JPWO2008114709A1 - Biomarkers specific for blood cells or specific for osteoclast differentiation - Google Patents

Abstract

The present invention provides a diagnostic reagent for a disease based on a disorder of blood cells or osteoclasts, and a useful means for development of a medicine for such a disease having a new mechanism of action. More specifically, the present invention relates to a novel polypeptide and its specific partial peptide, and a novel polynucleotide and its specific, which can be used as a biomarker specific to blood cells or specific to osteoclast differentiation. Partial nucleotides; expression vectors for such polynucleotides and their specific partial peptides; transformants into which such expression vectors have been introduced; antisense molecules to such biomarkers, RNAi-inducible nucleic acids (eg, siRNA) ), Aptamers, antibodies, and compositions containing them; mammalian cells or non-human mammals in which the expression or function of such biomarkers is regulated; means for measuring such biomarkers (eg, primer sets) , Nucleic acid probes, antibodies, aptamers), and reagents containing them.

Description

  The present invention relates to a polypeptide and a partial peptide thereof, and a polynucleotide and a partial nucleotide thereof, which can be used as a biomarker specific to blood cells or to osteoclast differentiation; an expression vector; a transformant Antisense molecules, RNAi-inducible nucleic acids (eg, siRNA), aptamers, antibodies, and compositions containing them; mammalian cells or non-human mammals; Means for measuring a marker (eg, primer set, nucleic acid probe, antibody, aptamer), a measuring method, and the like are provided.

  Advances in human genome research have greatly advanced the analysis of human chromosomal sequences, but not all of the functions of human genes have been revealed. In humans, gene diversity is largely associated with changes in the function of the gene. In fact, in humans, it is known that multiple mRNAs are transcribed from specific regions of the chromosome, resulting in different variants.

  A series of genes found by the present inventors that can be used as a biomarker specific for blood cell lineage or specific for osteoclast differentiation (if necessary, “blood cell line / osteoclast specific gene”) For abbreviated), known variants have been reported. For example, as such known variants, blood cell / osteoclast-specific gene 1 (Genbank accession number: NM_012252.1; Non-patent Documents 1 and 2), blood cell / osteoclast-specific gene 2 (Genbank access) Session number: NM_001637.1; Non-patent document 3, 4), Blood cell / osteoclast specific gene 3 (Genbank accession number: NM_002314.2; Non-patent document 5, 6), Blood cell / osteoclast specific Gene 4 (Genbank accession number: NM_002830.2; Non-patent documents 7, 8), blood cell line / osteoclast specific gene 5 (Genbank accession number: NM_006153.3; Non-patent documents 9, 10), blood cell System / osteoclast-specific gene 6 (Genbank accession number: NM_000480.1; Non-patent documents 11 and 12), blood cell system / osteoclast-specific gene 7 (Genbank accession number: NM_024850.1; non-patent document) 13, 14), blood cell line, osteoclast Specific gene 8 (Genbank accession number: NM_015864.2; Non-patent documents 15, 16), blood cell line / osteoclast specific gene 9 (Genbank accession number: NM_139241.1; Non-patent documents 17, 18), Blood cell / osteoclast specific gene 10 (Genbank accession number: NM_178833.3; Non-patent document 19), Blood cell / osteoclast specific gene 11 (Genbank accession number: NM_005807.1; Non-patent document 20) 21), a known variant of the blood cell line / osteoclast specific gene 12 (Genbank accession number: NM_031443.2; non-patent documents 22, 23) has been reported.

However, the blood cell / osteoclast-specific gene 1-12 may be useful as a biomarker specific for the blood cell system or specific for osteoclast differentiation, and the blood cell / osteoclast-specific gene 1-12. For 12, it is not known that there are specific variants found by the inventors.
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  Analysis of biomarkers specific for blood cell lineage or osteoclast differentiation can be performed by, for example, reagents for identifying blood cell line or osteoclasts or determining the differentiation state of osteoclasts, disorders of blood cell line or osteoclasts It leads to the development of diagnostic reagents for diseases based on the disease, and drugs for diseases based on disorders of blood cells or osteoclasts having a new mechanism of action. The present invention provides such reagents and medicines based on the knowledge obtained by analyzing the expression profile of a predetermined gene, and provides means useful for the development of such reagents and medicines, etc. With the goal.

As a result of intensive studies, the present inventors have found blood cell / osteoclast-specific genes 1 to 12 as biomarkers specific to the blood cell lineage or specific to osteoclast differentiation. The present inventors have also found a novel variant of blood cell / osteoclast-specific genes 1 to 12 that can be used as a biomarker specific for blood cells or specific for osteoclast differentiation. Therefore, by using the blood cell line / osteoclast specific gene 1-12 and / or a novel variant thereof, identification of the blood cell line or osteoclast, determination of the differentiation state of the blood cell line or osteoclast, Diagnosis of diseases based on osteoclast damage is possible. In particular, since the blood cell / osteoclast-specific genes 1 to 12 and / or the novel variant thereof are specifically expressed in a specific differentiation stage of the blood cell or osteoclast, such a specific differentiation stage It is possible to improve the accuracy of determination of blood cells or osteoclasts. In addition, by using blood cell / osteoclast-specific genes 1 to 12 and / or novel variants thereof, it is possible to develop new medicines for predetermined diseases such as diseases based on disorders of blood cells or osteoclasts. It is thought to become.
Based on the above findings, the present inventors have completed the present invention.

That is, the present invention relates to the following inventions and the like:
[1] The polypeptide of any one of 1) to 12) below or a specific partial peptide thereof:
1) a polypeptide having the amino acid sequence represented by SEQ ID NO: 10 or an amino acid sequence substantially identical thereto;
2) An amino acid sequence represented by SEQ ID NO: 30 or 37, an amino acid sequence consisting of amino acid residues 24 to 543 in the amino acid sequence represented by SEQ ID NO: 30, or an amino acid sequence substantially identical to them A polypeptide having;
3) a polypeptide having the amino acid sequence represented by SEQ ID NO: 57 or a substantially identical amino acid sequence thereto;
4) a polypeptide having the amino acid sequence represented by SEQ ID NO: 73 or an amino acid sequence substantially identical thereto;
5) A polypeptide having the amino acid sequence represented by SEQ ID NO: 88, or a substantially identical amino acid sequence thereof;
6) a polypeptide having the amino acid sequence represented by SEQ ID NO: 104 or a substantially identical amino acid sequence thereof;
7) A polypeptide having the amino acid sequence represented by SEQ ID NO: 120 or a substantially identical amino acid sequence thereof;
8) A polypeptide having the amino acid sequence represented by SEQ ID NO: 143 or SEQ ID NO: 153, or a substantially identical amino acid sequence thereto;
9) A polypeptide having the amino acid sequence represented by SEQ ID NO: 173 or SEQ ID NO: 180, or a substantially identical amino acid sequence thereof;
10) a polypeptide having the amino acid sequence represented by SEQ ID NO: 201, or a substantially identical amino acid sequence thereof;
11) 25 in the amino acid sequence represented by SEQ ID NO: 226 or SEQ ID NO: 233, the amino acid sequence consisting of the 25th to 853rd amino acid residues in the amino acid sequence represented by SEQ ID NO: 226, or the amino acid sequence represented by SEQ ID NO: 233 An amino acid sequence consisting of the 216th amino acid residue, or a polypeptide having an amino acid sequence substantially identical to them; or 12) an amino acid sequence represented by SEQ ID NO: 251, SEQ ID NO: 266 or SEQ ID NO: 272, or thereof A polypeptide having substantially the same amino acid sequence as
[2] The polypeptide of the above [1] or a specific partial peptide thereof, wherein the polypeptide is a polypeptide of any one of 1) to 12) below:
1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 10;
2) A polypeptide comprising an amino acid sequence consisting of amino acid residues 24 to 543 in the amino acid sequence represented by SEQ ID NO: 30 or SEQ ID NO: 37 or the amino acid sequence represented by SEQ ID NO: 30;
3) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57;
4) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 73;
5) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 88;
6) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 104;
7) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 120;
8) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 143 or SEQ ID NO: 153;
9) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 173 or SEQ ID NO: 180;
10) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 201;
11) 25 in the amino acid sequence represented by SEQ ID NO: 226 or SEQ ID NO: 233, the amino acid sequence consisting of the 25th to 853rd amino acid residues in the amino acid sequence represented by SEQ ID NO: 226, or the amino acid sequence represented by SEQ ID NO: 233 A polypeptide comprising an amino acid sequence comprising the 216th amino acid residue; or 12) a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 251, SEQ ID NO: 266 or SEQ ID NO: 272.
[3] The polypeptide of [1] or [2] above or a specific partial peptide thereof fused to a polypeptide consisting of a heterologous amino acid sequence.
[4] A partial peptide specific to a polypeptide encoded by a blood cell / osteoclast specific gene 1-12, which is any partial peptide of 1) to 12) below:
1) the amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 14, or a partial peptide comprising these partial amino acid sequences, or a partial peptide comprising the amino acid sequence represented by SEQ ID NO: 16;
2) the amino acid sequence represented by SEQ ID NO: 32 or SEQ ID NO: 34, or a partial peptide comprising these partial amino acid sequences, or a partial peptide comprising the amino acid sequence represented by SEQ ID NO: 39;
3) A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 59 or a partial amino acid sequence thereof;
4) A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 290 or a partial amino acid sequence thereof;
5) A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 90 or a partial amino acid sequence thereof;
6) A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 106 or a partial amino acid sequence thereof;
7) A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 124, SEQ ID NO: 125 or SEQ ID NO: 127, or a partial amino acid sequence thereof;
8) SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 155, SEQ ID NO: 157 or SEQ ID NO: 159, or a partial peptide consisting of these partial amino acid sequences;
9) The amino acid sequence represented by SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 182 or SEQ ID NO: 184, or a partial peptide comprising these partial amino acid sequences;
10) A partial peptide comprising the amino acid sequence represented by SEQ ID NO: 204 or SEQ ID NO: 206, or a partial amino acid sequence thereof;
11) the amino acid sequence represented by SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 235 or SEQ ID NO: 237, or a partial peptide consisting of a partial amino acid sequence thereof; or 12) SEQ ID NO: 253, SEQ ID NO: 256, SEQ ID NO: 258, A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 268, SEQ ID NO: 275, SEQ ID NO: 276, or SEQ ID NO: 278, or a partial amino acid sequence thereof.
[5] A polynucleotide encoding the polypeptide of any one of [1] to [3] above or the specific partial peptide of any of [1] to [4] above.
[6] The polynucleotide according to any one of 1) to 12) below or a specific partial nucleotide thereof:
1) a nucleic acid sequence represented by SEQ ID NO: 8, a nucleic acid sequence corresponding to the ORF, or a polynucleotide having a nucleic acid sequence substantially identical to them;
2) Nucleic acid sequence represented by SEQ ID NO: 28 or SEQ ID NO: 35, or an ORF corresponding nucleic acid sequence thereof, or a nucleic acid sequence consisting of nucleotide residues 357 to 1919 in the nucleic acid sequence represented by SEQ ID NO: 28, or thereof A polynucleotide having a nucleic acid sequence substantially identical to said;
3) a polynucleotide having the nucleic acid sequence represented by SEQ ID NO: 55, the ORF-compatible nucleic acid sequence, or a nucleic acid sequence substantially the same as them;
4) a nucleic acid sequence represented by SEQ ID NO: 71, a nucleic acid sequence corresponding to the ORF, or a polynucleotide having a nucleic acid sequence substantially the same as them;
5) a polynucleotide having the nucleic acid sequence represented by SEQ ID NO: 86, the ORF-corresponding nucleic acid sequence, or a nucleic acid sequence substantially identical thereto;
6) a polynucleotide having the nucleic acid sequence represented by SEQ ID NO: 102, the ORF-corresponding nucleic acid sequence, or a nucleic acid sequence substantially identical thereto;
7) a nucleic acid sequence represented by SEQ ID NO: 118, a nucleic acid sequence corresponding to the ORF, or a polynucleotide having a nucleic acid sequence substantially the same as them;
8) a polynucleotide having SEQ ID NO: 141 or 151, or an ORF corresponding nucleic acid sequence thereof, or a nucleic acid sequence substantially identical thereto;
9) a nucleic acid sequence represented by SEQ ID NO: 171 or SEQ ID NO: 178, or an ORF corresponding nucleic acid sequence thereof, or a polynucleotide having a nucleic acid sequence substantially identical to them;
10) a polynucleotide having the nucleic acid sequence represented by SEQ ID NO: 199, or an ORF-corresponding nucleic acid sequence, or a nucleic acid sequence substantially identical thereto;
11) The nucleic acid sequence represented by SEQ ID NO: 224 or SEQ ID NO: 231 or the ORF-compatible nucleic acid sequence thereof, or the nucleic acid sequence consisting of 118 to 2604th nucleotide residues in the nucleic acid sequence represented by SEQ ID NO: 224 or SEQ ID NO: A nucleic acid sequence consisting of nucleotide residues 118 to 693 in the nucleic acid sequence represented by 231; or a polynucleotide having a nucleic acid sequence substantially the same as them; or 12) SEQ ID NO: 249, SEQ ID NO: 264, or SEQ ID NO: 270 A polynucleotide having the nucleic acid sequence represented by the above, or the ORF corresponding nucleic acid sequence thereof, or a nucleic acid sequence substantially identical to them.
[7] The polynucleotide of [6] above or a specific partial nucleotide thereof, wherein the polynucleotide of any one of 1) to 12) is the polynucleotide of any one of 1) to 12) below:
1) a nucleic acid sequence represented by SEQ ID NO: 8, or a polynucleotide comprising the nucleic acid sequence corresponding to the ORF;
2) a nucleic acid sequence represented by SEQ ID NO: 28 or SEQ ID NO: 35, or an ORF corresponding nucleic acid sequence thereof, or a nucleic acid sequence comprising a nucleic acid sequence consisting of nucleotide residues 357 to 1919 in the nucleic acid sequence represented by SEQ ID NO: 28 nucleotide;
3) a nucleic acid sequence represented by SEQ ID NO: 55 or a polynucleotide comprising the ORF-compatible nucleic acid sequence;
4) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 71 or a nucleic acid sequence corresponding to the ORF;
5) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 86 or a nucleic acid sequence corresponding to the ORF;
6) a polynucleotide consisting of the nucleic acid sequence represented by SEQ ID NO: 102, or a nucleic acid sequence corresponding to the ORF thereof;
7) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 118 or a nucleic acid sequence corresponding to the ORF;
8) A polynucleotide comprising SEQ ID NO: 141 or SEQ ID NO: 151, or a nucleic acid sequence corresponding to the ORF thereof;
9) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 171 or SEQ ID NO: 178, or an ORF-compatible nucleic acid sequence thereof;
10) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 199 or a nucleic acid sequence corresponding to the ORF;
11) The nucleic acid sequence represented by SEQ ID NO: 224 or SEQ ID NO: 231 or the ORF-compatible nucleic acid sequence thereof, or the nucleic acid sequence consisting of 118 to 2604th nucleotide residues in the nucleic acid sequence represented by SEQ ID NO: 224 or SEQ ID NO: A polynucleotide comprising the nucleic acid sequence consisting of nucleotide residues 118 to 693 in the nucleic acid sequence represented by 231; or 12) the nucleic acid sequence represented by SEQ ID NO: 249, SEQ ID NO: 264 or SEQ ID NO: 270, or an ORF thereof A polynucleotide comprising a corresponding nucleic acid sequence.
[8] Partial nucleotides specific to the polynucleotide encoded by the blood cell / osteoclast specific gene 1-12, which is any partial nucleotide of 1) to 12) below:
1) a nucleic acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 27, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
2) Nucleic acid sequence represented by SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 53 or SEQ ID NO: 54, or a partial nucleic acid sequence thereof A partial nucleotide consisting of;
3) a nucleic acid sequence represented by SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66 or SEQ ID NO: 70, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
4) a nucleic acid sequence represented by SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 81 or SEQ ID NO: 85, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
5) a nucleic acid sequence represented by SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 97 or SEQ ID NO: 101, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
6) a nucleic acid sequence represented by SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 113 or SEQ ID NO: 117, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
7) A nucleic acid sequence represented by SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134 or SEQ ID NO: 140, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
8) Nucleic acid represented by SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 163, SEQ ID NO: 166 or SEQ ID NO: 170 A partial nucleotide consisting of a sequence or a partial nucleic acid sequence thereof;
9) Nucleic acid sequence represented by SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 190, SEQ ID NO: 193, SEQ ID NO: 197 or SEQ ID NO: 198, or a partial nucleic acid sequence thereof A partial nucleotide consisting of;
10) Nucleic acid sequence represented by SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 209, SEQ ID NO: 212, SEQ ID NO: 215, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 223, or a partial nucleic acid sequence thereof A partial nucleotide consisting of;
11) A nucleic acid sequence represented by SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 240, SEQ ID NO: 243, SEQ ID NO: 247, or SEQ ID NO: 248, or a partial nucleotide consisting of a partial nucleic acid sequence thereof Or 12) SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 277, SEQ ID NO: 281, a nucleic acid sequence represented by SEQ ID NO: 284, SEQ ID NO: 288 or SEQ ID NO: 289, or a partial nucleotide consisting of a partial nucleic acid sequence thereof.
[9] The polynucleotide according to any one of [5] to [7] above, or the specific partial nucleotide according to any one of [6] to [8] above, and a promoter operably linked thereto, An expression vector for the polypeptide of any one of [1] to [3] or the specific partial peptide of any of [1] to [4] above.
[10] A transformant into which the expression vector of [9] has been introduced.
[11] Any one of the polymorphisms comprising a nucleic acid sequence complementary to the nucleic acid sequence of the specific partial nucleotide of [7] or [8] above and encoded by a blood cell / osteoclast specific gene 1-12 An antisense molecule capable of suppressing the expression of a peptide.
[12] A sense strand comprising the nucleic acid sequence of the specific partial nucleotide of [7] or [8] above and an antisense strand comprising the complementary nucleic acid sequence, and one of the sense strand and the antisense strand Alternatively, the expression of any polypeptide encoded by the hematopoietic / osteoclast specific gene 1-12, which may have an overhang at both the 5 ′ end and / or the 3 ′ end, may be suppressed. RNAi-inducible nucleic acids.
[13] The RNAi-inducible nucleic acid according to [12] above, wherein the RNAi-inducible nucleic acid is siRNA.
[14] To any polypeptide encoded by the blood cell line / osteoclast specific gene 1-12 through a region corresponding to the specific partial peptide of any one of [2] to [4] above Aptamers that can bind.
[15] To any of the polypeptides encoded by the blood cell / osteoclast-specific genes 1 to 12 via a region corresponding to the specific partial peptide of any of [2] to [4] above An antibody that can bind.
[16] The antibody according to [15] above, wherein the antibody is any one of i) to iii) below:
i) polyclonal antibodies;
ii) a monoclonal antibody or part thereof;
iii) chimeric, humanized or human antibodies.
[17] A cell that produces the antibody of [15] or [16] above.
[18] The cell according to [17] above, wherein the cell is a hybridoma.
[19] The polypeptide of any one of [1] to [3], the antisense molecule of [11], the RNAi-inducible nucleic acid of [12] or [13], the aptamer of [14], [ [15] A composition comprising the antibody of [16], or an expression vector thereof, and a pharmaceutically acceptable carrier.
[20] A mammalian cell or a non-human mammal in which the expression or function of the polypeptide of any one of [1] to [3] is regulated.
[21] A primer set specific for any polynucleotide encoded by the blood cell / osteoclast specific gene 1-12 or a specific partial nucleotide thereof, including the following (a) or (b):
(A) a sense primer corresponding to the first nucleic acid sequence of the polynucleotide of [7] above, or a specific partial nucleotide of [7] or [8] above; and (b) the polynucleotide of [7] above, Alternatively, an antisense primer corresponding to a nucleic acid sequence complementary to the second nucleic acid sequence of the specific partial nucleotide of [7] or [8] above.
[22] A nucleic acid specific for any polynucleotide encoded by the blood cell / osteoclast-specific gene 1-12, or a specific partial nucleotide thereof, which is either (a) or (b) below probe:
(A) a single-stranded polynucleotide comprising a nucleic acid sequence complementary to the nucleic acid sequence of the specific partial nucleotide of [7] or [8] above; or (b) the specific of [7] or [8] above A double-stranded polynucleotide comprising a sense strand containing a partial nucleotide nucleic acid sequence and an antisense strand containing a complementary nucleic acid sequence.
[23] A blood cell system comprising one or more substances or sets selected from the aptamer of [14], the antibody of [15] or [16], the primer set of [21] and the nucleic acid probe of [22] A reagent or kit for detection or quantification of any polypeptide or polynucleotide encoded by the osteoclast-specific gene 1-12.
[24] The reagent or kit according to [23] above, which is a reagent or kit for identifying blood cells or osteoclasts or determining their differentiation.
[25] Measuring the expression of any polypeptide or polynucleotide encoded by a blood cell / osteoclast specific gene 1-12 in a biological sample obtained from a mammal, or in a cell or tissue culture. And the method for detecting or quantifying the polypeptide or polynucleotide, wherein the biological sample or the culture contains blood cells, osteoclasts, hematopoietic tissue, lymphoid tissue or synovial tissue.
[26] The method comprising measuring the expression of the polypeptide of [2] or [3] or the polynucleotide of [7] in a biological sample obtained from a mammal, or a cell or tissue culture. A method for detecting or quantifying a peptide or the polynucleotide.
[27] The detection or quantification method according to [26] above, wherein the biological sample or the culture contains blood cells, osteoclasts, hematopoietic tissue, lymphoid tissue or synovial tissue.

The polypeptide of the present invention and the partial peptide of the present invention can specifically recognize the polypeptide of the present invention or a known polypeptide, for example, as a biomarker specific for blood cells or specific for osteoclast differentiation. A substance, a substance capable of comprehensively recognizing both the polypeptide of the present invention and a known polypeptide, and a substance capable of specifically regulating the function of the polypeptide of the present invention or a known polypeptide, or the polypeptide of the present invention And can be useful in the development of substances that can comprehensively regulate the function of both known polypeptides.
The polynucleotide of the present invention and the partial nucleotide of the present invention can specifically recognize the polynucleotide of the present invention or a known polynucleotide, for example, as a biomarker specific for blood cells or specific for osteoclast differentiation. A substance, a substance capable of comprehensively recognizing both the polynucleotide of the present invention and a known polynucleotide, and a substance capable of specifically regulating the expression of the polypeptide of the present invention or a known polypeptide, or the polypeptide of the present invention And can be useful in the development of substances that can comprehensively regulate the expression of both known polypeptides.
The related substances of the present invention (eg, antisense molecules, RNAi-inducible nucleic acids such as siRNA, aptamers and antibodies, and expression vectors thereof) can be useful as, for example, drugs or reagents.
The cells of the present invention can be useful, for example, for the production of the polypeptides of the present invention and partial peptides of the present invention, and antibodies of the present invention. The cells of the present invention can also be used to develop pharmaceuticals (eg, preventive or therapeutic agents for diseases based on disorders of blood cells or osteoclasts), to identify additional marker genes specific to blood cells or specific to osteoclast differentiation. , And in the analysis of mechanisms involved in the differentiation of blood cells or osteoclasts.
The animals of the present invention may be useful, for example, in drug development, identification of additional marker genes that are specific for the blood cell lineage or specific for osteoclast differentiation, and analysis of mechanisms associated with osteoclast differentiation.
The measurement means (eg, primer set, nucleic acid probe, antibody, aptamer) and measurement method of the present invention are, for example, specific for the polynucleotide of the present invention or the known polynucleotide, or the polypeptide of the present invention or the known polypeptide. It may be useful for detection and quantification, or for comprehensive detection and quantification of polynucleotides of the invention and known polynucleotides, or both polypeptides of the invention and known polypeptides. Such means and methods can also be used for identification of blood cells or osteoclasts, determination of osteoclast differentiation, and screening of drugs, reagents or foods.

1. Blood cell line / osteoclast specific gene The gene of the present invention may be a gene derived from any mammal. Examples of mammals include primates and rodents, laboratory animals, farm animals, working animals, pets and the like. Specifically, examples of mammals include humans, monkeys, rats, mice, rabbits, horses, cows, goats, sheep, dogs, cats and the like. Preferably the mammal is a human.

  The gene of the present invention may be specifically expressed in hematopoietic / lymphoid tissue or synovial tissue, or may not be specifically expressed. The gene of the present invention can also be expressed at high or low levels in hematopoietic / lymphoid tissues or synovial tissues as compared to known polynucleotides and / or known polypeptides. Examples of such hematopoietic / lymphoid tissues include bone marrow, thymus, and spleen.

  The gene of the present invention may be specifically expressed in blood cells or osteoclasts or may not be specifically expressed. The gene of the present invention can also be expressed at high or low levels in blood cells or osteoclasts compared to known polynucleotides and / or known polypeptides. Examples of such blood cells include macrophages, lymphocytes (eg, T cells, B cells, NK cells), monocytes, granulocytes, and the like.

  Hereinafter, the polypeptide and its partial peptide, and the polynucleotide and its partial nucleotide provided by the present invention will be described.

1.1. Polypeptide and partial peptide thereof The present invention has an amino acid sequence represented by SEQ ID NO: X or an amino acid sequence substantially the same as the amino acid sequence (abbreviated as “amino acid sequence represented by SEQ ID NO: X” as necessary). A polypeptide is provided.

  “SEQ ID NO: X” indicates the SEQ ID NO of any amino acid sequence disclosed herein. In addition, the polypeptide having “having” the amino acid sequence represented by SEQ ID NO: X is a “consisting of” polypeptide such as the amino acid sequence represented by SEQ ID NO: X, and the amino acid sequence, etc. Means a “comprising” polypeptide.

  In one embodiment, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: X may be an amino acid sequence having a predetermined amino acid sequence identity with the amino acid sequence represented by SEQ ID NO: X. The degree of amino acid sequence identity is about 90% or higher, preferably about 92% or higher, more preferably about 95% or higher, even more preferably about 96% or higher, most preferably about 97% or higher, about 98% or higher, or It can be about 99% or more. Amino acid sequence identity can be determined by a method known per se. Unless otherwise specified, the amino acid sequence identity (%) is calculated by executing a command of the maximum matching method, for example, using DNASIS (Hitachi Software Engineering), which is sequence analysis software. The parameters at that time are default settings (initial settings). In addition, the amino acid sequence identity (%) can be determined using a program commonly used in the field (for example, BLAST, FASTA, etc.) in the initial setting without depending on the above. In another aspect, identity (%) is determined by any algorithm known in the art, such as Needleman et al. (1970) (J. Mol. Biol. 48: 444-453), Myers and Miller (CABIOS, 1988, 4: It can be determined using the algorithm of 11-17). Needleman et al.'S algorithm is incorporated into the GCG program in the GCG software package, and the percent identity is, for example, BLOSUM 62 matrix or PAM250 matrix, and gap weight: 16, 14, 12, 10, 8, 6 or 4 and length weight: can be determined by using any of 1, 2, 3, 4, 5 or 6. The Myers and Miller algorithms are also incorporated into the ALIGN program that is part of the GCG sequence alignment software package. When the ALIGN program is used to compare amino acid sequences, for example, PAM120 weight residue table, gap length penalty 12, and gap penalty 4 can be used. For the calculation of amino acid sequence identity, a method showing the lowest value among the above methods may be adopted.

  In another embodiment, the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: X is selected from substitution, addition, deletion and insertion of one or more amino acids in the amino acid sequence represented by SEQ ID NO: X It may be an amino acid sequence having one or more modifications. The number of amino acids to be modified is not particularly limited as long as it is 1 or more. For example, 1 to about 50, preferably 1 to about 30, more preferably 1 to about 20, even more preferably 1 to about 10 is used. May be from 1 to about 5 (eg, 1 or 2).

  The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: X completely retains its characteristic part (eg, part corresponding to the specific partial polypeptide described below), and other than that A portion (eg, a portion present in a known polypeptide) may be substantially the same as the corresponding portion of the amino acid sequence represented by SEQ ID NO: X. Alternatively, in the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: X, the non-characteristic part is the same as the corresponding part of the amino acid sequence represented by SEQ ID NO: X, and the characteristic part is , May be substantially the same as the corresponding portion of the amino acid sequence represented by SEQ ID NO: X.

  The polypeptide of the present invention may have the same or different function as a known polypeptide (eg, known variant). The polypeptides of the present invention may also have an enhanced or reduced function compared to known polypeptides (eg, known variants).

  Specifically, novel polypeptides of blood cell / osteoclast specific genes 1 to 12 are as follows.

1) Blood cell line / osteoclast specific gene 1
D-SYNOV4003692.1 (SEQ ID NO: 10)
Examples of known variants of blood cell / osteoclast-specific gene 1 include, for example, variants disclosed in Examples (human transcription factor EC (TFEC); total number of nucleotides in ORF nucleic acid sequence: 1044; A total of 347 amino acids; GenBank accession number: NM_012252.1) has been reported. It has been reported that a known variant of blood cell / osteoclast specific gene 1 has a predetermined function (eg, transcriptional regulatory ability) (eg, Zhao, GQ et al., Mol. Cell. Biol. 13). (8), 4505-4512 (1993); Chung, MC et al., Biochemistry 40 (30), 8887-8897 (2001)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of blood cell / osteoclast specific gene 1 may also have these functions.

2) Blood cell / osteoclast specific gene 2
D-D3OST2001534.1 (SEQ ID NO: 30)
D-NETRP1000046.1 (SEQ ID NO: 37)
Examples of known variants of blood cell / osteoclast-specific gene 2 include, for example, variants disclosed in Examples (human acyloxyacyl hydrolase (AOAH); total number of nucleotides in ORF nucleic acid sequence: 1728); A total of 575 amino acids; GenBank accession number: NM_001637.1) has been reported. It is reported that a known variant of blood cell / osteoclast specific gene 2 has a predetermined function (eg, acyloxyacyl hydrolase activity) (eg, Hagen, FS et al., Biochemistry 30 (34), 8415-8423 (1991)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of blood cell / osteoclast specific gene 2 may also have these functions.
The region consisting of the 1st to 23rd amino acid residues in the amino acid sequence represented by SEQ ID NO: 30 is SignalP (http://www.cbs.dtu.dk/services/SignalP/) which is a signal sequence prediction tool. And PSORT II (http://psort.nibb.ac.jp/), it is predicted as a signal region. In addition, NM_001637.1, a known variant of blood cell / osteoclast-specific gene 2, is a signal sequence prediction tool in addition to being described in the database as known information that the same region corresponds to the signal sequence. Signal sequences are also predicted in the same region in SignalP and PSORT II. Therefore, the present invention also provides a polypeptide from which such a signal region has been removed (that is, an amino acid sequence consisting of amino acid residues 24 to 543 in the amino acid sequence represented by SEQ ID NO: 30 or substantially the same as that). A polypeptide having the amino acid sequence of

3) Blood cell / osteoclast specific gene 3
D-PLACE7010497.1 (SEQ ID NO: 57)
Examples of known variants of blood cell / osteoclast-specific gene 3 include, for example, variants disclosed in Examples (human LIM domain kinase 1 (LIMK1); total number of nucleotides in ORF nucleic acid sequence: 1944); The total number of amino acids of 647; GenBank accession number: NM_002314.2) has been reported. It is reported that a known variant of blood cell / osteoclast specific gene 3 has a predetermined function (eg, kinase activity) (eg, Okano, I. et al., J. Biol. Chem. 270). (52), 31321-31330 (1995); Mizuno, K. et al., Oncogene 9 (6), 1605-1612 (1994)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of blood cell / osteoclast specific gene 3 may also have these functions.

4) Blood cell / osteoclast specific gene 4
D-SPLEN2030304.1 (SEQ ID NO: 73)
Examples of known variants of blood cell / osteoclast-specific gene 4 include, for example, variants disclosed in the Examples (human protein tyrosine phosphatase, non-receptor type 4 (PTPN4); total number of nucleotides in ORF nucleic acid sequence: 2781 A total of 926 amino acids in the protein; GenBank accession number: NM_002830.2) has been reported. It is reported that a known variant of blood cell / osteoclast specific gene 4 has a predetermined function (eg, tyrosine phosphatase activity) (eg, Gu, M. et al., J. Biol. Chem. 271 (44), 27751-27759 (1996)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of blood cell / osteoclast specific gene 4 may also have these functions.

5) Blood cell / osteoclast specific gene 5
D-SYNOV4000324.1 (SEQ ID NO: 88)
Examples of known variants of blood cell / osteoclast specific gene 5 include, for example, the variants disclosed in the Examples (human NCK adapter protein 1 (NCK1); total number of nucleotides in ORF nucleic acid sequence: 1134; in protein 377 amino acids; GenBank accession number: NM_006153.3) has been reported. It has been reported that a known variant of blood cell / osteoclast specific gene 5 has a predetermined function (eg, ability to interact with Fas ligand or nephrin) (eg, Lettau, M. et al., Proc. Natl. Acad. Sci. USA 103 (15), 5911-5916 (2006); Jones, N. et al., Nature 440 (7085), 818-823 (2006)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of blood cell / osteoclast specific gene 5 may also have these functions.

6) Blood cell / osteoclast specific gene 6
D-SYNOV4007243.1 (SEQ ID NO: 104)
Examples of known variants of the blood cell / osteoclast specific gene 6 include, for example, the variant disclosed in the Examples (human adenosine monophosphate deaminase (isoform E) (AMPD3); the total number of nucleotides in the ORF nucleic acid sequence: 2331 A total of 776 amino acids in the protein; GenBank accession number: NM_000480.1) has been reported. Known variants of the blood cell / osteoclast specific gene 6 have been reported to have a predetermined function (eg, adenosine monophosphate deaminase activity) (eg, Zydowo, MM, Acta Biochim. Pol. 40 (4 ), 429-432 (1993); Yamada, Y. et al., Biochim. Biophys. Acta 1171 (1), 125-128 (1992)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of blood cell / osteoclast specific gene 6 may also have these functions.

7) Blood cell / osteoclast specific gene 7
D-NETRP2004161.1 (SEQ ID NO: 120)
Examples of known variants of the hematopoietic / osteoclast specific gene 7 include, for example, the variants disclosed in the examples (human butyrophilin-like 8 (BTNL8), transcription variant 1; total number of nucleotides in the ORF nucleic acid sequence: 1044; total number of amino acids 347 in protein; GenBank accession number: NM_024850.1) has been reported. Known variants of blood cell / osteoclast-specific gene 7 have been reported to have predetermined functions (eg, functions of immunoglobulin molecules including functions as immune system, plasma membrane receptor and adhesion molecule). (See, eg, Ogg, SL et al., Mammalian Genome 7: 900-905 (1996); Shibui, A. et al., J. Hum. Genet. 44: 249-252 (1999)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of blood cell / osteoclast specific gene 7 may also have these functions.

8) Blood cell / osteoclast specific gene 8
D-NT2RI3001993.1 (SEQ ID NO: 143)
D-SMINT2013276.1 (SEQ ID NO: 153)
Examples of known variants of the hematopoietic / osteoclast specific gene 8 include, for example, the variant disclosed in the Examples (human chromosome 6 open reading frame 32 (C6orf32); total number of nucleotides in ORF nucleic acid sequence: 1776) A total number of 591 amino acids in the protein; GenBank accession number: NM_015864.2) has been reported. A known variant of hematopoietic / osteoclast specific gene 8 is responsible for the formation of a non-mitotic polynuclear syncytium from a proliferative cytotrophoblast in a given function (eg, trophoblast differentiation). (Eg, Morrish, DW et al., Curr. Protein Pept. Sci. 2 (3), 245-259 (2001); Morrish, DW et al., J. Reprod. Immunol. 39 (1-2), 179-195 (1998); Dakour, J. et al., Gene 185 (2), 153-157 (1997)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of blood cell / osteoclast specific gene 8 may also have these functions.

9) Blood cell / osteoclast specific gene 9
D-THYMU3006588.1 (SEQ ID NO: 173)
D-D3OST2003177.1 (SEQ ID NO: 180)
Examples of known variants of the blood cell / osteoclast specific gene 9 include, for example, the variants disclosed in the examples (human FYVE, RhoGEF and PH domain-containing 4 (FGD4); the total number of nucleotides in the ORF nucleic acid sequence: 2301. A total of 766 amino acids in the protein; GenBank accession number: NM_139241.1) has been reported. Known variants of the hematopoietic / osteoclast specific gene 9 have specific functions (eg, specific binding ability to Rho GTPase cdc42 via PH and DH homology domains, and GDP-GTP exchange of isoprenylated cdc42). (See, for example, Zheng, Y. et al., J. Biol. Chem. 271: 33169-33172 (1996)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of blood cell / osteoclast specific gene 9 may also have these functions.

10) Blood cell / osteoclast specific gene 10
D-D3OST2000831.1 (SEQ ID NO: 201)
Examples of known variants of the blood cell / osteoclast specific gene 10 include, for example, the variant disclosed in the examples (human hypothetical protein BC009732 (LOC133308); the total number of nucleotides in the ORF nucleic acid sequence: 1614; protein 537 amino acids in total; GenBank accession number: see NM_178833.3) has been reported. Known variants of blood cell / osteoclast specific gene 10 have been reported to have a predetermined function (eg apical Na ⁺ / H ⁺ exchange activity) (eg Klanke, CA et al. , Genomics 25: 615-622 (1995)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of the blood cell / osteoclast specific gene 10 may also have these functions.

11) Blood cell / osteoclast specific gene 11
D-HCASM2001914.1 (SEQ ID NO: 226)
D-SYNOV4003291.1 (SEQ ID NO: 233)
Examples of known variants of the blood cell / osteoclast specific gene 11 include, for example, the variants disclosed in the Examples (human proteoglycan 4 (PRG4); total number of nucleotides in ORF nucleic acid sequence: 4215; amino acids in protein) 1404; GenBank accession number: see NM_005807.1) has been reported. Known variants of the blood cell / osteoclast-specific gene 11 have a predetermined function (for example, the ability to promote differentiation of bone marrow mononuclear cells into hematopoietic cells and / or endothelial cells, the ability to promote proliferation in cartilage metabolism, the ability to protect cells) (Eg, Liu, YJ et al., Blood 103 (12), 4449-4456 (2004); Flannery, CR et al., Biochem. Biophys. Res. Commun. 254 (3), 535-541 (1999)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of the blood cell / osteoclast specific gene 11 may also have these functions.
A region consisting of amino acid residues 1 to 24 in the amino acid sequences represented by SEQ ID NO: 226 and SEQ ID NO: 233 is predicted as a signal region by SignalP and PSORT II, which are signal sequence prediction tools. In NM_005807.1, which is a known variant of the blood cell / osteoclast specific gene 11, the same region is predicted as a signal sequence by SignalP and PSORT II. Therefore, the present invention also provides a polypeptide from which such a signal region has been removed (that is, an amino acid sequence consisting of amino acid residues 25 to 853 in the amino acid sequence represented by SEQ ID NO: 226, or SEQ ID NO: 233). A polypeptide having an amino acid sequence consisting of amino acid residues 25 to 216 in the amino acid sequence or a substantially identical amino acid sequence thereto.

12) Blood cell / osteoclast specific gene 12
D-NETRP2002051.1 (SEQ ID NO: 251)
D-THYMU2031383.1 (SEQ ID NO: 266)
D-THYMU3032318.1 (SEQ ID NO: 272)
Examples of known variants of the blood cell / osteoclast specific gene 12 include, for example, the variant disclosed in the Examples (human cerebral cavernous malformation 2 (CCM2); nucleotides in the ORF nucleic acid sequence) The total number: 1335; the total number of amino acids in the protein: 444; GenBank accession number: NM_031443.2) has been reported. A known variant of the hematopoietic / osteoclast specific gene 12 may have a predetermined function (eg, phosphotyrosine binding ability resulting from a phosphotyrosine binding domain) (eg, Liquori, CL et al., Am. J. Hum Genet. 73 (6), 1459-1464 (2003)). In general, it is known that a plurality of variants (splicing variants) generated from a single locus have similar functions, although the degree may vary. Therefore, a novel variant of blood cell / osteoclast specific gene 12 may also have these functions.

  The polypeptide of the present invention is, for example, a substance that can specifically recognize the polypeptide of the present invention, a substance that cannot specifically recognize the polypeptide of the present invention, or both of the polypeptide of the present invention and a known polypeptide. Development of substances capable of comprehensively recognizing, and substances that can specifically regulate the function of the polypeptide of the present invention, substances that cannot specifically regulate the function of the polypeptide of the present invention, It may be useful for the development of substances that can comprehensively recognize the functions of both peptides and known polypeptides.

  The present invention also provides partial peptides.

