WO2004065633A1

WO2004065633A1 - Method of using peptidyl arginine deiminase type iv

Info

Publication number: WO2004065633A1
Application number: PCT/JP2004/000430
Authority: WO
Inventors: Kazuhiko Yamamoto; Ryo Yamada; Akari Horiguchi; Xiaotian Chang; Hidehiko Furukawa; Mitsuru Ono; Reimi Mikami; Makiko Nakayama; Masahiko Ohtsuki; Masakatsu Suzuki; Miyuki Nagasaki
Original assignee: Riken; Sankyo Company, Limited
Priority date: 2003-01-21
Filing date: 2004-01-20
Publication date: 2004-08-05
Also published as: JPWO2004065633A1; JP4503531B2; TW200502398A

Abstract

A method of evaluating the therapeutic effect of a remedy for rheumatoid arthritis and a method of estimating the risk for onset of rheumatoid arthritis by using peptidyl arginine deiminase type IV. Namely, it is intended to provide a method of evaluating the effect of a test substance as a remedy for rheumatoid arthritis by using its inhibitory effect on a peptidyl arginine deiminase type IV gene, an orthologous gene thereof or a protein encoded by the gene as an indication. It is also intended to provide a method of estimating the risk for onset of rheumatoid arthritis in a subject based on the expression amount of a mutant peptidyl arginine deiminase type IV protein comprising the amino acid sequence represented by SEQ ID NO:21 or its gene (SEQ ID NO:20) in a specimen which is provided by the subject.

Description

Peptidyl arginine · Diminase · Type IV usage technology Technology

The present invention relates to a method for evaluating the effect of a therapeutic agent for rheumatoid arthritis, and a method for predicting the risk of developing rheumatoid arthritis, using peptidylarginine diminase 'type IV.

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Background technology

Rheumatoid arthritis (RA) is a chronic inflammatory disease that mainly involves the synovial tissue of the joints, with a prevalence of about 1% of the population. RA initially develops synovitis, gradually invades cartilage and bones, and as it progresses, the joints rupture and deform. The course of symptoms varies, including repeated cures and relapses of arthritis, complete cure, and rapid progression. Early detection and early treatment are also important for improving the quality of life of RA patients.

RA pathologically begins with a specific immune response to an unknown antigen that has reached the joints, causing chronic inflammation and synovial proliferation by blood lymphocytes, macrophages, and neutrophils that have infiltrated the joints, Osteoclasts derived from inflammatory granulation tissue and bone and cartilage destruction by proteases occur. Although the etiology of RA is unknown, both genetic and environmental factors, such as microbial infection, are thought to be involved.

The diagnosis of RA is mainly based on symptoms, but detection of autoantibodies found in the blood of RA patients is currently useful for early diagnosis. As such autoantibodies, for example, rheumatoid factor, anti-Sa protein antibody, anti-perinuclear factor antibody, anti-keratin antibody, anti-filaggrin antibody, anti-citrullinated peptide antibody and the like are known (Mart inus AM van Boekel). et al., Arthr it is Research (2002) 4 (2), p87-93)

Anti-citrullinated peptide antibody is detected in about 76% of sera of RA patients, but only about 4% is detected in sera of patients who show rheumatic-like symptoms but are not determined to be RA. It is an autoantibody (Gerard A. Schel lekens et al., The Journal of Clinical Investigation (1998) 101 (1), 273-281; ZhiJie Zhou and Henri-Andre Menard, Current Opinion in Rheumatology (2002) 14 (3), p250-253 Citrullinated proteins are detected in the synovium of RA patients, suggesting that epitopes containing citrulline may be involved in the pathogenesis of RA (Walter J van Venrooij and Ger JM Pruijn, Arthrit is Research (2000) 2 (4), p249-251; Christine Masson-BessiD et al., Journal of Immunology (2001) 166 (6), p4177-4184) ₀

Peptidyl citrulline is an amino acid produced by the conversion of arginine residues in proteins by peptidyl arginine diminase (hereinafter referred to as “PADI”). To date, four subtypes of PADI are known in rodents: type I is epidermis and uterus, type II is various tissues such as muscle, uterus, salivary glands, and knees; type II I is In epidermal and hair root cysts, type IV is expressed in various tissues (Ahied Abu Rus'd et al., European Journal of Biochemistry (1999) 259 (3), p660-669). In humans, genes of type I, II, III, and V have the highest homology to rodent types I, II, III, and IV, respectively, and are registered on Gene Information Bank. However, type I II (Takuya Kanno et al., Journal of Investigative Dermatology (2000) 115 (5), p813-823) and type V (Katsuhiko Nakashiia et al., Journal of Biological Chemistry (1999) ) Other than 274 (39), p27786-27792), no detailed report has been made. Type III of human PADI is the same as that of rodents in hair root cysts (Takuya Kanno et al., Journal of Investigative Dermatology (2000) 115 (5), p813-823), and evening V is favorable (Hiroaki Asaga et al., Journal of Leukocyte Biology (2001) 70 (1), p46-51), respectively, but direct association between PADI and RA has been reported. Unknown.

In addition, since the gene which was said to be type V at the time of the basic application of the present application was subsequently confirmed to be a mouse type IV ortholog, the type V human PADI is referred to as type IV in this specification. And

A major factor that determines the constitution of an individual is a genetic polymorphism. Recent advances in pharmacogenomics research have elucidated the relationship between genetic polymorphisms and the effects or side effects of drugs, and the effects or side effects of the drugs using genetic diagnosis It is becoming possible to predict before administration. In addition, by examining the relationship between genetic polymorphisms and diseases, it will be possible to make preliminary diagnoses and determine prognosis for some diseases.

An example of the former is a gene polymorphism of a drug metabolizing enzyme. For example, drug metabolizing enzymes whose activity is increased or decreased in association with polymorphism include cytochrome P4501A2, cytochrome P4502A6, cytochrome P4502C9, cytochrome P4502C19, cytochrome P4502D6, Cytochrome P450 2 E 1 and the like are known. Genetic polymorphisms also occur in a group of enzymes called conjugating enzymes, such as thiopurine methyltransferase, N-acetyltransferase, UDP-dalcuronosyltransferase, and daryuthion S-transferase. It has been reported that the activity is reduced by polymorphism (Yusuke Nakamura, ed., “Strategy for SNP gene polymorphism”, (2000), Nakayama Shoten, Tokyo).

An example of the latter is a disease-causing gene found by a polymorphism analysis study. For example, (1) HLA as a causative gene for ulcerative colitis, (2) TCR as a causative gene for rheumatoid arthritis, (3) AP0E4 as a causative gene for Alzheimer's disease, (4) mental health Dopamine D3 receptor as a causative gene for schizophrenia, (5) tributophan hydroxylase as a causative gene for manic depression, (6) angiotensin precursor as a causative gene for albuminuria, (7) myocardial infarction Blood coagulation factor VII is known as a causative gene for leukemia, and (8) lebutin is known as a causative gene for obesity (L. Kruglyak, Nature Genetics (1999) 22 (2), pl39-144).

On the other hand, TNF-a is thought to be an important substance involved in the pathogenesis of various inflammatory diseases. Recently, however, the expression of the TNF-h gene has occurred in the 5'-terminal upstream region of the TNF-α gene. (T. Higuchi et al., Tissue Antigens (1998) 51 (6), p605-612). Since these polymorphisms enhance the expression level of the TNF-a gene, they can be diagnostic markers for diseases involving TNF-0; such as juvenile rheumatoid arthritis, rheumatoid arthritis, and diabetes. It is believed that. Disclosure of the invention

The present invention relates to the effects of a therapeutic agent for rheumatoid arthritis or the onset of rheumatoid arthritis. The purpose is to provide a new method for assessing risk.

As a result of intensive studies to solve the above problems, the present inventors found that PADI4 protein and its mRNA are highly expressed specifically in the lesion site of RA patients, and that the expression of PADI4 Increased levels and contributed to RA development. Based on these findings, the present inventors considered that it is possible to evaluate a therapeutic agent for rheumatoid arthritis using the inhibitory effect on the expression level or activity of PADI4 protein or its mRNA as an index.

That is, the present invention relates to a method for evaluating the effect of a test substance as a therapeutic agent for chronic rheumatoid arthritis, using as an index the inhibitory effect on the PADI4 gene or its orthologous gene, or the protein encoded by the gene.

In one embodiment, the evaluation method of the present invention includes the following steps:

1) keep the animals under the conditions of administration or non-administration of the test substance;

2) detecting the expression level of the PADI4 gene or its orthologous gene in the blood or cells of the animal;

3) The effect of the test substance as a therapeutic agent for rheumatoid arthritis is evaluated based on the difference in the expression amount under the conditions of administration or non-administration of the test substance.

In the above method, the expression level of the gene can be determined, for example, by using a nucleic acid hybridization method, an RT-PCR method, a real-time PCR using a solid-phased sample selected from a gene chip, a cDNA array, and a membrane filter. , A subtraction method, a differential display method, a differential hybridization method, and a cross-hybridization method.

In another embodiment, the evaluation method of the present invention comprises the following steps:

2) detecting the expression level of the protein encoded by the PADI4 gene or its orthologous gene in the blood or cells of the animal using an antibody that specifically binds to the protein;

In the above method, the expression level of the protein is determined by, for example, Detected by one of the following methods: blot plot, slot blot, ELISA, and RIA.

The cells used in the evaluation method of the present invention are preferably blood-derived cells, synovial cells, spleen cells, and peritoneal cells. In addition, the animal used in the evaluation method of the present invention is preferably a mouse. In this case, the detection target is preferably the mouse ortholog of the PADI4 gene: the mouse PADI4 gene, or a protein encoded by the gene.

In still another embodiment, the evaluation method of the present invention comprises the following steps:

1) Add PADI4 protein to the reaction mixture containing the arginine-containing compound with or without the test substance.

2) The activity of PADI4 protein is measured by quantifying the amount of the reaction product in the above reaction solution.

3) The effect of the test substance as a therapeutic agent for rheumatoid arthritis is evaluated based on the difference in the above activities under the conditions of addition or non-addition of the test substance.

In the above method, for example, the activity of the protein is preferably measured by quantification of the reaction product using a fluorescence method or an absorption method. The PADI4 protein is preferably a human-derived PADI4 protein. In addition, the present inventors have found that there is a haplotype on the PADI4 gene that frequently appears in RA patients. They found that this haplotype encodes a mutant PAD 14 protein having a different amino acid sequence and enzyme activity from the normal PAD 14 protein, and that this difference is closely related to the onset of RA. Based on these findings, the present inventors thought that the use of the mutant PADI4 protein and its gene would enable prediction of the risk of developing RA and evaluation of the therapeutic effect on RA.

That is, the present invention provides a method for chronically treating a subject who has provided a specimen based on the expression level of a mutant PADI4 protein having the amino acid sequence shown in SEQ ID NO: 21 or the gene (SEQ ID NO: 20) in the specimen. A method for predicting the risk of developing rheumatoid arthritis is provided.

In one embodiment, the prediction method of the present invention comprises the following steps:

1) Prepare total RNA from each sample isolated from subjects and normal persons; 2) detecting the expression level of the mutant PADI4 gene mRNA in the total RNA;

3) Analyze the difference in the expression level between the subject and normal person and predict the risk of developing rheumatoid arthritis in the subject.

In the above method, the expression level of the gene may be determined, for example, by using a nucleic acid hybridization method using a solid-phased sample selected from a gene chip, a cDNA array, and a membrane fill sample, an RT-PCR method, a real-time PCR method, It is detected by any one of the subtraction method, the differential display method, the differential hybridization method, and the cross-hybridization method.

In another embodiment, the prediction method of the invention comprises the following steps:

1) detecting the expression level of the mutant PADI4 protein in a sample isolated from a subject and a normal person using an antibody capable of specifically binding to the protein;

2) Analyze the difference in the expression level between the test subject and normal subject, and predict the risk of developing rheumatoid arthritis in the test subject.

In the above method, the expression level of the protein is detected by, for example, any one method selected from Western blotting, dot blotting, slot blotting, ELISA, and RIA.

Further, the present invention provides a kit for predicting the risk of developing rheumatoid arthritis, comprising at least one selected from the group consisting of the following a) to e). a) Mutant type consisting of the nucleotide sequence shown in SEQ ID NO: 20 A continuous oligonucleotide primer having a length of 15 to 30 nucleotides for specifically amplifying the PADI4 gene b) Mutant type consisting of the nucleotide sequence shown in SEQ ID NO: 20 A continuous polynucleotide probe of 20-1500 base length that specifically hybridizes to the PADI4 gene and detects the gene.

c) An immobilized sample on which the polynucleotide probe described in b) above is immobilized.d) An antibody specifically binding to the mutant PADI4 protein having the amino acid sequence shown in SEQ ID NO: 21 and detecting the protein.

e) secondary antibody capable of specifically binding to the antibody described in d) above

Further, the present invention relates to a variant comprising the amino acid sequence of SEQ ID NO: 21. Provided is a method for evaluating the effect of a test substance as a therapeutic drug for rheumatoid arthritis using an inhibitory effect on PADI4 protein as an index.

The method includes, for example, the following steps:

1) Add the mutant PADI4 protein to the reaction solution containing the arginine-containing compound, with or without the addition of the test substance;

2) measuring the activity of the mutant PADI4 protein by quantifying the amount of the reaction product in the reaction solution;

Furthermore, the present invention provides a mutant PADI4 protein consisting of the amino acid sequence shown in SEQ ID NO: 21. The protein is useful as a predictive marker for predicting the risk of developing rheumatoid arthritis. ADVANTAGE OF THE INVENTION According to this invention, the simple screening of the therapeutic agent for rheumatoid arthritis is possible. In addition, the risk of developing a subject with rheumatoid arthritis can be determined at an early stage by detecting an indicator of the expression level of the mutant PADI4 protein and the gene according to the present invention. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph comparing the mRNA stability (resolution) of one haplotype of human PADI4: V9 and V18.

FIG. 2 is a graph showing (A) a result of PADI4 enzyme activity measurement by a fluorescence method, and (B) a graph showing a result of PADI4 enzyme activity measurement by an absorption method.

FIG. 3 is a graph showing the results of comparing the enzyme activities of two haplotypes of PADI4: V9 and V18 by an absorption method.

FIG. 4 is a photograph showing the results of In situ RT-PCR in which the expression of PADI4 mA in RA patient synovial tissues was observed. The two right ones in the figure are negative controls.

