WO2006109695A1 - Composition comprising genetically engineered haptoglobin - Google Patents

Composition comprising genetically engineered haptoglobin Download PDF

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Publication number
WO2006109695A1
WO2006109695A1 PCT/JP2006/307374 JP2006307374W WO2006109695A1 WO 2006109695 A1 WO2006109695 A1 WO 2006109695A1 JP 2006307374 W JP2006307374 W JP 2006307374W WO 2006109695 A1 WO2006109695 A1 WO 2006109695A1
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Prior art keywords
haptoglobin
seq
amino acid
acid sequence
dna
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PCT/JP2006/307374
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French (fr)
Japanese (ja)
Inventor
Kazuya Yamano
Yutaka Kanda
Mitsuo Satoh
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Kyowa Hakko Kogyo Co., Ltd.
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Publication of WO2006109695A1 publication Critical patent/WO2006109695A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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 having a recombinant haptoglobin molecular force having an N-glycoside-linked complex sugar chain, wherein the N-glycoside-bonded complex sugar chain is N-acetyl at the reducing end of the sugar chain.
  • the present invention relates to a haptoglobin composition which is a sugar chain and has its use, wherein fucose is bound to darcosamine.
  • Haptoglobin is a plasma glycoprotein with a molecular weight of 85,000 to 400,000 produced by mature granular leukocytes represented by eosinophils present in hepatocytes and lymph nodes. Usually, it exists in blood at a concentration of 0.2 to 3 g / L, and when hemoglobin is released into the blood by hemolysis, it specifically binds to form a complex (Non-patent Document 1). This complex is transported to the liver and then taken up into the cell by binding to receptors such as CD163 expressed in cells of the reticuloendothelial cell line, and to hepatoxylase by hemoxygenase. Metabolized (Non-Patent Documents 2 to 4).
  • the blood half-life of human haptoglobin is 3.5 to 5 days.
  • the half-life of the complex bound to hemoglobin is 10-30 minutes, and it is known that it is rapidly taken up and degraded. .
  • hemolysis occurs due to the occurrence of pathological conditions such as burns, burns, blood transfusions, open-heart surgery under extracorporeal circulation such as cardiopulmonary bypass, and hemoglobin in the blood exceeds haptoglobin, hemoglobin leaks from the glomeruli. This causes iron depletion from the body and oxidative vascular and renal tubule damage caused by free hemoglobin.
  • Haptoglobin preparations are used as therapeutic agents for such hemoglobinemia and hemoglobinuria.
  • haptoglobin has characteristics as an acute phase reaction protein. In infection, inflammation, tissue disruption, malignant tumor, etc., serum haptoglobin is markedly increased due to ectopic production other than liver such as eosinophils. It is known to be accepted (Non-patent Document 5).
  • Haptoglobin has a tetramer structure (hereinafter referred to as (a
  • This is a glycoprotein that can take a multimeric structure based on this tetrameric structure (FIG. 2) (Non-patent Document 6).
  • the alpha chain and the j8 chain of ptoglobin are single genes encoded in one open reading frame and exist in human chromosome 6 long arm 22.1 (Non-patent Documents 7 and 8).
  • the haptoglobin precursor which is the translation product of this single gene, is cleaved at one site in the cell that expresses haptoglobin, thereby generating an ⁇ chain and a j8 chain.
  • Hp 1 and Hp2 There are two types of human habutoglobin genes, Hp 1 and Hp2, and there are genetic polymorphisms (Fig. 1), so each human has three types of haptoglobin, Hpl-1, Hp2-1 and Hp2-2. One of them ( Figure 2). There is a racial difference in the genotype distribution of this haptoglobin.
  • Hp2-1 is 45-55%
  • Hp2-2 is 30-40%
  • Hpl-1 is 10-20%
  • Japanese is Hp2- It is known that 1 is 58%
  • Hp2-2 is 35%
  • Hpl-1 is 7%
  • about half of Africans are Hpl-1 (Non-Patent Documents 9 and 10).
