WO2003102182A1 - Gene slurp-2 associe a une maladie de peau inflammatoire et utilisation de ce gene - Google Patents
Gene slurp-2 associe a une maladie de peau inflammatoire et utilisation de ce gene Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- 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
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/20—Dermatological disorders
Definitions
- the present invention relates to an inflammatory skin disease-related gene and its use.
- Background Art-Psoriasis vulgaris (MIM 177900) is a chronic inflammatory skin disease with a prevalence of 2-3% in Caucasians (Nevitt, G. J. and Hutchinson, PE (1996). Br. J. Dermatol. 135: 533-538.), 1-2% in the UK and Europe (Hellgren, L. (1967) .Psoriasis: The Prevalence in Sex, Age and Occupational Groups in Total Populations in
- Psoriatic lesions have been suggested to be triggered by antigens z superantigens or autoantigens supplied by noncutaneous inducers (Freedberg, IM, Eisen, AZ, Wolff, K, Goldsmith, LA, Katz, SI , Fitzpatrick, TB. (1998) .FITZPATRIC ' S Dermatology in general medicine, 5th edition, vol. 1, 510.).
- T cells play a pivotal role in the immunopathology of this disease (Gotenden, SL, Gilleaudeau, P., Johnson, R., Estes,
- Psoriasis The Prevalence in Sex, Age, and Occupational Group in Total Populatio n in Sweden: Morphology, Inheritance and Association with Other Skin and
- the susceptibility locus for psoriasis is chromosome 6p (PS0RS1 [MIM 177900]) (Trembath, RC, et al. (1997). Hum Mol. Genet. 6: 813-820., Nair, RP, et al. (1997). Hum. Mol. Genet. 6: 1349-1356 ⁇ , Oka, A. et al. (1999). Hum. Mol. Genet. 8: 2165-2170.), 17q (PS0RS2 [MIM 602723]) (Tomfohrde, J et al. (199 4). Science 264: 1141-1145.) and 4q (PS0RS3 [MIM 601454]) (Matthews,
- Ly-6 superfamily has 8-10 conserved cysteine residues showing a characteristic arrangement pattern, and is uniquely attached to the cell surface via glycosyl-phosphatidylinositol (GPI) -anchor. Ly-6 domain proteins-new insights and new members' ⁇ a C-terminal Ly-6 domain in sperm acrosomal protein SP-10. Tis (Palfree, RG (1996). sue antigens 48: 71-79., "Low, ⁇ . G. (1989). The glycosyl-phosphatidylinositol anchor of membrane proteins. Biochim. Biophy. Acta 988: 427-454.). (Adermann, K., Wattler, F., Wattler, S.
- Ly-6 superfamily members have been first identified in mice (McKenzie, ⁇ . R, Gardiner, J., Cherry, Perry and Snell, GD (1977). Lymphocyte antigens: Ly-4, Ly-6, and Ly-7. Transplant. Proc. 9: 667-669.) Many family proteins have been isolated not only in mice but also in humans. Co-localization of the human CD59 (Bickmore, WA, Longbottom, D., Oghene, k., Fletcher, JM, and van Heyningen, V. (1993). gene to llpl3 with the MICH cell surface antigen. Genomics 17: 129-135.), Perokinase plasminogen activator receptor (uPAR) (Suh, T.
- uPAR Perokinase plasminogen activator receptor
- chromosome 8 Large of that member The segment is found on chromosome 8, especially 8q24.3, which is homologous to mouse chromosome 15; the mouse Ly-6 locus on D-pan (Kamiura, S., Nolan, CM, and Meruel o, D. (1992). Long-range physical map of the Ly-6 complex: mapping the 1 y-6 multigene family by field-inversion and two-dimensional gel electrophoresis. Genomics 12: 89-105. Live Ly—6th / Family! The function of the 5-minute member has not yet been elucidated. Disclosure of the invention
- an object of the present invention is to isolate and identify a novel inflammatory skin disease-related gene and to provide a method for using the gene. More specifically, an object of the present invention is to provide a novel psoriasis recurrent gene and a method of using the same.
- the present inventors performed a comprehensive analysis using microarrays to identify transcriptional changes occurring in the skin of affected and unaffected areas of psoriatic patients with normal controls in order to identify novel psoriasis-related genes.
- 86 ESTs were found to be up-regulated or down-regulated about 10 times or more in psoriatic lesions compared to normal tissues.
- the protein encoded from the cDNA was a novel member of Ly-6 / uPAR superamylase.
- the present inventors named the protein encoded from the cDNA SLURP-2 (secreted Ly-6 / uPAR-related protein-2).
- RT-PCR expression analysis revealed that the SLURP-2 gene was expressed in many tissues, mainly in epithelial tissues.
- the SLURP-2 gene was not detected in bone marrow and spleen, but was detected in thymus.
- SLURP-2 gene was significantly upregulated in psoriatic lesional skin. It was shown that. Therefore,
- the SLURP-2 gene can be a diagnostic marker for inflammatory skin diseases such as psoriasis.
- the present invention relates to an inflammatory skin disease-related gene and its use, more specifically,
- polypeptide comprising the amino acid sequence of SEQ ID NO: 2 with one or more amino acids substituted, deleted, inserted, and / or added, and comprising the amino acid sequence of SEQ ID NO: 2; DNA encoding a polypeptide that is functionally equivalent to the peptide,
- a monoclonal antibody the antibody according to (7), (9) an oligonucleotide comprising at least 15 nucleotides complementary to the DNA or the complementary strand thereof according to (1),
- a method for detecting an inflammatory skin disease comprising the following steps (a) to (c):
- test agent for an inflammatory skin disease comprising the oligonucleotide according to (9), (16) a test agent for an inflammatory skin disease, comprising the antibody according to (7) or (8), [17] The test agent according to [15] or [16], wherein the inflammatory skin disease is psoriasis.
- the present invention provides a novel gene “SLURP-2 (secretory Ly-6ZuPAR-related protein-2) gene”.
- SLURP-2 secretory Ly-6ZuPAR-related protein-2 gene.
- the nucleotide sequence of the cDNA of the human SLURP-2 gene is shown in SEQ ID NO: 1
- amino acid sequence of the polypeptide encoded by the cDNA is shown in SEQ ID NO: 2.
- the SLURP-2 gene was isolated as a gene expressed in affected areas of inflammatory skin diseases. Therefore, the gene can be used as a diagnostic marker for inflammatory skin diseases. The gene is also expected to be applied to treatment and prevention of inflammatory skin diseases.
- the inflammatory skin disease is characterized by infiltration of inflammatory cells such as activated helper T cells and mononuclear cells, and the occurrence of skin lesions caused by epidermal hyperplasia and abnormal keratinocyte differentiation.
- inflammatory skin diseases include, but are not limited to, for example, psoriasis, lichen planus, pityriasis rufosa and palmar pustulosis.
- Psoriasis usually means psoriasis vulgaris, but the psoriasis in the present invention includes not only psoriasis vulgaris but also other psoriasis such as pustular psoriasis and psoriatic arthritis.
- the present invention also includes a DNA encoding a polypeptide functionally equivalent to the polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
- DNAs include, for example, DNAs encoding mutants, alleles, palants, homologs and the like of polypeptides containing the amino acid sequence of SEQ ID NO: 2.
- “functionally equivalent” means that the target polypeptide has a biological function and a biochemical function equivalent to SLURP-2. Further, it is preferable that SLURP-2 in the present invention has a property that its expression is increased in an affected area of an inflammatory skin disease as compared with an unaffected area. Whether or not the polypeptide encoded by the subject DM has similar properties can be measured by a method well known to those skilled in the art.
- the present invention includes DNAs that hybridize under stringent conditions to DNAs encoding SLURP-2 and encode polypeptides that are functionally equivalent to SLURP-2.
- DNAs include, but are not limited to, human, monkey, mouse, rat, septa, and homologues derived from Pseudomonas.
- Hybridization conditions for isolating DNA encoding a polypeptide functionally equivalent to SLURP-2 can be appropriately selected by those skilled in the art.
- Hybridization conditions include, for example, low stringency conditions.
- the low stringent conditions are, for example, conditions of 42 ° C, 5 XSSC, 0.1% SDS, and preferably 50 ° C, 5 XSSC, 0.1% SDS in washing after hybridization.
- More preferable conditions for the hybridization are high stringency conditions.
- Highly stringent conditions are, for example, 65. C, 0.1 X SSC and 0.1% SDS. Under these conditions, it is expected that DNA with higher homology can be efficiently obtained as the temperature is increased.
- multiple factors such as temperature and salt concentration can be considered as factors affecting the stringency of high predication, and those skilled in the art can realize the same stringency by appropriately selecting these factors. It is possible.
- a gene amplification method using a primer synthesized based on the sequence information of DNA (SEQ ID NO: 1) encoding SLURP-2 for example, a polymerase chain reaction (PCR) method is used to generate SLURP-2 and SLURP-2. It is also possible to isolate DNA encoding functionally equivalent polypeptides. -1 o-Polypeptides functionally equivalent to SLURP-2, encoded by DNA isolated by these hybridization and gene amplification techniques, usually have high homology in amino acid sequence with SLURP-2 .
- the polypeptides of the present invention also include polypeptides that are functionally equivalent to SLURP-2 and have high homology to the amino acid sequence of the polypeptide. High homology usually means at least 50% identity, preferably at least 75% identity, more preferably at least 85% identity, and even more preferably at least 95% identity at the amino acid level. Point to.
- the present invention also includes a polypeptide comprising an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of SLURP-2, and functionally equivalent to the polypeptide.
- amino acid mutations can also occur in nature.
- the number of amino acids to be mutated is usually within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids (eg, within 3 amino acids), and even more preferably within 2 amino acids. .
