WO1997020929A1 - Fibroblast growth factor fgf-10 - Google Patents

Fibroblast growth factor fgf-10 Download PDF

Info

Publication number
WO1997020929A1
WO1997020929A1 PCT/JP1996/003579 JP9603579W WO9720929A1 WO 1997020929 A1 WO1997020929 A1 WO 1997020929A1 JP 9603579 W JP9603579 W JP 9603579W WO 9720929 A1 WO9720929 A1 WO 9720929A1
Authority
WO
WIPO (PCT)
Prior art keywords
fgf
primer
seq
dna
sequence
Prior art date
Application number
PCT/JP1996/003579
Other languages
French (fr)
Japanese (ja)
Inventor
Nobuyukia Itoh
Takaharu Negoro
Takashi Katsumata
Shuzo Tagashira
Original Assignee
Sumitomo Pharmaceuticals Company, Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sumitomo Pharmaceuticals Company, Limited filed Critical Sumitomo Pharmaceuticals Company, Limited
Priority to AU10412/97A priority Critical patent/AU1041297A/en
Publication of WO1997020929A1 publication Critical patent/WO1997020929A1/en

Links

Classifications

    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • 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 fibroblast growth factor (hereinafter, abbreviated as FGF) and a method for recombinantly producing the same. Further, the present invention relates to a pharmaceutical use of the factor.
  • FGF novel fibroblast growth factor
  • FGF was discovered as an angiogenic factor in the 1970s. Initially, acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) were studied,
  • bFGF and a FGF are being studied as potential therapeutic agents for diseases of the nervous system, vascular system, and bone metabolic system, utilizing their widespread cell growth promoting effect.
  • its usefulness in clinical trials has not been proven. Similar research in the new FGF is anticipated.
  • An object of the present invention is to provide a method for industrially producing a recombinant protein by discovering and analyzing a novel FGF gene.
  • FGF-10 a completely new type of FGF (hereinafter abbreviated as FGF-10) DNA, and completed the present invention. Reached.
  • the present invention encodes FGF-10 as shown in the following (1)-(15).
  • the present invention relates to DNA, an expression vector carrying the DNA, a transformant, a method for producing a recombinant protein using the DNA, a recombinant protein, and a pharmaceutical use of the FGF-10 recombinant protein.
  • a recombinant DNA comprising a nucleotide sequence encoding a fibroblast growth factor which is a polypeptide of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence complementary thereto.
  • a method for producing a recombinant fibroblast growth factor which comprises using the transformant of (4).
  • a recombinant fibroblast growth factor which is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a main part thereof.
  • a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a main part thereof, which is produced by the transformant of (5) and exhibits cell growth activity.
  • a recombinant fibroblast growth factor A recombinant fibroblast growth factor.
  • a medicament comprising the recombinant fibroblast growth factor according to (7) or (8) as an active ingredient.
  • a method for treating bone Z cartilage disease or bone Z cartilage damage which comprises administering an effective amount of the recombinant fibroblast growth factor of (7) or (8) to an animal including a human.
  • the effective amount of the recombinant fibroblast growth factor of (7) or (8) A method for promoting wound healing comprising administering to animals.
  • Figure 1 shows the two types of FGF-3, FGF-7, and FGF-10 common primers used for cloning the FGF-10 gene, and (A) Ty r—L eu—A la—Met—A sn — Lys, (B) Tyr—A sn—Thr—Tyr—A1a-Ser.
  • FIG. 2 shows a primer for isolating FGF-10c DNA used in the Rapid Amplifo ncat o Endo Fc DNA Ends (RACE) method.
  • FIG. 3 shows an outline of a plasmid construction method for obtaining plasmids pFGF-10F, plasmids pCDM8-F10SP and pCDM8-F10HX.
  • FIG. 4 shows the primer and PCR reaction conditions used to replace the translation initiation codon upstream with the Kozak consensus sequence.
  • Fig. 5 is a photograph showing the expression of FGF-1 OmRNA in rat joint tissues by the in situ hybridization method.
  • A is a micrograph of an articular cartilage specimen
  • B is an epiphyseal cartilage. It is a microscope picture of a board.
  • FIG. 6 is a graph showing the uptake of tritiated thymidine into FRSK cultured cells.
  • the abscissa Bq indicates the control, and Sp and Hx indicate the cases where a culture solution of FGF-10 expressing COS cells was added.
  • the vertical axis shows the radioactivity in the cell.
  • FIG. 7 is an image-processed image of a tibial soft radiograph of the human FGF-10 administration group in the test example.
  • FIG. 8 is an image-processed image of a tibial soft radiograph of the control group in the test example.
  • FIG. 9 is a conceptual diagram of the construction of an FGF-10 variant expression plasmid, which shows the production of a mutant DNA fragment.
  • FIG. 10 is a graph showing the biological activity of human FGF-10 expressing COS cells and its variants, and showing the uptake of tritium-labeled thymidine into FRSK cultured cells.
  • the symbol on the horizontal axis is WT, natural human FGF-10, DN 1-5, DC 1-4, C84ZS106, S150ZS106, A51 / A196.SA, A51, A 196, S150 and S106 (Avr is the undiluted culture supernatant, Avr (1Z10) is the 1Z10 dilution of the culture supernatant) were the respective FGF-10 variants.
  • Means, the vertical axis is F Shows radioactivity in RSK cells.
  • FGF-10 means a fibroblast growth factor produced by a mammal, including the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a major portion thereof.
  • the main part means a mature protein amino acid sequence obtained by removing a signal (pre) sequence or a pro sequence from the above sequence. That is, an amino acid sequence consisting of 179 amino acid residues ranging from glutamine at position 37 (Gin) to serine at position 21 (Ser) in SEQ ID NO: 1, or 3 in SEQ ID NO: 2. This is an amino acid sequence consisting of 1711 amino acid residues from glutamine (Gin) at position 8 to serine (Ser) at position 20-8.
  • FGF-10 is an A sn—Ser—S er located at position 50-52 of the amino acid sequence shown in SEQ ID NO: 1, and an A sn—Th r at position 203—205.
  • the FGF-10 variants of the following types (1) to (5) also exhibit the same physiological actions as the natural type FGF-10, and belong to the technical scope of the present invention.
  • FGF-10 variants of (1) to (19) are exemplified. As described in detail in Examples, these can be created by a known technique of genetic engineering today.
  • Fibroblast growth factor activity refers to cell growth stimulating action, hematopoietic stem cell growth action, angiogenesis action, cell growth promotion action for various cells, cell differentiation induction action, extracellular matrix modification action, etc. , At least one of a wide variety of physiological activities of FGFs, such as the effect of maintaining the survival of nerve cells [Clinical Inspection, Vol. 38, No. 11, pp. 219-221 (1994 extra number) )].
  • Epithelium including rat fetal epidermal cells (FRSK cells), as seen in FGF-7
  • the cell growth stimulating action of the cell-derived cell line is also included in the activity.
  • Bone / cartilage damage treatment or bone Z cartilage disease treatment '' is a pharmaceutical preparation that promotes the healing of physical damage to bone cartilage, such as accidental fractures and bone / cartilage resection due to surgery.
  • it is a pharmaceutical preparation for treating a disease whose main symptom is a decrease in bone Z cartilage formation, and is directed to the following pharmaceutical uses (1) to (6).
  • Bone defect treatment (2) Bone fracture treatment, (3) Osteoporosis treatment, (4) Cartilage tissue repair promoter, (5) Articular cartilage tissue repair treatment, and (6) Osteoarthritis Therapeutic agent.
  • Wild healing promoter means a pharmaceutical preparation that promotes the healing of trauma, frostbite and burns due to physical and chemical factors caused by accident. Also includes healing accelerators for intractable skin and muscle tissue disorders such as radiation damage, floor rubbing and skin ulcers caused by diabetes.
  • the DNA encoding FGF-10 of the present invention can be produced by a known genetic engineering method. That is, mRNA can be isolated from mammalian living tissue or cultured cells, and then double-stranded cDNA can be obtained. Furthermore, using this cDNA as a primer, PCR can be performed, amplified, and the sequence can be determined as appropriate. Each of these kits has a dedicated kit on the market.
  • the type of biological tissue or cultured cells as the raw material of mRNA is not particularly limited, but a method using a rat fetus about 14 days old is particularly preferable. Since mRNA expression is relatively high in lung and joint tissues, lung cells, cultured cells derived from bone-Z cartilage-derived cells, and the like can also be used.
  • a method using commercially available adult human lung-derived poly (A) + RNA [Clontech] is also simple and preferable. Further, from the DNA sequence encoding FGF-10 disclosed in the present specification, it is possible to clone an appropriate sequence as a DNA probe and clone it from various living organism-derived cDNA or genomic gene libraries. it can.
  • the gene library is prepared as follows according to a conventional method.
  • the DNA probe selects a highly specific sequence from the DNA sequences encoding the FGF family proteins disclosed in the present specification. It can be chemically synthesized by conventional methods and labeled with 32 P or the like.
  • Expression vectors incorporating the cDNA of FGF-10 thus obtained include, for example, a plasmid or phage capable of growing in a suitable host such as Escherichia coli, Bacillus subtilis, yeast, and animal insect cells.
  • a suitable host such as Escherichia coli, Bacillus subtilis, yeast, and animal insect cells.
  • the host is not particularly limited as long as it is an organism or a cultured cell that can be transformed by introduction of the vector and can produce FGF-10.
  • Typical examples of bacteria include Escherichia coli and Bacillus subtilis (Bacillus), yeasts include Saccharomyces, Torula and Pichia, and animal cells include COS cells, CHO cells, and NSO cells.
  • cultured insect cells, fungi, plant cells, and unicellular cells, insects, mammals, and plants into which the target protein gene has been incorporated fall into the category of hosts.
  • Escherichia coli or bacteria of the genus Bacillus are generally used as a prokaryotic cell production system.
  • Bacillus 11 usbrevis which has a reduced protease production ability, is useful as a host for secretory expression (see : Japanese Patent Application Laid-Open No. 6-2966485, Japanese Patent Application Laid-Open No. 6-133878, Y. Sagiyaeta 1 .; Ap plied
  • clones can be transiently expressed in COS cells and cloned by evaluating the physiological activity of the culture supernatant.
  • the physiological activity of the expressed FGF-10 protein can be easily detected by a conventional method. For example, it can be evaluated by measuring the growth promoting effect of epithelial cells such as known FRSK cells.
  • the cloned DNA-containing plasmid can be used as it is or cut out with restriction enzymes, and it is incorporated into an expression vector suitable for various hosts and expressed to produce a large amount of FGF-10 protein.
  • the expression method is not particularly limited, and recombinant protein production techniques known in the art can be applied as appropriate, such as fusion expression, secretory expression, and direct expression using bacteria, and expression using eukaryotic cells.
  • the FGF-10 protein produced by recombinant technology can be purified by a purification method commonly used in the field of biochemistry. Ion exchange chromatography, gel filtration, reverse phase HPLC, ammonium sulfate precipitation, ultrafiltration, SDS-PAGE, etc. are used in combination as appropriate.
  • FGFs affinity using ligands such as heparin is particularly important.
  • Monochromatography, antibody column chromatography and the like are suitable for large-scale purification.
  • Antibodies to the FGF-10 protein can be prepared by a method known per se for both polyclonal and monoclonal antibodies. FGF-10-specific antibodies can be used not only for antibody columns but also for immunochemical quantification such as ELISA.
  • the FGF-10 protein obtained by the above-described method has various physiological actions including a cell growth promoting action, and is used as a wound healing promoting agent, a therapeutic agent for circulatory insufficiency, an agent for maintaining nerve survival, and hair growth. It can be used for pharmaceutical applications such as accelerators. In particular, its expression in cartilage tissue of adult mammals has been observed, and its application to the treatment of bone diseases such as fracture healing and the treatment of cartilage and connective tissue damage is conceivable. It can also be used as an experimental reagent for promoting cell proliferation.
  • Administration of the FGF-I0 protein to animals and humans can be carried out by a usual administration route, for example, intramuscular, intravenous, subcutaneous, intraperitoneal, transdermal administration and the like. Dosage and administration The number of doses varies depending on the subject of administration, route of administration, degree of symptoms, body weight, etc., and is not particularly limited. The number of times is administered. Examples of the dosage form include an injection. At the time of formulation, it is manufactured by a usual method using an ordinary formulation carrier. That is, when preparing an injection, a freeze-dried product of FGF-10 protein is dissolved in physiological saline, and a pH adjuster, a buffer, a stabilizer, a solubilizer, and the like are added as necessary. Use it as a propellant in the usual way.
  • Rat fetal cDNA was prepared using Moroni murine leukemia virus reverse transcriptase using the rat fetal mRNA as a type II and a random primer (6mer) as a primer. That is, rat fetal poly (A) + RNA (5 g) was added to 300 units of Moloney muriine.
  • the cDNA was obtained by incubating at 37 ° C for 60 minutes in a reaction solution containing RNaseinhibbitor (Wako Pure Chemical Industries) and 0.5 g of random primer (6mer).
  • the amino acid sequences of seven known human FGFs were compared, and two amino acid sequences identical between FGF_3 and FGF-7 (Tyr-Leu—Ala—Met—Asn—Ly s, Tyr—Asn—Thr—Tyr—Ala-Ser) were selected, and two types of FGF primers shown in FIG. 1 were prepared.
  • Rat fetal cDNA was converted to type II, and FGF family DNA was amplified by the polymase chain reaction (PCR) method using the two types of FGF primers and Taq DNA polymase. That is, it contains an appropriate amount of cDNA, 0.05 unit /// 1 TaQ DNA polymerase (Wako Pure Chemical Industries) and 5 pmo 1/1 of the above-mentioned sense- or antisense primer.
  • the reaction solution 25 ⁇ 1 was subjected to 30 cycles of PCR. After the reaction, the solution was subjected to 8% polyacrylamide gel electrophoresis, and a fraction having a desired size ( ⁇ 110 base pairs) was eluted by electrophoresis.
  • the FGF family DNA amplified by the FGF primer was inserted into a pGEM-T DNA vector (Promega), and the resulting recombinant vector was infected into E. coli (XL1-b1ue strain). A DNA clone was obtained.
  • c DNA sequence analysis includes DNA sequencer 373 A (Ap p 1 i e d
  • FGF-10 a peptide having a similar amino acid sequence structure to the known FGF family peptide ( ⁇ 50%) was also encoded.
  • New FGF cDNA was isolated. This was designated as FGF-10.
  • cDNA was synthesized by reverse transcriptase using primer-X with rat fetal mRNA as type III. Using the cDNA thus obtained as type I, PCR was performed using primers C and Y. Further, PCR was performed using primers D and Y. The obtained amplified fragment was introduced into pGEM-T and cloned by transforming Escherichia coli (XL1b1ue strain). When the nucleotide sequences of several clones were determined, a clone containing a part of the above partial sequence was obtained. The clone was named pFGF-10 (3 ').
  • E and F (FIG. 2, SEQ ID NOS: 11 and 12) were generated.
  • the first strand of cDNA was obtained by reverse transcriptase using rat fetal mRNA as type I and oligo dT as primer. Using this as a type I, PCR was performed using Primers E and F. The obtained amplified fragment was inserted into pGEM-T, and cloned by transforming Escherichia coli (XL1-b1ue strain).
  • Plasmid pFGF-10 (FIG. 3) was digested with Sphl and PstI, and a fragment containing the entire cDNA was isolated by polyacrylamide electrophoresis. This fragment was ligated with an SphI and Pstl digest of PUC19, and Escherichia coli JM109 strain was transformed to obtain a plasmid pUC-F10 containing FGF-10 cDNA. A fragment containing FGF-10 cDNA was excised by digesting pUC-F10 with HindIII and XbaI, and the mammalian cell expression vector ⁇ 01 ⁇ 8 1 ⁇ 11 By ligating with the 0,11,13 I digest and transforming the E. coli MC1061P3 strain, plasmid p CDM8 with FG F-10c DNA under the control of the CMV promoter was obtained. — I got F10 SP.
  • Mutagenesis is introduced by PCR, using p FG F-10 as a ⁇ type as shown in Fig. 4 and having a Hind III cleavage site at the 5 'end and a Cossack consensus sequence.
  • the reaction was carried out by using a primer and an antisense primer having an XbaI cleavage site at the 5 'end (see FIG. 4 for reaction conditions).
  • the PCR product was treated with phenol-mouth form, treated with ether, precipitated with ethanol, digested with Hindlli and XbaI, and then subjected to polyacrylamide gel electrophoresis. Released.
  • This fragment was ligated with the H1ndllUXbaI digest of PCDM8, which is a mammalian cell expression vector, and transformed into colonies obtained by transforming Escherichia coli MC1061ZP3. Four clones were selected from, and the nucleotide sequence was analyzed using a DNA sequencer (Perkin-Elmer 373).
  • Transformation of C0S-1 cells with a rat FGF-10-expressable brassmid A rat FGF-10-expressable brassmid constructed in Example 2, pCDM8-F108? A large amount of 01 ⁇ 8—F10HX was prepared according to the usual method, and purified by twice performing cesium chloride density gradient ultracentrifugation. Using these two kinds of plasmids and pCDM8 as a control, COS-1 cells were transformed by an electric pulse method. The transformed cells were cultured for 24 hours in DMEM containing 10% fetal bovine serum treated with lysine cephalo-chromatography, the medium was replaced with serum-free DMEM, and further cultured for 96 hours. After centrifuging the culture solution thus obtained, the supernatant was dispensed and stored frozen at 80 ° C.
  • the FGF-10c DNA was incorporated into the pGEM-T vector, the plasmid was transfected into E. coli JM109, and then mass-cultured.
  • the highly pure FGF-10c DNA was purified using Flexi Prekit of the company. After confirming the sequence using a Perkin Elmer 373 A / DNA sequencer, a cRNA probe was prepared using a DIG / RNA labeling kit (SP6 / T7) from Boehringer.
  • Wistar female rats were sacrificed at the age of 3 weeks, and the femur and tibia were excised while holding the joints, the soft tissues were removed, trimmed to an appropriate size, and then fixed immediately (4% 3. soak in paraformaldehyde) Fixed overnight at C. After dehydration, they were immersed in a demineralized solution (10% EDTA, 15% glycerol—PBS) and decalcified for 4 to 5 days (the solution was changed every day). Then, it was trimmed to about 2 cm before and after the knee joint, immersed in a 0. C. T compound, frozen with liquid nitrogen, and made a 10-zm-thick joint tissue section using a cryostat. Mounted on Tingslide glass.
  • su 1 fate 600 mM NaC 0.25% SDS
  • dilute 10-fold put 50 ⁇ 1 per piece, cover with small pieces of parafilm, and place at 50 ° C 16 Time Incubation.
  • Unnecessary probes were digested with RNase A, washed with SSC, and then subjected to an antibody reaction and a color reaction.
  • Rat epithelial cells were cultured at 37 ° C in 15 ml of F-12 medium containing 10% 10% fetal serum per 75 square cm culture flask. The cells were cultured under a gas phase of 5% carbon dioxide / 95% air. Cells were subcultured once every 7 days at a rate of 1.Z10.
  • FGF-10 was transiently expressed in COS-1 cells (see Example 3), and the culture supernatant was subjected to the following assay (hereinafter, obtained using pCDM8-F10SP). Culture supernatant obtained using FGF-10Sp, pCDM8-F10HX was used for the culture supernatant, and culture supernatant obtained using FGF-10ZHx and control plasmid pCDM8. The supernatant is indicated as Bq).
  • DNA-synthesizing assay incorporation of tritium-labeled thymidine: After culturing the cells to subconfluents, detach the cells by trypsin treatment, adjust to 1000 cells / m1 using the above medium, and adjust to 96 1001 was inoculated into the well plate, and cultured at 37 ° C in a gas phase of 5% carbon dioxide and 95% air. Once every two days, the medium is replaced with the above-mentioned medium 100 Z1.After culturing for 7 days, the medium is changed to medium F-012 containing 0.1% 0 serum albumin. Replaced.
  • the FGF-10 expression group: Sp, Hx greatly increased the incorporation of tritium-labeled thymidine into FRSK cells (each 2 86%, 50 1%). It is suggested that FGF-10 is a factor that promotes the proliferation of epithelial cells.
  • human lung poly (A) + RNA [clontech (C1ontech): Catalog No. 6524, derived from adult male whole lung] as type III, using a random primer (6mer) as a primer
  • Human lung cDNA was prepared using mouse leukemia virus reverse transcriptase. That is, human lung poly (A) + RNA (5 g) was converted to 300 units of M 0 10 n e y mu r i n e
  • Example 1 The two types of FGF primers used in Example 1 shown in FIG. 1 (Tyr—Leu-A1a-Met_Asn—Lys, TyrAsn—Thr—Tyr-A1a— Ser) was used to amplify the human FGF-10 gene.
  • the human lung cDNA was converted into type II, and the pol ymerase chain using the above two FGF primers and Taq DNA pol ymerase.
  • FGF family DNA was amplified by the chain (PCR) method. That is, a reaction solution containing an appropriate amount of cDNA, 0.05 units // 1 of TaqDNA polymerase (Wako Pure Chemical Industries) and 5 pmo1Z // 1 of the above-mentioned sense-1 or antisense primer ( 25/1) was subjected to 30 cycles of PCR. After the reaction, the solution was subjected to 8% polyacrylamide gel electrophoresis, and a fraction having a desired size ( ⁇ 110 base pairs) was eluted by electrophoresis.
  • Primers A ′ and D ′ (FIG. 2, SEQ ID NOS: 13 and 14) were prepared from the partial structure of human FGF-10 cDNA. Primers B, C, X and Y
  • phFGF-10 (5 ').
  • cDNA was synthesized by reverse transcriptase using human lung mRNA as type II. Using the cDNA thus obtained as type I, PCR was performed using primers C and X. Furthermore, PCR was performed using primers D 'and Y. The obtained amplified fragment was inserted into pGEM-T, and cloned by transforming Escherichia coli (XL1-b1ue strain). When the nucleotide sequence of several clones was determined, a clone containing a part of the above partial sequence was obtained, and this was named phFGF-10 (3 ′).
  • primer E (Fig. 2: SEQ ID NO: 12) was diverted. From the base sequence of the most downstream base sequence obtained from phFGF_10 (3 '), a new primer 1F' (FIG. 2: SEQ ID NO: 15) was newly prepared as a 3'-side primer.
  • the first strand of cDNA was obtained by reverse transcriptase using human lung mRNA as type II and oligo dT as primer. PCR was carried out using the primers E and F 'as a type III. The obtained amplified fragment was introduced into PGEM-T, and cloned by transforming Escherichia coli (XL1-b1ue strain). When the nucleotide sequences of several clones were determined, the nucleotide sequence of the upstream portion of the translation region obtained from phFGF-10 (5 ') and the nucleotide sequence of the most downstream portion obtained from phFGF-10 (3') were obtained. A clone was obtained which continuously retained the nucleotide sequence of the sequence. One of these clones was selected and named phFGF-10. Human FGF-10 cDNA containing the entire translation region carried by this plasmid was analyzed.
  • nucleotide sequence (690 bp) of SEQ ID NO: 4 was determined.
  • the determination of the entire amino acid sequence of c-human FGF-10 was determined.
  • the translated region of human FGF-10 cDNA comprises 624 bp, and human FGF-10 is represented by SEQ ID NO: 2. It was revealed that the polypeptide was composed of amino acids. Investigation of the amino acid sequence revealed that the protein was a secretory protein having a signal sequence at the N-terminus. The mature part is 38-208, a polypeptide consisting of 17 1 amino acids It is regarded as C. 5 A sn—Ser—Ser at position 1—53 and A sn—Thr_Ser at position 196-198 are N-linked glycan-binding sequences. May have
  • p FGF-10 was used as type II, 15 cycles of PCR were carried out using the following primer pair (SEQ ID NO: 16 and SEQ ID NO: 17), and ethanol precipitation was performed after processing with phenol noclo mouth form.
  • the DNA fragment corresponding to the mature amino acid sequence of human FGF-10 cDNA is obtained by digesting with NdeI and BamHI and separating a band of a desired size by polyacrylamide gel electrophoresis. (A) was obtained.
  • PET-11c (Stratagene), an Escherichia coli expression vector, was digested with NdeI and BamHI and fractionated by agarose gel electrophoresis to obtain linearized vector-DNA (b). Obtained.
  • BL 2 1 (DE 3) / p ET- h FG F-10 was inoculated into 1 Om 1 of 18 medium containing 100 g of ampicillin / '111 1 and prepared at 37 ° C. Pre-culture overnight. On the following day, the whole amount was inoculated into four 500 mL x 1 TB media containing 100 ⁇ g / ml, and cultured with shaking at 37 ° C. When the OD 600 reached 0.8, IPTG was added to a final concentration of 1 mM, the culture temperature was lowered to 28 ° C, and the culture was continued for another 6 hours.
  • the culture solution was centrifuged, and the obtained cells were washed once with 5 OmMT ris—HC1, H8.0, and then washed with 1 mM EDTA, 2 / g nom 1 leptin, 2 / igZm l ⁇ Pustatin was suspended in 5 OmMT ris-HC1, H8.0 containing 1 mM PMS F.
  • the cells are disrupted by sonication, and centrifuged at 1500 rpm for 1 hour using a JA-20 rotor with a Beckman J2-21 MZE high-speed cooling centrifuge. Then, the supernatant was collected.
  • HiTrap Heparin (5 ml, Pharmacia) was equilibrated with 5 OmMTris-HC1, H8.0, and the cell lysate supernatant prepared above was applied. Subsequently, the protein was washed with 5 OmMT ris—HC1, H8.0 until the eluate A 260 returned to the base line, and then the NaC1 concentration gradient was continuously increased to 3 M to increase the protein content. Eluted. A protein of about 19 kDa corresponding to the recombinant human FGF-10 was eluted at a position of about 1.2M NaC1. The flow rate was set at 2 m1Z.
  • the above eluted fraction was diluted two-fold with 50 mM Tris-HCl, H8.0 and applied to HiTrapAP SP (5 ml, Pharmacia). After washing with 50 mM Tris-HC1, pH 8.0, the protein was eluted by continuously increasing the NaCl concentration gradient to 2M. A protein of about 19 kDa corresponding to recombinant human FGF-10 was eluted at about 1.2 M NaCl. The flow rate was set at 2 m1Z. Next, the above eluted fraction is replaced with PBS (-) by dialysis, and 1Z10 volume of Pi-mouth Sepp 1 C (Daicel Industries) is added to remove endotoxin.
  • Three groups of 4-week-old male Wistar rats (weight: 94 to 120 g) were prepared as 3 to 4 individuals per group.
  • 27 syringe needle micro syringe under ether anesthesia Using the FGF-10 aqueous solution prepared in Preparation Example 1 above, a liquid volume corresponding to 10.6 and 21.2 g of the medullary cavity was administered into the medullary cavity of the tibia using, respectively.
  • One group received saline as a control group.
  • FIG. 7 shows the observation results of the FGF-10 administration group
  • FIG. 8 shows the observation results of the control group.
  • Table 1 shows the results of evaluation of bone formation in the medullary cavity on day 4 of the injection of human FGF-10 from soft X-ray images.
  • human FGF-10 showed a clear promoting action of formation and repair of bone Z cartilage tissue, which is important in the treatment of bone Z cartilage disease.
  • FGF-10 variant Construction of a plasmid that can be expressed in FGF-10 variant mammalian cells
  • the construction was performed in the same manner as in the construction of pCDM8-F10HX described in Examples. That is, the plasmid phFGF- ⁇ 0 having a natural human FGF-10 sequence was used as a type I, and the following primer hF10HX was used instead of the supplier F10HS shown in FIG. A PCR reaction was performed using the following primer hF10XR instead of F10XR, and the reaction was digested with Hind IE and XbaI. An approximately 700 bp fragment was isolated by electrophoresis.
  • This fragment was ligated with Hindfl and XbaI digests of pCDM8, and transformed into Escherichia coli MC1061 / p3 to transform the desired human FGF-10 into mammalian cells.
  • An expressible plasmid p CDM8—hF10HX was obtained.
  • Plasmid p CDM8-hF10HX having the natural human FGF-10 gene sequence was designated as type III, and a DNA fragment 1 having the replacement sequence was obtained by PCR using primers 1 and 13 did.
  • DNA fragment 2 having the same sequence as the replacement sequence and partially overlapping with DNA fragment 1 was obtained. did.
  • the DNA fragment 1 and the DNA fragment 2 thus obtained were converted into type III, and a PCR reaction was performed using primers 13 and 14 to obtain a DNA fragment 3.
  • DNA fragment 3 was digested with two types of restriction enzymes Hi 1 (1111 and aI), and pCDM8—hF 10 HX was digested with the same restriction enzymes and ligated with dephosphorylated, and E. coli MC 1
  • a plasmid PCDM8-F10 S106 capable of expressing a protein having the desired amino acid substitution was obtained.
  • Primers 5 were used instead of Primer 1 and Primer 6 was used instead of Primer 2, but the desired mutation was found by a two-step PCR reaction similar to the construction of pCDM8-F10 (S106). After obtaining the introduced DNA fragment, it was cleaved with two restriction enzymes Hindffl and XbaI, and pCDM8-hF10HX was digested with the same restriction enzyme and dephosphorylated, and A plasmid capable of expressing a protein having the desired amino acid substitution by transforming E. coli MC1061 / p3 p CDM8 -F10 (SI50) was obtained.
  • SI50 A plasmid capable of expressing a protein having the desired amino acid substitution by transforming E. coli MC1061 / p3 p CDM8 -F10
  • Primer 7 was used in place of Primer 1 and Primer 8 was used in place of Primer 2 except that primer CD8 was used in the same two-step PCR reaction as in the construction of pCDM8-F10 (S106).
  • the DNA fragment was cut with two kinds of restriction enzymes Hindm and XbaI, and pCDM8-hF10HX was digested with the same restriction enzyme and dephosphorylated.
  • a plasmid p CDM8 -F10 (S150) capable of expressing a protein having the amino acid substitution of interest by transforming E. coli MC1061 / 'p3 strain I got it.
  • Approximately 270 bp DNA fragment containing the A51 mutation was obtained by digesting pCDM8-F10 (A51) with Hindm and ScaI. Also, pCDM8-F10 (A196) was digested with Seal and XbaI to obtain a DNA fragment of about 390 bp containing the A196 mutation. These two DNA fragments were ligated with pCDM8-hF101 digested with restriction enzymes 11i11 (3111 and & I) and dephosphorylated, and E. coli MC1061 / p3 By transforming the strain, a plasmid PCDM8-F10 (A51ZA196) capable of expressing a protein having the desired amino acid substitution was obtained.
  • Primer 13 having a sequence complementary to the outer sequence sandwiching the region to be deleted and primer 14 complementary to primer 13 were prepared. Plasmid pCDM8-hF10HX was used as type II, and DNA fragment 4 was obtained by PCR reaction using primer 13 and primer 3. Separately, a DNA fragment 5 was obtained from the same type by a PCR reaction using primers 14 and 14. Next, The DNA fragments 4 and 5 obtained as described above were used as type I, and a PCR reaction was performed using primers 13 and 14 to obtain the DNA fragment into which the desired deletion was introduced. Acquired 6.
  • DNA fragment 6 was digested with two kinds of restriction enzymes Hindill and XbaI, and pCDM8-hF10HX was digested with the same restriction enzymes and ligated with dephosphorylated E. coli MC1.
  • DN1 plasmid PCDM8-F10
  • Primer 15 was used in place of primer 13 and primer 16 was used in place of primer 14
  • the DNA fragment was digested with two restriction enzymes Hindm and Xba1, digested with pCDM8-hF10HX and dephosphorylated by the same restriction enzyme.
  • DN2 plasmid p CDM8-F10
  • the DNA fragment is cleaved with two types of restriction enzymes, Hindm and XbaI, and pCDM8-hF10HX is digested with the same restriction enzymes.
  • a plasmid capable of expressing the protein having the desired amino acid substitution is 01 ⁇ 8-? 1 0 (DN 3) was obtained.
  • the DNA fragment is digested with the two restriction enzymes Hindin and XbaI, digested with pCDM8-hF10HX, and dephosphorylated.
  • DN4 plasmid pCDM8-F10 (DN4) capable of expressing a protein having the desired amino acid substitution was obtained.
  • pCDM8-F10 The same two-step PCR reaction as in the construction of pCDM8-F10 (DN1) was performed except that primer 21 was used instead of primer 13 and primer 22 was used instead of primer 14
  • primer 21 was used instead of primer 13
  • primer 22 was used instead of primer 14
  • the DNA fragment was digested with two restriction enzymes, Hind DI and XbaI, and pCDM8-hF10HX was digested with the same restriction enzyme and dephosphorylated.
  • the plasmid pCDM8-F10 (DN5) capable of expressing a protein having the amino acid substitution of interest was obtained by transforming E. coli MC1061 / p3 strain.
  • Primer 123 having a nucleotide sequence in which the codon encoding Asn at position 196 was replaced with a stop codon and having a recognition sequence for XbaI was prepared. Plasmid p CDM8 -hF10HX was used as type I, and a DNA fragment 7 was obtained by a PCR reaction using primers 13 and 14. Next, the DNA fragment 7 obtained in this manner was digested with two types of restriction enzymes Hindm and XbaI, and pCDM8-hF10HX was digested with the same restriction enzymes and dephosphorylated.
  • the target deletion was introduced by a single-step PCR reaction similar to the construction of pCDM8-F10 (DC1) except that primer 25 was used instead of primer 23 D
  • primer 25 was used instead of primer 23 D
  • a plasmid pCDM8-F10 (DC3) capable of expressing a protein having the desired amino acid substitution was obtained.
  • a DNA fragment containing the target deletion was obtained by a single-step PCR reaction similar to the construction of pCDM8-F10 (DC1) except that primer 126 was used instead of primer 23. after this was digested with two restriction enzymes H in dm and Xb a I, P CDM8-hF 1 0 the HX was digested with the same restriction enzymes were those with Raige one to Bok dephosphorylated, E. coli MC 1 0 6 By transforming the IZP3 strain, a plasmid PCDM8-F10 (DC4) capable of expressing a protein having the desired amino acid substitution was obtained.
  • DC4 plasmid PCDM8-F10
  • SAIF oligonucleotides containing mutations
  • the SA1R fragment and the SA2F / SA2R fragment were obtained. These two fragments were digested with Hind HI and BstX1, pCDM8—hF10HX was digested with the same restriction enzymes and ligated with dephosphorylated, and E. coli MC106. Plasmid P capable of expressing a protein having the desired amino acid substitution by transforming 1p3 strain
  • Primer 7 AGAGGCCACCGCCTCTTCTTCCTCCTCC Primer 8 AGGAAGAAGAGGCGGTGGCCTCTGGTGA
  • Primer 1 AAAGCTATTCTGTTTCACCAAGTACTn
  • Primer 1 AAGAAGAGTTGGCnGGCAGGTGACAGGGA
  • Primer-2 2 AGAGTTTCCCGGCTTGGCAGGTGACAGGGA
  • Primer-2 3 TTTTCTAGACTATTTCCnCGTGTTTT
  • Primer 2 4 TTTTCTAGACTATCTCCTTGGAGCTCC
  • Primer-2 6 TTTTCTAGACTACTTCTTGTTCATGGC
  • Transformation of COS-1 cells with a plasmid capable of expressing the FGF10 variant Prepare a large amount of plasmid DNA capable of expressing the above 17 types of FGF10 variants in a conventional manner, and use a cesium chloride density gradient. Purification was performed by performing centrifugation twice. These 1 7 kinds COS-1 cells were transformed by an electric pulse method using pCDM8-hF10HX and pCDM8 as a plasmid and a control. After the transformed cells are cultured in DMEM or IMEM medium containing 10% fetal calf serum for 24 hours, the medium is replaced with serum-free IMEM medium containing 10 gZm1 of heparin. Further culturing was continued.
  • the FGF10 variant (DN1, DN2, DC2, SA, A51, A196, S150, S106, C84 / S106) , S150 / S106 and A51 / A196) were compared with the culture supernatant obtained using pCDM8. As a result, it clearly showed the action of promoting the uptake of tritium-labeled thymidine.
  • the therapeutic agent for bone / cartilage disease of the present invention can be used, for example, to repair (1) repair of cartilage defects caused by arthritis associated with autoimmune diseases such as osteoarthritis and rheumatoid arthritis, (2) bone loss due to trauma, Repair of cartilage defects caused by osteochondritis dissecans, promotion of cartilage formation during bone preserving osteotomy, (5) repair after fracture, (6) repair after bone loss, (7) osteoporosis, etc. It is useful for promoting osteogenesis at sites where local bone loss has been observed, and is used for treatment of various bone / cartilage tissue diseases.
  • the present invention provides a DNA encoding FGF-10, an expression vector carrying the DNA, a transformant, a method for producing a recombinant protein using them, and a recombinant protein.
  • the present invention provides a medicine using various factors. Sequence listing
  • Trp Lys Trp lie Leu Thr His Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15
  • Glu Arg lie Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn Trp
  • Organism name human
  • Trp Lys Trp lie Leu Thr His Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Organism name human
  • AGG AAA AAC ACC TCT GCT CAC TTT CTT CCA ATG GTG GTA CAC TCA TAGAG 684
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence length 20 bp Sequence type: nucleic acid
  • Sequence length 20 bp Sequence type: nucleic acid
  • Sequence length 22bp Sequence type: nucleic acid
  • Sequence length 22 bp Sequence type: nucleic acid Number of chains: single strand
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

