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WO1997047172A1 - Proteine isoforme du recepteur de la vitamine d - Google Patents

Proteine isoforme du recepteur de la vitamine d Download PDF

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Publication number
WO1997047172A1
WO1997047172A1 PCT/IB1997/000947 IB9700947W WO9747172A1 WO 1997047172 A1 WO1997047172 A1 WO 1997047172A1 IB 9700947 W IB9700947 W IB 9700947W WO 9747172 A1 WO9747172 A1 WO 9747172A1
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Prior art keywords
vitamin
protein
vdr
gene
nucleotide sequence
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PCT/IB1997/000947
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English (en)
Japanese (ja)
Inventor
Shigeaki Kato
Kenju Ueno
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Chugai Seiyaku Kabushiki Kaisha
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Priority to AU35550/97A priority Critical patent/AU3555097A/en
Publication of WO1997047172A1 publication Critical patent/WO1997047172A1/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
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors

Definitions

  • the present invention provides a gene encoding a novel vitamin D receptor isoform protein, a recombinant vector containing the gene, a host cell transformed with the recombinant vector, and culturing the host cell.
  • 1,25-Dihydroxyvitamin D 3 [1,25 (OH) 2 D 3 ] has biological activities such as controlling calcium homeostasis and cell differentiation, but most of its biological activities are nuclear. Acts by the expression of a gene mediated by the internal vitamin D receptor (VDR) (Darwisnana der De uca, Crit. Rev. Eukaryotic Gene express., 3: 89-116, 1993). It is known to be a member of nuclear receptors and half-amilies that function as ligand-inducible transcription factors (Green and Chambon, Trends Genet., 4: 309-314, 1988; Parker, Cur, Op in.
  • VDR vitamin D receptor
  • This family includes nuclear receptors for steroid hormones, thyroid hormone and retinoic acid, as well as a ligand called Ophan's Resebuta. Based on similarities in structure and function, including unknown receptors VDR forms subfamilies within the nuclear receptor bar family with retinoic acid receptor (RAR), 9-cis retinoic acid receptor (RXR) and thyroid hormone receptor (TR) .
  • RAR retinoic acid receptor
  • RXR 9-cis retinoic acid receptor
  • TR thyroid hormone receptor
  • VDK is known to form a heterodimer with RXR. These heterodimers are different but similar target enhancers — which bind to the element.
  • One element consists of two repetitive commands.
  • the AGGTCA motif (or a related 6-base motif) consists of: The space between the two core motifs is 3 bp (RXR / VDR) It is 4 (DR4) for 13 ⁇ 413 ⁇ 413 ⁇ 41 ⁇ and 2 (DR2) and 5 bp (DR5) for ⁇ 3 ⁇ 4A1 111113 ⁇ 4. Based on this difference, the nuclear receptor for recognizing the target enhancer element is distinguished (Umesono et al., Cell 65: 1255-1266, 1991; Rastinejad et al., Ature 375: 203- 211, 1995).
  • VDR forms a homodimer in several potent vitamin D response elements (VDREs), indicating that vitamin D has two signaling pathways. Suggest (Carber et al., Ature 361: ⁇ 7-66 ⁇ , 1993; Towers et al., Proc. Natl. Acad. Sci. USA 90: 6310-6311, 1993).
  • exons containing a termination codon in many nuclear receptors are longer than other exons.
  • Various genetic polymorphisms are known in the nucleotide sequence of the final exon and the introductory front and rear of the exon.
  • mutations in bases due to polymorphisms alter mRNA stability and expression level.
  • For aberrant splicing associated with disease due to base changes 1) exon skimming, 2) activation of hidden splice city i, 3) generation of pseudo-exons within introns, 4) intronic exons. Is known to be associated with many diseases. Abnormal splicing is particularly likely to occur around long exons.
  • an object of the present invention is to examine the presence or absence of an isoform caused by alternative splicing of VDR, and to examine its function.
