WO2006109696A1 - Composition comprising genetically engineered follicle-stimulating hormone - Google Patents

Composition comprising genetically engineered follicle-stimulating hormone Download PDF

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Publication number
WO2006109696A1
WO2006109696A1 PCT/JP2006/307375 JP2006307375W WO2006109696A1 WO 2006109696 A1 WO2006109696 A1 WO 2006109696A1 JP 2006307375 W JP2006307375 W JP 2006307375W WO 2006109696 A1 WO2006109696 A1 WO 2006109696A1
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Prior art keywords
stimulating hormone
seq
dna
amino acid
follicle
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PCT/JP2006/307375
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French (fr)
Japanese (ja)
Inventor
Yutaka Kanda
Mitsuo Satoh
Tsuyoshi Yamada
Kazuya Yamano
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Kyowa Hakko Kogyo Co., Ltd.
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Publication of WO2006109696A1 publication Critical patent/WO2006109696A1/en

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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
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • 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 composition comprising a recombinant follicle-stimulating hormone molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is a reducing end of the sugar chain.
  • the present invention relates to a follicle-stimulating hormone composition, which is a sugar chain, which has fucose bound to N-acetylcylcosamine and its use.
  • Follicle stimulating hormone (hereinafter also referred to as FSH) is mainly used for infertility treatment.
  • Infertility means that (1) men and women of reproductive age have a normal sexual life, but no pregnancy is found after 2 years excluding the contraceptive period, and (2) pregnancy is established. However, it means the situation where a live child cannot be obtained due to miscarriage and stillbirth. Of these, (2) is also called infertility, and the term infertility is generally used to include infertility. Infertility is classified as primary infertility with no pregnancy experience and secondary infertility with at least one pregnancy experience.
  • Infertility is a complex factor that rarely develops from a single cause.
  • the main treatment method for infertility is medical practice such as surgery on the fallopian tube and uterus.
  • Drugs such as ovulation disorders are administered especially for ovulation disorders (Non-patent Document 1).
  • ovulation disorder therapeutic agents gonadotropic hormones such as FSH, luteinizing hormone (Luteinizing hormone, LH) and ciliary gonadotropin (CG) are widely used. Used in medical settings.
  • LH is a glycoprotein with a molecular weight of 29,000 and promotes estrogen secretion from the ovary and progesterone production from the corpus luteum in women. In men, it is also called Interstitial cell stimulating hormone (ICSH) because it acts on Leydig stromal cells to promote testosterone secretion.
  • ICSH Interstitial cell stimulating hormone
  • FSH is a glycoprotein with a molecular weight of 32,000 that promotes follicular development in women and spermatogenesis in the testicles in men.
  • TSH is a glycoprotein with a molecular weight of 28,000 that stimulates the thyroid gland Promotes secretory synthesis of glandular hormones.
  • CG is a glycoprotein with a molecular weight of 37,000. It is secreted from the placenta during pregnancy and exhibits the same physiological activity as LH.
  • FSH, LH, and TSH are hormones synthesized and secreted in the anterior pituitary gland.
  • FSH FSH, LH, CG and thyroid stimulating hormone (hereinafter referred to as TSH) all have a heterodimeric structure consisting of an ⁇ subunit and a j8 subunit.
  • TSH thyroid stimulating hormone
  • all molecules are made of the same precursor protein, and have the same amino acid sequence except for the difference in the length of the 5 amino acids on the heel side (Non-patent Document 2). . Therefore, it can be said that these four types of holmons belong to the same family structurally.
  • each j8 subunit is different, and it determines the specificity of each hormone.
  • the ⁇ subunits of human FSH, human LH, and human TSH are all encoded by a single gene on human chromosome 6.
  • the human FSH 13 subunit gene is encoded on chromosome 11
  • the human LH ⁇ subunit gene is encoded on chromosome 19
  • the human TSH ⁇ subunit gene is encoded on chromosome 1. Yes.
  • FSH When FSH is secreted from the anterior pituitary gland, females promote follicular development and maturation by specifically binding to the FSH receptor expressed on the surface of follicular cells. FS also promotes estrogen production and secretion. In men, FSH promotes seminiferous tubule development and spermatogenesis in the testicles by specifically binding to the FSH receptor expressed on the seminiferous cell surface.
  • FSH binds to the FSH receptor, as with many peptide hormones and receptors, intracellular adenylate cyclase activity increases via GTP-binding proteins, and intracellular cyclic adenosine monophosphate (cAMP). Synthesis is promoted.
  • the generated cA MP functions as an intracellular signal transmitter, and activates intracellular protein kinases.
  • FSH is a heterodimer in which an a subunit composed of 92 amino acid residues and a ⁇ subunit composed of 118 amino acid residues are associated.
  • Sugar accounts for 20% of the molecular weight of FSH, and each heterodimer has four ⁇ -glycoside-linked sugar chains.
  • the sugar chains bound to FSH are complex, and two (Asn52, Asn78) sugar chains bind to the ⁇ subunit, and two (Asn7, Asn24) sugar chains bind to the 0 subunit (Non-patent Document 2).
  • the typical sugar chain structure of N-glycoside-linked sugar chains found in human urine-derived FSH is shown below. [0006] [Chemical 1]
  • Non-Patent Documents 3 and 4 The modification of the non-reducing end of the sugar chain is based on the same anterior pituitary hormones LH and TSH, which is predominantly sulfated N-acetylgalatatosamine. In FSH, sialic acid is predominant! /, (Non-Patent Documents 3 and 4).
  • LH and TSH sulfated N-acetylgalatatosamine.
  • FSH sialic acid is predominant! /
  • Non-Patent Documents 3 and 4 the sugar chain part bound to the polypeptide is important for the expression of biological activity in vivo. Removing the sugar chain maintains the ability to bind to the receptor, but it is halved in blood. Biological activity is greatly lost due to the shortened period (Non-patent Documents 4 to 6).
  • Non-patent Documents 7 and 8 it is known that removal of sialic acid or sulfated N-acetylylgalatatosamine at the non-reducing terminal portion of the sugar chain has a significant effect on blood half-life.
  • This decrease in in vivo activity associated with the cationization occurs because galactose on the non-reducing end of the sugar chain is exposed by sialic acid removal, and follicle-stimulating hormone is captured and degraded by the liver galactose-binding protein. It has been clarified! (Non-patent document 9).
  • Non-patent Document 10 Non-patent Document 10
  • FSH FSH is mainly modified with sialic acid in the living body
  • removal of the sugar chain attached to Asn52 in the ⁇ - subject affects the intracellular signal transduction, and the importance of the sugar chain that binds to the polypeptide has been pointed out ( Non-patent literature 11-13).
  • placental gonadotropin extracted from pregnant woman's urine or placenta, not extracted from the pituitary gland has LH-like activity.
  • Pituitary gonadotropin extracted from menopausal women's urine as a ciliary gonadotropin (hCG preparation) is FSH-like It is used as an active menopausal gonadotropin preparation (human menopausal gonadotropin; hMG preparation), respectively.
  • Pregnantmare serum gonadotropin (PMSG; trade name Serotropin) is also available on the market as an FSH preparation.
  • Gonadotropin with animal power is also antigenic to humans. Has the disadvantage of producing anti-hormone antibodies.
  • ovarian hyperstimulation and ovarian rupture are problematic as side effects, and the use of high-purity products and appropriate use are recommended.
  • gonadotropin preparations are prepared by purifying human tissues or urine as described above, it is difficult to completely isolate FSH and LH having similar chemical structures.
  • the sugar chain structure of the recombinant FSH preparation produced in CHO cells is not observed in the non-reducing terminal part of the sugar chain at the non-reducing end of the sugar chain.
  • there are differences such as a small proportion of multi-branched sugar chains with many N-glycoside-bonded complex double-chain sugar chains.
  • the rate of fucose modification to the sugar chain is equivalent to about 50%, and it has a complete N-glycoside-linked complex type sugar chain.
  • Non-Patent Documents 14 to 17 Although there are differences in the sugar chain structure, there is no significant difference in biological activity between human urine-derived FSH preparations and genetically modified FSH preparations. (Non-patent documents 18, 19).
  • mutant FSH has been constructed.
  • the amino acid sequence of Ala- Asn-lie- Thr- Va ⁇ Asn-lie- Thr- Val at the N-terminus of the ⁇ subunit The amino acid sequence of the four 0-glycoside-linked glycosylation domains existing at the C-terminal of the ⁇ -subunit C-terminal of CG ⁇ -subunit CSH of the FSH ⁇ -subunit CG and FSH chimeric protein bound to the terminal (Non-patent Document 22), N-terminal of FSH a subunit and C-terminal of 13 subunit, Ser-Gly- Ser- Asn- Ala- Thr-Gly -Ser-Gly-Ser-Asn-Ala-Thr-Ser-Gly-Ser Amino acid sequence of single-chain FSH (Non-patent Document 23) linked by a peptide linker is being developed!
  • the mutant FSH described above has a blood half-life of 2 to 4 times longer than that of conventional recombinant FSH.
  • these mutant FSHs are artificial non-natural proteins, it is necessary to consider antigenicity issues when used as pharmaceuticals.
  • anti-FSH antibodies Once patients have developed anti-FSH antibodies, not only is it impossible to administer FSH preparations, but the patient's endogenous FSH concentration may decrease, which is a serious problem.
  • attempts to extend the blood half-life of FSH preparations include synthetic components such as polyethylene glycol (PEG).
  • Non-patent Documents 24 and 25 A method has been examined in which FSH kidney glomerular force is also suppressed from being released into the urine by artificially linking the pups to FSH protein to increase the molecular weight.
  • PEG PEG-maleimidomase
  • the affinity of FSH for the FSH receptor is greatly reduced.
  • FSH to which PEG is bound exists as a huge molecule, there is a possibility that the distribution in the living body may change, and the risk of showing an activity different from the physiological activity inherent to FSH itself is not considered.
  • Hanana Non-patent Document 26).
  • Non-Patent Document 1 99 Drug Data Book IV, 175 (1999)
  • Non-Patent Document 2 European Journal of Biochemistry 242, 608 (1996)
  • Non-Patent Document 3 Endocrinology 128, 341 (1991)
  • Non-Patent Document 4 Recent Progress in Hormone Research 54, 271 (1999)
  • Non-Patent Document 5 Molecular Endocrinology 3, 2011 (1989)
  • Non-Patent Document 6 Endocrinology 136, 2635 (1995)
  • Non-Patent Document 7 Human Reproduction 14, 1160 (1999)
  • Non-Patent Document 8 Human Reproduction 3, 491 (1988)
  • Non-Patent Document 9 Blood 73, 84 (1989)
  • Non-Patent Document 10 Recent Progress in Hormone Research 54, 271 (1999)
  • Non-Patent Document 11 Molecular and Cellular Endocrinology 199, 73 (2003)
  • Non-Patent Document 12 Journal of Biological Chemistry 264, 17113 (1989)
  • Non-Patent Document 13 Biology of Reproduction 65, 1686 (2001)
  • Non-Patent Document 14 Pharmacotherapy Vol.18, No.5 (1998)
  • Non-Patent Document 15 Biochimica et Biophysica Acta 947, 287 (1988)
  • Non-Patent Document 16 Biochem. J. 287, 665 (1992)
  • Non-Patent Document 17 Molecular Human Reproduction 2, 371 (1996)
  • Non-Patent Document 18 Endocrinology, 129, 2623-2630
  • Non-Patent Document 19 Recent Progress in Hormone Research 54, 271 (1999)
  • Non-Patent Document 20 Novel Therapeutic Proteins Selected Case Studies WILEY- VCH (200
  • Non-Patent Literature 21 Journal of Clinical Endocrinology and Metabolism 88, 3227 (2002)
  • Non-Patent Document 22 Recent Progress in Hormone Research 54, 271 (1999)
  • Non-Patent Document 23 Human Reproduction 18, 50 (2003)
  • Non-patent document 24 Nephrology and Dialysis and Transplantation Suppl4, 166 (2003) Non-patent document 25 Journal of Pharmaceutical Science 93, 3027 (2004)
  • Non-patent document 26 Development of follow-up drugs Noo-conjugate drugs Yodogawa Shoten (1993) Invention disclosure
  • the object is to provide a follicle stimulating hormone preparation that is safe and has an increased blood half-life so that the number of administrations can be reduced.
  • the present invention relates to the following (1) to (27).
  • a composition comprising a genetically modified follicle-stimulating hormone molecule having an N-glycoside-linked complex sugar chain, wherein the N-glycoside-linked complex sugar chain is the N-acetyl dalcosamine at the reducing end of the sugar chain
  • a follicle stimulating hormone composition in which fucose is bound to a sugar chain.
  • N-glycoside-linked complex type sugar chain is an N-glycoside-linked complex type sugar chain that is bound to the ex subunit and j8 subunit constituting the follicle stimulating hormone. Stimulating hormone composition.
  • the N-glycoside-bonded complex type sugar chain is a sugar chain in which the 1-position of fucose is not ⁇ - bonded to the 6-position of N-acetylyldarcosamine at the reducing end of the sugar chain.
  • the ex subunit that constitutes the follicle stimulating hormone is a polypeptide in which the following (a), (b), (c), (d), (and (1) a group force having a force is also selected.
  • the follicle-stimulating hormone composition according to any one of (1) to (3).
  • amino acid sequence represented by SEQ ID NO: 5 one or more amino acids are deleted, substituted, or inserted.
  • polypeptide comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 5 and having substantially the same activity as that of the follicle stimulating hormone OC subunit;
  • the ex subunit constituting the follicle-stimulating hormone is a polypeptide encoded by a DNA selected from the group forces consisting of the following (a), (b), (c) and (d), (1) The follicle-stimulating hormone composition according to item 1 of ⁇ (3).
  • the ⁇ subunit that constitutes the follicle stimulating hormone is a polypeptide in which the following (a), (b), (c), (d), (and (1) powerful group force is also selected.
  • the follicle-stimulating hormone composition according to any one of (1) to (3).
  • polypeptide comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 7, and having substantially the same activity as that of the follicle stimulating hormone j8 subunit;
  • the ⁇ subunit constituting the follicle stimulating hormone is a polypeptide encoded by a DNA selected from the group consisting of the following (a), (b), (c) and (d) (1
  • (d) encodes a polypeptide that hybridizes under stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 4 and has substantially the same activity as that of the follicle stimulating hormone j8 subunit. DNA.
  • the host cell is responsible for the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose,
  • the genome was modified so that the activity of the enzyme involved in sugar chain modification in which the 1-position of fucose was ⁇ -linked to the 6-position of N-glycidyl darcosamine at the N-glycoside-linked complex sugar chain reducing end was deleted.
  • the transformant according to (8) above which is a cell.
  • amino acid sequence represented by SEQ ID NO: 10 one or more amino acids are deleted, substituted, inserted and Z or added, and the amino acid sequence capacity is GDP-mannose 4,6-dehydrata.
  • a protein having lyase activity A protein having lyase activity
  • GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is a protein in which the following (a), (b) and (c) group forces are selected (11) ).
  • (c) It consists of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 12, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity protein.
  • GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is a protein encoded by a DNA selected from the following (a) and (b) group forces that also have power (11 Cell)
  • N-glycoside-linked complex sugar chain reducing terminal N-acetylcylcosamine has an enzyme involved in sugar chain modification in which the 1-position of fucose is a-linked to the 6-position of a 1,6-fucosyltransferase
  • a 1,6-fucosyltransferase is a protein for which a group force consisting of the following (a), (b), (c), (d), (e) and (1) is also selected (16 ).
  • amino acid sequence represented by SEQ ID NO: 16 one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have ⁇ 1,6-fucosyltransferase activity.
  • a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 15 and having ⁇ 1,6-fucosyltransferase activity;
  • D consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by D SEQ ID NO: 16, and a protein having a1,6-fucosyltransferase activity;
  • a 1, 6-fucosyltransferase is a protein encoded by a DNA selected from the following groups (a), (b), (c) and (d) which also has a force: Transformant.
  • Lentil lectin LCA Lientil Agglutinin from Lens Culinaris
  • Herochawantake lectin AAL Lectin from Aleuria aurantia
  • the host cell has the following (a), (b), (c), (d), (e), (£), (g), (h), (i) and (j) forces The transformant according to any one of (8) to (21) above, which is a cell selected from a group.
  • the transformant according to any one of (8) to (22) above is cultured in a medium, a follicle stimulating hormone composition is produced and accumulated in the culture, and a follicle stimulating hormone composition is produced from the culture.
  • the manufacturing method of a follicle stimulating hormone composition including the process of extract
  • a medicament comprising as an active ingredient the follicle-stimulating hormone composition described in (1) to (7) and (24) above.
  • a therapeutic agent for follicular maturation and ovulation disorders comprising the follicle-stimulating hormone composition described in (1) to (7) and (24) as an active ingredient.
  • a therapeutic agent for spermatogenesis and maturation disorder comprising the follicle-stimulating hormone composition described in (1) to (7) and (24) as an active ingredient.
  • a composition comprising a recombinant follicle-stimulating hormone molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is a reduced powder of the sugar chain.
  • a follicle stimulating hormone composition which is a sugar chain in which fucose is bound to N-acetildarcosamine at the end.
  • FIG. 1 shows the production flow of plasmid pBS-FSH a.
  • FIG. 2 shows the production flow of plasmid pKAN-FSHA.
  • FIG. 3 shows the production flow of plasmid pBS-FSH ⁇ .
  • FIG. 4 shows the production flow of plasmid pKAN-FSH ⁇ .
  • FIG. 5 shows the production flow of plasmid pT7-FSHR.
  • FIG. 6 shows the production flow of plasmid pKAN-FSHR.
  • FIG. 7 shows the intracellular cAMP induction effect by FSH stimulation.
  • FIG. 8 shows changes in blood concentration of FSH.
  • the present invention is a composition comprising a recombinant follicle-stimulating hormone molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is a reducing end of the sugar chain.
  • the present invention relates to a follicle-stimulating hormone composition that is a sugar chain in which fucose is bonded to N-acetylyldarcosamine.
  • Follicle stimulating hormone includes follicle stimulating hormone composed of OC subunit and ⁇ subunit. Therefore, the follicle-stimulating hormone composition of the present invention has a ⁇ -daricoside-bonded complex sugar chain strength that is bound to each of the ⁇ subunit and ⁇ subunit constituting the follicle stimulating hormone.
  • -A follicle-stimulating hormone composition comprising a sugar chain in which fucose is bound to acetylyldarcosamine.
  • a composition comprising a recombinant follicle-stimulating hormone molecule having a ⁇ -glycoside-bonded complex type sugar chain according to the present invention, wherein the ⁇ -glycoside-linked complex type sugar chain is a reducing end of the sugar chain.
  • a follicle stimulating hormone composition (hereinafter also referred to as the “composition of the present invention”), which is a sugar chain in which fucose is bound to ⁇ -acetylyldarcosamine, is a ⁇ -glycoside-linked complex type sugar chain reduction Any composition is included as long as it is a follicle-stimulating hormone composition having a genetically modified follicle-stimulating hormone molecular force in which fucose is bound to the terminal ⁇ -acetyltilcosamine.
  • the follicle stimulating hormone is expressed on the surface of follicle cells in women, specifically binds to the follicle stimulating hormone receptor, and has the activity of promoting follicular growth and maturation.
  • Glycoprotein in males, is a glycoprotein that specifically binds to the follicle-stimulating hormone receptor expressed on the surface of seminiferous tubules and has the activity of promoting seminiferous tube development and spermatogenesis in the testicles ! / Evil things are also included.
  • follicle stimulating hormone As specific follicle stimulating hormone, the following (a), (b), (c) and (d) force group force selection Follicle stimulating hormone a subunit polypeptide encoded by DNA and (e), (£), (g), and (h) follicle stimulating hormone 13 subunit polypeptide encoded by DNA that is also selected for force Or the following (0, (j), 00, (1), (m) and (n) group forces that also have a force selected follicle stimulating hormone a subunit polypeptide, and (o), (p) , (Q), (r), (and (t) force group forces, such as heterodimers with selected follicle stimulating hormone / 3 subunit polypeptides.
  • (d) Encodes a protein that is substantially identical to the activity of the follicle stimulating hormone OC subunit and that hybridizes under stringent conditions with DNA having the nucleotide sequence shown in SEQ ID NO: 2 DNA to do;
  • (h) encodes a protein that has the same activity as that of the follicle-stimulating hormone j8 subunit, and is hybridized under stringent conditions with DNA having the nucleotide sequence shown in SEQ ID NO: 4. DNA to do;
  • amino acid sequence represented by SEQ ID NO: 6 one or more amino acids are deleted, substituted, or inserted
  • a protein having the ability to sequence amino acids added and Z or added, and having substantially the same activity as that of the follicle stimulating hormone ⁇ -subunit;
  • a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6 and having substantially the same activity as that of the follicle stimulating hormone OC subunit;
  • a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, and having substantially the same activity as that of the follicle stimulating hormone j8 subunit.
  • DNA that hybridizes under stringent conditions refers to, for example, DNA such as DNA having the base sequence represented by SEQ ID NO: 1, 2, 3, or 4 or a fragment thereof.
  • DNA such as DNA having the base sequence represented by SEQ ID NO: 1, 2, 3, or 4 or a fragment thereof.
  • it means DNA obtained by using the Koguchi-1 'hybridization method, plaque' hybridization method, Southern blot hybridization method, etc., specifically, colonies or plaques. Hive at 65 ° C in the presence of 0.7 to 1.0 M sodium chloride using a filter with immobilized DNA After rehydration, 0.1 to 2 times the concentration of SSC solution (the composition of 1 time concentration of SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate), and the filter was used at 65 ° C.
  • DNA that can be identified by washing can be raised.
  • HYBRIDISE ⁇ Nyon Yong, MolecularCloning, A Laboratory Manual, Second Edition, old Spring Harbor Laboratory Press, (1989) (hereinafter abbreviated as Molecular ⁇ ⁇ Cloning 2nd Edition), Current Protocols in Molecular Biology, John Wiley & Sons, (1987-1997) (hereinafter referred to as Current 'Protocols'in' Molecular ⁇ ⁇ Biology), DNA Cloning 1: ColeTechniques, A Practical Approach, Second Edition, Oxford University (1995), etc. It can be performed according to the method described in 1.
  • DNA capable of hybridizing DNA having at least 60% or more homology with the base sequence represented by SEQ ID NO: 1, 2, 3 or 4, preferably 70% or more, more preferably 80% or more More preferred is a DNA having a homology of 90% or more, particularly preferably 95% or more, and most preferably 98% or more.
  • one or more amino acids in the amino acid sequence represented by SEQ ID NO: 5 or 6 are deleted, substituted, inserted and Z or added, and the follicle stimulating hormone subunit is Proteins that have substantially the same activity as those of Molecular ⁇ ⁇ Cloning 2nd Edition, Current 'Protocols' In' Molecular ⁇ ⁇ ⁇ Biologics, Nucleic Acids Research, 10, 6487 (1982 ), Proc. Natl. Acad. Sci., USA, 7 9,6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad.
  • site-directed mutagenesis is introduced into DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 5 or 6.
  • the amino acid sequence represented by SEQ ID NO: 5 or 6 has a homology of 80% or more and substantially the same activity as the follicle stimulating hormone ⁇ subunit.
  • the protein having the activity of BLAST (J. Mol. Biol, 215, 403 (1990)) and FASTA (Methods in Enzymology, 183, 63 (1990)) At least 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more, most preferably, a protein having the amino acid sequence shown in SEQ ID NO: 5 or 6 It means that the protein is 99% or more.
  • a heterodimer is formed with the follicle-stimulating hormone 13 subunit at a molar ratio of 1: 1. It specifically binds to the follicle-stimulating hormone receptor and has substantially the same activity to promote follicular development and maturation in women, and to promote microtubule development and spermatogenesis in testis in men. Protein. “Substantially the same” means that the activities are the same in nature. Therefore, quantitative factors such as the strength of activity may be different.
  • the amino acid sequence having one or more amino acids deleted, substituted, inserted and Z or added in the amino acid sequence represented by SEQ ID NO: 7 or 8 also has an amino acid sequence ability, and follicle stimulating hormone ⁇ subunit Proteins that have substantially the same activity as that of the molecule are as follows: Molecular ⁇ ⁇ Cloning 2nd Edition, Current 'Protocols' In' Molecular ⁇ ⁇ Biologics, Nucleic Acids Research, 10, 6487 ( 1982), Proc. Natl. Acad. Sci., USA, 7 9,6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad.
  • site-directed mutagenesis is introduced into DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 7 or 8.
  • Means a protein that can be obtained by The number of amino acids to be deleted, substituted, inserted and Z or added is 1 or more, and the number is not particularly limited. However, deletion, substitution or addition can be performed by well-known techniques such as the above-mentioned site-directed mutagenesis. The number is as much as possible, for example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.
  • the amino acid sequence represented by SEQ ID NO: 7 or 8 has a homology of 80% or more and is substantially the same as the activity of the follicle stimulating hormone j8 subunit.
  • Proteins having activity include BLAST [J. Mol. Biol, 215. 403 (1990)] and FASTA [Me thods in Enzymology, 183, 63 (1990)) or the like, and a protein having the amino acid sequence of SEQ ID NO: 7 or 8 and at least 80% or more, preferably 85% or more, more preferably Means 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more.
  • a heterodimer is formed with the follicle stimulating hormone a subunit at a molar ratio of 1: 1. It specifically binds to the follicle-stimulating hormone receptor and has substantially the same activity to promote follicular development and maturation in women, and to promote microtubule development and spermatogenesis in testis in men. Protein. “Substantially the same” means that the activities are the same in nature. Therefore, quantitative factors such as the strength of activity may be different.
  • N-glycoside-linked glycans bound to glycoproteins may have a common core structure represented by the following structural formula (I) in any case having various structures.
  • Structural Formula (I) In Structural Formula (I), the end of the sugar chain that binds to asparagine is called the reducing end, and the opposite side is called the non-reducing end.
  • the N-glycoside-linked sugar chain is a high mannose type in which only mannose binds to the non-reducing end of the core structure, and galactose —N-acetyldarcosamine (hereinafter referred to as Ga ⁇ GlcNAc) on the non-reducing end of the core structure. 1) in parallel!
  • the follicle-stimulating hormone molecule constituting the composition of the present invention has an additional sequence of N-glycoside-linked sugar chains at at least 4 sites, and N-glycoside-linked sugar chains are bound to these sites.
  • Specific examples of the N-glycoside-linked sugar chain that binds to follicle-stimulating hormone include the aforementioned N-glycoside-linked complex sugar chain.
  • the N-dalcoside-linked complex sugar chain that binds to the follicle-stimulating hormone molecule includes any sugar chain containing the core structure represented by the structural formula (I).
  • the composition of the present invention may be composed of follicle stimulating hormone molecules having a single sugar chain structure, as long as the effects of the present invention are obtained.
  • the composition of the present invention may be composed of follicle-stimulating hormone molecules having different sugar chain structures
  • the composition of the present invention has an N-glycoside-linked complex type sugar chain, even if the sugar structure is V. Fucose is bound to the reducing terminal N-acetyl darcosamine! /, N! /, And has a sugar chain.
  • N-glycoside-bonded complex type sugar chain N-acetylyldarcosamine of the reducing end is linked to fucose. Any sugar chain may be included as long as the sugar chain is not bound to a non-reducing terminal sugar chain.
  • the 1st position of fucose is the 6th position of the N-glycidyl darcosamine of the N-glycoside-bonded complex sugar chain (X-linked!
  • a sugar chain in which fucose is not bound to ⁇ ⁇ -acetylyldarcosamine at the sugar chain reducing end means that fucose is not substantially bound to the sugar chain.
  • the fucose content is 0%.
  • the fact that fucose is not substantially bound means that fucose cannot be substantially detected in the sugar chain analysis described in 4 below. “Substantially undetectable” means below the detection limit of measurement.
  • composition of the present invention is a follicle-stimulating hormone in which fucose is bound to N-acetylside darcosamine at the N-glycosidic complex reducing end, such as human urine-derived FSH, which is also known in the art. In comparison, it has an equivalent affinity for the follicle-stimulating hormone receptor and has a long blood half-life when administered in vivo.
  • the transformant of the present invention includes any transformant as long as it is capable of producing the composition of the present invention.
  • DNA encoding each subunit protein that forms a follicle-stimulating hormone molecule is represented by the following (a) or (b
  • Enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose include GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose-3,5 -Epimerase (FX).
  • the GDP-mannose 4,6-dehydratase includes a protein encoded by the following DNA (a) or (b), or a protein (c), (d) or (e) below. .
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 10;
  • one or more amino acids are deleted, substituted, inserted and / or added, and have an amino acid sequence ability and have GDP-mannose 4,6-dehydratase activity protein;
  • a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, and having GDP-mannose 4,6-dehydratase activity.
  • GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase includes a protein encoded by the following DNA (a) or (b), or the following (c), (D) or (e) protein.
  • DNA which has a base sequence ability represented by SEQ ID NO: 11, is hybridized under stringent conditions and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity.
  • (c) It comprises an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 12, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity Protein.
  • N-glycoside-linked complex type sugar chain reducing terminal N-acetylcylcosamine 6-position of the fucose 1-position the enzyme involved in sugar chain modification is a 1,6-fucosyltransferase For example.
  • ⁇ 1,6-fucosyltransferase is a protein encoded by the following DNA (a), (b), (c) or (d), or (, (£), (g), (H), (0 or (j) protein, etc.).
  • a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 16, and having a 1,6-fucosyltransferase activity.
  • DNA that hybridizes under stringent conditions means, for example, DNA such as DNA having a nucleotide sequence represented by SEQ ID NO: 9, 11, 13, or 14, or a fragment thereof.
  • DNA obtained by using the Koguchi-1 'hybridization method, plaque' hybridization method, Southern hybridization method, etc. as a probe, and specifically, colonies or plaques.
  • OM sodium chloride using a filter to which DNA derived from DNA was immobilized 0.1 to 2 times the concentration of SSC solution (1x
  • the composition of the SSC solution with the concentration is 150 mM sodium chloride and 15 mM sodium citrate), and DNA can be identified by washing the filter under 65 ° C conditions.
  • DNA that can be hybridized under stringent conditions is specifically DNA having at least 60% homology with the nucleotide sequence represented by SEQ ID NO: 9, 11, 13, or 14, preferably 70% or more More preferred is DNA having a homology of 80% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.
  • the amino acid sequence represented by SEQ ID NO: 10 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or added, and GDP-mannose 4,6-dehydratase
  • an active protein In the amino acid sequence represented by SEQ ID NO: 12, an active protein
  • a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or added in the amino acid sequence represented by 15 or 16, and having ⁇ 1,6-fucosyltransferase activity is molecular. 'Crowung 2nd Edition, Current Protocols in Molecular Biology, Nucleic Acids Research, 10, 648 7 (1982), Proc. Natl. Acad.
  • the number of amino acids to be deleted, substituted, inserted, and Z or added is 1 or more, and the number is not particularly limited. However, deletion and substitution can be performed by well-known techniques such as the above-described site-directed mutagenesis. Or it is the number which can be added, for example, 1 to several tens, Preferably it is 1-20, More preferably, it is 1-10, More preferably, it is 1-5.
  • the present invention comprises an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, 12, 15 or 16, and GDP-mannose 4,6-dehydrata In order to have asease activity, GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity or ⁇ 1,6-fucosyltransferase activity, respectively in SEQ ID NO: 10, 12, 15 or 16 And at least 80% when calculated using analysis software such as BLAST [J. Mol. Biol, 215, 403 (1990)] and FAS TA [Methods in Enzymology, 183, 63 (1990)].
  • This means that the protein has a homology of 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more.
  • a host cell lacking the above-mentioned enzyme activity that is, an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-acetylyldarcosamine at the reducing end of N-glycoside-linked complex type sugar chain Encodes the a and j8 subunits of the follicle-stimulating hormone molecule in a host cell whose genome has been altered so that the activity of the enzyme involved in the glycosylation modification in which the 1-position of fucose is linked to a at position 6
  • a transformant producing the composition of the present invention can be obtained.
  • the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-acetylcolcamine at the reducing end of N-darcoside-linked complex sugar chain, the 6-position of fucose, the 1-position of fucose is ⁇ -linked
  • a mutation is introduced into the expression regulatory region of the gene so as to eliminate the expression of the enzyme, or the function of the enzyme It means that a mutation is introduced into the amino acid sequence of the gene so as to disappear.
  • Introducing mutation means that the base sequence on the genome is deleted, substituted, inserted, and deleted or added, and the base sequence is modified, completely suppressing the expression or function of the modified genomic gene. Knock out to do it.
  • a specific example of knocking out a genomic gene is one in which all or part of the target gene has been deleted from the genome. It can be knocked out by removing the genomic region of the etason containing the start codon of the target gene.
  • any method can be used as long as the target genome can be modified.
  • any lectin that can recognize the sugar chain structure can be used. Specific examples of this are: Lentil lectin LCA (Lentil Agglutinin from Lens Culinaris), Endumame lectin PS A (Peum sativum-derived PeaLectin), Broad bean lectin VFA (Agglutini n from Vicia faba), Hirochawantake lectin AAL ( Lectin from Aleuria aurantia).
  • a cell resistant to lectin refers to a cell whose growth is not inhibited even when an effective concentration of lectin is given.
  • the effective concentration is not less than the concentration at which cells before the genomic gene is modified (hereinafter also referred to as “parent cell”) cannot grow normally, preferably the concentration at which cells before the modified genomic gene cannot grow , More preferably 2 to 5 times, still more preferably 10 times, and most preferably 20 times or more.
  • the effective concentration of a lectin whose growth is not inhibited may be appropriately determined depending on the cell line, but is usually 10 / zg / ml to 10mg / ml, preferably 0.5mg / ml to 2.0mg. / ml.
  • any cell that can express the composition of the present invention can be used.
  • yeast, animal cells, insect cells, plant cells and the like can be mentioned. Specific examples include those described in 3. below.
  • animal cells include CHO cells derived from Chinese omster ovary tissue, rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cell, mouse myeloma cell line NS0 cell, mouse myeloma cell line SP2 / 0- Examples include Agl4 cells, Syrian hamster kidney tissue-derived BHK cells, human leukemia cell lines Namalba cells, embryonic stem cells, and fertilized egg cells.
  • genetic Host cells used to produce recombinant glycoprotein drugs include embryonic stem cells or fertilized egg cells used to produce non-human transgenic animals that produce genetically modified glycoprotein drugs, and recombinant sugars Examples include plant cells used to produce transgene plants that produce protein drugs.
  • the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-glycidyl-linked glycan reducing end N-acetylcylcosamine at position 6 of fucose It includes cells prior to the application of a technique for altering the genomic gene of an enzyme involved in sugar chain modification in which position 1 is OC-linked. For example, the following cells are preferable.
  • NS0 cell parent cell lines are described in the literature such as Bio / Technology (BIO / TECHNOLOGY), 10, 169 (1992), Biotechnology No. 1 Bioengineering (Biotechnol. Bioeng.), 73, 261, (2 001), etc. NS0 cells are listed.
  • NS0 cell line (RCB0213) registered with the RIKEN Cell Development Bank, or sub-strains obtained by acclimatizing these strains to various serum-free media are also included.
  • SP2 / 0-Agl4 cells As a parent cell of SP2 / 0-Agl4 cells, Journal 'Ob' Immunology (J. Immunol.) .126. 317, (1981), Nature (Nature), 276, 269, (1978), Human SP2 / 0-Agl4 cells described in documents such as “Antibody's” and “HumanAnt3 ⁇ 4odies and Hybridomas”, 3, 129, (1992).
  • SP2 / 0-Agl4 cells (ATCC CRL-1581) registered in ATCC or sub-strains (ATCC CRL-1581.1) in which these strains are conditioned in various serum-free media are also included.
  • CHO-K1 strain ATCC CCL-61
  • DUXB11 strain ATCC CRL-9096
  • Pro-5 strain ATCC CRL-1781 registered in ATCC
  • commercially available CHO-S strain (Lifetechnologi es Cat # l 1619), or sub-strains made by adapting these strains to various serum-free media can give.
  • the parent cell of rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cell includes a cell line established from Y3 / Ag 1.2.3 cell (ATCC CRL-1631). Specific examples are YB2 / 3HL.P2.G11.16Ag. Described in documents such as J. Cell. Biol., 93, 576 (1982), Methods Enzymol. 73B, 1 (1981). There are 20 cells. In addition, YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL-1662) registered in ATCC or sub-strains obtained by acclimating these strains to various serum-free media are also included.
  • the cell producing the composition of the present invention introduces a gene encoding follicle-stimulating hormone into CHO cells into which a gene encoding ⁇ 1,6-fucosyltransferase has been knocked out.
  • PKAN-FSH9-3 AFMS705 a strain that has been conditioned to a serum-free medium, and a gene encoding follicle-stimulating hormone introduced into CHO cells in which the gene encoding GDP-mannose 4,6-dehydratase has been knocked out PKAN- FSH2 GMDKO strain, which is a transformed strain.
  • the transformant of the present invention Compared to the erythropoietin composition obtained from the parent cell line, the transformant of the present invention has an affinity for the equivalent follicle-stimulating hormone receptor and has an increased half-life in blood clots. A follicle stimulating hormone composition can be produced.
  • the binding activity of the follicle stimulating hormone composition to the follicle stimulating hormone receptor, induction of intracellular signal transduction, estrogen production inducing activity, follicle maturation inducing activity, and blood half-life are already known follicle stimulating hormone receptor binding.
  • the host cell used for producing follicle stimulating hormone composition of the present invention can be produced by the method described below.
  • the host cell used to prepare the follicle stimulating hormone composition of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or N-glycidyl-linked complex N-acetylyldarcosamine at the reducing end of the sugar chain. It must be prepared by targeting the gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is ⁇ -linked at position 6 (hereinafter referred to as “enzyme related to fucose modification”) and using a gene disruption method. Can do.
  • GDP-fucose examples include GDP-mannose 4,6-dehydratase (hereinafter referred to as “GMD”), GDP-4-keto-6- Deoxy-D-mannose-3,5-epimerase (hereinafter referred to as “Fx”).
  • GMD GDP-mannose 4,6-dehydratase
  • Fx GDP-4-keto-6- Deoxy-D-mannose-3,5-epimerase
  • N-glycoside bond As an enzyme involved in sugar chain modification in which the 1-position of fucose is a-linked to the 6-position of N-acetylyldarcosamine at the reducing end of the complex type sugar chain, ⁇ 1,6- Examples include fucosyltransferase and ⁇ -L-fucosidase.
  • the gene herein includes DNA or RNA.
  • any method can be used as the gene disruption method as long as it can destroy the gene of the target enzyme.
  • Examples include the antisense method, ribozyme method, homologous recombination method, RNA-DNA oligonucleotide method (hereinafter referred to as “RDO method”), RNA interference method (hereinafter referred to as “RNAi method”). ), A method using a retrovirus, a method using a transposon, and the like. These will be specifically described below.
  • the host cell used to produce the follicle stimulating hormone composition of the present invention targets an enzyme gene related to fucose modification, and is described in Cell Engineering, 12, 239 (1993), Bio Z Technolo. GIO (BIO / TECHNOLOGY), 17, 1097 (1999), Human Molecular ⁇ ⁇ ⁇ Genetics (Hum. Mol. Genet.), 5, 1083 (1995), Cell Engineering, 13, 255 (1994), Proceding 'Ob The National' Academy ⁇ ⁇ Ob 'Science (Proc. Natl. Acad. Sci. USA),
  • cDNA or genomic DNA encoding an enzyme related to fucose modification is prepared.
  • an antisense gene or ribozyme construct of appropriate length including the DNA part encoding the enzyme related to fucose modification, the part of the untranslated region or the intron part.
  • D prepared in order to express the antisense gene or ribozyme in cells
  • a recombinant vector is prepared by inserting the NA fragment or full length downstream of the promoter of an appropriate expression vector.
  • a transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • a host cell used for preparing the follicle-stimulating hormone composition of the present invention can be obtained.
  • a host cell used for preparing the follicle stimulating hormone composition of the present invention is selected by selecting a transformant using the sugar chain structure of the glycoprotein on the cell membrane or the sugar chain structure of the produced glycoprotein molecule as an index. It can also be obtained.
  • the host cell used for producing the follicle-stimulating hormone composition of the present invention has an enzyme gene related to target fucose modification such as yeast, animal cell, insect cell, plant cell and the like. Any of these can be used. Specifically, the host cell described in 3 below can be mentioned.
  • the expression vector is capable of autonomous replication in the above host cell, or can be integrated into the chromosome and contains a designed antisense gene or a promoter at a position where a ribozyme can be transcribed. .
  • the expression vector described in 3 below can be mentioned.
  • the method for introducing a gene into various host cells the method for introducing a recombinant vector suitable for various host cells described in 3 below can be used.
  • Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the following methods.
  • biochemical method examples include a method for evaluating enzyme activity using an enzyme-specific substrate.
  • genetic engineering methods include Northern analysis for measuring the amount of mRNA of an enzyme gene and RT-PCR method.
  • Examples of a method for selecting a transformant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (1) below. Examples of the method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
  • Examples of a method for preparing cDNA encoding an enzyme related to fucose modification include the methods described below.
  • Total RNA or mRNA is prepared from the tissues or cell strength of various host cells.
  • a cDNA library is prepared from the prepared total RNA or mRNA.
  • a degenerative primer is prepared, and a gene fragment encoding the enzyme related to fucose modification is obtained by PCR using the prepared cDNA library as a saddle type To do. [0069] Using the obtained gene fragment as a probe, a cDNA library can be screened to obtain DNA encoding an enzyme related to fucose modification.
  • Human or non-human animal thread and tissue or cell mRNA may be commercially available (for example, Clontech) V, and human or non-human animal tissue or cell force may also be prepared as follows. It's good.
  • thiocyanic acid As a method for preparing total RNA of human or non-human animal tissues or cells, thiocyanic acid can be used.
  • Examples of a method for preparing mRNA as total RNA poly (A) + RNA include an oligo (dT) -fixed cellulose column method (Molecular 'Cloung 2nd edition).
  • mRNA can be prepared by using a commercially available kit such as Fast Track mRNA Isolation Kit (Invitrogen) or Quick Prep mRNA Purification Kit (Pharmacia).
  • kit such as Fast Track mRNA Isolation Kit (Invitrogen) or Quick Prep mRNA Purification Kit (Pharmacia).
  • a cDNA library is prepared from the prepared human or non-human animal tissue or cell mRNA.
  • the cDNA library can be prepared by a method described in Molecular 'Crowing 2nd Edition, Current Protocols in Molecular Biology, ALaboratory Manual, 2nd Ed. (l 989), or commercially available kits. For example, a method using SuperScriptPlasmid System for cDNA Synthesis and Plasmid (Life Technologies Neeri, ZAP-cDNA byn is is Kit (STRATAGENE)) can be mentioned.
  • a cloning vector for constructing a cDNA library is the Escherichia coli K12 strain.
  • Any phage vector or plasmid vector can be used as long as it can replicate autonomously.
  • ZAP Express [STRATAGENE, Strategies,, 58 (1992)]
  • pBluescript II SK (+) [Nucleic Acids Rese arch, 17,9494 (1989)]
  • ⁇ ZAP II (STRATAGENE)
  • gtl0, e gtl l Dienue 1.
  • Escherichia coli is preferably used as a host microorganism for preparing a cDNA library. Specifically, Escherichia coli XL1- Blue MRF '[STRATAGENE, Strategies, 5, 81 (1992)], Escherichiacoli C600 [Genetics, 39, 440 (1954) 1, Escherichia coli Y10 88 [Science, 222, 778 (1983) 1, Escherichia coli Y1090 [Science, 222,778 (1983) 1, Escherichia coli NM522 "Journal, Ob, Molecura ⁇ Biology 0. Mol.BioL ), 166, 1 (1983) 1. Escherichia coli K802 (J. Mol. Biol.), 16, 118 (1966) ⁇ and Escherichia coli ⁇ 105 ⁇ Gene (Gene) , 38,275 (1985)].
  • the obtained gene fragment is DNA encoding an enzyme related to fucose modification.
  • nucleotide sequence analysis methods such as the Sidi et al. Dideoxy method [pro Seeds ⁇ Ob ⁇ The ⁇ National ⁇ Academia ⁇ ⁇ ⁇ Ob ⁇ Science (Pro Natl. Acad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377DNA Sequencer (Applied Biosystems) and other bases This can be confirmed by analysis using a sequence analyzer.
  • colony hybridization or plaque hybridization (molecular molecular clones) from cDNA or cDNA library synthesized from mRNA contained in tissues or cells of human or non-human animals. -Nu 2nd edition) etc. can be used to obtain DNA for enzymes related to fucose modification.
  • a cDNA or cDNA library synthesized from mRNA contained in human or non-human animal tissues or cells using the primers used to obtain a gene fragment encoding an enzyme related to fucose modification as a saddle type.
  • the cDNA of the enzyme related to fucose modification can also be obtained by amplification using PCR.
  • the base sequence of the obtained DNA encoding the enzyme related to the fucose modification can be determined by a commonly used base sequence analysis method, for example, Sanger et al.'S dideoxy method [Procedurals ⁇ Ob ⁇ The 'National ⁇ Academia ⁇ ⁇ Ob Science (Pro Natl. Acad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems), etc.
  • the base sequence of DNA can be determined.
  • a homology search program such as BLAST is used to search base sequence databases such as Genbank, EMBL, and DDBJ. It can also be confirmed that the gene encodes an enzyme related to fucose modification.
  • nucleotide sequence of the gene encoding the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose obtained by the above method include the nucleotide sequence set forth in SEQ ID NO: 9 or 11.
  • N-glycoside-linked complex-type sugar chain-reducing terminal N-acetylcylcosamine obtained by the above-described method encodes an enzyme that encodes an enzyme involved in sugar chain modification in which position 1 of fucose is oc-bonded to position 6.
  • Examples of the base sequence include the base sequence described in SEQ ID NO: 13 or 14. It is done.
  • Examples of a method for preparing genomic DNA of an enzyme related to fucose modification include the methods described below.
  • genomic DNA of an enzyme related to fucose modification can be obtained by using a genomic DNA library screening system (GenomeSystems) or Unigen GenomeWalker TM Kits (CLONTECH).
  • the base sequence of the DNA can be determined.
  • nucleotide sequence of the genomic DNA of the enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose obtained by the above method is, for example, those described in SEQ ID NOs: 17, 18, 19, and 20. A base sequence is mentioned.
  • the base sequence of the genomic DNA of the enzyme involved in the sugar chain modification in which the 1-position of fucose is ⁇ -bonded to the 6-position of N-acetyldylcosamine at the N-glycoside-linked complex sugar chain reducing end obtained by the above method is, for example, Examples include the nucleotide sequence set forth in SEQ ID NO: 21.
  • the follicle stimulating hormone composition of the present invention can be obtained by directly introducing an antisense oligonucleotide or ribozyme designed based on the base sequence of an enzyme related to fucose modification without using an expression vector into a host cell. Obtaining host cells to be used for production.
  • Antisense oligonucleotides or ribozymes can be prepared by conventional methods or DNA synthesizers. Specifically, it corresponds to a continuous 5 to 150 bases, preferably 5 to 60 bases, more preferably 10 to 40 bases in the base sequences of cDNA and genomic DNA encoding the enzyme related to fucose modification. Based on the sequence information of the oligonucleotide having the sequence, an oligonucleotide (antisense oligonucleotide) corresponding to a sequence complementary to the oligonucleotide or a ribozyme containing the sequence of the oligonucleotide can be synthesized and prepared.
  • oligonucleotides include oligo RNA and derivatives of the oligonucleotide (hereinafter referred to as oligonucleotide derivatives).
  • Oligonucleotide derivatives include oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides are converted to ⁇ 3'- ⁇ 5 'phosphoramidate bonds.
  • Oligonucleotide derivatives oligonucleotide derivatives in which the ribose and phosphodiester bonds in the oligonucleotide are converted to peptide nucleic acid bonds, oligonucleotide derivatives in which the uracil in the oligonucleotide is replaced with C-5 propylene uracil, in the oligonucleotide Derivative uracil in which uracil is substituted with C-5 thiazoleuracil, cytosine in the oligonucleotide is substituted with C-5 propylcytosine, and the derivative is cytosine in the oligonucleotide.
  • Oligonucleotide derivatives substituted with enoxazine-modified cytosine oligonucleotide derivatives substituted with 2'-0-propylribose in the oligonucleotide, or Examples include oligonucleotide derivatives in which the ribose in the nucleotide is substituted with methoxyethoxyribose [Cell engineering, 16, 1463 (1997)].
  • a host cell used for preparing the follicle stimulating hormone composition of the present invention is prepared by targeting a gene of an enzyme related to fucose modification and modifying the target gene on the chromosome using a homologous recombination method. Can do.
  • Genomic DNA of an enzyme related to fucose modification is prepared.
  • Target gene to be modified based on the genomic DNA base sequence eg, related to fucose modification
  • a target vector for homologous recombination of the enzyme's structural gene or promoter gene is provided.
  • a host used for preparing the follicle stimulating hormone composition of the present invention by introducing the prepared target vector into a host cell and selecting a cell that has undergone homologous recombination between the target gene on the chromosome and the target vector. Cells can be made.
  • any yeast cell, animal cell, insect cell, plant cell, etc. can be used as long as it has a gene for an enzyme related to the target fucose modification.
  • Examples of the method for preparing genomic DNA of an enzyme related to fucose modification include the method for preparing genomic DNA described in (1) (a) of 1 above.
  • Enzyme involved in synthesis of intracellular sugar nucleotide GDP-fucose obtained by the above method As a base sequence of the genomic DNA of, for example, the base sequences described in SEQ ID NOs: 17, 18, 19, and 20
  • Target vectors for homologous replacement of the target gene on the chromosome were Gene Targ eting, A Practical Approach, IRLPress at Oxford University Press (1993), Noyoma-series 8 gene targeting, ES cells were used. Production of mutant mice (Yodosha) (1995) and the like.
  • the target vector can be either a replacement type or an insertion type.
  • Methods for efficiently selecting homologous recombinants include, for example, Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8 Gene Targeting, Production of Mutant Mice Using ES Cells (Sheep Methods such as positive selection, promoter selection, negative selection, poly A selection, etc. described in (Satoshisha) (1995) etc. can be used.
  • Methods for selecting the desired homologous recombinants from the selected cell lines include the Southern Hybridization Method (Molequila's Cloning 2nd Edition) for genomic DNA and the PCR method [PCR Protocols. (PCR Protocols), Academic Press (1990)].
  • the host cell used for producing the follicle-stimulating hormone composition of the present invention can be produced, for example, as follows using the RDO method targeting an enzyme gene related to fucose modification.
  • a host cell for producing the composition of the present invention is prepared by introducing a synthesized RDO into a host cell and selecting a transformant in which a target enzyme, ie, an enzyme related to fucose modification has been mutated. Can be produced.
  • a target enzyme ie, an enzyme related to fucose modification has been mutated.
  • a yeast cell As a host cell, a yeast cell, an animal cell, an insect cell, a plant cell, etc. can be used as long as it has a gene for an enzyme related to the target fucose modification.
  • Examples of the method for preparing cDNA of the enzyme related to fucose modification include the method for preparing cDNA described in (1) (a) of 1 above.
  • Examples thereof include a method for preparing genomic DNA as described in (1) (a).
  • the DNA base sequence is cleaved with an appropriate restriction enzyme, and then subcloned into a plasmid such as pBluescript SK (-) (Stratagene), and a commonly used base sequence analysis method such as Sanger ( Sanger) et al. [Procedures of the National Academia Sci., USA), 74, 5463 (1977)] This can be confirmed by analysis using an automatic base sequence analyzer, for example, a base sequence analyzer such as ABI PRISM377 DNA Sequencer (Applied Biosystems).
  • a base sequence analyzer such as ABI PRISM377 DNA Sequencer (Applied Biosystems).
  • RDO can be prepared by a conventional method or using a DNA synthesizer.
  • the method for selecting a transformant using the activity of an enzyme related to the introduced fucose modification described in (1) (a) of 1 above as an index, and the cell membrane described in (1) (5) below A method for selecting a transformant using the sugar chain structure of the above glycoprotein as an index, or a method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule described in 5 or 6 below as an index. Can also be used.
  • RDO constructs are described in Science, 273, 1386 (1996); Nichiya's Medicine (Nature Medicine), 4, 285 (1998); Hepatology, 25, 1462 (1997); Gene Therapy, 5, 1960 (1999); Gene Therapy, 5, 1960 (1999); Journal 'Ob' Molequila 'Medine 0. Mol. Med.), 75, 829 ( 199 7); Procedures 'Ob The National' Academy ⁇ ⁇ Ob Science (Proc. Natl. Acad. Sci. USA), 96, 8774 (1999); 'National Academia ⁇ ⁇ Ob' Science (Proc. Natl. Acad. Sci. USA), 96, 8768 (1999); Nuclidec 'A Sid' Research (Nuc. Acids.
  • the host cell used for preparing the follicle stimulating hormone composition of the present invention can be prepared as follows by targeting the gene of an enzyme related to fucose modification and using the RNAi method.
  • RNAi gene construct of appropriate length that includes the untranslated region.
  • a thread recombination vector is prepared by inserting the prepared cDNA fragment or full length downstream of the promoter of an appropriate expression vector.
  • a transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • the follicle stimulating hormone composition of the present invention is prepared by selecting a transformant using as an index the activity of the enzyme related to the introduced fucose modification or the glycoprotein structure of the produced glycoprotein molecule or cell surface glycoprotein. Host cells used for the purpose can be obtained.
  • any yeast cell, animal cell, insect cell, plant cell, etc. having an enzyme gene related to the target fucose modification can be used.
  • the host cells described in 3 below can be mentioned.
  • RNAi gene a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where the designed RNAi gene can be transcribed is used.
  • the expression vector described in 3 below can be mentioned.
  • Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the method described in (a) of (1) in this section 1.
  • Examples of a method for selecting a transformant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (5) of this section 1. Examples of the method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
  • Examples of a method for preparing cDNA of an enzyme related to fucose modification include the method for preparing cDNA described in (1) (a) of this section 1.
  • the follicle stimulating hormone of the present invention is used. It is also possible to obtain host cells that are used to make the Hmong composition.
  • RNAi gene can be prepared by a conventional method or using a DNA synthesizer.
  • RNAi gene construct is described in (Nature, 391,806 (1998); Proc. Of the Ob. The 'National' De force of Ob 'Science (Proc. Natl. Acad. Sci. USA), 95. , 15502 (1998); Nature, 395, 854 (1998); Proceedings 'Ob' The National 'De force 1' Science (Proc. Natl. Acad. Sci. USA), 96, 5049 (19 99); Cell, 95, 1017 (1998); Procedurals 'Ob The National' A Power Demi ⁇ Ob Science (Proc. Natl. Acad. Sci. USA ), 96, 1451 (1999); Proceedings 'Ob The' National 'Academia ⁇ ⁇ Ob' Science (Proc. Natl. Acad. Sci. USA), 95, 13959 (1998); The cell can be designed in accordance with the description of Nature Cell Biol, 2, 70 (2000)].
  • the host cell used for preparing the follicle stimulating hormone composition of the present invention is an enzyme related to fucose modification using the transposon system described in Nature Genet., 25, 35 (2000), etc.
  • a host cell used to produce the follicle-stimulating hormone composition of the present invention by selecting a mutant using as an index the activity of the protein, or the glycoprotein structure of the produced glycoprotein molecule or the glycoprotein on the cell membrane. be able to.
  • the transposon system is a system that induces mutations by randomly inserting foreign genes onto the chromosome, and is usually used as a vector to induce mutations in foreign genes inserted into transposons.
  • a transposase expression vector for randomly inserting the gene into the chromosome is introduced into the cell at the same time.
  • a transposase can be used if it is suitable for the transposon sequence used!
  • any gene can be used as long as it induces mutation in the DNA of the host cell.
  • Host cells include yeast, animal cells, insect cells, plant cells, etc. If you have a gene for an enzyme related to the modification, you can also use the deviation
  • the host cells described in 3 below can be mentioned.
  • the recombinant vector introduction method suitable for various host cells described in 3 below can be used.
  • Examples of a method for selecting a mutant using as an index the activity of an enzyme related to fucose modification include the method described in (1) (a) of this section 1.
  • Examples of a method for selecting a mutant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (5) of this section 1. Examples of the method for selecting a mutant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
  • a host cell used for preparing the follicle stimulating hormone composition of the present invention can be prepared by using a technique for targeting a gene of an enzyme related to fucose modification and introducing a dominant negative form of the enzyme.
  • Specific examples of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx. N-glycoside-linked complex sugar
  • enzymes involved in sugar chain modification in which the 1-position of fucose is ⁇ -linked to the 6-position of N-acetyl darcosamine at the chain-reducing end include 1,6-fucosyltransferase, a -L- Fuco
  • These enzymes are enzymes that catalyze a specific reaction having substrate specificity, and these enzymes are destroyed by destroying the active center of the catalytic activity having substrate specificity. A dominant negative form of the enzyme can be prepared. G of target enzymes
  • a dominant negative By substituting these four amino acids, a dominant negative can be produced. Based on the results of the preparation of dominant negative GMD derived from E. coli, by comparing homology and predicting the three-dimensional structure based on amino acid sequence information, for example, GMD derived from CHO cells (SEQ ID NO: 10) By replacing the 155th threonine, the 157th glutamic acid, the 179th tyrosine, and the 183rd lysine with other amino acids, a dominant negative body can be prepared. Genes with such amino acid substitutions were created by site-specific mutations described in Molecular 2 ⁇ Cloning 2nd edition, Current 'Protocols' in' Molecular ⁇ ⁇ This can be done using the introduction method.
  • the host cell used to produce the follicle-stimulating hormone composition of the present invention uses a gene encoding a dominant negative form of the target enzyme produced as described above (hereinafter abbreviated as a dominant negative form gene). According to the method of gene transfer described in Molecular 'Crowing 2nd Edition, Current' Protocorenoles' In 'Molecular' Biology, Manipulating 'Mouse' Enbrio 2nd Edition, etc. Can be produced.
  • a dominant negative gene of an enzyme related to fucose modification is prepared.
  • a DNA fragment of an appropriate length containing a portion encoding the protein is prepared.
  • a recombinant vector is prepared by inserting the DNA fragment or full-length DNA downstream of the promoter of an appropriate expression vector.
  • a transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • the follicle stimulation of the present invention is selected by selecting a transformant using as an index the activity of the enzyme related to the fucose modification, or the glycoprotein structure of the glycoprotein molecule or glycoprotein on the cell membrane. Host cells used to make the hormone composition can be made.
  • any yeast cell, animal cell, insect cell, plant cell, etc. having an enzyme gene related to the target fucose modification can be used.
  • the host cells described in 3 below can be mentioned.
  • the expression vector is capable of autonomous replication in the above host cell or can be inserted into the chromosome, and can be transcribed at a position where the DNA encoding the desired dominant negative body can be transcribed.
  • Those containing a promoter are used. Specifically, the expression vector described in 3 below can be mentioned.
  • Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the method described in (a) of (1) below.
  • Examples of the method for selecting a transformant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (1) below. Examples of the method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
  • the host cell used for producing the follicle-stimulating hormone composition of the present invention is a method of introducing a mutation into a gene of an enzyme related to fucose modification and selecting a desired cell line in which the enzyme is mutated. Can be produced.
  • GMD As an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, specifically, GMD
  • [0121] As a method for introducing a mutation into an enzyme related to fucose modification, 1) from a mutant in which a parent strain was treated by a mutagenesis treatment or a naturally occurring mutant. , A method of selecting a desired cell line using as an index the activity of an enzyme related to fucose modification, 2) a production sugar from a mutant in which the parent strain was treated by mutagenesis treatment or a spontaneously occurring mutant. A method for selecting a desired cell line using the sugar chain structure of a protein molecule as an index, 3) from a mutant in which the parent strain was treated by mutagenesis or a naturally occurring mutant on the cell membrane of the cell. And a method of selecting a desired cell line by using the sugar chain structure of the glycoprotein as an index.
  • any treatment can be used as long as it induces a frameshift mutation in the DNA of the parent cell line.
  • Specific examples include treatment with ethyl nitrosourea, nitrosoguanidine, benzopyrene, and atalidine dye, and irradiation with radiation.
  • Various alkylating agents and carcinogens can also be used as mutagens. Examples of methods for causing a mutagenic agent to act on cells include, for example, tissue culture technology 3rd edition (Asakura Shoten) edited by the Japanese Society for Tissue Culture (1996), Nature Genet., 24, 314, (2000) and the like.
  • Examples of the method for identifying the sugar chain structure of the produced glycoprotein molecule include the methods described in 5 and 6 below.
  • Examples of the method for identifying the sugar chain structure of a glycoprotein on the cell membrane include the method described in 1 (5) of this section.
  • the host cell used to produce the follicle-stimulating hormone composition of the present invention targets the gene of an enzyme related to fucose modification, and antisense RNAZDNA technology [Bioscience and Industry, ⁇ , 322 (1992), Chemical , 681 (1991), Biotechnology, ⁇ , 358 (1 992), Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology, 10, 152 (1992), Cell engineering, 16, 1463 (1997)], Triple ⁇ Helix technology [Trends in Biotechnology, 10 , 132 (1992)] etc., and can be produced by suppressing transcription or translation of the target gene.
  • Specific examples of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx.
  • Specific examples of the enzyme involved in the sugar chain modification in which the 1-position of fucose is ⁇ - linked to the 6-position of N-glycidyl lucosamine at the N-glycoside-linked complex sugar reducing end include ⁇ ⁇ , 6-fucosyltransferase, Examples include ⁇ -L-fucosidase.
  • the host cell used to produce the stimulating hormone composition is a lectin that recognizes the sugar chain structure in which the N-glycidyl-linked sugar chain reducing terminal N-acetylcylcosamine 6-position and fucose 1-position are linked.
  • the strain can be produced by using a technique for selecting a strain resistant to.
  • any lectin can be used as long as it recognizes a sugar chain structure in which the N-glycidylcolcamine 6-position of the N-glycoside-linked sugar chain reducing end and the 1-position of fucose are a- linked.
  • Specific examples include Lentil lectin LCA (Lentil Agglutinin from L ⁇ Culinaris) Endumame lectin PSA (Peum sativum-derived Pe a Lectin), Broad bean lectin VFA (Agglutinin from Viciafaba), and Hirochawantake lectin AAL (Lectin derived from Aleuria aurantia) and the like.
  • the cells are cultured in a medium containing the above-mentioned lectin at a concentration of 1 ⁇ g / mL to 1 mg / mL for 1 to 2 weeks, preferably 1 to 1 week, and surviving cells are passed.
  • Subculture or colonies are picked up, transferred to another culture vessel, and further cultured in a medium containing lectin, whereby the N-glycidyl-linked sugar chain reducing terminal N-acetylyldarcosamine of the present invention and position 1 of fucose Resistant to lectins that recognize a-linked sugar chain structures Stocks can be selected.
  • Transgenic non-human animals or plants or their progeny whose genomic genes have been modified so that the activity of the enzyme involved in the modification of the follicle-stimulating hormone sugar chain is controlled, synthesis of intracellular sugar nucleotide GDP-fucose Targeting the gene of the enzyme involved in glycosylation, in which the 1-position of fucose is ⁇ -linked to the 6-position of the N-glycosidic complexed sugar chain reducing N-acetylyldarcosamine From the embryonic stem cells, fertilized egg cells, and plant callus cells of the present invention produced using the above, for example, they can be produced as follows.
  • target non-human animals such as ushi, hidge, goats, pigs, horses, mice, rats, -embryonic stem cells such as rabbits, monkeys, and rabbits.
  • target non-human animals such as ushi, hidge, goats, pigs, horses, mice, rats, -embryonic stem cells such as rabbits, monkeys, and rabbits.
  • a chimera that has normal cell strength and embryonic stem cell clones by a method such as an injection chimera method or an assembly chimera method into a blastcyst of an animal fertilized egg Individuals can be prepared.
  • a method such as an injection chimera method or an assembly chimera method into a blastcyst of an animal fertilized egg
  • Individuals can be prepared.
  • Transgenic non-human animals can be obtained in which the activity of the enzyme involved in sugar chain modification in which position 1 of fucose is a- linked is reduced.
  • target non-human animals such as ushi, hidge, goat, pig, horse, mouse, rat, two
  • fertilized egg cells such as chickens, monkeys, and rabbits
  • the activity of enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose or N-glycoside-linked complex sugars The fertilized egg cell of the present invention in which the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is oc-bonded to the 6-position of N-acetyl darcosamine at the chain reducing end can be produced.
  • the produced fertilized egg cells are transplanted into the oviduct or uterus of a pseudopregnant female using the embryo transfer method described in the Mapurating 'Mouse' Embryo 2nd Edition, etc., to give birth to intracellular sugar.
  • Nucleotide GDP-enzyme activity involved in the synthesis of fucose or N-glycoside-linked complex sugar chain-reducing terminal N-acetylyldarcosamine Transgenic non-human animals with reduced activity can be produced.
  • the enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose is used for the target plant strength or cells.
  • the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is a- linked to the 6-position of the N-glycidarcosamine at the reducing end of the active or N-glycoside-linked complex sugar chain Can do.
  • the follicle-stimulating hormone composition of the present invention is composed of a molecular 'clawing 2nd edition, current' protocorores 'in' molecular.Nology, Antibodies, ALaboratory manual, Cold Spring Harbor Laboratory, 1988 (hereinafter abbreviated as antibodies).
  • antibodies Monoclonal An tioodies: principles and practice, Third Edition, Acaa.Press, 1993 (below, Monochrome ⁇ Nanole Antibodies), AntibodyEngineering, A Practical Approach, IRL Press Using the method described in at Oxford University Press, 1996 (hereinafter abbreviated as antibody engineering), etc., for example, it can be expressed in a host cell as follows.
  • a full-length cDNA encoding each subunit of the follicle-stimulating hormone molecule is prepared, and a DNA fragment of an appropriate length containing a portion encoding each subunit of the follicle-stimulating hormone molecule is prepared.
  • a recombinant vector may be prepared by introducing cDNA encoding each subunit separately, or a recombinant vector in which cDNA encoding each subunit is connected in tandem may be prepared.
  • a transformant producing a follicle stimulating hormone molecule By introducing the recombinant vector into a host cell suitable for the expression vector, a transformant producing a follicle stimulating hormone molecule can be obtained.
  • any yeast cell, animal cell, insect cell, plant cell, etc. that can express the target gene can be used.
  • the expression vector can replicate autonomously in the above host cell or can be integrated into the chromosome, and can be promoted to a position where DNA encoding the desired follicle stimulating hormone molecule can be transcribed.
  • the one containing 1 ter is used.
  • a probe specific to the target follicle stimulating hormone molecule is obtained from a tissue or cell of a human or non-human animal. Etc. can be used.
  • yeast When yeast is used as a host cell, examples of the expression vector include YEP13 (ATC C37115), YEp24 (ATCC37051), YCp50 (ATCC37419) and the like. Any promoter can be used as long as it can be expressed in yeast strains. For example, promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP promoter, ADH promoter Gall promoter, gal 10 promoter, heat shock protein promoter, MF al promoter, CUP 1 promoter and the like.
  • promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP promoter, ADH promoter Gall promoter, gal 10 promoter, heat shock protein promoter, MF al promoter, CUP 1 promoter and the like.
  • Examples of host cells include microorganisms belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth genus, Trichosporon, Schu-omyces, Pichia, etc., for example, Saccharom vces cerevisiae. Achizosaccharomvces pombe, Kluweromvces lactis. Tnchosporon pullulans, Schwanniomvces alluvius, Pichia pastoris, etc.
  • the method is to introduce DNA into yeast, the deviation is used.
  • the electo mouth position method [Met hods. Enzymol., 194, 182 (1990)]
  • the spheroplast method [Procedinas' of the National. Sci. USA, 84, 1929 (1978)]
  • Lithium acetate method [Journal of Bacteriology, ⁇ 53, 163 (1983)]
  • Proceedings 'Ob The National' Academia Sob. (Proc. Natl. Acad. Sci. USA), 75, 1929 (1978)].
  • examples of expression vectors include pcDNAU pcD M8 (commercially available from Funakoshi), pAGE107 [JP 3-22979; Cytotechno logy, 3, 133, ( 1990)], pAS3-3 [JP-A-2-227075], pCDM8 [Nature, 22 £, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 (Journal) ⁇ Ob 'Biochemistry 0. Biochemistry), dish, 1307 (1987)], pAGE210, etc.
  • Any promoter can be used as long as it can be expressed in animal cells.
  • a promoter of the cytomegalovirus (CMV) IE (immediateearly) gene an early promoter of SV40, a retroinores promoter , Meta-mouthone promoter, heat shock promoter, SRa promoter, and the like.
  • CMV cytomegalovirus
  • Any recombinant vector can be introduced by introducing DNA into animal cells.
  • the electopore position method [Cytotechnology, 3, 133 (1990)]
  • the calcium phosphate method Japanese Patent Laid-Open No. 2-227075
  • the lipofuxion method [Proceedings 'Ob The' National 'Academia ⁇ Science' (Proc. Natl. Acad. Sci. USA), 84, 7413 (1987)]
  • injection method [Mapleating the 'Mouse' Embryo Laboratory Laboratory Manual]
  • Particle Method of using a cancer (gene gun) Patent No. 2606856, Patent No.
  • the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the protein. it can.
  • Examples of the gene transfer vector used in the method include pVL1392, pVLl393, pBlueBacIII (both from Invitorogen) and the like.
  • the baculovirus e.g., burglar Gaka insect is a virus that infects Autogu La 'Karifuoru - Power Nuclear one poly to Doroshisu virus (Autographacalifornica nucl ear polyhedrosis virus) force 21 e monkey be used like.
  • Insect cells are Spodopterafrugiperda ovarian cells S19, S1 1 [Current 'Pro Toko ⁇ Norezu in 'Molekiyura 1 ⁇ ' Noroji 1 ⁇ Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)], Trichoplusiani's nest cell, High 5 (Invitrogen ) Etc. can be used.
  • Examples of methods for co-introducing the above recombinant gene transfer vector and the above baculovirus into insect cells for preparing recombinant viruses include, for example, the calcium phosphate method (JP-A-2-227075), the lipofusion method [Proceedings' ⁇ The 'National' Academia's Science (Pro Natl. Acad. Sci. USA), 84, 7413 (1987)].
  • expression vectors include Ti plasmids and tobacco mosaic virus vectors. Any promoter can be used as long as it can be expressed in plant cells. Examples thereof include the 35S promoter of califlora mosaic virus (CaMV) and the actin 1 promoter.
  • CaMV califlora mosaic virus
  • host cells include tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, barley, and other plant cells.
  • any method can be used as long as it is a method for introducing DNA into plant cells.
  • Agrobacterium JP 59-140 885, JP No. 60-70080, WO94 / 00977
  • an elect mouth position method Japanese Patent Laid-Open No. 60-251887
  • a method using a particle gun (gene gun) Japanese Patent No. 25 17813.
  • the transformant of the present invention obtained as described above is cultured in a medium, the follicle stimulating hormone composition of the present invention is produced and accumulated in the culture, and the composition is collected from the strength of the culture. By doing so, a follicle stimulating hormone composition can be produced.
  • Transformant into medium The method for culturing can be carried out according to a conventional method used for culturing host cells.
  • a medium for culturing a transformant obtained by using a eukaryote such as yeast as a host it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism, so that the transformant can be cultured efficiently. If the medium can be used, the difference between natural and synthetic media can be used.
  • the carbon source may be glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolyzate, acetic acid, propionic acid, etc. as long as the organism can assimilate. Alcohols such as organic acids, ethanol, and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium salts, and other nitrogen-containing elements.
  • Compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof, and the like can be used.
  • inorganic salts monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride salt, ferrous sulfate, mangan sulfate, copper sulfate, calcium carbonate, etc. are used. be able to.
  • the culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is 15-40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH during the culture is maintained at 3.0 to 9.0.
  • the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
  • antibiotics such as ampicillin and tetracycline to the medium during culture.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium as necessary.
  • a microorganism transformed with a recombinant vector using the lac promoter when cultivating a microorganism transformed with isopropyl- ⁇ -D-thiogalatatopyranoside or the like with a recombinant vector using the trp promoter. Cultivate indole acrylic acid, etc. You may add it to the ground.
  • the culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C, and 5% CO.
  • antibiotics such as kanamycin and penicillin may be added to the medium as needed during the culture.
  • the culture media for transformants obtained using insect cells as hosts include the commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (LifeTechnologies), ExCell400, and ExCell405 (all JRH Biosciences), Grace's Insect Medium [Nature, I2S, 788 (1962)] and the like can be used.
  • Cultivation is usually carried out under conditions of pH 6-7, 25-30 ° C, etc. for 1-5 days.
  • antibiotics such as gentamicin may be added to the medium as needed during the culture.
  • Transformants obtained using plant cells as hosts are cultured as cells or differentiated into plant cells and organs. can do.
  • a medium for culturing the transformant commonly used Murashige 'and' Sturg (MS) medium, White medium, or these mediums are used.
  • a medium supplemented with plant hormones such as auxin and cytokinin can be used.
  • Cultivation is usually carried out under conditions of pH 5-9 and 20-40 ° C for 3-60 days.
  • antibiotics such as kanamycin and hygromycin are added to the medium. You may add to
  • a microorganism, animal cell, or plant cell-derived transformant having a recombinant vector incorporating a DNA encoding a follicle-stimulating hormone molecule is cultured according to a normal culture method, and follicle-stimulating hormone is obtained.
  • the follicle stimulating hormone composition can be produced by producing and accumulating the composition and collecting the follicle stimulating hormone composition from the culture.
  • the follicle-stimulating hormone composition can be produced in a host cell, secreted outside the host cell, or produced on the host cell membrane.
  • the method can be selected by changing the structure of the follicle stimulating hormone molecule to be produced.
