JP2006067829A - POLYPEPTIDE BINDABLE TO d-DOMAIN OF DR3 GENE AND USE OF THE POLYPEPTIDE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polypeptide useful for elucidation of the mechanism of onset of human articular rheumatism, to provide a polynucleotide encoding the same, and to provide a diagnostic kit and a diagnostic method capable of determining in high accuracy human articular rheumatism onset or its possibility. <P>SOLUTION: The polypeptide bindable to the SNP(single nucleotide polymorphism) site of a DR3 gene involved in human articular rheumatism is retrieved, thus identifying the polypeptide bindable to the d-domain of the DR3 gene and a polynucleotide encoding the polypeptide. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a polypeptide involved in the onset of rheumatoid arthritis, a polynucleotide encoding the polypeptide, a diagnostic kit for the onset or the possibility of the onset of rheumatoid arthritis using these, a diagnostic method, and a therapeutic agent for rheumatoid arthritis. Is.

  Rheumatoid arthritis (hereinafter also referred to as “RA”) is a systemic inflammatory disease whose main cause is multiple erosive arthritis, but at the same time causes many organs to be damaged. RA progresses chronically with repeated remissions and exacerbations. If left untreated, it causes destruction and deformation of joints, and eventually exhibits dysfunction of motor organs. Sometimes life threatens. Therefore, RA patients will suffer great physical and mental pain throughout their lives.

  RA has various ways of onset, and the diagnostic criteria of the American College of Rheumatology are widely used for its diagnosis. However, the onset of RA is usually slow, ranging from weeks to months, and the presence of rheumatoid factor as an objective index in the diagnostic criteria of the American College of Rheumatology is 33% within 3 months, 12 months Also in the above, it is about 88% (for example, refer nonpatent literature 1), and it has not reached a reliable diagnosis with RA. Therefore, an attempt has been made to detect rheumatoid arthritis-related antibody IgM antibody in patient serum that reacts with a recombinant antigen to diagnose rheumatoid arthritis (see, for example, Patent Document 1).

  In addition, for RA treatment, the treatment means to be selected usually depends on the progression of the pathology of the RA pathology. In general, a non-steroidal anti-inflammatory drug (NSAID) is administered at an early stage when a definitive diagnosis cannot be made, and a disease-modifying rheumatic drug (DMARD) is administered in addition to the NSAID when a definitive diagnosis can be made. Particularly in the early stage of RA onset, it is difficult to make a definitive diagnosis. At present, NSAID is administered, and differentiation from other rheumatic diseases including collagen disease is performed simultaneously while carefully monitoring the course. If symptoms progress further, steroid drugs may be administered, and physical therapy and brace therapy are performed for maintenance and recovery of joint function as well as drug therapy for pain. In addition, when daily life becomes inconvenient due to joint destruction, surgical therapy may be performed.

In Patent Document 2, the inventors of the present application specify three loci where rheumatoid arthritis disease genes are located by conducting linkage analysis using microsatellite markers on RA patients and their relatives. The following disease genes have been identified.
(1) Rheumatoid arthritis disease gene located within ± 1 centmorgan from the DNA sequence of the human chromosome 1 to which microsatellite markers D1S214 and / or D1S253 hybridize.
(2) Rheumatoid arthritis disease gene located within ± 1 centmorgan from the DNA sequence of human chromosome 8 to which the microsatellite marker D8S556 hybridizes.
(3) Rheumatoid arthritis disease gene located within ± 1 cm Morgan from the DNA sequence to which the microsatellite markers DXSl001, DXS1047, DXS1205, DXS1227 and / or DXS1232 hybridize on the human X chromosome.

  Further, Non-Patent Document 2 discloses that the present inventors include death receptors as disease-related genes of markers D1S214 and D1S253 related to the disease-related genes of (1) above among the disease-related genes of the prior invention. 3 (death receptor 3: also referred to as “DR3” or “DR3 gene”), a restriction fragment length polymorphism of DR3 was confirmed between healthy individuals and RA patients, and DR3 may be a disease-related gene in RA Suggests sex.

Furthermore, in Patent Document 3, the inventors of the present application disclose the relationship between the genomic mutation of the DR3 gene and the onset of rheumatoid arthritis (chronic rheumatoid arthritis).
JP 10-513257 (release date: December 15, 1998) International Publication No. WO 98/51791 (International Publication Date: November 19, 1998) International Publication WO02 / 34912 (International Publication Date: May 2, 2002) Therapeutic, Vol. 73, No. 3, pp. 23-27, 1991 Rheumatism, No. 39, No. 2, pages 444 and 445 (1999)

  As described above, many positive research results have been reported on various autoimmune diseases, but techniques for objectively diagnosing the onset of autoimmune diseases (particularly rheumatoid arthritis) are still well established. That's not true. Therefore, establishment of a new technology that can contribute to an objective and accurate diagnosis of the onset of autoimmune diseases such as rheumatoid arthritis has been strongly desired.

