WO2003091435A1 - Novel proteins and dnas encoding the same - Google Patents

Novel proteins and dnas encoding the same Download PDF

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Publication number
WO2003091435A1
WO2003091435A1 PCT/JP2003/005174 JP0305174W WO03091435A1 WO 2003091435 A1 WO2003091435 A1 WO 2003091435A1 JP 0305174 W JP0305174 W JP 0305174W WO 03091435 A1 WO03091435 A1 WO 03091435A1
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protein
seq
dna
amino acid
sequence
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PCT/JP2003/005174
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French (fr)
Japanese (ja)
Inventor
Yoshihide Hayashizaki
Mamoru Kamiya
Hideo Kubodera
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Riken
K. K. Dnaform
Mitsubishi Chemical Corporation
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Priority to AU2003235099A priority Critical patent/AU2003235099A1/en
Publication of WO2003091435A1 publication Critical patent/WO2003091435A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators

Definitions

  • the present invention introduces a novel protein, a DNA encoding the protein, a full-length cDNA encoding the protein, a recombinant vector having the DNA, an oligonucleotide comprising a partial sequence of the DNA, and the DNA.
  • a cataloged library means that there is no overlap in the cDNAs contained in the library, and refers to a library containing one type of each cDNA.
  • the full-length cDNA cloning method is described in JP-A-9-248187 and JP-A-10-127291.
  • a tag molecule is bound to the diol structure present in the 5 ′ cap site of the mRNA, the mRNA bound to the tag molecule is type- ⁇ , and the RNA is obtained by reverse transcription using o1igo dT as a primer.
  • a method comprising the steps of: preparing a DNA complex, and separating a complex having a DNA corresponding to the full length of the mRNA using the function of a tag molecule.
  • the full-length cDNA library produced by such a technique does not contain all the elements that are different evenly among the individual elements of the library. Some clones do not. Since a clone existing only in such a trace amount is highly likely to be novel, a subtraction method for enriching such a clone, ie, a normalization method, has also been developed (Japanese Patent Application Laid-Open No. 2000-325080; Carninci). , P. et al., Genomics, 37, 327-336 (1996)). The nucleotide sequence of each clone of the cataloged full-length cDNA library thus obtained can be identified by a known method, but the physiological activity of the protein encoded by the cDNA is still unknown. Remains. Disclosure of the invention
  • the present invention analyzes the nucleotide sequence of a cDNA clone contained in a cataloged full-length cDNA library, and among those having a novel sequence, specifies the physiological activity of the protein encoded by the sequence.
  • the purpose of the present invention is to propose a method of using a protein based on a physiological activity and a DNA encoding the protein.
  • the present inventors analyzed the nucleotide sequence of the cDNA clone in the mouse full-length cDNA library and searched a database based on the homology of the sequence, and found that the sequence has a specific function. A protein-specific sequence was found. The expression levels of these cDNAs in each tissue were analyzed. The present invention has been accomplished based on these findings.
  • (b) consists of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence of SEQ ID NOs: 2 and 3, and has an activity of binding to a TGF / 3 receptor family.
  • Protein. (2) DNA encoding the protein of (1) above.
  • a protein having an activity of binding to L A protein having an activity of binding to L.
  • a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 29 to 41;
  • a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of any of SEQ ID NOs: 29 to 41, and which has ATP-binding carrier activity;
  • nucleotide sequence of any one of SEQ ID NOs: 16 to 28 one or several nucleotides have a nucleotide sequence in which deletion, substitution, Z or addition is performed, and ATP-binding transport DNA encoding a protein having body activity.
  • nucleotide sequence capable of hybridizing under stringent conditions with a DNA having the nucleotide sequence of any of SEQ ID NOs: 16 to 28 or a sequence complementary thereto, and an ATP-binding carrier activity DNA encoding a protein having
  • a base capable of hybridizing under stringent conditions with a DNA having the base sequence of any one of SEQ ID NOs: 42 to 46 or a complementary sequence thereof DNA encoding a protein having a sequence and having immunoglobulin-like protein activity.
  • a recombinant vector comprising the DNA according to any of (2) to (4), (6) to (8), (10) to (12), and (14) to (16).
  • oligonucleotide selected from the group consisting of a sense oligonucleotide having the same sequence as 100 bases, an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide, and an oligonucleotide derivative of the sense or antisense oligonucleotide .
  • the protein according to (1), (5), (9), (13), or (19) is brought into contact with a test substance, and the activity of the protein by the test substance is measured. Measuring the change in the activity of the protein.
  • the DNA of the present invention comprises a protein consisting of the amino acid sequence described in SEQ ID NOs: 2, 3, 14, 15, 29 to 41, and 47 to 51, or one or several amino acids in the amino acid sequence (hereinafter referred to as Although the number is not particularly limited, it means, for example, substitution of 20 or less, preferably 15 or less, more preferably 10 or less, and still more preferably 5 or less amino acid residues. Any protein may be used as long as it comprises an amino acid sequence containing a deletion, insertion, addition, or inversion and encodes a protein having the specific activity described below. Specifically, it may be only the translation region encoding the amino acid sequence, or may include the entire length of the cDNA.
  • examples of the DNA containing the full-length cDNA include, for example, DNAs comprising the nucleotide sequences of SEQ ID NOs: 1, 12, 13, 16 to 28, and 42 to 46. Also, as the translation area,
  • the DNA of the present invention may also include the above-described translation region and a region adjacent to the 3 ′ and / or 5 ′ end thereof, which contains the minimum necessary portion for the expression of the translation region. include.
  • the DNA of the present invention may be obtained by any method as long as it can be obtained. Specifically, it can be obtained, for example, by the method described below.
  • mRNA is prepared from a suitable animal, preferably a mammalian tissue or the like, by a method known per se and generally used.
  • the mRNA of c DNA synthesis Suruga as ⁇ become tag specific diol structure at the 5 'cap (TM e G ppp N) site for the synthesis of full-length c DNA this time
  • the function of the tag molecule It is preferable to use a method of separating only full-length cDNA using U.S.
  • thermostable reverse transcriptase in the presence of trehalose, etc.
  • high temperature means 40 to 80 ° C.
  • the cDNA obtained in this manner is inserted into an appropriate closing vector to perform closing.
  • the vector used here has a recombinase recognition sequence at both ends of a cloning site capable of uniformly closing DNAs of various chain lengths, and is directly inserted into a host by a method other than infection.
  • a chain vector (JP-A-11-9273) is preferably used.
  • JP-A-11-9273 is preferably used in the thus obtained cDNA library.
  • not all clones are uniformly present (hereinafter, this may be referred to as "tagged").
  • the subtraction method and the normalization method JP-A-2000-325080; Carninci, P. et al., Genomics, 37, 327-336 (1996) for enriching such clones are used. Is preferred.
  • the cataloged cDNA library is subjected to nucleotide sequence analysis by a commonly used method known per se.
  • the DNA of the present invention is obtained by combining the base sequence obtained for the 100-base sequence at the
  • Examples of the DNA having such a full-length cDNA base sequence and its translation region include those described above.
  • a sufficiently significant hit sequence is e-V a1 ue when the degree of coincidence between the catalytic domain portion of the registered amino acid sequence and the corresponding portion of the amino acid sequence encoded by the DNA of the present invention is e-V a1 ue. 10- 4 or less things as, or show a more than 30%.
  • HMMPFAM an analysis is performed by a method of checking whether or not the sequence to be analyzed has the characteristics of the sequence of an entry in a database in which a protein profile called Pfam is accumulated. Profiles are extracted from a series of proteins with the same characteristics, and even if a function cannot be clarified by comparing the full length of one-to-one sequences, if there is a characteristic region in the sequence, it will be found and its function predicted. it can. A specific example of the prediction of the function of a protein thus performed will be described below.
  • amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 was determined by BLAST search to be Homo sapiensputativetran smemb ranerotein NMA precursor, e-value: 3 X 10 and 138 amino acid residues with 67% identity, — Defici ent TGF betasuperf am ilyreceptorsub un it force e-value: l Xl (T 48 , 67% of the 153 amino acid residues over 67% of 1 ⁇ !
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 has a binding activity to TGF receptor family, and is kinasse—deficinetTGFbeetasupeperfamlilyreceptortsubunit.
  • TGF] 3 receptor families are known to have a serine-threonine kinase domain in the intracellular domain, penetrating the cell membrane once, and are classified into type 1 and type 2 receptors based on their structure and function. can do.
  • Type 1 receptors have a glycine- and serine-rich GS domain N-terminal to the kinase region.
  • the receptor forms a tetramer consisting of two molecules of type 1 receptor and two molecules of type 2 receptor, and the type 1 receptor is kinased by type 2 receptor.
  • GS domain is phosphorylated, and as a result, type 1 receptor kinase is activated and a signal is transmitted into cells.
  • BAMBI BMP and activin membrane-bound inhibitor; Nature Vol.401, 480-483 (1999)
  • TGF TGF 3 family
  • the protein of the present invention has high homology with the pseudo-receptors BAMB I and Nma of TGF family (J. Dent. Res. 80 (10), 1895-1902 (2001)), and normal TGF
  • the lack of a common serine threonine kinase domain in the [3] family suggests that it is a pseudoreceptor for the TGF] 3 family.
  • (1-2) Protein having binding activity to denatured LDL (low-density lipoprotein)
  • the amino acid sequence encoded by the base sequence described in SEQ ID NO: 12 is obtained by using BLAST.
  • lectin one like oxidized LDL receptor (Oryctolagus cuniculus) is, e- va 1 ue: 29% of the degree of match over the 1 X 10- 21, 235 amino acid residues, was good, oxidised low density lipoprotein (lectin - like) receptor 1
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 12 or the protein consisting of the amino acid sequence shown in SEQ ID NO: 14 has a binding activity with denatured LDL, and It can be inferred that it is a type of modified LDL receptor involved in the incorporation of LDL and the like into cells and cell dysfunction.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 13, by 7 this homology ⁇ using B LAST, lectin - like oxidized LDL receptor (Oryctolagus cuniculus) is, e- value: 1 X 1 0- 19, 235 Amino acid residue 28% over - in ⁇ , also, oxidised low density lipoprotein (lectin - liKe) receptor 1 (Homo sapiens) power e- value: 6 X l 0- 18 , 235 to Amino acid residue Hits with a 26% match rate.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 13 or the protein consisting of the amino acid sequence shown in SEQ ID NO: 15 has a binding activity to denatured LDL, It can be speculated that this is one of the modified LDL receptors involved in the incorporation of LDL into cells and cell dysfunction.
  • the protein of the present invention is one type of modified LDL receptor.
  • Known denatured LDLs include oxidized LDL related to arteriosclerosis, glycated LDL related to diabetes, etc., advanced glycation endproducts (AGE) -LDL, and malondialdehyde-modified LDL related to coronary artery disease, etc.
  • the protein of the present invention has high homology with lectin-like oxidized LDL receptor 1 (L0X-1), which is an oxidized LDL receptor, and has a cysteine repeating structure in the extracellular lectin region. Is conserved, it can be inferred to be a family molecule of oxidized LDL receptor.
  • the oxidized LDL receptor has an activity to bind oxidized LDL and take it up into cells, and is considered to play an important role in the etiology of arteriosclerosis, and its structure is a one-time transmembrane type. It is a type of glycoprotein that is known to have a short N-terminal protruding into the cell and a C-terminal protruding out of the cell.
  • the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 16 was obtained from human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER (ABC transporter)) 3) shows that the mouse ATP-binding cassette transporter ABCA3 has an e-va 1 ue of 0, 791 amino acid residues and 46% identity over the 791 amino acid residues. % in the degree of coincidence, also, mouse ATP- binding cassette transporter ⁇ Ka, e- va 1 ue: hits in 5 X 10_ 117, 791 37% degree of coincidence over the amino acid residues.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 16 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER 3), and has ATP binding property. It can be inferred to have carrier activity. Therefore, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 16 is an ABC transporter involved in extracellular excretion of drugs, etc., and foreign substances such as drugs, endogenous substances such as calcium, phospholipids, and amphiphilic substances It can be inferred that it is involved in the transport of toxic substances.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 17 shows that the ATP-binding cassette protein of the (ABCA subfamily) product shows e-va 1 ue: 0, 671 amino acid residues by homology search using BLAST.
  • Human ATP-binding cassette transporter ABCA3 force e-va 1 ue with 90% identity over all groups: 0, 671 amino acids 90% degree of coincidence over the residue, addition, Homo sapiens cDNA FLJ31971 fis, clone NT2RP7008137, weakly similar to ATP- BINDING CASSETTE, SUB-FAMILY A, MEMBER 1 is, e- va 1 ue: 5X10- m , 417 Hits with 87% identity across amino acid residues.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 17 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER 3), and has ATP-binding transport It can be presumed to have body activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 17 is an ABC transporter involved in the extracellular excretion of drugs, etc., and the foreign substances such as drugs and intrinsic factors such as calcium, phospholipids and amphiphiles It can be inferred that it is involved in the transport of toxic substances.
  • the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 18 shows that the human ATP-binding cassette protein of the (ABCA subfamily) product has an e-va 1 ue of 0, 1250 amino acid residues by homology search using BLAST.
  • e_value 0, hits with 38% identity over 1251 amino acid residues.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 18 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A and has ATP-binding carrier activity. From this, the protein encoded by the nucleotide sequence of SEQ ID NO: 18 is an ABC transporter involved in the extracellular excretion of drugs, etc., and foreign substances such as drugs, calcium, phospholipids, amphipathic substances, etc. Transport of endogenous substances It can be guessed that it is related to the above.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 19 was obtained by using BLAST in the same order as the 7th order! "Homo sapiens ATP—binding cassette A9 power, e-va 1 ue: 0, 948, with 79% identity over 948 amino acid residues, KIAA0822, e_va 1 ue: 0, 948 amino A hit with a 67% concordance over acid residues and a 60% concordance over Homo sapiens ATP-binding cassette A10 1S e-value: 0, 958 amino acid residues.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 19 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A9, and can be inferred to have ATP-binding carrier activity.
  • the nucleotide sequence represented by No. 19 A A B C transporters click protein is involved in the extracellular discharge, etc. of the drug, and foreign matter such as a drug, calcium, phospholipids, be involved in transportation, etc. of endogenous substances such as amphiphiles can be inferred.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 20 was identified by homology search using BLAST as Homo sapiens cDNA FLJ32506 fis, clone SMINT1000042, weakly similar to ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 However, e-va 1 ue: 5 X 1 (T " 3 , Homo sapiens with 75% identity over 332 amino acid residues
  • ATP-binding cassette A9 is, e _ va 1 ue: at 5 X 10- 143, 332 75% degree of coincidence over the amino acid residues, also, Homo sapiens ATP- binding cassette A10 force e - va 1 ue: 5 X 10 124, 330 hits in 65% of the degree of coincidence over the amino acid residues.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 20 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, and It can be presumed to have body activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 20 is an ABC transporter involved in extracellular excretion of a drug, etc. It can be inferred that it is involved in the transport of volatile substances.
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 21 was found by homology search using BLAST to find that Mus rausculus, clone IMAGE: 4505946 shows e-va 1 ue: 0, 591 amino acid residues.
  • Homo sapiens cDNA KIM0822 has 100% concordance, e_va 1 ue: 0, 73% concordance over 587 amino acid residues, and
  • Homo sapiens ATP-binding cassette A9 has e-va 1 ue : 0% hits at 590 amino acid residues with 68%-lethality.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 21 is an ABC transporter involved in extracellular excretion of drugs, etc., and foreign substances such as drugs, and intrinsic factors such as calcium, phospholipids, amphiphiles, etc. It can be inferred that it is involved in the transport of sex substances.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 22 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A and has ATP-binding carrier activity. Therefore, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 22 is an ABC transporter involved in the extracellular excretion of drugs, etc., and foreign substances such as drugs, intrinsic factors such as calcium, phospholipids, amphipathic substances, etc. It can be inferred that it is involved in the transport of toxic substances.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 23 shows that, by homology search using BLAST, human ATP11C gene for ATPase, Class VI, type 11C has e-va 1 ue: 0, 436 amino acid residues. 91% coincidence, Potential
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 23 has a sequence similar to the transporter ATPase family, and has ATP-binding transporter activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 23 is a carrier ATPase family, which is involved in the transport of foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphiphilic substances. This can be inferred.
  • the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 24, by homology search using B LAST, human KIM1939 is, e- va 1 ue: 5X10- 155 , 489 85% match over Amino acid residue time in, Homo sapiens cDNA FLJ30324weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3 is, e- va 1 ue: in 5 ⁇ 1 ( ⁇ 154, 489 ⁇ amino acid residues 78% degree of coincidence over, also, Potential phospholipid - transporting ATPase IC is, e- va 1 ue: 5X10- 136 , 489 hits in 57% of the degree of coincidence over the amino acid residues.
  • the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 25, B by homology search using LAS T, human KIM1939 is, e _ va 1 ue: 5X10- 150, 300 Amino acid residues 1 Ri 86% in the matching degree, Homo sapiens otential phospholipid - transporting ATPase IC is, e- va 1 ue: 5X10- " , with 57% degree of coincidence over the 309 amino acid residues, also," CG14741 "; Drosophila melanogaster genomic scaffold force s , e- va 1 ue:.
  • the protein comprising the amino acid sequence nucleotide sequence coding for SEQ ID NO: 25 is transported It can be inferred that the protein has a sequence similar to that of the ATPase family and has ATP-binding transporter activity. Foreign substances such as drugs, calcium and phosphorus It is speculated that the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 26 can be determined by Homo sapiens, by homology search using BLAST. clone IMAGE: 4111596, e-va 1 ue: 0, human potential with 54% identity over 600 amino acid residues
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 26 was found to be a carrier A TP as It has a sequence similar to the e-family and can be inferred to have ATP-binding transporter activity.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 26 is the transporter ATPase family, and is used for transporting foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphiphilic substances. It can be guessed to be involved.
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 27 was found to be Homo sapiens cDNA FLJ30324 weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3 by elasta: in ⁇ 1 ( ⁇ 101, 271 amino acids 63% degree of coincidence over the residue, human Potential phospholipid- transporting ATPase IC power S, e- va 1 ue: 1 ⁇ over 1 ( ⁇ 85, 290 amino acid residues 51 in% degree of coincidence, also, gene:;: from 2 X 10- 84, 280 hits in 55% of the degree of coincidence over the amino acid residues of these results "CG14741 Drosophila melanogaster genomic scaftolc ⁇ e- va 1 ue.
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 27 has a sequence similar to the ATPase family of the carrier and has ATP-binding carrier activity.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 27 is the carrier ATPase It can be assumed that it is involved in the transport of foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphipathic substances.
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 28 was obtained by homology search using BLAST, gene: CG14741; Drosophila melanogaster genomic scaffold, e-va 1 ue: 5X1 (T 136 , 421 Potential phospholipid-"transporting ATPase IC force s , e- va 1 ue: 5 X 1 (T 122 , 53% identity over 431 amino acid residues, with 56% identity over amino acid residues, and , Homo sapiens cDNA FLJ30324 fis, clone BRACE2007138, weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3 is, e- va 1 ue:.
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 28 has a sequence similar to the transporter ATPase family and has ATP-binding transporter activity.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 28 Carrier ATP ase family, foreign substances such as drugs, It can be inferred that it is involved in the transport of endogenous substances such as calcium, phospholipids and amphiphiles.
  • the ABC (ATP-binding cassette) transporter is a member of the ATPase family of transporters that transports sugars, amino acids, polypeptides, long-chain fatty acids, hydrophobic substances, etc. using the energy of ATP degradation. Its structure is characterized by having two hydrophobic regions that penetrate the cell membrane several times and determine substrate specificity, and two intracellular ATP-binding regions.
  • the protein of the present invention has a high homology to the ABC transporter and the carrier ATPase family, and therefore, the foreign substances such as drugs, endogenous substances such as calcium, phospholipids, amphipathic substances and the like. It can be inferred that this is an ATP-binding carrier involved in the transport of sex substances.
  • a protein characteristic search using HMM PF AM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 42 shows a sequence that shows the characteristics of Alpha-2-macroglobulin (sequence that is entered as A2M-N in P f am) Is found.
  • the P01031, Complement C5 precursor (HUMAN) protein is considered to be involved in the inflammatory reaction based on the literature information in the database (Biochemistry 27: 3568-3580 (1988)).
  • MOUSE protein is related to inflammatory reactions based on literature information in the database (j. Biol. Chem. 265: 2435-2440 (1990)).
  • CAVPO Complement C3 precursor
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 42 is an immunoglobulin-like protein.
  • the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 43 was obtained by BLAST search using database registration code P01031, Complement C5 precursor (HUMAN) power e-va 1 ue: 3X1 CT 84 , 63% over 241 amino acid residues in the matching degree, and the data base over scan registration mark P06684, Complement C5 precursor (MOUSE) force e - va 1 ue: 2 X 10- 83, 242 with 59% degree of coincidence over the amino acid residues, more database registration mark P12387, Complement C3 precursor (CAVPO) power e ⁇ value: 3X 10 _17 , hits 234 amino acid residues with 30% match.
  • database registration code P01031 Complement C5 precursor
  • MOUSE Complement C5 precursor
  • CAVPO Complement C3 precursor
  • RAT Alpha- 1- inhibitor III precursor
  • CHICK Ovostatin precursor
  • P20742 Pregnancy zone protein precursor (HUMAN ) force e- value: hits in 29% of the degree of match over the 5 X 10- 1M, 995 amino acid residues.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 44 is an immunoglobulin-like protein involved in inhibition of protease activity.
  • HMMP FAM When a protein characteristic search is performed by HMMP FAM on the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 45, a sequence showing the characteristics of immunoglobulin (a sequence which is entered as 18 into P f 3111) is found.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 45 is an immunoglobulin-like protein.
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 46 was identified by the BLAST search according to the database database registration symbol AF329485 as having e_va 1 ue: 0.0, 99% coincidence over 343 amino acid residues, and database registration symbol AF459634 is, e -value: 9 in X l ( ⁇ 99, 327 58 % degree of coincidence over the amino acid residues, more database registration mark AL356276, e- value: 1 X 10- 73, 298 amino acids To the residue Hits with 51% match over time.
  • HMMP FAM When a protein characteristic search is performed by HMMP FAM on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 46, a sequence showing the characteristics of immunoglobulin (a sequence that is entered as ig in P f am) is found.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 46 is an immunoglobulin-like protein.
  • the DNA of the present invention may be obtained with a base deletion or insertion in the translation region, but as a result of the homology search or the protein feature search as described above, the DNA base of the DNA is obtained.
  • a known method such as library screening or PCR closing, which is generally used by those skilled in the art, can be used to obtain the full-length without deletion or insertion of the base.
  • c Can obtain DNA.
  • the full-length cDNA thus obtained is used to express the protein of the present invention, which can be used for functional analysis and the like.
  • the DNA of the present invention which is obtained by force, whose nucleotide sequence is determined, and whose function is estimated is the nucleotide sequence of SEQ ID NOS: 1, 12, 13, 16 to 28, 42 to 46, or Not only those having the base sequences shown above as the translation regions, but also one or several bases in these base sequences (the number here is not particularly limited, for example, a force S, for example, 60 or less, preferably 30 Or less, more preferably 20 or less, still more preferably 10 or less, particularly preferably 5 or less.) which has a base sequence in which the base is deleted, substituted and / or added, and Also included are DNAs encoding proteins having each activity, and DNAs which hybridize with these under stringent conditions and encode proteins having the above-mentioned activities.
  • DNA that hybridizes under stringent conditions refers to the nucleotide sequence shown in SEQ ID NO: 1, 12, 13, 16, 16-28, or 42-46 or its complementary sequence in a BLAST analysis of 80% or more, preferably 90% or more. % Or more, more preferably 95% or more having a homologous nucleotide sequence.
  • Hybridization under stringent conditions means that the reaction is carried out in a normal hybridization buffer at a temperature of 40 to 70 ° C, preferably 60 to 65 ° C, etc. Can be performed in a washing solution of 15 mM to 300 mM, preferably 15 mM to 60 mM.
  • DNA of the present invention may be obtained by the above-described method or may be synthesized.
  • the DNA base sequence can be easily replaced with a commercially available kit such as a site-directed mutagenesis kit (Takara Shuzo) or a quick change site-directed mutagenesis kit (Stratagene). Can be.
  • SEQ ID NO: 1, 12, 13, 16 to 28 the nucleotide sequence according to 42-46, the force is intended to be derived from the mouse s, cDNA library of human according to Preparation of cDNA library described above Is prepared and subjected to hybridization using a DNA fragment having the nucleotide sequence of SEQ ID NO: 1, 12, 13, 16 to 28 or 42 to 46 as a probe, whereby SEQ ID NO: 1, 12, 13 , 16-28, and 42-46, a DNA encoding a human homolog protein of the protein encoded by the nucleotide sequence can also be obtained.
  • DNAs that hybridize under stringent conditions with the DNAs of SEQ ID NOs: 1, 12, 13, 16 to 28, and 42 to 46 of the present invention also include DNAs encoding such human homologs. .
  • nucleotide sequence of the human homolog DNA can be predicted using informatics, and the human homologous DNA can be obtained from the above human cDNA library based on the nucleotide sequence. .
  • methods for predicting a nucleotide sequence encoding a homologous protein of a target protein by using informatics include, for example, (i) a method for estimating a target protein; A method of performing a homology search using BLAST etc.
  • any of the above methods can be used, and the method of the present invention can be applied to any of SEQ ID NOs: 1, 12, 13, 16 to
  • any of the cDNAs having the nucleotide sequences described in Nos. 28 and 42 to 46 is novel, and it is considered that the method (i) cannot obtain the nucleotide sequence of the human homologous DNA. ) Are preferably used.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 1, 12, 13, 16, 16-28, 42-46 was obtained from the above human cDNA library.
  • DNA encoding a human homolog protein can also be obtained.
  • a primer having a nucleotide sequence complementary to the nucleotide sequence at the 5 ′ end and 3 ′ end of the predicted human homolog DNA is used.
  • a method of performing hybridization on the human cDNA library using a partial sequence of the predicted human homolog DNA as a probe is performed using a partial sequence of the predicted human homolog DNA as a probe.
  • a similar gene having a nucleotide sequence having a higher homology to the nucleotide sequence of the target gene is called a “homolog”, and the above-mentioned method also aims to obtain a human homolog, but in the function analysis of the gene, Is only due to the similarity of base sequences
  • it is important to confirm that the gene obtained as a homolog is a family member of the target gene.
  • Genes acquired as “homologs” between two species of organisms are likely to be “o / resologs”, which are the same genes evolved from a common ancestral gene, and also caused by duplication from a common ancestral gene. It could be a different gene, a “paralog”.
  • the human-derived DNA obtained as a homologue in order for the human-derived DNA obtained as a homologue to have the same function as the protein of the present invention, it is necessary to use the DNA of the protein encoded by the human-derived DNA.
  • the human homolog is an ortholog of a closely related species of the mouse gene of the present invention.
  • the following method is used as a method for confirming the ortholog.
  • (2) homology is analyzed for the nucleotide sequence of the obtained human homolog DNA and the corresponding nucleotide sequence of the cDNA of the present invention.
  • the obtained human homolog DNA base sequence as a query, we performed a homology search on international base sequence databases such as DDB J, EMBL, Gen Bank, and mouse base sequences contained in patent databases. It is confirmed that the degree of matching between the cDNA of the present invention and the base sequence of the query is higher than the degree of matching between the base sequence obtained from the database and the base sequence of the query.
  • the obtained human homolog can be identified as a human ortholog corresponding to the cDNA of the present invention.
  • the homology analysis described in (1) and (2) above may be performed by comparing amino acid sequences, or by drawing a molecular evolutionary phylogenetic tree.
  • Such a human homologue is contained in DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence of SEQ ID NOS: 1, 12, 13, 16 to 28, or 42 to 46 or a sequence complementary thereto.
  • DNAs encoding orthologous proteins are also included.
  • the translation region of the protein encoded by the DNA of the present invention may be, for example, a base sequence of the DNA, which is converted into amino acids by three types of reading frames, and the range in which the longest polypeptide is encoded is determined.
  • the amino acid sequence can be determined as the translation region of the invention. Examples of such an amino acid sequence include those described in SEQ ID NOs: 2, 3, 14, 15, 29 to 41, and 47 to 51.
  • the protein of the present invention is not limited to the above-mentioned amino acid sequence, but comprises an amino acid sequence in which one or several amino acids have been substituted, deleted and / or added in the amino acid sequence, and Those having activity are also included.
  • the method of transcription / translation of the DNA of the present invention described in (1) by an appropriate method is preferably used.
  • a suitable expression vector or a recombinant vector inserted into a suitable vector together with a suitable promoter is prepared, and this recombinant vector is used to transform a suitable host microorganism or introduced into a suitable cultured cell. And then can be obtained by purifying it.
  • the protein thus obtained is obtained in a free form, a known method or It can be converted to a salt by a method according to it, and conversely, if it is obtained as a salt, it can be converted to a free form or another salt.
  • Such salts of the protein of the present invention are also included in the protein of the present invention.
  • the protein produced by the above-mentioned transformant can be modified before or after purification by the action of an appropriate protein-modifying enzyme to arbitrarily modify the protein or partially remove the polypeptide. can do.
  • These modified proteins are also included in the scope of the present invention as long as they have the above activity.
  • the vector used for the production of the recombinant vector containing the DNA of the present invention is not particularly limited as long as the DNA is expressed in the transformant.
  • phage vectors a commercially available protein expression vector into which an expression control region DNA such as a promoter suitable for the host into which the DNA is introduced has already been inserted is used.
  • Specific examples of such a protein expression vector include pET3 and pETll (manufactured by Stratagene) p GEX (manufactured by Amersham Pharmacia Biotech) when the host is Escherichia coli, and yeast.
  • p ESP-I expression vector manufactured by Stratagene
  • Bac PAK6 manufactured by Clontech
  • examples include ZAP Express (manufactured by Stratagene) and pSVK3 (manufactured by Amersham Armasia Biotech).
  • the promoter used herein may be a promoter contained in a host microorganism or a cultured cell, but is not limited thereto.
  • a promoter contained in a host microorganism or a cultured cell, but is not limited thereto.
  • the host is Escherichia coli, T3, T7 , it can be used tac, 1 ac promoter, and the like, in the case of yeast can be used nm t 1 promoter, G a 1 1 promoter.
  • SV40 promoter, CMV promoter and the like are preferably used.
  • the present invention When a host capable of functioning as a mammalian-derived promoter is used, the present invention A promoter specific to the gene can also be used. Insertion of the DNA of the present invention into these vectors is performed by linking the DNA or the DNA fragment containing the DNA to the amino acid sequence of the protein encoded by the gene DNA downstream of the promoter in the vector. Good.
  • the recombinant vector thus prepared can be transformed into a host described below by a method known per se to prepare a DNA-introduced body.
  • a method for introducing the vector into a host specifically, a heat shock method (J. Mol. Biol., 53, 154, (1970)), a calcium phosphate method (Science, 221, 551, (1983)), DEAE Dextran method (Science, 215, 166, (1982)), in vitro packaging method (Proc. Natl. Acad. Sci. USA, 72, 581, (1975)), virus vector method (Cell, 37, 1053, (1984) )) And electric pulse method (Chu. Et al., Nuc. Acids Res., 15, 1331 (1987)).
  • the host for producing the DNA-introduced host is not particularly limited as long as the DNA of the present invention is expressed in the body.
  • Escherichia coli, yeast, baculovirus (arthropod polyhedrosis virus) -one insect cell Or animal cells Specifically, B L21, XL-2B 1 ue (Stratagene) for E. coli, SP-Q01 (Stratagene) for yeast, AcNPV for baculovirus, etc.
  • African green monkey kidney-derived COS-7 (ATCCCRL1651: cells stored in the American Type Culture Collection) is preferably used.
  • the protein of the present invention is obtained.
  • protein expression is induced by subjecting the DNA of the present invention obtained in the above (1) to a cell-free transcription / translation system, thereby obtaining a protein of the present invention.
  • the cell-free transcription / translation system used in the present invention is a system containing all the elements necessary for transcription of DNA to mRNA and translation of mRNA to protein, and by adding DNA thereto. Any system in which the protein encoded by the DNA is synthesized.
  • the cell-free transcription / translation system include a transcription / translation system prepared based on an eukaryotic cell, a bacterial cell, or an extract from a part thereof, and a particularly preferred example is Egret A transcription translation system prepared based on extracts from reticulocytes, wheat germ, and Escherichia coli (Escherichia coli S30 extract) may be mentioned.
  • Separation and purification of the protein of the present invention from the obtained transcription / translation product of the cell-free transcription / translation system can be performed by a method known per se and generally used. Specifically, for example, a DNA region encoding an epitope peptide, a polyhistidine peptide, daltathione-1 S-transferase (GST), a maltose binding protein, or the like is introduced into the DNA to be transcribed and translated. It can be expressed as described above and purified using the affinity of the protein with a substance having affinity.
  • the expression of the target protein is separated by SDS-polyacrylamide gel electrophoresis or the like, and stained with Coomassie Priliant Blue (manufactured by Sigma), or specifically binds to the protein of the present invention described later. It can be confirmed by the detection method using an antibody.
  • the expressed protein is a protein existing in vivo. It is known that it is cleaved by a degrading enzyme (processing).
  • the protein of the present invention is, of course, included in the protein of the present invention as long as it has the above activity, even if it is a partial fragment of the cleaved amino acid sequence.
  • a method for analyzing the interaction a conventional method known per se can be used. Specifically, for example, a yeast two-hybrid method, a fluorescence depolarization method, a surface plasmon method, a phage display method, and a liposome method.
  • a yeast two-hybrid method a fluorescence depolarization method, a surface plasmon method, a phage display method, and a liposome method
  • a multiple display method is the multiple display method.
  • the DNA is prepared by a conventional method using a DNA synthesizer or the like.
  • Oligonucleotides such as antisense oligonucleotides and sense oligonucleotides having a partial sequence of the DNA of the present invention can be prepared.
  • the oligonucleotide examples include a DNA having the same sequence as the consecutive 5 to 100 bases in the base sequence of the DNA or a DNA having a sequence complementary to the DNA.
  • the above oligonucleotides in which the melting temperature (Tm) and the number of bases of both do not extremely change are preferred.
  • the length of the sequence is generally 5 to 100 bases, preferably 10 to 60 bases, and more preferably 15 to 50 bases.
  • oligonucleotide derivatives of these oligonucleotides can also be used as the oligonucleotide of the present invention.
  • the oligonucleotide derivative include an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide having N3, -P5 ' Oligonucleotide derivatives converted to phosphoamidate bonds, oligonucleotide derivatives in which ribose and phosphodiester bonds in oligonucleotides are converted to peptide nucleic acid bonds, and peracyl in oligonucleotides are C-5 propynyl peracyl.
  • a substituted oligonucleotide derivative an oligonucleotide derivative in which peracyl in an oligonucleotide is substituted with C_5 thiazoleperacyl, an oligonucleotide derivative in which cytosine in an oligonucleotide is substituted with C-15 probucytosine, Oligonucleotide derivatives in which cytosine in the oligonucleotide has been replaced with phenoxazine-modified cytosine), oligonucleotide derivatives in which ribose in the oligonucleotide has been replaced with 2,1O-propylribose, There is ribose in the oligonucleotide is 2 '- Ru can be mentioned oligonucleotide derivatives substituted with main butoxy ethoxy ribose.
  • RNAi method The oligonucleotide of the present invention can be applied to the RA interference method (hereinafter, this may be referred to as “RNAi method”) by preparing it as a double-stranded RA. .
  • RNAi method for the method for preparing double-stranded RNA and the RNA interference method, for example, the method described in (Elbashir, S., et al., Nature, 411, 494-498 (2)) is used. Can be.
  • RNAs need not all be RNAs. Specifically, as a part of which is a DNA, those described in WO 02/13774 can be used.
  • any target gene may be used as long as it is the DNA of the present invention.
  • a double-stranded polynucleotide consisting of RNA having a sequence substantially identical to at least a part of the base sequence of these DNAs (hereinafter sometimes referred to as “double-stranded polynucleotide”) is a target It comprises a sequence substantially the same as a sequence of 15 bp or more, which may be any part of the nucleotide sequence of the gene.
  • “substantially the same” means that it has 80% or more homology with the sequence of the target gene. Nucleotide lengths range from 15 bp to the full length of the open reading frame (0RF) of the target gene.
  • the length may be any length up to about 15 to 50 Obp.
  • mammalian cells have a signal transduction system that activates in response to long double-stranded RNA of 30 bp or more. This is called the interfering reaction (Mareus, PI, et al., Interferon, 5, 115-180 (1983)), and when the double-stranded RNA enters the cell, PKR (dsRNA-responsive protein) Kinase: Non-specific inhibition of translation initiation of many genes via Bass, BL, Nature, 411, 428-429 (2001)), and at the same time, 2 'and 5' oligoadenylate synthetase (Bass, BL, Nature, 411, 428-429 (2001)), which activates RNaseL and causes nonspecific degradation of intracellular RNA.
  • PKR dsRNA-responsive protein
  • the double-stranded polynucleotide does not need to be entirely double-stranded, and includes those having a partially protruding 5 ′ or 3 ′ end, but those having a 3 ′ end protruding two bases are preferred.
  • the double-stranded polynucleotide means a double-stranded polynucleotide having complementarity, but may be a self-annealed single-stranded polynucleotide having self-complementarity.
  • Single-stranded polynucleotides having self-complementarity include, for example, those having an inverted repeat sequence.
  • the method for preparing the double-stranded polynucleotide is not particularly limited, it is preferable to use a known chemical synthesis method.
  • chemical synthesis a single-stranded polynucleotide having complementarity can be separately synthesized, and can be converted into a double-stranded strand by associating them by an appropriate method.
  • Examples of the method of association include a method in which the above polynucleotides are mixed, heated to a temperature at which the double strand dissociates, and then gradually cooled.
  • the associated double-stranded polynucleotide is confirmed using an agarose gel or the like, and the remaining single-stranded polynucleotide is removed by, for example, decomposing with a suitable enzyme.
  • the transfectant into which the double-stranded polynucleotide prepared in this way is introduced is one in which the target gene can be transcribed into RNA or translated into protein in the cell.
  • Any substance may be used, but specific examples include those belonging to plant, animal, protozoan, virus, bacterial, or fungal species.
  • the plant can be a monocotyledonous, dicotyledonous or gymnosperm, and the animal can be a vertebrate or invertebrate.
  • Preferred microorganisms are those used in agriculture or by industry, and are pathogenic to plants or animals. Fungi include organisms in both mold and yeast forms.
  • vertebrates examples include mammals, including fish, sea lions, goats, pigs, sheep, hamsters, mice, rats and humans, and invertebrates include nematodes and other reptiles. , Drosophila, and other insects.
  • the cells are vertebrate cells.
  • the transductant means a cell, tissue, or individual.
  • the cell may be from germline or somatic, totipotent or pluripotent, split or non-split, parenchymal or epithelial, immortalized or transformed, and the like.
  • the cell can be a gamete or an embryo, in the case of an embryo, a single cell embryo or a constitutive cell, or a cell from a multi-cell embryo, including fetal tissue.
  • they may be undifferentiated cells, such as stem cells, or differentiated cells, such as from cells of an organ or tissue, including fetal tissue, or any other cells present in an organism.
  • Differentiating cell types include adipocytes, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, nerve cells, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, Includes basophils, mast cells, leukocytes, granulocytes, keratinocytes, osteoblasts, osteoclasts, hepatocytes and cells of the endocrine or exocrine glands.
  • a method for introducing a double-stranded polynucleotide into a recipient when the recipient is a cell or tissue, calcium phosphate method, electroporation method, lipofection method, virus infection, two Immersion in a strand polynucleotide solution or a transformation method is used. Examples of the method for introducing the gene into the embryo include microinjection, electoral poration, and virus infection.
  • a method of injecting or perfusing the plant into the body cavity or stromal cells, or spraying is used.
  • the double-stranded polynucleotide can be mixed directly with the food of the organism. Further, when introduced into an individual, it can be administered, for example, by administration as an implanted long-term release preparation or the like, or by ingesting an introduced body into which a double-stranded polynucleotide has been introduced.
  • the amount of the double-stranded polynucleotide to be introduced is preferably an amount sufficient to introduce at least one copy per force cell that can be appropriately selected depending on the transductant and the target gene.
  • the transfectant is a human cultured cell and the double-stranded polynucleotide is introduced by a calcium phosphate method, 0.1 to 100 OnM is preferable.
  • RNA interference By suppressing the expression of the gene of the present invention in the transfection body by RNA interference, it is possible to confirm the function of the protein encoded by the gene of the present invention or to analyze a new function.
  • an antibody that specifically binds to the protein of the present invention As a method for preparing an antibody that specifically binds to the protein of the present invention, a commonly used known method can be used.
  • epitope antigen
  • a suitable sequence can be selected and used as the determinant.
  • commercially available software such as Epitope Adviser (manufactured by Fujitsu Kyushu System Engineering Co., Ltd.) can be used.
  • polypeptide used as the above antigen a synthetic peptide synthesized according to a known method, or the protein itself of the present invention can be used.
  • a polypeptide serving as an antigen can be prepared in an appropriate solution according to a known method to immunize a mammal, for example, a heron, a mouse, a rat, or the like. Conjugate the antigen peptide to a suitable carrier protein to increase It is preferable to perform immunization by using or adjuvant.
  • the route of administration of the antigen upon immunization is not particularly limited, and any route such as subcutaneous, intraperitoneal, intravenous, or intramuscular may be used. Specifically, for example, a method of inoculating a BALB mouse several times every several days to several weeks with an antigen polypeptide is used.
  • the antigen intake is preferably about 0.3 to 0.5 mg Zl when the antigen is a polypeptide, but is appropriately adjusted depending on the type of the polypeptide and the animal species to be immunized.
  • test blood is collected as appropriate, and an increase in antibody titer is confirmed by enzyme-linked immunosorbent assay (hereinafter sometimes referred to as “ELISA”) or Western blotting.
  • ELISA enzyme-linked immunosorbent assay
  • Blood is collected from animals with elevated antibody titers.
  • a polyclonal antibody can be obtained by subjecting this to an appropriate treatment used for antibody preparation. Specific examples include a method of obtaining a purified antibody obtained by purifying an antibody component from serum according to a known method. For the purification of the antibody component, methods such as ion separation, ion exchange chromatography, affinity mouth chromatography, etc. can be used.
  • a hybridoma fused with spleen cells of the animal and myeoma cells according to a known method is used (Milstein, et al., Nature, 256, 495 (1975)). Can also be prepared.
  • a monoclonal antibody can be obtained, for example, by the following method.
  • antibody-producing cells are obtained from an animal whose antibody titer has been increased by immunization with the above-mentioned antigen.
  • the antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any of the individuals, but is preferably obtained from spleen, lymph nodes, peripheral blood and the like.
  • the myeloma to be fused with these cells is generally a cell line obtained from a mouse, for example, an 8-azaguanine-resistant mouse (BALB / c-derived etc.) myeloma cell line P3X63-Ag8.653 (ATCC: CRL -1580), P3-NSl / lAg4.1 (RIKEN cell bank: RCB0095) and the like are preferably used.
  • an appropriate cell fusion medium such as RPMI1640 Discov's modified Dalbecco's medium (IMDM) or Dulbecco's modified idal is used. 50% in medium (DMEM) It can be performed by using a solution in which polyethylene glycol (PEG) is dissolved.
  • PEG polyethylene glycol
  • Hybridoma is myeloma cell line 8 Azaguanin resistance by utilizing a and this is strain suitable amount of hypoxanthine 'aminopterin-thymidine (HAT) normal medium containing liquid (HAT medium) in 5% C0 2 was used, It can be selected by culturing at 37 ° C for an appropriate time. This selection method can be appropriately selected and used depending on the myeloma cell line to be used.
  • the antibody titer of the antibody produced by the selected hybridoma is analyzed by the method described above, the hybridoma producing the antibody with a high antibody titer is separated by limiting dilution, etc., and the separated fused cells are separated into an appropriate medium.
  • a monoclonal antibody can be obtained by purifying from a culture supernatant obtained by culturing with an appropriate method such as ammonium sulfate fractionation or affinity chromatography.
  • an appropriate method such as ammonium sulfate fractionation or affinity chromatography.
  • a commercially available monoclonal antibody purification kit can also be used.
  • by growing the antibody-producing hybridoma obtained above in the abdominal cavity of an animal of the same strain as the immunized animal or nude mouse, etc. it is possible to obtain ascites containing a large amount of the monoclonal antibody of the present invention. You can also.
  • human peripheral blood lymphocytes are transplanted using the polypeptide or a partial peptide thereof as an antigen, and transplanted into Severe combined immune deficiency (SCID) mice.
  • a human antibody can also be prepared by immunization using the above method and
  • RA is extracted from the obtained hybridoma producing the human antibody, the gene encoding the desired human antibody is cloned, this gene is inserted into an appropriate vector, and this is inserted into an appropriate host.
  • human antibodies can be produced in larger quantities.
  • an antibody with low binding to an antigen can be obtained as an antibody with higher binding by using an evolutionary engineering technique known per se. You can also.
  • a partial fragment such as a monovalent antibody can be prepared using, for example, papain.
  • It can be prepared by cutting the Fab portion and the Fc portion and collecting the Fab portion using an affinity column or the like.
  • the thus-obtained antibody that specifically binds to the protein of the present invention can also be used as a neutral antibody that specifically binds to the protein of the present invention and thereby inhibits the activity of the protein.
  • There is no particular limitation on the method for selecting a substance that inhibits the activity of the protein For example, whether the function of the target protein in the introduced substance is inhibited by contacting the antibody with the DNA transfectant prepared in (2) above There is a method of analyzing whether or not to do so.
  • Such a neutralizing antibody can be used alone for the clinical application, but can also be used as a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight.
  • a powerful drug can be administered in various forms, such as tablets, capsules, granules, powders, or syrups, orally, or injections, drops, ribosomes. And parenteral administration with suppositories and the like. In addition, the dose can be appropriately selected depending on symptoms, age, weight, and the like.
  • the protein of the present invention is prepared as a recombinant protein as described in (2) above, and by analyzing this, it can be confirmed that it has the activity estimated in (1). Furthermore, analysis can also be performed by combining with an antibody or the like prepared as described in (4) above.
  • the activity of the protein of the present invention is, for example, a force S that can be analyzed by the following method, and is not limited thereto.
  • these analysis methods can also be used for screening for a function activator or a function inhibitor of the protein of the present invention and a screening for a protein expression regulator of the present invention, which will be described later.
  • the binding activity to the TGF ⁇ receptor family was determined, for example, by preparing a clone in which the C tag was bound to the C-terminus of the TGF / 3 receptor family and a clone in which a flag tag was bound to the C-terminus of the protein of the present invention. After co-expression in cosl cells, etc., and the action of the TGF / 3 family molecule, a cell lysate was obtained, immunoprecipitated using an anti-flag antibody, electrophoresed, and used for anti-HA antibody. By conducting the procedure, the binding between both proteins can be confirmed (Nature Vol. 401, 480-483 (1999)).
  • the effect of the protein of the present invention on signal transduction by TGF) 3 family molecules can be evaluated, for example, by an Atsushi system using a reporter gene having a BMP response element (BRE) or an activin response element (ARE). it can. Specifically, a reporter gene in which a luciferase gene is linked downstream of the BRE sequence is used.
  • the protein of the present invention, the TGF] 3 receptor family, and the reporter gene are shared with P19 cells or force oocytes. The biological activity can be assayed by measuring the amount of luminescence when expressed and allowed to act on one molecule of the TGF) 3 family.
  • binding activity of the protein of the present invention to denatured LDL or the incorporation activity into cells can be assayed by those skilled in the art, for example, as follows.
  • oxidized LDL For example, in the case of oxidized LDL, first, cDNA is cloned into an expression vector for eukaryotic cells, and introduced into CH0-K1 cells or HEK293 cells using a conventional gene transfer method. Express the protein. Next, this is a radioactively labeled oxidized LDL
  • the activity of the protein of the present invention is measured using denatured LDL labeled with fluorescence. It may be measured by low cytometry.
  • the function of the protein of the present invention can be confirmed by measuring the ATP consuming activity (JBC, 267, 7, 4854-4858 (1992)). That is, a membrane fraction (containing a membrane protein of lOig) was extracted from cells expressing the protein of the present invention, and 50 mM Tris-Mes (pH 6.8), 2 raM EGTA, 2 raM DTT, 50 mM KCl, and 5 mM Suspend in 0.1 ml of a solution consisting of mM sodium azide and keep at 37 ° C. Next, this is supplemented with 5 mM ATP, and treated with a drug, sugar, or fatty acid. After 20 minutes, the reaction is stopped with 5% SDS at 0.1 raM, and the consumed inorganic phosphoric acid is quantified using a color reaction, whereby the consumption activity can be measured.
  • JBC ATP consuming activity
  • lipid excretion atsey (BBRC, 290, 713-721 (2002)) introduced cDNA into cos cells, etc. using a known gene transfer reagent, and then used 1 / Ci / ml [ 3 H] cholesterol the next day. After labeling for 18 hours, wash with PBS. Next, after culturing in a DMEM medium containing no essential fatty acids for 2 hours, the medium was replaced with fresh DMEM medium, and cultivation was continued at 37 ° C for 4 hours in the presence or absence of 15 wg / ml apoA-1. It can be performed by measuring the radioactivity in the medium. In addition, the ability to excrete a drug such as an anticancer drug can be examined using a similar technique.
  • immunoglobulin-like domains consist of hundreds of proteins with different functions, such as antibodies, complement, the giant muscle protein titin, and tyrosine kinase-type receptors.
  • the immunoglobulin-like domains are resilient and are thought to be involved in cell adhesion, protein-protein interactions, and protein-ligand interactions.
  • the evaluation of the function of the immunoglobulin-like protein is not uniform, but can be performed by a commonly used method known per se based on each interaction. For example, binding test, surface plasmon resonance, two-hybrid method, fluorescent energy Methods include, but are not limited to, the energy transfer method and the specific heat measurement method.
  • the activity of complement one of the immunoglobulin-like proteins
  • Hedge-sensitized erythrocytes in which antibodies (hemolysin) are reacted with hedge erythrocytes, activate the classical pathway, but lyse as complement is activated.
  • EA Hedge-sensitized erythrocytes
  • serum or this protein activate the classical pathway, but lyse as complement is activated.
  • ⁇ 1 inhibitor 3 a type of immunoglobulin-like protein
  • ⁇ 113 is a member of the ⁇ 2 macroglobulin family and exists at high concentrations in blood but has a physiological role.
  • ⁇ 113 is known to inhibit the activity by covalently binding to the target protease via a thiol ester, and the function of this protein can be examined in a normal protease inhibitor evaluation system. It is also thought that ⁇ ⁇ I3 binds to cellular proteins that are incomplete and need to be removed and is involved in clearance via ⁇ -macroglobulin receptor. It can also be measured and evaluated using the system (Biochemistry 1989 Feb 7; 28 (3): 1406-12).
  • methods for analyzing the function of the protein of the present invention include, for example, (i) a method of comparatively analyzing the expression state of each tissue, disease, or developmental stage, and (ii) the interaction with other proteins and DNA. (I ii) a method of transducing into a suitable cell or individual and analyzing a phenotypic change, and (iv) a phenotype by inhibiting the expression of the protein in a suitable cell or individual. And a method of analyzing the change in According to such a method, the activity specific to the target protein can be analyzed from many aspects. In the method (i), expression of the protein of the present invention can be analyzed at the mRNA or protein level.
  • the in situ hybridization method In situ hybridization: Application to Developmental Biology & Medicine., nd. by Harris, N. and Wilkinson, DG, Cambridge University Press (1990)
  • a hybridization method using a DNA chip a quantitative PCR method, and the like.
  • a tissue staining method using an antibody that specifically binds to the protein of the present invention described later an ELISA method, a Western blot method, and the like can be mentioned.
  • the protein to be analyzed is a splicing variant in which a known variant is present
  • a cDNA that exists only in the cDNA encoding the protein to be analyzed and that encodes a known variant is It is preferable to use a non-hybridizing probe.
  • the quantitative PCR method a method in which primers that can generate amplified fragments of different lengths between the target variant and the known variant are selected and performed (Wong, Y., Neuroscience Let., 320: 141-145 (2002)), etc. Is mentioned.
  • the function of the protein of the present invention can be analyzed by examining the presence or absence of interaction between the protein of the present invention and a known protein.
  • a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, ribosome method
  • yeast two-hybrid method fluorescence depolarization method
  • surface plasmon method phage display method
  • ribosome method One example is the multiple display method.
  • the protein to be analyzed is a splicing variant in which a known variant is present
  • the known variant is analyzed for interacting substances in the same manner, and a substance that specifically interacts with the target protein is analyzed. Identification is preferred.
  • the cells into which the cDNA of the present invention is introduced are not particularly limited, but human cultured cells are particularly preferably used.
  • the method for introducing DNA into cells is described above.
  • the phenotype of the transfected cells includes cell viability, Cell growth rate, cell differentiation, if the cell is a neuron, neurite outgrowth, localization and translocation of intracellular proteins, etc., which can be observed with a microscope, etc., and expression of specific proteins in cells Includes those that can be analyzed by biochemical experiments such as changes.
  • these phenotypes can be similarly introduced into cells, and a phenotype related to the variant to be analyzed can be identified by comparative analysis.
  • the method can be efficiently performed by a method using an oligonucleotide described below or an RNA interference method.
  • a known variant is present in the target protein to be analyzed
  • the same analysis is performed on the known variant and other variants, and the target protein is analyzed by comparative analysis. Function can be identified.
  • This method of screening for a regulatory substance may be any method as long as it can obtain a substance that specifically binds to the protein of the present invention and has an activity of inhibiting, antagonizing or enhancing the activity of the protein.
  • the protein of the present invention is brought into contact with a test substance, and the test substance is selected by using the change in the activity of the protein of the present invention as an index, after selecting by using the binding property to the protein as an index.
  • a method can be used.
  • the test substance may be any substance that can interact with the protein of the present invention and affect the activity of the protein. Physically, for example, peptides, proteins, non-peptidic compounds, low molecular weight compounds, synthetic compounds, fermentation products, cell extracts, animal tissue extracts and the like can be mentioned. These substances may be new substances or known substances.
  • a method for analyzing the interaction between the test substance and the protein of the present invention a conventional method known per se can be used.
  • the plasmon method, the phage display method, the ribosomal display method, or the competition analysis method with the antibody described in the above (4) can be used.
  • the substance found to bind to the protein of the present invention by such a method is then analyzed by analyzing how the activity of the protein of the present invention is affected in the presence of the substance. Whether it is used as a modulator or not is identified.
  • the above-mentioned human homologous protein or orthologous protein for the DNA or recombinant protein of the present invention to be used. Further, the substances screened by the above method may be selected as drug candidates by screening in vivo.
  • the analysis of the change in the activity of the protein of the present invention can be carried out by a method known per se and generally used, based on the method for analyzing the function of the protein of the present invention (confirmation method of activity).
  • confirmation method of activity a method for analyzing a substance that regulates each activity of the protein of the present invention will be described with reference to specific examples.
  • the DNA introduced A protein serving as a substrate is introduced in the same manner.
  • the dephosphorylation of the substrate protein in the presence / absence of the selected substance with respect to this transductant is analyzed by a commonly used method known per se. Specifically, it can be performed using the method described in (5-1) above.
  • the substance may function as an activator of binding to the TGF] 3 receptor family, and may decrease, Alternatively, when inhibited, the substance can be identified as possibly functioning as an inhibitor of the binding activity to the TGF) 3 receptor family.
  • the protein of the present invention has a binding activity to the TGF / 3 receptor family, but the TGF] 3 receptor family includes, for example, fibers of tissues related to renal fibrosis, pulmonary fibrosis, myocardial infarction and the like.
  • the compounds identified as modulators of the expression of this receptor by the present screening method can be used as therapeutic agents for various fibrotic diseases and the like.
  • Specific types of diseases include, for example, glomerulonephritis, scarring of nerves, scarring of skin, scarring of eyes, pulmonary fibrosis, arterial injury, proliferative retinopathy, retinal detachment, respiratory distress syndrome , Cirrhosis, Bosto myocardial infarction, Bosto angioplasty restenosis, keloid scar formation, scleroderma, vascular disorders, cataracts, glaucoma, osteoporosis and the like.
  • the therapeutic agent for such a disease is for treating a condition in which the effect of the TGF family molecule is harmful to an individual, and the effect is an effect of promoting fibrosis.
  • Specific methods for analyzing a substance that regulates the binding activity with modified t ⁇ LDL include, for example, when analyzing a substance that regulates the binding activity with modified LDL, introducing the modified LDL into the DNA transductant. I do.
  • the interaction between the protein of the present invention and denatured LDL in the presence / absence of the selected substance is analyzed with respect to this transductant by a method known per se and used in a usual manner. Specifically, it can be performed using the method described in (5-2) above. If the binding to denatured LDL is increased as compared to the absence of the substance, the substance may function as an activator of binding to denatured LDL and may be reduced or inhibited.
  • the substance can be identified as possibly acting as an inhibitor of binding to denatured LDL.
  • the protein of the present invention has a binding activity to denatured LDL, it can be estimated that the protein is involved in the incorporation of oxidized LDL-acetylated LDL into cells and cell dysfunction. Therefore, compounds that can be identified by this screening method include hyperlipidemia, the onset and progression of atherosclerosis, atherosclerosis, genetic diseases associated with atherosclerosis, familial hypercholesterolemia, myocardial infarction, cerebral infarction, etc. It can be used as a therapeutic agent.
  • a specific analysis method of a substance that modulates ATP-binding transporter activity for example, when analyzing a substance that modulates ATP-binding transporter activity, specifically, as described in (5-3) above, It can be performed by using the method described in the above. If the binding to ATP is increased compared to the absence of the substance, the substance may function as an ATP-binding transporter activator and if reduced or inhibited Can identify that the substance may function as an ATP binding inhibitor.
  • substances screened by the above method may be selected as drug candidates by screening in vivo.
  • the ABC transporter is known to be involved in various diseases, drug metabolism and the like.
  • the ABC transporter subtype ABCA 1 is a gene that causes HDL metabolism and causes Tangier disease
  • ABCB 1 is responsible for the extracellular excretion of anticancer drugs
  • ABCC 7 is a gene that causes cystic fibrosis
  • ABCC 8 is a treatment for diabetes It is known to be a receptor for the drug sulfonylprea.
  • compounds that can be identified by this screening method include, for example, diabetes mellitus, atherosclerosis ⁇ coronary heart aisease, cystic flDrosis ⁇ adrenoleukodystrophy ⁇ Stargardt 's disease ⁇ drug-resistant tumours
  • Dubin- Johnson syndrome ⁇ Byler 's disease ⁇ progressive familiar intrahepatic cholestasis ⁇ X- linked siderblastic anemia and ataxia ⁇ persistent
  • the method described in the above (5-4) can be used as a specific method for analyzing such a substance that regulates the activity of the immunoglobulin-like protein.
  • the method of analyzing the function of an imnoglobulin-like protein is not uniform, it can be carried out by a commonly used method known per se, based on each interaction. Examples include, but are not limited to, binding tests, surface plasmon resonance, two-hybrid methods, fluorescence energy transfer methods, and specific heat measurements. 'More preferably, it is preferable to measure and evaluate the function specific to the member of the superfamily having the immunoglobulin-like domain, for example, as described in (5-4) above, according to the evaluation method suitable for each. .
  • the substance may function as an activator of the immunoglobulin-like protein, When reduced or inhibited, it can be identified as having the potential to function as an immunoglobulin-like protein activity inhibitor.
  • the protein of the present invention has immunoglobulin-like protein activity and is presumed to be involved in immune reactions such as complement activation, inflammatory reactions, allergic reactions, protease inhibitory activities, receptor or adhesion molecule actions. Is done. Therefore, substances identified by the above analysis method include systemic erythematosus, congenital complement component deficiency, rheumatoid arthritis, immune diseases such as autoimmune diseases, inflammatory diseases such as glomerulonephritis and hepatitis. It can be used as a remedy for diseases such as infectious diseases, cancer and infertility.
  • modulators can be used alone as the above active ingredient when clinically applied, or can be used as a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier.
  • the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight.
  • the drug can be administered in various forms. Examples of the dosage form include oral administration of tablets, capsules, granules, powders, syrups, and the like, or injections, drops, ribosomes. And parenteral administration using suppositories and the like. The dose can be appropriately selected depending on the condition, age, weight, and the like.
  • Examples of the screening method include a method of analyzing the expression level of the protein of the present invention or the mRNA encoding the protein in the presence of a test substance.
  • a method of analyzing the expression level of the protein of the present invention or the mRNA encoding the protein in the presence of a test substance Specifically, for example, cells expressing the protein of the present invention described in (2) are cultured in an appropriate medium containing a test substance, and the amount of the protein of the present invention expressed in the cells is determined by ELISA. And the like, or the amount of mRNA encoding the protein of the present invention in the cells can be analyzed by quantitative reverse transcription PCR, Northern plotting, or the like.
