KR101216379B1 - Screening methods for candidate materials having a prophylactic or treating activity of multiple sclerosis - Google Patents

Abstract

The present invention comprises the steps of (a) treating the candidate substance to a protein (DRG2) having the amino acid sequence of SEQ ID NO: 1; And (b) measuring the change in activity of DRG2 obtained in step (a) and selecting a substance that increases the activity of DRG2, the method of screening for a substance having a prophylactic or therapeutic activity of multiple sclerosis. ; Or (a ') treating and culturing the candidate substance in cells overexpressing DRG2; And (b ') measuring the amount of DRG2 produced in the culture in step (a') and selecting a substance that increases the amount of DRG2 produced, the method of screening a substance having a prophylactic or therapeutic activity of multiple sclerosis. To provide. In addition, the present invention is a diagnostic kit for multiple sclerosis comprising a molecule capable of measuring the expression amount of a polynucleotide encoding DRG2, antibody, substrate, ligand, or cofactor (which the molecule specifically binds to the protein) cofactor); Or it provides a kit for diagnosing multiple sclerosis, which is a primer having a complementary sequence specific to the gene encoding the protein.

DRG2, multiple sclerosis

Description

Screening methods for candidate materials having a prophylactic or treating activity of multiple sclerosis}

The present invention relates to a method for screening a substance having a prophylactic or therapeutic activity for multiple sclerosis. The present invention also relates to a kit for diagnosing multiple sclerosis.

GTP-binding protein (G protein) is a protein involved in various regulation in the cell, such as trimeric G protein (e.g. transducin), monomeric G protein (e.g. ras), and GTPase (eg, elongation factor Tu), which is involved in protein synthesis, are known to exist in three important subtypes.

Recently, new types of G proteins with different sequencing and functions have been reported. Among these, the developmentally regulated GTP-binding protein (DRG) has all five sites from G1 to G5 required for binding to GTP, and is a trimmer in size. Similar to Rick G protein. However, the amino acid sequence is classified as a new subtype because it has completely different characteristics from existing G protein subtypes as well as trimeric G protein.

DRG also has DRG1 and DRG2 that differ in sequence and function (Schenker & Trueb (1997) Cytogenet. Cell Genet . 79, 274-275) and are present in almost all types of cells, from bacteria to humans. DRG proteins are evolutionarily highly conserved, for example, the human DRG2 amino acid sequence is 98% with mouse and bovine DRG2, 95% with Xenopus DRG2, and zebrafish DRG2 with 91% homology. The fact that these proteins are common to various cell types and is an evolutionarily conserved protein suggests that DRG proteins play an important role in regulating cell growth and differentiation.

DRG proteins have been discovered relatively recently and most of them are accidentally discovered during genomic DNA or cDNA cloning, so their function is still unknown. In mice and Xenopus, DRG protein was observed to increase in the early stages of differentiation of the brain and decrease in expression as the differentiation progressed (Sazuka et al., (1992a) Biochem. Biophys. Res. Commun. 189 , 363-370 and Sazuka et al., (1992b) Biochem.Biophys.Res.Commun. 189, 371-377), in humans expressed in normal fibroblasts and then in fibroblasts transformed by SV40 The phenomenon of decreased expression was confirmed (Schenker et al ., (1994) J. Biol. Chem. 269, 25447-25453).

We have reported an increase in the expression of DRG2 in fish cells infected with rhabdovirus when the virus begins to be released out of the cell (Lee et al ., (1998) FEBS Lett 429, 407-411 In addition, it has been reported that the cell cycle of Jurkat cells, which are human T cells overexpressing DRG2, is inhibited in the G2 / M phase (Song et al., (2004) J. Biochem . 135, 331). -335). In addition, it has been reported that the inhibition of the cell cycle inhibits the growth of cells while reducing the apoptosis of the cells (Ko MS et al. (2004) Arch. Biochem. Biophys . 422, 137-144 ). These results suggest that DRG2 is likely to play an important role in cell differentiation by inhibiting the cell cycle of cells and inhibiting apoptosis. In addition, the present inventors have prepared a transgenic mouse (TG) overexpressing DRG2 and using the obtained DRG2-overexpressing TG to analyze the DRG2 function in thymic differentiation, especially in estrogen-induced thymic atrophy. It has been reported that DRG2 is involved in T-cell differentiation of the thymus (Korean Society for Molecular and Cell Biology Fall Conference, F-110, October 2006).