  The present invention has been made in view of the above problems, and its purpose is to provide a factor for enabling an objective and accurate diagnosis of the onset of rheumatoid arthritis, a diagnostic kit using the factor, and a diagnosis It is to provide a method and medicament.

  The present inventors have identified four SNPs (SNPa, SNPc, SNPd, and SNPe, respectively) and 14 bases of DR3 (Death Receptor 3) identified as one of the disease genes for RA, and rheumatoid arthritis (RA). It has been found that it is significantly observed in family-onset cases (see Patent Document 3). Among these, it has been suggested that SNPd produces DR3 splicing variants with functional mutations. However, it is still unclear on what mechanism these splicing variants are produced. Therefore, the present inventors have attempted to search for a factor that binds to the DR3 mutation region for the purpose of elucidating the production mechanism of the variant. Specifically, oligonucleotide probes each containing a normal sequence or a mutant sequence at polymorphic sites for four SNPs (SNPa, SNPc, SNPd, and SNPe) are prepared, and a DR3 mutation region is obtained by gel shift method for DR3 gene. The existence of a factor that binds to was investigated.

  As a result, a band shift was observed only when a probe containing SNPd was used, indicating that there was a factor that specifically binds to SNPd.

  Thus, the present inventors have found that there is a factor that binds to the d region in DR3 genomic DNA, and showed that the factor is one of the factors that produce DR3 splicing variants. Moreover, it discovered that the onset of RA based on the variation | mutation of DR3 in the test subject or a sample (for example, cell) derived from a test subject, or its onset possibility can be determined by using the said factor. Furthermore, the present inventors have found that the factor is useful as a new preventive method, therapeutic method and therapeutic agent for rheumatoid arthritis. Based on these findings, the present inventors have completed the present invention.

  The polypeptide according to the present invention is characterized by binding to the d region of the DR3 gene.

  The polypeptide according to the present invention is characterized in that it binds to a polynucleotide comprising the base sequence represented by SEQ ID NO: 7.

  The polypeptide according to the present invention preferably does not bind to the polynucleotide consisting of the base sequence represented by SEQ ID NO: 8.

  The polynucleotide according to the present invention is characterized by encoding the above-mentioned polypeptide.

  The diagnostic kit according to the present invention is characterized by comprising the above-mentioned polypeptide in order to determine the onset of rheumatoid arthritis or the possibility of the onset thereof.

  The diagnostic kit according to the present invention is characterized by comprising the above-mentioned polynucleotide in order to determine the onset of rheumatoid arthritis or the possibility of the onset thereof.

  The diagnostic method according to the present invention is characterized by using the above polypeptide in order to determine the onset of rheumatoid arthritis or the possibility of its onset.

  The diagnostic method according to the present invention is characterized by using the above-mentioned polynucleotide in order to determine the onset of or possibility of the onset of rheumatoid arthritis.

  The medicament according to the present invention is characterized by containing the above-mentioned polypeptide in order to treat rheumatoid arthritis.

  The drug according to the present invention is characterized by containing the above-mentioned polynucleotide for treating rheumatoid arthritis.

  The screening kit according to the present invention is characterized by comprising a polynucleotide comprising the base sequence represented by SEQ ID NO: 7.

  The screening kit according to the present invention preferably further comprises a polynucleotide comprising the base sequence represented by SEQ ID NO: 8.

  The screening method according to the present invention is characterized by using a polynucleotide comprising the base sequence represented by SEQ ID NO: 7.

  The screening kit according to the present invention preferably further uses a polynucleotide comprising the base sequence represented by SEQ ID NO: 8.

  The polypeptide or polynucleotide according to the present invention can be effectively used for elucidation of the onset mechanism of human rheumatoid arthritis. Furthermore, the polypeptide or polynucleotide of the present invention can be used for diagnosis of rheumatoid arthritis, determination of the possibility of developing rheumatoid arthritis, and development of an appropriate therapeutic agent for rheumatoid arthritis.

