WO2000073440A1 - Pollinosis-associated gene 787 - Google Patents

Abstract

A novel gene undergoing significantly lowered expression after the pollen scattering has been successfully isolated by preparing T cells from the subjects before and after the pollen-scattering season and searching the gene by the differential display method. It is found out that this gene is usable in examining allergic diseases and screening candidate compounds for remedies capable of regulating the response of T cells to the stimulus by an antigen.

Description

 Hay fever-related gene, 7 8 7 Technical field

 The present invention relates to a gene associated with an antigen stimulatory response, a method for testing an allergic disease using the expression of the gene as an index, and a method for screening a candidate therapeutic compound for suppressing a T cell antigen stimulatory response. Background art

 Allergic diseases, including hay fever, are considered multifactorial diseases. These diseases are caused by the interaction of the expression of many different genes, and the expression of these individual genes is affected by multiple environmental factors. Therefore, it is very difficult to elucidate the specific genes that cause specific diseases.

 It is thought that the onset of allergic disease involves mutations or defects in genes expressed in response to antigenic stimulation, or overexpression or decreased expression levels. To understand the role of gene expression in disease, it is necessary to understand how genes are involved in pathogenesis and how external stimuli, such as antigens and drugs, alter gene expression.

Recent developments in gene expression analysis technology have made it possible to analyze and compare gene expression in many clinical samples. The differential display (DD) method is useful as such a method. The differential display method was first developed in 1992 by Liang and Pardee (Science, 1992, 257: 967-971). By using this method, dozens or more of samples can be screened at a time, and It is possible to detect a gene whose expression has changed. Using such a method to examine genes with mutations or genes whose expression changes with time or environment is expected to provide important information for elucidating pathogenic genes. These genes include those whose expression is affected by environmental factors.

 Allergic diseases such as hay fever are one of the diseases seen by many people in recent years. The pathogenesis of hay fever may be related to several genes whose expression is affected by pollen, one of the environmental factors. Under such circumstances, it has been desired to isolate genes associated with allergic diseases such as hay fever. Disclosure of the invention

 An object of the present invention is to provide a gene associated with an allergic disease. Further, the present invention provides a method for testing an allergic disease, using the expression of the gene as an index.

It is an object of the present invention to provide a method for screening a therapeutic drug candidate compound that suppresses T cell antigen-stimulated response.

 The present inventors have proposed a method for treating a plurality of humans based on the already established “Fluorescent DD (Fluorescent DD) method” (T. I. to et al., 1994, FEBS Lett. 351: 231-236). We have developed a new DD system that can analyze T cell RNA samples prepared from blood. Using this system, the present inventors collected T cells from blood before and after pollen dispersal in a plurality of subjects including pollinosis patients and expressed them between subjects with different cedar pollen-specific IgE values and before and after pollen dispersal. We screened for genes with varying amounts and isolated a novel gene (the “787” gene).

The present inventors performed a comparative analysis of the expression level of the isolated 787J gene in lymphocytes isolated from subjects before and after pollen scattering, and found that the gene showed a significantly lower value after cedar pollen scattering. Therefore, the present inventors used the expression level of the gene as an indicator to test for allergic diseases and to suppress antigen-stimulated response of T cells. It has been found that screening of drug candidate compounds is possible.

 That is, the present invention relates to a gene showing a low value after pollen scattering, a method for testing allergic disease using the expression of the gene as an index, and a method for screening a candidate therapeutic compound for suppressing T cell antigen stimulation response. More specifically,

 [1] a nucleic acid molecule comprising the base sequence of SEQ ID NO: 20,

 [2] a nucleic acid molecule comprising the protein coding region of the nucleotide sequence of SEQ ID NO: 20, [3] a nucleic acid molecule specifically hybridized to the nucleic acid molecule of [1] or [2], and a chain length of at least 15 nucleotides DNA having

 (4) The method for detecting a nucleic acid molecule according to (1), which comprises using the DNA according to (3).

 [5] a method for testing an allergic disease,

 (a) preparing T cells from a subject,

 (b) preparing an RNA sample from the T cells,

 (c) a step of performing hybridization on the RNA sample using the labeled DNA according to (3) as a probe,

 (d) measuring the amount of RNA derived from a subject that hybridizes to the labeled DNA according to [3], and comparing it with a control (in the case of a healthy subject).

 [6] a method for testing an allergic disease,

 (a) preparing T cells from a subject,

 (b) preparing an RNA sample from the T cells,

 (c) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;

 (d) a step of performing a polymerase chain reaction (PCR) using the cDNA as a type III and the DNA according to [3] as a primer,

 (e) comparing the amount of DNA amplified by the polymerase chain reaction with a control (in the case of a healthy subject).

[7] The method according to [6], wherein the polymerase chain reaction is performed by a PCR amplification monitor method. Law,

 (8) the method according to any of (5) to (7), wherein T cells are prepared from peripheral blood of the subject;

 (9) the method according to any one of (5) to (8), wherein the allergic disease is cedar pollinosis;

 [10] 方法 a method of screening for a candidate therapeutic compound that suppresses the antigen-stimulated response of cells,

 (a) Step of administering a test compound to a model animal and stimulating it with pollen antigen

(b) preparing T cells from the model animal,

 (c) preparing an RNA sample from the T cells,

 (d) performing a hybridization on the RNA sample using the labeled DNA according to (3) as a probe,

 (e) measuring the amount of RNA derived from the T cells that hybridizes to the labeled DNA according to (3),

 (f) selecting a compound that suppresses the decrease in the amount of RNA measured in step (e) as compared to a control (in the case where the test compound is not administered);

 (11) A method for screening a candidate therapeutic compound that suppresses the antigen-stimulated response of T cells,

 (a) administering a test compound to a model animal and stimulating with a pollen antigen,

(b) preparing T cells from the model animal,

 (c) preparing an RNA sample from the T cells,

 (d) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;

 (e) a step of performing a polymerase chain reaction (PCR) using the cDNA as a type III and the DNA according to [3] as a primer,

(f) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (e) as compared to a control (in the case where no test compound is administered); [12] A method for screening a candidate therapeutic compound that suppresses the antigen-stimulated response of T cells,

 (a) administering a test compound to a model animal,

 (b) preparing lymphocytes from the model animal,

 (c) stimulating the lymphocytes with a pollen antigen,

 (d) separating T cells from the antigen-stimulated lymphocytes,

 (e) preparing an RNA sample from the T cells,

 (f) a step of performing hybridization on the RNA sample using the labeled DNA of (3) as a probe,

 (g) a step of measuring the amount of RNA derived from the T cells that hybridizes to the labeled DNA according to (3),

 (h) selecting a compound that suppresses the decrease in the amount of RNA measured in step (g) compared to a control (in the case where the test compound is not administered),

 (13) A method for screening a candidate therapeutic compound that suppresses the antigen stimulation response of T cells,

