WO2003078624A1 - Gene llpl et nouvelle utilisation de ses produits geniques - Google Patents
Gene llpl et nouvelle utilisation de ses produits geniques Download PDFInfo
- Publication number
- WO2003078624A1 WO2003078624A1 PCT/JP2003/003136 JP0303136W WO03078624A1 WO 2003078624 A1 WO2003078624 A1 WO 2003078624A1 JP 0303136 W JP0303136 W JP 0303136W WO 03078624 A1 WO03078624 A1 WO 03078624A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- salt
- amino acid
- acid sequence
- seq
- protein
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2510/00—Detection of programmed cell death, i.e. apoptosis
Definitions
- the present invention relates to a lecithin: cholesterol acyltransferase-like lysophospholipase (LCAT-like lysophospholipase; hereinafter referred to as “LLPL”) containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1.
- Screening method for a compound that changes the activity of the compound, a compound or a salt thereof obtainable by the screening method, a drug containing the compound or the salt, apoptosis using a non-human mammal deficient in LLPL gene expression.
- the present invention relates to a screening method for induced-regulation drugs. Background art
- Apoprotein [Srivastava, R. & A., Srivastava, N. (2000) Mol. Cell Biochem., 209, 131-144], enzymes and receptors [Hiltunen, T., P. & Yla- Herttuala, S. (1998) Atherosclerosis, 137, S81-88], lipid transfer protein [Yamashita, S. et al., (2000) Atherosclerosis, 152, 271-285, 0 ram, J., F. & Vaughan, A , M. (2000) Curr. Op in. Lipidol., 11, 253-260], but individual differences caused by differences in the genotype of these proteins and dietary differences are caused by arterial changes. It is closely related to sclerosis.
- HDL high-density lipoprotein
- Lecithin cholesterol acyltransferase (LCAT), the rate-limiting enzyme in the reverse cholesterol transfer system, is mainly produced in the liver [Warden, C. et al. (1989) J. Biol. Chem., 264, 21573-21581. ], It is present in HDL particles in blood [Kuivenhoven, J. et al., (1997) J. Lipid Res., 38, 191-205]. Free cholesterol in peripheral cells extracted by Pre / 3HD L is esterified by LCAT and suppresses cholesterol influx into cells, resulting in a cholesterol extraction promoting effect. It is thought to work atherosclerotically [Czarnecka, H. & Yokoyama, S. (1995) Biochemistry, 34, 4385-92].
- the enzyme LLP which has 47% homology at the amino acid level to human LCAT, was discovered by subtraction PCR using the cDNA library of human macrophage-like cells, and the entire amino acid sequence was identified. Sequences have also been determined from cDNA analysis [Taniyama et al., Biochem. Biophys. Res. Co Fr n., 257: 50-56 (1999)].
- LLPL is secreted by macrophage-like cells in humans and peritoneal macula phage in mice; (2) 88% homology in the amino acid sequence; 3) The lipase motif is similar in that it has the same AHSMG sequence (JP-A-11-269199), and (4) the expression specificity of the LLPL gene in the entire individual is in peripheral tissues. Furthermore, since the expression of the LLPL gene was confirmed in the atherosclerotic lesions of ApoE-deficient mice, the effect on atherosclerotic lesions and plasma lipid profile was considered.
- Oxidized LDL not only foams macrophage cells, but also has an arteriosclerogenic effect on vascular endothelial cells, smooth muscle cells, and macrophage cells, one of which is the induction of apoptosis. .
- LLPL-knockout mice are protective against arteriosclerosis due to the progression of atherosclerotic lesions in genetically modified mice that cause hyperlipidemia, such as apolipoprotein E (ApoE) -knockout mice.
- ApoE apolipoprotein E
- Japanese Patent Application No. 2001-152520, Japanese Patent Application No. 2001-31 1971, Japanese Patent Application No. 2002-145876 and PCT / JP 02/04876 WO 02/102998.
- LLPL has an anti-apoptotic activity.
- a drug having an apoptosis-inducing regulatory action targeting LLPL has the potential to prevent and / or treat arteriosclerosis, It is thought to be effective as an action and cancer treatment, As a result of further studies, the present invention has been completed.
- a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide thereof or a salt thereof, Or a partial peptide thereof or a lecithin salt thereof: a cholesterol acyltransferase-like lysophospholipase (LLPL) -altering compound or a salt thereof, and a screening kit thereof.
- SEQ ID NO: 1 amino acid sequence represented by SEQ ID NO: 1
- a partial peptide thereof or a salt thereof Or a partial peptide thereof or a lecithin salt thereof: a cholesterol acyltransferase-like lysophospholipase (LLPL) -altering compound or a salt thereof, and a screening kit thereof.
- LLPL cholesterol acyltransferase-like lysophospholipase
- the screening kit according to the above (4) which comprises a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof.
- an amino acid represented by SEQ ID NO: 1 which can be obtained using the screening method according to any one of the above (1) to (3) or the screening kit according to the above (4) or (5) A compound or a salt thereof, which alters the LLPL activity of a protein having the same or substantially the same amino acid sequence as the sequence, a partial peptide thereof or a salt thereof,
- the medicament according to the above (7) which is an agent for preventing and treating neurodegenerative diseases
- (11) An amino acid identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, characterized by using a polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 2 or a part thereof.
- a method for screening a compound or a salt thereof which alters the expression level of a protein comprising a sequence or a partial peptide thereof or a salt thereof,
- a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2 or a part thereof, which is the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 1
- (22) a protein or a partial peptide thereof or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8; Peptide or it A method for screening a compound or salt thereof that alters the activity of lecithin: cholesterol acyltransferase-like lysophospholipase (LLPL);
- LLPL cholesterol acyltransferase-like lysophospholipase
- a protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 8, a partial peptide thereof, or a salt thereof, Lecithin of a protein or its partial peptide or a salt thereof: a cholesterol acyltransferase-like lysophospholipase (LLPL) activity-modifying compound or a salt thereof, a screening kit,
- a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9 or a part thereof, wherein the amino acid is the same as or substantially the same as the amino acid sequence of SEQ ID NO: 8
- a method for screening a compound or a salt thereof which alters the expression level of a protein comprising a sequence or a partial peptide thereof or a salt thereof (33) a polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 9 or a part thereof, which is the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 8
- a method for regulating apoptosis induction which comprises administering to a mammal an effective amount of the compound or a salt thereof according to (27) or (34),
- a medicament comprising a compound having an apoptotic induction-regulating effect or a salt thereof, which can be obtained by using the screening method according to (43).
- (50) a method for regulating apoptosis induction, which comprises administering to a mammal an effective amount of the compound or a salt thereof having the activity of regulating apoptosis induction according to (45);
- (51) a method for preventing and / or treating cancer, which comprises administering to a mammal an effective amount of the compound or a salt thereof having the apoptosis-inducing regulatory activity according to (45);
- an apoptosis induction inhibitor comprising a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8, or a partial peptide thereof, or a salt thereof;
- an apoptosis induction inhibitor comprising a DNA encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or a partial peptide thereof,
- amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 8 An apoptosis induction promoter comprising an antibody against a protein containing a amino acid sequence, a partial peptide thereof, or a salt thereof;
- an agent for diagnosing cancer comprising an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8, a partial peptide thereof, or a salt thereof;
- an apoptosis induction promoter comprising an antisense polynucleotide comprising a nucleotide sequence complementary to the polynucleotide represented by SEQ ID NO: 9 or a part thereof,
- a diagnostic agent for cancer comprising an antisense polynucleotide comprising a nucleotide sequence complementary to the polynucleotide represented by SEQ ID NO: 9 or a part thereof,
- (63) a method for diagnosing cancer, which comprises using an antisense polynucleotide comprising a nucleotide sequence complementary to the polynucleotide represented by SEQ ID NO: 9 or a part thereof;
- (64) contains siRNA for a polynucleotide containing a polynucleotide containing a protein encoding an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 8 or a partial peptide thereof Apoptosis induction promoter,
- a method for diagnosing cancer which comprises using siRNA for a polynucleotide containing a polynucleotide encoding a protein containing a amino acid sequence or a partial peptide thereof,
- SEQ ID NO: 8 protein or comprising an amino acid sequence identical or substantially identical ⁇ amino acid sequence represented by comprising a partial peptide thereof or a salt thereof acidic phospholipase eight 2 (APLA 2) Enhancer (aPLA 2 activity enhancer),
- a protein or a partial peptide thereof comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 for producing an apoptosis-inducing regulator, or a partial peptide thereof It is also intended to provide a use of a compound or a salt thereof that changes the LLPL activity or expression level of a salt.
- FIG. 1 shows the exon-intron structure of the LLP L gene on the mouse genome.
- the exon part is indicated by a box, and the intron part is indicated by a solid line.
- the five DNA fragments obtained by restriction enzyme treatment and subcloned are indicated by thick solid lines.
- FIG. 2 shows a construction diagram of one evening targeting vector obtained in Reference Example 3.
- Ne o r is the neomycin resistance gene
- HS V- TK shows Le Paix scan virus one thymidine kinase gene to simple.
- FIG. 3 shows the results of Southern hybridization of the L L P L knockout ES cell line obtained in Reference Example 4.
- FIG. 4 shows a standard curve for the measurement of recombinant human LLPL by the sandwich ELISA method.
- FIG. 5 shows the measurement results of the LLPL concentration in normal human serum.
- FIG. 6 shows the results of measuring the amount of human LLPL in the culture supernatant of macrophage-like THP-1 cells.
- FIG. 7 shows the measurement results of acid phospholipase VIII- 2 (aPLA 2 ) activity in liver, kidney and brain tissues of LLPL homozygous, heterozygous, and wild-type mice in the background of ApoE deficiency.
- aPLA 2 acid phospholipase VIII- 2
- FIG. 8 shows the results of measuring the acid phospholipase 2 activity using a fluorescent substrate for liver tissue lysate proteins of LLPL homozygous, heterozygous, and wild-type mice in the background of ApoE deficiency.
- FIG. 10 shows the results of measuring the activity of transylceramide synthase in liver, kidney and brain tissues prepared from LLPLZApoE double-deficient mice.
- the protein used in the present invention is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 8 (hereinafter abbreviated as LLPL), or a partial peptide thereof.
- the salt The amino acid sequence of human LLPL is known, and is disclosed, for example, in JP-A-11-269199. And the amino acid sequence represented by SEQ ID NO: 1. Further, the amino acid sequence of mouse LLPL is known, and includes, for example, the amino acid sequence represented by SEQ ID NO: 8 in JP-A-11-269199, which is registered as GenBanld Accessioii No. AB017494 (Reference: Biocliem. Biophys. Res. Conunun., 257 (1), 50-56 (1999)). This is the first time that LLPL has been implicated in apoptosis.
- the "LLPL activity” as used herein refers to (i) Rizohosufuoripa Ichize activity, (ii) PLA 2 activity, (iii) Ashiru transfer activity, and the activity of the protective effect of anti-apoptotic activity and cell based on these.
