WO2010005067A1 - Reagent for detecting type of allele based on polymorphism of siglec14 gene - Google Patents

Reagent for detecting type of allele based on polymorphism of siglec14 gene Download PDF

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WO2010005067A1
WO2010005067A1 PCT/JP2009/062572 JP2009062572W WO2010005067A1 WO 2010005067 A1 WO2010005067 A1 WO 2010005067A1 JP 2009062572 W JP2009062572 W JP 2009062572W WO 2010005067 A1 WO2010005067 A1 WO 2010005067A1
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siglec
gene
protein
antibody
seq
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PCT/JP2009/062572
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French (fr)
Japanese (ja)
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将敬 山中
高志 安形
久 成松
貴文 木村
一太 保井
文也 平山
慶彦 谷
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独立行政法人産業技術総合研究所
日本赤十字社
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Publication of WO2010005067A1 publication Critical patent/WO2010005067A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the present invention relates to an allelic detection reagent based on the Siglec 14 gene polymorphism and the detection method.
  • the present invention also relates to a reagent for detecting anti-Siglec 14 antibody and a method for detecting anti-Siglec 14 antibody in a human blood sample using the same.
  • Siglec family proteins are a group of type I transmembrane proteins belonging to the immunoglobulin superfamily, and have lectin activity that specifically recognizes various sugar chain structures including sialic acid (Non-patent Document 1). Most of the molecules belonging to the Siglec family are known to bind to sugar chains containing sialic acid in the extracellular region, and to participate in signal transduction by binding to signal transduction molecules in the intracellular region.
  • Patent Document 1 proteins that specifically recognize sugar chain structures expressed in human cells or tissues are also considered useful for drug delivery such as detection of cancer cells and antibody medicine.
  • Patent Document 2 and Patent Document 3 proteins belonging to the Siglec family such as Siglec 2 protein (also known as CD22) and Siglec 3 protein (also known as CD33) are known to be useful as targets for antibody medicine (Non-Patent Document 2, Non-patent Document 2, Patent Document 3).
  • Siglec 5 protein has been reported to be useful for detection of myeloid leukemia cells (Non-patent Document 4).
  • Siglec 14 protein and its gene is an immunoreceptor tyrosine-containing suppression motif (ITIM) found in the conventional Siglec family protein.
  • ITIM immunoreceptor tyrosine-containing suppression motif
  • Non-hemolytic transfusion side effects are side effects due to transfusion therapy, and are collectively referred to as side effects excluding hemolytic and infectious side effects and post-transfusion GVHD.
  • the causes are assumed to be anti-plasma component antibodies, anti-leukocyte antibodies, inflammatory cytokines in blood products, etc., but have not been fully elucidated.
  • the frequency of non-hemolytic transfusion side effects accounts for about 90% of the transfusion side effect reports (approximately 2,000 cases per year) reported by the medical institution to the Japanese Red Cross Society, and minor side effects cases are not reported. Is expected to greatly exceed this.
  • the object of the present invention is to investigate whether there is a difference in the expression status of Siglec 14 protein in individuals, or whether there is any relationship between similar Siglec 5 protein and its function, or at the gene level.
  • new knowledge related to Siglec 14 protein and gene is obtained, and medically useful means are provided based on this knowledge.
  • the object of the present invention is also to provide a method for identifying a blood product involved in non-hemolytic transfusion side effects and a method for preventing non-hemolytic transfusion side effects and ensuring the safety of transfusion.
  • the present inventors have found that there are individuals who do not express the Siglec 14 protein and those who do not express the Siglec 14 protein.
  • Those who have a homozygous form of this fusion gene express the Siglec 14/5 fusion protein (having the same amino acid sequence as the Siglec 5 protein), but do not express the Siglec 14 protein and are produced according to individual differences. Obtained that the antibody against Siglec 14 can contribute to transfusion side effects, and there is a significant difference in the amount of TNF- ⁇ produced between cells expressing Siglec 14 protein and cells not expressing Siglec 14 protein. The knowledge that it has was also obtained.
  • the inventors have completed the present invention based on these findings.
  • the present invention is as follows.
  • a reagent for detecting an allelic form based on a polymorphism of the Siglec 14 gene comprising:
  • the primer pair that specifically amplifies the Siglec 14 gene has a sequence corresponding to the base sequence of the region outside the conserved region in the base sequence of the Siglec 14 gene and the Siglec 5 gene (1) The reagent for detection as described in).
  • the primer pair that specifically amplifies the Siglec 14/5 fusion gene has a sequence corresponding to the base sequence outside the conserved region in the base sequence of the Siglec 14 gene and the Siglec 5 gene, The reagent kit for detection according to (4) above.
  • a primer pair for specifically amplifying the Siglec 14 gene according to any one of (1) to (3) above using human genomic DNA as a template, or the detection reagent kit according to (4) or (5) above A method for detecting an allelic type based on a polymorphism of the siglec 14 gene, wherein PCR is performed to detect the presence or absence of the siglec 14 gene or further the presence or absence of the siglec 14/5 gene.
  • the Siglec 14 gene characterized in that a probe that specifically hybridizes to the Siglec 14 gene according to (1) is hybridized to human genomic DNA, and the presence or absence of the Siglec 14 gene is detected. Allele type detection method based on polymorphism of
  • a cell that expresses a protein having an amino acid sequence containing at least the 238th to 358th sequence of SEQ ID NO: 4 or a protein having an amino acid sequence containing at least the 238th to 358th sequence of SEQ ID NO: 4 A reagent for detecting an anti-Siglec 14 antibody, characterized in that
  • the present invention is based on the first discovery that a polymorphism exists in the Siglec 14 gene.
  • the reagent of the present invention produced based on this finding can distinguish and detect an allelic type having the Siglec 14 gene and a homozygous type of the Siglec 14/5 fusion gene not having the Siglec 14 gene.
  • Such allele-type detection results can be used to eliminate risks such as blood transfusion side effects in advance or to determine a treatment policy such as an infectious disease.
  • the detection reagent for anti-Siglec 14 antibody containing Siglec 14 protein or Siglec 14 protein-expressing cell of the present invention it is possible to detect anti-Siglec 14 antibody in a human blood sample.
  • detection results of the anti-Siglec 14 antibody can be used to prevent non-hemolytic transfusion side effects and to ensure the safety of transfusion.
  • FIG. 1 is a graph showing the results of leukocyte flow cytometry analysis using a Siglec 14 protein-specific antibody.
  • FIG. 2A is a diagram showing the results of polymorphism analysis by flow cytometry of individual differences in Siglec 14 protein expression and Siglec 5 protein expression.
  • FIG. 2B is a diagram showing a schematic diagram of genomic PCR analysis concerning the Siglec 14 gene and its polymorphism. Arrowheads in the figure indicate the position and orientation of each primer.
  • FIG. 2C is a schematic diagram showing the results of genomic PCR analysis on the Siglec 14 gene and its polymorphism.
  • FIG. 2D is a diagram showing the results of genomic PCR analysis for the Siglec 14 gene and its polymorphism.
  • FIG. 1 is a graph showing the results of leukocyte flow cytometry analysis using a Siglec 14 protein-specific antibody.
  • FIG. 2A is a diagram showing the results of polymorphism analysis by flow cytometry of individual differences in Siglec 14 protein expression
  • FIG. 3A is a diagram showing the results of analyzing the relationship between forced expression of Siglec 14 protein and the expression level of TNF- ⁇ .
  • FIG. 3B is a diagram showing the analysis results of the expression level of TNF- ⁇ by introduction of siRNA that suppresses forced expression of Siglec 14 protein and expression of DAP12 protein.
  • FIG. 4 is a schematic diagram showing a method for detecting an anti-Siglec 14 alloantibody using a Siglec 14-expressing cell line.
  • FIG. 5 shows the results of transgene expression analysis in a siglec 5 or siglec 14-expressing cell line.
  • FIG. 6 is a view showing the detection results of anti-Siglec 14 alloantibodies in a preparation causing non-hemolytic transfusion side effects using Siglec 5 or Siglec 14-expressing cell lines.
  • FIG. 7 is a diagram showing the analysis results of the expression level of Mac-1 antigen in neutrophils stimulated with anti-Siglec 14 antibody-positive serum.
  • FIG. 8 is a diagram showing the analysis result of the amount of HBP released by neutrophils stimulated with anti-Siglec 14 antibody-positive serum.
  • the present invention relates to a reagent for detecting an allelic type based on a polymorphism of the Siglec 14 gene, and a method for detecting the allelic type using the same.
  • Siglec 5 SIGLEC5
  • Siglec 14 SIGLEC14
  • This fusion gene found for the first time in the present invention lacks a region encoding a specific amino acid sequence portion of Siglec 14 (SIGLEC14) protein, and the amino acid sequence of the product is identical to Siglec 5 (SIGLEC5) protein. .
  • the amino acid sequences of these Siglec 14 proteins and the base sequences of the Siglec 14 gene are shown in SEQ ID Nos.
  • the amino acid sequence of the fusion gene product (same as Siglec 5 protein) and the base sequence of the fusion gene are the same as SEQ ID NO: 6 and SEQ ID NO: 3 (of which 1001 to 19105 are the regions encoding the protein) ).
  • Siglec 14 protein when the expression status of the Siglec 14 protein is seen for each individual, there are those who express the Siglec 14 protein and those who do not express it, and those who have the Siglec 14 gene are Siglec 14 Those who express protein and Siglec 5 protein but have homozygous allelic form of the Siglec 14/5 fusion gene lack Siglec 14 protein and have the same amino acid as Siglec 14/5 fusion protein. Having a sequence). In addition, those who have a Siglec 14/5 fusion gene, but who have a heterozygous allele of the Siglec 14 gene, have Siglec 14 protein, Siglec 5 protein, and Siglec 14/5 fusion protein (the same amino acid sequence as Siglec 5 protein). Having a). Interesting results have also been obtained that the proportion of persons with homozygous allelic forms of this Siglec 14/5 fusion gene varies from region to region in the world (see Table 1 in Example 4).
  • Siglec 14 protein is expressed on leukocytes, and when blood of an individual expressing Siglec 14 protein is transfused into an individual lacking Siglec 14 protein, Siglec 14 protein presented on the surface of leukocytes in the transfused blood is Recognized as a blood group antigen, antibodies against leukocytes may be produced.
  • cells expressing Siglec 14 protein have a stronger inflammatory cytokine (TNF- ⁇ ) response to bacterial lipopolysaccharide than cells not expressing Siglec 14 including cells expressing Siglec 5 protein. Is obtained. This result is considered to indicate that the siglec 14 protein regulates the production of inflammatory cytokines associated with infection of viruses, bacteria, fungi, protozoa, etc., and the susceptibility to infection by the polymorphism of the siglec 14 gene. Therefore, it is important to detect the polymorphism of the Siglec 14 gene in determining the treatment strategy for infectious diseases.
  • TNF- ⁇ inflammatory cytokine
  • the allele types based on the polymorphism of the Siglec 14 gene are homozygous ⁇ wild type> having only the Siglec 14 gene, heterozygous having Siglec 14 and Siglec 14/5 genes, and Siglec 14 There are three types of homozygous types that have only the / 5 fusion gene.
  • Both of the former express siglec 14 protein and siglec 5 protein (or siglec 14/5 fusion protein having the same amino acid sequence as siglec 5 protein), while the homozygous form of the latter siglec 14/5 fusion gene is: Since the Siglec 14/5 fusion protein having the same amino acid sequence as that of the Siglec 5 protein is expressed without expressing the Siglec 14 protein, for example, in the response to the above-mentioned hemocompatibility and infectious diseases, the former 2 and Siglec 14 are expressed. Therefore, it is only necessary to distinguish the homozygous type having only the / 5 fusion gene, and for this purpose, the presence or absence of the Siglec 14 gene or the presence or absence of the Siglec 14 protein may be detected.
  • the allelic detection reagent based on the polymorphism of the siglec 14 gene of the present invention detects the presence or absence of the siglec 14 gene or siglec 14 protein.
  • this detection means Uses a primer pair that specifically amplifies the Siglec 14 gene by PCR.
  • Each primer that specifically amplifies the Siglec 14 gene is designed based on the nucleotide sequence in the Siglec 14 gene, but it is necessary not to amplify the Siglec 14/5 fusion gene or the Siglec 5 gene.
  • a base sequence outside the conserved region (common sequence portion) of 14 genes and Siglec 5 gene (5 ′ side and 3 ′ side from the conserved region) is selected and designed.
  • the conserved region of the Siglec 14 gene is located at positions 753 to 2110 in the nucleotide sequence of SEQ ID NO: 1.
  • the forward primer of the primer pair for detection of the Siglec 14 gene is the region 5′-terminal from the 753rd and reverse.
  • the primer is designed by selecting a region 3 ′ end side from the 2110th position.
  • each primer is not particularly limited, but it is 15 to 40 bases, preferably 20 to 35 bases from the viewpoint of annealing temperature and annealing specificity.
  • More specific primers include those shown in SEQ ID NO: 7 as forward primers and those shown in SEQ ID NO: 8 as reverse primers.
  • the method for detecting the Siglec 14 gene using these primers is, for example, as follows.
  • Siglec 14 gene is determined by PCR using genomic DNA as a template.
  • genomic DNA is extracted and purified from a human-derived sample such as peripheral blood collected from a subject or a mouth-rubbed sample, for example.
  • the purification method may be a combination of proteinase digestion, phenol / chloroform extraction, ethanol precipitation, or a commercially available genomic DNA purification kit.
  • a PCR reaction is performed using the purified genomic DNA as a template.
  • a heat-resistant DNA polymerase, deoxyribonucleoside triphosphate mixture, a buffer suitable for PCR reaction, a genomic DNA serving as a template, and a primer pair that specifically amplifies the Siglec 14 gene are added to prepare a reaction solution To do.
  • a double-stranded DNA detection reagent eg SYBR Green or ethidium bromide
  • SYBR Green or ethidium bromide a double-stranded DNA detection reagent
  • a double-stranded DNA detection reagent such as SYBR Green or ethidium bromide
  • the genotype is determined to be a homozygous type of the Siglec 14/5 fusion gene when no reaction product specific to the Siglec 14 gene is detected from a specific human sample.
  • a probe specific for the Siglec 14 gene can be mentioned in the present invention.
  • the nucleic acid probe that specifically detects the Siglec 14 gene is designed based on the nucleotide sequence in the Siglec 14 gene, but it is necessary not to anneal to the Siglec 14/5 fusion gene or the Siglec 5 gene.
  • the conserved region (common sequence portion) of the Siglec 14 gene and the Siglec 5 gene is avoided, and the region specific to the Siglec 14 gene is on the 3 ′ side of the conserved region in the Siglec 14 gene, and 5 of the conserved region in the Siglec 5 gene. 'The side is. Therefore, a nucleic acid probe that specifically detects the Siglec 14 gene is designed by selecting such a base sequence.
  • the conserved region of the Siglec 14 gene is located at the 753rd to 2110th positions in the nucleotide sequence of SEQ ID NO: 1, and the nucleic acid probe for detecting the Siglec 14 gene is designed by selecting a region 3 'end side from the 2110th position.
  • the base length of the nucleic acid probe is not particularly limited, but is about 20 to 10,000 base pairs, preferably about 20 to 1000 base pairs, from the annealing temperature and the specificity of annealing.
  • nucleic acid probes include those shown in SEQ ID NOs: 15 to 17, but naturally, a nucleic acid probe having a sequence complementary to these may be used.
  • a method for detecting the Siglec 14 gene using such a nucleic acid probe is, for example, as follows.
  • the presence or absence of the Siglec 14 gene is determined by Southern blotting using human genomic DNA.
  • genomic DNA is extracted and purified from a human-derived sample such as peripheral blood collected from a subject or a mouth-rubbed sample, for example.
  • the purification method may be a combination of proteinase digestion, phenol / chloroform extraction, ethanol precipitation, or a commercially available genomic DNA purification kit.
  • the genomic DNA is directly immobilized on a thin film such as a nylon membrane, or is fragmented by an appropriate method (such as digestion with a restriction enzyme) and separated by agarose gel electrophoresis. Transfer and immobilize.
  • a nucleic acid probe that specifically hybridizes with the Siglec 14 gene is prepared, a label is introduced, and it is hybridized with the genomic DNA on the thin film.
  • the label used in this case uses a radioisotope (32P, 33P, 35S, etc.), a fluorescent dye (Cy3, Cy5, etc.), or an epitope such as biotin or digoxigenin.
  • a secondary detection reagent such as streptavidin or anti-digoxigenin antibody bound with a fluorescent dye
  • the hybridized nucleic acid probe is detected by an optimum method according to the label method.
  • a Siglec 14 gene-specific nucleic acid probe is solid-phased on a thin film or glass surface, and is fragmented by an appropriate method (such as mechanical shearing or digestion with a restriction enzyme) and hybridized with genomic DNA into which a label has been introduced.
  • the label used in this case uses a radioisotope (32P, 33P, 35S, etc.), a fluorescent dye (Cy3, Cy5, etc.), or an epitope such as biotin or digoxigenin.
  • a secondary detection reagent streptavidin or anti-digoxigenin antibody bound with a fluorescent dye
  • the hybridized genomic DNA is detected by an optimum method according to the label method.
  • the genotype is determined to be a homozygous type of the Siglec 14/5 fusion gene when a hybridization signal between the Siglec 14 gene-specific nucleic acid probe and genomic DNA cannot be detected from a specific human sample.
  • an antibody that specifically recognizes Siglec 14 protein can be mentioned.
  • the antibody may be a polyclonal antibody or a monoclonal antibody.
  • a polyclonal antibody uses a Siglec 14 protein to immunize animals such as mice, rats, rabbits, goats, sheep, donkeys, etc., and serum is collected after a lapse of a certain period of time, and protein A, protein G, antigen protein, It is obtained by purifying with a column or the like on which an antigen peptide is immobilized.
  • the monoclonal antibody is immunized in the same manner as described above, and after a certain period of time, for example, spleen cells are collected, the cells are fused with myeloma cells, the resulting hybridoma is screened, and the antibody against Siglec 14 protein is obtained. Collected from antibody-producing hybridoma.
  • an antibody that does not cross-react with other Siglec proteins including Siglec 5 protein is obtained by using a portion specific to Siglec 14 protein as an antigen used for immunization.
  • an antibody that does not cross-react with Siglec 5 protein is obtained by using a portion specific to Siglec 14 protein as an antigen used for immunization.
  • a hybridoma that cross-reacts with part or all of the Siglec 5 protein is prepared. Excluding by screening, an antibody specific for Siglec 14 protein is obtained.
  • Siglec 14 protein In order to detect Siglec 14 protein using such an antibody, the presence or absence of Siglec 14 protein is determined by analysis of leukocytes by flow cytometry.
  • Siglec 14 protein is detected, it is homozygous ⁇ wild type> having only Siglec 14 gene or heterozygous having Siglec 14 gene and Siglec 14/5 fusion gene, and Siglec 14 protein is not detected.
  • the homozygous type of the Siglec 14/5 fusion gene can be determined.
  • the blood of the subject is collected in a blood collection tube containing an anticoagulant (EDTA, citric acid, heparin, etc.) and used as a sample.
  • an anticoagulant EDTA, citric acid, heparin, etc.
  • White blood cells may be isolated from the blood by hemolysis of red blood cells using ammonium chloride or the like.
  • White blood cells may be isolated by the hemagglutination sedimentation method using hydroxyethyl starch or the like.
  • Anti-Siglec 14 antibody is added to a human leukocyte (or whole blood) sample, incubated for a certain period of time, and then the antibody that has not been bound by washing is removed.
  • the antibody may be an unlabeled one, or a fluorescent dye (a fluorescent dye that can be detected by a device such as fluorescein, R-phycoerythrin, or allophycocyanin) or a biotin-labeled one.
  • a secondary antibody that recognizes the animal's immunoglobulin used to produce this antibody (labeled with a fluorescent dye or biotin), an antibody labeled with biotin, etc. Is detected using a probe that specifically binds to this label (such as streptavidin labeled with a fluorescent dye) as a secondary reagent.
  • the antibody that did not bind was removed by washing, and the presence or absence of Siglec 14 protein on leukocytes was verified using a flow cytometer. At this time, it is possible to examine the presence or absence of Siglec 14 protein using any leukocyte fraction other than T lymphocytes, but considering the high number of cells and the high expression level of Siglec 14 protein, granulocytes Alternatively, it is desirable to examine the expression of Siglec 14 protein on monocytes.
  • All of the above-mentioned inspection means are homozygous type having only the Siglec 14 gene ⁇ wild type>, heterozygous type having the Siglec 14 gene and the Siglec 14/5 fusion gene, and homozygous type having only the Siglec fusion 14/5 gene.
  • the former two and the latter are identified. In the present invention, each of these three parties can be identified.
  • PCR is further performed using a primer pair for specific amplification of the Siglec 14/5 fusion gene.
  • the primer pair for Siglec 14/5 fusion gene amplification is designed based on the base sequence derived from the Siglec 14 gene and the base sequence portion derived from the Siglec 5 gene in the fusion gene. It is preferable to select and design a base sequence outside the conserved region (common sequence portion) in the Siglec 14 gene and Siglec 5 gene (5 ′ side and 3 ′ side from the conserved region).
  • the conserved region of the Siglec 14 gene is located at positions 753 to 2110 in the base sequence of SEQ ID NO: 1.
  • the forward primer of the primer pair for detection of the Siglec 14/5 fusion gene has a region 5′-terminal from the 753rd. Select and design. That is, the forward primer may be the same as the Siglec 14 gene detection forward primer.
  • the conserved region of the Siglec 5 fusion gene is located at positions 753 to 2114 in the base sequence of SEQ ID NO: 2 (of which the protein coding region is the 1001st to 19105th base sequence), and is used for detection of the Siglec 14/5 fusion gene
  • the reverse primer of the primer pair is designed by selecting a region 3 ′ end side from the 2114th position.
  • each primer may be the same as that of the above-mentioned Siglec 14 gene amplification primer, and the method for detecting the Siglec 14/5 fusion gene using these primers is the same as that of the above-mentioned Siglec 14 gene amplification primer. It's okay.
  • the siglec 14 gene or siglec 14 protein is detected using the siglec 14 gene amplification primer, the same gene detection probe, or the siglec 14 protein recognition antibody, is it homozygous for the siglec 14 gene? Alternatively, it is one of the heterojunction type of Siglec 14 and Siglec 14/5.
  • the Siglec 14/5 fusion gene is detected using the Siglec 14/5 fusion gene amplification primer for the sample thus determined, the heterozygous type of Siglec 14 and Siglec 14/5 is used. It can be determined that there is a homozygous type of Siglec 14 gene and a heterozygous type of Siglec 14 and Siglec 14/5.
  • the siglec 14/5 fusion gene is analyzed by analyzing the presence or absence of the siglec 14/5 fusion gene using this siglec 14/5 fusion gene amplification primer. It can be confirmed that it is a homozygous type, and the accuracy is increased.
  • Such a primer pair for amplifying Siglec 14/5 fusion gene is used in combination with the primer pair for amplifying Siglec 14 gene, the same probe or an antibody of Siglec 14 protein, and for detecting an allelic type based on the polymorphism of Siglec 14 gene. It can be used as a reagent kit. Further, the primer pair for amplifying the siglec 14/5 fusion gene can be used for detecting a polymorphism of the siglec 14 gene.
  • the copy number of the conserved region per diploid genome is 4 for wild type: heterozygote: homozygote. : 3: 2 may be used to determine the genotype.
  • genomic DNA derived from a human sample as a template
  • quantitative PCR was performed using a primer pair that amplifies the above conserved region, and the same amount of human genomic DNA of known genotype (wild type, heterozygous) Genotypes are determined by quantitative comparison of reaction products using a standard product as a quantitative standard.
  • the present invention relates to a reagent for detecting an antibody against Siglec 14 protein and a method for detecting an antibody against Siglec 14 protein using the same.
  • the antibody against Siglec 14 protein is preferably an alloantibody against Siglec 14 protein contained in a human blood sample, preferably a blood product.
  • a human blood sample preferably a blood product.
  • the frequency of containing alloantibodies against Siglec 14 is significantly higher in the former, and neutrophils Is stimulated with anti-Siglec 14 antibody-positive plasma, the neutrophils are activated.
  • the reagent for detecting an antibody against the Siglec 14 protein of the present invention includes Siglec 14 protein or Siglec 14 protein-expressing cells.
  • Siglec 14 is publicly known, and sequence information is disclosed in databases such as GenBank.
