WO2000075315A1 - Nouveau gene transporteur exprime dans le foie - Google Patents

Nouveau gene transporteur exprime dans le foie Download PDF

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Publication number
WO2000075315A1
WO2000075315A1 PCT/JP2000/003586 JP0003586W WO0075315A1 WO 2000075315 A1 WO2000075315 A1 WO 2000075315A1 JP 0003586 W JP0003586 W JP 0003586W WO 0075315 A1 WO0075315 A1 WO 0075315A1
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protein
dna
liver
cells
amino acid
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PCT/JP2000/003586
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English (en)
Japanese (ja)
Inventor
Takaaki Abe
Masayuki Kakyo
Taro Tokui
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Sankyo Company, Limited
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Priority to AU49533/00A priority Critical patent/AU4953300A/en
Publication of WO2000075315A1 publication Critical patent/WO2000075315A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention relates to a novel protein involved in transporting organic anions, DNA encoding the protein, a recombinant vector containing the DNA, a host cell carrying the recombinant vector, and a use of the protein.
  • the liver plays a crucial role in maintaining the homeostasis of the body, controlling the mechanism that takes in endogenous and exogenous substances from the blood, metabolizes, detoxifies, and eliminates it into bile.
  • organic anions such as bile acids
  • the liver plays a crucial role in maintaining the homeostasis of the body, controlling the mechanism that takes in endogenous and exogenous substances from the blood, metabolizes, detoxifies, and eliminates it into bile.
  • organic anions such as bile acids
  • an object of the present invention is to provide a novel organic anion transporter gene that can be used as described above and that is involved in organic anion transport in the vascular apical membrane of human liver, and that the polyenzyme encoded by the gene can be used.
  • An object of the present invention is to provide an organic anion transporter which is a peptide.
  • the present inventors cloned a gene for a novel protein capable of transporting organic anions from human liver, and expressed the product of this gene in oocytes of Xenopus laevis to produce an organic anion.
  • the inventors succeeded in confirming the ion transport ability, and completed the present invention. That is, the present invention relates to a protein comprising the amino acid sequence represented by amino acid numbers 1 to 691 of SEQ ID NO: 2 in the sequence listing, or the amino acid sequence represented by amino acid numbers 1 to 691 of SEQ ID NO: 2 in the sequence listing.
  • a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted and Z- or added, and having the ability to transport organic anions preferably, the protein is derived from liver tissue -, And the protein is preferably derived from human. More preferably, the transformed E. coli strain E.co1ipH] SANK 7109 (FERM BP-6 743) is a protein consisting of the amino acid sequence encoded by DNA inserted into the recombinant vector held by the recombinant vector. The present invention also relates to a DNA encoding a protein having the ability to transport organic anions.
  • the DNA is a nucleotide sequence represented by nucleotide numbers 92 to 2164 of SEQ ID NO: 1 in the sequence listing. Or hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 1 in the Sequence Listing, and the organic anion Although it is a DNA encoding a protein having an ability, it is preferably derived from liver tissue, and more preferably from human. As the most preferred such DNA of the present invention, a DNA comprising the nucleotide sequence represented by nucleotide number 92 to 246 of SEQ ID NO: 1 in the sequence listing or a transformed E.
  • E.co1 ip H1 SANK 7109 can include a DNA inserted into a recombinant vector maintained in the present invention.
  • the present invention relates to a vector and a transformed host cell harboring the recombinant vector.
  • Such a transformed host cell includes, as of June 3, 1999, Tsukuba East 1 Ibaraki, Japan
  • the transformed Escherichia coli strain E.co 1 ip H1 SANK 710909 (Accession No. F ERM BP—6743) deposited internationally with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology (Chome 1-3) be able to.
  • the present invention relates to an antibody that specifically recognizes a protein having the ability to transport organic anions.
  • the present invention provides a method for expressing a protein having the ability to transport organic anions in animal cells, and assaying the liver permeability of a test substance using the ability of the protein to transport organic anions. On how to W
  • the ability to transport organic anions refers to the activity of taking an organic anion, such as taurocholate (taurocholate), from the outside of a cell to the inside of a cell by passing through the cell membrane. . That is, the amount of taurocholate taken up into cells expressing the protein of the present invention is significantly higher than that of cells not expressing the protein of the present invention.
  • the organic anions are bile acids (taurocholate, cholic acid), conjugated steroids (dehydroepiandrosterone sulfate conjugate, estradiol dalcuronic acid conjugate, estrone sulfate conjugate).