- the amino acid residue to be mutated is desirably mutated to another amino acid that preserves the properties of the amino acid side chain.
- the properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C ;, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, ⁇ , ⁇ ) Amino acids having side chains containing sulfur atoms (C, M); amino acids having side chains containing carboxylic acids and amides (D, N, E, Q); amino acids having side chains containing base groups (R, K, ⁇ ) And amino acids having an aromatic-containing side chain ( ⁇ , F, Y, W) (one parenthesis of each amino acid is shown in parentheses).
- a method of introducing a mutation into a polypeptide is known.
- those skilled in the art can use site-directed mutagenesis (Gotoh, T. et al. (1995) Gene 152, 27 275, Zoller, MJ, and Smith, M, (1983) Methods Enzymo 1.100, 468-500, Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987) Methods.Enzymol. 154, 350-367, Kunkel, TA (1985) Proc Natl Acad Sci USA. 82, 488-492, Kunkel (1988) Methods Enzymol. 85, 2763-2766), etc., to introduce mutations into the SLURP-2 amino acids as appropriate to It is possible to prepare functionally equivalent polypeptides.
- Polypeptides obtained by adding a plurality of amino acid residues to the amino acid sequence of SLURP-2 include fusion polypeptides containing these polypeptides.
- a fusion polypeptide is a fusion of these polypeptides with other polypeptides. included.
- DNA encoding SLURP-2 and DNA encoding another polypeptide are ligated in frame so that they are introduced into an expression vector, and expressed in a host. I just need.
- Other polypeptides to be fused with the polypeptide of the present invention are not particularly limited.
- peptides to be fused to the polypeptide of the present invention include, for example, FL AG (Hopp, T. P. et al., BioTechnology (1988) 6, 1204-1210), 6 His
- Histidine residue consisting of 6XHis, 10XHis, influenza agglutinin (HA), human C-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, SV40T
- HA agglutinin
- human C-myc fragment VSV-GP fragment
- pl8HIV fragment T7-tag
- HSV-tag HSV-tag
- E-tag SV40T
- peptides such as antigen fragments, lck tags, hy-tubulin fragments, B-tags, and Protein C fragments can be used.
- polypeptides to be fused with the polypeptide of the present invention include, for example, GST (daltathione-S-transferase), HA (influenza agglutinin), immunoglobulin constant region, ⁇ -gal Cuctosidase, ⁇ (maltose binding protein) and the like.
- the commercially available DNAs encoding these polypeptides are fused with the polypeptide encoding the polypeptide of the present invention!), And the fusion DM prepared thereby is expressed to thereby form the fusion polypeptide. Can be prepared.
- the DNA of the present invention may be in any form as long as it can encode the polypeptide of the present invention. That is, it does not matter whether it is cDNA synthesized from mRNA, genomic DNA, chemically synthesized DNA, or the like.
- DNAs having an arbitrary nucleotide sequence based on the degeneracy of the genetic code are included as long as they can encode the polypeptide of the present invention.
- the DNA of the present invention can be prepared by a method known to those skilled in the art.
- a cDNA library is prepared from cells expressing the polypeptide of the present invention, and a portion of the DNA sequence of the present invention (for example, SEQ ID NO: 1) is used as a probe to perform hybridization.
- SEQ ID NO: 1 a portion of the DNA sequence of the present invention (for example, SEQ ID NO: 1) is used as a probe to perform hybridization.
- SEQ ID NO: 1 a portion of the DNA sequence of the present invention
- the cDNA library may be prepared, for example, by the method described in the literature (Sambrook, J. et al., Molecular Clonings Cold Spring Harbor Laboratory Press (1989)), or commercially available DNA Ripley may be used.
- RNA is prepared from cells expressing the polypeptide of the present invention
- cDNA is synthesized using reverse transcriptase
- oligo DNA is synthesized based on the sequence of the DNA of the present invention (eg, SEQ ID NO: 1). It can also be prepared by synthesizing and performing a PCR reaction using this as a primer to amplify the cDNA encoding the polypeptide of the present invention.
- the translation region encoded by the cDNA can be determined, and the amino acid sequence of the polypeptide of the present invention can be obtained.
- genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA as a probe.
- cells expressing the polypeptide of the present invention such as cells, ligaments (e.g., brain, lung, esophagus, stomach, small intestine, colon, rectum, testis, ovary, cervix, thymus, skin, fetal skin, epidermal keratinocytes Etc.) or mRNA isolated from the affected area of inflammatory skin disease. Isolation of mRNA is performed by known methods, for example, guar-gin ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. and Sacchi, N. , Anal. Biochem.
- ligaments e.g., brain, lung, esophagus, stomach, small intestine, colon, rectum, testis, ovary, cervix, thymus, skin, fetal skin, epidermal keratinocytes Etc.
- mRNA is purified from the total RNA using the mRNA Purification Kit (Pharmacia).
- mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).
- CDNA is synthesized from the obtained mRNA using reverse transcriptase.
- cDNA can also be synthesized using a reverse transcriptase first-strand cDNA synthesis kit (such as Iidairogaku-e IJ), 5'-Ampli FINDER RACE Kit (manufactured by Clontech) and polymerase. 5, -RACE method using polymerase chain reaction (PCR) (Frohman, MA et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al. According to Nucleic Acids Res. (1989) 17, 2919-2932), cDNA can be synthesized and amplified.
- PCR polymerase chain reaction
- a target DNA fragment is prepared from the obtained PCR product, and ligated to the vector DNA. Furthermore, a recombinant vector is prepared from this, and introduced into E. coli or the like, and a colony is selected to prepare a desired recombinant vector.
- the nucleotide sequence of the target DNA can be confirmed by a known method, for example, the dideoxynucleotide chain termination method.
- a nucleotide sequence having higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et al., Nucelic Acids Research (1981)). ) 9, r43-74).
- the DNA of the present invention can be modified by a commercially available kit or a known method. Modifications include, for example, digestion with restriction enzymes, insertion of synthetic oligonucleotides, insertion of appropriate DNA fragments, addition of linkers, initiation codons (ATG) and insertion of Z or stop codons (TAA, TGA, or TAG). And the like.
- the present invention also includes a DNA that hybridizes under stringent conditions with a DNA encoding SLURP-2 (SEQ ID NO: 1).
- DNA that hybridizes with the SLURP-2 gene under stringent conditions can be used to measure the expression level of the SLURP-2 gene.
- DNA that hybridizes under stringent conditions with the SLURP-2 gene does not necessarily need to encode a polypeptide.
- Such a DNA preferably hybridizes to the code region of the nucleotide sequence set forth in SEQ ID NO: 1. Further, it preferably has a length of 100 bases or more, more preferably 200 bases or more, and most preferably 250 bases or more.
- the stringent conditions for the hybridization can be the same as those described above.
- DNA that hybridizes under such stringent conditions usually has a high degree of identity (eg, greater than 70% identity, preferably greater than 80% identity, more preferably greater than 90% identity). Have.
- the present invention provides a polypeptide encoded by the DNA of the present invention.
- the polypeptide of the present invention may vary in amino acid sequence, molecular weight, isoelectric point, presence / absence and form of sugar chains, etc., depending on the cell or host producing the polypeptide described below or the purification method. However, as long as the obtained polypeptide has a function equivalent to that of SLURP-2, it is included in the present invention.
- a prokaryotic cell for example, Escherichia coli
- a methionine residue is added to the N-terminal of the original amino acid sequence of the polypeptide.
- the polypeptide of the present invention also includes such a polypeptide.
- the polypeptide of the present invention can be prepared as a recombinant polypeptide or a natural polypeptide by methods known to those skilled in the art.
- a DNA encoding the polypeptide of the present invention is incorporated into an appropriate expression vector, and the resulting transformant is introduced into an appropriate host cell.
- the resulting transformant is recovered to obtain an extract.
- chromatography such as ion exchange, reversed phase, gel filtration, or ab init mouth chromatography in which an antibody against the polypeptide of the present invention is immobilized on a column, or a plurality of these columns. It can be purified and prepared by combining them.
- polypeptide of the present invention was expressed in a host cell (for example, an animal cell or Escherichia coli) as a fusion polypeptide with daltathione-S-transferase protein or as a recombinant polypeptide to which a plurality of histidines were added.
- a host cell for example, an animal cell or Escherichia coli
- the expressed recombinant polypeptide can be purified using a glutathione column or a nickel column. After purification of the fusion polypeptide, if necessary, a region other than the target polypeptide in the fusion polypeptide can be cleaved with thrombin, Fukuta Xa, or the like and removed.
- an antibody that binds to the polypeptide of the present invention described below against an extract of a tissue or a cell expressing the polypeptide of the present invention can be isolated by allowing it to act on an affinity column to which is bound.
- the antibody may be a polyclonal antibody or a monoclonal antibody.
- the present invention also includes partial peptides of the polypeptide of the present invention.
- the partial peptide of the present invention can be prepared, for example, by preparing an antibody against the polypeptide of the present invention, It can be used for screening of a compound that binds to a polypeptide, and for screening for a promoter or inhibitor of the polypeptide of the present invention. Further, it can be an antagonist of the polypeptide of the present invention.
- the partial peptide of the present invention When used as an immunogen, the partial peptide of the present invention has an amino acid sequence of at least 7 amino acids or more, preferably 8 or more amino acids, more preferably 9 or more amino acids. When used as a competitive inhibitor of the polypeptide of the present invention, it comprises an amino acid sequence of at least 100 amino acids or more, preferably 200 amino acids or more, more preferably 300 amino acids or more (for example, 400 amino acids or more).
- the partial peptide of the present invention can be produced by a genetic engineering technique, a known peptide synthesis method, or by cleaving the polypeptide of the present invention with an appropriate peptidase.
