilin-2, the expression vector of Stabilin-2 was used as a template, and the primers shown in Table 3 (SEQ ID NOs: 19 to 20) were used. Using the same polymerase chain reaction (PCR) as in <Example 1-1>. The product amplified by the PCR was digested with XhoI and BamHI (TaKaRa) restriction enzymes, and then inserted into the same restriction enzyme site of the pcDNA-S-TMC vector using T4 ligase (Invitrogen) to stabilize the Stabilin- The recombinant vector was replaced by replacing the extracellular portion of 2 with the repeat site (EGFrp) of the third EGF-like domain, which was named 'pcDNA-EGF3'.

The plasmids prepared above were analyzed in a sequencing analyzer (Applied Biosystems AB13700) to confirm that they were correctly cloned (results not shown).

Primers Used for Cloning of EGF3 Expression Vectors primer order SEQ ID NO: Signal sense 5’-AAAATGCTAGCTCAGCCTGACAGGTGC-3 ’ 15 Signal antisense 5'-AAAATCTCGAGTCTTGCCTGCCCTGTG-3 ' 16 TMC Sense 5’-AAAAAGGATCCACCCACACTGGCTTG-3 ’ 17 TMC Antisense 5’-CCCGTCCAAGCTTGCACAGTGTCCT-3 ’ 18 EGF3 sense 5’-AAAACCTCGAGAGATGTGATAATAATGAC-3 ’ 19 EGF3 antisense 5’-AAAAAGGATCCGCGGCAGGTAAATCC-3 ’ 20

<3-2> Transformation of L Cells

The expression vector pcDNA-EGF3 prepared in <Example 3-1> was transformed into L cells (ATCC CCL-1, provided by Dr. Takechi, Department of Biophysics, University of Tokyo), which are mouse fibroblasts. Specifically, the L cells were cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% heat-inactivated fetal bovine serum (FBS), penicillin G and streptomycin, and the L cells selected above were transformed with recombinant vector pcDNA-EGF3. Lipofectamine was used for the transformation and transformed according to the manufacturer's instructions. 36 hours after the transformation, each colony exhibiting resistance was isolated by incubating for 10 to 12 days with G418 (400 µg / ml). The cells transformed in the above manner were named 'L / EGF3', and as the negative control, cells transformed with pcDNA3.1 (-) / Myc-His, which are vectors containing no EGF3 gene, were used. Named 'L / Mock'.

<3-3> at the cell surface EGF3 The expression of

In order to confirm whether EGF3 is expressed on the L / EGF3 cell surface prepared in Example <3-2>, it was analyzed by flow cytometry using a monoclonal antibody (5G3) that recognizes EGF3.

Specifically, the L / EGF3 cells were completely confluent in a plate containing DMEM (Dulbecco's modified Eagle's medium) medium, and then treated with a phosphate buffer solution containing 0.25% trypsin and 0.05% EDTA on the plate surface. Cells were isolated from. The separated cells were washed twice with phosphate buffer solution and then resuspended in phosphate buffer solution, and then added with Stabilin-2 monoclonal antibody (5G3) and incubated at 4 ° C. for 1 hour. Secondary anti-mouse immunoglobulin G (Santa Cruz Biotechnology, Inc., CA) coupled with 10 μg / ml of fluorescein isothiocyanate (FITC) was added to the culture and further incubated at 4 ° C. for 1 hour. After incubation, the cells were analyzed at 488 nm using a 5-watt laser-equipped flow cytometer (Becton Dickinson, San Jose, Calif.), In which a mouse immunoglobulin (subtype-matched subtype) instead of the Stabilin-2 monoclonal antibody was used as a control IgG1) was used and the results are shown in FIG.

As a result, as shown in Figure 10, it was confirmed that EGF3 is expressed on the surface of the L / EGF3 cells, while it was found that EGF3 is not expressed on the surface of the L / Mock cells. In addition, expression of EGF3 was not detected even when mouse immunoglobulins were used instead of the Stabilin-2 monoclonal antibody.

< Example  4>

Stabilin -2 EGF Of repeat regions of the analogous domain Phosphatidylserine  Recognition and the Attachment and Phagocytosis of Aged Red Blood Cells

<4-1> L / EGF3  By cell Phosphatidylserine  recognition

To determine whether L / EGF3 cells recognize phosphatidylserine, the adhesion activity of L / EGF3 cells to phosphatidylcholine liposomes and phosphatidylserine liposomes was examined.

Specifically liposomes comprising phosphatidlycholine (PC) or phosphatidlyserine (PS) were prepared using known methods (Oka, K. et al., Proc . Natl . Acad . Sci . USA. 95: 9535-9540, 1998; Fadok, VA et al., Nature 405: 85-90, 2000). More specifically, phosphatidylserine: phosphatidylcholine was mixed at a molar ratio of 50:50, respectively, to prepare liposomes (hereinafter referred to as 'phosphatidylserine liposomes'), and liposomes containing only phosphatidylcholine (hereinafter referred to as 'phosphatidylcholine liposomes'). . The prepared liposome mixture was mixed with chloroform, dried under nitrogen gas, resuspended in phosphate buffer solution to a final concentration of 5 mM, and sonicated on ice for 10 minutes. In addition, the liposomes were prepared with fluorescently labeled phospholipid liposomes by adding 1% N- (lissamin rhodamine B sulfonyl) -L-α-phosphatidylethanolamine (Avanti Polar Lipids) to total phospholipids.

After fully culturing the L / EGF3 cells prepared in <Example 3-2> in DMEM medium, various phospholipid liposomes prepared above were added to each of 0, 10, and 100 μM and incubated at 37 ° C. for 1 hour. . After the incubation was completed, the phosphate buffer solution was sufficiently washed and the degree of adhesion of phospholipid liposomes to L / EGF3 was analyzed using a flow cytometer (Becton Dickinson, San Jose, Calif.) And the results are shown in FIG. 11.

As a result, as shown in FIG. 11, L / EGF3 cells were found to increase binding to phosphatidylserine liposomes by adding more phosphatidylserine liposomes, but did not bind to phosphatidylcholine liposomes even at high concentrations.

<4-2> Stabilin -2 EGF By repeat regions of the analogous domain Phosphatidylserine  recognition

In order to determine whether the repeat region (EGFrp) of the EGF-like domain of Stabilin-2 recognizes phosphatidylserine, the following cell adhesion experiment was performed.

Specifically, phosphatidylserine and phosphatidylcholine as a control group were coated on an ELISA plate at the concentrations of 0.1, 1, 10, and 100 μM in the same manner as in <Example 2-3>, L / stab-2 and <Example 3-2. L / EGF3 prepared in>, and L / Mock cells were added 10 ⁴ to each well and incubated for 1 hour. After incubation, the cells that did not bind phospholipids coated on the plate surface were removed using phosphate buffer, followed by further incubation for 1 hour by adding 90 µl of reaction buffer. After the further incubation, 60 μl of stop buffer was added to stop the color reaction, and then the absorbance was measured at 405 nm using a microplate reader (BioRad). The results are shown in FIG. 12.

As a result, as shown in FIG. 12, L / EGF3 cells and L / Stab-2 cells having a repeat site (EGFrp) of the EGF-like domain of Stabilin-2 adhered well to phosphatidylserine, but were used as a control. L / Mock cells did not attach to phosphatidylserine. Meanwhile, no cells were attached to phosphatidylcholine. From the above results, it was confirmed that the repeat region (EGFrp) of the EGF-like domain of Stabilin-2 recognizes phosphatidylserine.

<4-3> L / EGF3  Attachment and Phagocytosis of Aged Red Blood Cells by Cells

In the L / EGF3 cells prepared in <Example 3-2>, whether the aged red blood cells were attached and phagocytic were compared with the control L / Mock cells.

Specifically, after the aging red blood cells were prepared in the same manner as in <Example 2-1>, the L / EGF3 cells and L / Mock cells prepared in <Example 3-2> were each prepared with DMEM medium. Aged erythrocytes were added to 1% final hematocrit after further incubation in well plates and further incubated for 1 hour at 37 ° C. After the further incubation, the culture solution was washed five times with phosphate buffer solution, and the adhesion degree of aged red blood cells and normal red blood cells was observed with an optical microscope, and the results are shown in FIG. 13.

As a result of the experiment, the average value was obtained by counting the number of red blood cells attached to 100 randomly selected L / EGF3 cells, and the cells were washed with phosphate buffer solution for phagocytosis, and then treated with water for 10 seconds to dissolve all unproven cells. After staining, the L / EGF3 cells were stained using a Diff quick staining kit (IMEM Ins., San Marcos, Calif., USA), and the number of cells that phagocytized erythrocytes in five randomly selected cells was counted. It was. Based on the counting results, the adhesion and phagocytic index (%) was calculated by expressing the number of cells attached and phagocytic with red blood cells in percentage in total L / EGF3 cells and the results are shown in FIG. 14.

As a result, as shown in Figure 13 and 14, the aging red blood cells attached to the L / EGF3 cells, but it can be seen that the phagocytosis of the aged erythrocytes by the L / EGF3 cells does not occur, the aging in the control L / Mock cells It was found that neither attachment nor phagocytosis of the red blood cells occurred.

<4-4> Stabilin -2 EGF Attachment of Aged Red Blood Cells by Repeat Sites of Similar Domains

In <Example 4-3>, further experiments were conducted to confirm that the adhesion of aged red blood cells by L / EGF3 cells was due to the repetition site (EGFrp) of the EGF-like domain.

Specifically, 100 μM of phosphatidylserine liposomes or phosphatidylcholine liposomes, stabilin-2 monoclonal antibody (5G3), and 100 μg / ml of mouse immunoglobulin (IgG) were respectively added to the L / EGF3 cells, and cultured at 37 ° C. for 1 hour, followed by aging. After adding the added red blood cells, the degree of adhesion was examined in the same manner as in <Example 4-3>, and the results are shown in FIG. 15.

As a result, as shown in Figure 15, the adhesion of aged red blood cells by L / EGF3 cells was found to be inhibited by phosphatidylserine liposomes (PC) and Stabilin-2 monoclonal antibody (mAb 5G3), from the results It was found that the repetition site (EGFrp) of the EGF-like domain mediates the selective attachment of aged red blood cells in the context of expression on the cell surface.

< Example  5>

Stabilin -2 EGF Effect of Inhibiting Apoptotic Cell Phagocytosis by Repeat Sites of Similar Domains

<5-1> ex vivo ( in in vitro )in EGF Inhibition of Phagocytosis of Aged Erythrocytes by Repeated Sites of Similar Domains

To determine whether the repeat site (EGFrp) of the EGF-like domain of Stabilin-2 can inhibit phagocytosis of aged erythrocytes in vitro, first blood was drawn from a spontaneous donor of a normal adult and the monocytes from the blood. Human monocyte-derived macrophage (HMDM) derived from human monocytes was prepared by culturing for 7 days and then incubating in X-Vivo 10 (Bio Whitaker) containing 10% human serum to differentiate into macrophages. . After the preparation, the recombinant proteins Stab-U3, E3 and F5 prepared in <Example 1-2> and 100 μM of the phosphatidylserine liposomes prepared in <Example 4-1> were added and pre-cultured, respectively. After the pre-incubation, aged erythrocytes were added, and further cultured at 37 ° C. for 1 hour, and the culture was completed, and then washed five times with phosphate buffer solution in the same manner as in <Example 2-1>. ) And the results are shown in FIG.

As a result, as shown in FIG. 16, phagocytosis of aged erythrocytes by the macrophages was inhibited by Stab-U3 and E3 proteins containing repeat sites (EGFrp) of the EGF-like domain. The effect was found to show a similar result to that by phosphatidylserine liposomes.

<5-2> In vivo ( in vivo )in EGF Effect of Inhibiting Apoptotic Cell Phagocytosis by Repeat Sites of Similar Domains

To determine whether the repeat site (EGFrp) of the EGF-like domain of Stabilin-2 can inhibit phagocytosis of natural killer cells in vivo, it was performed as follows. First, macrophages were induced in the abdominal cavity of rats using known methods (Taylor, PR et al., J Exp Med . 192: 359-366, 2000), specifically, macrophages were induced by injecting 4 ml of 3% thioglycolate into the abdominal cavity of mice 4 days before the experiment.

On the other hand, in order to separate the thymus cells of the rats to be used as dead cells, the rat thymus was separated, chopped with scissors and forceps on a plate containing RPMI-1640 medium, and after one day, the cell strainer (BD) The tissue was filtered with Falcon). After filtration, the cells were allowed to settle by centrifugation at 1500 rpm for 3 minutes, and the cells were washed several times using phosphate buffer solution. The prepared thymic cells were prepared to include 1 × 10 ⁶ cells in 100 μl of medium, and then 3 μl of CFSE (Carboxyfluorescein diacetate succinimidyl ester, Invitrogen), a fluorescent reagent, was incubated at 37 ° C. for 30 minutes and stained. After the staining, 1 μM of dexamethasone (dexamethasone) was added to 10 ml of medium containing no 1 × 10 ⁷ cells (no serum was added) to induce thymic cell death and cultured at 37 ° C. for 4 hours. Cell death was induced.

After killing the induced thymus cells 5 μM of the E3 recombinant protein prepared in Example 1-2 at room temperature for 30 minutes, proteins not bound to the cells were removed with a phosphate buffer solution and the abdominal cavity of rats. The culture was injected into the syringe. At this time, the control group was used to pre-culture Nus protein for the same time. After injecting 5 ml of phosphate buffer solution into the abdominal cavity again 5, 15 and 30 minutes, the cells in the abdominal cavity are immediately separated using a lavage. The separated cells were centrifuged at 1500 rpm for 10 minutes, washed several times with phosphate buffer solution, and then resuspended in phosphate buffer solution. Cy5.5-bound anti-Mac-1 antibody was added to the cell suspension and incubated at 4 ° C. for 1 hour, and analyzed using a flow cytometer equipped with a 5-watt laser (Becton Dickinson, San Jose, CA). The results are shown in FIGS. 17 and 18.

As a result, as shown in FIG. 17, in the group pretreated with the E3 recombinant protein containing the repeat site (EGFrp) of the Stabilin-2 EGF-like domain, dead cells in the mouse abdominal cavity at 5 minutes after the injection of the dead cells The percentage of macrophages phagocytic was reduced, and the effect lasted for 30 minutes. In addition, as shown in Figure 18, it was found that the proportion of undeveloped dead cells was also increased in the group pretreated with E3 recombinant protein.

From the above results, it can be seen that there is an activity of inhibiting phagocytosis of macrophages to aged erythrocytes or dead cells by the repetition site (EGFrp) of EGF-like domain in vivo and ex vivo.

< Example  6>

EGF By repeat regions of the analogous domain Phosphatidylserine  Effects of Calcium in the Recognition Process

<6-1> Stabilin Effect of Calcium on Phagocytosis of Aged Red Blood Cells by -2

Proteins that bind to phosphatidylserine are known to be dependent on calcium ions (Swairjo, MA et al., Nat Struct Biol , 2: 968-74, 1995; Verdaguer, N. et al., Embo J , 18: 6329-38, 1999), whereby the inventors of the present invention investigated the effect of calcium on the medium to investigate the effect of calcium on stabilin-2 mediated phagocytosis. , 5 and 10mM were measured to measure the degree of phagocytosis of aged erythrocytes using the same method as in Example 2-1, and the results are shown in FIG. 19.

As a result, as shown in FIG. 19, the aging of red blood cells was hardly observed in the medium containing no calcium, but the concentration increased depending on the concentration of 2, 5, and 10 mM calcium. Therefore, it can be seen from the results that the stabilization process by Stabilin-2 is a calcium-dependent reaction.

<6-2> EGF By repeat regions of the analogous domain Phosphatidylserine  Effects of Calcium in the Recognition Process

On the basis of the <Example 6-1> it was investigated whether calcium is involved in the process of recognizing phosphatidylserine by the repetition site (EGFrp) of the EGF-like domain.

Specifically, NBD-phosphatidylcholine and NBD-phosphatidylserine (mixture ratio: 0.5 mol%) are mixed with 500 µg of phosphatidylcholine and phosphatidylcholine / phosphatidylserine (80/20) dissolved in chloroform, and then chloroform is used for a speed-back dryer (Speed-Vac). ) And then suspended in 300 μl phosphate buffer solution and ground for 3 minutes in an ultrasonic grinder (10/50 duration cycle) to prepare a fluorescent liposome.

In addition, an expression vector comprising Stabilin-2 cDNA used in Example <1-1> to prepare a recombinant protein for a repeating site of an EGF-like domain that does not contain a Nus-tag was used. The primer pairs of SEQ ID NO: 21 and SEQ ID NO: 22 of Table 4 are the same as in Example <1-1>. A polymerase chain reaction (PCR) was performed. As a result, each amplified product was digested with restriction enzymes BamHI and XhoI (TaKaRa), and then inserted into the same restriction enzyme site of the pET28a (Novagen) vector using T4 ligase (Invitrogen). pET-E3-16 'was prepared. Thereafter, a recombinant protein named 'E3-16' was prepared, each containing only the repeating site (EGFrp) of the third EGF-like domain of Stabilin-2.