Multiple sclerosis (MS), on the other hand, is an inflammatory demyelinating disorder in the central nervous system (CNS) that involves sensory, motor, and cognitive impairments (Ransohoff, RM (2006) Trends Immunol. 27: 167-168. Although multiple sclerosis has not yet been accurately identified at the cellular or molecular level, experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has been developed and developed by many researchers over the past 80 years.

EAE is an adjuvant for myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG), a kind of myelin antigens. by injection with adjuvants (Zamvil, SS, et al. (1986) Nature. 324 : 258-260; Sobel, RA, et al. (1994) Neurochem. Res. 19 : 915-921; Mendel, I., et al. (1995) Eur. J. Immunol. 25 : 1951-1959). EAE is an autoimmune disease induced by myelin antigen specific Th-17 cells (Langrish, CL, et al. (2005) J. Exp. Med. 201 : 233-240; Park, H., et al. ( Nat.Imunmunol . 6 : 1133-1141).

Chemokines and cytokines, which play important roles in EAE pathology, have been reported using genetically-deficient or transgenic mice, and have been reported to immune cells infiltrating the central nervous system and to surrounding glia cells. Inflammatory cytokines produced by (TNF, IL1-1β, Fas ligand, and IFN-γ) and chemokines (MCP-1, MIP-1α, Mig, IP-10 and RANTES) are organized and in pathological order Inflammation, demyelination and axsonal damage have been reported (Lock, C. et al. (2002) Nat. Med. 8: 500-508; Ibrahim, SM et. al. (2001) Brain 124: 1927-1938).

While the present inventors have conducted various studies to discover the function of DRG2, surprisingly, when overexpressing DRG2, IL-17 expression of immune cells is reduced, resulting in inhibition of immune cell penetration into the spinal cord, resulting in demyelination. This inhibition was found to alleviate the symptoms of EAE. This indicates that when DRG2 is overexpressed, the occurrence of multiple sclerosis can be suppressed, and the incidence of multiple sclerosis can be diagnosed by measuring the amount of DRG2 expression in vivo.

Accordingly, the present invention provides a method for screening a substance capable of increasing the activity of DRG2 or increasing the amount of DRG2 expression (ie, the amount of production) in a cell overexpressing DRG2, that is, a substance having a prophylactic or therapeutic activity for multiple sclerosis. It aims to do it.

Another object of the present invention is to provide a kit for diagnosing multiple sclerosis comprising a molecule capable of measuring the expression level of a polynucleotide encoding DRG2.

According to one aspect of the invention, there is provided a method for treating a protein having an amino acid sequence of SEQ ID NO: 1; And (b) measuring a change in activity of the protein obtained in step (a) and selecting a substance that increases the activity of the protein, the method of screening a substance having a prophylactic or therapeutic activity of multiple sclerosis. This is provided.

According to another aspect of the present invention, there is provided a method of treating a cell comprising (a ') treating and culturing a candidate substance in a cell that overexpresses a protein having the amino acid sequence of SEQ ID NO: 1; And (b ') measuring the amount of protein produced in the culture in step (a') and selecting a substance that increases the amount of protein produced. Screening of a substance having a prophylactic or therapeutic activity for multiple sclerosis A method is provided.

According to another aspect of the present invention, there is provided a diagnostic kit for multiple sclerosis comprising a molecule capable of measuring the amount of expression of a polynucleotide encoding a protein having an amino acid sequence of SEQ ID NO: 1, wherein said molecule is specific for said protein. Binding antibody, substrate, ligand, or cofactor; Or there is provided a kit for diagnosing multiple sclerosis, which is a primer having a complementary sequence specific for the gene encoding the protein.

When DRG2, which is one of the subtypes of GPT-binding protein, is overexpressed by the present invention, IL-17 expression of immune cells is reduced, and as a result, immune cell infiltration into the spinal cord is inhibited and demyelination is inhibited, thereby causing multiple sclerosis. It has been found that the occurrence of can be suppressed. Therefore, according to the screening method of the present invention, it is possible to search for a substance capable of increasing the activity of DRG2 or increasing the expression (or production) of DRG2, that is, a candidate substance capable of preventing or treating multiple sclerosis. In addition, when measuring the amount of DRG2 expression in a sample isolated from the patient (for example, blood, hair, etc.), multiple sclerosis can be diagnosed.

