WO2003089644A1 - Nouvelles proteines et adn codant pour elles - Google Patents

Nouvelles proteines et adn codant pour elles Download PDF

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WO2003089644A1
WO2003089644A1 PCT/JP2003/004983 JP0304983W WO03089644A1 WO 2003089644 A1 WO2003089644 A1 WO 2003089644A1 JP 0304983 W JP0304983 W JP 0304983W WO 03089644 A1 WO03089644 A1 WO 03089644A1
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protein
seq
amino acid
dna
sequence
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PCT/JP2003/004983
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English (en)
Japanese (ja)
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Yoshihide Hayashizaki
Mamoru Kamiya
Hideo Kubodera
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Riken
K. K. Dnaform
Mitsubishi Chemical Corporation
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Priority to AU2003227428A priority Critical patent/AU2003227428A1/en
Publication of WO2003089644A1 publication Critical patent/WO2003089644A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors

Definitions

  • the present invention relates to 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 a gene into which the DNA has been introduced.
  • the present invention relates to an introduced cell, and an antibody specifically binding to the protein. Background art.
  • cDNA and its nucleotide sequence analysis are indispensable for analyzing the physiological activity of a protein expressed in a living body and developing a method for utilizing the protein based on the activity.
  • creating a library that catalogs full-length cDNAs for all genotypes is an important issue of the Human Genome Project.
  • the cataloged library means that the cDNAs contained in the library have no duplication, and refers to a library containing one type of each cDNA.
  • the full-length cDNA closing method is described in JP-A-9-1248187 and JP-A-10-127291.
  • This method comprises the steps of binding a tag molecule to the diol structure present at the 5, capsite of mRNA, converting the mRNA bound to the tag molecule to type II, and using RNA-dT as a primer to reverse-transcribe RNA-DNA complex.
  • a method comprising preparing a body, and separating a complex having a DNA corresponding to the full-length mRNA from the complex using the function of a tag molecule.
  • the full-length cDNA library produced by such a technique does not necessarily include all the elements that are different evenly as individual elements of the library. Some clones do not exist. Since clones that exist in only a trace amount are likely to be novel, a subtraction method—a normalization method—for enriching such clones has also been developed (Japanese Patent Laid-Open No. 2000-325080; Carninci, P. et al., Genomics, 37, 327-336 (1996)).
  • nucleotide sequence of each clone of the full-length cDNA library obtained as described above can be identified by a known method, and the nucleotide sequence can be identified, but the physiology of the protein encoded by the cDNA can be identified. Activity remains unknown. 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, identifies the physiological activity of the protein encoded by the nucleotide sequence.
  • the purpose of the present invention is to propose a method of using a protein based on activity and a DNA encoding the protein.
  • the present inventors analyzed the nucleotide sequence of a cDNA clone in a mouse full-length cDNA library and searched a database based on the homology of the sequence, and found that the sequence showed an enzymatic activity (eg, a protease activity). , Phosphodiesterase activity, transferase activity, or isomerase activity), proteolytic enzyme inhibitory activity or a sequence specific to a protein having a physiological activity other than enzyme activity. It was identified as having a proteolytic enzyme inhibitory activity or a physiological activity other than the enzymatic activity.
  • a protein comprising an amino acid sequence in which several amino acids have been deleted, substituted and / or added, and having an enzyme activity or a protease inhibitory activity.
  • a protein comprising the amino acid sequence of any one of SEQ ID NOs: 72 to 76, wherein one or several amino acids are deleted, substituted, Z- or added, and has phosphodiesterase activity.
  • a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 80 to 82; (b) one or several amino acids in the amino acid sequence of any one of SEQ ID NOs: 80 to 82, wherein the amino acid sequence is composed of a deleted, substituted and / or Z- or added amino acid sequence; A protein having an inhibitory activity.
  • a protein comprising an amino acid sequence of any one of SEQ ID NOs: 94 to 96 in which one or several amino acids are deleted, substituted and / or added, and has isomerase activity .
  • a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted and / or modified in the amino acid sequence of SEQ ID NO: 88, and which has a carrier activity.
  • amino acid sequence according to any one of SEQ ID NOs: 108 to 111 or 1116, wherein the amino acid sequence comprises one or several amino acids deleted, substituted, Z-added, or And a protein having protein interaction activity.
  • a DNA having a base sequence capable of hybridizing with DNA under stringent conditions and encoding a protein having transferase activity is provided.
  • one or more bases in the nucleotide sequence of any one of SEQ ID NO: 85, one or more bases have a base sequence in which deletion, substitution, Z or addition is performed, and have a carrier activity.
  • DNA that encodes a protein in the nucleotide sequence of any one of SEQ ID NO: 85, one or more bases have a base sequence in which deletion, substitution, Z or addition is performed, and have a carrier activity.
  • (c) encodes a protein having a base sequence capable of hybridizing under stringent conditions with DNA having the base sequence of SEQ ID NO: 92 or 93 or its complementary sequence, and having an intracellular organelle activity DNA.
  • a sense oligonucleotide having the same sequence as 5 to 100 consecutive nucleotides in the nucleotide sequence of DNA according to any one of (11) to (22), having a sequence complementary to the sense oligonucleotide.
  • the protein according to (1) to (10) or (25) is brought into contact with a test substance, and a change in the activity of the protein caused by the test substance is measured.
  • a method for screening an activity modulator is provided.
  • a computer-readable recording medium storing at least one or more nucleotide sequence information selected from the group consisting of:
  • FIG. 1 is a conceptual diagram showing a method for inducing a transgene in an RNAi individual.
  • the DNA of the present invention is a protein comprising an amino acid sequence represented by SEQ ID NOS: 25 to 48, 60 to 62, 65, 66, 72 to 76, 80 to 82, 86 to 88, 94 to 98 or 108 to 116. Or in the amino acid sequence, one or several (the number is not particularly limited, for example, 20 or less, preferably 15 or less, more preferably 10 or less, and still more preferably 5 or less.
  • the amino acid sequence of the amino acid residue including substitution, deletion, insertion, addition, or inversion of the amino acid residue, and enzymatic activity (eg, protease activity, phosphodiesterase activity, transferase activity, or isomerase activity).
  • a protein having a proteolytic enzyme inhibitory activity or a physiological activity other than the enzymatic activity may be only the translation region encoding the amino acid sequence, or may include the full length of the cDNA.
  • examples of the DNA containing the full-length cDNA include SEQ ID NOS: 25 to 48, 60 to 62, 65, 66, 72 to 76, 80 to 82, 86 to 88, 94 to 98, or 108 to 1 And DNA comprising the nucleotide sequence described in 16 above.
  • the translation regions include;
  • nucleotide numbers 10 to 411 of SEQ ID NO: 5 nucleotide numbers 75 to 848 of SEQ ID NO: 6
  • nucleotide numbers 98 to 1351 of SEQ ID NO: 7 nucleotide numbers 526 to 2244 of SEQ ID NO: 8, SEQ ID NO: 9
  • the DNA of the present invention also includes the above-mentioned translation region and a region adjacent to the 3, or Z or 5 ′ end thereof, which contains the minimum necessary for expression of the translation region.
  • the DNA of the present invention may be obtained by any method as long as it can be obtained, and specifically, for example, can be obtained by the method described below.
  • mRNA is prepared from a suitable animal, preferably a mammal, such as a fibrous tissue, by a method known per se and generally used.
  • cDNA is synthesized using this mRNA as type II.
  • a molecule that is tagged with a diol structure specific to the 5, cap ( 7MeG ppp N) site is chemically synthesized.
  • the cDNA thus obtained is inserted into an appropriate cloning vector for cloning.
  • the vector used here has a recombinase recognition sequence at both ends of the cloning site that can uniformly clone DNA of various chain lengths, and is a linear vector that can be inserted into the host by a method other than infection.
  • the vector JP-A-119273
  • JP-A-119273 is preferably used.
  • not all clones exist uniformly hereinafter, this may be referred to as “cataloged”.
  • a clone that exists only in a plant has a high probability of being new. Therefore, a subtraction method for enriching such clones and a normalization method (Japanese Unexamined Patent Publication No. 2000-325080, Carninci, P. et al., Genomics, 37, 327-336 (1996)) can be used. preferable.
  • the base sequence is analyzed by the method.
  • the base sequence obtained from the sequence based on the terminal 100 is obtained by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/; National Center). of Biotechnology Information) using databases such as NCB I's Genbank, EMBL, DDBJ, and PDB, and the homology is 30% or less even for the sequence with the highest homology. Even a sequence showing the highest homology with respect to the full length of the translation region of the DNA, whose homology is 40% or less, was newly subjected to the following analysis.
  • DNA having the full-length cDNA base sequence examples include, for example, SEQ ID NOS: 1 to 24, 57 to 59, 63, 64, 67 to 71, 77 to 79, 83 to 85, 89 to 93 or 99.
  • the translation regions include;
  • the novel nucleotide sequence obtained in this manner is subjected to homology search (homology search) by BLAST (Basic local alignment search tool; Altschul, SF, et al., J. Mol. Biol., 215, 403-410 (1990)).
  • HMMPFAM http://pfam.wustl.edu
  • HMME R array analysis method using hidden Markov model
  • the function of the protein encoded by the base sequence can be estimated by performing a protein characteristic search (profile search) or the like using the above method.
  • the function of the clone to be analyzed can be estimated from various annotation information accompanying the hit sequence whose homology obtained as a result of the search is sufficiently significant.
  • the sufficient significant hit sequences, or not identity with the corresponding portion to the DNA of the catalytic domain portion of the sequence registered present invention is 10 4 or less as E_v alue, or 3 0% The above is shown. .
  • the HMMP FAM is an analysis based on a method of identifying whether or not a nucleotide sequence to be analyzed has a characteristic of a nucleotide 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 a single sequence to a single sequence, if there is a characteristic region in the sequence, the function is identified and predicted. Can be. A specific example of the protein function prediction performed in this manner will be described below.
  • the amino acid sequence (SEQ ID NO: 25) encoded by the nucleotide sequence of SEQ ID NO: 1 was determined by BLAST search with ADAMTS18 protein, e-va 1 ue: 0.0, 708 amino acid residue 3 ⁇ 475% homology ⁇ f, metalloprotease disintegrin 16 with thrombospon din type I motif and e- value: the 0.0, 671 homology 61% with an amino acid residue, and a disintegrin and metalloproteinase with thrombospondin motifs 12 e- value: at 5 X 10 one 152, 702 It has 42% homology with amino acid residues and also with A disintegrin ana metalloproteinase with thrombospondin motifs 10.
  • nucleotide numbers 412-1113 are encoded A sequence (characterized as Reprolysin at P f am) is found in the amino acid sequence.
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 is metalloproteinases.
  • this protein is presumed to be involved in arthritis and cancer from literature information (Gene 2002 Jan 23; 283 (1-2): 49-62).
  • amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2 was found by BLAST search to have an e-value of 0.0% with ADAMTS18 protein, 90% homology with 807 amino acid residues, and an e-value with ADAMTS16 protein. : 0.0, 816 amino acid residues have 61% homology, and e-value: 0.0, 786 amino acid residues have 43% homology with ADAMTS12.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2 was searched for the protein characteristics using HMM PFAM, the amino acid sequence encoded by nucleotide numbers 1355-1969 showed a sequence (Pf am is entered as a Reprolysin).
  • a sequence base sequence that is entered as Pep_M12B_propep in Pfam showing characteristics of metalloprotease is found in the amino acid sequence encoded by base numbers 796-1172.
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2 is metalloproteinases. This protein is presumed to be involved in arthritis and cancer based on literature information (Gene 2002 Jan 23; 283 (1-2): 49-62).
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 3 was found by BLAST search to have 67% homology with Probable serine protease HTRA4 precursor and e-va 1 ue: 5X10-175, an amino acid residue.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 3 When a protein feature search is performed by PFAM, a sequence (trypsin entry in P fam) indicating a characteristic of trypsin, which is a serine protease, is found in the amino acid sequence encoded by base number 559-111.
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 3 is a serine protease.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 4 was searched for protein characteristics using HMM PFAM, the amino acid sequence encoded by the nucleotide number 559-1152 showed the amino acid sequence encoded by peptidase reprolysin (P
  • the nucleotide sequence 190-528 encodes the amino acid sequence of Peptidase family M12B (sequence that is entered as Pep-M12B_propep in the Pfam). Found.
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 4 is a meta-oral protease.
  • this protein is presumed to be involved in sperm production from literature information (Mol Hum Reprod 1998 May; 4 (5): 29-37).
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 5 was searched for protein characteristics using HMM PFAM, the amino acid sequence encoded by nucleotide numbers 199-472 was found to have the characteristics of peptidase family M12B. (A sequence that is entered in P fam as Pep_M12B_propep) is found.
  • the protein consisting of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 5 is a meta-oral protease. This protein is presumed to be involved in spermatogenesis from literature information (Mol Hum Reprod 1998 May; 4 (5): 429-37).
  • a protein characteristic search using HMM PFAM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 6 shows that the amino acid sequence encoded by nucleotide numbers 150 to 827 shows the amino acid sequence encoded by the trypsin which is a serine protease in the amino acid sequence encoded by the nucleotide sequence. (Sequence that is entered as trypsin in P fam).
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 7 was subjected to protein characteristic search by HMM PFAM, and the amino acid sequence encoded by nucleotides 653-1330 showed a sequence (P sequence that is entered as a trypsin in the fam).
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 8 was searched for protein characteristics using HMM PFAM, the amino acid sequence encoded by nucleotide numbers 1528-2217 showed a sequence exhibiting the characteristics of trypsin, a serine protease. (Sequence that is entered as rypsin in P f am).
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 9 was subjected to protein characteristic search by HMM PFAM. (A sequence that is entered as an Astacin at P f am).
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 10 was searched for protein characteristics using HMMP FAM, the amino acid sequence encoded by nucleotides 392-739 showed a sequence (P fam Sequence that is entered as 0TU in
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 10 is a cysteine protease.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 11 was searched for protein characteristics using HMMP FAM, the amino acid sequence encoded by nucleotide numbers 604-951 showed a sequence (P fam To OTU Sequence to be entered).
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 11 is a cysteine protease.
  • HMMP FAM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 12 shows that the amino acid sequence encoded by nucleotides 398-745 shows a characteristic of cysteine protease (Pfam 0TU).
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 12 is a cysteine protease.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 13 was searched for the protein characteristics using HMMP FAM, the amino acid sequence encoded by nucleotide numbers 603-950 showed a sequence exhibiting the characteristics of cysteine protease (Pfam 0TU).
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 13 is a cysteine protease.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 14 was obtained from the BLAST site using Drosophila melanogaster genomic scaffold and e-value: 3 X 1 0 54, 20 1 Amino acid residues and 5 2% homology, Hypothetical 33.5 kDa protein and e- value: having 4 X 10- 31, 203 amino acid residues and 36% homology sex.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 14 was searched for protein characteristics using HMMP FAM, the amino acid sequence encoded by nucleotide numbers 392 to 739 showed a sequence exhibiting characteristics of cysteine protease (Pfam 0TU).
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 14 is a cysteine protease.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 15 was compared with Sequence 5 from Patent W00157081 by e-value: 0.0, 469 amino acid residues at 94% homology with Aqualysin by BLAST search.
  • I precursor and e- va 1 ue having 5 X 1 0- 45, 355 amino acid residues and 34% homology.
  • HMMP FAM protein characteristic search using HMMP FAM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 15 showed that the amino acid sequence encoded by nucleotides 714 to 1602 showed a sequence (Pfam Peptidase __S8).
  • the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 16 is: Serine protease DES precursor and e-va 1 ue: 2 X ⁇ 0-1, according to the BLAST search. 6, the 421 amino acid residues and 43% homology, airway trypsin- like protease and e- value: the 2 X 1 0- 89, 41 7 amino acid residues and 40% homology, further Airway trypsin- like protease precursor and e_va 1 ue: 1 x 10 one. 8, has a 40% homology over a 4 39 Amino acid residues.
  • nucleotide sequence shown in SEQ ID NO: 16 is searched for protein characteristics by HMMP FAM, nucleotide numbers 795-1472 are encoded.
  • amino acid sequence a sequence characteristic of trypsin, a serine protease (sequence that is entered as trypsin in P f am), is found.
  • amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 17 was searched for protein characteristics using HMMP FAM, the amino acid sequence encoded by nucleotide numbers 755-1432 showed the characteristics of trypsin, a serine protease. The sequence (the sequence that is entered as a trypsin at Pfam) is found.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 17 is It is presumed to be a serine protease having a function related to tumors and inflammation.
  • HMMP FAM protein characteristic search by HMMP FAM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 18 shows that the amino acid sequence encoded by nucleotides 788 to 1465 shows the amino acid sequence encoded by nucleotides of trypsin, a serine protease. (A sequence that is entered as a trypsin at P fam).
  • nucleotide numbers 637 to 1314 were encoded.
  • a sequence characteristic of trypsin a serine protease (sequence that is entered as trypsin in P f am), is found.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 20 was searched for protein characteristics using HMMP FAM, the amino acid sequence encoded by nucleotide numbers 628-1305 showed the characteristics of trypsin, a serine protease.
  • the sequence (the sequence that is entered as a trypsin at Pfam) is found.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 20 is a serine protease. From the literature information (JBiolChem 1998 May 8; 273 (19): 11895-901), this protein is presumed to be a trypsin-like serine protease isolated from sputum of a patient with chronic airway disease. From these facts, it is presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 20 is a serine protease having a function related to epithelial inflammation.
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 21 is a BLAST server By Chi, testes- specif ic protein TSP50 and e- value: 5 X 10 _128, 439 amino acid residues and 54% homology, Brain- specific serine protease 4 precursor and e- va 1 ue: 2 X 10- 36 , 28% amino acid residue and 33% homology or raw " ⁇ , and Marapsin precursor and e-value: 1 X 10-35 , 253 amino acid residue with 32% homology, Testisin precursor and e _ va 1 ue: having 9X 10- 35, 273 amino acid residues and 32% homology.
  • HMMP FAM protein characteristic search by HMMP FAM was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 21, the amino acid sequence encoded by nucleotide numbers 522-1246 showed a sequence (trypsin, a serine protease) having the characteristics ( (A sequence that is entered as a trypsin in P fam).
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 21 is a serine protease.
  • This protein was isolated from eosinophils according to literature information (BiochemBiophys Res Commun 1998 Nov 18; 252 (2): 307-12), and is found in many tissues other than skeletal muscle and kidney. ⁇ It is estimated that this is a serine protease that is most frequently expressed in the prostate and is moderately expressed in the lungs ⁇ spleen ⁇ kidney. From these facts, it is presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 21 is a serine protease having a function in more tissues, particularly testis / prostate.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 22 was found to have a homology of 98% with e-Va1ue: 0.0, 355 amino acid residues, and a KIM1203 protein, by BLAST search. If e- value: a 4X 10- 23, 1 18 ⁇ amino Torimugi residues and 50% homology ten raw, Homo sapiens deubiquitinating enzyme UnpES and e- value: over 2 X 10- 8, 66 amino acid residues 48 % of homology, is et to Ubiquitin carboxyl- terminal hydrolase 4 and e- value: having 2 X 10 one 8, 66 amino acid residues and 48% homology.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 22 is a protease. From the literature (Oncogene 1995 Jun 1; 10 (11): 2179-83), this protein is a nuclear ubiquitin protease, and it is speculated that its overexpression may be involved in tumorigenesis. From these facts, it is presumed that the protein encoded by the nucleotide sequence of SEQ ID NO: 22 is further a thiol protease involved in tumor formation.
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 23 has a protein of unknown function, e-Va1ue: 0.0, and a 99% homology with the amino acid residues of 827 by BLAST search.
  • KIM1203 protein and e- value l X 1 0- 67
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 23 was searched for protein characteristics by HMMP FAM, the amino acid sequence encoded by nucleotide numbers 244 to 337 showed a thiol protease, ubiquitin carbyl terminal.
  • a sequence showing the characteristics of hydrolace (a sequence that is entered as UCH-1 in P fam) is found.
  • amino acid sequence encoded by base number 1885-2700 shows a sequence characteristic of the thiol protease ubiquitin carbon terminal hydrolase (sequence entered as UCH-2 in P fam). .
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 23 is a protease. Based on literature information (Oncogene 1995 Jun 1; 10 (11): 2179-83), this protein is a nuclear ubiquitin protease, and it is speculated that its overexpression may be involved in tumorigenesis. From these facts, it is presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 23 is a thiol protease further involved in tumor formation.
  • Amino acid sequences which nucleotide sequence is encoded according to SEQ ID NO: 24, the BLAST server switch, carboxypeptidase homolog and e_v alue: a 1 X 10- 93, 331 Amino acid residues and 48% homology, and carboxypeptidase B precursor e- va 1 ue: a 2 X 1 0- 9 2, 3 2 7 Amino acid residues 4-8% homology, further carboxypeptidase B precursor and e_v alue: 4X 10- 90, 330 Amino acid residue and 46 Has% homology.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 24 was searched for protein characteristics using HM MPFAM, the amino acid sequence encoded by nucleotide numbers 509-1357 showed a sequence (Pfam The sequence is entered as Zn-carbOpept.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24 is lipoxypeptidase. According to literature information (JBiol Chem 1988 Nov 25; 263 (33): 17837-45), this protein is found to be a carboxypeptidase expressed in the knee, and other literature information (J Biol Chem 1992 Feb 5; 267 (4): 2575-81), it is presumed to be a carboxypeptidase involved in osteomyelitis / knee transplant rejection. From these facts, it is presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24 is a potent lipoxypeptidase involved in knee disease.
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 58 is a BLAST server By Chi, X. laevis mRNA for xolloid and e- value: 2 X 10 one 58, 436 with 30% degree of coincidence over the completion amino acid residues, also Dorsal- ventral patterning tolloid protein precursor and e- value: 4 X 10 _58, 30% degree of coincidence over the 445 amino acid residues, more Bone morphogenetic protein 1 precursor and e- va 1 ue: 7X 10- 57 , 436 hits with 30% degree of coincidence over the amino acid residues.