- the term “regulation of induction of apoptosis” refers to direct regulation (promotion or inhibition) of apoptosis or apoptosis.
- It may be any of indirect regulation (enhancement or inhibition) of cis.
- the protein used in the present invention includes, for example, all cells of humans and other mammals (for example, guinea pig, rat, mouse, mouse egret, pig, sheep, horsetail, monkey, etc.) [eg, retinal cells, splenocytes , Nerve cells, glial cells, knee jS cells, bone marrow cells, mesangial cells, Langer's cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, Macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes, leukocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts Cells, osteoclasts, breast cells, hepatocytes or stromal cells, or their precursors, stem cells or cancer cells (eg, breast cancer cell lines (GI_101),
- Cells or their culture cells eg, MEL, Ml, CTLL-2, HT-2, WEHI-3, HL-6 0, J ⁇ SK-1, K562, ML-1, MOLT-3, MOLT-4, MOL T-10, CCRF-CEM, TALL-1, Jurkat, CCRT-HS B-2, KE-37, SKW-3, HUT-78, HUT-102, H9, U937, THP-1, HEL, JK-1, CMK, KO-812, MEG-01, etc.
- It may be a synthetic protein.
- substantially identical amino acid sequence refers to, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably the amino acid sequence to be compared.
- An amino acid sequence having about 95% or more homology is also considered.
- Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1; Preferred are proteins having substantially the same activity as the protein containing the amino acid sequence represented by the following formula: Specifically, the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 8 includes, for example, a mouse-derived protein described in JP-A-11-269199, and the like. The amino acid sequence represented by 11 is exemplified.
- Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 8 include, for example, a protein containing the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8; A protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 8 is preferred.
- examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 8 include a mouse-derived protein described in JP-A-11-269199, and the like. : An amino acid sequence represented by 10, 12 to 15 can be mentioned.
- substantially the same activity examples include lysophospholipase activity and apoptosis inhibitory activity. Substantially the same means that their activities are the same in nature. Therefore, activities such as lysophospholipase activity and apoptosis inhibitory activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times). (More preferably about 0.5 to 2 times), but the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
- the activity such as lysophospholipase activity and apoptosis inhibitory activity can be measured according to a known method, but can be measured according to a screening method described later.
- Examples of the protein used in the present invention include: (1) one or more (preferably about 1 to 30 and more preferably 1 to 10) amino acids in the amino acid sequence represented by SEQ ID NO: 1; About 2 amino acids, more preferably several (1 to 5) amino acids, and (2) one or more amino acids (preferably 1 to 30 amino acids) in the amino acid sequence represented by SEQ ID NO: 1.
- amino acids More preferably about 1 to 10 amino acids, and even more preferably several (1 to 5) amino acids; (3) the amino acid sequence represented by SEQ ID NO: 1; Or an amino acid sequence in which two or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5)) amino acids are substituted with another amino acid, Or (4) a protein containing an amino acid sequence obtained by combining them. Click quality, such as can also be used.
- the protein has an N-terminus (an amino terminus) on the left end and a C-terminus (a lipoxyl terminus) on the right end according to the convention of peptide labeling.
- the protein used in the present invention containing the amino acid sequence represented by SEQ ID NO: 1 has a C-terminal hydroxyl group (—COOH), a carboxylate (one COO—), an amide (one CONH 2 ) Or an ester (one COOR).
- R in the ester e.g., methyl, Echiru, n- propyl, C ⁇ 6 alkyl group such as isopropyl or n- butyl, Shikurobe pentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl, C 6, such as a one-naphthyl - 12 Ariru group, e.g., benzyl, full of such phenethyl Eniru - C et alkyl or ⁇ - naphthylmethyl etc. ⁇ - Nafuchiru C!
- Ararukiru group such _ 2 alkyl groups, such as pivaloyl I Ruo carboxymethyl group which is generally used as an oral ester. .
- the lipoxyl group is amidated or esterified.
- Such proteins are also included in the protein used in the present invention.
- ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
- the protein used in the present invention in the protein described above, in the Amino group protecting groups Mechionin residues of N-terminal (e.g., formyl group, etc. Ashiru groups such as any C 2 _ 6 Arukanoiru group of Asechiru) Protected, N-terminally cleaved in vivo, Dalmin mill group generated by pidal dimmin oxidation, Substituent on side chain of amino acid in molecule (eg, 1H, 1SH) , an amino group, I Midazo Ichiru group, indole group, etc.
- Mechionin residues of N-terminal e.g., formyl group, etc.
- Ashiru groups such as any C 2 _ 6 Arukanoiru group of Asechiru
- Protected N-terminally cleaved in vivo, Dalmin mill group generated by pidal dimmin oxidation, Substituent on side chain of amino acid in molecule (eg, 1H,
- Guanijino group is protected with a suitable protecting group (e.g., ho mill group, such as C ⁇ 6 Ashiru group such as C 2 6 Arukanoiru group such Asechiru) Or complex proteins such as so-called glycoproteins to which sugar chains are bound.
- a suitable protecting group e.g., ho mill group, such as C ⁇ 6 Ashiru group such as C 2 6 Arukanoiru group such Asechiru
- complex proteins such as so-called glycoproteins to which sugar chains are bound.
- protein used in the present invention include, for example, a protein containing an amino acid sequence represented by SEQ ID NO: 1. (Partial peptide)
- the partial peptide used in the present invention may be any peptide as long as it is a partial peptide of the protein used in the present invention. Those having substantially the same activity are preferred.
- the number of amino acids in the partial peptide is at least 20 or more, preferably 50 or more, more preferably 100 or more of the amino acid sequences constituting the protein used in the present invention. Peptides and the like are preferred.
- one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence are deleted.
- 1 or 2 or more (preferably, about 1 to 20; more preferably, about 1 to 10; more preferably, several (1 to 5)) amino acids are added to the amino acid sequence.
- 1 or 2 or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the amino acid sequence have been substituted with another amino acid. You may.
- the partial peptide used in the present invention the C-terminus force Rupokishiru group (one COOH), carboxylate Ichito (- CO O-), even an amide (_ CO NH 2) or ester le (one COOR) Good.
- the partial peptide used in the present invention has an N-terminal methionine residue whose amino group is protected with a protecting group, and the N-terminal is cleaved in vivo.
- Py Glutamine-oxidized G 1n produced, the amino acid in the molecule has a substituent on the side chain protected by an appropriate protecting group, or a complex peptide such as a so-called glycopeptide linked to a sugar chain And so on.
- Examples of the salt of a protein or a partial peptide thereof used in the present invention include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable.
- Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
- inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
- organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
- Succinic acid tartaric acid, citric acid, malic acid,
- the protein or salt thereof used in the present invention can be produced from the above-mentioned human or other mammalian cells or tissues by a known protein purification method, or the protein used in the present invention described later is used. It can also be produced by culturing a transformant containing the encoding DNA. Also, the protein can be produced by a protein synthesis method described later or according to the method.
- the human or other mammalian tissues or cells are homogenized, and then extracted with an acid or the like. Can be purified and isolated by combining chromatography such as reverse phase chromatography and ion exchange chromatography.
- the partial peptide of the protein used in the present invention or a salt thereof may be a known peptide.
- a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein used in the present invention with the remaining portion, and when the product has a protecting group, removing the protecting group. it can.
- any polynucleotide containing a nucleotide sequence (DNA or RNA, preferably DNA) encoding the protein described above is used. Any thing may be used.
- the polynucleotide is RNA such as DNA or mRNA encoding the protein used in the present invention, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (that is, a coding strand) or an antisense strand (that is, a non-coding strand).
- the protein used in the present invention can be obtained by a known method described in Experimental Medicine Special Edition “New PCR and Its Application” 15 (7), 1997 or a method analogous thereto. MRNA can be quantified.
- the DNA encoding the protein used in the present invention may be any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells or tissues, cDNA library derived from the above-mentioned cells or tissues, and synthetic DNA.
- the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
- amplification can also be carried out directly by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells or tissues described above.
- RT-PCR method Reverse Transcriptase Polymerase Chain Reaction
- the DNA encoding the protein used in the present invention includes, for example, a DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9, or SEQ ID NO: 2 or SEQ ID NO: 9
- a DNA containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 8, which has a DNA that hybridizes under high stringent conditions with a DNA containing the nucleotide sequence represented by Any DNA may be used as long as it encodes a protein having the same activity (eg, apoptosis inhibitory activity).
- Examples of the DNA that hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 70% or more, preferably about 80%, of the nucleotide sequence represented by SEQ ID NO: 2. % Or more, more preferably about 90% or more, and still more preferably about 95% or more.
- Hybridization is performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). be able to. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.
- High stringency conditions refers to, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 ° C. The conditions at ⁇ 65 ° C are shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.
- the ⁇ polynucleotide comprising a part of the base sequence of DNA encoding a protein or a part of a base sequence complementary to the DNA '' used in the present invention is used in the present invention described below. It is used to include not only DNA encoding a partial peptide but also RNA.
- an antisense polynucleotide capable of inhibiting the replication or expression of a protein gene is obtained by DNA encoding an amino acid sequence-determined protein, or cloned. It can be designed and synthesized based on DNA base sequence information.
- a polynucleotide can hybridize with RNA of the protein gene used in the present invention and can inhibit the synthesis or function of the RNA, or the protein-related RNA used in the present invention.
- the expression of the protein gene used in the present invention can be regulated and controlled through the interaction with.
- the polynucleotide complementary to the selected sequence of the protein-related RNA used in the present invention and the polynucleotide capable of specifically hybridizing with the protein-related RNA used in the present invention include the protein used in the present invention. It is useful for regulating and controlling the expression of genes in vivo and in vitro, and is also useful for treating or diagnosing diseases and the like.
- Antisense polynucleotides are 2-deoxy_D-report-containing polydeoxyliponucleotides, D-report-containing polyliponucleotides, N-glycosides of purine or pyrimidine bases, and other types. Or other polymers with non-nucleotide backbones (eg, commercially available proteins, nucleic acids, and synthetic sequence-specific nucleic acid polymers) or other polymers containing specialized linkages, such as DNA or DNA.
- RNA contains nucleotides with a configuration that allows base attachment
- They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can also be unmodified polynucleotides (or unmodified oligonucleotides). ), As well as those with known modifications, such as labeled, capped, methylated, and one or more natural nucleotides as analogs known in the art.
- Substituted, modified with an intramolecular nucleotide for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramide, phorbamate, etc.), a charged bond or a sulfur-containing bond (eg, those having phosphorothioate, phosphorodithioate, etc., for example, proteins (nuclease, nuclease 'inhibitor, Compounds with side-chain groups such as toxins, antibodies, signal peptides, poly-L-lysine, etc.
- an intramolecular nucleotide for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramide, phorbamate, etc.), a charged bond or a sulfur-containing bond (eg, those having phosphorothioate, phosphorodithioate, etc., for example, proteins (nuclease, nuclease 'inhibi
- nucleoside may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles.
- Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or ethers, amines, etc. May be converted to a functional group.
- the antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA, or a modified nucleic acid (RNA, DNA).
- modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides.
- the antisense nucleic acid of the present invention (1) makes the antisense nucleic acid more stable in a cell, (2) enhances the cell permeability of the antisense nucleic acid, and (3) has an affinity for a target sense strand. (4) If it is toxic, it can be preferably designed based on the policy of making the antisense nucleic acid less toxic.
- the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, are provided in special forms such as ribosomes and microspheres, are applied to gene therapy, It could be given in additional form.
- additional forms include polycations such as polylysine, which acts to neutralize the charge on the phosphate backbone, and lipids that enhance interaction with cell membranes or increase uptake of nucleic acids (eg, , Phospholipids, cholesterol, etc.).
- Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate). , Cholic acid, etc.).
- Such a substance can be attached to the 3 ′ end or 5 ′ end of a nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond.
- Other groups include capping groups specifically located at the 3 'or 5' end of nucleic acids that prevent degradation by nucleases such as exonucleases and RNases.
- Can be Examples of such a capping group include, but are not limited to, hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene glycol.
- the inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vivo gene expression system or translation system of the protein used in the present invention.
- the nucleic acid can be applied to cells by various known methods.
- the antisense polynucleotide of the present invention can suppress the function of the LLL or LLPL gene in a living body, it can be used, for example, as an agent for preventing and / or treating a disease associated with LLPL dysfunction. Can be. Furthermore, the antisense polynucleotide of the present invention can also be used as a diagnostic oligonucleotide probe for examining the presence of DNA encoding LLPL in tissues or cells and its expression status. It can be used to diagnose diseases related to (DNA encoding partial peptide)
- the DNA encoding the partial peptide used in the present invention may be any DNA containing the nucleotide sequence encoding the partial peptide used in the present invention described above. Further, it may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA.
- the vector used for the library may be any of bacteriophages, plasmids, cosmids, phagemids, and the like.
- the reverse transcriptase may be directly prepared by using an mRNA fraction prepared from the cells or tissues described above. It can also be amplified by Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method).
- RT-PCR method Polymerase Chain Reaction
- an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 8 which has a DNA that hybridizes under high stringency conditions with a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9;
- a DNA having a partial nucleotide sequence of DNA encoding a protein having substantially the same activity (eg, apoptosis-inhibiting activity, etc.) as a protein peptide containing is used.
- DNA that hybridizes with DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or 9 under high stringency conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 9 And DNA containing a nucleotide sequence having a homology of about 0% or more, preferably about 80% or more, more preferably about 90% or more, and still more preferably about 95% or more.
- the screening means for cloning DNA that completely encodes the protein used in the present invention or its partial peptide includes (1) the present invention.
- the DNA base sequence can be replaced using PCR or a known kit, for example, Mutan TM -supper Express Ks (Takara Shuzo), Mutan TM -K (Takara Shuzo), or the like. , ODA-LA PCR method, Gap ed dulex method, Kunke method, etc., or a method similar thereto. be able to.
- the cloned DNA encoding the protein used in the present invention can be used as it is or after digesting with a restriction enzyme or adding a linker, if desired.
- the DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
- the expression vector of the protein used in the present invention may be prepared, for example, by (a) cutting out a DNA fragment encoding a protein used in the present invention, for example, a cDNA, and extracting a target DNA fragment from the cDNA; It can be produced by ligating downstream of a promoter in a suitable expression vector.
- vectors examples include Escherichia coli-derived plasmids (eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13) and Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194) , Yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as ⁇ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., ⁇ A1-11, pXT1, Rc / CMV , pRc / RSV, pcDNA I / Neo, and the like.
- Escherichia coli-derived plasmids eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13
- Bacillus subtilis-derived plasmids eg, pUB110
- the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
- SRo! Promoter SV40 promoter, LTR open motor, CMV promoter, HSV-TK promoter and the like can be mentioned.
- CMV promoter SRO; promoter, and the like.
- the host When the host is Eshierihia genus bacterium, trp promoter, lac promoter evening one, re cA promoter evening one, AP L promoter, lpp promoter, etc.
- the host When the host is Bacillus, SPO l promoter, SP02 promoter
- yeast such as the promoter and penP promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable.
- the host When the host is an insect cell, a polyhedrin promoter, P10 promoter, etc. are preferred.
- the expression vector contains, in addition to the promoter, an enhancer, a splicing signal, a poly-A addition signal, a selection primer, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like, if desired.
- selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Ampr), neomycin resistant gene (hereinafter sometimes abbreviated as Ne o r, G418 resistance).
- dhfr gene when used as a selection marker using CHO (dhfr_) cells, the target gene can also be selected using a thymidine-free medium.
- a signal sequence suitable for the host is added to the N-terminal side of the protein used in the present invention. If the host is Escherichia, PhoA • signal sequence, OmpA ⁇ signal sequence, etc. If the host is Bacillus, a-amylase ⁇ signal sequence, subtilisin ⁇ signal sequence, etc. For yeast, use MFa signal sequence, SUC2 signal sequence, etc.For host animal cells, use insulin 'signal sequence, ⁇ -interferon signal sequence, antibody molecule, signal sequence, etc. it can.
- a transformant can be produced.
- Hosts include, for example, Escherichia, Bacillus, yeast, insect cells,
- Hirats animal cells and the like are used.
- Escherichia examples include Escherichia coli Kl 2 DH1 [Processing's Op The National Academy ' Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research], 9 309 (1981)], J A221 [Journal of Molecular Biology, 120, 517 (1978)], HB 101 [Journal of Molecular Biology] Biology, 41, 459 (1969)], C 600 [Genetics, 39, 440 (1954)], DH5a [Inoue, E, Nojima, H. and Okayama, H., Gene, 96, 23-28 (1990)], DH10B (Proc. Natl. Acad. Sci. USA), Processings of the National Academy of Sciences of the USA 87, 4645-4649 (1990)].
- Bacillus bacteria examples include, for example, Bacillus subtilis MI 114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95] , 87 (1 984)].
- yeast examples include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC 1913, NCYC 2036, Pichia * Pichia pastoris is used.
- insect cells for example, when the virus is AcNP V, a cell line derived from a larva of Spodoptera (Spodoptera frugiperda cell; S f cell); MG1 cell derived from the midgut of Trichoplusia ni; High Five derived from egg of Trichoplusia ni TM cells, cells derived from Mamestra brass icae or cells derived from Est igmena acrea. If the virus is BmNPV, a silkworm-derived cell line (Bombyx mod N; BmN cell) or the like is used. Examples of the Sf cell include Sf9 cell (ATCC CRL1711) and Sf21 cell (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) Are used.
- insects for example, silkworm larvae are used [Maeda et al. (Nature), vol. 315, 592 (1985)].
- animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient Chinese muscular cell CHO (hereinafter CHO (dh fr ”) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.
- Transformation of a Bacillus bacterium can be performed, for example, according to the method described in Molecular & General Genetics (Molecular & General Genetics), vol. 168, 111 (1979).
- Suitable media and culturing conditions for various hosts are known (in particular, WO 00/14227, page 24, line 24 to page 26, line 8, EP 1 111047 A2, paragraph [0090] to [0096])
- the protein used in the present invention of the present invention can be produced in the cell, in the cell membrane, or outside the cell of the transformant.
- the protein used in the present invention can be separated and purified from the culture by, for example, the following method.
- the cells or cells are collected by a known method after culturing, and suspended in an appropriate buffer. After disrupting the cells or cells by ultrasonication, lysozyme, and / or freeze-thawing, a method of obtaining a crude extract of the protein used in the present invention by centrifugation or filtration is used as appropriate.
- the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM. If secreted, after the culture is completed, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.
- the protein used in the present invention contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods.
- These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly for differences in molecular weight.
- Methods that use charge differences such as ion exchange chromatography, methods that use specific affinity, such as affinity chromatography, and hydrophobic methods, such as reversed-phase high-performance liquid chromatography.
- a method using a difference, a method using an isoelectric point difference such as an isoelectric focusing method, and the like are used.
- the protein thus obtained When the protein thus obtained is obtained as 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 as a salt, a known method or analogous method Depending on the method, it can be converted into a free form or another salt.
- the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be modified before or after purification by the action of an appropriate protein-modifying enzyme. It can also be partially removed.
- an appropriate protein-modifying enzyme for example, trypsin, chymotribcin, arginylendopeptidase, protein kinase, glycosidase and the like are used.
- the activity of the protein or a salt thereof used in the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody.
- the antibody against the protein or its partial peptide or its salt used in the present invention may be a polyclonal antibody or a monoclonal antibody as long as it can recognize the protein or its partial peptide or its salt used in the present invention. There may be.
- An antibody against the protein used in the present invention or a partial peptide thereof or a salt thereof may be a known antibody using the protein used in the present invention as an antigen. Alternatively, it can be produced according to a method for producing an antiserum.
- the protein or the like used in the present invention is administered to a mammal at a site capable of producing an antibody by administration, itself or together with a carrier or a diluent.
- complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability.
- the administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times. Examples of mammals to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.
- a mammal immunized with the antigen for example, an individual having an antibody titer is selected from a mouse, and the spleen, gut or lymph node is collected 2 to 5 days after the final immunization.
- monoclonal antibody-producing hybridomas can be prepared. Wear.
- the measurement of the antibody titer in the antiserum is performed, for example, by reacting the antiserum with the labeled protein used in the present invention described below and measuring the activity of the labeling agent bound to the antibody. be able to.
- the fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)].
- the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
- myeloma cells examples include NS-1, P3U1, SP 2/0 and the like, and P3U1 is preferably used.
- the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and the concentration of PEG (preferably PEG1000 to PEG6000) is about 10 to 80%.
- PEG preferably PEG1000 to PEG6000
- hybridoma culture on a solid phase onto which antigens such as proteins used in the present invention are directly or adsorbed together with a carrier is used.
- the supernatant is added, and then an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice) or protein ⁇ ⁇ labeled with a radioactive substance, enzyme, etc.
- a method for detecting monoclonal antibodies bound to a protein a method of adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A has been adsorbed, and labeling the protein used in the present invention with a radioactive substance, an enzyme, or the like And a method for detecting a monoclonal antibody bound to a solid phase.
- the selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, the selection can be performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added.
- HAT hyperxanthine, aminopterin, thymidine
- any medium can be used as long as it can grow a hybridoma.
- RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.)
- serum-free medium for hybridoma culture SFM-101, Nissui Pharmaceutical Co., Ltd.
- the culture temperature is usually 20 to 40, preferably about 37 ° C.
- the culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks.
- the culture can be usually performed under 5% carbon dioxide gas.
