JP2002186485A - Method for assaying nucleic acid encoding eotaxin, rantes or beta-defensin-2, reagent therefor, and method for screening antiinflammatory agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a specific combination of primers and a probe effective for assaying a nucleic acid encoding eotaxin, RANTES or β-defensin-2 that are known to promote the short-circuited consumption of energy. SOLUTION: A method for assaying a gene as a template by PCR using a pair of PCR primers and a probe that hybridizes to a site between the primers on a template and is bound to a reporter and a quencher, wherein the method permits assaying a gene, particularly an mRNA, encoding eotaxin, RANTES or β-defensin-2 and a kit therefor are provided. These are useful for evaluating or selecting a substance having an antiinflammatory activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to eotaxin, RA
The present invention relates to a method for measuring mRNA or cDNA of NTES or β-defensin-2 and a kit therefor. The present invention is useful, for example, as a method for screening an anti-inflammatory agent or a method for screening a therapeutic agent for atopic dermatitis.

[0002]

2. Description of the Related Art In the case of irritative or allergic inflammation of the skin, it is known that inflammatory cells such as neutrophils and eosinophils migrate to the site of inflammation, are activated, and are involved in the generation of inflammation. In this case, interleukin- is a chemokine involved in the migration and activation of inflammatory cells.
8 (IL-8), eotaxin (eotaxin), RANTES (Regulat
ed upon Activation, Normal T Expressed and Secrete
d), β-defensin-2 and the like are known.

[0003] Therefore, by selecting a substance that suppresses the production of these chemokines by inflammatory cells, it becomes possible to select an anti-inflammatory agent. In this case, as a method of measuring the production of chemokines, a method of measuring the produced IL-8, eotaxin, etc. can be considered.However, to measure the expression of these chemotaxins more directly and with high sensitivity, it is necessary to use these methods. Is preferably measured. However, quantitative measurement of gene expression has traditionally been difficult,
It was semi-quantified by the comparative PCR method, the competitive PCR method, the Northern blotting method and the like.

The polymerase chain reaction (PCR) method is widely used as a nucleic acid amplification method. One use of this PCR method is to reporter dye 1 and quencher dye 2
There is a method of measuring a nucleic acid using a probe to which is bound. In this method, the primer hybridizes to a site on the template sandwiched between a site on the template to which the forward primer used in the PCR method hybridizes and a site on the template to which the reverse primer hybridizes, and the reporter dye 1 Using a probe to which quencher dye 2 is bound, a PCR reaction is performed using a DNA polymerase having 5 ′ → 3 ′ exonuclease activity.

[0005] For example, in FIGS.
When the forward primer is extended by the use of DNA polymerase to the probe, the oligonucleotide constituting the probe becomes 5 ′ →
It is degraded by the action of 3 'exonuclease activity, followed by the generation of extension products of the forward primer.

In this case, a reporter 1 and a quencher 2
While the is bound to the probe, it does not emit fluorescence due to the interaction between the two, but if the oligonucleotide constituting the probe is degraded with the extension of the primer, the reporter 1 and the quencher 2 are separated, and the reporter No. 1 does not receive the action of the quencher 2 and emits fluorescence upon irradiation with ultraviolet light. Therefore, by selecting primers and probes that specifically hybridize to a specific DNA, the specific nucleic acid can be selectively measured based on the fluorescence intensity. This method has already been used for Taq ManPCR
(Trademark) etc.

[0007]

In the above-mentioned method, it is necessary to select a pair of probes and a probe having the above-described positional relationship on the test nucleic acid, that is, the template nucleic acid. The probe must hybridize to the template nucleic acid faster than the primer under the above hybridization conditions. If the extension product of the primer (single-stranded nucleic acid) extends beyond the position where the probe should hybridize, the probe can no longer hybridize, and thus the DN
This is because it cannot be degraded by the 5 ′ → 3 ′ exonuclease activity of A polymerase.

[0008] When two nucleic acids whose specific nucleotide sequences are known hybridize, the likelihood of hybridization can be estimated to some extent by calculation of the melting point (Tm). However, the combination of the primer and the probe selected by this estimation is not always
It does not give good results in the assay and it is necessary to select a probe and primer combination by trial and error for the specific nucleic acid to be measured.

[0009] The present invention aims to provide a simple, efficient and means for evaluating a large number of test substances in a short time, for example, for the evaluation or selection of drugs having anti-inflammatory activity. And for that, eotaxin,
The PCR method described above is used as a means for measuring the expression level of the gene encoding RANTES or β-defensin-2, that is, the amount of mRNA. And, the present invention
It is intended to provide a primer pair and a probe particularly suitable for performing a PCR method. Therefore, the present invention relates to a specific set of primers and probes effective for measuring nucleic acid encoding eotaxin, RANTES or β-defensin-2, which are known to promote short-circuit consumption of energy. It is intended to provide matching.

[0010]

In order to solve the above-mentioned problems, the present invention provides a forward primer and a reverse primer, and a hybridizer with a template nucleic acid in a region having a reporter and a quencher and sandwiched between the two primers. The following primer pair and probe are used in a method for measuring mRNA or cDNA by performing a polymerase chain reaction (PCR) with a DNA polymerase having 5 ′ → 3 ′ exonuclease activity using a probe to be used.