  • test substance those described in (6) can be used. According to this analysis, if the amount of the protein or mRNA expressed in the cells cultured in the absence of the test substance increases as compared to the amount of the protein or mRNA, the substance functions as the substance for promoting expression of the DNA of the present invention. If it is possible and conversely decreases, it can be determined that the substance can be used as a substance for inhibiting the expression of the DNA of the present invention.
  • the above-mentioned active ingredient can be used alone for clinical application, but it can also be used as a pharmaceutical composition by mixing it with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier is between 1 and 90% by weight. Can be varied.
  • powerful drugs can be administered in various forms, such as tablets, capsules, granules, powders, or syrups, orally, or injections, drops, ribosomes And parenteral administration with suppositories and suppositories. The dose can be appropriately selected depending on the condition, age, weight, and the like.
  • the transfected DNA containing the DNA of the present invention described in (1) above is constructed, introduced into a fertilized egg of a mammal other than human, and transplanted into a female individual uterus to generate the present invention.
  • a non-human mammal into which the DNA has been introduced can be produced. More specifically, for example, after superovulation of a female individual by hormone administration, it is mated with a male, a fertilized egg is excised from the oviduct on the first day after mating, and microinjection of the introduced DNA into the fertilized egg is performed. It will be introduced by such methods.
  • the surviving fertilized eggs are transplanted into the uterus of a pseudopregnant female individual (foster parent) to give birth.
  • a pseudopregnant female individual foster parent
  • Whether or not the desired DNA has been introduced into the neonate can be identified by performing Southern blot analysis on DNA extracted from cells of the individual. Examples of mammals other than humans include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats, and the like.
  • the thus-obtained DNA-introduced animal of the present invention is used to breed this individual and subculture them in a normal breeding environment while confirming that the introduced DNA is stably maintained, thereby obtaining the offspring. Obtainable. In addition, by repeating in vitro fertilization, the offspring can be obtained and the strain can be maintained.
  • the non-human mammal into which the DNA of the present invention has been introduced can be used as an analysis of the function of the DNA of the present invention in a living body, or as a screening system for a substance regulating the function.
  • a resin substrate such as a nylon film or a polypropylene film, a nitrocellulose film, a glass plate, a silicon plate, or the like is used as a base for binding proteins and DNA, but the detection of hybridization is non-RI.
  • a glass plate or a silicon plate containing no fluorescent substance is preferably used.
  • the binding of the protein or DNA to the substrate can be easily carried out by a commonly used method known per se.
  • the amino acid sequence of the protein of the present invention and the nucleotide sequence of DNA can also be used as sequence information.
  • the nucleotide sequence of the DNA includes the nucleotide sequence of the corresponding RNA. That is, a database of amino acid sequences and nucleotide sequences can be constructed by storing the obtained amino acid sequences and nucleotide sequences in an appropriate recording medium in a computer-readable predetermined format. This database may contain other types of proteins and the base sequences of the DNA that encodes them. In the present invention, the database also means a computer system that writes the above-mentioned sequence on an appropriate recording medium and performs a search according to a predetermined program.
  • Suitable recording media include, for example, magnetic media such as flexible disks, hard disks, and magnetic tapes, and optical media such as CD-ROM, MO, CD-R, CD-RW, DVD-R, and DVD-RW. And semiconductor memories. Example Hereinafter, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited to these examples.
  • mRNA-prepared mouse (C57BL / 6) 0.5 or more of each organ or tissue: 1 g was homogenized with a 10 ml suspension, and 1 ml of 2M sodium acetate at pH 4.0 was added to the same amount of phenol / A mixed solution of black-mouthed form (5: 1 by volume) was added for extraction. When the same amount of isopropanol was added to the aqueous layer after the extraction, RNA separated and precipitated from the aqueous phase. After incubating the sample on ice for 1 hour, the precipitate was collected in a refrigerated centrifuge at 4,000 rpm for 15 minutes.
  • 5-methyl_dCTP, dATP, dTTP, and dGTP were each diluted with 3,000 units of reverse transcriptase in a reaction volume of 165 ⁇ l in a final volume of 165 ⁇ l.
  • HC1 pH 8.3
  • KC1 75 mM KC1
  • 3 mM MgC12 10 mM DTT
  • 52 ng / ⁇ 1 BSA RNase inhibitor 5 units
  • Oligonucleotide containing the recognition sequence of the restriction enzyme Xho I (SEQ ID NO: 4) (in the sequence, V represents A, G, or C, and N represents A, G, C, or T) 12. 6 ⁇ l was used as a primer.
  • RNase-free water 5 ⁇ EDTA 8 ⁇ 1, 1 0% SDS 2 ju 1, proteinase (P roteinase) ⁇ 20 ⁇ g was added, in 4 5 ° C Heated for 15 minutes. After extraction with phenol Z-cloth form and precipitation with ethanol, the precipitate was dissolved in 47 ⁇ l of RNase-free water (hereinafter referred to as RNase-free water).
  • Biotinylation of RN ⁇ diol A two-step reaction to bind biotin to the diol site of RN ((present at both the 5 'end of the Cap structure and the 3' end of the poly A chain ribose) was done. These are the oxidation of the diol group followed by the coupling reaction of the oxidized RNA with the biotin hydrazide. First, 15 // g of the RNA-first strand cDNA complex obtained by the reverse transcription reaction was used with 6.6 mM sodium acetate buffer (pH 4.5) and sodium periodate as an oxidizing agent. In a 50 ⁇ l reaction. This oxidation reaction was performed on ice for 45 minutes under light-shielded conditions.
  • yeast tRNA treated with DNase I was added to 5 mg (500 ⁇ l) of magnetic beads. After adding to beads (magneticporous glass (MPG) particles coated with st reptavidin (CPG, NJ)), leaving it on ice for 1 hour, it was washed with a solution of 5 OmM EDTA and 2 M NaC1.
  • the beads were suspended in 500 ⁇ l of a solution of 50 mM EDTA and 2 M NaCl, and the RNase I-treated cDNA obtained in (4) was added. By stirring for 30 minutes at room temperature, the magnetic beads and the full-length cDNA were bound.
  • the beads capturing the full-length cDNA were washed 4 times with a solution of 5 OmM EDTA and 2 M NaCI, 0.4% SDS, 50 g / ⁇ l once with yeast tRNA, and 1 OmM NaCl, 0.2 mM EDTA, 1 OmM Tris-HC1 (pH 7.5), once with 20% glycerol, once with 50 ⁇ g no ⁇ 1 yeast tRNA aqueous solution, once with RNAse H buffer 2 OmM T ris- HC 1 (p H 7.
  • the single-stranded full-length cDNA recovered in this manner is extracted with phenol Z-chloroform, and the volume is reduced to 100 // 1 or less in a speed bag, and then G 25 / G 1 OOS ephadex chromatography Attached.
  • the fraction having RI activity was collected in a silicon-treated microtube, 2 ⁇ g of dalycogen was added, and the precipitate obtained by ethanol precipitation was dissolved in 30 ⁇ l of ultrapure water.
  • the single-stranded cDNA 30/1 recovered in (5) above was used in a final volume of 50 ⁇ l of the reaction solution at 20 OmM sodium sodium codylate (pH 6.9), 1 mM MgCl 2 , 1 mM mM C o C l 2, 1 mM 2- mercaptoethanol, 100 / M under the conditions of dGTP, terminal de o carboxymethyl nucleotidyl transferase (T a Ka R a, Ltd.) 3 2 with Unit 3 7 ° C For 30 minutes for oligo dG addition reaction. At the end of the reaction, EDTA was added to 5 OmM, and after a series of extraction with phenol / chloroform and ethanol precipitation, it was dissolved in 31 ⁇ l of ultrapure water.
  • the synthesis of the second-strand cDNA obtained by converting the first-strand cDNA into a type II was carried out as follows. In a final reaction volume of 60 ⁇ l, use a second strand low buffer (200 mM Tris-HC1 (pH 8.75), 10 OmM KC1, 10 OmM (NH 4 ) 2 SO 4 , 2 OmM M g S0 4, 1% T riton X- 1 00, lmg / ⁇ 1 BSA) 3 ⁇ 1, second Kusaridaka buffer (20 OmM T ris- HC 1 ( pH9.
  • the double-stranded full-length cDNA obtained by the above method was inserted into an LZAPIII vector and recovered as a library.
  • the ⁇ II vector is obtained by modifying SEQ ID NO: 6, which is a partial sequence of the multiple cloning site of the ZAP II (manufactured by ST RAT AG EN ⁇ ) vector, to SEQ ID NO: 7 and adding two SfiI sites. It was introduced in
  • ⁇ PS (RI KEN) (named ⁇ —FLC_1 (FLC means FULL—LENGTH cDNA)) is a modification of the ⁇ PS vector from MoBiTec (Germany) for cDNA. It was done.
  • BamHI and Sa1I which are convenient for cDNA insertion, are introduced into the close-up site located on both sides of 10 kbpstuffer, and cDNA from 0.5 kb to about 13 kb can be cloned.
  • a 6 kb DNA fragment inserted into the XbaI site Japanese Patent Application Laid-Open No. 2000-325080).
  • RNA driver The mRNA prepared in Example 1 (1)
  • RNA prepared by the invitro transcription reaction were used as drivers.
  • the latter RNA is further divided into two types (hereinafter referred to as “(b) RNA driver” and “(c) RNA driver”).
  • cDNA was recovered from RNA-cDNA removed by normalization and cloned into a phage vector. After infection with Escherichia coli, 1000 to 2000 plaques are mixed per starting material into one library (mini-library), which is converted into plasmid DNA by a conventional method. Infect, transform into phagemids, and infect again to obtain plasmid DNA).
  • RNA driver was obtained by extraction of phenol Z-cloth form.
  • mini-libraries are prepared from nine types of tissues (pancreas, liver, lung, kidney, brain, spleen, testis, small intestine, stomach), and the nine types of mini-libraries are mixed. To obtain RNA.
  • RNA For another RNA, a library (about 20,000 clones) already stored as a non-overlapping clone is cultured, and the resulting DNA is used for (b) in vitro transcription reaction in the same manner as the RNA driver. (C) The RNA driver was selected.
  • RNA was labeled with a biotin label using the Label-ITB iotin Labeling Kit (manufactured by Mirus Corporation), and then added to the tester cDNA at a ratio of 1: 1: 1. Reaction at Rot 10 (4 2 ° C), and the second strand was synthesized from the supernatant collected by streptavidin bead (CPG) treatment.
  • CPG streptavidin bead
  • One representative clone was selected from each cluster. Representative clones were selected by Q-bot (manufactured by GENETIXLIMITED), and were placed on a 384-well plate. At that time, E. coli was cultured in 50 ⁇ l of L ⁇ medium at 30 for 18 to 24 hours. At this time, if the cDNA library has been introduced into the PS vector and transformed Escherichia coli DH10 ⁇ , add 10 Omg / m1 ampicillin and 5 Omg / m1 kanamycin and add it to the Zap vector. When introduced into the SOLR system, ampicillin at 10 OmgZm1 and streptavidin at 25 mg / m1 were added.
  • Each of the clones cultured in the above (1) is further cultured in 1.3 ml HT solution containing lOOmgZml of ampicillin, and the cells are collected by centrifugation. Then, QIAprep 96 Turbo (QI AG The plasmid DNA was collected and purified using EN. To check the length of the cDNA inserted into the obtained plasmid, 130 of the plasmid DNA obtained above was digested with the restriction enzyme PVuII and subjected to 1% agarose gel electrophoresis. Was.
  • Plasmids were divided into two categories: those with insertion sequences shorter than 2.5 kb and those with longer insertion sequences. Of these clones, the clone having an insertion sequence shorter than 2.5 kb was analyzed for the nucleotide sequence from both ends. In this case, the plasmid was prepared using the primers shown in SEQ ID NOS: 8 (sense strand) and 9 (antisense strand) when the vector was PS, and when the vector was Zap.
  • Gaps that could not be analyzed by the above nucleotide sequence analysis were determined by the primer walking method.
  • AB IPris m377 and Z or AB IPris sm3700 manufactured by Applied Biosystems Inc.
  • Big Dy ete rm inatorkit Big Dy ete rm inatorkit
  • Cyclic Sequencing FS ready Reaction Kit Applied B iosyst ems Inc.
  • sequencing of clones having an inserted cDNA longer than 2.5 kb was performed by the shotgun method.
  • ShimadzuRISA384 and DYE namicETTerminea torrccyclesequenecinigkit were used.
  • 48 DNA fragments grown by PCR from 48 independent representative clones were used to generate a shotgun library. The ends of the amplified DNA fragments were blunt-ended with T4 DNA polymerase.
  • This DNA fragment was inserted into a pUC18 vector, and Escherichia coli DH10B was transformed with the recombinant vector.
  • a plasmid was prepared from this E. coli in the same manner as in (2) above.
  • nucleotide sequence was determined by nucleotide sequence analysis from both ends, and the nucleotide sequences were ligated on a computer, followed by Double Stroke Shearing Device (manufactured by Fire Inc. ) Shearing was performed. Nucleotide sequencing by the shotgun method was performed with duplication of 12 to 15 clones. The gap whose sequence could not be determined by the nucleotide sequence determination was determined by primer walking in the same manner as described above.
  • dnafo rm51839 consists of 1156 bases as shown in SEQ ID NO: 1.
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 1 using BLAST, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database).
  • database registration mark AJ 277487 Homo sapiensputativetran smemb raneprotein NMA precursor force ev alue: 3 X l 0- 70 , 138 with 67% degree of coincidence over the amino acid residues
  • database registration mark AF three hundred eighty-seven thousand five hundred and thirteen kinase- deficient TG betasuperf am ilyreceptorsub un it forces e_v a 1 ue: 1 X 10- 48, 1 53 in Amino acid residue 67% degree of coincidence over the group
  • a database registration mark B CO 19378, Mu smuscu 1 us, BMP andactivin memb rane- boundi nh ibitor, h omo 1 og (X enopuslaevis) is, e _ va 1 ue: were hits in the IX 10- 48, 153 68% of the degree of match over the amino acid residues.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 1 had a binding activity to the TGF] 3 receptor family and was a kinase-deficient TGF betasuperfamilyreceptor subunit.
  • the homology of (i) and (ii) was observed.
  • the protein encoded by this gene is considered to be translated from the region of base number 218 to 869 in SEQ ID NO: 1.A few bases deleted at about several bases at base number 221 and about a few bases at base number 447 or two salts It was thought that there was an insertion of the group.
  • TGF receptor families are associated with bone formation, autoimmune diseases, renal fibrosis, pulmonary fibrosis, myocardial infarction, etc., and activators or inhibitors of this receptor may be used as therapeutics for these diseases. It was speculated that it could be developed.
  • dnafo rm34810 consists of 3294 bases, of which base numbers 95 to 976 constitute an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 293 amino acid residues (SEQ ID NO: 14).
  • a homology search was performed on the amino acid sequence encoded by SEQ ID NO: 12 using BLAST, and a SPTR protein database (swi SS-PROT protein sequence database and a TrEMBL nucleic acid translation database were integrated).
  • database registration code AB016237 lectin-like oxidized LDL receptor (Oryctolagus cuniculus) force e— value: 1 X 10 " 2 ⁇ 235 29% identity over amino acid residues
  • database registration mark BC022295 oxidised low density lipoprotein (lectin -like) receptor 1 (Homo sapiens) force s, e- value: hit in the 27% degree of coincidence over the 2 X 10- 18, 2 37 amino acid residues.
  • the family of denatured LDL receptors is involved in the incorporation of oxidized LDL-acetylated LDL into cells and cell dysfunction. Activators and inhibitors of this family receptor are hyperlipidemia. It was speculated that it would be useful as a therapeutic agent for various diseases such as the onset and progression of arteriosclerosis, atherosclerosis, genetic diseases associated with arteriosclerosis, familial hypercholesterolemia, myocardial infarction and cerebral infarction.
  • dnafo rm35841 consists of 1021 bases, of which base Nos. 94 to 810 have an open reading frame (terminated). (Including codons).
  • the amino acid sequence predicted from the open reading frame consists of 238 amino acid residues (SEQ ID NO: 15).
  • a homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 13, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database).
  • the family of modified LDL receptors is involved in the uptake of cells such as oxidized LDL-acetylated LDL and cell dysfunction. Activators and inhibitors of this family receptor are hyperlipidemia. It was speculated that it would be useful as a therapeutic agent for various diseases such as the onset and progression of arteriosclerosis, atherosclerosis, genetic diseases associated with arteriosclerosis, familial hypercholesterolemia, myocardial infarction and cerebral infarction.
  • dnaform26225 was composed of 2656 bases, of which nucleotides 163 to 2520 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 785 amino acid residues (SEQ ID NO: 29).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 16 using BLAST, and a SPTR protein database (SWISS—PROT protein sequence database and TrEMBL nucleic acid translation database integrated) In the (i) database registration symbol
  • the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 16 was searched for protein characteristics by HMM PFAM, a sequence showing the characteristics of ABC transporter (sequence entered as ABC-tran in P fam) was found. was found. From these facts, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 16 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER 3), and has ATP binding. Presumed to have sex transporter activity.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 16 is an ABC transporter involved in the extracellular excretion of drugs, etc., and foreign substances such as drugs, and endogenous substances such as calcium, phospholipids and amphiphiles. It was speculated that this would be involved in the transportation of refuse. (5) dnaform41412 (SEQ ID NOS: 17, 30)
  • dnafo rm41412 was composed of 3831 bases, of which base numbers 1817 to 3829 were open reading frames.
  • the amino acid sequence predicted from the open reading frame consists of 671 amino acid residues (SEQ ID NO: 30).
  • SEQ ID NO: 1 7 Homology searches were performed using the BLA ST for the amino acid sequence encoded by that combined S PTR protein database (SWI S 1 S- PROT protein sequence database and T r EMB L nucleic translation database integration (I) Database registration symbol: trembl
  • the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 17 was searched for protein characteristics by HMM PFAM, a sequence showing the characteristics of ABC transporter (sequence entered as ABC_tran in P f am) was found. From these facts, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 17 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER 3), and has ATP binding. Presumed to have sex transporter activity.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 17 is an ABC transporter involved in extracellular excretion of drugs, and foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphiphiles. It was speculated that this would be involved in the transportation of refuse. (6) dnaform43395 (SEQ ID NOS: 18, 31)
  • dnaform43395 was composed of 3950 bases, of which base numbers 201 to 3950 were open reading frames.
  • the amino acid sequence predicted from the open reading frame consists of 1250 amino acid residues (SEQ ID NO: 31).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 18 using BLAST. The results were in the S PTR protein database (SWI SS—PROT protein sequence database and Tr EMB L nucleic acid translation database).
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 18 A protein characteristic search by PFAM was performed, and a sequence showing the characteristics of ABC transporter (sequence that was entered as Pfan ⁇ ABC-tran) was found.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 18 had a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A and had ATP-binding carrier activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 18 is an ABC transporter involved in extracellular excretion of drugs, etc., and foreign substances such as drugs, and intrinsic factors such as calcium, phospholipids, amphiphiles, etc. It was speculated that it might be involved in the transport of toxic substances.
  • dnaform33133 was composed of 4119 bases, of which base numbers 68 to 2914 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 948 amino acid residues (SEQ ID NO: 32).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 19 using BLAST. The results were in the SPTR protein database (integrating the SWI S_PROT protein sequence database and the TrEMBL nucleic acid translation database). (I) Database registration symbol
  • AY028900-1, Homo sapiens ATP—binding cassette A10 — Value 0, 958 amino acid residues (there were hits with a 60% match.
  • the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 19 was searched for protein characteristics using HMM PFAM, a sequence showing the characteristics of ABC transporter (a sequence entered as ABC-tran in P f am) ) was found. From these facts, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 19 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A9, and has Presumed to have carrier activity. From this, the protein whose nucleotide sequence is shown in SEQ ID NO: 19 is an ABC transporter involved in the extracellular excretion of drugs, etc., and foreign substances such as drugs, calcium, phospholipids, amphiphilic substances, etc. It was speculated that it might be involved in the transport of endogenous substances.
  • dnaform63577 was composed of 3300 bases, and among them, bases from 1382 to 2458 had an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 358 amino acid residues (SEQ ID NO: 33).
  • a homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 20, and it was found in the SPTR protein database (integrated SWI S_P ROT protein sequence database and TrEMBL nucleic acid translation database). , (I) Database registration symbol
  • SMINT1000042 weakly similar to ATP - BINDING CASSETTE, SUB-FAMILY A, is MEMBER 3, e - va 1 ue : 5X10- 143, 75% degree of coincidence over the 332 amino acid residues, (ii) tremblnew
  • AY0288991 AY028899_1 , Homo sapiens ATP - binding cassette A9 is, E - value at 5Xl (T 143, 332 75% degree of coincidence over the amino acid residues, (iii) a database registration symbol tremblnew
  • AY0289001 AY028900_1, Homo sapiens ATP -binding cassette A10 but hit in E-value 5Xl (T 124 , 330 65% degree of coincidence over the amino acid residues.
  • amino acid sequence nucleotide sequence is co one de shown in SEQ ID NO: 20, proteins wherein the search by HMM PFAM As a result, a sequence exhibiting the characteristics of ABC transporter (sequence that is entered as ABC-tran in P f am) was found. Proteins have sequences similar to human ATP-BINDING CASSETTE, SUB-FAMILY A Therefore, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 20 is an ABC transporter involved in the extracellular excretion of the drug, and the like. And transport of endogenous substances such as calcium, phospholipids and amphiphiles It was speculated that it would be overpowered.
  • dnaform30449 was composed of 2844 bases, of which base numbers 397 to 2235 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 612 amino acid residues (SEQ ID NO: 34).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 21 using BLAST. The results were in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). (I) Database registration symbol
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 21 had a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A9, and was presumed to have ATP-binding carrier activity.
  • the protein whose base sequence shown in SEQ ID NO: 21 is encoded is an ABC transporter involved in the extracellular excretion of a drug, such as a foreign substance such as a drug, calcium, phospholipid, and an amphipathic substance. It was speculated that it might be involved in the transport of endogenous substances.
  • dnafo rm42393 consists of 2537 bases, of which base numbers 163 to 1674 have an open reading frame (including a stop codon). Mu).
  • the amino acid sequence predicted from the open reading frame is
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 22 was searched for protein characteristics using HMM PFAM.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 22 had a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, and had ATP-binding carrier activity. Therefore, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 22 is an ABC transporter involved in the extracellular excretion of drugs, etc., and foreign substances such as drugs, endogenous substances such as calcium, phospholipids, amphiphiles, etc. It was presumed to be involved in the transport of substances.
  • dnafor m39112 was composed of 2247 bases, of which base numbers 940 to 2247 were open reading frames.
  • the amino acid sequence predicted from the open reading frame consists of 436 amino acid residues (SEQ ID NO: 36).
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 23 had a sequence similar to the ATPase family of transporters and had ATP-binding transporter activity. From this fact, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 23 is a carrier ATPase family, which is involved in the transport of foreign substances such as drugs and endogenous substances such as canolesum, phospholipids and amphiphiles. It was inferred.
  • dnaform m43238 was composed of 1554 bases, of which base numbers 277 to 1554 were open reading frames.
  • the amino acid sequence predicted from the open reading frame consists of 426 amino acid residues (SEQ ID NO: 37).
  • a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 24.
  • the results were in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database).
  • AB075819-l is human KIM1939, e- va 1 ue: 5X10- 155, 489 Amino 85% degree of coincidence over the acid residues,
  • AK0548861 AK054886_1, Homo sapiens cDNA FLJ30324weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3 is, E- value at 5 X 1 (T 154, 489 78% degree of coincidence over the amino acid residues, (iii) a database registration mark
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24 had a sequence similar to human ATP-BINDING CASSETTE and had ATP-binding carrier activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24 is an ABC transporter involved in the extracellular excretion of a drug and the like. It was estimated to be involved in the transport of substances.
  • dnaform60061 was composed of 2825 bases, of which base numbers 526 to 1449 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 307 amino acid residues (SEQ ID NO: 38).
  • a homology search was performed on the amino acid sequence encoded by SEQ ID NO: 25 using BLAST, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). , (I) Database registration symbol
  • AB075819- 1 humanKIM1939 is, e- va 1 ue: in 5 ⁇ 1 ( ⁇ 150, 300 86% over amino acid residues matching degree, (ii) trembl
  • AF0380071 AF038007_1 , Homo sapiens potential phospholipid- transporting AiPase AE3691 1 AE003694_20, gene: "CG14741"; Drosophila melanogaster genomic scaffold E-value 8X " 97 , and 292 amino acid residues were hit with 60% concordance.
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 25 has a sequence similar to the transporter ATPase family, and has ATP binding. It was presumed to have sex carrier activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 25 is a carrier ATPase family, which is involved in the transport of foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphiphiles. It was inferred.
  • dnaform49889 was composed of 4705 bases, of which base numbers 2015 to 3883 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 622 amino acid residues (SEQ ID NO: 39).
  • a homology search was performed on the amino acid sequence encoded by SEQ ID NO: 26 using BLAST, and it was found in the SPTR protein database (integrated swiSS-PROT protein sequence database and TrEMBL nucleic acid translation database).
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 26 had a sequence similar to the ATPase family of transporters and had ATP-binding transporter activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 26 is a carrier ATPase family, and is used for transporting foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphipathic substances. It was speculated that this would be involved.
  • dnafo rm60440 consists of 2611 bases, as shown in SEQ ID NO: 27, of which base numbers 70 to 987 contain an open reading frame (including a stop codon). Mu).
  • the amino acid sequence predicted from the open reading frame is
  • trembl AK0548861 AK054886_1 Homo sapiens cDNA FLJ30324 weakly similar to PROBABLE CALCIUM- TRANSPORTING ATPASE 3, Power e - va 1 ue: 5X10- 101 , 271 ⁇ Mino 63% degree of coincidence over the acid residues, (ii) trembl
  • AF0380071 AF038007_1, human Potential phospholipid- transporting ATPase IC is in E_value l X 10- 85, 290 amino acid residues 51% degree of coincidence over the group, (iii) a database registration mark
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 27 had a sequence similar to the ATPase family of carriers and had ATP-binding carrier activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 27 is a carrier ATPase family, which is involved in transport of foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphipathic substances. It was inferred.
  • dnaform m67267 was composed of 1569 bases, of which base numbers 102 to 1484 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 460 amino acid residues (SEQ ID NO: 41).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 28 using BLAST.
  • AE003694- 20, Homo sapiens cDNA FLJ30324 f is , clone BRACE2007138, weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3 is, E - value was hit with 62% degree of coincidence over the 2 X 10- 84, 338 Amino acid residues.
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 28 had a sequence similar to the transporter ATPase family and had ATP-binding transporter activity.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 28 is a carrier ATPase family, which is used for transporting foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphiphilic substances. It was speculated that this would be involved.
  • dnaform m38308 was composed of 1648 bases, of which nucleotides 4 to 1062 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 35 amino acid residues (SEQ ID NO: 47).
  • a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 42.
  • the SPTR protein database (the SW ISS-PROT protein sequence database and the TrEMBL nucleic acid translation database were integrated).
  • the protein (i) may be involved in the inflammatory reaction from the literature information (Biochemistry 27: 3568-3580 (1988)) in the database, and the protein (ii) may be related to the literature information ( J. Biol. Chem. 265: 2435-2440 (1990)), and the protein of the above (iii) can be found in the literature information in the database (J. Clin. Invest. 86: 96-106). (1990)), it has been clarified that they are involved in complement activity.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 42 was subjected to protein characteristic search using HMM PFAM. The sequence that is entered as 2-1 ⁇ in f 3111) was found.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 42 was an immunoglobulin-like protein having a function related to the inflammation and diarrhea reaction.
  • dnafo rm38407 was composed of 1700 bases, of which base numbers 8 to 1078 were open reading lames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 356 amino acid residues (SEQ ID NO: 48).
  • a homology search was performed using BLAST on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 43, and the SPTR protein database (SWI SS-PROT protein sequence database and the TrEMBL nucleic acid translation database were integrated).
  • database registration mark P01031 Complement C5 precursor (HUMAN) force e - va 1 ue: at 3 X 10- 84, 241 63% degree of coincidence over the amino acid residues
  • database registration code P06684 Complement C5 precursor (MOUSE) force e— value: 2 X 1 ( ⁇ 59% identity over 83 and 242 amino acid residues
  • CAVPO Complement C3 precursor
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 43 was subjected to a protein feature search using HMM PFAM.
  • the amino acid sequence 6 to 230 in SEQ ID NO: 48 showed a sequence exhibiting the characteristics of Alpha-2-macroglobulin (P fam A2M—N was identified as a sequence.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 43 was an immunoglobulin-like protein having functions related to inflammation and allergic reactions.
  • dnafo rm36427 was composed of 4725 bases, of which base numbers 1340 to 4429 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 1029 amino acid residues (SEQ ID NO: 49).
  • a homology search was performed using BLAST on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 44.
  • the SPTR protein database (SW ISS—PROT protein sequence database and TrEMBL nucleic acid translation database were integrated) during, (i) a database registration mark P14046, alpha- 1- inhibitor III precursor (RAT) force e - va 1 ue: 5 X 10- 117, 1098 at 31% degree of coincidence over the amino acid residues, and (ii ) Database registration code P20740, Ovostatin precursor (CHICK) force S, e-va 1 ue: 5 X 10- U , with 29% identity over 981 amino acid residues, and (iii) database registration C " ⁇ P20742, Pregnancy zone protein precursor (HUMAN) force s, e- va 1 ue: was hit with 29% of the degree of match over the 5 X 10- 109, 995 amino acid residues.
  • a database registration mark P14046 alpha- 1- inhibitor III precursor (RAT) force e - va 1 ue: 5 X 10- 117, 1098 at 31% degree of coincidence over the amino acid residues
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 44 was subjected to protein characteristic search using HMM PFAM. Enter as A2M in fam Sequence).
  • the protein encoded by the nucleotide sequence represented by SEQ ID NO: 44 was an imnoglobulin-like protein having a function related to the inhibition of protease activity.
  • dnafor m36949 was composed of 1650 bases, of which base numbers 111 to 488 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 125 amino acid residues (SEQ ID NO: 50).
  • a homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 45.
  • the SPTR protein database (SWISS—PROT protein sequence database and TrEMBL nucleic acid translation database integrated) during, (i) a database registration mark AF329485 1S e - V a 1 ue : 3 X 10- 52, 121 with 82% degree of coincidence over the amino acid residues, also (ii) a database registration mark AL356276 is, e- value : in 2 X 10 '2 84 amino acid residues 61% degree of coincidence over the further (iii) a database registration mark AF459634 I ev alue: at 2X 10 one 26, 84 61% of the degree of coincidence over the amino acid residues It was hit.
  • AF329485 has a structure similar to leukocyte Fc receptors and cell adhesion molecule PECAM-1 based on literature information (Igen unogenetics, 2002, May, 54 (2) 87-95), and is called IFGP family. I understood that.
  • dnafor m44492 was composed of 1922 bases, of which base numbers 49 to 1080 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 343 amino acid residues (SEQ ID NO: 51).
  • a homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 46, and the S PTR protein database (SWI SS-PROT protein sequence database and Tr EMB L nucleic acid translation database integrated) Among them, (i) database registration code AF329485 force e—va 1 ue: 0.0, 99% identity over 343 amino acid residues, and (ii) database registration code AF459634, e _value: 9 X 10 "", 327 Amino acid residues in the 58% degree of coincidence over the group, further (iii) a database registration mark AL356276, e -value: l with X l ( ⁇ 73, 51% of matches over 298 Amino acid residue AF329485 has a structure similar to leukocyte Fc receptors and cell adhesion molecule PECAM-1 based on literature information (Iramunogenetics, 2002, May, 54 (2) 87-95). It turned out to be called.
  • Tissue expression analysis was performed as described in Miki, R., et al., Proc. Natl. Acad. Sci. USA, 98, 2199-2204 (2001).
  • Nucleotide sequences of two mouse full-length cDNAs (dnafor m33 133, dnaf or m49889) and four kinds of mouse cDNA libraries FANTOM (http: // fantom. gsc. riken.) belonging to the same cluster as the full-length mouse cDNA to be analyzed (having a base sequence homologous to the cDNA). go.jp/)
  • the base sequence of cDNA derived from cDNA (FANTOM NO: 2310069H21, 5430413J22, 9430067009, 4831440K17) was amplified using Ml3 forward and reverse primers, and the PCR product was then treated with isopropanol.
  • the detection sensitivity of this DNA microarray was 1 to 3 copies of mRNA per cell.
  • the signal intensity of clones with approximately 80% match with the target sequence was 1/10 that of clones with perfect sequence match.
  • the signal intensity of clones with less than 80% match with the target sequence was at the background level.
  • C57BLZ6 J mouse fetus 49 tissues of C57BLZ6 J mouse fetus, neonate, adult (kidney, brain, spleen, lung, liver, testis, knee, stomach, small intestine, colon, cecum, placenta, heart, tongue, thymus, thymus Day), cerebellum, medulla oblongata, olfactory brain, epididymis, eyeball, cortex, follicular gland, uterus, ovary and uterus (1st day of pregnancy), bone, muscle, mammary gland (10th day of lactation), 10-day-old fetus Whole body, 11 day old fetal whole body, 13 day old fetal whole body, 11 day old fetal head, 12 day old fetal head, 13 day old fetal head, 15 day old fetal head, 16 day old fetal head Part, 17-day-old fetal head, 16-day-old fetal lung, 13-day-old
  • the above-mentioned probe solution was added to the DNA microarray, covered with a cover slip, and hybridized at 65 ° C- ⁇ in Hybricasete (ArrayaIt).
  • the DNA microarray was washed with 2 ⁇ SSC, 0.1% SDS, and then rinsed with 1 ⁇ SSC for 2 minutes and with 0.1 ⁇ SSC for 2 minutes.
  • the microarray was scanned using a ScanAnaray 500 000 confocal laser scanner, and the images were subjected to angular analysis using I MAGENE (BioDiversive).
  • the mRNA level (Cy3 labeling) in each tissue is expressed as the logarithm (1 og 2 ). Experiments were performed twice independently to increase data accuracy and reproducible results were obtained. Used. The results are shown in Table 1 below.
  • an increase or decrease of about 2 times is regarded as an experimental error. From this, when the value of the result shown in Table 1 is 1 or more, the amount of mRNA in a certain tissue is a control. Yes, it was interpreted as a significant increase. Conversely, if the value of the result is 11 or less, the amount of mRNA in a certain tissue is less than one-half that of the control, and the value of mRNA in the whole fetal body at 17.5 days of age is significant. It was interpreted as a decrease.
  • the mRNA quantity is 2 times, if it is 2, the mRNA quantity is 4 times, and conversely If the difference between the values of the tissues is 11, the amount of mRNA is 1/2 times, and if the difference is _2, the amount of mRNA is 1Z 4 times.
  • a mouse cDNA clone (dnafo rm30449, dnaform 51839, dnafo) belonging to the same cluster as the DNA spotted on the microarray and having a region having a nucleotide sequence identity of at least 80% over at least 200 bases.
  • rm41412, dnafo rm43395, dnafo rm60440, dnafo rm67267, dnafo rm36427) are also described in Table 1 as the cDNA to be analyzed, and the numerical values of the measurement results of the DNA spotted on the microarray are described instead.
  • DNA _ body _ whole ⁇ whole body head
  • dnafo rm33133 was attenuated overall as compared to the control, but equivalent or higher expression was observed in the heart, epididymis-derived fat cells, stomach, and uterus. Since dnafo rm41412 and dnafo rm43395 have high homology to FANTOMNO: 9430067009 belonging to the same cluster, when this is examined as a probe, testes, cerebellum, stomach, ⁇ ! However, the expression tended to increase in the cerebellum of a 10-day-old newborn.
  • dnafo rm49889 was enhanced as a whole as compared with the control, but the expression was increased in adipocytes and knees, and a tendency of increased expression was observed in kidney, liver, testis and the like.
  • mice C57BL / KsJ- + m / + m Jcl (female, 8 weeks old) whole brain, thalamus, lungs, kidneys, bone marrow, bone marrow, lentils, fat cells, liver, eyes
  • the expression of the following seven mRNAs encoding the protein of the present invention was determined using a light cycler constant-quantity PCR device (Roche's Diagnostics).
  • 3'-side primer MGGTCACCTCTCGGACTACA (SEQ ID NO: 57)
  • 5'-side primer TTGCGCAAAGATTTTGTGAT (SEQ ID NO: 58)
  • 5'-side primer GCACTACCCTACAGGACGGTTA (SEQ ID NO: 60)
  • dnafor m34810 was strongly expressed in lung, and also strongly expressed in bone marrow, knee and fat.
  • dnafo rm35841 was strongly expressed in lung, bone marrow and spleen, and was attenuated in diabetic knee and colon cancer.
  • the expression level of dnafo rm26225 was low, and specific expression was observed in adipose tissue.
  • dnafo rm391 12 was strongly expressed in the whole body, especially in liver, lung and knee. Although expression level of dnafo rm42393 was low, expression was observed in adipocytes and diabetic kidney.
  • dnafo rm432 38 and dnafo rm60061 were strongly expressed in bone marrow, strongly expressed in lung, and increased in diabetic fat.
  • the cDNA of the above clone and the cDNA Can be applied to the treatment and diagnosis of diabetes and cancer. Further, the protein encoded by the cDNA may be involved in a disease relating to a tissue in which the mRNA expression is varied as described above or a tissue having a high mRNA expression level.
  • the protein encoded by this cDNA is similar to the kinase-deficient human TGF] 3 receptor superamylysubunit AF387513, and compared to the control (17.5-day-old fetal whole body), bone and 10-day-old Expression increased in neonatal skin, testes, and lungs.
  • this protein is used for osteoporosis, autoimmune diseases and other immune diseases, inflammatory diseases, cancer, glomerulonephritis, nerve scar formation, skin scar formation, eye scar formation, lung fibrosis, arterial injury, Functions related to proliferative retinopathy, retinal detachment, respiratory distress syndrome, cirrhosis, lost myocardial infarction, postangioplastic restenosis, keloid scarring, scleroderma, vascular disorders, cataract, glaucoma, and infertility It was considered useful for the development of these therapeutic agents.
  • the protein encoded by this cDNA is similar to the oxidized low density lipoprotein (lectin-like) receptor and is predicted to be a family member of the denatured LDL receptor.It is strongly expressed in the lung, and is expressed in bone marrow and kidney. However, fat was also strongly expressed.
  • this protein is useful for the development of hyperlipidemia, atherosclerosis, atherosclerosis, genetic disease associated with arteriosclerosis, familial hypercholesterolemia, myocardial infarction, cerebral infarction, pulmonary embolism It has functions related to diabetes, arteriosclerosis, obesity, etc., and was considered to be useful for the development of these therapeutic agents.
  • This cDNA is similar to the oxidized low density lipoprotein (lectin-like) receptor and is predicted to be a family member of the modified LDL receptor, and is strongly expressed in lung, bone marrow and kidney. The expression was diminished in diabetic knee and colon cancer. Based on these findings, this protein is used for the development and development of hyperlipidemia, arteriosclerosis, atherosclerosis, atherosclerosis-associated genetic disease, familial hypercholesterolemia, myocardial infarction, cerebral infarction, pulmonary embolism It has functions related to diabetes, arteriosclerosis, obesity, cancer, etc., and was considered to be useful for the development of these therapeutic drugs.
  • the protein encoded by this cDNA is similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER 3) and is an ABC transporter involved in the extracellular excretion of drugs, etc.
  • the expression level was low, and specific expression was observed in adipose tissue.
  • this protein has functions related to cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral macular degeneration, jaundice, etc. It was considered useful for development.
  • This cDNA is similar to the S-encoded protein and human ATP—binding cassette protein of the (ABCA subfamily) product, and is presumed to be an ABC transporter involved in drug extracellular efflux.
  • FANT0M belonging to the same cluster
  • this protein has functions related to multidrug resistance of cancer, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, infertility, etc. It was considered useful in drug development.
  • the protein encoded by this cDNA is a human ATP-binding cassette protein of It is similar to the (ABCA subfamily) product, and is presumed to be an ABC transporter involved in the extracellular elimination of drugs, etc., and belongs to the same cluster FANT0M
  • NO: 9430067009 is highly homologous, and when this is examined as a probe, its expression in testis, cerebellum, stomach, lung, 10-day-old neonatal cerebellum, etc. is higher than that of control (17.5-day-old fetal whole body). There was a tendency to increase.
  • the protein encoded by this cDNA is similar to human ATP-binding cassette A9, and is presumed to be an ABC transporter involved in the extracellular excretion of the drug.
  • Control (17.5-day-old fetal whole body) Although the expression was attenuated as a whole compared with that of E. coli, equivalent or higher expression was observed in the heart, epididymis-derived adipocytes, stomach, and uterus.
  • this protein has functions related to cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral macular degeneration, jaundice, infertility, gastric ulcer, etc. It was considered useful for the development of these therapeutic agents.
  • the protein encoded by this cDNA has a sequence similar to that of human ATP-BINDING CASSETTE, SUB-FAMILY A, and was presumed to be an ABC transporter involved in drug extracellular efflux.
  • the protein encoded by this cDNA has a sequence similar to that of human ATP-binding cassette A, is presumed to be an ABC transporter involved in the extracellular excretion of drugs, etc., and belongs to the same cluster FANT0MN0: Consider using 2310069H21 as a probe As a result, the expression was strongly enhanced in the whole head and thymus of the 0-day-old newborn compared to the control, and increased in the skin, liver, muscle, ovary, and SV40-infected tissues of the 0-day and 10-day old newborns.
  • this protein is used in multidrug resistance of cancer, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, infertility, immune disease, inflammatory disease, infectious disease, etc. It has related functions and was considered useful for the development of these therapeutic agents.
  • the protein encoded by this cDNA is similar to mouse ATP-binding cassette transporter ABCA3, and is presumed to be an ABC transporter involved in the extracellular excretion of drugs, etc., and its expression level is low, but fat cells and diabetic spleen Expression was observed in.
  • this protein has functions related to cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, etc. It was considered useful in drug development.
  • the protein encoded by this cDNA is similar to human ATP11C gene for ATPase, Class VI, type 11C, and is presumed to be an ABC transporter involved in the transport of drugs, phospholipids, amphiphiles, etc. It was strongly expressed in the whole body, especially in liver, lung and knee.
  • this protein has functions related to cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral macular degeneration, jaundice, hypertension, etc. It was considered useful in drug development.
  • the protein encoded by this cDNA has a sequence similar to that of the human ATP-binding cassette, and is presumed to be involved in the transport of drugs, calcium, phospholipids, etc., and is strongly expressed in the bone marrow, And increased expression in diabetic fat.
  • this protein is useful for cancer multidrug resistance, cystic fibrosis, diabetes, It has functions related to pulse sclerosis, peroxisome disease, lateral macular degeneration, jaundice, hypertension, immune diseases, inflammatory diseases, etc., and was considered to be useful for the development of these therapeutic agents.
  • the protein encoded by this cDNA has a sequence similar to that of the human ATP-binding cassette and is presumed to be involved in the transport of drugs, calcium, phospholipids, etc., and is strongly expressed in the bone marrow, And increased expression in diabetic fat.
  • this protein has functions related to cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral macular degeneration, jaundice, hypertension, immune diseases, inflammatory diseases, etc. It was considered useful for the development of these therapeutic agents.
  • the protein encoded by this cDNA is human Potential
  • this protein is useful for cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, high blood pressure, infertility, immune diseases, inflammatory diseases, etc. It has related functions and was considered useful for the development of these therapeutic agents.
  • the protein encoded by this cDNA has a sequence similar to CALCIUM-TRANSPORTING ATPASE 3 and is presumed to be involved in the transport of drugs, calcium, phospholipids, etc., and has homology to dnafo rm49889 belonging to the same cluster. When this was used as a probe, expression was enhanced in adipocytes and spleen, and increased in kidney, liver and testis.
  • this protein is effective for cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, hypertension, infertility, immune disease, inflammation It has functions related to diseases, etc., and was considered useful for the development of these therapeutic agents.
  • This cDNA has a sequence similar to that of the S-encoded protein f, Potential phospho 1 ipid-transporting ATPase, and is presumed to be involved in the transport of drugs, calcium, phospholipids, etc. Since it has high homology to dnafo rm49889 belonging to the same cluster, when this was used as a probe, expression was enhanced in adipocytes and spleen, and increased in kidney, liver and testis.
  • this protein is related to multidrug resistance of cancer, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, high blood pressure, infertility, immune disease, inflammatory disease, etc. It is considered to be useful for the development of these therapeutic agents.
  • the protein encoded by this cDNA was similar to the human complement C5 precursor, and was estimated to be an immunoglobulin-like protein having a function related to the inflammatory allergy reaction.
  • this protein is useful for the treatment of systemic lupus erythematosus, congenital complement deficiency, rheumatoid arthritis, immune diseases such as autoimmune diseases, inflammatory diseases such as glomerulonephritis, hepatitis, infectious diseases, cancer, etc. It has related functions and is considered useful for the development of these diagnostics and therapeutics.
  • the protein encoded by this cDNA was similar to the human complement C5 precursor, and was estimated to be an immunoglobulin-like protein having a function related to the inflammatory allergy reaction.
  • this protein is useful for the treatment of systemic lupus erythematosus, congenital complement deficiency, rheumatoid arthritis, immune diseases such as autoimmune diseases, inflammatory diseases such as glomerulonephritis, hepatitis, infectious diseases, cancer, etc. With related functions, the development of these diagnostics and therapeutics It was considered useful for departure.
  • the protein encoded by this cDNA has a sequence exhibiting the characteristics of macroglobulin, and when FANTOMNO: 4831440K17 belonging to the same cluster was examined as a probe, the overall expression was reduced compared to the control. However, expression similar to that of the control was observed in the placenta, the skin of the 10-day-old newborn baby, the lung, the testis, and the like.
  • this protein is used for systemic lupus erythematosus, congenital complement deficiency, rheumatoid arthritis, immune diseases such as autoimmune diseases, glomerulonephritis, inflammatory diseases such as hepatitis, infectious diseases, cancer, infertility It has functions related to the above, and is considered to be useful for the development of these diagnostics and therapeutics.
  • the protein encoded by this cDNA is a member of the IFGP family that has a structure similar to leukocyte Fc receptors and the cell adhesion molecule PECAM-1.It is speculated that this protein is a receptor having an immunoglobulin-like structure or a protein involved in adhesion. Was done.
  • this protein has functions related to immune diseases, inflammatory diseases, cell adhesion, etc., and is useful for the development of these diagnostics and therapeutics.
  • the protein encoded by this cDNA is a member of the IFGP family that has a structure similar to leukocyte Fc receptors and the cell adhesion molecule PECAM-1.It is speculated that this protein is a receptor having an immunoglobulin-like structure or a protein involved in adhesion. Was done.
  • Japanese patent application Japanese Patent Application 2002-125934
  • Japanese patent application dated April 30, 2002 Japanese patent application dated December 4, 2002
  • Japanese Patent Application 2002-352619 Japanese patent application dated December 4, 2002
  • Japanese Patent Application 2002-352730 Based on a Japanese patent application filed on May 2, 2002 (Japanese Patent Application No. 2002-130914) and a Japanese patent application filed on January 4, 2002 (Japanese Patent Application No. 2002-352730). Captured as a reference. The contents of the documents cited in the present specification are also incorporated herein by reference.

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Abstract

The base sequences of cDNA clones involved in a catalogued full-length cDNA library are analyzed. Concerning cDNA clones having novel sequences from among those analyzed above, the physiological activities (functions) of proteins encoded thereby are specified. Based on these physiological activities (functions), a method of utilizing the proteins and DNAs encoding the same is proposed. Namely, the following proteins (a) and (b), DNAs encoding the same and a method of utilizing the same are provided: (a) a protein comprising an amino acid sequence represented by any of SEQ ID NOS:2, 3, 14, 15, 29 to 41 and 47 to 51; and b) a protein comprising an amino acid sequence derived from an amino sequence represented by any of SEQ ID NOS: 2, 3, 14, 15, 29 to 41 and 47 to 51 by deletion, substitution and/or addition of one to several amino acids and having an activity of binding to the TGFβ receptor family, an activity of binding to a modified LDL, an ATP-binding transporter activity or an immunoglobulin-like protein activity.

Description

明細書  Specification
新規タンパク質及びそれをコ一ドする DNA 技術分野  Novel protein and DNA technology that encodes it
本発明は、 新規なタンパク質、 該タンパク質をコードする DNA、該タンパク質 をコードする完全長 c DNA、該 DNAを有する組換えべクタ一、該 DNAの部分 配列から成るオリゴヌクレオチド、該 DN Aを導入した遺伝子導入細胞、及び該タ ンパク質に特異的に結合する抗体等に関する。 背景技術  The present invention introduces a novel protein, a DNA encoding the protein, a full-length cDNA encoding the protein, a recombinant vector having the DNA, an oligonucleotide comprising a partial sequence of the DNA, and the DNA. Transfected cells, antibodies that specifically bind to the protein, and the like. Background art
c DN Aの取得及びその塩基配列解析は、生体内に発現するタンパク質の生理活 性を解析し、その活性に基づくタンパク質の利用方法を開発するうえで不可欠であ る。 さらに、全遺伝子種に対応する完全長 cDNAをカタログ化したライブラリー の作製は、 ヒ トゲノムプロジェクトの重要な課題の一つである。 カタログ化したラ イブラリーとは、ライブラリーに含まれる cDNAに重複がないという意味であり、 各 c DN Aが 1種類ずつ含まれているライブラリーのことである。  Acquisition of cDNA and its nucleotide sequence analysis are indispensable for analyzing the physiological activity of a protein expressed in a living body and developing a method for utilizing the protein based on the activity. In addition, creating a library that catalogs full-length cDNAs for all gene types is one of the important issues of the human genome project. A cataloged library means that there is no overlap in the cDNAs contained in the library, and refers to a library containing one type of each cDNA.
完全長 cDNAクローニング法については、特開平 9— 248 187号公報及び 特開平 10— 127291号公報に記載されている。 この方法は、 mRNAの 5' キヤップサイ トに存在するジオール構造にタグになる分子を結合させる工程、前記 タグ分子を結合させた mRNAを踌型とし、 o 1 i g o dTをプライマーとして 逆転写により RNA— DNA複合体を作製し、 この複合体の内、 mRNAの完全長 に対応する DNAを有するものをタグ分子の機能を利用して分離する工程を含む ことを特徴とする方法である。  The full-length cDNA cloning method is described in JP-A-9-248187 and JP-A-10-127291. In this method, a tag molecule is bound to the diol structure present in the 5 ′ cap site of the mRNA, the mRNA bound to the tag molecule is type- 踌, and the RNA is obtained by reverse transcription using o1igo dT as a primer. A method comprising the steps of: preparing a DNA complex, and separating a complex having a DNA corresponding to the full length of the mRNA using the function of a tag molecule.
また効率のよい逆転写法として、铸型が高次構造を形成しないような高温で行う ための方法も開発されている （特開平 10— 84961号公報） 。 さらに、 合成さ れた完全長 c DNAライブラリーに含まれる DNA断片についてその鎖長に関わ らず一律にクローニングすることができるクローニングベクターも開発されてい る （特開平 1 1— 9273号公報） 。 Further, as an efficient reverse transfer method, a method for performing the method at a high temperature so that the 铸 type does not form a higher-order structure has been developed (Japanese Patent Application Laid-Open No. 10-84961). Furthermore, cloning vectors have been developed that can uniformly clone DNA fragments contained in a synthesized full-length cDNA library regardless of their length. (JP-A-11-9273).
このような技術により作製された完全長 cDN Aライブラリ一は、ライブラリー の個々の要素として全て均等に異なるものが含まれている訳ではなく、存在割合の 高いクローンや逆に極微量にしか存在しないクローンもある。この極微量にしか存 在しないクローンは新規である可能性が高いため、このようなクローンを濃縮する ためのサブトラクション法ゃノーマライゼーシヨン法も開発されている（特開 20 00-325080号公報； Carninci, P. et al. , Genomics, 37, 327-336 (1996) )。 かくして得られるカタログ化された完全長 cDNAライブラリーの各クローン について、公知の方法により塩基配列の解析を行えばその塩基配列は同定されるが、 該 c DNAがコードするタンパク質の生理活性は依然不明のままである。 発明の開示  The full-length cDNA library produced by such a technique does not contain all the elements that are different evenly among the individual elements of the library. Some clones do not. Since a clone existing only in such a trace amount is highly likely to be novel, a subtraction method for enriching such a clone, ie, a normalization method, has also been developed (Japanese Patent Application Laid-Open No. 2000-325080; Carninci). , P. et al., Genomics, 37, 327-336 (1996)). The nucleotide sequence of each clone of the cataloged full-length cDNA library thus obtained can be identified by a known method, but the physiological activity of the protein encoded by the cDNA is still unknown. Remains. Disclosure of the invention
本発明は、カタログ化された完全長 c DNAライブラリーに含まれる c DNAク ローンの塩基配列を解析し、 このうち配列が新規なものについては、 これがコード するタンパク質の生理活性を特定し、該生理活性に基づくタンパク質およびそれを コードする DN Aの利用方法を提案することを目的とする。  The present invention analyzes the nucleotide sequence of a cDNA clone contained in a cataloged full-length cDNA library, and among those having a novel sequence, specifies the physiological activity of the protein encoded by the sequence. The purpose of the present invention is to propose a method of using a protein based on a physiological activity and a DNA encoding the protein.
本発明者らは、マウス完全長 c DNAライブラリ一中の c DNAクローンが有す る塩基配列を解析し、該配列の相同性に基づきデータベースを検索したところ、該 配列中に特定の機能を有するタンパク質に特異的な配列を見出した。 また、 これら の cDN Aの各組織における発現量を解析した。本発明は、 これらの知見に基^ 5い て成し遂げられたものである。  The present inventors analyzed the nucleotide sequence of the cDNA clone in the mouse full-length cDNA library and searched a database based on the homology of the sequence, and found that the sequence has a specific function. A protein-specific sequence was found. The expression levels of these cDNAs in each tissue were analyzed. The present invention has been accomplished based on these findings.
すなわち本発明によれば、 以下の （1) 〜 （27) に記載の発明が提供される。 (1) 以下の （a) または （b) のタンパク質。  That is, according to the present invention, the following inventions (1) to (27) are provided. (1) The following protein (a) or (b):
( a ) 配列番号 2および 3に記載のァミノ酸配列からなるタンパク質。  (a) a protein comprising the amino acid sequence of SEQ ID NO: 2 or 3;
( b )配列番号 2および 3に記載のァミノ酸配列において 1若しくは数個のァミノ 酸が欠失、置換及び/または付加されたアミノ酸配列からなり、 かつ TGF/3受容 体フアミリーとの結合活性を有するタンパク質。 (2) 上記 （1) に記載のタンパク質をコードする DNA。 (b) consists of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence of SEQ ID NOs: 2 and 3, and has an activity of binding to a TGF / 3 receptor family. Protein. (2) DNA encoding the protein of (1) above.
(3) 上記 （1) に記載のタンパク質をコードする完全長 cDNA。  (3) A full-length cDNA encoding the protein described in (1) above.
(4) 以下の （a) 、 （b)または （c) の何れかの DNA。  (4) DNA of any of the following (a), (b) or (c):
( a ) 配列番号 1に記載の塩基配列を有する D N A。  (a) DNA having the nucleotide sequence of SEQ ID NO: 1.
(b) 配列番号 1に記載の塩基配列において、 1若しくは数個の塩基が欠失、 置換 及び または付加された塩基配列を有し、かつ TGF /3受容体ファミリーとの結合 活性を有するタンパク質をコードする DNA。  (b) a protein having a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added in the nucleotide sequence of SEQ ID NO: 1, and which has a binding activity to a TGF / 3 receptor family; The encoding DNA.
( c )配列番号 1に記載の塩基配列あるいはその相補配列を有する D N Aとストリ ンジェントな条件下でハイブリダイズすることができる塩基配列を有し、かつ T G F ]3受容体フアミリーとの結合活性を有するタンパク質をコードする DNA。 (c) having a base sequence capable of hybridizing under stringent conditions to DNA having the base sequence of SEQ ID NO: 1 or its complementary sequence, and having a binding activity to TGF] 3 receptor family DNA that encodes a protein.
(5) 以下の （a) または （b) のタンパク質。 (5) The following protein (a) or (b):
( a ) 配列番号 14または 15に記載のァミノ酸配列からなるタンパク質。  (a) a protein comprising the amino acid sequence of SEQ ID NO: 14 or 15;
(b)配列番号 14または 15に記載のアミノ酸配列において 1若しくは数個のァ ミノ酸が欠失、置換及び/または付加されたァミノ酸配列からなり、かつ変性 LD (b) an amino acid sequence represented by SEQ ID NO: 14 or 15 wherein one or several amino acids are deleted, substituted and / or added, and
Lとの結合活性を有するタンパク質。 A protein having an activity of binding to L.
(6) 上記 （5) に記載のタンパク質をコードする DNA。  (6) DNA encoding the protein of (5) above.
(7) 上記 （5) に記載のタンパク質をコードする完全長 c DNA。  (7) A full-length cDNA encoding the protein of (5).
(8) 以下の （a) 、 （b)又は （c) の何れかの DNA。  (8) DNA of any of the following (a), (b) or (c):
( a ) 配列番号 12または 1 3に記載の塩基配列を有する D NA。  (a) DNA having the nucleotide sequence of SEQ ID NO: 12 or 13.
(b)配列番号 12または 13に記載の塩基配列において、 1若しくは数個の塩基 が欠失、置換及び Zまたは付加された塩基配列を有し、 かつ変性 LDLとの結合活 性を有するタンパク質をコードする DNA。  (b) a protein having the nucleotide sequence of SEQ ID NO: 12 or 13 in which one or several nucleotides are deleted, substituted, Z or added, and which has a binding activity to denatured LDL; The encoding DNA.
(c)配列番号 12または 13に記載の塩基配列あるいはその相補配列を有する D NAとストリンジェントな条件下でハイプリダイズすることができる塩基配列を 有し、 かつ変性 LDLとの結合活性を有するタンパク質をコードする DNA。  (c) a protein having a nucleotide sequence capable of hybridizing with DNA having the nucleotide sequence of SEQ ID NO: 12 or 13 or its complementary sequence under stringent conditions, and having a binding activity with denatured LDL DNA that encodes
(9) 以下の （a) または （b) のタンパク質。  (9) The following protein (a) or (b):
(a) 配列番号 29〜41のいずれかに記載のアミノ酸配列からなるタンパク質。 ( b )配列番号 29〜 41のいずれかに記載のァミノ酸配列において 1若しくは数 個のアミノ酸が欠失、置換及び または付加されたアミノ酸配列からなり、 かつ A T P結合性運搬体活性を有するタンパク質。 (a) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 29 to 41; (b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of any of SEQ ID NOs: 29 to 41, and which has ATP-binding carrier activity;
(10) 上記 （9) に記載のタンパク質をコードする DNA。  (10) A DNA encoding the protein of (9).
(1 1) 上記 （9) に記載のタンパク質をコードする完全長 c DNA。  (1 1) A full-length cDNA encoding the protein of (9).
(1 2) 以下の （a) 、 （b)又は （c) の何れかの DNA。  (12) DNA of any of the following (a), (b) or (c):
( a ) 配列番号 16〜 28のいずれかに記載の塩基配列を有する DNA。  (a) DNA having the nucleotide sequence of any one of SEQ ID NOs: 16 to 28.
( b )配列番号 16〜 28のいずれかに記載の塩基配列において、 1若しくは数個 の塩基が欠失、置換及び Zまたは付カ卩された塩基配列を有し、 かつ AT P結合性運 搬体活性を有するタンパク質をコードする DNA。  (b) In the nucleotide sequence of any one of SEQ ID NOs: 16 to 28, one or several nucleotides have a nucleotide sequence in which deletion, substitution, Z or addition is performed, and ATP-binding transport DNA encoding a protein having body activity.
( c )配列番号 16〜 28のいずれかに記載の塩基配列あるいはその相補配列を有 する DNAとストリンジェントな条件下でハイブリダィズすることができる塩基 配列を有し、かつ AT P結合性運搬体活性を有するタンパク質をコ一ドする DNA。  (c) a nucleotide sequence capable of hybridizing under stringent conditions with a DNA having the nucleotide sequence of any of SEQ ID NOs: 16 to 28 or a sequence complementary thereto, and an ATP-binding carrier activity DNA encoding a protein having
(1 3) 以下の （a) または （b) のタンパク質。  (13) The following protein (a) or (b):
(a) 配列番号 47〜51のいずれかに記載のアミノ酸配列からなるタンパク質。 ( b )配列番号 47〜 51のいずれかに記載のァミノ酸配列において 1若しくは数 個のアミノ酸が欠失、置換及ぴノまたは付加されたアミノ酸配列からなり、 力っィ ムノグロブリン様タンパク質活性を有するタンパク質。  (a) a protein comprising the amino acid sequence of any one of SEQ ID NOs: 47 to 51; (b) an amino acid sequence represented by any one of SEQ ID NOs: 47 to 51, wherein one or several amino acids are deleted, substituted and / or added to the amino acid sequence, and the amino acid sequence has potent immunoglobulin-like protein activity. Protein.