  • eicosanoids brostaglandin E2, thromboxane B2, leukotriene C4, leukotrien D4, leukotrien E4
  • thyroid hormone T4, ⁇ 3
  • folate derivatives folate , Methotrexate
  • enkephalin derivatives DPD ⁇ ⁇
  • drugs having a carboxylic acid HMG-C ⁇ reductase inhibitor: bravastatin, ACE inhibitor: temocapril.
  • taurocholic acid More preferably, taurocholic acid, dehydrodrepiandrosterone sulfate conjugate, estradiol glucuronic acid conjugate, estrone sulfate conjugate, brostaglandin E2, thromboxane B2, leucotriene C4 , Leukotriene E4, thyroid hormone T4, thyroid hormone 3, and pravastatin.
  • novel protein having the ability to transport organic anions of the present invention ie, human liver organic anion transporter (LST-1: Liver Specific Transporter-1), is expressed mainly in the liver in vivo. It is a so-called sodium ion-independent organic anion transporter whose organic anion transport ability (organic anion uptake into expressing cells) is not affected by the presence of sodium ions outside the cells.
  • LST-1 human liver organic anion transporter
  • the human organic anion transporter LS11-1 of the present invention is a human organic anion transporter OATP and a prostaglandin transporter PGT (Kanai et al., Science, 268, p. 866, 1995).
  • human kidney OAT1 Hosoyamada et al., Am. J. Physiol., 276, F122, 19
  • the DNA encoding the protein of the present invention can be isolated and obtained by performing screening using mammalian liver tissue or cells as a gene source.
  • the mammal used herein preferably includes dogs, magpies, magpies, goats, sheep, monkeys, monkeys, butters, magpies, rats, mice or humans, and most preferably. Is a hit.
  • Screening and isolation of the DNA encoding the protein of the present invention can be performed by a library screening method: for example, using a database containing ESTs, amino acids of human OATP and human PGT. Search for sequences homologous to the sequence.
  • PCR primers for amplifying the obtained sequence are prepared, and a PCR reaction is performed using cDNA obtained by reverse-transcription of human liver mRNA (Clontech) as a template. Subjected to electrophoresis the Sanfuru anti ⁇ at Agarosugeru, the amplified bands excised Ri O Gel, p B 1 uescript (manufactured by scan Toratajin Inc.) To subclone Nin grayed cut out subcloned inserts, respectively, 32 Radiolabel with P to use as a screening probe.
  • the human liver c DNA library is dispersed so that the diameter 1 5 cm 2 X 1 0 4 plaques per Bureto of are made form: according to a conventional method, the nylon filters, Nikki transferred Fajipura over click - obtained The filter thus obtained is subjected to a neutralization using the above : i2P-labeled probe. After the reaction, perform autoradiography to select positive clones. The clones obtained are further subjected to secondary or tertiary screening to isolate clones having full length cDNA.
  • the nucleotide sequence of the DNA obtained in this manner can be determined, for example, by the chemical modification method of Maxa Mougilvert (Maxam, AM and Gilbert, W. (1980): “Metho ds in Enzymology” 65, 499-559). It can be performed by the dideoxynucleotide chain termination method using M13 phage (Messing, J. and Vieira, J. (1982) Gene 19, 269-276). In addition, an automatic DNA sequence analyzer using a fluorescent dye instead of the radioisotope (for example, Model 3773A manufactured by PerkinElmer Japan Applied Biosystems) can be used. The transformed E. coli strain E.co1ipHIS ANK 7 carrying the plasmid into which the cDNA encoding the most preferred protein of the present invention was inserted was inserted.
  • the gene encoding the protein of the present invention can be obtained from the strain.
  • the protein of the present invention can be produced by genetic recombination using, for example, the cDNA obtained as described above.
  • cDNA can be incorporated into a suitable expression vector, and the resulting recombinant DNA can be introduced into a suitable host cell: an expression system for producing polybutide (a host-vector system) Examples thereof include bacterial, yeast, insect cell, and mammalian cell expression systems. Of these, insect cells and mammalian cells are preferably used to obtain functional proteins.
  • prokaryotic host cells examples include Escherichia coli and Bacillus subtilis. Transformation of the gene of interest in these host cells involves transformation of the host cells with a plasmid vector containing regulatory elements and a replicon or origin of replication from a species compatible with the host.
  • a vector it is necessary to confer phenotypic (phenotype) selectivity to transformed cells.
  • K12 strain and the like are often used as Escherichia coli, and pBR322 and pUC-based plasmids are generally used as vectors. Not limited thereto, any of various known strains and vector can be used.
