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WO1996001843A1 - Polypeptide produit par des cellules du stroma - Google Patents

Polypeptide produit par des cellules du stroma Download PDF

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Publication number
WO1996001843A1
WO1996001843A1 PCT/JP1995/001342 JP9501342W WO9601843A1 WO 1996001843 A1 WO1996001843 A1 WO 1996001843A1 JP 9501342 W JP9501342 W JP 9501342W WO 9601843 A1 WO9601843 A1 WO 9601843A1
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WIPO (PCT)
Prior art keywords
polypeptide
dna
fragment
seq
cells
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PCT/JP1995/001342
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English (en)
Japanese (ja)
Inventor
Tasuku Honjo
Tohru Nakano
Atsushi Hirano
Shiro Sibayama
Hiroyuki Ohno
Original Assignee
Ono Pharmaceutical Co., Ltd.
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Publication of WO1996001843A1 publication Critical patent/WO1996001843A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel polypeptide produced by a stromal cell, a method for producing the same, a DNA encoding the polypeptide, a vector comprising the DNA, a host cell transformed with the vector,
  • the present invention relates to an antibody of a polypeptide, and a pharmaceutical composition containing the peptide or antibody. More specifically, a novel polypeptide produced by a certain mouse stromal cell line, a human homolog of the polypeptide, a method for producing the polypeptide, a DNA encoding the polypeptide, and a vector comprising the DNA
  • the present invention relates to host cells transformed with the vectors, antibodies of the polypeptides, and pharmaceutical compositions containing the polypeptides or the antibodies. Background art
  • Bone marrow stromal cells form the bone marrow microenvironment such as the immune system and hematopoietic system, and are factors that are indispensable for inducing the proliferation and differentiation of their stem cells, such as IL-17, SCF, IL-11, M—CSF, G— It is known that it produces and secretes factors such as CSF, GM—CSF, IL-16, TGF— / 3, and LIF. It has also been shown that some bone marrow stromal cells are involved in bone metabolism. However, only the group of factors isolated so far cannot completely fulfill the role of stromal cells. This suggests that some factors have not yet been isolated. Disclosure of the invention
  • the present inventors have paid attention to this point, and made intensive studies to find out a novel factor (polypeptide) produced by a certain type of stromal cells, particularly a secretory protein.
  • the desired biological activity is confirmed in a tissue or cell culture, and then the polypeptide is isolated and purified, followed by gene expression. Or a method of expressing and cloning a gene using its biological activity as an index has been generally used.
  • biologically active polypeptides in vivo often have various biological activities, and as a result of cloning a gene using a certain activity as an index, it is the same as a known polypeptide. More and more cases are found later.
  • most of the factors produced by bone marrow stromal cells produce only trace amounts, which makes isolation, purification and confirmation of biological activity difficult.
  • cDNA libraries are produced from various cells and tissues, the cDNA is randomly cloned, the nucleotide sequence is determined, and the corresponding polypeptide is expressed.
  • the method of analyzing physiological functions is being developed. This method has the characteristic that the gene can be cloned without any biochemical or genetic analysis and information on its nucleotide sequence can be obtained. Has a large accidental factor.
  • the present inventors have studied the cloning of genes for growth and differentiation factors that work in the hematopoietic and immune systems. And growth differentiation factors (eg, various Most of secreted proteins such as cytokins and membrane proteins such as their receptors (hereinafter collectively referred to as secreted proteins, etc.) have a sequence called signal peptide at the N-terminus. Focusing on the fact that the method of the present invention has been carried out, the present inventors have conducted intensive studies on a method for efficiently and selectively closing a gene encoding a signal peptide. As a result, we found a method to efficiently amplify the N-terminal fragment and to easily search for the presence or absence of a signal peptide (see Japanese Patent Publication No. Hei 6-315380 or European Patent Application Publication No. 0607054A2). The inventors have succeeded in finding a novel factor (peptide) produced by bone marrow stromal cells and a DNA encoding the same, and have completed the present invention.
  • the amino acid sequence of a known polypeptide registered in Swiss Prot Release 26 was investigated, but none of the polypeptides of the present invention had a sequence identical or highly homologous to that of the polypeptide of the present invention. None. Furthermore, the nucleotide sequence registered in DDBB (DNA Data Bank of Japan Release 17) was also examined, but no sequence identical or highly homologous to cDNA encoding the polypeptide of the present invention was found. Therefore, it was confirmed that the polypeptide of the present invention was completely novel.
  • a replication or expression vector comprising the DNA according to any one of the above items 4 to 6,
  • a method for producing the polypeptide comprising:
  • a pharmaceutical composition comprising the polypeptide according to 2 or 3 or the antibody according to 10 and a pharmaceutically acceptable excipient and Z or a carrier;
  • a method for producing the polypeptide according to the above 12 or 13, comprising culturing the host cell according to the above 18 under conditions for expressing the polypeptide.
  • a pharmaceutical composition comprising the polypeptide according to 12 or 13 or the antibody according to 20 and a pharmaceutically acceptable excipient and / or carrier. is there.