  The “partial peptide” has at least 6 selected from the target polypeptide, preferably having a predetermined utility (eg, use as an immunogenic or antigenic peptide, a functional peptide having a specific domain, etc.), preferably Consists of at least 8, more preferably at least 10, even more preferably at least 12, most preferably at least 15 consecutive amino acid residues.

  “Inserted amino acid sequence of the polypeptide of the present invention” means an amino acid sequence that is inserted in the polypeptide of the present invention (eg, a new variant) but is deleted in a known polypeptide (eg, a known variant). Say. On the other hand, the “inserted amino acid sequence of a known polypeptide” means an amino acid sequence that is inserted in a known polypeptide (eg, known variant) but is deleted in the polypeptide of the present invention (eg, new variant). Say. These inserted amino acid sequences are apparent from the disclosure herein.

  “Deleted amino acid sequence of the polypeptide of the present invention” means an amino acid that is deleted in a polypeptide of the present invention (eg, a new variant) but inserted in a known polypeptide (eg, a known variant). An array. On the other hand, the “deleted amino acid sequence of a known polypeptide” means an amino acid that is deleted in a known polypeptide (eg, known variant) but inserted in the polypeptide of the present invention (eg, new variant). An array. These deleted amino acid sequences are apparent from the disclosure herein. The “deleted amino acid sequence of the polypeptide of the present invention” may be synonymous with the “inserted amino acid sequence of the known polypeptide”, and the “deleted amino acid sequence of the known polypeptide” It can be synonymous with “sequence”.

  The partial peptide of the present invention is a) a specific partial peptide of the polypeptide of the present invention (abbreviated as “specific partial peptide A” if necessary), which can distinguish the polypeptide of the present invention from known polypeptides. B) a specific partial peptide of the known polypeptide that can be distinguished from the polypeptide of the present invention (abbreviated as “specific partial peptide B” if necessary), or c) the present invention. A partial peptide common to both the polypeptide and the known polypeptide (abbreviated as “common partial peptide” if necessary). Although these specific partial peptides are motivated to be produced or used as markers by the knowledge of the present inventors, without such knowledge, the motivation to produce them or be used as markers There is no. Specific partial peptides A and B, which are partial peptides specific to the polypeptide encoded by the blood cell / osteoclast specific genes 1 to 12, may be referred to as “specific partial peptides of the present invention”. Or “specific partial peptide”.

  The specific partial peptide A of the present invention is a partial peptide that exists only in a polypeptide having the amino acid sequence represented by SEQ ID NO: X and does not exist in a known polypeptide. Examples of such specific partial peptide A include i) a partial peptide consisting of the inserted amino acid sequence of the polypeptide of the present invention or a partial amino acid sequence thereof, and ii) an inserted amino acid sequence of the polypeptide of the present invention or a terminal portion thereof. A partial peptide consisting of an amino acid sequence and its adjacent amino acid sequence, and iii) both amino acids present on the N-terminal side and C-terminal side with respect to the inserted amino acid sequence of a known polypeptide resulting from the deletion of an exon Examples include partial peptides consisting of amino acid sequences linked in sequence.

  The specific partial peptide A of i) above consists of an inserted amino acid sequence of the polypeptide of the present invention or a partial amino acid sequence thereof. Such partial amino acid sequences are apparent from the disclosure herein.

  The specific partial peptide A of ii) above consists of the inserted amino acid sequence of the polypeptide of the present invention or the terminal partial amino acid sequence thereof and the adjacent amino acid sequence thereof. Examples of such a terminal partial amino acid sequence include an amino acid sequence corresponding to the N-terminal portion in the insertion amino acid sequence of the polypeptide of the present invention (abbreviated as “N-terminal partial amino acid sequence A” if necessary), An amino acid sequence corresponding to the C-terminal portion in the inserted amino acid sequence of the peptide (abbreviated as “C-terminal partial amino acid sequence A” if necessary) can be mentioned. Examples of such an adjacent amino acid sequence include an amino acid sequence existing on the N-terminal side with respect to the insertion amino acid sequence of the polypeptide of the present invention (abbreviated as “N-terminal adjacent amino acid sequence A” if necessary), An amino acid sequence existing on the C-terminal side with respect to the inserted amino acid sequence of the peptide (abbreviated as “C-terminal adjacent amino acid sequence A” if necessary). Therefore, the specific partial peptide A of the above ii) is a partial peptide consisting of an amino acid sequence extending from a predetermined position of the N-terminal adjacent amino acid sequence A to a predetermined position of the inserted amino acid sequence of the polypeptide of the present invention. A partial peptide consisting of an amino acid sequence extending from a predetermined position of the inserted amino acid sequence of the peptide to a predetermined position of the C-terminal adjacent amino acid sequence A, or a predetermined position of the C-terminal adjacent amino acid sequence A from a predetermined position of the N-terminal adjacent amino acid sequence A It can be a partial peptide consisting of an amino acid sequence including the entire insertion amino acid sequence of the polypeptide of the present invention. Amino acid residues in the inserted amino acid sequence (or N-terminal or C-terminal partial amino acid sequence A) or the adjacent amino acid sequence (or N-terminal or C-terminal adjacent amino acid sequence A) contained in the specific partial peptide A of ii) above The number is not particularly limited as long as it is a number that can ensure the specificity of the specific partial peptide A of ii) above, for example, at least 3, preferably at least 4, more preferably at least 5, even more preferably There may be at least 6, most preferably at least 7, 8, 9, or 10. Such terminal partial amino acid sequences and such adjacent amino acid sequences are apparent from the disclosure herein.

  The specific partial peptide A in the above iii) is an amino acid sequence in which both amino acid sequences existing on the N-terminal side and the C-terminal side are linked to the insertion amino acid sequence of a known polypeptide (exon in the polypeptide of the present invention). These amino acid sequences are linked to each other due to the deletion of (1), and is a partial peptide that does not exist in a known polypeptide. The number of amino acid residues in each amino acid sequence present on the N-terminal side and C-terminal side with respect to the inserted amino acid sequence of the known polypeptide contained in the specific partial peptide A of iii) is the same as that of iii) above. Although it is not particularly limited as long as it is a number that can ensure the specificity of a specific partial peptide A, for example, at least 3, preferably at least 4, more preferably at least 5, even more preferably at least 6, most preferably May be at least 7, 8, 9, or 10.

  The specific partial peptide A of the present invention can be useful, for example, as a target for specifically detecting the polypeptide of the present invention, and as a marker specific for blood cells or specific for osteoclast differentiation. . The specific partial peptide A of the present invention is also a substance that can specifically recognize the polypeptide of the present invention, or a substance that cannot specifically recognize the polypeptide of the present invention, or the polypeptide of the present invention. It may be useful for the development of a substance that can specifically regulate the function of or a substance that cannot specifically regulate the function of the polypeptide of the present invention.

  The specific partial peptide B of the present invention is a partial peptide that exists only in a known polypeptide and does not exist in a polypeptide having the amino acid sequence represented by SEQ ID NO: X or the like. Examples of such a specific partial peptide B include i) a partial peptide consisting of an inserted amino acid sequence of a known polypeptide or a partial amino acid sequence thereof; ii) an inserted amino acid sequence of a known polypeptide or a terminal partial amino acid sequence thereof; A partial peptide consisting of an adjacent amino acid sequence, iii) both amino acid sequences present on the N-terminal side and C-terminal side of the inserted amino acid sequence of the polypeptide of the present invention formed due to the deletion of an exon are linked A partial peptide consisting of the amino acid sequence.

  The specific partial peptide B of i) above consists of an inserted amino acid sequence of a known polypeptide or a partial amino acid sequence thereof. Such partial amino acid sequences are apparent from the disclosure herein.

  The specific partial peptide B of ii) above consists of an inserted amino acid sequence of a known polypeptide or its terminal partial amino acid sequence and its adjacent amino acid sequence. Examples of such terminal partial amino acid sequences include amino acid sequences corresponding to the N-terminal portion in the inserted amino acid sequence of known polypeptides (abbreviated as “N-terminal partial amino acid sequence B” if necessary), inserted amino acids of known polypeptides. An amino acid sequence corresponding to the C-terminal portion in the sequence (abbreviated as “C-terminal partial amino acid sequence B” if necessary) can be mentioned. As such an adjacent amino acid sequence, an amino acid sequence present on the N-terminal side with respect to an inserted amino acid sequence of a known polypeptide (abbreviated as “N-terminal adjacent amino acid sequence B” as necessary) An amino acid sequence existing on the C-terminal side with respect to the sequence (abbreviated as “C-terminal adjacent amino acid sequence B” if necessary) can be mentioned. Therefore, the specific partial peptide B of the above ii) is a partial peptide consisting of an amino acid sequence extending from a predetermined position of the N-terminal adjacent amino acid sequence B to a predetermined position of an inserted amino acid sequence of a known polypeptide, an inserted amino acid of a known polypeptide A partial peptide consisting of an amino acid sequence extending from a predetermined position of the sequence to a predetermined position of the C-terminal adjacent amino acid sequence B, or a known polypeptide extending from a predetermined position of the N-terminal adjacent amino acid sequence B to a predetermined position of the C-terminal adjacent amino acid sequence B; It may be a partial peptide consisting of an amino acid sequence including the entire inserted amino acid sequence of the peptide. Amino acid residues in the inserted amino acid sequence (or N-terminal or C-terminal partial amino acid sequence B) or the adjacent amino acid sequence (or N-terminal or C-terminal adjacent amino acid sequence B) contained in the specific partial peptide B of ii) above The number is not particularly limited as long as it is a number that can ensure the specificity of the specific partial peptide B of ii) above, for example, at least 3, preferably at least 4, more preferably at least 5, even more preferably There may be at least 6, most preferably at least 7, 8, 9, or 10. Such terminal partial amino acid sequences and such adjacent amino acid sequences are apparent from the disclosure herein.

  The specific partial peptide B of the above iii) is an amino acid sequence in which both amino acid sequences existing on the N-terminal side and C-terminal side are linked to the insertion amino acid sequence of the polypeptide of the present invention (exon in the known polypeptide) These amino acid sequences are linked to each other due to the deletion of), and is a partial peptide that does not exist in the polypeptide of the present invention. The number of amino acid residues in each amino acid sequence present on the N-terminal side and the C-terminal side with respect to the inserted amino acid sequence of the polypeptide of the present invention contained in the specific partial peptide B of iii) above is respectively iii Is not particularly limited as long as the specificity of the specific partial peptide B can be ensured, for example, at least 3, preferably at least 4, more preferably at least 5, even more preferably at least 6, Most preferably it may be at least 7, 8, 9, or 10.

  The specific partial peptide B of the present invention is, for example, as a target for specifically detecting a known polypeptide, and as a marker specific to the blood cell system or osteoclast differentiation, or non-specific to these. As useful markers. The specific partial peptide B of the present invention also specifically develops a substance that can specifically recognize a known polypeptide, or a substance that cannot specifically recognize a known polypeptide, or specifically functions of a known polypeptide. It may be useful in the development of substances that can be modulated or substances that cannot specifically regulate the function of a known polypeptide.

  The common partial peptide of the present invention may be a non-specific partial peptide that exists in both the polypeptide of the present invention and the known polypeptide. Such partial peptides are apparent from the disclosure herein. The common partial peptide of the present invention, for example, as a target for comprehensive detection of both the polypeptide of the present invention and known polypeptides, and as a marker specific for blood cell lineage or specific for osteoclast differentiation, Alternatively, it may be useful as a non-specific marker for these. The consensus peptide of the present invention can also comprehensively regulate the substance capable of comprehensively recognizing both the polypeptide of the present invention and the known polypeptide, or the functions of both the polypeptide of the present invention and the known polypeptide. Can be useful in material development.

  The polypeptide of the present invention or a specific partial peptide thereof may be fused with a polypeptide consisting of a heterologous amino acid sequence. Such polypeptides include polypeptides that facilitate purification or solubilization. Specifically, such polypeptides include histidine tag, maltose binding protein (MBP), glutathione-S-transferase (GST), calmodulin binding peptide (CBP), FLAG, Fc region of IgG molecule.

  The polypeptide of the present invention and its partial peptide may be provided in the form of a salt. Examples of the salt include inorganic bases (eg, alkali metals such as sodium and potassium; alkaline earth metals such as calcium and magnesium; aluminum and ammonium), organic bases (eg, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, Diethanolamine, triethanolamine, dicyclohexylamine, N, N-dibenzylethylenediamine), inorganic acids (eg, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid), organic acids (eg, formic acid, acetic acid, trifluoroacetic acid) , Fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid), basic amino acids (eg, arginine, lysine, ornithine) or acidic Amino acids (eg, aspartic acid, And salts with amine acid).

  The polypeptide of the present invention and its partial peptide can be prepared by a method known per se. For example, the polypeptide of the present invention and its partial peptide may be recovered from 1) the expression site, or 2) recovered from the transformant described below that expresses the polypeptide of the present invention and its partial peptide or its culture supernatant. 3) may be synthesized by a cell-free system using rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, or the like, or 4) may be synthesized organically (eg, solid phase synthesis). . The polypeptide of the present invention and its partial peptide are mainly molecular weights such as a method using solubility such as salting out and solvent precipitation; dialysis, ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis, etc. A method utilizing a difference in charge; a method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography or use of an antibody; a hydrophobic method such as reverse phase high performance liquid chromatography. It is appropriately purified by a method using a difference; a method using a difference in isoelectric point such as isoelectric focusing method; a method combining these, and the like.

1.2. Polynucleotide and its partial nucleotides The present invention relates to a nucleic acid sequence represented by SEQ ID NO: Y, a nucleic acid sequence Y1 or a nucleic acid sequence Y2, or a nucleic acid sequence substantially identical thereto (if necessary, represented by “SEQ ID NO: Y”). A polynucleotide having the abbreviation "Nucleic acid sequence etc."

  “SEQ ID NO: Y” represents the SEQ ID NO of any nucleic acid sequence disclosed herein. The polynucleotide having “SEQ ID NO: Y” means a polynucleotide “consisting of” SEQ ID NO: Y or the like, or a polynucleotide “including” the nucleic acid sequence or the like.

  “Nucleic acid sequence Y1” indicates a nucleic acid sequence corresponding to the coding portion (that is, the entire open reading frame (ORF) or a part thereof) in the nucleic acid sequence represented by SEQ ID NO: Y. In other words, “nucleic acid sequence Y1” indicates the nucleic acid sequence represented by SEQ ID NO: Y when the nucleic acid sequence represented by SEQ ID NO: Y consists of a nucleic acid sequence corresponding only to the coding portion. Where the nucleic acid sequence represented includes nucleic acid sequences that correspond to both the coding and non-coding portions, the nucleic acid sequence that corresponds only to the coding portion is indicated.

  “Nucleic acid sequence Y2” refers to a nucleic acid sequence corresponding to a non-coding portion (eg, 5 ′ or 3 ′ untranslated region) in the nucleic acid sequence represented by SEQ ID NO: Y. In other words, “nucleic acid sequence Y2” indicates the nucleic acid sequence represented by SEQ ID NO: Y when the nucleic acid sequence represented by SEQ ID NO: Y consists of a nucleic acid sequence corresponding only to a non-coding portion, When the nucleic acid sequence represented by (1) includes a nucleic acid sequence corresponding to both the non-coding portion and the coding portion, the nucleic acid sequence corresponding to only the non-coding portion is indicated.

  Accordingly, the nucleic acid sequence represented by “SEQ ID NO: Y” is i) nucleic acid sequence Y1 (when the entire nucleic acid sequence represented by SEQ ID NO: Y is a nucleic acid sequence corresponding to the coding portion), ii) nucleic acid sequence Y2 ( The entire nucleic acid sequence represented by SEQ ID NO: Y is a nucleic acid sequence corresponding to a non-coding portion), or iii) a nucleic acid sequence comprising nucleic acid sequence Y1 and nucleic acid sequence Y2 (the nucleic acid sequence represented by SEQ ID NO: Y is coding) The nucleic acid sequence corresponding to the portion and the non-coding portion).

  In one embodiment, the nucleic acid sequence represented by SEQ ID NO: Y, or the nucleic acid sequence substantially identical to the nucleic acid sequence Y1 or nucleic acid sequence Y2, is the nucleic acid sequence represented by SEQ ID NO: Y, or the nucleic acid sequence Y1 or nucleic acid sequence It may be a nucleic acid sequence having a predetermined sequence identity with Y2. The degree of nucleic acid sequence identity is about 90% or higher, preferably about 92% or higher, more preferably about 95% or higher, even more preferably about 96% or higher, most preferably about 97% or higher, about 98% or higher, or It can be about 99% or more. Nucleic acid sequence identity can be determined by methods known per se. For example, the nucleic acid sequence identity (%) can be determined by the same method as the amino acid sequence identity (%) described above.

  In another embodiment, the nucleic acid sequence represented by SEQ ID NO: Y, or the nucleic acid sequence substantially identical to the nucleic acid sequence Y1 or nucleic acid sequence Y2, is the nucleic acid sequence represented by SEQ ID NO: Y, or the nucleic acid sequence Y1 or nucleic acid In the sequence Y2, one or more nucleotides may be a nucleic acid sequence subjected to one or more modifications selected from substitution, addition, deletion and insertion. The number of nucleotides to be modified is not particularly limited as long as it is 1 or more. For example, 1 to about 100, preferably 1 to about 70, more preferably 1 to about 50, and even more preferably 1 to about 30. And most preferably 1 to about 20, 1 to about 10 or 1 to about 5 (eg, 1 or 2).

  In yet another embodiment, the nucleic acid sequence represented by SEQ ID NO: Y, or the nucleic acid sequence substantially identical to the nucleic acid sequence Y1 or nucleic acid sequence Y2, is the nucleic acid sequence represented by SEQ ID NO: Y, or the nucleic acid sequence Y1 or It can be a polynucleotide that can hybridize under high stringency conditions to a nucleic acid sequence complementary to the nucleic acid sequence Y2. Hybridization conditions under high stringency conditions can be set with reference to previously reported conditions (eg, see Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6 (1999)). . For example, hybridization conditions under high stringent conditions include hybridization at 6 × SSC (sodium chloride / sodium citrate) / 45 ° C., then 0.2 × SSC / 0.1% SDS / 50 to 65 ° C. Or two or more washes.

  The nucleic acid sequence represented by SEQ ID NO: Y, or a nucleic acid sequence substantially identical to the nucleic acid sequence Y1 or the nucleic acid sequence Y2, has its characteristic part (eg, part corresponding to a specific partial nucleotide described later) completely And the other portion (eg, the portion present in the known polynucleotide) is substantially the same as the nucleic acid sequence represented by SEQ ID NO: Y or the corresponding portion of the nucleic acid sequence Y1 or the nucleic acid sequence Y2. There may be. Alternatively, the nucleic acid sequence represented by SEQ ID NO: Y, the nucleic acid sequence substantially identical to the nucleic acid sequence Y1 or the nucleic acid sequence Y2, the nucleic acid sequence represented by SEQ ID NO: Y, or the nucleic acid sequence Y1 Alternatively, the corresponding portion of the nucleic acid sequence Y2 may be the same and the characteristic portion may be substantially the same as the nucleic acid sequence represented by SEQ ID NO: Y, or the corresponding portion of the nucleic acid sequence Y1 or the nucleic acid sequence Y2. .

  The polynucleotide of the present invention may encode the polypeptide of the present invention. Therefore, the polynucleotide of the present invention can be a polynucleotide such that the polypeptide encoded thereby can exhibit the same function as the polypeptide of the present invention.

  Specifically, the nucleic acid sequence Y of the polynucleotide for the hematopoietic / osteoclast specific gene 1-12, and the SEQ ID NO: Y of the ORF corresponding portion (the Yath to Ybth nucleotide residues in the nucleic acid sequence Y), and The Yath to Ybth are as follows.

1) Specific gene 1 of the present invention
D-SYNOV4003692.1 (SEQ ID NO: 8 or SEQ ID NO: 9, and positions 176 to 1126)

2) Specific gene 2 of the present invention
D-D3OST2001534.1 (SEQ ID NO: 28 or SEQ ID NO: 29, and 288 to 1919th)
D-NETRP1000046.1 (SEQ ID NO: 35 or SEQ ID NO: 36, and 57th to 950th positions)
The region consisting of nucleotide residues 288 to 356 in the nucleic acid sequence represented by SEQ ID NO: 28 or 29 encodes the signal region by SignalP and PSORT II (see above) which are signal sequence prediction tools. It is predicted that In addition, NM_001637.1, a known variant of blood cell / osteoclast-specific gene 2, is a signal sequence prediction tool in addition to being described in the database as known information that the same region corresponds to the signal sequence. Signal sequences are also predicted in the same region in SignalP and PSORT II. Therefore, the present invention also provides a polynucleotide in which the polynucleotide encoding such a putative signal region is removed from the ORF corresponding portion (that is, nucleotide residues 357 to 1919 in the nucleic acid sequence represented by SEQ ID NO: 28). Or a polynucleotide having a nucleic acid sequence substantially identical to the nucleic acid sequence.

3) Specific gene 3 of the present invention
D-PLACE7010497.1 (SEQ ID NO: 55 or SEQ ID NO: 56, and positions 34 to 1875)

4) Specific gene 4 of the present invention
D-SPLEN2030304.1 (SEQ ID NO: 71 or 72, and 89th to 1768th)

5) Specific gene 5 of the present invention
D-SYNOV4000324.1 (SEQ ID NO: 86 or SEQ ID NO: 87, and positions 45 to 986)

6) Specific gene 6 of the present invention
D-SYNOV4007243.1 (SEQ ID NO: 102 or SEQ ID NO: 103, and 202 to 2526th)

7) Specific gene 7 of the present invention
D-NETRP2004161.1 (SEQ ID NO: 118 or SEQ ID NO: 119, and positions 290 to 1240)

8) Specific gene 8 of the present invention
D-NT2RI3001993.1 (SEQ ID NO: 141 or SEQ ID NO: 142, and positions 63 to 3206)
D-SMINT2013276.1 (SEQ ID NO: 151 or SEQ ID NO: 152, and 93 to 2036)

9) Specific gene 9 of the present invention
D-THYMU3006588.1 (SEQ ID NO: 171 or SEQ ID NO: 172 and positions 87 to 2723)
D-D3OST2003177.1 (SEQ ID NO: 178 or SEQ ID NO: 179, and positions 163 to 2718)

10) Specific gene 10 of the present invention
D-D3OST2000831.1 (SEQ ID NO: 199 or SEQ ID NO: 200, and positions 178 to 1620)

11) Specific gene 11 of the present invention
D-HCASM2001914.1 (SEQ ID NO: 224 or SEQ ID NO: 225, and 46 to 2604th)
D-SYNOV4003291.1 (SEQ ID NO: 231 or SEQ ID NO: 232, and 46th to 693rd)
The region consisting of the 46th to 117th nucleotide residues in the nucleic acid sequence represented by SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 231 or SEQ ID NO: 232 is a signal sequence prediction tool SignalP and PSORT II (see above) ) Is predicted to encode a signal region. In NM_005807.1, which is a known variant of the blood cell / osteoclast specific gene 11, the same region is predicted as a signal sequence by SignalP and PSORT II. Therefore, the present invention also provides a polynucleotide in which the polynucleotide encoding such a putative signal region is removed from the ORF-corresponding portion (that is, the nucleotide residues 118 to 2604 in the nucleic acid sequence represented by SEQ ID NO: 224). A nucleic acid sequence comprising a group, a nucleic acid sequence comprising nucleotide residues 118 to 693 in the nucleic acid sequence represented by SEQ ID NO: 231, or a polynucleotide having a nucleic acid sequence substantially identical thereto.

12) Specific gene 12 of the present invention
D-NETRP2002051.1 (SEQ ID NO: 249 or SEQ ID NO: 250, and positions 34 to 579)
D-THYMU2031383.1 (SEQ ID NO: 264 or SEQ ID NO: 265, and positions 34 to 816)
D-THYMU3032318.1 (SEQ ID NO: 270 or SEQ ID NO: 271, and positions 36 to 470)

  The polynucleotide of the present invention is, for example, a substance that can specifically recognize the polynucleotide of the present invention, a substance that cannot specifically recognize the polynucleotide of the present invention, or both the polynucleotide of the present invention and a known polynucleotide. Development of a substance capable of comprehensively recognizing a substance, a substance capable of specifically regulating the expression of a polypeptide of the present invention, a substance capable of not specifically regulating the expression of a polypeptide of the present invention, or a polypeptide of the present invention It may be useful for the development of substances that can comprehensively regulate the expression of both peptides and known polypeptides.

  The present invention also provides partial nucleotides.

  “Partial nucleotide” means predetermined utility (eg, use as a probe, primer, polynucleotide encoding an immunogenic or antigenic peptide, polynucleotide encoding a functional peptide having a specific domain, etc.) At least 15, preferably at least 16, more preferably at least 18, even more preferably at least 20, most preferably at least 22, 23, 24 or 25 selected from the polynucleotide of interest Of consecutive nucleotide residues.

  The “inserted nucleic acid sequence of the polynucleotide of the present invention” refers to a nucleic acid sequence that is inserted into a polynucleotide of the present invention (eg, a novel variant) but is deleted in a known polynucleotide (eg, a known variant). Say. On the other hand, the “inserted nucleic acid sequence of a known polynucleotide” refers to a nucleic acid sequence that is inserted in a known polynucleotide (eg, known variant) but is deleted in the polynucleotide (eg, novel variant) of the present invention. Say. These inserted nucleic acid sequences will be apparent from the disclosure herein.

  “Deleted nucleic acid sequence of the polynucleotide of the present invention” means a nucleic acid that is deleted in a polynucleotide of the present invention (eg, a novel variant) but inserted in a known polynucleotide (eg, a known variant). An array. On the other hand, a “deleted nucleic acid sequence of a known polynucleotide” refers to a nucleic acid that is deleted in a known polynucleotide (eg, known variant) but inserted in the polynucleotide (eg, novel variant) of the present invention. An array. These deleted nucleic acid sequences are apparent from the disclosure herein. The “deleted nucleic acid sequence of the polynucleotide of the present invention” may be synonymous with the “inserted nucleic acid sequence of the known polynucleotide”, and the “deleted nucleic acid sequence of the known polynucleotide” It can be synonymous with “sequence”.

  The partial nucleotide of the present invention is a) a specific partial nucleotide of the polynucleotide of the present invention (abbreviated as “specific partial nucleotide A” if necessary) that can distinguish the polynucleotide of the present invention from known polynucleotides. B) a specific partial nucleotide of the known polynucleotide (abbreviated as “specific partial nucleotide B” if necessary), or c) the present invention, wherein the known polynucleotide can be distinguished from the polynucleotide of the present invention. Partial nucleotides common to both the polynucleotide and known polynucleotides (abbreviated as “common partial nucleotide” as necessary). Although these specific partial nucleotides are motivated to be produced or used as markers by the knowledge of the present inventors, without such knowledge, the motivation to produce or use them as markers is provided. There is no. Specific partial nucleotides A and B, which are specific partial nucleotides of the polynucleotide encoded by the hemocyte / osteoclast-specific genes 1 to 12, may be referred to as “specific partial nucleotides of the present invention”. "Or" specific partial nucleotide ".

  The specific partial nucleotide A of the present invention is a partial nucleotide that exists only in a polynucleotide having the nucleic acid sequence represented by SEQ ID NO: Y and does not exist in a known polynucleotide. Examples of such specific partial nucleotide A include: i) a partial nucleotide consisting of the inserted nucleic acid sequence of the polynucleotide of the present invention or a partial nucleic acid sequence thereof; ii) an inserted nucleic acid sequence of the polynucleotide of the present invention or a terminal portion thereof. A partial nucleotide consisting of a nucleic acid sequence and its adjacent nucleic acid sequence, and iii) both nucleic acid sequences present 5 ′ and 3 ′ to the inserted nucleic acid sequence of a known polynucleotide resulting from the deletion of an exon. Examples include partial nucleotides consisting of linked nucleic acid sequences.

  The specific partial nucleotide A in i) above consists of the inserted nucleic acid sequence of the polynucleotide of the present invention or a partial nucleic acid sequence thereof. Such partial nucleic acid sequences are apparent from the disclosure herein.

  The specific partial nucleotide A of ii) above consists of the inserted nucleic acid sequence of the polynucleotide of the present invention or its terminal partial nucleic acid sequence and its adjacent nucleic acid sequence. As such a terminal partial nucleic acid sequence, a nucleic acid sequence corresponding to the 5 ′ terminal part in the inserted nucleic acid sequence of the polynucleotide of the present invention (abbreviated as “5 ′ terminal partial nucleic acid sequence A” if necessary), the present invention And a nucleic acid sequence corresponding to the 3 ′ end portion of the inserted nucleic acid sequence of the polypeptide (abbreviated as “3 ′ end portion nucleic acid sequence A” if necessary). As such an adjacent nucleic acid sequence, a nucleic acid sequence existing 5 ′ to the inserted nucleic acid sequence of the polynucleotide of the present invention (abbreviated as “5 ′ adjacent nucleic acid sequence A” if necessary), the polynucleotide of the present invention A nucleic acid sequence existing 3 ′ to the inserted nucleic acid sequence (abbreviated as “3 ′ adjacent nucleic acid sequence A” if necessary). Therefore, the specific partial nucleotide A of ii) above is a partial nucleotide consisting of a nucleic acid sequence extending from a predetermined position of the 5 ′ adjacent nucleic acid sequence A to a predetermined position of the inserted nucleic acid sequence of the polynucleotide of the present invention. Partial nucleotide consisting of a nucleic acid sequence extending from a predetermined position of a nucleotide insertion nucleic acid sequence to a predetermined position of 3 ′ adjacent nucleic acid sequence A, or a predetermined position of 3 ′ adjacent nucleic acid sequence A from a predetermined position of 5 ′ adjacent nucleic acid sequence A Can be a partial nucleotide consisting of a nucleic acid sequence comprising the entire inserted nucleic acid sequence of a polynucleotide of the invention. In the inserted partial nucleic acid sequence (or 5 ′ terminal or 3 ′ terminal partial nucleic acid sequence A) or adjacent nucleic acid sequence (or 5 ′ terminal or 3 ′ terminal adjacent nucleic acid sequence A) included in the specific partial nucleotide A of ii) above The number of nucleotide residues is not particularly limited as long as it is a number that can ensure the specificity of the specific partial nucleotide A in the above ii), for example, at least 3, preferably at least 4, more preferably at least 5, Even more preferably it may be at least 6, most preferably at least 7, 8, 9, or 10. Such terminal partial nucleic acid sequences and such flanking nucleic acid sequences will be apparent from the disclosure herein.

  The specific partial nucleotide A in the above iii) is a nucleic acid sequence in which both 5 ′ and 3 ′ nucleic acid sequences are linked to an inserted nucleic acid sequence of a known polynucleotide (in the polynucleotide of the present invention, an exon is missing). These nucleic acid sequences are linked to each other due to loss, and are partial nucleotides not present in known polynucleotides. The number of nucleotide residues in each nucleic acid sequence present 5 ′ and 3 ′ with respect to the inserted nucleic acid sequence of the known polynucleotide contained in the specific partial nucleotide A of iii) is the specific number of the above iii) Is not particularly limited as long as the specificity of the partial nucleotide A can be ensured, but for example, at least 3, preferably at least 4, more preferably at least 5, even more preferably at least 6, most preferably at least 7 May be 8, 9, 9 or 10.

  The specific partial nucleotide A of the present invention can be useful, for example, as a target for specifically detecting the polynucleotide of the present invention, and as a marker specific for blood cells or specific for osteoclast differentiation. . The specific partial nucleotide A of the present invention can also be used to develop a substance that can specifically recognize the polynucleotide of the present invention, or a substance that cannot specifically recognize the polynucleotide of the present invention, or the polypeptide of the present invention. It may be useful for the development of a substance that can specifically regulate the expression of or a substance that cannot specifically regulate the expression of the polypeptide of the present invention.

  The specific partial nucleotide B of the present invention is a partial nucleotide that exists only in a known polynucleotide and does not exist in a polynucleotide having the nucleic acid sequence represented by SEQ ID NO: Y or the like. Examples of such a specific partial nucleotide B include i) a partial nucleotide consisting of an inserted nucleic acid sequence of a known polynucleotide or a partial nucleic acid sequence thereof; ii) an inserted nucleic acid sequence of a known polynucleotide or a terminal partial nucleic acid sequence thereof; A partial nucleotide consisting of an adjacent nucleic acid sequence, iii) a nucleic acid formed by ligation of both nucleic acid sequences 5 ′ and 3 ′ to the inserted nucleic acid sequence of the polynucleotide of the present invention formed by exon deletion Examples include partial nucleotides consisting of sequences.

  The specific partial nucleotide B in i) above consists of an inserted nucleic acid sequence of a known polynucleotide or a partial nucleic acid sequence thereof. Such partial nucleic acid sequences are apparent from the disclosure herein.

  The specific partial nucleotide B of ii) above consists of an inserted nucleic acid sequence of a known polynucleotide or its terminal partial nucleic acid sequence and its adjacent nucleic acid sequence. As such a terminal partial nucleic acid sequence, a nucleic acid sequence corresponding to the 5 ′ terminal portion of the inserted nucleic acid sequence of a known polynucleotide (abbreviated as “5 ′ terminal partial nucleic acid sequence B” if necessary), Examples include a nucleic acid sequence corresponding to the 3 ′ end portion in the inserted nucleic acid sequence (abbreviated as “3 ′ end portion nucleic acid sequence B” if necessary). As such an adjacent nucleic acid sequence, a nucleic acid sequence existing 5 ′ to an inserted nucleic acid sequence of a known polynucleotide (abbreviated as “5 ′ adjacent nucleic acid sequence B” if necessary), an inserted nucleic acid sequence of a known polynucleotide In contrast, a nucleic acid sequence existing 3 ′ (abbreviated as “3 ′ adjacent nucleic acid sequence B” if necessary). Therefore, the specific partial nucleotide B of the above ii) is a partial nucleotide consisting of a nucleic acid sequence extending from a predetermined position of the 5 ′ adjacent nucleic acid sequence B to a predetermined position of the inserted nucleic acid sequence of the known polynucleotide, an inserted nucleic acid of the known polynucleotide A partial nucleotide consisting of a nucleic acid sequence extending from a predetermined position of the sequence to a predetermined position of the 3 ′ adjacent nucleic acid sequence B, or a known polymorphism extending from a predetermined position of the 5 ′ adjacent nucleic acid sequence B to a predetermined position of the 3 ′ adjacent nucleic acid sequence B; It may be a partial nucleotide consisting of a nucleic acid sequence comprising the entire inserted nucleic acid sequence of nucleotides. In the inserted nucleic acid sequence (or 5′-end or 3′-end partial nucleic acid sequence B) or adjacent nucleic acid sequence (or 5′-end or 3′-end adjacent nucleic acid sequence B) contained in the specific partial nucleotide B of ii) above The number of nucleotide residues is not particularly limited as long as it is a number that can ensure the specificity of the specific partial nucleotide B in the above ii), for example, at least 3, preferably at least 4, more preferably at least 5, Even more preferably it may be at least 6, most preferably at least 7, 8, 9, or 10. Such terminal partial nucleic acid sequences and such flanking nucleic acid sequences will be apparent from the disclosure herein.

  The specific partial nucleotide B in the above iii) is a nucleic acid sequence in which both 5 ′ and 3 ′ nucleic acid sequences are linked to the inserted nucleic acid sequence of the polynucleotide of the present invention (in the known polynucleotide, the exon is missing). These nucleic acid sequences are linked to each other due to loss, and are partial nucleotides not present in the polynucleotide of the present invention. The number of nucleotide residues in each nucleic acid sequence present 5 ′ and 3 ′ with respect to the inserted nucleic acid sequence of the polynucleotide of the present invention contained in the specific partial nucleotide B of iii) is the same as that of iii) above. Although it is not particularly limited as long as it is a number that can ensure the specificity of a specific partial nucleotide B, for example, at least 3, preferably at least 4, more preferably at least 5, even more preferably at least 6, most preferably There may be at least 7, 8, 9, or 10.

  The specific partial nucleotide B of the present invention is used, for example, as a target for specifically detecting a known polynucleotide, and as a marker specific for blood cells or as a marker specific for osteoclast differentiation, or non-specific to these. As useful markers. The specific partial nucleotide B of the present invention also specifically develops a substance that can specifically recognize a known polynucleotide, or a substance that cannot specifically recognize a known polynucleotide, or expresses a known polypeptide. It may be useful in the development of substances that can be modulated or substances that cannot specifically regulate the expression of known polypeptides.

  The common partial nucleotide of the present invention may be a non-specific partial nucleotide present in both the polynucleotide of the present invention and the known polynucleotide. Such partial nucleotides are apparent from the disclosure herein. The consensus nucleotides of the present invention, for example, as a target for comprehensive detection of both the polynucleotides of the present invention and known polynucleotides, and as a marker specific for blood cell lineage or specific for osteoclast differentiation, Alternatively, it may be useful as a non-specific marker for these. The consensus nucleotides of the present invention can also comprehensively regulate the substance that can comprehensively recognize both the polynucleotides of the present invention and known polynucleotides, or the expression of both the polypeptides of the present invention and known polypeptides. Can be useful in material development.

  The polynucleotide of the present invention and the partial nucleotide thereof can encode the polypeptide of the present invention or the partial peptide of the present invention. The polynucleotide of the present invention or the partial nucleotide of the present invention may also be fused with a polynucleotide consisting of a heterologous nucleic acid sequence. Such heterologous nucleic acid sequences include those encoding the above heterologous amino acid sequences.

  The polynucleotide of the present invention and its partial nucleotide may be provided in the form of a salt. Examples of the salt include those described above.

  The polynucleotide of the present invention and its partial nucleotide can be prepared by a method known per se. For example, the nucleic acid sequence represented by SEQ ID NO: Y or the same nucleic acid sequence as the nucleic acid sequence Y1 or the nucleic acid sequence Y2 can be cloned using a predetermined tissue or cell. In addition, the nucleic acid sequence represented by SEQ ID NO: Y, or a nucleic acid sequence substantially identical to the nucleic acid sequence Y1 or the nucleic acid sequence Y2 can be prepared by introducing mutations into the cloned polynucleotide as described above. Examples of the mutagenesis method include synthetic oligonucleotide-directed mutagenesis method, gapped duplex method, method of randomly introducing point mutations (for example, treatment with nitrous acid or sulfite), cassette mutagenesis method, linker scanning method, A mismatch primer method is exemplified.

2. Related Substances The present invention provides a series of related substances that can be developed based on the polypeptide of the present invention and the partial peptide of the present invention, and the polynucleotide of the present invention and the partial nucleotide of the present invention. The related substances of the present invention described below can be useful, for example, as a medicine. When the related substance of the present invention is a medicine, the target disease can be a disease based on a disorder of blood cells or osteoclasts, and examples thereof include immune diseases, inflammatory diseases, and allergic diseases. Specifically, such diseases include immunodeficiency, leukemia, lymphoma, myelodysplastic syndrome, myeloma, neutrophil dysfunction, autoimmune disease, rheumatoid arthritis, collagen disease, atopic dermatitis, spleen Examples include hyperfunction, thymus species, bone metastasis of cancer, and osteoporosis. Diseases based on osteoclast damage include rheumatoid arthritis, bone metastasis of cancer, osteoporosis and the like.

2.1. Antisense molecules The present invention provides antisense molecules.

  The kind of antisense molecule may be DNA or RNA, or may be a DNA / RNA chimera. Even if the antisense molecule has a natural phosphodiester bond, the thiophosphate type (P = O in the phosphate bond is replaced by P = S) or 2′-O-methyl, which is stable to a degrading enzyme It may be a modified nucleotide such as a type. Other important factors for the design of antisense molecules include increasing water solubility and cell membrane permeability, but these can be overcome by devising dosage forms such as the use of liposomes and microspheres. The length of the antisense molecule is not particularly limited as long as it can specifically hybridize with the transcript. The length of the antisense molecule is about 15 nucleotides as short and includes a sequence that is long and complementary to the entire sequence of the transcript. It may be an array. From the viewpoint of ease of synthesis, antigenicity problems, etc., for example, oligonucleotides comprising about 15 nucleotides or more, preferably about 15 to about 100 nucleotides, more preferably about 18 to about 50 nucleotides are exemplified. The Furthermore, antisense molecules can not only hybridize with transcripts and inhibit translation, but also bind to double-stranded DNA to form triplex and inhibit transcription to mRNA. There may be.

