WO2003070937A1 - Proteine ayant une zone de fixation d'une proteine osteogenique et gene codant pour cette proteine - Google Patents

Proteine ayant une zone de fixation d'une proteine osteogenique et gene codant pour cette proteine Download PDF

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Publication number
WO2003070937A1
WO2003070937A1 PCT/JP2003/001850 JP0301850W WO03070937A1 WO 2003070937 A1 WO2003070937 A1 WO 2003070937A1 JP 0301850 W JP0301850 W JP 0301850W WO 03070937 A1 WO03070937 A1 WO 03070937A1
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seq
protein
gene
sequence
amino acid
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PCT/JP2003/001850
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English (en)
Japanese (ja)
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Kohji Sato
Takatoshi Ueki
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Taisho Pharmaceutical Co., Ltd.
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Priority to AU2003211554A priority Critical patent/AU2003211554A1/en
Publication of WO2003070937A1 publication Critical patent/WO2003070937A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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 protein having a bone morphogenetic protein binding region and a gene encoding the same.
  • the present invention relates to a novel protein derived from primary cells of ratast mouth site having a bone morphogenetic protein binding region (hereinafter also referred to as KOHJIRIN or Kjr), a gene encoding it (hereinafter also referred to as kohjirin or kjr), The present invention relates to homologous proteins and genes thereof, and their use.
  • KOHJIRIN bone morphogenetic protein binding region
  • kohjirin or kjr a gene encoding it
  • Astrocytes which are one of the major cells that make up the central nervous system, not only support neurons mechanically, but also produce various proteinaceous factors such as neurotrophic factors, which help to differentiate and differentiate neurons. It has become clear in recent years that it is involved in the regulation of function. In particular, it is known that in ischemic encephalopathy, traumatic encephalopathy, or neurodegenerative disease, the proliferation of reactive astrocytes, that is, dariosis, occurs in the lesion. The gliosis is originally thought to have an important role in protecting the central nervous system.
  • neural stem cells have also been shown to exist in the adult brain, and are expected to lead to treatment of a wide range of neurological diseases by effectively differentiating them into neural cells. Furthermore, it has recently been shown that Bone Morphogenetic Protein (BMP) force promotes epidermal ectodermal cell differentiation or promotes differentiation of pluripotent neural stem cells into glia. However, it has been shown that a bone morphogenetic protein inhibitor (BMP antagonist) having a physiological function that promotes neurogenesis of ectodermal cells or differentiation of neural stem cells into neurons by inhibiting its activity.
  • BMP Bone Morphogenetic Protein
  • astrocytes are actively proliferated and exhibit reactive dariosis, ie, astrocytosis, in cerebral ischemia and cerebral disorders such as head trauma. It is known to produce various neuroprotective factors You. However, it has not yet been reported that bone morphogenetic protein inhibitors, which act on neural stem cells and promote neurogenesis, are produced in mature astrocytes. Disclosure of the invention
  • an osteogenic protein inhibitory protein may itself be an effective drug. Further, such a protein is extremely useful in developing a substance having a function similar to the function of the protein and a substance having an action of inhibiting or promoting the function as a medicine. In view of the above, it is desired to identify a protein that inhibits bone morphogenetic protein secreted from astrocytes that have been separated and to elucidate its function.
  • the present invention solves the problem of identifying a bone morphogenetic protein inhibitory protein secreted from astrocytes separated and providing a novel protein having a bone morphogenetic protein binding region and a gene encoding the same. It should be a task to be done.
  • the present inventors aimed at identifying a bone morphogenetic protein-inhibiting protein secreted from astrocytes that had been subjected to shunting, to identify a protein encoded by a gene expressed in primary cultured cells of rat ostium. From among them, we identified the desired bone morphogenetic protein inhibitory protein.
  • KOHJIRIN has a bone morphogenetic protein binding domain, is expressed in astrocytes in the developing brain and in the area where neural stem cells exist, and It has been found that neural stem cells can be promoted to nerve cells.
  • the present invention has been completed based on these findings.
  • a protein according to any one of the following (a), (b) or (c). (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing;
  • a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and Z or added in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, and having an osteogenic protein binding region;
  • Examples of the gene of the present invention include the gene described in any of the following (a), (b) or (c).
  • the gene of the present invention further includes a gene described in any of the following (a), (b) or (c).
  • nucleotide numbers 238 to 1803 of the nucleotide sequence described in SEQ ID NO: 2 in the sequence listing are deleted, substituted and / or composed of a nucleotide sequence, and bind to bone morphogenetic protein A gene encoding a protein having a region;
  • a bone sequence-forming protein binding region comprising a base sequence hybridizable under stringent conditions to a base sequence of base numbers 238 to 1803 of the base sequence set forth in SEQ ID NO: 2 in the sequence listing, and A gene encoding a protein having:
  • a method for producing the protein of the present invention comprising culturing the above-described transformant of the present invention and collecting the protein of the present invention from the obtained culture.
  • the protein is expressed in the presence of a test substance using the above-described expression system for expressing the protein of the present invention, and the produced protein or the protein is encoded.
  • a method of screening for a substance that regulates the expression of the protein of the present invention comprising detecting or measuring mRNA.
  • an agent for promoting stem cell division comprising a protein according to any one of the following (a), (b) or (c):
  • the stem cell described above comprising using the gene according to any one of the following (a), (b) or (c) to produce a protein encoded by the gene: And a method for producing a differentiation promoting agent.
  • SEQ ID NO: 2 SEQ ID NO: 2 nucleotide numbers 238-1803, SEQ ID NO: 2 nucleotide numbers 529-1803, SEQ ID NO: 10, or SEQ ID NO: 10 nucleotide numbers 67-1
  • a gene comprising a base sequence in which one or several bases are deleted, substituted and / or Z- or added in the base sequence of 353, and encoding a protein having a bone-forming protein binding region; or
  • a gene therapy agent for promoting differentiation of a stem cell comprising the gene according to any one of (a), (b) and (c) is provided.
  • nucleotide numbers 238 to 1803 of SEQ ID NO: 2, SEQ ID NO: 2, nucleotide numbers 529 to 1803 of SEQ ID NO: 2, SEQ ID NO: 10, or nucleotide numbers 67 to of SEQ ID NO: 10: Encodes a protein consisting of a nucleotide sequence in which one or several bases have been deleted, substituted and / or added in the nucleotide sequence described in I353, and having a bone morphogenetic protein binding region.
  • FIG. 1 shows the nucleotide sequence of Fragment 1, which is a DNA fragment whose expression is increased in primary cultured cells of rat east mouth site showing contact inhibition after culturing for 3 weeks.
  • Figure 2 shows the identification of kohjirin mRNA in primary cultured rat astrocyte cells by Northern hybridization, and the increased expression in primary rat astrocyte cultured cells that have been cultured for 3 weeks and exhibit contact inhibition. Show.