  • FIG. 1 is a conceptual diagram of a method for preparing a cDNA library according to the present invention.
  • FIG. 2 is a construction diagram of the plasmid vector pUCSRaML2.
  • Figure 3 shows the construction of ML2-hT ac with pUCSR.
  • FIG. 4 shows the hydrophobicity profile of the polypeptide of the present invention (mouse, part).
  • FIG. 5 shows the hydrophobic profile of the polypeptide of the present invention (human and partial).
  • the present invention is a.
  • the present invention relates to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 5 in a substantially pure form, a homolog thereof, a fragment of the sequence and a homolog thereof.
  • the invention further relates to DNAs encoding those polypeptides. More specifically, a DNA having the nucleotide sequence of SEQ ID NO: 2, 3, 6, or 7 and a fragment that selectively hybridizes to the nucleotide sequence of SEQ ID NO: 2, 3, 6, or 7 Having DNA.
  • a polypeptide having the amino acid sequence of SEQ ID NO: 1 or 5 in substantially pure form is generally defined as at least 90%, for example, 95, 98 or 99%, of the polypeptide at the time of production. This means that the polypeptide has the amino acid sequence shown in SEQ ID NO: 1.
  • a homologue of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or 5 generally means at least 20, preferably at least 30, for example, 40, 60 or 100 contiguous amino acid regions. It is at least 70%, preferably at least 80 or 90%, more preferably 95% or more homologous, and such homologs are hereinafter described as polypeptides of the present invention.
  • a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or 5 A fragment of a peptide, or a fragment of a homologue thereof, means a portion of at least 10 amino acids, preferably at least 15 amino acids, for example a 20, 25, 30, 40, 50 or 60 amino acid portion.
  • DNAs that selectively hybridize to DNA having the nucleotide sequence shown in SEQ ID NO: 2, 3, 6, or 7 are generally at least 20, preferably at least 30, for example, 40, 60 or It is at least 70%, preferably at least 80 or 90%, more preferably 95% or more homologous in 100 contiguous nucleotide sequence regions. Described as DNA.
  • the fragment of DNA having the nucleotide sequence represented by SEQ ID NO: 2, 3, 6, or 7 means at least 10 bases, preferably at least 15 bases, for example, 20, 25, 30 or 40 bases, and Such a fragment is also included in the DNA of the present invention.
  • the present invention includes a replication or expression vector comprising the DNA of the present invention.
  • the vector include a plasmid, a virus or a phage vector comprising an ori region and, if necessary, a promoter for expression of the above DNA, a promoter and a control element, and the like.
  • the vector may contain one or more selectable marker genes, for example, an ampicillin resistance gene.
  • the vector can be used in vitro, for example, for the production of RNA corresponding to DNA and for the transformation of host cells.
  • the present invention provides a vector for replicating or expressing the DNA of the present invention, which comprises the nucleotide sequence represented by SEQ ID NO: 2, 3, 6, or 7, or a DNA having an open reading frame thereof.
  • host cells transformed with include, for example, bacteria, yeast, insect cells or mammalian cells.
  • the present invention also includes a method for producing the polypeptide of the present invention, which comprises culturing the host cell of the present invention under conditions for expressing the polypeptide of the present invention. The cultivation is preferably performed under conditions in which the polypeptide of the present invention is expressed and produced from host cells.
  • An antisense RNA can also be produced by inserting the DNA of the present invention into the antisense region of a vector as described above. Such antisense RNA can also be used to control the level of the polypeptide of the present invention in cells.
  • the present invention also includes the monoclonal or polyclonal antibodies of the polypeptide of the present invention. Furthermore, the present invention includes a method for producing a monoclonal or polyclonal antibody of the polypeptide of the present invention. Monoclonal antibodies can be produced by ordinary hybridoma technology using the peptides of the present invention or fragments thereof as antigens. The polyclonal antibody can be produced by an ordinary method in which a host animal (for example, rat egret) is inoculated with the polypeptide of the present invention and immune serum is collected. The present invention also includes a pharmaceutical composition comprising the polypeptide of the present invention, its antibody, and a pharmaceutically acceptable excipient and / or carrier.
  • polypeptide of the present invention of (1) or (5) examples include those having a part of the amino acid sequence shown in SEQ ID NO: 1 or 5 and a part of which is deleted (for example, SEQ ID NO: 1).
  • SEQ ID NO: 1 or 5 a polypeptide consisting of only a part essential for the expression of biological activity and a soluble polypeptide lacking the cytoplasmic domain and transmembrane domain, etc.), some of which are combined with other amino acids
  • Substitutions for example, substitutions with amino acids having similar properties
  • those in which other amino acids are added or inserted into a part thereof are also included.
  • one codon codes for one amino acid.
  • ⁇ 6 types for example, 1 type for Met and 6 types for Leu. Therefore, the base sequence of DNA can be changed without changing the amino acid sequence of the polypeptide.
  • the DNA of the present invention specified by (2) or (6) includes all base sequence groups encoding the polypeptide represented by SEQ ID NO: 1 of (1) or SEQ ID NO: 5 of (5). Is included. By changing the nucleotide sequence, the productivity of the polypeptide may be improved.