  • tryptophan (trp) promoter In Escherichia coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan 'lactose (tac) promoter, lipoprotein (lpp) promoter, polypeptide elongation factor Tu
  • eukaryotic host cells include vertebrates, insects, Examples of such vertebrate cells include cells such as yeast, such as monkey COS cells (Gluzman, Y.
  • the cDNA may be expressed in an appropriate expression vector (for example, a retrovirus-based vector, a papillomavirus vector, a vaccinia virus vector, or an SV40-based vector).
  • an appropriate expression vector for example, a retrovirus-based vector, a papillomavirus vector, a vaccinia virus vector, or an SV40-based vector.
  • Promoters eg, SV40 flow motor, LTR flow: mouth motor, elongation 1 ⁇ pro Motor, etc.
  • an appropriate animal cell is transformed with the obtained expression vector, and the transformant is cultured in an appropriate medium to produce a target polypeptide.
  • the mammalian cells used as the host include monkey COS-7 cells, Chinese hamster CHO cells, human HeLa cells, primary cultured cells derived from kidney tissue, LLC-PK1 cells derived from porcine kidney, and porcine rats. Cell lines such as kidney-derived OK cells are included.
  • the expression vector has an SV40 replication origin, and is capable of autonomous growth in CO S cells. Those having a transcription promoter, a transcription termination signal, and an RNA splice site can be used.
  • the expression vector was prepared by the following method: getylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res, 11, 1295-1308), and calcium phosphate-DNA coprecipitation method. (Graham, F.L. and van der Eb, AJ (1973) Virology 52, 456-457), and electric pulse drilling (Neumann, E. et al. (1982) EMBO J.
  • getylaminoethyl (DEAE) -dextran method Lithman, H. and Magnusson, G. (1983) Nucleic Acids Res, 11, 1295-1308
  • calcium phosphate-DNA coprecipitation method Graham, F.L. and van der Eb, AJ
  • the desired transformed cells can be obtained.
  • the antibacterial substance G4 is used together with the expression vector.
  • 18 Vectors that can express the neo gene that functions as a resistance marker such as pR SV neo (Sambrook, J. et al. (1989): “Molecular Cloning A Laboratory Manual” Cold Spring Harbor Laboratory, NY) and p SV2—neo (Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341) and the like.
  • pR SV neo Standardbrook, J. et al. (1989): “Molecular Cloning A Laboratory Manual” Cold Spring Harbor Laboratory, NY
  • p SV2—neo Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341
  • the protein of the present invention can be obtained transformed cells stably producing.
  • cell lines derived from ovarian cells of Spodoptera frugi perda (S f — 9 or S f — 21) of the family Lepidoptera, or High Five cells derived from egg cells of Trichoplusia ni (Wickham, TJ et al, (1992) Biotechnol. Prog. 1: 391-396) is frequently used as a host cell, and the baculovirus transfer vector is autographa nucleopolyhedrovirus (AcNPV).
  • PVLl392 / 1393 utilizing hedrin protein promoter is frequently used (Kidd, IM and VC Emery (1993) The use of baculoviruses as expression vectors.Applied Biochemistry and Biotechnology 42, 137-159).
  • a vector using the promoter of baculovirus P10 or an abasic protein can be used:
  • the secretory signal sequence of Ac NPV envelope surface protein GP67 can be used as the target protein
  • N Recombinant protein can also be expressed as a secreted protein by exposing it to the terminal side (Zhe-raei Wang, et al. (1998) Biol. Chem., 379, 167-174).
  • yeast As an expression system using a eukaryotic microorganism as a host cell, yeast is generally well-known, and among them, yeast of the genus Saccharomyces, for example, ⁇ . Saccharomyces cerevisiae and petroleum yeast Pichia pastoris are preferred.
  • expression vectors for eukaryotic microorganisms such as the yeast include, for example, a promoter for an alcohol dehydrogenase gene (Bennetzen,
  • a secretory signal sequence and a cleavage site of an endogenous protease or a known protease of the host cell are added to the N-terminal side. It can also be expressed as a recombinant.
  • the transformant obtained as described above can be cultured according to a conventional method, and the culture produces the protein of the present invention intracellularly or extracellularly.
  • the medium used for the culture can be appropriately selected from those commonly used depending on the host cell used.
  • RPMI 164 medium or Dulbecco's medium can be used.
  • a medium such as a modified Eagle's medium (hereinafter referred to as “DMEM”) to which serum components such as fetal calf serum are added as necessary can be used.
  • DMEM modified Eagle's medium
  • the cDNA obtained as described above is a cDNA encoding an organic anion transporter, that is, the gene product encoded by the cDNA is an organic anion transporter.
  • cRNA prepared from the obtained cDNA was introduced into oocytes of African clawed frog and expressed, and taurocholate, an organic anion, was used as a substrate.