  • FIG. 3 shows the amino acid sequence of the K0HJIRIN protein.
  • the underline indicates the signal peptide, and the actual underline indicates the cysteine-rich region.
  • Figure 4 shows that the amino acid sequences of the three cysteine-rich regions (Kjr-CR1, Kjr-CR2, and Kjr-CR3) of the K0HJIRIN protein have already been shown to bind to bone morphogenetic cysteine.
  • 2 shows the results of comparison with the amino acid sequence of a region rich in (Chd-CR1, Chd-CR3). This shows that WHP sequences important for binding to osteogenic factors are present in Kjr-CRl and Kjr-CR3.
  • Figure 5 shows the structural features of the protein K0HJIRIN.
  • FIG. 6 shows identification of kohjirin mRNA by RT-PCR in rat brain.
  • FIG. 7 shows the localization of the K0HJIRIN-EGFP fusion protein in C0S-7 cells.
  • FIG. 8 shows the results obtained by introducing the kjr gene into Xenopus fertilized eggs and examining the effect on secondary axis formation.
  • FIG. 9 shows the results of analyzing the expression of Kjr mRNA in the rat brain during development by in situ hybridization.
  • Figure 10 shows the analysis of Kjr protein expression in astrocytes in the area where neural stem cells exist. The results are shown.
  • FIG. 11 shows the results of analyzing the effect of Kjr on the differentiation of neural stem cells into neural cells.
  • FIG. 12 shows a comparison of the amino acid sequences of rat and human Kjr.
  • FIG. 13 shows the results of analyzing the expression of the Kjr gene in the 18th day rat embryo.
  • the protein of the present invention is any one of the following (a), (b) or (c):
  • a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, and Z or added in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and having an osteogenic protein binding region;
  • ⁇ one to several '' in the ⁇ amino acid sequence in which one or several amino acids are deleted, substituted and Z or added in the amino acid sequence '' referred to in the present specification is not particularly limited. It means 1 to 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, and particularly preferably about 1 to 3.
  • amino acid sequence having a homology of 95% or more means that the homology of the amino acid is at least 95% or more, and the homology is preferably 96% or more. And more preferably 97% or more.
  • a protein encoded by a mutant gene having a high homology to the gene kohjirin having the nucleotide sequence of SEQ ID NO: 2 in the sequence listing, wherein the bone morphogenetic protein binding region is All of the physiologically active proteins possessed are within the scope of the present invention.
  • the side chains of the amino acids that are the constituents of proteins differ in hydrophobicity, charge, size, etc., but do not substantially affect the three-dimensional structure (also called three-dimensional structure) of the entire protein.
  • the mutant protein is a mutant protein due to substitution, insertion, deletion, etc. on the amino acid sequence of K0HJIRIN described in SEQ ID NO: 1 in the sequence listing, the mutation is highly conserved in the three-dimensional structure of K0HJIRIN.
  • the mutant protein is a physiologically active protein having a bone morphogenetic protein binding domain, similar to K0HJIRIN, these all belong to the scope of the present invention.
  • the term “protein having a bone morphogenetic protein binding region” refers to the three cysteine-rich regions (Kjr-CR1, Kjr-CR2, and Kjr-CR3) of the K0HJIRIN protein of the present invention described in FIG.
  • the method for obtaining the protein of the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis or a naturally occurring protein isolated from a biological sample or cultured cells. Or a recombinant protein produced by a gene recombination technique.
  • genes encoding the proteins described in the above (1) belong to the scope of the present invention.
  • Specific examples of the gene of the present invention include the genes described in any of the following (a), (b) or (c).
  • nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of nucleotide numbers 238 to 1803 of SEQ ID NO: 2 which hybridizes under stringent conditions, and ⁇ .1 ⁇ - ⁇ which encodes a protein having a protein-binding region:
  • the range of "one or several" in the "base sequence in which one or several bases are deleted, substituted and Z or added in the base sequence” as used herein is not particularly limited. It means about 60, preferably 1 to 30, more preferably 1 to 20, more preferably 1 to 10, and particularly preferably about 1 to 5.
  • Examples of the degree of the above-mentioned DNA mutation include those having 80% or more homology with the nucleotide sequence of the gene kohjirin described in SEQ ID NO: 2 in the sequence listing.
  • hybridize under stringent conditions refers to under ordinary conditions (eg, DIG DNA La belingkit (Boehringer's man)).
  • the probe is labeled with Heim Cat No. 11 75033
  • DIGE asy Hyb solution Boehringer Mannheim Cat No. 1 603558
  • wash the membrane in a 33 ⁇ solution (containing 0.1% [w / v] SDS) under conditions (1 XS SC is 0.15 M Na CI, 0.015 M sodium citrate).
  • XS SC is 0.15 M Na CI, 0.015 M sodium citrate.
  • DNAs that hybridize under stringent conditions include DNAs having a certain degree of homology with the base sequence of the DNA used as a probe, for example, 80% or more, preferably 85% or more, more preferably DNAs having a homology of 90% or more, more preferably 93% or more, particularly preferably 95% or more, and most preferably 98% or more are mentioned.
  • the method for obtaining the gene of the present invention is not particularly limited. Proper probes and primers are prepared based on the amino acid sequence and base sequence information shown in SEQ ID NOs: 1 and 2 of the Sequence Listing of this specification, and cDNAs derived from rat astrocyte primary culture cells are used.
  • the gene of the present invention can be isolated by screening a library or a genomic DNA library.
  • the gene of the present invention can also be obtained by the PCR method.
  • the PCR reaction conditions can be set as appropriate, for example, at 94 ° C. for 30 seconds (denaturation),
  • a reaction process consisting of 30 seconds to 1 minute at 55 (annealing) and 2 minutes at 72 ° C (elongation) is performed as one cycle. For example, 30 cycles are performed, followed by a reaction at 72 ° C for 7 minutes. Conditions. Then, the amplified DNA fragment can be cloned into an appropriate vector that can be amplified in a host such as E. coli.
  • a protein comprising a base sequence in which one or several bases are deleted, substituted and Z or added in the base sequence of SEQ ID NO: 2 described above in the present specification, and having an osteogenic protein binding region; (Mutant gene) can also be produced by any method known to those skilled in the art, such as chemical synthesis, genetic engineering, or mutagenesis.
  • a mutant DNA can be obtained by using a DNA having the nucleotide sequence of SEQ ID NO: 2 and introducing a mutation into the DNA.
  • the method can be carried out by using a method in which DNA having the nucleotide sequence of SEQ ID NO: 2 is brought into contact with a mutagenic agent, a method of irradiating ultraviolet rays, a genetic engineering technique, or the like.
  • Site-directed mutagenesis which is one of the genetic engineering techniques, is useful because it can introduce a specific mutation at a specific position, and is useful in molecular cloning, 2nd edition, Current 'Protocols' in' Molecular. 'Can be performed according to the method described in Biology and the like.