  • the DNA specified by (3) or (7) is one embodiment of the DNA represented by (2) or (6), and represents a natural sequence.
  • the DNA shown in (4) or (8) represents the sequence obtained by adding the natural untranslated portion to the DNA specified in (3) or (7), respectively.
  • Preparation of DNA having the base sequence of SEQ ID NO: 3 is performed according to the following method.
  • the double-stranded DNA for the single-stranded DNA obtained in (1) is synthesized using a poly C oligomer to which a specific restriction enzyme (enzyme I) site is linked as a primer,
  • the amplified cDNA was subjected to polymerase chain reaction (hereinafter abbreviated as PCR) using a primer containing the enzyme I site and a primer containing the enzyme II site, and the amplified cDNA I was added to the enzyme I-enzyme II. After digestion with 1 42
  • step (1) after stimulating the target cells with an appropriate stimulant if necessary, a method such as Okayama H. (Method in Enzymology, 154, _3 ( 1987)).
  • the target cell may be any cell that may be producing a secretory protein or the like.
  • neural cells and hematopoietic cells can be mentioned.
  • the synthesis of single-stranded cDNA using a random primer is performed by a known method. Commercially available random primers are used.
  • oligo dT is added to the 3 'end of the single-stranded cDNA by terminal hydroxytransferase.
  • the synthesis of the double-stranded DNA in step (2) is also performed by a known method.
  • the restriction enzyme (enzyme I) site linked to the poly-G oligomer serving as a primer and the restriction enzyme (enzyme II) site used in the next step (3) can be any one as long as they are different from each other. Is also good.
  • Sa1I is used as enzyme I and SacI is used as enzyme II.
  • step (3) the cDNA is fragmented by sonication so that the cDNA length becomes 500 bp on average, fractionated into 400-800 bp cDNA by agarose electrophoresis (AGE), and then T4 DNA polymerase This is carried out by blunting the ends with ligation, ligating an enzyme II adapter, and fractionating again into a 400-800 bp DNA by agarose electrophoresis.
  • Enzyme II can be anything different from enzyme I as described above.
  • step (4) enzyme I, which may contain a signal peptide, and enzyme To further increase the probability of the existence of the part sandwiched by II, it is attached to PCR.
  • PCR is a well-known method, and automated devices for it are commercially available. Twenty-five to thirty amplifications are sufficient.
  • the amplified cDNA is digested with Enzyme I-Enzyme II and fractionated into 400-800 bp cDNA by agarose gel electrophoresis.
  • step (5) a known secretory protein or other gene (called a reporter gene) obtained by removing a signal peptide from a plasmid vector for eukaryotic cell expression, and the cDN obtained in (4) upstream thereof
  • a reporter gene obtained by removing a signal peptide from a plasmid vector for eukaryotic cell expression, and the cDN obtained in (4) upstream thereof
  • various plasmid vectors for eukaryotic cell expression are known. For example, pcDL-SR chick pcEV-4, which functions in Escherichia coli, is used.
  • the reporter gene genes of all kinds of soluble secreted proteins and mature proteins of membrane proteins are used. Furthermore, the expression of these reporter genes must be confirmed by any method such as an antibody method.
  • the human IL-2 receptor ⁇ gene is used. Many Escherichia coli strains for transformation are already known, and any of them may be used, but a DH5 competent cell (described in Gene, 96, 23 (1990)) is preferred. The transformant is cultured by a conventional method to obtain the cDNA library of the present invention (a conceptual diagram is shown in FIG. 1).
  • a library contains a gene fragment encoding a signal peptide, but not all clones contain the fragment. Not all gene fragments encode an unknown (new) signal peptide. Then, it is necessary to screen a gene fragment encoding an unknown signal peptide from the library. That is, the cDNA library is subdivided into pools of an appropriate size and incorporated into an expression system.
  • Expression systems for producing polypeptides include mammalian cells (eg, monkey COS-7 cells, Chinese hamster CHO cells, mouse L cells, etc.). Transfection is performed by known methods, for example the DEAE-Dextran method. After culture, the presence or absence of reporter gene expression is determined.
  • the reporter gene is known to be expressed even if the signal peptide is specific to other secretory proteins. That is, expression of the reporter gene indicates that a signal peptide of some secretory protein was incorporated into the library. Positive pools are further subdivided, and expression and determination are repeated until a single clone is obtained.
  • the expression of the reporter gene is determined depending on the type of the reporter gene, but is determined by a fluorescent-labeled antibody method, an enzyme-labeled antibody method (ELISA), a radiolabeled antibody method (RIA), or the like.
  • nucleotide sequence of the isolated positive clone was determined, and for cDNA that was found to encode an unknown protein, a full-length clone was isolated using it as a probe, and the full-length nucleotide sequence was sequenced. You can decide. These operations are performed by methods known to those skilled in the art. For example, the nucleotide sequence is determined by the Maxam-Gilbert method and the dideoxy terminator method. The full-length sequence is performed according to the method described in Molecular Cloning (published by Cold Spring Harbor Laboratory Press, 1989, by Sambrook, J., Fritsch, EF, and Maniatis, T.).
