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WO2004113533A1 - Proteine a variant d'epissage du recepteur alpha x du foie, gene codant pour ladite proteine et utilisation associee - Google Patents

Proteine a variant d'epissage du recepteur alpha x du foie, gene codant pour ladite proteine et utilisation associee Download PDF

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Publication number
WO2004113533A1
WO2004113533A1 PCT/JP2004/009227 JP2004009227W WO2004113533A1 WO 2004113533 A1 WO2004113533 A1 WO 2004113533A1 JP 2004009227 W JP2004009227 W JP 2004009227W WO 2004113533 A1 WO2004113533 A1 WO 2004113533A1
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receptor
liver
mutant protein
amino acid
polynucleotide
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Japanese (ja)
Inventor
Yoshikazu Naito
Kenji Oeda
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Sumitomo Chemical Co Ltd
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Sumitomo Chemical Co Ltd
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a liver X receptor splicing mutant protein, which is an isoform of liver X receptor, its gene and use thereof.
  • Cholesterol is an important lipid in the body and is also a component of various lipids. Cholesterol is absorbed from the intestinal tract by ingestion or biosynthesized by acetyl-Co A in the liver. Biosynthesized cholesterol is excreted from the liver, then reabsorbed in the small intestine, transported to the liver via blood, and reused. In M, some cholesterol is metabolized to bile acids. If the cholesterol level in the body rises due to any abnormality, hyperlipidemia derived from hypercholesterolemia causes atherosclerosis. The pathogenesis of these high cholesterol-derived diseases has not been fully elucidated. At present, cholesterol synthesis inhibitors (sutatins) and fibrates are used as therapeutic agents for diseases caused by hypercholesterolemia. However, treatment with these drugs is not always satisfactory in some cases.
  • Cholesterol reabsorption and conversion to bile acids are carried out by each specific transporter or enzyme. Furthermore, it is known that the liver X receptor, which is one of the nuclear receptors, is involved in the regulation of the expression of genes encoding these proteins.
  • Liver X receptor has been reported to have two subtypes (liver X receptor a, liver X receptor i3) (eg, Peet et al., Curr. Op in. Genet. Dev. 8; 571-575, 1998).
  • the liver X receptor forms a heterodimer with one of the nuclear receptors, retinoid X receptor (RXR), and regulates transcription of the target gene by binding to the transcriptional regulatory region of the target gene. It has been known.
  • Liver X Subtypes and isoforms have also been found in nuclear receptors other than the body.
  • RAR, RXR and PPAR three subtypes and isoforms in which the proteins encoded by these subtypes are altered (See, for example, Mange 1 s do rf and Evans, Cell, 83: 841-850, 1995).
  • RAR and PPAR isoforms are thought to result from alternative splicing and Z or control of different promoters and are known to differ in their tissue specificity and role in their expression (e.g., (See Takeyama et al., Biochem. Biophys. Res. Co. un. 222; 395-400, 1996).
  • liver X receptor which plays an important role in the regulation of cholesterol metabolism, if there is an isoform that functions to alter normal cholesterol metabolism by normal liver X receptor Isoforms are important for elucidating the association with pathological symptoms whose function is directly or indirectly involved, and for developing pharmaceuticals for the prevention or treatment of those diseases. Disclosure of the invention.
  • the present invention relates to a liver X receptor splicing mutant protein, a gene thereof and use thereof, which is a novel liver X receptor isoform involved in inhibition of normal cholesterol metabolism by normal liver X receptor. provide.
  • Liver X receptor splicing which is a glycoform of liver X receptor ⁇ , wherein at least a part of the amino acid sequence encoded by exon 5 of liver X receptor ⁇ gene is deleted.
  • Mutant protein hereinafter, also referred to as splicing mutant protein of the present invention
  • the amino acid sequence encoded by exons other than exon 5 is substantially the same as that of normal liver X receptor, and the liver X receptor splicing variant protein according to item 1 above;
  • liver X receptor according to item 2 above, which has any of the following amino acid sequences: Splice variant protein;
  • liver X receptor according to any one of items 1 to 3, wherein the normal iff organ X receptor ⁇ has a transcription activation ability lower than that of a ligand, Splicing variant protein;
  • a polypeptide comprising an amino acid sequence comprising two amino acid residues newly linked by deletion of a partial region of the encoded amino acid sequence hereinafter sometimes referred to as the polypeptide of the present invention
  • a method for producing a vector comprising a step of introducing into a vector capable of autonomous replication in a cell;
  • a method for producing a JR visceral X receptor ⁇ -splicing mutant protein comprising culturing the transformant according to the preceding paragraph 1 2 to produce a liver X receptor splicing mutant protein;
  • a polynucleotide having a base sequence encoding the amino acid sequence of the polypeptide according to 7 or 8 above (hereinafter also referred to as the polypeptide gene of the present invention), or the polynucleotide according to 9 or 10 above,
  • a recombinant vector comprising:
  • the method includes contacting a test substance with the liver X receptor ⁇ -splicing mutant protein according to any one of 1 to 4 above, and measuring a change in the liver X receptor splicing mutant protein.
  • a screening method for substances acting on liver X receptor splice variant protein hereinafter referred to as the screening method of the present invention fc;
  • the liver X receptor according to any one of 1 to 4 above, 0: a step of bringing a test substance into contact with a cell producing a splicing mutant protein, the liver X receptor in the cell measuring the production of other proteins whose expression is regulated by the ⁇ -splicing mutant protein, and evaluating the action of the test substance on the JFF visceral X receptor ⁇ -splicing mutant protein based on the production.
  • a cell that produces the published X receptor splicing mutant protein according to any one of 1 to 4 above A first step of contacting the liver X receptor a splicing mutant protein with a ligand and measuring the production amount of the other protein whose expression is regulated by the liver X receptor ⁇ splicing mutant protein; Any of the liver X receptor ⁇ splicing mutant protein described in 4 above, Liver X-receptor splicing mutant protein ligand and test substance are contacted, and the amount of other proteins that are regulated by the monthly X-receptor ⁇ -splicing mutant protein.
  • a method for screening a substance that acts on a liver X receptor a splicing mutant protein comprising:
  • liver X receptor ⁇ -splicing mutant protein selected by the screening method described in 18 above;
  • RNA probe for the nucleotide sequence of the polynucleotide according to item 9 or 10 for gene therapy;
  • a method for detecting the presence or absence of a Jff visceral X receptor splicing mutant protein according to any one of 1 to 4 above, wherein the messenger RNA (DiRNA) is present in a biological sample A first step of synthesizing a complementary DNA (cDNA), encoding the amino acid sequence of any one of the liver X receptor splicing mutant proteins according to 1 to 4 or the polypeptide according to any of 7 or 8 above. A second step of amplifying a partial region of the complementary DNA (cDNA) corresponding to the polynucleotide having the base sequence or the polynucleotide of the above item 9, and a third step of detecting the amplified partial region Method;
  • cDNA complementary DNA
  • the second step it is selected from the polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
  • a polynucleotide having the base sequence encoding the amino acid sequence of the moon spleen X receptor splicing mutant protein according to any one of 1 to 4 or the polypeptide according to 7 or 8 above, or 9 above The method according to item 27, wherein the polynucleotide is a polynucleotide having the base sequence represented by SEQ ID NO: 4;
  • a method for detecting the presence or absence of a jff visceral X receptor ⁇ -splicing mutant protein according to any one of 1 to 4 above, wherein the liver X receptor splicing mutant according to any one of 1 to 4 above A method comprising detecting the liver X receptor ⁇ splicing mutant protein based on an antigen-antibody reaction using an antibody or an antibody capable of specifically detecting the polypeptide of item 7 or 8;
  • the second step of binding the liver X receptor splicing variant protein to the first antibody on the microarray-well, after the second step, removing all unbound biological samples in the microarraywell Three steps, labeled liver X receptor 0; a labeled second antibody capable of binding to an epitope different from the first antibody in the splicing mutant protein , Fourth step of binding to a liver X receptor-splicing mutant proteins bound to the first antibody After the fourth step, after removing the second unbound antibody in the microtiter well, the fifth step, and after the fifth step, the label of
  • liver X receptor splicing variant protein is the liver X receptor ⁇ splicing variant protein according to the above items 2 to 4;
  • polynucleotide having the base sequence encoding the amino acid sequence of liver X receptor ⁇ -splicing mutant protein is the polynucleotide according to 5, 6, or 10 above. ;
  • FIG. 1 is a diagram schematically showing the structural domains of a normal liver X receptor gene and a liver X receptor gene variant protein gene found by the present invention. The translation area is shown in black.