 (a) administering a test compound to a model animal,

 (b) preparing lymphocytes from the model animal,

 (c) stimulating the lymphocytes with a pollen antigen,

 (d) separating T cells from the antigen-stimulated lymphocytes,

 (e) preparing an RNA sample from the T cells,

 (f) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;

 (g) performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to [3] as a primer,

 (h) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (g) as compared to a control (in the case where the test compound is not administered);

[14] Screen for candidate therapeutic compounds that suppress T cell antigen-stimulatory response Method

 (a) preparing lymphocytes from a model animal or human mosquito,

 (b) stimulating the lymphocytes with a pollen antigen in the presence of a test compound,

 (c) separating T cells from the antigen-stimulated lymphocytes,

 (d) preparing an RNA sample from the T cells,

 (e) a step of subjecting the RNA sample to professional hybridization with the labeled DNA according to (3),

 (f) measuring the amount of RNA derived from the T cells that hybridizes to the labeled DNA according to (3),

 (g) selecting a compound that suppresses the decrease in the amount of RNA measured in step (: f) as compared to a control (in the case where no test compound is administered);

 (15) a method for screening a candidate therapeutic compound that suppresses the antigen stimulation response of T cells,

 (a) preparing lymphocytes from a model animal or human,

 (b) stimulating the lymphocytes with a pollen antigen in the presence of a test compound,

 (c) separating T cells from the antigen-stimulated lymphocytes,

 (d) preparing an RNA sample from the T cells,

 (e) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;

 (f) performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to [3] as a primer,

 (g) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (f) as compared to a control (in the case where the test compound is not administered);

 (16) a method for screening a candidate therapeutic compound that suppresses the antigen stimulation response of T cells,

 (a) stimulating the established T cells with a lymphocyte stimulating substance in the presence of the test compound,

(b) preparing an RNA sample from the stimulated established T cells, (c) a step of performing hybridization on the RM sample using the labeled DNA according to (3) as a probe,

 (d) measuring the amount of RNA derived from the established T cells that hybridizes to the labeled DNA according to (3),

 (e) selecting a compound that suppresses the decrease in the amount of RNA measured in step (d) compared to a control (in the case where the test compound is not administered),

 (17) a method for screening a candidate therapeutic compound that suppresses the antigen stimulation response of T cells,

 (a) stimulating the established T cells with a lymphocyte stimulating substance in the presence of the test compound,

(b) preparing an RNA sample from the stimulated established T cells,

 (c) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;

 (d) a step of performing a polymerase chain reaction (PCR) using the cDNA as a type III and the DNA according to [3] as a primer,

 (e) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (d) compared to a control (in the case where no test compound is administered),

 (18) the method of (10) or (11), wherein the T cell is prepared from peripheral blood of a model animal;

 (19) the method of any one of (12) to (15), wherein the lymphocytes are prepared from peripheral blood,

 (20) the method of any one of (10) to (19), wherein the antigen is a cedar pollen antigen,

 About.

In the present invention, allergic disease is a general term for diseases associated with allergic reactions. More specifically, it can be defined as identifying the allergen, demonstrating a deep link between exposure to the allergen and the development of the lesion, and demonstrating an immunological mechanism for the lesion. Here, the immunological mechanism is the allele Means that T cells show an immune response upon the stimulation of the gene. Typical allergic diseases can include bronchial asthma, allergic rhinitis, atopic dermatitis, hay fever, or insect allergy. Allergic predisposition (allergic diathes is) is a genetic factor transmitted from parents to children with allergic diseases. Allergic diseases that occur familially are also called atopic diseases, and the genetic factors that cause them are atopic predispositions.

 The “nucleic acid molecule” in the present invention includes DNA and RNA.

 BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention relates to a novel gene “787J” correlated with the response of lymphocytes to cedar pollen antigen. The nucleotide sequence of the “787” cDNA found by the present inventors is shown in SEQ ID NO: 20.

 The nucleotide sequence of the “787” cDNA isolated by the present inventors is a partial distribution sequence of the “787” cDNA, and those skilled in the art will recognize the sequence information of the “787” cDNA described in SEQ ID NO: 20. Isolation of full-length cDNA for “787” based on the information can be usually performed. That is, a method for screening a T cell cDNA library or the like by hybridization using a sequence derived from “787” as a probe, or a method for screening a T cell cDNA library for a T cell cDNA library or the like using a sequence derived from “787” as a primer. There is a method of obtaining the full-length cDNA by screening the library using the 铸 as a marker and obtaining an amplification product having a size specific to the primer as an index. The RACE method (Frohman, MA et al .: Proc.), In which a sequence derived from “787” is used as a primer to convert mRNA from T cells or the like into single-stranded cDNA, add oligomers to the ends, and then perform PCR. Natl. Acad. Sci. USA, 85: 8992, 1988). There is a method of extending the sequence of "787".

 The “nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 20” in the present invention includes “78” which can be isolated based on the sequence information of “787” cDNA described in SEQ ID NO: 20 as described above. 7 "full-length cDNA.

“787” indicates that lymphocytes in subjects were more consistent after pollen antigen exposure than before exposure. The expression was statistically significantly lower. Therefore, using the expression of the “787” gene (including transcription into mRNA and translation into protein) as an index, testing for allergic diseases and screening for candidate therapeutic compounds that suppress T cell antigen stimulation response It is considered possible. Decreased expression of “787” indicates the response of T cells to stimulation of antigens such as pollen.In patients with a history of allergic disease, expression of “787” is used as an index to determine the response of the patient to specific antigen exposure. Monitoring the transition is helpful in understanding the state of the disease and examining response to treatment.

 As the allergic disease to be tested and treated in the present invention, cedar pollinosis is particularly preferred.

 Detection of the expression of the "787" gene in the test for an allergic disease according to the present invention can be performed by using an hybridization technique using a nucleic acid that hybridizes to the "787" gene as a probe, or by hybridizing to the gene of the present invention. It can be performed using gene amplification technology using DNA as a primer.

 As the probe or primer used in the test of the present invention, a nucleic acid molecule that specifically hybridizes to the “787” gene and has a chain length of at least 15 nucleotides is used. The term "specifically hybridizes" as used herein means that under normal hybridization conditions, preferably under stringent hybridization conditions, DNA and / or Z or RNA encoding other genes are cross-hybridized. Indicates that it does not occur significantly. For example, the probe and the transfer membrane were hybridized at 68 in Express Hydidi zat ion on Solut ion (manufactured by CL0NTECH), and finally, 0.1 X SS 0.05% SDS solution was used. Stringent conditions can be achieved by washing at ° C.