  In addition, according to the diagnostic kit and diagnostic method for rheumatoid arthritis according to the present invention, the determination of the onset of rheumatoid arthritis or the possibility of its onset can be performed with high accuracy and simplicity. Therefore, it can be used for prevention and treatment of rheumatoid arthritis.

As shown in Patent Document 3, the present inventors have found the following mutation in the genomic DNA sequence of DR3 having the base sequence represented by SEQ ID NO: 1 in cells obtained from a subject:
(A) substitution of the base at position 1755 from adenine (A) to guanine (G) (SNPa);
(B) deletion of bases at positions 2443 to 2456;
(C) substitution of the base at position 2531 from cytosine (C) to thymine (T) (SNPc);
(D) substitution of the base at position 2678 from adenine (A) to thymine (T) (SNPd); and (e) substitution of the base at position 2826 from adenine (A) to guanine (G) (SNPe). .

  As shown in FIG. 1, the base at position 1755 is in the exon region (exon 5) of DR3 genomic DNA. In the DR3 polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, the mutation of (a) above As a result, the amino acid at position 159 is mutated from aspartic acid to glycine. On the other hand, the mutations (b) to (e) are in the intron region of the DR3 genome.

  As used herein, the term “a region” intends a region containing the mutation of (a) above in the genomic DNA of DR3. The terms “b region” to “e region” are also used herein as well.

  The a region preferably consists of positions 1744 to 1767 of the base sequence shown in SEQ ID NO: 1, and more preferably consists of the base sequence shown in SEQ ID NO: 3. In the SNP (SNPa) in the a region, the base at position 1755 of the base sequence shown in SEQ ID NO: 1 is substituted from adenine (A) to guanine (G) (SEQ ID NO: 4). The c region preferably consists of positions 2520 to 2543 of the base sequence shown in SEQ ID NO: 1, more preferably the base sequence shown in SEQ ID NO: 5. In the SNP (SNPc) in the c region, the base at position 2531 of the base sequence shown in SEQ ID NO: 1 is substituted from cytosine (C) to thymine (T) (SEQ ID NO: 6). The d region preferably consists of positions 2667 to 2690 of the base sequence shown in SEQ ID NO: 1, and more preferably consists of the base sequence shown in SEQ ID NO: 7. In the SNP (SNPd) in the d region, the base at position 2678 of the base sequence shown in SEQ ID NO: 1 is substituted from adenine (A) to thymine (T) (SEQ ID NO: 8). The e region preferably consists of positions 2815 to 2838 of the base sequence shown in SEQ ID NO: 1, more preferably the base sequence shown in SEQ ID NO: 9. In the SNP (SNPe) in the e region, the base at position 2826 of the base sequence shown in SEQ ID NO: 1 is substituted from adenine (A) to guanine (G) (SEQ ID NO: 10).

  Unless otherwise specified, A, C, G, and T represent adenine, cytosine, guanine, and thymine bases unless otherwise specified. For amino acids and amino acid residues, the one-letter code or three-letter code defined by IUPAC and IUB is used. Moreover, a transcription product is a product produced as a result of transcription and translation of a genome, and examples thereof include mRNA, cDNA, and protein (polypeptide).

  The 1755th base in the base sequence shown in SEQ ID NO: 1 corresponds to the 564th base of the sequence (accession number NM_003790) registered in the gene bank as cDNA. If the base of the 3 ′ end of exon 5 of this genomic DNA is used as a reference and the base of the intron one base later is the first, the deletion at positions 2443 to 2456 is caused by the bases 622 to 635 from the 3 ′ end of exon 5. Corresponds to a defect. Further, with reference to the base at the 5 ′ end of exon 6, the base number of the intron one base before is −1, the mutation at position 2531 is −538, the mutation at position 2678 is −391, the position 2826 This mutation corresponds to the −243rd base (FIG. 1). In the region where T is 28 bases continuous (positions 2443 to 2470), the number may increase or decrease by 3 bases, and the region where the T of the mutant genome is 14 bases is similarly 3 May increase or decrease base content.

  Hereinafter, although this invention is demonstrated in detail, this invention is not limited to this description.

[1] Polypeptide The present invention provides a polypeptide that binds to the d region of the DR3 gene. In one embodiment, the polypeptide according to the present invention preferably binds to a polynucleotide consisting of the base sequence shown in SEQ ID NO: 7. In a further embodiment, the polynucleotide according to the present invention binds to the polynucleotide consisting of the base sequence shown in SEQ ID NO: 7, but preferably does not bind to the polynucleotide consisting of the base sequence shown in SEQ ID NO: 8.