(1) mRNA encoding eotaxin or
To measure the cDNA, nucleotides 170 to 189 of the nucleic acid encoding eotaxin are used as the one primer.
Using an oligonucleotide that hybridizes to the region, nucleotide 30 of the nucleic acid as the other primer
An oligonucleotide that hybridizes to the region from 1 to 320 is used, and an oligonucleotide that hybridizes to the region from nucleotide 191 to 223 in the nucleic acid is used as the probe.

FIG. 2 shows the positional relationship between the primer binding region and the probe binding region on the gene encoding eotaxin. This sequence is described in Kitaura, M. et al., J. Biol. Chem. Vol. 2
71 is a nucleotide sequence of cDNA encoding human eotaxin described in No. 13, p. 7725-7730 (1996). This sequence is shown in SEQ ID NO: 1, and position 1 of the sequence in FIG. 2 corresponds to position 1 in the sequence of SEQ ID NO: 1. In FIG. 2, the underlined portion in the upstream is the region where the forward primer hybridizes, the underlined portion in the downstream is the region where the reverse primer hybridizes, and the underlined portion between them is the region where the probe hybridizes. is there.

The preferred nucleotide sequences of the primer pair and the probe for measuring the eotaxin gene are as follows (in FIG. 2, underlined with rightward arrow, underlined with leftward arrow, and arrow between them, respectively). (Indicated by an underline without). Forward primer 5'-GCCAGCTTCTGTCCCAACCA-
3 '(SEQ ID NO: 4) Reverse primer 5'-GGCACAGATATCCTTGGCCA-3'
(SEQ ID NO: 5) Probe 5'-CTGCTGCTTTAACCTGGCCAATAGGAAGATACC-
3 '(SEQ ID NO: 6).

(2) In order to measure mRNA or cDNA encoding RANTES, an oligonucleotide that hybridizes to a region of nucleotides 50 to 71 of the nucleic acid encoding RANTES is used as the one primer, and the other primer is used as the other primer. An oligonucleotide that hybridizes to the region of nucleotides 274 to 300 of the nucleic acid is used, and an oligonucleotide that hybridizes to the region of nucleotides 73 to 94 in the nucleic acid is used as the probe.

FIG. 3 shows the positional relationship between the primer binding region and the probe binding region on the gene encoding RANTES.
This sequence is described in Schall, TJ et al., J. Immunol. Vol. 141, p. 1
018-1025 (1988) is the nucleotide sequence of cDNA encoding human RANTES. This sequence is represented by SEQ ID NO: 2.
3 corresponds to position 1 in the sequence of SEQ ID NO: 2. In FIG. 3, the underlined portion upstream is the region to which the forward primer hybridizes, the underlined portion downstream is the region to which the reverse primer hybridizes, and the underlined portion between them is the region to which the probe hybridizes. is there.

Preferred nucleotide sequences of the primer pair and the probe for measuring the RANTES gene are as follows (in FIG. 3, underlined with rightward arrow, underlined with leftward arrow, and arrow between them, respectively) (Indicated by an underline without). Forward primer 5'-CGCTCTCATCCTCATTGCTACT-
3 '(SEQ ID NO: 7) Reverse primer 5'-AGCTCATCTCCAAAGAGTTGATGTA
CT-3 '(SEQ ID NO: 8) Probe 5'-CCCTCTGCGCTCCTGCATCTGC-3' (SEQ ID NO: 9).

(3) In order to measure mRNA or cDNA encoding β-defensin-2, an oligo that hybridizes to the region of nucleotides 84 to 104 in the nucleic acid encoding β-defensin-2 is used as the one primer. Nucleotides, the other primer is an oligonucleotide that hybridizes to the region of nucleotides 276 to 295 in the nucleic acid, and the probe is an oligonucleotide that hybridizes to the region of nucleotides 108 to 139 in the nucleic acid.

FIG. 4 shows the positional relationship between the primer binding region and the probe binding region on the gene encoding β-defensin-2. This sequence is shown in SEQ ID NO: 3, and position 1 in the sequence of FIG. 4 corresponds to position 1 in the sequence of SEQ ID NO: 3. In FIG. 4, the underlined portion upstream is the region to which the forward primer hybridizes, the underlined portion downstream is the region to which the reverse primer hybridizes,
The underlined portion between them is the region where the probe hybridizes.

Preferred nucleotide sequences of the primer pair and the probe for measuring the β-defensin-2 gene are as follows (in FIG. 4, underlined with rightward arrow and underlined with leftward arrow, respectively). As well as underlined with no arrows between them). Preferred nucleotide sequences of primers and probes for measuring the β-defensin-2 gene are as follows. Forward primer 5'-GCCTCTTCCAGGTGTTTTTGG-
3 '(SEQ ID NO: 10) Reverse primer 5' CGCACGTCTCTGATGAGGGA3 '
(SEQ ID NO: 11) Probe 5'-TATAGGCGATCCTGTTACCTGCCTTAAGAGTGG-
3 '(SEQ ID NO: 12).