  • a protein characteristic search using the HMMPFAM for the amino acid sequence shown in SEQ ID NO: 61 shows that the amino acid sequence of amino acid number 30 to 370 in SEQ ID NO: 61 shows a sequence showing CUB domain characteristics at three positions (Pfam (A sequence that is entered as a CUB).
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 58 is a meta-oral protease involved in sashimi, bone formation, spermatogenesis and the like.
  • the Amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 59, the BLAST server switch, Mus musculus CSMD1 (Csmdl) and e_v a 1 ue: 5 X 10- 108, 257 68% match over Amino acid residue And Homo sapiens CUB and sushi multiple domains protein 1 short form and e-va 1 ue: 1 X 10 _48 , 153 amino acid residues.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 67 was obtained from BLAST search using Ectonucleotide pyrophospnate / phpsphodiesterase 1 and e-va 1 u e: 1 X 1 0- 4 8 , 4 0 3 with an amino acid residue indicates 3 1% homology, also Phosphodiesterase I and e- value: at 1 X 10- ° 4, for 6 1 amino acid residue 36 Has% homology.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 67 is a protein that functions as a phosphodiesterase.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 68 is a protein that functions as a phosphodiesterase.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 69 is a protein that functions as a phosphodiesterase.
  • a protein characteristic search using HM MPF AM with respect to the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 70 shows a sequence that shows the characteristics of glycoprotein hormone (base sequence that is entered as “phosphodiest” in P f am) Is found.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 70 is a protein that functions as a phosphodiesterase.
  • a protein characteristic search by HMMP FAM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 71 shows a sequence showing the characteristics of a glycoprotein hormone (a nucleotide sequence entered as “phosphodiest” in P i am). ) Is found.
  • the protein encoded by the nucleotide sequence represented by SEQ ID NO: 71 is a protein that functions as a phosphodiesterase.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 77 is a protease, Inhibita, having a function related to serine protease inhibition.
  • Amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 78 when a homology search using the BLAS T, Placental thrombin inhibitor and e- va 1 ue: a 5 X 1 0- 176, 379 amino acid residues E-value: 5 x 10-1 150 , 379 amino acid residue with 70% concordance, and Cytoplasmic antiproteinase 2 and e- va 1 e : 5 X 10 - 126, 380 hits with 60% degree of coincidence over the amino acid residues.
  • the protein consisting of the amino acid sequence shown in SEQ ID NO: 82 was a protease inhibitor.
  • this protein has been shown to be involved in the inhibition of thrombin from literature information (J. Biol. Chem. 270: 16089-16096 (1995)), and has been reported to be related to other literature information (Brain Res. Mol. Brain Res. 42: 293). -300 (1996)), it is presumed to be involved in the regulation of serine protease.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 79 is not It is presumed to be a protease inhibitor having a function related to the inhibition of phosphoprotease.
  • the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 83 has the database registration code AL031678, Human DNA sequence from clone RP4-816K17 on chromosome 20pl2.2-13.Contains the TGM3 gene for transglutaminase 3 e-va 1 e: 0.0, 87% homology over 442 amino acid residues, and database registration code Q08188, Human Protein-glutamine glutamyltransf erase E3 precursor, e- va 1 ue: 0. At 0, a homology of 49% over 705 amino acid residues was obtained.
  • database registration code Q08189 mouse Protein-glutamine glutamyltransferase E3 precursor power S, e- va 1 ue: 0.0, 705 amino acid residues With a homology of 48%.
  • a protein characteristic search using HM MPF AM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 83 shows a sequence showing the characteristics of transglutaminase-like superfamily (a sequence that is entered as “Transglutamin-N” in P fam).
  • J transglutaminase-like superfamily
  • J sequence to be entered as “Transglut-core” in P fam
  • Transglutaminase family C-terminal ig
  • the characteristics of the like domain are found in the non-T-robot system (J (sequence entered as “Transglutamin-C” in P fam).)
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 83 Among the transferases, the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 84 can be estimated to have AF357970 and Homo sapiens c by BLAST search.
  • arnitine palmitoyltransferase IC power, e-value: 0.0, and 83% over 802 amino acid residues, and database registration At 0, the similarity was 53% over 765 amino acid residues, and according to the protein feature search by HMM PF AM, Choline / Carnitine o-acyltransf erase Sequences exhibiting characteristics (sequences entered as “Carn-acyltransfj” at P fam) and sequences exhibiting characteristics of Dehydrin (sequences entered as “dehydrin” at P fam) are found. From these facts, it is presumed that the protein encoded by the nucleotide sequence of SEQ ID NO: 84 has palmitoyltransferase activity among transfelase.
  • the Amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 89, the BLAST server Ji (i) a database registration mark AK003402 force ev alue: at 5 X 10 one 43, also at 100% over 85 amino acid residues, also (Ii) Database registration number Q08752, 40 kDa peptidyl-prolyl cis-trans isomerase (HUMAN) power, e-va 1 ue: 0.0, 94% over 370 amino acid residues, and (iii) data base No. P26882, 40 kDa peptidyl-prolyl cis-trans isomerase (B0VIN) 1Se_va 1 ue: 0.0, having 91% homology over 370 amino acid residues.
  • the protein of the above (ii) is considered to be involved in the activity of the steroid hormone receptor complex from the literature information in the database (Genomics 35: 448-455 (1996)). References in Biol. Chem. 268: 13187-13192 (1993)) show that they are involved in estrogen receptor activity.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 89 was searched for protein characteristics using HMMP FAM. As a result, the amino acid sequence of SEQ ID NO: 94 was identified as amino acid number 16—241 ⁇ . Peptidyl-prolyl cis-trans isomerase. An unidentified system [J (base sequence that is entered as pro_isomerase in P fam)] is found.
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 90 was determined by BLAST search.
  • the protein of (ii) is considered to be involved in the activity of the steroid hormone receptor complex based on the literature information in the database (Genomics 35: 448-455 (1996)). References in the literature (J. Biol. Chem. 268: 13187-13192 (1993)) show that they are involved in estrogen receptor activity.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 90 is an isomerase protein having a function relating to steroid hormone receptor.
  • a protein feature search using HMMPF AM was performed, and found to be a chicken (J (a base sequence sequenced as a pro-isomerase in P fam)) that lacks the characteristics of peptidyl-prolyl cis-trans isomerase.
  • J a base sequence sequenced as a pro-isomerase in P fam
  • the protein of the above (ii) is considered to be involved in the activity of estrogen receptor based on literature information (J. Biol. Chem. 268: 13187-13192 (1993)) in the database.
  • Literature information in the database indicates that proteins are involved in the activity of steroid hormone receptor complexes.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 91 is an isomerase having a function related to a steroid hormone receptor.
  • the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 104 has a database registration code of P42436, Assimilatory nitrite reductase and an e-value of 3 ⁇ 10- ° 5 , 32% over 78 amino acid residues.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 104 is an Fe-mediated acid ligase reductase.
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 105 has a database registration code of P42436, Assimilatory nitrite reductase, and an e-value of 2X10- ° 5 , 34% of which covers 81 amino acid residues. in coincidence degree and the database registration symbol AE006738 e- value: 8X 10- 05, 114 with 31% degree of coincidence over the amino acid residues, yet the database registration symbol AP003000 e- V a 1 ue: 0.005 , 79 amino acid residues Hits with 30% match over base.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 105 is a reductase having a function of oxidative phosphorylation.
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 106 was obtained from BLAST search using the database registration symbol P42436, Assimilatory nitrite reductase. If e- value: 1 X 10- 05, 81 35% of the degree of coincidence over the amino acid residues, also a database registration symbol AE006738 e- value: 1 X 10- ° 4, 31% of over 114 amino acid residues Hits with a match degree of 30% match over 79 amino acid residues with e-Va 1 ue: 0.005 with database registration code AP003000.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 106 is a reductase having a function of oxidative phosphorylation.
  • Protein with carrier activity (carrier protein, transporter)
  • SEQ ID NO: 85 database registration mark by BL AS T search Amino acid sequence encoded by the nucleotide sequence according to AC004832, Homo sapiens PAC clone RP4 - 539M6 from 22 ( human SPF) 1S e- value: in 5X 10- 109, also similar in 85% over 221 Amino acid residues, also database registration mark AF309558, Rattus norvegicus supernatant protein factor (rack Bok SPF) force S, e- va 1 ue: with 2 X 10 one 86, 2 2 1 6 4% over amino acid residues, is et to AF487977, Bos taurus tocopherol- associated protein force S, e- va 1 ue: with 2 X 10 one 84, in 62% over 221 amino acid residues Showed similarity.
  • rack Bok SPF Rattus norvegicus supernatant protein factor
  • human SPF binds squalene and transports it to the microsomal squalene epoxide, catalyzing its conversion to squalene 2,3-oxide and initiating the later stages of sterol biosynthesis.
  • Human SPF is also shown to have an alpha-tocopherol-dependent transcription promoting activity from literature information (BBRC 2001, 285, 295-299). From these facts, it can be inferred that the nucleotide sequence represented by SEQ ID NO: 85 has a carrier protein such as protein squalene, retinaldehyde, phosphatidylcholine or f alpha-tocopherol, or has a transcription promoting activity.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 92 was identified by BLAST search (i database entry ⁇ ⁇ "AJ005766, lysosome—associated membrane protein (LAMP) power e-va 1 ue: 5 X 10 - 4 1, and in 42% over 250 Amino acid residues, also (ii) a database registration mark AB013924, "TSC403" product ⁇ e - ⁇ a 1 ue: in 6 X 1 0 one 4 2, 250 Amino acid It shows 42% over the residues and (iii) 30% homology over 130 amino acid residues with database registration code P28968, Glycoprotein X precursor power e-va1ue: 2X10- ° 4 .
  • TSC403 product is increased in lung cancer
  • i protein
  • LAMP protein
  • S-IV replication in the acute phase of infection is suppressed in CD68 + cells of the lung expressing DC-LAMP, and the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 92 is also known.
  • Tm HMM S. Moller, MDR Croning, R.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 92 is a glycoprotein having a transmembrane region and is involved in the functions of organelles such as lysosomes, and is associated with lysosomal diseases, mucopolysaccharidosis, etc. It is speculated that it is related to metabolic diseases and HIV infection.
  • SEQ ID NO: 9 3 amino acid sequence encoded by the nucleotide sequence according to the BLAST search by (i) Atabesu registered words himself AJO05766, lysosome- associated membrane protein (LAMP ) force e- va 1 ue: 5 X 1 0- 1 1 2 in, in Wataru Ri 51% to 423 amino acid residues, also (ii) a database registration mark AB013924, "TSC403" product is, e - V a 1 ue: at 5 X 1 0 one 1 1 2, the 423 amino acid residues 51% over further (iii Ryo '- "" data base over scan Noboru ball el No.
  • TSC403 protein LAMP of (i)
  • CD68 + cells in the lung expressing DC-LAMP suppress SIV replication during the acute phase of infection.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 93 is a glycoprotein having a transmembrane region, which is involved in the function of organelles such as lysosome, and is associated with lysosomal disease, mucopolysaccharidosis, etc. May be related to metabolic diseases and HIV infection.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 99 was identified by the BLAST search as having the database registration code of AK074067 at 6: & lt ; 6: 5 10 _148 , 84% identity over 310 amino acid residues, the database registration mark BC007206 e- V a 1 ue: 5 X 10- 49, 286 with 39% degree of coincidence over the amino acid residues, and database registration symbol AL390078 La is e- value: 5 X 10- 44, Hits with 37% concordance over 286 amino acid residues.
  • the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 99 was searched for protein characteristics by HMMP FAM. (Base sequence that is entered as SH2 in P f am) is found.
  • the protein encoded by the nucleotide sequence represented by SEQ ID NO: 99 is a protein having a function related to the interaction with the cleft synthetase protein.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 100 was identified by the BLAST search according to database registration symbols AK027856 and e_va1ue: 0.0, at an identity of 80% over 470 amino acid residues, and a database registration symbol Z18529 e- va 1 ue: IX 10- 84, 536 with 38% degree of coincidence over the amino acid residues, more data base over scan registration mark Q04205, Tensin and e- va 1 e: IX 10- 84 Hits with 38% identity over 536 amino acid residues.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 100 is A protein characteristic search using MM PF AM revealed that the amino acid sequence 429-521 of SEQ ID NO: 109 shows the sequence characteristic of the module structure required for binding to a signal transduction factor (base sequence entered as SH2 in P f am). Found.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 100 is a protein having a function relating to the interaction with the tyrosine phosphorylated protein.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 101 was subjected to protein characteristic search using HMMPFAM. (Base sequence that is entered as PSH in SH2) is found.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 101 is an adapter protein having a function relating to the interaction with the tyrosine phosphorylated protein.
  • amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 102 can be obtained by BLAST search using the database registration code P29353, SHC transforming protein (HUMAN) (known to be involved in cell carcinogenesis (Cell 1992).
  • HUMAN SHC transforming protein
  • HMMP FAM protein characteristic search by HMMP FAM was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 102, a module required for binding to a signal transduction factor was found at amino acids 72 to 147 of SEQ ID NO: 11 A sequence exhibiting structural features (base sequence that is entered as PSH in SH2) is found.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 102 is a protein having a function relating to the interaction with the tyrosine phosphorylated protein, and is involved in canceration.
  • the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 107 was converted to a database registration symbol AF479747, PYRIN-containing APAF1-like protein 4, e-va 1 ue: 0.0, 868 amino acid residue by BLAST search. 42% degree of coincidence over, or the database registration mark AF482706, ribonuclease inhibitor 2 and (RNH2) e- value: 5 X 10- 167, 780 at 41% degree of coincidence over the amino acid residues, more database registration mark AF298547, nucleotide - binding site protein 1 and e- va 1 ue: 2 X 10- 97, 911 hits in 29% of the degree of coincidence over the amino acid residues.
  • a protein characteristic search using HMMPFAM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 107 shows that a sequence exhibiting the characteristics of Leucine Rich Repeat at amino acid numbers 714-766 of SEQ ID NO: 116 (Pfa as LRR Entry sequence) is found.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 107 is a protein having a function related to protein-protein interaction.
  • the DNA of the present invention may be obtained in a state where a base sequence has been deleted or inserted in the translated sequence, but the results of the homology search and the protein feature search as described above were obtained.
  • the nucleotide sequence can be determined by a method commonly used by those skilled in the art, such as library cloning and PCR cloning.
  • a full-length cDNA with no deletion or insertion can be obtained.
  • the protein of the present invention is expressed using the full-length cDNA thus obtained, and can be used for functional analysis.
  • the DNAs of the present invention thus obtained, whose nucleotide sequence is determined, and whose functions are estimated are the above-mentioned SEQ ID NOS: 1 to 24, 57 to 59, 63, 64, 67 to 71, 77 to 79, 83 to 85 , 89-93 or 99-107, or those having the above-mentioned base sequences as their translation regions, but also one or several (the several The number is not particularly limited, but means, for example, 60 or less, preferably 30 or less, more preferably 20 or less, still more preferably 10 or less, and particularly preferably 5 or less.) , A DNA having a substitution, a Z, or an added base sequence, and a protein having an enzymatic activity, a proteolytic enzyme inhibitory activity or a physiological activity other than the enzymatic activity, and hybridizing with these under stringent conditions.
  • Soybean And enzymatic activity DNA or the like encoding a protein having a physiological activity other than protease inhibitory activity or enzyme activity are also included. As described above, these DNAs have the amino acid sequence of the protein described in SEQ ID NOS: 25 to 48, 60 to 62, 65, 66, 72 to 76, 80 to 82, 86 to 88, 94 to 98, or 108 to 116.
  • One or several amino acid sequences consisting of a deleted, substituted, or Z-added amino acid sequence, and encoding a protein having an enzymatic activity, a protease inhibitory activity, or a physiological activity other than the enzymatic activity Is included.
  • DNAs that hybridize under stringent conditions are described in SEQ ID NOs: 1 to 24, 57 to 59, 63, 64, 67 to 71, 77 to 79, 83 to 85, 89 to 93, or 99 to 107. And DNA having a homology of 80% or more, preferably 90% or more, more preferably 95% or more with BLAST analysis.
  • Hybridization under stringent conditions Zession is a reaction performed in a normal hybridization buffer at a temperature of 40 to 70 ° (preferably 60 to 65 ° C), and a salt concentration of 15 mM to 300 mM, preferably 15 mM to 6 mM.
  • the washing can be performed according to a method of washing in a washing solution such as OmM.
  • the DNA of the present invention may be obtained by the above-described method or may be synthesized.
  • the DNA base sequence can be easily replaced with a commercially available kit such as a site-directed mutagenesis kit (Takara Shuzo) or a quick change site-directed mutagenesis kit (Stratagene).
  • nucleotide sequences described in 83-85, 89-93 or 99-107 are derived from mouse, but a human cDNA library was prepared according to the above-described method for preparing a cDNA library.
  • DNA encoding a human homolog protein of the protein encoded by the base sequence described in L07 (hereinafter, may be referred to as "human homolog DNA”) can also be obtained. .
  • a DNA having the nucleotide sequence of SEQ ID NOS: 1 to 24, 57 to 59, 63, 64, 67 to 71, 77 to 79, 83 to 85, 89 to 93, or 99 to 107 or a complementary sequence thereof, DNA that hybridizes under gentle conditions includes such human homolog DNA.
  • nucleotide sequence of the human homolog DNA can be predicted using informatics, and the human homolog DNA can be obtained from the above human cDNA library or the like based on the nucleotide sequence.
  • homologues of the target protein using informatics As a method for predicting a nucleotide sequence encoding a protein, for example, (i) a cDNA database of a human or the like (a cDNA database that can be predicted by informatics) using a query of a nucleotide sequence of a target cDNA. And (ii) a homology search using BLAST or the like to a human or other EST database using the base sequence of the target cDNA as a query.
  • PCR is performed using the above human cDNA library as a ⁇ type using a primer having a nucleotide sequence complementary to the nucleotide sequence at the 5 'end and the 3' end of the predicted human homolog DNA.
  • a method of performing hybridization on the above-mentioned human cDNA library using a partial sequence of the predicted human homolog DNA as a probe Generally, a similar gene having a nucleotide sequence having the highest homologous nucleotide sequence to the nucleotide sequence of the target gene is called a “homolog”, and the above-mentioned method also aims to obtain human homolog DNA. In, it is important to confirm not only that the nucleotide sequences are similar, but also that the gene obtained as a homolog is a family member of the target gene.
  • Genes acquired as “homologs” between two species of organisms may be “orthologs”, which are the same genes evolved from a common ancestral gene, and also because of duplication from a common ancestral gene. The resulting different gene could be a "paralog”.
  • the function of the protein encoded by the human-derived DNA must be In order to verify and presume the same function in the mouse of the protein of the present invention, it is preferable to confirm that the human homolog is an ortholog of a closely related species of the mouse gene of the present invention.
  • the following method is used as a method for confirming the ortholog.
  • the homology analysis described in (i) and (ii.) Above may be performed by comparing amino acid sequences, or by drawing a molecular evolutionary phylogenetic tree.
  • the protein of the present invention is expressed using the obtained full-length cDNA of the human homolog DNA, and can be used for activity confirmation and functional analysis.
  • Examples of the human ortholog DNA of the cDNA of the present invention include DNA having the nucleotide sequence of SEQ ID NO: 63 or 64, and amino acids of SEQ ID NO: 65 or 66 as the human ortholog proteins of the protein of the present invention. And a protein having an acid sequence.
  • amino acid sequence (SEQ ID NO: 65) encoded by the nucleotide sequence set forth in SEQ ID NO: 63 was searched for protein characteristics using HMMPF AM, amino acids 92-283 were replaced with Astacin (Peptidase family M12A) (Pfam as Astacin). Amino acid sequence to be entered).
  • a protein belonging to the Peptidase family M12A is a peptide-degrading enzyme that requires zinc. Therefore, it was inferred that the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 63 is a peptide-degrading enzyme belonging to Peptidase family M12A and requiring zinc.
  • a protein characteristic search using the HMMPFAM for the amino acid sequence of SEQ ID NO: 66 revealed that the amino acid numbers 30 to 140, 145 to 252, and 257 to 370 show the CUB domain characteristics (entry as CUB in Pfam). Amino acid sequence).
  • the CUB domain is an extracellular domain of 110 amino acid residues, and is functionally found in various proteins (protease of the complement system, etc.), especially in proteins that are regulated at developmental stages.
  • a program for predicting membrane engulfment helix t mHMM S. Moller, MDR Croning, R.
  • amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 64 is one of the membrane proteins controlled by the developmental stage, particularly the protease of the capture system.
  • the translation region of the protein encoded by the DNA of the present invention converts the nucleotide sequence of the DNA into amino acids using three reading frames, and defines the range in which the longest polypeptide is encoded.
  • the amino acid sequence can be determined as the translation region of the invention.
  • Such amino acid sequences include, for example, SEQ ID NOS: 25-48, 60-62, 65, 66, 72-76, 80-82, 86-88, 94- 98 or 108 to 116 and the like.
  • the protein of the present invention is not limited to the above amino acid sequence, but comprises an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence, In addition, those having a physiological activity other than the enzyme activity, the protease inhibitory activity or the enzyme activity are also included.
  • the method of transcribing and translating the DNA of the present invention described in the above (1) by an appropriate method is preferably used.
  • a recombinant vector prepared by inserting a suitable expression vector or a suitable vector together with a suitable promoter is prepared, and a suitable host microorganism is transformed with the recombinant vector, or a suitable culture is performed. It can be expressed by introducing it into cells, and can be obtained by purifying it.
  • the protein thus obtained when the protein thus obtained is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained in a salt form, the free form or another salt Can be converted to 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 transformant may be modified before or after purification with an appropriate protein modifying enzyme to optionally modify the protein or partially remove the polypeptide to modify the protein. Quality.
  • modified proteins are also included in the scope of the present invention as long as they have a physiological activity other than the above-mentioned various enzyme activities, proteolytic enzyme inhibitory activities or enzyme activities.
  • the vector used for preparing the recombinant vector containing the DNA of the present invention is not particularly limited as long as the DNA is expressed in the transformant, and the plasmid may be used.
  • the vector or the phage vector may be shifted.