- the peptide may be synthesized by, for example, either a solid phase synthesis method or a liquid phase synthesis method.
- the present invention also provides a vector into which the DNA of the present invention has been inserted.
- the vector of the present invention is useful for retaining the DNA of the present invention in a host cell and expressing the polypeptide of the present invention.
- E. coli when E. coli is used as a host, the vector is amplified in E. coli (e.g., J1109, DH5a, HB101, XLlBlue), etc. It is not particularly limited as long as it has an ori and further has a transformed gene selected from Escherichia coli (eg, a drug-resistant gene that can be identified by any drug (ampicillin, tetracycline, kanamycin, chloramphenicol)).
- E. coli e.g., J1109, DH5a, HB101, XLlBlue
- Escherichia coli eg, a drug-resistant gene that can be identified by any drug (ampicillin, tetracycline, kanamycin, chloramphenicol)).
- vectors examples include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, and the like.
- pGEM-T pDIRECT
- pT7 pT7 and the like can be mentioned.
- an expression vector is useful.
- the expression vector when the expression vector is intended for expression in Escherichia coli, in addition to having the above-mentioned characteristics such that the vector is amplified in Escherichia coli, the host may be E. coli such as JM109, DH5o ;, HB101, XL1-Blue, etc.
- a promoter that can be efficiently expressed in E. coli such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J.
- Such vectors include pGEX-5X-1 (manufactured by Pharmacia), "QIAexpress systemj" in addition to the above vectors.
- the host is preferably BL21 which expresses T7 RNA polymerase), and the like.
- the vector may also include a signal sequence for polypeptide secretion.
- a signal sequence for polypeptide secretion the pelB signal sequence (Lei, SP, et al J. Bacteriol. (1987) 169, 4379) may be used for production in E. coli periplasm.
- the introduction of the vector into the host cell can be performed using, for example, a calcium chloride method or an electroporation method.
- vectors for producing the polypeptide of the present invention include mammalian expression vectors (for example, pcDNA3 (manufactured by Invitrogen) and EGF-BOS (Nucleic Acids. Res.
- insect cell-derived expression vectors eg, "Bac-to-BAC baculovairus expression syste mj (manufactured by Gibco BRL), BacPAK8), plant-derived expression vectors (Eg, ⁇ 1, pM H2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, “ Pichia Expression Kit “(Invitrogen), pNVll, SP-Q01), and Bacillus subtilis-derived expression vectors (eg, pPL608, pKTH50).
- insect cell-derived expression vectors eg, "Bac-to-BAC baculovairus expression syste mj (manufactured by Gibco BRL), BacPAK8
- plant-derived expression vectors Eg, ⁇ 1, pM H2
- animal virus-derived expression vectors
- promoters required for expression in cells for example, SV40 promoter (Mull igan et al., Nature (1979) 277, 108), having the MMLV-LTR promoter, EFla promoter-1 (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc. are essential.
- vectors with such characteristics include, pMAM, pDR2, pBK - RSV , P BK - CMV, pOPRSV, pOP13 , and the like.
- a vector having a DHFR gene that complements a nucleic acid synthesis pathway-deficient CH0 cell is used.
- pCHOI a vector having a DHFR gene that complements a nucleic acid synthesis pathway-deficient CH0 cell
- MTX transient expression of a gene
- the origin of replication of SV40 is determined using COS cells having a gene expressing the SV40 T antigen on the chromosome. Transformation with a vector (such as pcD).
- a vector such as pcD
- origin of replication those derived from poliovirus, adenovirus, pipapima-mavirus (BPV) and the like can also be used.
- the expression vector is selected as a selection marker for amplification of gene copy number in the host cell system, such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthinguanine phospholiposyltransferase (Ecogpt). Gene, dihydrofolate reductase (dhfr) gene and the like.
- APH aminoglycoside transferase
- TK thymidine kinase
- Ecogpt Escherichia coli xanthinguanine phospholiposyltransferase
- dhfr dihydrofolate reductase
- the DNA of the present invention is incorporated into an appropriate vector, and for example, a retrovirus method, a ribosome method, a Cathoyck ribosome method, an adenovirus method, etc. And other methods.
- a retrovirus method for example, a ribosome method, a Cathoyck ribosome method, an adenovirus method, etc.
- the vector to be used include, but are not limited to, an adenovirus vector (for example, pAdexlcw) and a letto-mouth virus vector (for example, p ZIPneo).
- General genetic manipulations such as insertion of the DNA of the present invention into a vector can be performed according to a conventional method (Mole cular Cloning, 5.61-5.63).
- Administration into a living body may be an ex vivo method or an in vivo method.
- the present invention also provides a host cell into which the vector of the present invention has been introduced.
- the host cell into which the vector of the present invention is introduced is not particularly limited, and for example, Escherichia coli and various animal cells can be used.
- the host cell of the present invention can be used, for example, as a production system for producing or expressing the polypeptide of the present invention.
- Production systems for polypeptide production include in iiro and i / 2 vivo production systems. Examples of the in Z'iro production system include a production system using eukaryotic cells and a production system using prokaryotic cells.
- animal cells for example, animal cells, plant cells, and fungal cells can be used as hosts.
- animal cells mammalian cells, for example, CHO (J. Exp. Med.
- CH0 cells in particular, dhfr-CHO (Proc. Natl.Acad.Sci. USA (1980) 77, 4216-4220) and CHO K-1 (Proc.
- the vector can be introduced into a host cell by, for example, the calcium phosphate method, the DEAE dextran method, the method using Cationic ribosome D0TAP (Boehringer Mannheim), the electoporation method, or the Lipofexion method. is there.
- a cell derived from Nicotiana tabacum is known as a polypeptide production system, and it may be callus cultured.
- yeasts for example, Saccharomyces, Saccha fan yces
- Saccharomyces' cerevisiae Saccharomyces cerevisiae
- filamentous fungi for example, the genus Aspergillus is known, for example, Aspergillus niger.
- bacterial cells When prokaryotic cells are used, there are production systems using bacterial cells.
- bacterial cells include Escherichia coli (R coli), for example, JM109, DH5a, HB101, and the like, and Bacillus subtilis.
- DMEM fetal calf serum
- FCS fetal calf serum
- FCS fetal calf serum
- the pH during culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary.
- examples of a system for producing a polypeptide in ⁇ ′ ⁇ ⁇ ⁇ include a production system using animals and a production system using plants.
- the desired DNA is introduced into these animals or plants, and the polypeptide is produced and recovered in the animals or plants.
- the “host” in the present invention includes these animals and plants.
- mice When using animals, there are production systems using mammals and insects. Goats, stags, sheep, mice, and mice can be used as mammals (Vicki Glasser, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used.
- the target DNA is prepared as a fusion gene with a gene encoding a polypeptide uniquely produced in milk, such as goat ⁇ -casein. Then, a DNA fragment containing the fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat.
- the desired polypeptide can be obtained from milk produced by the transgenic goat born from the goat that has received the embryo or its progeny. Appropriate hormones may be added to increase the amount of milk containing polypeptides produced by transgenic goats. It may also be used for Lanzienix goats (Ebert, KM et al., Bio / Technology (1994) 12, 699-702).
- silkworms can be used as insects, for example.
- the target polypeptide can be obtained from the body fluid of the silkworm by infecting the silkworm with a Pacu virus infected with DNA encoding the target polypeptide (Susumu, M. et al., Nature (1985) 315, 592-594).
- tobacco when using a plant, for example, tobacco can be used.
- DNA encoding the polypeptide of interest is introduced into a plant expression vector, for example, pMON530, and this vector is introduced into a vector such as iAgrobacterium tumefaciens. I do.
- This pacteria is infected with tobacco, for example, Nicotiana tabacum, and the desired polypeptide can be obtained from the leaves of this tobacco (Ma JK, et al., Eur. J. Immunol. (1994) 24, 131-138).
- the polypeptide of the present invention thus obtained can be isolated from the inside or outside of the host cell (such as a medium) and purified as a substantially pure and homogeneous polypeptide.
- the separation and purification of the polypeptide may be carried out by using the separation and purification methods used in ordinary polypeptide purification, and is not limited at all. For example, chromatography column chromatography, finoletter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc.
- the polypeptide can be separated and purified by appropriate selection and combination.
- chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory). Course Manual. Ed D aniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996) '. This These chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC. The present invention also encompasses highly purified polypeptides using these purification methods.
- the polypeptide can be arbitrarily modified or partially removed by reacting the polypeptide with a suitable protein modifying enzyme before or after purification.
- a suitable protein modifying enzyme for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, dalcosidase and the like are used.
- Examples of the antibody that binds to the polypeptide of the present invention include a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, and an antibody variant.
- the term "monoclonal antibody” in the present invention refers to a substantially homogeneous population of antibodies, that is, an antibody population in which the individual antibodies constituting the population are uniform except for a small amount of a naturally occurring variant that can occur in nature. Refers to antibodies obtained from Monoclonal antibodies are highly specific, acting against a single antigenic site.
- each monoclonal antibody is directed against a single antigenic determinant on the antigen, as compared to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes).
- the monoclonal antibodies are advantageous in that they are synthesized by hybridoma cultures that are not contaminated with other immunoglobulins.
- the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and does not limit the antibody from being produced by any particular method.
- the monoclonal antibody used in the present invention can be used, for example, by the hybridoma method described later (Kohler and Milstein, Nature
- the monoclonal antibodies used in the present invention may also be isolated from a phage antibody library (Clackson et al., Nature 352: 624-628 (1991)).
- a partial antibody comprises a portion of the heavy and / or light chains derived from a particular species, or particular antibody class or subclass, and the remainder of the chain comprising another species, or another antibody class or subclass. Included are "chimeric" antibodies (immunoglobulins) of subclass, as well as fragments of such antibodies as long as they have the desired biological activity (US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81: 685 1-6855 (1984)).