  • FIG. 2 shows the results of examining the expression of normal liver X receptor ⁇ in normal tissues.
  • the figure shows a primer composed of a base sequence represented by SEQ ID NO: 9 and a primer composed of a base sequence represented by SEQ ID NO: 10 designed to specifically amplify normal liver X receptor. It shows the PCR product amplified by 30 cycles of PCR.
  • the PCR product derived from the normal liver X receptor is a DNA fragment with 430 base pairs.
  • the PCR product obtained by the PCR reaction was subjected to cloning and the nucleotide sequence was determined to confirm that the target product was amplified.
  • Figure 3 shows the liver X receptor splicing variant protein found by the present invention. It is the figure which showed the result of having investigated the expression in normal tissue.
  • the figure shows a primer consisting of the nucleotide sequence shown in SEQ ID NO: 9 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 11 designed to specifically amplify the liver X receptor splice variant protein.
  • It shows the PCR product amplified by 30 or 35 cycles of PCR performed using.
  • the PCR product derived from the liver X receptor splicing mutant protein is a DNA fragment having 340 base pairs.
  • the PCR product obtained by the PCR reaction was subjected to TA cloning and then its nucleotide sequence was determined to confirm that the target product was amplified.
  • Figure 4 shows the results of examining the expression of normal liver X receptor in normal tissues. This figure is carried out using a primer consisting of the base sequence represented by SEQ ID NO: 9 and a primer consisting of the base sequence represented by SEQ ID NO: 10 designed to specifically amplify normal liver X receptor.
  • PCR products amplified by 30 cycles of PCR are shown.
  • the PCR product derived from normal liver X receptor is a DNA fragment with 430 base pairs.
  • the PCR product obtained by the PCR reaction was subjected to TA cloning, and the nucleotide sequence was determined to confirm that the target product was amplified.
  • FIG. 5 is a view showing the results of examining the expression in a normal tissue of the liver X receptor ⁇ -splicing mutant protein found by the present invention.
  • the figure consists of a primer consisting of the base sequence shown by SEQ ID NO: 9 and the base sequence shown by SEQ ID NO: 11 designed to specifically amplify the liver X receptor ⁇ splicing mutant protein. It shows the PCR product amplified by PCR of 30 or 35 cycles performed using primers.
  • the PCR product derived from the liver X receptor splice variant protein is a DNA fragment having 340 base pairs.
  • the PCR product obtained by the PCR reaction was subjected to TA cloning and then its nucleotide sequence was determined to confirm that the target product was amplified. .
  • Figure 6 shows the results of the functional evaluation of the F : X-receptor splice variant protein found in the present invention by reporter assembly in a transient expression system in HEK293 cells. is there. TATA- LXREx2-luci f erase vector and normal liver X receptor (L XR aWT) expression vector or JfF visceral X receptor splicing mutant protein (LXR Fig. 6 shows the results of measuring the transcriptional activation ability when the expression vector was introduced into HEK293 cells and the liver X receptor synthetic agonist T0901317 was added. The number below the graph represents the final concentration of ⁇ 09013 ⁇ added. Each test plot contains 1 ⁇ 9 9-cis retinoic acid. The error line represents the standard deviation.
  • Fig. 7 shows that in HEK293 cells, the normal liver X receptor (LXR a WT) expression vector coexists with the liver X receptor ⁇ -splicing mutant protein (LXR a Vl) expression vector found by the present invention. It is the figure which showed the result of having measured the transcriptional activation ability when letting it be made.
  • Each test plot contains 1 pM 9-cis retinoic acid.
  • the luciferase activity value when using cells transfected with the vector 1 plasmid PFLAG-CMV2 was shown as 1, and the luciferase activity value in each test group was shown as a relative value.
  • the left figure in Fig. 7 shows the results when ethanol was added to the cells, and the right figure in Fig. 7 shows the results when 5090 g / ml T0901317 was added to the cells.
  • the error line represents the standard deviation.
  • the present invention relates to a liver X receptor splicing variant protein which is a glycoform of liver X receptor, a gene thereof, and use thereof.
  • the splicing variant protein of the present invention is an isoform of liver X receptor ⁇ , and has an amino acid sequence in which at least a partial region of the amino acid sequence encoded by exon 5 is deleted. More specifically, the splicing variant protein of the present invention differs from the normal liver X receptor at least with respect to the amino acid sequence encoded by exon 5, and is a partial region of the amino acid sequence encoded by exon 5. And has an amino acid sequence including at least a partial region of the amino acid sequence encoded by exon 5 other than the partial region.
  • the reading frame of the translation region downstream of exon 5 is exactly the same as that of the normal type, that is, the amino acid sequence encoded in the translation region downstream of exon 5 is the same as that of the normal type Jff organ X receptor. Preferably it is an amino acid sequence.
  • a typical example is the liver X receptor ⁇ isoform encoded in the translation region lacking the 5'-terminal 90 base pairs of exon 5 of the liver X receptor gene. Can do.
  • the splicing variant protein of the present invention includes (1) an amino acid sequence represented by SEQ ID NO: 1, (2) an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, and (3 ) Those having any one of the amino acid sequences having 95% or more amino acid identity to the amino acid sequence represented by SEQ ID NO: 1
  • Such a splicing variant protein of the present invention has a transcription activation ability lower than that of a ligand-activated transcription activation of a normal liver X receptor.
  • T0901317 for example, one of the ligands of liver X receptor ⁇ possessed by the ffF visceral X receptor ⁇ splicing mutant protein shown in (1) above
  • the transcriptional activation ability by) was very weak compared to the transcriptional activation ability of normal liver X receptor tracts.
  • liver X receptor splicing mutant protein is involved in the inhibition of normal cholesterol metabolism by normal liver X receptor a in a dominant negative manner in vivo.
  • substantially identical amino acid sequence in general, when the amino acid sequence of a physiologically active protein is slightly changed, for example, one or more amino acids in the amino acid sequence are deleted.
  • substantially identical As long as the biological activity substantially equivalent to a specific amino acid sequence (that is, the amino acid sequence represented by SEQ ID NO: 1) is maintained, one or more amino acids in the amino acid sequence are deleted. It is meant that a liver X receptor splice variant protein substituted or added (eg, derived from human) is also included in the scope of the present invention. As for the number of amino acids modified in the above, Both are 1 residue, specifically 1 or several (here, “several” is about 2 to about 10), or more.
  • the number of such modifications may be in a range where the physiological activity of the protein is maintained. More specifically, 1 to 20 amino acids, preferably 1 to 10 amino acids, more preferably 1 to 5 amino acids in the amino acid sequence represented by SEQ ID NO: 1 are deleted, substituted or added.
  • a liver X receptor splicing variant protein eg, from human. Such a mutation may be a naturally occurring mutation due to, for example, the processing that the protein undergoes in the cell, the species difference of the organism from which the protein is derived, the individual difference, the organ, the tissue difference, etc.
  • Amino acid mutations for example, amino acids present in the amino acid sequence of proteins produced by introducing and expressing mutations in DNA encoding natural proteins by site-directed mutagenesis or mutagenesis
  • Mutation Such a mutant protein resulting from deletion, substitution or addition of amino acids may contain a conservatively substituted amino acid sequence.
  • a specific amino acid residue may be replaced by a residue having physical-physical similarity (for example, hydrophobicity, charge, pK: similar characteristics such as conformational features, etc.) Means.