After the preparation, 500 μl of the reaction solution (50 mM Hepes (pH 7.4), 100 mM NaCl, 25 mM KCl, 2.5 mM CaCl ₂ ) was prepared at a concentration of 0.0, 0.156, 0.032, 0.064, 0.126, 0.25, 0.5, 1 μM. ) And 2 μl of the prepared liposome solution were mixed to measure the emission spectrum from 460 nm to 600 nm with a fluorescence spectrometer (Perkin Elmer L55, excitation wavelength: 460 nm, excitation slits: 5 nm, emission slits: 10 nm). The results are shown in FIG. 20.

As a result, as shown in FIG. 20, it was found that the E3 recombinant protein including only the repeating site of the EGF-like domain binds to phosphatidylserine liposome considerably more than phosphatidylcholine liposome, and also EGTA, which is a calcium chelator in the reaction solution. It was found that the binding to phosphatidylserine liposomes is inhibited. Thus, it was also found that the recognition of phosphatidylserine by the repeat site of the EGF-like domain was a calcium dependent response.

< Example  7>

EGF -Repetition of similar domains Within the site With phosphatidylserine  Identification of motifs that mediate binding

<7-1> EGF Preparation of C-terminal or N-terminal Deletion Recombinant Protein Using Repeat Sites of Similar Domains

In order to specifically identify motifs that bind to phosphatidylserine of apoptotic cells in the repeating site (EGFrp) of the EGF-like domain, it is known that the repeating site (EGFrp) of each EGF-like domain consists of 6 EGF-like domains ( However, the repetition site of the fourth EGF-like domain is composed of five EGF-like domains.) Using the repetition site (EGFrp) of the EGF-like domain, a recombinant protein having N- or C-terminal deletions was prepared. . Specifically, the C-terminal deletion recombinant protein or N-terminus consisting of 6 to 1 EGF-like domains, using 6 EGF-like domains present at the repeat site (EGFrp) of the third EGF-like domain. The deleted recombinant protein was prepared as follows (see FIG. 21).

First, to prepare an expression vector for a C-terminal deletion recombinant protein composed of 5 to 1 EGF-like domains, the pET-E3-16 expression vector used in Example <6-2> was used as a template. The following primer pairs of SEQ ID NO: 21 and SEQ ID NO: 23 (E3-15), primer pairs of SEQ ID NO: 21 and SEQ ID NO: 24 (E3-14), primer pairs of SEQ ID NO: 21 and SEQ ID NO: 25 (E3-13), sequence The primer pair (E3-12) of SEQ ID NO: 21 and SEQ ID NO: 26 and the primer pair (E3-1) of SEQ ID NO: 21 and SEQ ID NO: 27 (see Table 4) are the same as those of <Example 1-1>. A polymerase chain reaction (PCR) was performed. As a result, each amplified product was digested with restriction enzymes BamHI and XhoI (TaKaRa), and then inserted into the same restriction enzyme site of the pET28a (Novagen) vector using T4 ligase (Invitrogen). pET-E3-15, 'pET-E3-14', 'pET-E3-13', 'pET-E3-12', and 'pET-E3-1' were prepared.

In addition, to prepare an expression vector for an N-terminal deletion recombinant protein consisting of 5 to 1 EGF-like domains, primer pairs of SEQ ID NO: 28 and SEQ ID NO: 22 (E3-26), SEQ ID NO: 29 and sequence Primer pair (E3-36) of SEQ ID NO: 22, primer pair (E3-46) of SEQ ID NO: 30 and SEQ ID NO: 22, primer pair (E3-56) of SEQ ID NO: 31 and SEQ ID NO: 22, and SEQ ID NO: 32 and SEQ ID NO: 22 Primer pairs (E3-6) (see Table 4) were used in the same manner as described above. As a result, the recombinant vectors' pET-E3-26, 'pET-E3-36', 'pET-E3-46', 'pET-E3-56', and 'pET-E3-6' were prepared.

In order to make a C-terminal deleted recombinant protein or N-terminal deleted recombinant protein using the expression vectors, the recombinant protein was isolated and purified using the same method as in <Example 1-2>. The C-terminal deletion recombinant proteins prepared as described above were named 'E3-15', 'E3-14', 'E3-13', 'E3-12', and 'E3-1', respectively, and the N-terminal protein. They were named 'E3-26', 'E3-36', 'E3-46', 'E3-56' and 'E3-6', respectively.

Primers used for cloning expression vectors to prepare C-terminal or N-terminal deleted recombinant proteins using repeat sites of EGF-like domains primer order SEQ ID NO: E3-16 sense 5’-AAAAAGGATCCACGAGAGAATGCTGTGCC-3 ’ 21 E3-16 Antisense 5’-AAAACTCGAGTCAGCGGCAGGTAAATCCATC-3 ’ 22 E3-15 Antisense 5’-GATCTCGAGTTATGTGCAGACCTTTCCATC-3 ’ 23 E3-14 Antisense 5’-GATCTCGAGTTACAGGCACACAATGCC-3 ’ 24 E3-13 Antisense 5’-GATCTCGAGTTATGTGCAGATGGTCCC-3 ’ 25 E3-12 Antisense 5’-GATCTCGAGTTAGGTTGCATTGTCAC-3 ’ 26 E3-1 antisense 5’-GATCTCGAGTTAGCAGGCTGTGCCGC-3 ’ 27 E3-26 sense 5'-AAAAAGGATCCGAGACCTGCACCGAG -3 ' 28 E3-36 sense 5’-AAAAGGATCCACAGAAGACAACTGCAATG-3 ’ 29 E3-46 sense 5’-AAAAGGATCCGCAATCAATGCCTGTGAG-3 ’ 30 E3-56 sense 5’-AAAAGGATCCGGAATCAACCCGTGTTTG-3 ’ 31 E3-6 sense 5’-AAAAGGATCCCTCATCAATGTCTGCTTAAC-3 ’ 32

<7-2> EGF Within the repeat region of the pseudo domain With phosphatidylserine  Identification of motifs that mediate binding

In order to examine the binding of the deleted recombinant protein prepared in Example <6-2> and Example <7-1> with phosphatidylserine, as in Example <6-2>, NBD- The quenching effect of fluorescence using phosphatidylcholine and NBD-phosphatidylserine was observed and the results are shown in FIGS. 22 and 23.

As a result, as shown in Fig. 22, in the experiment using the C-terminal deletion proteins, it can be seen that E3-15 and E3-14 recombinant proteins including wild-type E3-16 bind well with phosphatidylserine. there was. Therefore, it can be seen that more than four EGF-like domains are required for binding to phosphatidylserine.

In addition, as shown in FIG. 23, in the experiment using N-terminal deletion proteins, only wild-type E3-16 and E-26 recombinant proteins were found to bind well with phosphatidylserine. Therefore, it was found that the atypical EGF-like domain plays an important role in binding to phosphatidylserine.

On the other hand, using the deletion recombinant protein prepared in <Example 7-1> by performing a phagocytic experiment for aging red blood cells in the same manner as in <Example 2-1> and the results are shown in Figures 24 and 25 Indicated.

As a result, as shown in FIG. 24, among the C-terminal deletion recombinant proteins, E-16, E-15, and E-14 recombinant proteins having four or more EGF-like domains inhibited phagocytosis of aged red blood cells. In addition, as shown in FIG. 25, among the N-terminal deletion recombinant proteins, only E3-16 and E3-26 recombinant proteins having atypical EGF-like domains inhibited phagocytosis of aged erythrocytes. Appeared to be.

Therefore, based on the experimental results, it can be seen that the repeat region (EGFrp) of the EGF-like domain having at least four EGF-like domains with atypical EGF-like domains plays an important role in the recognition of phosphatidylserine. there was.

<7-3> Atypical ( atypical ) EGF Preparation of deleted recombinant protein lacking similar domains

A deletion recombinant protein lacking an atypical EGF-like domain was prepared to identify which atypical EGF-like domain is important for interaction with phosphatidylserine in the repeat region (EGFrp) of the EGF-like domain. .

Specifically, a recombinant protein consisting of four EGF-like domains (E3-25 in FIG. 26) was prepared, in which the first atypical EGF-like domain was deleted in the repeat region (EGFrp) of the third EGF-like domain. In order to prepare an expression vector for use, the recombinant vector 'pET-E3-25' was prepared using the primers set forth in Table 4 (SEQ ID NO: 28 and SEQ ID NO: 23) and the same method as in <Example 7-1>. Was prepared.

In addition, to prepare an expression vector for preparing a recombinant protein consisting of four EGF-like domains (E3-15Δ2 in FIG. 26), which is deleted from a second atypical EGF-like domain, the sequence described in Table 4 above. The primer pairs of SEQ ID NO: 21 and SEQ ID NO: 27 (amplifying the first EGF-like domain) and primer pairs of SEQ ID NO: 23 and SEQ ID NO: 29 (amplifying the third to fifth EGF-like domains), respectively, Same as Example 1-1> A polymerase chain reaction (PCR) was performed. As a result, the first EGF-like domain of the amplified product was digested with restriction enzymes BamHI and EcoRI (TaKaRa), and then T4 ligase (Invitrogen) was used in the same restriction enzyme site of pET28a (Novagen) vector. After insertion, the third to fifth EGF-like domains of the amplified products were cleaved with restriction enzymes EcoRI (TaKaRa) and XhoI (TaKaRa), and then T4 ligase in the same restriction enzyme site of the pET28a (Novagen) vector. , Invitrogen) to prepare a recombinant vector 'pET-E3-15Δ2'.

By using the expression vectors described above, the recombinant protein was isolated and purified using the same method as in <Example 1-2>, and the first atypical EGF- in the repeat region (EGFrp) of the third EGF-like domain. 'E3-25', a recombinant protein lacking a similar domain, was prepared and 'E3-15Δ2', a recombinant protein lacking a second atypical EGF-like domain, was prepared.

<7-4> Atypical ( atypical ) EGF -Of similar domains With phosphatidylserine  Identification of motifs that mediate binding

To determine which atypical EGF-like domain plays an important role in binding to phosphatidylserine in the repeat region (EGFrp) of the EGF-like domain, the C-terminal deletion proteins E3-14 and N-terminal deletion proteins In addition to E3-36, the binding to phosphatidylserine was compared using the recombinant proteins E3-25 and E3-15Δ2 prepared in Example <7-3>. At this time, in order to prevent the error caused by the size of the recombinant protein, the four proteins were used that consists of four EGF-like domains (see Fig. 26).

First, the attenuation effect of fluorescence using NBD-phosphatidylcholine and NBD-phosphatidylserine was observed for the recombinant protein using the same method as in Example 6-2, and the results are shown in FIG. 27.

As a result, as shown in Figure 27 it can be seen that the binding to phosphatidylserine is significantly reduced in E3-15Δ2 where the second atypical EGF-like domain is deleted.

In addition, to investigate whether the second atypical EGF-like domain is involved in calcium-dependent reactions, an experiment was conducted in the presence of calcium chelator EGTA to observe the attenuation effect of fluorescence using NBD-phosphatidylserine. The results are shown as dynamic quenching constants (K) according to Stern-Volmer law Fo / (Fo-F) = 1 / fa + 1 / (faK [Mt]). As a result, when EGTA was added, it was observed that the reactivity with PS was significantly decreased.

As a result, as shown in FIG. 28, it was found that the second atypical EGF-like domain plays an important role in binding to phosphatidylserine and its calcium dependency.

In addition, by using the recombinant protein in the same manner as in <Example 2-1> was carried out a phagocytic experiment for aging red blood cells and the results are shown in Figure 29. As shown in FIG. 29, the inhibitory effect on the phagocytosis of aged erythrocytes was significantly reduced in E3-15Δ2 in which the second atypical EGF-like domain was deleted.

The results showed that the second atypical EGF-like domain in the repeating site (EGFrp) of the EGF-like domain plays an important role in binding to phosphatidylserine.

As described above, the second atypical EGF-like domain and three or more EGF-like domains in the EGF-like domain repeat protein of stabilin-2 of the present invention. Sequentially linked polypeptides can specifically bind phosphatidylserine on the cell surface. Therefore, the present invention can be used in various ways, such as drug delivery to cells expressing phosphatidylserine on the cell surface, prevention, treatment or imaging of tumor diseases through delivery of drugs or markers to tumor cells.

1 is a schematic diagram showing the structure of the recombinant protein of derived from each domain of the stabilin-2 protein and the Stabilin-2 protein.

(Stab-U1, Stab-U2, Stab-U3, Stab-U4: recombinant protein for each extracellular portion of Stabilin-2 protein;

E1, E2, E3, E4: recombinant proteins for the repeat portion (EGFrp) of the EGF-like domain of Stabilin-2 protein;

F1, F2, F3, F4, F5, F6, F7: recombinant proteins for the FAS1 domain of Stabilin-2 protein;

Heavily filled pentagram: atypical EGFrp, lightly filled pentagram: standard EGFrp, square: FAS1 domain)

Figure 2 is a graph showing the effect on the phagocytosis of the aged red blood cells of Stab-U1, Stab-U2, Stab-U3, Stab-U4.

(None: untreated; Nus: containing only Nus protein (control))

Figure 3 is a photograph showing the degree of phagocytosis of apoptotic cells (apoptotic cells) in the presence of Stab-U1, Stab-U2, Stab-U3, Stab-U4 in L / Stab-2 cells.

4 is a graph showing the effect on the phagocytosis of aged erythrocytes when pretreated with different amounts of the third subunit (Stab-U3) and their fragments (E3, F5) (0.1, 1, 10 μg). .

Figure 5 is a photograph showing the effect on the phagocytosis of dead cells of the third subunit (Stab-U3) and fragments thereof (E3, F5).

FIG. 6 is a graph showing the effect of repeating portions (EGFrp; E1, E2, E3, E4) of four EGF-like domains in Stabilin-2 protein on phagocytosis of aged erythrocytes.

Figure 7 is a graph of the binding force of the phosphatidylserine and the third subunit (Stab-U3) and their fragments (E3, F5).

FIG. 8 is a graph illustrating the binding force between various kinds of phospholipids and E3 fragments (PS: phosphatidylserine; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol)

9 is a schematic diagram showing the structure of the membrane protein (Stab-EGF3) in which the repeating portion of the third EGF-like domain of the human Stabilin-2 protein is expressed on the cell surface.

10 is a result of analyzing the expression of Stab-EGF3 protein in L / EGF3 cells by flow cytometry using a Stabilin-2 monoclonal antibody.

11 is a result of analyzing the extent of L / EGF3 cells to phosphatidylserine (PS) liposomes by flow cytometry.

12 is a graph showing the degree of adhesion of L / Stab-2 cells, L / EGF3 cells, and L / Mock cells to phosphatidylserine.

Figure 13 is a photograph comparing the degree of adhesion of aged red blood cells by L / EGF3 cells and L / Mock cells.

14 is a graph comparing the degree of adhesion and phagocytosis of aged erythrocytes by L / EGF3 cells with L / Mock cells.

15 is a graph showing the degree of inhibition of adhesion of aged erythrocytes of L / EGF3 cells by phosphatidylserine or stabilin-2 monoclonal antibody and PS liposomes.

(None: nothing processed; PS: phosphatidylserine liposomes; PC: phosphatidylcholine liposomes; mAb 5G3: Stabilin-2 monoclonal antibody; Isotype cont Ab: mouse immunoglobulin with matching subtypes)

16 is a graph showing the effects of Stab-U3, E3, F5 protein and phosphatidylserine liposomes on phagocytosis of aged erythrocytes by macrophages derived from human monocytes.

17 is a graph showing the effect of E3 fragments over time on phagocytosis by macrophages in rat intraperitoneal animals in an animal model.

18 is a result of analyzing the effect of the E3 fragment on the removal of dead cells in the rat abdominal cavity in the animal model by flow cytometry.

19 is a graph showing the effect of calcium on phagocytosis of aged erythrocytes by L / Stab-2 cells.

20 is a graph showing the effect of calcium on the binding of E3 fragment and phosphatidylserine using the disappearance of NBD fluorescence.

Fig. 21 is a schematic diagram showing the structures of the N-terminal and C-terminal deleted proteins of the E3 fragment.

Figure 22 is a graph showing the extent of binding of the E3 fragment to the phosphatidylserine liposome of the C-terminal deletion protein through the disappearance of NBD fluorescence.

Figure 23 is a graph showing the degree of binding of the N-terminal deletion protein of the E3 fragment to the phosphatidylserine liposome through NBD fluorescence extinction.

24 is a graph showing the effect of C-terminal defective proteins of the E3 fragment on phagocytosis of aged erythrocytes by L / Stab-2 cells.

25 is a graph showing the effect of N-terminal defective proteins of the E3 fragment on phagocytosis of aged erythrocytes by L / Stab-2 cells.

Fig. 26 is a schematic diagram showing the structure of a recombinant protein in which an atypical EGF-like domain is deleted as an EGFrp consisting of four EGF-like domains.