These nucleic acid molecules used in the test of the present invention may be synthetic or natural. The probe DNA used for hybridization is usually labeled. Labels include, for example, nick translation labeling using DNA polymerase I, end labeling using polynucleotide kinase, Fill-in labeling with Reno fragment (Berger SL, Ki-band el AR. (1987) Guide to Molecular Cloning Techniques, Method in Enzymology, Academic Press; Hames BD, Higgins SJ (1985) Genes Probes: A Practical Approach. IRL . Press; Sambrook J, Fr i tsch EF, Maniat is T. (1989) Molecular Cloning: a Laboratory Manu l, 2nd Edn Cold Spring Harbor Laboratory Press) labeling with transcription with _¾ Momopo polymerase (Melton DA, Krieg, PA , Rebagkiati MR, Maniatis T, Zinn K, Green MR. (1984) Nucleic Acid Res., 12, 7035-7056), a method of incorporating modified nucleotides without radioisotopes into DNA (Kricka LJ. 992) Nonisotopic DNA Probing Techniques. Academic Press). Testing of allergic diseases using the hybridization technology can be performed using, for example, a Northern hybridization method, a dot blot method, a method using a DNA microarray, or the like.

 On the other hand, as a method using gene amplification technology, for example, the RT-KR method can be used. In the RT-PCR method, the expression of the “787” gene can be more accurately quantified by using the PCR amplification monitor method as shown in Example 8 in the process of gene amplification.

In the PCR gene amplification monitor method, probes that are labeled with different fluorescent dyes that cancel each other's fluorescence at both ends are used to hybridize to the detection target (DNA or reverse transcript of RNA). When the PCR proceeds and the probe is degraded by the 5'-3 'exonuclease activity of Taq polymerase, the two fluorescent dyes are separated and the fluorescence is detected. This fluorescence is detected in real time. The number of copies of the target in the target sample is determined based on the number of linear cycles of PCR amplification by simultaneously measuring a standard sample with a clear copy number for the target (Holland, PM et al., 1991, Pro Natl. Acad. Sci. USA 88:72 76-7280; Livak, KJ et al., 1995, PCR Methods and Applications 4 (6): 35 7-362; Heid, CA et al., Genome Research 6: 986. -994; Gibson, EMU e tal., 1996, Genome Research 6: 995-1001). In the PCR amplification monitor method, for example, ABI PRISM7700 (PerkinElmer) can be used.

 In addition, the test for an allergic disease of the present invention may be performed by detecting the protein encoded by “787”. As such a test method, for example, a Western blotting method using an antibody that binds to a protein encoded by “787”, an immunoprecipitation method, an ELISA method, and the like can be used. Antibodies to the protein encoded by the "787" of the present invention can be obtained as polyclonal antibodies or monoclonal antibodies using techniques well known to those skilled in the art (Milstein in C, et al., 1983, Nature 305 (5934): 537-40). For example, the protein or its partial peptide used as an antigen is prepared by incorporating the “787” gene or a part thereof into an expression vector, introducing this into an appropriate host cell, and preparing a transformant. It can be obtained by culturing the transformant to express the recombinant protein, and purifying the expressed recombinant protein from the culture or the culture supernatant. As a result of the test for an allergic disease according to the present invention, if the expression of the gene of the present invention is significantly low, it can be determined that the subject has developed an immune response to an allergen such as cedar pollen antigen. The measurement of the expression level of the gene of the present invention, together with the pollen-specific antibody titer, symptoms, etc., can be used for testing for allergic diseases.

The expression of the “787” gene expressed in T cells is reduced after pollen antigen exposure. The “787” gene is a gene whose expression level decreases as a response of the living body to cedar pollen antigen stimulation, and screening of a therapeutic agent for hay fever can be performed by monitoring the expression of the “787” gene. The expression level of the “787” gene is reduced by pollen antigen exposure in both healthy and hay fever patients. Presence or absence of hay fever symptoms is presumed to be due to the difference since the response of the "787J gene" to antigen stimulation. However, even in such cases, decreased expression of the "787" gene corresponds to increased T cell responsiveness Therefore, by monitoring the expression of the “7 87” gene, Can do it.

 The method of the present invention for screening a candidate therapeutic compound for suppressing a T cell antigen-stimulated response can be performed in vivo or in vitro. For in vivo screening, for example, after administering a candidate drug and stimulating with a pollen antigen to a model animal such as a mouse, T cells are separated from peripheral blood and the transcript of “787” is measured. I do. Alternatively, after administering a candidate drug to a model animal such as a mouse, lymphocytes are separated from peripheral blood, and the lymphocytes are stimulated with cedar pollen antigen or the like with in vitro. T cells are separated from the lymphocytes after the stimulation, and the transcript of the “787” gene is measured. As a result of these measurements, a compound that suppresses a decrease in the transcription level of the “787” gene compared to a control (when no candidate drug is administered) is selected. Here, the stimulation with the pollen antigen is performed for the purpose of eliciting an antigen-specific allergic reaction in T cells and determining the therapeutic effect of the candidate compound on it. In the present invention, the term “suppressing the decrease in the transcription amount of the“ 787 ”gene” means that a higher level of transcription amount is maintained by contact with a candidate compound as compared to antigen-stimulated T cells. Say Therefore, if a candidate compound induces a level that exceeds the transcription level of the “787” gene before being subjected to antigenic stimulation (ie, if transcription is increased), the compound is a compound to be selected in the screening method of the present invention. .

 In the in vitro screening, for example, peripheral blood lymphocytes are collected from a human mouse or the like, and the peripheral blood lymphocytes are stimulated with in vitro with a cedar pollen antigen or the like. Candidate compounds are added during in vitro stimulation. The T cells are then separated from the stimulated peripheral blood lymphocytes and the transcript of “787” is measured. As a result of this measurement, a compound that suppresses a decrease in the transcription amount of the “787” gene compared to a control (when the candidate drug is not contacted) is selected.

In addition, the screening of candidate therapeutic compounds that suppress the antigen-stimulated response of T cells of the present invention can also be performed using established T cells. For example, established T cells such as Molt4 cells and Jurka t cells are stimulated in vitro with a lymphocyte stimulator. Lymphocyte sting Examples of the intense substance include calcium ionophore (A23187), PMA, and phytohemagglutinin (PHA). Add candidate drug during invitro stimulation. Thereafter, the transcription amount of the “787” gene in the established T cells is measured. As a result of this measurement, a compound that suppresses a decrease in the transcription amount of the “787” gene compared to a control (when the candidate drug is not contacted) is selected.

 Detection of the expression of the "787" gene in the screening of a therapeutic drug candidate compound that suppresses the T cell antigen-stimulatory response according to the present invention can be performed by detecting the nucleic acid hybridizing to the "787" gene in the same manner as in the test for allergic disease of the present invention. The hybridization can be carried out using a hybridization technique using DNA as a probe, or a gene amplification technique using DNA that hybridizes to the gene of the present invention as a primer.