  • DNA encoding a follicle stimulating hormone molecule and DNA encoding a signal peptide appropriate for the expression of a follicle stimulating hormone molecule are inserted into an expression vector, By expressing the follicle stimulating hormone molecule after introducing the expression vector into the host cell, the target follicle stimulating hormone molecule can be actively secreted outside the host cell.
  • the production amount can also be increased using a gene amplification system using a dihydrofolate reductase gene or the like. Furthermore, by redifferentiating the cells of the animal or plant into which the gene was introduced, an animal individual (transgenic non-human animal) or plant individual (transgenic plant) into which the gene was introduced was created, and these individuals were used. Thus, a follicle stimulating hormone composition can also be produced.
  • the transformant is an animal individual or a plant individual, it is reared or cultivated according to a usual method to produce and accumulate a follicle stimulating hormone composition, and the follicle stimulating hormone composition is produced from the animal individual or plant individual. By collecting the product, the follicle stimulating hormone composition can be produced.
  • a transgenic non-human animal introduced with DNA encoding a follicle-stimulating hormone molecule is bred, and a follicle-stimulating hormone composition is produced and accumulated in the animal.
  • a follicle stimulating hormone composition can be produced.
  • the place to be produced and accumulated in the animal include milk of the animal (JP-A 63-309192), eggs and the like.
  • Any promoter can be used as long as it can be expressed in animals.
  • ⁇ -casein promoter, ⁇ -force zein promoter, ⁇ -lactoglobulin promoter, A whey acidic protein promoter or the like is preferably used.
  • a method for producing a follicle-stimulating hormone composition using an individual plant for example, a known method for transgenic plants into which DNA encoding a follicle-stimulating hormone molecule has been introduced [tissue culture, 2fi (1994); Culture, 21 (1995); Trends in Biotechnology, 15, 45 (1997)], and the follicle-stimulating hormone composition is produced and accumulated in the plant.
  • a method for producing a follicle stimulating hormone composition by collecting the follicle stimulating hormone composition can be mentioned.
  • the follicle stimulating hormone composition produced by the transformant into which the gene encoding the follicle stimulating hormone molecule is introduced for example, when expressed in a dissolved state in the cell, Cells are collected by centrifugation and aqueous buffer After suspending, the cells are crushed with an ultrasonic crusher, French press, Manton Gaurin homogenizer, dyno mill, etc. to obtain a cell-free extract.
  • an ordinary enzyme isolation and purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion-exchange chromatography using resin such as tilaminoethyl (DEAE) -Sepharose and DIAIONHPA-75 (Mitsubishi Chemical Corporation), and cation using resin such as S-Sepharose FF (Pharmacia) Exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and ferrule sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing, etc.
  • a solvent extraction method e.g., a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent
  • Anion-exchange chromatography using resin such as tilaminoethyl (DEAE
  • a purified preparation of the follicle-stimulating hormone composition can be obtained by using methods such as electrophoresis methods described above alone or in combination. Specifically, a method that combines the anion exchange chromatography method and the affinity chromatography method developed in the production of recombinant FSH preparations produced in CHO cells can be cited (Human Reproduction Update 4, 862). (1998)).
  • the follicle-stimulating hormone composition When expressed in the form of an insoluble substance in the cells, the cells are similarly collected, disrupted, and centrifuged to obtain a follicle-stimulating hormone as a precipitate fraction. The insoluble material of the composition is recovered. The recovered insoluble material of the follicle stimulating hormone composition is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed to return the follicle stimulating hormone composition to a normal three-dimensional structure, and then a purified preparation of the follicle stimulating hormone composition is obtained by the same isolation and purification method as described above. be able to.
  • the follicle-stimulating hormone composition or a derivative thereof can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by the same method such as centrifugation as described above, and the follicle stimulating hormone is obtained from the culture supernatant by using the same isolation and purification method as described above. A fine preparation of the composition can be obtained.
  • the cell is prepared using the method described in 1 above, after preparing the cell having the ability to express a follicle stimulating hormone molecule.
  • the follicle stimulating hormone composition of the present invention can be produced by culturing and purifying the target follicle stimulating hormone composition from the culture. 4. Activity evaluation of follicle stimulating hormone composition
  • the biological activity including the follicle maturation-inducing activity of the purified follicle-stimulating hormone composition can be measured using various known methods. Specifically, binding activity to follicle-stimulating hormone receptor, induction of intracellular signal transduction, estrogen production-inducing activity and follicle maturation-inducing activity were measured in follicle-stimulating hormone receptor binding activity measurement test, intracellular signal transduction activity measurement.
  • a purified follicle stimulating hormone composition as a test substance and a commercially available follicle stimulating hormone of known concentration and specific activity are used as standard products.
  • Cells in which the expression vector pcDNA3 (Invitrogen) incorporating follicle-stimulating hormone receptor cDNA is stably transfected into the HEK293 cell line according to the method of Sohn et al. (Journal of Biological Chemistry 278, 47868 (2003)) Create a strain. After recovering the follicle-stimulating hormone receptor-expressing HEK293 cells from the incubator, the cells are washed with ice-cooled buffer A (150 mM NaCl, 20 mM HEPES, pH 7.4).
  • Cells are lysed in buffer A containing l% Nonidet P-40 and 20% glycerol and protease inhibitors (ImM phenylmethylsulfonyl fluoride, 5 mM ethlmaleimide and lOmM EDTA) to solubilize the ovarian stimulating hormone receptor protein.
  • glycerol and protease inhibitors ImM phenylmethylsulfonyl fluoride, 5 mM ethlmaleimide and lOmM EDTA
  • buffer ⁇ containing 5 ⁇ g / ml ushi gamma globulin and 20% polyethylene glycol 8000. Incubate for 10 minutes at 4 ° C. Measure the radioactivity of the pellet fraction obtained by centrifugation. The radioactivity in the absence of competitor is taken as 100, and the relative radioactivity of each sample is calculated. This relative radioactivity indicates a relative affinity for the solubilized follicle stimulating hormone receptor, and the follicle stimulating hormone receptor binding activity of the test substance can be measured.
  • Rat ovarian force Prepared granulosa cells, seminiferous tubule cells prepared from rat testis (Sertri cells) or Sertoli cell-derived strain TM4 (ATCC CRL-1715) are cultured, and the follicle stimulating hormone composition is used as the test substance. Add commercially available follicle stimulating hormone of known concentration and specific activity as a standard. After incubating at 37 ° C for several minutes to several hours, the concentration of cAMP, one of the intracellular signal mediators, can be determined by using commercially available cAMP ELISA kit, cAMP Biotrak Enzyme immunoassay system (Amersham Biosciences), etc. Internal signal transduction activity can be measured.
  • the estrogen production-inducing activity of follicle stimulating hormone can be measured according to the method of Jia et al. (Neuroendocrinolog y 41, 445 (1985)). Rat ovarian force Prepared granulosa cells, seminiferous tubule cells (Sertoli cells) prepared from rat testis or Sertoli cell-derived strain TM4 (ATCC CRL-1715), etc. are cultured, and follicle stimulating hormone composition is used as a test substance. A commercially available follicle stimulating hormone with a known concentration and specific activity is added as a product. Then, add 500 nM androstenedione (Sigma) as an estrogen synthesis substrate and incubate at 37 ° C for several hours.
  • estrogen production-inducing activity of the test substance is determined by quantifying the concentration of estrogen secreted in the culture supernatant with a commercially available estrogenizer kit, Estrone ELISA kit (Nippon Enviguchi Chemicals), etc. ] Can be determined.
  • the follicle maturation-inducing activity by follicle-stimulating hormone can be measured by examining the change in the weight of the ovary or uterus in an in vivo test using an animal model using a purified follicle-stimulating hormone composition or the like as a test substance.
  • Model animals include young rats and mice Or hypophysectomized rats and mice (Endocrinology 53,604 (1953), Journal of Endocrinology 35, 199 (1966), Endocrinology 74,440 (1964)). Concretely
  • the test can be performed by the following procedure.
  • a test substance prepared in 0.25% rat serum albumin or a non-test substance-containing placebo (PBS containing 0.25% rat serum albumin) is administered once by intravenous injection.
  • the dosage of the follicle stimulating hormone composition is, for example, 0.1 ⁇ g / heac! It can be set between ⁇ 1.0 ⁇ g / head.
  • Blood is collected every 12 hours after administration, blood estrogen concentration is measured, and ovary or eclampsia is removed 48 to 72 hours after the administration of follicle stimulating hormone and weighed.
  • serum is collected from the rat administered with test substance, and the anti-follicle-stimulating hormone antibody appears by administration of the test substance, and it is determined that it is possible to test it.
  • Measurement of the blood half-life using the purified follicle-stimulating hormone composition can be carried out using a model animal such as a rat. Contains no test substance or test substance prepared in 0.25% rat serum albumin! / Acupuncture placebo (PBS containing 0.25% rat serum albumin) is administered as a single dose by intravenous injection or subcutaneous injection using carotid artery force-Eure.
  • the amount of follicle stimulating hormone composition to be administered is, for example, 1 ⁇ g / heac! Can be set between ⁇ 10 ⁇ g / head.
  • test substance in rat blood is obtained by ELISA method (such as the human FSH ELISA kit manufactured by Alpha Diagnostic International al) that can specifically detect and quantify human follicle-stimulating hormone. Measure the concentration.
  • the obtained data can be analyzed for pharmacokinetic parameters using, for example, PCNONLIN nonlinear regression analysis (Statistical Consultants, 1992), and the blood half-life can be calculated.
  • the clearance test for follicle-stimulating hormone compositions can also be evaluated using rodents other than rats, such as mice, and animal species models such as primates that are more closely related to humans, such as force-quizal. .
  • sugar chain structure of follicle stimulating hormone molecules expressed in various cells is the same as that of normal glycoproteins. It can be performed according to the analysis of the sugar chain structure.
  • sugar chains bound to follicle-stimulating hormone molecules are composed of neutral sugars such as galactose and mannose, amino sugars such as N-acetyldarcosamine, and acidic sugars such as sialic acid. It can be performed using a method such as a sugar chain structure analysis using a two-dimensional sugar chain map method.
  • neutral sugar or amino sugar can be liberated by performing acid-hydrolysis of the sugar chain with trifluoroacetic acid or the like, and the composition ratio can be analyzed.
  • composition ratio can also be analyzed by a fluorescent labeling method using 2-aminoviridine. Specifically, a sample hydrolyzed according to a known method [Agricultural 'and' Biological Chemistry (Agric. Biol. Chem.), 55il), 283-284 (1991)] was converted to 2-aminobilidyl. Fluorescent labeling can be performed using HPLC analysis and the composition ratio can be calculated.
  • Structural analysis of glycans in follicle-stimulating hormone molecules is based on two-dimensional glycan mapping method [Anal. Biochem., 171, 73 (1988), Biochemical Experimental Methods 23-Glycoprotein Glycan Research Law (Academic Publishing Center) Etsuko Takahashi (1989)].
  • 2D glycan mapping method for example, the retention time or elution position of glycans by reverse phase chromatography is plotted on the X axis, and the retention time or elution position of glycans by normal phase chromatography is plotted on the vertical axis. It is a method to estimate the sugar chain structure by plotting and comparing with the results of known sugar chains.
  • hydrazine decomposition of the follicle stimulating hormone composition releases sugar chains from the follicle stimulating hormone molecules, and the sugar chain fluorescence by 2-aminoviridine (hereinafter abbreviated as “ ⁇ ”).
  • ⁇ 2-aminoviridine
  • the glycan is separated from excess PA reagent by gel filtration and reverse phase chromatography is performed. .
  • normal phase chromatography is performed on each peak of the separated sugar chain.
  • mass analysis such as MALDI-TOF-MS of each sugar chain can be performed to confirm the structure estimated by the two-dimensional sugar chain mapping method.
  • the follicle stimulating hormone composition is composed of follicle stimulating hormone molecules having different sugar chain structures.
  • the genetically modified follicle stimulating hormone composition of the present invention is characterized in that fucose is not bound to ⁇ -glycylside reducing end ⁇ -acetyldarcosamine and exhibits a long-term half-life in blood. is doing.
  • Such a follicle stimulating hormone composition can be identified by using the method for analyzing the sugar chain structure of the follicle stimulating hormone molecule described in 5. above. It can also be identified by using an immunological quantification method using a lectin.
  • a lectin that recognizes the sugar chain structure of the follicle-stimulating hormone molecule constituting the follicle-stimulating hormone composition is labeled, and the labeled lectin is reacted with the sample follicle-stimulating hormone composition. Next, the amount of the complex of labeled lectin and follicle stimulating hormone molecule is measured.
  • Examples of lectins used to identify the sugar chain structure of follicle-stimulating hormone molecules include WGA u '. Vulgaris-derived wheat— germ agglutinin) ⁇ ConA (and concana valin A from ensiformis), RIC (R.
  • L— PHA leukoagglutinin from P. vulgaris
  • LCA lentil agglutinin from L. culinaris
  • PSA Pa lectin from P. sativum
  • AAL Aleuriaaurantia Lectin with ACL (Amaranthus caudatus Lectin) ⁇ BPL (Bauninia purpure a Lectin) ⁇ DSL (Datura stramonium Lectin) ⁇ DBA (Dolichos biflorus Agglutinin) ⁇ EB "Elderberry Balk Lectin) ⁇ ECL (Erythrina cristagalli Lectin) ⁇ EEL (Euonymuseurop aeus Lectin) ⁇ GNL (Galanthus nivalis Lectin) ⁇ GSL (imp Lectin) ⁇ H PA (Helix pomatia Agglutinin
  • a lectin that specifically recognizes a sugar chain structure in which fucose is bound to N-acetylcolcamine at the N-darcoside-linked complex sugar chain reducing end examples include lentil lectin LCA (Lentil Agglutinin from Lens Culinaris), endangered lectin PSA (Peasum sativum-derived Pea Lectin), broad bean lectin VFA (Agglutinin from Vicia faba), hirochawantake lectin AAL (Aleuria aurantia) Lectin).
  • the recombinant follicle-stimulating hormone composition of the present invention is conventionally known! / Fucose binds to N-glycidyl-linked N-acetyl darcosamine at the N-glycoside-linked complex sugar chain reducing end such as human urine-derived follicle stimulating hormone. Therefore, the present invention has the same affinity for the follicle stimulating hormone receptor as compared to the follicle stimulating hormone, and has a long half-life in blood when administered in vivo. In the treatment of various diseases using the above follicle stimulating hormone composition, the number of administration without increasing the dose can be reduced.
  • the follicle-stimulating hormone composition of the present invention enables treatment with fewer administrations without increasing the dose in various diseases to which treatment with follicle-stimulating hormone is indicated. This alleviates the burden on the patient and the medical site and must be administered frequently, resulting in accidents resulting from current treatment, or ovarian hyperstimulation syndrome or anti-follicles. It can contribute to the reduction of side effects such as the appearance of stimulating hormone antibodies.
  • Diseases to which treatment with the follicle-stimulating hormone composition of the present invention is indicated include diseases caused by a decrease in the production of follicle stimulating hormone.
  • follicle-stimulating hormone Diseases that develop due to a decrease in the production of follicle-stimulating hormone include infertility, and specifically include follicular maturation disorders, ovulation disorders, spermatogenesis disorders, and sperm maturation disorders.
  • the medicament containing the follicle-stimulating hormone composition of the present invention can be administered alone as a prophylactic or therapeutic agent S, usually one or more pharmacologically acceptable. It is desirable to provide it as a pharmaceutical formulation produced by any method well known in the pharmaceutical arts, mixed with a carrier.
  • the route of administration includes oral administration, where it is desirable to use the most effective treatment, or parenteral administration, such as buccal, airway, rectal, subcutaneous, intramuscular and intravenous.
  • parenteral administration such as buccal, airway, rectal, subcutaneous, intramuscular and intravenous.
  • a follicle-stimulating hormone preparation it can be preferably administered subcutaneously or intravenously.
  • dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes, gums and the like.
  • Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  • Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol, and fructose, Daricols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, P- Preservatives such as hydroxybenzoates
  • Flavors such as laver and peppermint can be used as additives.
  • Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc. It can be produced using a binder such as an agent, polybulal alcohol, hydroxypropylcellulose, gelatin, a surfactant such as a fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • Suitable preparations for parenteral administration include injections, suppositories, sprays and the like.
  • the injection is prepared using a carrier such as a salt solution, a glucose solution, or a mixture of both.
  • a powder injection can be prepared by freeze-drying a follicle stimulating hormone composition according to a conventional method and adding sodium chloride thereto.
  • Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  • the spray does not irritate the follicle stimulating hormone composition itself, or the recipient's oral cavity and airway mucosa, and the follicle stimulating hormone composition is dispersed as fine particles for absorption. It is prepared using a carrier or the like that facilitates.
  • the carrier include lactose and glycerin.
  • these parenteral agents can be obtained by adding the components exemplified as additives in the oral agent.
  • the dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, weight, etc., but the amount of active ingredient is usually 50 to 500 international units per day for an adult.
  • methods for measuring biological activity including follicle maturation-inducing activity of follicle-stimulating hormone compositions include in vitro experiments for binding activity to follicle-stimulating hormone receptor, measurement of intracellular signal transduction activity, estrogen production-inducing activity.
  • in vivo tests using model animals include follicle maturation-inducing activity measurement tests.
  • Example 1 Expression of human follicle stimulating hormone by FUT8 gene double knockout cell line
  • a serum-free conditioned FUT8 gene double knockout cell line producing the follicle stimulating hormone composition of the present invention was prepared by the method described below.
  • the solution is subjected to 1.5% (W / V) agarose gel electrophoresis to confirm the DNA fragment of the approximately 350 bp FSH ⁇ gene.
  • a 20 L reaction solution was prepared by adding 10 units of BamHI (Takara Bio) and 2 ⁇ L of 10 X H buffer, followed by digestion at 37 ° C for 16 hours. Subsequently, 3 ⁇ g of plasmid p BluescriptIIKS (+) (Stratagene) is dissolved in 17.5 ⁇ L of water, 10 units of EcoRI and 2 L of 10 XH buffer are added to the solution, and 20 L of reaction solution is added. After preparation, digestion reaction was performed at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 L of water. Further, 10 units of BamHI and 2 L of 10 X K buffer were added to the solution to prepare a 20 L reaction solution, followed by digestion at 37 ° C for 16 hours.
  • FSH a DNA fragment (EcoRI-BamHI) and pBluescriptll KS (+) fragment (EcoR) obtained above I-BamHI) was subjected to 1.5% (W / V) agarose gel electrophoresis, and DNA fragments of about 350 bp and 3 kbp were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
  • E. coli DH5 a strain manufactured by Toyo Boseki Co., Ltd.
  • E. coli DH5 a strain was transformed by heatshock method.
  • Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDyeTerminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM377 (Applied Biosystems) ) was used to analyze the nucleotide sequence.
  • QIAprep® Spin Miniprep Kit QIAGEN
  • BigDyeTerminator Cycle Sequencing Ready Reaction Kit v2.0 QIAGEN
  • DNA sequencer ABI PRISM377 Applied Biosystems
  • the FSH a DNA fragment (EcoRI-BsiWI) and pKANTEX93 fragment (EcoRI-BsiWI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and about 350 bp and 13 kbp DNA fragments were obtained. It refine
  • a reaction solution 20 / z L containing 50 ng of FSH a DNA fragment (EcoRI-BsiWI), 30 ng of pKANTEX93 fragment (EcoRI-BsiWI) and LigationHigh (manufactured by Toyobo) was prepared, and ligation reaction was performed at 16 ° C for 16 hours.
  • Obtained Escherichia coli DH5 ⁇ strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method using the resulting plasmid DNA. From the transformation, plasmid DNA was prepared using QIAprep® Spin Miniprep Kit (manufactured by QIAGEN) to obtain pKAN-FSHA.
  • FSH ⁇ Two types of FSH ⁇ added with restriction enzyme sites (EcoRI / NotI, BamHI) and Kozak sequence from human follicle stimulating hormone ⁇ subunit (hereinafter referred to as FSH ⁇ ) gene sequence (UniGene: Hs.36975, SEQ ID NO: 3) Gene-specific primers (SEQ ID NO: 24 and SEQ ID NO: 25) were prepared and subjected to the following PCR.
  • reaction solution containing human pituitary cDNA as a template (HotstarTaq (R) DNA polymerase (QIAGEN), 10 X PCR buffer, 0.2 mmol / L dNTP mixture, 0.5 ⁇ mol / L above) Primer (SEQ ID NO: 24 and SEQ ID NO: 25)], heated at 95 ° C for 15 minutes, then 1 cycle at 94 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 1 minute PCR was performed in 35 cycles of the reaction.
  • HotstarTaq (R) DNA polymerase (QIAGEN) QIAGEN
  • 10 X PCR buffer 0.2 mmol / L dNTP mixture, 0.5 ⁇ mol / L above
  • Primer SEQ ID NO: 24 and SEQ ID NO: 25
  • reaction solution was subjected to 1.5% (W / V) agarose gel electrophoresis to confirm a DNA fragment of about 400 bp FSH ⁇ subunit gene and purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
  • Reaction I did it.
  • E. coli DH5 a strain (manufactured by Toyo Boseki Co., Ltd.) was transformed by heatshock method.
  • Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDyeTerminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM377 (Applied Biosystems) ) was used to analyze the nucleotide sequence.
  • QIAprep® Spin Miniprep Kit QIAGEN
  • BigDyeTerminator Cycle Sequencing Ready Reaction Kit v2.0 QIAGEN
  • DNA sequencer ABI PRISM377 (Applied Biosystems)
  • PBS-FSH 8 3 / ⁇ obtained in 3 above is dissolved in 13.5 L of water, 10 units of NotI (Takarabio), 2 ⁇ L of 0.1% BSA, 2 ⁇ L of 0.1% TritonX-100, 2 ⁇ L of 10 XH buffer was added to prepare a reaction solution of L, and digestion reaction was performed at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 ⁇ L of water. Further, 10 units of BamHI and 2 ⁇ L of 10 ⁇ K buffer were added to the solution to prepare a 20 L reaction solution, followed by digestion reaction at 37 ° C. for 16 hours.
  • the plasmid pKAN-FSH ⁇ obtained in Example 4 was dissolved in 27 ⁇ L of water, and 20 units of Notl (manufactured by Takara Bio Inc.), 4 ⁇ L of 0.1% BSA B 4 ⁇ L of 0.1% TritonX—100 and 4 ⁇ L of 10 XH buffer were added to prepare a 40 L reaction solution, which was digested at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 ⁇ L of water. Further, 10 units of BamHI and 2 ⁇ L of 10 ⁇ K buffer were added to the solution to prepare a 20 ⁇ L reaction solution, followed by digestion reaction at 37 ° C. for 16 hours.
  • the FSH ⁇ DNA fragment (Notl-BamHI) and the pKAN-FSHA fragment (Notl-BamHI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and each of about 400 bp and 12 kbp.
  • the DNA fragment was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, prepare a 20 / zL reaction solution containing 50 ng of FSH jS DNA fragment (Notl-BamHI), 30 ng of pKAN-FSHA fragment (Notl-BamHI) and Ligation High (manufactured by Toyobo) at 16 ° C for 16 hours.
  • FUT8 gene double knockout cells described in the literature (Biotechnology and Bioengineering 87, 614 (2004)) were mixed with K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8.1 mmol / L). Na HPO, 1.5mmol / L KH PO
  • IMDM medium supplemented with 100 ⁇ l / well was added.
  • the culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days.
  • culture was repeated for 18 days by repeating the medium exchange operation using IMDM medium supplemented with 10% urine fetal dialyzed serum, 50 ⁇ g / mL gentamicin and 200 nM MTX, every 3-4 days.
  • the mouthpiece that was finally formed was replanted to a 24 well plate (Sigma).
  • 10% Ushi fetal dialysis serum, Medium replacement using IMDM medium supplemented with 50 ⁇ g / mL gentamicin and 500 nM MTX is repeated every 3-4 days, and cultured for 19 days with appropriate expansion to obtain a 500 nM MTX resistant strain did.
  • IMDM medium supplemented with 1.0 mL of 10 6 cells each containing 5 mL of 10% permeating fetal serum, 50 ⁇ g / mL gentamicin and 500 nM MTX The suspension was then seeded in a T25 flask and cultured. After 3 days of culture, the culture supernatant was collected, and the amount of FSH contained in the supernatant was measured using Human FSH ELISA Kit (manufactured by Antigenix America) to select a high-producing strain. The measuring method followed the manual attached to the kit.
  • the FSH high-producing strain obtained in the previous section was added to 4 mM L-Glutamine (Invitrogen), 50 g / ml gentamicin and 500 nM MTX in 15 ml EX-CELL302 medium (JRH, hereinafter, Suspended at 5 ⁇ 10 5 cells / ml in a serum-free medium), seeded in a 125 ml Erlenmeyer flask (manufactured by Corning), and subjected to floating swirl culture. Cultivation is performed at 35 ° C and swirl speed of 90-100 rpm. During passage, 4% or more of 5% CO in the culture vessel is aerated on the top of the medium,
  • the obtained strain was suspended in 15 mL of serum-free medium at a concentration of 3.0 X 10 5 cells / mL, seeded on a 12 mL flask, and cultured. After 3 days of culture, the culture supernatant was collected, and the amount of FSH contained in the supernatant was measured using HumanFSH ELISA Kit (manufactured by Antigenix America), and 14.8 in pKA N-FSH9-3 AFMS705 culture supernatant. It was confirmed that it was expressed at a concentration of IU / mL.
  • the pKAN-FSH9-3 AFMS705 strain is the pKAN-FSH9-3 AFMS705 share name, and the National Institute of Advanced Industrial Science and Technology (AIST) It is deposited as FERM BP-10086 at 1-chome, 1-no.
  • AIST National Institute of Advanced Industrial Science and Technology
  • CHO / DG44 cells Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)
  • IMDM—FBS 10 -HT (1) medium [Ushi Fetal Serum (FBS) (Invitrogen) In IMDM medium (Invitrogen)] containing 10% HT supplement (Invitrogen) at a 1-fold concentration in an incubation culture flask 75cm 2 (Grainer) and proliferate until just before confluence I let you. After washing the cells with 5 mL Dulbecco's PBS (hereinafter referred to as PBS) (Invitrogen), add 1.5 mL of 0.05% trypsin (Invitrogen) diluted with PBS at 37 ° C.
  • PBS Dulbecco's PBS
  • the cells were allowed to stand for minutes, and the cells were detached from the bottom of the incubator.
  • the detached cells are collected by centrifugation performed in normal cell culture, and supplemented with IMDM-FBS (10) -HT (1) medium to a density of 1 X 10 5 cells / mL.
  • MNNG manufactured by Sigma
  • MNNG manufactured by Sigma
  • a 96-well plate (Asahi Techno Glass Co., Ltd.) was seeded at a density of 1000 cells / well. Each well was supplemented with Img / mL lentil lectin (Lens culinaris agglutinin; hereinafter "", L and A, from Vector, Inc.) at 37 ° C in a CO incubator. Colonies that appeared after weekly culture
  • GDP-mannose 4, 6- an enzyme that catalyzes the dehydration reaction of converting GDP-mannose into GDP-4-keto, 6-deoxy- GDP-mannose in each lectin-resistant CHO / DG44 cell line obtained in the previous section
  • the expression level of dehydratase was calculated using the RT-PCR method as follows.
  • RNA was prepared according to the instructions for use.
  • SUPER SCRIPT First -Strand synthesis system for RT-PC R manufactured by Invitrogen was used to synthesize single-stranded cDNA from each RNA5 / Zg in a reaction solution of L according to the attached instruction manual.
  • DNA thermal cycler 480 Perkin Elma Co., Ltd.
  • the CHO SM strain When the resistance of the obtained CHO SM strain to various lectins was examined, the CHO SM strain was found to be a lectin that recognizes the same sugar chain structure as that recognized by LCA, that is, the N-glycoside-linked sugar chain reducing end. It was also resistant to other lectins that recognize sugar chain structures in which the N-acetyldarcosamine residue at position 6 and fucose at position 1 are attached by an a bond.
  • a culture medium supplemented with lum / mL endumame lectin (Pisum sativum Agglutinin; hereinafter referred to as PSA, manufactured by Vector) or a leek / mL high-growth bamboo lectin (final concentration lmg / mL).
  • PSA endumame lectin
  • AAL Aleuria aurantia Lectin
  • CHO / DG44 cells and the CHO SM strain obtained in the previous section were cultured using IMDM-FBS (IO) -HT (1) medium in a T75 flask for adherent cell culture (manufactured by Grainer) until just before reaching confluence. Later, genomic DNA was prepared according to the method described in the literature [Nuccleic Acid Research, 3, 2303, (1976)], and the obtained genomic DNA was added to TE-RNase buffer (pH 8.0) [ 10 mmol / l Tris—HC1, lmmol / 1 EDTA, 200 ⁇ g / ml RNase A] 30 0 1 was dissolved overnight.
  • TE-RNase buffer pH 8.0
  • Genomic DNA was transferred to the membrane. After the transfer, the nylon membrane was heat treated at 80 ° C for 2 hours. Next, for the purpose of confirming the quality of the genomic DNA transferred to the nylon membrane, it is considered to exist evenly in the genome regardless of the cell line. Southern hybridization was performed using the erase (FUT8) gene as a probe. A probe for detecting the FU T8 gene was prepared as follows. First, 10 ⁇ g of plasmid m!
  • FUT8-pCR2.1 containing mouse FUT8 cDNA described in Example 11 of WO02 / 31140 was dissolved in 50 ⁇ l M buffer (Takara Shuzo), and restriction enzyme Hindlll (Takara Shuzo) After overnight digestion, the reaction solution was replaced with H buffer (Takara Shuzo), and the digestion reaction was further performed overnight with the restriction enzyme EcoRI (Takara Shuzo). After completion of the reaction, the reaction solution was subjected to 2% agarose electrophoresis, and a 156 bp EcoRI-Hindlll fragment containing FUT8 gene exon 2 was purified.
  • the obtained DNA fragment (25 ng) was radiolabeled using [a- 32 P] dCTP 1.75 MBq and Megaprime DNA labeling system, dCTP (Amersham Biosciences).
  • hybridization was performed as follows. First, the above nylon membrane is sealed in a roller bottle, and 15 mL of a hybridization solution [4 X SSPE, 5 X Denhaldt, s solution, 0.5% (w / v) SDS, 0.1 mg / mL salmon sperm DNA] A prehybridization was performed at 65 ° C for 3 hours. Next, the 32 P-labeled probe DNA was heat denatured, put into a bottle, and heated at 65 ° C.
  • the nylon membrane was immersed in 50 mL of 2 X SSC-0.1% (w / v) SDS and heated at 65 ° C. for 15 minutes. After the above washing operation was repeated twice, the membrane was immersed in 50 mL of 0.2 X SSC-0.1% (w / v) S DS and heated at 65 ° C for 15 minutes. After washing, the nylon membrane was exposed to X-ray film at -80 ° C and developed. After development, the nylon membrane was boiled in a stripping solution [1% SDS, 0.1 X SSC] to peel off the probe and again subjected to hybridization with a different probe.
  • a stripping solution [1% SDS, 0.1 X SSC]
  • a probe specific for GMD gene exon 5 was prepared as follows. First, based on the known human GMD genomic DNA sequence (NCBI accession number NT_034880), 5X more oligo DNA primers (SEQ ID NO: 30 and SEQ ID NO: 31) that specifically bind to Etason 5 are added.
  • This region corresponds to nucleotide numbers 346 to 538 of the CHO GMD cDNA sequence shown in SEQ ID NO: 9.
  • the plasmid pAGE249GMD described in Example 15 of WO02 / 31140 is Prepare 100 ⁇ L reaction solution [ExTaq buffer (Takara Shuzo), 0.2 mmol / L dNTPs, 2.5 ⁇ mol / L above gene-specific primer (SEQ ID NO: 30 and SEQ ID NO: 31)] containing Polymerase chain Reaction (PCR) was performed.
  • PCR was performed under conditions of 30 cycles of heating at 94 ° C for 5 minutes, followed by 1 cycle of reaction at 94 ° C for 1 minute, 58 ° C for 2 minutes, and 72 ° C for 3 minutes. .
  • the reaction solution was subjected to 2% agarose electrophoresis, and an approximately 200 bp DNA fragment was purified.
  • [a- 32 P] dCTP 1.75 MBq and Megaprime DNA lab eling system, dCTP manufactured by Amersham Biosciences
  • the probe was subjected to hybridization on the nylon membrane shown above.
  • a specific fragment of GMD gene exon 5 was found in genomic DNA derived from CHO / DG44 cells, whereas a specific fragment of GMD gene exon 5 was completely detected in genomic DNA derived from CHO SM strain. The power was not. From the above results, it was shown that the C HO SM strain is a GMD knockout cell lacking at least the region containing exon 5 among the genomic region encoding GMD.
  • the plasmid pKAN-FSHo; ⁇ prepared in Example 1 was introduced into the CHO SM strain prepared in Example 2. These gene introductions were carried out by the following procedure using a known electoral position method [Cytotechnology, 3, 133 (1990)].
  • IMDM medium supplemented with baby dialysis serum, 50 ⁇ g / mL gentamicin and 50 nM methotrexate (MTX: Sigma) was added at 100 ⁇ L / well.
  • the culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days.
  • the colonies formed in 1 were replanted into 24 well plates (manufactured by Sigma).
  • 10% ⁇ shea dialyzed fetal serum, 50 ⁇ ⁇ / mL gentamicin and repeated medium exchange work using the IMDM medium ⁇ Ka ⁇ a 500 nM of MTX to 3-4 days, the 19 day culture while appropriately enlarged To obtain a 500 nM MTX resistant strain.
  • the FSH-producing strain pKAN-FS H2 GMDKO was expressed at a concentration of 1.27 IU / mL in the culture supernatant.
  • the pKAN-FSH2 GMDKO strain is the pKAN-FSH2 GMDKO stock name, and the National Institute of Advanced Industrial Science and Technology (AIST) It is deposited as FERMBP-10081 in East 1-chome, 1-No. 1 center 6).
  • the follicle-stimulating hormone obtained from the pKAN-FSH2 GMDKO strain prepared in this way was found to have an increased blood half-life compared to the follicle-stimulating hormone produced by the normal CHO / D G44 strain.
  • yeasts Many types of yeast are known, but typical yeasts often used as hosts for expressing recombinant proteins include yeasts of the genera Pichia and Saccaromyces. . Normally, the main structure of N-linked sugar chains added to recombinant proteins expressed by these yeasts has a 2-residue N-acetyl darcosamine in the core part on the reducing end, and the non-reducing end side. It is known that this is a mannose-type sugar chain having 9 to several tens of mannose residues and several to several tens of mannose 6-phosphate residues in the branched portion (Yeastl ⁇ , 1191 (2002)). Further, a high mannose type sugar chain having such a structure is often called a no-permannose type sugar chain.
  • the structure of the N-linked sugar chain to be added is mainly a hybrid sugar chain, which is an intermediate structure between a high-mannose sugar chain and a complex sugar chain.
  • the methods for producing Pichia yeast strains and Saccharomyces yeast strains expressing follicle-stimulating hormone that have been carotenized are described below.
  • Pichia yeast strains that have disrupted the PN01 enzyme gene present on the genome Pichia yeast strains such as Pichia pastoris GTS115 (manufactured by Invitrogen Corp.) are used as genomic DNA, and PCR is used to perform PNOKphosphomannosylationof Pichia yeast.
  • N-linked oligosaccharides 1 Amplify the entire translation region of the gene (GenBank accession number: AB099514).
  • the amplified PN01 gene sequence with a length of about 3200 bases was replaced with the yeast orotidine-5'-phosphate decarboxylase (UR A3) gene (GenBank accession number: AF321098).
  • a plasmid for PN01 gene disruption is prepared by inserting into a vector such as pCR2.1-TO PO vector (Invitrogen).
  • a vector such as pCR2.1-TO PO vector (Invitrogen).
  • 100 g of this plasmid is linearized with a restriction enzyme, and then the gene is stably introduced into a Pichia yeast such as GTS115 strain, for example, by the electoral position method described in PichiaExpressionKit (manufactured by Invitrogen). .
  • the remains The transferred yeast is cultured at room temperature in YPD medium (Invitrogen) deficient in uracil, and genomic DNA is extracted from each of the grown colonies.
  • a yeast clone in which the PN01 locus is disrupted by homologous recombination is selected by amplifying the yeast PN01 locus sequence by PCR using this genomic DNA as a saddle type.
  • the structure of the main N-linked sugar chain expressed in Pichia yeast has 9 residues on the non-reducing end side, with 2 residues of N-acetylyldarcosamine in the core part on the reducing end side.
  • Pichia yeast strains such as Pichia pastoris X-33 (manufactured by Invitrogen), are used in a vertical form, and by PCR, Pichia yeast ⁇ -1,6-mannose transferase (OCH1) gene (GenBank accession) Number: AF540063) is amplified. Amplified about 2 800 salt
  • the OCH1 gene sequence of the base length was replaced with the yeast's orotidine-5'-phosphate decarboxylase (URA3) gene (GenBank accession number: AF3210 98) after replacing the 5 'terminal half sequence with pCR2.1 -A vector for disrupting the OCH1 gene is prepared by inserting into a vector such as TOPO vector (Invitrogen). Next, 100 g of this vector was linearly digested with the restriction enzyme Sfil (manufactured by New England Biolabs), and then the yeast yeast strain, for example, the above-mentioned item was obtained by the electoral position method described in Pichia Expression Kit (manufactured by Invitrogen).
  • Sfil restriction enzyme
  • Stable gene transfer is carried out to the PN01 gene disruption strain described in 1) or the Pichia pastoris JC308 strain.
  • the transfected yeast is cultured at room temperature in YPD medium (Invitrogen) lacking uracil, and genomic DNA is extracted from each colony that has grown.
  • YPD medium Invitrogen
  • a yeast clonal strain in which the OCH1 locus is destroyed by homologous recombination is selected by amplifying the yeast OCH1 locus sequence by PCR using this genomic DNA as a saddle type.
  • the structure of the major N-linked sugar chain expressed in Pichia yeast is converted into a 2-residue N-case in the core at the reducing end. It can be modified to a Man8 type high mannose type sugar chain having tildarcosamine and having a structure in which 8 mannose residues are bonded to the non-reducing end.
  • the cDNA encoding the active domain of nematode ⁇ -1,2-mannosidase was obtained by PCR using specific primers and KOD polymerase (Toyobo Co., Ltd.). Amplify specifically.
  • the amplified cDNA is ligated to the 5 'end of the cDNA sequence encoding the yeast ⁇ -mannosidase (MNS1) gene (GenBank accession number: M63598) leader peptide, and then the yeast expression vector pPICZ. (Invitrogen) and other vectors are inserted into the yeast endoplasmic reticulum for expression of -1,2-mannosidase.
  • this vector is stably introduced into the Pichia yeast strain in which both the PN01 gene and the OCH1 gene described in the previous section have been disrupted by homologous recombination by the electopore method.
  • the yeast after gene transfer is cultured at room temperature in a YPD medium (Invitrogen) containing zeosin (Invitrogen) and lacking uracil, and total RNA is extracted from each of the grown colonies.
  • a yeast clonal strain in which expression of the recombinant chimeric ⁇ -1,2-mannosidase is observed is selected by PCR using the first-strand cDNA prepared with this total RNA strength as a saddle type.
  • the structure of the main ⁇ -linked glycan expressed in Pichia yeast has 2 residues ⁇ -acetildarcosamine in the core part on the reducing end side and 5 on the non-reducing end side. It can be modified to a Man5 type high mannose type sugar chain having a structure in which the mannose residues are bound.
  • RNA is extracted from yeast (Kluyveromyces lactis) using the RNeasy Mini Kit (Qiagen), and then this RNA is used as a cocoon for Superscript TM first-strand cDNA synthesis kit (in CDNA is prepared using Vitrogen). Next, this cDNA is used as a saddle, and PCR is performed using a specific primer and KOD polymerase (Toyobo Co., Ltd.).
  • a cDNA encoding the entire translation region of the -N-acetylcylcosamine transporter (GenBank accession number: AF106080) is specifically amplified. Next, the amplified about
  • a 3700 base-long cDNA is inserted between the restriction enzyme EcoRI and Not I cleavage sites located downstream of the alcohol oxygenase promoter sequence in vectors such as the yeast expression vector pPIC3.5K (Invitrogen).
  • the vector is inserted to express the UDP-N-acetyldarcosamine transporter in the Golgi apparatus of yeast.
  • this vector is stably introduced into the Pichia yeast strain into which the ⁇ -1,2-mannosidase gene has been introduced as described in the previous section by the electopore method.
  • the yeast after gene introduction is cultured at room temperature in a YPD medium containing the drug G418 (manufactured by Nacalai Testa), and total RNA is extracted from each of the grown colonies.
  • a yeast clonal strain in which expression of the recombinant UDP-N-acetylyldarcosamine transporter is observed is selected by the CR method using the cDNA prepared from this total RNA as a cage
  • N-Acetyldarcosaminyltransferase-1 (GenBank accession number) was obtained by performing PCR using human liver cDNA (Clontech) in a cage and using specific primers and KOD polymerase (Toyobo). : Amplify specifically the cDNA encoding the active domain of M55621). The amplified cDNA is linked to the 5 'end of the cDNA sequence encoding the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752), and then expressed for yeast.
  • MNN9 yeast mannose transferase
  • vector pAUR123 manufactured by Tacarano
  • N-acetylyldarcosamine transferase- is inserted into the yeast Golgi.
  • a vector for expressing 1 is prepared.
  • this vector is introduced into the Pichia yeast strain introduced with the UDP-N-acetylyldarcosamine transporter gene described in the previous section by the lithium acetate method described in the manual attached to the expression vector pAUR123.
  • the yeast Incubate at room temperature in YPD medium containing Caranoio), and extract total RNA from each of the grown colonies.
  • a yeast clonal strain in which expression of recombinant N-acetylyldarcosamine transferase-1 is observed is selected by PCR using the cDNA prepared from this total RNA as a saddle type.
  • the structure of the main N-linked sugar chain expressed in Pichia yeast has 2 residues of N-acetyldarcosamine in the core at the reducing end and 5 at the non-reducing end. It can be modified to a noblebrid sugar chain with a structure in which one N-acetylyldarcosamine residue is added to the non-reducing end of the Man5 type high mannose sugar chain to which the mannose residue is attached. .
  • Pichia yeast strain that mainly expresses a hybrid sugar chain, which is an intermediate structure between a high mannose sugar chain and a complex sugar chain, as an N-linked sugar chain has been described.
  • yeasts of the genus Saccharomyces can be mentioned as yeasts that are often used as hosts for expressing recombinant proteins.
  • a method for producing a Saccharomyces yeast strain that mainly expresses N-linked sugar chains and hybrid sugar chains as follows is described.
  • a yeast clone in which the OCH1 locus is destroyed by homologous recombination is selected.
  • the obtained Saccharomyces yeast strain in which the OCH1 gene was disrupted was derived from haploid cells according to the method of Sherman et al. (Methods'In'Enzymology 1 194, 21 (1991)), and then ⁇ -1,3-mannose.
  • a diploid zygote is formed by mixing with haploid cells of the mutant yeast strain LB1-10B (University of California Yeast Genetic Stock Center) in which the transferase (MNN1) gene is disrupted and culturing under nitrogen-deficient conditions .
  • the obtained zygote is cultured at room temperature in YPD medium lacking uracil and leucine, and genomic DNA is extracted from each colony force that has grown.
  • the yeast OCH1 locus sequence (GenBank accession number: AF540063) and the MNN1 locus sequence (GenBank accession number: AF540063L23753) are amplified by PCR using this genomic DNA as a saddle type.
  • a yeast clonal strain in which both the OCH1 locus and the MNN1 locus are disrupted is selected.
  • the structure of the major N-linked sugar chain expressed in Saccharomyces yeast has 2 residues of N-acetyldarcosamine in the core of the reducing end and 8 mannose residues on the non-reducing end. It can be modified to a Man8 type high mannose sugar chain having a bound structure.
  • RNA extract total RNA from mold (Aspergillus saitoi) using RNeasy Mini Kit (Qiagen), and then prepare cDNA using Superscript TM first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is converted into a saddle type, and PCR using a specific primer and KOD polymerase (Toyobo Co., Ltd.) is performed to obtain a cDNA encoding the entire translation region of mold ⁇ -1,2-mannosidase ( GenBank accession number: D 49827) is specifically amplified.
  • the amplified cDNA has a yeast endoplasmic reticulum localization signal peptide (embombonal 7, 913 (1988)), that is, histidine-aspartate, at the 3 'end from which the translation termination codon was deleted.
  • yeast endoplasmic reticulum localization signal peptide embombonal 7, 913 (1988)
  • histidine-aspartate at the 3 'end from which the translation termination codon was deleted.
  • this vector was stably introduced into the Saccharomyces yeast strain in which the a-1,6-mannose transferase gene and the a-1,3-mannose transferase gene were disrupted, as described in the previous section, by the electopore method.
  • the yeast after gene transfer is cultured at room temperature in a YPD medium (Invitrogen) containing zeocin (Invitrogen) and lacking uracil, and total RNA is extracted from each of the grown colonies.
  • a recombinant chimeric type yeast clone strain in which the expression of X-1,2-mannosidase was observed was selected by PCR using this cDNA with the total RNA strength prepared as a saddle type.
  • the structure of the major N-linked sugar chain expressed in Saccharomyces yeast has 2 residues of N-acetylyldarcosamine in the core portion on the reducing end and 5 mannose residues on the non-reducing end It can be modified into a Man5 type high mannose type sugar chain having a structure in which
  • RNA extract total RNA from yeast (Kluyveromyces lactis) using RNeasy Mini Kit (Qiagen), and then prepare cDNA using Superscript TM first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is used as a saddle, and PCR is performed using a specific primer and KOD polymerase (Toyobo Co., Ltd.).
  • a cDNA encoding the entire translation region of the -N-acetylcylcosamine transporter (GenBank accession number: AF106080) is specifically amplified. Next, the amplified about
  • a 3700 base-long cDNA is inserted between the restriction enzyme EcoRI and Not I cleavage sites located downstream of the alcohol oxygenase promoter sequence in vectors such as the yeast expression vector pPIC3.5K (Invitrogen).
  • the vector is inserted to express the UDP-N-acetyldarcosamine transporter in the Golgi apparatus of yeast.
  • this vector is stably introduced into the Saccharomyces fermentation mother strain into which the ⁇ -1,2-mannosidase gene has been introduced as described in the previous section by the electopore method.
  • the fermented mother after the gene transfer is cultured at room temperature in a YPD medium containing the drug G418 (manufactured by Leitesta Co., Ltd.).
  • a yeast clonal strain in which expression of the recombinant UDP-N-acetylyldarcosamine transporter is observed is selected by a PCR method using the prepared cDNA with the prepared total RNA strength.
  • N-acetylylcosamine transferase-1 (GenBank accession number) is obtained by PCR using human liver cDNA (manufactured by Clontech) as a cage and using specific primers and KOD polymerase (manufactured by Toyobo). : Amplify specifically the cDNA encoding the active domain of M55621). The amplified cDNA is linked to the 5 'end of the cDNA sequence encoding the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752), and then expressed for yeast.
  • MNN9 yeast mannose transferase
  • vector pAUR123 manufactured by Tacarano
  • N-acetylyldarcosamine transferase- is inserted into the yeast Golgi.
  • a vector for expressing 1 is prepared.
  • this vector One is introduced into the Saccharomyces yeast strain into which the UDP-N-acetylyldarcosamine transporter gene described above has been introduced by the lithium acetate method described in the manual attached to the expression vector pAUR123.
  • the yeast after the gene introduction is cultured at room temperature in a YPD medium containing a drug mouthful brassin A (manufactured by Takara Bio Inc.), and total RNA is extracted from each grown mouthpiece.
  • a yeast clone strain in which the expression of recombinant N-acetyl dalcosamine transferase-1 has been observed is selected by PCR using this cDNA, which has also been prepared for total RNA, in a vertical form.
  • the structure of the main N-linked sugar chain expressed in Saccharomyces yeast has a 2-residue N-acetylyldarcosamine in the core portion on the reducing end side and 5 in the non-reducing end side. It can be modified to a nodule type sugar chain in which one N-acetylyldarcosamine residue is added to the non-reducing terminal side of the Man5 type high mannose type sugar chain having a structure in which one mannose residue is bonded.
  • hybrid sugar chains are mainly expressed as an N-linked sugar chain with one N-acetylyldarcosamine residue added to the non-reducing end of Man5 type high mannose type sugar chain.
  • a method for producing the Pichia yeast strain or the Saccharomyces yeast strain was described.
  • a method for preparing a recombinant human follicle-stimulating hormone mainly using hybrid sugar chains as N-linked sugar chains using these yeast strains as hosts will be described.
  • human pituitary cDNA (manufactured by Clontech) was used as a cage and PCR using KOD polymerase (manufactured by Toyobo Co., Ltd.) as an amplification enzyme.
  • KOD polymerase manufactured by Toyobo Co., Ltd.
  • the reaction specifically amplifies mature human follicle-stimulating hormone ex and j8 subunit full-length cDNAs.
  • the alcohol oxygenase promoter sequence of the yeast expression vector PA0815 (Invitrogen) was inserted into the immediately downstream, making the vectors pA0815 / hFSH a for secretory expression of mature human follicle stimulating hormone a subunit, a vector p A0815 / hFSH ⁇ for secretory expression of mature human follicle stimulating hormone beta subunit .
  • the above-mentioned vector pA0815 / hFSH ⁇ ⁇ 100 g that secretes and expresses the mature human follicle-stimulating hormone is cleaved within the HIS4 gene with the restriction enzyme Sail (manufactured by New England Biolabs). Prepare the linearized vector. Next, according to the method of Mochizuki et al. (ProteinExpression and Purification 23, 55 (2001)), this linearly follicle-stimulating hormone expression vector was transformed into the N-linked sugar described in Example 5 above.
  • Pichia yeast strains that mainly express hybrid sugar chains as chains or Saccharomyces yeast strains that mainly express hybrid sugar chains as N-linked sugar chains described in Section 9 of this Example Introduced by the lithium acetate method.
  • the yeast after the gene introduction is cultured at room temperature in a YPD medium (Invitrogen) containing the drug blasticidin (Invitrogen) to obtain blasticidin-resistant colonies.
  • the blasticidin-resistant colonies are transplanted into liquid YPD medium (Invitrogen) and cultured at 30 ° C for 24 hours or longer.
  • the FSH concentration contained in the culture supernatant obtained after the culture is measured using Human FSH ELISAKit (manufactured by Antigenix America) using the follicle stimulating hormone drug Gona ⁇ F (manufactured by Serono) as a standard product.
  • a recombinant follicle-stimulating hormone having a hybrid sugar chain that does not contain fucose as an N-linked sugar chain and is secreted into this yeast culture supernatant is the method of Gadkari et al. (Protein Expression and Purification 32, 175 (2003)).
  • the purified follicle-stimulating hormone protein is analyzed for the sugar chain structure according to the method of Skibe li et al.
  • a Pichia yeast strain that mainly expresses a hybrid sugar chain in which one N-acetylethylcosamine residue is added to the non-reducing end of the Man5 type high mannose type sugar chain as an N-linked sugar chain.
  • a similarly modified Saccharomyces yeast strain can be used as a host, and a recombinant human follicle-stimulating hormone mainly having a hybrid sugar chain not containing fucose as an N-linked sugar chain can be prepared.
  • a yeast strain that expresses a recombinant human follicle-stimulating hormone that mainly has a hybrid sugar chain as this N-linked sugar chain a complex double chain that does not contain fucose as an N-linked sugar chain.
  • Genetically modified humans mainly having chain-type sugar chains A method for producing a yeast strain that expresses follicle-stimulating hormone is described below.
  • human manosidase II By performing PCR using human tissue-derived cDNA, such as liver-derived cDNA (Clontech), in a vertical form, and using a specific primer and KOD polymerase (Toyobo Co., Ltd.) V, human manosidase II (GenBank It specifically amplifies the cDNA encoding the active domain of the session number: U31520).
  • the amplified cDNA is linked to the 5 'end of the cDNA sequence encoding the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752), followed by expression for yeast.
  • the vector is inserted downstream of the promoter sequence of the vector to produce a vector that expresses ⁇ -mannosidase II in the yeast Golgi apparatus.
  • this vector was stably used for the yeast strain described in paragraph 11 of this Example, which expresses a recombinant human follicle-stimulating hormone mainly having a hybrid sugar chain as a ⁇ -linked sugar chain. Introduce. For the yeast after gene introduction, clones are selected using auxotrophy and drug resistance as indicators, and then the expression of chimeric mannoseidase I I is confirmed by RT-PCR.
  • N-acetyldarcosaminyltransferase--using human tissue for example, liver-derived cDNA (Clontech) in a saddle shape and PCR using a specific primer and KOD polymerase (Toyobo) V CDNA Amplify the cDNA encoding the active domain of GenBank accession number: U15128.
  • the amplified cDNA is ligated to the 5 'end of the cDNA sequence encoding the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L2375 2), and then expressed for yeast.
  • MNN9 yeast mannose transferase
  • the structure of the main N-linked sugar chain of the recombinant follicle-stimulating hormone expressed by the yeast strain stably incorporating the chimeric N-acetylyldarcosamine transferase II is obtained. It has two residues of N-acetyl darcosamine in the core part on the reducing end side, and its mannose residue is bifurcated on the non-reducing end side, and is attached to each of the two non-reducing ends. It can be modified into a complex double-stranded sugar chain that contains one N-acetylyldarcosamine residue and does not contain fucose.
  • yeast strain into which recombinant UDP-galactose-4-epimerase gene has been introduced
  • Human cDNA derived from human tissue, such as liver (Clontech) is used as a saddle type, with specific primers and KOD polymerase (Toyo By performing PCR using Spinning Co., Ltd., the cDNA encoding the entire translation region of UDP-galactose-4-epimerase (UniGene number Hs.76057) is specifically amplified.
  • the amplified cDNA is inserted downstream of the promoter sequence of the expression vector for yeast to produce a vector that expresses UDP-galactose-4-epimerase in the yeast cytosol.
  • this vector is stably introduced into the yeast strain described above, which expresses a recombinant human follicle-stimulating hormone mainly having an immature complex double-stranded sugar chain as an N-linked sugar chain.
  • yeast after gene transfer clones are selected using auxotrophy and drug resistance as indicators, and then the expression of UDP-galactose-4-epimelase is confirmed by RT-PCR.
  • clones are selected using auxotrophy and drug resistance as indicators, and then the expression of chimeric j8 1,4 galactose transferase is confirmed by RT-PCR.
  • RT-PCR the structure of the major N-linked sugar chain of the recombinant follicle-stimulating hormone expressed by the yeast strain stably incorporating the chimeric j8 1,4 galactosyltransferase is reduced to the reducing end.
  • N-acetyl darcosamine in the core part of the core, 3 mannose residues are linked in a bifurcated structure on the non-reducing end side, and N-to each of the 2 non-reducing ends It can be modified into a complex double-stranded sugar chain in which one acetylyldarcosamine residue and one galactose residue are added.
  • Yeast strain expressing the genetically modified follicle-stimulating hormone mainly having a complex double-stranded sugar chain having no fucose residue on the reducing end and galactose added on the non-reducing end. Is seeded in a liquid YPD medium (Invitrogen) and cultured at 30 ° C for 24 hours or longer to secrete recombinant follicle-stimulating hormone into the culture supernatant.
  • the FSH concentration contained in the culture supernatant obtained after culturing is measured using HumanFSH ELISA Kit (manufactured by Antigenix America) using Gona ⁇ F (manufactured by Serono) as a standard product.
  • Follicle stimulating hormone is purified according to the method of Gadkari et al. (Protein Expression and Purification 32, 175 (2003)). The purified follicle-stimulating hormone protein is analyzed for the sugar chain structure according to the method of SWbeli et al. (Bloody, 3626 (2001)).
  • the CHO / DG44 cell line obtained by double knockout of the FUT8 gene obtained in Example 1 was mixed with K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8.1 mmol / L Na HPO, 1.5 mmo).
  • the suspension was suspended in 1 / L KH PO, 4.0 mmol / L MgCl 2) to give 8 ⁇ 10 7 cells / mL.
  • Cell suspension 200
  • IMDM medium was added at 100 L / well. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Next, the medium exchange operation using IMDM medium supplemented with 10% urine fetal dialysis serum, 50 ⁇ g / mLgentamicin and 200 nM MTX was similarly repeated every 3 to 4 days, and cultured for 18 days.
  • the colonies formed were replanted in a 24 well plate (manufactured by Sigma). Repeat the medium change operation using IMDM medium supplemented with Sarako, 10% urine fetal dialysate serum, 50 g / m Lgentamicin and 500 nM MTX every 3-4 days, and culture for 19 days with appropriate expansion And remove the 500nMMTX resistant strain. Got.
  • a plurality of 500 nM MTX-resistant cell lines obtained in the previous section were suspended each 1.0 X 10 6 cells 5 mL of 10% ⁇ shea dialyzed fetal serum, in IMDM medium ⁇ Ka ⁇ a 50 g / mLgentamicin and 500 nM of MTX, Culturing was performed by seeding in a T25 flask. After 3 days of culture, the culture supernatant was collected, and the amount of FSH contained in the supernatant was measured using HumanFSH ELISA Kit (manufactured by Antigenix America). The method followed the attached manual. As a result, it was confirmed that the culture supernatant of pKAN-FSHKC895 strain derived from the normal CHO / DG44 cell line contained FSH at a concentration of 8.55 IU / mL.
  • pKAN-FSH KC895 strain FUT8 gene double knockout obtained in Example 1 Section 7 FSH production strain (pKAN- FSHMS705) strain derived from the HO cell strain was added to 10% urine fetal dialyzed serum, 50 g / mLgentamicin and 500 nM MTX The suspension was suspended in IMDM medium supplemented with seeds and seeded in a T175 flask (manufactured by Greiner).
  • the supernatant was removed, washed twice with PBS10 mL, and cultured with 30 mL of ExCELL 302 medium containing 6 mM L-Glutamine, 500 nM MTX, 50 ⁇ MNeu5Ac2en. After approximately 5 days of culture, the culture supernatant was collected and filtered through a 0.22 m bottle top filter (IWAKI) to obtain a culture supernatant containing KC895-derived FSH and an MS705-derived FSH. The amount of FSH contained in the supernatant was measured using a HumanFSH ELISA Kit.
  • FSHR Human follicle-stimulating hormone receptor
  • L reaction solution containing human testis cDNA as a template [ExTa q TM DNA polymerase (Takara Bio Inc.), 10 X PCR buffer ⁇ 0.2 mmol / L dNTP mixture, 0.5 / z mol / L above primer (SEQ ID NO: 33 And SEQ ID NO: 34)], heated at 94 ° C for 3 minutes, then 30 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1 minute. PCR was performed on the reaction.
  • Plasmid DNA was prepared using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDyeTerminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAG EN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) was used to analyze the nucleotide sequence.
  • the plasmid DNA obtained from the obtained transformants was treated with restriction enzymes MfeI (New England Biolabs) and BbsI (New England Biolabs). It was repaired by recombination in the same way as the procedure.
  • the base sequence of the repaired plasmid DNA was analyzed in the same manner as described above. As a result, it was confirmed that the base sequence of the repaired plasmid DNA was the same as the gene sequence encoding FSHR described in SEQ ID NO: 32, and pT7-FSHR was obtained (FIG. 5).
  • 8 obtained in Example 1 was dissolved in 17.5 ⁇ L of water, and 6 units of EcoRI (manufactured by Takara Bio Inc.) and 2 ⁇ L of 10 XH buffer were added to the solution.
  • a 20 L reaction solution was prepared and digested at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 ⁇ L of water. Further, 7.5 units of Apal and 2 ⁇ L of 10 ⁇ L buffer were added to the solution to prepare a 20 ⁇ L reaction solution, followed by digestion reaction at 37 ° C. for 16 hours.
  • E. coli DH5 ⁇ strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (manufactured by QIAGEN) to obtain pKAN-FSHR (FIG. 6).
  • the plasmid pKAN-FSHR prepared in the previous section was introduced into a normal CHO / DG44 cell line. These gene introductions were performed by the following procedure according to a known electoral position method [Cytotechnology, 3, 133 (1990)].
  • a linear digestion was performed by performing a time digestion reaction. After the reaction, the reaction mixture was purified by phenol / chloroform extraction treatment and ethanol precipitation to recover the linear plasmid.
  • the CHO / DG44 cell line was added to K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8.1 mmol / L Na HPO, 1.5 mmol / L KH PO, 4.0 mmol / L MgCl 2). Suspend 8 x 10 6 cells /
  • IMDM medium (LifeTechnologies) supplemented with 10% urine fetal serum (Life Technologies) and 50 g / mL gentamicin (National Power Tester) 10%
  • the suspension was suspended in mL, and seeded in an adherent cell culture T75 flask (manufactured by Grainer).
  • the medium was changed to 10 mL of IMDM medium supplemented with 10% urine fetal serum, 500 ⁇ g / mL G418 (Nacalai Testa) and 50 ⁇ g / mL gentamicin. Subculture or medium change was performed every 3-4 days.
  • the cells are suspended in IMDM medium supplemented with 10% fetal bovine serum, 50 nMMTX, 500 ⁇ g / mL G418 and 50 g / mL gentamicin, and 96-well One cell / well was seeded on the plate. After 5 days, the medium was changed with the same medium, and on day 13, the medium was changed to IMDM medium supplemented with 10% ushi fetal serum, 200 nM MTX, 500 ⁇ g / mL G418 and 50 ⁇ g / mL gentamicin. Was done. Thereafter, this culture medium was used to expand, subculture, or change the medium every 3 to 4 days. The passage was repeated for about one month to obtain a cloned FSHR-expressing CHO cell line. All cultures were performed at 5% CO and 37 ° C.
  • FSHR-expressing CHO cells prepared in the previous section are suspended in IM DM medium supplemented with 10% dialyzed fetal bovine serum, g / mLgentamicin, 500 ⁇ g / mL G418 and 200 nM methotrexate (MTX: Sigma). 2 x 10 5 cells / well in a 24-well plate. 5% CO, 37 ° C
  • the medium was changed to a medium containing 0.05 ng / mL to 400 ng / mL FSH sample and 100 uM 3-1 sobuty 1-methylxanthine.
  • EC50 was determined from the obtained results.As a result, Gona F was 4.68 (3.10-7.06) ng / mL, phFSI ⁇ ⁇ , 5.77 (4.89-6.81) ng / mL, KC895-derived FSH-containing culture supernatant was 2.478 (1.13 -5.45) ng / mL, 3.705 (2.84-5.00) ng / mL in the MS705-derived FSH-containing culture supernatant (Fig. 7). EC50 was calculated using analysis software GraphPad prism 4 (manufactured by Graphpad).
  • FSH purification from the culture supernatant was performed using a affinity column (1.3 mL) in which mouse anti-human FSH antibody (cat: MCA1028, manufactured by SEROTEC) was phased into agarose.
  • mice aged 11-12 weeks were used in this study. Mice were administered MS705-derived FSH and KC895-derived FSH samples from the tail vein at 10 g / mouse, respectively, 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours later. Blood was collected from the tail vein using a heparin-coated hematocrit tube (IWAKI). The obtained blood sample was immediately centrifuged at 3000 rpm for 10 minutes at 4 ° C using a refrigerated centrifuge and stored as plasma. The amount of FSH contained in the obtained plasma sample was measured using HumanFSH ELISA Kit (manufactured by Antigenix America). The measurement method followed the attached manual.
  • IWAKI heparin-coated hematocrit tube
  • composition comprising a recombinant follicle-stimulating hormone molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is a reduced end of the sugar chain. It is possible to provide a follicle stimulating hormone composition which is a sugar chain in which fucose is bound to the end N-acetylcylcosamine.

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Abstract

It is intended to provide a follicle-stimulating hormone composition comprising a genetically engineered follicle-stimulating hormone molecule having an N-glycoside bond type complex sugar chain, wherein the N-glycoside bond type complex sugar chain is a sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain and the use thereof.

Description

明 細 書  Specification
遺伝子組換え卵胞刺激ホルモン組成物  Genetically modified follicle stimulating hormone composition
技術分野  Technical field
[0001] 本発明は、 N-グリコシド結合複合型糖鎖を有する遺伝子組換え卵胞刺激ホルモン 分子カゝらなる組成物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末端の N-ァセチルダルコサミンにフコースが結合して ヽな 、糖鎖である卵胞刺激ホルモン 組成物及びその用途に関する。  [0001] The present invention relates to a composition comprising a recombinant follicle-stimulating hormone molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is a reducing end of the sugar chain. The present invention relates to a follicle-stimulating hormone composition, which is a sugar chain, which has fucose bound to N-acetylcylcosamine and its use.
背景技術  Background art
[0002] 卵胞刺激ホルモン（Follicle stimulating hormone,以下 FSHとも称す）は主に不妊治 療に用いられている。不妊とは、（1)生殖可能な年齢にある男女が正常な性生活を 営んでいるが、避妊期間を除いて 2年以上経過しても妊娠成立を見ない状態、（2)妊 娠成立をみるが、流産死産により生児を得られない状態などをいう。そのうち、（2)は 不育症ともいい、不妊症という言葉は、一般に、不育症も含む形で使われている。不 妊は、妊娠経験の全くない原発性不妊と少なくとも 1回以上の妊娠経験のある続発性 不妊とに分類される。不妊の原因について、男女割合としては、原発性不妊では女 性:男性 = 2 : 1、続発性不妊では女性側の要因が主である場合がほとんどである。不 妊症は単一の原因で発症することは少なぐ複合因子による疾患である。不妊症の 主な治療方法は、卵管や子宮に手術を施す等の医療行為であり、特に排卵障害に 対しては、排卵障害治療剤等の薬物を投与する (非特許文献 1)。排卵障害治療剤と しては、 FSH、黄体开成ホルモン（Luteinizing hormone, LHと称す）および繊毛性性 腺刺激ホルモン（Chorionic gonadotropin,以下 CGと称す）などの性腺刺激ホルモン (Gonadotropic hormone)が広く医療の現場で用いられている。  [0002] Follicle stimulating hormone (hereinafter also referred to as FSH) is mainly used for infertility treatment. Infertility means that (1) men and women of reproductive age have a normal sexual life, but no pregnancy is found after 2 years excluding the contraceptive period, and (2) pregnancy is established. However, it means the situation where a live child cannot be obtained due to miscarriage and stillbirth. Of these, (2) is also called infertility, and the term infertility is generally used to include infertility. Infertility is classified as primary infertility with no pregnancy experience and secondary infertility with at least one pregnancy experience. Regarding the cause of infertility, the ratio of men and women in primary infertility is mostly female: 2 = 2: 1, and in secondary infertility, the female side is the main factor. Infertility is a complex factor that rarely develops from a single cause. The main treatment method for infertility is medical practice such as surgery on the fallopian tube and uterus. Drugs such as ovulation disorders are administered especially for ovulation disorders (Non-patent Document 1). As ovulation disorder therapeutic agents, gonadotropic hormones such as FSH, luteinizing hormone (Luteinizing hormone, LH) and ciliary gonadotropin (CG) are widely used. Used in medical settings.
[0003] LHは分子量 29,000の糖蛋白質で、女性では卵巣からのエストロゲン分泌、黄体か らのプロゲステロン産生を促す。男性では、 Leydig間質細胞に作用してテストステロン 分泌を促進するため、 Interstitial cell stimulating hormone (ICSH)とも呼ばれている。  [0003] LH is a glycoprotein with a molecular weight of 29,000 and promotes estrogen secretion from the ovary and progesterone production from the corpus luteum in women. In men, it is also called Interstitial cell stimulating hormone (ICSH) because it acts on Leydig stromal cells to promote testosterone secretion.
FSHは分子量 32,000の糖蛋白質で、女性では卵胞の発育を、男性では睾丸での精 子形成を促進している。 TSHは分子量 28,000の糖蛋白質で、甲状腺を刺激して甲状 腺ホルモンの分泌合成を促進している。 CGは分子量 37,000の糖蛋白質で、妊娠時 に胎盤より分泌され LHと同様な生理活性を示す。 FSH、 LH、 TSHは脳下垂体前葉で 合成され分泌されるホルモンである。 FSH、 LH、 CGおよび甲状腺刺激ホルモン（Thy roid stimulating hormone,以下 TSHと称す）は、いずれの分子も αサブユニットと j8サ ブユニットからなるヘテロダイマー構造をとる。このうち、 αサブユニットに関しては、 いずれの分子も同じ前駆体蛋白質力 作られており、 Ν末端側 5アミノ酸の長さの違 い以外は同じアミノ酸配列を有している（非特許文献 2)。従って、これらの 4種類のホ ルモンは構造上同じファミリーに属しているといえる。しかしながら、 j8サブユニットは それぞれ異なっており、それが各々のホルモンの特異性を決定している。ヒト FSH、ヒ ト LHおよびヒト TSHの αサブユニットはいずれもヒト第 6番染色体の単一遺伝子にコ ードされている。ヒト FSHの 13サブユニット遺伝子は第 11番染色体上に、ヒト LHの βサ ブユニット遺伝子は第 19番染色体上に、ヒト TSHの βサブユニット遺伝子は第 1番染 色体上にそれぞれコードされている。 FSH is a glycoprotein with a molecular weight of 32,000 that promotes follicular development in women and spermatogenesis in the testicles in men. TSH is a glycoprotein with a molecular weight of 28,000 that stimulates the thyroid gland Promotes secretory synthesis of glandular hormones. CG is a glycoprotein with a molecular weight of 37,000. It is secreted from the placenta during pregnancy and exhibits the same physiological activity as LH. FSH, LH, and TSH are hormones synthesized and secreted in the anterior pituitary gland. FSH, LH, CG and thyroid stimulating hormone (hereinafter referred to as TSH) all have a heterodimeric structure consisting of an α subunit and a j8 subunit. Among these, for the α subunit, all molecules are made of the same precursor protein, and have the same amino acid sequence except for the difference in the length of the 5 amino acids on the heel side (Non-patent Document 2). . Therefore, it can be said that these four types of holmons belong to the same family structurally. However, each j8 subunit is different, and it determines the specificity of each hormone. The α subunits of human FSH, human LH, and human TSH are all encoded by a single gene on human chromosome 6. The human FSH 13 subunit gene is encoded on chromosome 11, the human LH β subunit gene is encoded on chromosome 19, and the human TSH β subunit gene is encoded on chromosome 1. Yes.
[0004] FSHが脳下垂体前葉から分泌されると、女性では、卵胞細胞表面上に発現して 、 る FSH受容体に特異的に結合することにより卵胞の発育と成熟を促進する。また、 FS Ηはエストロゲンの生成と分泌も促進する。男性では、 FSHは、精細管細胞表面上に 発現している FSH受容体に特異的に結合することにより、睾丸における精細管の発 育と精子形成を促進する。 FSHが FSH受容体に結合すると、多くのペプチドホルモン と受容体と同様に、 GTP結合蛋白質を介して細胞内のアデ-ル酸シクラーゼ活性が 上昇し、細胞内サイクリックアデノシン 1リン酸 (cAMP)合成が促進される。生成した cA MPは細胞内情報伝達物質として機能し、細胞内プロテインキナーゼを活性ィ匕させる などの作用を発現させる。  [0004] When FSH is secreted from the anterior pituitary gland, females promote follicular development and maturation by specifically binding to the FSH receptor expressed on the surface of follicular cells. FS also promotes estrogen production and secretion. In men, FSH promotes seminiferous tubule development and spermatogenesis in the testicles by specifically binding to the FSH receptor expressed on the seminiferous cell surface. When FSH binds to the FSH receptor, as with many peptide hormones and receptors, intracellular adenylate cyclase activity increases via GTP-binding proteins, and intracellular cyclic adenosine monophosphate (cAMP). Synthesis is promoted. The generated cA MP functions as an intracellular signal transmitter, and activates intracellular protein kinases.