  • the sequence information of human Siglec 14 is registered in GenBank, for example, as accession number AAX47338, NP_001092082, and these can be used.
  • Siglec 14 protein having a known amino acid sequence registered in GenBank or the like can be used as Siglec 14 protein, but Siglec 14 protein having an amino acid sequence represented by SEQ ID NO: 4 is preferably used.
  • the sequence represented by SEQ ID NO: 4 includes an N-terminal V-set immunoglobulin-like region and a C2-set immunoglobulin-like region, a transmembrane region, and an intracellular region, which are extracellular regions.
  • the Siglec 14 protein in the present invention includes at least a region specific to Siglec 14 included in the extracellular region (in SEQ ID NO: 4, the first to 358th sequences correspond to the extracellular region portion). Just do it.
  • the “region specific to Siglec 14” is included only in the extracellular region of Siglec 14 protein when the amino acid sequence of the extracellular region of Siglec 14 protein is compared with the amino acid sequence of the extracellular region of Siglec 5 protein. Means amino acid sequence.
  • the Siglec 14 protein in the present invention includes a protein consisting of a sequence comprising at least the 238th to 358th sequences of SEQ ID NO: 4.
  • the siglec 14 protein in the present invention includes siglec 14 mutants.
  • Such mutants include both natural mutants and artificial mutants as long as they can be recognized and bound by the anti-Siglec 14 antibody.
  • SEQ ID NO: 4 or at least the 238th to 358th sequences of SEQ ID NO: 4 Or a protein having a deletion, substitution, addition or insertion of one to several amino acids and capable of being recognized and bound by the anti-Siglec 14 antibody, or the amino acid sequence and BLAST (BasicmentLocal Alignment) 80% or more, preferably 85 when calculated using Search Tool at the National Center) for (Biological Information (e.g., US National Biological Information Center basic local alignment search tool)) % Or more, more preferably 90% or more, eg 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity
  • the range of “1 to several” is not particularly limited. For example, 1 to
  • the Siglec 14 protein in the present invention can be produced by a general gene recombination technique. Briefly, the production of Siglec 14 protein involves preparing a DNA encoding the protein, constructing an expression vector containing the DNA, and prokaryotic or eukaryotic cells (such as, but not limited to, E Transforming or transfecting coli, yeast, SF9 cells, SF21 cells, COS1 cells, COS7 cells, CHO cells, HEK293 cells, etc.) and recovering the desired recombinant protein from the resulting cell culture .
  • prokaryotic or eukaryotic cells such as, but not limited to, E Transforming or transfecting coli, yeast, SF9 cells, SF21 cells, COS1 cells, COS7 cells, CHO cells, HEK293 cells, etc.
  • Protein purification is performed by appropriately combining general protein purification methods such as ammonium sulfate precipitation, organic solvent precipitation, dialysis, electrophoresis, chromatofocusing, gel filtration chromatography, ion exchange chromatography, affinity chromatography, and HPLC. It can be implemented.
  • the Siglec 14 protein in the present invention may be in the form of a fusion protein in which a labeled peptide is bound to the C-terminal or N-terminal of a polypeptide.
  • Representative labeled peptides include histidine repeats (His tag) of 6 to 10 residues, FLAG, myc peptide, GFP polypeptide, etc., but the labeled peptide is not limited to these.
  • the Siglec 14 protein in the present invention may be chemically modified as long as the anti-Siglec 14 antibody can be recognized and bound.
  • Chemical modifications include, but are not limited to, for example, glycosylation, PEGylation, acetylation, amidation, phosphorylation and the like.
  • the Siglec 14 protein in the present invention may be bound to a solid phase carrier.
  • the shape and material of the solid support are not particularly limited. Examples of the shape include particle shapes such as a plate shape, a film shape, a spherical shape, and an indefinite shape. Examples of the material include glass, ceramics, and various plastics.
  • Siglec 14 protein-expressing cells express Siglec 14 protein on their cell membranes.
  • the cells can be obtained by preparing DNA encoding Siglec 14 protein, constructing an expression vector containing this DNA, and transforming or transfecting host cells with the vector.
  • DNA having the base sequence represented by SEQ ID NO: 20 is preferably used as the DNA encoding Siglec 14 protein.
  • the base sequence represented by SEQ ID NO: 20 includes an extracellular region, a transmembrane region, and an intracellular region of Siglec 14 protein.
  • the DNA encoding Siglec 14 protein may contain at least a base sequence encoding a region specific to Siglec 14, and preferably contains at least a region specific to Siglec 14 and a transmembrane region. It only needs to contain the base sequence to be encoded.
  • the 712th to 1074th sequences correspond to a base sequence encoding a region specific to Siglec 14, and the 1075th to 1143th sequences correspond to a base sequence encoding a transmembrane region. That is, the DNA encoding the Siglec 14 protein in the present invention includes a base sequence including at least the 712th to 1074th and 1075th to 1143th sequences of SEQ ID NO: 20.
  • the DNA encoding Siglec 14 protein includes one to several nucleotides in DNA having the sequence shown in SEQ ID NO: 20, or at least the 712th to 1074th and 1075th to 1143th sequences of SEQ ID NO: 20. Also included are DNAs that encode proteins that have the following deletions, substitutions, additions or insertions and that can be recognized and bound by anti-Siglec 14 antibodies.
  • the range of “1 to several” is not particularly limited, but for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 1. Three, or one or two.
  • the DNA encoding Siglec 14 protein is composed of a sequence complementary to the DNA consisting of the sequence shown in SEQ ID NO: 20, or at least the 712th to 1074th and 1075th to 1143th sequences of SEQ ID NO: 20. Also included are DNAs that encode proteins that hybridize with DNA under stringent conditions and that can be recognized and bound by anti-Siglec 14 antibodies.
  • the stringent condition refers to a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • the sodium concentration is 10 mM to 300 mM, preferably 20 to 100 mM
  • the temperature Refers to conditions at 25 ° C. to 70 ° C., preferably 42 ° C. to 55 ° C.
  • DNA encoding Siglec 14 protein SEQ ID NO: 20, or at least the 712th to 1074th and 1075th to 1143th sequences of SEQ ID NO: 20, BLAST and the like (for example, default or default parameters) are used. Also included is a DNA encoding a protein comprising a base sequence having an identity of 80% or more, more preferably 90% or more, and most preferably 95% or more when calculated, and which can be recognized and bound by an anti-Siglec 14 antibody. It is.
  • the DNA encoding the Siglec 14 protein is inserted into a known expression vector.
  • a plasmid vector for example, an adenoviral vector, a retroviral vector, a lentiviral vector), a non-viral vector, and the like can be used, and a viral vector is preferable.
  • a viral vector for example, an adenoviral vector, a retroviral vector, a lentiviral vector, a non-viral vector, and the like
  • a viral vector is preferable.
  • the expression vector can contain several regulatory elements to express the Siglec 14 protein.
  • Control elements include, for example, promoters, enhancers, poly A addition signals, replication origins, selectable markers, ribosome binding sequences, terminators, and the like.
  • a mammalian cell preferably a human cell having low reactivity with blood, serum and / or plasma of a healthy person can be used, and K562 cell is particularly preferably used.
  • the reagent for detecting an antibody against the Siglec 14 protein of the present invention includes commonly used excipients, extenders, binders, infiltrant, Disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, preservatives, colorants, stabilizers, and the like may also be included.
  • the reagent for detecting an antibody against the Siglec 14 protein of the present invention it is possible to detect an anti-Siglec 14 antibody contained in the blood of a human blood sample, preferably a blood product for blood transfusion or a non-hemolytic blood transfusion patient. it can.
  • a general technique for detecting antibody binding can be used. For example, immunohistochemistry, Western blot, enzyme immunoassay (EIA) (enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometer (FCM) and the like.
  • EIA enzyme immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FCM flow cytometer
  • a reagent for detecting an antibody against the Siglec 14 protein of the present invention and a blood sample, preferably a blood product are incubated at 4 ° C. to 37 ° C. for 0.25 hour to overnight, preferably at 4 ° C. for 15 minutes.
  • Siglec 14 protein or Siglec 14 protein-expressing cells are reacted with a blood sample, preferably an anti-Siglec 14 antibody contained in a blood product.
  • a blood sample preferably an anti-Siglec 14 antibody contained in a blood product.
  • the Siglec 14 protein or the anti-Siglec 14 antibody bound to the Siglec 14 protein-expressing cell is detected by the above method.
  • Siglec 14 protein was expressed in a wide range of leukocyte subsets excluding T cells.
  • the expression pattern of Siglec 14 protein overlapped with that of Siglec 5 protein.
  • Anti-Siglec 14 antibody (rabbit polyclonal antibody) was prepared by the following method.
  • a siglec 14-Fc fusion protein comprising the extracellular region of human Siglec 14 protein (amino acid residues 1 to 342 of SEQ ID NO: 4), the FLAG sequence (DYKDDDDK) (SEQ ID NO: 18), and the hinge + Fc region of human immunoglobulin G
  • a vector to be expressed was prepared and introduced into 293 cells or CHO cells, and several milligrams of the desired fusion protein was obtained from the culture supernatant (Patent Document 4).
  • the Siglec 5-Fc fusion consisting of the extracellular region of human Siglec 5 protein (amino acid residues 1 to 334 or 1-427 of SEQ ID NO: 5), the FLAG sequence and the hinge + Fc region of human immunoglobulin G
  • a vector expressing the protein was prepared and introduced into 293 cells or CHO cells, and several milligrams of the desired fusion protein were obtained from the culture supernatant.
  • These Siglec 14-Fc fusion protein and Siglec 5-Fc fusion protein were each immobilized on AffiGel 15 (Bio-Rad) (1 mg / 0.5 ml) to prepare a carrier for affinity purification.
  • the hinge + Fc region of human immunoglobulin G was removed by incubation with protein A-Sepharose, and the partial protein consisting of the extracellular region of human Siglec 14 protein and the FLAG sequence Got. Rabbits were immunized with this partial protein according to a conventional method to obtain antiserum.
  • the carrier for affinity purification on which the Siglec 14-Fc fusion protein prepared above was immobilized and antiserum were mixed and incubated at 4 ° C. overnight.
  • a washing buffer Dulbecco PBS
  • the antibody bound to the carrier is eluted with an elution buffer (0.1 M glycine hydrochloride buffer, pH 3.0), and neutralized buffer (1 Neutralized with M Tris-HCl, pH 8.0).
  • the buffer of the obtained antibody fraction (an antibody recognizing the Siglec 14-Fc fusion protein) was replaced with Dulbecco's PBS by ultrafiltration, and then the Siglec 5-Fc fusion protein prepared above was immobilized for affinity purification.
  • Anti-Siglec 5 antibody (mouse monoclonal antibody) was prepared by the following method.
  • Siglec 5- consisting of human Siglec 5 protein signal peptide, linker region, immunoglobulin-like regions 3 and 4 (amino acid residues 1 to 19 + 234 to 427 of SEQ ID NO: 5), FLAG sequence and human immunoglobulin G hinge + Fc region
  • a vector expressing Fc fusion protein was prepared and introduced into 293 cells or CHO cells, and several milligrams of the desired fusion protein were obtained from the culture supernatant. After this siglec 5-Fc fusion protein is treated with enterokinase, the hinge + Fc region of human immunoglobulin G is removed by incubation with protein A-Sepharose, and the immunoglobulin-like regions 3 and 4 of human siglec 5 protein and the FLAG sequence are removed.
  • a partial protein was obtained.
  • several BALB / c mice were immunized with this partial protein, spleens were collected, and spleen cells and mouse-derived myeloma cells (P3U1 cell line) were fused using polyethylene glycol.
  • P3U1 cell line mouse-derived myeloma cells
  • the reactivity to Siglec 5 protein and Siglec 14 protein was examined, and a strain producing an antibody that recognizes only Siglec 5 protein was selected.
  • the cells were further cloned by the limiting dilution method to obtain a hybridoma clone (4H7) that produces an antibody that recognizes Siglec 5 protein but does not recognize Siglec 14 protein.
  • Leukocyte staining and analysis Tens of milliliters of peripheral blood was collected from a healthy donor with consent, and erythrocytes were hemolyzed using ACK hemolyzed blood (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM ethylenediaminetetraacetic acid). Leukocytes obtained by centrifugation from this solution are washed several times with a staining buffer (Dulbecco PBS containing 1% bovine serum albumin and 0.02% sodium azide), and then stained for human IgG (1 mg / ml). The suspension was suspended at 10 7 cells / ml in a buffer and incubated on an ice bath to block the Fc ⁇ receptor.
  • ACK hemolyzed blood 150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM ethylenediaminetetraacetic acid.
  • Leukocytes obtained by centrifugation from this solution are washed several times with
  • Cells were collected by centrifugation and further washed with a staining buffer, and then each fraction was divided into cell lineage marker antibodies (anti-CD4 antibody, anti-CD8 antibody, anti-CD19 antibody, anti-CD56 antibody, anti-CD14 antibody labeled with phycoerythrin) Anti-BDCA-1 antibody or anti-BDCA-2 antibody) was added and incubated on an ice bath. Cells were collected by centrifugation, further washed with a staining buffer, suspended in the staining buffer, and analyzed using a flow cytometer (FACSAria manufactured by BD Flowcytometry) equipped with 488 nm and 633 nm lasers.
  • FACSAria manufactured by BD Flowcytometry
  • Primer pair 14F + 5R amplifies the approximately 1.7 kb region of this gene when the Siglec 14/5 fusion gene is present.
  • An approximately 17 kb fragment can be generated from wild-type allele using the same primer pair, but this product does not occur under the reaction conditions used in this experiment.
  • FIG. 2D shows the results of genotyping using the same individual genomic DNA as FIG. 2A. As expected, donors 3 and 5 were homozygous for the Siglec 14/5 fusion gene.
  • Primer primers for genomic PCR were designed as follows. Primers for specifically amplifying the Siglec 14 gene and the Siglec 5 gene respectively are upstream (forward primer) and downstream (reverse primer) of a region showing a high degree of sequence homology between the Siglec 14 gene and the Siglec 5 gene. Must be set. The highly conserved regions are the 753rd to 2110th bases of SEQ ID NO: 1 for the Siglec 14 gene, and the 753rd to 2114th bases of SEQ ID NO: 2 for the Siglec 5 gene. In view of DNA amplification efficiency, it is desirable that the region to be amplified by the primer pair is short.
  • the forward primer is the complement of the nucleotide sequence from 588 to 610 of SEQ ID NO: 1
  • the reverse primer is the complement of the nucleotide from 2238 to 2258 of SEQ ID NO: 1. It was an array.
  • the forward primer was the nucleotide sequence of the 605th to 628th nucleotides of SEQ ID NO: 2
  • the reverse primer was the complementary sequence of the 2268th to 2288th nucleotides of the SEQ ID NO: 2.
  • the forward primer used for the amplification of the Siglec 14 gene and the reverse primer used for the amplification of the Siglec 5 gene were used.
  • Genomic DNA preparation About 10 ml of peripheral blood was collected from 5 healthy donors with consent, and erythrocytes were hemolyzed using ACK hemolyzed blood (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM ethylenediaminetetraacetic acid). White blood cells were obtained from this solution by centrifugation. Genomic DNA was purified from the obtained white blood cells (5 ⁇ 10 6 cells) using DNeasy Blood & Tissue Kit (QIAGEN).
  • PCR For the PCR reaction, a reaction solution (20 ⁇ l) having the following composition was used. That is, genomic DNA 100 ng, each primer 0.3 ⁇ M, deoxyribonucleoside triphosphate (dATP, dCTP, dGTP, dTTP) 0.2 mM each, thermostable DNA polymerase Expand High Fidelity enzyme (Roche Diagnostics) 0.5 unit, magnesium chloride 1.5 mM, 1 x Expand High Fidelity Buffer (Roche Diagnostics).
  • Enhanced expression of TNF- ⁇ by forced expression of Siglec 14 protein 1 Expression of Siglec 5 protein, Siglec 14 protein wild type or point mutant (R119A or R362A) in THP-1 cells, a human monocytic cell line, respectively After introducing the cDNA to be differentiated into macrophage-like cells together with empty vector-introduced cells, lipopolysaccharide was added and the amount of TNF- ⁇ produced was measured. The results are shown in FIG. 3A.
  • TNF- ⁇ production was increased about twice in cells expressing Siglec 14 protein compared to control cells.
  • TNF- ⁇ production was suppressed by suppressing expression of DAP12 protein in cells expressing wild-type Siglec 14 protein. That is, it is strongly suggested that the DAP12 protein is involved in the enhancement of TNF- ⁇ production via the Siglec 14 protein.
  • the same siRNA did not suppress TNF- ⁇ production from control cells. Control siRNA did not affect the amount of TNF- ⁇ produced.
  • siglec 14 protein and siglec 5 protein expression vectors The full-length cDNA of wild-type Siglec 14 was used as a template.
  • the siglec 14 R119A point mutant (119th arginine to alanine) cDNA was prepared by using the oligonucleotides of SEQ ID NOs: 11 and 12 and the Siglec 14 R362A point mutation.
  • the mutant (the 362nd arginine to alanine) cDNA was prepared using the oligonucleotides of SEQ ID NOs: 13 and 14, and the mutation was introduced according to the protocol attached to the QuickChange II (Stragtagene) product.
  • both cDNA and EGFP can be introduced by introducing the cDNA of the gene to be expressed upstream of IRES. Can be expressed from a single mRNA. Therefore, it is possible to identify and select cells that express the target gene based on the expression of EGFP.
  • THP-1 cells human monocytic cell line
  • RPMI 1640 medium + 10% fetal bovine serum + antibiotics penicillin and streptomycin
  • Introducing the above construct or vector pMSCV-IRES-EGFP into the retrovirus packaging cells PLAT-A cells (benefited by Prof. Toshio Kitamura, University of Tokyo) by lipofection and containing retroviral particles A culture supernatant was obtained.
  • Gene transfer into THP-1 cells was carried out by incubating with the culture supernatant containing the retroviral particles in the presence of RetroNectin (Takara Bio) for 24 hours.
  • EGFP positive cells were selected and collected using a fluorescent cell sorter. Sorting was repeated as necessary to establish a cell population in which 95% or more of the cells consisted of EGFP positive cells (that is, cells expressing the target protein).
  • THP-1 cell differentiation into macrophages LPS stimulation and measurement of TNF- ⁇ production
  • Transfected THP-1 cells were seeded at 2 x 10 4 cells in a 96-well plate, and 50 nM 12-O-tetradeca Macrophage-like cells were differentiated by culturing in 0.1 ml of a medium containing noylphorbol 13-acetate for 4 days. After washing several times with a normal medium, the cells were cultured for 24 hours in 0.1 ml of a medium containing 50 ng / ml of lipopolysaccharide derived from E. coli (O111: B4). TNF- ⁇ secreted into the medium was quantified using BD OptEIA Human TNF ELISA Set (BD Bioscience).
  • RNA interference Stealth RNA and control Stealth RNA targeting 5′-UAGAGCAACUGCAAUCGCUCUGGGC-3 ′ (SEQ ID NO: 19), a partial sequence of DAP12 mRNA, were purchased from Invitrogen.
  • THP-1 cells were induced to differentiate into macrophage-like cells, and then Stealth RNA (final concentration 100 nM) was introduced into the cells using Lipofectamine RNAi MAX and cultured for 48 hours. After washing several times with a normal medium, the cells were cultured for 24 hours in 0.1 ml of a medium containing 50 ng / ml of lipopolysaccharide derived from E. coli (O111: B4).
  • TNF- ⁇ secreted into the medium was quantified using BD OptEIA Human TNF ELISA Set (BD Bioscience).
  • the results are shown in Table 1.
  • the homozygous form of the Siglec 14/5 fusion gene was frequently found in Asian human populations such as Southeast Asia and China, and it was found that the results differed from one human population to another.
  • siglec 5 protein or Siglec 14 protein expressing cells Selection of siglec 5 and siglec 14 gene-introduced cells
  • 6 types of non-adherent cells, 5 types of adherent cells and 3 types of normal human serum (normal serum) Were measured with a flow cytometer and analyzed for the presence or absence of an increase in background signal.
  • L-cells, Hela cells, 293T cells, 3T3 cells, and CHO cells showed high reactivity with normal serum and increased background signal.
  • K562 cells five types of non-adherent cells (K562 cells, Jurkat cells, THP-1 cells, Namalwa cells, and CMK cells) and one type of adherent cells (Cos7 cells) are almost reactive to normal serum. Not shown, low background signal.
  • HLA Human Neutrophil Antigen
  • Siglec 5 and Siglec 14 is reacted with anti-HLA antibody, anti-HNA antibody, anti-Siglec 5 antibody or anti-Siglec 14 antibody, respectively.
  • K562 cells were employed as a cell line for introducing Siglec 5 or Siglec 14 gene.
  • Anti-Siglec 14 alloantibodies contained in human serum or plasma were detected as the KY-Siglec 5 cell line and KY-Siglec. Detection was performed in 14 cell lines. Specifically, plasma derived from a non-hemolytic transfusion-causing drug (obtained from the Japanese Red Cross) was added to KY-Siglec 5 cells, KY-Siglec 14 cells, and KY-mock cells, and reacted at 4 ° C. for 15 minutes.
  • the detection ratios of anti-Siglec 14 alloantibodies were compared between plasma (168 types) and non-hemolytic transfusion-causing drug-derived plasma (500 types).
  • the positive rate of anti-Siglec 14 alloantibody was 2.4% (12/500) in the serum of healthy volunteers, whereas it was significantly higher at 11.9% (20/168) in the non-hemolytic transfusion-causing agent (p ⁇ 0.0001, Fisher test).
  • Mac-1 antigen which is a neutrophil activation marker
  • FCM flow cytometer
  • HBP Heparin Binding Protein
  • Mac-1 antigen was increased on the surface of neutrophils stimulated with fMLP (positive control).
  • fMLP neutrophils stimulated with anti-Siglec 14 antibody-positive plasma
  • the expression level of Mac-1 antigen was significantly increased compared to when stimulated with anti-Siglec 14 antibody-negative plasma ( FIG. 7).
  • HBP concentration in supernatant by ELISA was measured using the ELISA method of Tapper et al. (Blood 2002; 99: 1785-93.). 50 ⁇ l of an anti-HBP monoclonal antibody (R & D, 50 ⁇ g / mL) was added to an ELISA microplate (Nunc, C8 Maxisorp) and incubated overnight at 4 ° C. to adsorb the antibody.
  • T-PBS-BSA 0.5% Tween20, 1% BSA added to PBS solution
  • 1/6 diluted supernatant above a
  • various Recombinant human HBP R & D
  • Can Get Signal I solution Toyobo
  • T-PBS 0.5% Tween20 added to PBS solution
  • anti-HBP polyclonal antibody R & D, 50 ⁇ g / mL, 50 ⁇ L / well
  • the allelic detection reagent based on the polymorphism of the Siglec 14 gene of the present invention By using the allelic detection reagent based on the polymorphism of the Siglec 14 gene of the present invention, the allelic type having the Siglec 14 gene and the homozygous type of the Siglec 14/5 fusion gene not having the Siglec 14 gene are distinguished. It can be detected and can be used to eliminate risks such as blood transfusion side effects in advance or to determine a treatment policy such as infection.
  • anti-Siglec 14 antibody in a human blood sample can be detected, in order to prevent non-hemolytic transfusion side effects and ensure the safety of transfusion. Can be used.

Abstract

SIGLEC14 protein is expressed in some persons but is not expressed in some persons.  Persons in which SIGLEC14 protein is not expressed have a SIGLEC14/5 fusion gene.  SIGLEC14 gene has polymorphisms. Thus, disclosed is a reagent for detecting a polymorphism of SIGLEC14 gene, which comprises a primer pair capable of amplifying SIGLEC14 gene specifically, a probe capable of hybridizing with SIGLEC14 gene specifically or an antibody capable of recognizing specifically SIGLEC14 protein depicted in SEQ ID NO:4.  Also disclosed is a reagent for detecting an anti-SIGLEC14 antibody, which is characterized by comprising a protein having the amino acid sequence depicted in SEQ ID NO:4 or a cell capable of expressing a protein having the amino acid sequence depicted in SEQ ID NO:4.