A recombinant FGF-10 obtained by introducing an expression vector into which a DNA encoding a specific amino acid sequence has been integrated into a host cell, incubating the transformant thus obtained, and allowing it to produce the aimed protein. This recombinant FGF-10 is applicable to drugs and reagents utilizing the cell growth-promoting effect thereof.

Description

明 線維芽細胞増殖因子 F G F- 1 0 技術分野  Ming Fibroblast growth factor F G F- 10
本発明は、 新規な線維芽細胞増殖因子 （以下、 FGFと略称する） およびその 組換え製法に関する。 更には、 当該因子の医薬用途に関する。  The present invention relates to a novel fibroblast growth factor (hereinafter, abbreviated as FGF) and a method for recombinantly producing the same. Further, the present invention relates to a pharmaceutical use of the factor.
背景技術  Background art
FGFは、 1 97 0年代に血管新生因子として発見された。 当初、 酸性線維芽 細胞増殖因子 （a FGF) や塩基性線維芽細胞増殖因子 （bFGF) が研究され、 糸  FGF was discovered as an angiogenic factor in the 1970s. Initially, acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) were studied,
その構造や広範な細胞増殖促進作用が明田らかにされてきた 〔 D . Akita et al. Have been elucidated for its structure and its widespread cell growth promoting action [
Go s p o d a r ow i c s他； Na t u r e 24 9卷 1 2 3頁 （1 97 4) 、 Go spoda ro ow i c s and others; Natu r e 24 9 Vol. 1 23 (1974),
Bu r g e s s, W. H. およひ Ma c i a g, T. ； A n n u. Re v.Burgess, W. H. and Maciag, T .; Annuu. Rev.
B i o c h em. 58巻 57 5— 606頁 （1 989) 、 SUZUK I, F. ； C l i n i c a l C a l c i um. 4巻 1 5 1 6— 1 5 1 7頁 （1 994) 〕 。 現在では、 計 9種類の FGF類が報告されており、 それぞれのクロ一ニング、 構 造解析も成されている 〔細胞、 27巻 9号 34 1〜3 44頁 （1 99 5 ) 〕 力 \ それ以外の F G F類の存在については可能性のみ示唆されていた。 Biochem. 58, 575-606 (1 989); SUZUK I, F .; ClinicalCalcium. 4, 1516- 1517 (1994)]. At present, a total of nine types of FGFs have been reported, and their respective cloning and structural analyzes have also been performed. [Cells, Vol. 27, No. 9, pp. 34-344 (19995)] Only the possibility of other FGFs was suggested.
一方、 bFGFや a FGFはその広範な細胞増殖促進作用を利用して、 神経系、 血管系や骨代謝系の疾患において、 有望な治療剤となる可能性が検討されている 力 \ 現在もまだ、 臨床治験に於ける有用性が証明されるには至っていない。 新規 な F G Fでの同様の研究が待望されている。  On the other hand, bFGF and a FGF are being studied as potential therapeutic agents for diseases of the nervous system, vascular system, and bone metabolic system, utilizing their widespread cell growth promoting effect. However, its usefulness in clinical trials has not been proven. Similar research in the new FGF is anticipated.
本発明の目的は、 新規な FGFの遺伝子を発見し解析することによって、 その 組換え蛋白の工業的生産方法を提供することである。  An object of the present invention is to provide a method for industrially producing a recombinant protein by discovering and analyzing a novel FGF gene.
発明の開示  Disclosure of the invention
発明者は、 未知の FGFの DNAについて鋭意検討を行った結果、 全く新しい タイプの FGF (以下、 FGF— 1 0と略称する） の DNAを取得することに成 功し、 本発明を完成するに至った。  As a result of intensive studies on the DNA of an unknown FGF, the inventors succeeded in obtaining a completely new type of FGF (hereinafter abbreviated as FGF-10) DNA, and completed the present invention. Reached.
本発明は、 以下の （ 1) 一 （1 5) に示すように、 FGF— 1 0をコ一ドする DNA、 当該 DNAを担持した発現ベクター、 形質転換体、 それらを用いた組換 え蛋白の製造方法、 および組換え蛋白、 更には、 FGF— 1 0組換え蛋白の医薬 用途に関するものである。 The present invention encodes FGF-10 as shown in the following (1)-(15). The present invention relates to DNA, an expression vector carrying the DNA, a transformant, a method for producing a recombinant protein using the DNA, a recombinant protein, and a pharmaceutical use of the FGF-10 recombinant protein.
( I ) 配列番号： 1もしくは配列番号： 2に示すァミノ酸配列のポリぺプチドで ある線維芽細胞増殖因子をコードする塩基配列、 またはこれに相補的な塩基配列 を包含する組み換え DNA。  (I) A recombinant DNA comprising a nucleotide sequence encoding a fibroblast growth factor which is a polypeptide of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence complementary thereto.
( 2 ) 配列番号： 3もしくは配列番号： 4に示す塩基配列またはこれに相補的な 塩基配列を包含する （1) 記載の DNA。  (2) The DNA according to (1), which comprises the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence complementary thereto.
(3) (1 ) の DNAを担持する発現ベクター。  (3) An expression vector carrying the DNA of (1).
(4) (3) の発現ベクターを宿主に導入して得られる形質転換体。  (4) A transformant obtained by introducing the expression vector of (3) into a host.
(5) 宿主が動物細胞または大腸菌である （4) の形質転換体。  (5) The transformant according to (4), wherein the host is an animal cell or Escherichia coli.
(6) (4) の形質転換体を使用することを特徴とする組換え線維芽細胞増殖因 子の製造方法。  (6) A method for producing a recombinant fibroblast growth factor, which comprises using the transformant of (4).
(7) 配列番号： 1 もしくは配列番号： 2に示されるァミノ酸配列またはその主 要部分を包含するポリぺプチドである組換え線維芽細胞増殖因子。  (7) A recombinant fibroblast growth factor which is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a main part thereof.
(8) (5) の形質転換体が生産し、 細胞増殖活性を示す事を特徴とする、 配列 番号： 1もしくは配列番号： 2に示すァミノ酸配列またはその主要部分を包含す るポリべプチドである組換え線維芽細胞増殖因子。  (8) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a main part thereof, which is produced by the transformant of (5) and exhibits cell growth activity. A recombinant fibroblast growth factor.
(9) (7) もしくは （8) の組換え線維芽細胞増殖因子を有効成分として含有 する医薬。  (9) A medicament comprising the recombinant fibroblast growth factor according to (7) or (8) as an active ingredient.
(1 0) 骨/軟骨疾患または骨 Z軟骨損傷治療剤である （9) の医薬。  (10) The medicament according to (9), which is a therapeutic agent for bone / cartilage disease or bone Z cartilage damage.
( I I) 創傷治癒促進剤である （9) の医薬。  (II) The medicament according to (9), which is a wound healing promoter.
(1 2) 骨/軟骨疾患または骨 /軟骨損傷治療剤の製造のための （7) もしくは (8) の組換え線維芽細胞増殖因子の使用。  (1 2) Use of the recombinant fibroblast growth factor of (7) or (8) for the manufacture of a therapeutic agent for bone / cartilage disease or bone / cartilage damage.
( 1 3) 創傷治癒促進剤の製造のための （ 7 ) もしくは （ 8 ) の組換え線維芽細 胞増殖因子の使用。  (13) Use of the recombinant fibroblast growth factor of (7) or (8) for the manufacture of a wound healing promoter.
(1 4) ( 7 ) もしくは （ 8 ) の組み換え線維芽細胞増殖因子の有効量をヒトを 含む動物に投与することを含む骨 Z軟骨疾患または骨 Z軟骨損傷の治療方法。 (14) A method for treating bone Z cartilage disease or bone Z cartilage damage, which comprises administering an effective amount of the recombinant fibroblast growth factor of (7) or (8) to an animal including a human.
( 1 5) ( 7 ) もしくは （ 8 ) の^;且換え線維芽細胞増殖因子の有効量をヒ トを含 む動物に投与ことを含む創傷治癒促進方法。 (15) The effective amount of the recombinant fibroblast growth factor of (7) or (8) A method for promoting wound healing comprising administering to animals.
図面の簡単な説明  BRIEF DESCRIPTION OF THE FIGURES
図 1は、 FGF— 1 0遺伝子のクローニングに用いた 2種類の FGF— 3、 FGF— 7、 FGF— 1 0共通プライマ一、 （A) Ty r— L e u— A l a 一 Me t— A s n— L y s、 （B) T y r— A s n—T h r— T y r— A 1 a - S e rを示す。  Figure 1 shows the two types of FGF-3, FGF-7, and FGF-10 common primers used for cloning the FGF-10 gene, and (A) Ty r—L eu—A la—Met—A sn — Lys, (B) Tyr—A sn—Thr—Tyr—A1a-Ser.
図 2は、 R a p i d Amp l i f i c a t i o n o f c DNA End s (RACE) 法に用いた FGF— 1 0 c D N A単離用プライマ一を示す。 図 3は、 プラスミ ド pFGF— 1 0力ヽら、 プラスミ ド pCDM8— F 1 0 SP および pCDM8— F 1 0 HXを得るまでのプラスミ ド構築方法の概略を示す。 図 4は、 翻訳開始コドン上流をコザックのコンセンサス配列に置換するために 用いたプライマ一および P CR反応条件を示す。  FIG. 2 shows a primer for isolating FGF-10c DNA used in the Rapid Amplifo ncat o Endo Fc DNA Ends (RACE) method. FIG. 3 shows an outline of a plasmid construction method for obtaining plasmids pFGF-10F, plasmids pCDM8-F10SP and pCDM8-F10HX. FIG. 4 shows the primer and PCR reaction conditions used to replace the translation initiation codon upstream with the Kozak consensus sequence.
図 5は、 i n s i t u ハイブリダィゼーシヨン法によるラッ ト関節組織に おける FGF— 1 OmRNAの発現を示す写真であり、 （A) は、 関節軟骨標本 の顕微鏡写真、 （B) は、 骨端軟骨板の顕微鏡写真である。  Fig. 5 is a photograph showing the expression of FGF-1 OmRNA in rat joint tissues by the in situ hybridization method. (A) is a micrograph of an articular cartilage specimen, and (B) is an epiphyseal cartilage. It is a microscope picture of a board.
図 6は、 卜リチウム標識チミジンの FRSK培養細胞への取込みを示すグラフ である。 横軸 B qはコントロール、 S p、 Hxはそれぞれ FGF— 1 0発現 CO S細胞の培養液を加えた場合を示す。 縦軸は細胞内の放射活性を示す。  FIG. 6 is a graph showing the uptake of tritiated thymidine into FRSK cultured cells. The abscissa Bq indicates the control, and Sp and Hx indicate the cases where a culture solution of FGF-10 expressing COS cells was added. The vertical axis shows the radioactivity in the cell.
図 7は、 試験例におけるヒト FGF— 1 0投与群の脛骨軟 X線写真の画像処理 像である。  FIG. 7 is an image-processed image of a tibial soft radiograph of the human FGF-10 administration group in the test example.
図 8は、 試験例における対照群の脛骨軟 X線写真の画像処理像である。  FIG. 8 is an image-processed image of a tibial soft radiograph of the control group in the test example.
図 9は、 FGF— 1 0改変体発現プラスミ ドの構築概念図であり、 変異 DNA 断片の作成を示す。  FIG. 9 is a conceptual diagram of the construction of an FGF-10 variant expression plasmid, which shows the production of a mutant DNA fragment.
図 1 0は、 COS細胞発現ヒト FGF— 1 0、 およびその改変体の生理活性を 示し、 トリチウム標識チミジンの FRSK培養細胞への取り込みを示すグラフで ある。 横軸の記号 WTは天然型ヒト FGF— 1 0、 DN 1〜5、 DC 1〜4、 C 84ZS 1 06、 S 1 50ZS 1 06、 A 5 1 /A 1 96. SA、 A 5 1 , A 1 9 6、 S 1 50及び S 1 06 (A v rは培養上清原液、 A v r ( 1 Z 1 0 ) は培 養上清の 1 Z1 0希釈液） はそれぞれの FGF - 1 0改変体を意味し、 縦軸は F R S K細胞中の放射活性を表す。 FIG. 10 is a graph showing the biological activity of human FGF-10 expressing COS cells and its variants, and showing the uptake of tritium-labeled thymidine into FRSK cultured cells. The symbol on the horizontal axis is WT, natural human FGF-10, DN 1-5, DC 1-4, C84ZS106, S150ZS106, A51 / A196.SA, A51, A 196, S150 and S106 (Avr is the undiluted culture supernatant, Avr (1Z10) is the 1Z10 dilution of the culture supernatant) were the respective FGF-10 variants. Means, the vertical axis is F Shows radioactivity in RSK cells.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
本明細書において、 用語の意味または定義は下記の通りである。  In this specification, the meanings or definitions of the terms are as follows.
「FGF— 1 0」 は、 配列番号： 1 もしくは配列番号： 2に示されるアミノ酸 配列またはその主要部分を包含する哺乳類の産生する線維芽細胞増殖因子を意味 する。 主要部分とは、 前記配列から、 シグナル （プレ） 配列またはプロ配列が除 かれたマチュア蛋白アミノ酸配列を意味する。 すなわち、 配列番号： 1における 3 7位グルタミ ン （G i n) から 2 1 5位セリ ン （S e r) に到る 1 7 9ァミノ 酸残基からなるァミノ酸配列、 または配列番号： 2における 3 8位グルタミン (G i n) から 2 0 8位セリ ン （S e r) に到る 1 7 1アミノ酸残基からなるァ ミノ酸配列である。  “FGF-10” means a fibroblast growth factor produced by a mammal, including the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a major portion thereof. The main part means a mature protein amino acid sequence obtained by removing a signal (pre) sequence or a pro sequence from the above sequence. That is, an amino acid sequence consisting of 179 amino acid residues ranging from glutamine at position 37 (Gin) to serine at position 21 (Ser) in SEQ ID NO: 1, or 3 in SEQ ID NO: 2. This is an amino acid sequence consisting of 1711 amino acid residues from glutamine (Gin) at position 8 to serine (Ser) at position 20-8.
なお、 現在公知の技術により、 配列番号： 1 もしくは配列番号： 2に示される ァミノ酸配列またはその主要部分の一部のァミノ酸残基を欠失、 置換あるいは付 加させた蛋白質を製造することが可能であり、 ランダムスクリーニングあるいは 他の FGFファミリー蛋白に於ける配列改変研究の知見から、 本件発明の FGF - 1 0と生理活性の点で同質の改変蛋白質を創製することが可能である。 これら の改変蛋白質も線維芽細胞増殖因子活性を示すかぎり、 本件発明の技術範囲に属 する物である。 また、 FGF— 1 0は、 配列番号： 1に示されるアミノ酸配列の 5 0 - 5 2位に存在する A s n— S e r— S e r、 2 0 3— 2 0 5位の A s n— Th r— S e r、 配列番号： 2に示されるァミノ酸配列の 5 1 一 5 3位に存在す る A s n— S e r— S e r、 1 9 6— 1 9 8位の A s n— Th r— S e r、 二か 所に N結合型糖鎖結合部位を有する蛋白質であるが、 一般的には、 糖鎖の有無に 関わり無く生理活性を有する。 公知の宿主の選択により糖鎖の種類を変更するこ と、 あるいは除去することができるが、 これらの糖鎖改変蛋白質も下記の線維芽 細胞増殖因子活性を示すかぎり、 本件発明の技術範囲に属する物である。  It is to be noted that a protein in which the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a part of the amino acid residue of the main part thereof has been deleted, substituted or added by a currently known technique. It is possible to create a modified protein having the same biological activity as the FGF-10 of the present invention from the knowledge of random screening or sequence modification studies on other FGF family proteins. These modified proteins also belong to the technical scope of the present invention as long as they show fibroblast growth factor activity. FGF-10 is an A sn—Ser—S er located at position 50-52 of the amino acid sequence shown in SEQ ID NO: 1, and an A sn—Th r at position 203—205. — Ser, A sn—Ser—Ser located at position 51-53 of the amino acid sequence shown in SEQ ID NO: 2; A sn—Thr—S at position 1996—198 er, a protein having an N-linked sugar chain binding site in two places, but generally has a physiological activity regardless of the presence or absence of the sugar chain. The type of sugar chain can be changed or removed by selecting a known host, but these sugar chain-modified proteins also belong to the technical scope of the present invention as long as they exhibit the following fibroblast growth factor activity. Things.
なお、 下記 （ 1 ) から （5 ) のタイプの FGF— 1 0改変体も、 天然型 FG F 一 1 0と同様の生理作用を示し、 本件発明の技術範囲に属するものである。 ( 1 ) 天然の F G F— 1 0が有する C y s残基が他のァミノ酸 （ S e r o r A l a) に置換された、 分子内および分子間ジスルフィ ド結合を生じない FG F 一 1 0改変体、 In addition, the FGF-10 variants of the following types (1) to (5) also exhibit the same physiological actions as the natural type FGF-10, and belong to the technical scope of the present invention. (1) FG F in which intramolecular and intermolecular disulfide bonds are not formed, in which the Cys residue of natural FGF-10 is substituted by another amino acid (Seror Ala) 110 variants,
(2) FG F— 1 0の成熟蛋白質領域内の 2 ケ所の C y s残基の内の iつ (Cy s - 1 0 6) は他の FGF類で保存されている Cy sの位置と異なるため、 他の FGF類で保存されている位置のアミノ酸 （S e r— 8 4 i n FGF— 1 0) が Cy sに置換され、 保存されていない位置の C y s— 1 0 6が他のアミ ノ酸 （S e r または A 1 a) に置換された、 FGF類で保存された位置 に C y s残基を有する FGF— 1 0改変体、  (2) i (Cy s-106) of the two Cys residues in the mature protein region of FG F-10 differs from the Cys position conserved in other FGFs Therefore, amino acids at positions conserved in other FGFs (Ser-84 in FGF-10) are substituted with Cys, and Cys-106 at positions not conserved is substituted with other amino acids. An FGF-10 variant having a Cys residue at a position conserved by FGFs, substituted by an acid (Ser or A1a),
(3) 糖鎖付加配列 （As n— X— Y : Xは Cy s, P r o以外の任意のァミノ 酸、 Yは S e rまたは Th r) を、 他のァミノ酸配列に置換することにより、 糖 鎖付加を生じない F G F— 1 0改変体、 (3) By substituting a sugar chain addition sequence (As n—X—Y : X is any amino acid other than Cys and Pro, and Y is Ser or Thr) with another amino acid sequence FGF-10 variant that does not cause glycosylation,
(4) N末および/あるいは C末の数〜 1 0 0アミノ酸が欠乏した FGF— 1 0 改変体、  (4) N-terminal and / or C-terminal number to 100 amino acids deficient FGF-10 variant,
(5) プロテア一ゼにより切断を受ける可能性のあるァミノ酸配列が他のァミノ 酸配列に置換された FGF— 1 0改変体。  (5) An FGF-10 variant in which an amino acid sequence which may be cleaved by protease is replaced with another amino acid sequence.
より具体的に下記 （ 1 ) から （ 1 9) の FGF— 1 0改変体を例示する。 これ らは、 実施例にて詳述するように、 今日の遺伝子工学公知の手法により、 作成す ることができる。  More specifically, the following FGF-10 variants of (1) to (19) are exemplified. As described in detail in Examples, these can be created by a known technique of genetic engineering today.
( 1 ) リ一ダ一領域内の 7ケ所の C y s残基内の 6力所 （C y s— 9， — 1 9， - 2 0, 一 2 1， — 2 2， 及び— 2 3) が全て A 1 aあるいは S e rに置換され た F G F— 1 0改変体、  (1) The six force points (C ys—9, —19, −20, —21, —22, and —23) in the seven Cys residues in the leader region are FGF-10 variant with all A1a or Ser substitutions,
(2) C y s— 1 0 6が A 1 aあるいは S e rに置換された FGF— 1 0改変体、 (2) an FGF-10 variant in which Cys-106 is substituted with A1a or Ser,
(3) C y s— 1 5 0が A 1 aあるいは S e rに置換された FGF— 1 0改変体、 (4 ) C y s - 1 0 6および C y s— 1 5 0が共に A 1 aあるいは S e rに置換 された FGF— 1 0改変体、 (3) FGF-10 variant in which Cys-150 is substituted with A1a or Ser, (4) Cys-106 and Cys-150 are both A1a or S FGF-10 variant substituted with er,
(5) S e r— 8 4が Cy sに置換され、 さらに C y s— 1 0 6が A 1 aあるい は S e rに置換された FGF_ 1 0改変体、  (5) a modified FGF_10 in which Ser-84 is substituted with Cys, and Cys-106 is further substituted with A1a or Ser;
(6) Cy s - 9, 一 1 9， 一 2 0， 一 2 1， 一 2 2， 一 2 3， 一 3 7, — 1 0 (6) Cy s-9, 1 19, 1 20, 1 2 1, 1 2 2, 1 2 3, 1 3 7, — 1 0
6および— 1 5 0が全て A 1 aあるいは S e rに置換された FGF— 1 0改変体、FGF-10 variant in which 6 and -150 are all substituted with A1a or Ser,
(7) A s n- 5 1が A 1 aに置換された FGF— 1 0改変体、 (8) A s n— 1 9 6が A 1 aに置換された FGF— 1 0改変体、 (7) an FGF-10 variant in which A s n-51 is substituted with A 1 a, (8) FGF-10 variant in which A sn—196 is substituted by A 1a,
(9) A s n— 5 1および A s n— 1 9 6が共に A 1 aに置換された FGF— 1 0改変体、  (9) an FGF-10 variant in which A s n—51 and A s n—196 are both substituted with A 1a,
( 1 0) 成熟蛋白質の N末端のァミノ酸 （L e u— 4 0) から Th r— 5 0まで のアミノ酸配列が欠失した FGF— 1 0改変体、  (10) an FGF-10 variant in which the amino acid sequence from the amino acid at the N-terminus (Leu-40) to Thr-50 of the mature protein has been deleted,
( 1 1) 成熟蛋白質の N末端のアミノ酸 （L e u— 4 0) から S e r— 6 2まで のァミノ酸配列が欠失した F G F— 1 0改変体、  (11) an FGF-10 variant in which the amino acid sequence from the N-terminal amino acid (Leu-40) to Ser-62 of the mature protein has been deleted,
(1 ) 成熟蛋白質の N末端のァミノ酸 （L e u— 4 0) から Ly s— 8 1まで のァミノ酸配列が欠失した F G F— 1 0改変体、  (1) an FGF-10 variant in which the amino acid sequence from the N-terminal amino acid (Leu-40) to Lys81 of the mature protein is deleted,
(1 3) 成熟蛋白質の N末端のアミノ酸 （L e u— 4 0) から L y s— 1 0 3ま でのアミノ酸配列が欠失した F G F— 1 0改変体、  (13) an FGF-10 variant in which the amino acid sequence from the N-terminal amino acid (Leu-40) to Lys103 of the mature protein has been deleted,
( 1 4) 成熟蛋白質の N末端のァミノ酸 （L e u— 4 0) から Ly s— 1 3 7ま でのアミノ酸配列が欠失した F G F— 1 0改変体、  (14) a modified FGF-10 mutant in which the amino acid sequence from the N-terminal amino acid (Leu-40) to Lys137 of the mature protein is deleted,
( 1 5) A s n— 1 9 6から S e r— 2 0 8までのァミノ酸配列が欠失した F G F— 1 0改変体、  (15) an FGF-10 mutant in which the amino acid sequence from Asn-196 to Ser-208 is deleted,
( 1 6) G 1 y - 1 8 9から S e r— 2 0 8までのァミ ノ酸配列が欠失した F G F- 1 0改変体、  (16) an FGF-10 variant in which the amino acid sequence from G1y-189 to Ser-208 is deleted,
( 1 7) G 1 u— 1 5 4から S e r - 2 0 8までのァミノ酸配列が欠失した FG F - 1 0改変体、  (17) an FG F-10 variant in which the amino acid sequence from G 1 u—154 to Ser-208 has been deleted,
( 1 8) G 1 y— 1 3 8から S e r— 2 0 8までのァミノ酸配列が欠失した FG F- 1 0改変体、  (18) an FG F-10 variant in which the amino acid sequence from G 1 y—138 to Ser—208 is deleted,
( 1 9) ( 1 0) ~ ( 1 4 ) のいずれかのアミノ酸配列の欠失と （ 1 5) 〜 （ 1 8) のいずれかのアミノ酸配列の欠失が組み合わされた FGF - 1 0改変体。  (19) An FGF-10 modification in which a deletion of any of the amino acid sequences of (10) to (14) is combined with a deletion of the amino acid sequence of any of (15) to (18) body.