  • a commercially available protein expression vector into which an expression control region DNA such as a promoter suitable for a host into which the DNA is introduced has already been inserted is used.
  • a protein expression vector specifically, for example, when the host is Escherichia coli, pET3, pETll (manufactured by Stratagene) p GEX (Amersha PESP-I expression vector (manufactured by Stratagene) in the case of yeast, and Bac PAK6 (manufactured by Clontech) in the case of insect cells. And the like are used.
  • the host is an animal cell, examples include ZAP Exress (Stratagene), SVK3 (Amersham Pharmacia Biotech) and the like.
  • the promoter may be a promoter contained in a host microorganism or a cultured cell.
  • the promoter is not limited to this.
  • the host is Escherichia coli, T3, T7, tac, 1
  • An ac promoter or the like can be used.
  • yeast an nmt1 promoter, a Ga11 promoter or the like can be used.
  • SV40 promoter, CMV promoter and the like are preferably used.
  • a promoter specific to the gene of the present invention can also be used.
  • the DNA of the present invention may be inserted into these vectors by linking the DNA or a 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. Les ,.
  • 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)) and 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 the electric pulse method (Chu. Et al., Nuc. Acids Res., 15, 1331 (1987)).
  • the host for preparing the DNA transfectant is not particularly limited as long as the DNA of the present invention is expressed in the body.
  • E. coli E. coli, yeast, baculovirus (section Pod Polyhedrovirus) Insect cells or animal cells.
  • BL21 and XL-2B 1 ue (Stratagene) for E. coli, SP-Q01 (Stratagene) for yeast, and AcNPV (J. Biol. Chem., 263) for baculovirus. , 7406, (1988)
  • As animal cells mouse fibroblast C127 (J.
  • COS-7 American Type Culture Collection Cell
  • African green monkey kidney is preferably used from the viewpoint of expression level and simplicity of screening.
  • a homologous recombination technique (AA Vertes et al., Biosci.) In which a DNA fragment of the present invention linked to a promoter is directly inserted into the chromosome of a host microorganism. Biotechnol. Biochem., 57, 2036, (1993)), or using a transposon or insertion sequence (AA Vertes et al., Molecular Microbiol., 11, 739, (1994)) to produce a DNA transductant You can also.
  • the resulting culture is harvested by centrifuging the cells or cells, suspending the cells or cells in an appropriate buffer, and sonicating, lysozyme, and / or freezing and thawing. After disruption by the method, a crude protein solution is obtained by centrifugation, filtration, or the like, and further purified by a combination of appropriate purification methods. Thus, the protein of the present invention is obtained.
  • the protein of the present invention is induced by subjecting the DNA of the present invention obtained in (1) above to a cell-free transcription / translation system. Can be obtained.
  • the cell-free transcription / translation system used in the present invention is a system containing all the elements necessary for transcription from DNA to mRNA and translation of mRNA to protein, and by adding DNA thereto. Any system in which the protein encoded by the DNA is synthesized.
  • Specific examples of the cell-free transcription / translation system include eukaryotic cells, and bacterial cells, or a part thereof.
  • a transcription / translation system prepared based on the extract of Escherichia coli is particularly preferable.
  • a particularly preferred example is a extract prepared from Escherichia coli reticulocytes, wheat germ, and Escherichia coli (Escherichia coli S30 extract) Transcription and translation systems.
  • 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 carried out by a commonly used method known per se. 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, as described above. It can be expressed and purified using the affinity of the protein with a substance having affinity.
  • GST daltathione-1 S-transferase
  • the expression of the target protein is separated by SDS-polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue (Sigma), or detected by an antibody that specifically binds to the protein of the present invention described later. It can be confirmed by the method of doing so. It is generally known that the expressed protein is cleaved (processed) by a proteolytic enzyme present in the living body.
  • the protein of the present invention is, of course, a partial fragment of the cleaved amino acid sequence as long as it has an enzymatic activity, a protease inhibitory activity, or a physiological activity other than the enzymatic activity. Included in proteins.
  • a method for analyzing the interaction a known method per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, liposome A multiple display method and the like can be mentioned.
  • oligonucleotide examples include a DNA having the same sequence as 5 to 100 consecutive nucleotides in the base sequence of the DNA or a DNA having a sequence complementary to the DNA.
  • the above-mentioned oligonucleotides are preferred, in which the melting temperature (Tm) and the number of bases of both do not extremely change.
  • 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 in which an N 3, 1 P 5 ′ phosphoramidate bond is used.
  • O 03/089644 can do.
  • the oligonucleotide of the present invention is prepared as a double-stranded RNA, introduced into a recipient, and inhibits the expression of a target gene by the RA interference method (hereinafter referred to as the RNAi method). May be referred to).
  • the RA interference method for example, the method described in (Elbashir, S., et al., Ature, 411, 494-498 (2001)) can be used.
  • the double-stranded RNA does not necessarily have to be all RNA, and for example, those described in WOO 2Z10374 can be used.
  • the target gene may be any as long as it is the DNA of the present invention.
  • a double-stranded RNA having a sequence substantially identical to at least a part of the base sequence of these DNAs (hereinafter, may be referred to as “double-stranded polynucleotide”) is defined as the base sequence of the target gene. It is composed of a sequence substantially identical to a sequence of 15 bp or more, which may be any part.
  • substantially identical means that the sequence has 80% or more homology with the sequence of the target gene.
  • the nucleotide chain length may be any length from 15 bp to the entire length of the open reading frame (0RF) of the target gene, but a length of about 15 to 50 Obp is preferably used.
  • mammalian-derived cells have a signal transduction system that activates in response to a long double-stranded RNA of 3 Obp or more. This is called the interferon reaction (Mareus, PI, et al., Interferon, 5, 115-180 (1983)).
  • PKR dsRNA-responsive protein
  • the translation initiation of many genes is non-specifically inhibited via Bass, BL, Nature, 411, 428-429 (2001)), and at the same time, 2, 5, oligoadenylate synthetase (Bass, BL, Nature, 411, 428-429 (2001)), activation of Rnase L occurs, and nonspecific degradation of intracellular RNA is induced.
  • These non-specific reactions mask the specific response of the target gene. Therefore mammalian or 15 to 3 OBP in the case of using cells from the animal, or a tissue as an object to be transductant, preferably more preferably 1.
  • the double-stranded polynucleotide does not need to be entirely double-stranded, including those with a partially protruding 5 ′ or 3, but those with a partially protruding 3 ′ end. Is preferred.
  • the double-stranded polynucleotide means a complementary double-stranded polynucleotide, but may be a self-annealed single-stranded polynucleotide having self-complementarity. Examples of the single-stranded polynucleotide having self-complementarity include those having an inverted repeat sequence.
  • the method for preparing the double-stranded polynucleotide is not particularly limited, but it is preferable to use a chemical synthesis method known per se.
  • 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.
  • Specific examples of the method of association include a method in which the synthesized single-stranded polynucleotide is mixed, heated to a temperature at which the double-strand is dissociated, 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 may be any as long as the target gene can be transcribed into RA or translated into protein in the cell.
  • Specific examples include those belonging to plant, animal, protozoan, virus, bacterial, or fungal species.
  • the plant may be a monocotyledonous, dicotyledonous or gymnosperm, and the animal may be a vertebrate or invertebrate.
  • Preferred microorganisms are those used in agriculture or industry, and are pathogenic to plants or animals. Fungi include organisms in both power 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 animals.
  • Reptiles including Drosophila, and other insects.
  • the cells are vertebrate cells.
  • the transductant means a cell, tissue, or individual.
  • a cell refers to a germ line or somatic, totipotent or pluripotent, divided or undivided, parenchymal or It may be from epithelium, immortalized or transformed, etc.
  • the cells may be gametes or embryos, in the case of embryos, single cell embryos or constitutive cells, or cells from multicellular embryos, including fetal tissue. Further, 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 fat cells, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, neurons, glial, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, and eosinophils Basophils, mast cells, leukocytes, granulocytes, keratinocytes, osteoblasts, osteoclasts, liver cells and cells of the endocrine or exocrine glands.
  • a method for introducing the double-stranded polynucleotide into the recipient when the recipient is a cell or tissue, calcium phosphate method, electroporation method, lipofection method, virus infection, two For example, immersion in a strand polynucleotide solution or a transformation method is used. Examples of the method for introduction into an embryo include microinjection, electoral poration, and viral 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.
  • 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 can be appropriately selected depending on the introduced substance and the target gene, but it is preferable to introduce an amount sufficient to introduce at least one copy per cell.
  • the transfectant is a cultured human cell and a double-stranded polynucleotide is introduced by a calcium phosphate method, 0.1 to 100 OnM is preferable.
  • 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.
  • the polypeptide used as an antigen also has high antigenicity according to a known method, and can be used for epitope ( An appropriate sequence can be selected and used as the antigenic determinant.
  • An appropriate sequence can be selected and used as the antigenic determinant.
  • commercially available software such as Epitope Adviser (manufactured by Fujitsu Kyushu System Engineering Co., Ltd.) can be used.
  • the polypeptide used as the above antigen may be a synthetic peptide synthesized according to a known method, or the protein of the present invention itself.
  • the polypeptide serving as an antigen may be prepared in an appropriate solution or the like according to a known method and immunized to a mammal, for example, a heron, a mouse, a rat, or the like. It is preferable to use the antigen peptide as a conjugate with an appropriate carrier protein or to use an adjuvant or the like for immunization in order to enhance the immunity.
  • the route of administration of the antigen upon immunization is not particularly limited.
  • any route such as subcutaneous, intraperitoneal, intravenous, or intramuscular route may be used.
  • a method of inoculating a BALB / c 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 times 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 a rise 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. Specifically examples thereof 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 distance analysis, ion-exchange chromatography, and affinity chromatography can be used.
  • a monoclonal antibody can be prepared by using a hybridoma fused with spleen cells and myeloma cells of the animal according to a known method (Milstein, et al., Nature, 256, 495 (1975)). .
  • 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 raised 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 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 (such as BALB / c-derived) myeloma cell line P3X63-Ag8.653 (ATCC: CRL-1580), P3-NSl / lAg4.1 (RIKEN Cellpunk: RCB0095) and the like are preferably used.
  • an appropriate cell fusion medium such as RPMI 1640, Iskov's modified Dulbecco's medium (IMDM), or Dulbecco's modified Eagle's medium (DMEM).
  • % By dissolving polyethylene glycol (PEG). It can also be performed by the electrofusion method (U. Zimmermann. Et al., Natur issenschaften, 68, 577 (1981)).
  • Hybridoma is myeloma cell line 8 Azaguanin by utilizing the resistance of a strain with an appropriate amount of hypoxanthine-aminopterin-thymidine (HAT) solution in including normal medium (HAT medium) 5% C0 2 using At 37 ° C. for an appropriate time.
  • HAT hypoxanthine-aminopterin-thymidine
  • HAT medium normal medium
  • This selection method can be appropriately selected and used depending on the myeoma cell line 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 a limiting dilution method, etc., and the separated fused cells are cultured in an appropriate medium.
  • a monoclonal antibody can be obtained.
  • a commercially available monoclonal antibody purification kit can also be used.
  • ascites containing a large amount of the monoclonal antibody of the present invention can be obtained by growing the antibody-producing hybridoma obtained above in an animal of the same strain as the immunized animal or in the abdominal cavity of a nude mouse or the like. it can.
  • a human-derived protein is obtained as the protein of the present invention
  • the above-described method is applied to a Severe combined immune deficiency (SCID) mouse transplanted with human peripheral blood lymphocytes using the polypeptide or a partial peptide thereof as an antigen.
  • a human antibody can also be produced by immunizing in the same manner as described above and producing a hybridoma between the antibody-producing cells of the immunized animal and the myeoma cells of the human (Mosier, DE, et al. al. Nature, 335, 256-259 (1988); Duchosal, MA, et al., Nature, 355, 258-262 (1992)).
  • RA is extracted from the obtained hybridoma producing the human antibody, the gene encoding the target human antibody is cloned, this gene is inserted into an appropriate vector, and the gene is introduced into an appropriate host.
  • human antibodies can be produced in larger quantities.
  • an antibody having a low binding property to an antigen can be obtained as an antibody having a higher binding property by using an evolutionary engineering technique known per se.
  • a partial fragment such as a monovalent antibody can be prepared by, for example, cleaving the Fab portion and the Fc portion using papain or the like and collecting the Fab portion using an affinity column or the like.
  • the antibody that specifically binds to the protein of the present invention thus obtained can be used to specifically bind to the protein of the present invention so that the antibody has an enzymatic activity, a proteolytic enzyme inhibitory activity, or a physiological activity other than the enzymatic activity possessed by the protein. It can also be used as a neutralizing antibody that inhibits the activity.
  • a neutralizing antibody There is no particular limitation on the method for selecting one that inhibits the activity of the protein.For example, the function of the target protein in the transductant is inhibited by contacting the antibody with the DNA transfectant prepared in (2) above. There is a method of analyzing whether or not.
  • 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.
  • the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight.
  • Such drugs can be administered in various forms, such as oral administration with coagulants, capsules, granules, powders, syrups, etc., or injections, infusions, ribosomes And parenteral administration using suppositories and the like.
  • 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 the above (2), and by analyzing this, it can be confirmed that it has the activity estimated in the above (1). Furthermore, analysis can also be performed by combination with an antibody or the like prepared as described in (4) above.
  • the activity of the protein of the present invention can be confirmed, for example, as follows. Force is not limited to these methods.
  • these activity measurement systems are also used for screening of a function activator (eg, agonist) or a function inhibitor (eg, antagonist) of the protein of the present invention and a screening of a protein expression regulator of the present invention, which will be described later. be able to.
  • the fact that the protein of the present invention has protease activity can be analyzed based on the properties of various proteases by a known activity measurement method which is known per se.
  • the reaction solution used for measuring the protease activity includes a neutral to weakly basic buffer solution for serine protease to which trypsin, chymotrypsin, elastase, etc. belong, and a cysteine proteinase to which a papain, cathepsin, etc. belongs.
  • Asparagine to which neutral to weakly acidic buffers, pepsin, chymosin, etc. belong Acid protease is used for acid protease, and carboxypeptidases A, B, and thermolysin belong to metal protease.
  • Neutral buffer is used, and metal ions such as Ca 2+ and Zn 2+ are required. If added, add them.
  • peptides and proteins are used as substrates.
  • 7_am ino— 4 -methy 1— co uma ri ii or p—nitroani 1 ine force is released when the peptide bond is hydrolyzed.
  • a substrate is designed with a 4-metocoumary 1- 7-amide group and a 1-nitroaniide group attached to the carboxyl-terminal side of, the former will be fluorescence and the latter will be visible spectroscopic measurements. It is possible.
  • the enzyme When a protein substrate is used, the enzyme is allowed to act on the substrate protein or a protein that can serve as a protease substrate such as serum albumin or casein, and the presence or absence of cleavage is confirmed by electrophoresis or chromatography.
  • the fact that the protein of the present invention has phosphodiesterase activity can be analyzed by measuring the activity of hydrolyzing a phosphodiester bond such as a cyclic nucleotide (cAMP or cGMP) that is a substrate of phosphodiesterase or ATP. can do.
  • a phosphodiester bond such as a cyclic nucleotide (cAMP or cGMP) that is a substrate of phosphodiesterase or ATP.
  • cAMP or cGMP cyclic nucleotide
  • General methods include Thompson, et al., Adv. Cyclic Nucleotide Res., 10, 69-92 (1979); Yanaka, et al., Eur. J. Biochem., 255, 391-399 (1998). Etc. can be used.
  • cyclic nucleotides such as cAMP and cGMP and derivatives thereof, pyrophosphate and the like can be used.
  • the fact that the protein of the present invention has a protease inhibitory activity can be analyzed based on the properties of various protease inhibitors by a commonly used activity measurement method known per se.
  • protease inhibitor in the protease reaction system targeted by the protein of the present invention, that is, the protease inhibitor of protein, inhibits the protease activity.
  • the inhibitory activity is measured using the above as an index.
  • ⁇ 1-protease inhibitor typical proteases targeted by this inhibitor are trypsin and elastase, and the inhibitor is added to the reaction system. Incubate trypsin and elastase with various concentrations of ⁇ 1-protease inhibitor under basic conditions ( ⁇ 7.5-8.5). After a certain period of incubation, the substrates (commercially available peptides, eg trypsin: Boc—G1 ⁇ — ⁇ 1a—Arg—MCA, elastase: Suc-A1a-A1a-A1 a-MCA, or substrate protein) and further incubate, then measure enzyme activity.
  • substrates commercially available peptides, eg trypsin: Boc—G1 ⁇ — ⁇ 1a—Arg—MCA
  • elastase Suc-A1a-A1a-A1 a-MCA, or substrate protein
  • Identify and quantitate by fluorescence intensity for peptide MC A substrate, and electrophoresis etc. for protein substrate are used for the amount ratio of the enzyme to the substrate, the incubation temperature, the time, and the like.
  • the difference between the activity when no inhibitor is added and the activity when the inhibitor is added is defined as the inhibitory activity.
  • the fact that the protein of the present invention has a transferase activity can be analyzed by a commonly used activity measurement method known per se. Specifically, a method of contacting a substance serving as a substrate with the recombinant protein and measuring that the transferase of the substrate is transferred to another group by the transferase activity of the recombinant protein may be mentioned.
  • a method of contacting a substance serving as a substrate with the recombinant protein and measuring that the transferase of the substrate is transferred to another group by the transferase activity of the recombinant protein may be mentioned.
  • neutralization is performed using UDP-galactose and pyridylamino-labeled G1cNAc2Man3G1cNAc2 as a substrate.
  • the fact that the protein of the present invention has isomerase activity can be analyzed by a commonly used activity measurement method known per se.
  • the measurement method include a method for measuring the activity of a substance serving as a substrate, and a method for measuring a change in physicochemical properties due to a structural change.
  • DN DN forming a supercoil is used as a substrate, and in a neutral (pH 7 to 8) buffer, lmMEDTA, 1 mM dithiothreitol, 20% glycerol, 0.05 M NaC 1 Contact with the protein in the presence and detect the change from type I to type II by 0.8% agarose gel electrophoresis (Dynan, WS et al. (1981) J. Biol. Chem. , 256, 5860).
  • the protein of the present invention has the activity of a protein such as a protein or an enzyme which does not belong to the above classification can be analyzed by a commonly used activity measurement method known per se.
  • a protein such as a protein or an enzyme which does not belong to the above classification
  • there are many proton acceptors and donors that use NADH as a donor so that the amount of NADH may be measured.
  • NADH has an absorption at 340 nm and no NAD +, it is possible to measure the amount of NADH generated or reduced by the redox reaction using the change in absorbance at 340 nm as an index. it can.
  • the protein of the present invention has a carrier activity as a carrier protein (transporter) can be analyzed by a commonly used activity measurement method known per se.
  • the activity of transporting Squalene to microsomal squalene epoxide can be measured using the method described in the literature (PNAS 2000, 98, 2244-2249), using the conversion of Squalene to squalene 2,3-oxide as an index. Good.
  • the product is purified, lipids are extracted, and thin-layer chromatography is performed. Perform the chromatography and measure with an image analyzer to detect the conversion of 14C-labeled lipid.
  • the protein of the present invention has intracellular organelle activity can be analyzed by a commonly used activity measurement method known per se.
  • the activity of lysosomes is to perform metabolic rotation of cells and various biological functions as an intracellular recycling system that decomposes and reuses a part of the lysosome.
  • the expression of the protein of the present invention can be suppressed using the RNAi method, and lysosomal degradation activity and cell metabolic activity can be measured.
  • the fact that the protein of the present invention has a protein interaction can be analyzed by a commonly used activity measurement method known per se.
  • the protein-protein binding activity may be measured.
  • the protein-protein binding activity can be confirmed by, for example, in vitro translation and protein-protein binding assay as follows (Journal of Biological Chemistry 275, 7894-7901 (2000)).
  • the protein whose binding activity is to be measured is A and its partner is B.
  • Plasmid pGEM-A and plasmid pGST-B (Trends Biochem. Sci. 21, 208-214 (1996)) containing the full-length cDNA of A and the full-length cDNA of B were converted to TNT SP6 polymerase from Promega, Inc. Used for in vitro transcription and translation.
  • radioactive substance labeling can be performed by adding 40 ⁇ l of [ 35 S] methionine (Amersham) to a total volume of 50 ⁇ l.
  • the translation product is subjected to in vitro protein-protein binding.
  • in vitro binding for example, 50 ⁇ l of [ 35 S] methionine-labeled protein A is incubated with a glutathione resin containing GST-B 4-5 // g fusion protein of GST and B.
  • the reaction was performed slowly using a binding buffer (150 mM NaCl, 0.1% Nonidet P-40, 50 mM Hepes (pH 7.5)), 1 mM PMSF, and a protease inhibitor for 4 ⁇ , 4 hours. This can be done by following the steps below. ⁇
  • the protein-protein complex formed on the resin surface is ultracentrifuged at 14,000 rpm for 1 minute at 4 and the resin is washed 5 times with 1 ml cold binding buffer at 4 ° C.
  • Protein A bound to GST fusion protein B bound to resin is separated on SDS-12% polyacrylamide gel, and after drying the gel, autoradiography is performed to examine the binding activity between protein A and B be able to.
  • methods for analyzing the function of the protein of the present invention include, for example, (i) a method for comparing and analyzing the expression state in each tissue, disease, or developmental stage; (Iii) a method of analyzing the phenotype by introducing the protein into an appropriate cell or individual, and (iv) inhibiting the expression of the protein in the appropriate cell or individual to There is a method of analyzing the change. Further, according to such a method, the activity specific to the target protein can be analyzed from many aspects.
  • the expression of the protein of the present invention can be analyzed at the mRNA level or the protein level.
  • Analysis of expression level at mRNA level In situ hybridization (Application to Developmental Biology & Medicine., Ed. By Harris, N. and Wilkinson, DG, Cambridge University Press (1990)), DNA A hybridization method using a chip, a quantitative PCR method, and the like are used.
  • a tissue staining method using an antibody that specifically binds to the protein of the present invention described later, an ELISA method, a Western plot method, and the like can be mentioned.