  • taurocholate an organic anion
  • cDNA obtained as described above to screen an appropriate cDNA library or genomic DNA library made from a different gene source, homology from different tissues and different organisms can be screened. Genes and chromosomal genes can be isolated.
  • a synthetic primer designed based on the information of the disclosed nucleotide sequence of the gene of the present invention (the nucleotide sequence shown in SEQ ID NO: 1 or a part thereof)
  • ordinary PCR Polymerase Chain Reaction
  • the method can be used to isolate genes from a cDNA library or a genomic DNA library.
  • DNA libraries such as a cDNA library and a genomic DNA library include, for example, Molecular Cloning "[Sara Brooks,.]., Fritsch, EF, and Maniatis, T., published in Cold Spring Harbor and Abora tory Press in 1989]. Alternatively, if there is a commercially available library, this may be used.
  • the DNA of the present invention includes, in addition to a DNA having the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing, a DNA having the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions. Those containing DNA that can be hybridized are included.
  • the DNA capable of hybridizing in this manner may be any DNA as long as the protein encoded by the DNA has the ability to transport organic anions.
  • Such DNA has a homology of 70% or more, preferably 80% or more with the nucleotide sequence shown in SEQ ID NO: 1.
  • Such DNAs include mutant genes found in nature, artificially modified mutant genes, and homologous genes derived from heterologous organisms.
  • hybridization is carried out under the conditions of 5 XSSC (0.75 M sodium chloride). , 0.075 M sodium citrate) or equivalent salt concentration in a hybridization solution.
  • washing can be carried out in the above solution. Further, under conditions having a higher stringency (high stringent conditions), in the above, washing is performed at 0.
  • nucleotides oligonucleotides or polynucleotides
  • the nucleotides that hybridize under stringent conditions to the organic anion transporter gene of the present invention can be used as probes for detecting the organic anion transferer gene.
  • they can be used as antisense oligonucleotides, ribozymes, decoys, etc. to modulate the expression of organic anion transporter genes.
  • a nucleotide for example, a nucleotide containing a partial sequence of usually 14 nucleotides or more or a complementary sequence thereof in the nucleotide sequence represented by SEQ ID NO: 1 or 2 can be used.
  • a longer sequence may be used as the partial sequence, for example, a sequence of 20 nucleotides or more or 30 nucleotides or more.
  • the amino acid number 1 in SEQ ID NO: 2 in the sequence listing is not necessarily required. It is not necessary to have all of the amino acid sequence of the present invention, for example, even if it is a partial sequence, as long as it shows the ability to transport organic anions, those amino acid sequences are also included in the present invention. Included in proteins. Further, DNA encoding the protein is also included in the present invention. Examples of such a protein having the ability to transport organic anions include a protein consisting of 691 amino acids having the methionine residue of amino acid number 1 in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing as the N-terminal. Can be exemplified. Preferred examples of DNA encoding the protein include nucleotide numbers of SEQ ID NO: 1 in the sequence listing.
  • Examples include DNA consisting of the nucleotide sequence represented by 92 to 2164:
  • eukaryotic genes include polymorphism (poll), as is known for interfluenza genes.
  • ymorphi sm (see, for example, Ni shi, T. et al. (1985) J. Biochem. 97, 153-159), and this polymorphism results in one or more amino acids In some cases, substitutions may be made, and in others nucleotides may be substituted but the amino acids remain the same. Amino acid number of SEQ ID NO: 2 in Sequence Listing
  • a precursor of the protein of the present invention consisting of the amino acid sequence represented by 691 or amino acids of a mature form of the protein of the present invention consisting of the amino acid sequence represented by amino acid numbers 1 to 691
  • a protein in which one or more amino acid residues have been deleted, added, inserted or substituted at one or more sites in the sequence However, they often have the ability to transport organic anions.
  • a protein having an amino acid sequence in which a natural amino acid sequence is substituted has an activity equivalent to that of a natural protein.
  • interleukin 2 IL — 2
  • IL-2 activity Wang, A. et al. 198
  • Science 224, 1431-1433 All such proteins are included in the present invention as long as they have the ability to transport organic anions.
  • the present invention also includes all DNAs having the same nucleotide sequence encoding these proteins.
  • Such various DNAs of the present invention can be prepared, for example, by the phosphite triester method (Hunkapiller, M. et al.
  • the codon corresponding to the desired amino acid may be selected arbitrarily. For example, it can be determined according to a conventional method in consideration of the codon usage of the host to be used. (Grantham, R, et al. (1981) Nucleic Acids Res. 9, 143-174) :: Furthermore, partial modification of the codons of these nucleotide sequences can be carried out according to a conventional method. Site-specific mutagenesis using primers composed of nucleotides (site spe cific mutagenesis) (Mark, DF et al. (1984) Proc.