  • Non-limiting examples of such conservative substitutions are: (1) glycine, alanine; (2) parin, isoleucine, oral isine; (3) aspartic acid, glutamic acid, asparagine, glutamine, (4) serine, threonine; (5) lysine, arginine; Examples include substitution between aliphatic chain-containing amino acid residues and substitution between polar groups, such as substitution within the group of phenylalanine and tyrosine.
  • Mutant proteins resulting from amino acid deletions, substitutions or additions can be obtained by, for example, site-directed mutagenesis, which is a known technique for a gene having a base sequence encoding the amino acid sequence (for example, Nel son and McCllell and, Methods EnzymoK 216; 279, 1992, etc., method using amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456, 1984), PCR method using a primer for mutagenesis) It can be obtained by performing. Site-directed mutagenesis can be performed using a synthetic primer containing the mutation to be introduced.
  • site-directed mutagenesis which is a known technique for a gene having a base sequence encoding the amino acid sequence (for example, Nel son and McCllell and, Methods EnzymoK 216; 279, 1992, etc., method using amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456, 1984),
  • an amplification reaction is carried out using a plasmid containing the gene for the normal liver X receptor ⁇ as a cage.
  • Dpn I a methylation sensitive restriction enzyme
  • leaves only DNA with the newly formed mutation Using this reaction solution, E. coli XLI_Blue is transformed and plated on LB agar medium containing ampicillin. Incubate at 37 ° C and isolate the plasmid from the grown colonies. Thereby, a plasmid containing the mutated DNA can be obtained.
  • kits for example QuickChange Site-Dimuted Mutagenes is Kit (manufactured by Stratagene) is sold, and these may be used. The introduction of the target mutation can be confirmed by determining its nucleotide sequence.
  • methods for deleting, substituting, or adding amino acid sequences include the methods of treating genes with mutagens or the genes selected by cleaving genes with restriction enzymes. Examples thereof include a method of removing, adding or replacing fragments, and then ligating.
  • the term “normal liver X receptor” means the monthly collection X receptor that consists of the most frequently occurring amino acid sequence in the amino acid sequence of the receptor protein derived from the same species of organism. .
  • liver X receptor consisting of an amino acid sequence (GenBank Accession No. NM-005693) registered in a public database can be mentioned.
  • amino acid identity and “base identity” refer to sequence identity and homology between two proteins or two DNAs. The “identity” is determined by comparing two sequences aligned optimally over the entire region of the sequences to be compared.
  • the protein or DNA to be compared is the optimal sequence of the two sequences.
  • the inment may have an addition or a deletion (for example, a gap).
  • identity is calculated, for example, by creating an alignment using the Clus talW algorithm (Nucleic Acid Res.
  • amino acid identity in the present invention is an amino acid sequence standard, for example, about 95% or more.
  • base identity is based on the base sequence, and is preferably about 95% or more, for example. It can be obtained by the usual genetic engineering method such as.
  • RNA is extracted according to the genetic engineering method, and single-stranded cDNA is synthesized. Specifically, for example, tissue such as liver is crushed in a solution containing a protein denaturant such as guanidine thiocyanate, and protein is denatured by adding black mouth form or the like to the crushed material. Remove the denatured protein by centrifugation, etc., and then extract total RNA from the collected supernatant fraction using phenol, chloroform, etc. Examples of commercially available kits based on these methods include IS0GEN (manufactured by Futtsubon Gene) and TRIZ0L reagent (manufactured by Invitrogen).
  • IS0GEN manufactured by Futtsubon Gene
  • TRIZ0L reagent manufactured by Invitrogen
  • RNA is in a saddle shape, and an oligo dT primer is bound to the mRNA polyA sequence, and a reverse transcriptase such as RNaseH-Superscript II Reverse Transcriptase (Invitrogen) and the attached buffer and oligo are attached.
  • a reverse transcriptase such as RNaseH-Superscript II Reverse Transcriptase (Invitrogen) and the attached buffer and oligo are attached.
  • nick the mRNA strand with RNase H synthesize the double-stranded cDNA with E. coli DNA polymerase I using the single-stranded cDNA as a saddle.
  • the ends of the resulting double-stranded cDNA are smoothed with T4 DNA polymerase.
  • the blunted double-stranded cDNA is inserted into a vector such as pBluescript II vector Bacteriophage, eg, Agtll, EMBL3, etc., using T4 ligase to create a cDNA library.
  • a vector such as pBluescript II vector Bacteriophage, eg, Agtll, EMBL3, etc.
  • T4 ligase to create a cDNA library.
  • Examples of commercially available kits based on these methods include cDNA synthesis system plus (Amersham Biosciences) and TimeSaver cDNA synthesis kit (Amersham Biosciences).
  • Hybridization is performed from the cDNA library thus prepared using, for example, a DNA having a partial base sequence of the base sequence represented by SEQ ID NO: 2 or 4 as a probe.
  • Hybridization conditions may include conditions that allow hybridization under stringent conditions.
  • Hybridization is described in, for example, Sambrook J., Frisch EF, Maniatis S., Molecular Cloning 2nd edition, and Co Id Spring Harbor Laboratory press. This can be done according to the usual method. “Stringent conditions” means, for example, that a hybrid was formed at 45 ° C. in a solution containing 6XSSC (a solution containing 1.5M NaCK 0.15M trisodium quenate as 10 XSSC). Then wash with 50x 2X SSC at 50 (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.
  • the salt concentration in the washing step is, for example, from about 50 ° C for 2 XSSC (low stringency) to about 50 ° C for 0.2 X SSC (high stringency). Condition).
  • the temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C (high stringency conditions). Also change both salt concentration and temperature You can also.
  • the signal is detected by an X-ray film (for example, Hyperfilm-MP; manufactured by Amersham Bioscience) or a bioimaging system (BAS-2000; manufactured by Fuji Film), and a vector having a base sequence that binds to the probe is obtained. Recombinants containing can be obtained.
  • an X-ray film for example, Hyperfilm-MP; manufactured by Amersham Bioscience
  • a bioimaging system for example, BAS-2000; manufactured by Fuji Film
  • two may be selected from the base sequence represented by SEQ ID NO: 2 or 4 so as to satisfy the following conditions, for example.
  • the primer length is 15 to 40 bases, preferably 20 to 30 bases.
  • the ratio of guanine to cytosine in the primer is 40% to 60%, preferably 45% to 55%, more preferably 50% to 55%.
  • the distance on the base sequence of the gene corresponding to the selected primer is preferably 100 to 3000 bases, more preferably 100 to 500 bases.
  • each primer itself or two primers. If the base sequence of the primer is selected, it may be chemically synthesized using a commercially available DNA synthesizer.
  • a combination using a primer consisting of the base sequence shown in SEQ ID NO: 7 as the sense primer and a primer consisting of the base sequence shown in SEQ ID NO: 8 as the antisense primer can be mentioned.
  • primers are added to the reaction solution to 200 nM, and the single-stranded cDNA synthesized above is converted into a cage shape.
  • LA Taq DNA polymerase (Takara Shuzo) PCR) using the reaction buffer supplied with the enzyme.
  • 94 cycles at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute are performed for about 35 cycles.
  • cDNA synthesized as described above commercially available cDNA derived from various animals such as Clontech's Quick Clone cDNA may be used. Obtained reaction liquid A part of the sample is analyzed by agarose gel electrophoresis, and the target band is cut out directly or from the gel, and then cloned into pGEM-T Easy (Promega) using the TA cloning system. The nucleotide sequence of the inserted DNA fragment can be determined and confirmed by the dye terminator method.
  • the polynucleotide having the nucleotide sequence encoding the amino acid sequence of the splicing variant protein of the present invention thus obtained (that is, the splicing variant protein gene of the present invention) is used as a vector that can be used in the host cell to be transformed.
  • the splicing mutant protein gene of the present invention is a vector that can replicate autonomously in a host cell, can be isolated and purified from host cells, and has a detectable marker.
  • a vector containing the splicing mutant protein gene of the present invention can be constructed by integration according to a normal genetic engineering technique.