- antibody variant refers to an amino acid sequence parant of an antibody in which one or more amino acid residues have been modified. Regardless of how the modified amino acid parant has the same binding specificity as the original antibody, it is included in the “antibody variant” of the present invention.
- Such variants may have at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and at least 75%, the amino acid sequence of the variable domain of the antibody heavy or light chain. Most preferably has less than 100% sequence homology or similarity with an amino acid sequence having at least 95% amino acid sequence homology or similarity.
- Polyclonal antibodies are preferably made in non-human mammals by multiple subcutaneous (sc) or intraperitoneal (i P ) injections of the relevant antigen and adjuvant.
- Proteins that are immunogenic to the species to be immunized such as keyhole limpet hemosinin, serum anolebumin, pup thyroglobulin, or soybean trypsin inhibitor, for example, maleimidobenzoyl sulfosuccinimide Ester (linked via cysteine residue), N-hydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride, thionyl chloride, (Wherein R and R 1 are different alkyl groups), or a related agent can be bound using a bifunctional agent or inducer.
- an animal can be prepared by combining 100 / zg or 5 g of protein or conjugate (amount for the egret or mouse, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution multiple times intradermally.
- the antigen, immunogenic conjugate Immunize against a salt or derivative.
- the animals are boosted with 1/5 to 1/10 the amount of peptide or conjugate in the original Freund's complete adjuvant by subcutaneous injection at multiple sites.
- 7- After 14 days, animals are bled and serum is analyzed for antibody titer.
- a booster of the animal uses a conjugate linked to the same antigen but to a different protein and via Z or a different cross-linking reagent.
- Conjugates can also be made by recombinant cell culture protein fusion.
- a flocculant such as pan-meal is preferably used.
- the selected mammalian antibody usually has a sufficiently strong binding affinity for the antigen.
- Antibody affinity can be determined by saturation binding, enzyme-linked immunosorbent assay (ELISA), and competition analysis (eg, radioimmunoassay).
- the screening method for the desired polyclonal nano-antibody includes conventional cross-linking analysis as described in Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratoriey ⁇ Harlow and David Lane ed it. (1988)). It can be performed. Alternatively, for example, epitope mapping (Champe et al., J. Biol. Chem. 270: 1388-1394 (1995)) may be performed.
- a preferred method for measuring the efficacy of a polypeptide or antibody is a method using quantification of antibody binding affinity, but in other embodiments, in addition to or instead of binding affinity measurement, one or more of the antibodies may be used. Includes methods for assessing further biological properties. Such assays are particularly useful because they show the therapeutic efficacy of antibodies.
- Monoclonal antibodies are antibodies that recognize a single antigenic site and, because of their uniform specificity, are generally more useful than polyclonal antibodies that contain antibodies that recognize many different antigenic sites.
- Monoclonal antibodies can be produced by the hybridoma method (Kohler et al., Nature 256: 495 (1975)) or the recombinant DM method (US Pat. No. 4,816,567). In the hybridoma method, mice or other suitable host animals such as hamsters or rhesus monkeys are produced antibodies that specifically bind to the protein used for immunization, or lymphocytes that can be produced are induced. Immunization as described above.
- lymphocytes can be immunized in vitro. Thereafter, the lymphocytes are fused with myeloma cells using an appropriate fusing agent such as polyethylene dalicol to form hybridoma cells (Goding, Monoclonal Antibodies: Prints and Practice, pp. 590-103, Academic Press, (1986). the)) 0 manufactured Ha Ipuridoma cells, preferably, planted in an appropriate culture medium containing many substances 1 or than inhibiting the growth or growth of the parental myeloma cells unfused, grow.
- an appropriate fusing agent such as polyethylene dalicol
- the culture medium for the hybridoma will typically contain a substance that inhibits the growth of HGPRT-deficient cells. Contains hypoxanthine, aminopterin, and thymidine (HAT medium).
- HAT medium hypoxanthine, aminopterin, and thymidine
- Preferred myeloma cells are those that fuse efficiently, produce antibody at a stable and high level in the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
- preferred myeloma cell lines are MOPC-21 and MPC-11 mouse tumor-derived cells available from the Salk Institute Cell Distribution Center (San Diego, Calif.
- the culture medium in which the hybridoma cells grow is analyzed for the production of monoclonal antibodies against the antigen.
- the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or radioimmunoassay (RI A) or enzyme-linked immunosorbent assay (ELISA), etc. (measured by Dinyiiro binding assay.)
- RI A radioimmunoassay
- ELISA enzyme-linked immunosorbent assay
- hybridoma cells can also grow as ascites tumors in animals with in> 0.
- Monoclonal antibodies secreted by subclones are preferably From culture medium, ascites fluid, or serum, e.g., protein ⁇ -sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, Or affinity chromatography.
- a conventional immunoglobulin purification method such as one.
- the DNA encoding the monoclonal antibody can be readily isolated and sequenced using conventional methods (eg, using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody). Can decide. Hybridoma cells are a preferred starting material for such DNA.
- the DNA is introduced into an expression vector and A. eo / i cells, monkey COS cells, Chinese hamster single ovary (CH0) cells or myeloma cells that do not produce immunoglobulins if not transformed Etc., and produce monoclonal antibodies from the recombinant host cells.
- DNA encoding a monoclonal antibody can also be obtained, for example, by substituting the human heavy and light chain constant domain coding sequences for the corresponding mouse sequences (US Pat. No. 4,816,567; Morrison et al.). Natl. Acad. Sci. USA 81: 6851 (1984)), or immunoglobulin polypeptides by covalent attachment.
- non-immune glopurin polypeptides are chimeras that have an antigen-binding site that has specificity for one antigen and an antigen-binding site that has specificity for a different antigen.
- the antibody is replaced with a constant domain or a variable domain of an antibody antigen-binding site.
- Antibodies that bind to the polypeptides of the present invention also include human antibodies, humanized antibodies, chimeric antibodies, and antibody fragments (eg, Fab, F (ab ') 2 , and Fv).
- humanized antibody is also called a reshaped human antibody.
- the complementarity-determining region (CDR) of a non-human mammal such as a mouse antibody, is converted to the complementarity-determining region of a human antibody. It has been transplanted, and its general genetic recombination technique is also known.
- CDR complementarity-determining region
- FR framework region
- the obtained DNA is ligated with DM encoding the constant region of a human antibody, then inserted into an expression vector, and then introduced into a host to produce it (European Patent Application Publication No. EP 239400; Application Publication No. W0 96/02576).
- FR of human antibody linked via CDR is complementary Those whose determinant regions form a favorable antigen-binding site are selected. If necessary, amino acids in the framework region of the antibody variable region may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al., Cane er Res. (1993) 53, 851-856).
- a “chimeric antibody” is one in which a portion of the heavy and / or light chain is derived from a particular species, or a particular antibody class or subclass, and the remainder of the chain is another species, or another antibody class or subclass. Means an antibody of origin.
- antibody fragment refers to a part of a full-length antibody, and generally refers to an antigen-binding region or a variable region.
- the antibody fragments include Fab, Fab 's F (ab ,) 2, and Fv fragments.
- Papain digestion of antibodies yields two identical antigen-binding fragments, each with one antigen-binding site, called Fab fragments, and the remainder, a fragment called "Fc" for easy crystallization.
- Pepsin digestion yields an F (ab ') 2 fragment that has two antigen-binding sites and is capable of cross-linking antigen, and another fragment (called pFc').
- Other fragments include diabodies (diabodies), linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
- the “Fv” fragment is the smallest antibody fragment and contains a complete antigen recognition and binding site.
- This region is a dimer in which the variable domains of one heavy and light chain are tightly linked by a non-covalent bond ( VH - VL dimer).
- the three CDRs of each variable domain interact to form an antigen-binding site on the surface of the VH - VL dimer.
- the six CDRs provide the antibody with an antigen-binding site.
- a single variable domain or half of an Fv containing only three CDRs specific for an antigen
- the Fab fragment (also called F (ab)) further contains the constant domain of the light chain, and the constant domain of the cell of the heavy chain (CH1).
- Fab 'fragments consist of a Fab fragment and the carboxy terminus of the heavy chain CH1 domain containing one or more cysteines from the antibody hinge region. It differs in that it additionally has several residues from its origin.
- diabody refers to a small antibody fragment having two antigen-binding sites, and the fragment comprises a heavy chain variable domain (VL) linked to a light chain variable domain (VL) in the same polypeptide chain. Including the domain (VH) (VH-VL). Using a short linker in the same chain that cannot join between the two domains causes the two domains to pair with the constant domains of the other chain, creating two antigen-binding sites. Diabodies are described in more detail, for example, in EP404,097, W093 / 11161, and Holliner et al. (Proc. Natl. Acad. Sci. USA 9.0: 6444-6448 (1993)).
- Single-chain antibodies (hereinafter also referred to as single-chain Fv or sFv) or sFv antibody fragments contain the VH and VL domains of the antibody, and these domains are present in a single polypeptide chain .
- Fv polypeptides further comprise a polypeptide linker between the VH and VL domains, such that the sFv can form the necessary structure for antigen binding.
- Multispecific antibodies are antibodies that have specificity for at least two different antigens. Usually, such a molecule binds two antigens (ie, a bispecific antibody), but a “multispecific antibody” in the present invention refers to more (eg, three) antigens. Includes antibodies that have specificity for A multispecific antibody can be a full-length antibody, or a fragment of such an antibody (eg, a F (ab ') 2 bispecific antibody).
- antibody fragments have been produced by protease digestion of natural antibodies (Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); Brennan et al., Science 229: 81 (1985)). ), But it is now possible to produce it by recombinant technology.
- antibody fragments can be isolated from the antibody phage libraries described above.