  • the frequency is low, but the back mutation from the Hp2 gene to the Hpl gene due to intramolecular recombination occurs constantly in somatic cells, and it has been reported that it is in a chimeric state. (Non-patent document 11).
  • the human Hp2 gene is a gene produced by gene duplication of the exon 3 and 4 regions of the a-chain gene containing many cysteine residues of the Hpl gene (Fig. 1) (Non-patent Document 12). Therefore, ( ⁇ ) 2 having an Hp2 a chain can form a multimer via the free SH group of the cysteine residue generated by duplication (Fig. 2). This further increases the number of haptoglobin molecules. It is a cause of existence as a mixture of molecular species with molecular weights ranging from 85,000 to 400,000 in human plasma, and a wide range of physical and physical heterogeneity.
  • ( ⁇ ⁇ ) 2 having an Hpl a chain has a higher amount of hemoglobin binding per unit protein than ( ⁇ ⁇ ) 2 having an Hp2 a chain or a multimer (Non-patent Documents 13 and 10)
  • the haptoglobin-hemoglobin complex binds to the receptor expressed in reticuloendothelial cell line cells and is then taken up into the cell and metabolizes the bound hemoglobin. It has been reported that it is superior to ( ⁇ ) 2 having an a chain (Non-Patent Document 14).
  • haptoglobin can function as an antioxidant by suppressing the production of active oxygen radicals via iron in hemoglobin by binding to free hemoglobin.
  • Non-patent Document 10 The function as an antioxidant is also Hp2 a chain.
  • a j8) 2 having an Hpl a chain is higher than ( ⁇ ) 2 having a phenotype and multimers.
  • Non-patent Document 10 it has been reported that in humans of the Hp2-2 type gene polymorphism, vitamin C, which has a high abundance of iron ions in serum, has poor stability (Non-patent Documents 15 and 16).
  • haptoglobulin having an Hp2 a chain is sufficient to transfer to yarn and tissue. I can not expect. Therefore, in order to provide a uniform bioactive haptoglobin preparation, it is desired to adjust the amount of hemoglobin binding per unit preparation protein and make the molecular species composition constant.
  • Non-patent Documents 17 to 20 It has been reported that the sugar chain of haptoglobin has a low fucose modification rate of a few percent or less in healthy individuals, but it increases with canceration (Non-patent Documents). 21, 22).
  • haptoglobin preparations are produced from the plasma of many thousands of healthy individuals. For this reason, these preparations may cause transmission of infections such as various hepatitis viruses, AIDS viruses, human parvoviruses, and prions that cause mutant Creutzfeldt's Jacob disease. This cannot be completely denied. In addition, it is difficult to completely overcome the physical and physiological non-uniformity because it is necessary to use plasma that can be obtained from unspecified different healthy human power as a raw material. . From this point of view, it is instructed that the use of haptoglobin preparations should be kept to the minimum necessary after careful examination of the therapeutic need. The name and address, the date of administration, and the serial number of the administered product must be recorded and stored for at least 20 years.
  • Patent Document 1 Non-patent Document 1
  • Patent Documents 23 and 24 the sugar chain structure bound to haptoglobin produced by these host cells is significantly different from the sugar chain structure of human plasma-derived haptoglobin, and has the same activity as human plasma-derived haptoglobin.
  • haptoglobins having different sugar chain structures are not preferable as pharmaceuticals in terms of antigenicity, blood dynamics, and stability.
  • Non-patent Document 25 CHO cells derived from Chinese hamster ovary that have already been used in pharmaceutical manufacturing and have been proven to have no sugar chain structure that causes problems when administered to humans are used as host cells to produce recombinant haptoglobin.
