WO2000073435A1 - Pollinosis-associated gene 441 - Google Patents

Abstract

A novel gene undergoing significantly lowered expression after the pollen scattering has been successfully isolated by preparing T cells from 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

 Description Pollen allergy-related gene, 441

 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 diseases is related to mutations or defects in genes expressed in response to antigen stimulation, or overexpression or decreased expression. 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 advances 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 of species at a time More than one sample can be screened, and genes with altered expression in those samples can be detected. 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. Furthermore, another object of the present invention is to provide a method for testing an allergic disease and a method for screening a candidate compound for a therapeutic agent that suppresses the antigen-stimulatory response of T cells, using the expression of the gene as an index.

 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 scattering in multiple subjects including pollinosis patients, and expressed T cells between subjects with different cedar pollen-specific IgE values and before and after pollen scattering. We screened for genes with varying amounts and isolated a novel gene (the “441” gene).

The present inventors performed comparative analysis on the expression level of the isolated 441J gene in lymphocytes isolated from subjects before and after pollen scattering, and found that the gene showed a significantly low value after cedar pollen scattering. Therefore, the present inventors pointed out the expression level of the gene. As a standard, we found that it is possible to test for allergic diseases and to screen for therapeutic drug candidate compounds that suppress the T cell antigen-stimulated response.

 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 nucleotide sequence of SEQ ID NO: 1;

 [2] a nucleic acid molecule comprising the coding region of the nucleotide sequence of SEQ ID NO: 1;

 [3] A DNA that specifically hybridizes to the nucleic acid molecule of [1] or [2] and has a chain length of at least 15 nucleotides.

 [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 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 I 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.

 [8] The method according to any one of [5] to [7], wherein T cells are prepared from peripheral blood of the subject.

 [9] The method of any one of [5] to [8], wherein the allergic disease is cedar pollinosis.

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

 (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 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 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).

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

(f) Amplification in step (e) compared to control (without test compound administration) Selecting a compound that suppresses the decrease in the amount of DM to be obtained.

 (12) 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 hybridization on the RNA sample using the labeled DNA according to (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) as compared to a control (in the case where no test compound is administered).

 (13) a method for screening a candidate compound for a therapeutic agent 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 no test compound is administered). (14) 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 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) performing a hybridization on the RNA sample using the labeled DNA according to (3) as a probe,

 (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 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) 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 (II) as compared to a control (in the case where no test compound is administered).

 [16] 方法 a method for screening a candidate therapeutic compound that suppresses the antigen-stimulated response of 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 RNA 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) as compared to a control (in the case where no test compound is 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) as 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, allergens have been identified, demonstrated a deep link between exposure to allergens and the development of lesions, and the immunological mechanisms involved in the lesions. It can be defined as being proved. Here, the immunological mechanism means that T cells show an immune response by allergen stimulation. Representative allergic diseases can include bronchial asthma, allergic rhinitis, atopic dermatitis, hay fever, or insect allergy. Allergic predisposition (all ergi cdi athes 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 predisposition.

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

 BEST MODE FOR CARRYING OUT THE INVENTION

 TECHNICAL FIELD The present invention relates to a novel gene “441” correlated with a response of lymphocytes to a cedar pollen antigen. The nucleotide sequence of "441" cDNA found by the present inventors is shown in SEQ ID NO: 1.

 The nucleotide sequence of the "441" cDNA isolated by the present inventors is a partial distribution sequence of the "441" cDNA, and those skilled in the art will be able to obtain the sequence information of the "441" cDNA described in SEQ ID NO: 1. On the other hand, isolation of the full-length cDNA of "441" 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 “441” as a probe, or a method for screening a T cell cDNA library using a sequence derived from “441” as a primer. There is a method of obtaining a full-length cDNA by screening a library using DNA as a type II and using as an index that an amplification product of a size specific to the primer is obtained. The RACE method (Frohman, MA et al .: Pro Nat), in which a sequence derived from “441” is used as a primer to convert mRNA from T cells and the like into single-stranded cDNA, add an oligomer to the end, and then perform PCR. l. Acad. Sc i. USA,

85: 8992, 1988), there is a method of extending the sequence of "441".