  • the gene encoding a protein comprising a nucleotide sequence hybridizing under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing and encoding a protein having a bone morphogenetic protein binding region is as described above.
  • the colonies can be obtained by performing colony hybridization, plaque hybridization, Southern blot hybridization, etc. under certain hybridization conditions. it can.
  • the gene of the present invention can be isolated as a cDNA fragment containing the gene from the cDNA library derived from primary cells of the rat ostium primary site.
  • the cDNA library used by the present inventors was prepared based on mRNA obtained from primary cultured rat astrocyte cells for a long period of time and from cells showing contact inhibition. It is.
  • differential display- The PCR method can be used as a method for identifying a cDNA which seems to have a gene specifically expressed in a differentiated astrocyte culture cell at the time of contact inhibition.
  • mRNA was extracted from primary cultured rat astrocyte cells that had been cultured for 3 days, and from primary cultured rat astrocyte cells that had been cultured for 3 weeks and exhibited contact inhibition. Are synthesized.
  • a primer having an arbitrary sequence it is possible to obtain a partial PCR product of a gene specifically upregulated in primary rat astrocyte culture cells that have been cultured for 3 weeks and exhibit contact inhibition. it can.
  • the PCR product is subcloned into a pGEM-TA vector from Clonetech, and the entire nucleotide sequence is determined. This method is a method for identifying genes with different expression levels under different conditions in this way.
  • the present inventors carried out the above method using a primer having the sequence of 5′-GACCTCTMGMACCAT-3 ′ (SEQ ID NO: 3), and cultured for 3 weeks to show rat astrocytes exhibiting contact inhibition.
  • a partial PCR product of this gene whose expression was specifically increased in primary cultured cells was obtained.
  • the above PCR product was subcloned into pGEM-TA vector of Clonetech, and the entire nucleotide sequence was determined.
  • the above-mentioned cDNA fragment did not include the partial region of the 3 end of the mRNA.
  • RNA oligo primer from the known base sequence of the cDNA fragment. They were synthesized and the nucleotide sequences at their 5, 3 and 3 sides were determined by the usual RACE method.
  • total RNA prepared from the ostium site of primary culture from rat fetal cerebral cortex using TRIzol reagent from Invitrogen was used.
  • the 5 ′ terminal nucleotide sequence was determined using the GeneRacer kit of Invitrogen according to the conventional method of oligo capping.
  • the templates for calf intestinal phosphatase, tobacco acid pyrophosphatase ⁇ T4 RNA ligase and Superscript II Reverse Transcriptase provided by this kit include rat ophthalmology, otsuki ⁇ ⁇ , astrocytes cultured primarily from the cortex, and TRIzol from Invitrogen. Total RNA prepared using reagent was used.
  • the expression of the gene obtained by the above method in brain tissue can be confirmed by using the RT-PCR method.
  • the present inventors examined the expression of this gene in the rat brain using this method and found that it was specifically expressed in the rat brain at 14 days old, 1 day old, and 14 days old. It became clear.
  • the presence of a protein-encoding region (0RF, open reading frame) in the base sequence The presence can be confirmed by a general-purpose method of analyzing a nucleotide sequence using a computer program.
  • the present inventors who were convinced that the target gene was present in the cDNA sequence, found an ORF in the rooster train using a computer, identified this gene as kohjirin, and identified the protein encoded by the gene as kohjirin. Named KOHJIRIN. Further, in this protein, a region having an amino acid sequence known to be capable of binding to the bone morphogenetic protein was discovered at two places, and it was revealed that KOHJIRIN has a bone morphogenetic protein binding region.
  • the recombinant vector of the present invention can be prepared from the gene of the present invention by a general gene recombination technique using an appropriate host vector system. That is, the gene of the present invention can be used by inserting it into an appropriate vector.
  • the type of vector used in the present invention is not particularly limited, and may be, for example, an autonomously replicating vector (for example, a plasmid or the like), or may be integrated into the host cell genome when introduced into the host cell. And may be replicated together with the integrated chromosome.
  • An expression vector can also be used as a recombinant vector.
  • Elements required for transcription (for example, a promoter) are operably linked to the gene of the present invention incorporated in the expression vector.
  • the promoter is a DNA sequence showing transcription activity in a host cell, and can be appropriately selected depending on the type of the host.
  • Suitable vectors include plasmids derived from Escherichia coli (eg, pBR322, pUC118, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pSH19, etc.), and animal viruses such as batteriophage retrovirus and vaccinia virus. it can. Upon recombination, a translation initiation codon and a translation termination codon can be added using an appropriate synthetic DNA adapter.
  • the promoter used in the expression vector may be appropriately selected depending on the host. For example, when the host is Escherichia coli, the T7 promoter, lac promoter, trp promoter, and LPL promoter are used. When the host is Bacillus, the SOP promoter is used, and when the host is yeast. When the host is an animal cell, the SV40 promoter, retrovirus promoter, MT-1 (metamouth thionine gene) promoter, etc.
  • the polyhedrin promoter In the case of insect cells, the polyhedrin promoter, the P10 promoter, the autographer, the refolding force, the polyhedrosic basic protein mouth motor, the pakiurovirus immediate early gene 1 promoter, or the baculovirus 39 A K-delayed early gene promoter or the like can be used.
  • the gene of the invention may also be operably linked to a suitable terminator, such as, for example, a human growth hormone terminator or, for a fungal host, a TPI1 terminator or an ADH3 terminator, if desired.
  • a suitable terminator such as, for example, a human growth hormone terminator or, for a fungal host, a TPI1 terminator or an ADH3 terminator, if desired.
  • the recombinant vector of the present invention further comprises a polyadenylation signal (eg, from the SV40 or adenovirus 5E1b region), a transcription enhancer sequence (eg, the SV40 enhancer) and a translation enhancer sequence (eg, the adenovirus (Encoding the virus VA RNA).
  • the recombinant vector of the present invention may further comprise a DNA sequence enabling the vector to replicate in a host cell, such as the SV40 origin of replication (when the host cell is a mammalian cell). Is mentioned.
  • the recombinant vector of the present invention may further contain a selection marker.
  • Selectable markers include, for example, genes whose capture cells are deficient in host cells, such as dihydrofolate reductase (DHFR) or Schizosaccharomyces bomb TPI gene or the like, or ampicillin, kanamycin, tetracycline, chloramphenicol, A drug resistance gene for neomycin or hygromycin can be mentioned.
  • DHFR dihydrofolate reductase
  • Schizosaccharomyces bomb TPI gene or the like or ampicillin, kanamycin, tetracycline, chloramphenicol,
  • a drug resistance gene for neomycin or hygromycin can be mentioned.
  • the method of ligating the gene, promoter and, if desired, the terminator Z or secretory signal sequence of the present invention, respectively, and inserting these into an appropriate vector is as follows. It is well known to those skilled in the art.