(14) 上記 （13) に記載のタンパク質をコードする DNA。  (14) A DNA encoding the protein of (13).
(15) 上記 （13) に記載のタンパク質をコードする完全長 cDNA。  (15) A full-length cDNA encoding the protein according to (13).
(16) 以下の （a) 、 （b)又は （c) の何れかの DNA。  (16) Any one of the following DNAs (a), (b) or (c):
(a) 配列番号 42〜46のいずれかに記載の塩基配列を有する DNA。  (a) DNA having the nucleotide sequence of any one of SEQ ID NOs: 42 to 46.
(b)配列番号 42〜46のいずれかに記載の塩基配列において、 1若しくは数個 の塩基が欠失、置換及び Zまたは付加された塩基配列を有し、 かつィムノグロプリ ン様タンパク質活性を有するタンパク質をコードする D N A。  (b) a protein having a base sequence of any one of SEQ ID NOs: 42 to 46, wherein one or several bases have a base sequence in which deletion, substitution, Z or addition has been performed, and which has an immunoglobulin-like protein activity; DNA that encodes
(c)配列番号 42〜46のいずれかに記載の塩基配列あるいはその相補配列を有 する DNAとストリンジェントな条件下でハイブリダィズすることができる塩基 配列を有し、かつィムノグロブリン様タンパク質活性を有するタンパク質をコード する DNA。 (c) a base capable of hybridizing under stringent conditions with a DNA having the base sequence of any one of SEQ ID NOs: 42 to 46 or a complementary sequence thereof DNA encoding a protein having a sequence and having immunoglobulin-like protein activity.
(1 7) 上記 （2) 〜 （4) 、 （6) 〜 （8) 、 （10) 〜 （12) 、 (14) 〜 （16) のいずれかに記載の DNAを含む組換えベクター。  (17) A recombinant vector comprising the DNA according to any of (2) to (4), (6) to (8), (10) to (12), and (14) to (16).
(18) 上記 （2) 〜 （4) 、 （6) 〜 （8) 、 （10) 〜 （12) 、 （14) 〜（16) のいずれかに記載の DN Aまたは上記 （1 7) に記載の組み換えべクタ 一を導入した遺伝子導入細胞または該細胞からなる個体。  (18) The DNA described in any of the above (2) to (4), (6) to (8), (10) to (12), (14) to (16) or the above (17) A transgenic cell into which the recombinant vector described above has been introduced, or an individual comprising the cell.
(19) 上記（18)に記載の細胞により産生される、上記（1)、 （5)、 （9)、 または （13) に記載のタンパク質。  (19) The protein according to (1), (5), (9), or (13), which is produced by the cell according to (18).
(20) 上記 （2) 〜 （4) 、 (6) 〜 （8) 、 （10) 〜 （ 2) 、 （14) 〜（16) のいずれかに記載の DNAの塩基配列中の連続した 5〜100塩基と同 じ配列を有するセンスオリゴヌクレオチド、当該センスオリゴヌクレオチドと相補 的な配列を有するアンチセンスオリゴヌクレオチド、及び、 当該センス又はアンチ センスオリゴヌクレオチドのオリゴヌクレオチド誘導体から成る群から選ばれる オリゴヌクレオチド。  (20) 5 consecutive nucleotides in the DNA base sequence according to any one of the above (2) to (4), (6) to (8), (10) to (2), and (14) to (16). An oligonucleotide selected from the group consisting of a sense oligonucleotide having the same sequence as 100 bases, an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide, and an oligonucleotide derivative of the sense or antisense oligonucleotide .
(21) 上記 （1) 、 （5) 、 （9) 、 （1 3) 、 または （19) に記載のタン パク質に特異的に結合する抗体あるいはその部分フラグメント。  (21) An antibody or a partial fragment thereof that specifically binds to the protein of (1), (5), (9), (13), or (19).
(22) 抗体がモノクローナル抗体である上記 （21) に記載の抗体。  (22) The antibody according to (21), wherein the antibody is a monoclonal antibody.
(23) モノクローナル抗体が上記 （1) 、 （5) 、 （9) 、 （13) 、 または (19)に記載のタンパク質の活性を中和する作用を有することを特徴とする上記 (22) に記載の抗体。  (23) The method according to (22), wherein the monoclonal antibody has an action of neutralizing the activity of the protein according to (1), (5), (9), (13), or (19). The described antibody.
(24) 上記 （1) 、 （5) 、 （9) 、 （1 3) 、 または （19) に記載のタン パク質と被検物質を接触させ、該被検物質による該タンパク質が有する活性の変化 を測定することを特徴とする、該タンパク質の活性調節物質のスクリ一ユング方法。 (24) The protein according to (1), (5), (9), (13), or (19) is brought into contact with a test substance, and the activity of the protein by the test substance is measured. Measuring the change in the activity of the protein.
(25) 上記 （18) に記載の遺伝子導入細胞と被検物質を接触させ、 該細胞に 導入されている DN Aの発現レベルの変化を検出することを特徴とする、該 DN A の発現調節物質のスクリ一ユング方法。 (26) 上記 （1) 、 （5) 、 （9) 、 または （13) に記載のタンパク質のァ ミノ酸配列から選択される少なくとも 1以上のァミノ酸配列情報、および/または 上記 （2) 〜 （4) 、 (6) 〜 （8) 、 （10) 〜 （12) 、 （14) 〜 （16) のいずれかに記載の DN Aの塩基配列から選択される少なくとも 1以上の塩基配 列情報を保存したコンピュータ読み取り可能記録媒体。 (25) Expression control of the DNA, wherein the test substance is brought into contact with the gene-transfected cell according to (18), and a change in the expression level of the DNA introduced into the cell is detected. How to screen material. (26) at least one or more amino acid sequence information selected from the amino acid sequences of the proteins according to (1), (5), (9) or (13), and / or (2) to (4), (6) to (8), (10) to (12), (14) to (16), at least one or more nucleotide sequence information selected from the nucleotide sequence of DNA A computer-readable recording medium storing a computer.
(27) 上記 （1) 、 （5) 、 （9) 、 または （13) に記載のタンパク質、 お よび Zまたは上記 （2) 〜 （4) 、 (6) 〜 （8) 、 （10) 〜 （12) 、 (14) 〜 （1 6) のいずれかに記載の DNAを結合させた担体。 発明を実施するための最良の形態  (27) The protein described in (1), (5), (9), or (13) above, and Z or (2) to (4), (6) to (8), (10) to (12) A carrier to which the DNA according to any one of (14) to (16) is bound. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明をさらに詳細に説明する。  Hereinafter, the present invention will be described in more detail.
( 1 ) 完全長 c D N Aの取得及ぴ塩基配列の解析  (1) Acquisition of full-length cDNA and analysis of nucleotide sequence
本発明の DN Aは、 配列番号 2、 3、 14、 15、 29〜41、 47〜 51に記 載のアミノ酸配列からなるタンパク質、または該アミノ酸配列において、 1若しく は数個 （ここで言う数個の数は特には限定されないが、例えば 20個以下、好まし くは 1 5個以下、 より好ましくは 10個以下、 さらに好ましくは 5個以下を意味す る） のアミノ酸残基の置換、 欠失、 挿入、 付加、 若しくは逆位を含むアミノ酸配列 からなり、かつ後記の特定の活性を有するタンパク質をコードし得るものであれば 如何なるものであってもよい。具体的には、該アミノ酸配列をコードする翻訳領域 のみでも、 あるいはその c DN Aの全長を含むものでもよい。  The DNA of the present invention comprises a protein consisting of the amino acid sequence described in SEQ ID NOs: 2, 3, 14, 15, 29 to 41, and 47 to 51, or one or several amino acids in the amino acid sequence (hereinafter referred to as Although the number is not particularly limited, it means, for example, substitution of 20 or less, preferably 15 or less, more preferably 10 or less, and still more preferably 5 or less amino acid residues. Any protein may be used as long as it comprises an amino acid sequence containing a deletion, insertion, addition, or inversion and encodes a protein having the specific activity described below. Specifically, it may be only the translation region encoding the amino acid sequence, or may include the entire length of the cDNA.
具体的には、 c DNAの全長を含む DNAとしては、 例えば配列番号 1、 1 2、 13、 16〜28、 42〜46に記載の塩基配列からなる DNA等が挙げられる。 また、 その翻訳領域としては、  Specifically, examples of the DNA containing the full-length cDNA include, for example, DNAs comprising the nucleotide sequences of SEQ ID NOs: 1, 12, 13, 16 to 28, and 42 to 46. Also, as the translation area,
配列番号 1の塩基番号 218〜869に示される配列、 A sequence represented by base numbers 218 to 869 of SEQ ID NO: 1,
配列番号 12の塩基番号 95〜976に示される配列、 A sequence represented by base numbers 95 to 976 of SEQ ID NO: 12,
配列番号 13の塩基番号 94〜810に示される配列、 A sequence represented by base numbers 94 to 810 of SEQ ID NO: 13,
配列番号 16の塩基番号 163〜 2520に示される配列、 配列番号 17の塩基番号 181 7〜3829に示される配列、 A sequence represented by base numbers 163 to 2520 of SEQ ID NO: 16, A sequence represented by base numbers 181 7 to 3829 of SEQ ID NO: 17,
配列番号 18の塩基番号 201〜 3950に示される配列、 A sequence represented by base numbers 201 to 3950 of SEQ ID NO: 18,
配列番号 19の塩基番号 68〜2914に示される配列、 A sequence represented by base numbers 68 to 2914 of SEQ ID NO: 19,
配列番号 20の塩基番号 1382〜2458に示される配列、 A sequence represented by base numbers 1382 to 2458 of SEQ ID NO: 20,
配列番号 21の塩基番号 397〜2235に示される配列、 A sequence represented by base numbers 397 to 2235 of SEQ ID NO: 21,
配列番号 22の塩基番号 163〜 1674に示される配列、 A sequence represented by base numbers 163 to 1674 of SEQ ID NO: 22,
配列番号 23の塩基番号 940〜 2247に示される配列、 A sequence represented by base numbers 940 to 2247 of SEQ ID NO: 23,
配列番号 24の塩基番号 277〜 1554に示される配列、 A sequence represented by base numbers 277 to 1554 of SEQ ID NO: 24,
配列番号 25の塩基番号 526〜 1449に示される配列、 A sequence represented by base numbers 526 to 1449 of SEQ ID NO: 25,
配列番号 26の塩基番号 2015〜3883に示される配列、 A sequence represented by base numbers 2015 to 3883 of SEQ ID NO: 26,
配列番号 27の塩基番号 70〜 987に示される配列、 A sequence represented by base numbers 70 to 987 of SEQ ID NO: 27,
配列番号 28の塩基番号 102〜 1484に示される配列、 A sequence represented by base numbers 102 to 1484 of SEQ ID NO: 28,
配列番号 42の塩基番号 4〜1062に示される配列、 A sequence represented by base numbers 4 to 1062 of SEQ ID NO: 42,
配列番号 43の塩基番号 8〜1078に示される配列、 A sequence represented by base numbers 8 to 1078 of SEQ ID NO: 43,
配列番号 44の塩基番号 1340〜4429に示される配列、 A sequence represented by base numbers 1340 to 4429 of SEQ ID NO: 44,
配列番号 45の塩基番号 1 1 1〜488に示される配列、 A sequence represented by base numbers 11 1 to 488 of SEQ ID NO: 45,
配列番号 46の塩基番号 49〜 1080に示される配列 Sequence represented by base numbers 49 to 1080 of SEQ ID NO: 46
を有するものが挙げられる。 さらに上記の c DNAの全長でなくても、上記翻訳領 域とその 3' 及び/または 5'端に隣接する、翻訳領域の発現に最低限必要な部分 を含むもの等も本発明の DN Aに含まれる。 And the like. Furthermore, even if the cDNA is not the full-length cDNA described above, the DNA of the present invention may also include the above-described translation region and a region adjacent to the 3 ′ and / or 5 ′ end thereof, which contains the minimum necessary portion for the expression of the translation region. include.
本発明の DN Aは、これを取得できる方法であれば如何なる方法により取得した ものでもよいが、 具体的には、 例えば、 下述の方法により取得することができる。 まず、適当な動物、好ましくは哺乳動物の組織等からそれ自体既知の通常用いられ る方法により mRN Aを調製する。次に、 この mRNAを铸型として c DNAを合 成するが、 このとき完全長の c DNAを合成するために 5' キャップ （™eGpppN) サイトに特異的なジオール構造にタグになる分子を化学結合させ、この mRNAを 铸型として o l i g o dTをプライマーとして逆転写した後に、タグ分子の機能 を利用して完全長の cDN Aのみを分離する方法 （特開平 9— 248187号公 報；特開平 10— 127291号公報） を用いることが好ましい。 また、 逆転写の 際には、铸型が高次構造を形成して逆転写の効率が低下することを阻止するために、 トレハロース等の存在下で、耐熱性逆転写酵素を用いて高温下で逆転写を行う方法The DNA of the present invention may be obtained by any method as long as it can be obtained. Specifically, it can be obtained, for example, by the method described below. First, mRNA is prepared from a suitable animal, preferably a mammalian tissue or the like, by a method known per se and generally used. Next, the mRNA of c DNA synthesis Suruga as铸型become tag specific diol structure at the 5 'cap (™ e G ppp N) site for the synthesis of full-length c DNA this time After chemically binding the molecule and performing reverse transcription using this mRNA as type 铸 and oligo dT as a primer, the function of the tag molecule It is preferable to use a method of separating only full-length cDNA using U.S. Pat. In addition, in the case of reverse transcription, in order to prevent the type III from forming a higher-order structure and lowering the efficiency of reverse transcription, use of a thermostable reverse transcriptase in the presence of trehalose, etc. To perform reverse transcription with
(特開平 10— 84961号公報） を用いるのが好ましい。 ここで、 高温下とは 4 0〜80°Cを意味する。 (JP-A-10-84961) is preferably used. Here, high temperature means 40 to 80 ° C.
このようにして取得された cDNAは、これを適当なクローユングベクターに挿 入してクローユングを行う。 ここで用いられるベクターとしては、様々な鎖長の D NAを一律にクローユングすることが可能な、クローニングサイトの両末端にリコ ンビナーゼ認識配列を有し、感染以外の方法で宿主に挿入される直鎖状のベクター (特開平 1 1— 9273号公報） が好ましく用いられる。 かくして得られる cDN Aライブラリ一は、 全てのクローンが均一に存在している （以下、 これを 「カタ口 グ化されている」 と称することがある） 訳ではなく、 このライブラリ一中に極微量 にしか存在しないクローンこそ新規である確率が高レ、。 そこで、 このようなクロー ンを濃縮するためのサブトラクシヨン法やノーマライゼーシヨン法（特開 2000 -325080公報； Carninci, P. et al. , Genomics, 37, 327—336(1996)) を用 いることが好ましい。  The cDNA obtained in this manner is inserted into an appropriate closing vector to perform closing. The vector used here has a recombinase recognition sequence at both ends of a cloning site capable of uniformly closing DNAs of various chain lengths, and is directly inserted into a host by a method other than infection. A chain vector (JP-A-11-9273) is preferably used. In the thus obtained cDNA library, not all clones are uniformly present (hereinafter, this may be referred to as "tagged"). There is a high probability that a clone that exists only in Japan is new. Therefore, the subtraction method and the normalization method (JP-A-2000-325080; Carninci, P. et al., Genomics, 37, 327-336 (1996)) for enriching such clones are used. Is preferred.
カタログ化された c DN Aライブラリ一は、それ自体既知の通常用いられる方法 により塩基配列の解析を行う。本発明の DNAは、 cDNA全長の場合にはその末 端 100ベースの配列について得られた塩基配列を、 BLAST  The cataloged cDNA library is subjected to nucleotide sequence analysis by a commonly used method known per se. The DNA of the present invention is obtained by combining the base sequence obtained for the 100-base sequence at the
(http://www.ncbi.nlm.nih.gov/BLAbf/； National Center of Biotechnology Information) を用いて、 NCB Iの Ge n b a n k、 EMBL、 DDB J等のデ ータベースについて検索し、最も高い相同性を示す配列でも一致度が 30%以下で あるものを新規として以下の解析に供することとした。  (http://www.ncbi.nlm.nih.gov/BLAbf/; National Center of Biotechnology Information) to search databases such as NCBI I's Genbank, EMBL, DDB J, etc. A sequence with a degree of coincidence of 30% or less was newly submitted to the following analysis.
このような完全長 c D N Aの塩基配列を有する D N Aおよぴその翻訳領域とし ては、 例えば、 上記したものが挙げられる。  Examples of the DNA having such a full-length cDNA base sequence and its translation region include those described above.
かくして取得された新規な塩基配列を、 BLAST (Basic local alignment search tool； Altschul, S. F. , et al. , J. Mol. Biol., 215， 403-410(1990)) に よる相同性検索 （homology search)や、 HMME R (隠れ Markovモデルによる配列 解析手法； Eddy, S. R. , Bioinformatics 14, 755-763 (1998)) の機能群のひとつ である HMMPFAMによるタンパク質特徴検索 （profile search： The novel base sequence thus obtained is sequenced using BLAST (Basic local alignment Search tool; Homology search by Altschul, SF, et al., J. Mol. Biol., 215, 403-410 (1990)) and HMME R (sequence analysis method by hidden Markov model; Eddy) , SR, Bioinformatics 14, 755-763 (1998))
http://pfam. wustl. edu)等を行うことにより、該塩基配列がコードするタンパク質 の機能を推定することができる。 http: // pfam. wustl. edu) and the like, the function of the protein encoded by the nucleotide sequence can be estimated.
BLASTによる相同性検索においては、検索の結果得られた相同性が十分有意 なヒット配列に付随する種々のァノテーシヨン情報から、解析対象としているクロ ーンの機能を推定することができる。 ここで、 十分有意なヒット配列とは、 登録さ れているアミノ酸配列の触媒ドメイン部分と本発明の DNAがコードするアミノ 酸配列のこれに対応する部分との一致度が e— V a 1 u eとして 10— 4以下のもの か、 あるいは 30%以上のものを示す。 In the homology search by BLAST, the function of the clone to be analyzed can be estimated from various annotation information associated with hit sequences whose homology is sufficiently significant as a result of the search. Here, a sufficiently significant hit sequence is e-V a1 ue when the degree of coincidence between the catalytic domain portion of the registered amino acid sequence and the corresponding portion of the amino acid sequence encoded by the DNA of the present invention is e-V a1 ue. 10- 4 or less things as, or show a more than 30%.
例えば、上位にヒッ トした触媒ドメィン配列の多くが後記の特定の活性を確認さ れたものであるならば、それと配列上類似である解析対象クローンもまた同じ機能 を持つであろうという予測が成り立つ。  For example, if many of the top-hit catalytic domain sequences have been confirmed to have the specific activity described below, the prediction that the clone to be analyzed that is sequence-similar to that will also have the same function. Holds.
HMMPFAMでは、 P f a mというタンパク質プロファイルを集積したデータ ベース中にあるェントリーが有する配列の特徴を、解析対象である配列が有するか どうかを洗い出す方法による解析が行われる。プロファイルは一連の同一特徴を持 つタンパク質群から抽出されており、一配列対一配列の全長に亘る比較では明確化 できない機能でも、配列中にその特徴領域があればこれを見出し、機能予測ができ る。 かくして行われるタンパク質の機能予測の具体的な例として以下に説明する。  In HMMPFAM, an analysis is performed by a method of checking whether or not the sequence to be analyzed has the characteristics of the sequence of an entry in a database in which a protein profile called Pfam is accumulated. Profiles are extracted from a series of proteins with the same characteristics, and even if a function cannot be clarified by comparing the full length of one-to-one sequences, if there is a characteristic region in the sequence, it will be found and its function predicted. it can. A specific example of the prediction of the function of a protein thus performed will be described below.
(1-1) TGF ]3受容体ファミリーとの結合活性を有するタンパク質 (1-1) Protein having binding activity to TGF] 3 receptor family
配列番号 1に記載の塩基配列がコードするアミノ酸配列は B LAS Tサーチに より、 Homo s a p i e n s p u t a t i v e t r a n smemb r a n e r o t e i n NMA p r e c u r s o r 、 e— v a l u e : 3 X 10 、 138ァミノ酸残基にわたり 67 %の一致度で、 k i n a s e— d e f i c i e n t TGF b e t a s u p e r f am i l y r e c e p t o r s u b un i t力 e -v a l u e : l X l (T48、 153ァミノ酸残基にわたり 67 %の 一^!度 \ さらに Mu s m u s c u 1 u s , BMP a n d a c t i v i n memb r a n e— b o un d i nh i b i t o r, h omo 1 o g (Xe n o p u s l a e v i s) 、 e - v a 1 u e : 1X 10— 48、 153ァミノ酸残基 にわたり 68%の一致度でヒットする。 The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 was determined by BLAST search to be Homo sapiensputativetran smemb ranerotein NMA precursor, e-value: 3 X 10 and 138 amino acid residues with 67% identity, — Defici ent TGF betasuperf am ilyreceptorsub un it force e-value: l Xl (T 48 , 67% of the 153 amino acid residues over 67% of 1 ^! degree \ more Mu smuscu 1 us, BMP andactivin memb rane— bo un di nh ibitor, h omo 1 og (Xe nopuslaevis) , e - va 1 ue: 1X 10- 48, 153 hits in 68% of the degree of coincidence over Amino acid residues.
これらのことから、配列番号 1に示す塩基配列がコードするタンパク質は T G F 受容体フアミリーとの結合活性を有し、 k i n a s e— d e f i c i e n t T GFb e t a s u p e r f am i l y r e c e p t o r s u b un i tで あると推測できる。  From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 has a binding activity to TGF receptor family, and is kinasse—deficinetTGFbeetasupeperfamlilyreceptortsubunit.
TGF ]3受容体フアミリーとは、細胞膜を一回貫通し、細胞内ドメインにセリン スレオニンキナーゼ領域を持つことで知られる受容体であり、 構造と機能から 1型 受容体と 2型受容体に分類することができる。 1型受容体はキナーゼ領域の N端側 にグリシンとセリンに富んだ GSドメインを有する。リガンドである TGF ファ ミリ一が結合すると、受容体は 1型受容体 2分子と 2型受容体 2分子からなるへテ 口 4量体を形成し、 2型受容体のキナーゼにより 1型受容体の GSドメインがリン 酸化され、その結果、 1型受容体のキナーゼが活性化され細胞内に信号が伝達され る。 近年報告された TGF )3ファミリーの偽受容体である B AM B I (BMP and activin membrane-bound inhibitor； Nature Vol.401, 480-483(1999)) は、 1型 受容体とホモロジ一が高いが、通常の TGF /3受容体フアミリーに共通のセリンス レオニンキナーゼドメインを有さないことから、 TGF i3フアミリーによる信号が 伝達できなくなつている。  TGF] 3 receptor families are known to have a serine-threonine kinase domain in the intracellular domain, penetrating the cell membrane once, and are classified into type 1 and type 2 receptors based on their structure and function. can do. Type 1 receptors have a glycine- and serine-rich GS domain N-terminal to the kinase region. When the ligand TGF family binds, the receptor forms a tetramer consisting of two molecules of type 1 receptor and two molecules of type 2 receptor, and the type 1 receptor is kinased by type 2 receptor. GS domain is phosphorylated, and as a result, type 1 receptor kinase is activated and a signal is transmitted into cells. BAMBI (BMP and activin membrane-bound inhibitor; Nature Vol.401, 480-483 (1999)), a pseudoreceptor of the TGF) 3 family reported recently, has a high homology with the type 1 receptor. However, the lack of a common serine / threonine kinase domain in the normal TGF / 3 receptor family prevents the transmission of signals by the TGF i3 family.
本発明のタンパク質は、上記のとおり、 TGF フアミリーの偽受容体 BAMB Iや Nma (J. Dent. Res. 80 (10) , 1895-1902 (2001) ) と高い相同性を有し、 通常の TGF ]3受容体フアミリーに共通のセリンスレオニンキナーゼドメインを有さな いことから、 TGF ]3フアミリーの偽受容体であると推測できる。 (1 -2) 変性 LDL (低密度リポタンパク質） との結合活性を有するタンパク質 配列番号 12に記載の塩基配列がコードするァミノ酸配列は、 B L A S Tを用い 7こ†目 |PJ'I4検索により、 lectin一 like oxidized LDL receptor (Oryctolagus cuniculus)が、 e— v a 1 u e ： 1 X 10— 21、 235アミノ酸残基に亘り 29 %の 一致度で、 よた、 oxidised low density lipoprotein (lectin - like) receptor 1As described above, the protein of the present invention has high homology with the pseudo-receptors BAMB I and Nma of TGF family (J. Dent. Res. 80 (10), 1895-1902 (2001)), and normal TGF The lack of a common serine threonine kinase domain in the [3] family suggests that it is a pseudoreceptor for the TGF] 3 family. (1-2) Protein having binding activity to denatured LDL (low-density lipoprotein) The amino acid sequence encoded by the base sequence described in SEQ ID NO: 12 is obtained by using BLAST. lectin one like oxidized LDL receptor (Oryctolagus cuniculus) is, e- va 1 ue: 29% of the degree of match over the 1 X 10- 21, 235 amino acid residues, was good, oxidised low density lipoprotein (lectin - like) receptor 1
(Homo sapiens) 力 e-v a l u e : 2 X l 0一18、 237ァミノ酸残基に亘り 2 7%の一致度でヒットする。 これらの結果より、配列番号 12に示す塩基配列がコ ードするタンパク質、または配列番号 14に示したァミノ酸配列からなるタンパク 質は、 変性 LDLとの結合活性を有し、 酸化 LDLゃァセチル化 LDL等の細胞内へ の取りこみや細胞機能障害に関係する変性 LDL受容体の 1種であることが推測でき る。 (Homo sapiens) force ev alue: 2 X l hits in 0 one 18, 237 Amino over acid residue 2 7% degree of coincidence. Based on these results, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 12 or the protein consisting of the amino acid sequence shown in SEQ ID NO: 14 has a binding activity with denatured LDL, and It can be inferred that it is a type of modified LDL receptor involved in the incorporation of LDL and the like into cells and cell dysfunction.
配列番号 13に記載の塩基配列がコードするアミノ酸配列は、 B LASTを用い 7こ相同性恢索により、 lectin - like oxidized LDL receptor (Oryctolagus cuniculus)が、 e— v a l u e : 1 X 1 0—19、 235ァミノ酸残基に亘り 28 %の ―致度で、 また、 oxidised low density lipoprotein (lectin - liKe) receptor 1 (Homo sapiens) 力 e— v a l u e : 6 X l 0— 18、 235ァミノ酸残基に亘り 2 6%の一致度でヒットする。 これらの結果より、配列番号 13に示す塩基配列がコ 一ドするタンパク質、または配列番号 1 5に示したアミノ酸配列からなるタンパク 質は、 変性 LDLとの結合活性を有し、 変性 LDLゃァセチル化 LDLなどの細胞内 への取りこみや細胞機能障害に関係する変性 LDL受容体の 1種であることが推測で きる。 The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 13, by 7 this homology恢索using B LAST, lectin - like oxidized LDL receptor (Oryctolagus cuniculus) is, e- value: 1 X 1 0- 19, 235 Amino acid residue 28% over - in致度, also, oxidised low density lipoprotein (lectin - liKe) receptor 1 (Homo sapiens) power e- value: 6 X l 0- 18 , 235 to Amino acid residue Hits with a 26% match rate. From these results, it can be seen that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 13 or the protein consisting of the amino acid sequence shown in SEQ ID NO: 15 has a binding activity to denatured LDL, It can be speculated that this is one of the modified LDL receptors involved in the incorporation of LDL into cells and cell dysfunction.
上記のように、 本発明のタンパク質は変性 LDL受容体の 1種であることが推測で きる。 変性 LDLには、 動脈硬化等に関連する酸化 LDL、糖尿病等に関連する糖 化 LDLや AGE (advanced glycation endproducts) -LDL, 冠動脈疾患等に関連する malondialdehyde- modified LDL等が知られているが、 上記本発明のタンパク質は、 いずれも酸化 LDL受容体である lectin - like oxidized LDL受容体 1 (L0X- 1) と それぞれ高い相同性を有し、かつ細胞外レクチン領域のシスティンの繰り返し構造 が保存されていることから、酸化 L D L受容体のファミリー分子と推測することが できる。酸化 L D L受容体は、酸化 L D Lを結合して細胞内に取りこむ活性を有し、 動脈硬化の成因等に重要な役割を果たすと考えられており、その構造は、膜一回貫 通型の Π型糖タンパクであって短い N末端を細胞内に C末端を細胞外に突出させて いることが知られている。 As described above, it can be inferred that the protein of the present invention is one type of modified LDL receptor. Known denatured LDLs include oxidized LDL related to arteriosclerosis, glycated LDL related to diabetes, etc., advanced glycation endproducts (AGE) -LDL, and malondialdehyde-modified LDL related to coronary artery disease, etc. The protein of the present invention has high homology with lectin-like oxidized LDL receptor 1 (L0X-1), which is an oxidized LDL receptor, and has a cysteine repeating structure in the extracellular lectin region. Is conserved, it can be inferred to be a family molecule of oxidized LDL receptor. The oxidized LDL receptor has an activity to bind oxidized LDL and take it up into cells, and is considered to play an important role in the etiology of arteriosclerosis, and its structure is a one-time transmembrane type. It is a type of glycoprotein that is known to have a short N-terminal protruding into the cell and a C-terminal protruding out of the cell.
( 1 — 3 ) A T P結合性運搬体活性を有するタンパク質 (1 — 3) Protein with ATP-binding transporter activity
配列番号 1 6に記載の塩基配列がコードするァミノ酸配列は、 B L A S Tを用い た相同性検索により、 human ATP- BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP- BINDING CASSETTE TRANSPORTER (A B Cトランスポーター） 3)が、 e— v a 1 u e : 0、 791アミノ酸残基に亘り 46%の一致度で、 mouse ATP- binding cassette transporter ABCA3が、 e— v a 1 u e ： 0、 791ァミノ酸残基に亘り 46%の一致度 で、 また、 mouse ATP— binding cassette transporter 丄カ 、 e— v a 1 u e ： 5 X 10_117、 791アミノ酸残基に亘り 37%の一致度でヒットする。 The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 16 was obtained from human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER (ABC transporter)) 3) shows that the mouse ATP-binding cassette transporter ABCA3 has an e-va 1 ue of 0, 791 amino acid residues and 46% identity over the 791 amino acid residues. % in the degree of coincidence, also, mouse ATP- binding cassette transporter丄Ka, e- va 1 ue: hits in 5 X 10_ 117, 791 37% degree of coincidence over the amino acid residues.
また、配列番号 1 6に示す塩基配列がコードするアミノ酸配列について、 HMM P F AMによるタンパク質特徴検索を行うと、 ABC transporterの特徴を示す配列 ( P f a mに A B C— t r a nとしてェントリーされる配列） が見出される。  When a protein feature search was performed by HMM PFAM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 16, a sequence exhibiting the characteristics of ABC transporter (a sequence entry as ABC-tran in P fam) was found. It is.
これらのことから、 配列番号 1 6に示した塩基配列がコードするタンパク質は human ATP- BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP- BINDING CASSETTE TRANSPORTER 3)類似の配列を有し、 A T P結合性運搬体活性を有すると推測できる。 このことから、配列番号 1 6に示す塩基配列がコードするタンパク質が薬物の細胞 外排出等に関わる A B Cトランスポーターであり、 薬物等の異物や、 カルシウム、 リン脂質、 两親媒性物質等の内因性物質の輸送等にかかわることが推測できる。 配列番号 1 7に記載の塩基配列がコードするアミノ酸配列は、 B L A S Tを用い た相同性検索により、 ATP— binding cassette protein of the (ABCA subfamily) productが、 e— v a 1 u e ： 0、 671アミノ酸残基に亘り 90%の一致度で、 human ATP- binding cassette transporter ABCA3力 e - v a 1 u e ： 0、 671アミノ酸 残基に亘り 90%の一致度で、 また、 Homo sapiens cDNA FLJ31971 fis, clone NT2RP7008137, weakly similar to ATP- BINDING CASSETTE, SUB-FAMILY A, MEMBER 1が、 e— v a 1 u e : 5X10— m、 417アミノ酸残基に亘り 87%の一致度でヒットす る。 From these facts, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 16 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER 3), and has ATP binding property. It can be inferred to have carrier activity. Therefore, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 16 is an ABC transporter involved in extracellular excretion of drugs, etc., and foreign substances such as drugs, endogenous substances such as calcium, phospholipids, and amphiphilic substances It can be inferred that it is involved in the transport of toxic substances. The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 17 shows that the ATP-binding cassette protein of the (ABCA subfamily) product shows e-va 1 ue: 0, 671 amino acid residues by homology search using BLAST. Human ATP-binding cassette transporter ABCA3 force e-va 1 ue with 90% identity over all groups: 0, 671 amino acids 90% degree of coincidence over the residue, addition, Homo sapiens cDNA FLJ31971 fis, clone NT2RP7008137, weakly similar to ATP- BINDING CASSETTE, SUB-FAMILY A, MEMBER 1 is, e- va 1 ue: 5X10- m , 417 Hits with 87% identity across amino acid residues.
また、配列番号 17に示す塩基配列がコードするアミノ酸配列について、 HMM P F AMによるタンパク質特徴検索を行うと、 ABC transporterの特徴を示す配列 (P f an^ ABC—t r a nとしてェントリーされる配列） が見出される。  When a protein feature search was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 17 using HMM PFAM, a sequence showing the characteristics of ABC transporter (a sequence entry as Pfan ^ ABC-tran) was found. It is.
これらのことから、 配列番号 17に示した塩基配列がコードするタンパク質は human ATP- BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP- BINDING CASSETTE TRANSPORTER 3)類似の配列を有し、 ATP結合性運搬体活性を有すると推測できる。 このことから、配列番号 1 7に示す塩基配列がコードするタンパク質が薬物の細胞 外排出等にかかわる AB Cトランスポーターであり、薬物等の異物や、カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送等にかかわることが推測できる。 配列番号 18に記載の塩基配列がコードするアミノ酸配列は、 BLASTを用い た相同性検索により、 human ATP - binding cassette protein of the (ABCA subfamily) productが、 e— v a 1 u e : 0、 1250アミノ酸残基に亘り 89%の一致 度で、 Homo sapiens ATP- binding cassette A5»ゝ、 e— v a l u e : 0、 1250フ ノ酸残基に亘り 89%の一致度で、 また、 Homo sapiens ATP- binding cassette A9が、 e_v a l u e ： 0、 1251アミノ酸残基に亘り 38%の一致度でヒットする。  From these facts, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 17 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER 3), and has ATP-binding transport It can be presumed to have body activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 17 is an ABC transporter involved in the extracellular excretion of drugs, etc., and the foreign substances such as drugs and intrinsic factors such as calcium, phospholipids and amphiphiles It can be inferred that it is involved in the transport of toxic substances. The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 18 shows that the human ATP-binding cassette protein of the (ABCA subfamily) product has an e-va 1 ue of 0, 1250 amino acid residues by homology search using BLAST. Homo sapiens ATP-binding cassette A5 »ゝ, e-value: 0, 1250 homonic acid residues with 89% concordance, and Homo sapiens ATP-binding cassette A9 But e_value: 0, hits with 38% identity over 1251 amino acid residues.
また、配列番号 18に示す塩基配列がコードするアミノ酸配列について、 HMM P F AMによるタンパク質特徴検索を行うと、 ABC transporterの特徴を示す配列 (P f a mに AB C— t r a nとしてェントリーされる配列） が見出される。  When a protein feature search was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 18 using HMM PFAM, a sequence showing the characteristics of ABC transporter (sequence entry as ABC-tran in P fam) was found. It is.
これらのこと力 ら、 配列番号 18に示した塩基配列がコードするタンパク質は human ATP- BINDING CASSETTE, SUB-FAMILY A類似の配列を有し、 ATP結合性運搬 体活性を有すると推測できる。 このことから、配列番号 18に示す塩基配列がコー ドするタンパク質が薬物の細胞外排出等にかかわる AB Cトランスポーターであ り、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送 等にかかわることが推測できる。 From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 18 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A and has ATP-binding carrier activity. From this, the protein encoded by the nucleotide sequence of SEQ ID NO: 18 is an ABC transporter involved in the extracellular excretion of drugs, etc., and foreign substances such as drugs, calcium, phospholipids, amphipathic substances, etc. Transport of endogenous substances It can be guessed that it is related to the above.
配列番号 1 9に記載の塩基配列がコードするァミノ酸配列は、 B L A S Tを用い 7こ卞目同'！"生恢索により、 Homo sapiens ATP— binding cassette A9力、、 e— v a 1 u e ： 0、 948アミノ酸残基に亘り 79%の一致度で、 KIAA0822が、 e _ v a 1 u e ： 0、 948 ァミノ酸残基に亘り 67%の一致度で、 また、 Homo sapiens ATP- binding cassette A10 1S e— v a l u e : 0、 958アミノ酸残基に亘り 60%の一致度でヒットする。 また、配列番号 1 9に示す塩基配列がコードするアミノ酸配列について、 HMM P F AMによるタンパク質特徴検索を行うと、 ABC transporterの特徴を示す配列 ( P f a mに A B C— t r a nとしてェントリーされる配列） が見出される。 これらのこと力 ら、 配列番号 1 9に示した塩基配列がコードするタンパク質は human ATP-BINDING CASSETTE, SUB-FAMILY A9類似の配列を有し、 A T P結合性運 搬体活性を有すると推測できる。 このことから、配列番号 1 9に示す塩基配列がコ 一ドするタンパク質が薬物の細胞外排出等にかかわる A B Cトランスポーターで あり、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸 送等にかかわることが推測できる。  The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 19 was obtained by using BLAST in the same order as the 7th order! "Homo sapiens ATP—binding cassette A9 power, e-va 1 ue: 0, 948, with 79% identity over 948 amino acid residues, KIAA0822, e_va 1 ue: 0, 948 amino A hit with a 67% concordance over acid residues and a 60% concordance over Homo sapiens ATP-binding cassette A10 1S e-value: 0, 958 amino acid residues. When the amino acid sequence encoded by the nucleotide sequence shown is searched for protein characteristics using HMM PFAM, a sequence exhibiting ABC transporter characteristics (a sequence that is entered as ABC-tran in P fam) is found. The protein encoded by the nucleotide sequence shown in SEQ ID NO: 19 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A9, and can be inferred to have ATP-binding carrier activity. The nucleotide sequence represented by No. 19 A A B C transporters click protein is involved in the extracellular discharge, etc. of the drug, and foreign matter such as a drug, calcium, phospholipids, be involved in transportation, etc. of endogenous substances such as amphiphiles can be inferred.
配列番号 2 0に記載の塩基配列がコードするァミノ酸配列は、 B L A S Tを用い た相同性検索により、 Homo sapiens cDNA FLJ32506 fis, clone SMINT1000042, weakly similar to ATP- BINDING CASSETTE, SUB-FAMILY A, MEMBER 3が、 e— v a 1 u e : 5 X 1(T"3、 332アミノ酸残基に亘り 75%の一致度で、 Homo sapiens The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 20 was identified by homology search using BLAST as Homo sapiens cDNA FLJ32506 fis, clone SMINT1000042, weakly similar to ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 However, e-va 1 ue: 5 X 1 (T " 3 , Homo sapiens with 75% identity over 332 amino acid residues
ATP-binding cassette A9が、 e _ v a 1 u e ： 5 X 10— 143、 332アミノ酸残基に亘り 75%の一致度で、 また、 Homo sapiens ATP- binding cassette A10力 e - v a 1 u e : 5 X 10— 124、 330アミノ酸残基に亘り 65%の一致度でヒットする。 ATP-binding cassette A9 is, e _ va 1 ue: at 5 X 10- 143, 332 75% degree of coincidence over the amino acid residues, also, Homo sapiens ATP- binding cassette A10 force e - va 1 ue: 5 X 10 124, 330 hits in 65% of the degree of coincidence over the amino acid residues.
また、配列番号 2 0に示す塩基配列がコードするアミノ酸配列について、 HMM P F AMによるタンパク質特徴検索を行うと、 ABC transporterの特徴を示す配列 ( P f a mに A B C— t r a nとしてェントリーされる配列） が見出される。 これらのことから、 配列番号 2 0に示した塩基配列がコードするタンパク質は human ATP-BINDING CASSETTE, SUB- FAMILY A類似の配列を有し、 A T P結合性運搬 体活性を有すると推測できる。 このことから、配列番号 20に示す塩基配列がコー ドするタンパク質が薬物の細胞外排出等にかかわる ABCトランスポーターであ り、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送 等にかかわることが推測できる。 In addition, a protein characteristic search using HMM PFAM was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 20. It is. Thus, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 20 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, and It can be presumed to have body activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 20 is an ABC transporter involved in extracellular excretion of a drug, etc. It can be inferred that it is involved in the transport of volatile substances.
配列番号 21に記載の塩基配列がコ一ドするァミノ酸配列は、 B L A S Tを用い た相同性検索により、 Mus rausculus, clone IMAGE :4505946が、 e— v a 1 u e ： 0、 591アミノ酸残基に亘り 100%の一致度で、 Homo sapiens cDNA KIM0822が、 e _v a 1 u e ： 0、 587アミノ酸残基に亘り 73%の一致度で、 また、 Homo sapiens ATP- binding cassette A9が、 e— v a 1 u e ： 0、 590アミノ酸残基に亘り 68%の —致度でヒットする。  The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 21 was found by homology search using BLAST to find that Mus rausculus, clone IMAGE: 4505946 shows e-va 1 ue: 0, 591 amino acid residues. Homo sapiens cDNA KIM0822 has 100% concordance, e_va 1 ue: 0, 73% concordance over 587 amino acid residues, and Homo sapiens ATP-binding cassette A9 has e-va 1 ue : 0% hits at 590 amino acid residues with 68%-lethality.
また、配列番号 21に示す塩基配列がコードするアミノ酸配列について、 HMM P F AMによるタンパク質特徴検索を行うと、 ABC transporterの特徴を示す配列 (P f an^ ABC—t r a nとしてェントリーされる配列） が見出される。 これらのことから、 配列番号 21に示した塩基配列がコードするタンパク質は human ATP- BINDING CASSETTE, SUB-FAMILY A9類似の配列を有し、 AT P結合性運 搬体活性を有すると推測できる。 このことから、配列番号 21に示す塩基配列がコ 一ドするタンパク質が薬物の細胞外排出等にかかわる ABCトランスポーターで あり、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸 送等にかかわることが推測できる。  When a protein characteristic search was performed by HMM PFAM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 21, a sequence showing the characteristics of ABC transporter (a sequence entry as Pfan ^ ABC-tran) was found. It is. From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 21 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A9 and has ATP-binding carrier activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 21 is an ABC transporter involved in extracellular excretion of drugs, etc., and foreign substances such as drugs, and intrinsic factors such as calcium, phospholipids, amphiphiles, etc. It can be inferred that it is involved in the transport of sex substances.
配列番号 22に記載の塩基配列がコードするアミノ酸配列は、 BLASTを用い たネ目同'性検索により、 Mus musculus ATP— binding cassette transporter ABCA3力 e - v a 1 u e :5X10— 10。、 489アミノ酸残基に亘り 41%の一致度で、 lamellar body membrane specific ATP - binding cassette protein (ABし A3)力、 e— v a l u e : 2 X 10—96、 489アミノ酸残基に亘り 40%の一致度で、 また、 HumanABC3が、 e - v a 1 u e ： 1X10— 94、 489アミノ酸残基に亘り 40%の一致度でヒットする。 The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 22, the Ne th same 'of search using BLAST, Mus musculus ATP- binding cassette transporter ABCA3 force e - va 1 ue: 5X10- 10 . , 41% degree of coincidence over the 489 amino acid residues, lamellar body membrane specific ATP - binding cassette protein (AB and A3) force, e- value: 2 X 10- 96 , 489 40% match over the amino acid residues in degrees, also, HumanABC3 is, e - va 1 ue: 1X10- 94, 489 hits with 40% degree of coincidence over the amino acid residues.
また、配列番号 22に示す塩基配列がコードするアミノ酸配列について、 HMM PF AMによるタンパク質特徴検索を行うと、 ABC transporterの特徴を示す配列 (P f aii^ ABC—t r a nとしてェントリーされる配列） が見出される。 In addition, a protein characteristic search using HMM PFAM was performed on the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 22, and the sequence exhibiting ABC transporter characteristics was found. (P f aii ^ ABC—the sequence that is entered as tran).
これらのこと力 ら、 配列番号 22に示した塩基配列がコードするタンパク質は human ATP- BINDING CASSETTE, SUB- FAMILY A類似の配列を有し、 ATP結合性運搬 体活性を有すると推測できる。 このことから、配列番号 22に示す塩基配列がコー ドするタンパク質が薬物の細胞外排出等にかかわる ABCトランスポーターであ り、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送 等にかかわることが推測できる。  From these results, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 22 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A and has ATP-binding carrier activity. Therefore, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 22 is an ABC transporter involved in the extracellular excretion of drugs, etc., and foreign substances such as drugs, intrinsic factors such as calcium, phospholipids, amphipathic substances, etc. It can be inferred that it is involved in the transport of toxic substances.
配列番号 23に記載の塩基配列がコードするアミノ酸配列は、 BLASTを用い た相同性検索により、 human ATP11C gene for ATPase, Class VI, type 11Cが、 e — v a 1 u e : 0、 436アミノ酸残基に亘り 91%の一致度で、 Potential  The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 23 shows that, by homology search using BLAST, human ATP11C gene for ATPase, Class VI, type 11C has e-va 1 ue: 0, 436 amino acid residues. 91% coincidence, Potential
phospholipid - transpor ing ATPase IH力 e— v a 1 u e ： 5X10— 155、 449アミノ 酸残基に亘り 60%の一致度で、 また、 Potential phospholipid- transporting ATPase ISが、 e_v a 1 u e : 5X10— 127、 375アミノ酸残基に亘り 40%の一致度でヒットす る。 phospholipid - transpor ing ATPase IH force e- va 1 ue: 5X10- 155, 60% degree of coincidence over the 449 amino acid residues, also, Potential phospholipid- transporting ATPase IS is, e_v a 1 ue: 5X10- 127 , Hits with 40% identity over 375 amino acid residues.
これらの結果より、配列番号 23に記載の塩基配列がコードするアミノ酸配列か らなるタンパク質が運搬体 AT P a s eフアミリー類似の配列を有し、 AT P結合 性運搬体活性を有すると推測できる。 このことから、配列番号 23に示す塩基配列 がコードするタンパク質が運搬体 AT P a s eフアミリーであり、薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送等にかかわることが推 測できる。  From these results, it can be inferred that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 23 has a sequence similar to the transporter ATPase family, and has ATP-binding transporter activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 23 is a carrier ATPase family, which is involved in the transport of foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphiphilic substances. This can be inferred.
配列番号 24に記載の塩基配列がコードするアミノ酸配列は、 B LASTを用い た相同性検索により、 human KIM1939が、 e— v a 1 u e ： 5X10— 155、 489ァミノ 酸残基に亘り 85%の一致度で、 Homo sapiens cDNA FLJ30324weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3が、 e— v a 1 u e ： 5Χ1(Γ154、 489ァ ミノ酸残基に亘り 78%の一致度で、 また、 Potential phospholipid - transporting ATPase ICが、 e— v a 1 u e : 5X10— 136、 489アミノ酸残基に亘り 57%の一致度で ヒットする。 また、配列番号 24に示す塩基配列がコードするアミノ酸配列について、 HMM P F AMによるタンパク質特徴検索を行うと、 ABC transporterの特徴を示す配列 (P f air^ ABC—t r a nとしてェントリーされる配列） が見出される。 これらのこと力 ら、 配列番号 24に示した塩基配列がコードするタンパク質は human ATP-BINDING CASSETTE類似の配列を有し、 A T P結合性運搬体活性を有する と推測できる。 このことから、配列番号 24に示す塩基配列がコードするタンパク 質が薬物の細胞外排出等にかかわる A B Cトランスポーターであり、薬物等の異物 や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送等にかかわること が推測できる。 The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 24, by homology search using B LAST, human KIM1939 is, e- va 1 ue: 5X10- 155 , 489 85% match over Amino acid residue time in, Homo sapiens cDNA FLJ30324weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3 is, e- va 1 ue: in 5Χ1 (Γ 154, 489 § amino acid residues 78% degree of coincidence over, also, Potential phospholipid - transporting ATPase IC is, e- va 1 ue: 5X10- 136 , 489 hits in 57% of the degree of coincidence over the amino acid residues. When a protein feature search was performed by HMM PFAM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 24, a sequence showing the characteristics of ABC transporter (a sequence entry as P f air ^ ABC-tran) was found. It is. From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24 has a sequence similar to human ATP-BINDING CASSETTE and has ATP-binding carrier activity. Based on this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24 is an ABC transporter involved in the extracellular excretion of drugs, etc. It can be inferred that it is involved in the transport of substances.
配列番号 25に記載の塩基配列がコードするアミノ酸配列は、 B LAS Tを用い た相同性検索により、 human KIM1939が、 e _ v a 1 u e ： 5X10— 150、 300ァミノ 酸残基に 1り 86%の一致度で、 Homo sapiens otential phospholipid - transporting ATPase ICが、 e— v a 1 u e ： 5X10—"、 309アミノ酸残基に亘り 57%の一致度で、 また、 "CG14741"； Drosophila melanogaster genomic scaffold力 s、 e— v a 1 u e ： 8X10-97, 292アミノ酸残基に亘り 60%の一致度でヒットする。 これらの結果よ り、配列番号 25に記載の塩基配列がコードするアミノ酸配列からなるタンパク質 が運搬体 ATP a s eファミリー類似の配列を有し、 ATP結合性運搬体活性を有 すると推測できる。 このことから、配列番号 25に示す塩基配列がコードするタン パク質が運搬体 AT P a s eファミリーであり、 薬物等の異物や、 カルシウム、 リ ン脂質、 両親媒性物質等の内因性物質の輸送等にかかわることが推測できる。 配列番号 26に記載の塩基配列がコードするアミノ酸配列は、 B LAS Tを用い た相同性検索により、 Homo sapiens, clone IMAGE :4111596が、 e - v a 1 u e ： 0、 600アミノ酸残基に亘り 54%の一致度で、 human Potential The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 25, B by homology search using LAS T, human KIM1939 is, e _ va 1 ue: 5X10- 150, 300 Amino acid residues 1 Ri 86% in the matching degree, Homo sapiens otential phospholipid - transporting ATPase IC is, e- va 1 ue: 5X10- " , with 57% degree of coincidence over the 309 amino acid residues, also," CG14741 "; Drosophila melanogaster genomic scaffold force s , e- va 1 ue:. 8X10- 97, 292 hits with 60% degree of coincidence over the amino acid residues Ri by these results, the protein comprising the amino acid sequence nucleotide sequence coding for SEQ ID NO: 25 is transported It can be inferred that the protein has a sequence similar to that of the ATPase family and has ATP-binding transporter activity. Foreign substances such as drugs, calcium and phosphorus It is speculated that the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 26 can be determined by Homo sapiens, by homology search using BLAST. clone IMAGE: 4111596, e-va 1 ue: 0, human potential with 54% identity over 600 amino acid residues
phospholipid-transporting ATPase ID力 S、 e_v a 1 u e : 0、 600アミノ酸残 に亘り 53%の一致度で、 また、 human KIM1939が、 e— v a 1 u e ： 0、 595アミ ノ酸残基に亘り 52%の一致度でヒットする。 これらの結果より、 配列番号 26に記 載の塩基配列がコードするァミノ酸配列からなるタンパク質が運搬体 A TP a s eファミリ一類似の配列を有し、 AT P結合性運搬体活性を有すると推測できる。 このことから、配列番号 2 6に示す塩基配列がコードするタンパク質が運搬体 A T P a s eファミリーであり、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物 質等の内因性物質の輸送等にかかわることが推測できる。 phospholipid-transporting ATPase ID force S, e_va 1 ue: 0, 53% identity over 600 amino acid residues, and human KIM1939, e-va 1 ue: 0, 595 over amino acid residues 52 Hit with% match. From these results, the protein consisting of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 26 was found to be a carrier A TP as It has a sequence similar to the e-family and can be inferred to have ATP-binding transporter activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 26 is the transporter ATPase family, and is used for transporting foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphiphilic substances. It can be guessed to be involved.
配列番号 2 7に記載の塩基配列がコ一ドするァミノ酸配列は、 B L A S Tを用い た相同性検索により、 Homo sapiens cDNA FLJ30324 weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3が、 e— v a 1 u e : 5 Χ 1(Γ101、 271ァミノ酸残基 に亘り 63%の一致度で、 human Potential phospholipid- transporting ATPase IC 力 S、 e— v a 1 u e : 1 Χ 1(Γ85、 290アミノ酸残基に亘り 51%の一致度で、また、 gene : "CG14741 ； Drosophila melanogaster genomic scaftolc^^ e— v a 1 u e ： 2 X 10— 84、 280アミノ酸残基に亘り 55%の一致度でヒットする。 これらの結果より、 配 列番号 2 7に記載の塩基配列がコードするァミノ酸配列からなるタンパク質が運 搬体 A T P a s eファミリー類似の配列を有し、 A T P結合性運搬体活性を有する と推測できる。 このことから、配列番号 2 7に示す塩基配列がコードするタンパク 質が運搬体 A T P a s eフアミリ一であり、薬物等の異物や、 カルシウム、 リン脂 質、 両親媒性物質等の内因性物質の輸送等にかかわることが推測できる。 The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 27 was found to be Homo sapiens cDNA FLJ30324 weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3 by elasta: in Χ 1 (Γ 101, 271 amino acids 63% degree of coincidence over the residue, human Potential phospholipid- transporting ATPase IC power S, e- va 1 ue: 1 Χ over 1 (Γ 85, 290 amino acid residues 51 in% degree of coincidence, also, gene:;: from 2 X 10- 84, 280 hits in 55% of the degree of coincidence over the amino acid residues of these results "CG14741 Drosophila melanogaster genomic scaftolc ^^ e- va 1 ue. It can be inferred that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 27 has a sequence similar to the ATPase family of the carrier and has ATP-binding carrier activity. The protein encoded by the nucleotide sequence of SEQ ID NO: 27 is the carrier ATPase It can be assumed that it is involved in the transport of foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphipathic substances.
配列番号 2 8に記載の塩基配列がコードするアミノ酸配列は、 B L A S Tを用い ァこ相同性検索により、 gene : CG14741 ； Drosophila melanogaster genomic scaffoldが、 e— v a 1 u e : 5 X 1(T136、 421ァミノ酸残基に亘り 56%の一致度で、 Potential phospholipid -" transporting ATPase IC力 s、 e— v a 1 u e : 5 X 1(T122、 431アミノ酸残基に亘り 53 %の一致度で、 また、 Homo sapiens cDNA FLJ30324 fis, clone BRACE2007138, weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3が、 e— v a 1 u e : 2 X 10—84、 338アミノ酸残基に亘り 62%の一致度でヒットする。 これらの結果より、配列番号 2 8に記載の塩基配列がコードするァミノ酸配列か らなるタンパク質が運搬体 A T P a s eファミリー類似の配列を有し、 A T P結合 性運搬体活性を有すると推測できる。 このことから、配列番号 2 8に示す塩基配列 がコードするタンパク質が運搬体 A T P a s eフアミリーであり、薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送等にかかわることが推 測できる。 The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 28 was obtained by homology search using BLAST, gene: CG14741; Drosophila melanogaster genomic scaffold, e-va 1 ue: 5X1 (T 136 , 421 Potential phospholipid-"transporting ATPase IC force s , e- va 1 ue: 5 X 1 (T 122 , 53% identity over 431 amino acid residues, with 56% identity over amino acid residues, and , Homo sapiens cDNA FLJ30324 fis, clone BRACE2007138, weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3 is, e- va 1 ue:. 2 X 10- 84, 338 hits in 62% of the degree of coincidence over the amino acid residues of From the results, it can be inferred that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 28 has a sequence similar to the transporter ATPase family and has ATP-binding transporter activity. The protein encoded by the nucleotide sequence shown in SEQ ID NO: 28 Carrier ATP ase family, foreign substances such as drugs, It can be inferred that it is involved in the transport of endogenous substances such as calcium, phospholipids and amphiphiles.
ABC (ATP- binding cassette) トランスポーターは、 ATP分解のエネルギー を用いて、 糖、 アミノ酸、 ポリペプチド、 長鎖脂肪酸、 疎水性物質などを輸送する 運搬体 AT P a s eファミリ一の一つであり、その構造は細胞膜を数回貫通し基質 特異性を決定している疎水性領域 2つと、細胞内の ATP結合領域 2つを有するこ とを特徴としている。  The ABC (ATP-binding cassette) transporter is a member of the ATPase family of transporters that transports sugars, amino acids, polypeptides, long-chain fatty acids, hydrophobic substances, etc. using the energy of ATP degradation. Its structure is characterized by having two hydrophobic regions that penetrate the cell membrane several times and determine substrate specificity, and two intracellular ATP-binding regions.
本発明のタンパク質は、上記のとおり、 ABCトランスポーターや運搬体 ATP a s eフアミリーと高い相同性を有していることから、薬物等の異物や、 カルシゥ ム、 リン脂質、両親媒性物質等の内因性物質の輸送等に関与している AT P結合性 運搬体であると推測できる。  As described above, the protein of the present invention has a high homology to the ABC transporter and the carrier ATPase family, and therefore, the foreign substances such as drugs, endogenous substances such as calcium, phospholipids, amphipathic substances and the like. It can be inferred that this is an ATP-binding carrier involved in the transport of sex substances.
(1-4) ィムノグロブリン様タンパク質活性を有するタンパク質 (1-4) Protein having immunoglobulin-like protein activity
配列番号 42に記載の塩基配列がコードするアミノ酸配列は、 B LAS Tサーチ によりデータベース登録記号 P01031、 Complement C5 precursor (HUMAN) 1) e - v a l u e ： 5 X 10_82、 242アミノ酸残基に亘り 62%の一致度で、 またデータべ ース登録記号 P06684、 Complement C5 precursor (MOUSE) iS e - v a 1 u e : 2 X 10— 81、 243アミノ酸残基に亘り 59%の一致度で、 またデータベース登録記号 P12387、 Complement C3 precursor (CAVPO) 力 e— v a 1 u e ： 2 X 10-18、 236 アミノ酸残基に亘り 30%の一致度でヒットする。 The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 42, B LAS T searched by the database registration mark P01031, Complement C5 precursor (HUMAN) 1) e - value: 5 X 10_ 82, 242 62% over amino acid residues in the matching degree, and the data base over scan registration mark P06684, Complement C5 precursor (MOUSE) iS e - va 1 ue: 2 X 10- 81, 243 with 59% degree of coincidence over the amino acid residues, also database registration mark P12387, Complement C3 precursor (CAVPO) force e- va 1 ue: 2 X 10 -18, hits with 30% degree of coincidence over the 236 amino acid residues.
また、配列番号 42に示す塩基配列がコードするアミノ酸配列について、 HMM P F AMによる蛋白質特徴検索を行うと Alpha-2-macroglobulinの特徴を示す配列 (P f amに A2M—Nとしてエントリーされる配列） が見出される。  In addition, a protein characteristic search using HMM PF AM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 42 shows a sequence that shows the characteristics of Alpha-2-macroglobulin (sequence that is entered as A2M-N in P f am) Is found.
また、 上記 P01031、 Complement C5 precursor (HUMAN) タンパク質は、 データべ ース中の文献情報 (Biochemistry 27 :3568-3580 (1988)) から炎症反応に関わるこ とが、 また上記 P06684、 Complement C5 precursor (MOUSE) のタンパク質は、 デー タベース中の文献情報 （j. Biol. Chem. 265 :2435-2440 (1990)) から炎症反応に関 わることが、 さらに上記 P12387、 Complement C3 precursor (CAVPO) のタンパク質 は、 データベース中の文献情報 (J. Clin. Invest. 86:96-106(1990)) から補体の 活性に関わることがそれぞれ示されている。 The P01031, Complement C5 precursor (HUMAN) protein is considered to be involved in the inflammatory reaction based on the literature information in the database (Biochemistry 27: 3568-3580 (1988)). MOUSE) protein is related to inflammatory reactions based on literature information in the database (j. Biol. Chem. 265: 2435-2440 (1990)). In addition, the above P12387 and Complement C3 precursor (CAVPO) proteins are shown to be involved in complement activity based on literature information in the database (J. Clin. Invest. 86: 96-106 (1990)). Have been.
これらのこと力 ら、配列番号 42に示した塩基配列がコードするタンパク質はィ ムノグロプリン様タンパク質であると推測できる。  From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 42 is an immunoglobulin-like protein.
