WO2001021775A1 - Gene encoding novel human secretory phospholipase a¿2? - Google Patents
Gene encoding novel human secretory phospholipase a¿2? Download PDFInfo
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- WO2001021775A1 WO2001021775A1 PCT/JP2000/006344 JP0006344W WO0121775A1 WO 2001021775 A1 WO2001021775 A1 WO 2001021775A1 JP 0006344 W JP0006344 W JP 0006344W WO 0121775 A1 WO0121775 A1 WO 0121775A1
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- C—CHEMISTRY; METALLURGY
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- 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)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to a human secretory phospholipase; a DM encoding the protein of the present invention; an expression vector containing the DNA of the present invention; a transformant having the expression vector of the present invention; A method for producing human secretory phospholipase 2 ; an antibody that specifically recognizes the protein of the present invention; a method for screening a compound using the protein of the present invention; a compound obtained by the screening method of the present invention About. Background art
- Phospholipase 2 (PLA 2 ; EC 3.1.1.4) is a general term for phospholipidases that hydrolyze the 2-acyl ester bond of 3-sn-phosphoglyceride.
- PLA 2 is involved in the digestion of phospholipids in food, the generation and metabolism of biological membrane phospholipids, and also functions as an initiator enzyme of arachidonic acid cascade, which leads to the production of lipid mediators such as prostaglandin.
- PLA family one is a PLA 2 molecular groups of the low molecular weight secreted extracellularly (13, 000 ⁇ 15,000), IB type, IIA type, IID type, IIC-type, V-type,
- six types of X type are known. Each molecular species has 12 to 16 Cys residues in its structure, forms an intramolecular disulfide bond, and has a conserved active center site consisting of His-Asp residues.
- they have a common Ca "binding region, Requires Ca on the order of mM (Tischfield et al., J. Biol. Chen. 272, 17247-17250 (1997), Cupillard et al., J. Biol. Chem. 272, 15745-15752 (1997), and Ishizaki et al. J. Biol. Chem. 274, 24973-24979 (1999)).
- IB type not only functions as a digestive enzyme in the brain and the like, but also participates in the formation of inflammatory conditions such as endotoxin shock through binding to its specific receptor.
- type IIA is present in platelets and synovial cells, etc., and its expression is enhanced by stimulation of inflammatory cytokines. Therefore, it is considered that it is involved in various inflammatory reactions.
- Type V is expressed on inflammatory cells along with the heart.
- Type X is also expressed in immune-related tissues such as spleen and thymus. Both have been suggested to be involved in host defense and inflammatory responses, but their significance in vivo has not been fully elucidated (Hanazaki et al., Cell Engineering, 17, 694-701 (1998)). Disclosure of the invention
- An object of the present invention is to provide a novel human secretory phospholipase; MA encoding the protein of the present invention; an expression vector containing the DNA of the present invention; a transformant having the expression vector of the present invention; A method for producing human secretory phospholipase 2 using the transformant; an antibody that specifically recognizes the protein of the present invention; a method of screening a compound using the protein of the present invention; a screening method of the present invention To provide a compound obtained by the method.
- the present inventors have conducted intensive studies with the aim of elucidating the physiological function of mouse X-type PLA; but in the process, the mouse X-type PLA; A partial sequence homologous to PLA 2 was found, and based on that sequence, an MA sequence encoding a novel secreted PLA 2 protein from the mouse spleen cDNA library was found. Further we found a DNA sequence encoding the human small intestine cMA library from human secreted PLA 2 proteins (IIE type) and the nucleotide sequence based on, and have you completed the present invention. That is, the present invention
- (II) a step of culturing the transformant according to (9) or (10), and culturing the protein according to any of (1), (2) or (3) produced above;
- a method for producing the protein comprising a step of recovering from the medium; (12) an antibody that specifically recognizes the protein according to any of (1), (2) or (3) above;
- a diagnostic agent for a secretory phospholipase 2- related disease which comprises the antibody according to (12) above;
- a kit for measuring secretory phospholipase which comprises the antibody according to (12) above;
- a secretory phospholipase comprising the antibody according to (12) ; a therapeutic agent for a related disease;
- a ring is carbocyclic ring which may be substituted, or substituted optionally heterocyclic ring which may;
- Ri is, -COOH, one S0 3 H, or one PO (OH) 2;
- R2 is one COCONH 2, -CH2CONH2, or one CH2CONHNH2;
- n is an integer from 0 to 6)
- the protein of the present invention is "a protein containing an amino acid sequence from Asn at position 1 of SEQ ID NO: 30 to Cys at position 123", and "Met at position 19 of SEQ ID NO: 30". From 1 Proteins containing amino acid sequences up to the Cys at position 23 "are also included.
- the protein of the present invention includes "a protein having an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted or added in the amino acid sequence, and having phospholipase! Activity".
- phospholipase! Activity means “phospholipid degrading activity of hydrolyzing the 2-acyl ester bond of 3-sn-phosphoglyceride in a Ca 2+ -dependent manner”.
- the protein of the present invention includes proteins encoded by all the modified DNAs having the above-mentioned properties irrespective of the cause and means of such modification and mutation.
- the DNA of the present invention refers to “DNA encoding the protein of the present invention”.
- Preferred examples of the DNA of the present invention include a DNA comprising the nucleotide sequence from A at position 116 to C at position 484 in SEQ ID NO: 29, which encodes the mature protein of the present invention.
- MAs that hybridize with the DNA of the present invention under stringent conditions and encode proteins having phospholipase activity are also included in the MA of the present invention.