Description

シグレック１４遺伝子の多型に基づく対立遺伝子型の検出用試薬Reagent for detection of allelic type based on polymorphism of siglec 14 gene
　本発明は、シグレック１４（SIGLEC14）遺伝子多型に基づく対立遺伝子型の検出試薬及び該検出方法に関する。本発明はまた、抗シグレック１４抗体を検出するため試薬、及びそれを用いたヒト血液試料中の抗シグレック１４抗体の検出方法に関する。 The present invention relates to an allelic detection reagent based on the Siglec 14 gene polymorphism and the detection method. The present invention also relates to a reagent for detecting anti-Siglec 14 antibody and a method for detecting anti-Siglec 14 antibody in a human blood sample using the same.
　シグレックファミリータンパク質は免疫グロブリンスーパーファミリーに属するI型膜貫通タンパク質の一群であり、シアル酸を含む各種糖鎖構造を特異的に認識するレクチン活性を有する（非特許文献１）。シグレックファミリーに属する分子はそのほとんどが細胞外領域でシアル酸を含む糖鎖と結合すること、また細胞内領域でシグナル伝達分子と結合してシグナル伝達に関与することが知られている。 Siglec family proteins are a group of type I transmembrane proteins belonging to the immunoglobulin superfamily, and have lectin activity that specifically recognizes various sugar chain structures including sialic acid (Non-patent Document 1). Most of the molecules belonging to the Siglec family are known to bind to sugar chains containing sialic acid in the extracellular region, and to participate in signal transduction by binding to signal transduction molecules in the intracellular region.
　一方、細胞の正常分化、癌性変化や免疫疾患などに伴って細胞表面の糖鎖構造が変化することが知られており、これらの変化を検出するものには、糖鎖を認識する抗体がよく用いられている（特許文献１）。また、このような抗体以外の、ヒト細胞或いは組織に発現する糖鎖構造を特異的に認識する蛋白質も、ガン細胞等の検出や抗体医療に類するドラッグデリバリーに有用であると考えられている（特許文献２、特許文献３）。一方、例えば、シグレック２タンパク質（別名ＣＤ２２）、シグレック３タンパク質（別名ＣＤ３３）等のシグレックファミリーに属する蛋白質は、抗体医療の標的として有用であることが知られている（非特許文献２、非特許文献３）。また、シグレック５タンパク質に関しても、骨髄性白血病細胞の検出に有用であることが報告されている（非特許文献４）。 On the other hand, it is known that the sugar chain structure on the cell surface changes with normal differentiation of cells, cancerous changes, immune diseases, etc., and those that detect these changes include antibodies that recognize sugar chains It is often used (Patent Document 1). In addition to such antibodies, proteins that specifically recognize sugar chain structures expressed in human cells or tissues are also considered useful for drug delivery such as detection of cancer cells and antibody medicine ( Patent Document 2 and Patent Document 3). On the other hand, for example, proteins belonging to the Siglec family such as Siglec 2 protein (also known as CD22) and Siglec 3 protein (also known as CD33) are known to be useful as targets for antibody medicine (Non-Patent Document 2, Non-patent Document 2, Patent Document 3). Also, Siglec 5 protein has been reported to be useful for detection of myeloid leukemia cells (Non-patent Document 4).
　本発明者は、このような状況下、シグレックファミリータンパク質としてシグレック１４タンパク質及びその遺伝子を見いだしており、シグレック１４タンパク質は上記従来のシグレックファミリータンパク質にみられる免疫受容体チロシン含有抑制モチーフ（ITIM）を有せず、また、良好な糖鎖認識能を有すること、及びこのシグレック１４タンパク質は、シグレック５タンパク質と部分的に９９％以上の相同性を示すが、これら遺伝子の染色体上の所在及び糖鎖に対する活性が異なり別タンパク質であるとの知見を得ている（特許文献４）。 Under such circumstances, the present inventor has found Siglec 14 protein and its gene as Siglec family protein, and Siglec 14 protein is an immunoreceptor tyrosine-containing suppression motif (ITIM) found in the conventional Siglec family protein. ) And good sugar chain recognition ability, and this Siglec 14 protein partially shows 99% or more homology with Siglec 5 protein, but the location of these genes on the chromosome and The knowledge that the activity with respect to a sugar chain differs and it is another protein has been acquired (patent document 4).
　しかし、このシグレック１４タンパク質については、その発現状況と生体に対する影響或いは類似するシグレック５タンパク質との関係等についての詳細は未だ明らかにはなっていない。 However, details of the expression status and the influence on the living body or the relationship with the similar Siglec 5 protein have not yet been clarified.
　非溶血性輸血副作用は、輸血療法による副作用であって、溶血性副作用および感染性副作用ならびに輸血後GVHDなどを除いた副作用に総称される。その原因は、抗血漿成分抗体、抗白血球抗体、血液製剤中の炎症性サイトカインなどが想定されているが、未だ十分には解明されていない。非溶血性輸血副作用の頻度は、医療機関から日本赤十字社に報告される輸血副作用報告（年間約2,000例）のうち約90％を占め、軽微な副作用症例は報告されないことから、実際の症例数はこれを大幅に上回ると予想される。非溶血性輸血副作用の症例は、蕁麻疹や発熱などの軽症例が大部分を占めるが、重篤なアナフィラキシー反応、輸血関連急性肺障害（TRALI）による死亡例も報告されている（非特許文献５、６）。当該分野においては、非溶血性輸血副作用の原因特定と、非溶血性輸血副作用を防止し輸血の安全性を確保する方法が望まれている。 Non-hemolytic transfusion side effects are side effects due to transfusion therapy, and are collectively referred to as side effects excluding hemolytic and infectious side effects and post-transfusion GVHD. The causes are assumed to be anti-plasma component antibodies, anti-leukocyte antibodies, inflammatory cytokines in blood products, etc., but have not been fully elucidated. The frequency of non-hemolytic transfusion side effects accounts for about 90% of the transfusion side effect reports (approximately 2,000 cases per year) reported by the medical institution to the Japanese Red Cross Society, and minor side effects cases are not reported. Is expected to greatly exceed this. Most cases of non-hemolytic transfusion side effects are mild cases such as urticaria and fever, but deaths due to severe anaphylactic reactions and transfusion-related acute lung injury (TRALI) have also been reported (Non-patent literature) 5, 6). In this field, a method for identifying the cause of non-hemolytic transfusion side effects and preventing non-hemolytic transfusion side effects and ensuring the safety of transfusion is desired.
特開平８-６７７００号公報JP-A-8-67700 特開２００４-２４４４１１号公報JP 2004-244411 A 特開２００３-２４６７９９号公報JP 2003-246799 A 特開２００６-３４５７８６号公報JP 2006-345786 A
　本発明の課題は、シグレック１４タンパク質が個人においてその発現状況に違いがあるか否か、或いは類似するシグレック５タンパク質とその機能において、或いはその遺伝子レベルで何らかの関係があるのか否か等について調べることにより、シグレック１４タンパク質及び遺伝子に関連する新たな知見を得て、この知見に基づき医学的に有用な手段を提供する点にある。 The object of the present invention is to investigate whether there is a difference in the expression status of Siglec 14 protein in individuals, or whether there is any relationship between similar Siglec 5 protein and its function, or at the gene level. Thus, new knowledge related to Siglec 14 protein and gene is obtained, and medically useful means are provided based on this knowledge.
　本発明の課題はまた、非溶血性輸血副作用に関与する血液製剤の特定方法、および非溶血性輸血副作用を防止し輸血の安全性を確保するための方法を提供する点にある。 The object of the present invention is also to provide a method for identifying a blood product involved in non-hemolytic transfusion side effects and a method for preventing non-hemolytic transfusion side effects and ensuring the safety of transfusion.
　本発明者は上記課題を解決すべく鋭意研究した結果、シグレック１４タンパク質は個人によって発現している者と発現していない者が存在し、シグレック１４タンパク質を発現していない者は、シグレック１４／５融合遺伝子を有し、シグレック１４遺伝子には多型が存在することを見いだした。そして、この融合遺伝子のホモ接合型を有する者はシグレック１４／５融合タンパク質（シグレック５タンパク質と同じアミノ酸配列を有する）を発現するが、シグレック１４タンパク質を発現しておらず、この個人差によって産生されるシグレック１４に対する抗体が輸血副作用の一因になりうるとの知見を得るとともに、シグレック１４タンパク質を発現する細胞とシグレック１４タンパク質を発現しない細胞とではTNF-αの産生量において顕著な差違を有するとの知見も得た。 As a result of intensive studies to solve the above problems, the present inventors have found that there are individuals who do not express the Siglec 14 protein and those who do not express the Siglec 14 protein. We have 5 fusion genes and found that polymorphism exists in Siglec 14 gene. Those who have a homozygous form of this fusion gene express the Siglec 14/5 fusion protein (having the same amino acid sequence as the Siglec 5 protein), but do not express the Siglec 14 protein and are produced according to individual differences. Obtained that the antibody against Siglec 14 can contribute to transfusion side effects, and there is a significant difference in the amount of TNF-α produced between cells expressing Siglec 14 protein and cells not expressing Siglec 14 protein. The knowledge that it has was also obtained.
　したがって、このシグレック１４遺伝子の多型或いはこれに基づく対立遺伝子型を検出することは、上記輸血副作用の防止或いは感染症に対する治療方針の決定において極めて重要であることを見出した。 Therefore, it has been found that detecting this polymorphism of the Siglec 14 gene or an allelic form based on this polymorphism is extremely important in preventing the above-mentioned transfusion side effects or determining a treatment policy for infectious diseases.
　また、本発明者は、非溶血性輸血副作用に関与した血液と一般献血を比較すると、シグレック１４に対するアロ抗体を含む頻度が前者において有意に高い事、また好中球を抗シグレック１４抗体で刺激すると、好中球が活性化される事を見出した。これらの事実は、抗シグレック１４アロ抗体が非溶血性輸血副作用に関与する可能性を強く示唆し、抗シグレック１４アロ抗体を検出する事は、非溶血性輸血副作用を防止し輸血の安全性を確保する上で極めて重要であることを見出した。 In addition, when comparing the blood involved in non-hemolytic transfusion side effects and general blood donation, the present inventor shows that the frequency of alloantibodies against Siglec 14 is significantly higher in the former, and that neutrophils are stimulated with anti-Siglec 14 antibodies Then, it discovered that a neutrophil was activated. These facts strongly suggest that anti-Siglec 14 alloantibodies may be involved in non-hemolytic transfusion side effects, and detection of anti-Siglec 14 allo-antibodies prevents non-hemolytic transfusion side effects and improves transfusion safety. It was found to be extremely important in securing.
　本発明者はこれらの知見を基に本発明を完成するに至った。 The inventors have completed the present invention based on these findings.
　すなわち本発明は以下のとおりである。 That is, the present invention is as follows.
　（１）配列番号１に示されるシグレック１４遺伝子を特異的に増幅するプライマー対、該シグレック１４遺伝子に特異的にハイブリダイズするプローブ又は配列番号４に示されるシグレック１４タンパク質を特異的に認識する抗体を含有することを特徴とする、シグレック１４遺伝子の多型に基づく対立遺伝子型の検出用試薬。 (1) A primer pair that specifically amplifies the Siglec 14 gene represented by SEQ ID NO: 1, a probe that specifically hybridizes to the Siglec 14 gene, or an antibody that specifically recognizes the Siglec 14 protein represented by SEQ ID NO: 4. A reagent for detecting an allelic form based on a polymorphism of the Siglec 14 gene, comprising:
　（２）シグレック１４遺伝子を特異的に増幅するプライマー対が、シグレック１４遺伝子とシグレック５遺伝子の塩基配列における保存領域の外側領域の塩基配列に対応する配列を有することを特徴とする、上記（１）に記載の検出用試薬。 (2) The primer pair that specifically amplifies the Siglec 14 gene has a sequence corresponding to the base sequence of the region outside the conserved region in the base sequence of the Siglec 14 gene and the Siglec 5 gene (1) The reagent for detection as described in).
　（３）シグレック１４遺伝子を特異的に増幅するプライマー対が、配列番号７及び配列番号８で示される塩基配列を有するものであることを特徴とする、上記（２）に記載の検出用試薬。 (3) The detection reagent according to (2) above, wherein the primer pair that specifically amplifies the Siglec 14 gene has a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8.
　（４）上記（１）～（３）のいずれかに記載の検出用試薬と、配列番号３で示されるシグレック１４／５融合遺伝子を特異的に増幅するプライマー対とを組み合わせてなることを特徴とする、シグレック１４遺伝子の多型に基づく対立遺伝子型の検出用試薬キット。 (4) The detection reagent according to any one of (1) to (3) above and a primer pair that specifically amplifies the Siglec 14/5 fusion gene represented by SEQ ID NO: 3 A reagent kit for detecting an allelic type based on a polymorphism of the Siglec 14 gene.
　（５）シグレック１４／５融合遺伝子を特異的に増幅するプライマー対が、シグレック１４遺伝子とシグレック５遺伝子の塩基配列における保存領域の外側領域の塩基配列に対応する配列を有することを特徴とする、上記（４）に記載の検出用試薬キット。 (5) The primer pair that specifically amplifies the Siglec 14/5 fusion gene has a sequence corresponding to the base sequence outside the conserved region in the base sequence of the Siglec 14 gene and the Siglec 5 gene, The reagent kit for detection according to (4) above.
　（６）シグレック１４／５融合遺伝子を特異的に増幅するプライマー対が配列番号７及び配列番号１０で示される塩基配列を有するものであることを特徴とする、上記（５）に記載の検出用試薬キット。 (6) The detection pair according to (5) above, wherein the primer pair that specifically amplifies the Siglec 14/5 fusion gene has a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 10. Reagent kit.
　（７）血液適合性検査薬として使用することを特徴とする、上記（１）～（３）のいずれかに記載の検出用試薬。 (7) The detection reagent according to any one of (1) to (3) above, which is used as a blood compatibility test agent.
　（８）血液適合性検査薬として使用することを特徴とする、上記（４）～（６）のいずれかに記載の検出用試薬キット。 (8) The detection reagent kit according to any one of (4) to (6) above, which is used as a blood compatibility test agent.
　（９）感染症の治療方針の決定のために使用することを特徴とする、上記（７）に記載の検出用試薬。 (9) The detection reagent according to (7) above, which is used for determining a treatment policy for an infectious disease.
　（１０）感染症の治療方針の決定のために使用することを特徴とする、上記（８）に記載の検出用試薬キット。 (10) The reagent kit for detection according to (8) above, which is used for determining a treatment policy for an infectious disease.
　（１１）配列番号３に示されるシグレック１４／５融合遺伝子。 (11) Siglec 14/5 fusion gene shown in SEQ ID NO: 3.
　（１２）上記（１１）に記載のシグレック１４／５融合遺伝子を特異的に増幅するプライマー対。 (12) A primer pair that specifically amplifies the Siglec 14/5 fusion gene according to (11) above.
　（１３）シグレック１４遺伝子とシグレック５遺伝子の塩基配列における保存領域の外側領域の塩基配列に対応する配列を有することを特徴とする、上記（１２）に記載のプライマー対。 (13) The primer pair according to (12) above, which has a sequence corresponding to a base sequence outside the conserved region in the base sequences of the Siglec 14 gene and the Siglec 5 gene.
　（１４）配列番号７及び配列番号１０で示される塩基配列を有する、上記（１１）に記載のプライマー対。 (14) The primer pair according to (11) above, which has the base sequences represented by SEQ ID NO: 7 and SEQ ID NO: 10.
　（１５）ヒトゲノムＤＮＡを鋳型とし、上記（１）～（３）のいずれかに記載のシグレック１４遺伝子を特異的に増幅するプライマー対或いは上記（４）又は（５）に記載の検出用試薬キットを用いてＰＣＲを行い、シグレック１４遺伝子の存在の有無或いはさらにシグレック１４／５遺伝子の存在の有無を検出することを特徴とする、シグレック１４遺伝子の多型に基づく対立遺伝子型の検出方法。 (15) A primer pair for specifically amplifying the Siglec 14 gene according to any one of (1) to (3) above using human genomic DNA as a template, or the detection reagent kit according to (4) or (5) above A method for detecting an allelic type based on a polymorphism of the siglec 14 gene, wherein PCR is performed to detect the presence or absence of the siglec 14 gene or further the presence or absence of the siglec 14/5 gene.
　（１６）上記（１）に記載のシグレック１４遺伝子に特異的にハイブリダイズするプローブを、ヒトゲノムＤＮＡに対しハイブリダイズさせ、シグレック１４遺伝子の存在の有無を検出することを特徴とする、シグレック１４遺伝子の多型に基づく対立遺伝子型の検出方法。 (16) The Siglec 14 gene, characterized in that a probe that specifically hybridizes to the Siglec 14 gene according to (1) is hybridized to human genomic DNA, and the presence or absence of the Siglec 14 gene is detected. Allele type detection method based on polymorphism of
　（１７）上記（１）に記載のシグレック１４タンパク質を特異的に認識する抗体を用いて、ヒト血液試料中のシグレック１４タンパク質を検出することを特徴とする、シグレック１４遺伝子の多型に基づく対立遺伝子型の検出方法。 (17) An allele based on a polymorphism of the Siglec 14 gene, wherein the antibody specifically recognizes the Siglec 14 protein described in (1) above is used to detect the Siglec 14 protein in a human blood sample. Genotype detection method.
　（１８）配列番号４の少なくとも238番目から358番目の配列を含むアミノ酸配列を有するタンパク質、又は配列番号４の少なくとも238番目から358番目の配列を含むアミノ酸配列を有するタンパク質を発現する細胞を含有することを特徴とする、抗シグレック１４抗体の検出用試薬。 (18) A cell that expresses a protein having an amino acid sequence containing at least the 238th to 358th sequence of SEQ ID NO: 4 or a protein having an amino acid sequence containing at least the 238th to 358th sequence of SEQ ID NO: 4 A reagent for detecting an anti-Siglec 14 antibody, characterized in that
　（１９）上記（１８）に記載の抗シグレック１４抗体の検出用試薬を用いた、ヒト血液試料中の抗シグレック１４抗体の検出方法。 (19) A method for detecting an anti-Siglec 14 antibody in a human blood sample using the reagent for detecting an anti-Siglec 14 antibody described in (18) above.
　（２０）上記（１８）に記載の抗シグレック１４抗体の検出用試薬を用いた、抗シグレック１４抗体を含む血液製剤の特定方法。 (20) A method for identifying a blood product containing an anti-Siglec 14 antibody using the reagent for detecting an anti-Siglec 14 antibody according to (18) above.
　本発明は、シグレック１４遺伝子に多型が存在することを初めて見いだしたことに基づく。この知見に基づき作製された本発明の試薬は、シグレック１４遺伝子を有する対立遺伝子型とシグレック１４遺伝子を有しないシグレック１４／５融合遺伝子のホモ接合型とを識別して検出することができる。この様な対立遺伝子型の検出結果は、輸血副作用等の危険性を事前に排除し、或いは感染症などの治療方針を決定するために利用することができる。 The present invention is based on the first discovery that a polymorphism exists in the Siglec 14 gene. The reagent of the present invention produced based on this finding can distinguish and detect an allelic type having the Siglec 14 gene and a homozygous type of the Siglec 14/5 fusion gene not having the Siglec 14 gene. Such allele-type detection results can be used to eliminate risks such as blood transfusion side effects in advance or to determine a treatment policy such as an infectious disease.
　また本発明のシグレック１４タンパク質又はシグレック１４タンパク質発現細胞を含む抗シグレック１４抗体の検出用試薬を用いることによって、ヒト血液試料中の抗シグレック１４抗体を検出することが可能である。この様な抗シグレック１４抗体の検出結果は、非溶血性輸血副作用を防止し輸血の安全性を確保するために利用することができる。 Further, by using the detection reagent for anti-Siglec 14 antibody containing Siglec 14 protein or Siglec 14 protein-expressing cell of the present invention, it is possible to detect anti-Siglec 14 antibody in a human blood sample. Such detection results of the anti-Siglec 14 antibody can be used to prevent non-hemolytic transfusion side effects and to ensure the safety of transfusion.
　本明細書は本願の優先権の基礎である日本国特許出願2008-179791号、2009-134590号の明細書および／または図面に記載される内容を包含する。 This specification includes the contents described in the specification and / or drawings of Japanese Patent Application Nos. 2008-179791 and 2009-134590 which are the basis of the priority of the present application.
図１は、シグレック１４タンパク質特異的な抗体を用いた白血球のフローサイトメトリーによる解析の結果を示す図である。FIG. 1 is a graph showing the results of leukocyte flow cytometry analysis using a Siglec 14 protein-specific antibody. 図２Ａは、シグレック１４タンパク質発現及びシグレック５タンパク質発現における個人差のフローサイトメトリーによる多型解析結果を示す図である。FIG. 2A is a diagram showing the results of polymorphism analysis by flow cytometry of individual differences in Siglec 14 protein expression and Siglec 5 protein expression. 図２Ｂは、シグレック１４遺伝子及びその多型に関するゲノムＰＣＲ解析の概略図を示す図である。図中の矢頭は各プライマーの位置と向きを示す。FIG. 2B is a diagram showing a schematic diagram of genomic PCR analysis concerning the Siglec 14 gene and its polymorphism. Arrowheads in the figure indicate the position and orientation of each primer. 図２Ｃは、シグレック１４遺伝子及びその多型に関するゲノムＰＣＲ解析の結果を示す模式図である。FIG. 2C is a schematic diagram showing the results of genomic PCR analysis on the Siglec 14 gene and its polymorphism. 図２Ｄは、シグレック１４遺伝子及びその多型に関するゲノムＰＣＲ解析の結果を示す図である。FIG. 2D is a diagram showing the results of genomic PCR analysis for the Siglec 14 gene and its polymorphism. 図３Ａは、シグレック１４タンパク質の強制発現とTNF-αの発現量の関係を解析した結果を示す図である。FIG. 3A is a diagram showing the results of analyzing the relationship between forced expression of Siglec 14 protein and the expression level of TNF-α. 図３Ｂは、シグレック１４タンパク質の強制発現とDAP12タンパク質の発現を抑制するsiRNAの導入によるTNF-αの発現量の解析結果を示す図である。FIG. 3B is a diagram showing the analysis results of the expression level of TNF-α by introduction of siRNA that suppresses forced expression of Siglec 14 protein and expression of DAP12 protein. 図４は、シグレック１４発現細胞株を用いた抗シグレック１４アロ抗体の検出方法を示す模式図である。FIG. 4 is a schematic diagram showing a method for detecting an anti-Siglec 14 alloantibody using a Siglec 14-expressing cell line. 図５は、シグレック５又はシグレック１４発現細胞株における導入遺伝子の発現解析結果を示す図である。FIG. 5 shows the results of transgene expression analysis in a siglec 5 or siglec 14-expressing cell line. 図６は、シグレック５又はシグレック１４発現細胞株を用いた非溶血性輸血副作用の原因製剤中の抗シグレック１４アロ抗体の検出結果を示す図である。FIG. 6 is a view showing the detection results of anti-Siglec 14 alloantibodies in a preparation causing non-hemolytic transfusion side effects using Siglec 5 or Siglec 14-expressing cell lines. 図７は、抗シグレック１４抗体陽性血清を用いて刺激した好中球におけるMac-1抗原発現量の解析結果を示す図である。FIG. 7 is a diagram showing the analysis results of the expression level of Mac-1 antigen in neutrophils stimulated with anti-Siglec 14 antibody-positive serum. 図８は、抗シグレック１４抗体陽性血清を用いて刺激した好中球によるHBP放出量の解析結果を示す図である。FIG. 8 is a diagram showing the analysis result of the amount of HBP released by neutrophils stimulated with anti-Siglec 14 antibody-positive serum.
　本発明は、シグレック１４遺伝子の多型に基づく対立遺伝子型を検出するための試薬、及びこれを使用した同対立遺伝子型の検出方法に関する。 The present invention relates to a reagent for detecting an allelic type based on a polymorphism of the Siglec 14 gene, and a method for detecting the allelic type using the same.