  • the gene of the present invention can be expressed as a fusion protein with another protein (for example, daltathione S transferase, protein A and the like).
  • the fusion-type protein of the present invention thus expressed can be excised using an appropriate protease.
  • Transformants can be prepared by introducing the gene or the recombinant vector of the present invention into an appropriate host.
  • the host cell into which the gene or the recombinant vector of the present invention is introduced can be any cell as long as it can express the DNA construct of the present invention, and examples include bacteria, yeast, fungi, and higher eukaryotic cells.
  • bacterial cells examples include gram-positive bacteria such as Bacillus bacterium (eg, Bacillus ssubti1is) or Streptomyces bacterium, or gram-negative bacteria such as Escherichia coli (Escherichicchiacoli). Transformation of these bacteria may be performed by using a protoplast method or a known method by using a competent cell.
  • mammalian cells examples include HEK293 cells, HeLa cells, COS cells (for example, COS-7 cells), BHK cells, CHL cells, CHO cells, and the like.
  • Methods for transforming mammalian cells and expressing the introduced genes in the cells are also known, and for example, an electroporation method, a calcium phosphate method, a lipofection method and the like can be used.
  • yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, such as Saccharomyces cerevisiae or Saccharomyces kluyveri.
  • Examples of a method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroblast method, and a lithium acetate method. You.
  • filamentous fungi such as Aspergillus, Neurospora, Fusarium, or Trichoderma.
  • transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to a known method, for example, by homologous recombination or heterologous recombination.
  • a recombinant gene transfer vector and a baculovirus are co-transfected into the insect cell to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further introduced. Infects insect cells and expresses proteins. (See Baculovirus Expression Vectors, A Laboratory Manual; current, 'proto-conoles', 'molecular' biology, Bio / Technology, 6, 47 (1988) etc.).
  • the baculovirus may be, for example, an autographa calif ornica nuclear polyhedrosis virus, which is a virus that infects the insects of the family Spodoptera, Autographa calif ornica nuclear polyhedrosis virus.
  • Insect cells include Spodoptera frugiperda ovary cells, Sf9 and Sf21 [Paculo Innores' Expression. Vectors, A Laboratory's Manual, WH. WH Freeman and Company), New York (New York), (1992)], and Trichoplusia ni ovary cells, Hi Five (Invitrogen) can be used.
  • Examples of a method of co-introducing the recombinant gene transfer vector and the above-mentioned paculovirus into insect cells to prepare a recombinant virus include a calcium phosphate method and a lipofection method.
  • the antibody may be a polyclonal antibody or a monoclonal antibody. It can be prepared by a method known to those skilled in the art.
  • the protein of the present invention or a partial peptide thereof, which is an antigen is administered to a mammal to immunize the mammal, blood is collected from the mammal, and the antibody is collected from the collected blood. It can be obtained by separation and purification.
  • mammals such as mice, hamsters, monoremots, rats, egrets, dogs, goats, sheep, and birds, and birds such as chickens can be immunized.
  • the administration route of the antigen is not particularly limited, and subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like can be appropriately selected.
  • the antigen may be used by dissolving it in a suitable buffer, for example, a suitable buffer containing an adjuvant such as complete Freund's adjuvant. After raising the immunized mammal for a certain period, the serum of the mammal is collected and the antibody titer is measured. When the antibody titer increases, booster may be appropriately performed. Since last dose
  • a monoclonal antibody can be obtained by preparing a hybridoma.
  • a hybridoma can be obtained by cell fusion between an antibody-producing cell and a myeloma cell line.
  • the hybridoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.
  • spleen cells As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes, etc. from immunized animals are used.
  • the antigen the protein of the present invention or a partial peptide thereof is used.
  • Mice, rats, and the like can be used as immunized animals, and administration of the antigen to these animals is performed by a conventional method.
  • a suspension or emulsion of an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant and the protein of the present invention, which is an antigen
  • an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant
  • the protein of the present invention which is an antigen
  • spleen cells are obtained as antibody-producing cells from the immunized animal, and The cells can be fused with cells by a known method (G. Kohler et al-, Nature, 256 495 (1975)) to produce a hybridoma.
  • myeloma cell lines used for cell fusion include P3X63Ag8, P3U1 strain, and Sp2ZO strain in mice.
  • a fusion promoter such as polyethylene glycol or Sendai virus is used.
  • hypoxanthine / aminopterin'thymidine (HAT) medium is used according to a conventional method.
  • Hybridomas obtained by cell fusion are cloned by a limiting dilution method or the like. If necessary, screening can be performed by enzyme immunoassay using the protein of the present invention to obtain a cell line that produces a monoclonal antibody specifically recognizing the protein of the present invention. it can.
  • the hybridoma is cultured by a conventional cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites.
  • Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography can be used in an appropriate combination.
  • Antibody fragments include F (ab,) 2 fragment, Fab 'fragment and the like.
  • the antibody of the present invention can be used as an immobilized antibody immobilized on an insoluble carrier such as a solid phase carrier, or as a labeled antibody labeled with a labeling substance. All such immobilized antibodies and labeled antibodies are within the scope of the present invention.
  • the above transformant is cultured in an appropriate nutrient medium under conditions that allow expression of the introduced gene.
  • a conventional protein isolation and purification method may be used.
  • a normal protein isolation and purification method that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion-exchange chromatography using a resin such as getylaminoethyl (DEAE) Sepharose, cation-exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia), butyl sepharose, phenyl Hydrophobic chromatography using a resin such as sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, electrophoresis such as isoelectric focusing
  • the protein of the present invention is expressed by expressing the protein using an expression system that expresses the protein of the present invention in the presence of a test substance, and detecting or measuring the produced protein or mRNA encoding the protein. Can be used to screen substances that regulate the expression of E. coli. Similarly, by localizing the protein of the present invention by expressing the protein in the presence of a test substance using an expression system that expresses the protein of the present invention and measuring the localization of the protein produced, The substance that changes the sex can be screened. All of these screening methods belong to the scope of the present invention.
  • test substance Any substance can be used as a test substance to be subjected to the screening method of the present invention.
  • the type of the test substance is not particularly limited, and may be an individual low-molecular synthetic compound, a compound present in a natural product extract, or a synthetic peptide.
  • the test compound may also be a compound library, a phage display library, or a combinatorial library.
  • the test substance is preferably a low molecular compound, and a compound library of low molecular compounds is preferable.
  • the construction of compound libraries is known to those skilled in the art, and commercially available compound libraries can also be used.
  • the detection or measurement of the produced protein of the present invention or the mRNA encoding the same is known to those skilled in the art as described in Molecular Cloning, Second Edition or Current 'Protocols'in' Molecular Leuca. Can be performed by a conventional method.