- the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
- Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies. [Examples: salting out, alcohol precipitation, isoelectric focusing, electrophoresis, ion exchangers (ex. , DEAE), ultracentrifugation, gel filtration, antigen-binding solid phase or specific antibody that is collected by using an active adsorbent such as protein A or protein G to dissociate the bond and obtain the antibody. Purification method].
- the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (an antigen such as the protein used in the present invention) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody.
- the antibody can be produced by collecting an antibody-containing substance for the protein or the like to be used and separating and purifying the antibody.
- the type of carrier protein and the mixture ratio of carrier and hapten are determined by the antibody against hapten immunized by cross-linking with the carrier. Any efficiency can be achieved by cross-linking any substance at any ratio.For example, weighing serum albumin, thyroglobulin, keyhole, lindet, hemocyanin, etc. A method of pulling the hapten at a ratio of about 0.1 to 20 and preferably about 1 to 5 with respect to hapten 1 is used.
- various condensing agents can be used for force coupling between the hapten and the carrier, but daltaraldehyde ⁇ carbodiimide, maleimide active ester, An active ester reagent containing an all group or a dithioviridyl group is used.
- the condensation product is administered to a mammal at a site capable of producing an antibody itself or together with a carrier and a diluent.
- Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
- the administration can usually be made once every about 2 to 6 weeks, for a total of about 3 to 10 times.
- the polyclonal antibody can be collected from blood, ascites, etc., preferably from blood, of the mammal immunized by the above method.
- the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.
- Human LLPL does not have LCAT activity to esterify free cholesterol, but exhibits lysophospholipase activity in vitro, which decomposes to free fatty acids and glycerol phosphorylcholine using lysophosphatidylcholine as a substrate [Taniyama et al .; And 'Biophysical ⁇ Research ⁇ Communications ( Biochem. Biophys. Res. Commun.), 257: 50-56 (1999)].
- the L LPL gene product has such apoptosis-inducing and apoptotic inhibitory (suppressive) activities including apoptosis induction.
- LLPL is useful as a reagent for screening a compound or a salt thereof that alters (eg, promotes or inhibits) LLPL activity (eg, apoptosis inhibitory activity).
- a compound is useful, for example, for apo] ⁇ It is considered to be useful as a monocis induction regulator (apoptosis induction promoter or apoptosis induction inhibitor).
- modulation of apoptosis induction may be either direct regulation (promotion or inhibition) of apoptosis or indirect regulation (promotion or inhibition) of apoptosis.
- the present invention provides a method for screening for a compound that changes LLPL activity or a salt thereof.
- the present invention provides
- Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
- cells having the ability to produce the protein used in the present invention are prepared by suspending them in a buffer suitable for screening.
- a buffer suitable for screening a buffer suitable for screening.
- Any buffer, such as fur or borate buffer, that does not inhibit LLPL activity may be used.
- a host transformed with a vector containing a DNA encoding the protein used in the present invention described above is used.
- the host for example, animal cells such as CHO cells are preferably used.
- a transformant in which the protein used in the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.
- the LLPL activity (eg, lysophospholipase activity, apoptosis inhibitory activity, etc.) in the case (ii) above is about 20% or more, preferably 30% or more, compared to the case (i). More preferably, a test compound that promotes about 50% or more can be selected as a compound or a salt thereof that promotes the activity of the protein used in the present invention.
- the LLPL activity in the case of the above (ii) is inhibited by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (i).
- the test compound to be inhibited) can be selected as a compound that inhibits the activity of the protein used in the present invention or a salt thereof.
- LLPL activity in the case of inserting a gene such as secretory lipolytic phosphatase or luciferase at the downstream of the promoter of the LLPL gene and expressing it in the various cells described above, and bringing the test compound into contact with the cells.
- a compound or a salt thereof that promotes or suppresses the expression of LLPL ie, promotes or inhibits the activity of the protein used in the present invention
- searching for a compound or a salt thereof that activates or inhibits the activity of LLPL You can screen.
- the screening kit of the present invention contains cells capable of producing the protein or partial peptide used in the present invention or a salt thereof, or the protein or partial peptide used in the present invention.
- the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a test compound as described above, for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, or a plant extract.
- the animal A compound or a salt thereof selected from a tissue extract, plasma, or the like, and a compound or a salt thereof that promotes or inhibits LLPL activity (eg, lysophospholipase activity, apoptosis inhibitory activity, etc.).
- LLPL activity eg, lysophospholipase activity, apoptosis inhibitory activity, etc.
- compounds or salts thereof that promote the activity of LLPL include, for example, modulators of apoptosis induction (in particular, inhibitors of apoptosis induction), diseases caused by apoptosis (eg, neurodegenerative diseases such as Alzheimer's disease). , Arteriosclerosis, hyperlipidemia, etc.).
- Compounds or salts thereof that inhibit the activity of LLPL include, for example, drugs such as agents for preventing and / or treating apoptosis induction modulators (particularly, apoptosis induction promoters) and diseases caused by apoptosis (eg, cancer, etc.). It is useful as a pile cancer drug, especially as a drug sensitivity enhancer.
- LLPL or a partial peptide thereof or (2) DNA encoding LLPL or a partial peptide thereof can be used as a drug for preventing and / or treating a disease associated with LLPL dysfunction. .
- LLPL deficiency when there is a patient who cannot expect a physiological effect due to a decrease in LLPL in the living body (LLPL deficiency), (1) LLPL is administered to the patient to supplement the amount of the protein, (2a) administering the LLPL-encoding DNA to the patient and expressing it; or (2b) inserting the LLPL-encoding DNA into the target cell and expressing it, and then transplanting the cell into the patient.
- DNA encoding LLPL is useful as a preventive and / or therapeutic agent for diseases associated with LLPL dysfunction (eg, neurodegenerative diseases such as Alzheimer's disease, arteriosclerosis, hyperlipidemia, etc.) .
- LLPL or DNA encoding LLPL is useful for, for example, prevention and / or treatment of diseases caused by apoptosis (eg, cancer).
- LLPL antisense DNA is It is useful for prevention and Z or treatment of diseases (eg, cancer).
- a polynucleotide encoding LLPL or its partial peptide (also referred to as LLPL DNA) or a polynucleotide corresponding thereto (also referred to as LLPL * antisense DNA) can be used as a probe to produce human or other mammals.
- Quantification or abnormality of the DNA or mRNA encoding the protein used in the present invention or a partial peptide thereof in a rat eg, rat, mouse, egret, sheep, pigeon, bush, cat, dog, monkey, etc.
- Abnormalities can be detected, for example, if the DNA or mRNA is damaged, mutated or decreased in expression, the DNA or mRNA is increased or overexpressed, or a gene diagnostic agent or a related gene is detected. It is useful as a diagnostic for related diseases (eg, cancer).
- LLPL DNA is performed, for example, by the well-known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Procaging). Proceedings of the National Academy of Sciences of the United States of America; 86, 2766-2770 (1989)
- a disease associated with LLPL dysfunction eg, a neurological disorder such as Alzheimer's disease
- Degenerative diseases, arteriosclerosis, hyperlipidemia, etc. or have a high possibility of contracting in the future.
- LLPL.DNA By using LLPL.DNA as a probe, it can be used for screening for a compound that changes the expression level of LLPL or a partial peptide thereof.
- the present invention includes, for example, ( ⁇ ) non-human mammals (1) blood, (2) specific organs, (3) tissues or cells isolated from the organs, or (ii) transformants and the like.
- the measurement of the mRNA amount of LLPL or its partial peptide is specifically performed as follows.
- non-human mammals for example, mice, rats, rabbits, higgs, bushus, horses, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerosis ⁇ Drugs (eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flooding stress, electric shock, light / dark, low temperature, etc.)
- Drugs eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
- physical stress eg, flooding stress, electric shock, light / dark, low temperature, etc.
- the mRNA of LLPL or its partial peptide contained in the obtained cells can be quantified, for example, by extracting mRNA from cells or the like by a conventional method and using, for example, a technique such as TaqManPCR, The analysis can also be performed by performing a Northern blot by the means described in (1).
- a transformant expressing LLPL or its partial peptide can be prepared according to the above method, and mRNA of LLPL or its partial peptide contained in the transformant can be quantified and analyzed in the same manner. it can.
- Drugs or physical agents for normal or disease model non-human mammals A certain time before giving stress etc. (30 minutes to 24 hours ago, preferably 30 minutes to 12 hours ago, more preferably 1 hour to 6 hours ago) or after a certain time (30 minutes to 3 days later, preferably Is administered 1 hour to 2 days later, more preferably 1 hour to 24 hours later) or a test compound is administered simultaneously with a drug or physical stress, and after a certain period of time after administration (30 minutes to 3 days later, Preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), by quantifying and analyzing the mRNA amount of LLPL or its partial peptide contained in the cells,
- test compound When culturing the transformant according to a conventional method, the test compound is mixed in a medium and cultured for a certain period of time (after 1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days). After that, the amount can be determined by quantifying and analyzing the mRNA amount of LLPL or its partial peptide contained in the transformant.
- the compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the expression level of LLP L or a partial peptide thereof, and specifically, (a) LL PL or A compound that enhances the protein-mediated cell stimulating activity (eg, apoptosis-inhibiting activity) used in the present invention by increasing the expression level of the partial peptide; (mouth) LLPL or its partial peptide
- the compounds that reduce the cell stimulating activity by reducing the expression level of the peptide include peptides, proteins, non-peptidic compounds, synthetic compounds, and fermentation products. It may be a compound or a known compound.
- the compound that enhances the cell stimulating activity is useful as a drug for enhancing a physiological activity such as LLPL.
- the compound that attenuates the cell stimulating activity is useful as a drug for decreasing a physiological activity such as LLPL. '
- a prophylactic and / or therapeutic agent for various diseases containing a compound that changes the expression level of LLPL or its partial peptide
- LLPL has some important roles in vivo, such as central functions. It is thought that it plays. Therefore, a compound that alters the expression level of LLPL or a partial peptide thereof can be used as an agent for preventing and / or treating a disease associated with LLPL dysfunction.
- a compound that reduces the expression level of LLLPL or its partial peptide ⁇ is useful as a prophylactic and / or therapeutic agent for cancer, for example.
- the compound When used as a prophylactic and / or therapeutic agent for a disease associated with LLPL dysfunction, it can be formulated according to conventional means.
- the antibody of the present invention can specifically recognize LLPL and the like, quantification of LLPL and the like in a test solution, particularly quantification by a sandwich immunoassay, a competition method, an immunometric method, or a nephrometry, etc. Can be used for
- the measurement system for LLPL or a salt thereof may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method.
- the measurement system for LLPL or a salt thereof may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method.
- reviews and compendiums it is possible to refer to reviews and compendiums.
- LLPL or a salt thereof can be quantified with high sensitivity by using the antibody of the present invention. Furthermore, various diseases associated with LLPL dysfunction can be diagnosed by using the in vivo quantification method of LLPL or a salt thereof using the antibody of the present invention.