FIG. 5 is a graph showing the expression levels of mPADI4 gene in synovial tissue and spleen tissue in the mouse collagen-induced arthritis (CIA) group and the normal group. Figure 6 shows (A) immunohistochemical staining of RA patient synovium with anti-PADI4 antibody, and (B) is a photograph showing immunohistochemical staining of the synovium of an OA patient using both an antibody that specifically recognizes PADI4 and an antibody that recognizes all subtypes of PADI.

Figure 7 shows (A) immunohistochemical staining of RA patient synovium using anti-PADI4 antibody and (B) immunohistochemical staining of RA patient synovium using anti-citrullinated protein antibody. It is a photograph shown.

FIG. 8 is a graph comparing the stability of human PADI4 V9 and PADI4 V18 mRNA. This description includes part or all of the contents as disclosed in the description of Japanese Patent Application No. 2003-12738 and Japanese Patent Application No. 2003-12774, which are the basis of the priority of the present application. BEST MODE FOR CARRYING OUT THE INVENTION

1. Peptidyl arginine diminase type IV

I.1 Peptidylarginine diminase type IV (PAD 14)

Peptidyl arginine diminase (hereinafter referred to as “PADI”) is an enzyme that converts arginine residues in a protein to citrulline to produce peptidyl citrulline. There are several subtypes of PADI, including type I and

Four subtype genes, II, II I, and V, are registered on GenBank, but their detailed functions are unknown. In mice, four subtypes have been reported, type I, II, III, and IV, of which type IV is the mouse ortholog of human PADI4.

The present inventors have confirmed that the PADI4 protein and the PADI4 gene (mRNA) are specifically highly expressed in the lesion tissue of RA patients, and that the citrullinated peptide is detected at a high level. Citrullinated peptides can cause RA lesions through the production of autoantibodies (anti-citrullinated peptide antibodies). Therefore, it is possible to evaluate the effect of a test substance as a therapeutic agent for rheumatoid arthritis using the inhibitory effect on the expression level and activity of the PADI4 protein or its gene as an index.

(1) PADI4 gene of the present invention

The genes used in the evaluation method of the present invention include not only the PADI4 gene but also its orthologous gene. "Osolog gene" means "gene of common ancestry" Genes of different species having the same function and evolved from them ”. For example, orthologs of PADI4 include mouse PADI4. Hereinafter, the PADI4 gene and its ortholog gene are collectively referred to as “PADI4 gene of the present invention”.

The origin of the gene is not particularly limited, but is preferably derived from mammals, more preferably primates or rodents. In particular, the most preferred examples include the human PADI4 gene and the mouse PADI4 gene.

Among the PADI4 genes of the present invention, the sequence of cDNA encoding human PADI4 is shown in SEQ ID NO: 16, but is not limited to this sequence, and as long as it encodes human PADI4, the gene is included in the human PADI4 gene. Shall be For example, the present inventors have confirmed that human PADI4 has a plurality of haplotypes, and these haplotypes are also included in the human PADI4 gene. Similarly, among the orthologous genes according to the present invention, the sequence of the cDNA encoding mouse PADI4 is shown in SEQ ID NO: 18, but is not limited to this sequence. Is included in the mouse PADI4 gene.

In this specification, the term “gene” includes not only DNA but also its mRNA, cDNA and cDNA. Therefore, the gene of the present invention includes all of these DNAs, mRNAs, cDNAs, and cDNAs. In addition, all nucleotide sequences in the sequence listing are described as DNA sequences. When the sequence indicates RNA, the base symbol “t” in the sequence listing is replaced with “u”.

(2) PADI4 protein of the present invention

The protein (PADI4 protein) used in the evaluation method of the present invention includes, in addition to the PADI4 protein encoded by the PADI4 gene, the protein encoded by the orthologous gene. Hereinafter, these proteins are collectively referred to as “PADI4 protein of the present invention”.

The origin of the protein is not particularly limited, but is preferably derived from mammals, and more preferably derived from primates and rodents. In particular, the most preferred examples of the present invention include human PADI4 protein and mouse PADI4 protein. Among the proteins of the present invention, the amino acid sequence of human PADI4 is shown in SEQ ID NO: 17, but is not limited thereto, and is represented by a sequence in which one or several amino acids have been deleted, substituted or added. As long as the peptide also has a function as human PADI4, it is included in the human PADI4 protein. Similarly, the amino acid sequence of mouse PADI4 is shown in SEQ ID NO: 18, but is not limited thereto, and the peptide is represented by a sequence in which one or several amino acids have been deleted, substituted or added in the sequence. As long as they also have a function as mouse PADI4, they are included in the mouse PADI4 protein. 1.2 Mutant beptidyl arginine, diminase, type IV

The present inventors have found that there are a plurality of polymorphisms that frequently appear in patients with rheumatoid arthritis on the human PADI4 gene, and these are almost completely linked (98.9 to 100%). To make up one haplotype. Furthermore, it was confirmed that this haplotype encodes a mutant PADI4 protein having a different amino acid sequence and enzyme activity from a normal PADI4 protein, and that the difference is closely related to the onset of RA.

That is, the “mutant PADI4 protein” according to the present invention is a protein having the amino acid sequence represented by SEQ ID NO: 21, and the amino acid sequence of normal PADI4 (SEQ ID NO: 17) Amino acid 5 (substituted from Ser to G1y), Amino acid 82 (substituted from A1a to Va1), Amino acid 112 (substituted from A1a to G1y) Are different. Due to this difference, mutant PADI4 protein has higher enzymatic activity than normal PAD protein and enhances the production of citrullinated protein that contributes to RA lesions.

Further, the “mutant PADI4 gene” according to the present invention has a cDNA sequence represented by SEQ ID NO: 20. A normal PADI4 gene is defined as the third intron of the third intron in the genome sequence. 6th base (substitute cytosine for thymine), 2nd exon 7 1st base (substitute adenine for guanine), 2nd exon 15 3rd base (substitute cytosine for thymine), 3rd exon The second base (substitution of cytosine to guanine) of the fourth and the ninth base of exon 4 (substitution of cytosine to thymine: this substitution does not involve amino acid substitution) are different. Due to this difference, The mRNA of the mutant PADI4 gene is more stable than the mRNA of the normal PADI4 gene, and may increase the production of citrullinated proteins that contribute to RA lesions. 2 Methods for evaluating the effects of test substances as therapeutics for rheumatoid arthritis

The present invention provides a method for evaluating the effect of a test substance as a therapeutic agent for rheumatoid arthritis, using an inhibitory effect on the PADI4 gene of the present invention or the protein encoded by the gene as an index.

The above method may be a method for comparatively evaluating the inhibitory effect of one test substance under the administration (addition) and non-administration conditions, or a similar comparative evaluation of two or more test substances. You may. Alternatively, if a correlation between the expression level or activity of the gene or protein and the RA therapeutic effect is established empirically, an absolute evaluation without a comparative control may be performed based on the relationship.

In the evaluation method of the present invention, the inhibitory effect on the PADI4 gene or PADI4 protein of the present invention may be evaluated using the expression level of the PADI4 gene or PADI4 protein of the present invention as an index, or the inhibitory effect of the PADI4 gene or PADI4 protein of the present invention. Activity (including stability) may be evaluated as an index. The evaluation system may be an in vivo system or an in vitro system. Hereinafter, a specific implementation method of the evaluation method of the present invention will be described.

2.1 Evaluation method using expression level of PADI4 gene of the present invention as an index

The in vivo evaluation method of the present invention using the PADI4 gene expression level as an index includes, for example, the following steps.

Step 1: Animals are bred under the conditions of administration or non-administration of the test substance.

Step 2: detecting the expression level of the PADI4 gene of the present invention in the blood or cells of the above animal.

Step 3: Evaluate the effect of the test substance as a therapeutic agent for RA on the basis of the difference in the expression level under the conditions of administration or non-administration of the test substance. Process 1: Animal rearing The “animal” used in the evaluation method of the present invention is not particularly limited, but a model animal exhibiting RA pathology is preferable. Such animals may be commercially available or may be produced according to known methods. Models exhibiting such RA pathology include, for example, spontaneous arthritis model, MRL mouse arthritis, NZB / KN mouse arthritis, SKG mouse arthritis; inducible arthritis model, rat adjuvant arthritis, rat mouse collagen Arthritis (CIA rat 'mouse), pristine arthritis, SCID cell-transplanted arthritis, SCID mouse tissue transplant arthritis; and HTLV-1 transgenic mice, IL-1 receptor Yuichi antagonist K0 mice, etc. Model supervised by Nippon Medical Center, Hakuyo Kyogoku, editor, Chiyuki Abe, Takashi Sawai). The most common are RL mouse arthritis, rat adjuvant arthritis, rat-mouse collagen arthritis, and pristane arthritis.

The animals are bred for an appropriate period under the conditions of administration or non-administration of the test substance. The dose of the test substance to the animal is not particularly limited, and an appropriate dose may be set according to the properties of the test substance and the weight of the animal. The method and period of administration of the test substance to the animal are also not particularly limited, and the administration route and the administration period may be appropriately set according to the properties of the test substance. Step 2: Detection of the PADI4 gene of the present invention

Next, blood or cells are isolated from animals bred under the conditions of administration or non-administration of the test substance, and the expression level of the PADI4 gene of the present invention in the blood or cells is detected.

As the cells to be detected, cells in which the PADI4 gene of the present invention is highly expressed are preferable. preferable. The method for detecting the PADI4 gene of the present invention includes, for example, a method of first extracting total RNA from isolated blood or cells and detecting the expression level of mRNA in the total RNA.

(1) Extraction of total RNA

Extraction of total RNA is performed according to known methods from isolated blood or cells. Extract using the RNA extraction solvent. Examples of the extraction solvent include those containing a component having an action to inactivate liponucleases such as phenol (for example,

TRIzol reagent: manufactured by Gibco PRL) is preferable. The method of extracting RNA is not particularly limited. For example, guanidine thiocyanate / cesium chloride ultracentrifugation method, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, guanidine acid thiocyanate phenol / clonal form method (Chomczynski, P. and Sacchi, N., (1987) Anal. Bioche., 162, 156-159) and the like can be employed. Of these, the guanidine acid thiocyanate / phenol / chloroform method is preferred.

The extracted total RNA may be further purified only to mRNA if necessary. Although the purification method is not particularly limited, most mRNAs present in the cytoplasm of eukaryotic cells have a poly (A) sequence at the 3 ′ end, and this characteristic can be used, for example, as follows. can do. First, a biotinylated oligo (dT) probe is added to all the extracted RNA to adsorb poly (A) + RNA. Next, a paramagnetic particle carrier on which streptavidin is immobilized is added, and poly (A) + RNA is captured using the binding between biotin and streptavidin. After washing, poly (A) + RNA is eluted from the oligo (dT) probe. In addition to this method, a method of adsorbing poly (A) ⁺ RNA using an oligo (dT) cellulose column and eluting the same may be used for purification. The eluted poly (A) + RNA may be further fractionated by sucrose density gradient centrifugation or the like.

(2) Detection of PADI4 gene of the present invention

Next, the expression level of the PADI4 gene of the present invention in the total RNA under the conditions of administration or non-administration of the test substance is detected. The gene expression level can be detected as signal intensity by preparing cRNA or cDNA from the obtained total RNA and labeling it with an appropriate labeling compound. The method of detecting the expression level of the gene is described below in detail by dividing it into i) an analysis method using a solid-phased sample, ii) RT-PCR method (real-time PCR method), and iii) other analysis methods. . ii)) Analytical method using solid-phase sample preparation

A solid-phased sample material in which a known genetic gene has been fixed and fixed to the solid-phase-phased sample material, with or without administration. Cc DD NNAA or cc RR NNAA (hereinafter referred to as "" "" "). Under the same conditions, separately and / or mixed together at the same time so that they can be combined at the same time (( BBrroowwnn ,, PP .. 00 .. eett aall .. ((11..999999)) NNaattuurree ggeenneett .. 2211 ,, ssuupppplliimmeenntt ,, 3333--3377)). . The mark labeling probe described above, even with the mmRR NNAA chlorochloroform of PPAADDII44 of the present invention, labels all mmRR NNAA expressing and expressing. You may be aware of it. . As a starting and starting material for making prop rooves, it is possible to use mmRR NN AA, which may or may not be purified. However, it is better to use the poporily ((AA)) ++ RR NN AA purified and produced by the method described above. . In the following, the analysis and analysis method using various types of test samples for solid-phase solidification are explained. .

aa)) Genetic Gene Chichipfoff ::

The genetic gene chip used in the present invention is a method in which a genetic gene, which is an object to be detected and detected, is solid-phase-phased. If it is a thing that is sold, even if it is a thing that is marketed and sold, it is a publicly known method ((LLiippsshhuuttzz ,, RR .. JJ .. eett aall .. ((11999999)) NNaattuurree ggeenneett .. 2211 ,, ssuuppppll iimmeenntt ,, 2200--2244)). . Detection and detection by genetic genetic test can be performed in accordance with the conventional method. . For example, if a chip manufactured by Affiliate Metrics Rix Co., Ltd. is used, the protocol attached to the product may be used. Prepare a cc RR NN AA probe labeled with pipiootintin and prepare it. . Next, follow the procedure described in the above, and perform the following procedure, and perform the following steps. By detecting, analyzing and analyzing the emitted light, it is possible to obtain the amount of expression of the genetic gene. .

The array or membrane fill used in the present invention may be a commercially available array (for example, Intelligin (Takara Shuzo), a filter microarray atlas system) as long as the gene to be detected is immobilized. (Clontech, etc.)) or may be produced based on a known method. The gene to be immobilized is a cDNA or RT-PCR product cloned by performing a reverse transcriptase reaction or PCR with primers created based on sequence information such as Gen Bank. .

In the detection using an array, when producing cDNA from poly (A) + RNA by reverse transcriptase reaction, d- UTP, etc. labeled with a fluorescent dye (for example, Cy3, Cy5, etc.) To prepare a labeled probe. Test substance administration conditions In the above, poly (A) + RNA and poly (A) + RNA under the non-administration condition of the test substance are respectively labeled with different dyes, the two are mixed and hybridized, and detected using a fluorescent signal detector. The resulting fluorescence signal indicates the relative ratio of gene expression under the administration and non-administration conditions (Brown, P. 0. et al. (1999) Nature genet. 1, s liment, 33-37).