  The antisense molecule of the present invention may comprise a nucleic acid sequence complementary to a nucleic acid sequence corresponding to a partial nucleotide of the present invention (eg, specific partial nucleotides A and B of the present invention, common partial nucleotide of the present invention). Accordingly, an antisense molecule of the present invention is an antisense molecule specific for a polynucleotide of the present invention, an antisense molecule specific for a known polynucleotide, or an antisense common to both a polynucleotide of the present invention and a known polynucleotide. It can be a sense molecule. The antisense molecules of the present invention may be useful for specific suppression of expression of a polypeptide of the present invention or a known polypeptide, or for comprehensive suppression of expression of both a polypeptide of the present invention and a known polypeptide.

2.2. RNAi Inducible Nucleic Acid The present invention provides an RNAi inducible nucleic acid.

  RNAi-inducible nucleic acid refers to a polynucleotide that can induce an RNA interference (RNAi) effect when introduced into a cell, and is preferably RNA. The RNAi effect refers to a phenomenon in which a double-stranded RNA containing the same nucleic acid sequence as mRNA or a partial sequence thereof suppresses the expression of the mRNA. In order to obtain the RNAi effect, for example, it is preferable to use RNA having a double-stranded structure having the same nucleic acid sequence (or a partial sequence thereof) as at least 20 or more consecutive target mRNAs. The double-stranded structure may be composed of different strands, or may be a double-stranded structure provided by a single RNA stem-loop structure. Examples of RNAi-inducible nucleic acids include siRNA and miRNA, and siRNA is preferred. The siRNA is not particularly limited as long as it can induce RNAi, but may be, for example, 21 to 27 bases long, preferably 21 to 25 bases long.

  The RNAi-inducible nucleic acid of the present invention comprises a sense strand consisting of a nucleic acid sequence corresponding to a partial nucleotide of the present invention (eg, specific partial nucleotides A and B of the present invention, a common partial nucleotide of the present invention), and complementary thereto. It can be a double-stranded polynucleotide composed of an antisense strand consisting of a simple nucleic acid sequence. The RNAi-inducible nucleic acid of the present invention may also have an overhang at the 5 'end and / or the 3' end of one or both of the sense strand and the antisense strand. The overhang may be formed by the addition of one to several (eg, 1, 2 or 3) bases at the 5 'end and / or the 3' end of the sense and / or antisense strand. The RNAi-inducible nucleic acid of the present invention is common to the RNAi-inducible nucleic acid specific to the polynucleotide of the present invention, the RNAi-inducible nucleic acid specific to the known polynucleotide, or both the polynucleotide of the present invention and the known polynucleotide. It can be an RNAi-inducible nucleic acid. The RNAi-inducible nucleic acid of the present invention may be useful for specific suppression of expression of the polypeptide of the present invention or a known polypeptide, or for comprehensive suppression of expression of both the polypeptide of the present invention and a known polypeptide. .

2.3. Aptamer The present invention provides an aptamer.

  An aptamer refers to a polynucleotide having binding activity (or inhibitory activity) for a predetermined target molecule. The aptamers of the present invention can be RNA, DNA, modified nucleotides or mixtures thereof. The aptamer of the present invention can also be in a linear or cyclic form. The length of the aptamer is not particularly limited, and can generally be about 16 to about 200 nucleotides, for example, about 100 nucleotides or less, preferably about 50 nucleotides or less, more preferably about It can be up to 40 nucleotides. The length of the aptamer of the present invention may be, for example, about 18, about 20, about 25, or about 30 nucleotides or more. The aptamer may be one in which the sugar residue (eg, ribose) of each nucleotide is modified in order to enhance binding properties, stability, drug delivery properties, and the like. Examples of the site modified in the sugar residue include those in which the oxygen atom at the 2′-position, 3′-position and / or 4′-position of the sugar residue is replaced with another atom. Types of modifications include, for example, fluorination, O-alkylation, O-allylation, S-alkylation, S-allylation and amination (eg, Sproat et al., (1991) Nucl. Acid Res. 19, 733-738; Cotton et al., (1991) Nucl. Acid. Res. 19, 2629-2635). Aptamers may also be modified purines and pyrimidines. Examples of such modifications include 5-position pyrimidine modification, 8-position purine modification, modification with exocyclic amine, substitution with 4-thiouridine, and substitution with 5-bromo or 5-iodo-uracil. Moreover, the phosphate group contained in the aptamer of the present invention may be modified so as to be resistant to nuclease and hydrolysis. For example, the phosphate group may be substituted with thioate, dithioate or amidate. Aptamers can be made according to previous reports (eg, Ellington et al., (1990) Nature, 346, 818-822; Tuerk et al., (1990) Science, 249, 505-510).

  The aptamer of the present invention can specifically bind to the polypeptide of the present invention, the known polypeptide, or both the polypeptide of the present invention and the known polypeptide via a region corresponding to the partial peptide of the present invention. Therefore, the aptamer of the present invention can be an aptamer specific to the polypeptide of the present invention, an aptamer specific to a known polypeptide, or an aptamer common to both the polypeptide of the present invention and the known polypeptide. Such specific aptamers include, for example, (a) a polynucleotide that binds to the polypeptide of the present invention or a specific partial peptide thereof and does not bind to a known polypeptide, (b) a known polypeptide or a specific thereof. A polynucleotide that binds to a partial peptide and does not bind to the polypeptide of the present invention, or (c) a polynucleotide that binds to both the polypeptide of the present invention and a known polypeptide or the common partial peptide of the present invention. It can produce by selecting by.

2.4. Antibodies The present invention provides antibodies.

  The antibody of the present invention may be either a polyclonal antibody (antiserum) or a monoclonal antibody, and can be prepared by a well-known immunological technique. The monoclonal antibody may be any isotype such as IgG, IgM, IgA, IgD or IgE, but is preferably IgG or IgM.

For example, polyclonal antibodies, the antigen (if necessary, bovine serum albumin, KLH (may be a K eyhole L impet H emocyanin) complexes crosslinked to a carrier protein, etc.), commercial adjuvants (e.g., (Complete or incomplete Freund's adjuvant) is administered 2 to 4 times subcutaneously or intraperitoneally in animals every 2 to 3 weeks (the antibody titer of partially collected serum is measured by a known antigen-antibody reaction) It can be obtained by collecting the whole blood about 3 to 10 days after the final immunization and purifying the antiserum. Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

  A monoclonal antibody can be prepared by a cell fusion method. For example, the above antigen is administered to mice subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and spleen or lymph nodes are collected 3 days after the final administration, and white blood cells are collected. This leukocyte and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the factor. Cell fusion may be PEG or voltage pulse. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method. The hybridoma producing the monoclonal antibody can be cultured in vitro or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.

  The antibodies of the present invention may also be chimeric antibodies, humanized antibodies or human antibodies.

  A chimeric antibody means a monoclonal antibody derived from immunoglobulins of animal species whose variable regions and constant regions are different from each other. For example, the chimeric antibody can be a mouse / human chimeric monoclonal antibody whose variable region is a variable region derived from mouse immunoglobulin and whose constant region is a constant region derived from human immunoglobulin. The constant region derived from human immunoglobulin has a unique amino acid sequence depending on isotypes such as IgG, IgM, IgA, IgD, and IgE. However, the constant region of the recombinant chimeric monoclonal antibody of the present invention belongs to any isotype. It may be a constant region of groggrin. Preferably, it is a constant region of human IgG.

A chimeric antibody can be prepared by a method known per se. For example, a mouse / human chimeric monoclonal antibody can be prepared according to a previous report (eg, experimental medicine (special issue), Vol. 6, No. 10, 1988 and Japanese Patent Publication No. 3-73280). Specifically, downstream of an active V _H gene (rearranged VDJ gene encoding a heavy chain variable region) obtained from DNA encoding the mouse monoclonal antibody isolated from a hybridoma producing a mouse monoclonal antibody, C _H gene (H-chain constant region C gene encoding), also active V _L genes obtained from DNA encoding the mouse monoclonal antibody isolated from the hybridoma obtained from the DNA encoding human immunoglobulin (L the C gene) encoding the C _L gene (L-chain constant region obtained from DNA encoding human immunoglobulin downstream of rearranged VJ gene) encoding the chain variable region, arranged so that each can express Are inserted into one or a separate expression vector, and host cells are transformed with the expression vector. It can be prepared by culturing the transformed cell.

  A humanized antibody is a monoclonal antibody produced by genetic engineering. For example, a part or all of the complementarity determining region of the hypervariable region is derived from a mouse monoclonal antibody, and the framework region of the variable region and It means a human monoclonal antibody whose constant region is derived from human immunoglobulin. The complementarity-determining regions of the hypervariable region are the three regions (Complementarity-determining regions; CDR1, CDR2, and CDR3) that are present in the hypervariable region of the antibody variable region and that directly bind complementarily to the antigen. Yes, the frame region of the variable region is four regions (Framework: FR1, FR2, FR3, FR4) that are relatively conserved before and after the three complementarity determining regions. In other words, for example, a monoclonal antibody in which all regions other than part or all of the complementarity determining region of the hypervariable region of a mouse monoclonal antibody are replaced with the corresponding region of human immunoglobulin.

  A humanized antibody can be produced by a method known per se. For example, a recombinant humanized antibody derived from a mouse monoclonal antibody can be prepared in accordance with previous reports (eg, Japanese translations of PCT publication No. 4-506458 and JP-A-62-296890). Specifically, at least one mouse H chain CDR gene and at least one mouse L chain CDR gene corresponding to the mouse H chain CDR gene are isolated from a hybridoma producing a mouse monoclonal antibody, and the human immunoglobulin gene A human H chain gene encoding the entire region other than the human H chain CDR corresponding to the mouse H chain CDR and a human L chain gene encoding the entire region other than the human L chain CDR corresponding to the mouse L chain CDR are isolated. . The isolated mouse H chain CDR gene and the human H chain gene are introduced into an appropriate expression vector so that they can be expressed, and similarly, the mouse L chain CDR gene and the human L chain gene are also expressed appropriately. Into another expression vector. Alternatively, the mouse H chain CDR gene / human H chain gene and the mouse L chain CDR gene / human L chain gene can be introduced into the same expression vector so that they can be expressed. A humanized antibody-producing cell can be obtained by transforming a host cell with the expression vector thus prepared, and the desired humanized antibody can be obtained from the culture supernatant by culturing the cell.

  A human antibody means an antibody in which all regions including the variable region and constant region of H and L chains constituting immunoglobulin are derived from a gene encoding human immunoglobulin.

  Human antibodies can be produced by methods known per se. For example, a human antibody can be prepared by immunizing a transgenic animal produced by incorporating at least a human immunoglobulin gene into a genetic locus of a mammal other than human, such as a mouse. It can be produced in the same manner as the production method of the monoclonal antibody. For example, transgenic mice producing human antibodies have been reported (Nature Genetics, Vol. 15, p. 146-156, 1997; Nature Genetics, Vol. 7, p. 13-21, 1994; International Publication No. WO94 / 25585; Nature, Vol.368, p.856-859, 1994; and Japanese National Publication No. Hei 6-500233).

The antibody of the present invention can also be a part of the above-described antibody (eg, monoclonal antibody) of the present invention. Examples of such antibodies include F (ab ′) ₂ , Fab ′, Fab, Fv fragments, and single chain antibodies.

  The antibody of the present invention can specifically bind to the polypeptide of the present invention, the known polypeptide, or both the polypeptide of the present invention and the known polypeptide via a region corresponding to the partial peptide of the present invention. Accordingly, the antibody of the present invention may be an antibody specific for the polypeptide of the present invention, an antibody specific for a known polypeptide, or an antibody common to both the polypeptide of the present invention and the known polypeptide. Such a specific antibody can be produced, for example, by using a specific partial peptide of the polypeptide of the present invention, a specific partial peptide of a known polypeptide, or a common partial peptide of the present invention as an antigen.

2.5. Expression Vector The present invention provides an expression vector for the aforementioned substance.

  The expression vector of the present invention may comprise a polynucleotide encoding a target polypeptide to be expressed or a target polynucleotide to be expressed, and a promoter operably linked to the polynucleotide. “Promoter is operably linked to a polynucleotide” means that the promoter is capable of expressing the polynucleotide itself under its control or the expression of a polypeptide encoded by the polynucleotide. Is bound to a polynucleotide encoding.

  The backbone of the expression vector of the present invention is not particularly limited as long as it can produce a target substance in a predetermined cell, and examples thereof include a plasmid vector and a virus vector. When the expression vector is used as a medicine, suitable vectors for administration to mammals include adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, poliovirus, Sindbis virus, Sendai virus, etc. A viral vector is mentioned.

  When prokaryotic cells are used as host cells, expression vectors that can use prokaryotic cells as host cells can be used. Such an expression vector may contain elements such as a promoter-operator region, a start codon, a polynucleotide encoding the polypeptide of the present invention or a partial peptide thereof, a stop codon, a terminator region, and an origin of replication. The promoter-operator region for expressing the polypeptide of the present invention in bacteria contains a promoter, an operator and a Shine-Dalgarno (SD) sequence. As these elements, those known per se can be used.

  When eukaryotic cells are used as host cells, expression vectors that can use eukaryotic cells as host cells can be used. In this case, the promoter used is not particularly limited as long as it can function in eukaryotes such as mammals. For the purpose of polypeptide expression, such promoters include, for example, SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus-derived early promoter and the like, and β -Mammal constituent protein gene promoters such as actin gene promoter, PGK gene promoter, transferrin gene promoter and the like. When the expression of the polynucleotide is intended, the promoter can be a polIII promoter (eg, tRNA promoter, U6 promoter, H1 promoter).

The expression vectors of the present invention further comprise sites for transcription initiation and termination, and ribosome binding sites, replication origins and selectable marker genes (eg, ampicillin, tetracycline, kanamycin, spectinomycin that may be required for translation in the transcription region). Erythromycin, chloramphenicol) and the like. Expression vectors of the present invention can be prepared by a method known per se ^{(e.g., Molecular Cloning, 2 nd edition,} Sambrook et al., See Cold Spring Harbor Lab. Press (1989 )).

3. Composition The present invention provides a composition comprising the aforementioned substances.

  The compositions of the present invention can include any carrier, such as a pharmaceutically acceptable carrier, in addition to the materials described above. Examples of pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone. , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Aerosil, Talc, Sodium lauryl sulfate lubricant, Citric acid, Menthol, Glycyllysine / Ammonium salt, Glycine, Orange powder, Fragrance, Sodium benzoate Preservatives such as lithium, sodium hydrogen sulfite, methyl paraben, propyl paraben, stabilizers such as citric acid, sodium citrate, acetic acid, suspension agents such as methyl cellulose, polyvinyl pyrrolidone, aluminum stearate, dispersants such as surfactants, Examples include, but are not limited to, water, physiological saline, diluents such as orange juice, base waxes such as cacao butter, polyethylene glycol, and white kerosene.

  Preparations suitable for oral administration include a solution in which an effective amount of a substance is dissolved in a diluent such as water, physiological saline, orange juice, a capsule containing an effective amount of the substance as a solid or granule, a sachet or Examples thereof include tablets, suspensions in which an effective amount of a substance is suspended in an appropriate dispersion medium, and emulsions in which a solution in which an effective amount of a substance is dissolved is dispersed in an appropriate dispersion medium and emulsified.

  Formulations suitable for parenteral administration (eg, intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions. May contain antioxidants, buffers, antibacterial agents, isotonic agents and the like. Also, aqueous and non-aqueous sterile suspensions can be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. In addition, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

  The dose of the composition of the present invention varies depending on the activity and type of the active ingredient, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age, etc. The amount of active ingredient per day for an adult is about 0.001 to about 500 mg / kg.

  The composition of the present invention enables the regulation (eg, promotion or suppression) of the expression or function of the polypeptide of the present invention. The composition of the present invention can be useful, for example, as a medicine (eg, a prophylactic or therapeutic drug for the diseases as described above), a reagent, or a food.

4). Cell The present invention comprises a transformant producing the polypeptide of the present invention or the partial peptide of the present invention, a cell producing the antibody of the present invention, and a cell in which the expression or function of the polynucleotide or polypeptide of the present invention is regulated. provide.

4.1. Transformant The transformant of the present invention may be a cell transformed with the expression vector of the present invention, which expresses the polypeptide of the present invention or the partial peptide of the present invention. The host cell used for the production of the transformant is not particularly limited as long as it is compatible with the expression vector and can express the target polynucleotide or polypeptide, and examples thereof include primary cultured cells or cell lines. It is done. Specifically, examples of such host cells include prokaryotic cells such as Escherichia coli, Bacillus (eg, Bacillus subtilis), actinomycetes, yeast, insect cells, avian cells, and mammalian cells (eg, the above-described examples). Eukaryotic cells such as cells derived from mammals: eg, CHO cells). The transformant of the present invention can be prepared by a method known per se ^{(e.g., Molecular Cloning, 2 nd edition,} Sambrook et al., See Cold Spring Harbor Lab. Press (1989 )).

  The transformant can be cultured in a nutrient medium such as a liquid medium by a method known per se. The medium preferably contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. Here, examples of the carbon source include glucose, dextrin, soluble starch, and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, large extract, and the like. Examples include inorganic or organic substances such as bean paste and potato extract, and examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. Moreover, you may add a yeast extract, vitamins, etc. to a culture medium. Culture conditions such as temperature, medium pH and culture time are appropriately selected so that the polypeptide of the present invention is produced in large quantities. The culture temperature is, for example, 30 to 37 ° C.

4.2. Antibody-Producing Cell The antibody-producing cell of the present invention can be any cell that produces the antibody of the present invention. Examples of the antibody-producing cell of the present invention include the above-described hybridoma and a transformed cell into which the above-described antibody expression vector has been introduced. When the antibody-producing cell of the present invention is a transformed cell, details of the expression vector and host cell used for the production of the transformed cell, cell culture, and the like can be the same as described above.

4.3. Polypeptide expression or function regulated cellular invention of the present invention, the expression or function of the polypeptides of the present invention is to provide a regulated cellular.

  The cells of the present invention can be isolated and / or purified. The cell of the present invention may be a cell derived from the above-mentioned tissue or the above-mentioned type of cell. The cells of the present invention can be derived from the mammals described above. The cell of the present invention may be a primary cultured cell or cell line, or a normal cell, or a cell derived from a mammal having the above-mentioned diseases. The cell of the present invention may be a cell in which the expression or function of the polypeptide of the present invention is specifically regulated. The cell of the present invention has an action related to blood cell line or osteoclast, or a phenotype related to blood cell line or osteoclast by regulating (eg, promoting or suppressing) the expression or function of the polypeptide of the present invention. Can change. The cell of the present invention can be a cell in which the expression of the polypeptide of the present invention is transiently regulated, or a cell in which the expression is constitutively regulated (eg, homozygous or heterozygous defective cells). . The cells of the invention can also be transformants or non-transformants.

  The cell of the present invention may contain, for example, the above-mentioned substances that can regulate the expression or function of the polynucleotide of the present invention or the polypeptide of the present invention (eg, the polypeptide of the present invention, antisense molecule, RNAi-inducible nucleic acid, antibody, Alternatively, it can be prepared by treating cells with these expression vectors. The cell of the present invention can also be produced by isolating and / or purifying a cell from a transgenic animal or a gene-deficient (so-called knockout) animal described below.

  Cells in which the expression or function of the polypeptide of the present invention is regulated include, for example, development of drugs (eg, preventive or therapeutic agents for the diseases described above), reagents or foods, blood cell specific or osteoclast differentiation It may be useful for the identification of additional marker genes specific for, and for the analysis of mechanisms associated with blood cell lineage or osteoclast differentiation. For example, the expression profile in the cell of the present invention is measured using, for example, a microarray, a protein chip (eg, an antibody chip or a non-antibody chip such as a chip manufactured by Cyphergen), and compared with the expression profile of a control cell. Can be performed by expression profile analysis. The cells of the present invention may also be useful as model cells for the diseases described above.

5). Animals The present invention provides animals in which the expression or function of the polypeptide of the present invention is regulated.

  The animals of the present invention can be animals with or without genomic alterations. The animal species of the present invention can be, for example, similar to the non-human mammal described above.

  In one embodiment, the animal of the present invention may be a transgenic animal with genomic modification. The transgenic animal of the present invention can express the polypeptide of the present invention. The transgenic animal of the present invention can also express the polypeptide of the present invention in the aforementioned cell or tissue-specific manner.

  The transgenic animal of the present invention can be prepared by a method known per se. More specifically, the transgenic animal of the present invention can be applied to a predetermined promoter (eg, an unfertilized egg, sperm or a progenitor cell thereof), for example, at an early stage of development. The polynucleotide of the present invention (eg, may be in the form of the expression vector of the present invention) operably linked to the above-described cell or tissue can be prepared. Examples of gene introduction methods include electroporation, lipofection, aggregation, calcium phosphate coprecipitation, and microinjection. The transgenic animal of the present invention may also be an animal produced by crossing an animal produced in this way with another animal of the same species (eg, a model animal of the disease as described above).

  In another embodiment, the animal of the present invention may be a gene-deficient animal with genomic modification. The gene-deficient animal of the present invention cannot express the polypeptide of the present invention. The gene-deficient animal of the present invention is also unable to express the polypeptide of the present invention in the aforementioned cell or tissue-specific manner.

  The gene-deficient animal of the present invention can be prepared by a method known per se. More specifically, the gene-deficient animal of the present invention can be prepared using embryonic stem cells (ES cells) that specifically lack a blood cell line / osteoclast-specific gene. Such an ES cell can be prepared, for example, by introducing a predetermined targeting vector into an ES cell and selecting an ES cell that has undergone homologous recombination from the ES cell into which the targeting vector has been introduced.

  As the targeting vector, a targeting vector capable of inducing homologous recombination that causes specific expression failure of the polynucleotide or polypeptide of the present invention can be used. Such a targeting vector includes a first polynucleotide and a second polynucleotide that are homologous or specifically homologous to a blood cell / osteoclast-specific gene (at least one of the polynucleotides is a blood cell / osteoclast). A splicing donor signal for a cell-specific gene, and including a mutation in the signal that disables splicing to produce at least one isoform), and optionally a selectable marker. A person skilled in the art can easily determine a splicing donor signal of a blood cell / osteoclast specific gene and a mutation that disables splicing to generate at least one isoform in the signal. The first and second polynucleotides are polynucleotides having sufficient sequence identity and length to cause homologous recombination with genomic DNA related to blood cell / osteoclast specific genes. is there. The first and second polynucleotides are selected so as to result in a specific deletion of a particular isoform. Selection markers include positive selection markers (eg, neomycin resistance gene, hygromycin B phosphotransferase (BPH) gene, blasticidin S deaminase gene, puromycin resistance gene), negative selection markers (eg, herpes simplex virus (HSV)). Thymidine kinase (tk) gene, diphtheria toxin A fragment (DTA) gene) and the like. The targeting vector can include either a positive selection marker, a negative selection marker, or both. The targeting vector may also include two or more recombinase target sequences (eg, loxP sequences used in Cre / loxP systems from bacteriophage P1, FRT sequences used in yeast-derived FLP / FRT systems). The present invention also provides such a targeting vector.

  As a method for introducing the targeting vector into ES cells, a method known per se can be used. Examples of such a method include a calcium phosphate method, a lipofection method / liposome method, and an electroporation method. When the targeting vector is introduced into a cell, homologous recombination of genomic DNA related to a blood cell / osteoclast specific gene occurs in the cell. ES cells may be established by culturing inner cell mass separated from blastocysts of any animal on feeder cells, but existing ES cells may be used.

  In order to select ES cells in which homologous recombination has occurred, cells after introduction of the targeting vector are screened. For example, after selection by positive selection, negative selection, etc., screening based on genotype (for example, PCR method, Southern blot hybridization method) is performed. It is also preferable to further perform karyotype analysis of the obtained ES cells. Karyotype analysis confirms that there are no chromosomal abnormalities in the sorted ES cells. Karyotype analysis can be performed by a method known per se. The karyotype of the ES cell is preferably confirmed in advance before introducing the targeting vector.

  The gene-deficient animal of the present invention can be prepared by transplanting a chimeric embryo obtained by introducing the ES cells obtained as described above into an embryo, and then mating the obtained chimeric animal. Examples of embryos include blastocysts and 8-cell stage embryos. Embryos can be obtained by mating female animals that have undergone superovulation treatment with hormone agents (for example, using PMSG having FSH-like action and hCG having LH action) and the like. Examples of methods for introducing ES cells into the embryo include micromanipulation methods and aggregation methods.

  The animal into which the chimeric embryo is transferred is preferably a pseudopregnant animal. A pseudopregnant animal can be obtained by mating a female animal having a normal cycle with a male animal castrated by vagina ligation or the like. The animal into which the chimeric embryo has been introduced becomes pregnant and gives birth to the chimeric animal. Next, it is confirmed whether the born animal is a chimeric animal. Whether or not the animal born is a chimeric animal can be confirmed by a method known per se, for example, by body color or coat color. For discrimination, DNA may be extracted from a part of the body, and Southern blot analysis or PCR assay may be performed. The mating can preferably take place between wild-type animals and chimeric animals or between chimeric animals. It is known per se whether or not a deficiency of a blood cell / osteoclast-specific gene was introduced into a germ line cell of a chimeric animal to obtain a heterozygous offspring that lacks the blood cell / osteoclast-specific gene. Depending on the method, various traits can be confirmed as indicators, and for example, it can be discriminated by the body color or coat color of the offspring animals. For discrimination, DNA may be extracted from a part of the body, and Southern blot analysis or PCR assay may be performed. Furthermore, a homozygote can be produced by crossing the heterozygotes thus obtained. The gene-deficient animal of the present invention is also produced by mating an animal produced in this way with another animal of the same species (eg, a model animal of a disease based on a disorder of blood cell line or osteoclast, a genetically modified animal). May be an animal.

  In yet another embodiment, the animal of the present invention may be an animal without genomic modification. Such an animal is a substance as described above that can regulate the expression or function of the polynucleotide of the present invention or the polypeptide of the present invention (eg, the polypeptide of the present invention, antisense molecule, RNAi-inducible nucleic acid, antibody, or these). Of the expression vector). Such animals can also be animals that can or cannot express the above-described tissue-specific polypeptides of the invention by topical treatment. Animal treatment may be performed using methods such as those mentioned in the compositions of the present invention.

  The animal of the present invention can be used, for example, for the development of drugs (eg, preventive or therapeutic agents for diseases as described above), reagents or foods, identification of additional marker genes specific for the blood cell system or osteoclast differentiation, And may be useful in the analysis of mechanisms involved in the differentiation of blood cells or osteoclasts. These include, for example, expression profiles (particularly blood cells or osteoclasts or hematopoietic / lymphoid cells) in animals of the present invention using microarrays, protein chips (eg, antibody chips or non-antibody chips such as Cyphergen's chips), etc. Tissue expression profile) can be measured and compared with the expression profile of a control animal. The animal of the present invention may also be useful as a model animal for the diseases as described above.

6). Means for Measurement and Measuring Method The present invention provides means for measuring target polynucleotides and polypeptides (eg, primer sets, nucleic acid probes, antibodies, aptamers) and measuring methods.

6.1. Primer set and method of use thereof The primer set of the present invention is used for specific detection and quantification of the polynucleotide of the present invention or known polynucleotide, or for comprehensive detection and quantification of both the polynucleotide of the present invention and known polynucleotide. Can be used. For example, such detection and quantification can be performed by preparing total RNA from a biological sample, followed by PCR (eg, RT-PCR, real-time PCR, quantitative PCR), LAMP (Loop-mediated isothermal amplification) (eg, WO00 / 28082). And a gene amplification method such as ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) (eg, see WO00 / 56877). Since the number of primers required differs depending on the type of gene amplification method, the number of primers is not particularly limited. For example, the primer set of the present invention includes two or more primers composed of sense and antisense primers. obtain. Two or more primers may be mixed in advance or may not be mixed. Each of the sense and antisense primers is not particularly limited as long as it has a size capable of specifically amplifying the target region, but 12 (eg, at least about 15, preferably at least about 18, more preferably at least about 20). Etc.) of consecutive nucleotide residues. Sense and antisense primers can be designed to be visually detectable if the size of the polynucleotide amplified by them is visually detected. The visually detectable size is not particularly limited, but for example, at least about 50, preferably at least 70, more preferably at least about 100, even more preferably at least about 150, and most preferably at least about 200. , About 300, about 400, about 500 or more nucleotide residues in length. The sense and antisense primers do not need to be visually detected by the polynucleotide amplified by them, and may be detected by a fluorescent signal or the like as commonly used in real-time PCR.

  The primer set of the present invention is a) a primer set specific to the polynucleotide of the present invention that can distinguish the polynucleotide of the present invention from known polynucleotides (abbreviated as “specific primer set A” if necessary). ), B) a primer set specific to the known polynucleotide that can distinguish the known polynucleotide from the polynucleotide of the present invention (abbreviated as “specific primer set B” if necessary), c) of the present invention It may be a primer set common to both the polynucleotide of the present invention and the known polynucleotide that does not distinguish the polynucleotide and the known polynucleotide from each other (abbreviated as “common primer set” if necessary).

  Specific primer set A of the present invention is a sense designed so that i) the size of the amplified polynucleotide of the present invention or a partial nucleotide thereof can be distinguished from the size of the known polynucleotide or the partial nucleotide to be amplified. And ii) only sense and antisense primers designed such that only the polynucleotide of the invention or a partial nucleotide thereof is amplified and no known polynucleotide is amplified.

  Examples of the sense and antisense primers of i) above correspond to, for example, a) a nucleic acid sequence that is present 5 ′ above the specific partial nucleotide A nucleic acid sequence (particularly, the inserted nucleic acid sequence of the polynucleotide of the present invention). A sense primer, and an antisense primer corresponding to a nucleic acid sequence complementary to a nucleic acid sequence present 3 'from the nucleic acid sequence, or b) a nucleic acid sequence of the above-mentioned specific partial nucleotide B (especially of a known polynucleotide) A sense primer corresponding to a nucleic acid sequence present 5 ′ more than the inserted nucleic acid sequence) and an antisense primer corresponding to a nucleic acid sequence complementary to the nucleic acid sequence present 3 ′ to the nucleic acid sequence are preferred.

  Examples of the sense and antisense primers of the above ii) include: a) a sense primer corresponding to the above-mentioned specific partial nucleotide A nucleic acid sequence (in particular, the inserted nucleic acid sequence of the polynucleotide of the present invention), and a predetermined antisense A primer, b) a predetermined sense primer, and a sense primer corresponding to the nucleic acid sequence of the specific partial nucleotide A (particularly, the inserted nucleic acid sequence of the polynucleotide of the present invention), or c) the specific partial nucleotide A of the above Sense and antisense primers corresponding to these nucleic acid sequences (especially the inserted nucleic acid sequence of the polynucleotide of the present invention) are preferred.

  The specific primer set B of the present invention is a sense designed so that i) the size of the amplified known polynucleotide or a partial nucleotide thereof can be distinguished from the size of the amplified polynucleotide of the present invention or a partial nucleotide thereof. And ii) sense and antisense primers designed such that only known polynucleotides or partial nucleotides thereof are amplified and the polynucleotides of the invention are not amplified.

  Examples of the i) sense and antisense primers include: a) a sense corresponding to a nucleic acid sequence present 5 'from the specific partial nucleotide B nucleic acid sequence (particularly, an inserted nucleic acid sequence of a known polynucleotide). A primer and an antisense primer corresponding to a nucleic acid sequence complementary to a nucleic acid sequence present 3 'from the nucleic acid sequence, or b) a nucleic acid sequence of the above-mentioned specific partial nucleotide A (especially of the polynucleotide of the present invention) A sense primer corresponding to a nucleic acid sequence present 5 ′ more than the inserted nucleic acid sequence) and an antisense primer corresponding to a nucleic acid sequence complementary to the nucleic acid sequence present 3 ′ to the nucleic acid sequence are preferred.

  Examples of the sense and antisense primers of ii) include: a) a sense primer corresponding to the nucleic acid sequence of the specific partial nucleotide B (particularly, an inserted nucleic acid sequence of a known polynucleotide), and a predetermined antisense primer, b) a predetermined sense primer and a sense primer corresponding to the nucleic acid sequence of the specific partial nucleotide B (particularly, an inserted nucleic acid sequence of a known polynucleotide), or c) the nucleic acid sequence of the specific partial nucleotide B ( In particular, sense and antisense primers corresponding to known polynucleotide insert nucleic acid sequences) are preferred.

  The common primer set of the present invention is a sense and antisense primer designed so that the size of the amplified known polynucleotide or a partial nucleotide thereof is the same as the size of the amplified polynucleotide of the present invention or a partial nucleotide thereof. Can be included. As such sense and antisense primers, for example, the polynucleotide of the present invention to be amplified or its partial nucleotide, and the known polynucleotide to be amplified or its partial nucleotide are those of the above-mentioned specific partial nucleotides A and B. Sense and antisense primers designed to contain no nucleic acid sequences are preferred.

6.2. Nucleic acid probes and methods of use thereof The nucleic acid probes of the present invention can be used for specific detection and quantification of the polynucleotides of the present invention or known polynucleotides, or for comprehensive detection and quantification of both the polynucleotides of the present invention and known polynucleotides. Can be used. For example, such detection and quantification can be performed using Northern blotting, a nucleic acid array on which the nucleic acid probe of the present invention is immobilized, etc. after the preparation of total RNA from a biological sample. The nucleic acid probe can be DNA, RNA, modified nucleic acid, or a chimeric molecule thereof, but DNA is preferable in consideration of stability, convenience and the like. The nucleic acid probe may also be either a single stranded or double stranded polynucleotide. The size of the nucleic acid probe is not particularly limited as long as it can specifically hybridize to the transcription product of the target gene. For example, at least about 15 or 16, preferably about 15 to about 1000, more preferably about 20 To about 500, and even more preferably from about 25 to about 300. When the nucleic acid probe of the present invention is a single-stranded polynucleotide, the nucleic acid probe of the present invention can be the same as the antisense molecule of the present invention. When the nucleic acid probe of the present invention is a double-stranded polynucleotide, the nucleic acid probe of the present invention can be composed of the antisense molecule of the present invention and a polynucleotide molecule complementary thereto.

  The nucleic acid probe of the present invention is a) a nucleic acid probe specific to the polynucleotide of the present invention that can distinguish the polynucleotide of the present invention from known polynucleotides (abbreviated as “specific nucleic acid probe A” if necessary). B) Nucleic acid probe specific to a known polynucleotide that can distinguish a known polynucleotide from the polynucleotide of the present invention (abbreviated as “specific nucleic acid probe B” if necessary), c) of the present invention It may be a nucleic acid probe (abbreviated as “common nucleic acid probe” if necessary) common to both the polynucleotide of the present invention and the known polynucleotide, which does not distinguish the polynucleotide and the known polynucleotide from each other.

  The specific nucleic acid probe A of the present invention is a polynucleotide (single-stranded polynucleotide) having a nucleic acid sequence complementary to the nucleic acid sequence of the specific partial nucleotide A (particularly, the inserted nucleic acid sequence of the polynucleotide of the present invention). ), Or a polynucleotide having a nucleic acid sequence complementary to the nucleic acid sequence of the specific partial nucleotide A (particularly, the inserted nucleic acid sequence of the polynucleotide of the present invention) and the nucleic acid sequence (double-stranded polynucleotide). .

  The specific nucleic acid probe B of the present invention is a polynucleotide (single-stranded polynucleotide) having a nucleic acid sequence complementary to the nucleic acid sequence of the specific partial nucleotide B (particularly, an inserted nucleic acid sequence of a known polynucleotide), Alternatively, it may be a nucleic acid sequence (particularly an inserted nucleic acid sequence of a known polynucleotide) of the specific partial nucleotide B and a polynucleotide having a nucleic acid sequence complementary to the nucleic acid sequence (double-stranded polynucleotide).

  The common nucleic acid probe of the present invention is a polynucleotide having a nucleic acid sequence complementary to the nucleic acid sequence of the common partial nucleotide (single-stranded polynucleotide), or the nucleic acid sequence of the common partial nucleotide and complementary to the nucleic acid sequence. A polynucleotide having a specific nucleic acid sequence (double-stranded polynucleotide).

  The nucleic acid probe of the present invention may be provided in a form immobilized on a support (ie, as an array). The support for such a nucleic acid array is not particularly limited as long as it is a support commonly used in the art, and examples thereof include membranes (for example, nylon membrane), glass, plastic, metal, plates, and the like. . As a form of the nucleic acid array in the present invention, a form known per se can be used. For example, an array in which nucleic acids are directly synthesized on a support (so-called affiliometric method), an array in which nucleic acids are immobilized on a support (So-called Stanford method), fiber type array, and electrochemical array (ECA).

6.3. Antibodies and aptamers and methods of use thereof The antibodies and aptamers of the invention can be used for specific detection and quantification of the polypeptides of the invention, known polypeptides, or both polypeptides of the invention and known polypeptides. For example, such detection and quantification can be performed after preparation of an extract from a biological sample, or using a biological sample by an immunological technique or a method utilizing affinity. Examples of such immunological techniques include enzyme immunoassay (EIA) (eg, direct competition ELISA, indirect competition ELISA, sandwich ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Examples include immunochromatography, luminescence immunoassay, spin immunoassay, western blotting, and immunohistochemical staining. The method utilizing affinity can be performed according to the above-described immunological technique. The antibody and aptamer used for the measurement of the polypeptide of the present invention, the known polypeptide, or both the polypeptide of the present invention and the known polypeptide may be the same as the antibody and aptamer of the present invention described above.

  The antibodies and aptamers of the present invention are: a) an antibody and an aptamer specific to the polypeptide of the present invention, which can distinguish the polypeptide of the present invention from a known polypeptide (if necessary, “specific antibody and aptamer A A) and b) antibodies and aptamers specific to known polypeptides (abbreviated as “specific antibodies and aptamers B” if necessary), which can distinguish known polypeptides from the polypeptides of the present invention, c) It may be an antibody and an aptamer that do not distinguish the polypeptide of the present invention and the known polypeptide from each other and are common to both the polypeptide of the present invention and the known polypeptide (abbreviated as “common antibody and aptamer” as necessary). . The specific antibody and aptamer A of the present invention can bind to the above-mentioned specific partial peptide A (particularly a partial peptide consisting of the inserted amino acid sequence of the polypeptide of the present invention). The specific antibody and aptamer B of the present invention can bind to the above-mentioned specific partial peptide B (particularly, a partial peptide consisting of an inserted amino acid sequence of a known polypeptide). The common antibody and aptamer of the present invention can bind to the above-mentioned common partial peptide.

  The antibodies and aptamers of the invention may be provided in a form immobilized on a support (ie, as an array). The support for such a nucleic acid array is not particularly limited as long as it is a support that is usually used in the art. Multiwell plate).

6.4. Supplementary matter on the measuring means of the present invention The measuring means of the present invention can be provided in a form labeled with a labeling substance, if necessary. Examples of the labeling substance include fluorescent substances such as FITC and FAM, luminescent substances such as luminol, luciferin, and lucigenin, radioisotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S, and ¹²³ I, biotin, streptavidin, and the like. And the like, and the like.

  The measurement means of the present invention may be provided in the form of a kit including additional components in addition to the measurement means. In this case, each component included in the kit may be provided in an isolated form, for example, stored in a different container. For example, if the measuring means is not labeled with a labeling substance, such a kit may further comprise a labeling substance. The kit of the present invention comprises two or more measurements for two or more target genes (for example, a combination of a blood cell / osteoclast specific gene and a known gene, or a combination of two or more blood cell / osteoclast specific genes). Means may be included. When the measurement means of the present invention is provided in the form of an array, the array of the present invention may be one in which two or more measurement means for two or more target genes are fixed. The kits and arrays of the present invention can also include a means for measurement as described above for housekeeping genes (eg, GAPDH, β-actin).

6.5. Measurement Method of the Present Invention The present invention also provides a method for detecting or quantifying a target polypeptide or polynucleotide using the measurement means of the present invention.

  The measurement of the target polynucleotide and polypeptide can be appropriately performed according to the type of measuring means by the above-described method.

  In the method of the present invention, the expression level of a target polynucleotide or polypeptide in a biological sample obtained from the above-described mammal (eg, human) or in a culture (eg, cell or tissue culture) can be measured. The biological sample includes, for example, a sample containing a target polynucleotide or a polynucleotide-expressing cell or tissue, or, if the target polynucleotide or polypeptide is secreted or leaked, such a secreted or leaked polynucleotide. It is not particularly limited as long as it is an animal-derived sample (eg, blood, plasma, serum, saliva, cerebrospinal fluid, tears, urine) containing nucleotides or polypeptides. The biological sample may also contain the cells or tissues described above (eg, blood cells or osteoclasts or hematopoietic / lymphoid tissues or synovial tissues). The biological sample used in the present invention can be a biological sample collected in advance from a mammal unless otherwise specified, but in a specific aspect, the method of the present invention includes collecting a biological sample from a mammal. obtain.

