WO2005103256A1

WO2005103256A1 - Gene encoding gtpase activating protein and gene product thereof

Info

Publication number: WO2005103256A1
Application number: PCT/JP2005/007313
Authority: WO
Inventors: Osamu Ohara; Takahiro Nagase; Michio Ohishi; Hiroshi Yokota; Osamu Kamida
Original assignee: Daiichi Pharmaceutical Co., Ltd.; Kazusa Dna Research Institute Foundation
Priority date: 2004-04-23
Filing date: 2005-04-15
Publication date: 2005-11-03
Also published as: JPWO2005103256A1

Abstract

It is intended to find a novel protein capable of serving as a GTPase activating protein (GAP) to Rho family proteins belonging to a group of low-molecular weight GTP-binding proteins and a gene encoding this protein, and provide a method of identifying a compound inhibiting the function and/or expression of the GAP, a medicinal composition usable for a disease based on abnormality in the function and/or expression of the GAP, a method of judging the disease, and a reagent kit. A polynucleotide comprising the base sequence represented by SEQ ID NO:1 or its complementary chain; a homolog of the polynucleotide; a protein encoded by the polynucleotide; a vector containing the polynucleotide; a transformant containing the vector; an antibody against the above protein; a method of identifying a compound inhibiting the function and/or expression of the above protein and a method of judging a disease characterized by comprising using the same; and a medicinal composition and a reagent kit containing the same.

Description

Specification

TECHNICAL FIELD The gene encoding the GTPase activity protein and its gene product

The present invention relates to a protein capable of acting as a GTPase activating protein on a Rho family protein, which is a group of low molecular weight GTP-binding proteins, and a polynucleotide encoding the protein. More specifically, a protein that binds to Racl, one of the Rho family low-molecular-weight GTP-binding proteins, a polynucleotide encoding the protein, a recombinant vector containing the polynucleotide, and a transformant transformed with the recombinant vector It relates to a transformant. Further, the present invention relates to a method for producing the protein, and an antibody against the protein. Furthermore, the present invention relates to a method for identifying a compound that inhibits the function of the protein and the expression of Z or the polynucleotide. Further, the present invention relates to a method for diagnosing a chronic inflammatory disease, which comprises measuring the expression level of the polynucleotide. Further, a preventive and / or therapeutic agent for chronic inflammatory diseases comprising a protein function inhibitor and / or an expression inhibitor of the polynucleotide as an active ingredient, a protein function inhibitor and / or a Z or the polyfunctional inhibitor. The present invention relates to a method for preventing a chronic inflammatory disease and a method for treating Z or a therapeutic method, characterized by using a nucleotide expression inhibitor. The present invention also relates to a reagent kit comprising at least one of the protein, the polynucleotide, the recombinant vector, the transformant, and the antibody.

Background art

[0002] The Rho family low molecular weight GTP-binding protein (hereinafter sometimes simply referred to as Rho family protein) is a protein belonging to one group of low molecular weight GTP-binding proteins (hereinafter simply referred to as low molecular weight G protein). Low-molecular-weight G proteins act as signal amplification factors between cell membrane receptors and effectors involved in intracellular signaling pathways. In addition, the low-molecular-weight G protein specifically binds to guanosine ^ triphosphate (GTP) or guanosine ^ diphosphate (GDP) and has an enzymatic activity to hydrolyze the bound GTP to GDP. GTPase activity refers to the enzyme activity that hydrolyzes GTP to GDP. When an extracellular information substance binds to a cell membrane receptor, its signal is converted to a low molecular weight G protein Then, an exchange reaction occurs between GDP bound to the low molecular weight G protein and GTP present in the cell (hereinafter referred to as GDPZGTP exchange reaction). As a result, an active GTP-linked low molecular weight G protein is generated. Active low-molecular-weight G proteins act on effectors to amplify signals. Thereafter, the GTP-linked low-molecular-weight G protein hydrolyzes the bound GTP to GDP and inactivates it due to its GTPase activity. Thus, low-molecular-weight G proteins act as molecular switches in intracellular signaling pathways by exchanging guanine nucleotides.

[0003] One of the molecules involved in the action of a low molecular weight G protein as a molecular switch is GTPase activating protein (hereinafter abbreviated as GAP). GAP has the function of promoting the GTPase activity of low molecular weight G proteins. With this function, GAP promotes the change from GTP-linked active low-molecular-weight G protein to GDP-linked inactive low-molecular-weight G-protein. GAP has a characteristic domain structure called GAP domain. The GAP domain is the active domain of GAP.

[0004] Among GAPs, those that act on Rho family proteins are called Rho-GAPs.

As Rho family proteins, Cdc42, Racl, RhoA and the like are known. Cdc42 regulates the formation of filopodia in fibroblasts. Racl regulates the production of superoxide in leukocytes and macrophages, and regulates the formation of ruffling-lamellipodia in cell membranes in fibroblasts. Cdc42 and Racl can also activate the c Jun N-terminal kinase signaling pathway. Thus, Rho family proteins are involved in various cell functions through the control of intracellular signaling. As cellular functions involving the Rho family protein, for example, cytoskeletal rearrangement, cell adhesion, gene expression and the like are known. Such actions through Rho family proteins are thought to act on morphogenesis during development, leukocyte migration, neurite retraction, and metastasis and invasion of cancer cells.

[0005] Rho-GAP is thought to play an important role in controlling intracellular signaling involved in Rho family proteins by its function of promoting GTPase activity. The genes isolated as Rho-GAP include Oligophrenin-1 (non-patent document 1) involved in nonspecific X-linked mental retardation, Lowe syndrome OCRL-1 (Non-Patent Document 2) and the like involved in (Lowe's syndrome).

[0006] Non-Patent Document 1: "Nature", 1998, Vol. 392, No. 6679, p. 923-926.

Non-Patent Document 2: "Proceedings of the National Academy oi sciences oi The United States oi America", 1995, Vol. 92, Issue 11, p. 4853—4856.

Disclosure of the invention

Problems to be solved by the invention

An object of the present invention is to provide a novel Rho-GAP and a gene encoding the Rho-GAP. Another object of the present invention is to provide a recombinant vector containing the gene, and a transformant transformed by the recombinant vector. Further, the object of the present invention includes providing a method for producing the Rho-GAP and an antibody recognizing the Rho-GAP. Another object of the present invention is to provide a method for identifying a compound that inhibits the function of Rho-GAP and the expression of Z or the gene. Furthermore, the object of the present invention is to provide a method for preventing and treating a disease based on an abnormality in the function of Rho-GAP and an abnormality in the expression of Z or the gene, a method for diagnosing the disease, a diagnostic method for the disease, and a reagent kit. It also includes providing.

Means for solving the problem

[0008] The present inventors have made intensive efforts to solve the above-mentioned problems, have found a novel gene having a region encoding a GAP domain characteristic of Rho-GAP, and have further obtained a protein encoded by the gene. succeeded in. Then, it was experimentally revealed that the protein encoded by the gene binds to Racl, one of the Rho family proteins. Furthermore, it was found that the tissue expression of the gene was at least 4.5 times and at least 3.5 times higher than that of normal tissues in cases of chronic inflammation of the thyroid gland and chronic inflammation of the kidney, respectively. The present invention has been achieved based on these findings.

That is, the present invention provides

1. represented by the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence A polynucleotide encoding a protein represented by the amino acid sequence of SEQ ID NO: 2 or a polynucleotide represented by a nucleotide sequence complementary to the polynucleotide,

2. A polynucleotide selected from the group consisting of: a polynucleotide encoding a protein that binds to Racl:

i) a polynucleotide represented by a nucleotide sequence having at least 70% homology with the nucleotide sequence of the polynucleotide according to 1.

ii) a polynucleotide having a mutation such as deletion, substitution, addition or the like of one or several nucleotides or an induced mutation in the nucleotide sequence of the polynucleotide according to the item 1, and iii) the polynucleotide according to the item 1. A polynucleotide that hybridizes with the nucleotide under stringent conditions,

3. A polynucleotide selected from the following group, which encodes a protein having a function of promoting Racl GTPase activity (GAP activity):

4. A recombinant vector containing the polynucleotide according to any one of 1.

5. a transformant obtained by transformation with the recombinant vector according to 4.

6. The transformant according to 5 above, which is transformed with the recombinant vector according to 4 above and a recombinant vector comprising a polynucleotide encoding Racl.

7.A protein represented by the amino acid sequence described in SEQ ID NO: 2 in the sequence listing,

8. a protein encoded by the polynucleotide according to 2 or 3 above,

9. The method for producing a protein according to the above 7 or 8, comprising a step of culturing the transformant according to the above 5 or 6. 10. an antibody that recognizes the protein according to the above 7. or 8.

11. A method for identifying a compound that inhibits the function of the protein according to 7 or 8 and the expression of the polynucleotide according to any one of Z or 1. force 3. By detecting the presence, absence or alteration of said function and Z or said expression under conditions that allow interaction with the protein and Z or said polynucleotide.

An identification method, which comprises determining whether the compound inhibits the function of the protein and the expression of Z or the polynucleotide,

12. The method for identification according to the above 11, which is GAP activity against Racl,

13. A method for determining whether or not a test tissue derived from a human thyroid tissue is a tissue derived from a chronic inflammatory disease of the human thyroid gland, the method comprising: A determination method characterized by measuring the expression level of the described polynucleotide,

14. The expression of the polynucleotide according to any one of the above items 1 to 3 in the test tissue is at least 4.5 times the expression level of the polynucleotide in the normal human thyroid-derived fibrous tissue as a control. In some cases, the test tissue is determined to be a tissue derived from a chronic inflammatory disease of the human thyroid gland, wherein the determination method according to the above 13,

15. A method for determining whether or not a test tissue derived from a human kidney tissue is a tissue derived from a chronic inflammatory disease of a human kidney, the method comprising: A determination method characterized by measuring the expression level of the described polynucleotide,

16. Expression strength of the polynucleotide according to any one of 3. above in the test tissue is at least 3.5 times or more the expression level of the polynucleotide in a normal human kidney-derived tissue as a control. In some cases, the test tissue is determined to be a tissue derived from a chronic inflammatory disease of a human kidney,

17. As an active ingredient, a compound that inhibits the function of the protein described in 7 or 8 above and a compound that inhibits the expression of the polynucleotide described in any one of Z or 1. Preventive and Z or therapeutic agents for chronic inflammatory diseases of the thyroid and Z or kidney

18. A compound that inhibits the function of a protein according to the above 7. or 8. 1. A method for preventing a chronic inflammatory disease of the thyroid gland and Z or a chronic inflammatory disease of the kidney, characterized by using a compound that inhibits the expression of the polynucleotide according to any one of 1. Or treatment method,

19. The protein described in 7 or 8 above, the polynucleotide described in any one of 1. above, the recombinant vector described in 4. above, and the transformation described in 5. or 6. above. A reagent kit comprising at least one of the recombinant and the antibody according to 10.

Consists of

The invention's effect

In the present invention, it is possible to provide a polynucleotide encoding a novel protein that binds to a Rho family protein and a protein encoded by the polynucleotide. This protein bound to Racl, one of the Rho family proteins. In addition, this protein has a GAP domain characteristic of Rho-GAP in its amino acid sequence. From this, it is considered that the present protein binds to a Rho protein, for example, Racl, and acts as Rho-GAP. Further, in the present invention, it is possible to provide a recombinant vector containing the polynucleotide, and a transformant transformed by the recombinant vector. Furthermore, it is possible to provide a method for producing the protein, and an antibody against the protein. Further, a method for identifying a compound that inhibits the function of the protein and the expression of Z or the polynucleotide can be provided. Further, the present invention can provide a method for diagnosing a chronic inflammatory disease, such as chronic thyroiditis and chronic nephritis, which comprises measuring the expression level of the polynucleotide. In addition, an agent for preventing and treating a chronic inflammatory disease, for example, chronic thyroiditis and Z or chronic nephritis, comprising a protein function inhibitor and Z or an expression inhibitor of the polynucleotide as an active ingredient, It is possible to provide a method for preventing a chronic inflammatory disease, for example, chronic thyroiditis and Z or chronic nephritis, and a method for treating Z or chronic nephritis, which comprise using the protein function inhibitor and the Z or expression inhibitor of the polynucleotide. is there. Further, it is possible to provide a reagent kit comprising at least one of the protein, the polynucleotide, the recombinant vector, the transformant, and the antibody. [0011] The present invention makes it possible to elucidate and regulate signaling pathways and cell functions involving Rho family proteins, for example, Racl. Further, diseases based on abnormal function of the protein encoded by the present polynucleotide and abnormal expression of Z or the present polynucleotide, such as chronic inflammatory diseases, such as chronic thyroiditis and Z or chronic nephritis, prevention, and diagnosis of Z. Or treatment becomes possible.