In the detection using a membrane filter, in making cDNA from poly (A) + R NA with reverse transcriptase reaction, a radioisotope (e.g., ³² P, ³³ P) at the-labeled the d-CTP, etc. A labeled probe is prepared by the addition, and hybridization is performed by a conventional method. After performing hybridization and washing using an atlas system (manufactured by Clontech), which is a commercially available filter microarray, detection is performed using an analyzer (for example, atlas image: manufactured by Clontech). Perform analysis. ii) RT—PCR method (Real-time PCR method)

The RT-PCR method and one of them, the real-time PCR (TaciMan PCR) method, are suitable for the evaluation method of the present invention in that they can detect minute amounts of DNA with high sensitivity and quantitatively. In the real-time PCR (TaqMan PCR) method, an oligonucleotide probe that hybridizes to a specific region of the target gene and has a 5 'end labeled with a fluorescent dye (Repo Ichiichi) and a 3' end labeled with a fluorescent dye (Quenchia I) is used. used. In this probe, the fluorescence of the reporter is normally suppressed by quenching. With the fluorescent probe completely hybridized to the target gene, PCR is performed from outside using Tad DNA polymerase. As the elongation reaction of the Taci DNA polymerase progresses, the exonuclease activity hydrolyzes the fluorescent probe from the 5 'end, releasing the repo dye overnight to emit fluorescence. The real-time PCR method can accurately quantify the initial amount of type I DNA by monitoring the fluorescence intensity in real time. iii) Other analysis methods

In addition to the above, methods for analyzing gene expression levels include, for example, the subtraction method (Sive, HL and John, T. St. (1988) Nucleic Acids Research 16, 10937, Wang, Z., and Brown, DD (1991) Proc. Natl. Acad. Sci. USA 88, 11505-11509), Differential 'Display Method (Liang, P., and Pardee, AB (1992) Science 257, 967-971, Liang, P., Averboukh, L., Keyomarsi, K., Sager, R., and Pardee, AB (1992) Cancer Research 52, 6966-6968), differential 'hybridization' Method (John, T. St., and Davis, RW Cell (1979) 16, 443-452) and a cross-hybridization method using an appropriate probe ("Molecular Cloning, A Laboratory Manual" Maniatis, T. , Fritsch, EF, Sambrook, J. (1982) Cold Spring Harbor Laboratory Press).

a) Subtraction cloning method:

This is a method in which cDNA of a gene specifically expressed in a specific cell is obtained, and the gene is cloned by screening a cDNA library using the cDNA as a probe. As a subtraction method, a single-stranded cDNA was prepared from total RNA, and this was hybridized with all RNA obtained from another cell, and then a single-stranded cDNA was not hybridized with a hydroxyapatite column. A method for isolating strand DNA and preparing a cDNA library from this cDNA (Bio Manual Series 3, Genetic Cloning Experiments, Yodosha (1993), Current 'Protocols in' Molecular · Biology) or a cDNA library was prepared first, and single-stranded DNA was prepared from this library using helper phage, etc., and biotin was added to this single-stranded DNA and all RNA obtained from another cell. After hybridizing with the labeled product, single-stranded DNA that did not hybridize was isolated using avidin, returned to double-stranded DNA by DNA polymerase, and cDNA A method for preparing a slurry (Tanaka, H., Yoshimura, Y., Nishina, Υ., Nozaki, zaki., Nojiia, Η., And Nishimune, Υ. (1994) FEBS Lett. 355, 4-10) And the like.

Specifically, first, mRNA or total RNA is purified for each sample under the administration or non-administration conditions of the test substance, the total RNA purified from the sample under the administration condition is converted into type II, and cDNA is synthesized using reverse transcriptase. I do. CDNA can also be labeled by adding [a- ³² P] dNTP during synthesis. The labeled cDNA and total RNA in the form of a 铸 form a stable double-stranded DNA-RNA hybrid. However, high-temperature treatment in the presence of heat degrades only RNA and generates single-stranded cDNA. When this single-stranded cDNA and RNA extracted from a sample under non-administration conditions are mixed and allowed to stand under appropriate conditions, a double-stranded DNA-RNA hybrid is formed that is more stable than the nucleotide sequence complementarity. . That is, the cDNA expressed under non-administration conditions forms a hybrid, but the cDNA expressed specifically as RNA under the administration conditions remains single-stranded. Next, the double-stranded DNA-RNA hybrid and the single-stranded cDNA are separated on a hydroxyapatite column, and only the single-stranded cDNA is purified. By repeating this step, cDNA specific for the target tissue can be concentrated. When the concentrated specific cDNA is labeled with a radioisotope or the like, it can be used as a probe for screening a cDNA library. This operation can also be performed using a commercially available kit (for example, PCR Select cDNA Subtraction Kit: manufactured by Clontech, etc.).

b) Differential 'display method:

According to the method of Liang et al. (Science (1992) 257, 967-971), for example, it can be carried out as follows. First, mRNA or total RNA is extracted from two samples to be compared (in the case of the present invention, a sample under the administration or non-administration of the test substance), and this is converted into single-stranded cDNA using reverse transcriptase. Convert. Next, PCR is performed using the obtained single-stranded cDNA as a type II and using appropriate primers. As the primer, for example, a random primer (a primer of about 10 to 12 mers consisting of an arbitrary sequence) can be used. Alternatively, an uncard primer (anchored primer) and an arbitrary primer (arbitrary primer) may be used in combination. As an anchor primer, use a primer consisting of oligo d (T) _n VX [n = 11 to 12; V = guanine, adenine or cytosine; X == guanine, adenine, thymine or cytosine] Can be done. In addition, as an arbitrary primer, a random primer of about 1 Omer having an arbitrary sequence can be used. By performing such PCR by combining various primers, it becomes possible to screen a wider range of gene groups. Subsequently, the obtained PCR product is subjected to gel electrophoresis, and the expression pattern (fins) of all RNAs spread (displayed) on the gel is displayed. By performing a comparative analysis of the garprint, a gene (PADI4 gene of the present invention) that is specifically expressed in any of the specimens can be selected, and a cDNA fragment thereof can be isolated. This method can also be performed using a commercially available kit (for example, RNA Image Kit 1, manufactured by Jenhan Yuichi).

c) Differential hybridization method:

The cDNA library one prepared from total RNA purified from the tissue of interest, and screened with ³² P-labeled c DNA probe synthesized from total RNA of eye tissue and a control tissue, and clones are selected that seen Haiburidizu the probe of target tissue Is the way. For example, first, a cDNA library is prepared from total RNA purified from a sample under non-administration conditions according to a conventional method, and two sets of replica filters are prepared from the library. Next, cDNA is synthesized with reverse transcriptase using the total RNA purified from the sample under the non-administration condition as type III. During synthesis [- ³² P] labeling c DNA by adding dNTPs. The labeled cDNA and total RNA that has become type II are degraded to single-stranded DNA by high-temperature treatment in the presence of alkali that forms a stable double-stranded DNA-RNA hybrid. Purify the cDNA. Similarly, a single-stranded cDNA is prepared in which all RNA purified from a sample under the test substance administration conditions is labeled with ³² P in a small form. Using both labeled cDNAs as probes, perform hybridization with a filter prepared from a sample under non-administration conditions. By comparing the autoradiographic images of the X-ray film and selecting a clone that hybridizes only to one of the cDNA probes under the administration or non-administration conditions, a gene that is specifically expressed under the administration conditions of the test substance can be identified. Can be cloned.

d) Cross-hybridization method:

Positive clones were obtained by hybridizing the cDNA library derived from either the test substance-administered or non-administered sample with low DNA stringency using appropriate DNA as a probe. obtain. Using the obtained positive clone as a probe, Northern hybridization is performed on all RNAs derived from each sample, and a clone expressing only one of them is selected.

Using the cDNA obtained in this way as a probe, testing under administration or non-administration conditions By performing Northern plotting on all RNAs of the body, it can be confirmed that all the RNAs of the selected gene are specifically expressed under the administration conditions. Step 3: Evaluation of test substance

Finally, the effect of the test substance as an RA therapeutic is evaluated based on the difference in the expression level of the PADI4 gene of the present invention between administration and non-administration of the test substance. That is, when the expression level of the PADI4 gene of the present invention is significantly reduced under the administration condition of the test substance as compared with the non-administration condition, it can be evaluated that the test substance has an effect as a therapeutic agent for RA. Here, “significantly reduced” means that, for example, a statistically significant difference (p <0.05) in the expression level of the PADI4 gene of the present invention under the administration and non-administration conditions of the test substance. It means there is.

2.2 Evaluation method using expression level of PADI4 protein of the present invention as an index

The in vivo evaluation method using the expression level of the PADI4 protein of the present invention as an index includes, for example, the following steps.

Step 1: The animals are bred under the conditions of administration or non-administration of the test substance.

Step 2: The expression level of the PADI4 protein of the present invention in the blood or cells of the animal is detected using an antibody that specifically binds to the protein.

Step 3: The effect of the test substance as an RA therapeutic agent is evaluated based on the difference in the expression level between the administration and non-administration of the test substance. Process 1: Animal rearing

Animals are to be bred under the conditions of administration or non-administration of the test substance according to the method described in 2.1 above. Step 2: Detection of expression level of PADI4 protein of the present invention

Next, the expression level of the PADI4 protein of the present invention in the blood or cells of the animal is detected using an antibody that specifically binds to the protein.

Methods for detecting proteins using antibodies are not particularly limited, but include Western blot, dot blot, slot blot, ELISA, and RIA. Preferably, the method is any one selected from the following methods. Hereinafter, these detection methods will be described specifically from sample preparation to detection.

(1) Sample preparation

As the specimen, blood or cells in which the PADI4 protein of the present invention is highly expressed are preferable, and therefore, blood-derived cells such as neutrophils and eosinophils, synovial cells, spleen cells, and intraperitoneal cells are preferable. . These blood or cells (used as a cell extract) are subjected to high-speed centrifugation as necessary to remove insoluble substances, and then used as ELISA / RIA samples or Western blot samples as follows. To be prepared.

As the sample for ELISA / RIA, for example, the collected serum may be used as it is, or may be appropriately diluted with a buffer. For Western blot (for electrophoresis), for example, use a cell extract as it is or dilute appropriately with a buffer to prepare a sample buffer containing 2_mercatorethanol for SDS-polyacrylamide gel electrophoresis ( Sigma). As the sample for dot / slot plotting, for example, a recovered cell extract itself or a sample appropriately diluted with a buffer is directly adsorbed to a membrane using a blotting apparatus.

(2) Immobilization of sample

In the above method, first, polypeptide in a sample containing the PADI4 protein of the present invention is immobilized on a membrane or the bottom surface of a well of a 96-well plate.

There are two methods for immobilizing the sample on the membrane: transferring the polypeptide to the membrane via polyacrylamide gel electrophoresis of the sample (Western plot method), and directly impregnating the sample or its diluent into the membrane (dot prototyping). Slot method or slot plot method). Examples of the membrane to be used include a nitrocellulose membrane (for example, manufactured by Bio-Rad), a nylon membrane (for example, Hybond-ECL (manufactured by Amersham-Pharmacia)), a cotton membrane (for example, blotting sorbent filter (for example, Biorad) and polyvinylidene difluoride (PVDF) membrane (for example, Biorad). In addition, the plotting method used is an 式 ET-type blotting method (CURRENT PROTOCOLS IN IMMUNOLOGY volume 2 ed by JE Coligan, AM Krui sbeek, DH Margulies, EM Shevach, W. Strober), semi-dry blotting method (see CURRENT PROTOCOLS IN IMMUNOLOGY volume 2 above), and the like.

On the other hand, examples of the method for immobilization on a 96-well plate include enzyme-linked immunosorbent assay (ELISA) and radioisotope immunoassay (RIA). The immobilization may be performed, for example, by subjecting the 96-well plate (for example, Imnoplate Maxi Soap (manufactured by Nunc) or the like) to a sample or a dilution thereof (for example, phosphate buffered saline containing 0.05% sodium azide). Add a saline solution (hereinafter, referred to as “PBS”) diluted, and incubate at 4 to room temperature overnight or at 37 ° C for 1 to 3 hours to adsorb the polypeptide on the bottom of the well. You can do it.

(3) an antibody that specifically binds to the PADI4 protein of the present invention

The “antibody that specifically binds to the Δ4 protein of the present invention (hereinafter referred to as“ anti-PADI4 antibody ”)” used in this step may be prepared according to a known method, or may be commercially available. May be used.

The antibody can be prepared by a conventional method (for example, Shinsei Kagaku Kenkyusho 1, Protein 1, P. 389-397, 1992), using the PADI4 protein of the present invention as an antigen or any polypeptide selected from the amino acid sequence thereof. It can be obtained by immunizing an animal with the antibody and collecting and purifying an antibody produced in the living body of the animal. In addition, publicly known methods (for example, Kohler and Children, Nature 256, 495-497, 1975, Kennet, R. ed., Monoclonal Ant ibody p. 365-367, 1980, Prenum Press, NY) were used. Thus, a hybridoma can be established by fusing the antibody-producing cells producing the anti-PADI4 antibody with myeloma cells, and a monoclonal antibody can be obtained therefrom.

Antigens for producing antibodies include the PADI4 protein of the present invention or a polypeptide comprising at least six consecutive partial amino acid sequences thereof, or any amino acid sequence or carrier thereof (for example, a keyhole limpet to which an N-terminal is added). And derivatives to which mosyanine has been added.

The antigen polypeptide can be obtained by producing a PADI4 protein of the present invention in a host cell by genetic manipulation. Specifically, a vector capable of expressing the PADI4 gene of the present invention is prepared, and this is introduced into a host cell to transform the gene. It may be expressed.

Examples of the host cell include prokaryotic cells, such as Escherichia coli and Bacillus subtilis. To transform a gene of interest into these host cells, the host cells are transformed with a plasmid vector that contains a regulatory element and a replicon, or replication origin, from a species compatible with the host. The vector is preferably one having a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.

For example, in the case of Escherichia coli, the K12 strain or the like is often used, and the vector is generally a pBR322 or pUC-type plasmid, but is not limited thereto, and may use various known strains or vectors. it can. In addition, examples of a promoter used in Escherichia coli include a tryptophan (trp) promoter, a lactose (lac) promoter, a tryptophan lactose (tac) promoter, a lipoprotein (lpp) promoter, and a polypeptide. Chain elongation factor Tu (tufB) peptide motor and the like can be mentioned, and any of them can be suitably used.