  As used herein, the term “polypeptide” is used interchangeably with “peptide” or “protein”. The polypeptides according to the invention may also be isolated from natural sources, recombinantly produced, or chemically synthesized.

  The term “isolated” polypeptide or protein is intended to be a polypeptide or protein that has been removed from its natural environment. For example, recombinantly produced polypeptides and proteins expressed in a host cell can be isolated in the same manner as natural or recombinant polypeptides and proteins that have been substantially purified by any suitable technique. It is thought that there is.

  Polypeptides according to the present invention may be purified products from natural cells or tissues, products of chemical synthesis procedures, or prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells, insect cells, and It is preferably produced by recombinant technology from mammalian cells).

  In one embodiment, the polypeptide according to the present invention is preferably purified from an extract of a cell or tissue derived from a subject. The step of purifying the polypeptide is to prepare a cell extract (eg, nuclear extract) from the cells or tissue by a well-known method (eg, centrifuge the cell or tissue and then collect the soluble fraction by centrifugation). After that, well-known methods (for example, ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite The step of purification by chromatography and lectin chromatography) is preferred, but is not limited thereto.

  The cells to be used can be obtained from all the cells of the subject (for example, peripheral lymphocytes, synovial cells, organs, etc.) according to a known method. The obtained cells can be appropriately cultured and expanded for use. Preferable cells include Jurkat cells, Hela cells, TF-1 cells, MOLT4 cells and the like.

  It is also suggested that the polypeptide according to the present invention is involved in DR3 signaling. Therefore, it is preferable to stimulate the cells used to drive the DR3 signaling pathway. Examples of reagents used for stimulation include death receptor ligands (eg, Fas ligand, TNF, DR3 ligand, etc.), anti-Fas antibody, actinomycin D, radiation, glucocorticoid, phorbol 12-myristate 13-acetate (PMA). ), And phytohemagglutinin (PHA), but are not limited thereto. Further, two or more of these may be used in combination. Of these reagents, DR3 ligand, PMA, and PHA are preferable, and DR3 ligand is more preferable.

  The time for adding the reagent is not particularly limited, but may be, for example, about 1 to 72 hours, and preferably 12 to 48 hours. In addition, it is necessary to prepare control cells that do not give stimulation.

  In another embodiment, the polypeptide according to the present invention introduces a polynucleotide according to the present invention described later (polynucleotide encoding the polypeptide according to the present invention) into a host cell, and expresses the polypeptide in the cell. It may be in a state of being allowed to stand, or may be isolated and purified from expressed cells or tissues.

  Various vectors can be used to introduce the polynucleotide into a host cell. The specific type of the vector is not particularly limited, and a vector that can be expressed in the host cell may be appropriately selected. That is, according to the type of the host cell, a promoter sequence may be appropriately selected in order to reliably express the polynucleotide, and a vector in which this and the polynucleotide are incorporated into various plasmids may be used as an expression vector.

  The expression vector preferably includes at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and E. coli. Examples of culture in E. coli and other bacteria include a tetracycline resistance gene or an ampicillin resistance gene.

  By using the selectable marker, it can be confirmed whether or not the polynucleotide has been introduced into the host cell, and whether or not it is reliably expressed in the host cell.

  The host cell is not particularly limited, and various conventionally known cells can be suitably used. Specifically, for example, bacteria such as Escherichia coli, yeasts (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), and Xenopus laevis mothers such as Xenopus laevis However, it is not particularly limited. Appropriate culture media and conditions for the above-described host cells are well known in the art.

  A method for introducing the expression vector into a host cell, that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be suitably used. . In addition, for example, when the polypeptide according to the present invention is transferred and expressed in insects, an expression system using baculovirus may be used.

  In addition, the polypeptide according to this embodiment may include an additional polypeptide. Examples of the additional polypeptide include epitope-tagged polypeptides such as His, Myc, and Flag. In a preferred embodiment, the polypeptides according to the invention can be expressed recombinantly in a modified form such as a fusion protein. For example, additional amino acids, particularly charged amino acid regions, of the polypeptides according to the invention may be used in host cells to improve stability and persistence during purification or subsequent manipulation and storage. It can be added to the N-terminus of the polypeptide.

  Whether or not a gene has been introduced into a cell can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from transformed cells, PCR is performed by designing DNA-specific primers. PCR can be performed under the same conditions as those used for preparing the plasmid. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, It can be confirmed that it has been transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, it is possible to employ a method in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or enzymatic reaction.