The present invention is a useful invention that contributes widely to the field of basic science and pharmaceutical development. Brief Description of Drawings

[FIG. 1] Panel A is a diagram showing the detection of binding between a protein encoded by sj04085 and Racl. In a cell lysate of a cell obtained by cotransfection of the sj04085 expression vector and the Racl expression vector, a band indicating the binding of the protein encoded by sj04085 to Racl was detected (lane 6 in panel A). On the other hand, in the cell lysate of cells transfected with the sj04085 expression vector and j8-Glucuronidase (GUS) expression vector, such a band was not detected (panel A lane). Five). Such bands were not detected in any of the following cell lysates (Panel A): Cells transfected with sj04085 expression vector alone (lane 4); GUS expression vector or Rac l Cells transfected only with the expression vector (lanes 2 and 3); and cells transfected only with Lipofectamine ^™ 2000 without the introduction of each vector (lane 1). The measurement of the binding was performed by a pull-down method. Specifically, sj04085 was expressed in cells as FLAG fusion protein, and Racl and GUS were expressed as GST fusion proteins. Each cell lysate was incubated with glutathione sepharose, after deploying the protein bound to glutathione ceph Arosu in SDS-PAGE, and detected binding of _s J04085 and Rac l by I beam knob blotting using anti-FLAG antibody. Panel B is a diagram in which SDS-PAGE was performed using a sample of each cell lysate, and the protein encoded by sj04085 was detected using an anti-FLAG antibody. Panel C is a diagram in which SDS-PAGE was performed using a sample of each cell lysate, and GST-fused GUS was detected using an anti-GST antibody. Panel D is a diagram in which SDS-PAGE was performed using a sample of each cell lysate, and GST-fused Racl was detected using an anti-GST antibody. In each cell lysate, the amount of protein encoded by sj04085 was almost the same (panel B, lanes 46). In each cell lysate The amount of Racl or GUS was approximately the same (lanes 3 and 6 in panel D or lanes 2 and 5 in panel C). (Example 3)

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in more detail.

In the present invention, isolated full-length DNA and Z or RNA; synthetic full-length DNA and Z or RNA; isolated DNA oligonucleotides and Z or RNA oligonucleotides; The term "polynucleotide" is used as a generic term to refer to and Z or RNA oligonucleotides, where such DNA and Z or RNA have a minimum size of 2 nucleotides.

In the present invention, a generic term is used to mean an isolated or synthetic full-length protein; an isolated or synthetic full-length polypeptide; or an isolated or synthetic full-length oligopeptide. The term "protein" is used, where a protein, polypeptide or oligopeptide has a minimum size of 2 amino acids. Hereinafter, when describing amino acids, they may be represented by one or three letters.

[0014] (polynucleotide)

One aspect of the present invention relates to novel polynucleotides. This polynucleotide was identified and isolated from a human spleen-derived cDNA library as a gene having a region encoding a GAP domain, which is a characteristic domain of Rho-GAP.

[0015] A specific embodiment of the polynucleotide according to the present invention may be, for example, a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 in the sequence listing or a complementary nucleotide sequence thereof. The polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is a 4393 bp polynucleotide and contains an open reading frame (ORF) encoding 1101 amino acid residues (SEQ ID NO: 2). In the nucleotide sequence of SEQ ID NO: 1, the region from the 421st to the 879th nucleotide has the nucleotide force from the 34th amino acid (Pro) to the 186th amino acid in the amino acid sequence shown in SEQ ID NO: 2. It encodes a GAP domain consisting of 153 amino acid residues up to the serine (Ser). Within the scope of the present invention, a polynucleotide encoding the protein represented by the amino acid sequence set forth in SEQ ID NO: 2 or a complementary base sequence of the polynucleotide. Polynucleotides are also included.

[0016] The polynucleotide according to the present invention is preferably a polynucleotide encoding a protein that binds to a Rho family protein or a polynucleotide represented by a complementary nucleotide sequence to the polynucleotide. In the present invention, the term "binding between proteins" means that a certain protein (protein A) and another certain protein (protein B) form a complex, such as a hydrogen bond, a hydrophobic bond, or an electrostatic interaction. It means that protein A and protein B interact by non-covalent bond. Here, the binding is sufficient if protein A and protein B bind at a part of those molecules. For example, the amino acids constituting protein A or protein B may contain amino acids that are not involved in the binding between protein A and protein B!

[0017] The protein encoded by the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 bound to Racl, one of Rho family proteins. Specifically, in animal cells in which both the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 and the gene encoding Racl are expressed, the binding between the protein encoded by the polynucleotide and Racl is detected. (See Example 3).

The protein encoded by the polynucleotide according to the present invention has a GAP domain in its structure and binds to a Rho family protein. The GAP domain is the active domain of GAP. Therefore, the protein encoded by the present polynucleotide is considered to have GAP activity against Rho family proteins. “GAP activity on Rho family protein” means the activity of the Rho family protein to promote GTPase activity. As a result, the protein encoded by this polynucleotide promotes the GTPase activity of Rho family proteins, and the GTP-linked active Rho family protein also changes into a GDP-linked inactive Rho family protein. Facilitate. That is, the protein encoded by the present polynucleotide can promote the inactivation of Rho family proteins. The “GAP activity for Rho family proteins” includes an activity of promoting the change of the Rho family protein from an active form to an inactive form and an activity of promoting the inactivation of the Rho family protein.

[0019] The protein encoded by the polynucleotide of the present invention is preferably a Rho family protein capable of exhibiting GAP activity, for example, Racl. Rho family proteins are not limited to these, as long as the protein encoded by the present polynucleotide can exhibit GAP activity. In this case, the Rho family protein may be, for example, as long as it can bind. In the present invention, the term Rho family protein preferably refers to Racl. The binding between the protein encoded by the polynucleotide and the Rho family protein can be measured using, for example, the Burdun method (see Example 3). Further, promoting the inactivation of promotion and Rho family protein of Rho family proteins GTP Aze activity by proteins present polynucleotide encodes, for example as described below - measured by a method using [γ ³² P] GTP, etc. it can. Such a measuring method can be carried out by referring to a known document [“Journal of Biological Chemistry J, 1998, Vol. 273, No. 44, p. 29172-29177” or the like. It is.

[0020] The amino acid sequence of Racl and the nucleotide sequence of the Racl gene are described in SEQ ID NO: 6 and SEQ ID NO: 5 in the sequence listing, respectively. Racl and its gene are not limited to those represented by each of the above sequences, but are proteins and genes having one to several mutations in each of the above sequences as long as they have a generally known Racl function. be able to. Further, for desired purposes such as promoting or deleting these functions, mutants in which one or several mutations have been introduced into each of the above sequences can also be used. Racl is obtained, for example, by preparing a transformant by transfection of a recombinant vector containing the gene into an appropriate host by a genetic engineering method known per se, and culturing the transformant. Is possible.

[0021] The polynucleotide according to the present invention is prepared based on the specific information disclosed by the present invention, for example, the sequence information on the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. , Known genetic engineering methods (Sambrook et al., Eds., “Moleki Yura-Cloating, Laboratory Ma- ual, 2nd Edition”, 1989, Cold Spring Laboratories; and Masami Muramatsu, “ Lab Manual Genetic Engineering ", ₁₉₈₈

, Maruzen Co., Ltd.).

[0022] Specifically, a cDNA library is prepared from an appropriate source in which expression of the polynucleotide according to the present invention has been confirmed according to a conventional method, and a desired clone is also selected using the cDNA library. Thus, the present polynucleotide can be obtained. The source of the cDNA may be various cells or tissues in which expression of the present polynucleotide has been confirmed, or may be based on these. Examples of such cultured cells include, for example, cells derived from human spleen. Isolation of total RNA of these origins, isolation and purification of mRNA, acquisition of cDNA and its closing can all be carried out according to ordinary methods. Also, commercially available polyA + RNA derived from human spleen can be used by constructing a cDNA library. The method of selecting a desired clone and the method of selecting a desired clone are not particularly limited, and a conventional method can be used. For example, a desired clone can be selected by using a probe or a primer which selectively and / or selectively hybridizes with the present polynucleotide. Specifically, examples include a plaque hybridization method using a probe that selectively hybridizes to the present polynucleotide, a co-hybridization method, a method combining these methods, and the like. it can. Here for

V. As the probe, a polynucleotide or the like chemically synthesized based on the sequence information of the present polynucleotide can be generally used. In addition, the polynucleotide of the present invention or a polynucleotide represented by a partial base sequence thereof which has already been obtained can also be preferably used. Furthermore, sense primers and antisense primers designed based on the sequence information of the present polynucleotide can also be used as such a probe.

[0023] Selection of a desired clone from the cDNA library is performed by, for example, confirming the expressed protein of each clone using a known protein expression system, and further using the function of the protein as an indicator. Can be implemented. As the protein expression system, any expression system known per se can be used. Specifically, a cell-free protein expression system can be exemplified [Madin, K. et al., “Proceedings of The National Academy of the National Academy of the National Academy of the United States”. of Sciences of The United States of America), 2000, Vol. 97, p. 559-564].

[0024] The function of the protein encoded by the polynucleotide of the present invention or the function of the protein of the present invention preferably refers to GAP activity against a Rho family protein, more preferably

It means the GAP activity on the Rho family protein shown by the protein of the present invention bound to the Rho family protein. More preferably, the function is G

It means the AP activity, and more preferably the GAP activity on the Rho family protein shown by the protein of the present invention bound to Racl. [0025] The polynucleotide of the present invention can be obtained by the polymerase chain reaction [hereinafter, abbreviated as PCR, Ulmer, KM, "Science", 1983, Vol. 219, p. 666—671; Ehrlich, HA, eds., "PCR Technology, Principles and Applications of DNA Amplification", 1989, Stockton Press; and Saiki, RK, et al., Science, 1985. 230, p. 1350-1354]. If it is difficult to obtain a full-length cDNA from a cDNA library, the RACE method ("Experimental Medicine", 1994, Vol. 12, No. 6, p. 615-618), especially 5'- The RACE method (Frohman, MA) et al., "Proceedings of the National Academy of sciences of the United States of America", "Proceedings of the National Academy of Sciences of the United States of America," 1988, Vol. 85, No. 23, p. 8998-9002] is suitable. Primers used for PCR can be appropriately designed based on the base sequence information of the polynucleotide, and can be obtained by synthesis according to a conventional method. Isolation and purification of the amplified DNAZRNA fragment can be performed by a conventional method. For example, it can be carried out by a gel electrophoresis method or the like.

[0026] The nucleotide sequence of the polynucleotide obtained by such a method is determined by a conventional method, for example, the didoxy method [“Proceedings of the National Academy of Sciences of the United States, United States (Proceedings of the United States). The National Academy of Sciences of United States of America), 1977, Vol. 74, p. 5463-5467) and the Maxam-Gilbert method ("Methods in Enzymology", 1980, Vol. 65, p. 499-560], or simply using a commercially available sequence kit or the like.

[0027] The polynucleotide according to the present invention is not limited to the above-described polynucleotide, and may be a polynucleotide encoding a protein having sequence homology to the above-mentioned polynucleotide and having GAP activity against a Rho family protein, or a polynucleotide of the polynucleotide. Includes polynucleotides represented by complementary base sequences. Preferably, it is a polynucleotide having sequence homology to the present polynucleotide and encoding a protein that binds to a Rho family protein, and a polynucleotide represented by a complementary nucleotide sequence of the polynucleotide. Sequence homology is usually It is appropriate that the total nucleotide sequence is 50% or more, preferably at least 70%. It is more preferably at least 70%, further preferably at least 80%, even more preferably at least 90%. Preferably, a polynucleotide having a GAP domain coding region is preferred. Preferably, the sequence homology in the GAP domain coding region is at least 70%. It is more preferably 70% or more, further preferably 80% or more, and still more preferably 90% or more. It is further preferred that the GAP domain retains its function.