For Bacillus subtilis, strain 207-25 is preferable, and pT UB 228 (Ohmura, K. et al. (1984) J. Biochem. 95, 87-93) is used as a vector. It is not limited to this. In addition, extracellular secretory expression can be achieved by ligating the DNΑ sequence encoding the signal peptide sequence of α-amylase of Bacillus subtilis to the vector.

Eukaryotic host cells include cells such as vertebrates, insects, and yeast. Vertebrate cells include, for example, dihydrofolate reduction of monkey COS cells (Gluzman, Y. (1981) Cell 23, 175-182, ATCC CL-1650) and CHO cells (ATCC CCL-61). Enzyme-deficient strains (Urlaub, G. and Chasin, LA (1980) Pro Natl. Acad. Sci. USA 77, 4126-4220) are frequently used, but are not limited thereto.

As an expression vector for a vertebrate cell, a vector having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like located upstream of a gene to be normally expressed can be used. In addition, it may have a replication origin if desired. Examples of the expression vector include pCR3.1 (manufactured by Invitrogen) having the cytomegalovirus early promoter and the SV40 V2 dhί r having an early promoter (Subramani, S. et al. (1981) Mol. Cell. Biol. 1, 854-864), and the like, but are not limited thereto.

Taking the case where COS cells are used as host cells as an example, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and has a transcription promoter, a transcription termination signal, and Those having an RNA splice site can be suitably used. The expression vector is prepared by the method of getylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnus son, G. (1983) Nucleic Acids Res, 11, 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FL) and van der Eb, AJ (1973) Virology 52, 456-457) and electric pulse perforation (Neumann, E. et al. (1982) EMB0 J. 1, 841-845) Thus, desired transformed cells can be obtained.

When CH〇 cells are used as host cells, vectors that can express the neo gene functioning as an antibiotic G418 resistance marker together with the expression vector, for example, pRSVneo (Saibrook, J. et. al. (1989): "Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory, NY) and p S V2 neo (Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341) Transfected cells that stably produce the desired polypeptide can be obtained by co-transfecting them and selecting G418-resistant colonies.

When insect cells are used as host cells, cell lines derived from ovarian cells of Spodoptera frugiperda (S f — 9 or S ί − 21) of the family Lepidoptera and the High Five cells derived from Trichoplusia ni eggs (Wickham, TJ) et al, (1992) Biotechnol. Prog, i: 391-396) is often used as a host cell, and as a Bacchus virus transfer vector, the polyhedrin protein of autographa nucleopolyhedrovirus (AcNPV) is used. PVL 1392/1393 using the Promo overnight is often used (Kidd, i. Μ · and VC Emery (1993) The use of baculoviruses as expression vectors. Applied Biochemistry and Biotechnology 420, 137-159). In addition, vectors using baculovirus P10 and a promoter of the same basic protein can also be used. In addition, AcN The recombinant protein can be expressed as a secreted protein by connecting the secretory signal sequence of the PV envelope surface protein GP67 to the N-terminal side of the target protein (Zhe-mei Wang, et al. (1998) Biol. Chem., 379, 167-174).

As an expression system using a eukaryotic microorganism as a host cell, yeast is generally well known. Among them, yeast of the genus Saccharomyces, for example, baker's yeast Saccharomyces cerevis iae and petroleum yeast Pichia pastoris are preferable. Examples of expression vectors for eukaryotic microorganisms such as the yeast include, for example, a promoter for an alcohol dehydrogenase gene (Bennettzen, JL and Hall, BD (1982) J. Biol. Chem. 257, 3018-3025) and Acid phosphatase gene promoter (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1-5) can be preferably used. When expressed as a secretory protein, it can also be expressed as a recombinant having a secretory signal sequence and a cleavage site for an endogenous protease or a known protease of the host cell at the N-terminal side. . For example, in a system in which human mast cell tryptase, a trypsin-type serine protease, is expressed in petroleum yeast, expression must be performed by connecting the secretory signal sequence of yeast en factor-1 to the N-terminal end of the KEX2 protease cleavage site of petroleum yeast. It is known that activated trypsinase is secreted into the culture medium (Andrew, L. Niles, et al. (1998) Biotechnol. Appl. Biochem. 28, 125-131).

The transformant obtained as described above can be cultured according to a conventional method, and the culture produces the target polypeptide inside or outside the cell. As the medium used for the culture, various types of commonly used media can be appropriately selected depending on the host cell used. For example, in the case of the above COS cells, serum components such as fetal calf serum were added to a medium such as RPMI 1640 medium or Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”) as necessary. Anything can be used. The recombinant protein produced inside or outside the cells of the transformant by the above culture is separated and purified by a known separation operation method utilizing the physical and chemical properties of the protein. be able to. Examples of the method include treatment with a protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPLC ) Etc. Raffy and dialysis can be used alone or in combination. If a histidine consisting of 6 residues is linked to the recombinant protein to be expressed, it can be purified efficiently with a nickel affinity column. The target PADI4 protein of the present invention can be easily produced in high yield and high purity by appropriately combining the methods described above.

(4) Detection ''

The anti-PADI4 antibody obtained by the method described in the above (3) may be directly labeled, or may be used as a primary antibody to specifically recognize the primary antibody (antibody derived from the animal from which the antibody was prepared) Recognition) Used for detection in cooperation with a labeled secondary antibody.

Preferable examples of the type of the label include an enzyme (alkaline phosphatase or horseradish peroxidase) or piotin (however, an operation of binding an enzyme-labeled streptavidin to the secondary antibody piotin is added). Not limited. As the labeled secondary antibody (or labeled streptavidin), various types of pre-labeled antibodies (or streptavidin) are commercially available. In the case of RIA using antibodies labeled with a radioactive isotope such as ¹²⁵ 1, the measurement is performed using a liquid scintillation one Shiyonkaun evening first prize.

By detecting the activity of these labeled enzymes, the expression level of the antigen is measured. In the case of labeling with alkaline phosphatase or horseradish peroxidase, substrates that emit color or emit light by the catalyst of these enzymes are commercially available. When a color-developing substrate is used, it can be detected visually using Western plotting or dot / slot plotting. In the ELISA method, it is preferable to measure and quantify the absorbance (measurement wavelength varies depending on the substrate) of each well using a commercially available microplate reader. Also, by preparing a dilution series of the antigen used in the above antibody preparation, using this as a standard antigen sample, performing detection operations simultaneously with other samples, and creating a standard curve plotting the standard antigen concentration and the measured value, It is also possible to quantify the concentration of antigen in other samples.

On the other hand, when a substrate that emits light is used, detection can be performed by Western radiography or dot / slot plotting by autoradiography using an X-ray film or imaging plate, or by photographing using an instant camera. Can be. Also, densitometry and molecular imagers — Quantification using Fx system (manufactured by Bio-Rad) is also possible. When a luminescent substrate is used in the ELISA method, the enzyme activity is measured using a luminescent microplate reader (for example, Bio-Rad).

(5) Measurement operation

a) In the case of Western blot, dot plot or slot blot First, in order to prevent non-specific adsorption of antibodies, the membrane must be treated with a substance that inhibits such non-specific adsorption (skim milk, casein, serum albumin, gelatin, etc.). (Polyvinylpyrrolidone, etc.) for a certain period of time (blocking). As the composition of the blocking solution, for example, phosphate buffered saline (PBS) or Tris buffered saline (TBS) containing 5% skim milk, 0.05 to 0.1% Tween 20 is used. Instead of skim milk, Block Ace (Dainippon Pharmaceutical); -10% serum albumin, 0.5-3% gelatin or 1% polyvinylpyrrolidone may be used. The blocking time is 4 to 16 hours at 4 or 1 to 3 hours at room temperature.

Next, the membrane is washed with PBS or TBS containing 0.05 to 0.1% Tween 20 (hereinafter referred to as “washing solution”) to remove excess blocking solution, and then diluted appropriately with the blocking solution. An anti-PAD14 antibody is immersed in the solution for a certain period of time to allow the antibody to bind to the antigen on the membrane. The dilution ratio of the antibody at this time can be determined, for example, by performing a preliminary pastelotting experiment using a serial dilution of the recombinant antigen as a sample. This antibody reaction operation is preferably performed at room temperature for 2 hours. After the antibody reaction operation, wash the membrane with washing solution. Here, if the antibody used is labeled, the detection operation can be performed immediately. If an unlabeled antibody is used, perform a secondary antibody reaction. For example, when using a commercially available labeled secondary antibody, dilute it with a blocking solution at a ratio of 2000 to 20000 times. (If a suitable dilution ratio is described in the attached instruction sheet, follow the description.) ). After washing and removing the primary antibody, immerse the membrane in the secondary antibody solution at room temperature for 45 minutes to 1 hour, wash with the washing solution, and perform the detection operation according to the labeling method. The washing operation is performed, for example, by first shaking the membrane in the washing solution for 15 minutes, then replacing the washing solution with a new one. After shaking for 5 minutes, change the washing solution again and shake for 5 minutes. If necessary, the washing solution may be further exchanged for washing.

b) E L I S A / R I A

First, in order to prevent non-specific adsorption of the antibody to the inner bottom surface of the plate on which the sample is immobilized, blocking is performed in advance as in the case of Western blot. The blocking conditions are as described in the section of Western plot.

Next, the inside of the well was washed with PBS or TBS containing 0.05 to 0.1% Tween 20 (hereinafter referred to as “washing solution”) to remove excess blocking solution, and then appropriately diluted with the washing solution. An anti-PADI4 antibody is dispensed and incubated for a certain period of time to bind the antibody to the antigen. The dilution ratio of the antibody at this time can be determined, for example, by conducting a preliminary ELISA experiment using a sample obtained by serially diluting the recombinant antigen. This antibody reaction operation is preferably performed at room temperature for about 1 hour. After completion of the antibody reaction procedure, wash the inside of the well with a washing solution. Here, if the antibody used is labeled, the detection operation can be performed immediately. If an unlabeled antibody is used, perform a secondary antibody reaction. When using a commercially available labeled secondary antibody, for example, dilute it to 2000 to 2000 times with a washing solution and use it (when a suitable dilution ratio is described in the attached instruction sheet). Is subject to that statement). Dispense the secondary antibody solution into the wells after washing and removing the primary antibody, incubate at room temperature for 1 to 3 hours, wash with the washing solution, and perform the detection operation according to the labeling method. The washing operation is performed, for example, by first dispensing the washing solution into the well, shaking for 5 minutes, replacing the washing solution with a new one, shaking for 5 minutes, replacing the washing solution again, and shaking for 5 minutes. . If necessary, the washing solution may be further exchanged for washing.

For example, in the present invention, the so-called sandwich ELISA can be performed by the method described below. First, two hydrophilic regions are selected from each amino acid sequence of the PADI4 protein of the present invention. Next, a partial peptide comprising at least 6 amino acid residues in each region is synthesized, and two types of antibodies using the partial peptide as an antigen are obtained. One of the antibodies is labeled. The unlabeled antibody was immobilized on the bottom of the well of a 96-well ELISA plate. I do. After blocking, place the sample solution in a well and incubate at room temperature for 1 hour. After washing the wells, dispense the labeled antibody diluent into each well and incubate. After washing the inside of the well again, perform the detection operation according to the labeling method. Step 3: Evaluation of test substance

Finally, based on the difference in the expression level of the PADI4 protein of the present invention under the conditions of administration or non-administration of the test substance, the effect of the test substance as an RA therapeutic is evaluated.

That is, when the expression level of the protein is significantly reduced under the test substance administration condition compared to the non-administration condition, the test substance can be evaluated as having an effect as a RA therapeutic agent. Here, “significantly reduced” means, for example, that there is a statistically significant difference (p <0.05) in the expression level of the PADI4 protein of the present invention under the test substance administration and non-administration conditions. Means that.

2.3 Evaluation method based on PADI4 protein activity

A method for evaluating the effect of a test substance as an RA therapeutic agent in vitro using PADI4 activity as an index includes, for example, the following steps. , Step 1: Add PADI4 protein to the reaction mixture containing the arginine-containing compound with or without the addition of the test substance.

Step 2: PADI4 protein activity is measured by quantifying the amount of the reaction product in the reaction solution.

Step 3: The effect of the test substance as a therapeutic agent for rheumatoid arthritis is evaluated based on the difference in the activity under the conditions of addition or non-addition of the test substance. Step 1: Preparation of reaction solution

The reaction solution used in this method contains an arginine-containing compound that can serve as an enzyme substrate for PADI4 protein in a suitable buffer. Arginine-containing compounds include, for example, arginine such as BAEE (Na-benzoyl-L-arginine e thyl ester), N--Benzoyl-L-arginine, Dansyl-glycy L-citruline, etc. Compound If it is a thing, it will not be specifically limited. The amount of the test substance to be added to the reaction solution and the method of addition are not particularly limited, and may be appropriately set according to the properties of the test substance. PAD 14 and the substrate are allowed to react at 25 to 5 (around TC for about 1 to 2 hours. Step 2:

Next, the activity of the PADI4 protein is measured by quantifying the amount of a reaction product generated by the conversion of arginine residues in the substrate to citrulline by PADI4. Here, the “reaction product” may be a reaction product directly generated from a substrate by an enzymatic reaction, or may be a reaction product generated as a by-product. Hereinafter, two methods, a fluorescence method and an absorption method, will be described as preferable measurement methods to be used in the present method. However, the present invention is not limited thereto, and other known methods: for example, fluorescently labeled dansyl-glycyl -Using L-arginine to quantitatively determine the enzymatic reaction product by HPLC (T. Cikura et al., Analytical Biochemistry 285, p230-234, (2000)) be able to.

(1) Fluorescence method

As a reaction solution, for example, a solution is prepared by adding lOmM BAEE or Να-benzoy arginine as a substrate to a lOOmM Tris / HCl (pH 7.8) buffer containing 10 mM CaCl ₂ and 5 M DTT. To this reaction solution, an appropriate amount of PADI4 protein is added, and the reaction is performed, for example, at 37 ° C. for about 1 to 2 hours. After the reaction, stop the reaction by adding an appropriate amount of EDTA, and measure the wavelengths of the excitation wavelength of 413ηηι and the fluorescence wavelength of 476 nm using a fluorescence microplate reader. The measurement results were obtained by subtracting the measured value of the reaction solution containing no PADI4 protein as a background, and drawing a calibration curve from the measured values of the reaction solution to which ammonium sulfate was added for the preparation of a calibration curve. Determine the concentration. The activity of the PADI4 protein is evaluated using the ammonium ion concentration as an index.