Figure 27 is a graph showing the degree of binding of phosphatidylserine liposomes of EGFrp proteins lacking the atypical EGF-like domain through the disappearance of NBD fluorescence.

FIG. 28 is a graph showing the disappearance of NBD fluorescence by EGFrp protein lacking an atypical EGF-like domain as a dynamic quenching constant proportional to the rate of fluorescence reduction [Fo / (Fo-F)]. K: K (dynamic quenching constant)

29 is a graph showing the effect of EGFrp protein deficient in atypical EGF-like domains on phagocytosis of aged erythrocytes by L / Stab-2 cells.

<110> Kyungpook national university industry-academic cooperation foundation <120> Composition for drug delivery comprising the polypeptide fragment          of EGF-like domain repeat protein of stabilin-2 and use <130> NP08-0001 <160> 116 <170> KopatentIn 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Stab-U1 sense <400> 1 aaaaggatcc gtaggggttc gagattg 27 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Stab-U1 antisense <400> 2 aaaactcgag tcagggagga atgagaac 28 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Stab-U2 sense <400> 3 aaaaggatcc aactctgagc ccacag 26 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Stab-U2 antisense <400> 4 aaaactcgag tcactgaatt tccag 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Stab-U3 sense <400> 5 aaaaggatcc gagaagagga gatgc 25 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Stab-U3 antisense <400> 6 aaaactcgag tcaatcaatc agcagac 27 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Stab-U4 sense <400> 7 aaaaggatcc aggtggagta aaccaaag 28 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Stab-U4 antisense <400> 8 aaaactcgag tcagggtgct tttaaaggc 29 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> E1 antisense <400> 9 aaaaactcga gtcaataaca cgttaagcc 29 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> E2 antisense <400> 10 aaaaactcga gtcaatagca cagaaagcc 29 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> E3 antisense <400> 11 aaaaactcga gtcagcggca ggtaaatcc 29 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> E4 antisense <400> 12 aaaaactcga gtcactcaca gttcagccc 29 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> F5 sense <400> 13 aaaaggatcc actgtttttg cacc 24 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> F5 antisense <400> 14 aaaactcgag tcatttggga gatagcaa 28 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> signal sense <400> 15 aaaatgctag ctcagcctga caggtgc 27 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> signal antisense <400> 16 aaaatctcga gtcttgcctg ccctgtg 27 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> TMC sense <400> 17 aaaaaggatc cacccacact ggcttg 26 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> TMC antisense <400> 18 cccgtccaag cttgcacagt gtcct 25 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> EGF3 sense <400> 19 aaaacctcga gagatgtgat aataatgac 29 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> EGF3 antisense <400> 20 aaaaaggatc cgcggcaggt aaatcc 26 <210> 21 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> E3-16 sense <400> 21 aaaaaggatc cacgagagaa tgctgtgcc 29 <210> 22 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> E3-16 antisense <400> 22 aaaactcgag tcagcggcag gtaaatccat c 31 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> E3-15 antisense <400> 23 gatctcgagt tatgtgcaga cctttccatc 30 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> E3-14 antisense <400> 24 gatctcgagt tacaggcaca caatgcc 27 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> E3-13 antisense <400> 25 gatctcgagt tatgtgcaga tggtccc 27 <210> 26 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> E3-12 antisense <400> 26 gatctcgagt taggttgcat tgtcac 26 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> E3-1 antisense <400> 27 gatctcgagt tagcaggctg tgccgc 26 <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> E3-26 sense <400> 28 aaaaaggatc cgagacctgc accgag 26 <210> 29 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> E3-36 sense <400> 29 aaaaggatcc acagaagaca actgcaatg 29 <210> 30 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> E3-46 sense <400> 30 aaaaggatcc gcaatcaatg cctgtgag 28 <210> 31 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> E3-56 sense <400> 31 aaaaggatcc ggaatcaacc cgtgtttg 28 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> E3-6 sense <400> 32 aaaaggatcc ctcatcaatg tctgcttaac 30 <210> 33 <211> 1770 <212> DNA <213> Homo sapiens <400> 33 gtaggggttc gagattgcag gtacaccttt gaggtcagaa catactctct gtctctcccc 60 ggatgccgcc atatttgtag gaaggactat ctccaacctc ggtgttgtcc tggccgctgg 120 ggcccagact gtatagagtg cccaggtgga gcggggtcac cctgcaatgg cagaggcagt 180 tgtgctgaag gcatggaagg aaatggaacc tgctcctgcc aagaagggtt tggtggaaca 240 gcctgtgaaa cctgtgctga cgacaactta tttggaccca gctgttcatc agtgtgcaac 300 tgtgtgcatg gggtgtgcaa cagtggacta gatggcgatg gaacctgtga gtgctactct 360 gcgtacactg gccccaagtg tgacaagccc atccctgaat gtgcagcctt gctctgccca 420 gaaaattcca gatgttcgcc ttccactgaa gatgaaaaca aactggaatg caaatgcctt 480 cccaattacc gaggcgatgg caaatactgc gaccccatca atccatgttt acgaaaaatc 540 tgccaccctc atgctcattg tacgtacctg ggaccaaatc ggcacagttg tacatgccaa 600 gaaggctacc gtggggatgg ccaagtgtgc ttgcctgtgg acccctgcca aattaacttt 660 ggaaactgcc ctacaaagtc tacagtgtgc aaatatgatg ggcctggaca gtctcactgc 720 gagtgtaagg agcattacca gaatttcgta cctggagtgg ggtgcagtat gactgatata 780 tgtaaatcag ataacccgtg tcataggaat gcaaattgca ccaccgtcgc accaggccga 840 actgaatgca tttgccagaa aggttacgtg ggtgatggct taacgtgtta tggaaacatt 900 atggagcgac tcagagaatt aaatactgaa cccagaggaa aatggcaagg aaggctgacc 960 tctttcatct cactcctaga caaagcttat gcctggccac tgagtaagct gggacccttc 1020 acggtgctgt tacctacaga caagggactg aaaggattca atgtaaatga gcttttggtg 1080 gataataaag ctgctcaata ctttgtgaaa ctccacataa ttgctggtca gatgaacatc 1140 gaatatatga ataacacaga catgttctac accttgactg gaaagtcggg ggaaatcttc 1200 aacagcgata aggacaatca aataaagctt aaactccatg gaggcaaaaa gaaggtaaaa 1260 attatacaag gggacatcat tgcttccaat gggcttctgc acatccttga cagagccatg 1320 gacaagttag aacccacatt tgagagcaac aatgagcaaa ccataatgac aatgctacaa 1380 ccaaggtaca gcaagttcag atctttgtta gaggaaacca atttgggaca tgccttagat 1440 gaggatggag ttggtggacc atacaccatt tttgttccaa ataatgaagc attgaataac 1500 atgaaggacg gcactctcga ttacctcctt tctccagagg gatctcggaa gcttctggaa 1560 ctcgtcagat accacattgt cccatttacc cagcttgaag tggccactct catctccacc 1620 cctcacatca ggagcatggc caaccagctc atacagttca acaccaccga caatggacag 1680 attctggcaa atgatgtggc aatggaagaa attgagatca ctgccaaaaa tggccgaatt 1740 tacacactga caggagttct cattcctccc 1770 <210> 34 <211> 1734 <212> DNA <213> Homo sapiens <400> 34 aactctgagc ccacagcact cttcacacac agatgtgtct acagtggcag gtttgggagc 60 ctgaagagcg gctgtgcccg gtactgcaat gccactgtga agattccaaa gtgctgcaaa 120 ggcttctatg gacctgactg caaccagtgt ccaggaggct tctcaaatcc atgctcagga 180 aatggacagt gtgcagatag cctcggcggc aacgggacat gcatttgtga ggagggcttc 240 caaggctccc agtgtcagtt ctgctctgat cccaataaat acggacctcg gtgtaacaaa 300 aaatgcctgt gcgttcacgg aacatgcaat aacaggatag acagcgatgg ggcctgcctc 360 actggcacat gcagagacgg ctctgccggg agactctgtg ataagcagac ctcagcctgt 420 gggccctacg tgcagttctg tcacatccac gccacctgtg aatacagcaa tgggacagcc 480 agttgtattt gcaaagcagg atatgaagga gatggaactc tgtgttctga gatggaccct 540 tgcacaggac taactccagg aggctgtagc cgcaatgcag aatgcatcaa aactggcacg 600 ggcacccaca cctgcgtgtg tcagcagggt tggacaggga atgggagaga ctgctcggag 660 atcaacaact gcctgctgcc cagtgcaggc ggctgccacg acaacgcatc ctgtttgtat 720 gtgggtcccg ggcagaatga gtgtgagtgc aagaaaggat ttcgaggaaa tgggattgac 780 tgtgaaccaa taacttcatg cttggaacaa accgggaaat gtcatccatt ggcaagctgt 840 caatctactt cgtctggtgt ctggagctgt gtttgtcaag agggctatga aggagatggc 900 tttctgtgct atggaaacgc agcagtggaa ttgtcatttc tctccgaagc agctatattt 960 aaccgatgga taaataatgc ttctctacaa cccacactgt cagccacctc aaacctcact 1020 gtcctcgtgc cttcccaaca agctactgag gacatggacc aggatgagaa aagcttctgg 1080 ttgtcacaga gcaatattcc agccctaata aagtaccata tgctactagg cacatacaga 1140 gtggcagatc tgcagaccct gtcttcttct gacatgttgg caacatcttt gcagggcaac 1200 ttccttcact tggcaaaggt ggatgggaat atcacaattg aaggggcctc cattgtcgat 1260 ggggacaacg cagccacaaa tggagtgata cacatcatca acaaggtgct ggtcccacaa 1320 agacgtctaa ctggctcctt accaaacctg ctcatgcggc tggaacagat gcctgactat 1380 tccatcttcc ggggctacat cattcaatat aatctggcga atgcaattga ggctgccgat 1440 gcctacacag tgtttgctcc aaacaacaat gccatcgaga attacatcag ggagaagaaa 1500 gtcttgtctc tagaggagga cgtcctccgg tatcatgtgg tcctggagga gaaactcctg 1560 aagaatgacc tgcacaatgg catgcatcgt gagaccatgc tgggtttctc ctatttcctt 1620 agcttctttc tccataatga ccagctctat gtaaatgagg ctccaataaa ctacaccaat 1680 gtagccactg ataagggagt gatccatggc ttgggaaaag ttctggaaat tcag 1734 <210> 35 <211> 1743 <212> DNA <213> Homo sapiens <400> 35 gagaagagga gatgcatcta tacctcctat ttcatgggaa gacgaaccct gtttattggg 60 tgccagccaa aatgtgtgag aaccgtcatt acgagagaat gctgtgccgg cttctttggc 120 ccccaatgcc agccctgccc agggaatgcc cagaatgtct gctttggtaa tggcatctgt 180 ttggatggag tgaatggcac aggtgtgtgt gagtgtgggg agggcttcag cggcacagcc 240 tgcgagacct gcaccgaggg caagtacggc atccactgtg accaagcatg ttcttgtgtc 300 catgggagat gcaaccaagg acccttggga gatggctcct gtgactgtga tgttggctgg 360 cgaggagtgc attgtgacaa tgcaaccaca gaagacaact gcaatgggac atgccatacc 420 agcgccaact gcctcaccaa ctcagatggt acagcttcat gcaagtgtgc agcaggattc 480 caaggaaacg ggaccatctg cacagcaatc aatgcctgtg agatcagcaa tggaggttgc 540 tctgccaagg ctgactgtaa gagaaccacc ccaggaaggc gagtgtgcac gtgcaaagca 600 ggctacacgg gtgatggcat tgtgtgcctg gaaatcaacc cgtgtttgga gaaccatggt 660 ggctgtgaca agaatgcgga gtgcacacag acaggaccca accaggctgc ctgtaactgt 720 ttgccagcat acactggaga tggaaaggtc tgcacactca tcaatgtctg cttaactaaa 780 aatggcggct gtagtgaatt tgccatctgc aaccacactg ggcaagtaga aaggacttgt 840 acttgcaagc caaactacat tggagatgga tttacctgcc gcggcagcat ttatcaggag 900 cttcccaaga acccgaaaac ttcccagtat ttcttccagt tgcaggagca tttcgtgaaa 960 gatctggtcg gcccaggccc cttcactgtt tttgcacctt tatctgcagc ctttgatgag 1020 gaagctcggg ttaaagactg ggacaaatac ggtttaatgc cccaggttct tcggtaccat 1080 gtggtcgcct gccaccagct gcttctggaa aacctgaaat tgatctcaaa tgctacttcc 1140 ctccaaggag agccaatagt catctccgtc tctcagagca cggtgtatat aaacaataag 1200 gctaagatca tatccagtga tatcatcagt actaatggga ttgttcatat catagacaaa 1260 ttgctatctc ccaaaaattt gcttatcact cccaaagaca actctggaag aattctgcaa 1320 aatcttacga ctttggcaac aaacaatggc tacatcaaat ttagcaactt aatacaggac 1380 tcaggtttgc tgagtgtcat caccgatccc atccacaccc cagtcactct cttctggccc 1440 accgaccaag ccctccatgc cctacctgct gaacaacagg acttcctgtt caaccaagac 1500 aacaaggaca agctgaagga gtatttgaag tttcatgtga tacgagatgc caaggtttta 1560 gctgtggatc ttcccacatc cactgcctgg aagaccctgc aaggttcaga gctgagtgtg 1620 aaatgtggag ctggcaggga catcggtgac ctctttctga atggccaaac ctgcagaatt 1680 gtgcagcggg agctcttgtt tgacctgggt gtggcctacg gcattgactg tctgctgatt 1740 gat 1743 <210> 36 <211> 1611 <212> DNA <213> Homo sapiens <400> 36 aggtggagta aaccaaaggg tgtgaagcag aagtgtctct acaacctgcc cttcaagagg 60 aacctggaag gctgccggga gcggtgcagc ctggtgatac agatccccag gtgctgcaag 120 ggctacttcg ggcgagactg tcaggcctgc cctggaggac cagatgcccc gtgtaataac 180 cggggtgtct gccttgatca gtactcggcc accggagagt gtaaatgcaa caccggcttc 240 aatgggacgg cgtgtgagat gtgctggccg gggagattcg ggcctgattg tctgccctgt 300 ggctgctcag accacggaca gtgcgatgat ggcatcacgg gctccgggca gtgcctctgt 360 gaaacggggt ggacaggccc ctcgtgtgac actcaggcag ttttgcctgc agtgtgtacg 420 cctccttgtt ctgctcatgc cacctgtaag gagaacaaca cgtgtgagtg taacctggat 480 tatgaaggtg acggaatcac atgcacagtt gtggatttct gcaaacagga caacgggggc 540 tgtgcaaagg tggccagatg ctcccagaag ggcacgaagg tctcctgcag ctgccagaag 600 ggatacaaag gggacgggca cagctgcaca gagatagacc cctgtgcaga cggccttaac 660 ggagggtgtc acgagcacgc cacctgtaag atgacaggcc cgggcaagca caagtgtgag 720 tgtaaaagtc actatgtcgg agatgggctg aactgtgagc cggagcagct gcccattgac 780 cgctgcttac aggacaatgg gcagtgccat gcagacgcca aatgtgtcga cctccacttc 840 caggatacca ctgttggggt gttccatcta cgctccccac tgggccagta taagctgacc 900 tttgacaaag ccagagaggc ctgtgccaac gaagctgcga ccatggcaac ctacaaccag 960 ctctcctatg cccagaaggc caagtaccac ctgtgctcag caggctggct ggagaccggg 1020 cgggttgcct accccacagc cttcgcctcc cagaactgtg gctctggtgt ggttgggata 1080 gtggactatg gacctagacc caacaagagt gaaatgtggg atgtcttctg ctatcggatg 1140 aaagatgtga actgcacctg caaggtgggc tatgtgggag atggcttctc atgcagtggg 1200 aacctgctgc aggtcctgat gtccttcccc tcactcacaa acttcctgac ggaagtgctg 1260 gcctattcca acagctcagc tcgaggccgt gcatttctag aacacctgac tgacctgtcc 1320 atccgcggca ccctctttgt gccacagaac agtgggctgg gggagaatga gaccttgtct 1380 gggcgggaca tcgagcacca cctcgccaat gtcagcatgt ttttctacaa tgaccttgtc 1440 aatggcacca ccctgcaaac gaggctggga agcaagctgc tcatcactgc cagccaggac 1500 ccactccaac cgacggagac caggtttgtt gatggaagag ccattctgca gtgggacatc 1560 tttgcctcca atgggatcat tcatgtcatt tccaggcctt taaaagcacc c 1611 <210> 37 <211> 891 <212> DNA <213> Homo sapiens <400> 37 gtaggggttc gagattgcag gtacaccttt gaggtcagaa catactctct gtctctcccc 60 ggatgccgcc atatttgtag gaaggactat ctccaacctc ggtgttgtcc tggccgctgg 120 ggcccagact gtatagagtg cccaggtgga gcggggtcac cctgcaatgg cagaggcagt 180 tgtgctgaag gcatggaagg aaatggaacc tgctcctgcc aagaagggtt tggtggaaca 240 gcctgtgaaa cctgtgctga cgacaactta tttggaccca gctgttcatc agtgtgcaac 300 tgtgtgcatg gggtgtgcaa cagtggacta gatggcgatg gaacctgtga gtgctactct 360 