  In one embodiment, the method of the invention comprises identification of a blood cell line or osteoclast, determination of the differentiation state of the blood cell line or osteoclast, diagnosis for a disease based on a disorder of the blood cell line or osteoclast (eg, onset or Can be used to determine the likelihood of onset). Such a method measures the expression level of a target polynucleotide or polypeptide in a biological sample collected from an animal, and evaluates the onset or possibility of onset of the target disease based on the measured expression level or relative expression ratio. Can include. For example, the measured expression level or the relative expression ratio is compared with the expression level in a mammal (eg, normal animal) not suffering from the target disease. The expression level or expression ratio in a mammal not affected by the target disease can be determined by a method known per se. Based on such comparison, it is determined whether or not the animal is likely to be affected by the target disease, or whether or not the animal is likely to be affected in the future. It is known that changes in the expression of genes associated with a particular disease are often observed in mammals that have developed a particular disease. It is also known that changes in the expression of specific genes are often observed before the onset of specific diseases. Therefore, it is possible to determine the onset or possibility of onset of the target disease by such analysis. Such a method may be useful, for example, for simple determination of the target disease and early detection. Of course, the measuring means of the present invention and the reagent or kit of the present invention can also be used for such determination.

  Specifically, changes in the expression profile of blood cell / osteoclast-specific genes 1 to 12 in blood cells or osteoclasts or hematopoietic / lymphoid tissues are as described in Examples. Accordingly, the polynucleotide of the present invention and the partial nucleotide of the present invention (eg, the specific partial nucleotide A of the present invention, the specific partial nucleotide B of the present invention, the common partial nucleotide of the present invention), and the polypeptide of the present invention And the measurement of the present invention that enables specific measurement of the partial peptide of the present invention (eg, specific partial peptide A of the present invention, specific partial peptide B of the present invention, common partial peptide of the present invention) By evaluating the degree of expression of blood cell line / osteoclast-specific genes 1 to 12 and / or their relative expression ratios using means, identification of blood cell line or osteoclast, blood cell line or osteoclast It is possible to determine the differentiation state of bone cells, or to diagnose a disease based on a disorder of blood cells or osteoclasts.

  In another embodiment, the method of the present invention can be used for screening pharmaceuticals, reagents or foods. For example, in one methodology, the screening method can include evaluating whether the test substance can modulate (eg, increase, decrease) the blood cell line or osteoclast number. The number of blood cells or osteoclasts can be correlated with the expression level of blood cell / osteoclast-specific genes. be able to. In another methodology, the screening method can include assessing whether the test substance can modulate the expression or function of the target polynucleotide or polypeptide. Such a screening method is effective for a predetermined disease (eg, a disease based on a disorder of blood cell line or osteoclast) including, for example, selecting a test substance that can modulate the expression or function of a target. Drugs with reduced predetermined effects (eg, side effects such as blood cell or osteoclast differentiation-regulating effects) including screening methods for drugs and the like, and selecting test substances that cannot regulate target expression or function It can be used as a screening method. The subject to be subjected to the screening method can be a known or novel compound or composition, for example, a nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, compound prepared using combinatorial chemistry technology Examples include libraries, random peptide libraries prepared by solid-phase synthesis and phage display methods, natural components derived from microorganisms, animals and plants, marine organisms, existing pharmaceuticals, reagents, foods, and the like. In the screening method, mammals, cells and tissues (for example, blood cells or osteoclasts, hematopoietic / lymphoid tissues or synovial tissues) as described above, or reconstitution systems (non-cell systems) can be used. A medicament obtained by the screening method is also provided by the present invention.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1: Preparation and sequence analysis of human cDNA library (1) Preparation and sequence analysis of cDNA library by improved oligocap method 1) Extraction and purchase of mRNA From human tissues (shown below), literature (J. Sambrook, EF Fritsch & T. Maniatis, Molecular Cloning Second Edition, Cold Spring harbor Laboratory Press, 1989), mRNA was extracted as total RNA. In addition, after culturing human cultured cells and primary human cultured cells (shown below) by the method described in the catalog, literature (J. Sambrook, EF Fritsch & T. Maniatis, Molecular Cloning Second edition, Cold Spring harbor Laboratory Press, 1989) MRNA was extracted as total RNA by the method described.
The relationship between the library name and its origin is shown below in the order of “library name: origin”. For subtracted ones, we also showed how to create a subtract library.

<MRNA extraction from human tissue>
NTONG: normal tongue (Tongue); CTONG: tongue cancer (Tongue, Tumor); FCBBF: fetal brain (Brain, Fetal); OCBBF: fetal brain (Brain, Fetal); PLACE: placenta (Placenta); SYNOV: synovial tissue (Synovial membrane tissue from rheumatioid arthritis); CORDB: Cord blood.

<MRNA extraction from cultured cells>
BNGH4: H4 cells (ATCC # HTB-148); IMR32: IMR32 cells (ATCC # CCL-127); SKNMC: SK-N-MC cells (ATCC # HTB-10); 3NB69: NB69 cells (RCB # RCB0480); BGGI1: GI1 cells (RCB # RCB0763); NB9N4: NB9 cells (RCB # RCB0477); SKNSH: SK-N-SH cells (RCB # RCB0426); AHMSC: HMSC cells (mesenchymal cells, human mesenchymal cells); CHONS: Chondrocytes; ERLTF: TF-1 cells (erythroleukemia cells, erythroleukemia); HERAC: HeLa cells; JCMLC: leukemia cells (Leukemia, myelogenous); MESTC: Mesenchyme stem cells; N1ESE: between Mesenchymal stem cells; NCRRM: Embryonal carcinoma cells; NCRRP: Embryonal carcinoma cells induced by retinoic acid (RA) treatment; T1ESE: Mesenchymal stem cells Induced with trichostatin and 5-azacytidine treatment; NT2RM: NT2 cells (STARATAGENE # 204101); NT2RP: NT2 cells were treated with retinoic acid (RA) for 5 weeks; NT2RI: NT2 cells were treated with RA after 5 weeks of induction of RA treatment 2 Week: NT2NE: NT2 cells are differentiated by RA treatment and growth inhibitor treatment, then nerves are concentrated and recovered (NT2 Neuron); NTISM: NT2 cells (STARATAGENE # 204101) are treated with growth inhibitor 5 weeks after induction of RA treatment A library (NT2RI-NT2RM) obtained by subtracting cDNA overlapping with mRNA of undifferentiated NT2 cells from a cDNA library prepared from 2 weeks of mRNA using Subtract Kit (Invitrogen # K4320-01). RCB was purchased from RIKEN Genebank and Cell Development Bank, and ATCC was purchased from the American Type Culture Collection.

<MRNA extraction from primary cultured cells>
ASTRO: Normal Human Astrocyte NHA5732, Takara Shuzo # CC2565; DFNES: Normal Human Dermal Fibroblasts (Neonatal Skin); NHDF-Neo NHDF2564, Takara Shuzo # CC2509; MESAN: Normal Normal human mesangial cells NHMC56046-2, Takara Shuzo # CC2559; NHNPC: Normal human neural progenitor cells NHNP5958, Takara Shuzo # CC2599; PEBLM: Normal peripheral blood mononuclear cells HPBMC5939, Takara Shuzo # CC2702; HSYRA: Synovial cells HS-RA (Human synoviocytes from rheumatioid arthritis), Toyobo # T404K-05; PUAEN: Normal pulmonary artery endothelial cells, Toyobo # T302K-05; UMVEN: Normal umbilical vein endothelial cells HUVEC, Toyobo # T200K-05; HCASM: Normal coronary artery smooth muscle cells HCASMC, Toyobo # T305K-05; HCHON: Normal chondrocytes HC ( Human Chondrocytes), Toyobo # T402K-05; HHDPC: Normal hair papilla cells HDPC (Human dermal papilla cells), Toyobo # THPCK-001; CD34C: CD34 + cells (AllCells, LLC # CB14435M); D3OST: CD34 + cells osteoclast differentiation factor ( ODF) treatment induction for 3 days; D6OST: CD34 + cells induced for ODF treatment for 6 days; D9OST: CD34 + cells induced for ODF treatment for 9 days; ACTVT: Activated T-cells; LYMPB: lymphoblasts (Lymphoblast, EB virus transferred B cell); NETRP: Neutrophil.

  Subsequently, mRNA extracted as total RNA from human tissues shown below was purchased. The relationship between the library name and its origin is shown below in the order of “library name: origin”. For subtracted ones, we also showed how to create a subtract library.

<Purchase of mRNA from human tissue with total RNA>
ADRGL: Adrenal gland, CLONTECH # 64016-1; BRACE: Cerebellum (Brain, cerebellum), CLONTECH # 64035-1; BRAWH: Whole brain (Brain, whole), CLONTECH # 64020-1; FEBRA: Fetal brain ( Brain, Fetal), CLONTECH # 64019-1; FELIV: Liver, Fetal, CLONTECH # 64018-1; HEART: Heart, CLONTECH # 64025-1; HLUNG: Lung, CLONTECH # 64023 -1; KIDNE: Kidney, CLONTECH # 64030-1; LIVER: Liver, CLONTECH # 64022-1; MAMGL: Mammary Gland, CLONTECH # 64037-1; PANCR: Pancreas, CLONTECH # 64031-1; PROST: Prostate, CLONTECH # 64038-1; SALGL: Salivary Gland, CLONTECH # 64026-1; SKMUS: Skeletal Muscle, CLONTECH # 64033-1; SMINT: Small Intestine, CLONTECH # 64039-1; SPLEN: Spleen, CLONTECH # 64034-1; STOMA: Stomach, CLONTECH # 64090-1; TBAES: Breast cancer (Breast, Tumor), CLONTECH # 64015 -1; TCERX: cervical cancer (Cervix, Tumor), CLONTECH # 64010-1; TCOLN: colon cancer (Colon, Tumor), CLONTECH # 64014-1; TESTI Testis, CLONTECH # 64027-1; THYMU: Thymus, CLONTECH # 64028-1; TRUNG: Lung cancer (Lung, Tumor), CLONTECH # 64013-1; TOVAR: Ovarian cancer (Ovary, Tumor), CLONTECH # 64011-1; TRACH: Trachea, CLONTECH # 64091-1; TUTER: Uterus cancer (Uterus, Tumor), CLONTECH # 64008-1; UTERU: Uterus, CLONTECH # 64029-1; ADIPS: Fat Tissue (Adipose), Invitrogen # D6005-01; BLADE: Bladder, Invitrogen # D6020-01; BRALZ: Brain cortex (Brain, cortex, Alzheimer), Invitrogen # D6830-01; CERVX: Cervix ), Invitrogen # D6047-01; COLON: Colon, Invitrogen # D6050-0; NESOP: Esophagus, Invitrogen # D6060-01; PERIC: Pericardium, Invitrogen # D6105-01; RECTM: Rectum (Rectum), Invitrogen # D6110-01; TESOP: Esophageal cancer (Esophageal, Tumor), Invitrogen # D6860-01; TKIDN: Kidney cancer (Kidney, Tumor), Invitrogen # D6870-01; TLIVE: Liver cancer (Liver, Tumor), Invitrogen # D6880-01; TSTOM: Stomach, Tumor, Invitrogen # D6920-01 ： BEAST: Adult Breast, STARATAGENE # 735044; FEHRT: Fetal heart (Heart, Fetal), STARATAGENE # 738012; FEKID: Fetal kidney (Kidney, Fetal), STARATAGENE # 738014; FELNG: Fetal lung (Lung, Fetal) ), STARATAGENE # 738020; NOVAR: Adult Ovary, STARATAGENE # 735260; BRASW: Alzheimer patient cerebral cortex [BRALZ: Alzheimer patient cerebral cortex (Brain, cortex, Alzheimer), Invitrogen # D6830-01] mRNA A library obtained by subtracting cDNA overlapping with mRNA of whole brain tissue [BRAWH: Brain, whole, CLONTECH # 64020-1] from the prepared cDNA library using Subtract Kit (Invitrogen # K4320-01) (BRALZ-BRAWH).

  Furthermore, mRNA extracted and purified as poly A (+) RNA was purchased from the following human tissues. A cDNA library was prepared from RNA obtained by mixing poly A (+) RNA derived from each tissue with poly A (−) RNA. Poly A (−) RNA was prepared by removing poly A (+) RNA from whole RNA of brain, whole, CLONTECH # 64020-1 with oligo dT cellulose. The relationship between the library name and its origin is shown below in the order of “library name: origin”.

<Purchase of mRNA from human tissue with poly A (+) RNA>
BRAMY: Tonsils (Brain, amygdala), CLONTECH # 6574-1; BRCAN: Brain, caudate nucleus, CLONTECH # 6575-1; BRCOC: Brain, corpus callosum, CLONTECH # 6577-1; BRHIP: Hippocampus, CLONTECH # 6578-1; BRSSN: Black matter (Brain, substantianigra), CLONTECH # 6580-1; BRSTN: Brain, subthalamic nucleus, CLONTECH # 6581-1; BRTHA : Thalamus (Brain, thalamus), CLONTECH # 6582-1.

2) Preparation of cDNA library by improved oligo-capping method Oligocap method [M. Maruyama and S. Sugano, Gene, 138: 171-174 (1994)] was improved from each RNA (WO 01/04286). A cDNA library was prepared. Using Oligo-cap linker (SEQ ID NO: 1) and Oligo dT primer (SEQ ID NO: 2), BAP (Bacterial Alkaline Phosphatase) treatment, TAP (Tobacco Acid Pyrophosphatase) treatment, RNA as described in WO 01/04286 Ligation, first strand cDNA synthesis and RNA removal were performed. Next, it was converted into double-stranded cDNA by PCR (polymerase chain reaction) using PCR primers of 5 ′ (SEQ ID NO: 3) and 3 ′ (SEQ ID NO: 4), and cleaved with SfiI. Next, the cDNA fragment was usually cloned into a vector pME18SFL3 (GenBank AB009864, Expression vector) obtained by cleaving the cDNA fragment fractionated to 2 kb or more (in some cases 3 kb or more) with DraIII to prepare a cDNA library. .
Tables 1-1 to 1-6 show the names of the cDNA libraries used for the 5′-end sequence analysis of cDNA and their origins. Tables 1-1 to 1-6 also show the numbers of 5′-terminal sequences of cDNAs in each cDNA library after mapping to the human genome.

3) Analysis of 5'-terminal sequence of cDNA from cDNA library prepared by improved oligo-cap method DNA sequencing reagent (BigDye Terminator Cycle Sequencing FS Ready) Using a Reaction Kit (manufactured by PE Biosystems), a sequencing reaction was performed according to the manual, followed by analysis with a DNA sequencer (ABI PRISM 3700, PE Biosystems). The obtained data was made into a database. The total length ratio of the 5′-end of each cDNA library prepared by the improved oligocap method was 90% on average and high in total length (calculated using the protein coding region of known mRNA as an index).

4) Full-length cDNA nucleic acid sequence analysis The full-length cDNA nucleic acid sequence was determined for each of the cDNAs selected for full-length cDNA nucleic acid sequence analysis. The nucleic acid sequence was determined mainly by the primer walking method by the dideoxy terminator method using custom synthetic DNA primers. That is, using a custom synthetic DNA primer, a sequencing reaction was performed according to the manual using a DNA sequencing reagent manufactured by PE Biosystems, and then a DNA nucleic acid sequence was analyzed using a sequencer manufactured by the same company. The full-length nucleic acid sequence was finally determined by completely overlapping the partial nucleic acid sequence determined by the above method. Next, from the determined full-length cDNA nucleic acid sequence, the translation region into the protein was estimated and the amino acid sequence was determined.

(2) Preparation and sequence analysis of cDNA library by oligocap method 1) Preparation of cDNA library by oligocap method NT-2 neurons that can be differentiated into neurons by treatment with retinoic acid in teratocarcinoma cells derived from human fetal testis Progenitor cells (purchased from Stratagene) were treated as follows according to the attached manual.
・ Cultivate NT-2 cells without retinoic acid induction (NT2RM)
・ After culturing NT-2 cells, add retinoic acid and induce, then culture for 2 days and 2 weeks (NT2RP)
In addition, human cultured cells SK-N-MC (ATCC HTB-10) (SKNMC), human cultured cells Y79 (ATCC HTB-18) (Y79AA), human cultured cells GI1 (RCB RCB0763) (BGGI1), human cultured cells H4 (ATCC HTB-148) (BNGH4), human cultured cells IMR32 (ATCC CCL-127) (IMR32), and human cultured cells NB9 (RCB # RCB0477) (NB9N4) were cultured by the method described in the catalog. RCB was purchased from RIKEN Genebank and Cell Development Bank, and ATCC was purchased from the American Type Culture Collection.
The above cultured cells were collected, and mRNA was extracted by the method described in the literature (J. Sambrook, EF Fritsch & T. Maniatis, Molecular Cloning Second Edition, Cold Spring Harbor Laboratory Press 1989). Furthermore, poly (A) + RNA was purified with oligo dT cellulose.
Similarly, human placenta tissue (PLACE), human ovarian cancer tissue (OVARC), tissue containing more head than human 10-week-old fetus (HEMBA), tissue containing more body part than human 10-week-old fetus (HEMBB), Human mammary tissue (MAMMA), human thyroid tissue (THYRO), human vascular endothelial tissue primary cultured cells (VESEN), literature (J. Sambrook, EF Fritsch & T. Maniatis, Molecular Cloning Second edition, Cold Spring harbor Laboratory Press , 1989), and mRNA was extracted. Furthermore, poly (A) + RNA was purified with oligo dT cellulose.
A cDNA library was prepared from each of the above poly (A) + RNAs by the oligocap method [M. Maruyama and S. Sugano, Gene, 138: 171-174 (1994)]. Using the Oligo-cap linker (SEQ ID NO: 1) and Oligo dT primer (SEQ ID NO: 2), literature [Suzuki and Sugano, Protein Nucleic Acid Enzyme, 41: 197-201 (1996), Y. Suzuki et al., Gene , 200: 149-156 (1997)], BAP (Bacterial Alkaline Phosphatase) treatment, TAP (Tobacco Acid Phosphatase) treatment, RNA ligation, first-strand cDNA synthesis and RNA removal were performed. Subsequently, it was converted into double-stranded cDNA by PCR (polymerase chain reaction) using PCR primers of 5 ′ (SEQ ID NO: 3) and 3 ′ (SEQ ID NO: 4), and SfiI was cleaved. Next, the cDNA was cloned into the vector pUC19FL3 (part of NT2RM and NT2RP) or pME18SFL3 (GenBank AB009864, Expression vector) cleaved with DraIII to prepare a cDNA library.
Tables 1-1 to 1-6 show the names of the cDNA libraries used for the 5′-end sequence analysis of cDNA and their origins. Tables 1-1 to 1-6 also show the numbers of 5′-terminal sequences of cDNAs in each cDNA library after mapping to the human genome.

２）オリゴキャップ法で作製したcDNAライブラリーからのcDNAの5'-末端配列解析
各cDNAライブラリーより取得したcDNAの5’-端又は3'-端の核酸配列をDNAシーケンシング試薬（Dye Terminator Cycle Sequencing FS Ready Reaction Kit, dRhodamine Terminator Cycle Sequencing FS Ready Reaction Kit又はBigDye Terminator Cycle Sequencing FS Ready Reaction Kit, PE Biosystems社製）を用い、マニュアルに従ってシーケンシング反応後、DNAシーケンサー（ABI PRISM 377, PE Biosystems社製）でDNA核酸配列を解析した。得られたデータをデータベース化した。オリゴキャップ法で作製した各cDNAライブラリーの5'-末端の全長率は、平均６０％であった（既知mRNAのタンパク質コード領域を指標にして算出した）。

2) Analysis of 5'-terminal sequence of cDNA from cDNA library prepared by oligocap method DNA sequencing reagent (Dye Terminator) Cycle Sequencing FS Ready Reaction Kit, dRhodamine Terminator Cycle Sequencing FS Ready Reaction Kit or BigDye Terminator Cycle Sequencing FS Ready Reaction Kit, manufactured by PE Biosystems) DNA nucleic acid sequence was analyzed. The obtained data was made into a database. The total length ratio of the 5′-end of each cDNA library prepared by the oligocap method was 60% on average (calculated using the protein coding region of a known mRNA as an index).

3) Full-length cDNA nucleic acid sequence analysis The full-length cDNA nucleic acid sequence was determined for each of the cDNAs selected for full-length cDNA nucleic acid sequence analysis. The nucleic acid sequence was determined mainly by the primer walking method by the dideoxy terminator method using custom synthetic DNA primers. That is, using a custom synthetic DNA primer, a sequencing reaction was performed according to the manual using a DNA sequencing reagent manufactured by PE Biosystems, and then a DNA nucleic acid sequence was analyzed using a sequencer manufactured by the same company. For some clones, Licor DNA sequencer was also used. For some cDNAs, DNA nucleic acid sequences were similarly determined with a DNA sequencer using a shotgun method that randomly cuts a plasmid containing cDNA without using a custom primer. The full-length nucleic acid sequence was finally determined by completely overlapping the partial nucleic acid sequence determined by the above method. Next, from the determined full-length nucleic acid sequence, the translation region into the protein was estimated and the amino acid sequence was determined.

Example 2: Genome mapping and clustering (1) Sequence data set The following sequences were used as a data set.
-Human genome sequence: UCSC hg 17 (NCBI Build 35) (http://www.genome.ucsc.edu/)
・ Human full-length cDNA sequence that we newly acquired and performed full-length cDNA sequence analysis 19,265 sequence ・ Human full-length cDNA sequence that we acquired and performed full-length cDNA sequence analysis and registered in existing public databases (DDBJ / GenBank / EMBL) ( Accession numbers: AB038269, AB045981, AB056476, AB056477, AK000001 to AK002212, AK021413 to AK027260, AK027263 to AK027902, AK054561 to AK058202, AK074029 to AK074481, AK074483 to AK075325, AK075326 to AK075 368 AK131107, AK131190 to AK131575, AK160364 to AK160386, AK172724 to AK172740, AK172741 to AK172866), which can be used for genome mapping 30,754 sequences. 2039 sequences (http://www.kazusa.or.jp/huge/)
・ It will be listed in the Full Length Clone List on the Mammalian Gene Collection (http://mgc.nci.nih.gov/) web page by January 30, 2005, and GenBenk gbpri (ftp: //ftp.ncbi. nih.gov/genbank/) Human full-length cDNA 20,878 sequence ・ Human full-length cDNA 9,280 sequence registered as Deutsches Krebsforschungszentrum (DKFZ) in GenBenk gbpri until January 30, 2005 ・ January 31, 2005 Human full-length cDNA 13,984 sequence that was registered as mRNA and included in GenBenk gbpri with the sequence of the Japanese version of human RefSeq sequence (http://www.ncbi.nlm.nih.gov/RefSeq/) The 31st version of the human RefSeq sequence (http://www.ncbi.nlm.nih.gov/RefSeq/) 28,931 sequence of the February 10, 2005 Ensembl (http://www.ensembl.org/) Of the human genome assembly sequence (NCBI35.nov_26.35), NCBI35.nov_26. Which had been mapped to the hg 17 human genome at UCSC (University of California, Santa Cruz, http://www.genome.ucsc.edu/). 35 33,666 sequences Sequence analysis in human cDNA 5 'terminal sequence 1,456,213 sequence and 3' terminal sequence 109,283 sequence (including published sequence accession numbers: AU116788 to AU160826, AU279383 to AU280837, DA000001 to DA999999, DB000001 to DB384947)

(2) Genome mapping Using the above data set using BLASTN (ftp://ftp.ncbi.nih.gov/blast/), the homology (Identity) is 95% or more and the consensus length is 50 base pairs (bp) or more. Genome mapping was performed. Approximately 99% of the sequences used in the mapping were genome mapped.

(3) Clustering After genome mapping, clusters were formed with a group of sequences contained in the genomic region as one cluster. That is, each cluster is selected so that there is no overlap between the sequences mapped to each genomic region on the outer sides of each genomic region of the sequence group. As a result, a total of 87,173 clusters existed. Excluding the 17,535 clusters that consisted of only the 3 'terminal sequence of human cDNA obtained and sequence-analyzed in our project, there were 69,638 clusters. Of these, the 36,782 cluster was excluded because it consisted only of the 5 'terminal sequence of human cDNA obtained and sequenced in our project (excluded are those that do not have full-length cDNA, RefSeq, or Ensembl sequences). As a result, 32,856 clusters contained at least one full-length cDNA, RefSeq, and Ensembl sequence. Among them, 21,703 clusters were obtained by selecting clusters containing one or more full-length cDNAs, RefSeq, and Ensembl sequences that are predicted to have a certain level of reliable ORF (open reading frame, translation region). The expression specificity of these 21,703 clusters was determined.

Example 3: Experimental method for real-time PCR (1) Template Synthesis of cDNA 1) When human mRNA (Human Total RNA) is used as a template
The reaction was performed in a system of 150 μl against 50 μg of human total RNA.
10 μl random primer (concentration 65 ng / μl) and 7.5 μl dNTP Mix (concentration 10 mM each dNTP mix) were added to 50 μg total RNA dissolved in 87 μl H ₂ O. Incubated at 65 ° C. for 5 minutes and incubated on ice for 1 minute. 30 μl of 5 × reaction buffer (attached to Invitrogen SuperScript III RT kit), 7.5 μl of 0.1 M DTT, 3 μl of RNase Inhibitor (STRATAGENE) and 5 μl of SuperScript III RT (Invitrogen) were added. Incubated at 25 ° C for 5 minutes, incubated at 50 ° C for 60 minutes, and incubated at 70 ° C for 15 minutes. After the reaction, phenol / chloroform extraction was performed to inactivate the enzyme. RNA was degraded by adding 3 μl of EDTA (0.5M) and 22.5 μl of 0.1N NaOH to perform alkali treatment. 30 μl of Tris (1M pH 7.8) was added to neutralize the reaction solution, followed by ethanol precipitation and dissolution in 100 μl of TE buffer.
Human mRNA (Human Total RNA) from an mRNA source was obtained by the method described in Example 1.
A list of human mRNAs used in the experiments is shown in Table 2.

(2) Primer and probe design
CDNA with other splice patterns transcribed from the same chromosomal region as the cDNA you want to compare using Primer Design software Primer Express software 3.0 included with Applied Biosystems real-time PCR 7500 Fast Primer and Probe were designed using the sequence of the part that becomes the boundary of the change region. Moreover, real-time PCR was performed using those designed primers, and it was confirmed that these bands were single and that only one kind of cDNA could be specifically detected.

(3) Expression analysis using real-time PCR 1) mRNA used
All the mRNAs used are of human origin.
Among 12 experimental systems, cluster chr7 + 1084, chr7-1842, chr7 + 710, chr2 + 1780, chr3 + 1925, chr11 + 275, chr1 + 3150, chr7-669, chr6-1987, chr12 + 613, chr4-770 In 11-cluster experiments, SYBR GREEN was used as the reaction system for real-time PCR, and bone marrow (Bone Marrow), thymus (Thymus), spleen (Spleen), CD34 + cells (CD34 +), and CD34 + cells were osteoclasts as template cDNAs. Differentiation factor (ODF) treatment induction 3 days (CD34 + ODF 3days), CD34 + cells induced osteoclast differentiation factor (ODF) treatment 6 days (CD34 + ODF 6days), CD34 + cells induced osteoclast differentiation factor (ODF) treatment 9 Days (CD34 + ODF 9days), synovial (Synovial), Jurkat cells (Jurkat), spinal cord, brain (Brain, whole), visceral tissue mixture (Mix, viscus tissues) [heart, kidney (Kidney ), Liver (Lung), lung (Colon), stomach (Stomach)], a total of 12 samples were used.
Of the 12 experimental systems, one cluster chr5 + 2320 experiment uses TaqMan (Applied Biosystems) as the reaction system for real-time PCR, and the bone marrow (Bone Marrow), thymus (Thymus), and spleen (Spleen) are used as template cDNAs. , CD34 + cells (CD34 +), CD34 + cells treated with osteoclast differentiation factor (ODF) 3 days (CD34 + ODF 3 days), CD34 + cells treated with osteoclast differentiation factor (ODF) 6 days (CD34 + ODF 6days), CD34 + Induction of osteoclast differentiation factor (ODF) treatment for 9 days (CD34 + ODF 9 days), synovial (Synovial), Jurkat cells (Jurkat), spinal cord (Binal, whole), visceral tissue mixture (Mix , viscus tissues) [Heart, Kidney, Liver, Lung, Colon, Stomach] A total of 12 samples were used.

2) Reaction system using SYBR GREEN
The SYBR GREEN I Dye assay chemistry is an experimental system that utilizes the feature of strong fluorescence when SYBR GREEN binds to double-stranded DNA. During the PCR reaction, when the DNA is denatured into single strands, SYBR GREEN is released from the DNA and the fluorescence decreases rapidly. The SYBR GREEN I Dye assay chemistry detects fluorescence intensity that increases with each PCR cycle.
As a template, add Forward Primer (final concentration 250 nM), Reverse Primer (final concentration 250 nM), and SYBR Green PCR Master Mix (ABI 4309155) to 0.2 μl of cDNA from each tissue (equivalent to 100 ng in terms of total RNA) as a template. The total volume was 20 μl. As an endogenous control, GAPDH (Accession No; NM_002046.2) was always used as a reaction control for all templates.
PCR was performed under the following conditions, which are standard protocols for real-time PCR 7500 Fast from Applied Biosystems. Initial step 50 ° C. for 2 minutes, 95 ° C. for 10 minutes, denaturation at 95 ° C. for 15 seconds, and annealing extension at 60 ° C. for 1 minute were repeated 40 cycles.
GAPDH-F (SEQ ID NO: 5): Forward Primer of endogenous control GAPDH
GAPDH-R (SEQ ID NO: 6): Reverse Primer of endogenous control GAPDH

3) Reaction system using TaqMan
TaqMan assay chemistry is an experimental system that uses a TaqMan probe labeled with a fluorescent dye (reporter) at the 5 'end and a Rhodamine fluorescent dye (quencher) at the 3' end. is there. When TaqMan probe is present alone, the fluorescence wavelength of the TaqMan probe is close to that of the quencher even if it is irradiated with the excitation light of the reporter. Is done. However, if the TaqMan probe is degraded by the 5'-3 'exonuclease activity of the DNA polymerase during the extension reaction from the primer during the PCR reaction, the reporter fluorescent dye is released from the 5' end of the TaqMan probe, and the quencher fluorescent dye And emits fluorescence when the distance increases. TaqMan assay chemistry detects the intensity of the fluorescence emitted by a reporter that increases with each PCR cycle.
As a template, 0.2μl of cDNA derived from each tissue (equivalent to 100 ng in terms of total RNA), Forward Primer (final concentration 900 nM), Reverse Primer (final concentration 900 nM), TaqMan Probe (final concentration 250 nM), TaqMan Fast Universal PCR Master Mix (ABI 466073) was added to make a total volume of 20 μl. As an endogenous control, GAPDH reaction control was always applied to all templates.
PCR was performed under the following conditions, which is the Fast protocol of Applied Biosystems Real-time PCR 7500 Fast. After 20 seconds of enzyme activation at 95 ° C., 40 cycles of denaturation at 95 ° C. for 3 seconds and annealing extension at 60 ° C. for 30 seconds were repeated.
GAPDH-Probe (SEQ ID NO: 7): TaqMan Probe of endogenous control GAPDH

(4) Statistical analysis method of data The results were analyzed using a relative quantification method.
Threshold is set in the exponential amplification region of the amplification curve using Applied Biosystems real-time PCR 7500 Fast RQ study software. The number of cycles at that time was Ct (Threshold cycle). In order to correct the initial RNA amount, a value obtained by subtracting Ct of GAPDH, which is an endogenous control, from the obtained Ct was calculated, and this was defined as dCt. [dCt = Target Ct−ENDOGENUS Ct (GAPDH)] A value obtained by subtracting the dCt of the reference sample (control) from the obtained dCt was calculated, and this was taken as ddCt. [ddCt = Target dCt-Control dCt] Based on this value, a relative value was calculated and used as RQ. [RQ = 2 ^-ddCt ] Based on the results, graphs were created in the log system, and the amount of amplification, that is, the amount of expression in each Primer and Probe was compared.
In each example, analysis results of RQ and Log ₁₀ RQ are shown. The numerical value of RQ is displayed with one decimal point. Those that could not be detected by real-time PCR were described as “Undet.” In the RQ value and Log ₁₀ RQ values. Log ₁₀ RQ values are displayed with two decimal places. However, the sample (Mix, viscus tissues) (RQ numerical value “1.0”) mixed with the normal visceral tissue of the control was described as “0.0” in the numerical value of Log ₁₀ RQ (only brain of Example 9 ( Brain, whole) are used as controls).

Example 4: Cluster chr7-669 (Data set: 047)
(1) Cluster analysis 1) Cluster characteristics Cluster full-length cDNA of chr7-669 (Human genome UCSC hg18 (NCBI Build34) chromosome 7, 115,150,000 bp to 115,400,000 bp) [D-SYNOV4003692.1 , AL110232.1, BC029891.1, BX538223.1, D43945.1, ENST00000265440, ENST00000320239, NM_012252.1] were analyzed. They are classified into four types according to the difference in expression pattern, but the following two types existed mainly when focusing on the transcription start point.
[1] D-SYNOV4003692.1
[2] D43945.1, ENST00000265440, NM_012252.1
[1] is a cDNA we newly acquired and performed full-length cDNA sequence analysis, and the ORF was different from [2] registered in the existing public DB.
Since [1] is expressed from a chromosomal region upstream of the known [2], the ORF region was different.
The ORF regions of the two types of cDNA patterns [1] to [2] are expressed from the same chromosomal region from different transcription start sites, resulting in changes in amino acid sequences, resulting in diverse proteins and mRNAs. I found out that it was.

2) Features of D-SYNOV4003692.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
047_ [1] _1-N0 (SEQ ID NO: 8): D-SYNOV4003692.1 whole region of nucleic acid sequence
047_ [1] _1-NA0 (SEQ ID NO: 9): Combination of D-SYNOV4003692.1 entire region and amino acid sequence
047_ [1] _1-A0 (SEQ ID NO: 10): D-SYNOV4003692.1 full amino acid region
1-373 bases (SEQ ID NO: 11) of D-SYNOV4003692.1 are variants inserted by exons that do not exist in NM_012252.1, which is a control registered in the existing public DB, and have no homology with NM_012252.1. Due to the presence of the translation start point on this inserted exon, the N-terminal amino acid was 90 residues (SEQ ID NO: 12) different from NM_012252.1.
In addition, 1,110 to 1,245 bases (SEQ ID NO: 15) of D-SYNOV4003692.1 is a variant inserted by an exon that does not exist in NM_012252.1, which is a control registered in the existing public DB, and has no homology with NM_012252.1 . Due to the presence of a translation termination point on this inserted exon, the C-terminal amino acid was 5 residues (SEQ ID NO: 16) different from NM_012252.1.
047_ [1] _1-N1 (SEQ ID NO: 11): D-SYNOV4003692.1 373-base inserted nucleic acid sequence region
047_ [1] _1-A1 (SEQ ID NO: 12): 90-residue insertion amino acid sequence region of D-SYNOV4003692.1
047_ [1] _1-N2 (SEQ ID NO: 13): ORF nucleic acid sequence region in the 373 base insertion region of D-SYNOV4003692.1
047_ [1] _1-A2 (SEQ ID NO: 14): ORF amino acid region related to 373 base insertion region of D-SYNOV4003692.1
047_ [1] _1-N3 (SEQ ID NO: 15): 136-base inserted nucleic acid sequence region of D-SYNOV4003692.1
047_ [1] _1-A3 (SEQ ID NO: 16): 5-residue insertion amino acid sequence region of D-SYNOV4003692.1
047_ [1] _1-N4 (SEQ ID NO: 17): ORF nucleic acid sequence region in the 136-base insertion region of D-SYNOV4003692.1
047_ [1] _1-A4 (same as SEQ ID NO: 16): ORF amino acid region related to 136-base insertion region of D-SYNOV4003692.1

3) Expression specificity analysis and real-time PCR primer design In order to clearly distinguish the characteristic regions as shown above and examine their expression levels, the following regions were used as detection regions. By comparing the expression levels of the detection regions, it was considered possible to compare the individual expression levels.
047_01-Specific region existing on the N-terminal side of the cDNA pattern of [1]: This is the transcription start region of the cDNA pattern of [1] that we newly analyzed and registered in the existing public DB Novel region that has not been processed → Fragment 047_01 (SEQ ID NO: 20) amplified by Primer047_01F (SEQ ID NO: 18) and Primer047_01R (SEQ ID NO: 19)
047_02-Control to compare the transcription start point region [2] registered in the existing public DB, [1] → Fragment 047_02 amplified by Primer047_02F (SEQ ID NO: 21) and Primer047_02R (SEQ ID NO: 22) (SEQ ID NO: 23)
047_05-Common region shared by [1] and [2]: [1] cDNA pattern we newly performed full-length cDNA sequence analysis, and [2] cDNA pattern registered in the existing public DB A region where all the patterns that serve as a control are in common to compare the expression level of the whole of the fragment → Fragment 047_05 (SEQ ID NO: 26) amplified by Primer047_05F (SEQ ID NO: 24) and Primer047_05R (SEQ ID NO: 25)
The exon regions specific to each of the cDNA patterns [1] and [2] shown above are mapped and compared to the human genome sequence of approximately 1.44 million 5 'end sequences obtained using the oligocap method. As a result, the following results were obtained.
In the newly analyzed cDNA pattern [1], there are two 5 'end sequences, and the origin is two synovial membrane tissue from rheumatioid arthritis (analysis parameter 27,489). .
In the pattern of [2], which is a control registered in the existing public DB, there are 8 5 'terminal sequences, and the origin is that CD34 + cells are induced by osteoclast differentiation factor (ODF) treatment for 9 days (D9OST). 2 sequences (analysis parameter 4,407), CD34 + cells treated with osteoclast differentiation factor (ODF) treatment for 3 days (D3OST) (analysis parameter 5,092), normal peripheral blood mononuclear cells 1 sequence (analysis parameter 7,900), bladder (Bladder) 1 sequence (analysis parameter 8,431), stomach (Stomach) 1 sequence (analysis parameter 8,685), and lung (Lung) 1 sequence (analysis parameter 16,146) ), Trachea (Trachea) was one sequence (analysis parameter 52,352).
From this result, it was found that the transcription start point of [1] is highly expressed in synovial membrane tissue from rheumatioid arthritis. In addition, at the transcription start point of [2], expression was observed in various tissues. As a result, it was considered that the mechanism of transcription differs between the synovial membrane tissue from rheumatioid arthritis and other tissues, and the transcription start point used may be different in this chromosomal region.

(2) Analysis of expression specificity by real-time PCR In order to know what kind of tissue in the blood cell system tissue and in what state the transcription start point used for expression changes, details of the expression level are real-time Analyzed by PCR. The results are shown in Table 3.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As experimental controls, a sample (Mix, viscus tissues) mixed with normal visceral tissue described in Example 3 and a whole brain (Brain, whole) were used for comparison.
Depending on the selectivity of the transcription start site compared between 047_01 (SEQ ID NO: 20) and 047_02 (SEQ ID NO: 23), the mRNA was changed, and as a result, the rate at which the ORF changed greatly changed in the following tissues.
Bone Marrow, CD34 + cells (CD34 +), CD34 + cells treated with osteoclast differentiation factor (ODF) 3 days induction (CD34 + ODF 3days), CD34 + cells induced ODF treatment 6 days (CD34 + ODF 6days), CD34 + cells In the ODF treatment induction 9 days (CD34 + ODF 9days) and the spinal cord, there are many transcripts (mRNA) from the known downstream transcription start point represented by 047_02 (SEQ ID NO: 23), but the synovial (Synovial ) In tissues (rheumatic patients), there were many transcripts (mRNA) from the newly acquired upstream transcription start point represented by 047_01 (SEQ ID NO: 20) (Table 3).
In other words, in synovial tissues (rheumatoid patients), unlike other tissues, transcription factors or transcription mechanisms have a variety of transcription start points, and mRNAs with different ORF regions were expressed.
From these results, the 5′-terminal region of the newly obtained cDNA region indicated by the detection region 047_01 (SEQ ID NO: 20) (region near the transcription start point) 047_ [1] _1-N1 (SEQ ID NO: 11) By comparing the expression, it was proved that it can be used as a therapeutic / diagnostic marker specific to the synovial tissue of rheumatic patients. It is also possible to develop new drugs using compounds, antibodies, siRNA, etc. that target specific regions.
The following areas may also be useful as therapeutic / diagnostic markers specific to the synovial tissue of rheumatic patients.
The upstream sequence 047_ [1] _1-N5 (SEQ ID NO: 27) containing the 361th to 380th bases primed by Primer047_01R (SEQ ID NO: 19) in D-SYNOV4003692.1 of the cDNA pattern of [1].
Region 047_01 (SEQ ID NO: 20) amplified by Primer047_01F (SEQ ID NO: 18) and Primer047_01R (SEQ ID NO: 19) in the cDNA pattern of [1]

Example 5: Cluster chr7-1842 (Data set: 002)
(1) Cluster analysis
1) Cluster characteristics Six full-length cDNA genome-mapped on cluster chr7-1842 (Human genome UCSC hg18 (NCBI Build34) chromosome 7, 36,325,000 bp to 36,550,000 bp) [D-D3OST2001534.1, D-NETRP1000046.1 , BC025698.1, ENST00000258749, M62840.1, NM_001637.1]. They could be classified into three types according to the difference in expression pattern as follows.
[1] D-D3OST2001534.1
[2] D-NETRP1000046.1
[3] BC025698.1, ENST00000258749, M62840.1, NM_001637.1
[1] and [2] are cDNAs we newly acquired and subjected to full-length cDNA sequence analysis, and the ORF is different from [3] registered in the existing public DB (DDBJ / Genbank / EMBL). It was.
[1] has the same transcription start point and translation start point as known [3], but the ORF region was different because the region corresponding to the second exon of [3] was deleted.
[2] was expressed from a chromosomal region downstream of [1] and [3] and had a translation initiation point on a non-homologous exon, so the ORF region was different.
The ORF regions of the three types of cDNA patterns [1] to [3] vary in amino acid sequence due to deletion of exons from the same chromosomal region or expression from different transcription start sites, resulting in diverse It turned out that protein and mRNA with the nature were produced.

