JP2008109915A - Testing method of immune disease based on polymorphism of TSLP gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for examining immunologic diseases from genetic information of immunological allergic diseases such as asthma. <P>SOLUTION: The present invention provides a method for diagnosing immunologic diseases, particularly asthma, from a result of analysis by analyzing genetic polymorphism which exists on IL-7-like cytokine, thymic stromal lymphopoietin (TSLP) gene by using a probe or primer having a specific base sequence, and a method for determining onset risk/morbidity possibility of immunologic diseases by using an expression level of long isoform of TSLP as index, and a method for screening candidates of therapeutic agents for these diseases. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a test method for immune diseases such as asthma and a test reagent used in the test method.

Currently, about 3% of Japanese adults and about 6% of children are said to have bronchial asthma. This bronchial asthma is clinically characterized by chronic reversible airway obstruction with wheezing, coughing, shortness of breath and the like. The bronchial asthma is one of the diseases which has been increasing in recent years, and elucidation of the pathological condition is awaited. In recent years, numerous genetic predispositions have been actively identified for such bronchial asthma.
T-bet gene (Patent Document 1), Iκβ-related protein gene (Patent Document 2), IL-12B gene promoter (Patent Document 3), TRAIL receptor gene (Patent Document 4), ASTH1 gene (Patent Literature 5), a method for examining the risk of bronchial asthma based on polymorphisms such as 5-lipoxygenase activating protein gene (Patent Literature 6) and IL-4R gene (Patent Literature 7) is known.

On the other hand, TSLP is an IL-7-like cytokine and is known to induce differentiation of T cells into Th2 cells (Non-patent Document 1). It is also known that TSLP expression increases in the airways of asthmatic patients (Non-patent Document 2). However, the polymorphism of TSLP gene associated with bronchial asthma has not been known.
JP 2006-149276 JP JP 2002-218997 A Japanese Unexamined Patent Publication No. 2006-101847 JP 2005-027595 Special Table 2002-500895 Special Table 2002-511242 Special table 2002-532073 gazette J Exp Med. 2005 Nov 7; 202 (9): 1213-23 J Immunol. 2005 Jun 15; 174 (12): 8183-90

  An object of the present invention is to provide a method for accurately examining the onset risk and presence of an immune disease such as asthma, and a test reagent used in the method.

  The present inventors have made extensive studies to solve the above problems. As a result, they discovered that single nucleotide polymorphisms on the TSLP gene are deeply related to the development of immune diseases such as asthma. Then, by examining polymorphisms on the TSLP gene, it was found that an immune disease test can be performed accurately and efficiently, and the present invention has been completed.

That is, the present invention is as follows.
(1) A method of analyzing a single nucleotide polymorphism of a thymic stromal lymphopoietin (TSLP) gene and examining an immune disease based on the analysis result.
(2) The method according to (1), wherein the single nucleotide polymorphism is a base at position -82 or a single nucleotide polymorphism in a base in linkage disequilibrium with the base.
(3) The method according to (2), wherein the base in linkage disequilibrium with the base at position -82 is a base selected from the positions -1914, -847, 1117, 1479 and 5901.
(4) The method according to any one of (1) to (3), wherein the immune disease is an allergic disease.
(5) The method of (4), wherein the allergic disease is asthma.
(6) A diagnostic reagent for immune diseases comprising one or more probes selected from the following group:
(A) a probe having a sequence of 10 bases or more including the 1600th base in the base sequence of SEQ ID NO: 1, or a complementary sequence thereof,
(B) a probe having a sequence of 10 bases or more including the 2667th base in the base sequence of SEQ ID NO: 1, or a complementary sequence thereof,
(C) a probe having a sequence of 10 bases or more including the 3432th base in the base sequence of SEQ ID NO: 1, or a complementary sequence thereof,
(D) a probe having a sequence of 10 bases or more including the 4630th base in the base sequence of SEQ ID NO: 1, or a complementary sequence thereof,
(E) a probe having a sequence of 10 bases or more including the 4992th base in the base sequence of SEQ ID NO: 1, or a complementary sequence thereof,
(F) A probe having a sequence of 10 bases or more including the 9414th base in the base sequence of SEQ ID NO: 1 or a complementary sequence thereof.
(7) A diagnostic reagent for immune diseases comprising one or more primers selected from the following group:
(A) a primer for detecting a region containing the 1600th base of the base sequence of SEQ ID NO: 1,
(B) a primer for detecting a region containing the 2667th base of the base sequence of SEQ ID NO: 1,
(C) a primer for detecting a region containing the 3432th base of the base sequence of SEQ ID NO: 1,
(D) a primer for detecting a region containing the 4630th base of the base sequence of SEQ ID NO: 1,
(E) a primer for detecting a region containing the 4992th base of the base sequence of SEQ ID NO: 1,
(F) A primer for detecting a region containing the 9414th base of the base sequence of SEQ ID NO: 1.
(8) A method of analyzing an expression level of a long isoform of a thymic stromal lymphopoietin (TSLP) gene and examining an immune disease based on the analysis result.
(9) The test method according to (8), wherein a case where the expression level of long isoform is higher than that of a healthy subject is determined as having a high risk of developing an immune disease or a high possibility of having a disease.
(10) A method for determining the effect of a therapeutic agent for an immune disease, comprising a step of comparing the expression level of a long isoform of a thymic stromal lymphopoietin (TSLP) gene in the presence and absence of the therapeutic agent for the immune disease.
(11) a step of measuring the expression level of thymic stromal lymphopoietin (TSLP) gene long isoform in the presence and absence of the candidate substance, and then when the expression level is suppressed in the presence of the candidate substance, A screening method for an immune disease therapeutic drug, comprising a step of selecting the candidate substance as an immune disease therapeutic drug.