- “MA that hybridizes to DNA under stringent conditions” can be obtained by using DNA in the coding region as a probe. I can do it.
- hybridize under stringent conditions means, for example, after heating at 42 ° C in a solution of 6 x SSC, 0.5% SDS and 50% formamide, 0.1 x SSC This indicates that a positive hybridizing signal was still observed under the conditions of washing at 68 ° C. in a solution of 0.5 SDS.
- Production of a recombinant protein using the DNA of the present invention can be carried out, for example, based on many textbooks and documents such as Molecular Cloning. Specifically, a translation start codon is added upstream of the DNA to be expressed, and a translation stop codon is added downstream.
- control gene such as a promoter sequence that controls transcription (eg, trp, lac, T7, SV40 early promoter) is added, and an appropriate vector (eg, pBR322, pUC19, pSV • SP0RT1) is added. )
- a control gene such as a promoter sequence that controls transcription (eg, trp, lac, T7, SV40 early promoter) is added, and an appropriate vector (eg, pBR322, pUC19, pSV • SP0RT1) is added. )
- an appropriate vector eg, pBR322, pUC19, pSV • SP0RT1
- the expression plasmid is introduced into a suitable host cell to obtain a transformant.
- the host cell include prokaryotes such as Escherichia coli, unicellular eukaryotes such as yeast, and cells of multicellular organisms such as insects and mammals.
- it is a mammalian cell, and examples of the mammalian cell include a CH0 cell, a 2993 cell, and a C0S-7 cell.
- the antibody of the present invention is an antibody against a polypeptide fragment or a peptide fragment capable of constituting the peptide of the present invention, and includes both a monoclonal antibody and a polyclonal antibody.
- Secreted PLA 2 is involved in the release of fatty acids (eg, arachidonic acid). Excessive fatty acid release causes diseases such as septic shock, adult respiratory distress syndrome, inflammation, trauma, bronchial asthma, allergic rhinitis, and rheumatoid arthritis. Thus, by measuring the concentration of secreted PLA 2, diagnosis of these diseases is possible.
- the antibody of the present invention provides a diagnostic agent and a measurement kit for secretory phospholipase-related disease. Further, when the antibody of the present invention inhibit the activity of PLA 2, the therapeutic agent for a disease to which an antibody itself attributable to PLA 2.
- the present invention relates to IIE phospholipase A 2 inhibitor which a compound obtainable from the screening method of the present invention as an active ingredient.
- the compound has structural features represented by the following general formula (I). (Where
- Ring A is an optionally substituted carbocycle, or an optionally substituted heterocycle
- R2 is one COCONH 2, one CH 2 CONH 2, or one CH2CONHNH2;
- n is an integer from 0 to 6)
- the term "carbocycle” refers to a saturated or unsaturated, substituted or unsubstituted ring-forming atom other than a hydrogen atom, which is a carbon atom other than a hydrogen atom.
- a membered ring preferably a 5- to 10-membered ring, more preferably a 5- to 7-membered organic skeleton. Also included are those in which two or three carbon rings are continuous.
- carbocycles include cycloalkanes (eg, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane), cycloalkenes (cyclobutene, cyclopentene, cyclohexene, cycloheptene, and cyclooptene) Benzene, naphthalene, indene, phenanthrene, and anthracene.
- cycloalkanes eg, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, and cyclooctane
- cycloalkenes cyclobutene, cyclopentene, cyclohexene, cycloheptene, and cyclooptene
- Benzene naphthalene, indene
- heterocycle is monocyclic or polycyclic, is saturated or unsaturated, and is one to three selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom.
- substituents in the “optionally substituted carbocycle” and the “optionally substituted heterocycle” include alkyl, haloalkyl, alkenyl, alkynyl, alkyl, carboxy, and halogen (F, Cl, Br, I ), Hydroxyalkyl, hydroxy, nitro, cyano, mercapto, thioformyl, thioacetyl, thiocarpoxy, dithiocarboxy, thiocarpamoyl, sulfino, sulfo, sulfamoyl, sulfamoyl, optionally substituted aryl, substituted Aralkyl which may be substituted, heteroaryl which may be substituted, and heteroarylalkyl which may be substituted. These substituents can be substituted at any of the substitutable 1-4 positions on the ring-constituting atoms of the carbocycle and heterocycle.
- Alkyl means a straight or branched C1-C8 alkyl or C3-C8 cyclic alkyl. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, cyclopropyl, cyclobutyl, cyclopentyl And cyclohexyl, cycloheptyl and the like.
- Haloalkyl means a straight or branched C 1 -C 8 haloalkyl or a C 3 -C 8 cyclic haloalkyl substituted with one or more halogens. For example, chloromethyl, fluoromethyl, trifluoromethyl, 2,2,2—Trifluoroethyl, 1,1 difluoroethyl, trichloromethyl, 2,2,2—Trichloroethyl, 1,1 dichloroethyl and the like.
- Alkenyl means a straight or branched C 2 -C 8 alkenyl or C 3 -C 8 cyclic alkenyl having one or more double bonds. For example, vinyl, 1-propenyl, 2-propenyl, isopropyl, 2-cyclobutene-11-yl, 2-cyclopentene-1-yl, 3-cyclopentene-1-yl, 2-cyclo Hexene-1-yl and the like.
- Alkynyl means a straight or branched C2-C8 alkynyl or C3-C8 cyclic alkynyl having one or more triple bonds. For example, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl and the like can be mentioned.
- Halogen refers to fluorine, chlorine, bromine and iodine.