2) Features of D-D3OST2001534.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
002_ [1] _1-N0 (SEQ ID NO: 28): D-D3OST2001534.1 whole region of nucleic acid sequence
002_ [1] _1-NA0 (SEQ ID NO: 29): Combination of D-D3OST2001534.1 full-length nucleic acid sequence and amino acid sequence
002_ [1] _1-A0 (SEQ ID NO: 30): All amino acid region of D-D3OST2001534.1 96 base exon existing in 402 to 497 bases of NM_001637.1 as a control registered in existing public DB (SEQ ID NO: 33) is not present in the region of 414 to 415 bases of D-D3OST2001534.1 and is deleted. Therefore, the amino acid length was 32 residues shorter than NM_001637.1.
002_ [1] _1-N1 (SEQ ID NO: 31): D-D3OST2001534.1 deleted nucleic acid sequence region
002_ [1] _1-A1 (SEQ ID NO: 32): D-D3OST2001534.1 changing amino sequence region
002_ [1] _1-N2 (same as SEQ ID NO: 31): ORF nucleic acid sequence region in the deleted region of D-D3OST2001534.1
002_ [1] _1-A2 (same as SEQ ID NO: 32): ORF amino acid region related to the deletion region of D-D3OST2001534.1
002_ [1] _C-N1 (SEQ ID NO: 33): 96-base inserted nucleic acid sequence present in 402-497 bases of NM_001637.1 inserted in the region of 414-415 bases of D-D3OST2001534.1
002_ [1] _C-A1 (SEQ ID NO: 34): Amino acid region related to the 96-base inserted nucleic acid sequence present in 402-497 bases of NM_001637.1 to be inserted into the 414-415 base region of D-D3OST2001534.1 .

3) Features of D-NETRP1000046.1 ([2]) that we newly acquired and performed full-length cDNA sequence analysis
002_ [2] _1-N0 (SEQ ID NO: 35): D-NETRP1000046.1 whole region of nucleic acid sequence
002_ [2] _1-NA0 (SEQ ID NO: 36): Combination of the entire nucleic acid sequence and amino acid sequence of D-NETRP1000046.1
002_ [2] _1-A0 (SEQ ID NO: 37): D-NETRP1000046.1 full amino acid region
1-67 bases (SEQ ID NO: 38) of D-NETRP1000046.1 are exons that do not exist in NM_001637.1 as a control registered in the existing public DB, and have no homology with NM_001637.1. Since there was a translation start point on this exon, the amino acid on the N-terminal side was changed by 4 residues (SEQ ID NO: 39).
002_ [2] _1-N1 (SEQ ID NO: 38): 67-base inserted nucleic acid sequence region of D-NETRP1000046.1
002_ [2] _1-A1 (SEQ ID NO: 39): 4-residue insertion amino acid sequence region of D-NETRP1000046.1
002_ [2] _1-N2 (SEQ ID NO: 40): ORF nucleic acid sequence region in the 67-base insertion region of D-NETRP1000046.1
002_ [2] _1-A2 (same as SEQ ID NO: 39): ORF amino acid sequence region in the 67 base insertion region of D-NETRP1000046.1 SignalP (http://www.cbs.dtu.dk/ services / SignalP /) and PSORT (http://psort.nibb.ac.jp/), the N-terminal secretion signal sequence predicted to exist at amino acids 1 to 22 of NM_001637.1 was lost.

4) Expression specificity analysis and design of primers for real-time PCR In order to clearly distinguish the characteristic regions as shown above and examine their expression levels, the following regions were used as detection regions. By comparing the expression levels of the detection regions, it was considered possible to compare the individual expression levels.
002_01-[1] cDNA pattern specifically extracted using sequence information at the boundary of the region where exon is deleted: cDNA of [1] we newly analyzed full-length cDNA In the pattern, deletion region of exon changing ORF → Fragment 002_01 (SEQ ID NO: 43) amplified by Primer002_01F (SEQ ID NO: 41) and Primer002_01R (SEQ ID NO: 42)
002_02-This is a specific region corresponding to the insertion region when compared with the deleted region of [1] in the cDNA pattern of [3] registered in the existing public DB, and to compare [1] Control → Fragment 002_02 (SEQ ID NO: 46) amplified by Primer002_02F (SEQ ID NO: 44) and Primer002_02R (SEQ ID NO: 45)
002_03-Specific region existing on the N-terminal side of the cDNA pattern of [2]: This is the transcription start region of the cDNA pattern of [2] that we newly analyzed and registered in the existing public DB Novel region that has not been processed → Fragment 002_03 (SEQ ID NO: 49) amplified by Primer002_03F (SEQ ID NO: 47) and Primer002_03R (SEQ ID NO: 48)
002_04-[1]-[3] Common region shared by all: [1], [2] cDNA patterns we have newly analyzed, and registered in the existing public DB [ 3] The region where all the control patterns are common to compare the expression level of the cDNA pattern as a whole → Fragment 002_04 (sequence) amplified by Primer002_04F (SEQ ID NO: 50) and Primer002_04R (SEQ ID NO: 51) (Number 52)
The exon region specific to each of the cDNA patterns [1] to [2] shown above is a comparative analysis by mapping about 1.44 million 5 'end sequences obtained using the oligo cap method to the human genome sequence. As a result, the following results were obtained.
In the newly acquired and analyzed cDNA [1] pattern, there are 7 5 'terminal sequences, the origin of which is 5 sequences of spleen (analysis parameter 33,950), and CD34 + cells are differentiated into osteoclasts. Factor (ODF) treatment induction 3 days (D3OST) was 2 sequences (analysis parameter 5,092).
In the newly analyzed cDNA [2] pattern, there was one 5 'end sequence, and the origin was one neutrophil sequence (analysis parameter 9170).
In the pattern of control [3] registered in the existing public DB, there are 49 5 'terminal sequences, and the origin is 20 sequences of spleen (analysis parameter 33,950) and thymus (Thymus). 5 sequences (analysis parameter 70,578), CD34 + cells treated with osteoclast differentiation factor (ODF) treatment 9 days (D9OST) 4 sequences (analysis parameter 4,407), synovial membrane tissue from rheumatioid arthritis 3 Sequence (analysis parameter 27,489), CD34 + cells treated with osteoclast differentiation factor (ODF) treatment 3 days (D3OST) 2 sequences (analysis parameter 5,092), breast cancer (Breast, Tumor) 2 sequences (analysis parameter 8,416 ), Lung (Lung) 2 sequences (analysis parameter 16,146), and uterus (Uterus) 2 sequences (analysis parameter 49,561).
From this result, it was found that the expression was high in blood cell tissues, and in the pattern [1], the expression was high in the spleen and CD34 + cells induced by ODF treatment. In the pattern [2], there was much expression in neutrophils. As a result, it was considered that there is a possibility that expression in a specific cell state such as expression in a specific tissue or in a specific cell state such as cell differentiation increases in some blood cell tissues by some expression mechanism.

(2) Analysis of expression specificity by real-time PCR In order to know what kind of state changes occur in the blood cell system, the details of the expression level were analyzed by real-time PCR. The results are shown in Tables 4 and 5.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As experimental controls, a sample (Mix, viscus tissues) mixed with normal visceral tissue described in Example 3 and a whole brain (Brain, whole) were used for comparison.
The ratio of ORF changes due to the deletion in the mRNA compared with 002_01 (SEQ ID NO: 43) and 002_02 (SEQ ID NO: 46) is almost the same for CD34 + cells in blood cell tissues other than those induced by osteoclast differentiation factor (ODF) treatment. It didn't change. In other words, it seems that two ORFs are expressed at the same rate in any blood cell tissue. However, the ratio changed greatly in the differentiation of CD34 + cells into osteoclasts. In the stage of CD34 + cells induced by ODF treatment for 3 days (CD34 + ODF 3days) and CD34 + cells in ODF treatment induced for 9 days (CD34 + ODF 9days), the rate of deletion in the mRNA shown by 002_01 (SEQ ID NO: 43) is high. (Tables 4 and 5).
Expression from the new transcription start point shown in 002_03 (SEQ ID NO: 29) is as follows: Synovial tissue (rheumatic patients), CD34 + cells induced to ODF treatment for 3 days (CD34 + ODF 3days), CD34 + cells induced to ODF treatment9 Increased in days (CD34 + ODF 9days) and bone marrow (Table 4 and Table 5).
In other words, in synovial tissues (rheumatoid patients) and bone marrow (Bone Marrow), the diversity of transcripts (mRNA) caused diversity in ORF. Furthermore, a mechanism that changes the transcript (mRNA) depending on the stage of osteoclast differentiation worked, resulting in diversity in the transcription start point and exon selectivity.
From these results, newly obtained cDNA regions 002_ [1] _1-N1 (SEQ ID NO: 31), 002_ [2] _1-N1 indicated by the detection regions 002_01 (SEQ ID NO: 43) and 002_03 (SEQ ID NO: 46) By comparing the expression of (SEQ ID NO: 38), as a therapeutic / diagnostic marker specific for the blood cell system of synovial tissue of rheumatic patients, as a therapeutic / diagnostic marker specific for osteoclast differentiation, It was also demonstrated that it can be used as a marker specific to the bone marrow blood cell system. In addition, new drug development using compounds, antibodies, siRNA, etc. that target specific regions is also possible.
The following areas are also considered as therapeutic / diagnostic markers specific to the blood cell system of synovial tissues of rheumatic patients, as therapeutic / diagnostic markers specific to osteoclast differentiation, and to bone marrow (Bone Marrow). It seems to be useful as a marker specific to the blood cell system.
The upstream sequence 002_ [1] _1-N3 (SEQ ID NO: 53) containing the 396th to 420th bases primed by Primer002_01R (SEQ ID NO: 42) in D-D3OST2001534.1 of the cDNA pattern of [1].
The upstream sequence 002_ [2] _1-N3 (SEQ ID NO: 54) containing the 53rd to 77th bases primed by Primer002_03R (SEQ ID NO: 48) in D-NETRP1000046.1 of the cDNA pattern of [2].
Region 002_01 (SEQ ID NO: 43) amplified by Primer002_01F (SEQ ID NO: 41) and Primer002_01R (SEQ ID NO: 42) in the cDNA pattern of [1]
Region 002_03 (SEQ ID NO: 49) amplified by Primer002_03F (SEQ ID NO: 47) and Primer002_03R (SEQ ID NO: 48) in the cDNA pattern of [2]

Example 6: Cluster chr7 + 1084 (data set: 012)
(1) Cluster analysis
1) Features of the cluster Nine full-length cDNA genome-mapped on the cluster chr7 + 1084 (Human genome UCSC hg18 (NCBI Build34) chromosome 7, 72,940,000 bp to 72,985,000 bp) [D-PLACE7010497.1, AF134379.1, C -FCBBF3003373, C-KAT04587, D26309.1, ENST00000336180, ENST00000355120, NM_002314.2, NM_016735.1] were analyzed. They are classified into 5 types according to the difference in expression pattern.
[1] D-PLACE7010497.1
[2] D26309.1, ENST00000336180, ENST00000355120, NM_002314.2
[3] AF134379.1, NM_016735.1
[1] is a cDNA we newly acquired and performed full-length cDNA sequence analysis, and the ORF was different from [2], [3] registered in the existing public DB.
[1] is expressed from a chromosomal region downstream of the known [2], [3] and has a translation start point on a new exon that has no homology to [2], [3], so the ORF region Was different.
[2] and [3] had the same transcription start point and translation start point, and different ORF regions due to splicing pattern changes.
The ORF region of the three types of cDNA patterns [1] to [3] is expressed from different transcription start points from the same chromosomal region, and the splicing pattern changes, thereby changing the amino acid sequence, It was found that diverse proteins and mRNAs were produced.

2) Features of D-PLACE7010497.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
012_ [1] _1-N0 (SEQ ID NO: 55): The entire nucleic acid sequence of D-PLACE7010497.1
012_ [1] _1-NA0 (SEQ ID NO: 56): Combination of D-PLACE7010497.1 full-length nucleic acid sequence and amino acid sequence
012_ [1] _1-A0 (SEQ ID NO: 57): Full amino acid region of D-PLACE7010497.1
1-83 bases (SEQ ID NO: 58) of D-PLACE7010497.1 are variants inserted by exons that do not exist in NM_002314.2, which is a control registered in the existing public DB, and have no homology with NM_002314.2 . Since there was a translation start point on the exon to be inserted, the N-terminal amino acid was 17 residues (SEQ ID NO: 59) different from NM_002314.2.
012_ [1] _1-N1 (SEQ ID NO: 58): 83-nucleotide inserted nucleic acid sequence region of D-PLACE7010497.1
012_ [1] _1-A1 (SEQ ID NO: 59): 17-residue insertion amino acid sequence region of D-PLACE7010497.1
012_ [1] _1-N2 (SEQ ID NO: 60): ORF nucleic acid sequence region in the 83-base insertion region of D-PLACE7010497.1
012_ [1] _1-A2 (same as SEQ ID NO: 59): ORF amino acid region related to the 83 base insertion region of D-PLACE7010497.1

3) Expression specificity analysis and design of primers for real-time PCR In order to clearly distinguish the characteristic regions as shown above and examine their expression levels, the following regions were used as detection regions. By comparing the expression levels of the detection regions, it was considered possible to compare the individual expression levels.
012_01-Specific region existing on the N-terminal side of the cDNA pattern of [1]: This is the transcription start region of the cDNA pattern of [1] that we newly analyzed, and is registered in the existing public DB Novel region that has not been changed → Fragment 012_01 (SEQ ID NO: 63) amplified by Primer012_01F (SEQ ID NO: 61) and Primer012_01R (SEQ ID NO: 62)
012_02-Control to compare transcription start point region [1] shared by [2] and [3] registered in the existing public DB → Primer012_02F (SEQ ID NO: 64) and Primer012_02R (SEQ ID NO: 65) Fragment 012_02 amplified by (SEQ ID NO: 66)
002_03-[1]-[3] Common area shared by all: [1] cDNA pattern we newly performed full-length cDNA sequence analysis, and also registered in the existing public DB [2], [ 3] A region where all patterns serving as a control are in common to compare the expression levels of the cDNA pattern as a whole → Fragment 012_03 (sequence) amplified by Primer012_03F (SEQ ID NO: 67) and Primer012_03R (SEQ ID NO: 68) (Number 69)
The exon regions specific to each of the cDNA patterns [1] and [2] shown above are mapped and compared to the human genome sequence of approximately 1.44 million 5 'end sequences obtained using the oligocap method. As a result, the following results were obtained.
In the newly analyzed cDNA [1] pattern, there are 12 5'-terminal sequences, 3 of which are synovial membrane tissue from rheumatioid arthritis (analysis parameter 27,489), thymus ( (Thymus) has 3 sequences (analysis parameter 70,578), spleen (Spleen) has 2 sequences (analysis parameter 33,950), CD34 + cells (CD34 +) has 1 sequence (analysis parameter 1,420), leukemia cells (Leukemia, myelogenous) has 1 sequence The sequence (analysis parameter 2,156), the substantia nigra (Brain, substantia nigra) was 1 sequence (analysis parameter 15,897), and the placenta (Placenta) was 1 sequence (analysis parameter 46,090).
In the pattern [2] or [3], which is a control registered in the existing public DB, there are 9 5 'terminal sequences, and the origin is 2 sequences of the brain (Brain, Fetal) (analysis parameter 47,574). ), Thalamus (Brain, thalamus) 2 sequences (analysis parameter 53,267), tonsils (Brain, amygdala) 2 sequences (analysis parameter 58,640), SK-N-SH cells (Neuroblastoma) 1 sequence (analysis parameter) 8,662), hippocampus (Brain, hippocampus) had one sequence (analysis parameter 57,918), and whole brain (Brain, whole) had one sequence (analysis parameter 59,069).
From this result, it was found that the transcription start point of [1] is highly expressed in blood cell tissues such as Synovial membrane tissue from rheumatioid arthritis and Thymus. Moreover, at the transcription start point shared by [2] and [3], there was much expression in the brain. As a result, it was found that in the chromosomal region, the transcriptional mechanism is different and the transcription start point used is different between the blood cell system tissue and other tissues (particularly the brain).

(2) Analysis of expression specificity by real-time PCR In order to know what kind of tissue in the blood cell system tissue and what state the transcription start point used for expression changes, details of the expression level are real-time PCR. Analyzed with The results are shown in Tables 6 and 7.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As an experimental control, a sample (Mix, viscus tissues) mixed with normal visceral tissue described in Example 3 and a whole brain (Brain, whole) were used for comparison.
The mRNA changes depending on the selectivity of the transcription start site compared between 012_01 (SEQ ID NO: 63) and 012_02 (SEQ ID NO: 66). As a result, the rate of change in ORF greatly changed in the following tissues and cells.
Spleen (Spleen), synovial tissue (rheumatoid patients), CD34 + cells induced by osteoclast differentiation factor (ODF) treatment for 3 days (CD34 + ODF 3days), CD34 + cells induced by ODF treatment for 9 days (CD34 + ODF 9days) There were many transcripts (mRNA) from the downstream transcription start point represented by 012_01 (SEQ ID NO: 63) (Tables 6 and 7). Furthermore, when comparing CD34 + cells induced by treatment with ODF, CD34 + cells, the initial stage of differentiation, showed the largest difference in 3 days of ODF treatment induction (CD34 + ODF 3days) (Tables 6 and 7).
In the whole brain (Brain, whole) and spinal cord (Spinal Cord), there were many transcripts (mRNA) from the transcription start point registered in the existing public DB represented by 012_02 (SEQ ID NO: 66).
In other words, in the spleen (Spleen), synovial tissue (rheumatoid patients), and CD34 + cells induced by treatment with ODF, the transcription start point varies due to transcription factors or transcription mechanisms different from those in the brain, resulting in different ORF regions. MRNA is expressed. Furthermore, changes were observed in the stage of differentiation into osteoclasts, and a mechanism for changing the transcript (mRNA) worked particularly at the early stage of differentiation, resulting in diversity at the transcription start point.
From these results, the 5′-terminal region of the newly obtained cDNA region indicated by the detection region 012_01 (SEQ ID NO: 63) (region near the transcription start point) 012_ [1] _1-N1 (SEQ ID NO: 58) By comparing the expression, as a therapeutic / diagnostic marker specific to the blood system of the synovial tissue of rheumatic patients, as a therapeutic / diagnostic marker to detect differentiation into osteoclasts, especially the early stages of differentiation, It was also proved that it can be used as a marker specific to the spleen blood system. In addition, new drug development using compounds, antibodies, siRNA, etc. that target specific regions is also possible.
In addition, the following areas are also used as therapeutic / diagnostic markers specific to the blood cell system of the synovial tissue of rheumatic patients, as a therapeutic / diagnostic marker for detecting differentiation into osteoclasts, particularly the early stages of differentiation, and It seems to be useful as a marker specific to the blood system of the spleen (Spleen).
The upstream sequence 012_ [1] _1-N3 (SEQ ID NO: 70) containing the 67th to 87th bases primed by Primer002_01R (SEQ ID NO: 62) in D-PLACE7010497.1 of the cDNA pattern of [1].
Region 012_01 (SEQ ID NO: 63) amplified by Primer012_01F (SEQ ID NO: 61) and Primer012_01R (SEQ ID NO: 62) in the cDNA pattern of [1]

Example 7: Cluster chr2 + 1780 (Data set: 009)
(1) Cluster analysis 1) Cluster characteristics Six full-length cDNA genome-mapped to cluster chr2 + 1780 (Human genome UCSC hg18 (NCBI Build34) chromosome 2, 120,200,000 bp to 120,500,000 bp) [D-SPLEN2030304.1 , BC010674.1, BX648614.1, ENST00000263708, M68941.1, NM_002830.2] was analyzed. They are classified into three types according to the difference in expression pattern, and the following two types exist as main ones.
[1] D-SPLEN2030304.1
[2] BC010674.1, ENST00000263708, M68941.1, NM_002830.2
[1] is a cDNA we newly acquired and performed full-length cDNA sequence analysis, and the ORF was different from [2] registered in the existing public DB.
Since [1] is expressed from a chromosomal region downstream from the known [2], the ORF region was different.
The ORF regions of the two types of cDNA patterns [1] to [2] are expressed from different transcription start sites in the same chromosomal region, so that the amino acid sequence changes, and diverse proteins and mRNAs are produced. It turns out that it is.

2) Features of D-SPLEN2030304.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
009_ [1] _1-N0 (SEQ ID NO: 71): D-SPLEN2030304.1 whole region of nucleic acid sequence
009_ [1] _1-NA0 (SEQ ID NO: 72): Combination of the entire nucleic acid sequence and amino acid sequence of D-SPLEN2030304.1
009_ [1] _1-A0 (SEQ ID NO: 73): D-SPLEN2030304.1 full amino acid region
The exon (first exon) of 1 to 57 bases (SEQ ID NO: 74) of D-SPLEN2030304.1 is an exon that does not exist in NM_002830.2, which is a control registered in the existing public DB, and has no homology. The second exon of D-SPLEN2030304.1 corresponds to the 14th exon of NM_002830.2, and the exons thereafter are common to both cDNAs. Since the translation start point of ORF of NM_002830.2 exists on the second exon, the translation start point of D-SPLEN2030304.1 exists in the region of the second exon (14th exon of NM_002830.2), Compared to NM_002830.2, the N-terminal amino acid was 367 residues shorter. (SEQ ID NO: 290).
009_ [1] _1-N1 (SEQ ID NO: 74): 57-base inserted nucleic acid sequence region of D-SPLEN2030304.1
009_ [1] _1-N2 (SEQ ID NO: 75): ORF 5'UTR region 88 bases starting from the 89th base of D-SPLEN2030304.1
009_ [1] _C-A1 (SEQ ID NO: 290): D-SPLEN2030304.1 367-residue deleted amino acid sequence region present in NM_002830.2 Along with this change, it exists in amino acids 31-222 of NM_002830.2 Pfam motif “FERM domain (Band 4.1 family)” disappeared (http://pfam.janelia.org/).

3) Expression specificity analysis and design of primers for real-time PCR In order to clearly distinguish the characteristic regions as shown above and examine their expression levels, the following regions were used as detection regions. By comparing the expression levels of the detection regions, it was considered possible to compare the individual expression levels.
009_01-Specific region existing on the N-terminal side of the cDNA pattern of [1]: This is the transcription start region of the cDNA pattern of [1] that we newly analyzed and registered in the existing public DB Novel region that has not been processed → Fragment 009_01 (SEQ ID NO: 78) amplified by Primer009_01F (SEQ ID NO: 76) and Primer009_01R (SEQ ID NO: 77)
009_02-Control to compare and examine [2] transcription start site region [1] registered in the existing public DB → Fragment 009_02 amplified by Primer009_02F (SEQ ID NO: 79) and Primer009_02R (SEQ ID NO: 80) (SEQ ID NO: 81)
009_03-Common region shared by [1] and [2]: cDNA pattern of [1] that we newly analyzed full-length cDNA sequence, and also [2] cDNA pattern registered in the existing public DB A region where all patterns serving as a control are in common to compare the expression level of the entire expression of the fragment → Fragment 009_03 (SEQ ID NO: 84) amplified by Primer009_03F (SEQ ID NO: 82) and Primer009_03R (SEQ ID NO: 83)
The exon regions specific to each of the cDNA patterns [1] and [2] shown above map the approximately 1.44 million 5 'end sequences obtained using the oligo cap method to human genome sequences for comparative analysis. As a result, the following results were obtained.
In the newly analyzed cDNA [1] pattern, there are 4 5 'terminal sequences, 3 of which are derived from the spleen (analysis parameter 33,950) and 1 from the lung (analysis). The parameter was 16,146).
In the pattern of [2], which is the control registered in the existing public DB, there are 24 5 'terminal sequences, and the origin is 6 sequences of the thalamus (Brain, thalamus) (analysis parameter 53,267), whole brain ( 3 sequences (Brain, whole) (analysis parameter 59,069), 2 sequences of normal astrocytes (analysis parameter 17,162), 2 sequences of retinoic acid treatment induction (NT2RP) of NT2 cells (analysis) 39,242), 3 sequences of fetal brain (Brain, Fetal) (analysis parameter 79,560), 2 sequences of trachea (analysis parameter 52,352), 2 sequences of thymus (analysis parameter 70,578), 1 sequence of kidney cancer (Kidney, Tumor) (analysis parameter 15,970), 1 sequence of kidney (Kidney) (analysis parameter 17,008), NT2 cells 5 weeks after induction of retinoic acid treatment, 2 weeks of growth inhibitor treatment (NT2RI ) Was 1 sequence (analysis parameter 32,662) and cerebellum (Brain, cerebellum) was 1 sequence (analysis parameter 82,880).
From this result, it was found that the transcription start point of [1] is highly expressed in the spleen. Moreover, at the transcription start point of [2], expression was observed in various tissues such as the brain. As a result, it was considered that the transcriptional mechanism is different in the chromosomal region between the blood cell tissue and the other tissue, and the transcription start point used may be different.

(2) Analysis of expression specificity by real-time PCR In order to know what kind of tissue in the blood cell system tissue and what state the transcription start point used for expression changes, details of the expression level are real-time PCR. Analyzed with The results are shown in Table 8 and Table 9.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As an experimental control, a sample (Mix, viscus tissues) mixed with normal visceral tissue described in Example 3 and a whole brain (Brain, whole) were used for comparison.
Depending on the selectivity of the transcription start site compared between 009_01 (SEQ ID NO: 78) and 009_02 (SEQ ID NO: 81), the mRNA was changed, and as a result, the ratio of change in ORF was greatly changed in the following tissues and cells.
In the spleen (Spleen) and bone marrow (Bone Marrow), there were many transcripts (mRNA) from the downstream transcription start point represented by 009_01 (SEQ ID NO: 78) (Tables 8 and 9).
In addition, when comparing CD34 + cells induced with osteoclast differentiation factor (ODF) in detail, transcripts (mRNA) from the transcription start point registered in the existing public DB represented by 009_02 (SEQ ID NO: 81) ) Is almost unchanged in all differentiation stages, whereas the transcript (mRNA) from the downstream transcription start point represented by 009_01 (SEQ ID NO: 78) is the state of undifferentiated CD34 + cells. The expression level of CD34 + cell ODF treatment was induced for 3 days (CD34 + ODF 3 days) and CD34 + cell ODF treatment was induced for 6 days (CD34 + ODF 6days). No expression was observed for 9 days (CD34 + ODF 9days) (Tables 8 and 9).
In addition, in 009_02 (SEQ ID NO: 81), more expression was observed in the whole brain (Brain, whole) and spinal cord (Spinal Cord) than 009_01 (SEQ ID NO: 78).
In other words, in the spleen and bone marrow, unlike other tissues, transcription factors or transcription mechanisms caused diversity in the transcription start point, and mRNAs with different ORF regions were expressed. Furthermore, it was found that by differentiating into osteoclasts, the mechanism that changes the transcript (mRNA) does not function, and the diversity of transcription start points is lost.
From these results, the 5′-terminal region of the newly obtained cDNA region indicated by the detection region of 009_01 (SEQ ID NO: 78) (region near the transcription start point) 009_ [1] _1-N1 (SEQ ID NO: 74) By comparing the expression, it is specific as a differentiation marker (diagnosis / treatment marker for osteoporosis, rheumatism and the process leading to it) as well as to the spleen and bone marrow (Bone Marrow) blood cells. It was proved that it can be used as a typical marker. It is also possible to develop new drugs using compounds, antibodies, siRNA, etc. that target specific regions.
The following regions are also useful as differentiation markers for detecting the differentiation stage of osteoclasts and as markers specific to the spleen and bone marrow blood cell systems.
The upstream sequence 009_ [1] _1-N 3 (SEQ ID NO: 85) containing the 89th to 112th bases primed by Primer009_01R (SEQ ID NO: 77) in D-SPLEN2030304.1 of the cDNA pattern of [1].
Region 009_01 (SEQ ID NO: 78) amplified by Primer009_01F (SEQ ID NO: 76) and Primer009_01R (SEQ ID NO: 77) in the cDNA pattern of [1]

Example 8: Cluster chr3 + 1925 (data set: 045)
(1) Cluster analysis 1) Cluster characteristics Four full-length cDNA genome-mapped to cluster chr3 + 1925 (Human genome UCSC hg18 (NCBI Build34) chromosome 3, 138,060,000 bp to 138,160,000 bp) [D-SYNOV4000324.1 , BC006403.2, ENST00000288986, NM_006153.3]. They could be classified into two types according to the difference in expression pattern as follows.
[1] D-SYNOV4000324.1
[2] BC006403.2, ENST00000288986, NM_006153.3
[1] is a cDNA we newly acquired and performed full-length cDNA sequence analysis, and the ORF was different from [2] registered in the existing public DB.
[1] was expressed from a chromosomal region downstream of the known [2], and the ORF region was different because it had a translation start on a new exon that had no homology to [2].
The ORF regions of the two types of cDNA patterns [1] to [2] are expressed from the same chromosomal region from different transcription initiation sites, resulting in altered amino acid sequences and the generation of diverse proteins and mRNAs. I found out.

2) Features of D-SYNOV4000324.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
045_ [1] _1-N0 (SEQ ID NO: 86): Entire region of the nucleic acid sequence of D-SYNOV4000324.1
045_ [1] _1-NA0 (SEQ ID NO: 87): Combination of D-SYNOV4000324.1 nucleic acid sequence and amino acid sequence
045_ [1] _1-A0 (SEQ ID NO: 88): Full amino acid region of D-SYNOV4000324.1
1-78 bases (SEQ ID NO: 89) of D-SYNOV4000324.1 are exons that do not exist in NM_006153.3, which is a control registered in the existing public DB, and have no homology with NM_006153.3. Since there was a translation start point on this exon, the amino acid on the N-terminal side changed 12 residues (SEQ ID NO: 90).
045_ [1] _1-N1 (SEQ ID NO: 89): 78-base inserted nucleic acid sequence region of D-SYNOV4000324.1
045_ [1] _1-A1 (SEQ ID NO: 90): 12-residue insertion amino acid sequence region of D-SYNOV4000324.1
045_ [1] _1-N2 (SEQ ID NO: 91): ORF nucleic acid sequence region in the 78-base insertion region of D-SYNOV4000324.1
045_ [1] _1-A2 (same as SEQ ID NO: 90): ORF amino acid sequence region in the 78-base insertion region of D-SYNOV4000324.1 Along with this change, Pfam exists at amino acids 5 to 59 of NM_006153.3 The motif “SH3 domain” disappeared.

3) Expression specificity analysis and design of primers for real-time PCR In order to clearly distinguish the characteristic regions as shown above and examine their expression levels, the following regions were used as detection regions. By comparing the expression levels of the detection regions, it was considered possible to compare the individual expression levels.
045_01-Specific region existing on the N-terminal side of the cDNA pattern of [1]: This is the transcription start region of the cDNA pattern of [1] that we newly analyzed, and is registered in the existing public DB Novel region that has not been processed → Fragment 045_01 (SEQ ID NO: 94) amplified by Primer045_01F (SEQ ID NO: 92) and Primer045_01R (SEQ ID NO: 93)
045_02-Transcription start region where [2] registered in the existing public DB exists, control to compare [1] → amplified by Primer045_02F (SEQ ID NO: 95) and Primer045_02R (SEQ ID NO: 96) Fragment 045_02 (SEQ ID NO: 97)
045_03-[1]-[2] Common region shared by all: [1] cDNA pattern we newly performed full-length cDNA sequence analysis, and [2] cDNA registered in the existing public DB A region where all the control patterns are in common to compare the expression level of the entire pattern → Fragment 045_03 (SEQ ID NO: 100) amplified by Primer045_03F (SEQ ID NO: 98) and Primer045_03R (SEQ ID NO: 99)
The exon regions specific to each of the cDNA patterns [1] and [2] shown above are mapped and compared to the human genome sequence of approximately 1.44 million 5 'end sequences obtained using the oligocap method. As a result, the following results were obtained.
In the newly analyzed cDNA [1] pattern, there are 3 sequences of 5 'terminal sequence, and the origin is 1 sequence of synovial membrane tissue from rheumatioid arthritis (analysis parameter 27,489), colon ( Colon) had 1 sequence (analysis parameter 8,398), NT2 cells were 5 weeks after induction of retinoic acid treatment, and growth inhibitor treatment 2 weeks (NT2RI) had 1 sequence (analysis parameter 32,662).
In the pattern of [2], which is a control registered in the existing public DB, there are 34 5 'terminal sequences, the origin of which is 9 sequences of thymus (analysis parameter 70,578), pericardium (Pericardium) 3 sequences (analysis parameter 8,781), 2 sequences of caudate nucleus (Brain, caudate nucleus) (analysis parameter 25,786), 2 sequences of trachea (analysis parameter 52,352), hippocampus (Brain, hippocampus) Two sequences (analysis parameter 57,918) and whole brain (Brain, whole) were expressed in various tissues such as two sequences (analysis parameter 59,069).
From this result, it was found that the transcription start point of [1] was expressed in synovial membrane tissue from rheumatioid arthritis. Moreover, cDNA derived from various tissues was expressed from the transcription start point of [2] as compared to the transcription start point of [1]. In other words, in this chromosomal region, there is a possibility that the selection mechanism of mRNA pattern change that causes different proteins to be expressed in specific tissues changes in amino acid sequence due to exon selectivity like the pattern of [1]. It was.

(2) Analysis of expression specificity by real-time PCR In order to know how the transcription start point used for expression changes between tissues, the details of the expression level were analyzed by real-time PCR. The results are shown in Table 10 and Table 11.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As experimental controls, a sample (Mix, viscus tissues) mixed with normal visceral tissue described in Example 3 and a whole brain (Brain, whole) were used for comparison.
Depending on the selectivity of the transcription start site compared between 045_01 (SEQ ID NO: 94) and 045_02 (SEQ ID NO: 97), the mRNA was changed, and as a result, the rate at which the ORF was changed greatly in the following tissues and cells.
Synovial tissue (rheumatic patients) and CD34 + cell ODF (osteoclast differentiation factor) treatment induction 3 days (CD34 + ODF 3days), CD34 + cell ODF treatment induction 6 days (CD34 + ODF 6days), CD34 + cell ODF treatment induction 9 There were many transcripts (mRNA) from the downstream transcription start point represented by 045_01 (SEQ ID NO: 94) in all stages in which CD34 + cells were induced with ODF for a day (CD34 + ODF 9days) (Table 10 and Tables). 11).
In the whole brain (Brain, whole), there was much transcription from the transcription start point registered in the existing public DB represented by 045_02 (SEQ ID NO: 97).
In other words, in synovial tissues (rheumatoid patients) and CD34 + cells induced by treatment with ODF, mRNAs with different ORF regions are expressed due to the diversity of transcription start points due to transcription factors or transcription mechanisms different from those in the brain. I found out that
From these results, the 5'-terminal region (region near the transcription start point) 045_ [1] _1-N1 (SEQ ID NO: 89) of the newly obtained cDNA region indicated by the detection region 045_01 (SEQ ID NO: 94) By comparing the expression, therapeutic and diagnostic markers specific to the blood cell system, especially synovial tissues of rheumatic patients, and specific therapeutic and diagnostic markers for osteoclast differentiation (osteoporosis, rheumatism and It has been proved that it can be used as a diagnostic and therapeutic marker for all processes. It is also possible to develop new drugs using compounds, antibodies, siRNA, etc. that target specific regions.
The following areas are also useful as therapeutic / diagnostic markers specific for the blood cell system, particularly the synovial tissue of rheumatic patients, and for therapeutic / diagnostic markers specific for osteoclast differentiation.
The upstream sequence 045_ [1] _1-N3 (SEQ ID NO: 101) containing the 130th to 153rd bases primed by Primer045_01R (SEQ ID NO: 93) in D-SYNOV4000324.1 of the cDNA pattern of [1].
Region 045_01 (SEQ ID NO: 94) amplified by Primer045_01F (SEQ ID NO: 92) and Primer045_01R (SEQ ID NO: 93) in the cDNA pattern of [1]

Example 9: cluster chr11 + 275 (data set: 064)
(1) Cluster analysis 1) Cluster characteristics 13 full-length cDNA genome mapped to cluster chr11 + 275 (Human genome UCSC hg18 (NCBI Build34) chromosome 11, 10,275,000 bp to 10,500,000 bp) [D-SYNOV4007243.1 , Z-TRACH3025625-01, D-BRAMY3006431.1, D-BRAWH3035395.1, D-NT2RI3008791.1, D-PLACE7015823.1, D-TESTI4045452.1, Z-CD34C3000530-01, Z-NT2RI3008366-01, Z -TESTI4015893-01, NM_000480.1, ENST00000256183, ENST00000356959,] was analyzed. They are classified into six types according to the difference in expression pattern, but the following two types existed mainly when focusing on the transcription start point.
[1] D-SYNOV4007243.1
[2] NM_000480.1, ENST00000256183
[1] is a cDNA we newly acquired and performed full-length cDNA sequence analysis, and the ORF was different from [2] registered in the existing public DB.
Since [1] is expressed from a chromosomal region downstream from the known [2], the ORF region was different.
The ORF regions of the two types of cDNA patterns [1] to [2] are expressed from the same chromosomal region from different transcription start sites, resulting in changes in amino acid sequences, resulting in diverse proteins and mRNAs. I found out that it was.