  • a known pariant is present in the protein to be analyzed, use a probe that exists only in the cDNA encoding the protein to be analyzed and does not hybridize with the cDNA encoding the known variant. Is preferred.
  • a method is used in which primers capable of producing amplified fragments of different lengths between the target cDNA and a known variant are selected (Wong, Y., Neuroscience Let., 320: 141-145 (2002)). )) And the like.
  • 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 method for analyzing interaction a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, liposome A multiple display method and the like can be mentioned.
  • yeast two-hybrid method fluorescence depolarization method, surface plasmon method, phage display method, liposome A multiple display method and the like can be mentioned.
  • the interacting substance of the known variant should be analyzed in the same manner to identify a substance that interacts specifically with the target protein. Is preferred.
  • the cell into which the cDNA of the present invention is introduced is not particularly limited, but human cultured cells and the like are particularly preferably used. Methods for introducing DNA into cells include those described in (2) above.
  • the phenotype of the transfected cells can be observed with a microscope, such as cell viability, cell growth rate, cell differentiation, neurite outgrowth when cells are neurons, and localization and migration of intracellular proteins. Stuff and intracellular Includes those that can be analyzed by biochemical experiments, such as changes in the expression of specific proteins. When known variants of the target protein exist, these phenotypes can also be introduced into cells in the same manner, and the phenotype associated with the target protein can be identified by comparative analysis. it can.
  • the protein of the present invention has an enzymatic activity, a proteolytic enzyme inhibitory activity, or a physiological activity other than the enzymatic activity. It is also preferable to analyze by paying attention to the phenotype and the like found in diseases associated with proteins having physiological activities.
  • the method (iv) can be efficiently performed by the method using the oligonucleotide described in the above (3) or the RNA interference method.
  • the same analysis is performed for the known variant and other variants, and a comparative analysis is performed to determine the specificity of the target protein. 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 selection is carried out using the binding property of the protein as an index.
  • the test substance can be used based on the change in the enzyme activity, proteolytic enzyme inhibitory activity, or physiological activity other than the enzyme activity).
  • the test substance may be any substance as long as it can interact with the protein of the present invention and affect the activity of the protein.
  • a conventional method known per se can be used as a method for analyzing the interaction between the test substance and the protein of the present invention. Specifically, for example, a yeast two-hybrid method, a fluorescence depolarization method, Examples include the surface plasmon method, the phage display method, the liposomal display method, and the competition analysis method with the antibody described in the above (4).
  • a substance found to bind to the protein of the present invention by such a method is then analyzed for how the activity of the protein of the present invention is affected in the presence of the substance.
  • the above-mentioned human homolog DNA or human homolog protein for the DNA or recombinant protein of the present invention to be used. Further, the substance screened by the above method may be further selected as a drug candidate by screening in vivo.
  • Analysis of the change in protease activity can be carried out by a known method per se, based on the properties of various proteases. Specifically, it can be carried out by using the method described in (5-1) above.
  • the protein having the protease activity of the present invention has an important function as a regulatory factor involved in various physiological functions, and the abnormality of the protein in vivo may cause various diseases.
  • the modulator of protease activity obtained by the above screening method can be used as a therapeutic agent for various diseases, for example, inflammatory diseases such as arthritis, immunological diseases, lung cancer, spleen cancer, ovarian cancer, squamous cell carcinoma, retina Central diseases such as cancer such as blastoma, motor integration dysfunction, neurodegenerative disease, spinocerebellar degeneration, It can be used as a therapeutic or contraceptive for endocrine diseases such as diabetes and obesity, respiratory diseases such as asthma, osteoporosis and infertility. .
  • a substrate is introduced into the DNA-introduced product described in (2) above.
  • the hydrolysis of the phosphate ester bond of the substrate in the presence or absence of the selected substance is analyzed by commonly used methods known per se. Specifically, it can be performed using the method described in (5-2) above. If the hydrolysis activity of a suitable substrate having a phosphodiester bond is increased as compared with the absence of the substance, the substance may function as a phosphodiesterase activator, or may decrease. Or, if inhibited, the substance can be identified as potentially functioning as a phosphodiesterase inhibitor.
  • the protein having phosphodiesterase activity possessed by the protein of the present invention hydrolyzes a phosphodiester bond.
  • those using cyclic nucleotides such as cAMP and cGMP as substrates serve as intracellular signaling As a second messenger of, it is involved in the regulation of many functions in the body, and the intracellular cAMP and cGMP concentrations that fluctuate in response to extracellular signals are It is regulated by the balance between the involved adenyl cyclase and guanyl cyclase and the phosphodiesterase involved in cyclic nucleotide degradation.
  • substances that can be identified by this screening method that inhibit or antagonize phosphodiesterase activity include cardiovascular diseases such as hypertension, kidney diseases, respiratory diseases such as ED (erectile dysfunction), bronchial asthma, and chronic bronchitis, Diseases related to neurological dysfunctions such as Alzheimer's disease, Parkinson's disease, diseases related to mental dysfunction such as manic depression, inflammatory diseases such as atopic dermatitis, acquired immunodeficiency syndrome, and deformation Systemic or local joint disease such as osteoarthritis of the knee, rheumatoid arthritis, tumor necrosis factor (TNF) such as Crohn's disease, septic shock, endotoxin shock, and other cytotoxic agents (IL-1, Diseases involving IL_6) Can be used as a pharmaceutical composition for the prevention or treatment of.
  • cardiovascular diseases such as hypertension, kidney diseases, respiratory diseases such as ED (erectile dysfunction), bronchial asthma, and chronic bronchitis
  • Diseases related to neurological dysfunctions such as Alzheimer's disease,
  • the analysis of the change in the protease inhibitory activity can be carried out by a method known per se and generally used, based on the properties of various protease inhibitors.
  • protease inhibitor coexistence of the protein of the present invention (proteolytic enzyme inhibitor) in the protease reaction system targeted by the protein of the present invention, that is, the protease inhibitor of protein, inhibits the protease activity.
  • the inhibitory activity is measured using the above as an index. Specifically, it can be performed using the method described in the above (5-3).
  • the protein having the protease inhibitory activity of the present invention has an important function as a control factor involved in various physiological functions, and abnormalities of the protein in a living body can be caused by various diseases.
  • the modulator of the protease inhibitory activity obtained by the above screening method can be used as a therapeutic agent for various diseases, for example, blood coagulation diseases, cardiovascular diseases, viral diseases such as HIV and influenza virus, and cancer. , DIC and the like. (6-4) Analysis of substances that regulate transferase activity
  • a protein serving as a substrate is introduced into the DNA-introduced substance described in (2) in the same manner.
  • whether or not the target group of the transferase in the substrate protein is transferred to the transductant is analyzed by a commonly known method. . Specifically, it can be performed using the method described in (5-4) above. If the transfer of the target group of the transferase is increased as compared to the absence of the substance, the substance may function as a transferase active substance and may be reduced or inhibited. In some cases, the substance can be identified as potentially functioning as a transferase inhibitor.
  • glycosyltransferase has a function of activating oncogene products, a function of controlling nerve function, immune function, inflammation, cell differentiation, virus infection, and the like. There is something. Therefore, substances that can be identified by this screening method are therapeutic drugs for cancer, diabetes, circulatory disease, Alzheimer's disease, Parkinson's disease, chorea, ischemic brain disease, diabetic peripheral neuropathy, infertility, etc. It can be used as a diagnostic agent or a regenerative tissue inducer.
  • a substrate is also introduced into the DNA-introduced product described in (2) by the same method. Changes in the substrate in the presence / absence of the selected substance for this transductant are analyzed by commonly used methods known per se. Specifically, it can be performed using the method described in (5-5) above. If the activity of the protein is increased as compared to the absence of the substance, the substance may function as an active substance, and if reduced or inhibited, the substance is an inhibitor. Can be identified.
  • Examples of the protein having isomerase activity of the protein of the present invention include those having an immunity function, a function of controlling inflammation, virus infection, and the like. Therefore, substances that can be identified by this screening method include immunological diseases, inflammatory diseases, viral diseases, cancer, diabetes, Alzheimer's dementia, Parkinson's disease, chorea, ischemic brain disease, diabetic peripheral neuropathy, infertility It can be used as a remedy for diseases.
  • Analysis of changes in the enzymatic activity of enzymes that do not belong to the above-mentioned classification can be performed by a commonly used method known per se based on the properties of the protein of the present invention. Specifically, it can be performed using the method described in (5-6) above.
  • the protein of the present invention has an important function as an enzyme involved in various physiological functions.
  • the modulator of enzyme activity obtained by the above screening method can be used as a therapeutic agent for various diseases, for example, anticancer agents (antitumor agents), anti-inflammatory agents, neurodegenerative diseases, cardiovascular diseases such as hypertension, metabolic It can be used as a therapeutic agent for diseases such as diseases, immune diseases and blood coagulation diseases.
  • the analysis of the change in the carrier activity can be performed by a commonly used method known per se, based on the properties of the protein of the present invention. Specifically, it can be carried out using the method described in the above (5-7).
  • the protein of the present invention has an important function as a transporter protein involved in various physiological functions, and abnormalities of the protein in vivo cause various diseases. Therefore, the modulator of the carrier activity obtained by the above screening method can be used as a therapeutic agent for various diseases, for example, metabolic disease, arteriosclerosis, vitamin deficiency, night blindness, diseases caused by peroxidation, aging, dementia. It can be used as a therapeutic agent such as.
  • Analysis of changes in intracellular organelle activity can be carried out by a known method per se, based on the properties of the protein of the present invention. Specifically, it can be carried out using the method described in the above (518).
  • the protein of the present invention has an important function as a protein having intracellular organelle activity involved in various physiological functions, and abnormalities of the protein in vivo cause various diseases. Becomes Therefore, the modulator of intracellular organelle activity obtained by the above screening method is used as a therapeutic agent for various diseases, for example, circulatory system diseases such as cancer and hypertension, immunological diseases, inflammatory diseases, and lysosomal diseases. It can be used as a therapeutic drug for metabolic diseases such as mucopolysaccharidosis and HIV infection.
  • the analysis of the change in the protein interaction activity can be carried out based on the properties of the protein of the present invention by a method known per se and generally used. In particular, It can be performed using the method described in (5-9) above.
  • the protein of the present invention has an important function as a protein having a protein interacting activity involved in various physiological functions, and abnormality of the protein in a living body may cause various diseases.
  • the modulators of protein interaction activity obtained by the above screening method are therapeutic agents for various diseases, for example, cancer, inflammatory diseases, immune diseases, cardiovascular diseases such as arteriosclerosis, psychiatric and neurological disorders. It can be used as a therapeutic agent for diseases such as diseases, metabolic diseases, and respiratory diseases such as bronchial asthma.
  • composition containing a modulator of the activity of the protein of the present invention When applied to pharmaceuticals, the above-mentioned active ingredient can be used alone, but can also be used as a pharmaceutical composition by blending 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, injections, drops, Parenteral administration with ribosomes, suppositories and the like can be mentioned.
  • the dose can be appropriately selected depending on symptoms, 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 it 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 it in the presence of a test substance Specifically, for example, cells expressing the protein of the present invention described in (2) above are cultured in a suitable medium containing a test substance, and the amount of the protein of the present invention expressed in the cells is determined.
  • the analysis can be performed using a conventional method such as ELISA, or by analyzing the amount of mRNA encoding the protein of the present invention in the cells by quantitative reverse transcription PCR, Northern blotting, or the like.
  • the test substance those described in the above (6) can be used.
  • the substance functions as the DNA expression promoting substance of the present invention. If it is possible and conversely decreases, it can be determined that the substance can be used as a DNA expression inhibitor 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 blending it with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight.
  • the drug can be administered in various forms. Examples of the dosage form include tablet, capsule, granule, powder, syrup, and the like, oral administration, injection, drip, ribosome 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 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 a human, and transplanted into a female individual uterus to generate the DNA.
  • a non-human mammal into which DNA has been introduced can be produced. More specifically, for example, a female individual is superovulated by hormone administration, then mated with a male, a fertilized egg is extracted from the oviduct on the first day after mating, and the introduced DNA is microinjected into the fertilized egg. It is introduced by a method such as extrusion. After culturing by an appropriate method, the surviving fertilized eggs are transplanted into the uterus of a pseudopregnant female individual (foster parent) to give birth.
  • Non-human mammals include, for example, mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats, and the like.
  • the DNA-introduced animal of the present invention thus obtained is obtained by breeding this individual and introducing the introduced DNA.
  • the offspring can be obtained by subculturing them in a normal breeding environment while confirming that they are maintained stably. Also, 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.
  • the protein of the present invention can be used as a carrier having it bound on a substrate.
  • a nucleotide sequence encoding the protein of the present invention for example, any one of SEQ ID NOS: 1 to 24, 57 to 59, 63, 64, 67 to 71, 77 to 79, 83 to 85, 89 to 93, or 99 to 107 DNA and its partial fragments having the nucleotide sequence described in (1) can be used as a carrier to which they are bound on a substrate.
  • proteins proteins
  • DNA chips DNA arrays
  • DNA microarrays and DNA macroarrays DNA microarrays and DNA macroarrays
  • 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 substrate for binding proteins and DNA, but the detection of hybridization is not performed.
  • 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 substrate for binding proteins and DNA, but the detection of hybridization is not performed.
  • RI for example, when using a fluorescent substance or the like, a glass plate or a silicon plate containing no fluorescent substance is preferably used.
  • the binding of the protein or DNA to the substrate can be easily performed by a commonly used method known per se.
  • These protein chips, DNA chips, or DNA arrays are also included in the scope of the present invention.
  • amino acid sequence of the protein of the present invention and the nucleotide sequence of the DNA are sequence information. Can also be used.
  • the base sequence of this DNA also includes the base sequence of the corresponding RNA. That is, a database of amino acid sequences and base sequences can be constructed by storing the obtained amino acid sequences and base sequences in an appropriate recording medium in a predetermined format readable by a computer. This database may contain the nucleotide sequences of other types of proteins and the DNA that encodes them. Further, 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; optical disks such as CD-ROM, MO, CD-R, CD-RW, DVD-R, and DVD-RAM; and semiconductors. Memory and the like can be mentioned.
  • mRNA-prepared mouse C57B LZ6
  • Each organ or tissue 0.5 to: Lg is homogenized with a 10 ml suspension, and the same amount of phenol / clonal solution as lm1 of 2M sodium acetate pH 4.0.
  • a mixture of mouth form (volume ratio 5: 1) was added for extraction.
  • RNA was separated from the aqueous phase and precipitated. After incubating the sample on ice for 1 hour, the precipitate was collected in a refrigerated centrifuge at 4,000 rpm for 15 minutes.
  • RNA M guanidine was dissolved in C1. After adding 2 volumes of ethanol, the mixture was incubated on ice for 1 hour, centrifuged at 4,000 rpm for 15 minutes, and the resulting precipitate was washed with 70% ethanol to collect RNA. Was dissolved in water again and the purity of the RNA was determined by reading the OD ratio 260Z280 (> 1.8) and 230 260 «0.45).
  • Oligonucleotide containing the recognition sequence of the restriction enzyme XhoI (SEQ ID NO: 49) (in the sequence, V indicates A, G or C, and N indicates A, G, C or T) 12.6 ⁇ 1 was used as a primer.
  • RNase-free water RNase-free water
  • Biotinylation of RNA dionole Binding of biotin to the diol site of RNA both at the 5, terminal end of the Cap structure and at the 3 'end ribose of the poly A chain
  • a two-step reaction was performed. These are the oxidation of the diol group followed by the coupling reaction of biotin hydrazide with the oxidized RNA.
  • 15 // g of the RNA-first strand cDNA complex obtained in the reverse transcription reaction was combined with 6.6 mM sodium acetate buffer (PH4.5) and sodium periodate as an oxidizing agent. Processing was performed in 50 ⁇ l of the reaction solution. This oxidation reaction was performed on ice for 45 minutes under light-shielded conditions.
  • Nonspecific adsorption of cDNA to streptavidin-coated magnetic beads 100 ⁇ g of yeast tRNA (treated with DNase I) was added to 5 mg (500 ju 1) of magnetic beads (magneticporousglass (MP G) particlescoatedithstreptavidin (CPG, NJ)) to prevent the adhesion. After leaving on ice for 1 hour, the plate was washed with a solution of 50 mM EDTA and 2 M NaCl.
  • 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 NaC1, 0.4% SDS, once with 50 ⁇ g / 1 yeast tRNA, and 10 mM NaCl.
  • the single-stranded full-length cDNA recovered in this manner was extracted with phenol / chloroform, and the volume of the solution was reduced to 100 1 or less with a speed bag, and then subjected to G25ZGlOOSE Sephadex chromatography. Fractions having RI activity were collected in siliconized microtubes, 2 ⁇ g of dalycogen was added, and the precipitate obtained by ethanol precipitation was dissolved in 30 ⁇ l of ultrapure water. (6) Add oligo d G to single-stranded cDNA
  • the single-stranded cDNA 30 / Z1 recovered in (5) above was used in a final volume of 50 ⁇ l of the reaction mixture at 20 OmM sodium codylate ( ⁇ 6.9), 1 mM MgCl 2 , 1 mM Co Cl 2 , 1 mM 2—Mercaptoethanol, ⁇ ⁇ ⁇ ⁇ d GT Under the conditions of P, using 32 units of terminal deoxynucleotidyl transferase (TaKaRa) 37. (: 30 min. To add the oligo dG addition reaction. At the end of the reaction, EDTA was added to 5 OmM, followed by a series of extractions with phenol / chloroform, ethanol precipitation, and 31 ⁇ l of ultrapure Dissolved in water.
  • the second-strand cDNA obtained by converting the first-strand cDNA into ⁇ was performed as follows.
  • the second strand low buffer 200mM T ris-HC 1 ( p H8. 75), l O OmM KC 1, 1 00 mM (NH 4) 2 S0 4, 20 mM Mg S0 4 , 1% Triton X—100, lmg / jul BSA) 3 ⁇ 1
  • second strand high buffer (20 OmM Tris_HCl (pH9.2), 600 mM KC1, 2 OmM MgC 1 2 ) 3 1, d CTP, dATP, d TTP, (1 & D each 0.25 mM, ⁇ -NADH 6 ⁇ 1, oligo dG-added first strand ⁇ ⁇ 31 1 ⁇ 1, second strand primer
  • Add 1 ⁇ one adapter 600 ⁇ g and add 15 units of ExTaq DNA polymerase (T
  • the reaction was stopped by adding 1 ⁇ l of 0.5 M EDTA, and further dissolved in 1 ⁇ l of 10% SDS and 10 ⁇ g of proteinase K to dissolve the protein components.
  • the mixture was heated at 15 ° C for 15 minutes, and finally, a double-stranded full-length cDNA was purified by extraction with phenol Z-cloth form and ethanol precipitation.
  • ⁇ ZAP III vector is ⁇ ZAP I SEQ ID NO: 51 of the multiple cloning site of the I (S TRATAGENE) vector was modified from SEQ ID NO: 51 to SEQ ID NO: 52, and two SfiI sites were newly introduced.
  • a PS (RI KEN) vector was prepared and cDNA was inserted.
  • LPS (RI KEN) (named ⁇ -FLC-1 (FLC stands for FULL—LENGTH cDNA)) is an LPS vector from Mo Bitec (Germany); It has been modified. That is introduced respectively l O kbpstuffer sides Conveniently the c DNA ⁇ the cloning site that exists in B a mH I and S a 1 I, c DNA from 0. 5 kb to about 1 3 kb is claw A 6 kb DNA fragment was inserted into the XbaI site so that it could be read (Japanese Patent Laid-Open No. 2000-325080).
  • L-FLC-1 for example, in the case of a lung cDNA library, the average chain length of the insert is 2.57 kb, and the actual insert from 0.5 kb to 12 kb is cloned. I was able to do it. In the case of the conventional method ⁇ ZAP, the average insert length was 0.97 kb. Therefore, the use of ⁇ -FLC-1 allows efficient cloning of large cDNAs compared to L ZAP. We can see that we can do it.
  • Example 2 Normalization of a full-length cDNA library No subtraction
  • RNA driver The mRNA prepared in Example 1 (1) (hereinafter sometimes referred to as “(a) RNA driver”) and the RNA prepared by the in vitro 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”).
  • One is a cDNA recovered from RNA-cDNA that has been removed by normalization and cloned into a phage vector. After infection with E. coli, 1000-2000 plaques are mixed per starting material into one library (Mura Phagemid) and convert it to plasmid DNA by a conventional method (the phage is again infected with E. coli together with the helper phage, converted to phagemid, and further infected again to obtain plasmid DNA).
  • RNA drier RNA drier.
  • mini-libraries are created from nine types of tissues (pancreas, liver, lung, kidney, brain, spleen, testes, small intestine, and stomach), and nine types of mini-libraries are mixed. To obtain RNA.
  • RNA is cultured from a library (about 20,000 clones) that has already been stored as a non-overlapping clone, and the resulting DNA is subjected to in vitro transcription reaction in the same manner as (b) RNA dripper (c ) RNA Dryer.
  • RNAs were labeled with biotin using the Label-ITB iotin labeling kit (Mirrus Corporation) and then added to tester cDNA at a ratio of 1: 1: 1. Reaction at Rot 10
  • One representative clone was selected from each cluster. Representative clones were selected using Q-bot (manufactured by G ENET IXLIMI TED) and arrayed 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 Escherichia coli DH10B has been transformed, add 10 Omg / ml of ampicillin and 5 Omg / ml of kanamycin and introduce it into the Zap vector. Led to SOLR system If so, 100 Omg / ml of ampicillin and 25 mg / ml of streptavidin were added.
  • Each clone cultured in the above (1) is further cultured in 1.3 ml HT solution containing 10 OmgZm1 ampicillin, and the cells are collected by centrifugation. Then, QI Aprep 96 Turbo (QI AGEN The plasmid DNA was recovered and purified using the following method. To check the chain length of the cDNA inserted in the obtained plasmid, 1/30 of the plasmid DNA obtained above was digested with the restriction enzyme Pvu II and subjected to 1% agarose gel electrophoresis. Was.