In the present invention, the nucleic acid whose nucleotide sequence is known can be specifically measured using primers and probes which have been confirmed to be able to be specifically measured, and this nucleic acid can be used as a control. it can. As such a control, a gene encoding glyceraldehyde-3-phosphate dehydrogenase can be used. In this case, glyceraldehyde-3-
In the gene encoding phosphate dehydrogenase, an oligonucleotide that hybridizes to the region of nucleotides 66 to 84 is used as one primer, and an oligonucleotide that hybridizes to the region of nucleotides 272 to 291 is used as the other primer, The glyceraldehyde-3-phosphate dehydrogenase gene is measured using an oligonucleotide in the region of nucleotides 242 to 262 as a probe.

Glyceraldehyde-3-phosphate
FIG. 5 shows the positional relationship between the primer and the probe on the gene encoding dehydrogenase. In this figure,
The meaning of the three underlined areas is as described for FIG. Glyceraldehyde-3-phosphate
Preferred nucleotide sequences of primers and probes for measuring the dehydrogenase gene are as follows.

Forward primer 5'GAAGGTGAAGGT
CGGAGTC (SEQ ID NO: 13) Reverse primer 5'GAAGATGGTGATGGGATTTC (SEQ ID NO: 14) Probe 5 'AGGCTGAGAACGGGAAGCTTG (SEQ ID NO: 1
5) The oligonucleotide number for the various primers and probes described above is 15 to 40 nucleotides, and preferably
20-30. This is because if the size of the primer and the probe is long, it is difficult to hybridize to the single-stranded DNA, and if the size is too short, the specificity of the hybridization decreases.

The above specific nucleotide sequences of primers and probes are particularly preferred sequences,
It is known that a primer or a probe consisting of nucleotides hybridizes even if a small number of mismatches exist between the primer and the template strand, and can function as a primer for PCR or as a detection probe. Therefore, the primers and probes of the present invention are not limited to those having the specific nucleotide sequence described above. For example, substitution of 4 or less nucleotides with respect to the specific nucleotide sequence,
Primers and probes modified by deletion and / or addition and capable of hybridizing to a predetermined region are also included in the present invention.

The probe used in the present invention has a reporter dye attached to one end, eg, the 5'-end, and a quencher dye attached to the other end, eg, the 3'-end. Whereas a reporter dye is a substance that emits fluorescence upon irradiation with ultraviolet light, for example, a quencher acts on the reporter dye when present in close proximity to the reporter dye to eliminate the generation of fluorescence. Have Reporter dyes include, for example, 6-carboxy-fluorescein (FAM), tetrachloro-6-
Carboxyfluorescein (TET), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE), hexochloro-6-carboxyfluorescein (HEX) and the like; As 6-carboxy-tetramethyl-rhodamine (TAMRA)
Etc. are used.

The binding of a reporter dye and a quencher dye to the probe oligonucleotide is performed, for example, by using several methylene chains as linkers on the 5 'side of the probe, and adding a FAM molecule to the terminal phosphate group in the form of a phosphate ester. And on the 3 ′ side via a structural unit shown below,
The TAMRA molecule is linked by an amide bond.

[0026]

Embedded image

The method of the present invention is useful for evaluating or selecting a drug to which the present invention is directed, for example, having an anti-inflammatory effect, for eotaxin, RANTES or β-defensin-2.
It is particularly useful when measuring mRNA as a method for measuring the expression level of. In this case, eotaxin, RANTES or β
-Culturing the tissue or cells of the organism whose defensin expression is to be measured in the presence of the test substance, extracting mRNA from the cultured cells according to a conventional method, and then complementing the mRNA.
After synthesizing cDNA according to a conventional method, PCR may be performed using the primer and probe of the present invention. The present invention further provides kits used for performing the above methods. This kit comprises the primers and probes defined above. The kit of the present invention may further include a nucleic acid to be used as a control and primers and probes for measuring the nucleic acid.

The method of the present invention is useful for evaluating or selecting a drug to which the present invention is directed, for example, having an anti-inflammatory effect, for eotaxin, RANTES or β-defensin-2.
It is particularly useful when measuring mRNA as a method for measuring the expression level of. In this case, a tissue of the organism for which the expression of a specific cytokine is to be measured is collected, and is subjected to an ordinary method.
After extracting mRNA and then synthesizing cDNA complementary thereto according to a conventional method, PCR may be performed using the primer and probe of the present invention. The present invention further provides kits used for performing the above methods. This kit comprises the primers and probes defined above. The kit of the present invention may further include a nucleic acid to be used as a control and primers and probes for measuring the nucleic acid.

As the cells used for carrying out the method of the present invention, any animal cells capable of expressing eotaxin, RANTES or β-defensin-2 can be used, for example, fibroblasts or these cells. Cultured cell lines and the like can be used, and as the origin of these cells and tissues, cells and tissues from any animal such as mouse, rat and human can be used, but human cells are used. Is preferred.

Human skin fibroblasts are commercially available,
For example, it is available from Kurabo Industries (Kurashiki Spinning Co., Ltd.). Culture of human fibroblasts may be performed according to a standard method for culturing animal cells, and includes 10% fetal bovine serum.
DMEM medium (Dulbecco's modified Eagle medium) is particularly preferred.

In the experiment of the screening method of the present invention, cells are cultured in the above-described medium in the presence of a test substance and in the absence of a control. Culture may be performed at 37 ° C. for 5 to 24 hours. After culturing, isolate RNA, synthesize cDNA, and perform PCR. RNA isolation and
cDNA synthesis is described in the Examples section.
PCR may be performed according to a conventional method.