  • the detection or measurement of the protein can be performed, for example, by a Western blotting method using an antibody against the protein or ELISA.
  • the detection or measurement of mRNA can be performed by Northern blotting, RT-PCR, or the like.
  • RT-PCR instead of PCR using all lnRNA and pre-produced cDNA as primers and PCR using primers specific to the gene of the present invention to detect or measure the mRNA of the gene of the present invention You can also.
  • the localization of the produced protein can also be measured by a conventional method known to those skilled in the art.
  • the protein of the present invention is expressed in a cell in the form of a fusion protein with a labeling substance for detection (preferably, a fluorescent protein such as GFP or EGFP).
  • a labeling substance for detection preferably, a fluorescent protein such as GFP or EGFP.
  • the localization of the expressed fusion protein can be measured based on the labeling substance. For example, when a fluorescent protein such as GFP or EGFP is used as the labeling substance, the localization of the fusion protein in the cell can be measured by observing the cell with a fluorescence microscope.
  • the protein described in any one of the following (a), (b) or (c) can be used as a stem cell differentiation promoter.
  • a protein comprising an amino acid sequence in which an acid has been deleted, substituted, and Z or added, and having an osteogenic protein binding region; or (c) It has a homology of 95% or more with the amino acid sequence described in SEQ ID NO: 1, SEQ ID NO: 1 in amino acid numbers 98 to 521, SEQ ID NO: 9 or amino acid numbers in SEQ ID NO: 9 in amino acid numbers 23 to 450.
  • a protein consisting of an amino acid sequence having a bone morphogenetic protein binding region
  • Amino acid numbers 98 to 521 of SEQ ID NO: 1 and amino acid numbers 23 to 450 of SEQ ID NO: 9 are amino acid sequences of proteins excluding signal peptides, respectively. Further, by producing a protein encoded by the gene according to any of the following (a), (b) or (c), the above-mentioned stem cell differentiation promoter can be produced. .
  • SEQ ID NO: 2 SEQ ID NO: 238 to 1803, SEQ ID NO: 2, nucleotide numbers 529 to 1803, SEQ ID NO: 10, or SEQ ID NO: 10, or nucleotide numbers 67 to 1353 of SEQ ID NO: 10
  • a gene consisting of a nucleotide sequence in which one or several bases are deleted, substituted and Z or added, and encoding a protein having a bone morphogenetic protein binding region; or
  • SEQ ID NO: 2 SEQ ID NO: 2
  • nucleotide numbers 238 to 1803 of SEQ ID NO: 2 nucleotide numbers 529 to 1803 of SEQ ID NO: 2, SEQ ID NO: 10, or the nucleotide sequence of nucleotide numbers 67 to 1353 of SEQ ID NO: 10
  • SEQ ID NO: 9 is an amino acid sequence of human KOHJIRIN (Kjr) protein;
  • the nucleotide sequence described in No. 10 is a nucleotide sequence encoding the same. As shown in FIG.
  • the human-derived KOHJIRIN (Kjr) protein described in SEQ ID NO: 9 has almost the same functional domain as rat Kjr, and is presumed to be functionally similar. Acquisition and utilization of the human-derived genes and proteins described in (a), (b) or (c) above can be performed in the same manner as in the case of the rat-derived genes described above in this specification.
  • the protein described in the above (a), (b) or (c) is used as a medicine, it is generally in the form of a pharmaceutical composition containing the protein as an active ingredient and a pharmaceutical additive. Can be provided.
  • the above protein can be administered as a medicine to mammals including humans.
  • the administration route of the medicament of the present invention is not particularly limited, and oral or parenteral administration (for example, intramuscular administration, intravenous administration, subcutaneous administration, intraperitoneal administration, mucosal administration to nasal cavity, or inhalation administration, etc.) Any of them.
  • the form of the medicament of the present invention is not particularly limited, and examples of formulations for oral administration include tablets, capsules, fine granules, powders, granules, solutions, syrups, and the like. Preparations include, for example, injections, drops, suppositories, inhalants, transmucosal absorbers, transdermal absorbers, nasal drops, ear drops and the like.
  • the dose of the medicament of the present invention can be appropriately selected in consideration of the sex, age or weight of the patient, the severity of symptoms, the purpose of administration such as prevention or treatment, and the like.
  • the dose is generally 0.001 / gZkg body weight Z days to 1 000 ⁇ g Zkg body weight Z days, preferably 0.001 ⁇ g / kg body weight days to 100 ⁇ g / kg body weight / day. Day.
  • the gene described in any of the following (a), (b) or (c) can be used as a gene therapy agent for promoting differentiation of stem cells.
  • nucleotide numbers 238 to 1803 of SEQ ID NO: 2, SEQ ID NO: 2, nucleotide numbers 529 to 1803 of SEQ ID NO: 2, nucleotide numbers 67 to 803 of SEQ ID NO: 2, or nucleotide numbers 67 to of SEQ ID NO: 10 A gene consisting of the nucleotide sequence of 1353;
  • SEQ ID NO: 2 SEQ ID NO: 2, Nucleotide Number 2338 to 1803, SEQ ID NO: 2, Nucleotide Number 529 to 1803, SEQ ID NO: 10, or SEQ ID NO: 10
  • a gene comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence described in Nucleotide Nos. 67 to 1353, and encoding a protein having an osteogenic protein binding region: shown in the following Examples.
  • the gene of the present invention since the gene of the present invention has a stem cell differentiation promoting action, it can be used as a gene therapy agent for promoting stem cell differentiation by administering it to a living body.
  • the gene therapy agent of the present invention comprises a recombinant gene vector containing the gene according to any one of the above (a), (b) or (c) as an active ingredient together with a base used for a gene therapy agent. It can be manufactured by the following.
  • the base used for the gene therapy agent those usually used for injections can be used.
  • the base include distilled water, salt solutions such as sodium chloride or salt mixture of sodium chloride and inorganic salt, mannitol, ratatose. Dextran, glucose, etc., amino acid solutions such as daricin and arginine, organic acid solutions or mixed solutions of salt solutions and glucose solutions, and the like.
  • these bases may be injected as a solution, suspension, or dispersion using an auxiliary agent such as an osmotic pressure adjusting agent, an H adjusting agent, a vegetable oil, or a surfactant.
  • An agent can also be prepared.
  • the administration form of the gene therapy agent of the present invention may be systemic administration such as usual intravenous or intraarterial administration, or local administration such as local injection or oral administration. Further, the gene therapy agent of the present invention can be in a form of administration in combination with catheter technology, gene transfer technology, surgical operation or the like.
  • the dosage of the gene therapy agent of the present invention varies depending on the age, sex, symptom, administration route, administration frequency, and dosage form.
  • the weight of the recombinant gene per day is from 1 ⁇ g / kg to 100 O mg / kg range, preferably 10 ⁇ g / kg It is in the range of about 10 OmgZkg.
  • the frequency of administration is not particularly limited.