「線維芽細胞増殖因子活性」 とは、 細胞増殖刺激作用、 造血幹細胞増殖作用、 血管新生作用など種々の細胞に対する細胞増殖促進作用、 細胞分化誘導作用や細 胞外マトリックス改変作用などの分化調節作用、 神経細胞の生存維持作用等、 多 岐に渡る FGF類の生理活性のうちの少なくとも一種 〔臨床検査 3 8巻、 1 1 号、 2 1 9— 2 2 1頁 （ 1 9 9 4年 増刊号） 〕 を意味する。 FGF - 7におい て見られるような、 ラッ ト胎児由来表皮細胞 （FRSK細胞） を始めとする上皮 細胞由来細胞株の細胞増殖刺激作用も、 当該活性に含まれるものとする。 "Fibroblast growth factor activity" refers to cell growth stimulating action, hematopoietic stem cell growth action, angiogenesis action, cell growth promotion action for various cells, cell differentiation induction action, extracellular matrix modification action, etc. , At least one of a wide variety of physiological activities of FGFs, such as the effect of maintaining the survival of nerve cells [Clinical Inspection, Vol. 38, No. 11, pp. 219-221 (1994 extra number) )]. Epithelium, including rat fetal epidermal cells (FRSK cells), as seen in FGF-7 The cell growth stimulating action of the cell-derived cell line is also included in the activity.
「骨/軟骨損傷治療剤または骨 Z軟骨疾患治療剤」 とは、 事故による骨折や手 術に伴う骨/軟骨の切除など、 骨ノ軟骨の物理的な損傷の治癒を促進するための 医薬製剤または、 骨 Z軟骨形成の低下を主たる症状とする疾患を治療するための 医薬製剤であり、 下記 （1 ) から （6) の医薬用途を指向するものである。  `` Bone / cartilage damage treatment or bone Z cartilage disease treatment '' is a pharmaceutical preparation that promotes the healing of physical damage to bone cartilage, such as accidental fractures and bone / cartilage resection due to surgery. Alternatively, it is a pharmaceutical preparation for treating a disease whose main symptom is a decrease in bone Z cartilage formation, and is directed to the following pharmaceutical uses (1) to (6).
( 1 ) 骨欠損治療剤、 （ 2 ) 骨折治療剤、 （ 3 ) 骨粗鬆症治療剤、 （ 4 ) 軟骨組 織修復促進剤、 （5) 関節軟骨組織修復治療剤、 および、 （6) 変形関節症治療 剤。  (1) Bone defect treatment, (2) Bone fracture treatment, (3) Osteoporosis treatment, (4) Cartilage tissue repair promoter, (5) Articular cartilage tissue repair treatment, and (6) Osteoarthritis Therapeutic agent.
「創傷治癒促進剤」 とは、 事故による物理的 Z化学的要因による外傷や凍傷、 熱傷の治癒促進のための医薬製剤を意味する。 放射線障害、 床擦れや糖尿病によ つて生じる皮膚潰瘍など、 難治性の皮膚筋肉組織障害の治癒促進剤も含む。  “Wound healing promoter” means a pharmaceutical preparation that promotes the healing of trauma, frostbite and burns due to physical and chemical factors caused by accident. Also includes healing accelerators for intractable skin and muscle tissue disorders such as radiation damage, floor rubbing and skin ulcers caused by diabetes.
以下、 本発明をより詳細に説明する。  Hereinafter, the present invention will be described in more detail.
FGF- 1 0遺伝子の取得  Acquisition of FGF-10 gene
本発明の FGF— 1 0をコードする DNAは、 公知の遺伝子工学的方法にて製 造出来る。 すなわち、 哺乳類の生体組織あるいは培養細胞から、 mRNAを単離 し、 それから二本鎖 c DNAを得ることができる。 さらに、 この cDNAをプラ イマ一に用いて、 PCR法を行い、 増幅し、 適宜配列を決定できる。 これらはい ずれも専用キッ トが市販されている。 なお、 mRNAの原料の生体組織や培養細 胞は特に種類を限定されるものではないが、 特に、 約 1 4日令のラッ ト胎児を用 いる方法が好適である。 肺や関節組織における mRNA発現が比較的多いため、 肺細胞、 骨 Z軟骨由来細胞由来の培養細胞等も用いうる。 市販の成人ヒト肺由来 ポリ （A) +RNA 〔クローンテック社〕 などを使用する方法も簡便で好ましい。 また、 本特許明細書に開示の FGF— 1 0をコードする DNA配列の中から、 適当な配列を DN Aプローブとして用い、 種々の生体由来の c DNAまたはゲノ ム遺伝子ライブラリ一からクローニングすることができる。  The DNA encoding FGF-10 of the present invention can be produced by a known genetic engineering method. That is, mRNA can be isolated from mammalian living tissue or cultured cells, and then double-stranded cDNA can be obtained. Furthermore, using this cDNA as a primer, PCR can be performed, amplified, and the sequence can be determined as appropriate. Each of these kits has a dedicated kit on the market. The type of biological tissue or cultured cells as the raw material of mRNA is not particularly limited, but a method using a rat fetus about 14 days old is particularly preferable. Since mRNA expression is relatively high in lung and joint tissues, lung cells, cultured cells derived from bone-Z cartilage-derived cells, and the like can also be used. A method using commercially available adult human lung-derived poly (A) + RNA [Clontech] is also simple and preferable. Further, from the DNA sequence encoding FGF-10 disclosed in the present specification, it is possible to clone an appropriate sequence as a DNA probe and clone it from various living organism-derived cDNA or genomic gene libraries. it can.
遺伝子ライブラリーは常法に従って、 下記のように調製する。  The gene library is prepared as follows according to a conventional method.
1. 動物組織を凍結粉末化したものを RN a s e及びプロテア一ゼ処理し、 高分 子量 DN Aを沈澱させて得る。 DN A抽出物については、 市販のものが利用でき る 〔クローンテック （c 1 o n t e c h) 社等〕 ： 2. 制限酵素 （E c oR I等） で部分的に切断し、 エタノール沈澱で DNA断片 を得る ； 1. Frozen and powdered animal tissue is treated with RNase and protease to precipitate high molecular weight DNA. Commercially available DNA extracts can be used [Clontech, etc.]: 2. Obtain DNA fragments by partial digestion with restriction enzymes (EcoRI, etc.) and ethanol precipitation;
3. DNAリガーゼを用いて、 スファージに DNA断片を揷入し；  3. Introduce the DNA fragment into the sphage using DNA ligase;
4. 市販の i n v i t r oパッケージングキッ トを用いて、 ノ ッケージングを 行い、 遺伝子ライブラリーを作る。  4. Using a commercially available invitro packaging kit, perform knocking to create a gene library.
DN Aプローブは、 本特許明細書に開示の FGFファミ リー蛋白をコ一ドする DN A配列の中から、 特異性の高い配列を選ぶ。 常法により、 化学合成し、 32 P 等でラベルすることができる。 The DNA probe selects a highly specific sequence from the DNA sequences encoding the FGF family proteins disclosed in the present specification. It can be chemically synthesized by conventional methods and labeled with 32 P or the like.
FGF- 1 0蛋白質の製造  Production of FGF-10 protein
こうして得られた FGF— 1 0の c DN Aを組み込む発現ベクターとしては、 適当な大腸菌、 枯草菌、 酵母、 動物昆虫細胞等の宿主内で増殖できるプラスミ ド やファージが選ばれる力 <、 例えば、 大腸菌由来の pBR 3 2 2、 p BR 3 2 5 [Ge n e 4巻 1 2 1頁 （1 978) 〕 、 枯草菌由来 p UB 1 1 0  Expression vectors incorporating the cDNA of FGF-10 thus obtained include, for example, a plasmid or phage capable of growing in a suitable host such as Escherichia coli, Bacillus subtilis, yeast, and animal insect cells. PBR32, pBR32 from Escherichia coli [Gene 4, pp. 121 (1 978)], p UB1 10 from Bacillus subtilis
〔B i o c h em. B i o ph y s. Re s. C ommu n. ) 1 1 2巻， 67 8頁 （ 1 98 3 ) 〕 、 COS細胞に好適な p C DM 8等が挙げられる。 c DNA をプラスミ ドに組み込む方法としては、 常法が、 T. Ma n i a t i s他、 o l e c u l a r c l o n i n g、 Co l d s p r i n h a r b a r l a b, 2 3 9頁 （ 1 9 82 ) に記載されている。  Commun.) 112, p. 678 (19983)], pCDM8 suitable for COS cells, and the like. As a method for incorporating cDNA into plasmid, the usual method is described in T. Maniatis, et al., olecuulcarcloninng, Coldsprininharharbarb, 239, p. 239 (1992).
宿主は、 ベクタ一の導入により形質転換され、 FGF— 1 0を産生できる生物 や培養細胞であれば、 特に限定されない。 細菌としては、 大腸菌、 枯草菌 （バチ ルス類） 等、 酵母としては、 サッカロマイセス属、 トルラ属、 ピキア属等、 動物 細胞としては、 COS細胞、 CHO細胞、 N SO細胞等が代表例である。 培養昆 虫細胞、 真菌、 植物細胞、 単細胞系だけでなく、 目的蛋白質遺伝子を組み込まれ た昆虫や哺乳類、 植物も宿主の範疇に入る。  The host is not particularly limited as long as it is an organism or a cultured cell that can be transformed by introduction of the vector and can produce FGF-10. Typical examples of bacteria include Escherichia coli and Bacillus subtilis (Bacillus), yeasts include Saccharomyces, Torula and Pichia, and animal cells include COS cells, CHO cells, and NSO cells. In addition to cultured insect cells, fungi, plant cells, and unicellular cells, insects, mammals, and plants into which the target protein gene has been incorporated fall into the category of hosts.
原核細胞産生系としては、 大腸菌またはバチルス属細菌が一般的に用いられ、 特に、 プロテア一ゼ産生能を低下させた B a c i 1 1 u s b r e v i sは、 分 泌発現のための宿主として有用である （参照：特開平 6— 29 64 8 5、 特開平 6 - 1 3 3 7 8 2 , Y. S a g i y a e t a 1. ； Ap p l i e d Escherichia coli or bacteria of the genus Bacillus are generally used as a prokaryotic cell production system. In particular, Bacillus 11 usbrevis, which has a reduced protease production ability, is useful as a host for secretory expression (see : Japanese Patent Application Laid-Open No. 6-2966485, Japanese Patent Application Laid-Open No. 6-133878, Y. Sagiyaeta 1 .; Ap plied
i c r o b i o l. B i o t e c h n o l . 4 2巻， 3 5 8— 36 3頁 （1 9 94 ) など） 。 icrobio l. Biotechnol. 42, 35 8—36 3 (1 9 94)).
形質転換体から、 公知の方法、 例えば、 コロニー 'ハイブリダイゼィション法 〔ジーン G e n e, 1 0巻 6 3頁 （ 1 9 8 0 ) 〕 および D N A塩基配列決定 法 〔P r o c. Na t l. Ac a d. S c i. USA, 7 4巻 560頁 （1 97 7) 〕 を用い、 所望のクローンを選出する。 また、 COS細胞にて一過性に発現 させ、 培養上清の生理活性を評価してクローン選択することも可能である。  From the transformant, a known method, for example, a colony 'hybridization method [Gene Gene, 10:63 (1980)]] and a DNA sequencing method [Proc. Ac ad. Sci. USA, 74, 560 (1977)] to select a desired clone. Alternatively, clones can be transiently expressed in COS cells and cloned by evaluating the physiological activity of the culture supernatant.
発現された FGF— 1 0蛋白の生理活性は、 常法により容易に検出できる。 た とえば、 公知の FRSK細胞など、 上皮細胞の増殖促進作用を測定することによ り評価できる。  The physiological activity of the expressed FGF-10 protein can be easily detected by a conventional method. For example, it can be evaluated by measuring the growth promoting effect of epithelial cells such as known FRSK cells.
クローン化された DNA含有プラスミ ドは、 そのままあるいは制限酵素で切り 出して利用することが可能であり、 種々の宿主に適応した発現べクタ一に組み込 んで発現させ、 FGF— 1 0蛋白を大量に製造することができる。 発現方法は特 に制限されず、 細菌を用いた融合発現、 分泌発現および直接発現、 また真核細胞 を用いた発現など適宜、 当該分野で公知の組換え蛋白生産技術が応用しうる。 組換え技術により生産された FGF— 1 0蛋白は、 生化学の分野で常用される 精製方法にて精製が可能である。 イオン交換クロマトグラフィー、 ゲル濾過、 逆 相 HPLC、 硫安沈澱、 限外濾過、 SDS— PAGEなどが適宜組み合わせて用 いられる力く、 FGF類の場合、 特にへパリン等のリガンドを用いたァフィ二ティ 一クロマトグラフィー、 抗体カラムクロマ卜グラフィーなどが大量精製に好適で ある。 FGF— 1 0蛋白に対する抗体は、 ポリクロ一ナル、 モノクローナル共に、 自体公知の方法で作製し得る。 FGF— 1 0特異的抗体は抗体カラムに使用出来 るだけでなく、 EL I S A等の免疫化学的定量法に使用できる。  The cloned DNA-containing plasmid can be used as it is or cut out with restriction enzymes, and it is incorporated into an expression vector suitable for various hosts and expressed to produce a large amount of FGF-10 protein. Can be manufactured. The expression method is not particularly limited, and recombinant protein production techniques known in the art can be applied as appropriate, such as fusion expression, secretory expression, and direct expression using bacteria, and expression using eukaryotic cells. The FGF-10 protein produced by recombinant technology can be purified by a purification method commonly used in the field of biochemistry. Ion exchange chromatography, gel filtration, reverse phase HPLC, ammonium sulfate precipitation, ultrafiltration, SDS-PAGE, etc. are used in combination as appropriate. In the case of FGFs, affinity using ligands such as heparin is particularly important. Monochromatography, antibody column chromatography and the like are suitable for large-scale purification. Antibodies to the FGF-10 protein can be prepared by a method known per se for both polyclonal and monoclonal antibodies. FGF-10-specific antibodies can be used not only for antibody columns but also for immunochemical quantification such as ELISA.
上述の方法で得られた FGF— 1 0蛋白は、 細胞増殖促進作用を始めとする種 種の生理作用を有しており、 創傷治癒促進剤、 循環不全治療剤、 神経生存維持剤、 発毛促進剤などの医薬用途に用いうる。 特に、 成体哺乳類の軟骨組織での発現が 認められており、 骨折治癒等の骨疾患治療剤、 軟骨，結合組織の損傷治療剤への 応用が考えられる。 また、 細胞増殖促進用の実験試薬としても使用しうる。  The FGF-10 protein obtained by the above-described method has various physiological actions including a cell growth promoting action, and is used as a wound healing promoting agent, a therapeutic agent for circulatory insufficiency, an agent for maintaining nerve survival, and hair growth. It can be used for pharmaceutical applications such as accelerators. In particular, its expression in cartilage tissue of adult mammals has been observed, and its application to the treatment of bone diseases such as fracture healing and the treatment of cartilage and connective tissue damage is conceivable. It can also be used as an experimental reagent for promoting cell proliferation.
FGF— I 0蛋白の動物およびヒ卜への投与は通常の投与経路、 例えば筋肉内、 静脈内、 皮下、 腹腔内、 経皮投与等により行うことができる。 投与量および投与 回数は投与対象、 投与経路、 症状の程度、 体重等によって異なり特に限定されな いが、 ヒトにおいては、 通常成人 1日あたり約 1 g〜0. 1 gを 1日 1回もし くはそれ以上の回数で投与される。 投与剤形としては、 例えば注射剤などが挙げ られる。 製剤化の際は、 通常の製剤担体を用い、 常法により製造する。 即ち、 注 射剤を調製する場合は、 FGF— 1 0蛋白の凍結乾品を生理食塩水に溶解し、 必 要により pH調製剤、 緩衝剤、 安定化剤、 可溶化剤などを添加し、 常法により注 射剤とする。 Administration of the FGF-I0 protein to animals and humans can be carried out by a usual administration route, for example, intramuscular, intravenous, subcutaneous, intraperitoneal, transdermal administration and the like. Dosage and administration The number of doses varies depending on the subject of administration, route of administration, degree of symptoms, body weight, etc., and is not particularly limited. The number of times is administered. Examples of the dosage form include an injection. At the time of formulation, it is manufactured by a usual method using an ordinary formulation carrier. That is, when preparing an injection, a freeze-dried product of FGF-10 protein is dissolved in physiological saline, and a pH adjuster, a buffer, a stabilizer, a solubilizer, and the like are added as necessary. Use it as a propellant in the usual way.
以下の実施例によって、 本発明をさらに詳細に説明するが、 本発明はそれに限 定されるものではない。  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.
実施例 1  Example 1
F G F— 1 0遺伝子の構造解析  Structural analysis of FGF-10 gene
ラッ 卜遺伝子ラィブラリ一の調製  Preparation of rat gene library
1 4日齢ウィスターラッ ト胎児全組織から、 常法 〔Ch omo c z yn s k i 他、 An a l . B i o c h em. 1 62巻， 1 5 6— 1 5 9頁 （】 9 87) 〕 に 従い、 mRNAを調製した。 そのラッ ト胎児 mR N Aを铸型に、 ランダムプライ マー （6me r ) をプライマ一として、 モロニ一マウス白血病ウィルス逆転写酵 素を用いてラッ 卜胎児 c DN Aを調製した。 すなわち、 ラッ ト胎児ポリ （A) + RN A ( 5 g) を 3 0 0ュニッ トの Mo l o n e y mu r i n e  From the whole tissue of a 14-day-old Wistar rat fetus, according to the conventional method [Chomo czynski et al., An al. Bioch em. 162, 156—159 (p. 987)], mRNA was prepared. Rat fetal cDNA was prepared using Moroni murine leukemia virus reverse transcriptase using the rat fetal mRNA as a type II and a random primer (6mer) as a primer. That is, rat fetal poly (A) + RNA (5 g) was added to 300 units of Moloney muriine.
l e u k em i a v i r u s r e v e r s e t r a n s c r i p t a s e (G I BCO— BRL) 、 1 5ユニッ トの h uma n l a c e n t a l e u k em i a v i r u s r e v e r s e t r a n s c r i p t a s e (GI BCO— BRL), 15 units of huma n l a c e n t a
RN a s e i n h i b i t o r (和光純薬工業） および 0. 5 gのランダム プライマ一 （6me r) を含む反応溶液中で 3 7°C60分ィンキュベー卜して、 c DNAを得た。 The cDNA was obtained by incubating at 37 ° C for 60 minutes in a reaction solution containing RNaseinhibbitor (Wako Pure Chemical Industries) and 0.5 g of random primer (6mer).
FGF- 3及び FGF— 7に共通なプライマ一の作成  Creation of a common primer for FGF-3 and FGF-7
既知の 7種類のヒ ト FGFのァミノ酸配列を比較し、 FGF_ 3、 FGF- 7 間でァミノ酸配列が同一である 2箇所 （Ty r - L e u— A l a— Me t— A s n— Ly s、 Ty r— A s n— Th r— Ty r— A l a - S e r) を選び、 図 1に示す 2種類の FGFプライマーを作成した。  The amino acid sequences of seven known human FGFs were compared, and two amino acid sequences identical between FGF_3 and FGF-7 (Tyr-Leu—Ala—Met—Asn—Ly s, Tyr—Asn—Thr—Tyr—Ala-Ser) were selected, and two types of FGF primers shown in FIG. 1 were prepared.
ラッ ト F G Fファミ リ一 D N Aの増幅 ラッ 卜胎児 c DNAを铸型にし、 上記の 2種類の FGFプライマ一と Ta q D N A p o l yme r a s e 用いた p o l yme r a s e c h a i n r e a c t i o n (PCR) 法により FGFファミ リ一DNAを増幅した。 即ち、 適当量の cDNA、 0. 0 5ユニッ ト// / 1の T a Q DNAボリメラ一ゼ （和光 純薬工業） および 5 pmo 1 / 1の前述のセンス—またはアンチセンスプライ マ一を含む反応溶液 （2 5〃 1) を 30サイクルの PCRに供した。 反応後、 溶 液を 8 %ポリアクリルァミ ドゲル電気泳動にかけ、 所望のサイズ （〜 1 1 0塩基 対） のフラクションを電気泳動で溶出した。 Amplification of rat FGF family DNA Rat fetal cDNA was converted to type II, and FGF family DNA was amplified by the polymase chain reaction (PCR) method using the two types of FGF primers and Taq DNA polymase. That is, it contains an appropriate amount of cDNA, 0.05 unit /// 1 TaQ DNA polymerase (Wako Pure Chemical Industries) and 5 pmo 1/1 of the above-mentioned sense- or antisense primer. The reaction solution (25〃1) was subjected to 30 cycles of PCR. After the reaction, the solution was subjected to 8% polyacrylamide gel electrophoresis, and a fraction having a desired size (〜110 base pairs) was eluted by electrophoresis.
ラッ ト FGFファ ミ リ一 DNAのスクリ一ニング  Rat FGF Family DNA Screening
FGFプライマ一により增幅した FGFファ ミ リー DNAを pGEM— T DNA v e c t o r (P r ome g a) に挿入し、 得られた組換えべクタ一を 大腸菌 （XL 1— b 1 u e株） に感染させ、 DNAクローンを得た。 c DNA配 列の解析には、 DN Aシークェンサ一373 A (Ap p 1 i e d  The FGF family DNA amplified by the FGF primer was inserted into a pGEM-T DNA vector (Promega), and the resulting recombinant vector was infected into E. coli (XL1-b1ue strain). A DNA clone was obtained. c DNA sequence analysis includes DNA sequencer 373 A (Ap p 1 i e d
B i o s y s t ems I n c. ) を用いた。  BiosystemsInc.) Was used.
各 DNAクローンの塩基配列を決定したところ、 既知の FGF— 3、 FGF— 7の c DNA以外にも、 既知の FGFファミリーペプチドと類似のァミノ酸配列 構造を持つ （〜50%) ペプチドをコードしている新規な FGF c DNAが単離 された。 これを、 FGF— 1 0と命名した。  When the nucleotide sequence of each DNA clone was determined, in addition to the known FGF-3 and FGF-7 cDNAs, a peptide having a similar amino acid sequence structure to the known FGF family peptide (~ 50%) was also encoded. New FGF cDNA was isolated. This was designated as FGF-10.
ラッ ト FGF— 1 0 cD N Aの全翻訳領域の構造解析  Structural analysis of all translated regions of rat FGF—10 cD NA
上記実験にて判明した FGF— 1 0 cDNAの部分構造から、 プライマーを作 成し、 Ra p i d Amp l i f i c a t i on o f c D Ν Λ En d s (RAC E) 法 〔F r o hma n， PCR P r o t o c o l s— A  Primers were prepared from the partial structure of the FGF- 10 cDNA identified in the above experiment, and the primers were assembled using the Rapid Amplifi ica tio n o f c D Ν Λ Ends (RACE) method [Fro hman, PCR P ro t o co l s — A
gu i d e t o me t h o d s an d a pp l i c a t i o n s, Ac a d em i c P r e s s, p p. 28 - 38 (1 9 90) 〕 を利用して全 翻訳領域を取得した。 詳細は以下 〔1〕 〜 〔6〕 に記述する。  All translation regions were obtained using gu i d e t o me t ho d s and a d a pp l i c a t i o n s s, Ac a d e m i c P R e ss, p p. 28-38 (1 990)]. Details are described in [1] to [6] below.
〔1〕 FGF- 1 0 c DNAの部分構造からプライマ一 A〜D (図 2、 配列番 号： 5、 6、 7および 8) を作成し、 また、 RACE法用として、 プライマ一 X 及び Y (図 2、 配列番号： 9および 1 0) を作成した。  [1] Primers A to D (FIG. 2, SEQ ID NOs: 5, 6, 7, and 8) were prepared from the partial structure of FGF-10 cDNA, and primers X and Y were used for the RACE method. (FIG. 2, SEQ ID NOS: 9 and 10).