  According to the test method of the present invention, the risk of developing an immune disease such as asthma and the presence / absence of the onset can be determined at an early stage, and thus there is an advantage that early detection of an immune disease and prompt determination of a treatment policy can be performed.

<1> Test Method of the Present Invention The test method of the present invention is a method of analyzing a genetic polymorphism related to an immune disease on the thymic stromal lymphopoietin (TSLP) gene and testing the immune disease based on the analysis result. . Although it does not specifically limit as an immune disease, For example, allergic diseases, such as asthma and atopic dermatitis, are preferable, and asthma is more preferable. Asthma includes adult asthma, childhood asthma, and atopic asthma. In the present invention, “test” includes a test for the risk of developing an immune disease and a test for the presence or absence of the disease.

  The TSLP gene is preferably a human TSLP gene, and examples thereof include a gene having the base sequence of SEQ ID NO: 1. Note that the sequence of the TSLP gene is not limited to the gene of the above sequence because there is a possibility that substitutions, deletions, etc. may exist in bases other than those related to immune diseases due to differences in race.

The type of gene polymorphism is not particularly limited as long as it is related to an immune disease, and includes single nucleotide polymorphisms (SNPs), VNTR (Variable Number of Tandem Repeat) and the like. Among them, single nucleotide polymorphism is more preferable.
The single nucleotide polymorphism of the TSLP gene associated with immune diseases is not particularly limited, and preferred examples include a base at position -82 (position 3432 of SEQ ID NO: 1) or a base in linkage disequilibrium with this base. Here, “position −82” means a position when the translation start point of the long isoform of the TSLP gene (position 3514 in SEQ ID NO: 1) is counted as 1. The same applies to the following. For example, in the case of “−82”, when an insertion or deletion occurs between the position and the translation start point, the number may be mixed, but the sequence is compared and the “ The base corresponding to “−82” is also included in the “−82” base. The same applies to other positions.
The bases that are in linkage disequilibrium with the base at position -82 are the base at position -1914 (position 1600 of SEQ ID NO. 1), the base at position -847 (position 2667 of SEQ ID NO. 1), and position 1117 (of SEQ ID NO. 1). 4630th base), 1479 (4992th of SEQ ID NO: 1) base, 5901 (9414th of SEQ ID NO: 1) base.

In the TSLP gene on the human chromosome, there is a polymorphism of A (adenine)> G (guanine) at the base (a) -1914 (position 1600 of SEQ ID NO: 1) (A and G polymorphisms). Indicates that it is a major allele, and so on).
In addition, a polymorphism of C (cytosine)> T (thymine) exists at the position (b) -847 (position 2667 of SEQ ID NO: 1).
In addition, a polymorphism of C> T exists at the base at position (c) -82 (position 3432 of SEQ ID NO: 1).
Further, (d) a polymorphism of C> T exists at the 1117th position (position 4630 of SEQ ID NO: 1).
In addition, a polymorphism of C> G exists at the base of (e) 1479 (4992th in SEQ ID NO: 1).
In addition, a polymorphism of G> A exists at the base of (f) position 5901 (the 9414th position of SEQ ID NO: 1).

  By analyzing polymorphisms of these bases alone or in combination, immune diseases can be examined. The TSLP gene sequence may be analyzed for the sense strand or the antisense strand.

  The sample used for analysis of the gene polymorphism of the TSLP gene is not particularly limited as long as it is a sample containing chromosomal DNA. Examples thereof include body fluid samples such as blood and urine, cells such as hepatocytes, body hair such as hair, and the like. It is done. Although these samples can be used directly for the analysis of gene polymorphism, it is preferable to isolate chromosomal DNA from these samples by a conventional method and analyze them.

  Analysis of the gene polymorphism of the TSLP gene can be performed by a normal gene polymorphism analysis method. Examples include, but are not limited to, sequence analysis, PCR, hybridization, and the like.

  The sequence can be performed by a usual method. Specifically, a sequence reaction is performed using a primer set at a position of several tens of bases on the 5 ′ side of a base showing polymorphism, and the type of base at the corresponding position is determined from the analysis result. can do. In addition, when performing a sequence, it is preferable to amplify the fragment | piece containing a polymorphism by PCR etc. previously.