　従来、シグレック５（SIGLEC5）遺伝子とシグレック１４（SIGLEC14）遺伝子の存在は知られていたが、その両者の融合遺伝子であるシグレック１４／５遺伝子（SIGLEC14/5）の存在は知られていなかった。本発明においてはじめて見いだされたこの融合遺伝子は、シグレック１４（SIGLEC14）タンパク質の特異的アミノ酸配列部分をコードする領域を欠損しており、その産物のアミノ酸配列はシグレック５（SIGLEC5）タンパク質と同一である。これらのシグレック１４タンパク質のアミノ酸配列及びシグレック１４遺伝子の塩基配列を、配列表の配列番号４及び１（うちタンパク質をコードする領域は塩基配列中１００１番目～４４０５番目）にそれぞれ示し、また、シグレック１４／５融合遺伝子の産物（シグレック５タンパク質と同じ）のアミノ酸配列、及び該融合遺伝子の塩基配列を、同配列番号６及び配列番号３（うちタンパク質をコードする領域は塩基配列中１００１番目～１９１０５番目）に示す。 Previously, the existence of the Siglec 5 (SIGLEC5) gene and the Siglec 14 (SIGLEC14) gene was known, but the existence of the Siglec 14/5 gene (SIGLEC14 / 5), which was a fusion of both, was not known. This fusion gene found for the first time in the present invention lacks a region encoding a specific amino acid sequence portion of Siglec 14 (SIGLEC14) protein, and the amino acid sequence of the product is identical to Siglec 5 (SIGLEC5) protein. . The amino acid sequences of these Siglec 14 proteins and the base sequences of the Siglec 14 gene are shown in SEQ ID Nos. 4 and 1 (of which the region encoding the protein is the 1001st to 4405th in the base sequence), respectively. / 5 The amino acid sequence of the fusion gene product (same as Siglec 5 protein) and the base sequence of the fusion gene are the same as SEQ ID NO: 6 and SEQ ID NO: 3 (of which 1001 to 19105 are the regions encoding the protein) ).
　本発明者が得た知見によれば、シグレック１４タンパク質の発現状況を個人毎に見れば、シグレック１４タンパク質を発現している者と発現しない者が存在し、シグレック１４遺伝子を有する者はシグレック１４タンパク質及びシグレック５タンパク質を発現するが、上記シグレック１４／５融合遺伝子のホモ接合型の対立遺伝子型を有する者は、シグレック１４タンパク質を欠損し、シグレック１４／５融合タンパク質（シグレック５タンパク質と同じアミノ酸配列を有する）を発現する。また、シグレック１４／５融合遺伝子を有するが、シグレック１４遺伝子とのヘテロ接合型の対立遺伝子型を有する者はシグレック１４タンパク質とシグレック５タンパク質及びシグレック１４／５融合タンパク質（シグレック５タンパク質と同じアミノ酸配列を有する）を発現する。このシグレック１４／５融合遺伝子のホモ接合型の対立遺伝子型を有する者の割合は世界において地域毎に異なるという興味深い結果も得られている（実施例４表１参照）。 According to the knowledge obtained by the present inventor, when the expression status of the Siglec 14 protein is seen for each individual, there are those who express the Siglec 14 protein and those who do not express it, and those who have the Siglec 14 gene are Siglec 14 Those who express protein and Siglec 5 protein but have homozygous allelic form of the Siglec 14/5 fusion gene lack Siglec 14 protein and have the same amino acid as Siglec 14/5 fusion protein. Having a sequence). In addition, those who have a Siglec 14/5 fusion gene, but who have a heterozygous allele of the Siglec 14 gene, have Siglec 14 protein, Siglec 5 protein, and Siglec 14/5 fusion protein (the same amino acid sequence as Siglec 5 protein). Having a). Interesting results have also been obtained that the proportion of persons with homozygous allelic forms of this Siglec 14/5 fusion gene varies from region to region in the world (see Table 1 in Example 4).
　一方、シグレック１４タンパク質は白血球上に発現しており、シグレック１４タンパク質を欠損する個人にシグレック１４タンパク質を発現する個人の血液を輸血すると、該輸血血液中の白血球表面に提示されたシグレック１４タンパク質が血液型抗原として認識され、白血球に対する抗体が産生される可能性がある。 On the other hand, Siglec 14 protein is expressed on leukocytes, and when blood of an individual expressing Siglec 14 protein is transfused into an individual lacking Siglec 14 protein, Siglec 14 protein presented on the surface of leukocytes in the transfused blood is Recognized as a blood group antigen, antibodies against leukocytes may be produced.
　非溶血性輸血副作用の発症に種々の白血球抗原とそれに対するアロ抗体との抗原抗体反応が報告されている。現在、その原因となる抗原が多数知られているものの、全容は明らかではない。そこで、上記のようなシグレック１４遺伝子の多型は、輸血副作用の一因になり得、このような危険性はなるべく排除しておくことが必要である。 Antigen-antibody reactions between various leukocyte antigens and alloantibodies against them have been reported in the development of non-hemolytic transfusion side effects. Although many of the antigens that cause it are currently known, the complete picture is not clear. Therefore, the polymorphism of the Siglec 14 gene as described above can contribute to blood transfusion side effects, and it is necessary to eliminate such a risk as much as possible.
　また、シグレック１４タンパク質を発現する細胞は、細菌由来のリポ多糖への炎症性サイトカイン（TNF-α）応答が、シグレック５タンパク質を発現する細胞を含めてシグレック１４を発現しない細胞よりも強いというデータが得られている。この結果は、ウイルス、細菌、真菌、原虫などの感染に伴う炎症性サイトカインの産生をシグレック１４タンパク質が制御していることを示すものと考えられ、シグレック１４遺伝子の多型により感染症への感受性が変わる可能性が示唆され、シグレック１４遺伝子の多型を検出することは、感染症に対する治療方針を決定する上で重要である。 Further, cells expressing Siglec 14 protein have a stronger inflammatory cytokine (TNF-α) response to bacterial lipopolysaccharide than cells not expressing Siglec 14 including cells expressing Siglec 5 protein. Is obtained. This result is considered to indicate that the siglec 14 protein regulates the production of inflammatory cytokines associated with infection of viruses, bacteria, fungi, protozoa, etc., and the susceptibility to infection by the polymorphism of the siglec 14 gene. Therefore, it is important to detect the polymorphism of the Siglec 14 gene in determining the treatment strategy for infectious diseases.
　上記知見によれば、シグレック１４遺伝子の多型に基づく対立遺伝子の型は、シグレック１４遺伝子のみ有するホモ接合型〈野生型〉、シグレック１４遺伝子とシグレック１４／５遺伝子を有するヘテロ接合型及びシグレック１４／５融合遺伝子のみを有するホモ接合型の３者に分けられる。前２者はともにシグレック１４タンパク質とシグレック５タンパク質（又はシグレック５タンパク質と同じアミノ酸配列を有するシグレック１４／５融合タンパク質）を発現し、一方、後者のシグレック１４／５融合遺伝子のホモ接合型は、シグレック１４タンパク質を発現せず、シグレック５タンパク質と同じアミノ酸配列を有するシグレック１４／５融合タンパク質を発現するから、例えば、上記血液適合性及び感染症への対応においては、上記前２者とシグレック１４／５融合遺伝子のみを有するホモ接合型とを識別できればよいことになり、このためには、シグレック１４遺伝子の有無或いはシグレック１４タンパクの有無を検出すれば良いことになる。 According to the above findings, the allele types based on the polymorphism of the Siglec 14 gene are homozygous <wild type> having only the Siglec 14 gene, heterozygous having Siglec 14 and Siglec 14/5 genes, and Siglec 14 There are three types of homozygous types that have only the / 5 fusion gene. Both of the former express siglec 14 protein and siglec 5 protein (or siglec 14/5 fusion protein having the same amino acid sequence as siglec 5 protein), while the homozygous form of the latter siglec 14/5 fusion gene is: Since the Siglec 14/5 fusion protein having the same amino acid sequence as that of the Siglec 5 protein is expressed without expressing the Siglec 14 protein, for example, in the response to the above-mentioned hemocompatibility and infectious diseases, the former 2 and Siglec 14 are expressed. Therefore, it is only necessary to distinguish the homozygous type having only the / 5 fusion gene, and for this purpose, the presence or absence of the Siglec 14 gene or the presence or absence of the Siglec 14 protein may be detected.
　すなわち、本発明のシグレック１４遺伝子の多型に基づく対立遺伝子型検出用試薬は、シグレック１４遺伝子或いはシグレック１４タンパク質の有無を検出するものであり、この検出手段の第一の態様として、本発明においてはシグレック１４遺伝子をＰＣＲにより特異的に増幅するプライマー対を使用するものである。 That is, the allelic detection reagent based on the polymorphism of the siglec 14 gene of the present invention detects the presence or absence of the siglec 14 gene or siglec 14 protein. As a first aspect of this detection means, Uses a primer pair that specifically amplifies the Siglec 14 gene by PCR.
　シグレック１４遺伝子を特異的に増幅する各プライマーは、シグレック１４遺伝子中の塩基配列に基づき設計するが、シグレック１４／５融合遺伝子或いはシグレック５遺伝子を増幅させないことが必要であり、各プライマーは、シグレック１４遺伝子とシグレック５遺伝子における保存領域（共通配列部分）の外側（保存領域より５’側及び３’側）の塩基配列を選択して設計する。シグレック１４遺伝子の保存領域は配列番号１の塩基配列中、７５３番目～２１１０番目にあり、シグレック１４遺伝子の検出用プライマー対のうちフォワードプライマーは、７５３番目より５’末端側の領域を、またリバースプライマーは、２１１０番目より３’末端側の領域を選択して設計する。 Each primer that specifically amplifies the Siglec 14 gene is designed based on the nucleotide sequence in the Siglec 14 gene, but it is necessary not to amplify the Siglec 14/5 fusion gene or the Siglec 5 gene. A base sequence outside the conserved region (common sequence portion) of 14 genes and Siglec 5 gene (5 ′ side and 3 ′ side from the conserved region) is selected and designed. The conserved region of the Siglec 14 gene is located at positions 753 to 2110 in the nucleotide sequence of SEQ ID NO: 1. The forward primer of the primer pair for detection of the Siglec 14 gene is the region 5′-terminal from the 753rd and reverse. The primer is designed by selecting a region 3 ′ end side from the 2110th position.
　これら各プライマーの塩基長については特に制限はないが、アニーリング温度並びにアニーリングの特異性等の点から、15～40塩基、好ましくは20～35塩基である。 The base length of each primer is not particularly limited, but it is 15 to 40 bases, preferably 20 to 35 bases from the viewpoint of annealing temperature and annealing specificity.
　より具体的なプライマーとしては、フォワードプライマーとしては、配列番号７に示されるもの、リバースプライマーとしては配列番号８に示すものが挙げられる。 More specific primers include those shown in SEQ ID NO: 7 as forward primers and those shown in SEQ ID NO: 8 as reverse primers.
　これらプライマーを用いて、シグレック１４遺伝子を検出する手法は、例えば以下のとおりである。 The method for detecting the Siglec 14 gene using these primers is, for example, as follows.
　すなわち、ゲノムDNAをテンプレートに用いたPCRによりシグレック１４遺伝子の有無を判定する。 That is, the presence or absence of Siglec 14 gene is determined by PCR using genomic DNA as a template.
　このためには、まず、例えば、被験者から採取された末梢血或いは口腔擦過試料などのヒト由来試料からゲノムDNAを抽出・精製する。精製法はプロテイナーゼ消化、フェノール・クロロホルム抽出、エタノール沈殿などの組み合わせによるものでもよく、また市販のゲノムDNA精製キットなどを用いてもよい。 For this purpose, first, genomic DNA is extracted and purified from a human-derived sample such as peripheral blood collected from a subject or a mouth-rubbed sample, for example. The purification method may be a combination of proteinase digestion, phenol / chloroform extraction, ethanol precipitation, or a commercially available genomic DNA purification kit.
　次いで、精製したゲノムDNAを鋳型に用いたPCR反応を行う。具体的には、耐熱性DNAポリメラーゼ、デオキシリボヌクレオシド三リン酸混合物、PCR反応に適したバッファー、鋳型となるゲノムDNAと、シグレック１４遺伝子を特異的に増幅するプライマー対を添加し、反応液を調製する。反応液を適当な反応容器（反応液の蒸散を防ぐ物が望ましい）に入れ、鋳型DNAの熱溶解、鋳型DNAとプライマーのアニーリング、DNAポリメラーゼによるDNA伸長反応からなるPCR反応を行う。この反応にはサーマルサイクラーを用いるのが望ましい。 Next, a PCR reaction is performed using the purified genomic DNA as a template. Specifically, a heat-resistant DNA polymerase, deoxyribonucleoside triphosphate mixture, a buffer suitable for PCR reaction, a genomic DNA serving as a template, and a primer pair that specifically amplifies the Siglec 14 gene are added to prepare a reaction solution To do. Place the reaction solution in a suitable reaction vessel (preferably one that prevents evaporation of the reaction solution), and perform a PCR reaction consisting of heat-dissolving the template DNA, annealing the template DNA and primer, and DNA extension reaction with DNA polymerase. It is desirable to use a thermal cycler for this reaction.
　PCR反応後、生成したPCR反応産物を検出する。これには反応液に二本鎖DNA検出試薬（例えばSYBR Greenやエチジウムブロマイドなど）を直接添加し、適当な光源の存在下で反応産物を検出するか、或いは反応産物をアガロースゲル電気泳動やキャピラリー電気泳動などで分離し、二本鎖DNA検出試薬（例えばSYBR Greenやエチジウムブロマイドなど）を用いてPCR反応産物を検出する。 After PCR reaction, the generated PCR reaction product is detected. For this purpose, a double-stranded DNA detection reagent (eg SYBR Green or ethidium bromide) is added directly to the reaction solution, and the reaction product is detected in the presence of an appropriate light source, or the reaction product is detected by agarose gel electrophoresis or capillary Separate by electrophoresis, etc., and detect the PCR reaction product using a double-stranded DNA detection reagent (such as SYBR Green or ethidium bromide).
　遺伝子型の判定は、ある特定のヒト試料からシグレック１４遺伝子特異的な反応産物が検出されない場合にシグレック１４／５融合遺伝子のホモ接合型と判定する。 The genotype is determined to be a homozygous type of the Siglec 14/5 fusion gene when no reaction product specific to the Siglec 14 gene is detected from a specific human sample.
　本発明の検出手段の第二の態様として、本発明においてはシグレック１４遺伝子に特異的なプローブを挙げることができる。 As a second embodiment of the detection means of the present invention, a probe specific for the Siglec 14 gene can be mentioned in the present invention.
　シグレック１４遺伝子を特異的に検出する核酸プローブは、シグレック１４遺伝子中の塩基配列に基づき設計するが、シグレック１４／５融合遺伝子或いはシグレック５遺伝子にアニールしないことが必要である。シグレック１４遺伝子とシグレック５遺伝子の保存領域（共通配列部分）を避け、かつシグレック１４遺伝子に特異的な領域はシグレック１４遺伝子における保存領域の３’側であり、かつシグレック５遺伝子における保存領域の５’側である。従ってシグレック１４遺伝子を特異的に検出する核酸プローブは、かかる塩基配列を選択して設計する。シグレック１４遺伝子の保存領域は配列番号１の塩基配列中、７５３番目～２１１０番目にあり、シグレック１４遺伝子の検出用核酸プローブは２１１０番目より３’末端側の領域を選択して設計する。 The nucleic acid probe that specifically detects the Siglec 14 gene is designed based on the nucleotide sequence in the Siglec 14 gene, but it is necessary not to anneal to the Siglec 14/5 fusion gene or the Siglec 5 gene. The conserved region (common sequence portion) of the Siglec 14 gene and the Siglec 5 gene is avoided, and the region specific to the Siglec 14 gene is on the 3 ′ side of the conserved region in the Siglec 14 gene, and 5 of the conserved region in the Siglec 5 gene. 'The side is. Therefore, a nucleic acid probe that specifically detects the Siglec 14 gene is designed by selecting such a base sequence. The conserved region of the Siglec 14 gene is located at the 753rd to 2110th positions in the nucleotide sequence of SEQ ID NO: 1, and the nucleic acid probe for detecting the Siglec 14 gene is designed by selecting a region 3 'end side from the 2110th position.
　核酸プローブの塩基長については特に制限はないが、アニーリング温度やアニーリングの特異性等から、２０～１００００塩基対程度、好ましくは２０～１０００塩基対程度である。 The base length of the nucleic acid probe is not particularly limited, but is about 20 to 10,000 base pairs, preferably about 20 to 1000 base pairs, from the annealing temperature and the specificity of annealing.
　より具体的な核酸プローブとしては、配列番号１５～１７に示されるものが挙げられるが、当然これらと相補の配列を有する物であっても良い。 More specific nucleic acid probes include those shown in SEQ ID NOs: 15 to 17, but naturally, a nucleic acid probe having a sequence complementary to these may be used.
　かかる核酸プローブを用いて、シグレック１４遺伝子を検出する手法は、例えば以下のとおりである。 A method for detecting the Siglec 14 gene using such a nucleic acid probe is, for example, as follows.
　すなわち、ヒトゲノムDNAを用いたサザンブロッティングによりシグレック１４遺伝子の有無を判定する。 That is, the presence or absence of the Siglec 14 gene is determined by Southern blotting using human genomic DNA.
　このためには、まず、例えば、被験者から採取された末梢血或いは口腔擦過試料などのヒト由来試料からゲノムDNAを抽出・精製する。精製法はプロテイナーゼ消化、フェノール・クロロホルム抽出、エタノール沈殿などの組み合わせによるものでもよく、また市販のゲノムDNA精製キットなどを用いてもよい。 For this purpose, first, genomic DNA is extracted and purified from a human-derived sample such as peripheral blood collected from a subject or a mouth-rubbed sample, for example. The purification method may be a combination of proteinase digestion, phenol / chloroform extraction, ethanol precipitation, or a commercially available genomic DNA purification kit.
　次いで、かかるゲノムDNAを直接ナイロン膜などの薄膜に固相化するか、或いは適当な方法（制限酵素等による消化など）で断片化しアガロースゲル電気泳動などにより分離した後、ナイロン膜などの薄膜に転写し固相化する。 Next, the genomic DNA is directly immobilized on a thin film such as a nylon membrane, or is fragmented by an appropriate method (such as digestion with a restriction enzyme) and separated by agarose gel electrophoresis. Transfer and immobilize.
　次いで、シグレック１４遺伝子と特異的にハイブリダイズする核酸プローブを調製し、ラベルを導入し、薄膜上のゲノムDNAとハイブリダイズさせる。この際に用いるラベルは放射性同位元素（32P、33P、35S等）、蛍光色素（Cy3やCy5等）、或いはビオチンやジゴキシゲニンなどのエピトープを用いる。ビオチンやジゴキシゲニンなどのエピトープを導入した場合は、核酸プローブの検出に二次検出試薬（蛍光色素を結合したストレプトアビジンや抗ジゴキシゲニン抗体など）を必要とする。ハイブリダイズしなかったプローブを洗浄操作により除いた後、ハイブリダイズした核酸プローブをラベル法に応じた至適の方法で検出する。 Next, a nucleic acid probe that specifically hybridizes with the Siglec 14 gene is prepared, a label is introduced, and it is hybridized with the genomic DNA on the thin film. The label used in this case uses a radioisotope (32P, 33P, 35S, etc.), a fluorescent dye (Cy3, Cy5, etc.), or an epitope such as biotin or digoxigenin. When an epitope such as biotin or digoxigenin is introduced, a secondary detection reagent (such as streptavidin or anti-digoxigenin antibody bound with a fluorescent dye) is required for detection of the nucleic acid probe. After removing the unhybridized probe by a washing operation, the hybridized nucleic acid probe is detected by an optimum method according to the label method.
　或いは、シグレック１４遺伝子特異的な核酸プローブを薄膜或いはガラス面などに固相化し、適当な方法（機械的剪断、制限酵素等による消化など）で断片化しラベルを導入したゲノムDNAをハイブリダイズさせる。この際に用いるラベルは放射性同位元素（32P、33P、35S等）、蛍光色素（Cy3やCy5等）、或いはビオチンやジゴキシゲニンなどのエピトープを用いる。ビオチンやジゴキシゲニンなどのエピトープを導入した場合は、核酸プローブの検出に二次検出試薬（蛍光色素を結合したストレプトアビジンや抗ジゴキシゲニン抗体）を必要とする。ハイブリダイズしなかったプローブを洗浄操作により除いた後、ハイブリダイズしたゲノムDNAをラベル法に応じた至適の方法で検出する。 Alternatively, a Siglec 14 gene-specific nucleic acid probe is solid-phased on a thin film or glass surface, and is fragmented by an appropriate method (such as mechanical shearing or digestion with a restriction enzyme) and hybridized with genomic DNA into which a label has been introduced. The label used in this case uses a radioisotope (32P, 33P, 35S, etc.), a fluorescent dye (Cy3, Cy5, etc.), or an epitope such as biotin or digoxigenin. When an epitope such as biotin or digoxigenin is introduced, a secondary detection reagent (streptavidin or anti-digoxigenin antibody bound with a fluorescent dye) is required for detection of the nucleic acid probe. After removing the unhybridized probe by washing, the hybridized genomic DNA is detected by an optimum method according to the label method.
　遺伝子型の判定は、ある特定のヒト試料からシグレック１４遺伝子特異的な核酸プローブとゲノムDNAのハイブリダイゼーションシグナルが検出できない場合にシグレック１４／５融合遺伝子のホモ接合型と判定する。 The genotype is determined to be a homozygous type of the Siglec 14/5 fusion gene when a hybridization signal between the Siglec 14 gene-specific nucleic acid probe and genomic DNA cannot be detected from a specific human sample.
　本発明の検出手段の第三の態様としては、シグレック１４タンパク質を特異的に認識する抗体を挙げることができる。該抗体は、ポリクローナル抗体でもモノクローナル抗体でも良い。 As a third aspect of the detection means of the present invention, an antibody that specifically recognizes Siglec 14 protein can be mentioned. The antibody may be a polyclonal antibody or a monoclonal antibody.
　これら抗体は従来周知の方法で調製できる。例えばポリクローナル抗体は、シグレック１４タンパク質を用いて、マウス、ラット、ウサギ、ヤギ、ヒツジ、ロバ等の動物を免疫感作し、一定期間経過後血清を採取し、プロテインA、プロテインG、抗原タンパク質、抗原ペプチドなどを固相化したカラムなどで精製して得る。また、モノクローナル抗体は、上記と同様に免疫感作し、一定期間経過後、例えば脾臓細胞を採取し、該細胞とミエローマ細胞とを細胞融合し、得られたハイブリドーマをスクリーニングし、シグレック１４タンパク質に対する抗体産生ハイブリドーマから採取する。 These antibodies can be prepared by a conventionally known method. For example, a polyclonal antibody uses a Siglec 14 protein to immunize animals such as mice, rats, rabbits, goats, sheep, donkeys, etc., and serum is collected after a lapse of a certain period of time, and protein A, protein G, antigen protein, It is obtained by purifying with a column or the like on which an antigen peptide is immobilized. The monoclonal antibody is immunized in the same manner as described above, and after a certain period of time, for example, spleen cells are collected, the cells are fused with myeloma cells, the resulting hybridoma is screened, and the antibody against Siglec 14 protein is obtained. Collected from antibody-producing hybridoma.
　ポリクローナル抗体の場合は、免疫に用いる抗原としてシグレック１４タンパク質に特有の部分を用いることにより、シグレック５タンパク質を含む他のシグレックタンパク質と交差反応しない抗体を得る。或いは、シグレック１４タンパク質の一部又は全体を抗原に用いて抗体を作成した後、シグレック５タンパク質の一部（シグレック１４タンパク質と高い配列相同性を示す部分を含む）又は全部と交差反応する画分を吸着などにより除き、シグレック１４タンパク質に特異的な抗体を得る。 In the case of a polyclonal antibody, an antibody that does not cross-react with other Siglec proteins including Siglec 5 protein is obtained by using a portion specific to Siglec 14 protein as an antigen used for immunization. Alternatively, a fraction that cross-reacts with a part of Siglec 5 protein (including a part showing high sequence homology with Siglec 14 protein) or after making an antibody using part or whole of Siglec 14 protein as an antigen Is removed by adsorption or the like to obtain an antibody specific for Siglec 14 protein.
　モノクローナル抗体の場合は、免疫に用いる抗原としてシグレック１４タンパク質に特有の部分を用いることにより、シグレック５タンパク質と交差反応しない抗体を得る。或いは、シグレック１４タンパク質の一部又は全体を抗原に用いてハイブリドーマを作成した後、シグレック５タンパク質の一部（シグレック１４タンパク質と高い配列相同性を示す部分を含む）又は全部と交差反応するハイブリドーマをスクリーニングにより除き、シグレック１４タンパク質に特異的な抗体を得る。 In the case of a monoclonal antibody, an antibody that does not cross-react with Siglec 5 protein is obtained by using a portion specific to Siglec 14 protein as an antigen used for immunization. Alternatively, after preparing a hybridoma using part or all of the Siglec 14 protein as an antigen, a hybridoma that cross-reacts with part or all of the Siglec 5 protein (including a part showing high sequence homology with the Siglec 14 protein) is prepared. Excluding by screening, an antibody specific for Siglec 14 protein is obtained.