[0028] In the polynucleotide according to the present invention, one or more, for example, 1 to: L00, preferably 1 to 30, more preferably 1 to 20, and still more preferably 1 to 100 nucleotides in the base sequence of the polynucleotide. 1 to: A polynucleotide represented by a nucleotide sequence having a mutation such as deletion, substitution, addition or insertion of L0, particularly preferably 1 to several nucleotides, or a nucleotide sequence complementary thereto is included. The degree of mutation, their position, and the like are not particularly limited as long as the polynucleotide having the mutation is a protein having GAP activity against Rho family proteins, more preferably a protein that binds to Rho family proteins. More preferably, it is a polynucleotide encoding a protein having a GAP domain. A polynucleotide having such a mutation may be a naturally-occurring polynucleotide and a polynucleotide having an induced mutation. Further, it may be a polynucleotide obtained by introducing a mutation based on a naturally-occurring gene. Methods for introducing mutations are known per se, and for example, site-directed mutagenesis, homologous recombination, primer extension, PCR and the like can be used alone or in an appropriate combination. For example, the method described in a compendium [Sambrook et al., Eds., "Molecular Cloning, Laboratory Manual, Second Edition", 1989, Cold Spring Harbor Laboratory; and Masamura Muramatsu, "Lab Manual Genetic Engineering" 1988, Maruzen Co., Ltd.] or by modifying those methods, and can be carried out using Ulmer's technology [Ulmer, K.M., “Science”, 1983. 219, p. 666—671].

[0029] Examples of the polynucleotide according to the present invention include a polynucleotide that hybridizes to the above-mentioned polynucleotide under stringent conditions. Hybridize The conditions for the screening are according to, for example, the method described in a compendium (edited by Sambrook et al., "Molecular Closing, Laboratory Machines, 2nd Edition", Cold Spring Novell Laboratory, 1989). be able to. Specifically, "under stringent conditions" means, for example, after heating at 42 ° C. in a solution of 6 × SSC, 0.5% SDS and 50% honolemamide, 0.1 × Conditions for washing at 68 ° C in a solution of SSC, 0.5% SDS are as follows. These polynucleotides need not be polynucleotides having a complementary sequence as long as they hybridize to the present polynucleotide. Preferably, it is a polynucleotide encoding a protein having a GAP domain. More preferably, the encoded protein is a protein having GAP activity against a Rho family protein, and even more preferably, the encoded protein is a protein that binds to a Rho family protein.

[0030] The polynucleotide according to the present invention includes an oligonucleotide represented by a partial base sequence existing in a designated region of the polynucleotide. Such an oligonucleotide is preferably composed of 5 or more nucleotides, more preferably 10 or more, more preferably 20 or more nucleotides continuous in the region as a minimum unit. Specifically, an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 3 or 4 in the sequence listing can be preferably exemplified. These oligonucleotides can be used as a primer for amplifying the present gene or the present gene fragment, a probe for detecting the present gene or a transcription product thereof, and the like. These oligonucleotides can be produced by designing a desired sequence according to the nucleotide sequence information of the polynucleotide according to the present invention, and by a chemical synthesis method known per se. Conveniently, it can be obtained using a DNAZRNA automatic synthesizer.

[0031] The polynucleotide according to the present invention is preferably a human-derived polynucleotide. However, as long as it is a protein having sequence homology to the present polynucleotide and having a GAP activity against a Rho family protein, preferably a polynucleotide encoding a protein that binds to a Rho family protein, a polynucleotide derived from a mammal, For example, polynucleotides derived from mice, horses, sheep, horses, dogs, dogs, monkeys, cats, bears, rats, and egrets are also included in the present invention. Sequence homology is usually preferred to be 50% or more of the entire base sequence. Or at least 70%. It is more preferably 70% or more, further preferably 80% or more, and still more preferably 90% or more. Preferably, a polynucleotide having a GAP domain coding region is desirable. Preferably, sequence homology in the GAP domain coding region is at least 70%. More preferably, it is at least 70%, further preferably at least 80%, even more preferably at least 90%.

[0032] The polynucleotide according to the present invention may be a polynucleotide having a desired gene added to its 5, terminal or ^ terminal as long as its expression or the function of the protein encoded thereby is not inhibited. !,. Specific examples of genes that can be added to the polynucleotide include daltathione S-transferase (GST), monogalactosidase (j8-Gal), horseradish peroxidase (HRP), and alkaline phosphatase. Examples include enzymes such as (ALP), and genes such as tag peptides such as His-tag, Myc-tag, HA-tag, FLAG-tag and Xpress-tag. One or more types of genes selected from these genes can be added in combination. The addition of these genes can be performed by conventional genetic engineering techniques, and is useful for facilitating the detection of genes and mRNA.

[0033] (vector)

One aspect of the present invention relates to a recombinant vector containing the polynucleotide according to the present invention. The present recombinant vector can be obtained by inserting the present polynucleotide into an appropriate vector DNA.

[0034] The vector DNA is not particularly limited as long as it can be replicated in the host, and is appropriately selected depending on the type of the host and the purpose of use. The vector DNA may be a vector DNA obtained by extracting a naturally-occurring DNA, or a vector DNA lacking a part of a DNA other than a part necessary for replication. Representative vector DNAs include, for example, vector DNAs derived from plasmids, batteriophages and viruses. Examples of the plasmid DNA include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, and a plasmid derived from yeast. Batatheriophage DNA includes λ phage and the like. Examples of Winnores-derived vector DNAs include, for example, Retro Pinoles, Vaccinia Pinoles, Examples include vectors derived from animal viruses such as adenovirus, papovavirus, SV40, fowlpox virus, and pseudorabies virus, and some vectors include vectors derived from insect viruses such as baculovirus. Other examples include transposon-derived, insertion element-derived, and yeast chromosome element-derived vector DNA. Alternatively, vector DNAs prepared by combining them, for example, vector DNAs (cosmids / phagemids etc.) prepared by combining genetic elements of plasmids and batteriophages can be exemplified.

[0035] Any vector DNA, such as an expression vector or a closing vector, can be used depending on the purpose. The recombinant expression vector containing the polynucleotide according to the present invention can be used for producing a protein encoded by the present polynucleotide.

[0036] It is necessary to incorporate the polynucleotide into the vector DNA so that the function of the polynucleotide according to the present invention is exhibited, and at least the present polynucleotide and a promoter are constituent elements thereof. In addition to these elements, if desired, a gene sequence carrying information on replication and control can be combined and incorporated into vector DNA by a method known per se. Such gene sequences include, for example, ribosome binding sequences, terminators, signal sequences, cis elements such as enhancers, splicing signals, and selectable markers (dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, etc. ) And the like. One or more gene sequences selected from these can be incorporated into the vector DNA.

As a method for incorporating the polynucleotide according to the present invention into a vector DNA, a method known per se can be applied. For example, a method is used in which a gene containing the present polynucleotide is treated with an appropriate restriction enzyme, cut at a specific site, then mixed with a similarly treated vector DNA, and religated with ligase. Alternatively, a desired recombinant vector can also be obtained by ligating a suitable linker to the present polynucleotide and inserting it into a multicloning site of a vector suitable for the purpose.

[0038] (Transformant)

One aspect of the present invention relates to a transformant obtained by transforming a host with the recombinant vector of the present invention. A transformant into which the recombinant expression vector containing the polynucleotide according to the present invention has been introduced is useful for producing a protein encoded by the present polynucleotide. It is. One or more kinds of vector DNAs containing a desired gene other than the present polynucleotide can be further introduced into the present transformant. As a vector DNA containing a desired gene other than the present polynucleotide, for example, a vector DNA containing a gene encoding a Rho family protein such as Racl can be mentioned. A transformant obtained by transformation with an expression vector containing the present polynucleotide and an expression vector containing a gene encoding a Rho family protein has a GAP for the Rho family protein of the protein encoded by the present polynucleotide. It can be used in a method for identifying a compound that inhibits the activity. Specifically, it is used for the method of identifying a compound that inhibits the binding of the present protein to the Rho family protein, the promotion of the GTPase activity of the Rho family protein by the present protein, and the promotion of the inactivation of the Rho family protein by the Z or the present protein. it can. Such a transformant is preferably a transformant obtained by transforming the recombinant vector according to the present invention with a recombinant vector containing a gene encoding Racl.

[0039] Prokaryotic and eukaryotic organisms can also be used as hosts. Examples of prokaryotes include genus Escherichia such as Escherichia coli, genus Bacillus such as Bacillus subtilis, genus Pseudomonas such as Pseudomonas putida, and Rhizobium meliloti. And bacteria belonging to the genus Rhizobium. Examples of eukaryotes include animal cells such as yeast, insect cells, and mammalian cells. Examples of yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and the like. Examples of insect cells include Sf9 cells and Sf21 cells. Examples of the mammalian cells include monkey kidney-derived cells (such as COS cells and Vero cells), Chinese nose, Muster ovary cells (CHO cells), mouse L cells, rat GH3 cells, human FL cells, and 293EBNA cells. Preferably, mammalian cells are used. Most preferably, 293EBNA cells are used.

[0040] Transformation of a host cell with a recombinant vector can be carried out by applying a means known per se. For example, the method can be carried out by a standard method described in a compendium (Sambrook et al., Edited by "Molecular Cloning, Laboratory Machines, Second Edition", 1989, Cold Spring Nover Laboratory). A more preferable method is to use gene safety. If qualitative considerations are taken into account, an integration method into the chromosome can be mentioned. For convenience, an autonomous replication system using extranuclear genes can be used. Specifically, calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, cationic lipid-mediated transfection, electoral poration, transduction, scrape loading, ballistic introduction and ballistic introduction Infection.

When a prokaryote is used as a host, the recombinant vector must be capable of autonomous replication in the prokaryote and, at the same time, be composed of a promoter, a ribosome binding sequence, the polynucleotide of the present invention, and a transcription termination sequence. preferable. In addition, a gene that controls a promoter may be included. When a bacterium is used as a host, any promoter can be used as long as it can be expressed in bacteria such as Escherichia coli. For example, promoters derived from the Otsuki Bacillus phage, such as the trp motor, the same promoter, the pL promoter, and the pR promoter, are used. An artificially designed and modified promoter such as the tac promoter may be used. The method for introducing the recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria, and any method can be used. Preferably, for example, in the case of using a mammalian cell as a host which can use a method using calcium ion, an electoral poration method, or the like, the recombinant vector can be autonomously replicated in the cell, and at the same time, a promoter and a price site can be used. , A polynucleotide according to the present invention, a polyadenylation site, and a transcription termination sequence. Further, a replication origin may be included if desired. As the promoter, an SRa promoter, an SV40 promoter, an LTR promoter, a CMV promoter or the like may be used, or an early gene promoter of cytomegalovirus may be used. As a method for introducing the recombinant vector into mammalian cells, preferably, for example, an electoral port method, a calcium phosphate method, a lipofection method, or the like can be used. Most preferably, the Lipofection method is used.

When yeast is used as a host, the promoter is not particularly limited as long as it can be expressed in yeast, and examples thereof include gall promoter, gallO promoter, and heat shock. Examples include a protein promoter, an MFa1 promoter, a PH05 promoter, a PGK promoter, a GAP promoter, an ADH promoter, and an AOX1 promoter. The method for introducing the recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and preferably, for example, an electoporation method, a spheroplast method, a lithium acetate method, or the like can be used.

When an insect cell is used as a host, the recombinant vector can be preferably introduced by, for example, a calcium phosphate method, a lipofection method, an electoral poration method, or the like.

[0042] (protein)

One embodiment of the present invention relates to a protein encoded by the polynucleotide of the present invention.

[0043] A specific embodiment of the protein according to the present invention includes, for example, a protein encoded by a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1. More specifically, a protein represented by the amino acid sequence of SEQ ID NO: 2 can be exemplified as such a protein. In this protein, the amino acid residues from the 34th proline (Pro) to the 186th serine (Ser) constitute the GAP domain.

[0044] The protein according to the present invention is preferably a protein having GAP activity against Rho family proteins. The protein encoded by the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 bound to Racl, one of Rho family proteins. Specifically, in animal cells in which the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 and the gene encoding Racl are both expressed, the binding between the protein encoded by the polynucleotide and Rac1 was detected. (See Example 3). The polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 encodes the protein represented by the amino acid sequence of SEQ ID NO: 2. Therefore, it is considered that the protein represented by the amino acid sequence of SEQ ID NO: 2 bound to Racl.