- Aryl means a C 6 to C 14 aromatic monocyclic or fused aromatic ring, for example, phenyl, naphthyl (eg, 1-naphthyl, 2-naphthyl), anthryl (eg, 1-naphthyl) 1-anthryl, 2-anthryl, 91-anthryl) and the like.
- the “aralkyl” includes those in which the above “alkyl” has been substituted by one or more of the above “alkyl”, and these may be substituted at all possible positions.
- benzyl, phenylethyl eg, 2-phenylethyl
- phenylpropyl eg, 3-phenylpropyl
- naphthylmethyl eg, 1-naphthylmethyl, 2-naphthylmethyl
- anthrylmethyl eg, 2-naphthylmethyl
- 9.1 anthryl methyl etc.
- benzyl and phenylethyl are exemplified.
- heteroaryl includes a 5- to 6-membered aromatic ring optionally containing one or more oxygen, sulfur or nitrogen atoms in the ring.
- Heteroarylalkyl includes those in which one or more of the above “heteroaryl” are substituted at any position of the above “alkyl”, and these are all possible Position.
- chenylmethyl for example, 2-phenylmethyl
- chenylethyl for example, 2- (thiophene-2-yl) ethyl
- furylmethyl for example, 2-furylmethyl
- furylethyl for example, 2- (furan-2-y) Le
- pyrrolylmethyl for example, 2-pyrrolylmethyl
- pyrrolylethyl for example, 2— (pyrroyl-2-yl) ethyl
- imidazolylmethyl for example, 2-imidazolylmethyl, 4-imidazolylmethyl
- Imidazolylethyl for example, 2- (imidazoyl-2-yl) ethyl
- pyrazolylmethyl for example, 3—virazolylmethyl
- pyrazolylmethyl for example, 2-
- substituents in the “optionally substituted aryl”, the “optionally substituted aralkyl”, the “optionally substituted heteroaryl” and the “optionally substituted heteroarylalkyl” include: Alkyl, haloalkyl, alkenyl, alkynyl, alkyl, carboxy, halogen (F, Cl, Br, I), hydroxyalkyl, hydroxy, nitro, cyano, mercapto, thioformyl, thioacetyl, thiocarboxy, dithiocarboxy Thiocarbamoyl, sulfino, sulfo, sulfamoyl, sulfoamino and the like.
- FIG. 1 shows the relative positions of the oligomers of SEQ ID NOs: 2, 3, 4, and 5. It is a schematic diagram.
- Figure 2 is a transformant having genes encoding human PLA 2 of the present invention, in cells of transformant having no PLA 2 (Lysate) and PLA 2 activity in the cell supernatant (Supernatant)
- FIG. 1 A transformant having genes encoding human PLA 2 of the present invention, in cells of transformant having no PLA 2 (Lysate) and PLA 2 activity in the cell supernatant (Supernatant)
- FIG. 1 A transformant having genes encoding human PLA 2 of the present invention, in cells of transformant having no PLA 2 (Lysate) and PLA 2 activity in the cell supernatant (Supernatant)
- PLA of culture Youe brass of transformants having the gene co one de a human secretory PLAi of the present invention; illustrates a Ca "dependent activity.
- Figure 4 is a diagram showing the inhibitory activity of the secretory PLA 2 containing the present invention secreted PLA 2 by Indoxam. BEST MODE FOR CARRYING OUT THE INVENTION
- the present invention relates mainly to novel human secretory phospholipases.
- the DNA of the present invention can be easily produced and obtained by general genetic engineering techniques based on the sequence information taught by the present invention (see, for example, Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989)). . Specifically, a cDNA library is prepared from an appropriate source expressing the DNA of the present invention in accordance with a conventional method, and a desired clone is cloned from the library using an appropriate probe or antibody specific to the DNA of the present invention. Selection can be performed (see Pro Natl. Acad. Sci., USA., 78, 6613 (1981); Science, 22, 778 (1983), etc.). The source of cDNA includes various cells expressing the DNA of the present invention, Examples include tissues and cultured cells derived therefrom.
- cDNA libraries are commercially available, and in the present invention, those cMA libraries, for example, various cDNA libraries available from Clontech can also be used.
- the method for screening the DNA of the present invention from the cDNA library is not particularly limited, either, and any method can be used. Specifically, for example, for a protein produced by cDNA, a method for selecting a corresponding cDNA clone by immunoscreening using a protein-specific fang, selectively for a target DNA sequence Examples include plaque hybridization, colony hybridization using a probe to be bound, and combinations thereof.
- DNAs chemically synthesized based on information on the nucleotide sequence of the DNA of the present invention can be generally exemplified, but of course, the MA itself and fragments of the present invention which have already been obtained can also be preferably used. Available.
- the sense primer and antisense primer set based on the nucleotide sequence information of the DNA of the present invention can also be used as screening probes.
- the PCR method (Science, 230, 1350 (1985) )))can also be used preferably.
- the primer used when employing such a PCR method can be appropriately set based on the sequence information of the DNA of the present invention which has already been revealed by the present invention.
- a primer can be synthesized according to a conventional method.
- Isolation and purification of the amplified DNA / RNA fragment can be performed according to a conventional method as described above. For example, it can be performed by a gel electrophoresis method or the like.
- the DNA or various fragments of the present invention obtained above can be obtained by a conventional method such as the dideoxy method (Proc.