- F (ab ') 2 -SH fragment directly from a host such as E. coli Can be recovered and chemically coupled to the form of F (ab ') 2 fragment (Carter et al., Bio / Technology 10: 163-167 (1992)).
- the F (ab ') 2 fragment can be isolated directly from recombinant host culture.
- methods for producing fragments such as single-chain antibodies are also known. Methods for making single chain antibodies are well known in the art (eg, US Pat. No. 4,946,778, US Pat. No. 5,260,203, US Pat. No. 5,091,513). And US Pat. No. 5,455,030).
- bispecific antibodies Methods for producing multispecific antibodies are known in the art.
- the production of full-length bispecific antibodies involves the co-expression of two immunoglobulin heavy-light chains with different specificities (Millstein et al., Nature 305: 537-539 (1983)). Since the heavy and light chains of immunoglobulin are randomly combined, the resulting multiple hybridomas (quadromas) that co-express are a mixture of hybridomas that express different antibody molecules, each of which is correct. It is necessary to select one that produces a bispecific antibody. Selection can be performed by a method such as affinity chromatography. Alternatively, the variable region of the antibody with the desired binding specificity is fused to the constant domain sequence of immunoglobulin.
- the constant domain sequence preferably includes at least a part of hinge, CH2 and CH3 regions of the constant region of the heavy chain of immunoglobulin. Preferably, it further includes the CH1 region of the heavy chain necessary for binding to the light chain.
- the DNA encoding the immunoglobulin heavy chain fusion and, if desired, the DNA encoding the immunoglobulin light chain are inserted into separate expression vectors, and transformed into an appropriate host organism. By inserting each gene into a separate expression vector, it is convenient to control the expression ratio of each chain if the presence ratio of each chain is not the same and the yield of the resulting antibody increases. However, it goes without saying that genes encoding multiple chains can be inserted into one vector and used.
- Specific antibodies are preferred.
- a light chain on only one arm In the presence of, the bispecific antibody can be easily separated from other immunoglobulins. See W094 / 04690 for the separation method.
- the method of preparing a bispecific antibody the method of Suresh et al. (Methods in Enzymology 121: 210 (1986)) can be further referred to.
- one of the antibody molecules which contains the constant domain CH3, binds to the other molecule in one antibody molecule.
- several small side chain amino acids are changed to large side chain amino acids (eg, tyrosine or tryptophan), and the corresponding large side chain amino acids of the other antibody molecule are changed to small ones (eg, alanine-threonine).
- small ones eg, alanine-threonine. It is also known to provide a cavity corresponding to the large side chain of the first antibody molecule (W096 / 27011).
- Bispecific antibodies include, for example, heteroconjugate antibodies in which one antibody is bound to avidin and the other is bound to biotin or the like (US Pat. No. 4,676,980; W091). / 00360; W092 / 00373; EP03089).
- Crosslinking agents used to make such heteroconjugated antibodies are well known, and such examples are described, for example, in US Pat. No. 4,676,980.
- a method for producing a bispecific antibody from an antibody fragment has been reported.
- it can be manufactured using a chemical bond.
- an F (ab ') 2 fragment is first prepared, and the fragment is reduced in the presence of the dithiol complexing agent arsanyl sodium to prevent difluid formation in the same molecule.
- the F (ab ') 2 fragment is converted to a thionitrobenzoate (TNB) derivative.
- F (ab ') 2 -TNB derivative is reduced again to Fab'-thiol using mercaptoethylamine, equal amounts of the F (ab') 2 -TNB derivative and Fab'-thiol are mixed.
- the heavy chain variable domain (VH) is linked to the light chain variable domain (VL) via a short linker at a position where the two domains cannot be paired with each other, and is combined with another complementary VL and VH domain. to form a pair, whereby there is a method of forming two antigen-binding sites (Hollinger et al, Proc Natl Acad Sci USA 90:à 6444-6448 (1993)) 0
- single chain Fv A dimer using sFV
- trispecific rather than bispecific antibodies have been reported (Tutt et al., J. Immunol. 147: 60 (1991)).
- Human antibodies can be obtained by methods known to those skilled in the art.
- human lymphocytes can be sensitized with in ⁇ ' ⁇ , and the sensitized lymphocytes can be fused with human-derived myeloma cells capable of permanent division to obtain a desired human antibody (Japanese Patent Publication No. 1-59878). No.).
- an antigen may be administered to a transgenic animal having the entire repertoire of human antibody genes to obtain antibody-producing cells, and human antibodies may be obtained from the immortalized cells (see International Patent Application No. Publication numbers W094 / 25585, W093 / 12227, W092 / 03918, and W094 / 02602).
- a recombinant host obtained by transforming a host with a cDNA encoding each of the heavy chain and light chain of such a human antibody, preferably with a vector containing the cDNA, by a genetic recombination technique.
- it can be obtained from the culture supernatant by culturing a host producing the recombinant human monoclonal antibody.
- the host is a eukaryotic cell other than a fertilized egg, preferably a CH0 cell, or a mammalian cell such as a lymphocyte or myeoma.
- the obtained antibody can be purified to homogeneity.
- the separation and purification of the antibody may be performed by the separation and purification methods used for ordinary proteins. For example, chromatography columns such as abundance chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc.
- Antibodies can be separated and purified by appropriate selection and combination (Antibodies: A Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor Labora tory, 1988).
- Columns used for affinity chromatography include a protein A column and a protein G column.
- columns using a protein A column include Hyper D, P0R0S, Separose FF (Pharmacia), and the like.
- Antibodies may be used in combination with various reagents for therapeutic purposes.
- reagents include chemotherapeutic agents such as doxorubicin, methotrexate and taxol, heavy metals, radionuclides, and toxins such as Pseudomonas detoxin.
- the present invention also provides an oligonucleotide comprising at least 15 nucleotides complementary to the DNA of the present invention or its complementary strand.
- complementary strand refers to one strand of a double-stranded nucleic acid consisting of A: T (U for RNA) and G: C base pairs with respect to the other strand.
- the term “complementary” is not limited to a completely complementary sequence in at least 15 contiguous nucleotide regions, but is at least 70%, preferably at least 80%, more preferably 90%, Preferably, it should have 95% or more homology on the base sequence. The algorithm for determining homology may use the algorithm described in this specification. “Oligonucleotide” also includes polynucleotides.
- the oligonucleotide of the present invention includes a probe and a primer used for detection and amplification of a DNA encoding the polypeptide of the present invention, a primer for detecting the expression of the DNA, and the expression of the polypeptide of the present invention. It can be used as a controlling nucleotide or a nucleotide derivative (for example, antisense oligonucleotide, ribozyme, or DNA encoding them). In addition, the oligonucleotide of the present invention can be used in the form of a DNA array substrate. When the oligonucleotides are used as primers, their length is usually 15b P ⁇ 100bp, preferably 17Bp ⁇ 30bp.
- the primer is not particularly limited as long as it can amplify at least a part of the DNA of the present invention or its complementary strand.
- the 3 ′ region can be made complementary, and a restriction enzyme recognition sequence tag can be added to the 5 ′ region.
- the probe is not particularly limited as long as it specifically hybridizes to at least a part of the DNA of the present invention or its complementary strand.
- the probe may be a synthetic oligonucleotide and usually has a chain length of at least 15 bp or more.
- the oligonucleotide of the present invention is used as a probe, it is preferable to appropriately label it. Labeling can be performed by using T4 polynucleotide kinase to label the oligonucleotide by phosphorylating the 5, 5 ends with 32 P, or by using a random polymerase oligonucleotide using DNA polymerase such as Klenow enzyme.
- a method of incorporating a substrate base labeled with an isotope such as 32 P or the like, a fluorescent dye, or biotin using nucleotide or the like as a primer can be exemplified.
- the oligonucleotide of the present invention can be produced, for example, using a commercially available oligonucleotide synthesizer.
- the probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like.
- the antisense oligonucleotide examples include an antisense oligonucleotide that hybridizes at any position in the base sequence of SEQ ID NO: 1.
- the antisense oligonucleotide is preferably an antisense oligonucleotide for at least 15 or more consecutive nucleotides in the base sequence of SEQ ID NO: 1. More preferably, at least 15 or more consecutive nucleotides are antisense oligonucleotides containing a translation initiation codon.
- the antisense oligonucleotide use derivatives or modifications thereof.
- the modified form include a modified lower alkylphosphonate such as a methylphosphonate type or an ethylphosphonate type, a phosphorothioate modified form or a phosphoroamidate modified form.
- Antisense oligonucleotides include not only those whose nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are all complementary sequences, but also those having nucleotides represented by SEQ ID NO: 1 in which DNA or mRNA and oligonucleotides are complementary. As long as it can specifically hybridize to a DNA, it includes one or more nucleotide mismatches.
- the antisense oligonucleotide derivative of the present invention acts on cells producing the polypeptide of the present invention to inhibit transcription or translation of the polypeptide by binding to the DNA or mRNA encoding the polypeptide.
- By suppressing the expression of the polypeptide of the present invention for example, by promoting the degradation of mRNA, it has the effect of suppressing the action of the polypeptide of the present invention.
- the antisense oligonucleotide derivative of the present invention can be mixed with a suitable base material which is inactive against the derivative to prepare an external preparation such as a liniment or a poultice.
- excipients may be added to tablets, splinters, granules, capsules, ribosome capsules, It can be a lyophilized agent such as a propellant, a liquid, a nasal drop and the like. These can be prepared according to a conventional method.
- the antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient, or is applied to the patient so as to be able to reach the affected area as a result of intravenous administration.
- an antisense-encapsulated material that enhances durability and membrane permeability can be used.
- ribosome, poly-L-lysine, lipid, cholesterol, ribofectin or a derivative thereof can be mentioned.