The “nucleic acid molecule comprising the base sequence of SEQ ID NO: 1” in the present invention includes the full length of “441” which can be isolated based on the sequence information of “441” cDNA described in SEQ ID NO: 1. cDNA is included. Five

“441” was statistically significantly lower in subjects' lymphocytes after exposure to pollen antigen than before exposure. Therefore, using the expression of “441” gene (including transcription into mRNA and translation into protein) as an index, we will conduct tests for allergic diseases and screen candidate therapeutic compounds that suppress antigen-stimulated response of T cells. It is considered possible. Decreased expression of “441” indicates the response of T cells to antigen stimulation such as pollen, so that in patients with a history of allergic disease, the response of that patient to specific antigen exposure using the expression of “441” as an index Monitoring the progress is useful for assessing disease status and examining response to treatment.

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

 The detection of the expression of the "441" gene in the test for allergic disease according to the present invention can be carried out by a hybridization technique using a nucleic acid hybridizing to the "441" gene as a probe, or a DNA hybridizing to the gene of the present invention as a primer. It is possible to use the gene amplification technology.

 As the probe or primer used in the test of the present invention, a nucleic acid molecule that specifically hybridizes to the “441” gene and has a chain length of at least 15 nucleotides is used. The term "specifically hybridizes" as used herein refers to a DNA that is cross-hybridized with DNA and Z or RNA encoding another gene under ordinary hybridization conditions, preferably under stringent hybridization conditions. It indicates that one shot does not occur significantly. For example, the probe and the transfer membrane are hybridized at 68 in Express Hydidi zation on Solution (manufactured by CL0NTECH), and finally mixed with 50% IX SS 0.05% SDS solution at 50%. By washing with, stringent conditions can be achieved.

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, nicks using DNA polymerase I. Labeling with lance label, end labeling with polynucleotide kinase, fill-in labeling with cleno fragment (Berger SL, Kimmel AR. (1987) Guide to Molecular Cloning Techniques, Method in Enzymology, Academic Press; Hames BD, Higgins SJ (1985) Genes Probes: A Practical Approach. IR L Press; Sambrook J, Fritsch EF, Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press) Labeling by transcription (Melton DA, Krieg, PA, Rebagkiati MR, Maniatis T, Zinn K, Green MR. (1984) Nucleic Acid Res., 12, 7035-7056), modified nucleotides without radioisotopes (Kricka LJ. (1992) Nonisotopic DNA Probing Techniques. Academic Press). Testing for 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, and the like.

 On the other hand, as a method using the gene amplification technique, for example, an RT-PCR method can be used. In the RT-PCR method, the expression of the “441” gene can be more accurately quantified by using a PCR amplification monitor method as shown in Example 8 in the process of gene amplification.

In the PCR gene amplification monitoring method, probes that are labeled with different fluorescent dyes at both ends to cancel each other's fluorescence are used to hybridize to the detection target (DNA or RNA reverse transcript). When the PCR proceeds and the probe is decomposed 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 by the number of linear cycles of PCR amplification by simultaneously measuring the standard sample whose copy number is clear for the target (Holland, PM et al., 1991, Proc. Natl. Acad. Sci. USA 88:72 76-7280; Livak, KJ et al., 1995, PCR Methods and Applications 4 (6): 35. Heid, CA et al., Genome Research 6: 986-994; Gibson, EMU et al., 1996, Genome Research 6: 995-1001). In the PCR amplification monitoring 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 “441”. As such a test method, for example, a Western blotting method using an antibody that binds to the protein encoded by “441”, an immunoprecipitation method, an EUSA method, and the like can be used. The antibody of the protein encoded by "441" of the present invention can be obtained as a polyclonal antibody or a monoclonal antibody using techniques well known to those skilled in the art (Milisten C, et al., 1983, Nature 305 (5934): 537-40). For example, a protein or a partial peptide thereof used as an antigen is prepared by incorporating the "441" gene or a part thereof into an expression vector, introducing the gene into an appropriate host cell, and preparing a transformant. Is cultured to express a recombinant protein, and the expressed recombinant protein is purified from a culture or a 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 "441" gene expressed in T cells is reduced after pollen antigen exposure. The "441" gene is a gene whose expression level decreases as a response of the living body to the stimulation of the cedar pollen antigen. By monitoring the expression of the "441" gene, a therapeutic agent for hay fever can be screened. The expression level of the “441” gene is decreased by pollen antigen exposure in both healthy and hay fever patients. Presence or absence of hay fever symptoms is presumed to be due to differences since the response of the "441" gene to antigen stimulation. However, even in such cases, decreased expression of the “441” gene corresponds to enhanced T cell responsiveness. 41 "By monitoring gene expression, it is possible to screen for drugs for treating allergic diseases.