- Such a fragment can be isolated, for example, from a primer prepared based on the nucleotide sequence of the EST database by PCR using a mouse tissue-derived cDNA sample as type III. Obtained cDNA fragment (SEQ ID NO: 1) was used as a probe for Northern analysis. As a result, it was found that the gene was highly expressed in the mouse heart.
- the sequence of the upstream and downstream portions of the cMA, including the 5 'and 3' ends, is, for example, an adapter DNA consisting of a specific sequence at the 5 'and 3' ends in advance.
- the cDNA sample which is added in the ⁇ , PCR can be obtained by performing at O Li sesame one and PLA 2 Origoma one pair one specific for cDNA fragments corresponding to the specific sequence (apid mpl ifi cation of cDNA End: RACE method).
- a mouse spleen-derived Marathon-ready cDNA (Clontech) was used as a cDNA sample.
- SEQ ID NO: 14 and SEQ ID NO: 15 in the sequence listing show that the mouse secreted form obtained in this manner?
- the sequence of the DNA having the 1 ⁇ 2 protein gene and the amino acid sequence encoded thereby are shown.
- cDNA sequence of the mouse of the present invention I IA type, since it has been found that a high homology with I ID type, human secretory PLA 2 gene also I IA forms of the invention, the I ID type High homology can be expected.
- oligomers were designed based on the consensus sequences stored in these PLAs; and PCR was performed using human Genomic DNA (BOEHRINGER MANNHEIM) as type III. , Corresponding hit The partial sequence of secreted PLA2 was cloned.
- the protein of the present invention can be obtained by a genetic engineering technique (Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sci., USA., 80, 5990 (1983)). More specifically, a gene encoding a desired protein is inserted into an appropriate vector. The vector is introduced into a host cell to prepare a transformant. By culturing the transformant, a recombinant protein can be obtained.
- the eukaryotic cells include cells such as vertebrates and yeast.
- the vertebrate cells include COS cells (Cell, 23, 175 (1981)) which are monkey cells and Chinese vertebrate cells. ⁇ Hamster ovary cells are frequently used.
- An expression vector is usually a promoter located upstream of the gene to be expressed. Every night, those having an RNA splice site, polyadenylation site, transcription termination sequence and the like can be used, and may further have an origin of replication if necessary.
- An example of the expression vector is pSV2dhfr (Mol. Cell. Biol., 1, 854 (1981)), which has an initial promoter of SV40.
- yeasts are generally used, and Saccharomyces yeasts can be used.
- the base Kuta one expression of the yeast, for example, promoter for the acid phosphatase Ichize gene - evening - ().
- Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts.
- a plasmid vector that can replicate in the host bacterium is used, and a promoter and an SD sequence, and a promoter sequence upstream of the gene, so that the desired gene can be expressed during the reaction.
- E. coli K12 strain or the like is used as the above host.
- pBR322 and its improved vector are often used as vectors, but not limited thereto, and various known strains and vectors can also be used.
- the promoter for example, a trp promoter, an lpp promoter, an lac promoter, a PL / PR open motor, etc. can be used.
- various general methods can be adopted.
- the obtained transformant can be cultured according to a conventional method, and the desired protein is produced by the culture.
- the medium used for the culture various types commonly used depending on the host cell can be appropriately selected and used, and the culture can be performed under conditions suitable for the host cell.
- recombinant secreted PLA protein can be produced.
- Recombinant proteins are subjected to various separation operations utilizing their physical and chemical properties (Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987), etc.). Can be separated and purified.
- Such methods include salting out, centrifugation, osmotic shock, sonication, ultrafiltration, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, and high-performance liquid chromatography.
- Examples include first-class liquid chromatography, dialysis, and combinations thereof.
- the amino acid sequence is replaced by deleting any amino acid sequence and introducing a desired amino acid or amino acid sequence.
- a method known as protein engineering can be widely used for the substitution treatment of the amino acid sequence.
- Site-diredted deletion (Site-specific deletion) method (Nucl. Acids Res., 11, 1645, 1983)
- Site-specific mutagenesis (site-specific mutation) method (Zoller, MJ et al., Methods in Enzymol., 100, 468, 1983, Kunkel. TA et al., Methods in Enzymol., 154, 367-382, 1987)
- PCR Methods include mutagenesis, restriction enzyme treatment and the use of synthetic genes.
- site-directed mutagenesis for example, site-directed mutagenesis described in Molecuar Cloning: A Laboratory Manual, 2nd Edition, Volume 1-3, Sambrook, J. et al., Cold Spring Harber Laboratory Press, New York, 1989
- a mutation is introduced into the DNA sequence of the present invention by a method such as the PCR method or the PCR method.
- the A sequence mutated by these methods can be used, for example, by using an appropriate vector and host system, for example, Molecuar Cloning: A Laboratory Manual, 2nd edition, Vol. It may be expressed genetically by the method described in Sambrook, J. et al., Cold Spring Harber Laboratory Press, New York, 1989.
- kits such as Mutan TM -SuperExpress Km, Mutan TM -K (Takara Shuzo) and Quik Change Site-Directed Mutagenesis Kit (Stratagene) can be used.
- site-directed mutagenesis can be performed by first obtaining a single-stranded vector containing a DNA sequence encoding a protein in its sequence.
- Oligonucleotide primers having the desired mutagenized sequence are generally synthesized, for example, by Claire et al. (Crea, R. et al., Proc. Natl. Acsd. Sci. USA, 75, 5765, 1978).
- this primer is aligned with a single-stranded vector containing the present DNA sequence, and a DNA polymerase such as Escherichia coli polymerase I Klenow fragment is allowed to act on the primer to synthesize the mutation-containing strand.