Moreover, it can analyze by investigating the presence or absence of amplification by PCR. For example, a primer having a sequence corresponding to a region containing a base showing a polymorphism and corresponding to each polymorphism is prepared. PCR can be performed using each primer, and the type of polymorphism can be determined depending on the presence or absence of the amplification product.
Further, the presence or absence of amplification may be examined by the LAMP method (Japanese Patent No. 3313358), NASBA method (Nucleic Acid Sequence-Based Amplification; Japanese Patent No. 2843586), ICAN method (Japanese Patent Laid-Open No. 2002-233379), etc. it can. In addition, a single-strand amplification method may be used.

  It is also possible to amplify a DNA fragment containing a polymorphism and determine which type of polymorphism is based on the difference in mobility in electrophoresis of the amplified product. Examples of such a method include a PCR-SSCP (single-strand conformation polymorphism) method (Genomics. 1992 Jan 1; 12 (1): 139-146.). Specifically, first, DNA containing a polymorphic site of the TSLP gene is amplified, and the amplified DNA is dissociated into single-stranded DNA. Next, the dissociated single-stranded DNA is separated on a non-denaturing gel, and the type of polymorphism can be determined by the difference in mobility of the separated single-stranded DNA on the gel.

  Furthermore, when a base showing polymorphism is included in the restriction enzyme recognition sequence, it can be analyzed by the presence or absence of cleavage by a restriction enzyme (RFLP method). In this case, the DNA sample is first cleaved with a restriction enzyme. The DNA fragments can then be separated and the type of polymorphism determined by the size of the detected DNA fragment.

Next, based on the polymorphism analyzed by the method as described above, an immune disease is examined.
For example, when judging based on the polymorphism of the base at position -82 of the TSLP gene, when the base is T, it is judged that there is a high risk of developing an immune disease or a high possibility of suffering from an immune disease. can do. In addition, allele polymorphisms may be considered, for example, when the genotype is TT, there is a higher risk of developing an immune disease than in the case of CT or CC. It can be determined that the property is high.
Since the polymorphisms at positions -1914, -847, 1117, 1479, and 5901 are in linkage disequilibrium with the polymorphism at -82, one of these is substituted for -82. Base polymorphisms may be analyzed.

  In the test method of the present invention, in addition to polymorphisms of the TSLP gene, polymorphisms of other genes may be analyzed, and an immune disease test may be performed based on the combination of the polymorphisms of those genes. Examples of other genes include T-bet gene (JP 2006-149276), IL-12B gene promoter (JP 2006-101847), TRAIL receptor gene (JP 2005-027595) and the like.

In the present invention, TSLP contains two types of expression products, Long isoform (translation start point is 3514th A of SEQ ID NO: 1) and Short isoform (translation start point is 5215th A of SEQ ID NO: 1). However, since it was shown that the expression of long isoform of TSLP is induced in response to viral stimulation, examination of immune diseases such as asthma is performed using the expression level of long isoform of TSLP as an index. It is also possible. For example, when the expression level of the long isoform of TSLP is larger than that in healthy subjects, it can be determined that the risk of developing an immune disease is high, the possibility of suffering from an immune disease is high, and the like. In order to specifically detect the expression of long isoform, a region specific to long isoform may be detected by RT-PCR or hybridization. For example, quantitative analysis can be performed using primers of SEQ ID NOs: 8 and 9. RT-PCR may be performed.

In the present invention, the expression level of the TSLP gene long isoform can also be compared between the presence and absence of an immunological disease therapeutic agent to determine the effect of the immunological disease therapeutic agent. For example, by adding an immune disease therapeutic agent to airway epithelial cells, etc., and determining whether the induction of long isoform upon stimulation with poly (I: C) is suppressed compared to when no immune disease therapeutic agent is added Can do.
In the present invention, the expression level of the long isoform of the TSLP gene is measured in the presence and absence of the candidate substance, and when the expression level is suppressed in the presence of the candidate substance, the candidate substance is added. By selecting as an immune disease therapeutic agent, screening for an immune disease therapeutic agent can also be performed. For example, a candidate substance is added to airway epithelial cells, etc., and screening for therapeutic agents for immune diseases is performed using as an index whether induction of long isoform upon stimulation with poly (I: C) is suppressed compared to when no candidate substance is added be able to.

<2> Reagent for test | inspection of this invention This invention also provides the test reagent containing the primer or probe for test | inspecting an immune disease. Examples of such a probe include a probe having a sequence containing the 1600th base (position 1914) in the base sequence of SEQ ID NO: 1 or a complementary sequence thereof, and a base having the 2667th position (position -847) in the base sequence of SEQ ID NO: 1. ), A probe having a complementary sequence thereof, a sequence containing the 3432th base (position -82) in the base sequence of SEQ ID NO: 1, or a probe having a complementary sequence thereof, the 4630th position in the base sequence of SEQ ID NO: 1 A probe having a base sequence (position 1117) or a complementary sequence thereof, a sequence containing the 4992 base (position 1479) in the base sequence of SEQ ID NO: 1, or a probe having a complementary sequence thereof, or a base of SEQ ID NO: 1 Probes having a sequence containing the 9414th base (position 5901) in the sequence or a complementary sequence thereof are listed. It is.
As these probes, one type of probe having a sequence corresponding to one of the polymorphisms may be used, or two types of probes having sequences corresponding to the respective polymorphisms may be used.