 As a method using the hybridization technology, for example, a Northern hybridization method, a dot blot method, a method using a DNA microarray, or the like can be used. On the other hand, as a method utilizing the gene amplification technique, an RT-PCR method can be used. In the RT-PCR method, the expression of the “787” gene can be more accurately quantified by using a PCR amplification monitoring method as shown in Example 8 in the gene amplification process.

 The test compounds used in these screenings include compound preparations synthesized by existing chemical methods such as steroid derivatives, compound preparations synthesized by combinatorial chemistry, extracts of animal and plant tissues, or microbial cultures. A mixture containing a plurality of compounds such as a product, a sample purified from the mixture, and the like. The compound isolated by the method of the present invention for screening a candidate therapeutic compound for suppressing a T cell antigen-stimulated response is a candidate for a drug that suppresses an immune response.

When the compound isolated by the screening method of the present invention is used as a pharmaceutical, it can be used as a pharmaceutical preparation by a known pharmaceutical production method. For example, it is administered to a patient together with a pharmacologically acceptable carrier or vehicle (such as saline, vegetable oils, suspensions, surfactants, stabilizers, etc.). Dosage depends on the nature of the compound Performed percutaneously, intranasally, transbronchially, intramuscularly, intravenously, or orally. The dose varies depending on the patient's age, body weight, symptoms, administration method and the like, but those skilled in the art can appropriately select an appropriate dose. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing the antibody titers of cedar pollen-specific IgE antibodies in a total of 18 blood samples from 10 subjects who collected blood. The values of cedar pollen-specific IgE antibodies in each blood sample of Test Subjects A to (sample numbers 1 to 18) were expressed in AU / ml. The pair before pollen scattering is shown on the left (white column), and the one after scattering is shown on the right (black column). Subjects A and B collected only blood after pollen scattering.

 FIG. 2 is a diagram showing changes in the expression of “787” in the pre-scattering group and the post-scattering group when grouping was performed before and after the cedar pollen scattering time. Error bars represent standard deviation. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

 [Example 1] Blood collection from 10 adult volunteers

Blood samples of 10 adult volunteers (A ~; 0 to 10 ml) were collected before and after pollen dispersal to collect T cells before and after pollen dispersal. The first blood sample was Japanese cedar pollen dispersal And before the season (January and February 1997) and the second after pollen dispersal in Japan (March, April and May 1997). For the remaining two volunteers, only samples obtained after pollen dispersal were obtained, and a part of these blood samples was used to measure the amount of cedar pollen-specific IgE. IgE is measured by the CAP RAST method (Pharmacia), which is an improved version of the RAST method (radio allergo sorbent test, Wide, L. et, a1: Lancet 2: 1105-1107, 1967) using a paper disk as a solid phase. I went. Using serum containing a standard antibody titer manufactured by Pharmacia, the IgE antibody titer (unit is also indicated as Pharmacia RAST Unit, PRU or AU (arbitrary unit)) of each sample was determined based on the serum.

 Fig. 1 shows the measured cedar pollen-specific IgE values before and after pollen scattering in each subject. As shown, most of the 10 subjects had increased serum levels of cedar pollen-specific IgE after pollen exposure. The presence of atopic predisposition was determined by whether the value of the CAP RAST test for cedar pollen-specific IgE was greater than 2. That is, eight subjects A to G and I were considered to be atopic predisposition groups (hereinafter also referred to as “patients”), and two subjects H and were considered to be healthy subjects (hereinafter also referred to as “normal groups”). Of the eight subjects with an atopic predisposition, seven exhibited symptoms of allergic rhinitis after pollen dispersal.

 [Example 2] Preparation of lymphocyte fraction from blood sample

When preparing T cells from 10 ml of blood, the procedure was as follows. First, the wall of the syringe was uniformly treated with 1 ml of heparin from Novo, etc., and blood was collected in a 10 ml syringe containing a final concentration of 50 unit / ml heparin. At this time, two 22G needles were prepared for one blood sample. The injection needle was removed and transferred to a 50 ml centrifuge tube (made of polypropylene). After centrifugation at 1500 rpm for 5 minutes at room temperature, 1.1 ml was collected from the surface as close as possible, and centrifuged at 15000 rpm for 5 minutes at 4 to collect 1 ml of the supernatant as plasma. An equal volume (9 ml) of 0.9% NaCl containing 3% dextran (manufactured by Nacalai) was added to the rest of the collected plasma, and the mixture was gently inverted several times to mix. Then, it was left still at room temperature for 30 minutes. PRP (Pla teletrich plasma, platelet-rich plasma) was transferred to another 15 ml centrifuge tube, and centrifuged at 1200 rpm (equivalent to 150 Xg in a Tommy centrifuge) for 5 minutes at room temperature. After centrifugation, platelets were in the supernatant. The precipitated cells were suspended in 5 ml of Ca- and Mg-free HBSS obtained from Gibco or the like. This was overlaid on one tube (Falcon tube: 2006 or 2059; made of polypropylene) containing 5 ml of Ficol Paque (Pharmacia) using a Pasteur pipe. After centrifugation at 1200 i "pm for 5 minutes, 1500 The mixture was centrifuged at room temperature for 30 minutes at rpm (equivalent to 400 × g in a centrifuge manufactured by Tomy). As a result, granulocytes and erythrocytes precipitated, and lymphocytes (ly immediate hocytes), monocytes (monocytes;), and platelets (platelets) were contained in the middle layer with the ficoll layer interposed therebetween. Collect the middle layer with a Pasteur pipette, add 2 to 3 volumes of BSA / PBS (0.5% BSA, 2 mM EDTA in PBS, pH 7.2; degas immediately before use), and add 1200 rpm, 4 rpm. Centrifuged at ° C for 5 minutes. The precipitate was collected and washed once with BSA / PBS. After the first washing, cells were suspended in 5 ml, and a part thereof was diluted 2-fold with trypan blue, and the number of cells was counted. Total cell number was about IX 10 ^7. This was used as the lymphocyte fraction.

 [Example 3] Separation of T cells from lymphocyte fraction

The lymphocyte fraction obtained in Example 2 was centrifuged at 1200 i "pm for 4 and 5 minutes, and suspended in BSA / PBS at 10 ⁸ per 100 / xl. The volume became about 201. This was transferred to an Eppendorf tube (1.5 ml), to which the CD3 microbead solution was added, and then left for 30 minutes at 4 to 10 ° C (not placed on ice at this time). The treatment was performed as follows using Yuichi (MACS) (manufactured by Miltenyi Biotech Inc.).

 The MS + / RS + column was mounted on a Mini MACS or Vario MACS separation unit (without needles). 500 1 of BSA / PBS was gently applied to the column, and the buffer was poured out. Next, cells labeled with CD3 microbeads were applied to the column. The column was washed three times with 500 ^ 1 (B cell fraction). The column was removed from the separation unit and placed on a tube collecting the eluate. 1 ml of BSA / PBS was applied to the column, and positive cells were rapidly flushed out using a plunger attached to the column. This was used as the T cell fraction.