gcgtacactg gccccaagtg tgacaagccc atccctgaat gtgcagcctt gctctgccca 420 gaaaattcca gatgttcgcc ttccactgaa gatgaaaaca aactggaatg caaatgcctt 480 cccaattacc gaggcgatgg caaatactgc gaccccatca atccatgttt acgaaaaatc 540 tgccaccctc atgctcattg tacgtacctg ggaccaaatc ggcacagttg tacatgccaa 600 gaaggctacc gtggggatgg ccaagtgtgc ttgcctgtgg acccctgcca aattaacttt 660 ggaaactgcc ctacaaagtc tacagtgtgc aaatatgatg ggcctggaca gtctcactgc 720 gagtgtaagg agcattacca gaatttcgta cctggagtgg ggtgcagtat gactgatata 780 tgtaaatcag ataacccgtg tcataggaat gcaaattgca ccaccgtcgc accaggccga 840 actgaatgca tttgccagaa aggttacgtg ggtgatggct taacgtgtta t 891 <210> 38 <211> 912 <212> DNA <213> Homo sapiens <400> 38 aactctgagc ccacagcact cttcacacac agatgtgtct acagtggcag gtttgggagc 60 ctgaagagcg gctgtgcccg gtactgcaat gccactgtga agattccaaa gtgctgcaaa 120 ggcttctatg gacctgactg caaccagtgt ccaggaggct tctcaaatcc atgctcagga 180 aatggacagt gtgcagatag cctcggcggc aacgggacat gcatttgtga ggagggcttc 240 caaggctccc agtgtcagtt ctgctctgat cccaataaat acggacctcg gtgtaacaaa 300 aaatgcctgt gcgttcacgg aacatgcaat aacaggatag acagcgatgg ggcctgcctc 360 actggcacat gcagagacgg ctctgccggg agactctgtg ataagcagac ctcagcctgt 420 gggccctacg tgcagttctg tcacatccac gccacctgtg aatacagcaa tgggacagcc 480 agttgtattt gcaaagcagg atatgaagga gatggaactc tgtgttctga gatggaccct 540 tgcacaggac taactccagg aggctgtagc cgcaatgcag aatgcatcaa aactggcacg 600 ggcacccaca cctgcgtgtg tcagcagggt tggacaggga atgggagaga ctgctcggag 660 atcaacaact gcctgctgcc cagtgcaggc ggctgccacg acaacgcatc ctgtttgtat 720 gtgggtcccg ggcagaatga gtgtgagtgc aagaaaggat ttcgaggaaa tgggattgac 780 tgtgaaccaa taacttcatg cttggaacaa accgggaaat gtcatccatt ggcaagctgt 840 caatctactt cgtctggtgt ctggagctgt gtttgtcaag agggctatga aggagatggc 900 tttctgtgct at 912 <210> 39 <211> 882 <212> DNA <213> Homo sapiens <400> 39 gagaagagga gatgcatcta tacctcctat ttcatgggaa gacgaaccct gtttattggg 60 tgccagccaa aatgtgtgag aaccgtcatt acgagagaat gctgtgccgg cttctttggc 120 ccccaatgcc agccctgccc agggaatgcc cagaatgtct gctttggtaa tggcatctgt 180 ttggatggag tgaatggcac aggtgtgtgt gagtgtgggg agggcttcag cggcacagcc 240 tgcgagacct gcaccgaggg caagtacggc atccactgtg accaagcatg ttcttgtgtc 300 catgggagat gcaaccaagg acccttggga gatggctcct gtgactgtga tgttggctgg 360 cgaggagtgc attgtgacaa tgcaaccaca gaagacaact gcaatgggac atgccatacc 420 agcgccaact gcctcaccaa ctcagatggt acagcttcat gcaagtgtgc agcaggattc 480 caaggaaacg ggaccatctg cacagcaatc aatgcctgtg agatcagcaa tggaggttgc 540 tctgccaagg ctgactgtaa gagaaccacc ccaggaaggc gagtgtgcac gtgcaaagca 600 ggctacacgg gtgatggcat tgtgtgcctg gaaatcaacc cgtgtttgga gaaccatggt 660 ggctgtgaca agaatgcgga gtgcacacag acaggaccca accaggctgc ctgtaactgt 720 ttgccagcat acactggaga tggaaaggtc tgcacactca tcaatgtctg cttaactaaa 780 aatggcggct gtagtgaatt tgccatctgc aaccacactg ggcaagtaga aaggacttgt 840 acttgcaagc caaactacat tggagatgga tttacctgcc gc 882 <210> 40 <211> 759 <212> DNA <213> Homo sapiens <400> 40 aggtggagta aaccaaaggg tgtgaagcag aagtgtctct acaacctgcc cttcaagagg 60 aacctggaag gctgccggga gcggtgcagc ctggtgatac agatccccag gtgctgcaag 120 ggctacttcg ggcgagactg tcaggcctgc cctggaggac cagatgcccc gtgtaataac 180 cggggtgtct gccttgatca gtactcggcc accggagagt gtaaatgcaa caccggcttc 240 aatgggacgg cgtgtgagat gtgctggccg gggagattcg ggcctgattg tctgccctgt 300 ggctgctcag accacggaca gtgcgatgat ggcatcacgg gctccgggca gtgcctctgt 360 gaaacggggt ggacaggccc ctcgtgtgac actcaggcag ttttgcctgc agtgtgtacg 420 cctccttgtt ctgctcatgc cacctgtaag gagaacaaca cgtgtgagtg taacctggat 480 tatgaaggtg acggaatcac atgcacagtt gtggatttct gcaaacagga caacgggggc 540 tgtgcaaagg tggccagatg ctcccagaag ggcacgaagg tctcctgcag ctgccagaag 600 ggatacaaag gggacgggca cagctgcaca gagatagacc cctgtgcaga cggccttaac 660 ggagggtgtc acgagcacgc cacctgtaag atgacaggcc cgggcaagca caagtgtgag 720 tgtaaaagtc actatgtcgg agatgggctg aactgtgag 759 <210> 41 <211> 153 <212> DNA <213> Homo sapiens <400> 41 caacctcggt gttgtcctgg ccgctggggc ccagactgta tagagtgccc aggtggagcg 60 gggtcaccct gcaatggcag aggcagttgt gctgaaggca tggaaggaaa tggaacctgc 120 tcctgccaag aagggtttgg tggaacagcc tgt 153 <210> 42 <211> 141 <212> DNA <213> Homo sapiens <400> 42 gaaacctgtg ctgacgacaa cttatttgga cccagctgtt catcagtgtg caactgtgtg 60 catggggtgt gcaacagtgg actagatggc gatggaacct gtgagtgcta ctctgcgtac 120 actggcccca agtgtgacaa g 141 <210> 43 <211> 126 <212> DNA <213> Homo sapiens <400> 43 cccatccctg aatgtgcagc cttgctctgc ccagaaaatt ccagatgttc gccttccact 60 gaagatgaaa acaaactgga atgcaaatgc cttcccaatt accgaggcga tggcaaatac 120 tgcgac 126 <210> 44 <211> 120 <212> DNA <213> Homo sapiens <400> 44 cccatcaatc catgtttacg aaaaatctgc caccctcatg ctcattgtac gtacctggga 60 ccaaatcggc acagttgtac atgccaagaa ggctaccgtg gggatggcca agtgtgcttg 120                                                                          120 <210> 45 <211> 135 <212> DNA <213> Homo sapiens <400> 45 cctgtggacc cctgccaaat taactttgga aactgcccta caaagtctac agtgtgcaaa 60 tatgatgggc ctggacagtc tcactgcgag tgtaaggagc attaccagaa tttcgtacct 120 ggagtggggt gcagt 135 <210> 46 <211> 123 <212> DNA <213> Homo sapiens <400> 46 atgactgata tatgtaaatc agataacccg tgtcatagga atgcaaattg caccaccgtc 60 gcaccaggcc gaactgaatg catttgccag aaaggttacg tgggtgatgg cttaacgtgt 120 tat 123 <210> 47 <211> 153 <212> DNA <213> Homo sapiens <400> 47 attccaaagt gctgcaaagg cttctatgga cctgactgca accagtgtcc aggaggcttc 60 tcaaatccat gctcaggaaa tggacagtgt gcagatagcc tcggcggcaa cgggacatgc 120 atttgtgagg agggcttcca aggctcccag tgt 153 <210> 48 <211> 150 <212> DNA <213> Homo sapiens <400> 48 cagttctgct ctgatcccaa taaatacgga cctcggtgta acaaaaaatg cctgtgcgtt 60 cacggaacat gcaataacag gatagacagc gatggggcct gcctcactgg cacatgcaga 120 gacggctctg ccgggagact ctgtgataag 150 <210> 49 <211> 123 <212> DNA <213> Homo sapiens <400> 49 cagacctcag cctgtgggcc ctacgtgcag ttctgtcaca tccacgccac ctgtgaatac 60 agcaatggga cagccagttg tatttgcaaa gcaggatatg aaggagatgg aactctgtgt 120 tct 123 <210> 50 <211> 129 <212> DNA <213> Homo sapiens <400> 50 gagatggacc cttgcacagg actaactcca ggaggctgta gccgcaatgc agaatgcatc 60 aaaactggca cgggcaccca cacctgcgtg tgtcagcagg gttggacagg gaatgggaga 120 gactgctcg 129 <210> 51 <211> 129 <212> DNA <213> Homo sapiens <400> 51 gagatcaaca actgcctgct gcccagtgca ggcggctgcc acgacaacgc atcctgtttg 60 tatgtgggtc ccgggcagaa tgagtgtgag tgcaagaaag gatttcgagg aaatgggatt 120 gactgtgaa 129 <210> 52 <211> 126 <212> DNA <213> Homo sapiens <400> 52 ccaataactt catgcttgga acaaaccggg aaatgtcatc cattggcaag ctgtcaatct 60 acttcgtctg gtgtctggag ctgtgtttgt caagagggct atgaaggaga tggctttctg 120 tgctat 126 <210> 53 <211> 153 <212> DNA <213> Homo sapiens <400> 53 acgagagaat gctgtgccgg cttctttggc ccccaatgcc agccctgccc agggaatgcc 60 cagaatgtct gctttggtaa tggcatctgt ttggatggag tgaatggcac aggtgtgtgt 120 gagtgtgggg agggcttcag cggcacagcc tgc 153 <210> 54 <211> 144 <212> DNA <213> Homo sapiens <400> 54 gagacctgca ccgagggcaa gtacggcatc cactgtgacc aagcatgttc ttgtgtccat 60 gggagatgca accaaggacc cttgggagat ggctcctgtg actgtgatgt tggctggcga 120 ggagtgcatt gtgacaatgc aacc 144 <210> 55 <211> 117 <212> DNA <213> Homo sapiens <400> 55 acagaagaca actgcaatgg gacatgccat accagcgcca actgcctcac caactcagat 60 ggtacagctt catgcaagtg tgcagcagga ttccaaggaa acgggaccat ctgcaca 117 <210> 56 <211> 126 <212> DNA <213> Homo sapiens <400> 56 gcaatcaatg cctgtgagat cagcaatgga ggttgctctg ccaaggctga ctgtaagaga 60 accaccccag gaaggcgagt gtgcacgtgc aaagcaggct acacgggtga tggcattgtg 120 tgcctg 126 <210> 57 <211> 126 <212> DNA <213> Homo sapiens <400> 57 gaaatcaacc cgtgtttgga gaaccatggt ggctgtgaca agaatgcgga gtgcacacag 60 acaggaccca accaggctgc ctgtaactgt ttgccagcat acactggaga tggaaaggtc 120 tgcaca 126 <210> 58 <211> 126 <212> DNA <213> Homo sapiens <400> 58 ctcatcaatg tctgcttaac taaaaatggc ggctgtagtg aatttgccat ctgcaaccac 60 actgggcaag tagaaaggac ttgtacttgc aagccaaact acattggaga tggatttacc 120 tgccgc 126 <210> 59 <211> 153 <212> DNA <213> Homo sapiens <400> 59 atccccaggt gctgcaaggg ctacttcggg cgagactgtc aggcctgccc tggaggacca 60 gatgccccgt gtaataaccg gggtgtctgc cttgatcagt actcggccac cggagagtgt 120 aaatgcaaca ccggcttcaa tgggacggcg tgt 153 <210> 60 <211> 147 <212> DNA <213> Homo sapiens <400> 60 gagatgtgct ggccggggag attcgggcct gattgtctgc cctgtggctg ctcagaccac 60 ggacagtgcg atgatggcat cacgggctcc gggcagtgcc tctgtgaaac ggggtggaca 120 ggcccctcgt gtgacactca ggcagtt 147 <210> 61 <211> 105 <212> DNA <213> Homo sapiens <400> 61 ttgcctgcag tgtgtacgcc tccttgttct gctcatgcca cctgtaagga gaacaacacg 60 tgtgagtgta acctggatta tgaaggtgac ggaatcacat gcaca 105 <210> 62 <211> 123 <212> DNA <213> Homo sapiens <400> 62 gttgtggatt tctgcaaaca ggacaacggg ggctgtgcaa aggtggccag atgctcccag 60 aagggcacga aggtctcctg cagctgccag aagggataca aaggggacgg gcacagctgc 120 aca 123 <210> 63 <211> 129 <212> DNA <213> Homo sapiens <400> 63 gagatagacc cctgtgcaga cggccttaac ggagggtgtc acgagcacgc cacctgtaag 60 atgacaggcc cgggcaagca caagtgtgag tgtaaaagtc actatgtcgg agatgggctg 120 aactgtgag 129 <210> 64 <211> 540 <212> DNA <213> Homo sapiens <400> 64 caacctcggt gttgtcctgg ccgctggggc ccagactgta tagagtgccc aggtggagcg 60 gggtcaccct gcaatggcag aggcagttgt gctgaaggca tggaaggaaa tggaacctgc 120 tcctgccaag aagggtttgg tggaacagcc tgtgaaacct gtgctgacga caacttattt 180 ggacccagct gttcatcagt gtgcaactgt gtgcatgggg tgtgcaacag tggactagat 240 ggcgatggaa cctgtgagtg ctactctgcg tacactggcc ccaagtgtga caagcccatc 300 cctgaatgtg cagccttgct ctgcccagaa aattccagat gttcgccttc cactgaagat 360 gaaaacaaac tggaatgcaa atgccttccc aattaccgag gcgatggcaa atactgcgac 420 cccatcaatc catgtttacg aaaaatctgc caccctcatg ctcattgtac gtacctggga 480 ccaaatcggc acagttgtac atgccaagaa ggctaccgtg gggatggcca agtgtgcttg 540                                                                          540 <210> 65 <211> 675 <212> DNA <213> Homo sapiens <400> 65 caacctcggt gttgtcctgg ccgctggggc ccagactgta tagagtgccc aggtggagcg 60 gggtcaccct gcaatggcag aggcagttgt gctgaaggca tggaaggaaa tggaacctgc 120 tcctgccaag aagggtttgg tggaacagcc tgtgaaacct gtgctgacga caacttattt 180 ggacccagct gttcatcagt gtgcaactgt gtgcatgggg tgtgcaacag tggactagat 240 ggcgatggaa cctgtgagtg ctactctgcg tacactggcc ccaagtgtga caagcccatc 300 cctgaatgtg cagccttgct ctgcccagaa aattccagat gttcgccttc cactgaagat 360 gaaaacaaac tggaatgcaa atgccttccc aattaccgag gcgatggcaa atactgcgac 420 cccatcaatc catgtttacg aaaaatctgc caccctcatg ctcattgtac gtacctggga 480 ccaaatcggc acagttgtac atgccaagaa ggctaccgtg gggatggcca agtgtgcttg 540 cctgtggacc cctgccaaat taactttgga aactgcccta caaagtctac agtgtgcaaa 600 tatgatgggc ctggacagtc tcactgcgag tgtaaggagc attaccagaa tttcgtacct 660 ggagtggggt gcagt 675 <210> 66 <211> 522 <212> DNA <213> Homo sapiens <400> 66 gaaacctgtg ctgacgacaa cttatttgga cccagctgtt catcagtgtg caactgtgtg 60 catggggtgt gcaacagtgg actagatggc gatggaacct gtgagtgcta ctctgcgtac 120 actggcccca agtgtgacaa gcccatccct gaatgtgcag ccttgctctg cccagaaaat 180 tccagatgtt cgccttccac tgaagatgaa aacaaactgg aatgcaaatg ccttcccaat 240 taccgaggcg atggcaaata ctgcgacccc atcaatccat gtttacgaaa aatctgccac 300 cctcatgctc attgtacgta cctgggacca aatcggcaca gttgtacatg ccaagaaggc 360 taccgtgggg atggccaagt gtgcttgcct gtggacccct gccaaattaa ctttggaaac 420 tgccctacaa agtctacagt gtgcaaatat gatgggcctg gacagtctca ctgcgagtgt 480 aaggagcatt accagaattt cgtacctgga gtggggtgca gt 522 <210> 67 <211> 555 <212> DNA <213> Homo sapiens <400> 67 attccaaagt gctgcaaagg cttctatgga cctgactgca accagtgtcc aggaggcttc 60 tcaaatccat gctcaggaaa tggacagtgt gcagatagcc tcggcggcaa cgggacatgc 120 atttgtgagg agggcttcca aggctcccag tgtcagttct gctctgatcc caataaatac 180 ggacctcggt gtaacaaaaa atgcctgtgc gttcacggaa catgcaataa caggatagac 240 agcgatgggg cctgcctcac tggcacatgc agagacggct ctgccgggag actctgtgat 300 aagcagacct cagcctgtgg gccctacgtg cagttctgtc acatccacgc cacctgtgaa 360 tacagcaatg ggacagccag ttgtatttgc aaagcaggat atgaaggaga tggaactctg 420 tgttctgaga tggacccttg cacaggacta actccaggag gctgtagccg caatgcagaa 480 tgcatcaaaa ctggcacggg cacccacacc tgcgtgtgtc agcagggttg gacagggaat 540 gggagagact gctcg 555 <210> 68 <211> 684 <212> DNA <213> Homo sapiens <400> 68 attccaaagt gctgcaaagg cttctatgga cctgactgca accagtgtcc aggaggcttc 60 tcaaatccat gctcaggaaa tggacagtgt gcagatagcc tcggcggcaa cgggacatgc 120 atttgtgagg agggcttcca aggctcccag tgtcagttct gctctgatcc caataaatac 180 ggacctcggt gtaacaaaaa atgcctgtgc gttcacggaa catgcaataa caggatagac 240 agcgatgggg cctgcctcac tggcacatgc agagacggct ctgccgggag actctgtgat 300 aagcagacct cagcctgtgg gccctacgtg cagttctgtc acatccacgc cacctgtgaa 360 tacagcaatg ggacagccag ttgtatttgc aaagcaggat atgaaggaga tggaactctg 420 tgttctgaga tggacccttg cacaggacta actccaggag gctgtagccg caatgcagaa 480 tgcatcaaaa ctggcacggg cacccacacc tgcgtgtgtc agcagggttg gacagggaat 540 gggagagact gctcggagat caacaactgc ctgctgccca gtgcaggcgg ctgccacgac 600 aacgcatcct gtttgtatgt gggtcccggg cagaatgagt gtgagtgcaa gaaaggattt 660 cgaggaaatg ggattgactg tgaa 684 <210> 69 <211> 531 <212> DNA <213> Homo sapiens <400> 69 cagttctgct ctgatcccaa taaatacgga cctcggtgta acaaaaaatg cctgtgcgtt 60 cacggaacat gcaataacag gatagacagc gatggggcct gcctcactgg cacatgcaga 120 gacggctctg ccgggagact ctgtgataag cagacctcag cctgtgggcc ctacgtgcag 180 ttctgtcaca tccacgccac ctgtgaatac agcaatggga cagccagttg tatttgcaaa 240 gcaggatatg aaggagatgg aactctgtgt tctgagatgg acccttgcac aggactaact 300 ccaggaggct gtagccgcaa tgcagaatgc atcaaaactg gcacgggcac ccacacctgc 360 gtgtgtcagc agggttggac agggaatggg agagactgct cggagatcaa caactgcctg 420 ctgcccagtg caggcggctg ccacgacaac