  • the present inventors have designed a DNA fragment encoding various regions of canonical rat VDR (hereinafter referred to as rVDR0) and a P-box in the DNA binding domain.
  • rVDR0 canonical rat VDR
  • rVDR0 canonical rat VDR
  • P-box in the DNA binding domain.
  • the present invention provides a gene encoding a VDR isoform protein-a preferred gene of the present invention is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1.
  • a preferred gene derived from such a rat is that shown in SEQ ID NO: 2.
  • a preferred gene of the present invention is also a nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide obtained by partially substituting, deleting or adding the nucleotide sequence.
  • the present invention also provides a recombinant vector comprising a gene encoding a VDR isoform protein.
  • the present invention further provides a recombinant vector comprising a gene encoding a VDR isoform protein.
  • Prokaryotic or eukaryotic host cells are provided:
  • the invention further provides for culturing transformants obtained by transformation with a recombinant vector containing a gene encoding a VDR isoform protein.
  • the present invention further provides an antibody recognizing a VDR isoform protein.
  • the present invention further provides a method for diagnosing bone density using a VDR isoform protein.
  • the present invention further provides a method for screening a vitamin D-like substance using a VDR isoform protein.
  • FIG. 1 shows the nucleotide and amino acid sequences of rVDR0 and rVDR1 cDNA isolated from a rat kidney cDNA library.
  • FIG. 2 shows the rat VDR genomic region near exons 7-9 and the protein structure of the two rVDR isoforms (rVDRO and rVDR1) generated by alternative slicing.
  • r V [) R schematically shows the R to ⁇ regions of R and their amino acid residues.
  • Figure 3 shows the expression of rVDR0 and rVDR1 transcripts in rat intestine and kidney.
  • FIG. 3 is a diagram (photograph of electrophoresis) analyzed by Northern Plot using these poly A (+) mRNAs.
  • FIG. 4 shows the results of a dominant negative activity test of rVDRO against rVDR1 using CAT technology.
  • FIG. 5 shows the results of a dose-dependent activity test of rVDR1 using CAT assay.
  • FIG. 6 shows the results of a test using CAT Atsee on rVDR1 on thyroid hormone and retinoic acid signaling pathways.
  • Figure 7 is a graph showing that the dominant negative activity of rVDR1 is sequence-specific
  • Figure 8 shows samples obtained by expressing rVDR0 and rVDR1 as GST fusion proteins in Escherichia coli (GST_rVDRO and GST-rVDRl), and digested with trombin and purified. The molecular weight measured on a polyacrylamide-SDS gel using rVDRO and rVDR1 samples was shown (photograph of electrophoresis).
  • Figure 9 shows the results of gel shift assay performed on various amounts of purified mouse RXR (RXR) and purified rVDR0 and rVDR1 using three DRs as probes. ).
  • FIG. 10 shows the results of gel shift assay performed on various amounts of purified rVDR1, nitrile T Ra (TR) and mouse RAR (RAR) using DR4 and DR5 as probes. Photo of electrophoresis).
  • Figure 1 1 is a synthetic recombinant r VD R protein in E. coli (r VDR O or r VDR 1), 1, 2 5 (OH), and Dn 1 n M labeled with [3 H], and label in addition no 1, 2 5 (OH) at various concentrations, 4 '5 C, 1 6 hour ink Interview base and one Bok, except vitamin D not bound by centrifugation, the recombinant r VD R data Showing the results of measuring the radioactivity of a ligand bound to a substance Best mode for carrying out the invention
  • a gene encoding a VDR form protein is prepared by the following procedure.
  • the gene encoding the cloned VDR isoform protein can be transformed into other prokaryotic or eukaryotic host cells by incorporating it into the appropriate vector DNA.
  • the gene can be expressed in each host cell:
  • the polypeptide encodes a polypeptide in addition to the target gene.
  • eukaryotic genes are considered to exhibit polymorphism as is known in human interferon genes (eg, Nishi et al., J. Biochem. 97 15 198 85).