  • Escherichia coli when Escherichia coli is used as a host cell, for example, plasmid pUC19 (Takara Shuzo), pBluescript II (Stratagene) Etc.).
  • plasmids PACT2 (Clontech), PYES2 (Invitrogen) and the like can be mentioned.
  • plasmids such as pRc / RSV and pRc / CMV (Invitrogen) can be used.
  • a splicing mutant protein of the present invention is linked upstream of the splicing mutant protein gene of the present invention in a functional form in a host cell that can function in a host cell, and incorporated into the above-described vector.
  • a vector capable of expressing a quality gene in a host cell can be constructed.
  • “to bind in a functional manner” means that the splicing mutant protein gene of the present invention is expressed under the control of a promoter in a host cell into which the splicing mutant protein gene of the present invention is introduced. It means that the promoter and the splicing mutant protein gene of the present invention are combined.
  • the promoter used is the promoter in the host cell to be transformed.
  • the host cell when the host cell is Escherichia coli, one of the promoters such as Lactobacillus operoper promoter, tac promoter, T7 promoter, etc. of Escherichia coli can be mentioned.
  • the host cell when the host cell is an animal cell, examples thereof include a rous sarcoma virus (RSV) promoter, a cytomegalovirus (CMV) promoter, and a simian virus (SV40) promoter.
  • examples include alcohol dehydrogenase (ADH) 1 gene promoter and galactose metabolizing enzyme (GAL) 1 gene promoter.
  • the promoter possessed by the vector and the splicing variant protein gene of the present invention are linked in a functional manner.
  • the splicing variant protein gene of the present invention may be inserted downstream of the promoter.
  • the plasmids pRc / SV, pRc / CMV, etc. described above have a cloning site downstream of a promoter that can function in animal cells, and the splicing mutant protein gene of the present invention is inserted into the cloning site.
  • the splicing mutant protein gene of the present invention can be expressed.
  • the aforementioned budding yeast plasmid PACT2 has the ADH1 promoter, and the splicing mutant of the present invention can be obtained by inserting the splicing mutant protein gene of the present invention downstream of the ADH1 promoter of the plasmid or its derivative.
  • the recombinant vector of the present invention capable of expressing a large amount of protein genes in budding yeast such as CG1945 strain (Clontech) can be constructed.
  • the transformant of the present invention can be obtained by introducing the splicing mutant protein gene of the present invention or the recombinant vector of the present invention into the host cell.
  • a normal introduction method according to the host cell to be transformed can be applied.
  • E. coli a microorganism
  • the calcium chloride method described in Molecular Cloning 3rd Edition (Sambrook and Russel K Cold 'Spring' Hyper Laboratory, 2001), etc.
  • a usual method such as an electro-boration method can be used.
  • It also contains mammalian cells or insect cells.
  • it can be introduced into the cell by a general gene transfer method such as a calcium phosphate method, an electopore position method, or a lipofection method.
  • the splicing mutant protein gene of the present invention may be expressed using yeast as a host cell.
  • budding yeast eg, Saccharomyces cerevisiae
  • yeast such as Pichia may also be used.
  • Examples of a method for transforming yeast include the method of Ito et al. (J. Bacter io. 153; 163-168, 1983).
  • a transfer vector containing a base sequence homologous to the genome of the virus to be used can be used.
  • transfer vectors include plasmids such as pVL1392 and PVL1393 (Invitrogen).
  • viral genomes such as baculovirus and adenovirus can be used.
  • a virus When a virus is used in a vector, it is possible to introduce viral DNA into a host cell by a general gene transfer method as described above, or by infecting a recombinant virus directly into the host cell. Viral DNA can be introduced into host cells.
  • the splicing mutant protein of the present invention can be prepared as a natural protein by operations such as extraction and purification from a naturally occurring organism, or can be prepared as a recombinant protein using genetic engineering techniques. it can.
  • a purified protein can be prepared by preparing a crude extract from human cells or tissues and using various columns.
  • the cells here are not particularly limited as long as they produce and express the splicing mutant protein of the present invention.
  • liver-derived cells, kidney-derived cells and the like can be used.
  • the splicing mutant protein of the present invention Those that are produced and expressed in non-human organisms can be prepared from the organism.
  • the splicing mutant protein gene of the present invention or the recombinant vector of the present invention is transformed into an appropriate host cell as described above, and the transformant (that is, the present invention).
  • the transformant may be cultured to produce the liver X receptor ⁇ -splicing mutant protein.
  • the produced liver X receptor splice variant protein is recovered according to the usual method.
  • the recovered splicing mutant protein of the present invention is purified by an appropriate method according to the purpose.
  • the transformant of the present invention when the transformant of the present invention is a microorganism, the transformant includes various media appropriately containing a carbon source, a nitrogen source, an organic salt, an inorganic salt, and the like used for normal culture in general microorganisms. Is used to culture. Cultivation is performed according to the usual method for general microorganisms, and solid culture, liquid culture (test tube shaking culture, reciprocating shaking culture, Jar Fermenter culture, tank culture, etc.) are possible. It is.
  • the culture temperature can be appropriately changed within the range where the microorganism grows. For example, the culture temperature is generally about 15 ° C.
  • the culture time varies depending on the culture conditions, but is usually about 1 hour to about 24 hours. If you are using an inductive promo, the induction time should be within one day, usually a few hours.
  • the transformant when the transformant is an animal cell such as a mammal or an insect, the transformant can be cultured using a medium used for normal culture in general cultured cells.
  • a medium used for normal culture in general cultured cells In the case of animal cells, for example, a liquid medium supplemented with fetal bovine serum (FBS) so that the final concentration is about 5% (v / v) to about 10% (v / v). (Invitrogen, etc.) may be used under the conditions of 37 ° C, 5% CO 2 and the like.
  • FBS fetal bovine serum
  • the cells have grown to confluence, for example, add approximately 0.25 (v / v) trypsin / PBS solution to disperse the cells, dilute several times, seed in a new petri dish, and continue culturing. .
  • a culture temperature of about 25 ° C. to about 30 Incubate at ° C.
  • recombinant viral vectors such as baculovirus
  • the splicing mutant protein of the present invention produced by the transformant of the present invention may be collected by appropriately combining ordinary isolation and purification methods. For example, after completion of the culture, the cells of the transformant are centrifuged. The collected cells are suspended in a normal buffer, for example, PBS containing an appropriate protease inhibitor, and then disrupted by sonication, Dounce homogenizer, etc., and the disrupted solution is 20,000 X g Then, the fraction containing the splicing mutant protein of the present invention can be obtained by centrifuging for about 1 hour to tens of minutes and collecting the supernatant fraction.
  • a normal buffer for example, PBS containing an appropriate protease inhibitor
  • the spliced variant protein of the present invention which is a more purified object, can be recovered from the supernatant fraction by subjecting it to various chromatographies by a normal protein purification technique.
  • the polypeptide of the present invention can be recovered by the same method as described above instead of the splicing mutant protein of the present invention.
  • the splicing mutant protein of the present invention produced in this way can be used, for example, as a ligand / receptor binding assay for evaluating the binding ability and binding amount of the test substance and the splicing mutant protein of the present invention. Can be used.
  • the screening method for substances acting on the iff visceral X receptor splicing mutant protein includes the steps of contacting the test substance with the F visceral X receptor splicing mutant protein and the liver X receptor splicing mutant. A step of measuring a change in the protein.
  • the splicing mutant protein of the present invention can be used as the liver X receptor ⁇ -splicing mutant protein.
  • the concentration of the test substance to be contacted with the liver X receptor splicing mutant protein is usually about 0.1 M to about 10 and preferably 1 / ⁇ to 10 ⁇ .
  • the contact time between the liver X receptor splicing variant protein and the test substance is usually 18 hours to 60 hours, preferably 24 hours. About 40 hours.
  • the ligand 'receptor binding assay using the splicing variant protein of the present invention is a test method capable of measuring the binding ability of a chemical substance to the protein and quantifying the binding amount, as well as analyzing the binding specificity and binding force. is there.