配列番号 43に記載の塩基配列がコードするアミノ酸配列は、 BLASTサーチ によりデータベース登録記号 P01031、 Complement C5 precursor (HUMAN) 力 e - v a 1 u e ： 3 X 1 CT84、 241アミノ酸残基に亘り 63%の一致度で、 またデータべ ース登録記号 P06684、 Complement C5 precursor (MOUSE) 力 e - v a 1 u e ： 2 X 10—83、 242アミノ酸残基に亘り 59%の一致度で、 さらにデータベース登録記号 P12387、 Complement C3 precursor (CAVPO) 力 e~v a l u e ： 3X 10_17、 234 アミノ酸残基に亘り 30%の一致度でヒットする。 The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 43 was obtained by BLAST search using database registration code P01031, Complement C5 precursor (HUMAN) power e-va 1 ue: 3X1 CT 84 , 63% over 241 amino acid residues in the matching degree, and the data base over scan registration mark P06684, Complement C5 precursor (MOUSE) force e - va 1 ue: 2 X 10- 83, 242 with 59% degree of coincidence over the amino acid residues, more database registration mark P12387, Complement C3 precursor (CAVPO) power e ~ value: 3X 10 _17 , hits 234 amino acid residues with 30% match.
また、配列番号 43に示す塩基配列がコードするアミノ酸配列について HMMP F AMによる蛋白質特徴検索を行うと、 Alpha- 2- macroglobulinの特徴を示す配列 (P f & 111に八2¾1—}としてェントリーされる配歹 IJ) が見出される。 これらのことか ら、配列番号 43に示す塩基配列がコードするタンパク質はィムノグロプリン様タ ンパク質であることが推測できる。  When a protein feature search using HMMP FAM was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 43, a sequence showing the characteristics of Alpha-2-macroglobulin (Pf & 111 was entered as 182-1--) System IJ) is found. From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 43 is an immunoglobulin-like protein.
配列番号 44に記載の塩基配列がコードするアミノ酸配列は、 B LASTサーチ によりデータベース登録記号 P14046、 Alpha- 1- inhibitor III precursor (RAT)力 e - v a 1 u e : 5 X 10— 117、 1098アミノ酸残基に亘り 31%の一致度で、 また、 データベース登録記号 P20740、 Ovostatin precursor (CHICK)力 e— v a 1 u e ： 5 X 10-U、 981アミノ酸残基に亘り 29%の一致度で、 さらにデータベース登録記 号 P20742、 Pregnancy zone protein precursor (HUMAN) 力 e— v a l u e : 5 X 10— 1M、 995アミノ酸残基に亘り 29%の一致度でヒットする。 The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 44, the database registration mark P14046 by B LAST search, Alpha- 1- inhibitor III precursor (RAT ) force e - va 1 ue: 5 X 10- 117, 1098 amino acid residues The database registration code P20740, Ovostatin precursor (CHICK) force e—va 1 ue: 5 X 10- U , 29% agreement over 981 amino acid residues, and database registration Symbol P20742, Pregnancy zone protein precursor (HUMAN ) force e- value: hits in 29% of the degree of match over the 5 X 10- 1M, 995 amino acid residues.
また、配列番号 44に示す塩基配列がコードするアミノ酸配列について HMMP F AMによる蛋白質特徴検索を行うと、 Alpha- 2- macroglobulinの特徴を示す配列 (P f amに A2Mとしてエントリーされる配列） が見出される。 また、 上記 P14046、 Alpha- l-inhibitor III precursor (RAT) のタンパク質は、 データベース中の文献情報 ひ. Biol. Chem. 263： 3999-4012 (1988) . ) からプロテ ァーゼの活性阻害に関わることが、また上記 P20740、 Ovostat in precursor (CHICK) のタンパク質は、 データベース中の文献情報 （J. Biol. Chem. When a protein feature search was performed by HMMP FAM on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 44, a sequence showing the characteristics of Alpha-2-macroglobulin (sequence to be entered as A2M in P f am) was found. It is. In addition, P14046, the protein of Alpha-l-inhibitor III precursor (RAT), may be involved in the inhibition of protease activity based on literature information in the database [Biol. Chem. 263: 3999-4012 (1988). Also, P20740 and the protein of Ovostat in precursor (CHICK) described above are available from the literature information (J. Biol. Chem.
258：7481-7489(1983)) からプロテアーゼの活性阻害に関わることが、 さらに上記 P20742, Pregnancy zone protein precursor (HUMAN) のタンノ ク質は、 データべ ース中の文献情報 （J. Biol. Chem. 260： 15723-15735 (1985) ) からプロテアーゼの 活性阻害に関わることがそれぞれわかる。 258: 7481-7489 (1983)), and that the protein of P20742, Pregnancy zone protein precursor (HUMAN), described above was identified in the literature information in the database (J. Biol. Chem. 260: 15723-15735 (1985)), respectively.
これらのことから、配列番号 44に示す塩基配列がコードするタンパク質は、プ 口テアーゼ活性阻害に関わるィムノグロブリン様タンパク質であることが推測で きる。  From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 44 is an immunoglobulin-like protein involved in inhibition of protease activity.
配列番号 45に記載の塩基配列がコードするアミノ酸配列は、 B L A S Tサーチ によりデータベース登録記号 AF329485が、 e— v a 1 u e ： 3 X 1 (Γ52、 121アミ ノ酸残基に亘り 82%の一致度で、 また、 データベース登録記号 AL356276が、 e— ν a 1 u e ： 2 X 1 (Γ26、 84アミノ酸残基に亘り 61%の一致度で、 さらに AF459634 力 e-v a l u e : 2X l CT26、 84アミノ酸残基に亘り 61%の一致度でヒットす る。 The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 45, a database registration mark AF329485 by BLAST search, e- va 1 ue: 3 X 1 (Γ 52, 121 82% degree of coincidence over the amino acid residues in, addition, database registration symbol AL356276 is, e- ν a 1 ue: in 2 X 1 (Γ 26, 84 61% of the degree of coincidence over the amino acid residues, further AF459634 force ev alue: 2X l CT 26, 84 amino acids Hits with 61% match across residues.
また、配列番号 45に示す塩基配列がコードするアミノ酸配列について HMMP FAMによる蛋白質特徴検索を行うと、ィムノグロブリンの特徴を示す配列 （P f 3111に18としてエントリーされる配列） が見出される。 When a protein characteristic search is performed by HMMP FAM on the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 45, a sequence showing the characteristics of immunoglobulin (a sequence which is entered as 18 into P f 3111) is found.
これらのこと力 ら、配列番号 45に示す塩基配列がコードするタンパク質はィム ノグロブリン様タンパク質であることが推測できる。  From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 45 is an immunoglobulin-like protein.
配列番号 46に記載の塩基配列がコードするアミノ酸配列は、 B LAS Tサーチ によりデータベースデータベース登録記号 AF329485が、 e _ v a 1 u e ： 0.0、 343 アミノ酸残基に亘り 99%の一致度で、 また、 データベース登録記号 AF459634が、 e -v a l u e : 9 X l (Γ99、 327アミノ酸残基に亘り 58%の一致度で、 さらにデー タベース登録記号 AL356276が、 e— v a l u e : 1 X 10—73、 298アミノ酸残基に 亘り 51%の一致度でヒッ トする。 The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 46 was identified by the BLAST search according to the database database registration symbol AF329485 as having e_va 1 ue: 0.0, 99% coincidence over 343 amino acid residues, and database registration symbol AF459634 is, e -value: 9 in X l (Γ 99, 327 58 % degree of coincidence over the amino acid residues, more database registration mark AL356276, e- value: 1 X 10- 73, 298 amino acids To the residue Hits with 51% match over time.
また、配列番号 46に示す塩基配列がコードするアミノ酸配列について HMMP F AMによる蛋白質特徴検索を行うと、ィムノグロプリンの特徴を示す配列 （P f amに igとしてエントリーされる配列） が見出される。  When a protein characteristic search is performed by HMMP FAM on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 46, a sequence showing the characteristics of immunoglobulin (a sequence that is entered as ig in P f am) is found.
これらのこと力 ら、配列番号 46に示す塩基配列がコードするタンパク質はィム ノグロブリン様タンパク質であることが推測できる。  From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 46 is an immunoglobulin-like protein.
本発明の D N Aは、翻訳領域中に塩基の欠失もしくは挿入を有した状態で取得さ れることがあるが、 上記のような相同性検索やタンパク質特徴検索を行った結果、 該 DN Aの塩基配列中の塩基の欠失もしくは挿入が推測された場合には、当業者に おいて通常用いられるライブラリースクリーニングや PCRクローユング等の公 知の方法を用いて塩基の欠失もしくは挿入のない完全長 c DN Aを取得すること ができる。かくして得られる完全長 c DNAを用いて本発明のタンパク質を発現さ せ、 これを機能解析等に用いることができる。 The DNA of the present invention may be obtained with a base deletion or insertion in the translation region, but as a result of the homology search or the protein feature search as described above, the DNA base of the DNA is obtained. When a deletion or insertion of a base in the sequence is estimated, a known method such as library screening or PCR closing, which is generally used by those skilled in the art, can be used to obtain the full-length without deletion or insertion of the base. c Can obtain DNA. The full-length cDNA thus obtained is used to express the protein of the present invention, which can be used for functional analysis and the like.
力べして取得され、塩基配列が決定され、 また機能が推定される本発明の DNA は上記の配列番号 1、 12、 13、 16〜28、 42〜46に記載の塩基配列、 あ るいはその翻訳領域として上記に示した塩基配列を有するものだけでなく、これら の塩基配列において、 1若しくは数個 （ここで言う数個の数は特には限定されない 力 S、 例えば 60個以下、 好ましくは 30個以下、 より好ましくは 20個以下、 さら に好ましくは 10個以下、 特に好ましくは 5個以下を意味する。 ） の塩基が欠失、 置換及び または付加された塩基配列を有し、かつ上記した各活性を有するタンパ ク質をコードする DNA、並びに、 これらとストリンジェントな条件下でハイプリ ダイズし、かつ上記した各活性を有するタンパク質をコードする D N A等も含まれ る。 これら DN入には前記したとおり、配列番号 2、 3、 14、 1 5、 29〜41、 47〜51に記載のタンパク質のアミノ酸配列において 1若しくは数個のァミノ 酸配列が欠失、置換及び または付加されたアミノ酸配列からなり、かつ上記した 各活性を有するタンパク質をコードするものが含まれる。 ここで、ストリンジヱントな条件でハイブリダィズする D N Aとは、配列番号 1、 12、 1 3、 16〜28、 42〜46に示される塩基配列あるいはその相補配列と BLAS T解析で 80 %以上、好ましくは 90 %以上、 さらに好ましくは 95 %以 上の相同性を有する塩基配列を含む DN A等が挙げられる。 また、ストリンジェン トな条件下のハイブリダイゼーシヨンとは、通常のハイブリダイゼーション緩衝液 中で、 温度が 40〜70°C、 好ましくは 60〜65°C等で反応を行レ、、塩濃度が 1 5 mM〜 300 mM、好ましくは 15 mM〜 60 mM等の洗浄液中で洗浄を行う方 法に従って行うことができる。 The DNA of the present invention which is obtained by force, whose nucleotide sequence is determined, and whose function is estimated is the nucleotide sequence of SEQ ID NOS: 1, 12, 13, 16 to 28, 42 to 46, or Not only those having the base sequences shown above as the translation regions, but also one or several bases in these base sequences (the number here is not particularly limited, for example, a force S, for example, 60 or less, preferably 30 Or less, more preferably 20 or less, still more preferably 10 or less, particularly preferably 5 or less.) Which has a base sequence in which the base is deleted, substituted and / or added, and Also included are DNAs encoding proteins having each activity, and DNAs which hybridize with these under stringent conditions and encode proteins having the above-mentioned activities. As described above, in these DNs, one or several amino acid sequences are deleted, substituted, and / or in the amino acid sequence of the protein of SEQ ID NO: 2, 3, 14, 15, 29 to 41, or 47 to 51. Included are those comprising an added amino acid sequence and encoding a protein having each of the above-mentioned activities. Here, DNA that hybridizes under stringent conditions refers to the nucleotide sequence shown in SEQ ID NO: 1, 12, 13, 16, 16-28, or 42-46 or its complementary sequence in a BLAST analysis of 80% or more, preferably 90% or more. % Or more, more preferably 95% or more having a homologous nucleotide sequence. Hybridization under stringent conditions means that the reaction is carried out in a normal hybridization buffer at a temperature of 40 to 70 ° C, preferably 60 to 65 ° C, etc. Can be performed in a washing solution of 15 mM to 300 mM, preferably 15 mM to 60 mM.
さらに、 本発明の DNAは、 上述の方法により取得されたものでも、 また合成さ れたものでもよい。 DNAの塩基配列の置換は、例えばサイ トダイレクテツドミュ ータジエネシスキット （宝酒造社製） や、 クイックチェンジサイ トダイレクテツド ミュータジエネシスキット （ストラタジーン社製）等の市販キットで容易に行うこ とができる。  Further, the DNA of the present invention may be obtained by the above-described method or may be synthesized. The DNA base sequence can be easily replaced with a commercially available kit such as a site-directed mutagenesis kit (Takara Shuzo) or a quick change site-directed mutagenesis kit (Stratagene). Can be.
また、 配列番号 1、 12、 13、 16〜28、 42〜 46に記載の塩基配列は、 マウスを由来とするものである力 s、上記した cDNAライブラリーの作製法に従ってヒ トの cDNAライブラリーを作製し、該ライブラリーに対して配列番号 1、 12、 13、 16〜28、 42〜 46の塩基配列を有する DNA断片をプローブとしたハイブリダ ィゼーシヨンを行うことにより、 配列番号 1、 12、 13、 16〜28、 42〜4 6に記載の塩基配列がコードするタンパク質のヒ トのホモログタンパク質をコー ドする DNAを取得することもできる。 本発明の配列番号 1、 12、 13、 16〜2 8、 42〜 46に記載の DNAとストリンジェントな条件でハイブリダイズする DNA には、 このようなヒ トのホモログをコードする DNAも含まれる。 See also SEQ ID NO: 1, 12, 13, 16 to 28, the nucleotide sequence according to 42-46, the force is intended to be derived from the mouse s, cDNA library of human according to Preparation of cDNA library described above Is prepared and subjected to hybridization using a DNA fragment having the nucleotide sequence of SEQ ID NO: 1, 12, 13, 16 to 28 or 42 to 46 as a probe, whereby SEQ ID NO: 1, 12, 13 , 16-28, and 42-46, a DNA encoding a human homolog protein of the protein encoded by the nucleotide sequence can also be obtained. DNAs that hybridize under stringent conditions with the DNAs of SEQ ID NOs: 1, 12, 13, 16 to 28, and 42 to 46 of the present invention also include DNAs encoding such human homologs. .
また、 インフォマティックスを利用して、 ヒ トホモログ DNAが有する塩基配列 を予測し、該塩基配列を基に上記のヒ ト c DN Aライブラリーなどからヒ トホモ口 グ DN Aを取得することもできる。  In addition, the nucleotide sequence of the human homolog DNA can be predicted using informatics, and the human homologous DNA can be obtained from the above human cDNA library based on the nucleotide sequence. .
一般的に、インフォマティックスを利用して目的とするタンパク質のホモログタ ンパク質をコードする塩基配列を予測する方法としては、 例えば、 （i) 目的とす る c DNAの塩基配列をクエリーとして、 ヒ ト等の c DNAデータベース （インフ ォマティックスにより予測される c DNAデータベースを含む）に対し B LAST などを用いて相同性検索を行う方法や、 （i i) 目的とする c DNAの塩基配列を クエリーとして、ヒ ト等の ESTデータベースに対し BLASTなどを用いて相同 性検索を行い、ヒットした ESTが有する配列を目的とする cDNAの塩基配列を 参照して連結する方法、 さらに （ i i i) 目的とする c DNAの塩基配列をクエリ 一として、ヒ トなどのゲノムデータベースに対し BLASTなどを用いて相同性検 索を行い、 目的とする c DNAの遺伝子が存在するゲノム上の位置を特定し、その ゲノム領域に対して G e n s c a n (http:// genes, mit. edu/GENSCAN. html) や SIn general, methods for predicting a nucleotide sequence encoding a homologous protein of a target protein by using informatics include, for example, (i) a method for estimating a target protein; A method of performing a homology search using BLAST etc. on a cDNA database of humans or the like (including a cDNA database predicted by informatics) using the base sequence of the cDNA as a query, and (ii) Using the base sequence of cDNA as a query, perform a homology search using an EST database such as human using BLAST, etc., and link the sequence of the hit EST with reference to the base sequence of the target cDNA And (iii) using the base sequence of the target cDNA as a query, performing a homology search on a genomic database such as humans using BLAST or the like, and searching for the genome in which the gene of the target cDNA exists. Genscan (http: // genes, mit.edu/GENSCAN.html) or S
1 m4 (Genome Res. , 8： 976-74 (1998)) 等を用いて、 該ゲノム中の遺伝子部 分の塩基配列を予測する方法等が挙げられる。 1 m4 (Genome Res., 8: 976-74 (1998)), etc., and a method of predicting the nucleotide sequence of the gene portion in the genome.
マウス由来の c DNAのヒ トホモログ DN Aの塩基配列を予測する場合、上記の 方法のいずれも用いることができるが、 本発明の配列番号 1、 12、 13、 16〜 When predicting the nucleotide sequence of the human homologue DNA of the mouse-derived cDNA, any of the above methods can be used, and the method of the present invention can be applied to any of SEQ ID NOs: 1, 12, 13, 16 to
28、 42〜46に記載の塩基配列を有する c DNAはいずれも新規であり、上記 (i) の方法では、 ヒ トホモログ DNAの塩基配列を取得できないと考えられるた め、 （ii) あるいは （iii) に記載の方法などが好ましく用いられる。 Any of the cDNAs having the nucleotide sequences described in Nos. 28 and 42 to 46 is novel, and it is considered that the method (i) cannot obtain the nucleotide sequence of the human homologous DNA. ) Are preferably used.
かくして予測されたヒ トホモログ DNAの塩基配列を基に、上記のヒ ト cDNA ライブラリーから、 配列番号 1、 1 2、 13、 16〜28、 42〜46に記載の塩 基配列がコードするタンパク質のヒ トのホモログタンパク質をコードする DNA を取得することもできる。 具体的な取得方法としては、 例えば、 予測されたヒ トホ モログ DNAの 5' 端、 および 3' 端の塩基配列に相補的な塩基配列を有するブラ イマ一を用いて、上記ヒ ト cDNAライブラリーを铸型として PC Rを行う方法や、 予測されたヒ トホモログ DNAの一部の配列をプローブとして、上記ヒ ト cDNA ライブラリーに対してハイブリダィゼーシヨンを行う方法等が挙げられる。  Based on the nucleotide sequence of the human homolog DNA predicted as described above, the protein encoded by the nucleotide sequence of SEQ ID NO: 1, 12, 13, 16, 16-28, 42-46 was obtained from the above human cDNA library. DNA encoding a human homolog protein can also be obtained. As a specific method for obtaining the above cDNA library, for example, a primer having a nucleotide sequence complementary to the nucleotide sequence at the 5 ′ end and 3 ′ end of the predicted human homolog DNA is used. And a method of performing hybridization on the human cDNA library using a partial sequence of the predicted human homolog DNA as a probe.
一般的に、目的遺伝子が有する塩基配列とホモロジ一の高い塩基配列を有する類 似遺伝子を 「ホモログ」 と呼ぴ、 上記の方法においてもヒ トホモログの取得を目的 としているが、遺伝子の機能解析においては、塩基配列が類似していることだけで はなく、 ホモログとして取得された遺伝子が、 目的遺伝子のファミリーメンバーで あることを確認することが重要である。 2種類の生物間で 「ホモログ」 として取得 された遺伝子は、共通の祖先遺伝子から進化した同一の遺伝子である「ォ /レソログ」 である可能性と、 また、共通の祖先遺伝子からの重複によって生じた異なる遺伝子 である 「パラログ」 である可能性がある。 In general, a similar gene having a nucleotide sequence having a higher homology to the nucleotide sequence of the target gene is called a “homolog”, and the above-mentioned method also aims to obtain a human homolog, but in the function analysis of the gene, Is only due to the similarity of base sequences However, it is important to confirm that the gene obtained as a homolog is a family member of the target gene. Genes acquired as “homologs” between two species of organisms are likely to be “o / resologs”, which are the same genes evolved from a common ancestral gene, and also caused by duplication from a common ancestral gene. It could be a different gene, a “paralog”.
つまり、 上記でホモログとして取得されたヒ ト由来の DNAは、 これを、 本発明 のタンパク質と同一の機能を有すると解するには、 また、該ヒ ト由来の DNAがコ 一ドするタンパク質の機能を、本発明のタンパク質のマウスにおける機能として推 定検証するには、上記ヒ トホモログが本発明のマウス遺伝子の近縁種のオルソログ であることを確認することが好ましい。  In other words, in order for the human-derived DNA obtained as a homologue to have the same function as the protein of the present invention, it is necessary to use the DNA of the protein encoded by the human-derived DNA. To estimate and verify the function of the protein of the present invention as a mouse function, it is preferable to confirm that the human homolog is an ortholog of a closely related species of the mouse gene of the present invention.
オルソログであることの確認方法は、 例えば、 以下の方法などが用いられる。  For example, the following method is used as a method for confirming the ortholog.
(1) まず、 本発明の c DN Aの塩基配列と、 取得されたヒ トホモログ DN Aの塩 基配列について相同性を解析する。次に、本発明の cDN Aの塩基配列をクエリー として、 DDB J、 EMB L、G e n B a n kなどの国際塩基配列データベースや、 特許データベースに含まれるヒ ト塩基配列について相同性検索を行レ、、取得された ヒ トホモログ DNAとクエリーの塩基配列の一致度が、データベースから得られた 塩基配列とクエリーの塩基配列の一致度より高いことを確認する。 さらに、 （2) 取得されたヒ トホモログ DNAの塩基配列と、対応する本発明の c DNAの塩基配 列について相同性を解析する。 次に、取得されたヒ トホモログ DNAの塩基配列を クエリーとして、 DDB J、 EMBL、 G e n B a n kなどの国際塩基配列データ ベースや、特許データベースに含まれるマウス塩基配列について相同性検索を行レ、、 本発明の cDNAとクエリーの塩基配列の一致度が、データベースから得られた塩 基配列とクエリ一の塩基配列との一致度より高いことを確認する。 上記 （ 1 ) およ び （2) を確認することにより、 取得されたヒ トホモログが、 本発明の cDNAに 対応するヒ トオルソログであると同定することができる。 上記 （1) および （2) に記載した相同性の解析はアミ ノ酸配列の比較を用いても良く 、 また、 分 子進化系統樹を描いて検討すること もできる。 また、 上記 （ 1 ) およ び （ 2 ) に記載した相同性解析による一致度は、 クエリーの全長にわ たる一致度と して解析することが好ましい。 (1) First, homology is analyzed for the nucleotide sequence of the cDNA of the present invention and the nucleotide sequence of the obtained human homolog DNA. Next, using the nucleotide sequence of the cDNA of the present invention as a query, a homology search was performed on the international nucleotide sequence database such as DDB J, EMB L, Gen Bank, and the human nucleotide sequence contained in the patent database. Confirm that the degree of matching between the obtained human homolog DNA and the base sequence of the query is higher than the degree of matching between the base sequence obtained from the database and the base sequence of the query. Further, (2) homology is analyzed for the nucleotide sequence of the obtained human homolog DNA and the corresponding nucleotide sequence of the cDNA of the present invention. Next, using the obtained human homolog DNA base sequence as a query, we performed a homology search on international base sequence databases such as DDB J, EMBL, Gen Bank, and mouse base sequences contained in patent databases. It is confirmed that the degree of matching between the cDNA of the present invention and the base sequence of the query is higher than the degree of matching between the base sequence obtained from the database and the base sequence of the query. By confirming the above (1) and (2), the obtained human homolog can be identified as a human ortholog corresponding to the cDNA of the present invention. The homology analysis described in (1) and (2) above may be performed by comparing amino acid sequences, or by drawing a molecular evolutionary phylogenetic tree. In addition, the above (1) and It is preferable to analyze the degree of coincidence by the homology analysis described in (2) as the degree of coincidence over the entire length of the query.
かくして取得されたヒ トホモログ DNA、あるいはオルソログ DNAの塩基配列 を、 B LASTによる相同性検索や HMMPF AMによる蛋白質特徴検索等を行う ことにより、該塩基配列がコードするタンパク質の機能を推定および確認すること ができる。  Estimating and confirming the function of the protein encoded by the nucleotide sequence of the human homologous DNA or orthologous DNA obtained by performing homology search by BLAST or protein characteristic search by HMMPFAM, etc. Can be.
本発明の配列番号 1、 12、 13、 16〜28、 42〜46に記載の塩基配列ま たはその相補配列を有する D N Aとストリンジェントな条件でハイブリダイズす る DNAには、 このようなヒトホモログ、 あるいはオルソログタンパク質をコード する DNAも含まれる。  Such a human homologue is contained in DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence of SEQ ID NOS: 1, 12, 13, 16 to 28, or 42 to 46 or a sequence complementary thereto. Alternatively, DNAs encoding orthologous proteins are also included.
(2) 新規 cDNAがコードするタンパク質 (2) Protein encoded by the new cDNA
本発明の DNAがコードするタンパク質の翻訳領域は、例えば、該 DNAが有す る塩基配列について 3種類の読み枠によりアミノ酸に変換していき、最も長いポリ ぺプチドをコ一ドする範囲を本発明の翻訳領域としてそのァミノ酸配列を決定す ること等ができる。このようなアミノ酸配列として例えば、配列番号 2、 3、 14、 15、 29〜41、 47〜 51に記載のもの等が挙げられる。 また、 本発明のタン パク質は、上記のアミノ酸配列に限られるものではなく、該アミノ酸配列において 1若しくは数個のアミノ酸が置換、欠失、及び または付加されたアミノ酸配列か らなり、 かつ上記活性を有するものも含まれる。  The translation region of the protein encoded by the DNA of the present invention may be, for example, a base sequence of the DNA, which is converted into amino acids by three types of reading frames, and the range in which the longest polypeptide is encoded is determined. The amino acid sequence can be determined as the translation region of the invention. Examples of such an amino acid sequence include those described in SEQ ID NOs: 2, 3, 14, 15, 29 to 41, and 47 to 51. Further, the protein of the present invention is not limited to the above-mentioned amino acid sequence, but comprises an amino acid sequence in which one or several amino acids have been substituted, deleted and / or added in the amino acid sequence, and Those having activity are also included.
本発明のタンパク質の取得方法としては、 （1) に記載の本発明の DNAを適当 な方法により転写/翻訳する方法が好ましく用いられる。具体的には、適当な発現 用ベクター若しくは適当なベクターに適当なプロモーターとともに挿入した組換 えベクターを作製し、 この組換えベクターで適当な宿主微生物を形質転換したり、 適当な培養細胞に導入することにより発現させ、これを精製することにより取得す ることができる。  As a method for obtaining the protein of the present invention, the method of transcription / translation of the DNA of the present invention described in (1) by an appropriate method is preferably used. Specifically, a suitable expression vector or a recombinant vector inserted into a suitable vector together with a suitable promoter is prepared, and this recombinant vector is used to transform a suitable host microorganism or introduced into a suitable cultured cell. And then can be obtained by purifying it.
かくして得られるタンパク質が遊離体で得られた場合には、公知の方法あるいは それに準じる方法によつて塩に変換することができ、逆に塩で得られた場合には遊 離体、又は他の塩に変換することができる。 この様な本発明のタンパク質の塩も本 発明のタンパク質に含まれる。 また、 上記形質転換体が産生するタンパク質を、 精 製前、又は後に適当なタンパク質修飾酵素を作用させることにより、任意に修飾を 加えたり、ポリぺプチドを部分的に除去することにより修飾タンパク質とすること ができる。これらの修飾タンパク質も上記活性を有するものであれば本発明の範囲 に含まれる。 When the protein thus obtained is obtained in a free form, a known method or It can be converted to a salt by a method according to it, and conversely, if it is obtained as a salt, it can be converted to a free form or another salt. Such salts of the protein of the present invention are also included in the protein of the present invention. In addition, the protein produced by the above-mentioned transformant can be modified before or after purification by the action of an appropriate protein-modifying enzyme to arbitrarily modify the protein or partially remove the polypeptide. can do. These modified proteins are also included in the scope of the present invention as long as they have the above activity.
本発明のタンパク質の産生を行う際、本発明の DN Aを含む組換えベクターの作 製に用いるベクターとしては、形質転換体内で該 DN Aが発現されるものであれば 特に制限はなく、 プラスミ ドベクター、 ファージベクターのいずれでもよい。 これ らのうち通常は、該 DN Aが導入される宿主に適したプロモーター等の発現制御領 域 DNAが既に挿入されている市販のタンパク質発現用ベクターを用いる。このよ うなタンパク質発現用ベクターとして、具体的には例えば、宿主が大腸菌の場合で は、 pET3、 pET l l (ストラタジーン社製） p GEX (アマシャムフアルマ シァバイオテク社製）等が挙げられ、酵母の場合では p E S P— Iェクスプレツシ ヨンベクター （ストラタジーン社製） 等が挙げられ、 さらに昆虫細胞の場合では B a c PAK6 (クロンテック社製） 等が用いられる。 また宿主が動物細胞の場合で は、 ZAP Ex p r e s s (ストラタジーン社製） 、 p S VK3 (アマシャムフ アルマシアバイオテク社製） 等が挙げられる。  When producing the protein of the present invention, the vector used for the production of the recombinant vector containing the DNA of the present invention is not particularly limited as long as the DNA is expressed in the transformant. And phage vectors. Usually, a commercially available protein expression vector into which an expression control region DNA such as a promoter suitable for the host into which the DNA is introduced has already been inserted is used. Specific examples of such a protein expression vector include pET3 and pETll (manufactured by Stratagene) p GEX (manufactured by Amersham Pharmacia Biotech) when the host is Escherichia coli, and yeast. In the case of (b), p ESP-I expression vector (manufactured by Stratagene) and the like are used. In the case of insect cells, Bac PAK6 (manufactured by Clontech) is used. When the host is an animal cell, examples include ZAP Express (manufactured by Stratagene) and pSVK3 (manufactured by Amersham Armasia Biotech).
発現制御領域が挿入されていないベクターを用いる場合には、発現制御領域とし て少なくともプロモーターを挿入する必要がある。 ここでプロモーターとしては、 宿主微生物、 または培養細胞が保有するプロモーターを用いることができるが、 こ れに限られるものではなく、 具体的には例えば、 宿主が大腸菌の場合には T 3、 T 7、 t a c、 1 a cプロモーター等を用いることができ、 酵母の場合には nm t 1 プロモーター、 G a 1 1プロモーター等を用いることができる。 また宿主が動物細 胞の場合には SV40プロモーター、 CMVプロモーター等が好ましく用いられる。 また哺乳動物由来のプロモーターが機能可能な宿主を用いる場合には、本発明の 遺伝子に固有のプロモーターを用いることもできる。これらのベクターへの本発明 の DN Aの挿入は、該 DN Aまたはこれを含む DN A断片をベクター中のプロモー ターの下流に該遺伝子 DN Aがコードするタンパク質のアミノ酸配列を連結して 行えばよい。 When using a vector into which an expression control region has not been inserted, it is necessary to insert at least a promoter as the expression control region. The promoter used herein may be a promoter contained in a host microorganism or a cultured cell, but is not limited thereto. For example, when the host is Escherichia coli, T3, T7 , it can be used tac, 1 ac promoter, and the like, in the case of yeast can be used nm t 1 promoter, G a 1 1 promoter. When the host is an animal cell, SV40 promoter, CMV promoter and the like are preferably used. When a host capable of functioning as a mammalian-derived promoter is used, the present invention A promoter specific to the gene can also be used. Insertion of the DNA of the present invention into these vectors is performed by linking the DNA or the DNA fragment containing the DNA to the amino acid sequence of the protein encoded by the gene DNA downstream of the promoter in the vector. Good.
このようにして作製した組換えベクターは、それ自体既知の方法により後述する 宿主を形質転換して、 DN A導入体を作製することができる。宿主への該ベクター の導入方法として、具体的には、ヒートショック法（J. Mol. Biol. , 53, 154, (1970))、 リン酸カルシウム法 (Science, 221, 551, (1983)) 、 DEAEデキストラン法 (Science, 215, 166, (1982)) 、 インビトロパッケージング法 （Proc. Natl. Acad. Sci.USA, 72, 581, (1975)) 、 ウィルスベクター法 (Cell, 37, 1053, (1984)) 、 および 電気パルス法 (Chu. et al. ,Nuc. Acids Res.， 15, 1331 (1987) ) 等が挙げられる。  The recombinant vector thus prepared can be transformed into a host described below by a method known per se to prepare a DNA-introduced body. As a method for introducing the vector into a host, specifically, a heat shock method (J. Mol. Biol., 53, 154, (1970)), a calcium phosphate method (Science, 221, 551, (1983)), DEAE Dextran method (Science, 215, 166, (1982)), in vitro packaging method (Proc. Natl. Acad. Sci. USA, 72, 581, (1975)), virus vector method (Cell, 37, 1053, (1984) )) And electric pulse method (Chu. Et al., Nuc. Acids Res., 15, 1331 (1987)).
DN A導入体を作製するための宿主としては、本発明の DN Aが体内で発現する ものであれば特に限定されないが、 例えば大腸菌、 酵母、 バキュロウィルス （節足 動物多角体ウィルス）一昆虫細胞、あるいは動物細胞等が挙げられる。具体的には、 大腸菌では B L 21, XL-2B 1 u e (ス トラタジーン社製） 等、 酵母では S P -Q01 (ストラタジーン社製） 等、 バキュロウィルスでは AcNPV  The host for producing the DNA-introduced host is not particularly limited as long as the DNA of the present invention is expressed in the body. For example, Escherichia coli, yeast, baculovirus (arthropod polyhedrosis virus) -one insect cell Or animal cells. Specifically, B L21, XL-2B 1 ue (Stratagene) for E. coli, SP-Q01 (Stratagene) for yeast, AcNPV for baculovirus, etc.
(J. Biol. Chem. , 263, 7406, (1988)) とその宿主である S f - 9  (J. Biol. Chem., 263, 7406, (1988)) and its host, S f-9
(J. Biol. Chera. , 263, 7406, (1988))等が挙げられる。 また動物細胞としてはマウス 繊維芽細胞 C 127 (J. Viol. , 26, 291, (1978)) やチャイニーズハムスター卵巣細 胞 CHO細胞 (Proc. Natl. Acad. Sci. USA, 77, 4216, (1980)) 等が挙げられるが、 発現量やスクリーニングの簡便さから好ましくはアフリカミ ドリザル腎臓由来 C OS— 7 (ATCCCRL1651：アメリカン タイプ カルチャー コレクション保存細 胞） が用いられる。  (J. Biol. Chera., 263, 7406, (1988)). Examples of animal cells include mouse fibroblast C127 (J. Viol., 26, 291, (1978)) and Chinese hamster ovary cell CHO cells (Proc. Natl. Acad. Sci. USA, 77, 4216, (1980). )), Etc., but from the viewpoint of expression level and simplicity of screening, African green monkey kidney-derived COS-7 (ATCCCRL1651: cells stored in the American Type Culture Collection) is preferably used.
上記したようなタンパク質発現用ベクターを用いる発現方法の他に、プロモータ 一を連結した本発明の D N A断片を宿主微生物の染色体中に直接挿入する相同組 換え技術 (A. A. Vertes et al. , Biosci. Biotechnol. Biochem. , 57, 2036, (1993) ) 、 あるいはトランスポゾンや挿入配列 （A.A. Vertes et al. , Molecular Microbiol. , 11, 739, (1994) ) 等を用いて D N A導入体を作製することもできる。 得られた培養物は細胞、 あるいは菌体を遠心分離等の方法により収集し、 これを 適当な緩衝液に懸濁し、 超音波、 リゾチーム、 および Zまたは凍結融解等のそれ自 体既知の適当な方法により破壊した後、遠心分離や濾過等によりタンパク質粗精製 液を得、 さらに適当な精製方法を組み合わせることにより精製することができる。 かくして、本発明のタンパク質が取得される。上記したタンパク質発現組換えべク ターを用いる発現方法の他に、 上記 （1 ) で取得された本発明の D N Aを無細胞転 写翻訳系に供することによりタンパク質発現を誘導し、本発明のタンパク質を取得 することができる。本発明で用いられる無細胞転写翻訳系とは、 D NAから m R N Aへの転写、および m R N Aからタンパク質への翻訳に必要な全ての要素を含む系 であり、そこに D NAを加えることによってその D N Aがコードしているタンパク 質が合成されるようなあらゆる系を指す。無細胞転写翻訳系の具体例としては、真 核細胞、およびバクテリア細胞、又はそれらの一部からの抽出液に基づいて調製さ れた転写翻訳系が挙げられ、 特に好ましい具体例としては、 ゥサギ網状赤血球、小 麦胚芽、 大腸菌からの抽出液 （大腸菌 S 3 0抽出液） に基づいて調製された転写翻 訳系が挙げられる。 In addition to the above-described expression methods using protein expression vectors, homologous recombination techniques (AA Vertes et al., Biosci. Biotechnol. Biochem., 57, 2036, (1993)), or a transposon or insertion sequence (AA Vertes et al., Molecular Microbiol., 11, 739, (1994)) and the like. The obtained culture is collected by centrifugation or the like to collect cells or cells, suspended in an appropriate buffer, and then sonicated, lysozyme, and Z or freeze-thaw, etc. After disruption by the method, a crude protein solution is obtained by centrifugation, filtration, etc., and can be further purified by a combination of appropriate purification methods. Thus, the protein of the present invention is obtained. In addition to the above-described expression method using a protein expression recombinant vector, protein expression is induced by subjecting the DNA of the present invention obtained in the above (1) to a cell-free transcription / translation system, thereby obtaining a protein of the present invention. Can be obtained. The cell-free transcription / translation system used in the present invention is a system containing all the elements necessary for transcription of DNA to mRNA and translation of mRNA to protein, and by adding DNA thereto. Any system in which the protein encoded by the DNA is synthesized. Specific examples of the cell-free transcription / translation system include a transcription / translation system prepared based on an eukaryotic cell, a bacterial cell, or an extract from a part thereof, and a particularly preferred example is Egret A transcription translation system prepared based on extracts from reticulocytes, wheat germ, and Escherichia coli (Escherichia coli S30 extract) may be mentioned.
得られた無細胞転写翻訳系の転写翻訳産物からの、 本発明のタンパク質の分離、 および精製は、それ自体既知の通常用いられる方法で行うことができる。 具体的に は、 例えばェピトープペプチド、 ポリヒスチジンペプチド、 ダルタチオン一 S—ト ランスフ-ラーゼ （G S T) 、 マルトース結合タンパク質等をコードする D N A領 域を、 前記した転写翻訳されるべき D N Aに導入し、 前記の通り発現させ、該タン パク質と親和性を有する物質とのァフィ二ティーを利用して精製することができ る。  Separation and purification of the protein of the present invention from the obtained transcription / translation product of the cell-free transcription / translation system can be performed by a method known per se and generally used. Specifically, for example, a DNA region encoding an epitope peptide, a polyhistidine peptide, daltathione-1 S-transferase (GST), a maltose binding protein, or the like is introduced into the DNA to be transcribed and translated. It can be expressed as described above and purified using the affinity of the protein with a substance having affinity.
目的とするタンパク質の発現は、 S D S—ポリアクリルアミ ドゲル電気泳動等で 分離し、 クマシ一プリリアントブルー （シグマ社製） 等で染色するか、 または後述 する本発明のタンパク質に特異的に結合する抗体により検出する方法等によって 確認できる。 また一般的に、発現されたタンパク質は生体内に存在するタンパク質 分解酵素により切断されること （プロセッシング） が知られている。 本発明のタン パク質も当然のことながら切断されたァミノ酸配列の部分断片であっても、上記活 性を有するものであれば、 本発明のタンパク質に含まれる。 The expression of the target protein is separated by SDS-polyacrylamide gel electrophoresis or the like, and stained with Coomassie Priliant Blue (manufactured by Sigma), or specifically binds to the protein of the present invention described later. It can be confirmed by the detection method using an antibody. In general, the expressed protein is a protein existing in vivo. It is known that it is cleaved by a degrading enzyme (processing). The protein of the present invention is, of course, included in the protein of the present invention as long as it has the above activity, even if it is a partial fragment of the cleaved amino acid sequence.
かくして得られたタンパク質は、他のタンパク質、 D NAとの相互作用等を解析 することにより、 生体内における多面的な機能を知ることができる。上記相互作用 の解析法としては、 それ自体既知の常法を用いることができるが、 具体的には、 例 えば、 酵母ツーハイプリッド法、 蛍光偏光解消法、 表面プラズモン法、 ファージデ イスプレイ法、 リポソ一マルディスプレイ法等が挙げられる。  By analyzing the interaction between the thus obtained protein and other proteins and DNA, it is possible to know the multifaceted functions in the living body. As a method for analyzing the interaction, a conventional method known per se can be used. Specifically, for example, a yeast two-hybrid method, a fluorescence depolarization method, a surface plasmon method, a phage display method, and a liposome method One example is the multiple display method.
( 3 ) オリゴヌクレオチドの調製及び該オリゴヌクレオチドを用いる機能解析 上記 （1 ) に記載の方法で取得した本発明の D NAまたはその断片を用いて、 D NA合成機などを用いる常法により、本発明の D N Aの一部の配列を有するアンチ センス ·オリゴヌクレオチド、 センス ·オリゴヌクレオチド等のォリゴヌクレオチ ドを調製することができる。 (3) Preparation of Oligonucleotide and Functional Analysis Using the Oligonucleotide Using the DNA of the present invention or a fragment thereof obtained by the method described in (1) above, the DNA is prepared by a conventional method using a DNA synthesizer or the like. Oligonucleotides such as antisense oligonucleotides and sense oligonucleotides having a partial sequence of the DNA of the present invention can be prepared.
該オリゴヌクレオチドとしては、上記 D N Aの有する塩基配列中の連続した 5〜 1 0 0塩基と同じ配列を有する D NAまたは該 D N Aと相補的な配列を有する D NAを挙げることができる。 具体例としては、 配列番号 1、 1 2、 1 3、 1 6〜2 8、 4 2〜4 6に記載の塩基配列中の連続した 5〜1 0 0塩基と同じ配列を有する D N Aまたは該 D N Aと相補的な配列を有する D N Aを挙げることができる。セン スプライマーおよびアンチセンスプライマーとして用いる場合には、両者の融解温 度（T m)および塩基数が極端に変わることのない上記のオリゴヌクレオチドが好 ましい。 また、 配列の長さは、 一般的には 5〜1 0 0塩基であり、 好ましくは 1 0 〜6 0塩基であり、 より好ましくは 1 5〜5 0塩基である。  Examples of the oligonucleotide include a DNA having the same sequence as the consecutive 5 to 100 bases in the base sequence of the DNA or a DNA having a sequence complementary to the DNA. As a specific example, DNA having the same sequence as 5 to 100 consecutive bases in the base sequence described in SEQ ID NO: 1, 12, 13, 13, 16 to 28, 42 to 46 or the DNA And a DNA having a sequence complementary to the above. When used as a sense primer and an antisense primer, the above oligonucleotides in which the melting temperature (Tm) and the number of bases of both do not extremely change are preferred. The length of the sequence is generally 5 to 100 bases, preferably 10 to 60 bases, and more preferably 15 to 50 bases.
また、これらオリゴヌクレオチドの誘導体も本発明のオリゴヌクレオチドとして 利用することができる。該オリゴヌクレオチド誘導体としては、オリゴヌクレオチ ド中のリン酸ジエステル結合がホスホロチォエート結合に変換されたオリゴヌク レオチド誘導体、 オリゴヌクレオチド中のリン酸ジエステル結合が N 3， - P 5 ' ホスフォアミデート結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオ チド中のリボースとリン酸ジエステル結合がぺプチド核酸結合に変換されたオリ ゴヌクレオチド誘導体、オリゴヌクレオチド中のゥラシルが C— 5プロピニルゥラ シルで置換されたォリゴヌクレオチド誘導体、オリゴヌクレオチド中のゥラシルが C _ 5チアゾールゥラシルで置換されたォリゴヌクレオチド誘導体、オリゴヌクレ ォチド中のシトシンが C一 5プロビュルシトシンで置換されたオリゴヌクレオチ ド誘導体、 オリゴヌクレオチド中のシトシンがフエノキサジン修飾シトシン iphenoxazine-modified cytosine) で置換されたオリゴヌクレオチド誘導体、 ォ リゴヌクレオチド中のリボースが 2，一O—プロピルリボースで置換されたオリゴ ヌクレオチド誘導体、 あるいはオリゴヌクレオチド中のリボースが 2 ' —メ トキシ エトキシリボースで置換されたオリゴヌクレオチド誘導体等を挙げることができ る。 In addition, derivatives of these oligonucleotides can also be used as the oligonucleotide of the present invention. Examples of the oligonucleotide derivative include an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide having N3, -P5 ' Oligonucleotide derivatives converted to phosphoamidate bonds, oligonucleotide derivatives in which ribose and phosphodiester bonds in oligonucleotides are converted to peptide nucleic acid bonds, and peracyl in oligonucleotides are C-5 propynyl peracyl. A substituted oligonucleotide derivative, an oligonucleotide derivative in which peracyl in an oligonucleotide is substituted with C_5 thiazoleperacyl, an oligonucleotide derivative in which cytosine in an oligonucleotide is substituted with C-15 probucytosine, Oligonucleotide derivatives in which cytosine in the oligonucleotide has been replaced with phenoxazine-modified cytosine), oligonucleotide derivatives in which ribose in the oligonucleotide has been replaced with 2,1O-propylribose, There is ribose in the oligonucleotide is 2 '- Ru can be mentioned oligonucleotide derivatives substituted with main butoxy ethoxy ribose.
また、 本発明のオリゴヌクレオチドは、 これを 2本鎖 R Aとして調製することに より、 R Aインターフェアレンス法 （以下、 これを 「R NA i法」 と称することが ある） に適用することができる。 2本鎖 RNAの作製方法、 及び RNAインターフェアレ ンス法については、例えば、 (Elbashir, S.， et al. , Nature, 411, 494- 498 (2謝）) に記載の方法等を用いることができる。  The oligonucleotide of the present invention can be applied to the RA interference method (hereinafter, this may be referred to as “RNAi method”) by preparing it as a double-stranded RA. . For the method for preparing double-stranded RNA and the RNA interference method, for example, the method described in (Elbashir, S., et al., Nature, 411, 494-498 (2)) is used. Can be.
上記 2本鎖 R N Aは、 そのすべてが R NAである必要はない。 具体的には、 その 一部が D NAであるものとして、 WO O 2 / 1 0 3 7 4公報に記載のものを用いる ことができる。  The double-stranded RNAs need not all be RNAs. Specifically, as a part of which is a DNA, those described in WO 02/13774 can be used.
ここで、 標的遺伝子としては、 本発明の DNAであれば、 如何なるものであっても よレ、。これらの DNAの少なくとも一部の塩基配列と実質的に同一な配列を有する RNA からなる 2本鎖ポリヌクレオチド （以下、 これを 「2本鎖ポリヌクレオチド」 と称 することがある） とは、標的遺伝子の塩基配列のうち、 いずれの部分でもよい 1 5 bp以上の配列と実質的に同一な配列からなるものである。 ここで、実質的に同一と は、標的遺伝子の配列と 8 0 %以上の相同性を有することを意味する。 ヌクレオチ ドの鎖長は 1 5 bpから標的遺伝子のオープンリーディングフレーム （0RF) の全長 までの如何なる長さでもよいが、 1 5〜5 0 O bp程度のものが好ましく用いられる。 ただし、 哺乳類動物由来の細胞おいては、 3 0 bp以上の長い 2本鎖 RNAに反応して 活性化するシグナル伝達系の存在が知られている。これはィンターフェ口ン反応と 呼ばれており （Mareus, P. I. , et al. , Interferon, 5, 115 - 180 (1983) ) 、 該 2 本鎖 RNAが細胞内に侵入すると、 PKR (dsRNA-responsive protein kinase： Bass, B. L. , Nature, 411, 428-429 (2001) ) を介して多くの遺伝子の翻訳開始が非特異的 に阻害され、それと同時に 2 '、 5 ' oligoadenylate synthetase (Bass, B. L. , Nature, 411, 428-429 (2001) ) を介して RNaseLの活性化が起こり、 細胞内の RNAの非特異的 な分解が惹起される。 これらの非特異的な反応のために、標的遺伝子の特異的反応 が隠蔽されてしまう。 従って哺乳類動物、 または該動物由来の細胞、 あるいは組織 を被導入体として用いる場合には 1 5〜3 O bp、好ましくは 1 9〜2 4 b p、最も 好ましくは 2 1 b pの 2本鎖ポリヌクレオチドを用いることが好ましレ、。 2本鎖ポ リヌクレオチドはその全体が 2本鎖である必要はなく、 5 ' 、 または 3 ' 末端が一 部突出したものも含むが、 3 '末端が 2塩基突出したものを用いることが好ましレ、。 Here, any target gene may be used as long as it is the DNA of the present invention. A double-stranded polynucleotide consisting of RNA having a sequence substantially identical to at least a part of the base sequence of these DNAs (hereinafter sometimes referred to as “double-stranded polynucleotide”) is a target It comprises a sequence substantially the same as a sequence of 15 bp or more, which may be any part of the nucleotide sequence of the gene. Here, “substantially the same” means that it has 80% or more homology with the sequence of the target gene. Nucleotide lengths range from 15 bp to the full length of the open reading frame (0RF) of the target gene. The length may be any length up to about 15 to 50 Obp. However, it is known that mammalian cells have a signal transduction system that activates in response to long double-stranded RNA of 30 bp or more. This is called the interfering reaction (Mareus, PI, et al., Interferon, 5, 115-180 (1983)), and when the double-stranded RNA enters the cell, PKR (dsRNA-responsive protein) Kinase: Non-specific inhibition of translation initiation of many genes via Bass, BL, Nature, 411, 428-429 (2001)), and at the same time, 2 'and 5' oligoadenylate synthetase (Bass, BL, Nature, 411, 428-429 (2001)), which activates RNaseL and causes nonspecific degradation of intracellular RNA. These non-specific reactions mask the specific response of the target gene. Accordingly, when a mammal, or a cell or tissue derived from the animal is used as the transfectant, a double-stranded polynucleotide of 15 to 30 bp, preferably 19 to 24 bp, most preferably 21 bp. It is preferable to use. The double-stranded polynucleotide does not need to be entirely double-stranded, and includes those having a partially protruding 5 ′ or 3 ′ end, but those having a 3 ′ end protruding two bases are preferred. Masure,
2本鎖ポリヌクレオチドは相補性を有する 2本鎖のポリヌクレオチドを意味す るが、自己相補性を有する 1本鎖ポリヌクレオチドが自己アニーリングしたもので もよレ、。 自己相補性を有する 1本鎖ポリヌクレオチドとしては、 例えば、 逆方向反 復配列を有するもの等が挙げられる。  The double-stranded polynucleotide means a double-stranded polynucleotide having complementarity, but may be a self-annealed single-stranded polynucleotide having self-complementarity. Single-stranded polynucleotides having self-complementarity include, for example, those having an inverted repeat sequence.
2本鎖ポリヌクレオチドの調製方法としては、特に制限はなレ、が、それ自体既知 の化学合成方法を用いることが好ましい。化学合成は、相補性を有する 1本鎖ポリ ヌクレオチドを別個に合成し、これを適当な方法で会合させることにより 2本鎖と することができる。 会合の方法としては上記ポリヌクレオチドを混合し、 2本鎖が 解離する温度にまで加熱し、その後徐々に冷却する方法等が挙げられる。会合した 2本鎖ポリヌクレオチドは、 ァガロースゲル等を用いて確認し、残存する 1本鎖ポ リヌクレオチドを適当な酵素により分解する等して除去する。  Although the method for preparing the double-stranded polynucleotide is not particularly limited, it is preferable to use a known chemical synthesis method. In chemical synthesis, a single-stranded polynucleotide having complementarity can be separately synthesized, and can be converted into a double-stranded strand by associating them by an appropriate method. Examples of the method of association include a method in which the above polynucleotides are mixed, heated to a temperature at which the double strand dissociates, and then gradually cooled. The associated double-stranded polynucleotide is confirmed using an agarose gel or the like, and the remaining single-stranded polynucleotide is removed by, for example, decomposing with a suitable enzyme.
このようにして調製した 2本鎖ポリヌクレオチドを導入する被導入体としては、 標的遺伝子がその細胞内で RNAに転写、 またはタンパク質に翻訳を受け得るもので あれば如何なるものであってもよいが、 具体的には、 植物、 動物、 原生動物、 ウイ ルス、 バクテリア、 または真菌種に属するものが挙げられる。 植物は単子葉植物、 双子葉植物または裸子植物であってよく、動物は、脊椎動物または無脊椎動物であ つてよレ、。好ましい微生物は、農業で、または工業によつて使用されるものであり、 そして植物または動物に対して病原性のものである。真菌には、 カビ及び酵母形態 両方での生物体が含まれる。脊椎動物の例には、魚類、 ゥシ、ャギ、ブタ、 ヒッジ、 ハムスター、 マウス、 ラット及ぴヒ トを含む哺乳動物が含まれ、 無脊椎動物には、 線虫類及び他の虫類、 キイ口ショウジヨウバエ （Drosophila) 、 及び他の昆虫が含 まれる。 好ましくは、 細胞は脊椎動物細胞である。 The transfectant into which the double-stranded polynucleotide prepared in this way is introduced is one in which the target gene can be transcribed into RNA or translated into protein in the cell. Any substance may be used, but specific examples include those belonging to plant, animal, protozoan, virus, bacterial, or fungal species. The plant can be a monocotyledonous, dicotyledonous or gymnosperm, and the animal can be a vertebrate or invertebrate. Preferred microorganisms are those used in agriculture or by industry, and are pathogenic to plants or animals. Fungi include organisms in both mold and yeast forms. Examples of vertebrates include mammals, including fish, sea lions, goats, pigs, sheep, hamsters, mice, rats and humans, and invertebrates include nematodes and other reptiles. , Drosophila, and other insects. Preferably, the cells are vertebrate cells.
被導入体は、 細胞、 組織、 あるいは個体を意味する。 ここで細胞とは、 生殖系列 または体性、分化全能、または多分化能、分割または非分割、実質組織または上皮、 不滅化したものまたは形質転換したもの等からであってよい。細胞は、配偶子また は胚であってよく、胚の場合、 単一細胞胚または構成性細胞、 または多重細胞胚か らの細胞であり、 胎児組織を含む。 さらには、 幹細胞のような未分化細胞、 または 胎児組織を含む器官または組織の細胞からのような分化細胞、または生物内に存在 する任意の他の細胞であってよい。 分化している細胞型には、 脂肪細胞、 繊維芽細 胞、 筋細胞、 心筋細胞、 内皮細胞、 神経細胞、 グリア、 血液細胞、 巨核球、 リンパ 球、 マクロファージ、 好中球、 好酸球、 好塩基球、 マス ト細胞、 白血球、 顆粒球、 ケラチン生成細胞、 軟骨細胞、 骨芽細胞、 破骨細胞、 肝細胞及び内分泌腺または外 分泌腺の細胞が含まれる。  The transductant means a cell, tissue, or individual. Here, the cell may be from germline or somatic, totipotent or pluripotent, split or non-split, parenchymal or epithelial, immortalized or transformed, and the like. The cell can be a gamete or an embryo, in the case of an embryo, a single cell embryo or a constitutive cell, or a cell from a multi-cell embryo, including fetal tissue. Furthermore, they may be undifferentiated cells, such as stem cells, or differentiated cells, such as from cells of an organ or tissue, including fetal tissue, or any other cells present in an organism. Differentiating cell types include adipocytes, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, nerve cells, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, Includes basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes and cells of the endocrine or exocrine glands.
被導入体への 2本鎖ポリヌクレオチドの導入法としては、被導入体が細胞、 ある いは組織の場合は、 カルシウムフォスフェート法、 エレクトロポレーシヨン法、 リ ポフエクシヨン法、 ウィルス感染、 2本鎖ポリヌクレオチド溶液への浸漬、 あるい は形質転換法等が用いられる。 また、 胚に導入する方法としては、 マイクロインジ ェクシヨン、 エレクト口ポレーシヨン法、 あるいはウィルス感染等が挙げられる。 被導入体が植物の場合には、 植物体の体腔または間質細胞等への注入または灌流、 あるいは噴霧による方法が用いられる。 また、 動物個体の場合には、 経口、 局所、 非経口 （皮下、 筋肉内及び静脈内投与を含む） 、 経膣、 経直腸、 経鼻、 経眼、 腹膜 内投与等によって全身的に導入する方法、あるいはエレクトロポレーシヨン法ゃゥ ィルス感染等が用いられる。経口導入のための方法には、 2本鎖ポリヌクレオチド を生物の食物と直接混合することができる。 さらに、個体に導入する場合には、 例 えば埋め込み長期放出製剤等として投与することや、 2本鎖ポリヌクレオチドを導 入した導入体を摂取させることにより行うこともできる。 As a method for introducing a double-stranded polynucleotide into a recipient, when the recipient is a cell or tissue, calcium phosphate method, electroporation method, lipofection method, virus infection, two Immersion in a strand polynucleotide solution or a transformation method is used. Examples of the method for introducing the gene into the embryo include microinjection, electoral poration, and virus infection. When the recipient is a plant, a method of injecting or perfusing the plant into the body cavity or stromal cells, or spraying is used. Oral, topical, Parenteral (including subcutaneous, intramuscular and intravenous administration), vaginal, rectal, nasal, ophthalmic, intraperitoneal administration, etc., systemic introduction, or electroporation virus infection, etc. Is used. For methods for oral introduction, the double-stranded polynucleotide can be mixed directly with the food of the organism. Further, when introduced into an individual, it can be administered, for example, by administration as an implanted long-term release preparation or the like, or by ingesting an introduced body into which a double-stranded polynucleotide has been introduced.
導入する 2本鎖ポリヌクレオチドの量は、導入体や、標的遺伝子によって適宜選 択することができる力 細胞あたり少なくとも 1コピー導入されるに充分量を導入 することが好ましい。 具体的には、 例えば、 被導入体がヒ ト培養細胞で、 カルシゥ ムフォスフェート法により 2本鎖ポリヌクレオチドを導入する場合、 0 . 1〜1 0 0 O nMが好ましい。  The amount of the double-stranded polynucleotide to be introduced is preferably an amount sufficient to introduce at least one copy per force cell that can be appropriately selected depending on the transductant and the target gene. Specifically, for example, when the transfectant is a human cultured cell and the double-stranded polynucleotide is introduced by a calcium phosphate method, 0.1 to 100 OnM is preferable.
RNAィンターフェアレンスによる本発明の遺伝子の導入体内での発現抑制により、 本発明の遺伝子がコードするタンパク質の機能の確認、あるいは新たな機能の解析 等を行うことができる。  By suppressing the expression of the gene of the present invention in the transfection body by RNA interference, it is possible to confirm the function of the protein encoded by the gene of the present invention or to analyze a new function.
( 4 ) 本発明のタンパク質に特異的に結合する抗体 (4) an antibody that specifically binds to the protein of the present invention
本発明のタンパク質と特異的に結合する抗体の調製方法としては、通常用いられ る公知の方法を用いることができ、抗原として用いられるポリペプチドについても、 公知の方法に従って抗原性が高くェピトープ（抗原決定基） として適した配列を選 択して用いることができる。 ェピトープの選択方法としては、 例えば Epitope Adviser (富士通九州システムエンジニアリング社製） 等の市販のソフトウェアを 用いることができる。  As a method for preparing an antibody that specifically binds to the protein of the present invention, a commonly used known method can be used. For a polypeptide used as an antigen, epitope (antigen) A suitable sequence can be selected and used as the determinant. As a method for selecting an epitope, for example, commercially available software such as Epitope Adviser (manufactured by Fujitsu Kyushu System Engineering Co., Ltd.) can be used.
上記の抗原として用いるポリぺプチドは、公知の方法に従って合成した合成ぺプ チドでも、 また本発明のタンパク質そのものを用いることもできる。抗原となるポ リペプチドは、 公知の方法に従って適当な溶液等に調製して、 哺乳動物、 例えばゥ サギ、 マウス、 ラット等に免疫を行えばょレ、が、 安定的な免疫を行ったり抗体価を 高めるために抗原べプチドを適当なキヤリァタンパク質とのコンジュゲートにし て用いたり、 アジュバント等を加えて免疫を行うのが好ましい。 As the polypeptide used as the above antigen, a synthetic peptide synthesized according to a known method, or the protein itself of the present invention can be used. A polypeptide serving as an antigen can be prepared in an appropriate solution according to a known method to immunize a mammal, for example, a heron, a mouse, a rat, or the like. Conjugate the antigen peptide to a suitable carrier protein to increase It is preferable to perform immunization by using or adjuvant.