 The method for screening a candidate therapeutic compound that suppresses the antigen-stimulated response of T cells according to the present invention can be performed in vivo or in vitro. 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 “441” is measured. . Alternatively, after administering the candidate drug to a model animal such as a mouse, lymphocytes are separated from peripheral blood, and the lymphocytes are stimulated in vitro with cedar pollen antigen or the like. T cells are separated from the lymphocytes after the stimulation, and the transcript of the “441” gene is measured. As a result of these measurements, a compound that suppresses a decrease in the transcription level of the “441” 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 level of the“ 441 ”gene” means that a higher level of transcription level is maintained by contact with a candidate compound when compared with antigen-stimulated T cells. Say Therefore, if a candidate compound induces a level that exceeds the transcription level of the “441” gene before being subjected to antigen stimulation (ie, if transcription is increased), that 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 in vitro with cedar pollen antigen or the like. Candidate compounds are added during in vitro stimulation. Then, T cells are separated from the stimulated peripheral blood lymphocytes, and the “441” transcript is measured. As a result of this measurement, a compound that suppresses a decrease in the transcription level of the “441” gene compared to a control (when no candidate drug is 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, Molt4 cells, Jurka Cell lines such as t cells T cells are stimulated in vitro with a lymphocyte stimulator. Examples of lymphocyte stimulants include calcium ionophore (A23187), PMA, and phytohemagglutinin (PHA). Candidate drugs are added during in vitro stimulation. Thereafter, the transcription amount of the “441” gene in the established T cells is measured. As a result of this measurement, a compound that suppresses a decrease in the transcription level of the “441” gene compared to a control (when no candidate drug is contacted) is selected.

 Detection of the expression of the "441" gene in the screening of a candidate therapeutic drug that suppresses the T cell antigen-stimulatory response according to the present invention can be performed by detecting the nucleic acid hybridizing to the "441" gene in the same manner as in the test for an 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 utilizing 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, more accurate quantification of the expression of the “441” gene can be performed by using a PCR amplification monitor 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, and extracts of animal and plant tissues or Examples thereof include a mixture containing a plurality of compounds such as a microorganism culture, and a sample purified therefrom. 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, pharmacologically acceptable carriers or vehicles (saline, vegetable oils, suspensions, (Activators, stabilizers, etc.). Administration will be transdermal, intranasal, transbronchial, intramuscular, intravenous, or oral, depending on the nature of the compound. 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. Subject A ~ :! The values of cedar pollen-specific IgE antibodies of each blood sample (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 “441” 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

In order to collect T cells before and after pollen dispersal, 10 ml blood samples were collected from 10 adult volunteers (A to I) before and after pollen dispersal. The first blood sample was collected before the Japanese cedar pollen season (January and February 1997), and the second after the Japanese cedar pollen season (March, April and May 1997). Collected. Eight of the volunteers obtained samples from two periods. For the remaining two volunteers, only samples after pollen scattering were available. An aliquot of these blood samples was used to determine the amount of cedar pollen-specific IgE. Specific IgE measurement was performed using a paper disk as a solid phase by the RAST method (radio all ergo sorbent test, Wide, L. et. Al .: Lancet 2: 1105-1107, 1967) by the improved CAP RAST method (Pharmacia). Using a serum containing a standard antibody titer manufactured by Pharmacia, and using that as a reference, the IgE antibody titer of each sample (the unit is also indicated as Pharmacia RAST Unit, PRU, or AU (arbitrary unit)) It was determined.