  • One or more amino acids may be replaced by a polymorphism, or the nucleotide sequence may change but the amino acid may not change at all.
  • amino acids in the amino acid sequence of SEQ ID NO: 1 that lack or add one or more amino acids have amino acids replaced by one or more amino acids.
  • Polypeptides may also have VDR isoform protein activity. For example, it has already been known that a polypeptide obtained by converting a nucleotide sequence corresponding to the cysteine of the human interleukin 2 (IL-2) gene to a sequence corresponding to serine retains more than 12 activities. (Wang et al., Science, 224 1431 1984) ⁇
  • VDR isoform protein May have activity
  • the obtained polypeptide has VDR isoform protein activity
  • the present invention also includes a gene that encodes a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or a gene that hybridizes with the nucleotide sequence shown in SEQ ID NO: 2 or 3.
  • the conditions may be the same as those used in ordinary probe hybridization (eg, Molecular Cloning: A and aboratory Manual, SaniDrook et al., And old spring Habor and aboratorv Press, 1989).
  • the expression vector of the present invention contains an origin of replication, a selection marker, a promoter located in front of the gene to be expressed, an RNA slice site, a polyadenylation signal, and the like.
  • Prokaryotic host cells among the hosts used in the expression system of the present invention include, for example, Escherichia coli, Nosinoles zuno, thyris (Baci 1 us subtil is), Mofilus (Bacillus thermophi lus) and the like.
  • examples of host cells for eukaryotic microorganisms include Saccharomyces cerevisiae
  • examples of mammalian host cells include COS cells and Chinese hamster ovary ( CHO) cells, C127 cells, 3T3 cells, Hela cells, BHK cells, Nabarba cells, etc.
  • the transformant of the present invention is cultured under culture conditions suitable for host cells. Can be selected as appropriate
  • the transformant transformed with the gene encoding the desired VDR isoform protein is cultured, and the produced VDR isoform protein is isolated from the inside or outside of the cell and homogenized. Can be purified.
  • the separation and purification of the VDR isoform protein which is the target protein of the present invention, may be carried out by using the separation and purification methods used for ordinary proteins, and is not limited at all.
  • various types of chromatography, Reliance, salting, dialysis, etc. can be appropriately selected and combined.
  • ligand (1 ⁇ l of alltrans retinoic acid or 0.1 ⁇ m of thyroid hormone or 0.1% of vitamin D) to the medium.
  • calcium phosphate-precipitated DNA After incubating with calcium phosphate-precipitated DNA for 20 hours, wash the cells with fresh medium and incubate for another 20 to 24 hours.
  • a cell extract is prepared by lyophilization, and the galactosidase activity is normalized by the method described in the literature (Sasaki et al., Biochemistry 34: 370-377, 1995), and the CAT is assayed.
  • the antibody recognizing the VDR isoform protein of the present invention may be a polyclonal antibody or a monoclonal antibody.
  • a conventional method for example, Neogene Chemistry Laboratory Course 1, Protein According to I, p389-3997, 1992
  • the antigen isoform protein
  • animals such as egrets, rats, goats, sheep, mice, etc. It can be obtained by collecting the produced antibody.
  • the titer of the obtained antibody can be measured by a method known in the art.
  • Monoclonal antibodies can also be prepared according to a conventional method (eg, Kohler et al., Feature 256: 496, 175; Kohlcr et al., Eur. J. Immunol. 6: 511, 1976).
  • the animals are immunized as described above to obtain antibody secreting somatic cells, which are fused with a myeloma cell line, and a hybridoma producing the antibody is selected.
  • binding fragments of such a monoclonal antibody and a monoclonal antibody for example, Fab, F (ab ') Fv fragment can also be used as the antibody of the present invention.
  • the antibody can be obtained by a conventional method by digesting the antibody with papain or pepsin.