  • Non-patent Document 25 An example has been reported (Non-patent Document 25). However, it is bound to haptoglobin obtained by the production method and fucose is bound to most of the sugar chains, and this structure is similar to the abnormal haptoglobin observed in the plasma of cancer-bearing patients. It is known that The causal relationship between fucose modification to sugar chains of haptoglobin and the carcinogenicity is still unknown.
  • haptoglobin In the treatment of patients, the single dose of haptoglobin preparation is about 5.7 g, so haptoglobin In order to administer to patients, it must be serious. In addition, because of the high physiological activity of hemoglobin, buttoglobin, not only is it related to canceration, but also considers the effect on the biodistribution of haptoglobin itself and its complex with hemoglobin due to a significant change in sugar chain structure. It is left as an issue to be done. Therefore, there is a need for a recombinant haptoglobin that has a pharmacological effect equivalent to or higher than that of human plasma-derived haptoglobin, and that can reduce the risk of virus infection, which is a drawback of current blood products, and heterogeneity. It is. Patent Document 1: Japanese Patent Publication No. 46068 2001
  • Non-Patent Document 2 Proc. Natl. Acad. Sci. USA, 61, 748 (1968)
  • Non-Patent Document 3 J. Biol. Chem., 255, 9616 (1980)
  • Non-Patent Document 4 Circ. Res., 92, 1193 (2003)
  • Non-Patent Document 5 Clin. Lab., 46, 547 (2000)
  • Non-Patent Document 6 Redox Rep., 6, 379 (2001)
  • Non-Patent Document 7 Hum. Genet., 42, 61 (1978)
  • Non-Patent Document 8 Proc. Natl. Acad. Sci. USA, 80, 5875 (1983)
  • Non-Patent Document 9 The Second Series of Pharmaceutical Research and Development 'Bio od Product "20, 215 (1992)
  • Non-Patent Document 10 Clin. Lab., 46, 547 (2000)
  • Non-Patent Document 12 Redox Report, 6, 119 (2001)
  • Non-Patent Document 13 Vox Sang, 10, 320 (1965)
  • Non-Patent Document 14 Circ. Res., 92, 1193 (2003)
  • Non-Patent Document 15 Am. J. Clin. Nutr., 66, 606 (1997)
  • Non-Patent Document 16 AIDS 12, 1027 (1998)
  • Non-Patent Document 17 J. Biol. Chem., 258, 7858 (1983)
  • Non-Patent Document 18 Clin. Chim. Acta, 227, 69 (1994)
  • Non-Patent Document 19 Biochim. Biophys. Acta, 953, 345 (1988)
  • Non-Patent Document 20 Glycoconjugates. Proc. 6th Int. Sym. Glyco. Tokyo, 275 (1981)
  • Non-Patent Document 21 Cancer Letters, 66, 43 (1992)
  • Non-Patent Document 22 The Second Series of Pharmaceutical Research and Development 'BL oodProduct "20, 215 (1992)
  • Non-Patent Document 23 DNA, 5, 129 (1986)
  • Non-Patent Document 24 Mol. Biol. Rep., 13, 225 (1989)
  • Non-Patent Document 25 Proteomics, 4, 2221 (2004) Disclosure of the invention
  • the present invention relates to the following (1) to (24).
  • a composition comprising a recombinant haptoglobin molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain binds N-acetyldarcosamine at the reducing end of the sugar chain.
  • a haptoglobin composition which is a sugar chain.
  • the N-glycoside-linked complex type sugar chain is a sugar chain in which N-acetylyldarcosamine at the reducing end of the sugar chain is a sugar chain in which position 1 of fucose is not ⁇ -bonded.
  • Haptoglobin composition is a sugar chain in which N-acetylyldarcosamine at the reducing end of the sugar chain is a sugar chain in which position 1 of fucose is not ⁇ -bonded.
  • Haptoglobin force A protein consisting of the following (a) to (i) group forces that also have forces, and the following (j) to (l) polypeptides that also have group forces that have selected forces.