gcatcctgtt tgtatgtggg tcccgggcag 480 aatgagtgtg agtgcaagaa aggatttcga ggaaatggga ttgactgtga a 531 <210> 70 <211> 540 <212> DNA <213> Homo sapiens <400> 70 acgagagaat gctgtgccgg cttctttggc ccccaatgcc agccctgccc agggaatgcc 60 cagaatgtct gctttggtaa tggcatctgt ttggatggag tgaatggcac aggtgtgtgt 120 gagtgtgggg agggcttcag cggcacagcc tgcgagacct gcaccgaggg caagtacggc 180 atccactgtg accaagcatg ttcttgtgtc catgggagat gcaaccaagg acccttggga 240 gatggctcct gtgactgtga tgttggctgg cgaggagtgc attgtgacaa tgcaaccaca 300 gaagacaact gcaatgggac atgccatacc agcgccaact gcctcaccaa ctcagatggt 360 acagcttcat gcaagtgtgc agcaggattc caaggaaacg ggaccatctg cacagcaatc 420 aatgcctgtg agatcagcaa tggaggttgc tctgccaagg ctgactgtaa gagaaccacc 480 ccaggaaggc gagtgtgcac gtgcaaagca ggctacacgg gtgatggcat tgtgtgcctg 540                                                                          540 <210> 71 <211> 666 <212> DNA <213> Homo sapiens <400> 71 acgagagaat gctgtgccgg cttctttggc ccccaatgcc agccctgccc agggaatgcc 60 cagaatgtct gctttggtaa tggcatctgt ttggatggag tgaatggcac aggtgtgtgt 120 gagtgtgggg agggcttcag cggcacagcc tgcgagacct gcaccgaggg caagtacggc 180 atccactgtg accaagcatg ttcttgtgtc catgggagat gcaaccaagg acccttggga 240 gatggctcct gtgactgtga tgttggctgg cgaggagtgc attgtgacaa tgcaaccaca 300 gaagacaact gcaatgggac atgccatacc agcgccaact gcctcaccaa ctcagatggt 360 acagcttcat gcaagtgtgc agcaggattc caaggaaacg ggaccatctg cacagcaatc 420 aatgcctgtg agatcagcaa tggaggttgc tctgccaagg ctgactgtaa gagaaccacc 480 ccaggaaggc gagtgtgcac gtgcaaagca ggctacacgg gtgatggcat tgtgtgcctg 540 gaaatcaacc cgtgtttgga gaaccatggt ggctgtgaca agaatgcgga gtgcacacag 600 acaggaccca accaggctgc ctgtaactgt ttgccagcat acactggaga tggaaaggtc 660 tgcaca 666 <210> 72 <211> 513 <212> DNA <213> Homo sapiens <400> 72 gagacctgca ccgagggcaa gtacggcatc cactgtgacc aagcatgttc ttgtgtccat 60 gggagatgca accaaggacc cttgggagat ggctcctgtg actgtgatgt tggctggcga 120 ggagtgcatt gtgacaatgc aaccacagaa gacaactgca atgggacatg ccataccagc 180 gccaactgcc tcaccaactc agatggtaca gcttcatgca agtgtgcagc aggattccaa 240 ggaaacggga ccatctgcac agcaatcaat gcctgtgaga tcagcaatgg aggttgctct 300 gccaaggctg actgtaagag aaccacccca ggaaggcgag tgtgcacgtg caaagcaggc 360 tacacgggtg atggcattgt gtgcctggaa atcaacccgt gtttggagaa ccatggtggc 420 tgtgacaaga atgcggagtg cacacagaca ggacccaacc aggctgcctg taactgtttg 480 ccagcataca ctggagatgg aaaggtctgc aca 513 <210> 73 <211> 528 <212> DNA <213> Homo sapiens <400> 73 atccccaggt gctgcaaggg ctacttcggg cgagactgtc aggcctgccc tggaggacca 60 gatgccccgt gtaataaccg gggtgtctgc cttgatcagt actcggccac cggagagtgt 120 aaatgcaaca ccggcttcaa tgggacggcg tgtgagatgt gctggccggg gagattcggg 180 cctgattgtc tgccctgtgg ctgctcagac cacggacagt gcgatgatgg catcacgggc 240 tccgggcagt gcctctgtga aacggggtgg acaggcccct cgtgtgacac tcaggcagtt 300 ttgcctgcag tgtgtacgcc tccttgttct gctcatgcca cctgtaagga gaacaacacg 360 tgtgagtgta acctggatta tgaaggtgac ggaatcacat gcacagttgt ggatttctgc 420 aaacaggaca acgggggctg tgcaaaggtg gccagatgct cccagaaggg cacgaaggtc 480 tcctgcagct gccagaaggg atacaaaggg gacgggcaca gctgcaca 528 <210> 74 <211> 504 <212> DNA <213> Homo sapiens <400> 74 gagatgtgct ggccggggag attcgggcct gattgtctgc cctgtggctg ctcagaccac 60 ggacagtgcg atgatggcat cacgggctcc gggcagtgcc tctgtgaaac ggggtggaca 120 ggcccctcgt gtgacactca ggcagttttg cctgcagtgt gtacgcctcc ttgttctgct 180 catgccacct gtaaggagaa caacacgtgt gagtgtaacc tggattatga aggtgacgga 240 atcacatgca cagttgtgga tttctgcaaa caggacaacg ggggctgtgc aaaggtggcc 300 agatgctccc agaagggcac gaaggtctcc tgcagctgcc agaagggata caaaggggac 360 gggcacagct gcacagagat agacccctgt gcagacggcc ttaacggagg gtgtcacgag 420 cacgccacct gtaagatgac aggcccgggc aagcacaagt gtgagtgtaa aagtcactat 480 gtcggagatg ggctgaactg tgag 504 <210> 75 <211> 590 <212> PRT <213> Homo sapiens <400> 75 Val Gly Val Arg Asp Cys Arg Tyr Thr Phe Glu Val Arg Thr Tyr Ser   1 5 10 15 Leu Ser Leu Pro Gly Cys Arg His Ile Cys Arg Lys Asp Tyr Leu Gln              20 25 30 Pro Arg Cys Cys Pro Gly Arg Trp Gly Pro Asp Cys Ile Glu Cys Pro          35 40 45 Gly Gly Ala Gly Ser Pro Cys Asn Gly Arg Gly Ser Cys Ala Glu Gly      50 55 60 Met Glu Gly Asn Gly Thr Cys Ser Cys Gln Glu Gly Phe Gly Gly Thr  65 70 75 80 Ala Cys Glu Thr Cys Ala Asp Asp Asn Leu Phe Gly Pro Ser Cys Ser                  85 90 95 Ser Val Cys Asn Cys Val His Gly Val Cys Asn Ser Gly Leu Asp Gly             100 105 110 Asp Gly Thr Cys Glu Cys Tyr Ser Ala Tyr Thr Gly Pro Lys Cys Asp         115 120 125 Lys Pro Ile Pro Glu Cys Ala Ala Leu Leu Cys Pro Glu Asn Ser Arg     130 135 140 Cys Ser Pro Ser Thr Glu Asp Glu Asn Lys Leu Glu Cys Lys Cys Leu 145 150 155 160 Pro Asn Tyr Arg Gly Asp Gly Lys Tyr Cys Asp Pro Ile Asn Pro Cys                 165 170 175 Leu Arg Lys Ile Cys His Pro His Ala His Cys Thr Tyr Leu Gly Pro             180 185 190 Asn Arg His Ser Cys Thr Cys Gln Glu Gly Tyr Arg Gly Asp Gly Gln         195 200 205 Val Cys Leu Pro Val Asp Pro Cys Gln Ile Asn Phe Gly Asn Cys Pro     210 215 220 Thr Lys Ser Thr Val Cys Lys Tyr Asp Gly Pro Gly Gln Ser His Cys 225 230 235 240 Glu Cys Lys Glu His Tyr Gln Asn Phe Val Pro Gly Val Gly Cys Ser                 245 250 255 Met Thr Asp Ile Cys Lys Ser Asp Asn Pro Cys His Arg Asn Ala Asn             260 265 270 Cys Thr Thr Val Ala Pro Gly Arg Thr Glu Cys Ile Cys Gln Lys Gly         275 280 285 Tyr Val Gly Asp Gly Leu Thr Cys Tyr Gly Asn Ile Met Glu Arg Leu     290 295 300 Arg Glu Leu Asn Thr Glu Pro Arg Gly Lys Trp Gln Gly Arg Leu Thr 305 310 315 320 Ser Phe Ile Ser Leu Leu Asp Lys Ala Tyr Ala Trp Pro Leu Ser Lys                 325 330 335 Leu Gly Pro Phe Thr Val Leu Leu Pro Thr Asp Lys Gly Leu Lys Gly             340 345 350 Phe Asn Val Asn Glu Leu Leu Val Asp Asn Lys Ala Ala Gln Tyr Phe         355 360 365 Val Lys Leu His Ile Ile Ala Gly Gln Met Asn Ile Glu Tyr Met Asn     370 375 380 Asn Thr Asp Met Phe Tyr Thr Leu Thr Gly Lys Ser Gly Glu Ile Phe 385 390 395 400 Asn Ser Asp Lys Asp Asn Gln Ile Lys Leu Lys Leu His Gly Gly Lys                 405 410 415 Lys Lys Val Lys Ile Ile Gln Gly Asp Ile Ile Ala Ser Asn Gly Leu             420 425 430 Leu His Ile Leu Asp Arg Ala Met Asp Lys Leu Glu Pro Thr Phe Glu         435 440 445 Ser Asn Asn Glu Gln Thr Ile Met Thr Met Leu Gln Pro Arg Tyr Ser     450 455 460 Lys Phe Arg Ser Leu Leu Glu Glu Thr Asn Leu Gly His Ala Leu Asp 465 470 475 480 Glu Asp Gly Val Gly Gly Pro Tyr Thr Ile Phe Val Pro Asn Asn Glu                 485 490 495 Ala Leu Asn Asn Met Lys Asp Gly Thr Leu Asp Tyr Leu Leu Ser Pro             500 505 510 Glu Gly Ser Arg Lys Leu Leu Glu Leu Val Arg Tyr His Ile Val Pro         515 520 525 Phe Thr Gln Leu Glu Val Ala Thr Leu Ile Ser Thr Pro His Ile Arg     530 535 540 Ser Met Ala Asn Gln Leu Ile Gln Phe Asn Thr Thr Asp Asn Gly Gln 545 550 555 560 Ile Leu Ala Asn Asp Val Ala Met Glu Glu Ile Glu Ile Thr Ala Lys                 565 570 575 Asn Gly Arg Ile Tyr Thr Leu Thr Gly Val Leu Ile Pro Pro             580 585 590 <210> 76 <211> 578 <212> PRT <213> Homo sapiens <400> 76 Asn Ser Glu Pro Thr Ala Leu Phe Thr His Arg Cys Val Tyr Ser Gly   1 5 10 15 Arg Phe Gly Ser Leu Lys Ser Gly Cys Ala Arg Tyr Cys Asn Ala Thr              20 25 30 Val Lys Ile Pro Lys Cys Cys Lys Gly Phe Tyr Gly Pro Asp Cys Asn          35 40 45 Gln Cys Pro Gly Gly Phe Ser Asn Pro Cys Ser Gly Asn Gly Gln Cys      50 55 60 Ala Asp Ser Leu Gly Gly Asn Gly Thr Cys Ile Cys Glu Glu Gly Phe  65 70 75 80 Gln Gly Ser Gln Cys Gln Phe Cys Ser Asp Pro Asn Lys Tyr Gly Pro                  85 90 95 Arg Cys Asn Lys Lys Cys Leu Cys Val His Gly Thr Cys Asn Asn Arg             100 105 110 Ile Asp Ser Asp Gly Ala Cys Leu Thr Gly Thr Cys Arg Asp Gly Ser         115 120 125 Ala Gly Arg Leu Cys Asp Lys Gln Thr Ser Ala Cys Gly Pro Tyr Val     130 135 140 Gln Phe Cys His Ile His Ala Thr Cys Glu Tyr Ser Asn Gly Thr Ala 145 150 155 160 Ser Cys Ile Cys Lys Ala Gly Tyr Glu Gly Asp Gly Thr Leu Cys Ser                 165 170 175 Glu Met Asp Pro Cys Thr Gly Leu Thr Pro Gly Gly Cys Ser Arg Asn             180 185 190 Ala Glu Cys Ile Lys Thr Gly Thr Gly Thr His Thr Cys Val Cys Gln         195 200 205 Gln Gly Trp Thr Gly Asn Gly Arg Asp Cys Ser Glu Ile Asn Asn Cys     210 215 220 Leu Leu Pro Ser Ala Gly Gly Cys His Asp Asn Ala Ser Cys Leu Tyr 225 230 235 240 Val Gly Pro Gly Gln Asn Glu Cys Glu Cys Lys Lys Gly Phe Arg Gly                 245 250 255 Asn Gly Ile Asp Cys Glu Pro Ile Thr Ser Cys Leu Glu Gln Thr Gly             260 265 270 Lys Cys His Pro Leu Ala Ser Cys Gln Ser Thr Ser Ser Gly Val Trp         275 280 285 Ser Cys Val Cys Gln Glu Gly Tyr Glu Gly Asp Gly Phe Leu Cys Tyr     290 295 300 Gly Asn Ala Ala Val Glu Leu Ser Phe Leu Ser Glu Ala Ala Ile Phe 305 310 315 320 Asn Arg Trp Ile Asn Asn Ala Ser Leu Gln Pro Thr Leu Ser Ala Thr                 325 330 335 Ser Asn Leu Thr Val Leu Val Pro Ser Gln Gln Ala Thr Glu Asp Met             340 345 350 Asp Gln Asp Glu Lys Ser Phe Trp Leu Ser Gln Ser Asn Ile Pro Ala         355 360 365 Leu Ile Lys Tyr His Met Leu Leu Gly Thr Tyr Arg Val Ala Asp Leu     370 375 380 Gln Thr Leu Ser Ser Ser Asp Met Leu Ala Thr Ser Leu Gln Gly Asn 385 390 395 400 Phe Leu His Leu Ala Lys Val Asp Gly Asn Ile Thr Ile Glu Gly Ala                 405 410 415 Ser Ile Val Asp Gly Asp Asn Ala Ala Thr Asn Gly Val Ile His Ile             420 425 430 Ile Asn Lys Val Leu Val Pro Gln Arg Arg Leu Thr Gly Ser Leu Pro         435 440 445 Asn Leu Leu Met Arg Leu Glu Gln Met Pro Asp Tyr Ser Ile Phe Arg     450 455 460 Gly Tyr Ile Ile Gln Tyr Asn Leu Ala Asn Ala Ile Glu Ala Ala Asp 465 470 475 480 Ala Tyr Thr Val Phe Ala Pro Asn Asn Asn Ala Ile Glu Asn Tyr Ile                 485 490 495 Arg Glu Lys Lys Val Leu Ser Leu Glu Glu Asp Val Leu Arg Tyr His             500 505 510 Val Val Leu Glu Glu Lys Leu Leu Lys Asn Asp Leu His Asn Gly Met         515 520 525 His Arg Glu Thr Met Leu Gly Phe Ser Tyr Phe Leu Ser Phe Phe Leu     530 535 540 His Asn Asp Gln Leu Tyr Val Asn Glu Ala Pro Ile Asn Tyr Thr Asn 545 550 555 560 Val Ala Thr Asp Lys Gly Val Ile His Gly Leu Gly Lys Val Leu Glu                 565 570 575 Ile gln         <210> 77 <211> 581 <212> PRT <213> Homo sapiens <400> 77 Glu Lys Arg Arg Cys Ile Tyr Thr Ser Tyr Phe Met Gly Arg Arg Thr   1 5 10 15 Leu Phe Ile Gly Cys Gln Pro Lys Cys Val Arg Thr Val Ile Thr Arg              20 25 30 Glu Cys Cys Ala Gly Phe Ghe Pro Gln Cys Gln Pro Cys Pro Gly          35 40 45 Asn Ala Gln Asn Val Cys Phe Gly Asn Gly Ile Cys Leu Asp Gly Val      50 55 60 Asn Gly Thr Gly Val Cys Glu Cys Gly Glu Gly Phe Ser Gly Thr Ala  65 70 75 80 Cys Glu Thr Cys Thr Glu Gly Lys Tyr Gly Ile His Cys Asp Gln Ala                  85 90 95 Cys Ser Cys Val His Gly Arg Cys Asn Gln Gly Pro Leu Gly Asp Gly             100 105 110 Ser Cys Asp Cys Asp Val Gly Trp Arg Gly Val His Cys Asp Asn Ala         115 120 125 Thr Thr Glu Asp Asn Cys Asn Gly Thr Cys His Thr Ser Ala Asn Cys     130 135 140 Leu Thr Asn Ser Asp Gly Thr Ala Ser Cys Lys Cys Ala Ala Gly Phe 145 150 155 160 Gln Gly Asn Gly Thr Ile Cys Thr Ala Ile Asn Ala Cys Glu Ile Ser                 165 170 175 Asn Gly Gly Cys Ser Ala Lys Ala Asp Cys Lys Arg Thr Thr Pro Gly             180 185 190 Arg Arg Val Cys Thr Cys Lys Ala Gly Tyr Thr Gly Asp Gly Ile Val         195 200 205 Cys Leu Glu Ile Asn Pro Cys Leu Glu Asn His Gly Gly Cys Asp Lys     210 215 220 Asn Ala Glu Cys Thr Gln Thr Gly Pro Asn Gln Ala Ala Cys Asn Cys 225 230 235 240 Leu Pro Ala Tyr Thr Gly Asp Gly Lys Val Cys Thr Leu Ile Asn Val                 245 250 255 Cys Leu Thr Lys Asn Gly Gly Cys Ser Glu Phe Ala Ile Cys Asn His             260 265 270 Thr Gly Gln Val Glu Arg Thr Cys Thr Cys Lys Pro Asn Tyr Ile Gly         275 280 285 Asp Gly Phe Thr Cys Arg Gly Ser Ile Tyr Gln Glu Leu Pro Lys Asn     290 295 300 Pro Lys Thr Ser Gln Tyr Phe Phe Gln Leu Gln Glu His Phe Val Lys 305 310 315 320 Asp Leu Val Gly Pro Gly Pro Phe Thr Val Phe Ala Pro Leu Ser Ala                 325 330 335 Ala Phe Asp Glu Glu Ala Arg Val Lys Asp Trp Asp Lys Tyr Gly Leu             340 345 350 Met Pro Gln Val Leu Arg Tyr His Val Val Ala Cys His Gln Leu Leu         355 360 365 Leu Glu Asn Leu Lys Leu Ile Ser Asn Ala Thr Ser Leu Gln Gly Glu     370 375 380 Pro Ile Val Ile Ser Val Ser Gln Ser Thr Val Tyr Ile Asn Asn Lys 385 390 395 400 Ala Lys Ile Ile Ser Ser Asp Ile Ile Ser Thr Asn Gly Ile Val His                 405 410 415 Ile Ile Asp Lys Leu Leu Ser Pro Lys Asn Leu Leu Ile Thr Pro Lys             420 425 430 Asp Asn Ser Gly Arg Ile Leu Gln Asn Leu Thr Thr Leu Ala Thr Asn         435 440 445 Asn Gly Tyr Ile Lys Phe Ser Asn Leu Ile Gln Asp Ser Gly Leu Leu     450 455 460 Ser Val Ile Thr Asp Pro Ile His Thr Pro Val Thr Leu Phe Trp Pro 465 470 475 480 Thr Asp Gln Ala Leu His Ala Leu Pro Ala Glu Gln Gln Asp Phe Leu                 485 490 495 Phe Asn Gln Asp Asn Lys Asp Lys Leu Lys Glu Tyr Leu Lys Phe His             500 505 510 Val Ile Arg Asp Ala Lys Val Leu Ala Val Asp Leu Pro Thr Ser Thr         515 520 525 Ala Trp Lys Thr Leu Gln Gly Ser Glu Leu Ser Val Lys Cys Gly Ala     530 535 540 Gly Arg Asp Ile Gly Asp Leu Phe Leu Asn Gly Gln Thr Cys Arg Ile 545 550 555 560 Val Gln Arg Glu Leu Leu Phe Asp Leu Gly Val Ala Tyr Gly Ile Asp                 565 570 575 Cys Leu Leu Ile Asp             580 <210> 78 <211> 537 <212> PRT <213> Homo sapiens <400> 78 Arg Trp Ser Lys Pro Lys Gly Val Lys Gln Lys Cys Leu Tyr Asn Leu   1 5 10 15 