  In another embodiment, the polypeptides according to the invention can be synthesized using known methods of chemical synthesis. For example, Houghten is prepared for the synthesis of multiple peptides such as 10-20 mg of 248 different 13 residue peptides that represent a single amino acid variant of the HA1 polypeptide segment prepared and characterized in less than 4 weeks. A simple method is described. Houghten, R.A. A. , Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985). This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in US Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure, individual resins for solid phase synthesis of various peptides are contained in separate solvent permeable packets, allowing optimal use of many identical iteration steps associated with solid phase methods. Complete manual procedures allow 500-1000 or more syntheses to be performed simultaneously (Houghten et al., Supra, 5134). These documents are incorporated herein by reference.

  The polypeptides according to the present invention are useful in methods and kits for determining the onset or likelihood of onset of rheumatoid arthritis, and medicaments for treating rheumatoid arthritis.

  Thus, it can be said that the polypeptide according to the present invention only needs to be a polypeptide that binds to at least the d region of the DR3 gene (preferably, a polynucleotide comprising the base sequence shown in SEQ ID NO: 7). That is, it should be noted that the present invention also includes a case where the polypeptide is linked to a polypeptide consisting of an arbitrary amino acid sequence having a specific function (for example, a tag). Moreover, the said polypeptide and the said arbitrary amino acid sequences may be connected with the appropriate linker peptide so that each function may not be inhibited.

  That is, an object of the present invention is to provide a polypeptide that binds to the d region of the DR3 gene, and does not exist in the method for producing a polypeptide specifically described in the present specification. Therefore, it should be noted that polypeptides obtained by methods other than the above methods also belong to the scope of the present invention.

[2] Polynucleotide The present invention provides a polynucleotide encoding the polypeptide according to the present invention. As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. As used herein, the term “base sequence” is used interchangeably with “gene sequence”, “nucleic acid sequence” or “nucleotide sequence” and is abbreviated as deoxyribonucleotides (A, G, C and T). )).

  The polynucleotide according to the present invention may be generated as a cleaved fragment of a longer polynucleotide (for example, a polynucleotide comprising the full-length cDNA encoding the DR3 binding protein according to the present invention) or may be synthesized by synthesis. For example, restriction endonuclease cleavage or ultrasonic shearing can be readily used to create fragments of various sizes. Alternatively, such fragments can be made synthetically. Appropriate fragments (oligonucleotides) are synthesized by Applied Biosystems Incorporated (ABI, 850 Lincoln Center Dr., Foster City, CA 94404) type 392 synthesizer and the like.

  Examples of the method for obtaining the polynucleotide according to the present invention include a method using an amplification means such as PCR. For example, among the cDNA of the polynucleotide according to the present invention, primers are prepared from the 5 ′ side and 3 ′ side sequences (or their complementary sequences), and genomic DNA (or cDNA) or the like is prepared using these primers. By performing PCR or the like as a template and amplifying a DNA region sandwiched between both primers, a large amount of DNA fragments containing the polynucleotide according to the present invention can be obtained.

  Further, the polynucleotide according to the present invention can be fused to the polynucleotide encoding the tag tag (tag sequence or marker sequence) described above on the 5 'side or 3' side.

  That is, an object of the present invention is to provide a polynucleotide that encodes a polypeptide that binds to the d region of DR3, and resides in a method for producing a polynucleotide specifically described in the present specification. Not. Therefore, it should be noted that polynucleotides obtained by methods other than the above methods also belong to the scope of the present invention.

[3] Diagnostic Kit and Diagnostic Method The present invention provides a diagnostic kit for determining the onset of rheumatoid arthritis or the possibility of its onset. As used herein, “probability of onset” of rheumatoid arthritis refers to an index indicating the risk of developing rheumatoid arthritis (RA). Indicates that it is difficult to become RA if the possibility of onset is low. In one embodiment, the diagnostic kit according to the present invention preferably comprises the polypeptide according to the present invention. In another embodiment, the diagnostic kit according to the present invention preferably comprises the polynucleotide according to the present invention. In addition, the diagnostic kit according to the present invention may be provided with other reagents.

  The present invention also provides a diagnostic method for determining the onset or the likelihood of developing rheumatoid arthritis. In one embodiment, the diagnostic kit according to the present invention preferably uses the polypeptide according to the present invention. In another embodiment, the diagnostic kit according to the present invention preferably uses the polynucleotide according to the present invention. In addition, the diagnostic method according to the present invention may include other steps.

  By using the diagnosis (determination) kit and diagnosis (determination) method according to the present invention, RA diagnosis (determination of the onset or the possibility of its onset) can be performed with high accuracy.