  • Haptoglobin force The following (a), (b), (c), (d), (e) and (1) Group force of force The above-mentioned (1) or ( The haptoglobin composition described in 2).
  • the host cell is the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose, or the N-glycidyl-linked N-acetylyldarcosamine at the 6-position of the reducing glycan is located at position 1 of fucose ⁇
  • the transformant according to (5) above, which is a cell whose genome has been altered so that the activity of the enzyme involved in the modification of the sugar chain to be bound is lost.
  • the host cell is the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose, or the first position of fucose at position 6 of ⁇ ⁇ -acetylcylcosamine at the reducing end of ⁇ -glycosidic complex glycan
  • the transformant according to (6) above, wherein the allele on the genome of the enzyme involved in the sugar chain modification to be bound is a knocked-out cell.
  • amino acid sequence represented by SEQ ID NO: 8 one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have GDP-mannose 4,6-dehydratase activity protein;
  • 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 (8) ).
  • 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 power is GDP-4-keto-6-deoxy-D- A protein having mannose-3,5-epimerase activity;
  • (c) It consists of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity protein.
  • the above-mentioned (8) GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is a protein encoded by DNA selected from the following (a) and (b) group forces that also include forces (8) ).
  • N-glycoside-linked complex type sugar chain N-acetylyldarcosamine at the reducing end is an enzyme involved in sugar chain modification in which the 1-position of fucose is a-linked to 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 (13 ).
  • amino acid sequence represented by SEQ ID NO: 14 one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have an ⁇ 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: 14, and having a 1,6-fucosyltransferase activity.
  • a 1, 6-fucosyltransferase is a protein encoded by DNA selected from the group consisting of the following (a), (b), (c) and (d) 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 (5) to (18) above, which is a cell selected from a group.
  • the transformant according to any one of (5) to (19) above is cultured in a medium, a haptoglobin composition is produced and accumulated in the culture, and the haptoglobin composition is collected from the culture
  • the manufacturing method of a haptoglobin composition including a process.
  • a medicament comprising the haptoglobin composition according to any one of (1) to (4) and (21) as an active ingredient.
  • a therapeutic agent for hemoglobinemia and hemoglobinuria comprising the haptoglobin composition according to any one of (1) to (4) and (21) as an active ingredient.
  • a disease associated with acid-induced vascular injury due to blood free hemoglobin comprising as an active ingredient the haptoglobin composition according to item 1 of (1) to (4) and (21) above Therapeutic agent.
  • composition comprising a recombinant haptoglobin molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is N- A haptoglobin composition which is a sugar chain in which fucose is bound to acetylyldarcosamine is provided.
  • FIG. 1 shows the structure of HP-1 type and HP-2 type haptoglobin.
  • FIG. 2 shows the structure of the haptoglobin complex in HP1-1 type, HP2-1 type, and HP2-2 type.
  • FIG. 3 shows a method for preparing plasmid pKAN-HP1.
  • FIG. 4 shows a method for preparing plasmid pKAN-HP2.
  • the composition of the present invention also comprises a recombinant haptoglobin molecule having an N-glycoside-bonded complex sugar chain, wherein the N-glycoside-bonded complex sugar chain is N-acetyl at the reducing end of the sugar chain.
  • a haptoglobin composition (hereinafter referred to as “the composition of the present invention”), which is a sugar chain in which fucose is bound to darcosamine, is an N-glycoside-linked complex sugar chain reducing end N-a. Any composition is included as long as it is a haptoglobin composition in which fucose is bound to cetyldarcosamine! /, Na! / And also has a recombinant haptoglobin molecular force.
  • haptoglobin includes any glycoprotein having affinity for hemoglobin and a haptoglobin receptor and having hemoglobin metabolic activity.
  • haptoglobin examples include a polypeptide selected from the group consisting of (a) to (i) below the haptoglobin power and a polypeptide selected from the group consisting of (j) to (l) below: Examples include proteins.