  • Plasmids were also 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 of SEQ ID NOS: 53 (sense strand) and 54 (antisense strand) when the vector was PS, and SEQ ID NO: 55 (sense strand) when the vector was Zap. Strands) and 56 (antisense strand). ⁇ Thermo sequenase Primer Cycl Sequencing Kit It Am ersh am Ph arma cia Bi
  • the reaction was performed using a DNA polymerase 4100 (LonggrEadseqeqencer).
  • Gaps that could not be analyzed by the above nucleotide sequence analysis were determined by the primer walking method.
  • AB I P ri sni377 and Z or AB I P ri s m3700 manufactured by Appli ed Bi os y s terns I n c.
  • 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 Stro Ke S hearing De ice (Fiore Inc. Was performed. The nucleotide sequence determination by the shotgun method was performed with an overlap of 12 to 15 clones. The gap whose sequence could not be determined by the nucleotide sequence determination was determined by primer walking in the same manner as described above.
  • Example 4 Analysis of base sequence of each full-length cDNA clone
  • dnaform37901 consists of 4593 bases, of which base numbers 1531 to 2631 are open reading frames.
  • the amino acid sequence predicted from the open reading frame consists of 366 amino acid residues (SEQ ID NO: 25).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 1 using BLAST.
  • S PTR protein database a combination of the SWI SS—PROT protein sequence database and the TrEMBL nucleic acid translation database
  • the above-mentioned protein is known to be involved in arthritis and cancer from literature information (Gene 2002 Jan 23; 283 (1-2): 49-62).
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 was subjected to protein characteristic search using HMM PFAM, and the amino acid sequence encoded by nucleotide numbers 41 2-1 137 in SEQ ID NO: 1 showed the characteristics of the meta-mouth protease.
  • sequence shown sequence that is entered as Reprolysin in P fam was found.
  • dnafo rm46350 was composed of 4335 bases, of which base numbers 992 to 4270 were open reading frames (including a termination codon).
  • the amino acid sequence predicted from the open reading frame consists of 1092 amino acid residues (SEQ ID NO: 26).
  • the above-mentioned protein is known to be involved in arthritis and cancer from literature information (Gene 2002 Jan 23; 283 (1-2): 9-62).
  • the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2 was subjected to a protein feature search using HMM PFAM. As a result, the amino acid sequence encoded by the nucleotide number 1354—19669 of SEQ ID NO: 2 was meta- A sequence showing the characteristics of the protease (sequence as a reprolysin in P fam) was found. In addition, a sequence (a base sequence that is entered as Pep-M12B-propep at Pfam) showing the characteristics of meta-oral protease was found in the amino acid sequence encoded by base numbers 796-1118 of SEQ ID NO: 2.
  • dnafor m51325 was composed of 1577 bases, of which base numbers 8 to 1459 had an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 483 amino acid residues (SEQ ID NO: 27).
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 3 was subjected to a protein feature search using HMM PFAM. (A sequence that is entered as trypsin in P f am) was found.
  • dnaform m9578 was composed of 2235 bases, of which base numbers 1 to 2034 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 677 amino acid residues (SEQ ID NO: 28).
  • a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 4. The results were in the S PTR protein database (integrated swi SS-PROT protein sequence database and Tr EMB L nucleic acid translation database).
  • the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 4 was subjected to a protein feature search using HMM PFAM, which revealed that the amino acid sequence encoded by nucleotide numbers 559-1152 in SEQ ID NO: 4 showed the characteristics of reprolysin, a peptidase.
  • the sequence (base sequence entered as Reprolysin in P fam) is added to the amino acid sequence encoded by base Nos. 190-528, which shows the characteristics of peptidase family M12B (Pep-M12B_propep in Pfam). (Entry sequence). '
  • dnafor ml3298 was composed of 746 bases, of which base numbers 10 to 411 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 133 amino acid residues (SEQ ID NO: 29).
  • a homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 5, and it was found in the S PTR protein database (swi SS—PROT protein sequence database and Tr EMB L nucleic acid translation database integrated).
  • dnafo rm28983 was composed of 2373 bases, of which base numbers 75 to 848 had an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 257 amino acid residues (SEQ ID NO: 30).
  • a homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 6. The results were in the S PTR protein database (integrated swi SS-PRO T protein sequence database and Tr EMB L nucleic acid translation database).
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 6 was subjected to protein characteristic search using HMM PFAM. The sequence shown (the sequence entered as trypsin in P f am) was found.
  • dnafo rm36451 consists of 2877 bases, of which base numbers 98 to 1351 constitute an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 417 amino acid residues (SEQ ID NO: 31).
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 7 was subjected to a protein feature search using HMM PFAM, and the amino acid sequence encoded by nucleotides 653-1330 in SEQ ID NO: 7 was characterized by the characteristics of trypsin, a serine protease. (A sequence that is entered as a trypsin in P fam) was found.
  • dnafo rm30375 was composed of 2335 bases, of which base numbers 526 to 2244 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 572 amino acid residues (SEQ ID NO: 32).
  • dnafor m26163 consists of 2377 bases, of which base numbers 256 to 1500 constitute an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 414 amino acid residues (SEQ ID NO: 33).
  • a homology search was performed on the amino acid sequence encoded by SEQ ID NO: 9 using BLAST. The results were obtained from the SPTR protein database (sw i SS—PROT protein sequence database and Tr EMBL nucleic acid translation database).
  • the above-mentioned protein (i i) was found to belong to astacin (protease) from literature information (Dev Biol 1992 Oct; 153 (2): 250-8) in the database.
  • the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 9 was searched for protein characteristics using HMM PFAM.When the amino acid sequence encoded by nucleotide numbers 466-1 1041 in SEQ ID NO: 9 was found to have the amino acid sequence encoded by A sequence (a sequence that is entered as Astacin in P fam) was found to exhibit characteristics.
  • dnafor m33042 was composed of 3614 bases, of which base numbers 68 to 1042 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 324 amino acid residues (SEQ ID NO: 34).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 10 using BLAST.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 10 was subjected to protein characteristic search by HMMP FAM, and the amino acid sequence encoded by nucleotide numbers 392 to 739 in SEQ ID NO: 10 showed characteristics of cysteine protease.
  • a sequence (a sequence that is entered as 0TU at Pfam) was found.
  • dnafor m37103 was composed of 3793 bases, of which base numbers 280 to 1191 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 303 amino acid residues (SEQ ID NO: 35).
  • a homology search was performed using the BLAST for the amino acid sequence encoded by SEQ ID NO: 11.
  • dnafor m44990 consists of 3646 bases, of which base numbers 344 to 985 constitute an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 213 amino acid residues (SEQ ID NO: 36).
  • a homology search was performed using the BLAST for the amino acid sequence encoded by SEQ ID NO: 12, and it was found in the S PTR protein database (integrated SWI SS-PROT protein sequence database and Tr EM BL nucleic acid translation database).
  • dnafor m45367 was composed of 3418 bases, of which base numbers 159 to 1190 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: 37).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 13 using BLAST.
  • Database registration code AE003567 Drosophila melanogaster genomic scaffold force S, e-va 1 ue: 7 XI 0-63, with 47 o / 0 identity over 343 amino acid residues
  • database registration symbol P43558 Hypothetical 33.5 kDa protein force e - va 1 ue: at 3 X 10- 33, hit in 35% of the degree of coincidence over the 278 amino acid residues.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 13 was subjected to protein characteristic search using HMMP FAM. (Sequence that is entered as 0TU at Pfam).
  • dnafor m51872 was composed of 3617 bases, of which base numbers 476 to 979 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 167 amino acid residues (SEQ ID NO: 38).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 14 using BLAST.
  • dnafor m39531 was composed of 2803 bases, of which base numbers 227 to 1582 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 451 amino acid residues (SEQ ID NO: 39).
  • a homology search was performed using the BLAST for the amino acid sequence encoded by SEQ ID NO: 15.
  • database registration code AX207688 Sequence 5 from Patent W00157081 e-va 1 ue: 0.0, and 94% identity over 469 amino acid residues
  • database registration code P08594 Aqualysin I precursor force e -value: at 5 X l 0- 45, hit 34% identity between over 355 amino acid residues.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 15 was subjected to protein characteristic search by HMMP FAM, and the amino acid sequence encoded by nucleotide numbers 71-1602 in SEQ ID NO: 15 showed the characteristics of serine protease.
  • the sequence shown (a sequence that is entered as Peptidase-S8 in P f am) was found.
  • dnaform m25900 was composed of 2222 bases, of which base numbers 174 to 1493 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 439 amino acid residues (SEQ ID NO: 40).
  • the S PTR protein database (integrating the SWI SS-PROT protein sequence database and the TrEM BL nucleic acid translation database) (i) a database registration mark Q9UL52, Serine protease DESC1 precursor force S, e- va 1 ue: 2 X 1 0 in ⁇ 9 °, and in 43% of the degree of coincidence over the 421 amino acid residues, also (ii) a database registration g his own " ⁇ " Ai 53776, airway trypsin liKe protease force S, e- va 1 ue: in 2X 1 0-8 9, at 40 o / 0 of the degree of match for 4 17 amino acid residues, and further (iii) data base over to register his own ⁇ "060235, Airway trypsin- like protease precursor i) e- va 1 ue:. 1
  • the protein (i) is characteristically expressed in the head, neck, epithelium, prostate, testis and squamous epithelium according to literature information (Br J Cancer 2001 Jan; 84 (2): 237-43).
  • the expression of (iii) was isolated from the sputum of a patient with chronic airway disease based on the fact that its expression was suppressed in tumors, and the above-mentioned protein (iii) was isolated from literature information (JBiolChem 1998 May 8; 273 (19): 11895-901) It is known to be a trypsin-like serine protease.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 16 was subjected to a protein feature search using HMMP FAM, and the amino acid sequence encoded by nucleotide numbers 795 to 1472 in SEQ ID NO: 16 was compared with the amino acid sequence encoded by the serine protease trypsin. A sequence exhibiting characteristics (sequence entered as trypsin in P f am) was found. From these facts, it is speculated that the protein encoded by the nucleotide sequence of SEQ ID NO: 16 is a serine protease having a function related to tumor and inflammation in epithelium. Was done.
  • dnafor m26560 was composed of 3568 bases, and among them, bases 134 to 1453 had an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 439 amino acid residues (SEQ ID NO: 41).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 17 using BLAST, and it was found in the S PTR protein database (integrated SWI SS-PROT protein sequence database and Tr EM BL nucleic acid translation database).
  • the protein of the above (i) is characteristically expressed in the head, neck, epithelium, prostate and testis from literature information (Br J Cancer 2001 Jan; 84 (2): 237-43), and squamous epithelium
  • the expression of (iii) was isolated from the sputum of a patient with chronic airway disease based on the fact that its expression was suppressed in tumors and that the protein of (iii) above was isolated from the literature (JBiolChem 1998 May 8; 273 (19): 11895-901). It is known to be a trypsin-like serine protease.
  • the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 17 was searched for protein characteristics by HMMP FAM. (A sequence that is entered as trypsin in P f am) was found. Therefore, the protein encoded by the nucleotide sequence of SEQ ID NO: 17 is epithelial It was presumed that it was a serine protease having a function related to tumor and inflammation in.
  • dnaform m32827 was composed of 3599 bases, of which base numbers 167 to 1486 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 439 amino acid residues (SEQ ID NO: 42).
  • the S PTR protein database (integrating the SWI SS-PROT protein sequence database and the TrEM BL nucleic acid translation database) (i) a database registration mark Q9UL52, Serine protease DESC1 precursor force e- value: 3 with X 10 one 96, and in 43% of the degree of coincidence over the 421 amino acid residues, also (ii) a database registration fi ⁇ "AF453776, airway trypsin- like protease X), e- value : at 3X 1 0 one 89, with 40% of the degree of match over the 417 amino acid residues, and further (iii) data base Ichisu registered Corps eyes his own No.
  • the protein of the above (i) is characteristically expressed in the head, neck, epithelium, prostate, testis, and squamous epithelium according to literature information (Br J Cancer 2001 Jan; 84 (2): 237-43).
  • the expression of (iii) was isolated from the sputum of a patient with chronic airway disease according to literature information (JBiolChem 1998 May 8; 273 (19): 11895-901). It is known to be a trypsin-like serine protease.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 18 was searched for protein characteristics by HMMP FAM. A sequence exhibiting characteristics (sequence entered as trypsin in P f am) was found. From these facts, it was presumed that the protein encoded by the nucleotide sequence of SEQ ID NO: 18 was a serine protease having a function related to tumor and inflammation in epithelium.
  • dnafo rm33661 consists of 3817 bases, of which base numbers 16 to 1335 constitute an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 439 amino acid residues (SEQ ID NO: 43).
  • the protein of the above (i) is characteristically expressed in the head, neck, epithelium, prostate and testis from literature information (Br J Cancer 2001 Jan; 84 (2): 237-43), and squamous epithelium
  • the expression of (iii) was isolated from the sputum of a patient with chronic airway disease based on the fact that its expression was suppressed in tumors, and that the protein of (iii) above was isolated from literature information (JBiolChem 1998 May 8; 273 (19): 11895-901). It is known to be a trypsin-like serine protease.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 19 was subjected to a protein feature search by HM MPFAM. A sequence exhibiting characteristics (sequences that are entered as trypsin in P fam) was found. From these facts, it was presumed that the protein encoded by the nucleotide sequence of SEQ ID NO: 19 was a serine protease having a function related to tumor and inflammation in epithelium.
  • dnaform m34950 was composed of 3355 bases, of which base numbers 76 to 1326 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 416 amino acid residues (SEQ ID NO: 44).
  • the S PTR protein database (integrating the SWI SS-PROT protein sequence database and the TrEM BL nucleic acid translation database) (i) a database registration mark AX262410, Sequence 1 from Patent W00173074 I e- va 1 ue: at 5 X 10- 122, also at 51% degree of coincidence over the 417 amino acid residues, also (ii) data base over the scan register yourself 060235, Airway trypsin- like protease precursor e- va 1 ue: at 5 X 10- 93, with 40% degree of coincidence over the 420 amino acid residues, yet (iii; data base over the scan register ti No.
  • the protein of the above (ii) is a trypsin-like serine protease isolated from sputum of a patient with chronic airway disease according to literature information (J Biol Chem 1998 May 8; 273 (19): 11895-901). It has been known.
  • the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 20 was subjected to a protein feature search using HMMP FAM. (A sequence entered as trypsin in P f am) was found. From these facts, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 20 was a serine protease having a function related to inflammation in epithelium. (21) dnafor m40997 (SEQ ID NOs: 21, 45)
  • dnafor m40997 was composed of 1456 bases, of which nucleotides 26 to 1345 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 439 amino acid residues (SEQ ID NO: 45).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 21 using BLAST. The results were as follows: i) database registration mark AF100707, testes- specific protein TSP50 force e - va 1 ue: at 5 X 10- 128, also at 54% degree of coincidence over the 439 amino acid residues, also (ii) a database registration mark AF100707, testes- specific protein TSP50 force e - va 1 ue: at 5 X 10- 128, also at 54% degree of coincidence over the 439 amino acid residues, also (ii) a database
  • the protein of (iv) above was isolated from eosinophils from literature information (Biochem Biophys Res Coramun 1998 Nov 18; 252 (2): 307-12), and many proteins other than skeletal muscle and kidney It is known to be a serine protease that is found in the testis and prostate, most commonly in testis and prostate, and moderately expressed in lung, spleen and knee.
  • the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 21 was subjected to a protein feature search using HMMP FAM. of A sequence exhibiting characteristics (sequence entered as trypsin in P f am) was found. From these facts, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 21 is a serine protease having a function in many tissues, particularly testis / prostate.
  • dnafor m43509 was composed of 1600 bases, of which base numbers 466 to 1476 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 336 amino acid residues (SEQ ID NO: 46).
  • a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 22.
  • the protein of (iii) and (iv) is a nuclear ubiquitin protease from the literature (Oncogene 1995 Jun 1; 10 (11): 2179-83), and its overexpression may be involved in tumorigenesis. It is known that there is.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 22 was subjected to a protein feature search using HMMP FAM.
  • amino acid sequence encoded by 05 a sequence (a sequence that is entered as UCH-2 at P f am) showing the characteristics of the thiol protease ubiquitin carbon terminal hydrolase was found.
  • dnaform m55017 consists of 4298 bases, of which base numbers 277 to 3342 constitute an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 1021 amino acid residues (SEQ ID NO: 47).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 23 using BLAST, and the SPTR protein database (SWI SS—PROT protein sequence database and the TrEMBL nucleic acid translation database were integrated.
  • the database entry code BC021474 a protein whose function is unknown, has an e-V a1 ue of 0.0, a 99% identity over 827 amino acid residues, and (ii) a database entry.
  • KIM1203 protein is, e _v a 1 ue: at 1 X 10- 67, 55% over 234 amino acid residues - in ⁇ further (iii) a database registration mark AF048705, ubiquitin- specific protease force e - va 1 ue: at 8 X 10- 56, hit 29% degree of coincidence over the 653 Amino acid residues. From these results, it was presumed that the protein consisting of the amino acid sequence shown in SEQ ID NO: 47 was a protease.
  • the protein of (iii) above is a nuclear ubiquitin protease from the literature (Oncogene 1995 Jun 1; 10 (11): 2179-83), and its overproduction (J expression may be involved in fl It is known that there is.
  • the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 23 was subjected to a protein feature search using HMMP FAM, and the amino acid sequence encoded by nucleotide numbers 244 to 337 of SEQ ID NO: 23 was compared with the amino acid sequence encoded by the thiol protease ubiquitin force bonyl. Sequence showing characteristics of terminal hydro race (PCH as UCH-1 Sequence to be stored) was found.
  • a sequence (a sequence which is entered as UCH-2 in P f am) was found in the amino acid sequence encoded by base numbers 1885-2070 in SEQ ID NO: 23, which is a characteristic of ubiquitin-potency rubonyl terminal hydrolace as a thiol protease.
  • dnafor m65027 was composed of 1816 bases, of which base numbers 623 to 1411 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 262 amino acid residues (SEQ ID NO: 48).
  • a homology search was performed using the BLAST for the amino acid sequence encoded by SEQ ID NO: 24.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24 was a carboxypeptidase involved in a kidney disease.
  • dnaform 35225 was composed of 1036 bases, of which base numbers 332 to 826 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 164 amino acid residues (SEQ ID NO: 60).
  • dnafor 146870 was composed of 2613 bases, of which bases 819 to 2102 constitute an open reading frame (including a stop codon). Amino acids predicted from open reading frames The sequence consists of 427 amino acid residues (SEQ ID NO: 58).
  • a homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 58. The results were as follows: i) database registration mark Y09661, X.
  • laevis mRNA for xolloid force e - va 1 ue: at 2 X 10 58, 30% of the degree of coincidence over or 436 amino acid residues also (ii) a database registration SL 057460, Dorsal-ventral patterning tolloid protein precursor mosquito e- va 1 ue: in the 4 X 10- 58, with 30% of the degree of match over the 445 amino acid residues, further (i 1 1) Ata to 1 to scan registration g himself No. P980 Ri 3, Bone morphogenetic protein 1 precursor ⁇ e- va 1 ue: at 7 X 10 57, hit with 30% degree of coincidence over the 436 Amino acid residues.
  • dnafor m41583 was composed of 2841 bases, of which base numbers 1110 to 1889 were open reading frames (including the termination codon).
  • the amino acid sequence predicted from the open reading frame consists of 259 amino acid residues (SEQ ID NO: 62).
  • a homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 59, and the SPTR protein database (SWI SS—PROT protein sequence database and TrEMBL nucleic acid translation database integrated) during, (i) a database registration mark AY017475, Mus mus cuius CSMD1 (Csmdl ) force e- va 1 ue: at 5 X 10- 108, In 68% of the degree of coincidence over the 257 amino acid residues, also (ii) the database registering Symbol AYO17307, Homo sapiens CUB ana sushi multiple domains protein 1 short form force 6 & 1 116: 1 10 48, 153 amino acids Hits were found with 67% identity across residues.
  • amino acid sequence shown in SEQ ID NO: 62 was subjected to protein characteristic search using HMMPFAM, and it was found that the complement control protein (CCP) modules or short A sequence exhibiting the characteristics of consensus repeats (SCR) (sequence entered as sushi in P fam) was found, and a sequence exhibiting the characteristics of the CUB domain in the amino acid sequence of amino acids 87-195 (entry as CUB in P fam) Ru system (J).
  • CCP complement control protein
  • SCR consensus repeats
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 59 was a meta-oral protease of the capture system.
  • dn afo rm31526 is composed of 301 1 bases, as shown in SEQ ID NO: 67, of which base numbers 52 to 1 374 are open reading frames
  • the amino acid sequence predicted from the open reading frame consists of 440 amino acid residues (SEQ ID NO: 72).
  • SEQ ID NO: 72 The amino acid sequence predicted from the open reading frame consists of 440 amino acid residues (SEQ ID NO: 72).
  • SEQ ID NO: 67 the amino acid sequence encoded by SEQ ID NO: 67
  • the SPTR protein database SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database integrated
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 67 was subjected to a protein feature search using HMMP FAM. Amino acid sequence entered as "Phosphodiest").
  • dnafo rm32312 was composed of 3066 bases, of which base numbers 48 to 1370 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 440 amino acid residues (SEQ ID NO: 73).
  • a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 68.
  • the S PTR protein database (SWI SS—PROT protein sequence database and TrEMBL nucleic acid translation database integrated ), (I) Database registration code P06802, Ectonucleotidepyrophospnate / phosphodiesterase 1 force e—va 1 ue: 1 XI 0-48, and 31% identity over 4 o 3 amino acid residues (Ii) Database registration symbol p 15396, Phosphodiesterase I force e—va 1 ue: 1 X 10_. At 4 , hits were made with 36% identity over 61 amino acid residues. From these results, it was presumed that the protein consisting of the amino acid sequence shown in SEQ ID NO: 73 had phosphodiesterase activity.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 68 was subjected to protein characteristic search using HMMP FAM. As a result, a sequence exhibiting phosphodiesterase characteristics at amino acid numbers 5-381 of SEQ ID NO: 73 ("P fam" Phosphodiest ”).