[4] Therapeutic drug and therapeutic method The present invention provides a drug for treating rheumatoid arthritis. In one embodiment, the therapeutic agent according to the present invention preferably comprises a polypeptide according to the present invention. In another embodiment, the diagnostic kit according to the present invention preferably includes the polynucleotide according to the present invention. The diagnostic kit according to the present invention may also contain a pharmaceutically acceptable carrier.

  The present invention also provides a method for treating rheumatoid arthritis. In one embodiment, the therapeutic method according to the present invention preferably uses the polypeptide according to the present invention. In another embodiment, the treatment method according to the present invention preferably uses the polynucleotide according to the present invention. Moreover, the treatment method according to the present invention may include other steps.

  By using the therapeutic agent and method according to the present invention, RA can be treated significantly.

[5] Screening kit and screening method The present invention provides a screening kit for screening a polypeptide that binds to the d region of the DR3 gene. In one embodiment, the screening kit according to the present invention preferably comprises a polynucleotide consisting of the base sequence shown in SEQ ID NO: 7. In a further embodiment, the screening kit according to the present invention preferably further comprises a polynucleotide consisting of the base sequence represented by SEQ ID NO: 8.

  The present invention also provides a screening method for screening a polypeptide that binds to the d region of the DR3 gene. In one embodiment, the screening method according to the present invention preferably uses a polynucleotide comprising the base sequence shown in SEQ ID NO: 7. In a further embodiment, the screening method according to the present invention preferably further uses a polynucleotide consisting of the base sequence represented by SEQ ID NO: 8.

  EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, it is needless to say that the present invention is not limited to the following examples, and various modes are possible for details. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention also relates to embodiments obtained by appropriately combining the disclosed technical means. Is included in the technical scope.

[Example 1: Analysis by gel shift method using oligonucleotides for mutation regions a, c, d, and e of DR3 gene]
The present inventors prepared 24 bp oligonucleotide probes each containing a normal sequence or a mutant sequence for four SNPs (SNPa, SNPc, SNPd, and SNPe), and placed them in the DR3 mutant region by gel shift method for DR3 gene. The presence of the binding factor was examined.

Jurkat cells (2 × 10 ⁶ cells) were stimulated (or unstimulated) with 20 ng / ml phorbol 12-myristate 13-acetate (PMA) and 1 μg / ml phytohemagglutinin (PHA).

24 hours after stimulation, the above cells were mixed with 500 μl of hypotonic buffer (10 mM Hepes-KOH (pH 7.9), 1.5 mM MgCl ₂ , 10 mM KCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM dithiotray. It was suspended in Toll (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM leupeptin, 1 μM aprotinin) and allowed to stand for 10 minutes under ice cooling. Further, 5 μl of 10% Nonidet P-40 (NP-40) was added, incubated for 20 minutes under ice cooling, and then centrifuged at 2,000 × g for 3 minutes to obtain a nuclear fraction.

  In this nuclear fraction, 200 μl of elution buffer (20 mM Hepes-KOH (pH 7.9), 10% glycerol, 420 mM NaCl, 1.5 mM MgCl 2, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM dithiothreitol ( DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM leupeptin, 1 μM aprotinin), shaken on ice for 30 minutes, and then centrifuged at 15,000 × g for 15 minutes to obtain a supernatant. A nuclear extract was obtained.

  A 24 bp oligonucleotide probe was prepared, each containing normal or mutated sequences at polymorphic sites for four SNPs (SNPa, SNPc, SNPd, and SNPe).

These probes were end-labeled using DIG-11-ddUTP (Roche Diagnostics) to obtain digoxigenin (DIG) -labeled double-stranded oligonucleotide probes.

  20 mM Hepes-KOH (pH 7.9), 10% glycerol, 20 mM KCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg In a 20 μl total reaction system containing BSA and 0.5 μg poly (dI-dC) (Amersham Biosciences), 5 μg of the nuclear extract and the above DIG-labeled double-stranded oligonucleotide probe (each 0.5 pmole) were reacted. .

The sample after the reaction was electrophoresed on 4% polyacrylamide gel using 1 × TGE buffer (pH 8.5) (50 mM Tris-HCl, 380 mM glycine, 2 mM ethylenediaminetetraacetic acid), and then nylon membrane plus charge. (Roche Diagnostics). The membrane to which the DIG-labeled oligonucleotide was transferred was reacted with alkaline phosphatase-labeled F (ab) ₂ anti-DIG antibody (Roche Diagnostics), and then 100 μg / ml CSPD substrate (Roche Diagnostics). It visualized by the chemiluminescent reaction using (Figure 2).