[0045] The protein according to the present invention has a GAP domain in its structure and binds to a Rho family protein. The GAP domain is the active domain of GAP. Therefore, the present protein is considered to have GAP activity against the Rh family protein. More specifically, the protein represented by the amino acid sequence of SEQ ID NO: 2 is considered to have GAP activity against Racl, one of Rho family proteins. [0046] The protein according to the present invention is not limited to the above proteins, and any protein encoded by the polynucleotide according to the present invention is included in the scope of the present invention. Preferably, a protein encoded by the present polynucleotide, which has GAP activity against a Rho family protein, is included. More preferably, it includes a protein encoded by the present polynucleotide, which protein binds to a Rho family protein. As such a protein, for example, a polynucleotide encoding the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence and the polynucleotide encoding the protein represented by the amino acid sequence of SEQ ID NO: 2 or A polynucleotide represented by a nucleotide sequence having at least 70% homology with the nucleotide sequence of any one of the polynucleotides selected from the polynucleotides represented by the complementary nucleotide sequences of the polynucleotide, wherein the Rho family protein And a protein that encodes a polynucleotide that encodes a protein that binds to a protein that has GAP activity against Rho or a Rho family protein. Also, for example, a polynucleotide represented by a nucleotide sequence having a mutation such as deletion, substitution, addition or the like of one or several nucleotides or an induced mutation in the nucleotide sequence of any one of the polynucleotides selected from the above-mentioned polynucleotide group A protein having GAP activity against a Rho family protein or a protein encoded by a polynucleotide encoding a protein that binds to a Rho family protein. Furthermore, a polynucleotide that hybridizes under stringent conditions with a polynucleotide of any force selected from the above-mentioned polynucleotide groups, and binds to a protein having GAP activity against a Rho family protein or to a Rho family protein. It may be a protein encoded by a polynucleotide encoding the protein.

[0047] More specifically, such a protein according to the present invention has sequence homology to the protein represented by the amino acid sequence of SEQ ID NO: 2 and has a GAP activity against a Rho family protein. Can be exemplified. More preferably, it is a protein having sequence homology to the protein represented by the amino acid sequence of SEQ ID NO: 2 and binding to a Rho family protein. The sequence homology is usually at least 50%, preferably at least 70%, of the entire amino acid sequence. It is more preferably 70% or more, further preferably 80% or more, and still more preferably 90% or more. Also preferably, GAP A polypeptide having a domain is desirable. Preferably, the sequence homology in the GAP domain is at least 70%. It is more preferably at least 70%, further preferably at least 80%, even more preferably at least 90%. More preferably, the GAP domain retains its function. In addition, as the present protein, one or more amino acids in the amino acid sequence of SEQ ID NO: 2, for example, 1 to: L00, preferably 1 to 30, more preferably 1 to 20, still more preferably 1 to 10, Particularly preferred is a protein represented by an amino acid sequence having a mutation such as deletion, substitution, addition or insertion of one or several amino acids and having GAP activity against Rho family protein. More preferably, it is a protein represented by the amino acid sequence having the above mutation in the amino acid sequence of SEQ ID NO: 2, and which binds to a Rho family protein. The degree of amino acid mutation and the position thereof are not particularly limited as long as the protein having the mutation is a protein having GAP activity against a Rho family protein, more preferably a protein that binds to a Rho family protein. More preferably, it is a protein having a GAP domain. Proteins having such mutations may be those naturally produced by, for example, mutation or post-translational modification, and those obtained by introducing mutations based on naturally-occurring genes. Good. Methods for introducing mutations are known per se, and can be carried out using, for example, known genetic engineering techniques. The point of view that the mutation does not alter the basic properties (physical properties, function, physiological activity or immunological activity, etc.) of the protein is, for example, related amino acids (polar amino acids, non-polar amino acids, hydrophobic amino acids). Mutual substitutions between amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids and aromatic amino acids) are readily envisioned.

[0048] The protein according to the present invention further includes a protein represented by a partial sequence of the above protein. For example, a protein represented by the partial sequence of the protein represented by the amino acid sequence of SEQ ID NO: 2 is also included in the scope of the present invention. Such a protein is represented by a minimum unit of preferably 5 or more, more preferably 8 or more, still more preferably 12 or more, particularly preferably 15 or more continuous amino acids.

[0049] The protein according to the present invention is preferably a human-derived protein. However, proteins having sequence homology with the present protein and having GAP activity against Rho family proteins are preferred. Preferably, as long as it is a protein that binds to a Rho family protein, it is a protein derived from a mammal, such as a mouse, a horse, a sheep, a horse, a dog, a monkey, a cat, a bear, a rat, or a rabbit. Derived proteins are also included in the present invention. The sequence homology is usually at least 50%, preferably at least 70%, as a whole of the amino acid sequence. More preferably, it is at least 70%, further preferably at least 80%, even more preferably at least 90%. Preferably, a protein having a GAP domain is desirable. Preferably, the sequence homology in the GAP domain is at least 70%. It is more preferably at least 70%, further preferably at least 80%, even more preferably at least 90%.

[0050] The protein according to the present invention may be a cell, a cell-free synthetic product, a chemical synthetic product, or a biological sample derived from the cell or the organism, in which a gene encoding the protein is expressed by a genetic engineering technique. The power was also prepared, and these powers were also more refined.

[0051] The protein according to the present invention can be further modified as long as its function is not remarkably changed, for example, by modifying its constituent amino group or carboxyl group or the like by amidation modification. Labeling was performed by adding another protein or the like to the N-terminal side or C-terminal side directly or indirectly via a linker peptide or the like using genetic engineering techniques or the like. May be something. Preferably, labeling is desired so that the function of the present protein is not inhibited. Substances (labeling substances) used for labeling such as enzymes such as GST, β-Gal, HRP or ALp, His-tag, Myc-tag, HA-tag, FLAG-tag or Xpress-tag Tag peptides, fluorescent substances such as fluorescein isothiocynate or phycoerythrin, maltose-binding protein, Fc fragment of immunoglobulin or biotin. Not. Labeling with a radioisotope is also possible. The labeling substance can be added to the present protein in one kind or in combination of two or more kinds. By measuring the labeling substance itself or its function, the present protein can be easily detected or purified.For example, it is possible to detect the binding of the present protein to another protein or to measure the function of the present protein. .

(Method for Producing Protein)

One embodiment of the present invention relates to a method for producing the protein of the present invention. This protein, for example, For example, a general genetic engineering method based on the nucleotide sequence information of the gene encoding the present protein [Sambrook et al., Edited by Molecular Cloning, Laboratory Manual, 2nd Edition, 1989, Cold Spring Harbour Laboratory; Masanori Muramatsu, "La Boma-Yual Genetic Engineering", 1988, Maruzen Co .; Ulmer, KM, "Science", 1983, Vol. 219, p. 666—671; And Enolichich (HA), edited by PCR Technology, Principles and Applications of DNA Amplification, 1989, Stockton Press, etc.]. For example, a cDNA library is first prepared from various cells and tissues in which expression of the polynucleotide according to the present invention has been confirmed, or cultured cells derived therefrom in accordance with a conventional method. Next, the cDNA library is also amplified using the primers that selectively hybridize to the gene encoding the present protein. The present protein can be obtained by inducing the expression of the obtained polynucleotide using a known gene engineering technique.

[0053] Specifically, for example, the present protein can be produced by culturing the transformant according to the present invention and then recovering the present protein from the obtained culture. The present transformant can be cultured under culture conditions and methods known per se that are optimal for the host used to prepare the transformant. The culturing can be carried out using the present protein itself expressed by the transformant or the function of the present protein as an index. Alternatively, subculture or batch culture may be performed using the amount of the transformant in the medium, which can be cultured using the present protein itself or the amount of the protein produced in or outside the host as an index. .

When the protein of the present invention is expressed in the cells of the transformant or on the cell membrane, the transformant is crushed to extract the present protein. When the protein is secreted outside the transformant, use the culture solution as it is or use a culture solution from which the transformant has been removed by centrifugation or the like.

[0055] The protein according to the present invention can be separated and Z- or purified from a culture solution or a transformant in which the transformant has been cultured, if desired. Separation and Z or purification can be performed by various separation procedures utilizing the physical properties, chemical properties, etc. of the present protein. Specific examples of the separation operation method include ammonium sulfate precipitation, ultrafiltration, gel chromatography, ion exchange chromatography, affinity chromatography, and high-speed separation. Examples thereof include liquid chromatography and dialysis. Further, these methods can be appropriately combined and used. Preferably, it is recommended to use affinity chromatography using a column to which a specific antibody against the present protein is bound. A specific antibody to the present protein can be obtained by a known antibody preparation method based on the amino acid sequence information of the present protein. When performing separation and Z or purification, the function of the present protein can be used as an index for obtaining the present protein.

[0056] The protein according to the present invention can also be produced by a general chemical synthesis method. As a method for chemically synthesizing a protein, for example, a solid phase synthesis method, a liquid phase synthesis method and the like are known. Either method can be used. More specifically, such a protein synthesis method is based on amino acid sequence information, in which each amino acid is successively linked one by one to extend the chain, which is referred to as the so-called stepwise longation method. A fragment condensing method in which a fragment having several amino acids is synthesized in advance, and then the respective fragments are subjected to a coupling reaction. The synthesis of the present protein can be performed by any of them. The condensation method used in the above protein synthesis method can also follow a conventional method. Examples of the condensation method include azide method, mixed acid anhydride method, DCC method, active ester method, oxidation-reduction method, DPPA (diphenylphosphoryl azide) method, DCC + Sol, N-hydroxysuccinamide, N-hydroxy-15-norbornene-1,2,3-dicarboximide) method, Woodward method and the like. The present protein obtained by chemical synthesis can be further appropriately purified by various conventional purification methods as described above.

[0057] The protein represented by the partial sequence of the protein according to the present invention can also be obtained by cleaving the present protein with an appropriate peptidase.

[0058] (Antibody)

One embodiment of the present invention relates to an antibody that recognizes the protein of the present invention. The present antibody can be prepared using the present protein as an antigen. The antigen is composed of at least 8, preferably at least 10, more preferably at least 12, and even more preferably 15 or more amino acids, which may be the present protein or a fragment thereof. In order to prepare an antibody specific to the present protein, it is preferable to use, as an antigen, a region having amino acid sequence power unique to the present protein. The amino acid sequence of this region is not necessarily the amino acid sequence of the present protein or a fragment thereof. It need not be the same as the sequence. As such an amino acid sequence, an amino acid sequence at an exposed site on the three-dimensional structure of a protein is preferable. Even if the amino acid sequence at the exposed site is discontinuous on the primary structure, the amino acid sequence should be continuous at the exposed site. The present antibody is not particularly limited as long as it is an antibody that specifically recognizes the present protein. Recognizing the present protein specifically means recognizing the present protein, for example, binding to the present protein, but not recognizing or weakly recognizing a protein other than the present protein. The presence or absence of recognition can be determined by a known antigen-antibody binding reaction.

[0059] Antibodies can be produced by a known antibody production method. For example, antibody production is achieved by administering an antigen to an animal in the presence or absence of an adjuvant, alone or in combination with a carrier, to induce immunity such as humoral response and Z or cellular response. Can be applied. As the carrier, any known carrier can be used as long as it has no adverse effect on the host itself and enhances antigenicity. Specific examples include cellulose, polymerized amino acids, albumin, and keyhole limpet hemocyanin. As adjuvants, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL + TDM), pertussis vaccine (Bordet ella pertussis vaccine), muramyl dipeptide (MDP) ), Aluminum adjuvant (ALUM), and combinations thereof. As an animal to be immunized, a mouse, a rat, a heron, a goat, a horse, and the like are suitably used.

[0060] The polyclonal antibody can be obtained from the serum of an animal to which immunization has been applied by a known antibody recovery method. Preferred antibody recovery means includes immunoaffinity chromatography.

[0061] Monoclonal antibodies include antibody-producing cells (for example, lymphocytes derived from spleen or lymph node) collected from an animal to which immunization has been applied, and perpetually proliferating cells known per se (for example, P3-X63- Ag8 strains and other myeloma strains) can be produced using a hybridoma. For example, a hybridoma is prepared by fusing antibody-producing cells and perpetually proliferating cells in a manner known per se, and then cloned. Hybridomas that produce an antibody that specifically recognizes the protein according to the present invention are selected from the cloned hybridomas, and the antibody in the culture solution of the hybridomas is also recovered. [0062] A polyclonal antibody or a monoclonal antibody capable of recognizing or binding to the protein according to the present invention can be used as an antibody for purifying the protein, a reagent, a label marker, or the like. In particular, antibodies that inhibit the function of this protein can be used to regulate the function of this protein, and are useful for elucidation, prevention, improvement, and Z or treatment of various diseases caused by abnormal or quantitative abnormalities of this protein. It is.