　ポリクローナル抗体、モノクローナル抗体いずれの場合も、ヒト由来の他のシグレック（例えばシグレック６）に交差反応しないことを確認することが望ましい。 It is desirable to confirm that neither a polyclonal antibody nor a monoclonal antibody cross-reacts with other human-derived siglecs (eg, siglec 6).
　このような抗体を使用してシグレック１４タンパク質を検出するには、白血球のフローサイトメトリーによる解析によりシグレック１４タンパク質の有無を判定する。 In order to detect Siglec 14 protein using such an antibody, the presence or absence of Siglec 14 protein is determined by analysis of leukocytes by flow cytometry.
　シグレック１４タンパク質が検出されれば、シグレック１４遺伝子のみ有するホモ接合型〈野生型〉か、或いはシグレック１４遺伝子とシグレック１４／５融合遺伝子を有するヘテロ接合型であり、シグレック１４タンパク質が検出されなければ、シグレック１４／５融合遺伝子のホモ接合型と判定できる。 If Siglec 14 protein is detected, it is homozygous <wild type> having only Siglec 14 gene or heterozygous having Siglec 14 gene and Siglec 14/5 fusion gene, and Siglec 14 protein is not detected. The homozygous type of the Siglec 14/5 fusion gene can be determined.
　具体的には、被験者の血液を抗凝固剤（EDTA、クエン酸、ヘパリンなど）の入った採血管に採取し、試料として用いる。血液から塩化アンモニウムなどを用いた赤血球の溶血により白血球を単離してもよく、また望ましい。ヒドロキシエチルスターチなどを用いた赤血球凝集沈降法などにより白血球を単離してもよい。 Specifically, the blood of the subject is collected in a blood collection tube containing an anticoagulant (EDTA, citric acid, heparin, etc.) and used as a sample. White blood cells may be isolated from the blood by hemolysis of red blood cells using ammonium chloride or the like. White blood cells may be isolated by the hemagglutination sedimentation method using hydroxyethyl starch or the like.
　ヒト白血球（又は全血）試料に抗シグレック１４抗体を添加し、一定時間保温した後、洗浄により結合しなかった抗体を除く。抗体はラベルなしの物でも良く、また蛍光色素（フルオレセイン、R-フィコエリスリン、アロフィコシアニンなど、使用する機器で検出可能な蛍光色素）或いはビオチンなどでラベルした物を用いても良い。ラベルなしの抗シグレック１４抗体を用いる場合は、この抗体の作製に用いた動物の免疫グロブリンを認識する二次抗体（蛍光色素又はビオチンなどでラベルした物）、ビオチンなどでラベルした抗体を用いる場合はこのラベルと特異的に結合するプローブ（蛍光色素ラベルしたストレプトアビジンなど）を二次試薬に用いて検出する。 Anti-Siglec 14 antibody is added to a human leukocyte (or whole blood) sample, incubated for a certain period of time, and then the antibody that has not been bound by washing is removed. The antibody may be an unlabeled one, or a fluorescent dye (a fluorescent dye that can be detected by a device such as fluorescein, R-phycoerythrin, or allophycocyanin) or a biotin-labeled one. When using an unlabeled anti-Siglec 14 antibody, a secondary antibody that recognizes the animal's immunoglobulin used to produce this antibody (labeled with a fluorescent dye or biotin), an antibody labeled with biotin, etc. Is detected using a probe that specifically binds to this label (such as streptavidin labeled with a fluorescent dye) as a secondary reagent.
　結合しなかった抗体を洗浄操作により除き、フローサイトメーターを用いて白血球上のシグレック１４タンパク質の有無を検証する。この際、Ｔリンパ球以外の白血球画分のいずれを用いてもシグレック１４タンパク質の有無を検討可能であるが、細胞数の多さとシグレック１４タンパク質の発現レベルの高さなどを考慮し、顆粒球、或いは単球上でのシグレック１４タンパク質の発現を検討することが望ましい。 The antibody that did not bind was removed by washing, and the presence or absence of Siglec 14 protein on leukocytes was verified using a flow cytometer. At this time, it is possible to examine the presence or absence of Siglec 14 protein using any leukocyte fraction other than T lymphocytes, but considering the high number of cells and the high expression level of Siglec 14 protein, granulocytes Alternatively, it is desirable to examine the expression of Siglec 14 protein on monocytes.
　上記した検査手段は、いずれも、シグレック１４遺伝子のみ有するホモ接合型〈野生型〉、シグレック１４遺伝子とシグレック１４／５融合遺伝子を有するヘテロ接合型及びシグレック融合１４／５遺伝子のみ有するホモ接合型の３者のうち、前２者と後一者を識別するものであるが、本発明においてはこれら３者のそれぞれを識別することも可能である。 All of the above-mentioned inspection means are homozygous type having only the Siglec 14 gene <wild type>, heterozygous type having the Siglec 14 gene and the Siglec 14/5 fusion gene, and homozygous type having only the Siglec fusion 14/5 gene. Among the three parties, the former two and the latter are identified. In the present invention, each of these three parties can be identified.
　これには、さらに、シグレック１４／５融合遺伝子の特異的増幅用プライマー対を用いてPCRを行う。 For this, PCR is further performed using a primer pair for specific amplification of the Siglec 14/5 fusion gene.
　シグレック１４／５融合遺伝子増幅用プライマー対は、該融合遺伝子におけるシグレック１４遺伝子に由来する塩基配列とシグレック５遺伝子に由来する塩基配列部分に基づき設計するが、これらプライマー対を構成する各プライマーは、シグレック１４遺伝子とシグレック５遺伝子における保存領域（共通配列部分）の外側（保存領域より５’側及び３’側）の塩基配列を選択して設計することが好ましい。 The primer pair for Siglec 14/5 fusion gene amplification is designed based on the base sequence derived from the Siglec 14 gene and the base sequence portion derived from the Siglec 5 gene in the fusion gene. It is preferable to select and design a base sequence outside the conserved region (common sequence portion) in the Siglec 14 gene and Siglec 5 gene (5 ′ side and 3 ′ side from the conserved region).
　シグレック１４遺伝子の保存領域は配列番号１の塩基配列中、７５３番目～２１１０番目にあり、シグレック１４／５融合遺伝子の検出用プライマー対のうちフォワードプライマーは、７５３番目より５’末端側の領域を選択して設計する。すなわち、フォワードプライマーはシグレック１４遺伝子検出用フォワードプライマーと同様でよい。 The conserved region of the Siglec 14 gene is located at positions 753 to 2110 in the base sequence of SEQ ID NO: 1. The forward primer of the primer pair for detection of the Siglec 14/5 fusion gene has a region 5′-terminal from the 753rd. Select and design. That is, the forward primer may be the same as the Siglec 14 gene detection forward primer.
　シグレック５融合遺伝子の保存領域は配列番号２（うちタンパク質をコードする領域は塩基配列中１００１番目～１９１０５番目）の塩基配列中、７５３番目～２１１４番目にあり、シグレック１４／５融合遺伝子の検出用プライマー対のうちリバースプライマーは、２１１４番目より３’末端側の領域を選択して設計する。 The conserved region of the Siglec 5 fusion gene is located at positions 753 to 2114 in the base sequence of SEQ ID NO: 2 (of which the protein coding region is the 1001st to 19105th base sequence), and is used for detection of the Siglec 14/5 fusion gene The reverse primer of the primer pair is designed by selecting a region 3 ′ end side from the 2114th position.
　これら各プライマーの塩基長については、上記シグレック１４遺伝子増幅用プライマーと同様でよく、また、これらプライマーを用いて、シグレック１４／５融合遺伝子を検出する手法も、上記シグレック１４遺伝子増幅用プライマーと同様でよい。 The base length of each primer may be the same as that of the above-mentioned Siglec 14 gene amplification primer, and the method for detecting the Siglec 14/5 fusion gene using these primers is the same as that of the above-mentioned Siglec 14 gene amplification primer. It's okay.
　このプライマー対の具体的塩基配列は、配列番号７及び１０に示される。 The specific base sequences of this primer pair are shown in SEQ ID NOs: 7 and 10.
　上記したように、シグレック１４遺伝子増幅用プライマー、同遺伝子検出用プローブ或いはシグレック１４タンパク質の認識抗体を使用してシグレック１４遺伝子或いはシグレック１４タンパク質が検出された場合は、シグレック１４遺伝子のホモ接合型か或いはシグレック１４とシグレック１４／５のヘテロ接合型のいずれかである。このように判定された試料について、さらに、シグレック１４／５融合遺伝子増幅用プライマーを用いて、シグレック１４／５融合遺伝子が検出された場合には、シグレック１４とシグレック１４／５のヘテロ接合型であると判定でき、シグレック１４遺伝子のホモ接合型と、シグレック１４とシグレック１４／５のヘテロ接合型とを識別できる。 As described above, when the siglec 14 gene or siglec 14 protein is detected using the siglec 14 gene amplification primer, the same gene detection probe, or the siglec 14 protein recognition antibody, is it homozygous for the siglec 14 gene? Alternatively, it is one of the heterojunction type of Siglec 14 and Siglec 14/5. When the Siglec 14/5 fusion gene is detected using the Siglec 14/5 fusion gene amplification primer for the sample thus determined, the heterozygous type of Siglec 14 and Siglec 14/5 is used. It can be determined that there is a homozygous type of Siglec 14 gene and a heterozygous type of Siglec 14 and Siglec 14/5.
　また、シグレック１４遺伝子及びシグレック１４タンパク質が検出されなかった場合、このシグレック１４／５融合遺伝子増幅用プライマーを用いて、シグレック１４／５融合遺伝子の有無を解析することにより、シグレック１４／５融合遺伝子のホモ接合型であることを確認でき、正確性を増す。 Further, when the siglec 14 gene and siglec 14 protein are not detected, the siglec 14/5 fusion gene is analyzed by analyzing the presence or absence of the siglec 14/5 fusion gene using this siglec 14/5 fusion gene amplification primer. It can be confirmed that it is a homozygous type, and the accuracy is increased.
　このようなシグレック１４／５融合遺伝子増幅用プライマー対は、上記シグレック１４遺伝子増幅用プライマー対、同プローブ或いはシグレック１４タンパク質の抗体と組み合わされ、シグレック１４遺伝子の多型に基づく対立遺伝子型の検出用試薬キットとして使用できる。また、シグレック１４／５融合遺伝子増幅用プライマー対は、シグレック１４遺伝子の多型検出に用いることができる。 Such a primer pair for amplifying Siglec 14/5 fusion gene is used in combination with the primer pair for amplifying Siglec 14 gene, the same probe or an antibody of Siglec 14 protein, and for detecting an allelic type based on the polymorphism of Siglec 14 gene. It can be used as a reagent kit. Further, the primer pair for amplifying the siglec 14/5 fusion gene can be used for detecting a polymorphism of the siglec 14 gene.
　一方、シグレック５遺伝子、シグレック１４遺伝子、シグレック１４／５融合遺伝子に保存された配列領域に注目し、二倍体ゲノムあたりの保存領域のコピー数が野生型：ヘテロ接合体：ホモ接合体で４：３：２であることを利用して遺伝子型を判定しても良い。具体的には、ヒト試料由来のゲノムDNAを鋳型に用い、上記の保存領域を増幅するプライマー対を用いて定量的PCRを実施し、遺伝子型既知の同量のヒトゲノムDNA（野生型、ヘテロ接合体、ホモ接合体）を定量標準品として用いた反応産物の定量的な比較により、遺伝子型を判定する。 On the other hand, paying attention to the sequence regions conserved in the siglec 5 gene, siglec 14 gene, and siglec 14/5 fusion gene, the copy number of the conserved region per diploid genome is 4 for wild type: heterozygote: homozygote. : 3: 2 may be used to determine the genotype. Specifically, using genomic DNA derived from a human sample as a template, quantitative PCR was performed using a primer pair that amplifies the above conserved region, and the same amount of human genomic DNA of known genotype (wild type, heterozygous) Genotypes are determined by quantitative comparison of reaction products using a standard product as a quantitative standard.
　本発明は、シグレック１４タンパク質に対する抗体を検出するための試薬、及びこれを使用したシグレック１４タンパク質に対する抗体の検出方法に関する。 The present invention relates to a reagent for detecting an antibody against Siglec 14 protein and a method for detecting an antibody against Siglec 14 protein using the same.
　本発明において、シグレック１４タンパク質に対する抗体とは、ヒト血液試料、好ましくは血液製剤中に含まれるシグレック１４タンパク質に対するアロ抗体が好ましい。下記実施例にて詳述されるように、非溶血性輸血副作用に関与した血液試料と一般献血試料とを比較すると、シグレック１４に対するアロ抗体を含む頻度が前者において有意に高く、また好中球を抗シグレック１４抗体陽性血漿で刺激すると、当該好中球は活性化される。これらの事実は、抗シグレック１４アロ抗体が非溶血性輸血副作用に関与する可能性を強く示唆する。したがって、本発明により血液試料中の抗シグレック１４アロ抗体を検出することによって、非溶血性輸血副作用に関与し得る血液試料を排除して、非溶血性輸血副作用を防止し輸血の安全性を確保することができる。 In the present invention, the antibody against Siglec 14 protein is preferably an alloantibody against Siglec 14 protein contained in a human blood sample, preferably a blood product. As will be described in detail in the following examples, when comparing blood samples involved in non-hemolytic transfusion side effects and general blood donated samples, the frequency of containing alloantibodies against Siglec 14 is significantly higher in the former, and neutrophils Is stimulated with anti-Siglec 14 antibody-positive plasma, the neutrophils are activated. These facts strongly suggest that anti-Siglec 14 alloantibodies may be involved in non-hemolytic transfusion side effects. Therefore, by detecting anti-Siglec 14 alloantibodies in blood samples according to the present invention, blood samples that can be involved in non-hemolytic transfusion side effects are eliminated, non-hemolytic transfusion side effects are prevented, and transfusion safety is ensured. can do.
　本発明のシグレック１４タンパク質に対する抗体を検出するための試薬は、シグレック１４タンパク質又はシグレック１４タンパク質発現細胞を含む。 The reagent for detecting an antibody against the Siglec 14 protein of the present invention includes Siglec 14 protein or Siglec 14 protein-expressing cells.
　シグレック１４は公知であり、配列情報がGenBankなどのデータベースに公開されている。ヒトシグレック１４の配列情報は、例えばアクセッション番号AAX47338, NP_001092082等として、GenBankに登録されておりこれらを利用することが可能である。 Siglec 14 is publicly known, and sequence information is disclosed in databases such as GenBank. The sequence information of human Siglec 14 is registered in GenBank, for example, as accession number AAX47338, NP_001092082, and these can be used.
　シグレック１４タンパク質は、GenBankなどに登録された公知のアミノ酸配列を有するシグレック１４タンパク質を用いることができるが、好ましくは配列番号４で示されたアミノ酸配列を有するシグレック１４タンパク質を用いる。 Siglec 14 protein having a known amino acid sequence registered in GenBank or the like can be used as Siglec 14 protein, but Siglec 14 protein having an amino acid sequence represented by SEQ ID NO: 4 is preferably used.
　また、配列番号４で示される配列は、細胞外領域であるN末端Vセット免疫グロブリン様領域及びC2セット免疫グロブリン様領域、膜貫通領域並びに細胞内領域を含む。本発明におけるシグレック１４タンパク質には少なくとも、細胞外領域（配列番号４においては、1番目から358番目の配列が細胞外領域部分に相当する）に含まれるシグレック１４に特異的な領域が含まれていれば良い。「シグレック１４に特異的な領域」とは、シグレック１４タンパク質の細胞外領域のアミノ酸配列とシグレック５タンパク質の細胞外領域のアミノ酸配列を比較した場合に、シグレック１４タンパク質の細胞外領域にのみ含まれるアミノ酸配列を意味する。配列番号４においては、238番目から358番目のアミノ酸配列がシグレック１４に特異的な領域に相当する。したがって、本発明におけるシグレック１４タンパク質は、配列番号４の少なくとも238番目から358番目の配列を含む配列からなるタンパク質を含む。 The sequence represented by SEQ ID NO: 4 includes an N-terminal V-set immunoglobulin-like region and a C2-set immunoglobulin-like region, a transmembrane region, and an intracellular region, which are extracellular regions. The Siglec 14 protein in the present invention includes at least a region specific to Siglec 14 included in the extracellular region (in SEQ ID NO: 4, the first to 358th sequences correspond to the extracellular region portion). Just do it. The “region specific to Siglec 14” is included only in the extracellular region of Siglec 14 protein when the amino acid sequence of the extracellular region of Siglec 14 protein is compared with the amino acid sequence of the extracellular region of Siglec 5 protein. Means amino acid sequence. In SEQ ID NO: 4, the amino acid sequence from position 238 to position 358 corresponds to a region specific to siglec 14. Therefore, the Siglec 14 protein in the present invention includes a protein consisting of a sequence comprising at least the 238th to 358th sequences of SEQ ID NO: 4.
　さらに、本発明におけるシグレック１４タンパク質には、シグレック１４の変異体も包含される。このような変異体は、抗シグレック１４抗体に認識され結合され得る限り、天然の突然変異体及び人工変異体のいずれも含まれ、配列番号４又は配列番号４の少なくとも238番目から358番目の配列に示されるアミノ酸配列において、１～数個のアミノ酸の欠失、置換、付加又は挿入を有し、かつ抗シグレック１４抗体に認識され結合され得るタンパク質、或いは、該アミノ酸配列とBLAST（Basic Local Alignment Search Tool at the National Center for Biological Information（米国国立生物学情報センターの基本ローカルアラインメント検索ツール））等（例えば、デフォルトすなわち初期設定のパラメータ）を用いて計算したときに、80％以上、好ましくは85％以上、より好ましくは90％以上、例えば93％以上、95％以上、97％以上、98％以上又は99％以上の同一性を有するアミノ酸配列を有し、かつ抗シグレック１４抗体に認識され結合され得るタンパク質が含まれる。「１から数個」の範囲は特には限定されないが、例えば、１から20個、好ましくは１から10個、より好ましくは1から7個、さらに好ましくは1から5個、特に好ましくは1から3個、或いは1個又は2個である。 Further, the siglec 14 protein in the present invention includes siglec 14 mutants. Such mutants include both natural mutants and artificial mutants as long as they can be recognized and bound by the anti-Siglec 14 antibody. SEQ ID NO: 4 or at least the 238th to 358th sequences of SEQ ID NO: 4 Or a protein having a deletion, substitution, addition or insertion of one to several amino acids and capable of being recognized and bound by the anti-Siglec 14 antibody, or the amino acid sequence and BLAST (BasicmentLocal Alignment) 80% or more, preferably 85 when calculated using Search Tool at the National Center） for （Biological Information (e.g., US National Biological Information Center basic local alignment search tool)) % Or more, more preferably 90% or more, eg 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity And a protein that can be recognized and bound by an anti-Siglec 14 antibody. The range of “1 to several” is not particularly limited. For example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 1. Three, or one or two.
　本発明におけるシグレック１４タンパク質は、一般的な遺伝子組換え技術によって作製することができる。簡単に説明すると、シグレック１４タンパク質の作製は、当該タンパク質をコードするDNAを用意し、このDNAを含む発現ベクターを構築し、当該ベクターで原核又は真核細胞（例えば、これらに限定されないが、E. coli、酵母、SF9細胞、SF21細胞、COS1細胞、COS7細胞、CHO細胞、HEK293細胞など）を形質転換又はトランスフェクションし、得られた細胞の培養から目的の組換えタンパク質を回収することを含む。タンパク質の精製は、タンパク質の一般的な精製法、例えば硫酸アンモニウム沈殿、有機溶媒沈殿、透析、電気泳動、クロマトフォーカシング、ゲルろ過クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー、HPLCなどを適宜組み合わせることによって実施可能である。 The Siglec 14 protein in the present invention can be produced by a general gene recombination technique. Briefly, the production of Siglec 14 protein involves preparing a DNA encoding the protein, constructing an expression vector containing the DNA, and prokaryotic or eukaryotic cells (such as, but not limited to, E Transforming or transfecting coli, yeast, SF9 cells, SF21 cells, COS1 cells, COS7 cells, CHO cells, HEK293 cells, etc.) and recovering the desired recombinant protein from the resulting cell culture . Protein purification is performed by appropriately combining general protein purification methods such as ammonium sulfate precipitation, organic solvent precipitation, dialysis, electrophoresis, chromatofocusing, gel filtration chromatography, ion exchange chromatography, affinity chromatography, and HPLC. It can be implemented.
　上記DNAや発現ベクターに関しては、後述のシグレック１４タンパク質発現細胞の作製についての記述、及び実施例等を参照することができる。 For the above DNA and expression vector, reference can be made to the description of the production of Siglec 14 protein-expressing cells described later, examples and the like.
　本発明におけるシグレック１４タンパク質は、ポリペプチドのC末端又はN末端に標識ペプチドを結合させた融合タンパク質の形態であっても良い。代表的な標識ペプチドには、6～10残基のヒスチジンリピート（Hisタグ）、FLAG、mycペプチド、GFPポリペプチドなどが挙げられるが、標識ペプチドはこれらに限られるものではない。 The Siglec 14 protein in the present invention may be in the form of a fusion protein in which a labeled peptide is bound to the C-terminal or N-terminal of a polypeptide. Representative labeled peptides include histidine repeats (His tag) of 6 to 10 residues, FLAG, myc peptide, GFP polypeptide, etc., but the labeled peptide is not limited to these.
　また、本発明におけるシグレック１４タンパク質は、抗シグレック１４抗体が認識し結合し得る限り化学修飾されていてもよい。化学修飾には、非限定的に、例えばグリコシル化、ペグ(PEG)化、アセチル化、アミド化、リン酸化などが挙げられる。 The Siglec 14 protein in the present invention may be chemically modified as long as the anti-Siglec 14 antibody can be recognized and bound. Chemical modifications include, but are not limited to, for example, glycosylation, PEGylation, acetylation, amidation, phosphorylation and the like.
　さらに、本発明におけるシグレック１４タンパク質は、固相担体に結合されていても良い。固相担体の形状や材質は特に限定されない。形状としてはプレート状、フィルム状、球状又は不定形状等の粒子状が挙げられる。また、材質としては、ガラス、セラミックス、各種プラスチックが挙げられる。 Furthermore, the Siglec 14 protein in the present invention may be bound to a solid phase carrier. The shape and material of the solid support are not particularly limited. Examples of the shape include particle shapes such as a plate shape, a film shape, a spherical shape, and an indefinite shape. Examples of the material include glass, ceramics, and various plastics.
　シグレック１４タンパク質発現細胞は、その細胞膜上にシグレック１４タンパク質を発現する。 Siglec 14 protein-expressing cells express Siglec 14 protein on their cell membranes.
　当該細胞は、シグレック１４タンパク質をコードするDNAを用意し、このDNAを含む発現ベクターを構築し、当該ベクターで宿主細胞を形質転換又はトランスフェクションすることによって得ることができる。 The cells can be obtained by preparing DNA encoding Siglec 14 protein, constructing an expression vector containing this DNA, and transforming or transfecting host cells with the vector.
　シグレック１４タンパク質をコードするDNAは、好ましくは配列番号２０で示された塩基配列を有するDNAを用いる。配列番号２０で示される塩基配列は、シグレック１４タンパク質の細胞外領域、膜貫通領域及び細胞内領域を含む。本発明においてシグレック１４タンパク質をコードするDNAには、少なくとも上記シグレック１４に特異的な領域をコードする塩基配列が含まれていれば良く、好ましくは少なくともシグレック１４に特異的な領域及び膜貫通領域をコードする塩基配列が含まれていればよい。配列番号２０においては、712番目から1074番目の配列がシグレック１４に特異的な領域コードする塩基配列、1075番目から1143番目の配列が膜貫通領域コードする塩基配列に相当する。すなわち、本発明におけるシグレック１４タンパク質をコードするDNAは、配列番号２０の少なくとも712番目から1074番目および1075番目から1143番目の配列を含む塩基配列を含む。 As the DNA encoding Siglec 14 protein, DNA having the base sequence represented by SEQ ID NO: 20 is preferably used. The base sequence represented by SEQ ID NO: 20 includes an extracellular region, a transmembrane region, and an intracellular region of Siglec 14 protein. In the present invention, the DNA encoding Siglec 14 protein may contain at least a base sequence encoding a region specific to Siglec 14, and preferably contains at least a region specific to Siglec 14 and a transmembrane region. It only needs to contain the base sequence to be encoded. In SEQ ID NO: 20, the 712th to 1074th sequences correspond to a base sequence encoding a region specific to Siglec 14, and the 1075th to 1143th sequences correspond to a base sequence encoding a transmembrane region. That is, the DNA encoding the Siglec 14 protein in the present invention includes a base sequence including at least the 712th to 1074th and 1075th to 1143th sequences of SEQ ID NO: 20.
