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WO1997020929A1 - Facteur de croissance des fibroblastes fgf-10 - Google Patents

Facteur de croissance des fibroblastes fgf-10 Download PDF

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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
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Prior art keywords
fgf
primer
seq
dna
sequence
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PCT/JP1996/003579
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English (en)
Japanese (ja)
Inventor
Nobuyukia Itoh
Takaharu Negoro
Takashi Katsumata
Shuzo Tagashira
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Sumitomo Pharmaceuticals Company, Limited
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Priority to AU10412/97A priority Critical patent/AU1041297A/en
Publication of WO1997020929A1 publication Critical patent/WO1997020929A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—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

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Abstract

L'invention concerne un FGF-10 de recombinaison obtenu en introduisant dans une cellule hôte un vecteur d'expression dans lequel est intégré un ADN codant pour une séquence spécifique d'aminoacides. La cellule hôte transformée est ensuite incubée pour lui permettre de produire la protéine recherchée. Ce FGF-10 de recombinaison est utile pour des médicaments et des réactifs utilisant sa capacité à promouvoir la croissance cellulaire.
PCT/JP1996/003579 1995-12-07 1996-12-06 Facteur de croissance des fibroblastes fgf-10 WO1997020929A1 (fr)

Priority Applications (1)

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AU10412/97A AU1041297A (en) 1995-12-07 1996-12-06 Fibroblast growth factor fgf-10

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JP34568995 1995-12-07
JP7/345689 1995-12-07
JP8/103240 1996-03-28
JP10324096 1996-03-28
JP8/214378 1996-07-24
JP21437896 1996-07-24

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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 (fr) * 2000-03-02 2001-09-07 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, facteur 1-25 analogue au facteur humain de croissance du fibroblaste, et polynucleotide codant pour ce polypeptide
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 (fr) * 1995-02-14 1996-08-22 Human Genome Sciences, Inc. Facteur-2 de croissance des keratinocytes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996025422A1 (fr) * 1995-02-14 1996-08-22 Human Genome Sciences, Inc. Facteur-2 de croissance des keratinocytes

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 (fr) * 2000-03-02 2001-09-07 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, facteur 1-25 analogue au facteur humain de croissance du fibroblaste, et polynucleotide codant pour ce polypeptide

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