(Method for identifying compound)

One embodiment of the present invention relates to a method for identifying a compound that inhibits the function of the protein according to the present invention or a compound that inhibits expression of the polynucleotide according to the present invention. The present identification method can be carried out using at least one of the protein, polynucleotide, recombinant vector, transformant and antibody according to the present invention, using a drug screening system known per se. . The identification method includes any method performed in vitro or in vivo. This identification method enables selection of antagonists by drag design based on the three-dimensional structure of the protein, selection of inhibitors of gene-level expression using a protein synthesis system, or selection of antibody-recognizing substances using antibodies. It is possible.

[0064] The method for identifying a compound that inhibits the function of a protein according to the present invention is characterized in that, in an experimental system capable of measuring the function of the protein, the interaction between the protein and the compound to be examined (test compound) is tested. The function of the present protein is measured in the presence of the test compound and the function of the present protein in the absence of the test compound. The present invention can be carried out by comparing the function of the protein and detecting the presence, absence, or change of the function of the present protein, for example, reduction, increase in calorie, disappearance, or appearance. When the function of the present protein in the presence of the test compound decreases or disappears as compared with the function of the present protein in the absence of the test compound, it can be determined that the test compound inhibits the function of the present protein. The function can be measured by directly detecting the function, or by, for example, introducing a signal as an indicator of the function into an experimental system and detecting the signal. Enzymes such as GST, tag peptides such as His-tag, Myc-tag, HA-tag, FLAG-tag or Xpress-tag, and fluorescent proteins can be used as the signal. Other than these exemplified substances, deviations can also be used as long as they are generally used in a compound identification method and are labeled substances. [0065] The protein according to the present invention bound to a Rho family protein. Since this protein has a GAP domain, it is considered that this protein has GAP activity against Rho family proteins. That is, the functions of the present protein include the activity of promoting the GTPase activity of the Rho family protein and the activity of promoting the inactivation of the Rho family protein.

[0066] The identification method using the binding of the protein to the Rho family protein according to the present invention as an index is, for example, obtained by expressing the present protein by a genetic engineering technique, in the presence or absence of a test compound. The detection can be performed by detecting the binding to the Rho family protein in the above. Specifically, for example, a Rho family protein is expressed as a GST-tag fusion protein by genetic engineering techniques, and then bound to daltathione sepharose, and the protein is combined with the present protein in the presence or absence of a test compound. Let react. By quantifying the present protein that binds to the Rho family protein bound to daltathione sepharose, it is possible to identify a compound that inhibits the binding of the present protein to the Rho family protein. When the binding of both proteins in the presence of the test compound is reduced or eliminated as compared to the binding of both proteins in the absence of the test compound, the test compound binds to the Rho family protein of the present protein. It can be determined that binding is inhibited. Quantification of the present protein can be performed, for example, using the antibody according to the present invention. As the antibody, an antibody labeled with an enzyme such as HRP or ALP, a radioisotope, a fluorescent substance, or a labeling substance such as biotin can be used. Alternatively, a labeled secondary antibody may be used. If a protein fused with a tag peptide is used as the present protein, quantification can be performed using an anti-tag antibody. Alternatively, the present protein may be directly labeled with a labeling substance such as the above enzyme, radioisotope, fluorescent substance, and biotin. In such a case, the present protein can be quantified by measuring the labeling substance.

More specifically, using appropriate cells in which a polynucleotide encoding the protein of the present invention and a polynucleotide encoding a Rho family protein are co-expressed, the binding between the two proteins is detected by a pull-down method. Using an experimental system, compounds that inhibit the binding can be identified (see Example 3).

[0068] In addition, a compound that inhibits the binding of the protein of the present invention to a Rho family protein is also included. The determination method can be performed using a known two-hybrid method. For example, it contains a plasmid expressing the protein of the present invention and a DNA binding protein as a fusion protein, a plasmid expressing a Rho family protein and a transcriptionally active protein as a fusion protein, and a reporter gene connected to an appropriate promoter gene. The resulting plasmid is introduced into yeast, eukaryotic cells, or the like. Next, by comparing the expression level of the reporter gene in the presence of the test compound with the expression level of the reporter gene in the absence of the test compound, the expression of the compound that inhibits the binding between the present protein and the Rho family protein was determined. The same can be achieved. When the expression level of the reporter gene in the presence of the test compound decreases or disappears as compared with the expression level of the reporter gene in the absence of the test compound, the test compound is compared with the Rho family protein of the present protein. It can be determined that binding is inhibited. As the reporter gene, a gene generally used in the reporter atssay can be used. Specifically, a gene having an enzymatic activity such as luciferase, β Gal or chloramue-co-acetylacetyl transferase can be exemplified. The expression of the reporter gene can be detected by detecting the activity of the gene product, for example, the enzyme activity in the case of the above-described reporter gene.

[0069] The method for identifying a compound that inhibits the binding between the protein and the Rho family protein according to the present invention can also be carried out using a surface plasmon resonance sensor such as a BIACORE system. Alternatively, the identification method is performed using a scintillation proximity assay (SPA) or a force method that applies optical resonance energy transfer (FRET). It is possible.

[0070] The method for identifying a compound that inhibits the GAP activity of the protein according to the present invention can be carried out, for example, by using a general method for measuring the GAP activity of Rho-GAP. The measurement method of the Rho-GAP of GAP activity, e.g., measurement method worn exemplified using [γ- ³² P] GTP [ "Journal O Bed Biological Chemistry (Journal of Biological

Chemistry, 1998, Vol. 273, No. 44, p. 29172-29177]. Rho-GAP method Measurement of GAP activity is specifically purified Rho protein is reacted with [γ- ³² P] GTP, to form a GTP-bound Rho protein. Then, [γ- ³² P] GTP-bound R The ho protein is reacted with the protein of the present invention. [γ-P] GTP-linked Rho family One protein can also measure the GAP activity of this protein by measuring the change to GDP-linked Rho family protein. [Γ- ³² P] measured changes to GTP-bound Rho family protein force also GDP-bound Rho family proteins, the change to [γ- ³² P] GTP-bound Rho family蛋HakuTadashiryoku also GDP-bound Rho family protein It can be carried out by measuring the released γ- ³²される. Specifically, after reacting the present protein with [γ ³² P] GTP-bound Rho protein, the reaction solution is filtered through a trocellulose filter or the like, and the trocellulose filter is washed, dried, and released. the _Ύ ³² p measurements can be measured by the amount of decrease in radioactivity of the filter. Since the protein is captured by the nitrocellulose filter, the Rho protein is captured by the filter, and the released ³² P passes through the filter. GTP-linked Rho protein also increases the amount of free ³² P as the reaction with the GDP-linked Rho protein progresses, and decreases the amount of ³² P captured by the trocellulose filter via the Rho protein. In other words, if the radioactivity of the nitrocellulose filter decreases, it can be said that the reaction of the GTP-bound Rho protein with the GDP-bound Rho protein is in progress. Therefore, when the radioactivity of the -trocellulose filter in the presence of the protein according to the present invention is reduced as compared to the case in the absence of the protein, the protein has GAP activity against the Rho protein. Then it can be determined.

[0071] The method of identifying a compound that inhibits the GAP activity protein has according to the present invention, for example, a method of measuring the GAP activity of Rho-GAP with [.gamma. ³² P] GTP, as described above, the test It can be carried out in the presence or absence of a compound. Specifically, after reaction in the presence or absence of [γ 3 ² P] GTP-bound Rho protein and the protein according to the present invention, the test compound, [γ- ³² P] GTP-bound Measure the change from Rho family protein to GDP-linked Rho family protein. Γ- ³² P] GTP-binding Rho family protein in the presence of test compound also decreases or disappears when changes to GDP-binding Rho family protein are reduced compared to changes in the absence of test compound It can be determined that the test compound inhibits the GAP activity of the present protein on the Rho family protein.

[0072] The Rho family protein used in the identification method of the present invention is a GTPase thereof. As long as the activity and the effect of the protein of the present invention on the GTPase activity are not affected, the protein may be partially defective or may be a protein to which the above-mentioned labeling substance has been added. .

[0073] The method for identifying a compound that inhibits the expression of a polynucleotide according to the present invention comprises a method for determining the interaction between the present polynucleotide and a test compound in an experimental system capable of measuring the expression of the present polynucleotide. The expression of the polynucleotide is measured in the presence of the test compound, and the expression of the polynucleotide is measured in the absence of the test compound. And the presence, absence or change of the expression of the present polynucleotide, for example, reduction, increase, disappearance, or appearance, can be detected. The test compound inhibits the expression of the present polynucleotide when the expression of the present polynucleotide in the presence of the test compound decreases or disappears compared to the expression of the present polynucleotide in the absence of the test compound. Then it can be determined. Specifically, for example, the present identification method comprises bringing the transformant into contact with a test compound in an experimental system for expressing the present polynucleotide using the transformant according to the present invention. Thereafter, the expression can be measured by measuring the expression of the present polynucleotide. The expression can be measured simply by using the amount of the expressed protein or the function of the protein as an index. In addition, for example, expression can be measured by introducing a signal as an index of expression into an experimental system and detecting the signal. Enzymes such as GST, tag peptides such as His-tag, Myc-tag, HA-tag, FLAG-tag or Xpress-tag, and fluorescent substances can be used as the signal. Methods for detecting these signals are well known to those skilled in the art.

[0074] The method for identifying a compound that inhibits the expression of a polynucleotide according to the present invention also includes, for example, preparing a vector in which a reporter gene is linked instead of the polynucleotide downstream of the promoter region of a gene containing the polynucleotide. The method can be carried out by contacting a test compound with a cell into which the vector has been introduced, for example, a eukaryotic cell, and measuring the presence, absence, or change in the expression of the reporter gene. As a reporter gene, a gene commonly used in reporter attesties can be used. Specifically, luciferase, β Gal or chloramphene-coal acetyltransferase, etc. A gene having the above enzyme activity can be used. The expression of the reporter gene can be detected by detecting the activity of the gene product, for example, the enzyme activity of the reporter gene exemplified above.

[0075] (Compound)

The compound obtained by the identification method according to the present invention can be used as a candidate compound such as an inhibitor or an antagonist of the function of the protein according to the present invention. Further, it can also be used as a candidate for the polynucleotide expression inhibitor according to the present invention. These candidate compounds can be prepared as a medicament by selecting them in consideration of the balance between their usefulness and toxicity, and can be used in various pathological conditions caused by abnormal function of the present protein and abnormal expression of Z or the present polynucleotide. The effect of preventing symptoms and Z or therapeutic effects can be expected. Further, the compound according to the present invention is a compound obtained by a method other than the present identification method, and includes a compound that inhibits the function of the present protein and the expression of Z or the present polynucleotide.

(Pharmaceutical composition)

One embodiment of the present invention comprises, as an active ingredient, the protein, polypeptide, recombinant vector, transformant, antibody, or compound according to the present invention, and inhibits the function of the protein and the expression of Z or the polypeptide. It relates to a medicament or a pharmaceutical composition based on antagonism.

[0077] The medicament according to the present invention does not include a medicament containing at least one of the protein, polynucleotide, recombinant vector, transformant, antibody, or compound according to the present invention as an active ingredient and an effective amount thereof. You may. Usually, it is preferable to produce a pharmaceutical composition using one or more kinds of pharmaceutically acceptable carriers (pharmaceutical carriers).

[0078] The amount of the active ingredient contained in the pharmaceutical preparation according to the present invention is appropriately selected from a wide range. Usually, it is suitably in the range of about 0.0001 to 70% by weight, preferably about 0.0001 to 5% by weight.

[0079] As the pharmaceutical carrier, diluents and excipients such as fillers, extenders, binders, humectants, disintegrants, lubricants and the like which are usually used depending on the use form of the preparation are used. Can be illustrated. These are appropriately selected and used depending on the administration form of the obtained preparation. [0080] For example, water, a pharmaceutically acceptable organic solvent, collagen, polybutyl alcohol, polybutylpyrrolidone, carboxybutyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, Examples include zein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, glycerin, paraffin, stearyl alcohol, stearic acid, human serum albumin, mantole, sorbitol, ratatose and the like. These are used singly or in combination of two or more depending on the dosage form according to the present invention.

[0081] If desired, various components that can be used in ordinary protein preparations, for example, a stabilizer, a bactericide, a buffer, a tonicity agent, a chelating agent, a pH adjuster, and a surfactant are appropriately used. To produce.