- a decrease in LLP L concentration may be a disease associated with LLPL dysfunction (eg, a neurodegenerative disease such as Alzheimer's disease, arteriosclerosis, hyperlipidemia, etc.). Can be diagnosed as being more likely or likely to be affected in the future.
- LLPL dysfunction eg, a neurodegenerative disease such as Alzheimer's disease, arteriosclerosis, hyperlipidemia, etc.
- LLPL for example, cancer
- the antibody of the present invention can be used for specifically detecting LLPL or the like present in a subject such as a body fluid or a tissue. It can also be used to prepare antibody columns used to purify LLPL, etc., to detect LLP L etc. in each fraction during purification, and to analyze the behavior of LLP L in test cells. it can.
- the neutralizing activity of an antibody against LLPL or a partial peptide thereof or a salt thereof against a protein or the like used in the present invention means an activity of inactivating a physiological activity involved in the protein. Therefore, when the antibody has a neutralizing activity, it can inactivate a cell-stimulating activity (eg, an apoptosis-inhibiting activity) mediated by the protein, for example. Therefore, such an antibody can be used for prevention and Z or treatment of a disease caused by overexpression of the protein, for example, a disease caused by apoptosis (eg, cancer).
- apoptosis eg, cancer
- transgenic animals expressing LLPL etc. can be created.
- animals include non-human mammals (eg, rats, mice, egrets, sheep, sheep, bush, elephants, cats, dogs, monkeys, etc.) (hereinafter abbreviated as animals). Particularly preferred are mice, egrets, and the like.
- LLPL DNA When introducing LLPL DNA into a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter capable of being expressed in animal cells.
- a ubiquitous expression promoter such as a virus-derived promoter or melothinonein can be used, but preferably, an NGF gene promoter specifically expressed in the brain, an enolase gene promoter, or the like is used.
- LLPL DNA is present in all germ cells and somatic cells of the target animal.
- the presence of LLPL and the like in the germ cells of the produced animal after DNA introduction means that all the offspring of the produced animal have LLPL and the like in all of its germ cells and somatic cells.
- the offspring of this type of animal that has inherited the gene have LLPL etc. in all of its germinal and somatic cells.
- the animal After confirming that the LLPL ′ DNA-introduced animal stably retains the gene by mating, the animal can be bred in a normal breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the target DNA, homozygous animals having the transgene on both homologous chromosomes are obtained, and by crossing the male and female animals, all progeny have the DNA. Breeding can be subcultured
- LLPL-DNA transgenic animals can also be used as a cell source for tissue culture.
- LLPL and the like can be analyzed by directly analyzing DNA or RNA in the tissues of LLPL / DNA-transfected mice, or by analyzing tissues in which LLPL expressed by a gene is present.
- Cells of a tissue having LLPL or the like can be cultured by standard tissue culture techniques, and these can be used to study the function of cells from tissues that are generally difficult to culture, such as those from brain or peripheral tissues.
- tissue culture techniques such as those from brain or peripheral tissues.
- LLPL and the like can be isolated and purified therefrom.
- the present invention provides a non-human mammal embryonic stem cell in which LLPL DNA has been inactivated and a non-human mammal deficient in LLPL-DNA expression.
- the DNA is inactivated by introducing a reporter gene (eg, a j8_galactosidase gene derived from Escherichia coli), and the reporter gene can be expressed under the control of a promoter for LLPL DNA.
- a reporter gene eg, a j8_galactosidase gene derived from Escherichia coli
- Mammalian ES cells in which the LLPL gene has been inactivated are artificially mutated LLPL genes in mammalian ES cells to suppress gene expression or to encode the gene. By substantially eliminating the activity of LLPL, the gene has been substantially inactivated without the ability to express LLPL (hereinafter sometimes referred to as the knockout gene of the present invention). ES cells.
- a mammal used as a material for the ES cells for example, human, porcupine, pig, sheep, goat, goat, canine, cat, guinea pig, hamster, mouse, rat and the like are used.
- the non-human animal may be any animal other than a human having the LLPL gene, but is preferably a non-human mammal.
- a non-human mammal for example, red sea lions, bushes, higgins, goats, blue herons, dogs, cats, guinea pigs, hamsters, mice, rats, and the like are used.
- mice for example, C57BLZ6 strain, DBA as pure strains
- 2 strains such as B6C3F1 strain, BDF1 strain, B6D2F1 strain, BALB / c strain, ICR strain, etc.
- pure strain C57BL / 6 strain, etc.
- crossing strain a BDF strain or an ICR strain
- a rat eg, Wistar, SD, etc.
- Examples of a method for artificially adding a mutation to the LLPL gene include deletion of part or all of the gene sequence by genetic engineering, or insertion or substitution of another gene.
- the knockout gene of the present invention can be prepared, for example, by disrupting the ability to shift the codon reading frame, the function of the promoter or exon, and the like.
- LLPL gene inactivated ES cells or knockout ES cells include, for example, drug resistance genes (eg, neomycin resistance gene, hygromycin resistance gene, or zeocin resistance gene, etc.).
- a neomycin resistance gene, etc. or a repo overnight gene (eg, 1 ac Z (Escherichia coli j3_galactosidase gene), cat (chloramphenicol acetylacetyltransferase gene), GUS (] 3-Dark mouth Nidase gene), luciferase gene, equorin gene, taumarin gene, GFP (Green Fluorescent Protein) gene, preferably 1 ac Z), etc., to destroy the exon function of the LLP L gene.
- 1 ac Z Escherichia coli j3_galactosidase gene
- cat chloramphenicol acetylacetyltransferase gene
- GUS 3-Dark mouth Nidase gene
- luciferase gene equorin gene
- taumarin gene equorin gene
- GFP Green Fluorescent Protein
- a DNA vector (hereinafter abbreviated as a targeting vector) having a DNA sequence constructed to insert a DNA sequence (for example, a polyA addition signal) so that complete mRNA cannot be synthesized, resulting in gene disruption. ) Is prepared.
- a reporter gene When a reporter gene is inserted to disrupt the function of exon, it is preferable to insert the reporter gene so that it is expressed under the control of the LLPL promoter.
- drug resistance gene refers to a gene involved in drug resistance of antibiotics and the like, and is used as a marker for selecting whether or not the introduced gene has been expressed in cells.
- the “repo overnight gene” refers to a group of genes that serve as indicators of gene expression, and usually, structural genes of enzymes that catalyze a luminescence reaction or a color reaction are often used. (1) Those with no genetic background, (2) High-sensitivity methods that can quantitatively express gene expression, (3) Those that have little effect on transformed cells, and (4) Those that show localization of the expression site are preferably used. (Plant Cell Engineering, Vol. 2, p. 721, 1990).
- the above-mentioned “drug resistance gene” is also used for the same purpose, but the “reporter gene” is not only whether or not the gene to be introduced is expressed in cells, but also in which tissue and when. Can be examined, and the expression level can be accurately and quantitatively examined.
- the evening targeting vector is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cells are placed on or near the LLP L gene.
- This DNA sequence was analyzed by Southern hybridization analysis using a DNA sequence as a probe or by the PCR method using the DNA sequence in the neighboring region other than the LLPL gene used in the preparation of the targeting vector and the DNA sequence on the targeting vector as a primer. It can be obtained by selecting the knockout ES cells of the invention.
- the targeting vector examples include plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13, etc.) and plasmids derived from Bacillus subtilis (eg, pUBllO, pTB5, pC194, etc.) , Yeast-derived plasmids (eg, pSH19, pSHI5, etc.), bacteriophages such as phage ⁇ , retroviruses such as Moroni leukemia virus, vaccinia virus or adenovirus vectors, baculovirus, papillomas Viruses, viruses from the herpes virus group, or animal viruses such as Epstein-Barr virus are used.
- Escherichia coli eg, pBR322, pBR325, pUC12, pUC13, etc.
- Bacillus subtilis eg, pUBllO, pTB5, pC194, etc.
- ES cells from which the DLL PL gene is inactivated by the homologous recombination method or the like for example, those already established as described above may be used, or the known ES cells of Evans and Kaufman may be used. It may be newly established according to the method.
- BDF1 mice C57BL / 6 mice
- C57BLZ6 mice or C57BL / 6 mice have reduced the number of eggs collected by hybridization with DBA / 2
- F1 mice have the advantage of high number of eggs collected and robust eggs
- C57BLZ6 mice have a genetic background. Then, it can be used advantageously in that it is possible to replace its genetic background with C57BL / 6 mice by backcrossing with C57BLZ6 mice.
- blastocysts 3.5 days after fertilization are generally used, but other 8 cell-stage embryos (8-cell embryos around 2.5 days after fertilization)
- a large number of early embryos can be obtained efficiently by collecting eggs (preferably) and culturing them up to blastocysts.
- the ES cell line obtained in this way usually has very good growth potential, but it must be carefully subcultured because it tends to lose its ability to generate individuals.
- a suitable feeder cell such as STO fibroblasts
- a carbon dioxide incubator preferably 5% carbon dioxide, 95% air or 5%
- LIF 1-1000 OU / ml
- trypsin / EDTA solution usually 0.001-0.5% trypsin 70 1-5 mM EDTA (preferably, about 0.1% trypsin / ImM EDTA) is treated to form a single cell, which is then seeded on a newly prepared feeder cell.
- Such subculture is usually performed every 11 to 13 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.
- ES cells can be cultured in monolayers at high densities or in suspension cultures to form cell clumps under appropriate conditions to produce various types of cells such as parietal, visceral, and cardiac muscles.
- MJ Evans and MH Kau fman Nature, Vol. 292, pp. 154, 1981; GR Martin Proceedings of National Academy of Sciences, Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Ebemblology 1 and Experimental Morphology 1, 87, 27, 1985
- the LLP L gene-deficient cells obtained by differentiating the ES cells of the present invention are useful in in vitro LLPL cell biology studies.
- DMEM Dulbecco's modified Eagle's medium
- the non-human animal deficient in LLPL gene expression of the present invention (hereinafter sometimes referred to as a non-human animal deficient in gene expression) is, for example, a cell derived from a mammalian ES cell in which the LLPL gene is inactivated. This is a non-human animal in which an inactivated LLPL gene sequence has been introduced into germ cells and somatic cells at an early stage of embryogenesis.
- the above-mentioned targeting vector can be transferred to a non-human animal ES cell or a non-human animal egg cell by a known method (for example, an electoral poration method, a microinjection method, a calcium phosphate method, a lipofection method, or an agglutination method). , Particle gun method, DEAE-dextran method, etc. (preferred introduction methods include the electoral poration method when introducing into ES cells, and the microinjection method when introducing into egg cells)
- the inactivating LLP L gene sequence of the targeting vector can be replaced with the LLPL gene on the chromosome of a non-human animal ES cell or non-human animal egg cell by homologous recombination.
- Cells in which the LLP L gene was knocked out were used for Southern hybridization analysis or the DNA sequence on the targeting vector using the DNA sequence on or near the LLP L gene as a probe and the targeting vector. It can be determined by PCR analysis using the DNA sequence of the neighboring region other than the mouse-derived LLPL gene as a primer.