  • rVDRO canonical rat VDR
  • rVDRO and rVDRI The sequence of rVDRO and rVDRI was identified to identify 285 nucleotides specific for rVDR1 ( Figure 1). In rVDR1, 1134 bp was inserted in the ligand binding domain of rVDR0, but the other sequences of rVDR1 were identical to the open reading frame of rVDR0 (Fig. 1)
  • VDR isoform protein (rVDR1) of the present invention is coded.
  • the gene to be loaded has the nucleotide sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 1.
  • the deduced amino acid sequence of rVDR1 lacks 86 amino acids at the C-terminus and has an additional 19 amino acids compared to rVDR0: this is the intron present between exon 8 and exon 9. This is probably because translation was stopped by the stop codon located at 1134 bp in (Fig. 1 and Fig. 2).
  • This exon of rVDR1 was then used as a Northern blot specific probe to detect the presence of the rVDR1 transcript (Sasaki et al., Biochemistry 34: 370-377, 1995).
  • this transcript was detected in the stomach and intestine where rVDR0 was expressed (Fig. 3): On the other hand, the nontranscript containing 1042 bp of intron 6 between exons 6 and 7 was detected. No specific transcripts could be detected using specific probes. Analysis of the specific band with a densitometer revealed that the amount of rVDR1 transcript was between 115 and 1Z20 of the amount of rVDR0. In addition, bor (A) + mRNA from various tissues was converted to cDNA using reverse transcriptase, and then amplified by PCR to detect rVDR1 transcript in the cytosolic mRNA fraction. The presence of the rVDR1 transcript was confirmed. Therefore, it was suggested that the rVDR1 transcript was localized in the cytosol as the mature mRN for transcription.
  • This consensus vitamin D response element was prepared from a vector expressing DRO, rVDR1 and rat RXR ⁇ , and a CAT reporter brasmid containing a consensus vitamin D response element (VDRE).
  • VDRE consensus vitamin D response element
  • two AGTTCA motifs are directly linked via the 3 bp spacer (DR3T) described above. This motif is said to be a stronger VDR binding motif than AGGTC A (Freedman et al., Mo]. Endcrinol., 8: 265-273, 1994).
  • rVDR1 itself did not have transcription promoting activity.
  • the dominant negative activity of rVDR1 may be sequence-specific.
  • the degree of inhibition of rVDR against rVDR0 was sequence-specific, which was more pronounced for mouse osteopontin than for human osteocalcin VDRE: the VDR homodimer was found to bind to the target VDRE of mouse osteohontin. (Cheskis et al., Mole Cell. Biol. 11: 3329-3338, 194), suggesting that the AGTTCA motif is a VDR binding core rather than the AGGTCA motif. Is better supported by the fact that DR3T is more active as VDR [': than DR3G (see Figure 7).
  • rVDR1 is a consensus response element of retinoic acid (DR5) and thyroid hormone consensus when the same receptor is present.
  • r VP R 1 binds to VDRE as a homodimer; does not form a heterodimer with RXR
  • the rVDR0 and rVDR1 proteins were produced by the genetic recombination method: 48 kDa and 40 kDa, respectively, from the open reading frame of the cDNA of rVDRO and rVDRl. It was predicted to be a KDa protein: The purified rVDR0 and rVDR1 proteins migrated to the expected molecular weight position on the SDS-PAGE gel ( Figure 8).
  • the purified recombinant rVDRO protein was found to be free of RXR, as described in the literature (Freedman et al., Viol. Endocrinol. Homodimer bound to DR3 (Fig. 9) r VDRl homodimer bound to DR3T to the same extent. Then, when mouse RXRa was added to rVDR0, DN ⁇ binding was significantly increased by heterodimer formation, and a specific monoclonal antibody against RXR ⁇ induced this D ⁇ ⁇ H ⁇ dimer. Recognition and binding shifted the electrophoretic band, confirming the presence of RXR in the complex.