2) Features of D-SYNOV4007243.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
064_ [1] _1-N0 (SEQ ID NO: 102): Nucleic acid sequence entire region of D-SYNOV4007243.1
064_ [1] _1-NA0 (SEQ ID NO: 103): Combination of the entire nucleic acid sequence and amino acid sequence of D-SYNOV4007243.1
064_ [1] _1-A0 (SEQ ID NO: 104): D-SYNOV4007243.1 full amino acid region
1-217 bases (SEQ ID NO: 105) of D-SYNOV4007243.1 are variants inserted by exons that do not exist in NM_000480.1, which is a control registered in the existing public DB, and have no homology with NM_000480.1. Since it exists as a translation start point on this inserted exon, the amino acid at the N-terminal was 6 residues (SEQ ID NO: 106) different from NM_000480.1.
064_ [1] _1-N1 (SEQ ID NO: 105): 217-base inserted nucleic acid sequence region of D-SYNOV4007243.1
064_ [1] _1-A1 (SEQ ID NO: 106): D-SYNOV4007243.1 6-residue insertion amino acid sequence region
064_ [1] _1-N2 (SEQ ID NO: 107): ORF nucleic acid sequence region in the 217-base insertion region of D-SYNOV4007243.1
064_ [1] _1-A2 (same as SEQ ID NO: 106): ORF amino acid region related to 217 base insertion region of D-SYNOV4007243.1

3) Expression specificity analysis and design of primers for real-time PCR In order to clearly distinguish the characteristic regions as shown above and examine their expression levels, the following regions were used as detection regions. By comparing the expression levels of the detection regions, it was considered possible to compare the individual expression levels.
064_01-Specific region existing on the N-terminal side of the cDNA pattern of [1]: This is the transcription start region of the cDNA pattern of [1] that we newly analyzed and registered in the existing public DB Novel region that has not been changed → Fragment 064_01 (SEQ ID NO: 110) amplified by Primer064_01F (SEQ ID NO: 108) and Primer064_01R (SEQ ID NO: 109)
064_02-Control for comparing the transcription start point region [2] registered in the existing public DB, [1] → Fragment 064_02 amplified by Primer064_02F (SEQ ID NO: 111) and Primer064_02R (SEQ ID NO: 112) (SEQ ID NO: 113)
064_03-Common region shared by [1] and [2]: cDNA pattern of [1] that we newly analyzed full-length cDNA sequence, and also [2] cDNA pattern registered in the existing public DB A region where all patterns serving as controls are in common to compare the expression level of the entire expression of the fragment → Fragment 064_03 (SEQ ID NO: 116) amplified by Primer064_03F (SEQ ID NO: 114) and Primer064_03R (SEQ ID NO: 115)
The exon regions specific to each of the cDNA patterns [1] and [2] shown above map the approximately 1.44 million 5 'end sequences obtained using the oligo cap method to human genome sequences for comparative analysis. As a result, the following results were obtained.
In the newly analyzed cDNA [1] pattern, there are 13 5 'terminal sequences, and the origin is 3 synovial membrane tissue from rheumatioid arthritis (analysis parameter 27,489), trachea ( Trachea) (3 sequences (analysis parameter 52,352), whole brain (Brain, whole) 2 sequences (analysis parameter 59,069), testis (Testis) 2 sequences (analysis parameter 90,188), spleen (Spleen) 1 sequence (Analysis parameter 33,950), 1 sequence of Uterus (analysis parameter 49,561), and 1 sequence of thymus (Thymus) (analysis parameter 70,578).
In the pattern of control [2], which is registered in the existing public DB, there are 11 5 'terminal sequences, which are derived from 4 sequences of hippocampus (Brain, hippocampus) (analysis parameter 57,918), whole brain ( Brain, whole) had 3 sequences (analysis parameter 59,069), thalamus (Brain, thalamus) had 2 sequences (analysis parameter 53,267), and tonsils (Brain, amygdala) had 2 sequences (analysis parameter 58,640).
From this result, it was found that the transcription start point of [1] is highly expressed in blood cell tissues such as synovial membrane tissue from rheumatioid arthritis. Moreover, at the transcription start point of [2], there was much expression in the brain. As a result, in the chromosomal region, the transcriptional mechanism is different between the blood cell tissue such as synovial membrane tissue from rheumatioid arthritis and the other tissue (especially the brain), and the transcription start point used is also different. The possibility was considered.

(2) Analysis of expression specificity by real-time PCR In order to know what kind of tissue in the blood cell system and in what state the transcription start point used for expression changes, details of the expression level are real-time. Analyzed by PCR. The results are shown in Tables 12 and 13.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As experimental controls, comparison was made using samples (Mix, viscus tissues) mixed with normal visceral tissues described in Example 3 and whole brain (Brain, whole).
Depending on the selectivity of the transcription start site compared between 064_01 (SEQ ID NO: 110) and 064_02 (SEQ ID NO: 113), the mRNA changes, and as a result, the rate at which the ORF changes greatly changes in the following tissues.
Bone marrow, Thymus, Spleen, CD34 + cells (CD34 +), CD34 + cells induced by osteoclast differentiation factor (ODF) 3 days (CD34 + ODF 3 days), CD34 + cells induced by ODF 6 days (CD34 + ODF 6 days), CD34 + cells were treated with ODF 9 days (CD34 + ODF 9 days), synovial tissue (rheumatic patients), Jurkat cells (Jurkat), spinal cord (064_01) The transcript (mRNA) from the downstream transcription start point represented by SEQ ID NO: 110) was particularly large (Tables 12 and 13).
In other words, unlike these other tissues, transcription factors or transcription mechanisms caused the diversity of transcription start points to express mRNA with different ORFs in these tissues.
From these results, the 5′-terminal region of the newly obtained cDNA region indicated by the detection region 064_01 (SEQ ID NO: 110) (region near the transcription start point) 064_ [1] _1-N1 (SEQ ID NO: 105) By comparing the expression, the bone marrow (Bone Marrow), thymus (Thymus), spleen (Spleen), Jurkat cell (Jurkat) blood cell lineage specific markers, and synovial tissue (rheumatic patients) It has been proved that it can be used as a marker or a differentiation marker specific for osteoclast differentiation. It is also possible to develop new drugs using compounds, antibodies, siRNA, etc. that target specific regions.
In the following areas, bone marrow (Bone Marrow), thymus (Thymus), spleen (Spleen), Jurkat cell (Jurkat) blood cell lineage specific markers, as well as synovial tissue (rheumatic patients) markers It seems to be useful as a differentiation marker specific for the differentiation of osteoclasts.
The upstream sequence 064_ [1] _1-N3 (SEQ ID NO: 117) containing the 185th to 205th bases primed by Primer064_01R (SEQ ID NO: 109) in the D-SYNOV4007243.1 of the cDNA pattern of [1].
Region 064_01 (SEQ ID NO: 110) amplified by Primer064_01F (SEQ ID NO: 108) and Primer064_01R (SEQ ID NO: 109) in the cDNA pattern of [1]

Example 10: Cluster chr5 + 2320 (data set: 074)
(1) Cluster analysis 1) Cluster characteristics 11 full-length cDNA genome-mapped to cluster chr5 + 2320 (Human genome UCSC hg18 (NCBI Build34) chromosome 5, 180,240,000 bp to 180,380,000 bp) [D-NETRP2004161.1 , D-COLON2000670.1, Z-NETRP2006158-01, AY358385.1, C-COL04713, C-COL07391, ENST00000231229, ENST00000342868, NM_006707.2, NM_024850.1, NM_197975.1]. They are classified into five types according to the difference in expression pattern, but the following two types existed mainly when focusing on the transcription start point.
[1] D-NETRP2004161.1
[2] C-COL04713 (AK025111.1), ENST00000231229, NM_024850.1
[1] is a cDNA we newly acquired and performed full-length cDNA sequence analysis, and the ORF was different from [2] registered in the existing public DB.
Since [1] is expressed from a chromosomal region downstream from the known [2], the ORF region was different.
The ORF regions of the two types of cDNA patterns [1] to [2] are expressed from the same chromosomal region from different transcription start sites, resulting in changes in amino acid sequences, resulting in diverse proteins and mRNAs. I found out that it was.

2) Features of D-NETRP2004161.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
074_ [1] _1-N0 (SEQ ID NO: 118): The entire nucleic acid sequence of D-NETRP2004161.1
074_ [1] _1-NA0 (SEQ ID NO: 119): Combination of the entire nucleic acid sequence and amino acid sequence of D-NETRP2004161.1
074_ [1] _1-A0 (SEQ ID NO: 120): The entire amino acid sequence of D-NETRP2004161.1
The exon (first exon) of 1 to 134 bases (SEQ ID NO: 121) of D-NETRP2004161.1 is an exon that does not exist in NM_024850.1 which is a control registered in the existing public DB and has no homology. The translation start point of ORF of NM_024850.1 exists on the first exon which is an exon that does not exist in D-NETRP2004161.1, whereas the translation start point of D-NETRP2004161.1 is the second exon (NM_024850.1 3rd exon). Therefore, the amino acid on the N-terminal side was 184 residues shorter than NM_024850.1.
With this change, D-NETRP2004161.1 does not have an N-terminal secretory signal sequence that is predicted to exist at amino acids 1 to 16 of NM_024850.1 by SignalP and PSORT.
In addition, the 130 base exon (SEQ ID NO: 126) present at 941 to 1,070 bases of NM_024850.1, which is the control registered in the existing public DB, should be present in the region of 525 to 526 bases of D-NETRP2004161.1. It is deleted. With this change, the translation frame of ORF changed, and the C-terminal amino acid changed by 238 residues compared to NM_024850.1.
074_ [1] _1-N 1 (SEQ ID NO: 121): 134-base inserted nucleic acid sequence region of D-NETRP2004161.1
074_ [1] _1-N 2 (SEQ ID NO: 122): ORF 5'UTR region 289 bases starting from the 290th base of D-NETRP2004161.1
074_ [1] _1-N3 (SEQ ID NO: 123): Deleted nucleic acid sequence region of D-NETRP2004161.1
074_ [1] _1-A1 (SEQ ID NO: 124): Amino sequence region changed with deletion of D-NETRP2004161.1
074_ [1] _1-N4 (same as SEQ ID NO: 123): ORF nucleic acid sequence region 074_ [1] _1-A2 (SEQ ID NO: 125) in the deleted region of D-NETRP2004161.1: lack of D-NETRP2004161.1 ORF amino acid region related to the loss region
074_ [1] _C-N1 (SEQ ID NO: 126): 130-base inserted nucleic acid sequence present in 941-1,070 bases of NM_024850.1 to be inserted into the 525-526 base region of D-NETRP2004161.1
074_ [1] _C-A1 (SEQ ID NO: 127): Amino acid region related to the 130-base inserted nucleic acid sequence present in 941-1,070 bases of NM_024850.1 to be inserted in the 525-526 base region of D-NETRP2004161.1

3) Expression specificity analysis and real-time PCR primers and TaqMan probe design In order to clearly distinguish the characteristic regions as shown above and examine their expression levels, the following regions are detected regions. It was. By comparing the expression levels of the detection regions, it was considered possible to compare the individual expression levels.
074_01-Specific region existing on the N-terminal side of the cDNA pattern of [1]: This is the transcription start region of the cDNA pattern of [1] that we newly analyzed, and is registered in the existing public DB Novel region that has not been processed → Fragment 074_01 (SEQ ID NO: 130) amplified by Primer074_01F (SEQ ID NO: 128) and Primer074_01R (SEQ ID NO: 129)
TaqMan probe used: 074_01TP: (SEQ ID NO: 131)
074_03-Control for comparing the transcription start point region [2] registered in the existing public DB, [1] → Fragment 074_03 amplified by Primer074_03F (SEQ ID NO: 132) and Primer074_03R (SEQ ID NO: 133) (SEQ ID NO: 134)
TaqMan probe used: 074_03TP: (SEQ ID NO: 135)
074_04-Common region shared by [1] and [2]: cDNA pattern of [1] that we newly analyzed full-length cDNA sequence, and also [2] cDNA pattern registered in the existing public DB A region where all the patterns that serve as a control are common to compare the expression level as a whole → Fragment 074_04 (SEQ ID NO: 138) amplified by Primer074_04F (SEQ ID NO: 136) and Primer074_04R (SEQ ID NO: 137)
TaqMan probe used: 074_04TP: (SEQ ID NO: 139)
The exon regions specific to each of the cDNA patterns [1] and [2] shown above map the approximately 1.44 million 5 'end sequences obtained using the oligo cap method to human genome sequences for comparative analysis. As a result, the following results were obtained.
In the newly analyzed cDNA [1] pattern, there are 6 5 'terminal sequences, which are derived from 5 sequences of neutrophil (analysis parameter 9,170) and 1 sequence of lung (Lung). The analysis parameter was 16,146.
In the pattern of control [2] registered in the existing public DB, there are 19 5 'terminal sequences, and the origin is 12 colons (analysis parameter 8,398), small intestine 6 sequences (analysis parameter 16,767) and rectum (Rectum) 1 sequence (analysis parameter 2,723).
From this result, it was found that the transcription start point of [1] is highly expressed in neutrophils. In addition, at the transcription start point in [2], expression was frequently observed in the colon and small intestine. As a result, it was considered that the transcriptional mechanism is different in the chromosomal region between the blood cell tissue and the other tissue, and the transcription start point used may be different.

(2) Analysis of expression specificity by real-time PCR In order to know what kind of tissue and in what state the transcription start point used for expression changes in blood cell tissue, details of the expression level are real-time. Analyzed by PCR. The results are shown in Table 14.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As experimental controls, a sample (Mix, viscus tissues) mixed with normal visceral tissue described in Example 3 and a whole brain (Brain, whole) were used for comparison.
The mRNA changes depending on the selectivity of the transcription start site compared between 074_01 (SEQ ID NO: 130) and 074_03 (SEQ ID NO: 134), and as a result, the rate at which the ORF changes greatly varies in the following tissues.
Bone marrow, spleen (spleen), synovial (synovial) tissue (rheumatic patients), CD34 + cells treated with osteoclast differentiation factor (ODF) treatment for 3 days (CD34 + ODF 3days) at 074_01 (SEQ ID NO: 130) The transcript (mRNA) from the downstream transcription start point represented was particularly large (Table 14). The bone marrow (Bone Marrow), spleen (Spleen), synovial (Synovial) tissue (rheumatoid patients), CD34 + cells induced ODF treatment for 3 days (CD34 + ODF 3days), the transcription factor or transcription mechanism is different from other tissues, Due to the diversity of transcription start sites, mRNAs with different ORF regions were expressed.
From these results, the 5′-terminal region of the newly obtained cDNA region indicated by the detection region 074_01 (SEQ ID NO: 130) (region near the transcription start point) 074_ [1] _1-N1 (SEQ ID NO: 121) As a therapeutic / diagnostic marker specific to the blood cell system of synovial tissue in rheumatic patients by comparing the expression, a therapeutic / diagnostic marker that detects differentiation into osteoclasts, particularly the early stages of differentiation (osteoporosis) -It has been proved that it can be used as a marker for diagnosis and treatment of rheumatism and the processes leading to it, and as a specific marker for the bone marrow (Bone Marrow) and spleen (Spleen) blood cells. In addition, new drug development using compounds, antibodies, siRNA, etc. that target specific regions is also possible.
In addition, the following areas are also used as therapeutic / diagnostic markers specific to the blood system of the synovial tissue of rheumatic patients, as a therapeutic / diagnostic marker for detecting differentiation into osteoclasts, particularly the early stages of differentiation, and It seems to be useful as a marker specific to the blood cell system of bone marrow and spleen.
Upstream sequence 074_ [1] _1-N5 (SEQ ID NO: 140) containing the 128th to 146th bases primed by Primer074_01R (SEQ ID NO: 129) in D-NETRP2004161.1 with the cDNA pattern of [1]
Region 074_01 (SEQ ID NO: 130) amplified by Primer074_01F (SEQ ID NO: 128) and Primer074_01R (SEQ ID NO: 129) in the cDNA pattern of [1]

Example 11: Cluster chr6-1987 (Data set: 026)
(1) Cluster analysis 1) Cluster characteristics 8 full-length cDNA genome-mapped on cluster chr6-1987 (Human genome UCSC hg18 (NCBI Build34) chromosome 6, 24,900,000 bp to 25,200,000 bp) [D-NT2RI3001993.1 , D-SMINT2013276.1, AB002384.1, BC001232.1, C-KIDNE2007944, ENST00000229635, ENST00000259698, NM_015864.2]. They are classified into five types depending on the expression pattern of the transcription start point, and the following four types exist as main ones.
[1] D-NT2RI3001993.1
[2] D-SMINT2013276.1
[3] AB002384.1
[4] NM_015864.2, BC001232.1
[1] and [2] are cDNAs that we newly acquired and performed full-length cDNA sequence analysis, and their ORFs were different from [3] and [4] registered in the existing public DB.
Since [1] and [2] are expressed from the chromosomal region upstream of the known [3] and [4], the ORF regions are different.
The ORF regions of the four types of cDNA patterns [1] to [4] are expressed from the same chromosomal region from different transcription start sites, resulting in changes in amino acid sequences, resulting in diverse proteins and mRNAs. I found out.

2) Features of D-NT2RI3001993.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
026_ [1] _1-N0 (SEQ ID NO: 141): D-NT2RI3001993.1 nucleic acid entire region
026_ [1] _1-NA0 (SEQ ID NO: 142): Combination of the entire nucleic acid sequence and amino acid sequence of D-NT2RI3001993.1
026_ [1] _1-A0 (SEQ ID NO: 143): D-NT2RI3001993.1 full amino acid region
1-123 bases (SEQ ID NO: 144) of D-NT2RI3001993.1 are variants inserted by exons that do not exist in NM_015864.2, which is a control registered in the existing public DB, and have no homology with NM_015864.2. Since there was a translation start point on this inserted exon, the N-terminal amino acid was 29 residues (SEQ ID NO: 145) different from NM_015864.2.
1,920-3,256 bases (SEQ ID NO: 148) of D-NT2RI3001993.1 are variants inserted by exons that do not exist in NM_015864.2, which is a control registered in the existing public DB, and have no homology with NM_015864.2. Due to the presence of a translation termination point on this inserted exon, the C-terminal amino acid was 428 residues (SEQ ID NO: 149) different from NM_015864.2.
026_ [1] _1-N1 (SEQ ID NO: 144): 123-base inserted nucleic acid sequence region of D-NT2RI3001993.1
026_ [1] _1-A1 (SEQ ID NO: 145): 29-residue insertion amino acid sequence region of D-NT2RI3001993.1
026_ [1] _1-N2 (SEQ ID NO: 146): ORF nucleic acid sequence region in the 123 base insertion region of D-NT2RI3001993.1
026_ [1] _1-A2 (SEQ ID NO: 147): ORF amino acid region related to the 123-base insertion region of D-NT2RI3001993.1
026_ [1] _1-N3 (SEQ ID NO: 148): 1,337-base inserted nucleic acid sequence region of D-NT2RI3001993.1
026_ [1] _1-A3 (SEQ ID NO: 149): D-NT2RI3001993.1 428-residue insertion amino acid sequence region
026_ [1] _1-N4 (SEQ ID NO: 150): ORF nucleic acid sequence region in the 1,337 base insertion region of D-NT2RI3001993.1
026_ [1] _1-A4 (same as SEQ ID NO: 149): ORF amino acid region related to 1,337 base insertion region of D-NT2RI3001993.1

3) Features of D-SMINT2013276.1 ([2]) that we newly acquired and performed full-length cDNA sequence analysis
026_ [2] _1-N0 (SEQ ID NO: 151): D-SMINT2013276.1 whole region of nucleic acid sequence
026_ [2] _1-NA0 (SEQ ID NO: 152): Combination of the entire nucleic acid sequence and amino acid sequence of D-SMINT2013276.1
026_ [2] _1-A0 (SEQ ID NO: 153): D-SMINT2013276.1 full amino acid region
1-168 bases (SEQ ID NO: 154) of D-SMINT2013276.1 are variants inserted by exons that do not exist in NM_015864.2, which is a control registered in the existing public DB, and have no homology with NM_015864.2. Since there was a translation start point on this inserted exon, the N-terminal amino acid was 34 residues (SEQ ID NO: 155) different from NM_015864.2.
1,965-2,415 bases (SEQ ID NO: 158) of D-SMINT2013276.1 are variants inserted by exons that do not exist in NM_015864.2, which is a control registered in the existing public DB, and have no homology with NM_015864.2. Due to the presence of a translation termination point on this inserted exon, the C-terminal amino acid was 23 residues (SEQ ID NO: 159) different from NM_015864.2.
026_ [2] _1-N1 (SEQ ID NO: 154): D-SMINT2013276.1 168 bases inserted nucleic acid sequence region
026_ [2] _1-A1 (SEQ ID NO: 155): 34 amino acid insertion region of D-SMINT2013276.1
026_ [2] _1-N2 (SEQ ID NO: 156): ORF nucleic acid sequence region in the 168 base insertion region of D-SMINT2013276.1
026_ [2] _1-A2 (SEQ ID NO: 157): ORF amino acid region related to 168 base insertion region of D-SMINT2013276.1
026_ [2] _1-N3 (SEQ ID NO: 158): D-SMINT2013276.1 451 base insertion nucleic acid sequence region
026_ [2] _1-A3 (SEQ ID NO: 159): 23-residue insertion amino acid sequence region of D-SMINT2013276.1
026_ [2] _1-N4 (SEQ ID NO: 160): ORF nucleic acid sequence region in the 451 base insertion region of D-SMINT2013276.1
026_ [2] _1-A4 (same as SEQ ID NO: 159): ORF amino acid region related to 451 base insertion region of D-SMINT2013276.1

4) Expression specificity analysis and design of primers for real-time PCR In order to clearly distinguish the characteristic regions as shown above and examine their expression levels, the following regions were used as detection regions. By comparing the expression levels of the detection regions, it was considered possible to compare the individual expression levels.
026_02-Specific region existing on the N-terminal side of the cDNA pattern of [1]: This is the transcription start region of the cDNA pattern of [1] that we newly analyzed and registered in the existing public DB Novel region that has not been processed → Fragment 026_02 (SEQ ID NO: 163) amplified by Primer026_02F (SEQ ID NO: 161) and Primer026_02R (SEQ ID NO: 162)
026_03-Control for comparing [1] transcription start site region [1] registered in existing public DB → Fragment 026_03 amplified by Primer026_03F (SEQ ID NO: 164) and Primer026_03R (SEQ ID NO: 165) (SEQ ID NO: 166)
026_05-Common region shared by [1] and [3]: cDNA pattern of [1] that we newly analyzed full-length cDNA sequence, and [3] cDNA pattern registered in the existing public DB A region where all patterns serving as controls are in common to compare the expression level as a whole → Fragment 026_05 (SEQ ID NO: 169) amplified by Primer026_05F (SEQ ID NO: 167) and Primer026_05R (SEQ ID NO: 168)
The exon regions specific to each of the cDNA patterns [1] and [3] shown above are mapped and compared with the human genome sequence of approximately 1.44 million 5 'end sequences obtained using the oligocap method. As a result, the following results were obtained.
In the newly analyzed cDNA [1] pattern, there are 95 5 'end sequences, 56 from neutrophils (analysis parameter 9,170) and 21 from spleen (Spleen). (Analysis parameter 33,950), 8 sequences of human peripheral blood mononuclear cells (analysis parameter 7,900), 5 sequences of thymus (analysis parameter 70,578), NT2 cells treated with retinoic acid 5 weeks after induction, 2 sequences for growth inhibitor treatment (NT2RI) (analysis parameter 32,662), 2 sequences for thalamus (Brain, thalamus) (analysis parameter 53,267), 1 sequence for synovial (analysis) The parameter was 27,489).
In the pattern of control [3] registered in the existing public DB, there are 26 5 'terminal sequences, and the origin is 6 sequences of hippocampus (Brain, hippocampus) (analysis parameter 57,918), tonsils (Brain , amygdala) (analysis parameter 58,640), uterus (Uterus) 3 sequences (analysis parameter 49,561), fetal brain (Brain, Fetal) 3 sequences (analysis parameter 47,574), NT2 cells treated with retinoic acid Five weeks after induction, 2 weeks of growth inhibitor treatment (NT2RI) were expressed in various tissues such as 2 sequences (analysis parameter 32,662).
From this result, it was found that the transcription start point of [1] is highly expressed in blood cell tissues such as neutrophil and spleen. In addition, at the transcription start point of [3], expression was observed in various tissues such as brain tissue. As a result, it was considered that the transcriptional mechanism is different in the chromosomal region between the blood cell tissue and the other tissue, and the transcription start point used may be different.

(2) Analysis of expression specificity by real-time PCR In order to know what kind of tissue in the blood cell system tissue and in what state the transcription start point used for expression changes, details of the expression level are real-time Analyzed by PCR. The results are shown in Table 15.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As experimental controls, a sample (Mix, viscus tissues) mixed with normal visceral tissue described in Example 3 and a whole brain (Brain, whole) were used for comparison.
Depending on the selectivity of the transcription start site compared between 026_02 (SEQ ID NO: 163) and 026_03 (SEQ ID NO: 166), the mRNA was changed, and as a result, the rate at which ORF was changed in the following tissues and cells.
In the spleen (Spleen) and bone marrow (Bone Marrow), there were many transcripts (mRNA) from the upstream transcription start point represented by 026_02 (SEQ ID NO: 163) (Table 15).
In other words, unlike the other tissues, spleen and bone marrow express mRNAs with different ORFs due to the diversity of transcription start sites depending on transcription factors or transcription mechanisms.
In addition, CD34 + cells were induced with osteoclast differentiation factor (ODF) treatment for 3 days (CD34 + ODF 3 days), CD34 + cells were induced with ODF treatment for 9 days (CD34 + ODF 9days), and whole brain (Brain, whole) was 026_03 (SEQ ID NO: 166). There were many transcripts (mRNA) from the transcription start point from the downstream known by ().
From these results, the 5′-terminal region of the newly obtained cDNA region indicated by the detection region 026_02 (SEQ ID NO: 163) (region near the transcription start point) 026_ [1] _1-N1 (SEQ ID NO: 144) By comparing the expression, it was proved that it can be used as a specific marker for the spleen and bone marrow blood cells. It is also possible to develop new drugs using compounds, antibodies, siRNA, etc. that target specific regions.
The following areas are also useful as markers specific to the spleen and bone marrow blood cells.
The upstream sequence 026_ [1] _1-N5 (SEQ ID NO: 170) containing the 99th to 122nd bases primed by Primer026_02R (SEQ ID NO: 162) in D-NT2RI3001993.1 of the cDNA pattern of [1].
Region 026_02 (SEQ ID NO: 163) amplified by Primer026_02F (SEQ ID NO: 161) and Primer026_02R (SEQ ID NO: 162) in the cDNA pattern of [1]

Example 12: cluster chr12 + 613 (data set: 040)
(1) Cluster analysis 1) Cluster characteristics Nine full-length cDNAs mapped to the cluster chr12 + 613 (Human genome UCSC hg18 (NCBI Build34) chromosome 12, 32,400,000 bp to 32,700,000 bp) [D-THYMU3006588.1 , D-D3OST2003177.1, AL713762.1, AL832064.1, C-BRAMY2019963, C-FEBRA2017154, C-TESTI2001141, ENST00000266482, NM_139241.1]. They are classified into four types depending on the expression pattern of the transcription start point, and the following three types exist as main ones.
[1] D-THYMU3006588.1
[2] D-D3OST2003177.1
[3] C-TESTI2001141 (AK057294.1), ENST00000266482, NM_139241.1
[1] and [2] are cDNAs we newly acquired and subjected to full-length cDNA sequence analysis, and the ORF was different from [3] registered in the existing public DB.
Since [1] is expressed from a chromosomal region downstream from the known [3], and [2] is expressed from a chromosomal region upstream from the known [3], the ORF region was different.
The ORF regions of the three types of cDNA patterns [1] to [3] are expressed from different transcription start sites from the same chromosomal region, thereby changing the amino acid sequence and producing diverse proteins and mRNAs. I found out that it was.

2) Features of D-THYMU3006588.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
040_ [1] _1-N0 (SEQ ID NO: 171): The entire nucleic acid sequence of D-THYMU3006588.1
040_ [1] _1-NA0 (SEQ ID NO: 172): Combination of the entire nucleic acid sequence and amino acid sequence of D-THYMU3006588.1
040_ [1] _1-A0 (SEQ ID NO: 173): D-THYMU3006588.1 full amino acid region
1-177 bases (SEQ ID NO: 174) of D-THYMU3006588.1 are variants inserted by exons that do not exist in NM_139241.1, which is a control registered in the existing public DB, and have no homology with NM_139241.1. Since there was a translation start point on this exon to be inserted, the amino acid at the N-terminal was 112 residues (SEQ ID NO: 175) different from NM_139241.1.
040_ [1] _1-N1 (SEQ ID NO: 174): 177 base insertion nucleic acid sequence region of D-THYMU3006588.1
040_ [1] _1-A1 (SEQ ID NO: 175): 112 amino acid insertion region of D-THYMU3006588.1
040_ [1] _1-N2 (SEQ ID NO: 176): ORF nucleic acid sequence region in the 177 base insertion region of D-THYMU3006588.1
040_ [1] _1-A2 (SEQ ID NO: 177): ORF amino acid region related to 177 base insertion region of D-THYMU3006588.1

3) Features of D-D3OST2003177.1 ([2]) that we newly acquired and performed full-length cDNA sequence analysis
040_ [2] _1-N0 (SEQ ID NO: 178): The entire nucleic acid sequence of D-D3OST2003177.1
040_ [2] _1-NA0 (SEQ ID NO: 179): Combination of D-D3OST2003177.1 nucleic acid sequence and amino acid sequence
040_ [2] _1-A0 (SEQ ID NO: 180): D-D3OST2003177.1 full amino acid sequence
1-172 bases (SEQ ID NO: 181) of D-D3OST2003177.1 are variants inserted by exons that do not exist in NM_139241.1, which is a control registered in the existing public DB, and have no homology with NM_139241.1. Since there was a translation start point on the exon to be inserted, the N-terminal amino acid was 85 residues (SEQ ID NO: 182) different from NM_139241.1.
040_ [2] _1-N1 (SEQ ID NO: 181): 172 bases inserted nucleic acid sequence region of D-D3OST2003177.1
040_ [2] _1-A1 (SEQ ID NO: 182): 85 amino acid insertion region of D-D3OST2003177.1
040_ [2] _1-N2 (SEQ ID NO: 183): ORF nucleic acid sequence region in the 172 base insertion region of D-D3OST2003177.1
040_ [2] _1-A2 (SEQ ID NO: 184): ORF amino acid region related to 172 base insertion region of D-D3OST2003177.1

4) Expression specificity analysis and design of primers for real-time PCR In order to clearly distinguish the characteristic regions as shown above and examine their expression levels, the following regions were used as detection regions. By comparing the expression levels of the detection regions, it was considered possible to compare the individual expression levels.
040_01-Specific region existing on the N-terminal side of the cDNA pattern of [1]: This is the transcription start region of the cDNA pattern of [1] that we newly analyzed and registered in the existing public DB Novel region that has not been changed → Fragment 040_01 (SEQ ID NO: 187) amplified by Primer040_01F (SEQ ID NO: 185) and Primer040_01R (SEQ ID NO: 186)
040_02-Specific region existing on the N-terminal side of the cDNA pattern of [2]: This is the transcription start region of the cDNA pattern of [2] that we newly analyzed, and is registered in the existing public DB Novel region that has not been processed → Fragment 040_02 (SEQ ID NO: 190) amplified by Primer040_02F (SEQ ID NO: 188) and Primer040_02R (SEQ ID NO: 189)
040_03-[3] transcription start region registered in the existing public DB, control to compare and examine [1], [2] → amplified by Primer040_03F (SEQ ID NO: 191) and Primer040_03R (SEQ ID NO: 192) Fragment 040_03 (SEQ ID NO: 193)
040_04-Common region shared by [1] to [3]: We newly performed full-length cDNA sequence analysis [1], [2] cDNA pattern, and also registered in the existing public DB [3] In order to compare the expression level of the entire cDNA pattern of [], a region in which all the control patterns are common → Fragment 040_04 (SEQ ID NO: 195) amplified by Primer040_04F (SEQ ID NO: 194) and Primer040_04R (SEQ ID NO: 195) 196)
The exon region specific to each of the cDNA patterns [1] to [3] shown above is a comparative analysis by mapping approximately 1.44 million 5 'end sequences obtained using the oligo cap method to the human genome sequence. As a result, the following results were obtained.
In the newly analyzed cDNA pattern [1], there are 4 5 'terminal sequences, and the origin is 1 sequence of normal thymus (Thymus) (analysis parameter 70,578), chondrocyte HC (Human Chondrocytes). 1 sequence (analysis parameter 9,397), 1 sequence of normal human mesangial cells (analysis parameter 16,053), NT2 cells were differentiated by retinoic acid treatment and growth inhibitor treatment, and nerves were concentrated and recovered (NT2 Neuron) was one sequence (analysis parameter 16,337).
In the newly analyzed cDNA [2] pattern, there are 3 5 'end sequences, 2 of which are neutrophils (analysis parameter 9,170), and CD34 + cells are differentiated into osteoclasts. Factor (ODF) treatment induction 3 days (D3OST) was 1 sequence (analysis parameter 5,092).
In the pattern of [3], which is a control registered in the existing public DB, there are 15 5 'terminal sequences, the origin of which is 12 sequences of testis (analysis parameter 90,188), fetal brain (Brain, Fetal) was 2 sequences (analysis parameter 55,564), and hippocampus (Brain, hippocampus) was 1 sequence (analysis parameter 57,918).
From these results, the transcription start sites of [1] and [2] were highly expressed in the thymus (Thymus), neutrophil (Neutrophil), and CD34 + cells induced by osteoclast differentiation factor (ODF) treatment. In addition, at the transcription start point of [3], the expression was high in the testis. As a result, it was considered that there is a possibility that the transcription start point used in the chromosomal region differs depending on the transcription mechanism used in the blood cell tissue and other tissues.

(2) Analysis of expression specificity by real-time PCR In order to know what kind of tissue in the blood cell system tissue and in what state the transcription start point used for expression changes, details of the expression level are real-time Analyzed by PCR. The results are shown in Table 16.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As an experimental control, a sample (Mix, viscus tissues) mixed with normal visceral tissue described in Example 3 and a whole brain (Brain, whole) were used for comparison.
040_01 (SEQ ID NO: 187), 040_02 (SEQ ID NO: 190) and 040_03 (SEQ ID NO: 193) mRNA changes depending on the selectivity of the transcription start point compared, and as a result, the rate of change of ORF is as follows: It has changed greatly.
Bone Marrow, Thymus, Spleen, CD34 + cells (CD34 +), CD34 + cells induced by osteoclast differentiation factor (ODF) treatment for 3 days (CD34 + ODF 3days), CD34 + cells induced by ODF treatment6 Days (CD34 + ODF 6days), CD34 + cells induced to ODF treatment 9 days (CD34 + ODF 9days), synovial tissue (rheumatic patients), spinal cord (040_01 (SEQ ID NO: 187), 040_02 (SEQ ID NO: 190) ) The transcript (mRNA) from the newly found transcription start point represented by) was higher than that of 040_03 (SEQ ID NO: 193) (Table 16).
In particular, at the transcription start point represented by 040_01 (SEQ ID NO: 187), thymus (Thymus), spleen (Spleen), CD34 + cells were induced with ODF treatment for 6 days (CD34 + ODF 6days), and CD34 + cells were induced with ODF treatment for 9 days (CD34 + ODF 9days), synovial tissues (rheumatoid patients), spinal cord (Spinal Cord), the expression is 040_02 (SEQ ID NO: 190) at the transcription start point, bone marrow (Bone Marrow), CD34 + cells (CD34 + ), CD34 + cells were highly expressed in ODF treatment-induced 3 days (CD34 + ODF 3days).
In other words, unlike blood cells, mRNAs with different ORFs were expressed in blood cell tissues because of the diversity of transcription start points depending on transcription factors or transcription mechanisms.
From these results, the 5′-terminal region of the newly obtained cDNA region indicated by the detection region 040_01 (SEQ ID NO: 187) (region near the transcription start point) 040_ [1] _1-N1 (SEQ ID NO: 174) By comparing the expression, a therapeutic / diagnostic marker that detects osteoclast differentiation, especially the middle and late stages of differentiation, as a specific therapeutic / diagnostic marker for the synovial tissues of rheumatic patients It has been proved that it can be used as a marker for diagnosis and treatment of osteoporosis and rheumatism and the process leading to it, and as a marker specific to the blood cell system of thymus and spleen.
Also, compare the expression of the 040_ [2] _1-N1 (SEQ ID NO: 181) 5'-terminal region (region near the transcription start site) of the newly obtained cDNA region indicated by the detection region 040_02 (SEQ ID NO: 190) Specific to osteoclast differentiation, especially as a therapeutic / diagnostic marker (diagnostic / therapeutic marker for osteoporosis / rheumatism and the process leading to it) and bone marrow (Bone Marrow) It was proved that it can be used as a typical marker.
In addition, new drug development using compounds, antibodies, siRNA, etc. that target specific regions is also possible.
In addition, as a therapeutic / diagnostic marker specific to the blood cell system of the synovial tissue of rheumatic patients in the following areas, osteoclast differentiation, especially as a therapeutic / diagnostic marker for detecting the middle and late stages of differentiation, In addition, it seems to be useful as a marker specific to the blood cell system of the thymus and spleen.
An upstream sequence 040_ [1] _1-N3 (SEQ ID NO: 197) containing nucleotides 182 to 202 primed by Primer040_01R (SEQ ID NO: 186) in D-THYMU3006588.1 of the cDNA pattern of [1].
An upstream sequence 040_ [2] _1-N3 (SEQ ID NO: 198) containing nucleotides 162 to 179 that Primer040_02R (SEQ ID NO: 189) primes in D-D3OST2003177.1 of the cDNA pattern of [2].
Region 040_01 (SEQ ID NO: 187) amplified by Primer040_01F (SEQ ID NO: 185) and Primer040_01R (SEQ ID NO: 186) in the cDNA pattern of [1]
Region 040_02 (SEQ ID NO: 190) amplified by Primer040_02F (SEQ ID NO: 188) and Primer040_02R (SEQ ID NO: 189) in the cDNA pattern of [2]

Example 13: Cluster chr4-770 (Data set: 069)
(1) Cluster analysis 1) Cluster characteristics 11 full-length cDNA genome mapped to cluster chr4-770 (Human genome UCSC hg18 (NCBI Build34) chromosome 4, 104,290,000 bp to 104,360,000 bp) [D-D3OST2000831.1 , D-MESAN2002178.1, D-OCBBF3022929.1, BC047447.1, BX640844.1, C-HEP22543, ENST00000339611, ENST00000355967, ENST00000358643, ENST00000360917, NM_178833.3] were analyzed. They are classified into seven types according to the difference in expression pattern, but the following two types existed mainly when focusing on the transcription start point.
[1] D-D3OST2000831.1
[2] C-HEP22543 (AK172823.1), ENST00000360917, NM_178833.3
[1] is a cDNA we newly acquired and performed full-length cDNA sequence analysis, and the ORF was different from [2] registered in the existing public DB.
[1] is expressed from a chromosomal region downstream of the known [2], but has a common translation start point, so there is no change in ORF due to a change in the transcription start point. However, the ORF region was different due to the presence of the deletion region.
The ORF regions of the two cDNA patterns [1] to [2] are expressed from the same chromosomal region and expressed from different transcription start sites, resulting in deletions that change the amino acid sequence, resulting in diverse proteins. And mRNA was found.