免疫に際しての抗原の投与経路は特に限定されず、例えば皮下、腹腔内、静脈内、 あるいは筋肉内等のいずれの経路を用いてもよい。具体的には、例えば BALBんマウ スに抗原ポリぺプチドを数日〜数週間おきに数回接種する方法等が用いられる。ま た、抗原の摂取量としては、抗原がポリべプチドの場合 0 . 3〜0 . 5 m g Z l回程 度が好ましいが、 ポリペプチドの種類、 また免疫する動物種によっては適宜調節さ れる。  The route of administration of the antigen upon immunization is not particularly limited, and any route such as subcutaneous, intraperitoneal, intravenous, or intramuscular may be used. Specifically, for example, a method of inoculating a BALB mouse several times every several days to several weeks with an antigen polypeptide is used. The antigen intake is preferably about 0.3 to 0.5 mg Zl when the antigen is a polypeptide, but is appropriately adjusted depending on the type of the polypeptide and the animal species to be immunized.
免疫後、 適宜試験的に採血を行って固相酵素免疫検定法 （以下、 これを 「ELISA 法」 と称することがある）やウェスタンブロッテイング等の方法で抗体価の上昇を 確認し、十分に抗体価の上昇した動物から採血を行う。 これに抗体の調製に用いら れる適当な処理を行えばポリクローナル抗体を得ることができる。具体的には、例 えば、公知の方法に従い血清から抗体成分を精製した精製抗体を取得する方法等が 挙げられる。 抗体成分の精製は、 遠析、 イオン交換クロマトグラフィー、 ァフィ二 ティーク口マトグラフィ一等の方法を用いることができる。  After immunization, test blood is collected as appropriate, and an increase in antibody titer is confirmed by enzyme-linked immunosorbent assay (hereinafter sometimes referred to as “ELISA”) or Western blotting. Blood is collected from animals with elevated antibody titers. A polyclonal antibody can be obtained by subjecting this to an appropriate treatment used for antibody preparation. Specific examples include a method of obtaining a purified antibody obtained by purifying an antibody component from serum according to a known method. For the purification of the antibody component, methods such as ion separation, ion exchange chromatography, affinity mouth chromatography, etc. can be used.
また、該動物の脾臓細胞とミエ口一マ細胞とを用いて公知の方法に従って融合さ せたハイブリ ドーマを用いる (Milstein, et al. , Nature, 256, 495 (1975) ) ことに よりモノクローナル抗体を作製することもできる。モノクローナル抗体は、例えば 以下の方法により取得することができる。  In addition, a hybridoma fused with spleen cells of the animal and myeoma cells according to a known method is used (Milstein, et al., Nature, 256, 495 (1975)). Can also be prepared. A monoclonal antibody can be obtained, for example, by the following method.
まず、上記した抗原の免疫により抗体価の高まった動物から抗体産生細胞を取得 する。 抗体産生細胞は、 形質細胞、 及びその前駆細胞であるリンパ球であり、 これ は個体の何れから取得してもよいが、 好ましくは脾臓、 リンパ節、 末梢血等から取 得する。 これらの細胞と融合させるミエローマとしては、一般的にはマウスから得 られた株化細胞、 例えば 8—ァザグァニン耐性マウス （BALB/c由来等） ミエローマ 細胞株である P3X63 - Ag8. 653 (ATCC:CRL- 1580)、 P3 - NSl/lAg4. 1 (理研セルバンク ： RCB0095) 等が好ましく用いられる。 細胞の融合は、 抗体産生細胞とミエローマ細 胞を適当な割合で混合し、適当な細胞融合培地、例えば RPMI1640ゃィスコフ改変ダ ルべッコ培地（IMDM)、あるいはダルべッコ改変ィ一ダル培地（DMEM)等に、 5 0 % ポリエチレングリコール （PEG) を溶解したもの等を用いることにより行うことが できる。 まに電 融合法 (U. Zimmer- mann. et ai. , Naturwissenschaften, 68,First, antibody-producing cells are obtained from an animal whose antibody titer has been increased by immunization with the above-mentioned antigen. The antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any of the individuals, but is preferably obtained from spleen, lymph nodes, peripheral blood and the like. The myeloma to be fused with these cells is generally a cell line obtained from a mouse, for example, an 8-azaguanine-resistant mouse (BALB / c-derived etc.) myeloma cell line P3X63-Ag8.653 (ATCC: CRL -1580), P3-NSl / lAg4.1 (RIKEN cell bank: RCB0095) and the like are preferably used. For cell fusion, antibody-producing cells and myeloma cells are mixed at an appropriate ratio, and an appropriate cell fusion medium such as RPMI1640 Discov's modified Dalbecco's medium (IMDM) or Dulbecco's modified idal is used. 50% in medium (DMEM) It can be performed by using a solution in which polyethylene glycol (PEG) is dissolved. U. Zimmermann et al., Naturwissenschaften, 68,
577 (1981) ) によっても行うことができる。 577 (1981)).
ハイブリ ドーマは、用いたミエローマ細胞株が 8—ァザグァニン耐性株であるこ とを利用して適量のヒポキサンチン 'アミノプテリン ·チミジン （HAT) 液を含む 正常培地 （HAT培地） 中で 5 %C02、 3 7 °Cで適当時間培養することにより選択する ことができる。この選択方法は用いるミエローマ細胞株によって適宜選択して用い ることができる。選択されたハイプリ ドーマが産生する抗体の抗体価を上記した方 法により解析し、抗体価の高い抗体を産生するハイプリ ドーマを限界希釈法等によ り分離し、分離した融合細胞を適当な培地で培養して得られる培養上清から硫安分 画、ァフィ二テイクロマトググラフィ一等の適当な方法により精製してモノクロ一 ナル抗体を得ることができる。また精製には市販のモノクローナル抗体精製キット を用いることもできる。 さらには、免疫した動物と同系統の動物、 またはヌードマ ウス等の腹腔内で上記で得られた抗体産生ハイプリ ドーマを増殖させることによ り、 本発明のモノクローナル抗体を大量に含む腹水を得ることもできる。 Hybridoma is myeloma cell line 8 Azaguanin resistance by utilizing a and this is strain suitable amount of hypoxanthine 'aminopterin-thymidine (HAT) normal medium containing liquid (HAT medium) in 5% C0 2 was used, It can be selected by culturing at 37 ° C for an appropriate time. This selection method can be appropriately selected and used depending on the myeloma cell line to be used. The antibody titer of the antibody produced by the selected hybridoma is analyzed by the method described above, the hybridoma producing the antibody with a high antibody titer is separated by limiting dilution, etc., and the separated fused cells are separated into an appropriate medium. A monoclonal antibody can be obtained by purifying from a culture supernatant obtained by culturing with an appropriate method such as ammonium sulfate fractionation or affinity chromatography. For purification, a commercially available monoclonal antibody purification kit can also be used. Furthermore, by growing the antibody-producing hybridoma obtained above in the abdominal cavity of an animal of the same strain as the immunized animal or nude mouse, etc., it is possible to obtain ascites containing a large amount of the monoclonal antibody of the present invention. You can also.
また、本発明のタンパク質としてヒ ト由来のものを取得した場合には、かかるポ リペプチド、 あるいはその部分ペプチドを抗原として、 ヒト末梢血リンパ球を移植 し 7こ Severe combined immune deficiency (SCID) マウスに上 elし 7こ方法と |PJ様に して免疫し、該免疫動物の抗体産生細胞とヒ トのミエ口一マ細胞とのハイブリ ドー マを作製することによってもヒ ト型抗体を作製することができる （Mosier, D. E. , et al. Nature, 335, 256—259 (1988)； Duchosal, M. A. , et al. , Nature, 355, 258-262 (1992) ) 。  In addition, when a human-derived protein is obtained as the protein of the present invention, human peripheral blood lymphocytes are transplanted using the polypeptide or a partial peptide thereof as an antigen, and transplanted into Severe combined immune deficiency (SCID) mice. A human antibody can also be prepared by immunization using the above method and | PJ, and then preparing a hybridoma between the antibody-producing cells of the immunized animal and the myeoma cells of the human. (Mosier, DE, et al. Nature, 335, 256-259 (1988); Duchosal, MA, et al., Nature, 355, 258-262 (1992)).
また、 取得したヒ ト型抗体を産生するハイプリ ドーマから R Aを抽出し、 目的の ヒ ト型抗体をコードする遺伝子をクローニングして、この遺伝子を適当なベクター に挿入し、 これを適当な宿主に導入して発現させることにより、 さらに大量にヒ ト 型抗体を作製することができる。 ここで、 抗原との結合性の低い抗体は、 それ自体 既知の進化工学的手法を用いることによりさらに結合性の高い抗体として取得す ることもできる。一価性抗体等の部分フラグメントは、例えばパパイン等を用いてIn addition, RA is extracted from the obtained hybridoma producing the human antibody, the gene encoding the desired human antibody is cloned, this gene is inserted into an appropriate vector, and this is inserted into an appropriate host. By introducing and expressing it, human antibodies can be produced in larger quantities. Here, an antibody with low binding to an antigen can be obtained as an antibody with higher binding by using an evolutionary engineering technique known per se. You can also. A partial fragment such as a monovalent antibody can be prepared using, for example, papain.
Fab部分と Fc部分を切断し、ァフィ二ティカラム等を用いて Fab部分を回収すること によって作製することができる。 It can be prepared by cutting the Fab portion and the Fc portion and collecting the Fab portion using an affinity column or the like.
かくして得られる本発明のタンパク質と特異的に結合する抗体は、本発明のタン パク質に特異的に結合することによって該タンパク質が有する活性を阻害する中 和抗体として用いることもできる。タンパク質が有する活性を阻害するものの選択 方法としては特に制限はないが、 例えば、 上記 （2 ) で作製した D N A導入体に抗 体を接触させ、導入体中の目的タンパク質の機能が阻害されるか否かを解析する方 法等が挙げられる。  The thus-obtained antibody that specifically binds to the protein of the present invention can also be used as a neutral antibody that specifically binds to the protein of the present invention and thereby inhibits the activity of the protein. There is no particular limitation on the method for selecting a substance that inhibits the activity of the protein. For example, whether the function of the target protein in the introduced substance is inhibited by contacting the antibody with the DNA transfectant prepared in (2) above There is a method of analyzing whether or not to do so.
かかる中和抗体は、 臨床へ応用するに際し、 上記有効成分を単独で用いることも 可能であるが、薬学的に許容され得る担体と配合して医薬品組成物として用いるこ ともできる。 この時の有効成分の担体に対する割合は、 1〜9 0重量％の間で変動 され得る。 また、 力かる薬剤は種々の形態で投与することができ、 それらの投与形 態としては、 錠剤、 カプセル剤、 顆粒剤、 散剤、 あるいはシロップ剤等による経口 投与、 または注射剤、 点滴剤、 リボソーム剤、 坐薬剤等による非経口投与を挙げる ことができる。 また、 その投与量は、 症状、 年齢、 体重等によって適宜選択するこ とができる。  Such a neutralizing antibody can be used alone for the clinical application, but can also be used as a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. In addition, a powerful drug can be administered in various forms, such as tablets, capsules, granules, powders, or syrups, orally, or injections, drops, ribosomes. And parenteral administration with suppositories and the like. In addition, the dose can be appropriately selected depending on symptoms, age, weight, and the like.
( 5 ) 本発明のタンパク質が有する活性の確認および解析 (5) Confirmation and analysis of the activity of the protein of the present invention
本発明のタンパク質は、 これを上記 （2 ) に記載のとおり組み換えタンパク質と して作製し、 これを解析することにより （1 ) で推測した活性を有していることを 確認することができる。 さらに上述 （4 ) のとおりに作製した抗体等との組み合わ せにより解析することもできる。  The protein of the present invention is prepared as a recombinant protein as described in (2) above, and by analyzing this, it can be confirmed that it has the activity estimated in (1). Furthermore, analysis can also be performed by combining with an antibody or the like prepared as described in (4) above.
本発明のタンパク質が有する活性は、例えば、次に示すような方法により解析す ることができる力 S、これらに限定されるものではなレ、。また、これらの解析方法は、 後述する本発明のタンパク質の機能賦活物資や機能阻害物質のスクリーニングや 本発明のタンパク質の発現調節物質のスクリーニングにも用いることができる。 (5 - 1) TGF ]3受容体ファミ リーとの結合活性 The activity of the protein of the present invention is, for example, a force S that can be analyzed by the following method, and is not limited thereto. In addition, these analysis methods can also be used for screening for a function activator or a function inhibitor of the protein of the present invention and a screening for a protein expression regulator of the present invention, which will be described later. (5-1) TGF] 3 Receptor Family Binding Activity
TGF β受容体フアミリーとの結合活性は、例えば、 T G F /3受容体フアミリー の C末端に ΗΑタグを結合させたクローンと本発明のタンパク質の C末端にフラ ッグタグを結合させたクローンとを作製し、 cosl細胞等に共発現させ TGF /3ファ ミリー分子を作用させた後、細胞ライゼートを得て、抗フラッグ抗体を用いて免疫 沈降させ、電気泳動を行って抗 HA抗体を用いたゥヱスタンプ口ットを実施するこ とにより、 両タンパク間の結合を確認できる （Nature Vol.401, 480-483(1999))。 また、本発明のタンパク質が TGF )3フアミリー分子によるシグナル伝達に及ぼ す影響は、 例えば、 BMP応答配列 （BRE) またはァクチビン応答配列 （ARE) を有 するレポータージーンを用いたアツセィ系により評価することができる。具体的に は、 BRE配列の下流にルシフェラーゼ遺伝子を結合させたレポータージーンを用レ、、 本発明のタンパク質、 TGF ]3受容体フアミリー、及びレポータージーンを P 1 9 細胞または力エル卵母細胞などに共発現させ、 TGF )3ファミリ一分子を作用させ た時の発光量を測定することにより、 生物活性を検定することができる。  The binding activity to the TGFβ receptor family was determined, for example, by preparing a clone in which the C tag was bound to the C-terminus of the TGF / 3 receptor family and a clone in which a flag tag was bound to the C-terminus of the protein of the present invention. After co-expression in cosl cells, etc., and the action of the TGF / 3 family molecule, a cell lysate was obtained, immunoprecipitated using an anti-flag antibody, electrophoresed, and used for anti-HA antibody. By conducting the procedure, the binding between both proteins can be confirmed (Nature Vol. 401, 480-483 (1999)). In addition, the effect of the protein of the present invention on signal transduction by TGF) 3 family molecules can be evaluated, for example, by an Atsushi system using a reporter gene having a BMP response element (BRE) or an activin response element (ARE). it can. Specifically, a reporter gene in which a luciferase gene is linked downstream of the BRE sequence is used. The protein of the present invention, the TGF] 3 receptor family, and the reporter gene are shared with P19 cells or force oocytes. The biological activity can be assayed by measuring the amount of luminescence when expressed and allowed to act on one molecule of the TGF) 3 family.
(5 - 2) 変性 LDLとの結合活性 (5-2) Binding activity to denatured LDL
本発明のタンパク質の変性 LD Lとの結合活性あるいは細胞への取りこみ活性 は、 当業者であれば例えば以下のようにして検定することができる。  The binding activity of the protein of the present invention to denatured LDL or the incorporation activity into cells can be assayed by those skilled in the art, for example, as follows.
例えば、 酸化 LDLの場合、 まず、 c DN Aを真核細胞での発現ベクターにクロ 一ユングし、通常の遺伝子導入法を用いて CH0- K1細胞または HEK293細胞等に導入し て、 本発明のタンパク質を発現させる。 次に、 これを放射性物質標識した酸化 LDL For example, in the case of oxidized LDL, first, cDNA is cloned into an expression vector for eukaryotic cells, and introduced into CH0-K1 cells or HEK293 cells using a conventional gene transfer method. Express the protein. Next, this is a radioactively labeled oxidized LDL
([125I]0x-LDL) の 5μ§/πι1とともに氷上で 1時間インキュベートした後、 PBSで 3回 洗浄し、 γカウンターを用いて放射能を測定する。 特異的結合量は、 100倍量の Ox-LDLを反応液中に添加した場合との差から計算で求めることができる ([125 I] 0x-LDL ) was incubated on ice for 1 hour with 5μ § / πι1 of, washed 3 times with PBS, and radioactivity is measured using a γ counter. The specific binding amount can be calculated from the difference from the case where 100 times the amount of Ox-LDL is added to the reaction solution.
(Nature, 386, 73- 77(1997)) 。  (Nature, 386, 73-77 (1997)).
また、本発明のタンパク質の活性測定には、蛍光標識した変性 LDLを用いてフ ローサイ トメ トリーにより測定してもよい。 In addition, the activity of the protein of the present invention is measured using denatured LDL labeled with fluorescence. It may be measured by low cytometry.
(5-3) AT P結合性運搬体活性 (5-3) ATP binding carrier activity
本発明のタンパク質が機能することは、 ATP消費活性の測定により確認すること ができる （JBC, 267， 7， 4854-4858 (1992)) 。 すなわち、 本発明のタンパク質を発現 させた細胞から、 膜画分 （lO igの膜タンパク含有） を抽出し、 50mMTris-Mes(pH 6.8), 2 raM EGTA, 2 raM DTT, 50 mM KCl, 及び 5 mM sodium azideからなる溶液 0.1 mlに懸濁して 37°Cに保温する。 次に、 これに 5 mMの ATPを添カ卩して、 薬剤あるいは 糖、 脂肪酸等で処理する。 20分後に 0.1 raMの 5% SDSにて反応を停止し、 遊離した 無機リン酸を呈色反応を用いて定量することにより、 ΑΤΡ消費活性を測定すること ができる。  The function of the protein of the present invention can be confirmed by measuring the ATP consuming activity (JBC, 267, 7, 4854-4858 (1992)). That is, a membrane fraction (containing a membrane protein of lOig) was extracted from cells expressing the protein of the present invention, and 50 mM Tris-Mes (pH 6.8), 2 raM EGTA, 2 raM DTT, 50 mM KCl, and 5 mM Suspend in 0.1 ml of a solution consisting of mM sodium azide and keep at 37 ° C. Next, this is supplemented with 5 mM ATP, and treated with a drug, sugar, or fatty acid. After 20 minutes, the reaction is stopped with 5% SDS at 0.1 raM, and the consumed inorganic phosphoric acid is quantified using a color reaction, whereby the consumption activity can be measured.
さらに、 脂質排出アツセィ (BBRC, 290, 713-721 (2002)) は、 cos細胞等に cDN Aを既知の遺伝子導入試薬を用いて導入し、 翌日 1 / Ci/mlの [3H] cholesterolで 18時間標識した後、 PBSで洗浄する。 次に、 必須脂肪酸を含まない DMEM培地にて 2 時間培養後、 新鮮な DMEM培地に入れ替えて 15 wg/mlの apoA- 1の存在または非存在 下でさらに 4時間 37°Cにて培養し、 培地中の放射活性を測定することにより行うこ とができる。 また、 同様な手法を用いて、抗癌剤等の薬剤排出能を検討することが できる。 In addition, lipid excretion atsey (BBRC, 290, 713-721 (2002)) introduced cDNA into cos cells, etc. using a known gene transfer reagent, and then used 1 / Ci / ml [ 3 H] cholesterol the next day. After labeling for 18 hours, wash with PBS. Next, after culturing in a DMEM medium containing no essential fatty acids for 2 hours, the medium was replaced with fresh DMEM medium, and cultivation was continued at 37 ° C for 4 hours in the presence or absence of 15 wg / ml apoA-1. It can be performed by measuring the radioactivity in the medium. In addition, the ability to excrete a drug such as an anticancer drug can be examined using a similar technique.
(5-4) ィムノグロブリン様タンパク質活性 (5-4) Immunoglobulin-like protein activity
ィムノグロプリン様ドメインを持つスーパーフアミリーのメンバーは、異なる機 能を有する数百のタンパク質、 例えば、 抗体、 補体、 巨大筋肉蛋白質チチン、 チロ シンキナーゼ型受容体などからなる。ィムノグロブリン様ドメインは弾力性に富み、 細胞接着、蛋白質間相互作用や蛋白質とリガンドとの相互作用に関連すると考えら れている。 ィムノグロブリン様蛋白質の機能評価は、 画一的ではないが、 それぞれ の相互作用に基づいて、それ自体既知の通常用いられる方法により実施することが できる。 例えば、 結合試験、 表面プラズモン共鳴、 ツーハイプリッド法、 蛍光エネ ルギー移動法、 比熱測定法、 などがあるがこれに限定されない。 Members of the superfamily that have an immunoglobulin-like domain consist of hundreds of proteins with different functions, such as antibodies, complement, the giant muscle protein titin, and tyrosine kinase-type receptors. The immunoglobulin-like domains are resilient and are thought to be involved in cell adhesion, protein-protein interactions, and protein-ligand interactions. The evaluation of the function of the immunoglobulin-like protein is not uniform, but can be performed by a commonly used method known per se based on each interaction. For example, binding test, surface plasmon resonance, two-hybrid method, fluorescent energy Methods include, but are not limited to, the energy transfer method and the specific heat measurement method.
例えば、ィムノグロプリン様蛋白質の 1種である補体の活性測定は以下の様に行 うことが出来る。ヒッジ赤血球に抗体 (溶血素）を反応させたヒッジ感作赤血球 (EA) は、 古典経路を活性化するが、補体の活性化が進むと溶血する。 一定量の EAを血清 あるいは本蛋白質と反応させると、補体量に応じて溶血程度の変化を定量すること が出来る。  For example, the activity of complement, one of the immunoglobulin-like proteins, can be measured as follows. Hedge-sensitized erythrocytes (EA), in which antibodies (hemolysin) are reacted with hedge erythrocytes, activate the classical pathway, but lyse as complement is activated. When a certain amount of EA is reacted with serum or this protein, the change in the degree of hemolysis can be quantified according to the amount of complement.
また、例えば、 ィムノグロブリン様蛋白質の 1種である α 1インヒビター 3 ( α 1 1 3 ) は、 α 2マクログロブリンファミリーのメンバーであり、 血中に高濃度存 在するが生理的な役割は不明である。 α 1 1 3はターゲットプロテアーゼとチォー ルエステルを介して共有結合して活性を阻害することが知られており、本蛋白質を 通常のプロテアーゼ阻害物質の評価系で機能を検討することが出来る。 また、 α ΐ I 3は不完全で除去する必要のある細胞蛋白質と結合し αマクログロプリン受容体 を介してクリアランスに関与するとも考えられており、本蛋白質を αマクログロブ リン受容体との結合試験系 (Biochemistry 1989 Feb 7 ; 28 (3)： 1406- 12) で測定評 価することもできる。 なお、本発明のタンパク質が有する活性の確認は、上記した方法に限定されるも のではない。 また、 これらの機能アツセィ系は、 後述する本発明のタンパク質の機 能賦活物質や機能阻害物質のスクリーニングゃ本発明のタンパク質の発現調節物 質のスクリーニングにも用いることができる。  Also, for example, α1 inhibitor 3 (α113), a type of immunoglobulin-like protein, is a member of the α2 macroglobulin family and exists at high concentrations in blood but has a physiological role. Unknown. α113 is known to inhibit the activity by covalently binding to the target protease via a thiol ester, and the function of this protein can be examined in a normal protease inhibitor evaluation system. It is also thought that α 結合 I3 binds to cellular proteins that are incomplete and need to be removed and is involved in clearance via α-macroglobulin receptor. It can also be measured and evaluated using the system (Biochemistry 1989 Feb 7; 28 (3): 1406-12). Note that confirmation of the activity of the protein of the present invention is not limited to the method described above. Further, these functional assay systems can also be used for screening of a function activator or a function inhibitor of the protein of the present invention, which will be described later, and a screening of an expression regulator of the protein of the present invention.
本発明のタンパク質の機能解析の方法として一般的には、例えば、 （i)各組織、 疾患、 あるいは発生段階における発現状態を比較解析する方法、 （ii) 他のタンパ ク質、 D N Aとの相互作用を解析する方法、 （i ii) 適当な細胞あるいは個体へ導 入し、表現型の変化を解析する方法、 （iv) 適当な細胞あるいは個体において該タ ンパク質の発現を阻害して表現型の変化を解析する方法などが挙げられる。このよ うな方法によれば、対象タンパク質に特異的な活性を多面的に解析することができ る。 (i) の方法においては、 本発明のタンパク質の発現を、 m R NAレベルあるい はタンパク質レベルで解析することができる。 m R N Aレベルで発現量を解析する 場合は、 例えば、 in situハイブリダィゼーシヨン法 (In situ hybridization: Application to Developmental Biology & Medicine. , nd. by Harris, N. and Wilkinson, D. G.， Cambridge University Press (1990) ) 、 D NAチップを利用 したハイブリダィゼーシヨン法、 定量 P C R法等が用いられる。 また、 タンパク質 レベルで解析する場合には、後述する本発明のタンパク質に特異的に結合する抗体 を用いた組織染色法、 E L I S A法、 ウェスタンブロット法などが挙げられる。 こ こで、解析の対象タンパク質が公知のバリアントが存在するスプライシングバリァ ントである場合には、解析対象タンパク質をコードする c D N Aにのみ存在し、公 知のバリアントをコードする c D NAとはハイブリダィズしないプローブを用い ることが好ましレ、。 定量 P C R法の場合には、対象バリアントと公知バリアント間 で異なる長さの増幅断片ができるプライマーを選択して行う方法 （Wong, Y. , Neuroscience Let. , 320： 141-145 (2002) ) 等が挙げられる。 また、 タンパク質 レベルで解析する場合にも、対象タンパク質にのみ反応し、公知のバリアントには 反応しない抗体を用いることが好ましい。 In general, methods for analyzing the function of the protein of the present invention include, for example, (i) a method of comparatively analyzing the expression state of each tissue, disease, or developmental stage, and (ii) the interaction with other proteins and DNA. (I ii) a method of transducing into a suitable cell or individual and analyzing a phenotypic change, and (iv) a phenotype by inhibiting the expression of the protein in a suitable cell or individual. And a method of analyzing the change in According to such a method, the activity specific to the target protein can be analyzed from many aspects. In the method (i), expression of the protein of the present invention can be analyzed at the mRNA or protein level. When analyzing the expression level at the mRNA level, for example, the in situ hybridization method (In situ hybridization: Application to Developmental Biology & Medicine., nd. by Harris, N. and Wilkinson, DG, Cambridge University Press) (1990)), a hybridization method using a DNA chip, a quantitative PCR method, and the like. When the analysis is performed at the protein level, a tissue staining method using an antibody that specifically binds to the protein of the present invention described later, an ELISA method, a Western blot method, and the like can be mentioned. Here, when the protein to be analyzed is a splicing variant in which a known variant is present, a cDNA that exists only in the cDNA encoding the protein to be analyzed and that encodes a known variant is It is preferable to use a non-hybridizing probe. In the case of the quantitative PCR method, a method in which primers that can generate amplified fragments of different lengths between the target variant and the known variant are selected and performed (Wong, Y., Neuroscience Let., 320: 141-145 (2002)), etc. Is mentioned. Also, when analyzing at the protein level, it is preferable to use an antibody that reacts only with the target protein and does not react with a known variant.
(ii) の方法においては、 本発明のタンパク質と既知のタンパク質との相互作用 の有無を調べて、本発明のタンパク質の機能を解析することができる。相互作用の 解析法としては、 それ自体既知の常法を用いることができるが、 具体的には、 例え ば、 酵母ツーハイプリッド法、 蛍光偏光解消法、 表面プラズモン法、 ファージディ スプレイ法、 リボソ一マルディスプレイ法等が挙げられる。 該方法においても、解 析対象タンパク質が公知のバリアントが存在するスプライシングバリアントの場 合には、公知のバリアントも同様にして相互作用する物質を解析し、対象タンパク 質特異的に相互作用する物質を同定することが好ましい。  In the method (ii), the function of the protein of the present invention can be analyzed by examining the presence or absence of interaction between the protein of the present invention and a known protein. As a method for analyzing the interaction, a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, ribosome method One example is the multiple display method. In this method as well, when the protein to be analyzed is a splicing variant in which a known variant is present, the known variant is analyzed for interacting substances in the same manner, and a substance that specifically interacts with the target protein is analyzed. Identification is preferred.
(iii) の方法では、 本発明の c D NAを導入する細胞は特に制限はないが、 ヒ ト培養細胞等が特に好ましく用いられる。 D NAの細胞への導入法としては、上記 In the method (iii), the cells into which the cDNA of the present invention is introduced are not particularly limited, but human cultured cells are particularly preferably used. The method for introducing DNA into cells is described above.
( 2 )に記載のものが挙げられる。さらに導入細胞の表現型としては、細胞の生死、 細胞の増殖速度、 細胞の分化、 細胞が神経細胞の場合には神経突起の伸長度、 細胞 内タンパク質の局在や移行など顕微鏡等で観察可能なものや、細胞内の特定タンパ ク質の発現変化など生化学的実験により解析可能なものも含む。これらの表現型は、 公知のバリアントが存在するスプライシングバリアントの場合には、公知のものも 同様に細胞へ導入し、比較解析することにより解析対象バリアントに関連する表現 型を同定することができる。 また、本発明のタンパク質は上記活性を有するもので あることがわかっているので、上記活性を有するタンパク質が関連する疾患に見ら れる表現型等に注目して解析することも好ましい。 Examples described in (2) are given. Furthermore, the phenotype of the transfected cells includes cell viability, Cell growth rate, cell differentiation, if the cell is a neuron, neurite outgrowth, localization and translocation of intracellular proteins, etc., which can be observed with a microscope, etc., and expression of specific proteins in cells Includes those that can be analyzed by biochemical experiments such as changes. In the case of a splicing variant in which a known variant exists, these phenotypes can be similarly introduced into cells, and a phenotype related to the variant to be analyzed can be identified by comparative analysis. In addition, since it is known that the protein of the present invention has the above activity, it is also preferable to analyze the protein by focusing on a phenotype or the like found in a disease associated with the protein having the above activity.
(iv) の方法では、 後述するオリゴヌクレオチドを用いた方法や、 R NAインタ 一フェアレンス法により効率的に行うことができる。 この方法においても、解析す る対象タンパク質に公知のバリアントが存在する場合には、公知のバリアントやそ の他のバリアントについても同様の解析を行レ、、比較解析することにより対象タン パク質特異的な機能を同定することができる。  In the method (iv), the method can be efficiently performed by a method using an oligonucleotide described below or an RNA interference method. In this method, when a known variant is present in the target protein to be analyzed, the same analysis is performed on the known variant and other variants, and the target protein is analyzed by comparative analysis. Function can be identified.
( 6 ) 本発明のタンパク質が有する活性を調節する分子のスクリーニング 本発明のタンパク質に特異的に結合し、 かつ本発明のタンパク質の機能 （活性） を阻害、拮抗または増強する作用を有する物質をスクリーニングすることにより本 発明のタンパク質の機能調節物質（以下、 これを「調節物質」 と称することがある） を得ることができる。 (6) Screening for a molecule that regulates the activity of the protein of the present invention Screening for a substance that specifically binds to the protein of the present invention and has an effect of inhibiting, antagonizing or enhancing the function (activity) of the protein of the present invention By doing so, a function modulator of the protein of the present invention (hereinafter sometimes referred to as “modulator”) can be obtained.
この調節物質のスクリーニング方法は、 本発明のタンパク質に特異的に結合し、 且つ該タンパク質の活性を阻害、拮抗または増強する作用を有する物質が得られる 方法であれば如何なるものであってもよい。例えば、 まずはじめに本発明のタンパ ク質と被検物質とを接触させ、該タンパク質との結合性を指標として選抜した後に、 本発明のタンパク質が有する活性の変化を指標として被検物質を選抜する方法を 用いることができる。  This method of screening for a regulatory substance may be any method as long as it can obtain a substance that specifically binds to the protein of the present invention and has an activity of inhibiting, antagonizing or enhancing the activity of the protein. For example, first, the protein of the present invention is brought into contact with a test substance, and the test substance is selected by using the change in the activity of the protein of the present invention as an index, after selecting by using the binding property to the protein as an index. A method can be used.
被検物質としては、本発明のタンパク質と相互作用して、該タンパク質が有する 活性に影響を及ぼす可能性のある物質であれば如何なるものであってもよレ、が、具 体的には、 例えば、 ペプチド、 タンパク質、 非ペプチド性化合物、 低分子化合物、 合成化合物、 発酵生産物、 細胞抽出液、 動物組織抽出液等が挙げられる。 これらの 物質は新規な物質であってもよいし、公知の物質であってもよい。被検物質と本発 明のタンパク質の相互作用の解析法としては、それ自体既知の常法を用いることが できるが、 具体的には、 例えば、 酵母ツーハイプリッド法、 蛍光偏光解消法、 表面 プラズモン法、 ファージディスプレイ法、 リボソ一マルディスプレイ法、 あるいは 上記（ 4 )に記載した抗体との競合解析法等が挙げられる。このような方法により、 本発明のタンパク質に結合する活性を見いだされた物質は、次に該物質の存在下で 本発明のタンパク質が有する活性がどのような影響を受けるかを解析することに よって、 調節物質として用いられるか否かが同定される。 The test substance may be any substance that can interact with the protein of the present invention and affect the activity of the protein. Physically, for example, peptides, proteins, non-peptidic compounds, low molecular weight compounds, synthetic compounds, fermentation products, cell extracts, animal tissue extracts and the like can be mentioned. These substances may be new substances or known substances. As a method for analyzing the interaction between the test substance and the protein of the present invention, a conventional method known per se can be used. The plasmon method, the phage display method, the ribosomal display method, or the competition analysis method with the antibody described in the above (4) can be used. The substance found to bind to the protein of the present invention by such a method is then analyzed by analyzing how the activity of the protein of the present invention is affected in the presence of the substance. Whether it is used as a modulator or not is identified.
ここで、 医薬活性成分のスクリーニングを目的とするため、用いる本発明の D N A、 あるいは組み換えタンパク質については、上記したヒ トのホモログタンパク質 またはオルソログタンパク質を用いることが好ましい。さらに上記方法によってス クリーニングされた物質は、これらの生体内でのスクリーニングによって医薬候補 としての選択を行ってもよい。  Here, for the purpose of screening a pharmaceutically active ingredient, it is preferable to use the above-mentioned human homologous protein or orthologous protein for the DNA or recombinant protein of the present invention to be used. Further, the substances screened by the above method may be selected as drug candidates by screening in vivo.
本発明のタンパク質の活性の変化の解析は、上記した本発明のタンパク質の機能 解析方法 （活性の確認方法） に基づき、 それ自体公知の通常用いられる方法により 行うことができる。以下に、本発明のタンパク質の各活性を調節する物質の解析方 法について、 具体例を挙げて説明する。  The analysis of the change in the activity of the protein of the present invention can be carried out by a method known per se and generally used, based on the method for analyzing the function of the protein of the present invention (confirmation method of activity). Hereinafter, a method for analyzing a substance that regulates each activity of the protein of the present invention will be described with reference to specific examples.
( 6 - 1 ) T G F 受容体フアミリーとの結合活性を調節する物質の解析 (6-1) Analysis of substances that regulate binding activity to TGF receptor family
T G F 受容体フアミリーとの結合活性を調節する物質の具体的な解析方法と しては、例えば、 T G F 受容体フアミリーとの結合活性を調節する物質を解析す る場合には、 前記 D NA導入体に基質となるタンパク質も同様の方法で導入する。 この導入体について選択された物質の存在下/まだは非存在下で基質となるタン パク質の脱リン酸化をそれ自体既知の通常用いられる方法により解析する。具体的 には、 上記 （5— 1 ) に記載の方法等を用いて行うことができる。 T G F ]3受容体 フアミリーとの結合活性が、物質の非存在下の場合と比べて増加した場合には、該 物質は TGF ]3受容体ファミリーとの結合活性化物質として機能する可能性があ り、 また低下、 または阻害された場合には物質は TGF )3受容体フアミリーとの結 合活性阻害物質として機能する可能性があると同定できる。 As a specific method for analyzing a substance that regulates the binding activity to the TGF receptor family, for example, when analyzing a substance that regulates the binding activity to the TGF receptor family, the DNA introduced A protein serving as a substrate is introduced in the same manner. The dephosphorylation of the substrate protein in the presence / absence of the selected substance with respect to this transductant is analyzed by a commonly used method known per se. Specifically, it can be performed using the method described in (5-1) above. TGF] 3 receptor If the binding activity to the family is increased compared to the absence of the substance, the substance may function as an activator of binding to the TGF] 3 receptor family, and may decrease, Alternatively, when inhibited, the substance can be identified as possibly functioning as an inhibitor of the binding activity to the TGF) 3 receptor family.
本発明のタンパク質は TGF /3受容体フアミリーとの結合活性を有するが、 TG F ]3受容体ファミリ一は、 例えば、 腎臓の繊維化、 肺繊維症、 心筋梗塞等に関連し た組織の繊維化に関係しており、本スクリーニング方法によりこの受容体の発現調 節薬等として同定された化合物は、様々な繊維化疾患等の治療薬として用いられ得 るものである。  The protein of the present invention has a binding activity to the TGF / 3 receptor family, but the TGF] 3 receptor family includes, for example, fibers of tissues related to renal fibrosis, pulmonary fibrosis, myocardial infarction and the like. The compounds identified as modulators of the expression of this receptor by the present screening method can be used as therapeutic agents for various fibrotic diseases and the like.
疾患の種類としては、 具体的には、 例えば、 糸球体腎炎、 神経の瘢痕形成、 皮膚 の瘢痕形成、 眼の瘢痕形成、 肺線維症、 動脈傷害、 増殖性網膜症、 網膜剥離、 呼吸 促進症候群、肝硬変、ボスト心筋梗塞、ボスト脈管形成再狭窄、ケロイド瘢痕形成、 強皮症、 脈管障害、 白内障、 及び緑内障、 骨粗鬆症等が挙げられる。 このような疾 患の治療薬は、すなわち、 TGF フアミリー分子の効果が個体に有害である病状 を治療するためのものであって、効果が線維症を促進する効果であることを特徴と してレヽる。  Specific types of diseases include, for example, glomerulonephritis, scarring of nerves, scarring of skin, scarring of eyes, pulmonary fibrosis, arterial injury, proliferative retinopathy, retinal detachment, respiratory distress syndrome , Cirrhosis, Bosto myocardial infarction, Bosto angioplasty restenosis, keloid scar formation, scleroderma, vascular disorders, cataracts, glaucoma, osteoporosis and the like. The therapeutic agent for such a disease is for treating a condition in which the effect of the TGF family molecule is harmful to an individual, and the effect is an effect of promoting fibrosis. Reply
(6-2) 変性 LDLとの結合活性を調節する物質の解析 (6-2) Analysis of substances that regulate binding activity with modified LDL
変 t^LD Lとの結合活性を調節する物質の具体的な解析方法としては、 例えば、 変性 LDLとの結合活性を調節する物質を解析する場合には、前記 DNA導入体に 変性 L D Lを導入する。この導入体について選択された物質の存在下/または非存 在下で本発明のタンパク質と変性 L D Lとの相互作用をそれ自体既知の通常用レ、 られる方法により解析する。 具体的には、 上記 （5— 2) に記載の方法等を用いて 行うことができる。変性 LDLとの結合性が、物質の非存在下の場合と比べて増加 した場合には、該物質は変性 LDLとの結合の活性化物質として機能する可能性が あり、 また低下、 または阻害された場合には物質は変性 LDLとの結合の阻害物質 として機能する可能性があると同定できる。 本発明のタンパク質は変性 L D Lとの結合活性を有することから、例えば、酸化 LDLゃァセチル化 LDL等の細胞内への取りこみや細胞機能障害に関係することが推 測できる。 そこで、 本スクリーニング方法により同定できる化合物は、 高脂血症、 動脈硬化の発症 ·進展、 粥状動脈硬化、 動脈硬化を伴う遺伝疾患、 家族性高コレス テロール血症、 心筋梗塞、 脳梗塞等の治療薬として用いられ得るものである。 Specific methods for analyzing a substance that regulates the binding activity with modified t ^ LDL include, for example, when analyzing a substance that regulates the binding activity with modified LDL, introducing the modified LDL into the DNA transductant. I do. The interaction between the protein of the present invention and denatured LDL in the presence / absence of the selected substance is analyzed with respect to this transductant by a method known per se and used in a usual manner. Specifically, it can be performed using the method described in (5-2) above. If the binding to denatured LDL is increased as compared to the absence of the substance, the substance may function as an activator of binding to denatured LDL and may be reduced or inhibited. In this case, the substance can be identified as possibly acting as an inhibitor of binding to denatured LDL. Since the protein of the present invention has a binding activity to denatured LDL, it can be estimated that the protein is involved in the incorporation of oxidized LDL-acetylated LDL into cells and cell dysfunction. Therefore, compounds that can be identified by this screening method include hyperlipidemia, the onset and progression of atherosclerosis, atherosclerosis, genetic diseases associated with atherosclerosis, familial hypercholesterolemia, myocardial infarction, cerebral infarction, etc. It can be used as a therapeutic agent.
( 6 - 3 ) AT P結合性運搬体活性を調節する物質の解析 (6-3) Analysis of substances that regulate ATP-binding transporter activity
A T P結合性運搬体活性を調節する物質の具体的な解析方法としては、 例えば、 A T P結合性運搬体活性を調節する物質を解析する場合には、具体的には、上記（5 —3 ) に記載の方法等を用いて行うことができる。 A T Pとの結合性が、 物質の非 存在下の場合と比べて増加した場合には、該物質は A T P結合性運搬体活性化物質 として機能する可能性があり、また低下、 または阻害された場合には物質は A T P 結合阻害物質として機能する可能性があると同定できる。 ここで、 医薬活性成分の スクリーニングを目的とするため、用いる本発明の D NA、 あるいは組み換えタン パク質については、上記したヒ トのホモログを用いることが好ましい。 さらに上記 方法によってスクリーユングされた物質は、これらの生体内でのスクリーニングに よって医薬候補としての選択を行ってもよい。  As a specific analysis method of a substance that modulates ATP-binding transporter activity, for example, when analyzing a substance that modulates ATP-binding transporter activity, specifically, as described in (5-3) above, It can be performed by using the method described in the above. If the binding to ATP is increased compared to the absence of the substance, the substance may function as an ATP-binding transporter activator and if reduced or inhibited Can identify that the substance may function as an ATP binding inhibitor. Here, for the purpose of screening for a pharmaceutically active ingredient, it is preferable to use the above-mentioned human homolog for the DNA or recombinant protein of the present invention to be used. Furthermore, substances screened by the above method may be selected as drug candidates by screening in vivo.
本発明の A T P結合性運搬体活性を有するタンパク質の中でも、 A B C トランス ポーターは、種々の疾患や薬物代謝等に関与していることが知られている。例えば、 A B C トランスポーターのサブタイプ ABCA 1は HDL代謝を行いタンジール病の原因 遺伝子であり、 ABCB 1は抗癌剤の細胞外排出を担い、 ABCC7は cystic fibrosisの原 因遺伝子であり、 ABCC 8は糖尿病治療薬であるスルホニルゥレアの受容体であるこ とが知られている。  Among the proteins having the ATP-binding carrier activity of the present invention, the ABC transporter is known to be involved in various diseases, drug metabolism and the like. For example, the ABC transporter subtype ABCA 1 is a gene that causes HDL metabolism and causes Tangier disease, ABCB 1 is responsible for the extracellular excretion of anticancer drugs, ABCC 7 is a gene that causes cystic fibrosis, and ABCC 8 is a treatment for diabetes It is known to be a receptor for the drug sulfonylprea.
従って、 本スクリーニング方法により同定できる化合物は、 例えば、 diabetes mellitus、 atherosclerosis^ coronary heart aisease、 cystic f lDrosis^ adrenoleukodystrophy^ Stargardt' s disease^ drug-resistant tumours  Therefore, compounds that can be identified by this screening method include, for example, diabetes mellitus, atherosclerosis ^ coronary heart aisease, cystic flDrosis ^ adrenoleukodystrophy ^ Stargardt 's disease ^ drug-resistant tumours
Dubin- Johnson syndrome^ Byler' s disease^ progressive familiar intrahepatic cholestasis^ X- linked siderblastic anemia and ataxia^ persistent Dubin- Johnson syndrome ^ Byler 's disease ^ progressive familiar intrahepatic cholestasis ^ X- linked siderblastic anemia and ataxia ^ persistent
hyperinsulimenic hypoglycemia of infancy等の疾患治療薬として用レヽられ得るも のである。 It can be used as a therapeutic agent for diseases such as hyperinsulimenic hypoglycemia of infancy.
( 6— 4 ) ィムノグロブリン様タンパク質活性を調節する物質の解析 (6-4) Analysis of substances regulating immunoglobulin-like protein activity
ィムノグロブリン様ドメインを持つスーパーフアミリーのメンバーは、異なる機 能を有する数百のタンパク質、 例えば、 抗体、 補体、 巨大筋肉蛋白質チチン、 チロ シンキナーゼ型受容体などからなり、細胞接着、蛋白質間相互作用や蛋白質とリガ ンドとの相互作用に関連すると考えられている。  Members of the superfamily, which have immunoglobulin-like domains, consist of hundreds of proteins with different functions, such as antibodies, complement, giant muscle protein titin, and tyrosine kinase-type receptors. It is thought to be related to the interaction and interaction of the protein with the ligand.
このようなィムノグロプリン様タンパク質の活性を調節する物質の具体的な解 析方法としては、 例えば、 上記 （5— 4 ) に記載の方法等を用いることができる。 ィムノグロプリン様蛋白質の機能解析方法は、画一的ではないが、それぞれの相互 作用に基づいて、それ自体既知の通常用いられる方法により実施することができる。 例えば、 結合試験、 表面プラズモン共鳴、 ツーハイプリッド法、 蛍光エネルギー移 動法、 比熱測定法、 などがあるがこれに限定されない。' より好ましくは、 ィムノグ ロブリン様ドメインを持つスーパーフアミリーのメンバー固有の機能を、例えば上 記 （5— 4 ) に記載したように、 それぞれに適した評価法に従い測定評価すること が好ましレ、。解析の結果、本発明のタンパク質のィムノグロプリン様タンパク質活 性が、物質の非存在下の場合と比べて増加した場合には、該物質はィムノグロプリ ン様タンパク質活性化物質として機能する可能性があり、 また、低下もしくは阻害 された場合には、ィムノグロブリン様タンパク質活性阻害物質として機能する可能 性があると同定することができる。  As a specific method for analyzing such a substance that regulates the activity of the immunoglobulin-like protein, for example, the method described in the above (5-4) can be used. Although the method of analyzing the function of an imnoglobulin-like protein is not uniform, it can be carried out by a commonly used method known per se, based on each interaction. Examples include, but are not limited to, binding tests, surface plasmon resonance, two-hybrid methods, fluorescence energy transfer methods, and specific heat measurements. 'More preferably, it is preferable to measure and evaluate the function specific to the member of the superfamily having the immunoglobulin-like domain, for example, as described in (5-4) above, according to the evaluation method suitable for each. . As a result of the analysis, if the activity of the protein of the present invention is increased as compared with the absence of the substance, the substance may function as an activator of the immunoglobulin-like protein, When reduced or inhibited, it can be identified as having the potential to function as an immunoglobulin-like protein activity inhibitor.
本発明のタンパク質はィムノグロブリン様タンパク質活性を有するものであつ て、 補体の活性化などの免疫反応、 炎症反応、 アレルギー反応、 プロテアーゼ阻害 活性、 受容体あるいは接着分子作用に関与することが推測される。 従って、 上記解 析方法により同定される物質は、 全身性ェリ トマトーデス ·先天性補体成分欠損 症 ·関節リゥマチ ·自己免疫疾患等の免疫疾患、糸球体腎炎'肝炎等の炎症性疾患、 感染症、 癌、 不妊等の疾患の治療薬として用い得るものである。 かかる調節物質は、臨床へ応用するに際し、上記有効成分を単独で用いることも 可能であるが、薬学的に許容され得る担体と配合して医薬品組成物として用いるこ ともできる。 この時の有効成分の担体に対する割合は、 1〜9 0重量％の間で変動 され得る。 また、 かかる薬剤は種々の形態で投与することができ、 それらの投与形 態としては、 錠剤、 カプセル剤、 顆粒剤、 散剤、 あるいはシロップ剤等による経口 投与、 または注射剤、 点滴剤、 リボソーム剤、 坐薬剤等による非経口投与を挙げる ことができる。 また、 その投与量は、 症状、 年齢、 体重等によって適宜選択するこ とができる。 The protein of the present invention has immunoglobulin-like protein activity and is presumed to be involved in immune reactions such as complement activation, inflammatory reactions, allergic reactions, protease inhibitory activities, receptor or adhesion molecule actions. Is done. Therefore, substances identified by the above analysis method include systemic erythematosus, congenital complement component deficiency, rheumatoid arthritis, immune diseases such as autoimmune diseases, inflammatory diseases such as glomerulonephritis and hepatitis. It can be used as a remedy for diseases such as infectious diseases, cancer and infertility. Such modulators can be used alone as the above active ingredient when clinically applied, or can be used as a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. The drug can be administered in various forms. Examples of the dosage form include oral administration of tablets, capsules, granules, powders, syrups, and the like, or injections, drops, ribosomes. And parenteral administration using suppositories and the like. The dose can be appropriately selected depending on the condition, age, weight, and the like.
( 7 ) 本発明の D N Aの発現調節物質のスクリーニング (7) Screening of the DNA expression regulator of the present invention
スクリーニングの方法としては、被検物質の存在下で本発明のタンパク質、 ある いはそれをコードする mRNAの発現量を解析する方法等が挙げられる。 具体的には、 例えば、 （2 ) に記載した本発明のタンパク質を発現する細胞を被検物質を含む適 当な培地で培養し、 該細胞内に発現している本発明のタンパク質量を ELISA等の常 法を用いて解析する力、あるいは該細胞内の本発明のタンパク質をコードする mRNA 量を、 定量的逆転写 PCR法や、 ノーザンプロット法等により解析することにより行 うことができる。  Examples of the screening method include a method of analyzing the expression level of the protein of the present invention or the mRNA encoding the protein in the presence of a test substance. Specifically, for example, cells expressing the protein of the present invention described in (2) are cultured in an appropriate medium containing a test substance, and the amount of the protein of the present invention expressed in the cells is determined by ELISA. And the like, or the amount of mRNA encoding the protein of the present invention in the cells can be analyzed by quantitative reverse transcription PCR, Northern plotting, or the like.
被検物質としては、 （6 )に記載のものを用いることができる。この解析により、 被検物質の非存在下で培養された当該細胞内で発現されたタンパク質、 あるいは mRNA量と比べてその量が増加すれば、物質は本発明の D N Aの発現促進物質として 機能する可能性があり、逆に減少した場合には、物質は本発明の D N Aの発現阻害 物質として用いられ得ると判断することができる。  As the test substance, those described in (6) can be used. According to this analysis, if the amount of the protein or mRNA expressed in the cells cultured in the absence of the test substance increases as compared to the amount of the protein or mRNA, the substance functions as the substance for promoting expression of the DNA of the present invention. If it is possible and conversely decreases, it can be determined that the substance can be used as a substance for inhibiting the expression of the DNA of the present invention.
かかる発現調節物質は、 臨床へ応用するに際し、上記有効成分を単独で用いるこ とも可能であるが、薬学的に許容され得る担体と配合して医薬品組成物として用い ることもできる。 この時の有効成分の担体に対する割合は、 1〜 9 0重量％の間で 変動され得る。 また、 力かる薬剤は種々の形態で投与することができ、 それらの投 与形態としては、 錠剤、 カプセル剤、 顆粒剤、 散剤、 あるいはシロップ剤等による 経口投与、 または注射剤、 点滴剤、 リボソーム剤、 坐薬剤等による非経口投与を挙 げることができる。 また、 その投与量は、 症状、 年齢、 体重等によって適宜選択す ることができる。 The above-mentioned active ingredient can be used alone for clinical application, but it can also be used as a pharmaceutical composition by mixing it with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier is between 1 and 90% by weight. Can be varied. In addition, powerful drugs can be administered in various forms, such as tablets, capsules, granules, powders, or syrups, orally, or injections, drops, ribosomes And parenteral administration with suppositories and suppositories. The dose can be appropriately selected depending on the condition, age, weight, and the like.
( 8 ) 本発明の D N A導入動物 (8) The DNA-introduced animal of the present invention
上記 （1 ) に記載の、 本発明の DNAを含む導入 DNAを構築し、 ヒ ト以外の哺乳動物 の受精卵に導入して、 これを雌個体子宮に移植して発生させることにより、本発明 の DNAが導入された非ヒ ト哺乳動物を作製することができる。 より、 具体的には、 例えば、雌個体をホルモン投与により過剰排卵させた後、雄と交配し、 交配後 1日 目の卵管から受精卵を摘出し、 該受精卵に導入 DNAをマイクロインジェクション等 の方法により導入する。この後、適当な方法で培養した後、生存している受精卵を、 偽妊娠させた雌個体 （仮親） の子宮に移植して出産させる。 新生仔に目的の DNAが 導入されているか否かは、 該個体の細胞から抽出した DNAのサザンブロット解析を 行うことにより同定することができる。 ヒ ト以外の哺乳動物としては、例えばマウ ス、 ラット、 モルモット、 ハムスター、 ゥサギ、 ャギ、 ブタ、 ィヌ、 ネコ等が挙げ られる。  The transfected DNA containing the DNA of the present invention described in (1) above is constructed, introduced into a fertilized egg of a mammal other than human, and transplanted into a female individual uterus to generate the present invention. A non-human mammal into which the DNA has been introduced can be produced. More specifically, for example, after superovulation of a female individual by hormone administration, it is mated with a male, a fertilized egg is excised from the oviduct on the first day after mating, and microinjection of the introduced DNA into the fertilized egg is performed. It will be introduced by such methods. Then, after culturing by an appropriate method, the surviving fertilized eggs are transplanted into the uterus of a pseudopregnant female individual (foster parent) to give birth. Whether or not the desired DNA has been introduced into the neonate can be identified by performing Southern blot analysis on DNA extracted from cells of the individual. Examples of mammals other than humans include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats, and the like.
かくして得られた本発明の DNA導入動物は、 この個体を交配し、 導入された DNA が安定的に保持されていることを確認しながら通常の飼育環境で継代飼育するこ とによりその子孫を得ることができる。 また、体外受精を繰り返すことによりその 子孫を得て、 系統を維持することもできる。  The thus-obtained DNA-introduced animal of the present invention is used to breed this individual and subculture them in a normal breeding environment while confirming that the introduced DNA is stably maintained, thereby obtaining the offspring. Obtainable. In addition, by repeating in vitro fertilization, the offspring can be obtained and the strain can be maintained.
本発明の DNAが導入された非ヒ ト哺乳動物は、本発明の DNAの生体内における機能 の解析や、またこれを調節する物質のスクリーニング系等として用いることができ る。  The non-human mammal into which the DNA of the present invention has been introduced can be used as an analysis of the function of the DNA of the present invention in a living body, or as a screening system for a substance regulating the function.
( 9 )本発明のタンパク質及びそれをコ一ドする塩基配列を含む D N Aの他の利用 本発明のタンパク質は、それを基盤上に結合させた担体として利用することがで きる。 また、 本発明のタンパク質をコードする塩基配列、 例えば、 配列番号 1に記 載の塩基配列を有する DNA及びその部分断片は、 それらを基板上に結合させた担体 としてもちいられ得る。 これらを、 以下、 「プロテインチップ」 、 「DNAチップ」 または 「DNAアレイ」 （DNAマイクロアレイ及び DNAマクロアレイ） と称することが ある。 これらのプロテインチップ、 又は D NAチップもしくはアレイには、 本発明 のタンパク質や DNA以外に、 他のタンパク質や DNAが含まれていてもよい。 (9) Other uses of the protein of the present invention and DNA containing a base sequence encoding the same The protein of the present invention can be used as a carrier on which it is bound. In addition, a nucleotide sequence encoding the protein of the present invention, for example, a DNA having the nucleotide sequence shown in SEQ ID NO: 1 and a partial fragment thereof can be used as a carrier having them bound on a substrate. Hereinafter, these may be referred to as “protein chips”, “DNA chips” or “DNA arrays” (DNA microarrays and DNA macroarrays). These protein chips or DNA chips or arrays may contain other proteins and DNAs in addition to the proteins and DNAs of the present invention.
ここで、 タンパク質や DNAを結合させる基盤としては、 ナイロン膜、 ポリプロピ レン膜等の樹脂基板、 ニトロセルロース膜、 ガラスプレート、 シリコンプレート等 が用いられるが、 ハイブリダィゼーシヨンの検出を非 R I的に、 例えば、 蛍光物質 等を用いて行う場合には、蛍光物質を含まないガラスプレート、 シリコンプレート 等が好適に用いられる。 また該基盤へのタンパク質、 あるいは DNAの結合は、 それ 自体公知の通常用いられる方法により容易に行うことができる。これらのプロティ ンチップ、 DNAチップ、 あるいは DNAアレイも、 本発明の範囲に含まれる。  Here, a resin substrate such as a nylon film or a polypropylene film, a nitrocellulose film, a glass plate, a silicon plate, or the like is used as a base for binding proteins and DNA, but the detection of hybridization is non-RI. In the case of using a fluorescent substance or the like, a glass plate or a silicon plate containing no fluorescent substance is preferably used. The binding of the protein or DNA to the substrate can be easily carried out by a commonly used method known per se. These protein chips, DNA chips, or DNA arrays are also included in the scope of the present invention.
また、 本発明のタンパク質のアミノ酸配列及び DNAの塩基配列は、 配列情報とし ても用いることができる。 ここで、 D N Aの塩基配列には、 対応する R N Aの塩基 配列も含まれる。すなわち、得られたアミノ酸配列や塩基配列をコンピューターが 読みとり可能な所定の形式で適当な記録媒体に格納することにより、ァミノ酸配列 や塩基配列のデータベースが構築できる。 このデータベースには、他の種類のタン パク質やそれをコードする DNAの塩基配列が含まれていてもよい。 また、 本発明に おいてデータベースとは、上記配列を適当な記録媒体に書き込み、所定のプロダラ ムに従って検索を行うコンピューターシステムをも意味する。ここで適当な記録媒 体としては、 例えば、 フレキシブルディスク、 ハードディスク、 磁気テープ等の磁 気媒体、 C D— R OM、 MO、 C D - R , C D - RW, D V D - R , D V D - RW 等の光ディスク、 半導体メモリ等を挙げることができる。 実施例 以下、実施例を挙げて本発明を詳細に説明するが、本発明の範囲はこれらの実施 例により限定されるものではない。 In addition, the amino acid sequence of the protein of the present invention and the nucleotide sequence of DNA can also be used as sequence information. Here, the nucleotide sequence of the DNA includes the nucleotide sequence of the corresponding RNA. That is, a database of amino acid sequences and nucleotide sequences can be constructed by storing the obtained amino acid sequences and nucleotide sequences in an appropriate recording medium in a computer-readable predetermined format. This database may contain other types of proteins and the base sequences of the DNA that encodes them. In the present invention, the database also means a computer system that writes the above-mentioned sequence on an appropriate recording medium and performs a search according to a predetermined program. Suitable recording media include, for example, magnetic media such as flexible disks, hard disks, and magnetic tapes, and optical media such as CD-ROM, MO, CD-R, CD-RW, DVD-R, and DVD-RW. And semiconductor memories. Example Hereinafter, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited to these examples.
実施例 1 cDNAライブラリーの調製 Example 1 Preparation of cDNA Library
(1) mRNAの調製  (1) Preparation of mRNA
mRNA調製マウス （C 57 B L/6) 各器官または組織 0. 5〜： 1 gを 10 m 1の懸濁液でホモジェナイズし、 pH4. 0 の 2M 酢酸ナトリウム 1 m 1 と、 同 量のフエノール/クロ口ホルム（体積比 5 ： 1)混液を加え抽出した。 抽出後水層に 同量のイソプロパノールを加えると、 RNAが水相から分離沈澱した。 この試料を 氷の上で 1時間インキュベーションした後、 15分間 4, 000 r pmで冷却遠心 機にかけ、 沈澱物を回収した。 この検体を 70%エタノールで洗い、 8m lの水に 溶解後、 2m lの 5M Na C l、 l % CTAB (cetyltrimethy- laramonium bromide)、 4M尿素、 50 mM 丁 1： 1 3を含む 117. 0 の水溶液 16 m 1を 加えることで RNAを沈澱させ、 ポリサッカライドを除いた（CTAB沈澱） 。 続いて室温で 4， 000 r pm、 15分間遠心機にかけ、 R N Aを 4 m 1の 7 M グァニジン一 C 1に溶解した。 そして 2倍量のエタノールを加えた後、氷上で 1時 間インキュベーションし、 4, 000 r pm、 15分間遠心機にかけ、 生じた沈澱 物を 70%エタノールで洗い RN Aを回収した、 これを再度水に溶解し、 RNAの 純度を OD比 260 280 (>1. 8) と 230/260 « 0. 45) を読む ことによって計測した。  mRNA-prepared mouse (C57BL / 6) 0.5 or more of each organ or tissue: 1 g was homogenized with a 10 ml suspension, and 1 ml of 2M sodium acetate at pH 4.0 was added to the same amount of phenol / A mixed solution of black-mouthed form (5: 1 by volume) was added for extraction. When the same amount of isopropanol was added to the aqueous layer after the extraction, RNA separated and precipitated from the aqueous phase. After incubating the sample on ice for 1 hour, the precipitate was collected in a refrigerated centrifuge at 4,000 rpm for 15 minutes. This sample was washed with 70% ethanol, dissolved in 8 ml of water, and then contained 2 ml of 5 M NaCl, l% CTAB (cetyltrimethy- laramonium bromide), 4 M urea, and 50 mM 1: 1 13 RNA was precipitated by adding 16 ml of an aqueous solution of the polysaccharide to remove polysaccharide (CTAB precipitation). The RNA was then centrifuged at 4,000 rpm for 15 minutes at room temperature to dissolve the RNA in 4 ml of 7 M guanidine-C1. After adding twice the volume of ethanol, the mixture was incubated on ice for 1 hour, centrifuged at 4,000 rpm for 15 minutes, and the resulting precipitate was washed with 70% ethanol to collect RNA. The RNA was dissolved in water and the purity of the RNA was determined by reading the OD ratio 260 280 (> 1.8) and 230/260 «0.45).