〔2〕 ランダムへキサオリゴヌクレオチドをプライマーとして用いてラッ 卜胎児 mRNAを铸型として逆転写酵素により c DNAを合成した後、 デォキシアデ二 ン三リン酸の存在下に 3' デォキシヌクレオチジルトランスフヱラーゼを作用さ せて 3， 末端にポリ （A) 配列を付加した。 このようにして得られた c DNAを 铸型としてプライマ一 Bおよび Xを用いて PCRを行った。 さらに、 プライマ一 Aおよび Yを用いて PC Rを行った。 得られた増幅断片を p GEM—丁に挿入し、 大腸菌 （XL 1 -b 1 u e) を形質転換することによりクローン化した。 数クロ ―ンの塩基配列を決定したところ、 上記の部分配列の一部を含むク口一ンが得ら れたので、 これを pFGF— 1 0 (5' ) と命名した。 [2] Rat embryos using random hexaoligonucleotides as primers After synthesizing cDNA by reverse transcriptase using the mRNA as type III, 3'-deoxynucleotidyltransferase is allowed to act in the presence of deoxyadenyltriphosphate to generate a poly (A) sequence at the 3 'end. Was added. Using the cDNA thus obtained as a 铸 type, PCR was performed using Primers B and X. In addition, PCR was performed using primers A and Y. The obtained amplified fragment was inserted into pGEM-clone and cloned by transforming Escherichia coli (XL1-b1ue). When the nucleotide sequence of several clones was determined, a clone containing a part of the above partial sequence was obtained, and this was named pFGF-10 (5 ').
〔3〕 プライマ一 Xを用いてラッ 卜胎児 mRNAを鋅型として逆転写酵素により cDNAを合成した。 このようにして得られた c DNAを铸型として、 プライマ — Cおよび Yを用いて P CRを行った。 さらに、 プライマ一 Dおよび Yを用いて PCRを行った。 得られた増幅断片を pGEM— Tに揷入し、 大腸菌 （XL 1 b 1 u e株） を形質転換することによりクローン化した。 数クローンの塩基配列 を決定したところ、 上記の部分配列の一部を含むクローンが得られたので、 これ を pFGF— 1 0 (3' ) と命名した。  [3] cDNA was synthesized by reverse transcriptase using primer-X with rat fetal mRNA as type III. Using the cDNA thus obtained as type I, PCR was performed using primers C and Y. Further, PCR was performed using primers D and Y. The obtained amplified fragment was introduced into pGEM-T and cloned by transforming Escherichia coli (XL1b1ue strain). When the nucleotide sequences of several clones were determined, a clone containing a part of the above partial sequence was obtained. The clone was named pFGF-10 (3 ').
〔4〕 p FGF- 1 0 (5' ) より得られた最も上流部分の塩基配列と、 pFGF- 1 0 (3' ) より得られた最も下流部分の塩基配列の塩基配列より、 各々プライマ一 Eおよび F (図 2、 配列番号： 1 1および 1 2) を作成した。 〔5〕 ラッ 卜胎児 mRNAを铸型として、 オリゴ dTをプライマ一として逆転写 酵素により、 cDNA第一鎖を得た。 これを铸型としてプライマ一 Eおよび Fを 用いて PCRを行った。 得られた増幅断片を pGEM— Tに挿入し、 大腸菌 （X L 1— b 1 u e株） を形質転換することによりクローン化した。 数クローンの塩 基配列を決定したところ、 p FGF— 1 0 (5' ) より得られた最も上流部分の 塩基配列と、 p FGF— 1 0 (3' ) より得られた最も下流部分の塩基配列の塩 基配列を連続して保持するクローンが得られた。 この中の 1クローンを選び、 p FGF- 1 0と命名した。 このプラスミ ドに担持される全翻訳領域を含む FG F— 1 0 c DNAを解析した。  [4] From the base sequence of the most upstream part obtained from pFGF-10 (5 ') and the base sequence of the most downstream part obtained from pFGF-10 (3'), E and F (FIG. 2, SEQ ID NOS: 11 and 12) were generated. [5] The first strand of cDNA was obtained by reverse transcriptase using rat fetal mRNA as type I and oligo dT as primer. Using this as a type I, PCR was performed using Primers E and F. The obtained amplified fragment was inserted into pGEM-T, and cloned by transforming Escherichia coli (XL1-b1ue strain). When the base sequences of several clones were determined, the base sequence of the most upstream part obtained from pFGF-10 (5 ') and the base sequence of the most downstream part obtained from pFGF-10 (3') were obtained. A clone was obtained which continuously retained the base sequence of the sequence. One of these clones was selected and named pFGF-10. The FGF-10 cDNA containing the entire translation region carried by this plasmid was analyzed.
〔6〕 以上の結果を総合して配列番号： 3の塩基配列 （804 bp) を決定した c ラッ ト F G F— 1 0の全ァミノ酸配列の決定 〔 1〕 上記実験で得られた F G F— 1 0 c D N Aの塩基配列より、 F G F— 1 0 c DNAの翻訳領域は 6 4 5 b pからなり、 F G F— 1 0は配列番号： 1で示さ れる 2 1 5アミノ酸からなる新規 FG Fであることが明らかになった。 [6] more comprehensively the results SEQ ID NO: Determination of the 3 base sequence (804 bp) c rat FGF 1 total Amino acid sequence of zeros were determined [1] From the nucleotide sequence of FGF-10 cDNA obtained in the above experiment, the translation region of FGF-10 cDNA is composed of 645 bp, and FGF-10 is represented by SEQ ID NO: 1. It was revealed that this is a novel FGF consisting of 15 amino acids.
〔2〕 アミノ酸配列の検討から、 N末端にシグナル配列を有する分泌型の蛋白で あることが分かった。 マチュア部分は、 3 7— 2 1 5位で、 1 7 9アミノ酸から なるポリべプチドと目される。 5 0— 5 2位に存在する A s n— S e r— S e r、 2 0 3— 2 0 5位の A s n—Th r— S e rは、 N結合型糖鎖結合配列であり、 FGF- 1 0は糖鎖を有している可能性がある。  [2] From the examination of the amino acid sequence, it was found to be a secretory protein having a signal sequence at the N-terminus. The mature part is considered to be a polypeptide consisting of 179 amino acids at position 37-215. A sn—S er—S er at position 5 0—5 2 and A sn—Th r—S er at position 20 5—20 5 are N-linked sugar chain binding sequences, and FGF-1 0 may have a sugar chain.
実施例 2  Example 2
ラッ ト F G F— 1 0の哺乳動物細胞での発現  Expression of rat FGF-10 in mammalian cells
プラスミ ドの構築  Build plasmid
プラスミ ド p FGF— 1 0 (図 3 ) を S p h lと P s t Iで消化し、 ポリアク リルアミ ド電気泳動により c DNA全長を含む断片を単離した。 この断片を PUC 1 9の S p h l、 P s t l消化物とライゲーションし、 大腸菌 JM 1 0 9 株を形質転換することにより、 FGF— 1 0 c DNAを含むプラスミ ド pUC— F 1 0を得た。 pUC— F 1 0を H i n d lll 及び X b a Iで消化することによ り FGF— 1 0 c DNAを含む断片を切出し、 哺乳動物細胞発現べクタ一である 〇01^8の1^ 1 1 011【 、 13 3 I消化物とライゲ一ションし、 大腸菌 MC 1 0 6 1ノ P 3株を形質転換することにより、 CMVプロモーターの支配下に FG F- 1 0 c DNAを有するプラスミ ド p CDM8— F 1 0 S Pを得た。  Plasmid pFGF-10 (FIG. 3) was digested with Sphl and PstI, and a fragment containing the entire cDNA was isolated by polyacrylamide electrophoresis. This fragment was ligated with an SphI and Pstl digest of PUC19, and Escherichia coli JM109 strain was transformed to obtain a plasmid pUC-F10 containing FGF-10 cDNA. A fragment containing FGF-10 cDNA was excised by digesting pUC-F10 with HindIII and XbaI, and the mammalian cell expression vector ^ 01 ^ 8 1 ^ 11 By ligating with the 0,11,13 I digest and transforming the E. coli MC1061P3 strain, plasmid p CDM8 with FG F-10c DNA under the control of the CMV promoter was obtained. — I got F10 SP.
一方、 FGF— 1 0 c DNAにおける推定翻訳開始コ ドン上流の塩基配列がコ サックのコンセンサス配列からはずれていることから、 本 mRNAの翻訳効率が 良くない可能性があることが考えられた。 そこで、 翻訳効率の向上を目的として、 推定翻訳開始コ ドン上流をコザックのコンセンサス配列に置換する変異を導入す ることとした 〔マリ リ ン コザック （M. K o z a k ) 、 ザ · ジャーナル.ォブ •セル ·バイオロジー （Th e J o u r n a l o f C e l l  On the other hand, since the nucleotide sequence upstream of the putative translation initiation codon in the FGF-10 cDNA deviated from the Cossack consensus sequence, it was considered that the translation efficiency of this mRNA may not be good. Therefore, for the purpose of improving translation efficiency, a mutation was introduced to replace the putative translation initiation codon upstream of the consensus sequence of Kozak [Marilin Kozak (M. Kozak), The Journal of Ob. • Cell biology (Th e J ournalof Cell
B i o l o y) 1 0 8巻 2 2 9— 2 4 1頁 （ 1 9 8 9. 2) 〕 。  Bioloy) 108 Volume 2 2 9—2 41 Page (1 899.2)].
変異の導入は P C Rを利用し、 p FG F— 1 0を铸型として図 4に示す、 5' 末端に H i n d III 切断部位およびコサックのコンセンサス配列を有するセンス プライマーと、 5' 末端に Xb a I切断部位を有するアンチセンスプライマーを 用いることにより行った （反応条件は図 4参照） 。 Mutagenesis is introduced by PCR, using p FG F-10 as a 铸 type as shown in Fig. 4 and having a Hind III cleavage site at the 5 'end and a Cossack consensus sequence. The reaction was carried out by using a primer and an antisense primer having an XbaI cleavage site at the 5 'end (see FIG. 4 for reaction conditions).
反応終了後、 PCR産物をフヱノールクロ口ホルム処理、 エーテル処理、 つい でエタノール沈澱を行い、 H i ndlli および X b a Iで消化した後、 ポリアク リルアミ ド電気泳動により約 7 0 0塩基対の断片を単離した。 この断片を哺乳動 物細胞発現べクタ一である P C DM 8の H 1 n dllU Xb a I消化物とライゲ ーシヨンし、 大腸菌 MC 1 06 1 ZP 3株を形質転換して得られたコロニーの中 から 4クローンを選び、 DNAシークェンサ一 （パーキンエルマ一 37 3型） を 用し、て塩基配列の解析を行つた。  After completion of the reaction, the PCR product was treated with phenol-mouth form, treated with ether, precipitated with ethanol, digested with Hindlli and XbaI, and then subjected to polyacrylamide gel electrophoresis. Released. This fragment was ligated with the H1ndllUXbaI digest of PCDM8, which is a mammalian cell expression vector, and transformed into colonies obtained by transforming Escherichia coli MC1061ZP3. Four clones were selected from, and the nucleotide sequence was analyzed using a DNA sequencer (Perkin-Elmer 373).
その結果、 いずれのクローンも推定翻訳開始コ ドン上流の塩基配列はコザック のコンセンサス配列に置換されており、 かつコ一ドするァミノ酸配列に影響する 変異は起きていなかつたので、 これらの中から 1クローンを選び、 本プラスミ ド を p CDM8— F 1 0 HXと命名した。  As a result, in all clones, the nucleotide sequence upstream of the putative translation initiation codon was replaced with the Kozak consensus sequence, and no mutation affecting the coding amino acid sequence occurred. One clone was selected and this plasmid was designated as pCDM8-F10HX.
実施例 3  Example 3
ラッ ト FGF— 1 0発現可能なブラスミ ドによる C 0 S— 1細胞の形質転換 実施例 2で構築したラッ ト F G F— 1 0発現可能なブラスミ ド、 p C DM 8 - F 1 08?及び じ01^8— F 1 0 HXを通常の方法に従って大量に調製し、 塩 化セシウム密度勾配超遠心を 2回行って精製した。 これら 2種類のプラスミ ド及 びコントロールとして p C DM 8を用いて、 電気パルス法により COS— 1細胞 の形質転換を行った。 形質転換された細胞は、 リジンセファロ一スクロマト処理 した牛胎児血清 1 0 %を含有する DMEMで 24時間培養された後、 培地を無血 清の DMEMに交換、 さらに継続して 96時間培養された。 このようにして得た 培養液を遠心後、 その上清を分注し 80°Cで凍結保存した。  Transformation of C0S-1 cells with a rat FGF-10-expressable brassmid A rat FGF-10-expressable brassmid constructed in Example 2, pCDM8-F108? A large amount of 01 ^ 8—F10HX was prepared according to the usual method, and purified by twice performing cesium chloride density gradient ultracentrifugation. Using these two kinds of plasmids and pCDM8 as a control, COS-1 cells were transformed by an electric pulse method. The transformed cells were cultured for 24 hours in DMEM containing 10% fetal bovine serum treated with lysine cephalo-chromatography, the medium was replaced with serum-free DMEM, and further cultured for 96 hours. After centrifuging the culture solution thus obtained, the supernatant was dispensed and stored frozen at 80 ° C.
実施例 4  Example 4
i n s i t u ハイブリダィゼ一シヨン法による軟骨における F G F— 1 0 mRNAの発現の確認  Confirmation of FGF-10 mRNA expression in cartilage by insitu hybridization method
プローブの 製  Probe manufacture
FGF— 1 0 c DN Aを p G EM— Tベクターに組み込み、 そのプラスミ ドを 大腸菌 J M 1 0 9に卜ランスフヱク 卜した後、 大量培養を行い、 フアルマシア 社の F l e x i P r e k i tを用いて純度の高い F G F— 1 0 c DN Aを 精製した。 パーキンエルマ一 3 7 3 A/DN Aシークェンサ一を用いて配列を確 認したのち、 ベ一リンガー社の D I G/RNAラベリング キッ ト （S P 6/T 7) を用いて c RNAプローブを作成した。 The FGF-10c DNA was incorporated into the pGEM-T vector, the plasmid was transfected into E. coli JM109, and then mass-cultured. The highly pure FGF-10c DNA was purified using Flexi Prekit of the company. After confirming the sequence using a Perkin Elmer 373 A / DNA sequencer, a cRNA probe was prepared using a DIG / RNA labeling kit (SP6 / T7) from Boehringer.
切片作成  Section creation
ウィスター系雌性ラッ トを 3週令で屠殺後、 大腿骨と脛骨を関節を保持した状 態で摘出し、 軟部組織を除去し、 適当な大きさにトリ ミ ング後、 素早く固定液 (4 %パラホルムアルデヒド） に浸し、 4。Cで一晩固定した。 脱水後、 脱灰液 ( 1 0 %EDTA、 1 5 %グリセロール— PB S) に浸し、 4〜5日間脱灰を行 つた （毎日、 液は交換した） 。 その後、 膝関節前後約 2 cmにトリ ミングし、 0. C. Tコンパウンドに浸し、 液体窒素にて凍結させ、 ク リオスタツ 卜を用いて厚 さ 1 0 zmの関節組織切片を作製し、 シランコ一ティングスライ ドグラスにマウ ントした。  Wistar female rats were sacrificed at the age of 3 weeks, and the femur and tibia were excised while holding the joints, the soft tissues were removed, trimmed to an appropriate size, and then fixed immediately (4% 3. soak in paraformaldehyde) Fixed overnight at C. After dehydration, they were immersed in a demineralized solution (10% EDTA, 15% glycerol—PBS) and decalcified for 4 to 5 days (the solution was changed every day). Then, it was trimmed to about 2 cm before and after the knee joint, immersed in a 0. C. T compound, frozen with liquid nitrogen, and made a 10-zm-thick joint tissue section using a cryostat. Mounted on Tingslide glass.
ハイブリダイゼーション  Hybridization
前述の関節組織切片の前処理 （プロティナーゼ K消化、 0. 2 M HC 1で内 在性のアルカリフォスファタ一ゼを不活化、 0. 1 M TE A ： 0. 2 5 %無水 酢酸でァセチル化） を行った後、 エタノール系列で脱水し風乾した。 そして前述 のプローブをハイブリダィゼーシヨン液 （5 0 %ホルムアミ ド、 1 O mM  Pretreatment of joint tissue sections as described above (digestion with proteinase K, inactivating endogenous alkaline phosphatase with 0.2 M HC1, 0.1 M TEA: acetylation with 0.25% acetic anhydride ), Followed by dehydration with an ethanol series and air drying. Then, the aforementioned probe was mixed with hybridization solution (50% formamide, 1 OmM
T r i s -HC 1 /pH 7. 6、 2 0 0 g 1 t RNA, 1 x  T ris -HC 1 / pH 7.6, 200 g 1 t RNA, 1 x
D e n h a r d t' s s o l u t i o n、 1 0 %D e x t r a n  D e n h a r d t 's s o l u t i o n, 10% D e x t r a n
s u 1 f a t e, 6 0 0 mM N a C 0. 2 5 %S D S) で 1 0倍に希釈し、 一切片当り 5 0 ^ 1載せ、 小さく切ったパラフィルムで覆い、 5 0°Cで 1 6時間 ィンキュベーシヨンした。 RN a s e Aで不要なプローブを消化し S S Cで洗浄 した後、 抗体反応、 発色反応を行った。  su 1 fate, 600 mM NaC 0.25% SDS), dilute 10-fold, put 50 ^ 1 per piece, cover with small pieces of parafilm, and place at 50 ° C 16 Time Incubation. Unnecessary probes were digested with RNase A, washed with SSC, and then subjected to an antibody reaction and a color reaction.
抗体反応および発色反応  Antibody reaction and color reaction
プローブの洗浄が終わった切片をブロッキング液に 6 0分浸した後、 アルカリ フォスファタ一ゼ標識抗ジゴキシゲニン抗体 （An t i —  After washing the probe, the section was immersed in the blocking solution for 60 minutes.
D i g o x i g e n i n—AP : F a b f r a gme n t :ベ一リ ンガ一 マ ンハイム） を乗せ、 3 7 °Cで 1時間インキュベーションした。 抗体液を洗った後、 NBT、 X—フォスフヱイ トを加え、 3 7 °Cでインキュベーションし発色反応を 行った （1 2時間） 。 発色を確認した後、 発色停止液 （ 1 OmM T r i s— HC l /pH 7. 6、 1 mM EDTA/pH 8. 0 ) に漬け、 蒸留水で洗った 後、 水性封入した。 図 5 (A) (B) に示すように軟骨細胞に発色が確認された。 FGF— 1 0の mRNAが軟骨細胞に発現していることから、 FGF— 1 0は軟骨に対する活性 を有する骨および軟骨の損傷修復等に関与する因子であると推定される。 Digoxigenin-AP: Fab gramment: Berlinger Mannheim) and incubated at 37 ° C for 1 hour. After washing the antibody solution, NBT and X-phosphite were added, and the mixture was incubated at 37 ° C to perform a color reaction (12 hours). After confirming color development, it was immersed in a color stop solution (1 OmM Tris—HCl / pH 7.6, 1 mM EDTA / pH 8.0), washed with distilled water, and then sealed with water. As shown in FIGS. 5 (A) and 5 (B), color development was confirmed in chondrocytes. Since FGF-10 mRNA is expressed in chondrocytes, FGF-10 is presumed to be a factor having activity on cartilage and involved in repair of damaged bone and cartilage.
実施例 5  Example 5
FRS K細胞を用いた細胞増殖活性の検討  Examination of cell proliferation activity using FRS K cells
細胞培養  Cell culture
ラッ 卜の上皮細胞である FR SK細胞は、 培養面積 7 5平方 cmの培養フラス コ当り 1 5 m 1の 1 0 %ゥシ胎児血清を含む F— 1 2培地を用いて、 3 7 °C、 5 %二酸化炭素 / 9 5 %空気の気相下で培養した。 細胞は 7日に一度、 1.Z 1 0の 割合で継代を行った。  Rat epithelial cells, FR SK cells, were cultured at 37 ° C in 15 ml of F-12 medium containing 10% 10% fetal serum per 75 square cm culture flask. The cells were cultured under a gas phase of 5% carbon dioxide / 95% air. Cells were subcultured once every 7 days at a rate of 1.Z10.
FGF— 1 0蛋白の発現  Expression of FGF-10 protein
FGF— 1 0を COS— 1細胞で一過性に発現させ （実施例 3参照） 、 その培 養上清を以下のアツセィに供した （以下、 p CDM8— F l 0 S Pを用いて得ら れた培養上清を FGF— 1 0 S p , p C D M 8 ~ F 1 0 HXを用いて得られた 培養上清を FGF— 1 0ZHx、 コントロールプラスミ ド p C DM 8を用いて得 られた培養上清を Bqと表示する） 。  FGF-10 was transiently expressed in COS-1 cells (see Example 3), and the culture supernatant was subjected to the following assay (hereinafter, obtained using pCDM8-F10SP). Culture supernatant obtained using FGF-10Sp, pCDM8-F10HX was used for the culture supernatant, and culture supernatant obtained using FGF-10ZHx and control plasmid pCDM8. The supernatant is indicated as Bq).
• DN A合成アツセィ （トリチウム標識チミジンの取込み） ：細胞をサブコンフ ルェン卜まで培養した後、 トリプシン処理により細胞を剥がし、 上記培地を用い て 1 0 0 0 0細胞/ m 1に調整し、 9 6穴プレートに 1 0 0 1ずつ播き込み、 3 7 °C、 5 %二酸化炭素ノ 9 5 %空気の気相下で培養を行った。 2日に一度、 培 地を新しい上記培地 1 0 0 Z 1に交換し、 7日間培養を行った後、 培地を 0. 1 %ゥシ血清アルブミンを含む F— 1 2培地 1 0 0 1に交換した。 2 4時間後、 COS上清を 2 5 1添加し、 1 8時間3 7で、 5 %二酸化炭素/ 9 5 %空気の 気相下で培養を行 L、、 0. 2 C iのトリチウム標識チミジンを含む F— 1 2培 地 20 1を添加、 引続き同条件下で培養した。 4時間後培地を除去し、 2N NaOHを 50 1添加して 30分静置、 細胞を死滅させた。 I N HC 1で中 和後、 細胞をセルハーべスターで回収し、 ベ一夕プレートにてカウントを計測し o • DNA-synthesizing assay (incorporation of tritium-labeled thymidine): After culturing the cells to subconfluents, detach the cells by trypsin treatment, adjust to 1000 cells / m1 using the above medium, and adjust to 96 1001 was inoculated into the well plate, and cultured at 37 ° C in a gas phase of 5% carbon dioxide and 95% air. Once every two days, the medium is replaced with the above-mentioned medium 100 Z1.After culturing for 7 days, the medium is changed to medium F-012 containing 0.1% 0 serum albumin. Replaced. After 24 hours, add COS supernatant to 251, and incubate for 18 hours at 37 in the gas phase of 5% carbon dioxide / 95% air L, 0.2Ci tritium labeling F-12 containing thymidine The ground 201 was added, followed by culturing under the same conditions. After 4 hours, the medium was removed, and 501 of 2N NaOH was added, and the mixture was allowed to stand for 30 minutes to kill the cells. After neutralization with IN HC 1, cells were collected with a cell harvester, and counted on a plate overnight.
輕  Light
図 6に示すように、 コントロール群： B q ( 1 00%) と比較して、 FGF— 1 0発現群： Sp、 Hxは FRS K細胞のトリチウム標識チミジンの取込みを大 きく増加させた （各々 2 86 %、 50 1 %) 。 F G F— 1 0は上皮細胞の増殖を 促進する因子であることが示唆される。  As shown in FIG. 6, compared with the control group: Bq (100%), the FGF-10 expression group: Sp, Hx greatly increased the incorporation of tritium-labeled thymidine into FRSK cells (each 2 86%, 50 1%). It is suggested that FGF-10 is a factor that promotes the proliferation of epithelial cells.
実施例 6  Example 6
ヒト F G F— 1 0遺伝子の構造解析  Structural analysis of human FGF-10 gene
ヒト遺伝子ライブラリーの調製  Preparation of human gene library
市販のヒ ト肺ポリ （A) +RNA 〔クローンテック （c 1 o n t e c h) 社： カタログ番号 652 4、 成人男性全肺由来 〕 を铸型に、 ランダム プライマー (6 me r ) をプライマーとして、 モロニ一マウス白血病ウィルス逆転写酵素を 用いてヒ 卜肺 cDNAを調製した。 すなわち、 ヒ ト肺ポリ （A) +RNA (5 g) を 3 0 0ュニッ トの M 0 1 0 n e y mu r i n e  Using a commercially available human lung poly (A) + RNA [clontech (C1ontech): Catalog No. 6524, derived from adult male whole lung] as type III, using a random primer (6mer) as a primer Human lung cDNA was prepared using mouse leukemia virus reverse transcriptase. That is, human lung poly (A) + RNA (5 g) was converted to 300 units of M 0 10 n e y mu r i n e
l e u k em i a v i r u s r e v e r s e t r a n s c r i p t a s e (G I BCO— BRL) 、 1 5ユニッ トの huma n l a c e n t a RN a s e i n h i b i t o r (和光純薬工業） および 0. 5〃gのランダム プライマー （6 me r) を含む反応溶液中で 37 °C 6 0分ィンキュベ一トして、 c DNAを得た。  37 ° C 6 in a reaction solution containing leuk em iavirusreversetranscr iptase (GI BCO—BRL), 15 units of huma nlacenta RNase inhibitor (Wako Pure Chemical Industries) and 0.5 μg of random primers (6 mer) After incubating for 0 minutes, cDNA was obtained.
ヒト FGF— 1 0遺伝子増幅用プライマーの作成およびヒ ト FGFファミ リー DNAの増幅  Preparation of primers for amplification of human FGF-10 gene and amplification of human FGF family DNA
実施例 1で使用した、 図 1に示す 2種類の FGFプライマー （Ty r— L e u -A 1 a -Me t _A s n— Ly s、 Ty r A s n— Th r— Ty r - A 1 a — S e r) を選び、 ヒ ト FGF— 1 0遺伝子増幅に用いた。  The two types of FGF primers used in Example 1 shown in FIG. 1 (Tyr—Leu-A1a-Met_Asn—Lys, TyrAsn—Thr—Tyr-A1a— Ser) was used to amplify the human FGF-10 gene.