  • the test substance and Labeled ligand is released from the splicing mutant protein of the present invention and binds to the splicing mutant protein of the present invention depending on the affinity for both liver X receptor ⁇ splicing mutant protein due to competition with the labeled ligand.
  • the amount of labeled ligand thus reduced is reduced, and hence the amount of label bound to the splicing variant protein of the present invention is reduced. Therefore, the ability of the test substance to bind to the splicing variant protein of the present invention can be indirectly determined by monitoring the amount of labeled labeled ligand or the amount of labeled labeled ligand.
  • the labeled ligand for example, tritium-labeled oxycholesterol can be used.
  • the reaction system is roughly divided into three groups.
  • One system is a group in which only the solvent is added to the place where the labeled ligand is bound to the splicing mutant protein of the present invention, which corresponds to a system in which the concentration of the test substance is zero, and obtained from this system.
  • the labeling amount of the binding-type labeling ligand obtained represents the total binding amount of the labeling ligand to the splicing variant protein of the present invention.
  • the other system is where the labeled ligand is bound to the splicing mutant protein of the present invention.
  • an unlabeled oxycholesterol sufficiently saturates and labels the splicing mutant protein of the present invention.
  • the concentration of the labeled ligand obtained from this system is such that the concentration of the labeled ligand against the splicing mutant protein of the present invention is such that the concentration is such that the ligand cannot be bound (for example, 10 ⁇ ).
  • Specific binding amount is determined. Therefore, the specific binding amount of the labeled ligand to the splicing mutant protein of the present invention is a value obtained by subtracting the latter non-specific binding amount from the former total binding amount.
  • the third system is where the labeled ligand is bound to the splicing variant protein of the present invention. (This concentration is arbitrarily changed according to the purpose.)
  • the amount of the labeled labeled ligand obtained from this system is zero when the concentration of the test substance determined as described above is zero.
  • the specific binding amount of the labeled ligand to the splicing variant protein of the present invention at this time is smaller.
  • the binding ability of the test substance to the splicing variant protein of the present invention can be examined, and when the test substance contains a plurality of substances, It is also possible to examine whether or not there is a substance showing affinity for the splicing mutant protein of the present invention.
  • the binding concentration of the test substance in the third system is changed in the same manner and the binding label is used. Measure the labeled amount of the ligand. Based on the measured values, the amount of bound and free ligands in each assembly can be calculated to evaluate the binding affinity, binding specificity, binding capacity, etc.
  • liver X receptor ⁇ -splicing mutant protein activates the transcription of a gene under the control of the transcriptional regulatory region containing the binding sequence of liver X receptor is, for example, the liver X receptor Using a reporter gene ligated downstream of the transcriptional control region containing the binding sequence, it can be evaluated by a test method such as reporter assay described later.
  • Liver X receptor ⁇ forms a heterodimer with retinoid X receptor (RXR), one of the nuclear receptors, and binds to the transcriptional regulatory region containing the binding sequence of liver X receptor ⁇ . Transcription of a gene under the control of the transcription control region is controlled.
  • RXR retinoid X receptor
  • the binding sequence of the liver X receptor chain is a specific sequence (cis sequence) of a transcriptional regulatory region containing a core motif having a structure in which "AGGTCA" (or a related 6 nucleotide motif) is aligned in the same direction.
  • AGGTCA a specific 6 nucleotide motif
  • the spacing between core motifs varies depending on the heterodimer combination, and is 4 bp (DR4) in the case of a heterodimer of liver X receptor and retinoid receptor (eg, Lehman et al., J. Biol. Chem., 272; 3137-3140, 1997.).
  • the liver X receptor splicing mutant protein of the present invention is a pathological condition in which the liver X receptor splicing mutant protein and its function are directly or indirectly involved. It is extremely useful for elucidating the relevance of drugs and developing methods and drugs useful for the prevention, diagnosis and treatment of these pathological symptoms.
  • By the screening method as described above it is possible to search for substances that act on the liver X receptor splicing mutant protein and antagonists to the substance, and to provide these substances as drug candidates. It becomes possible.
  • An effective amount of a drug selected from the screening method of the present invention or an antagonist to the substance as an active ingredient can be orally or parenterally administered to a mammal such as a human.
  • the drug when administered orally, can be used in a normal form such as a tablet, capsule, syrup, suspension or the like.
  • the drug when administered parenterally, can be used in the form of a normal solution such as a solution, emulsion, suspension or the like.
  • a method for parenterally administering the drug in the form of the present fat accumulation regulator include a method of injection and a method of administering to the rectum in the form of a suppository.
  • the appropriate dosage form is obtained by blending a substance selected by the screening method of the present invention or an antagonist to the substance into an acceptable normal carrier, excipient, binder, stabilizer, diluent and the like. Can be manufactured.
  • acceptable buffering agents, solubilizing agents, isotonic agents and the like can be added.
  • the dose varies depending on the age, sex, weight, disease level of the mammal to be administered, type of the drug, dosage form, etc. lmg to about 2 g, preferably about 5 mg to about 1 g as the active ingredient amount, and in the case of injection, about 0.1 mg to about 50 O mg as the active ingredient amount in an adult Good.
  • the daily dose can be administered once or divided into several times. Monkey.
  • the relationship with the pathological condition in which the splicing variant protein of the present invention is directly or indirectly involved is that the ligand is first identified, and then the genes that are transcriptionally regulated by the ligand and the splicing variant protein of the present invention are identified. This can be clarified.
  • the ligand is identified by the expression level of a gene group that is transcriptionally regulated by the splicing variant protein of the present invention or the polypeptide of the present invention by bringing the test substance into contact with the splicing variant protein of the present invention or the polypeptide of the present invention. This can be done by detecting the change in.
  • a method for screening a substance that acts on a liver X receptor splicing mutant protein includes a step of bringing a test substance into contact with a cell producing liver X receptor ⁇ -splicing mutant protein, the JfF visceral X receptor splicing mutation
  • a method comprising measuring the production of another protein whose expression is regulated by a body protein, and evaluating the action of the test substance on the liver X receptor splice variant protein based on the production. It may be.
  • examples of cells that produce the liver X receptor ⁇ -splicing mutant protein include cells that produce the splicing mutant protein of the present invention.
  • the concentration of the test substance to be contacted with the cell producing the liver X receptor splicing mutant protein is usually about 0.1 M to about 10 M, and preferably 1 M to 10 M.
  • Liver X receptor ⁇ -splicing mutant Protein is usually contacted with the test substance for 18 hours to 60 hours, preferably about 24 hours to 40 hours. It is done.
  • the liver X receptor splicing mutant protein is added to the cell producing the liver X receptor ⁇ splicing mutant protein.
  • the first step of measuring the production amount of other proteins whose expression is regulated by the liver X receptor splicing mutant protein, the splicing mutant tamper of the present invention A cell producing a protein is contacted with both the liver X receptor splicing mutant protein ligand and the test substance, and the liver X receptor splicing mutant protein undergoes expression regulation by the liver X receptor splicing mutant protein.
  • a method having a process can also be mentioned.
  • Examples of the liver X receptor splicing mutant protein include the splicing mutant protein of the present invention.
  • the concentration of the ligand or test substance that is contacted with the cell producing the liver X receptor ⁇ -splicing mutant protein is usually about 0.1 ⁇ to about 10 M, and 1 ⁇ M to 10 M preferable.
  • the contact time between the cell producing the liver X receptor splice variant protein and the ligand or test substance is usually from 18 hours to 60 hours, preferably from 24 hours to 40 hours. Can be mentioned.
  • binding assays and the like can be performed using the splicing mutant protein of the present invention or a fragment thereof.
  • an expression system of the splicing mutant protein of the present invention and a target gene to which the liver X receptor splicing mutant protein binds is constructed, and an increase in the protein that is the target gene product due to the addition of a ligand is detected.
  • a reporter gene that expresses a reporter protein such as luciferase, chloramphenicol luciferyl transferase,) 3-galactosidase, etc. under the control of the promoter of the target gene, Presence or absence of expression of the target gene can be easily detected.