(2) Absorption method

As the reaction solution, for example, in lOOmM Tr is / HCl (pH7. 6) buffer containing 10iM CaCl ₂ 5mM DTT, a solution is prepared by adding lOmM BAEE (Sigma) as substrate. To this reaction solution, an appropriate amount of PADI4 protein is added, and the reaction is performed, for example, at 37 ° C. for about 1 to 2 hours. After the reaction, δΟπιΜ Diacetyl monoxime (3-hydroxyimino 2-butane), 2 mM and A solution containing _{_{thiosemicarbaz ide, 3M H 3 P0 4}} , 6M H 2 S0 4, 2mM N¾Fe (S0 4) the B solution containing ₂ 1: adding an appropriate amount of mixed solution at a ratio of 3, a thermal cycler one (Eg, GeneAmp ™ PCR System 9700 (Applied Biosystems)), for example, and react at 95 ° C for 15 minutes and then at room temperature for 10 minutes. After the reaction, the absorbance at 540 nm is measured using an absorbance meter (eg, Spectra max 250 (Molecular Devices)). The measurement result was calculated by subtracting the measured value of the reaction solution containing no enzyme as a background, and drawing a calibration curve from the measured value of the reaction solution to which citrulline for preparing a calibration curve was added, to obtain the amount of citrulline generated by the reaction. Find the concentration. The activity of PADI4 protein is evaluated using the citrulline concentration as an index. Step 3: Evaluation of test substance

Finally, based on the difference in the activity of the PADI4 gene of the present invention or the PADI4 protein of the present invention under the conditions of adding or not adding a test substance, the effect of the test substance as an RA therapeutic drug is evaluated.

That is, when the activity of the gene or the protein is significantly reduced under the conditions of the addition of the test substance as compared with the non-addition conditions, the test substance can be evaluated as having an effect as a therapeutic agent for RA. Here, the term “significantly reduced” refers to, for example, a statistically significant difference (p <0) between the expression level of the PADI4 gene of the present invention or the PADI4 protein of the present invention under the conditions of adding or not adding a test substance. 0 5).

2.4 Evaluation method using cultured cells

A method for evaluating the effect of a test substance as an RA therapeutic agent in vivo using cultured cells includes, for example, the following steps.

The evaluation method preferably includes the following steps.

Step 1: Cells are cultured under the condition of adding or not adding a test substance.

Step 2: The expression level of the PADI4 gene of the present invention in the above cells is detected, or the expression level of the PADI4 protein of the present invention is detected using an antibody that specifically binds to the protein.

Step 3: The effect of the test substance as a therapeutic agent for RA is evaluated based on the difference in the expression level under the conditions of addition or non-addition of the test substance. Step 1: Cell culture

The cells used in the evaluation method of the present invention are not particularly limited as long as they are mammalian cells that highly express the PADI4 gene of the present invention. Cultured cells derived from mammals are preferred, and cultured cells derived from blood, synovial tissue, and spleen tissue (preferably, primary cultured cells) are particularly preferred. The mammal is preferably a human, a mouse, a rat or a hamster, and more preferably a human or a mouse. Alternatively, artificially transformed cells, such as a cell into which the PADI4 gene of the present invention is introduced together with its promoter region and which is highly expressed, may be used. The cells are cultured with or without the addition of the test substance. The culture method is not particularly limited, and a culture method suitable for the cells may be appropriately selected. The method and amount of the test substance to be added to the cultured cells are not particularly limited either. The test substance is added to the culture medium and cultured for a certain period of time before or at an appropriate time during the culture of the cells. The culture period after the addition of the test substance is not particularly limited, but is preferably 30 minutes to 24 hours. Step 2: Detection of the expression level of the PADI4 gene of the present invention or the PADI4 protein of the present invention Next, the difference in the expression level of the PADI4 gene of the present invention in the above cells under the conditions of addition or non-addition of the test substance is detected. Alternatively, the difference in the expression level of the PADI4 protein of the present invention is detected using an antibody that specifically binds to the protein.

The detection of the gene may be basically performed according to the method described in 2.1. Detection using an antibody may be performed according to the method described in 2.2. In addition to the above-described methods, the present invention uses a gene (hereinafter referred to as “reporter gene”) that allows detection of the promoter activity under the control of the promoter of the PADI4 gene of the present invention. The expression of the PADI4 gene and the PADI4 protein of the present invention can also be detected. Hereinafter, a detection method using the repo overnight gene will be described.

(1) Reporter gene

The repo overnight gene encodes a reporter protein that is specifically distinguishable from any other protein that the host cell may produce in the course of this test method. Anything can be used. Preferably, the cells before transformation do not have a gene encoding the same or similar protein as the reporter protein. For example, even when the reporter protein is toxic to the cell or confers resistance to a susceptible antibiotic, the presence or absence of expression of the repo overnight gene can be determined. It can be determined by the survival rate of the cells. However, a more preferable reporter gene used in the present invention can specifically and quantitatively detect the expression level (for example, when a specific antibody against the protein encoded by the reporter gene is obtained). It is a structural gene. More preferably, it is a gene encoding an enzyme or the like which specifically reacts with a foreign substrate to produce a metabolite which can be easily measured quantitatively. Examples of such a reporter gene include, for example, genes encoding the following proteins, but the present invention is not limited thereto.

a) Chloramphenicol acetyltransferase:

An enzyme that adds an acetyl group to chloramphenicol, and can be detected by so-called CAT technology. PCAT3-BASIC vector (Promega) is commercially available as a vector that can be used to prepare a vector for repo assay simply by incorporating a promoter.

b) Firefly luciferase:

It can be quantified by measuring the bioluminescence generated when luciferin is metabolized. As a vector for repo overnight, pGL3-BASIC vector-1 (promega) is commercially available.

c) β-galactosidase:

Some substrates can be measured by color reaction, fluorescence or chemiluminescence, respectively. As a vector for repo overnight, p / 3gal-Basic (promega) is commercially available.

d) Secreted alkaline phosphatase:

There are substrates that can be measured by color reaction, bioluminescence, or chemiluminescence, respectively. As a vector for repo overnight, pSEAP2-Basic (Clontech) is commercially available.

e) Green-fluorescent protein: Although it is not an enzyme, it can quantify directly because it emits fluorescence. Similarly, pEGFP-1 (Clontech) is commercially available as a vector for repo overnight.

(2) Introduction of reporter gene

According to a known method, a reporter gene expression plasmid and a recombinant vector in which the PADI4 gene of the present invention of the present invention can be expressed in mammalian cells are prepared, and these are cotransfected into cells. As the vector, pCR3.1 (manufactured by Invitrogen), pCMV-Script (manufactured by Straugene), and the like can be preferably used, but are not limited thereto.

Methods for introducing an expression plasmid into cells include the DEAE-dextran method (Luthman, H. and Magnus son, G. (1983) Nucleic Acids Res. 11, 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FL and van der Eb, AJ (1973) Virology 52, 456-457), electric pulse perforation (Neumann, E. eUl. (1982) EMBO J. 1, 841-845), ribofection (Lopata et al. (1984) ) Nucl. Acids Res. 12, 5707-5717, Sussman and Milman (1984) Mol. Cell. Biol. 4, 1641-1643). However, if the cells are so-called floating cells, it is preferable to use a method other than the calcium phosphate-DNA coprecipitation method. In any of these methods, optimized transfection conditions are used according to the cells used. It is necessary to use.

(3) Evaluation

When cells transfected with the PADI4 gene expression vector of the present invention and a reporter expression vector are co-transfected, transcription of the repo overnight gene is promoted depending on the expression of the gene. Therefore, the change in the expression level of the reporter gene with and without the addition of any test substance in the medium under conditions that allow for the expression of the repo overnight gene indicates the PADI4 gene of the present invention. The expression level can be evaluated. Here, the “conditions under which the reporter gene can be expressed” are defined as conditions under which the cells transfected by the repo overnight expression vector survive and can produce a transcript (reporter protein) of the reporter gene. I just need. Preferably, the medium is suitable for the cell line used (or may contain serum components such as fetal bovine serum) and contains 4 to 6% (most preferably 5%) carbon dioxide. Air presence Incubate at 36-38 ° C (most preferably 37 ° C) for 2-3 days (most preferably 2 days).

(4) Others (establishment of transformed cell line)

Apart from the test method using the transient gene transfer method as described above, a cell obtained by double-transforming a host cell with the expression vector for the repo allelic gene and the PADI4 gene of the present invention is used. Test methods that have been adopted can also be adopted. In this case, an expression vector such as pIND (manufactured by Invitrogen) or pTet-On (manufactured by Clontech) is used to induce the expression of the reporter gene under conditions that induce the expression of the PADI4 gene of the present invention. It is necessary to establish a cell line that promotes expression of the gene. In producing transformed cells, it is desirable that the gene to be introduced be integrated into the chromosome of the host cell and be stably retained in the host cell even after repeated passages. In order to select cells transformed in such a manner, a transgene is ligated with a selectable marker such as an antibiotic resistance or the like (for example, neomycin (or G418) resistance gene neo or the like) and transfected. Alternatively, it is preferable to simultaneously transfect the separately prepared selectable marker and the transgene. Thereafter, by utilizing the characteristics of the selectable marker, stably transformed cells can be selected.

When the cell line thus obtained is placed under conditions that induce the expression of the PADI4 gene of the present invention, the transcription of the reporter gene is promoted in a manner dependent on the expression of the gene. Therefore, under the conditions where the reporter gene can be expressed, the expression level of the PADI4 of the present invention can be seen from the change in the expression level of the reporter gene with and without the addition of any test substance in the medium. Can be evaluated. Step 3: Evaluation of test substance

Finally, the effect of the test substance as a therapeutic agent for RA is evaluated based on the difference in the expression level of the PADI4 gene of the present invention or the PADI4 protein of the present invention under the conditions of adding or not adding the test substance.

That is, when the expression level of the gene or protein is significantly reduced under the conditions of the addition of the test substance as compared with the non-addition conditions, the test substance can be evaluated as having an effect as a therapeutic agent for RA. Here, “significantly decreases” means, for example, It means that there is a statistically significant difference (p <0.05) in the expression level of the PADI4 gene of the present invention or the PADI4 protein of the present invention under the conditions of adding or not adding the test substance.

2.5 Kits for evaluating the effects of test substances as RA therapeutics

Examples of the kit for performing the evaluation method of the present invention include a kit containing at least one of the following a) to: O 2.

a) Human PADI4 gene (SEQ ID NO: 16) or mouse PADI4 gene (SEQ ID NO: 16)

17) A continuous oligonucleotide primer of 15 to 30 bases in length to specifically amplify

b) A continuous polynucleotide probe of 20 to 1500 bases in length that specifically binds to the human PADI4 gene or mouse PADI4 gene and detects the gene c) The polynucleotide probe described in b) above was immobilized Immobilized sample d) Human PADI4 protein (SEQ ID NO: 17) or mouse PADI4 protein (SEQ ID NO:

18) an antibody that specifically binds to and detects the protein

f) Human PADI4 protein

The primer described in a) above can be readily designed and amplified according to a conventional method, for example, using commercially available primer design software, based on the nucleotide sequence of the above gene (SEQ ID NO: 16 or 17). Examples of such primers include, for example, oligonucleotides having the nucleotide sequences of SEQ ID NOs: 7 to 10.

The probe described in b) above is preferably a single-stranded oligonucleotide or double-stranded DNA having a length of about 20 bases in the case of the Northern hybridization method. In the case of a microarray, a double-stranded DNA having a length of about 100 to 1500 bases or a single-stranded oligonucleotide having a length of about 20 to 100 bases is suitably used. For the Affimetrix Gene Chip system, a single-stranded oligo with a length of about 25 bases is preferred. These are preferably designed as probes that specifically hybridize to a portion of the PADI4 gene of the present invention having high sequence specificity. Examples of such a probe include, for example, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 11. These probes and the aforementioned primers May be labeled with an appropriate label (for example, an enzyme label, a radioactive label, a fluorescent label, or the like), or may be modified with biotin, phosphoric acid, an amine, or the like.

The solid-phased sample described in c) above is produced by fixing the probe described in b) above to a solid phase such as a glass plate, a nylon membrane, microbeads, or a silicon chip. The immobilized sample and the method for preparing the same have already been described in 2.1, but examples thereof include a gene chip, a cDNA array, an oligo array, and a membrane filter.

The antibodies described in d) and e) and the protein in f) can be prepared by the method described in 2.2. The antibody may be labeled with a suitable label (for example, an enzyme label, a radioactive label, a fluorescent label, or the like), or may be appropriately modified with biotin or the like.

In addition to the components described above, the kit may be used for evaluation of the present invention, such as hybridization, probe labeling, or a reagent for detecting a label, a reaction buffer, and an enzyme substrate, if necessary. It may optionally contain other reagents and the like necessary for carrying out the method.

3. Method for Predicting Risk of Onset of Rheumatoid Arthritis Using Mutant PADI4 The present invention relates to a mutant PADI4 protein comprising the amino acid sequence shown in SEQ ID NO: 21 in a sample, or a gene thereof (SEQ ID NO: 20). The present invention provides a method for predicting the risk of developing rheumatoid arthritis (hereinafter referred to as “RA”) in a subject who has provided the test sample, based on the expression level of the test sample.

The term “specimen” used in the above-mentioned method means any sample such as blood, body fluid, tissue, or excrement isolated from a subject, and includes blood (blood-derived cells), synovial tissue (synovial cells) ), Spleen tissue (spleen cells), and cells containing peritoneal cells are preferred, and blood cells (neutrophils, eosinophils) and synovial tissue (synovial cells) are more preferred. The risk of onset may be predicted using the expression level of the mutant PADI4 protein as an index or the expression level of the mutant PADI4 gene (mRNA) as an index. 3.1 Method based on the expression level of mutant PADI4 gene The prediction method using the expression level of the PADI4 gene as an index includes, for example, the following steps. Step 1: preparing all RNAs from each sample isolated from a subject and a normal person; Step 2: detecting the expression level of mRNA of the mutant PADI4 gene in all the above RNAs;

Step 3: Analyze the difference in the above expression level between the subject and the normal person, and predict the risk of developing the subject's chronic joint rheumatism.