 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, subjects A to G and I were eight subjects as atopic predisposition group (hereinafter also referred to as “patient”), and subjects H and〗 were regarded as healthy subjects (hereinafter also referred to as “normal group”). 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 Nopo, 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). The mixture was centrifuged 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. Using a Pasteur pipette, place the tube containing 5 ml of Ficol Paque (Pharmacia) (Falcon tube: 2006 or Is 2059; made of polypropylene). After centrifugation at 1200 rpm for 5 minutes, the mixture was centrifuged at 1500 rpm (equivalent to 400 Xg in a Tomy centrifuge) for 30 minutes at room temperature. As a result, granulocytes and erythrocytes precipitated, and lymphocytes (lymphocytes), monocytes, and platelets were contained in the middle layer with the ficoll layer in between. Collect the intermediate 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 for 5 minutes. The precipitate was collected and washed twice with BSA / PBS. After the second washing, the 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 ι · ριη at 4 for 5 minutes, and suspended in BSA / PBS at 10 ⁸ per 100 ^ 1. The capacity became about 201. This was transferred to an Eppendorf tube (1.5 ml), and the CD3 microbead solution was added. Then, it was left at 4-10 for 30 minutes (at this time, it was not placed on ice). This sample was treated with a magnetic cell saw Yuichi (MACS) (manufactured by Miltenyi Biotech Inc.) as follows.

 The MS + / RS + column was attached to 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 drained. 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 for 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 at 4 for 5 minutes. The precipitate was washed twice with BSA / PBS. After the second washing, the cells were suspended in 1 ml, a part thereof was diluted 2-fold with trypan blue, and the number of cells was counted. The total cell number was about 4 × 10 ⁶ .

[Example 4] Preparation of total RNA from T cells Preparation of total RNA from T cells was performed using RNeasy Mini (manufactured by Qiagen) according to the attached manual in principle. All operations were performed at room temperature, wearing gloves. Four times the volume of ethanol was added to Posh Buffer-RPE. 10 w 1 / ml 2-mercaptoethanol was added to the lysis buffer RLT. The cell suspension was centrifuged at 1000-1200 rpm for 5 minutes, and the supernatant was removed by evaporation. To the precipitate was added 350 1 lysis buffer RLT (containing 2-mercaptoethanol) solution. At this stage, the lysate of cells in the RLT buffer could be stored at _70 ° C. If the cell lysate had been stored frozen, incubate at 37 for 10-15 minutes, and if insolubles were visible, centrifuge for 3 minutes at maximum speed to collect only the supernatant. The lysate was homogenized with a syringe equipped with a 20 G force terran needle and then treated with Q IAshredder. (That is, a normal cell lysate was applied to a Kyaschlets unit using a Pittman. This was centrifuged at 1500 i "pm for 2 minutes, and the effluent was collected.) 3501 70% ethanol was added. Add the RNeasy spin column to the attached 2 ml tube, apply the cell lysate mixture, centrifuge at 8000 X g (11500 rpm) for 1 minute, and discard the effluent. Posh buffer RW1 700 ^ 1 was applied to the column, and the tube was capped for 5 minutes, centrifuged at 11500 n> m for 15 seconds, and the effluent was discarded. Attached to the tube, applied Posh Buffer RPE (containing ethanol) 500 1 to the column, centrifuged at 11500 rpm for 15 seconds, and discarded the effluent. At maximum speed for 2 minutes The column was placed in a new 1.5 ml tube, DEPC-treated water was applied, the lid was capped, and the tube was allowed to stand for 10 minutes, and centrifuged at 11,500 rpm for 10 minutes to obtain total RNA. If the volume was low, re-attach the column to a new 1.5 ml tube, apply DEPC-treated water30, cap the lid for 10 minutes, and centrifuge at 11500 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. The reaction was performed with 2 units of DNase (Futtsubon Gene) and 50 units of RNase inhibitor