  • a chimeric gene consisting of a ligand binding region of the protein and a DM binding protein is expressed, and a base sequence to which a DNA binding protein binds as a repo overnight gene.
  • a plasmid in which a minimally active promoter and a gene encoding the aforementioned reporter protein are linked downstream can be used.
  • the DNA binding protein for example, GAL4, LexA and the like can be used.
  • An expression system of the splicing variant protein of the present invention and a target gene to which the liver X receptor splicing variant protein binds and a chimeric gene of the ligand binding region of the protein and a DM binding protein are generated.
  • the plasmid to be expressed can be prepared by ordinary gene recombination techniques.
  • expression was controlled by the splicing mutant protein of the present invention by transcribing labeled DM from mRNA expressed in the cells and hybridizing the obtained labeled DNA with a DNA library chip.
  • a gene can also be specified.
  • the relationship with the pathological condition in which the splicing mutant protein of the present invention is directly or indirectly involved is that the target gene when the antigenic or agonist is brought into contact with the cell expressing the splicing mutant protein of the present invention. It is possible to elucidate by identifying the gene to be activated by comparing the expression level of the target gene in the cell expressing the splicing variant protein of the present invention not contacted with the test substance.
  • examples include a second step of amplifying a partial region of the complementary DNA (cDNA) corresponding to the polypeptide gene of the invention, and a third step of detecting the amplified partial region.
  • Specific examples of the splicing mutant protein gene of the present invention or the polypeptide gene of the present invention include a polynucleotide having the base sequence represented by SEQ ID NO: 2 or 4.
  • a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
  • a step of amplification by a PCR method using one or a plurality of primers as primers can also be preferably mentioned.
  • the nucleotide sequence encoding the amino acid sequence of the splicing variant protein of the present invention Regarding experimental animals such as gene-deficient animals that lack the ability to express a polynucleotide having a sequence, etc., and their use, the gene having a base sequence that codes for the amino acid sequence of the splicing mutant protein of the present invention
  • a model animal is created by destroying (inactivating) a gene having a nucleotide sequence that encodes the amino acid sequence of an endogenous splicing mutant derived from an experimental animal that has a function.
  • the human-derived gene of the present invention into a model animal in which a gene having a base sequence encoding the amino acid sequence of the endogenous liver X receptor ⁇ -splicing mutant protein described above is destroyed (inactivated),
  • a drug compound etc.
  • the present invention further includes the use of a specific DNA probe for the base sequence of the polypeptide gene of the present invention for gene therapy, and the base sequence of the polypeptide gene of the present invention for gene therapy.
  • specific RNA probes are also provided.
  • the present invention also provides a diagnostic probe for a disease associated with inhibition of normal cholesterol metabolism by type I liver X receptor ⁇ , and a diagnostic agent for a disease associated with the disorder, comprising the probe. For example, by performing in situ hybridization of the splicing mutant protein gene of the present invention on a pathological section, it is possible to detect the presence or progression of a disease associated with the disorder.
  • the diagnostic probe is the whole or a part of the antisense strand of the splicing mutant protein gene of the present invention (DNA, RNA, cDNA), and has a length that is sufficient as a probe (at least 2). There is no particular limitation as long as it has 0 base or more.
  • the probe In order to use the probe as an active ingredient of a diagnostic agent, It is preferable to dissolve in an appropriate buffer or sterilized water that does not decompose.
  • Examples of the in situ hybridization method include the method described in J. Neurobiol. 29, 1-17 (1996). It is also possible to use the in situ PCR method.
  • a probe In the diagnosis, not only a probe but also an antibody (see below) that specifically recognizes the splicing variant protein of the present invention can be used.
  • normal liver X receptor can be obtained by immunostaining. It is possible to detect the presence or progression of diseases involved in the inhibition of normal cholesterol metabolism by ⁇ (Dev. Biol. 170, 207-222 (1995); J. Neurobio l. 29, 1-17 (1996). See)).
  • the antibody to be used can be prepared by a conventional method described in, for example, Ant ibodie s; A Laboratory Manual, Lane, H.D. et al., Col d Springing Harber Lboratory Press published New York 1989.
  • the splicing variant protein gene of the present invention or the polypeptide gene of the present invention itself is useful for use in gene therapy as an antisense drug that controls the function of the splicing mutant protein of the present invention at the gene level.
  • a base sequence of 20 bases or more contained in SEQ ID NO: 4, preferably a base sequence of about 30 bases can be used.
  • the above-mentioned antisense drugs include not only DNA but also RNA.
  • the base sequence may be a sense sequence or an antisense sequence (that is, a base sequence having complementarity to the sense sequence).
  • the polypeptide of the present invention and “the polypeptide gene of the present invention” are, as described above, the partial region (partial polypeptide) of the splicing mutant protein of the present invention or the splicing mutant protein gene of the present invention corresponding thereto.
  • a polypeptide comprising an amino acid sequence comprising two amino acid residues newly linked by deletion of at least a partial region of the amino acid sequence encoded by exon 5, which is a partial region (partial polynucleotide) Means the corresponding polynucleotide. Preferably it contains at least 6 consecutive amino acids or at least 18 consecutive bases. Good.
  • a typical example is the amino acid sequence of the liver X receptor ⁇ . Isoform encoded in the translation region of the liver X receptor ⁇ gene, exon 5 5 ′ end 90 base pairs deleted. Among them, a polypeptide consisting of a region not present in the normal iff organ X receptor ⁇ , a polynucleotide corresponding thereto, and the like can be mentioned.
  • polypeptide having the amino acid sequence represented by SEQ ID NO: 3 or a polynucleotide having the corresponding base sequence can be used.
  • a polynucleotide having a nucleotide sequence encoding the amino acid sequence of the above polypeptide or a recombinant vector containing the above polynucleotide may be prepared according to the above-described method for preparing the recombinant vector of the present invention.
  • a transformant can also be prepared by introducing the recombinant vector thus prepared into a host cell according to the aforementioned method for preparing a transformant of the present invention.
  • the nucleotide sequence represented by SEQ ID NO: 4 is a probe or primer that is a liver other than the splicing variant protein of the present invention. It can be used as a protein search tool.
  • the length is preferably a base sequence containing at least 15 consecutive bases.
  • the probe can be labeled by a conventional method, for example, with a radioisotope, digoxigenin, piotin, or a detectable enzyme.
  • the probe labeled in this manner is hybridized with a cDNA library for cloning. Hybridization can be carried out by conventional methods and conditions. For example, cleaning at 1XSSC, 0.5% (w / v) SDS, 65 ° C.
  • the cDNA library may be derived from animals including mammals, but is preferably derived from human tissues / cells. An antibody or a part of an antibody that specifically recognizes the splicing variant protein of the present invention can be prepared.
  • the splicing mutant protein of the present invention having the amino acid sequence represented by SEQ ID NO: 1 or 3 specifically recognizes the splicing mutant protein of the present invention obtained by, for example, the usual method of gene recombination described above. Or a partial region thereof (polypeptide), or a peptide fragment having the amino acid sequence represented by SEQ ID NO: 3, and animals such as rabbits, guinea pigs, rats, mice, goats, or hidges that are usually used to produce antibodies. It can be obtained by immunizing. A monoclonal antibody can also be obtained by fusing spleen cells and myeloma cells of an immunized mammal.
  • an antibody capable of specifically detecting the splicing mutant protein of the present invention or the polypeptide of the present invention is used.
  • examples thereof include a method comprising a step of detecting the liver X receptor splicing mutant protein based on the antigen-antibody reaction used.
  • a first step of binding a first antibody capable of specifically detecting the splicing variant protein of the present invention or the polypeptide of the present invention to a microarray well The second step of binding the splicing variant protein of the present invention to the first antibody on the microtiter well, after the second step, is in all unbound states within the microwell (ie free )
  • the third step of removing the biological sample, the labeled second antibody capable of binding to an epitope different from the first antibody in the liver X receptor splice variant protein is bound to the first antibody.