  • the DNA encoding the protein of the present invention present in mammals is also included.
  • a DNA encoding a homolog and a subset of the protein of the present invention can be obtained.
  • Oligonucleotides having the appropriate mouse nucleotide sequence are synthesized and used to hybridize from a mammalian cDNA library or mRNA by the PCR method, or using a fragment of the appropriate mouse nucleotide sequence as a probe. By doing so, a mammalian type DNA encoding the protein can be obtained from one of the mammalian cDNA libraries or the genomic library.
  • cells producing the peptide can be selected by Northern blot analysis or PCR.
  • a phage DNA was prepared from the obtained positive clone, and the excised cDNA was subcloned into a plasmid vector.
  • step (viii) It can be created by sequencing the entire length. More specifically, in step (i), a human glioblastoma cell line is applied. Stimulation with an appropriate stimulant (eg, IL-1 or the like) or no stimulation is performed according to the method of Okayama (H.) (described in Method in Enzymology, 154, 3 (1987)). It is.
  • a human glioblastoma cell line T98G ATCC strain number, CRL-1690
  • any cell producing the peptide can be used. Any cell may be used.
  • the step (ii) is performed according to the method of Okayama (H) or the like (see Method in Enzymology, 154, 3 (1987)).
  • Steps (iii), (iv) and (V) are steps for preparing a cDNA library and are performed according to the modified Gubler & Hoffman method (described in Gene, 25, 263 (1983)). .
  • plasmid vector Yuichi eg, pBR322, pBluescript II, etc.
  • phage vectors eg, ⁇ £ £ 10, ⁇ DAS ⁇ II, etc.
  • Many phage vectors are known, but preferably a phage vector ⁇ gt22A (42.73 kbp sold by BRL) is used.
  • Escherichia coli Y 1090 (r-1) (sold by BEL) is preferably used.
  • Steps (vi) and (vii) are performed according to the method described in Molecular Cloning (Sambrook, J, Frits, E, F, and Maniatis, T, Cold Spring Harbor Laboratory Press (1989)).
  • step (viii) is performed by the Maxam-Gilbert method, the didexy, and the Yuichi Minei Yuichi method.
  • the size of the mRNA obtained from the hybridized band and the size of the cDNA are compared, and if they are almost the same, the cDNA is considered to be almost full length.
  • the present invention is carried out by chemical synthesis or by chemically synthesizing a fragment of the nucleotide sequence and hybridizing this as a probe.
  • DNA can be obtained.
  • the required amount of the desired DNA can be obtained by introducing the vector containing the present DNA into an appropriate host and growing it.
  • Examples of (host-vector system) include bacterial, yeast, insect cell, and mammalian cell expression systems.
  • an initiation codon is added to the 5 'end of the DNA encoding the mature protein portion, and the resulting DNA is ligated to an appropriate promoter (eg, trp promoter, 1 ac promoter overnight;; iPL promoter, T7 promoter, etc.) and inserted into a vector that functions in E. coli (eg, pBR322, pUC18, pUC19, etc.) to insert the expression vector.
  • an appropriate promoter eg, trp promoter, 1 ac promoter overnight;; iPL promoter, T7 promoter, etc.
  • a vector that functions in E. coli eg, pBR322, pUC18, pUC19, etc.
  • Escherichia coli eg, E. Coli DH1, E. Coli J Ml09, E. Coli HB101, etc.
  • Escherichia coli transformed with this expression vector is cultured in a suitable medium, and the cells are used for the purpose. Can be obtained.
  • a bacterial signal peptide for example, the signal peptide of pe1B
  • the target polypeptide can be secreted into periplasm.
  • it can produce fusion proteins with other polypeptides.
  • the DNA encoding the nucleotide sequence represented by 7 is inserted into an appropriate vector (for example, a retrovirus vector, a herpes pilomavirus vector, a vaccinia virus vector, an SV40 vector vector, etc.).
  • An expression vector is prepared by inserting it downstream of a suitable promoter (eg, the SV40 promoter, LTR promoter, meta-mouth thionein promoter, etc.).
  • appropriate mammalian cells eg, monkey COS-7 cells, Chinese hamster CHO cells, mouse L cells, etc.
  • the transformants are cultured in an appropriate medium.
  • the desired polypeptide is secreted into the culture solution.
  • the polypeptide obtained as described above can be isolated and purified by a general biochemical method. Industrial applicability
  • polypeptide of the present invention is produced and secreted by a stromal cell line, biological activity related to the differentiation, proliferation, growth or maintenance of hematopoietic cells, biological activity related to the function of the immune system, It is thought to have biological activities related to proliferation, growth or inflammation, and also to bone metabolism.
  • any factor that acts on the immune system is also expected to act on the nervous system. Promoting proliferation of neurites, extending neurites, maintaining survival of ganglion cells, promoting proliferation or differentiation of astrocytosis, proliferation or maintaining survival of peripheral nerves, proliferation of Schwann cells, growth or survival of motor nerves. There may be.