  • the antibody can be obtained using the organic anion transporter of the present invention or a polypeptide having immunological equivalence with the organic anion transporter of the present invention. It can be used for detection and purification of ion transporters.
  • Antibodies can be produced using the organic anion transporter of the present invention, a fragment thereof, or a synthetic peptide having a partial sequence thereof as an antigen.
  • a polyclonal antibody can be produced by a conventional method in which a host animal (for example, a rat or a heron) is inoculated with an antigen and immune serum is collected.
  • a monoclonal antibody that specifically binds to the protein of the present invention can be obtained by the method described below.
  • the production of monoclonal antibodies generally requires the following steps:
  • an antibody-producing cell other than splenocytes and myeloma are used. You can also:
  • the protein of the present invention or a part thereof prepared by the method described above can be used. Further, since the entire primary structure of the protein of the present invention has been elucidated by the present invention, By a well-known method, a partial peptide of the amino acid sequence represented by amino acid number 1 to 691 in SEQ ID NO: 2 in the sequence listing can be chemically synthesized and used as an antigen.
  • the antigen obtained in the step (a) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant, or Rimijoban, and the animal is immunized as an immunogen.
  • an adjuvant such as Freund's complete or incomplete adjuvant, or Rimijoban
  • the method of immunogen administration for mouse immunization may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, and intramuscular injection, but subcutaneous injection or intraperitoneal injection is preferred.
  • Immunization can be performed once or several times at appropriate intervals (preferably at intervals of 1 to 5 weeks) —then the titer of the immunized animal against the antigen in the serum is determined, If an animal with a sufficiently high antibody titer is used as a source of antibody-producing cells, the effects of subsequent operations can be enhanced.
  • antibody-producing cells derived from animals 3 to 5 days after the final immunization can be used. Is preferably used for cell fusion.
  • the antibody titers used here include radioisotope immunoassay (hereinafter referred to as "RIA"), solid-phase enzyme immunoassay (hereinafter referred to as "ELISA”), fluorescent antibody assay, and passive immunoassay.
  • RIA radioisotope immunoassay
  • ELISA solid-phase enzyme immunoassay
  • fluorescent antibody assay and passive immunoassay.
  • passive immunoassay There are various known techniques such as the hemagglutination method, but from the viewpoints of detection
  • the antibody titer in the present invention can be measured, for example, by the following procedure according to the ELISA method.
  • the purified or partially purified antigen is adsorbed on the surface of a solid phase such as a 96-well plate for ELISA.
  • the solid surface is covered with a protein unrelated to the antigen, for example, serum albumin (hereinafter referred to as “BSA”).
  • BSA serum albumin
  • a serially diluted sample eg, mouse serum
  • a monoclonal antibody in the sample is bound to the above antigen:
  • an antibody against an enzyme-labeled mouse antibody is added as a secondary antibody to bind to the mouse antibody.
  • a substrate of the enzyme is added, and the substrate is decomposed.
  • the antibody titer is calculated by measuring the change in absorbance and the like due to color development based on this.
  • a cell line generally obtained from a mouse for example, an 8-azaguanine-resistant mouse (derived from BAL BZc) myeloma strain P3X63Ag8 or 1 (P3-U1) (Yelton, DE et al. urrent Topics in Microbiology and Immunology, 81, 1-7 (1978)), P3ZNSI Zl-Ag4-1 (NS-1) (Kohler, G. et al. European J. Immunology, 6, 511-519 (1976) ), Sp2 Z0-Agl4 (SP-2) (Shulman, M. et al. Nature, 276, 269-270 (1978)), P3X63Ag8.653 (653) (Kearney, JF et al. J.
  • Iscove's Modified Dulbecco's Medium hereinafter referred to as "IMDM"
  • DMEM normal medium 3-4 days before fusion
  • 5 1 04 medium e.g., 5 1 04 medium (Ajinomoto Co., Ltd.) containing 5 Ji 1 0% de
  • Antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any part of an individual, such as spleen, lymph node, peripheral blood, or a combination thereof as appropriate. Splenocytes are most commonly used. After the final immunization, a site where antibody-producing cells are present, for example, a spleen is removed from a mouse having a predetermined antibody titer, and spleen cells as antibody-producing cells are prepared. Currently, the most commonly used means for fusing the spleen cells with the mye obtained in step (c) is to use polyethylene glycol, which has relatively low cytotoxicity and simple fusion operation. This method includes, for example, the following procedure.