2) Features of D-D3OST2000831.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
069_ [1] _1-N0 (SEQ ID NO: 199): The entire nucleic acid sequence of D-D3OST2000831.1
069_ [1] _1-NA0 (SEQ ID NO: 200): Combination of the entire nucleic acid sequence and amino acid sequence of D-D3OST2000831.1
069_ [1] _1-A0 (SEQ ID NO: 201): D-D3OST2000831.1 full amino acid region
D-D3OST2000831.1 base sequence 1-135 (SEQ ID NO: 202) is a variant inserted by an exon that does not exist in NM_178833.3, which is a control registered in the existing public DB, and has homology to NM_178833.3 Absent. In addition, a 171 base exon (SEQ ID NO: 205) present in 904 to 1,074 bases of NM_178833.3, which is a control registered in an existing public DB, is present in the region of 448 to 449 bases in D-D3OST2000831.1. It is deleted without. D-D3OST2000831.1 and NM_178833.3 share the same translation start point and translation end point, but the ORF differs due to the presence of a 171 base deletion, and the amino acid length of D-D3OST2000831.1 is NM_178833.3 57 residues were shorter than
With this change, two transmembrane domains predicted by SOSUI to exist at amino acids 80 to 102 and 114 to 136 of NM_178833.3 were lost in D-D3OST2000831.1. 069_ [1] _1-N1 (SEQ ID NO: 202): 135-base inserted nucleic acid sequence region of D-D3OST2000831.1
069_ [1] _1-N2 (SEQ ID NO: 203): D-D3OST2000831.1 deleted nucleic acid sequence region
069_ [1] _1-N3 (identical to SEQ ID NO: 203): ORF nucleic acid sequence region 069_ [1] _1-A1 (SEQ ID NO: 204) in the deleted region of D-D3OST2000831.1: lack of D-D3OST2000831.1 ORF amino acid region related to the loss region
069_ [1] _C-N1 (SEQ ID NO: 205): 171 base inserted nucleic acid sequence present in 904 to 1,074 bases of NM_178833.3 inserted in the region of 448 to 449 bases in D-D3OST2000831.1
069_ [1] _C-A1 (SEQ ID NO: 206): Amino acid region related to the 171-base inserted nucleic acid sequence present in 904-1,074 bases of NM_178833.3 inserted in the 448-449 base region of D-D3OST2000831.1

3) Expression specificity analysis and design of primers for real-time PCR In order to clearly distinguish the characteristic regions as shown above and examine their expression levels, the following regions were used as detection regions. By comparing the expression levels of the detection regions, it was considered possible to compare the individual expression levels.
069_01-Specific region existing on the N-terminal side of the cDNA pattern of [1]: This is the transcription start region of the cDNA pattern of [1] that we newly analyzed, and is registered in the existing public DB Novel region that has not been processed → Fragment 069_01 (SEQ ID NO: 209) amplified by Primer069_01F (SEQ ID NO: 207) and Primer069_01R (SEQ ID NO: 208)
069_02-Control for comparing and examining [2] transcription start point region registered in the existing public DB → Fragment 069_02 amplified by Primer069_02F (SEQ ID NO: 210) and Primer069_02R (SEQ ID NO: 211) (SEQ ID NO: 212)
069_03-The region extracted specifically using the sequence information of the exon deletion region of the cDNA pattern of [1]: In the cDNA pattern of [1] that we newly performed full-length cDNA sequence analysis, Exon deletion region changing ORF → Fragment 069_03 (SEQ ID NO: 215) amplified by Primer069_03F (SEQ ID NO: 213) and Primer069_03R (SEQ ID NO: 214)
069_04 -This is a specific region that can be distinguished from the deletion region of [1] in the cDNA pattern of [2] registered in the existing public DB. Compare [1]. Control for → Fragment 069_04 (SEQ ID NO: 218) amplified by Primer069_04F (SEQ ID NO: 216) and Primer069_04R (SEQ ID NO: 217)
069_05-[1], [2] Common region shared by all: [1] cDNA pattern we newly analyzed full-length cDNA sequence, and [2] cDNA registered in the existing public DB A region in which all the patterns to be used as a control are in common to compare the expression level of the entire pattern → fragment 069_05 (SEQ ID NO: 221) amplified by Primer069_05F (SEQ ID NO: 219) and Primer069_05R (SEQ ID NO: 220)
The exon regions specific to each of the cDNA patterns [1] and [2] shown above map the approximately 1.44 million 5 'end sequences obtained using the oligo cap method to human genome sequences for comparative analysis. As a result, the following results were obtained.
In the newly analyzed cDNA [1] pattern, there are 14 5 'terminal sequences, and the origin is CD34 + cells that are induced by 5 days of osteoclast differentiation factor (ODF) treatment induction (D9OST). (4,407), CD34 + cells treated with osteoclast differentiation factor (ODF) treatment 3 days (D3OST) 2 sequences (analysis parameter 5,092), CD34 + cells treated with osteoclast differentiation factor (ODF) treatment 6 days (D6OST) ) Is 2 sequences (analysis parameter 888), caudate nucleus (Brain, caudate nucleus) is 2 sequences (analysis parameter 25,786), thalamus (Brain, thalamus) is 1 sequence (analysis parameter 53,267), and NT2 cells are RA After nerve differentiation by treatment and growth inhibitor treatment, the nerve was concentrated and recovered (NT2 Neuron) in one sequence (analysis parameter 16,337), and chondrocyte was in one sequence (analysis parameter 2,687).
In the control [2] pattern registered in the existing public DB, there are 79 5 'terminal sequences, the origin of which is 19 sequences of fetal brain (Brain, Fetal) (analysis parameter 71,152), cerebellum ( 9 sequences (Brain, cerebellum) (analysis parameter 82,880), 8 sequences (Brain, amygdala) (analysis parameter 58,640), 5 sequences (Brain, whole) (analysis parameter 59,069), thalamus (Brain) , thalamus) 4 sequences (analysis parameter 53,267), kidney cancer (Kidney, Tumor) 3 sequences (analysis parameter 15,970), NT2 cells 5 weeks after induction of retinoic acid treatment, growth inhibitor treatment 2 weeks (NT2RI) 3 sequences (analysis parameter 32,662) and testis (Testis) 3 sequences (analysis parameter 90,188).
From this result, it was found that the pattern [1] is highly expressed in CD34 + cells induced by osteoclast differentiation factor (ODF) treatment. The pattern [2] was expressed in various tissues, mainly in the brain. As a result, it was considered that the mechanism of transcription differs between the osteoclasts and other tissues (particularly the brain), and the transcription start point used may be different.

(2) Analysis of expression specificity by real-time PCR In order to know what kind of tissue in the blood cell system tissue and in what state the transcription start point used for expression changes, details of the expression level are real-time Analyzed by PCR. The results are shown in Tables 17-1 and 17-2 and Tables 18-1 and 18-2.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As an experimental control, a sample (Mix, viscus tissues) mixed with normal visceral tissue described in Example 3 and a whole brain (Brain, whole) were used for comparison.
The mRNA changes depending on the selectivity of the transcription start site compared between 069_01 (SEQ ID NO: 209) and 069_02 (SEQ ID NO: 212) and the exon selectivity compared between 069_03 (SEQ ID NO: 215) and 069_04 (SEQ ID NO: 218), As a result, the rate at which ORF changes has changed significantly in the following organizations:
Bone Marrow, Thymus, Spleen, CD34 + cells (CD34 +), CD34 + cells induced by osteoclast differentiation factor (ODF) treatment for 3 days (CD34 + ODF 3days), CD34 + cells induced by ODF treatment6 069_01 (SEQ ID NO: 209) for days (CD34 + ODF 6days), CD34 + cells treated with ODF treatment for 9 days (CD34 + ODF 9days), synovial tissues (rheumatic patients), Jurkat cells (Jurkat), and spinal cord (Spinal Cord) ) In the pattern in which the exon represented by 069_03 (SEQ ID NO: 215) is deleted (Table 17-1 and 17-2). 18-1, 18-2).
In other words, in these blood cell tissues, unlike other tissues, there are diversity in the transcription start point depending on transcription factors or transcription mechanisms, and exon selectivity is also diverse, so that mRNAs with different ORF regions are different. It was expressed.
From these results, the 5′-terminal region of the newly obtained cDNA region indicated by the detection region 069_01 (SEQ ID NO: 209) (region near the transcription start point) 069_ [1] _1-N1 (SEQ ID NO: 202) By comparing the expression frequency with the expression frequency of the exon deletion region 069_ [1] _1-N2 (SEQ ID NO: 203) indicated by the detection region of 069_03 (SEQ ID NO: 215) (either individually or in combination) Bone Marrow, Thymus, Spleen, CD34 + cells (CD34 +), Jurkat cells (Jurkat), as a specific marker for blood cell lineage, and synovial tissues (Rheumatoid patients) It has been proved that it can be used as a marker for osteoclasts and as a differentiation marker for osteoclasts. In addition, new drug development using compounds, antibodies, siRNA, etc. that target specific regions is also possible.
In the following areas, bone marrow (Bone Marrow), thymus (Thymus), spleen (Spleen), CD34 + cells (CD34 +), Jurkat cells (Jurkat) blood cell lineage specific markers, and synovial tissue It seems to be useful as a marker for rheumatic patients and a marker for osteoclast differentiation.
The upstream sequence 069_ [1] _1-N4 (SEQ ID NO: 222) comprising the 88th to 109th bases primed by Primer069_01R (SEQ ID NO: 208) in D-D3OST2000831.1 having the cDNA pattern of [1].
The upstream sequence 069_ [1] _1-N5 (SEQ ID NO: 223) containing the 530th to 551th bases primed by Primer069_03R (SEQ ID NO: 214) in D-D3OST2000831.1 of the cDNA pattern of [1].
Region 069_01 (SEQ ID NO: 209) amplified by Primer069_01F (SEQ ID NO: 207) and Primer069_01R (SEQ ID NO: 208) in the cDNA pattern of [1]
Region 069_03 (SEQ ID NO: 215) amplified by Primer069_03F (SEQ ID NO: 213) and Primer069_03R (SEQ ID NO: 214) in the cDNA pattern of [1]

Example 14: cluster chr1 + 3150 (data set: 108)
(1) Cluster analysis 1) Cluster characteristics Eight full-length cDNAs mapped to the cluster chr1 + 3150 (Human genome UCSC hg18 (NCBI Build34) chromosome 1, 182,997,500 bp to 183,017,500 bp) [D-HCASM2001914.1 , D-SYNOV4003291.1, D-SYNOV4005198.1, C-SYNOV2000178, C-SYNOV4003981, ENST00000251819, NM_005807.1, U70136.1] were analyzed. They are classified into six types depending on the expression pattern, but there are two main types.
[1] D-HCASM2001914.1, D-SYNOV4003291.1
[2] ENST00000251819, NM_005807.1, U70136.1
[1] is a cDNA we newly acquired and performed full-length cDNA sequence analysis, and the ORF was different from [2] registered in the existing public DB.
Compared with the known [2], [1] had an ORF change and a different ORF region because an exon different from other patterns in the ORF region was deleted.
The ORF regions of the two cDNA patterns [1] and [2] have different splicing patterns from the same chromosomal region, so that the amino acid sequence changes, and diverse proteins and mRNAs are produced. I found out.

2) Features of D-HCASM2001914.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
108_ [1] _1-N0 (SEQ ID NO: 224): The entire nucleic acid sequence of D-HCASM2001914.1
108_ [1] _1-NA0 (SEQ ID NO: 225): Combination of D-HCASM2001914.1 whole region and amino acid sequence
108_ [1] _1-A0 (SEQ ID NO: 226): Full amino acid region of D-HCASM2001914.1 279 base exons present in 353 to 631 bases of NM_005807.1 as a control registered in the existing public DB (SEQ ID NO: 229) does not exist in the region of 364 to 365 bases of D-HCASM2001914.1 and is deleted. Therefore, compared with NM_005807.1, 93 amino acids were deleted.
108_ [1] _1-N1 (SEQ ID NO: 227): D-HCASM2001914.1 deleted nucleic acid sequence region
108_ [1] _1-A1 (SEQ ID NO: 228): D-HCASM2001914.1 changing amino sequence region
108_ [1] _1-N2 (identical to SEQ ID NO: 227): ORF nucleic acid sequence region 108_ [1] _1-A2 (identical to SEQ ID NO: 228) in the deleted region of D-HCASM2001914.1: D-HCASM2001914.1 ORF amino acid region related to the deletion region of
108_ [1] _C-N1 (SEQ ID NO: 229): 279 base inserted nucleic acid sequence present in 353 to 631 bases of NM_005807.1 inserted in the region of 364 to 365 bases of D-HCASM2001914.1
108_ [1] _C-A1 (SEQ ID NO: 230): Amino acid region related to the inserted nucleic acid sequence of 279 bases present in 353 to 631 bases of NM_005807.1 inserted in the region of 364 to 365 bases of D-HCASM2001914.1 .

3) Features of D-SYNOV4003291.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
108_ [1] _2-N0 (SEQ ID NO: 231): D-SYNOV4003291.1 entire region of nucleic acid sequence
108_ [1] _2-NA0 (SEQ ID NO: 232): Combination of D-SYNOV4003291.1 nucleic acid sequence and amino acid sequence
108_ [1] _2-A0 (SEQ ID NO: 233): The entire amino acid sequence of D-SYNOV4003291.1 A 123-base exon in 109-231 bases of NM_005807.1, which is a control registered in an existing public DB (SEQ ID NO: 236) is not present in the region of 120 to 121 bases of D-SYNOV4003291.1 and is deleted. Therefore, 41 amino acid residues were deleted compared with NM_005807.1.
108_ [1] _2-N1 (SEQ ID NO: 234): D-SYNOV4003291.1 deleted nucleic acid sequence region, part 1
108_ [1] _2-A1 (SEQ ID NO: 235): D-SYNOV4003291.1 changing amino sequence region
108_ [1] _2-N2 (identical to SEQ ID NO: 234): ORF nucleic acid sequence region 108_ [1] _2-A2 (identical to SEQ ID NO: 235) in the deleted region of D-SYNOV4003291.1: D-SYNOV4003291.1 ORF amino acid region related to the deletion region of
108_ [1] _C-N1 (SEQ ID NO: 236): 123-base inserted nucleic acid sequence present in 109-231 bases of NM_005807.1 inserted in the 120-121 base region of D-SYNOV4003291.1
108_ [1] _C-A1 (SEQ ID NO: 237): Amino acid region related to the 123-base inserted nucleic acid sequence present in 109-231 bases of NM_005807.1 inserted in the 120-121 base region of D-SYNOV4003291.1 .
With this change, the Pfam motif “Somatomedin B domain” present at amino acids 28 to 69 of NM_005807.1 disappeared.
In addition, the 279-base exon (SEQ ID NO: 229) present in bases 353 to 631 of NM_005807.1, which is a control registered in the existing public DB, is present in the region of bases 241 to 242 in D-SYNOV4003291.1. Is missing. Therefore, compared with NM_005807.1, 93 amino acids were deleted.
108_ [1] _2-N3 (identical to SEQ ID NO: 227): D-SYNOV4003291.1 deleted nucleic acid sequence region, part 2
108_ [1] _2-A3 (same as SEQ ID NO: 228): D-SYNOV4003291.1 changing amino sequence region
108_ [1] _2-N4 (identical to SEQ ID NO: 227): ORF nucleic acid sequence region 108_ [1] _2-A4 (identical to SEQ ID NO: 228) in the deleted region of D-SYNOV4003291.1: D-SYNOV4003291.1 ORF amino acid region related to the deletion region of
108_ [1] _C-N3 (same as SEQ ID NO: 229): 279 base inserted nucleic acid sequence present in 353 to 631 bases of NM_005807.1 inserted in the region of 241 to 242 bases of D-SYNOV4003291.1
108_ [1] _C-A3 (same as SEQ ID NO: 230): involved in the inserted nucleic acid sequence of 279 bases present in 353 to 631 bases of NM_005807.1 inserted in the region of 241 to 242 bases of D-SYNOV4003291.1 Amino acid region.

4) Expression specificity analysis and design of primers for real-time PCR In order to clearly distinguish the characteristic regions as shown above and examine their expression levels, the following regions were used as detection regions. By comparing the expression levels of the detection regions, it was considered possible to compare the individual expression levels.
108_01-A region specifically extracted using sequence information at the boundary of exon regions that are commonly deleted in the cDNA pattern of [1]: We newly performed full-length cDNA sequence analysis [1] In the cDNA pattern of: deletion region of exon changing ORF → Fragment 108_01 (SEQ ID NO: 240) amplified by Primer108_01F (SEQ ID NO: 238) and Primer108_01R (SEQ ID NO: 239)
108_03-Specific region corresponding to the insertion region when compared with the cDNA pattern of [2] registered in the existing public DB and the deleted region of [1] → Primer108_03F (SEQ ID NO: 241) and Primer108_03R (sequence) No. 242) amplified by fragment 108_03 (SEQ ID NO: 243)
108_04-[1], [2] Common area shared by all: [1] cDNA pattern we newly analyzed full-length cDNA sequence, and [2] cDNA registered in the existing public DB A region in which all patterns serving as controls are in common to compare the expression level of the entire pattern → Fragment 108_04 (SEQ ID NO: 246) amplified by Primer108_04F (SEQ ID NO: 244) and Primer108_04R (SEQ ID NO: 245)
The exon regions specific to each of the cDNA patterns [1] and [2] shown above are mapped and compared to the human genome sequence of approximately 1.44 million 5 'end sequences obtained using the oligocap method. As a result, the following results were obtained.
In the newly analyzed cDNA [1] pattern, there are 17 5'-terminal sequences, 14 of which are derived from Synovial membrane tissue from rheumatioid arthritis (analysis parameter 27,489), thymus ( Thymus) had 2 sequences (analysis parameter 70,578), and normal coronary artery smooth muscle cells HCASMC (Human coronary artery smooth muscle cells) had 1 sequence (analysis parameter 8,949).
In the pattern of [2], which is a control registered in the existing public DB, there were 42 5 'terminal sequences, and 42 synovial membrane tissue from rheumatioid arthritis (analysis parameter 27,489) .
From this result, it was found that the expression in synovial membrane tissue from rheumatioid arthritis is high in both patterns [1] and [2]. In other words, in Synovial membrane tissue from rheumatioid arthritis, ORF changes due to the diversity of exon splicing patterns, such as the patterns of [1] and [2], in this chromosomal region, It was found that a mechanism to express different proteins works.

(2) Analysis of expression specificity by real-time PCR In order to know how the diversity of protein expression due to exon selectivity changes in various tissues, the details of the expression level were analyzed by real-time PCR. The results are shown in Table 19 and Table 20.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As an experimental control, a sample (Mix, viscus tissues) mixed with normal visceral tissue described in Example 3 and a whole brain (Brain, whole) were used for comparison.
The newly obtained mRNA expression pattern represented by 108_01 (SEQ ID NO: 240) and the known mRNA expression pattern represented by 108_03 (SEQ ID NO: 243) are both in synovial tissue (rheumatic patients). It was found that splicing selectivity in exon insertion / deletion mRNA was diverse in synovial tissues (rheumatic patients) (Tables 19 and 20). In addition, in the known mRNA expression pattern represented by 108_03 (SEQ ID NO: 243), CD34 + cell ODF (osteoclast differentiation factor) treatment induction 3 days (CD34 + ODF 3days), CD34 + cell ODF treatment induction 9 days (CD34 + ODF) 9 days), and expression was high in the spinal cord.
From these results, selective exon regions 108_ [1] _1-N1 (SEQ ID NO: 227) and 108_ [1] _2-N3 (SEQ ID NO: 227) of the newly obtained cDNA indicated by the detection region 108_01 (SEQ ID NO: 240) By detecting the expression of No. 227), it was proved that it can be used as a diagnostic and therapeutic marker specific to the synovial tissue of rheumatic patients in the blood cell system. It is also possible to develop new drugs using compounds, antibodies, siRNA, etc. that target specific regions.
The following areas are also useful as diagnostic and therapeutic markers specific to the synovial tissue of rheumatic patients, especially in the blood cell system.
The upstream sequence 108_ [1] _1-N3 (SEQ ID NO: 247) containing the 438th to 458th bases primed by Primer108_01R (SEQ ID NO: 239) in D-HCASM2001914.1 of the cDNA pattern of [1].
The upstream sequence 108_ [1] _2-N5 (SEQ ID NO: 248) containing the 315th to 335th bases primed by Primer108_01R (SEQ ID NO: 239) in D-SYNOV4003291.1 of the cDNA pattern of [1].
Region 108_01 (SEQ ID NO: 240) amplified by Primer108_01F (SEQ ID NO: 238) and Primer108_01R (SEQ ID NO: 239) in the cDNA pattern of [1]

Example 15: Cluster chr7 + 710 (data set: 076)
(1) Cluster analysis 1) Cluster characteristics 14 full-length cDNA genome mapped to cluster chr7 + 710 (Human genome UCSC hg18 (NCBI Build34) chromosome 7, 44,810,000 bp to 44,890,000 bp) [D-NETRP2002051.1 , D-THYMU2031383.1, D-THYMU3032318.1, AL832992.1, BC004903.2, BC008859.2, BC016832.2, BC063663.1, BX641167.1, C-HRC00201, C-SYNOV2007758, C-THYMU2024071, ENST00000258781 , NM_031443.2] was analyzed. They are classified into nine types of ORFs depending on the expression pattern, but the main ones are the three types we newly performed full-length cDNA sequence analysis, and one type as a pattern registered in the existing public DB. There were a total of 4 types.
[1] D-NETRP2002051.1
[2] D-THYMU2031383.1
[3] D-THYMU3032318.1
[4] BC004903.2, BC008859.2, BC016832.2, ENST00000258781, NM_031443.2
[1], [2], and [3] are cDNAs that we newly acquired and performed full-length cDNA sequence analysis. The ORF was different from [4] registered in the existing public DB.
[1], [2], [3] are expressed from a chromosomal region downstream of the known [4], and the ORF is located on a new exon that has no homology with [4]. The area was different. [1], [2], and [3] have the same transcription start point and translation start point because of different splicing patterns, but different ORFs.
The ORF regions of the four types of cDNA patterns [1] to [4] have different transcription start points and different splicing patterns from the same chromosomal region. Was found to have been created.

2) Features of D-NETRP2002051.1 ([1]) that we newly acquired and performed full-length cDNA sequence analysis
076_ [1] _1-N0 (SEQ ID NO: 249): The entire nucleic acid sequence of D-NETRP2002051.1
076_ [1] _1-NA0 (SEQ ID NO: 250): Combination of the entire nucleic acid sequence and amino acid sequence of D-NETRP2002051.1
076_ [1] _1-A0 (SEQ ID NO: 251): Full amino acid region of D-NETRP2002051.1
1-126 bases (SEQ ID NO: 252) of D-NETRP2002051.1 is a variant inserted by an exon that does not exist in NM_0314443.2, which is a control registered in the existing public DB, and has homology to NM_0314443.2 The N-terminal amino acid differs by 31 residues (SEQ ID NO: 253) compared to NM_0314443.2 because it exists as a translation start point on the inserted exon.
076_ [1] _1-N1 (SEQ ID NO: 252): 126-base inserted nucleic acid sequence region of D-NETRP2002051.1
076_ [1] _1-A1 (SEQ ID NO: 253): 31-amino acid insertion region of D-NETRP2002051.1
076_ [1] _1-N2 (SEQ ID NO: 254): ORF nucleic acid sequence region in the 126 base insertion region of D-NETRP2002051.1
076_ [1] _1-A2 (same as SEQ ID NO: 253): ORF amino acid region related to 126-base insertion region of D-NETRP2002051.1 Also, NM_0314443.2 286 as a control registered in the existing public DB An exon of 84 bases (SEQ ID NO: 260) present at ˜369 bases is not present in the region of 300-301 bases of D-NETRP2002051.1 and is deleted. Therefore, compared with NM_0314443.2, 28 amino acid residues (SEQ ID NO: 261) were deleted.
076_ [1] _1-N3 (SEQ ID NO: 255): D-NETRP2002051.1 deleted nucleic acid sequence region, part 1
076_ [1] _1-A3 (SEQ ID NO: 256): D-NETRP2002051.1 changing amino acid sequence region
076_ [1] _1-N4 (same as SEQ ID NO: 255): ORF nucleic acid sequence region 076_ [1] _1-A4 (same as SEQ ID NO: 256) in the deleted region of D-NETRP2002051.1: D-NETRP2002051.1 ORF amino acid region related to the deletion region of
076_ [1] _C-N1 (SEQ ID NO: 260): An inserted nucleic acid sequence of 84 bases present in 286 to 369 bases of NM_0314443.2 to be inserted into the region of 300 to 301 bases of D-NETRP2002051.1
076_ [1] _C-A1 (SEQ ID NO: 261): Amino acid region related to the 84-base inserted nucleic acid sequence present in 286-369 bases of NM_0314443.2 inserted in the 300-301 base region of D-NETRP2002051.1 .
In addition, a 137 base exon (SEQ ID NO: 262) present in 554 to 690 bases of NM_0314443.2, which is a control registered in the existing public DB, is present in the region of 484 to 485 bases in D-NETRP2002051.1. It is deleted without. With this change, the translation frame of ORF changes, so the amino acid at the C-terminal differs by 38 residues (SEQ ID NO: 258) compared to NM_0314443.2.
076_ [1] _1-N5 (SEQ ID NO: 257): D-NETRP2002051.1 deleted nucleic acid sequence region, part 2
076_ [1] _1-A5 (SEQ ID NO: 258): D-NETRP2002051.1 changing amino acid sequence region
076_ [1] _1-N6 (identical to SEQ ID NO: 257): ORF nucleic acid sequence region 076_ [1] _1-A6 (SEQ ID NO: 259) in the deleted region of D-NETRP2002051.1: lack of D-NETRP2002051.1 ORF amino acid region related to the loss region
076_ [1] _C-N2 (SEQ ID NO: 262): Nucleic acid sequence of 137 bases present in 554 to 690 bases of NM_0314443.2 inserted in the region of 484 to 485 bases of D-NETRP2002051.1
076_ [1] _C-A2 (SEQ ID NO: 263): Amino acid region related to the 137-base inserted nucleic acid sequence present in 554-690 bases of NM_0314443.2 inserted in the 484-485 base region of D-NETRP2002051.1 .

3) Features of D-THYMU2031383.1 ([2]) that we newly acquired and performed full-length cDNA sequence analysis
076_ [2] _1-N0 (SEQ ID NO: 264): The entire nucleic acid sequence of D-THYMU2031383.1
076_ [2] _1-NA0 (SEQ ID NO: 265): Combination of the entire nucleic acid sequence and amino acid sequence of D-THYMU2031383.1
076_ [2] _1-A0 (SEQ ID NO: 266): D-THYMU2031383.1 entire amino acid sequence region
1-126 bases (SEQ ID NO: 252) of D-THYMU2031383.1 are variants inserted by exons that do not exist in NM_0314443.2, which is a control registered in the existing public DB, and have homology to NM_0314443.2 The N-terminal amino acid differs by 31 residues (SEQ ID NO: 253) compared to NM_0314443.2 because it exists as a translation start point on the inserted exon.
076_ [2] _1-N1 (same as SEQ ID NO: 252): 126-base inserted nucleic acid sequence region of D-THYMU2031383.1
076_ [2] _1-A1 (same as SEQ ID NO: 253): D-THYMU2031383.1 31-residue amino acid sequence region
076_ [2] _1-N2 (same as SEQ ID NO: 254): ORF nucleic acid sequence region in the 126 base insertion region of D-THYMU2031383.1
076_ [2] _1-A2 (same as SEQ ID NO: 253): ORF amino acid region related to 126 base insertion region of D-THYMU2031383.1 Also, 668 to 1,459 bases (SEQ ID NO: 267) of D-THYMU2031383.1 are NM_0331443.2 is a variant inserted by an exon that does not exist in NM_0314443.2, which is a control registered in the existing public DB.There is no homology with NM_0314443.2, and there is a translation termination point on the inserted exon. Compared to 2, the C-terminal amino acid was 49 residues (SEQ ID NO: 268) different.
076_ [2] _1-N3 (SEQ ID NO: 267): 792-base inserted nucleic acid sequence region of D-THYMU2031383.1
076_ [2] _1-A3 (SEQ ID NO: 268): 49-residue insertion amino acid sequence region of D-THYMU2031383.1
076_ [2] _1-N4 (SEQ ID NO: 269): ORF nucleic acid sequence region in the 792-base insertion region of D-THYMU2031383.1
076_ [2] _1-A4 (same as SEQ ID NO: 268): ORF amino acid region related to the insertion region of 792 bases in D-THYMU2031383.1

4) Features of D-THYMU3032318.1 ([3]) that we newly acquired and performed full-length cDNA sequence analysis
076_ [3] _1-N0 (SEQ ID NO: 270): D-THYMU3032318.1 entire nucleic acid sequence region
076_ [3] _1-NA0 (SEQ ID NO: 271): Combination of the entire nucleic acid sequence and amino acid sequence of D-THYMU3032318.1
076_ [3] _1-A0 (SEQ ID NO: 272): D-THYMU3032318.1 whole amino acid sequence region
D-THYMU3032318.1, 1-128 bases (SEQ ID NO: 273) is a variant inserted by an exon that does not exist in NM_0314443.2, which is a control registered in the existing public DB, and has homology to NM_0314443.2 The N-terminal amino acid differs by 31 residues (SEQ ID NO: 253) compared to NM_0314443.2 because it exists as a translation start point on the inserted exon.
076_ [3] _1-N1 (SEQ ID NO: 273): 128-base inserted nucleic acid sequence region of D-THYMU3032318.1
076_ [3] _1-A1 (same as SEQ ID NO: 253): 31-amino acid sequence region of D-THYMU3032318.1
076_ [3] _1-N2 (same as SEQ ID NO: 254): ORF nucleic acid sequence region in the 128-base insertion region of D-THYMU3032318.1
076_ [3] _1-A2 (same as SEQ ID NO: 253): ORF amino acid region related to the 128-base insertion region of D-THYMU3032318.1 Also, 286 of NM_0314443.2, which is a control registered in the existing public DB A 268-base exon (SEQ ID NO: 277) existing at ~ 553 bases is not present in the 302-303 base region of D-THYMU3032318.1 and is deleted. With this change, the translation frame of ORF changes, so the amino acid at the C-terminal was 55 residues (SEQ ID NO: 275) different from NM_0314443.2.
076_ [3] _1-N3 (SEQ ID NO: 274): D-THYMU3032318.1 deleted nucleic acid sequence region
076_ [3] _1-A3 (SEQ ID NO: 275): D-THYMU3032318.1 changing amino acid sequence region
076_ [3] _1-N4 (identical to SEQ ID NO: 274): ORF nucleic acid sequence region 076_ [3] _1-A4 (SEQ ID NO: 276) in the deleted region of D-THYMU3032318.1: lack of D-THYMU3032318.1 ORF amino acid region related to the loss region
076_ [3] _C-N1 (SEQ ID NO: 277): 268-base inserted nucleic acid sequence present in 286-553 bases of NM_0314443.2 inserted in the 302-303 base region of D-THYMU3032318.1
076_ [3] _C-A1 (SEQ ID NO: 278): Amino acid region related to the 268-base inserted nucleic acid sequence present in 286-553 bases of NM_0314443.2 inserted in the 302-303 base region of D-THYMU3032318.1 .

5) Expression specificity analysis and real-time PCR primer design
The ORF regions of the four types of cDNA patterns [1] to [4] can be classified into two types, focusing on the transcription start site region. It seemed possible to compare the expression from the starting point.
076_01-Specific region existing on the N-terminal side of the cDNA pattern of [1] and [3]: In the transcription start region of the cDNA pattern of [1] and [3] that we newly performed full-length cDNA sequence analysis There is a novel region that is not registered in the existing public DB → Fragment 076_01 (SEQ ID NO: 281) amplified by Primer076_01F (SEQ ID NO: 279) and Primer076_01R (SEQ ID NO: 280)
076_02-Control to compare transcription start point region [1], [2], [3] with [4] registered in existing public DB → Primer076_02F (SEQ ID NO: 282) and Primer076_02R ( SEQ ID NO: 283) Fragment 076_02 amplified by (SEQ ID NO: 284)
076_04-Common region shared by [1], [2], [4]: The cDNA pattern of [1], [2], which we newly performed full-length cDNA sequence analysis, and registered in the existing public DB A common region serving as a control for comparing the expression levels of the cDNA patterns of [4] → Fragment 076_04 (SEQ ID NO: 287) amplified by Primer076_04F (SEQ ID NO: 285) and Primer076_04R (SEQ ID NO: 286)
The exon regions specific to each of the cDNA patterns [1] to [4] shown above are mapped and compared with the human genome sequence of approximately 1.44 million 5 'end sequences obtained using the oligocap method. As a result, the following results were obtained.
In the newly analyzed cDNA [1] pattern, there are two 5 'end sequences, one from neutrophils (analysis parameter 9,170) and one from spleen (Spleen). (Analysis parameter 33,950).
In the newly analyzed cDNA [2] pattern, there are 7 5 'terminal sequences, and the origin is 5 sequences of thymus (analysis parameter 70,578) and 1 sequence of spleen (analysis). The parameter was 33,950), and the thalamus (Brain, thalamus) was one sequence (analysis parameter 53,267).
In the newly analyzed cDNA pattern [3], there was one 5 'terminal sequence, which was derived from one thymus (analytical parameter 70,578).
In the pattern [4], which is a control registered in the existing public DB, there was no 5 'terminal sequence.
From these results, it was found that the transcription start points of [1], [2], and [3] are highly expressed in the thymus (Symus) and spleen (Spleen). In other words, in the thymus (Thymus) and spleen (Spleen), in this chromosomal region, the transcription start site located downstream on the chromosome like the patterns [1], [2], [3] should be used selectively. It was found that the transcription mechanism works.

(2) Analysis of expression specificity by real-time PCR In order to know what kind of tissue in the blood cell system tissue and in what state the transcription start point used for expression changes, details of the expression level are real-time Analyzed by PCR. The results are shown in Table 21.

Expression levels were compared using 12 types of samples such as blood cell tissues described in Example 3, CD34 + cells, and cells in which CD34 + cells were induced to be treated with osteoclast differentiation factor (ODF). As an experimental control, a sample (Mix, viscus tissues) mixed with normal visceral tissue described in Example 3 and a whole brain (Brain, whole) were used for comparison.
Depending on the selectivity of the transcription start site compared between 076_01 (SEQ ID NO: 281) and 076_02 (SEQ ID NO: 284), the mRNA changes, and as a result, the ratio of ORF changes greatly in the following tissues and cells.
Synovial tissue (rheumatoid patients), thymus (Bone Marrow), spleen (Spleen) blood cell tissue, from the downstream transcription start point represented by 076_01 (SEQ ID NO: 281) There were many transcripts (mRNA) (Table 21). In the brain, there were many transcripts (mRNA) from the known transcription start point represented by 076_02 (SEQ ID NO: 284) (Table 21).
When CD34 + cells were induced by osteoclast differentiation factor (ODF) treatment, the differentiation stage was compared, and it was found that the selectivity of the transcription start point greatly changed depending on the stage of differentiation. There were many transcripts (mRNA) from the downstream transcription start point represented by 076_01 (SEQ ID NO: 281) until 3 days (CD34 + ODF 3days) of CD34 + cells in the early stage of differentiation. CD34 + cells in ODF treatment induction 6 days (CD34 + ODF 6days) and CD34 + cells in ODF treatment induction 9 days (CD34 + ODF 9days) reversed the selectivity, and the known transcription start point represented by 076_02 (SEQ ID NO: 284) There were many transcripts (mRNA) from (Table 21).
In other words, mRNAs with different ORFs were expressed by different transcription factors or transcription mechanisms in blood cell tissues and brains due to the diversity of transcription start points. Furthermore, in osteoclasts, it was found that the selected transcription start point changes as differentiation progresses.
From these results, the 5′-terminal region of the newly obtained cDNA region indicated by the detection region of 076_01 (SEQ ID NO: 281) (region near the transcription start point) 076_ [1] _1-N1 (SEQ ID NO: 252) , 076_ [2] _1-N1 (SEQ ID NO: 252), 076_ [3] _1-N1 (SEQ ID NO: 273) are compared with each other in the synovial tissue of the rheumatic patients. As a therapeutic / diagnostic marker, differentiation into osteoclasts, especially as a therapeutic / diagnostic marker (diagnostic / therapeutic marker for osteoporosis / rheumatism and the process leading to it) to detect the early stages of differentiation, as well as thymus, bone marrow It has been proved that it can be used as a marker specific to the spleen blood cell system. In addition, new drug development using compounds, antibodies, siRNA, etc. that target specific regions is also possible.
In addition, the following areas are also considered as therapeutic / diagnostic markers specific to the blood cell system of synovial tissue in rheumatic patients, as differentiation / intestinal differentiation, particularly as a therapeutic / diagnostic marker for detecting the early stages of differentiation, and It seems to be useful as a specific marker for the blood cell system of thymus, bone marrow, and spleen.
The upstream sequence 076_ [1] _1-N7 (SEQ ID NO: 288) containing the 149th to 174th bases primed by Primer076_01R (SEQ ID NO: 280) in D-NETRP2002051.1 having the cDNA pattern of [1].
The upstream sequence 076_ [3] _1-N5 (SEQ ID NO: 289) containing the 151st to 176th bases primed by Primer076_01R (SEQ ID NO: 280) in D-THYMU3032318.1 of the cDNA pattern of [3].
Region 076_01 (SEQ ID NO: 281) amplified by Primer076_01F (SEQ ID NO: 279) and Primer076_01R (SEQ ID NO: 280) in the cDNA pattern of [1] and [3]

Example 16: Results of ORF information and homology analysis of full-length cDNA sequences and analysis results of motifs, etc. In order to know the functions of 18 full-length cDNAs that we newly acquired and performed full-length cDNA sequence analysis, ORF prediction and annotation analysis were performed. went. The annotation analysis result can be updated when the database or analysis software to be compared is upgraded. As a result, it may be possible to add a new annotation under the same condition even if no annotation is described.