(2) 第 1鎖 cDNAの調製  (2) Preparation of first strand cDNA
上記 （1) で調製した mRNA 15 // gを使って逆転写酵素 3, 000 u n i t により、最終容量 165 μ 1の反応液中で、 5—メチル _dCTP、 d ATP, dTTP、 dGTPを各々 0. 54mM、 0. 6Mトレハロース、 50 mM T r i s— HC 1 (pH8. 3) 、 75 mM KC 1、 3 mM MgC 1 2、 10 mM DTT, 52 n g/ μ 1 B S A、 RN a s eインヒビター 5 u n i tの条件下 で逆転写反応を行った。 制限酵素 Xh o Iの認識配列を含むオリゴヌクレオチド (配列番号 4) (配列中、 Vは A,G,又は Cを示し、 Nは A, G, C,又は Tを示す） 12. 6 μ 1をプライマーとして用いた。 Using 15 / g of the mRNA prepared in (1) above, 5-methyl_dCTP, dATP, dTTP, and dGTP were each diluted with 3,000 units of reverse transcriptase in a reaction volume of 165 μl in a final volume of 165 μl. 54 mM, 0.6 M trehalose, 50 mM Tris—HC1 (pH 8.3), 75 mM KC1, 3 mM MgC12, 10 mM DTT, 52 ng / μ1 BSA, RNase inhibitor 5 units To perform a reverse transcription reaction. Oligonucleotide containing the recognition sequence of the restriction enzyme Xho I (SEQ ID NO: 4) (in the sequence, V represents A, G, or C, and N represents A, G, C, or T) 12. 6 μl was used as a primer.
この反応を始める際、 反応液の 1Z4を採取し、 それに 1. 5 μ 1の [α—32 Ρ] - d GTP (30 00 C i /mm o 1 0 μ C i /μ 1 ； Ame r s h a m社製） を加えるこことにより、第 1鎖 c DNAの合成効率を測定した。 R I標識した反応 液の 0. 5 μ 1を DE— 8 1ペーパー上にスポットし、 0. 5Μリン酸ナトリウム 緩衝液 （ρ Η 7. 0) で 3回洗った前後の R I活性を測定し、 計算した。 その後、 R I標識した反応液と非標識の反応液を混合し、 0. 5Μ EDTA 8 μ 1、 1 0% S D S 2 ju 1、 プロティナーゼ （P r o t e i n a s e) Κ 20 μ gを 加え、 4 5°Cで 1 5分間加熱した。 フエノール Zクロ口ホルムによる抽出、 ェタノ ール沈澱後、沈澱を RN a s eフリ一に処理してある水（以下 RN a s eフリ一水 とする） 4 7 μ 1に溶解した。 At the beginning of this reaction, the 1Z4 of the reaction solution was taken and thereto 1. 5 μ 1 [α- 32 Ρ] - d GTP (30 00 C i / mm o 1 0 μ C i / μ 1; Ame rsham Inc. Thus, the synthesis efficiency of the first-strand cDNA was measured. 0.5 μl of the RI-labeled reaction solution was spotted on DE-81 paper, and the RI activity before and after washing three times with 0.5% sodium phosphate buffer (ρ Η 7.0) was measured. Calculated. Then, by mixing RI-labeled reaction liquid and non-labeled reaction solution, 0. 5Μ EDTA 8 μ 1, 1 0% SDS 2 ju 1, proteinase (P roteinase) Κ 20 μ g was added, in 4 5 ° C Heated for 15 minutes. After extraction with phenol Z-cloth form and precipitation with ethanol, the precipitate was dissolved in 47 µl of RNase-free water (hereinafter referred to as RNase-free water).
(3) 5， キャップ構造及び 3， 末端へのピオチン付カロ  (3) 5, cap structure and 3, carotene with biotin
RN Αジオールのビォチン化 RN Αのジオール部位（C a p構造のある 5，末端 と、 ポリ A鎖のある 3， 末端のリボースの双方に存在） にピオチンを結合させるた めに、 2段階の反応を行った。 それらは、 ジオール基の酸化とそれに続くピオチン ヒ ドラジドと酸化 RNA体のカップリング反応である。 まず、逆転写反応で得られ た RNA—第 1鎖 c DNA複合体 1 5 // gを、 6. 6 mM酢酸ナトリゥム緩衝液（p H4. 5) と、 酸化剤として過ヨウ素酸ナトリウムを用いて 50 μ 1の反応液中で 処理した。 この酸化反応は遮光条件の下、 氷上で 4 5分間行った。  Biotinylation of RN Αdiol A two-step reaction to bind biotin to the diol site of RN （(present at both the 5 'end of the Cap structure and the 3' end of the poly A chain ribose) Was done. These are the oxidation of the diol group followed by the coupling reaction of the oxidized RNA with the biotin hydrazide. First, 15 // g of the RNA-first strand cDNA complex obtained by the reverse transcription reaction was used with 6.6 mM sodium acetate buffer (pH 4.5) and sodium periodate as an oxidizing agent. In a 50 μl reaction. This oxidation reaction was performed on ice for 45 minutes under light-shielded conditions.
続いて、 5Μ塩化ナトリウム 1 1 // 1、 1 0% SD S 0. 5 1、 そして同 量のイソプロパノールを加え、 6 0分間氷上に放置した後、 4°Cで 1 5分間 1 5, 0 00 r pm遠心し沈澱を取得した。 沈澱物は 70 %エタノールで洗レ、、 RN a s eフリー水 5 0 / 1に再溶解させた。その試料に 1M酢酸ナトリウム（p H6. 1 ) 5 μ 1 0 % S D S 5 μ 1、 1 OmMピオチンヒドラジド （S i gma社製） 1 5 0 1を加え、 室温 （2 2〜2 6°C) で終夜反応させた。 最後に、 5 μ 1の 5 M N a C 1、 1 M酢酸ナトリウム （p H6. 1) 7 5 μ 1、 及ぴ 2. 5倍量のェ タノ一ルを加え、 1時間の氷上冷却後、 4 °Cにおいて 1 5分間遠心し、 ビォチン化 した。反応後、反応液を 1 5分間遠心し、再度 R N A— D N A複合体を沈澱させた。 沈澱物は 70%エタノールで 1回、更に 8 0%エタノールで 1回洗い、 RN a s e フリー水 70 μ 1に溶角早した。 Then, add 5 ナ ト リ ウ ム sodium chloride 1 1 // 1, 10% SD S 0.51, and the same amount of isopropanol, leave on ice for 60 minutes, then 15 minutes at 4 ° C for 15 minutes. The precipitate was obtained by centrifugation at 00 rpm. The precipitate was washed with 70% ethanol and redissolved in 50/1 RNase-free water. To the sample was added 5 μl of 1 M sodium acetate (pH 6.1), 5 μl of 10% SDS, and 150 μm of 1 OmM biotin hydrazide (manufactured by Sigma) at room temperature (22 to 26 ° C). ) Overnight. Finally, add 5 μl of 5 M NaC1, 1 M sodium acetate (pH 6.1), 75 μl, and 2.5 times the volume of ethanol, and cool on ice for 1 hour. Centrifuge at 4 ° C for 15 minutes to biotin did. After the reaction, the reaction solution was centrifuged for 15 minutes to precipitate the RNA-DNA complex again. The precipitate was washed once with 70% ethanol, and once with 80% ethanol, and was rapidly dissolved in 70 μl of RNase-free water.
(4) RN a s e Iによる完全長 c D N Aの選択  (4) Selection of full-length cDNA by RNaseI
上記（3) で取得したビォチン化 RNA— DN A複合体について、 1本鎖 RNA を消化する RN a s e Iで処理することにより、逆転写反応時に完全な c DN A の伸長が得られなかった mRNA、および mRNAの 3 '末端に標識されたビォチ ン残基を取り除いた。 具体的には、 上記 （3) で得られた試料 70 μ 1に 1 0 X R N a s e Iバッファー （1 00 mM T r i s _HC l (p H 7. 5) 、 5 0m M EDTA、 2M N a OA c) 1 0 /i l、 RN a s e I (RN a s e On e™ ; P r ome g a社製） 200 u n i tを加えて、 3 7 °Cで 1 5分間 1本鎖 R N Aを消化した。 By treating the biotinylated RNA-DNA complex obtained in (3) above with RNase I that digests single-stranded RNA, mRNA that did not achieve complete cDNA extension during the reverse transcription reaction , And a labeled biotin residue at the 3 ′ end of the mRNA were removed. Specifically, the (3) 1 0 to the sample 70 mu 1 obtained in XRN ase I buffer (1 00 mM T ris _HC l (p H 7. 5), 5 0m M EDTA, 2M N a OA c ) 10 / il, 200 units of RNase I (RNase One ™; manufactured by Promega) were added, and the single-stranded RNA was digested at 37 ° C for 15 minutes.
(5) 完全長 c DNAの採取  (5) Collection of full-length cDNA
ストレプトアビジンコートしたマグネティックビーズに c DNAが非特異的吸 着するのを防止するため、 1 00 μ gの酵母 t RNA (DN a s e I処理したも の） を 5mg (5 00 μ 1 ) のマグネテイツクビーズ (ma g n e t i c p o r o u s g l a s s (MP G) p a r t i c l e s c o a t e d w i t h s t r e p t a v i d i n (C PG, N J ) ) に加え、 1時間氷上に放置した後、 5 OmM EDTA、 2M N a C 1の溶液にて洗った。  To prevent nonspecific adsorption of cDNA to magnetic beads coated with streptavidin, 100 μg of yeast tRNA (treated with DNase I) was added to 5 mg (500 μl) of magnetic beads. After adding to beads (magneticporous glass (MPG) particles coated with st reptavidin (CPG, NJ)), leaving it on ice for 1 hour, it was washed with a solution of 5 OmM EDTA and 2 M NaC1.
このビーズを 5 0 mM EDTA、 2M N a C 1の溶液 5 00 μ 1中に懸濁し、 (4) で取得した RN a s e I処理を施された c DNAを加えた。室温にて 3 0 分間撹拌することで、マグネティックビーズと完全長 c D N Aを結合させた。完全 長 c DNAを捕獲したビーズを 5 OmM EDTA、 2M N a C Iの溶液で 4回、 0. 4% SD S、 5 0 g/μ 1酵母 t RNAで 1回、 1 OmM N a C l、 0. 2mM EDTA、 1 OmM T r i s— HC 1 (p H 7. 5) 、 20%グリセ口 ールで 1回、 50 μ gノ μ 1酵母 t RNA水溶液で1回、 RN a s e Hバッファ 一 (2 OmM T r i s— HC 1 (p H 7. 5) 、 1 OmM M g C 12、 2 0m M KC 1、 0. 1 mM EDTA、 0. 1 mM ジチオスレィトール （D T T) ) で 1回洗浄した後、 RNa s e H用バッファー 1 00 1に懸濁し、 RNa s e H 3 u n i tを加え、 37 °C下 30分間加温した。 その後、 10% SDS 1 // 1、 0. 5M EDTA 2 μ 1を加えて、 1 0分間、 6 5°Cに曝し、 その上清 を回収した。 The beads were suspended in 500 μl of a solution of 50 mM EDTA and 2 M NaCl, and the RNase I-treated cDNA obtained in (4) was added. By stirring for 30 minutes at room temperature, the magnetic beads and the full-length cDNA were bound. The beads capturing the full-length cDNA were washed 4 times with a solution of 5 OmM EDTA and 2 M NaCI, 0.4% SDS, 50 g / μl once with yeast tRNA, and 1 OmM NaCl, 0.2 mM EDTA, 1 OmM Tris-HC1 (pH 7.5), once with 20% glycerol, once with 50 μg noμ1 yeast tRNA aqueous solution, once with RNAse H buffer 2 OmM T ris- HC 1 (p H 7. 5), 1 OmM M g C 1 2, 2 0m M KC1, 0.1 mM EDTA, 0.1 mM dithiothreitol (DTT)), wash once, suspend in RNase H buffer 1001, add 3 units of RNase H, add Heated at ° C for 30 minutes. Thereafter, 2 μl of 10% SDS 1 // 1, 0.5 M EDTA was added, and the mixture was exposed to 65 ° C. for 10 minutes, and the supernatant was collected.
このようにして回収された 1本鎖完全長 c DN Aはフエノール Zクロロホルム で抽出され、スピードバッグにて液量を 1 00 // 1以下に減じてから G 2 5/G 1 O O S e p h a d e xクロマトグラフィーに付した。 R I活性を持つた分画はシリ コン処理したマイクロチューブに収集するとともに、 ダリコーゲン 2 μ gを加え、 エタノール沈澱にて得られた沈澱物を 30 μ 1の超純水に溶解した。  The single-stranded full-length cDNA recovered in this manner is extracted with phenol Z-chloroform, and the volume is reduced to 100 // 1 or less in a speed bag, and then G 25 / G 1 OOS ephadex chromatography Attached. The fraction having RI activity was collected in a silicon-treated microtube, 2 μg of dalycogen was added, and the precipitate obtained by ethanol precipitation was dissolved in 30 μl of ultrapure water.
(6) 1本鎖 c DNAへのオリゴ d G付加  (6) Add oligo d G to single-stranded cDNA
上記 （ 5 ) で回収された 1本鎖 c D N A 30 / 1は、 最終容量 50 μ 1の反応液 中で、 20 OmM力コジル酸ナトリウム （pH6. 9) 、 1 mM M g C 12、 1 mM C o C l 2、 1 mM 2—メルカプトエタノール、 100 / M dGTPの 条件のもと、 ターミナルデォキシヌクレオチジルトランスフェラーゼ（T a Ka R a社製） 3 2 u n i tを用いて 3 7 °Cで 30分間のオリゴ d G付加反応に付した。 反応終了時に EDTAを 5 OmMとなるように加え、一連のフヱノール/クロロホ ルムによる抽出、 エタノール沈澱を経て、 3 1 μ 1の超純水に溶解した。 The single-stranded cDNA 30/1 recovered in (5) above was used in a final volume of 50 μl of the reaction solution at 20 OmM sodium sodium codylate (pH 6.9), 1 mM MgCl 2 , 1 mM mM C o C l 2, 1 mM 2- mercaptoethanol, 100 / M under the conditions of dGTP, terminal de o carboxymethyl nucleotidyl transferase (T a Ka R a, Ltd.) 3 2 with Unit 3 7 ° C For 30 minutes for oligo dG addition reaction. At the end of the reaction, EDTA was added to 5 OmM, and after a series of extraction with phenol / chloroform and ethanol precipitation, it was dissolved in 31 μl of ultrapure water.
(7) 第 2鎖 c DNA合成  (7) Second strand cDNA synthesis
第 1鎖 c DNAを铸型にした第 2鎖 c DNAの合成は以下のように行った。最終 容量 60 μ 1の反応系で、第 2鎖低バッファー（ 200 mM T r i s— H C 1 (p H8. 75) 、 1 0 OmM KC 1、 1 0 OmM (NH4) 2SO4、 2 OmM M g S04、 1 %T r i t o n X— 1 00、 lmg/μ 1 B S A) 3 μ 1、 第 2 鎖高バッファー （20 OmM T r i s— HC 1 (pH9. 2) 、 60 OmM K C l、 2 OmM M g C 12) 3 μ 1、 d CTP、 d ATP, dTTP、 dGTP 各々 0. 25mM、 3— NADH 6 / 1、 オリゴ d G付加された第 1鎖 c D N A 3 1 μ 1、 第 2鎖プライマ一—アダプター （配列番号 5) 600 n gを加え、 E x T a q DNAポリメラーゼ （T a K a R a Ex T a q ; T a K a R a社製） 1 5 u n i t、 而熱性 DNAリガーゼ (Amp 1 i g a s e ; Ep i c e n t r e 社製） 1 50 u n i t、 而ォ熱性 R N a s e H (Hy b r i d a s e ; Ep i c e n t r e社製） 3 u n i tによって第 2鎖 c D N Aを合成した。 The synthesis of the second-strand cDNA obtained by converting the first-strand cDNA into a type II was carried out as follows. In a final reaction volume of 60 μl, use a second strand low buffer (200 mM Tris-HC1 (pH 8.75), 10 OmM KC1, 10 OmM (NH 4 ) 2 SO 4 , 2 OmM M g S0 4, 1% T riton X- 1 00, lmg / μ 1 BSA) 3 μ 1, second Kusaridaka buffer (20 OmM T ris- HC 1 ( pH9. 2), 60 OmM KC l, 2 OmM M g C 1 2) 3 μ 1 , d CTP, d ATP, dTTP, dGTP each 0. 25mM, 3- NADH 6/1 , first strand c DNA 3 1 mu 1 which is added oligo d G, second strand primer One—Add 600 ng of the adapter (SEQ ID NO: 5) and add E x Taq DNA polymerase (TaKaRa Ex Taq; TaKaRa) 15 units, thermophilic DNA ligase (Amp 1 igase; Epcentre) 150 units, thermophilic RN Second strand cDNA was synthesized using 3 units of ase H (Hybridase; manufactured by Epcentre).
0. 5M EDTAを 1 1加えることで反応を停止させ、更にタンパク成分を 溶解するために、 10% SDS 1 μ 1、 プロティナーゼ （P r o t e i n a s e) K 10 μ gの存在下に 45°Cで 15分間加熱し、最終的にフエノールノク口 口ホルムによる抽出、 エタノール沈澱にて精製した 2本鎖完全長 c DN Aを得た。 (8) ライブラリーの調製  Stop the reaction by adding 1 1 of 0.5M EDTA, and further dissolve the protein components by removing 1 μl of 10% SDS and 10 μg of proteinase K at 45 ° C for 15 minutes. The mixture was heated, and finally, a double-stranded full-length cDNA was purified by phenol extraction and ethanol precipitation. (8) Library preparation
以上の方法により得られた二本鎖完全長 c DN Aは、； L ZAP I I Iベクターに 挿入し、ライブラリ一として回収した。 λ ΖΑΡ Ι I Iベクターは； ZAP I I (S T RAT AG EN Ε社製）ベクターのマルチクローニングサイトの一部の配列であ る配列番号 6を配列番号 7に改変し、二つの S f i Iサイトを新たに導入したもの である。  The double-stranded full-length cDNA obtained by the above method was inserted into an LZAPIII vector and recovered as a library. The λΖΑΡII vector is obtained by modifying SEQ ID NO: 6, which is a partial sequence of the multiple cloning site of the ZAP II (manufactured by ST RAT AG ENΕ) vector, to SEQ ID NO: 7 and adding two SfiI sites. It was introduced in
さらに; I P S (R I KEN)ベクターを作製し、 c DNAを挿入した。 λ P S (R I KEN) (λ— FLC_ 1と命名 （FLCとは FULL— LENGTH c DN Aを意味する） ） とは、 Mo B i T e c社 （ドイツ） の λ P Sベクターを c DNA 用に改変したものである。即ち 10 k b p s t u f f e rの両側に存在するクロ 一ユングサイ トに c DNA挿入に便利な B a mH Iならびに S a 1 Iを各々導入 するとともに、 0. 5 k bから 13 k b程度までの c DNAがクローニングできる ように Xb a Iサイトに 6 k bの DNA断片を挿入したものである（特開 2000 - 325080号公報） 。 この； L— FLC—1を用いると、 例えば肺臓 c DNAラ イブラリ一の場合には、 ィンサートの平均鎖長は 2. 57 k bとなり、 実際に 0. 5 k bから 1 2 k bまでのインサートをクローニングすることが出来た。従来法の L ZAPの場合には、 インサートの平均鎖長は 0. 97 k bであったことから、 λ -F LC- 1を用いることによって、サイズの大きな c DNAも； L ZAPに比べて 効率よくクローユングできることがわかる。 実施例 2 完全長 c D N Aライブラリーのノーマライゼ一ション Zサブトラクシ ョン Further; an IPS (RI KEN) vector was prepared and cDNA was inserted. λ PS (RI KEN) (named λ—FLC_1 (FLC means FULL—LENGTH cDNA)) is a modification of the λ PS vector from MoBiTec (Germany) for cDNA. It was done. In other words, BamHI and Sa1I, which are convenient for cDNA insertion, are introduced into the close-up site located on both sides of 10 kbpstuffer, and cDNA from 0.5 kb to about 13 kb can be cloned. And a 6 kb DNA fragment inserted into the XbaI site (Japanese Patent Application Laid-Open No. 2000-325080). When L-FLC-1 is used, for example, in the case of the lung cDNA library, the average insert length is 2.57 kb, and the insert from 0.5 kb to 12 kb is actually cloned. I was able to do it. In the case of the conventional method, LZAP, the average length of the insert was 0.97 kb, so using λ-FLC-1 enables the use of large-sized cDNAs; efficiency compared to LZAP You can see that crawling can be done well. Example 2 Normalization of a full-length cDNA library Z subtraction
(1) ドライバーの調製  (1) Preparation of driver
実施例 1 (1) で作製した mRNA (以下、 これを 「 （a) RNAドライバー」 と称することがある） 、及び i n v i t r o転写反応で作成した RNAをドライ バーとして用いた。 後者の RNAはさらに 2種類 （以下、 これを 「 （b) RNAド ライバー」 、 及ぴ 「 （c) RNAドライバー」 と称する） に分けられる。 1っはノ 一マライゼーシヨンにより除かれた RNA— c DNAから c DNAを回収し、ファ ージベクターにクローニングしたものである。大腸菌に感染後 1つの出発材料あた り 1000から 2000プラークを混ぜ合わせて 1つのライブラリー（ミニライブ ラリー） とし、 常法によりプラスミ ド DNAに変換する （ファージをヘルパーファ ージとともに再度大腸菌に感染させ、 ファージミ ドとし、 さらにもう一度感染させ てプラスミ ド DNAを得る） 。  The mRNA prepared in Example 1 (1) (hereinafter sometimes referred to as “(a) RNA driver”) and the RNA prepared by the invitro transcription reaction were used as drivers. The latter RNA is further divided into two types (hereinafter referred to as “(b) RNA driver” and “(c) RNA driver”). First, cDNA was recovered from RNA-cDNA removed by normalization and cloned into a phage vector. After infection with Escherichia coli, 1000 to 2000 plaques are mixed per starting material into one library (mini-library), which is converted into plasmid DNA by a conventional method. Infect, transform into phagemids, and infect again to obtain plasmid DNA).
得られた DNAについて i n V i t r o転写反応（T 3 RNAポリメラーゼま たは T7RNAポリメラーゼを用いる） を行い、 DNa s e I (RQ 1 -RN a s e f r e e ; P r ome g a社製） 、 P r o t e i n a s e K処理後、 フェ ノール Zクロ口ホルム抽出をして RNA (b) RNAドライバーを得た。 この際、 通常出発材料としては 9種類 （すい臓、 肝臓、 肺、 腎臓、 脳、 脾臓、 睾丸、 小腸、 胃） の組織からそれぞれミニライブラリーを作成して、 9種類のミニライブラリー を混合して RNAを得る。もう一つの RNAはすでに重複のないクローンとして保 存されているライブラリー （クローン数約 2万個） を培養し、 得られた DNAにつ いて （b) RNAドライバーと同様に i n V i t r o転写反応を行い （c) RN Aドライバ一とした。  The obtained DNA was subjected to an in vitro transcription reaction (using T3 RNA polymerase or T7 RNA polymerase), followed by treatment with DNase I (RQ 1 -RNasefree; Promega) and Proteinase K. RNA (b) RNA driver was obtained by extraction of phenol Z-cloth form. At this time, as a starting material, mini-libraries are prepared from nine types of tissues (pancreas, liver, lung, kidney, brain, spleen, testis, small intestine, stomach), and the nine types of mini-libraries are mixed. To obtain RNA. For another RNA, a library (about 20,000 clones) already stored as a non-overlapping clone is cultured, and the resulting DNA is used for (b) in vitro transcription reaction in the same manner as the RNA driver. (C) The RNA driver was selected.
これら 3種の RNAは、 La b e l— I T B i o t i n L a b e l i n g K i t (M i r u s Co r p o r a t i o n製） を用いてピオチン化標識を行つ たあと、 1 : 1 : 1の割合でテスター cDN Aに添加し、 Ro t 10での反応 （4 2°C) を行い、 ス トレプトアビジンビーズ （CPG) 処理を行って回収した上清に ついて、 第 2鎖の合成を行った。 実施例 3 完全長 c D N Aクローンの塩基配列決定 These three types of RNA were labeled with a biotin label using the Label-ITB iotin Labeling Kit (manufactured by Mirus Corporation), and then added to the tester cDNA at a ratio of 1: 1: 1. Reaction at Rot 10 (4 2 ° C), and the second strand was synthesized from the supernatant collected by streptavidin bead (CPG) treatment. Example 3 Determination of base sequence of full-length cDNA clone
( 1 ) クローンの r e a r r a y  (1) r e a r r a y of the clone
各クラスタからひとつの代表クローンを選んだ。代表クローンは Q— b o t (G ENET I X L I M I TED製） で選択し、 384穴プレートに a r r a yィ匕し た。 その際、 大腸菌は 30でで 18〜 24時間、 50 μ 1の L Β培地で培養した。 このとき、 c DNAライブラリーが P Sベクターに導入され大腸菌 DH 10 Βを形 質転換している場合には 10 Omg/m 1のアンピシリン及び 5 Omg/m 1の カナマイシンを添加し、 Z a pベクターに導入し、 SOLRシステムに導入してい る場合には 10 OmgZm 1のアンピシリン及び 25mg/m 1のストレプトァ ビジンを添加して行った。  One representative clone was selected from each cluster. Representative clones were selected by Q-bot (manufactured by GENETIXLIMITED), and were placed on a 384-well plate. At that time, E. coli was cultured in 50 µl of LΒ medium at 30 for 18 to 24 hours. At this time, if the cDNA library has been introduced into the PS vector and transformed Escherichia coli DH10Β, add 10 Omg / m1 ampicillin and 5 Omg / m1 kanamycin and add it to the Zap vector. When introduced into the SOLR system, ampicillin at 10 OmgZm1 and streptavidin at 25 mg / m1 were added.
(2) プラスミ ドの抽出と I n s S i z i n g  (2) Plasmid extraction and Ins S i z i n g
上記 （1) で培養した各クローンは、 さらに l O OmgZm lのアンピシリンを 含む 1. 3m 1の HT液中で培養され、 遠心分離により菌体を回収した後、 Q I A p r e p 96 T u r b o (Q I AG E N社製） を用いてプラスミド D N Aを回 収、精製した。取得されたプラスミ ド中に挿入されている c DNAの鎖長を調べる ために、上記で取得したプラスミ ド DNAの 1 30を制限酵素 P V u I Iで消化 し、 1 %の a g a r o s eゲル電気泳動を行った。  Each of the clones cultured in the above (1) is further cultured in 1.3 ml HT solution containing lOOmgZml of ampicillin, and the cells are collected by centrifugation. Then, QIAprep 96 Turbo (QI AG The plasmid DNA was collected and purified using EN. To check the length of the cDNA inserted into the obtained plasmid, 130 of the plasmid DNA obtained above was digested with the restriction enzyme PVuII and subjected to 1% agarose gel electrophoresis. Was.
(3) 配列決定  (3) Sequence determination
かくして取得されたプラスミ ド中に挿入された完全長 c DNAの全長の塩基配 列解析には、 3種類のシークェンサを用いた。 また、 プラスミ ドは挿入配列の長さ が 2. 5 k bより短いものと長いものの 2つのカテゴリに分けた。 このうち 2. 5 k bより短い挿入配列を有するクローンについては両端から塩基配列を解析した。 その際、 プラスミ ドはベクターが PSの場合には配列番号 8 (センス鎖） 、 及び 9 (アンチセンス鎖） に記載のプライマーを用いて、 またベクターが Z a pの場合に は配列番号 10 (センス鎖） 、 及び 1 1 (アンチセンス鎖） に記載のプライマーを 用レヽて I h e rmo s e q u e n a s e P r i me r Cy c l e ¾ e q u e n c i n g K i t (Am e r s h am Ph a rma c i a B i o t e c h 社製） で反応し、 L i c o r DNA4200 (l o n g r e a d s e q u e n c e r ) を用いて解析した。 Three types of sequencers were used for full-length nucleotide sequence analysis of the full-length cDNA inserted into the plasmid thus obtained. Plasmids were divided into two categories: those with insertion sequences shorter than 2.5 kb and those with longer insertion sequences. Of these clones, the clone having an insertion sequence shorter than 2.5 kb was analyzed for the nucleotide sequence from both ends. In this case, the plasmid was prepared using the primers shown in SEQ ID NOS: 8 (sense strand) and 9 (antisense strand) when the vector was PS, and when the vector was Zap. Using the primers described in SEQ ID NOs: 10 (sense strand) and 11 (antisense strand), use the primers described in Ihermosequenase Primer Cyclic Equencing Kit (Amersham Pharmacia Biotech). And analyzed using Licor DNA4200 (longreadsequencer).
上記塩基配列解析により解析ができなかったギャップは、プライマウォーキング 法により決定した。 その際、 AB I P r i s m377及び Zまたは AB I P r i sm3700 (Ap p l i e d B i o s y s t erns I n c. 製） と B i g Dy e t e rm i n a t o r k i tと Cy c l e S e qu e n c i n g F S r e a d y Re a c t i o n K i t (Ap p l i e d B i o s y s t ems I n c . 製) を用レヽた。  Gaps that could not be analyzed by the above nucleotide sequence analysis were determined by the primer walking method. At this time, AB IPris m377 and Z or AB IPris sm3700 (manufactured by Applied Biosystems Inc.), Big Dy ete rm inatorkit, and Cyclic Sequencing FS ready Reaction Kit (Applied B iosyst ems Inc.).
また、 挿入されている c DNAが 2. 5 k bより長いクローンの配列決定は、 シ ヨットガン法によった。その際、 S h i ma d z u R I S A 384と DYE n am i c E T t e rm i n a t o r c y c l e s e qu e n c i n g k i t (Am e r s h am Ph a rma c i a B i o t e c h社製)を用レヽた。 ショットガンライブラリを作製するために、 48の独立な代表クローンから PCR で増殖した 48の DNAフラグメントを用いた。増幅された DNA断片の末端を T 4 DNAポリメラーゼによって平滑化した。  In addition, sequencing of clones having an inserted cDNA longer than 2.5 kb was performed by the shotgun method. At that time, ShimadzuRISA384 and DYE namicETTerminea torrccyclesequenecinigkit (Amersham PharmaciaBiotech) were used. 48 DNA fragments grown by PCR from 48 independent representative clones were used to generate a shotgun library. The ends of the amplified DNA fragments were blunt-ended with T4 DNA polymerase.
この DNA断片を、 pUC 18ベクターへ挿入し、更に該組み換えベクターによ り大腸菌 DH10Bを形質転換した。 この大腸菌から上記 （2) と同様にしてブラ スミ ドを調製した。  This DNA fragment was inserted into a pUC18 vector, and Escherichia coli DH10B was transformed with the recombinant vector. A plasmid was prepared from this E. coli in the same manner as in (2) above.
それらの代表クローンについては、両末端からの塩基配列解析によつて塩基配列 を決定し、該塩基配列をコンピューター上で連結した後、 Do u b l e S t r o k e Sh e a r i n g De v i c e (F i o r e I n c. 製) による s h e a r i n gを行った。 ショットガン法による塩基配列決定は、 12〜 15クローン の重複をもって行った。この塩基配列決定により配列が決定できなかったギャップ は、 上記と同様にプライマウォーキングによって決定した。 実施例 4 塩基配列の解析 For those representative clones, the nucleotide sequence was determined by nucleotide sequence analysis from both ends, and the nucleotide sequences were ligated on a computer, followed by Double Stroke Shearing Device (manufactured by Fire Inc. ) Shearing was performed. Nucleotide sequencing by the shotgun method was performed with duplication of 12 to 15 clones. The gap whose sequence could not be determined by the nucleotide sequence determination was determined by primer walking in the same manner as described above. Example 4 Analysis of base sequence
(1) d n a f o rm51839 (配列番号 1、 2、 3)  (1) d n a f o rm51839 (SEQ ID NO: 1, 2, 3)
d n a f o rm51839は、配列番号 1に示すように、 1 156塩基から成る。 配列番号 1がコードするアミノ酸配列について B LASTを用いて相同性検索を 行ったところ、 S PTR蛋白質データベース （SWI S S— PROT蛋白質配列デ ータベースと T r EMB L核酸翻訳データベースを統合したもの） 中に、 （i) デ ータベース登録記号 A J 277487, Homo s a p i e n s p u t a t i v e t r a n smemb r a n e p r o t e i n NMA p r e c u r s o r力 e-v a l u e : 3 X l 0— 70、 138アミノ酸残基に亘り 67%の一致度 で、 また （ i i ) データベース登録記号 A F 387513、 k i n a s e— d e f i c i e n t T G b e t a s u p e r f am i l y r e c e p t o r s u b un i t力 e_v a 1 u e : 1 X 10—48、 1 53ァミノ酸残基に亘り 67 % の一致度で、さらに（ i i i)データベース登録記号 B CO 19378、Mu s m u s c u 1 u s , BMP a n d a c t i v i n memb r a n e— b o u n d i nh i b i t o r, h omo 1 o g (X e n o p u s l a e v i s) が、 e _ v a 1 u e ： IX 10— 48、 153アミノ酸残基に亘り 68 %の一致度でヒ ットした。 これらの結果より、配列番号 1に示す塩基配列がコードするタンパク質 は TGF ]3受容体フアミリーとの結合活性を有し、 k i n a s e— d e f i c i e n t TGF b e t a s u p e r f am i l y r e c e p t o r s u b u n i tであることが推測された。 dnafo rm51839 consists of 1156 bases as shown in SEQ ID NO: 1. A homology search was performed for the amino acid sequence encoded by SEQ ID NO: 1 using BLAST, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). , (i) database registration mark AJ 277487, Homo sapiensputativetran smemb raneprotein NMA precursor force ev alue: 3 X l 0- 70 , 138 with 67% degree of coincidence over the amino acid residues, also (ii) a database registration mark AF three hundred eighty-seven thousand five hundred and thirteen , kinase- deficient TG betasuperf am ilyreceptorsub un it forces e_v a 1 ue: 1 X 10- 48, 1 53 in Amino acid residue 67% degree of coincidence over the group, further (iii) a database registration mark B CO 19378, Mu smuscu 1 us, BMP andactivin memb rane- boundi nh ibitor, h omo 1 og (X enopuslaevis) is, e _ va 1 ue: were hits in the IX 10- 48, 153 68% of the degree of match over the amino acid residues. From these results, it was inferred that the protein encoded by the nucleotide sequence of SEQ ID NO: 1 had a binding activity to the TGF] 3 receptor family and was a kinase-deficient TGF betasuperfamilyreceptor subunit.
配列番号 1の塩基番号 223〜 444がコードするァミノ酸配列（配列番号 2 )、 塩基番号 450〜 869がコードするァミノ酸配列 （配列番号 3 ) に対して、 上記 With respect to the amino acid sequence encoded by base numbers 223 to 444 of SEQ ID NO: 1 (SEQ ID NO: 2) and the amino acid sequence encoded by base numbers 450 to 869 (SEQ ID NO: 3),
( i) および （ i i) の相同性がみられた。 本遺伝子がコードするタンパク質は、 配列番号 1の塩基番号 218〜869の領域から翻訳されると考えられる力 塩基 番号 221の数塩基前後、塩基番号 447の数塩基前後に 1塩基欠失もしくは 2塩 基の挿入があると考えられた。 T G F 受容体フアミリーは、骨形成、自己免疫疾患、腎臓の繊維化、肺繊維症、 心筋梗塞等に関連しており、この受容体の活性化剤または抑制剤等はこれらの疾患 の治療薬として開発できると推測された。 The homology of (i) and (ii) was observed. The protein encoded by this gene is considered to be translated from the region of base number 218 to 869 in SEQ ID NO: 1.A few bases deleted at about several bases at base number 221 and about a few bases at base number 447 or two salts It was thought that there was an insertion of the group. TGF receptor families are associated with bone formation, autoimmune diseases, renal fibrosis, pulmonary fibrosis, myocardial infarction, etc., and activators or inhibitors of this receptor may be used as therapeutics for these diseases. It was speculated that it could be developed.
(2) d n a f o rm34810 (配列番号 12、 14)  (2) d n a f o rm34810 (SEQ ID NOs: 12, 14)
d n a f o rm34810は、 配列番号 12に示すように、 3294塩基から成り、 そのうち塩基番号 95から 976までがオープンリーディングフレーム （終止コドンを 含む） になっていた。 オープンリーディングフレームから予測されるアミノ酸配列 は、 293アミノ酸残基から成る （配列番号 14)。 配列番号 12がコードするァ ミノ酸配列について B LASTを用いて相同性検索を行ったところ、 S PTR蛋白 質データベース （swi S S— PROT蛋白質配列データベースと T r EMB L 核酸翻訳データベースを統合したもの） 中に、 （ i) データベース登録記号 AB016237、 lectin- like oxidized LDL receptor (Oryctolagus cuniculus) 力 e— v a l u e : 1 X 1 0"2\ 235ァミノ酸残基に亘り 29 %の一致度で、 また ( i i ) データベース登録記号 BC022295、 oxidised low density lipoprotein (lectin-like) receptor 1 (Homo sapiens) 力 s、 e— v a l u e : 2 X 10— 18、 2 37アミノ酸残基に亘り 27%の一致度でヒットした。 これらの結果より、配列番 号 12に示す塩基配列がコードするタンパク質、または配列番号 14に示したアミ ノ酸配列からなるタンパク質は、 変性 LDLとの結合活性を有し、 変性 LDL受容体 の 1種であることが推測された。 As shown in SEQ ID NO: 12, dnafo rm34810 consists of 3294 bases, of which base numbers 95 to 976 constitute an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 293 amino acid residues (SEQ ID NO: 14). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 12 using BLAST, and a SPTR protein database (swi SS-PROT protein sequence database and a TrEMBL nucleic acid translation database were integrated). Among them, (i) database registration code AB016237, lectin-like oxidized LDL receptor (Oryctolagus cuniculus) force e— value: 1 X 10 " 2 \ 235 29% identity over amino acid residues, and (ii ) database registration mark BC022295, oxidised low density lipoprotein (lectin -like) receptor 1 (Homo sapiens) force s, e- value: hit in the 27% degree of coincidence over the 2 X 10- 18, 2 37 amino acid residues. From these results, it can be seen that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 12 or the protein consisting of the amino acid sequence shown in SEQ ID NO: 14 has a binding activity with denatured LDL. Speculated to be a species It was.
変性 LDL受容体のファミリーは、 酸化 LDLゃァセチル化 LDL等の細胞内への取りこ みや細胞機能障害に関係しており、 このフアミリーのレセプターの活性化剤、阻害 剤等は、 高脂血症、 動脈硬化の発症 ·進展、 粥状動脈硬化、 動脈硬化を伴う遺伝疾 患、 家族性高コレステロール血症、 心筋梗塞、脳梗塞等の様々な治療薬として有用 であると推測された。  The family of denatured LDL receptors is involved in the incorporation of oxidized LDL-acetylated LDL into cells and cell dysfunction.Activators and inhibitors of this family receptor are hyperlipidemia. It was speculated that it would be useful as a therapeutic agent for various diseases such as the onset and progression of arteriosclerosis, atherosclerosis, genetic diseases associated with arteriosclerosis, familial hypercholesterolemia, myocardial infarction and cerebral infarction.
(3) d n a f o rm35841 (配列番号 13、 15)  (3) d n a f o rm35841 (SEQ ID NOs: 13, 15)
d n a f o rm35841は、配列番号 13に示すように、 1021塩基から成 り、そのうち塩基番号 94から 810までがオープンリーディングフレーム （終止 コドンを含む） になっていた。 オープンリーディングフレームから予測されるアミ ノ酸配列は、 238ァミノ酸残基から成る （配列番号 15)。 配列番号 13がコー ドするアミノ酸配列について BLASTを用いて相同性検索を行つたところ、 S P TR蛋白質データベース （SWI S S—P ROT蛋白質配列データベースと T r EMB L核酸翻訳データベースを統合したもの） 中に、 （ i ) データベース登録記 号 AB016237、 lectin— like oxidized LDL receptor (Oryctolagus cunicu丄 usノ 、 e - v a 1 u e ： 1 10— 19、 235アミノ酸残基に亘り 28%の一致度で、 またAs shown in SEQ ID NO: 13, dnafo rm35841 consists of 1021 bases, of which base Nos. 94 to 810 have an open reading frame (terminated). (Including codons). The amino acid sequence predicted from the open reading frame consists of 238 amino acid residues (SEQ ID NO: 15). A homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 13, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). , (i) database registration Symbol AB016237, lectin- like oxidized LDL receptor ( Oryctolagus cunicu丄us Roh, e - va 1 ue: at 28% of the degree of match over one 10- 19, 235 amino acid residues, also
( i i ) データベース登録記号 BC022295、 oxidised low density lipoprotein (lectin- like) receptor 1 (Homo sapiens) ゝ、 e— v a 1 u e ： 6X 10一18、 2 35ァミノ酸残基に亘り 26 %の一致度でヒットした。 これらの結果より、配列番 号 1 3に示す塩基配列がコードするタンパク質、または配列番号 15に示したアミ ノ酸配列からなるタンパク質は、 変性 LDLとの結合活性を有し、 変性 LDL受容体 の 1種であることが推測された。 (Ii) a database registration mark BC022295, oxidised low density lipoprotein (lectin- like) receptor 1 (Homo sapiens)ゝ, e- va 1 ue: in 6X 10 one 18, 2 35 Amino acid residue 26% degree of coincidence over the base It was hit. From these results, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 13 or the protein consisting of the amino acid sequence shown in SEQ ID NO: 15 has a binding activity with denatured LDL, and It was speculated that it was one.
変性 LDL受容体のフアミリ一は、 酸化 LDLゃァセチル化 LDL等の細胞内への取りこ みや細胞機能障害に関係しており、 このフアミリーのレセプターの活性化剤、 阻害 剤等は高脂血症、動脈硬化の発症 ·進展、粥状動脈硬化、動脈硬化を伴う遺伝疾患、 家族性高コレステロール血症、心筋梗塞、脳梗塞等の様々な治療薬として有用であ ると推測された。  The family of modified LDL receptors is involved in the uptake of cells such as oxidized LDL-acetylated LDL and cell dysfunction.Activators and inhibitors of this family receptor are hyperlipidemia. It was speculated that it would be useful as a therapeutic agent for various diseases such as the onset and progression of arteriosclerosis, atherosclerosis, genetic diseases associated with arteriosclerosis, familial hypercholesterolemia, myocardial infarction and cerebral infarction.
(4) dn a f o rm26225 (配列番号 16、 29)  (4) dn a f o rm26225 (SEQ ID NOS: 16, 29)
d n a f o r m26225は、 配列番号 16に示すように、 2656塩基から成り、 その うち塩基番号 163から 2520までがオープンリーディングフレーム （終止コドンを含 む） になっていた。 オープンリーディングフレームから予測されるアミノ酸配列は、 785アミノ酸残基から成る （配列番号 29)。配列番号 16がコードするアミノ酸配 列について B LASTを用いて相同性検索を行ったところ、 S PTR蛋白質データ ベース （SWI S S— P ROT蛋白質配列データベースと T r EMB L核酸翻訳 データベースを統合したもの） 中に、 （ i) データベース登録記号  As shown in SEQ ID NO: 16, dnaform26225 was composed of 2656 bases, of which nucleotides 163 to 2520 were an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 785 amino acid residues (SEQ ID NO: 29). A homology search was performed for the amino acid sequence encoded by SEQ ID NO: 16 using BLAST, and a SPTR protein database (SWISS—PROT protein sequence database and TrEMBL nucleic acid translation database integrated) In the (i) database registration symbol
trembl | U787351 HSU78735_1, human ATP- BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP - BINDING CASSETTE TRANSPORTER 3)が、 e _ v a 1 u e : 0、 791アミノ酸 残基に亘り 46%の一致度で 、 （ii) trerablnew | AY0836161 AY083616_1, mouse ATP - binding cassette transporter ABCA3力 E - value=0、 791アミノ酸残基に亘 り 46%の一致度で、 （iii) データベース登録記号 trembl|X75926|MMABCl— 1 mouse ATP-binding cassette transporter 1が、 E- value=5 X 10— 、 791アミノ酸残基に 亘り 37%の一致度でヒットした。 trembl | U787351 HSU78735_1, human ATP- BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER 3), e_va 1 ue: 0, 791 amino acid residues with 46% identity, (ii) trerablnew | AY0836161 AY083616_1, mouse ATP-binding cassette transporter ABCA3 force E- value = 0, with 46% identity over 791 amino acid residues. (iii) Database entry symbol trembl | X75926 | MMABCl—1 mouse Hits were found with 37% identity over amino acid residues.
また、配列番号 16に示す塩基配列がコードするアミノ酸配列について、 HMM PF AMによる蛋白質特徴検索を行ったところ、 ABC transporterの特徴を示す配 列 （P f a mに ABC— t r a nとしてエントリーされる配列） が見出された。 これらのこと力 ら、 配列番号 16に示した塩基配列がコードするタンパク質は human ATP- BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP- BINDING CASSETTE TRANSPORTER 3)類似の配列を有し、 AT P結合性運搬体活性を有すると推測された。 このことから、配列番号 16に示す塩基配列がコードするタンパク質が薬物の細胞 外排出等に関わる ABCトランスポーターであり、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送等にかかわることが推測された。 (5) d n a f o r m41412 (配列番号 17、 30)  When the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 16 was searched for protein characteristics by HMM PFAM, a sequence showing the characteristics of ABC transporter (sequence entered as ABC-tran in P fam) was found. Was found. From these facts, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 16 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER 3), and has ATP binding. Presumed to have sex transporter activity. Thus, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 16 is an ABC transporter involved in the extracellular excretion of drugs, etc., and foreign substances such as drugs, and endogenous substances such as calcium, phospholipids and amphiphiles. It was speculated that this would be involved in the transportation of refuse. (5) dnaform41412 (SEQ ID NOS: 17, 30)
d n a f o rm41412は、 配列番号 1 7に示すように、 3831塩基から成り、 その うち塩基番号 1817から 3829までがオープンリーディングフレームになっていた。ォ ープンリーディングフレームから予測されるアミノ酸配列は、 671アミノ酸残基か ら成る （配列番号 30)。 配列番号 1 7がコードするアミノ酸配列について BLA STを用いて相同性検索を行ったところ、 S PTR蛋白質データベース （SWI S1 S— PROT蛋白質配列データベースと T r EMB L核酸翻訳データベースを統 合したもの） 中に、 （ i ) データベース登録記号 trembl | AJ2759731 HSA275973— 1、 ATP— binding cassette protein or the (ABし A subfamily) product力、 e— v a 1 u e : 0、 671ァミノ残基に亘り 90%の一致度で 、 (ii) As shown in SEQ ID NO: 17, dnafo rm41412 was composed of 3831 bases, of which base numbers 1817 to 3829 were open reading frames. The amino acid sequence predicted from the open reading frame consists of 671 amino acid residues (SEQ ID NO: 30). When SEQ ID NO: 1 7 Homology searches were performed using the BLA ST for the amino acid sequence encoded by that combined S PTR protein database (SWI S 1 S- PROT protein sequence database and T r EMB L nucleic translation database integration (I) Database registration symbol: trembl | (Ii)
tremblne | AY0288971 AY028897_1, human ATP - binding cassette transporter ABCA3 力 E- value=0、 671アミノ酸残基に亘り 90%の一致度で、 （iii)データベース登録 記号 trembl | AK0565331 AK056533_1 Homo sapiens cDNA FLJ31971 fis, clone NT2RP7008137, weakly similar to ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 1.力 E- value=5X 10—117、 417ァミノ酸残基に亘り 87%の一致度でヒットした。 tremblne | AY0288971 AY028897_1, human ATP-binding cassette transporter ABCA3 force E-value = 0, with 90% identity over 671 amino acid residues, (iii) database registration Symbol trembl | AK0565331 AK056533_1 Homo sapiens cDNA FLJ31971 fis, clone NT2RP7008137, weakly similar to ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 1. force E- value = 5X 10- 117, 417 Amino acid residues 87% over Hits with the degree of match.
また、配列番号 1 7に示す塩基配列がコードするアミノ酸配列について、 HMM P F AMによる蛋白質特徴検索を行ったところ、 ABC transporterの特徴を示す配 列 （P f amに ABC_t r a nとしてエントリーされる配列） が見出された。 これらのことから、 配列番号 1 7に示した塩基配列がコードするタンパク質は human ATP- BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP- BINDING CASSETTE TRANSPORTER 3)類似の配列を有し、 AT P結合性運搬体活性を有すると推測された。 このことから、配列番号 17に示す塩基配列がコードするタンパク質が薬物の細胞 外排出等に関わる ABCトランスポーターであり、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送等にかかわることが推測された。 (6) d n a f o r m43395 (配列番号 18、 31)  In addition, when the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 17 was searched for protein characteristics by HMM PFAM, a sequence showing the characteristics of ABC transporter (sequence entered as ABC_tran in P f am) Was found. From these facts, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 17 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER 3), and has ATP binding. Presumed to have sex transporter activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 17 is an ABC transporter involved in extracellular excretion of drugs, and foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphiphiles. It was speculated that this would be involved in the transportation of refuse. (6) dnaform43395 (SEQ ID NOS: 18, 31)
d n a f o r m43395は、 配列番号 18に示すように、 3950塩基から成り、 その うち塩基番号 201から 3950までがオープンリーディングフレームになっていた。 ォ ープンリーディングフレームから予測されるアミノ酸配列は、 1250ァミノ酸残基か ら成る （配列番号 31)。 配列番号 18がコードするアミノ酸配列について B L A STを用いて相同性検索を行ったところ、 S PTR蛋白質データベース （SWI S S— P ROT蛋白質配列データベースと T r EMB L核酸翻訳データベースを統 合したもの） 中に、 （ i ) データベース登録記号 trembl | AJ2759731 HSA275973— 1、 human ATP - binding cassette protein of the (ABCA subfamily) product力、 e _v a 1 u e : 0、 1250アミノ酸残基に亘り 89%の一致度で、 （ii)  As shown in SEQ ID NO: 18, dnaform43395 was composed of 3950 bases, of which base numbers 201 to 3950 were open reading frames. The amino acid sequence predicted from the open reading frame consists of 1250 amino acid residues (SEQ ID NO: 31). A homology search was performed for the amino acid sequence encoded by SEQ ID NO: 18 using BLAST. The results were in the S PTR protein database (SWI SS—PROT protein sequence database and Tr EMB L nucleic acid translation database). (I) Database registration symbol trembl | AJ2759731 HSA275973—1, human ATP-binding cassette protein of the (ABCA subfamily) product power, e_va 1 ue: 0, with 1250 amino acid residues with 89% concordance , (Ii)
trerablnew | AY0288971 AY028897_1, Homo sapiens ATP - binding cassette A5力 E - value=0、 1250アミノ酸残基に亘り 89%の一致度で、 （iii) データベース登録記 号 tremblnew|AY028899|AY028899— 1, Homo sapiens ATP- binding cassette A9 が、 E_value=0、 1251ァミノ酸残基に亘り 38%の一致度でヒットした。 trerablnew | AY0288971 AY028897_1, Homo sapiens ATP-binding cassette A5 force E-value = 0, 89% identity over 1250 amino acid residues, (iii) Database registration symbol tremblnew | AY028899 | AY028899—1, Homo sapiens ATP -Binding cassette A9 hit with 38% identity over E_value = 0, 1251 amino acid residues.
また、配列番号 18に示す塩基配列がコードするアミノ酸配列について、 HMM P F AMによる蛋白質特徴検索を行ったところ ABC transporterの特徴を示す配列 (P f an^ ABC—t r a nとしてェントリーされる配列） が見出された。 In addition, the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 18 A protein characteristic search by PFAM was performed, and a sequence showing the characteristics of ABC transporter (sequence that was entered as Pfan ^ ABC-tran) was found.
これらのことから、 配列番号 18に示した塩基配列がコードするタンパク質は human ATP- BINDING CASSETTE, SUB- FAMILY A類似の配列を有し、 ATP結合性運搬 体活性を有すると推測された。 このことから、配列番号 18に示す塩基配列がコー ドするタンパク質が薬物の細胞外排出等に関わる A BCトランスポーターであり、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送等に かかわることが推測された。  From these facts, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 18 had a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A and had ATP-binding carrier activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 18 is an ABC transporter involved in extracellular excretion of drugs, etc., and foreign substances such as drugs, and intrinsic factors such as calcium, phospholipids, amphiphiles, etc. It was speculated that it might be involved in the transport of toxic substances.
(7) d n a f o r m33133 (配列番号 19、 32)  (7) dnaform33133 (SEQ ID NOs: 19 and 32)
d n a f o rm33133は、 配列番号 19に示すように、 4119塩基から成り、 その うち塩基番号 68から 2914までがオープンリーディングフレーム （終止コドンを含 む）になっていた。 オープンリーディングフレームから予測されるアミノ酸配列は、 948ァミノ酸残基から成る （配列番号 32)。配列番号 19がコードするアミノ酸配 列について B LASTを用いて相同性検索を行ったところ、 S PTR蛋白質データ ベース （SWI S S_ PRO T蛋白質配列データベースと T r EMBL核酸翻訳 データベースを統合したもの） 中に、 （i) データベース登録記号  As shown in SEQ ID NO: 19, dnaform33133 was composed of 4119 bases, of which base numbers 68 to 2914 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 948 amino acid residues (SEQ ID NO: 32). A homology search was performed for the amino acid sequence encoded by SEQ ID NO: 19 using BLAST. The results were in the SPTR protein database (integrating the SWI S_PROT protein sequence database and the TrEMBL nucleic acid translation database). (I) Database registration symbol
tremblnew | AY0288991 AY028899_1 , Homo sapiens ATP - binding cassette A9力 e 一 v a 1 u e : 0、 948アミノ酸残基に亘り 79%の一致度で、 （ii) tremblnew | AY0288991 AY028899_1, Homo sapiens ATP-binding cassette A9 e e v a 1 u e: 0, 79% identity over 948 amino acid residues, (ii)
trembl | AB0206291 AB020629_1, KIM0822が、 E- value=0、 948アミノ酸残基に亘り 67%の一致度で、 （iii) データベース登録記号 tremblnew |AY028900|AY028900—1， Homo sapiens ATP—binding cassette A10力 E— value=0、 958アミノ酸残基 (こ互り 60%の一致度でヒットした。 trembl | AB0206291 AB020629_1, KIM0822 has E-value = 0, 67% identity over 948 amino acid residues, and (iii) database registration symbol tremblnew | AY028900 | AY028900-1, Homo sapiens ATP—binding cassette A10 — Value = 0, 958 amino acid residues (there were hits with a 60% match.
また、配列番号 1 9に示す塩基配列がコードするアミノ酸配列について、 HMM PF AMによる蛋白質特徴検索を行ったところ、 ABC transporterの特徴を示す配 列 （P f amに ABC— t r a nとしてエントリーされる配列） が見出された。 これらのことから、 配列番号 19に示した塩基配列がコードするタンパク質は human ATP- BINDING CASSETTE, SUB-FAMILY A9類似の配列を有し、 AT P結合性運 搬体活性を有すると推測された。 このことから、配列番号 19に示す塩基配列がコ 一ドするタンパク質が薬物の細胞外排出等に関わる ABCトランスポーターであ り、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送 等にかかわることが推測された。 When the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 19 was searched for protein characteristics using HMM PFAM, a sequence showing the characteristics of ABC transporter (a sequence entered as ABC-tran in P f am) ) Was found. From these facts, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 19 has a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A9, and has Presumed to have carrier activity. From this, the protein whose nucleotide sequence is shown in SEQ ID NO: 19 is an ABC transporter involved in the extracellular excretion of drugs, etc., and foreign substances such as drugs, calcium, phospholipids, amphiphilic substances, etc. It was speculated that it might be involved in the transport of endogenous substances.
(8) d n a f o r m63577 (配列番号 20、 33)  (8) dnaform63577 (SEQ ID NOs: 20, 33)
d n a f o r m63577は、 配列番号 20に示すように、 3300塩基から成り、 その うち塩基番号 1382から 2458までがオープンリーディングフレーム （終止コドンを 含む） になっていた。 オープンリーディングフレームから予測されるアミノ酸配列 は、 358アミノ酸残基から成る （配列番号 33)。配列番号 20がコードするァミノ 酸配列について B LASTを用いて相同性検索を行ったところ、 S PTR蛋白質デ ータベース （SWI S S_P ROT蛋白質配列データベースと T r EMBL核酸 翻訳データベースを統合したもの） 中に、 （ i) データベース登録記号  As shown in SEQ ID NO: 20, dnaform63577 was composed of 3300 bases, and among them, bases from 1382 to 2458 had an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 358 amino acid residues (SEQ ID NO: 33). A homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 20, and it was found in the SPTR protein database (integrated SWI S_P ROT protein sequence database and TrEMBL nucleic acid translation database). , (I) Database registration symbol
trembl | AK0570681 AK057068_1, Homo sapiens cDNA FLJ32506 fis, clone trembl | AK0570681 AK057068_1, Homo sapiens cDNA FLJ32506 fis, clone
SMINT1000042, weakly similar to ATP - BINDING CASSETTE, SUB-FAMILY A, MEMBER 3が、 e - v a 1 u e : 5X10—143、 332アミノ酸残基に亘り 75%の一致度で 、 (ii) tremblnew | AY0288991 AY028899_1, Homo sapiens ATP - binding cassette A9が、 E - value=5Xl(T143、 332アミノ酸残基に亘り 75%の一致度で、 （iii) データベース登 録記号 tremblnew | AY0289001 AY028900_1, Homo sapiens ATP-binding cassette A10が、 E-value=5Xl(T124、 330アミノ酸残基に亘り 65%の一致度でヒットした。 また、配列番号 20に示す塩基配列がコ一ドするアミノ酸配列について、 HMM PFAMによる蛋白質特徴検索を行ったところ、 ABC transporterの特徴を示す配 列 （P f amに ABC— t r a nとしてエントリーされる配列） が見出された。 これらのこと力ゝら、 配列番号 20に示した塩基配列がコードするタンパク質は human ATP- BINDING CASSETTE, SUB-FAMILY A類似の配列を有し、 ATP結合性運搬 体活性を有すると推測された。 このことから、配列番号 20に示す塩基配列がコ一 ドするタンパク質が薬物の細胞外排出等に関わる ABCトランスポーターであり、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送等に 力かわることが推測された。 SMINT1000042, weakly similar to ATP - BINDING CASSETTE, SUB-FAMILY A, is MEMBER 3, e - va 1 ue : 5X10- 143, 75% degree of coincidence over the 332 amino acid residues, (ii) tremblnew | AY0288991 AY028899_1 , Homo sapiens ATP - binding cassette A9 is, E - value = at 5Xl (T 143, 332 75% degree of coincidence over the amino acid residues, (iii) a database registration symbol tremblnew | AY0289001 AY028900_1, Homo sapiens ATP -binding cassette A10 but hit in E-value = 5Xl (T 124 , 330 65% degree of coincidence over the amino acid residues. Furthermore, the amino acid sequence nucleotide sequence is co one de shown in SEQ ID NO: 20, proteins wherein the search by HMM PFAM As a result, a sequence exhibiting the characteristics of ABC transporter (sequence that is entered as ABC-tran in P f am) was found. Proteins have sequences similar to human ATP-BINDING CASSETTE, SUB-FAMILY A Therefore, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 20 is an ABC transporter involved in the extracellular excretion of the drug, and the like. And transport of endogenous substances such as calcium, phospholipids and amphiphiles It was speculated that it would be overpowered.