  • polypeptide having an amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4 (j) a polypeptide having an amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4; (k) one or more amino acids deleted in the amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4, A polypeptide having a substitution, insertion and Z or added amino acid sequence, and having hemoglobin metabolic activity in combination with the above-mentioned (a) to (polypeptides having a group strength of 0);
  • DNA encoding the amino acid sequence of haptoglobin is stringent with DNA having the base sequence represented by SEQ ID NO: 1, 2 or 3, and DNA having the base sequence represented by SEQ ID NO: 1, 2 or 3.
  • Examples include DNA that encodes a protein that undergoes nobbreviation and hybridization under certain conditions and has hemoglobin metabolic activity.
  • the DNA that hybridizes under stringent conditions is, for example, DNA such as DNA having the base sequence represented by SEQ ID NO: 1, 2, or 3, or a fragment thereof.
  • Koguchi-1 'hybridization method plaque' hybridization method is "means DNA obtained by using Southern blot hybridization method, etc., specifically, colony or Hybridization was performed at 65 ° C in the presence of 0.7 to 1.
  • OM sodium chloride using a filter on which plaque-derived DNA was immobilized, and then an SSC solution (1 to 2 times in concentration) was added.
  • the concentration of SSC solution is 150mM sodium chloride and 15mM sodium citrate), and DNA can be identified by washing the filter under 65 ° C conditions. .
  • DNA capable of hybridizing is DNA having at least 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3, preferably 70% or more, more preferably 80% or more, and still more preferably May be DNA having a homology of 90% or more, particularly preferably 95% or more, and most preferably 98% or more.
  • amino acids in the amino acid sequence represented by SEQ ID NO: 4, 5, or 6, one or more amino acids
  • a protein that has amino acid sequence ability with deletion, substitution, insertion and Z or addition of mino acid, and also has a hemoglobin metabolic activity is the molecular 'Clooung 2nd edition, current' Protocols in Molecular Biology, Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34,315 (1985), Nucleic Acids Research, 13 , 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc., for example, represented by SEQ ID NO: 4, 5, or 6 It means a protein that can be obtained by introducing a site-specific mutation into DNA encoding a protein having an amino acid sequence.
  • 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. For example, it is 1 to several tens, preferably 1 to 20, more preferably 1 to: L0, and further preferably 1 to 5.
  • the protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4, 5, or 6 and having hemoglobin metabolic activity is BLAST [J. Mol. Biol, 215, 403 (1990)] and FASTA [Methods in Enzymology, 183, 63 (1990)], etc., and the amino acid sequence described in SEQ ID NO: 4, 5 or 6 At least 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more. Means that.
  • Hemoglobin metabolic activity refers to the binding of haptoglobin force to free hemoglobin to form a complex, and the complex is expressed as a receptor for the hemoglobin-habu globin complex expressed in the reticuloendothelial cell line of the liver. It binds and is taken up into cells after binding, and has the activity of metabolizing heme generated by the degradation of hemoglobin into pyrilbin.
  • N-glycoside-linked sugar chains bound to glycoproteins are known to have a common core structure represented by the following structural formula (I) in any of the various structures. .
  • the end of the sugar chain that binds to asparagine is the reducing end, and the opposite side is non- It is called the 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) 1) in parallel, and more than one branch, and a complex type having a structure such as sialic acid, Neusecting N-acetylethylcosamine on the non-reducing end side of Ga ⁇ GlcNAc, It is known that there are hybrid types having both a hymannose type and a complex type branch on the non-reducing end side of the core structure.
  • the j8 chain of the haptoglobin molecule constituting the composition of the present invention has four additional sequences of N-glycoside-linked sugar chains, and N-glycoside-linked sugar chains are bound to these sites.
  • Specific examples of the N-glycoside-bonded sugar chain that binds to habtoglobin include the aforementioned N-glycoside-bonded complex sugar chains.