  • Example 1 Cloning of gene kohjirin
  • RNA is extracted from primary cultured ratast mouth site cells cultured for 3 days and primary cultured ratast mouth site cells showing contact inhibition for 3 weeks, and cDNA is synthesized using oligo dT as a primer. did.
  • the following oligonucleotides (oligonucleotide 1) were synthesized using a DNA synthesizer (380 B, manufactured by PE Applied Biosystems).
  • Oligonucleotide 1 GACGTCTMGAAACCAT (SEQ ID NO: 3)
  • the PCR cycle was held at 95 ° C for 2 minutes, then held at 35 for 40 seconds, at 72 for 1 minute and 30 seconds, and at 95 ° C for 40 seconds. After holding for 40 seconds, holding at 72 ° C for 1 minute and 30 seconds, and holding at 95 ° C for 40 seconds 32 times, the reaction was carried out at 72 ° C for 5 minutes.
  • the above PCR product is developed on an acrylamide gel by a conventional method, cultured for 3 weeks, and the band of the gene specifically upregulated in the cDNA derived from the primary culture of ratast mouth site cells showing contact inhibition is removed.
  • the DNA was cut out and extracted from the gel by a conventional method to obtain a PCR product.
  • the PCR product was subcloned into the pGEM-TA vector of Clonetech, and the entire nucleotide sequence was determined to obtain a gene (fragment-1) having the sequence shown in FIG. Sequencing was performed using a DNA applicator (ABIPR ISM377) manufactured by PE Applied Biosystems and a reaction kit manufactured by the company.
  • a probe was synthesized with Ready to go kit (Pharmacia) in the presence of 32P-dCTP. Hybridization was carried out at 42 in a solution having the following composition (all concentrations were final concentrations).
  • composition of the hybridization solution 50% formamide, 5X SSC, 0.05M sodium phosphate, 10X Denhardt's solution, 1% SDS, 200 ⁇ g / ml Salmon sperm
  • membrane washed twice with 2 ⁇ SSC, 0.1% SDS solution at room temperature.
  • X-0MAT manufactured by Kodak film was used for signal detection.
  • the expression of the gene was increased in the primary culture cells of the rat mouth mouth site showing contact inhibition after culturing for 3 weeks.
  • Cloning of the cDNA fragment containing fragment 11 was performed by the following method.
  • the cDNA fragment contained only a part of the region at the end of the mRNA. Therefore, based on the base information of the relevant region (hereinafter referred to as 3 ′ fragment), the present inventors deduced from the known base sequence of the cDNA fragment the 5, 5 and 3 nucleotides (oligonucleotide 2), 3 and 3 nucleotides. Ligonucleotides (oligonucleotides 3) were synthesized using a DNA synthesizer (380 B, manufactured by PE Applied Biosystems).
  • Oligonucleotide 2 ATGCAGCTCAGAGACTCC (SEQ ID NO: 4)
  • Oligonucleotide 3 GAAATCAGCCATAGTCTTAGA (SEQ ID NO: 5)
  • an oligonucleotide (oligonucleotide 4) was synthesized from a known nucleotide sequence using a DNA synthesizer (380B manufactured by PE Applied Biosystems). .
  • Oligonucleotide 4 CAAGGTGTGCTGTGACTGGAT (SEQ ID NO: 6)
  • the 5'-terminal nucleotide sequence was determined using a GeneRacer kit from Invitrogen according to the standard method of oligo capping.
  • Calf intestinal phospnatase N tobacco acid pyrophosphatase ⁇ T4 RNA ligase provided by the kit Superscript II Reverse Transcriptase template includes the foot moon daisies Otsuki ⁇ ⁇ Invitrogen TRIzol reagent from astrocytes originally cultured from the cortex Total RNA prepared using the method was used.
  • the cDNA contains an ORF encoding a protein (K0HJIRIN) consisting of 521 residues. A termination codon appeared at the same reading frame in the upstream region of the methionine residue which is the initiation codon of the protein.
  • the amino acid sequence of the protein encoded by the cDNA fragment is shown in SEQ ID NO: 1 in the sequence listing. Things were the only ones.
  • the primary structural features of K0HJIRIN are shown in Fig. 3, Fig. 4 and Fig. 5.
  • the amino acid structure analysis software SOSUI Nemiki Miyake: Protein Nucleic Acid Enzyme, 1999, VOL42, p3020-3025 was used.
  • K0HJIRIN has a protein secretion signal sequence at the N-terminal amino acid moiety.
  • nucleotide sequence of cDNA containing ORF of gene kohjirin is shown in SEQ ID NO: 1 in the sequence listing.
  • Example 3 Identification of kohjirin mRNA in rat brain
  • RNA was extracted from rat brain by a conventional method, and cDNA was synthesized using oligo dT as a primer. Oligonucleotides with sequences contained in kohjirin cDNA (Orico Oligonucleotides (oligonucleotides 6) having nucleotides 5) and reverse complementary strands and sequences were each synthesized using a DNA synthesizer (AB I3 ⁇ 4380 B).
  • Oligonucleotide 5 TAGGACTTTGTGGTACAGCTC (SEQ ID NO: 7)
  • Oligonucleotide 6 TTACAACCTGCCTCAMGGCT (SEQ ID NO: 8)
  • cDNA (2 1 (40 ⁇ ⁇ )), 10X PCR buffer (including 25mMg ++) (1.5 ⁇ 1), 2.5mM dNTP (2 ⁇ 1), ⁇ ⁇ ⁇ oligonucleotide 5 (0.4 / 1), ⁇ ⁇ ⁇ oligonucleotide 6 ( 0. ⁇ 1), water (8.15 ⁇ 1), LATaq polymerase (0.15 / zl), total amount 15 / z1.
  • PCR cycle hold at 95 ° C for 5 minutes, react at 95 ° C for 35 seconds, hold at 57 ° C for 35 seconds, and hold at 72 ° C for 1 minute 3 times I went.
  • a plasmid (pGEM-kohjirin) containing the coding region of the gene kohjirin having the base sequence of the cDNA containing the ORF (SEQ ID NO: 2 in the sequence listing) was treated with a restriction enzyme for 1 hour under the following reaction conditions.
  • EcoRI (15U / ⁇ 1) ( 1 ⁇ I), buffer (500mM Tris-HCl (pH8.5), lOOraM MgCl 2, lOmM Dithiothreitol, 1M NaCl) (2 / zl), plasmid (2 ⁇ g / ⁇ ) ( ⁇ ), water (16 ⁇ 1), total volume 20 ⁇ 1.
  • the desired PCR product of about 1.6 kb was cut out and purified using genecleankit (Funakoshi). Update the purified product Then, treatment was performed at 37 ° C for 20 minutes under the following reaction conditions, and subsequently at 50 ° C for 20 minutes.