  • the fourth step to bind to the liver X receptor ⁇ -splicing mutant protein, after the fourth step, all non-binding in the microtiter well A fifth step of removing the second antibody in a state (ie, free), and a sixth step of detecting the presence or absence of the second antibody using the label of the second antibody as an indicator after the fifth step.
  • Examples include a method for detecting the presence or absence of the splicing mutant protein of the present invention.
  • Example The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
  • Example 1 (Cloning of Splicing Mutant Protein Gene of the Present Invention) Human Liver-derived cDNA library (SUPERSCRIPT Human Liver cDNA Library manufactured by Impitrogen) was used as a saddle type and human liver X receptor ⁇ gene exon 3 To 8 (region containing the ligand binding region) was amplified by PCR using a programmable thermal cycler. The primer is based on the nucleotide sequence of human JfF visceral X receptor ⁇ gene (Wily et al., Genes Dev. 9: 1033-1045, 1995; GenBank Accession number, NM—005693).
  • a primer having the base sequence of the sense strand (SEQ ID NO: 5, hLXR aFl) and a primer having the base sequence of the antisense strand of Exon 8 (SEQ ID NO: 6, hLXR a Rl) were designed and chemically synthesized. .
  • the reaction conditions for the PCR method were as follows.
  • reaction solution containing the single-stranded cDNA is ⁇ (M gS0 4, including de-O alkoxy-lipoic nucleotide mixture) was prepared.
  • This reaction solution was heat-denatured at 94 ° C for 2 minutes, and then subjected to 30 cycles of reaction at 94 ° C for 15 seconds, 55 ° C for 30 seconds and 68 at 90 seconds.
  • the obtained PCR product was electrophoresed on a 1% agarose gel, and the amplified DNA fragment was recovered.
  • the recovered DNA fragment was cloned into pM (Clontech) previously cut with restriction enzymes Mlul and Xbal. That is, after mixing pM (about 100 ng) cleaved with restriction enzymes Mlul and Xbal and the above insert DNA (about 100 ng), T4 ligase was added thereto, and the mixture was heated at 16 ° C. Incubated for 120 minutes. The ligation was performed according to the method recommended by the kit used. The resulting ligation reaction solution was used to transform E. coli DH5 strain competent cells (manufactured by Toyobo Co., Ltd.).
  • the obtained cells were seeded on an LB agar medium containing ampicillin and cultured overnight at 37 ° C. After culture, plasmid DNA was prepared from ampicillin resistant colonies formed on the agar medium, and the nucleotide sequence of the DNA fragment inserted into the plasmid was determined. The base sequence is different from the normal type. A clone lacking the base pair was obtained. The clone (plasmid) was named pM-hLXR aVl. In this way, a partial fragment of the splicing mutant protein gene of the present invention was cloned.
  • Example 2 (Identification of a liver X receptor splice variant protein in which a partial region of the amino acid sequence encoded by exon 5 is deleted)
  • the entire translation region can be amplified.
  • a unique primer was designed. Using hLXR a (0RF) —FZH (SEQ ID NO: 7) as the sense primer and hLXR (ORF) -R / B (SEQ ID NO: 8) as the antisense primer, and using human colon cancer-derived cDNA (Clontech) The DNA fragment was amplified by PCR using the mold. The reaction conditions for the PCR method were as follows.
  • a portion of the resulting PCR product was electrophoresed on a 1% agarose gel containing bromide zyme, and after confirming amplification of the DNA fragment, there was a recognition site in exon 5 of the liver X receptor gene.
  • the PCR product derived from the normal liver X receptor gene was cleaved by treatment with restriction enzyme Aor 51AI (Takara Shuzo).
  • the reaction product was ligated to pGEM-TE Easy vec tor (Promega) using a TA cloning system. Ligation was performed according to the method recommended by the kit used.
  • the obtained liage suspension reaction solution transformed E. coli DH5a strain cell (manufactured by Toyobo Co., Ltd.).
  • the obtained cells were seeded on an LB agar medium containing ampicillin coated with X-gal and isopropyl / 3-D-galactopyranoside (IPTG), and cultured at 37 ° C. for a while. After culturing, colony PCR was performed using a transformant in which ampicillin-resistant white colonies were formed on the agar medium, so that the amino acid encoded by exon 5 A clone containing the entire translation region encoding the liver X receptor splicing mutant protein in which a partial region of the acid sequence was deleted was detected.
  • the reaction conditions for the colony PCR method were as follows.
  • FIG. 1 shows a comparison between the translation region encoding baboon normal liver X receptor a and the translation region encoding the liver X receptor ⁇ -splicing mutant protein obtained in this example.
  • the difference in the translation region encoding the liver X receptor splice variant protein is that the 5 ′ end 90 base pair of exon 5 is missing. There was no difference in other nucleotide sequences. Since the number of bases deleted in exon 5 is 90 (encoding 30 amino acids), that is, a multiple of 3, the reading frame of the translation region downstream from exon 5 was exactly the same as the normal type.
  • the translation region encoding the liver X receptor splicing mutant protein encodes an amino acid sequence (SEQ ID NO: 1) of a protein consisting of 417 amino acids (1251 bases).
  • SEQ ID NO: 1 amino acid sequence of a protein consisting of 417 amino acids (1251 bases).
  • RNA derived from normal rabbit chick tissue RNA derived from normal rabbit chick tissue.
  • Normal liver X receptor a cDNA for human normal tissue using primers shown in SEQ ID NO: 9, 10 or 11 that specifically amplify the protein gene and the liver X receptor ⁇ -splicing mutant protein gene PCR was performed using the cage type.
  • hLXRo; Ex3-F (SEQ ID NO: 9) and JRo! R2 (SEQ ID NO: 10) are used as primers, amplification of the DNA fragment from the transcript of the normal liver X receptor gene is not possible. This results in amplification of a DNA fragment with 430 base pairs.
  • hLXRo; Ex3-F (SEQ ID NO: 9) and hLXRaVlRl (SEQ ID NO: 11), which are combinations of primers that specifically amplify the liver X receptor splice variant protein gene, were used in the same manner as described above.
  • PCR amplification of a DNA fragment derived from the transcript of the liver X receptor splice variant protein gene results in amplification of a DNA fragment having 340 base pairs.
  • PCR was performed without addition of vertical DNA to check for DNA contamination in the reagent. The results are shown in Figs. As is clear from these figures,
  • liver X receptor splicing mutant protein gene was expressed at a high level in heart tissue. In heart tissue, it was expressed at a high level, particularly in the left ventricle.
  • Example 4 Construction of a vector expressing the splicing mutant protein of the present invention
  • the solution treated with the enzyme was subjected to agarose gel electrophoresis, and the DNA fragment corresponding to the insert DNA was recovered from the gel.
  • the recovered DNA fragment was cloned into pFLAG-CMV2 (Sigma) previously cut with Hind III and BamH I.
  • PFLAG-CMV2 (about 300 ng) that had been cleaved with Hind III and BamlH I was mixed with the above insert DNA (about 500 ng).
  • T4 ligase was added thereto, and the mixture was ligated at 16 ° C for 30 minutes. Ligation was performed according to the method recommended by the kit used.
  • the resulting ligation reaction solution was used to transform E. coli DH5 Q! Strain competent cells (manufactured by Toyobo Co., Ltd.) according to the method recommended by the manufacturer.
  • the obtained cells were seeded on an LB agar medium containing ampicillin and cultured overnight at 37 ° C. After culturing, plasmid DNA was prepared from ampicillin resistant colonies formed on the agar medium, and the nucleotide sequence of the DNA fragment inserted into the plasmid was determined. The obtained base sequence is compared with the base sequence obtained by the above-mentioned direct sequence, and a plasmid in which the base sequence of the translation region is confirmed to be completely matched is selected. Were named pCMV 2-hLXR aWT and pCMV2-hLXR a Vl.