Pro Phe Lys Arg Asn Leu Glu Gly Cys Arg Glu Arg Cys Ser Leu Val              20 25 30 Ile Gln Ile Pro Arg Cys Cys Lys Gly Tyr Phe Gly Arg Asp Cys Gln          35 40 45 Ala Cys Pro Gly Gly Pro Asp Ala Pro Cys Asn Asn Arg Gly Val Cys      50 55 60 Leu Asp Gln Tyr Ser Ala Thr Gly Glu Cys Lys Cys Asn Thr Gly Phe  65 70 75 80 Asn Gly Thr Ala Cys Glu Met Cys Trp Pro Gly Arg Phe Gly Pro Asp                  85 90 95 Cys Leu Pro Cys Gly Cys Ser Asp His Gly Gln Cys Asp Asp Gly Ile             100 105 110 Thr Gly Ser Gly Gln Cys Leu Cys Glu Thr Gly Trp Thr Gly Pro Ser         115 120 125 Cys Asp Thr Gln Ala Val Leu Pro Ala Val Cys Thr Pro Pro Cys Ser     130 135 140 Ala His Ala Thr Cys Lys Glu Asn Asn Thr Cys Glu Cys Asn Leu Asp 145 150 155 160 Tyr Glu Gly Asp Gly Ile Thr Cys Thr Val Val Asp Phe Cys Lys Gln                 165 170 175 Asp Asn Gly Gly Cys Ala Lys Val Ala Arg Cys Ser Gln Lys Gly Thr             180 185 190 Lys Val Ser Cys Ser Cys Gln Lys Gly Tyr Lys Gly Asp Gly His Ser         195 200 205 Cys Thr Glu Ile Asp Pro Cys Ala Asp Gly Leu Asn Gly Gly Cys His     210 215 220 Glu His Ala Thr Cys Lys Met Thr Gly Pro Gly Lys His Lys Cys Glu 225 230 235 240 Cys Lys Ser His Tyr Val Gly Asp Gly Leu Asn Cys Glu Pro Glu Gln                 245 250 255 Leu Pro Ile Asp Arg Cys Leu Gln Asp Asn Gly Gln Cys His Ala Asp             260 265 270 Ala Lys Cys Val Asp Leu His Phe Gln Asp Thr Thr Val Gly Val Phe         275 280 285 His Leu Arg Ser Pro Leu Gly Gln Tyr Lys Leu Thr Phe Asp Lys Ala     290 295 300 Arg Glu Ala Cys Ala Asn Glu Ala Ala Thr Met Ala Thr Tyr Asn Gln 305 310 315 320 Leu Ser Tyr Ala Gln Lys Ala Lys Tyr His Leu Cys Ser Ala Gly Trp                 325 330 335 Leu Glu Thr Gly Arg Val Ala Tyr Pro Thr Ala Phe Ala Ser Gln Asn             340 345 350 Cys Gly Ser Gly Val Val Gly Ile Val Asp Tyr Gly Pro Arg Pro Asn         355 360 365 Lys Ser Glu Met Trp Asp Val Phe Cys Tyr Arg Met Lys Asp Val Asn     370 375 380 Cys Thr Cys Lys Val Gly Tyr Val Gly Asp Gly Phe Ser Cys Ser Gly 385 390 395 400 Asn Leu Leu Gln Val Leu Met Ser Phe Pro Ser Leu Thr Asn Phe Leu                 405 410 415 Thr Glu Val Leu Ala Tyr Ser Asn Ser Ser Ala Arg Gly Arg Ala Phe             420 425 430 Leu Glu His Leu Thr Asp Leu Ser Ile Arg Gly Thr Leu Phe Val Pro         435 440 445 Gln Asn Ser Gly Leu Gly Glu Asn Glu Thr Leu Ser Gly Arg Asp Ile     450 455 460 Glu His His Leu Ala Asn Val Ser Met Phe Phe Tyr Asn Asp Leu Val 465 470 475 480 Asn Gly Thr Thr Leu Gln Thr Arg Leu Gly Ser Lys Leu Leu Ile Thr                 485 490 495 Ala Ser Gln Asp Pro Leu Gln Pro Thr Glu Thr Arg Phe Val Asp Gly             500 505 510 Arg Ala Ile Leu Gln Trp Asp Ile Phe Ala Ser Asn Gly Ile Ile His         515 520 525 Val Ile Ser Arg Pro Leu Lys Ala Pro     530 535 <210> 79 <211> 266 <212> PRT <213> Homo sapiens <400> 79 Gln Pro Arg Cys Cys Pro Gly Arg Trp Gly Pro Asp Cys Ile Glu Cys   1 5 10 15 Pro Gly Gly Ala Gly Ser Pro Cys Asn Gly Arg Gly Ser Cys Ala Glu              20 25 30 Gly Met Glu Gly Asn Gly Thr Cys Ser Cys Gln Glu Gly Phe Gly Gly          35 40 45 Thr Ala Cys Glu Thr Cys Ala Asp Asp Asn Leu Phe Gly Pro Ser Cys      50 55 60 Ser Ser Val Cys Asn Cys Val His Gly Val Cys Asn Ser Gly Leu Asp  65 70 75 80 Gly Asp Gly Thr Cys Glu Cys Tyr Ser Ala Tyr Thr Gly Pro Lys Cys                  85 90 95 Asp Lys Pro Ile Pro Glu Cys Ala Ala Leu Leu Cys Pro Glu Asn Ser             100 105 110 Arg Cys Ser Pro Ser Thr Glu Asp Glu Asn Lys Leu Glu Cys Lys Cys         115 120 125 Leu Pro Asn Tyr Arg Gly Asp Gly Lys Tyr Cys Asp Pro Ile Asn Pro     130 135 140 Cys Leu Arg Lys Ile Cys His Pro His Ala His Cys Thr Tyr Leu Gly 145 150 155 160 Pro Asn Arg His Ser Cys Thr Cys Gln Glu Gly Tyr Arg Gly Asp Gly                 165 170 175 Gln Val Cys Leu Pro Val Asp Pro Cys Gln Ile Asn Phe Gly Asn Cys             180 185 190 Pro Thr Lys Ser Thr Val Cys Lys Tyr Asp Gly Pro Gly Gln Ser His         195 200 205 Cys Glu Cys Lys Glu His Tyr Gln Asn Phe Val Pro Gly Val Gly Cys     210 215 220 Ser Met Thr Asp Ile Cys Lys Ser Asp Asn Pro Cys His Arg Asn Ala 225 230 235 240 Asn Cys Thr Thr Val Ala Pro Gly Arg Thr Glu Cys Ile Cys Gln Lys                 245 250 255 Gly Tyr Val Gly Asp Gly Leu Thr Cys Tyr             260 265 <210> 80 <211> 270 <212> PRT <213> Homo sapiens <400> 80 Ile Pro Lys Cys Cys Lys Gly Phe Tyr Gly Pro Asp Cys Asn Gln Cys   1 5 10 15 Pro Gly Gly Phe Ser Asn Pro Cys Ser Gly Asn Gly Gln Cys Ala Asp              20 25 30 Ser Leu Gly Gly Asn Gly Thr Cys Ile Cys Glu Glu Gly Phe Gln Gly          35 40 45 Ser Gln Cys Gln Phe Cys Ser Asp Pro Asn Lys Tyr Gly Pro Arg Cys      50 55 60 Asn Lys Lys Cys Leu Cys Val His Gly Thr Cys Asn Asn Arg Ile Asp  65 70 75 80 Ser Asp Gly Ala Cys Leu Thr Gly Thr Cys Arg Asp Gly Ser Ala Gly                  85 90 95 Arg Leu Cys Asp Lys Gln Thr Ser Ala Cys Gly Pro Tyr Val Gln Phe             100 105 110 Cys His Ile His Ala Thr Cys Glu Tyr Ser Asn Gly Thr Ala Ser Cys         115 120 125 Ile Cys Lys Ala Gly Tyr Glu Gly Asp Gly Thr Leu Cys Ser Glu Met     130 135 140 Asp Pro Cys Thr Gly Leu Thr Pro Gly Gly Cys Ser Arg Asn Ala Glu 145 150 155 160 Cys Ile Lys Thr Gly Thr Gly Thr His Thr Cys Val Cys Gln Gln Gly                 165 170 175 Trp Thr Gly Asn Gly Arg Asp Cys Ser Glu Ile Asn Asn Cys Leu Leu             180 185 190 Pro Ser Ala Gly Gly Cys His Asp Asn Ala Ser Cys Leu Tyr Val Gly         195 200 205 Pro Gly Gln Asn Glu Cys Glu Cys Lys Lys Gly Phe Arg Gly Asn Gly     210 215 220 Ile Asp Cys Glu Pro Ile Thr Ser Cys Leu Glu Gln Thr Gly Lys Cys 225 230 235 240 His Pro Leu Ala Ser Cys Gln Ser Thr Ser Ser Gly Val Trp Ser Cys                 245 250 255 Val Cys Gln Glu Gly Tyr Glu Gly Asp Gly Phe Leu Cys Tyr             260 265 270 <210> 81 <211> 264 <212> PRT <213> Homo sapiens <400> 81 Thr Arg Glu Cys Cys Ala Gly Phe Phe Gly Pro Gln Cys Gln Pro Cys   1 5 10 15 Pro Gly Asn Ala Gln Asn Val Cys Phe Gly Asn Gly Ile Cys Leu Asp              20 25 30 Gly Val Asn Gly Thr Gly Val Cys Glu Cys Gly Glu Gly Phe Ser Gly          35 40 45 Thr Ala Cys Glu Thr Cys Thr Glu Gly Lys Tyr Gly Ile His Cys Asp      50 55 60 Gln Ala Cys Ser Cys Val His Gly Arg Cys Asn Gln Gly Pro Leu Gly  65 70 75 80 Asp Gly Ser Cys Asp Cys Asp Val Gly Trp Arg Gly Val His Cys Asp                  85 90 95 Asn Ala Thr Thr Glu Asp Asn Cys Asn Gly Thr Cys His Thr Ser Ala             100 105 110 Asn Cys Leu Thr Asn Ser Asp Gly Thr Ala Ser Cys Lys Cys Ala Ala         115 120 125 Gly Phe Gln Gly Asn Gly Thr Ile Cys Thr Ala Ile Asn Ala Cys Glu     130 135 140 Ile Ser Asn Gly Gly Cys Ser Ala Lys Ala Asp Cys Lys Arg Thr Thr 145 150 155 160 Pro Gly Arg Arg Val Cys Thr Cys Lys Ala Gly Tyr Thr Gly Asp Gly                 165 170 175 Ile Val Cys Leu Glu Ile Asn Pro Cys Leu Glu Asn His Gly Gly Cys             180 185 190 Asp Lys Asn Ala Glu Cys Thr Gln Thr Gly Pro Asn Gln Ala Ala Cys         195 200 205 Asn Cys Leu Pro Ala Tyr Thr Gly Asp Gly Lys Val Cys Thr Leu Ile     210 215 220 Asn Val Cys Leu Thr Lys Asn Gly Gly Cys Ser Glu Phe Ala Ile Cys 225 230 235 240 Asn His Thr Gly Gln Val Glu Arg Thr Cys Thr Cys Lys Pro Asn Tyr                 245 250 255 Ile Gly Asp Gly Phe Thr Cys Arg             260 <210> 82 <211> 219 <212> PRT <213> Homo sapiens <400> 82 Ile Pro Arg Cys Cys Lys Gly Tyr Phe Gly Arg Asp Cys Gln Ala Cys   1 5 10 15 Pro Gly Gly Pro Asp Ala Pro Cys Asn Asn Arg Gly Val Cys Leu Asp              20 25 30 Gln Tyr Ser Ala Thr Gly Glu Cys Lys Cys Asn Thr Gly Phe Asn Gly          35 40 45 Thr Ala Cys Glu Met Cys Trp Pro Gly Arg Phe Gly Pro Asp Cys Leu      50 55 60 Pro Cys Gly Cys Ser Asp His Gly Gln Cys Asp Asp Gly Ile Thr Gly  65 70 75 80 Ser Gly Gln Cys Leu Cys Glu Thr Gly Trp Thr Gly Pro Ser Cys Asp                  85 90 95 Thr Gln Ala Val Leu Pro Ala Val Cys Thr Pro Pro Cys Ser Ala His             100 105 110 Ala Thr Cys Lys Glu Asn Asn Thr Cys Glu Cys Asn Leu Asp Tyr Glu         115 120 125 Gly Asp Gly Ile Thr Cys Thr Val Val Asp Phe Cys Lys Gln Asp Asn     130 135 140 Gly Gly Cys Ala Lys Val Ala Arg Cys Ser Gln Lys Gly Thr Lys Val 145 150 155 160 Ser Cys Ser Cys Gln Lys Gly Tyr Lys Gly Asp Gly His Ser Cys Thr                 165 170 175 Glu Ile Asp Pro Cys Ala Asp Gly Leu Asn Gly Gly Cys His Glu His             180 185 190 Ala Thr Cys Lys Met Thr Gly Pro Gly Lys His Lys Cys Glu Cys Lys         195 200 205 Ser His Tyr Val Gly Asp Gly Leu Asn Cys Glu     210 215 <210> 83 <211> 51 <212> PRT <213> Homo sapiens <400> 83 Gln Pro Arg Cys Cys Pro Gly Arg Trp Gly Pro Asp Cys Ile Glu Cys   1 5 10 15 Pro Gly Gly Ala Gly Ser Pro Cys Asn Gly Arg Gly Ser Cys Ala Glu              20 25 30 Gly Met Glu Gly Asn Gly Thr Cys Ser Cys Gln Glu Gly Phe Gly Gly          35 40 45 Thr ala cys      50 <210> 84 <211> 47 <212> PRT <213> Homo sapiens <400> 84 Glu Thr Cys Ala Asp Asp Asn Leu Phe Gly Pro Ser Cys Ser Ser Val   1 5 10 15 Cys Asn Cys Val His Gly Val Cys Asn Ser Gly Leu Asp Gly Asp Gly              20 25 30 Thr Cys Glu Cys Tyr Ser Ala Tyr Thr Gly Pro Lys Cys Asp Lys          35 40 45 <210> 85 <211> 42 <212> PRT <213> Homo sapiens <400> 85 Pro Ile Pro Glu Cys Ala Ala Leu Leu Cys Pro Glu Asn Ser Arg Cys   1 5 10 15 Ser Pro Ser Thr Glu Asp Glu Asn Lys Leu Glu Cys Lys Cys Leu Pro              20 25 30 Asn Tyr Arg Gly Asp Gly Lys Tyr Cys Asp          35 40 <210> 86 <211> 40 <212> PRT <213> Homo sapiens <400> 86 Pro Ile Asn Pro Cys Leu Arg Lys Ile Cys His Pro His Ala His Cys   1 5 10 15 Thr Tyr Leu Gly Pro Asn Arg His Ser Cys Thr Cys Gln Glu Gly Tyr              20 25 30 Arg Gly Asp Gly Gln Val Cys Leu          35 40 <210> 87 <211> 45 <212> PRT <213> Homo sapiens <400> 87 Pro Val Asp Pro Cys Gln Ile Asn Phe Gly Asn Cys Pro Thr Lys Ser   1 5 10 15 Thr Val Cys Lys Tyr Asp Gly Pro Gly Gln Ser His Cys Glu Cys Lys              20 25 30 Glu His Tyr Gln Asn Phe Val Pro Gly Val Gly Cys Ser          35 40 45 <210> 88 <211> 41 <212> PRT <213> Homo sapiens <400> 88 Met Thr Asp Ile Cys Lys Ser Asp Asn Pro Cys His Arg Asn Ala Asn   1 5 10 15 Cys Thr Thr Val Ala Pro Gly Arg Thr Glu Cys Ile Cys Gln Lys Gly              20 25 30 Tyr Val Gly Asp Gly Leu Thr Cys Tyr          35 40 <210> 89 <211> 51 <212> PRT <213> Homo sapiens <400> 89 Ile Pro Lys Cys Cys Lys Gly Phe Tyr Gly Pro Asp Cys Asn Gln Cys   1 5 10 15 Pro Gly Gly Phe Ser Asn Pro Cys Ser Gly Asn Gly Gln Cys Ala Asp              20 25 30 Ser Leu Gly Gly Asn Gly Thr Cys Ile Cys Glu Glu Gly Phe Gln Gly          35 40 45 Ser Gln Cys      50 <210> 90 <211> 50 <212> PRT <213> Homo sapiens <400> 90 Gln Phe Cys Ser Asp Pro Asn Lys Tyr Gly Pro Arg Cys Asn Lys Lys   1 5 10 15 Cys Leu Cys Val His Gly Thr Cys Asn Asn Arg Ile Asp Ser Asp Gly              20 25 30 Ala Cys Leu Thr Gly Thr Cys Arg Asp Gly Ser Ala Gly Arg Leu Cys          35 40 45 Asp lys      50 <210> 91 <211> 41 <212> PRT <213> Homo sapiens <400> 91 Gln Thr Ser Ala Cys Gly Pro Tyr Val Gln Phe Cys His Ile His Ala   1 5 10 15 Thr Cys Glu Tyr Ser Asn Gly Thr Ala Ser Cys Ile Cys Lys Ala Gly              20 25 30 Tyr Glu Gly Asp Gly Thr Leu Cys Ser          35 40 <210> 92 <211> 43 <212> PRT <213> Homo sapiens <400> 92 Glu Met Asp Pro Cys Thr Gly Leu Thr Pro Gly Gly Cys Ser Arg Asn   1 5 10 15 Ala Glu Cys Ile Lys Thr Gly Thr Gly Thr His Thr Cys Val Cys Gln              20 25 30 Gln Gly Trp Thr Gly Asn Gly Arg Asp Cys Ser          35 40 <210> 93 <211> 43 <212> PRT <213> Homo sapiens <400> 93 Glu Ile Asn Asn Cys Leu Leu Pro Ser Ala Gly Gly Cys His Asp Asn   1 5 10 15 Ala Ser Cys Leu Tyr Val Gly Pro Gly Gln Asn Glu Cys Glu Cys Lys              20 25 30 Lys Gly Phe Arg Gly Asn Gly Ile Asp Cys Glu          35 40 <210> 94 <211> 42 <212> PRT <213> Homo sapiens <400> 94 Pro Ile Thr Ser Cys Leu Glu Gln Thr Gly Lys Cys His Pro Leu Ala   1 5 10 15 Ser Cys Gln Ser Thr Ser Ser Gly Val Trp Ser Cys Val Cys Gln Glu              20 25 30 Gly Tyr Glu Gly Asp Gly Phe Leu Cys Tyr          35 40 <210> 95 <211> 51 <212> PRT <213> Homo sapiens <400> 95 Thr Arg Glu Cys Cys Ala Gly Phe Phe Gly Pro Gln Cys Gln Pro Cys   1 5 10 15 Pro Gly Asn Ala Gln Asn Val Cys Phe Gly Asn Gly Ile Cys Leu Asp              20 25 30 Gly Val Asn Gly Thr Gly Val Cys Glu Cys Gly Glu Gly Phe Ser Gly          35 40 45 Thr ala cys      50 <210> 96 <211> 48 <212> PRT <213> Homo sapiens <400> 96 Glu Thr Cys Thr Glu Gly Lys Tyr Gly Ile His Cys Asp Gln Ala Cys   1 5 10 15 Ser Cys Val His Gly Arg Cys Asn Gln Gly Pro Leu Gly Asp Gly Ser              20 25 30 Cys Asp Cys Asp Val Gly Trp Arg Gly Val His Cys Asp Asn Ala Thr          35 40 45 <210> 97 <211> 39 <212> PRT <213> Homo sapiens <400> 97 Thr Glu Asp Asn Cys Asn Gly Thr Cys His Thr Ser Ala Asn Cys Leu   1 5 10 15 Thr Asn Ser Asp Gly Thr Ala Ser Cys Lys Cys Ala Ala Gly Phe Gln              20 25 30 Gly Asn Gly Thr Ile Cys Thr          35 <210> 98 <211> 42 <212> PRT <213> Homo sapiens <400> 98 Ala Ile Asn Ala Cys Glu Ile Ser Asn Gly Gly Cys Ser Ala Lys Ala   1 5 10 15 Asp Cys Lys Arg Thr Thr Pro Gly Arg Arg Val Cys Thr Cys Lys Ala              20 25 30 Gly Tyr Thr Gly Asp Gly Ile Val Cys Leu          35 40 <210> 99 <211> 42 <212> PRT <213> Homo sapiens <400> 99 Glu Ile Asn Pro Cys Leu Glu Asn His Gly Gly Cys Asp Lys Asn Ala   1 5 10 15 Glu Cys Thr Gln Thr Gly Pro Asn Gln Ala Ala Cys Asn Cys Leu Pro              20 25 30 Ala Tyr Thr Gly Asp Gly Lys Val Cys Thr          35 40 <210> 100 <211> 