- a DNA polymerase such as Escherichia coli polymerase I Klenow fragment
- the first strand encodes the original non-mutated sequence and the second strand forms a heteroduplex with the desired mutation.
- the double-stranded vector is then used to transform appropriate bacteria or cells and cloned via hybridization to a radioactive probe consisting of a "P-labeled mutagenic primer.” Select (Wallace, RB, Nucleic Acids Res., 9, 3647, 1981)
- the selected clone contains a recombinant vector having a mutated sequence. After that, the mutated region of the protein can be placed in an expression vector of the type used for transformation.
- a mutant can be produced in a host cell according to the method for preparing a recombinant protein described in (1).
- the antibody against the protein of the present invention is produced by the following method.
- the produced protein is purified by a usual protein chemical method, and these are used as an immunogen.
- the immunogen is used to immunize animals by an appropriate method according to the method described in Antibodies; A Laboratory Manual, edited by Lane, HD et al., Cold Spring Harber Laboratory Press, New York, 1989.
- a polyclonal antibody that specifically recognizes a protein can be easily prepared and purified. Animals such as mice, rats, hamsters, and egrets can be immunized to produce polyclonal antibodies derived from their sera.
- Lymphocytes are extracted from the spleen or lymph node of mouse rats immunized with the above-mentioned immunogen, fused with myeloma cells, and Kohler and Milstein's method (Nature, 256, 495-497 (1975))
- a monoclonal antibody can be produced from the hybridoma.
- a monoclonal antibody of the present protein can be obtained by the following steps:
- the protein of the present invention can be measured using a monoclonal antibody against the protein of the present invention.
- the assay using the antibody against the protein of the present invention measures the amount of the antibody, antigen or antibody-antigen complex corresponding to the amount of the antigen (eg, the amount of the protein of the present invention) in the test solution by chemical or physical means. Any measurement method may be used as long as it is detected and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition, immunometric, and sandwich techniques can be used.
- a chemical bond usually used for immobilizing a protein or an enzyme can be used.
- the carrier insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used.
- the amount of the protein of the present invention in the liquid to be measured can be measured by the following steps. (1) The monoclonal antibody of the present invention which has been immobilized is reacted with the liquid to be measured, and another labeled monoclonal antibody of the present invention is reacted.
- the immobilized antibody and the labeled antibody need not be the same antibody.
- the immobilized antibody is an antibody that recognizes the N-terminus of the present protein
- an antibody that recognizes the C-terminus can be used as the labeled antibody.
- Radiolabels, enzymes, and fluorescent substances are used as labeling agents. Radioisotopes, such as m I, 3 H, 1 4 C is used.
- enzymes peroxidase, 5-galactosidase,? -Glucosidase, alkaline phosphatase and the like are used.
- this protein can be diagnosed as a disease such as septic shock, adult respiratory distress syndrome, inflammation, trauma, bronchial asthma, allergic rhinitis, rheumatoid arthritis, etc. Can be.
- the antibody of the present invention provides a “diagnostic agent” and a “measurement kit” that enable such a diagnosis.
- the antibodies of the present invention are useful for treating these diseases.
- the antibodies of the present invention also provide "therapeutic agents.”
- examples of the “screening method” include high-throughput screening using the protein of the present invention.
- a sample and a phospholipid substrate (racemic diheptanoyl thio-phosphatidylcholine) and a coloring reagent (5,5'- Add dithiobis (2-nitrobenzoic acid)).
- a coloring reagent (5,5'- Add dithiobis (2-nitrobenzoic acid)
- Add this protein incubate at 40 ° C for an appropriate time, and measure the change in absorbance (OD: 405 nm). By comparing with the value without the sample added, the The inhibitory activity on PLA2 can be evaluated.
- Secreted PLA 2 is involved in the release of fatty acids (eg, arachidonic acid). Excessive fatty acid release causes diseases such as septic shock, adult dyspnea syndrome, inflammation, trauma, bronchial asthma, allergic rhinitis, and rheumatoid arthritis. Compounds obtained by disk re-learning method of the present invention inhibit the activity of secreted PLA 2. Therefore, the compound is useful for treating septic shock caused by excessive secretory PLA2 production.
- fatty acids eg, arachidonic acid
- the compounds also include pharmaceutically acceptable salts.
- Such salts include, for example, non-toxic alkali metal salts such as sodium, potassium, lithium, calcium, magnesium, barium, ammonium and the like, alkaline earth metal salts and ammonium salts, which are prepared by well-known methods.
- a pharmaceutical formulation is prepared using a pharmaceutically effective amount of the compound as the active ingredient.
- various forms can be selected according to the purpose of treatment, and typical examples are solid dosage forms such as tablets, pills, powders, powders, granules, capsules and the like.
- Dosage forms and liquid dosage forms such as solutions, suspensions, emulsions, syrups and elixirs. These are further classified into oral preparations, parenteral preparations, nasal preparations, vaginal preparations, suppositories, sublingual preparations, ointments, etc. according to the route of administration, and can be prepared, formed or prepared according to the usual methods. can do.
- the administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease, and the like.
- solid dosage forms such as tablets, pills, granules and capsules, and liquids, suspensions and emulsions are orally administered.
- Injectables are administered intravenously, alone or in admixture with ordinary replenishers such as glucose and amino acids. (Injections are also administered alone, if necessary, intramuscularly, intradermally, subcutaneously, or intraperitoneally.