- a cell line in which the LLPL gene is inactivated is cloned by homologous recombination, and the cells are cloned at an appropriate time during the early stage of embryogenesis, for example, at the 8-cell stage non-human Chimeric embryos are prepared by injecting them into animal embryos or blastocysts (injection method), or by inserting the ES cell mass in which the LLPL gene is inactivated between two 8-cell stage embryos (assembly chimera method). Implantation into the uterus of the pseudopregnant non-human animal.
- the animals produced were cells with normal LLPL loci and artificially mutated LL It is a chimeric animal composed of both cells having the PL locus.
- all tissues were artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the LLPL locus, for example, by determining coat color.
- the individuals obtained in this manner are usually LLPL heterozygous expression-deficient individuals, and can be obtained by mating individuals with LTE expression-deficient to LLPL and obtaining LLPL homo-expression-defective individuals from their offspring.
- a transgenic non-human animal having a targeting vector introduced into a chromosome can be obtained by injecting a gene solution into a nucleus of an egg cell by a microinjection method. It can be obtained by comparing transgenic non-human animals and selecting those with mutations in the LLPL locus by homologous recombination.
- a non-human animal deficient in LLLPL gene expression can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
- the animal individual obtained by the crossing can confirm that the gene has been knocked out, and can carry out rearing in an ordinary rearing environment.
- the germline can be obtained and maintained according to a conventional method. That is, by mating male and female animals having the inactivated gene sequence, a homozygous animal having the inactivated gene sequence on both homologous chromosomes can be obtained.
- the obtained homozygote animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and a plurality of homozygotes are obtained.
- homozygous and heterozygous animals having the inactivated gene sequence can be bred and passaged.
- the progeny of the animal having the inactivated gene sequence thus obtained is also included in the non-human animal deficient in expression of the LLPL gene of the present invention.
- LLPL genes mammalian ES cells in which the LLPL gene has been inactivated are LLPL genes. It is very useful in creating non-human animals with insufficient gene expression.
- non-human abdominal rostral expression deficient in LLPL gene or its tissue or cells derived therefrom are not affected by diseases caused by LLPL deficiency, such as loss of various biological activities that can be induced by LLPL.
- diseases caused by inactivation of the biological activity of LLPL eg, diseases caused by cebutic shock, apoptosis, etc.
- the non-human animal deficient in LLPL gene expression of the present invention or a tissue thereof or a cell derived therefrom can be used for screening for a preventive and / or therapeutic drug for the disease.
- examples of the above-mentioned tissues and cells derived therefrom include measuring the specific activity using a homogenate such as liver or kidney, or measuring the activity or production amount of a specific product using peritoneal macrophages. It can be used for screening.
- a non-human mammal deficient in LLPL • DNA expression can be used for screening for a compound that has a prophylactic and / or therapeutic effect on diseases caused by deficiency or damage of LLPL • DNA.
- the present invention provides a method for administering a test compound to a non-human mammal deficient in LLPL / DNA expression, observing and measuring changes in the animal, and treating diseases caused by deficiency or damage of LLPL / DNA.
- a method for screening a compound having a prophylactic and / or therapeutic effect or a salt thereof is provided.
- Examples of the non-human mammal deficient in LLPL / DNA expression used in the screening method include those described above.
- Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma, and these compounds are novel compounds. Or a known compound.
- a non-human mammal deficient in LLPL / DNA expression is treated with a test compound
- a test animal treated with a test compound can be used to test the preventive and / or therapeutic effects of the test compound using changes in various organs, tissues, disease symptoms, etc. of the animal as an index, as compared to an untreated control animal.
- oral administration, intravenous injection and the like are used, and the method can be appropriately selected according to the condition of the test animal, the properties of the test compound, and the like.
- the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
- a test compound when screening for a compound having a preventive and / or therapeutic effect on a disease caused by apoptosis, a test compound is brought into contact with a primary cultured cell derived from a non-human mammal deficient in DNA expression of the present invention to induce apoptosis.
- Stimuli induction of intracellular calcium ion increase, TNF- or Fas ligand, radiation, ultraviolet light, heat, stress such as hydrogen peroxide, LPS, ceramide, etc.
- apoptotic cell biology or Measure biochemical markers caspase activity, chromatin aggregation, chromosome fragmentation, etc.
- test compound when a test compound is administered to a test animal, the survival rate of the test animal is improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more.
- a test compound can be selected as a compound having a prophylactic and / or therapeutic effect on the above-mentioned diseases.
- the compound obtained by using the screening method is a compound selected from the test compounds described above, and has a preventive and / or therapeutic effect on a disease caused by deficiency or damage of the polypeptide of the present invention. Therefore, it can be used as a medicament such as a safe and low toxic preventive and / or therapeutic agent for the disease. Further, compounds derived from the compounds obtained by the above screening can be used in the same manner.
- the present invention relates to LLPL ′ DNA, which comprises administering a test compound to a non-human mammal deficient in LLPL DNA expression and detecting reporter gene expression.
- LLPLDNA expression-defective non-human mammal among the aforementioned LLPLDNA expression-deficient non-human mammals, LLP LDNA is inactivated by introducing a reporter gene, One in which the repo overnight gene can be expressed under the control of promoter overnight for LLPL • DNA is used.
- test compound examples include the same compounds as described above.
- the same ones as described above are used, and the jS_galactosidase gene (1 ac Z.), the soluble alkaline phosphatase gene or the luciferase gene are suitable.
- 3-galactosidase when a part of the DNA region encoding the polypeptide of the present invention is replaced with a / 3-galactosidase gene (1 ac Z) derived from Escherichia coli, a tissue expressing the polypeptide of the present invention originally Instead of the polypeptide of the invention,; 3-galactosidase is expressed. Therefore, for example, by staining with a reagent that is a base of j3-galactosidase such as 5_bromo-4-monocloth_3-indolyl i3-galactopyranoside (X-gal), it is easy to carry out staining. The expression state of the polypeptide of the present invention in an animal can be observed.
- a polypeptide-deficient mouse of the present invention or a tissue section thereof is fixed with dartalaldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature.
- PBS phosphate buffered saline
- the tissue sample is washed with an ImMEDTA / PBS solution to stop the] -galactosidase reaction, and color development may be observed.
- mRNA encoding 1acZ may be detected according to a conventional method.
- Compounds or salts thereof obtained by the above screening method are A compound selected from the test compounds, which promotes or inhibits the overnight activity of LLPL DNA.
- a compound or a salt thereof that promotes the activity of a promoter against LLPL DNA can promote the expression of the polypeptide of the present invention and promote the function of the polypeptide.
- a disease caused by apoptosis For example, it is useful as a drug for neurodegenerative diseases such as Alzheimer's disease, arteriosclerosis, hyperlipidemia, etc.
- a compound or a salt thereof that inhibits the promoter activity against LLPL DNA can inhibit the expression of the polypeptide of the present invention and inhibit the function of the polypeptide.
- a medicament containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned medicament containing the polypeptide of the present invention or a salt thereof.
- non-human mammals deficient in LLPL DNA expression are extremely useful for screening compounds or salts thereof that promote or inhibit the activity of the promoter for LLPL DNA, It can greatly contribute to the investigation of the cause of various diseases caused by the disease or the development of preventive and / or therapeutic drugs.
- transgenic animal In addition, using a DNA containing the promoter region of the polypeptide of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). ) Makes it possible to specifically synthesize the polypeptide and examine its effects on living organisms. Furthermore, by binding an appropriate repo overnight gene to the above promoter portion and establishing a cell line that expresses this gene, the polypeptide of the present invention has the action of specifically promoting or suppressing the ability of the polypeptide itself to be produced in the body. It can be used as a search system for low molecular compounds. (12) Formulation, administration method, dosage, etc. of active ingredients such as proteins used in the present invention
- LLPL or its partial peptide a compound that alters LLPL activity, a polynucleotide encoding an LLP L protein, an antisense polynucleotide, an antibody against LLPL or its partial peptide, obtained by any of the above screening methods
- a compound hereinafter sometimes abbreviated as an active ingredient
- it can be formulated according to conventional means.
- LLPL / DNA polynucleotide or antisense polynucleotide
- LLPL / DNA polynucleotide or antisense polynucleotide
- LLPL / DNA can be administered as is, or with an adjuvant to enhance uptake, by means of a gene gun or a catheter such as a Hydmouth gel catheter.
- the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.
- the present invention further provides
- RNA interference in which double-stranded RNA specifically suppresses gene expression
- siRNA mammalian cells
- ribozyme an RNA molecule that undergoes a self-cleavage reaction, is also involved in the suppression of gene expression.
- the double-stranded RNA, lipozyme, and the like can suppress the expression of the polynucleotide (eg, DNA) of the present invention, Since the activity of the polynucleotide (eg, DNA) of the present invention can be inhibited, it can be used as an agent for preventing and treating various cancers.
- the double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
- the lipozyme can be produced by designing based on the sequence of the polynucleotide encoding the protein of the present invention according to a known method (eg, TRENDS in Molecular Medicine, 7, 221, 2001). For example, it can be produced by linking a known lipozyme to a part of R N ⁇ encoding the protein of the present invention.
- a part of the RNA encoding the protein of the present invention includes a portion (RNA fragment) close to the cleavage site on the RNA of the present invention which can be cleaved by a known lipozyme.
- the active ingredient (or compound) which can be formulated and administered in the same manner as an antisense polynucleotide is pharmaceutically acceptable.
- a salt that can be formed can be formed, such a salt may be formed.
- physiologically acceptable acids eg, inorganic acids, organic acids, etc.
- bases eg, alkali metals, etc.
- Addition salts are preferred.
- salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
- inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
- organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
- Succinic acid tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.
- the active ingredient (including its salts) is orally administered as tablets, capsules, elixirs, microcapsules and the like, if necessary, or water or other pharmaceutically acceptable sugar-coated tablets It can be used parenterally in the form of injectable solutions, such as sterile solutions with liquid or suspensions.
- the active compound is physiologically recognized Known carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in a unit dosage form generally required for the practice of pharmaceutical preparations. .
- the amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
- Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin ', alginic acid Swelling agents such as sucrose, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
- binders such as gelatin, corn starch, tragacanth, gum arabic
- excipients such as crystalline cellulose, corn starch, gelatin '
- alginic acid Swelling agents such as sucrose, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
- a liquid carrier such as an oil or fat.
- Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
- aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like.
- an alcohol e.g., ethanol
- polyalcohol e.g., propylene glycol, polyethylene Dali call
- a nonionic surfactant eg, polysorbate WINCH 8 0 TM, HCO- 5 0
- the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
- the prophylactic and Z or therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, proforce hydrochloride, etc.), stabilizers (eg, , Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
- buffers eg, phosphate buffer, sodium acetate buffer
- soothing agents eg, benzalkonium chloride, proforce hydrochloride, etc.
- stabilizers eg, Human serum albumin, polyethylene glycol, etc.