(9) d n a f o r m 30449 (配列番号 21、 34)  (9) dnaform30449 (SEQ ID NOS: 21, 34)
d n a f o r m30449は、 配列番号 21に示すように、 2844塩基から成り、 その うち塩基番号 397から 2235までがオープンリーディングフレーム （終止コドンを含 む）になっていた。 オープンリーディングフレームから予測されるアミノ酸配列は、 612アミノ酸残基から成る （配列番号 34)。配列番号 2 1がコードするアミノ酸配 列について BLASTを用いて相同性検索を行ったところ、 S PTR蛋白質データ ベース （SWI S S— P ROT蛋白質配列データベースと T r EMBL核酸翻訳 データベースを統合したもの） 中に、 （ i) データベース登録記号  As shown in SEQ ID NO: 21, dnaform30449 was composed of 2844 bases, of which base numbers 397 to 2235 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 612 amino acid residues (SEQ ID NO: 34). A homology search was performed for the amino acid sequence encoded by SEQ ID NO: 21 using BLAST. The results were in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). (I) Database registration symbol
gpnew I BC026496120071480, Mus musculus, clone IMAGE :4505946力 e - v a 1 u e ： 0、 591アミノ酸残基に亘り 100%の一致度で 、 （ii) trembl | AB0206291 AB020629 — 1, Homo sapiens cDNA KIM0822.が、 E-value=0、 587アミノ酸残基に亘り 73% の一致度で、 （iii) データベース登録記号 tremblnew|AY028899|AY028899—l, Homo sapiens ATP— binding cassette A9力 E_value=0、 590アミノ酸残基 tこ互り 68%の一致度でヒットした。 gpnew I BC026496120071480, Mus musculus, clone IMAGE: 4505946 force e-va 1 ue: 0, 591 amino acid residues with 100% identity, (ii) trembl | AB0206291 AB020629 — 1, Homo sapiens cDNA KIM0822. E-value = 0, 73% identity over 587 amino acid residues, (iii) Database registration code tremblnew | AY028899 | AY028899-l, Homo sapiens ATP—binding cassette A9 force They hit with a 68% match.
また、配列番号 21に示す塩基配列がコードするアミノ酸配列について、 HMM PFAMによる蛋白質特徴検索を行ったところ ABC transporterの特徴を示す配列 (P f an^ ABC—t r a nとしてェントリーされる配列） が見出された。  In addition, a protein characteristic search using HMM PFAM was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 21, and a sequence showing the characteristics of ABC transporter (sequence entry as Pfan ^ ABC-tran) was found. Was done.
これらのこと力、ら、 配列番号 21に示した塩基配列がコードするタンパク質は human ATP- BINDING CASSETTE, SUB- FAMILY A9類似の配列を有し、 ATP結合性運 搬体活性を有すると推測された。 このことから、配列番号 21に示す塩基配列がコ 一ドするタンパク質が薬物の細胞外排出等に関わる ABCトランスポーターであ り、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送 等にかかわることが推測された。  Thus, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 21 had a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A9, and was presumed to have ATP-binding carrier activity. . From this, the protein whose base sequence shown in SEQ ID NO: 21 is encoded is an ABC transporter involved in the extracellular excretion of a drug, such as a foreign substance such as a drug, calcium, phospholipid, and an amphipathic substance. It was speculated that it might be involved in the transport of endogenous substances.
(10) d n a f o r m42393 (配列番号 22、 35)  (10) dnaform42393 (SEQ ID NOS: 22, 35)
d n a f o rm42393は、 配列番号 22に示すように、 2537塩基から成り、 その うち塩基番号 163から 1674までがオープンリーディングフレーム （終止コドンを含 む） になっていた。 オープンリーディングフレームから予測されるアミノ酸配列は、As shown in SEQ ID NO: 22, dnafo rm42393 consists of 2537 bases, of which base numbers 163 to 1674 have an open reading frame (including a stop codon). Mu). The amino acid sequence predicted from the open reading frame is
503アミノ酸残基から成る （配列番号 35) 。 配列番号 22がコードするアミノ酸 配列について B LASTを用いて相同性検索を行ったところ、 S PTR蛋白質デー タベース （SWI S S— PROT蛋白質配列データベースと T r EMB L核酸翻 訳データベースを統合したもの） 中に、 （i) データベース登録記号 It consists of 503 amino acid residues (SEQ ID NO: 35). A homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 22. The results were in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). (I) Database registration symbol
tremblnew | AY0836161 AY083616_1 , Mus musculus ATP - binding cassette transporter ABCA3が、 e— v a 1 u e : 5X1(T10°、 489アミノ酸残基に亘り 41%の 一致度で、 （ii) tremblnew | AB0709291 AB070929_1, lamellar body membrane specific ATP— binding cassette protein (ABCA3)力 S、 E-value=2 X 10"96, 489アミノ 酸残基に亘り 40%の一致度で、 （iii) データベース登録記号 tremblnew | AY0836161 AY083616_1, Mus musculus ATP-binding cassette transporter ABCA3, e- va 1 ue: 5X1 (T 10 °, 41% identity over 489 amino acid residues, (ii) tremblnew | AB0709291 AB070929_1, lamellar body membrane specific ATP- binding cassette protein (ABCA3 ) force S, at E-value = 2 X 10 " 96, 40% of the degree of coincidence over the 489 amino acid residues, (iii) a database registration mark
trembl | U787351 HSU78735_1, Human ABC3が、 E-value=l X 10"9\ 489アミノ酸残基 に亘り 40%の一致度でヒットした。 trembl | U787351 HSU78735_1, Human ABC3 was hit with 40% identity over E-value = l X 10 " 9 \ 489 amino acid residues.
また、配列番号 22に示す塩基配列がコードするアミノ酸配列について、 HMM P F AMによる蛋白質特徴検索を行ったところ ABC transporterの特徴を示す配列 The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 22 was searched for protein characteristics using HMM PFAM.
(P f an^ ABC—t r a nとしてエントリーされる配列） が見出された。 (P f an ^ ABC—sequence entered as tran) was found.
これらのこと力ゝら、 配列番号 22に示した塩基配列がコードするタンパク質は human ATP-BINDING CASSETTE, SUB- FAMILY A類似の配列を有し、 ATP結合性運搬 体活性を有すると推測された。 このことから、配列番号 22に示す塩基配列がコー ドするタンパク質が薬物の細胞外排出等に関わる ABCトランスポーターであり、 薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送等に かかわることが推測された。  From these facts, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 22 had a sequence similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, and had ATP-binding carrier activity. Therefore, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 22 is an ABC transporter involved in the extracellular excretion of drugs, etc., and foreign substances such as drugs, endogenous substances such as calcium, phospholipids, amphiphiles, etc. It was presumed to be involved in the transport of substances.
(1 1) d n a f o r m39112 (配列番号 23、 36)  (1 1) d n a f o r m39112 (SEQ ID NOS: 23 and 36)
d n a f o r m39112は、 配列番号 23に示すように、 2247塩基から成り、 その うち塩基番号 940から 2247までがオープンリーディングフレームになっていた。 ォ 一プンリ一ディングフレームから予測されるアミノ酸配列は 436アミノ酸残基から 成る （配列番号 36)。 配列番号 23がコードするアミノ酸配列について BLAS Tを用いて相同性検索を行ったところ、 S PTR蛋白質データベース （SWI S S 一 P R O T蛋白質配列データベースと Tr EMBL核酸翻訳データベースを統合 したもの） 中に、 （i) データベース登録記号 tremblnew|AL356785|AL356785—l、 human ATP11C gene for ATPase, Class VI, type 11Cが、 e— v a 1 u e=0、 436 アミノ酸残基に亘り 91%の一致度で、 （ii) trembl |AF1565511 AF156551_1, As shown in SEQ ID NO: 23, dnafor m39112 was composed of 2247 bases, of which base numbers 940 to 2247 were open reading frames. The amino acid sequence predicted from the open reading frame consists of 436 amino acid residues (SEQ ID NO: 36). A homology search was performed for the amino acid sequence encoded by SEQ ID NO: 23 using BLAST. (A combination of the PROT protein sequence database and the Tr EMBL nucleic acid translation database). ue = 0, 91% identity over 436 amino acid residues, (ii) trembl | AF1565511 AF156551_1,
Potential phospholipid-transporting ATPase IH力 E - value=5 X 10— 155、 449アミ ノ酸残基に亘り 60%の一致度で、 （iii) データベース登録記号 Potential phospholipid-transporting ATPase IH force E-value = 5 X 10— 155 , 449 amino acid residues with 60% identity, (iii) Database registration code
trembl | AB028944 | AB028944_1, Potential phospholipid-transporting ATPase IS が、 Ε=5Χ1(Γ127、 375アミノ酸残基に亘り 40%の一致度でヒットした。 trembl | AB028944 | AB028944_1, Potential phospholipid -transporting ATPase IS is found as 40% of the degree of coincidence over the Ε = 5Χ1 (Γ 127, 375 amino acid residues.
これらの結果より、配列番号 23に記載の塩基配列がコードするアミノ酸配列か らなるタンパク質が運搬体 AT P a s eファミリー類似の配列を有し、 ATP結合 性運搬体活性を有すると推測された。 このことから、配列番号 23に示す塩基配列 がコードするタンパク質が運搬体 AT P a s eフアミリーであり、薬物等の異物や、 カノレシゥム、 リン脂質、 両親媒性物質等の内因性物質の輸送等にかかわることが推 測された。  From these results, it was inferred that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 23 had a sequence similar to the ATPase family of transporters and had ATP-binding transporter activity. From this fact, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 23 is a carrier ATPase family, which is involved in the transport of foreign substances such as drugs and endogenous substances such as canolesum, phospholipids and amphiphiles. It was inferred.
(1 2) d n a f o r m43238 (配列番号 24、 37)  (1 2) d n a f o r m43238 (SEQ ID NOS: 24 and 37)
d n a f o r m43238は、 配列番号 24に示すように、 1554塩基から成り、 その うち塩基番号 277から 1554までがオープンリーディングフレームになっていた。 ォ ープンリーディングフレームから予測されるアミノ酸配列は、 426アミノ酸残基か ら成る （配列番号 37)。 配列番号 24がコードするアミノ酸配列について B LA STを用いて相同性検索を行ったところ、 S PTR蛋白質データベース （SWI S S— PROT蛋白質配列データベースと T r EMB L核酸翻訳データベースを統 合したもの） 中に、 （ i )データベース登録記号 tremblnew|AB075819|AB075819—l、 human KIM1939が、 e— v a 1 u e ： 5X10— 155、 489ァミノ酸残基に亘り 85%の一致 度で 、 （ii) trembl | AK0548861 AK054886_1, Homo sapiens cDNA FLJ30324weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3が、 E- value=5 X 1(T154、 489 アミノ酸残基に亘り 78%の一致度で、 （iii) データベース登録記号 As shown in SEQ ID NO: 24, dnaform m43238 was composed of 1554 bases, of which base numbers 277 to 1554 were open reading frames. The amino acid sequence predicted from the open reading frame consists of 426 amino acid residues (SEQ ID NO: 37). A homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 24. The results were in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). to, (i) a database registration mark tremblnew | AB075819 | AB075819-l, is human KIM1939, e- va 1 ue: 5X10- 155, 489 Amino 85% degree of coincidence over the acid residues, (ii) trembl | AK0548861 AK054886_1, Homo sapiens cDNA FLJ30324weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3 is, E- value = at 5 X 1 (T 154, 489 78% degree of coincidence over the amino acid residues, (iii) a database registration mark
trembl | AF0380071 AF038007_1, Potential phospholipid-transporting ATPase IC が、 E- value=5Xl(T136、 489アミノ酸残基に亘り 57% の一致度でヒットした。 trembl | AF0380071 AF038007_1, Potential phospholipid-transporting ATPase IC But hit in E- value = 5Xl (T 136, 489 57% degree of coincidence over the amino acid residues.
また、配列番号 24に示す塩基配列がコードするアミノ酸配列について、 HMM PF AMによる蛋白質特徴検索を行ったところ ABC transporterの特徴を示す配列 (P f an ABC—t r a nとしてェ.ントリーされる配列） が見出された。  In addition, a protein characteristic search using HMM PFAM was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 24. As a result, a sequence showing the characteristics of ABC transporter (sequence entered as Pfan ABC-tran) was found. Was found.
これらのこと力 ら、 配列番号 24に示した塩基配列がコードするタンパク質は human ATP-BINDING CASSETTE類似の配列を有し、 AT P結合性運搬体活性を有する と推測された。 このことから、配列番号 24に示す塩基配列がコードするタンパク 質が薬物の細胞外排出等に関わる ABCトランスポーターであり、薬物等の異物や、 カルシウム、 リン脂質、両親媒性物質等の内因性物質の輸送等にかかわることが推 測された。  From these results, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24 had a sequence similar to human ATP-BINDING CASSETTE and had ATP-binding carrier activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24 is an ABC transporter involved in the extracellular excretion of a drug and the like. It was estimated to be involved in the transport of substances.
(13) d n a f o r m 60061 (配列番号 25、 38)  (13) dnaform60061 (SEQ ID NOS: 25 and 38)
d n a f o r m60061は、 配列番号 25に示すように、 2825塩基から成り、 その うち塩基番号 526から 1449までがオープンリーディングフレーム （終止コドンを含 む）になっていた。 オープンリーディングフレームから予測されるアミノ酸配列は、 307ァミノ酸残基から成る （配列番号 38)。配列番号 25がコードするァミノ酸配 列について BLASTを用いて相同性検索を行ったところ、 S PTR蛋白質データ ベース （SWI S S— PROT蛋白質配列データベースと T r EMBL核酸翻訳 データベースを統合したもの） 中に、 （i) データベース登録記号  As shown in SEQ ID NO: 25, dnaform60061 was composed of 2825 bases, of which base numbers 526 to 1449 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 307 amino acid residues (SEQ ID NO: 38). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 25 using BLAST, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). , (I) Database registration symbol
tremblnew|AB075819|AB075819— 1、 humanKIM1939が、 e— v a 1 u e :5Χ1(Γ150、 300アミノ酸残基に亘り 86%の一致度で、 （ii) trembl | AF0380071 AF038007_1, Homo sapiens potential phospholipid— transporting AiPase Iし力、 E-value=5 X 10"", 309アミノ酸残基に亘り 57%の一致度で、 （iii) データベース登録記号 trembl | AE00369 1 AE003694_20, gene: "CG14741"; Drosophila melanogaster genomic scaffoldが、 E- value=8X "97、 292アミノ酸残基に亘り 60%の一致度でヒ ットした。 tremblnew | AB075819 | AB075819- 1, humanKIM1939 is, e- va 1 ue: in 5Χ1 (Γ 150, 300 86% over amino acid residues matching degree, (ii) trembl | AF0380071 AF038007_1 , Homo sapiens potential phospholipid- transporting AiPase AE3691 1 AE003694_20, gene: "CG14741"; Drosophila melanogaster genomic scaffold E-value = 8X " 97 , and 292 amino acid residues were hit with 60% concordance.
これらの結果より、配列番号 25に記載の塩基配列がコードするァミノ酸配列か らなるタンパク質が運搬体 AT P a s eファミリー類似の配列を有し、 A TP結合 性運搬体活性を有すると推測された。 このことから、配列番号 25に示す塩基配列 がコードするタンパク質が運搬体 AT P a s eフアミリーであり、薬物等の異物や、 カルシウム、 リン脂質、両親媒性物質等の内因性物質の輸送等にかかわることが推 測された。 From these results, it can be seen that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 25 has a sequence similar to the transporter ATPase family, and has ATP binding. It was presumed to have sex carrier activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 25 is a carrier ATPase family, which is involved in the transport of foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphiphiles. It was inferred.
(14) d n a f o r m49889 (配列番号 26、 39)  (14) dnaform49889 (SEQ ID NOs: 26 and 39)
d n a f o r m49889は、 配列番号 26に示すように、 4705塩基から成り、 その うち塩基番号 2015から 3883までがオープンリーディングフレーム （終止コドンを 含む）になっていた。 オープンリーディングフレームから予測されるアミノ酸配列 は、 622アミノ酸残基から成る （配列番号 39) 。 配列番号 26がコードするアミ ノ酸配列について BLASTを用いて相同性検索を行ったところ、 S PTR蛋白質 データベース （swi S S— P ROT蛋白質配列データベースと Tr EMBL核 酸翻訳データベースを統合したもの） 中に、 （ i) データベース登録記号 trembl | BC0078371 BC007837_U Homo sapiens, clone IMAGE :4111596,が、 e— v a 1 u e : 0、 600ァミノ酸残基に亘り 54%の一致度で、 （ii)  As shown in SEQ ID NO: 26, dnaform49889 was composed of 4705 bases, of which base numbers 2015 to 3883 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 622 amino acid residues (SEQ ID NO: 39). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 26 using BLAST, and it was found in the SPTR protein database (integrated swiSS-PROT protein sequence database and TrEMBL nucleic acid translation database). (I) Database registration symbol trembl | BC0078371 BC007837_U Homo sapiens, clone IMAGE: 4111596, e-va 1 ue: 0, 54% identity over 600 amino acid residues, (ii)
trembl |扁32963|崖 32963— 1, human Potential phospholipid - transporting ATPase IDが、 E- value=0、 600アミノ酸残基に亘り 53%の一致度で、 （iii) データ ベース登録記号 tremblnew|AB075819|AB075819—l,human KIM1939が、 E- value= 0、 595ァミノ酸残基に亘り 52%の一致度でヒッ トした。 trembl. -l, human KIM1 9 3 9 is, E- value = 0, and hit with 52% degree of coincidence over the 595 Amino acid residues.
これらの結果より、配列番号 26に記載の塩基配列がコードするアミノ酸配列か らなるタンパク質が運搬体 AT P a s eファミリー類似の配列を有し、 ATP結合 性運搬体活性を有すると推測された。 このことから、配列番号 26に示す塩基配列 がコードするタンパク質が運搬体 AT P a s eフアミリ一であり、薬物等の異物や、 カルシウム、 リン脂質、両親媒性物質等の内因性物質の輸送等にかかわることが推 測された。  From these results, it was inferred that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 26 had a sequence similar to the ATPase family of transporters and had ATP-binding transporter activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 26 is a carrier ATPase family, and is used for transporting foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphipathic substances. It was speculated that this would be involved.
(15) d n a f o r m60440 (配列番号 27、 40)  (15) dnaform60440 (SEQ ID NOS: 27 and 40)
d n a f o rm60440は、 配列番号 27に示すように、 2611塩基から成り、 その うち塩基番号 70から 987までがオープンリーディングフレーム （終止コドンを含 む） になっていた。 オープンリーディングフレームから予測されるアミノ酸配列は、dnafo rm60440 consists of 2611 bases, as shown in SEQ ID NO: 27, of which base numbers 70 to 987 contain an open reading frame (including a stop codon). Mu). The amino acid sequence predicted from the open reading frame is
305ァミノ酸残基から成る （配列番号 40)。配列番号 27がコードするアミノ酸配 列について B LASTを用いて相同性検索を行ったところ、 S PTR蛋白質データ ベース （SWI S S— P ROT蛋白質配列データベースと T r EMBL核酸翻訳 データベースを統合したもの） 中に、 （ i) データベース登録記号 Consists of 305 amino acid residues (SEQ ID NO: 40). A homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 27. The results were in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). (I) Database registration symbol
trembl | AK0548861 AK054886_1 Homo sapiens cDNA FLJ30324 weakly similar to PROBABLE CALCIUM— TRANSPORTING ATPASE 3,力 e - v a 1 u e : 5X10— 101、 271ァ ミノ酸残基に亘り 63%の一致度で 、 （ii) trembl | AF0380071 AF038007_1, human Potential phospholipid- transporting ATPase ICが、 E_value=l X 10— 85、 290アミ ノ酸残基に亘り 51%の一致度で、 （iii) データベース登録記号 trembl | AK0548861 AK054886_1 Homo sapiens cDNA FLJ30324 weakly similar to PROBABLE CALCIUM- TRANSPORTING ATPASE 3, Power e - va 1 ue: 5X10- 101 , 271 § Mino 63% degree of coincidence over the acid residues, (ii) trembl | AF0380071 AF038007_1, human Potential phospholipid- transporting ATPase IC is in E_value = l X 10- 85, 290 amino acid residues 51% degree of coincidence over the group, (iii) a database registration mark
trembl | AE0036941 AE003694_20, gene: "CG14741"; Drosophila melanogaster genomic scaffoldが、 E_value=2 X 10—84、 280ァミノ酸残基に亘り 55%の一致度でヒ ットした。 trembl | AE0036941 AE003694_20, gene: "CG14741"; Drosophila melanogaster genomic scaffold was then hits with 55% degree of coincidence over the E_value = 2 X 10- 84, 280 Amino acid residues.
これらの結果より、配列番号 27に記載の塩基配列がコードするァミノ酸配列か らなるタンパク質が運搬体 AT P a s eファミリー類似の配列を有し、 ATP結合 性運搬体活性を有すると推測された。 このことから、配列番号 27に示す塩基配列 がコードするタンパク質が運搬体 AT P a s eフアミリーであり、薬物等の異物や、 カルシウム、 リン脂質、両親媒性物質等の内因性物質の輸送等にかかわることが推 測された。  From these results, it was inferred that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 27 had a sequence similar to the ATPase family of carriers and had ATP-binding carrier activity. From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 27 is a carrier ATPase family, which is involved in transport of foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphipathic substances. It was inferred.
(16) dn a f o r m67267 (配列番号 28、 41)  (16) dn a for m67267 (SEQ ID NOs: 28 and 41)
d n a f o r m67267は、 配列番号 28に示すように、 1569塩基から成り、 その うち塩基番号 102から 1484までがオープンリーディングフレーム （終止コドンを含 む） になっていた。 オープンリーディングフレームから予測されるアミノ酸配列は、 460アミノ酸残基から成る （配列番号 41)。配列番号 28がコードするアミノ酸配 列について B L A S Tを用いて相同性検索を行ったところ、 SPTR蛋白質データ ベース （SWI S S— P ROT蛋白質配列データベースと T r EMBL核酸翻訳 データベースを統合したもの） 中に、 （ i) データベース登録記号 trembl | AE003694 | AE003694_20, gene: "CG14741"; Drosophila melanogaster genomic scaffold,が、 e— v a 1 u e : 5X 1(T136、 421ァミノ酸残基に亘り 56%の 一致度で、 （ii) trembl | AK0548861 AK054886_1, Potential As shown in SEQ ID NO: 28, dnaform m67267 was composed of 1569 bases, of which base numbers 102 to 1484 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 460 amino acid residues (SEQ ID NO: 41). A homology search was performed for the amino acid sequence encoded by SEQ ID NO: 28 using BLAST. (I) Database registration symbol AE003694 | AE003694_20, gene: "CG14741"; Drosophila melanogaster genomic scaffold, but e- va 1 ue: 5X 1 (T 136 , 421 amino acid residues with 56% concordance, (ii) trembl | AK0548861 AK054886_1, Potential
phospholipid-transporting ATPase ICが、 E- value=5 X 10—122、 431アミノ酸残基に 亘り 53%の一致度で、 (iii)データベース登録記号 trembl |AE003694|AE003694— 20, Homo sapiens cDNA FLJ30324 f is, clone BRACE2007138, weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3が、 E - value=2 X 10— 84、 338ァミノ酸残基に亘り 62% の一致度でヒッ トした。 phospholipid-transporting ATPase IC is, E- value = at 5 X 10- 122, 431 53% degree of coincidence over the amino acid residues, (iii) a database registration mark trembl | AE003694 | AE003694- 20, Homo sapiens cDNA FLJ30324 f is , clone BRACE2007138, weakly similar to PROBABLE CALCIUM-TRANSPORTING ATPASE 3 is, E - value = was hit with 62% degree of coincidence over the 2 X 10- 84, 338 Amino acid residues.
これらの結果より、配列番号 2 8に記載の塩基配列がコードするァミノ酸配列か らなるタンパク質が運搬体 AT P a s eファミリー類似の配列を有し、 ATP結合 性運搬体活性を有すると推測された。 このことから、配列番号 2 8に示す塩基配列 がコードするタンパク質が運搬体 AT P a s eフアミリーであり、薬物等の異物や、 カルシウム、 リン脂質、 両親媒性物質等の内因性物質の輸送等にかかわることが推 測された。  From these results, it was inferred that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 28 had a sequence similar to the transporter ATPase family and had ATP-binding transporter activity. . From this, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 28 is a carrier ATPase family, which is used for transporting foreign substances such as drugs and endogenous substances such as calcium, phospholipids and amphiphilic substances. It was speculated that this would be involved.
(1 7) d n a f o r m3 8 3 0 8 (配列番号 4 2、 4 7)  (1 7) d n a f o r m3 8 3 0 8 (SEQ ID NOs: 42, 4 7)
d n a f o r m3 8 30 8は、配列番号 4 2に示すように、 1 6 48塩基から成 り、そのうち塩基番号 4から 1 0 6 2までがオープンリーディングフレーム （終止 コドンを含む）になっていた。 オープンリーディングフレームから予測されるアミ ノ酸配列は、 3 5 2アミノ酸残基から成る （配列番号 4 7) 。 配列番号 4 2がコー ドするアミノ酸配列について B LAS Tを用いて相同性検索を行ったところ、 S P TR蛋白質データベース （SW I S S—PROT蛋白質配列データベースと T r EMB L核酸翻訳データベースを統合したもの） 中に、 （ i ) ( i ) データベース 登録記号 P01031、 Complement C5 precursor (HUMAN) 力 e— v a l u e : 5 X 1 0—82、 242アミノ酸残基に亘り 62%の一致度で、 また （ i i ) データベース登録記 号 P06684、 Complement C5 precursor (MOUSE) 力 e— v a l u e : 2 X l 0—81、 243アミノ酸残基に亘り 59%の一致度で、 さらに （ i i i ) データベース登録記号 P12387、 Complement C3 precursor (CAVPO) 力 e— v a l u e : 2 X 1 0—18、 236 ァミノ酸残基に亘り 30%の一致度でヒッ トした。 As shown in SEQ ID NO: 42, dnaform m38308 was composed of 1648 bases, of which nucleotides 4 to 1062 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 35 amino acid residues (SEQ ID NO: 47). A homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 42. The SPTR protein database (the SW ISS-PROT protein sequence database and the TrEMBL nucleic acid translation database were integrated). during, (i) (i) a database registration mark P01031, Complement C5 precursor (HUMAN) power e- value: at 5 X 1 0- 82, 242 62 % degree of coincidence over the amino acid residues, also (ii) a database registration symbol P06684, Complement C5 precursor (MOUSE) power e- value: 2 X l 0- 81 , 243 with 59% degree of coincidence over the amino acid residues, yet (iii) a database registration mark P12387, Complement C3 precursor (CAVPO ) power e- value: 2 X 1 0- 18 , 236 Hits were made with 30% identity over amino acid residues.
これらの結果より、配列番号 47に示したアミノ酸配列からなるタンパク質はィ ムノグロブリン様タンパク質であることが推測された。  From these results, it was presumed that the protein consisting of the amino acid sequence represented by SEQ ID NO: 47 was an immunoglobulin-like protein.
また、 上記 （ i) のタンパク質は、 データベース中の文献情報 （Biochemistry 27:3568-3580(1988)) から炎症反応に関わることが、 また上記 （i i) のタンパク 質は、 データベース中の文献情報 （J. Biol. Chem. 265 :2435-2440 (1990)) から炎 症反応に関わることが、 さらに上記 (i i i) のタンパク質は、 データベース中の 文献情報 (J. Clin. Invest. 86:96-106(1990)) から補体の活性に関わることがそ れぞれ明らかとなった。  The protein (i) may be involved in the inflammatory reaction from the literature information (Biochemistry 27: 3568-3580 (1988)) in the database, and the protein (ii) may be related to the literature information ( J. Biol. Chem. 265: 2435-2440 (1990)), and the protein of the above (iii) can be found in the literature information in the database (J. Clin. Invest. 86: 96-106). (1990)), it has been clarified that they are involved in complement activity.
また、配列番号 42に示す塩基配列がコードするアミノ酸配列について、 HMM PF AMによる蛋白質特徴検索を行ったところ、 配列番号 47のアミノ酸番号 16 —241に Alpha- 2- macroglobulinの特徴を示す配列（P f 3111に 2—1^としてェント リーされる配列） を見出した。  In addition, the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 42 was subjected to protein characteristic search using HMM PFAM. The sequence that is entered as 2-1 ^ in f 3111) was found.
これらのこと力 ら、配列番号 42に示す塩基配列がコードするタンパク質は炎症 ゃァレルギ一反応に関わる機能を有するィムノグロブリン様タンパク質であるこ とが推測された。  From these facts, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 42 was an immunoglobulin-like protein having a function related to the inflammation and diarrhea reaction.
(18) d n a f o rm38407 (配列番号 43、 48)  (18) d n a f o rm38407 (SEQ ID NOs: 43 and 48)
d n a f o rm38407は、配列番号 43に示すように、 1700塩基から成 り、 そのうち塩基番号 8から 1078までがオープンリーディングラレーム（終止 コドンを含む）になっていた。 オープンリーディングフレームから予測されるアミ ノ酸配列は、 356アミノ酸残基から成る（配列番号 48) 。 配列番号 43に示す 塩基配列がコードするアミノ酸配列について B LASTを用いて相同性検索を行 つたところ、 S PTR蛋白質データベース（SWI S S— PROT蛋白質配列デー タベースと T r EMB L核酸翻訳データベースを統合したもの）中に、 （ i) デー タベース登録記号 P01031、 Complement C5 precursor (HUMAN) 力 e - v a 1 u e ： 3 X 10—84、 241アミノ酸残基に亘り 63%の一致度で、 また （ i i ) データベース 登録記号 P06684、 Complement C5 precursor (MOUSE) 力 e— v a l u e : 2 X 1 (Γ83、 242アミノ酸残基に亘り 59%の一致度で、 さらに （i i i) データベース登 録記号 P12387、 Complement C3 precursor (CAVPO) 力 e— v a l u e : 3 X 10 17、 234アミノ酸残基に亘り 30%の一致度でヒットした。 As shown in SEQ ID NO: 43, dnafo rm38407 was composed of 1700 bases, of which base numbers 8 to 1078 were open reading lames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 356 amino acid residues (SEQ ID NO: 48). A homology search was performed using BLAST on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 43, and the SPTR protein database (SWI SS-PROT protein sequence database and the TrEMBL nucleic acid translation database were integrated). during things), (i) database registration mark P01031, Complement C5 precursor (HUMAN) force e - va 1 ue: at 3 X 10- 84, 241 63% degree of coincidence over the amino acid residues, also (ii) Database registration code P06684, Complement C5 precursor (MOUSE) force e— value: 2 X 1 (Γ 59% identity over 83 and 242 amino acid residues, plus (iii) Database registration code P12387, Complement C3 precursor (CAVPO) force e—value: 3 × 10 17 and 234 amino acid residues Hit with 30% match.
また、配列番号 43に示す塩基配列がコードするアミノ酸配列について、 HMM PF AMによる蛋白質特徴検索を行ったところ、 配列番号 48のアミノ酸番号 6_ 230に Alpha- 2-macroglobulinの特徴を示す配列（P f a mに A2M— Nとしてェントリ 一される配列） を見出した。  In addition, the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 43 was subjected to a protein feature search using HMM PFAM. The amino acid sequence 6 to 230 in SEQ ID NO: 48 showed a sequence exhibiting the characteristics of Alpha-2-macroglobulin (P fam A2M—N was identified as a sequence.
これらのことから配列番号 43に示す塩基配列がコードするタンパク質は炎症 やアレルギー反応に関わる機能を有するィムノグロブリン様タンパク質であるこ とが推測された。  From these facts, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 43 was an immunoglobulin-like protein having functions related to inflammation and allergic reactions.
(19) dn a f o rm36427 (配列番号 44、 49)  (19) dn a f o rm36427 (SEQ ID NOs: 44 and 49)
d n a f o rm36427は、配列番号 44に示すように、 4725塩基から成 り、そのうち塩基番号 1340から 4429までがオープンリーディングフレーム (終止コドンを含む）になっていた。オープンリーディングフレームから予測される ァミノ酸配列は、 1029ァミノ酸残基から成る（配列番号 49 )。配列番号 44に 示す塩基配列がコードするアミノ酸配列について B LASTを用いて相同性検索 を行ったところ、 S PTR蛋白質データベース（SW I S S— PROT蛋白質配列 データベースと T r EMBL核酸翻訳データベースを統合したもの）中に、 （ i ) データベース登録記号 P14046、 Alpha- 1- inhibitor III precursor (RAT) 力 e - v a 1 u e : 5 X 10—117、 1098アミノ酸残基に亘り 31%の一致度で、 また （ i i ) データベース登録記号 P20740、 Ovostatin precursor (CHICK)力 S、 e— v a 1 u e ： 5 X 1 0-U、 981アミノ酸残基に亘り 29%の一致度で、 さらに （ i i i ) データべ 一ス登録 C"^P20742、 Pregnancy zone protein precursor (HUMAN) 力 s、 e— v a 1 u e : 5 X 10— 109、 995アミノ酸残基に亘り 29%の一致度でヒットした。 As shown in SEQ ID NO: 44, dnafo rm36427 was composed of 4725 bases, of which base numbers 1340 to 4429 were an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 1029 amino acid residues (SEQ ID NO: 49). A homology search was performed using BLAST on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 44. The SPTR protein database (SW ISS—PROT protein sequence database and TrEMBL nucleic acid translation database were integrated) during, (i) a database registration mark P14046, alpha- 1- inhibitor III precursor ( RAT) force e - va 1 ue: 5 X 10- 117, 1098 at 31% degree of coincidence over the amino acid residues, and (ii ) Database registration code P20740, Ovostatin precursor (CHICK) force S, e-va 1 ue: 5 X 10- U , with 29% identity over 981 amino acid residues, and (iii) database registration C "^ P20742, Pregnancy zone protein precursor (HUMAN) force s, e- va 1 ue: was hit with 29% of the degree of match over the 5 X 10- 109, 995 amino acid residues.
また、配列番号 44に示す塩基配列がコードするアミノ酸配列について、 HMM PF AMによる蛋白質特徴検索を行ったところ、 配列番号 49のアミノ酸番号 268 —982に Alpha- 2-macroglobulinの特徴を示す配列 (P f a mに A2Mとしてェントリ 一される配列） を見出した。 The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 44 was subjected to protein characteristic search using HMM PFAM. Enter as A2M in fam Sequence).
これらのことから、配列番号 44に示す塩基配列がコードするタンパク質はプロ テアーゼ活性の阻害に関わる機能を有するィムノグロプリン様タンパク質である ことが推測された。  From these facts, it was presumed that the protein encoded by the nucleotide sequence represented by SEQ ID NO: 44 was an imnoglobulin-like protein having a function related to the inhibition of protease activity.
(20) d n a f o r m36949 (配列番号 45、 50)  (20) d n a f o r m36949 (SEQ ID NOs: 45 and 50)
d n a f o r m36949は、 配列番号 45に示すように、 1650塩基から成り、 その うち塩基番号 111から 488までがオープンリーディングフレーム （終止コドンを含 む）になっていた。 オープンリーディングフレームから予測されるアミノ酸配列は、 125アミノ酸残基から成る （配列番号 50)。 配列番号 45がコードするァミノ 酸配列について B LASTを用いて相同性検索を行ったところ、 S PTR蛋白質デ ータベース （SWI S S— P ROT蛋白質配列データベースと T r EMB L核酸 翻訳データベースを統合したもの） 中に、 （ i) データベース登録記号 AF329485 1S e - V a 1 u e ： 3 X 10—52、 121アミノ酸残基に亘り 82%の一致度で、 また ( i i ) データベース登録記号 AL356276が、 e— v a l u e : 2 X 10'2 84アミ ノ酸残基に亘り 61%の一致度で、 さらに （i i i) データベース登録記号 AF459634 I e-v a l u e ： 2X 10一26、 84アミノ酸残基に亘り 61%の一致度でヒットし た。 As shown in SEQ ID NO: 45, dnafor m36949 was composed of 1650 bases, of which base numbers 111 to 488 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 125 amino acid residues (SEQ ID NO: 50). A homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 45. The SPTR protein database (SWISS—PROT protein sequence database and TrEMBL nucleic acid translation database integrated) during, (i) a database registration mark AF329485 1S e - V a 1 ue : 3 X 10- 52, 121 with 82% degree of coincidence over the amino acid residues, also (ii) a database registration mark AL356276 is, e- value : in 2 X 10 '2 84 amino acid residues 61% degree of coincidence over the further (iii) a database registration mark AF459634 I ev alue: at 2X 10 one 26, 84 61% of the degree of coincidence over the amino acid residues It was hit.
また、 上記 AF329485は、 文献情報 （I議 unogenetics, 2002, May, 54(2)87-95) から、 leukocyte Fc receptorsや、 細胞接着分子 PECAM - 1と類似する構造であり、 IFGPフアミリーと呼ばれていることがわかった。  The above-mentioned AF329485 has a structure similar to leukocyte Fc receptors and cell adhesion molecule PECAM-1 based on literature information (Igen unogenetics, 2002, May, 54 (2) 87-95), and is called IFGP family. I understood that.
また、配列番号 45に示す塩基配列がコードするアミノ酸配列について、 HMM PF AMによる蛋白質特徴検索を行ったところ、 配列番号 50のアミノ酸番号 56 — 114にィムノグロブリンの特徴を示す配列 （P f & 111に1§としてェントリーされ る配列） を見出した。 In addition, a protein characteristic search was performed on the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 45 using HMM PFAM, and the sequence (P f & (A sequence that is entered as 1 § in 111) was found.
これらのこと力 ら、配列番号 45に示す塩基配列がコードするタンパク質はィム ノグロプリン様構造を有する受容体または接着に関連するタンパク質であること が推測された。 (2 1) d n a f o r m44492 (配列番号 46、 5 1) From these results, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 45 was a receptor having an immunoglobulin-like structure or a protein related to adhesion. (2 1) dnafor m44492 (SEQ ID NO: 46, 5 1)
d n a f o r m44492は、 配列番号 46に示すように、 1922塩基から成り、 その うち塩基番号 49から 1080までがオープンリーディングフレーム （終止コドンを含 む） になっていた。 オープンリーディングフレームから予測されるアミノ酸配列は、 34 3アミノ酸残基から成る （配列番号 5 1)。 配列番号 46がコードするァミノ 酸配列について B LASTを用いて相同性検索を行ったところ、 S PTR蛋白質デ —タベース （SWI S S— PROT蛋白質配列データベースと T r EMB L核酸 翻訳データベースを統合したもの） 中に、 （ i ) データベース登録記号 AF329485 力 e— v a 1 u e : 0.0、 343アミノ酸残基に亘り 99%の一致度で、 また （i i ) データベース登録記号 AF459634が、 e _v a l u e : 9 X 1 0""、 327ァミノ酸残 基に亘り 58%の一致度で、 さらに （ i i i ) データベース登録記号 AL356276が、 e -v a l u e : l X l (Γ73で、 298ァミノ酸残基に亘り 51%の一致度でヒットした。 また、 上記 AF329485は、 文献情報 （Iramunogenetics, 2002, May, 54 (2) 87 - 95) から、 leukocyte Fc receptorsや、 細胞接着分子 PECAM-1と類似する構造であり、 IFGPフアミリーと呼ばれていることがわかった。 As shown in SEQ ID NO: 46, dnafor m44492 was composed of 1922 bases, of which base numbers 49 to 1080 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 343 amino acid residues (SEQ ID NO: 51). A homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 46, and the S PTR protein database (SWI SS-PROT protein sequence database and Tr EMB L nucleic acid translation database integrated) Among them, (i) database registration code AF329485 force e—va 1 ue: 0.0, 99% identity over 343 amino acid residues, and (ii) database registration code AF459634, e _value: 9 X 10 "", 327 Amino acid residues in the 58% degree of coincidence over the group, further (iii) a database registration mark AL356276, e -value: l with X l (Γ 73, 51% of matches over 298 Amino acid residue AF329485 has a structure similar to leukocyte Fc receptors and cell adhesion molecule PECAM-1 based on literature information (Iramunogenetics, 2002, May, 54 (2) 87-95). It turned out to be called.
また、配列番号 46に示す塩基配列がコードするアミノ酸配列について、 HMM PF AMによる蛋白質特徴検索を行ったところ、 配列番号 5 1のアミノ酸番号 36 —94にィムノグロブリンの特徴を示す配列（P f 3 111に1§としてェントリーされる 配列） を見出した。 In addition, a protein characteristic search was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 46 using HMM PFAM. 3 111 Entori the array to be as 1 §) we found.
これらのこと力 ら、配列番号 46に示す塩基配列がコードするタンパク質はィム ノグロブリン様受容体タンパク質であることが推測された。 実施例 5 DN Aマイクロアレーを用いた組織発現解析  From these facts, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 46 was an immunoglobulin-like receptor protein. Example 5 Analysis of tissue expression using DNA microarray
組織発現解析は、 Miki, R. , et al. , Proc. Natl. Acad. Sci. USA, 98, 2199-2204 (2001)の記載に従って行った。  Tissue expression analysis was performed as described in Miki, R., et al., Proc. Natl. Acad. Sci. USA, 98, 2199-2204 (2001).
(1) DN Aマイクロアレーの作成  (1) Creation of DN A microarray
2種類のマウス全長 c DNAの塩基配列（d n a f o r m33 1 33、 d n a f o r m49889 )および解析対象のマウス全長 c D N Aと同じクラスタに属する (該 c DNAと相同な塩基配列を有する） 4種類のマウス c DN Aライブラリー F A N T O M (http： //f antom. gsc. riken. go. jp/)由来の c D N Aの塩基配歹 lj (FANTOM NO:2310069H21、 5430413J22, 9430067009、 4831440K17) を、 Ml 3フォワードお よびリバースプライマーを用いて増幅後、この PCR産物をィソプロパノールにて 沈澱させ 15 μ 1の 3 X S SC液に溶解した。これらの 6種類の DN Α溶液をポリ Lリジンコートしたガラススライドに、 16チップ（SMP 3、Te l e Ch eIn I n t e r n a t i o n a 1、 Sunny v a 1 e、 C A) の DNAアレイヤーを用 いてスポットし、 DNAマイクロアレーを作成した （方法の詳細は Nucleotide sequences of two mouse full-length cDNAs (dnafor m33 133, dnaf or m49889) and four kinds of mouse cDNA libraries FANTOM (http: // fantom. gsc. riken.) belonging to the same cluster as the full-length mouse cDNA to be analyzed (having a base sequence homologous to the cDNA). go.jp/) The base sequence of cDNA derived from cDNA (FANTOM NO: 2310069H21, 5430413J22, 9430067009, 4831440K17) was amplified using Ml3 forward and reverse primers, and the PCR product was then treated with isopropanol. The precipitate was precipitated and dissolved in 15 μl of 3 XS SC solution. These 6 types of DNΑ solutions were spotted on a poly-L-lysine-coated glass slide using a DNA arrayer of 16 chips (SMP3, TeleChina International, Sunnyva 1e, CA), and DNA microscopy was performed. I created an array (for details on how
http://cmgm. Stanford, edu/pbrown/raguide/ inaex. htmlに gci载され飞レヽ >) 。 マソ ス 3ァクチンとグリセルアルデヒド- 3 -フォスフェートデヒ ドロゲナーゼの c DNAをポジティブコントロールとし、シロイヌナズナの c DNAをネガティブコ ントロールとして用いた。 http: // cmgm. Stanford, edu / pbrown / raguide / inaex. The cDNA of masos 3 actin and glyceraldehyde-3-phosphate dehydrogenase was used as a positive control, and the Arabidopsis thaliana cDNA was used as a negative control.
この DNAマイクロアレーの検出感度は、 1細胞当たり mRNA 1ないし 3コピ 一であった。ターゲット配列との一致度がおよそ 80%のクローンのシグナル強度 は、完全に配列が一致するクローンの 10分の 1であった。 ターゲット配列との一 致度が 80%未満のクローンのシグナル強度は、 バックグランドレベルであった。  The detection sensitivity of this DNA microarray was 1 to 3 copies of mRNA per cell. The signal intensity of clones with approximately 80% match with the target sequence was 1/10 that of clones with perfect sequence match. The signal intensity of clones with less than 80% match with the target sequence was at the background level.
(2) プローブの調製  (2) Preparation of probe
C57BLZ6 Jマウスの胎児、新生仔、アダルトの 49組織（腎臓、脳、脾臓、 肺、 肝臓、 精巣、 膝臓、 胃、 小腸、 結腸、 盲腸、 胎盤、 心臓、 舌、 胸腺、 胸腺 （妊 娠 1日目） 、 小脳、 延髄、 嗅脳、 副精巣、 眼球、 皮質、 小胞腺、 子宮、 卵巣および 子宮 （妊娠 1 1日目） 、 骨、 筋肉、 乳腺 （授乳 10日目） 、 10日齢胎児全身、 1 1日齢胎児全身、 13日齢胎児全身、 1 1日齢胎児頭部、 12日齢胎児頭部、 13 日齢胎児頭部、 1 5日齢胎児頭部、 16日齢胎児頭部、 1 7日齢胎児頭部、 16日 齢胎児肺、 1 3日齢胎児肝臓、 14日齢胎児肝臓、 0日齢新生児全頭部、 6日齢新 生児全頭部、 10日齢新生児全頭部、 10日齢新生児腸、 0日齢新生児肺、 10日 齢新生児小脳、 0日齢新生児皮膚、 10日齢新生児皮膚、 S V40感染）、または、 2 2組織 （腎臓、脳、 脾臓、 肺、 肝臓、 精巣、 脖臓、 胃、 小腸、 結腸、 胎盤、 心臓、 胸腺、 小脳、 子宮、 骨、 筋肉、 背側腎臓由来脂肪細胞、 副精巣由来脂肪細胞、 内臓 脂肪、 1 0日齢新生児小脳、 1 0日齢新生児皮膚） から抽出した mRNA 1 gを 定法に従いランダムプライム逆転写反応を行い蛍光色素 C y 3 (Ame r s h a m P h a r ma c i a社製） を取りこませた。 他方、 1 7. 5日齢の胎児全身から抽 出した mRNA l μ gをランダムプライム逆転写反応を行い、蛍光色素 C y 5を取 りこませ発現解析のリファレンスとした。 C y D y e標識 c DNAプローブは、 C y S c r i b e GFX P u r i f i c a t i o n K i t 、Am e r s h a m P h a r ma c i a社製） を用いて精製し、滅菌水 1 7 μ 1にてカラムから溶出し た。 これに 3 μ 1の 1 0 μ g/μ 1 ο 1 i g ο ( d A) , 3 μ 1の酵母 t RNA 2 0 μ g/μ 1 , 1 /ζ 1の 20 μ g/μ 1マウス C ο t 1 DNA, 5. 1 μ Iの 20 X S S C, および 0. 9 /i lの 1 0% SD Sからなるブロッキング溶液を混 和して C y D y e標識 c DNAプローブを調製した。 49 tissues of C57BLZ6 J mouse fetus, neonate, adult (kidney, brain, spleen, lung, liver, testis, knee, stomach, small intestine, colon, cecum, placenta, heart, tongue, thymus, thymus Day), cerebellum, medulla oblongata, olfactory brain, epididymis, eyeball, cortex, follicular gland, uterus, ovary and uterus (1st day of pregnancy), bone, muscle, mammary gland (10th day of lactation), 10-day-old fetus Whole body, 11 day old fetal whole body, 13 day old fetal whole body, 11 day old fetal head, 12 day old fetal head, 13 day old fetal head, 15 day old fetal head, 16 day old fetal head Part, 17-day-old fetal head, 16-day-old fetal lung, 13-day-old fetal liver, 14-day-old fetal liver, 0-day-old newborn whole head, 6-day-old newborn whole head, 10-day-old Whole neonatal head, 10-day-old neonatal intestine, 0-day-old neonatal lung, 10-day-old neonatal cerebellum, 0-day-old neonatal skin, 10-day-old neonatal skin, SV40 infection), or 2 2 tissues (kidney, brain, spleen, lung, liver, testis, stomach, stomach, small intestine, colon, placenta, heart, thymus, cerebellum, uterus, bone, muscle, dorsal kidney-derived fat cells, accessory testes-derived fat 1 g of mRNA extracted from cells, visceral fat, 10-day-old neonatal cerebellum, and 10-day-old neonatal skin) were subjected to random prime reverse transcription according to a standard method, and a fluorescent dye Cy3 (Amersham Pharmacia) was used. I took in. On the other hand, 1 μg of mRNA extracted from the whole body of a 17.5-day-old fetus was subjected to a random prime reverse transcription reaction to incorporate the fluorescent dye Cy5, which was used as a reference for expression analysis. The CyDye-labeled cDNA probe was purified using a CyScribe GFX Purification Kit (Amersham Pharmacia) and eluted from the column with 17 μl of sterile water. 3 μl of 10 μg / μ 1 ο 1 ig ο (dA), 3 μl of yeast tRNA 20 μg / μ 1, 1 / ζ 1 of 20 μg / μ 1 mouse C A blocking solution consisting of ot1 DNA, 5.1 μI of 20 XSSC, and 0.9 / il of 10% SDS was mixed to prepare a CyDye-labeled cDNA probe.
(3) DNAマイクロアレーのハイブリダィゼイシヨン  (3) DNA microarray hybridization
発現解析対象組織由来 c DN Aプローブ （C y 3標識） とリファレンスの 1 7. 5日齢胎児由来 c DN Aプローブ（C y 5標識） を混和した溶液 3 0 μ 1を 9 5 °C にて 1分間熱処理を行い室温にて冷却した。 DNAマイクロアレーに上記プローブ 溶液を添加しカバースリップを被せ、 Hy b r i c a s e t t e (A r r a y I t 社製） 中にて 6 5 °C—晚ハイブリダィズさせた。 次に、 DNAマイクロアレーを 2 X S S C, 0. 1 % SD Sを用いて洗浄し、 続いて 1 X S S Cにて 2分間、 0. 1 X S S Cにて 2分間リンスした。マイクロアレーは S c a nA r r a y 5 00 0 共焦点レーザースキャナーを用いてスキャンし、画像を I MAGENE (B i o D i s c o v e r y社製） で角军析した。  30 μl of a solution obtained by mixing a cDNA probe (Cy3 label) derived from the tissue to be analyzed for expression with a reference DNA probe (Cy5 label) derived from a 17.5-day-old embryo at 95 ° C For 1 minute and cooled at room temperature. The above-mentioned probe solution was added to the DNA microarray, covered with a cover slip, and hybridized at 65 ° C- 晚 in Hybricasete (ArrayaIt). Next, the DNA microarray was washed with 2 × SSC, 0.1% SDS, and then rinsed with 1 × SSC for 2 minutes and with 0.1 × SSC for 2 minutes. The microarray was scanned using a ScanAnaray 500 000 confocal laser scanner, and the images were subjected to angular analysis using I MAGENE (BioDiversive).
(4) データ解析  (4) Data analysis
各組織中の mRNA量 （C y 3標識） は、 リファレンスの 1 7. 5日齢の胎児全 身 mRNA量 （C y 5標識） との比 （C y 3/C y 5) を対数 （ 1 o g2) で表示 した。データの正確性を増すために実験は独立に 2回行い、再現性の有る結果を採 用した。 結果を以下の表 1に示す。 The mRNA level (Cy3 labeling) in each tissue is expressed as the logarithm (1 og 2 ). Experiments were performed twice independently to increase data accuracy and reproducible results were obtained. Used. The results are shown in Table 1 below.
一般的に、 DN Aアレーを使用した発現解析では、 2倍程度の増減は実験誤差と みなされる。 このことから、表 1に示す結果の数値が 1以上の場合にはある組織中 の mRN A量が対照である 17. 5日齢の胎児全身の mR N A量と比較して 2倍以 上であり、有意に増加していると解釈した。逆に、結果の数値が一 1以下の場合は、 ある組織中の mRN A量が対照である 17. 5日齢の胎児全身の mRN A量と比較 して 2分の 1以下であり、有意に減少していると解釈した。 また、任意の組織間の mRN A発現量を比較検討する際は、各組織における数値の差が 1であれば mRN A量は 2倍、 2であれば mRNA量は 4倍であり、逆に、組織間の数値の差が一 1 であれば mR N A量は 1 / 2倍、 _ 2であれば mR N A量は 1 Z 4倍であることを 意味する。  Generally, in an expression analysis using a DNA array, an increase or decrease of about 2 times is regarded as an experimental error. From this, when the value of the result shown in Table 1 is 1 or more, the amount of mRNA in a certain tissue is a control. Yes, it was interpreted as a significant increase. Conversely, if the value of the result is 11 or less, the amount of mRNA in a certain tissue is less than one-half that of the control, and the value of mRNA in the whole fetal body at 17.5 days of age is significant. It was interpreted as a decrease. Also, when comparing the mRNA expression levels between any tissues, if the difference between the values in each tissue is 1, the mRNA quantity is 2 times, if it is 2, the mRNA quantity is 4 times, and conversely If the difference between the values of the tissues is 11, the amount of mRNA is 1/2 times, and if the difference is _2, the amount of mRNA is 1Z 4 times.
なお、マイクロアレイにスポットした DNAと同じクラスタに属し、該 DNAと 少なくとも 200塩基に亘り 80%以上の塩基配列の一致度を有する領域を有す るマウス c DNAクローン（d n a f o rm30449、 d n a f o r m 5183 9、 d n a f o rm41412、 d n a f o rm43395、 d n a f o rm60 440、 d n a f o rm67267、 dn a f o rm36427) についても、 表 1に解析対象 c DNAとして記載し、マイクロアレイにスポットした該 DNAの測 定結果の数値を代用して記載した。 A mouse cDNA clone (dnafo rm30449, dnaform 51839, dnafo) belonging to the same cluster as the DNA spotted on the microarray and having a region having a nucleotide sequence identity of at least 80% over at least 200 bases. rm41412, dnafo rm43395, dnafo rm60440, dnafo rm67267, dnafo rm36427) are also described in Table 1 as the cDNA to be analyzed, and the numerical values of the measurement results of the DNA spotted on the microarray are described instead.
解析対象 cDNA マイクロアレーにスポットした腎臓 凶 脾臓 肺 Analysis target cDNA spotted on microarray Kidney spleen Lung
DNA  DNA
dnaform30449 FANTOM NO:2310069H21 -0.107719 0.006037 0.079898 0.620773 解析対象 cDNAマイクロアレーにスポットした肝臓 ^Fh巢 降疏 dnaform30449 FANTOM NO: 2310069H21 -0.107719 0.006037 0.079898 0.620773 Liver spotted on cDNA microarray to be analyzed ^ Fh 巢
DNA  DNA
dnaform30449 FANTOM NO:2310069H21 1.66912 -0.861087 0.338814 -0.092846 解析対象 cDNA マイクロアレーにスポットした小腸 胎盤 dnaform30449 FANTOM NO: 2310069H21 1.66912 -0.861087 0.338814 -0.092846 Small intestine placenta spotted on cDNA microarray to be analyzed
DNA  DNA
dnaform30449 FANTOM NO:2310069H21 -0.124527 0.084441 -0.448783 -0.690754 解析対象 cDNA マイクロアレ一にスポットした心臓 舌 胸腺 胸腺（妊娠 1 dnaform30449 FANTOM NO: 2310069H21 -0.124527 0.084441 -0.448783 -0.690754 Target cDNA Micro spot spotted on microarray Tongue Thymus Thymus (pregnancy 1
DNA 曰 § ) dnaform30449 FANTOM Nひ 2310069H21 0.527034 0.9128 3.41084 0.747104 解析対象 cDNA マイクロアレ一にスポットした小脳 延髄 嗅脳 副精巣  DNA §) dnaform30449 FANTOM N 2310069H21 0.527034 0.9128 3.41084 0.747104 cDNA to be analyzed Cerebellum spotted on microarray Medullary medulla Olfactory brain Epididymis
DNA  DNA
dnaform30449 FANTOM Nひ 2310069H21 -0.24943 -0.724039 -0.32742 0.147158 解析対象 cDNAマイクロアレーにスポットした眼球 皮質 小胞腺 子宮 dnaform30449 FANTOM N 2310069H21 -0.24943 -0.724039 -0.32742 0.147158 Target eyeball spotted on cDNA microarray Cortical cortical follicular gland Uterus
DNA  DNA
dnaform30449 FANTOM Nひ 2310069H21 0.301515 -0.390903 0.226586 0.558371 解析対象 cDNAマイクロアレーにスポットした卵巣および 骨 筋肉 乳腺 (授乳 dnaform30449 FANTOM N 2310069H21 0.301515 -0.390903 0.226586 0.558371 Ovarian and bone muscle spotted on cDNA microarray to be analyzed
DNA 子宮（妊娠 10日目）  DNA uterus (day 10 of pregnancy)
11曰目）  11 statements)
dnaform30449 ""~ FANTOM NO:2310069H21 1.13407 ~ 0.108588 1.50513 -0.253459 解析対象 cDNAマイクロアレーにスポットした 10日齢胎児 1 1日齢胎 13日齢胎児 1 1日齢胎児 dnaform30449 "" ~ FANTOM NO: 2310069H21 1.13407 ~ 0.108588 1.50513 -0.253459 Target 10-day-old fetus 1 1-day-old fetus 13-day-old fetus 1 1-day-old fetus spotted on cDNA microarray to be analyzed
DNA _ 身 _児全： ¾ 全身 頭部 dnaform30449 FANTOM Nひ 2310069H21 -0.341017 -0.094889 0 0.073223 解析対象 cDNA マイクロアレーにスポットした 12曰齢胎児 13日齢胎 15曰齢胎児 16日齢胎児  DNA _ body _ whole: 全身 whole body head
DNA _ 部 児頭部 頭部 頭部  DNA _ part Child head Head Head
dnaform30449 FANTOM Nひ 2310069H21 0.404469 -0.87302 -0.102219 0.239442 解析対象 cDNA マイクロアレーにスポットした 17曰齢胎児 16曰齢胎 13曰齢胎児 14曰齢胎児 dnaform 30449 FANTOM N 2310069H21 0.404469 -0.87302 -0.102219 0.239442 Target cDNA 17 spotted fetus 16 spotted fetus 13 spotted fetus 14 spotted fetus
DNA p部 児肺 ― 肝臓 肝臓  DNA p part infant lung-liver liver
dnaform30449 FANTOM Nひ 2310069H21 1.28003 0.500602 0.394619 -0.995414 解析対象 cDNA マイクロアレーにスポットした 0日齢新生 6日齢新生 10日齢新生 10日齢新生 dnaform30449 FANTOM N 2310069H21 1.28003 0.500602 0.394619 -0.995414 0-day newborn 6-day newborn 10-day newborn 10-day newborn spotted on the cDNA microarray to be analyzed
DNA 児全頭部 児全頭部 児全頭部 児腸 dnaform30449 ~~ FANTOM NO:2310069H21 6.3662 1.06955 ~ 0.906365 ~~ 0.723262 表 1 (続き) DNA whole child head whole child child whole head child intestine dnaform30449 ~~ FANTOM NO: 2310069H21 6.3662 1.06955 ~ 0.906365 ~~ 0.723262 Table 1 (continued)
解析対象 cDNA マイクロアレ一にスポットした 0日齢新生 10曰齢新 0日齢新生 10曰齢新生 0 days old new 10 days old new 10 days old 10 new days old spotted on the analysis target cDNA microarray
DNA 児肺 生] ¾小 ¾| 児皮 w i¾皮膚 dnaform30449 FANTOM NO:2310069H21 0 -0.696454 1.92827 2.34609 解析対象 cDNAマイクロアレ一にスポットした sv40感染  DNA infant lung] ¾small ¾ | scalp w i¾skin dnaform30449 FANTOM NO: 2310069H21 0 -0.696454 1.92827 2.34609 sv40 infection spotted on cDNA microarray
DNA  DNA
dnaform30449 FANTOM NO:2310069H21 1.45411 解析対象 cDNAマイクロアレ一にスポットした腎臓 脾臓 肺 dnaform30449 FANTOM NO: 2310069H21 1.45411 Analysis target Kidney spotted on cDNA microarray Spleen Lung
DNA  DNA
dnaform51839 FANTOM Nひ 5430413J22 -0.291107 0.690953 0 1.01092 dnaform41412 FANTOM NO:9430067O09 -0.022349 0.296793 -0.465093 0.641874 dnaform43395 FANTOM NO:9430067O09 -0.022349 0.296793 -0.465093 0.641874 dnaform33133 dnaform33133 -0.678764 -1.38616 -0.122963 -0.347474 dnaform49889 dnaform49889 0.816437 - 0.002556 O.353306 0.432307 dnaform60440 dnaform49889 0.816437 -0.002556 0.353306 0.432307 dnaform6 /2b7 dnaform49889 0.816437 - 0.002556 0.353306 0.432307 dnaform36427 FANTOM Nひ 4831440K17 -0.740519 -1.7679 - 2.17688 0 解析対象 cDNAマイクロアレ一にスポットした肝臓 精巣 膝臓 冃 dnaform51839 FANTOM N HI 5430413J22 -0.291107 0.690953 0 1.01092 dnaform41412 FANTOM NO: 9430067O09 -0.022349 0.296793 -0.465093 0.641874 dnaform43395 FANTOM NO: 9430067O09 -0.022349 0.296793 -0.465093 0.641874 dnaform33133 dnaform33133 -0.678749 -1.367 -0.37874 -0.87 0.432307 dnaform60440 dnaform49889 0.816437 -0.002556 0.353306 0.432307 dnaform6 / 2b7 dnaform49889 0.816437-0.002556 0.353306 0.432307 dnaform36427 FANTOM Nh 4831440K17 -0.740519 -1.7679-2.17688 0 Analysis target
DNA  DNA
dnaform51839 FANTOM Nひ 5430413J22 0.74515 0.951565 -0.095124 0.085743 dnaform41412 FANTOM Nひ 9430067009 -0.227092 0.835839 -0.170522 0.689348 dnaform43395 FANTOM Nひ 9430067009 -0.227092 0.835839 -0.170522 0.689348 dnaformj3133 dnaform33l 33 -1.22005 -0.259036 -1.6569 0 dnaform49889 dnaform49889 0.550258 0.564867 0.993312 -0.231643 dnaform60440 dnaform49889 0.550258 0.564867 0.993312 -0.231643 dnaform67267 dnaform49889 0.550258 0.564867 0.993312 -0.231643 dnaform36427 FANTOM NO:4831440K17 -1.55599 -0.055264 -1.76778 -0.868013 解析対象 cDNAマイクロアレ一にスポットした小腸 胎盤 心臓 dnaform51839 FANTOM N 5430413J22 0.74515 0.951565 -0.095124 0.085743 dnaform41412 FANTOM N N 9430067009 -0.227092 0.835839 -0.170522 0.689348 dnaform43395 FANTOM N N 9430067009 -0.227092 0.835839 -0.170522 0.689348 dnaformj3133 -dnaform3329033 -1.2. dnaform49889 0.550258 0.564867 0.993312 -0.231643 dnaform67267 dnaform49889 0.550258 0.564867 0.993312 -0.231643 dnaform36427 FANTOM NO: 4831440K17 -1.55599 -0.055264 -1.76778 -0.868013 Target for analysis Small intestine Placenta heart spotted on cDNA microarray
DNA  DNA
dnaform51839 FANTOM Nひ 5430413J22 0.087427 0.781469 0.719051 0.61274 dnaform41412 FANTOM Nひ 9430067009 -0.826974 -0.619294 0.403315 -0.409725 dnaform43395 FANTOM Nひ 9430067009 -0.826974 -0.619294 0.403315 -0.409725 dnaform33133 dnaform33133 -0.907237 -1.60368 -1.855 0.345903 dnaform49889 dnaform49889 0.384719 0.528775 0.627352 0.146455 dnaform60440 dnaform49889 0.384719 0.528775 0.627352 0.146455 dnaform67267 dnaform49889 0.384719 0.528775 0.627352 0.146455 dnaform36427 FANTOM NO:4831440K17 -1.27601 -1.69523 0.169097 -1.51891 解析対象 cDNAマイクロアレーにスポットした胸腺 小 to ナ呂 骨 dnaform51839 FANTOM N N 5430413J22 0.087427 0.781469 0.719051 0.61274 dnaform41412 FANTOM N N 9430067009 -0.826974 -0.619294 0.403315 -0.409725 dnaform43395 FANTOM N N 9430067009 -0.826974 -0.619294 0.403315 -0.409725 dnaform33133 dnaform334933 -0.919237 0.48 0.384719 0.528775 0.627352 0.146455 dnaform67267 dnaform49889 0.384719 0.528775 0.627352 0.146455 dnaform36427 FANTOM NO: 4831440K17 -1.27601 -1.69523 0.169097 -1.51891 Thymus spotted on the cDNA microarray to be analyzed.