Further, as a primer, a primer capable of discriminating the polymorphism of the 1600th base of the base sequence of SEQ ID NO: 1, for example, a region having a sequence containing the 1600th base of the base sequence of SEQ ID NO: 1 is amplified. Primer capable of discriminating the polymorphism of the 2667th base of the base sequence of SEQ ID NO: 1, for example, amplifying a region having a sequence containing the 2667th base of the base sequence of SEQ ID NO: 1 A primer capable of discriminating the polymorphism of the 3432th base of the base sequence of SEQ ID NO: 1, for example, amplifying a region having a sequence containing the 3432th base of the base sequence of SEQ ID NO: 1 A primer capable of discriminating the polymorphism of the 4630th base of the base sequence of SEQ ID NO: 1, for example, A primer capable of amplifying a region having a sequence containing the 4630th base of the base sequence of column No. 1; a primer capable of discriminating the polymorphism of the 4992th base of the base sequence of SEQ ID NO: 1, for example, a sequence Primer capable of amplifying a region having a sequence containing the 4992th base of the base sequence of No. 1; a primer capable of discriminating the polymorphism of the 9414th base of the base sequence of SEQ ID NO: 1, for example, SEQ ID NO: A primer that can amplify a region having a sequence containing the 9414th base of one base sequence is mentioned. These primers may be a primer set of a forward primer and a reverse primer set at both ends of a region containing a polymorphism (preferably a region having a length of 50 to 1000 bases).
In addition, when used for sequence analysis or single-strand amplification, a primer having a sequence 50 to 100 bases upstream of a polymorphic base can be used. In this case, a primer that amplifies the complementary strand of the above region may be used.
In the case where the presence or absence of amplification is used as an indicator, primers having a sequence of a region containing a polymorphism or a complementary sequence thereof may be used.
Although the length of such a primer or probe is not particularly limited, for example, an oligonucleotide having 10 to 100 bases is preferable, and an oligonucleotide having 15 to 50 bases is more preferable. The test reagent of the present invention may contain a polymerase for PCR, a buffer for hybridization, and the like in addition to these primers and probes.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

<1> Polymorphism analysis subject (subject)
All subject patients with asthma were diagnosed using the American Thoracic Society diagnostic criteria (1962). Diagnosis of atopic asthma is based on whether a prick test is positive or one or more RAST positive for one or more allergens (Hirota T. et. Al., Functional haplotypes of IL-12B are associated with childhood atopic asthma. J Allergy Clin Immunol; 116, 789-795, 2005). As controls (control group), 717 individuals were randomly selected from the population with no atopic related diseases (age range 20-75 years, average age 49.2 years; male: female = 2.64: 1.0). All races are Japanese, and all of them are in writing to participate in the research in accordance with the rules of the Research Ethics Committee at the Institute of Physical and Chemical Research (RIKEN), SNP Research Center. By in-farm consent.
To evaluate whether genetic variants of TSLP are associated with susceptibility to bronchial asthma, TSLP gene polymorphisms were analyzed.

Create linkage disequilibrium map First, to find genetic polymorphisms in the region of TSLP gene in 12 control specimens, 12 pediatric bronchial asthma specimens, and 12 adult bronchial asthma specimens for allele comparison The gene information from about 3 kbp upstream of the exon to about 2 kbp downstream of the last exon was decoded using a gene analyzer. As a result, 19 SNPs were found, of which 7 SNPs with a minor allele frequency of 10% or more (-1914th (SNP2), -847th (SNP4), -82th from the translation start of Long isoform) (SNP5), 1117th (SNP8), 1479th (SNP10), 1560th (SNP11), 5901th (SNP17)) were identified (Table 1).
Next, in order to investigate linkage disequilibrium between these SNPs, the found SNPs were analyzed using the Haploview 3.2 program (http://www.broad.mit.edu/mpg/haploview/). D '| and r ² . As a result, as shown in FIG. 1A and Table 2, ^two tag SNPs satisfying r ² > 0.8 were selected: −82C> T and 1560C> G. It was found that SNPs (SNP2, SNP4, SNP5, SNP8, SNP10, SNP17) other than the marker SNP6 (1560C / G) showed the same behavior as one block.

表１．24人の喘息患者と12人の健常人の比較により見出されたTSLP遺伝子における多型の位置と頻度

Table 1. Location and frequency of polymorphisms in the TSLP gene found by comparing 24 asthma patients with 12 healthy individuals

表２．７種類の多型の連鎖不平衡解析

Table 2. Linkage disequilibrium analysis of 7 polymorphisms

Detection of SNPs Polymorphisms were detected in two (1479, -82) Tag SNPs by genotyping gene mutations using the TaqMan method. That is, the following forward primer, reverse primer and Taqman probe were used for the SNP at position -82 from the long isoform translation start point. TaqManR SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) were used for the 1560th SNP. The specific method of genotyping followed the manual attached to the TaqMan system.