　また、シグレック１４タンパク質をコードするDNAには、配列番号２０、または配列番号２０の少なくとも712番目から1074番目および1075番目から1143番目の配列に示される配列を有するDNAにおいて、１から数個のヌクレオチドの欠失、置換、付加又は挿入を有し、かつ抗シグレック１４抗体に認識され結合され得るタンパク質をコードするDNAも含まれる。「１から数個」の範囲は特には限定されないが、例えば、１から20個、好ましくは１から10個、より好ましくは１から７個、さらに好ましくは１から５個、特に好ましくは１から３個、あるいは１個又は２個である。また、シグレック１４タンパク質をコードするDNAには、配配列番号２０、または配列番号２０の少なくとも712番目から1074番目および1075番目から1143番目の配列に示される配列からなるDNAに相補的な配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ抗シグレック１４抗体に認識され結合され得るタンパク質をコードするDNAも含まれる。ここで、ストリンジェントな条件とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいい、例えば、ナトリウム濃度が、10mM～300mM、好ましくは20～100mMであり、温度が25℃～70℃、好ましくは42℃～55℃での条件をいう。さらに、シグレック１４タンパク質をコードするDNAには配列番号２０、または配列番号２０の少なくとも712番目から1074番目および1075番目から1143番目の配列とBLAST等（例えば、デフォルトすなわち初期設定のパラメータ）を用いて計算したときに、80％以上、より好ましくは90％以上、最も好ましくは95％以上の同一性を有する塩基配列からなり、かつ抗シグレック１４抗体に認識され結合され得るタンパク質をコードするDNAも含まれる。 In addition, the DNA encoding Siglec 14 protein includes one to several nucleotides in DNA having the sequence shown in SEQ ID NO: 20, or at least the 712th to 1074th and 1075th to 1143th sequences of SEQ ID NO: 20. Also included are DNAs that encode proteins that have the following deletions, substitutions, additions or insertions and that can be recognized and bound by anti-Siglec 14 antibodies. The range of “1 to several” is not particularly limited, but for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 1. Three, or one or two. Further, the DNA encoding Siglec 14 protein is composed of a sequence complementary to the DNA consisting of the sequence shown in SEQ ID NO: 20, or at least the 712th to 1074th and 1075th to 1143th sequences of SEQ ID NO: 20. Also included are DNAs that encode proteins that hybridize with DNA under stringent conditions and that can be recognized and bound by anti-Siglec 14 antibodies. Here, the stringent condition refers to a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, the sodium concentration is 10 mM to 300 mM, preferably 20 to 100 mM, and the temperature Refers to conditions at 25 ° C. to 70 ° C., preferably 42 ° C. to 55 ° C. Further, for DNA encoding Siglec 14 protein, SEQ ID NO: 20, or at least the 712th to 1074th and 1075th to 1143th sequences of SEQ ID NO: 20, BLAST and the like (for example, default or default parameters) are used. Also included is a DNA encoding a protein comprising a base sequence having an identity of 80% or more, more preferably 90% or more, and most preferably 95% or more when calculated, and which can be recognized and bound by an anti-Siglec 14 antibody. It is.
　当該シグレック１４タンパク質をコードするDNAを、公知の発現ベクターに挿入する。 The DNA encoding the Siglec 14 protein is inserted into a known expression vector.
　本発明に用いることができるベクターとしては、プラスミドベクター、ウイルスベクター(例えば、アデノウイルスベクター、レトロウイルスベクター、レンチウイルスベクター)、非ウイルスベクターなどを用いることができるが、好ましくはウイルスベクターである。 As a vector that can be used in the present invention, a plasmid vector, a viral vector (for example, an adenoviral vector, a retroviral vector, a lentiviral vector), a non-viral vector, and the like can be used, and a viral vector is preferable.
　発現ベクターには、シグレック１４タンパク質を発現するためにいくつかの制御エレメントを含むことができる。制御エレメントには、例えばプロモーター、エンハンサー、ポリＡ付加シグナル、複製開始点、選択マーカー、リボソーム結合配列、ターミネーターなどが含まれる。 The expression vector can contain several regulatory elements to express the Siglec 14 protein. Control elements include, for example, promoters, enhancers, poly A addition signals, replication origins, selectable markers, ribosome binding sequences, terminators, and the like.
　発現ベクターを用いた宿主細胞の形質転換又はトランスフェクションとしては、当業者に公知の一般的な手法を用いることが可能であり、例えばエレクトロポレーション法、スフェロプラスト法、酢酸リチウム法、リン酸カルシウム法、アグロバクテリウム法、ウイルス感染法、リポソーム法、マイクロインジェクション法、遺伝子銃法、リポフェクション法などが挙げられる。 For transformation or transfection of host cells using an expression vector, general techniques known to those skilled in the art can be used. For example, electroporation method, spheroplast method, lithium acetate method, calcium phosphate method , Agrobacterium method, virus infection method, liposome method, microinjection method, gene gun method, lipofection method and the like.
　宿主細胞としては、健常人の血液、血清および／または血漿との反応性が低い哺乳動物細胞（好ましくはヒト細胞）を利用することが可能であり、特に好ましくはK562細胞を用いる。 As a host cell, a mammalian cell (preferably a human cell) having low reactivity with blood, serum and / or plasma of a healthy person can be used, and K562 cell is particularly preferably used.
　本発明のシグレック１４タンパク質に対する抗体を検出するための試薬には、上記シグレック１４タンパク質又は上記シグレック１４タンパク質発現細胞の他に、通常用いられている賦形剤、増量剤、結合剤、浸潤剤、崩壊剤、表面活性剤、滑沢剤、分散剤、緩衝剤、保存剤、溶解補助剤、防腐剤、着色料、及び安定化剤などを含んでも良い。 In addition to the Siglec 14 protein or the Siglec 14 protein-expressing cells, the reagent for detecting an antibody against the Siglec 14 protein of the present invention includes commonly used excipients, extenders, binders, infiltrant, Disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, preservatives, colorants, stabilizers, and the like may also be included.
　本発明のシグレック１４タンパク質に対する抗体を検出するための試薬を用いて、ヒト血液試料、好ましくは輸血用血液製剤或いは非溶血性輸血副作用患者等の血液に含まれる抗シグレック１４抗体を検出することができる。 Using the reagent for detecting an antibody against the Siglec 14 protein of the present invention, it is possible to detect an anti-Siglec 14 antibody contained in the blood of a human blood sample, preferably a blood product for blood transfusion or a non-hemolytic blood transfusion patient. it can.
　また、本発明のシグレック１４タンパク質に対する抗体を検出するための試薬を用いて、抗シグレック１４抗体を含むヒト血液試料、好ましくは輸血用血液製剤を判別し、除去することが可能であり、非溶血性輸血副作用を防止し輸血の安全性を確保するために利用することができる。 In addition, it is possible to discriminate and remove a human blood sample containing anti-Siglec 14 antibody, preferably a blood product for blood transfusion, using the reagent for detecting an antibody against Siglec 14 protein of the present invention. It can be used to prevent sexual transfusion side effects and ensure the safety of transfusion.
　当該抗体の検出には、抗体結合を検出するための一般的な手法を用いることが可能であり、例えば、免疫組織化学、ウェスタンブロット、酵素免疫測定法（EIA）(酵素結合イムノソルベント検定法(ELISA)等)、ラジオイムノアッセイ（RIA）及びフローサイトメーター(FCM)等を用いることができる。例えば、本発明のシグレック１４タンパク質に対する抗体を検出するための試薬と血液試料、好ましくは血液製剤とを、4℃～37℃にて0.25時間～一晩、好ましくは4℃にて15分間インキュベートして、シグレック１４タンパク質又はシグレック１４タンパク質発現細胞と血液試料、好ましくは血液製剤中に含まれる抗シグレック１４抗体とを反応させる。次に、シグレック１４タンパク質又はシグレック１４タンパク質発現細胞へ結合した抗シグレック１４抗体を上記方法により検出する。 For detection of the antibody, a general technique for detecting antibody binding can be used. For example, immunohistochemistry, Western blot, enzyme immunoassay (EIA) (enzyme-linked immunosorbent assay ( ELISA), radioimmunoassay (RIA), flow cytometer (FCM) and the like. For example, a reagent for detecting an antibody against the Siglec 14 protein of the present invention and a blood sample, preferably a blood product, are incubated at 4 ° C. to 37 ° C. for 0.25 hour to overnight, preferably at 4 ° C. for 15 minutes. Then, Siglec 14 protein or Siglec 14 protein-expressing cells are reacted with a blood sample, preferably an anti-Siglec 14 antibody contained in a blood product. Next, the Siglec 14 protein or the anti-Siglec 14 antibody bound to the Siglec 14 protein-expressing cell is detected by the above method.
　下記実施例にて詳述されるように、非溶血性輸血副作用に関与した血液試料と一般献血試料を比較すると、シグレック１４に対するアロ抗体を含む頻度が前者において有意に高い。 As described in detail in the Examples below, when comparing blood samples involved in non-hemolytic transfusion side effects and general blood donated samples, the frequency of containing alloantibodies against Siglec 14 is significantly higher in the former.
　以下に本発明の実施例を示すが、本発明はこれら実施例により限定されるものではない。 Examples of the present invention are shown below, but the present invention is not limited to these examples.
　シグレック１４特異的な抗体を用いた白血球のフローサイトメトリーによる解析
　ヒト末梢血由来白血球をシグレック５タンパク質とシグレック１４タンパク質の両者を認識する抗体（clone 194128, R&D Systems;上段）、シグレック５タンパク質を特異的に認識する抗体（clone 4H7;中段）、シグレック１４タンパク質を特異的に認識する抗体（下段）でそれぞれ染色し、フローサイトメトリーで解析した。結果を図１に示す。なお、白血球の副集団（各系統マーカーによる染色性に基づいて判定）ごとに表示した。図中、灰色はコントロール抗体で染色した細胞集団のヒストグラムである。mDC：骨髄球系樹状細胞、pDC：形質細胞様樹状細胞である。 Analysis of leukocytes using Siglec 14 specific antibodies by flow cytometry Human peripheral blood leukocytes recognize both Siglec 5 protein and Siglec 14 protein (clone 194128, R & D Systems; upper), Siglec 5 protein specific Which were specifically recognized (clone 4H7; middle panel) and an antibody specifically recognizing Siglec 14 protein (bottom panel) were stained and analyzed by flow cytometry. The results are shown in FIG. In addition, it displayed for every subpopulation of leukocytes (determined based on the stainability by each system | strain marker). In the figure, gray is a histogram of the cell population stained with the control antibody. mDC: myeloid dendritic cell, pDC: plasmacytoid dendritic cell.
　これによれば、シグレック１４タンパク質はT細胞を除く広範な白血球サブセットに発現していた。シグレック１４タンパク質の発現パターンはシグレック５タンパク質のそれと重複していた。 According to this, Siglec 14 protein was expressed in a wide range of leukocyte subsets excluding T cells. The expression pattern of Siglec 14 protein overlapped with that of Siglec 5 protein.
　本実施例で行った実験手法の詳細は以下のとおりである。 The details of the experimental method performed in this example are as follows.
　抗体の作製：
　抗シグレック１４抗体（ウサギポリクローナル抗体）は以下の方法で作製した。 Antibody production:
Anti-Siglec 14 antibody (rabbit polyclonal antibody) was prepared by the following method.
　ヒトシグレック１４タンパク質の細胞外領域（配列番号４のアミノ酸残基1～342番目）とFLAG配列（DYKDDDDK）(配列番号１８)及びヒト免疫グロブリンGのヒンジ＋Fc領域からなるシグレック１４-Fc融合タンパク質を発現するベクターを作製し、293細胞又はCHO細胞に導入し、培養上清から目的の融合タンパク質数ミリグラムを得た（特許文献４）。同様の方法により、ヒトシグレック５タンパク質の細胞外領域（配列番号５のアミノ酸残基1～334番目又は1～427番目）とFLAG配列及びヒト免疫グロブリンGのヒンジ＋Fc領域からなるシグレック５-Fc融合タンパク質を発現するベクターを作製し、293細胞又はCHO細胞に導入し、培養上清から目的の融合タンパク質数ミリグラムを得た。これらシグレック１４-Fc融合タンパク質、シグレック５-Fc融合タンパク質をそれぞれAffiGel 15（Bio-Rad社）に固相化（1 mg/0.5 ml）し、アフィニティ精製用の担体を作製した。 A siglec 14-Fc fusion protein comprising the extracellular region of human Siglec 14 protein (amino acid residues 1 to 342 of SEQ ID NO: 4), the FLAG sequence (DYKDDDDK) (SEQ ID NO: 18), and the hinge + Fc region of human immunoglobulin G A vector to be expressed was prepared and introduced into 293 cells or CHO cells, and several milligrams of the desired fusion protein was obtained from the culture supernatant (Patent Document 4). In a similar manner, the Siglec 5-Fc fusion consisting of the extracellular region of human Siglec 5 protein (amino acid residues 1 to 334 or 1-427 of SEQ ID NO: 5), the FLAG sequence and the hinge + Fc region of human immunoglobulin G A vector expressing the protein was prepared and introduced into 293 cells or CHO cells, and several milligrams of the desired fusion protein were obtained from the culture supernatant. These Siglec 14-Fc fusion protein and Siglec 5-Fc fusion protein were each immobilized on AffiGel 15 (Bio-Rad) (1 mg / 0.5 ml) to prepare a carrier for affinity purification.
　一方、シグレック１４-Fc融合タンパク質をエンテロキナーゼ処理したのち、ヒト免疫グロブリンGのヒンジ＋Fc領域をprotein A-Sepharoseとのインキュベーションにより除去し、ヒトシグレック１４タンパク質の細胞外領域とFLAG配列からなる部分タンパク質を得た。この部分タンパク質を常法に従ってウサギに免疫し、抗血清を得た。 On the other hand, after treating the Siglec 14-Fc fusion protein with enterokinase, the hinge + Fc region of human immunoglobulin G was removed by incubation with protein A-Sepharose, and the partial protein consisting of the extracellular region of human Siglec 14 protein and the FLAG sequence Got. Rabbits were immunized with this partial protein according to a conventional method to obtain antiserum.
　上記で作製したシグレック１４-Fc融合タンパク質を固相化したアフィニティ精製用の担体と抗血清を混合し、4℃で終夜インキュベートした。担体に結合しなかったタンパク質を洗浄用バッファー（ダルベッコPBS）により除去したのち、担体に結合した抗体を溶出用バッファー（0.1 Mグリシン塩酸バッファー、pH 3.0）を用いて溶出し、中和バッファー（1 Mトリス塩酸、pH 8.0）で中和した。得られた抗体画分（シグレック１４-Fc融合タンパク質を認識する抗体）のバッファーを限外濾過によりダルベッコPBSに置換した後、上記で作製したシグレック５-Fc融合タンパク質を固相化したアフィニティ精製用の担体と混合し、4℃で終夜インキュベートした。担体に結合しなかった抗体（すなわちシグレック１４-Fc融合タンパク質を認識するがシグレック５-Fc融合タンパク質を認識しない抗体）を回収し、シグレック１４タンパク質特異的な抗体とした。必要な場合はシグレック５-Fc融合タンパク質を固相化したアフィニティ精製用の担体と再度インキュベートし、シグレック５タンパク質と交差反応する抗体画分を除去した。 The carrier for affinity purification on which the Siglec 14-Fc fusion protein prepared above was immobilized and antiserum were mixed and incubated at 4 ° C. overnight. After removing the protein not bound to the carrier with a washing buffer (Dulbecco PBS), the antibody bound to the carrier is eluted with an elution buffer (0.1 M glycine hydrochloride buffer, pH 3.0), and neutralized buffer (1 Neutralized with M Tris-HCl, pH 8.0). The buffer of the obtained antibody fraction (an antibody recognizing the Siglec 14-Fc fusion protein) was replaced with Dulbecco's PBS by ultrafiltration, and then the Siglec 5-Fc fusion protein prepared above was immobilized for affinity purification. And incubated at 4 ° C. overnight. An antibody that did not bind to the carrier (that is, an antibody that recognizes the Siglec 14-Fc fusion protein but does not recognize the Siglec 5-Fc fusion protein) was recovered and used as an antibody specific to the Siglec 14 protein. When necessary, the antibody fraction that cross-reacts with the Siglec 5 protein was removed by incubating again with the carrier for affinity purification in which the Siglec 5-Fc fusion protein was immobilized.
　抗シグレック５抗体（マウスモノクローナル抗体）は以下のような方法で作製した。 Anti-Siglec 5 antibody (mouse monoclonal antibody) was prepared by the following method.
　ヒトシグレック５タンパク質のシグナルペプチド、リンカー領域、免疫グロブリン様領域3及び4（配列番号５のアミノ酸残基1～19＋234～427番目）とFLAG配列及びヒト免疫グロブリンGのヒンジ＋Fc領域からなるシグレック５-Fc融合タンパク質を発現するベクターを作製し、293細胞又はCHO細胞に導入し、培養上清から目的の融合タンパク質数ミリグラムを得た。かかるシグレック５-Fc融合タンパク質をエンテロキナーゼ処理したのち、ヒト免疫グロブリンGのヒンジ＋Fc領域をprotein A-Sepharoseとのインキュベーションにより除去し、ヒトシグレック５タンパク質の免疫グロブリン様領域3及び4とFLAG配列からなる部分タンパク質を得た。この部分タンパク質を常法に従ってBALB/cマウス数匹に免疫し、脾臓を採取し、ポリエチレングリコールを用いて脾細胞とマウス由来ミエローマ細胞（P3U1細胞株）を融合した。HAT選択により得られたハイブリドーマの上清を用いてシグレック５タンパク質及びシグレック１４タンパク質に対する反応性を検討し、シグレック５タンパク質のみを認識する抗体を産生する株を選択した。更に限界希釈法により細胞を単クローン化し、シグレック５タンパク質を認識しシグレック１４タンパク質を認識しない抗体を産生するハイブリドーマのクローン（4H7）を得た。 Siglec 5- consisting of human Siglec 5 protein signal peptide, linker region, immunoglobulin-like regions 3 and 4 (amino acid residues 1 to 19 + 234 to 427 of SEQ ID NO: 5), FLAG sequence and human immunoglobulin G hinge + Fc region A vector expressing Fc fusion protein was prepared and introduced into 293 cells or CHO cells, and several milligrams of the desired fusion protein were obtained from the culture supernatant. After this siglec 5-Fc fusion protein is treated with enterokinase, the hinge + Fc region of human immunoglobulin G is removed by incubation with protein A-Sepharose, and the immunoglobulin-like regions 3 and 4 of human siglec 5 protein and the FLAG sequence are removed. A partial protein was obtained. According to a conventional method, several BALB / c mice were immunized with this partial protein, spleens were collected, and spleen cells and mouse-derived myeloma cells (P3U1 cell line) were fused using polyethylene glycol. Using the supernatant of the hybridoma obtained by HAT selection, the reactivity to Siglec 5 protein and Siglec 14 protein was examined, and a strain producing an antibody that recognizes only Siglec 5 protein was selected. The cells were further cloned by the limiting dilution method to obtain a hybridoma clone (4H7) that produces an antibody that recognizes Siglec 5 protein but does not recognize Siglec 14 protein.
　白血球の染色と解析：
　健常人ドナーから同意のもと末梢血数十mlを採取し、ACK溶血液（150 mM塩化アンモニウム、10 mM炭酸水素カリウム、0.1 mMエチレンジアミン四酢酸）を用いて赤血球を溶血させた。この液から遠心操作により得られた白血球を数回染色用バッファー（1%ウシ血清アルブミン、0.02%アジ化ナトリウムを含むダルベッコPBS）で洗浄した後、ヒトIgG（1 mg/ml）を含む染色用バッファーに107細胞/mlになるよう懸濁して氷浴上でインキュベートし、Fcγ受容体をブロックした。細胞懸濁液を3等分し、抗シグレック５抗体（クローン4H7）とアロフィコシアニン標識抗マウスIgG1抗体Fab断片（Zenon Mouse IgG1 Labeling Kit、Invitrogen社）の複合体、又はシグレック５とシグレック１４の両者を認識する市販抗体（R&D Systems社、クローン194128）とアロフィコシアニン標識抗マウスIgG1抗体Fab断片（Zenon Mouse IgG1 Labeling Kit、Invitrogen社）の複合体、又は抗シグレック１４ウサギポリクローナル抗体とアロフィコシアニン標識抗ウサギIgG抗体Fab断片（Zenon Rabbit IgG Labeling Kit、Invitrogen社）の複合体と氷浴上でインキュベートした。遠心操作により細胞を集め、さらに染色用バッファーで洗浄した後、各画分を細胞系譜マーカー抗体（フィコエリスリン標識した抗CD4抗体、抗CD8抗体、抗CD19抗体、抗CD56抗体、抗CD14抗体、抗BDCA-1抗体、又は抗BDCA-2抗体）を添加し、氷浴上でインキュベートした。遠心操作により細胞を集め、さらに染色用バッファーで洗浄した後、染色用バッファーに懸濁し、488 nm及び633 nmのレーザーを搭載したフローサイトメーター（BD Flowcytometry社製FACSAria）を用いて解析した。 Leukocyte staining and analysis:
Tens of milliliters of peripheral blood was collected from a healthy donor with consent, and erythrocytes were hemolyzed using ACK hemolyzed blood (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM ethylenediaminetetraacetic acid). Leukocytes obtained by centrifugation from this solution are washed several times with a staining buffer (Dulbecco PBS containing 1% bovine serum albumin and 0.02% sodium azide), and then stained for human IgG (1 mg / ml). The suspension was suspended at 10 7 cells / ml in a buffer and incubated on an ice bath to block the Fcγ receptor. Divide the cell suspension into 3 equal parts, and a complex of anti-Siglec 5 antibody (clone 4H7) and allophycocyanin-labeled anti-mouse IgG1 antibody Fab fragment (Zenon Mouse IgG1 Labeling Kit, Invitrogen), or both Siglec 5 and Siglec 14 A complex of a commercially available antibody (R & D Systems, clone 194128) that recognizes and an allophycocyanin-labeled anti-mouse IgG1 antibody Fab fragment (Zenon Mouse IgG1 Labeling Kit, Invitrogen), or an anti-Siglec 14 rabbit polyclonal antibody and an allophycocyanin-labeled anti-rabbit IgG antibody Fab fragment (Zenon Rabbit IgG Labeling Kit, Invitrogen) was incubated with an ice bath. Cells were collected by centrifugation and further washed with a staining buffer, and then each fraction was divided into cell lineage marker antibodies (anti-CD4 antibody, anti-CD8 antibody, anti-CD19 antibody, anti-CD56 antibody, anti-CD14 antibody labeled with phycoerythrin) Anti-BDCA-1 antibody or anti-BDCA-2 antibody) was added and incubated on an ice bath. Cells were collected by centrifugation, further washed with a staining buffer, suspended in the staining buffer, and analyzed using a flow cytometer (FACSAria manufactured by BD Flowcytometry) equipped with 488 nm and 633 nm lasers.
シグレック１４遺伝子の多型の解析
　１）シグレック１４タンパク質を発現しない個人の同定
　複数の健常人ドナーから末梢血を採取し、実施例１と同様の方法を用いて顆粒球におけるシグレック１４タンパク質及びシグレック５タンパク質発現のフローサイトメトリーによる解析を行った。結果を図２Aに示す。これによれば、全ての個人はシグレック５タンパク質（又はこれと同一のアミノ酸配列を有するタンパク質）を発現するが、ドナー3と5はシグレック１４タンパク質を発現していない。 Analysis of polymorphisms of siglec 14 gene 1) Identification of individuals who do not express siglec 14 protein Peripheral blood was collected from a plurality of healthy donors and siglec 14 protein and siglec 5 in granulocytes were collected using the same method as in Example 1. Analysis of protein expression by flow cytometry was performed. The results are shown in FIG. 2A. According to this, all individuals express Siglec 5 protein (or a protein having the same amino acid sequence), but donors 3 and 5 do not express Siglec 14 protein.