  • Example 5 (Preparation of plasmid containing luciferase repo gene having binding sequence of liver X receptor)
  • oligonucleotides consisting of the base sequence near the TATA pox of the mouse metachinin I gene and the base sequence derived from the leader sequence, namely, the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 12 and SEQ ID NO: 13
  • An oligonucleotide consisting of the nucleotide sequence shown below is annealed to form double-stranded DNA, and this is allowed to act on T 4 polynucleotide kinase to phosphorylate both ends (hereinafter, the DNA is referred to as TA TA DM).
  • the plasmid pGL3-Basic vector (Promega) containing the protein luciferase gene was digested with restriction enzymes Bgl II and Hind III, followed by addition of bacterial alkaline phosphatase (BAP). And kept at 65 ° C for 1 hour. Next, the incubated solution was subjected to electrophoresis using a low melting point agarose (Agarose L; manufactured by Nibonbon Gene Co., Ltd.), and then the DNA fragment was recovered from the detected gel of the band part. About 100 ng of the recovered DNA fragment and about 1 g of the TATA DNA were mixed and ligated with T4 ligase to prepare plasmid PGL3-TATA.
  • BAP bacterial alkaline phosphatase
  • Oligonucleotide consisting of base sequence (SEQ ID NO: 14) upstream of cholesterol 7 ⁇ -hydroxylase (CYP7A1) gene containing the binding sequence (LXRE) of lunar spleen X receptor a and a base complementary to the base sequence
  • Oligonucleotide consisting of sequence (SEQ ID NO: 15) Were synthesized and annealed to form double-stranded DNA (hereinafter referred to as LXRE DNA), and then T4 ligase was allowed to act on this to bind LXRE DNA in tandem. By phosphorylating both ends by allowing T4 polynucleotide kinase to act on the obtained DNA fragment, a phosphorylated tandem-linked DNA fragment was obtained.
  • E. coli-derived alkaline phosphatase was added and incubated at 65 ° C. for 1 hour.
  • the heat retaining solution was subjected to low melting point agarose gel electrophoresis, and then a DNA fragment was recovered from the gel of the detected band portion.
  • About 100 ng of the recovered DNA and about 1 g of the above phosphorylated tandem-binding DNA fragment were mixed, and T4 ligase was added thereto, and the mixture was mixed at 16 ° C for 30 minutes. I went on a raiguessillon.
  • the ligation was performed according to the method recommended by the kit used.
  • the obtained ligation reaction solution was used to transform E.
  • coli DH5a competent cell (Takara Shuzo) according to the method recommended by the manufacturer.
  • the obtained cells were plated on LB agar medium containing ampicillin coated with X-gal and isopropyl / 3-D-galactopyranoside (IPTG), and cultured overnight at 37 ° C.
  • IPTG isopropyl / 3-D-galactopyranoside
  • plasmid DNA was prepared from ampicillin resistant white colonies formed on the agar medium. Further, the base sequence of the inserted DNA fragment was determined. From the obtained base sequence, a plasmid having a DM fragment in which four LXR E DNAs were linked in tandem at the Mlul site of PGL3-TATA was selected and named plasmid pGL3-TATA-LXREx4.
  • Example 6 Ferctional Evaluation of Liver X Receptor Splicing Mutant Protein by Reporter Assembly in Transient Expression System
  • HEK293 cell line derived from human fetal kidney (ATCC; CRL-1573), fetal bovine serum (FBS) 10% from which low molecular weight substance was removed by thiacoldextran, penicillin (100 u ni t / ml) and streptomycin as antibiotics includes a (100 g / ml) and were subcultured in 5% C0 2 present under 37 in Fueno one Rureddo not including Dulbecco's modified Eagle's medium (DMEM). The subcultured HEK293 cell line was seeded in a 10 cm cell culture dish to about 2 ⁇ 10 6 cells.
  • FBS fetal bovine serum
  • penicillin 100 u ni t / ml
  • streptomycin as antibiotics includes a (100 g / ml) and were subcultured in 5% C0 2 present under 37 in Fueno one Rureddo not including Dulbecco's modified Eagle's medium (DMEM
  • Example 4 After culturing the cells at 37 ° C for 1 day, the cells were prepared in Example 4 using Lipofectamine (Impertogen) according to the method recommended by the kit. 1 vector each expressing a normal liver X receptor vector and a vector expressing a human liver X receptor splice variant protein and the reporter plasmid pGL3-TATA LXRE x4 prepared in Example 5 were introduced. did. This was cultured at 37 ° C. for 24 hours, and then the cells were collected by pipetting. The collected cells were uniformly suspended and then seeded in a 96-well plate. In addition, various compounds dissolved in ethanol were added to the plate.
  • Lipofectamine Impertogen
  • liver X receptor ⁇ ligands for normal liver X receptor ⁇ or liver X receptor ⁇ splicing mutant protein
  • the measurement results are shown in Figs. 6 and 7, respectively.
  • the transcriptional activation ability by T0901317 which is one of the ligands for visceral X receptor nuclei, was very weak or unrecognized compared to the transcriptional activation ability of normal ffF visceral X receptor phenotypes ( Figure 6).
  • the normal liver X receptor ⁇ and the liver X receptor splicing mutant protein were co-expressed, the ligand-dependent transcriptional activity observed when only the normal liver X receptor was expressed. was significantly suppressed (Fig. 7).
  • liver X receptor splicing mutant protein a gene thereof, and a novel isoform of liver X receptor involved in the inhibition of normal cholesterol metabolism by normal fF visceral X receptor Can be provided. Sequence listing free text
  • Oligonucleotide designed to synthesize lunar spleen X receptor binding element SEQ ID NO: 15
  • Oligonucleotides designed to synthesize lunar spleen X receptor binding elements Oligonucleotides designed to synthesize lunar spleen X receptor binding elements

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Abstract

L'invention concerne une protéine à variant d'épissage du récepteur alpha X du foie constituant une isoforme du récepteur alpha X du foie et comprenant au moins une séquence d'acides aminés codée au niveau de l'exon 5 du gène du récepteur alpha X du foie, une région partielle étant supprimée ; ledit gène ; et leur utilisation. Lesdits éléments peuvent être utilisés dans le développement d'un médicament ou d'une méthode utile pour la prévention, le diagnostic, le traitement, etc. de maladies et de troubles participant à l'inhibition du métabolisme du cholestérol normal par un récepteur alpha X du foie normal.
PCT/JP2004/009227 2003-06-24 2004-06-23 Proteine a variant d'epissage du recepteur alpha x du foie, gene codant pour ladite proteine et utilisation associee Ceased WO2004113533A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002077229A2 (fr) * 2001-03-01 2002-10-03 Lion Bioscience Ag Nouveaux cofacteurs du recepteur alpha x du foie et techniques d'utilisation
WO2003060078A2 (fr) * 2001-12-21 2003-07-24 X-Ceptor Therapeutics, Inc. Modulateurs heterocycliques de recepteurs nucleaires
WO2003082198A2 (fr) * 2002-03-27 2003-10-09 Smithkline Beecham Corporation Procedes de traitement au moyen de modulateurs lxr

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002077229A2 (fr) * 2001-03-01 2002-10-03 Lion Bioscience Ag Nouveaux cofacteurs du recepteur alpha x du foie et techniques d'utilisation
WO2003060078A2 (fr) * 2001-12-21 2003-07-24 X-Ceptor Therapeutics, Inc. Modulateurs heterocycliques de recepteurs nucleaires
WO2003082198A2 (fr) * 2002-03-27 2003-10-09 Smithkline Beecham Corporation Procedes de traitement au moyen de modulateurs lxr

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FUJITA A. ET AL: "Differential Regulation of Ligand-Dependent and Ligand-Independent Functions of the Mouse Retinoid X Receptor Beta by Alternative Splicing", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 255, 1999, pages 625 - 630, XP002981307 *
ZHANG Z. ET AL: "Genomic Analysis of the Nuclear Receptor Family: New Insights Into Structure, Regulation, and Evolution From the Rat Genome", GENOME RESEARCH, vol. 14, no. 4, April 2004 (2004-04-01), pages 580 - 590, XP002981306 *

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