  • rVDR1 formed a heterodimer with rRXRct. It is a mutant that does not have the C-terminal end of human VDR (hVD) (Nakajima ct al., Mo. Endocrinol. 8: 159-172, 199 1). Similarly, it may be because they do not have the C-terminal domain required for heterodimer formation.
  • Replacement form (Rule 26) rVDR1 does not specifically bind to the consensus thyroid response element (DR4) or retinoic acid response element (DR5) ( Figure 10), indicating that rVDR1 binds to the target enhancer element. It suggests that the specificity is the same as rVDR0.
  • VDR similar to RAR, 1 ⁇ 13 ⁇ 4 ⁇ , the conventional observation that the dimer interface formed between the DN ⁇ binding domains specifies the recognition of receptor dimer binding to its cognate response element (eg, Rastinejad et al., Nature 375: 203-211, 1995) and this result agree well.
  • rVDR1 competitively binds to the target VDRE (vitamin D response element) as a homodimer, and as a dominant negative receptor in the vitamin D signaling pathway. It shows that it works.
  • the novel rat VDR isoform (rVDRl) obtained in the present invention is a primary rVDR tiller produced by alternative splicing, but rVDR1 is present at intron 8 in rVDR0. Has extra exons. This new exon
  • the stop codon of (intron 8 in rVDRO) loses part of the ligand-binding domain (86 amino acids) at the C-terminus, but adds 19 amino acids.
  • a primer was designed to specifically enhance intron 8 based on the structure of the intron 8 (W094-03633), and intron 8 was amplified from human genomic DNA.
  • this intron 8 as a probe incorporating 32 P-d CTP by a random primer, screening of human leukocyte cDNII library (C1 oruech) was performed, and human VDR A cDNA fragment was obtained that contained the long intron 7, the exon 8, and the intron 8.
  • the cDNA was ligated to the exon 8 so that the intron 7 full-length frame was aligned during amino acid translation.
  • the fragment of isoform c ⁇ which also retained intron 7 could be cloned ..,
  • the nucleotide sequence of the obtained DNA fragment was determined, and the nucleotide sequence shown in SEQ ID NO: i was determined.
  • the VDR is unique in this subfamily because the isoform of ⁇ consists of various exons that are combined by alternative splicing and / or use of different promoters. In some of these genes, it is already known that retaining introns as exons results in functionally distinct isoform proteins (Nakamura et al., Science 257: 1138-1142, 1992). This is the first example of a receptor isoform of a gene superfamily, '' unlike the RAR, RXR and TR, the VDR isoform has its cDNA clone.
  • VDR isoform protein of the present invention According to a recent report that acts as a dominant negative receptor in the pathway (Morrison et al., Proc. Natl. Acad. Sci. USA 89: 6665-6669, 1994), allelic changes in the human VDR gene are It is closely related to blood osteocalcin levels and bone density. Bone density can be a criterion for predicting the risk of osteoporotic fracture. The report states that allelic changes in the human VDR gene that predict bone density are located in intron 8. It is very interesting that the rVDR1 obtained in the present invention was generated by retaining the rat VDR gene intron 8. VDR isoform protein of the present invention
  • the expression level of the VDR isoform of the present invention is related to various diseases.
  • the diseases include VDR such as osteoporosis, fracture, secondary hyperthyroidism, immune disease, and skin sickness disease.
  • the magnitude of expression of the isoform of the present invention may be related to the onset. Therefore, the isolation and characterization of the VDR isoform of the present invention has great significance in elucidating and treating these diseases.
  • a vitamin D-like substance can be screened using the VDR isoform protein of the present invention.
  • a gene encoding the VDR protein of the present invention is transfected into an appropriate cell, and a vitamin D-like action (eg, calcium / bone metabolism action, differentiation induction action, immunosuppressive action, antitumor (Eg, steroids, retinoic acid) that have at least one of the following actions:
  • the nucleotide sequence of the obtained rVDR1 is shown in SEQ ID NO: 2, and the deduced amino acid sequence is shown in SEQ ID NO: 1. Also, the sequence compared with rVDR0 is shown in Figure]. The mapping of rVDR1 resulting from splicing is shown in FIG. 2 in comparison to rVDR0.