The obtained T cell fraction was centrifuged at 1200 rpm for 5 minutes at 4 ° C. The precipitate was washed twice with BSA / PBS. After the second washing, the cells were suspended in 1 ml, and a part thereof was diluted 2-fold with trypan blue to count the number of cells. The total cell number was about 4 × 10 ⁶ .

 [Example 4] Preparation of total RNA from T cells

For preparation of total RNA from T cells, use RNeasy Mini (manufactured by Qiagen). Performed according to the attached manual. All operations were performed at room temperature, wearing gloves. Four volumes of ethanol were added to Posh Buffer RPE. Lysis buffer RLT was supplemented with 10 1 / ml 2-mercaptoethanol. The cell suspension was centrifuged at 1000-1200 i "pm for 5 minutes, and the supernatant was removed by aspirating. A 350 // 1 lysis buffer RLT (containing 2-mercaptoethanol) solution was added to the precipitate. At this stage, the lysate of the cells in the RLT buffer could be stored at -70 ° C. If the lysate of the cells had been stored frozen, 10-15 at 37 ° C. After incubating for 3 min, if insolubles were visible, centrifugation was performed at maximum speed for 3 min, and only the supernatant was collected.The lysate was homogenized with a syringe equipped with a 20 G force teran needle, and then purified with Kia Shredda ( (That is, the lysate of 3501 cells was usually applied to a Kyaschlets d'Aunit using a Pittman. This was centrifuged at 1500 µm for 2 minutes, and the effluent was collected.) Add 350 1 of 70% ethanol, pipet Attach the RNeasy spin column to the attached 2 ml tube, apply the lysate mixture of cells, centrifuge at 8000 X g (11500 ι · ρπι) for 1 minute, and discard the effluent. Wash buffer RW1 700 1 was applied to the column and the tube was capped for 5 minutes, centrifuged at 11500 rpm for 15 seconds, and the effluent was discarded. After applying Wash Buffer RPE (containing ethanol) 500 1 to the column, centrifuged at 11500 rpm for 15 seconds and discarded the effluent Wash Buffer RPE 500 1 was applied to the column and centrifuged at maximum speed for 2 minutes The column was placed in a new 1.5 ml tube, DEPC-treated water 301 was applied, the lid was put on the tube, and the tube was allowed to stand for 10 minutes, followed by centrifugation at 11500 卬 m for 10 minutes to obtain total RNA. Measure and if the volume is low, The sample was placed in a new 1.5 ml tube, water 301 treated with DEPC was applied, the lid was put on the tube, the tube was set for 10 minutes, and centrifuged at 11,500 rpm for 10 minutes.

 [Example 5] DNase treatment of total RNA

DNase treatment was performed to remove DNA from total RNA prepared from T cells. Reaction 2 Unit DNase (Nitsubon Gene) and 50 units RNase inhibitor Yuichi

(Pharmacia) in 100 l lXDNase buffer (Futan Gene). After incubating this at 37 ° C for 15 minutes, an equal volume of PCI (phenol: black form: isoamyl alcohol = 25: 24: 1) was added and vortexed. The mixture was centrifuged at 12,000 rpm at room temperature for 10 minutes, and the upper layer (aqueous layer) was transferred to a new 1.5 ml tube. One-tenth volume of 3M sodium acetate (pH 5.2) was added, and 2.5 volumes of 100% ethanol and 1 liter of eta-precipitated mate were added and mixed by inversion. After leaving at −20 ° C. for 15 minutes, the mixture was centrifuged at 12,000 rpm at 4 ° C. for 15 minutes, the supernatant was removed, and 70% ethanol was added. After tapping to the extent that the precipitate was detached, the supernatant was removed cleanly. Dried for 3 minutes and dissolved in 10-201 DDW (no DNase and RNase). The concentration was measured and stored at -80 ° C until use.

 [Example 6] Differential display (DD) analysis using total RNA prepared from T cells

 Fluorescence differential display (F1 uorescent Differential Display, abbreviated as “DD”) using total RNA prepared from T cells is described in the literature (T. Ito et al., 1994, FEBS Lett. 351: 231–236). Performed according to the method. Total RNA prepared from T cells was reverse transcribed to obtain cDNA. For the primary DD-PCR reaction, cDNA was prepared using 0.2 g of total RNA for each of the three anchor primers. For the secondary DD-PCR reaction, cDNA was prepared using 0.4 zg of RNA for each of the three anchor primers. All cDNAs were diluted to a final concentration equivalent to 0.4 ng / l RNA and used for experiments. A DD-PCR reaction was performed using cDNA equivalent to 1 ng RNA per reaction. Table 1 shows the composition of the reaction solution.

 Table 1 cDNA (equivalent to 0.4 ng / ^ 1 RNA) 2.5 1

Optional primer (2 / zM) .5 ^ 1 lOXAmpliTaq PCR buffer 1.0 x 1

 2.5mM dNTP 0.8 1

 50 M anchor per primer 0.1 1

 (GT15A, GT15C, GT15G)

 Gene Taq (5U / 1) 0.05 1

 Am liTaq (5U // z 1) 0.05 1

 dH, 0 3.0 1

10.0M

The PCR reaction conditions were as follows: 1 cycle of 95 ° C for 3 minutes, 40 ° C for 5 minutes, 72 ° C for 5 minutes, followed by 30 cycles of 94 ° C for 15 seconds, 40 ° C for 2 minutes, and 72 ° C for 1 minute. Thereafter, the temperature was kept at 72 ° C for 5 minutes, and then continuously 4T :. The primer pairs used were primers AG1 to 110 and AG111 to GT15A (SEQ ID NO: 2), GT15C (SEQ ID NO: 3), and GT15G (SEQ ID NO: 4), which are anchor primers, respectively. A total of 287 reactions were performed by combining 199 and AG 200-287. As an optional primer, an oligomer composed of 10 nucleotides having a GC content of 50% was designed, synthesized, and used.

 For gel electrophoresis, a 6% denaturing polyacrylamide gel was prepared, 2.5 1 samples were applied, and electrophoresed at 40 W for 210 minutes. Thereafter, the gel plate was scanned using Hitachi Fluorescence Image Analyzer -FMBI0 II, and electrophoretic images were obtained by fluorescence detection.

 [Example 7] Amplification and sequencing of band cut out by DD analysis

 Two DD analyzes were performed using a number of arbitrary primers. Bands that differed before and after pollen dispersal or between the patient and healthy groups were selected and reproducible bands were excised from the gel in two experiments.