  • dn afo rm40586 consists of 1393 bases, as shown in SEQ ID NO: 69, of which base numbers 19 to 191 are open reading frames
  • the amino acid sequence predicted from the open reading frame consists of 390 amino acid residues (SEQ ID NO: 74).
  • SEQ ID NO: 74 The amino acid sequence encoded by SEQ ID NO: 69, the SPTR protein database (SWI SS-PROT protein sequence database and the TrEMBL nucleic acid translation database were integrated).
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 69 was subjected to protein characteristic search using HMMP FAM. As a result, a sequence exhibiting phosphodiesterase characteristics at amino acid numbers 5-381 of SEQ ID NO: 74 (P fam (an amino acid sequence that is entered as a hosphodiest).
  • dn a fo rm64786 consists of 1760 bases, as shown in SEQ ID NO: 70, of which base numbers 29 to 727 are open reading frames
  • the amino acid sequence predicted from the open reading frame consists of 232 amino acid residues (SEQ ID NO: 75).
  • SEQ ID NO: 7 When a homology search was performed using BLAST for the amino acid sequence encoded by No. 0, the SPTR protein database (integrating the SWI SS-PROT protein sequence database and the TrEMBL nucleic acid translation database) contained: i) data base over scan registration mark P 06802, E cto nu cleotidepyrophos ph ate / phosphodiesterase 1 force, e- value: at 4 XI 0 23, and in 3 1% degree of coincidence over the 202 amino acid residues, and (ii ) Database registration symbol p1 5396, Phosphodiesterase I, e—va1ue: 7 ⁇ 10—. 5 hits with 36% identity over 61 amino acid residues. From these results, it was presumed that the protein consisting of the amino acid sequence shown in SEQ ID NO: 75 had phosphodiesterase activity.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 70 was searched for protein characteristics by HM MPFAM. As a result, a sequence (Pf osphodiest). .
  • dnafo rm64814 was composed of 1573 bases, of which base numbers 383 to 1369 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 328 amino acid residues (SEQ ID NO: 76).
  • a homology search was performed for the amino acid sequence encoded by SEQ ID NO: 71 using BLAST.
  • amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 71 was subjected to protein characteristic search using HMMP FAM. As a result, a sequence exhibiting phosphodiesterase characteristics at amino acids 1 to 269 of SEQ ID NO: 76 (“P f am Ph osphodiest ”).
  • dnaform m29662 was composed of 1404 bases, of which base numbers 66 to 1271 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 401 amino acid residues (SEQ ID NO: 80).
  • SEQ ID NO: 80 amino acid residues
  • the protein (i) is considered to be involved in serine protease inhibition based on literature information (EMB0 J. 12: 1871-1898 (1993)) in the database.
  • the protein (ii) can be obtained from the literature information (Biochem. J. 345: 595-601 (2000)), and that the protein of (iii) is intracellular based on literature information in the database (Gene. 1999, 234 (1): 127-37). It has been clarified that they are involved in protein turnover.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 77 was subjected to a protein feature search using HMMP FAM. As a result, it was found that amino acid numbers 218 to 399 of SEQ ID NO: 80 showed a sequence (P fam base sequence entered as serpin).
  • dnafo rm62602 consisted of 1422 bases, of which base numbers 50 to 1183 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 377 amino acid residues (SEQ ID NO: 81).
  • SEQ ID NO: 81 When a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 78, the SPTR protein database (SWI SS—PROT protein sequence database and TrEMBL nucleic acid translation database were integrated).
  • the protein consisting of the amino acid sequence shown in SEQ ID NO: 81 was a protease inhibitor.
  • the protein of (i) is based on the literature information (J. Biol. Chem. 270: 16089-16096 (1995)) in the database, and has the ability to inhibit thrombin.
  • the protein of (ii) is Mol. Brain Res. 42: 293-300 (1996)), which is involved in the regulation of serine protease. J. Biol. Chem. 270: 29854-29861 (1995)), respectively, has been found to be involved in serine protease inhibition.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 78 was subjected to protein characteristic search using HMMP FAM. As a result, a sequence (Pf am, a nucleotide sequence entered as serpin).
  • SEQ ID NO: 79 dnafor m66120 was composed of 1962 bases, of which base numbers 112 to 744 were open reading frames (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 210 amino acid residues (SEQ ID NO: 82).
  • a homology search was performed on the amino acid sequence encoded by SEQ ID NO: 79 using BLAST.
  • the protein of the above (i) is related to the inhibition of thrombin based on the literature information (J. Biol. Chera. 270: 16089-16096 (1995)) in the database, and the protein of the above (ii) is From the literature information in the book (Brain Res. Mol. Brain Res. 42: 293-300 (1996)), it was clarified that they are involved in the regulation of serine protease.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 79 was searched for protein characteristics using HM MPFAM. As a result, it was found that amino acid numbers 4 to 193 in SEQ ID NO: 82 showed a sequence (P fam base sequence entered as serpin).
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 79 is a protease inhibitor having a function related to serine protease inhibition, and the protein or the expression control substance of the protein, the function activation
  • the substance or function inhibitor may be a therapeutic agent for viral diseases such as HIV and influenza, blood clotting diseases, cancer, and DIC.
  • dnafo rm27698 was composed of 3331 bases, of which base numbers 78 to 2198 were an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 706 amino acid residues (SEQ ID NO: 86).
  • dnafo rm50441 was composed of 2757 bases, of which base numbers 235 to 2631 were open reading frames (including the termination codon).
  • the amino acid sequence predicted from the open reading frame consists of 798 amino acid residues (SEQ ID NO: 87).
  • amino acid sequence of SEQ ID NO: 87 was searched for the protein characteristics by HMMPFAM.
  • amino acid sequence of amino acid No. 170-758 of SEQ ID NO: 87 showed the amino acid sequence of Choline / Carnitine o-acyl ransferase with the amino acid sequence of We found a system (sequence entered as rCarn_acyltransfJ in Pfam), and found a sequence showing the characteristics of Dehydrin in the amino acid sequence of amino acids 82-98 (sequence entered as "dehydrin” in Pfam). .
  • dnafo rm28700 consists of 3184 bases, of which base numbers 26 to 940 constitute an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 304 amino acid residues (SEQ ID NO: 88).
  • a homology search was performed using BLAST on the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 85.
  • SPF binds squalene and transports it to the squalene epoxide of the microsome, thereby catalyzing the conversion to squalene 2,3-oxide and initiating the late stage of sterol biosynthesis. Furthermore, human SPF is shown to have an alpha-tocopherol-dependent transcription promoting activity from literature information (BBRC 2001, 285, 295-299).
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 85 was squalene, It is presumed that phosphatidy cnoline alpha-tocopherol has a carrier protein or a transcription promoting activity, and the protein or its expression regulator, function activator, or function inhibitor is used for It may be a therapeutic drug for arteriosclerosis, vitamin deficiency, night blindness, diseases caused by peroxidation, aging, dementia, etc.
  • dnafor m6659 consisted of 1346 bases, of which base numbers 76 to 1188 were an open reading frame (including a stop codon).
  • the amino acid sequence 3 I IJ predicted from the open reading frame consists of 370 amino acid residues (SEQ ID NO: 94).
  • the protein of (ii) is considered to be involved in the activity of the steroid hormone receptor complex from the literature information in the database (Genomics 35: 448-455 (1996)).
  • Literature information in the database J. Biol. Chem. 268: 13187-13192 (1993) has revealed that it is involved in estrogen receptor activity.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 89 was subjected to protein characteristic search using HMMP FAM.
  • the amino acid numbers 16 to 241 in SEQ ID NO: 94 show the characteristics of peptidyl-prolyl cis-trans isomerase I found a stem (J (base sequence that is entered as a pro-isomerase at P f am)).
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 89 is an isomerase having a function related to a steroid hormone receptor.
  • dnafor m6776 was composed of 480 bases, of which base numbers 75 to 332 had an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 85 amino acid residues (SEQ ID NO: 95).
  • the amino acid sequence encoded by SEQ ID NO: 90 was subjected to a homology search using BLAST to find that the S PTR protein During the quality database (SWI SS- PROT protein sequence database as the integration of Tr EMB L nucleic translation database), (i) a database registration mark AK003402 ie- V a 1 ue: at 5 X 10_ 43, also 85 amino acid residues (Ii) database registration code Q08752, 40 kDa peptidyl-prolyl cis-trans isomerase (HUMAN) force e—value: X 10 — 40 , and 94% of 85 amino acid residues degree of coincidence, further (iii) a database registration mark P26882, 40 kDa peptidyl-prolyl cis -trans isomerase (BOVINE e -va 1 ue: with 2 X 10- 39, hit 91% degree of coincidence over the 85 amino acid residues From these results, it was inferred that the protein having the amino acid
  • the protein of the above (ii) is related to the activity of the steroid hormone receptor complex from the literature information in the database (Genomics 35: 448-455 (1996)).
  • Literature information in the database J. Biol. Chem. 268: 13187-13192 (1993) has revealed that each is involved in estrogen receptor activity.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 90 was searched for protein characteristics by HMMP FAM, and the amino acid numbers 22-55 of SEQ ID NO: 95 showed the characteristics of the tetratricopeptide repeat involved in protein-protein interaction.
  • the sequence shown base sequence entered as TPR at P f am) was found.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 90 is an isomerase having a function related to steroid hormone receptor.
  • dnafor ml7871 was composed of 1720 bases, of which base numbers 81 to 761 were an open reading frame (including a stop codon).
  • Amino acid sequence predicted from open reading frame The row consists of 226 amino acid residues (SEQ ID NO: 96).
  • a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 91.
  • the protein of the above (ii) may be involved in the activity of the estrogen receptor according to literature information (J. Biol. Chem. 268: 13187-13192 (1993)) in the database.
  • the literature information in the database revealed that it is involved in the activity of the steroid hormone receptor complex.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 91 was subjected to protein characteristic search using HMMP FAM.
  • amino acid numbers 17 to 185 of SEQ ID NO: 96 showed that In the G system U, J fam, a base sequence that is entrapped as a pro-isomerase was found.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 91 is an isomerase having a function related to a steroid receptor.
  • dnafor m26175 consists of 3291 bases, as shown in SEQ ID NO: 92, of which base numbers 54 to 830 contain open reading frames (with a stop codon (Including).
  • the amino acid sequence predicted from the open reading frame consists of 258 amino acid residues (SEQ ID NO: 97).
  • a database registration mark AJ005766, lysosome- associated membrane protein (LAMP ) is, e- va 1 ue: in 5X 10- 41, and in 42% of the degree of coincidence over the 250 amino acid residues, also (ii) a database registration symbol AB013924, ⁇ TSC403 ⁇ product is, e- value: at 6 X 10- 42, with 42% degree of coincidence over the 250 amino acid residues, yet (iii) database registration mark P28968, Glycoprotein X precursor force e- value : in 2X 1 0- 04, hit with 30% degree of coincidence over the 130 amino acid residues. It is known that expression of "TSC403"; product is increased in lung cancer.
  • the protein LAMP of the above (i) is a glycoprotein on the lysosomal membrane.
  • CD68 + cells in the lung expressing DC-LAMP suppress the SIV replication in the acute 1 "life stage of infection. Is known to be.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 92 is a glycoprotein having a transmembrane region and is involved in the functions of organelles such as lysosomes and metabolism such as lysosomal disease and mucopolysaccharidosis. It was speculated that it was related to disease and HIV infection.
  • dnafor m42159 (SEQ ID NOs: 93 and 98) As shown in SEQ ID NO: 93, dnaform m42159 was composed of 3290 bases, and among them, bases from 54 to 1289 had an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 411 amino acid residues (SEQ ID NO: 98).
  • a homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 93, and a SPTR protein database (integrated SWI SS—PROT protein sequence database and TrEMBL nucleic acid translation database) during, (i) a database registration mark AJ005766, lysosome - associated membrane protein ( LAMP) force, e- va 1 ue: at 5 X 10- 112, 51% degree of coincidence over the 423 amino acid residues, and (ii ) database registration mark AB013924, "TSC403" product, e -value: at 5 X l 0 one 112, 51% degree of coincidence over the 423 amino acid residues, yet (iii) data base Ichisu book ⁇ himself No.
  • the protein LAMP of the above (i) is a glycoprotein on the lysosomal membrane.
  • CD68 + cells in the lung expressing DC-LAMP suppress SIV replication during acute infection. It is known that
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 93 was subjected to protein characteristic search by HMMP FAM.
  • amino acid numbers 61 to 411 of SEQ ID NO: 98 lack the characteristics of Lysosome-associated membrane glycoprotein.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 93 has a Transmembrane using tmHMM (S. Moller, MDR Croning, R. Apweiler.Evaluation of methods for the prediction of membrane spanning regions.Bioinformatics, 17 (7): 646-653, 2001.) When the sites were predicted, transmembrane sites were predicted at amino acid numbers 5-24 and 375-397.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 93 is a glycoprotein having a transmembrane region and is involved in the functions of organelles such as lysosomes and metabolism such as lysosomal disease and mucopolysaccharidosis. It was estimated to be related to disease and HIV infection.
  • dnafor m34219 was composed of 2617 bases, of which base numbers 146 to 1624 were open reading frames (including the termination codon).
  • the amino acid sequence predicted from the open reading frame consists of 492 amino acid residues (SEQ ID NO: 108).
  • the SPTR protein database (integrating the SWI SS-PROT protein sequence database and the TrEMBL nucleic acid translation database) showed (i) database entries symbol AK074067 iK e- value: 5 X 10_ at 148, and in l Ri 84% degree of coincidence to 310 amino acid residues, also (ii) a database registration mark BC007206 force e - va 1 e: 5 in X 10- 49, in 39% of the degree of coincidence over the 286 amino acid residues, yet is (iii) a database registration mark AL390078, e- value: at 5 X l 0- 44, 37% over 286 amino acid residue Hits with a match degree of
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 99 was subjected to a protein feature search using HMMP FAM, which revealed that amino acid numbers 393 to 469 in SEQ ID NO: 108 had the module structure required for binding to a signal transduction factor.
  • a sequence base sequence that is entered as SH2 in P f am showing characteristics was found.
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 99 It was speculated that the protein has a function related to the interaction with the syn-phosphorylated protein.
  • dnafor m51548 consists of 4755 bases, of which base numbers 159 to 2249 constitute an open reading frame (including a termination codon).
  • the amino acid sequence predicted from the open reading frame consists of 696 amino acid residues (SEQ ID NO: 109).
  • a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 100. The homology was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database).
  • the database registration code AK027856 is e-va 1 ue: 0.0, and the identity is 80% over 470 amino acid residues.
  • the database registration symbol Z18529 is e-va 1 ue : 1 in XI 0- 84, with 38% degree of coincidence over the 536 amino acid residues, yet (iii) database registration mark Q04205, Tens in force e- value: in 1X 10- 84, 536 Ryo amino acid residues Hits with a 38% match score.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 100 was subjected to a protein feature search using HMMPFAM. As a result, amino acid numbers 429 to 521 of SEQ ID NO: 109 were found to have the modular structure required for binding to a signal transduction factor. A sequence exhibiting characteristics (a base sequence that is entered as SH2 in P fam) was found.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 100 was a protein having a function relating to the interaction with the caspase phosphorylation protein.
  • dnaform m46811 was composed of 3089 bases, of which base numbers 474 to 1010 were open reading frames (including a termination codon).
  • the amino acid sequence predicted from the open reading frame consists of 178 amino acid residues (SEQ ID NO: 110).
  • SEQ ID NO: 101 is When the homology search was performed using BLAST for the amino acid sequence to be read, the (i) registration symbol BC025237, hematopoietic SH2 protein force S, e- va 1 ue: 2 10 5 and 38% of the degree of coincidence over the 138 amino acid residues, also (ii) a database registration Q9QXK9, SH2 domain protein 2A, Lad , an adapter protein interacting with the SH2 domain of p561ck (involved in T cell differentiation) I e— va 1 ue: 2 ⁇ 10 10 with 37% identity over 105 amino acid residues, and (iii) database registration mark Q9NP31, SH2 domain protein 2A force ev alue: at 7X l 0- 08, hit 36% degree of coincidence over the 100 amino acid residues.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 101 was subjected to protein characteristic search using HMMPFAM. As a result, it was found that amino acid numbers 72 to 147 of SEQ ID NO: 110 are modules required for binding to a signal transduction factor. A sequence (base sequence entered as SH2 in Pfam) showing structural features was found.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 101 was an adapter protein having a function related to the interaction with the cleft synthetase protein.
  • dnafor m27114 was composed of 1990 bases, of which bases 535 to 1440 had an open reading frame (including a termination codon).
  • the amino acid sequence predicted from the open reading frame consists of 301 amino acid residues (SEQ ID NO: 11).
  • a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 102, and the results were obtained in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database).
  • database registration code P29353, SHC transforming protein (HUMAN) (known to be involved in cell carcinogenesis (Cell 1992 Jul 10; 70 (1): 93-104)) va 1 ue: 1 X 10- 55, and in 44% of the degree of coincidence over the 317 amino acid residues
  • data base registration mark P98083, SHC transforming protein (MOUSE) Fei known to be involved in canceration of cells Biol Chem 1994 Dec 23; 269 ( 51): 32031 - 4)
  • ie one va 1 ue: with 2 X 10-5 4, with 44% degree of coincidence over the 373 amino acid residues further to (iii) database entry Symbol P98077, Protein SCK (HUMAN) ⁇ e-va 1 ue: 2 X 10- ", with a hit of 39% over 305 amino acid residues.
  • amino acid encoded by the nucleotide sequence of SEQ ID NO: 102 The protein was searched for protein characteristics using HMMPFAM.
  • the amino acid numbers 72 to 147 of SEQ ID NO: 11 show a sequence showing the characteristics of the module structure required for binding to a signal transduction factor (Pf am Base sequence entered as).
  • the protein encoded by the nucleotide sequence represented by SEQ ID NO: 102 is a protein having a function relating to the interaction with the tyrosine phosphorylated protein, and is presumed to be involved in canceration.
  • a substance that regulates the onset of expression, a function activator, or a function inhibitor could be developed as a therapeutic agent for cancer, arteriosclerosis, etc.
  • dnafor m31796 was composed of 3004 bases, of which base numbers 50 to 1657 were an open reading frame (including a termination codon).
  • the amino acid sequence predicted from the open reading frame consists of 535 amino acid residues (SEQ ID NO: 112).
  • a homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 103, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database).
  • SPTR protein database integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database.
  • database registration No. d P46681, Actin interacting protein 2 force S e—value: 5 X 10 _1 36 , and 488 amino acid residues!
  • Ri with 52% degree of coincidence also is (ii) a database registration mark AE005999, e _v alue: at l X l 0- 64, in Wataru Ri 34% degree of coincidence to 437 amino acid residues, yet (iii) database Registration code P32891, D-lactate Dehydrogenase (YEAST) force e- va 1 ue: with 2 X 10- 38, hit 27% degree of coincidence over the 433 amino acid residues. From these results, it was presumed that the protein consisting of the amino acid sequence shown in SEQ ID NO: 112 was an enzyme.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 103 was subjected to a protein characteristic search using HMMPFAM. As a result, a sequence exhibiting a characteristic of binding to FAD at amino acid numbers 82 to 275 of SEQ ID NO: 12 ( Pfam, a base sequence entered as FAD—binding—4).
  • dnaform m25173 was composed of 1395 bases, of which bases 869 to 1174 had an open reading frame (including a termination codon).
  • the amino acid sequence predicted from the open reading frame consists of 101 amino acid residues (SEQ ID NO: 113).
  • SEQ ID NO: 113 101 amino acid residues.
  • SPTR protein database integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database.
  • the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 104 was subjected to protein characteristic search using HMMPFAM, and as a result, a sequence (P f am entry as Rieske Base sequence). '' Based on these facts, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 104 was presumed to be an Fe-mediated acid ligase reductase. Functional inhibitors may be developed as therapeutics for cancer, high blood pressure, immune disorders, inflammatory disorders, and others.
  • dnaform m4547 was composed of 1327 bases, of which base numbers 549 to 1022 were open reading frames (including a termination codon).
  • the amino acid sequence predicted from the open reading frame consists of 157 amino acid residues (SEQ ID NO: 114).
  • a homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 105, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). , (i) a database registration No.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 105 was subjected to protein characteristic search using HMMPFAM, and as a result, a sequence exhibiting the characteristic of binding to Fe at amino acid numbers 16 to 101 of SEQ ID NO: 114 (P The base sequence that was entered as Rieske on the fam) was found.
  • the protein encoded by the nucleotide sequence shown in SEQ ID NO: 105 was a reductase having a function of oxidative phosphorylation. Therefore, the protein or an expression control substance, a function activator, or a function inhibitor of the protein Harmful substances have the potential to be developed as therapeutics for cancer, hypertension, immune disorders, inflammatory disorders, etc.
  • dnafor m56923 consists of 2808 bases, of which base numbers 1045 to 1518 constitute an open reading frame (including a stop codon).
  • the amino acid sequence predicted from the open reading frame consists of 157 amino acid residues (SEQ ID NO: 115).
  • a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 106. , (i) a database registered 3 ⁇ 4 ⁇ No.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 106 was subjected to protein characteristic search using HMMPFAM. As a result, a sequence exhibiting the characteristics of binding to Fe at amino acid numbers 16 to 134 of SEQ ID NO: 115 (Pfam Rieske entry).
  • the protein encoded by the nucleotide sequence of SEQ ID NO: 106 was a reductase having the function of oxidative phosphoric acid. Therefore, there is a possibility that the protein or its expression controlling substance, function activating substance or function inhibiting substance can be developed as a therapeutic drug for cancer, hypertension, immune disease, inflammatory disease, etc.
  • dn afor m48770 (SEQ ID NO: 107, 1 16) As shown in SEQ ID NO: 107, dnafor m48770 was composed of 3275 bases, of which base numbers 169 to 2760 were open reading frames (including the termination codon). The amino acid sequence predicted from the open reading frame consists of 863 amino acid residues (SEQ ID NO: 116).