  • the spleen cells and myeloma are thoroughly washed with a serum-free medium (for example, RPMI 1640) or PBS, and the ratio of spleen cells to myeloma cells is about 5: 1 10: 1.
  • a serum-free medium for example, RPMI 1640
  • PBS for example, PBS
  • the ratio of spleen cells to myeloma cells is about 5: 1 10: 1.
  • Add serum-free medium dropwise. Then, slowly add 10 ml of serum-free medium and centrifuge.
  • HAT medium hypoxanthine.aminopterin 'thymidine
  • IL-12 mouse interleukin-12
  • the myeloma cell is an 8-azaguanine resistant strain, that is, a hypoxanthine 'guanine' phosphoribosyltransferase (HG PRT) deficient strain
  • HG PRT hypoxanthine 'guanine' phosphoribosyltransferase
  • HT medium a medium in which aminopterin has been removed from the HAT medium
  • a part of the culture supernatant is collected, and the antibody titer is measured, for example, by ELISA.
  • the method using an 8-azaguanine-resistant cell line has been described above, but other cell lines can also be used according to the selection method of the hybridoma, and in that case, the composition of the medium used also changes.
  • the antibody titer is measured in the same manner as described in step (b), and the hybridoma that has been found to produce a specific antibody is transferred to another plate and cloned.
  • the limiting method is a limiting dilution method in which one well of the plate is diluted so that one hybridoma is contained in each well, a soft agar method in which the colonies are collected by culturing in a soft agar medium, and a micro agar.
  • the limiting dilution method is simple and is often used.
  • cloning by the limiting dilution method is repeated 2 to 4 times, and those with a stable antibody titer are selected as the monoclonal antibody-producing hybridoma strain of the present invention.
  • the hybridomas are cultured by replacing the medium with HT medium and normal medium: Large-scale culture is performed by rotary culture using large culture bottles or spinner culture. By purifying the supernatant in this large-scale culture using a method known to those skilled in the art such as gel filtration, a monoclonal antibody that specifically binds to the protein of the present invention can be obtained.
  • the hybridoma can be grown intraperitoneally in a mouse strain of the strain (for example, BALB'c above) or a Nuu mouse. As a result, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained.
  • a commercially available monoclonal antibody purification kit for example, MAbTrap GII kit; manufactured by Pharmacia
  • the monoclonal antibody thus obtained has a high antigen specificity to the protein of the present invention.
  • the identification method includes the Ouchterlony method, the ELISA method, and the RIA method.
  • the Okterlony method is simple, but requires concentration if the concentration of monoclonal antibody is low.
  • the ELISA method or the RIA method is used, the culture supernatant is reacted with the antigen-adsorbed solid phase as it is, and the monoclonal antibody is used as a secondary antibody by using antibodies corresponding to various immunoglobulin isotypes and subclasses. It is possible to identify the isotype and subclass of the antibody.
  • the thus obtained monoclonal antibody of the present invention can be used for detection, separation and purification of the protein of the present invention utilizing its specificity.
  • FIG. 1 shows the results of Northern blot analysis using RNA derived from various human tissues.
  • the numbers represent the chain length (kb) of the marker RNA:
  • Figure 2 shows the results of Northern blot analysis using RNA from various rat tissues.
  • the numbers represent the chain length (kb) of the marker RNA.
  • Fig. 3 shows the results of Northern blot analysis using RNAs derived from various mouse tissues: The numbers represent the chain length (kb) of the marker RNA.
  • Figure 4 shows the results of organic anion uptake experiments using the African oocytes frog cell line:
  • CDNA obtained by reverse transcription of human liver mRNA (manufactured by Clontech) using a combination of three types of primers: Framers 1 and 2, primers 3 and 4, and primers 5 and 6. )) was performed, and PCR (30 times at 94 ° C for 1 minute, 50 ° C for 2 minutes, and 72 ° C for 2 minutes) was performed.
  • the three reaction solutions after the reaction were electrophoresed on a 0.8% agarose gel, the amplified band was cut out from the gel, and subcloned into pBluescript (Stratagene).
  • human liver cDNA library a human cDNA library prepared using human liver poly (A) + RNA (Clontech) as a gene source was used.
  • the cDNA library was prepared according to the Gub 1 er method. That is, 5 ⁇ g of Bol (A) + RNA and 24 units of reverse transcriptase (Superscript II: Gibco BRL) were added to 50 ⁇ l of a reaction solution (50 mM Tris-HCl (50 mM)).