Further analysis was performed on one of the cut-out bands (referred to as "787"). The “787” band is GT15G (SEQ ID NO: 4) as an anchor primer, DD analysis using AG274 (TGACCTAGCT / SEQ ID NO: 5) as an imaginary animal revealed that the expression of “787” was stronger in 4 out of 8 subjects before dispersal than after dispersal.

 To determine the nucleotide sequence of “787”, a gel containing the band of “787” was cut out, stored in a TE solution, and heated at 60 ° C. for 10 minutes to elute DNA from the gel. Using this TE solution as type II, PCR was performed under the same conditions as DD-PCR, and a DNA fragment of about 290 bp was amplified. GT15G was used as the anchor primer, and AG274 was used as the optional primer. The amplified DNA fragment was cloned into a plasmid vector pCR2.l (Invitrogen) to obtain a plasmid p787-5 having a DNA fragment of about 290 bp. Using the plasmid DNA, the nucleotide sequence of the DNA fragment was determined according to a conventional method.

 [Example 8] Quantification by ABI-7700

 The expression amount of "787" was quantified by the TaqMan method using ABI-PRISM7700. This method is a system for real-time quantitative detection of PCR-amplified DNA strands using fluorescent dyes. For the purpose of quantification, blood samples before and after cedar pollen scattering were newly collected from 22 volunteers in the spring of 1998, T cells were prepared, and total RNA was extracted. The expression level of the target gene was quantified using a total of 44 total RNA samples.

In the same manner as in Example 1, specific IgE values and total IgE values of cedar pollen, cypress pollen, Dermatophagoides farinae, and Dermatophagoides farinae were measured (Table 2).

Table 2

Specific IqE value

 Specific lgE (UA / ml)

 Subject Blood sampling time (UA / ml)

A Before flying 42.7 5.46 1 .09 <0.34 300 After flying 83.2 7.85 1.28 <0.34 460

B Before flying 31.9 4.33 72.5 52.6 770 After flying 36.8 3.56 78.8 47.9 840

Before Flying K 1 5.2 1.5 68.7 66.1 450 After Flying 20.3 1.32 64.3 49.7 330

D Before losing 13.9 1.1 .1 39.3 63.4 200 After splattering 1 8.4 0.81 31.9 54.7 1 20

E Before flying 5.25 0.48 <0.34 0.34 30 After flying 8.33 0.46 <0.34 <0.34 38

F Before enemy 6.64 0.39 <0.34 0.34 26 After flight 8.21 0.47 <0.34 <0.34 27

G before flying 1.29 0.34 <0.34 <0.34 26 after flying 4.02 0.34 <0.34 0.34 30

H Before losing 1.99 0.41 26.5 36.1 220 After splattering 3.65 0.53 23.2 29.5 1 50

1 Flying enemy 0.93 <0.34 54.6 51.7 1 30

 3.51 <0.34 43.3 39.9 1 00

J before 3.55 0.68 0.55 <0.34 96 after tt 2.77 0.42 0.39 <0.34 78 before losing 1.2 0.34 <0.34 <0.34 96 after losing 2.72 <0.34 <0.34 0.34 110 then before scatter 0.95 0.39 1.3 1.8 13 before Ittft 2.5 0.51 1.45 2.38

 Before flying 0.34 <0.34 <0.34 <0.34 36 Flying> After 2.08 <0.34 0.34 0.34 43

N fly 0.42 0.34 <0.34 0.34 22 After fly 1.67 0.34 0.45 0.34 73

0 Fly ttffi 0.54 <0.34 28.1 27.2 180 Fly tt 1.42 0.34 27.2 26.3 160

Before Flying 0.38 0,34 5.08 3.65 280 After Flying 0.68 0.34 4.49 3.02 240

Before Q fly 0.34 0.34 <0.34 0.34 5.0 Fly tt contact 0.34 0.34 <0.34 <0.34 5.0 Fly ttu 0.34 0.34 0.34 <0.34 53 Fly After IK <0.34 0.34 <0.34 0.34 62

0.34 0.34 <0.34 <0.34 420

 <0.34 0.34 <0.34 0.34 370

Before T fly tt 0.34 0.34 <0.34 <0.34 82 After fly 0.34 0.34 0.34 <0.34 62 u Before fly 0.34 0.34 0.34 <0.34 18 After fly <0.34 0.34 0.34 0.34 16

Before flying <0.34 0.34 0.79 0.81 180 After flying 0.34 0.34 0.78 0.9 160 Based on the nucleotide sequence of the DD band determined in Example 7, primers 787-5 ′ (AGCTTCTGGACAGCCCAGTC / SEQ ID NO: 6), 787-3 ′ (GGAGTTAGCCAGGCAGCAAAZ sequence ID: 7), and TaqMan probe 787-13SEQ (TGGTGATGGCTGGAGGGAGTGATTGZ Column number: 8) was designed, synthesized, and used for quantitative reactions. The TaqMan probe 787-13SEQ was used by fluorescently labeling the 5 end with FAM (6-carboxyfluorescein) and the 3 end with TAMRA (6-carboxy-tetramethytri rhodamine). For type II, reverse transcribed cDNA from 44 total RNAs using poly T (12 to 18 mer) as a primer was used. For the standard curve for calculating the copy number, a serial dilution of the plasmid p787-5 obtained in Example 7 was used as a type II reaction. Table 3 shows the composition of the reaction solution for monitoring PCR amplification. In addition, in order to correct the difference in cDNA concentration in the sample, the same quantitative analysis was performed on the β-actin (3-actin) gene, and correction was performed based on the copy number of those genes, and the target gene (787) was corrected. The number of copies was calculated.

 Table 3

 Reaction composition of ABI-PRISM 7700 (reaction volume per 1 liter) Sterile distilled water 25.66 (L)

 10x TaqMan Roof A5

25mM MgCl ₂ 7

 dATP (lOmM) 1.2

 dCTP (lOmM) 1.2

 dGTP (lOmM) 1.2

 dUTP (lOmM) 1.2

 Forward Primer (100 μΜ) 0.15

 Reverse Primer (lOO ^ M) 0.15

 787 TaqMan probe (6.7 M) 1.49

AmpliTaq Gold (W fil) 0.25 AmpErase UNG (WD 0.5

 Template solution 5

50

Table 4 shows the number (copy number) of “787” in each sample corrected for the copy number of / 3-actin. For the correction, the average copy of -actin in all samples was determined, and the copy number of "787" in each sample was divided by the relative value of / 3-actin in each sample when it was set to 1.