ヒ ト肺 cDNAを铸型にし、 上記の 2種類の FGFプライマーと T a q DNA p o l yme r a s eを用いた p o l yme r a s e c h a i n c h a i n (PCR) 法により FGFファミリ一 DN Aを增幅した。 即ち、 適当 量の cDNA、 0. 05ユニッ ト// 1の T a qDNAポリメラ一ゼ （和光純薬 工業） および 5 pmo 1 Z// 1の前述のセンス一またはアンチセンスプライマー を含む反応溶液 （25 / 1 ) を 30サイクルの PCRに供した。 反応後、 溶液を 8 %ポリアクリルアミ ドゲル電気泳動にかけ、 所望のサイズ （〜 1 1 0塩基対） のフラクションを電気泳動で溶出した。 The human lung cDNA was converted into type II, and the pol ymerase chain using the above two FGF primers and Taq DNA pol ymerase. FGF family DNA was amplified by the chain (PCR) method. That is, a reaction solution containing an appropriate amount of cDNA, 0.05 units // 1 of TaqDNA polymerase (Wako Pure Chemical Industries) and 5 pmo1Z // 1 of the above-mentioned sense-1 or antisense primer ( 25/1) was subjected to 30 cycles of PCR. After the reaction, the solution was subjected to 8% polyacrylamide gel electrophoresis, and a fraction having a desired size (〜110 base pairs) was eluted by electrophoresis.
ヒ ト F G Fファミリー DNAのスクリ一二ング Screening of human FGF family DNA
0?プラィマ一にょり増幅した 0?ファミリ一 DNAを pGEM— T DNA v e c t o r (P r ome g a) に挿入し、 得られた組換えべクタ一を 大腸菌 （XL 1—b 1 u e株） に感染させ、 DNAクローンを得た。 cDNA配 列の解析には、 DN Aシークェンサ一 373 A (A p 1 i e d  Insert the 0-family DNA amplified in 0-primer into pGEM-T DNA vector (Promomega), and infect the resulting recombinant vector with E. coli (XL1-b1ue strain) To obtain a DNA clone. For analysis of the cDNA sequence, the DNA sequencer 373 A (Ap1 ied
B i o s y s t ems I n c. ) を用いた。 BiosystemsInc.) Was used.
各 DNAクローンの塩基配列を決定したところ、 ラッ ト型 FGF— 1 0と同一 の配列を有するぺプチドをコ一ドする c DNAが增幅されていた。 この遺伝子は ヒ 卜 FGF— 1 0をコードするものと考えられた。  When the nucleotide sequence of each DNA clone was determined, cDNA encoding a peptide having the same sequence as that of the rat FGF-10 was found to be wide. This gene was thought to encode human FGF-10.
ヒ ト FGF— 1 0 cD N Aの全翻訳領域の構造解析  Structural analysis of the entire translation region of human FGF—10 cD NA
実施例 1と同じ方法でヒ 卜 F G F— 1 0全翻訳領域を含む c D N Aを増幅及び 解析し、 配列番号： 4の塩基配列を決定した。 詳細は以下 〔1〕 〜 〔6〕 に示す。 CDNA containing the whole human FGF-10 translation region was amplified and analyzed in the same manner as in Example 1, and the nucleotide sequence of SEQ ID NO: 4 was determined. Details are shown in [1] to [6] below.
〔1〕 ヒ 卜 FGF— 1 0 c DNAの部分構造からプライマー A' および D' (図 2、 配列番号： 1 3および 1 4) を作成した。 プライマー B、 C、 Xおよび Y[1] Primers A ′ and D ′ (FIG. 2, SEQ ID NOS: 13 and 14) were prepared from the partial structure of human FGF-10 cDNA. Primers B, C, X and Y
(図 2、 配列番号： 5、 6、 7および 8) は実施例 1で使用したものを流用した。 用いた。 (FIG. 2, SEQ ID NOS: 5, 6, 7, and 8) used in Example 1 were diverted. Using.
〔2〕 ランダムへキサオリゴヌクレオチドをプライマーとして用いてヒ 卜肺 mRNAを铸型として逆転写酵素により cDNAを合成した後、 デォキンアデ二 ン三リン酸の存在下に 3' デォキシヌクレオチジル卜ランスフェラ一ゼを作用さ せて 3' 末端にポリ （A) 配列を付加した。 このようにして得られた c DNAを 铸型としてプライマ一 A' および Xを用いて PCRを行い、 さらに、 プライマ一 Bおよび Yを用いて P CRを行った。 得られた増幅断片を p GEM— Tに挿入し、 大腸菌 （XL 1— b 1 u e) を形質転換することにより、 上記の部分配列の一部 を含むクローンが得られたので、 これを p hFGF— 1 0 (5' ) と命名した。 〔3〕 プライマー Xを用いてヒ ト肺 mRNAを铸型として逆転写酵素により c DNAを合成した。 このようにして得られた c DNAを铸型として、 プライマ — Cおよび Xを用いて P CRを行った。 さらに、 プライマー D' および Yを用い て PCRを行った。 得られた増幅断片を pGEM— Tに挿入し、 大腸菌 （XL 1 一 b 1 u e株） を形質転換することによりクローン化した。 数クローンの塩基配 列を決定したところ、 上記の部分配列の一部を含むクローンが得られたので、 こ れを p hFGF— 1 0 (3' ) と命名した。 [2] After synthesizing cDNA with reverse transcriptase using human lung mRNA as type I using a random hexoligonucleotide as a primer, 3'-deoxynucleotidyltransferase was added in the presence of deokinadenidin triphosphate. The poly (A) sequence was added to the 3 'end by the action of ferulase. Using the thus obtained cDNA as type I, PCR was performed using primers A 'and X, and PCR was performed using primers B and Y. The amplified fragment obtained is inserted into pGEM-T and transformed into E. coli (XL1-b1ue) to obtain a part of the above partial sequence. Was obtained, and this was named phFGF-10 (5 '). [3] Using primer X, cDNA was synthesized by reverse transcriptase using human lung mRNA as type II. Using the cDNA thus obtained as type I, PCR was performed using primers C and X. Furthermore, PCR was performed using primers D 'and Y. The obtained amplified fragment was inserted into pGEM-T, and cloned by transforming Escherichia coli (XL1-b1ue strain). When the nucleotide sequence of several clones was determined, a clone containing a part of the above partial sequence was obtained, and this was named phFGF-10 (3 ′).
〔4〕 p hFGF— 1 0 (5' ) より得られた翻訳領域上流部分の塩基配列は、 ラッ ト型遺伝子と同じ配列を含んでいたので、 5' 側のプライマーとしてプライ マ一 E (図 2 ：配列番号 1 2) を流用した。 また、 phFGF_ l 0 (3' ) よ り得られた最も下流部分の塩基配列の塩基配列から、 3' 側のプライマーとして 新たにプライマ一 F' (図 2 ：配列番号 1 5) を作成した。  [4] Since the nucleotide sequence upstream of the translation region obtained from phFGF-10 (5 ') contained the same sequence as the rat gene, primer E (Fig. 2: SEQ ID NO: 12) was diverted. From the base sequence of the most downstream base sequence obtained from phFGF_10 (3 '), a new primer 1F' (FIG. 2: SEQ ID NO: 15) was newly prepared as a 3'-side primer.
〔5〕 ヒ卜肺 mRNAを铸型として、 オリゴ dTをプライマ一として逆転写酵素 により、 cDNA第一鎖を得た。 これを铸型としてプライマ一 Eおよび F' を用 いて PCRを行った。 得られた増幅断片を PGEM— Tに揷入し、 大腸菌 （XL 1一 b 1 u e株） を形質転換することによりクローン化した。 数クローンの塩基 配列を決定したところ、 phFGF— 1 0 (5' ) より得られた翻訳領域上流部 分の塩基配列と、 p hFGF— 1 0 (3' ) より得られた最も下流部分の塩基配 列の塩基配列を連続して保持するクローンが得られた。 この中の 1クローンを選 び、 p hFGF— 1 0と命名した。 このプラスミ ドに担持される全翻訳領域を含 むヒ ト FGF— 1 0 c DNAを解析した。  [5] The first strand of cDNA was obtained by reverse transcriptase using human lung mRNA as type II and oligo dT as primer. PCR was carried out using the primers E and F 'as a type III. The obtained amplified fragment was introduced into PGEM-T, and cloned by transforming Escherichia coli (XL1-b1ue strain). When the nucleotide sequences of several clones were determined, the nucleotide sequence of the upstream portion of the translation region obtained from phFGF-10 (5 ') and the nucleotide sequence of the most downstream portion obtained from phFGF-10 (3') were obtained. A clone was obtained which continuously retained the nucleotide sequence of the sequence. One of these clones was selected and named phFGF-10. Human FGF-10 cDNA containing the entire translation region carried by this plasmid was analyzed.
〔6〕 以上の結果を総合し、 配列番号： 4の塩基配列 （6 9 0 b p) を決定した c ヒ ト FGF— 1 0の全ァミノ酸配列の決定  [6] Based on the above results, the nucleotide sequence (690 bp) of SEQ ID NO: 4 was determined. The determination of the entire amino acid sequence of c-human FGF-10 was determined.
上記実験で得られた c DN Aの塩基配列より、 ヒト FG F - 1 0 c DN Aの翻 訳領域は 6 24 b pからなり、 ヒ 卜 FGF— 1 0は配列番号： 2で示される 20 8アミノ酸からなるポリペプチドであることが明らかになった。 また、 アミノ酸 配列の検討から、 N末端にシグナル配列を有する分泌型の蛋白であることが分か つた。 マチュア部分は、 38— 2 08位で、 1 7 1アミノ酸からなるポリべプチ ドと目される。 5 1— 53位に存在する A s n— S e r— S e r、 1 9 6 - 1 9 8位の A s n— Th r _S e rは、 N結合型糖鎖結合配列であり、 この部位に糖 鎖を有している可能性がある。 From the nucleotide sequence of cDNA obtained in the above experiment, the translated region of human FGF-10 cDNA comprises 624 bp, and human FGF-10 is represented by SEQ ID NO: 2. It was revealed that the polypeptide was composed of amino acids. Investigation of the amino acid sequence revealed that the protein was a secretory protein having a signal sequence at the N-terminus. The mature part is 38-208, a polypeptide consisting of 17 1 amino acids It is regarded as C. 5 A sn—Ser—Ser at position 1—53 and A sn—Thr_Ser at position 196-198 are N-linked glycan-binding sequences. May have
実施例 7  Example 7
ヒト FGF— 1 0成熟蛋白の発現と精製  Expression and purification of human FGF-10 mature protein
p FGF- 1 0を铸型として以下のプライマ一ペア （配列番号： 1 6、 配列番 号： 1 7) を用いて 1 5サイクルの P CRを行い、 フヱノールノクロ口ホルム処 理後エタノール沈殿を行い、 さらに Nd e Iおよび B amH Iで消化し、 ポリア クリルアミ ドゲル電気泳動にて目的の大きさのバンドを分取することにより、 ヒ ト FGF— 1 0 c DNAの成熟アミノ酸配列に相当する DNA断片 （a) を得た。 一方、 大腸菌発現ベクターである P ET 1 1 c (ストラタジーン） を Nd e Iお よび B amH Iで消化し、 ァガロースゲル電気泳動にて分取することにより直鎖 化したベクタ一 DNA (b) を得た。 これら （a) と （b) をライゲ一シヨンし、 大腸菌 JM1 09を形質転換することによりクローン化した。 これらの中から (a) が正しい方向に挿入されたプラスミ ドを単離し、 塩基配列の確認を行い、 p ET-h FGF- 1 0を得た。 これを用いて大腸菌 BL 2 1 (DE 3) を形質 転換した。 得られた組換えクローンのうちの 1つを BL 2 1 (DE 3) / ET 一 h FGF— 1 0と名づけ、 これを用いてヒト FGF— 1 0の発現生産を行った。  p FGF-10 was used as type II, 15 cycles of PCR were carried out using the following primer pair (SEQ ID NO: 16 and SEQ ID NO: 17), and ethanol precipitation was performed after processing with phenol noclo mouth form. The DNA fragment corresponding to the mature amino acid sequence of human FGF-10 cDNA is obtained by digesting with NdeI and BamHI and separating a band of a desired size by polyacrylamide gel electrophoresis. (A) was obtained. On the other hand, PET-11c (Stratagene), an Escherichia coli expression vector, was digested with NdeI and BamHI and fractionated by agarose gel electrophoresis to obtain linearized vector-DNA (b). Obtained. These (a) and (b) were ligated and cloned by transforming Escherichia coli JM109. From these, the plasmid in which (a) was inserted in the correct direction was isolated, and the nucleotide sequence was confirmed. Thus, pET-hFGF-10 was obtained. This was used to transform E. coli BL21 (DE3). One of the obtained recombinant clones was named BL21 (DE3) / ET-1hFGF-10, and was used to produce and produce human FGF-10.
B L 2 1 (DE 3) /p ET— h FG F— 1 0をアンピシリ ン 1 0 0 g /'111 1を含む1 8培地1 Om 1に植菌したものを 4本用意し、 37 °Cで一晩前培 養を行った。 翌日それぞれ全量を 1 00〃 g/m 1を含む TB培地 50 0 m 1 x 4本に植え込み 37 °Cで振とう培養した。 OD 600 = 0. 8に達した時点で I PTGを最終濃度が 1 mMになるように添加し、 培養温度を 28 °Cに下げてさら に 6時間培養を継続した。  BL 2 1 (DE 3) / p ET- h FG F-10 was inoculated into 1 Om 1 of 18 medium containing 100 g of ampicillin / '111 1 and prepared at 37 ° C. Pre-culture overnight. On the following day, the whole amount was inoculated into four 500 mL x 1 TB media containing 100 µg / ml, and cultured with shaking at 37 ° C. When the OD 600 reached 0.8, IPTG was added to a final concentration of 1 mM, the culture temperature was lowered to 28 ° C, and the culture was continued for another 6 hours.
培養液を遠心分離し、 得られた菌体を 5 OmMT r i s— HC 1， H 8. 0 にて 1回洗浄し、 1 mM EDTA、 2 / gノ m 1ロイぺプチン、 2 /igZm l ぺプスタチン、 1 mM PMS Fを含む 5 OmMT r i s— HC 1 , H 8. 0 に懸濁した。 超音波破砕により菌体を破砕し、 ベックマン J 2— 2 1 MZE高速 冷却遠心機にて J A— 20ロータ一を用いて、 1 50 00回転で 1時間遠心分離 することにより上清を採取した。 H i T r a p He p a r i n (5m l， ファ ルマシア） を 5 OmMT r i s— HC 1， H 8. 0で平衡化し、 先に調製した 菌体破砕上清をアプライした。 続いて 5 OmMT r i s— HC 1， H 8. 0で 溶出液の A 260がべ一スラインに戻るまで洗浄した後、 連続的に N a C 1濃度 勾配を 3 Mまで増加させることにより、 蛋白を溶出した。 組換えヒ ト FGF— 1 0に相当する約 1 9 kDaの蛋白は約 1. 2M N a C 1の位置に溶出された。 なお、 流速は 2 m 1 Z分で行った。 The culture solution was centrifuged, and the obtained cells were washed once with 5 OmMT ris—HC1, H8.0, and then washed with 1 mM EDTA, 2 / g nom 1 leptin, 2 / igZm l ぺPustatin was suspended in 5 OmMT ris-HC1, H8.0 containing 1 mM PMS F. The cells are disrupted by sonication, and centrifuged at 1500 rpm for 1 hour using a JA-20 rotor with a Beckman J2-21 MZE high-speed cooling centrifuge. Then, the supernatant was collected. HiTrap Heparin (5 ml, Pharmacia) was equilibrated with 5 OmMTris-HC1, H8.0, and the cell lysate supernatant prepared above was applied. Subsequently, the protein was washed with 5 OmMT ris—HC1, H8.0 until the eluate A 260 returned to the base line, and then the NaC1 concentration gradient was continuously increased to 3 M to increase the protein content. Eluted. A protein of about 19 kDa corresponding to the recombinant human FGF-10 was eluted at a position of about 1.2M NaC1. The flow rate was set at 2 m1Z.
続いて、 上記溶出画分を 5 0 mMT r i s— H C 1， H 8. 0で 2倍希 釈し、 H i T r ap S P ( 5 m 1， フアルマシア） にアプライした。 50 mMT r i s— HC 1, pH 8. 0で洗浄した後、 連続的に N a C 1濃度勾配を 2 Mまで増加させることにより、 蛋白を溶出した。 組換えヒト FGF— 1 0に相 当する約 1 9 kD aの蛋白は約 1. 2M N a C 1の位置に溶出された。 なお、 流速は 2 m 1 Z分で行った。 次に、 上記溶出画分を透析により PBS (—） に置 換し、 さらにエンドトキシンを除去するために 1 Z 1 0量のパイ口セップ 1 C (ダイセル工業） を添加し、 2時間 4 °Cで振とうした後、 上清を回収した。 ェン ドトキシン濃度をエンドスぺ一シー ES— 6 (生化学工業） を用いて定量した結 果、 検出限界以下であった。 得られた蛋白をプロテインアツセィキッ 卜 （バイオ ラッ ド） を用いて定量した結果、 総計 3. 5mgの蛋白が得られたことが明らか となつた。  Subsequently, the above eluted fraction was diluted two-fold with 50 mM Tris-HCl, H8.0 and applied to HiTrapAP SP (5 ml, Pharmacia). After washing with 50 mM Tris-HC1, pH 8.0, the protein was eluted by continuously increasing the NaCl concentration gradient to 2M. A protein of about 19 kDa corresponding to recombinant human FGF-10 was eluted at about 1.2 M NaCl. The flow rate was set at 2 m1Z. Next, the above eluted fraction is replaced with PBS (-) by dialysis, and 1Z10 volume of Pi-mouth Sepp 1 C (Daicel Industries) is added to remove endotoxin. After shaking with, the supernatant was recovered. The endotoxin concentration was determined using Endosc ES ES-6 (Seikagaku Corporation) and found to be below the detection limit. As a result of quantifying the obtained protein using a protein assay kit (BioRad), it was found that a total of 3.5 mg of the protein was obtained.
実施例 8  Example 8
骨組織に及ぼす F G F— 1 0の影響  Effect of FGF-10 on bone tissue
以下、 製剤例および試験例により本発明の骨 （軟骨） 疾患治療剤の製剤および 骨 （軟骨） 組織形成 ·修復作用効果について説明する。  Hereinafter, the formulation of the therapeutic agent for bone (cartilage) disease of the present invention and the effect of bone (cartilage) tissue formation and repair will be described with reference to formulation examples and test examples.
•製剤：実施例 7の方法で作成した精製ヒ ト FGF— 1 0成熟蛋白を用いて、 注 射用水溶液を調製した。 即ち、 ヒ ト FGF— 1 0 (2. 1 2mg) を生理食塩液 (l m l ) に溶解させ、 下記の実験に供した。  • Formulation: An aqueous injection solution was prepared using the purified human FGF-10 mature protein prepared by the method of Example 7. That is, human FGF-10 (2.12 mg) was dissolved in physiological saline (lml) and subjected to the following experiment.
i n V i v o薬効評価試験：骨組織に及ぼす F G F— 1 0の影響  In Vivo efficacy evaluation test: Effect of FGF-10 on bone tissue
4週齢の Wi s t a r系雄性ラッ 卜 （体重 94〜 1 2 0 g) を 1群 3ないし 4 個体として計 3群用意した。 エーテル麻酔下で 27ゲージ針のマイクロシリ ンジ を用いて脛骨骨髄腔内に前記製剤例 1で調製した FGF— 1 0水溶液剤を用いて、 それぞれ 1 0. 6、 2 1. 2 g骨髄腔に相当する液量を投与した。 一群には対 照群として生理食塩液を投与した。 Three groups of 4-week-old male Wistar rats (weight: 94 to 120 g) were prepared as 3 to 4 individuals per group. 27 syringe needle micro syringe under ether anesthesia Using the FGF-10 aqueous solution prepared in Preparation Example 1 above, a liquid volume corresponding to 10.6 and 21.2 g of the medullary cavity was administered into the medullary cavity of the tibia using, respectively. One group received saline as a control group.
4日間飼育した後、 屠殺し、 脛骨を摘出した。 脛骨の軟 X線写真を撮影し写真 像をスキャナ一を用いてパーソナルコンピュータ一に取り込み、 画像処理プログ ラムを使用して、 骨形成の像を観察した。  After breeding for 4 days, the animals were sacrificed and the tibia was removed. A soft radiograph of the tibia was taken, the image was taken into a personal computer using a scanner, and an image of bone formation was observed using an image processing program.
FGF- 1 0投与群の観察結果を図 7に対照群の観察結果を図 8に示した。 ヒ 卜 FGF— 1 0投与群においては、 骨形成の亢進を示す石灰化部分の増加が認め られる。 ヒ 卜 FGF— 1 0注入 4日目の骨髄腔内の骨形成について、 軟 X線写真 の画像解析像から評価した結果を表 1に示す。  FIG. 7 shows the observation results of the FGF-10 administration group, and FIG. 8 shows the observation results of the control group. In the human FGF-10 administration group, an increase in the calcified portion showing enhanced bone formation is observed. Table 1 shows the results of evaluation of bone formation in the medullary cavity on day 4 of the injection of human FGF-10 from soft X-ray images.
表 1 table 1
FGF - 1 0 ( g) 0 1 0. 6 2 1. 2 FGF-10 (g) 0 1 0.6.2 1.2
匹数 3 4 4 対照群と比較して骨形成 4 4  Number of animals 3 4 4 Osteogenesis 4 4 compared to control group
亢進が認められた匹数 これらの結果、 ヒ ト F G F— 1 0は骨 Z軟骨疾患治療で重要な骨 Z軟骨組織の 明らかな形成 ·修復促進作用を示した。  As a result, human FGF-10 showed a clear promoting action of formation and repair of bone Z cartilage tissue, which is important in the treatment of bone Z cartilage disease.
実施例 9  Example 9
FGF- 1 0改変体の作成： F G F - 1 0改変体哺乳類細胞で発現可能なブラ スミ ドの構築  Preparation of FGF-10 variant: Construction of a plasmid that can be expressed in FGF-10 variant mammalian cells
ヒ ト F G F— 1 0を哺乳類細胞で発現可能なブラスミ ドの構築  Construction of a plasmid capable of expressing human FGF-10 in mammalian cells
実施例に記載の p CDM8— F 1 0 H Xの構築と同様に行なった。 即ち、 天然 のヒ ト FGF— 1 0配列を有するプラスミ ド p hFGF—〖 0を铸型として図 4 に示すサプライマ一 F 1 0 H Sの代わりに下記プライマ一 h F 1 0 HXを、 また プライマ一 F 1 0 XRの代わりに下記プライマ一 h F 1 0 XRを用いた PCR反 応を行し、、 反応物を H i n d IEおよび X b a Iで消化後、 ボリァクリルァミ ドゲ ル電気泳動により約 700 b pの断片を単離した。 この断片を p C DM 8の H i ndffl、 Xb a I消化物とライゲーションし、 大腸菌 MC 1 06 1/p 3株を形 質転換することにより、 目的のヒ ト FGF— 1 0を哺乳類細胞で発現可能なブラ スミ ド p CDM8— h F l 0 H Xを得た。 The construction was performed in the same manner as in the construction of pCDM8-F10HX described in Examples. That is, the plasmid phFGF- 〖0 having a natural human FGF-10 sequence was used as a type I, and the following primer hF10HX was used instead of the supplier F10HS shown in FIG. A PCR reaction was performed using the following primer hF10XR instead of F10XR, and the reaction was digested with Hind IE and XbaI. An approximately 700 bp fragment was isolated by electrophoresis. This fragment was ligated with Hindfl and XbaI digests of pCDM8, and transformed into Escherichia coli MC1061 / p3 to transform the desired human FGF-10 into mammalian cells. An expressible plasmid p CDM8—hF10HX was obtained.
プライマ一 5' — 3' Primer 5 '— 3'
hF l 0 HS nTAAGCTTCCACCATGTGGAAATGGATACTGAC hF l 0 HS nTAAGCTTCCACCATGTGGAAATGGATACTGAC
hF l 0 XR TTnCTAGAACAAACGGTGCCTTCCTCTATG hF l 0 XR TTnCTAGAACAAACGGTGCCTTCCTCTATG
p CDM8 -F 1 0 (S I 0 6) の構築 （図 9 A)  Construction of p CDM8 -F 10 (S I 06) (Figure 9A)