- the amount of the active ingredient of the compound of the present invention to be contained in the above pharmaceutical preparation and the dose thereof are not particularly limited, and are appropriately determined according to the desired therapeutic effect, administration method, treatment period, patient age, sex, and other conditions. Selected. In general, the dose is preferably about l to 10 mg per kg of body weight per day, and the preparation can be administered once or several times a day.
- the antibody of the present invention inhibits the PLA 2 activity of the secreted PLA 2 of the present invention by specifically binding to the secreted PLA 2 of the present invention.
- antibodies of the present invention, as well as the compounds obtained by the scan click re-learning method of the present invention are useful in the treatment of such septic shock caused by the production of excess secreted PLA 2.
- cDNA samples were prepared by reverse transcription of mRNA extracted from tissues of 129 / SvJ strain mice (heart, kidney, lung). PCR was performed on this cDNA sample using type I oligonucleotides of SEQ ID NOS: 2 and 3.
- the enzyme used was TaKaRa Ex Taq from Takara Shuzo Co., Ltd., and the reaction composition was in accordance with the instruction manual for this enzyme.
- the amplification equipment used was a Thermal Cycler from Perkin Elmer Cetus, and amplification was performed at 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1 minute in 30 cycles.
- a second PCR was carried out with the oligos of SEQ ID NOS: 4 and 5 using type 11 as the type I reaction solution.
- the reaction conditions are the same as the first.
- the PCR reaction solution was subjected to 2% agarose gel electrophoresis to separate amplification products.
- DNA was amplified to the expected size from cDNAs from all examined organs. This DNA fragment was cut out from the gel, and the DNA was purified using a GenElute Agarose spin column manufactured by Spelco. This DNA was ligated with Invitrogen vector pCRI I. After ligation, Escherichia coli DH5 ⁇ strain (T0Y0B0) was transformed with the ligation solution.
- Example 3 Determination of the Primary Structure of Mouse Secreted PLA: cDNA
- the upstream and downstream portions of the cDNA can be isolated by the following method.
- Several antisense oligomers and sense oligomers are prepared based on the known sequence.
- As cDNA samples previously the 5, and what is the adapter one A comprising a specific sequence is added at the end 3 'end to ⁇ , Origoma one and PLA 2 specific oligomer corresponding to this specific sequence
- RACE method Marathon-ready cDNA (Mouse spleen) from Clontech was used as a cMA sample.
- SEQ ID NOs: 6 to 9 Sequences used in the RACE method are shown in SEQ ID NOs: 6 to 9. Of these, SEQ ID NOs: 6 and 7 are 5, antisense oligomers for isolating the upstream region, and SEQ ID NOs: 8 and 9 are senseoligomers for isolating the downstream region.
- the RACE method was also performed by nested PCR similarly to the PCR amplification at the center.
- the oligomer of SEQ ID NO: 6 and the oligomer AP-1 attached to the Clontech cDNA sample were used in the first PCR, and the oligomer of SEQ ID NO: 7 and the cDNA sample (Clontech ) The attached AP-2 was used. 3.
- the oligo sequence of SEQ ID NO: 8 and AP-1 were used the first time, and the oligomer of sequence ID 9 and AP-2 were used the second time.
- the enzyme uses TaKaRa Ex Taq, the first time is 30 cycles of 94 ° C 1 minute, 60 ° C 1 minute, 72 ° C 2 minutes, the second time is 94 ° C 1 minute, 62 ° C Amplification was carried out for 30 minutes at C for 1 minute and at 72 ° C for 2 minutes. After PCR, electrophoresis was performed to separate amplification products. As a result, a band of about 450 base pairs was detected from the 5 side RACE and about 470 base pairs was detected from the 3 side RACE. These MAs were cloned in the same manner as in Example 1, and their nucleotide sequences were analyzed and determined using a PRISM 310 Genetic Analyzer from PE Applied Biosystems.
- the upstream region of the PLA 2 cDNA central, partially isolated three areas downstream zone.
- a second PCR was carried out with oligomers 11 and 13 using 1 n1 of the reaction solution as ⁇ type.
- TaKaRa Ex Taq was used as the enzyme, and amplification was performed at 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes in 30 cycles.
- the amplified MA was cloned in the same manner as above, and its nucleotide sequence was determined. It has been determined The sequence of the cDNA is shown in SEQ ID NO: 14, and the amino acid sequence encoded thereby is shown in SEQ ID NO : 15.o From the sequence encoding the protein, PLA ; the number of amino acid residues in the protein is 1 It turned out to be 4 2. This protein retained all the structural features (PLA ; active center site, Ca "binding site sequence, and number of Cys residues) conserved between the two secretory PLA family molecules.
- this evening protein is composed of a known secreted PLA 2 different sequences, homology at the amino acid level, the mouse type IB and 35,8%, the mouse type IIA and 47.7%, the mouse I IC Type 38.0%, mouse IID type 41.8%, mouse type V 41.0%, mouse type X 35.8%
- Human secretory PLA
- the mouse and human sequences in genes in the same Taib can be expected to have a high homology to each other, cDNA sequences corresponding to the consensus sequence, especially highly homologous with the cDNA sequence of the human type PLA 2
- the mouse cDNA sequence determined in Example 3 is considered to be a region of high value when it is useful to clone a human homologous gene based on the sequence information of the mouse PLA! Gene.
- type since it has been found that a high homology with the IID type, can be predicted corresponding human secretory PLA 2 gene also human type IIA for a high homology with human IID type.