- the dosage of the antisense oligonucleotide derivative of the present invention can be appropriately adjusted according to the condition of the patient, and a preferred amount can be used. For example, 0.1 to; 100 mg / kg, Preferably 0.:! It can be administered in the range of up to 50 mg / kg.
- the antisense oligonucleotide of the present invention inhibits the expression of the polypeptide of the present invention and is therefore useful in suppressing the biological activity of the polypeptide of the present invention.
- the expression inhibitor containing the antisense oligonucleotide of the present invention is useful in that it can suppress the biological activity of the polypeptide of the present invention.
- substrate means a plate-like material on which oligonucleotides can be immobilized.
- the substrate of the present invention is not particularly limited as long as the oligonucleotide can be immobilized thereon, but a substrate generally used in DNA array technology can be suitably used.
- DNA arrays are composed of thousands of oligonucleotides printed on a substrate at high density. Usually these DNAs are printed on the surface of a non-porous substrate.
- the surface of the substrate is typically glass, but a permeable membrane, such as a two-cell, one-membrane, can be used.
- examples of the oligonucleotide immobilization (array) method include an oligonucleotide-based array developed by Affymetrix.
- the oligonucleotides are usually synthesized in situ.
- in situ synthesis of oligonucleotides by photolithographic technology (Affymetrix) and ink jet (Rosetta Inpharmatics) technology for immobilizing chemical substances are already known, and any of these technologies is the present invention. It can be used for the production of a substrate.
- the length of the nucleotide probe to be bound to the substrate in the present invention, when fixing the Origonuku Reochido is usually 10: a OObp, preferably 10 ⁇ 50B P, more preferably from 15 ⁇ 25Bp.
- the present invention also provides a method for screening for a compound that binds to the polypeptide of the present invention.
- This method includes the polypeptide of the present invention and a compound binding thereto. Contacting the test sample with the test sample, detecting the binding activity between the polypeptide and the test sample, and selecting a compound having an activity of binding to the polypeptide of the present invention.
- the polypeptide of the present invention used for screening may be a recombinant polypeptide or a naturally occurring polypeptide. It may be a partial peptide. It may also be in the form expressed on the cell surface or as a membrane fraction.
- the test sample is not particularly limited and includes, for example, a cell extract, a cell culture supernatant, an enzymatic microorganism product, a marine organism extract, a plant extract, a purified or crudely purified polypeptide, a non-peptide compound, Synthetic low molecular weight compounds and natural compounds.
- the polypeptide of the present invention in contact with the test sample may be, for example, a purified polypeptide, a soluble polypeptide, a form bound to a carrier, a fusion polypeptide with another polypeptide, a cell membrane, As a form expressed above, it can be brought into contact with a test sample as a membrane fraction.
- a method for screening the polypeptide binding to the polypeptide of the present invention many methods known to those skilled in the art can be used. Such screening can be performed, for example, by immunoprecipitation. Specifically, it can be performed as follows.
- the DN A encoding a polypeptide of the present invention, pSV 2 neo such as an animal cell to express the gene in an insertion child into a vector for foreign gene expression, such as pcDNA I, pCD8.
- Promoters used for expression include the SV40 early promoter (Rigby In Williamson, ed.), Genetic Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982), EF-1a promoter (Kim et al. Gene 91). , P.
- CAG promoter Niwa et al. Gene 108, p. 193-200 (1991)
- RSV LTR promoter Cullen Methods in Enzymology 152, p. 684-704
- SRa promoter Takebe et al. Mol. Cell. Biol. 8, p. 466 (198 8>), CMV immediate early promoter (Seed and Aruffo Proc. Natl. Acad. Sci.
- SV40 late promoter Gheysen and Fiers J. M. ol. Appl. Genet. 1, p. 385-394 (1982)
- Adenovirus late promoter Kaufman et al. ol. Cell. Biol. 9, p. 946 (1989)
- HSV TK promoter etc. Any promoter that can be used may be used.
- the present invention provides a fusion polypeptide having a recognition site for a monoclonal antibody of the present invention.
- a recognition site epitope of a monoclonal antibody whose specificity is known to the N-terminal or C-terminal of the polypeptide of the present invention.
- a commercially available epitope-antibody system can be used (Experimental Medicine 13, 85-90 (1995)).
- Vectors capable of expressing a fusion polypeptide with ⁇ -galactosidase, maltose-binding protein, daltathione-S-transferase, green fluorescent protein (GFP), and the like via a multicloning site are commercially available.
- the polypeptide of the present invention In order to minimize the properties of the polypeptide of the present invention by changing it into a fusion polypeptide, only a small epitope portion consisting of several to several tens of amino acids is introduced, and the fusion polypeptide is introduced. Preparation methods have also been reported.
- polyhistidine His-tag
- influenza agglutinin HA human c-myc, FLAG
- VSV-GP Vesicular stomatitis vinoles glycoprotein
- T7 genelO protein T7-tag
- human simple herb ⁇ virus glycoprotein HSV-tag
- E-tag Epitopes such as (epitopes on a monoclonal phage) and a monoclonal antibody recognizing it can be used as an epitope antibody system for scripting of polypeptides that bind to the polypeptides of the present invention (Experimental Medicine 13 , 85-90 (1995)).
- an immune complex is formed by adding these antibodies to a cell lysate prepared using an appropriate surfactant.
- This immune complex comprises the polypeptide of the present invention, a polypeptide capable of binding thereto, and an antibody.
- immunoprecipitation can also be performed using an antibody against the polypeptide of the present invention.
- An antibody against the polypeptide of the present invention can be obtained, for example, by introducing a gene encoding the polypeptide of the present invention into an appropriate Escherichia coli expression vector, expressing it in Escherichia coli, purifying the expressed polypeptide, ⁇ It can be prepared by immunizing mice, rats, goats, and chickens. Alternatively, it can be prepared by immunizing the above animal with the synthesized partial peptide of the polypeptide of the present invention.
- the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose.
- the polypeptide of the present invention is prepared as a fusion polypeptide with, for example, an epitope such as GST, a substance specifically binding to these epitopes such as daltathione-Sepharose 4B may be used to prepare the polypeptide.
- An immune complex can be formed in the same manner as when the antibody of the polypeptide of the present invention is used.
- SDS-PAGE is generally used for the analysis of immunoprecipitated polypeptides.
- the bound polypeptides can be analyzed by the molecular weight of the polypeptides.
- the polypeptide binds to the polypeptide of the present invention.
- the polypeptides since it is difficult to detect in normal staining of polypeptides such as Coomassie one staining or silver staining, 35 is a radioactive isotope S - Mechionin and 3 3 ⁇ 4 - containing cysteine broth By culturing the cells in the cell, and labeling the polypeptide in the cell and detecting the same, the detection sensitivity can be improved. Once the molecular weight of the polypeptide is known, the desired polypeptide can be purified directly from the SDS-polyacrylamide gel and its sequence determined.
- a method for isolating a polypeptide that binds to the polypeptide using the polypeptide of the present invention for example, a West Western blotting method (Skolnik, EY et al., Cell (1991) 65 83-90). That is, a cDNA library using a phage vector (gtll, ZAP, etc.) was prepared from a cell, which is expected to express a polypeptide that binds to the polypeptide of the present invention, such as ito-tori. The polypeptide expressed on LB-agarose and expressed on a filter was immobilized, and the purified and labeled polypeptide of the present invention was reacted with the filter to bind to the polypeptide of the present invention.
- a West Western blotting method Skolnik, EY et al., Cell (1991) 65 83-90. That is, a cDNA library using a phage vector (gtll, ZAP, etc.) was prepared from a cell, which
- Plaques expressing the polypeptide may be detected by labeling.
- a method for labeling the polypeptide of the present invention a method utilizing the binding property between biotin and avidin, a polypeptide fused to the polypeptide of the present invention or the polypeptide of the present invention (for example, GST, etc.) may be used. Examples thereof include a method using an antibody that specifically binds, a method using a radioisotope, and a method using fluorescence.
- a 2-hybrid system using cells Yields, S. and Sternglanz, R., Trends. Genet. (1994) 10, 286-292, Dalton S. , And Treisman R (1992) Characterization of SAP-1, aprotein recruited by serum response factor to the c-fos serum response el ement.Cell 68, 597-612, "MATCHMARKER Two-Hybrid System”, “Mammalian M ATCHMAKER Two" -Hybrid Assay Kit ”,“ MATCHMAKER One-Hybrid System] (both made by Clontech), fHybriZAP Two-Hybrid Vector System] (Stratagy )).
- the polypeptide of the present invention or a partial peptide thereof is fused with an SRF DNA binding region or a GAL4 DNA binding region and expressed in yeast cells, and the polypeptide that binds to the polypeptide of the present invention is obtained.
- a cDNA library was prepared that would be expressed in a form fused with the VP16 or GAL4 transcription activation region, and this was introduced into the yeast cells described above, and the detected positive A cDNA derived from the library is isolated from the clone (if the polypeptide that binds to the polypeptide of the present invention is expressed in yeast cells, the binding of the two activates the reporter gene, and a positive clone can be confirmed).
- the isolated cDNA into Escherichia coli for expression, the polypeptide encoded by the cDNA can be obtained. As a result, it is possible to prepare a polypeptide that binds to the polypeptide of the present invention or a gene thereof.
- Reporter genes used in the 2-hybrid system include, for example, HIS3 gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (Plasminogen activator inhibitor typel) gene, etc. However, it is not limited to these. Screening by the 2-hybrid method can also be performed using mammalian cells in addition to yeast.
- Screening for a compound that binds to the polypeptide of the present invention can also be performed using affinity chromatography.
- the polypeptide of the present invention is immobilized on a carrier of an abundity column, and a test sample which is expected to express a polypeptide that binds to the polypeptide of the present invention is applied thereto.