Table 4

Quantitative signal by ABI7700 (copy / ngRNA) beta actin correction data Subject Blood collection time Band ID

 787

A Before losing 147

 After flying 119

B before scattering 970

 After scattering 98

C Hitoumae 165

 After scattering 81

D Before defeat 433

 After defeat 66

E Hitoumae 149

 After dispersal 160

F Before Defeat 195

 After defeat 493

G before scattering 130

 After splashing 72

H flying κ front 387

 After defeat 60

1 Before the Defeat 293

 After 286

J Tobimae 444

 After defeat 155

K before scattering 128

 After flying 118 and before losing 612

 After speed 207

M Before Defeat 191

 After splash 82

N Before defeat 190

 After splashing 84

0 Before defeat 186

 After defeat 71

P Before Defeat 134

 After speed 177

Q Before losing 369

 After defeat 219

R Flying before 95

 After defeat 13

S before scattering 123

 After splashing 84

T Hitoshi Mae 181

 After defeat 92 u Before defeat 212

 After defeat 128

V Before Defeat 191

After dispersal 88 Using this value, a paired t-test was performed. StatView software (Abacuus Concepts, Inc.) was used for the assay. The results showed that the expression of “787” was significantly higher in the pre-scatter group than in the post-scatter group (P value = 0.0098) when divided into groups before and after cedar pollen scattering (Fig. 2). The expression level of “787” in the group before pollen dispersal and the drop after dispersal was 269.2 ± 205.0 and 134.2 ± 101.4 copies of Zng RNA (mean soil standard deviation), respectively.

 [Example 9] Cloning of "787" and analysis of nucleotide sequence

 To clone “787”, a specific primer prepared from DD band sequence using Yeast Two Hybrid Leukemia cDNA Library (CLONTECH) as type II — 787-12 (CCAGGCTGGATTCTTGGTTTCCTAZ SEQ ID NO: 9) and vector ( Screening by PCR was carried out using GAL4 Activation Domain Cloning vector) primer (CTATTCGATGATGAAGATACCCCACCAAACC SEQ ID NO: 10). After heating at 94 ° C for 30 seconds, 5 cycles of `` 94 ° C for 5 seconds, 72 ° C for 4 minutes '', 5 cycles of `` 94 ° C for 5 seconds, 70 ° C for 4 minutes '', `` 94 ° C for 5 seconds, 68 ^ 4 minutes "for 25 cycles. As a result, an amplification product of about l.lkb was obtained.

 Furthermore, a primer was designed in the determined "787" sequence, and further upstream sequencing was attempted using a Marathon cDNA Amplification Kit (CLONTECH). Using cDNA from Human Leukemia K-562 cell line (CLONTECH), a cDNA for Marathon was prepared and type II. PCR was carried out using the primer 787_M1U (TTCACGCGGT CTCTTGTAMGTTZ SEQ ID NO: 11) designed in a known sequence region and the adapter primer attached to the kit. The PCR reaction conditions were as follows: after heating at 94 ° C for 30 seconds, 5 cycles of `` 945 seconds, 704 minutes '', 5 cycles of `` 94 ° C for 5 seconds, 68 minutes for 4 minutes, '' `` 94 ° C for 5 seconds, 66 4 minutes "for 25 cycles. As a result, an amplification product of about 1.3 kb was obtained. When this DNA sequence was determined, a 1305 bp sequence including the previously determined “787” sequence was obtained. This sequence is shown in SEQ ID NO: 1.

[Example 10] Analysis by 5 'RACE method 1 5 'RACE analysis was performed using Marathon-Ready cDNA (CLONTECH) Bone Marrow as type II. For the primary 5 'RACE-PCR reaction, PCR was performed using the attached API Primer (CCATCCTAATACGACTC ACTATAGGGCZ SEQ ID NO: 12) and the 787-specific 787-Rl Primer (ACTGATTGA GTCTATCTCTGATG NO SEQ ID NO: 13). The reaction was performed. For the second (nested) 5 'RACE-PCR reaction, the attached AP2 Primer (ACTCACTATAGGGCTCGAGCGGCZ sequence number: 14) and the 787-specific 787-2R Primer (GCTCCCTAGTCTCTCTCACCTCCT sequence number: 15) Was used to perform a PCR reaction. The reaction conditions for the PCR were as follows: primary: one cycle of `` 94 ° C for 3 minutes '', followed by `` 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 3 minutes '' after 35 cycles of `` 72 ° C One cycle of “C5 min” was followed by continuous 4 ° C. Second, one cycle of `` 94 ° C for 3 minutes '', followed by one cycle of `` 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 2 minutes '' followed by one cycle of `` 72 ° C for 5 minutes '' Thereafter, the temperature was continuously raised to 4 ° C. In both reactions, TaKaRa Ex Taq (TaKaRa) was used as a Taq enzyme, and reaction solutions were prepared from the attached reaction reagents according to the attached manual.

 For gel electrophoresis, a 1.5% agarose gel was prepared, 5 / X1 sample was applied, and electrophoresis was performed at 100 V constant voltage for 30 minutes. Thereafter, electrophoretic images were obtained by UV transillumination.

 [Example 1 1] Collection and sequencing of band excised by 5 'RACE analysis 1

In order to determine the nucleotide sequence of the band obtained by 5 'RACE analysis, the amplified DNA fragment was excised from the gel, cloned with plasmid vector PCR2.1 (Invitrogen), and a plasmid containing a DNA fragment of about 600 bp was cloned. Obtained. Using a plasmid DNA, the nucleotide sequence of the DNA fragment was determined according to a conventional method. As a result, about 200 bp of the upstream sequence could be obtained. The sequence is shown in SEQ ID NO: 16.

 [Example 12] Analysis by 5 'RACE method 2

5 'RACE analysis was performed using Marathon-Ready cDNA (CLONTECH) Bone Marrow as type II. For the primary 5 'RACE-PCR reaction, the attached API Primer (CCATCCTAATACGACTC ACTATAGGGCZ SEQ ID NO: 12) and 787-yRl Primer (AGCCCTCT A PCR reaction was performed using GAATCTCCACTCTCTAZ SEQ ID NO: 17). For the second (nested) 5 'RACE-PCR reaction, the attached AP2 Primer (ACTCACTATAGGGCTCGAGCGGC / S2 column number: 14) and the 787-specific 787-yR2 Primer (TCCCTTACCAGATACTACCTCG TG / SEQ ID NO: A PCR reaction was performed using (18). The 787-yRl Primer and 787-yR2 Primer were designed based on the nucleotide sequence obtained from "Recovery of band excised by 5 'RACE analysis and sequencing 1". The reaction conditions for the PCR were as follows: primary: one cycle of `` 94 ° C for 3 minutes '' followed by `` 94 ° C for 30 seconds, 6 CTC for 30 seconds, 72 ° C for 1 minute '' after 35 cycles of `` 72 ° C Min) for one cycle and then continuously at 4 ° C. The second is one cycle of `` 94 ° C for 3 minutes '' followed by one cycle of `` 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for one minute '' followed by one cycle of `` 72 ° C for 5 minutes '' Thereafter, the temperature was continuously raised to 4 ° C. In both reactions, TaKaRa Ex Taq (TaKaRa) was used as a Taq enzyme, and reaction solutions were prepared from the attached reaction reagents according to the attached manual.