- oligomers were designed based on the mouse cDNA sequence, human ⁇ type, and human IID type consensus sequence portions determined in Example 3. Based on the cDNA sequence corresponding to Tyr at position 23 to Gly at position 28 described in SEQ ID NO: 14 and taking into account the cDNA sequences corresponding to human IIA type and human IID type, respectively.
- An oligomer composed of 16 was prepared. It also consists of SEQ ID NO: 17 corresponding to the sequence from His at position 46 to Arg at position 52 in SEQ ID NO: 14. Rigoma-1 (antisense oligomer) was also prepared.
- PCR was performed at 30 cycles of 94 ° C for 1 minute, 50 ° C for 1 minute, and 72 ° C for 2 minutes, and the other conditions were the same as in Example 1.
- Human Genomi cDNA of BOEHRINGER MANNHE IM was used for type II of PCR.
- DNA fragments of about 800 bp was found to be human I IA type PLA 2
- MA fragment of about 350 base pairs is a human IID type PLA 2. Since these DNA fragments contained one intron sequence at the same position, it can be expected that the new PLAi also contains an int sequence at the same position.
- the nucleotide sequence of a DNA fragment of about 300 base pairs is shown in SEQ ID NO: 18.
- the nucleotide sequence includes I IA type, expected can sequence Intoron of 2 1 2 base pairs in the same position as I ID type PLA 2 (2 6 9 position from the 5 8 of SEQ ID NO: 1 8) There was 85.1% homology between the expected exon sequence and the corresponding region of the mouse cDNA sequence determined in Example 3.
- this fragment could be expected to be a part of the novel human PLA gene.
- three sense oligomers and three antisense oligomers were designed. The sequences of those oligomers are shown in SEQ ID NOS: 191-24.
- nested-PCR was performed using these oligomers, using human cMA derived from various tissues as type III in the same manner as described in Example 1. That is, in the first PCR, the oligomers of SEQ ID NOs: 19 and 22 were used, and the amplified product was designated as ⁇ . In the second PCR, the oligomers of SEQ ID NOs: 20 and 23 were used. Was. In each of the first and second PCR, 30 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1 minute were performed, and the other conditions were the same as in Example 1.
- Clontech® ⁇ Marathon-ready cDNA Human kidney pancreas, small 1 intestine spleen, lung, heart, leukocyte ⁇ liver
- the obtained DNA fragment was cloned in the same manner as in Example 1, and its nucleotide sequence was determined.
- the cDNA sequence was composed of the exon region as expected in the previous section.
- the upstream portion including the 5 'end was isolated by the RACE method.
- SEQ ID NOS: 23 and 24 were used as antisense oligonucleotides for isolating the upstream region. As a result, a DNA fragment of about 120 base pairs was obtained, and the nucleotide sequence was determined.
- Two antisense oligomers SEQ ID NOS: 25 and 26 were designed to further isolate the upstream region from the sequence, and the RACE method was performed in the same manner. As a result, a DNA fragment of about 150 base pairs was obtained, and the nucleotide sequence was determined.
- the downstream portion including the 3 'end was isolated by nested PCR as follows. That is, the nucleotide sequence obtained so far revealed that the novel human PLA ⁇ had a very high homology between the corresponding mouse and PLA ; and thus the mouse PLA ⁇ determined in Example 3 was used. 2 antisense oligonucleotides corresponding to positions 595 to 615, and 601 to 621 based on the cDNA sequence (SEQ ID NO: 14: 7, 28) were prepared and PCR was performed using these oligomers as antisense primers. In the first PCR, the oligomers of SEQ ID NOs: 20 and 28 were used, and the amplified product was designated as type III.
- the oligomers of SEQ ID NOs: 21 and 27 were used.
- the first PCR In the second run, 30 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes were performed under the same conditions as in Example 1.
- the results using approximately 3 0 0 bp DNA fragment was obtained, in the same manner as in Example 1 base The sequence was determined.
- PLA 2 cDNA was partially isolated by dividing it into a central region, two upstream regions, and a downstream region. These nucleotide sequences have continuity, and the sequence obtained by linking them is shown in SEQ ID NO: 29. From the chain length of the portion encoding the protein, it was found that the number of amino acid residues of PLA; protein was 142.
- This protein (SEQ ID NO: 30) contains all of the structural features conserved between single molecules of the secreted PLAi family (PLAi active center and Ca " However, this protein is composed of a sequence different from that of known secreted PLA, and has homology at the amino acid level of 39.0% with human IB and 51.4% with human IIA. The ratio was 39.2% for human IID, 45.7% for human V, and 38.2% for human X.
- Example 5 Expression of recombinant PLA;
- oligosaccharide SEQ ID NOS: 31-34
- Nested PCR Nested PCR to amplify the amino acid coding portion (coding region) in the cDNA sequence.
- For the first PCR use the oligomers shown in SEQ ID NOs: 31 and 33
- for the second PCR use the oligomers shown in SEQ ID NOs: 32 and 34.
- For type II use Clontech's Marathon-ready PCR cDNA (Human Small Intestine) was used.
- the oligomer shown in SEQ ID NO: 32 has a sequence recognized by the restriction enzyme Notl and a sequence that promotes translation of mRNA into protein (Kozak sequence), and the oligomer shown in SEQ ID NO: 34 is recognized by the restriction enzyme Xbal. With an array that is There are no sequences recognized by Notl and Xbal in the coding region. Therefore, digestion of the cDNA fragment amplified by PCR with these restriction enzymes The cDNA corresponding to the coding region contains a Kozak sequence upstream of the translation initiation site, and is prepared as a cDNA having cuts cut by Notl and Xbal at the upstream and downstream ends, respectively.