The chemokine or β-defensin-2 encoded by the gene to be measured of the present invention is
It is associated with the inflammatory response of the skin or the antibacterial properties of the skin as follows. Eotaxin: specifically acts on eosinophils among chemokines that mainly act on eosinophils and basophils. Causes eosinophil migration and degranulation. It plays an important role in chronic allergies such as atopic dermatitis and parasite infection. It is known that dermal fibroblasts produce.

RANTES: A chemokine that has attracted attention for its effect on eosinophils, like eotaxin. More receptor species than eotaxin, CD45 positive memory-T
It is considered to be important for the activation of T cells because it also acts as a chemotactic factor for cells. It is known that dermal fibroblasts and keratinocytes produce them. β-defensin-2: an antimicrobial peptide produced by epithelial cells such as skin keratinocytes. Has broad antibacterial activity against many types of bacteria. It was first found in the skin of psoriatic patients. Attention has been paid to the relationship between atopic dermatitis patients and Staphylococcus aureus.

[0034]

Next, the present invention will be described more specifically with reference to examples. General Methods RNA isolation Place the cells in a 2.2 ml tube, 2000Xg, 5 minutes, 4 ° C
And remove the culture solution by centrifugation. ISOGEN to remaining cells
(Nippon Gene) (1 ml) was added, stirred, and allowed to stand at room temperature for 5 minutes.

Add 0.2 ml of chloroform and stir for 15 seconds. After leaving at room temperature for 2-3 minutes, 12000Xg, 15 minutes,
After centrifugation at 4 ° C, transfer the aqueous layer to another tube. And 0.5ml
Of isopropanol, and left at room temperature for 5-10 minutes.
Centrifuge at 000 × g for 10 minutes at 4 ° C. to obtain a precipitate. 75% ethanol is added to the obtained precipitate, and the mixture is centrifuged at 12,000 × g for 5 minutes at 4 ° C. to obtain a precipitate. After air-drying the precipitate, dissolve it in distilled water (this solution is referred to as RNA solution).

Synthesis of cDNA 5XFirst Strand was added to 10.5 μl of RNA solution containing 1 μg of RNA.
Buffer (Gibco BRL) 4 μl, 0.1 M DTT (Gibco BRL)
2 μl, 0.5 mg / ml oligo (dT) _12-18 Primer (Gibco
BRL) 1 μl, 2.5 mM dNTP (dATP, dTTP, dGTP, dCTP)
(Takara Shuzo) 1 μl, 124 U / μl RNase Inhibitor (Takara Shuzo) 0.5 μl, 200 U / μl M-MLV Reverse Transcr
After leaving 1 μl of a mixture of iptase (Gibco BRL) at room temperature for 10 minutes, the mixture is incubated at 37 ° C. for 50 minutes.

Measurement of cDNA 5 μl of cDNA, 5 μl of 10 × PCR buffer (Perkin Elmer), 25
7 μl of mM MgCl ₂ (Perkin Elmer), 0.75 μl of 20 μM forward primer, 0.75 μm of 20 μM reverse primer
l, 3 μM probe 5 μl, 2.5 mM dATP (Perkin Elme
r) 1 μl, 2.5 mM dGTP (Perkin Elmer) 1 μl, 2.5 mM
dCTP (Perkin Elmer) 1 μl, 5 mM dUTP (Perkin Elme)
r) 1 μl, distilled water 21.75 μl, AmpErase ^R UNG (Perkin
Elmer) 0.5 μl, AmpliTaq ^R DNA Polymerase (Perkin E
lmer) 0.25 μl of the mixture was mixed with ABI PRISM7700 (Perkin Elme
Set to r) and perform PCR reaction. Temperature condition is 50 ℃ 2
After incubating at 95 ° C for 10 minutes, (at 95 ° C for 15 seconds,
(For 1 minute at 60 ° C.) 40 times, and the fluorescence intensity is measured for each cycle.

Embodiment 1 Remains encoding eotaxin
The initial molecular weight of the gene (template) that encodes a standard curve eotaxin in measuring gene and (concentration), PCR cycle number until the fluorescence intensity starts to increase disengaged from baseline (Threshold Cycle) C _T
The relationship was tested. The template gene, forward primer, reverse primer and probe were as follows.

Template: Eotaxin (Kitaura et al. JB
iol. Chem. Vol.271, No.13, p.7725-7730 (1996)) Forward primer: SEQ ID NO: 4 Reverse primer: SEQ ID NO: 5 Probe: SEQ ID NO: 6 Reporter dye: FAM Quencher dye: TAMRA FIG. 6 shows the results. These results, there is a linear relationship between C _T and the number of molecules of the nucleic acid (concentration) is also in the measurement of gene, it was found to be capable of accurate measurement of nucleic acid by the method of the present invention.

[0040]Embodiment 2. FIG. Of the gene encoding RANTES
Standard curve for measurement The initial molecular weight (concentration) of the gene (template) encoding RANTES
Degree), and the fluorescence intensity starts to deviate from the baseline and increase.
PCR cycle (Threshold Cycle) C _T With
The relationship was tested. Template gene, forward primer,
Reverse primers and probes were as follows.