In the above method, extraction of all RNAs and detection and analysis of the mRNA expression level of the mutant PADI4 gene can be performed according to the description in 2.1. As a result, if the expression level of the mutant PADI4 mRNA is significantly higher in a subject than in a normal person, the subject can be evaluated as having a high risk of developing RA. Here, “significantly higher” means that, for example, there is a statistically significant difference (p <0.05) in the expression level of the mutant PADI4 mRNA between the subject and a normal person. As described above, the evaluation was performed using the ratio of the expression level of the mutant PADI4 gene and the appropriate control gene (for example, 3-actin gene ゃ liposomal RNA, etc.) in the sample, and subjected to the above analysis. Is also good.

3.2 Evaluation method based on the expression level of mutant PADI4 protein

The method using the expression level of the mutant PADI4 protein as an index includes, for example, the following steps. Step 1: detecting the expression level of the mutant PADI4 protein in a sample isolated from a subject and a normal person using an antibody capable of specifically binding to the protein; Step 2: detecting the expression level in the subject and a normal person The difference in the expression level is analyzed to predict the risk of developing RA in the subject.

In the above method, detection and analysis of the expression level of the mutant PADI4 protein can be performed according to the description in 2.2. As a result, when the expression level of the mutant PADI4 protein in the subject is significantly higher than that in a normal person, the subject can be evaluated as having a high risk of developing RA. Here, “significantly high” means, for example, a statistically significant difference in the expression level of mutant PADI4 protein between a subject and a normal person (p <0.

0 5). 3.3 Kit for predicting the risk of developing rheumatoid arthritis The present invention also provides a kit for predicting the risk of developing rheumatoid arthritis. The kit includes, for example, at least one of the following a) to e). a) a 15- to 30-base-length continuous oligonucleotide primer for specifically amplifying a mutant PADI4 gene consisting of the base sequence shown in SEQ ID NO: 20 b) the base shown in SEQ ID NO: 20 A continuous polynucleotide probe with a length of 20 to 150 bases for specifically hybridizing to the mutant PADI4 gene comprising the sequence and detecting the gene.

c) An immobilized sample on which the polynucleotide probe described in b) above is immobilized d) An antibody specifically binding to the mutant PADI4 protein having the amino acid sequence shown in SEQ ID NO: 21 and detecting the protein

e) A secondary antibody capable of specifically binding to the antibody described in d) above. The primer described in a) is a commercially available primer design software based on the nucleotide sequence of the above gene (SEQ ID NO: 20 or 21). It can be easily designed and amplified according to the usual method such as using. In the Northern hybridization method, a single-stranded oligonucleotide or double-stranded DNA having a length of about 20 bases is suitably used as the probe described in b) above. In the case of a microarray, a double-stranded DNA having a length of about 100 to 150 bases or a single-stranded oligonucleotide having a length of about 200 to 100 bases is suitably used. In the case of the Gene Chip system of Afmetrix, a single-stranded oligo with a length of about 25 bases is preferred. These are preferably designed as probes that specifically hybridize to a portion having high sequence specificity of the mutant PADI4 gene. Examples of such a probe include, for example, an oligonucleotide having the base sequence of SEQ ID NO: 4. These probes and the aforementioned primers may be labeled with an appropriate label (for example, an enzyme label, a radioactive label, a fluorescent label, or the like), or may be modified with biotin, phosphoric acid, an amine, or the like.

The solid-phased sample described in c) above is prepared by fixing the probe described in b) above to a solid phase such as a glass plate, a nylon membrane, micropease, or a silicon chip. Such immobilized samples and their preparation methods have already been described in 2.1. For example, gene chips, cDNA arrays, oligo arrays, One example is a plain filter. The antibody according to d) and e),

It can be produced by the method described in 2.2. The antibody may be labeled with an appropriate label (for example, an enzyme label, a radioactive label, a fluorescent label, or the like), or may be appropriately modified with biotin or the like.

The kit is, in addition to the components described above, necessary for the implementation of the method of the present invention, such as a hybridization, a reagent for detecting the label of the probe or a label, a reaction buffer, etc. Further, other reagents and the like may be appropriately contained.

3.4 RA diagnostic marker.

Mutant PADI4 protein ゃ The gene was newly discovered by the present inventors, and the functional difference between these mutant PADI4 protein ゃ gene and normal PADI4 protein ゃ gene is It is closely related to RA onset. That is, a mutant PADI4 protein consisting of the amino acid sequence shown in SEQ ID NO: 21 and a mutant PADI4 gene consisting of the nucleotide sequence shown in SEQ ID NO: 20 are used as diagnostic markers for RA (for example, anti-mutant PADI4 Useful as an antigen for producing antibodies). The present invention also provides RA diagnostic markers using such mutant PADI4.

4. Screening method of RA therapeutic drug using mutant PADI4

The mutant PADI4 protein has a higher enzyme activity than the normal PADI4 protein, and may be one of the causes of RA onset through enhanced production of citrullinated peptides. Therefore, by using the specific inhibitory effect on the mutant PADI4 protein as an index, screening for RA therapeutic agents is possible.

That is, the present invention provides a method for evaluating the effect of a test substance as a therapeutic drug for rheumatoid arthritis using the inhibitory effect on the mutant PADI4 protein consisting of the amino acid sequence shown in SEQ ID NO: 21 as an index.

For example, the method includes the following steps.

Step 1: Add the mutant PADI4 protein to the reaction solution containing the arginine-containing compound, with or without the addition of the test substance;

Step 2: measuring the activity of the mutant PADI4 protein by quantifying the amount of the reaction product in the reaction solution; Step 3: The effect of the test substance as a therapeutic agent for rheumatoid arthritis is evaluated based on the difference in the activity under the conditions of addition or non-addition of the test substance.

In the above method, the mutant PADI4 protein can be prepared as described in 2.2, and steps 1 to 3 can be performed as described in 2.3.

5. Other

5.1 Prediction method of RA disease state using PADI4

The human PADI4 gene and PADI4 protein are specifically highly expressed in diseased tissues (eg, synovial tissue) of RA patients, and may cause RA through the production of citrullinated proteins. Therefore, by measuring the expression levels of the PADI4 gene and PADI4 protein in the specimen isolated from the subject as well as the mutant PADI4 described above, the RA pathology of the subject can be predicted. 5.2 RA model animal

By artificially expressing the PADI4 gene or mutant PADI4 gene of the present invention at a high level, an animal (eg, a mouse or the like) exhibiting an RA pathological condition can be produced. For example, the PADI4 gene or the mutant PADI4 gene of the present invention is incorporated into an appropriate vector and introduced into an animal, and if phenotypic changes such as inflammatory symptoms typified by human RA appear, the animal is used. Research on the condition of RA and its remedies. Similarly, by introducing the PADI4 protein or the mutant PADI4 protein of the present invention directly into an animal, a model animal exhibiting an RA disease state can be produced. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

[Example 1] Association analysis between single nucleotide polymorphism in PADI4 third intron and RA disease

1.Select patient for analysis.

Genomes from 846 RA patients and 658 non-patients from clinical facilities around Japan Blood for MA extraction was collected. All sample providers are Japanese. The donors gave their consent for informed consent by the Senyuichi Ethics Committee for Genetic Polymorphism Research at RIKEN. For RA patients, those who met at least three of the following seven criteria of the revised criteria of the American College of Rheumatology for the classification of rheumatoid arthritis were selected as RA patients.

(1) Morning stiffness

(2) Arthritis in three or more areas

(3) Wrist arthritis

(4) symmetric arthritis

(5) Rheumatoid nodules

(6) Rheumatoid factor

(7) X-ray findings

The non-patient group was selected from the general Japanese population independently of patients. Sampling was performed so that there was no difference between the RA patient group and the non-patient group except for the presence or absence of the eight diagnosis.

2. Preparation of genomic DNA

Genomic DNA was extracted from leukocytes separated from peripheral blood of RA patients and non-patients as a starting material. For extraction of genomic DNA, blood was collected in a blood collection tube (Benoject) containing EDTA'2Na, and the QIAa DNA Blood Kit (QIAGEN) was used. The absorbance at 260 nm of each sample was measured, and the DNA concentration of each sample was calculated. Thereafter, the prepared DNA was dissolved in lOmM Tris-HCl buffer (pH 8.0) containing lmMEDTA'2Na, and stored at -80 until use.

3. Detection of single nucleotide polymorphism in the third intron

Using the three types of probes shown below, we detected whether the base at position 2136 (-15) of the third intron of PADI4 (position 6177 in SEQ ID NO: 1) was cytosine or thymine. .

Invader probe: 5'-CTGCGCACAGGGAGATTTCTGAAATCCCATCAT-3 '(SEQ ID NO: 2) FAM probe for cytosine detection:

5'-ATGACGTGGCAGACGGTAAGAGGAGAGGTTGGY-3 '(SEQ ID NO: 3)

VIC probe for thymine detection:

5'-CGCGCCGAGGAGTAAGAGGAGAGGTTGGY-3 '(SEQ ID NO: 4)

Each probe was commissioned to Third Wave Technologies, Inc., designed and synthesized, and used as a mixed reagent. Invader Atsushi is prepared by adding an invader reaction solution (Applied Biosystems, Inc.) containing 96-well (Nihon Genetics) or 384-well plates (ABI) containing a 4-fold dilution of the PCR reaction solution 21 and the above three probes. ) Was added, and the mixture was incubated at 95 ° C for 5 minutes, and then reacted at 63 ° C for 20 to 80 minutes until a signal was separated. The signal was detected using the ABI PRISM 7700 Seauence Detector System (Applied Biosystems) for 96-well, and the ABI PRISM 7900HT Sequence Detector System (Applied Biosystems) for 384-well.

4. Statistical analysis

A case-control-related test was conducted for 846 case patients (RA patients) and 658 controls (non-patients) for the single nucleotide polymorphism at the 3rd intron 2 13 6 Was. The test was performed by comparing the allele frequency distribution between the two groups, comparing the genotype distribution using the dominant inheritance model, and comparing the dienotype frequency using the recessive inheritance model. As a test method, a 2 × 2 contingency table χ2 test was used. Table 1 shows the analysis results.

〔table 1〕

Analysis results (PADI4 3rd intron Single nucleotide polymorphism at base No. 2136)

Odds Ratio TT / CC 1.97 (95% CI 1.44-2.69)

TT / TC 1.38 (95% CI 1.03-1.86)

TC / CC 1.43 (95% CI 1.13-1,80)

As described above, a statistically significant correlation was found between the polymorphism at position 2136 of the third intron of the PADI4 gene (position 6177 in SEQ ID NO: 1) and the incidence of rheumatoid arthritis.

[Example 2] Detection of novel exon-nucleotide polymorphism in PADI4 gene

1. Detection method

GenBank accession number: An attempt was made to detect a novel gene polymorphism on the PADI4 gene, which is present at nucleotides 415,085 to 470,600 in the genome sequence shown in NTJ30584.2 by the following method.

First, the entire exon, the intron near the exon, and the 5'-flanking region of the gene were divided every about 500 base pairs and set as an amplification region, followed by PCR amplification. Primers for PCR were designed using Primer3 (http: // www-genome, wi. Mit. Edu / ge band e_software / oter / primer3.html) software. PCR conditions were optimized by changing the concentration of SO and the annealing temperature.

PCR was performed in 96-well PCR plates, and 1 ng of 4 ng / ^ 1 genomic DNA was used for each well. The reaction _{_{_{26.5mM (NH 4) 2 S0 4}}} , 7.2mM Tris-HCl (pH8.8), 3.2 mM Mg ²⁺ , 1.5 mM dNTPs, 1.6 mM b-Mercapto ethanol, 10% DMSO (If DMSO is not used, substitute with にて 0), IM primers, 2.OU DNA polymerase (ExTaa (TaKaRa) ),

The experiment was performed under the solution conditions of 0.04 xM Taa Start Ant ibody (Clontech). After denaturation at 94 ° C for 2 minutes, the extension reaction was performed at 94T for 15 seconds, at an annealing temperature optimized for each reaction for 45 seconds, and at 72 for 1 or 3 minutes. 37 cycles were performed using an ABI9700 thermal cycler (Applied Biosystems). The amplified DNA sequence was reacted using BigDye ™ Terminator RR Mix (Applied Biosystems) and the nucleotide sequence was determined using ABI 3700 seciuencer. 2. Result

The nucleotide sequences of 48 regions were compared for each region, and the nucleotides with polymorphism were regarded as single nucleotide polymorphisms. As a result, the following three new exon single nucleotide polymorphisms with amino acid substitution and one new exon polymorphism without amino acid substitution were found.

1) Polymorphism with substitution of amino acid 5 (Ser / Gly):

Single nucleotide polymorphism in which the 71st base of the second exon (1071st in SEQ ID NO: 1) is adenine or guanine

2) Polymorphism with substitution of amino acid 8 (Al a / Val):

Single nucleotide polymorphism in which the 153rd base of the second exon (115rd base in SEQ ID NO: 1) is cytosine or thymine

3) Polymorphism involving substitution of amino acids 1 and 2 (Al a / Gly):

Single nucleotide polymorphism in which the 3rd exon's 6 2nd base (SEQ ID NO: 1 at 4036) is cytosine or guanine

4) Polymorphism without amino acid substitution in the 117th amino acid (Leu): Single nucleotide polymorphism in which the 9th base of the fourth exon (620000th in SEQ ID NO: 1) is cytosine or thymine Type

[Example 3] Linkage analysis of each single nucleotide polymorphism on PADI4 gene

1. Analysis method

PADI4 3rd intron 2 1 3 6th (1-15) strongly associated with RA Pairwise Linkage Disequilibrium Index (D ') between the nucleotide single nucleotide polymorphism (PADI4 intron 3-15 T / C) and four new single nucleotide polymorphisms on PADI4 Was calculated using the EM algorithm. 2. Result

As a result, the EM algorithm shows that the four single nucleotide polymorphisms are almost completely linked to each other (98.9 to 100%), and are also almost completely linked to PADI4 intron 3 -15 T / C. Haplotype analysis. On the other hand, the known single nucleotide polymorphisms in the PADI2 and PADI1 genes near PADI4 and the PADI4 intron 3-15 T / C almost reached linkage equilibrium.

In other words, in the Japanese population, 55 (Ser) -82 (Al a) -112 (Al a) type and 55 (Gly)-82 (Val)-112 (Gly It was confirmed that only the 2 type peptide exists, and that the latter type and RA have the same association as PADI4 intron 3 -15 T / C. Hereinafter, the former is referred to as “PADI4 V18” (or simply “V18”), and the latter is referred to as “PADI4 V9” (or simply “V9”). The cDNA sequence of V18 is shown in SEQ ID NO: 16 and the amino acid sequence is shown in SEQ ID NO: 17 in the sequence listing. The cDNA sequence of V9 is shown in SEQ ID NO: 20 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 21.