42 <212> PRT <213> Homo sapiens <400> 100 Leu Ile Asn Val Cys Leu Thr Lys Asn Gly Gly Cys Ser Glu Phe Ala   1 5 10 15 Ile Cys Asn His Thr Gly Gln Val Glu Arg Thr Cys Thr Cys Lys Pro              20 25 30 Asn Tyr Ile Gly Asp Gly Phe Thr Cys Arg          35 40 <210> 101 <211> 51 <212> PRT <213> Homo sapiens <400> 101 Ile Pro Arg Cys Cys Lys Gly Tyr Phe Gly Arg Asp Cys Gln Ala Cys   1 5 10 15 Pro Gly Gly Pro Asp Ala Pro Cys Asn Asn Arg Gly Val Cys Leu Asp              20 25 30 Gln Tyr Ser Ala Thr Gly Glu Cys Lys Cys Asn Thr Gly Phe Asn Gly          35 40 45 Thr ala cys      50 <210> 102 <211> 49 <212> PRT <213> Homo sapiens <400> 102 Glu Met Cys Trp Pro Gly Arg Phe Gly Pro Asp Cys Leu Pro Cys Gly   1 5 10 15 Cys Ser Asp His Gly Gln Cys Asp Asp Gly Ile Thr Gly Ser Gly Gln              20 25 30 Cys Leu Cys Glu Thr Gly Trp Thr Gly Pro Ser Cys Asp Thr Gln Ala          35 40 45 Val     <210> 103 <211> 35 <212> PRT <213> Homo sapiens <400> 103 Leu Pro Ala Val Cys Thr Pro Pro Cys Ser Ala His Ala Thr Cys Lys   1 5 10 15 Glu Asn Asn Thr Cys Glu Cys Asn Leu Asp Tyr Glu Gly Asp Gly Ile              20 25 30 Thr Cys Thr          35 <210> 104 <211> 41 <212> PRT <213> Homo sapiens <400> 104 Val Val Asp Phe Cys Lys Gln Asp Asn Gly Gly Cys Ala Lys Val Ala   1 5 10 15 Arg Cys Ser Gln Lys Gly Thr Lys Val Ser Cys Ser Cys Gln Lys Gly              20 25 30 Tyr Lys Gly Asp Gly His Ser Cys Thr          35 40 <210> 105 <211> 43 <212> PRT <213> Homo sapiens <400> 105 Glu Ile Asp Pro Cys Ala Asp Gly Leu Asn Gly Gly Cys His Glu His   1 5 10 15 Ala Thr Cys Lys Met Thr Gly Pro Gly Lys His Lys Cys Glu Cys Lys              20 25 30 Ser His Tyr Val Gly Asp Gly Leu Asn Cys Glu          35 40 <210> 106 <211> 180 <212> PRT <213> Homo sapiens <400> 106 Gln Pro Arg Cys Cys Pro Gly Arg Trp Gly Pro Asp Cys Ile Glu Cys   1 5 10 15 Pro Gly Gly Ala Gly Ser Pro Cys Asn Gly Arg Gly Ser Cys Ala Glu              20 25 30 Gly Met Glu Gly Asn Gly Thr Cys Ser Cys Gln Glu Gly Phe Gly Gly          35 40 45 Thr Ala Cys Glu Thr Cys Ala Asp Asp Asn Leu Phe Gly Pro Ser Cys      50 55 60 Ser Ser Val Cys Asn Cys Val His Gly Val Cys Asn Ser Gly Leu Asp  65 70 75 80 Gly Asp Gly Thr Cys Glu Cys Tyr Ser Ala Tyr Thr Gly Pro Lys Cys                  85 90 95 Asp Lys Pro Ile Pro Glu Cys Ala Ala Leu Leu Cys Pro Glu Asn Ser             100 105 110 Arg Cys Ser Pro Ser Thr Glu Asp Glu Asn Lys Leu Glu Cys Lys Cys         115 120 125 Leu Pro Asn Tyr Arg Gly Asp Gly Lys Tyr Cys Asp Pro Ile Asn Pro     130 135 140 Cys Leu Arg Lys Ile Cys His Pro His Ala His Cys Thr Tyr Leu Gly 145 150 155 160 Pro Asn Arg His Ser Cys Thr Cys Gln Glu Gly Tyr Arg Gly Asp Gly                 165 170 175 Gln val cys leu             180 <210> 107 <211> 225 <212> PRT <213> Homo sapiens <400> 107 Gln Pro Arg Cys Cys Pro Gly Arg Trp Gly Pro Asp Cys Ile Glu Cys   1 5 10 15 Pro Gly Gly Ala Gly Ser Pro Cys Asn Gly Arg Gly Ser Cys Ala Glu              20 25 30 Gly Met Glu Gly Asn Gly Thr Cys Ser Cys Gln Glu Gly Phe Gly Gly          35 40 45 Thr Ala Cys Glu Thr Cys Ala Asp Asp Asn Leu Phe Gly Pro Ser Cys      50 55 60 Ser Ser Val Cys Asn Cys Val His Gly Val Cys Asn Ser Gly Leu Asp  65 70 75 80 Gly Asp Gly Thr Cys Glu Cys Tyr Ser Ala Tyr Thr Gly Pro Lys Cys                  85 90 95 Asp Lys Pro Ile Pro Glu Cys Ala Ala Leu Leu Cys Pro Glu Asn Ser             100 105 110 Arg Cys Ser Pro Ser Thr Glu Asp Glu Asn Lys Leu Glu Cys Lys Cys         115 120 125 Leu Pro Asn Tyr Arg Gly Asp Gly Lys Tyr Cys Asp Pro Ile Asn Pro     130 135 140 Cys Leu Arg Lys Ile Cys His Pro His Ala His Cys Thr Tyr Leu Gly 145 150 155 160 Pro Asn Arg His Ser Cys Thr Cys Gln Glu Gly Tyr Arg Gly Asp Gly                 165 170 175 Gln Val Cys Leu Pro Val Asp Pro Cys Gln Ile Asn Phe Gly Asn Cys             180 185 190 Pro Thr Lys Ser Thr Val Cys Lys Tyr Asp Gly Pro Gly Gln Ser His         195 200 205 Cys Glu Cys Lys Glu His Tyr Gln Asn Phe Val Pro Gly Val Gly Cys     210 215 220 Ser 225 <210> 108 <211> 174 <212> PRT <213> Homo sapiens <400> 108 Glu Thr Cys Ala Asp Asp Asn Leu Phe Gly Pro Ser Cys Ser Ser Val   1 5 10 15 Cys Asn Cys Val His Gly Val Cys Asn Ser Gly Leu Asp Gly Asp Gly              20 25 30 Thr Cys Glu Cys Tyr Ser Ala Tyr Thr Gly Pro Lys Cys Asp Lys Pro          35 40 45 Ile Pro Glu Cys Ala Ala Leu Leu Cys Pro Glu Asn Ser Arg Cys Ser      50 55 60 Pro Ser Thr Glu Asp Glu Asn Lys Leu Glu Cys Lys Cys Leu Pro Asn  65 70 75 80 Tyr Arg Gly Asp Gly Lys Tyr Cys Asp Pro Ile Asn Pro Cys Leu Arg                  85 90 95 Lys Ile Cys His Pro His Ala His Cys Thr Tyr Leu Gly Pro Asn Arg             100 105 110 His Ser Cys Thr Cys Gln Glu Gly Tyr Arg Gly Asp Gly Gln Val Cys         115 120 125 Leu Pro Val Asp Pro Cys Gln Ile Asn Phe Gly Asn Cys Pro Thr Lys     130 135 140 Ser Thr Val Cys Lys Tyr Asp Gly Pro Gly Gln Ser His Cys Glu Cys 145 150 155 160 Lys Glu His Tyr Gln Asn Phe Val Pro Gly Val Gly Cys Ser                 165 170 <210> 109 <211> 185 <212> PRT <213> Homo sapiens <400> 109 Ile Pro Lys Cys Cys Lys Gly Phe Tyr Gly Pro Asp Cys Asn Gln Cys   1 5 10 15 Pro Gly Gly Phe Ser Asn Pro Cys Ser Gly Asn Gly Gln Cys Ala Asp              20 25 30 Ser Leu Gly Gly Asn Gly Thr Cys Ile Cys Glu Glu Gly Phe Gln Gly          35 40 45 Ser Gln Cys Gln Phe Cys Ser Asp Pro Asn Lys Tyr Gly Pro Arg Cys      50 55 60 Asn Lys Lys Cys Leu Cys Val His Gly Thr Cys Asn Asn Arg Ile Asp  65 70 75 80 Ser Asp Gly Ala Cys Leu Thr Gly Thr Cys Arg Asp Gly Ser Ala Gly                  85 90 95 Arg Leu Cys Asp Lys Gln Thr Ser Ala Cys Gly Pro Tyr Val Gln Phe             100 105 110 Cys His Ile His Ala Thr Cys Glu Tyr Ser Asn Gly Thr Ala Ser Cys         115 120 125 Ile Cys Lys Ala Gly Tyr Glu Gly Asp Gly Thr Leu Cys Ser Glu Met     130 135 140 Asp Pro Cys Thr Gly Leu Thr Pro Gly Gly Cys Ser Arg Asn Ala Glu 145 150 155 160 Cys Ile Lys Thr Gly Thr Gly Thr His Thr Cys Val Cys Gln Gln Gly                 165 170 175 Trp Thr Gly Asn Gly Arg Asp Cys Ser             180 185 <210> 110 <211> 228 <212> PRT <213> Homo sapiens <400> 110 Ile Pro Lys Cys Cys Lys Gly Phe Tyr Gly Pro Asp Cys Asn Gln Cys   1 5 10 15 Pro Gly Gly Phe Ser Asn Pro Cys Ser Gly Asn Gly Gln Cys Ala Asp              20 25 30 Ser Leu Gly Gly Asn Gly Thr Cys Ile Cys Glu Glu Gly Phe Gln Gly          35 40 45 Ser Gln Cys Gln Phe Cys Ser Asp Pro Asn Lys Tyr Gly Pro Arg Cys      50 55 60 Asn Lys Lys Cys Leu Cys Val His Gly Thr Cys Asn Asn Arg Ile Asp  65 70 75 80 Ser Asp Gly Ala Cys Leu Thr Gly Thr Cys Arg Asp Gly Ser Ala Gly                  85 90 95 Arg Leu Cys Asp Lys Gln Thr Ser Ala Cys Gly Pro Tyr Val Gln Phe             100 105 110 Cys His Ile His Ala Thr Cys Glu Tyr Ser Asn Gly Thr Ala Ser Cys         115 120 125 Ile Cys Lys Ala Gly Tyr Glu Gly Asp Gly Thr Leu Cys Ser Glu Met     130 135 140 Asp Pro Cys Thr Gly Leu Thr Pro Gly Gly Cys Ser Arg Asn Ala Glu 145 150 155 160 Cys Ile Lys Thr Gly Thr Gly Thr His Thr Cys Val Cys Gln Gln Gly                 165 170 175 Trp Thr Gly Asn Gly Arg Asp Cys Ser Glu Ile Asn Asn Cys Leu Leu             180 185 190 Pro Ser Ala Gly Gly Cys His Asp Asn Ala Ser Cys Leu Tyr Val Gly         195 200 205 Pro Gly Gln Asn Glu Cys Glu Cys Lys Lys Gly Phe Arg Gly Asn Gly     210 215 220 Ile Asp Cys Glu 225 <210> 111 <211> 177 <212> PRT <213> Homo sapiens <400> 111 Gln Phe Cys Ser Asp Pro Asn Lys Tyr Gly Pro Arg Cys Asn Lys Lys   1 5 10 15 Cys Leu Cys Val His Gly Thr Cys Asn Asn Arg Ile Asp Ser Asp Gly              20 25 30 Ala Cys Leu Thr Gly Thr Cys Arg Asp Gly Ser Ala Gly Arg Leu Cys          35 40 45 Asp Lys Gln Thr Ser Ala Cys Gly Pro Tyr Val Gln Phe Cys His Ile      50 55 60 His Ala Thr Cys Glu Tyr Ser Asn Gly Thr Ala Ser Cys Ile Cys Lys  65 70 75 80 Ala Gly Tyr Glu Gly Asp Gly Thr Leu Cys Ser Glu Met Asp Pro Cys                  85 90 95 Thr Gly Leu Thr Pro Gly Gly Cys Ser Arg Asn Ala Glu Cys Ile Lys             100 105 110 Thr Gly Thr Gly Thr His Thr Cys Val Cys Gln Gln Gly Trp Thr Gly         115 120 125 Asn Gly Arg Asp Cys Ser Glu Ile Asn Asn Cys Leu Leu Pro Ser Ala     130 135 140 Gly Gly Cys His Asp Asn Ala Ser Cys Leu Tyr Val Gly Pro Gly Gln 145 150 155 160 Asn Glu Cys Glu Cys Lys Lys Gly Phe Arg Gly Asn Gly Ile Asp Cys                 165 170 175 Glu     <210> 112 <211> 180 <212> PRT <213> Homo sapiens <400> 112 Thr Arg Glu Cys Cys Ala Gly Phe Phe Gly Pro Gln Cys Gln Pro Cys   1 5 10 15 Pro Gly Asn Ala Gln Asn Val Cys Phe Gly Asn Gly Ile Cys Leu Asp              20 25 30 Gly Val Asn Gly Thr Gly Val Cys Glu Cys Gly Glu Gly Phe Ser Gly          35 40 45 Thr Ala Cys Glu Thr Cys Thr Glu Gly Lys Tyr Gly Ile His Cys Asp      50 55 60 Gln Ala Cys Ser Cys Val His Gly Arg Cys Asn Gln Gly Pro Leu Gly  65 70 75 80 Asp Gly Ser Cys Asp Cys Asp Val Gly Trp Arg Gly Val His Cys Asp                  85 90 95 Asn Ala Thr Thr Glu Asp Asn Cys Asn Gly Thr Cys His Thr Ser Ala             100 105 110 Asn Cys Leu Thr Asn Ser Asp Gly Thr Ala Ser Cys Lys Cys Ala Ala         115 120 125 Gly Phe Gln Gly Asn Gly Thr Ile Cys Thr Ala Ile Asn Ala Cys Glu     130 135 140 Ile Ser Asn Gly Gly Cys Ser Ala Lys Ala Asp Cys Lys Arg Thr Thr 145 150 155 160 Pro Gly Arg Arg Val Cys Thr Cys Lys Ala Gly Tyr Thr Gly Asp Gly                 165 170 175 Ile val cys leu             180 <210> 113 <211> 222 <212> PRT <213> Homo sapiens <400> 113 Thr Arg Glu Cys Cys Ala Gly Phe Phe Gly Pro Gln Cys Gln Pro Cys   1 5 10 15 Pro Gly Asn Ala Gln Asn Val Cys Phe Gly Asn Gly Ile Cys Leu Asp              20 25 30 Gly Val Asn Gly Thr Gly Val Cys Glu Cys Gly Glu Gly Phe Ser Gly          35 40 45 Thr Ala Cys Glu Thr Cys Thr Glu Gly Lys Tyr Gly Ile His Cys Asp      50 55 60 Gln Ala Cys Ser Cys Val His Gly Arg Cys Asn Gln Gly Pro Leu Gly  65 70 75 80 Asp Gly Ser Cys Asp Cys Asp Val Gly Trp Arg Gly Val His Cys Asp                  85 90 95 Asn Ala Thr Thr Glu Asp Asn Cys Asn Gly Thr Cys His Thr Ser Ala             100 105 110 Asn Cys Leu Thr Asn Ser Asp Gly Thr Ala Ser Cys Lys Cys Ala Ala         115 120 125 Gly Phe Gln Gly Asn Gly Thr Ile Cys Thr Ala Ile Asn Ala Cys Glu     130 135 140 Ile Ser Asn Gly Gly Cys Ser Ala Lys Ala Asp Cys Lys Arg Thr Thr 145 150 155 160 Pro Gly Arg Arg Val Cys Thr Cys Lys Ala Gly Tyr Thr Gly Asp Gly                 165 170 175 Ile Val Cys Leu Glu Ile Asn Pro Cys Leu Glu Asn His Gly Gly Cys             180 185 190 Asp Lys Asn Ala Glu Cys Thr Gln Thr Gly Pro Asn Gln Ala Ala Cys         195 200 205 Asn Cys Leu Pro Ala Tyr Thr Gly Asp Gly Lys Val Cys Thr     210 215 220 <210> 114 <211> 171 <212> PRT <213> Homo sapiens <400> 114 Glu Thr Cys Thr Glu Gly Lys Tyr Gly Ile His Cys Asp Gln Ala Cys   1 5 10 15 Ser Cys Val His Gly Arg Cys Asn Gln Gly Pro Leu Gly Asp Gly Ser              20 25 30 Cys Asp Cys Asp Val Gly Trp Arg Gly Val His Cys Asp Asn Ala Thr          35 40 45 Thr Glu Asp Asn Cys Asn Gly Thr Cys His Thr Ser Ala Asn Cys Leu      50 55 60 Thr Asn Ser Asp Gly Thr Ala Ser Cys Lys Cys Ala Ala Gly Phe Gln  65 70 75 80 Gly Asn Gly Thr Ile Cys Thr Ala Ile Asn Ala Cys Glu Ile Ser Asn                  85 90 95 Gly Gly Cys Ser Ala Lys Ala Asp Cys Lys Arg Thr Thr Pro Gly Arg             100 105 110 Arg Val Cys Thr Cys Lys Ala Gly Tyr Thr Gly Asp Gly Ile Val Cys         115 120 125 Leu Glu Ile Asn Pro Cys Leu Glu Asn His Gly Gly Cys Asp Lys Asn     130 135 140 Ala Glu Cys Thr Gln Thr Gly Pro Asn Gln Ala Ala Cys Asn Cys Leu 145 150 155 160 Pro Ala Tyr Thr Gly Asp Gly Lys Val Cys Thr                 165 170 <210> 115 <211> 176 <212> PRT <213> Homo sapiens <400> 115 Ile Pro Arg Cys Cys Lys Gly Tyr Phe Gly Arg Asp Cys Gln Ala Cys   1 5 10 15 Pro Gly Gly Pro Asp Ala Pro Cys Asn Asn Arg Gly Val Cys Leu Asp              20 25 30 Gln Tyr Ser Ala Thr Gly Glu Cys Lys Cys Asn Thr Gly Phe Asn Gly          35 40 45 Thr Ala Cys Glu Met Cys Trp Pro Gly Arg Phe Gly Pro Asp Cys Leu      50 55 60 Pro Cys Gly Cys Ser Asp His Gly Gln Cys Asp Asp Gly Ile Thr Gly  65 70 75 80 Ser Gly Gln Cys Leu Cys Glu Thr Gly Trp Thr Gly Pro Ser Cys Asp                  85 90 95 Thr Gln Ala Val Leu Pro Ala Val Cys Thr Pro Pro Cys Ser Ala His             100 105 110 Ala Thr Cys Lys Glu Asn Asn Thr Cys Glu Cys Asn Leu Asp Tyr Glu         115 120 125 Gly Asp Gly Ile Thr Cys Thr Val Val Asp Phe Cys Lys Gln Asp Asn     130 135 140 Gly Gly Cys Ala Lys Val Ala Arg Cys Ser Gln Lys Gly Thr Lys Val 145 150 155 160 Ser Cys Ser Cys Gln Lys Gly Tyr Lys Gly Asp Gly His Ser Cys Thr                 165 170 175 <210> 116 <211> 168 <212> PRT <213> Homo sapiens <400> 116 Glu Met Cys Trp Pro Gly Arg Phe Gly Pro Asp Cys Leu Pro Cys Gly   1 5 10 15 Cys Ser Asp His Gly Gln Cys Asp Asp Gly Ile Thr Gly Ser Gly Gln              20 25 30 Cys Leu Cys Glu Thr Gly Trp Thr Gly Pro Ser Cys Asp Thr Gln Ala          35 40 45 Val Leu Pro Ala Val Cys Thr Pro Pro Cys Ser Ala His Ala Thr Cys      50 55 60 Lys Glu Asn Asn Thr Cys Glu Cys Asn Leu Asp Tyr Glu Gly Asp Gly  65 70 75 80 Ile Thr Cys Thr Val Val Asp Phe Cys Lys Gln Asp Asn Gly Cys                  85 90 95 Ala Lys Val Ala Arg Cys Ser Gln Lys Gly Thr Lys Val Ser Cys Ser             100 105 110 Cys Gln Lys Gly Tyr Lys Gly Asp Gly His Ser Cys Thr Glu Ile Asp         115 120 125 Pro Cys Ala Asp Gly Leu Asn Gly Gly Cys His Glu His Ala Thr Cys     130 135 140 Lys Met Thr Gly Pro Gly Lys His Lys Cys Glu Cys Lys Ser His Tyr 145 150 155 160 Val Gly Asp Gly Leu Asn Cys Glu                 165  