Template: RANTES (Schall, TJ et al., J. im
munol. Vol. 141, p. 1018-1025 (1988)) Forward primer: SEQ ID NO: 7 Reverse primer: SEQ ID NO: 8 Probe: SEQ ID NO: 9 Reporter dye: FAM Quencher dye: TAMRA The results are shown in FIG. . These results, there is a linear relationship between C _T and the number of molecules of the nucleic acid (concentration) is also in the measurement of gene, it was found to be capable of accurate measurement of nucleic acid by the method of the present invention.

Embodiment 3 FIG . β-Defensin-2
Standard curve for measurement of genes to be loaded The initial molecular weight (concentration) of the gene (template) encoding β-defensin-2 and the number of PCR cycles (Threshold) until the fluorescence intensity departs from the baseline and begins to increase
Cycle) were tested the relationship between the C _T. The template gene, forward primer, reverse primer and probe were as follows.

Template: β-defensin-2 Forward primer: SEQ ID NO: 10 Reverse primer: SEQ ID NO: 11 Probe: SEQ ID NO: 12 Reporter dye: FAM quencher dye: TAMRA The results are shown in FIG. These results, there is a linear relationship between C _T and the number of molecules of the nucleic acid (concentration) is also in the measurement of gene, it was found to be capable of accurate measurement of nucleic acid by the method of the present invention.

Embodiment 4 FIG . For eotaxin expression
Effect of IFN-γ and IL-4 Human fibroblasts were cultured in a 6 cm diameter culture dish in DMEM medium containing 10% fetal bovine serum until confluent,
It was used for the experiment. Various concentrations of TNF-α, IFN-γ, and IL-4 were added and cultured for 24 hours at 37 ° C. Next, RNA was isolated from the cells, cDNA was synthesized, and the expression level of the eotaxin gene was measured according to the method of the present invention. As a result, as shown in FIG. 9, IL-4 exhibited an enhancing effect and IFN-γ exhibited an inhibiting effect on the expression of eotaxin.

Embodiment 5 FIG . IFN-γ for RANTES expression
Influence of and IL-4 Human fibroblasts were cultured in a 6 cm diameter culture dish in DMEM medium containing 10% fetal bovine serum until confluence,
It was used for the experiment. Various concentrations of TNF-α, IFN-γ, and IL-4 were added and cultured for 24 hours at 37 ° C. Next, RNA is isolated from the cells and cDNA is synthesized.
The expression level of the ES gene was measured. As a result, as shown in FIG. 10, IL-4 shows an inhibitory effect on the expression of RANTES,
IFN-γ showed an enhancing effect.

Embodiment 6 FIG . For expression of β-defensin
Effect of IFN and IL on human fibroblasts were cultured in a 6 cm diameter culture dish in DMEM medium containing 10% fetal bovine serum until confluent.
It was used for the experiment. Various concentrations of TNF-α, IFN-γ, and IL-1α were added and cultured for 24 hours at 37 ° C. Next, RNA is isolated from the cells, cDNA is synthesized, and RA is synthesized according to the method of the present invention.
The expression level of the NTES gene was measured. As a result, as shown in FIG. 11, the expression of β-defensin 2 was increased between TNF-α and IL-1.
Induced by α.

[0047]

[Sequence List] SEQUENCE LISTING <110> Shiseido <120> Method for masuring nucleic acid encoding eotaxin, RANTES or beta- defensin-2 and kit therefor <130> <160>

<210> 1 <211> 807 <212> DNA <213> Homo sapiens <223> Nucleotide sequence of DNA encoding human eotaxin <400> gctgccttca gcccccaggg gctcgctggg ccagcttctg 180 tcccaaccac ctgctgcttt aacctggcca ataggaagat accccttcag cgactagaga 240 gctacaggag aatcaccagt ggcaaatgtc cccagaaagc tgtgatcttc aagaccaaac 300 tggccaagga tatctgtgcc gaccccaaga agaagtgggt gcaggattcc atgaagtatc 360 tggaccaaaa atctccaact ccaaagccat aaataatcac catttttgaa accaaaccag 420 agcctgagtg ttgcctaatt tgttttccct tcttacaatg cattctgagg taacctcatt 480 atcagtccaa agggcatggg ttttattata tatatatata tttttttttt aaaaaaaaac 540 gtattgcatt taatttattg aggctttaaa acttatcctc catgaatatc agttattttt 600 aaactgtaaa gctttgtgca gattctttac cccctgggag ccccaattcg atcccctgtc 660 acgtgtgggc aatgttcccc ctctcctctc ttcctccctg gaatcttgta aaggtccatgt 720 cagtatgatat t tcttgtgaac ccaaagtgtg actcattaaa 780 tggaagtaaa tgttgtttta ggaatac 807