  • the polypeptide is used to induce the formation of epidermis, brain, spine, and nerves by ectoderm-inducing action, dorsal cord connective tissue (bone, muscle, tendon), blood cells, It promotes or inhibits the formation of heart, kidney, gonad organs, or the formation of digestive organs (stomach, intestine, liver, kidney) and respiratory system (lung, trachea) by endoderm induction In addition to its potential, it may also have the effect of inhibiting the growth or growth of the above organs in living organisms.
  • the polypeptide of the present invention itself is a disease relating to hypoplasia or abnormal proliferation of hematopoietic cells, a decrease or increase in the function of the immune or nervous system or bone metabolism, such as inflammatory diseases (rheumatism, ulcers). Ulcerative colitis ), Hematopoietic stem cell depletion after bone marrow transplantation, Leukocyte, platelet, B cell or T cell depletion after radiation or chemotherapy for cancer, leukemia, anemia, infectious disease, cancer, leukemia, AIDS, It is expected to be used as a therapeutic agent for various degenerative diseases (Alheimer's disease, multiple sclerosis, etc.), nerve damage, and bone metabolism disorder (osteoporosis, etc.).
  • the polypeptide may have an action of differentiating or proliferating ectoderm, mesoderm, or endoderm-derived organs
  • the polypeptide epidermal, bone, muscle, tendon, heart, kidney, stomach, intestine, liver, It is also expected to be used as a tissue repair agent for the viscera, lung, trachea, etc.
  • polypeptide can be quantified in a living body using a polypeptide monoclonal antibody or a monoclonal antibody of the polypeptide, whereby the polypeptide can be used for studying the relationship between the polypeptide and a disease or diagnosing a disease.
  • Can be Polyclonal and monoclonal antibodies can be prepared by a conventional method using the polypeptide or a fragment thereof as an antigen.
  • the DNA of the present invention is not only an important and essential type II for producing the polypeptide of the present invention, which is expected to have tremendous utility, but also the diagnosis and treatment of genetic diseases (treatment of gene deficiency or It can be used for antisense DNA (RNA) to stop the expression of polypeptides and other treatments.
  • genomic DNA can be separated using the DNA of the present invention as a probe.
  • the polypeptides of the present invention may be used for diseases related to hypoplasia or abnormal growth of hematopoietic cells, abnormal or increased nervous system function, or increased or decreased immune system function, such as inflammatory diseases (rheumatism, ulcerative colitis, etc.), bone marrow Decrease in hematopoietic stem cells after transplantation Disease, leukocyte, platelet, B-cell or T-cell depletion after radiation therapy or chemotherapy, anemia, infectious disease, cancer, leukemia, AIDS, various degenerative diseases (Alzheimer's disease, multiple sclerosis) ) Or for preventing or treating nerve damage, for preventing or treating bone metabolic disorders (such as osteoporosis), or for repairing tissues, etc., usually systemically or locally, generally orally or parenterally. It is administered in the form of Preferred are oral administration, intravenous administration and intraventricular administration.
  • Dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually in the range of 1 to 10 mg per adult, once to several times a day. It is administered orally or parenterally, once to several times a day, in the range of 10 tg to 100 mg / adult per adult.
  • the dose varies depending on various conditions, so that a dose smaller than the above dose may be sufficient, or may be required beyond the range.
  • it is used as a solid composition, a liquid composition and other compositions for oral administration, an injection, an external preparation, a suppository and the like for parenteral administration.
  • Solid compositions for oral administration include tablets, pills, capsules, powders, granules and the like.
  • Capsules include soft capsules and hard carbs.
  • the one or more active substances include at least one inert diluent (e.g., lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch). , Polyvinylpyrrolidone, magnesium aluminate metasilicate, etc.).
  • the composition is prepared according to a conventional method, using additives other than inert diluents, such as lubricants (magnesium stearate, etc.), disintegrants (cellulose It may contain a calcium cholate, etc.), a stabilizer (human serum albumin, lactose, etc.) and a solubilizing agent (arginine, aspartic acid, etc.).
  • Tablets or pills may be coated with gastric or enteric film such as sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate if necessary, or two or more layers. May be applied. Also included are capsules of absorbable materials, such as gelatin.
  • Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, and commonly used inert diluents (eg, purified water, Ethanol etc.). Such compositions may contain, in addition to the inert diluent, adjuvants such as wetting agents and suspending agents, sweetening agents, flavoring agents, flavoring agents and preservatives.
  • adjuvants such as wetting agents and suspending agents, sweetening agents, flavoring agents, flavoring agents and preservatives.
  • compositions for oral administration include sprays which contain one or more active substances and are formulated in a manner known per se.
  • This composition contains, in addition to the inert diluent, a stabilizer such as sodium bisulfite to provide isotonicity, an isotonic agent such as sodium chloride, sodium citrate or citric acid. It may be.
  • a stabilizer such as sodium bisulfite to provide isotonicity
  • an isotonic agent such as sodium chloride, sodium citrate or citric acid. It may be.
  • the preparation of sprays is described in detail, for example, in U.S. Pat. Nos. 2,868,691 and 3,095,355.
  • Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • aqueous or non-aqueous solutions and suspensions one or more active substances are mixed with at least one inert diluent.
  • Aqueous diluents include, for example, distilled water for injection and physiological saline.
  • Non-aqueous diluents include, for example, propylene glycol, polyethylene glycol, and olive oil. Vegetable oils, alcohols such as ethanol, polysorbate 80 (registered trademark), and the like.
  • compositions may further include preservatives, wetting agents, emulsifiers, dispersants, stabilizers (eg, human serum albumin, lactose, etc.), solubilizing agents (eg, arginine, aspartic acid, etc.) ) May be included.
  • preservatives wetting agents, emulsifiers, dispersants, stabilizers (eg, human serum albumin, lactose, etc.), solubilizing agents (eg, arginine, aspartic acid, etc.) ) May be included.
  • compositions for parenteral administration include, for example, topical solutions, softeners, liniments, suppositories and pessaries for rectal administration, which contain one or more active substances and are formulated in a conventional manner. included.
  • Example 1 Construction of a plasmid for producing an expression vector
  • the P c D_SR 296 vector (described in Mol. Cell. Biol., 8, 966 (1988)) prepared by Takebe et al.
  • pc D—SRa 296 vector (provided by Takebe, National Institute of Health) was digested with Sa1I and the 1.7 kb P digestion containing the SRa promoter was separated using agarose gel electrophoresis. After recovery, the fragment was treated with Klenow to obtain blunt ends.
  • a 2.4 kbp fragment containing the AmpR and pUCori regions was separated and recovered using agarose electrophoresis, and treated with Klenow to make blunt ends. Thereafter, the cells were further treated with BAP (bacterial alkaline phosphatase) to remove the phosphate group at the 5 'end.
  • BAP bacterial alkaline phosphatase
  • the 1.7 kbp fragment containing the SRa promoter and the 2.4 kbp fragment containing pUC ori thus obtained were circularized by ligation to construct a new vector.
  • the PstI-KpnI fragment was removed from the obtained vector, and the following synthetic polylinker having the following T7 and sp6 promoters was synthesized.
  • the plasmid vector thus constructed (about 3.9 kbp, shown in Fig. 2) was named pUCSRaML2.
  • pUCSRML2 has the following features as a multipurpose plasmid vector.
  • Single-stranded DNA can also be prepared.
  • Example 2 cDNA library with selectivity for signal peptides —Construction of construction vector
  • a plasmid was constructed in which the cDNA encoding hT ac (used as a reporter gene, such as human IL-2 receptor) was transcribed into pUCSR ML2 as described above. And named pUC SRa-hT ac. Incorporates cDNA into the vector downstream of the hTac gene, downstream of the SR ⁇ promoter of the vector, and forms a fusion protein with hTac by translation of the protein from the cDNA having a signal sequence. Then, the fusion protein is expressed on the membrane.
  • hT a was added to the HindIII site of pBluescript SK (+) (sold by Stratagene, pBS). of. After digestion of pBS-hTac into which DNA has been incorporated, digestion with KpnI, blunting with T4 DNA polymerase, digestion with SacI, removal of leader sequence, The I-Eco RI adapter was ligated to obtain an Eco RI-blunt end fragment. This fragment was incorporated into the EcoRI-Smal site of pUCSRaML2, which had broken the SacI site, to obtain pUCSRML2-hTac (shown in FIG. 3).
  • Example 3 Generation of a cDNA library with selectivity for signal peptide
  • RNA was extracted by the AGPC (acid guanidine-funool-clo mouth form) method (described in detail in the Cell Engineering Experiment Protocol (published by Shujunsha), pages 28-31). Poly A-RNA was purified using oligo (dT) -latex (Oligotex-dT30 (trade name, sold by Takara Shuzo Co., Ltd.)).
  • the primers were used as primers to synthesize double-stranded cDNA. Fragmented cDNA by sonication to an average length of 500 bp Thereafter, cDNA of 400 to 800 bp was fractionated by agarose electrophoresis. After blunting the ends with T4 DNA polymerase, the mouth containing the Sac I site
  • the colonies obtained from the library prepared in Example 3 were subdivided into pools (about 50 colonies / pool).
  • plasmid was isolated by the miniprep method and transfected into COS-7 cells by the DAE-dextran method (Current Protocol in Molecular Biology, ⁇ 9.2.1). After 48 hours, the cells were detached from the dish, and incubated with mouse anti-Tac IgG antibody for 20 minutes on ice. After removing the free antibodies, the goat anti-mouse IgG antibody labeled with FITC (fluorescein isothiosinate) was incubated for 20 minutes on ice, and the free antibodies were removed again. Then, fluorescent staining for Tac on the cell surface was performed.
  • FITC fluorescein isothiosinate
  • pUCSR ⁇ ML2-hTac vector Two types of synthetic primers specific to one
  • the cDNA library was prepared from mRNA derived from the mouse stromal cell line ST-2 using SuperScript (registered trademark) lambda system (sold by BRL). This cDNA was ligated to Sgt22A (available from BRL) having a phosphatase-treated Sa1I, N0tI arm.