[0005] FSHは、 92アミノ酸残基で構成される aサブユニットと 118アミノ酸残基で構成される βサブユニットが会合したヘテロダイマーである。 FSHの分子量の 20%は糖が占めて おり、ヘテロダイマー 1分子に 4本の Ν-グリコシド結合型糖鎖を有している。 FSHに結 合している糖鎖は複合型であり、 αサブユニットに 2本 (Asn52、 Asn78)、 0サブュニッ トに 2本 (Asn7、 Asn24)の糖鎖が結合する (非特許文献 2)。ヒト尿由来 FSHで見られる N -グリコシド結合型糖鎖の代表的な糖鎖構造を以下に示す。 [0006] [化 1] [0005] FSH is a heterodimer in which an a subunit composed of 92 amino acid residues and a β subunit composed of 118 amino acid residues are associated. Sugar accounts for 20% of the molecular weight of FSH, and each heterodimer has four Ν-glycoside-linked sugar chains. The sugar chains bound to FSH are complex, and two (Asn52, Asn78) sugar chains bind to the α subunit, and two (Asn7, Asn24) sugar chains bind to the 0 subunit (Non-patent Document 2). . The typical sugar chain structure of N-glycoside-linked sugar chains found in human urine-derived FSH is shown below. [0006] [Chemical 1]
Fucffl  Fucffl
I  I
6  6
土 Neu5Acfl2→ 3Galy91→ 4GlcNAc^l→ 2Manarl 6Μ&ηβι→ 4GlcNAc^l - → 4Glc NAc 土 Neu5Aci?2→ 3Gal/31→ 4GlcNAc 1→ 2Manorl 3 Sat Neu5Acfl2 → 3Galy91 → 4GlcNAc ^ l → 2Manarl 6 Μ & ηβι → 4G l c NAc ^ l-→ 4Glc NAc Sat Neu5Aci? 2 → 3Gal / 31 → 4GlcNAc 1 → 2Manorl 3
士 Neu5AC"2→ 3Gal^l→ 4GlcNAc^l→ 2M → 4(¾ΝΑ . > 4GlcNAc 土 Neu5Aca2→ 3Gal(31→ 4GicNAc 1→ 2Manal J Neu5A C "2 → 3Gal ^ l → 4GlcNAc ^ l → 2M → 4 (¾ΝΑ .> 4GlcNAc Sat Neu5Aca2 → 3Gal (31 → 4GicNAc 1 → 2Manal J
Manffl l→4Glc Ac^l ■ 4Glc Ac Manffl l → 4Glc Ac ^ l ■ 4Glc Ac
土 Neu5Aco2→ 3Gal^l→ 4GlcNAc,i91→ 2Man£rl ^ J Sat Neu5Aco2 → 3Gal ^ l → 4GlcNAc, i91 → 2Man £ rl ^ J
[0007] 糖鎖非還元末端の修飾は、同じ脳下垂体前葉ホルモンである LHと TSHでは硫酸 化 N-ァセチルガラタトサミンが主である力 FSHではシアル酸が主であると!/、う特徴を 有している（非特許文献 3、 4)。いずれのホルモンにおいても生体内での生物活性 発現のためにはポリペプチドに結合している糖鎖部分が重要で、糖鎖を除去すると 受容体との結合能は維持されるが、血液中半減期が短くなるため生物活性が大きく 失われる（非特許文献 4〜6)。特に、糖鎖非還元末端部分のシアル酸あるいは硫酸 化 N-ァセチルガラタトサミンの除去は血中半減期に大きな影響を及ぼすことが知られ ている (非特許文献 7、 8)。このようなァシァ口化に伴う in vivo活性の低下は、シアル 酸除去によって糖鎖非還元末端側のガラクトースがむき出しとなり、卵胞刺激ホルモ ンが肝臓のガラクトース結合蛋白質に補足され分解されるために起こることが明らか にされて!ヽる（非特許文献 9)。糖鎖非還元末端がシアル酸で修飾されて ヽる糖蛋白 質の方が硫酸ィ匕 N-ァセチルガラタトサミンで修飾されている糖蛋白質より有意に長い 血中半減期を示すことが明ら力となっており（非特許文献 10)、生体において FSHが 主にシアル酸で修飾されている生理的な意味が見直されている。また、 αサブュ-ッ トの Asn52に付加している糖鎖を除去すると細胞内シグナル伝達に影響を与えること も報告されており、ポリペプチドに結合する糖鎖の重要性が指摘されている (非特許 文献 11〜13)。 [0007] The modification of the non-reducing end of the sugar chain is based on the same anterior pituitary hormones LH and TSH, which is predominantly sulfated N-acetylgalatatosamine. In FSH, sialic acid is predominant! /, (Non-Patent Documents 3 and 4). For any hormone, the sugar chain part bound to the polypeptide is important for the expression of biological activity in vivo. Removing the sugar chain maintains the ability to bind to the receptor, but it is halved in blood. Biological activity is greatly lost due to the shortened period (Non-patent Documents 4 to 6). In particular, it is known that removal of sialic acid or sulfated N-acetylylgalatatosamine at the non-reducing terminal portion of the sugar chain has a significant effect on blood half-life (Non-patent Documents 7 and 8). This decrease in in vivo activity associated with the cationization occurs because galactose on the non-reducing end of the sugar chain is exposed by sialic acid removal, and follicle-stimulating hormone is captured and degraded by the liver galactose-binding protein. It has been clarified! (Non-patent document 9). It is clear that glycoproteins modified with sialic acid at the non-reducing end of the sugar chain show a significantly longer half-life in blood than glycoproteins modified with N-acetyl-galatatosamine sulfate. (Non-patent Document 10), and the physiological meaning that FSH is mainly modified with sialic acid in the living body has been reviewed. It has also been reported that removal of the sugar chain attached to Asn52 in the α- subject affects the intracellular signal transduction, and the importance of the sugar chain that binds to the polypeptide has been pointed out ( Non-patent literature 11-13).
[0008] 実際に医療の現場で使われている性腺刺激ホルモン製剤としては、下垂体から抽 出されたものではなぐ妊婦尿または胎盤カゝら取り出した胎盤性性腺刺激ホルモンが 、 LH様活性を有する繊毛性性腺刺激ホルモン製剤 (humanchorionic gonadotropin; hCG製剤）として、更年期婦人尿カゝら抽出した下垂体性性腺刺激ホルモンが、 FSH様 活性を有する閉経婦人尿性性腺刺激ホルモン製剤（human menopausal gonadotropi n; hMG製剤）としてそれぞれ用いられている。妊娠した馬の尿力も精製した血清性性 腺刺激ホルモン (Pregnantmare serum gonadotropin; PMSG;商品名 Serotropin)も FS H製剤として市販されている力 動物力も得られた性腺刺激ホルモンはヒトに対して 抗原性を有するため、抗ホルモン抗体を生じるという欠点がある。 hMG製剤では、副 作用として、卵巣過剰刺激や卵巣破裂などが問題となっており、高純度品の使用や 適正な使用が推奨されている。しかしながら、現行の性腺刺激ホルモン製剤は上述 のごとくヒト組織または尿力も精製して製剤化するため、化学構造が類似した FSHと L Hを完全に単離することは難 、。 [0008] As a gonadotropin preparation that is actually used in the medical field, placental gonadotropin extracted from pregnant woman's urine or placenta, not extracted from the pituitary gland, has LH-like activity. Pituitary gonadotropin extracted from menopausal women's urine as a ciliary gonadotropin (hCG preparation) is FSH-like It is used as an active menopausal gonadotropin preparation (human menopausal gonadotropin; hMG preparation), respectively. Pregnantmare serum gonadotropin (PMSG; trade name Serotropin) is also available on the market as an FSH preparation. Gonadotropin with animal power is also antigenic to humans. Has the disadvantage of producing anti-hormone antibodies. In hMG preparations, ovarian hyperstimulation and ovarian rupture are problematic as side effects, and the use of high-purity products and appropriate use are recommended. However, since the current gonadotropin preparations are prepared by purifying human tissues or urine as described above, it is difficult to completely isolate FSH and LH having similar chemical structures.
[0009] このような背景の中、 Organon社が遺伝子組換え体 FSHの開発を進め、 1997年 9月 に米国で初めて認可を獲得し商品名 Puregonの名で発売を開始した。また、 Serono 社も、同年、遺伝子組換え体 FSH製剤商品名 Gona卜 Fを発売した。これらはいずれも チャイニーズノ、ムスター卵巣細胞である CHO細胞を用いて製造された遺伝子組換え 体である。女性疾患に対する関心の高まりは不妊治療にとどまらず広がりを見せてお り、特に、ホルモンバランスの乱れ力もくる疾患は女性の現代病とも言われるように増 カロしている。高純度な遺伝子組換え体 FSH製剤は、このような潜在的な需要の拡大と 相まって、多くの患者に恩恵を与えている。  [0009] Against this background, Organon promoted the development of genetically modified FSH, and in September 1997, it was approved for the first time in the United States and launched under the name Puregon. In the same year, Serono also released the product name Gona 卜 F, which is a recombinant FSH product. These are all recombinants produced using CHO cells, which are Chinese and Muster ovary cells. Increasing interest in women's diseases is expanding beyond fertility treatment, and in particular, diseases that cause hormonal balance disruption are increasing as women are said to be modern diseases. Combined with this potential increase in demand, high-purity recombinant FSH products have benefited many patients.
[0010] CHO細胞で生産した遺伝子組換え FSH製剤の糖鎖構造は、ヒト由来の製剤と比べ て、糖鎖非還元末端部分に硫酸ィ匕 N-ァセチルガラタトサミンの構造が観察されな ヽ こと、 N-グリコシド結合複合型二本鎖糖鎖が多ぐ多分岐糖鎖の割合が少ないことな どの相違が認められる。また、糖鎖へのフコース修飾率は約 50%と同等であり、完全 な N-グリコシド結合複合型糖鎖を有して 、ることが確認されて 、る (非特許文献 14〜 17)。このような糖鎖構造の相違が認められるものの、ヒト尿由来 FSH製剤と遺伝子組 換え FSH製剤の生物活性に有意な差は認められない。（非特許文献 18、 19)。  [0010] The sugar chain structure of the recombinant FSH preparation produced in CHO cells is not observed in the non-reducing terminal part of the sugar chain at the non-reducing end of the sugar chain. In addition, there are differences such as a small proportion of multi-branched sugar chains with many N-glycoside-bonded complex double-chain sugar chains. Further, it has been confirmed that the rate of fucose modification to the sugar chain is equivalent to about 50%, and it has a complete N-glycoside-linked complex type sugar chain (Non-Patent Documents 14 to 17). Although there are differences in the sugar chain structure, there is no significant difference in biological activity between human urine-derived FSH preparations and genetically modified FSH preparations. (Non-patent documents 18, 19).
[0011] ヒト生体由来の FSH製剤力 CHO細胞由来遺伝子組換え FSH製剤への切り替えが 進むことで医療の向上がもたらされたが、更なる向上が求められている。ヒト生体由来 FSH製剤および CHO細胞由来遺伝子組換え FSH製剤は、ともに血中半減期が短 ヽ ことが課題として指摘されている。現行の不妊治療では、排卵障害による無月経患者 を治療するにあたり、 75〜150国際単位の組換え FSH製剤を、 1日 1回、 3週間にわた り皮下注射で投与し続ける必要がある。毎日の投与が必要となる理由は、 FSH製剤 の血中半減期が 24時間前後と短いことと [0011] The power of FSH preparations derived from human living bodies The improvement of medical treatment has been brought about by the switch to CHO cell-derived genetically modified FSH preparations, but further improvements are required. It has been pointed out that both human FSH preparations derived from living organisms and CHO cell-derived recombinant FSH preparations have a short half-life in blood. Current infertility treatment includes amenorrhea due to ovulation disorders 75-150 international units of recombinant FSH preparation should be administered by subcutaneous injection once a day for 3 weeks. The reason why daily administration is necessary is that the blood half-life of the FSH preparation is as short as around 24 hours.
、FSH製剤を一度に大量投与すると卵巣過剰刺激症候群などの副作用を起こす危 険性があるためである。実際の不妊治療では、超音波で卵胞の成熟をモニタリングし ながら、 3週間に渡って FSH製剤を投与した後、最終日に CG製剤を投与する。この 3 週間が治療の 1サイクルである。 1サイクルの治療によって妊娠が成立する確率は 15 〜16%であり、治療にお!、て卵巣過剰刺激症候群を発症する確率は約 1%である（ 非特許文献 20)。妊娠が成立しない場合には、妊娠が成立するまでこの治療サイク ルを引き続き繰り返すことになるが、連日の皮下注射は患者あるいは医療の現場に 大きな負担を強いることになる。また、注射部位に局部的な皮膚炎が生じるなど副作 用も問題となっている (非特許文献 21)。したがって、現行の FSH製剤より患者への 投与回数を減らすことのできる、血中半減期が延長された FSH製剤の開発が求めら れている。  This is because large doses of FSH preparations may cause side effects such as ovarian hyperstimulation syndrome. In actual fertility treatment, the FSH product is administered over 3 weeks while monitoring follicular maturation with ultrasound, and then the CG product is administered on the final day. These three weeks are one cycle of treatment. The probability that pregnancy will be established by one cycle of treatment is 15 to 16%, and the probability of developing ovarian hyperstimulation syndrome is about 1% (Non-patent Document 20). If pregnancy does not occur, this treatment cycle will continue to be repeated until pregnancy is established, but daily subcutaneous injections impose a heavy burden on the patient or in the medical setting. In addition, side effects such as local dermatitis occurring at the injection site are also problematic (Non-patent Document 21). Therefore, there is a need for the development of FSH products with an extended blood half-life that can reduce the number of doses to patients compared to current FSH products.
FSH製剤の血中半減期を延長させる試みとして、変異型 FSHの造成がなされており 、 αサブユニットの N末端に Ala- Asn- lie- Thr- Va卜 Asn- lie- Thr- Valのアミノ酸配列で 示されるペプチドを結合させた変異型 FSH (非特許文献 21)、 CGの βサブユニット C 末端に存在する 4つの 0-グリコシド結合型糖鎖付加ドメインのアミノ酸配列を FSHの βサブユニットの C末端に結合させた CGと FSHとのキメラ型蛋白質 (非特許文献 22) 、 FSH aサブユニットの N末端と 13サブユニットの C末端とを、 Ser- Gly- Ser- Asn- Ala- Thr-Gly-Ser-Gly-Ser-Asn- Ala- Thr-Ser-Gly-Serのアミノ酸配列で示されるペプチド リンカ一で連結した一本鎖型 FSH (非特許文献 23)などの開発が行われて!/ヽる。上述 の変異型 FSHは、従来の組換え FSHに比べ血中半減期が 2〜4倍に延長される。しか しながら、これらの変異型 FSHは、人工的な非天然型の蛋白質であるため、医薬品と して使用する場合には抗原性の問題を考慮する必要がある。一旦、抗 FSH抗体を生 じてしまった患者では、 FSH製剤の投与ができなくなるばかりか、患者の内在性 FSH 濃度が減少する可能性もあるため深刻な問題となる。アミノ酸改変以外に FSH製剤の 血中半減期を延長させる試みとしては、ポリエチレングリコール (PEG)などの合成分 子を FSH蛋白質に人工的に連結し分子量を増加させることにより、 FSHの腎臓糸球 体力も尿中への排出を抑制する方法が検討されている（非特許文献 24、 25)。し力し ながら、 FSHに PEGを結合させることにより、 FSHの FSH受容体への親和性が大幅に 低下してしまう。また、 PEGを結合させた FSHは巨大な分子として存在するため、生体 内での分布が変化する可能性があり FSH自身が本来有する生理活性とは異なる活 性を示す危険性も考慮しなくてはならな 、 (非特許文献 26)。 In an attempt to extend the blood half-life of FSH preparations, mutant FSH has been constructed. The amino acid sequence of Ala- Asn-lie- Thr- Va 卜 Asn-lie- Thr- Val at the N-terminus of the α subunit The amino acid sequence of the four 0-glycoside-linked glycosylation domains existing at the C-terminal of the β-subunit C-terminal of CG β-subunit CSH of the FSH β-subunit CG and FSH chimeric protein bound to the terminal (Non-patent Document 22), N-terminal of FSH a subunit and C-terminal of 13 subunit, Ser-Gly- Ser- Asn- Ala- Thr-Gly -Ser-Gly-Ser-Asn-Ala-Thr-Ser-Gly-Ser Amino acid sequence of single-chain FSH (Non-patent Document 23) linked by a peptide linker is being developed! / Speak. The mutant FSH described above has a blood half-life of 2 to 4 times longer than that of conventional recombinant FSH. However, since these mutant FSHs are artificial non-natural proteins, it is necessary to consider antigenicity issues when used as pharmaceuticals. Once patients have developed anti-FSH antibodies, not only is it impossible to administer FSH preparations, but the patient's endogenous FSH concentration may decrease, which is a serious problem. In addition to amino acid modification, attempts to extend the blood half-life of FSH preparations include synthetic components such as polyethylene glycol (PEG). A method has been examined in which FSH kidney glomerular force is also suppressed from being released into the urine by artificially linking the pups to FSH protein to increase the molecular weight (Non-patent Documents 24 and 25). However, by binding PEG to FSH, the affinity of FSH for the FSH receptor is greatly reduced. In addition, since FSH to which PEG is bound exists as a huge molecule, there is a possibility that the distribution in the living body may change, and the risk of showing an activity different from the physiological activity inherent to FSH itself is not considered. Hanana (Non-patent Document 26).
非特許文献 1 : 99医療用医薬品データブック IV, 175 (1999) Non-Patent Document 1: 99 Drug Data Book IV, 175 (1999)
非特許文献 2 : European Journal of Biochemistry 242, 608 (1996) Non-Patent Document 2: European Journal of Biochemistry 242, 608 (1996)
非特許文献 3 : Endocrinology 128, 341 (1991) Non-Patent Document 3: Endocrinology 128, 341 (1991)
非特許文献 4 : Recent Progress in Hormone Research 54, 271 (1999) Non-Patent Document 4: Recent Progress in Hormone Research 54, 271 (1999)
非特許文献 5 : Molecular Endocrinology 3, 2011 (1989) Non-Patent Document 5: Molecular Endocrinology 3, 2011 (1989)
非特許文献 6 : Endocrinology 136, 2635 (1995) Non-Patent Document 6: Endocrinology 136, 2635 (1995)
非特許文献 7 : Human Reproduction 14, 1160 (1999) Non-Patent Document 7: Human Reproduction 14, 1160 (1999)
非特許文献 8 : Human Reproduction 3, 491 (1988) Non-Patent Document 8: Human Reproduction 3, 491 (1988)
非特許文献 9 : Blood 73, 84 (1989) Non-Patent Document 9: Blood 73, 84 (1989)
非特許文献 10 : Recent Progress in Hormone Research 54, 271 (1999) Non-Patent Document 10: Recent Progress in Hormone Research 54, 271 (1999)
非特許文献 11 : Molecular and Cellular Endocrinology 199, 73 (2003) Non-Patent Document 11: Molecular and Cellular Endocrinology 199, 73 (2003)
非特許文献 12 Journal of Biological Chemistry 264, 17113 (1989) Non-Patent Document 12 Journal of Biological Chemistry 264, 17113 (1989)
非特許文献 13 : Biology of Reproduction 65, 1686 (2001) Non-Patent Document 13: Biology of Reproduction 65, 1686 (2001)
非特許文献 14 Pharmacotherapy Vol.18, No.5 (1998) Non-Patent Document 14 Pharmacotherapy Vol.18, No.5 (1998)
非特許文献 15 : Biochimica et Biophysica Acta 947, 287 (1988) Non-Patent Document 15: Biochimica et Biophysica Acta 947, 287 (1988)
非特許文献 16 : Biochem. J. 287, 665 (1992) Non-Patent Document 16: Biochem. J. 287, 665 (1992)
非特許文献 17 : Molecular Human Reproduction 2, 371 (1996) Non-Patent Document 17: Molecular Human Reproduction 2, 371 (1996)
非特許文献 18 : Endocrinology, 129, 2623-2630 Non-Patent Document 18: Endocrinology, 129, 2623-2630
非特許文献 19 : Recent Progress in Hormone Research 54, 271 (1999) Non-Patent Document 19: Recent Progress in Hormone Research 54, 271 (1999)
非特許文献 20 : Novel Therapeutic Proteins Selected Case Studies WILEY- VCH(200Non-Patent Document 20: Novel Therapeutic Proteins Selected Case Studies WILEY- VCH (200
0) 0)
非特許文献 21 Journal of Clinical Endocrinology and Metabolism 88, 3227 (2002) 非特許文献 22 : Recent Progress in Hormone Research 54, 271 (1999) 非特許文献 23 : Human Reproduction 18, 50 (2003) Non-Patent Literature 21 Journal of Clinical Endocrinology and Metabolism 88, 3227 (2002) Non-Patent Document 22: Recent Progress in Hormone Research 54, 271 (1999) Non-Patent Document 23: Human Reproduction 18, 50 (2003)
非特許文献 24 : Nephrology and Dialysis and Transplantation Suppl4, 166 (2003) 非特許文献 25 Journal of Pharmaceutical Science 93, 3027 (2004)  Non-patent document 24: Nephrology and Dialysis and Transplantation Suppl4, 166 (2003) Non-patent document 25 Journal of Pharmaceutical Science 93, 3027 (2004)
非特許文献 26 :続医薬品の開発 ノ^オコンジュゲート医薬品 廣川書店 (1993) 発明の開示  Non-patent document 26: Development of follow-up drugs Noo-conjugate drugs Yodogawa Shoten (1993) Invention disclosure
発明が解決しょうとする課題  Problems to be solved by the invention
[0013] 医療現場での効率的な治療、医療事故や副作用の軽減、患者の負担軽減及び医 療経済の観点から、現行の製剤と比較して、ヒト FSHに構造及び活性が類似し、より 安全で、かつ投与回数を減らすことができるように血中半減期が延長された卵胞刺 激ホルモン製剤を提供することにある。 [0013] From the viewpoint of efficient treatment in the medical field, reduction of medical accidents and side effects, reduction of patient burden, and medical economy, the structure and activity of human FSH are similar and more The object is to provide a follicle stimulating hormone preparation that is safe and has an increased blood half-life so that the number of administrations can be reduced.
課題を解決するための手段  Means for solving the problem
[0014] 本発明は、以下の（1)〜（27)に関する。 The present invention relates to the following (1) to (27).
( 1) N-グリコシド結合複合型糖鎖を有する遺伝子組換え卵胞刺激ホルモン分子か らなる組成物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末端の N-ァセ チルダルコサミンにフコースが結合して 、な 、糖鎖である卵胞刺激ホルモン組成物。 (1) A composition comprising a genetically modified follicle-stimulating hormone molecule having an N-glycoside-linked complex sugar chain, wherein the N-glycoside-linked complex sugar chain is the N-acetyl dalcosamine at the reducing end of the sugar chain A follicle stimulating hormone composition in which fucose is bound to a sugar chain.
(2) N-グリコシド結合複合型糖鎖が、卵胞刺激ホルモンを構成する exサブユニット および j8サブユニットに結合している N-グリコシド結合複合型糖鎖である、上記（1) に記載の卵胞刺激ホルモン組成物。 (2) The follicle according to (1) above, wherein the N-glycoside-linked complex type sugar chain is an N-glycoside-linked complex type sugar chain that is bound to the ex subunit and j8 subunit constituting the follicle stimulating hormone. Stimulating hormone composition.
(3) N-グリコシド結合複合型糖鎖が、該糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が α結合していない糖鎖である、上記（1)または（2)項に記載 の卵胞刺激ホルモン組成物。 (3) The N-glycoside-bonded complex type sugar chain is a sugar chain in which the 1-position of fucose is not α- bonded to the 6-position of N-acetylyldarcosamine at the reducing end of the sugar chain. The follicle-stimulating hormone composition according to item 2).
(4) 卵胞刺激ホルモンを構成する exサブユニットが、以下の (a)、（b)、（c)、（d)、（ お よび (1)力もなる群力も選ばれるポリペプチドである、上記（1)〜（3)の 、ずれか 1項に 記載の卵胞刺激ホルモン組成物。  (4) The ex subunit that constitutes the follicle stimulating hormone is a polypeptide in which the following (a), (b), (c), (d), (and (1) a group force having a force is also selected. The follicle-stimulating hormone composition according to any one of (1) to (3).
(a) 配列番号 5で表されるァミノ配列からなるポリペプチド；  (a) a polypeptide comprising an amino sequence represented by SEQ ID NO: 5;
(b) 配列番号 6で表されるァミノ配列力もなるポリペプチド；  (b) a polypeptide having an amino acid sequence represented by SEQ ID NO: 6;
(c) 配列番号 5で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン αサブュ ニットが有する活性と実質的に同一の活性を有するポリペプチド； (c) In the amino acid sequence represented by SEQ ID NO: 5, one or more amino acids are deleted, substituted, or inserted. A polypeptide having an amino acid sequence ability added and Z or added, and having substantially the same activity as that of the follicle stimulating hormone α-subunit;
(d) 配列番号 6で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン aサブュ ニットが有する活性と実質的に同一の活性を有するポリペプチド；  (d) In the amino acid sequence represented by SEQ ID NO: 6, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and the activity and substantial activity of the follicle stimulating hormone a subunit A polypeptide having the same activity as
(e) 配列番号 5で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活性を 有するポリペプチド；  (e) a polypeptide comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 5 and having substantially the same activity as that of the follicle stimulating hormone OC subunit;
(D 配列番号 6で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活性を 有するポリペプチド；  (D a polypeptide comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6 and having substantially the same activity as that of the follicle stimulating hormone OC subunit;
(5) 卵胞刺激ホルモンを構成する exサブユニットが、以下の (a)、（b)、（c)および (d) からなる群力も選ばれる DNAがコードするポリペプチドである、上記（1)〜（3)の!、ず れカ 1項に記載の卵胞刺激ホルモン組成物。  (5) The ex subunit constituting the follicle-stimulating hormone is a polypeptide encoded by a DNA selected from the group forces consisting of the following (a), (b), (c) and (d), (1) The follicle-stimulating hormone composition according to item 1 of ~ (3).
(a) 配列番号 1で表される塩基配列からなる DNA ;  (a) DNA comprising the base sequence represented by SEQ ID NO: 1;
(b) 配列番号 2で表される塩基配列力 なる DNA；  (b) DNA having the nucleotide sequence represented by SEQ ID NO: 2;
(c) 配列番号 1で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活 性を有する蛋白質をコードする DNA ;  (c) It encodes a protein that has the same activity as that of the follicle stimulating hormone OC subunit, and is hybridized under stringent conditions with DNA having the nucleotide sequence shown by SEQ ID NO: 1. DNA to do;
(d) 配列番号 2で表される塩基配列力もなる DNAとストリンジェントな条件でハイプリ ダイズし、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活 性を有する蛋白質をコードする DNA ;  (d) DNA that encodes a protein that is hybridized under stringent conditions with DNA having the base sequence ability represented by SEQ ID NO: 2 and that has substantially the same activity as that of the follicle-stimulating hormone OC subunit ;
(6) 卵胞刺激ホルモンを構成する βサブユニットが、以下の (a)、（b)、（c)、（d)、（ お よび (1)力もなる群力も選ばれるポリペプチドである、上記（1)〜（3)の 、ずれか 1項に 記載の卵胞刺激ホルモン組成物。  (6) The β subunit that constitutes the follicle stimulating hormone is a polypeptide in which the following (a), (b), (c), (d), (and (1) powerful group force is also selected. The follicle-stimulating hormone composition according to any one of (1) to (3).
(a)配列番号 7で表されるァミノ配列からなるポリペプチド；  (a) a polypeptide comprising an amino sequence represented by SEQ ID NO: 7;
(b)配列番号 8で表されるァミノ配列力もなるポリペプチド；  (b) a polypeptide having an amino acid sequence represented by SEQ ID NO: 8;
(c)配列番号 7で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン j8サブュ ニットが有する活性と実質的に同一の活性を有するポリペプチド； (c) In the amino acid sequence represented by SEQ ID NO: 7, one or more amino acids are deleted, substituted, or inserted A polypeptide having an amino acid sequence ability added and Z or added, and having substantially the same activity as that of the follicle stimulating hormone j8 subunit;
(d)配列番号 8で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン j8サブュ ニットが有する活性と実質的に同一の活性を有するポリペプチド；  (d) In the amino acid sequence represented by SEQ ID NO: 8, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and the activity and substantial activity of the follicle stimulating hormone j8 subunit A polypeptide having the same activity as
(e)配列番号 7で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列から なり、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活性を 有するポリペプチド；  (e) a polypeptide comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 7, and having substantially the same activity as that of the follicle stimulating hormone j8 subunit;
(D配列番号 8で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列から なり、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活性を 有するポ  (D consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 8, and having a activity substantially the same as that of the follicle stimulating hormone j8 subunit.
リペプチド。 Repeptide.
(7) 卵胞刺激ホルモンを構成する βサブユニットが、以下の (a)、（b)、（c)および (d)か らなる群から選ばれる DNAがコードするポリペプチドである、上記（1)〜（3)の!、 ずれ力 1項に記載の卵胞刺激ホルモン組成物。  (7) The β subunit constituting the follicle stimulating hormone is a polypeptide encoded by a DNA selected from the group consisting of the following (a), (b), (c) and (d) (1 The follicle-stimulating hormone composition according to item 1, wherein!
(a)配列番号 3で表される塩基配列からなる DNA；  (a) DNA consisting of the base sequence represented by SEQ ID NO: 3;
(b)配列番号 4で表される塩基配列力 なる DNA；  (b) DNA having a nucleotide sequence represented by SEQ ID NO: 4;
(c)配列番号 3で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブリダ ィズし、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活性 を有するポリペプチドをコードする DNA ;  (c) a polypeptide that has substantially the same activity as that of the follicle-stimulating hormone j8 subunit, and that is hybridized under stringent conditions with DNA having the base sequence ability represented by SEQ ID NO: 3. Encoding DNA;
(d)配列番号 4で表される塩基配列力もなる DNAとストリンジェントな条件でハイブリダ ィズし、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活性 を有するポリペプチドをコードする DNA。  (d) encodes a polypeptide that hybridizes under stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 4 and has substantially the same activity as that of the follicle stimulating hormone j8 subunit. DNA.
(8) 卵胞刺激ホルモン exサブユニット及び βサブユニットをコードする DNAを宿主 細  (8) DNA encoding follicle stimulating hormone ex subunit and β subunit
胞に導入して得られる、上記（1)〜（7)の 、ずれ力 1項に記載の卵胞刺激ホルモン 組成物を生産する形質転換体。 A transformant that produces the follicle-stimulating hormone composition described in (1) above, which is obtained by introduction into a follicle.
(9) 宿主細胞が、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、また は N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコース の 1位が α結合する糖鎖修飾に関与する酵素の活性が欠失するようにゲノムが改変 された細胞である、上記（8)に記載の形質転換体。 (9) The host cell is responsible for the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, The genome was modified so that the activity of the enzyme involved in sugar chain modification in which the 1-position of fucose was α-linked to the 6-position of N-glycidyl darcosamine at the N-glycoside-linked complex sugar chain reducing end was deleted. The transformant according to (8) above, which is a cell.
(10) 宿主細胞が、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、ま たは Ν-グリコシド結合複合型糖鎖還元末端の Ν-ァセチルダルコサミンの 6位にフコ ースの 1位が OC結合する糖鎖修飾に関与する酵素のゲノム上の対立遺伝子のすべ てがノックアウトされた細胞である、上記（9)に記載の形質転換体。  (10) An enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or コ -glycidyl-linked complex glycan reducing terminal Ν-acetyldarcosamine at position 6 The transformant according to (9) above, wherein all of the alleles on the genome of an enzyme involved in sugar chain modification that binds to OC are knocked out.
(11) 細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素力 GDP-マンノ ース 4, 6-デヒドラターゼ及び GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラー ゼカもなる群力も選ばれる酵素である、上記（9)または（10)に記載の形質転換体。 (11) Intracellular sugar nucleotide GDP-Enzyme power involved in the synthesis of fucose GDP-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimelar The transformant according to (9) or (10) above, which is an enzyme for which group power is also selected.
(12) GDP-マンノース 4,6-デヒドラターゼカ 以下の（a)、（b)及び (c)からなる群から 選ばれる蛋白質である、上記（11)に記載の形質転換体。 (12) GDP-mannose 4,6-dehydratase The transformant according to (11) above, which is a protein selected from the group consisting of the following (a), (b) and (c).
(a)配列番号 10で表されるアミノ酸配列からなる蛋白質；  (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 10;
(b)配列番号 10で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ GDP-マンノース 4,6-デヒド ラタ  (b) In the amino acid sequence represented by SEQ ID NO: 10, one or more amino acids are deleted, substituted, inserted and Z or added, and the amino acid sequence capacity is GDP-mannose 4,6-dehydrata.
ーゼ活性を有する蛋白質； A protein having lyase activity;
(c)配列番号 10で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質；  (c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10 and having GDP-mannose 4,6-dehydratase activity;
(13) GDP-マンノース 4,6-デヒドラターゼカ 以下の (a)及び (b)からなる群から選ば れる DNAがコードする蛋白質である、上記（11)に記載の形質転換体。  (13) The transformant according to (11) above, which is a protein encoded by a DNA selected from the group consisting of the following (a) and (b): GDP-mannose 4,6-dehydratase
(a)配列番号 9で表される塩基配列からなる DNA；  (a) DNA comprising the base sequence represented by SEQ ID NO: 9;
(b)配列番号 9で表される塩基配列力もなる DNAとストリンジェントな条件でハイブリダ ィズし、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質をコードする DN A。  (b) DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence shown by SEQ ID NO: 9 and encodes a protein having GDP-mannose 4,6-dehydratase activity.
(14) GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼが、以下の（a)、 (b) および（c)力もなる群力も選ばれる蛋白質である、上記（11)に記載の形質転換体。 ( a)配列番号 12で表されるアミノ酸配列力 なる蛋白質； (b)配列番号 12で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ GDP-4-ケト -6-デォキシ -D -マンノース- 3,5-ェピメラーゼ活性を有する蛋白質； (14) GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is a protein in which the following (a), (b) and (c) group forces are selected (11) ). (a) a protein having the amino acid sequence ability represented by SEQ ID NO: 12; (b) In the amino acid sequence represented by SEQ ID NO: 12, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and GDP-4-keto-6-deoxy-D- A protein having mannose-3,5-epimerase activity;
(c)配列番号 12で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有する 蛋白質。  (c) It consists of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 12, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity protein.
(15) GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼが、以下の（a)及び（ b)力もなる群力 選ばれる DNAがコードする蛋白質である、上記（11)に記載の細胞  (15) GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is a protein encoded by a DNA selected from the following (a) and (b) group forces that also have power (11 Cell)
(a)配列番号 11で表される塩基配列からなる DNA； (a) DNA comprising the base sequence represented by SEQ ID NO: 11;
(b)配列番号 11で表される塩基配列力もなる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有す る蛋白質をコードする DNA。  (b) It is hybridized under stringent conditions with DNA having the nucleotide sequence shown by SEQ ID NO: 11, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity. DNA that encodes proteins.
(16) N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフ コースの 1位が a結合する糖鎖修飾に関与する酵素が a 1,6-フコシルトランスフェラ ーゼである上記（9)または（10)に記載の形質転換体。  (16) The N-glycoside-linked complex sugar chain reducing terminal N-acetylcylcosamine has an enzyme involved in sugar chain modification in which the 1-position of fucose is a-linked to the 6-position of a 1,6-fucosyltransferase The transformant according to (9) or (10) above, wherein
(17) a 1,6-フコシルトランスフェラーゼカ 以下の（a)、 (b)、（c)、（d)、（e)および (1)か らなる群力も選ばれる蛋白質である、上記（16)に記載の形質転換体。  (17) a 1,6-fucosyltransferase is a protein for which a group force consisting of the following (a), (b), (c), (d), (e) and (1) is also selected (16 ).
(a)配列番号 15で表されるアミノ酸配列からなる蛋白質；  (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 15;
(b)配列番号 16で表されるアミノ酸配列力もなる蛋白質；  (b) a protein having an amino acid sequence ability represented by SEQ ID NO: 16;
(c)配列番号 15で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ α 1,6-フコシルトランスフェラ ーゼ活性を有する蛋白質；  (c) In the amino acid sequence represented by SEQ ID NO: 15, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have α 1,6-fucosyltransferase activity. A protein having;
(d)配列番号 16で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ α 1,6-フコシルトランスフェラ ーゼ活性を有する蛋白質；  (d) In the amino acid sequence represented by SEQ ID NO: 16, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have α 1,6-fucosyltransferase activity. A protein having;
(e)配列番号 15で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ α 1,6-フコシルトランスフェラーゼ活性を有する蛋白質； (D配列番号 16で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ a 1 ,6-フコシルトランスフェラーゼ活性を有する蛋白質； (e) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 15 and having α1,6-fucosyltransferase activity; (D consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by D SEQ ID NO: 16, and a protein having a1,6-fucosyltransferase activity;
( 18) a 1 ,6-フコシルトランスフェラーゼカ 以下の（a)、（b)、（c)及び (d)力もなる群か ら選ばれる DNAがコードする蛋白質である、上記（16)に記載の形質転換体。  (18) a 1, 6-fucosyltransferase is a protein encoded by a DNA selected from the following groups (a), (b), (c) and (d) which also has a force: Transformant.
(a)配列番号 13で表される塩基配列力 なる DNA；  (a) DNA having a nucleotide sequence represented by SEQ ID NO: 13;
(b)配列番号 14で表される塩基配列力もなる DNA ;  (b) DNA having a nucleotide sequence represented by SEQ ID NO: 14;
(c)配列番号 13で表される塩基配列力もなる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ α 1 ,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする DN Α ;  (c) DN Α that encodes a protein that is hybridized under stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 13 and that has α 1, 6-fucosyltransferase activity;
(d)配列番号 14で表される塩基配列力もなる DNAとストリンジェントな条件でハイプリ ダイズし、かつ α 1 ,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする DN Α。  (d) DN Α that encodes a protein that is hybridized under stringent conditions with DNA having the nucleotide sequence shown by SEQ ID NO: 14 and that has α 1, 6-fucosyltransferase activity.
( 19) Ν-グリコシド結合複合型糖鎖還元末端の Ν-ァセチルダルコサミンの 6位とフコ ースの 1位が ex結合した糖鎖構造を認識するレクチンに耐性である、上記（8)〜（18 )の 、ずれか 1項に記載の形質転換体。  (19) Resistant to a lectin that recognizes a sugar chain structure in which the 6-position of 還 元 -acetyldarcosamine at the reducing end of Ν-glycoside-linked complex type sugar chain and the 1-position of fucose are linked to each other (8) The transformant according to any one of to (18).
(20) レクチン耐性力 Ν-グリコシド結合複合型糖鎖還元末端の Ν-ァセチルダルコ サミンの 6位とフコースの 1位が a結合した糖鎖構造を認識するレクチンを含む培地 で培養した場合に、ゲノムが改変される以前の細胞よりも高 、生存率を示すことであ る、上記（19)に記載の形質転換体。  (20) Resistance to lectin When cultured in a medium containing a lectin that recognizes a sugar chain structure in which the 6-position of 還 元 -acetyldarcosamine at the reducing end of Ν-glycoside-bonded complex and 1-position of fucose are a-linked The transformant according to (19) above, wherein the transformant is higher in viability than cells before being modified.
(21) 少なくとも、以下の (a)、（b)、（c)及び (d)力 なる群力 選ばれるレクチンの一つ に耐性である、上記（19)または（20)に記載の形質転換体。  (21) The transformation according to (19) or (20) above, which is resistant to at least one of the following (a), (b), (c) and (d) force group forces selected: body.
(a) レンズマメレクチン LCA (Lens Culinaris由来の Lentil Agglutinin) ；  (a) Lentil lectin LCA (Lentil Agglutinin from Lens Culinaris);
(b) エンドゥマメレクチン PSA (Pisum sativum由来の Pea Lectin) ；  (b) Endumen lectin PSA (Peasum sativum-derived Pea Lectin);
(c) ソラマメレクチン VFA (Vicia faba由来の Agglutinin)：  (c) Broad bean lectin VFA (Agglutinin from Vicia faba):
(d) ヒィロチャワンタケレクチン AAL (Aleuria aurantia由来の Lectin) .  (d) Herochawantake lectin AAL (Lectin from Aleuria aurantia).
(22) 宿主細胞が、下記の (a)、（b)、（c)、（d)、（e)、（£)、（g)、（h)、（i)及び (j)力 なる群か ら選ばれる細胞である上記（8)〜（21)の 、ずれか 1項に記載の形質転換体。  (22) The host cell has the following (a), (b), (c), (d), (e), (£), (g), (h), (i) and (j) forces The transformant according to any one of (8) to (21) above, which is a cell selected from a group.
(a)チャイニーズノヽムスター卵巣組織由来 CHO細胞； (b)ラットミエローマ細胞株 YB2/3HL.P2.G11.16Ag.20細胞； (a) Chinese nomstar ovarian tissue-derived CHO cells; (b) rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cells;
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0- Agl4細胞；  (d) mouse myeloma cell line SP2 / 0-Agl4 cells;
(e)シリアンノヽムスター腎臓組織由来 BHK細胞；  (e) Syrian Nomster kidney tissue-derived BHK cells;
(1)ヒト白血病細胞株ナマルバ細胞；  (1) human leukemia cell line Namalva cells;
(g)胚性幹細胞；  (g) embryonic stem cells;
(h)受精卵細胞；  (h) fertilized egg cells;
(0植物細胞；  (0 plant cells;
(j)酵母。  (j) Yeast.
(23) 上記（8)〜（22)のいずれか 1項に記載の形質転換体を培地に培養し、培養 物中に卵胞刺激ホルモン組成物を生成蓄積させ、該培養物から卵胞刺激ホルモン 組成物を採取する工程を含む、卵胞刺激ホルモン組成物の製造方法。  (23) The transformant according to any one of (8) to (22) above is cultured in a medium, a follicle stimulating hormone composition is produced and accumulated in the culture, and a follicle stimulating hormone composition is produced from the culture. The manufacturing method of a follicle stimulating hormone composition including the process of extract | collecting a thing.
(24) 上記（23)に記載の製造方法で得られた卵胞刺激ホルモン組成物。  (24) A follicle-stimulating hormone composition obtained by the production method according to (23) above.
(25) 上記（1)〜（7)および（24)に記載の卵胞刺激ホルモン組成物を有効成分と して含有する医薬。  (25) A medicament comprising as an active ingredient the follicle-stimulating hormone composition described in (1) to (7) and (24) above.
(26) 上記（1)〜（7)および（24)に記載の卵胞刺激ホルモン組成物を有効成分と して含有する卵胞成熟及び排卵障害の治療剤。  (26) A therapeutic agent for follicular maturation and ovulation disorders comprising the follicle-stimulating hormone composition described in (1) to (7) and (24) as an active ingredient.
(27) 上記（1)〜（7)および（24)に記載の卵胞刺激ホルモン組成物を有効成分と して含有する精子形成及び成熟障害の治療剤。  (27) A therapeutic agent for spermatogenesis and maturation disorder comprising the follicle-stimulating hormone composition described in (1) to (7) and (24) as an active ingredient.
発明の効果  The invention's effect
[0015] 本発明により、 N-グリコシド結合複合型糖鎖を有する遺伝子組換え卵胞刺激ホル モン分子カゝらなる組成物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末 端の N-ァセチルダルコサミンにフコースが結合して ヽな 、糖鎖である卵胞刺激ホル モン組成物が提供される。  [0015] According to the present invention, there is provided a composition comprising a recombinant follicle-stimulating hormone molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is a reduced powder of the sugar chain. Provided is a follicle stimulating hormone composition which is a sugar chain in which fucose is bound to N-acetildarcosamine at the end.
図面の簡単な説明  Brief Description of Drawings
[0016] [図 1]は、プラスミド pBS-FSH aの作製フローを示す。 [0016] [Fig. 1] shows the production flow of plasmid pBS-FSH a.
[図 2]は、プラスミド pKAN-FSH aの作製フローを示す。  [FIG. 2] shows the production flow of plasmid pKAN-FSHA.
[図 3]は、プラスミド pBS-FSH βの作製フローを示す。 [図 4]は、プラスミド pKAN-FSH α βの作製フローを示す。 [FIG. 3] shows the production flow of plasmid pBS-FSH β. [FIG. 4] shows the production flow of plasmid pKAN-FSH αβ.
[図 5]は、プラスミド pT7- FSHRの作製フローを示す。  [FIG. 5] shows the production flow of plasmid pT7-FSHR.
[図 6]は、プラスミド pKAN- FSHRの作製フローを示す。  [FIG. 6] shows the production flow of plasmid pKAN-FSHR.
[図 7]は、 FSH刺激による細胞内 cAMP誘導効果を示す。  [FIG. 7] shows the intracellular cAMP induction effect by FSH stimulation.
[図 8]は、 FSHの血中濃度推移を示す。  [Fig. 8] shows changes in blood concentration of FSH.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0017] 本発明は、 N-グリコシド結合複合型糖鎖を有する遺伝子組換え卵胞刺激ホルモン 分子カゝらなる組成物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末端の N-ァセチルダルコサミンにフコースが結合して ヽな 、糖鎖である卵胞刺激ホルモン 組成物に関する。 [0017] The present invention is a composition comprising a recombinant follicle-stimulating hormone molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is a reducing end of the sugar chain. The present invention relates to a follicle-stimulating hormone composition that is a sugar chain in which fucose is bonded to N-acetylyldarcosamine.
卵胞刺激ホルモンは OCサブユニットと βサブユニットとから構成される卵胞刺激ホル モンを包含する。したがって、本発明の卵胞刺激ホルモン組成物は、卵胞刺激ホル モンを構成する αサブユニットおよび βサブユニットにそれぞれ結合している Ν-ダリ コシド結合複合型糖鎖力 該糖鎖の還元末端の Ν-ァセチルダルコサミンににフコー スが結合して 、な 、糖鎖である、卵胞刺激ホルモン組成物を包含する。  Follicle stimulating hormone includes follicle stimulating hormone composed of OC subunit and β subunit. Therefore, the follicle-stimulating hormone composition of the present invention has a ダ -daricoside-bonded complex sugar chain strength that is bound to each of the α subunit and β subunit constituting the follicle stimulating hormone. -A follicle-stimulating hormone composition comprising a sugar chain in which fucose is bound to acetylyldarcosamine.
[0018] 本発明の、 Ν-グリコシド結合複合型糖鎖を有する遺伝子組換え卵胞刺激ホルモン 分子カゝらなる組成物であって、 Ν-グリコシド結合複合型糖鎖が該糖鎖の還元末端の Ν-ァセチルダルコサミンにフコースが結合して ヽな 、糖鎖である卵胞刺激ホルモン 組成物（以下、「本発明の組成物」とも表記する）とは、 Ν-グリコシド結合複合型糖鎖 還元末端の Ν-ァセチルダルコサミンにフコースが結合して ヽな 、遺伝子組換え卵胞 刺激ホルモン分子力 なる卵胞刺激ホルモン組成物であればいかなる組成物も包含 される。 [0018] A composition comprising a recombinant follicle-stimulating hormone molecule having a Ν-glycoside-bonded complex type sugar chain according to the present invention, wherein the Ν-glycoside-linked complex type sugar chain is a reducing end of the sugar chain. A follicle stimulating hormone composition (hereinafter also referred to as the “composition of the present invention”), which is a sugar chain in which fucose is bound to ァ -acetylyldarcosamine, is a Ν-glycoside-linked complex type sugar chain reduction Any composition is included as long as it is a follicle-stimulating hormone composition having a genetically modified follicle-stimulating hormone molecular force in which fucose is bound to the terminal ァ -acetyltilcosamine.
[0019] 本発明において、卵胞刺激ホルモンとは、女性においては卵胞細胞表面上に発現 して 、る卵胞刺激ホルモン受容体に特異的に結合し、かつ卵胞の発育と成熟を促進 する活性を有する糖蛋白質、男性においては精細管細胞表面上に発現している卵 胞刺激ホルモン受容体に特異的に結合し、かつ睾丸における精細管の発育と精子 形成を促進する活性を有する糖蛋白質であれば!/ヽかなるものも包含される。  [0019] In the present invention, the follicle stimulating hormone is expressed on the surface of follicle cells in women, specifically binds to the follicle stimulating hormone receptor, and has the activity of promoting follicular growth and maturation. Glycoprotein, in males, is a glycoprotein that specifically binds to the follicle-stimulating hormone receptor expressed on the surface of seminiferous tubules and has the activity of promoting seminiferous tube development and spermatogenesis in the testicles ! / Evil things are also included.
[0020] 具体的な卵胞刺激ホルモンとしては、下記 (a)、（b)、（c)および (d)力 なる群力 選ば れる DNAがコードする卵胞刺激ホルモン aサブユニットポリペプチドと、（e)、（£)、（g)お よび (h)力もなる群力も選ばれる DNAがコードする卵胞刺激ホルモン 13サブユニットポ リペプチドとのヘテロダイマー、または、下記 (0、（j)、 00、（1)、（m)および (n)力もなる群 力 選ばれる卵胞刺激ホルモン aサブユニットポリペプチドと、（o)、（p)、（q)、（r)、（ お よび (t)力 なる群力 選ばれる卵胞刺激ホルモン /3サブユニットポリペプチドとのへ テロダイマーなどがあげられる。 [0020] As specific follicle stimulating hormone, the following (a), (b), (c) and (d) force group force selection Follicle stimulating hormone a subunit polypeptide encoded by DNA and (e), (£), (g), and (h) follicle stimulating hormone 13 subunit polypeptide encoded by DNA that is also selected for force Or the following (0, (j), 00, (1), (m) and (n) group forces that also have a force selected follicle stimulating hormone a subunit polypeptide, and (o), (p) , (Q), (r), (and (t) force group forces, such as heterodimers with selected follicle stimulating hormone / 3 subunit polypeptides.
(a) 配列番号 1で表される塩基配列力 なる DNA ;  (a) DNA having a nucleotide sequence represented by SEQ ID NO: 1;
(b) 配列番号 2で表される塩基配列力 なる DNA；  (b) DNA having the nucleotide sequence represented by SEQ ID NO: 2;
(c) 配列番号 1で表される塩基配列からなる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活 性を有する蛋白質をコードする DNA ;  (c) A protein that is hybridized under stringent conditions with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 and that has substantially the same activity as that of the follicle stimulating hormone OC subunit. DNA to do;
(d) 配列番号 2で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活 性を有する蛋白質をコードする DNA ;  (d) Encodes a protein that is substantially identical to the activity of the follicle stimulating hormone OC subunit and that hybridizes under stringent conditions with DNA having the nucleotide sequence shown in SEQ ID NO: 2 DNA to do;
(e) 配列番号 3で表される塩基配列力 なる DNA；  (e) DNA having a nucleotide sequence represented by SEQ ID NO: 3;
(D 配列番号 4で表される塩基配列力 なる DNA； (D DNA having the nucleotide sequence represented by SEQ ID NO: 4;
(g) 配列番号 3で表される塩基配列力もなる DNAとストリンジヱントな条件でハイプリ ダイズし、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活 性を有する蛋白質をコードする DNA ;  (g) DNA that encodes a protein that is hybridized under stringent conditions with DNA having the base sequence ability represented by SEQ ID NO: 3 and that has substantially the same activity as that of the follicle stimulating hormone j8 subunit;
(h) 配列番 4で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブリダ ィズし、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活性 を有する蛋白質をコードする DNA ;  (h) encodes a protein that has the same activity as that of the follicle-stimulating hormone j8 subunit, and is hybridized under stringent conditions with DNA having the nucleotide sequence shown in SEQ ID NO: 4. DNA to do;
(0 配列番号 5で表されるァミノ配列力 なる蛋白質；  (0 a protein having amino sequence ability represented by SEQ ID NO: 5;
(j) 配列番号 6で表されるァミノ配列力 なる蛋白質； (j) a protein having an amino sequence ability represented by SEQ ID NO: 6;
(k) 配列番号 5で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン aサブュ ニットが有する活性と実質的に同一の活性を有する蛋白質；  (k) In the amino acid sequence represented by SEQ ID NO: 5, one or more amino acids are deleted, substituted, inserted, and Z or added, and the activity of the follicle stimulating hormone a subunit is substantially the same. A protein having the same activity as
(1) 配列番号 6で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン αサブュ ニットが有する活性と実質的に同一の活性を有する蛋白質； (1) In the amino acid sequence represented by SEQ ID NO: 6, one or more amino acids are deleted, substituted, or inserted A protein having the ability to sequence amino acids added and Z or added, and having substantially the same activity as that of the follicle stimulating hormone α-subunit;
(m) 配列番号 5で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活性 を有する蛋白質； (m) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 5 and having substantially the same activity as that of the follicle stimulating hormone OC subunit;
(n) 配列番号 6で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活性を 有する蛋白質；  (n) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6 and having substantially the same activity as that of the follicle stimulating hormone OC subunit;
(0) 配列番号 7で表されるァミノ配列力 なる蛋白質；  (0) a protein having an amino sequence ability represented by SEQ ID NO: 7;
(P) 配列番号 8で表されるァミノ配列力 なる蛋白質； (P) a protein having amino sequence ability represented by SEQ ID NO: 8;
(q) 配列番号 7で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン j8サブュ ニットが有する活性と実質的に同一の活性を有する蛋白質；  (q) In the amino acid sequence represented by SEQ ID NO: 7, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and the activity and substantial activity of the follicle stimulating hormone j8 subunit A protein having the same activity as
(r) 配列番号 8で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン j8サブュ ニットが有する活性と実質的に同一の活性を有する蛋白質；  (r) In the amino acid sequence represented by SEQ ID NO: 8, one or more amino acids are deleted, substituted, inserted and Z or added, and the amino acid sequence power is substantial, and the activity and substantial activity of the follicle stimulating hormone j8 subunit A protein having the same activity as
(s) 配列番号 7で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活性を 有する蛋白質；  (s) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 7, and having substantially the same activity as that of the follicle stimulating hormone j8 subunit;
(t) 配列番号 8で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活性を 有する蛋白質。  (t) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, and having substantially the same activity as that of the follicle stimulating hormone j8 subunit.
本発明にお!/、て、ストリンジヱントな条件下でハイブリダィズする DNAとは、例えば 配列番号 1、 2、 3または 4で表される塩基配列を有する DNAなどの DNAまたはその 一部の断片をプローブとして、コ口-一'ハイブリダィゼーシヨン法、プラーク 'ハイブリ ダイゼーシヨン法あるいはサザンブロットハイブリダィゼーシヨン法等を用いることによ り得られる DNAを意味し、具体的には、コロニーあるいはプラーク由来の DNAを固 定化したフィルターを用いて、 0. 7〜1. 0Mの塩化ナトリウム存在下、 65°Cでハイブ リダィゼーシヨンを行った後、 0. 1〜2倍濃度の SSC溶液（1倍濃度の SSC溶液の組 成は、 150mM塩化ナトリウム、 15mMクェン酸ナトリウムよりなる）を用い、 65°C条件 下でフィルターを洗浄することにより同定できる DNAをあげることができる。ハイブリ ダィセ ~~ンヨン ί 、 MolecularCloning, A Laboratory Manual, Second Edition,し old Sp ring Harbor LaboratoryPress, (1989) (以下、モレキュラ^ ~·クロー-ング第 2版と略す )、 Current Protocols in Molecular Biology, John Wiley & Sons,(1987- 1997) (以下、 カレント 'プロトコ一ルズ'イン'モレキュラ^ ~·バイオロジーと略す）、 DNA Cloning 1: C oreTechniques, A Practical Approach, Second Edition, Oxford University (1995)等 に記載されて 、る方法に準じて行うことができる。ハイブリダィズ可能な DNAとして具 体的には、配列番号 1、 2、 3または 4で表される塩基配列と少なくとも 60%以上の相 同性を有する DNA、好ましくは 70%以上、より好ましくは 80%以上、さらに好ましく は 90%以上、特に好ましくは 95%以上、最も好ましくは 98%以上の相同性を有する DNAをあげることができる。 In the present invention, DNA that hybridizes under stringent conditions refers to, for example, DNA such as DNA having the base sequence represented by SEQ ID NO: 1, 2, 3, or 4 or a fragment thereof. As used herein, it means DNA obtained by using the Koguchi-1 'hybridization method, plaque' hybridization method, Southern blot hybridization method, etc., specifically, colonies or plaques. Hive at 65 ° C in the presence of 0.7 to 1.0 M sodium chloride using a filter with immobilized DNA After rehydration, 0.1 to 2 times the concentration of SSC solution (the composition of 1 time concentration of SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate), and the filter was used at 65 ° C. DNA that can be identified by washing can be raised. HYBRIDISE ~~ Nyon Yong, MolecularCloning, A Laboratory Manual, Second Edition, old Spring Harbor Laboratory Press, (1989) (hereinafter abbreviated as Molecular ^ ~ Cloning 2nd Edition), Current Protocols in Molecular Biology, John Wiley & Sons, (1987-1997) (hereinafter referred to as Current 'Protocols'in' Molecular ^ ~ Biology), DNA Cloning 1: ColeTechniques, A Practical Approach, Second Edition, Oxford University (1995), etc. It can be performed according to the method described in 1. Specifically, as DNA capable of hybridizing, DNA having at least 60% or more homology with the base sequence represented by SEQ ID NO: 1, 2, 3 or 4, preferably 70% or more, more preferably 80% or more More preferred is a DNA having a homology of 90% or more, particularly preferably 95% or more, and most preferably 98% or more.
[0022] 本発明において、配列番号 5または 6で表されるアミノ酸配列において 1以上のアミ ノ酸が欠失、置換、挿入および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞 刺激ホルモン サブユニットが有する活性と実質的に同一の活性を有する蛋白質と は、モレキュラ^ ~ ·クロー-ング第 2版、カレント 'プロトコ一ルズ'イン'モレキュラ^ ~ ·バ ィォロジ一、 NucleicAcids Research, 10, 6487 (1982)、 Proc. Natl. Acad. Sci., USA, 7 9,6409 (1982)、 Gene, 34, 315 (1985)、 Nucleic Acids Research, 13, 4431(1985)、 Proc . Natl. Acad. Sci USA, 82, 488 (1985)等に記載の部位特異的変異導入法を用いて、 例えば、配列番号 5または 6で表されるアミノ酸配列を有する蛋白質をコードする DN Aに部位特異的変異を導入することにより取得することができる蛋白質を意味する。 欠失、置換、挿入および Zまたは付加されるアミノ酸の数は 1個以上でありその数は 特に限定されないが、上記の部位特異的変異導入法等の周知の技術により、欠失、 置換もしくは付加できる程度の数であり、例えば、 1〜数十個、好ましくは 1〜20個、 より好ましくは 1〜 10個、さらに好ましくは 1〜 5個である。  [0022] In the present invention, one or more amino acids in the amino acid sequence represented by SEQ ID NO: 5 or 6 are deleted, substituted, inserted and Z or added, and the follicle stimulating hormone subunit is Proteins that have substantially the same activity as those of Molecular ^ ~ Cloning 2nd Edition, Current 'Protocols' In' Molecular ^ ~ · Biologics, Nucleic Acids Research, 10, 6487 (1982 ), Proc. Natl. Acad. Sci., USA, 7 9,6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, Using site-directed mutagenesis described in 82, 488 (1985) etc., for example, site-directed mutagenesis is introduced into DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 5 or 6. Means a protein that can be obtained by The number of amino acids to be deleted, substituted, inserted and Z or added is 1 or more, and the number is not particularly limited. However, deletion, substitution or addition can be performed by well-known techniques such as the above-mentioned site-directed mutagenesis. The number is as much as possible, for example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.
[0023] また、本発明にお 、て、配列番号 5または 6で表されるアミノ酸配列と 80%以上の 相同性を有し、かつ卵胞刺激ホルモン αサブユニットが有する活性と実質的に同一 の活性を有する蛋白質とは、 BLAST〔J.Mol. Biol, 215, 403 (1990)〕や FASTA〔Me thods in Enzymology, 183, 63(1990)〕等の解析ソフトを用いて計算したときに、配列 番号 5または 6に記載のアミノ酸配列を有する蛋白質と少なくとも 80%以上、好ましく は 85%以上、より好ましくは 90%以上、さらに好ましくは 95%以上、特に好ましくは 9 7%以上、最も好ましくは 99%以上である蛋白質であることを意味する。 [0023] In the present invention, the amino acid sequence represented by SEQ ID NO: 5 or 6 has a homology of 80% or more and substantially the same activity as the follicle stimulating hormone α subunit. The protein having the activity of BLAST (J. Mol. Biol, 215, 403 (1990)) and FASTA (Methods in Enzymology, 183, 63 (1990)) At least 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more, most preferably, a protein having the amino acid sequence shown in SEQ ID NO: 5 or 6 It means that the protein is 99% or more.
[0024] 卵胞刺激ホルモン exサブユニットが有する活性と実質的に同一の活性を有する蛋 白質としては、卵胞刺激ホルモン 13サブユニットとモル比 1： 1でへテロダイマーを形 成し、該ヘテロダイマーが卵胞刺激ホルモン受容体に特異的に結合し、かつ、女性 においては卵胞の発育と成熟を促進する活性、男性においては睾丸における精細 管の発育と精子形成を促進する活性が実質的に同一である蛋白質があげられる。実 質的に同一とは活性が性質的に同一であることをいう。したがって、活性の強さなど の量的な要素は異なってもかまわない。  [0024] As a protein having substantially the same activity as that of the follicle-stimulating hormone ex subunit, a heterodimer is formed with the follicle-stimulating hormone 13 subunit at a molar ratio of 1: 1. It specifically binds to the follicle-stimulating hormone receptor and has substantially the same activity to promote follicular development and maturation in women, and to promote microtubule development and spermatogenesis in testis in men. Protein. “Substantially the same” means that the activities are the same in nature. Therefore, quantitative factors such as the strength of activity may be different.
[0025] 本発明において、配列番号 7または 8で表されるアミノ酸配列において 1以上のアミ ノ酸が欠失、置換、挿入および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞 刺激ホルモン βサブユニットが有する活性と実質的に同一の活性を有する蛋白質と は、モレキュラ^ ~ ·クロー-ング第 2版、カレント 'プロトコ一ルズ'イン'モレキュラ^ ~ ·バ ィォロジ一、 NucleicAcids Research, 10, 6487 (1982)、 Proc. Natl. Acad. Sci., USA, 7 9,6409 (1982)、 Gene, 34, 315 (1985)、 Nucleic Acids Research, 13, 4431(1985)、 Proc . Natl. Acad. Sci USA, 82, 488 (1985)等に記載の部位特異的変異導入法を用いて、 例えば、配列番号 7または 8で表されるアミノ酸配列を有する蛋白質をコードする DN Aに部位特異的変異を導入することにより取得することができる蛋白質を意味する。 欠失、置換、挿入および Zまたは付加されるアミノ酸の数は 1個以上でありその数は 特に限定されないが、上記の部位特異的変異導入法等の周知の技術により、欠失、 置換もしくは付加できる程度の数であり、例えば、 1〜数十個、好ましくは 1〜20個、 より好ましくは 1〜 10個、さらに好ましくは 1〜 5個である。  [0025] In the present invention, the amino acid sequence having one or more amino acids deleted, substituted, inserted and Z or added in the amino acid sequence represented by SEQ ID NO: 7 or 8 also has an amino acid sequence ability, and follicle stimulating hormone β subunit Proteins that have substantially the same activity as that of the molecule are as follows: Molecular ^ ~ Cloning 2nd Edition, Current 'Protocols' In' Molecular ^ ~ Biologics, Nucleic Acids Research, 10, 6487 ( 1982), Proc. Natl. Acad. Sci., USA, 7 9,6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA , 82, 488 (1985) etc., for example, site-directed mutagenesis is introduced into DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 7 or 8. Means a protein that can be obtained by The number of amino acids to be deleted, substituted, inserted and Z or added is 1 or more, and the number is not particularly limited. However, deletion, substitution or addition can be performed by well-known techniques such as the above-mentioned site-directed mutagenesis. The number is as much as possible, for example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.
[0026] また、本発明にお 、て、配列番号 7または 8で表されるアミノ酸配列と 80%以上の 相同性を有し、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一 の活性を有する蛋白質とは、 BLAST〔J.Mol. Biol, 215. 403 (1990)〕や FASTA〔Me thods in Enzymology, 183, 63(1990)〕等の解析ソフトを用いて計算したときに、配列 番号 7または 8に記載のアミノ酸配列を有する蛋白質と少なくとも 80%以上、好ましく は 85%以上、より好ましくは 90%以上、さらに好ましくは 95%以上、特に好ましくは 9 7%以上、最も好ましくは 99%以上である蛋白質であることを意味する。 [0026] Further, in the present invention, the amino acid sequence represented by SEQ ID NO: 7 or 8 has a homology of 80% or more and is substantially the same as the activity of the follicle stimulating hormone j8 subunit. Proteins having activity include BLAST [J. Mol. Biol, 215. 403 (1990)] and FASTA [Me thods in Enzymology, 183, 63 (1990)) or the like, and a protein having the amino acid sequence of SEQ ID NO: 7 or 8 and at least 80% or more, preferably 85% or more, more preferably Means 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more.
[0027] 卵胞刺激ホルモン βサブユニットが有する活性と実質的に同一の活性を有する蛋 白質としては、卵胞刺激ホルモン aサブユニットとモル比 1： 1でへテロダイマーを形 成し、該ヘテロダイマーが卵胞刺激ホルモン受容体に特異的に結合し、かつ、女性 においては卵胞の発育と成熟を促進する活性、男性においては睾丸における精細 管の発育と精子形成を促進する活性が実質的に同一である蛋白質があげられる。実 質的に同一とは活性が性質的に同一であることをいう。したがって、活性の強さなど の量的な要素は異なってもかまわない。  [0027] As a protein having substantially the same activity as that of the follicle stimulating hormone β subunit, a heterodimer is formed with the follicle stimulating hormone a subunit at a molar ratio of 1: 1. It specifically binds to the follicle-stimulating hormone receptor and has substantially the same activity to promote follicular development and maturation in women, and to promote microtubule development and spermatogenesis in testis in men. Protein. “Substantially the same” means that the activities are the same in nature. Therefore, quantitative factors such as the strength of activity may be different.
[0028] 糖蛋白質に結合している N-グリコシド結合糖鎖は、様々な構造を有している力 い ずれの場合にも以下の構造式 (I)に示す共通のコア構造を有することが知られて ヽる  [0028] N-glycoside-linked glycans bound to glycoproteins may have a common core structure represented by the following structural formula (I) in any case having various structures. Known
[0029] [化 2] [0029] [Chemical 2]
Man l- Man l-
3 Man 1■ 4GlcNAcp1 - 4GlcNAc 3 Man 1 ■ 4GlcNAcp1-4GlcNAc
Manal- Manal-
[0030] 構造式 (I) 構造式 (I)において、ァスパラギンと結合する糖鎖の末端が還元末端、反対側が非 還元末端と呼ばれている。 N-グリコシド結合糖鎖には、コア構造の非還元末端にマ ンノースのみが結合するハイマンノース型、コア構造の非還元末端側にガラクトース —N-ァセチルダルコサミン（以下、 Ga卜 GlcNAcと表記する）の枝を並行して 1な!、しは 複数本有し、更 Structural Formula (I) In Structural Formula (I), the end of the sugar chain that binds to asparagine is called the reducing end, and the opposite side is called the non-reducing end. The N-glycoside-linked sugar chain is a high mannose type in which only mannose binds to the non-reducing end of the core structure, and galactose —N-acetyldarcosamine (hereinafter referred to as Ga 卜 GlcNAc) on the non-reducing end of the core structure. 1) in parallel!
に Ga卜 GlcNAcの非還元末端側にシアル酸、バイセクティングの N-ァセチルダルコサ ミンなどの構造を有する複合型、コア構造の非還元末端側にノ、ィマンノース型と複合 型の両方の枝を持つハイブリッド型などがあることが知られている。 [0031] 本発明の組成物を構成する卵胞刺激ホルモン分子には少なくとも 4箇所に N-グリコ シド結合糖鎖の付加配列が存在し、これら部位に N-グリコシド結合糖鎖が結合する。 卵胞刺激ホルモンに結合する N-グリコシド結合糖鎖としては、具体的には、上述の N -グリコシド結合複合型糖鎖を挙げることができる。卵胞刺激ホルモン分子に結合す る N-ダルコシド結合複合型糖鎖としては、前記構造式 (I)で示されるコア構造を含む いかなる糖鎖も包含さ In addition, hybrids with structures such as sialic acid on the non-reducing end side of Ga 卜 GlcNAc and bisecting N-acetylethyldarcosamine, etc., and hybrids with both mannose-type and complex-type branches on the non-reducing end side of the core structure. It is known that there are types. [0031] The follicle-stimulating hormone molecule constituting the composition of the present invention has an additional sequence of N-glycoside-linked sugar chains at at least 4 sites, and N-glycoside-linked sugar chains are bound to these sites. Specific examples of the N-glycoside-linked sugar chain that binds to follicle-stimulating hormone include the aforementioned N-glycoside-linked complex sugar chain. The N-dalcoside-linked complex sugar chain that binds to the follicle-stimulating hormone molecule includes any sugar chain containing the core structure represented by the structural formula (I).
れるので、多数の糖鎖の組み合わせが存在することになる。  Therefore, there are many combinations of sugar chains.
[0032] したがって、本発明の組成物は、本発明の効果が得られる範囲であれば、単一の 糖鎖構造を有する卵胞刺激ホルモン分子から構成されて!ヽてもよ!ヽし、複数の異な る糖鎖構造を有する卵胞刺激ホルモン分子から構成されて ヽてもよ ヽが、本発明の 組成物は、 V、ずれの糖鎖構造にお ヽても N-グリコシド結合複合型糖鎖還元末端の N -ァセチルダルコサミンにフコースが結合して!/、な!/、糖鎖を有して 、る。  [0032] Therefore, the composition of the present invention may be composed of follicle stimulating hormone molecules having a single sugar chain structure, as long as the effects of the present invention are obtained. Although the composition of the present invention may be composed of follicle-stimulating hormone molecules having different sugar chain structures, the composition of the present invention has an N-glycoside-linked complex type sugar chain, even if the sugar structure is V. Fucose is bound to the reducing terminal N-acetyl darcosamine! /, N! /, And has a sugar chain.
[0033] N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンにフコースが結 合して 、な 、糖鎖とは、フコースが N-グリコシド結合複合型糖鎖の N-ァセチルダルコ サミンに結合していない糖鎖であれば、いかなるものも包含され、非還元末端の糖鎖 の構造に多様性があっても構わない。例えばフコースの 1位が N-グリコシド結合複合 型糖鎖の N-ァセチルダルコサミンの 6位に (X結合して!/ヽな ヽ糖鎖等があげられる。  [0033] N-glycoside-bonded complex type sugar chain N-acetylyldarcosamine of the reducing end is linked to fucose. Any sugar chain may be included as long as the sugar chain is not bound to a non-reducing terminal sugar chain. For example, the 1st position of fucose is the 6th position of the N-glycidyl darcosamine of the N-glycoside-bonded complex sugar chain (X-linked!
[0034] N-グリコシド結合複合型糖鎖を有する卵胞刺激ホルモン分子からなる組成物中の 糖鎖構造の解析は、卵胞刺激ホルモン分子からヒドラジン分解や酵素消化などの公 知の方法 [生  [0034] Analysis of the sugar chain structure in a composition comprising follicle-stimulating hormone molecules having N-glycoside-bonded complex sugar chains is based on known methods such as hydrazine degradation and enzymatic digestion from follicle-stimulating hormone molecules.
物化学実験法 23—糖タンパク質糖鎖研究法 (学会出版センター)高橋禮子編 (1989) ]を  Physical Chemistry Experimental Method 23—Glycoprotein Glycan Research Method (Academic Publishing Center) Takahashi Kyoko (1989)]
用い、糖鎖を遊離させ、遊離させた糖鎖を蛍光標識又は同位元素標識し、標識した 糖鎖をクロマトグラフィー法にて分離することによって決定することができる。また、遊 離させた糖鎖を HPAED- PAD法 [ジャーナル ·ォブ ·リキッド ·クロマトグラフィー（Jliq. Chromatogr.) , 6, 1577 (1983)]によって分析することで決定することもできる。  It can be determined by releasing the sugar chain, labeling the released sugar chain with a fluorescent label or an isotope label, and separating the labeled sugar chain by a chromatographic method. It can also be determined by analyzing the released sugar chain by the HPAED-PAD method [Jliq. Chromatogr., 6, 1577 (1983)].
[0035] 本発明にお 、て、糖鎖還元末端の Ν-ァセチルダルコサミンにフコースが結合して いない糖鎖とは、糖鎖に、実質的にフコースが結合していないことをいい、好ましくは フコース含有率が 0%であることをいう。実質的にフコースが結合していないとは、具 体的には、後述の 4に記載の糖鎖分析において、フコースが実質的に検出できない ことをいう。実質的に検出できないとは、測定の検出限界以下であることを意味する。 [0035] In the present invention, a sugar chain in which fucose is not bound to チ ル -acetylyldarcosamine at the sugar chain reducing end means that fucose is not substantially bound to the sugar chain. Preferably This means that the fucose content is 0%. The fact that fucose is not substantially bound means that fucose cannot be substantially detected in the sugar chain analysis described in 4 below. “Substantially undetectable” means below the detection limit of measurement.
[0036] 本発明の組成物は、従来力も知られているヒト尿由来 FSHなどの N-グリコシド結合 複合型糖鎖還元末端の N-ァセチルダルコサミンにフコースが結合している卵胞刺激 ホルモンに比べて、卵胞刺激ホルモン受容体に対する同等の親和性を有し、かつ生 体内に投与した場合に血中半減期が長い。 [0036] The composition of the present invention is a follicle-stimulating hormone in which fucose is bound to N-acetylside darcosamine at the N-glycosidic complex reducing end, such as human urine-derived FSH, which is also known in the art. In comparison, it has an equivalent affinity for the follicle-stimulating hormone receptor and has a long blood half-life when administered in vivo.
本発明の形質転換体としては、本発明の組成物を生産することができる形質転換 体であれば、いかなる形質転換体でも包含される。具体的な例としては、卵胞刺激ホ ルモン分子を形成する各サブユニット蛋白質をコードする DNAを、以下の (a)または (b The transformant of the present invention includes any transformant as long as it is capable of producing the composition of the present invention. As a specific example, DNA encoding each subunit protein that forms a follicle-stimulating hormone molecule is represented by the following (a) or (b
)などの宿主細胞に導入して得られる形質転換体あげられる。 ) And the like obtained by introduction into a host cell.
[0037] (a)細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素の活性が欠失する ようにゲノムが改変された細胞； [0037] (a) a cell whose genome has been modified to lack the activity of an enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose;
(b) N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコ ースの 1位が α結合する糖鎖修飾に関与する酵素の活性が欠失するようにゲノムが 改変された  (b) N-glycoside-linked complex-type sugar chain reducing N-acetylyldarcosamine at the 6-position of the genome so that the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked is deleted Has been modified
細胞。  cell.
[0038] 細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素としては、 GDP-マンノ ース 4,6-デヒドラターゼ（GMD)、 GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメ ラーゼ (FX)などがあげられる。  [0038] Enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose include GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose-3,5 -Epimerase (FX).
本発明において、 GDP-マンノース 4,6-デヒドラターゼとしては、下記 (a)あるいは (b) の DNAがコードする蛋白質、または下記 (c)、（d)あるいは (e)の蛋白質などがあげられ る。  In the present invention, the GDP-mannose 4,6-dehydratase includes a protein encoded by the following DNA (a) or (b), or a protein (c), (d) or (e) below. .