In the present specification, multiple sclerosis (MS) is an inflammatory demyelinating disorder that occurs in the central nervous system (CNS) and refers to a disease involving sensory, motor, and cognitive impairment. And autoimmune encephalomyelitis.

In order to elucidate the role of DRG2 in cell differentiation, the present inventors prepared transgenic mice (DRG2 TG mice) overexpressing DRG2 and analyzed the changes caused by DRG2 overexpression. Surprisingly, analysis of the effect of DRG2 overexpression on EAE generation induced by MOG treatment revealed that EAE development was significantly suppressed. EAE is induced by Th-17 cells that secrete IL-17. In DRG2 TG mice, the secretion of IL-17 in immune cells was significantly reduced. This indicates that DRG2 inhibits the development of EAE by inhibiting the differentiation of Th-17 cells. In addition, the results indicate that EAE can be predicted (ie diagnosed) based on the expression level of DRG2, and screening for substances that increase DRG2 expression or activity may be used to search for substances for the prevention or treatment of multiple sclerosis. That means you can.

The screening method of the present invention includes a process of selecting a substance that increases the activity of DRG2 by treating DRG2 or a cell overexpressing DRG2 with a candidate, and measuring the change in the activity of DRG2 or the amount of DRG2 produced from the cell. The amino acid sequence and base sequence of DRG2 are known from GenBank (accession number NM_001388), and each sequence is as SEQ ID NO: 1 and 2.

The present invention comprises the steps of (a) treating a candidate substance to a protein having the amino acid sequence of SEQ ID NO: 1; And (b) measuring a change in the activity of the protein obtained in step (a) and selecting a substance that increases the activity of the protein, the method for screening a substance having a prophylactic or therapeutic activity for multiple sclerosis.

Treatment of the candidate with DRG2 may be performed by dispersing DRG2 in a suitable medium (eg, PBS, etc.) and then adding the candidate.

After treatment with the candidate substance, measurement of DRG2 activity change can be used without limitation as long as it can measure DRG2 activity. See, eg, Sazuka, T. et al., "Expression of DRG During Murine Embryonic Development", Biochem. Biophys., Res. DRG2 activity can be measured by GPT-binding assay known in Comm., 189: 371-377 (1992) and the like. Specifically, purified DRG2 is bound to a binding buffer (eg, 20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 5 mM MgCl ₂ , 2 mM MnCl ₂ , 1 mM dithiothreitol, 10 uM). Incubated with [ ³² P] -GPT in ATP) and then irradiated with UV for about 30 minutes in ice. The obtained sample was incubated with anti-DRG2 antiserum, then incubated with protein A-Sepharose purified resin on ice, and protein A-antibody complexes were subjected to TNE buffer (10 mM Tris-HCL, pH 7.8, 1% as needed). Changes in DRG2 activity can be determined by washing with NP-40, 150 mN NaCl, 1 mM EDTA, 10 ug / ml aprotinin), and then electrophoresing on SDS-polyacrylamide gels.

In addition, the present invention comprises the steps of (a ') treating and culturing a candidate substance in cells overexpressing a protein having the amino acid sequence of SEQ ID NO: 1; And (b ') measuring the amount of the protein produced in the culture in step (a') and selecting a substance that increases the amount of the protein produced, the screening of the material having the prophylactic or therapeutic activity of multiple sclerosis. It includes a method.

The cell overexpressing DRG2 may be a cell transformed with a vector comprising a polynucleotide having a nucleotide sequence of SEQ ID NO: 2. Cells transformed with a vector comprising a polynucleotide having the nucleotide sequence of SEQ ID NO: 2 are described in the previous papers of the present inventors, ie Ko MS et al. (2004) Arch. Biochem. Biophys . 422, 137-144 and Song H et al. (2004) J. Biochem . The cells disclosed in 135, 331-335, ie Jurkat-LNCX2-DRG2, can be used. The cell is a cell transformed with Jurkat cells with a pLNCX2-DRG2 vector prepared by inserting the DRG2 gene of SEQ ID NO: 2 into a pLNCX2 vector. Those skilled in the art will appreciate that the DRG2 gene and Using pLNCX2 vector, etc., Jurkat-LNCX2-DRG2 can be produced without any difficulty.