- preservatives eg, benzyl alcohol, phenol, etc.
- antioxidants e.g, antioxidants and the like.
- the prepared injection solution is usually filled into a suitable ampoule.
- the prophylactic and / or Z- or therapeutic agent can be used in combination with an appropriate drug, for example, as a DDS preparation
- the dosage of the active ingredient of the present invention varies depending on the administration subject, target organ, symptom, administration method and the like.
- one dose per patient (as 60 kg) is generally used. It is about 0.1 mg to 100 mg per day, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg.
- the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc.
- it is usually used, for example, for patients (as 6 Okg).
- About 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day. is there.
- the dose can be administered in terms of 6 Okg.
- the preparations thus obtained can be administered to, for example, humans and other mammals (eg, rats, puppies, sheep, bush, puppies, cats, dogs, monkeys, etc.).
- mammals eg, rats, puppies, sheep, bush, puppies, cats, dogs, monkeys, etc.
- bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by IU PAC-IUB Coalition on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below.
- amino acids may have optical isomers
- L-form shall be indicated unless otherwise specified.
- G 1 n Glutamine
- primer P 101-4 The nucleotide sequence of primer P 101-4 is shown.
- 1 shows the nucleotide sequence of DNA encoding human-derived LLP L.
- FIG. 2 shows the amino acid sequence of LL PL (mature) derived from mouse kidney.
- FIG. 2 shows the amino acid sequence of human kidney-derived LLPL (mature). Between the 66th position (Leu) and the 67th position (Va1) of the amino acid sequence represented by SEQ ID NO: 12, the 67th position (Glu) to 67th position of the amino acid sequence represented by SEQ ID NO: 13 The 98th (Leu) 32 amino acid residues have been inserted.
- ES cells and feeder single cell is The suggested 15% FBS, 2mM L- G lut am i ne, 10 one 4 M / 3- mercaptoethanol, ESQ DMEM medium containing 50 UZM 1 penicillin, 50 g / m 1 streptomycin using (ES cell medium), and cultured at 37 ° C, 5% C_ ⁇ 2 presence.
- the ES cells were cultured on a feder cell layer arrested by culturing for 3 hours in a medium supplemented with 10 g / ml mitomycin C (Sigma). In order to suppress the differentiation of ES cells as much as possible, the medium was changed basically on a half-volume basis and subcultured every 2-3 days.
- Reference Example 2 Screening of mouse genomic DNA library
- the pCMV-SPORT plasmid (pTB2010) containing mLLPL cDNA was treated with SmaI restriction enzyme to isolate about 1 kbp from the vector MCS located at the N-terminus to the Smal site of mLLPL.
- SmaI restriction enzyme SmaI restriction enzyme to isolate about 1 kbp from the vector MCS located at the N-terminus to the Smal site of mLLPL.
- BAC ES Mouse Hybridization library screening services GEOME SYSTEMS
- the DNA fragments of these three clones which had been fragmented by treatment with EcoRI and PstI restriction enzymes, were analyzed by the Southern hybridization method using a DNA probe containing almost the entire region of mLLLPLORF. went.
- the DNA probe was prepared by PCR using pTB2010 as a template and sense strand primer P94-1 (SEQ ID NO: 3) and antisense strand primer P101-4 (SEQ ID NO: 4). About 11 kbp and about 7.5 kbp DNA fragments were obtained from the EcoRI-treated product, and about 6.5 and about 1.51 ⁇ and about 0.6 from the PstI-treated product.
- the obtained clone was found to cover about 13 kbp of the entire region of the mLLPL genome, and the structure of the exon intron on the 13 kbp mL LPL genome was determined (Fig. 1).
- the mouse LLPL ORF was composed of a total of six exons, and it was revealed that the lipase motif, which is the active site, is located on the fifth exon.
- Reference Example 3 Construction of the targeting vector pTB 2224
- DNA fragments to be short am and ion arm were prepared using a plasmid obtained by fragmenting mLLPL / pBe1oBac11 by treatment with a restriction enzyme and subcloning into pUC118 vector.
- An EcoRI fragment containing the first and second exons, with the HindIII / PstI fragment (1.1kbp) containing the sixth exon and a SalI site added to both ends as a short arm The following operation was performed in order to use a long arm having Not I sites added at both ends of (ll kbp).
- a HindIII fragment (1.3 kbp) was isolated from the EcoR I (7.5 kbp) / pUC118 plasmid and isolated from pB1116 3 1 1 "1 1; 11? ⁇ 11 (11 1 1 ).
- the clone was selected at the 5 'end of the MCS insert near the S a1 I. After cutting this plasmid with BamHI, the end was blunt-ended using T4 DNA polymerase. Then, a plasmid containing Sa1I linker was obtained, which was cleaved with PstI and self-cyclized to obtain a PstI fragment (0.2 kbp) at the 3 'end of the insert.
- the ES cells (T-75) are washed three times with 10 ml of PBS / EDTA, suspended in 3 ml of 0.25 trypsin ZlmM EDTA solution (Gibco BRL), and then 7 ml of D-PBS (-) is added. After adding and suspending the cells, the cells were collected by centrifugation. The collected cells were washed three times with 10 ml of D-PBS (-), and then re-suspended in D-PBS (-) lm 1 to prepare an ES cell suspension.
- the targeting vector DNA (about 120 ⁇ g) obtained in Reference Example 3 (about 120 ⁇ g) was linearized with SalI restriction enzyme, phenol-chloroform was extracted, and ethanol precipitation was performed.
- 0.9 ml of D-PBS (-) was added to make 1 ml, and the mixture was added to the previously prepared ES cell suspension and left for 3 minutes.
- Each lml of the solution was dispensed into a cuvette for electroporation, and electroporation of ES cells to ES cells was performed using Cell Porator Electroporation System I (Gibco BRL). After checking Low ⁇ , increase the monitor value to Fast-UP to about 300.
- the culture medium was changed every day, and colonies that had grown from day 9 to day 11 were collected and subjected to primary screening by Southern analysis of PCR products and secondary screening by genomic Southern analysis.
- the target clone was selected by the ning.
- LLP L gene deficiency of the obtained G418-resistant strain was determined using the cell lysate prepared from a part of each collected colony as a template and the sense strand primer SI-75 (SEQ ID NO: 5) on the neomycin resistance gene.
- PCR was performed using the antisense strand primer P 3 -t (SEQ ID NO: 6) located on the short arm.
- This PCR product was electrophoresed on agarose gel and then alkali-transferred to Hybond N + (Aniers am Pharmacia Biotech).
- the prepared Southern plot was analyzed by the Southern hybridization method using the 0.41 ⁇ ⁇ 1 II fragment in the short arm region as probe B.
- the genome DNA was extracted from the cells that had been subsequently scaled up and cultured, and treated with EcoRI restriction enzyme.
- Genomic Southern analysis was performed using the 0.5 kbp Hindlll / Pstl fragment of the outer region of the short arm in the LLPL cDNA 3, one untranslated region as probe C.
- mice for blastocyst collection were produced as follows. First, an 8-week-old C57BL / 6 mouse (Cr J) female mouse was purchased, and an SPF animal breeding facility controlled at room temperature of 25 ⁇ 1 ° C, humidity of 50 ⁇ 10%, and 7 to 19 o'clock for 12 hours was used. For one week. On the first day of the experiment, mice were cohabited with male mice of the same strain of 10 weeks or older, and natural mating was performed. On the following day, the female mice living together were checked for vaginal plugs, and the female mice with the confirmed vaginal plugs were bred for 3 days until a microphone-mouth injection experiment.
- mice On the day of the microinjection experiment, the mice were sacrificed by cervical dislocation, the abdomen was opened, and the ovaries, fallopian tubes, and uterus were removed. The upper part of the uterus was removed, and the uterus was cut right and left. Further, a 25 G injection needle was inserted from the uterine horn side and vaginal side, respectively, and perfused with an egg collection medium (DMEM containing 10% FCS, 100 U benicillin and 100 U streptomycin). 3. harvested 5 cm Petri dish, 37, 7% C0 2 was allowed to stand for 30 minutes or more under the conditions of the incubator, was recovered blastocyst only.
- DMEM egg collection medium
- the homologous recombinant ES cells that had been cryopreserved at a cell density of 5 ⁇ 10 5 or 1 ⁇ 10 6 were thawed on the second day of the experiment, and the ST ⁇ cells were immediately transferred to a Feeder medium (10% FCS, 20 OmM Lg 1 ut).
- the seeds were seeded on a 6 cm diameter feeder dish cultured in DMEM containing amine, lOmM'NEAA, 100 U penicillin and 10 OU streptomycin.
- the grown homologous recombinant ES cells were washed twice using a PBS solution. The washed cells were treated with a 0.025% trypsin solution for 5 minutes. After the addition of the ES medium, the cells were centrifuged at 1200 rpm for 2 minutes at 4 ° C. Next, the supernatant was removed, and 2 to 3 ml of ES medium was added. The cells were thoroughly loosened, and the same medium was further added to adjust the volume to 1 Om1.
- a microinjection medium (16% FCS, 20 OmM Lg 1 uta mine, 1 OmM NEAA, 100 U penicillin, 10 OU streptomycin, 2 OmM HEPES) is placed in the center of the lid of a 6 cm diameter dish dish as an injection chamber.
- DMEM containing 0.1 mM / 3-mercaptoethanol and transfer 5 to 10 blastocysts in the center of the drop, and transfer the above homologous recombinant ES cells to the periphery of the drop. did.
- Liquid paraffin was added to prevent the drops from drying, and the mixture was refrigerated at 4 ° C for 20 to 30 minutes. Microinjection experiments were performed by the method of Hogan et al.
- Blastocysts ended microinjection were transferred to DMEM medium supplemented with 10% FCS, blastocysts were cultured until returning to its original shape at 37 ° C, 7% C0 2 Inkyube one coater controlled in the condition.
- Eight to ten normal blastocysts were transplanted into the right and left horns of ICR (JcL) strain pseudopregnant female mice prepared by the method described below, and the room temperature was 25 ⁇ 1 ° C and the humidity was 50 ⁇ 10%.
- the transplanted mice were bred in a breeding room controlled under 12-hour sunshine conditions from 7 to 19 o'clock.
- ICR (J cL) strain female mice was purchased and kept for 1 week at SPF animal breeding facility controlled at room temperature 25 ⁇ 1 ° C, humidity 50 ⁇ 10%, and 7 to 19 o'clock for 12 hours. Had acclimated.
- natural mating was carried out by coexisting with previously prepared male mice of ICR (JcL) strain vasectomy strain of 10 weeks or older prepared.
- the female mice living together were checked for vaginal plugs, bred for 2 days until the day of the microinjection experiment, and used for the experiment.