[0039] (a)配列番号 9で表される塩基配列力 なる DNA;  [0039] (a) DNA having a nucleotide sequence represented by SEQ ID NO: 9;
(b)配列番号 9で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質をコードする DNA;  (b) a DNA that encodes a protein that has a base sequence ability represented by SEQ ID NO: 9 and hybridizes under stringent conditions and has a GDP-mannose 4,6-dehydratase activity;
(c)配列番号 10で表されるアミノ酸配列からなる蛋白質； (d)配列番号 10で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、 挿入および/または付加されたアミノ酸配列力もなり、かつ GDP-マンノース 4,6-デヒ ドラターゼ活性を有する蛋白質； (c) a protein comprising the amino acid sequence represented by SEQ ID NO: 10; (d) In the amino acid sequence represented by SEQ ID NO: 10, one or more amino acids are deleted, substituted, inserted and / or added, and have an amino acid sequence ability and have GDP-mannose 4,6-dehydratase activity protein;
(e)配列番号 10で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質。  (e) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, and having GDP-mannose 4,6-dehydratase activity.
[0040] 本発明において、 GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼとして は、下記 (a)あるいは (b)の DNAがコードする蛋白質、または下記 (c)、（d)あるいは (e)の 蛋白質などがあげられる。  [0040] In the present invention, GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase includes a protein encoded by the following DNA (a) or (b), or the following (c), (D) or (e) protein.
(a)配列番号 11で表される塩基配列力 なる DNA;  (a) DNA having a nucleotide sequence represented by SEQ ID NO: 11;
(b)配列番号 11で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブ リダィズし、かつ GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有 する蛋白質をコードする DNA;  (b) DNA, which has a base sequence ability represented by SEQ ID NO: 11, is hybridized under stringent conditions and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity. DNA encoding the protein to be
(a)配列番号 12で表されるアミノ酸配列力もなる蛋白質；  (a) a protein having an amino acid sequence ability represented by SEQ ID NO: 12;
(b)配列番号 12で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、 挿入および Zまたは付加されたアミノ酸配列力もなり、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有する蛋白質；  (b) In the amino acid sequence represented by SEQ ID NO: 12, one or more amino acids are deleted, substituted, inserted and Z or added amino acid sequence power, and GDP-4-keto-6-deoxy-D-mannose -A protein having 3,5-epimerase activity;
(c)配列番号 12で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有す る蛋白質。  (c) It comprises an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 12, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity Protein.
[0041] N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコース の 1位が a結合する糖鎖修飾に関与する酵素としては、 a 1,6-フコシルトランスフェラ ーゼなどがあげられる。  [0041] N-glycoside-linked complex type sugar chain reducing terminal N-acetylcylcosamine 6-position of the fucose 1-position, the enzyme involved in sugar chain modification is a 1,6-fucosyltransferase For example.
本発明において、 α 1,6-フコシルトランスフェラーゼとしては、下記 (a)、（b)、（c)ある いは (d)の DNAがコードする蛋白質、または ( 、（£)、（g)、（h)、（0あるいは (j)の蛋白質な どがあげられる。  In the present invention, α 1,6-fucosyltransferase is a protein encoded by the following DNA (a), (b), (c) or (d), or (, (£), (g), (H), (0 or (j) protein, etc.).
[0042] (a)配列番号 13で表される塩基配列力もなる DNA; [0042] (a) DNA having a nucleotide sequence represented by SEQ ID NO: 13;
(b)配列番号 14で表される塩基配列力もなる DNA;  (b) DNA having a nucleotide sequence represented by SEQ ID NO: 14;
(c)配列番号 13で表される塩基配列からなる DNAとストリンジェントな条件でノ、イブ リダィズし、かつ (X 1,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする D NA; (c) DNA having the nucleotide sequence represented by SEQ ID NO: 13 and stringent conditions under stringent conditions Redis and (XNA encoding a protein having 1,6-fucosyltransferase activity;
(d)配列番号 14で表される塩基配列力もなる DNAとストリンジェントな条件でノ、イブ リダィズし、かつ a 1,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする D NA;  (d) DNA that encodes a protein that has a base sequence ability represented by SEQ ID NO: 14 and is stringent under a stringent condition and has a 1,6-fucosyltransferase activity;
(e)配列番号 15で表されるアミノ酸配列力もなる蛋白質；  (e) a protein having an amino acid sequence ability represented by SEQ ID NO: 15;
(£)配列番号 16で表されるアミノ酸配列力もなる蛋白質；  (£) a protein having an amino acid sequence ability represented by SEQ ID NO: 16;
(g)配列番号 15で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、 挿入および Zまたは付加されたアミノ酸配列力もなり、かつ α 1,6-フコシルトランスフ エラーゼ活性を有する蛋白質；  (g) a protein having an amino acid sequence ability in which one or more amino acids are deleted, substituted, inserted and Z or added in the amino acid sequence represented by SEQ ID NO: 15 and having α1,6-fucosyltransferase activity;
(h)配列番号 16で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、 挿入および Zまたは付加されたアミノ酸配列力もなり、かつ α 1,6-フコシルトランスフ エラーゼ活性を有する蛋白質；  (h) a protein having an amino acid sequence ability in which one or more amino acids are deleted, substituted, inserted and Z or added in the amino acid sequence represented by SEQ ID NO: 16 and having α 1,6-fucosyltransferase activity;
(0配列番号 15で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつ a 1,6-フコシルトランスフェラーゼ活性を有する蛋白質；  (0 a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 15, and having a 1,6-fucosyltransferase activity;
0)配列番号 16で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつ a 1,6-フコシルトランスフェラーゼ活性を有する蛋白質。  0) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 16, and having a 1,6-fucosyltransferase activity.
本発明にお!/、て、ストリンジヱントな条件下でハイブリダィズする DNAとは、例えば配 列番号 9、 11、 13または 14で表される塩基配列力もなる DNAなどの DNAまたはその 一部の断片をプローブとして、コ口-一'ハイブリダィゼーシヨン法、プラーク 'ハイブリ ダイゼーシヨン法あるいはサザンノヽイブリダィゼーシヨン法等を用いることにより得られ る DNAを意味し、具体的には、コロニーあるいはプラーク由来の DNAを固定化したフ ィルターを用いて、 0. 7〜1. OMの塩化ナトリウム存在下、 65°Cでハイブリダィゼー シヨンを行った後、 0. 1〜2倍濃度の SSC溶液（1倍濃度の SSC溶液の組成は、 150 mM塩化ナトリウム、 15mMクェン酸ナトリウムよりなる）を用い、 65°C条件下でフィル ターを洗浄することにより同定できる DNAをあげることができる。ノ、イブリダィゼーショ ン【ま、 Molecularし loning, A Laooratory Manual, Second edition, Cold Spring Harbor Laboratory Press, 1989 (以下、モレキュラ^ ~ ·クロー-ング第 2版と略す）、 Current Pr otocols in MolecularBiology, John Wiley & Sons, 1987— 1997 (以下、カレント 'プロトコ 一ルズ'イン'モレキュラ^ ~ ·バイオロジーと略す）、 DNA Cloning 1: Core Techniques, A PracticalApproach, Second edition, Oxford University (1995)等に己载されている 方法に準じて行うことができる。ストリンジェントな条件下でノ、イブリダィズ可能な DNA として具体的には、配列番号 9、 11、 13または 14で表される塩基配列と少なくとも 60 %以上の相同性を有する DNA、好ましくは 70%以上、より好ましくは 80%以上、さら に好ましくは 90%以上、特に好ましくは 95%以上、最も好ましくは 98%以上の相同 性を有する DNAをあげることができる。 In the present invention, DNA that hybridizes under stringent conditions means, for example, DNA such as DNA having a nucleotide sequence represented by SEQ ID NO: 9, 11, 13, or 14, or a fragment thereof. This means DNA obtained by using the Koguchi-1 'hybridization method, plaque' hybridization method, Southern hybridization method, etc. as a probe, and specifically, colonies or plaques. After performing hybridization at 65 ° C in the presence of 0.7 to 1. OM sodium chloride using a filter to which DNA derived from DNA was immobilized, 0.1 to 2 times the concentration of SSC solution (1x The composition of the SSC solution with the concentration is 150 mM sodium chloride and 15 mM sodium citrate), and DNA can be identified by washing the filter under 65 ° C conditions. , Molecularization loning, A Laooratory Manual, Second edition, Cold Spring Harbor Laboratory Press, 1989 (hereinafter abbreviated as Molecular ^ ~ Cloning 2nd Edition), Current Pr otocols in MolecularBiology, John Wiley & Sons, 1987—1997 (hereinafter referred to as Current 'Protocol Ones In' Molecular ^ ~ Biology), DNA Cloning 1: Core Techniques, A PracticalApproach, Second edition, Oxford University (1995 ) Etc. can be carried out according to the method self-installed. Specifically, DNA that can be hybridized under stringent conditions is specifically DNA having at least 60% homology with the nucleotide sequence represented by SEQ ID NO: 9, 11, 13, or 14, preferably 70% or more More preferred is DNA having a homology of 80% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.
[0044] 本発明において、配列番号 10で表されるアミノ酸配列において 1以上のアミノ酸が 欠失、置換、挿入および Zまたは付加されたアミノ酸配列からなり、かつ GDP-マンノ ース 4,6-デヒドラターゼ活性を有する蛋白質、配列番号 12で表されるアミノ酸配列に おいて [0044] In the present invention, the amino acid sequence represented by SEQ ID NO: 10 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or added, and GDP-mannose 4,6-dehydratase In the amino acid sequence represented by SEQ ID NO: 12, an active protein
1以上のアミノ酸が欠失、置換、挿入および Zまたは付加されたアミノ酸配列力もなり 、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有する蛋白質 、または配列番号 15または 16で表されるアミノ酸配列にお 、て 1以上のアミノ酸が欠 失、置換、挿入および Zまたは付加されたアミノ酸配列からなり、かつ α 1,6-フコシル トランスフェラーゼ活性を有する蛋白質は、モレキュラー 'クローユング第 2版、カレン ト.プロトコールズ.イン.モレキュラー.バイオロジー、 NucleicAcids Research, 10, 648 7 (1982)、 Proc. Natl. Acad. Sci., USA, 79,6409 (1982)、 Gene, 34, 315 (1985)、 Nucle ic Acids Research, 13, 4431(1985)、 Proc. Natl. Acad. Sci USA, 82, 488 (1985)等に 記載の部位特異的変異導入法を用いて、例えば、配列番号 10、 12、 15または 16で 表される塩基配列を有する DNAに部位特異的変異を導入することにより取得すること ができる。欠失、置換、挿入および Zまたは付加されるアミノ酸の数は 1個以上であり その数は特に限定されないが、上記の部位特異的変異導入法等の周知の技術によ り、欠失、置換もしくは付加できる程度の数であり、例えば、 1〜数十個、好ましくは 1 〜20個、より好ましくは 1〜10個、さらに好ましくは 1〜5個である。  A protein having an amino acid sequence ability in which one or more amino acids are deleted, substituted, inserted and Z or added, and has a GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity, or SEQ ID NO: A protein having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or added in the amino acid sequence represented by 15 or 16, and having α1,6-fucosyltransferase activity, is molecular. 'Crowung 2nd Edition, Current Protocols in Molecular Biology, Nucleic Acids Research, 10, 648 7 (1982), Proc. Natl. Acad. Sci., USA, 79,6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), etc. D having the base sequence represented by SEQ ID NO: 10, 12, 15 or 16 It can be obtained by introducing site-specific mutations into NA. The number of amino acids to be deleted, substituted, inserted, and Z or added is 1 or more, and the number is not particularly limited. However, deletion and substitution can be performed by well-known techniques such as the above-described site-directed mutagenesis. Or it is the number which can be added, for example, 1 to several tens, Preferably it is 1-20, More preferably, it is 1-10, More preferably, it is 1-5.
[0045] また、本発明において、配列番号 10、 12、 15または 16で表されるアミノ酸配列と 8 0%以上の相同性を有するアミノ酸配列からなり、かつ GDP-マンノース 4,6-デヒドラタ ーゼ活性、 GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性または α 1, 6-フコシルトランスフェラーゼ活性を有するためには、それぞれ配列番号 10、 12、 15 または 16で表されるアミノ酸配列と BLAST〔J. Mol. Biol, 215, 403 (1990)〕や FAS TA [Methods inEnzymology, 183, 63 (1990)〕等の解析ソフトを用いて計算したときに 、少なくとも 80%以上、好ましくは 85%以上、より好ましくは 90%以上、さらに好ましく は 95%以上、特に好ましくは 97%以上、最も好ましくは 99%以上の相同性を有する 蛋白質であることを意味する。 [0045] Further, in the present invention, it comprises an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, 12, 15 or 16, and GDP-mannose 4,6-dehydrata In order to have asease activity, GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity or α 1,6-fucosyltransferase activity, respectively in SEQ ID NO: 10, 12, 15 or 16 And at least 80% when calculated using analysis software such as BLAST [J. Mol. Biol, 215, 403 (1990)] and FAS TA [Methods in Enzymology, 183, 63 (1990)]. This means that the protein has a homology of 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more.
[0046] また、上述の酵素活性が欠失した宿主細胞、すなわち細胞内糖ヌクレオチド GDP- フコースの合成に関与する酵素、または N-グリコシド結合複合型糖鎖還元末端の N- ァセチルダルコサミンの 6位にフコースの 1位が a結合する糖鎖修飾に関与する酵素 の活性が欠失するようにゲノムが改変された宿主細胞に、卵胞刺激ホルモン分子の aサブユニットおよび j8サブユニットをコードする DNAを導入することによって、本発 明の組成物を生産する形質転換体を得ることができる。 [0046] Further, a host cell lacking the above-mentioned enzyme activity, that is, an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-acetylyldarcosamine at the reducing end of N-glycoside-linked complex type sugar chain Encodes the a and j8 subunits of the follicle-stimulating hormone molecule in a host cell whose genome has been altered so that the activity of the enzyme involved in the glycosylation modification in which the 1-position of fucose is linked to a at position 6 By introducing DNA, a transformant producing the composition of the present invention can be obtained.
[0047] ここで、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、または N-ダリ コシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が α結合する糖鎖修飾に関与する酵素の活性が欠失するようにゲノムが改変されたと は、該酵素の発現を消失させるように該遺伝子の発現調節領域に変異を導入したり 、あるいは該酵素の機能を消失させるように該遺伝子のアミノ酸配列に変異を導入す ることを意味する。変異を導入するとは、ゲノム上の塩基配列に欠失、置換、挿入お よび Ζまたは付加と 、つた塩基配列の改変を行うことを意味し、改変したゲノム遺伝 子の発現または機能を完全に抑制することをノックアウトすると 、う。ゲノム遺伝子をノ ックアウトする具体的な例としては、標的となる遺伝子のすべてまたは一部がゲノムか ら削除された例が挙げられる。標的となる遺伝子の開始コドンを含むエタソンのゲノム 領域を染色体上力 除くことでノックアウトすることができる。  [0047] Here, the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-acetylcolcamine at the reducing end of N-darcoside-linked complex sugar chain, the 6-position of fucose, the 1-position of fucose is α-linked When the genome is modified so that the activity of the enzyme involved in the sugar chain modification is lost, a mutation is introduced into the expression regulatory region of the gene so as to eliminate the expression of the enzyme, or the function of the enzyme It means that a mutation is introduced into the amino acid sequence of the gene so as to disappear. Introducing mutation means that the base sequence on the genome is deleted, substituted, inserted, and deleted or added, and the base sequence is modified, completely suppressing the expression or function of the modified genomic gene. Knock out to do it. A specific example of knocking out a genomic gene is one in which all or part of the target gene has been deleted from the genome. It can be knocked out by removing the genomic region of the etason containing the start codon of the target gene.
[0048] このような細胞を取得する方法としては、目的とするゲノムの改変を行うことができれ ば、いずれの手法でも用いることができる。上述の酵素活性を欠失させる手法として  [0048] As a method for obtaining such cells, any method can be used as long as the target genome can be modified. As a method of deleting the above enzyme activity
(a)酵素の遺伝子を標的した遺伝子破壊の手法； (b)酵素の遺伝子のドミナントネガティブ体を導入する手法； (a) a gene disruption technique targeting an enzyme gene; (b) a method of introducing a dominant negative form of the enzyme gene;
(c)酵素につ 、ての突然変異を導入する手法；  (c) a method of introducing all mutations in the enzyme;
(d)酵素の遺伝子の転写又は翻訳を抑制する手法；  (d) a method for suppressing transcription or translation of an enzyme gene;
(e) N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1 位が oc結合した糖鎖構造を認識するレクチンに耐性である株を選択する手法などが あげられる。  (e) A method of selecting a strain resistant to a lectin that recognizes a sugar chain structure in which the N-glycidyl glycosamine 6-position of the N-glycoside-linked sugar chain and the 1-position of fucose are oc-linked .
[0049] N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1位が  [0049] N-glycoside-linked sugar chain reducing terminal N-acetylcylcosamine position 6 and fucose position 1
a結合した糖鎖構造を認識するレクチンとしては、該糖鎖構造を認識できるレクチン であれば、いずれのレクチンでも用いることができる。その具体的な例としては、レン ズマメレクチン LCA (Lens Culinaris由来の Lentil Agglutinin)、エンドゥマメレクチン PS A (Pisum sativum由来の PeaLectin)、ソラマメレクチン VFA (Vicia faba由来の Agglutini n)、ヒィロチャワンタケレクチン AAL (Aleuria aurantia由来の Lectin)等を挙げることが できる。  As the lectin that recognizes the a-linked sugar chain structure, any lectin that can recognize the sugar chain structure can be used. Specific examples of this are: Lentil lectin LCA (Lentil Agglutinin from Lens Culinaris), Endumame lectin PS A (Peum sativum-derived PeaLectin), Broad bean lectin VFA (Agglutini n from Vicia faba), Hirochawantake lectin AAL ( Lectin from Aleuria aurantia).
[0050] レクチンに耐性な細胞とは、レクチンを有効濃度与えたときにも、生育が阻害されな い細胞をいう。有効濃度とは、ゲノム遺伝子が改変される以前の細胞（以下、「親株 細胞」とも称す)が正常に生育できない濃度以上であり、好ましくは、ゲノム遺伝子が 改変される以前の細胞が生育できない濃度と同濃度、より好ましくは 2〜5倍、さらに 好ましくは 10倍、最も好ましくは 20倍以上である。  [0050] A cell resistant to lectin refers to a cell whose growth is not inhibited even when an effective concentration of lectin is given. The effective concentration is not less than the concentration at which cells before the genomic gene is modified (hereinafter also referred to as “parent cell”) cannot grow normally, preferably the concentration at which cells before the modified genomic gene cannot grow , More preferably 2 to 5 times, still more preferably 10 times, and most preferably 20 times or more.
[0051] 本発明において、生育が阻害されないレクチンの有効濃度は、細胞株に応じて適 宜定めればよいが、通常 10 /z g/ml〜10mg/ml、好ましくは 0.5mg/ml〜2.0mg/mlであ る。  [0051] In the present invention, the effective concentration of a lectin whose growth is not inhibited may be appropriately determined depending on the cell line, but is usually 10 / zg / ml to 10mg / ml, preferably 0.5mg / ml to 2.0mg. / ml.
本発明の形質転換体としては、本発明の組成物を発現できる細胞であれば!/ヽかな る細胞でもよいが、酵母、動物細胞、昆虫細胞、植物細胞などがあげられ、これらの 細胞の具体的な例としては、後述の 3. に記載のものがあげられる。動物細胞の具体 例としては、チャイニーズノヽムスター卵巣組織由来の CHO細胞、ラットミエローマ細胞 株 YB2/3HL.P2.G11.16Ag.20細胞、マウスミエローマ細胞株 NS0細胞、マウスミエロー マ細胞株 SP2/0-Agl4細胞、シリアンハムスター腎臓組織由来 BHK細胞、ヒト白血病 細胞株ナマルバ細胞、胚性幹細胞、受精卵細胞などがあげられる。好ましくは、遺伝 子組換え糖蛋白質医薬品を製造するために用いる宿主細胞、遺伝子組換え糖蛋白 質医薬品を生産するヒト以外のトランスジエニック動物を製造するために用いる胚性 幹細胞または受精卵細胞、ならびに遺伝子組換え糖蛋白質医薬品を生産するトラン スジ ニック植物を製造するために用いる植物細胞などがあげられる。 As the transformant of the present invention, any cell that can express the composition of the present invention can be used. However, yeast, animal cells, insect cells, plant cells and the like can be mentioned. Specific examples include those described in 3. below. Specific examples of animal cells include CHO cells derived from Chinese omster ovary tissue, rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cell, mouse myeloma cell line NS0 cell, mouse myeloma cell line SP2 / 0- Examples include Agl4 cells, Syrian hamster kidney tissue-derived BHK cells, human leukemia cell lines Namalba cells, embryonic stem cells, and fertilized egg cells. Preferably genetic Host cells used to produce recombinant glycoprotein drugs, embryonic stem cells or fertilized egg cells used to produce non-human transgenic animals that produce genetically modified glycoprotein drugs, and recombinant sugars Examples include plant cells used to produce transgene plants that produce protein drugs.
[0052] 親株細胞としては、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、ま たは N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコ ースの 1位が OC結合する糖鎖修飾に関与する酵素のゲノム遺伝子を改変させるため の手法を施す前の細胞を包含する。例えば、以下の細胞が好適にあげられる。  [0052] As the parent cell line, the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-glycidyl-linked glycan reducing end N-acetylcylcosamine at position 6 of fucose It includes cells prior to the application of a technique for altering the genomic gene of an enzyme involved in sugar chain modification in which position 1 is OC-linked. For example, the following cells are preferable.
NS0細胞の親株細胞としては、バイオ/テクノロジー (BIO/TECHNOLOGY), 10, 169 (1992)、バイオテクノロジ一'バイオエンジニアリング (Biotechnol.Bioeng.), 73, 261, (2 001)等の文献に記載されている NS0細胞があげられる。また、理化学研究所細胞開 発銀行に登録されている NS0細胞株（RCB0213)、あるいはこれら株を様々な無血清 培地に馴化させた亜株などもあげられる。  NS0 cell parent cell lines are described in the literature such as Bio / Technology (BIO / TECHNOLOGY), 10, 169 (1992), Biotechnology No. 1 Bioengineering (Biotechnol. Bioeng.), 73, 261, (2 001), etc. NS0 cells are listed. In addition, NS0 cell line (RCB0213) registered with the RIKEN Cell Development Bank, or sub-strains obtained by acclimatizing these strains to various serum-free media are also included.
[0053] SP2/0-Agl4細胞の親株細胞としては、ジャーナル'ォブ 'ィムノロジー (J. Immunol.) .126. 317, (1981)、ネイチヤー (Nature), 276, 269, (1978)、ヒューマン 'アンチイボディ ズ'アンド'ノ、イブリドーマズ (HumanAnt¾odies and Hybridomas), 3, 129, (1992)等の 文献に記載されている SP2/0-Agl4細胞があげられる。また、 ATCCに登録されてい る SP2/0-Agl4細胞（ATCC CRL-1581)あるいはこれら株を様々な無血清培地に馴 化させた亜株 (ATCC CRL-1581.1)などもあげられる。  [0053] As a parent cell of SP2 / 0-Agl4 cells, Journal 'Ob' Immunology (J. Immunol.) .126. 317, (1981), Nature (Nature), 276, 269, (1978), Human SP2 / 0-Agl4 cells described in documents such as “Antibody's” and “HumanAnt¾odies and Hybridomas”, 3, 129, (1992). In addition, SP2 / 0-Agl4 cells (ATCC CRL-1581) registered in ATCC or sub-strains (ATCC CRL-1581.1) in which these strains are conditioned in various serum-free media are also included.
[0054] チャイニーズノヽムスター卵巣組織由来 CHO細胞の親株細胞としては、 Journal of E xperimental Medicine, 108.945 (1958)、 Proc. Natl. Acad. Sci. USA, 60, 1275 (1968) 、 Genetics, 55,513 (1968)、 Chromosoma, 41, 129 (1973)、 Methods in Cell Science, 18,115 (1996)、 Radiation Research, 148, 260 (1997)、 Proc. Natl. Acad. Sci.USA, 77, 4216 (1980)、 Proc. Natl. Acad. Sci. 60, 1275 (1968)、 Cell,6, 121 (1975)、 Molecular Cell Genetics, Appendix 1,11 (p883- 900)等の文献に記載されている CHO細胞があげ られる。また、 ATCCに登録されている CHO- K1株（ATCC CCL-61)、 DUXB11株（A TCC CRL- 9096)、 Pro- 5株（ATCC CRL- 1781)や、市販の CHO- S株（Lifetechnologi es社製 Cat#l 1619)、あるいはこれら株を様々な無血清培地に馴化させた亜株なども あげられる。 [0054] As a parent cell of CHO cells derived from Chinese nomster ovary tissue, Journal of Experimental Medicine, 108.945 (1958), Proc. Natl. Acad. Sci. USA, 60, 1275 (1968), Genetics, 55,513 (1968) ), Chromosoma, 41, 129 (1973), Methods in Cell Science, 18,115 (1996), Radiation Research, 148, 260 (1997), Proc. Natl. Acad. Sci. USA, 77, 4216 (1980), Proc. Natl. Acad. Sci. 60, 1275 (1968), Cell, 6, 121 (1975), Molecular Cell Genetics, Appendix 1, 11 (p883-900) and the like. In addition, CHO-K1 strain (ATCC CCL-61), DUXB11 strain (ATCC CRL-9096), Pro-5 strain (ATCC CRL-1781) registered in ATCC, and commercially available CHO-S strain (Lifetechnologi es Cat # l 1619), or sub-strains made by adapting these strains to various serum-free media can give.
[0055] ラットミエローマ細胞株 YB2/3HL.P2.G11.16Ag.20細胞の親株細胞としては、 Y3/Ag 1.2.3細胞 (ATCC CRL-1631)から樹立された株化細胞が包含される。その具体的な 例としては、 J. Cell. Biol., 93, 576 (1982)、 Methods Enzymol. 73B, 1 (1981)等の文 献に記載されている YB2/3HL.P2.G11.16Ag.20細胞があげられる。また、 ATCCに登 録されている YB2/3HL.P2.G11.16Ag.20細胞（ATCC CRL-1662)あるいはこれら株を 様々な無血清培地に馴化させた亜株などもあげられる。  The parent cell of rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cell includes a cell line established from Y3 / Ag 1.2.3 cell (ATCC CRL-1631). Specific examples are YB2 / 3HL.P2.G11.16Ag. Described in documents such as J. Cell. Biol., 93, 576 (1982), Methods Enzymol. 73B, 1 (1981). There are 20 cells. In addition, YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL-1662) registered in ATCC or sub-strains obtained by acclimating these strains to various serum-free media are also included.
[0056] 本発明の組成物を生産する細胞としては、具体的には、 α 1,6-フコシルトランスフエ ラーゼをコードする遺伝子がノックアウトされた CHO細胞に卵胞刺激ホルモンをコー ドする遺伝子を導入した形質転 ·を無血清培地に馴化した株である PKAN-FSH9- 3 AFMS705株、 GDP-マンノース 4,6-デヒドラターゼをコードする遺伝子がノックアウト された CHO細胞に卵胞刺激ホルモンをコードする遺伝子を導入した形質転換株であ る pKAN- FSH2 GMDKO株などがあげられる。  [0056] Specifically, the cell producing the composition of the present invention introduces a gene encoding follicle-stimulating hormone into CHO cells into which a gene encoding α1,6-fucosyltransferase has been knocked out. PKAN-FSH9-3 AFMS705, a strain that has been conditioned to a serum-free medium, and a gene encoding follicle-stimulating hormone introduced into CHO cells in which the gene encoding GDP-mannose 4,6-dehydratase has been knocked out PKAN- FSH2 GMDKO strain, which is a transformed strain.
[0057] pKAN-FSH9-3 AFMS705株および pKAN- FSH2 GMDKO株は、平成 16年 8月 10日 付けで、独立行政法人産業技術総合研究所特許生物寄託センター（日本国茨城県 つくば巿東 1丁目 1番地 1中央第 6)にそれぞれ FERM BP-10086、 FERM BP-10081と してそれぞれ寄託されて 、る。  [0057] The pKAN-FSH9-3 AFMS705 strain and the pKAN-FSH2 GMDKO strain were issued on August 10, 2004, at the National Institute of Advanced Industrial Science and Technology (AIST) FERM BP-10086 and FERM BP-10081 are deposited at address 1 and center 6), respectively.
本発明の形質転換体は、親株細胞カゝら得られるエリスロポイエチン組成物に比べ て、同等の卵胞刺激ホルモン受容体への親和性を有しており、かっ血中半減期が延 長された卵胞刺激ホルモン組成物を製造することができる。  Compared to the erythropoietin composition obtained from the parent cell line, the transformant of the present invention has an affinity for the equivalent follicle-stimulating hormone receptor and has an increased half-life in blood clots. A follicle stimulating hormone composition can be produced.
[0058] 卵胞刺激ホルモン組成物の卵胞刺激ホルモン受容体に対する結合活性、細胞内 情報伝達の惹起、エストロゲン産生誘導活性、卵胞成熟誘導活性および血中半減 期は、既に公知の卵胞刺激ホルモン受容体結合活性測定試験、細胞内情報伝達活 性測定試験、エストロゲン産生誘導活性測定試験などの invitro試験、マウスやラット などのモデル動物を用いた卵胞成熟誘導活性測定試験などの in vivo試験、あるい はヒトを用いた臨床試験などを用いて測定することができる（Proceedings of the Natio nal Academy of Sciences UbA 78, 5465(1981)、 Journal of Biological Chemistry 278, 47868 (2003)、 Neuroendocrinology 41, 445 (1985)、 Journal of Clinical Endocrinology and Metabolism 88, 3227(2003)、 Human Reproduction 14, 2709 (1999))。 [0058] The binding activity of the follicle stimulating hormone composition to the follicle stimulating hormone receptor, induction of intracellular signal transduction, estrogen production inducing activity, follicle maturation inducing activity, and blood half-life are already known follicle stimulating hormone receptor binding. In vitro tests such as activity measurement tests, intracellular signal transduction activity measurement tests, estrogen production induction activity measurement tests, in vivo tests such as follicle maturation induction activity measurement tests using model animals such as mice and rats, or humans (Proceedings of the Ratio Academy of Sciences UbA 78, 5465 (1981), Journal of Biological Chemistry 278, 47868 (2003), Neuroendocrinology 41, 445 (1985), Journal of Clinical Endocrinology and Metabolism 88, 3227 (2003), Human Reproduction 14, 2709 (1999)).
[0059] 以下、本発明を具体的に詳細に説明する。 [0059] Hereinafter, the present invention will be described in detail.
1.本発明の卵胞刺激ホルモン組成物を生産するために用いる宿主細胞の作製 本発明の卵胞刺激ホルモン組成物を生産するために用いる宿主細胞は、以下に 述べる手法により作製することができる。  1. Production of host cell used for producing follicle stimulating hormone composition of the present invention The host cell used for producing follicle stimulating hormone composition of the present invention can be produced by the method described below.
(1)酵素の遺伝子を標的とした遺伝子破壊の手法  (1) Gene disruption methods targeting enzyme genes
本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞は、細胞内 糖ヌクレオチド GDP-フコースの合成に関与する酵素または N-グリコシド結合複合型 糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が α結合する糖鎖 修飾に関与する酵素（以下、「フコース修飾に関連する酵素」と表記する）の遺伝子を 標的とし、遺伝子破壊の方法を用いることにより作製することができる。細胞内糖ヌク レオチド GDP-フコースの合成に関与する酵素としては、具体的には、 GDP-マンノー ス 4,6-デヒドラターゼ（以下、「GMD」と表記する）、 GDP- 4-ケト- 6-デォキシ- D-マンノ ース -3,5-ェピメラーゼ (以下、「Fx」と表記する）などがあげられる。 N-グリコシド結合 複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が a結合す る糖鎖修飾に関与する酵素としては、具体的には、 α 1,6-フコシルトランスフェラーゼ 、 α -L-フコシダーゼなどがあげられる。  The host cell used to prepare the follicle stimulating hormone composition of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or N-glycidyl-linked complex N-acetylyldarcosamine at the reducing end of the sugar chain. It must be prepared by targeting the gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked at position 6 (hereinafter referred to as “enzyme related to fucose modification”) and using a gene disruption method. Can do. Specific examples of enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose include GDP-mannose 4,6-dehydratase (hereinafter referred to as “GMD”), GDP-4-keto-6- Deoxy-D-mannose-3,5-epimerase (hereinafter referred to as “Fx”). N-glycoside bond As an enzyme involved in sugar chain modification in which the 1-position of fucose is a-linked to the 6-position of N-acetylyldarcosamine at the reducing end of the complex type sugar chain, α 1,6- Examples include fucosyltransferase and α-L-fucosidase.
[0060] ここでいう遺伝子とは、 DNAまたは RNAを含む。  [0060] The gene herein includes DNA or RNA.
遺伝子破壊の方法としては、標的とする酵素の遺伝子を破壊することができる方法 であればいかなる方法も包含される。その例としては、アンチセンス法、リボザィム法 、相同組換え法、 RNA-DNAオリゴヌクレオチド法（以下、「RDO法」と表記する）、 RNA インターフェアレンス法（以下、「RNAi法」と表記する）、レトロウイルスを用いた方法、 トランスポゾンを用いた方法等があげられる。以下これらを具体的に説明する。  Any method can be used as the gene disruption method as long as it can destroy the gene of the target enzyme. Examples include the antisense method, ribozyme method, homologous recombination method, RNA-DNA oligonucleotide method (hereinafter referred to as “RDO method”), RNA interference method (hereinafter referred to as “RNAi method”). ), A method using a retrovirus, a method using a transposon, and the like. These will be specifically described below.
[0061] (a)アンチセンス法又はリボザィム法による本発明の卵胞刺激ホルモン組成物を作 製  [0061] (a) Production of follicle stimulating hormone composition of the present invention by antisense method or ribozyme method
するための宿主細胞の作製  A host cell to do
本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞は、フコース 修飾に関連する酵素遺伝子を標的とし、細胞工学， 12, 239(1993)、バイオ Zテクノロ ジー (BIO/TECHNOLOGY), 17, 1097 (1999)、ヒューマン ·モレキュラ^ ~ ·ジエネテイク ス (Hum.Mol. Genet.), 5, 1083 (1995)、細胞工学， 13, 255 (1994)、プロシーデイング ス'ォブ ·ザ'ナショナル 'アカデミ^ ~·ォブ 'サイエンス (Proc. Natl. Acad. Sci. U.S.A.),The host cell used to produce the follicle stimulating hormone composition of the present invention targets an enzyme gene related to fucose modification, and is described in Cell Engineering, 12, 239 (1993), Bio Z Technolo. GIO (BIO / TECHNOLOGY), 17, 1097 (1999), Human Molecular ^ ~ · Genetics (Hum. Mol. Genet.), 5, 1083 (1995), Cell Engineering, 13, 255 (1994), Proceding 'Ob The National' Academy ^ ~ Ob 'Science (Proc. Natl. Acad. Sci. USA),
96,1886 (1999)等に記載されたアンチセンス法またはリボザィム法を用いて、例えばUsing the antisense method or ribozyme method described in 96,1886 (1999) etc., for example
、以下のように作製することができる。 It can be produced as follows.
[0062] フコース修飾に関連する酵素をコードする cDNAあるいはゲノム DNAを調製する。 [0062] cDNA or genomic DNA encoding an enzyme related to fucose modification is prepared.
調製した cDNAあるいはゲノム DNAの塩基配列を決定する。  Determine the base sequence of the prepared cDNA or genomic DNA.
決定した DNAの配列に基づき、フコース修飾に関連する酵素をコードする DNA部 分、非翻訳領域の部分あるいはイントロン部分を含む適当な長さのアンチセンス遺伝 子またはリボザィムのコンストラクトを設計する。  Based on the determined DNA sequence, construct an antisense gene or ribozyme construct of appropriate length including the DNA part encoding the enzyme related to fucose modification, the part of the untranslated region or the intron part.
[0063] 該アンチセンス遺伝子、またはリボザィムを細胞内で発現させるために、調製した D[0063] D prepared in order to express the antisense gene or ribozyme in cells
NAの断片、または全長を適当な発現ベクターのプロモーターの下流に挿入すること により、組換えベクターを作製する。 A recombinant vector is prepared by inserting the NA fragment or full length downstream of the promoter of an appropriate expression vector.
該組換えベクターを、該発現べクタ一に適合した宿主細胞に導入することにより形 質転換体を得る。  A transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
[0064] フコース修飾に関連する酵素の活性を指標として形質転換体を選択することにより 、本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞を得ることが できる。また、細胞膜上の糖蛋白質の糖鎖構造または産生糖蛋白質分子の糖鎖構 造を指標として形質転換体を選択することにより、本発明の卵胞刺激ホルモン組成 物を作製するために用いる宿主細胞を得ることもできる。  [0064] By selecting a transformant using as an index the activity of an enzyme related to fucose modification, a host cell used for preparing the follicle-stimulating hormone composition of the present invention can be obtained. In addition, a host cell used for preparing the follicle stimulating hormone composition of the present invention is selected by selecting a transformant using the sugar chain structure of the glycoprotein on the cell membrane or the sugar chain structure of the produced glycoprotein molecule as an index. It can also be obtained.
[0065] 本発明の卵胞刺激ホルモン組成物を作製するために用いられる宿主細胞としては 、酵母、動物細胞、昆虫細胞、植物細胞など、標的とするフコース修飾に関連する酵 素の遺伝子を有しているものであればいずれも用いることができる。具体的には、後 述の 3に記載の宿主細胞があげられる。  [0065] The host cell used for producing the follicle-stimulating hormone composition of the present invention has an enzyme gene related to target fucose modification such as yeast, animal cell, insect cell, plant cell and the like. Any of these can be used. Specifically, the host cell described in 3 below can be mentioned.
発現ベクターとしては、上記宿主細胞において自立複製が可能である力、ないしは 染色体中への組み込みが可能で、設計したアンチセンス遺伝子、またはリボザィムを 転写できる位置にプロモーターを含有しているものが用いられる。具体的には、後述 3に記載の発現ベクターがあげられる。 [0066] 各種宿主細胞への遺伝子の導入方法としては、後述 3に記載の各種宿主細胞に 適した組換えベクターの導入方法を用いることができる。 The expression vector is capable of autonomous replication in the above host cell, or can be integrated into the chromosome and contains a designed antisense gene or a promoter at a position where a ribozyme can be transcribed. . Specifically, the expression vector described in 3 below can be mentioned. [0066] As a method for introducing a gene into various host cells, the method for introducing a recombinant vector suitable for various host cells described in 3 below can be used.
フコース修飾に関連する酵素の活性を指標として形質転換体を選択する方法とし ては、例えば、以下の方法があげられる。  Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the following methods.
形皙転椽体を選択する方法  How to select a shape
フコース修飾に関連する酵素の活性が欠失した細胞を選択する方法としては、文 献 if生化学実験講座 3—糖質 I,糖タンパク質 (東京化学同人)日本生化学会編 (198 8)]、文献 [細胞工学,別冊，実験プロトコールシリーズ,グライコバイオロジー実験プロト コール,糖タンパク質 ·糖脂質 ·プロテオダリカン (秀潤社製)谷口直之 ·鈴木明美 ·古川 清.菅原一幸監修（1996)]、モレキュラー 'クローユング第 2版、カレント.プロトコール ズ，ィ  As a method of selecting cells lacking the activity of the enzyme related to fucose modification, refer to the literature if biochemistry experiment course 3-carbohydrate I, glycoprotein (Tokyo Kagaku Dojin) edited by the Japanese Biochemical Society (198 8)], Literature [Cell engineering, separate volume, experimental protocol series, glycobiology experimental protocol, glycoproteins · glycolipids · proteodaricans (manufactured by Shujunsha) Naoyuki Taniguchi · Akemi Suzuki · Kiyoshi Furukawa (supervised by Kazuyuki Sugawara (1996)), Molecular 'Crowing 2nd Edition, Current Protocols,
ン 'モレキュラー 'バイオロジー等に記載された生化学的な方法あるいは遺伝子工学 的な方法などを用いて、フコース修飾に関連する酵素の活性を測定する方法があげ られる。生化学的な方法としては、例えば、酵素特異的な基質を用いて酵素活性を 評価する方法があげられる。遺伝子工学的な方法としては、例えば、酵素遺伝子の m RNA量を測定するノーザン解析や RT- PCR法等があげられる。  There are methods for measuring the activity of an enzyme related to fucose modification using biochemical methods or genetic engineering methods described in “Molecular” biology and the like. Examples of the biochemical method include a method for evaluating enzyme activity using an enzyme-specific substrate. Examples of genetic engineering methods include Northern analysis for measuring the amount of mRNA of an enzyme gene and RT-PCR method.
[0067] 細胞膜上の糖蛋白質の糖鎖構造を指標として形質転換体を選択する方法としては 、例えば、後述 1の（5)に記載の方法があげられる。産生糖蛋白質分子の糖鎖構造 を指標として形質転換体を選択する方法としては、例えば、後述 5または後述 6に記 載の方法があげられる。 [0067] Examples of a method for selecting a transformant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (1) below. Examples of the method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
フコース修飾に関連する酵素をコードする cDNAを調製する方法としては、例えば、 下記に記載の方法があげられる。 Examples of a method for preparing cDNA encoding an enzyme related to fucose modification include the methods described below.
[0068] cDNAの調製方法 [0068] cDNA preparation method
各種宿主細胞の組織又は細胞力ゝら全 RNA又は mRNAを調製する。  Total RNA or mRNA is prepared from the tissues or cell strength of various host cells.
調製した全 RNA又は mRNAから cDNAライブラリーを作製する。  A cDNA library is prepared from the prepared total RNA or mRNA.
フコース修飾に関連する酵素のアミノ酸配列に基づ 、て、デジエネレイティブプライ マーを作製し、作製した cDNAライブラリーを铸型として PCR法でフコース修飾に関連 する酵素をコードする遺伝子断片を取得する。 [0069] 取得した遺伝子断片をプローブとして用い、 cDNAライブラリーをスクリーニングし、 フコース修飾に関連する酵素をコードする DNAを取得することができる。 Based on the amino acid sequence of the enzyme related to fucose modification, a degenerative primer is prepared, and a gene fragment encoding the enzyme related to fucose modification is obtained by PCR using the prepared cDNA library as a saddle type To do. [0069] Using the obtained gene fragment as a probe, a cDNA library can be screened to obtain DNA encoding an enzyme related to fucose modification.
ヒト又は非ヒト動物の糸且織又は細胞の mRNAは市販のもの (例えば Clontech社)を用 V、てもよ 、し、以下のようにしてヒト又は非ヒト動物の組織又は細胞力も調製してもよ い。  Human or non-human animal thread and tissue or cell mRNA may be commercially available (for example, Clontech) V, and human or non-human animal tissue or cell force may also be prepared as follows. It's good.
ヒト又は非ヒト動物の組織又は細胞力も全 RNAを調製する方法としては、チォシアン 酸  As a method for preparing total RNA of human or non-human animal tissues or cells, thiocyanic acid can be used.
グァ-ジン-トリフルォロ酢酸セシウム法 [メソッズ ·イン ·ェンザィモロジ一 (Methods in Enzymology), 154, 3(1987)]、酸性チォシアン酸グァ-ジン'フエノール'クロ口ホルム (AGPC)法 [アナリティカル 'バイオケミストリー (Analytical Biochemistry), 162, 156 (1 987);実験医学、 9,1937 (1991)]などがあげられる。  Guazin-trifluoroacetate method [Methods in Enzymology, 154, 3 (1987)], Guazin acid thiocyanate 'Phenol' black mouth form (AGPC) method [Analytical Bio Chemistry (Analytical Biochemistry), 162, 156 (1 987); experimental medicine, 9,1937 (1991)].
[0070] また、全 RNA力 poly(A)+ RNAとして mRNAを調製する方法としては、オリゴ（dT)固 定ィ匕セルロースカラム法 (モレキュラー 'クローユング第 2版)等があげられる。 [0070] Examples of a method for preparing mRNA as total RNA poly (A) + RNA include an oligo (dT) -fixed cellulose column method (Molecular 'Cloung 2nd edition).
さらに、 Fast Track mRNA Isolation Kit (Invitrogen社)、 Quick Prep mRNA Purificat ion Kit (Pharmacia社）などの市販のキットを用いることにより mRNAを調製することがで きる。  Furthermore, mRNA can be prepared by using a commercially available kit such as Fast Track mRNA Isolation Kit (Invitrogen) or Quick Prep mRNA Purification Kit (Pharmacia).
[0071] 次に、調製したヒト又は非ヒト動物の組織又は細胞 mRNAから cDNAライブラリーを作 製する。 cDNAライブラリー作製法としては、モレキュラー 'クローユング第 2版、カレン ト.プロトコールズ.イン.モレキュラー.バイオロジー、 ALaboratory Manual, 2 nd Ed.(l 989)等に記載された方法、あるいは市販のキット、例えば SuperScriptPlasmid System for cDNA Synthesis and Plasmidし loning (Life Technologiesネエリ、 ZAP- cDNA bynthes is Kit (STRATAGENE社）を用いる方法などがあげられる。  [0071] Next, a cDNA library is prepared from the prepared human or non-human animal tissue or cell mRNA. The cDNA library can be prepared by a method described in Molecular 'Crowing 2nd Edition, Current Protocols in Molecular Biology, ALaboratory Manual, 2nd Ed. (l 989), or commercially available kits. For example, a method using SuperScriptPlasmid System for cDNA Synthesis and Plasmid (Life Technologies Neeri, ZAP-cDNA byn is is Kit (STRATAGENE)) can be mentioned.
[0072] cDNAライブラリーを作製するためのクローユングベクターとしては、大腸菌 K12株中 で  [0072] A cloning vector for constructing a cDNA library is the Escherichia coli K12 strain.
自立複製できるものであれば、ファージベクター、プラスミドベクター等いずれでも使 用できる。具体的には、 ZAP Express [STRATAGENE社、ストラテジーズ (Strategies), , 58 (1992)]、 pBluescript II SK(+) [ヌクレイツク'アシッド'リサーチ (Nucleic Acids Rese arch), 17,9494 (1989)]、 λ ZAP II (STRATAGENE社）、 gtl0、え gtl l [ディーェヌェ 一.クロー-ング.ァ.プラクティカノレ.アプローチ (DNAcloning, A Practical Approach), 1, 49 (1985)]、 TriplEx (Clontech社）、 λ ExCell (Pharmacia社）、 pT7T318U (Pharm acia社）、 pcD2 [モレキユラ一'セルラ一'バイオロジー (Mol.Cell. Biol), 3, 280 (1983)] および pUC18 [ジーン (Gene), 33, 103 (1985)]等をあげることができる。 Any phage vector or plasmid vector can be used as long as it can replicate autonomously. Specifically, ZAP Express [STRATAGENE, Strategies,, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Rese arch, 17,9494 (1989)] , Λ ZAP II (STRATAGENE), gtl0, e gtl l [Dienue 1. Cloning a Practical Approach, 1, 49 (1985)], TriplEx (Clontech), λ ExCell (Pharmacia), pT7T318U (Pharm acia), pcD2 [ Moleculera 'Cellular Biology, 3, 280 (1983)] and pUC18 [Gene, 33, 103 (1985)].
[0073] cDNAライブラリーを作製するための宿主微生物としては、微生物であればいずれ でも用いることができるが、好ましくは大腸菌が用いられる。具体的には、 Escherichia coli XL1- Blue MRF' [STRATAGENE社、ストラテジーズ (Strategies), 5, 81 (1992)] , E scherichiacoli C600 [ジエネテイクス (Genetics), 39, 440 (1954)1 , Escherichia coli Y10 88 [サイエンス (Science) ,222, 778 (1983)1、 Escherichia coli Y1090 [サイエンス (Scienc e), 222,778 (1983)1、 Escherichia coli NM522「ジャーナル，ォブ，モレキユラ ~ ·バイオ ロジー 0. Mol.BioL), 166, 1 (1983)1. Escherichia coli K802 [ジャーナル'ォブ 'モレ キユラ ~ ·バイオロジー (J.Mol. Biol.), 16, 118 (1966)Ίおよび Escherichia coli ΤΜ105 Γ ジーン (Gene), 38,275 (1985)]等が用いられる。  [0073] As a host microorganism for preparing a cDNA library, any microorganism can be used, but Escherichia coli is preferably used. Specifically, Escherichia coli XL1- Blue MRF '[STRATAGENE, Strategies, 5, 81 (1992)], Escherichiacoli C600 [Genetics, 39, 440 (1954) 1, Escherichia coli Y10 88 [Science, 222, 778 (1983) 1, Escherichia coli Y1090 [Science, 222,778 (1983) 1, Escherichia coli NM522 "Journal, Ob, Molecura ~ Biology 0. Mol.BioL ), 166, 1 (1983) 1. Escherichia coli K802 (J. Mol. Biol.), 16, 118 (1966) Ί and Escherichia coli ΤΜ105 Γ Gene (Gene) , 38,275 (1985)].
[0074] この cDNAライブラリ一は、そのまま以降の解析に用いてもよいが、不完全長 cDNA の割合を下げ、なるべく完全長 cDNAを効率よく取得するために、菅野らが開発した オリゴキャップ法 [ジーン (Gene),12S, 171 (1994);ジーン (Gene), 200, 149 (1997);蛋 白質核酸酵素， 41,603 (1996);実験医学， U, 2491 (1993); cDNAクローユング (羊土 社) (1996);遺伝子ライブラリーの作製法 (羊土社）（1994)]を用いて調製して以下の解 祈に用いてもよい。  [0074] Although this cDNA library can be used as it is for the subsequent analysis, the oligo cap method developed by Kanno et al. Has been developed in order to reduce the proportion of incomplete-length cDNA and obtain full-length cDNA as efficiently as possible. Gene (Gene), 12S, 171 (1994); Gene (Gene), 200, 149 (1997); Protein Nucleic Acid Enzyme, 41,603 (1996); Experimental Medicine, U, 2491 (1993); ) (1996); Preparation method of gene library (Yodosha) (1994)].
[0075] フコース修飾に関連する酵素のアミノ酸配列に基づいて、該アミノ酸配列をコード することが予測される塩基配列の 5'末端および 3'末端の塩基配列に特異的なデジ ネレイティブプライマーを作製し、作製した cDNAライブラリーを铸型として PCR法 [ピ 一シーア一ノレ'プロトコーノレズ (PCR Protocols), Academic Press (1990)]を用いて DN Aの増幅を行うことにより、フコース修飾に関連する酵素をコードする遺伝子断片を取 得することができる。  [0075] Based on the amino acid sequence of the enzyme related to the fucose modification, the creation of a degenerative primer specific to the 5'-end and 3'-end base sequences predicted to encode the amino acid sequence Using the prepared cDNA library as a cage, amplification of DNA was performed by PCR using the PCR method [PCR Protocols, Academic Press (1990)]. A gene fragment encoding the enzyme to be obtained can be obtained.
[0076] 取得した遺伝子断片がフコース修飾に関連する酵素をコードする DNAであることは 、通  [0076] It is common that the obtained gene fragment is DNA encoding an enzyme related to fucose modification.
常用いられる塩基配列解析方法、例えばサンガー (Sanger)らのジデォキシ法 [プロ シーディングス ·ォブ ·ザ ·ナショナル ·アカデミ^ ~ ·ォブ ·サイエンス (Pro Natl. Acad. Sci. U.S.A.), 74, 5463 (1977)]あるいは ABI PRISM377DNAシークェンサ一（Applied Biosystems社製)等の塩基配列分析装置を用いて分析することにより、確認すること ができる。 Commonly used nucleotide sequence analysis methods such as the Sidi et al. Dideoxy method [pro Seeds · Ob · The · National · Academia ^ ~ · Ob · Science (Pro Natl. Acad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377DNA Sequencer (Applied Biosystems) and other bases This can be confirmed by analysis using a sequence analyzer.
[0077] 該遺伝子断片をプローブとして、ヒト又は非ヒト動物の組織又は細胞に含まれる mR NAから合成した cDNAあるいは cDNAライブラリーからコロニーハイブリダィゼーシヨン やプラークハイブリダィゼーシヨン (モレキュラー ·クロー-ング第 2版)等を用いて、フ コース修飾に関連する酵素の DNAを取得することができる。  [0077] Using the gene fragment as a probe, colony hybridization or plaque hybridization (molecular molecular clones) from cDNA or cDNA library synthesized from mRNA contained in tissues or cells of human or non-human animals. -Nu 2nd edition) etc. can be used to obtain DNA for enzymes related to fucose modification.
また、フコース修飾に関連する酵素をコードする遺伝子断片を取得するために用い たプライマーを用い、ヒト又は非ヒト動物の組織又は細胞に含まれる mRNAから合成し た cDNAあるいは cDNAライブラリーを铸型として、 PCR法を用いて増幅することにより 、フコース修飾に関連する酵素の cDNAを取得することもできる。  In addition, a cDNA or cDNA library synthesized from mRNA contained in human or non-human animal tissues or cells using the primers used to obtain a gene fragment encoding an enzyme related to fucose modification as a saddle type. The cDNA of the enzyme related to fucose modification can also be obtained by amplification using PCR.
[0078] 取得したフコース修飾に関連する酵素をコードする DNAの塩基配列は、通常用い られる塩基配列解析方法、例えばサンガー（Sanger)らのジデォキシ法 [プロシーディ ングス ·ォブ ·ザ'ナショナル ·アカデミ^ ~ ·ォブ ·サイエンス (Pro Natl. Acad. Sci. U.S. A.), 74, 5463 (1977)]あるいは ABI PRISM377DNAシークェンサ一（AppliedBiosystem s社製)等の塩基配列分析装置を用いて分析することにより、該 DNAの塩基配列を決 定することができる。  [0078] The base sequence of the obtained DNA encoding the enzyme related to the fucose modification can be determined by a commonly used base sequence analysis method, for example, Sanger et al.'S dideoxy method [Procedurals · Ob · The 'National · Academia ^ ~ Ob Science (Pro Natl. Acad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems), etc. The base sequence of DNA can be determined.
[0079] 決定した cDNAの塩基配列をもとに、 BLAST等の相同性検索プログラムを用いて、 Genbank、 EMBLおよび DDBJなどの塩基配列データベースを検索することにより、取 得した DNAがデータベース中の遺伝子の中でフコース修飾に関連する酵素をコード して 、る遺伝子であることを確認することもできる。  [0079] Based on the determined cDNA base sequence, a homology search program such as BLAST is used to search base sequence databases such as Genbank, EMBL, and DDBJ. It can also be confirmed that the gene encodes an enzyme related to fucose modification.
上記の方法で得られる細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素 をコードする遺伝子の塩基配列としては、例えば、配列番号 9または 11に記載の塩 基配列があげられる。  Examples of the nucleotide sequence of the gene encoding the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose obtained by the above method include the nucleotide sequence set forth in SEQ ID NO: 9 or 11.
[0080] 上記の方法で得られる N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコ サミンの 6位にフコースの 1位が oc結合する糖鎖修飾に関与する酵素をコードする遺 伝子の塩基配列としては、例えば、配列番号 13または 14に記載の塩基配列があげ られる。 [0080] An N-glycoside-linked complex-type sugar chain-reducing terminal N-acetylcylcosamine obtained by the above-described method encodes an enzyme that encodes an enzyme involved in sugar chain modification in which position 1 of fucose is oc-bonded to position 6. Examples of the base sequence include the base sequence described in SEQ ID NO: 13 or 14. It is done.
決定された DNAの塩基配列に基づ 、て、フォスフォアミダイト法を利用した DNA合 成機 model 392 (Perkin Elmer社製）等の DNA合成機で化学合成することにより、フコ ース修飾に関連する酵素の cDNAを取得することもできる。  Based on the determined DNA base sequence, it is related to fucose modification by chemical synthesis using a DNA synthesizer such as the DNA synthesizer model 392 (Perkin Elmer) using the phosphoramidite method. It is also possible to obtain the cDNA of the enzyme.
[0081] フコース修飾に関連する酵素のゲノム DNAを調製する方法としては、例えば、以下 に記載の方法があげられる。 [0081] Examples of a method for preparing genomic DNA of an enzyme related to fucose modification include the methods described below.
ゲノム DNAの調製方法  Method for preparing genomic DNA
ゲノム DNAを調製する方法としては、モレキュラー ·クローユング第 2版やカレント · プロトコールズ ·イン.モレキュラー.バイオロジー等に記載された公知の方法があげら れる。また、ゲノム DNAライブラリースクリーニングシステム（GenomeSystems社）や Uni versal GenomeWalker™ Kits (CLONTECH社）などを用いることにより、フコース修飾 に関連する酵素のゲノム DNAを取得することもできる。  Examples of methods for preparing genomic DNA include known methods described in Molecular Cloning 2nd Edition and Current Protocols in Molecular Biology. In addition, genomic DNA of an enzyme related to fucose modification can be obtained by using a genomic DNA library screening system (GenomeSystems) or Unigen GenomeWalker ™ Kits (CLONTECH).
[0082] 取得したフコース修飾に関連する酵素をコードする DNAの塩基配列は、通常用い られる [0082] The obtained base sequence of DNA encoding the enzyme related to fucose modification is usually used.
塩基配列解析方法、例えばサンガー (Sanger)らのジデォキシ法 [プロシーディンダス •ォブ 'ザ'ナショナル 'ァ力デミ一'ォブ 'サイエンス (Proc.Natl. Acad. Sci. U.S.A.), 74 Nucleotide sequence analysis methods, such as Sanger et al.'S dideoxy method [Procedinas Abb. Scad. U.S.A., 74
, 5463 (1977)]あるいは ABI PRISM377DNAシークェンサ一（AppliedBiosystems社製） 等の塩基配列分析装置を用いて分析することにより、該 DNAの塩基配列を決定する ことができる。 , 5463 (1977)] or by using a base sequence analyzer such as ABI PRISM377 DNA Sequencer (Applied Biosystems), the base sequence of the DNA can be determined.
[0083] 決定したゲノム DNAの塩基配列をもとに、 BLAST等の相同性検索プログラムを用い て、 Genbank、 EMBLおよび DDBJなどの塩基配列データベースを検索することにより 、取得した DNAがデータベース中の遺伝子の中でフコース修飾に関連する酵素をコ ードしている遺伝子であることを確認することもできる。  [0083] Based on the determined base sequence of genomic DNA, using a homology search program such as BLAST to search base sequence databases such as Genbank, EMBL and DDBJ, It is also possible to confirm that the gene encodes an enzyme related to fucose modification.
決定された DNAの塩基配列に基づ 、て、フォスフォアミダイト法を利用した DNA合 成機 model 392 (Perkin Elmer社製）等の DNA合成機で化学合成することにより、フコ ース修飾に関連する酵素のゲノム DNAを取得することもできる。  Based on the determined DNA base sequence, it is related to fucose modification by chemical synthesis using a DNA synthesizer such as the DNA synthesizer model 392 (Perkin Elmer) using the phosphoramidite method. It is also possible to obtain genomic DNA of the enzyme.
[0084] 上記の方法で得られる細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素 のゲノム DNAの塩基配列としては、例えば配列番号 17、 18、 19および 20に記載の 塩基配列があげられる。 [0084] The nucleotide sequence of the genomic DNA of the enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose obtained by the above method is, for example, those described in SEQ ID NOs: 17, 18, 19, and 20. A base sequence is mentioned.
上記の方法で得られる N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコ サミンの 6位にフコースの 1位が α結合する糖鎖修飾に関与する酵素のゲノム DNAの 塩基配列としては、例えば配列番号 21に記載の塩基配列があげられる。  The base sequence of the genomic DNA of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-bonded to the 6-position of N-acetyldylcosamine at the N-glycoside-linked complex sugar chain reducing end obtained by the above method is, for example, Examples include the nucleotide sequence set forth in SEQ ID NO: 21.
[0085] また、発現ベクターを用いず、フコース修飾に関連する酵素の塩基配列に基づい て設計したアンチセンスオリゴヌクレオチドまたはリボザィムを、直接宿主細胞に導入 することで、本発明の卵胞刺激ホルモン組成物作製のために用いる宿主細胞を得る ことちでさる。 [0085] Furthermore, the follicle stimulating hormone composition of the present invention can be obtained by directly introducing an antisense oligonucleotide or ribozyme designed based on the base sequence of an enzyme related to fucose modification without using an expression vector into a host cell. Obtaining host cells to be used for production.
アンチセンスオリゴヌクレオチドまたはリボザィムは、常法または DNA合成機により調 製することができる。具体的には、フコース修飾に関連する酵素をコードする cDNAお よびゲノム DNAの塩基配列のうち、連続した 5〜150塩基、好ましくは 5〜60塩基、よ り好ましくは 10〜40塩基に相当する配列を有するオリゴヌクレオチドの配列情報に 基づき、該オリゴヌクレオチドと相補的な配列に相当するオリゴヌクレオチド (アンチセ ンスオリゴヌクレオチド)または該オリゴヌクレオチドの配列を含むリボザィムを合成し て調製することができる。  Antisense oligonucleotides or ribozymes can be prepared by conventional methods or DNA synthesizers. Specifically, it corresponds to a continuous 5 to 150 bases, preferably 5 to 60 bases, more preferably 10 to 40 bases in the base sequences of cDNA and genomic DNA encoding the enzyme related to fucose modification. Based on the sequence information of the oligonucleotide having the sequence, an oligonucleotide (antisense oligonucleotide) corresponding to a sequence complementary to the oligonucleotide or a ribozyme containing the sequence of the oligonucleotide can be synthesized and prepared.
[0086] オリゴヌクレオチドとしては、オリゴ RNAおよび該オリゴヌクレオチドの誘導体 (以下、 オリゴヌクレオチド誘導体と ヽぅ）等があげられる。 [0086] Examples of oligonucleotides include oligo RNA and derivatives of the oligonucleotide (hereinafter referred to as oligonucleotide derivatives).
オリゴヌクレオチド誘導体としては、オリゴヌクレオチド中のリン酸ジエステル結合が ホスフォロチォエート結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド 中のリン酸ジエステル結合が Ν3'-Ρ5'ホスフォアミデート結合に変換されたオリゴヌク レオチド誘導体、オリゴヌクレオチド中のリボースとリン酸ジエステル結合がペプチド 核酸結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のゥラシルが C-5プロピ-ルゥラシルで置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中 のゥラシルが C-5チアゾールゥラシルで置換された誘導体オリゴヌクレオチド、オリゴ ヌクレオチド中のシトシンが C-5プロピ-ルシトシンで置換されたオリゴヌクレオチド誘 導体、オリゴヌクレオチド中のシトシンがフエノキサジン修飾シトシン（phenoxazine-mo dified cytosine)で置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のリボ ースが 2'-0-プロピルリボースで置換されたオリゴヌクレオチド誘導体、あるいはオリ ゴヌクレオチド中のリボースが 2しメトキシエトキシリボースで置換されたオリゴヌクレオ チド誘導体等があげられる [細胞工学， 16, 1463(1997)]。 Oligonucleotide derivatives include oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides are converted to に 3'-Ρ5 'phosphoramidate bonds. Oligonucleotide derivatives, oligonucleotide derivatives in which the ribose and phosphodiester bonds in the oligonucleotide are converted to peptide nucleic acid bonds, oligonucleotide derivatives in which the uracil in the oligonucleotide is replaced with C-5 propylene uracil, in the oligonucleotide Derivative uracil in which uracil is substituted with C-5 thiazoleuracil, cytosine in the oligonucleotide is substituted with C-5 propylcytosine, and the derivative is cytosine in the oligonucleotide. Oligonucleotide derivatives substituted with enoxazine-modified cytosine, oligonucleotide derivatives substituted with 2'-0-propylribose in the oligonucleotide, or Examples include oligonucleotide derivatives in which the ribose in the nucleotide is substituted with methoxyethoxyribose [Cell engineering, 16, 1463 (1997)].
[0087] (b)相同組換え法による本発明の卵胞刺激ホルモン組成物を作製するために用い る宿主細胞の作製 [0087] (b) Production of host cell used for producing follicle-stimulating hormone composition of the present invention by homologous recombination method
本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞は、フコース 修飾に関連する酵素の遺伝子を標的とし、染色体上の標的遺伝子を相同組換え法 を用いて改変することによって作製することができる。  A host cell used for preparing the follicle stimulating hormone composition of the present invention is prepared by targeting a gene of an enzyme related to fucose modification and modifying the target gene on the chromosome using a homologous recombination method. Can do.
[0088] 染色体上の標的遺伝子の改変は、 Manipulating the Mouse Embryo A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1994) (以下、「マ-ピ ュレイティング 'ザ 'マウス'ェンブリオ 'ァ'ラボラトリ一'マニュアル」と略す）、 Gene Tar geting, A Practical Approach, IRL Press at Oxford University Press (1993)、ノヽィォマ -ュアルシリーズ 8ジーンターゲッティング， ES細胞を用いた変異マウスの作製,羊 土社（1995) (以下、「ES細胞を用いた変異マウスの作製」と略す)等に記載の方法を 用い、例えば以下のように行うことができる。  [0088] Modification of target genes on chromosomes is described in Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (hereinafter referred to as "MuPurating 'The' Mouse 'Embryo' A 'Laboratory Laboratory"). Abbreviated 'manual'), Gene Tar geting, A Practical Approach, IRL Press at Oxford University Press (1993), Noyoma-8 Series Gene Targeting, Production of Mutant Mice Using ES Cells, Yodosha (1995) ( Hereinafter, the method described in “abbreviation of mutant mouse using ES cell”) can be used, for example, as follows.
[0089] フコース修飾に関連する酵素のゲノム DNAを調製する。  [0089] Genomic DNA of an enzyme related to fucose modification is prepared.
ゲノム DNAの塩基配列にも基づき、改変する標的遺伝子 (例えば、フコース修飾に 関連  Target gene to be modified based on the genomic DNA base sequence (eg, related to fucose modification)
する酵素の構造遺伝子、あるいはプロモーター遺伝子）を相同組換えするためのタ 一ゲットベクターを作製する。  A target vector for homologous recombination of the enzyme's structural gene or promoter gene.
作製したターゲットベクターを宿主細胞に導入し、染色体上の標的遺伝子とターゲ ットベクターの間で相同組換えを起こした細胞を選択することにより、本発明の卵胞 刺激ホルモン組成物を作製するために用いる宿主細胞を作製することができる。  A host used for preparing the follicle stimulating hormone composition of the present invention by introducing the prepared target vector into a host cell and selecting a cell that has undergone homologous recombination between the target gene on the chromosome and the target vector. Cells can be made.
[0090] 宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、標的とするフコース 修飾に関連する酵素の遺伝子を有して 、るものであれば 、ずれも用いることができる[0090] As the host cell, any yeast cell, animal cell, insect cell, plant cell, etc. can be used as long as it has a gene for an enzyme related to the target fucose modification.
。具体的には、後述 3に記載の宿主細胞があげられる。 . Specifically, the host cells described in 3 below can be mentioned.
フコース修飾に関連する酵素のゲノム DNAを調製する方法としては、上記 1の（1) の（a)に記載のゲノム DNAの調製方法などがあげられる。  Examples of the method for preparing genomic DNA of an enzyme related to fucose modification include the method for preparing genomic DNA described in (1) (a) of 1 above.
[0091] 上記の方法で得られる細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素 のゲノム DNAの塩基配列として、例えば配列番号 17、 18、 19および 20に記載の塩 基配列 [0091] Enzyme involved in synthesis of intracellular sugar nucleotide GDP-fucose obtained by the above method As a base sequence of the genomic DNA of, for example, the base sequences described in SEQ ID NOs: 17, 18, 19, and 20
があげられる。  Can be given.
上記の方法で得られる N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコ サミンの 6位にフコースの 1位が α結合する糖鎖修飾に関与する酵素のゲノム DNAの 塩基配列と  The base sequence of the genomic DNA of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of the N-glycidyl-linked N-acetylyldarcosamine at the reducing end of the N-glycoside-linked complex
して、例えば配列番号 21に記載の塩基配列があげられる。  For example, the base sequence shown in SEQ ID NO: 21 can be mentioned.
[0092] 染色体上の標的遺伝子を相同糸且換えするためのターゲットベクターは、 Gene Targ eting, A Practical Approach, IRLPress at Oxford University Press (1993)、ノヽィォマ -ュアルシリーズ 8ジーンターゲッティング， ES細胞を用いた変異マウスの作製 (羊 土社) (1995)等に記載の方法にしたがって作製することができる。ターゲットベクター は、リプレースメント型、インサーシヨン型いずれでも用いることができる。  [0092] Target vectors for homologous replacement of the target gene on the chromosome were Gene Targ eting, A Practical Approach, IRLPress at Oxford University Press (1993), Noyoma-series 8 gene targeting, ES cells were used. Production of mutant mice (Yodosha) (1995) and the like. The target vector can be either a replacement type or an insertion type.
[0093] 各種宿主細胞へのターゲットベクターの導入には、後述の 3に記載の各種宿主細 胞に適した組換えベクターの導入方法を用いることができる。  [0093] For introducing the target vector into various host cells, the method for introducing a recombinant vector suitable for various host cells described in 3 below can be used.
相同組換え体を効率的に選別する方法として、例えば、 Gene Targeting, A Practic al Approach, IRL Press atOxford University Press (1993)、バイオマニュアルシリーズ 8ジーンターゲッティング， ES細胞を用いた変異マウスの作製 (羊土社) (1995)等に記 載のポジティブ選択、プロモーター選択、ネガティブ選択、ポリ A選択などの方法を用 いることができる。選別した細胞株の中から目的とする相同組換え体を選択する方法 としては、ゲノム DNAに対するサザンハイブリダィゼーシヨン法（モレキユラ一'クロー ユング第 2版）や PCR法 [ピーシーアール 'プロトコールズ (PCR Protocols), Academic Press (1990)]等があげられる。  Methods for efficiently selecting homologous recombinants include, for example, Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8 Gene Targeting, Production of Mutant Mice Using ES Cells (Sheep Methods such as positive selection, promoter selection, negative selection, poly A selection, etc. described in (Satoshisha) (1995) etc. can be used. Methods for selecting the desired homologous recombinants from the selected cell lines include the Southern Hybridization Method (Molequila's Cloning 2nd Edition) for genomic DNA and the PCR method [PCR Protocols. (PCR Protocols), Academic Press (1990)].
[0094] (c) RDO方法による本発明の卵胞刺激ホルモン組成物を作製するために用いる宿 主細胞の作製  [0094] (c) Preparation of host cells used for preparing follicle stimulating hormone composition of the present invention by RDO method
本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞は、フコース 修飾に関連する酵素の遺伝子を標的とし、 RDO法を用い、例えば、以下のように作 製することができる。  The host cell used for producing the follicle-stimulating hormone composition of the present invention can be produced, for example, as follows using the RDO method targeting an enzyme gene related to fucose modification.
[0095] フコース修飾に関連する酵素の cDNAあるいはゲノム DNAを上記 1の（1)の（a)に記 載の方法を用い、調製する。 [0095] The cDNA or genomic DNA of the enzyme related to fucose modification is described in (1) (a) above. Prepare using the method described.
調製した cDNAあるいはゲノム DNAの塩基配列を決定する。  Determine the base sequence of the prepared cDNA or genomic DNA.
決定した DNAの配列に基づき、フコース修飾に関連する酵素をコードする部分、非 翻訳領域の部分あるいはイントロン部分を含む適当な長さの RDOのコンストラクトを設 計し合成する。  Based on the determined DNA sequence, design and synthesize an RDO construct of an appropriate length that includes the enzyme coding for fucose modification, the untranslated region, or the intron.
[0096] 合成した RDOを宿主細胞に導入し、標的とした酵素、すなわちフコース修飾に関連 する酵素に変異が生じた形質転換体を選択することにより、本発明の組成物作製の ための宿主細胞を作製することができる。  [0096] A host cell for producing the composition of the present invention is prepared by introducing a synthesized RDO into a host cell and selecting a transformant in which a target enzyme, ie, an enzyme related to fucose modification has been mutated. Can be produced.
宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、標的とするフコース 修飾に関連する酵素の遺伝子を有して 、るものであれば 、ずれも用いることができる As a host cell, a yeast cell, an animal cell, an insect cell, a plant cell, etc. can be used as long as it has a gene for an enzyme related to the target fucose modification.
。具体的には、後述 3に記載の宿主細胞があげられる。 . Specifically, the host cells described in 3 below can be mentioned.
[0097] 各種宿主細胞への RDOの導入には、後述 3に記載の各種宿主細胞に適した組換 えベクターの導入方法を用いることができる。  [0097] For introduction of RDO into various host cells, the method for introducing a recombinant vector suitable for various host cells described in 3 below can be used.
フコース修飾に関連する酵素の cDNAを調製する方法としては、例えば、上記 1の（ 1)の（a)に記載の cDNAの調製方法などがあげられる。 Examples of the method for preparing cDNA of the enzyme related to fucose modification include the method for preparing cDNA described in (1) (a) of 1 above.
フコース修飾に関連する酵素のゲノム DNAを調製する方法としては、例えば、上記 1の  As a method for preparing genomic DNA of an enzyme related to fucose modification, for example,
(1)の（a)に記載のゲノム DNAの調製方法などがあげられる。  Examples thereof include a method for preparing genomic DNA as described in (1) (a).