[0082] Examples of the stabilizer include human serum albumin, ordinary L-amino acids, saccharides, cellulose derivatives and the like, and these can be used alone or in combination with a surfactant or the like. In particular, this combination may improve the stability of the active ingredient in some cases. The L amino acid is not particularly limited, and may be, for example, glycine, cysteine, glutamic acid, or the like. The sugars are not particularly limited, for example, monosaccharides such as glucose, mannose, galactose and fructose, sugar alcohols such as mantol, inositol and xylitol, disaccharides such as sucrose, maltose and lactose, dextran, hydroxypropyl starch and chondroitin. Any of polysaccharides such as sulfuric acid and hyaluronic acid and derivatives thereof may be used. Cellulose derivatives are also not particularly limited, such as methylcellulose, ethylcellulose, hydroxyethylenosenorelose, hydroxypropinoresenorelose, hydroxypropinolemethinoresenorelose, sodium carboxymethylcellulose and the like.

[0083] The surfactant is not particularly limited, and the difference between ionic and nonionic surfactants can be used. This includes, for example, polyoxyethylene glycol sorbitan alkyl ester type, polyoxyethylene alkyl ether type, sorbitan monoacyl ester type, fatty acid glyceride type and the like.

[0084] As a buffer, boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid and 対応 or a salt thereof (for example, alkali salts such as sodium salt, potassium salt, calcium salt, magnesium salt and the like) Metal salts and alkaline earth metal salts). The

[0085] Examples of the tonicity agent include sodium salt, potassium salt, saccharides, glycerin and the like.

[0086] Examples of the chelating agent include sodium edetate and citric acid.

[0087] The medicament and the pharmaceutical composition according to the present invention can be used as a solution preparation. Alternatively, it can be used after freeze-drying it to make it storable and dissolving it in a buffer solution containing water or a saline solution, etc. to prepare an appropriate concentration before use.

[0088] The medicament and the pharmaceutical composition according to the present invention may be used as a preventive agent and a Z or therapeutic agent for diseases based on abnormal function of the protein according to the present invention and abnormal expression of Z or the present polynucleotide. Can be. In addition, it can be used for a method for preventing and Z or treating the disease.

[0089] For abnormal symptoms associated with excessive expression of the function and Z of the protein of the present invention or the expression of the polynucleotide of the present invention, for example, the function of the present protein and the expression of Z or the present polynucleotide are inhibited. Administering an effective amount of an inhibitor to a subject together with a pharmaceutically acceptable pharmaceutical carrier can prevent, ameliorate or treat abnormal symptoms. Alternatively, the expression of the endogenous polynucleotide may be inhibited using an expression blocking method. For example, an oligonucleotide that is a partial sequence of the present polynucleotide can be used as an antisense oligonucleotide in gene therapy to inhibit the expression of the present polynucleotide. Oligonucleotides used as antisense oligonucleotides are useful even if they correspond to untranslated regions other than only the translated regions of the present polynucleotide. In order to specifically inhibit the expression of the present polynucleotide, it is preferable to use a nucleotide sequence of a region unique to the polynucleotide.

[0090] Tissue expression of the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 in the Sequence Listing, which is one embodiment of the polynucleotide according to the present invention, was examined using disease database information BioExpress (GeneLogic). The expression level in thyroid tissue of patients with chronic inflammation of the thyroid gland was found to be 4.65 times higher than that in normal thyroid tissue. When the expression of the polynucleotide was examined using BioExpress (GeneLogic), it was found that the expression level in renal tissues of patients with chronic inflammation of the kidney was 3.55 times higher than that in normal kidney tissues. found. From these results, it is considered that high expression of the present polynucleotide is associated with chronic inflammatory diseases, for example, thyroid and kidney chronic inflammatory diseases. Specific examples of chronic inflammatory diseases of the thyroid gland and kidney include chronic thyroiditis and chronic nephritis. The medicament and the pharmaceutical composition according to the present invention are useful as a preventive agent and a Z or therapeutic agent for chronic inflammatory diseases such as chronic thyroiditis and chronic nephritis. Further, it can be used for a method for preventing and treating Z or treating chronic inflammatory diseases such as chronic thyroiditis and chronic nephritis.

[0091] The dose range of the medicament and the pharmaceutical composition according to the present invention is not particularly limited. And the like, whether or not a drug is used), and the judgment of the attending physician. In general, a suitable dose is, for example, about 0. Ol ^ g-lOOmg, preferably about 0: g to lmg per kg of the subject's body weight. Whilst, these dosages can be varied using routine experimentation for optimization well known in the art. The above dose can be administered once or several times a day, or may be administered intermittently once every few days or weeks.

[0092] When administering the medicament or pharmaceutical composition according to the present invention, the medicament or pharmaceutical composition may be used alone or in combination with other compounds or medicaments necessary for treatment.

[0093] As for the administration route, the difference between systemic administration and local administration can be selected. In this case, an appropriate administration route is selected according to the disease, symptom and the like. For example, parenteral routes include normal intravenous administration and intraarterial administration, as well as subcutaneous, intradermal and intramuscular administration. Alternatively, oral administration is also possible. Further, transmucosal administration or transdermal administration is also possible.

[0094] Various dosage forms can be appropriately selected according to the purpose of treatment. Typical examples are solid dosage forms such as tablets, pills, powders, powders, fine granules, granules, capsules, aqueous solutions, ethanol solutions, suspensions, fat emulsions, ribosomes Formulations, inclusion forms such as cyclodextrin, liquid dosage forms such as syrups and elixirs are included. Depending on the route of administration, these may be oral, parenteral (drip, injection), nasal, inhalant, vaginal, suppository, sublingual, eye drops, ear drops, ointment, cream Agent, transdermal absorbent, transmucosal They are classified into absorbents and the like, and can be prepared, molded and prepared according to the usual methods.

[0095] (Diagnosis method)

The protein, polynucleotide, recombinant vector, transformant, antibody or compound according to the present invention can be used per se as a means for diagnosing a disease such as a diagnostic marker or a diagnostic reagent.

According to the present invention, for example, by utilizing a part or all of the polynucleotide of the polynucleotide of the present invention, the presence or absence of abnormality in the polynucleotide or the gene containing the polynucleotide in an individual or various kinds of fibrous tissue is determined. The presence or absence of expression can be specifically detected. By detecting the polynucleotide according to the present invention, it is possible to diagnose the susceptibility, onset, and Z or prognosis of a disease based on the quantitative abnormality and the Z or functional abnormality of the polynucleotide or a gene containing the polynucleotide. is there.

[0096] Diagnosis of a disease is, for example, to detect the presence of the polynucleotide according to the present invention, determine its abundance, and identify Z or its mutation in a sample to be examined (test sample). It can be implemented by doing. In comparison with a normal control sample, a change in the presence of the present polynucleotide and a quantitative change thereof can be detected. Alternatively, a mutation such as a deletion or insertion can be detected by, for example, measuring a change in size of an amplification product obtained by amplifying the present polynucleotide by a known method in comparison with a normal genotype. In addition, a point mutation can be identified by hybridizing a polynucleotide whose test sample strength has also been amplified with, for example, a labeled present polynucleotide. By detecting such changes and mutations, the above diagnosis can be performed.

[0097] In the present invention, a method for qualitatively or quantitatively measuring the polynucleotide of the present invention in a test sample, or qualitatively or quantitatively detecting a mutation in a specific region of the polynucleotide. Measurement methods can also be provided.

[0098] The tissue expression of the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1 was examined using the disease database information BioExpress (GeneLogic). The expression level in the thyroid tissue of a case of chronic inflammation of the thyroid gland was determined. It was found to be 4.65 times higher than normal thyroid tissue. In addition, the expression of the polynucleotide is based on the disease database information BioExpress ( Examination using GeneLogic) revealed that the expression level in kidney tissue of patients with chronic inflammation of the kidney was 3.55 times higher than that in normal kidney tissue. From these results, it is considered that high expression of the present polynucleotide is associated with chronic inflammatory diseases, for example, thyroid and kidney chronic inflammatory diseases. Therefore, a method for determining whether the test sample is a test tissue derived from a chronic inflammatory disease by detecting an increase in the expression level of the polynucleotide in the test sample is performed. It is possible. Such a determination method is also included in the scope of the present invention. In this determination method, an increase in the expression level of the polynucleotide can be detected by comparing the test sample with a normal control sample. The test sample preferably includes a test tissue derived from human thyroid tissue or human kidney tissue. Control samples preferably include human normal thyroid-derived tissue or human normal kidney-derived tissue. In a sample derived from human thyroid tissue, if the expression level of the polynucleotide is increased as compared to the control sample, the caloric increase is preferably at least 4.5 times or more, more preferably at least 5 times or more. If so, it can be determined that the test sample is a sample derived from a chronic inflammatory disease of the thyroid gland. In addition, when the expression level of the present polynucleotide in a sample derived from human kidney tissue is increased in caloric level as compared to the control sample, it is preferably increased by at least 3.5 times or more, more preferably by at least 4 times or more! In this case, it can be determined that the test sample is a sample derived from a chronic inflammatory disease of the kidney. The expression level of the polynucleotide according to the present invention means the level of the transcription product of the polynucleotide.

[0099] The test sample is not particularly limited as long as it contains the polynucleotide of the present invention, the gene containing the polynucleotide or the nucleic acid of the mutant gene thereof. Examples thereof include cells, blood, urine, saliva, cerebrospinal fluid, A sample derived from a living organism such as a tissue biopsy or autopsy material can be exemplified. Alternatively, if necessary, a nucleic acid sample can be prepared by extracting a nucleic acid from the sample and used. Nucleic acids may be used directly for detection of genomic DNA or may be amplified enzymatically by using PCR or other amplification methods prior to analysis. RNA or cDNA may be used as well. Nucleic acid samples may also be prepared by various methods that facilitate detection of the target sequence, such as denaturation, restriction enzyme digestion, electrophoresis or dot blotting.

[0100] For detection of the polynucleotide according to the present invention or a gene containing the polynucleotide, Any known gene detection method can be used. Specifically, plaque hybridization, colony hybridization, Southern blotting, Northern blotting

, NASBA method, reverse transcription polymerase chain reaction (RT-PCR) and the like. It can also be detected by measurement at the cell level using in situ RT-PCR or in situ hybridization. In such a gene detection method, in order to identify the polynucleotide according to the present invention, a gene containing the polynucleotide or a mutant gene thereof, and to carry out Z or amplification thereof, an oligonucleotide having a partial sequence ability of the present polynucleotide is used. However, those having properties as a probe or those having properties as a primer are useful. The oligonucleotide having the property as a probe refers to an oligonucleotide having a sequence specific to the polynucleotide which can specifically hybridize only to the present polynucleotide. The one having the property as a primer means one having specific sequence power capable of specifically amplifying only the present polynucleotide. When detecting a mutant gene that can be amplified, a primer or probe having a sequence of a predetermined length including a site having a mutation in the gene is prepared and used. Probes or primers generally have a nucleotide sequence length of about 5 to 50 nucleotides, more preferably about 10 to 35 nucleotides, and more preferably about 15 to 30 nucleotides. preferable. As a primer for amplifying the polynucleotide of the present invention or a fragment thereof, or a probe for detecting the present polynucleotide, specifically, an oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 3 or 4 Reotide is a preferred example. Usually, a labeled probe is used. However, unlabeled probes can also be used. Alternatively, detection may be performed by direct or indirect specific binding with a labeled ligand. Various methods are known for labeling a probe and a ligand, and examples thereof include a method using nick translation, random priming, and kinase treatment. Suitable labels include radioisotopes, biotin, fluorescent substances, chemiluminescent substances, enzymes, antibodies and the like.

In the gene detection method, PCR is also preferable in terms of sensitivity. As long as the PCR is a method using a primer capable of specifically amplifying the polynucleotide according to the present invention, a gene containing the polynucleotide or a mutant gene thereof, any deviation from a conventionally known method can be used. For example, RT-PCR can be exemplified. In addition, various modifications of PCR used in the art can be applied.

[0102] In addition to the detection of the gene, the DNA of the polynucleotide of the present invention, the gene containing the polynucleotide or the mutant gene thereof can be quantified by PCR. Examples of such analysis methods include the competitive quantification method such as the MSSA method, and the PCR-SSCP method known as a mutation detection method that utilizes the change in mobility due to the change in the higher-order structure of single-stranded DNA. Can be shown.

[0103] According to the present invention, for example, by using the protein according to the present invention, the presence or absence of abnormality in the protein and its function in an individual or various tissues can be specifically detected. Detection of abnormalities in the protein and its function according to the present invention makes it possible to diagnose the susceptibility, onset, and Z or prognosis of a disease based on the quantitative abnormality of the protein and Z or function abnormality.