<210> 2 <211> 1160 <212> DNA <213> Homo sapiens <223> Nucleotide sequence of DNA encoding human RANTES <400> 2 cctccgacag cctctccaca ggtaccatga aggtctccgc ggcacgcctc gctgtcatcc 60 tcattgctac tgccctgccctccctgcctccctccctccctgcc gcccgcccac tgccccgtgc ccacatcaag gagtatttct 180 acaccagtgg caagtgctcc aacccagcag tcgtctttgt cacccgaaag aaccgccaag 240 tgtgtgccaa cccagagaag aaatgggttc gggagtacat caactctttg gagatgagct 300 aggatggaga gtccttgaac ctgaacttac acaaatttgc ctgtttctgc ttgctcttgt 360 cctagcttgg gaggcttccc ctcactatcc taccccaccc gctccttgaa gggcccagat 420 tctgaccacg acgagcagca gttacaaaaa ccttccccag gctggacgtg gtggctcagc 480 cttgtaatcc cagcactttg ggaggccaag gtgggtggat cacttgaggt caggagttcg 540 agacagcctg gccaacatga tgaaacccca tgtgtactaa aaatacaaaa aattagccgg 600 gcgtggtagc gggcgcctgt agtcccagct actcgggagg ctgaggcagg agaatggcgt 660 gaacccggga gcggagcttg cagtgagccg agatcgcgcc actgcactcc agcctgggcg aaaagaga gacgac gag cc a aaaaaaaaaa aaaaaataca aaaattagcc 780 gcgtggtggc ccacgcctgt aatcccagct actcgggagg ctaaggcagg aaaattgttt 840 gaacccagga ggtggaggct gcagtgagct gagattgtgc cacttcactc cagcctgggt 900 gacaaagtga gactccgtca caacaacaac aacaaaaagc ttccccaact aaagcctaga 960 agagcttctg aggcgctgct ttgtcaaaag gaagtctcta ggttctgagc tctggctttg 1020 ccttggcttt gcaagggctc tgtgacaagg aaggaagtca gcatgcctct agaggcaagg 1080 aagggaggaa cactgcactc ttaagcttcc gccgtctcaa cccctcacag gagcttactg 1140 gcaaacatga aaaatcgggg 1160

<210> 3 <211> 336 <212> DNA <213> Homo sapiens <223> Nucleotide sequence of DNA encoding human beta-defencin-2 <400> 3 agactcagct cctggtgaag ctcccagcca tcagccatga gggtcttgta tctcctcttc 60 tcgttcctcttgttcattgttgttgatt aggcgatcct 120 gttacctgcc ttaagagtgg agccatatgt catccagtct tttgccctag aaggtataaa 180 caaattggca cctgtggtct ccctggaaca aaatgctgca aaaagccatg aggaggccaa 240 gaagctgctg tggctgatgc ggattcagaa agggctccct catcagagac gtgcgacatg 300 taaaccaaat taaactatgg tgtccaaaga tacgca 336

<210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplification of human gene for eotaxin <400> 4 gccagcttct gtcccaacca 20

<210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for amplification of human gene for eotaxin <400> 5 ggcacagata tccttggcca 20

<210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Probe for detection of human eotaxin <400> 6 ctgctgcttt aacctggcca ataggaagat acc 33

<210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplification of human gene for RANTES <400> 7 cgctctcatc ctcattgcta ct 22

<210> 8 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for amplification of human gene for RANTES <400> 8 agctcatctc caaagagttg atgtact 27

<210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe for detection of human gene for RANTES <400> 9 ccctctgcgc tcctgcatct gc 22

<210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplification of human gene for beta-defensin-2 <400> 10 gcctcttcca ggtgtttttg g 21

<210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplification of human gene for beta-defensin-2 <400> 11 cgcacgtctc tgatgaggga 20

<210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Probe for detection of human gene for beta-defensin-2 <400> 12 tataggcgat cctgttacct gccttaagag tgg 33

<210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplification of gene for glyceraldehide-3-phos phate dehydrogenase <400> 13 gaaggtgaag gtcggagtc 19

<210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplification of gene for glyceroldehyde-3-phos phate dehydrogenase <400> 14 gaagatggtg atgggatttc 20

<210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe for detection of gene for glyceraldehyde-3-phosphate dehydrogenase <400> 15 aggctgagaa cgggaagctt g 21

<210> 16 <211> 360 <212> DNA <213> Homo sapiens <223> Patial nucleotide sequence of rat gene for glyceraldehyde-3-phosphate dehydrogenase <400> 16 gctcggctgg cgacgcaaaa gaagatgcgg ctgactgtcg agccacatcg ctcagacgg tggagggag atggggaggg agtcaacgga tttggtcgta ttgggcgcct ggtcaccagg 120 gctgctttta actctggtaa agtggatatt gttgccatca atgacccctt cattgacctc 180 aactacatgg tttacatgtt ccaatatgat tccacccatg gcaaattcca tggcaccgtc 240 aaggctgaga acgggaagct tgtcatcaat ggaaatccca tcaccatctt ccaggagcga 300 gatccctcca aaatcaagtg gggcgatgct ggcgctgagt acgtcgtgga gtccactggc 360

[Brief description of the drawings]

FIG. 1 is a diagram showing the principle of the measurement method of the present invention.

FIG. 2 shows human-derived eotaxin encoding
FIG. 3 is a diagram showing the nucleotide sequence of cDNA and the positions of primers and probes corresponding thereto.

FIG. 3 is a diagram showing the nucleotide sequence of human-derived cDNA encoding RANTES and the positions of primers and probes corresponding thereto.

FIG. 4 is a diagram showing a part of the nucleotide sequence of human-derived cDNA encoding β-defensin-2 and the positions of primers and probes corresponding thereto.