[0098] DNAの塩基配列は、適当な制限酵素などで切断後、 pBluescript SK (-) (Stratagene 社製)等のプラスミドにサブクローユングし、通常用いられる塩基配列解析方法、例え ば、サンガー（Sanger)らのジデォキシ法 [プロシーディングス ·ォブ ·ザ ·ナショナル · ァカデミ一'ォブ 'サイエンス (Proc.Natl. Acad. Sci.,U.S.A.), 74, 5463 (1977)]等の反 応を行い、塩基配列自動分析装置、例えば、 ABI PRISM377DNAシークェンサ一（Ap plied Biosystems社製)等の塩基配列分析装置を用いて分析することにより、確認す ることがでさる。 [0098] The DNA base sequence is cleaved with an appropriate restriction enzyme, and then subcloned into a plasmid such as pBluescript SK (-) (Stratagene), and a commonly used base sequence analysis method such as Sanger ( Sanger) et al. [Procedures of the National Academia Sci., USA), 74, 5463 (1977)] This can be confirmed by analysis using an automatic base sequence analyzer, for example, a base sequence analyzer such as ABI PRISM377 DNA Sequencer (Applied Biosystems).
[0099] RDOは、常法または DNA合成機を用いることにより調製することができる。  [0099] RDO can be prepared by a conventional method or using a DNA synthesizer.
RDOを宿主細胞に導入し、標的とした酵素、フコース修飾に関連する酵素の遺伝 子に変異が生じた細胞を選択する方法としては、モレキュラー 'クローユング第 2版、 カレント.プロトコールズ.イン.モレキュラー.バイオロジー等に記載された染色体上の 遺伝子の変異を直接検出する方法があげられる。 As a method of introducing RDO into host cells and selecting cells in which the genes of the target enzyme and the enzyme related to fucose modification are mutated, Molecular 'Crowing 2nd Edition, Examples include methods for directly detecting gene mutations on chromosomes described in Current Protocols in Molecular Biology.
[0100] また、前記 1の（1)の（a)に記載の、導入したフコース修飾に関連する酵素の活性 を指標として形質転換体を選択する方法、後述 1の（5)に記載の細胞膜上の糖蛋白 質の糖鎖構造を指標として形質転換体を選択する方法、あるいは、後述 5または後 述 6に記載の産生糖蛋白質分子の糖鎖構造を指標として形質転換体を選択する方 法も用 、ることができる。  [0100] Further, the method for selecting a transformant using the activity of an enzyme related to the introduced fucose modification described in (1) (a) of 1 above as an index, and the cell membrane described in (1) (5) below A method for selecting a transformant using the sugar chain structure of the above glycoprotein as an index, or a method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule described in 5 or 6 below as an index. Can also be used.
[0101] RDOのコンストラクトは、サイエンス (Science), 273, 1386 (1996);ネィチヤ一'メディ シン (Nature Medicine), 4, 285 (1998);へパトロジー (Hepatology), 25, 1462 (1997); ジーン'セラピー (GeneTherapy), 5, 1960 (1999);ジーン'セラピー (Gene Therapy), 5, 1960 (1999);ジャーナル'ォブ 'モレキユラ一'メデイシン 0. Mol. Med.), 75, 829 (199 7);プロシーデイングス'ォブ ·ザ'ナショナル ·アカデミ^ ~ ·ォブ ·サイエンス (Proc. Natl . Acad. Sci. USA), 96, 8774 (1999);プロシーデイングス'ォブ ·ザ'ナショナル ·ァカデ ミ^ ~ ·ォブ'サイエンス (Proc. Natl. Acad. Sci. USA), 96, 8768(1999);ヌクレイック 'ァ シッド 'リサーチ (Nuc. Acids. Res.), 27, 1323 (1999);インべスティゲーシヨン'ォブ 'ダ 一マトロジー (Invest.Dematol.), U1, 1172 (1998);ネイチヤ^ ~ ·バイオテクノロジー (Na ture Biotech.), 16, 1343 (1998);ネイチヤ^ ~ ·バイオテクノロジー (Nature Biotech.), 18, 43 (2000);ネィチヤ一'バイオテクノロジー (NatureBiotech.), 18, 555 (2000)等の記載 に従って設計することができる。  [0101] RDO constructs are described in Science, 273, 1386 (1996); Nichiya's Medicine (Nature Medicine), 4, 285 (1998); Hepatology, 25, 1462 (1997); Gene Therapy, 5, 1960 (1999); Gene Therapy, 5, 1960 (1999); Journal 'Ob' Molequila 'Medine 0. Mol. Med.), 75, 829 ( 199 7); Procedures 'Ob The National' Academy ^ ~ Ob Science (Proc. Natl. Acad. Sci. USA), 96, 8774 (1999); 'National Academia ^ ~ Ob' Science (Proc. Natl. Acad. Sci. USA), 96, 8768 (1999); Nuclidec 'A Sid' Research (Nuc. Acids. Res.), 27, 1323 ( 1999); Investigation 'Ob' Da Matology (Invest.Dematol.), U1, 1172 (1998); Nature 〜 ^ · Biotechnology (Nature Biotech.), 16, 1343 (1998); Nature ^ ~ · Biotechnology -(Nature Biotech.), 18, 43 (2000); Nichiya's Biotechnology (Nature Biotech.), 18, 555 (2000).
[0102] (d) RNAi法による本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主 細胞の作製  [0102] (d) Preparation of host cell used for preparing follicle stimulating hormone composition of the present invention by RNAi method
本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞は、フコース 修飾に関連する酵素の遺伝子を標的とし、 RNAi法を用い、例えば、以下のように作 製することができる。  The host cell used for preparing the follicle stimulating hormone composition of the present invention can be prepared as follows by targeting the gene of an enzyme related to fucose modification and using the RNAi method.
[0103] フコース修飾に関連する酵素の上記 1の（1)の（a)に記載の方法を用い、 cDNAを 調製する。  [0103] Using the method described in (1) (a) of 1 above of the enzyme related to fucose modification, cDNA is prepared.
調製した cDNAの塩基配列を決定する。  Determine the base sequence of the prepared cDNA.
決定した cDNAの配列に基づき、フコース修飾に関連する酵素をコードする部分あ るいは非翻訳領域の部分を含む適当な長さの RNAi遺伝子のコンストラクトを設計す る。 Based on the determined cDNA sequence, the portion encoding the enzyme related to fucose modification Alternatively, construct an RNAi gene construct of appropriate length that includes the untranslated region.
[0104] 該 RNAi遺伝子を細胞内で発現させるために、調製した cDNAの断片、または全長 を適当な発現ベクターのプロモーターの下流に挿入することにより、糸且換えベクター を作製する。  [0104] In order to express the RNAi gene in a cell, a thread recombination vector is prepared by inserting the prepared cDNA fragment or full length downstream of the promoter of an appropriate expression vector.
該組換えベクターを、該発現べクタ一に適合した宿主細胞に導入することにより形 質転換体を得る。  A transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
導入したフコース修飾に関連する酵素の活性、あるいは産生糖蛋白質分子または 細胞表面上の糖蛋白質の糖鎖構造を指標に形質転換体を選択することで、本発明 の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞を得ることができる。  The follicle stimulating hormone composition of the present invention is prepared by selecting a transformant using as an index the activity of the enzyme related to the introduced fucose modification or the glycoprotein structure of the produced glycoprotein molecule or cell surface glycoprotein. Host cells used for the purpose can be obtained.
[0105] 宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、標的とするフコース 修飾に関連する酵素の遺伝子を有して 、るものであれば 、ずれも用いることができる 。具体的には、後述 3に記載の宿主細胞があげられる。 [0105] As the host cell, any yeast cell, animal cell, insect cell, plant cell, etc. having an enzyme gene related to the target fucose modification can be used. Specifically, the host cells described in 3 below can be mentioned.
発現ベクターとしては、上記宿主細胞において自立複製可能ないしは染色体への 組み込みが可能で、設計した RNAi遺伝子を転写できる位置にプロモーターを含有し ているものが用いられる。具体的には、後述 3に記載の発現ベクターがあげられる。  As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where the designed RNAi gene can be transcribed is used. Specifically, the expression vector described in 3 below can be mentioned.
[0106] 各種宿主細胞への遺伝子の導入には、後述 3に記載の各種宿主細胞に適した組 換えベクターの導入方法を用いることができる。 [0106] For the introduction of genes into various host cells, the recombinant vector introduction method suitable for various host cells described in 3 below can be used.
フコース修飾に関連する酵素の活性を指標として形質転換体を選択する方法とし ては、例えば、本項 1の（1)の（a)に記載の方法があげられる。  Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the method described in (a) of (1) in this section 1.
細胞膜上の糖蛋白質の糖鎖構造を指標として形質転換体を選択する方法としては 、例えば、本項 1の（5)に記載の方法があげられる。産生糖蛋白質分子の糖鎖構造 を指標として形質転換体を選択する方法としては、例えば、後述 5または後述 6に記 載の方法があげられる。  Examples of a method for selecting a transformant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (5) of this section 1. Examples of the method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
[0107] フコース修飾に関連する酵素の cDNAを調製する方法としては、例えば、本項 1の（ 1)の（a)に記載された cDNAの調製方法などがあげられる。 [0107] Examples of a method for preparing cDNA of an enzyme related to fucose modification include the method for preparing cDNA described in (1) (a) of this section 1.
また、発現ベクターを用いず、フコース修飾に関連する酵素の塩基配列に基づい て設計した RNAi遺伝子を、直接宿主細胞に導入することで、本発明の卵胞刺激ホル モン組成物を作製するために用いる宿主細胞を得ることもできる。 In addition, by introducing an RNAi gene designed based on the base sequence of an enzyme related to fucose modification directly into a host cell without using an expression vector, the follicle stimulating hormone of the present invention is used. It is also possible to obtain host cells that are used to make the Hmong composition.
[0108] RNAi遺伝子は、常法または DNA合成機を用いることにより調製することができる。  [0108] The RNAi gene can be prepared by a conventional method or using a DNA synthesizer.
RNAi遺伝子のコンストラクトは、 [ネイチヤー (Nature), 391,806 (1998);プロシーディン グス ·ォブ ·ザ'ナショナル 'ァ力デミ一 ·ォブ 'サイエンス (Proc. Natl. Acad. Sci. USA), 95, 15502(1998);ネイチヤー (Nature), 395, 854 (1998);プロシーデイングス'ォブ 'ザ •ナショナル 'ァ力デミ一 ·ォブ 'サイエンス (Proc. Natl. Acad. Sci. USA), 96, 5049 (19 99);セル (Cell), 95, 1017 (1998);プロシーデイングス'ォブ ·ザ'ナショナル 'ァ力デミ ~ ·ォブ 'サイエンス (Proc.Natl. Acad. Sci. USA), 96, 1451 (1999);プロシーデイング ス'ォブ ·ザ'ナショナル 'アカデミ^ ~ ·ォブ 'サイエンス (Proc.Natl. Acad. Sci. USA), 95 , 13959 (1998);ネィチヤ一'セル'バイオロジー (Nature Cell Biol), 2, 70 (2000)]等の 記載に従って設計することができる。  The RNAi gene construct is described in (Nature, 391,806 (1998); Proc. Of the Ob. The 'National' De force of Ob 'Science (Proc. Natl. Acad. Sci. USA), 95. , 15502 (1998); Nature, 395, 854 (1998); Proceedings 'Ob' The National 'De force 1' Science (Proc. Natl. Acad. Sci. USA), 96, 5049 (19 99); Cell, 95, 1017 (1998); Procedurals 'Ob The National' A Power Demi ~ Ob Science (Proc. Natl. Acad. Sci. USA ), 96, 1451 (1999); Proceedings 'Ob The' National 'Academia ^ ~ Ob' Science (Proc. Natl. Acad. Sci. USA), 95, 13959 (1998); The cell can be designed in accordance with the description of Nature Cell Biol, 2, 70 (2000)].
[0109] (e)トランスポゾンを用いた方法による、本発明の卵胞刺激ホルモン組成物を作製 するために用いる宿主細胞の作製  [0109] (e) Production of host cells used for producing the follicle-stimulating hormone composition of the present invention by a method using a transposon
本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞は、ネィチヤ ~ ·ジエネテイク (Nature Genet.), 25, 35(2000)等に記載のトランスポゾンのシステムを 用い、フコース修飾に関連する酵素の活性、あるいは産生糖蛋白質分子または細胞 膜上の糖蛋白質の糖鎖構造を指標に突然変異体を選択することで、本発明の卵胞 刺激ホルモン組成物を作製するために用いる宿主細胞を作製することができる。  The host cell used for preparing the follicle stimulating hormone composition of the present invention is an enzyme related to fucose modification using the transposon system described in Nature Genet., 25, 35 (2000), etc. A host cell used to produce the follicle-stimulating hormone composition of the present invention by selecting a mutant using as an index the activity of the protein, or the glycoprotein structure of the produced glycoprotein molecule or the glycoprotein on the cell membrane. be able to.
[0110] トランスポゾンのシステムとは、外来遺伝子をランダムに染色体上に挿入させること で突然変異を誘発させるシステムであり、通常、トランスポゾンに挿まれた外来遺伝子 に突然変異を誘発させるベクターとして用い、この遺伝子を染色体上にランダムに挿 入させるためのトランスポゼースの発現ベクターを同時に細胞の中に導入する。 トランスポゼースは、用いるトランスポゾンの配列に適したものであれば!/、かなるもの ち用いることがでさる。  [0110] The transposon system is a system that induces mutations by randomly inserting foreign genes onto the chromosome, and is usually used as a vector to induce mutations in foreign genes inserted into transposons. A transposase expression vector for randomly inserting the gene into the chromosome is introduced into the cell at the same time. A transposase can be used if it is suitable for the transposon sequence used!
[0111] 外来遺伝子としては、宿主細胞の DNAに変異を誘起するものであればいかなる遺 伝子も  [0111] As the foreign gene, any gene can be used as long as it induces mutation in the DNA of the host cell.
用!/、ることができる。  You can!
宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、標的とするフコース 修飾に関連する酵素の遺伝子を有して 、るものであれば 、ずれも用いることができるHost cells include yeast, animal cells, insect cells, plant cells, etc. If you have a gene for an enzyme related to the modification, you can also use the deviation
。具体的には、後述 3に記載の宿主細胞があげられる。各種宿主細胞への遺伝子の 導入には、後述 3に記載の各種宿主細胞に適した組み換えベクターの導入方法を 用!/、ることができる。 . Specifically, the host cells described in 3 below can be mentioned. For introduction of genes into various host cells, the recombinant vector introduction method suitable for various host cells described in 3 below can be used.
[0112] フコース修飾に関連する酵素の活性を指標として突然変異体を選択する方法とし ては、例えば、本項 1の（1)の（a)に記載の方法があげられる。  [0112] Examples of a method for selecting a mutant using as an index the activity of an enzyme related to fucose modification include the method described in (1) (a) of this section 1.
細胞膜上の糖蛋白質の糖鎖構造を指標として突然変異体を選択する方法としては 、例えば、本項 1の（5)に記載の方法があげられる。産生糖蛋白質分子の糖鎖構造 を指標として突然変異体を選択する方法としては、例えば、後述 5または後述 6に記 載の方法があげられる。  Examples of a method for selecting a mutant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (5) of this section 1. Examples of the method for selecting a mutant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
[0113] (2)酵素の遺伝子のドミナントネガティブ体を導入する手法  [0113] (2) Method of introducing dominant negative form of enzyme gene
本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞は、フコース 修飾に関連する酵素の遺伝子を標的とし、該酵素のドミナントネガティブ体を導入す る手法を用いることにより作製することができる。細胞内糖ヌクレオチド GDP-フコース の合成に関与する酵素としては、具体的には、 GMD、 Fxなどがあげられる。 N-グリコ シド結合複合型糖  A host cell used for preparing the follicle stimulating hormone composition of the present invention can be prepared by using a technique for targeting a gene of an enzyme related to fucose modification and introducing a dominant negative form of the enzyme. . Specific examples of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx. N-glycoside-linked complex sugar
鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が α結合する糖鎖修 飾に関与する酵素としては、具体的には、 ひ1, 6—フコシルトランスフェラーゼ、 a -L -フコ Specific examples of enzymes involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetyl darcosamine at the chain-reducing end include 1,6-fucosyltransferase, a -L- Fuco
シダーゼなどがあげられる。  Sidase and so on.
[0114] これらの酵素は、基質特異性を有したある特定の反応を触媒する酵素であり、この ような基質特異性を有した触媒作用を有する酵素の活性中心を破壊することで、これ らの酵素のドミナントネガティブ体を作製することができる。標的とする酵素のうち、 G[0114] These enzymes are enzymes that catalyze a specific reaction having substrate specificity, and these enzymes are destroyed by destroying the active center of the catalytic activity having substrate specificity. A dominant negative form of the enzyme can be prepared. G of target enzymes
MDを伊と MD to Italy
して、そのドミナントネガティブ体に作製について具体的に以下に述べる。  The production of the dominant negative body will be specifically described below.
大腸菌由来の GMDの立体構造を解析した結果、 4つのアミノ酸（133番目のトレオ- ン、 135番目のグルタミン酸、 157番目のチロシン、 161番目のリシン）が酵素活性に重 要な機能を担っていることが明らかにされている（Structure, 8, 2, 2000)。すなわち、 立体構造の情報にもとづきこれら 4つのアミノ酸を異なる他のアミノ酸に置換した変異 体を作製した結果、 、ずれの変異体にぉ ヽても有意に酵素活性が低下して 、たこと が示されている。一方、 GMDの補酵素 NADPや基質である GDP-マンノースとの結合 能に関しては、いずれの変異体においてもほとんど変化が観察されていない。従って 、 GMDの酵素活性を担うこ As a result of analyzing the three-dimensional structure of GMD derived from E. coli, four amino acids (133th threonine, 135th glutamic acid, 157th tyrosine, 161th lysine) have important functions for enzyme activity. (Structure, 8, 2, 2000). That is, Based on the three-dimensional structure information, it was shown that, as a result of producing mutants in which these four amino acids were substituted with other different amino acids, the enzyme activity was significantly reduced even when the mutants were different. Yes. On the other hand, almost no change was observed in the GMD coenzyme NADP and its ability to bind to its substrate, GDP-mannose. Therefore, GMD is responsible for the enzyme activity
れら 4つのアミノ酸を置換することによりドミナントネガティブ体を作製することができる 。大腸菌由来の GMDのドミナントネガティブ体の作製の結果に基づき、アミノ酸配列 情報をもとにした相同性比較や立体構造予測を行うことにより、例えば、 CHO細胞由 来の GMD (配列番号 10)では、 155番目のトレオニン、 157番目のグルタミン酸、 179番 目のチロシン、 183番目のリシンを他のアミノ酸に置換することによりドミナントネガティ ブ体を作製することができる。このようなアミノ酸置換を導入した遺伝子の作製は、モ レキユラ^ ~ ·クロー-ング第 2版、カレント 'プロトコ一ルズ'イン'モレキュラ^ ~ ·バイオ口 ジ一等に記載された部位特異的変異導入法を用いて行うことができる。  By substituting these four amino acids, a dominant negative can be produced. Based on the results of the preparation of dominant negative GMD derived from E. coli, by comparing homology and predicting the three-dimensional structure based on amino acid sequence information, for example, GMD derived from CHO cells (SEQ ID NO: 10) By replacing the 155th threonine, the 157th glutamic acid, the 179th tyrosine, and the 183rd lysine with other amino acids, a dominant negative body can be prepared. Genes with such amino acid substitutions were created by site-specific mutations described in Molecular 2 ~ Cloning 2nd edition, Current 'Protocols' in' Molecular ^ ~ This can be done using the introduction method.
[0115] 本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞は、上述のよ うに作製した標的酵素のドミナントネガティブ体をコードする遺伝子 (以下、ドミナント ネガティブ体遺伝子と略記する）を用い、モレキュラー 'クローユング第 2版、カレント' プロトコーノレズ 'イン'モレキュラー'バイオロジー、マニピユレ一ティング 'マウス'ェン ブリオ第 2版等に記載された遺伝子導入の方法に従って、例えば、以下のように作製 することができる。 [0115] The host cell used to produce the follicle-stimulating hormone composition of the present invention uses a gene encoding a dominant negative form of the target enzyme produced as described above (hereinafter abbreviated as a dominant negative form gene). According to the method of gene transfer described in Molecular 'Crowing 2nd Edition, Current' Protocorenoles' In 'Molecular' Biology, Manipulating 'Mouse' Enbrio 2nd Edition, etc. Can be produced.
[0116] フコース修飾に関連する酵素のドミナントネガティブ体遺伝子を調製する。  [0116] A dominant negative gene of an enzyme related to fucose modification is prepared.
調製したドミナントネガティブ体遺伝子の全長 DNAをもとにして、必要に応じて、該 蛋白質をコードする部分を含む適当な長さの DNA断片を調製する。  Based on the prepared full length DNA of the dominant negative body gene, if necessary, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared.
該 DNA断片、または全長 DNAを適当な発現ベクターのプロモーターの下流に挿入 することにより、組換えベクターを作製する。  A recombinant vector is prepared by inserting the DNA fragment or full-length DNA downstream of the promoter of an appropriate expression vector.
[0117] 該組換えベクターを、該発現べクタ一に適合した宿主細胞に導入することにより、 形質転換体を得る。 [0117] A transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
フコース修飾に関連する酵素の活性、あるいは産生糖蛋白質分子または細胞膜上 の糖蛋白質の糖鎖構造を指標に形質転換体を選択することで、本発明の卵胞刺激 ホルモン組成物を作製するために用いる宿主細胞を作製することができる。 The follicle stimulation of the present invention is selected by selecting a transformant using as an index the activity of the enzyme related to the fucose modification, or the glycoprotein structure of the glycoprotein molecule or glycoprotein on the cell membrane. Host cells used to make the hormone composition can be made.
[0118] 宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、標的とするフコース 修飾に関連する酵素の遺伝子を有して 、るものであれば 、ずれも用いることができる 。具体的には、後述 3に記載の宿主細胞があげられる。  [0118] As the host cell, any yeast cell, animal cell, insect cell, plant cell, etc. having an enzyme gene related to the target fucose modification can be used. Specifically, the host cells described in 3 below can be mentioned.
発現ベクターとしては、上記宿主細胞において自立複製可能ないしは染色体中へ の^ aみ込みが可能で、目的とするドミナントネガティブ体をコードする DNAを転写でき る位置に  The expression vector is capable of autonomous replication in the above host cell or can be inserted into the chromosome, and can be transcribed at a position where the DNA encoding the desired dominant negative body can be transcribed.
プロモーターを含有しているものが用いられる。具体的には、後述 3に記載の発現べ クタ一があげられる。  Those containing a promoter are used. Specifically, the expression vector described in 3 below can be mentioned.
[0119] 各種宿主細胞への遺伝子の導入には、後述 3に記載の各種宿主細胞に適した組 換えベクターの導入方法を用いることができる。  [0119] For introduction of genes into various host cells, the method for introducing recombinant vectors suitable for various host cells described in 3 below can be used.
フコース修飾に関連する酵素の活性を指標として形質転換体を選択する方法とし ては、例えば、後述 1の（1)の（a)に記載の方法があげられる。  Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the method described in (a) of (1) below.
細胞膜上の糖蛋白質の糖鎖構造を指標として形質転換体を選択する方法としては 、例えば、後述 1の（5)に記載の方法があげられる。産生糖蛋白質分子の糖鎖構造 を指標として形質転換体を選択する方法としては、例えば、後述 5または後述 6に記 載の方法があげられる。  Examples of the method for selecting a transformant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (1) below. Examples of the method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
[0120] (3)酵素に突然変異を導入する手法 [0120] (3) Methods for introducing mutations into enzymes
本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞は、フコース 修飾に関連する酵素の遺伝子に突然変異を導入し、該酵素に突然変異を生じた所 望の細胞株を選択する手法を用いることにより作製できる。  The host cell used for producing the follicle-stimulating hormone composition of the present invention is a method of introducing a mutation into a gene of an enzyme related to fucose modification and selecting a desired cell line in which the enzyme is mutated. Can be produced.
細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素としては、具体的には、 GMD  As an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, specifically, GMD
、 Fxなどがあげられる。 N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコ サミンの 6位にフコースの 1位が α結合する糖鎖修飾に関与する酵素としては、具体 的には、 α ΐ, 6—フコシルトランスフェラーゼ、 α -L-フコシダーゼなどがあげられる。  , Fx, etc. Specific examples of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-glycidylcolacamine at the N-glycoside-bonded sugar chain reducing terminal include α ΐ, 6-fucosyltransferase, Examples include α-L-fucosidase.
[0121] フコース修飾に関連する酵素に突然変異を導入する方法としては、 1)突然変異誘 発処理で親株を処理した突然変異体あるいは自然発生的に生じた突然変異体から 、フコース修飾に関連する酵素の活性を指標として所望の細胞株を選択する方法、 2 )突然変異誘発処理で親株を処理した突然変異体あるいは自然発生的に生じた突 然変異体から、生産糖蛋白質分子の糖鎖構造を指標として所望の細胞株を選択す る方法、 3)突然変異誘発処理で親株を処理した突然変異体あるいは自然発生的に 生じた突然変異体から、該細胞の細胞膜上の糖蛋白質の糖鎖構造を指標として所 望の細胞株を選択する方法などがあげられる。 [0121] As a method for introducing a mutation into an enzyme related to fucose modification, 1) from a mutant in which a parent strain was treated by a mutagenesis treatment or a naturally occurring mutant. , A method of selecting a desired cell line using as an index the activity of an enzyme related to fucose modification, 2) a production sugar from a mutant in which the parent strain was treated by mutagenesis treatment or a spontaneously occurring mutant. A method for selecting a desired cell line using the sugar chain structure of a protein molecule as an index, 3) from a mutant in which the parent strain was treated by mutagenesis or a naturally occurring mutant on the cell membrane of the cell. And a method of selecting a desired cell line by using the sugar chain structure of the glycoprotein as an index.
[0122] 突然変異誘発処理としては、親株の細胞の DNAに点突然変異、欠失ある!/、はフレ ームシフト突然変異を誘起するものであればいかなる処理も用いることができる。 具体的には、ェチルニトロソゥレア、ニトロソグァ-ジン、ベンゾピレン、アタリジン色 素による処理、放射線の照射などがあげられる。また、種々のアルキル化剤や発癌 物質も突然変異誘発物質として用いることができる。突然変異誘発物質を細胞に作 用させる方法としては、例えば、組織培養の技術第三版 (朝倉書店）日本組織培養 学会編 (1996)、ネィチヤ一'ジエネテイクス (Nature Genet.), 24, 314, (2000)等に記載 の方法を挙げることができる。  [0122] As the mutagenesis treatment, any treatment can be used as long as it induces a frameshift mutation in the DNA of the parent cell line. Specific examples include treatment with ethyl nitrosourea, nitrosoguanidine, benzopyrene, and atalidine dye, and irradiation with radiation. Various alkylating agents and carcinogens can also be used as mutagens. Examples of methods for causing a mutagenic agent to act on cells include, for example, tissue culture technology 3rd edition (Asakura Shoten) edited by the Japanese Society for Tissue Culture (1996), Nature Genet., 24, 314, (2000) and the like.
[0123] 自然発生的に生じた突然変異体としては、特別な突然変異誘発処理を施さな!/、で [0123] As a naturally occurring mutant, do not perform special mutagenesis!
、通常の細胞培養の条件で継代培養を続けることによって自然発生的に生じる突然 変異体を挙げることができる。 A sudden mutant that occurs spontaneously by continuing subculture under normal cell culture conditions can be mentioned.
フコース修飾に関連する酵素の活性を測定する方法としては、例えば、本項 1の（1 As a method for measuring the activity of an enzyme related to fucose modification, for example, (1 in this section)
)の（a)に記載の方法があげられる。産生糖蛋白質分子の糖鎖構造を識別する方法 としては、例えば、後述 5または後述 6に記載の方法があげられる。細胞膜上の糖蛋 白質の糖鎖構造を識別する方法としては、例えば、本項の 1の（5)に記載の方法が あげられる。 ) Of (a). Examples of the method for identifying the sugar chain structure of the produced glycoprotein molecule include the methods described in 5 and 6 below. Examples of the method for identifying the sugar chain structure of a glycoprotein on the cell membrane include the method described in 1 (5) of this section.
[0124] (4)酵素の遺伝子の転写又は翻訳を抑制する手法  [0124] (4) Technique for suppressing transcription or translation of enzyme gene
本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞は、フコース 修飾に関連する酵素の遺伝子を標的とし、アンチセンス RNAZDNA技術 [バイオサイ エンスとインダストリ一,^, 322 (1992)、化学， 681 (1991)、 Biotechnology,^, 358(1 992)、 Trends in Biotechnology ,10, 87 (1992)、 Trends in Biotechnology, 10, 152 (1992 )、細胞工学， 16, 1463 (1997)]、トリプル ·ヘリックス技術 [Trends in Biotechnology ,10 , 132 (1992)]等を用い、標的とする遺伝子の転写または翻訳を抑制することで作製 することができる。 The host cell used to produce the follicle-stimulating hormone composition of the present invention targets the gene of an enzyme related to fucose modification, and antisense RNAZDNA technology [Bioscience and Industry, ^, 322 (1992), Chemical , 681 (1991), Biotechnology, ^, 358 (1 992), Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology, 10, 152 (1992), Cell engineering, 16, 1463 (1997)], Triple · Helix technology [Trends in Biotechnology, 10 , 132 (1992)] etc., and can be produced by suppressing transcription or translation of the target gene.
[0125] 細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素としては、具体的には、 GMD、 Fxなどがあげられる。 N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダ ルコサミンの 6位にフコースの 1位が α結合する糖鎖修飾に関与する酵素としては、 具体的には、 α ΐ , 6—フコシルトランスフェラーゼ、 α -L-フコシダーゼなどがあげら れる。 [0125] Specific examples of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx. Specific examples of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α- linked to the 6-position of N-glycidyl lucosamine at the N-glycoside-linked complex sugar reducing end include α ΐ, 6-fucosyltransferase, Examples include α-L-fucosidase.
(5) N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1 位が oc結合した糖鎖構造を認識するレクチンに耐性である株を選択する手法 本発明の卵胞刺激ホルモン組成物を作製するために用いる宿主細胞は、 N-グリコ シド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1位がひ結合し た糖鎖構造を認識するレクチンに耐性である株を選択する手法を用いることにより作 製することができる。  (5) A method for selecting a strain resistant to a lectin that recognizes a sugar chain structure in which the 6-position of N-glycidyl darcosamine at the reducing end of N-glycoside-linked sugar chain and the 1-position of fucose are oc-linked The host cell used to produce the stimulating hormone composition is a lectin that recognizes the sugar chain structure in which the N-glycidyl-linked sugar chain reducing terminal N-acetylcylcosamine 6-position and fucose 1-position are linked. The strain can be produced by using a technique for selecting a strain resistant to.
[0126] N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1位が a結合した糖鎖構造を認識するレクチンに耐性である株を選択する手法としては、例 えば、ソマテイク'セル 'アンド'モレキュラ^ ~ ·ジエネテイクス（SomaticCell Mol. Genet. ) , 12, 51 (1986)等に記載のレクチンを用いた方法があげられる。  [0126] As a technique for selecting a strain that is resistant to a lectin that recognizes a sugar chain structure in which the N-glycidylcolcamine 6-position of the N-glycoside-linked sugar chain and the 1-position of fucose are a-linked For example, there is a method using a lectin described in Somatic Cell Mol. Genet., 12, 51 (1986).
レクチンとしては、 N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位 とフコースの 1位が a結合した糖鎖構造を認識するレクチンであればいずれのレクチ ンでも用いることができる力 その具体的な例としては、レンズマメレクチン LCA(L§ Culinaris由来の Lentil Agglutinin)エンドゥマメレクチン PSA (Pisum sativum由来の Pe a Lectin) ,ソラマメレクチン VFA (Viciafaba由来の Agglutinin)、ヒィロチャワンタケレク チン AAL (Aleuria aurantia由来の Lectin)等を挙げることができる。 As the lectin, any lectin can be used as long as it recognizes a sugar chain structure in which the N-glycidylcolcamine 6-position of the N-glycoside-linked sugar chain reducing end and the 1-position of fucose are a- linked. Specific examples include Lentil lectin LCA (Lentil Agglutinin from L§ Culinaris) Endumame lectin PSA (Peum sativum-derived Pe a Lectin), Broad bean lectin VFA (Agglutinin from Viciafaba), and Hirochawantake lectin AAL (Lectin derived from Aleuria aurantia) and the like.
[0127] 具体的には、 1 μ g/mL〜lmg/mLの濃度の上述のレクチンを含む培地で 1日〜2 週間、好ましくは 1日〜 1週間培養し、生存している細胞を継代培養あるいはコロニー をピックアップし別の培養容器に移し、さらに引き続きレクチンを含む培地で培養を 続けることによって、本発明の N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサ ミンの 6位とフコースの 1位が a結合した糖鎖構造を認識するレクチンに耐性である 株を選択することができる。 [0127] Specifically, the cells are cultured in a medium containing the above-mentioned lectin at a concentration of 1 μg / mL to 1 mg / mL for 1 to 2 weeks, preferably 1 to 1 week, and surviving cells are passed. Subculture or colonies are picked up, transferred to another culture vessel, and further cultured in a medium containing lectin, whereby the N-glycidyl-linked sugar chain reducing terminal N-acetylyldarcosamine of the present invention and position 1 of fucose Resistant to lectins that recognize a-linked sugar chain structures Stocks can be selected.
[0128] 2.トランスジエニック非ヒト動物あるいは植物またはそれら子孫の作製  [0128] 2. Production of transgenic non-human animals or plants or their progeny
卵胞刺激ホルモンの糖鎖の修飾に係わる酵素の活性が制御されるようにゲノム遺 伝子が改変されたトランスジエニック非ヒト動物あるいは植物またはそれら子孫は、細 胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素または N-グリコシド結合複 合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が α結合する 糖鎖修飾に関与する酵素の遺伝子を標的として、前記 1を用いて作製した本発明の 胚性幹細胞、受精卵細胞、植物カルス細胞より、例えば以下のように作製することが できる。  Transgenic non-human animals or plants or their progeny whose genomic genes have been modified so that the activity of the enzyme involved in the modification of the follicle-stimulating hormone sugar chain is controlled, synthesis of intracellular sugar nucleotide GDP-fucose Targeting the gene of the enzyme involved in glycosylation, in which the 1-position of fucose is α-linked to the 6-position of the N-glycosidic complexed sugar chain reducing N-acetylyldarcosamine From the embryonic stem cells, fertilized egg cells, and plant callus cells of the present invention produced using the above, for example, they can be produced as follows.
[0129] トランスジヱニック非ヒト動物の場合、 目的とする非ヒト動物、例えばゥシ、ヒッジ、ャ ギ、ブタ、ゥマ、マウス、ラット、 -ヮトリ、サル、ゥサギ等の胚性幹細胞に、前記 1. に記 載の手法を用いることにより、細胞内糖ヌクレオチド GDP-フコースの合成に関与する 酵素の活性または Ν-グリコシド結合複合型糖鎖還元末端の Ν-ァセチルダルコサミン の 6位にフコースの 1位が a結合する糖鎖修飾に関与する酵素の活性が制御された 本発明の胚性幹細胞を作製することができる。  [0129] In the case of transgenic non-human animals, target non-human animals such as ushi, hidge, goats, pigs, horses, mice, rats, -embryonic stem cells such as rabbits, monkeys, and rabbits. By using the method described in 1. above, the activity of the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or the 6th position of 還 元 -acetylcyldarcosamine at the reducing end of Ν-glycoside-linked complex type sugar chain Thus, the embryonic stem cell of the present invention in which the activity of an enzyme involved in sugar chain modification in which position 1 of fucose is a-linked can be produced.
[0130] 具体的は、染色体上の細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素 の活性または N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6 位にフコースの 1位が a結合する糖鎖修飾に関与する酵素をコードする遺伝子を公 知の相同組換えの手法 [例えば、 Nature,22£, 6110, 295 (1987)、 Cell,51, 3, 503 (19 87)等]により不活ィ匕または任意の配列と置換した変異クローンを作製する。作製した 胚性幹細胞 (例えば、該変異クローン)を用い、動物の受精卵の胚盤胞 (blastcyst)へ の注入キメラ法または集合キメラ法等の手法により、胚性幹細胞クローンと正常細胞 力 なるキメラ個体を調製することができる。このキメラ個体と正常個体の掛け合わせ により、全身の細胞で細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素の 活性または N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位 にフコースの 1位が a結合する糖鎖修飾に関与する酵素の活性が低下したトランス ジエニック非ヒト動物を得ることができる。 [0130] Specifically, the activity of an enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose on the chromosome or N-acetylyldarcosamine at the 6th position of N-glycidyl-linked glycan reducing terminal 1 of fucose A known homologous recombination method is used to encode a gene encoding an enzyme involved in modification of a sugar chain whose position is a [for example, Nature, 22 £, 6110, 295 (1987), Cell, 51, 3, 503 (19 87 ) Etc.] to produce a mutant clone substituted with inactive or any sequence. Using the prepared embryonic stem cells (for example, the mutant clones), a chimera that has normal cell strength and embryonic stem cell clones by a method such as an injection chimera method or an assembly chimera method into a blastcyst of an animal fertilized egg Individuals can be prepared. By combining this chimeric individual with a normal individual, the activity of an enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose in whole body cells or the N-glycidyl-linked N-acetylyldarcosamine 6 Transgenic non-human animals can be obtained in which the activity of the enzyme involved in sugar chain modification in which position 1 of fucose is a- linked is reduced.
[0131] また、 目的とする非ヒト動物、例えばゥシ、ヒッジ、ャギ、ブタ、ゥマ、マウス、ラット、二 ヮトリ、サル、ゥサギ等の受精卵細胞に、前記 1.に記載の手法と同様の手法を用いる ことにより、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素の活性または N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が oc結合する糖鎖修飾に関与する酵素の活性が低下した本発明の受精卵細胞 を作製することができる。 [0131] In addition, target non-human animals such as ushi, hidge, goat, pig, horse, mouse, rat, two By using a technique similar to that described in 1. above for fertilized egg cells such as chickens, monkeys, and rabbits, the activity of enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose or N-glycoside-linked complex sugars The fertilized egg cell of the present invention in which the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is oc-bonded to the 6-position of N-acetyl darcosamine at the chain reducing end can be produced.
[0132] 作製した受精卵細胞を、マ-ピューレーティング'マウス'ェンブリオ第 2版等に記載 の胚移植の方法を用いて偽妊娠雌の卵管あるいは子宮に移植し出産させることで、 細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素の活性または N-グリコシ ド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位がひ 結合する糖鎖修飾に関与する酵素の活性が低下したトランスジエニック非ヒト動物を 作製することができる。 [0132] The produced fertilized egg cells are transplanted into the oviduct or uterus of a pseudopregnant female using the embryo transfer method described in the Mapurating 'Mouse' Embryo 2nd Edition, etc., to give birth to intracellular sugar. Nucleotide GDP-enzyme activity involved in the synthesis of fucose or N-glycoside-linked complex sugar chain-reducing terminal N-acetylyldarcosamine Transgenic non-human animals with reduced activity can be produced.
[0133] トランスジエニック植物の場合、 目的とする植物体力ルスまたは細胞に、前記 1.に 記載の手法と同様の手法を用いることにより、細胞内糖ヌクレオチド GDP-フコースの 合成に関与する酵素の活性または N-グリコシド結合複合型糖鎖還元末端の N-ァセ チルダルコサミンの 6位にフコースの 1位が a結合する糖鎖修飾に関与する酵素の 活性が低下した本発明のカルスを作製することができる。 [0133] In the case of a transgenic plant, by using a method similar to the method described in 1. above, the enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose is used for the target plant strength or cells. To produce a callus of the present invention in which the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is a- linked to the 6-position of the N-glycidarcosamine at the reducing end of the active or N-glycoside-linked complex sugar chain Can do.
[0134] 作製したカルスを、公知の方法 [組織培養， 20 (1994);組織培養， 21 (1995);トレン ズ'イン'バイオテクノロジー (Trends in Biotechnology), 15, 45 (1997)]に準じてォーキ シン及びサイトカイニンを含む培地で培養することで再分ィ匕させ、細胞内糖ヌクレオ チド GDP-フコースの合成に関与する酵素の活性または N-グリコシド結合複合型糖 鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が α結合する糖鎖修 飾に関与する酵素の活性が低下したトランスジエニック植物を作製することができる。  [0134] According to a known method [tissue culture, 20 (1994); tissue culture, 21 (1995); Trends in Biotechnology, 15, 45 (1997)] Incubate in a medium containing carboxycin and cytokinin for re-separation, and the activity of enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose or N-glycosides at the N-glycosidic complex glycan reducing end. Transgenic plants can be produced in which the activity of the enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of cetyldarcosamine is reduced.
[0135] 3.本発明の卵胞刺激ホルモン組成物の製造方法  [0135] 3. Method for producing follicle-stimulating hormone composition of the present invention
本発明の卵胞刺激ホルモン組成物は、モレキュラー 'クローユング第 2版、カレント' プロトコーノレズ 'イン'モレキュラー.ノィォロジ一、 Antibodies, ALaboratory manual, Cold Spring Harbor Laboratory, 1988 (以下、アンチボディズと略す）、 Monoclonal An tioodies: principles and practice, Third Edition, Acaa. Press, 1993 ( 下、モノクロ ~~ ナノレアンチボディズと略す)、 AntibodyEngineering, A Practical Approach, IRL Press at Oxford University Press, 1996 (以下、アンチボディエンジニアリングと略す）等に 記載された方法を用い、例えば、以下のように宿主細胞中で発現させて取得すること ができる。 The follicle-stimulating hormone composition of the present invention is composed of a molecular 'clawing 2nd edition, current' protocorores 'in' molecular.Nology, Antibodies, ALaboratory manual, Cold Spring Harbor Laboratory, 1988 (hereinafter abbreviated as antibodies). , Monoclonal An tioodies: principles and practice, Third Edition, Acaa.Press, 1993 (below, Monochrome ~~ Nanole Antibodies), AntibodyEngineering, A Practical Approach, IRL Press Using the method described in at Oxford University Press, 1996 (hereinafter abbreviated as antibody engineering), etc., for example, it can be expressed in a host cell as follows.
[0136] 卵胞刺激ホルモン分子の各サブユニットをコードする全長 cDNAを調製し、該卵胞 刺激ホルモン分子の各サブユニットをコードする部分を含む適当な長さの DNA断片 を調製する。  [0136] A full-length cDNA encoding each subunit of the follicle-stimulating hormone molecule is prepared, and a DNA fragment of an appropriate length containing a portion encoding each subunit of the follicle-stimulating hormone molecule is prepared.
該 DNA断片、または全長 cDNAを適当な発現ベクターのプロモーターの下流に揷 入するこ  Insert the DNA fragment or full-length cDNA downstream of the appropriate expression vector promoter.
とにより、組換えベクターを作製する。組換えベクターは、各サブユニットをコードする cDNAを別々に導入したものを作製してもよいし、各サブユニットをコードする cDNAを タンデムに繋 ヽだ組換えベクターを作製してもよ 、。  To produce a recombinant vector. A recombinant vector may be prepared by introducing cDNA encoding each subunit separately, or a recombinant vector in which cDNA encoding each subunit is connected in tandem may be prepared.
[0137] 該組換えベクターを、該発現べクタ一に適合した宿主細胞に導入することにより、 卵胞刺激ホルモン分子を生産する形質転換体を得ることができる。 [0137] By introducing the recombinant vector into a host cell suitable for the expression vector, a transformant producing a follicle stimulating hormone molecule can be obtained.
宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、目的とする遺伝子を 発現できるものであれば 、ずれも用いることができる。  As the host cell, any yeast cell, animal cell, insect cell, plant cell, etc. that can express the target gene can be used.
卵胞刺激ホルモン分子に結合する N-グリコシド結合糖鎖の修飾に係わる酵素、す なわちフコース修飾に関連する酵素の活性が欠失した細胞を選択するか、または前 述 1に示された種々の人為的手法により得られた細胞を宿主細胞として用いることも できる。  Select an enzyme involved in the modification of the N-glycoside-linked sugar chain that binds to the follicle-stimulating hormone molecule, that is, a cell deficient in the activity of the enzyme related to fucose modification, or select one of the various types described in 1 above. Cells obtained by an artificial method can also be used as host cells.
[0138] 発現ベクターとしては、上記宿主細胞において自立複製可能ないしは染色体中へ の組込が可能で、目的とする卵胞刺激ホルモン分子をコードする DNAを転写できる 位置にプロモ  [0138] The expression vector can replicate autonomously in the above host cell or can be integrated into the chromosome, and can be promoted to a position where DNA encoding the desired follicle stimulating hormone molecule can be transcribed.
一ターを含有して ヽるものが用いられる。  The one containing 1 ter is used.
cDNAは、前記 1.の（1)の（a)に記載の cDNAの調製方法に従い、ヒト又は非ヒト動 物の組織又は細胞より、目的とする卵胞刺激ホルモン分子に特異的なプローブゃプ ライマー等を用いて調製することができる。  In accordance with the cDNA preparation method described in 1. (1) (a) of 1. above, a probe specific to the target follicle stimulating hormone molecule is obtained from a tissue or cell of a human or non-human animal. Etc. can be used.
[0139] 酵母を宿主細胞として用いる場合には、発現ベクターとして、例えば、 YEP13 (ATC C37115)、 YEp24 (ATCC37051)、 YCp50 (ATCC37419)等をあげることができる。 プロモーターとしては、酵母菌株中で発現できるものであればいずれのものを用い てもよく、例えば、へキソースキナーゼ等の解糖系の遺伝子のプロモーター、 PH05 プロモーター、 PGKプロモーター、 GAPプロモーター、 ADHプロモーター、 gallプロモ 一ター、 gal 10プロモーター、ヒートショックタンパク質プロモーター、 MF a lプロモー ター、 CUP 1プロモーター等をあげることができる。 [0139] When yeast is used as a host cell, examples of the expression vector include YEP13 (ATC C37115), YEp24 (ATCC37051), YCp50 (ATCC37419) and the like. Any promoter can be used as long as it can be expressed in yeast strains. For example, promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP promoter, ADH promoter Gall promoter, gal 10 promoter, heat shock protein promoter, MF al promoter, CUP 1 promoter and the like.
[0140] 宿主細胞としては、サッカロミセス属、シゾサッカロミセス属、クリュイべ口ミセス属、ト リコスポロン属、シュヮ-ォミセス属、ピキア属等に属する微生物、例えば、 Saccharom vces cerevisiae. achizosaccharomvces pombe、 Kluweromvces lactis. Tnchosporon pullulans、 Schwanniomvces alluvius, Pichia pastoris等 あけ こと力できる。 [0140] Examples of host cells include microorganisms belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth genus, Trichosporon, Schu-omyces, Pichia, etc., for example, Saccharom vces cerevisiae. Achizosaccharomvces pombe, Kluweromvces lactis. Tnchosporon pullulans, Schwanniomvces alluvius, Pichia pastoris, etc.
組換えベクターの導入方法としては、酵母に DNAを導入する方法であれば 、ずれ も用い  As a method for introducing the recombinant vector, if the method is to introduce DNA into yeast, the deviation is used.
ることができ、例えば、エレクト口ポレーシヨン法 [メソッズ'イン'ェンザィモロジ一 (Met hods. Enzymol.),194, 182(1990)]、スフエロプラスト法 [プロシーディンダス' ォブ ·ザ.ナショナル .ァ力デミ一 ·ォブ ·サイエンス (Proc. Natl. Acad. Sci. U.S.A), 84, 1929 (1978)]、酢酸リチウム法 [ジャーナル ·ォブ 'バタテリォロジ一 (J.Bacteriology),丄 53, 163 (1983)]、プロシーディングス 'ォブ ·ザ'ナショナル'ァカデミ一'ォブ 'サイェ ンス (Proc.Natl. Acad. Sci. U.S.A), 75, 1929 (1978)]に記載の方法等をあげることが できる。  For example, the electo mouth position method [Met hods. Enzymol., 194, 182 (1990)], the spheroplast method [Procedinas' of the National. Sci. USA, 84, 1929 (1978)], Lithium acetate method [Journal of Bacteriology, 丄 53, 163 (1983)], Proceedings 'Ob The National' Academia Sob. (Proc. Natl. Acad. Sci. USA), 75, 1929 (1978)]. Is possible.
[0141] 動物細胞を宿主として用いる場合には、発現ベクターとして、例えば、 pcDNAU pcD M8 (フナコシ社より巿販）、 pAGE107 [特開平 3- 22979 ;サイトテクノロジー (Cytotechno logy), 3, 133, (1990)]、 pAS3- 3 [特開平 2- 227075]、 pCDM8 [ネイチヤー (Nature),22£ , 840, (1987)]、 pcDNAI/Amp (Invitrogen社）、 pREP4 (Invitrogen社）、 pAGE103 [ジャ ーナル ·ォブ 'バイオケミストリー 0. Biochemistry),皿， 1307 (1987)]、 pAGE210等を あげることができる。  [0141] When animal cells are used as hosts, examples of expression vectors include pcDNAU pcD M8 (commercially available from Funakoshi), pAGE107 [JP 3-22979; Cytotechno logy, 3, 133, ( 1990)], pAS3-3 [JP-A-2-227075], pCDM8 [Nature, 22 £, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 (Journal) · Ob 'Biochemistry 0. Biochemistry), dish, 1307 (1987)], pAGE210, etc.
[0142] プロモーターとしては、動物細胞中で発現できるものであればいずれも用いることが でき、例えば、サイトメガロウィルス（CMV)の IE (immediateearly)遺伝子のプロモータ 一、 SV40の初期プロモーター、レトロゥイノレスのプロモーター、メタ口チォネインプロモ 一ター、ヒートショックプロモーター、 SR aプロモーター等をあげることができる。また、 ヒト CMVの IE遺伝子のェンハンサーをプロモーターと共に用いてもよ!、。 [0142] Any promoter can be used as long as it can be expressed in animal cells. For example, a promoter of the cytomegalovirus (CMV) IE (immediateearly) gene, an early promoter of SV40, a retroinores promoter , Meta-mouthone promoter, heat shock promoter, SRa promoter, and the like. Also, You can use the enhancer of the IE gene of human CMV with a promoter!
[0143] 宿主細胞としては、ヒトの細胞であるナマルバ（Namalwa)細胞、サルの細胞である C OS細胞、チャイニーズ'ノ、ムスターの細胞である CHO細胞、 HBT5637 (特開昭 63- 29 9)、ラットミエローマ細胞、マウスミエローマ細胞、シリアンノヽムスター腎臓由来細胞、 胚性幹細胞、受精卵細胞等をあげることができる。 [0143] As host cells, Namalwa cells that are human cells, COS cells that are monkey cells, CHO cells that are Chinese cells and Muster cells, HBT5637 (Japanese Patent Laid-Open No. 63-299) Rat myeloma cells, mouse myeloma cells, Syrian omster kidney-derived cells, embryonic stem cells, fertilized egg cells, and the like.
組換えベクターの導入方法としては、動物細胞に DNAを導入する方法であれば ヽ ずれも  Any recombinant vector can be introduced by introducing DNA into animal cells.
用いることができ、例えば、エレクト口ポレーシヨン法 [サイトテクノロジー (Cytotechnolo gy), 3, 133 (1990)]、リン酸カルシウム法 [特開平 2-227075]、リポフエクシヨン法 [プロ シーディングス 'ォブ ·ザ'ナショナル 'アカデミ^ ォブ 'サイエンス (Proc. Natl. Acad. Sci. U.S.A.), 84, 7413 (1987)]、インジェクション法 [マ-ピュレイティング ·ザ'マウス' ェンブリオ ·ァ ·ラボラトリ一 ·マニュアル]、パーティクルガン (遺伝子銃）を用 、る方法 [特許第 2606856、特許第 2517813]、 DEAE-デキストラン法 [バイオマ-ユアルシリ ーズ 4一遺伝子導入と発現 ·解析法 (羊土社)横田崇 ·新井賢一編 (1994)]、ウィルス ベクター法 [マ-ピュレーティング ·マウス ·ェンブリオ第 2版]等をあげることができる。  For example, the electopore position method [Cytotechnology, 3, 133 (1990)], the calcium phosphate method [Japanese Patent Laid-Open No. 2-227075], the lipofuxion method [Proceedings 'Ob The' National 'Academia ^ Science' (Proc. Natl. Acad. Sci. USA), 84, 7413 (1987)], injection method [Mapleating the 'Mouse' Embryo Laboratory Laboratory Manual], Particle Method of using a cancer (gene gun) [Patent No. 2606856, Patent No. 2517813], DEAE-dextran method [Bio-Yar series 4 gene transfer and expression / analysis method (Yodosha) Takashi Yokota, Kenichi Arai (1994)], and the virus vector method [Mupureating, Mouse, Embryo 2nd edition].
[0144] 昆虫細胞を宿主として用いる場合には、例えばカレント 'プロトコールズ'イン'モレ =Τュフ ~~ 'ノヽィォロン ~~ Baculovirus ExpressionVectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992)、ノィォ Zテクノロジー (Bio/Technology), 6 , 47(1988)等に記載された方法によって、タンパク質を発現することができる。  [0144] When using insect cells as a host, for example, Current 'Protocols' in 'More = Nyuf ~~' Nyoron ~~ Baculovirus ExpressionVectors, A Laboratory Manual, WH Freeman and Company, New York (1992) Proteins can be expressed by the method described in Neu Z Technology (Bio / Technology), 6, 47 (1988).
即ち、組換え遺伝子導入ベクターおよびバキュロウィルスを昆虫細胞に共導入して 昆虫細胞培養上清中に組換えウィルスを得た後、さらに組換えウィルスを昆虫細胞 に感染させ、タンパク質を発現させることができる。  That is, the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the protein. it can.
[0145] 該方法において用いられる遺伝子導入べクタ一としては、例えば、 pVL1392、 pVLl 393、 pBlueBacIII (ともに Invitorogen社）等をあげることができる。  [0145] Examples of the gene transfer vector used in the method include pVL1392, pVLl393, pBlueBacIII (both from Invitorogen) and the like.
バキュロウィルスとしては、例えば、夜盗蛾科昆虫に感染するウィルスであるアウトグ ラファ 'カリフオル-力 ·ヌクレア一 ·ポリへドロシス ·ウィルス (Autographacalifornica nucl ear polyhedrosis virus)等を用いること力 21 eさる。 The baculovirus, e.g., burglar Gaka insect is a virus that infects Autogu La 'Karifuoru - Power Nuclear one poly to Doroshisu virus (Autographacalifornica nucl ear polyhedrosis virus) force 21 e monkey be used like.
[0146] 昆虫細胞としては、 Spodopterafrugiperdaの卵巣細胞である S19、 S1 1 [カレント 'プロ トコ ~~ノレズ ·イン'モレキユラ1 ~~ 'ノ ィォロジ1 ~~ Baculovirus Expression Vectors, A Labo ratory Manual, W.H. Freeman and Company, New York (1992)]、 Trichoplusianiの卵 巣細胞である High 5 (Invitrogen社)等を用いることができる。 [0146] Insect cells are Spodopterafrugiperda ovarian cells S19, S1 1 [Current 'Pro Toko ~~ Norezu in 'Molekiyura 1 ~~' Noroji 1 ~~ Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)], Trichoplusiani's nest cell, High 5 (Invitrogen ) Etc. can be used.
組換えウィルスを調製するための、昆虫細胞への上記組換え遺伝子導入ベクター と上記バキュロウィルスの共導入方法としては、例えば、リン酸カルシウム法 (特開平 2 -227075)、リポフエクシヨン法 [プロシーディングス 'ォブ ·ザ'ナショナル'ァカデミ一' ォブ ·サイエンス (Pro Natl. Acad. Sci. U.S.A.), 84, 7413 (1987)]等をあげることがで きる。  Examples of methods for co-introducing the above recombinant gene transfer vector and the above baculovirus into insect cells for preparing recombinant viruses include, for example, the calcium phosphate method (JP-A-2-227075), the lipofusion method [Proceedings' · The 'National' Academia's Science (Pro Natl. Acad. Sci. USA), 84, 7413 (1987)].
[0147] 植物細胞を宿主細胞として用いる場合には、発現ベクターとして、例えば、 Tiプラス ミド、タバコモザイクウィルスベクター等をあげることができる。 プロモーターとしては 、植物細胞中で発現できるものであればいずれのものを用いてもよぐ例えば、カリフ ラヮーモザイクウィルス (CaMV)の 35Sプロモーター、ィネアクチン 1プロモーター等を あげることができる。  [0147] When plant cells are used as host cells, examples of expression vectors include Ti plasmids and tobacco mosaic virus vectors. Any promoter can be used as long as it can be expressed in plant cells. Examples thereof include the 35S promoter of califlora mosaic virus (CaMV) and the actin 1 promoter.
[0148] 宿主細胞としては、タバコ、ジャガイモ、トマト、ニンジン、ダイズ、アブラナ、アルファ ルファ、イネ、コムギ、ォォムギ等の植物細胞等をあげることができる。  [0148] Examples of host cells include tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, barley, and other plant cells.
組換えベクターの導入方法としては、植物細胞に DNAを導入する方法であれば ヽ ずれも用いることができ、例えば、ァグロバタテリゥム (Agrobacterium) [特開昭 59- 140 885、特開昭 60-70080、 WO94/00977] ,エレクト口ポレーシヨン法 [特開昭 60-251887 ]、パーティクルガン (遺伝子銃）を用いる方法 [日本特許第 2606856、 日本特許第 25 17813]等をあげることができる。  As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into plant cells. For example, Agrobacterium [JP 59-140 885, JP No. 60-70080, WO94 / 00977], an elect mouth position method [Japanese Patent Laid-Open No. 60-251887], a method using a particle gun (gene gun) [Japanese Patent No. 2606856, Japanese Patent No. 25 17813].
[0149] 遺伝子の発現方法としては、直接発現以外に、モレキュラー ·クロー-ング第 2版に 記載されている方法等に準じて、分泌生産等を行うことができる。  [0149] As a gene expression method, in addition to direct expression, secretory production or the like can be performed according to the method described in Molecular Cloning 2nd edition.
糖鎖の合成に関与する遺伝子を導入した、酵母、動物細胞、昆虫細胞または植物 細胞により発現させた場合には、導入した遺伝子によって糖あるいは糖鎖が付加さ れた卵胞刺激ホルモン分子を得ることができる。  When expressed in yeast, animal cells, insect cells, or plant cells into which genes involved in sugar chain synthesis have been introduced, obtain follicle stimulating hormone molecules to which sugars or sugar chains have been added by the introduced genes. Can do.
[0150] 以上のようにして得られる本発明の形質転換体を培地に培養し、培養物中に本発 明の卵胞刺激ホルモン組成物を生成蓄積させ、該培養物力ゝら該組成物を採取する ことにより、卵胞刺激ホルモン組成物を製造することができる。形質転換体を培地に 培養する方法は、宿主細胞の培養に用いられる通常の方法に従って行うことができ る。 [0150] The transformant of the present invention obtained as described above is cultured in a medium, the follicle stimulating hormone composition of the present invention is produced and accumulated in the culture, and the composition is collected from the strength of the culture. By doing so, a follicle stimulating hormone composition can be produced. Transformant into medium The method for culturing can be carried out according to a conventional method used for culturing host cells.
酵母等の真核生物を宿主として得られた形質転換体を培養する培地としては、該 生物が資化し得る炭素源、窒素源、無機塩類等を含有し、形質転換体の培養を効率 的に行える培地であれば天然培地、合成培地の ヽずれを用いてもょ ヽ。  As a medium for culturing a transformant obtained by using a eukaryote such as yeast as a host, it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism, so that the transformant can be cultured efficiently. If the medium can be used, the difference between natural and synthetic media can be used.
[0151] 炭素源としては、該生物が資化し得るものであればよぐグルコース、フラクトース、 スクロース、これらを含有する糖蜜、デンプンあるいはデンプン加水分解物等の炭水 化物、酢酸、プロピオン酸等の有機酸、エタノール、プロパノールなどのアルコール 類等を用いることができる。  [0151] The carbon source may be glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolyzate, acetic acid, propionic acid, etc. as long as the organism can assimilate. Alcohols such as organic acids, ethanol, and propanol can be used.
窒素源としては、アンモニア、塩化アンモ-ゥム、硫酸アンモ-ゥム、酢酸アンモ- ゥム、リン酸アンモ-ゥム等の無機酸もしくは有機酸のアンモ-ゥム塩、その他の含窒 素化合物、ならびに、ペプトン、肉エキス、酵母エキス、コーンスチープリカー、カゼィ ン加水分解物、大豆粕および大豆粕加水分解物、各種発酵菌体およびその消化物 等を用いることができる。  Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium salts, and other nitrogen-containing elements. Compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof, and the like can be used.
[0152] 無機塩類としては、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫 酸マグネシウム、塩ィ匕ナトリウム、硫酸第一鉄、硫酸マン癌、硫酸銅、炭酸カルシウム 等を用いることができる。  [0152] As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride salt, ferrous sulfate, mangan sulfate, copper sulfate, calcium carbonate, etc. are used. be able to.
培養は、通常振盪培養または深部通気攪拌培養などの好気的条件下で行う。培養 温度は 15〜40°Cがよぐ培養時間は、通常 16時間〜 7日間である。培養中の pHは 3. 0〜9. 0に保持する。 pHの調製は、無機または有機の酸、アルカリ溶液、尿素、 炭酸カルシウム、アンモニアなどを用いて行う。  The culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is 15-40 ° C, and the culture time is usually 16 hours to 7 days. The pH during the culture is maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
[0153] また、培養中必要に応じて、アンピシリンやテトラサイクリン等の抗生物質を培地に 添カロしてちょい。  [0153] If necessary, add antibiotics such as ampicillin and tetracycline to the medium during culture.
プロモーターとして誘導性のプロモーターを用いた糸且換えベクターで形質転換した 微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。 例えば、 lacプロモーターを用いた組換えベクターで形質転換した微生物を培養する ときにはイソプロピル- β -D-チォガラタトピラノシド等を、 trpプロモーターを用いた組 換えベクターで形質転換した微生物を培養するときにはインドールアクリル酸等を培 地に添カ卩してもよい。 When cultivating a microorganism transformed with a thread-replacement vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using the lac promoter, when cultivating a microorganism transformed with isopropyl-β-D-thiogalatatopyranoside or the like with a recombinant vector using the trp promoter. Cultivate indole acrylic acid, etc. You may add it to the ground.
[0154] 動物細胞を宿主として得られた形質転換体を培養する培地としては、一般に使用さ れて 、る RPMI 1640培地 [ザ ·ジャーナル ·ォブ ·ザ ·アメリカン'メディカル ·ァソシエイ シヨン (The Journal of the American Medical Association),塑， 519 (1967)]、 Eagleの MEM培地 [サイエンス (Science), 12^,501 (1952)]、ダルベッコ改変 MEM培地 [ヴユウ口 ロジー (Virology), 8, 396 (1959)]、 199培地 [プロシーデイング'ォブ ·ザ'ソサ イエティ ·フォア.ザ.バイオロジカル ·メディスン (Proceeding of the Society for the Biol ogicalMedicine), 73, 1 (1950)]、 Whitten培地 [発生工学実験マニュアル-トランスジェ ニック'マウスの作り方 (講談社)勝木元也編（1987)ほたはこれら培地に牛胎児血清 等を添加した培地等を用いることができる。  [0154] As a medium for culturing a transformant obtained using animal cells as a host, the RPMI 1640 medium [The Journal of the American American Medical Association (The Journal of the American Medical Association), Plastic, 519 (1967)], Eagle's MEM medium [Science, 12 ^, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 ( 1959)], 199 medium [Proceeding of the Society for the Biomedical Medicine, 73, 1 (1950)], Whitten medium [Developmental engineering] Experimental Manual-How to Make Transgenic 'Mice (Kodansha) Motoya Katsaki (1987) Hota can use media such as fetal bovine serum added to these media.
[0155] 培養は、通常 pH6〜8、 30〜40°C、 5%CO存在下等の条件下で 1〜7日間行う。  [0155] The culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C, and 5% CO.
2  2
また、培養中必要に応じて、カナマイシン、ペニシリン等の抗生物質を培地に添カロ してちよい。  In addition, antibiotics such as kanamycin and penicillin may be added to the medium as needed during the culture.
昆虫細胞を宿主として得られた形質転換体を培養する培地としては、一般に使用さ れている TNM- FH培地（Pharmingen社）、 Sf- 900 II SFM培地（LifeTechnologies社）、 ExCell400、 ExCell405 (いずれも JRH Biosciences社）、 Grace's Insect Medium [ネイチ ヤー (Nature),I2S, 788 (1962)]等を用いることができる。  The culture media for transformants obtained using insect cells as hosts include the commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (LifeTechnologies), ExCell400, and ExCell405 (all JRH Biosciences), Grace's Insect Medium [Nature, I2S, 788 (1962)] and the like can be used.
[0156] 培養は、通常 pH6〜7、 25〜30°C等の条件下で、 1〜5日間行う。 [0156] Cultivation is usually carried out under conditions of pH 6-7, 25-30 ° C, etc. for 1-5 days.
また、培養中必要に応じて、ゲンタマイシン等の抗生物質を培地に添加してもよい 植物細胞を宿主として得られた形質転換体は、細胞として、または植物の細胞や器 官に分化させて培養することができる。該形質転換体を培養する培地としては、一般 に使用されているムラシゲ'アンド'スターグ (MS)培地、ホワイト (White)培地、またはこ れら培  In addition, antibiotics such as gentamicin may be added to the medium as needed during the culture.Transformants obtained using plant cells as hosts are cultured as cells or differentiated into plant cells and organs. can do. As a medium for culturing the transformant, commonly used Murashige 'and' Sturg (MS) medium, White medium, or these mediums are used.
地にオーキシン、サイトカイニン等、植物ホルモンを添カ卩した培地等を用いることがで きる。  A medium supplemented with plant hormones such as auxin and cytokinin can be used.
[0157] 培養は、通常 pH5〜9、 20〜40°Cの条件下で 3〜60日間行う。  [0157] Cultivation is usually carried out under conditions of pH 5-9 and 20-40 ° C for 3-60 days.
また、培養中必要に応じて、カナマイシン、ハイグロマイシン等の抗生物質を培地 に添カ卩してもよい。 In addition, if necessary during culture, antibiotics such as kanamycin and hygromycin are added to the medium. You may add to
上記のとおり、卵胞刺激ホルモン分子をコードする DNAを組み込んだ組換え体べク ターを保有する微生物、動物細胞、あるいは植物細胞由来の形質転換体を、通常の 培養方法に従って培養し、卵胞刺激ホルモン組成物を生成蓄積させ、該培養物より 卵胞刺激ホルモン組成物を採取することにより、卵胞刺激ホルモン組成物を製造す ることがでさる。  As described above, a microorganism, animal cell, or plant cell-derived transformant having a recombinant vector incorporating a DNA encoding a follicle-stimulating hormone molecule is cultured according to a normal culture method, and follicle-stimulating hormone is obtained. The follicle stimulating hormone composition can be produced by producing and accumulating the composition and collecting the follicle stimulating hormone composition from the culture.
[0158] 卵胞刺激ホルモン組成物の生産方法としては、宿主細胞内に生産させる方法、宿 主細胞外に分泌させる方法、あるいは宿主細胞外膜上に生産させる方法があり、使 用する宿主細胞や、生産させる卵胞刺激ホルモン分子の構造を変えることにより、該 方法を選択することができる。  [0158] The follicle-stimulating hormone composition can be produced in a host cell, secreted outside the host cell, or produced on the host cell membrane. The method can be selected by changing the structure of the follicle stimulating hormone molecule to be produced.
卵胞刺激ホルモン組成物が宿主細胞内あるいは宿主細胞外膜上に生産される場 合、ポールソンらの方法 [ジャーナル'ォブ 'バイオロジカル 'ケミストリー (J. Biol. Che m.), 264, 17619 (1989)]、ロウらの方法 [プロシーディングス 'ォブ 'ザ'ナショナル'ァ 力デミ一'ォブ 'サイエンス (Proc. Natl. Acad. Sci. U.S.A.), 86, 8227 (1989);ジーン' デベロップメント (Genes Develop.), 4, 1288 (1990) ]、または特開平 05-336963、特開 平 06-823021等に記載の方法を準用することにより、該卵胞刺激ホルモン組成物を 宿  When the follicle-stimulating hormone composition is produced in the host cell or on the outer membrane of the host cell, the method of Paulson et al. [Journal 'Ob' Biological 'Chemistry (J. Biol. Chem.), 264, 17619 ( 1989)], Law et al. [Proceedings' Ob 'The' National 'Ability Demi 1' Ob 'Science (Proc. Natl. Acad. Sci. USA), 86, 8227 (1989); Gene' Development (Genes Develop.), 4, 1288 (1990)], or by applying the method described in JP-A-05-336963, JP-A-06-823021, etc.
主細胞外に積極的に分泌させることができる。  It can be actively secreted outside the main cell.
[0159] すなわち、遺伝子組換えの手法を用いて、発現ベクターに、卵胞刺激ホルモン分 子をコードする DNA、および卵胞刺激ホルモン分子の発現に適切なシグナルぺプチ ドをコードする DNAを挿入し、該発現ベクターを宿主細胞へ導入の後に卵胞刺激ホ ルモン分子を発現させることにより、目的とする卵胞刺激ホルモン分子を宿主細胞外 に積極的に分泌させることができる。  [0159] That is, using genetic recombination techniques, DNA encoding a follicle stimulating hormone molecule and DNA encoding a signal peptide appropriate for the expression of a follicle stimulating hormone molecule are inserted into an expression vector, By expressing the follicle stimulating hormone molecule after introducing the expression vector into the host cell, the target follicle stimulating hormone molecule can be actively secreted outside the host cell.
[0160] また、特開平 2-227075に記載されている方法に準じて、ジヒドロ葉酸還元酵素遺伝 子等を用いた遺伝子増幅系を利用して生産量を上昇させることもできる。 さらに、遺 伝子導入した動物または植物の細胞を再分化させることにより、遺伝子が導入された 動物個体 (トランスジ ニック非ヒト動物)または植物個体 (トランスジ ニック植物）を 造成し、これらの個体を用いて卵胞刺激ホルモン組成物を製造することもできる。 [0161] 形質転換体が動物個体または植物個体の場合は、通常の方法に従って、飼育また は栽培し、卵胞刺激ホルモン組成物を生成蓄積させ、該動物個体または植物個体よ り該卵胞刺激ホルモン組成物を採取することにより、該卵胞刺激ホルモン組成物を 製造することができる。 [0160] Further, according to the method described in Japanese Patent Laid-Open No. 2-227075, the production amount can also be increased using a gene amplification system using a dihydrofolate reductase gene or the like. Furthermore, by redifferentiating the cells of the animal or plant into which the gene was introduced, an animal individual (transgenic non-human animal) or plant individual (transgenic plant) into which the gene was introduced was created, and these individuals were used. Thus, a follicle stimulating hormone composition can also be produced. [0161] When the transformant is an animal individual or a plant individual, it is reared or cultivated according to a usual method to produce and accumulate a follicle stimulating hormone composition, and the follicle stimulating hormone composition is produced from the animal individual or plant individual. By collecting the product, the follicle stimulating hormone composition can be produced.
動物個体を用いて卵胞刺激ホルモン組成物を製造する方法としては、例えば公知 の方法 [アメリカン'ジャーナル ·ォブ ·タリ-カル ·二ユートリシヨン (Americanjournal of Clinical Nutrition), 63, 639S (1996);アメリカン 'ジャーナル'ォブ 'タリ-カル ·ニュー トリシヨン (American Journal of Clinical Nutrition), 63, 627S (1996);バイオ/テクノロ ジー (Bio/Technology), 9, 830 (1991)]に準じて遺伝子を導入して造成した動物中に 目的とする卵胞刺激ホルモン組成物を生産させる方法があげられる。  As a method for producing a follicle stimulating hormone composition using an animal individual, for example, a known method [American's Journal of Clinical Nutrition, 63, 639S (1996); American Introduced gene according to 'Journal' of 'National Journal of Clinical Nutrition, 63, 627S (1996); Bio / Technology, 9, 830 (1991)] Thus, there is a method for producing a target follicle stimulating hormone composition in an animal constructed.
[0162] 動物個体の場合は、例えば、卵胞刺激ホルモン分子をコードする DNAを導入したト ランスジ ニック非ヒト動物を飼育し、卵胞刺激ホルモン組成物を該動物中に生成蓄 積させ、該動物中より卵胞刺激ホルモン組成物を採取することにより、卵胞刺激ホル モン組成物を製造することができる。該動物中に生成蓄積させる場所としては、例え ば、該動物のミルク (特開昭 63-309192)、卵等をあげることができる。この際に用いら れるプロモーターとしては、動物で発現できるものであればいずれも用いることができ る力 例えば、乳腺細胞特異的なプロモーターである αカゼインプロモーター、 β力 ゼインプロモーター、 βラクトグロブリンプロモーター、ホエー酸性プロテインプロモー ター等が好適に用いられる。  [0162] In the case of an animal individual, for example, a transgenic non-human animal introduced with DNA encoding a follicle-stimulating hormone molecule is bred, and a follicle-stimulating hormone composition is produced and accumulated in the animal. By collecting the follicle stimulating hormone composition, a follicle stimulating hormone composition can be produced. Examples of the place to be produced and accumulated in the animal include milk of the animal (JP-A 63-309192), eggs and the like. Any promoter can be used as long as it can be expressed in animals. For example, α-casein promoter, β-force zein promoter, β-lactoglobulin promoter, A whey acidic protein promoter or the like is preferably used.
[0163] 植物個体を用いて卵胞刺激ホルモン組成物を製造する方法としては、例えば卵胞 刺激ホルモン分子をコードする DNAを導入したトランスジエニック植物を公知の方法 [ 組織培養, 2fi (1994);組織培養， 21 (1995);トレンド'イン'バイオテクノロジー (Trends inBiotechnology), 15, 45 (1997)]に準じて栽培し、卵胞刺激ホルモン組成物を該植 物中に生成蓄積させ、該植物中より該卵胞刺激ホルモン組成物を採取することにより 、卵胞刺激ホルモン組成物を生産する方法があげられる。  [0163] As a method for producing a follicle-stimulating hormone composition using an individual plant, for example, a known method for transgenic plants into which DNA encoding a follicle-stimulating hormone molecule has been introduced [tissue culture, 2fi (1994); Culture, 21 (1995); Trends in Biotechnology, 15, 45 (1997)], and the follicle-stimulating hormone composition is produced and accumulated in the plant. A method for producing a follicle stimulating hormone composition by collecting the follicle stimulating hormone composition can be mentioned.