DNA  DNA
dnaform51839 FANTOM Nひ 5430413J22 -0.000584 -0.79966 0.329673 1.94213 dnaform41412 FANTOM NO:9430067O09 - 1.04358 0.828028 -0.178942 -0.048341 dnaform43395 FANTOM Nひ 9430067009 - 1.04358 0.828028 -0.178942 -0.048341 dnaformj3133 dnaform33133 -2.00959 -1.29028 0 -0.397246 dnaform49889 dnaform49889 -0.033134 -0.793352 0.257493 0.122818 dnaform60440 dnaform49889 -0.033134 -0.793352 0.257493 0.122818 dnaform67267 dnaform49889 -0.033134 -0.793352 0.257493 0.122818 dnaform36427 FANTOM Nひ 4831440K17 -2.98904 -2.36494 —1.52262 -0.522517 表 1 (続き） 解析対象 cDNAマイクロアレ一にスポットした筋肉 背側腎臓 副精巣由来内臓脂肪 dnaform51839 FANTOM N N 5430413J22 -0.000584 -0.79966 0.329673 1.94213 dnaform41412 FANTOM NO: 9430067O09-1.04358 0.828028 -0.178942 -0.048341 dnaform43395 FANTOM N N 9430067009-1.04358 0.828028 -0.178942 -0.048341 dnaformj3133 dnaform 33. 0.257493 0.122818 dnaform60440 dnaform49889 -0.033134 -0.793352 0.257493 0.122818 dnaform67267 dnaform49889 -0.033134 -0.793352 0.257493 0.122818 dnaform36427 FANTOM Nh 4831440K17 -2.98904 -2.36494 -1.52262 -0.522517 Table 1 (Continued) Analyzed muscle muscle spotted on cDNA microarray Dorsal kidney Visceral fat derived from epididymis
DNA _ 由来脂肪 脂肪細胞 dnaform51839 FANTO NO:5430413J22 0.484558 0.120568 0.666206 0.714305 dnaform41412 FANTOM Nひ 9430067009 -0.387452 -0.0619 -0.574951 -0.580364 dnaform43395 FANTOM NO:9430067O09 -0.387452 -0.0619 -0.574951 -0.580364 dnaform33133 dnaform33133 -0.94046 -0.717601 0.154913 -0.472842 dnaform49889 dnaform49889 -0.035625 1.69206 0.363326 0.739609 dnaTormo0440 dnaform49889 -0.035625 1.69206 0.363326 0.739609 dnaform67267 dnaform49889 -0.035625 1.69206 0.363326 0.739609 dnaform36427 FANTOM NO:4831440K17 -1.94176 -1.12891 -1.14336 -1.52155 解析対象 cDNAマイクロアレーにスポットした 10日齢新生 10日齢新  DNA _ derived fats Adipocytes dnaform51839 FANTO NO: 5430413J22 0.484558 0.120568 0.666206 0.714305 dnaform41412 FANTOM NH 9430067009 -0.387452 -0.0619 -0.574951 -0.580364 dnaform43395 FANTOM NO: 9430067O09 -0.387452 -0.0619 -0.574951 -0.313301na -0.574951 -0.51338041d dnaform49889 dnaform49889 -0.035625 1.69206 0.363326 0.739609 dnaTormo0440 dnaform49889 -0.035625 1.69206 0.363326 0.739609 dnaform67267 dnaform49889 -0.035625 1.69206 0.363326 0.739609 dnaform36427 FANTOM NO: 4831440K17 -1.94176 -1.12891 microday
DNA 児小脳 ―生児皮虜  DNA cerebellum
dnaform51839 FANTOM NO:5430413J22 -0.018364 1.19823 dnaform51839 FANTOM NO: 5430413J22 -0.018364 1.19823
dnaTorm41412 FANTOM NO:9430067O09 0.552192 -0.265812 dnaTorm41412 FANTOM NO: 9430067O09 0.552192 -0.265812
dnaform43395 FANTOM Nひ 9430067009 0.552192 -0.265812 dnaform43395 FANTOM Nh 9430067009 0.552192 -0.265812
dnaform3j I J3 dnaform33133 -1.25078 -0.468986 dnaform3j I J3 dnaform33133 -1.25078 -0.468986
dnaform49889 dnaform49889 0 0.622091 dnaform49889 dnaform49889 0 0.622091
dnaform60440 dnaform49889 0 0.622091 dnaform60440 dnaform49889 0 0.622091
dnaform67267 dnaform49889 0 0.622091 dnaform67267 dnaform49889 0 0.622091
dnaform36427 FANTOM NO:4831440K17 -1.29051 0.199098 dnaform36427 FANTOM NO: 4831440K17 -1.29051 0.199098
表 1から明らかなように、 dn a f o rm51839は同一クラスターに属する FANTOMNO:5430413J22をプローブとして発現組織を検討すると、 対照 （17. 5日 齢胎児全身） と比較して骨、 10日齢新生児皮膚、 精巣、 および、 肺で発現が増加し た。 d n a f o rm30449は同一クラスターに属する FANTOM N0:2310069H21 をプローブとして検討すると、対照と比較して 0日齢新生児全頭部、胸腺で強く発 現増強し、 0日齢 · 10日齢新生児皮膚、 肝臓、 筋肉、 卵巣、 SV40感染組織など で発現が増加した。 d n a f o rm33133は対照と比較して全体的に発現が減 弱していたが、 心臓、 副精巣由来脂肪細胞、 胃、 子宮で同等あるいはそれ以上の発 現が観察された。 dn a f o rm41412および d n a f o rm43395は同 一クラスターに属する FANTOMNO:9430067009と相同性が高いので、 これをプローブ として検討すると、 対照と比較して、 精巣、 小脳、 胃、 ^！、 10日齢新生児小脳な どで発現が増加する傾向があった。 d n a f o rm49889は対照と比較して全 体的に発現が増強していたが、 脂肪細胞、 膝臓で発現が増強し、 腎臓、 肝臓、 精巣 などで発現増加傾向が観察された。 d n a f o rm60440および d n a f o r m67267は、同一クラスターに属する dn a f o rm49889と相同性が高 いため、これをプローブとして検討すると、脂肪細胞、膝臓で発現が増強し、腎臓、 肝臓、精巣などで発現増加傾向が見られた。 d n a f o rm36427は同一クラ スターに属する FANTOMN0:4831440K17をプローブとして検討すると、対照と比較し て全体的に発現が減弱していたが、胎盤、 10日齢新生児皮膚、肺、精巣などで対 照と同程度の発現が観察された。 As is clear from Table 1, when dn afo rm51839 belongs to the same cluster, and FANTOMNO: 5430413J22 is used as a probe to examine the expression tissue, bone, 10-day-old neonatal skin, Expression increased in testis and lung. When dnafo rm30449 is examined using FANTOM N0: 2310069H21, which belongs to the same cluster, as a probe, it strongly enhances the expression in the whole head and thymus of the 0-day-old neonate as compared to the control, and the 0-day-old and 10-day-old neonatal skin, liver, Expression increased in muscle, ovary, SV40 infected tissues, and so on. The expression of dnafo rm33133 was attenuated overall as compared to the control, but equivalent or higher expression was observed in the heart, epididymis-derived fat cells, stomach, and uterus. Since dnafo rm41412 and dnafo rm43395 have high homology to FANTOMNO: 9430067009 belonging to the same cluster, when this is examined as a probe, testes, cerebellum, stomach, ^! However, the expression tended to increase in the cerebellum of a 10-day-old newborn. The expression of dnafo rm49889 was enhanced as a whole as compared with the control, but the expression was increased in adipocytes and knees, and a tendency of increased expression was observed in kidney, liver, testis and the like. dnafo rm60440 and dnafor Since m67267 has high homology to dnafo rm49889 belonging to the same cluster, when this was examined as a probe, expression was enhanced in adipocytes and knees, and increased in kidney, liver, testis and the like. When dnafo rm36427 was examined using FANTOMN0: 4831440K17, which belongs to the same cluster, as a probe, its expression was reduced overall as compared with the control, but it was the same as in the control in placenta, 10-day-old newborn skin, lung, testis, etc. A degree of expression was observed.
実施例 6 P C R法を用いた組織発現解析 Example 6 Tissue expression analysis using PCR method
本発明のタンパク質をコ一ドする mRN Aの正常マウスおょぴ疾患マウスでの 組織発現変動を検討するために、 定法 （Higuchi R, et al. , Biotechnology, 11: 1026-30 (1993)) に従い、 P C R法を用いた組織発現解析を行った。  In order to examine the change in tissue expression of mRNA encoding the protein of the present invention in normal and diseased mice, a conventional method (Higuchi R, et al., Biotechnology, 11: 1026-30 (1993)) And tissue expression analysis using the PCR method was performed.
(1) cDNA合成  (1) cDNA synthesis
以下のマウス （森脇和郎、 外 1名編、 Molecular Medicine別冊、 Vol. 36 「自然 発症疾患モデル動物 」、中山書店、 1999年）の 19組織からトータル RNAを抽出し、 オリゴ dTをプライマーとして逆転写酵素を用いて cDNA合成を行った。  Extracted total RNA from 19 tissues of the following mice (Kazuo Moriwaki, 1 other edition, Molecular Medicine, Supplement, Vol. 36 “Spontaneous Disease Model Animal”, Nakayama Shoten, 1999), reverse transcription using oligo dT as primer CDNA synthesis was performed using the enzyme.
( a ) 正常マゥスの組織および糖尿病モデルマゥスの組織  (a) Tissue of normal mice and tissue of diabetes model mice
①対照マウス C57BL/KsJ- +m/+m Jcl (メス、 8週齢） の全脳、 視床、 肺、 腎臓、 骨 髄、 滕臓、 脂肪細胞、 肝臓、 眼  ① Control mice C57BL / KsJ- + m / + m Jcl (female, 8 weeks old) whole brain, thalamus, lungs, kidneys, bone marrow, bone marrow, lentils, fat cells, liver, eyes
②糖尿病モデルマウス C57BL/KsJ - db/db Jcl (メス、 8週齢） の脾臓、 脂肪細胞、 肝臓、 眼  ② Diabetes model mouse C57BL / KsJ-db / db Jcl (female, 8 weeks old) spleen, fat cells, liver, eyes
(b) 老化促進マウスの組織  (b) Aging-promoting mouse tissue
①正常老化マウス SAM Rl/TA Sic (ォス、 13週齢） の海馬、 前頭葉皮質 (1) Hippocampus and frontal cortex of normal aging mouse SAM Rl / TA Sic (13 weeks old)
②老化促進マウス SAM P8/Ta Sic (ォス、 15週齢） の海馬、 前頭葉皮質 ② Senescence-accelerated mouse SAM P8 / Ta Sic (Oss, 15 weeks old) hippocampus, frontal cortex
(c) 癌転移モデルマウスの組織  (c) Tissue of cancer metastasis model mouse
①対照マウス Balb/c (メス、 5週齢） の正常結腸  ① Normal colon of control mouse Balb / c (female, 5 weeks old)
②癌転移モデルマウス Balb/c (メス、 6週齢） の結腸癌 （マウス腹腔に結腸癌細胞 Colon26を移植し、 2週間後に結腸癌を摘出） (2) PCR法による定量 (2) Colon cancer of a cancer metastasis model mouse Balb / c (female, 6 weeks old) (Colony 26 colon cancer cells were transplanted into the abdominal cavity of the mouse, and colon cancer was removed 2 weeks later) (2) Quantification by PCR method
下記の 7個の、本発明のタンパク質をコードしている mRNAの発現は、 ライ ト サイクラ一定量 PCR装置 （ロシュ 'ダイァグノステイクス社製） と  The expression of the following seven mRNAs encoding the protein of the present invention was determined using a light cycler constant-quantity PCR device (Roche's Diagnostics).
LightCycler-FastStart DNAマスタ一 SYBR Green I試薬を用いて、 製品に添付さ れているプロトコールに従い定量した。 定量 PCRに用いた合成 DN A配列を以下に 示す。 LightCycler-FastStart DNA Master-Quantified using SYBR Green I reagent according to the protocol attached to the product. The synthetic DNA sequences used for quantitative PCR are shown below.
(a) d n a f o rm348 1 0  (a) d n a f o rm348 1 0
5， 側プライマー ： TGCTTGGCTGATAGCTTGM (配列番号 52)  5, side primer: TGCTTGGCTGATAGCTTGM (SEQ ID NO: 52)
3 ' 側プライマー ： CTACAGCTTTGGGGCAGMG (配列番号 53) 3'-side primer: CTACAGCTTTGGGGCAGMG (SEQ ID NO: 53)
(b) d n a f o r m3584 1  (b) d n a f o r m3584 1
5， 側プライマ一： ATTGGGTGGGAGTGTTTCM (配列番号 54)  5. Side primer: ATTGGGTGGGAGTGTTTCM (SEQ ID NO: 54)
3， 側プライマー ： TGATCGCTGTCTTTCTGCTG (配列番号 5 5) 3, primer: TGATCGCTGTCTTTCTGCTG (SEQ ID NO: 55)
(c) d n a f o rm26 22 5  (c) d n a f o rm 26 22 5
5 ' 側プライマー ： GGTTGGCTAGAGCGTTGAGA (配列番号 56)  5 'side primer: GGTTGGCTAGAGCGTTGAGA (SEQ ID NO: 56)
3 ' 側プライマー ： MGGTCACCTCTCGGACTACA (配列番号 5 7 ) 3'-side primer: MGGTCACCTCTCGGACTACA (SEQ ID NO: 57)
(d) d n a f o r m39 1 1 2  (d) d n a f o r m39 1 1 2
5 ' 側プライマー ： TTGCGCAAAGATTTTGTGAT (配列番号 5 8)  5'-side primer: TTGCGCAAAGATTTTGTGAT (SEQ ID NO: 58)
3 ' 側プライマー ： GGTTGGTGTGTCTACTGTGACC (配列番号 59) 3'-side primer: GGTTGGTGTGTCTACTGTGACC (SEQ ID NO: 59)
(e ) d n a f o rm42 39 3  (e) d n a f o rm42 39 3
5 ' 側プライマー ： GCACTACCCTACAGGACGGTTA (配列番号 60)  5'-side primer: GCACTACCCTACAGGACGGTTA (SEQ ID NO: 60)
3， 側プライマー ： ACCACTTAGCGCCTTATCCA (配列番号 6 1) 3. Side primer: ACCACTTAGCGCCTTATCCA (SEQ ID NO: 61)
(f ) d n a f o rm43 238  (f) d n a f o rm 43 238
5 ' 側プライマー ： ACCCATGACGTGGTGAAAAC (配列番号 6 2)  5 'primer: ACCCATGACGTGGTGAAAAC (SEQ ID NO: 62)
3 ' 側プライマー ： CGTTGTACTTGGACGTATGGA (配列番号 6 3) 3 'primer: CGTTGTACTTGGACGTATGGA (SEQ ID NO: 63)
( g d n a I o r mo 006 1  (g d n a I o r mo 006 1
5 ' 側プライマー ： CTACGGAAAAGGAGGCTACG (配列番号 64)  5'-side primer: CTACGGAAAAGGAGGCTACG (SEQ ID NO: 64)
3 ' 側プライマー ： CAGTATGGCAGAGTGCGAGT (配列番号 6 5) 定量結果は Glyceraldehyde 3- phosphate dehydrogenase (GAPDH) を内部標 準として、 補正した。 即ち、 各組織での対象遺伝子の発現量 （コピー数/ μ 1 ) を GAPDHの発現量 （コピー数/ μ 1 ) で除し、 定数 （ 1 X 1 06) (注： 1 0の 6乗） を乗して表示した。 表 2 3 'primer: CAGTATGGCAGAGTGCGAGT (SEQ ID NO: 65) The quantitative results were corrected using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal standard. That is, the expression level of the gene of interest in each tissue (copy number / mu 1) divided by the expression amount of GAPDH (copy number / mu 1), the constant (1 X 1 0 6) (Note: 1 0 6 square of ) Is displayed. Table 2
Figure imgf000086_0001
結果をまとめると、 d n a f o r m34810は肺に強力に発現し、骨髄、膝臓、 脂肪でも強く発現した。 d n a f o rm35841は肺、骨髄、脾臓に強く発現し、 糖尿病膝臓と結腸癌で発現が減弱した。 d n a f o rm26225は発現量が少な く、脂肪組織で特異的な発現が観察された。 d n a f o rm391 12は全身で強 く発現し、 特に肝臓、肺、 膝臓で強力に発現した。 d n a f o rm42393は発 現量が低いが、脂肪細胞、糖尿病脖臓で発現が観察された。 d n a f o rm432 38および d n a f o rm60061は、 骨髄に強力に発現し、 肺に強く発現し、 糖尿病の脂肪で発現が増加した。上記クローンの c DNAおよび該 c DNAによつ てコードされるタンパク質は、糖尿病や癌などの治療や診断に応用できる。 また該 c DNAによってコードされるタンパク質は、上記のような mRNA発現の変動が 見られる組織あるいは mRN A発現量の多い組織に関わる疾患に関与している可 能性がある。 実施例 7 各完全長 c DNAがコードするタンパク質の総合解析
Figure imgf000086_0001
In summary, dnafor m34810 was strongly expressed in lung, and also strongly expressed in bone marrow, knee and fat. dnafo rm35841 was strongly expressed in lung, bone marrow and spleen, and was attenuated in diabetic knee and colon cancer. The expression level of dnafo rm26225 was low, and specific expression was observed in adipose tissue. dnafo rm391 12 was strongly expressed in the whole body, especially in liver, lung and knee. Although expression level of dnafo rm42393 was low, expression was observed in adipocytes and diabetic kidney. dnafo rm432 38 and dnafo rm60061 were strongly expressed in bone marrow, strongly expressed in lung, and increased in diabetic fat. The cDNA of the above clone and the cDNA Can be applied to the treatment and diagnosis of diabetes and cancer. Further, the protein encoded by the cDNA may be involved in a disease relating to a tissue in which the mRNA expression is varied as described above or a tissue having a high mRNA expression level. Example 7 Comprehensive analysis of proteins encoded by each full-length cDNA
上記実施例 4、 実施例 5、 およぴ実施例 6の結果より、 下記のとおり、 各完全長 c DNAがコードするタンパク質の総合解析を行った。  Based on the results of Examples 4, 5, and 6, a comprehensive analysis of the proteins encoded by each full-length cDNA was performed as follows.
(1) d n a f o rm51839  (1) d n a f o rm51839
本 c DNAがコードするタンパク質は、キナーゼ欠損ヒ ト TGF ]3受容体スーパ ーフアミリーサブュニット AF387513と類似しており、対照（17. 5日齢胎児全身） と比較して骨、 10日齢新生児皮膚、 精巣、 および、 肺で発現が増加した。  The protein encoded by this cDNA is similar to the kinase-deficient human TGF] 3 receptor superamylysubunit AF387513, and compared to the control (17.5-day-old fetal whole body), bone and 10-day-old Expression increased in neonatal skin, testes, and lungs.
これらのことから、 本タンパク質は、 骨粗鬆症、 自己免疫疾患などの免疫疾患 · 炎症疾患、 癌、 糸球体腎炎、 神経の瘢痕形成、 皮膚の瘢痕形成、 眼の瘢痕形成、 肺 線維症、 動脈傷害、 増殖性網膜症、 網膜剥離、 呼吸促進症候群、 肝硬変、 ボスト心 筋梗塞、 ポスト脈管形成再狭窄、 ケロイド瘢痕形成、 強皮症、 脈管障害、 白内障、 緑内障、及び不妊などに関連する機能を有し、 これらの治療薬の開発に有用である と考えられた。  From these facts, this protein is used for osteoporosis, autoimmune diseases and other immune diseases, inflammatory diseases, cancer, glomerulonephritis, nerve scar formation, skin scar formation, eye scar formation, lung fibrosis, arterial injury, Functions related to proliferative retinopathy, retinal detachment, respiratory distress syndrome, cirrhosis, lost myocardial infarction, postangioplastic restenosis, keloid scarring, scleroderma, vascular disorders, cataract, glaucoma, and infertility It was considered useful for the development of these therapeutic agents.
(2) d n a f o rm34810  (2) d n a f o rm34810
本 c DNAカ コードするタンノ ク質 ίま、 oxidised low density lipoprotein (lectin- like) receptorと類似しており変性 LDL受容体のフアミリーメンバーと推 測され、 肺に強力に発現し、 骨髄、 脖臓、 脂肪でも強く発現した。  The protein encoded by this cDNA is similar to the oxidized low density lipoprotein (lectin-like) receptor and is predicted to be a family member of the denatured LDL receptor.It is strongly expressed in the lung, and is expressed in bone marrow and kidney. However, fat was also strongly expressed.
これらのことから、 本タンパク質は、 高脂血症、 動脈硬化の発症'進展、 粥状動 脈硬化、 動脈硬化を伴う遺伝疾患、 家族性高コレステロール血症、 心筋梗塞、 脳梗 塞、 肺塞栓、 糖尿病、 動脈硬化、 肥満などに関連する機能を有し、 これらの治療薬 の開発に有用であると考えられた。  From these facts, this protein is useful for the development of hyperlipidemia, atherosclerosis, atherosclerosis, genetic disease associated with arteriosclerosis, familial hypercholesterolemia, myocardial infarction, cerebral infarction, pulmonary embolism It has functions related to diabetes, arteriosclerosis, obesity, etc., and was considered to be useful for the development of these therapeutic agents.
(3) d n a f o rm35841 本 c DNA力 Sコードするタンノ ク質 fま、 oxidised low density lipoprotein (lectin - like) receptorと類似しており変性 LDL受容体のフアミリーメンバーと推 測され、 肺、 骨髄、 脖臓に強く発現し、 糖尿病膝臓と結腸癌で発現が減弱した。 これらのことから、 本タンパク質は、 高脂血症、 動脈硬化の発症 ·進展、 粥状動 脈硬化、 動脈硬化を伴う遺伝疾患、 家族性高コレステロール血症、 心筋梗塞、 脳梗 塞、 肺塞栓、 糖尿病、 動脈硬化、 肥満、 癌などに関連する機能を有し、 これらの治 療薬の開発に有用であると考えられた。 (3) dnafo rm35841 This cDNA is similar to the oxidized low density lipoprotein (lectin-like) receptor and is predicted to be a family member of the modified LDL receptor, and is strongly expressed in lung, bone marrow and kidney. The expression was diminished in diabetic knee and colon cancer. Based on these findings, this protein is used for the development and development of hyperlipidemia, arteriosclerosis, atherosclerosis, atherosclerosis-associated genetic disease, familial hypercholesterolemia, myocardial infarction, cerebral infarction, pulmonary embolism It has functions related to diabetes, arteriosclerosis, obesity, cancer, etc., and was considered to be useful for the development of these therapeutic drugs.
(4) d n a f o r m26225  (4) d n a f o r m26225
本 c D N Aがコードするタンパク質は、 ヒ ト ATP - BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP- BINDING CASSETTE TRANSPORTER 3)と類似しており、 薬物の細胞 外排出等にかかわる ABCトランスポーターであると推測され、 発現量が少なく、 脂肪組織で特異的な発現が観察された。  The protein encoded by this cDNA is similar to human ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBER 3 (ATP-BINDING CASSETTE TRANSPORTER 3) and is an ABC transporter involved in the extracellular excretion of drugs, etc. The expression level was low, and specific expression was observed in adipose tissue.
これらのこと力 、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、 ペルォキシソーム病、 横斑変性症、 黄疸などに関連する機能を有し、 これ らの治療薬の開発に有用であると考えられた。  These properties indicate that this protein has functions related to cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral macular degeneration, jaundice, etc. It was considered useful for development.
(5) dn a f o rm41412  (5) dn a f o rm41412
本 c DNA力 Sコードするタン/ ク質 ίま、 ヒ 卜 ATP— binding cassette protein of the (ABCA subfamily) productと類似しており、 薬物の細胞外排出等にかかわる A BCトランスポーターであると推測され、 同一クラスターに属する FANT0M  This cDNA is similar to the S-encoded protein and human ATP—binding cassette protein of the (ABCA subfamily) product, and is presumed to be an ABC transporter involved in drug extracellular efflux. FANT0M belonging to the same cluster
N0:9430067009と相同性が高いので、 これをプローブとして検討すると、 対照 （1 7. 5日齢胎児全身） と比較して、 精巣、 小脳、 胃、 肺、 10日齢新生児小脳など で発現が増加する傾向があった。 Because of high homology with N0: 9430067009, when this was examined as a probe, expression was found in testis, cerebellum, stomach, lung, 10-day-old neonatal cerebellum, etc., compared with control (17.5-day-old fetal whole body). There was a tendency to increase.
これらのこと力 ら、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、ペルォキシソーム病、横斑変性症、黄疸、不妊などに関連する機能を有し、 これらの治療薬の開発に有用であると考えられた。  From these facts, this protein has functions related to multidrug resistance of cancer, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, infertility, etc. It was considered useful in drug development.
(6) d n a f o r m43395  (6) d n a f o r m43395
本 c DNAがコードするタンパク質は、 ヒ ト ATP-binding cassette protein of the (ABCA subfamily) productと類似しており、 薬物の細胞外排出等にかかわる A BCトランスポーターであると推測され、 同一クラスターに属する FANT0M The protein encoded by this cDNA is a human ATP-binding cassette protein of It is similar to the (ABCA subfamily) product, and is presumed to be an ABC transporter involved in the extracellular elimination of drugs, etc., and belongs to the same cluster FANT0M
NO: 9430067009と相同性が高いので、 これをプローブとして検討すると、 対照 （1 7. 5日齢胎児全身） と比較して、 精巣、 小脳、 胃、 肺、 10日齢新生児小脳など で発現が増加する傾向があった。 NO: 9430067009 is highly homologous, and when this is examined as a probe, its expression in testis, cerebellum, stomach, lung, 10-day-old neonatal cerebellum, etc. is higher than that of control (17.5-day-old fetal whole body). There was a tendency to increase.
これらのこと力、ら、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、ペルォキシソーム病、横斑変性症、黄疸、不妊などに関連する機能を有し、 これらの治療薬の開発に有用であると考えられた。  These proteins have functions related to cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral macular degeneration, jaundice, infertility, etc. It was considered useful for the development of therapeutics.
(7) d n a f o rm33133  (7) d n a f o rm33133
本 c DNAがコードするタンパク質は、 ヒ ト ATP - binding cassette A9と類似し ており、薬物の細胞外排出等にかかわる ABCトランスポーターであると推測され、 対照 （1 7. 5日齢胎児全身） と比較して全体的に発現が減弱していたが、 心臓、 副精巣由来脂肪細胞、 胃、 子宮で同等あるいはそれ以上の発現が観察された。  The protein encoded by this cDNA is similar to human ATP-binding cassette A9, and is presumed to be an ABC transporter involved in the extracellular excretion of the drug. Control (17.5-day-old fetal whole body) Although the expression was attenuated as a whole compared with that of E. coli, equivalent or higher expression was observed in the heart, epididymis-derived adipocytes, stomach, and uterus.
これらのこと力 ら、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、 ペルォキシソーム病、 横斑変性症、 黄疸、 不妊、 胃潰瘍などに関連する機 能を有し、 これらの治療薬の開発に有用であると考えられた。  From these facts, this protein has functions related to cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral macular degeneration, jaundice, infertility, gastric ulcer, etc. It was considered useful for the development of these therapeutic agents.
(8) d n a f o rm63577  (8) d n a f o rm63577
本 cDNAがコードするタンパク質は、 ヒ ト ATP - BINDING CASSETTE, SUB-FAMILY Aと類似する配列を有しており、 薬物の細胞外排出等にかかわる ABCトランスポ 一ターであると推測された。  The protein encoded by this cDNA has a sequence similar to that of human ATP-BINDING CASSETTE, SUB-FAMILY A, and was presumed to be an ABC transporter involved in drug extracellular efflux.
これらのこと力、ら、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、 ペルォキシソーム病、 横斑変性症、 黄疸、 不妊、 胃潰瘍などに関連する機 能を有し、 これらの治療薬の開発に有用であると考えられた。  These proteins have functions related to multidrug resistance of cancer, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral macular degeneration, jaundice, infertility, gastric ulcer, etc. However, it was considered useful for the development of these therapeutic agents.
(9) d n a f o rm30449  (9) d n a f o rm30449
本 c DN Aがコードするタンパク質は、 ヒ ト ATP - binding cassette Aと類似する 配列を有しており、薬物の細胞外排出等にかかわる A B Cトランスポーターである と推測され、 同一クラスターに属する FANT0MN0:2310069H21をプローブとして検討 すると、 対照と比較して 0日齢新生児全頭部、 胸腺で強く発現増強し、 0日齢 · 1 0日齢新生児皮膚、 肝臓、 筋肉、 卵巣、 SV40感染組織などで発現が増加した。 これらのこと力 ら、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、 ペルォキシソーム病、 横斑変性症、 黄疸、 不妊、 免疫疾患、 炎症性疾患、 感染症などに関連する機能を有し、これらの治療薬の開発に有用であると考えられ た。 The protein encoded by this cDNA has a sequence similar to that of human ATP-binding cassette A, is presumed to be an ABC transporter involved in the extracellular excretion of drugs, etc., and belongs to the same cluster FANT0MN0: Consider using 2310069H21 as a probe As a result, the expression was strongly enhanced in the whole head and thymus of the 0-day-old newborn compared to the control, and increased in the skin, liver, muscle, ovary, and SV40-infected tissues of the 0-day and 10-day old newborns. Based on these facts, this protein is used in multidrug resistance of cancer, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, infertility, immune disease, inflammatory disease, infectious disease, etc. It has related functions and was considered useful for the development of these therapeutic agents.
(1 0) d n a f o r m4 23 9 3  (1 0) d n a f o r m4 23 9 3
本 c DNAがコードするタンパク質は、 マウス ATP-binding cassette transporter ABCA3と類似しており、 薬物の細胞外排出等にかかわる A B Cトラン スポーターであると推測され、発現量が低いが、 脂肪細胞、 糖尿病脾臓で発現が観 察された。  The protein encoded by this cDNA is similar to mouse ATP-binding cassette transporter ABCA3, and is presumed to be an ABC transporter involved in the extracellular excretion of drugs, etc., and its expression level is low, but fat cells and diabetic spleen Expression was observed in.
これらのこと力 ら、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、 ペルォキシソーム病、 横斑変性症、 黄疽などに関連する機能を有し、 これ らの治療薬の開発に有用であると考えられた。  From these facts, this protein has functions related to cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, etc. It was considered useful in drug development.
(1 1) d n a f o r m3 9 1 1 2  (1 1) d n a f o r m3 9 1 1 2
本 c DNAがコードするタンパク質は、 ヒ ト ATP11C gene for ATPase, Class VI, type 11Cと類似しており、 薬物やリン脂質、 両親媒性物質などの輸送にかかわる A BCトランスポーターであると推測され、 全身で強く発現し、 特に肝臓、 肺、 膝臓 で強力に発現した。  The protein encoded by this cDNA is similar to human ATP11C gene for ATPase, Class VI, type 11C, and is presumed to be an ABC transporter involved in the transport of drugs, phospholipids, amphiphiles, etc. It was strongly expressed in the whole body, especially in liver, lung and knee.
これらのこと力 ら、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、 ペルォキシソーム病、 横斑変性症、 黄疸、 高血圧などに関連する機能を有 し、 これらの治療薬の開発に有用であると考えられた。  From these facts, this protein has functions related to cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral macular degeneration, jaundice, hypertension, etc. It was considered useful in drug development.
(1 2) d n a f o r m4 32 38  (1 2) d n a f o r m4 32 38
本 c DNAがコードするタンパク質は、 ヒ ト ATP - binding cassetteと類似する配 列を有しており、 薬物、 カルシウム、 リン脂質などの輸送に関わると推測され、 骨 髄に強力に発現し、 肺に強く発現し、 糖尿病の脂肪で発現が増加した。  The protein encoded by this cDNA has a sequence similar to that of the human ATP-binding cassette, and is presumed to be involved in the transport of drugs, calcium, phospholipids, etc., and is strongly expressed in the bone marrow, And increased expression in diabetic fat.
これらのことから、 本タンパク質は、 癌の多剤耐性、 囊胞性繊維症、 糖尿病、 動 脈硬化、 ペルォキシソーム病、 横斑変性症、 黄疽、 高血圧、 免疫疾患、 炎症疾患、 などに関連する機能を有し、 これらの治療薬の開発に有用であると考えられた。These results indicate that this protein is useful for cancer multidrug resistance, cystic fibrosis, diabetes, It has functions related to pulse sclerosis, peroxisome disease, lateral macular degeneration, jaundice, hypertension, immune diseases, inflammatory diseases, etc., and was considered to be useful for the development of these therapeutic agents.
(13) d n a f o rm60061 (13) d n a f o rm60061
本 c DNAがコードするタンパク質は、 ヒ ト ATP- binding cassetteと類似する配 列を有しており、 薬物、 カルシウム、 リン脂質などの輸送に関わると推測され、 骨 髄に強力に発現し、 肺に強く発現し、 糖尿病の脂肪で発現が増加した。  The protein encoded by this cDNA has a sequence similar to that of the human ATP-binding cassette and is presumed to be involved in the transport of drugs, calcium, phospholipids, etc., and is strongly expressed in the bone marrow, And increased expression in diabetic fat.
これらのこと力 ら、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、 ペルォキシソーム病、 横斑変性症、 黄疸、 高血圧、 免疫疾患、 炎症疾患、 などに関連する機能を有し、 これらの治療薬の開発に有用であると考えられた。 Based on these facts, this protein has functions related to cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral macular degeneration, jaundice, hypertension, immune diseases, inflammatory diseases, etc. It was considered useful for the development of these therapeutic agents.
(14) d n a f o rm49889 (14) d n a f o rm49889
本 c DNAがコードするタンパク質は、 ヒ ト Potential  The protein encoded by this cDNA is human Potential
phospholipid- transporting ATPaseと類似する配列を有しており、 薬物、 カルシゥ ム、 リン脂質などの輸送に関わると推測され、対照と比較して全体的に発現が増強 していたが、 脂肪細胞、 膝臓で発現が増強し、 腎臓、 肝臓、 精巣などで発現増加傾 向が観察された。 It has a sequence similar to phospholipid-transporting ATPase, and is presumed to be involved in the transport of drugs, calcium, phospholipids, etc., and its expression was enhanced as a whole compared to controls. Expression was enhanced in the kidney, and a tendency for increased expression was observed in the kidney, liver and testis.
これらのこと力 ら、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、 ペルォキシソーム病、 横斑変性症、 黄疽、 高血圧、 不妊、 免疫疾患、 炎症 疾患、 などに関連する機能を有し、 これらの治療薬の開発に有用であると考えられ た。  From these facts, this protein is useful for cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, high blood pressure, infertility, immune diseases, inflammatory diseases, etc. It has related functions and was considered useful for the development of these therapeutic agents.
(1 5) d n a f o rm60440  (1 5) d n a f o rm60440
本 c D N Aがコードするタンパク質は、 CALCIUM-TRANSPORTING ATPASE 3と類似 する配列を有しており、 薬物、 カルシウム、 リン脂質などの輸送に関わると推測さ れ、 同一クラスターに属する d n a f o rm49889と相同性が高いため、 これ をプローブとして検討すると、 脂肪細胞、 脾臓で発現が増強し、 腎臓、 肝臓、 精巣 などで発現増加傾向が見られた。  The protein encoded by this cDNA has a sequence similar to CALCIUM-TRANSPORTING ATPASE 3 and is presumed to be involved in the transport of drugs, calcium, phospholipids, etc., and has homology to dnafo rm49889 belonging to the same cluster. When this was used as a probe, expression was enhanced in adipocytes and spleen, and increased in kidney, liver and testis.
これらのこと力 ら、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、 ペルォキシソーム病、 横斑変性症、 黄疽、 高血圧、 不妊、 免疫疾患、 炎症 疾患、 などに関連する機能を有し、 これらの治療薬の開発に有用であると考えられ た。 Based on these strengths, this protein is effective for cancer multidrug resistance, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, hypertension, infertility, immune disease, inflammation It has functions related to diseases, etc., and was considered useful for the development of these therapeutic agents.
(16) d n a f o rm67267  (16) d n a f o rm67267
本 c DNA力 Sコードするタンノ ク質 fま、 Potential pho spho 1 i p i d- transport i ng ATPaseと類似する配列を有しており、 薬物、 カルシウム、 リン脂質などの輸送に関 わると推測され、同一クラスターに属する d n a f o rm49889と相同性が高 いため、これをプローブとして検討すると、脂肪細胞、脾臓で発現が増強し、腎臓、 肝臓、 精巣などで発現増加傾向が見られた。  This cDNA has a sequence similar to that of the S-encoded protein f, Potential phospho 1 ipid-transporting ATPase, and is presumed to be involved in the transport of drugs, calcium, phospholipids, etc. Since it has high homology to dnafo rm49889 belonging to the same cluster, when this was used as a probe, expression was enhanced in adipocytes and spleen, and increased in kidney, liver and testis.
これらのことから、 本タンパク質は、 癌の多剤耐性、 嚢胞性繊維症、 糖尿病、 動 脈硬化、 ペルォキシソーム病、 横斑変性症、 黄疽、 高血圧、 不妊、 免疫疾患、 炎症 疾患、 などに関連する機能を有し、 これらの治療薬の開発に有用であると考えられ た。  Therefore, this protein is related to multidrug resistance of cancer, cystic fibrosis, diabetes, arteriosclerosis, peroxisome disease, lateral degeneration, jaundice, high blood pressure, infertility, immune disease, inflammatory disease, etc. It is considered to be useful for the development of these therapeutic agents.
(17) d n a f o rm38308  (17) d n a f o rm38308
本 c DNAがコードするタンパク質は、 ヒ ト補体 C5 precursorと類似しており、 炎症ゃァレルギ一反応に関わる機能を有するィムノグロブリン様タンパク質と推 測された。  The protein encoded by this cDNA was similar to the human complement C5 precursor, and was estimated to be an immunoglobulin-like protein having a function related to the inflammatory allergy reaction.
これらのことから、本タンパク質は、 全身性エリテマトーデス ·先天性補体成分 欠損症 ·関節リウマチ ·自己免疫疾患などの免疫性疾患、 糸球体腎炎 ·肝炎などの 炎症性疾患、 感染症、 癌などに関連する機能を有し、 これらの診断薬、 治療薬の開 発に有用であると考えられた。  From these facts, this protein is useful for the treatment of systemic lupus erythematosus, congenital complement deficiency, rheumatoid arthritis, immune diseases such as autoimmune diseases, inflammatory diseases such as glomerulonephritis, hepatitis, infectious diseases, cancer, etc. It has related functions and is considered useful for the development of these diagnostics and therapeutics.
(18) d n a f o rm38407  (18) d n a f o rm38407
本 c DNAがコードするタンパク質は、 ヒ ト補体 C5 precursorと類似しており、 炎症ゃァレルギ一反応に関わる機能を有するィムノグロブリン様タンパク質と推 測された。  The protein encoded by this cDNA was similar to the human complement C5 precursor, and was estimated to be an immunoglobulin-like protein having a function related to the inflammatory allergy reaction.
これらのことから、 本タンパク質は、 全身性エリテマトーデス ·先天性補体成分 欠損症 ·関節リウマチ ·自己免疫疾患などの免疫性疾患、 糸球体腎炎 ·肝炎などの 炎症性疾患、 感染症、 癌などに関連する機能を有し、 これらの診断薬、 治療薬の開 発に有用であると考えられた。 From these facts, this protein is useful for the treatment of systemic lupus erythematosus, congenital complement deficiency, rheumatoid arthritis, immune diseases such as autoimmune diseases, inflammatory diseases such as glomerulonephritis, hepatitis, infectious diseases, cancer, etc. With related functions, the development of these diagnostics and therapeutics It was considered useful for departure.
(19) d n a f o rm36427  (19) d n a f o rm36427
本 c DNAがコードするタンパク質は、マクログロブリンの特徴を示す配列が存 在しており、 同一クラスターに属する FANTOMNO:4831440K17をプローブとして検討 すると、 対照と比較して全体的に発現が減弱していたが、 胎盤、 10日齢新生児皮 膚、 肺、 精巣などで対照と同程度の発現が観察された。  The protein encoded by this cDNA has a sequence exhibiting the characteristics of macroglobulin, and when FANTOMNO: 4831440K17 belonging to the same cluster was examined as a probe, the overall expression was reduced compared to the control. However, expression similar to that of the control was observed in the placenta, the skin of the 10-day-old newborn baby, the lung, the testis, and the like.
これらのことから、 本タンパク質は、 全身性エリテマトーデス ·先天性補体成分 欠損症 ·関節リウマチ ·自己免疫疾患などの免疫性疾患、 糸球体腎炎 ·肝炎などの 炎症性疾患、 感染症、 癌、 不妊などに関連する機能を有し、 これらの診断薬、 治療 薬の開発に有用であると考えられた。  From these facts, this protein is used for systemic lupus erythematosus, congenital complement deficiency, rheumatoid arthritis, immune diseases such as autoimmune diseases, glomerulonephritis, inflammatory diseases such as hepatitis, infectious diseases, cancer, infertility It has functions related to the above, and is considered to be useful for the development of these diagnostics and therapeutics.
(20) d n a f o rm36949  (20) d n a f o rm36949
本 c DNAがコードするタンパク質は、 leukocyte Fc receptors や、 細胞接着 分子 PECAM- 1と類似する構造をもつ IFGPファミリ一であり、 ィムノグロプリン様構 造を持つ受容体あるいは接着に関わるタンパク質であることが推測された。  The protein encoded by this cDNA is a member of the IFGP family that has a structure similar to leukocyte Fc receptors and the cell adhesion molecule PECAM-1.It is speculated that this protein is a receptor having an immunoglobulin-like structure or a protein involved in adhesion. Was done.
これらのことから、 本タンパク質は、 免疫性疾患、 炎症性疾患、 細胞接着などに 関連する機能を有し、 これらの診断薬、 治療薬の開発に有用であると考えられた。 These results suggest that this protein has functions related to immune diseases, inflammatory diseases, cell adhesion, etc., and is useful for the development of these diagnostics and therapeutics.
(21) d n a f o r m44492 (21) d n a f o r m44492
本 c DNAがコードするタンパク質は、 leukocyte Fc receptors や、 細胞接着 分子 PECAM- 1と類似する構造をもつ IFGPファミリ一であり、 ィムノグロプリン様構 造を持つ受容体あるいは接着に関わるタンパク質であることが推測された。  The protein encoded by this cDNA is a member of the IFGP family that has a structure similar to leukocyte Fc receptors and the cell adhesion molecule PECAM-1.It is speculated that this protein is a receptor having an immunoglobulin-like structure or a protein involved in adhesion. Was done.
これらのことから、 本タンパク質は、 免疫性疾患、 炎症性疾患、 細胞接着などに 関連する機能を有し、 これらの診断薬、 治療薬の開発に有用であると考えられた。 産業上利用の可能性  These results suggest that this protein has functions related to immune diseases, inflammatory diseases, cell adhesion, etc., and is useful for the development of these diagnostics and therapeutics. Possibility of industrial use
本発明のタンパク質およびそれをコードする DNAは、 TGF i3受容体フアミリ 一との結合活性、 変性 L D Lとの結合活性、 A T P結合性運搬体活性、 ィムノグロ ブリン様タンパク質活性等を有することから、該タンパク質あるいはそれをコード する DN Aを用いてこれらの活性を調節する物質をスクリーニングすることがで き、 該タンパク質が関連する疾患等に作用し得る医薬の開発に有用である。 本出願は、 2002年 4月 23日付けの日本特許出願（特願 2002- 1208 53)、 2002年 12月 4日付けの日本特許出願（特願 2002— 352270)、 2002年 4月 26日付けの日本特許出願 （特願 2002— 125934) 、 20 02年 4月 30日付けの日本特許出願 （特願 2002— 128505) 、 2002 年 12月 4日付けの日本特許出願 （特願 2002— 352619) 、 2002年 5 月 2日付けの日本特許出願 （特願 2002— 130914) 、 および 2002年 1 2月 4日付けの日本特許出願（特願 2002-352730)に基づくものであり、 その内容はここに参照として取り込まれる。 また、本明細書にて引用した文献の内 容もここに参照として取り込まれる。 Since the protein of the present invention and DNA encoding the same have binding activity to TGF i3 receptor family, binding activity to denatured LDL, ATP-binding transporter activity, immunoglobulin-like protein activity, etc. Or code it Thus, a substance that regulates these activities can be screened using a DNA, and is useful for the development of a drug that can act on diseases and the like in which the protein is associated. This application was filed on April 23, 2002 (Japanese Patent Application 2002-120853), filed on December 4, 2002 (Japanese Patent Application 2002-352270), and filed on April 26, 2002. Japanese patent application (Japanese Patent Application 2002-125934), Japanese patent application dated April 30, 2002 (Japanese Patent Application 2002-128505), Japanese patent application dated December 4, 2002 (Japanese Patent Application 2002-352619) , Based on a Japanese patent application filed on May 2, 2002 (Japanese Patent Application No. 2002-130914) and a Japanese patent application filed on January 4, 2002 (Japanese Patent Application No. 2002-352730). Captured as a reference. The contents of the documents cited in the present specification are also incorporated herein by reference.

Claims

請求の範囲 The scope of the claims
1. 以下の （a) または （b) のタンパク質。 1. The following protein (a) or (b):
( a ) 配列番号 2および 3に記載のァミノ酸配列からなるタンパク質。  (a) a protein comprising the amino acid sequence of SEQ ID NO: 2 or 3;
( b )配列番号 2および 3に記載のァミノ酸配列において 1若しくは数個のァミノ 酸が欠失、置換及び または付加されたアミノ酸配列からなり、 かつ TGF J3受容 体フアミリーとの結合活性を有するタンパク質。  (b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NOs: 2 and 3, and which has binding activity to a TGF J3 receptor family .
2. 請求項 1に記載のタンパク質をコードする DNA。  2. A DNA encoding the protein of claim 1.
3. 請求項 1に記載のタンパク質をコードする完全長 c D N A。  3. A full-length cDNA encoding the protein of claim 1.
4. 以下の （a) 、 （b)または （c) の何れかの DNA。  4. DNA of any of the following (a), (b) or (c):
(a) 配列番号 1に記載の塩基配列を有する DNA。  (a) DNA having the nucleotide sequence of SEQ ID NO: 1.
(b) 配列番号 1に記載の塩基配列において、 1若しくは数個の塩基が欠失、 置換 及び または付加された塩基配列を有し、かつ T G F /3受容体フアミリーとの結合 活性を有するタンパク質をコードする DNA。  (b) a protein having the nucleotide sequence of SEQ ID NO: 1 in which one or several nucleotides are deleted, substituted and / or added, and which has a binding activity to a TGF / 3 receptor family; The encoding DNA.
(c)配列番号 1に記載の塩基配列あるいはその相補配列を有する DN Aとストリ ンジェントな条件下でハイブリダイズすることができる塩基配列を有し、かつ T G F 受容体フアミリーとの結合活性を有するタンパク質をコードする DNA。 (c) a protein having a nucleotide sequence capable of hybridizing under stringent conditions to DNA having the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence, and having a binding activity to a TGF receptor family DNA that encodes
5. 以下の （a) または （b) のタンパク質。 5. The following (a) or (b) protein:
( a ) 配列番号 14または 15に記載のァミノ酸配列からなるタンパク質。  (a) a protein comprising the amino acid sequence of SEQ ID NO: 14 or 15;
( b )配列番号 14または 15に記載のァミノ酸配列において 1若しくは数個のァ ミノ酸が欠失、置換及び/または付カ卩されたアミノ酸配列からなり、かつ変性 LD (b) an amino acid sequence represented by SEQ ID NO: 14 or 15, wherein one or several amino acids are deleted, substituted and / or added, and
Lとの結合活性を有するタンパク質。 A protein having an activity of binding to L.
6. 請求項 5に記載のタンパク質をコードする DNA。  6. A DNA encoding the protein of claim 5.
7. 請求項 5に記載のタンパク質をコードする完全長 c DNA。  7. A full-length cDNA encoding the protein of claim 5.
8. 以下の （a) 、 （b)又は （c) の何れかの DNA。  8. DNA of any of the following (a), (b) or (c):
(a) 配列番号 12または 13に記載の塩基配列を有する DNA。  (a) DNA having the nucleotide sequence of SEQ ID NO: 12 or 13.
( b )配列番号 12または 13に記載の塩基配列において、 1若しくは数個の塩基 が欠失、置換及び または付カ卩された塩基配列を有し、 かつ変性 LDLとの結合活 性を有するタンパク質をコードする DNA。 (b) in the base sequence of SEQ ID NO: 12 or 13, one or several bases DNA encoding a protein having a base sequence in which is deleted, substituted or added, and which has a binding activity with modified LDL.
(c)配列番号 12または 1 3に記載の塩基配列あるいはその相補配列を有する D NAとストリンジェントな条件下でハイブリダイズすることができる塩基配列を 有し、 かつ変性 LDLとの結合活性を有するタンパク質をコードする DNA。  (c) has a nucleotide sequence capable of hybridizing under stringent conditions with DNA having the nucleotide sequence of SEQ ID NO: 12 or 13 or its complementary sequence, and has a binding activity with denatured LDL DNA that encodes a protein.
9. 以下の （a) または （b) のタンパク質。  9. The following (a) or (b) protein:
(a) 配列番号 29〜41のいずれかに記載のアミノ酸配列からなるタンパク質。 (a) a protein comprising the amino acid sequence of any one of SEQ ID NOs: 29 to 41;
(b)配列番号 29〜41のいずれかに記載のアミノ酸配列において 1若しくは数 個のアミノ酸が欠失、置換及び/または付加されたアミノ酸配列からなり、かつ A T P結合性運搬体活性を有するタンパク質。 (b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted and / or added in the amino acid sequence of any of SEQ ID NOs: 29 to 41, and which has ATP-binding carrier activity;
10. 請求項 9に記載のタンパク質をコードする DNA。  10. A DNA encoding the protein of claim 9.
1 1. 請求項 9に記載のタンパク質をコードする完全長 cDNA。  1 1. A full-length cDNA encoding the protein of claim 9.
12. 以下の （a) 、 （b)又は （c) の何れかの DNA。  12. DNA of any of the following (a), (b) or (c):
(a) 配列番号 16〜28のいずれかに記載の塩基配列を有する DNA。  (a) DNA having the nucleotide sequence of any one of SEQ ID NOs: 16 to 28.
( b )配列番号 16〜 28のいずれかに記載の塩基配列において、 1若しくは数個 の塩基が欠失、置換及び Zまたは付加された塩基配列を有し、 かつ AT P結合性運 搬体活性を有するタンパク質をコードする DNA。  (b) the base sequence of any one of SEQ ID NOs: 16 to 28, wherein one or several bases have a base sequence with deletion, substitution, Z or addition, and ATP-binding carrier activity; DNA encoding a protein having
( c )配列番号 16〜 28のいずれかに記載の塩基配列あるいはその相補配列を有 する DNAとストリンジェントな条件下でハイブリダィズすることができる塩基 配列を有し、かつ ATP結合性運搬体活性を有するタンパク質をコードする DNA。 (c) having a base sequence capable of hybridizing under stringent conditions with a DNA having the base sequence of any of SEQ ID NOs: 16 to 28 or a complementary sequence thereof, and exhibiting ATP-binding carrier activity. DNA encoding a protein having
13. 以下の （a) または （b) のタンパク質。 13. The following protein (a) or (b):
(a) 配列番号 47〜51のいずれかに記載のアミノ酸配列からなるタンパク質。 (a) a protein comprising the amino acid sequence of any one of SEQ ID NOs: 47 to 51;
(b)配列番号 47〜51のいずれかに記載のアミノ酸配列において 1若しくは数 個のアミノ酸が欠失、置換及び/または付加されたアミノ酸配列からなり、かつィ ムノグロブリン様タンパク質活性を有するタンパク質。 (b) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 47 to 51 with one or several amino acids deleted, substituted and / or added, and having an immunoglobulin-like protein activity;
14. 請求項 13に記載のタンパク質をコードする DNA。  14. A DNA encoding the protein of claim 13.
15. 請求項 13に記載のタンパク質をコードする完全長 c DNA。 15. A full-length cDNA encoding the protein of claim 13.
16. 以下の （a) 、 （b)又は （c) の何れかの DNA。 16. DNA of any of the following (a), (b) or (c):
(a) 配列番号 42〜46のいずれかに記載の塩基配列を有する DNA。  (a) DNA having the nucleotide sequence of any one of SEQ ID NOs: 42 to 46.
(b)配列番号 42〜46のいずれかに記載の塩基配列において、 1若しくは数個 の塩基が欠失、置換及び/または付加された塩基配列を有し、かつィムノグロプリ ン様タンパク質活性を有するタンパク質をコードする D N A。  (b) a protein having a nucleotide sequence in which one or several bases are deleted, substituted and / or added in the nucleotide sequence set forth in any one of SEQ ID NOs: 42 to 46, and having an immunoglobulin-like protein activity DNA that encodes
(c)配列番号 42〜46のいずれかに記載の塩基配列あるいはその相補配列を有 する DNAとストリンジェントな条件下でハイブリダィズすることができる塩基 配列を有し、かつィムノグロプリン様タンパク質活性を有するタンパク質をコード する DNA。  (c) a protein having a nucleotide sequence capable of hybridizing under stringent conditions to a DNA having the nucleotide sequence of any of SEQ ID NOs: 42 to 46 or a sequence complementary thereto, and having an immunoglobulin-like protein activity DNA that encodes
1 7. 請求項 2〜4、 6〜8、 10〜： 1 2、 14〜 16のいずれかに記載の D N Aを含む組換えベクター。  1 7. A recombinant vector containing the DNA according to any one of claims 2 to 4, 6 to 8, 10 to: 12, 14 to 16.
18. 請求項 2〜4、 6〜8、 10〜： 12、 14〜 16のいずれかに記載の D N Aまたは請求項 17に記載の組み換えべクターを導入した遺伝子導入細胞または 該細胞からなる個体。  18. A transgenic cell into which the DNA according to any one of claims 2 to 4, 6 to 8, and 10 to 12 or 14 to 16 or the recombinant vector according to claim 17 has been introduced, or an individual comprising the cell.
19. 請求項 18に記載の細胞により産生される、 請求項 1、 5、 9、 または 1 3に記載のタンパク質。  19. The protein of claim 1, 5, 9, or 13, produced by the cell of claim 18.
20. 請求項 2〜4、 6〜8、 10〜： 1 2、 14〜： 16のいずれかに記載の D N Aの塩基配列中の連続した 5〜100塩基と同じ配列を有するセンスオリゴヌク レオチド、当該センスオリゴヌクレオチドと相補的な配列を有するアンチセンスォ リゴヌクレオチド、 及び、 当該センス又はアンチセンスオリゴヌクレオチドのオリ ゴヌクレオチド誘導体から成る群から選ばれるオリゴヌクレオチド。  20. A sense oligonucleotide having the same sequence as 5 to 100 consecutive bases in the base sequence of the DNA according to any one of claims 2 to 4, 6 to 8, 10 to 12, and 14 to 16, An oligonucleotide selected from the group consisting of an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide, and an oligonucleotide derivative of the sense or antisense oligonucleotide.
21. 請求項 1、 5、 9、 13、 または 19に記載のタンパク質に特異的に結合 する抗体あるいはその部分フラグメント。 21. An antibody or a partial fragment thereof that specifically binds to the protein of claim 1, 5, 9, 13, or 19.
22. 抗体がモノクローナル抗体である請求項 21に記載の抗体。  22. The antibody according to claim 21, wherein the antibody is a monoclonal antibody.
23. モノクローナル抗体が請求項 1、 5、 9、 13、 または 1 9に記載のタン パク質の活性を中和する作用を有することを特徴とする請求項 22に記載の抗体。 23. The antibody according to claim 22, wherein the monoclonal antibody has an action of neutralizing the activity of the protein according to claim 1, 5, 9, 13, or 19.
24. 請求項 1、 5、 9、 13、 または 19に記載のタンパク質と被検物質を接 触させ、該被検物質による該タンパク質が有する活性の変化を測定することを特徴 とする、 該タンパク質の活性調節物質のスクリーニング方法。 24. Contact the test substance with the protein of claim 1, 5, 9, 13, or 19. A method for screening a substance that modulates the activity of the protein, characterized by measuring the change in the activity of the protein caused by the test substance.
25. 請求項 18に記載の遺伝子導入細胞と被検物質を接触させ、該細胞に導入 されている DN Aの発現レベルの変化を検出することを特徴とする、該 DNAの発 現調節物質のスクリーニング方法。  25. Contacting the test substance with the gene-introduced cell according to claim 18, and detecting a change in the expression level of DNA introduced into the cell, wherein the expression of the DNA is regulated. Screening method.
26. 請求項 1、 5、 9、 または 13に記載のタンパク質のアミノ酸配列から選 択される少なくとも 1以上のアミノ酸配列情報、および Zまたは請求項 2〜4、 6 〜8、 10〜1 2、 14〜16のいずれかに記載の DN Aの塩基配列から選択され る少なくとも 1以上の塩基配列情報を保存したコンピュータ読み取り可能記録媒 体。  26. At least one or more amino acid sequence information selected from the amino acid sequence of the protein according to claim 1, 5, 9, or 13, and Z or claim 2 to 4, 6 to 8, 10 to 12, A computer-readable recording medium storing at least one or more nucleotide sequence information selected from the nucleotide sequence of DNA according to any one of 14 to 16.
27. 請求項 1、 5、 9、 または 13に記載のタンパク質、 および Zまたは請求 項 2〜4、 6〜8、 10〜12、 14〜 16のいずれ力に記載の DNAを結合させ た担体。  27. A carrier to which the protein according to claim 1, 5, 9, or 13 and Z or the DNA according to any one of claims 2 to 4, 6 to 8, 10 to 12, and 14 to 16 are bound.
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