  • Example 2 Northern blot analysis
  • rVDR0 and rVDR1 transcripts were expressed in the gut and gland where rVDK () was expressed. The relative amount of rVD ⁇ transcript was Calculated by scanning with densitometer
  • rVDR1 expression was achieved by substituting the SacI-BamylI fragment of rVDR0 with the rVDR1 fragment (91 1 to 1071 bp in FIG. 1) amplified by PCR. A vector was built.
  • Example 4 Expression and purification of recombinant VDR protein
  • the cDNA encoding rVDR () and rVDR1 is amplified by PCR using the BamHI and EcoR1 restriction sites to generate the corresponding plasmid pGEX-2T (Phar macia). Escherichia coli (DH5 ⁇ ) was transformed with these vectors inserted into the site, and induced with 1 PTG (0.1 mM).
  • r VDR c 3 bp tested dominant negative activity of r VDR 1 for 0 (DR 3 T) 5 scan the two via a colonel one into '- AGTTCA CAT reporter one data one plasmid containing the motif, and mouse HeLa cells were transformed with vectors expressing RXRa (0.5 ⁇ g), rVDR0 (0.5 ug) and rVDR1 (2 ⁇ g): , 25— (OH) 2 D (10 nM) in the presence ( ⁇ ) or absence (1) for 44 hours, ⁇ -force expressed by the pCHl10 internal control vector Toshida CAT activity was normalized by Ichize activity, the results obtained stand expressed as mean ⁇ standard deviation from the the three least independent testing Figure 4 c
  • the cells were cultured in the presence of 0.5 jii g of the rVDR0 expression vector and 1,25- (OH) 2D. (10 nM) in the amount of the VDR expression vector (rVDR0 CAT activity was calculated in the same manner as in Example 5. As is clear from FIG. 5, the amount of the rVDR1 expression vector added in the presence of rVDR0 was determined. With increasing, the inhibitory activity of rVDK1 became more pronounced. r Addition of 5 ⁇ g of VDRO expression vector did not suppress ligand-induced transcriptional promotion activity.
  • Example 7 Effect of rVDR1 on thyroid hormone and retinoic acid signaling pathway
  • the effect of rVDR1 was tested by co-transfection with rVDR1 expression vector (2 / g). CAT activity was calculated in the same manner as in Example 5.
  • r VDR1 was found not to suppress ligand-induced transcriptional promotion of retinoic acid consensus response element (DR5) or thyroid hormone consensus response element (DR4) in the presence of the same type of receptor.
  • DR5 retinoic acid consensus response element
  • DR4 thyroid hormone consensus response element
  • DR 3G, DR 3 CAT, CAT Revota-Plasmid including hyosteocalcin VDRE (OC) and mouse steobontin VDRE (OPN), as well as RXR and VDR (rVDRO or rVDR1) expression vectors ,
  • rVDR0 and rVDR1 were expressed as GST fusion proteins in Escherichia coli, purified with daltathione 'Sepharose 4B, and then digested with thrombin. The digested sample was applied to a Sephadex G-100 column to further purify the rVDRO and rVDRl proteins.
  • GST fusion protein shown as GST-rVDRO or GST-rVDR1 in Figure 8
  • the purified rVDR protein (shown as rVDRO or rVDR1 in Figure 8) was electrophoresed on a 5% polyacrylamide-SDS gel and the molecular weight was determined from the molecular weight marker: rVDR A band was observed at the position of each of the molecular weights expected from the open reading frames of 0 and rVDR1 (48 KDa for rVDR0; 4010a for 1 to 013 ⁇ 41).