FIG. 5 is a diagram showing a part of the nucleotide sequence of a gene encoding glyceraldehyde-3-phosphate dehydrogenase and the positions of primers and probes relative thereto.

Figure 6 is a graph showing as measured by the method of the present invention a gene coding for eotaxin, the relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

Figure 7, as measured by the method of the present invention the gene encoding RANTES, is a graph showing the relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

Figure 8 is a graph showing β- defensin-2 when measured by the method of the present invention a gene encoding, the relationship between the logarithm of C _T of the initial number molecular template nucleic acid.

FIG. 9 is a graph showing the effects of IFN-γ and IL-4 on eotaxin expression.

FIG. 10. IFN-γ and RANTES expression.
It is a graph which shows the influence of IL-4.

FIG. 11 is a graph showing the effects of TNF-α and IL-1α on β-defensin expression.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) G01N 33/15 G01N 33/50 Z 33/50 33/53 M 33/53 33/566 33/566 C12N 15 / 00 ZNAA (72) Inventor Hideyuki Ichikawa 2-12-1 Fukuura, Kanazawa-ku, Kanagawa Prefecture F-term in Shiseido Research Center Co., Ltd. (Reference) 2G045 AA34 AA40 CB01 CB30 DA12 DA13 DA14 DA36 DA80 FB02 FB12 FB20 GC15 4B024 AA01 AA11 BA08 BA21 CA04 CA09 FA10 GA11 HA12 4B063 QA01 QQ08 QQ24 QQ43 QQ53 QR32 QR55 QR62 QS25 QS32 4C084 AA17 NA14 ZB112 ZB132

Claims (12)