- PLA 2 expression base Kuta one is, pcDNA 3.1 the cDNA is expressed base Kuta one for mammalian cells (+) (Invitrogen Corp.) NotI so that in the forward direction to the promoter one downstream, was inserted between the Xbal site Created.
- Promo Isseki Re determining the nucleotide sequence of the subjected PLA ⁇ cDNA from scratch, after confirming that the building on artificial mutation is not introduced, is the PLA 2 expression base Kuta one cell cultures from monkey kidney COS -Transfected into 7 cells. For the introduction, FuGENE TM 6 Transfection Reagent from BOEHRINGER MANNHEIM was used, and the procedure was according to the instruction manual.
- PLA ⁇ activity in the culture supernatant and cell lysate was measured.
- Determination of PLA 2 activity the E. coli membrane phospholipid fraction labeled as a substrate in 3 H- Orein acid, Elsbach et al. Method (Methods Enzymol. 197, 24-31 ( 1991)) was carried out according to the method of.
- Rupura Sumi de not insert the PLA 2 cDNA, significantly higher PLA; activity was detected in the culture supernatant, confirming that a secreted PLA 2 ( Figure 2).
- the expression of the enzyme activity a high concentration of Ca "was essential ( Figure 3). Risk re-learning of the compounds to inhibit the Example 6 PLA 2 activity
- Indoxam a 1-oxamoyl indol idine derivative, which has been reported to strongly inhibit the enzymatic activity of type I and type IB secreted PLA ⁇ (Yokota et al., Biochim. Biophys. Acta (1999) 1438, 213-222) was examined for its inhibitory activity on human newly secreted PLA; PLA; activity, Draheim et al. Method (.. J. Med Chem, ( 1996) 39, 5159-5175) analogously to, from lf C-labeled 1-palmi toy 1-2-oleoyl -phosphatidyl choline (POPC) "The measurement was performed by measuring the amount of released C-oleic acid.
- POPC lf C-labeled 1-palmi toy 1-2-oleoyl -phosphatidyl choline
- human secretory PLA 2 DNA encoding human secretory PLA PL ; an expression vector containing the DNA; a transformant having the expression vector; There was method for producing a human secreted PLA 2 is provided.
- secretory PLA 2 of the present invention as well as providing an analysis means diseases secretory PLA 2 is involved, it also of a also provides a search means compounds that specifically inhibit the enzyme.
- the measurement system using the antibody can be applied to diagnosis of various diseases.
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Description
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU73147/00A AU7314700A (en) | 1999-09-21 | 2000-09-18 | Gene encoding novel human secretory phospholipase a2 |
CA002385406A CA2385406A1 (en) | 1999-09-21 | 2000-09-18 | Gene encoding novel human secretory phospholipase a2 |
US10/088,092 US6872557B1 (en) | 1999-09-21 | 2000-09-18 | Gene encoding novel human secretory phospholipase A2 |
EP00961047A EP1223214A4 (en) | 1999-09-21 | 2000-09-18 | GEN ENCODING A NEW HUMANE SECRETORIC PHOSPHOLIPASE A2 |
Applications Claiming Priority (2)
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JP11/266616 | 1999-09-21 | ||
JP26661699 | 1999-09-21 |
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WO2001021775A1 true WO2001021775A1 (en) | 2001-03-29 |
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PCT/JP2000/006344 WO2001021775A1 (en) | 1999-09-21 | 2000-09-18 | Gene encoding novel human secretory phospholipase a¿2? |
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US (1) | US6872557B1 (ja) |
EP (1) | EP1223214A4 (ja) |
AU (1) | AU7314700A (ja) |
CA (1) | CA2385406A1 (ja) |
WO (1) | WO2001021775A1 (ja) |
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AU2010241567B2 (en) | 2009-04-29 | 2013-10-31 | Amarin Pharmaceuticals Ireland Limited | Pharmaceutical compositions comprising EPA and a cardiovascular agent and methods of using the same |
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US5656602A (en) * | 1991-07-04 | 1997-08-12 | Garvan Institute Of Medical Research | PLA2 inhibitory compounds |
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2000
- 2000-09-18 CA CA002385406A patent/CA2385406A1/en not_active Abandoned
- 2000-09-18 EP EP00961047A patent/EP1223214A4/en not_active Withdrawn
- 2000-09-18 US US10/088,092 patent/US6872557B1/en not_active Expired - Fee Related
- 2000-09-18 WO PCT/JP2000/006344 patent/WO2001021775A1/ja not_active Application Discontinuation
- 2000-09-18 AU AU73147/00A patent/AU7314700A/en not_active Abandoned
Non-Patent Citations (3)
Title |
---|
BALSINDE J. ET AL.: "Function and inhibition of intracellular calcium-independent phospholipase A2", J. BIOL. CHEM., vol. 272, no. 26, 1997, pages 16069 - 16072, XP002935872 * |
See also references of EP1223214A4 * |
TISCHFIELD J.A. ET AL.: "A reassessment of the low molecular weight phospholipase A2 gene family in mammals", J. BIOL. CHEM., vol. 272, no. 28, 1997, pages 17247 - 17250, XP002935873 * |
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Publication number | Publication date |
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US6872557B1 (en) | 2005-03-29 |
CA2385406A1 (en) | 2001-03-29 |
EP1223214A4 (en) | 2003-03-26 |
EP1223214A1 (en) | 2002-07-17 |
AU7314700A (en) | 2001-04-24 |
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