  • Electrophoretic migration shift assay (EMSA) and antibody supershift were performed using the method described in the literature (Sasaki et al., Biochemistry 34: 370-377, 1995): The following purified receptors were also used for this assay. did :
  • R A partially purified mouse RAR ct lacking AB region generated in E. coli
  • RX R partially purified mouse lacking ⁇ region produced in E. coli RXR alpha
  • T Partially purified chicken T R ⁇ _ produced in E. coli
  • Monoclonal antibody 4 RX (for RX R) was used for the pile super shift. Resebuta first and [32 ⁇ ] - 5- end-labeled synthetic Origonuku Reochido (DR 3 chome, DR 3G, 01 4 Oyobi 131 ⁇ 5) coupled reaction mixture containing, and Helsingborg (did C) (Pharmacia, 2 M g ) In a binding buffer [10 mM Tris-HCI (pH 7.5), imM dithiothreitol, 1 mM EDT, 10 mM KCI, 10% glycerol] Ink incubated at 25 "C for 15 minutes
  • FIG. 9 shows the obtained results.
  • a supershift test using a monoclonal antibody (4 RX for mouse RX Rct) confirmed the presence of the DR3T-RXR-VDR complex.
  • the band in lane 6 shows the position of the complex super-shifted with the anti-RXR antibody: This shows that the rVDR1 homodimer binds to the consensus VDRE (DR3T).
  • VDR intron 8 was specifically amplified from human genomic DNA by PCR, radiolabeled by random prime method, and used as a probe to screen human leukocyte cDNA library-1 (Ciori tech). Haipuridaize one Deployment The mixture of 50% formamide, 5 x D enhardt 's solution, 5 x SSC, 32 P- labeled 0. 1% SDS, 200 ⁇ g / m I denatured salmon sperm DNA and I 0 beta cpm Nitrocellulose membrane.
  • the nucleotide sequence of the obtained clone was determined, and the nucleotide sequence of SEQ ID NO: 5 was determined.
  • TCCATGCTGC CCCACCTGGC TGACCTTGTC AGTTACAGCA TCCAAAAGGT CATCGGCTTT 720
  • Sequence type nucleic acid
  • CTGTCCCAGC TCTCCATGCT GCCCCACCTG GCTGACCTGG TCAGTTACAG CATCCAAAAC 720
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

Cette invention se rapporte: à une séquence nucléotidique codant pour la séquence d'acides aminés représentée par SEQ ID NO:1; à une séquence nucléotidique codant pour une protéine ayant des activités de protéine isoforme du récepteur de la vitamine D et comprenant une séquence d'acides aminés résultant de la substitution, de la suppression ou de l'addition d'une partie de la séquence d'acides aminés représentée par SEQ ID NO:1; à un gène codant pour cette protéine isoforme du récepteur de la vitamine D, qui est un ADN contenant une séquence nucléotidique s'hybridant avec les séquences nucléotidiques spécifiées ci-dessus; à un vecteur recombiné contenant ce gène; à des cellules hôtes procaryotes ou eucaryotes transformées par ce vecteur; à une protéine isoforme de récepteur de la vitamine D obtenue par culture de ces cellules hôtes; à un anticorps reconnaissant cette protéine; à un procédé pour diagnostiquer les densités osseuses à l'aide de cette protéine; et à un procédé pour cribler des substances apparentées à la vitamine D en utilisant cette protéine.
PCT/IB1997/000947 1996-06-10 1997-06-10 Proteine isoforme du recepteur de la vitamine d WO1997047172A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5260199A (en) * 1991-07-30 1993-11-09 Wisconsin Alumni Research Foundation Method of producing 1,25-dihydroxyvitamin D3 receptor protein
JPH08501933A (ja) * 1992-07-31 1996-03-05 ガーヴァン インスティチュート オブ メディカル リサーチ 作動因子における対立遺伝子の変動の評価

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5260199A (en) * 1991-07-30 1993-11-09 Wisconsin Alumni Research Foundation Method of producing 1,25-dihydroxyvitamin D3 receptor protein
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