[0104] Diagnosis of a disease by detecting a protein can be carried out, for example, by detecting the presence of the protein, determining its abundance, and detecting Z or its mutation in a test sample. That is, the protein of the present invention and Z or a mutant thereof are quantitatively or qualitatively measured. In comparison with a normal control sample, a change in the presence of the present protein and a quantitative change thereof can be detected. In comparison with a normal protein, for example, its mutation can be detected by determining the amino acid sequence. The above diagnosis can be carried out by detecting such changes and mutations. The test sample is not particularly limited as long as it contains the present protein and Z or a mutant thereof, and examples thereof include biological samples derived from living organisms such as blood, serum, urine, and biopsy tissue.

[0105] The measurement of the protein of the present invention and the protein having a mutation is performed on the protein of the present invention, for example, the protein represented by the amino acid sequence of SEQ ID NO: 2 in the sequence listing, or the amino acid sequence of the protein. This can be achieved by using a protein represented by an amino acid sequence in which one or several or a plurality of amino acids have been deleted, substituted, inserted or added, a fragment thereof, or an antibody against the protein or a fragment thereof.

[0106] Quantitative or qualitative measurement of a protein can be carried out using a protein detection method or a quantification method according to a conventional technique in this field. For example, the amino acid sequence of this protein Mutant proteins can be detected by column analysis. More preferably, by using an antibody (polyclonal or monoclonal antibody), a difference in protein sequence or the presence or absence of the protein can be detected.

[0107] In the present invention, it is also possible to provide a qualitative or quantitative method for measuring the present protein in a test sample, or a qualitative or quantitative method for measuring a mutation in a specific region of the protein.

[0108] Specifically, the above-described detection can be performed by subjecting a test sample to immunoprecipitation using a specific antibody against the present protein, and analyzing the present protein by Western blotting or immunoblotting. In addition, the present protein can be detected in paraffin or frozen tissue sections using an immunohistochemical technique using an antibody against the present protein.

[0109] Preferred specific examples of the method for detecting the present protein or a mutant thereof include an enzyme immunoassay (ELISA) and a radioimmunoassay (RIA), including a sandwich method using a monoclonal antibody and Z or a polyclonal antibody. , Immunoradiometric assay (IRMA), and immunoenzymatic assay (IEMA). In addition, it is also possible to use a radioimmunity / competition bond / attachment.

[0110] Any of the proteins, polynucleotides, recombinant vectors, transformants, and antibodies according to the present invention can be used alone or in combination as reagents or the like. The reagent may be at least one of the protein, polynucleotide, recombinant vector, transformant, and antibody according to the present invention, as well as substances such as buffers, salts, stabilizers, and / or preservatives. Can be included. In formulating, a known formulation means may be introduced according to each property. The reagent can be used, for example, in the determination method according to the present invention, the method for identifying a compound, or the method for measuring the present protein or the present polynucleotide. In addition, the reagent is useful for elucidation of intracellular signal transduction pathways involving the protein or polynucleotide of the present invention, and basic research on diseases or the like based on abnormalities of the protein or polynucleotide.

[0111] The present invention also provides a reagent kit comprising at least one of the protein, polynucleotide, recombinant vector, transformant, and antibody according to the present invention. The reagent kit also includes a label for detecting the protein or polynucleotide according to the present invention. It can contain substances required for the measurement, such as identification substances, label detection agents, reaction diluents, standard antibodies, buffers, detergents, and reaction stop solutions. Examples of the labeling substance include the aforementioned proteins and radioisotopes. The labeling substance may be added in advance to the protein or polynucleotide according to the present invention. The present reagent kit can be used for the determination method according to the present invention, the method for identifying a compound, or the method for measuring the present protein or the present polynucleotide. Further, the present reagent kit can be used as a test agent and a test kit in a test method using the above-mentioned measurement method. Also, the diagnostic method using the above-mentioned measurement method can be used as a diagnostic agent and a diagnostic kit.

[0112] Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not construed as being limited to these Examples.

Example 1

[0113] (Clothing of the gene)

A human spleen-derived cDNA library was constructed according to the method of Ohara et al. [Ohara, O., et al., "DNA Research", 1997, Vol. 4, p. 53-59). Specifically, an oligonucleotide having a Notl site [GACTAGTTCTAGA TCGCGAGCGGCCGCCC (T) 15] (SEQ ID NO: 9: GIBCO BRL) was used as a primer, and human spleen mRNA (Clontech: Catalog No. 6542-1) was used as a template. Double-stranded cDNA was synthesized using Superscriptll reverse transcriptase kit (GIBCO BRL) An adapter (GIBCO BRL) having a S all site was ligated with cDNA, Notl digested, and 1% low melting point A DNA fragment of 3 kb or more was purified by agarose electrophoresis, and the purified cDNA fragment was ligated with pBluescriptll SK + plasmid treated with Sail-Notl restriction enzyme, and E. coli ElectroMa X DH10B (GIBCO BRL) by the electoral poration method. Approximately 10,000 recombinants were selected from the cDNA library thus constructed, and the DNA clones from both ends of these clones were selected. From these, the total nucleotide sequence of cDNA of about 420 clones containing the novel gene was determined.The sequencing was performed using a DNA sequencer (RISA) manufactured by Shimadzu Corporation and PE Applied. A reaction kit from Biosystems was used.For most sequences, a shotgun clone was prepared using the dye terminator method (Terminator (One-label method).

[0114] For the cDNA clones for which the entire nucleotide sequence was determined, ORF was predicted by a general-purpose analysis method using a computer program. Next, a motif domain search was performed on the ORF region to identify cDNA containing a region encoding a GAP domain characteristic of Rho-GAP.

[0115] As a result, cDNA (hereinafter, referred to as sj04085) having a novel nucleotide sequence and containing a region encoding the GAP domain was identified.

[0116] As a result of sequence analysis, sj04085 was found to consist of the base sequence of SEQ ID NO: 1 (4393 bp) and encode the amino acid sequence of SEQ ID NO: 2 (1101 amino acids). In sjO 4085, the GAP domain is encoded in the region that also has the 879th nucleotide force from the 421st eyesight to the 879th nucleotide force in the base Irigami IJ number 1 shown in IJ number 1. In the protein encoded by sj04085, the GAP domain consists of 153 amino acid residues from the 34th peptide (Pro) to the 186th serine (Ser) in the amino acid sequence of SEQ ID NO: 2. .

Example 2

[0117] (Expression and purification of DNA)

The expression vector of sj04085 obtained in Example 1 was produced using Gateway ™ Cloning Technology (manufactured by GATEWAY ™ Cloning Technology, Invitrogen). Next, using the expression vector, the protein encoded by sj04085 was expressed as a FLAG-tag fusion protein in 293EBNA cells (Invitrogen). Expression was confirmed by Western blotting.

Specifically, the sj04085 (inserted into the Sall-Notl site of pBluescriptII SK +) obtained in Example 1 was used as a template, and the ORF region of sj04085 (STRATAGENE) was used by pfu turbo (manufactured by STRATAGENE). (Excluding the stop codon). Oligonucleotides represented by the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 were used as amplification primers. Thereafter, the amplified product was inserted into pENTRZ SD / D-TOPO by a reaction using a TOPO cloning system to prepare an entry-one vector. After ligation of the gene fragment obtained by cutting the prepared entry vector with KpnlZHindlllZNdel and the gene fragment obtained by cutting sj04085 obtained in Example 1 with HindlllZNdel, the combined (TOPlC lnvitrogen). DNA was purified from the transformed Escherichia coli using a purification kit (QI AGEN). It was confirmed by sequence analysis that the sequence of the amplified region and the sequence before and after the base sequence subjected to the restriction enzyme treatment were correct. And the cans reaction was performed using DYEnamic ET Terminator Cycle Sequencing Kit (manufactured by Amersham Biosciences), and electrophoresis and analysis were performed using ABI PRISM 377. Next, using sj04085 and the C-terminal FLAG-tag (3X) fusion protein expression vector cleaved with BspEI, an sj04085 expression plasmid was prepared by a recombination reaction using an LR clonase enzyme. By using this expression plasmid, the protein encoded by sj04085 is expressed as a protein with FLAG-tag (3X) added to the C-terminus.

[0119] As a result of sequence analysis, when sj04085 inserted into the expression vector was compared with the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, it was found that one nucleotide might be different. However, the amino acid substitution due to this single base difference was not recognized. Specifically, the 486th nucleotide in the nucleotide sequence of SEQ ID NO: 1 is cytosine (C). On the other hand, in the base sequence of sj04085 inserted into the expression vector, the 486th base was adenine (A) or cytosine (C). This revealed that there is a possibility that sj04085 inserted into the expression vector and the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 differ by one base. In the polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 1, the base up to the 486th position and the 486th position are CGC, and encode arginine (Arg). On the other hand, in sj04085 inserted into the expression vector, the base corresponding to the position is CGA or CGC, each of which encodes arginine. That is, amino acid substitution due to a single base difference was not observed. Nucleotide sequences other than the 486th were identical without error. Therefore, the amino acid sequence of the protein expressed by this expression vector and the amino acid sequence encoded by the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 are the same.

[0120] The sj04085 expression vector was transfected into 293EBNA cells by the Lipofux method. 293EBNA cells were seeded on a 24-well plate on the day before gene transfer at a cell count of 6 × 10 ⁴ Zwell, and cultured in a culture medium (IMDM medium, manufactured by SIGMA; 10% fetal bovine serum; 4%). mM glutamine; and 10 μg / ml gentamicin). The next day, gene transfer was performed by a method using Lipofectamine 2000 (Lipofectamine ^™ 2000, manufactured by Invitrogen). Specifically, first, serum-free DMEM to which each expression vector was added (manufactured by SIGMA) and DMEM to which Lipofectamine ^™ 2000 was added were mixed and incubated at room temperature for 20 minutes. The resulting mixture was seeded the day before and kept at 37 ° C with 5% CO.

2 was added to 293EBNA cells cultured in the presence. The cells subjected to the gene transfer treatment were further cultured at 37 ° C in the presence of 5% CO. Two days after gene transfer, remove the medium and add phosphate

2

The cells are washed with a buffered saline solution (PBS), and the cells are washed with a lysis buffer (Lysis buffer) containing 1% of a protease inhibitor cocktail (protease inhibitor cocktail, 1/100 concentration, manufactured by SIGMA). The cells were lysed to prepare a cell lysate. The lysis buffer consists of the following composition: 25 mM Tris-HCU pH 7.5; 150 mM NaCl; lm M CaCl; and 1% Triton X-100.

2

[0121] Each cell lysate was mixed with an equal volume of SDS-PAGE sample buffer, and heat-treated (at 100 ° C for 5 minutes) to prepare a sample for electrophoresis. After performing SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the electrophoresis gel was immersed in a blotting buffer for 5 minutes or more to equilibrate, and then the protein was transferred onto a PVDF membrane. After the completion of blotting, the PVDF membrane is blocked by soaking at 4 ° C in a solution (TBS-T + BA) in which Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) is mixed with TBS-T at a ratio of 3: 1. did. After the blocking was completed, the PVDF membrane was washed once with TBS-T for 10 minutes or more while shaking. As the above-mentioned SDS-PAGE sample buffer, Tris SDSβME sample treatment solution (manufactured by Daiichi Pure Chemicals) was used. The electrophoresis buffer used for SDS-PAGE also has the following compositional power: 100 mM Tris; 192 mM glycine; 0.1% SDS, pH 8.3 (Bio Rad). The blocking buffer has the following composition: 25 mM Tris; 40 mM ε-amino-n-caproic acid; 20% methanol; and 0.05% SDS. TBS-T consists of the following composition: 1 50 mM NaCl; 10 mM Tris-HC1, pH 7.5; and 0.05% Tween-20.

[0122] To the PVDF membrane, an anti-FLAG M2 monoclonal antibody (1000-fold diluted, manufactured by SIGMA) was added after dilution with TBS-T + BA, and the mixture was kept at 37 ° C for 1 hour or more. Then, wash the PVDF membrane three times with TBS-T (shake for at least 20 minutes per wash), and wash with TBS-T + BA for 1 hour. HRP-labeled goat anti-mouse IgG antibody (manufactured by Cell Signaling Technology) diluted 000-fold was added, and the mixture was kept at 37 ° C for 1 hour or more. Finally, after washing the PVDF membrane three times with TBS-T (for at least 20 minutes of shaking for one wash), anti-FLAG was detected using the ECL Plus Western Blotting Detection System (Amersham Biosciences). The expressed protein that reacts with the antibody was detected. The chemiluminescence signal was visualized by a detector (Lumino Imaging Analyzer, manufactured by Toyobo Co., Ltd.).