  • Calf intestinal phosphatase (15U / ⁇ 1) ( ⁇ ⁇ ), buffer (2 ⁇ 1), the above product (1 ⁇ g / ⁇ ) ( ⁇ ), water ( 16 ⁇ 1), total volume 20 ⁇ 1.
  • EGF P-N 1 (Clontech) was similarly treated with EcoRI for 1 hour. 2 (Takara Shuzo), the cDNA fragment treated with the restriction enzyme was inserted into pEGFP-N1 (Clontech), and the recombinant was isolated according to a conventional method. A pKOHJIRIN-EGFP plasmid containing the DNA of the sequence shown in 2 correctly was obtained.
  • COS-7 cells were cultured using D-MEM medium (manufactured by Lifetech) containing 10% inactivated fetal serum (Dainippon Pharmaceutical) and antibiotics as basic medium.
  • COS- 7 cells were seeded preliminarily 1 0 5 in a culture dish with a diameter of 3 5 mm. The cells were cultured for 2 hours in a basal medium containing a solution in which 101 LIPOFECTAMINE gene transfer reagents (in vitrogen) and 2 ⁇ g of: K0HJIRIN-EGFP plasmid were mixed in advance for 30 minutes, and gene transfer was performed. Cells were observed with a fluorescence microscope 24 hours after gene transfer.
  • K0HJIRIN-EGFP protein was found to be present mainly in the cytoplasm near the nucleus. This indicates that K0HJIRIN protein is a protein synthesized and secreted in cells. Furthermore, by introducing the pKOHJIRIN-EGFP plasmid into various cultured cell lines in the same manner and adding the compound at the same time, it is possible to screen for a compound that changes the localization without changing the protein expression level. Becomes possible.
  • Example 5 Cloning of human Kjr (Human and rat kjr amino acid sequence and base sequence)
  • cDNA was synthesized using Superstaripto RNase H-reverse transcriptase (Invitrogen).
  • two PCR primers were prepared (5'-tgc atg ttt caa gac aag aag tat aga gtt-3, (rooster sequence number 11) and 5, -tgg gca cac ctt gca gca ctt tec ate taa-3 ') (SEQ ID NO: 12) from human placenta PCR was performed using the cDNA of Example 1 as a template.
  • nucleotide sequences at the 5 'end and the 3' end were clarified using the 5, RACE system, version 2.0 and the 3 'RACE system (both from Invitrogen).
  • 5, RACE system, version 2.0 and the 3 'RACE system both from Invitrogen.
  • 5, Gene-Specific Primer (GSP) for the RACE system 5, -gca aag cac att ccc att etc tga -3, (SEQ ID NO: 13)
  • GSP for the 3 'RACE system 5,-aat gtc acc aag caa gaa tgt aag-3 ′ (SEQ ID NO: 14) was used.
  • the nucleotide sequence was determined using ABI PRISM310 (PE Applied Biosystems) using the instructions of the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech).
  • the human kjr amino acid sequence is shown in SEQ ID NO: 9, and the base sequence is shown in SEQ ID NO: 10.
  • Fig. 12 shows a comparison between the amino acid sequences of rat and human Kjr.
  • human Kjr and rat Kjr have a signal peptide rich in hydrophobic amino acids at the amino terminus (human Kjr (MGGMKYIFSLLFFLLLEGGKTE), rat Kjr (MFFLPPTFESPLPLRAHSSSRHNLRAPDLGGAPQGAQSVCKRGRRKRPVPSFSPPVLGVSELHFA VAQRRREWMWGI
  • human Kjr has rat Kjr and rat Kjr, both of which have three multicysteine regions and that the first and third multicysteine regions have BMP-4 binding amino acid sequences (hidden). It is strongly suggested that they have the same physiological activity.
  • Kjr has a physiological activity as a BMP antagonist, and is considered to be involved in the formation of neurons from neural stem cells, and in the regulation of bone metabolism.
  • Example 6 Effect of kjr gene transfer on fertilized Xenopus eggs on secondary axis formation
  • cap-added mRNA was synthesized by in vitro transcription.
  • the synthesized mRNA was microinjected (0.8 or 1.6 ng RNA per embryo) into the blastomere of Xenopus on the ventral side of the 8-cell stage embryo, and the effect on secondary axis formation at stage 36 was examined. .
  • Fig. 8 shows the results.
  • Fig. 8a injection of Kjr mRNA into the ventral blastomere of an 8-cell stage embryo resulted in The formation of a secondary axis was observed (arrow). That is, the formation of a secondary axis on the ventral side was observed depending on the amount of the microinjected mRNA. The uppermost embryo is a control.
  • FIG. 8b shows an embryo that gave rise to a typical secondary axis where cement gland (eg) can be seen.
  • FIG. 8c shows a cross-sectional histological image of the embryo into which Kjr mRNA was injected, at stage 36. notochord (no, somites (so), neural tube, nt) (left
  • Embryonic 17 days old, 1 day old, 7 days old, 14 days old rats were decapitated under anesthesia, and sections were prepared with a cryostat and mounted on silane-coated slide glasses.
  • the following oligo DNA probe capable of binding complementarily to rat Kjr mRNA was synthesized.
  • the oligo DNA probe is then labeled with 35S using terminal deoxytransf erase (Takara) and 35S-dATP (EN), hybridized with the above section at 42 ° C, and then washed. After drying, the emulsion was applied and autoradiography was performed at 4 ° C for 3 weeks. After development with a developer, the specimen was examined with a visual field microscope. Fig. 9 shows the results.
  • a to c show the expression of Kjr mRNA in the rat brain during development. 17 days after birth; b. 1 day after birth; c. 7 days after birth
  • d to f indicate the expression of Kjr in the hippocampal dentate gyrus. d. 1 day after birth; e. 7 days after birth; f. 14 days after birth;
  • CxP (.cortical plate), ne (neuroepithelium), i (lateral ventricle), Re (retina), Cor (cerebral cortex), Hi (hippocampus), RMS (rostral migratory stream), CPu (caudate putamen), Th (thalamus) ), DG (dentate gyrus), scale Are 400 ⁇ (ac) and 200 ⁇ (d ⁇ f).
  • Kjr mRNA was strongly expressed in the region where neural stem cells exist, such as neuroepithelium and hippocampal dentate gyrus, from embryonic stage to mature stage. This result suggests that this protein may be involved in the regulation of neural stem cells.
  • Example 8 Analysis of Kjr protein expression in astrocytes in the area where neural stem cells exist
  • Adult rats were decapitated under anesthesia, and sections were prepared with a cryostat and mounted on silane-coated slide glasses.
  • the cells were fixed with ice-cold 4% paraformaldehyde for 20 minutes, washed, and reacted with anti-Kjr antibody (1: 100) and anti-GFAP antibody (chemicon 1: 800) as primary antibodies at ⁇ and 4 ° C. .