  • In vitro packaging carried out in accordance with professional tocol of in vitro-Nono 0 Kkei Managing Kit Lambda Yi down (in vitro Packaging Kit LAMDA INN) ( Nippon Gene), the recombinant phage host E. coli Y 1 090 (r —) (Sold by BBL). The result was a cDNA library of 1 million plaques.
  • pBS-ST-T1 was digested with Sail and Notl, and an ST-T1 fragment was prepared by agarose electrophoresis. Using the oligo-labeled ST-Tlc DNA fragment as a probe, the library was screened to obtain a number of positive clones.
  • the insert is about 1.0k 96/01 43
  • the Sa1I-NotI fragment cut out from the igt22A vector was subcloned into pUCS RaML2 to obtain plasmid pUCSRML2-ST-T1.
  • the base sequence of 300 bp on the 5 'side of the STT1 cDNA was determined, and the sequence matching the ST-T1 of the probe was the most 5% of the ST-T1 from the phage library. 'I first confirmed that it was on the side.
  • Example 6 Determining the full-length sequence and open reading frame of cDNA
  • plasmid was recovered from the ST—T1 clone, and the cDNA insert was separated and purified. This was ligated and fragmented, the end of the DNA fragment was blunt-ended with T4 polymerase, and the DNA fragment having a length of around 400 bp was recovered.
  • the obtained DNA fragment was cloned into the SmaI site of a plasmid vector, BLUESCRIPT II (sold by Stratagene), and then transformed into E. coli. After randomly picking 20 colonies and preparing plasmid DNA, the DNA sequence of these 20 plasmids (all of which have a fragment of ST-T1 cDNA as an insert). Quensing was performed. Sequencing of DNA and reading of the sequence were performed by the method described in Example 4.
  • sequence data for the ST—T1 cDNA fragment is the DNA fragment of the DNAS
  • the human glioblastoma cell line T98G (ATCC strain number, CRL-1690) was homogenized and incubated with oligo dT-cellulose. After washing unnecessary substances, polyA-RNA was eluted and recovered (see Venn storm, B. et. Al., Cell, 28, 135 (1982)).
  • the RNA 2 / g and after 1.0% Agarosu electrophoresis, block tee Ngushi to nitrocellulose main Npuren, 32 P-labeled mouse ST- T lc DNA is High Priestess soybean as a probe permitted the expression of mRNA A of 1.4kb Was.
  • Example 8 Separation and purification of human mRNA
  • CsTFA cesium trifluoroacetate
  • CsTFA cesium trifluoroacetate
  • CsTFA cesium trifluoroacetate
  • the cDNA library was prepared by a modification of the Gubler & Hoffmann method (see Gene, 25, 263 (1983)). From p 0 1 y prepared in Example 8 (A) ⁇ RN A ( 5 g), using an oligo dT primer with No t I site, by reverse transcriptase to synthesize first strand. Subsequently, a second strand was synthesized, subjected to Sail adapter ligation and N0tI digestion, and then subjected to gel filtration column chromatography (Sephacryl S-500HR (Pharmacia)). Excluding the adapter and primers, 820 ng of the cDNA fraction was recovered. The above cDNA synthesis step was performed using a kit of Super Script Lambda System (Super Script System, sold by BRL).
  • Example 10 Screening of human ST-Tlc DNA by cross-hybridization from a human cDNA library
  • Example 5 a recombinant phage was prepared to obtain a cDNA library consisting of 1,000,000 plaques. Illustrated 1,000,000 plaques obtained on LB plates Purotti Ngushi to nitrocellulose main Npuren, with 32 P-labeled mouse ST- T lc DNA (Example 5 SEQ ID NO: 3 of the same fragments as prepared in Were used as probes, and 40 positive clones were obtained.
  • Example 11 Isolation of positive clones of human ST-Tlc DNA Phage DNA was prepared from 6 of the clones by a conventional method (see Cell Engineering Laboratory Protocol, Shujunsha, page 8).
  • an open reading frame (shown as SEQ ID NO: 6) was determined, and further translated into an amino acid sequence to obtain a sequence shown as SEQ ID NO: 5.
  • the hydrophobic profile of the obtained amino acid sequence was drawn, and the hydrophobic region at the N-terminal portion characteristic of the signal peptide was confirmed (shown in FIG. 5).
  • the signal peptide portion of this peptide was estimated (Von Heuane, G. Nucleic Acids Res. 14, 4683 (1986)), and the sequence shown in SEQ ID NO: 8 was obtained.
  • the nucleotide sequence of DNA was determined by a cycle sequence method using a fluorescent dye terminator (available from Applied Biosystems Inc.). The sequence was read using a DNA sequencer (Model 373A, available from Applied Biosystems Inc.).