-82 forward primer 5'-GGCAAAACCTGGTGCTTGAG-3 '(SEQ ID NO: 2)
-82 reverse primer 5'-CCACGAGTGTAAGAGATCTGTAAGG-3 '(SEQ ID NO: 3)
-82 reporter 1 VIC labeled 5'-CACTGGCCCCTAAGG-3 '(SEQ ID NO: 4)
-82 reporter 2 FAM labeled 5'-CACTGGCCTCTAAGG-3 '(SEQ ID NO: 5)

Genotyping was performed on 391 adult asthma, 487 childhood asthma, 453 atopic asthma, and non-asthma control group. About them, the chi-square test was performed about the frequency of the gene polymorphism and the allele frequency, and the significance of the frequency difference was tested.
The results are shown in Tables 3, 4, and 5, respectively. A significant difference was observed between the adult asthma group, the childhood asthma group and the atopic asthma group in the allele frequency of SNP5 (-82) in the TSLP gene and the healthy group.

表３．TSLP遺伝子の多型と成人喘息との相関

Table 3. Correlation between polymorphism of TSLP gene and adult asthma

表４．TSLP遺伝子の多型と小児喘息との相関

Table 4. Correlation between polymorphism of TSLP gene and childhood asthma

表５．TSLP遺伝子の多型とアトピー性喘息との相関

Table 5. Correlation between polymorphism of TSLP gene and atopic asthma

<2> Expression analysis of TSLP As shown in FIG. 1B, the present inventors have revealed that the TSLP gene produces two types of transcripts, Long isoform and Short isoform. The tissue specificity and stimulus inducibility of the expression of both these isoforms were examined by quantitative RT-PCR.

Quantitative RT-PCR
Total RNA was extracted from tissues or cells with RNAeasy (QIAGEN, Valencia, Calif.), And then cDNA was synthesized with a mixed primer of random primer and oligo dT primer using DNAse-treated total RNA.
The expression of TSLP mRNA (Long isoform (Long), Short isoform (Short) and both) (common)) was analyzed by quantitative RT-PCR using Syber ^R GreenI dye (SYBER ^R Green). In all experiments, cDNA was quantified and corrected for GAPDH, the human housekeeping gene. The primer sets used for PCR are as follows.

TSLP-common,
5'-CTAAGGCTGCCTTAGCTATC-3 '(SEQ ID NO: 6)
5'-AAGCGACGCCACAATCCTTG-3 '(SEQ ID NO: 7)
TSLP-long
5'-GATTACATATATGAGTGGGAC-3 '(SEQ ID NO: 8)
5'-TTCATTGCCTGAGTAGCAT-3 '(SEQ ID NO: 9)
TSLP-short
5'-CGTAAACTTTGCCGCCTATGA-3 '(SEQ ID NO: 10)
5'-TTCTTCATTGCCTGAGTAGCATTTAT-3 '(SEQ ID NO: 11)
GAPDH
5'-GCACCGTCAAGGCTGAGAAC-3 '(SEQ ID NO: 12)
5'-ATGGTGGTGAAGACGCCAGT-3 '(SEQ ID NO: 13)

  FIG. 2A shows TSLP gene expression in human tissues. In addition, cDNA derived from human tissues was purchased from Clonetech (Mountain View, CA). As a result, it was found that TSLP was highly expressed in the heart, liver, lungs, and trachea, and the short isoform was mainly expressed.

Next, normal airway epithelial cells (NHBE), normal lung fibroblasts (NHLF) and normal lung smooth muscle cells (BSMC) are stimulated with specific molecular groups (PAMPs) such as pathogenic bacteria, and expression of TSLP gene at the time of stimulation I investigated. For PAMPs, 100 ng / ml LPS, 10 μg / ml poly (I: C) and 1 μg / ml MALP-2 were used. Cells were collected 4 hours after stimulation and expression was analyzed using quantitative RT-PCR. As a result, Poly in NHBE cells
Long isoform was remarkably induced in response to (I: C) stimulation (FIG. 2B). On the other hand, Short isoform was not induced by PAMPs.

  Normal human airway epithelial cells (NHBE), normal human lung fibroblasts (NHLF) and normal human airway smooth muscle cells (BSMC) were purchased from Cambrex (Walkersvill, MD). The cells were cultured in a medium kit corresponding to each cell sold by Cambrex. Poly (I: C), LPS and Pam3CSK4 were purchased from InvivoGen (San Diego, CA), and MALP-2 was purchased from Alexis (Lausen, Switzerland).

  Next, the temporal change in the expression level of TSLP upon stimulation with PAMP in NHBE cells was examined. For PAMPs, 200 ng / ml Pam3CSK4, 1 μg / ml MALP-2, 100 ng / ml LPS, and 10 μg / ml poly (I: C) were used. Cells were collected at each time shown in FIG. Short isoform and common gene expression were detected by quantitative RT-PCR using SyberR Green method. Each sample was corrected by the expression level of GAPDH. The experiment was repeated twice and similar results were obtained. From FIG. 3A, it was found that Long isoform was induced 4 hours after poly (I: C) stimulation.