From the above, it was concluded that there was a relationship between the dienotype of the PADI4 gene and RA onset, and that the relationship was derived from two haplotypes present in the PADI4 gene. The haplotype associated with the onset of RA was V9, and the haplotype not associated with the onset of RA was V18.

[Example 4] Comparison of stability of human PADI4 mRNA

1. Degradation measurement

Genes encoding two PAD 14 haplotypes were prepared in the following manner. First, cDNA synthesized by first strand cDNA synthes is kit (Amersham Pharmacia) using bone marrow total RNA (Clontech) was cloned into DONR201 (Invitrogen). The obtained cDNA was further recloned into PDEST14 (Invitrogen) containing the T7 promoter, and pV18-T7 And PV9-T7 vectors were prepared and sequenced. Then, the pV18-T7 and pV9-T7 vectors were digested with Clal, and in vitro transcription was performed using RiboMax ™ Large Scale RNA Product ion System-T7 (Promega Chido) to produce V9 and V18 mRNA. .

To prepare Whole cell extract, wash HL-60 cells with PBS and extract buffer (0.5% NP-40; 20 mM HEPES pH 8.0; 20% glycerol (v / v); 400 mM NaCl containing 0.5 mM DTT; 0.2 mM EDTA; 1% proteinase inhibitor cocktail (Nacal ai)). After incubating on ice for 30 minutes, the mixture was centrifuged at 4 ° C, and the supernatant was stored at 80 ° C.

The above-mentioned Whole cell extract 3 xl diluted 1000-fold was mixed with 5 g of each V9 and V18 mRNA and incubated at room temperature. After reacting for 0, 5, and 10 minutes, the reaction was stopped by adding formamide dye, heated at 68 for 10 minutes, and cooled on ice. Transcripts were detected by Northern plot hybridization. Scanning was performed using DocuCentre color 500cp (Fuji iXerox), and the signal intensity was measured by obePhotoshop 6.0 (Adobe).

The results are shown in Figure 1. As is evident from FIG. 1, it was found that V9 mRNA associated with RA lesions was significantly less likely to be degraded than V18 mRNA not associated with RA lesions. (P = 0.048 5 minutes after * T test, p = 0.013 after 10 minutes). [Example 5] Preparation of human PADI4 protein

1. Expression of PAD 14 protein with Hi S-1 ag by Escherichia coli

An expression plasmid was prepared in which two PADI4 haplotype fragments to which His-tags were added: RGS6-HisV18 (SEQ ID NO: 5) or RGS6-HisV9 (SEQ ID NO: 6) were inserted into NcoI / BamHI of pETlld (Stratagene). Escherichia coli BL2 codonPlus (DE3) -RIL cell (Strategene) transformed with this plasmid was added to LB medium (ampicillin 50 g / ml (Sigma) and chloramphenicol 50 / g / ml (Sigma)). Invitrogen) and cultured overnight at 37 ° C. 10 mL of the culture solution was added to a double-concentration LB medium (Invitrogen) supplemented with 1 L of 2% glucose (Wako Pure Chemical Industries) and ampicillin 50 ig / ml (Sigma), and the absorbance at 600 nm was reduced to 0.9. The cells were cultured at 37 ° C until complete. Isopropylthio-0-D-galacto to a final concentration of 0.1 Cid (IPTG: Wako Pure Chemical Industries) was added, and the cells were cultured at 20 ° C for 4 hours. The culture was centrifuged at 6000 X g for 10 minutes to collect the cells. Bacterial cells were 30m1 of 50mM Tris-HCl (pH 7.6) (Wako Pure Chemical), lOOmM sodium chloride (Wako Pure Chemical), 2mM dithiothreitol (DTT: Wako Pure Chemical), specified amount of complete protease The suspension was suspended in a lysis buffer containing an inhibitor cocktail (Roche Diagnostics), sonicated at 4 ° C., and centrifuged at 97,000 × g for 30 minutes to collect the supernatant. The supernatant was applied to a Mi Ilex-HV (Millipore) filter.

2. Purification of His-tagged PADI4 protein

Sephadex-G25 (Amersham Biosciences, Inc.) previously equilibrated the supernatant prepared in the previous section with buffer 50 mM Tris-HCl (pH 7.6) (Wako Pure Chemical Industries) and lmM ethylenediaminetetraacetic acid (EDTA: Dojindo Chemical) 1 ) And fractionated with buffer 1A containing 0.1 M sodium chloride. The fraction in which the activity was observed was applied to DEAE-Sepharose (Amersham Bioscience) pre-equilibrated with buffer A, washed with buffer A containing 0.1 M sodium chloride, and then added to buffer A. Elution was performed by gradually changing the sodium chloride concentration from 0.1 M to 0.2 M, and fractionation was performed. A three-fold amount of buffer B (50 mM Tris-HCl (pH 7.6) (Wako Pure Chemical Industries), 0.1 M sodium chloride (Wako Pure Chemical Industries), 0.1% Triton X-100 } And added to a Ni-NTA super-flow column (QIAGEN). The column was washed with buffer B containing 20 mM imidazole and eluted with buffer B containing 200 mM imidazole. The fractions showing the activity were analyzed using buffer C {10 mM Tris-HCl (pH 7.6) (Wako Pure Chemical), 0.15 M sodium chloride (Wako Pure Chemical), 2 mM DTT (Wako Pure Chemical), lmM EDTA (Dojindo) After concentrating, glycerol (Sigma) was added to a final concentration of 10% and stored at _80 ° C.

[Example 6] Activity measurement of human PADI4 protein

1. Method for measuring PADI4 activity (fluorescence method)

As a buffer solution for enzyme reaction, lOOmM Tri s / HCl (pH7 8.), Flame _{CaCl 2, 5mM DTT, lOmM BAEE} (Sigma) or N-a - A solution containing Benzoy Bokuboku arginine (Tokyo Kasei) was prepared . This buffer solution 401 and the undiluted enzyme solution of ¥ 18 prepared in Example 5 (113 1111) Was diluted in a PCR reaction plate (Applied Biosystems) and reacted at 37 ° C for 2 hours. After the reaction, the reaction was stopped by adding lO / il of 200 mM EDTA. A portion of this was transferred to a deep well plate (Nihon Genetics). At the same time, a 0-50 nM ammonium sulfate solution was also added to another well to prepare a calibration curve. Further, 1 ml of 50 mM boric acid and 50 xl of an equal volume mixed solution of 50 mM o-phthalaldehyde and 50 mM DTT were added to these wells, and reacted at room temperature for 1 hour. The reaction solution was transferred to a white plate (Coaster Coating Co., Ltd.), and the excitation wavelength of 413 nm and the fluorescence wavelength of 476 nm were measured using Spectra max GEMINI (Molecular Devices). The measured value of the well without enzyme was subtracted as the background, and the concentration of ammonium ion generated by the reaction was determined by drawing a calibration curve from the measured value of the well with the addition of ammonium sulfate for preparing the calibration curve (Fig. 2A). ;).

2. PADI4 activity measurement method (absorption method)

A solution containing 100 mM Tris / HCl (pH 7.6), 10 mM CaCl ₂ , 5 mM DTT, and 10 mM BAEE (Sigma) was prepared as a buffer for the enzyme reaction. This buffer 40 1 and

10 l of the diluted series of V18 enzyme stock solution (113 / g / ml) prepared in Example 5 was mixed in a PCR reaction plate (Applied Biosystems), and the mixture was reacted at 50 ° C or 371: for 2 hours.ヽAfter the reaction, including solution A (SOmMDiacetyl monoxime (3- hydroxy imino 2- butane), 2mM t iosemicarbazide) containing) and B solution _{_{(3M H 3 P0 4, 6M}} ¾S0 4, 2mM NH 4 Fe (S0 4) 2 ) In a 1: 3 ratio were added in 150 1 increments. At the same time, to prepare a calibration curve, a 0-400 M L-citrulline solution was also added to another well, and a mixed solution of solution A and solution B was similarly added. The mixed solution was reacted using a thermal cycler (GeneAmp ™ PCR System 9700, Applied Biosystems) at 95 for 15 minutes and then at room temperature for 10 minutes. The solution after the reaction was transferred at 150 × 1 minute to a 96-well plate (Corning), and the absorbance at 540 nm was measured using Spectra max 250 (Molecular Devices). The concentration of citrulline produced by the reaction was determined by subtracting the measured value of the well containing no enzyme as a background and drawing a calibration curve from the measured value of the well to which citrulline for preparing the calibration curve was added (Fig. 2B). ). 3. Enzyme activity of human PADI4 haplotype V9 and V18

The enzymatic activities of human PAD 14 haplotypes V9 and V18 were measured using the absorbance method and compared (Figure 3). As is clear from FIG. 3, it was confirmed that V9 had significantly higher enzyme activity than V18 (P 0.01). Based on the above, PADI4 Eve (V9) associated with RA lesions had higher enzyme activity than normal PADI4 type (V18), suggesting that such a difference in enzyme activity might have some association with RA onset.

[Example 7] Association between PADI4 dienotype and anti-citrullinated filaggrin antibody titer in RA patients

1. Anti-citrullinated peptide antibody titer measurement method

The MESACUP ACF test for the anti-citrullinated peptide antibody titer of RA patient sera was performed using a kit commissioned by the Institute of Medical Biology (Nagoya). The MESACUP ACF test is a method for measuring antibody titer by ELISA using citrullinated filaggrin, which is obtained by converting recombinant human filadrin peptide into citrulline by PAD, as an antigen.

First, serum was prepared from peripheral blood of a patient, and 10 zl was added to 1 mL of a reaction buffer and diluted 101-fold. The anti-filaggrin antibody standard serum 2, the standard serum 1, and the diluted sample were added to the primary reaction preparation microplate at 150 / xL in the same manner as in the actual case. Thereafter, 100 L was transferred to the antigen-sensitized microcup using a multi-pip. After allowing the reaction to stand at room temperature (20 to 25 ° C) for 1 hour, the plate was washed four times with a washing solution, and the enzyme-labeled antibody was added by a multipipette. Thereafter, the reaction was allowed to stand at room temperature (20 to 25 ° C) for 1 hour, and the reaction terminating solution was added by a multipipette. The sample after each reaction was measured for OD450 using a spectrophotometer, and the Index value was calculated using the following equation.

A450 of sample—A450 of standard serum 1

Index value = X100 A450 of anti-filaggrin antibody standard serum 2—A450 of standard serum 1

2. Association between PAD 14 dienotype and titer of anti-citrullinated filaggrin

Serum anti-synthetic citrullinated filaggrin antibody titers in 57 RA patients with defined PADI4 intron 3 -15 T / C dienotype were measured. A contingency table test with a cutoff value of 10 gave the results shown in Table 2 below. (Table 2)

Comparison between the dienotype of affected homozygotes (TT) and other dienotypes by Fisher's exact test (One-sided) gave p = 0.0284. That is, among RA patients, it was shown that the patients with the affected homozygote (TT) dienotype had significantly higher anti-citrullinated filadarin antibody titers than the patients with other dienotypes. From the results of 5 o and the results of Examples 4 and 6, differences in the PADI4 genotype are likely to lead to changes in the PADI4 phenotype such as niRNA stability and enzyme activity, leading to an increase in citrullinated protein levels. That was speculated.

[Example 8] Examination of PADI4 expression level in synovial tissue of RA patient

1. In situ-RT-PCR

Pro STAR HF (Stratagenes) was added to a piece of tissue prepared from the synovial tissue of RA patient, and one step in situ-RT-PCR was performed under the following conditions using Hybaid Omni slide (Hybaid Ltd., Middlesex, UK). Implemented above.

(1) 42 ° C 30 minutes

(2) 94t: 2 minutes, 55 ° C 45 seconds, 68 ° C 2 minutes

(3) 94t 45 seconds, 55 ° C 45 seconds, 68 ° C 2 minutes, 25 cycles

The slide glass after the PCR reaction was washed twice with PBS for 5 minutes each.

PCR was performed on the hPADI4 V18 cDNA (SEQ ID NO: 16) using the following primers for PADI4 amplification, and the resulting 335-base amplification product was labeled with digoxigenin (DIG) to detect PADI4. A probe was used.

Forward Primer: 5 '-GACCAGAAGGTTCAGATTTCATACTAC-3' (SEQ ID NO: 7)

Reverse Primer: 5'-ACCAGTGTATGGTTTGTGAAGAAGT-3 '(SEQ ID NO: 8)

DIG labeling was performed using PCR DIG probe synthes is kit (Roche Diagnostics). To the sample after reverse transcription, the above-mentioned primer was added to a final concentration of 0.34 M, covered with a slide glass, and prehybridized at 37 ° C for 1 hour. Next, a DIG-labeled probe denatured at 94 ° C for 5 minutes was added, and hybridization was performed at 37 ° C for 12 hours. After the reaction, the slide was washed, and the PADI4 gene was detected using digoxygenin detect ion kit (Roche Diagnostics). As a negative control, detection was performed in the same manner with no primer added and no reaction enzyme added.

2. Result

The results are shown in FIG. The transcript of PAD 14 was localized in the synovial tissue of RA patients at the surface of the synovial tissue (lining) and near the subsurface of the synovium (sub-lining layer). The signal was also observed in fibroblast-like cells and in round-nucleated cells around blood vessels. [Example 9] Examination of PADI4 gene expression level in mouse collagen-induced arthritis (CIA)

Collagen-induced arthritis (CIA) was induced in mice, and the expression level of mouse peptidylarginine diminase 'type IV (mPADI4) gene in synovial tissue and spleen tissue was examined. MPADM is a mouse ortholog of human peptidylarginine diminase 'type IV (hPADI4). The cDNA sequence and amino acid sequence of mPADI4 are shown in SEQ ID NO: 18 and SEQ ID NO:

Described in 19.

1. Creation of mouse CIA model

Mice (five-week-old females, purchased from Nippon Charlriva Co., Ltd.) were divided into CIA group (N = l l) and healthy group (N = l l). CIA group after training, type II collagen

2mg / ml (collagen technology workshop) and ADJUVANT COMPLETE FREUND (DIFC0) mixed in equal proportions and emulsified by ultrasound, 100 1 per animal, 2 times at one week intervals twice It was intradermally administered to the part. Finally, the CIA group was subjected to the following experiment with 7 limbs of 5 animals with onset, and the healthy group with 22 limbs of 11 animals. 2. Measurement of mPADI4 gene expression in synovial tissue and spleen tissue

The mice were sacrificed 7 weeks after the second administration by decapitation and the synovial tissue and spleen tissue were removed with scissors and tweezers. Total RNA was prepared from the excised tissues using RNeasy mini kit or RNeasy midi kit (QIAGEN) according to the attached protocol. CDNA was synthesized from the prepared total RNA using TaKaRa RNA PCR ™ Kit (AMV) Ver. 2.1 (TaKaRa) according to the attached protocol.