After the candidate material is treated with the cells overexpressing DRG2, the culturing step may be carried out under conventional Jurkat cell culture conditions, and preferably, the culture conditions disclosed in the preceding articles of the present inventors, for example, 10%. It can be performed in Dulbecco modified Eagle medium (DMEM) medium supplemented with fetal bovine serum and 100 μg / ml penicillin-streptomycin.

Measuring the amount of DRG2 produced in the culture and selecting a substance that increases the amount of DRG2 produced (ie, step (b ')) can be used without limitation as long as the method can measure the amount of produced DRG2, for example It may also be carried out by the GTP-binding assay described above.

According to another aspect of the present invention, there is provided a diagnostic kit for multiple sclerosis comprising a molecule capable of measuring the expression level of a polynucleotide encoding a protein having an amino acid sequence of SEQ ID NO: 1, wherein the molecule is specifically bound to the protein. Antibodies, substrates, ligands, or cofactors; Or there is provided a kit for diagnosing multiple sclerosis, which is a primer having a complementary sequence specific for the gene encoding the protein.

The polynucleotide encoding the protein having the amino acid sequence of SEQ ID NO: 1 (that is, DRG2) preferably has the nucleotide sequence of SEQ ID NO: 2.

Examples of molecules capable of measuring the expression level of the gene encoding DRG2 include antibodies, substrates, ligands, or cofactors that specifically bind to the protein; Or a primer having a complementary sequence specific for the gene encoding the protein.

According to methods commonly used in the field of biotechnology, polyclonal antibodies or monoclonal antibodies that specifically recognize DRG2 may be prepared, and diagnostic kits containing the antibodies may be prepared. The kits of the invention can also be constructed to include substrates (eg, GTP), ligands, or cofactors for DRG2. In addition, a primer having a complementary sequence specific for a gene encoding DRG2 (eg, a gene having a nucleotide sequence of SEQ ID NO: 2) may be prepared according to a method commonly used in the field of biotechnology. Diagnostic kits may also be prepared.

In the diagnostic kit of the present invention, a molecule capable of measuring the expression level of the gene encoding DRG2 may be labeled with a detectable label (eg, a chromophore, etc.). In addition, the diagnostic kit of the present invention may be in the form of a microarray in which the primer or a probe made from the primer is immobilized on a substrate, that is, a chip such as a DNA chip.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are intended to illustrate the invention and should not be construed as limiting the invention.

<Materials and Test Methods>

1. Preparation of DRG2 Overexpressing Mouse

A cDNA synthesized from mRNA of mice C57BL6 splenocytes (spleen) as a template that contains a BamHI restriction enzyme mDRG2-U that contains the site (5'-CGC GGATCC ACTATGGGGATCTTGGA-3 ', SEQ ID NO: 3), and the XhoI restriction site The primers of mDRG2-D (5′-CCG CTCGAG CTACTTCTTCACAATCTGGA-3 ′, SEQ ID NO: 4) were used to amplify mouse DRG2 cDNA (Genbank accession number BC082564). The amplified PCR product was cut into BamHI and XhoI, and then subcloned into the BamHI / XhoI site of pcDNA3 (Invitrogen), a plasmid pcDNA / having a structure of CMV (cytomegalovirus) promoter-T7 promoter-mouse DRG2 cDNA-SP6 promoter-BGH polyA mDRG2 was constructed (see FIG. 1A). Using the plasmid construct, DRG2 overexpressing mice were commissioned by Macrogen Co., Ltd. to produce as follows. The plasmid construct was injected into the fertilized male pronucleus of C57BL mice and then transplanted into C57BL / 6 surrogate mothers. 58 mouse tails were cut to extract DNA, and then T7 primer (5'-AATACGACTCACTATAGGGA-3 ', SEQ ID NO: 5), a pcDNA specific primer, and mDRG2-694D (5'-TGTATTCATGTAGGATGAGC-3', a mouse DRG2 specific primer) were extracted. PCR was performed using SEQ ID NO: 6) (FIG. 1A). As a result, as shown in Figure 1B 8, 33, 38, 45, 48, 50 mice were confirmed to have a DRG2 transgene. CDNA was synthesized from their spleen mRNA, followed by mouse DRG2-specific PCR primers (mDRG2-570U, 5'-CTCAACAGTCACACTGACAC-3 '(SEQ ID NO: 7); mDRG2-817D, 5'-TACCGCAACTGATAACTACA-3' (SEQ ID NO: 8)). As a result of confirming DRG2 expression, it was confirmed that DRG2 expression was increased in 45 and 50 mice (Fig. 1C), and thus, 45 and 50 were mated with C57BL mice and used in subsequent studies.