  • the nucleotide sequence of the obtained human ST-T1 cDNA and the predicted amino acid sequence were compared with those of mouse ST-T1, and found to be 92.4% in the amino acid sequence and 92.4% in the DNA sequence. Has 76.9% homology Arrangement table
  • Tyr Leu Asp Asp Trp Tyr Val Leu Cys lie Gly Pro Tyr Trp Val Arg 130 135 140 Asp Gly Glu Val Arg Phe Lys His Ser Ser Tyr Asp Val Leu Leu Ser 145 150 155 160
  • Sequence type nucleic acid
  • CAGCCAAGTC AGAACAACTA CTGGAAGGCC ATGGAAGGCA TCTTCATGAA GCCCAGTGAG 600
  • Sequence type nucleic acid
  • GACAGTGCTC TGTATTGGAC CTTACTGGGT TAGAGATGGT GAGGTGAGGT TCAAACATTC 480 TTCCACTGAC GTACTGCTGT CTGTCACAGG AGAACAGTAC GGACGACCCA TAAGTGGACA 540
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • CAGCCAAGTC AGAACAACTA CTGGAAAGCC ATGGAAGGCA TCTTCATGAA GCCCAGTGAG 600 TTGTTGAAGG CAGAAGCCCA CCATGCAGAG CTGTGA 636 SEQ ID NO: 7
  • Sequence type nucleic acid
  • GTTTTCTTCG AAGATTTGGG GCTCCGCGAT ACAGTTAGGA TGGCTGTAGT ACCTCTGCTG 60 TTGTTGGGGG GTTTGTGGAG CGCTGTGGGA GCGTCCAGCC TGGGTGTCGT TACTTGCGGC 120 TCCGTGGTGA AGCTACTCAA TACGCGCCAC AACGTCCGAC TGCACTCACACGGACGCG
  • Sequence type nucleic acid
  • Organism name Homo Sapiens
  • Trp Arg lie Arg Arg Lys Ser Ala Thr Val Cys Glu Arg Gly Thr Pro

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Abstract

L'invention concerne un polypeptide produit par les cellules médullaires du stroma de souris et comprenant 211 restes d'acide aminé, ainsi qu'un homologue humain de ce polypeptide; un procédé de production dudit polypeptide; un ADN codant pour le polypeptide; un fragment s'hybridant sélectivement avec la séquence d'ADN; un vecteur de réplication ou d'expression comprenant ledit ADN; une cellule hôte transformée par ce vecteur; et un anticorps dirigé contre le polypeptide, ainsi qu'une composition pharmaceutique contenant ledit polypeptide ou ledit anticorps. Ce polypeptide peut être utilisé pour la prévention ou le traitement de l'hypoplasie ou la croissance anormale des cellules hématopoïétiques, l'hyperergasie ou l'hypergasie du système nerveux, les maladies liées à l'hyperergasie ou l'hypergasie du système immunitaire telles que les maladies inflammatoires, la diminution des cellules hématopoïétiques consécutive à une greffe de moelle osseuse, la diminution des leucocytes, des plaquettes, des lymphocytes B ou T après une radiothérapie, l'anémie et les maladies infectieuses; la prévention ou le traitement du cancer, de la leucémie ou du SIDA; la prévention ou le traitement de diverses maladies dégénératives et de lésions nerveuses; la prévention ou le traitement de désordres métaboliques des os, et la réparation tissulaire.
PCT/JP1995/001342 1994-07-08 1995-07-05 Polypeptide produit par des cellules du stroma WO1996001843A1 (fr)

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JP18079194 1994-07-08
JP6/180791 1994-07-08

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WO1996001843A1 true WO1996001843A1 (fr) 1996-01-25

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001061004A1 (fr) * 2000-02-15 2001-08-23 Chugai Seiyaku Kabushiki Kaisha Nouvelle proteine comportant une sequence signal
JP2002325573A (ja) * 2001-04-27 2002-11-12 Japan Science & Technology Corp ベクター

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06315380A (ja) * 1993-01-14 1994-11-15 Yuu Honshiyo ｃＤＮＡライブラリーの作製方法、および新規なポリペプチドとそれをコードするＤＮＡ

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06315380A (ja) * 1993-01-14 1994-11-15 Yuu Honshiyo ｃＤＮＡライブラリーの作製方法、および新規なポリペプチドとそれをコードするＤＮＡ

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EMBO J., (1988), Vol. 7, No. 5, OGAWA M. et al., "B Cell Ontogeny in Murine Embryo Studied by a Culture System With the Monolayer of a Stromal Cell Clone ST2 B Cell Progenitor Develops First in the Embryonal Body Rather Than in the Yolk Sac", pages 1337-1344. *
SCIENCE, (1993), Vol. 261, No. 5121, TASHIRO K. et al., "Signal Sequence Trap a Cloning Strategy for Secreted Proteins and Type I Membrane Proteins", pages 600-3. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001061004A1 (fr) * 2000-02-15 2001-08-23 Chugai Seiyaku Kabushiki Kaisha Nouvelle proteine comportant une sequence signal
US7067297B2 (en) 2000-02-15 2006-06-27 Chugai Seiyaku Kabushiki Kaisha Mannosyltransferase polypeptides and polynucleotides encoding them and methods for making and using them
JP2002325573A (ja) * 2001-04-27 2002-11-12 Japan Science & Technology Corp ベクター

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