Next, the expression level of TSLP upon stimulation with poly (I: C) in NHBE cells was measured by changing the concentration of poly (I: C). NHBE cells were stimulated with poly (I: C) (0.1 and 10 μg / ml) for 4 hours. Detection was performed by quantitative RT-PCR in the same manner as described above using NHBE derived from four different persons. Values were expressed as mean ± SEM. As a result, it was found that Long isoform was induced by 10 μg / ml poly (I: C) (FIG. 3B).

Next, changes in the amount of TSLP protein upon poly (I: C) stimulation in NHBE cells were examined. TSLP measurement ELISA kit (R & D, Minneapolis, MN) was used for quantification of TSLP protein. NHBE cells (1 × 10 ⁴ cells / well, x3 wells) were stimulated with poly (I: C) (10 μg / ml), and the TSLP concentration in the culture supernatant was measured for 4-24 hours (FIG. 3C left). Further, TSLP was quantified in the culture supernatant of NHBE cells stimulated with 10 μg / ml poly (I: C) or 100 ng / ml LPS for 24 hours (right in FIG. 3C). The experiment was repeated twice and similar results were obtained. Values are mean ± SD, asterisk (*) indicates below quantitation limit (<15.6 pg / ml). As a result, it was found that TSLP was induced by poly (I: C) even at the protein level.

<3> Promoter analysis
In order to examine the influence of SNPs present in the promoter region of the TSLP gene on the promoter activity, promoters of each allele were prepared, and the promoter activity was evaluated using the expression of the reporter gene as an indicator.
The arrangement of TSLP gene and SNP is shown in the upper part of FIG. 4A. As shown in the lower part of FIG. 4A, a reporter construct was prepared by targeting SNPs in the promoter regions of Long isoform and Short isoform.
The promoter regions of human TSLP-long isoform (-1041 to -12) and TSLP-short isoform (+842 to +1661) were cloned using the following primers with linkers added from the genomes of human asthma patients and healthy volunteers. The gene sequence was confirmed (KpnI and XhoI sites for forward and reverse primers, respectively). Then pGL3-enhancer as reporter vector
The promoter sequence was recombined into vector (Promega, Madison WI) and used for promoter assay.
TSLP-long promoter
5'- TGAGCTCAGGACAGCATCGT-3 '(SEQ ID NO: 14)
5'-CAATTCCACCCCAGTTTCAC-3 '(SEQ ID NO: 15)
TSLP-short promoter
5'-GGCTATTTCACTGCCTTGTC-3 '(SEQ ID NO: 16)
5'-GATTGAAGGTTAGGCTCTGG-3 '(SEQ ID NO: 17)
In the long isoform, polymorphisms at -847 (SNP4) and -82 (SNP5) were evaluated. On the other hand, short isoforms were evaluated for polymorphisms at positions -585 (SNP8) and -223 (SNP10). The numbers -585 and -223 are counted from the translation start point of the Short isoform.
The TSLP-long isoform promoter mutant was prepared using Gene Editor in vitro site-directed mutagenesis system (Q9280; Promega) according to the kit manual.
To convert -847C (Major) to -847T (Minor), 5'-AGGTGCCCCTAGTCACCAAGAG-3 '(SEQ ID NO: 18) was used. On the other hand, 5'-GAGCACTGGCCTCTAAGGCAGGCC-3 '(SEQ ID NO: 19) was used to convert -82C (Major) to -82T (Minor). Also, 5'-GAGTATCCTGCTATGTGCAGAACTCCG-3 '(SEQ ID NO: 28) was used to convert -585C (Major) to -585T (Minor). On the other hand, 5′-GCTCTACTCAACCGTGACCTCTTCTCTC-3 ′ (SEQ ID NO: 29) was used to convert −223C (Major) to −223G (Minor).

Both the reporter vector and Renilla luciferase vector (pRL-TK) were transfected into NHBE cells (5 × 10 ⁴ cells / 12 well plate) (500 ng and 10 ng DNA / well, respectively). After 24 hours, the cells were stimulated as necessary, and luciferase activity was measured with the Dual Luciferase Reporter Assay System based on the Promega kit manual. The promoter activity was corrected with pRL-TK.

That is, 500 ng TSLP reporter construct as shown in FIG. 4A, pGL3-TSLP-long isoform / major type (-847C, -82C) or pGL3-TSLP-long isoform / minor type (-847T, -82T) and pGL3- Gene transfer of TSLP-short isoform / major type (-585C, -223C) or pGL3-TSLP-short isoform / minor type (-585T, -223G) or pGL3-enhancer control vector (Mock) into NHBE (5x10 ⁴ cells) did. The 10 ng pRL-TK vector was also introduced at the same time as each reporter construct as an internal control. After 24 hours, the cells were stimulated with 10 μg / ml poly (I: C) for 4 hours, and the luciferase activity of NHBE was measured. The experiment was repeated three times with similar results. Values represent mean ± SD. As a result, as shown in FIG. 4B, it was found that the long isoform was more strongly induced by poly (I: C) in the minor type than in the major type. On the other hand, in the short isoform, there was no difference between Major type and minor type.