2. EAE Induction

DRG2 at 8-12 weeks of age was used for complete freund's adjuvant and 5 μg MOG (35-55) (MEVGWYRSPFSRVVHLYRNGK) peptides per mouse in DRG2 transgenic mice and wild-type mice. mg / ml of Mycobacterium tuberculosis H37RA was mixed and injected subcutaneously. On the same day and two days after MOG (35-55) injection, 200 ng of pertussis toxin per horse was diluted in 100 μl of phosphate buffered saline (PBS) and injected into the abdominal cavity of mice.

3. Histology and Immunohistochemistry

After 17 days of injection of MOG (35-55), spinal cords were isolated from rats, fixed with 10% neutral buffered formalin, embedded with paraffin, and 5 μm thick sections were prepared. Hematotoxin and eosin staining and Luxol fast blue staining confirmed the extent of spinal cord lesions.

4. Cytokine Expression Analysis

After 14 days of injection of MOG (35-55), cells were isolated from the spleen of the mouse and the cells were dispensed into 96-well cell culture plates, containing 0-100 μg / ml MOG (35-55) peptide with 10% FBS. Was added to RPMI-1640 medium and incubated for 48 hours. After harvesting the cell culture, the expression difference of IL-17 was confirmed by DuoSet ELISA development systems (R & D Systems), and the experimental method was carried out according to the protocol determined by each manufacturer.

Results and Discussion

1. Clinical score

MOG was injected into females (F) and males (M) of normal mice (WT) and DRG2 TG mice to determine whether DRG2 overexpression altered the immune response. Since then, the clinical score was analyzed at regular time intervals. The results are shown in Table 1 and FIG.

Genotype attack Onset day Maximum incidence index
Maximum clinical score WT male 5 out of 5 (100%) 13.4 2.6 ± 0.245 WT female 5 out of 5 (100%) 18.0 2.4 ± 0.307 DRG2TG male 4 out of 5 (80%) 16.3 0.83 ± 0.092 DRG2TG female 2 out of 5 (40%) 17.5 0.4 ± 0.063

As can be seen from the results of Table 1, both the female (0.4 ± 0.063) and male (0.83 ± 0.092) DRG2 overexpressing mice had a significantly higher incidence index (female, 2.4 ± 0.307; male, 2.6 ± 0.245) than in normal mice. Decreased. In addition, in the incidence frequency of normal mice, male and female were 100%, while DRG2 TG mice were reduced to 40% of females and 80% of males.

2. Analysis by H & E staining

Multiple sclerosis and EAE are autoimmune diseases caused by the removal of myelin sheath, which surrounds axons of nerve cells, under the attack of immune cells. In other words, an inflammatory reaction occurs in the central nervous system, causing immune cells to infiltrate the nerve cells, and the immune cells gathered around the nerve cells attack and destroy myelin sheath, which surrounds the axons of the nerve cells. H & E staining to see the infiltration of immune cells in the spinal cord by separating spinal cord from each mouse 17 days after EAE induction to determine whether EAE reduction in DRG2 TG mice was due to immune cell invasion differences around neurons. Was done. The result is shown in FIG. 3. As can be seen in Figure 3, EAE lesions did not appear in both normal mice and DRG2 TG mice when EAE was not induced, but a large number of immune cells invaded the spinal cord when EAE was induced. In DRG2 TG mice, however, the number of immune cells invading the spinal cord was significantly reduced.

3. Analysis by Luxol fast blue staining

Luxol fast blue staining was performed on sections of the spinal cord as described above to determine whether the number of immune cells collected around the neurons of the DRG2 TG rat spinal cord was suppressed as a result of demyelinating. The degree of demyelination was observed. The result is shown in FIG. 4. As can be seen in Figure 4, in the case of normal mice severe demyelination occurred by the MOG treatment, while in the DRG2 TG mice dehydration phenomenon was hardly observed.

4. Cytokine Analysis

Inflammatory response by IL-17 is known to be a major cause of both human multiple sclerosis and its animal model EAE. Therefore, in the case of DRG2 TG mice, immune cells were extracted from lymph nodes and spleen of DRG2 TG mice and normal mice, and then treated with MOG in order to determine whether there was a change in IL-17 expression. It was. Subsequently, proliferation of immune cells was analyzed through [3H] thymidine incorporation assay, and the amount of IL-17 present in the culture medium was analyzed by ELISA. The result is shown in FIG. From Figure 5, it can be seen that the proliferation of T cells and IL-17 expression in the splenocytes of DRG2.