The expression of the mPADI4 gene was examined by RT-PCR using ABI PRIZM 7700 System (Applied Biosystems). The used primers for amplifying the mPADI4 gene and the probe for detecting the mPADI4 gene are as follows.

Forward Primer: 5'-ACGCTGCCTGTGGTCTTTG-3 '(SEQ ID NO: 9)

Reverse Primer: 5'-CCAGCCCAGTGAGCTCTGA-3 '(SEQ ID NO: 10)

Probe: 5'-CCTGAAGGATTTCCCTGTCAAGCGAGTT-3 '(SEQ ID NO: 11)

The expression level of mPADI4 gene was corrected by the expression level of mouse / 3-glucuronidase (mGUS) gene used as an internal standard. The sequences of the primer for amplifying the MGUS4 gene and the probe for detecting the MGUS4 gene are as follows.

Forward Primer: 5'-TACGGGATTGTGGTCATCGA-3 '(SEQ ID NO: 12)

Reverse Primer: 5'-CGAACCAGCTCCTCCATCAC-3 '(SEQ ID NO: 13)

Probe: 5'-CAACGAGTCACTTCGGCACCACCTAGAG-3 '(SEQ ID NO: 14) 3. Results

The results are shown in FIG. The expression level of mPADI4 was increased 19.5-fold in the synovial tissue of the CIA group compared with the healthy group. In the spleen tissue of the CIA group, the expression level of mPAD was three times higher than in the spleen tissue of the healthy group. From the above, it was confirmed that mPAD gene expression was significantly increased in synovial tissue and spleen tissue in the CIA group.

[Example 10] Preparation of anti-PADI4 antibody (anti-PADI4 peptide antiserum)

Synthesize peptide sequence (PAKKKSTGSSTWP_Cys: SEQ ID NO: 15) derived from human PADI4 (Oriental Yeast Kitayama Labes, Peptide Synthesizer: Applied Biosystems Japan, Pioneer, Synthesis: Solid phase synthesis using Fmoc amino acid ), KHL (Keyhole l impet heinocyanin, CALBIOCHEM) using MBS (PIERCE) method It was conjugated.

For the immunized animals, a heron (Kbl'JW, Oss, age: retired) was used. In the first immunization, an equal mixture of FCA (Freund's complete adjuvant, DIFC0) and an antigen was injected subcutaneously in the back. The first immunization was performed using this antigen at 600; g peptide / animal. From the second dose onwards, an equal volume mixture of FIA (incomplete Freund's adjuvant, DIFC0) and antigen was injected subcutaneously three times every two weeks into the back. After the second immunization, this antigen was used at 300 / g peptide / animal. Two weeks after the final immunization, the whole blood was collected, centrifuged at 3000 rpm for 20 minutes, and the serum was separated and collected, and NaN ₃ (Wako Pure Chemical) was added at 0.05% (w / v) and the temperature was 4 ° C (Refrigerated).

[Example 11] Immunostaining of synovial tissue section of RA patient joint

1. Preparation of articular synovial tissue section

A synovial tissue piece was obtained from the dissected tissue obtained during the total knee arthroplasty. The obtained synovial tissue piece was immediately immersed in liquid nitrogen and stored at -80 until preparation of a thin section on a slide glass. For thin sectioning, fix the frozen tissue section with 10% neutralized buffered formalin, and add 70% ethanol, 85% ethanol, 90% ethanol, and 100% ethanol (twice for 100% ethanol only). After dehydration for 1 hour each, the tissue was treated once with 50% ethanol and 50% paraffin solution for 30 minutes and twice with 100% paraffin solution for 30 minutes to paraffinize the tissue pieces. Thereafter, the tissue pieces were embedded in paraffin. Paraffin-embedded tissue slices were sliced to a thickness of 4 m with Leica Microtome RM2165 and fixed on slide glasses.

2. Immunostaining

Prior to immunostaining, attach the paraffin-embedded section prepared in the previous section to xylene twice for 3 minutes each, and then deparaffinize.Then, 100% ethanol twice for 1 minute, and 95% ethanol twice for 1 minute Then, the cells were immersed twice in PBS (0.13 M NaCL 8.6 mM K2HP04, 1.5 mM KH2P04) for 3 minutes each and rehydrated. 30% ¾0 ₂ main evening treated with Nord solution 3 minutes to remove endogenous Piochin. After washing twice with PBS for 5 minutes each and diluting 1000-fold, add each antibody: anti-citrullinated protein antibody (Biogenesis) and the anti-PADI4 antibody prepared in Example 10 and 4 for 12 hours. Reacted. After the reaction, the plate was washed twice with PBS for 5 minutes each, and SimpleStain MAX-P0 (Nichirei) was added to each slide, followed by reaction at 25: for 30 minutes. Thereafter, the cells were washed twice with PBS for 5 minutes each. As a substrate, SimpleStain AEC (Nichirei) was added 100 per slide, and allowed to react for 5 to 20 minutes until a signal was detected. The reaction was stopped by immersing in ultrapure water. .

The results are shown in FIGS. In the synovial tissue of RA patients, a reaction with an antibody that specifically recognizes PADI4 was observed (red area in box A), but osteoarthritis (OA), a negative control for rheumatoid arthritis, was observed. ) No reaction was observed in the synovial tissue of the patient due to antibodies recognizing PADI4 (Fig. 6B). In the synovial tissue of RA patients, citrullinated protein was observed in almost the same area as PADI4 (Fig. 7) [Example 12] Comparison of human PADI4 mRNA stability

1. Preparation of mRNA

The mRNAs of the two PADI4 haplotypes were prepared by the following method.

A plasmid in which the gene encoding V9 or V18 was connected downstream of the CMV promoter was transfected into human K562 cells using Lipofectamine 2000 (Invitrogen) according to the attached protocol. After culturing at 37 ° C for 4 hours, PMA (Sigma) was added to a final concentration of 50 ng / ml. After culturing for 40 hours, actinomycin D (Sigma) was added to a final concentration of 5 x g / ml, and cells were collected after 0, 2, and 4 hours, respectively.

Next, total RNA was extracted using a total RNA extraction reagent (RNeasyMini and RNase-Free Dnase set: QIAGEN) according to the attached protocol. The collected total RNA was stored at -80 ° C. The remaining human PADI4 V9 or V18 mRNA is measured using ABI PRISM 7700 Sequence Detection System (Applied Biosystems) using TaqMan One-step RT-PCR Master Mix (Applied Biosystems) and following the specified protocol. did. The obtained data was corrected using the value of ribosomal RNA (18S). Otsu Holo Song

As shown in FIG. 8, it was found that PADI4 V9 niRNA tended to be significantly harder to degrade than PADI4 V18 (p = 0.03 after 4 hours from T test). All publications, patents and patent applications cited in, are incorporated herein by reference in their entirety. Industrial potential

INDUSTRIAL APPLICABILITY The present invention can be used for screening drugs for treating rheumatoid arthritis in drug development. In addition, the present invention can be used in the medical field for early determination of the potential risk of a subject to develop rheumatoid arthritis. Sequence listing free text

SEQ ID NO: 1—Human PADI4 gene fragment (first intron-1000 to fourth intron) Description of one mutation: Single nucleotide polymorphism due to substitution of (A) and (G)

Explanation of one mutation: Single nucleotide polymorphism due to substitution of (C) and (T)

Explanation of one mutation: Single nucleotide polymorphism due to substitution of (C) and (G)

SEQ ID NO: 2—Description of Artificial Sequence: Probe

SEQ ID NO: 3—Description of artificial sequence: probe

SEQ ID NO: 4 Description of Artificial Sequence: Probe

SEQ ID NO: 7—Description of artificial sequence: primer

SEQ ID NO: 8—Description of artificial sequence: primer

SEQ ID NO: 9 Description of artificial sequence: Primer

SEQ ID NO: 10—Description of artificial sequence: primer

SEQ ID NO: 1 Description of Artificial Sequence: Probe

SEQ ID NO: 12—Description of artificial sequence: primer

SEQ ID NO: 13—Description of artificial sequence: primer

SEQ ID NO: 14 Description of Artificial Sequence: Probe SEQ ID NO: 15—Description of artificial sequence: Human PADI4 derived peptide

Claims

1. A method for evaluating the effect of a test substance as a therapeutic agent for rheumatoid arthritis using an inhibitory effect on a peptidylarginine diminase type IV gene or an ortholog gene thereof, or a protein encoded by the gene as an index.

2. The method of claim 1, comprising the following steps:

1) breed animals under conditions that allow or prevent administration of the test substance;

2) detecting the expression level of the beptidylarginine deiminase type IV gene or its ortholog gene in the blood or cells of the animal;

3) The effect of the test substance as a therapeutic agent for rheumatoid arthritis is evaluated based on the difference in the expression level under the conditions of administration or non-administration of the test substance.

3. The nucleic acid hybridization method using a solid-phased sample selected from a gene chip, a cDNA array, and a membrane filter,

RT-PCR, real-time PCR, subtraction, differential display, differential hybridization, and cross-hybridization. The method of claim 2, wherein the method is characterized by:

4. The method according to claim 3, wherein the expression level of the gene is detected by an RT-PCR method.

5. The method of claim 1, comprising the following steps:

2) In the blood or cells of the above animal, the expression level of the protein encoded by the peptidylarginine diminase type IV gene or its ortholog gene is detected using an antibody that specifically binds to the protein. ;

6. The protein expression level is selected from one of the following: Western plot, dot plot, slot blot, ELISA, and RIA. The method according to claim 5, wherein the method is detected by a method.

7. The method according to claim 6, wherein the expression level of the protein is detected by an ELISA method.

8. The method according to any one of claims 2 to 7, wherein the cells are selected from blood-derived cells, synovial cells, spleen cells, and peritoneal cells.

9. The animal according to claim 2, wherein the animal is a mouse, and the expression level of a mouse beptidylarginine-diminase 'type IV gene or a protein encoded by the gene is detected. The method according to any one of the preceding claims.

10. The method of claim 1, comprising the following steps:

1) Add peptidylarginine / diminase / type IV protein to the reaction solution containing the arginine-containing compound with or without the test substance.

2) The activity of peptidylarginine / diminase / type IV protein is determined by quantifying the amount of the reaction product in the above reaction solution. 3) Evaluate the effect of the test substance as a therapeutic agent for rheumatoid arthritis based on the difference in the above activities under the conditions of addition or non-addition of the test substance.

11. The method according to claim 10, wherein the activity of the protein is measured by using a fluorescence method or an absorption method.

12. The method according to claim 10 or 11, wherein the peptidyl arginine diminase type IV protein is a human peptidyl arginine diminase type IV protein.

13. Provide the sample based on the expression level of the mutant beptidyl arginine, diminase, type IV protein consisting of the amino acid sequence shown in SEQ ID NO: 21 or the gene (SEQ ID NO: 20) in the sample or the gene To predict the risk of developing rheumatoid arthritis in affected subjects.

14. The method of claim 13, comprising the following steps:

1) Prepare total RNA from each sample isolated from subjects and normal persons;

2) detecting the expression level of mRNA of the mutant beptidylarginine / diminase / type IV gene in all the RNAs;

3) Analyze the difference between the above expression levels between the subjects and normal subjects, and Predict the risk of developing rheumatoid arthritis.

15. Gene expression level is determined by nucleic acid hybridization method, RT-PCR method, real-time PCR method, subtraction method using immobilized sample selected from gene chip, cDNA array and membrane filter. 15. The method according to claim 14, wherein the detection is performed by any one method selected from a differential display method, a differential hybridization method, and a cross-hybridization method.

16. The method according to claim 15, wherein the expression level of the gene is detected by an RT-PCR method.

17. The method of claim 13, comprising the following steps:

1) detecting the expression level of a mutant beptidylarginine / diminase / type IV protein in a sample isolated from a subject or a normal person using an antibody capable of binding specifically to the protein;

2) Analyze the difference in the expression level between the test subject and the normal person, and predict the risk of developing rheumatoid arthritis in the test subject.

18. The method according to claim 17, wherein the expression level of the protein is detected by any one method selected from Western plotting, dot plotting, slot blotting, ELISA, and RIA. The described method.

19. The method according to claim 18, wherein the expression level of the protein is detected by an ELISA method.

20. A kit for predicting the risk of developing rheumatoid arthritis, comprising at least one selected from the group consisting of: a) to e) below.

a) A continuous oligonucleotide primer having a length of 15 to 30 bases for specifically amplifying a mutant beptidylarginine-diminase * type IV gene consisting of the base sequence shown in SEQ ID NO: 20

b) A 20- to 1500-base-long continuous polynucleotide for specifically hybridizing to the mutant beptidyl arginine 'diminase' type IV gene having the nucleotide sequence shown in SEQ ID NO: 20 and detecting the gene. Nucleotide probe

c) Immobilized sample on which the polynucleotide probe described in b) above is immobilized d) an antibody that specifically binds to a mutant beptidylarginine deiminase type IV protein consisting of the amino acid sequence represented by SEQ ID NO: 21 and detects the protein

e) secondary antibody capable of specifically binding to the antibody according to d) above

21. A method for evaluating the effect of a test substance as a therapeutic agent for rheumatoid arthritis using an inhibitory effect on a mutant type beptidylarginine deiminase 'type IV protein having the amino acid sequence shown in SEQ ID NO: 21 as an index.

22. The method of claim 21 comprising the following steps:

1) To a reaction solution containing an arginine-containing compound, a mutant beptidylarginine-diminase 'type IV protein is added under the conditions of adding or not adding a test substance;

2) measuring the activity of the mutant beptidylarginine-diminase 'type IV protein by quantifying the amount of the reaction product in the reaction solution;

3) The effect of the test substance as a therapeutic agent for rheumatoid arthritis is evaluated based on the difference in the activity under the conditions of addition or non-addition of the test substance.

23. A mutant beptidylarginine deiminase type IV protein consisting of the amino acid sequence shown in SEQ ID NO: 21.