  • a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 107, and a SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database) Among them, (i) database registration symbol AF479747, PYRIN-containing APAF G like protein 4 force e-va 1 ue: 0.0, and 42% identity over 868 amino acid residues, and (ii) data base one scan registration mark AF482706, ribonuclease inhibitor 2 (negation 2), e- va 1 ue: at 5 X 10- 167, 41% degree of coincidence over the 780 amino acid residues, yet (iii) database registration mark AF298547 , nucleotide- binding site protein 1 force s, e- va 1 ue: at 2 X 10 97, hit 29% degree of coincidence over the 911 amino acid residues.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 107 was subjected to protein characteristic search using HMMPFAM. As a result, a sequence exhibiting the characteristics of Leucine Rich Repeat at amino acid Nos. The base sequence that is entered as the LRR was found.
  • a tissue expression analysis using a DNA microarray was performed as described in Miki, R., et al., Proc. Natl. Acad. Sci. USA, 98, 2199-2204 (2001).
  • Nucleotide sequences of 6 mouse full-length cDNAs (dn afo rm9578, dnafo rm37901, dnafo rm64786, dnafo rm50441 d After nafo rm6659 and dnafo rm26 175) were amplified using M13 forward and reverse primers, the PCR product was precipitated with isopropanol and dissolved in 15 ⁇ l of 3XSSC solution.
  • 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 one-tenth 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.
  • Cy3 Fluorescent dye Cy3 (Amersham Phar ma cia).
  • 1 ⁇ g of mRNA extracted from the whole body of a 17.5-day-old fetus was subjected to random prime reverse transcription reaction, and the fluorescent dye Cy5 was taken in as a reference for expression analysis.
  • the CyDye-labeled cDNA probe is CyScribe GFX Purificati
  • the product was purified using 0 nKit (AmershamPharmacia) and eluted from the column with 17 ⁇ l of sterile water. 3 ⁇ 1 of 10 g / ⁇ 1 ⁇ 1 i g ⁇
  • 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 ScanArayay500 confocal laser scanner, and the images were analyzed on an IMAGENE (BioDiversive).
  • the mRNA level (Cy3 label) in each tissue is expressed as the ratio (Cy3 / Cy5) to the reference 17.5-day-old fetal whole-body mRNA level (Cy5 label) as a logarithm (1 og). Displayed in 2 ). That is, when the mRNA expression level corresponding to the full-length cDNA of each mouse to be analyzed is larger in each tissue than in the reference tissue, a positive numerical value is used. If not, it is a negative number, otherwise it is 0. Experiments were performed twice independently to increase the accuracy of the data, and reproducible results were used. The results are shown in Table 1.
  • the results of expression analysis using a DNA array are considered to be experimental errors if the results are about two-fold increase / decrease.
  • the result when the result is 1 or more, the mRNA level in a certain tissue is a control.17
  • the amount of mRNA is more than twice as large as that of the whole fetal body at 5 days of age and significantly increased, and conversely, if the result is less than 11, the amount of mRNA in one tissue is This was less than one-half that of the whole fetal mRNA at 17.5 days of age, which was interpreted as significantly reduced.
  • the raRNA level is 2 times, and if it is 2, the mRNA level is 4 times. If the difference between the tissues is 11, the mRNA amount is 1/2 times, and if it is 12, the mRNA amount is 1 to 4 times.
  • a mouse cDNA clone (dnafo rm46350, dnafo rm31 526, dnafo rm3231212) 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% with at least 200 bases.
  • Dnafo rm40586, dnafor m64814, dnafo rm6776, dnafo rml 7871, and dnafor m42159 are also described as the analysis target cDNA in Table 1, and the values of the measurement results of the DNA spotted on the microarray are substituted. Described.
  • 16-day-old 16-day-old 16-day-old 16-day-old 1-year-old 14-year-old fetal DNA DNA head dnaform6659 0.442715 -0.173972 -0.01888 0.590957 -0.139838 dnaform6776 dnaform6659 0.442715 -0.173972 one 0.01888 0.590957 -0.139838 dnaform17871 dnaform6659 0.442715 -0.173972 -0.01888 0.590957 -0.139838 Analysis target cDNA New age 0 0 Age newborn DNA whole child whole child whole child whole child whole child live intestine baby lung
  • dnaform37901 dnaform37901 -1.00373 0.255501 -0.108227 d 0.029519 -0.108285 dnaform46350 dnaform37901 -1.00373 0.255501 d 0.108227 -0.029519-0.108285 dnaform31526 dnaform64 / 86 -0.115941 1.31019 0.381869 0.000821 0.377181 dnaform32312 0.3nad1.
  • dnaform37901 dnaform37901 -0.503089 -0.740213 -0.79368 1.54568 -0.342865 dnaform46350 dnaform37901 -0.503089 i 0.740213 -0.79368 1.54568 -0.342865 dnaform31526 dnaform64786 -0.615982 0.561235 -0.488765 0.128262 0.450018 dnaform32312 0.48 -82.
  • dnaform42159 dnaform26175 -0.560286 0.08057 As is evident from Table 1, dnaform 37901 was spotted on a microarray and used as a target sequence. It can be seen that the expression is increased in the cerebellum and neonatal cerebellum as compared to). Thus, it shows a characteristic expression in the central nervous system. Expression analysis of dnaform 4650, which uses dnaform 37901 as a target sequence of the members in the cluster, shows that expression is increased in the cerebellum and neonatal cerebellum compared to the reference. Expression analysis using dnafo rm9578 as its target sequence revealed that it was more strongly expressed in the testis than in the reference, which is considered to be a remarkable feature.
  • dnaform31526 is characterized in that it is more strongly expressed in kidney than in reference by expression analysis using dnaform64786 of a member of the cluster as a target sequence. It can be seen that the expression is also increased in the kidney. A tendency to increase expression was also observed in adipocytes.
  • Expression of dnaform32312 is characterized in that it is more strongly expressed in kidney than in reference by expression analysis using dnaform64786, a member in the cluster, as a target sequence. It can be seen that the expression is also increased in the knee. An increase in expression was also observed in fat cells.
  • dnaform40586 in the kidney is stronger than that of the reference by expression analysis using dnaform64786, a member of the cluster, as a target sequence. It can be seen that the expression is also increased in the spleen. A tendency to increase expression was also observed in adipocytes.
  • the expression of dnaform64786 is characterized in that it is strongly expressed in the kidney compared to the reference by expression analysis using the target sequence itself. It can be seen that the expression is also increased in the knee. A tendency to increase expression was also observed in adipocytes.
  • dnaform64814 The expression of dnaform64814 is characterized in that it is strongly expressed in the kidney compared to the reference by expression analysis using dnaform64786, a member in the cluster, as the target sequence. It can be seen that the expression is also increased in the kidney. A tendency toward increased expression was also observed in adipocytes.
  • dnaform50441 itself as a target sequence shows that dnaform50441 is more strongly expressed in the brain, cerebellum, and testis than in the reference. It can be seen that expression is generally weakened in other organs.
  • dnafo rm6659 by itself has a tendency to be more strongly expressed in fetal whole body, neonatal lung and mammary gland than in reference.
  • dnafo rm6776 is a member of dnafo rm66 Expression analysis using 59 as the target sequence showed that the expression tended to be stronger in the whole fetal body, neonatal lung and gland compared to the reference.
  • Expression analysis of dnafo rml 7871 using the above dnafo rm6659 as a target sequence of a member in the cluster tends to be more strongly expressed in the whole fetus, neonatal lung, and mammary gland than in the reference.
  • dnaform26175 is more strongly expressed in the lungs than the reference. Expression is also increased in kidney and epididymal adipocytes. Expression analysis of dnafo rm42159, in which the dnafo rm26175 of the members in the cluster is the target IJ, shows that it is more strongly expressed in the lungs than the reference. Expression is also increased in kidney and epididymal adipocytes.
  • dnafo rm26163 human ortholog DNA (SEQ ID NO: 63, 65)
  • the dnafo rm26 163 nucleotide sequence (SEQ ID NO: 9) was queried to obtain a human genome draft sequence (NCBIB uild 30; http: / /ww.ncbi.nlm.nih.gov/About/Doc/hs_genomeintro.html), a BLA ST search showed that a highly homologous region from 94.65Mb of 2q11.2 on chromosome 2 An area of 94.68 Mb was found.
  • nucleotide sequence of SEQ ID NO: 9 had a homology of 73.6% over the length of about 1280 base pairs with the human nucleotide sequence of SEQ ID NO: 63.
  • nucleotide sequence of SEQ ID NO: 63 would be translated into the human amino acid sequence of SEQ ID NO: 65.
  • SEQ ID NO: 63 was considered to be the nucleotide sequence of a novel human ortholog DN ⁇ for SEQ ID NO: 9.
  • the open reading frame (including the termination codon) of the human ortholog DNA of dnaform26163 consists of 1296 bases, and the amino acid sequence predicted from the open reading frame consists of 431 amino acid residues.
  • amino acid sequence (SEQ ID NO: 65) encoded by the nucleotide sequence of SEQ ID NO: 63 was searched for protein characteristics by HMMP FAM, amino acid Nos. 92-283 were replaced with Astacin (Peptidase family M12A) (P Amino acid sequence entered as Astacin).
  • a protein belonging to the Peptidase family M12A is a peptide-degrading enzyme that requires zinc. Therefore, the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 63 was presumed to be a peptide-degrading enzyme that requires zinc belonging to Peptidase family M12A.
  • nucleotide sequence of SEQ ID NO: 64 would be translated into the amino acid sequence of SEQ ID NO: 66. There was 87% homology over the 372 amino acid sequence between the amino acid sequence of SEQ ID NO: 66 and the amino acid sequence predicted from the open reading frame of dnaform46870 (SEQ ID NO: 61).
  • the homology is higher than that of the nucleotide sequence of SEQ ID NO: 64.
  • the human base sequence could not be searched.
  • SEQ ID NO: 64 was considered to be the nucleotide sequence of the novel human ortholog DNA of SEQ ID NO: 58.
  • the open reading frame (including the stop codon) of the DNA ortholog DNA of dnaform46870 consists of 1680 bases, and the amino acid sequence predicted from the open reading frame consists of 559 amino acid residues.
  • the amino acid sequence described in SEQ ID NO: 66 was searched for a special protein using HMMPFAM.
  • the amino acid sequences 30 to 140, 145 to 252, and 257 to 370 show the characteristics of the CUB domain (entry as PUB as CUB) Amino acid sequence).
  • the CUB domain is an extracellular domain of 110 amino acid residues, and is functionally found in various proteins (protease of the complement system, etc.), and is particularly found in proteins regulated at developmental stages.
  • a program for predicting a transmembrane helix t mHMM S. Moller, MDR Croning, R.
  • amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 64 is one of the membrane proteins controlled by the developmental stage, particularly the protease of the complement system.
  • Example 7 Protein-protein interaction analysis
  • Two-hybrid experiments in mammalian cells were performed using the Ch ec kMa te ma mm a 1 ian two—hy bridsyst em (Promomega) was used.
  • the sample for protein-protein interaction analysis was a plasmid vector in which the DNA binding region of Ga14 gene was inserted downstream of the CMV promoter] BIND, and the transcription activation region of VP16 gene was inserted downstream of the CMV promoter
  • Plasmid vector pACT and plasmid vector pG51uc in which the reporter luciferase gene was inserted downstream of the five Ga14 binding regions and the TATA box were prepared as type III.
  • a fusion gene of the Ga14 gene and the nucleotide sequence of the mouse full-length cDNA (dnafo rm50441 s dnafo rm31 796 or dnafor m4681 1), and a VP16 gene and a mouse cDNA live Rally FANTOM
  • the fusion gene of the full-length cDNA with the protein coding sequence of each clone is basically in accordance with the protocol of promega. It was created by combining the ligation using the sequence portion and the two-step PCR method.
  • the BIND and ACT samples prepared by PCR were used directly without further purification. 0.25 ⁇ l, 30 ng of pG51 1 uc, and 9.5 ⁇ l of Opti-MEM medium (Lifetech) of the BIND sample and the ACT sample, respectively, were dispensed into a 384-well plate.
  • Opti-MEM medium Add 10 ⁇ l of LF2000 Transfection Reagent (Lifetech) diluted 32 times in a well, mix, incubate for 20 minutes, and suspend CHO-K in 1,300 cells 1 in F12 medium.
  • One Chinese hamster cell solution (20 ⁇ l) was added and the cells were suspended well. After incubating the Atsushi sample in a CO 2 incubator for 20 hours, the interaction was confirmed by measuring the luciferase activity using S tea dy—Glo Luciferase As say System (Promomega). did.
  • the results of the above (2) are shown in Table 2.
  • the proteins encoded by the three mouse full-length cDNA base sequences (dnafo rm50441, dnafo rm31796 dn aio rm468 l 1) are a mouse cDNA library FANTOM (http: // fantom. gsc. riken. go. jp /) It was clarified that each cDNA had an interaction with the protein encoded by the cDNA base sequence of the specific clone.
  • a protein having an amino acid sequence (SEQ ID NO: 87) predicted from the open reading frame of dnafo rm50441 (hereinafter referred to as “the present protein”) is palmitoyltransferase. It is known that palmitoyl transferase is involved in the transport of fatty acids from adipose tissue to fluid mitochondria (Int J Sports Med 199819 (4): 231-44), and is associated with lipid metabolism and diabetes Is suggested. As is evident from Table 2, this protein was found to interact with the hypothetical outer arm dynein light chain 1 structure containing protein.
  • Outer arm dynein is one of the motor proteins and belongs to the MA + family ATPase, which is responsible for ciliary and flagellar movements and is responsible for intracellular transport and cell division such as membrane vesicles There are two types of power, "cytoplasmic dynein". Since this protein / protein mosquito S interacted with hypothetical outer arm dynein light chain 1 structure containing protein, It was estimated that the activity of fatty acid-metabolizing enzymes as palmitoyl transferase is related to the movement of cilia and flagella, the transport of membrane vesicles and the like in cells, and cell division.
  • Aiolos zinc finger protein, subfamily 1A, 3
  • Ikaros Ikaros
  • Example 4 a protein having an amino acid sequence (SEQ ID NO: 110) predicted from the open reading frame of dnafo rm4681 1 (hereinafter referred to as “the present protein”) has an interaction with a tyrosine phosphorylated protein. It is presumed that the protein has a function related to. Many tyrosine phosphorylated proteins are known to have important functions in signal transduction.
  • this protein is (i) neural precursor cell expressed, development ally aown-regulated gene 9 (Nedd9) Z co-protein, 1 i) hypothetical outer arm dynein light chain 1 structure containing protein N , iii) melanoma antigen / family D, (iv) nuclear factor F-kappaB pl05, (v) OVARC1000148 PROTEIN homolog hypothetical protein FLJ10911 [Homo sapiens].
  • the hypothetical outer arm dynein light chain 1 structure containing protein is located in the cytoskeleton as a motor protein, and cytoplasmic dynein is known to be involved in transport of membrane vesicles and the like and cell division. It is also known that agonist stimulation of beta-Adrenergic receptor causes phosphorylation of Outer arm dynein in airway epithelial cells (J. Allergy Clin Immunol 2002 110 (6Suppl): S275-81). From the interaction with arm dynein, it was speculated that this protein is related to respiratory diseases such as bronchial asthma via tracheal ciliary movement. :
  • nuclear factor NF-kappaB pl05 is a transcriptional regulatory factor that regulates the expression of various genes (Exp Hematol, 30: 285-96 ( 2002)) 0
  • 0VARC1000148 PROTEIN homolog hypothetical protein FLJ10911 [Homo sapiens] has an RNA binding domain and is a clone obtained from a cDNA library of ovarian cancer.
  • this protein is located in the cytoskeleton and nucleus, is involved in cell proliferation and cell adhesion, and is related to respiratory diseases such as canceration, cell apoptosis, and bronchial asthma. .
  • PCR was performed according to a standard method (Higuchi R, et al., Biotechnology, 11: 1026-30 (1993)). Was used to perform tissue expression analysis.
  • the expression of the following four mRNAs encoding the protein of the present invention was carried out using a light cycler constant-quantity PCR device (Roche Diagnostics) and the LightCycler-FastStart DNA Master SYBR Green I reagent. ⁇ Quantification was performed according to the attached protocol. The synthetic DNA sequences used for quantitative PCR are shown below.
  • primer TGGCCAAMCCACCTTATTC (SEQ ID NO: 120)
  • Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was corrected. That is, the expression level (copy number / ⁇ 1) of the target gene in each tissue was divided by the expression level of GAPDH (copy number / 1) and multiplied by a constant (1 ⁇ 10 6 ). The results are shown in Table 3.
  • Table 3
  • dnafo rm28983 was strongly expressed in eyes and strongly expressed in adipose tissue, but decreased in diabetic adipose tissue.
  • dnaform 33042 was strongly expressed throughout the body, especially in lung and kidney, and decreased in diabetic liver and colon cancer.
  • the expression level of dn afo rm46870 was extremely low and weakly expressed in fat / tissue and thalamus.
  • dn aio rm31 796 was strongly expressed throughout the whole body, but decreased in colon cancer.
  • the cDNA of the clone and the protein encoded by the cDNA can be applied to the treatment and diagnosis of diabetes and cancer.
  • the protein encoded by the cDNA is useful for diseases involving tissues in which mRNA expression is fluctuated or tissues with high mRNA expression levels as described above. May be involved.
  • the homolog DNA of the fly gene for the nucleotide sequence of the full-length mouse cDNA obtained above was predicted by the following sequence analysis. Sequence analysis was performed at the amino acid sequence level, considering the protein translated from the ORF.
  • the mouse clones dnafo rm28983, dnafo rm33042, and dnafor m31 796 were designated as mouse full-length cDNA clones containing the amino acid sequences shown in SEQ ID NOs: 30, 34, and 112 (SEQ ID NOs: 6, 10, 103) and fly clones (Berkeley Drosophila).
  • the gene numbers of the fly homolog DNA are as follows: mouse: dnafo rm28983, flies: CG4821 (transcript number: CG4821—PB), mouse: dnafo rm33042, flies: CG4 603 (transcript number: CG4603—PA), Mouse: dn afo rm 3 1796, whereas flies: CG3835 (transcript number: pp—CT 4 2118).
  • ORF of the above fly cDNA (CG4821, CG4603, CG3835) 5.
  • a fragment of about 500 bp on the side (hereinafter sometimes referred to as "target cDNA"), cDNA library of / ⁇ (Berkeley Drosophila Genome Projects http: // w. Fruitfly.org) Invitrogen f ⁇ ) was amplified by PCR.
  • the PCR primers were Cpo I A7 (SEQ ID NO: 125, the base of the desired cDNA at the 3 'end of AAATTTCGGACCG linked to the 5'-end 21-base sequence) and S fi IA 3 (SEQ ID NO: 1).
  • a DNA fragment of about 500 bp amplified by PCR using the primers of each set described above is digested with SfiI, and this digested fragment is used as a fly cloning vector (pUAST CS1: Tatsu Ueda et al., Cell Engineering, vol. 21, vol. 8, 923-932 (2002)) was inserted between the sites digested with SfiI and cloned. Further, a DNA fragment obtained by digesting the DNA fragment amplified by the above PCR with CpoI was inserted between sites where this vector was digested with CpoI, and cloned.
  • the two rounds of subcloning resulted in an approximately 500 bp fragment of the fly cDNA ORF, inverted under the control of the heat shock protein 70 basic promoter downstream of the UAS sequence (GAL upstream activation sequence).
  • the obtained vector inverted repeat vector was obtained.
  • the pUAST vector a fly transformation vector used above, is a vector that uses the transposon P factor. By doing so, the vector can induce transcription of the inverted repeat sequence inserted downstream of the UAS sequence.
  • the inverted repeat vector prepared in (i) above was prepared according to the method described in Ryu Ueda et al., Cell Engineering, vol. 21, No. 8, 923-932 (2002), which is a conventional P factor transformation method.
  • the DNA W 1 1 1 8 system integration of Nono E in Indiana stock center:: http. ... // flybase bio indiana edu / stocks / fbstock hform) early embryos microinjected with Maikuroma Nyepi aerator. After this was cultured and hatched to obtain an adult, mating was carried out according to the procedure described in the above-mentioned literature.
  • Act 5 C— GAL 4 is a strain in which the fusion gene, in which the yeast GAL 4 gene is linked to the promoter of the cellular actin gene (Act5C) expressed in cells throughout the body, has been transfected. Therefore, the GAL4 protein is expressed in all cells in the fly having the Act5C-GAL4 transgene.
  • RNA i individual In the offspring (F1 generation, hereafter referred to as “RNA i individual”) in which the flies of the IR strain and the flies of the Act 5 C—GAL 4 strain were crossed, as shown in FIG. Gene is present. Therefore, the GAL4 protein is expressed in all cells, and since the I R vector is forcibly transcribed, ds RNA appears in the cells, exerts the RNAi effect, and degrades when the target mRNA is expressed. Therefore, the function of the target gene is inhibited in all cells of the individual. It has no effect on cells that do not express the target mRNA.
  • Phenotype analysis results The function of the protein encoded by the fly homolog DNA (CG4821, CG4603, CG3835) of each mouse cDNA (dnafo rm28983, dnafo rm33042, dnafo rm31 796) was inhibited by the RNAi effect induced in (3) above. Changes in the phenotype of flies were observed. As a result, CG4821 RNAi individuals and CG4603 RNAi individuals were lethal, that is, less than 0% of individuals became pupae but molted and became adults. However, it was found that the number of individuals that molted and became adults was less than 5%.
  • the fly individual By inhibiting the expression of the above fly gene using RNAi technology and inhibiting its function, the fly individual became lethal or semi-lethal, and this gene is important for development or survival of the individual and maintenance of its function. It turned out to be playing a role.
  • the protein (SEQ ID NO: 30) encoded by dn afo rm28983 (SEQ ID NO: 6) is estimated to be a protease having a tumor-controlling function.
  • fly homolog DNA It is probable that, due to the inhibition of the activity of the protease encoded by, cell growth did not proceed normally during the development and differentiation of the individual, and the individual died.
  • the protein (SEQ ID NO: 34) encoded by dnafo rm33042 (SEQ ID NO: 10) is predicted to be a cystine protease, and is strongly expressed in the whole body of mice, and its expression is reduced in cancer tissues.