As a result, a band shift was observed only with the probe containing SNPd, indicating that a factor that specifically binds to SNPd exists. This factor bound strongly to the normal sequence compared to the mutant sequence containing SNPd, and this binding was further enhanced by stimulating the cells with PHA and PMA (FIG. 2).
[Example 2: Analysis of molecular weight of d region binding protein using UV cross-linking method]
Unstimulated Jurkat cells (2 × 10 ⁶ cells) were added to 500 μl of hypotonic buffer (10 mM Hepes-KOH (pH 7.9), 1.5 mM magnesium chloride, 10 mM potassium chloride, 0.5 mM ethylenediaminetetraacetic acid, 0. The suspension was suspended in 5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM leupeptin, and 1 μM aprotinin), and allowed to stand for 10 minutes under ice cooling. Further, 5 μl of 10% Nonidet P-40 (NP-40) was added, incubated for 20 minutes under ice cooling, and then centrifuged at 2000 × g for 3 minutes. The nuclear fraction was collected and 200 μl of elution buffer (20 mM Hepes-KOH (pH 7.9), 10% glycerol, 420 mM sodium chloride, 1.5 mM magnesium chloride, 0.2 mM ethylenediaminetetraacetic acid, 0.5 mM dithiothreitol. , 0.5 mM phenylmethylsulfonyl fluoride, 1 μM leupeptin and 1 μM aprotinin), and the mixture was shaken on ice for 30 minutes and then centrifuged at 15000 × g for 15 minutes. The obtained supernatant was used as a nuclear extract.

  Biotin-labeled double stranded DNA probes (SEQ ID NOs: 7 and 8) for the d region were labeled using Psoralen-Biotin conjugate (Ambion). In a reaction system of a total volume of 20 μl, a reaction solution containing 5 μg of the nuclear extract and 40 fmol of the probe (100 mM Tris-HCl (pH 8.0), 5 mM ethylenediaminetetraacetic acid, 2 mM β-mercaptoethanol, 0.1 mM zinc chloride, 12 .5% glycerol) was allowed to react for 20 minutes at room temperature.

  The sample after the reaction was irradiated with UV light (254 nm) for 10 minutes under ice cooling, and then 2 μl each of 100 mM magnesium sulfate and 100 mM calcium chloride was added. Further, 5 μg of deoxyribonuclease I (manufactured by Wako Pure Chemical Industries, Ltd.) and 1 unit of micrococcal nuclease (manufactured by Fermentas) were added, and the mixture was reacted at 37 ° C. for 30 minutes. Add sodium dodecyl sulfate (SDS) -sample buffer (containing 0.062 M Tris-HCl (pH 6.8), 2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.002% bromophenol blue) The reaction was stopped and heated at 95 ° C. for 5 minutes to prepare a sample for SDS-polyacrylamide electrophoresis (PAGE).

  The obtained sample was electrophoresed on a 7.5-15% gradient SDS-polyacrylamide gel (Biocraft) together with a biotin-labeled molecular weight marker (Amersham Biosciences) according to a conventional method. After the electrophoresis, the sample separated by SDS-PAGE was transferred to a nylon membrane plus charge (manufactured by Roche Diagnostics). The biotin-labeled DNA probe was reacted with alkaline phosphatase-labeled streptavidin (Ambion) and then visualized by a chemiluminescence reaction using a CDP-Star substrate (Ambion) to detect the target protein.

[Example 3: Affinity purification of d region binding protein]
Unstimulated Jurkat cells (2 × 10 ⁶ cells) were added to 500 μl of hypotonic buffer (10 mM Hepes-KOH (pH 7.9), 1.5 mM magnesium chloride, 10 mM potassium chloride, 0.5 mM ethylenediaminetetraacetic acid, 0. The suspension was suspended in 5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM leupeptin, and 1 μM aprotinin), and allowed to stand for 10 minutes under ice cooling. Further, 5 μl of 10% Nonidet P-40 (NP-40) was added, incubated for 20 minutes under ice cooling, and then centrifuged at 2000 × g for 3 minutes. The nuclear fraction was collected and 200 μl of elution buffer (20 mM Hepes-KOH (pH 7.9), 10% glycerol, 420 mM sodium chloride, 1.5 mM magnesium chloride, 0.2 mM ethylenediaminetetraacetic acid, 0.5 mM dithiothreitol. , 0.5 mM phenylmethylsulfonyl fluoride, 1 μM leupeptin and 1 μM aprotinin), and the mixture was shaken on ice for 30 minutes and then centrifuged at 15000 × g for 15 minutes. The obtained supernatant was used as a nuclear extract.