(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. After centrifugation at 12 000 i "pm for 10 minutes at room temperature, the upper layer (aqueous layer) was transferred to a new 1.5 ml tube. 1/10 volume of 3M sodium acetate (pH 5, 2) was added, and 2.5 volumes of 100% Ethanol and eta precipitate mate 11 were added and mixed by inversion.Stand at -20 for 15 minutes, then centrifuge at 12000 2000m for 4 minutes at 4 ° C, remove supernatant and remove 70% Ethanol was added and the supernatant was tapped to remove the precipitate, and the supernatant was removed, dried for 3 minutes, and dissolved in 10-20 zl of DDW (without DNase and RNase). Saved up to -80.

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

 The analysis of fluorescent 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 jtig of the total RNA for each of the three anchor primers. For the secondary DD-PCR reaction, cDNA was prepared using 0.4 g of RNA for each of the three anchor primers. All cDNAs were diluted to a final concentration of 0.4 ng // _ il RNA and used for the 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 (0.4ng / zl RNA equivalent) 2.5μ1 Optional primer (2 \ 0 2.5 zl

 lOXAmpliTaq PCR buffer 1.0 1

 2.5mM dNTP 0.8 ^ 1

 50 ^ M anchor primer 0.1 1

 (GT15A, GT15C, GT15G)

 Gene Taq (5U / 1) 0.05 / 1

 Am liTaq (5U / 1) 0.05 / 1

dH ₂ 0 3.0 z 1

10.0 z

The PCR reaction conditions were as follows: 1 minute at 95 minutes, 3 minutes at 40, 5 minutes at 725, and 7 minutes at 72 minutes after 30 cycles of `` 95 seconds, 2 minutes at 40, and 1 minute at 72 ''. For 5 minutes, and then continuously at 4. The primer pair used was an arbitrary primer for each of the primers GT15A (SEQ ID NO: 2), GT15C (SEQ ID NO: 3), and GT15G (SEQ ID NO: 4), AG 1 to 110 and AG 111, respectively. 199 and AG 200-287 were combined for a total of 287 reactions. 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 excised bands (referred to as "441"). "Four The band “41” was found by DD analysis using GT15A (SEQ ID NO: 2) as an anchor primer and AG54 (CCTCTTAGTCZ SEQ ID NO: 5) as an arbitrary primer.

 To determine the nucleotide sequence of "441", the gel containing the "441" band was cut out, stored in a TE solution, and heated at 60 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 250 bp was amplified. GT15A was used as an anchor primer, and AG54 was used as an optional primer. The amplified DNA fragment was cloned into a plasmid vector pCR2.1 (Invitrogen) to obtain a plasmid p44-13 carrying a DNA fragment of about 250 bp. Using the plasmid DNA, the nucleotide sequence of the DNA fragment was determined according to a conventional method. SEQ ID NO: 1 shows the nucleotide sequence of "441"

 [Example 8] Quantification by ABI-7700

 The expression level of "441" was quantified by the TaqMan method using ABI-PRISM7700. This method uses a fluorescent dye to quantitatively detect the PCR-amplified DNA strand in real time. For the purpose of quantification, in the spring of 1998, a new blood sample before and after the cedar pollen was dispersed was collected from 22 volunteers, 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