Next, in order to investigate which of the long isoforms -847 (SNP4) and -82 (SNP5) is important for induction by poly (I: C), (-847C, -82C), (-847T, Four types of promoter constructs -82C), (-847C, -82T), and (-847T, -82T) were prepared, and the promoter activity was measured in the same manner as described above. The experiment was repeated twice and similar results were obtained. Values are mean ±
Indicates SD. As shown in FIG. 4C, a significant difference was observed between (−847T, −82T) and (−847C, −82T) in promoter activity upon stimulation with poly (I: C). It was found that polymorphism of the position affects the expression of long isoform of TSLP upon poly (I: C) stimulation. That is, it was found that when the −847 position is T, it is more easily induced by poly (I: C) stimulation than when the −847 position is C.

Biotinylated oligo DNA precipitation Next, biotinylated oligo DNA precipitation was performed to examine whether or not a transcription factor binds to a region containing the Long isoform promoter polymorphism. That is, -847C and -847T double-stranded oligonucleotides and -82C and -82T double-stranded oligonucleotides were prepared, and the binding of transcription factors to them was evaluated.
Extraction of nucleoprotein from NHBE cells was performed as follows. 1.5x10 ⁶ cells in buffer C '(20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl ₂ , 0.2 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, 1 mM Na ₂ V0 ₃ , 1 mM NaF, 1 mM sodium glycerophosphate, After solubilization with 1 mM phenylmethylsulfonyl fluoride and protease inhibitor mix), the mixture was allowed to stand for 15 minutes under ice cooling. Thereafter, the supernatant was collected by centrifugation at 15000 rpm and 4 ° C. for 15 minutes. Buffer D ′ (a solution obtained by removing NaCl from buffer C ′) was added to the supernatant so as to have a ratio of 1: 3, and this was used for biotinylated oligo DNA precipitation.
The following biotinylated double-stranded oligo DNA (3 μg) and 3 μg poly (dI-dC) were added to the nucleoprotein sample and reacted at 4 ° C. As a control, only biotinylated double-stranded oligo DNA (3 μg) was added and reacted at 4 ° C. One hour later, streptavidin-agarose was added to recover the biotinylated double-stranded oligo DNA and the factor bound thereto. Biotinylated oligo DNA precipitated samples were separated by SDS-PAGE, Western blotted, and anti-AP1 antibody (Oncogene, Sandiego, CA) or anti-AP2α antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
As shown in FIG. 5, it was found that AP1 specifically binds to -847T upon stimulation with poly (I: C). From this, it was considered that when the -847 position is T, AP1 binds in response to stimulation and transcription of Long isoform is activated.

-847C oligo
5′-GGTGCCCCTAGCCACCAAGAG-3 ′ (SEQ ID NO: 20)
5′-CTCTTGGTGGCTAGGGGCACC-3 ′ (SEQ ID NO: 21)
-847T oligo
5'-GGTGCCCCTAGTCACCAAGAG-3 '(SEQ ID NO: 22)
5′-CTCTTGGTGACTAGGGGCACC-3 ′ (SEQ ID NO: 23)
-82C oligo
5'- TGGCCCCTAAGGCAGGCCTTACAG -3 '(SEQ ID NO: 24)
5'-CTGTAAGGCCTGCCTTAGGGGCCA-3 '(SEQ ID NO: 25)
-82T oligo
5'- TGGCCTCTAAGGCAGGCCTTACAG -3 '(SEQ ID NO: 26)
5′-CTGTAAGGCCTGCCTTAGGGGCCA-3 ′ (SEQ ID NO: 27)