まず、 変異配列を有するプライマ一 1およびプライマー 1に部分的に相補的な 配列を有するプライマ一 2ならびにこれら 2種のプライマーではさまれる領域の 外側の領域にァニールする 2種のプライマ一 3および 4を作成した。 天然のヒト FGF— 1 0遺伝子配列を有するプラスミ ド p CDM8 - h F 1 0 HXを铸型と し、 プライマ一 1とプライマ一 3を用いた PCR反応により、 置換配列を有する DNA断片 1を取得した。 これとは別に、 同铸型に対しプライマー 2とプライマ —4を用いた PCR反応により、 同部位の置換配列を有し、 DNA断片 1と部分 的に重複した配列を有する DN A断片 2を取得した。 次に、 このようにして取得 された DNA断片 1と DN A断片 2を鋅型とし、 プライマ一 3とプライマ一 4を 用いた P CR反応を行うことにより、 DN A断片 3を取得した。 その後、 DNA 断片 3を 2種の制限酵素 H i 1 (1111ぉょび a Iで切断し、 pCDM8— hF 1 0 HXを同制限酵素で消化し脱リン酸化したものとライゲートし、 大腸菌 MC 1 06 1/p 3株を形質転換することにより、 目的のアミノ酸置換を有する蛋白 質を発現可能なプラスミ ド PCDM8— F 1 0 (S 1 06) を取得した。  First, the primer 1 having the mutant sequence and the primer 1 having a sequence partially complementary to the primer 1 and the two primers 3 and 4 which anneal to the region outside the region between the two primers It was created. Plasmid p CDM8-hF10HX having the natural human FGF-10 gene sequence was designated as type III, and a DNA fragment 1 having the replacement sequence was obtained by PCR using primers 1 and 13 did. Separately, by performing a PCR reaction on the same type using Primer 2 and Primer-4, DNA fragment 2 having the same sequence as the replacement sequence and partially overlapping with DNA fragment 1 was obtained. did. Next, the DNA fragment 1 and the DNA fragment 2 thus obtained were converted into type III, and a PCR reaction was performed using primers 13 and 14 to obtain a DNA fragment 3. Then, DNA fragment 3 was digested with two types of restriction enzymes Hi 1 (1111 and aI), and pCDM8—hF 10 HX was digested with the same restriction enzymes and ligated with dephosphorylated, and E. coli MC 1 By transforming the 061 / p3 strain, a plasmid PCDM8-F10 (S106) capable of expressing a protein having the desired amino acid substitution was obtained.
p CDM8 -F 1 0 (S 1 50) の構築  Construction of p CDM8 -F 1 0 (S 150)
プライマ一 1の代わりにプライマー 5を、 プライマ一 2の代わりにプライマ一 6を用いた以外は p CDM8— F 1 0 ( S 1 06 ) の構築と同様の 2段階の P C R反応により目的の変異が導入された D N A断片を取得後、 これを 2種の制限酵 素 H i n dfflと Xb a Iで切断し、 pCDM8— hF 1 0 HXを同制限酵素で消 ィ匕し脱リン酸化したものとライゲ一卜し、 大腸菌 MC 1 06 1 /p 3株を形質転 換することにより、 目的のアミノ酸置換を有する蛋白質を発現可能なプラスミ ド p CDM8 -F 1 0 (S I 50) を取得した。 Primers 5 were used instead of Primer 1 and Primer 6 was used instead of Primer 2, but the desired mutation was found by a two-step PCR reaction similar to the construction of pCDM8-F10 (S106). After obtaining the introduced DNA fragment, it was cleaved with two restriction enzymes Hindffl and XbaI, and pCDM8-hF10HX was digested with the same restriction enzyme and dephosphorylated, and A plasmid capable of expressing a protein having the desired amino acid substitution by transforming E. coli MC1061 / p3 p CDM8 -F10 (SI50) was obtained.
p CDM8 -F 1 0 (A 5 1 ) の構築  Construction of p CDM8 -F 10 (A 5 1)
プライマ一 1の代わりにプライマー 7を、 プライマ一 2の代わりにプライマ一 8を用いた以外は p C DM 8— F 1 0 (S 1 06) の構築と同様の 2段階の P C R反応により目的の変位が導入された DNA断片を取得後、 これを 2種の制限酵 素 H i ndmと Xba Iで切断し、 p CDM8— h F 1 0 HXを同制限酵素で消 ィ匕し脱リン酸化したものとライゲ一トし、 大腸菌 MC 1 06 1 /' p 3株を形質転 換することにより、 目的のアミノ酸置換を有する蛋白質を発現可能なプラスミ ド p CDM8 -F 1 0 (S 1 50) を取得した。  Primer 7 was used in place of Primer 1 and Primer 8 was used in place of Primer 2 except that primer CD8 was used in the same two-step PCR reaction as in the construction of pCDM8-F10 (S106). After obtaining the DNA fragment into which the displacement was introduced, the DNA fragment was cut with two kinds of restriction enzymes Hindm and XbaI, and pCDM8-hF10HX was digested with the same restriction enzyme and dephosphorylated. A plasmid p CDM8 -F10 (S150) capable of expressing a protein having the amino acid substitution of interest by transforming E. coli MC1061 / 'p3 strain I got it.
p CDM8 -F 1 0 (A 1 96) の構築  Construction of p CDM8 -F10 (A196)
プライマー 1の代わりにプライマー 9を、 プライマー 2の代わりにプライマ一 1 0を用いた以外は p CDM8 -F 1 0 (S 1 06) の構築と同様の 2段階の P CR反応により目的の変位が導入された DN A断片を取得後、 これを 2種の制限 酵素 H i n d Π1と Xb a Iで切断し、 p CDM8— h F 1 0 HXを同制限酵素で 消化し脱リン酸化したものとライゲ一トし、 大腸菌 MC 1 06 1 /p 3株を形質 転換することにより、 目的のアミノ酸置換を有する蛋白質を発現可能なプラスミ ド p CDM8— F 1 0 (A 1 96) を取得した。  Except that primer 9 was used in place of primer 1 and primer 10 was used in place of primer 2, the desired displacement was achieved by a two-step PCR reaction similar to the construction of pCDM8-F10 (S106). After obtaining the introduced DNA fragment, it was cleaved with two restriction enzymes Hind in1 and XbaI, digested with pCDM8-hF10HX with the same restriction enzymes and dephosphorylated By transforming the E. coli MC1061 / p3 strain, plasmid pCDM8-F10 (A196) capable of expressing a protein having the desired amino acid substitution was obtained.
p CDM8 -F 1 0 (C 84 ) の構築  Construction of p CDM8 -F 10 (C 84)
プライマー 1の代わりにプライマー 1 1を、 プライマー 2の代わりにプライマ 一 1 2を用いた以外は pCDM8— F 1 0 ( S 1 0 6 ) の構築と同様の 2段階の P C R反応により目的の変位が導入された D N A断片を取得後、 これを 2種の制 限酵素 H i n dlHと Xb a Iで切断し、 pCDM8— hF 1 0 HXを同制限酵素 で消化し脱リン酸化したものとライゲートし、 大腸菌 MC 1 06 1 /p 3株を形 質転換することにより、 目的のァミノ酸置換を有する蛋白質を発現可能なブラス ミ ド p CDM8— F i 0 (C 8 4) を取得した。  Except for using Primer 11 in place of Primer 1 and Primer 12 in place of Primer 2, the two-step PCR reaction similar to the construction of pCDM8-F10 (S106) produced the desired displacement. After obtaining the introduced DNA fragment, it is cleaved with two restriction enzymes HindlH and XbaI, ligated with pCDM8-hF10HX digested with the same restriction enzyme and dephosphorylated, By transforming the Escherichia coli MC1061 / p3 strain, a plasmid pCDM8-Fi0 (C84) capable of expressing a protein having the desired amino acid substitution was obtained.
p CDM 8 -F 1 0 (S 1 50/S 1 0 6) の構築  Construction of p CDM 8 -F 1 0 (S 150 / S 106)
p CDM8 -F 1 0 (S 1 06) を H i n d ΠΙおよび Mv a Iで消化すること により S 1 0 6変異を含む約 3 4 0 b pの DNA断片を取得した。 また p CDM 8 - F 1 0 (S 1 5 0) を Mv a lおよび Xb a Iで消化することにより S 1 5 0変異を含む約 320 b pの DNA断片を取得した。 これら 2種の DNA断片を p CDM8 -h F 1 0 HXを制限酵素 H i n dfflおよび Xb a Iで消化し脱リン 酸化したものとライゲ一卜し、 大腸菌 MC 1 06 1/p 3株を形質転換すること により、 目的のアミノ酸置換を有する蛋白質を発現可能ナプラスミ ド P CDM8 -F 1 0 (S 1 50/S 1 06) を取得した。 By digesting pCDM8-F10 (S106) with HindIII and MvaI, a DNA fragment of about 340 bp containing the S106 mutation was obtained. In addition, p CDM 8-F 10 (S 150) was digested with Mval and XbaI to give S 15 An approximately 320 bp DNA fragment containing 0 mutation was obtained. These two DNA fragments were ligated with pCDM8-hF10HX digested with restriction enzymes Hindffl and XbaI and dephosphorylated, and transformed into E. coli MC1061 / p3 strain. By the conversion, naplasmid PCDM8-F10 (S150 / S106) capable of expressing a protein having the desired amino acid substitution was obtained.
p CDM 8 -F 1 0 (C 8 4/S 1 06) の構築  Construction of p CDM 8 -F 10 (C 84 / S 106)
p CDM8— F 1 0 (C 84) を H i n dfflおよび S c a Iで消化することに より C 8 4変異を含む約 270 b pの DNA断片を取得した。 また p CDM8 - F 1 0 (S 1 06) を S e a lおよび Xb a Iで消化することにより S 1 0 6変 異を含む約 3 9 0 b pの DN A断片を取得した。 これら 2種の DNA断片を PCDM8— hF 1 0^1 を制限酵素11 i 110111ぉょび & & Iで消ィヒし脱リン 酸化したものとライゲートし、 大腸菌 MC 1 06 lZp 3株を形質転換すること により、 目的のアミノ酸置換を有する蛋白質を発現可能なプラスミ ド PCDM8 — F 1 0 (C 84/S 1 06 ) を取得した。  By digesting pCDM8-F10 (C84) with Hindffl and ScaI, an approximately 270 bp DNA fragment containing the C84 mutation was obtained. In addition, pCDM8-F10 (S106) was digested with Seal and XbaI to obtain a DNA fragment of about 390 bp containing the S106 mutation. These two DNA fragments were ligated with PCDM8-hF10 ^ 1 which had been dephosphorylated and digested with restriction enzymes 11i110111 and && I, and transformed into three Escherichia coli MC106 lZp3 strains. As a result, a plasmid PCDM8-F10 (C84 / S106) capable of expressing a protein having the desired amino acid substitution was obtained.
p CDM8 -F 1 0 ( A 5 1 Z A 1 96 ) の構築  Construction of p CDM8 -F10 (A51ZA196)
p CDM8 -F 1 0 (A 5 1 ) を H i n d mおよび S c a Iで消化することに より A 5 1変異を含む約 27 0 b pの DNA断片を取得した。 また p C DM 8— F 1 0 (A 1 96) を S e a lおよび Xb a Iで消化することにより A 1 9 6変 異を含む約 3 9 0 b pの DNA断片を取得した。 これら 2種の DN A断片を p CDM8 - h F 1 01 を制限酵素11 i 11 (3111ぉょび & Iで消化し脱リン 酸化したものとライゲ一卜し、 大腸菌 MC 1 06 1/p 3株を形質転換すること により、 目的のアミノ酸置換を有する蛋白質を発現可能なプラスミ ド PCDM8 — F 1 0 (A 5 1 ZA 1 96) を取得した。  Approximately 270 bp DNA fragment containing the A51 mutation was obtained by digesting pCDM8-F10 (A51) with Hindm and ScaI. Also, pCDM8-F10 (A196) was digested with Seal and XbaI to obtain a DNA fragment of about 390 bp containing the A196 mutation. These two DNA fragments were ligated with pCDM8-hF101 digested with restriction enzymes 11i11 (3111 and & I) and dephosphorylated, and E. coli MC1061 / p3 By transforming the strain, a plasmid PCDM8-F10 (A51ZA196) capable of expressing a protein having the desired amino acid substitution was obtained.
p CDM8 -F 1 0 (DN 1 ) の構築 （図 9 B)  Construction of p CDM8 -F 10 (DN 1) (Fig. 9B)
欠失させる領域をはさむ外側の配列に相補的な配列を有するプライマー 1 3お よびプライマー 1 3に相捕的なプライマー 1 4を作成した。 プラスミ ド p CDM 8 -hF 1 0HXを铸型とし、 プライマ一 1 3とプライマー 3を用いた PC R反 応により、 DN A断片 4を取得した。 これとは別に同铸型に対しプライマ一 1 4 とプライマ一 4を用いた PC R反応により、 DN A断片 5を取得した。 次に、 こ のようにして取得された DN A断片 4と DN A断片 5を铸型とし、 プライマ一 3 とプライマ一 4を用いた P CR反応を行なうことにより、 目的の欠失が導入され た DN A断片 6を取得した。 その後、 DNA断片 6を 2種の制限酵素 H i n dill と Xb a Iで切断し、 p CDM8— hF 1 0 H Xを同制限酵素で消化し脱リン酸 ィヒしたものとライゲートし、 大腸菌 MC 1 06 l/p 3株を形質転換することに より、 目的のアミノ酸置換を有する蛋白質を発現可能なプラスミ ド P CDM8— F 1 0 (DN 1 ) を取得した。 Primer 13 having a sequence complementary to the outer sequence sandwiching the region to be deleted and primer 14 complementary to primer 13 were prepared. Plasmid pCDM8-hF10HX was used as type II, and DNA fragment 4 was obtained by PCR reaction using primer 13 and primer 3. Separately, a DNA fragment 5 was obtained from the same type by a PCR reaction using primers 14 and 14. Next, The DNA fragments 4 and 5 obtained as described above were used as type I, and a PCR reaction was performed using primers 13 and 14 to obtain the DNA fragment into which the desired deletion was introduced. Acquired 6. Thereafter, DNA fragment 6 was digested with two kinds of restriction enzymes Hindill and XbaI, and pCDM8-hF10HX was digested with the same restriction enzymes and ligated with dephosphorylated E. coli MC1. By transforming the 06 l / p3 strain, a plasmid PCDM8-F10 (DN1) capable of expressing a protein having the desired amino acid substitution was obtained.
p CDM8 -F 10 (DN 2) の構築  Construction of p CDM8 -F 10 (DN 2)
プライマ一 1 3の代わりにプライマー 1 5を、 プライマ一 1 4の代わりにブラ イマー 1 6を用いた以外は PCDM8— F 1 0 (DN 1 ) の構築と同様の 2段階 の P C R反応により目的の欠失が導入された DN A断片を取得後、 これを 2種の 制限酵素 H i n dmと Xb a 1で切断し、 pCDM8—hF 1 0 HXを同制限酵 素で消化し脱リン酸化したものとライゲ一トし、 大腸菌 MC 1 06 1 /p 3株を 形質転換することにより、 目的のァミノ酸置換を有する蛋白質を発現可能なブラ スミ ド p CDM8— F 1 0 (DN 2) を取得した。  Primer 15 was used in place of primer 13 and primer 16 was used in place of primer 14 In the same two-step PCR reaction as in the construction of PCDM8-F10 (DN1), After obtaining the DNA fragment into which the deletion was introduced, the DNA fragment was digested with two restriction enzymes Hindm and Xba1, digested with pCDM8-hF10HX and dephosphorylated by the same restriction enzyme. By transforming E. coli MC1061 / p3 strain, a plasmid p CDM8-F10 (DN2) capable of expressing a protein having the desired amino acid substitution was obtained. .
p CDM8 -F 1 0 (DN 3 ) の構築  Construction of p CDM8 -F 10 (DN 3)
プライマ一 1 3の代わりにプライマ一 1 7を、 プライマ一 1 4の代わりにプラ イマ一 1 8を用いた以外は p CDM8— F 1 0 (DN 1 ) の構築と同様の 2段階 の P C R反応により目的の欠失が導入された DN A断片を取得後、 これを 2種の 制限酵素 H i n dmと Xb a Iで切断し、 p C DM 8— h F 1 0 H Xを同制限酵 素で消化し脱リン酸化したものとライゲートし、 大腸菌 MC 1 0 6 1/p 3株を 形質転換することにより、 目的のァミノ酸置換を有する蛋白質を発現可能なブラ スミ ド じ01^8—？ 1 0 (DN 3) を取得した。  A two-step PCR reaction similar to the construction of pCDM8-F10 (DN1) except that primer 17 was used instead of primer 13 and primer 18 was used instead of primer 14 After obtaining the DNA fragment into which the desired deletion has been introduced, the DNA fragment is cleaved with two types of restriction enzymes, Hindm and XbaI, and pCDM8-hF10HX is digested with the same restriction enzymes. By ligating the digested and dephosphorylated product and transforming Escherichia coli MC1061 / p3 strain, a plasmid capable of expressing the protein having the desired amino acid substitution is 01 ^ 8-? 1 0 (DN 3) was obtained.
p CDM8 - F 1 0 (DN 4 ) の構築  Construction of p CDM8-F 10 (DN 4)
プライマ一 1 3の代わりにプライマ一 1 9を、 プライマ一 1 4の代わりにブラ イマ一 2 0を用いた以外は p CDM8— F 1 0 (DN 1 ) の構築と同様の 2段階 の P C R反応により目的の欠失が導入された D N A断片を取得後、 これを 2種の 制限酵素 H i ndinと Xb a Iで切断し、 p CDM8— hF 1 0 HXを同制限酵 素で消化し脱リン酸化したものとライゲ一卜し、 大腸菌 MC 1 0 6 1 Zp 3株を 形質転換することにより、 目的のアミノ酸置換を有する蛋白質を発現可能なブラ スミ ド pC DM 8— F 1 0 (DN 4) を取得した。 A two-step PCR reaction similar to the construction of pCDM8-F10 (DN1), except that primer 19 was used instead of primer 13 and primer 20 was used instead of primer 14. After obtaining the DNA fragment into which the desired deletion has been introduced, the DNA fragment is digested with the two restriction enzymes Hindin and XbaI, digested with pCDM8-hF10HX, and dephosphorylated. Ligated with the oxidized one to transform E. coli MC1061 Zp3 By transformation, a plasmid pCDM8-F10 (DN4) capable of expressing a protein having the desired amino acid substitution was obtained.
p CDM8— F 1 0 (DN 5) の構築  p CDM8—construct F 10 (DN 5)
プライマー 1 3の代わりにプライマ一 2 1を、 プライマ一 1 4の代わりにブラ イマ一 22を用いた以外は pCDM8— F 1 0 (D N 1 ) の構築と同様の 2段階 の PCR反応により目的の欠失が導入された D N A断片を取得後、 これを 2種の 制限酵素 H i n d DIおよび Xb a Iで切断し、 p CDM8— h F 1 0 HXを同制 限酵素で消化し脱リン酸化したものとライゲ一トし、 大腸菌 MC 1 06 1/p 3 株を形質転換することにより、 目的のアミノ酸置換を有する蛋白質を発現可能な プラスミ ド p CDM8— F 1 0 (DN 5) を取得した。  The same two-step PCR reaction as in the construction of pCDM8-F10 (DN1) was performed except that primer 21 was used instead of primer 13 and primer 22 was used instead of primer 14 After obtaining the DNA fragment into which the deletion was introduced, the DNA fragment was digested with two restriction enzymes, Hind DI and XbaI, and pCDM8-hF10HX was digested with the same restriction enzyme and dephosphorylated. The plasmid pCDM8-F10 (DN5) capable of expressing a protein having the amino acid substitution of interest was obtained by transforming E. coli MC1061 / p3 strain.
p CDM8 -F 1 0 (DC 1) の構築 （図 9 C)  Construction of p CDM8 -F10 (DC1) (Fig. 9C)
1 96位の A s nをコードするコドンが終始コドンに置換された塩基配列を有 しかつ Xb a Iを認識配列を有するプライマ一 2 3を作成した。 プラスミ ド p CDM8 -h F 1 0 HXを铸型とし、 プライマ一 1 3とプライマ一 4を用いた PCR反応により、 DN A断片 7を取得した。 次に、 このようにして取得された DN A断片 7を 2種の制限酵素 H i n dmおよび Xb a Iで切断し、 p CDM8 -hF 1 0 HXを同制限酵素で消化し脱リン酸化したものとライゲ一トし、 大腸 菌 MC 1 06 1 /p 3株を形質転換することにより、 目的のァミノ酸置換を有す る蛋白質を発現可能なプラスミ ド PCDM8— F 1 0 (DC 1 ) を取得した。 p CDM8 -F 1 0 (DC 2) の構築  Primer 123 having a nucleotide sequence in which the codon encoding Asn at position 196 was replaced with a stop codon and having a recognition sequence for XbaI was prepared. Plasmid p CDM8 -hF10HX was used as type I, and a DNA fragment 7 was obtained by a PCR reaction using primers 13 and 14. Next, the DNA fragment 7 obtained in this manner was digested with two types of restriction enzymes Hindm and XbaI, and pCDM8-hF10HX was digested with the same restriction enzymes and dephosphorylated. By transforming Escherichia coli MC1061 / p3 strain, a plasmid PCDM8-F10 (DC1) capable of expressing a protein having the desired amino acid substitution was obtained. did. Construction of p CDM8 -F 1 0 (DC 2)
プライマ一 2 3の代わりにプライマ一 24を用いた以外は p CDM8— F 1 0 (DC 1) の構築と同様の 1段階の PCR反応により目的の欠失が導入された D N A断片を取得後、 これを 2種の制限酵素 H i n dmと Xb a Iで切断し、 pC DM8 -hF 1 0 HXを同制限酵素で消化し脱リン酸化したものとライゲートし、 大腸菌 MC 1 06 1/p 3株を形質転換することにより、 目的のアミノ酸置換を 有する蛋白質を発現可能なプラスミ ド PCDM8— F 1 0 (DC 2) を取得した c p CDM8 -F 1 0 (DC 3) の構築 After obtaining a DNA fragment into which the desired deletion has been introduced by a one-step PCR reaction similar to the construction of pCDM8-F10 (DC1) except that primer 24 was used instead of primer 23, This was cut with two kinds of restriction enzymes, Hindm and XbaI, and ligated with pCDM8-hF10HX digested with the same restriction enzyme and dephosphorylated, and E. coli MC1061 / p3 the by transformation, construction of c p CDM8 -F 1 0 obtained protein capable of expressing plasmid PCDM8- F 1 0 (DC 2) with amino acid substitutions of interest (DC 3)
プライマ一 2 3の代わりにプライマ一 25を用いた以外は p C DM 8— F 1 0 (DC 1 ) の構築と同様の 1段階の PCR反応により目的の欠失が導入された D N A断片を取得後、 これを 2種の制限酵素 H i n dfflと Xb a Iで切断し、 pC DM8—h F 1 0 HXを同制限酵素で消化し脱リン酸化したものとライゲ一トし、 大腸菌 MC 1 0 6 1 /p 3株を形質転換することにより、 目的のァミノ酸置換を 有する蛋白質を発現可能なプラスミ ド p CDM8— F 1 0 (DC 3) を取得した。 The target deletion was introduced by a single-step PCR reaction similar to the construction of pCDM8-F10 (DC1) except that primer 25 was used instead of primer 23 D After obtaining the NA fragment, it was cut with two kinds of restriction enzymes, Hindfl and XbaI, and ligated with pCDM8-hF10HX digested with the same restriction enzyme and dephosphorylated, By transforming the E. coli MC1061 / p3 strain, a plasmid pCDM8-F10 (DC3) capable of expressing a protein having the desired amino acid substitution was obtained.
p CDM 8— F 1 0 (DC 4) の構築  Construction of p CDM 8—F 10 (DC 4)
プライマ一 2 3の代わりにプライマ一 2 6を用いた以外は p CDM8— F 1 0 (DC 1 ) の構築と同様の 1段階の PC R反応により目的の欠失が導入された D N A断片を取得後、 これを 2種の制限酵素 H i n dmおよび Xb a Iで切断し、 P CDM8-hF 1 0 HXを同制限酵素で消化し脱リン酸化したものとライゲ一 卜し、 大腸菌 MC 1 0 6 I ZP 3株を形質転換することにより、 目的のアミノ酸 置換を有する蛋白質を発現可能なプラスミ ド PCDM8— F 1 0 (DC 4) を取 得した。 A DNA fragment containing the target deletion was obtained by a single-step PCR reaction similar to the construction of pCDM8-F10 (DC1) except that primer 126 was used instead of primer 23. after this was digested with two restriction enzymes H in dm and Xb a I, P CDM8-hF 1 0 the HX was digested with the same restriction enzymes were those with Raige one to Bok dephosphorylated, E. coli MC 1 0 6 By transforming the IZP3 strain, a plasmid PCDM8-F10 (DC4) capable of expressing a protein having the desired amino acid substitution was obtained.
p CDM8 -F 1 0 (SA) の構築 （図 9 D)  Construction of p CDM8 -F10 (SA) (Fig. 9D)
変異を含む 4本のオリゴヌクレオチド （S A 1 F， S A 1 R, SA 2 F, SA 2 R) を作製した。 これらのオリゴヌクレオチドの 5' 末端をリン酸化後、 SA Four oligonucleotides containing mutations (SAIF, SAIR, SA2F, SA2R) were prepared. After phosphorylation of the 5 'end of these oligonucleotides, SA
1 Fと S A 1 Rあるいは S A 2 Fと S A 2 Rをそれぞれァニールし、 S A 1 F/Anneal 1 F and S A 1 R or S A 2 F and S A 2 R respectively to obtain S A 1 F /
SA 1 R断片および S A 2 F/S A 2 R断片を得た。 これら 2種の断片を H i n d HIおよび B s t X 1で消化し、 p CDM 8— h F 1 0 HXを同制限酵素で消化 し脱リン酸化したものとライゲ一トし、 大腸菌 MC 1 0 6 1 p 3株を形質転換 することにより、 目的のアミノ酸置換を有する蛋白質を発現可能なプラスミ ド PThe SA1R fragment and the SA2F / SA2R fragment were obtained. These two fragments were digested with Hind HI and BstX1, pCDM8—hF10HX was digested with the same restriction enzymes and ligated with dephosphorylated, and E. coli MC106. Plasmid P capable of expressing a protein having the desired amino acid substitution by transforming 1p3 strain
CDM8 -F 1 0 (SA) を取得した。 CDM8 -F10 (SA) was obtained.
5' -→ 3'  5 '-→ 3'
プライマ一 1 GMGGAGMCAGCCCGTACAGC ATCCTG  Primer 1 GMGGAGMCAGCCCGTACAGC ATCCTG