- DNA was extracted from the tail of ES cell-derived offspring mice according to a standard method, and this genomic DNA was used as type ⁇ for the sense strand primer SI-75 (SEQ ID NO: 5) on the neomycin resistance gene and the sense strand primer on the LLPL gene PCR was performed using three primers, P 4_ s (SEQ ID NO: 7) and the antisense strand primer P 3 _t (SEQ ID NO: 6) located on the short arm. was analyzed. Using this primer, a 0.4 kbp band was amplified from the wild-type allele in which no mutation was introduced, and a 0.3 kb band was amplified from the disrupted allele in which the LLPL gene had been introduced. Can be seen.
- LLPL-deficient mice Using primary cultured cells derived from LLPL-deficient mice and their wild-type mice, apply stress such as TNF-cu, ultraviolet light, hydrogen peroxide, and apoptosis-stimulating stimuli such as LPS and ceramide to cells, and specific to apoptosis Detection of specific cell biological or biochemical markers (eg, caspase activity, chromatin aggregation, chromosome fragmentation, etc.). These phenomena are observed with less stimulation in LLPL-deficient mice than in wild-type mice. That is, it is suggested that the LLP L gene (or gene product) is involved in the expression of apoptosis. LLPL-deficient mice can be used to screen for drugs that compensate for apoptosis induction.
- Example 2 Sensitivity of Apoptosis Induction by Oxidized LDL and DL-PPMP
- Peritoneal macrophages were prepared from pig mice. Following the method of Yamamura et al. (Shinsei Kagaku Kenkyusho 4, pp.187-206 (1993)), LDL was prepared from ⁇ sagi serum and then used by Hakamada et al. (Cell Engineering Separate Volume Medical Experiment Manual Series 2, Arteriosclerosis + Lipids) Oxidized LDL was prepared according to the Hematology Research Strategy, pp. 36-41 (1996).
- Peritoneal macrophage cells contain oxidized LDL-containing DMEM—25mM HEPES (pH-MPMP) containing DL_threo-1-pheny-2- 3-palmitololino-l-propanol-HCl (DL-PPMP), a darcosylceramide synthase inhibitor at a certain concentration. 7) The cells were cultured for 24 hours using the medium. After washing twice with PBS, apoptotic cells were stained using Annex in-V-FLUOS Staining Kit (Roche). DAP I-containing VECTASHIELD (VECTOR) was mounted and apoptotic cells were detected under a fluorescence microscope.
- the ratio of Ann exin-V positive area to DAPI positive area was determined by image processing using Image-Pro Plus Ver.4.5 (MediaCybernetics).
- the relative ratio of each control condition was determined by setting the value of the control treatment condition in the peritoneal macrophage cells derived from the ApOE knockout mouse to 1. Table 1 shows the results.
- Coating buffer (0.1 M Carbonate buffer, pH9.6) containing 2.5 gZm1 of purified monoclonal antibody m7-1C is dispensed into a 96-well mic opening plate at 200 H1 each, and left at 4 ° C for 24 hours did.
- Wash buffer A PBS, 0.05% Tween20
- Blocking buffer PBS, 0.05% Tween20, 1% BSA
- the sandwich ELISA method using 7-1C as a solid-phase antibody and 4-1H-HRP as a labeling substance can detect human LLPL down to 0.5 ng / ml, which is extremely high. It was found that high-sensitivity detection was possible.
- Fig. 5 shows the results.
- the blood concentration of human LLPL was expected to be 8.4 ⁇ 8.0 ng gZm1.
- THP-1 cells were treated with polpolymyristate acetate (PMA), 100 nM for 3 days to make them macrophages, and then replaced with RPMI 1640 medium containing 1% FBS and 25 mM HE PES.
- PMA polpolymyristate acetate
- LLPL production was measured for the culture supernatant cultured for 3 days.
- Fig. 6 shows the results.
- Macrophage-like THP The LLPL secretion production per cell for 3 days was expected to be 2.3 ⁇ 1 • 2 ngXm1.
- Example 5 Measurement of Acid Phospholipase A 2 (aPLA 2 ) Activity in LLPL / ApoE Double Deficient Mice
- the aPLAg property in liver, kidney and brain tissues prepared from LLPLZApoE double-deficient mice was measured.
- Reaction buffer 50mM sodium Kuen acid, pH 4.5
- ribosomes phosphatidylcholine (70 mol%), l-acyl-2- [1-C 14] ho sphatidylet anolainine (2 mol%), dicetylphosphate (30 mol%)
- the final concentration was added as a substrate so as to be 128 M, and then each tissue lysate protein (final concentration: lOO ⁇ gZml) was added to make the total amount 500/1.
- the liver tissue lysate protein of the LLP LZA poE double-deficient mouse used in Example 5 was analyzed for the fluorescent phospholipid 1,2-bis- (l-pyrenebutanoyl) -sn-glycero-3-phospocholine (B-3781 was measured acidic phospholipase eight 2 activity using Molecular Probes). That is, a methanol solution of fluorescent phospholipid (final concentration: 0.9 M) is added as a substrate to a reaction buffer (50 sodium citrate, PH45), and a tissue lysate protein (final concentration: 10 ig / ml) is added. The total volume was set at 2001.
- transylceramide synthase The activity of transylceramide synthase in liver, kidney and brain tissues prepared from L L PL / Apo E double-deficient mice was measured.
- Reaction buffer i sodium 50 ⁇ Kuen acid, pH 4.5
- phosphatidylcholine 70 mol %
- dicetylphosphate 30 mo 1%)
- ⁇ acetyl-sphingosine final concentration: 20 M
- each tissue lysate protein final concentration: 100 ig / ml
- a preventive and / or therapeutic agent for various diseases caused by LLPL deficiency can be efficiently screened.
- the non-human animal deficient in LLPL expression of the present invention lacks various biological activities that can be induced by LLPL, and thus can serve as a model for a disease caused by LLPL deficiency. Therefore, according to this model, various symptoms caused by LLPL deficiency, For example, it is useful for screening of therapeutic and / or therapeutic agents for diseases caused by apoptosis, for investigating the causes of LLPL-related diseases, and for examining treatment methods.
- the compound obtained by the screening method of the present invention is useful as an agent for preventing and / or treating a disease caused by apoptosis.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2003221402A AU2003221402A1 (en) | 2002-03-18 | 2003-03-17 | Llpl gene and novel use of its gene product |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2002-074180 | 2002-03-18 | ||
JP2002074180 | 2002-03-18 | ||
JP2002314098 | 2002-10-29 | ||
JP2002-314098 | 2002-10-29 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2003078624A1 true WO2003078624A1 (fr) | 2003-09-25 |
Family
ID=28043749
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2003/003136 WO2003078624A1 (fr) | 2002-03-18 | 2003-03-17 | Gene llpl et nouvelle utilisation de ses produits geniques |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU2003221402A1 (fr) |
WO (1) | WO2003078624A1 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1391506A1 (fr) * | 2001-05-22 | 2004-02-25 | Takeda Chemical Industries, Ltd. | Animal avec hypoexpression genetique |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998046767A1 (fr) * | 1997-04-11 | 1998-10-22 | Takeda Chemical Industries, Ltd. | Proteines presentant une activite de lecithine-cholesterol semblable a acyltransferase, leur preparation et leur utilisation |
WO2002102998A1 (fr) * | 2001-05-22 | 2002-12-27 | Takeda Chemical Industries, Ltd. | Animal avec hypoexpression genetique |
-
2003
- 2003-03-17 WO PCT/JP2003/003136 patent/WO2003078624A1/fr active Application Filing
- 2003-03-17 AU AU2003221402A patent/AU2003221402A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998046767A1 (fr) * | 1997-04-11 | 1998-10-22 | Takeda Chemical Industries, Ltd. | Proteines presentant une activite de lecithine-cholesterol semblable a acyltransferase, leur preparation et leur utilisation |
WO2002102998A1 (fr) * | 2001-05-22 | 2002-12-27 | Takeda Chemical Industries, Ltd. | Animal avec hypoexpression genetique |
Non-Patent Citations (2)
Title |
---|
HIRAOKA M. ET AL.: "Cloning and characterization of a lysosomal phospholipase A2, 1-O-acylceramide synthase", J. BIOL. CHEM., vol. 277, no. 12, 22 March 2002 (2002-03-22), pages 10090 - 10099, XP002967695 * |
TANIYAMA Y. ET AL.: "Cloning and expression of a novel lysophospholipase which structurally resembles lecithin cholesterol acyltransferase", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 257, no. 1, 1999, pages 50 - 56, XP002161572 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1391506A1 (fr) * | 2001-05-22 | 2004-02-25 | Takeda Chemical Industries, Ltd. | Animal avec hypoexpression genetique |
EP1391506A4 (fr) * | 2001-05-22 | 2005-03-09 | Takeda Pharmaceutical | Animal avec hypoexpression genetique |
Also Published As
Publication number | Publication date |
---|---|
AU2003221402A1 (en) | 2003-09-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2004048565A1 (fr) | Proteine associee a l'apoptose et son utilisation | |
WO2003078624A1 (fr) | Gene llpl et nouvelle utilisation de ses produits geniques | |
JP4451158B2 (ja) | 転写制御シスエレメント及びそれに特異的に結合する転写調節因子並びにそれらの用途 | |
WO2001000799A1 (fr) | Nouvelle proteine et son adn | |
JP2004205480A (ja) | Llpl遺伝子およびその遺伝子産物の新規用途 | |
JP4488720B2 (ja) | アポトーシス関連蛋白質およびその用途 | |
JP2004075569A (ja) | Il20受容体およびil20の新規用途 | |
WO2004039405A1 (fr) | Utilisation de l'homologue de sglt | |
US20070032643A1 (en) | Cis-element regulating transcription, transcriptional regulatory factor binding specifically thereto and use of the same | |
EP1391506A1 (fr) | Animal avec hypoexpression genetique | |
JP4300008B2 (ja) | 新規タンパク質およびそのdna | |
WO2004078208A1 (fr) | Utilisation medicale d'un inhibiteur de mip-3$g(a) et procede de criblage d'un agent protecteur d'une cellule du cerveau/nerveuse | |
JP2004203868A (ja) | Edg受容体の新規用途 | |
JP2004026692A (ja) | Mepeの新規用途 | |
JP2001228146A (ja) | 疾患関連遺伝子の用途 | |
WO2003054190A1 (fr) | Nouvelles proteines et adn correspondants | |
WO2004014414A1 (fr) | Nouvelles utilisations de rfrp et de ot7t022 | |
JP2003189878A (ja) | 新規タンパク質およびそのdna | |
JP2003000272A (ja) | 新規タンパク質およびそのdna | |
JP2004131471A (ja) | Rfrpおよびot7t022の新規用途 | |
WO2002033082A1 (fr) | Nouveau gene associe a des maladies et son utilisation | |
JP2001048803A (ja) | 医 薬 | |
EP1308512A1 (fr) | Nouvelles proteines et adn correspondants | |
JP2004166691A (ja) | Hi7t213遺伝子導入動物および新規なhi7t213タンパク質およびそのdna | |
WO2002036772A1 (fr) | Nouveau gene associe a une maladie et son utilisation |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: JP |