　２）シグレック１４タンパク質の発現とシグレック１４遺伝子の多型の関連の解析
　シグレック１４遺伝子とシグレック５遺伝子は近接しており、両者の間に極めて相同性の高い領域が存在することから、この2つの遺伝子間で組み換えが起こり、シグレック１４／５融合遺伝子が生じた（その産物のアミノ酸配列はシグレック５タンパク質のそれと同一である）と仮定し、シグレック１４遺伝子、シグレック５遺伝子、シグレック１４／５融合遺伝子をそれぞれ特異的に増幅する以下に示すプライマーセットを用いたゲノムPCRを行った。ゲノムPCRの概略図を図２Bに、PCR産物の電気泳動像の模式図を図２Cに示す。 2) Analysis of the relationship between the expression of the Siglec 14 protein and the polymorphism of the Siglec 14 gene The Siglec 14 gene and the Siglec 5 gene are close to each other, and there is a region with extremely high homology between them. Assuming that recombination has occurred between the genes and a Siglec 14/5 fusion gene has been generated (the amino acid sequence of the product is the same as that of the Siglec 5 protein), the Siglec 14 gene, the Siglec 5 gene, the Siglec 14/5 fusion gene Genomic PCR was performed using the primer sets shown below that specifically amplify each of. A schematic diagram of genomic PCR is shown in FIG. 2B, and a schematic diagram of an electrophoresis image of the PCR product is shown in FIG. 2C.
　シグレック１４遺伝子増幅用プライマー
　フォワードプライマー（14F）
　AGGATTTATTCTCCCATCTCGCT（配列番号７）
　リバースプライマー（14R）
　GATGCTGATGGCGAGGTTCTG（配列番号８）
　シグレック５遺伝子増幅用プライマー
　フォワードプライマー（5F）
　GTGGTTCTGACATCTCACCTCATC（配列番号９）
　リバースプライマー（5R）
　CCTGAAGATGGTGATGGTCTG（配列番号１０）
　シグレック１４／５融合遺伝子増幅用プライマー
　フォワードプライマー（14F）
　AGGATTTATTCTCCCATCTCGCT（配列番号７）
　リバースプライマー（5R）
　CCTGAAGATGGTGATGGTCTG（配列番号１０）
　プライマー対5F＋5Rはシグレック５遺伝子の一部を、プライマー対14F＋14Rはシグレック１４遺伝子の一部を特異的に増幅する。プライマー対14F + 5Rはシグレック１４／５融合遺伝子が存在する場合にはこの遺伝子の約1.7 kbの領域を増幅する。同じプライマー対を用いて野生型アリルから約17 kbの断片が生じうるが、本実験で用いた反応条件下ではこの産物は生じない。 Siglec 14 gene amplification primer Forward primer (14F)
AGGATTTATTCTCCCATCTCGCT (SEQ ID NO: 7)
Reverse primer (14R)
GATGCTGATGGCGAGGTTCTG (SEQ ID NO: 8)
Siglec 5 gene amplification primer Forward primer (5F)
GTGGTTCTGACATCTCACCTCATC (SEQ ID NO: 9)
Reverse primer (5R)
CCTGAAGATGGTGATGGTCTG (SEQ ID NO: 10)
Siglec 14/5 Fusion Gene Amplification Primer Forward Primer (14F)
AGGATTTATTCTCCCATCTCGCT (SEQ ID NO: 7)
Reverse primer (5R)
CCTGAAGATGGTGATGGTCTG (SEQ ID NO: 10)
The primer pair 5F + 5R specifically amplifies a part of the Siglec 5 gene, and the primer pair 14F + 14R specifically amplifies a part of the Siglec 14 gene. Primer pair 14F + 5R amplifies the approximately 1.7 kb region of this gene when the Siglec 14/5 fusion gene is present. An approximately 17 kb fragment can be generated from wild-type allele using the same primer pair, but this product does not occur under the reaction conditions used in this experiment.
　図２Ａと同じ個人のゲノムDNAを用いた遺伝子型解析の結果を図２Ｄに示す。予想通りドナー3と5はシグレック１４／５融合遺伝子のホモ接合体であった。 FIG. 2D shows the results of genotyping using the same individual genomic DNA as FIG. 2A. As expected, donors 3 and 5 were homozygous for the Siglec 14/5 fusion gene.
　本実施例で行った実験手法の詳細は以下のとおりである。 The details of the experimental method performed in this example are as follows.
　プライマーのデザイン：
　ゲノムPCR用のプライマーのデザインは下記のように行った。シグレック１４遺伝子とシグレック５遺伝子をそれぞれ特異的に増幅するためのプライマーは、シグレック１４遺伝子とシグレック５遺伝子の間で高度の配列相同性を示す領域の上流（フォワードプライマー）と下流（リバースプライマー）に設定しなければならない。高度に保存された領域はシグレック１４遺伝子では配列番号１の７５３番目～２１１０番目の塩基、シグレック５遺伝子では配列番号２の７５３番目～２１１４番目の塩基である。またDNAの増幅効率に鑑み、プライマー対で増幅するべき領域は短い方が望ましい。以上の要件から、シグレック１４遺伝子を特異的に増幅するプライマー対としてはフォワードプライマーを配列番号１の５８８番目～６１０番目の塩基配列、リバースプライマーを配列番号１の２２３８番目～２２５８番目の塩基の相補配列とした。またシグレック５遺伝子を特異的に増幅するプライマー対としてはフォワードプライマーを配列番号２の６０５番目～６２８番目の塩基配列、リバースプライマーを配列番号２の２２６８番目～２２８８番目の塩基の相補配列とした。シグレック１４／５融合遺伝子の検出には、シグレック１４遺伝子の増幅に用いたフォワードプライマーとシグレック５遺伝子の増幅に用いたリバースプライマーを用いることとした。 Primer design:
Primer primers for genomic PCR were designed as follows. Primers for specifically amplifying the Siglec 14 gene and the Siglec 5 gene respectively are upstream (forward primer) and downstream (reverse primer) of a region showing a high degree of sequence homology between the Siglec 14 gene and the Siglec 5 gene. Must be set. The highly conserved regions are the 753rd to 2110th bases of SEQ ID NO: 1 for the Siglec 14 gene, and the 753rd to 2114th bases of SEQ ID NO: 2 for the Siglec 5 gene. In view of DNA amplification efficiency, it is desirable that the region to be amplified by the primer pair is short. Based on the above requirements, as a primer pair for specifically amplifying the Siglec 14 gene, the forward primer is the complement of the nucleotide sequence from 588 to 610 of SEQ ID NO: 1, and the reverse primer is the complement of the nucleotide from 2238 to 2258 of SEQ ID NO: 1. It was an array. In addition, as a primer pair for specifically amplifying the Siglec 5 gene, the forward primer was the nucleotide sequence of the 605th to 628th nucleotides of SEQ ID NO: 2, and the reverse primer was the complementary sequence of the 2268th to 2288th nucleotides of the SEQ ID NO: 2. For detection of the Siglec 14/5 fusion gene, the forward primer used for the amplification of the Siglec 14 gene and the reverse primer used for the amplification of the Siglec 5 gene were used.
　ゲノムDNAの調製：
　健常人ドナー5名から同意のもと末梢血約10 mlを採取し、ACK溶血液（150 mM塩化アンモニウム、10 mM炭酸水素カリウム、0.1 mMエチレンジアミン四酢酸）を用いて赤血球を溶血させた。この液から遠心操作により白血球を得た。得られた白血球（5 x 106細胞）からDNeasy Blood & Tissue Kit（QIAGEN社）を用いてゲノムDNAを精製した。 Genomic DNA preparation:
About 10 ml of peripheral blood was collected from 5 healthy donors with consent, and erythrocytes were hemolyzed using ACK hemolyzed blood (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM ethylenediaminetetraacetic acid). White blood cells were obtained from this solution by centrifugation. Genomic DNA was purified from the obtained white blood cells (5 × 10 6 cells) using DNeasy Blood & Tissue Kit (QIAGEN).
　PCR:
　PCR反応には下記のような組成の反応液（20μl）を用いた。すなわち、ゲノムDNA 100 ng、プライマー各0.3μM、デオキシリボヌクレオシド三リン酸（dATP、dCTP、dGTP、dTTP）各0.2 mM、耐熱性DNAポリメラーゼExpand High Fidelity enzyme（ロシュ・ダイアグノスティクス）0.5ユニット、塩化マグネシウム1.5 mM、1 x Expand High Fidelity Buffer（ロシュ・ダイアグノスティクス）。 PCR:
For the PCR reaction, a reaction solution (20 μl) having the following composition was used. That is, genomic DNA 100 ng, each primer 0.3 μM, deoxyribonucleoside triphosphate (dATP, dCTP, dGTP, dTTP) 0.2 mM each, thermostable DNA polymerase Expand High Fidelity enzyme (Roche Diagnostics) 0.5 unit, magnesium chloride 1.5 mM, 1 x Expand High Fidelity Buffer (Roche Diagnostics).
　反応サイクルは下記の条件を用いた。 The following conditions were used for the reaction cycle.
94℃、2分
続いて（94℃、15秒；56℃、30秒；72℃、90秒）を10サイクル
続いて（94℃、15秒；56℃、30秒；72℃、90秒＋各サイクル5秒ずつ延長）を20サイクル
続いて72℃、7分
　アガロースゲル電気泳動：
　上記のPCR反応産物（各レーン10μl）を、1%アガロースゲルを用いてTAEバッファー（40 mMトリス、40 mM酢酸、0.1 mMエチレンジアミン四酢酸）中で電気泳動し、反応産物を分離した。エチジウムブロマイドでゲルを染色し、紫外線ランプで照射してDNAを検出し、撮影した。 94 ° C, 2 minutes followed (94 ° C, 15 seconds; 56 ° C, 30 seconds; 72 ° C, 90 seconds) followed by 10 cycles (94 ° C, 15 seconds; 56 ° C, 30 seconds; 72 ° C, 90 seconds + Extend each cycle for 5 seconds) 20 cycles followed by 72 ° C, 7 minutes Agarose gel electrophoresis:
The PCR reaction product (10 μl in each lane) was electrophoresed in TAE buffer (40 mM Tris, 40 mM acetic acid, 0.1 mM ethylenediaminetetraacetic acid) using a 1% agarose gel to separate the reaction products. The gel was stained with ethidium bromide, and irradiated with an ultraviolet lamp to detect DNA and photographed.
シグレック１４タンパク質強制発現によるTNF-αの発現増強
　１）ヒト単球系細胞株であるTHP-1細胞に、それぞれシグレック５タンパク質、シグレック１４タンパク質の野生型又は点変異体（R119A又はR362A）を発現するcDNAを導入し、空ベクター導入細胞とともに、マクロファージ様細胞に分化させた後、リポ多糖を加えてTNF-αの産生量を測定した。結果を図３Ａに示す。 Enhanced expression of TNF-α by forced expression of Siglec 14 protein 1) Expression of Siglec 5 protein, Siglec 14 protein wild type or point mutant (R119A or R362A) in THP-1 cells, a human monocytic cell line, respectively After introducing the cDNA to be differentiated into macrophage-like cells together with empty vector-introduced cells, lipopolysaccharide was added and the amount of TNF-α produced was measured. The results are shown in FIG. 3A.
　これによれば、シグレック１４タンパク質を発現する細胞ではコントロールの細胞と比較してTNF-αの産生が約2倍に亢進していた。 According to this, TNF-α production was increased about twice in cells expressing Siglec 14 protein compared to control cells.
　なお、図中、統計的有意差（StudentのT検定にてP < 0.01）を示した組を***で示した。シグレック１４タンパク質の野生型及びR119A変異体を発現する細胞におけるTNF-α産生が他の細胞に比べて有意に高いことが明らかである。シグレック１４タンパク質のR119A変異体は糖鎖認識能を欠損し、R362A点変異体はシグナルアダプタータンパク質DAP12との結合能を欠損する。上記の結果はシグレック１４を介したTNF-α産生増強にはDAP12との会合が必須であるが、糖鎖認識能は必須ではないことを示唆する。 In the figure, *** indicates a statistically significant difference (P <0.01 by Student's T test). It is clear that TNF-α production in cells expressing the wild type of Siglec 14 protein and the R119A mutant is significantly higher than in other cells. The R119A mutant of Siglec 14 protein lacks the ability to recognize sugar chains, and the R362A point mutant lacks the ability to bind to the signal adapter protein DAP12. The above results suggest that the association with DAP12 is essential for enhancing TNF-α production via Siglec 14, but the ability to recognize sugar chains is not essential.
　２）空ベクター、シグレック１４タンパク質の野生型又はR362A点変異体を発現するTHP-1細胞をマクロファージ様細胞に分化させ、シグナルアダプタータンパク質DAP12の発現を抑制するsiRNAを添加した後、リポ多糖を加えてTNF-αの産生量を測定した。結果を図３Bに示す。これによれば、野生型シグレック１４タンパク質を発現する細胞で、DAP12タンパク質の発現を抑制することにより、TNF-αの産生が抑制された。すなわちシグレック１４タンパク質を介したTNF-αの産生亢進にDAP12タンパク質が関与していることが強く示唆される。一方、同じsiRNAはコントロール細胞からのTNF-α産生を抑制しなかった。またコントロールsiRNAはTNF-αの産生量に影響を及ぼさなかった。図中、統計的有意差（StudentのT検定にてP < 0.01）を示した組を***で示した（ただし、同じ細胞に異なる処理を行った場合のみを比較した）。野生型シグレック１４タンパク質を発現する細胞にDAP12タンパク質の発現を抑制するsiRNAを導入することにより、統計的に有為なTNF-αの産生抑制が認められることが明らかである。 2) Differentiate THP-1 cells expressing wild vector of siglec 14 protein or R362A point mutant into macrophage-like cells, add siRNA that suppresses expression of signal adapter protein DAP12, and then add lipopolysaccharide The amount of TNF-α produced was measured. The results are shown in FIG. 3B. According to this, TNF-α production was suppressed by suppressing expression of DAP12 protein in cells expressing wild-type Siglec 14 protein. That is, it is strongly suggested that the DAP12 protein is involved in the enhancement of TNF-α production via the Siglec 14 protein. On the other hand, the same siRNA did not suppress TNF-α production from control cells. Control siRNA did not affect the amount of TNF-α produced. In the figure, *** indicates a statistically significant difference (P <0.01 by Student's T test) (however, only when the same cells were treated differently). It is clear that statistically significant suppression of TNF-α production is observed by introducing siRNA that suppresses expression of DAP12 protein into cells expressing wild-type Siglec 14 protein.
　本実施例で行った実験手法の詳細は以下のとおりである。 The details of the experimental method performed in this example are as follows.
　シグレック１４タンパク質及びシグレック５タンパク質発現用ベクターの作製：
野生型シグレック１４の全長cDNAをテンプレートに用い、シグレック１４ R119A点変異体（119番目のアルギニンのアラニンへの変異体）cDNAの作製には配列番号１１と１２のオリゴヌクレオチドを、シグレック１４ R362A点変異体（362番目のアルギニンのアラニンへの変異体）cDNAの作製には配列番号１３と１４のオリゴヌクレオチドを用いて、QuickChange II（Stragtagene）製品添付のプロトコルに従って変異を導入した。 Preparation of siglec 14 protein and siglec 5 protein expression vectors:
The full-length cDNA of wild-type Siglec 14 was used as a template. The siglec 14 R119A point mutant (119th arginine to alanine) cDNA was prepared by using the oligonucleotides of SEQ ID NOs: 11 and 12 and the Siglec 14 R362A point mutation. The mutant (the 362nd arginine to alanine) cDNA was prepared using the oligonucleotides of SEQ ID NOs: 13 and 14, and the mutation was introduced according to the protocol attached to the QuickChange II (Stragtagene) product.
　野生型シグレック１４、シグレック１４ R119A点変異体、シグレック１４ R362A点変異体とシグレック５の全長cDNAをそれぞれレトロウイルスベクターであるpMSCV-IRES-EGFP（自治医科大学・久米晃啓先生より恵与）に導入した。 Introduced wild-type siglec 14, siglec 14 R119A point mutant, siglec 14 R362A point mutant and siglec 5 full-length cDNA into pMSCV-IRES-EGFP, a retroviral vector (given from Dr. Toshihiro Kume, Jichi Medical University) did.
　pMSCV-IRES-EGFPはIRES（Internal Ribosome Entry Site）の下流にEGFP（Enhanced Green Fluorescent Protein）cDNAを有するため、IRESの上流に発現するべき遺伝子のcDNAを導入することにより、該遺伝子とEGFPの両者を１つのmRNAから発現することができる。よってEGFPの発現に基づいて目的遺伝子を発現する細胞の同定と選別が可能である。 Since pMSCV-IRES-EGFP has EGFP (Enhanced Green Fluorescent Protein) cDNA downstream of IRES (Internal Ribosome Entry Site), both cDNA and EGFP can be introduced by introducing the cDNA of the gene to be expressed upstream of IRES. Can be expressed from a single mRNA. Therefore, it is possible to identify and select cells that express the target gene based on the expression of EGFP.
　THP-1細胞への遺伝子導入
　THP-1細胞（ヒト単球系細胞株）はRPMI 1640培地＋10%ウシ胎児血清＋抗生物質（ペニシリン及びストレプトマイシン）を用いて培養した。上記のコンストラクト或いはベクターであるpMSCV-IRES-EGFPをレトロウイルスパッケージング細胞であるPLAT-A細胞（東京大学医科学研究所・北村俊雄先生より恵与）にリポフェクションにより導入し、レトロウイルス粒子を含む培養上清を得た。THP-1細胞への遺伝子導入はRetroNectin（タカラバイオ）存在下で上記のレトロウイルス粒子を含む培養上清と24時間インキュベートすることにより実施した。感染後の細胞を通常の培地で数日間培養して回復させた後、蛍光セルソーターを用いてEGFP陽性の細胞を選別・回収した。必要に応じてソーティングを繰り返し、95％以上の細胞がEGFP陽性細胞（すなわち目的のタンパク質を発現する細胞）からなる細胞集団を樹立した。 Gene introduction into THP-1 cells THP-1 cells (human monocytic cell line) were cultured using RPMI 1640 medium + 10% fetal bovine serum + antibiotics (penicillin and streptomycin). Introducing the above construct or vector pMSCV-IRES-EGFP into the retrovirus packaging cells PLAT-A cells (benefited by Prof. Toshio Kitamura, University of Tokyo) by lipofection and containing retroviral particles A culture supernatant was obtained. Gene transfer into THP-1 cells was carried out by incubating with the culture supernatant containing the retroviral particles in the presence of RetroNectin (Takara Bio) for 24 hours. After the infected cells were recovered by culturing in a normal medium for several days, EGFP positive cells were selected and collected using a fluorescent cell sorter. Sorting was repeated as necessary to establish a cell population in which 95% or more of the cells consisted of EGFP positive cells (that is, cells expressing the target protein).
　THP-1細胞のマクロファージへの分化誘導、LPS刺激とTNF-α産生の測定
　遺伝子導入したTHP-1細胞を96ウェルプレートに2 x 104細胞ずつ播種し、50 nMの12-O-テトラデカノイルホルボール 13-アセテートを含む培地0.1 ml中で4日間培養することにより、マクロファージ様細胞に分化させた。通常の培地で数回洗った後、大腸菌（O111:B4）由来のリポ多糖50 ng/mlを含む培地0.1 ml中で24時間培養した。培地中に分泌されたTNF-αはBD OptEIA Human TNF ELISA Set（BD Bioscience）を用いて定量した。 Induction of THP-1 cell differentiation into macrophages, LPS stimulation and measurement of TNF-α production Transfected THP-1 cells were seeded at 2 x 10 4 cells in a 96-well plate, and 50 nM 12-O-tetradeca Macrophage-like cells were differentiated by culturing in 0.1 ml of a medium containing noylphorbol 13-acetate for 4 days. After washing several times with a normal medium, the cells were cultured for 24 hours in 0.1 ml of a medium containing 50 ng / ml of lipopolysaccharide derived from E. coli (O111: B4). TNF-α secreted into the medium was quantified using BD OptEIA Human TNF ELISA Set (BD Bioscience).
　RNA干渉
　DAP12 mRNAの部分配列である5'-UAGAGCAACUGCAAUCGCUCUGGGC-3'（配列番号１９）を標的とするStealth RNA及びコントロールStealth RNAはInvitrogen社から購入した。上記と同様にしてTHP-1細胞をマクロファージ様細胞に分化誘導した後、Stealth RNA（終濃度100 nM）をLipofectamine RNAi MAXを用いて細胞に導入し、48時間培養した。通常の培地で数回洗った後、大腸菌（O111:B4）由来のリポ多糖50 ng/mlを含む培地0.1 ml中で24時間培養した。培地中に分泌されたTNF-αはBD OptEIA Human TNF ELISA Set（BD Bioscience）を用いて定量した。 RNA interference Stealth RNA and control Stealth RNA targeting 5′-UAGAGCAACUGCAAUCGCUCUGGGC-3 ′ (SEQ ID NO: 19), a partial sequence of DAP12 mRNA, were purchased from Invitrogen. In the same manner as above, THP-1 cells were induced to differentiate into macrophage-like cells, and then Stealth RNA (final concentration 100 nM) was introduced into the cells using Lipofectamine RNAi MAX and cultured for 48 hours. After washing several times with a normal medium, the cells were cultured for 24 hours in 0.1 ml of a medium containing 50 ng / ml of lipopolysaccharide derived from E. coli (O111: B4). TNF-α secreted into the medium was quantified using BD OptEIA Human TNF ELISA Set (BD Bioscience).
　シグレック１４／５融合遺伝子の世界各地のヒト集団における分布
　世界各地のヒト集団由来のゲノムDNA（Coriell Institute for Medical Researchより入手）を用い、上記実施例２と同様のゲノムPCRを行った。 Distribution of Siglec 14/5 fusion gene in human populations around the world Genomic PCR similar to Example 2 was performed using genomic DNA derived from human populations around the world (obtained from Coriell Institute for Medical Research).
　結果を表１に示す。シグレック１４／５融合遺伝子のホモ接合型は東南アジア、中国などアジアのヒト集団で高頻度に見出され、ヒト集団毎に異なるという結果が得られた。
Figure JPOXMLDOC01-appb-T000001
 The results are shown in Table 1. The homozygous form of the Siglec 14/5 fusion gene was frequently found in Asian human populations such as Southeast Asia and China, and it was found that the results differed from one human population to another.
Figure JPOXMLDOC01-appb-T000001
（シグレック５タンパク質又はシグレック１４タンパク質発現細胞）
　シグレック５、シグレック１４遺伝子導入細胞の選択
　シグレック１４遺伝子を導入する細胞株を選択するために、６種類の非接着性細胞及び５種類の接着性細胞と３種類の健常人血清（ノーマル血清）との反応性をフローサイトメーターで測定し、バックグラウンドシグナルの増大の有無をそれぞれ解析した。L-細胞、Hela細胞、293T細胞、3T3細胞、CHO細胞は、ノーマル血清と高い反応性を示し、バックグラウンドシグナルが上昇した。しかし、５種類の非接着性細胞（K562細胞、Jurkat細胞、THP-1細胞、Namalwa細胞、及びCMK細胞）と１種類の接着性細胞（Cos7細胞）は、ノーマル血清に対して殆ど反応性を示さず低いバックグラウンドシグナルであった。次に、HLA、Human Neutrophil Antigen (HNA)、シグレック５及びシグレック１４の発現によるバックグラウンドシグナルの増大の可能性を抗HLA抗体、抗HNA抗体、抗シグレック５抗体或いは抗シグレック１４抗体とそれぞれ反応させ検討したところ、K562細胞のみがいずれの血清とも反応せず低いバックグラウンドシグナルであった。以上の結果から、シグレック５もしくはシグレック１４遺伝子を導入する細胞株としてK562細胞を採用した。 (Siglec 5 protein or Siglec 14 protein expressing cells)
Selection of siglec 5 and siglec 14 gene-introduced cells In order to select a cell line into which siglec 14 gene is introduced, 6 types of non-adherent cells, 5 types of adherent cells and 3 types of normal human serum (normal serum) Were measured with a flow cytometer and analyzed for the presence or absence of an increase in background signal. L-cells, Hela cells, 293T cells, 3T3 cells, and CHO cells showed high reactivity with normal serum and increased background signal. However, five types of non-adherent cells (K562 cells, Jurkat cells, THP-1 cells, Namalwa cells, and CMK cells) and one type of adherent cells (Cos7 cells) are almost reactive to normal serum. Not shown, low background signal. Next, the possibility of increased background signal due to expression of HLA, Human Neutrophil Antigen (HNA), Siglec 5 and Siglec 14 is reacted with anti-HLA antibody, anti-HNA antibody, anti-Siglec 5 antibody or anti-Siglec 14 antibody, respectively. When examined, only K562 cells did not react with any serum and had a low background signal. From the above results, K562 cells were employed as a cell line for introducing Siglec 5 or Siglec 14 gene.
