WO2006109696A1 - Composition comprenant une folliculo-stimuline genetiquement modifiee - Google Patents

Composition comprenant une folliculo-stimuline genetiquement modifiee 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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stimulating hormone
seq
dna
amino acid
follicle
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PCT/JP2006/307375
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English (en)
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/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—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

L'invention vise à procurer une composition de folliculo-stimuline comprenant une molécule de folliculo-stimuline génétiquement modifiée ayant une chaîne de sucre complexe de type à liaison N-glycoside, la chaîne de sucre complexe de type à liaison N-glycoside étant une chaîne de sucre dans laquelle le fucose n’est pas lié à la N-acétylglucosamine à l’extrémité réductrice de la chaîne de sucre, ainsi que son utilisation.
PCT/JP2006/307375 2005-04-06 2006-04-06 Composition comprenant une folliculo-stimuline genetiquement modifiee WO2006109696A1 (fr)

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JP2009273427A (ja) * 2008-05-16 2009-11-26 Jcr Pharmaceuticals Co Ltd 組換え体ヒトｆｓｈの製造方法
CN110121354A (zh) * 2016-08-29 2019-08-13 宾夕法尼亚大学理事会 靶向***受体(fshr)的最优化的合成共有免疫原性组合物

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