[0164] 卵胞刺激ホルモン分子をコードする遺伝子を導入した形質転換体により製造され た卵胞刺激ホルモン組成物は、例えば卵胞刺激ホルモン組成物力 細胞内に溶解 状態で発現した場合には、培養終了後、細胞を遠心分離により回収し、水系緩衝液 にけん濁後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー、ダイノ ミル等により細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離する ことにより得られる上清から、通常の酵素の単離精製法、即ち、溶媒抽出法、硫安等 による塩析法、脱塩法、有機溶媒による沈殿法、ジェチルアミノエチル (DEAE) -セフ ァロース、 DIAIONHPA-75 (三菱化学 (株)製)等レジンを用いた陰イオン交換クロマト グラフィ一法、 S- Sepharose FF (Pharmacia社）等のレジンを用いた陽イオン交換クロ マトグラフィ一法、ブチルセファロース、フエ-ルセファロース等のレジンを用いた疎水 性クロマトグラフィー法、分子篩を用いたゲルろ過法、ァフィユティークロマトグラフィ 一法、クロマトフォーカシング法、等電点電気泳動等の電気泳動法等の手法を単独 あるいは組み合わせて用い、卵胞刺激ホルモン組成物の精製標品を得ることができ る。具体的には、 CHO細胞で生産した組換え FSH製剤の製造に際し開発された、陰 イオン交換クロマトグラフィー法とァフィ二ティークロマトグラフィー法を組み合わせた 方法をあげることができる（Human Reproduction Update 4, 862 (1998))。 [0164] When the follicle stimulating hormone composition produced by the transformant into which the gene encoding the follicle stimulating hormone molecule is introduced, for example, when expressed in a dissolved state in the cell, Cells are collected by centrifugation and aqueous buffer After suspending, the cells are crushed with an ultrasonic crusher, French press, Manton Gaurin homogenizer, dyno mill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion-exchange chromatography using resin such as tilaminoethyl (DEAE) -Sepharose and DIAIONHPA-75 (Mitsubishi Chemical Corporation), and cation using resin such as S-Sepharose FF (Pharmacia) Exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and ferrule sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing, etc. A purified preparation of the follicle-stimulating hormone composition can be obtained by using methods such as electrophoresis methods described above alone or in combination. Specifically, a method that combines the anion exchange chromatography method and the affinity chromatography method developed in the production of recombinant FSH preparations produced in CHO cells can be cited (Human Reproduction Update 4, 862). (1998)).
[0165] また、卵胞刺激ホルモン組成物が細胞内に不溶体を形成して発現した場合は、同 様に細胞を回収後破砕し、遠心分離を行うことにより、沈殿画分として卵胞刺激ホル モン組成物の不溶体を回収する。回収した卵胞刺激ホルモン組成物の不溶体をタン パク質変性剤で可溶化する。該可溶化液を希釈または透析することにより、該卵胞刺 激ホルモン組成物を正常な立体構造に戻した後、上記と同様の単離精製法により該 卵胞刺激ホルモン組成物の精製標品を得ることができる。  [0165] When the follicle-stimulating hormone composition is expressed in the form of an insoluble substance in the cells, the cells are similarly collected, disrupted, and centrifuged to obtain a follicle-stimulating hormone as a precipitate fraction. The insoluble material of the composition is recovered. The recovered insoluble material of the follicle stimulating hormone composition is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed to return the follicle stimulating hormone composition to a normal three-dimensional structure, and then a purified preparation of the follicle stimulating hormone composition is obtained by the same isolation and purification method as described above. be able to.
[0166] 卵胞刺激ホルモン組成物が細胞外に分泌された場合には、培養上清に該卵胞刺 激ホルモン組成物あるいはその誘導体を回収することができる。即ち、該培養物を上 記と同様の遠心分離等の手法により処理することにより培養上清を取得し、該培養上 清から、上記と同様の単離精製法を用いることにより、卵胞刺激ホルモン組成物の精 製標品を得ることができる。  [0166] When the follicle-stimulating hormone composition is secreted extracellularly, the follicle-stimulating hormone composition or a derivative thereof can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by the same method such as centrifugation as described above, and the follicle stimulating hormone is obtained from the culture supernatant by using the same isolation and purification method as described above. A fine preparation of the composition can be obtained.
[0167] すでに宿主細胞が卵胞刺激ホルモン分子を発現する能力を有する場合には、上 記 1に記載した方法を用いて卵胞刺激ホルモン分子を発現する能力を有する細胞を 調製した後に、該細胞を培養し、該培養物から目的とする卵胞刺激ホルモン組成物 を精製することにより、本発明の卵胞刺激ホルモン組成物を製造することができる。 4.卵胞刺激ホルモン組成物の活性評価 [0167] If the host cell already has the ability to express a follicle stimulating hormone molecule, the cell is prepared using the method described in 1 above, after preparing the cell having the ability to express a follicle stimulating hormone molecule. The follicle stimulating hormone composition of the present invention can be produced by culturing and purifying the target follicle stimulating hormone composition from the culture. 4. Activity evaluation of follicle stimulating hormone composition
精製した卵胞刺激ホルモン組成物の卵胞成熟誘導活性をはじめとする生物活性 は、既に公知の各種方法を用いて測定することができる。具体的には、卵胞刺激ホ ルモン受容体に対する結合活性、細胞内情報伝達の惹起、エストロゲン産生誘導活 性および卵胞成熟誘導活性は、卵胞刺激ホルモン受容体結合活性測定試験、細胞 内情報伝達活性測定試験、エストロゲン産生誘導活性測定試験などの in vitro試験、 マウスやラットなどのモデル動物を用いた卵胞成熟誘導活性測定試験などの in vivo 試験、あるいはヒトを用いた臨床試験などを用いて測定することができる（Proceedings of the National Academy of Sciences UbA 78,5465 (1981)、 Journal of Biological Che mistry 278, 47868 (2003)、 Neuroendocrinology41, 445 (1985)、 Journal of Clinical En docrinology and Metabolism 88,3227 (2003))。また、精製した卵胞刺激ホルモン組成 物を用いて in vivo試験を行うことで、卵胞刺激ホルモン組成物の血中半減期を測定 することができる（Journalof Clinical Endocrinology and Metabolism 88, 3227 (2003)、 HumanReproduction 14, 2709 (1999))。以下に、その具体的な例を示す。  The biological activity including the follicle maturation-inducing activity of the purified follicle-stimulating hormone composition can be measured using various known methods. Specifically, binding activity to follicle-stimulating hormone receptor, induction of intracellular signal transduction, estrogen production-inducing activity and follicle maturation-inducing activity were measured in follicle-stimulating hormone receptor binding activity measurement test, intracellular signal transduction activity measurement. Measurement using in vitro tests such as tests, estrogen production-inducing activity measurement tests, in vivo tests such as follicle maturation-inducing activity measurement tests using model animals such as mice and rats, or clinical tests using humans (Proceedings of the National Academy of Sciences UbA 78,5465 (1981), Journal of Biological Chemistry 278, 47868 (2003), Neuroendocrinology 41, 445 (1985), Journal of Clinical En docrinology and Metabolism 88,3227 (2003) ). In vivo tests using purified follicle-stimulating hormone compositions can measure the blood half-life of follicle-stimulating hormone compositions (Journalof Clinical Endocrinology and Metabolism 88, 3227 (2003), HumanReproduction 14, 2709 (1999)). Specific examples are shown below.
(1)卵胞刺激ホルモン受容体結合活性測定法  (1) Follicle stimulating hormone receptor binding activity assay
被検物質として精製した卵胞刺激ホルモン組成物と、標準品として濃度及び比活 性既知の巿販卵胞刺激ホルモンとを用いる。 Sohnらの方法（Journal of Biological Ch emistry 278, 47868 (2003))に準じ、卵胞刺激ホルモン受容体 cDNAを組み込んだ発 現ベクター pcDNA3 (インビトロジェン社）を HEK293細胞株に安定的にトランスフエタト した細胞株を作製する。この卵胞刺激ホルモン受容体発現 HEK293細胞を培養器よ り回収後、細胞を氷冷したバッファー A (150mM NaCl、 20mM HEPES、 pH7.4)で洗浄 する。細胞を、 l%Nonidet P- 40と 20%グリセロールおよびプロテアーゼインヒビター（ ImM phenylmethylsulfonyl fluoride,、 5mM ethlmaleimideおよび lOmM EDTA)を含 有するバッファー A中で溶解させ、卵巣刺激ホルモン受容体蛋白質を可溶化する。 遠心分離により得られた上清画分に対し、質量数 125のヨウ素で放射標識した被検 物質あるいは標準品を lOOOOOOcpm相当量添加する。ここに放射標識していない標 準品の段階希釈系列を競合剤として添加後、 4°Cにて 12時間インキュベートする。次 に 5 μ g/mlのゥシ γグロブリンと 20%ポリエチレングリコール 8000を含むバッファー Αを 添して 4°Cにて 10分間インキュベートする。遠心分離により得られたペレット画分の放 射活性を測定する。競合剤非存在下での放射活性を 100とし、各検体の相対的放射 活性を算出する。この相対的放射活性は、可溶化卵胞刺激ホルモン受容体に対す る相対的親和性を示しており、被検物質の卵胞刺激ホルモン受容体結合活性を測 定することができる。 A purified follicle stimulating hormone composition as a test substance and a commercially available follicle stimulating hormone of known concentration and specific activity are used as standard products. Cells in which the expression vector pcDNA3 (Invitrogen) incorporating follicle-stimulating hormone receptor cDNA is stably transfected into the HEK293 cell line according to the method of Sohn et al. (Journal of Biological Chemistry 278, 47868 (2003)) Create a strain. After recovering the follicle-stimulating hormone receptor-expressing HEK293 cells from the incubator, the cells are washed with ice-cooled buffer A (150 mM NaCl, 20 mM HEPES, pH 7.4). Cells are lysed in buffer A containing l% Nonidet P-40 and 20% glycerol and protease inhibitors (ImM phenylmethylsulfonyl fluoride, 5 mM ethlmaleimide and lOmM EDTA) to solubilize the ovarian stimulating hormone receptor protein. To the supernatant fraction obtained by centrifugation, add a test substance or standard substance radiolabeled with 125 mass iodine in an amount equivalent to lOOOOOOcpm. After adding a serial dilution series of standard products that are not radiolabeled as competitors, incubate at 4 ° C for 12 hours. Next, add buffer Α containing 5 μg / ml ushi gamma globulin and 20% polyethylene glycol 8000. Incubate for 10 minutes at 4 ° C. Measure the radioactivity of the pellet fraction obtained by centrifugation. The radioactivity in the absence of competitor is taken as 100, and the relative radioactivity of each sample is calculated. This relative radioactivity indicates a relative affinity for the solubilized follicle stimulating hormone receptor, and the follicle stimulating hormone receptor binding activity of the test substance can be measured.
[0169] (2)細胞内情報伝達活性の測定法  [0169] (2) Measuring method of intracellular signal transduction activity
ラット卵巣力 調製した顆粒膜細胞、ラット精巣から調製した精細管細胞 (セルトリ細 胞)またはセルトリ細胞由来株 TM4 (ATCC CRL-1715)などを培養し、被検物質とし て卵胞刺激ホルモン組成物を、標準品として濃度及び比活性既知の市販卵胞刺激 ホルモンを添加する。 37°Cにて数分間から数時間インキュベート後、細胞内シグナル メディエーターのひとつ cAMPの濃度を、市販の cAMPェライザキット、 cAMP Biotrak Enzyme immunoassay system (Amersham Biosciences社)などで す ことによって 、卵胞刺激ホルモンの細胞内情報伝達活性を測定することができる。  Rat ovarian force Prepared granulosa cells, seminiferous tubule cells prepared from rat testis (Sertri cells) or Sertoli cell-derived strain TM4 (ATCC CRL-1715) are cultured, and the follicle stimulating hormone composition is used as the test substance. Add commercially available follicle stimulating hormone of known concentration and specific activity as a standard. After incubating at 37 ° C for several minutes to several hours, the concentration of cAMP, one of the intracellular signal mediators, can be determined by using commercially available cAMP ELISA kit, cAMP Biotrak Enzyme immunoassay system (Amersham Biosciences), etc. Internal signal transduction activity can be measured.
[0170] (3)エストロゲン産生誘導活性の測定法  [0170] (3) Method for measuring estrogen production-inducing activity
卵胞刺激ホルモンのエストロゲン産生誘導活性は、 Jiaらの方法（Neuroendocrinolog y 41, 445 (1985))に順じて測定することができる。ラット卵巣力 調製した顆粒膜細胞 、ラット精巣から調製した精細管細胞 (セルトリ細胞)またはセルトリ細胞由来株 TM4 ( ATCC CRL-1715)などを培養し、被検物質として卵胞刺激ホルモン組成物を、標準 品として濃度及び比活性既知の巿販卵胞刺激ホルモンを添加する。その後、ェスト ロゲン合成基質として 500nMのアンドロステネジオン（Sigma社）を添カ卩し 37°Cにて数 時間インキュベートする。インキュベーション後、培養上清中に分泌されたエストロゲ ン濃度を、市販のエストロゲンェライザキット、エストロン ELISAキット（日本エンバイ口 ケミカルズ社)などで定量することによって、被検物質のエストロゲン産生誘導活性を 柳』定することができる。  The estrogen production-inducing activity of follicle stimulating hormone can be measured according to the method of Jia et al. (Neuroendocrinolog y 41, 445 (1985)). Rat ovarian force Prepared granulosa cells, seminiferous tubule cells (Sertoli cells) prepared from rat testis or Sertoli cell-derived strain TM4 (ATCC CRL-1715), etc. are cultured, and follicle stimulating hormone composition is used as a test substance. A commercially available follicle stimulating hormone with a known concentration and specific activity is added as a product. Then, add 500 nM androstenedione (Sigma) as an estrogen synthesis substrate and incubate at 37 ° C for several hours. After incubation, estrogen production-inducing activity of the test substance is determined by quantifying the concentration of estrogen secreted in the culture supernatant with a commercially available estrogenizer kit, Estrone ELISA kit (Nippon Enviguchi Chemicals), etc. ] Can be determined.
[0171] (4)卵胞成熟誘導活性の測定法  [0171] (4) Measurement of follicle maturation-inducing activity
卵胞刺激ホルモンによる卵胞成熟誘導活性は、精製した卵胞刺激ホルモン組成物 などを被検物質とし、動物モデルを用いた in vivo試験において卵巣あるいは子宮の 重量変化を調べることで測定することがきる。モデル動物としては、幼若ラットやマウ ス、あるいは脳下垂体摘出ラットやマウスを用いることができる（Endocrinology 53,604 (1953)、 Journal of Endocrinology 35, 199 (1966)、 Endocrinology 74,440 (1964))。具 体的に The follicle maturation-inducing activity by follicle-stimulating hormone can be measured by examining the change in the weight of the ovary or uterus in an in vivo test using an animal model using a purified follicle-stimulating hormone composition or the like as a test substance. Model animals include young rats and mice Or hypophysectomized rats and mice (Endocrinology 53,604 (1953), Journal of Endocrinology 35, 199 (1966), Endocrinology 74,440 (1964)). Concretely
は、モデル動物にラットを用いる場合には、以下の手順で試験を行うことができる。  When rats are used as model animals, the test can be performed by the following procedure.
[0172] 0.25%ラット血清アルブミン中で調製された被検物質または被検物質を含まな ヽプ ラシーボ (0.25%ラット血清アルブミンを含む PBS)を静脈注射により単回投与する。そ の際の卵胞刺激ホルモン組成物の投与量は、例えば、 0.1 μ g/heac！〜 1.0 μ g/head の間で設定することができる。投与後 12時間ごとに採血を行い、血中エストロゲン濃 度を測定し、卵胞刺激ホルモン投与力 48ないし 72時間後に卵巣あるいは子宫を摘 出し重量を測定する。また、試験終了後、披検物質を投与したラットから血清を採取 し、被検物質の投与によって抗卵胞刺激ホルモン抗体が出現して ヽな ヽか検定する ことちでさる。 [0172] A test substance prepared in 0.25% rat serum albumin or a non-test substance-containing placebo (PBS containing 0.25% rat serum albumin) is administered once by intravenous injection. In this case, the dosage of the follicle stimulating hormone composition is, for example, 0.1 μg / heac! It can be set between ~ 1.0 μg / head. Blood is collected every 12 hours after administration, blood estrogen concentration is measured, and ovary or eclampsia is removed 48 to 72 hours after the administration of follicle stimulating hormone and weighed. In addition, after completion of the test, serum is collected from the rat administered with test substance, and the anti-follicle-stimulating hormone antibody appears by administration of the test substance, and it is determined that it is possible to test it.
[0173] (5)モデル動物を用いた卵胞刺激ホルモン血中半減期の測定  [0173] (5) Measurement of blood half-life of follicle-stimulating hormone using model animals
精製した卵胞刺激ホルモン組成物を用いた血中半減期の測定は、ラットなどのモ デル動物を用いて調べることができる。 0.25%ラット血清アルブミン中で調製された被 検物質または被検物質を含まな！/ヽプラシーボ (0.25%ラット血清アルブミンを含む PB S)を、頸動脈力-ユーレなどを用いた静脈注射あるいは皮下注射により単回投与す る。投与する卵胞刺激ホルモン組成物の量は、例えば、 1 μ g/heac！〜 10 μ g/headの 間で設定することができる。投与後の任意の時点で 300 L程度の血液を採取し、ヒト 卵胞刺激ホルモンを特異的に検出定量できる ELISA法（Alpha Diagnostic Internation al社製 human FSH ELISAキットなど）によりラット血液中の被検物質濃度を測定する。 得られたデータは、たとえば PCNONLIN非線形回帰分析（Statistical Consultants, 19 92)を用いて薬物動態パラメーターを解析することができ血中半減期を算出すること ができる。なお、卵胞刺激ホルモン組成物のクリアランス試験は、ラット以外のげつ歯 類、たとえばマウスや、ヒトにより近縁の霊長類、たとえば力-クイザル等の動物種モ デルを用いて評価することもできる。  Measurement of the blood half-life using the purified follicle-stimulating hormone composition can be carried out using a model animal such as a rat. Contains no test substance or test substance prepared in 0.25% rat serum albumin! / Acupuncture placebo (PBS containing 0.25% rat serum albumin) is administered as a single dose by intravenous injection or subcutaneous injection using carotid artery force-Eure. The amount of follicle stimulating hormone composition to be administered is, for example, 1 μg / heac! Can be set between ~ 10 μg / head. About 300 L of blood is collected at any time after administration, and the test substance in rat blood is obtained by ELISA method (such as the human FSH ELISA kit manufactured by Alpha Diagnostic International al) that can specifically detect and quantify human follicle-stimulating hormone. Measure the concentration. The obtained data can be analyzed for pharmacokinetic parameters using, for example, PCNONLIN nonlinear regression analysis (Statistical Consultants, 1992), and the blood half-life can be calculated. The clearance test for follicle-stimulating hormone compositions can also be evaluated using rodents other than rats, such as mice, and animal species models such as primates that are more closely related to humans, such as force-quizal. .
[0174] 5.卵胞刺激ホルモン組成物の糖鎖の分析  [0174] 5. Analysis of sugar chain of follicle stimulating hormone composition
各種細胞で発現させた卵胞刺激ホルモン分子の糖鎖構造は、通常の糖蛋白質の 糖鎖構造の解析に準じて行うことができる。例えば、卵胞刺激ホルモン分子に結合し ている糖鎖はガラクトース、マンノースなどの中性糖、 N-ァセチルダルコサミンなどの アミノ糖、シアル酸などの酸性糖から構成されており、糖組成分析および二次元糖鎖 マップ法などを用いた糖鎖構造解析等の手法を用いて行うことができる。 The sugar chain structure of follicle stimulating hormone molecules expressed in various cells is the same as that of normal glycoproteins. It can be performed according to the analysis of the sugar chain structure. For example, sugar chains bound to follicle-stimulating hormone molecules are composed of neutral sugars such as galactose and mannose, amino sugars such as N-acetyldarcosamine, and acidic sugars such as sialic acid. It can be performed using a method such as a sugar chain structure analysis using a two-dimensional sugar chain map method.
[0175] ( 1)中性糖'アミノ糖組成分析 [0175] (1) Neutral sugar 'amino sugar composition analysis
卵胞刺激ホルモン分子の糖鎖の組成分析は、トリフルォロ酢酸等で、糖鎖の酸カロ 水分解を行うことにより、中性糖またはアミノ糖を遊離し、その組成比を分析すること ができる。  In the composition analysis of the sugar chain of the follicle stimulating hormone molecule, neutral sugar or amino sugar can be liberated by performing acid-hydrolysis of the sugar chain with trifluoroacetic acid or the like, and the composition ratio can be analyzed.
具体的な方法として、 Dionex社製糖組成分析装置を用いる方法があげられる。 Bio LCま HPAEC— PAD (hign performanceanion— exchange chromatography— pulsed amper ometric detection)法 [ジャーナノレ ·ォブ ·リキッド ·クロマトグラフィー (j.Liq.Chromatog r.) ,6, 1577 (1983)]によって糖組成を分析する装置である。  As a specific method, there is a method using a sugar composition analyzer manufactured by Dionex. Bio LC or HPAEC—PAD (hign performanceanion—exchange chromatography—pulsed amperometric detection) method [j. Liq. Chromatog r., 6, 1577 (1983)] It is a device to analyze.
[0176] また、 2-アミノビリジンによる蛍光標識ィ匕法でも組成比を分析することができる。具体 的には、公知の方法 [ァグリカルチュラル 'アンド'バイオロジカル ·ケミストリー (Agric.B iol.Chem.),55il), 283-284 (1991)]に従って酸加水分解した試料を 2-アミノビリジル 化で蛍光ラベルイ匕し、 HPLC分析して組成比を算出することができる。 [0176] The composition ratio can also be analyzed by a fluorescent labeling method using 2-aminoviridine. Specifically, a sample hydrolyzed according to a known method [Agricultural 'and' Biological Chemistry (Agric. Biol. Chem.), 55il), 283-284 (1991)] was converted to 2-aminobilidyl. Fluorescent labeling can be performed using HPLC analysis and the composition ratio can be calculated.
(2)糖鎖構造解析  (2) Sugar chain structure analysis
卵胞刺激ホルモン分子の糖鎖の構造解析は、 2次元糖鎖マップ法 [アナリティカル · バイオケミストリー（Anal. Biochem.) , 171 , 73 (1988)、生物化学実験法 23-糖タンパク 質糖鎖研究法 (学会出版センター)高橋禮子編（1989年) ]により行うことができる。 2 次元糖鎖マップ法は、例えば、 X軸には逆相クロマトグラフィーによる糖鎖の保持時 間または溶出位置を、 Υ軸には順相クロマトグラフィーによる糖鎖の保持時間または 溶出位置を、それぞれプロットし、既知糖鎖のそれらの結果と比較することにより、糖 鎖構造を推定する方法である。  Structural analysis of glycans in follicle-stimulating hormone molecules is based on two-dimensional glycan mapping method [Anal. Biochem., 171, 73 (1988), Biochemical Experimental Methods 23-Glycoprotein Glycan Research Law (Academic Publishing Center) Etsuko Takahashi (1989)]. In the 2D glycan mapping method, for example, the retention time or elution position of glycans by reverse phase chromatography is plotted on the X axis, and the retention time or elution position of glycans by normal phase chromatography is plotted on the vertical axis. It is a method to estimate the sugar chain structure by plotting and comparing with the results of known sugar chains.
[0177] 具体的には、卵胞刺激ホルモン組成物をヒドラジン分解して、卵胞刺激ホルモン分 子から糖鎖を遊離し、 2-アミノビリジン (以下、「ΡΑ」と略記する）による糖鎖の蛍光標 識 [ジャーナル'ォブ 'バイオケミストリー（J.Biochem.) , 197 (1984)]を行った後、 ゲルろ過により糖鎖を過剰の PAィ匕試薬などと分離し、逆相クロマトグラフィーを行う。 次いで、分取した糖鎖の各ピークについて順相クロマトグラフィーを行う。これらの結 果をもとに、 2次元糖鎖マップ上にプロットし、糖鎖スタンダード (TaKaRa社製）、文献 [アナリティカル 'バイオケミストリー（Anal. Biochem.) , 171, 73 (1988)]とのスポットの 比較より糖鎖構造を推定することができる。 [0177] Specifically, hydrazine decomposition of the follicle stimulating hormone composition releases sugar chains from the follicle stimulating hormone molecules, and the sugar chain fluorescence by 2-aminoviridine (hereinafter abbreviated as “ΡΑ”). After labeling [Journal 'Ob' Biochemistry (J. Biochem.), 197 (1984)], the glycan is separated from excess PA reagent by gel filtration and reverse phase chromatography is performed. . Next, normal phase chromatography is performed on each peak of the separated sugar chain. Based on these results, a 2D glycan map was plotted and the glycan standard (TaKaRa), literature [Analytical Biochem., 171, 73 (1988)] The sugar chain structure can be estimated by comparing the spots.
[0178] さらに各糖鎖の MALDI-TOF-MSなどの質量分析を行い、 2次元糖鎖マップ法によ り推定される構造を確認することができる。  [0178] Furthermore, mass analysis such as MALDI-TOF-MS of each sugar chain can be performed to confirm the structure estimated by the two-dimensional sugar chain mapping method.
6.卵胞刺激ホルモン分子の糖鎖構造を識別する免疫学的定量方法  6. Immunological quantification method to identify sugar chain structure of follicle stimulating hormone molecule
卵胞刺激ホルモン組成物は、糖鎖構造が異なった卵胞刺激ホルモン分子から構成 されている。本発明の遺伝子組換え卵胞刺激ホルモン組成物は、 Ν-グリコシド結合 複合型糖鎖還元末端の Ν-ァセチルダルコサミンにフコースが結合しておらず、長 ヽ 血中半減期を示す特徴を有している。このような卵胞刺激ホルモン組成物は、上記 5 .に記載の卵胞刺激ホルモン分子の糖鎖構造の分析法を用いることにより識別でき る。また、レクチンを用いた免疫学的定量方法を用いることによつても識別できる。  The follicle stimulating hormone composition is composed of follicle stimulating hormone molecules having different sugar chain structures. The genetically modified follicle stimulating hormone composition of the present invention is characterized in that fucose is not bound to Ν-glycylside reducing end Ν-acetyldarcosamine and exhibits a long-term half-life in blood. is doing. Such a follicle stimulating hormone composition can be identified by using the method for analyzing the sugar chain structure of the follicle stimulating hormone molecule described in 5. above. It can also be identified by using an immunological quantification method using a lectin.
[0179] レクチンを用いた免疫学的定量方法を用いた卵胞刺激ホルモン分子の糖鎖構造 の識別は、文献 [モノクローナル .アンティボディズ：プリンシプルズ 'アンド'アプリケー ンヨンズ (MonoclonalAntibodies: Principles and Applications), Wiley- Liss, Inc., (1995 );酵素免疫測定法，第 3版，医学書院 (1987) ;改訂版，酵素抗体法，学際企画 (1985 ；) ]等に記載のウェスタン染色、 RIA (Radioimmunoassay)ゝ VIA (Viroimmunoassay)、 EI A ( Enzymoimmunoas s ay)、 FIA ( Fiuoroimmunoas s ay)、 MIA (Metalloimmunoassay)な どの免疫学的定量方法に準じて、例えば、以下のように行うことができる。  [0179] Identification of the glycan structure of follicle-stimulating hormone molecules using lectin-based immunological quantification methods can be found in the literature [Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., (1995); Enzyme immunoassay, 3rd edition, School of Medicine (1987); Revised edition, Enzyme antibody method, Interdisciplinary planning (1985;)] etc., RIA (Radioimmunoassay ) ゝ According to immunological quantification methods such as VIA (Viroimmunoassay), EIA (Enzymoimmunoas say), FIA (Fiuoroimmunoas say), MIA (Metalloimmunoassay), for example, the following can be carried out.
[0180] 卵胞刺激ホルモン組成物を構成する卵胞刺激ホルモン分子の糖鎖構造を認識す るレクチンを標識し、標識したレクチンと試料である卵胞刺激ホルモン組成物を反応 させる。次に、標識したレクチンと卵胞刺激ホルモン分子の複合体の量を測定する。 卵胞刺激ホルモン分子の糖鎖構造を識別に用いられるレクチンとしては、例えば、 WGA u'. vulgaris由来の wheat— germ agglutinin) ^ ConA (し. ensiformis由来の concana valin A)、 RIC (R. communis由来の毒素)、 L— PHA (P.vulgaris由来の leukoagglutinin) 、 LCA(L. culinaris由来の lentil agglutinin), PSA (P. sativum由来の Pea lectin), AAL ( Aleuriaaurantia Lectin入 ACL (Amaranthus caudatus Lectin) ^ BPL (Bauninia purpure a Lectin) ^ DSL (Datura stramonium Lectin) ^ DBA (Dolichos biflorus Agglutinin) ^ EB "Elderberry Balk Lectin) ^ ECL (Erythrina cristagalli Lectin) ^ EEL (Euonymuseurop aeus Lectin) ^ GNL (Galanthus nivalis Lectin) ^ GSL (Griffonia simplicifolia Lectin) ^ H PA (Helix pomatia Agglutinin) ^ HHL (Hippeastrum Hybrid Lectin) ^ Jacalin、 LTL(Lot us tetragonolobus Lectin) ^ LEL (Lycopersicon esculent um Lectin) ^ MAL (Maackiaa murensis Lectin) ^ MPL (Maclura pomifera Lectin) ^ NPL (Narcissus pseudonarcissusL ectin)、 PNA (Peanut Agglutinin) ^ E- PHA (Phaseolus vulgaris Erythroagglutinin)、 PT L(Psophocarpus tetragonolobus Lectin) ^ RCA (Ricinus communis Agglutinin) ^ STL(S olanum tuberosum Lectin)、 SJA (Sophora japonica Agglutinin)、 SBA (Soybean Agglut inin)、 UEA (Ulex europaeus Agglutinin) ^ WL (Vicia villosa Lectin) ^ WFA(Wisteria fl oribunda Agglutinin)力 Sあげられる。 [0180] A lectin that recognizes the sugar chain structure of the follicle-stimulating hormone molecule constituting the follicle-stimulating hormone composition is labeled, and the labeled lectin is reacted with the sample follicle-stimulating hormone composition. Next, the amount of the complex of labeled lectin and follicle stimulating hormone molecule is measured. Examples of lectins used to identify the sugar chain structure of follicle-stimulating hormone molecules include WGA u '. Vulgaris-derived wheat— germ agglutinin) ^ ConA (and concana valin A from ensiformis), RIC (R. communis-derived) ), L— PHA (leukoagglutinin from P. vulgaris), LCA (lentil agglutinin from L. culinaris), PSA (Pa lectin from P. sativum), AAL (Aleuriaaurantia Lectin with ACL (Amaranthus caudatus Lectin) ^ BPL (Bauninia purpure a Lectin) ^ DSL (Datura stramonium Lectin) ^ DBA (Dolichos biflorus Agglutinin) ^ EB "Elderberry Balk Lectin) ^ ECL (Erythrina cristagalli Lectin) ^ EEL (Euonymuseurop aeus Lectin) ^ GNL (Galanthus nivalis Lectin) ^ GSL (imp Lectin) ^ H PA (Helix pomatia Agglutinin) ^ HHL (Hippeastrum Hybrid Lectin) ^ Jacalin, LTL (Lot us tetragonolobus Lectin) ^ LEL (Lycopersicon esculent um Lectin) ^ MAL (Maackiaa murensis Lectin) ^ MPL (Maclura pomifera Lectin) ^ NPL (Narcissus pseudonarcissusL ectin), PNA (Peanut Agglutinin) ^ E- PHA (Phaseolus vulgaris Erythroagglutinin), PT L (Psophocarpus tetragonolobus Lectin) ^ RCA (Ricinus communis Agglutinin) ^ STL (S olanum tuberosum Agophinin, JJA) ), SBA (Soybean Agglutinin), UEA (Ulex europaeus Agglutinin) ^ WL (Vicia villosa Lectin) ^ WFA (Wisteria floribunda Agglutinin).
[0181] 上記レクチンのなかでも、 N-ダルコシド結合複合型糖鎖還元末端の N-ァセチルダ ルコサミンにフコースが結合している糖鎖構造を特異的に認識するレクチンを用いる ことが好ましぐその具体的な例としては、レンズマメレクチン LCA (Lens Culinaris由 来の Lentil Agglutinin)エンドゥマメレクチン PSA (Pisum sativum由来の Pea Lectin)、 ソラマメレクチン VFA (Vicia faba由来の Agglutinin)、ヒィロチャワンタケレクチン AAL ( Aleuria aurantia由来の Lectin)を挙げることができる。 [0181] Among the above-mentioned lectins, it is preferable to use a lectin that specifically recognizes a sugar chain structure in which fucose is bound to N-acetylcolcamine at the N-darcoside-linked complex sugar chain reducing end. Typical examples include lentil lectin LCA (Lentil Agglutinin from Lens Culinaris), endangered lectin PSA (Peasum sativum-derived Pea Lectin), broad bean lectin VFA (Agglutinin from Vicia faba), hirochawantake lectin AAL (Aleuria aurantia) Lectin).
[0182] 7.本発明の卵胞刺激ホルモン組成物の利用 [0182] 7. Use of follicle stimulating hormone composition of the present invention
本発明の遺伝子組換え卵胞刺激ホルモン組成物は、従来から知られて!/ヽるヒト尿 由来卵胞刺激ホルモンなどの N-グリコシド結合複合型糖鎖還元末端の N-ァセチル ダルコサミンにフコースが結合して 、る卵胞刺激ホルモンに比べて、卵胞刺激ホルモ ン受容体に対する同等の親和性を有し、かつ生体内に投与した場合に血中半減期 が長 、と 、う特徴を有するため、本発明の卵胞刺激ホルモン組成物を用いる各種疾 患の治療は、投与量を上げることなぐ投与回数をより少なくすることができる。従って 、本発明の卵胞刺激ホルモン組成物は、卵胞刺激ホルモンによる治療が適応される 各種疾患において、投与量を上げることなぐ投与回数をより少なくする治療を可能 にする。このことは、患者や医療現場での負担を軽減するとともに、頻繁に投与しな けばならな 、現行治療の結果生じる事故、あるいは卵巣過剰刺激症候群ゃ抗卵胞 刺激ホルモン抗体の出現といった副作用の軽減に貢献することができる。 The recombinant follicle-stimulating hormone composition of the present invention is conventionally known! / Fucose binds to N-glycidyl-linked N-acetyl darcosamine at the N-glycoside-linked complex sugar chain reducing end such as human urine-derived follicle stimulating hormone. Therefore, the present invention has the same affinity for the follicle stimulating hormone receptor as compared to the follicle stimulating hormone, and has a long half-life in blood when administered in vivo. In the treatment of various diseases using the above follicle stimulating hormone composition, the number of administration without increasing the dose can be reduced. Therefore, the follicle-stimulating hormone composition of the present invention enables treatment with fewer administrations without increasing the dose in various diseases to which treatment with follicle-stimulating hormone is indicated. This alleviates the burden on the patient and the medical site and must be administered frequently, resulting in accidents resulting from current treatment, or ovarian hyperstimulation syndrome or anti-follicles. It can contribute to the reduction of side effects such as the appearance of stimulating hormone antibodies.
[0183] 本発明の卵胞刺激ホルモン組成物による治療が適応される疾患としては、卵胞刺 激ホルモンの産生が低下が原因で発症する疾患があげられる。  [0183] Diseases to which treatment with the follicle-stimulating hormone composition of the present invention is indicated include diseases caused by a decrease in the production of follicle stimulating hormone.
卵胞刺激ホルモンの産生が低下が原因で発症する疾患としては、不妊症があげら れ、具体的には卵胞成熟障害、排卵障害、精子形成障害、精子成熟障害などがあ げられる。  Diseases that develop due to a decrease in the production of follicle-stimulating hormone include infertility, and specifically include follicular maturation disorders, ovulation disorders, spermatogenesis disorders, and sperm maturation disorders.
本発明の卵胞刺激ホルモン組成物を含有する医薬は、予防薬あるいは治療薬とし て単独で投与することも可能ではある力 S、通常は薬理学的に許容される一つあるい はそれ以上の担体と一緒に混合し、製剤学の技術分野においてよく知られる任意の 方法により製造した医薬製剤として提供するのが望ましい。  The medicament containing the follicle-stimulating hormone composition of the present invention can be administered alone as a prophylactic or therapeutic agent S, usually one or more pharmacologically acceptable. It is desirable to provide it as a pharmaceutical formulation produced by any method well known in the pharmaceutical arts, mixed with a carrier.
[0184] 投与経路は、治療に際して最も効果的なものを使用するのが望ましぐ経口投与、 または口腔内、気道内、直腸内、皮下、筋肉内および静脈内等の非経口投与をあげ ることができ、卵胞刺激ホルモン製剤の場合、望ましくは皮下もしくは静脈内投与を あげることができる。 [0184] The route of administration includes oral administration, where it is desirable to use the most effective treatment, or parenteral administration, such as buccal, airway, rectal, subcutaneous, intramuscular and intravenous. In the case of a follicle-stimulating hormone preparation, it can be preferably administered subcutaneously or intravenously.
投与形態としては、噴霧剤、カプセル剤、錠剤、顆粒剤、シロップ剤、乳剤、座剤、 注射剤、軟膏、テープ剤、ガム剤等があげられる。  Examples of dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes, gums and the like.
[0185] 経口投与に適当な製剤としては、乳剤、シロップ剤、カプセル剤、錠剤、散剤、顆粒 剤等があげられる。 [0185] Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
乳剤およびシロップ剤のような液体調製物は、水、ショ糖、ソルビトール、果糖等の 糖類、ポリエチレングリコール、プロピレングリコール等のダリコール類、ごま油、オリ ーブ油、大豆油等の油類、 P-ヒドロキシ安息香酸エステル類等の防腐剤、スト口ベリ ーフ  Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol, and fructose, Daricols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, P- Preservatives such as hydroxybenzoates
レーバー、ペパーミント等のフレーバー類等を添加剤として用いて製造できる。  Flavors such as laver and peppermint can be used as additives.
[0186] カプセル剤、錠剤、散剤、顆粒剤等は、乳糖、ブドウ糖、ショ糖、マン-トール等の 賦形剤、デンプン、アルギン酸ナトリウム等の崩壊剤、ステアリン酸マグネシウム、タル ク等の滑沢剤、ポリビュルアルコール、ヒドロキシプロピルセルロース、ゼラチン等の 結合剤、脂肪酸エステル等の界面活性剤、グリセリン等の可塑剤等を添加剤として 用いて製造できる。 [0187] 非経口投与に適当な製剤としては、注射剤、座剤、噴霧剤等があげられる。 [0186] Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc. It can be produced using a binder such as an agent, polybulal alcohol, hydroxypropylcellulose, gelatin, a surfactant such as a fatty acid ester, a plasticizer such as glycerin, and the like as additives. [0187] Suitable preparations for parenteral administration include injections, suppositories, sprays and the like.
注射剤は、塩溶液、ブドウ糖溶液、あるいは両者の混合物カゝらなる担体等を用いて 調製される。または、卵胞刺激ホルモン組成物を常法に従って凍結乾燥し、これに塩 化ナトリウムを加えることによって粉末注射剤を調製することもできる。  The injection is prepared using a carrier such as a salt solution, a glucose solution, or a mixture of both. Alternatively, a powder injection can be prepared by freeze-drying a follicle stimulating hormone composition according to a conventional method and adding sodium chloride thereto.
座剤はカカオ脂、水素化脂肪またはカルボン酸等の担体を用いて調製される。  Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
[0188] また、噴霧剤は該卵胞刺激ホルモン組成物そのもの、な 、しは受容者の口腔およ び気道粘膜を刺激せず、かつ該卵胞刺激ホルモン組成物を微細な粒子として分散 させ吸収を容易にさせる担体等を用いて調製される。 [0188] Further, the spray does not irritate the follicle stimulating hormone composition itself, or the recipient's oral cavity and airway mucosa, and the follicle stimulating hormone composition is dispersed as fine particles for absorption. It is prepared using a carrier or the like that facilitates.
担体として具体的には乳糖、グリセリン等が例示される。該卵胞刺激ホルモン組成 物および用いる担体の性質により、エアロゾル、ドライパウダー等の製剤が可能であ る。また、これらの非経口剤においても経口剤で添加剤として例示した成分を添加す ることちでさる。  Specific examples of the carrier include lactose and glycerin. Depending on the nature of the follicle-stimulating hormone composition and the carrier used, it is possible to prepare aerosols, dry powders and the like. In addition, these parenteral agents can be obtained by adding the components exemplified as additives in the oral agent.
[0189] 投与量または投与回数は、目的とする治療効果、投与方法、治療期間、年齢、体 重等により異なるが、有効成分の量として、通常成人 1日当たり 50〜500国際単位で ある。  [0189] The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, weight, etc., but the amount of active ingredient is usually 50 to 500 international units per day for an adult.
また、卵胞刺激ホルモン組成物の卵胞成熟誘導活性をはじめとする生物活性を測 定する方法は、インビトロ実験としては卵胞刺激ホルモン受容体に対する結合活性、 細胞内情報伝達活性の測定、エストロゲン産生誘導活性の測定などの試験、モデル 動物を用いた invivo試験としては卵胞成熟誘導活性測定試験などがあげられる。  In addition, methods for measuring biological activity including follicle maturation-inducing activity of follicle-stimulating hormone compositions include in vitro experiments for binding activity to follicle-stimulating hormone receptor, measurement of intracellular signal transduction activity, estrogen production-inducing activity. Examples of in vivo tests using model animals include follicle maturation-inducing activity measurement tests.
[0190] 卵胞刺激ホルモン受容体結合活性測定法、細胞内情報伝達活性の測定法、ェスト ロゲン産生誘導活性の測定法、モデル動物を用いた卵胞成熟誘導活性の測定法は 、文献 [Proceedmgsof the National Academy of sciences UbA 78, 5465 (1981)、 Jour nal of Biological Chemistry 278, 47868 (2003)、 Neuroendocrinology 41, 445(1985)、 J ournal of Clinical Endocrinology and Metabolism 88, 3227 (2003)等]記載の公知の 方法に従って行うことができる。  [0190] Follicle-stimulating hormone receptor binding activity measurement method, intracellular signal transduction activity measurement method, estrogen production induction activity measurement method, follicle maturation induction activity measurement method using model animals are described in the literature [Proceedmgsof the National Academy of sciences UbA 78, 5465 (1981), Journal of Biological Chemistry 278, 47868 (2003), Neuroendocrinology 41, 445 (1985), Journal of Clinical Endocrinology and Metabolism 88, 3227 (2003), etc.] Can be done according to the method.
[0191] 以下の実施例により、本発明をより具体的に説明する力 実施例は本発明の単なる 例示を示すものにすぎず、本発明の範囲を限定するものではない。  [0191] The following examples illustrate the present invention more specifically. The examples are merely illustrative of the present invention and do not limit the scope of the present invention.
実施例 1 [0192] FUT8遺伝子ダブルノックアウト細胞株によるヒト卵胞刺激ホルモンの発現 本発明の卵胞刺激ホルモン組成物を産生する、無血清馴化 FUT8遺伝子ダブルノ ックアウト細胞株を以下に示す方法で作製した。 Example 1 Expression of human follicle stimulating hormone by FUT8 gene double knockout cell line [0192] A serum-free conditioned FUT8 gene double knockout cell line producing the follicle stimulating hormone composition of the present invention was prepared by the method described below.
1.プラスミド pBS-FSH aの作製  1. Construction of plasmid pBS-FSH a
ヒト卵胞刺激ホルモン (Xサブユニット (I 下垂体前葉ホルモン (Xサブユニットと同義 以下、 FSH αと称する)の遺伝子配列 (UniGene: Hs. l l9689、配列番号 1)より制限酵 素サイト (EcoRI、 BsiWI&BamHI)及びコザック配列を付カ卩した二種類の FSH a遺伝子 特異的プライマー (配列番号 22および配列番号 23)を作製し、以下の PCRを行なった 。ヒト脳下垂体由来 cDNAをテンプレートとして含む Lの反応液 [Pyrobest(R)DNA Polymerase (タカラバイオ社製)、 lO X Pyrobest bufferゝ 0.2 mmol/L dNTP mixtureゝ 0.5 μ mol/L上記プライマー (配列番号 22および配列番号 23)]を調製し、 94°Cで 1分間加 熱した後、 98°Cで 30秒間、 50°Cで 1分間、 72°Cで 2分間を 1サイクルとした 30サイクル の反応で PCRを行なった。 PCR後、反応  From the gene sequence of human follicle stimulating hormone (X subunit (I anterior pituitary hormone (synonymous with X subunit, hereinafter referred to as FSH α)) (UniGene: Hs.l l9689, SEQ ID NO: 1), the restriction enzyme site (EcoRI, BsiWI & BamHI) and two FSH a gene-specific primers (SEQ ID NO: 22 and SEQ ID NO: 23) with Kozak sequences were prepared and PCR was performed as follows: L containing human pituitary-derived cDNA as a template L [Pyrobest (R) DNA Polymerase (Takara Bio Inc.), lO X Pyrobest bufferbest 0.2 mmol / L dNTP mixture ゝ 0.5 μmol / L the above primers (SEQ ID NO: 22 and SEQ ID NO: 23)] After heating at 94 ° C for 1 minute, PCR was performed in a 30-cycle reaction with 98 ° C for 30 seconds, 50 ° C for 1 minute, and 72 ° C for 2 minutes.
液を 1.5% (W/V)ァガロースゲル電気泳動に供し、約 350 bpの FSH α遺伝子の DNA 断片を確  The solution is subjected to 1.5% (W / V) agarose gel electrophoresis to confirm the DNA fragment of the approximately 350 bp FSH α gene.
認し、 QIAquick Gel Extraction Kit(QIAGEN社製)を用いて精製した。  And purified using QIAquick Gel Extraction Kit (QIAGEN).
[0193] 得られた精製 FSH a DNA断片を 17 Lの水に溶解した後、該液に 10単位の制限酵 素 EcoRI( [0193] The obtained purified FSH a DNA fragment was dissolved in 17 L of water, and 10 units of restriction enzyme EcoRI (
タカラバィォ社製)及び 10単位の BamHI (タカラバイオ社製)、 2 μ Lの 10 X H bufferを加 えて 20 Lの反応液を調製し、 37°Cで 16時間消化反応を行なった。続いてプラスミド p BluescriptIIKS(+)(Stratagene社製) 3 μ gを 17.5 μ Lの水に溶解し、該液に 10単位の Ec oRI、 2 Lの 10 X H bufferを加えて 20 Lの反応液を調製後、 37°Cで 16時間消化反 応を行なった。反応後、フエノール/クロ口ホルム抽出処理及びエタノール沈殿を行い 、回収したプラスミドを 17.5 Lの水に溶解した。さらに該液に 10単位の BamHI、 2 L の 10 X K bufferを加えて 20 Lの反応液を調製後、 37°Cで 16時間消化反応を行なつ た。  A 20 L reaction solution was prepared by adding 10 units of BamHI (Takara Bio) and 2 μL of 10 X H buffer, followed by digestion at 37 ° C for 16 hours. Subsequently, 3 μg of plasmid p BluescriptIIKS (+) (Stratagene) is dissolved in 17.5 μL of water, 10 units of EcoRI and 2 L of 10 XH buffer are added to the solution, and 20 L of reaction solution is added. After preparation, digestion reaction was performed at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 L of water. Further, 10 units of BamHI and 2 L of 10 X K buffer were added to the solution to prepare a 20 L reaction solution, followed by digestion at 37 ° C for 16 hours.
[0194] 上記で得られた FSH a DNA断片 (EcoRI- BamHI)及び pBluescriptll KS(+)断片 (EcoR I- BamHI)を 1.5% (W/V)ァガロースゲル電気泳動に供し、それぞれ約 350bp、 3 kbp の DNA断片を QIAquick Gel Extraction Kit(QIAGEN社製)を用いて精製した。次いで FSH a DNA断片 (EcoRI- BamHI)20ngゝ pBluescriptll KS(+)断片 (EcoRI- BamHI)80 ngゝ Ligation High (東洋紡社製)を含む反応液 20 Lを調製し、 16°Cで 16時間連結反応を 行なった。得られたプラスミド DNAを用い、 heatshock法により大腸菌 DH5 a株 (東洋 紡社製)を形質転換した。形質転換株より QIAprep(R) Spin Miniprep Kit(QIAGEN社 製)を用いてプラスミド DNAを調製し、 BigDyeTerminator Cycle Sequencing Ready Rea ction Kit v2.0(QIAGEN社製)と DNAシーケンサ ABI PRISM377(Applied Biosystems社 製)を用いて塩基配列を解析した。その結果、 FSH a遺伝子配列を含むプラスミド pBS -FSH aを得た。 [0194] FSH a DNA fragment (EcoRI-BamHI) and pBluescriptll KS (+) fragment (EcoR) obtained above I-BamHI) was subjected to 1.5% (W / V) agarose gel electrophoresis, and DNA fragments of about 350 bp and 3 kbp were purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, prepare 20 L of reaction solution containing 20 ng Blue pBluescriptll KS (+) fragment (EcoRI- BamHI) 80 ng ゝ Ligation High (Toyobo Co., Ltd.) with FSH a DNA fragment (EcoRI- BamHI), and ligate at 16 ° C for 16 hours. The reaction was performed. Using the obtained plasmid DNA, E. coli DH5 a strain (manufactured by Toyo Boseki Co., Ltd.) was transformed by heatshock method. Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDyeTerminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM377 (Applied Biosystems) ) Was used to analyze the nucleotide sequence. As a result, a plasmid pBS-FSH a containing the FSH a gene sequence was obtained.
[0195] 2.プラスミド pKAN- FSH aの作製  [0195] 2. Construction of plasmid pKAN- FSH a
上記 1項で得られた pBS-FSH aを 17.5 μ Lの水に溶解した後、該液に 5単位の BsiW I(New England Biolabs社製)、 2 μ Lの 10 X NEB3 bufferをカロえて 20 μ Lの反応液を調 製し、 55°Cで 16時間消化反応を行なった。反応後、フエノール/クロ口ホルム抽出処 理及びエタノール沈殿を行い、回収したプラスミドを 17.5 Lの水に溶解した。さらに 該液に 10単位の EcoRI、 2 μ Lの 10 X H bufferを加えて 20 μ Lの反応液を調製後、 37 °Cで 16時間消化反応を行なった。  After dissolving pBS-FSHa obtained in the above 1 in 17.5 μL of water, add 5 units of BsiW I (New England Biolabs) and 2 μL of 10 X NEB3 buffer to the solution. μL of the reaction solution was prepared and digested at 55 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 L of water. Further, 10 units of EcoRI and 2 μL of 10 X H buffer were added to the solution to prepare a 20 μL reaction solution, followed by digestion reaction at 37 ° C. for 16 hours.
[0196] 続いてプラスミド pKANTEX93(W097/ 10354)3 μ gを 17.5 μ Lの水に溶解し、該液に 5 単位の BsiWI(New England Biolabs社製)、 2 μ Lの 10 X NEB3 bufferをカ卩えて 20 μしの 反応液を調製し、 55°Cで 16時間消化反応を行なった。反応後、フエノール/クロロホ ルム抽出処理及びエタノール沈殿を行い、回収したプラスミドを 17.5 Lの水に溶解 した。さらに該液に 10単位の EcoRI、 2 μ Lの 10 X H bufferを加えて 20 μ Lの反応液を 調製後、 37°Cで 16時間消化反応を行なった。  [0196] Subsequently, 3 μg of plasmid pKANTEX93 (W097 / 10354) was dissolved in 17.5 μL of water, and 5 units of BsiWI (New England Biolabs) and 2 μL of 10 X NEB3 buffer were added to the solution. A 20 μl reaction solution was prepared and digested at 55 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 L of water. Further, 10 units of EcoRI and 2 μL of 10 X H buffer were added to the solution to prepare a 20 μL reaction solution, followed by digestion reaction at 37 ° C. for 16 hours.
[0197] 上記で得られた FSH a DNA断片 (EcoRI-BsiWI)及び pKANTEX93断片 (EcoRI-BsiW I)を 1.5% (W/V)ァガロースゲル電気泳動に供し、それぞれ約 350bp、 13 kbpの DNA 断片を QIAquick Gel Extraction Kit(QIAGEN社製)を用いて精製した。次いで FSH a DNA断片 (EcoRI- BsiWI)50ng、 pKANTEX93断片 (EcoRI- BsiWI)30ng、 LigationHigh( 東洋紡社製)を含む反応液 20 /z Lを調製し、 16°Cで 16時間連結反応を行なった。得ら れたプラスミド DNAを用い、 heat shock法により大腸菌 DH5 α株 (東洋紡社製)を形質 転換した。形質転 ·より QIAprep(R) Spin Miniprep Kit(QIAGEN社製)を用いてプラ スミド DNAを調製し、 pKAN-FSH aを得た。 [0197] The FSH a DNA fragment (EcoRI-BsiWI) and pKANTEX93 fragment (EcoRI-BsiWI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and about 350 bp and 13 kbp DNA fragments were obtained. It refine | purified using QIAquick Gel Extraction Kit (made by QIAGEN). Next, a reaction solution 20 / z L containing 50 ng of FSH a DNA fragment (EcoRI-BsiWI), 30 ng of pKANTEX93 fragment (EcoRI-BsiWI) and LigationHigh (manufactured by Toyobo) was prepared, and ligation reaction was performed at 16 ° C for 16 hours. . Obtained Escherichia coli DH5 α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method using the resulting plasmid DNA. From the transformation, plasmid DNA was prepared using QIAprep® Spin Miniprep Kit (manufactured by QIAGEN) to obtain pKAN-FSHA.
[0198] 3.プラスミド pBS- FSH βの作製  [0198] 3. Construction of plasmid pBS-FSH β
ヒト卵胞刺激ホルモン βサブユニット (以下 FSH βと称する)の遺伝子配列 (UniGene: Hs.36975、配列番号 3)より制限酵素サイト (EcoRI/NotI、 BamHI)及びコザック配列を 付加した二種類の FSH β遺伝子特異的プライマー (配列番号 24および配列番号 25) を作製し、以下の PCRを行なった。ヒト脳下垂体由来 cDNAをテンプレートとして含む 2 0 μ Lの反応液 [HotstarTaq(R) DNA polymerase(QIAGEN社製)、 10 X PCR buffer、 0. 2 mmol/L dNTP mixture, 0.5 μ mol/L上記プライマー (配列番号 24および配列番号 2 5)]を調製し、 95°Cで 15分間加熱した後、 94°Cで 1分間、 60°Cで 1分間、 72°Cで 1分間 を 1サイクルとした 35サイクルの反応で PCRを行なった。 PCR後、反応液を 1.5%(W/V )ァガロースゲル電気泳動に供し、約 400bpの FSH βサブユニット遺伝子の DNA断片 を確認し、 QIAquick Gel Extraction Kit(QIAGEN社製)を用いて精製した。  Two types of FSH β added with restriction enzyme sites (EcoRI / NotI, BamHI) and Kozak sequence from human follicle stimulating hormone β subunit (hereinafter referred to as FSH β) gene sequence (UniGene: Hs.36975, SEQ ID NO: 3) Gene-specific primers (SEQ ID NO: 24 and SEQ ID NO: 25) were prepared and subjected to the following PCR. 20 μL reaction solution containing human pituitary cDNA as a template (HotstarTaq (R) DNA polymerase (QIAGEN), 10 X PCR buffer, 0.2 mmol / L dNTP mixture, 0.5 μmol / L above) Primer (SEQ ID NO: 24 and SEQ ID NO: 25)], heated at 95 ° C for 15 minutes, then 1 cycle at 94 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 1 minute PCR was performed in 35 cycles of the reaction. After PCR, the reaction solution was subjected to 1.5% (W / V) agarose gel electrophoresis to confirm a DNA fragment of about 400 bp FSH β subunit gene and purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
[0199] 得られた精製 FSH β DNA断片を 17 μ Lの水に溶解した後、該液に 10単位の EcoRI( タカラバィォ社製)及び 10単位の BamHI (タカラバイオ社製)、 2 μ Lの 10 X H bufferを加 えて 20 Lの反応液を調製し、 37°Cで 16時間消化反応を行なった。続いてプラスミド p Bluescriptll KS(+)(Stratagene社製) 3 μ gを 17.5 μ Lの水に溶解し、該液に 10単位の Ec oRI、 2 Lの 10 X H bufferを加えて 20 Lの反応液を調製後、 37°Cで 16時間消化反 応を行なった。反応後、フエノール/クロ口ホルム抽出処理及びエタノール沈殿を行い 、回収したプラスミドを 17.5 Lの水に溶解した。さらに該液に 10単位の BamHI、 2 L の 10 X K bufferを加えて 20 Lの反応液を調製後、 37°Cで 16時間消化反応を行なつ た。  [0199] After the obtained purified FSH β DNA fragment was dissolved in 17 μL of water, 10 units of EcoRI (manufactured by Takara Bio) and 10 units of BamHI (manufactured by Takara Bio), 2 μL of A 20 L reaction solution was prepared by adding 10 XH buffer, and digestion was performed at 37 ° C for 16 hours. Subsequently, 3 μg of plasmid p Bluescriptll KS (+) (Stratagene) was dissolved in 17.5 μL of water, 10 units of EcoRI and 2 L of 10 XH buffer were added to the solution, and 20 L of reaction solution was added. After the preparation, digestion reaction was performed at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 L of water. Further, 10 units of BamHI and 2 L of 10 X K buffer were added to the solution to prepare a 20 L reaction solution, followed by digestion at 37 ° C for 16 hours.
[0200] 上記で得られた FSH β DNA断片 (EcoRI- BamHI)及び pBluescriptll KS(+)断片 (EcoR I- BamHI)を 1.5% (W/V)ァガロースゲル電気泳動に供し、それぞれ約 400bp、 3 kbp の DNA断片を QIAquick Gel Extraction Kit(QIAGEN社製)を用いて精製した。次いで FSH β DNA断片 (EcoRI— BamHI)20ngゝ pBluescriptll KS(+)断片 (EcoRI— BamHI)80 ngゝ Ligation High (東洋紡社製)を含む反応液 20 Lを調製し、 16°Cで 16時間連結反応を 行なった。得られたプラスミド DNAを用い、 heatshock法により大腸菌 DH5 a株 (東洋 紡社製)を形質転換した。形質転換株より QIAprep(R) Spin Miniprep Kit(QIAGEN社 製)を用いてプラスミド DNAを調製し、 BigDyeTerminator Cycle Sequencing Ready Rea ction Kit v2.0(QIAGEN社製)と DNAシーケンサ ABI PRISM377(Applied Biosystems社 製)を用いて塩基配列を解析した。その結果、 FSH |8遺伝子配列を含むプラスミド pBS -FSH βを得た。 [0200] The FSH β DNA fragment (EcoRI-BamHI) and pBluescriptll KS (+) fragment (EcoR I-BamHI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and about 400 bp and 3 kbp, respectively. The DNA fragment was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, prepare 20 L of a reaction solution containing 20 ng Blue pBluescriptll KS (+) fragment (EcoRI— BamHI) 80 ng ゝ Ligation High (Toyobo), FSH β DNA fragment (EcoRI— BamHI), and ligate at 16 ° C for 16 hours. Reaction I did it. Using the obtained plasmid DNA, E. coli DH5 a strain (manufactured by Toyo Boseki Co., Ltd.) was transformed by heatshock method. Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDyeTerminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM377 (Applied Biosystems) ) Was used to analyze the nucleotide sequence. As a result, a plasmid pBS-FSH β containing the FSH | 8 gene sequence was obtained.
[0201] 4.発現ベクター pKAN- FSH α βの作製  [0201] 4. Construction of expression vector pKAN- FSH α β
上記 3で得られた pBS- FSH |8 3 /^を13.5 Lの水に溶解し、該液に 10単位の NotI( タカラバィォ社製)、 2 μ Lの 0.1 % BSA、 2 μ Lの 0.1 %TritonX- 100、 2 μ Lの 10 X H bu fferを加えて Lの反応液を調製し、 37°Cで 16時間消化反応を行なった。反応後、 フエノール/クロ口ホルム抽出処理及びエタノール沈殿を行 、、回収したプラスミドを 1 7.5 μ Lの水に溶解した。さらに該液に 10単位の BamHI、 2 μ Lの 10 X K bufferをカ卩えて 20 Lの反応液を調製後、 37°Cで 16時間消化反応を行なった。続いて本実施例 4で 得られたプラスミド pKAN-FSH α βを 27 μ Lの水に溶解し、該液に 20単位の Notl (タカ ラバイオ社製)、 4 μ Lの 0.1 % BSAゝ 4 μ Lの 0.1 % TritonX— 100、 4 μ Lの 10 X H buffer を加えて 40 Lの反応液を調製し、 37°Cで 16時間消化反応を行なった。反応後、フエ ノール/クロ口ホルム抽出処理及びエタノール沈殿を行 、、回収したプラスミドを 17.5 μ Lの水に溶解した。さらに該液に 10単位の BamHI、 2 μ Lの 10 X K bufferをカ卩えて 20 μ Lの反応液を調製後、 37°Cで 16時間消化反応を行なった。  PBS-FSH | 8 3 / ^ obtained in 3 above is dissolved in 13.5 L of water, 10 units of NotI (Takarabio), 2 μL of 0.1% BSA, 2 μL of 0.1% TritonX-100, 2 μL of 10 XH buffer was added to prepare a reaction solution of L, and digestion reaction was performed at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 μL of water. Further, 10 units of BamHI and 2 μL of 10 × K buffer were added to the solution to prepare a 20 L reaction solution, followed by digestion reaction at 37 ° C. for 16 hours. Subsequently, the plasmid pKAN-FSH αβ obtained in Example 4 was dissolved in 27 μL of water, and 20 units of Notl (manufactured by Takara Bio Inc.), 4 μL of 0.1% BSA B 4 μ L of 0.1% TritonX—100 and 4 μL of 10 XH buffer were added to prepare a 40 L reaction solution, which was digested at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 μL of water. Further, 10 units of BamHI and 2 μL of 10 × K buffer were added to the solution to prepare a 20 μL reaction solution, followed by digestion reaction at 37 ° C. for 16 hours.
[0202] 上記で得られた FSH β DNA断片 (Notl- BamHI)及び pKAN- FSH a断片 (Notl- BamH I)を 1.5% (W/V)ァガロースゲル電気泳動に供し、それぞれ約 400bp、 12 kbpの DNA 断片を QIAquick Gel Extraction Kit(QIAGEN社製)を用いて精製した。次いで FSH jS DNA断片 (Notl- BamHI)50 ng、 pKAN- FSH a断片 (Notl- BamHI)30ng、 Ligation High( 東洋紡社製)を含む反応液 20 /z Lを調製し、 16°Cで 16時間連結反応を行なった。得ら れたプラスミド DNAを用い、 heat shock法により大腸菌 DH5 α株 (東洋紡社製)を形質 転換した。形質転 ·より QIAprep(R) Spin Miniprep Kit(QIAGEN社製)を用いてプラ スミド DNAを調製し、 pKAN-FSH α βを得た。 pKAN- FSH α βの作製については、図 1〜図 4に示した。 [0203] 5. FUT8遺伝子ダブルノックアウト細胞への FSH発現プラスミドの導入 文献（Biotechnology and Bioengineering 87, 614 (2004))に記載された FUT8遺伝 子ダブルノックアウト細胞に、上記 4で作製したプラスミド pKAN-FSH α |8を導入した。 これらの遺伝子導入は公知のエレクト口ポレーシヨン法 [サイトテクノロジー（Cytotech nology), 3, 133 (1990)]により以下の手順で行った。まず、プラスミド pKAN-FSH α β 30 μ gを NEBuffer 3 (New England Biolabs社製) 20 μ Lと 100単位の制限酵素 MluI(New England Biolabs社製）を含む 200 Lの反応液を調製し、 37°Cで 16時間消化反応を 行うことにより線状ィ匕した。反応後、該反応液に対しフ ノール/クロ口ホルム抽出処 理およびエタノール沈殿により精製を行い、線状ィ匕プラスミドを回収した。 [0202] The FSH β DNA fragment (Notl-BamHI) and the pKAN-FSHA fragment (Notl-BamHI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and each of about 400 bp and 12 kbp. The DNA fragment was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, prepare a 20 / zL reaction solution containing 50 ng of FSH jS DNA fragment (Notl-BamHI), 30 ng of pKAN-FSHA fragment (Notl-BamHI) and Ligation High (manufactured by Toyobo) at 16 ° C for 16 hours. A ligation reaction was performed. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. From the transformation, plasmid DNA was prepared using QIAprep® Spin Miniprep Kit (manufactured by QIAGEN) to obtain pKAN-FSH αβ. The preparation of pKAN-FSH α β is shown in FIGS. [0203] 5. Introduction of FSH expression plasmid into FUT8 gene double knockout cells The plasmid pKAN-FSH prepared in 4 above was added to the FUT8 gene double knockout cells described in the literature (Biotechnology and Bioengineering 87, 614 (2004)). α | 8 was introduced. These gene introductions were carried out by the following procedure according to a known electopore position method [Cytotechnology, 3, 133 (1990)]. First, prepare a 200 L reaction solution containing 30 μg of plasmid pKAN-FSH αβ 30 μg NEBuffer 3 (New England Biolabs) and 100 units of restriction enzyme MluI (New England Biolabs). Linear digestion was achieved by digestion at 16 ° C for 16 hours. After the reaction, the reaction mixture was purified by phenol / chloroform extraction and ethanol precipitation to recover the linear plasmid.
[0204] 次に、文献（Biotechnology and Bioengineering 87, 614 (2004))に記載された FUT8 遺伝子ダブルノックアウト細胞を K-PBS緩衝液 (137 mmol/L KC1、 2.7 mmol/L NaCl、 8.1 mmol/L Na HPO、 1.5mmol/L KH PO  [0204] Next, FUT8 gene double knockout cells described in the literature (Biotechnology and Bioengineering 87, 614 (2004)) were mixed with K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8.1 mmol / L). Na HPO, 1.5mmol / L KH PO
2 4、 4.0 mmol/L MgCl )に懸濁して 8 X 107 2 4, 4.0 mmol / L MgCl) 8 X 10 7
2 4 2 2 4 2
細胞/ mLとした。細胞懸濁液 200 L (1.6 X 106細胞）と上記の線状ィ匕プラスミド 9 g を混和した後、細胞- DNA混和液の全量を GenePulser Cuvette (電極間距離 2 mm) ( BIO- RAD社製)へ移し、細胞融合装置 Gene Pulser (BIO- RAD社製）を用いてパルス 電圧 350V、電気容量 250 Fの条件で遺伝子導入を行った。遺伝子導入を行ったの ち、細胞懸濁液を 10%ゥシ胎児血清（Life Technologies社製)および 50 g/mLgenta micin (ナカライテスタ社製）を添カ卩した IMDM培地 (Life Technologies社製) 30mLに懸 濁し、接着細胞培養 96ゥエルプレート (グライナ一社製) 3枚へ 100 L/ゥエルで播種 した。培養は 5% CO、 37°Cの条件下で行った。 Cells / mL. After mixing 200 L of cell suspension (1.6 X 10 6 cells) and 9 g of the above-mentioned linear plasmid, the total amount of the cell-DNA mixture is the GenePulser Cuvette (distance between electrodes: 2 mm) (BIO-RAD) Then, gene transfer was performed using a cell fusion device Gene Pulser (manufactured by BIO-RAD) under conditions of a pulse voltage of 350 V and an electric capacity of 250 F. After gene transfer, IMDM medium (Life Technologies) supplemented with 10% ushi fetal serum (Life Technologies) and 50 g / mLgenta micin (Nacalai Testa) was added to the cell suspension. The suspension was suspended in 30 mL, and seeded on 3 adherent cell culture 96 well plates (manufactured by Grainer) at 100 L / well. The culture was performed under conditions of 5% CO and 37 ° C.
2  2
[0205] 6. 500 nM MTX而性株の取得  [0205] 6. Acquisition of 500 nM MTX metastrain
前項で得た pKAN-FSH α β導入細胞を 6日間培養した後、培養上清を除去し、 10 %透析ゥシ胎児血清、 50 μ g/mL gentamicinおよび 50 nMmethotrexate (MTX:シグ マ社製)を添加した IMDM培地を 100 L/ゥエルずつ添加した。この培地交換作業を 3 〜4日毎に繰り返しながら 9日間の培養を行った。次いで、 10%ゥシ胎児透析血清、 5 0 μ g/mL gentamicinおよび 200 nMの MTXを添カ卩した IMDM培地を用いた培地交換 作業を同様に 3〜4日毎に繰り返しながら 18日間培養し、最終的に形成されたコ口- 一を 24ゥエルプレート (シグマ社製)に植え替えた。さらに、 10%ゥシ胎児透析血清、 50 μ g/mL gentamicinおよび 500 nMの MTXを添カ卩した IMDM培地を用いた培地交換 作業を 3〜4日毎に繰り返し、適宜拡大しながら 19日間培養を行い、 500 nM MTX耐 性株を取得した。 After culturing the pKAN-FSH αβ-introduced cells obtained in the previous section for 6 days, the culture supernatant is removed, and 10% dialyzed fetal bovine serum, 50 μg / mL gentamicin and 50 nMmethotrexate (MTX: manufactured by Sigma) IMDM medium supplemented with 100 μl / well was added. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Next, culture was repeated for 18 days by repeating the medium exchange operation using IMDM medium supplemented with 10% urine fetal dialyzed serum, 50 μg / mL gentamicin and 200 nM MTX, every 3-4 days. The mouthpiece that was finally formed was replanted to a 24 well plate (Sigma). In addition, 10% Ushi fetal dialysis serum, Medium replacement using IMDM medium supplemented with 50 μg / mL gentamicin and 500 nM MTX is repeated every 3-4 days, and cultured for 19 days with appropriate expansion to obtain a 500 nM MTX resistant strain did.
[0206] 7. FSH高生産株の選別  [0206] 7. Selection of high production strains of FSH
前項で取得した複数の 500 nM MTX耐性株より、各 1.0 X 106細胞を 5 mLの 10%透 析ゥシ胎児血清、 50 μ g/mL gentamicinおよび 500 nMの MTXを添カ卩した IMDM培地 に懸濁し、 T25フラスコへ播種して培養を行った。培養 3日後に培養上清を回収し、上 清中に含まれる FSH量を Human FSH ELISA Kit(AntigenixAmerica社製)を用いて測 定し、高生産株を選別した。測定方法はキット添付マニュアルに従った。 From the multiple 500 nM MTX-resistant strains obtained in the previous section, IMDM medium supplemented with 1.0 mL of 10 6 cells each containing 5 mL of 10% permeating fetal serum, 50 μg / mL gentamicin and 500 nM MTX The suspension was then seeded in a T25 flask and cultured. After 3 days of culture, the culture supernatant was collected, and the amount of FSH contained in the supernatant was measured using Human FSH ELISA Kit (manufactured by Antigenix America) to select a high-producing strain. The measuring method followed the manual attached to the kit.
[0207] 8.無血清培地への馴化  [0207] 8. Acclimatization to serum-free medium
前項で得られた FSH高生産株を 4 mMの L-Glutamine (インビトロジェン社製)、 50 g /ml gentamicinおよび 500 nMの MTXを添カ卩した 15mlの EX- CELL302培地 (JRH社製、 以下、無血清培地と称する)に 5 X 105細胞/ mlで懸濁して 125 ml三角フラスコ (コ一- ング社製)へ播種し、浮遊旋回培養を行なった。培養は、 35°C、旋回速度は 90〜100 rpmで行い、継代の際には培養容器の 4倍量以上の 5% COを培地上面に通気し、 The FSH high-producing strain obtained in the previous section was added to 4 mM L-Glutamine (Invitrogen), 50 g / ml gentamicin and 500 nM MTX in 15 ml EX-CELL302 medium (JRH, hereinafter, Suspended at 5 × 10 5 cells / ml in a serum-free medium), seeded in a 125 ml Erlenmeyer flask (manufactured by Corning), and subjected to floating swirl culture. Cultivation is performed at 35 ° C and swirl speed of 90-100 rpm. During passage, 4% or more of 5% CO in the culture vessel is aerated on the top of the medium,
2  2
三角フラスコ中の空気を置換した。 3日後に培地交換を行い、 6日目に 5 X 105細胞/ m 1で継代を行なった。以降、 3〜5 X 105細胞/ mlで 3〜4日毎に継代を二週間行なった 。この培養により無血清培地で増殖し、かつ凝集の起こらない細胞 PKAN-FSH9-3 A FMS705を得た。 The air in the Erlenmeyer flask was replaced. The medium was changed after 3 days, and passage was performed at 5 × 10 5 cells / m 1 on the 6th day. Thereafter, passage was performed every 3-4 days at 3-5 × 10 5 cells / ml for 2 weeks. This culture yielded cells PKAN-FSH9-3 A FMS705 that grew in serum-free medium and did not aggregate.