- the test sample in this case includes, for example, a cell extract, a cell lysate, and the like. After applying the test sample, the column is washed to prepare a polypeptide bound to the polypeptide of the present invention.
- the method for isolating not only polypeptides but also compounds (including agost and antagoust) binding to the polypeptide of the present invention includes, for example, immobilized polypeptide of the present invention, synthetic compound, and natural compound.
- a method for screening molecules that bind to the polypeptide of the present invention by using a puncture or random peptide dipeptide library, and a screening method using high-throughput by combinatorial chemistry technology (Wrighton NC; Farrell FX ;
- a biosensor utilizing the surface plasmon resonance phenomenon can be used as a means for detecting or measuring the bound compound.
- the biosensor utilizing the surface plasmon resonance phenomenon is capable of realizing the interaction between the polypeptide of the present invention and the test compound as a surface plasmon resonance signal in real time using a small amount of the polypeptide and without labeling. It is possible to observe (eg BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide of the present invention and a test compound by using a biosensor such as BIAcore.
- the compound that can be isolated by the scripting of the present invention is a candidate for a drug for regulating the activity of the polypeptide of the present invention, and is used for diseases or abnormalities caused by abnormal expression or function of the polypeptide of the present invention.
- the application of the polypeptide of the present invention to the treatment of diseases which can be treated by controlling the activity of the polypeptide can be considered.
- the disease to be treated or prevented is preferably an inflammatory skin disease.
- the present invention also includes compounds that can be isolated by such screening.
- the present invention provides a method for examining an inflammatory skin disease, comprising a step of measuring the expression level of the SLURP-2 gene. Specific embodiments of the inspection method are described below, but the inspection method of the present invention is not limited to those methods.
- RNA sample is prepared from a subject.
- the amount of RNA encoding the polypeptide of the present invention contained in the RNA sample is measured.
- the measured amount of RNA is then compared to a control.
- a cDNA sample is prepared from a subject.
- the amount of cDNA encoding the polypeptide of the present invention contained in the cDNA sample is measured.
- the measured amount of cDNA is then compared to a control. Examples of such methods include methods well known to those skilled in the art, such as Northern plotting, RT-PCR, and DNA array method.
- a cDNA sample is prepared by using RA prepared from a subject as a ⁇ type, and brought into contact with a substrate on which the oligonucleotide of the present invention is immobilized.
- the hybridization intensity By detecting the hybridization intensity, the expression level of the SLURP-2 gene contained in the cDNA sample is measured.
- the measured expression level of the SLURP-2 gene is compared with a control.
- Preparation of a cDNA sample from a subject can be performed by methods well known to those skilled in the art.
- total RA is extracted from cells or tissues of a subject (for example, skin keratinocytes).
- the extraction of total RNA can be performed by a method known to those skilled in the art, for example, as follows.
- existing methods and kits can be used as long as high-purity total RNA can be prepared.
- total RNA is extracted using "Isogen" from Nippon Gene. The specific method may be in accordance with those attached protocols.
- cDNA is synthesized using reverse transcriptase with all the extracted RA as type III, and a cDNA sample is prepared.
- Synthesis of cDNA from all RAs can be performed by methods well known to those skilled in the art. Label the prepared cDNA sample for detection, if necessary. You.
- the labeling substance is not particularly limited as long as it is detectable, and examples thereof include a fluorescent substance and a radioactive element. Labeling can be performed by methods commonly performed by those skilled in the art, such as Luo et al., Ene expression profiles of laser-capture dadjacent neuronal subtypes. Nat. Med. 1999, 117-122).
- the detection of the hybridization intensity between the nucleotide probe and the cDNA can be appropriately performed by those skilled in the art depending on the type of the substance that has labeled the cDNA sample. For example, if the cDNA is labeled with a fluorescent substance, it can be detected by reading the fluorescent signal with a scanner.
- a polypeptide sample is prepared from cells or tissues of a subject (for example, skin keratinocytes).
- a subject for example, skin keratinocytes.
- the amount of the polypeptide of the present invention contained in the polypeptide sample is measured.
- the measured amount of polypeptide is then compared to a control.
- Examples of such methods include SDS polyacrylamide electrophoresis, Western blotting, dot blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence using the antibodies of the present invention. Can be exemplified.
- the subject is suspected of developing inflammatory skin disease in the future (high risk), Alternatively, it is determined that the patient already has an inflammatory skin disease.
- the present invention also provides a test agent for use in a method for testing an inflammatory skin disease.
- test agents include a test agent containing the oligonucleotide of the present invention (including a substrate to which an oligonucleotide probe is immobilized) and a test agent containing the antibody of the present invention.
- the antibody is not particularly limited as long as it can be used for the test. The antibody is labeled if necessary.
- oligonucleotide ⁇ antibody in addition to the oligonucleotide ⁇ antibody as an active ingredient, for example, sterilized water, physiological saline, vegetable oil, surfactant, lipid, solubilizing agent, buffer, Protein stabilizers (such as BSA and gelatin), preservatives and the like may be mixed as necessary.
- sterilized water, physiological saline, vegetable oil, surfactant, lipid, solubilizing agent, buffer, Protein stabilizers (such as BSA and gelatin), preservatives and the like may be mixed as necessary.
- FIG. 1 is a diagram showing the cDNA nucleotide of SLURP-2, the deduced amino acid sequence, and the sequence of the promoter region. Arrows indicate transcription start sites.
- the nucleotide sequence and the amino acid sequence in the figure are the same as the sequences described in SEQ ID NOs: 1 and 2, respectively.
- FIG. 2 is a diagram in which the amino acid sequences of human and mouse Ly-6 superfamily are aligned. conserveed cysteine residues are boxed.
- the amino acid sequences of SLURP-1, E48, Ly6I (mouse) and RIG-E are the same as those described in SEQ ID NOs: 14 to 17.
- FIG. 3 is a photograph showing tissue expression analysis by RT-PCR.
- the SLURP-2 gene is expressed mainly in epithelial tissues, with the strongest expression in the esophagus and cervix, followed by skin and keratinocytes. Not found in bone marrow and spleen, but found in thymus. GAPDH was also used as a mouthing control.
- FIG. 4 is a photograph showing expression analysis of SLURP-2 mRNA by Northern hybridization.
- SLURP-2 mRNA is expressed in the esophagus and stomach and duodenum, but in different sizes.
- Tissues examined were: lane 2, esophagus; lane 3, stomach; lane 4, duodenum; lane 5, ileum; lane 6, ileum; lane 7, jejunum; lane 8, ascending colon; Descending colon; lane 10, transverse colon; lane 11, rectum; lane 12, cecum, lane 13, liver. 3.
- 3-actin probe was used as a control.
- FIG. 5 is a photograph showing a Southern blot analysis of the genome. 10 g of human genome DNA was digested with Kpnl, EcoRI + Bglll and Kpnl + BamHI, and separated on a 0.6% agarose gel. The probe for the plot was searched using the fragment corresponding to the exon 3 region.
- FIG. 7 is a diagram showing a physical map and a transcript map of human chromosome 8q24.3. BEST MODE FOR CARRYING OUT THE INVENTION
- Skin biopsy specimens were taken from the affected and unaffected skin of the patient and from control cases. In all cases, the presence or absence of psoriasis was determined by patient history and clinical evaluation. Informed consent was obtained from all individuals who underwent skin biopsy. The protocol for obtaining patient biopsy specimens has been approved by Tokai University School of Medicine. All patients were Japanese.
- RNA quality was assessed by agarose gel electrophoresis (no apparent degradation of 28S and 18S bands visible to the naked eye) and spectrophotometry.
- Double-stranded cDNA was synthesized using the Superscript Choice system (Life Technologies, Rockville MD) and the T7 (dT) 24 primer (GENSET). Done.
- cDNA transcription was performed in the presence of biotinylated nucleoside triphosphate. This biotinylated cRNA was hybridized with U95A to U95E arrays at 45 ° C for 16 hours.
- biotinylated cRNA was stained with streptavidin-buicoerythrin (Molecular Probes, Eugene, OR), followed by scanning with an HP Gene Array Scanner. The fluorescence intensity of each probe was quantified using the computer program GeneChip Analysis Suite 3.3 (Affymetrix).
- BLAST (t I shelf.ncbi.nlm.nih.gov/tI) was used to determine whether the selected ESTs were known or unknown according to data using microarray analysis with cDNA collected from the skin of patients with psoriasis vulgaris and healthy subjects. It was confirmed by homozygous search using BLAST). One of the unknowns (GenBank accession number AI829641) was cloned.
- gene-specific primers (5 for 3 'RACE, -CTAGGACMGCGGTGCTGGACGG-3' (SEQ ID NO: 3), 5, RACE PCR using 5'-CTAGGAC MGCGGTGCTGGACGG-3, (SEQ ID NO: 4)
- each general-purpose adapter primer AP-1 (Clontech) was used, and after purification, the pGEM-T easy vector (Promeg a) was used.
- AP-1 general-purpose adapter primer
- oligocapped cDNA libraries were prepared from cultured keratinocytes. 3 / il cDNA using gene-specific primers (5, -TAGGACMGCGGTGCTGGACG-3, (SEQ ID NO: 5), 5'-GGCAGCAAGCGATGGATACGTAG-3 '(SEQ ID NO: 6)) and 5' RACE Outer primer Amplified by a touchdown PCR reaction under the following conditions: 5 cycles of 96 ° C for 5 seconds and 69 ° C for 4 minutes, followed by 95 ° C for 10 minutes, then 96 ° C for 5 seconds, and 67 ° C for 10 seconds.