  • the activity of the protease encoded by the fly homolog DNA was inhibited, and cell growth did not proceed normally during the development and differentiation of the individual, and the individual died. it is conceivable that.
  • the protein (SEQ ID NO: 113) encoded by dnafo rm31 796 (SEQ ID NO: 103) is estimated to be an enzyme capable of binding to FAD.
  • a protein having an amino acid sequence (SEQ ID NO: 25) predicted from the open reading frame of dnafo rm37901 (hereinafter referred to as “the present protein”) is a meta-protein having functions related to arthritis and cancer. It was presumed to be a protease. Also, from Example 5, this protein shows characteristic expression in the central nervous system, and its expression is particularly increased in the cerebellum and neonatal cerebellum, which may be involved in cerebellar development and differentiation. There is. Therefore, the present protein or its expression regulator, function activator, or function inhibitor can be used for immunological diseases such as arthritis, inflammatory diseases, cancer, central diseases, such as motor integration dysfunction, spinal cord cerebellar degeneration, etc. Disease, etc.
  • a protein having an amino acid sequence (SEQ ID NO: 26) predicted from the open reading frame of dnafo rm46350 (hereinafter referred to as “the present protein”) has a meta-portion having a function related to arthritis and cancer. It was presumed to be a protease.
  • this protein shows characteristic expression in the central nervous system from Example 5, and its expression is particularly increased in the cerebellum and neonatal cerebellum, which may be involved in the development and differentiation of the brain.
  • the protein or protein Protein expression regulators, function activators, or function inhibitors can be involved in immune diseases such as arthritis, inflammatory diseases, cancer, central diseases, such as motor integration dysfunction, spinocerebellar degeneration, etc. There is.
  • a protein having an amino acid sequence (SEQ ID NO: 28) predicted from the open reading frame of dnafo rm9578 (hereinafter referred to as “the present protein”) has a function of controlling the production of germ cells and the like from Example 4. It was speculated to be a meta-mouth protease. In addition, Example 5 showed that the present protein tended to be more strongly expressed in all tissues as a whole, but was particularly strongly expressed in testis. From this, it is considered that the present protein or the expression regulator, function activator, or function inhibitor of the present protein is involved in controlling functions such as germ cell generation and adhesion, and is used for infertility, contraception, immune diseases, and inflammation. May be a therapeutic agent for sexual diseases.
  • Example 4 a protein having an amino acid sequence (SEQ ID NO: 30) predicted from the open reading frame of dnafo rm28983 (hereinafter referred to as “the present protein”) is a protease having a function of controlling tumors. It was speculated. In addition, Example 8 showed that the present protein was strongly expressed in eyes and strongly expressed in adipose tissue, but decreased in diabetic adipose tissue. Further, from Example 9, the phenotype of the fly individual was observed to be changed due to the inhibition of the function of the fly homolog protein of this protein (the protein encoded by CG4821). As a result, the RNAi individual of CG4821 was fatal.
  • SEQ ID NO: 30 amino acid sequence predicted from the open reading frame of dnafo rm28983
  • the present protein or an expression controlling substance, a function activator or a function inhibiting substance of the present protein may be used for cancer such as squamous cell carcinoma and retinoblastoma, endocrine diseases such as diabetes and obesity, and respiratory diseases such as asthma. Be a remedy for there is a possibility.
  • Example 4 a protein having an amino acid sequence (SEQ ID NO: 33) predicted from the open reading frame of dn afo rm26 163 (hereinafter referred to as “the present protein”) was obtained from Example 4 as one of metastasis proteases, astacin Was presumed to be a protease. From Example 6, it was inferred that the human ortholog protein for this protein is a peptide-degrading enzyme that requires zinc belonging to the Peptidase family M12A. Therefore, the present protein or an expression regulator, a function activator, or a function inhibitor of the present protein may be used as a therapeutic drug for cancer, infertility / contraception, diabetes, endocrine diseases such as obesity, and the like.
  • SEQ ID NO: 33 amino acid sequence predicted from the open reading frame of dn afo rm26 163
  • Example 4 a protein having an amino acid sequence (SEQ ID NO: 34) predicted from the open reading frame of dnaform33042 (hereinafter referred to as “the present protein”) was presumed to be a cysteine protease.
  • this protein is strongly expressed in the whole body, as shown in Example 8, and is particularly strongly expressed in the lungs and knees, while its expression is decreased in diabetic liver and colon cancer. Indicated. From Example 9, it was observed that the phenotype of a fly individual was changed due to the inhibition of the function of the fly homolog protein of the present protein (a protein encoded by CG4603).
  • the present protein or an expression regulator, a function activator, or a function inhibitor of the present protein may be a therapeutic drug for cancer such as lung, spleen, and ovary, and endocrine disease such as diabetes and obesity.
  • the protein having the amino acid sequence (SEQ ID NO: 61) (hereinafter referred to as “the present protein”) was estimated from Example 4 to be a meta-oral protease involved in development and differentiation, bone formation, spermatogenesis, etc. .
  • the present protein was presumed to be a membrane protein that is controlled particularly at the developmental stage.
  • the expression level was extremely low and was weakly expressed in adipose tissue and the thalamus. Therefore, the present protein or a substance that regulates the expression of the present protein, a function activator, or a function inhibitor may be a therapeutic agent for endocrine diseases such as cancer, diabetes and obesity, osteoporosis, and infertility.
  • Example 4 From Example 4, it was inferred from Example 4 that a protein having an amino acid sequence (SEQ ID NO: 72) predicted from the open reading frame of dnaform31526 (hereinafter referred to as “the present protein”) had phosphodiesterase activity.
  • the present protein was strongly expressed in the kidney and also strongly expressed in the knee.
  • Phosphodiesterase inhibitors have been developed as cardiovascular drugs such as inotropic drugs, diuretic drugs related to accessory sympathetic nerves, and drugs for ED (erectile dysfunction). New treatments such as cardiovascular drugs such as cardiotonic drugs and hypertensive drugs that have diuretic action related to the parasympathetic nerve, ED drugs, etc. May be a drug.
  • Example 4 a protein having an amino acid sequence (SEQ ID NO: 73) predicted from the open reading frame of dnafo rm32312 (hereinafter referred to as “the present protein”) was estimated to have phosphodiesterase activity.
  • Example 5 showed that the present protein was strongly expressed in the kidney and also strongly expressed in the musculus.
  • Phosphodiesterase inhibitors have been used as cardiovascular drugs such as inotropic drugs, diuretic drugs related to accessory sympathetic nerves, and ED drugs. Since it has been developed, substances that control the expression of this protein, function activators, or function-inhibiting substances are used in cardiovascular drugs, such as cardiotonic drugs and hypertensive drugs that have diuretic action related to the parasympathetic nerve. It may be a new therapeutic agent such as ED therapeutic agent.
  • Example 4 From Example 4, it was inferred from Example 4 that a protein having an amino acid sequence (SEQ ID NO: 74) predicted from the open reading frame of dnaform4086 (hereinafter referred to as “the present protein”) had phosphodiesterase activity.
  • the present protein was strongly expressed in the kidney and also strongly expressed in the spleen.
  • Phosphodiesterase inhibitors have been developed as cardiovascular drugs such as cardiotonic drugs, diuretic drugs related to accessory sympathetic nerves, and ED drugs. Regulatory substances, function activating substances, or function inhibiting substances become new therapeutic agents such as cardiovascular drugs, ED drugs, etc. such as cardiotonic drugs and hypertensive drugs that have diuretic action associated with parasympathetic nerves. there is a possibility.
  • Example 4 a protein having an amino acid sequence (SEQ ID NO: 75) predicted from the open reading frame of dnaform64786 (hereinafter referred to as “the present protein”) was presumed to have phosphodiesterase activity.
  • Example 5 showed that the present protein was strongly expressed in the kidney and also strongly expressed in the knee.
  • Phosphodiesterase inhibitors have been developed as cardiovascular drugs such as cardiotonic drugs, diuretic drugs related to accessory sympathetic nerves, and ED drugs. Regulatory substances, function activating substances, or function inhibiting substances become new therapeutic agents such as cardiovascular drugs, ED drugs, etc. such as cardiotonic drugs and hypertensive drugs that have diuretic action associated with parasympathetic nerves. there is a possibility.
  • Example 4 a protein having an amino acid sequence (SEQ ID NO: 76) predicted from the open reading frame of dnafo rm64814 (hereinafter referred to as “the present protein”) was estimated to have phosphodiesterase activity. Also, Example 5 shows that this protein is strongly expressed in the kidney and also strongly in the arm.
  • Phosphodiesterase inhibitors have been developed as cardiovascular drugs such as cardiotonic drugs, diuretic drugs related to accessory sympathetic nerves, and ED drugs. Regulatory substances, function activators, and, are new therapeutic agents such as cardiovascular drugs, ED drugs, etc., such as cardiotonic drugs and hypertensive drugs that have diuretic effects associated with parasympathetic nerves. there is a possibility.
  • a protein having an amino acid sequence (SEQ ID NO: 87) predicted from the open reading frame of dnaform. 50441 (hereinafter referred to as “the present protein”) was presumed to be palmitoyl transferase.
  • the present protein was strongly expressed in the brain, cerebellum, and testis. Palmitoyl transferase has been reported to be associated with lipid metabolism, cancer, apoptosis, PPAR, etc.In addition, lipid metabolism including its association with apolipoprotein E4 plays an important role in lipid metabolism in Alzheimer's disease Has been reported (Bioclinica, August 2002, August issue).
  • the expression controlling substance, function activating substance, or function inhibiting substance of the present protein or protein may be used for cancer, diabetes, Alzheimer's dementia, Parkinson's disease, chorea, ischemic brain disease, diabetic peripheral neuropathy There is a possibility that it can be developed as a treatment for infertility.
  • the protein having an amino acid sequence (SEQ ID NO: 94) predicted from the open reading frame of dnafo rm6659 (hereinafter referred to as “the present protein”) is an isomerase having a function related to steroid hormone receptor from Example 4. It was speculated. Example 5 shows that this protein is strongly expressed in the whole fetus, the lung of the newborn and the mammary gland.
  • this protein is suggested to be associated with immune diseases and inflammatory diseases by controlling the activity of steroid receptors such as steroid hormones, and this protein and its expression regulators, function activators, Or function inhibitors may be immune disorders, inflammatory disorders, hemolytic disorders, infertility, bone It may be developed as a treatment for osteoporosis, cancer and Alzheimer's disease.
  • the protein having an amino acid sequence (SEQ ID NO: 95) predicted from the open reading frame of dnafo rm6776 (hereinafter referred to as “the present protein”) is an isomerase having a function related to steroid hormone receptor from Example 4. It was speculated. In addition, Example 5 showed that the present protein was strongly expressed in whole fetuses, lungs of newborns, and mammary glands.
  • this protein is suggested to be related to immune diseases and inflammatory diseases, and it is suggested that this protein and its expression regulator, function activator, or Functional inhibitors could be developed as therapeutics for immune, inflammatory, hemolytic, infertility, osteoporosis, cancer, Alzheimer's disease, and others.
  • a protein having an amino acid sequence (SEQ ID NO: 96) predicted from the open reading frame of dnafo rml 7871 (hereinafter referred to as “the present protein”) has a function related to steroid hormone receptor from Example 4.
  • Example 5 showed that this protein, which was presumed to be an isomerase, was strongly expressed in whole fetuses, neonatal lungs, and mammary glands.
  • this protein is suggested to be related to immune diseases and inflammatory diseases, and this protein and its expression regulators, function activators, Alternatively, functional inhibitors could potentially be developed as therapeutics for immune, inflammatory, hemolytic, infertility, osteoporosis, cancer, Alzheimer's disease, and others.
  • the protein having an amino acid sequence (SEQ ID NO: 97) predicted from the open reading frame of dnafo rm26 175 (hereinafter referred to as “the present protein”) is a glycoprotein having a transmembrane region from Example 4. It was speculated. Ma In addition, Example 5 showed that the present protein was very strongly expressed in the lung and also strongly expressed in kidney and epididymal adipocytes. Therefore, there is a possibility that the expression controlling substance, function activating substance or function inhibiting substance of the present protein or the present protein can be developed as a therapeutic agent for cancer, hypertension, immune disease, inflammatory disease and the like.
  • the protein having an amino acid sequence (SEQ ID NO: 98) predicted from the open reading frame of dnafo rm42159 (hereinafter referred to as “the present protein”) is a glycoprotein having two transmembrane regions from Example 4. It was speculated. In addition, Example 5 showed that the present protein was very strongly expressed in the lung and also strongly expressed in kidney and epididymal adipocytes. Therefore, there is a possibility that the expression regulator, function activator, or function inhibitor of the present protein or the present protein can be developed as a therapeutic drug for cancer, hypertension, immune disease, inflammatory disease, and the like.
  • Example 4 a protein having an amino acid sequence (SEQ ID NO: 110) predicted from the open reading frame of dnafo rm4681 1 (hereinafter referred to as “the present protein”) was found to have an interaction with the tyrosine phosphorylation protein from Example 4. It is an adapter protein having a function related to the action, and it was speculated that it may be involved in the differentiation of blood cells. From Example 7, it was inferred from Example 7 that this protein is involved in cell proliferation and cell adhesion and is involved in canceration and cell apoptosis. Therefore, there is a possibility that the expression regulator, function activator or function inhibitor of the present protein can be developed as a therapeutic drug for cancer, inflammatory diseases, immune diseases, respiratory diseases such as bronchial asthma and the like.
  • SEQ ID NO: 110 predicted from the open reading frame of dnafo rm4681 1
  • this protein a protein having an amino acid sequence (SEQ ID NO: 112) predicted from the open reading frame of dnafo rm31 796 (hereinafter referred to as “this protein”) was determined to be an enzyme having a function of binding to FAD from Example 4. In addition, this protein was strongly expressed throughout the whole body as in Example 8, but its expression was decreased in colon cancer.From Example 9, the RNAi effect of the fly homolog DNA was semi-lethal. It is speculated that this enzyme is an enzyme that catalyzes a reaction utilizing FAD, which plays an important role in development and differentiation. Therefore, there is a possibility that the present protein or an expression regulator, a function activator, or a function inhibitor of the present protein can be developed as a therapeutic drug for cancer, metabolic disease, immune disease, inflammatory disease, and the like. Industrial applicability
  • the protein of the present invention and the DNA encoding the same have various enzyme activities or proteolytic enzyme inhibitory activities, a substance that regulates the activity is screened using the protein or the DNA encoding the protein.
  • the present invention is useful for the development of a medicament capable of acting on a disease or the like in which the protein is associated.
  • proteases function as important regulators of cell function.
  • Proper control of protease activity has shown that protease reactions are advantageous in order to ensure that biological reactions proceed in one direction, and that limited degradation of proteins is important for controlling many important cell functions. It has become clear that they are involved.
  • abnormal protease activity causes various diseases. For example, muscular dystrophy is caused by enhanced activity of a neutral protease called CANP.
  • CANP neutral protease
  • cysteine protease thiol protease
  • suppressing the activity of the related protease may lead to prevention of the progression of the related disease or treatment.
  • examples of progression of the disease due to suppression of protease activity include neurodegenerative diseases represented by Alzheimer's disease. In the case of Alzheimer's disease-due to the accumulation of amyloid peptide, which is thought to be due to abnormalities in the degrading enzyme neprilysin.
  • thrombus due to fibrin accumulation can be treated by increasing the amount of plasmin, a fibrinolytic enzyme.
  • the conversion of the plasmin precursor protein plasminogen to plasmin is promoted by the plasminogen activator, a species of serine protease.
  • the modulator of protease activity obtained by the screening method of the present invention can be a therapeutic agent for various diseases, for example, an anticancer agent (antitumor agent), an anti-inflammatory agent, a therapeutic agent for neurodegenerative disease, and the like.
  • protease inhibitors include serine proteinase inhibitors and cysteine protease inhibitors.
  • serine protease inhibitors include BPTI (Kunitz) type inhibitor (inter ⁇ -trypsin inhibitor), Kaza type 1 inhibitor (porcine semen acrosin inhibitor, egg white ovomucoid), and SSI type inhibitor (S treptin).
  • omy ces S ubutilisin Inhibitor plasmin inhibitor, BBI inhibitor (legume seed inhibitor), STI inhibitor (soybean trypsin inhibitor), lactate L animal blood inhibitor (a 1-protease inhibitor, a 1-anti-antibody) Chymotrypsin, inter-trypsin inhibitor, ⁇ 2-plasmin inhibitor, antithrombin III, C11 activator, ⁇ 2-macroglobulin, plasminogen activator inhibitor) and the like are known.
  • cystinp mouth inhibitor are, for example, leuptin, antipine, cystatin, etc. Of these, leuptin and antipine are peptide It is a sex inhibitor. Thus, the existence of various protease inhibitors is known.
  • Serum proteases of animal origin have been studied in detail in serum, and their physiological significance is also understood as a regulator of blood coagulation and cunnogenesis.
  • the existence of intracellular inhibitors has been elucidated, and the significance of their existence has been elucidated.
  • the relationship between abnormalities of these inhibitors and diseases has also been clarified.
  • tPA tissue plasminogen activator
  • PA1-1 plasminogen activator inhibitor-i
  • the blood flow is maintained by maintaining the balance of these activities, but when thrombus forms, the amount of PAI-1 increases and tPA activity is suppressed.
  • PAI_1 inhibitor instead of administering tPA.
  • control of the activation cascade by the protease always involves a proteolytic enzyme inhibitor, and it is thought that the cascade can be controlled by controlling the inhibitor.
  • the protein having protease inhibitory activity of the present invention can be a target protein for the development of various therapeutic agents, for example, therapeutic agents for coagulation diseases and cardiovascular diseases.
  • a substance regulating the activity is screened. It is useful for the development of a medicament capable of acting on diseases and the like in which the protein is related.
  • This application is a Japanese patent application filed on April 19, 2002 (Japanese Patent Application No. 2002-111).
  • Japanese patent application dated May 2, 2002 Japanese patent application dated May 1, 2002 (Japanese Patent Application 2002—352559), April 26, 2002 Japanese patent application (Japanese Patent Application 2002-126250), Japanese patent application dated February 4, 2002 (Japanese Patent Application 2002-352326), Japanese patent application dated April 30, 2002 (Japanese Patent Application 2002-128657) , Japanese patent application filed on December 4, 2002 (Japanese Patent Application 2002-352381), Japanese patent application filed on May 2, 2002 (Japanese Patent Application 2002-130702), Japanese patent application filed on December 4, 2002 (Japanese Patent Application 2002-352694), Japanese Patent Application dated May 2, 2002 (Japanese Patent Application 2002-1 30925), Japanese Patent Application filed on December 4, 2002 (Japanese Patent Application 2002-352808), 2002 It is based on a Japanese patent application filed on May 2, 2002 (Japanese Patent Application No. 2002-130918) and a Japanese patent application filed on February 4, 2002 (Japanese Patent Application 2002 (Japanese Patent Application No. 2002-1309

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Abstract

L'invention concerne un procédé d'utilisation de protéines et d'ADN codant pour celles-ci sur la base de leur activité physiologique par l'analyse des séquences de base des clones d'ADNc participant à la bibliothèque cataloguée d'ADNc de longueur entière, et d'indication de l'activité physiologique des protéines codées par les clones comportant des séquences nouvelles ainsi détectées. L'invention concerne nommément les protéines (a) et (b) suivantes: (a) une protéine comprenant une séquence d'acides aminés représentée par une des SEQ ID NOS:25 à 48, 60 à 62, 65, 66, 72 à 76, 80 à 82, 86, 87, 94 à 96 et 112 à 115; et (b) une protéine comprenant une séquence d'acides aminés représentée par une des SEQ ID NOS: 25 à 48, 60 à 62, 65, 66, 72 à 76, 80 à 82, 86, 87, 94 à 96 et 112 à 115 par délétion, substitution et/ou addition d'un ou plusieurs acides aminés et possédant une activité d'enzyme ou une activité d'inhibiteur de protéase.
PCT/JP2003/004983 2002-04-19 2003-04-18 Nouvelles proteines et adn codant pour elles WO2003089644A1 (fr)

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JP2002-117794 2002-04-19
JP2002117794 2002-04-19
JP2002-126250 2002-04-26
JP2002126250 2002-04-26
JP2002-128657 2002-04-30
JP2002128657 2002-04-30
JP2002130871 2002-05-02
JP2002-130871 2002-05-02
JP2002-130702 2002-05-02
JP2002130925 2002-05-02
JP2002-130925 2002-05-02
JP2002130702 2002-05-02
JP2002130918 2002-05-02
JP2002-130918 2002-05-02
JP2002-352808 2002-12-04
JP2002352694 2002-12-04
JP2002352808 2002-12-04
JP2002-352786 2002-12-04
JP2002352559 2002-12-04
JP2002-352326 2002-12-04
JP2002-352694 2002-12-04
JP2002352786 2002-12-04
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
US20100183617A1 (en) * 2005-02-23 2010-07-22 University Of Virginia Patent Foundation Compositions and methods for regulating sas1r

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WO2001083782A2 (fr) * 2000-05-04 2001-11-08 Sugen, Inc. Nouvelles proteases
WO2002050258A1 (fr) * 2000-12-18 2002-06-27 Yamanouchi Pharmaceutical Co., Ltd. Nouvelle aggrecanase

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WO2001083782A2 (fr) * 2000-05-04 2001-11-08 Sugen, Inc. Nouvelles proteases
WO2002050258A1 (fr) * 2000-12-18 2002-06-27 Yamanouchi Pharmaceutical Co., Ltd. Nouvelle aggrecanase

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CAL S. ET AL.: "Cloning, expression analysis and structural characterization of seven novel human ADAMTSs, a family of a metalloproteinases with disintegrin and thrombospondin-1 domains", GENE, vol. 283, January 2002 (2002-01-01), pages 49 - 62, XP004339892 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100183617A1 (en) * 2005-02-23 2010-07-22 University Of Virginia Patent Foundation Compositions and methods for regulating sas1r
US9803012B2 (en) 2005-02-23 2017-10-31 University Of Virginia Patent Foundation Compositions and methods for regulating SAS1R

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