[0208] 得られた株を 3.0 X 105細胞/ mLの濃度で 15 mLの無血清培地に懸濁し、 12 mLフラ スコへ播種して培養を行った。培養 3日後に培養上清を回収し、上清中に含まれる F SH量を HumanFSH ELISA Kit(Antigenix America社製)を用いて測定したところ、 pKA N-FSH9-3 AFMS705培養上清中に 14.8 IU/mLの濃度で発現していることを確認し た。なお、 pKAN- FSH9- 3 AFMS705株は pKAN- FSH9- 3 AFMS705の株名で、平成 1 6年 8月 10日付けで独立行政法人産業技術総合研究所 特許生物寄託センター (茨 城県つくば巿東 1丁目 1番地 1号中央第 6)に FERM BP-10086として寄託されている。 実施例 2 [0208] The obtained strain was suspended in 15 mL of serum-free medium at a concentration of 3.0 X 10 5 cells / mL, seeded on a 12 mL flask, and cultured. After 3 days of culture, the culture supernatant was collected, and the amount of FSH contained in the supernatant was measured using HumanFSH ELISA Kit (manufactured by Antigenix America), and 14.8 in pKA N-FSH9-3 AFMS705 culture supernatant. It was confirmed that it was expressed at a concentration of IU / mL. The pKAN-FSH9-3 AFMS705 strain is the pKAN-FSH9-3 AFMS705 share name, and the National Institute of Advanced Industrial Science and Technology (AIST) It is deposited as FERM BP-10086 at 1-chome, 1-no. Example 2
[0209] GDP-マンノースを GDP- 4-ケト, 6-デォキシ- GDP-マンノースに変換する脱水反応 を触媒する酵素の遺伝子が発現して!/ヽな ヽ細胞株の取得 [0209] Dehydration to convert GDP-mannose to GDP-4-keto, 6-deoxy- GDP-mannose The gene for the enzyme that catalyzes the expression of!
1.レクチン耐性 CHO/DG44株の取得  1. Acquisition of lectin resistant CHO / DG44 strain
CHO/DG44細胞（Proc. Natl. Acad. Sci. USA, 77, 4216 (1980))を、 IMDM— FBS(10 )- HT(1)培地 [ゥシ胎児血清 (FBS) (インビトロジ ン社製）を 10%、 HT supplement (ィ ンビトロジェン社製)を 1倍濃度で含む IMDM培地 (インビトロジェン社製) ]にて接着培 養用フラスコ 75cm 2 (グライナ一社製）中で培養し、コンフルェント直前まで増殖させた 。 5mLのダルベッコ PBS (以下、 PBSと表記する）（インビトロジェン社製）にて細胞を洗 浄後、 PBSで希釈した 0.05%トリプシン (インビトロジェン社製）を 1.5mL添カロして 37°C にて 5分間放置し、細胞を培養器底面から剥離させた。剥離させた細胞を通常の細 胞培養で行われる遠心分離操作により回収し、 1 X 105細胞/ mLの密度になるように I MDM-FBS(10)-HT(1)培地を添カ卩して懸濁後、未添カ卩又は 0.1 μ g/mLのアルキル化 剤である MNNG (シグマ社製）を添カ卩した。 COインキュベーター (TABAI製)内で 37°C CHO / DG44 cells (Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)), IMDM—FBS (10) -HT (1) medium [Ushi Fetal Serum (FBS) (Invitrogen) In IMDM medium (Invitrogen)] containing 10% HT supplement (Invitrogen) at a 1-fold concentration in an incubation culture flask 75cm 2 (Grainer) and proliferate until just before confluence I let you. After washing the cells with 5 mL Dulbecco's PBS (hereinafter referred to as PBS) (Invitrogen), add 1.5 mL of 0.05% trypsin (Invitrogen) diluted with PBS at 37 ° C. The cells were allowed to stand for minutes, and the cells were detached from the bottom of the incubator. The detached cells are collected by centrifugation performed in normal cell culture, and supplemented with IMDM-FBS (10) -HT (1) medium to a density of 1 X 10 5 cells / mL. Then, after suspension, MNNG (manufactured by Sigma), which was not added or 0.1 μg / mL alkylating agent, was added. 37 ° C in a CO incubator (made by TABAI)
2  2
にて 3日間放置後、培養上清を除き、再び上述した操作と同様の操作で細胞を洗浄 、剥離、回収し、 IMDM-FBS(IO)- HT(1)培地に懸濁後、接着培養用 96穴プレート (旭 テクノグラス社製)に 1000細胞/ゥエルの密度で播種した。各ゥエルには培地中終濃度 で Img/mLのレンズマメレクチン (Lens culinaris agglutinin;以「""、 Lし Aと表 己、 Vector 社製)を添加した。 COインキュベータ内で 37°Cにて 2週間培養後、出現したコロニー  After leaving for 3 days, remove the culture supernatant and wash, peel and collect the cells again in the same manner as described above, then suspend in IMDM-FBS (IO) -HT (1) medium and adhere to culture. A 96-well plate (Asahi Techno Glass Co., Ltd.) was seeded at a density of 1000 cells / well. Each well was supplemented with Img / mL lentil lectin (Lens culinaris agglutinin; hereinafter "", L and A, from Vector, Inc.) at 37 ° C in a CO incubator. Colonies that appeared after weekly culture
2  2
をレクチン耐性 CHO/DG44細胞株として取得した。  Was obtained as a lectin resistant CHO / DG44 cell line.
[0210] 2.取得したレクチン而ォ性 CHO/DG44細胞株の GDP-マンノース 4, 6-デヒドラターゼ mRNAの定量 [0210] 2. Quantification of GDP-mannose 4, 6-dehydratase mRNA in the obtained lectin metabolite CHO / DG44 cell line
前項で取得した各レクチン耐性 CHO/DG44細胞株における、 GDP-マンノースを G DP-4-ケト, 6-デォキシ- GDP-マンノースに変換する脱水反応を触媒する酵素である GDP-マンノース 4, 6-デヒドラターゼの発現量を、 RT-PCR法を用いて以下の様に解 祈した。  GDP-mannose 4, 6-, an enzyme that catalyzes the dehydration reaction of converting GDP-mannose into GDP-4-keto, 6-deoxy- GDP-mannose in each lectin-resistant CHO / DG44 cell line obtained in the previous section The expression level of dehydratase was calculated using the RT-PCR method as follows.
[0211] (1)レクチン耐性 CHO/DG44細胞株からの RNA調製と一本鎖 cDNAの調製  [0211] (1) Preparation of RNA from lectin-resistant CHO / DG44 cell line and preparation of single-stranded cDNA
親株である CHO/DG44細胞、および本実施例の 1項で取得した各レクチン耐性 CH 0/DG44細胞株それぞれ 1 X 107細胞より、 RNeasy Protect Mini kit (キアゲン社製)を 用いて、添付の使用説明書に従って RNAを調製した。続いて、 SUPER SCRIPT First -Strand synthesis system for RT- PC R (インビトロジェン社製）を用い、添付の使用説 明書に従って各 RNA5 /Z gより Lの反応液中にて一本鎖 cDNAを合成した。 Using the RNeasy Protect Mini kit (Qiagen) from the parental CHO / DG44 cells and each lectin-resistant CH 0 / DG44 cell line obtained in section 1 of this example, 1 X 10 7 cells, RNA was prepared according to the instructions for use. Next, SUPER SCRIPT First -Strand synthesis system for RT-PC R (manufactured by Invitrogen) was used to synthesize single-stranded cDNA from each RNA5 / Zg in a reaction solution of L according to the attached instruction manual.
[0212] (2) RT- PCR法を用いた β -ァクチン遺伝子の発現量解析 [0212] (2) Expression analysis of β-actin gene using RT-PCR
本項の (1)で作製した各細胞株由来の一本鎖 cDNAの品質を確かめる目的で、 /3 - ァクチン cDNA力 PCR法によって増幅されるかを、以下の様に検討した。  For the purpose of confirming the quality of single-stranded cDNA derived from each cell line prepared in (1) of this section, we examined whether it can be amplified by the / 3-actin cDNA force PCR method as follows.
本項の (1)で作製した各細胞株由来の一本鎖 cDNA 0.5 Lを铸型として含む 20 L の反応液 [1 X EX Taq Buffer (宝酒造社製）、 0.2mMの dNTP's、 0.5単位の EX Taq pol ymerase (宝酒造社製）、 0.5 μ Μの合成オリゴ DNAプライマー（配列番号 26、 27) ]を調 製し、 DNAサーマルサイクラ一 480 (パーキンエルマ一社製）を用いて、 94°Cにて 5分 間加熱した後、 94°Cにて 1分間、 68°Cにて 2分間のサイクルを 14サイクル行なった。上 記の該 PCR反応液 10 Lをァガロース電気泳動した後、サイバーグリーン (BMA社製 )を用いて DNA断片を染色し、予想される約 800bpの DNA断片量を Fluorlmager SI (モ レキユラ一ダイナミクス社製)を用いて測定した。その結果、調製したいずれの細胞株 由来の一本鎖 cDNAを用いても、同程度の β -ァクチンの発現を検出することができ た。  20 L reaction solution containing 0.5 L of single-stranded cDNA derived from each cell line prepared in (1) of this section as a cage [1 X EX Taq Buffer (Takara Shuzo), 0.2 mM dNTP's, 0.5 units EX Taq pol ymerase (Takara Shuzo), 0.5 μΜ synthetic oligo DNA primer (SEQ ID NO: 26, 27)] was prepared, and 94 ° C using DNA thermal cycler 480 (Perkin Elma) After heating for 5 minutes at 94 ° C, 14 cycles of 94 ° C for 1 minute and 68 ° C for 2 minutes were performed. After 10 L of the above PCR reaction solution was subjected to agarose electrophoresis, the DNA fragment was stained using Cyber Green (manufactured by BMA), and the expected DNA fragment amount of about 800 bp was obtained from Fluorlmager SI (Molecular Dynamics). ). As a result, the same level of β-actin expression could be detected using the single-stranded cDNA derived from any cell line prepared.
[0213] (3) RT-PCR法を用いた GDP-マンノース 4,6-デヒドラターゼ遺伝子の発現量解析 次に、本項 (1)で取得したそれぞれのレクチン耐性 CHO/DG44細胞株の GDP-マン ノース 4,6-デヒドラターゼ遺伝子の発現量解析を行った。 00?-マンノース4,6-デヒド ラターゼ遺伝子の cDNAを PCR法によって増幅するために、配列番号 9で示される CH 0細胞由来の GDP-マンノース 4,6-デヒドラターゼの cDNA配列より、配列番号 28で示 される塩基配列を有する 26merの合成オリゴ DNAプライマーと、配列番号 29で示され る塩基配列を有する 28merの合成オリゴ DNAプライマーを作製した。続いて、本項 (1) で作製した各細胞株由来の一本鎖 cDNA 0.5 Lを铸型として含む 20 Lの反応液 [ 1 X EX Taq Buffer (宝酒造社製）、 0.2mMの dNTP mixtureゝ 0.5単位の Ex Taqpolymer ase (宝酒造社製）、 0.5 μ Μの配列番号 28と 29の合成 DNAプライマー]を調製し、 DNA サーマルサイクラ一 480 (パーキンエルマ一社製）を用いて、 94°Cにて 5分間加熱した 後、 94°Cにて 1分間、 68°Cにて 2分間のサイクルを 30サイクル行なった。上記の該 PCR 反応液 10 Lをァガロース電気泳動した後、サイバーグリーン (BMA社製）を用いて D NA断片を染色し、予想される約 430bpの DNA断片量を Fluorlmager SI (モレキュラー ダイナミクス社製)を用いて測定した。その結果、取得したレクチン耐性 CHO/DG44 細胞株の中に、 GDP-マンノース 4,6-デヒドラターゼ遺伝子の発現が観察されない細 胞株が存在することを確認した。この GDP-マンノース 4,6-デヒドラターゼ遺伝子の発 現が観察されない株を CHO SM株と名付けた。なお、取得した CHO SM株の各種レク チンに対する耐性を調べたところ、 CHO SM株は、 LCAが認識する糖鎖構造と同じ糖 鎖構造を認識するレクチン、すなわち、 N-グリコシド結合糖鎖還元末端の N-ァセチ ルダルコサミン残基の 6位とフコースの 1位が a結合で付加された糖鎖構造を認識す る他のレクチンに対しても耐性を示した。具体的には、終濃度 lmg/mLのエンドゥマメ レクチン（Pisum sativum Agglutinin ;以下、 PSAと表記、 Vector社製）が添カ卩された培 地、あるいは終濃度 lmg/mLのヒイロチヤワンタケレクチン（Aleuria aurantia Lectin ;以 下、 AALと表記、 Vector社製）が添加された培地でも耐性を有していた。 [0213] (3) Expression level analysis of GDP-mannose 4,6-dehydratase gene using RT-PCR method Next, GDP-Mann of each lectin resistant CHO / DG44 cell line obtained in (1) of this section. The expression level of the north 4,6-dehydratase gene was analyzed. In order to amplify the cDNA of 00? -Mannose 4,6-dehydratase gene by PCR, the cDNA sequence of GDP-mannose 4,6-dehydratase derived from CH 0 cell shown in SEQ ID NO: 9 A 26-mer synthetic oligo DNA primer having the nucleotide sequence shown and a 28-mer synthetic oligo DNA primer having the nucleotide sequence shown in SEQ ID NO: 29 were prepared. Next, 20 L reaction solution containing 0.5 L of single-stranded cDNA derived from each cell line prepared in this section (1) as a cage [1 X EX Taq Buffer (Takara Shuzo), 0.2 mM dNTP mixture ゝPrepare 0.5 units of Ex Taqpolymerase (Takara Shuzo Co., Ltd.), 0.5 μΜ of synthetic DNA primer of SEQ ID NOS: 28 and 29, and use DNA thermal cycler 480 (Perkin Elma Co., Ltd.) at 94 ° C. After heating for 5 minutes, 30 cycles of 94 ° C for 1 minute and 68 ° C for 2 minutes were performed. After 10 L of the above PCR reaction solution was subjected to agarose electrophoresis, D was added using Cyber Green (manufactured by BMA). The NA fragment was stained, and the expected DNA fragment amount of about 430 bp was measured using Fluorlmager SI (manufactured by Molecular Dynamics). As a result, it was confirmed that there was a cell line in which the expression of the GDP-mannose 4,6-dehydratase gene was not observed in the obtained lectin-resistant CHO / DG44 cell line. The strain in which the expression of the GDP-mannose 4,6-dehydratase gene was not observed was named CHO SM strain. When the resistance of the obtained CHO SM strain to various lectins was examined, the CHO SM strain was found to be a lectin that recognizes the same sugar chain structure as that recognized by LCA, that is, the N-glycoside-linked sugar chain reducing end. It was also resistant to other lectins that recognize sugar chain structures in which the N-acetyldarcosamine residue at position 6 and fucose at position 1 are attached by an a bond. Specifically, a culture medium supplemented with lum / mL endumame lectin (Pisum sativum Agglutinin; hereinafter referred to as PSA, manufactured by Vector) or a leek / mL high-growth bamboo lectin (final concentration lmg / mL). Aleuria aurantia Lectin; hereinafter referred to as AAL (manufactured by Vector)) was also resistant.
3. GDP-マンノースを GDP- 4-ケト, 6-デォキシ- GDP-マンノースに変換する脱水反 応を触媒する酵素の遺伝子が発現して!/ヽな ヽ細胞株のゲノム解析  3. Gene analysis of an enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy- GDP-mannose!
CHO/DG44細胞、および前項で取得した CHO SM株を IMDM- FBS(IO)- HT(1)培地 を用いて接着細胞培養用 T75フラスコ（グライナ一社製)でコンフルェントに到達する 直前まで培養した後、文献 [ヌクレイック'アシッド'リサーチ (Nuccleic Acid Research), 3, 2303, (1976)]に記載の方法従ってゲノム DNAを調製し、取得したゲノム DNAを TE- RNase緩衝液（pH8.0) [10mmol/lTris— HC1、 lmmol/1 EDTA、 200 μ g/ml RNase A] 30 0 1に一晩溶解させた。上記で調製したゲノム DNA 12 gを、 3種の異なる制限酵素 、 EcoRI (宝酒造社製）、 Hindlll (宝酒造社製）、 Bglll (宝酒造社製)でそれぞれ消化し 、エタノール沈殿法を用いて DNA断片を回収した後、 TE緩衝液 (pH8.0) [10mmol/l T ris- HC1、 lmmol/1 EDTA ] 20 μ 1に溶解し、 0.8%(w/v)ァガロースゲル電気泳動に供 した。泳動後、文献 [プロシーディンダス 'ォブ ·ザ'ナショナル 'アカデミー'ォブ 'サイ エンス (Proc. Natl. Acad. Sci. USA), 76, 3683,(1979)]に記載の方法に従い、ナイロン 膜へゲノム DNAを転写した。転写終了後、ナイロン膜に対して 80°Cで 2時間の熱処理 を行った。次に、ナイロンメンブレンに転写されたゲノム DNAの品質を確認する目的 で、細胞株を問わずゲノム中に均等に存在すると考えられる a 1 ,6-フコシルトランスフ エラーゼ（FUT8)遺伝子をプローブとしたサザンハイブリダィゼーシヨンを行った。 FU T8遺伝子を検出するためのプローブは以下のように調製した。まず、 WO02/31140の 実施例 11に記載のマウス FUT8cDNAを含むプラスミド m!FUT8- pCR2.1 10 μ gを 50 μ しの M buffer (宝酒造社製）に溶解し、制限酵素 Hindlll (宝酒造社製)で一晩消化し た後、反応液を H buffer (宝酒造社製）に置換し、制限酵素 EcoRI (宝酒造社製)でさ らに一晩消化反応を行った。反応終了後、該反応液を 2%ァガロース電気泳動に供 し、 FUT8遺伝子ェクソン 2を含む 156bpの EcoRI-Hindlll断片を精製した。得られた DN A断片 25ngに対し、 [ a -32P]dCTP 1.75MBqおよび Megaprime DNA labelingsystem, dCTP (アマシャムバイオサイエンス社製)を用いて放射標識した。次に、ハイブリダィ ゼーシヨンを以下のように行った。まず、上記ナイロン膜をローラーボトルへ封入し、 ハイブリダィゼーシヨン液 [4 X SSPE、 5 X Denhaldt，s液、 0.5%(w/v)SDS、 0.1mg/mLサ ケ精子 DNA] 15mLをカ卩えて 65°Cで 3時間のプレハイブリダィゼーシヨンを行った。次 に32 P標識したプローブ DNAを熱変性してボトルへ投入し、 65°Cでー晚加温した。ノヽ イブリダィゼーシヨン後、ナイロン膜を 2 X SSC- 0.1%(w/v) SDS 50mLに浸漬し、 65°Cで 15分間加温した。上記の洗浄操作を 2回繰り返した後、膜を 0.2 X SSC-0.1 %(w/v) S DS 50mLに浸漬し、 65°Cで 15分間加温した。洗浄後、ナイロン膜を X線フィルムへ- 80 °Cでニ晚暴露し現像した。現像後、ナイロン膜をストリッピング液 [1 %SDS、 0.1 X SSC ]中で煮沸することにより、プローブを剥離させ、再度異なるプローブでのハイブリダィ ゼーシヨンに供することとした。上記の方法により、 CHO/DG44株および CHO SM株 いずれのゲノム DNAにおいても、 FUT8遺伝子ェクソン 2に特異的な断片が検出され た。以上の結果よりナイロン膜上に転写された CHO SM株および CHO/DG44株由来 のゲノム DNAは等しい品質を有して!/、ることが示された。 CHO / DG44 cells and the CHO SM strain obtained in the previous section were cultured using IMDM-FBS (IO) -HT (1) medium in a T75 flask for adherent cell culture (manufactured by Grainer) until just before reaching confluence. Later, genomic DNA was prepared according to the method described in the literature [Nuccleic Acid Research, 3, 2303, (1976)], and the obtained genomic DNA was added to TE-RNase buffer (pH 8.0) [ 10 mmol / l Tris—HC1, lmmol / 1 EDTA, 200 μg / ml RNase A] 30 0 1 was dissolved overnight. 12 g of the genomic DNA prepared above was digested with 3 different restriction enzymes, EcoRI (Takara Shuzo), Hindlll (Takara Shuzo), and Bglll (Takara Shuzo), respectively, and the DNA fragment was obtained using ethanol precipitation. After being collected, it was dissolved in TE buffer (pH 8.0) [10 mmol / l Tris-HC1, lmmol / 1 EDTA] 20 μl and subjected to 0.8% (w / v) agarose gel electrophoresis. After electrophoresis, nylon was produced according to the method described in the literature [Procedinas 'Ob The National' Academy 'Ob' Science (Proc. Natl. Acad. Sci. USA), 76, 3683, (1979)]. Genomic DNA was transferred to the membrane. After the transfer, the nylon membrane was heat treated at 80 ° C for 2 hours. Next, for the purpose of confirming the quality of the genomic DNA transferred to the nylon membrane, it is considered to exist evenly in the genome regardless of the cell line. Southern hybridization was performed using the erase (FUT8) gene as a probe. A probe for detecting the FU T8 gene was prepared as follows. First, 10 μg of plasmid m! FUT8-pCR2.1 containing mouse FUT8 cDNA described in Example 11 of WO02 / 31140 was dissolved in 50 μl M buffer (Takara Shuzo), and restriction enzyme Hindlll (Takara Shuzo) After overnight digestion, the reaction solution was replaced with H buffer (Takara Shuzo), and the digestion reaction was further performed overnight with the restriction enzyme EcoRI (Takara Shuzo). After completion of the reaction, the reaction solution was subjected to 2% agarose electrophoresis, and a 156 bp EcoRI-Hindlll fragment containing FUT8 gene exon 2 was purified. The obtained DNA fragment (25 ng) was radiolabeled using [a- 32 P] dCTP 1.75 MBq and Megaprime DNA labeling system, dCTP (Amersham Biosciences). Next, hybridization was performed as follows. First, the above nylon membrane is sealed in a roller bottle, and 15 mL of a hybridization solution [4 X SSPE, 5 X Denhaldt, s solution, 0.5% (w / v) SDS, 0.1 mg / mL salmon sperm DNA] A prehybridization was performed at 65 ° C for 3 hours. Next, the 32 P-labeled probe DNA was heat denatured, put into a bottle, and heated at 65 ° C. After the hybridization, the nylon membrane was immersed in 50 mL of 2 X SSC-0.1% (w / v) SDS and heated at 65 ° C. for 15 minutes. After the above washing operation was repeated twice, the membrane was immersed in 50 mL of 0.2 X SSC-0.1% (w / v) S DS and heated at 65 ° C for 15 minutes. After washing, the nylon membrane was exposed to X-ray film at -80 ° C and developed. After development, the nylon membrane was boiled in a stripping solution [1% SDS, 0.1 X SSC] to peel off the probe and again subjected to hybridization with a different probe. By the above method, a fragment specific to FUT8 gene exon 2 was detected in the genomic DNA of both CHO / DG44 strain and CHO SM strain. From the above results, it was shown that the genomic DNAs derived from CHO SM strain and CHO / DG44 strain transcribed on nylon membrane have equal quality! /.
一方、 GMD遺伝子ェクソン 5に特異的なプローブを以下のように調製した。まず、公 知であるヒト GMDゲノム DNA配列（NCBIァクセッション番号 NT_034880)を基に、エタ ソン 5に特異的に結合するオリゴ DNAプライマー（配列番号 30および配列番号 31)を 5X十し 7  On the other hand, a probe specific for GMD gene exon 5 was prepared as follows. First, based on the known human GMD genomic DNA sequence (NCBI accession number NT_034880), 5X more oligo DNA primers (SEQ ID NO: 30 and SEQ ID NO: 31) that specifically bind to Etason 5 are added.
該領域は配列番号 9に記載の CHO GMD cDNA配列の塩基番号 346から塩基番号 5 38に相当する。次に、 WO02/31140の実施例 15に記載のプラスミド pAGE249GMDを 1 Ong含む 100 μ Lの反応液 [ExTaq buffer (宝酒造社製）、 0.2mmol/L dNTPs、 2.5 μ mol /L上記遺伝子特異的プライマー (配列番号 30および配列番号 31) ]を調製し、ポリメ ラーゼ連鎖反応 (PCR)を行った。 PCRは、 94°Cで 5分間の加熱の後、 94°Cで 1分間、 5 8°Cで 2分間、 72°Cで 3分間力 なる反応を 1サイクルとした 30サイクルの条件で行った 。 PCR後、反応液を 2%ァガロース電気泳動に供し、約 200bpの DNA断片を精製した 。得られた DNA断片 25ngに対し、 [ a -32P]dCTP 1.75MBqおよび Megaprime DNA lab eling system, dCTP (アマシャムバイオサイエンス社製 This region corresponds to nucleotide numbers 346 to 538 of the CHO GMD cDNA sequence shown in SEQ ID NO: 9. Next, the plasmid pAGE249GMD described in Example 15 of WO02 / 31140 is Prepare 100 μL reaction solution [ExTaq buffer (Takara Shuzo), 0.2 mmol / L dNTPs, 2.5 μmol / L above gene-specific primer (SEQ ID NO: 30 and SEQ ID NO: 31)] containing Polymerase chain Reaction (PCR) was performed. PCR was performed under conditions of 30 cycles of heating at 94 ° C for 5 minutes, followed by 1 cycle of reaction at 94 ° C for 1 minute, 58 ° C for 2 minutes, and 72 ° C for 3 minutes. . After PCR, the reaction solution was subjected to 2% agarose electrophoresis, and an approximately 200 bp DNA fragment was purified. For 25 ng of the obtained DNA fragment, [a- 32 P] dCTP 1.75 MBq and Megaprime DNA lab eling system, dCTP (manufactured by Amersham Biosciences)
)を用いて放射標識した。該プローブを上記で示したナイロン膜に対してノ、イブリダイ ゼーシヨンを行った。その結果、 CHO/DG44細胞由来のゲノム DNAでは GMD遺伝子 ェクソン 5の特異的断片が見出されたのに対し、 CHO SM株由来のゲノム DNAにおい ては GMD遺伝子ェクソン 5の特異的断片が全く検出されな力つた。以上の結果から C HO SM株は GMDをコードするゲノム領域のうち、少なくともェクソン 5を含む領域を欠 損した GMDノックアウト細胞であることが示された。  ). The probe was subjected to hybridization on the nylon membrane shown above. As a result, a specific fragment of GMD gene exon 5 was found in genomic DNA derived from CHO / DG44 cells, whereas a specific fragment of GMD gene exon 5 was completely detected in genomic DNA derived from CHO SM strain. The power was not. From the above results, it was shown that the C HO SM strain is a GMD knockout cell lacking at least the region containing exon 5 among the genomic region encoding GMD.
実施例 3  Example 3
[0216] GMDノックアウト細胞による卵胞刺激ホルモンの発現  [0216] Expression of follicle-stimulating hormone by GMD knockout cells
l .CHO SM株への FSH発現プラスミドの導入  l Introduction of FSH expression plasmid into CHO SM strain
実施例 2にて作製した CHO SM株に、実施例 1で作製したプラスミド pKAN-FSH o; βを導入した。これらの遺伝子導入は公知のエレクト口ポレーシヨン法 [サイトテクノロ ジー (Cytotechnology), 3, 133 (1990)]により以下の手順で行った。まず、プラスミド p KAN— FSH α β 30 μ gを NEBuffer3 (New England Biolabs社製) 20 μ Lと 100単位の制 限酵素 MluI(New England Biolabs社製）を含む 200 μ Lの反応液を調製し、 37°Cで 16 時間消化反応を行うことにより線状化した。反応後、該反応液に対しフ ノール/クロ 口ホルム抽出処理およびエタノール沈殿により精製を行い、線状ィ匕プラスミドを回収 した。  The plasmid pKAN-FSHo; β prepared in Example 1 was introduced into the CHO SM strain prepared in Example 2. These gene introductions were carried out by the following procedure using a known electoral position method [Cytotechnology, 3, 133 (1990)]. First, prepare a reaction solution of 200 μL containing 30 μg of plasmid p KAN- FSH α β 20 μL of NEBuffer3 (New England Biolabs) and 100 units of restriction enzyme MluI (New England Biolabs). It was linearized by digestion at 37 ° C for 16 hours. After the reaction, the reaction mixture was purified by phenol / chloroform extraction treatment and ethanol precipitation, and a linear plasmid was recovered.
[0217] 次に、実施例 2で取得した CHO SM株を K- PBS緩衝液 (137 mmol/L KC1、 2.7 mmol /L NaCl  [0217] Next, the CHO SM strain obtained in Example 2 was added to K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl).
、 8.1 mmol/L Na HPO、 1.5 mmol/L KH PO、 4.0mmol/L MgCl )に懸濁して 8 X 107 , 8.1 mmol / L Na HPO, 1.5 mmol / L KH PO, 4.0mmol / L was suspended in MgCl) 8 X 10 7
2 4 2 4 2  2 4 2 4 2
細胞/ mLとした。細胞懸濁液 200 L (1.6 X 106個)と上記の線状ィ匕プラスミド 9 gを 混和した後、細胞- DNA混和液の全量を Gene Pulser Cuvette (電極間距離 2 mm) (BI 0- RAD社製)へ移し、細胞融合装置 GenePulser (BIO- RAD社製）を用いてパルス電 圧 350 V、電気容量 250 Fの条件で遺伝子導入を行った。遺伝子導入を行ったのち 、細胞懸濁液を 10%ゥシ胎児血清 (Life Technologies社製)および 50 μ g/mL gentami cin (ナカライテスタ社製）を添カ卩した IMDM培地 (Life Technologies社製) 30 mLに懸濁 し、接着細胞培養 96ゥエルプレート (グライナ一社製) 3枚へ 100 L/ゥエルで播種し た。培養は 5%CO、 37°Cの条件下で行った。 Cells / mL. 200 L of cell suspension (1.6 x 10 6 ) and 9 g of the above linear plasmid After mixing, transfer the entire cell-DNA mixture to Gene Pulser Cuvette (distance between electrodes: 2 mm) (BI 0-RAD) and pulse voltage using cell fusion device GenePulser (BIO-RAD). Gene transfer was performed under conditions of 350 V and electric capacity of 250 F. After gene transfer, IMDM medium (Life Technologies) supplemented with 10% Ushi fetal serum (Life Technologies) and 50 μg / mL gentamicin (Nacalai Testa) was added to the cell suspension. ) Suspended in 30 mL, and seeded 3 adherent cell culture 96 well plates (manufactured by Grainer) at 100 L / well. The culture was performed under conditions of 5% CO and 37 ° C.
2  2
[0218] 2. 500 nM MTXffif性株の取得  [0218] 2. Acquisition of 500 nM MTXffif stock
前項で得た pKAN-FSH α β導入細胞を 6日間培養した後、培養上清を除去し、 10 %ゥシ胎  After culturing the pKAN-FSH α β-introduced cells obtained in the previous section for 6 days, the culture supernatant is removed and 10%
児透析血清、 50 μ g/mL gentamicinおよび 50 nM methotrexate (MTX:シグマ社製)を 添カ卩した IMDM培地を 100 μ L/ゥエルずつ添カ卩した。この培地交換作業を 3〜4日毎 に繰り返しながら 9日間の培養を行った。次いで、 10%ゥシ胎児透析血清、 g/m L gentamicinおよび 200 nMの MTXを添カ卩した IMDM培地を用いた培地交換作業を 同様に 3〜4日毎に繰り返しながら 18日間培養し、最終的に形成されたコロニーを 24 ゥエルプレート (シグマ社製)に植え替えた。さらに、 10%ゥシ胎児透析血清、 50 μ §/ mL gentamicinおよび 500 nMの MTXを添カ卩した IMDM培地を用いた培地交換作業を 3〜4日毎に繰り返し、適宜拡大しながら 19日間培養を行い、 500 nM MTX耐性株を 取得した。 IMDM medium supplemented with baby dialysis serum, 50 μg / mL gentamicin and 50 nM methotrexate (MTX: Sigma) was added at 100 μL / well. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Next, repeat the medium exchange operation using IMDM medium supplemented with 10% urine fetal dialyzed serum, g / m L gentamicin and 200 nM MTX in the same manner every 3 to 4 days. The colonies formed in 1 were replanted into 24 well plates (manufactured by Sigma). Furthermore, 10% © shea dialyzed fetal serum, 50 μ § / mL gentamicin and repeated medium exchange work using the IMDM medium添Ka卩a 500 nM of MTX to 3-4 days, the 19 day culture while appropriately enlarged To obtain a 500 nM MTX resistant strain.
[0219] 3. FSH高生産株の選別 [0219] 3. Selection of high production strains of FSH
前項で取得した複数の 500 nM MTX耐性株より、各 1.0 X 106細胞を 5 mLの 10%ゥ シ胎児透析血清、 50 μ g/mL gentamicinおよび 500 nMの MTXを添カ卩した IMDM培地 に懸濁し、 T25フラスコへ播種して培養を行った。培養 3日後に培養上清を回収し、上 清中に含まれる FSH量を Human FSH ELISA Kit(AntigenixAmerica社製)を用いて測 定した。方法は添付マニュアルに従った。その結果、得られた FSH産生株 pKAN-FS H2 GMDKOの培養上清中に 1.27IU/mLの濃度で発現していることを確認した。なお 、 pKAN-FSH2 GMDKO株は pKAN- FSH2 GMDKOの株名で、平成 16年 8月 10日付 けで独立行政法人産業技術総合研究所 特許生物寄託センター (茨城県つくば巿 東 1丁目 1番地 1号中央第 6)に FERMBP-10081として寄託されている。このようにして 作製した pKAN- FSH2 GMDKO株より得られた卵胞刺激ホルモンは、通常の CHO/D G44株で生産した卵胞刺激ホルモンに比較して血中半減期の延長が認められた。 実施例 4 A plurality of 500 nM MTX-resistant strain obtained in the previous section, 10% © shea dialyzed fetal serum each 1.0 X 10 6 cells 5 mL, in IMDM medium添Ka卩the 50 μ g / mL gentamicin and 500 nM of MTX It was suspended and seeded in a T25 flask for culture. After 3 days of culture, the culture supernatant was collected, and the amount of FSH contained in the supernatant was measured using Human FSH ELISA Kit (manufactured by Antigenix America). The method followed the attached manual. As a result, it was confirmed that the FSH-producing strain pKAN-FS H2 GMDKO was expressed at a concentration of 1.27 IU / mL in the culture supernatant. The pKAN-FSH2 GMDKO strain is the pKAN-FSH2 GMDKO stock name, and the National Institute of Advanced Industrial Science and Technology (AIST) It is deposited as FERMBP-10081 in East 1-chome, 1-No. 1 center 6). The follicle-stimulating hormone obtained from the pKAN-FSH2 GMDKO strain prepared in this way was found to have an increased blood half-life compared to the follicle-stimulating hormone produced by the normal CHO / D G44 strain. Example 4
[0220] 酵母による遺伝子組換え卵胞刺激ホルモンの発現  [0220] Expression of transgenic follicle-stimulating hormone by yeast
酵母には多くの種類が知られているが、組換え蛋白質を発現させる宿主としてしば しば用いられる代表的な酵母として、ピキア（Pichia)属とサッカロマイセス (Saccaromy ces)属の酵母が挙げられる。通常、これらの酵母が発現する組換え蛋白質に付加さ れる N-結合型糖鎖の主要な構造は、還元末端側のコア部分に 2残基の N-ァセチル ダルコサミンを有し、非還元末端側の分岐部分に 9個から数十個のマンノース残基と 、数個から十数個のマンノース 6-リン酸残基を有する、ノ、ィマンノース型糖鎖であるこ とが知られている（Yeastl^, 1191 (2002))。また、このような構造を有するハイマンノー ス型糖鎖は、しばしばノヽィパーマンノース型糖鎖とも呼ばれる。  Many types of yeast are known, but typical yeasts often used as hosts for expressing recombinant proteins include yeasts of the genera Pichia and Saccaromyces. . Normally, the main structure of N-linked sugar chains added to recombinant proteins expressed by these yeasts has a 2-residue N-acetyl darcosamine in the core part on the reducing end, and the non-reducing end side. It is known that this is a mannose-type sugar chain having 9 to several tens of mannose residues and several to several tens of mannose 6-phosphate residues in the branched portion (Yeastl ^ , 1191 (2002)). Further, a high mannose type sugar chain having such a structure is often called a no-permannose type sugar chain.
[0221] 以下に記載する実施例ではまず、主に付加される N-結合型糖鎖の構造として、ハ ィマンノース型糖鎖と複合型糖鎖の中間の構造である、ハイブリッド型糖鎖が主に付 カロされた卵胞刺激ホルモンを発現するピキア酵母株とサッカロマイセス酵母株の作 製方法について記載する。 [0221] In the examples described below, first, the structure of the N-linked sugar chain to be added is mainly a hybrid sugar chain, which is an intermediate structure between a high-mannose sugar chain and a complex sugar chain. The methods for producing Pichia yeast strains and Saccharomyces yeast strains expressing follicle-stimulating hormone that have been carotenized are described below.
1.ゲノム上に存在する PN01酵素遺伝子を破壊したピキア酵母株の作製 ピキア酵母株、たとえば Pichia pastoris GTS115株（インビトロジェン社製）などのゲノ ム DNAを铸型とし、 PCR法によって、ピキア酵母の PNOKphosphomannosylationof N- linked oligosaccharides 1)遺伝子（GenBankァクセッションナンバー： AB099514)の翻 訳領域全長の配列を増幅させる。増幅させた約 3200塩基長の PN01遺伝子配列は、 その 5，末端側半分の配列を、酵母由来の orotidine- 5'- phosphate decarboxylase (UR A3)遺伝子（GenBankァクセッションナンバー： AF321098)に置換した後、 pCR2.1- TO POベクター（インビトロジェン社製）などのベクターに挿入することにより、 PN01遺伝 子破壊用のプラスミドを作製する。次に、このプラスミド 100 gを制限酵素で線状ィ匕し た後、 PichiaExpressionKit (インビトロジェン社製）記載のエレクト口ポレーシヨン法によ つて、たとえば GTS115株などのピキア酵母に安定的に遺伝子導入を行う。次に、遺 伝子導入した酵母を、ゥラシルを欠損させた YPD培地 (インビトロジェン社製)を用い て室温にて培養し、増殖してきた各コロニーカゝらゲノム DNAを抽出する。次に、このゲ ノム DNAを铸型とした PCR法によって、酵母 PN01遺伝子座の配列を増幅させること により、相同組換えによって PN01遺伝子座が破壊された酵母クローンを選択する。 上記の方法により、ピキア酵母が発現する主要な N-結合型糖鎖の構造を、還元末端 側のコア部分に 2残基の N-ァセチルダルコサミンを有し、非還元末端側に 9個から数 十個のマンノース残基が結合した構造を有したハイマンノース型糖鎖に改変すること ができる。 1. Preparation of Pichia yeast strains that have disrupted the PN01 enzyme gene present on the genome Pichia yeast strains such as Pichia pastoris GTS115 (manufactured by Invitrogen Corp.) are used as genomic DNA, and PCR is used to perform PNOKphosphomannosylationof Pichia yeast. N-linked oligosaccharides 1) Amplify the entire translation region of the gene (GenBank accession number: AB099514). The amplified PN01 gene sequence with a length of about 3200 bases was replaced with the yeast orotidine-5'-phosphate decarboxylase (UR A3) gene (GenBank accession number: AF321098). Then, a plasmid for PN01 gene disruption is prepared by inserting into a vector such as pCR2.1-TO PO vector (Invitrogen). Next, 100 g of this plasmid is linearized with a restriction enzyme, and then the gene is stably introduced into a Pichia yeast such as GTS115 strain, for example, by the electoral position method described in PichiaExpressionKit (manufactured by Invitrogen). . Next, the remains The transferred yeast is cultured at room temperature in YPD medium (Invitrogen) deficient in uracil, and genomic DNA is extracted from each of the grown colonies. Next, a yeast clone in which the PN01 locus is disrupted by homologous recombination is selected by amplifying the yeast PN01 locus sequence by PCR using this genomic DNA as a saddle type. By the above method, the structure of the main N-linked sugar chain expressed in Pichia yeast has 9 residues on the non-reducing end side, with 2 residues of N-acetylyldarcosamine in the core part on the reducing end side. To a high mannose sugar chain having a structure in which several tens of mannose residues are bonded.
2.ゲノム上に存在する a _1,6_マンノース転移酵素遺伝子を破壊したピキア酵母株 の  2.Pichia yeast strains that have disrupted the a_1,6_mannose transferase gene present on the genome
作製 Production
ピキア酵母株、たとえば Pichia pastoris X- 33株（インビトロジェン社製）などのゲノム DNAを铸型とし、 PCR法によって、ピキア酵母の α - 1,6-マンノース転移酵素（OCH1 )遺伝子（GenBankァクセッションナンバー： AF540063)を増幅させる。増幅させた約 2 800塩  Pichia yeast strains, such as Pichia pastoris X-33 (manufactured by Invitrogen), are used in a vertical form, and by PCR, Pichia yeast α-1,6-mannose transferase (OCH1) gene (GenBank accession) Number: AF540063) is amplified. Amplified about 2 800 salt
基長の OCH1遺伝子配列は、その 5 '末端側半分の配列を、酵母由来の orotidine-5' -phosphate decarboxylase (URA3)遺伝子（GenBankァクセッションナンバー： AF3210 98)に置換した後、 pCR2.1-TOPOベクター（インビトロジェン社製）などのベクターに 挿入することにより、 OCH1遺伝子破壊用ベクターが作製される。次に、このベクター 100 gを制限酵素 Sfil (ニューイングランドバイオラブズ社製)で線状ィ匕した後、 Pichia Expression Kit (インビトロジェン社製）記載のエレクト口ポレーシヨン法によって、ピキ ァ酵母株、例えば前項に記載した PN01遺伝子破壊株や、 Pichia pastorisJC308株な どに対し、安定的な遺伝子導入を行う。次に、遺伝子導入した酵母を、ゥラシルを欠 損させた YPD培地 (インビトロジェン社製)を用いて室温にて培養し、増殖してきた各 コロニー力もゲノム DNAを抽出する。次に、このゲノム DNAを铸型とした PCR法によつ て、酵母 OCH1遺伝子座の配列を増幅させることにより、相同組換えによって OCH1 遺伝子座が破壊された酵母クローン株を選択する。上記の方法により、ピキア酵母が 発現する主要な N-結合型糖鎖の構造を、還元末端側のコア部分に 2残基の N-ァセ チルダルコサミンを有し、非還元末端側に 8個のマンノース残基が結合した構造を有 した Man8型ハイマンノース型糖鎖に改変することができる。 The OCH1 gene sequence of the base length was replaced with the yeast's orotidine-5'-phosphate decarboxylase (URA3) gene (GenBank accession number: AF3210 98) after replacing the 5 'terminal half sequence with pCR2.1 -A vector for disrupting the OCH1 gene is prepared by inserting into a vector such as TOPO vector (Invitrogen). Next, 100 g of this vector was linearly digested with the restriction enzyme Sfil (manufactured by New England Biolabs), and then the yeast yeast strain, for example, the above-mentioned item was obtained by the electoral position method described in Pichia Expression Kit (manufactured by Invitrogen). Stable gene transfer is carried out to the PN01 gene disruption strain described in 1) or the Pichia pastoris JC308 strain. Next, the transfected yeast is cultured at room temperature in YPD medium (Invitrogen) lacking uracil, and genomic DNA is extracted from each colony that has grown. Next, a yeast clonal strain in which the OCH1 locus is destroyed by homologous recombination is selected by amplifying the yeast OCH1 locus sequence by PCR using this genomic DNA as a saddle type. By the above method, the structure of the major N-linked sugar chain expressed in Pichia yeast is converted into a 2-residue N-case in the core at the reducing end. It can be modified to a Man8 type high mannose type sugar chain having tildarcosamine and having a structure in which 8 mannose residues are bonded to the non-reducing end.
[0223] 3.組換えキメラ型 a -1,2-マンノシダーゼ遺伝子を導入したピキア酵母株の作製 線虫（Caenorhabditis elegans)から RNeasy Mini Kit (キアゲン社製）を用いて全 RNA を抽出し、次にこの RNAを铸型として Superscript™first- strandcDNA synthesis kit ( インビトロジェン社製）を用いて first- strand cDNAを調製する。次に、この cDNAを铸 型 [0223] 3. Preparation of Pichia yeast strain into which recombinant chimera type a-1,2-mannosidase gene has been introduced Total RNA was extracted from Caenorhabditis elegans using RNeasy Mini Kit (Qiagen). First, first-strand cDNA is prepared using Superscript ™ first-strand cDNA synthesis kit (manufactured by Invitrogen) using this RNA as a cage. Next, this cDNA
とし、特異的プライマーと KODポリメラーゼ (東洋紡績社製)を用いた PCRを行うことに より、線虫 α - 1,2-マンノシダーゼ（GenBankァクセッションナンバー： NM_073594)の 活性ドメインをコードする cDNAを特異的に増幅させる。増幅させた cDNAは、その 5 ' 末端側に、酵母の αマンノシダーゼ（MNS1)遺伝子（GenBankァクセッションナンパ 一： M63598)のリーダーペプチドをコードする cDNA配列を連結した後に、酵母用の 発現ベクター pPICZ (インビトロジェン社製)などのベクターに挿入し、酵母の小胞体 内にひ -1,2-マンノシダーゼを発現させるベクターを作製する。次にこのベクターを、 前項に記載した PN01遺伝子と OCH1遺伝子の両方の遺伝子を相同組換えで破壊し たピキア酵母株に対し、エレクト口ポレーシヨン法により安定的に導入する。遺伝子導 入後の酵母は、ゥラシルを欠損しゼォシン (インビトロジェン社製)を含有する YPD培 地 (インビトロジェン社製)で室温にて培養し、増殖してきた各コロニーから全 RNAを 抽出する。次に、この全 RNA力も調製した first-strand cDNAを铸型とした PCR法によ つて、組換えキメラ型 α - 1,2-マンノシダーゼの発現が認められた酵母クローン株を選 択する。上記の方法により、ピキア酵母が発現する主要な Ν-結合型糖鎖の構造を、 還元末端側のコア部分に 2残基の Ν-ァセチルダルコサミンを有し、非還元末端側に 5 個のマンノース残基が結合した構造を有した Man5型ハイマンノース型糖鎖に改変で きる。  The cDNA encoding the active domain of nematode α-1,2-mannosidase (GenBank accession number: NM_073594) was obtained by PCR using specific primers and KOD polymerase (Toyobo Co., Ltd.). Amplify specifically. The amplified cDNA is ligated to the 5 'end of the cDNA sequence encoding the yeast α-mannosidase (MNS1) gene (GenBank accession number: M63598) leader peptide, and then the yeast expression vector pPICZ. (Invitrogen) and other vectors are inserted into the yeast endoplasmic reticulum for expression of -1,2-mannosidase. Next, this vector is stably introduced into the Pichia yeast strain in which both the PN01 gene and the OCH1 gene described in the previous section have been disrupted by homologous recombination by the electopore method. The yeast after gene transfer is cultured at room temperature in a YPD medium (Invitrogen) containing zeosin (Invitrogen) and lacking uracil, and total RNA is extracted from each of the grown colonies. Next, a yeast clonal strain in which expression of the recombinant chimeric α-1,2-mannosidase is observed is selected by PCR using the first-strand cDNA prepared with this total RNA strength as a saddle type. By the above method, the structure of the main Ν-linked glycan expressed in Pichia yeast has 2 residues Ν-acetildarcosamine in the core part on the reducing end side and 5 on the non-reducing end side. It can be modified to a Man5 type high mannose type sugar chain having a structure in which the mannose residues are bound.
[0224] 4.組換え UDP-N-ァセチルダルコサミントランスポーター遺伝子を導入したピキア 酵母株の作製  [0224] 4. Production of Pichia yeast strains into which recombinant UDP-N-acetylcylcosamine transporter gene was introduced
酵母（Kluyveromyces lactis)から RNeasy Mini Kit (キアゲン社製）を用いて全 RNAを 抽出し、次にこの RNAを铸型として Superscript™first- strand cDNA synthesis kit (イン ビトロジェン社製)を用いて cDNAを調製する。次に、この cDNAを铸型とし、特異的プ ライマーと KODポリメラーゼ (東洋紡績社製)を用 V、た PCRを行うことにより、酵母 UDPTotal RNA is extracted from yeast (Kluyveromyces lactis) using the RNeasy Mini Kit (Qiagen), and then this RNA is used as a cocoon for Superscript ™ first-strand cDNA synthesis kit (in CDNA is prepared using Vitrogen). Next, this cDNA is used as a saddle, and PCR is performed using a specific primer and KOD polymerase (Toyobo Co., Ltd.).
-N-ァセチルダルコサミントランスポーターの翻訳領域全長をコードする cDNA(GenB ankァクセッションナンバー： AF106080)を特異的に増幅させる。次に、増幅させた約A cDNA encoding the entire translation region of the -N-acetylcylcosamine transporter (GenBank accession number: AF106080) is specifically amplified. Next, the amplified about
3700塩基長の cDNAを、酵母用の発現ベクター pPIC3.5K (インビトロジェン社製）など のベクターのアルコールォキシゲナーゼプロモーター配列の下流に位置する制限酵 素 EcoRI切断部位と Not I切断部位の間に挿入し、酵母のゴルジ体内に UDP-N-ァセ チルダルコサミントランスポーターを発現させるベクターを作製する。次にこのべクタ 一を、前項に記載した、 α -1,2-マンノシダーゼ遺伝子を導入したピキア酵母株に対 し、エレクト口ポレーシヨン法により安定的に導入する。遺伝子導入後の酵母は、薬剤 G418 (ナカライテスタ社製)を含有する YPD培地で室温にて培養し、増殖してきた各 コロニー力ゝら全 RNAを抽出する。次に、この全 RNAから調製した cDNAを铸型とした Ρ CR法によって、組換え UDP-N-ァセチルダルコサミントランスポーターの発現が認め られた酵母クローン株を選択する。 A 3700 base-long cDNA is inserted between the restriction enzyme EcoRI and Not I cleavage sites located downstream of the alcohol oxygenase promoter sequence in vectors such as the yeast expression vector pPIC3.5K (Invitrogen). The vector is inserted to express the UDP-N-acetyldarcosamine transporter in the Golgi apparatus of yeast. Next, this vector is stably introduced into the Pichia yeast strain into which the α-1,2-mannosidase gene has been introduced as described in the previous section by the electopore method. The yeast after gene introduction is cultured at room temperature in a YPD medium containing the drug G418 (manufactured by Nacalai Testa), and total RNA is extracted from each of the grown colonies. Next, a yeast clonal strain in which expression of the recombinant UDP-N-acetylyldarcosamine transporter is observed is selected by the CR method using the cDNA prepared from this total RNA as a cage.
5.組換えキメラ型 N-ァセチルダルコサミン転移酵素- 1遺伝子を導入したピキア酵 母株の作製  5. Production of a Pichia fermentation mother strain into which a recombinant chimeric N-acetyldylcosamine transferase-1 gene has been introduced
ヒト肝臓 cDNA (クロンテック社製)を铸型とし、特異的プライマーと KODポリメラーゼ (東洋紡績社製)を用いた PCRを行うことにより、 N-ァセチルダルコサミン転移酵素- 1 ( GenBankァクセッションナンバー： M55621)の活性ドメインをコードする cDNAを特異 的に増幅させる。増幅させた cDNAは、その 5'末端側に、酵母のマンノース転移酵素 (MNN9)遺伝子（GenBankァクセッションナンバー： L23752)のリーダーペプチドをコ ードする cDNA配列を連結した後に、酵母用の発現ベクター pAUR123 (タカラノィォ 社製）などのベクターのアルコールデヒドロゲナーゼプロモーター配列の下流に位置 する制限酵素 Kpnl切断部位と Xba I切断部位の間に挿入し、酵母のゴルジ体内に N- ァセチルダルコサミン転移酵素- 1を発現させるベクターを作製する。次にこのべクタ 一を、前項に記載した、 UDP-N-ァセチルダルコサミントランスポーター遺伝子を導入 したピキア酵母株に対し、発現ベクター pAUR123に添付のマニュアルに掲載された 酢酸リチウム法により導入する。遺伝子導入後の酵母は、薬剤ォ一口ブラシジン A (タ カラノィォ社製)を含有する YPD培地で室温にて培養し、増殖してきた各コロニーか ら全 RNAを抽出する。次に、この全 RNAから調製した cDNAを铸型とした PCR法によつ て、組換え N-ァセチルダルコサミン転移酵素- 1の発現が認められた酵母クローン株 を選択する。上記の方法により、ピキア酵母が発現する主要な N-結合型糖鎖の構造 を、還元末端側のコア部分に 2残基の N-ァセチルダルコサミンを有し、非還元末端側 に 5個のマンノース残基が結合した Man5型ハイマンノース型糖鎖の非還元末端側に 、 N-ァセチルダルコサミン残基が 1個付加された構造を有する、ノヽイブリツド型糖鎖に 改変することができる。 N-Acetyldarcosaminyltransferase-1 (GenBank accession number) was obtained by performing PCR using human liver cDNA (Clontech) in a cage and using specific primers and KOD polymerase (Toyobo). : Amplify specifically the cDNA encoding the active domain of M55621). The amplified cDNA is linked to the 5 'end of the cDNA sequence encoding the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752), and then expressed for yeast. It is inserted between the restriction enzyme Kpnl cleavage site and the Xba I cleavage site located downstream of the alcohol dehydrogenase promoter sequence of vectors such as vector pAUR123 (manufactured by Tacarano), and N-acetylyldarcosamine transferase- is inserted into the yeast Golgi. A vector for expressing 1 is prepared. Next, this vector is introduced into the Pichia yeast strain introduced with the UDP-N-acetylyldarcosamine transporter gene described in the previous section by the lithium acetate method described in the manual attached to the expression vector pAUR123. . After the gene transfer, the yeast Incubate at room temperature in YPD medium containing Caranoio), and extract total RNA from each of the grown colonies. Next, a yeast clonal strain in which expression of recombinant N-acetylyldarcosamine transferase-1 is observed is selected by PCR using the cDNA prepared from this total RNA as a saddle type. By the above method, the structure of the main N-linked sugar chain expressed in Pichia yeast has 2 residues of N-acetyldarcosamine in the core at the reducing end and 5 at the non-reducing end. It can be modified to a noblebrid sugar chain with a structure in which one N-acetylyldarcosamine residue is added to the non-reducing end of the Man5 type high mannose sugar chain to which the mannose residue is attached. .
[0226] 以上、 N-結合型糖鎖として、ハイマンノース型糖鎖と複合型糖鎖の中間の構造で ある、ハイブリッド型糖鎖を主要に発現するピキア酵母株の作製方法について記載し た。上述のピキア酵母以外に、組換え蛋白質を発現させる宿主としてしばしば用いら れる酵母として、サッカロマイセス（Saccharomyces)属の酵母が挙げられる。以下、 N- 結合型糖鎖としてノ、イブリツド型糖鎖を主要に発現するサッカロマイセス酵母株の作 製方法について述べる。  [0226] The method for producing a Pichia yeast strain that mainly expresses a hybrid sugar chain, which is an intermediate structure between a high mannose sugar chain and a complex sugar chain, as an N-linked sugar chain has been described. In addition to the above-mentioned Pichia yeast, yeasts of the genus Saccharomyces can be mentioned as yeasts that are often used as hosts for expressing recombinant proteins. A method for producing a Saccharomyces yeast strain that mainly expresses N-linked sugar chains and hybrid sugar chains as follows is described.
[0227] 6.ゲノム上に存在する α -1,6-マンノース転移酵素遺伝子とひ -1,3-マンノース転 移酵素遺伝子を破壊したサッカロマイセス酵母株の作製  [0227] 6. Production of Saccharomyces yeast strains with disrupted α-1,6-mannose transferase and -1,3-mannose transferase genes present on the genome
Nakayamaらの方法（EMBO Journal, 11, 2511 (1992))に従い、相同組換えによって OCH1遺伝子座が破壊された酵母クローンを選択する。得られた OCH1遺伝子が破 壊されたサッカロマイセス酵母株は、 Shermanらの方法 (メソッズ'イン'ェンザィモロジ 一 194, 21 (1991))に従い、半数体細胞を誘導した後、 α - 1,3-マンノース転移酵素（ MNN1)遺伝子が破壊された変異酵母株 LB1-10B (カリフォルニア大学 Yeast Genetic Stock Center)の半数体細胞と混合し、窒素欠乏条件で培養することにより、二倍体 の接合子を形成させる。次に、得られた接合子を、ゥラシルとロイシンを欠損させた Y PD培地で室温にて培養し、増殖してきた各コロニー力もゲノム DNAを抽出する。次に 、このゲノム DNAを铸型とした PCR法によって、酵母 OCH1遺伝子座の配列（GenBan kァクセッションナンバー： AF540063)と、 MNN1遺伝子座の配列（GenBankァクセッシ ヨンナンバー： AF540063L23753)をそれぞれ増幅させることにより、 OCH1遺伝子座と MNN1遺伝子座の両方が破壊された酵母クローン株を選択する。上記の方法により、 サッカロマイセス酵母が発現する主要な N-結合型糖鎖の構造を、還元末端側のコア 部分に 2残基の N-ァセチルダルコサミンを有し、非還元末端側に 8個のマンノース残 基が結合した構造を有する、 Man8型ハイマンノース型糖鎖に改変できる。 According to the method of Nakayama et al. (EMBO Journal, 11, 2511 (1992)), a yeast clone in which the OCH1 locus is destroyed by homologous recombination is selected. The obtained Saccharomyces yeast strain in which the OCH1 gene was disrupted was derived from haploid cells according to the method of Sherman et al. (Methods'In'Enzymology 1 194, 21 (1991)), and then α-1,3-mannose. A diploid zygote is formed by mixing with haploid cells of the mutant yeast strain LB1-10B (University of California Yeast Genetic Stock Center) in which the transferase (MNN1) gene is disrupted and culturing under nitrogen-deficient conditions . Next, the obtained zygote is cultured at room temperature in YPD medium lacking uracil and leucine, and genomic DNA is extracted from each colony force that has grown. Next, the yeast OCH1 locus sequence (GenBank accession number: AF540063) and the MNN1 locus sequence (GenBank accession number: AF540063L23753) are amplified by PCR using this genomic DNA as a saddle type. Thus, a yeast clonal strain in which both the OCH1 locus and the MNN1 locus are disrupted is selected. By the above method The structure of the major N-linked sugar chain expressed in Saccharomyces yeast has 2 residues of N-acetyldarcosamine in the core of the reducing end and 8 mannose residues on the non-reducing end. It can be modified to a Man8 type high mannose sugar chain having a bound structure.
[0228] 7.組換えキメラ型 a -1,2-マンノシダーゼ遺伝子を導入したサッカロマイセス酵母 株 [0228] 7. Saccharomyces yeast strain introduced with recombinant chimeric a-1,2-mannosidase gene
の作製  Making
カビ（Aspergillus saitoi)から RNeasy Mini Kit (キアゲン社製）を用いて全 RNAを抽出 し、次にこの RNAを铸型として Superscript™first- strandcDNA synthesis kit (インビトロ ジェン社製)を用いて cDNAを調製する。次に、この cDNAを铸型とし、特異的プライマ 一と KODポリメラーゼ (東洋紡績社製)を用いた PCRを行うことにより、カビ α -1,2-マ ンノシダーゼの翻訳領域全長をコードする cDNA (GenBankァクセッションナンバー： D 49827)を特異的に増幅させる。増幅させた約 1500塩基長の cDNAは、その翻訳終止 コドンを削除した 3 '末端側に、酵母の小胞体局在シグナルペプチド (ェンボジャーナ ル 7, 913 (1988))、すなわちヒスチジンーァスパラギン酸一グルタミン酸一口イシンを コードする cDNA配列と翻訳終止コドンを連結した後に、酵母用の発現ベクター pPIC Z (インビトロジェン社製）などのベクターに挿入し、酵母の小胞体内に α -1,2-マンノ シダーゼを発現させるベクターを作製する。次にこのベクターを、前項に記載した、 a -1,6-マンノース転移酵素遺伝子と a -1,3-マンノース転移酵素遺伝子を破壊した サッカロマイセス酵母株に対し、エレクト口ポレーシヨン法により安定的に導入する。 遺伝子導入後の酵母は、ゥラシルを欠損しゼォシン (インビトロジェン社製)を含有す る YPD培地 (インビトロジェン社製)で室温にて培養し、増殖してきた各コロニー力ゝら全 RNAを抽出する。次に、この全 RNA力も調製した cDNAを铸型とした PCR法によって、 組換えキメラ型 (X -1,2-マンノシダーゼの発現が認められた酵母クローン株を選択す る。上記の方法により、サッカロマイセス酵母が発現する主要な N-結合型糖鎖の構 造を、還元末端側のコア部分に 2残基の N-ァセチルダルコサミンを有し、非還元末端 側に 5個のマンノース残基が結合した構造を有する、 Man5型ハイマンノース型糖鎖に 改変することができる。  Extract total RNA from mold (Aspergillus saitoi) using RNeasy Mini Kit (Qiagen), and then prepare cDNA using Superscript ™ first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is converted into a saddle type, and PCR using a specific primer and KOD polymerase (Toyobo Co., Ltd.) is performed to obtain a cDNA encoding the entire translation region of mold α-1,2-mannosidase ( GenBank accession number: D 49827) is specifically amplified. The amplified cDNA, about 1500 bases long, has a yeast endoplasmic reticulum localization signal peptide (embombonal 7, 913 (1988)), that is, histidine-aspartate, at the 3 'end from which the translation termination codon was deleted. After ligating a cDNA sequence encoding a monoglutamate bite isine and a translation termination codon, it is inserted into a vector such as the yeast expression vector pPIC Z (manufactured by Invitrogen), and α-1,2-manno in the yeast endoplasmic reticulum. A vector for expressing a sidase is prepared. Next, this vector was stably introduced into the Saccharomyces yeast strain in which the a-1,6-mannose transferase gene and the a-1,3-mannose transferase gene were disrupted, as described in the previous section, by the electopore method. To do. The yeast after gene transfer is cultured at room temperature in a YPD medium (Invitrogen) containing zeocin (Invitrogen) and lacking uracil, and total RNA is extracted from each of the grown colonies. Next, a recombinant chimeric type (yeast clone strain in which the expression of X-1,2-mannosidase was observed was selected by PCR using this cDNA with the total RNA strength prepared as a saddle type. By the above method, The structure of the major N-linked sugar chain expressed in Saccharomyces yeast has 2 residues of N-acetylyldarcosamine in the core portion on the reducing end and 5 mannose residues on the non-reducing end It can be modified into a Man5 type high mannose type sugar chain having a structure in which
[0229] 8.組換え UDP-N-ァセチルダルコサミントランスポーター遺伝子を導入したサッカロ マイセス酵母株の作製 [0229] 8. Saccharo introduced with recombinant UDP-N-acetylcylcosamine transporter gene Production of Myces yeast strain
酵母（Kluyveromyces lactis)から RNeasy Mini Kit (キアゲン社製）を用いて全 RNAを 抽出し、次にこの RNAを铸型として Superscript™first- strandcDNA synthesis kit (イン ビトロジェン社製)を用いて cDNAを調製する。次に、この cDNAを铸型とし、特異的プ ライマーと KODポリメラーゼ (東洋紡績社製)を用 V、た PCRを行うことにより、酵母 UDP Extract total RNA from yeast (Kluyveromyces lactis) using RNeasy Mini Kit (Qiagen), and then prepare cDNA using Superscript ™ first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is used as a saddle, and PCR is performed using a specific primer and KOD polymerase (Toyobo Co., Ltd.).
-N-ァセチルダルコサミントランスポーターの翻訳領域全長をコードする cDNA(GenB ankァクセッションナンバー： AF106080)を特異的に増幅させる。次に、増幅させた約A cDNA encoding the entire translation region of the -N-acetylcylcosamine transporter (GenBank accession number: AF106080) is specifically amplified. Next, the amplified about
3700塩基長の cDNAを、酵母用の発現ベクター pPIC3.5K (インビトロジェン社製）など のベクターのアルコールォキシゲナーゼプロモーター配列の下流に位置する制限酵 素 EcoRI切断部位と Not I切断部位の間に挿入し、酵母のゴルジ体内に UDP-N-ァセ チルダルコサミントランスポーターを発現させるベクターを作製する。次にこのべクタ 一を、前項に記載した、 α - 1,2-マンノシダーゼ遺伝子を導入したサッカロマイセス酵 母株に対し、エレクト口ポレーシヨン法により安定的に導入する。遺伝子導入後の酵 母は、薬剤 G418け力ライテスタ社製)を含有する YPD培地で室温にて培養し、増殖 してきた各コロニー力も全 RNAを抽出する。次に、この全 RNA力も調製した cDNAを铸 型とした PCR法によって、組換え UDP-N-ァセチルダルコサミントランスポーターの発 現が認められた酵母クローン株を選択する。 A 3700 base-long cDNA is inserted between the restriction enzyme EcoRI and Not I cleavage sites located downstream of the alcohol oxygenase promoter sequence in vectors such as the yeast expression vector pPIC3.5K (Invitrogen). The vector is inserted to express the UDP-N-acetyldarcosamine transporter in the Golgi apparatus of yeast. Next, this vector is stably introduced into the Saccharomyces fermentation mother strain into which the α-1,2-mannosidase gene has been introduced as described in the previous section by the electopore method. The fermented mother after the gene transfer is cultured at room temperature in a YPD medium containing the drug G418 (manufactured by Leitesta Co., Ltd.). Next, a yeast clonal strain in which expression of the recombinant UDP-N-acetylyldarcosamine transporter is observed is selected by a PCR method using the prepared cDNA with the prepared total RNA strength.
9.組換えキメラ型 N-ァセチルダルコサミン転移酵素- 1遺伝子を導入したサッカロマ イセス酵母株の作製  9. Production of a Saccharomyces yeast strain into which a recombinant chimeric N-acetyldylcosamine transferase-1 gene has been introduced
ヒト肝臓 cDNA (クロンテック社製)を铸型とし、特異的プライマーと KODポリメラーゼ（ 東洋紡績社製)を用いた PCRを行うことにより、 N-ァセチルダルコサミン転移酵素- 1 ( GenBankァクセッションナンバー： M55621)の活性ドメインをコードする cDNAを特異 的に増幅させる。増幅させた cDNAは、その 5'末端側に、酵母のマンノース転移酵素 (MNN9)遺伝子（GenBankァクセッションナンバー： L23752)のリーダーペプチドをコ ードする cDNA配列を連結した後に、酵母用の発現ベクター pAUR123 (タカラノィォ 社製）などのベクターのアルコールデヒドロゲナーゼプロモーター配列の下流に位置 する制限酵素 Kpnl切断部位と Xba I切断部位の間に挿入し、酵母のゴルジ体内に N- ァセチルダルコサミン転移酵素- 1を発現させるベクターを作製する。次にこのべクタ 一を、前項に記載した、 UDP-N-ァセチルダルコサミントランスポーター遺伝子を導入 したサッカロマイセス酵母株に対し、発現ベクター pAUR123に添付のマニュアルに掲 載された酢酸リチウム法により導入する。遺伝子導入後の酵母は、薬剤ォ一口ブラシ ジン A (タカラバイオ社製)を含有する YPD培地で室温にて培養し、増殖してきた各コ 口-一から全 RNAを抽出する。次に、この全 RNA力も調製した cDNAを铸型とした PCR 法によって、組換え N-ァセチルダルコサミン転移酵素- 1の発現が認められた酵母クロ 一ン株を選択する。上記の方法により、サッカロマイセス酵母が発現する主要な N-結 合型糖鎖の構造を、還元末端側のコア部分に 2残基の N-ァセチルダルコサミンを有 し、非還元末端側に 5個のマンノース残基が結合した構造を有する Man5型ハイマンノ 一ス型糖鎖の非還元末端側に、 N-ァセチルダルコサミン残基が 1個付加された、ノヽ イブリツド型糖鎖に改変できる。 N-acetylylcosamine transferase-1 (GenBank accession number) is obtained by PCR using human liver cDNA (manufactured by Clontech) as a cage and using specific primers and KOD polymerase (manufactured by Toyobo). : Amplify specifically the cDNA encoding the active domain of M55621). The amplified cDNA is linked to the 5 'end of the cDNA sequence encoding the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752), and then expressed for yeast. It is inserted between the restriction enzyme Kpnl cleavage site and the Xba I cleavage site located downstream of the alcohol dehydrogenase promoter sequence of vectors such as vector pAUR123 (manufactured by Tacarano), and N-acetylyldarcosamine transferase- is inserted into the yeast Golgi. A vector for expressing 1 is prepared. Next, this vector One is introduced into the Saccharomyces yeast strain into which the UDP-N-acetylyldarcosamine transporter gene described above has been introduced by the lithium acetate method described in the manual attached to the expression vector pAUR123. The yeast after the gene introduction is cultured at room temperature in a YPD medium containing a drug mouthful brassin A (manufactured by Takara Bio Inc.), and total RNA is extracted from each grown mouthpiece. Next, a yeast clone strain in which the expression of recombinant N-acetyl dalcosamine transferase-1 has been observed is selected by PCR using this cDNA, which has also been prepared for total RNA, in a vertical form. According to the above method, the structure of the main N-linked sugar chain expressed in Saccharomyces yeast has a 2-residue N-acetylyldarcosamine in the core portion on the reducing end side and 5 in the non-reducing end side. It can be modified to a nodule type sugar chain in which one N-acetylyldarcosamine residue is added to the non-reducing terminal side of the Man5 type high mannose type sugar chain having a structure in which one mannose residue is bonded.
[0231] 以上の通り、 N-結合型糖鎖として Man5型ハイマンノース型糖鎖の非還元末端側に N-ァセチルダルコサミン残基が 1個付加された、ハイブリッド型糖鎖を主要に発現す るピキア酵母株、あるいはサッカロマイセス酵母株の作製方法について述べた。次に 、これらの酵母株を宿主として用い、 N-結合型糖鎖としてハイブリッド型糖鎖を主要 に有する組換えヒト卵胞刺激ホルモンの調製方法について述べる。  [0231] As described above, hybrid sugar chains are mainly expressed as an N-linked sugar chain with one N-acetylyldarcosamine residue added to the non-reducing end of Man5 type high mannose type sugar chain. A method for producing the Pichia yeast strain or the Saccharomyces yeast strain was described. Next, a method for preparing a recombinant human follicle-stimulating hormone mainly using hybrid sugar chains as N-linked sugar chains using these yeast strains as hosts will be described.
[0232] 10.組換えヒト卵胞刺激ホルモン発現ベクターの作製  [0232] 10. Construction of recombinant human follicle stimulating hormone expression vector
Fidlerらの方法（Journal of Molecular Endocrinology 21 , 327 (1998))に従い、ヒト脳 下垂体 cDNA (クロンテック社製)を铸型とし、増幅用酵素として KODポリメラーゼ (東 洋紡績社製)を用いた PCR反応により、成熟型ヒト卵胞刺激ホルモンの exサブュ-ッ トと j8サブユニットの全長をコードする cDNAをそれぞれ特異的に増幅させる。次に、 得られた各サブユニット cDNAの 5 '末端側に MF aリーダー配列（Cell 36,309 (1984)) を連結後、酵母用の発現ベクター PA0815 (インビトロジェン社製）のアルコールォキ シゲナーゼプロモーター配列の直下流に挿入し、成熟型ヒト卵胞刺激ホルモン aサ ブユニットを分泌発現させるベクター pA0815/hFSH aと、成熟型ヒト卵胞刺激ホルモ ン βサブユニットを分泌発現させるベクター pA0815/hFSH βを作製する。次に、 ρΑ 0815/hFSH βを制限酵素 Bglllと BamHIで消化して得られた成熟型ヒト卵胞刺激ホル モン 13サブユニット cDNAを含む DNA断片を、 pA0815/hFSH aの BamHIサイトに揷 入することにより、成熟型ヒト卵胞刺激ホルモンヘテロダイマーを分泌発現させるベタ ター pA0815/hFSH α βを作製する。 In accordance with the method of Fidler et al. (Journal of Molecular Endocrinology 21, 327 (1998)), human pituitary cDNA (manufactured by Clontech) was used as a cage and PCR using KOD polymerase (manufactured by Toyobo Co., Ltd.) as an amplification enzyme. The reaction specifically amplifies mature human follicle-stimulating hormone ex and j8 subunit full-length cDNAs. Next, after linking the MFa leader sequence (Cell 36,309 (1984)) to the 5 'end of each subunit cDNA obtained, the alcohol oxygenase promoter sequence of the yeast expression vector PA0815 (Invitrogen) was inserted into the immediately downstream, making the vectors pA0815 / hFSH a for secretory expression of mature human follicle stimulating hormone a subunit, a vector p A0815 / hFSH β for secretory expression of mature human follicle stimulating hormone beta subunit . Next, a DNA fragment containing the mature human follicle-stimulating hormone 13 subunit cDNA obtained by digesting ρΑ0815 / hFSH β with restriction enzymes Bglll and BamHI was added to the BamHI site of pA0815 / hFSH a. In this way, the beta pA0815 / hFSH αβ that secretes and expresses the mature human follicle stimulating hormone heterodimer is prepared.