  • the resulting filter was washed with 50% formamide, 5XSSC (0.7M sodium chloride, 0.75M sodium citrate), 5X Denhardt's solution (1% serum albumin, 1% polyalbumin). Binirupiro Li Dong, 1% Fuikoru 4 0 0), in a solution containing 1% SDS, 1 0 0 m gZm 1 of denatured salmon sperm DNA, 4 2 ° C for 1 2 hour after prehybridization, with 32 P In a reaction containing 50% formamide, 5XSS :, 5X Denhardt's solution, 1% SDS, 100 mgZm1 of denatured salmon sperm DNA containing the radiolabeled probe42. ⁇ --Hybridization was performed.
  • the filter was washed with a solution containing 0.1 XSSC and 1% SDS at 68 ° C. for 1 hour.
  • the hybridized filter was exposed to a film for autoradiography (XOMAT-AR, Kodak) at 180 ° C and developed. As a result, 52 positive plaques were detected in the primary screening. At this time, the signals obtained by screening using the probes generated from the three types of EST clones were all common.
  • the size was found to be about 3 kb from the calibration curve using the molecular weight marker.
  • Heart, Brain, Placenta, Lung, Skeletal muscle, Kidney, Kidney, Pancreas, Spleen, Thymus (Thymus), prostate (Prostate), testis (Testis), ovary (Ovary), small intestine (Small intestine), large intestine (Colon) and leukocyte (Leukocyte) show no band, and the protein of the present invention is specific to the liver.
  • a probe labeled with the full-length cDNA of Frasmid pH1 with : P a rat tissue Northern blot filter and a mouse tissue Northern blot filter were used.
  • the filter was pre-hybridized in the same manner as in the case of human tissue, and then 5 XSSC, including the probe labeled with 32 P, was used. 5 X Denhardt's solution 2 5% formamide, Hybridization was carried out at 42 ° C. in a solution containing 1% SDS and 10 Om gZm 1 of denatured salmon sperm DNA. After the reaction was completed, the filter was washed with a solution containing 2 XSSC and 1% SDS at 55 ° C for 1 hour, and then subjected to autoradiography. As a result, rats ( Figure 2) and mice ( Figure 3) In each case, one band was detected only in the liver, which revealed that the homologue of the protein of the present invention was specifically expressed in the liver.
  • the cRNA was prepared from the plasmid pH1 obtained as described above, and the eggs of African Megafrog were prepared according to the method of Abe et al. (Abe et al., J. Biol. Chem., 273, 22395, 1998).
  • the protein of the present invention was expressed by injection into mother cells, and a substrate uptake experiment was performed.
  • cRNA was synthesized using T7 RNA polymerase (manufactured by Stratagene) by converting plasmid pHI into a linear form by digesting it with the restriction enzyme NotI to form linear form: MCAP RNA caving kit (Stratagene) was used for the reaction.
  • a female Afume frog (Xenopus 1 aevis: purchased from Hamamatsu Biological Teaching Materials Co., Ltd.) was ice-cooled with 0.1% 3-aminoethyl benzoate (MS-222) /0.3% potassium bicarbonate solution. And anesthetized. After incising the abdomen and removing an appropriate amount of ovaries from one side, add OR2 buffer (82.5m sodium chloride, 2mM potassium chloride, 1mM magnesium chloride, 5mM HE PES, pH 7.5). The plate was placed in a soaked petri dish and cut into about 20 to 30 oocytes.
  • OR2 buffer 82.5m sodium chloride, 2mM potassium chloride, 1mM magnesium chloride, 5mM HE PES, pH 7.5.
  • Morphol. 136, 153, 1972) were selected under a stereomicroscope.
  • MN-153: NARISHIGE micromanipulator
  • the above cRNA solution or water alone was injected into the oocytes in 50 n1 increments, and then the berth solution was filled. It was transferred to a glass vial of Om 1 and cultured for 3 days. All operations were performed in a thermostatic chamber adjusted to 18 ° C.
  • the oocytes were tested for taurocholate uptake and the effect of salts added to the culture medium: 6 to 10 oocytes were transferred to Eiken tubes filled with Bath's solution. The sample was allowed to stand at room temperature (21 ° C. to 22 ′ : C).
  • thyroid hormone (T 4 and T 3) was Michaelis one Menten kinetics test uptake: from c 1 25 I one T 4 and '25 I one T 3 uptake of RN A to the injected oocytes
  • the value obtained by subtracting the incorporation into water-injected oocytes was taken as the dependence on the protein of the present invention.
  • the uptake was measured for 20 minutes.
  • the Km values of the protein-dependent incorporation of the present invention obtained from the tests of three cases were 3.0 ⁇ 1.3 ⁇ ⁇ and 2.7 ⁇ 1. ⁇ ⁇ , respectively.