  • Figure 10 shows the expression of Kjr protein (A, D) in hippocampal dentate gyrus (AC) and subventricular zone (DF) in adult rats. It is the result of the experiment which compared with GFAP (B, E) which is a marker protein by double staining. C and F superimpose the two images. Kjr expression and GFAP expression were observed in the same cells. From the above results, it was clarified that the Kjr protein was expressed in astrocytes in the region where neural stem cells exist.
  • Example 9 Analysis of the effect of Kjr on the differentiation of neural stem cells into neural cells
  • D-MEM / F-12 medium (Invitrogen) containing 20 ng / ml bFGF (GT) and 1% N-2 supplement (Invitrogen)
  • GT ng / ml bFGF
  • N-2 supplement Invitrogen
  • the Kjr translation region cDNA and the cDNA encoding the mutant Kjr lacking the BMP-4 binding site were each incorporated into a pCXN2 expression vector, and Lipofectamine PLUS reagent was added to C0S-7 cells in 0PTI-MEM I medium (Invitrogen).
  • 0PTI-MEM I medium Invitrogen.
  • Kjr (-WHP) A culture supernatant containing Kjr or mutant Kjr
  • Kjr or mutant Kjr was pre-administered, and 3 hours later, 20 ng / ral BMP-4 (Wako Pure Chemical Industries, Ltd.) was added.
  • Neural stem cells were cultured and observed to differentiate into anti-GFAP antibody-positive astrocytes or anti-MAP2a, b antibody-positive neurons. The results showed that Kjr inhibited BMP-induced differentiation of neural stem cells into astrocytes and promoted neuronal differentiation.
  • the hippocampus of a 14-day-old rat is suspended in papain, cultured in a D-MEM medium containing 10% FBS (Invitrogen) for 2 days, and then transferred to a D-MEM medium containing N- ⁇ supplement. After culturing for 3 days, the culture supernatant was collected. Furthermore, neural stem cells were cultured for 5 days in culture supernatants incubated for 2 hours with anti-Kjr antibody, and the effect on neural stem cell differentiation into neurons was examined. This indicates that the activity of the hippocampus-derived factor that induces neural stem cells to bind to neurons is significantly inhibited by anti-Kjr antibodies, confirming that Kjr contributes to neurogenesis in the adult hippocampus. Was done.
  • FIG. 11 shows the results of measuring the frequency of MAP-2a, b-positive neurons (a) and GFAP-positive astrocytes (b).
  • BMP A system in which neural stem cells were cultured with BMP- for 5 days.
  • BMP + Kjr A system in which neural stem cells are cultured for 3 hours after adding Kjr, and further cultured for 5 days after adding BMP-4.
  • BMP + Kjr (-WHP) A system in which Kjr deficient in the amino acid WHP is added, the neural stem cells are cultured for 3 hours, and then BMP-4 is added to the mixture, followed by culturing for 5 days. A system in which Kjr was added and neural stem cells were cultured for 5 days.
  • Fig. 11 shows the differentiation of neural stem cells into nuclei by the hippocampus culture supernatant and its inhibition by the addition of anti-Kjr antibody (frequency of differentiation into MAP-2a, b-positive neurons) (c).
  • CM-fibroblast A system in which neural stem cells are cultured in fibroblast culture supernatant for 5 days.
  • CM A system cultured for 5 days in the culture supernatant of hippocampal cells.
  • CM + ab A system in which anti-Kjr antibody was added, cultured for 2 hours, and then cultured in the culture supernatant of hippocampal cells for 5 days.
  • CM + ag + ab A system in which antigen oligopeptide is mixed with anti-Kjr antibody, incubated for 30 minutes, added, cultured for 2 hours, and cultured in the culture supernatant of hippocampal cells for 5 days.
  • CM + NRS A system in which normal rabbit serum was administered, cultured for 2 hours, and then cultured for 5 days in the culture supernatant of hippocampal cells.
  • the Kjr protein blocks BMP-induced neural stem cell division into glial cells and promotes differentiation into neurons.
  • Rat fetuses were rapidly frozen under anesthesia, and sections were prepared with a cryostat and mounted on silane-coated slide glasses. The following oligo DNA probe capable of complementary binding to rat kjr mRNA was synthesized.
  • the oligo DNA probe was labeled with 35S using terminal deoxytransf erase (Takara) and 35S-dATP (NEN), hybridized with the above section at 42 ° C, and then washed. After drying, the emulsion was applied and autoradiography was performed at 4 ° C for 3 weeks. After development with a developer, microscopy was performed using a dark-field microscope. The results are shown in FIG.
  • KOHJIRIN as a representative example
  • KOHJIRIN is considered to have an effect of inhibiting bone morphogenetic proteins that are expressed in the central nervous system and suppress differentiation of nerve cells, abnormal expression of the kohjirin gene, Alternatively, dysfunction of protein KOHJIRIN is presumed to be a serious obstacle in maintaining higher brain functions.
  • the protein of the present invention itself is considered to be useful as a therapeutic agent for ischemic encephalopathy'traumatic encephalopathy or neurodegenerative diseases such as Alheimer's disease and Parkinson's disease. Further, it can be used for creating a substance having a function similar to the function of the protein, a substance promoting the function, or a substance promoting the expression of the gene.

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Abstract

L'invention concerne un gène kohjirin codant pour une nouvelle protéine KHOJIRIN ayant une zone de fixation d'une protéine ostéogénique. On obtient ce gène en clonant un astrocyte de rat de culture primaire originaire d'une bibliothèque d'ADNc. On obtient la nouvelle protéine KHOJIRIN par la mise en culture d'un transformant transformé par un vecteur d'expression ayant le gène ci-dessus. On peut utiliser cette protéine qui a une zone de fixation de la protéine ostéogénique, dans des médicaments ou dans le développement de médicaments.
PCT/JP2003/001850 2002-02-20 2003-02-20 Proteine ayant une zone de fixation d'une proteine osteogenique et gene codant pour cette proteine WO2003070937A1 (fr)

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US10961305B2 (en) 2016-12-21 2021-03-30 Mereo Biopharma 3 Limited Use of anti-sclerostin antibodies in the treatment of osteogenesis imperfecta

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WO2000009551A1 (fr) * 1998-08-10 2000-02-24 Genetics Institute, Inc. Proteines humaines connexes a la chordine et polynucleotides les codant
WO2001042465A2 (fr) * 1999-12-07 2001-06-14 Amgen, Inc. Molecules de type chordine et leurs utilisations
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WO2000009551A1 (fr) * 1998-08-10 2000-02-24 Genetics Institute, Inc. Proteines humaines connexes a la chordine et polynucleotides les codant
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Publication number Priority date Publication date Assignee Title
US10961305B2 (en) 2016-12-21 2021-03-30 Mereo Biopharma 3 Limited Use of anti-sclerostin antibodies in the treatment of osteogenesis imperfecta

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