  Wash buffer (containing 20 mM Hepes-KOH (pH 7.6), 100 mM potassium chloride, 0.01% NP-40) was added to 100 μl of 50% streptavidin-agarose bead solution to wash the beads. Blocking buffer (20 mM Hepes-KOH (pH 7.6), 300 mM potassium chloride, 0.01% NP-40, 0.5 mg / ml bovine serum albumin (BSA)) was added and reacted at 4 ° C. for 30 minutes. The beads were then washed with wash buffer and suspended in 100 μl wash buffer.

  In a reaction system with a total volume of 100 μl, a reaction solution (20 mM Hepes-KOH (20 mM Hepes-KOH ( pH 7.6), 100 mM potassium chloride, 0.01% NP-40, 0.5 mg / ml BSA) was reacted at 4 ° C. for 1 hour.

  Subsequently, binding buffer 1 (10 mM Hepes-KOH (pH 7.9), 100 mM potassium chloride, 2 mM magnesium chloride, 0.5 mM ethylenediaminetetraacetic acid, 10% glycerol, 1 mM dithiothreitol, 0.2% Tween 20) was used. The beads were washed and suspended in 20 μl binding buffer 1. In a reaction system with a total volume of 200 μl, binding buffer 2 (10 mM Hepes-KOH (pH 7.9), 100 mM potassium chloride, 2 mM magnesium chloride, 0.5 mM ethylenediamine tetrachloride) containing 20 μl of the obtained bead suspension and 100 μg of the nuclear extract. Reaction with acetic acid, 10% glycerol, 1 mM dithiothreitol, 0.2% Tween 20, 50 ng / μl Poly (dI-dC) (Amersham Biosciences), 0.2 μg / μl bovine serum albumin) at 4 ° C. for 30 minutes I let you.

  After the reaction, the beads were washed with binding buffer 1, and protein extraction buffer (58 mM Tris-HCl (pH 6.8), 6% glycerol, 1.7% SDS, 0.0025% bromophenol blue, 0.8% β-mercaptoethanol was added) and heated at 95 ° C. for 5 minutes to prepare a sample for SDS-polyacrylamide electrophoresis (PAGE). The obtained sample was electrophoresed on a 7.5 to 15% gradient SDS-polyacrylamide gel (Biocraft) together with a protein marker (Approscience) according to a conventional method. After electrophoresis, the separated protein was silver stained using Silver Stain II Kit Wako (Wako Pure Chemical Industries) to detect the target protein.

  The polypeptide and polynucleotide according to the present invention can be effectively used for elucidating the mechanism of the onset of rheumatoid arthritis. Moreover, if this invention is used, the onset of rheumatoid arthritis or its onset possibility can be diagnosed (detected) easily and reliably with high accuracy. Therefore, the present invention has high applicability in the field of prevention and treatment of rheumatoid arthritis.

FIG. 1 is a diagram showing SNP sites in the DR3 genomic sequence. FIG. 2 is a diagram showing the results of a gel shift method using oligonucleotide probes each containing a normal sequence or a mutant sequence for four SNPs (SNPa, SNPc, SNPd, and SNPe).

Claims (10)

  A polypeptide characterized by binding to the d region of the DR3 gene.   A polypeptide that binds to a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7.   The polypeptide according to claim 1 or 2, wherein the polypeptide does not bind to a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 8.   A polynucleotide encoding the polypeptide according to any one of claims 1 to 3.   A diagnostic kit for determining the onset or possibility of onset of rheumatoid arthritis, comprising the polypeptide according to any one of claims 1 to 3 or the polynucleotide according to claim 4.   A diagnostic method for determining the onset or possibility of onset of rheumatoid arthritis, comprising using the polypeptide according to any one of claims 1 to 3 or the polynucleotide according to claim 4.   A therapeutic agent for rheumatoid arthritis, comprising the polypeptide according to any one of claims 1 to 3 or the polynucleotide according to claim 4.   A screening kit for screening for a polypeptide that binds to the d region of the DR3 gene, comprising a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7.   The screening kit according to claim 8, further comprising a polynucleotide comprising the base sequence represented by SEQ ID NO: 8.   A screening method for screening for a polypeptide that binds to the d region of the DR3 gene, which comprises using a polynucleotide having the base sequence represented by SEQ ID NO: 7.