  • the protein of the present invention has the ability to take in bile acids and conjugated steroids as well as eicosanoids, and is an intermediate between the organic anion transporter ⁇ AT ⁇ and the prostaglandin transporter (PGT). It has been found that the protein of the present invention is a thyroid hormone which is a physiologically active substance.
  • the expression vector for animal cells containing the cDNA obtained in Example 1 was used in human cultured hepatocytes HepG2 (American type 'Culture Collection (ATCC) HB-8065-HepG2).
  • HepG2 American type 'Culture Collection (ATCC) HB-8065-HepG2).
  • Fig. 2 the organic anion transporter activity already disappeared. Is known to have been lost) using a gene transfer device by electroporation, and selection and cloning using the expression of a vector-specific selectable marker (eg, antibiotic resistance to G418) as an index. And isolating the cells into which the DNA has been introduced.
  • Example 2 Construction of bioartificial liver
  • the expression vector for animal cells containing the DNA obtained in Example 1 was introduced into human cultured hepatocytes HepG2 using a gene transfer device by electroporation, and the expression of a vector selection marker (for example, Select and clone using the antibiotic (G4 18 resistance) as an index, and isolate cells into which the DNA has been introduced.
  • a vector selection marker for example, Select and clone using the antibiotic (G4 18 resistance) as an index
  • the expression of the DNA obtained in Example 1 in the transformed cell line thus isolated is confirmed by the Northern blot method and the Estanblot method.
  • a transformed cell line is cultured in a double-chamber system (for example, Transwell manufactured by Coaster), and various organic anions (radio-labeled or fluorescent-labeled, etc.) are placed in one of the chambers.
  • Uptake and secretion will be examined by measuring transport to the contralateral chamber.
  • serum from a common bile duct ligation model rat add serum from obstructive jaundice to the transformed cells, and evaluate the ability to excrete hepatic impairment substances such as pyrilvin, cholic acid, and deoxycholic acid in the culture medium.
  • hepatic impairment substances such as pyrilvin, cholic acid, and deoxycholic acid in the culture medium.
  • a holofiber mass culture system coated with extracellular matrix an artificial liver, a liver failure material excretion system that substitutes for living liver, will be constructed.
  • the present invention provides a novel liver-specific organic anion transporter according to the present invention. And the DNA encoding it were provided. INDUSTRIAL APPLICABILITY The present invention is useful for producing a liver-permeable drug screening artificial liver.

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Abstract

L'invention concerne une nouvelle protéine qui est utile au dépistage de drogues susceptibles de pénétrer dans le foie et au développement d'un foie artificiel. L'invention se rapporte également à un ADN codant pour cette protéine. Plus particulièrement, l'invention se rapporte à une protéine comportant dans sa molécule une séquence d'acides aminés s'étendant de la position 1 à la position 691 de SEQ ID NO:2 dans la liste des séquences. Cette protéine peut servir à dépister des drogues susceptibles de pénétrer dans le foie et à développer un foie artificiel.
PCT/JP2000/003586 1999-06-03 2000-06-02 Nouveau gene transporteur exprime dans le foie WO2000075315A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001072798A2 (fr) * 2000-03-27 2001-10-04 Bayer Aktiengesellschaft Regulation de la proteine humaine apparentee a oatp2
WO2003076617A1 (fr) * 2002-03-14 2003-09-18 Japan Science And Technology Agency Transporteur transportant selectivement un conjugue de sulfate et son gene

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000008157A2 (fr) * 1998-08-07 2000-02-17 Axys Pharmaceuticals, Inc. Genes humains transporteurs d'anions atnov

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000008157A2 (fr) * 1998-08-07 2000-02-17 Axys Pharmaceuticals, Inc. Genes humains transporteurs d'anions atnov

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EMMANUEL JACQUEMIN ET AL.: "Expression cloning of a rat liver Na+-independent organic anion transporter", PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 133 - 137, XP002931742 *
GERD A. KULLAK-UBLICK ET AL.: "Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver", GASTROENTEROLOGY, vol. 109, 1995, pages 1274 - 1282, XP002931741 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001072798A2 (fr) * 2000-03-27 2001-10-04 Bayer Aktiengesellschaft Regulation de la proteine humaine apparentee a oatp2
WO2001072798A3 (fr) * 2000-03-27 2002-07-25 Bayer Ag Regulation de la proteine humaine apparentee a oatp2
WO2003076617A1 (fr) * 2002-03-14 2003-09-18 Japan Science And Technology Agency Transporteur transportant selectivement un conjugue de sulfate et son gene

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