 For gel electrophoresis, a 1.2% agarose gel was prepared, 5 ^ 1 sample was applied, and electrophoresis was performed at 100 V constant voltage for 30 minutes. Thereafter, electrophoretic images were obtained by UV transillumination.

 [Example 13] Collection and sequencing of band excised by 5 'RACE analysis 2

In order to determine the nucleotide sequence of the band obtained by 5 'RACE analysis, the amplified DNA fragment was excised from the gel, cloned with the plasmid vector PCR2.1 (Invitrogen), and the plasmid containing the approximately 1.2 kbp DNA fragment was cloned. Obtained. Using the plasmid DNA, the nucleotide sequence of the DNA fragment was determined according to a conventional method. As a result, a further upstream sequence of about 1 kb was obtained. The sequence is shown in SEQ ID NO: 19. Based on these findings, the nucleotide sequence of “787” was finally 2565 bp. The nucleotide sequence of "787" is shown in SEQ ID NO: 20. Industrial applicability

According to the present invention, it is used as an indicator of the response of T cells to antigen stimulation such as cedar pollen. New genes have been provided. Using the expression of the gene of the present invention as an index, it has become possible to conduct a test for the presence or absence of a response of T cells to pollen antigen, or to screen a candidate therapeutic compound for suppressing the response of T cells to antigen stimulation.

Claims

The scope of the claims 1. A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 20.  2. A nucleic acid molecule comprising the protein coding region of the nucleotide sequence of SEQ ID NO: 20. 3. A DNA that specifically hybridizes to the nucleic acid molecule according to claim 1 or 2, and has a chain length of at least 15 nucleotides.  4. The method for detecting a nucleic acid molecule according to claim 1, wherein the DNA according to claim 3 is used.  5. a test method for an allergic disease,  (a) preparing T cells from a subject,  (b) preparing an RNA sample from the T cells,  (c) performing a hybridization on the RNA sample using the labeled DNA of claim 3 as a probe,  (d) measuring the amount of RNA from a subject that hybridizes to the labeled DNA of claim 3, and comparing the amount with a control (in the case of a healthy subject).  6. A test method for an allergic disease,  (a) preparing T cells from a subject,  (b) preparing an RNA sample from the T cells,  (c) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;  (d) a step of performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to claim 3 as a primer,  (e) comparing the amount of DNA amplified by the polymerase chain reaction with a control (in the case of a healthy person).  7. The method according to claim 6, wherein the polymerase chain reaction is performed by a PCR amplification monitor method. 8. The method according to claim 5, wherein the T cells are prepared from peripheral blood of the subject. The method described.  9. The method according to any one of claims 5 to 8, wherein the allergic disease is cedar pollinosis.  10. A method for screening a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response,  (a) Step of administering a test compound to a model animal and stimulating it with a pollen antigen (b) preparing T cells from the model animal,  (c) preparing an RNA sample from the T cells,  (d) performing a hybridization on the RNA sample, using the labeled DNA of claim 3 as a probe.  (e) a step of measuring the amount of RNA derived from the T cells that hybridizes to the labeled DNA of claim 3,  (f) selecting a compound that suppresses a decrease in the amount of RNA measured in step (e) as compared to a control (in the case where a test compound is not administered).  1 1. A method of screening for a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response,  (a) administering a test compound to a model animal and stimulating with a pollen antigen, (b) preparing T cells from the model animal,  (c) preparing an RNA sample from the T cells,  (d) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;  (e) performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to claim 3 as a primer,  (f) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (e) as compared to a control (in the case where no test compound is administered). 1 2. A method of screening for a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response, (a) administering a test compound to a model animal,  (b) preparing lymphocytes from the model animal,  (c) stimulating the lymphocytes with a pollen antigen,  (d) separating T cells from the antigen-stimulated lymphocytes,  (e) preparing an RNA sample from the T cells,  (f) performing a hybridization on the RNA sample, using the labeled DNA of claim 3 as a probe,  (g) measuring the amount of the RNA derived from the T cell that hybridizes to the labeled DNA according to claim 3,  (h) selecting a compound that suppresses the decrease in the amount of RNA measured in step (g) as compared to a control (in the case where no test compound is administered).  13. A method of screening for a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response,  (a) administering a test compound to a model animal,  (b) preparing lymphocytes from the model animal,  (c) stimulating the lymphocytes with a pollen antigen,  (d) separating T cells from the antigen-stimulated lymphocytes,  (e) preparing an RNA sample from the T cells,  (f) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;  (g) performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to claim 3 as a primer;  (h) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (g) as compared to a control (in the case where no test compound is administered).  14. A method for screening a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response, (a) preparing lymphocytes from a model animal or human mosquito, (b) stimulating the lymphocytes with a pollen antigen in the presence of a test compound,  (c) separating T cells from the antigen-stimulated lymphocytes,  (d) preparing an RNA sample from the T cells,  (e) a step of performing hybridization on the RNA sample using the labeled DNA of claim 3 as a probe.  (f) measuring the amount of the RNA derived from the T cell that hybridizes to the labeled DNA according to claim 3,  (g) selecting a compound that suppresses the decrease in the amount of RNA measured in step (f) as compared to a control (in the case where no test compound is administered).  15. A method for screening a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response,  (a) preparing a model animal or human mosquito lymphocytes,  (b) stimulating the lymphocytes with a pollen antigen in the presence of a test compound,  (c) separating T cells from the antigen-stimulated lymphocytes,  (d) preparing an RNA sample from the T cells,  (e) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;  (f) carrying out a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to claim 3 as a primer,  (g) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (f) compared to a control (in the case where no test compound is administered).  16. A method for screening for a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response,  (a) stimulating the established T cells with a lymphocyte stimulating substance in the presence of the test compound, (b) preparing an RNA sample from the stimulated established T cells, (c) performing a hybridization on the RNA sample, using the labeled DNA of claim 3 as a probe, (d) a step of measuring the amount of RNA derived from the established T cells that hybridizes to the labeled DNA according to claim 3;  (e) selecting a compound that suppresses the decrease in the amount of RNA measured in step (d) as compared to a control (in the case where no test compound is administered).  1 7. A method of screening for a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response,  (a) stimulating the established T cells with a lymphocyte stimulating substance in the presence of the test compound, (b) preparing an RNA sample from the stimulated established T cells,  (c) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;  (d) performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to claim 3 as a primer;  (e) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (d) as compared to a control (in the case where no test compound is administered).  18. The method according to claim 10 or 11, wherein the T cells are prepared from peripheral blood of a model animal.  19. The method according to any one of claims 12 to 15, wherein the lymphocytes are prepared from peripheral blood.  20. The method according to any one of claims 10 to 19, wherein the antigen is cedar pollen antigen.