[0123] The predicted molecular weight of the protein encoded by sj04085, expressed as a FLAG-tag fusion protein, is approximately 120 KDa. A major band was detected at the position indicating the expected molecular weight of the protein. On the other hand, such a band was not observed in cells added with only Lipofectamine ^™ 2000 without introducing each vector. With great effort, I was able to obtain the protein encoded by sj04085.

Example 3

[0124] (Detection of binding between the protein encoded by sj04085 and Rho family proteins)

Using the sj04085 expression vector constructed in Example 2, binding between the protein encoded by sj04085 and the Rho family protein was examined by a pull-down method. Rho family Racl was used as one protein. In order to confirm the specificity of the Rho family protein that binds to sj04085, | 8-Gluccuronidase (GUS) with GST-tag added to the N-terminal side was used as a negative control. An expression vector for expressing Racl as an N-terminal GST-tag fusion protein was prepared using Gateway ™ Cloning Technology (Invitrogen). Specifically, first, the Racl gene was amplified using pfu turbo using the spleen first strand DNA (Spleen first strand DNA) of Multiple Tissue cDNA Panels (Clontech) as a template. The amplification product was inserted into pENTRZD in a reaction using the TOPO cloning system to prepare an entry vector. Oligonucleotides represented by the nucleotide sequences of SEQ ID NOs: 7 and 8 were used as primers in the amplification reaction. Next, for the constructed entry vector, a GST-fused Rho expression plasmid was prepared by recombination with LR clonase using pDEST27, which is an N-terminal GST-tag fusion protein expression vector. Sequence confirms that the base sequence of the coding region of each gene has been inserted correctly. And confirmed it. The sequencing reaction was performed using DYEnamic ET Terminator Cycle Sequencing Kit (manufactured by Amersham Biosciences), and the electrophoresis and analysis were performed using ABI P RISM 377. The GST-fused GUS protein was prepared using two vectors attached to an expression vector preparation kit (CAT # 11826-021) manufactured by Invitorogen. Specifically, two vectors, pENTR-gus, in which the GUS gene is integrated, and PDEST27, in which the GST gene is integrated, undergo recombination using LR clonase, and a GST-tagged GUS vector (pDEST27-GUS) is obtained. It was constructed.

[0125] In order to detect the binding between the protein encoded by sj04085 and the Rho family protein, cells were first cotransfected with an sj04085 expression vector and a Racl gene expression vector. Specifically, serum-free DMEM to which both vectors were added and serum-free DMEM to which Lipofectamine ™ 2000 was added were mixed and incubated at room temperature for 20 minutes. The obtained mixture was added to 293EBNA cells. 293EBNA cells were seeded on a 24-well plate with 6.0 X 10 ⁴ Zwell cells the day before gene transfer, and 5% CO2 at 37 ° C.

2 This was used in this example after overnight culture in the presence. The transfected cells were incubated at 37 ° C in the presence of 5% CO for 2 days. After culture, wash cells with PBS

2

Then, the cells were lysed with a lysis buffer (see Example 2 for the composition) containing 1% of a protease inhibitor cocktail (protease inhibitor cocktail, lZlOO concentration, manufactured by SIGMA) to prepare a cell lysate. As a negative control, a cell lysate of cells transfected with only the Racl gene expression vector was used.

[0126] The binding between the protein encoded by sj04085 and Racl was detected by the Bourdund method using the above cell lysate. 300 1 of each cell lysate, 20 1 of glutathione sepharose 4B suspended in the lysis buffer, and 100 1 of the lysis buffer were mixed. Each sample was treated with MgCl and dithiothreitol (DTT).

2

Each was prepared to have a final concentration of ImM. After reacting at 4 ° C for 1 hour while rotating on a turntable, use 1 ml of cold lysis buffer (final concentration of MgCl ImM).

2

And washed three times by centrifugation (1, OOOrpm at 4 ° C for 15 seconds). To the Glutathione sepharose 4B from which the supernatant was removed after washing, a solution obtained by mixing a lysis buffer and an equal amount of SDS-PAGE sample buffer was added. (At 00 ° C for 5 minutes) to prepare a sample for electrophoresis. SDS polyacrylamide gel electrophoresis was performed, and proteins were transferred onto a PVDF membrane from a migration gel that had been immersed in a blotting buffer for 5 minutes or longer and equilibrated. After the completion of the blotting, the PVDF membrane was immersed in TBS-T + BA at 4 ° C. for blocking. After blocking, the PVDF membrane was washed with TBS-T (1 shaking for 20 minutes or more). For the SDS-PAGE sample buffer, the blotting buffer, the TBS-T and the TBS-T + BA, the same buffers as those used in Example 2 were used.

[0127] An anti-FLAG M2 monoclonal antibody (1000-fold diluted, manufactured by SIGMA) was diluted with TBS-T + BA and added to the PVDF membrane, and the mixture was kept at 37 ° C for 1 hour or more. Thereafter, the PVDF membrane was washed 3 times with TBS-T (shake for 20 minutes or more per wash), and HRP-labeled anti-mouse IgG antibody (Cell Signaling Technology) diluted 1,000 times with TBS-T + BA. ) And kept at 37 ° C for 1 hour or more. Finally, after washing the PVDF membrane 3 times with TBS-T (shake for 20 minutes or more per washing), the membrane is washed with ECL Plus Western Blotting Detection System (Amersham Biosciences). An expressed protein that reacts with the FLAG antibody was detected. The chemiluminescence signal was visualized by a detector (Lumino Imaging Analyzer, manufactured by Toyobo Co., Ltd.).

[0128] When a band was detected with the anti-FLAG antibody, it was determined that the protein encoded by sj04085 bound to Racl. As shown in FIG. 1, in a sample prepared from cells in which the protein encoded by sj04085 and Racl were co-expressed, a band indicating the protein was detected (lane 6 of panel A in FIG. 1). On the other hand, in the sample in which the cell strength in which the protein encoded by sj04085 and GUS were co-expressed was also prepared, no band showing the protein was detected (lane 5 in panel A in FIG. 1). No such band was observed in cells transfected with the sj04085 expression vector alone (lane 4 of panel A in FIG. 1). No such band was observed in cells transfected with only the GUS expression vector or the Racl expression vector (FIG. 1, panel A, lanes 2 and 3). No such band was observed even in cells supplemented with Lipofectamine ^™ 2000 without the introduction of each vector (lane 1 of panel A in FIG. 1). In addition, the expression of the protein encoded by sj04085 in each cell was confirmed. The amount of protein encoded by sj04085 in each sample is approximately The amount was almost the same (lanes 4-6 of panel B in Fig. 1). In addition, the amount of Racl or GUS contained in each sample was almost the same (lanes 3 and 6 in panel D or lanes 2 and 5 in panel C in Fig. 1).

[0129] Thus, it was found that the protein encoded by sj04085 binds to Racl. The protein encoded by sj04085 has a GAP domain, which is an active domain of GAP. From these, it is considered that the protein encoded by sj04085 acts as Rho-GAP by binding to Rho family proteins such as Racl.

Industrial applicability

[0130] The protein encoded by the polynucleotide according to the present invention has a GAP domain, which is a characteristic domain of GAP, and bound to Racl. From this, it is considered that this protein has the function of Rho-GAP. Use of the present proteins and polynucleotides will elucidate and regulate signaling pathways and cell functions involving Rho family proteins, and diseases based on abnormalities of the present proteins or polynucleotides, such as chronic inflammatory diseases, and more specifically It can diagnose, prevent and prevent or treat chronic inflammatory diseases of the thyroid and kidneys. Therefore

Sequence listing free text

[0131] SEQ ID NO: 1 (421): (879) Region encoding GAP domain.

SEQ ID NO: 3: oligonucleotide designed based on the sequence of SEQ ID NO: 1 for primers.

SEQ ID NO: 4: Oligonucleotide designed for primer based on the sequence of SEQ ID NO: 1.

SEQ ID NO: 5: polynucleotide encoding Racl.

SEQ ID NO: 6: amino acid sequence of Rac 1.

SEQ ID NO: 7: Designed based on the sequence of SEQ ID NO: 5 for use as a primer.

SEQ ID NO: 8: Designed based on the sequence of SEQ ID NO: 5 for use as a primer.

SEQ ID NO: 9: oligonucleotide designed for primer.

Claims

The scope of the claims

[1] a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 1 in the sequence listing or a complementary nucleotide sequence thereof, or a polynucleotide encoding a protein represented by the amino acid sequence of SEQ ID NO: 2 in the sequence listing Alternatively, a polynucleotide represented by a complementary base sequence of the polynucleotide.

[2] A polynucleotide selected from the group consisting of polynucleotides encoding a protein that binds to Racl:

i) a polynucleotide represented by a nucleotide sequence having at least 70% homology with the nucleotide sequence of the polynucleotide according to claim 1,

ii) the nucleotide sequence of the polynucleotide according to claim 1, wherein the polynucleotide has a mutation such as deletion, substitution, or addition of one or several nucleotides or an induced mutation, and

iii) a polynucleotide that hybridizes with the polynucleotide of claim 1 under stringent conditions.

[3] A polynucleotide selected from the group consisting of: a polynucleotide encoding a protein having a function of promoting GTPase activity of Racl (GAP activity):

ii) a polynucleotide having a mutation such as deletion, substitution, or addition of one or several nucleotides or an induced mutation in the nucleotide sequence of the polynucleotide according to claim 1, and iii) the polynucleotide according to claim 1. A polynucleotide that hybridizes under stringent conditions with nucleotides.

[4] A recombinant vector containing the polynucleotide according to claim 1 according to claim 1

[5] A transformant obtained by transformation with the recombinant vector according to claim 4.

[6] The transformant according to claim 5, which is transformed with the recombinant vector according to claim 4 and a recombinant vector containing a polynucleotide encoding Racl.

[7] A protein represented by the amino acid sequence of SEQ ID NO: 2 in the sequence listing.

[8] A protein encoded by the polynucleotide according to claim 2 or 3.

[9] The method for producing a protein according to claim 7 or 8, comprising a step of culturing the transformant according to claim 5 or 6.

[10] An antibody that recognizes the protein according to claim 7 or 8.

[11] The function of the protein according to claim 7 or 8, and Z or any of claims 1 to 3.

A method for identifying a compound that inhibits expression of the polynucleotide according to claim 1, wherein the compound and the protein or Z or the polynucleotide are allowed to interact with each other.

Detecting the presence, absence or alteration of the function and Z or the expression to determine whether the compound has the ability to inhibit the function of the protein and the expression of Z or the polynucleotide. Identification method.

[12] The identification method according to claim 11, which is a GAP activity against Racl, a functional ability of the protein.

[13] A method for determining whether or not a test tissue derived from a human thyroid tissue is a tissue derived from a chronic inflammatory disease of the human thyroid, wherein the method according to claim 1 or 2 in the test tissue is performed. 13. A determination method, comprising measuring the expression level of the polynucleotide according to item 13.

[14] Expression capacity of the polynucleotide according to any one of claims 1 to 3 in a test tissue At least 4.5 times the expression level of the polynucleotide in a human normal thyroid-derived fibrous tissue as a control 14. The method according to claim 13, wherein in the case described above, the test tissue is determined to be a tissue derived from a chronic inflammatory disease of human thyroid.

[15] A method for determining whether or not a test tissue derived from a human kidney tissue is a tissue derived from a chronic inflammatory disease of a human kidney, wherein the test tissue in the test tissue is any one of claims 1 to 3. A determination method comprising measuring the expression level of the polynucleotide described in (1).

[16] the expression level of the polynucleotide according to any one of claims 1 to 3 in a test tissue, at least 3.5 times or more the expression level of the polynucleotide in a normal human kidney-derived tissue as a control; 16. The method according to claim 15, wherein in some cases, the test tissue is determined to be a tissue derived from a chronic inflammatory disease of a human kidney.

[17] A compound that inhibits the function of the protein according to claim 7 or 8 and a compound that inhibits the expression of Z or the polynucleotide according to any one of claims 1 to 3 as effective components. Chronic inflammatory disease of the thyroid and chronic inflammatory disease of the Z or kidneys Disease preventive and Z or therapeutic agents.

[18] A compound which inhibits the function of the protein according to claim 7 or 8, and a compound which inhibits the expression of Z or the polynucleotide according to any one of claims 1 to 3 is used. Methods for preventing and Z or treating chronic inflammatory diseases of the thyroid and Z or kidney.

[19] The protein according to claim 7 or 8, the polynucleotide according to any one of claims 1 to 3, the recombinant vector according to claim 4, and the transformant according to claim 5 or 6. And a reagent kit comprising at least one of the antibodies according to claim 10.