11.組換えヒト卵胞刺激ホルモン遺伝子を導入した酵母株の作製  11. Production of yeast strain with recombinant human follicle-stimulating hormone gene
上述の成熟型ヒト卵胞刺激ホルモンを分泌発現させるベクター pA0815/hFSH α β 100 gを、制限酵素 Sail (ニューイングランドバイオラブズ社製)で HIS4遺伝子内を切 断し、フエノールクロ口ホルム抽出とエタノール沈殿によって、線状化ベクターを調製 する。次に Mochizukiらの方法（ProteinExpression and Purification 23, 55 (2001))に 従い、この線状ィ匕した卵胞刺激ホルモン発現ベクターを、上述の本実施例第 5項に 記載した、 N-結合型糖鎖として主にハイブリッド型糖鎖を発現するピキア酵母株、も しくは本実施例第 9項に記載した、 N-結合型糖鎖として主にハイブリッド型糖鎖を発 現するサッカロマイセス酵母株に対し、酢酸リチウム法により導入する。遺伝子導入 後の酵母は、薬剤ブラストシジン (インビトロジェン社製)を含有する YPD培地 (インビト ロジェン社製)で室温にて培養し、ブラストシジン耐性コロニーを取得する。次に、ブ ラストシジン耐性コロニーを液体 YPD培地 (インビトロジェン社製）に移植し、 30°Cにて 24時間以上の回分培養を行う。培養後に得られた培養上清中に含まれる FSH濃度 は、卵胞刺激ホルモン医薬品 Gona卜 F (Serono社製）などを標準品とし、 Human FSH ELISAKit(Antigenix America社製)を用いて測定する。この酵母培養上清中に分泌さ れた、 N-結合型糖鎖としてフコースを含まな 、ハイブリッド型糖鎖を有する遺伝子組 換え卵胞刺激ホルモンは、 Gadkariらの方法（Protein Expression and Purification 32, 175 (2003))に順じて精製する。また、精製された卵胞刺激ホルモン蛋白質は、 Skibe liらの方法 (Blood 98, 3626 (2001))に順じて糖鎖構造の解析を行う。以上の通り、 N- 結合型糖鎖として、 Man5型ハイマンノース型糖鎖の非還元末端側に N-ァセチルダ ルコサミン残基が 1個付加されたハイブリッド型糖鎖を主要に発現するピキア酵母株 The above-mentioned vector pA0815 / hFSH α β 100 g that secretes and expresses the mature human follicle-stimulating hormone is cleaved within the HIS4 gene with the restriction enzyme Sail (manufactured by New England Biolabs). Prepare the linearized vector. Next, according to the method of Mochizuki et al. (ProteinExpression and Purification 23, 55 (2001)), this linearly follicle-stimulating hormone expression vector was transformed into the N-linked sugar described in Example 5 above. Pichia yeast strains that mainly express hybrid sugar chains as chains, or Saccharomyces yeast strains that mainly express hybrid sugar chains as N-linked sugar chains described in Section 9 of this Example Introduced by the lithium acetate method. The yeast after the gene introduction is cultured at room temperature in a YPD medium (Invitrogen) containing the drug blasticidin (Invitrogen) to obtain blasticidin-resistant colonies. Next, the blasticidin-resistant colonies are transplanted into liquid YPD medium (Invitrogen) and cultured at 30 ° C for 24 hours or longer. The FSH concentration contained in the culture supernatant obtained after the culture is measured using Human FSH ELISAKit (manufactured by Antigenix America) using the follicle stimulating hormone drug Gona 卜 F (manufactured by Serono) as a standard product. A recombinant follicle-stimulating hormone having a hybrid sugar chain that does not contain fucose as an N-linked sugar chain and is secreted into this yeast culture supernatant is the method of Gadkari et al. (Protein Expression and Purification 32, 175 (2003)). The purified follicle-stimulating hormone protein is analyzed for the sugar chain structure according to the method of Skibe li et al. (Blood 98, 3626 (2001)). As described above, a Pichia yeast strain that mainly expresses a hybrid sugar chain in which one N-acetylethylcosamine residue is added to the non-reducing end of the Man5 type high mannose type sugar chain as an N-linked sugar chain.
、あるいは同様に改変されたサッカロマイセス酵母株を宿主として用い、 N-結合型糖 鎖としてフコースを含まないハイブリッド型糖鎖を主要に有する遺伝子組換えヒト卵胞 刺激ホルモンを調製できる。次に、この N-結合型糖鎖としてハイブリッド型糖鎖を主 要に有する遺伝子組換えヒト卵胞刺激ホルモンを発現する酵母株を用いて、 N-結合 型糖鎖としてフコースを含まない複合二本鎖型糖鎖を主要に有する遺伝子組換えヒ ト卵胞刺激ホルモンを発現する酵母株を作製する方法について以下に記載する。 Alternatively, a similarly modified Saccharomyces yeast strain can be used as a host, and a recombinant human follicle-stimulating hormone mainly having a hybrid sugar chain not containing fucose as an N-linked sugar chain can be prepared. Next, using a yeast strain that expresses a recombinant human follicle-stimulating hormone that mainly has a hybrid sugar chain as this N-linked sugar chain, a complex double chain that does not contain fucose as an N-linked sugar chain. Genetically modified humans mainly having chain-type sugar chains A method for producing a yeast strain that expresses follicle-stimulating hormone is described below.
[0234] 12.組換えキメラ型 aマンノシダーゼ II遺伝子を導入した酵母株の作製  [0234] 12. Production of yeast strains with recombinant chimeric a-mannosidase II gene
ヒト組織由来、たとえば肝臓由来の cDNA (クロンテック社製)を铸型とし、特異的プ ライマーと KODポリメラーゼ (東洋紡績社製)を用 V、た PCRを行うことにより、 ひマンノ シダーゼ II (GenBankァクセッションナンバー： U31520)の活性ドメインをコードする cD NAを特異的に増幅させる。増幅させた cDNAは、その 5 '末端側に、酵母のマンノース 転移酵素（MNN9)遺伝子（GenBankァクセッションナンバー： L23752)のリーダーぺプ チドをコードする cDNA配列を連結した後に、酵母用の発現ベクターのプロモーター 配列の下流に挿入し、酵母のゴルジ体内に αマンノシダーゼ IIを発現させるベクター を作製する。次にこのベクターを、本実施例第 11項に記載した、 Ν-結合型糖鎖とし てハイブリッド型糖鎖を主要に有する遺伝子組換えヒト卵胞刺激ホルモンを発現する 酵母株に対し、安定的に導入する。遺伝子導入後の酵母は、栄養要求性と薬剤耐 性を指標にしてクローンを選抜した後、 RT- PCRによって、キメラ型ひマンノシダーゼ I Iの発現を確認する。  By performing PCR using human tissue-derived cDNA, such as liver-derived cDNA (Clontech), in a vertical form, and using a specific primer and KOD polymerase (Toyobo Co., Ltd.) V, human manosidase II (GenBank It specifically amplifies the cDNA encoding the active domain of the session number: U31520). The amplified cDNA is linked to the 5 'end of the cDNA sequence encoding the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752), followed by expression for yeast. The vector is inserted downstream of the promoter sequence of the vector to produce a vector that expresses α-mannosidase II in the yeast Golgi apparatus. Next, this vector was stably used for the yeast strain described in paragraph 11 of this Example, which expresses a recombinant human follicle-stimulating hormone mainly having a hybrid sugar chain as a Ν-linked sugar chain. Introduce. For the yeast after gene introduction, clones are selected using auxotrophy and drug resistance as indicators, and then the expression of chimeric mannoseidase I I is confirmed by RT-PCR.
[0235] 13.組換えキメラ型 Ν-ァセチルダルコサミン転移酵素- II遺伝子を導入した酵母株 の  [0235] 13. Recombinant chimeric type Ν-acetylcylcosamine transferase- of yeast strain introduced with II gene
作製  Production
ヒト組織由来、たとえば肝臓由来の cDNA (クロンテック社製)を铸型とし、特異的プ ライマーと KODポリメラーゼ (東洋紡績社製)を用 V、た PCRを行うことにより、 N-ァセチ ルダルコサミン転移酵素- Π (GenBankァクセッションナンバー： U15128)の活性ドメィ ンをコードする cDNAを特異的に増幅させる。増幅させた cDNAは、その 5 '末端側に、 酵母のマンノース転移酵素（MNN9)遺伝子（GenBankァクセッションナンバー： L2375 2)のリーダーペプチドをコードする cDNA配列を連結した後に、酵母用の発現べクタ 一のプロモーター配列の下流に挿入し、酵母のゴルジ体内に N-ァセチルダルコサミ ン転移酵素- IIを発現させるベクターを作製する。次にこのベクターを、前項に記載し た、 N-結合型糖鎖としてハイブリッド型糖鎖を主要に有する組換えヒト卵胞刺激ホル モンを発現する酵母株にキメラ型 (Xマンノシダーゼ IIを安定的に導入した酵母株に 対し、安定的に導入する。遺伝子導入後の酵母は、栄養要求性と薬剤耐性を指標に してクローンを選抜した後、 RT- PCRによって、キメラ型 N-ァセチルダルコサミン転移 酵素- IIの発現を確認する。上記の方法により、キメラ型 N-ァセチルダルコサミン転移 酵素- IIが安定的に組み込まれた酵母株が発現する遺伝子組換え卵胞刺激ホルモ ンが有する主要な N-結合型糖鎖の構造を、還元末端側のコア部分に 2残基の N-ァ セチルダルコサミンを有し、その非還元末端側に 3個のマンノース残基が二分岐する 構造で結合し、二つの非還元末端のそれぞれに N-ァセチルダルコサミン残基が 1個 ずつ付加された、フコースを含まない複合二本鎖型糖鎖に改変することができる。 N-acetyldarcosaminyltransferase--using human tissue, for example, liver-derived cDNA (Clontech) in a saddle shape and PCR using a specific primer and KOD polymerase (Toyobo) V CDNA Amplify the cDNA encoding the active domain of GenBank accession number: U15128. The amplified cDNA is ligated to the 5 'end of the cDNA sequence encoding the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L2375 2), and then expressed for yeast. Create a vector that is inserted downstream of the promoter sequence and expresses N-acetyl dalcosamine transferase-II in the yeast Golgi apparatus. Next, this vector was stably introduced into the chimeric strain (X mannosidase II) stably expressing the recombinant human follicle-stimulating hormone mainly having a hybrid sugar chain as an N-linked sugar chain as described in the previous section. Introduce stably into the introduced yeast strain, and the yeast after gene introduction uses auxotrophy and drug resistance as indicators. After selecting the clones, the expression of the chimeric N-acetylyldarcosamine transferase-II is confirmed by RT-PCR. By the above method, the structure of the main N-linked sugar chain of the recombinant follicle-stimulating hormone expressed by the yeast strain stably incorporating the chimeric N-acetylyldarcosamine transferase II is obtained. It has two residues of N-acetyl darcosamine in the core part on the reducing end side, and its mannose residue is bifurcated on the non-reducing end side, and is attached to each of the two non-reducing ends. It can be modified into a complex double-stranded sugar chain that contains one N-acetylyldarcosamine residue and does not contain fucose.
[0236] 14.組換え UDP-ガラクトース- 4-ェピメラーゼ遺伝子を導入した酵母株の作製 ヒト組織由来、たとえば肝臓由来の cDNA (クロンテック社製)を铸型とし、特異的プ ライマーと KODポリメラーゼ (東洋紡績社製)を用いた PCRを行うことにより、 UDP-ガラ クトース- 4-ェピメラーゼ（UniGeneナンバー Hs.76057)の翻訳領域全長をコードする c DNAを特異的に増幅させる。増幅させた cDNAは、酵母用の発現ベクターのプロモー ター配列の下流に挿入し、酵母のサイトゾル内に UDP-ガラクトース- 4-ェピメラーゼ を発現させるベクターを作製する。次にこのベクターを、前項に記載した、 N-結合型 糖鎖として未熟な複合二本鎖型糖鎖を主要に有する組換えヒト卵胞刺激ホルモンを 発現する酵母株に対し、安定的に導入する。遺伝子導入後の酵母は、栄養要求性と 薬剤耐性を指標にしてクローンを選抜した後、 RT-PCRによって、 UDP-ガラクトース- 4-ェピメラーゼの発現を確認する。  [0236] 14. Preparation of yeast strain into which recombinant UDP-galactose-4-epimerase gene has been introduced Human cDNA derived from human tissue, such as liver (Clontech) is used as a saddle type, with specific primers and KOD polymerase (Toyo By performing PCR using Spinning Co., Ltd., the cDNA encoding the entire translation region of UDP-galactose-4-epimerase (UniGene number Hs.76057) is specifically amplified. The amplified cDNA is inserted downstream of the promoter sequence of the expression vector for yeast to produce a vector that expresses UDP-galactose-4-epimerase in the yeast cytosol. Next, this vector is stably introduced into the yeast strain described above, which expresses a recombinant human follicle-stimulating hormone mainly having an immature complex double-stranded sugar chain as an N-linked sugar chain. . For the yeast after gene transfer, clones are selected using auxotrophy and drug resistance as indicators, and then the expression of UDP-galactose-4-epimelase is confirmed by RT-PCR.
[0237] 15.組換えキメラ型 β 1,4ガラクトース転移酵素遺伝子を導入した酵母株の作製 ヒト組織由来、たとえば肝臓由来の cDNA (クロンテック社製)を铸型とし、特異的プ ライマーと KODポリメラーゼ (東洋紡績社製)を用いた PCRを行うことにより、 β 1,4ガラ クトース転移酵素（GenBankァクセッションナンバー： M22921)の活性ドメインをコード する cDNAを特異的に増幅させる。増幅させた cDNAは、その 5'末端側に、酵母のマ ンノース転移酵素（MNN9)遺伝子（GenBankァクセッションナンバー： L23752)のリー ダーぺプチ  [0237] 15. Production of Yeast Strain Introduced with Recombinant Chimeric β1,4 Galactosyltransferase Gene Human tissue-derived, for example, liver-derived cDNA (Clontech) is used as a cocoon-shaped, specific primer and KOD polymerase. By performing PCR using Toyobo Co., Ltd., cDNA encoding the active domain of β1,4 galactose transferase (GenBank accession number: M22921) is specifically amplified. The amplified cDNA has a leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752) at the 5 'end.
ドをコードする cDNA配列を連結した後に、酵母用の発現ベクターのプロモーター配 列の下流に挿入し、酵母のゴルジ体内に j8 1,4ガラクトース転移酵素を発現させるベ クタ一を作製する。次にこのベクターを、上述の前項に記載した、 N-結合型糖鎖とし て未熟な複合二本鎖型糖鎖を主要に有する組換えヒト卵胞刺激ホルモンを発現する 酵母株にキメラ型 ι8 1,4ガラクトース転移酵素を安定的に導入した酵母株に対し、安 定的に導入する。遺伝子導入後の酵母は、栄養要求性と薬剤耐性を指標にしてクロ ーンを選抜した後、 RT-PCRによって、キメラ型 j8 1,4ガラクトース転移酵素の発現を 確認する。以上の方法により、キメラ型 j8 1,4ガラクトース転移酵素が安定的に組み込 まれた酵母株が発現する遺伝子組換え卵胞刺激ホルモンが有する主要な N-結合型 糖鎖の構造を、還元末端側のコア部分に 2残基の N-ァセチルダルコサミンを有し、そ の非還元末端側に 3個のマンノース残基が二分岐する構造で結合し、二つの非還元 末端のそれぞれに N-ァセチルダルコサミン残基とガラクトース残基が 1個ずつ付加さ れた、複合二本鎖型糖鎖に改変することができる。 After ligating the cDNA sequence coding for the plasmid, it is inserted downstream of the promoter sequence of the expression vector for yeast to produce a vector that expresses j8 1,4 galactosyltransferase in the yeast Golgi apparatus. Next, this vector was changed to the N-linked sugar chain described in the previous section. Stable against a yeast strain that stably introduces a chimeric ι8 1,4 galactosyltransferase into a yeast strain that expresses recombinant human follicle-stimulating hormone, which is mainly composed of immature complex double-stranded sugar chains Introduce. In the yeast after gene transfer, clones are selected using auxotrophy and drug resistance as indicators, and then the expression of chimeric j8 1,4 galactose transferase is confirmed by RT-PCR. By the above method, the structure of the major N-linked sugar chain of the recombinant follicle-stimulating hormone expressed by the yeast strain stably incorporating the chimeric j8 1,4 galactosyltransferase is reduced to the reducing end. It has 2 residues of N-acetyl darcosamine in the core part of the core, 3 mannose residues are linked in a bifurcated structure on the non-reducing end side, and N-to each of the 2 non-reducing ends It can be modified into a complex double-stranded sugar chain in which one acetylyldarcosamine residue and one galactose residue are added.
[0238] 16.酵母を用いた遺伝子組換え卵胞刺激ホルモン蛋白質の調製 [0238] 16. Preparation of genetically modified follicle-stimulating hormone protein using yeast
前項で作製した、還元末端側にフコース残基を有さず、非還元末端側にガラクトー スが付加された複合二本鎖型糖鎖を主に有する遺伝子組換え卵胞刺激ホルモンを 発現する酵母株は、液体 YPD培地 (インビトロジェン社製）に播種し、 30°Cにて 24時 間以上の回分培養を行うことにより、培養上清中に遺伝子組換え卵胞刺激ホルモン を分泌させる。培養後に得られた培養上清に含まれる FSH濃度は、卵胞刺激ホルモ ン医薬品 Gona卜 F (Serono社製）などを標準品とし、 HumanFSH ELISA Kit(Antigenix America社製)を用いて測定する。この酵母培養上清中に分泌された、 N-結合型糖鎖 として還元末端側にフコース残基を持たず非還元末端側にガラクトースが付加された 複合二本鎖型糖鎖を有する遺伝子組換え卵胞刺激ホルモンは、 Gadkariらの方法 (P roteinExpression and Purification 32, 175 (2003))に順じて精製する。また、精製され た卵胞刺激ホルモン蛋白質は、 SWbeliらの方法（Bloody, 3626 (2001))に順じて糖 鎖構造の解析を行う。  Yeast strain expressing the genetically modified follicle-stimulating hormone mainly having a complex double-stranded sugar chain having no fucose residue on the reducing end and galactose added on the non-reducing end. Is seeded in a liquid YPD medium (Invitrogen) and cultured at 30 ° C for 24 hours or longer to secrete recombinant follicle-stimulating hormone into the culture supernatant. The FSH concentration contained in the culture supernatant obtained after culturing is measured using HumanFSH ELISA Kit (manufactured by Antigenix America) using Gona 卜 F (manufactured by Serono) as a standard product. Genetic recombination with a complex double-stranded sugar chain that is secreted into the yeast culture supernatant and has no fucose residue at the reducing end and galactose added at the non-reducing end as an N-linked sugar chain Follicle stimulating hormone is purified according to the method of Gadkari et al. (Protein Expression and Purification 32, 175 (2003)). The purified follicle-stimulating hormone protein is analyzed for the sugar chain structure according to the method of SWbeli et al. (Bloody, 3626 (2001)).
実施例 5  Example 5
[0239] フコースを含まな ヽ複合型糖鎖を有する卵胞刺激ホルモンの血中動態の解析  [0239] Analysis of blood fate of follicle-stimulating hormone with a complex sugar chain containing fucose
1.CHO/DG44細胞株（Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)への FSH発現 プラスミドの導入  1. Introduction of FSH expression plasmid into CHO / DG44 cell line (Proc. Natl. Acad. Sci. USA, 77, 4216 (1980))
通常の CHO/DG44細胞株に、実施例 1第 4項で作製したプラスミド pKAN- FSH o; β を導入した。これらの遺伝子導入は公知のエレクト口ポレーシヨン法 [サイトテクノロジIn a normal CHO / DG44 cell line, the plasmid pKAN-FSH o; β Was introduced. These gene introductions are performed using the well-known electoral position method [site technology
— (Cytotechnology), 3, 133 (1990)]により以下の手順で行った。まず、プラスミド pKA N-FSH a j8 30 ^ gを NEBuffer3 (New England Biolabs社製) 20 μ Lと 100単位の制限 酵素 MluI(NewEngland Biolabs社製）を含む 200 μ Lの反応液を調製し、 37°Cで 16時 間消化反応を行うことにより線状ィ匕した。反応後、該反応液に対しフ ノール/クロ口 ホルム抽出処理およびエタノール沈殿により精製を行い、線状ィ匕プラスミドを回収し た。 — (Cytotechnology), 3, 133 (1990)]. First, prepare a 200 μL reaction mixture containing 20 μL of plasmid pKA N-FSH a j8 30 ^ g and 20 μL of NEBuffer3 (New England Biolabs) and 100 units of restriction enzyme MluI (NewEngland Biolabs). Linear digestion was performed by digestion at 16 ° C for 16 hours. After the reaction, the reaction solution was purified by phenol / chloroform extraction treatment and ethanol precipitation, and a linear plasmid was recovered.
[0240] 次に、実施例 1で取得した FUT8遺伝子をダブルノックアウトした CHO/DG44細胞株 を K— PBS緩衝液 (137mmol/L KC1、 2.7 mmol/L NaCl、 8.1 mmol/L Na HPO、 1.5mmo  [0240] Next, the CHO / DG44 cell line obtained by double knockout of the FUT8 gene obtained in Example 1 was mixed with K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8.1 mmol / L Na HPO, 1.5 mmo).
2 4  twenty four
1/L KH PO、 4.0 mmol/L MgCl )に懸濁して 8 X 107細胞/ mLとした。細胞懸濁液 200The suspension was suspended in 1 / L KH PO, 4.0 mmol / L MgCl 2) to give 8 × 10 7 cells / mL. Cell suspension 200
2 4 2 2 4 2
μ L (1.6 X 106個）と上記の線状ィ匕プラスミド 9 gとを混和した後、細胞- DNA混和液 の全量を GenePulser Cuvette (電極間距離 2 mm) (BIO- RAD社製)へ移し、細胞融合 装置 Gene Pulser(BIO- RAD社製）を用いてパルス電圧 350 V、電気容量 250 μ Fの条 件で遺伝子導入を行った。遺伝子導入を行ったのち、細胞懸濁液を 10%ゥシ胎児 血清 (Life Technologies社製)および 50 μ g/mL gentamicin (ナカライテスタ社製）を添 加した IMDM培地 (LifeTechnologies社製) 30 mLに懸濁し、接着細胞培養 96ゥエルプ レート (グライナ一社製) 3枚へ 100 L/ゥエルで播種した。培養は 5% CO、 37°Cの条 After mixing μL (1.6 X 10 6 pieces) and 9 g of the above linear DNA plasmid, the total amount of the cell-DNA mixture is transferred to GenePulser Cuvette (distance between electrodes: 2 mm) (BIO-RAD) Then, gene transfer was performed using a cell fusion device Gene Pulser (manufactured by BIO-RAD) under the conditions of a pulse voltage of 350 V and an electric capacity of 250 μF. After gene transfer, IMDM medium (LifeTechnologies) 30 mL containing 10% urine fetal serum (Life Technologies) and 50 μg / mL gentamicin (Nacalai Testa) was added to the cell suspension. And then seeded on 3 adherent cell culture 96 well plates (manufactured by Grainer) at 100 L / well. Culture is 5% CO, 37 ° C
2  2
件下で行った。  I went under the matter.
[0241] 2. 500 nM MTXffif性株の取得 [0241] 2. Acquisition of 500 nM MTXffif stock
前項で得た pKAN-FSH α β導入細胞を 6日間培養した後、培養上清を除去し、 10% ゥシ胎児透析血清、 50 μ g/mLgentamicinおよび 50 nM methotrexate (MTX:シグマ 社製)を添加した IMDM培地を 100 L/ゥエルずつ添加した。この培地交換作業を 3〜 4日毎に繰り返しながら 9日間の培養を行った。次いで、 10%ゥシ胎児透析血清、 50 μ g/mLgentamicinおよび 200 nMの MTXを添カ卩した IMDM培地を用いた培地交換作 業を同様に 3〜4日毎に繰り返しながら 18日間培養し、最終的に形成されたコロニー を 24well plate (シグマ社製)に植え替えた。さら〖こ、 10%ゥシ胎児透析血清、 50 g/m Lgentamicinおよび 500 nMの MTXを添カ卩した IMDM培地を用いた培地交換作業を 3 〜4日毎に繰り返し、適宜拡大しながら 19日間培養を行い、 500nMMTX耐性株を取 得した。 After culturing the pKAN-FSH αβ-introduced cells obtained in the previous section for 6 days, the culture supernatant is removed, and 10% urine fetal dialyzed serum, 50 μg / mLgentamicin and 50 nM methotrexate (MTX: manufactured by Sigma) are used. Added IMDM medium was added at 100 L / well. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Next, the medium exchange operation using IMDM medium supplemented with 10% urine fetal dialysis serum, 50 μg / mLgentamicin and 200 nM MTX was similarly repeated every 3 to 4 days, and cultured for 18 days. The colonies formed were replanted in a 24 well plate (manufactured by Sigma). Repeat the medium change operation using IMDM medium supplemented with Sarako, 10% urine fetal dialysate serum, 50 g / m Lgentamicin and 500 nM MTX every 3-4 days, and culture for 19 days with appropriate expansion And remove the 500nMMTX resistant strain. Got.
[0242] 3.卵胞刺激ホルモン高生産株の選別  [0242] 3. Selection of high follicle stimulating hormone producing strains
前項で取得した複数の 500nM MTX耐性株より、各 1.0 X 106細胞を 5 mLの 10%ゥシ 胎児透析血清、 50 g/mLgentamicinおよび 500 nMの MTXを添カ卩した IMDM培地に 懸濁し、 T25フラスコへ播種して培養を行った。培養 3日後に培養上清を回収し、上 清中に含まれる FSH量を HumanFSH ELISA Kit(Antigenix America社製)を用いて測 定した。方法は添付マニュアルに従った。その結果、通常の CHO/DG44細胞株由来 である pKAN- FSHKC895株の培養上清中に 8.55 IU/mLの濃度で FSHが含まれて!/ヽ ることを確認した。 A plurality of 500 nM MTX-resistant cell lines obtained in the previous section, were suspended each 1.0 X 10 6 cells 5 mL of 10% © shea dialyzed fetal serum, in IMDM medium添Ka卩a 50 g / mLgentamicin and 500 nM of MTX, Culturing was performed by seeding in a T25 flask. After 3 days of culture, the culture supernatant was collected, and the amount of FSH contained in the supernatant was measured using HumanFSH ELISA Kit (manufactured by Antigenix America). The method followed the attached manual. As a result, it was confirmed that the culture supernatant of pKAN-FSHKC895 strain derived from the normal CHO / DG44 cell line contained FSH at a concentration of 8.55 IU / mL.
[0243] 4.卵胞刺激ホルモン含有培養上清の調製  [0243] 4. Preparation of culture supernatant containing follicle stimulating hormone
pKAN-FSH KC895株、実施例 1第 7項で取得した FUT8遺伝子ダブルノックアウト C HO細胞株由来 FSH産生株 (pKAN- FSHMS705)株を 10%ゥシ胎児透析血清、 50 g/ mLgentamicinおよび 500 nM MTXを添カ卩した IMDM培地に懸濁して T175フラスコ (グ ライナー社製)に播種した。コンフルェントまで培養した後、上清を除去して PBSlOmL で 2回洗浄し、 6 mM L- Glutamine、 500 nM MTX, 50 μ MNeu5Ac2enを含む ExCELL 302培地を 30 mLカ卩えて培養を行った。およそ 5日間の培養の後、培養上清を回収し て 0.22 mボトルトップフィルター (IWAKI社製)でろ過し、それぞれ KC895由来 FSH含 有培養上清、 MS705由来 FSH含有培養上清を得た。上清中に含まれる FSH量は Hu manFSH ELISA Kitを用いて測定した。  pKAN-FSH KC895 strain, FUT8 gene double knockout obtained in Example 1 Section 7 FSH production strain (pKAN- FSHMS705) strain derived from the HO cell strain was added to 10% urine fetal dialyzed serum, 50 g / mLgentamicin and 500 nM MTX The suspension was suspended in IMDM medium supplemented with seeds and seeded in a T175 flask (manufactured by Greiner). After culturing to confluence, the supernatant was removed, washed twice with PBS10 mL, and cultured with 30 mL of ExCELL 302 medium containing 6 mM L-Glutamine, 500 nM MTX, 50 μMNeu5Ac2en. After approximately 5 days of culture, the culture supernatant was collected and filtered through a 0.22 m bottle top filter (IWAKI) to obtain a culture supernatant containing KC895-derived FSH and an MS705-derived FSH. The amount of FSH contained in the supernatant was measured using a HumanFSH ELISA Kit.
[0244] 5.卵胞刺激ホルモンレセプター発現ベクターのクロー-ング  [0244] 5. Cloning of follicle stimulating hormone receptor expression vector
ヒト濾胞刺激ホルモン受容体 (以下 FSHRと略す)遺伝子配列 (UniGene:Hs.l428、配 列番号 32)より制限酵素サイト (EcoRIおよび Apal)及びコザック配列を付カ卩した二種類 の FSHR遺伝子特異的プライマー (配列番号 33および配列番号 34)を作製し、以下の PCRを行なった。即ち、ヒト精巣 cDNAをテンプレートとして含む Lの反応液 [ExTa q™DNA polymerase (タカラバイオ社製)、 10 X PCR bufferゝ 0.2 mmol/L dNTP mixture 、 0.5 /z mol/L上記プライマー (配列番号 33および配列番号 34)]を調製し、 94°Cで 3分 間加熱した後、 94°Cで 1分間、 55°Cで 1分間、 72°Cで 1分間を 1サイクルとした 30サイク ルの反応で PCRを行なった。 PCR後、反応液を 1.5%(W/V)ァガロースゲル電気泳動 に供し、約 2200bpの FSHR遺伝子の DNA断片を確認し、 QIAquickGel Extraction Kit( QIAGEN社製)を用いて精製した。 Human follicle-stimulating hormone receptor (hereinafter abbreviated as FSHR) gene sequence (UniGene: Hs.l428, SEQ ID NO: 32) specific to two FSHR genes with restriction enzyme sites (EcoRI and Apal) and Kozak sequences Primers (SEQ ID NO: 33 and SEQ ID NO: 34) were prepared, and the following PCR was performed. That is, L reaction solution containing human testis cDNA as a template [ExTa q ™ DNA polymerase (Takara Bio Inc.), 10 X PCR buffer ゝ 0.2 mmol / L dNTP mixture, 0.5 / z mol / L above primer (SEQ ID NO: 33 And SEQ ID NO: 34)], heated at 94 ° C for 3 minutes, then 30 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1 minute. PCR was performed on the reaction. After PCR, the reaction solution was subjected to 1.5% (W / V) agarose gel electrophoresis The DNA fragment of the FSHR gene of about 2200 bp was confirmed and purified using QIAquickGel Extraction Kit (manufactured by QIAGEN).
[0245] 得られた精製 FSHR DNA断片、 pT7Blue vector(Novagen社製)、 Ligation High (東洋 紡社製)を含む反応液 20 Lを調製し、 16°Cで 16時間連結反応を行なった。得られた プラスミド DNAを用い、 heatshock法により大腸菌 DH5 α株 (東洋紡社製)を形質転換 した。形質転 ·より QIAprep(R) Spin Miniprep Kit(QIAGEN社製)を用いてプラスミド DNAを調製し、 BigDyeTerminator Cycle Sequencing Ready Reaction Kit v2.0(QIAG EN社製)と DNAシーケンサ ABI PRISM 377(Applied Biosystems社製)を用いて塩基配 列を解析した。 PCRエラーに対しては、複数得られた形質転赚カゝら得たプラスミド D NAを、制限酵素 MfeI(NewEngland Biolabs社製)、 BbsI(New England Biolabs社製)で 処理し、実施例 1の手順と同様にして組み換えることで修復した。修復したプラスミド D NAの塩基配列は、上記と同様に解析を行なった。その結果、修復したプラスミド DNA の塩基配列は配列番号 32記載の FSHRをコードする遺伝子配列と同一であり、 pT7- FSHRが得られたことを確認した (図 5)。  [0245] 20 L of a reaction solution containing the obtained purified FSHR DNA fragment, pT7Blue vector (manufactured by Novagen), and Ligation High (manufactured by Toyobo) was prepared, and a ligation reaction was performed at 16 ° C for 16 hours. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heatshock method. Plasmid DNA was prepared using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDyeTerminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAG EN) and DNA sequencer ABI PRISM 377 (Applied Biosystems) Was used to analyze the nucleotide sequence. For PCR errors, the plasmid DNA obtained from the obtained transformants was treated with restriction enzymes MfeI (New England Biolabs) and BbsI (New England Biolabs). It was repaired by recombination in the same way as the procedure. The base sequence of the repaired plasmid DNA was analyzed in the same manner as described above. As a result, it was confirmed that the base sequence of the repaired plasmid DNA was the same as the gene sequence encoding FSHR described in SEQ ID NO: 32, and pT7-FSHR was obtained (FIG. 5).
[0246] 6.発現ベクター pKAN- FSHRの作製  [0246] 6. Construction of expression vector pKAN-FSHR
前項で得られた PT7-FSHRを 17.5 μ Lの水に溶解し、該液に 6単位の EcoRI (タカラ バイオ社製)、 2 μ Lの 10 X H bufferをカ卩えて 20 μ Lの反応液を調製し、 37°Cで 16時間 消化反応を行なった。反応後、フエノール/クロ口ホルム抽出処理及びエタノール沈 殿を行い、回収したプラスミドを 17.5 Lの水に溶解した。さらに該液に 7.5単位の Apa 1(タカラバィォ社製 )、 2 Lの 10 X L bufferをカ卩えて 20 Lの反応液を調製後、 37°Cで 16時間消化反応を行なった。続いて実施例 1で得られたプラスミド pKAN-FSH o; |8を 17.5 μ Lの水に溶解し、該液に 6単位の EcoRI (タカラバイオ社製)、 2 μ Lの 10 X H buff erを加えて 20 Lの反応液を調製し、 37°Cで 16時間消化反応を行なった。反応後、フ ェノール/クロ口ホルム抽出処理及びエタノール沈殿を行 、、回収したプラスミドを 17. 5 μ Lの水に溶解した。さらに該液に 7.5単位の Apal、 2 μ Lの 10 X L bufferをカ卩えて 20 μ Lの反応液を調製後、 37°Cで 16時間消化反応を行なった。  Dissolve the PT7-FSHR obtained in the previous section in 17.5 μL of water, add 6 units of EcoRI (Takara Bio) and 2 μL of 10 XH buffer to the solution, and add 20 μL of the reaction solution. Prepared and digested at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 L of water. Further, 7.5 units of Apa 1 (manufactured by Takarabio Co., Ltd.) and 2 L of 10 XL buffer were added to the solution to prepare a 20 L reaction solution, followed by digestion at 37 ° C for 16 hours. Subsequently, the plasmid pKAN-FSHo; | 8 obtained in Example 1 was dissolved in 17.5 μL of water, and 6 units of EcoRI (manufactured by Takara Bio Inc.) and 2 μL of 10 XH buffer were added to the solution. In addition, a 20 L reaction solution was prepared and digested at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 μL of water. Further, 7.5 units of Apal and 2 μL of 10 × L buffer were added to the solution to prepare a 20 μL reaction solution, followed by digestion reaction at 37 ° C. for 16 hours.
[0247] 上記で得られた FSHRDNA断片 (EcoRI-Apal)及び pKAN- FSH α β断片 (EcoRI-Apa I)を 1.5%(W/V)ァガロースゲル電気泳動に供し、それぞれ約 2200 bp、 10 kbpの DNA 断片を QIAquickGel Extraction Kit(QIAGEN社製)を用いて精製した。次いで FSHR DNA断片 (EcoRI- Apal)50ngゝ pKAN- FSH α β断片 (EcoRI- Apal)30ngゝ LigationHigh( 東洋紡社製)を含む反応液 20 /z Lを調製し、 16°Cで 16時間連結反応を行なった。得ら れたプラスミド DNAを用い、 heat shock法により大腸菌 DH5 α株 (東洋紡社製)を形質 転換した。形質転換株より QIAprep(R)Spin Miniprep Kit(QIAGEN社製)を用いてプラ スミド DNAを調製し、 pKAN- FSHRを得た (図 6)。 [0247] The FSHRDNA fragment (EcoRI-Apal) and the pKAN-FSH αβ fragment (EcoRI-ApaI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and each of about 2200 bp and 10 kbp. DNA The fragment was purified using QIAquickGel Extraction Kit (QIAGEN). Next, prepare a 20 / zL reaction solution containing 50ng ゝ pKAN- FSH αβ fragment (EcoRI-Apal) 30ng ゝ LigationHigh (Toyobo), FSHR DNA fragment (EcoRI-Apal), and ligation reaction at 16 ° C for 16 hours Was done. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (manufactured by QIAGEN) to obtain pKAN-FSHR (FIG. 6).
[0248] 7.CHO/DG44細胞株（Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)への FSHR発現 プラスミドの導入、 FSHR発現 CHO細胞の造成  [0248] 7. Introduction of FSHR expression plasmid into CHO / DG44 cell line (Proc. Natl. Acad. Sci. USA, 77, 4216 (1980), construction of FSHR expression CHO cells
通常の CHO/DG44細胞株に、前項で作製したプラスミド pKAN- FSHRを導入した。 これらの遺伝子導入は公知のエレクト口ポレーシヨン法 [サイトテクノロジー (Cytotech nology), 3, 133 (1990)]により以下の手順で行った。まず、プラスミド pKAN-FSHR30 μ gを NEBuffer4 (New England Biolabs社製) 20 μ Lと 100単位の制限酵素 AatII(NewE ngland Biolabs社製）を含む 200 Lの反応液を調製し、 37°Cで 16時間消化反応を行 うことにより線状ィ匕した。反応後、該反応液に対しフ ノール/クロ口ホルム抽出処理 およびエタノール沈殿により精製を行い、線状ィ匕プラスミドを回収した。  The plasmid pKAN-FSHR prepared in the previous section was introduced into a normal CHO / DG44 cell line. These gene introductions were performed by the following procedure according to a known electoral position method [Cytotechnology, 3, 133 (1990)]. First, prepare a 200 L reaction solution containing 30 μg of plasmid pKAN-FSHR, 20 μL of NEBuffer4 (New England Biolabs) and 100 units of restriction enzyme AatII (New England Biolabs) at 37 ° C. A linear digestion was performed by performing a time digestion reaction. After the reaction, the reaction mixture was purified by phenol / chloroform extraction treatment and ethanol precipitation to recover the linear plasmid.
[0249] 次に、 CHO/DG44細胞株を K- PBS緩衝液 (137mmol/L KC1、 2.7 mmol/L NaCl、 8.1 mmol/L Na HPO、 1.5mmol/L KH PO、 4.0 mmol/L MgCl )に懸濁して 8 X 106細胞/ [0249] Next, the CHO / DG44 cell line was added to K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8.1 mmol / L Na HPO, 1.5 mmol / L KH PO, 4.0 mmol / L MgCl 2). Suspend 8 x 10 6 cells /
2 4 2 4 2  2 4 2 4 2
mLとした。細胞懸濁液 L(1.6 X 106個）と上記の線状ィ匕プラスミド 9 gを混和し た後、細胞- DNA混和液の全量を GenePulser Cuvette (電極間距離 2 mm) (BIO-RA D社製)へ移し、細胞融合装置 Gene Pulser(BIO- RAD社製）を用いてパルス電圧 350 V、電気容量 250 Fの条件で遺伝子導入を行った。遺伝子導入を行ったのち、細胞 懸濁液を 10%ゥシ胎児血清 (Life Technologies社製)および 50 g/mL gentamicin (ナ 力ライテスタ社製）を添カ卩した IMDM培地 (LifeTechnologies社製) 10 mLに懸濁し、接 着細胞培養 T75フラスコ (グライナ一社製)へ播種した。 1日後に 10%ゥシ胎児血清、 5 00 μ g/mLG418(ナカライテスタ社製)および 50 μ g/mLgentamicinを添カ卩した IMDM培 地 10 mLに培地交換を行い、以降本培地を用いて 3〜4日毎に継代または培地交換 を行なった。一ヶ月程継代を繰り返した後、 10%ゥシ胎児血清、 50 nMMTX、 500 μ g/mL G418および 50 g/mL gentamicinを添カ卩した IMDM培地に細胞を懸濁し、 96穴 プレートに 1細胞/ゥエルずつ播種した。 5日後に同培地で培地交換を行い、 13日目 には 10%ゥシ胎児血清、 200nM MTX、 500 μ g/mL G418および 50 μ g/mL gentami cinを添カ卩した IMDM培地に培地交換を行なった。以降本培地を用いて 3〜4日毎に 拡大培養、継代または培地交換を行なった。一ヶ月程度継代を繰り返し、クローンィ匕 した FSHR発現 CHO細胞株を得た。全ての培養は 5% CO、 37°Cの条件下で行った mL. After the cell suspension L (1.6 X 10 6 cells) was mixed linear I spoon plasmid 9 g of the cell - the total amount of DNA mixed solution GenePulser Cuvette (electrode distance 2 mm) (BIO-RA D Then, gene transfer was performed using a cell fusion device Gene Pulser (manufactured by BIO-RAD) under the conditions of a pulse voltage of 350 V and an electric capacity of 250 F. After gene transfer, IMDM medium (LifeTechnologies) supplemented with 10% urine fetal serum (Life Technologies) and 50 g / mL gentamicin (National Power Tester) 10% The suspension was suspended in mL, and seeded in an adherent cell culture T75 flask (manufactured by Grainer). One day later, the medium was changed to 10 mL of IMDM medium supplemented with 10% urine fetal serum, 500 μg / mL G418 (Nacalai Testa) and 50 μg / mL gentamicin. Subculture or medium change was performed every 3-4 days. After repeated passage for about one month, the cells are suspended in IMDM medium supplemented with 10% fetal bovine serum, 50 nMMTX, 500 μg / mL G418 and 50 g / mL gentamicin, and 96-well One cell / well was seeded on the plate. After 5 days, the medium was changed with the same medium, and on day 13, the medium was changed to IMDM medium supplemented with 10% ushi fetal serum, 200 nM MTX, 500 μg / mL G418 and 50 μg / mL gentamicin. Was done. Thereafter, this culture medium was used to expand, subculture, or change the medium every 3 to 4 days. The passage was repeated for about one month to obtain a cloned FSHR-expressing CHO cell line. All cultures were performed at 5% CO and 37 ° C.
2  2
[0250] 8.細胞内 cAMP濃度の測定 [0250] 8. Measurement of intracellular cAMP concentration
前項で作製した FSHR発現 CHO細胞を 10%透析ゥシ胎児血清、 g/mLgentami cin、 500 μ g/mL G418および 200 nM methotrexate (MTX:シグマ社製)を添カ卩した IM DM培地に懸濁し、 2 X 105cells/ゥエルで 24穴プレートに播種した。 5% CO、 37°Cの FSHR-expressing CHO cells prepared in the previous section are suspended in IM DM medium supplemented with 10% dialyzed fetal bovine serum, g / mLgentamicin, 500 μg / mL G418 and 200 nM methotrexate (MTX: Sigma). 2 x 10 5 cells / well in a 24-well plate. 5% CO, 37 ° C
2 条件下で 1〜2日間培養した後、 0.05ng/mL〜400 ng/mL FSHサンプル、 100 uM 3-1 sobuty卜 1-methylxanthine入りの培地に培地交換を行った。  After culturing for 1 to 2 days under 2 conditions, the medium was changed to a medium containing 0.05 ng / mL to 400 ng / mL FSH sample and 100 uM 3-1 sobuty 1-methylxanthine.
[0251] さら〖こ、 15分後に上清を除去して PBSGnvitrogen社製)で細胞を洗浄、 cAMP測定キ ット添付の溶解液を用いて細胞を溶解した。 cAMP測定キットは cAMPBiotrak Enzyme immunoassay kit(Amersham社製)を用い、以降はキット添付手順書に従って測定を行 つた。陽性対照には 10 Mホルスコリン (Wako社製)を用いた。  [0251] After 15 minutes, the supernatant was removed and the cells were washed with PBSGnvitrogen), and the cells were lysed using the lysate attached to the cAMP measurement kit. cAMP Biotrak Enzyme immunoassay kit (manufactured by Amersham) was used as the cAMP measurement kit, and the measurement was subsequently performed according to the kit attachment procedure. As a positive control, 10 M forskolin (manufactured by Wako) was used.
KC895由来 FSH含有培養上清、 MS705由来 FSH含有培養上清、巿販 FSH製剤 Gon a卜 F(Serono社製)、巿販下垂体由来 FSH試薬 (CORTEX社製)の細胞内 cAMP測定の 結果、いずれのサンプルも濃度依存的な細胞内 cAMPの誘導効果を示した。また、い ずれのサンプルも同程度の細胞内 cAMPの誘導効果を示した。得られた結果より EC5 0を求めたところ、 Gona Fで 4.68(3.10-7.06)ng/mL、 phFSI~^、5.77 (4.89- 6.81)ng/mL 、 KC895由来 FSH含有培養上清で 2.478(1.13-5.45)ng/mL、 MS705由来 FSH含有培 養上清で 3.77 (2.84-5.00)ng/mLであった（図 7)。 EC50は、解析ソフトウェア GraphPad prism 4(グラフパッド社製)を用いて算出した。  KC895-derived FSH-containing culture supernatant, MS705-derived FSH-containing culture supernatant, commercial FSH preparation Gon a 卜 F (manufactured by Serono), and commercial pituitary-derived FSH reagent (CORTEX), as a result of intracellular cAMP measurement, All samples showed a concentration-dependent effect of inducing intracellular cAMP. In addition, both samples showed the same level of intracellular cAMP induction effect. EC50 was determined from the obtained results.As a result, Gona F was 4.68 (3.10-7.06) ng / mL, phFSI ~ ^, 5.77 (4.89-6.81) ng / mL, KC895-derived FSH-containing culture supernatant was 2.478 (1.13 -5.45) ng / mL, 3.705 (2.84-5.00) ng / mL in the MS705-derived FSH-containing culture supernatant (Fig. 7). EC50 was calculated using analysis software GraphPad prism 4 (manufactured by Graphpad).
[0252] 9.卵胞刺激ホルモンの精製  [0252] 9. Purification of follicle stimulating hormone
培養上清からの FSH精製は、マウス抗ヒト FSH抗体 (cat:MCA1028、 SEROTEC社製） をァガロースに個相化したァフィユティーカラム (1.3mL)を用いて行った。  FSH purification from the culture supernatant was performed using a affinity column (1.3 mL) in which mouse anti-human FSH antibody (cat: MCA1028, manufactured by SEROTEC) was phased into agarose.
カラムを 0.1 MNaHCO、 0.25 M NaClからなる平衡化バッファーで平衡化し、 100 μ g 以上の FSHを含む培養上清を添カ卩した。 Washは平衡化バッファー及び 0.2 MNaHC 0、 0.5 M NaClからなるバッファーで行い、 0.1 Mクェン酸、 0.5 MNaCl(pH 7.0)からなEquilibrate the column with equilibration buffer consisting of 0.1 M NaHCO, 0.25 M NaCl, 100 μg The culture supernatant containing the above FSH was added. Wash is performed with an equilibration buffer and a buffer consisting of 0.2 M NaHC0 and 0.5 M NaCl, and consists of 0.1 M citrate and 0.5 M NaCl (pH 7.0).
3 Three
る溶出バッファーで溶出させた。溶出後は直ちに 2 M Tris-HClで溶液を中和させた  Elute with elution buffer. Immediately after elution, the solution was neutralized with 2 M Tris-HCl.
[0253] 得られたサンプルは Amicon(R)Ultra-4遠心式フィルターユニット (NMWF 10,000)を用 いて PBS (インビトロジェン社製)に濃縮置換した。その結果、精製された MSFSHおよ び KCFSHが得られた。 [0253] The obtained sample was concentrated and replaced with PBS (manufactured by Invitrogen) using an Amicon (R) Ultra-4 centrifugal filter unit (NMWF 10,000). As a result, purified MSFSH and KCFSH were obtained.
10.卵胞刺激ホルモンサンプルの血中滞留性評価  10. Evaluation of retention in blood of follicle stimulating hormone samples
本試験には 11週〜 12週齢の Crlj:CDlマウスを使用した。マウスに対し、 10 g/1匹 で MS705由来 FSHおよび KC895由来 FSHサンプルをそれぞれ尾静脈より投与し、 5分 、 30分、 1時間、 2時間、 4時間、 8時間、 12時間、 24時間後にへパリンコートのへマトク リット管 (IWAKI社製)を用いて尾静脈採血を行った。得られた血液サンプルは、直ち に冷却遠心機を用いて 3000rpm、 10分間、 4°Cで遠心を行い、血漿として保存した。 得られた血漿サンプル中に含まれる FSH量は、 HumanFSH ELISA Kit(Antigenix Am erica社製)を用いて測定した。測定方法は添付マニュアルに従った。  Crlj: CDl mice aged 11-12 weeks were used in this study. Mice were administered MS705-derived FSH and KC895-derived FSH samples from the tail vein at 10 g / mouse, respectively, 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours later. Blood was collected from the tail vein using a heparin-coated hematocrit tube (IWAKI). The obtained blood sample was immediately centrifuged at 3000 rpm for 10 minutes at 4 ° C using a refrigerated centrifuge and stored as plasma. The amount of FSH contained in the obtained plasma sample was measured using HumanFSH ELISA Kit (manufactured by Antigenix America). The measurement method followed the attached manual.
[0254] 得られた血漿中の FSH濃度推移の解析を行なったところ、分布相の半減期は MSFS H、 KCFSHでそれぞれ 0.53時間、 0.35時間であり、フコースが付カ卩していない糖鎖を 有する MSFSHはフコースの付カ卩した糖鎖を有する KCFSHよりも 1.51倍長い血中半減 期を示した。また、消失相の半減期は MSFSH、 KCFSHでそれぞれ 4.62時間、 4.23時 間であり、同様に MSFSHは KCFSHよりも長い血中半減期を示した。さらに、 AUC(0- ∞)で比較すると、 MSFSHは KCFSHに対して 1.45倍になり、血中滞留性の向上が示さ れた (図 8)。 [0254] When the FSH concentration transition in the obtained plasma was analyzed, the half-life of the distribution phase was 0.53 hours and 0.35 hours for MSFS H and KCFSH, respectively, and sugar chains without fucose attached were observed. MSFSH had a half-life in blood that was 1.51 times longer than KCFSH, which had a sugar chain with fucose attached. The half-life of the elimination phase was 4.62 hours and 4.23 hours for MSFSH and KCFSH, respectively. Similarly, MSFSH showed a longer blood half-life than KCFSH. Furthermore, when compared with AUC (0-∞), MSFSH was 1.45 times that of KCFSH, indicating an improvement in blood retention (Fig. 8).
産業上の利用可能性  Industrial applicability
[0255] 本発明により、 N-グリコシド結合複合型糖鎖を有する遺伝子組換え卵胞刺激ホル モン分子カゝらなる組成物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末 端の N-ァセチルダルコサミンにフコースが結合して ヽな 、糖鎖である卵胞刺激ホル モン組成物を提供することができる。 [0255] According to the present invention, there is provided a composition comprising a recombinant follicle-stimulating hormone molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is a reduced end of the sugar chain. It is possible to provide a follicle stimulating hormone composition which is a sugar chain in which fucose is bound to the end N-acetylcylcosamine.
配列表フリーテキスト 配列番号 2-人工配列の説明：合成 DNA 配列番号 4-人工配列の説明：合成 DNA 配列番号 6-人工配列の説明：アミノ酸配列 配列番号 8-人工配列の説明：アミノ酸配列 配列番号 22- -人工配列の説明 ：合成 DNA 配列番号 23- -人工配列の説明 ：合成 DNA 配列番号 24- -人工配列の説明 ：合成 DNA 配列番号 25- -人工配列の説明 ：合成 DNA 配列番号 26- -人工配列の説明 ：合成 DNA 配列番号 27- -人工配列の説明 ：合成 DNA 配列番号 28- -人工配列の説明 ：合成 DNA 配列番号 29- -人工配列の説明 ：合成 DNA 配列番号 30- -人工配列の説明 ：合成 DNA 配列番号 31- -人工配列の説明 ：合成 DNA 配列番号 33- -人工配列の説明 ：合成 DNA 配列番号 34- -人工配列の説明 ：合成 DNA Sequence listing free text SEQ ID NO: 2-Description of artificial sequence: Synthetic DNA SEQ ID NO: 4-Description of artificial sequence: Synthetic DNA SEQ ID NO: 6-Description of artificial sequence: Amino acid sequence SEQ ID NO: 8-Description of artificial sequence: Amino acid sequence SEQ ID NO: 22- -Artificial Sequence Description: Synthetic DNA SEQ ID NO: 23- -Artificial Sequence Description: Synthetic DNA SEQ ID NO: 24- -Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 25- -Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 26- -Artificial Sequence Description: Synthetic DNA SEQ ID NO: 27- -Description of artificial sequence: Synthetic DNA SEQ ID NO: 28- -Description of artificial sequence: Synthetic DNA SEQ ID NO: 29- -Description of artificial sequence: Synthetic DNA SEQ ID NO: 30- -Description of artificial sequence: Synthetic DNA SEQ ID NO: 31- -Description of artificial sequence: Synthetic DNA SEQ ID NO: 33- -Description of artificial sequence: Synthetic DNA SEQ ID NO: 34- -Description of artificial sequence: Synthetic DNA

Claims

請求の範囲 The scope of the claims
[1] N-グリコシド結合複合型糖鎖を有する遺伝子組換え卵胞刺激ホルモン分子からなる 組成物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末端の N-ァセチルダ ルコサミンにフコースが結合して ヽな 、糖鎖である卵胞刺激ホルモン組成物。  [1] A composition comprising a genetically modified follicle-stimulating hormone molecule having an N-glycoside-linked complex sugar chain, wherein the N-glycoside-linked complex sugar chain is fucose to N-acetylylcosamine at the reducing end of the sugar chain. A follicle-stimulating hormone composition, which is a sugar chain that binds to ヽ.
[2] N-グリコシド結合複合型糖鎖力 卵胞刺激ホルモンを構成する exサブユニットおよび βサブユニットに結合している Ν-グリコシド結合複合型糖鎖である、請求項 1に記載 の卵胞刺激ホルモン組成物。  [2] N-glycoside-bonded complex sugar chain strength The follicle-stimulating hormone according to claim 1, which is a Ν-glycoside-bonded complex sugar chain bound to the ex subunit and β subunit constituting the follicle stimulating hormone. Composition.
[3] Ν-グリコシド結合複合型糖鎖力 該糖鎖還元末端の Ν-ァセチルダルコサミンの 6位 にフコースの 1位が α結合していない糖鎖である、請求項 1または 2項に記載の卵胞 刺激ホルモン組成物。 [3] Ν-glycoside-bonded complex sugar chain strength According to claim 1 or 2, wherein the sugar chain is not α- bonded at position 1 of fucose to position 6 of Ν-acetyldarcosamine at the reducing end of the sugar chain. The follicle stimulating hormone composition described.
[4] 卵胞刺激ホルモンを構成する exサブユニットが、以下の (a)、（b)、（c)、（d)、（e)および (1) 力もなる群力 選ばれるポリペプチドである、請求項 1〜3のいずれか 1項に記載の卵 胞刺激ホルモン組成物。  [4] The ex subunit that constitutes the follicle stimulating hormone is a polypeptide selected from the following (a), (b), (c), (d), (e) and (1) group forces that also have power, The follicle stimulating hormone composition according to any one of claims 1 to 3.
(a)配列番号 5で表されるァミノ配列からなるポリペプチド；  (a) a polypeptide consisting of an amino sequence represented by SEQ ID NO: 5;
(b)配列番号 6で表されるァミノ配列力もなるポリペプチド；  (b) a polypeptide having amino amino acid sequence represented by SEQ ID NO: 6;
(c)配列番号 5で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入  (c) In the amino acid sequence represented by SEQ ID NO: 5, one or more amino acids are deleted, substituted, or inserted
および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン aサブュ- ットが有する活性と実質的に同一の活性を有するポリペプチド；  And Z or a polypeptide having an added amino acid sequence and having substantially the same activity as that of the follicle stimulating hormone a subunit;
(d) 配列番号 6で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン aサブュ ニットが有する活性と実質的に同一の活性を有するポリペプチド；  (d) In the amino acid sequence represented by SEQ ID NO: 6, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and the activity and substantial activity of the follicle stimulating hormone a subunit A polypeptide having the same activity as
(e)配列番号 5で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列から なり、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活性を 有するポリペプチド；  (e) a polypeptide comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 5 and having substantially the same activity as that of the follicle stimulating hormone OC subunit;
(D配列番号 6で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列から なり、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活性を 有するポリペプチド； (D) a polypeptide comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by D SEQ ID NO: 6 and having substantially the same activity as that of the follicle stimulating hormone OC subunit;
[5] 卵胞刺激ホルモンを構成する αサブユニットが、以下の (a)、（b)、（c)および (d)力 な る群力も選ばれる DNAがコードするポリペプチドである、請求項 1〜3のいずれ力 1項 に [5] The α subunit constituting the follicle-stimulating hormone is a polypeptide encoded by a DNA selected from the following (a), (b), (c), and (d) a powerful group force: Any force of ~ 3 to 1 term
記載の卵胞刺激ホルモン組成物。  The follicle stimulating hormone composition described.
(a) 配列番号 1で表される塩基配列からなる DNA;  (a) DNA consisting of the base sequence represented by SEQ ID NO: 1;
(b) 配列番号 2で表される塩基配列力 なる DNA；  (b) DNA having the nucleotide sequence represented by SEQ ID NO: 2;
(c) 配列番号 1で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活 性を有する蛋白質をコードする DNA;  (c) It encodes a protein that has the same activity as that of the follicle stimulating hormone OC subunit, and is hybridized under stringent conditions with DNA having the nucleotide sequence shown by SEQ ID NO: 1. DNA to do;
(d) 配列番号 2で表される塩基配列力もなる DNAとストリンジェントな条件でハイプリ ダイズし、かつ卵胞刺激ホルモン OCサブユニットが有する活性と実質的に同一の活 性を有する蛋白質をコードする DNA;  (d) DNA that encodes a protein that is hybridized under stringent conditions with DNA having the base sequence ability represented by SEQ ID NO: 2 and that has substantially the same activity as that of the follicle-stimulating hormone OC subunit ;
[6] 卵胞刺激ホルモンを構成する βサブユニットが、以下の (a)、（b)、（c)、（d)、（e)および (1) 力もなる群力 選ばれるポリペプチドである、請求項 1〜3のいずれか 1項に記載の卵 胞刺激ホルモン組成物。  [6] The β subunit constituting the follicle-stimulating hormone is a polypeptide that is selected from the following (a), (b), (c), (d), (e) and (1) group forces that also have power, The follicle stimulating hormone composition according to any one of claims 1 to 3.
(a)配列番号 7で表されるァミノ配列からなるポリペプチド；  (a) a polypeptide comprising an amino sequence represented by SEQ ID NO: 7;
(b)配列番号 8で表されるァミノ配列力もなるポリペプチド；  (b) a polypeptide having an amino acid sequence represented by SEQ ID NO: 8;
(c)配列番号 7で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入  (c) In the amino acid sequence represented by SEQ ID NO: 7, one or more amino acids are deleted, substituted, or inserted
および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン j8サブュ- ットが有する活性と実質的に同一の活性を有するポリペプチド；  And Z or a polypeptide having an added amino acid sequence and having substantially the same activity as that of the follicle stimulating hormone j8 subunit;
(d)配列番号 8で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入  (d) In the amino acid sequence represented by SEQ ID NO: 8, one or more amino acids are deleted, substituted, or inserted
および Zまたは付加されたアミノ酸配列力もなり、かつ卵胞刺激ホルモン j8サブュ- ットが有する活性と実質的に同一の活性を有するポリペプチド；  And Z or a polypeptide having an added amino acid sequence and having substantially the same activity as that of the follicle stimulating hormone j8 subunit;
(e)配列番号 7で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列から なり、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活性を 有するポリペプチド； (D配列番号 8で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列から なり、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活性を 有するポリペプチド。 (e) a polypeptide comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 7 and having substantially the same activity as that of the follicle stimulating hormone j8 subunit; (D A polypeptide comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by D SEQ ID NO: 8, and having substantially the same activity as that of the follicle stimulating hormone j8 subunit.
[7] 卵胞刺激ホルモンを構成する βサブユニットが、以下の (a)、（b)、（c)および (d)力 なる 群から選ばれる DNAがコードするポリペプチドである、請求項 1〜3のいずれ力 1項に 記  [7] The β subunit constituting the follicle-stimulating hormone is a polypeptide encoded by a DNA selected from the following groups (a), (b), (c) and (d): Any force of 3
載の卵胞刺激ホルモン組成物。  The follicle stimulating hormone composition.
(a)配列番号 3で表される塩基配列からなる DNA；  (a) DNA consisting of the base sequence represented by SEQ ID NO: 3;
(b)配列番号 4で表される塩基配列力 なる DNA；  (b) DNA having a nucleotide sequence represented by SEQ ID NO: 4;
(c)配列番号 3で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブリダ ィズし、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活性 を有するポリペプチドをコードする DNA ;  (c) a polypeptide that has substantially the same activity as that of the follicle-stimulating hormone j8 subunit, and that is hybridized under stringent conditions with DNA having the base sequence ability represented by SEQ ID NO: 3. Encoding DNA;
(d)配列番号 4で表される塩基配列力もなる DNAとストリンジェントな条件でハイブリダ ィズし、かつ卵胞刺激ホルモン j8サブユニットが有する活性と実質的に同一の活性 を有するポリペプチドをコードする DNA。  (d) encodes a polypeptide that hybridizes under stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 4 and has substantially the same activity as that of the follicle stimulating hormone j8 subunit. DNA.
[8] 卵胞刺激ホルモン (Xサブユニット及び βサブユニットをコードする DNAを宿主細胞に 導入  [8] Follicle stimulating hormone (introducing DNA encoding X subunit and β subunit into host cells)
して得られる、請求項 1〜7の 、ずれか 1項に記載の卵胞刺激ホルモン組成物を生 産する形質転換体。  A transformant producing the follicle-stimulating hormone composition according to any one of claims 1 to 7, which is obtained as described above.
[9] 宿主細胞が、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、または N- グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1 位が oc結合する糖鎖修飾に関与する酵素の活性が欠失するようにゲノムが改変され た細胞である、請求項 8に記載の形質転換体。  [9] The host cell is oc-coupled to position 6 of fucose at the 6th position of the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or N-glycidyl-linked N-acetylyldarcosamine 9. The transformant according to claim 8, which is a cell whose genome has been modified so that the activity of an enzyme involved in sugar chain modification is lost.
[10] 宿主細胞が、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、または N- グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1 位が a結合する糖鎖修飾に関与する酵素のゲノム上の対立遺伝子のすべてがノック アウトされた細胞である、請求項 9に記載の形質転換体。  [10] The host cell is linked to the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose, or N-acetylyldarcosamine at the reducing end of N-glycoside-linked complex sugar chain at position 1 of fucose 10. The transformant according to claim 9, wherein all of the alleles on the genome of the enzyme involved in glycosylation are knocked out.
[11] 細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素力 GDP-マンノース 4,6 -デヒ [11] Intracellular sugar nucleotides GDP-enzyme power involved in the synthesis of fucose GDP-mannose 4,6 -Dehi
ドラターゼ及び GDP-4-ケト -6-デォキシ- D-マンノース- 3, 5-ェピメラーゼからなる群 から  From the group consisting of dratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase
選ばれる酵素である、請求項 9または 10に記載の形質転換体。  The transformant according to claim 9 or 10, which is an enzyme to be selected.
[12] GDP-マンノース 4,6-デヒドラターゼカ 以下の（a)、（b)及び (c)力 なる群から選ばれ る蛋白質である、請求項 11に記載の形質転換体。 [12] The transformant according to claim 11, which is a protein selected from the following groups (a), (b) and (c): GDP-mannose 4,6-dehydratase.
(a)配列番号 10で表されるアミノ酸配列からなる蛋白質；  (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 10;
(b)配列番号 10で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入  (b) In the amino acid sequence represented by SEQ ID NO: 10, one or more amino acids are deleted, substituted, or inserted
および Zまたは付加されたアミノ酸配列力もなり、かつ GDP-マンノース 4,6-デヒドラ タ  And Z or added amino acid sequence, and GDP-mannose 4,6-dehydrata
ーゼ活性を有する蛋白質；  A protein having lyase activity;
(c)配列番号 10で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質；  (c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10 and having GDP-mannose 4,6-dehydratase activity;
[13] GDP-マンノース 4,6-デヒドラターゼカ 以下の (a)及び (b)力 なる群力 選ばれる DN Aがコードする蛋白質である、請求項 11に記載の形質転換体。  [13] The transformant according to claim 11, which is a protein encoded by GDP selected from GDP-mannose 4,6-dehydratase (a) and (b) group power as follows.
(a)配列番号 9で表される塩基配列からなる DNA；  (a) DNA comprising the base sequence represented by SEQ ID NO: 9;
(b)配列番号 9で表される塩基配列力もなる DNAとストリンジェントな条件でハイブリダ ィズし、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質をコードする DN A。  (b) DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence shown by SEQ ID NO: 9 and encodes a protein having GDP-mannose 4,6-dehydratase activity.
[14] GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼカ 以下の（a)、 (b)および [14] GDP-4-keto-6-deoxy-D-mannose-3,5-epimelasca (a), (b) and
(c)からなる群から選ばれる蛋白質である、請求項 11に記載の形質転換体。 12. The transformant according to claim 11, which is a protein selected from the group consisting of (c).
(a)配列番号 12で表されるアミノ酸配列からなる蛋白質；  (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 12;
(b)配列番号 12で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入  (b) In the amino acid sequence represented by SEQ ID NO: 12, one or more amino acids are deleted, substituted, or inserted
および Zまたは付加されたアミノ酸配列力もなり、かつ GDP-4-ケト -6-デォキシ -D- マンノース- 3,5-ェピメラーゼ活性を有する蛋白質；  And Z or a protein having an added amino acid sequence ability and having GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity;
(c)配列番号 12で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有する 蛋白質。 (c) an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 12 And a protein having GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity.
[15] GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼが、以下の（a)及び (b)から な  [15] GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase consists of the following (a) and (b)
る群力 選ばれる DNAがコードする蛋白質である、請求項 11に記載の細胞。  12. The cell according to claim 11, which is a protein encoded by the selected DNA.
(a)配列番号 11で表される塩基配列からなる DNA；  (a) DNA comprising the base sequence represented by SEQ ID NO: 11;
(b)配列番号 11で表される塩基配列力もなる DNAとストリンジェントな条件でノ、イブリ ダ  (b) DNA having the nucleotide sequence represented by SEQ ID NO: 11 and stringers under stringent conditions
ィズし、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3, 5-ェピメラーゼ活性を有する 蛋白  And a protein with GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity
質をコードする DNA。  DNA that encodes quality.
[16] N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が a結合する糖鎖修飾に関与する酵素が a 1,6-フコシルトランスフェラーゼであ る請求項 9または 10に記載の形質転換体。  [16] N-glycoside-linked complex type sugar chain N-acetylyldarcosamine at the reducing end is a 1,6-fucosyltransferase that is involved in the sugar chain modification in which the 1-position of fucose is a-linked to the 6-position The transformant according to claim 9 or 10.
[17] a 1,6-フコシルトランスフェラーゼカ 以下の（a)、 (b)、（c)、（d)、（e)および (1)力らなる 群力 選ばれる蛋白質である、請求項 16に記載の形質転換体。  [17] a 1,6-fucosyltransferase is a protein selected from the following (a), (b), (c), (d), (e), and (1) group force consisting of forces: A transformant according to 1.
(a)配列番号 15で表されるアミノ酸配列からなる蛋白質；  (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 15;
(b)配列番号 16で表されるアミノ酸配列力もなる蛋白質；  (b) a protein having an amino acid sequence ability represented by SEQ ID NO: 16;
(c)配列番号 15で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ α 1,6-フコシルトランスフェラ ーゼ活性を有する蛋白質；  (c) In the amino acid sequence represented by SEQ ID NO: 15, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have α 1,6-fucosyltransferase activity. A protein having;
(d)配列番号 16で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ α 1,6-フコシルトランスフェラ ーゼ活性を有する蛋白質；  (d) In the amino acid sequence represented by SEQ ID NO: 16, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have α 1,6-fucosyltransferase activity. A protein having;
(e)配列番号 15で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ a 1,6-フコシルトランスフェラーゼ活性を有する蛋白質；  (e) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 15 and having a 1,6-fucosyltransferase activity;
(1)配列番号 16で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ α 1,6-フコシルトランスフェラーゼ活性を有する蛋白質； (1) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 16 and having α1,6-fucosyltransferase activity;
[18] a 1,6-フコシルトランスフェラーゼカ 以下の（a)、（b)、（c)及び (d)力 なる群力 選 ばれる DNAがコードする蛋白質である、請求項 16に記載の形質転換体。 [18] The transformation according to claim 16, which is a protein encoded by a DNA selected from the following (a), (b), (c) and (d) body.
(a)配列番号 13で表される塩基配列力もなる DNA；  (a) DNA having a nucleotide sequence represented by SEQ ID NO: 13;
(b)配列番号 14で表される塩基配列力もなる DNA;  (b) DNA having a nucleotide sequence represented by SEQ ID NO: 14;
(c)配列番号 13で表される塩基配列力もなる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ α 1,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする DN A  (c) DNA encoding a protein that is hybridized under stringent conditions with a DNA having the nucleotide sequence represented by SEQ ID NO: 13 and that has α1,6-fucosyltransferase activity
(d)配列番号 14で表される塩基配列力もなる DNAとストリンジェントな条件でハイプリ ダイズし、かつ α 1,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする DN A (d) DN A that is hybridized with a DNA having the nucleotide sequence represented by SEQ ID NO: 14 under stringent conditions and encodes a protein having α1,6-fucosyltransferase activity
[19] N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1位が α結合した糖鎖構造を認識するレクチンに耐性である、請求項 8〜18のいず れか 1項 [19] The N-glycoside-linked complex-type sugar chain reducing terminal N-acetylyldarcosamine at position 6 and fucose 1-position are resistant to a lectin that recognizes an α-linked sugar chain structure. Either one
に記載の形質転換体。  A transformant according to 1.
[20] レクチン耐性力 Ν-グリコシド結合複合型糖鎖還元末端の Ν-ァセチルダルコサミン の 6位とフコースの 1位が a結合した糖鎖構造を認識するレクチンを含む培地で培養 した場合に、ゲノムが改変される以前の細胞よりも高い生存率を示すことである、請求 項 19に記載の形質転換体。  [20] Resistance to lectin When cultured in a medium containing a lectin that recognizes a glycan structure in which Ν-glycosidic complex glycan reducing end Ν-acetyldarcosamine 6-position and fucose 1-position are a-linked The transformant according to claim 19, wherein the transformant has a higher survival rate than a cell before the genome is modified.
[21] 少なくとも、以下の (a)、（b)、（c)及び (d)力 なる群力 選ばれるレクチンの一つに耐性 である、請求項 19または 20に記載の形質転換体。  [21] The transformant according to claim 19 or 20, wherein the transformant is resistant to at least one of the following (a), (b), (c) and (d) force group forces selected.
(a) レンズマメレクチン LCA (Lens Culinaris由来の Lentil Agglutinin)；  (a) Lentil lectin LCA (Lentil Agglutinin from Lens Culinaris);
(b) エンドゥマメレクチン PSA (Pisum sativum由来の Pea Lectin)；  (b) Endo bean lectin PSA (Peasum sativum-derived Pea Lectin);
(c) ソラマメレクチン VFA (Vicia faba由来の Agglutinin)：  (c) Broad bean lectin VFA (Agglutinin from Vicia faba):
(d) ヒィロチャワンタケレクチン AAL (Aleuria aurantia由来の Lectin) .  (d) Herochawantake lectin AAL (Lectin from Aleuria aurantia).
[22] 宿主細胞が、下記の (a)、（b)、（c)、（d)、（ 、（1)、（g)、（h)、（i)及び (j)力 なる群力 選ば れる細胞である請求項 8〜21のいずれ力 1項に記載の形質転換体。 (a)チャイニーズノヽムスター卵巣組織由来 CHO細胞； [22] The host cell has the following group power (a), (b), (c), (d), (, (1), (g), (h), (i) and (j) The transformant according to any one of claims 8 to 21, which is a selected cell. (a) Chinese nomstar ovarian tissue-derived CHO cells;
(b)ラットミエローマ細胞株 YB2/3HL.P2.G11.16Ag.20細胞；  (b) rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cells;
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0- Agl4細胞；  (d) mouse myeloma cell line SP2 / 0-Agl4 cells;
(e)シリアンノヽムスター腎臓組織由来 BHK細胞；  (e) Syrian Nomster kidney tissue-derived BHK cells;
(1)ヒト白血病細胞株ナマルバ細胞；  (1) human leukemia cell line Namalva cells;
(g)胚性幹細胞；  (g) embryonic stem cells;
(h)受精卵細胞；  (h) fertilized egg cells;
(0植物細胞；  (0 plant cells;
(j)酵母。  (j) Yeast.
[23] 請求項 8〜22のいずれか 1項に記載の形質転換体を培地に培養し、培養物中に卵 胞刺激ホルモン組成物を生成蓄積させ、該培養物から卵胞刺激ホルモン組成物を 採取する工程を含む、卵胞刺激ホルモン組成物の製造方法。  [23] The transformant according to any one of claims 8 to 22 is cultured in a medium, a follicle stimulating hormone composition is produced and accumulated in the culture, and the follicle stimulating hormone composition is obtained from the culture. The manufacturing method of a follicle stimulating hormone composition including the process to extract | collect.
[24] 請求項 23に記載の製造方法で得られた卵胞刺激ホルモン組成物。 [24] A follicle-stimulating hormone composition obtained by the production method according to claim 23.
[25] 請求項 1〜7および 24に記載の卵胞刺激ホルモン組成物を有効成分として含有する 医薬。 [25] A medicament comprising the follicle stimulating hormone composition according to any one of [1] to [7] and [24] as an active ingredient.
[26] 請求項 1〜7および 24に記載の卵胞刺激ホルモン組成物を有効成分として含有する 卵胞成熟及び排卵障害の治療剤。  [26] A therapeutic agent for follicular maturation and ovulation disorder, comprising the follicle stimulating hormone composition according to any one of claims 1 to 7 and 24 as an active ingredient.
[27] 請求項 1〜7および 24に記載の卵胞刺激ホルモン組成物を有効成分として含有する 精子形成及び成熟障害の治療剤。 [27] A therapeutic agent for spermatogenesis and maturation disorder, comprising the follicle stimulating hormone composition according to any one of claims 1 to 7 and 24 as an active ingredient.
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