[Claims] 1. A DNA having 5 ′ → 3 ′ exonuclease activity using a forward primer and a reverse primer, and a probe having a reporter and a quencher and hybridizing with a template nucleic acid in a region sandwiched between the primers. In a method for measuring mRNA or cDNA of eotaxin, RANTES or β-defensin-2 by performing polymerase chain reaction (PCR) with a polymerase, (1) measuring mRNA or cDNA encoding eotaxin; An oligonucleotide that hybridizes to a region of nucleotides 170 to 189 of a nucleic acid encoding eotaxin is used as a primer, an oligonucleotide that hybridizes to a region of nucleotides 301 to 320 of the nucleic acid is used as the other primer, and the probe is used as the probe. Nucleotide 191 of the serial in the nucleic acid -
An oligonucleotide that hybridizes to the 223 region; or (2) hybridizes to the region of nucleotides 50 to 71 of the RANTES-encoding nucleic acid as the one primer to measure mRNA or cDNA encoding RANTES. An oligonucleotide that hybridizes to the region of nucleotides 274 to 300 of the nucleic acid is used as the other primer, and an oligonucleotide that hybridizes to the region of nucleotides 73 to 94 of the nucleic acid is used as the probe. Or (3) mRNA or cD encoding β-defensin-2
In order to measure NA, β-
Nucleotides in nucleic acid encoding defensin-2
An oligonucleotide that hybridizes to the region from nucleotides 276 to 295 in the nucleic acid is used as the other primer, and an oligonucleotide that hybridizes to the region from nucleotides 276 to 295 in the nucleic acid is used as the other primer, and nucleotides 108 to 139 in the nucleic acid are used as the probe. Using an oligonucleotide that hybridizes to the region;
Or a method for measuring cDNA. 2. The method of claim 1, wherein said primers and probes are oligonucleotides consisting of 20 to 40 nucleotides. 3. The primer and the probe have the following nucleotide sequence: forward primer for measuring eotaxin gene 5′-GCCAGCTTCTGTCCCAACCA-
3 '(SEQ ID NO: 4) Reverse primer 5'-GGCACAGATATCCTTGGCCA-3'
(SEQ ID NO: 5) Probe 5'-CTGCTGCTTTAACCTGGCCAATAGGAAGATACC-
3 '(SEQ ID NO: 6); or forward primer 5'-CGCTCTCATCCTCATTGCTACT-
3 '(SEQ ID NO: 7) Reverse primer 5'-AGCTCATCTCCAAAGAGTTGATGTA
CT-3 '(SEQ ID NO: 8) Probe 5'-CCCTCTGCGCTCCTGCATCTGC-3' (SEQ ID NO: 9); or forward primer 5'-GCCTCTTCCAGGTGTTTTTGG- for β-defensin gene measurement
3 '(SEQ ID NO: 10) Reverse primer 5' CGCACGTCTCTGATGAGGGA3 '
(SEQ ID NO: 11) Probe 5'-TATAGGCGATCCTGTTACCTGCCTTAAGAGTGG-
Has 3 '(SEQ ID NO: 12); or has been modified by substitution, deletion and / or addition of 4 or less nucleotides in the sequence,
The method according to claim 1 or 2, wherein the method has a nucleotide sequence capable of hybridizing to a corresponding region. 4. A control further comprising the nucleotides 66-84 as one primer in the gene encoding glyceraldehyde-3-phosphate dehydrogenase.
Using an oligonucleotide that hybridizes to the region from nucleotides 272 to 291 as the other primer and using an oligonucleotide that hybridizes to the region from nucleotides 242 to 262 as the probe. The method according to any one of claims 1 to 3, wherein a -3-phosphate dehydrogenase gene is measured. 5. The following nucleotide sequence for the measurement of glyceraldehyde-3-phosphate dehydrogenase: forward primer 5′GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 13) reverse primer 5′GAAGATGGTGATGGGATTTC (SEQ ID NO: 14) probe 5′AGGCTGAGAACGGGAAGCTTG ( SEQ ID NO: 1
5) The method according to claim 4, wherein a primer and a probe having or having been modified by substitution, deletion and / or addition of 4 or less nucleotides, and capable of hybridizing to the corresponding region are used. . 6. A 5 ′ → 3 ′ exonuclease activity comprising a forward primer and a reverse primer, and a probe having a reporter and a quencher and hybridizing with a template nucleic acid in a region sandwiched between the primers. Eotaxin, RA by performing polymerase chain reaction (PCR) with DNA polymerase
A kit for measuring mRNA or cDNA of NTES or β-defensin-2, comprising: (1) nucleotide 170 of a nucleic acid encoding eotaxin as said one primer to measure mRNA or cDNA encoding eotaxin; Oligonucleotides that hybridize to the nucleotides 301 to 320 of the nucleic acid as the other primer, and oligonucleotides that hybridize to the nucleotides 191 to 223 of the nucleic acid as the probe. Or (2) an oligonucleotide that hybridizes to the region of nucleotides 50 to 71 of the RANTES-encoding nucleic acid as the one primer to measure mRNA or cDNA encoding RANTES, and the nucleic acid as the other primer From nucleotide 274 of An oligonucleotide that hybridizes to the region of nucleotides 73 to 94 in the nucleic acid as the probe; or (3) mRNA or cD encoding β-defensin-2.
In order to measure NA, β-
Nucleotides in nucleic acids encoding defensin-2
Oligonucleotides that hybridize to regions 84-104, nucleotides in the nucleic acid as the other primer
An oligonucleotide hybridizing to the region of nucleotides 276 to 295, and an oligonucleotide hybridizing to the region of nucleotides 108 to 139 in the nucleic acid as the probe, wherein the primer and the probe are 15 to 40.
Oligonucleotides consisting of
A kit for measuring mRNA or cDNA, characterized in that: 7. The kit according to claim 6, wherein the primer and the probe are oligonucleotides consisting of 20 to 30 nucleotides. 8. The primer and the probe have the following nucleotide sequence: forward primer for measuring eotaxin gene 5′-GCCAGCTTCTGTCCCAACCA-
3 '(SEQ ID NO: 4) Reverse primer 5'-GGCACAGATATCCTTGGCCA-3'
(SEQ ID NO: 5) Probe 5'-CTGCTGCTTTAACCTGGCCAATAGGAAGATACC-
3 '(SEQ ID NO: 6); or forward primer for measuring RANTES gene 5'-CGCTCTCATCCTCATTGCTACT-
3 '(SEQ ID NO: 7) Reverse primer 5'-AGCTCATCTCCAAAGAGTTGATGTA
CT-3 '(SEQ ID NO: 8) Probe 5'-CCCTCTGCGCTCCTGCATCTGC-3' (SEQ ID NO: 9); or forward primer 5'-GCCTCTTCCAGGTGTTTTTGG- for measuring β-defensin-2 gene
3 '(SEQ ID NO: 10) Reverse primer 5' CGCACGTCTCTGATGAGGGA3 '
(SEQ ID NO: 11) Probe 5'-TATAGGCGATCCTGTTACCTGCCTTAAGAGTGG-
Has 3 '(SEQ ID NO: 12); or has been modified by substitution, deletion and / or addition of 4 or less nucleotides in the sequence,
The kit according to claim 6 or 7, which has a nucleotide sequence capable of hybridizing to a corresponding region. 9. As a control, nucleotides 66 to 84 as one primer in a gene encoding glyceraldehyde-3-phosphate dehydrogenase.
An oligonucleotide which hybridizes to the region of nucleotides 272 to 291 as the other primer, and an oligonucleotide of the region of nucleotides 242 to 262 as the probe as the other primer. The kit according to 1. 10. The following nucleotide sequence for the measurement of glyceraldehyde-3-phosphate dehydrogenase: forward primer 5 ′ GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 13) reverse primer 5 ′ GAAGATGGTGATGGGATTTC (SEQ ID NO: 14) probe 5 ′ AGGCTGAGAACGGGAAGCTTG ( SEQ ID NO: 1
5) The kit according to claim 9, wherein the kit uses primers and probes that have or have been modified by substitution, deletion and / or addition of 4 or less nucleotides, and are capable of hybridizing to the corresponding region. . 11. In a method for evaluating or selecting a drug having an anti-inflammatory action, cells capable of expressing eotaxin, RANTES or β-defensin-2 are cultured in the presence of a test sample, and eotaxin, RANTES or β-defensin-
A method characterized in that the amount of mRNA encoding 2 is measured by the method according to any one of claims 1 to 5 or the kit according to any one of claims 6 to 10. 12. An anti-inflammatory agent as a pharmaceutically active ingredient evaluated by the method according to any one of claims 1 to 5 or by the kit according to any one of claims 1 to 10.