Based on the base sequence of the DD band determined in Example 7, primer 44Π (C TTCTCTATGGACCAATTCAACTTTGZ SEQ ID NO: 6), 441r (AAGGGCCATTTTTTTACCATAATCAA / SEQ ID NO: 7), and TaqMan probe P441 (TCTGGATAATTAGTAGGATTTAAGCTG TGTTACAAGGCAZ SEQ ID NO: 8) were designed, synthesized and used for quantitative reaction. TaqMan probe P441 was used with its 5 'end labeled with FAM (6-carboxyfluorescein) and its 3' end labeled with TAMRA (6-carboxy-tetramethytri rhodamine). For type I, cDNA transcribed reversely from 44 kinds of total RNA using poly T (12 to 18) as a primer was used. For a standard curve for calculating the copy number, a serial dilution of plasmid p44-13 obtained in Example 7 was used as a type III reaction. Table 3 shows the composition of the reaction mixture for monitoring PCR amplification. In addition, to correct for differences 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 to obtain a copy of the target gene (441). The number was calculated.

 Table 3

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

 10x TaqMan buffer A 5

25mM MgCl ₂ 7

 dATP (lOmM) 1.2

 dCTP (lOmM) 1.2

 dGTP (lOmM) 1.2

 dUTP (lOmM) 1.2

 Forward Primer (100 M) 0.15

 Reverse Primer (lOO ^ M) 0.15

 441 TaqMan probe (6.7 M) 1.49

 Am liTaq Gold (5U / / L) 0.25

AmpErase UNG (1U / L) 0.5 Template solution 5

50

Table 4 shows the number (copy number) of “441” in each sample corrected for the copy number of 3) -actin. For the correction, the average copy of 3-actin in all samples was determined, and the copy number of "441" in each sample was divided by the relative value of -actin in each sample when it was set to 1.

Table 4

Quantification by ABI7700 fit (copy / ngRNA) beta-actin correction data Subject Blood collection time

 441

A Before scattering 21 After scattering 24

B Before losing 39 After scatter 9

C Before scattering 1 3 After losing 7

D Before flight 27 After flight 1 1

E Before flying 1 8 After flying 8

F Before defeat 1 7 After defeat 26

G ίΐΐβί Before 1 2 Flying IB [After 1 3

H Fly 1K ago 1 1 After defeat 9

1 1 2 After defeat 1 1

J before IK 1 9 scattering ¾ 1 2

K τϋιΐχΐυ 1 7 Fly 1Κ¾ 6 Fly Scatter StJ 1 1 0 Fly tt¾ 50

Mmim 2

 9

N30 ftMxi *

 0 Before IK

 After IK 3

 -*

P before flight 7

Q Before defeat 30 After defeat 29

R Before scattering 33 After scattering 4

S Before scattering 28 After defeat 21

T Before losing 20 After scatter 1 9 u Before losing 28 After scatter 1 9

V Before scattering 23 After scattering 1 3 Using this value, a paired t-test was performed. For the assay, StatView software (Abacuus Concepts, Inc.) was used. The results showed that the expression of “441” was significantly higher in the pre-scatter group than in the post-scatter group (P value = 0.0176) when grouped before and after the cedar pollen was dispersed (Fig. 2). The expression levels of “441” in the group before and after pollen dispersal were 25.6 soil 20.7 and 17.1 ± 10.2 copies ng RNA (mean soil standard deviation), respectively. Industrial applicability

 According to the present invention, a novel gene that can be used as an indicator of the response of T cells to antigen stimulation such as cedar pollen was provided. Using the expression of the gene of the present invention as an index, it has become possible to carry out a test for the presence or absence of a T cell response to pollen antigen, or a screening for a therapeutic drug candidate compound that suppresses the T cell response to antigen stimulation.

Claims

The scope of the claims 1. A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.  2. A nucleic acid molecule comprising the coding region of the nucleotide sequence of SEQ ID NO: 1.  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 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) 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 subject).  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) measuring the amount of the RNA derived from the T cells that hybridizes to the labeled DNA according to claim 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 no test compound is 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). 12. A method for 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) a step of 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) 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,  (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, (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 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) 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,  (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) measuring the amount of RNA derived from the established T cell that hybridizes to the labeled DNA of 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) 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) 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.