Human TSLP linkage disequilibrium map and gene schematic. (Top) Linkage disequilibrium map between SNPs found at a frequency of 10% or more. (Bottom) Schematic representation of 7SNPs and two isoforms present in the TSLP gene region. The figure which shows the expression of the TSLP gene of a human tissue and a cell. (A) Expression of Long Isoform (Long), Short Isoform (Short) and both (common) of TSLP was detected by quantitative RT-PCR using a cDNA panel of normal human tissue. (B) Normal airway epithelial cells (NHBE), normal lung fibroblasts (NHLF), and normal lung smooth muscle cells (BSMC) were stimulated with specific molecular groups (PAMPs) such as pathogenic bacteria, and the expression of TSLP gene was examined. . The figure which shows activation of NHBE by poly (I: C) stimulation, and expression of a TSLP gene. (A) PAMPs were added to the culture medium of NHBE. For PAMPs, 200 ng / ml Pam3CSK4, 1 μg / ml MALP-2, 100 ng / ml LPS and 10 μg / ml poly (I: C) were used, and the cells were collected at each time shown in the figure, and the LPLP Long isoform, Short Isoform and common gene expression were detected by quantitative RT-PCR. (B) NHBE was stimulated with poly (I: C) (0.1 and 10 μg / ml) for 4 hours and detected by quantitative RT-PCR. (C) Production of TSLP protein of NHBE was examined using ELISA. (Left) NHBE (1 × 10 ⁴ cells / well, x3 wells) was stimulated with poly (I: C) (10 μg / ml), and the TSLP concentration in the culture supernatant was measured for 4-24 hours. (Right) Quantification of TSLP in the culture supernatant of NHBE stimulated with 10 μg / ml poly (I: C) or 100 ng / ml LPS for 24 hours. The figure which shows the search of the function control SNP of a TSLP gene. (A) Schematic representation of human TSLP gene and TSLP reporter construct. (Upper) The arrangement of TSLP gene and SNP is shown. (Lower) A reporter construct was prepared targeting SNPs in the promoter region of Long isoform and Short isoform. (B) A 500 ng TSLP reporter construct as shown in the figure was introduced into NHBE (5 × 10 ⁴ cells). The 10 ng pRL-TK vector was also introduced at the same time as each reporter construct as an internal control. After 24 hours, stimulation with 10 μg / ml poly (I: C) was performed for 4 hours, and the luciferase activity of NHBE was measured. (C) Identification of regulatory SNPs in TSLP expression. Reporter constructs of various 1-gene mutants in the long isoform promoter region were prepared. Four types of reporter constructs, C / C, T / T, C / T, and T / C, were prepared for bases -847 / -82 at the long isoform SNP position, as in (B). Promoter activity was measured. Figure (photo) showing binding of transcription factors to regulatory SNPs. The nucleoprotein of NHBE (1.5 × 10 6 cells) stimulated with poly (I: C) (10 μg / ml) for 4 hours or unstimulated was extracted. Proteins bound to biotinylated double-stranded oligoDNA of -847C or -847T and -82C or -82T, which are sequences around the long isoform SNP, are precipitated with streptavidin-agarose, and the proteins are analyzed by SDS-PAGE. Expanded. The binding protein of -847C or -847T was detected by anti-AP1 antibody (left), and the binding protein of -82C or -82T was detected by western butting using anti-AP2α antibody (right).

Claims (11)

A method of analyzing a single nucleotide polymorphism of a thymic stromal lymphopoietin (TSLP) gene and examining an immune disease based on the analysis result. The method according to claim 1, wherein the single nucleotide polymorphism is a single nucleotide polymorphism of the base at position -82 or a base in linkage disequilibrium with the base. The method according to claim 2, wherein the base in linkage disequilibrium with the base at position -82 is a base selected from the positions -1914, -847, 1117, 1479 and 5901. The method according to any one of claims 1 to 3, wherein the immune disease is an allergic disease. The method according to claim 4, wherein the allergic disease is asthma. A test reagent for immune diseases comprising one or more probes selected from the following group:
(A) a probe having a sequence of 10 bases or more including the 1600th base in the base sequence of SEQ ID NO: 1, or a complementary sequence thereof,
(B) a probe having a sequence of 10 bases or more including the 2667th base in the base sequence of SEQ ID NO: 1, or a complementary sequence thereof,
(C) a probe having a sequence of 10 bases or more including the 3432th base in the base sequence of SEQ ID NO: 1, or a complementary sequence thereof,
(D) a probe having a sequence of 10 bases or more including the 4630th base in the base sequence of SEQ ID NO: 1, or a complementary sequence thereof,
(E) a probe having a sequence of 10 bases or more including the 4992th base in the base sequence of SEQ ID NO: 1, or a complementary sequence thereof,
(F) A probe having a sequence of 10 bases or more including the 9414th base in the base sequence of SEQ ID NO: 1 or a complementary sequence thereof. A test reagent for immune diseases comprising one or more primers selected from the following group:
(A) a primer for detecting a region containing the 1600th base of the base sequence of SEQ ID NO: 1,
(B) a primer for detecting a region containing the 2667th base of the base sequence of SEQ ID NO: 1,
(C) a primer for detecting a region containing the 3432th base of the base sequence of SEQ ID NO: 1,
(D) a primer for detecting a region containing the 4630th base of the base sequence of SEQ ID NO: 1,
(E) a primer for detecting a region containing the 4992th base of the base sequence of SEQ ID NO: 1,
(F) A primer for detecting a region containing the 9414th base of the base sequence of SEQ ID NO: 1. A method for analyzing the expression level of a long isoform of a thymic stromal lymphopoietin (TSLP) gene and examining an immune disease based on the analysis result. The test method according to claim 8, wherein when the expression level of the long isoform is higher than that of a healthy subject, it is determined that the risk of developing an immune disease is high or the possibility of having a disease is high. A method for determining the effect of an immunological disease therapeutic agent, comprising a step of comparing the expression level of a long isoform of a thymic stromal lymphopoietin (TSLP) gene in the presence and absence of the immunological disease therapeutic agent. A step of measuring the expression level of the long isoform of the thymic stromal lymphopoietin (TSLP) gene in the presence and absence of the candidate substance, and then when the expression level is suppressed in the presence of the candidate substance A method for screening an immune disease therapeutic drug, comprising a step of selecting as an immune disease therapeutic drug.

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