Claims (11)

The amino acid sequence selected from the group consisting of SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 96, and SEQ ID NO: 102 of the EGF-like domain repeat protein of stabilin-2 Tumorous comprising a second atypical EGF-like domain and 3 to 10 EGF-like domains sequentially represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 105 as an active ingredient Composition for drug delivery specific to the disease. delete delete delete delete The composition of claim 1, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 75 to SEQ ID NO: 82 and SEQ ID NO: 106 to SEQ ID NO: 116. delete According to claim 1, The neoplastic disease is colon cancer, lung cancer, gastric cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, chorionic cancer, ovarian cancer, breast cancer, thyroid cancer , Brain cancer, head and neck cancer, malignant melanoma, skin cancer, liver cancer, leukemia, lymphoma, multiple myeloma, chronic myelogenous leukemia, neuroblastoma, aplastic anemia Compositions selected from the group consisting of. The method of claim 1, wherein the polypeptide is paclitaxel, doxorubicin, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, docetaxel, etoposide ), Teniposide, bisantrene, homoharringtonine, Gleevec (STI-571), cisplain, 5-fluouracil, adriamycin ), Methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, nitrosourea A composition characterized in that it is combined with an anti-tumor disease agent selected from the group consisting of. A pharmaceutical composition for the prevention and treatment of tumor diseases, comprising the drug delivery composition of claim 1 and an anti-tumor disease agent coupled thereto as an active ingredient. 11. The method of claim 10, wherein the anti-tumor disease agent is paclitaxel, doxorubicin, vincristine, daunorubicin, vinblastine, actinomycin-D (actinomycin-D), docetaxel (docetaxel) , Etoposide, teniposide, bisantrene, homoharringtonine, gleevec (STI-571), cisplain, 5-fluouracil , Adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, nitroso Urea (nitrosourea) composition, characterized in that selected from the group consisting of.

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