The above results indicate that overexpression of DRG2 decreases IL-17 expression of immune cells. As a result, the infiltration of immune cells into the spinal cord is inhibited and demyelination is inhibited, thereby alleviating EAE symptoms. Therefore, when DRG2 is overexpressed, the occurrence of multiple sclerosis can be suppressed, and the incidence of multiple sclerosis can be diagnosed by measuring the amount of DRG2 expression in vivo.

1A shows the DRG2 plasmid construct used to construct DRG2 overexpressing mice. T7 primers and mDRG2-D694 primers were used for PCR to identify DRG2 transgene present in the DNA of mice, and mDRG2-U570 and mDRG2-D817 primers were used for RT-PCR for mRNA expression analysis of DRG2 in mouse cells. Figure 1B shows the results of PCR using T7 and mDRG2-694D primers on DNA of 58 mouse tails born from C57BL / 6 surrogate mothers implanted with fertilized eggs. Arrows indicate PCR products of DRG2. FIG. 1C shows the results of cDNA synthesis from spleen and thymus mRNA of DRG2 overexpressing mouse clone # 45, followed by RT-PCR using mDRG2-570U and mDRG2-817D primers.

FIG. 2 shows the clinical scores of female mice (F) and males (M) in normal mice (WT) and DRG2 TG mice after injection of MOG to determine whether the DRG2 overexpression changes in immune response. ) Is the result of analysis.

Figure 3 shows the result of H & E staining of immune cells in the spinal cord by separating spinal cord from normal mice and DRG2 TG mice 17 days after EAE induction.

Figure 4 shows the results of spinal cord separation from normal mice and DRG2 TG mice 17 days after EAE induction, and luxol fast blue staining of immune cells in the spinal cord.

5 is a result of analyzing the proliferation (A) and the amount of IL-17 (B) of splenocytes of DRG2 TG mice and normal mice.