プライマ一 2 TGCTGTACGGGCTGTTCTCCTTCTTGGT  Primer 2 TGCTGTACGGGCTGTTCTCCTTCTTGGT
プライマ一 3 GATCCTCTAGAAGAAACGGT  Primer 1 GATCCTCTAGAAGAAACGGT
プライマー 4 GTAATACGACTCACTATAGGGC  Primer 4 GTAATACGACTCACTATAGGGC
プライマ一 5 TAACAATGACAGTAAGCTGAAGGAGAGG  Primer 1 TAACAATGACAGTAAGCTGAAGGAGAGG
プライマ一 6 CCTTCAGCTTACTGTCATTGTTAAATTC  Primer 6 CCTTCAGCTTACTGTCATTGTTAAATTC
プライマ一 7 AGAGGCCACCGCCTCTTCTTCCTCCTCC プライマ― 8 AGGAAGAAGAGGCGGTGGCCTCTGGTGA Primer 7 AGAGGCCACCGCCTCTTCTTCCTCCTCC Primer 8 AGGAAGAAGAGGCGGTGGCCTCTGGTGA
プライマ― 9 ACGAAGGAAAGCCACCTCTGCTCACTTT Primer 9 ACGAAGGAAAGCCACCTCTGCTCACTTT
プライマ― 1 0 GAGCAGAGGTGGCTTTCCTTCGTGnn Primer 10 GAGCAGAGGTGGCTTTCCTTCGTGnn
プライマ― 1 1 AAAGCTATTCTGTTTCACCAAGTACTn Primer 1 1 AAAGCTATTCTGTTTCACCAAGTACTn
プライマ― 1 2 ACTTGGTGAAACAGAATAGCrrTCTCCA Primer 1 2 ACTTGGTGAAACAGAATAGCrrTCTCCA
プライマ一 1 3 CTGCCAAGCCAACTCTTCTTCCTCCTCCH Primer 1 3 CTGCCAAGCCAACTCTTCTTCCTCCTCCH
プライマ― 1 4 AAGAAGAGTTGGCnGGCAGGTGACAGGGA Primer 1 4 AAGAAGAGTTGGCnGGCAGGTGACAGGGA
プライマ― 1 5 CTGCCAAGCCGCGGGAAGGCATGTGCGGAG Primer 1 5 CTGCCAAGCCGCGGGAAGGCATGTGCGGAG
プライマ一 1 6 GCCTTCCCGCGGCTTGGCAGGTGACAGGGA Primer 1 6 GCCTTCCCGCGGCTTGGCAGGTGACAGGGA
プライマ一 1 7 CTGCCAAGCCCTATTCTCTTTCACCAAGTA Primer 1 7 CTGCCAAGCCCTATTCTCTTTCACCAAGTA
プライマ― 1 8 AAGAGAATAGGGCTTGGCAGGTGACAGGGA Primer 18 AAGAGAATAGGGCTTGGCAGGTGACAGGGA
プライマ― 1 9 CTGCCAAGCCGAGAACTGCCCGTACAGCAT Primer 1 9 CTGCCAAGCCGAGAACTGCCCGTACAGCAT
プライマ― 2 0 GGCAGTTCTCGGCTTGGCAGGTGACAGGGA Primer 20 GGCAGTTCTCGGCTTGGCAGGTGACAGGGA
プライマ― 2 1 CTGCCAAGCCGGGAAACTCTATGGCTCAAA Primer 2 1 CTGCCAAGCCGGGAAACTCTATGGCTCAAA
プライマ - 2 2 AGAGTTTCCCGGCTTGGCAGGTGACAGGGA Primer-2 2 AGAGTTTCCCGGCTTGGCAGGTGACAGGGA
プライマ - 2 3 TTTTCTAGACTATTTCCnCGTGTTTT Primer-2 3 TTTTCTAGACTATTTCCnCGTGTTTT
プライマ一 2 4 TTTTCTAGACTATCTCCTTGGAGCTCC Primer 2 4 TTTTCTAGACTATCTCCTTGGAGCTCC
プライマ― 2 5 TTTTCTAGACTACTTCAGCTTACAGTC Primer 25 TTTTCTAGACTACTTCAGCTTACAGTC
プライマ - 2 6 TTTTCTAGACTACTTCTTGTTCATGGC Primer-2 6 TTTTCTAGACTACTTCTTGTTCATGGC
S A 1 F AGCTTCCACCATGTGGAAATGGATACTGACACATGCTGCCTCAGCCTTTCCTCA  S A 1 F AGCTTCCACCATGTGGAAATGGATACTGACACATGCTGCCTCAGCCTTTCCTCA
CCTGCCTGGCGCTG CCTGCCTGGCGCTG
S A 1 R CAGGTGAGGAAAGGCTGAGGCAGCATGTGTCAGTATCCATTTCCACATGGTGGA S A 2 F CTGCCGCTGCCTTTTTGTTGCTGTTCTTGGTGTCTTCCGTACCTGTCACCTGCC S A 1 R CAGGTGAGGAAAGGCTGAGGCAGCATGTGTCAGTATCCATTTCCACATGGTGGA S A 2 F CTGCCGCTGCCTTTTTGTTGCTGTTCTTGGTGTCTTCCGTACCTGTCACCTGCC
AAGCCC S A 2 R TTGGCAGGTGACAGGTACGGAAGACACCAAGAACAGCAACAAAAAGGCAGCGGC  AAGCCC S A 2 R TTGGCAGGTGACAGGTACGGAAGACACCAAGAACAGCAACAAAAAGGCAGCGGC
AGCAGCGCCAGG AGCAGCGCCAGG
F G F 1 0改変体を発現しうるプラスミ ドによる C O S— 1細胞の形質転換 上記 1 7種の F G F 1 0改変体を発現可能なプラミ ド D N Aを常法に従い大量 に調製し、 塩化セシウム密度勾配超遠心を 2回行って精製した。 これら 1 7種の プラスミ ドおよびコントロールとして pC DM 8 - h F 1 0 HXおよび pCDM 8を用いて、 電気パルス法により COS - 1細胞の形質転換を行った。 形質転換 された細胞を、 1 0%の牛胎児血清を含有する DMEMあるいは I MEM培地で 24時間培養後、 培地を無血清で 1 0 gZm 1のへパリンを含有する I MEM 培地に交換し、 さらに培養を継続した。 培地交換後 96時間後に培養液を遠心後、 その上清を回収した。 このようにして得られた培養上清原液ならびにそれを 1 0 U g/m 1のへパリンを含有する I MEM培地で 1 0倍希釈した希釈サンプルを 分注し、 一 8 0°Cで凍結保存した。 Transformation of COS-1 cells with a plasmid capable of expressing the FGF10 variant Prepare a large amount of plasmid DNA capable of expressing the above 17 types of FGF10 variants in a conventional manner, and use a cesium chloride density gradient. Purification was performed by performing centrifugation twice. These 1 7 kinds COS-1 cells were transformed by an electric pulse method using pCDM8-hF10HX and pCDM8 as a plasmid and a control. After the transformed cells are cultured in DMEM or IMEM medium containing 10% fetal calf serum for 24 hours, the medium is replaced with serum-free IMEM medium containing 10 gZm1 of heparin. Further culturing was continued. 96 hours after medium replacement, the culture was centrifuged, and the supernatant was collected. Dispense the undiluted culture supernatant obtained in this way and a diluted sample obtained by diluting it 10 times with IMEM medium containing 10 Ug / m1 heparin and freeze at 180 ° C. saved.
FRS K細胞を用いた細胞増殖促進活性の検討  Examination of cell growth promoting activity using FRS K cells
結果：図 1 0に示すように、 FGF 1 0改変体 （DN 1， DN 2, DC 2, S A, A 5 1 , A 1 9 6 , S 1 50 , S 1 06, C 84 / S 1 06, S 1 50/S 1 06及び A 5 1/A 1 96) を発現可能なプラスミ ドを用いた得られた培養上 清はいずれも p C DM 8を用いた得られた培養上清と比較して明らかなトリチウ ム標識チミジンの取り込みの促進作用を示した。  Results: As shown in FIG. 10, the FGF10 variant (DN1, DN2, DC2, SA, A51, A196, S150, S106, C84 / S106) , S150 / S106 and A51 / A196) were compared with the culture supernatant obtained using pCDM8. As a result, it clearly showed the action of promoting the uptake of tritium-labeled thymidine.
産業上の利用の可能性  Industrial applicability
以上の結果から明らかなごとく、 ヒト FGF— 1 0は、 優れた骨 Z軟骨組織新 生ないし再生作用を有し、 骨 Z軟骨組織疾患の治療に有用であることを見い出し た。 このことより、 本発明の骨/軟骨疾患治療剤は、 たとえば （ 1 ) 変形性関節 症、 慢性関節リウマチ等自己免疫疾患に伴う関節炎により生じた軟骨欠損の修復、 (2) 外傷による软骨欠損、 離断性骨軟骨炎により生じた軟骨欠損の修復、 骨を温存する骨きり術時の軟骨形成促進、 （ 5 ) 骨折後の修復、 （ 6 ) 骨欠損後 の修復、 （7) 骨粗鬆症等で局所的に骨減少が認められた部位の骨形成促進等に 有用であり、 各種骨/軟骨組織疾患治療に用いられる。  As is clear from the above results, it has been found that human FGF-10 has an excellent bone Z cartilage tissue regeneration or regeneration action, and is useful for treating bone Z cartilage tissue diseases. Thus, the therapeutic agent for bone / cartilage disease of the present invention can be used, for example, to repair (1) repair of cartilage defects caused by arthritis associated with autoimmune diseases such as osteoarthritis and rheumatoid arthritis, (2) bone loss due to trauma, Repair of cartilage defects caused by osteochondritis dissecans, promotion of cartilage formation during bone preserving osteotomy, (5) repair after fracture, (6) repair after bone loss, (7) osteoporosis, etc. It is useful for promoting osteogenesis at sites where local bone loss has been observed, and is used for treatment of various bone / cartilage tissue diseases.
本発明は、 FGF— 1 0をコードする DNA、 当該 DN Aを担持した発現べク ター、 形質転換体、 それらを用いた組換え蛋白の製造方法、 および組換え蛋白を 提供すると共に、 この新規な因子を用いた医薬を提供するものである。 配列表 The present invention provides a DNA encoding FGF-10, an expression vector carrying the DNA, a transformant, a method for producing a recombinant protein using them, and a recombinant protein. The present invention provides a medicine using various factors. Sequence listing
配列番号： 1 SEQ ID NO: 1
配列の長さ ： 2 1 5 Array length: 2 1 5
配列の型：アミノ酸 Sequence type: amino acid
トポロジー：直鎖状  Topology: linear
配列の種類：ぺプチド Sequence type: peptide
起源 Origin
生物名：ラッ ト Organism name: Rat
配列 Array
Met Trp Lys Trp lie Leu Thr His Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15 Met Trp Lys Trp lie Leu Thr His Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15
Pro Gly Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser Val Pro Gly Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser Val
20 25 30  20 25 30
Pro Val Thr Cys Gin Ala Leu Gly Gin Asp Met Val Ser Pro Glu Ala  Pro Val Thr Cys Gin Ala Leu Gly Gin Asp Met Val Ser Pro Glu Ala
35 40 45  35 40 45
Thr Asn Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Phe Thr Asn Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Phe
50 55 60 50 55 60
Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr Asn His Leu 65 70 75 80 Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr Asn His Leu 65 70 75 80
Gin Gly Asp Val Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr Phe Gin Gly Asp Val Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr Phe
85 90 95 85 90 95
Leu Lys lie Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Leu Lys lie Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn
100 105 110  100 105 110
Cys Pro Tyr Ser lie Leu Glu He Thr Ser Val Glu lie Gly Val Val  Cys Pro Tyr Ser lie Leu Glu He Thr Ser Val Glu lie Gly Val Val
115 120 125  115 120 125
Ala Val Lys Ala lie Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys Ala Val Lys Ala lie Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys
130 135 140 130 135 140
Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys 145 150 155 160Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys 145 150 155 160
Glu Arg lie Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Glu Arg lie Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn Trp
165 170 175 165 170 175
Gin His Asn Gly Arg Gin Met Tyr Val Ala Leu Asn Gly Lys Gly Ala Gin His Asn Gly Arg Gin Met Tyr Val Ala Leu Asn Gly Lys Gly Ala
180 185 190  180 185 190
Pro Arg Arg Gly Gin Lys Thr Arg Arg Lys Asn Thr Ser Ala His Phe  Pro Arg Arg Gly Gin Lys Thr Arg Arg Lys Asn Thr Ser Ala His Phe
195 200 205  195 200 205
Leu Pro Met Val Val His Ser  Leu Pro Met Val Val His Ser
210 215 配列番号： 2  210 215 SEQ ID NO: 2
配列の長さ ： 2 0 8 Array length: 2 0 8
配列の型：アミノ酸 Sequence type: amino acid
トポロジー ：直鎖状  Topology: linear
配列の種類：ぺプチド Sequence type: peptide
起源 Origin
生物名： ヒ 卜 Organism name: human
配列 Array
Met Trp Lys Trp lie Leu Thr His Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15 Met Trp Lys Trp lie Leu Thr His Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15
Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser
20 25 30  20 25 30
Val Pro Val Thr Cys Gin Ala Leu Gly Gin Asp Met Val Ser Pro Glu  Val Pro Val Thr Cys Gin Ala Leu Gly Gin Asp Met Val Ser Pro Glu
35 40 45  35 40 45
Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly
50 55 60 50 55 60
Arg His Val Arg Ser Tyr Asn His Leu Gin Gly Asp Val Arg Trp Arg 65 70 75 80 Arg His Val Arg Ser Tyr Asn His Leu Gin Gly Asp Val Arg Trp Arg 65 70 75 80
Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly 85 90 95 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys He Glu Lys Asn Gly 85 90 95
Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser lie Leu Glu  Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser lie Leu Glu
100 105 110  100 105 110
He Thr Ser Val Glu lie Gly Val Val Ala Val Lys Ala lie Asn Ser  He Thr Ser Val Glu lie Gly Val Val Ala Val Lys Ala lie Asn Ser
115 120 125  115 120 125
Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys  Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys
130 135 140  130 135 140
Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg lie Glu Glu Asn Gly  Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg lie Glu Glu Glu Asn Gly
145 150 155 160 145 150 155 160
Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gin His Asn Gly Arg Gin Met  Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gin His Asn Gly Arg Gin Met
165 170 175  165 170 175
Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gin Lys Thr  Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gin Lys Thr
180 185 190  180 185 190
Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser  Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser
195 200 205 配列番号： 3  195 200 205 SEQ ID NO: 3
配列の長さ ： 8 0 4 b p Array length: 8 0 4 b p
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状  Topology: linear
配列の種類： c DNA Sequence type: cDNA
起源 Origin
生物名： ラッ ト Organism name: Rat
存在位置： 1 0 9 - 7 5 3 Location: 1 0 9-7 5 3
特徴を決定した方法： E How the features were determined: E
配列 Array
TAACCAGTAG CCATCACCTC CAGCTGTCTC TTTGCCTCGC ACCAGGTCTT ACCCTTCCAG 60 TATGTTCCTT CTGATGAGAC AATTTCCAGT GCCGAGAGTT TCAGTACA ATG TGG AAG 117 TGG ATA CTG ACA CAT TGT GCC TCA GCC TTT CCC CAC CTG CCG GGC TGC 165 TGT TGC TGC TTC TTG TTG CTC TTC TTG GTG TCT TCC GTC CCT GTC ACC 213 TGC CAA GCT CTT GGT CAG GAC ATG GTG TCA CCG GAG GCT ACC AAC TCC 261 TCT TCC TCC TCC TCT TCC TCC TCC TCG TCC TCT TCC TTC TCC TCT CCT 309 TCC AGC GCG GGG AGG CAT GTG CGG AGC TAC AAT CAC CTC CAG GGA GAT 357 GTC CGC TGG AGA AAG CTG TTC TCC TTC ACC AAG TAC TTT CTC AAG ATT 405 GAA AAG AAC GGC AAG GTC AGC GGG ACC AAG AAG GAA AAC TGT CCG TAC 453 AGT ATC CTA GAG ATA ACA TCA CTG GAA ATC GGA GTT GTT GCC GTC AAA 501 GCC ATT AAC AGC AAC TAT TAC TTA GCC ATG AAC AAG AAG GGG AAA CTC 549 TAT GGC TCA AAA GAA TTT AAC AAT GAC TGT AAA CTG AAA GAG AGG ATA 597 GAG GAA ΑΛΤ GGA TAC AAC ACC TAT GCA TCT TTT AAC TGG CAG CAC AAC 645 GGC AGG CAA ATG TAT GTG GCA TTG AAT GGA AAA GGA GCT CCC AGG AGA 693 GGA CAA AAA ACA AGA AGG AAA AAC ACC TCC GCT CAC TTC CTC CCC ATG 741 GTG GTC CAC TCA TAGAAGA AGGCACCGTT GGTGGATGCA GTACAACCAA TGACTCTTTG 800 CCAA 配列番号： 4 TAACCAGTAG CCATCACCTC CAGCTGTCTC TTTGCCTCGC ACCAGGTCTT ACCCTTCCAG 60 TATGTTCCTT CTGATGAGAC AATTTCCAGT GCCGAGAGTT TCAGTACA ATG TGG AAG 117 TGG ATA CTG ACA CAT TGT GCC TCA GCC TTT CCC CAC CTG CCG GGC TGC 165 TGT TGC TGC TTC TTG TTG CTC TTC TTG GTG TCT TCC GTC CCT GTC ACC 213 TGC CAA GCT CTT GGT CAG GAC ATG GTG TCA CCG GAG GCC ACC AAC 261 TCT TCC TCC TCC TCT TCC TCC TCC TCG TCC TCT TCC TTC TCC TCT CCT 309 TCC AGC GCG GGG AGG CAT GTG CGG AGC TAC AAT CAC CTC CAG GGA GAT 357 GTC CGC TGG AGA AAG CTG TTC TCC TTC ACC AAG TAC TTC ATT 405 GAA AAG AAC GGC AAG GTC AGC GGG ACC AAG AAG GAA AAC TGT CCG TAC 453 AGT ATC CTA GAG ATA ACA TCA CTG GAA ATC GGA GTT GTT GCC GTC AAA 501 GCC ATT AAC AGC AAC TAT TAC TTA GCC ATG AAC AAG GAG AAA CTC 549 TAT GGC TCA AAA GAA TTT AAC AAT GAC TGT AAA CTG AAA GAG AGG ATA 597 GAG GAA ΑΛΤ GGA TAC AAC ACC TAT GCA TCT TTT AAC TGG CAG CAC AAC 645 GGC AGG CAA ATG TAT GTG GCA TTG AAT GGA AAA GGA GCT CCC AGG AGA 693 GGA CAA AAA ACA AGA AGG AAA AAC ACC TCC GCT CAC TTC CTC CCC ATG 741 GTG GTC CAC TCA TAGAAGA AGGCACCGTT GGTGGATGCA GTACAACCAA TGACTCTTTG 800 CCAA SEQ ID NO: 4
配列の長さ ： 6 9 0 b p Sequence length: 6 9 0 b p
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖  Number of chains: double strand
トポロジー：直鎖状  Topology: linear
配列の種類： c D N A  Sequence type: c D N A
起源  Origin
生物名： ヒ 卜  Organism name: human
配列  Array
CTTCCAGTAT GTTCCTTCTG ATGAGACAAT TTCCAGTGCC GAGAGTTCCA GTACA ATG 58 TGG AAA TGG ATA CTG ACA CAT TGT GCC TCA GCC TTT CCC CAC CTG CCC 106 GGC TGC TGC TGC TGC TGC TTT TTG TTG CTG TTC TTG GTG TCT TCC GTC 154 CCT GTC ACC TGC CAA GCC CTT GGT CAG GAC ATG GTG TCA CCA GAG GCC 202 ACC AAC TCT TCT TCC TCC TCC TTC TCC TCT CCT TCC AGC GCG GGA AGG 250CTTCCAGTAT GTTCCTTCTG ATGAGACAAT TTCCAGTGCC GAGAGTTCCA GTACA ATG 58 TGG AAA TGG ATA CTG ACA CAT TGT GCC TCA GCC TTT CCC CAC CTG CCC 106 GGC TGC TGC TGC TGC TGC TTT TTG TTG CTG TTC TTG GTG TCT TCC GTC GTC TTC GTC TTC GTC TTC GTC TTC GTC TTC GTC TTC GTC CAG GAC ATG GTG TCA CCA GAG GCC 202 ACC AAC TCT TCT TCC TCC TCC TTC TCC TCT CCT TCC AGC GCG GGA AGG 250
CAT GTG CGG AGC TAC AAT CAC CTl CAA GGA GAT GTC CGC TGG AGA AAG 298CAT GTG CGG AGC TAC AAT CAC CTl CAA GGA GAT GTC CGC TGG AGA AAG 298
CTA TTC TCT TTC ACC AAG TAC TTT CTC AAG ATT GAG AAG AAC GGG AAG 346CTA TTC TCT TTC ACC AAG TAC TTT CTC AAG ATT GAG AAG AAC GGG AAG 346
GTC AGC GGG ACC AAG AAG GAG AAC TGC CCG TAC AGC ATC CTG GAG ATA 394GTC AGC GGG ACC AAG AAG GAG AAC TGC CCG TAC AGC ATC CTG GAG ATA 394
ACA TCA GTA GAA ATC GGA GTT GTT GCC GTC AAA GCC ATT AAC AGC AAC 442ACA TCA GTA GAA ATC GGA GTT GTT GCC GTC AAA GCC ATT AAC AGC AAC 442
TAT TAC ΠΑ GCC ATG AAC AAG AAG GGG AAA CTC TAT GGC TCA AAA GAA 490TAT TAC ΠΑ GCC ATG AAC AAG AAG GGG AAA CTC TAT GGC TCA AAA GAA 490
TTT AAC AAT GAC TGT AAG CTG AAG GAG AGG ATA GAG GAA AAT GGA TAC 538TTT AAC AAT GAC TGT AAG CTG AAG GAG AGG ATA GAG GAA AAT GGA TAC 538
AAT ACC TAT GCA TCA TTT AAC TGG CAG CAT AAT GGG AGG CAA ATG TAT 586AAT ACC TAT GCA TCA TTT AAC TGG CAG CAT AAT GGG AGG CAA ATG TAT 586
GTG GCA TTG AAT GGA AAA GGA GCT CCA AGG AGA GGA CAG AAA ACA CGA 634GTG GCA TTG AAT GGA AAA GGA GCT CCA AGG AGA GGA CAG AAA ACA CGA 634
AGG AAA AAC ACC TCT GCT CAC TTT CTT CCA ATG GTG GTA CAC TCA TAGAG 684AGG AAA AAC ACC TCT GCT CAC TTT CTT CCA ATG GTG GTA CAC TCA TAGAG 684
GAAGGC 690 配列番号： 5 GAAGGC 690 SEQ ID NO: 5
配列の長さ ： 2 2 b p Array length: 2 2 b p
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー：直鎖状  Topology: linear
酉己歹リ： Rooster system:
GATGCATAGG TATTGTATCC AT 配列番号： 6  GATGCATAGG TATTGTATCC AT SEQ ID NO: 6
配列の長さ ： 2 1 b p Array length: 2 1 b p
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー ：直鎖状  Topology: linear
酉己歹 IJ： Tori self IJ:
TCCATTTTCC TCTATCCTCT C 配列番号： 7 配列の長さ ： 2 0 b p TCCATTTTCC TCTATCCTCT C SEQ ID NO: 7 Sequence length: 20 bp
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー：直鎖状  Topology: linear
配列： Array:
AGAAGGGGAA ACTCTATGGC 配列番号： 8  AGAAGGGGAA ACTCTATGGC SEQ ID NO: 8
配列の長さ ： 2 1 b p Array length: 2 1 b p
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー：直鎖状  Topology: linear
配列： Array:
GACTGTAAAC TGAAAGAGAG G 配列番号： 9  GACTGTAAAC TGAAAGAGAG G SEQ ID NO: 9
配列の長さ ： 3 2 b p Array length: 3 2 b p
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー：直鎖状  Topology: linear
配歹り： Distribution:
GCGAGCTCAA GCITTTTTTT TTTTTTTTTT TT 配列番号： 1 0  GCGAGCTCAA GCITTTTTTT TTTTTTTTTT TT SEQ ID NO: 10
配列の長さ ： 1 8 b p  Array length: 1 8 b p
配列の型：核酸  Sequence type: nucleic acid
鎖の数：一本鎖  Number of chains: single strand
トポロジー：直鎖状  Topology: linear
酉己歹 II： GCGAGCTCAA GCTTTTTT Tori self II: GCGAGCTCAA GCTTTTTT
配列番号： 1 1 SEQ ID NO: 1 1
配列の長さ ： 2 0 b p 配列の型：核酸 Sequence length: 20 bp Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー：直鎖状 配列：  Topology: linear sequence:
CHCCAGTAT CATCCTTCTG 配列番号： 1 2  CHCCAGTAT CATCCTTCTG SEQ ID NO: 1 2
配列の長さ ： 2 0 b p 配列の型：核酸 Sequence length: 20 bp Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー：直鎖状 酉己歹 IJ：  Topology: Straight-chain IJ:
GGCAAAGAGT CATTGGTTGT 配列番号： 1 3  GGCAAAGAGT CATTGGTTGT SEQ ID NO: 1 3
配列の長さ ： 2 2 b p 配列の型：核酸 Sequence length: 22bp Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー ：直鎖状 配列：  Topology: linear sequence:
GATGCATAGG TATTGTATCC AT 配列番号： 1 4  GATGCATAGG TATTGTATCC AT SEQ ID NO: 1 4
配列の長さ ： 2 2 b p 配列の型：核酸 鎖の数：一本鎖 Sequence length: 22 bp Sequence type: nucleic acid Number of chains: single strand
トポロジー：直鎖状  Topology: linear
配列： Array:
GAAACTCTAT GGCTCAAAAG AA  GAAACTCTAT GGCTCAAAAG AA
配列番号： 1 5 SEQ ID NO: 1 5
配列の長さ ： 2 0 b p Array length: 2 0 b p
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー ：直鎖状  Topology: linear
配列： Array:
GTACACTCAT AGAGGAAGGC 配列番号： 1 6  GTACACTCAT AGAGGAAGGC SEQ ID NO: 16
配列の長さ ： 3 4 b p Array length: 3 4 b p
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー：直鎖状  Topology: linear
配列： Array:
GGGAATTCCA TATGCTTGGT CAGGACATGG TGTC 配列番号： 1 7  GGGAATTCCA TATGCTTGGT CAGGACATGG TGTC SEQ ID NO: 17
配列の長さ ： 2 9 b p Array length: 2 9 b p
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー ：直鎖状  Topology: linear
酉己歹リ： Rooster system:
CGCGGATCCG CTATGCATGC AACGCGTTG  CGCGGATCCG CTATGCATGC AACGCGTTG