1) Predicting the ORF of cDNA analyzed by full-length cDNA sequence
Full-length cDNA sequences using ORF prediction and evaluation systems such as ATGpr (A. Salamov et al. (1998) Bioinformatics 14: 384-390) and TRins (K. Kimura et al. (2003) Genome Informatics 14: 456-457) Predicted the ORF. The ORF region information predicted from the full-length cDNA sequence is shown below.
The description method of the ORF region is described in accordance with the rules of “DDBJ / EMBL / GenBank Feature Table Definition” (http://www.ncbi.nlm.nih.gov/collab/FT/index.html). The ORF start position is the first letter of “ATG”, which is the base encoding methionine, and the stop position is the third letter of the stop codon. This is delimited by “..”. However, for ORFs in which no stop codon appears, the stop position is described using “>” in accordance with the description rules.
--------------
cDNA sequence name ORF region
--------------
D-SYNOV4003692.1 176..1126
D-D3OST2001534.1 288..1919
D-NETRP1000046.1 57..950
D-PLACE7010497.1 34..1875
D-SPLEN2030304.1 89..1768
D-SYNOV4000324.1 45..986
D-SYNOV4007243.1 202..2526
D-NETRP2004161.1 290..1240
D-NT2RI3001993.1 63..3206
D-SMINT2013276.1 93..2036
D-THYMU3006588.1 87..2723
D-D3OST2003177.1 163..2718
D-D3OST2000831.1 178..1620
D-HCASM2001914.1 46 ..> 2604
D-SYNOV4003291.1 46 ..> 693
D-NETRP2002051.1 34..579
D-THYMU2031383.1 34..816
D-THYMU3032318.1 36..470
--------------

2) Results of homology analysis using BLASTP (SwissProt)
Using the BLASTP (blastall 2.2.6; ftp://ftp.ncbi.nih.gov/blast/) for the 18-sequence ORF described in 1) of Example 16, the version of SwissProt of August 22, 2006 ( Homology analysis was performed on ftp://us.expasy.org/databases/swiss-prot/). Those having the highest homology than the results of the homology analysis and showing the homology with an E-value of 1E-10 or less are described below. However, when the following conditions are met, the next candidate is described without selecting the corresponding candidate.
Definitions beginning with "ALU SUBFAMILY" Definitions starting with "Alu subfamily" Definitions starting with "!!!! ALU SUBFAMILY" Definitions starting with "B-CELL GROWTH FACTOR PRECURSOR" Definitions including "NRK2" Definitions starting with "PROLINE-RICH" Definitions starting with "GLYCINE-RICH" Definitions starting with "EXTENSIN PRECURSOR" Definitions starting with "COLLAGEN" Definitions starting with "100KD""RETROVIRUS- Definitions that start with "RELATED POL POLYPROTEIN" Definitions that start with "CUTICLE COLLAGEN" Definitions that start with "HYPOTHETICAL" Definitions that start with "Hypothetical" Definitions that start with "SALIVARY PROLINE-RICH PROTEIN""IMMEDIATE-EARLY" Definition data beginning with "PROTEIN" Accession number "P49646" Each data is the cDNA sequence name, ORF region, hit data accession number Of hit data Definition, keyword of hit data, E-value, consensus length (amino acids in length), as described, separated by "//" in the order of Identity.
D-SYNOV4003692.1 // 176..1126 // O75030 // Microphthalmia-associated transcription factor // Activator; Albinism; Alternative splicing; Deafness; Developmental protein; Disease mutation; DNA-binding; Nuclear protein; Phosphorylation; Transcription; Transcription regulation; Waardenburg syndrome.// 7E-60 // 158 // 46
D-D3OST2001534.1 // 288..1919 // P28039 // Acyloxyacyl hydrolase precursor (EC 3.1.1.77) [Contains: Acyloxyacyl hydrolase small subunit; Acyloxyacyl hydrolase large subunit] // Direct protein sequencing; Glycoprotein; Hydrolase; Polymorphism; Signal; Zymogen.// 0 // 543 // 94
D-NETRP1000046.1 // 57..950 // P28039 // Acyloxyacyl hydrolase precursor (EC 3.1.1.77) [Contains: Acyloxyacyl hydrolase small subunit; Acyloxyacyl hydrolase large subunit] // Direct protein sequencing; Glycoprotein; Hydrolase; Polymorphism; Signal; Zymogen.// 1E-176 // 293 // 100
D-PLACE7010497.1 // 34..1875 // P53667 // LIM domain kinase 1 (EC 2.7.11.1) (LIMK-1) // Alternative splicing; ATP-binding; Kinase; LIM domain; Metal-binding; Nucleotide -binding; Phosphorylation; Repeat; Serine / threonine-protein kinase; Transferase; Williams-Beuren syndrome; Zinc.// 0 // 596 // 99
D-SPLEN2030304.1 // 89..1768 // P29074 // Tyrosine-protein phosphatase non-receptor type4 (EC 3.1.3.48) (Protein-tyrosine phosphatase MEG1) (PTPase-MEG1) (MEG) // 3D-structure ; Cytoskeleton; Hydrolase; Protein phosphatase; Structural protein.// 0 // 558 // 99
D-SYNOV4000324.1 // 45..986 // P16333 // Cytoplasmic protein NCK1 (NCK adaptor protein1) (SH2 / SH3 adaptor protein NCK-alpha) // 3D-structure; Direct protein sequencing; Phosphorylation; Repeat; SH2 domain ; SH3 domain.// 1E-180 // 303 // 99
D-SYNOV4007243.1 // 202..2526 // Q01432 // AMP deaminase 3 (EC 3.5.4.6) (AMP deaminaseisoform E) (Erythrocyte AMP deaminase) // Alternative splicing; Hydrolase; Nucleotide metabolism; Polymorphism.// 0 // 767 // 100
D-NETRP2004161.1 // 290..1240 // Q6UX41 // Butyrophilin-like protein 8 precursor // Alternative splicing; Direct protein sequencing; Glycoprotein; Immunoglobulin domain; Membrane; Repeat; Signal; Transmembrane.// 0 // 315 // 99
D-NT2RI3001993.1 // 63..3206 // Q96MK2 // Uncharacterized protein C20orf175 // // 1E-99 // 245 // 38
D-SMINT2013276.1 // 93..2036 // Q96MK2 // Uncharacterized protein C20orf175 // // 7E-96 // 232 // 38
D-THYMU3006588.1 // 87..2723 // Q96M96 // FYVE, RhoGEF and PH domain-containing protein4 (Actin filament-binding protein frabin) (FGD1-relatedF-actin-binding protein) (Zinc finger FYVEdomain-containing protein 6) // Actin-binding; Alternative splicing; Cytoskeleton; Guanine-nucleotide releasing factor; Metal-binding; Repeat; Zinc; Zinc-finger.// 0 // 764 // 99
D-D3OST2003177.1 // 163..2718 // Q96M96 // FYVE, RhoGEF and PH domain-containing protein4 (Actin filament-binding protein frabin) (FGD1-relatedF-actin-binding protein) (Zinc finger FYVEdomain-containing protein 6) // Actin-binding; Alternative splicing; Cytoskeleton; Guanine-nucleotide releasing factor; Metal-binding; Repeat; Zinc; Zinc-finger.// 0 // 765 // 99
D-HCASM2001914.1 // 46 ..> 2604 // Q92954 // Proteoglycan-4 precursor (Lubricin) (Megakaryocyte-stimulating factor) (Superficial zoneproteoglycan) (Contains: Proteoglycan-4 C-terminal part] // Alternative splicing; Glycoprotein; Polymorphism; Proteoglycan; Repeat; Signal.// 0 // 744 // 99
D-SYNOV4003291.1 // 46 ..> 693 // Q92954 // Proteoglycan-4 precursor (Lubricin) (Megakaryocyte-stimulating factor) (Superficial zoneproteoglycan) (Contains: Proteoglycan-4 C-terminal part] // Alternative splicing; Glycoprotein; Polymorphism; Proteoglycan; Repeat; Signal.// 2E-82 // 151 // 99
D-NETRP2002051.1 // 34..579 // Q9BSQ5 // Malcavernin (Cerebral cavernous malformations2 protein) // Disease mutation; Polymorphism.// 9E-59 // 120 // 78
D-THYMU2031383.1 // 34..816 // Q9BSQ5 // Malcavernin (Cerebral cavernous malformations2 protein) // Disease mutation; Polymorphism.// 4E-99 // 181 // 98
D-THYMU3032318.1 // 36..470 // Q9BSQ5 // Malcavernin (Cerebral cavernousmalformations 2 protein) // Disease mutation; Polymorphism.// 5E-27 // 58 // 98

3) Results of homology analysis using BLASTP (RefSeq)
Using the BLASTP (blastall 2.2.6; ftp://ftp.ncbi.nih.gov/blast/) for the 18-sequence ORF described in 1) of Example 16, the version of RefSeq of July 15, 2006 ( Human, mouse, rat; ftp://ftp.ncbi.nih.gov/refseq/). Those having the highest homology than the results of the homology analysis and showing the homology with an E-value of 1E-10 or less are described below. However, when the following conditions are met, the next candidate is described without selecting the corresponding candidate.
Definitions starting with "hypothetical protein FLJ" Definitions starting with "KIAA" Definitions starting with "hypothetical protein DKFZ" Definitions starting with "DKFZ" Definitions starting with "RIKEN cDNA""hypothetical protein MGC" Definitions starting with "hypothetical protein" Definitions starting with "hypothetical protein PP" Definitions starting with "hypothetical protein PP" Definitions starting with "neuronal thread protein" Definitions starting with "clone FLB" Starting with "hypothetical protein PRO" Definitions including "PRO0483 protein" Definitions including "MNC" Definitions including "MOST-1" Definitions starting with "similar to" Definitions including "TPR gene on Y" Definition data beginning with “HSPC” Definition data starting with “CGI-” Each data includes cDNA sequence name, ORF region, hit data accession number, hit number Data Definition, E-value, consensus length (amino acids in length), as described, separated by "//" in the order of Identity.
D-SYNOV4003692.1 // 176..1126 // NP_036384.1 // transcription factor EC isoform a [Homo sapiens] // 1E-125 // 221 // 100
D-D3OST2001534.1 // 288..1919 // NP_001628.1 // acyloxyacyl hydrolase precursor [Homo sapiens] // 0 // 543 // 94
D-NETRP1000046.1 // 57..950 // NP_001628.1 // acyloxyacyl hydrolase precursor [Homo sapiens] // 1E-177 // 293 // 100
D-PLACE7010497.1 // 34..1875 // NP_002305.1 // LIM domain kinase 1 [Homo sapiens] // 0 // 596 // 99
D-SPLEN2030304.1 // 89..1768 // NP_002821.1 // protein tyrosine phosphatase, non-receptor type 4 [Homo sapiens] // 0 // 558 // 99
D-SYNOV4000324.1 // 45..986 // NP_006144.1 // NCK adaptor protein 1 [Homo sapiens] // 0 // 303 // 99
D-SYNOV4007243.1 // 202..2526 // NP_001020561.1 // erythrocyte adenosine monophosphate deaminase isoform 1C [Homo sapiens] // 0 // 774 // 100
D-NETRP2004161.1 // 290..1240 // NP_001035552.1 // butyrophilin-like 8 long form [Homo sapiens] // 0 // 315 // 99
D-THYMU3006588.1 // 87..2723 // NP_640334.1 // FYVE, RhoGEF and PH domain containing 4 [Homo sapiens] // 0 // 764 // 99
D-D3OST2003177.1 // 163..2718 // NP_640334.1 // FYVE, RhoGEF and PH domain containing 4 [Homo sapiens] // 0 // 765 // 99
D-HCASM2001914.1 // 46 ..> 2604 // NP_005798.2 // proteoglycan 4 [Homo sapiens] // 0 // 744 // 99
D-SYNOV4003291.1 // 46 ..> 693 // NP_005798.2 // proteoglycan 4 [Homo sapiens] // 1E-82 // 151 // 99
D-NETRP2002051.1 // 34..579 // NP_001025006.1 // cerebral cavernous malformation 2 isoform 1 [Homo sapiens] // 2E-78 // 151 // 82
D-THYMU2031383.1 // 34..816 // NP_113631.1 // cerebral cavernous malformation 2 isoform 2 [Homo sapiens] // 2E-99 // 181 // 98
D-THYMU3032318.1 // 36..470 // NP_001025006.1 // cerebral cavernous malformation 2 isoform 1 [Homo sapiens] // 9E-47 // 89 // 100

4) Results of motif homology analysis using Pfam Motif homology using Pfam (ftp://ftp.sanger.ac.uk/pub/databases/Pfam/) for the 18-sequence ORF described in Example 16 1) Analysis was performed. Hmmpfam v2.3.2 was used as the analysis program, and analysis was performed on the November 2005 version of Pfam19.0. Those having the highest homology than the results of the homology analysis and showing the homology with an E-value of 1E-10 or less are described below.
Each data consists of the cDNA sequence name, ORF region, hit data accession number, hit data name, hit data description, E-value, and InterPro ID in the order of “\”. It was repeated repeatedly with just “//” separators.
D-SYNOV4003692.1 // 176..1126 // PF00010.15 \ HLH \ Helix-loop-helix DNA-binding domain \ 1.8e-15 \ IPR001092
D-D3OST2001534.1 // 288..1919 // PF00657.11 \ Lipase_GDSL \ GDSL-like Lipase / Acylhydrolase \ 1e-39 \ IPR001087
D-NETRP1000046.1 // 57..950 // PF00657.11 \ Lipase_GDSL \ GDSL-like Lipase / Acylhydrolase \ 8.5e-29 \ IPR001087
D-PLACE7010497.1 // 34..1875 // PF00069.14 \ Pkinase \ Protein kinase domain \ 4.6e-49 \ IPR000719 // PF07714.5 \ Pkinase_Tyr \ Protein tyrosine kinase \ 1.8e-48 \ // PF00412. 11 \ LIM \ LIM domain \ 1e-21 \ IPR001781 // PF00595.12 \ PDZ \ PDZ domain (Also known as DHR or GLGF) \ 4.7e-19 \ IPR001478
D-SPLEN2030304.1 // 89..1768 // PF00102.16 \ Y_phosphatase \ Protein-tyrosine phosphatase \ 2.9e-110 \ IPR000242 // PF00595.12 \ PDZ \ PDZ domain (Also known as DHR or GLGF) \ 4.5 e-25 \ IPR001478
D-SYNOV4000324.1 // 45..986 // PF00017.12 \ SH2 \ SH2 domain \ 8.8e-34 \ IPR000980 // PF00018.16 \ SH3_1 \ SH3 domain \ 5.3e-23 \ IPR001452 // PF07653.5 \ SH3_2 \ Variant SH3 domain \ 1.3e-11 \ IPR011511
D-SYNOV4007243.1 // 202..2526 // PF00962.11 \ A_deaminase \ Adenosine / AMP deaminase \ 4.1e-232 \ IPR001365
D-NETRP2004161.1 // 290..1240 // PF00622.17 \ SPRY \ SPRY domain \ 6e-30 \ IPR003877
D-THYMU3006588.1 // 87..2723 // PF00621.10 \ RhoGEF \ RhoGEF domain \ 5e-59 \ IPR000219 // PF01363.12 \ FYVE \ FYVE zinc finger \ 4.9e-24 \ IPR000306 // PF00169.17 \ PH \ PH domain \ 4.3e-18 \ IPR001849
D-D3OST2003177.1 // 163..2718 // PF00621.10 \ RhoGEF \ RhoGEF domain \ 2.2e-61 \ IPR000219 // PF01363.12 \ FYVE \ FYVE zinc finger \ 4.9e-24 \ IPR000306 // PF00169. 17 \ PH \ PH domain \ 4.3e-18 \ IPR001849
D-HCASM2001914.1 // 46 ..> 2604 // PF01033.7 \ Somatomedin_B \ Somatomedin B domain \ 1.1e-19 \ IPR001212
D-SYNOV4003291.1 // 46 ..> 693 // PF01033.7 \ Somatomedin_B \ Somatomedin B domain \ 1.3e-16 \ IPR001212

5) Transmembrane domain prediction analysis by SOSUI Using SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/) for the 18-sequence ORF described in 1) of Example 16, transmembrane domain Predictive analysis was performed. SOSUI version 1.5 was used for the analysis. What can be predicted for the transmembrane domain in the SOSUI analysis is described below.
In each data, the cDNA sequence name, ORF region, and transmembrane domain number are described in “//” delimiters.
D-NETRP2004161.1 // 290..1240 // 1
D-D3OST2000831.1 // 178..1620 // 10
D-HCASM2001914.1 // 46 ..> 2604 // 1
D-SYNOV4003291.1 // 46 ..> 693 // 1

6) N-terminal secretion signal sequence prediction analysis by PSORT N-terminal secretion signal sequence prediction was performed using PSORT (http://psort.nibb.ac.jp/) for the 18-sequence ORF described in Example 16 1). went. PSORT II was used for the analysis. The following describes the N-terminal secretory signal sequence that could be predicted in the PSORT analysis.
Each data is described as a cDNA sequence name and an ORF region separated by “//”.
D-D3OST2001534.1 // 288..1919
D-HCASM2001914.1 // 46 ..> 2604
D-SYNOV4003291.1 // 46 ..> 693

7) N-terminal secretion signal sequence prediction analysis by SignalP ver.3.0 Using SignalP (http://www.cbs.dtu.dk/services/SignalP/) for the 18-sequence ORF described in Example 16 1), N-terminal secretion signal sequence prediction was performed. SignalP version 3.0 was used for the analysis. What can predict the N-terminal secretory signal sequence in SignalP analysis is described below.
Each data is described as a cDNA sequence name and an ORF region separated by “//”.
D-D3OST2001534.1 // 288..1919
D-HCASM2001914.1 // 46 ..> 2604
D-SYNOV4003291.1 // 46 ..> 693

(Overview for Examples 1-16)
Advances in human genome research have greatly advanced the analysis of human chromosome sequences, but not all of the functions of human genes have been revealed. By analyzing human genes with emphasis on their diversity, we have clarified that diversity is greatly related to changes in gene function.
By comparing the sequence information of the human genome with the human cDNA data transcribed from it, it was found that multiple mRNAs were transcribed from a certain region of the chromosome. There is a difference in the ORF region that is predicted to encode a protein and it is predicted that a different protein will be produced, and the difference is in the 5'UTR region and 3'UTR region that are untranslated regions, and the same protein as the protein There was a case. We searched and sequenced such cDNAs, with particular emphasis on cDNAs that are predicted to encode proteins that are different from known cDNAs that have already been analyzed. Then, it was found that the causes of diversity were mainly transcription start point selectivity and exon selectivity. In terms of the selectivity of the transcription start point, the transcription factor used in a certain chromosomal region changed, and thus the transcription start position was different, resulting in diversity in cDNA. In addition, in terms of exon selectivity, when transcription and splicing occurred, diversity occurred in cDNA by increasing or decreasing the number of exons used even if they were transcribed from the same chromosomal region.
We analyzed how gene diversity is related to function based on mRNA expression frequency information based on the 5 'end sequence (5'-onepass sequence) of about 1.5 million human cDNAs. We found many examples where diversity seems to have a significant effect on function, such as selective transcriptional regions differing only in certain organs or exon deletion only in certain pathological conditions. We found and analyzed in detail the genes that show changes in mRNA diversity in the differentiation stage of blood cell-derived osteoclasts, synovial tissue of rheumatic patients, and blood cell organ tissues.
As an analysis method, the expression levels were compared using real-time PCR (polymerase chain reaction). For example, if there is an exon that is predicted to be selectively inserted only when CD34-positive (CD34 +) cells that are blood cells differentiate into multinucleated osteoclasts, Primer that specifically detects the exon region ( 01) is designed, and Primer (02) that specifically detects only the pattern in which the exon is not inserted is designed, and Primer (03) is also designed to detect a region shared by both patterns. Using these three types of Primer, we compare the amount of amplification at each stage of osteoclast differentiation. By comparing the amount of amplification of 03 in the shared region as a control and comparing the amount of amplification by stage of osteoclast differentiation of 01 and 02, which is the detection result of specific regions, the selectivity of exon You can see how it has changed due to differentiation. In other words, the correlation between exon selectivity and tissue-specific expression can be observed.
With this method, we have found many genes whose gene diversity is associated with tissue-specific expression. The specificity of the expressed tissue suggests that diversity greatly affects the function of the gene. In other words, it is possible to detect in detail the differentiation stage of osteoclasts by using a specific region where diversity occurs as a genetic marker, or to investigate the pathology of blood cell tissues such as synovial tissue. It seems that it will be possible to know the situation. Furthermore, for example, by proceeding with the development of a drug targeting a protein having a specific region expressed from a blood cell system tissue, it is considered possible to develop a drug having a higher effect with fewer side effects.

<Explanation of in vitro osteoclast formation method and osteoclast>
1) Osteoclasts Osteoclasts are multinucleated cells derived from monocyte and macrophage progenitor cells, and not only in normal bone remodeling but also in pathological bone resorption such as rheumatoid arthritis and bone metastasis of cancer. It plays a central role (Miyamoto, T. and Suda, T., Keio J Med. (2003) 52: 1-7; Udagawa, N. et al., Arthritis Res. (2002) 4: 281-289 ). One reason for the lack of research on osteoclasts was that osteoclasts were non-dividing cells and there were no cell lines. Recently, however, it has become possible to differentiate single cells into osteoclasts.

2) Mouse osteoclast formation system
In 1988, Takahashi et al. Discovered that osteoclast-like multinucleated cells were formed by culturing mouse bone marrow cells in the presence of bone resorption factors such as active vitamin D3 and parathyroid hormone (Takahashi, N. et al., Endocrinology (1988) 123: 1504-1510). The multinucleated cells formed by this method are tartrate-resistant acid phosphatase (TRAP) -positive, have many calcitonin receptors, and have properties as osteoclasts such as forming resorption pits on dentin slices. We were all satisfied. The osteoclast-like cells formed at this time are formed close to alkaline phosphatase-positive osteoblast-like stromal cells, so they differentiate from osteocytic cells to osteoclasts. I thought that it might support. Therefore, when osteoblasts obtained from the mouse skull and splenocytes, which are purely blood cells, were cocultured, osteoclast-like cells were formed in the presence of bone resorption factor as expected (Takahashi , N. et al., Endocrinology (1988) 123: 2600-2602). Interestingly, when osteoblasts and spleen cells are cultured alone, osteoclasts are not formed even in the presence of bone resorption factor, and direct contact between osteoblasts and osteoclast precursor cells is osteoclast formation. It became clear that it was essential. The role of osteoblasts at this time has been unknown for some time, but recently the macrophage colony-stimulating factor (M-CSF) produced by osteoblasts (Yoshida, H. et al., Nature ( 1990) 345: 442-444) and Osteoclast Differentiation Factor (ODF) (Yasuda, H. et al., Proc. Natl. Acad. Sci. USA (1998) 95: 3597-3602) are extremely important in osteoclast differentiation. It became clear that it played an important role.

3) Human osteoclast formation system
Since 1998 when Yasuda et al. (Yasuda, H. et al., Proc. Natl. Acad. Sci. USA (1998) 95: 3597-3602) have cloned ODF, research into osteoclast differentiation by single cells has been conducted. Advanced, it was found by the group of Suda et al. That multinucleated osteoclasts were formed about 10 days after treatment of human cord blood-derived CD34 positive (CD34 +) cells with M-CSF and ODF. Although these cells proliferate as macrophages, they can be efficiently differentiated into osteoclasts (with high purity) by adjusting the differentiation conditions.

4) Preparation of cDNA library from differentiation-inducing cells and gene expression analysis When CD34 + cells derived from human cord blood are treated with M-CSF and ODF, multinucleated osteoclasts are formed on the 10th day. Human cord blood-derived CD34 + cells were purchased from AllCells, LLC, CB-CD34 +, cultured for 3 weeks, and then differentiated with ODF. After differentiation induction, looking at the status of each differentiation stage, cells that differentiated into multinucleated osteoclasts were seen on the 6th day, and most were differentiated into multinucleated osteoclasts on the 9th day. After this undifferentiation and differentiation induction, a cDNA library was prepared by the improved oligocap method from the 3rd, 6th and 9th, and the gene expression frequency analysis was performed by analyzing the 5'-end sequence. By comparing the results with other 5'-end sequences from other cDNA libraries, cDNA with expression specificity was obtained and further analysis was performed.

5) Genes and mRNAs related to osteoporosis
Osteoporosis is a condition in which the composition of bone has decreased as a whole and fractures have become easier, but the onset of the disease is the balance between the action of osteoblasts that produce bone and osteoclasts that absorb bone, that is, bone. Metabolism is involved. Therefore, it can be said that the gene / mRNA related to the increase of osteoclasts differentiated from monocyte / macrophage progenitor cells is a gene / mRNA related to osteoporosis related to bone metabolism.

<CD34 positive (CD34 +) cells>
CD34 + cells are a cell fraction rich in human blood stem cells. CD34 is a membrane protein having a molecular weight of about 110 kDa, and is expressed in about 1% of human bone marrow cells, and is considered to function as an adhesion molecule. About one-third of CD34 + cells form colonies in vitro. Human CD34 + cells can also differentiate into T cells, and human hematopoietic stem cells have been shown to express CD34. At present, hematopoietic stem cell transplantation for transplanting CD34 + cells is also in practical use. Hematopoietic stem cells are also present in human peripheral blood and umbilical cord blood, and CD34 + cells are said to be about 0.01% and 0.3%, respectively.

<Description of synovial tissue and rheumatoid arthritis>
The joint is the part that connects the bones, and the slight gap (joint cavity) is filled with sticky joint fluid (synovial fluid) so that the hard bones do not collide directly, and the tip of the bone is elastic A cartilage covers and acts as a cushion. Cartilage is a soft and soft bone containing 80% water, and contains a fiber component called collagen to maintain its strength. Cartilage changes with age and weight. The entire joint is wrapped in an articular follicle, and the synovium inside it secretes joint fluid, smoothing the movement of the joint and replenishing nutrition. When inflammation occurs in the synovium, swelling and pain occur in the joint.
Symptoms characteristic of rheumatoid arthritis are joint swelling and pain. The cause of this swelling and pain is inflammation in the joints. “Inflammatory response” is one of the protective responses to repair when a tissue is damaged. Synovial inflammation is caused by immune cells beginning to attack themselves. 1) Immune cells such as T cells and neutrophils enter the synovium. 2) Rheumatoid factor appears in the synovium and spills into the joint fluid. 3) Rheumatoid factor binds to an antibody called IgG to form an “immune complex”, and further binds to a protein called complement to cause neutrophils and macrophages. 4) Neutrophils attack the “immune complex” by producing substances such as lysosomes and prostaglandins. These substances and enzymes cause inflammation in the synovium and destroy bone and cartilage. A large amount of joint fluid is secreted and the joint swells and hurts. When T cells and B cells participate in the attack, the inflammation further progresses and becomes chronic. When the inflammation of the synovium becomes chronic, joint deformation gradually occurs. Since bone destruction and deformation cannot be reversed, it is important to treat with early detection and stop inflammation.
The stage of rheumatoid arthritis is as follows. Stage 1 (Initial): When the joint fluid increases in the joint cavity, the calcium content of the bones is lost, and bone destruction has not yet occurred, but there may be bone atrophy such that the bones are filled. Stage 2 (moderate): Synovial cells proliferate to form granulation (pannus), granulation begins to erode cartilage and subchondral bone, and bone erosion is observed. Atrophy is seen in the muscles around the joint. Stage 3 (advanced): The cartilage erodes and the bones are destroyed, causing the joints to not mesh properly, resulting in joint deformation and subluxation / dislocation. Strong atrophy is seen in the muscles, and the muscles and tendons stretch and become deformed. Stage 4 (end stage): The granulation becomes fibrotic and hard, the ends of the two bones stick together and become a bone fusion state, and the joint cannot move at all.

<Bone marrow>
Bone marrow is a soft tissue that exists in bone. The bone marrow is rich in blood, and there are hematopoietic stem cells that can differentiate into all blood cells (red blood cells, white blood cells, lymphocytes, megakaryocytes that form platelets, etc.). In mice, it has been proven that all hematopoietic cells can be reconstituted by transplanting a single hematopoietic stem cell. In humans, bone marrow transplantation is effective as a radical treatment for hematopoietic diseases such as leukemia. There may be.

<Thymus>
The thymus is an organ involved in the immune system that resides in the thoracic cavity. It works actively until puberty, but then becomes smaller and eventually fat. It has a role to differentiate T lymphocytes in the thymus. However, since T lymphocytes cannot gradually differentiate due to age-related atrophy, autoantibodies are made with age and destroy normal cells, which is considered to be a cause of aging.

<Spleen>
The spleen is an organ that intervenes in the splenic artery and splenic vein. The function of the spleen is as follows. Immune function: Making lymphocytes in the white pulp. Hematopoietic function: Red blood cells are made in the spleen during the embryonic period until hematopoiesis begins in the bone marrow. Its function is lost after birth, but hematopoiesis may occur again in the spleen if massive bleeding or bone marrow function is suppressed. Blood cell destruction: Destruction of old red blood cells. Hemoglobin in erythrocytes is also destroyed and has the function of recovering iron. Blood storage function: Has the function of storing blood. Humans do not store much blood. When exercising your muscles needing a lot of oxygen, you can deliver enough oxygen to your muscles by expelling the blood stored in your spleen.

<Jurkat cells>
It is a human T cell line (T-cell leukemia).

  This application is based on Japanese Patent Application No. 2007-066434 (filing date: March 15, 2007) filed in Japan, the contents of which are incorporated in full herein.

Claims (27)

The polypeptide of any one of 1) to 12) below or a specific partial peptide thereof:
1) a polypeptide having the amino acid sequence represented by SEQ ID NO: 10 or an amino acid sequence substantially identical thereto;
2) An amino acid sequence represented by SEQ ID NO: 30 or 37, an amino acid sequence consisting of amino acid residues 24 to 543 in the amino acid sequence represented by SEQ ID NO: 30, or an amino acid sequence substantially identical to them A polypeptide having;
3) a polypeptide having the amino acid sequence represented by SEQ ID NO: 57 or a substantially identical amino acid sequence thereto;
4) a polypeptide having the amino acid sequence represented by SEQ ID NO: 73 or an amino acid sequence substantially identical thereto;
5) A polypeptide having the amino acid sequence represented by SEQ ID NO: 88, or a substantially identical amino acid sequence thereof;
6) a polypeptide having the amino acid sequence represented by SEQ ID NO: 104 or a substantially identical amino acid sequence thereof;
7) A polypeptide having the amino acid sequence represented by SEQ ID NO: 120 or a substantially identical amino acid sequence thereof;
8) A polypeptide having the amino acid sequence represented by SEQ ID NO: 143 or SEQ ID NO: 153, or a substantially identical amino acid sequence thereto;
9) A polypeptide having the amino acid sequence represented by SEQ ID NO: 173 or SEQ ID NO: 180, or a substantially identical amino acid sequence thereof;
10) a polypeptide having the amino acid sequence represented by SEQ ID NO: 201, or a substantially identical amino acid sequence thereof;
11) 25 in the amino acid sequence represented by SEQ ID NO: 226 or SEQ ID NO: 233, the amino acid sequence consisting of the 25th to 853rd amino acid residues in the amino acid sequence represented by SEQ ID NO: 226, or the amino acid sequence represented by SEQ ID NO: 233 An amino acid sequence consisting of the 216th amino acid residue, or a polypeptide having an amino acid sequence substantially identical to them; or 12) an amino acid sequence represented by SEQ ID NO: 251, SEQ ID NO: 266 or SEQ ID NO: 272, or thereof A polypeptide having substantially the same amino acid sequence as The polypeptide according to claim 1 or a specific partial peptide thereof, wherein the polypeptide is a polypeptide of any one of 1) to 12) below:
1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 10;
2) A polypeptide comprising an amino acid sequence consisting of amino acid residues 24 to 543 in the amino acid sequence represented by SEQ ID NO: 30 or SEQ ID NO: 37 or the amino acid sequence represented by SEQ ID NO: 30;
3) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 57;
4) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 73;
5) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 88;
6) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 104;
7) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 120;
8) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 143 or SEQ ID NO: 153;
9) A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 173 or SEQ ID NO: 180;
10) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 201;
11) 25 in the amino acid sequence represented by SEQ ID NO: 226 or SEQ ID NO: 233, the amino acid sequence consisting of the 25th to 853rd amino acid residues in the amino acid sequence represented by SEQ ID NO: 226, or the amino acid sequence represented by SEQ ID NO: 233 A polypeptide comprising an amino acid sequence comprising the 216th amino acid residue; or 12) a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 251, SEQ ID NO: 266 or SEQ ID NO: 272.   The polypeptide according to claim 1 or 2, or a specific partial peptide thereof, which is fused with a polypeptide comprising a heterologous amino acid sequence. The partial peptide specific to the polypeptide encoded by the blood cell / osteoclast specific gene 1-12, which is a partial peptide of any one of 1) to 12) below:
1) the amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 14, or a partial peptide comprising these partial amino acid sequences, or a partial peptide comprising the amino acid sequence represented by SEQ ID NO: 16;
2) the amino acid sequence represented by SEQ ID NO: 32 or SEQ ID NO: 34, or a partial peptide comprising these partial amino acid sequences, or a partial peptide comprising the amino acid sequence represented by SEQ ID NO: 39;
3) A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 59 or a partial amino acid sequence thereof;
4) A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 290 or a partial amino acid sequence thereof;
5) A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 90 or a partial amino acid sequence thereof;
6) A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 106 or a partial amino acid sequence thereof;
7) A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 124, SEQ ID NO: 125 or SEQ ID NO: 127, or a partial amino acid sequence thereof;
8) SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 155, SEQ ID NO: 157 or SEQ ID NO: 159, or a partial peptide consisting of these partial amino acid sequences;
9) The amino acid sequence represented by SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 182 or SEQ ID NO: 184, or a partial peptide comprising these partial amino acid sequences;
10) A partial peptide comprising the amino acid sequence represented by SEQ ID NO: 204 or SEQ ID NO: 206, or a partial amino acid sequence thereof;
11) the amino acid sequence represented by SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 235 or SEQ ID NO: 237, or a partial peptide consisting of a partial amino acid sequence thereof; or 12) SEQ ID NO: 253, SEQ ID NO: 256, SEQ ID NO: 258, A partial peptide consisting of the amino acid sequence represented by SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 268, SEQ ID NO: 275, SEQ ID NO: 276, or SEQ ID NO: 278, or a partial amino acid sequence thereof.   A polynucleotide encoding the polypeptide according to any one of claims 1 to 3, or the specific partial peptide according to any one of claims 1 to 4. The polynucleotide of any one of 1) to 12) below or a specific partial nucleotide thereof:
1) a nucleic acid sequence represented by SEQ ID NO: 8, a nucleic acid sequence corresponding to the ORF, or a polynucleotide having a nucleic acid sequence substantially identical to them;
2) Nucleic acid sequence represented by SEQ ID NO: 28 or SEQ ID NO: 35, or an ORF corresponding nucleic acid sequence thereof, or a nucleic acid sequence consisting of nucleotide residues 357 to 1919 in the nucleic acid sequence represented by SEQ ID NO: 28, or thereof A polynucleotide having a nucleic acid sequence substantially identical to said;
3) a polynucleotide having the nucleic acid sequence represented by SEQ ID NO: 55, the ORF-compatible nucleic acid sequence, or a nucleic acid sequence substantially the same as them;
4) a nucleic acid sequence represented by SEQ ID NO: 71, a nucleic acid sequence corresponding to the ORF, or a polynucleotide having a nucleic acid sequence substantially the same as them;
5) a polynucleotide having the nucleic acid sequence represented by SEQ ID NO: 86, the ORF-corresponding nucleic acid sequence, or a nucleic acid sequence substantially identical thereto;
6) a polynucleotide having the nucleic acid sequence represented by SEQ ID NO: 102, the ORF-corresponding nucleic acid sequence, or a nucleic acid sequence substantially identical thereto;
7) a nucleic acid sequence represented by SEQ ID NO: 118, a nucleic acid sequence corresponding to the ORF, or a polynucleotide having a nucleic acid sequence substantially the same as them;
8) a polynucleotide having SEQ ID NO: 141 or 151, or an ORF corresponding nucleic acid sequence thereof, or a nucleic acid sequence substantially identical thereto;
9) a nucleic acid sequence represented by SEQ ID NO: 171 or SEQ ID NO: 178, or an ORF corresponding nucleic acid sequence thereof, or a polynucleotide having a nucleic acid sequence substantially identical to them;
10) a polynucleotide having the nucleic acid sequence represented by SEQ ID NO: 199, or an ORF-corresponding nucleic acid sequence, or a nucleic acid sequence substantially identical thereto;
11) The nucleic acid sequence represented by SEQ ID NO: 224 or SEQ ID NO: 231 or the ORF-compatible nucleic acid sequence thereof, or the nucleic acid sequence consisting of 118 to 2604th nucleotide residues in the nucleic acid sequence represented by SEQ ID NO: 224 or SEQ ID NO: A nucleic acid sequence consisting of nucleotide residues 118 to 693 in the nucleic acid sequence represented by 231; or a polynucleotide having a nucleic acid sequence substantially the same as them; or 12) SEQ ID NO: 249, SEQ ID NO: 264, or SEQ ID NO: 270 A polynucleotide having the nucleic acid sequence represented by the above, or the ORF corresponding nucleic acid sequence thereof, or a nucleic acid sequence substantially identical to them. The polynucleotide according to claim 6 or a specific partial nucleotide thereof, wherein the polynucleotide of any one of 1) to 12) is the polynucleotide of any one of 1) to 12) below:
1) a nucleic acid sequence represented by SEQ ID NO: 8, or a polynucleotide comprising the nucleic acid sequence corresponding to the ORF;
2) a nucleic acid sequence represented by SEQ ID NO: 28 or SEQ ID NO: 35, or an ORF corresponding nucleic acid sequence thereof, or a nucleic acid sequence comprising a nucleic acid sequence consisting of nucleotide residues 357 to 1919 in the nucleic acid sequence represented by SEQ ID NO: 28 nucleotide;
3) a nucleic acid sequence represented by SEQ ID NO: 55 or a polynucleotide comprising the ORF-compatible nucleic acid sequence;
4) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 71 or a nucleic acid sequence corresponding to the ORF;
5) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 86 or a nucleic acid sequence corresponding to the ORF;
6) a polynucleotide consisting of the nucleic acid sequence represented by SEQ ID NO: 102, or a nucleic acid sequence corresponding to the ORF thereof;
7) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 118 or a nucleic acid sequence corresponding to the ORF;
8) A polynucleotide comprising SEQ ID NO: 141 or SEQ ID NO: 151, or a nucleic acid sequence corresponding to the ORF thereof;
9) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 171 or SEQ ID NO: 178, or an ORF-compatible nucleic acid sequence thereof;
10) a polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO: 199 or a nucleic acid sequence corresponding to the ORF;
11) The nucleic acid sequence represented by SEQ ID NO: 224 or SEQ ID NO: 231 or the ORF-compatible nucleic acid sequence thereof, or the nucleic acid sequence consisting of 118 to 2604th nucleotide residues in the nucleic acid sequence represented by SEQ ID NO: 224 or SEQ ID NO: A polynucleotide comprising the nucleic acid sequence consisting of nucleotide residues 118 to 693 in the nucleic acid sequence represented by 231; or 12) the nucleic acid sequence represented by SEQ ID NO: 249, SEQ ID NO: 264 or SEQ ID NO: 270, or an ORF thereof A polynucleotide comprising a corresponding nucleic acid sequence. The following partial nucleotides specific to the polynucleotide encoded by the blood cell / osteoclast specific gene 1-12, which is any partial nucleotide of 1) to 12):
1) a nucleic acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 27, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
2) Nucleic acid sequence represented by SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 53 or SEQ ID NO: 54, or a partial nucleic acid sequence thereof A partial nucleotide consisting of;
3) a nucleic acid sequence represented by SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66 or SEQ ID NO: 70, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
4) a nucleic acid sequence represented by SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 81 or SEQ ID NO: 85, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
5) a nucleic acid sequence represented by SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 97 or SEQ ID NO: 101, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
6) a nucleic acid sequence represented by SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 113 or SEQ ID NO: 117, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
7) A nucleic acid sequence represented by SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134 or SEQ ID NO: 140, or a partial nucleotide consisting of a partial nucleic acid sequence thereof;
8) Nucleic acid represented by SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 163, SEQ ID NO: 166 or SEQ ID NO: 170 A partial nucleotide consisting of a sequence or a partial nucleic acid sequence thereof;
9) Nucleic acid sequence represented by SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 190, SEQ ID NO: 193, SEQ ID NO: 197 or SEQ ID NO: 198, or a partial nucleic acid sequence thereof A partial nucleotide consisting of;
10) Nucleic acid sequence represented by SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 209, SEQ ID NO: 212, SEQ ID NO: 215, SEQ ID NO: 218, SEQ ID NO: 222 or SEQ ID NO: 223, or a partial nucleic acid sequence thereof A partial nucleotide consisting of;
11) A nucleic acid sequence represented by SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 240, SEQ ID NO: 243, SEQ ID NO: 247, or SEQ ID NO: 248, or a partial nucleotide consisting of a partial nucleic acid sequence thereof Or 12) SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 277, SEQ ID NO: 281, a nucleic acid sequence represented by SEQ ID NO: 284, SEQ ID NO: 288 or SEQ ID NO: 289, or a partial nucleotide consisting of a partial nucleic acid sequence thereof.   The polynucleotide according to any one of claims 5 to 7, or the specific partial nucleotide according to any one of claims 6 to 8, and a promoter operably linked thereto. 5. An expression vector for the polypeptide according to any one of 3 or the specific partial peptide according to any one of claims 1 to 4.   A transformant into which the expression vector according to claim 9 has been introduced.   9. A nucleic acid sequence complementary to the nucleic acid sequence of the specific partial nucleotide according to claim 7 or 8, and suppressing the expression of any polypeptide encoded by a blood cell / osteoclast specific gene 1-12. Antisense molecules that can.   A sense strand comprising a nucleic acid sequence of the specific partial nucleotide according to claim 7 or 8, and an antisense strand comprising a nucleic acid sequence complementary thereto, and 5 'of one or both of the sense strand and the antisense strand RNAi-inducible nucleic acid capable of suppressing the expression of any polypeptide encoded by blood cell / osteoclast-specific genes 1-12, which may have an overhang at the terminal and / or 3 'terminal .   The RNAi-inducible nucleic acid according to claim 12, wherein the RNAi-inducible nucleic acid is siRNA.   It can bind to any polypeptide encoded by the blood cell line / osteoclast-specific gene 1-12 via a region corresponding to the specific partial peptide according to any one of claims 2 to 4. Aptamer.   It can bind to any polypeptide encoded by the blood cell line / osteoclast-specific gene 1-12 via a region corresponding to the specific partial peptide according to any one of claims 2 to 4. ,antibody. The antibody according to claim 15, wherein the antibody is any one of i) to iii) below:
i) polyclonal antibodies;
ii) a monoclonal antibody or part thereof;
iii) chimeric, humanized or human antibodies.   A cell that produces the antibody according to claim 15 or 16.   The cell according to claim 17, wherein the cell is a hybridoma.   The polypeptide according to any one of claims 1 to 3, the antisense molecule according to claim 11, the RNAi-inducible nucleic acid according to claim 12 or 13, the aptamer according to claim 14, and the claims 15 or 16. A composition comprising an antibody, or an expression vector thereof, and a pharmaceutically acceptable carrier.   A mammalian cell or a non-human mammal, wherein expression or function of the polypeptide according to any one of claims 1 to 3 is regulated. Primer set specific to any polynucleotide encoded by the blood cell / osteoclast specific gene 1-12 or its specific partial nucleotide, including the following (a) or (b):
(A) a sense primer corresponding to the first nucleic acid sequence of the polynucleotide according to claim 7 or the specific partial nucleotide according to claim 7 or 8; and (b) the polynucleotide according to claim 7 or claim. Item 9. An antisense primer corresponding to a nucleic acid sequence complementary to the second nucleic acid sequence of the specific partial nucleotide according to Item 7 or 8. A nucleic acid probe specific for any of the polynucleotides encoded by the blood cell line / osteoclast specific genes 1 to 12 or a specific partial nucleotide thereof, which is either of the following (a) or (b):
(A) a single-stranded polynucleotide comprising a nucleic acid sequence complementary to the nucleic acid sequence of the specific partial nucleotide according to claim 7 or 8, or (b) the nucleic acid of the specific partial nucleotide according to claim 7 or 8. A double-stranded polynucleotide comprising a sense strand comprising a sequence and an antisense strand comprising a nucleic acid sequence complementary thereto.   Blood cell line / osteoclast specific, comprising one or more substances or sets selected from the aptamer according to claim 14, the antibody according to claim 15 or 16, the primer set according to claim 21, and the nucleic acid probe according to claim 22. A reagent or kit for detection or quantification of any polypeptide or polynucleotide encoded by genetic genes 1-12.   24. The reagent or kit according to claim 23, which is a reagent or kit for identifying a blood cell line or osteoclast, or for determining differentiation thereof.   Measuring the expression of any polypeptide or polynucleotide encoded by a blood cell / osteoclast specific gene 1-12 in a biological sample obtained from a mammal, or in a cell or tissue culture, And a method of detecting or quantifying the polypeptide or polynucleotide, wherein the biological sample or the culture contains blood cells, osteoclasts, hematopoietic tissue, lymphoid tissue or synovial tissue.   The polypeptide or the polynucleotide comprising measuring the expression of the polypeptide according to claim 2 or 3 or the polynucleotide according to claim 7 in a biological sample obtained from a mammal, or a cell or tissue culture. Detection or quantification method.   27. The detection or quantification method according to claim 26, wherein the biological sample or the culture contains blood cells, osteoclasts, hematopoietic tissue, lymphoid tissue or synovial tissue.