<110> University of Ulsan Foundation For Industry Cooperation <120> Screening methods for candidate materials having a prophylactic          or treating activity of multiple sclerosis <130> PN0248 <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 364 <212> PRT <213> Homo sapiens <400> 1 Met Gly Ile Leu Glu Lys Ile Ser Glu Ile Glu Lys Glu Ile Ala Arg   1 5 10 15 Thr Gln Lys Asn Lys Ala Thr Glu Tyr His Leu Gly Leu Leu Lys Ala              20 25 30 Lys Leu Ala Lys Tyr Arg Ala Gln Leu Leu Glu Pro Ser Lys Ser Ala          35 40 45 Ser Ser Lys Gly Glu Gly Phe Asp Val Met Lys Ser Gly Asp Ala Arg      50 55 60 Val Ala Leu Ile Gly Phe Pro Ser Val Gly Lys Ser Thr Phe Leu Ser  65 70 75 80 Leu Met Thr Ser Thr Ala Ser Glu Ala Ala Ser Tyr Glu Phe Thr Thr                  85 90 95 Leu Thr Cys Ile Pro Gly Val Ile Glu Tyr Lys Gly Ala Asn Ile Gln             100 105 110 Leu Leu Asp Leu Pro Gly Ile Ile Glu Gly Ala Ala Gln Gly Lys Gly         115 120 125 Arg Gly Arg Gln Val Ile Ala Val Ala Arg Thr Ala Asp Val Ile Ile     130 135 140 Met Met Leu Asp Ala Thr Lys Gly Glu Val Gln Arg Ser Leu Leu Glu 145 150 155 160 Lys Glu Leu Glu Ser Val Gly Ile Arg Leu Asn Lys His Lys Pro Asn                 165 170 175 Ile Tyr Phe Lys Pro Lys Lys Gly Gly Gly Ile Ser Phe Asn Ser Thr             180 185 190 Val Thr Leu Thr Gln Cys Ser Glu Lys Leu Val Gln Leu Ile Leu His         195 200 205 Glu Tyr Lys Ile Phe Asn Ala Glu Val Leu Phe Arg Glu Asp Cys Ser     210 215 220 Pro Asp Glu Phe Ile Asp Val Ile Val Gly Asn Arg Val Tyr Met Pro 225 230 235 240 Cys Leu Tyr Val Tyr Asn Lys Ile Asp Gln Ile Ser Met Glu Glu Val                 245 250 255 Asp Arg Leu Ala Arg Lys Pro Asn Ser Val Val Ile Ser Cys Gly Met             260 265 270 Lys Leu Asn Leu Asp Tyr Leu Leu Glu Met Leu Trp Glu Tyr Leu Ala         275 280 285 Leu Thr Cys Ile Tyr Thr Lys Lys Arg Gly Gln Arg Pro Asp Phe Thr     290 295 300 Asp Ala Ile Ile Leu Arg Lys Gly Ala Ser Val Glu His Val Cys His 305 310 315 320 Arg Ile His Arg Ser Leu Ala Ser Gln Phe Lys Tyr Ala Leu Val Trp                 325 330 335 Gly Thr Ser Thr Lys Tyr Ser Pro Gln Arg Val Gly Leu Thr His Thr             340 345 350 Met Glu His Glu Asp Val Ile Gln Ile Val Lys Lys         355 360 <210> 2 <211> 1888 <212> DNA <213> Homo sapiens <400> 2 cggaattgcc ggtgccgccg ccaccgctgt ctgtgcgccc acctctgctg ctaccatggg 60 gatcttagag aagatctcgg agatcgagaa ggagatcgct cggacacaga agaacaaggc 120 cactgagtat catctgggcc tgctgaaagc taagctcgcc aagtatcggg cccagctcct 180 ggaaccgtcc aaatcggcct cgtccaaagg agagggcttt gatgtcatga agtcgggtga 240 tgcccgtgtg gcgctgattg gatttccctc tgtgggtaag tccacattct tgagtctgat 300 gacctccacg gccagcgagg cagcgtccta tgagttcacc actctgacgt gtattcctgg 360 ggtcattgaa tacaaaggtg ccaacatcca gctcctggac cttcctggaa tcattgaagg 420 cgcagcccaa ggaaaaggcc gtggccggca ggtgatcgct gtggcgcgca cggctgacgt 480 catcatcatg atgctggatg ccaccaaggg agaggtgcag aggtctctgc tggagaagga 540 gctggagtct gtgggcatcc gcctcaacaa gcacaagcct aacatctact tcaagcccaa 600 gaaaggtggt ggcatctcct ttaactcgac agtcacgctg acccagtgct cggaaaagct 660 ggtgcagctc atcctgcacg aatacaagat cttcaatgca gaagtgcttt tccgagaaga 720 ctgctccccg gacgagttca tcgatgtgat cgtgggcaac cgggtgtaca tgccctgcct 780 gtatgtttat aacaaaatcg accagatctc catggaagag gtggaccgcc tggcccgaaa 840 acccaacagt gtggtcatca gctgcggcat gaagctgaac ctggactatc tgctggagat 900 gctttgggag tacttggccc tgacctgcat ctacaccaag aagagaggac agaggccaga 960 cttcacagac gccatcattc tccggaaagg ggcctcagtg gagcacgtgt gccaccgcat 1020 ccaccggtca ctcgccagcc agttcaagta cgccctggtg tggggcacca gcaccaagta 1080 cagtccgcag cgggtgggcc tgacccacac catggagcat gaggacgtca tccagatcgt 1140 gaagaagtaa cggcgcctgc cgggcctccc gcccacctgc ctcgtctccc tggggaggtg 1200 gtcccactgg gacacacaaa cacccaaaca gaaaaataca aatacacgta ccccaggaag 1260 gggtccctca agtctctgct atttacagaa gtttcttcag taggcagacg aagagtgtgt 1320 tggggcaaag gggctcggtt ggaggcattt cccataagac tgagccctct catgggggtt 1380 ttgagtttgt agtgctgagc ctgcatctgt gcctcccagc cccctgcact gagggagcaa 1440 gttgcccaca tgcccgccag ccagggccta aagcagatgg catgctcagt 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ctcaacagtc acactgacac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 taccgcaact gataactaca 20  

Claims (7)

delete delete delete (a ') treating and culturing a candidate substance in a cell transformed with a vector comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2; And (b ') measuring the production amount of the protein consisting of the amino acid sequence of SEQ ID NO: 1 in the culture of step (a'), and selecting a substance that increases the production amount of the protein. Method of screening materials with therapeutic activity. delete delete delete

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