Claims

請 求 の 範 囲 The scope of the claims
1. 配列番号： 1 もしくは配列番号： 2に示すァミノ酸配列のポリぺプチドで ある線維芽細胞増殖因子をコ一ドする塩基配列、 またはこれに相補的な塩基配列 を包含する組換え D N A。 1. A recombinant DNA comprising a nucleotide sequence encoding fibroblast growth factor, which is a polypeptide of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence complementary thereto.
2. 配列番号： 3もしくは配列番号： 4に示す塩基配列またはこれに相捕的な 塩基配列を包含する請求の範囲 1の D N A。  2. The DNA of claim 1, which comprises the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence complementary thereto.
3. 請求の範囲 1の D N Aを担持する発現べクタ一。  3. An expression vector carrying the DNA of claim 1.
4. 請求の範囲 3の発現ベクターを宿主に導入して得られる形質転換体。  4. A transformant obtained by introducing the expression vector of claim 3 into a host.
5. 宿主が動物細胞または大腸菌である請求の範囲 4の形質転換体。  5. The transformant according to claim 4, wherein the host is an animal cell or Escherichia coli.
6. 請求の範囲 4の形質転換体を使用することを特徴とする組換え線維芽細胞 増殖因子の製造方法。  6. A method for producing a recombinant fibroblast growth factor, comprising using the transformant according to claim 4.
7. 配列番号： 1 もしくは配列番号： 2に示すァミノ酸配列またはその主要部 分を包含するポリぺプチドである組換え線維芽細胞増殖因子。  7. A recombinant fibroblast growth factor which is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a main part thereof.
8. 請求の範囲 5の形質転換体が生産し、 細胞増殖活性を示す事を特徴とする、 配列番号： 1 もしくは配列番号： 2に示すァミノ酸配列またはその主要部分を包 含するポリぺプチドである組換え線維芽細胞増殖因子。  8. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a main part thereof, which is produced by the transformant of claim 5 and exhibits a cell growth activity. A recombinant fibroblast growth factor.
9. 請求の範囲 7もしくは 8の組換え線維芽細胞増殖因子を有効成分として含 有する医薬。  9. A medicament comprising the recombinant fibroblast growth factor of claim 7 or 8 as an active ingredient.
1 0. 骨/軟骨疾患または骨ノ軟骨損傷治療剤である請求の範囲 9の医薬。  10. The medicament according to claim 9, which is a therapeutic agent for bone / cartilage disease or osteochondral damage.
1 1. 創傷治癒促進剤である請求の範囲 9の医薬。  1 1. The medicament according to claim 9, which is a wound healing promoter.
1 2. 骨/軟骨疾患または骨 Z軟骨損傷治療剤の製造のための請求の範囲 7もし くは 8の組換え線維芽細胞増殖因子の使用。  1 2. Use of the recombinant fibroblast growth factor of claim 7 or 8 for the manufacture of a therapeutic agent for bone / cartilage disease or bone-Z cartilage damage.
1 3. 創傷治癒促進剤の製造のための請求の範囲 7もしくは 8の組換え線維芽細 胞增殖因子の使用。  1 3. Use of the recombinant fibroblast growth factor of claim 7 or 8 for the manufacture of a wound healing promoter.
1 . 請求の範囲 7もしくは 8の組換え線維芽細胞増殖因子の有効量をヒ トを含 む動物に投与することを含む骨 Z軟骨疾患または骨 Z軟骨損傷の治療方法。  1. A method for treating bone Z cartilage disease or bone Z cartilage damage, which comprises administering an effective amount of the recombinant fibroblast growth factor of claim 7 or 8 to an animal containing human.
1 5. 請求の範囲 7もしくは 8の組換え線維芽細胞増殖因子の有効量をヒ トを含 む動物に投与することを含む創傷治癒促進方法。  1 5. A method for promoting wound healing, comprising administering an effective amount of the recombinant fibroblast growth factor of claim 7 or 8 to an animal containing human.
PCT/JP1996/003579 1995-12-07 1996-12-06 Fibroblast growth factor fgf-10 WO1997020929A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU10412/97A AU1041297A (en) 1995-12-07 1996-12-06 Fibroblast growth factor fgf-10

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP7/345689 1995-12-07
JP34568995 1995-12-07
JP10324096 1996-03-28
JP8/103240 1996-03-28
JP21437896 1996-07-24
JP8/214378 1996-07-24

Publications (1)

Publication Number Publication Date
WO1997020929A1 true WO1997020929A1 (en) 1997-06-12

Family

ID=27309932

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1996/003579 WO1997020929A1 (en) 1995-12-07 1996-12-06 Fibroblast growth factor fgf-10

Country Status (2)

Country Link
AU (1) AU1041297A (en)
WO (1) WO1997020929A1 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6077692A (en) * 1995-02-14 2000-06-20 Human Genome Sciences, Inc. Keratinocyte growth factor-2
US6238888B1 (en) 1997-12-22 2001-05-29 Human Genone Sciences, Inc. Keratinocyte growth factor-2 formulations
WO2001064915A1 (en) * 2000-03-02 2001-09-07 Biowindow Gene Development Inc. Shanghai Novel polypeptide---the factor 1-25 being homology with human fibroblast growth factor and polynucleotide encoding it
US6693077B1 (en) 1995-02-14 2004-02-17 Human Genome Sciences, Inc. Keratinocyte growth factor-2
US6743422B1 (en) 1996-10-15 2004-06-01 Amgen, Inc. Keratinocyte growth factor-2 products
US6869927B1 (en) 1997-12-22 2005-03-22 Human Genome Sciences, Inc. Keratinocyte growth factor-2 formulations
US7232667B2 (en) 1995-02-14 2007-06-19 Human Genome Sciences, Inc. Keratinocyte growth factor-2 polynucleotides

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996025422A1 (en) * 1995-02-14 1996-08-22 Human Genome Sciences, Inc. Keratinocyte growth factor-2

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996025422A1 (en) * 1995-02-14 1996-08-22 Human Genome Sciences, Inc. Keratinocyte growth factor-2

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CELL, Vol. 27, No. 9, (1995), pages 341-344. *
J. BIOL. CHEM., Vol. 271, No. 27, July 1996, YAMASAKI et al., "Structure and Expression of the Rat mRNA Encoding a Novel Member of the Fibroblast Growth Factor Family", pages 15918-15921 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6077692A (en) * 1995-02-14 2000-06-20 Human Genome Sciences, Inc. Keratinocyte growth factor-2
US6693077B1 (en) 1995-02-14 2004-02-17 Human Genome Sciences, Inc. Keratinocyte growth factor-2
US6903072B2 (en) 1995-02-14 2005-06-07 Human Genome Sciences, Inc. Keratinocyte growth factor-2
US6916786B2 (en) 1995-02-14 2005-07-12 Human Genome Sciences, Inc. Keratinocyte growth factor-2
US7232667B2 (en) 1995-02-14 2007-06-19 Human Genome Sciences, Inc. Keratinocyte growth factor-2 polynucleotides
US6743422B1 (en) 1996-10-15 2004-06-01 Amgen, Inc. Keratinocyte growth factor-2 products
US6238888B1 (en) 1997-12-22 2001-05-29 Human Genone Sciences, Inc. Keratinocyte growth factor-2 formulations
US6653284B2 (en) 1997-12-22 2003-11-25 Human Genome Sciences, Inc. Keratinocyte growth factor-2 formulations
US6869927B1 (en) 1997-12-22 2005-03-22 Human Genome Sciences, Inc. Keratinocyte growth factor-2 formulations
WO2001064915A1 (en) * 2000-03-02 2001-09-07 Biowindow Gene Development Inc. Shanghai Novel polypeptide---the factor 1-25 being homology with human fibroblast growth factor and polynucleotide encoding it

Also Published As

Publication number Publication date
AU1041297A (en) 1997-06-27

Similar Documents

Publication Publication Date Title
JP3045398B2 (en) Proteins, DNAs and their uses
EP0506477B1 (en) Vascular endothelial cell growth factor C subunit
KR100788219B1 (en) Protein for blocking platelet adhesion
Zwijsen et al. Characterization of a Rat C6 Glioma‐Secreted Follistatin‐Related Protein (FRP) Cloning and Sequence of the Human Homologue
WO1989006657A1 (en) Pulmonary surfactant protein and related polypeptide
JPH0565298A (en) Vascular endotheliocyte growth factor ii
WO2001049728A2 (en) HUMAN PROTEINS HAVING HYDROPHOBIC DOMAINS AND DNAs ENCODING THESE PROTEINS
JPH01501361A (en) Production of new T cell suppressor factors and their uses
JP2002510492A (en) Interleukin-1 receptor antagonist and use thereof
WO1997020929A1 (en) Fibroblast growth factor fgf-10
JP3395181B2 (en) Hematopoietic stem cell augmentation agent
WO1996011952A1 (en) Method for purifying keratinocyte growth factors
EP1856249B1 (en) Growth factor mutants with improved biological activity
WO2000009551A1 (en) Human chordin-related proteins and polynucleotides encoding them
CA2322711A1 (en) Human proteins having transmembrane domains and dnas encoding these proteins
WO1999055863A1 (en) NOVEL POLYPEPTIDE, cDNA ENCODING THE SAME AND UTILIZATION THEREOF
US5972652A (en) Polynucleotides encoding secreted proteins
JP3527760B2 (en) Novel osteogenesis-inducing protein, DNA encoding the same, method for producing the protein, and osteogenesis-inducing agent containing the same as an active ingredient
CA2305337A1 (en) Human proteins having secretory signal sequences and cdnas encoding these proteins
JP3488459B2 (en) Monocyte or macrophage migration factor
US20020165151A1 (en) Secreted proteins
JPH03180192A (en) Production of human pro-tgf-beta1 by genetic recombination
JP2816971B2 (en) Recombinant hepatocyte growth factor
JP3292873B2 (en) Recombinant hepatocyte growth factor
JP3318323B2 (en) Recombinant hepatocyte growth factor

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WR Later publication of a revised version of an international search report
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase