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WO2003091435A1 - Nouvelles proteines et differents adn les codant - Google Patents

Nouvelles proteines et differents adn les codant 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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Prior art keywords
protein
seq
dna
amino acid
sequence
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PCT/JP2003/005174
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English (en)
Japanese (ja)
Inventor
Yoshihide Hayashizaki
Mamoru Kamiya
Hideo Kubodera
Original Assignee
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/fr

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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

Les séquences de base de clones d'ADNc impliquées dans des échantillothèques d'ADNc pleine longueur catalogués. Concernant les clones d'ADNc comportant de nouvelles séquences provenant de parmi celles qui sont analysées ci-dessus, les activités physiologiques (fonctions) des protéines ainsi codées sont spécifiées. Sur la base de ces activités physiologiques (fonctions), on propose un procédé permettant d'utiliser les protéines et les ADN codant ces protéines. L'invention concerne plus particulièrement les protéines (a) et (b) suivantes, les ADN les codant et un procédé permettant de les utiliser. La protéine (a) est une protéine comprenant l'une des séquences d'acides aminés SEQ ID: 2, 3, 14, 15, 29 à 41 et 47 à 51. La protéine (b) est une protéine comprenant l'une des séquences d'acides aminés dérivant des SEQ ID: 2, 3, 14, 15, 29 à 41 et 47 à 51 par suppression, substitution et/ou adjonction d'au moins un acide aminé présentant une activité de liaison avec la famille des récepteurs TGFB, une activité de liaison à un LDL modifié, une activité de transporteur se liant à l'ATP, ou une activité de protéine de type immunoglobuline.
PCT/JP2003/005174 2002-04-23 2003-04-23 Nouvelles proteines et differents adn les codant WO2003091435A1 (fr)

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AU2003235099A AU2003235099A1 (en) 2002-04-23 2003-04-23 Novel proteins and dnas encoding the same

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JP2002-120853 2002-04-23
JP2002120853 2002-04-23
JP2002125934 2002-04-26
JP2002-125934 2002-04-26
JP2002-128505 2002-04-30
JP2002128505 2002-04-30
JP2002130914 2002-05-02
JP2002-130914 2002-05-02
JP2002352619 2002-12-04
JP2002352730 2002-12-04
JP2002-352730 2002-12-04
JP2002-352619 2002-12-04
JP2002-352270 2002-12-04
JP2002352270 2002-12-04

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009026660A1 (fr) 2007-08-30 2009-03-05 Walter And Eliza Hall Institute Of Medical Research Marqueur de cellules dendritiques et son utilisation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001258576A (ja) * 2000-05-22 2001-09-25 Hitachi Ltd プライマー設計システム
US20010039036A1 (en) * 1991-05-10 2001-11-08 The Salk Institute For Biological Studies. Cloning and recombinant production of receptor(s) of the activin/TGF-beta superfamily

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010039036A1 (en) * 1991-05-10 2001-11-08 The Salk Institute For Biological Studies. Cloning and recombinant production of receptor(s) of the activin/TGF-beta superfamily
JP2001258576A (ja) * 2000-05-22 2001-09-25 Hitachi Ltd プライマー設計システム

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KONDO S. ET AL.: "Computational analysis of full-length mouse cDNAs compared with human genome sequences", MAMM GENOME, vol. 12, no. 9, 2001, pages 673 - 677, XP002965278 *
THE FANTOM CONSORTIUM AND THE RIKEN GENOME EXPLORATION RESEARCH GROUP PHASE I & II TEAM: "Analysis of the mouse transcriptome based on functional annotation of 60, 770 full-length cDNAs", NATURE, vol. 420, 5 December 2002 (2002-12-05), pages 563 - 573, XP002965277 *
THE FANTOM CONSORTIUM AND THE RIKEN GENOME EXPLORATION RESEARCH GROUP PHASE II TEAM: "Functional annotation of a full-length mouse cDNA collection", NATURE, vol. 409, 8 February 2001 (2001-02-08), pages 685 - 690, XP002952962 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009026660A1 (fr) 2007-08-30 2009-03-05 Walter And Eliza Hall Institute Of Medical Research Marqueur de cellules dendritiques et son utilisation
EP2195346A4 (fr) * 2007-08-30 2013-01-23 Inst Medical W & E Hall Marqueur de cellules dendritiques et son utilisation
US8426565B2 (en) 2007-08-30 2013-04-23 Walter And Eliza Hall Institute Of Medical Research Dendritic cell marker and uses thereof
AU2008294074B2 (en) * 2007-08-30 2015-01-22 Walter And Eliza Hall Institute Of Medical Research Dendritic cell marker and uses thereof
US9988431B2 (en) 2007-08-30 2018-06-05 The Walter And Eliza Hall Institute Of Medical Research Dendritic cell marker and uses thereof

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AU2003235099A1 (en) 2003-11-10

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