- the cDNA in the PCR fragment was extracted from the agarose gel and purified. After ligating them into a pGEM-T easy vector, a transformant was introduced into a competent cell named J109 (T0Y0B0). Plasmid DNA was isolated using the protocol of the QIAprep Spin Miniprep Kit (QIAGEN). One-way sequencing was performed on each cDNA clone using the ABI PRISM 3700 DNA Analyzer (Applied Biosystems).
- SLURP-2 gene was evaluated by Northern hybridization RT-PCR. Regarding the former, Human Digestive System 12- Lane MTN Blot (Clontec) was purchased. Using a DIGRA labeling kit (Roche), 200 bp RNAs derived from exon 1 to exon 3 labeled with digoxigenin-11-d UTP were treated as probes. After washing twice with 2 ⁇ SSCZ0.1% SDS at room temperature and 0.5 ⁇ SSCZ0.1% SDS at 68 ° C., detection was performed according to the protocol of the DIG system.
- RNA from infant skin, cultured keratinocytes, and fibroblasts was used. Keratinocytes were extracted from the culture and all others were purchased. Primers for exon 1 and exon 3 were designed; 5'-GATTGAGGCAAGACTCCACG-3 '(SEQ ID NO: 9) and 5, -CTGGCTGCAGCCGMG-3, (SEQ ID NO: 10).
- cDNA was synthesized using the ThermoScript® RT-PCR System (Gibco BRL) and subsequently amplified using primers.
- Genomic DNA was isolated from human lymphocytes according to the protocol of Molecular Cloning. This was digested once or twice with the following restriction enzymes: Kpnl, EcoRI + Bglll and Kpnl + BamHI (NEB). Genomic DNA digested in this manner was separated on a 0.6% agarose gel and transferred to a positively charged nylon membrane (ROCHE). This was hybridized with a DNA probe labeled with DIG-ll-dUTP using a PCR DIG Labeling Mix (ROCHE) at 42 ° C for 16 hours in a DIG Easy Hy bri (ROCHE). This probe consists of Etherson 3.
- ROCHE PCR DIG Labeling Mix
- the membrane was washed twice with 2 ⁇ SSC / 10% SDS at room temperature for 5 minutes and with 0.5 ⁇ SSCZlO% SDS and 0.1 ⁇ SSC 0% SDS at 68 ° C. for 15 minutes each. Detection was performed using the DIG Nucleic Acid Detection Kit (ROCHE).
- ROCHE DIG Nucleic Acid Detection Kit
- the present inventors isolated the full-length cDNA using the RACE and oligocaving methods (FIG. 1). The sequencing revealed that this gene was about 5.6 kb long and consisted of three exons. Exxon-Intron boundary follows GT-AG rules (Breathnach, R., and Chambon P. Organization an express ion of eucaryotic split genes coding for proteins. (1981). Annu. Rev. Biochem. 50: 349-383.) (Table 1) .
- the open reading frame (0RF) encodes a protein consisting of 97 amino acids, including a Ly / uPAR domain of 10 cysteine residues that shows a conserved and distinctive pattern of arrangement ( Figure 2).
- Figure 2 A BLAST homology search showed that the human and mouse Ly-6 superfamily had 29-31% identity at the amino acid level with other members.
- signal IP prediction showed that this protein contained a 22 amino acid signal peptide. GPI-anchor was not recognized.
- RT-PCR Three transcription start sites were identified using the RT-PCR and oligocaving methods.
- RT-PCR was performed using gene-specific primers (the former primer was designed inside or around the initiator).
- the -80 nucleotide was shown to be the transcription initiation site ( Figure 1).
- Takaaki Hon et al. Determined the 5 'end using the oligocaving method.
- this gene has two transcription initiation sites.
- nucleotides indicated as +1 in Fig. 1 were found.
- Example 5 Expression analysis of SLURP-2 gene Expression analysis of SLURP-2 gene in various tissues was performed. RT-PCR showed that the SLURP-2 gene was highly expressed in the cervix and esophagus of the ovary, and subsequently in the skin and keratinocytes of adult fetuses. Weak expression was detected in brain, lung, stomach, small intestine, large intestine, rectum, uterus and thymus ( Figure 3). No expression was detected in spleen and bone marrow.
- the present inventors isolated from cultured keratinocytes a full-length cDNA of a novel human gene encoding a protein named SLURP-2, which is up-regulated in psoriatic lesions. It is 580 base pairs, consists of three exons and two introns, and contains an ORF that encodes 97 amino acids. Its amino acid sequence is unique and has 10 cysteine residues showing the same characteristic arrangement pattern as the Ly-6ZuPA R motif
- SLURP-1 secreted Ly-6 / uPAR-related protein ;!
- SLURP-1 secreted Ly-6 / uPAR-related protein ;!
- This clone 8q24. 3 is located in, this position is a murine chromosome 15 homologous to members of the Ly- 6 Super Fuamiri are clustered. Many members of the human Ly-6 superfamily also cluster at this location.
- mapping data from Gold en Path, 2001 DEC show that ARS components B and ⁇ 48, two other members of Ly-6 superfamily, both expressed in skin / keratinocytes, are SLURP- They were located side by side within the range of 2 and about 50 kb and were shown to form small clusters (Figure 7). E48 is found in head and neck SCC cell lines, suggesting that it may be identical to the cell adhesion molecule desmoglein IIlZdg4
- E48 antigen highly homologous with the muri ne Ly-6 antigen ThB, is a GPI—anchored molecule apparently involved in keratinocyte cell-cell adhesion. J. Cell Biol. 129: 1677-1689).
- Mouse homologue of E48 has significant homology to the EGF repeat of Notch family (Apostolopoulos, J., McKenzie, IFC, and Sandrin, MS (2000) LY6d-L, a cell surface ligand for mouse Ly6d. Immunity 12: 223-232.) It is presumed to be related to cell proliferation.
- SUR P-1 Secreted Lyu uPAR-related protein 1
- RT-PCR results also showed another feature that the SLURP-2 gene was not expressed in bone marrow and spleen, but was expressed in the thymus (Figure 3).
- Some members of the mouse Ljr-6 superfamily are known to be cell surface markers for hematopoietic and thymocyte segregation.
- Sea-1 (Ly6A / E) is used as a marker for hematopoietic stem cells (Spangrude GJ, Heimfeld S., and We is sman, IL (1988) .Purification and characterization of mouse hematopoietic stem cells. 241: 58-62 .; Spangrude G. and Jscollay, R. (199 0) .A simplified method for enrichment of mouse hematopoietic stem cells.
- Ly-6G is a peripheral T cell activating antigen (McCormack, JM, Leenen, PJM and Walker, WS ( Macrophage progenitors from mouse bone marrow and spleen differ in their expression of the Ly-6C differentiation antigen.
- thymus is an organ through which T lymphocytes mature through interaction with epithelial cells.
- SLURP-2 is a force associated with epithelial cell components, a specific T cell antigen in the thymus, or both. Suggest a possibility.
- 4-1BB is an inducible T cell antigen and belongs to the tumor necrosis factor (TNF) expressed on activated CD4 and CD8 (bmitn, CA, Davis, T., Anderson, D., So ⁇ am , L., and et al.
- TNF tumor necrosis factor
- a rec eptor for tumor necrosis factor defines an unsua ⁇ family of cellular and viral proteins. Science 248: 1019-1023 .; Loetshcer, H., Pan, Y-CE, Laha, H- W., Gentz, R. and et al. (1990).
- TNF has various functions such as thymocyte proliferation and differentiation, T cell proliferation, IL-2R induction, and IFN- ⁇ production. Because SLURP-2 is a secreted protein, it activates T cells It appears to be able to act as a ligand for an unknown receptor that plays a role in the metabolism.
- the present inventors have cloned this gene using the results of expression profiling using microarray technology to isolate a gene associated with psoriasis vulgaris.
- Quantitative real-time RT-PCR analysis showed that the SLURP-2 gene was significantly up-regulated in psoriatic lesions as compared to non-lesioned skin and normal skin (FIG. 6).
- Promoter analysis indicated that some AP-1 and E2F sites could be associated with keratinocyte hyperproliferation in psoriasis.
- Promoter One analysis also showed another site, GATA-3, far upstream of the promoter region, but interestingly, GATA-3 is a promoter that induces T cell sorting (Vinay , DS and Kwon, BS (1998). Role of 4 IBB in immune responses. Sem. Immunol. 10: 481-489. Available for industrial use! ⁇
- the present inventors have isolated the SLURP-2 gene as a gene expressed in affected areas of inflammatory skin diseases. Therefore, the gene can be used as a diagnostic marker for inflammatory skin diseases.
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US10/516,209 US20070009515A1 (en) | 2002-05-31 | 2003-05-30 | Inflammatory skin disease-related slurp-2 gene and utilization thereof |
JP2004510420A JPWO2003102182A1 (ja) | 2002-05-31 | 2003-05-30 | 炎症性皮膚疾患に関連するslurp−2遺伝子およびその利用 |
EP03730727A EP1514926A4 (en) | 2002-05-31 | 2003-05-30 | SLURP-2 GENE ASSOCIATED WITH INFLAMMATORY SKIN DISEASE AND USE THEREOF |
AU2003241979A AU2003241979A1 (en) | 2002-05-31 | 2003-05-30 | Inflammatory skin disease-related slurp-2 gene and utilization thereof |
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WO2001068848A2 (en) * | 2000-03-01 | 2001-09-20 | Genentech, Inc. | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
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US6488203B1 (en) * | 1999-10-26 | 2002-12-03 | First Data Corporation | Method and system for performing money transfer transactions |
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AU2003241979A1 (en) | 2003-12-19 |
EP1514926A1 (en) | 2005-03-16 |
JPWO2003102182A1 (ja) | 2005-09-29 |
US20070009515A1 (en) | 2007-01-11 |
EP1514926A4 (en) | 2006-01-11 |
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