　シグレック５、シグレック１４発現細胞の調製方法
　シグレック５及びシグレック１４のcDNAは、IMAGEコンソーシアムによる全長cDNAクローンIMAGE:5178889およびIMAGE:5756894をそれぞれ用いた。それぞれのcDNAは、市販のレトロウィルスベクターであるpQCXIP（Becton Dickinson, SanJose, CA）の制限酵素EcoRIとNotIサイトにサブクローニングし、pQCXIP-シグレック５及びpQCXIP-シグレック１４を得た。次に、Lipofetamine Plus試薬（Invitrogen）の標準プロトコールに従い、pQCXIP-シグレック５或いはpQCXIP-シグレック１４とpVSV-G（Becton Dickinson）をgp-293Tパッケージング細胞株（Becton Dickinson）に遺伝子導入した。この際、Siglec遺伝子を導入していないpQCXIPのみを同様の操作で遺伝子導入したものも作製した。上記の遺伝子導入したgp-293T細胞を48時間培養し、105/mlの組換えウイルス粒子を含む上清を得た。この上清0.1mlに0.9mlの感染用培地（10％ FBSを含むRPMI1640培地に終濃度8μg/mlになるようにpolybreneを添加）に懸濁した1ｘ106個のK562細胞を加えて、２時間培養した後、R10培地（10％ FBSを含むRPMI1640培地）で２回洗浄し、R10培地で２日間培養した。次に、組換えウイルスを感染させた細胞のうちピューロマイシン耐性のものを限界希釈法でクローニングし、シグレック５或いはシグレック１４をそれぞれ発現する、KY-シグレック５、KY-シグレック１４及び、ベクターのみが導入されたKY-mock細胞株をそれぞれ得た（図４）。 Preparation of Siglec 5 and Siglec 14-expressing cells The full-length cDNA clones IMAGE: 5178889 and IMAGE: 5756894 from the IMAGE consortium were used as the cDNA for Siglec 5 and Siglec 14, respectively. Each cDNA was subcloned into restriction enzyme EcoRI and NotI sites of a commercially available retroviral vector pQCXIP (Becton Dickinson, San Jose, Calif.) To obtain pQCXIP-Siglec 5 and pQCXIP-Siglec 14. Next, according to the standard protocol of Lipofetamine Plus reagent (Invitrogen), pQCXIP-Siglec 5 or pQCXIP-Siglec 14 and pVSV-G (Becton Dickinson) were introduced into the gp-293T packaging cell line (Becton Dickinson). At this time, a product in which only pQCXIP into which no Siglec gene was introduced was introduced by the same operation was also prepared. The above-introduced gp-293T cells were cultured for 48 hours to obtain a supernatant containing 10 5 / ml recombinant virus particles. Add 1 x 10 6 K562 cells suspended in 0.9 ml of this supernatant to 0.9 ml of infection medium (adding polybrene to RPMI1640 medium containing 10% FBS to a final concentration of 8 μg / ml) for 2 hours. After culturing, the cells were washed twice with R10 medium (RPMI1640 medium containing 10% FBS) and cultured for 2 days in R10 medium. Next, puromycin-resistant cells infected with the recombinant virus are cloned by limiting dilution, and express only Siglec 5 or Siglec 14, respectively. KY-Siglec 5, KY-Siglec 14 and only vectors Each of the introduced KY-mock cell lines was obtained (FIG. 4).
（フローサイトメーター（FCM）による解析）
　ａ．KY-シグレック５細胞とKY-シグレック１４細胞のシグレック抗原発現の検討
KY-シグレック５細胞、KY-シグレック１４細胞及びKY-mock細胞に抗シグレック５/14抗体（シグレック５及びシグレック１４に反応する）、抗シグレック１４抗体（シグレック１４に反応する）を加え４℃で１５分間反応させた。PBSにて洗浄後、FITC標識抗マウスIgG抗体もしくはFITC標識抗ウサギIgG抗体で4℃、15分間染色し、PBSにて再度洗浄し、FCMを用いて解析した。その結果を図５に示す。シグレック５遺伝子を導入したKY-シグレック５細胞はシグレック５抗原を、シグレック１４遺伝子を導入したKY-シグレック１４細胞はシグレック１４抗原をそれぞれ特異的に発現していることが確認された。また、KY-mock細胞には非特異的な反応は見られなかった。 (Analysis by flow cytometer (FCM))
a. Examination of Siglec antigen expression in KY-Siglec 5 cells and KY-Siglec 14 cells
Add anti-Siglec 5/14 antibody (reacts to Siglec 5 and Siglec 14) and anti-Siglec 14 antibody (reacts to Siglec 14) to KY-Siglec 5 cells, KY-Siglec 14 cells and KY-mock cells at 4 ° C. The reaction was allowed for 15 minutes. After washing with PBS, it was stained with FITC-labeled anti-mouse IgG antibody or FITC-labeled anti-rabbit IgG antibody at 4 ° C. for 15 minutes, washed again with PBS, and analyzed using FCM. The result is shown in FIG. It was confirmed that the KY-Siglec 5 cells introduced with the Siglec 5 gene specifically expressed the Siglec 5 antigen, and the KY-Siglec 14 cells introduced with the Siglec 14 gene specifically expressed the Siglec 14 antigen. Moreover, no non-specific reaction was observed in KY-mock cells.
　ｂ．KY-シグレック５細胞及びKY-シグレック１４細胞を用いたヒト血清または血漿中の抗シグレック１４抗体検出
　ヒト血清または血漿中に含まれる抗シグレック１４アロ抗体を上記KY-シグレック５細胞株及びKY-シグレック１４細胞株にて検出した。すなわち、KY-シグレック５細胞、KY-シグレック１４細胞及びKY-mock細胞に非溶血性輸血副作用の原因製剤由来血漿（日本赤十字社より入手）を加え、4℃で15分間反応させた。PBSにて洗浄後、FITC標識抗ヒトIgG抗体又はPE標識抗ヒトIgM抗体で4℃、15分間染色し、PBSにて再度洗浄し、FCMを用いて解析した。その代表的なヒストグラムを図６に示す。 b. Detection of anti-Siglec 14 antibody in human serum or plasma using KY-Siglec 5 cell and KY-Siglec 14 cell Anti-Siglec 14 alloantibodies contained in human serum or plasma were detected as the KY-Siglec 5 cell line and KY-Siglec. Detection was performed in 14 cell lines. Specifically, plasma derived from a non-hemolytic transfusion-causing drug (obtained from the Japanese Red Cross) was added to KY-Siglec 5 cells, KY-Siglec 14 cells, and KY-mock cells, and reacted at 4 ° C. for 15 minutes. After washing with PBS, it was stained with FITC-labeled anti-human IgG antibody or PE-labeled anti-human IgM antibody at 4 ° C. for 15 minutes, washed again with PBS, and analyzed using FCM. A typical histogram is shown in FIG.
　さらに、非溶血性輸血副作用の原因製剤由来血漿（168種類）と健常人血清（500種類）とで、抗シグレック１４アロ抗体の検出割合を比較した。抗シグレック１４アロ抗体の陽性率は、健常人血清で2.4%（12/500）であるのに対して非溶血性輸血副作用原因製剤では11.9%（20/168）と有意に高かった（p<0.0001、Fisher検定）。 Furthermore, the detection ratios of anti-Siglec 14 alloantibodies were compared between plasma (168 types) and non-hemolytic transfusion-causing drug-derived plasma (500 types). The positive rate of anti-Siglec 14 alloantibody was 2.4% (12/500) in the serum of healthy volunteers, whereas it was significantly higher at 11.9% (20/168) in the non-hemolytic transfusion-causing agent (p < 0.0001, Fisher test).
　一方、抗シグレック５アロ抗体の陽性率は健常人血清と非溶血性輸血副作用原因製剤とで差はなかった。 On the other hand, the positive rate of anti-Siglec 5 alloantibody was not different between normal human serum and non-hemolytic transfusion side-effect side preparation.
（抗シグレック１４抗体陽性血漿による好中球活性化）
　a. 抗シグレック１４抗体陽性血漿と好中球との反応
　全血200μLに種々の刺激剤（抗シグレック１４抗体陰性血漿（日本赤十字社より入手）、抗シグレック１４抗体陽性血漿（日本赤十字社より入手）、又はβ-acetyl-γ-o-alkyl-α-phosphatidylcholine、phorbol 12-myristate 13-acetate:fMLP）10μLを添加し、37℃で30分間インキュベートし、その後遠心分離（1,000g、3分間）して全血より血球と上清をそれぞれ分取した。血球についてはフローサイトメーター（FCM）により、好中球活性化マーカーであるMac-1抗原の発現量を測定し、上清についてはELISAにより、放出されたHeparin Binding Protein（HBP、炎症性血管作動性因子で、血液細胞では好中球のみが産生する顆粒タンパク質）の濃度を測定した（詳細は下記）。 (Neutrophil activation by anti-Siglec 14 antibody positive plasma)
a. Reaction of anti-Siglec 14 antibody-positive plasma with neutrophils Various stimulants (anti-Siglec 14 antibody-negative plasma (obtained from the Japanese Red Cross), anti-Siglec 14 antibody-positive plasma (obtained from the Japanese Red Cross) ), Or β-acetyl-γ-o-alkyl-α-phosphatidylcholine, phorbol 12-myristate 13-acetate: fMLP) 10 μL, incubate for 30 minutes at 37 ° C, then centrifuge (1,000 g, 3 minutes) Then, blood cells and supernatant were collected from whole blood. For blood cells, the expression level of Mac-1 antigen, which is a neutrophil activation marker, is measured using a flow cytometer (FCM). For the supernatant, released Heparin Binding Protein (HBP, inflammatory vasoactivity) is measured using ELISA. As a sex factor, the concentration of granule protein produced only by neutrophils in blood cells was measured (details are given below).
　b. FCMによる白血球におけるMac-1抗原発現量の測定
　各種刺激剤による刺激後の全血より白血球を分画し、PBS-BSA (10mM EDTA、0.5%BSA)で洗浄した後、同細胞をPE標識抗Mac-1モノクローナル抗体（Becton Dickinson、カタログ番号347557）で4℃、15分間染色した。PBS-BSA にて洗浄後、BD FACS Lysing Solution（Becton Dickinson社）で溶血させ、好中球分画でのMac-1の発現量をFCMで測定した。 b. Measurement of Mac-1 antigen expression level in leukocytes by FCM Leukocytes were fractionated from whole blood after stimulation with various stimulants, washed with PBS-BSA (10 mM EDTA, 0.5% BSA), and then the cells were treated with PE. Staining was performed at 4 ° C. for 15 minutes with a labeled anti-Mac-1 monoclonal antibody (Becton Dickinson, catalog number 347557). After washing with PBS-BSA, hemolysis was performed with BD FACS Lysing Solution (Becton Dickinson), and the expression level of Mac-1 in the neutrophil fraction was measured with FCM.
　fMLP（陽性コントロール）を用いて刺激した好中球表面では、Mac-1抗原の発現量が増加していることが確認された。また、抗シグレック１４抗体陽性血漿を用いて刺激した場合、抗シグレック１４抗体陰性血漿を用いて刺激した場合に比べ、Mac-1抗原の発現量が有意に増加していることが観察された（図７）。 It was confirmed that the expression level of Mac-1 antigen was increased on the surface of neutrophils stimulated with fMLP (positive control). In addition, when stimulated with anti-Siglec 14 antibody-positive plasma, it was observed that the expression level of Mac-1 antigen was significantly increased compared to when stimulated with anti-Siglec 14 antibody-negative plasma ( FIG. 7).
　c. ELISA法による上清中のHBP濃度の測定
　上清中のHBP濃度は、TapperらのELISA法を用いて測定した（Blood 2002; 99: 1785-93.）。ELISA用マイクロプレート（Nunc社、C8 Maxisorp）に抗HBP モノクローナル抗体（R＆D社、50μg/mL）を50μl添加し、4℃で一晩インキュベートし、抗体を吸着させた。同プレートをT-PBS-BSA（PBS溶液に0.5% Tween20、1% BSAを添加）でブロックした後（タンパク質の非特異結合阻害）、1/6に希釈した上清（上記a）もしくは種々の濃度に希釈した組換えヒトHBP（R＆D社）を加え、37℃で60分間インキュベートした。なお、血漿（血清）の希釈はCan Get Signal I溶液（Toyobo社）を用いた。T-PBS（PBS溶液に0.5% Tween20を添加）で洗浄後、抗HBP ポリクローナル抗体（R＆D社、50μg/mL、50μL/well）を37℃で45分間反応させ、洗浄後さらに結合した同ポリクローナル抗体をHRP標識抗ヤギIgG抗体（Promega社、1/5000希釈、50μL/well）で検出した。発色はTMB発色溶液（Kirkegaard＆Perry社）を用い、マイクロプレートリーダー（Corona社、MTP-120）で450nmの吸光度を測定した。抗シグレック１４抗体陽性血漿を用いて刺激した場合、抗シグレック１４抗体陰性血漿を用いて刺激した場合と比べて、上清中のHBP濃度が高いことが観察された（図８）。 c. Measurement of HBP concentration in supernatant by ELISA The HBP concentration in the supernatant was measured using the ELISA method of Tapper et al. (Blood 2002; 99: 1785-93.). 50 μl of an anti-HBP monoclonal antibody (R & D, 50 μg / mL) was added to an ELISA microplate (Nunc, C8 Maxisorp) and incubated overnight at 4 ° C. to adsorb the antibody. After blocking the plate with T-PBS-BSA (0.5% Tween20, 1% BSA added to PBS solution) (non-specific binding inhibition of protein), 1/6 diluted supernatant (above a) or various Recombinant human HBP (R & D) diluted to a concentration was added and incubated at 37 ° C. for 60 minutes. For plasma (serum) dilution, Can Get Signal I solution (Toyobo) was used. After washing with T-PBS (0.5% Tween20 added to PBS solution), anti-HBP polyclonal antibody (R & D, 50 μg / mL, 50 μL / well) was allowed to react at 37 ° C. for 45 minutes. Was detected with an HRP-labeled anti-goat IgG antibody (Promega, 1/5000 dilution, 50 μL / well). For color development, TMB color development solution (Kirkegaard & Perry) was used, and absorbance at 450 nm was measured with a microplate reader (Corona, MTP-120). When stimulated with anti-Siglec 14 antibody-positive plasma, it was observed that the HBP concentration in the supernatant was higher than when stimulated with anti-Siglec 14 antibody-negative plasma (FIG. 8).
　上記の結果は、抗シグレック１４抗体陽性血漿により好中球が活性化されることを示し、抗シグレック１４抗体陽性血漿が非溶血性輸血副作用を引き起こすことを示唆する。 The above results indicate that neutrophils are activated by anti-Siglec 14 antibody positive plasma, suggesting that anti-Siglec 14 antibody positive plasma causes non-hemolytic transfusion side effects.
　本発明のシグレック１４遺伝子の多型に基づく対立遺伝子型の検出用試薬により、シグレック１４遺伝子を有する対立遺伝子型とシグレック１４遺伝子を有しないシグレック１４／５融合遺伝子のホモ接合型とを識別して検出することができ、輸血副作用等の危険性を事前に排除し、或いは感染症などの治療方針を決定するために利用することができる。 By using the allelic detection reagent based on the polymorphism of the Siglec 14 gene of the present invention, the allelic type having the Siglec 14 gene and the homozygous type of the Siglec 14/5 fusion gene not having the Siglec 14 gene are distinguished. It can be detected and can be used to eliminate risks such as blood transfusion side effects in advance or to determine a treatment policy such as infection.
　また本発明の抗シグレック１４抗体の検出用試薬を用いることによって、ヒト血液試料中の抗シグレック１４抗体を検出することができ、非溶血性輸血副作用を防止し輸血の安全性を確保するために利用することができる。 Further, by using the reagent for detecting anti-Siglec 14 antibody of the present invention, anti-Siglec 14 antibody in a human blood sample can be detected, in order to prevent non-hemolytic transfusion side effects and ensure the safety of transfusion. Can be used.
　本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.

Claims (20)

  1. 　配列番号１に示されるシグレック１４遺伝子を特異的に増幅するプライマー対、該シグレック１４遺伝子に特異的にハイブリダイズするプローブ又は配列番号４に示されるシグレック１４タンパク質を特異的に認識する抗体を含有することを特徴とする、シグレック１４遺伝子の多型に基づく対立遺伝子型の検出用試薬。 A primer pair that specifically amplifies the Siglec 14 gene shown in SEQ ID NO: 1, a probe that specifically hybridizes to the Siglec 14 gene, or an antibody that specifically recognizes the Siglec 14 protein shown in SEQ ID NO: 4 An allelic detection reagent based on a polymorphism of the Siglec 14 gene,
  2. 　シグレック１４遺伝子を特異的に増幅するプライマー対が、シグレック１４遺伝子とシグレック５遺伝子の塩基配列における保存領域の外側領域の塩基配列に対応する配列を有することを特徴とする、請求項１に記載の検出用試薬。 2. The primer pair according to claim 1, wherein the primer pair that specifically amplifies the Siglec 14 gene has a sequence corresponding to a base sequence in a region outside the conserved region in the base sequences of the Siglec 14 gene and the Siglec 5 gene. Detection reagent.
  3. 　シグレック１４遺伝子を特異的に増幅するプライマー対が、配列番号７及び配列番号８で示される塩基配列を有するものであることを特徴とする、請求項２に記載の検出用試薬。 The detection reagent according to claim 2, wherein the primer pair that specifically amplifies the Siglec 14 gene has a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8.
  4. 　請求項１～３のいずれかに記載の検出用試薬と、配列番号３で示されるシグレック１４／５融合遺伝子を特異的に増幅するプライマー対とを組み合わせてなることを特徴とする、シグレック１４遺伝子の多型に基づく対立遺伝子型の検出用試薬キット。 A Siglec 14 gene comprising the detection reagent according to any one of Claims 1 to 3 and a primer pair that specifically amplifies the Siglec 14/5 fusion gene represented by SEQ ID NO: 3. Reagent kit for detection of allelic type based on polymorphism of
  5. 　シグレック１４／５融合遺伝子を特異的に増幅するプライマー対が、シグレック１４遺伝子とシグレック５遺伝子の塩基配列における保存領域の外側領域の塩基配列に対応する配列を有することを特徴とする、請求項４に記載の検出用試薬キット。 The primer pair that specifically amplifies the Siglec 14/5 fusion gene has a sequence corresponding to the base sequence of the region outside the conserved region in the base sequences of the Siglec 14 gene and the Siglec 5 gene. The reagent kit for detection according to 1.
  6. 　シグレック１４／５融合遺伝子を特異的に増幅するプライマー対が配列番号７及び配列番号１０で示される塩基配列を有するものであることを特徴とする、請求項５に記載の検出用試薬キット。 6. The detection reagent kit according to claim 5, wherein the primer pair that specifically amplifies the Siglec 14/5 fusion gene has a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 10.
  7. 　血液型適合性検査薬として使用することを特徴とする、請求項１～３のいずれかに記載の検出用試薬。 4. The detection reagent according to claim 1, which is used as a blood type compatibility test agent.
  8. 　血液型適合性検査薬として使用することを特徴とする、請求項４～６のいずれかに記載の検出用試薬キット。 The detection reagent kit according to any one of claims 4 to 6, which is used as a blood type compatibility test agent.
  9. 　感染症の治療方針の決定のために使用することを特徴とする、請求項７に記載の検出用試薬。 The detection reagent according to claim 7, wherein the detection reagent is used for determining a treatment policy for an infectious disease.
  10. 　感染症の治療方針の決定のために使用することを特徴とする、請求項８に記載の検出用試薬キット。 9. The detection reagent kit according to claim 8, wherein the detection reagent kit is used for determining a treatment policy for an infectious disease.
  11. 　配列番号３に示されるシグレック１４／５融合遺伝子。 Siglec 14/5 fusion gene shown in SEQ ID NO: 3.
  12. 　請求項１１に記載のシグレック１４／５融合遺伝子を特異的に増幅するプライマー対。 A primer pair that specifically amplifies the Siglec 14/5 fusion gene according to claim 11.
  13. 　シグレック１４遺伝子とシグレック５遺伝子の塩基配列における保存領域の外側領域の塩基配列に対応する配列を有することを特徴とする、請求項１２に記載のプライマー対。 The primer pair according to claim 12, wherein the primer pair has a sequence corresponding to the base sequence of the region outside the conserved region in the base sequences of the Siglec 14 gene and the Siglec 5 gene.
  14. 　配列番号７及び配列番号１０で示される塩基配列を有する、請求項１１に記載のプライマー対。 The primer pair according to claim 11, which has the base sequences represented by SEQ ID NO: 7 and SEQ ID NO: 10.
  15. 　ヒトゲノムＤＮＡを鋳型とし、請求項１～３のいずれかに記載のシグレック１４遺伝子を特異的に増幅するプライマー対又は請求項４又は５に記載の検出用試薬キットを用いてＰＣＲを行い、シグレック１４遺伝子の存在の有無、又はさらにシグレック１４／５遺伝子の存在の有無を検出することを特徴とする、シグレック１４遺伝子の多型に基づく対立遺伝子型の検出方法。 PCR is performed using human genomic DNA as a template and a primer pair that specifically amplifies the Siglec 14 gene according to any one of claims 1 to 3 or the detection reagent kit according to claim 4 or 5, and Siglec 14 A method for detecting an allelic type based on a polymorphism of a Siglec 14 gene, characterized by detecting the presence or absence of a gene, or further the presence or absence of a Siglec 14/5 gene.
  16. 　請求項１に記載のシグレック１４遺伝子に特異的にハイブリダイズするプローブを、ヒトゲノムＤＮＡに対しハイブリダイズさせ、シグレック１４遺伝子の存在の有無を検出することを特徴とする、シグレック１４遺伝子の多型に基づく対立遺伝子型の検出方法。 A probe that specifically hybridizes to the Siglec 14 gene according to claim 1 is hybridized to human genomic DNA to detect the presence or absence of the Siglec 14 gene. Allele type detection method based.
  17. 　請求項１に記載のシグレック１４タンパク質を特異的に認識する抗体を用いて、ヒト血液試料中のシグレック１４タンパク質を検出することを特徴とする、シグレック１４遺伝子の多型に基づく対立遺伝子型の検出方法。 Detection of allelic type based on polymorphism of siglec 14 gene, wherein the antibody specifically recognizes siglec 14 protein according to claim 1 is used to detect siglec 14 protein in a human blood sample. Method.
  18. 　配列番号４の少なくとも238番目から358番目の配列を含むアミノ酸配列を有するタンパク質、又は配列番号４の少なくとも238番目から358番目の配列を含むアミノ酸配列を有するタンパク質を発現する細胞を含有することを特徴とする、抗シグレック１４抗体の検出用試薬。 A cell that expresses a protein having an amino acid sequence comprising at least the 238th to 358th sequence of SEQ ID NO: 4 or a protein having an amino acid sequence comprising at least the 238th to 358th sequence of SEQ ID NO: 4 A reagent for detecting anti-Siglec 14 antibody.
  19. 　請求項１８に記載の抗シグレック１４抗体の検出用試薬を用いた、ヒト血液試料中の抗シグレック１４抗体の検出方法。 A method for detecting an anti-Siglec 14 antibody in a human blood sample using the reagent for detecting an anti-Siglec 14 antibody according to claim 18.
  20. 　請求項１８に記載の抗シグレック１４抗体の検出用試薬を用いた、抗シグレック１４抗体を含む血液製剤の特定方法。 A method for identifying a blood product comprising an anti-Siglec 14 antibody, wherein the reagent for detecting an anti-Siglec 14 antibody according to claim 18 is used.
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