  • the N-glycoside-bonded complex sugar chain that binds to the haptoglobin molecule includes a large number of sugar chains including the core structure represented by the structural formula (I), and therefore there are many combinations of sugar chains. It will be.
  • the composition of the present invention is a composition having a genetically modified haptoglobin molecular force having an N-glycoside-bonded complex sugar chain, wherein the N-glycoside-bonded complex sugar chain is the sugar chain reducing end.
  • a haptoglobin molecular force having a single sugar chain structure or a haptoglobin having a plurality of different sugar chain structures may be used. Consists of molecules.
  • N-glycoside-linked complex type sugar chain is a sugar chain in which fucose is not bound to N-acetylyldarcosamine at the reducing end. Specifically, position 1 of fucose is an N-glycoside-linked complex type sugar. The 6-position of the N-acetylyldarcosamine in the chain (X-bonded!
  • fucose is bonded to ⁇ ⁇ -acetyldarcosamine at the sugar chain reducing end.
  • An unacceptable sugar chain means that fucose is not substantially bound to the sugar chain, and preferably means that the fucose content is 0%.
  • the fact that fucose is not substantially bound means that fucose cannot be substantially detected in the sugar chain analysis described in 4 below. “Substantially undetectable” means below the detection limit of measurement.
  • the composition of the present invention has a long half-life in blood when administered in vivo compared to haptoglobin in which fucose is bound to N-glycidyl darcosamine at the N-glycoside-linked complex type sugar chain reducing end. .
  • the transformant of the present invention includes any transformant as long as it is capable of producing the composition of the present invention.
  • a specific example is a transformant obtained by introducing a DNA encoding a haptoglobin molecule into a host cell such as (a) or (b) below.
  • 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. .
  • amino acid sequence represented by SEQ ID NO: 8 one or more amino acids are deleted, substituted, or inserted A protein having amino acid sequence ability with added and Z or added, and having GDP-mannose 4,6-dehydratase activity;
  • 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 GDP-mannose 4,6-dehydratase activity.
  • GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is:
  • Examples include the protein encoded by DNA (a) or (b) below, or the protein (c), (d) or (e) below.
  • 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 power is GDP-4-keto-6-deoxy-D-mannose.
  • (e) It consists of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity Protein.
  • N-glycoside-linked complex type sugar chain N-acetylyldarcosamine at the reducing end 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 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: 14, and having a 1,6-fucosyltransferase activity.
  • DNA that hybridizes under stringent conditions is, for example, DNA such as DNA having a nucleotide sequence represented by SEQ ID NO: 7, 9, 11, or 12, or a fragment thereof.
  • DNA obtained by using the Koguchi-ichi 'hybridization method, plaque' hybridization method, Southern hybridization method, etc. as a probe, specifically derived from colonies or plaques.
  • OM sodium chloride 0.1 to 2 times the concentration of SSC solution (The composition of the SSC solution with a 1-fold concentration consists of 150 mM sodium chloride and 15 mM sodium quenate), 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: 7, 9, 11 or 12, 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: 8 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or added, and the GDP-mannose 4,6-dehydratase activity
  • GDP-4-keto-6-deoxy-D -A protein having mannose-3,5-epimerase activity or an 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: 13 or 14.
  • 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, but the above-mentioned site-specific mutagenesis method, etc.
  • the number is such that it can be deleted, substituted or added by, for example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5. is there.
  • the present invention comprises an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, 10, 13 or 14, and has GDP-mannose 4,6-dehydratase activity. , GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity or ⁇ 1,
  • the amino acid sequence represented by SEQ ID NO: 8, 10, 13 or 14 and BLAST [J. Mol.
  • 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 N-glycoside-linked complex sugar chain reducing end
  • an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-acetylyldarcosamine at the N-glycoside-linked complex sugar chain reducing end By introducing DNA encoding a haptoglobin molecule into a host cell whose genome has been altered so that the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is linked to a at position 6 is a It is possible to obtain a transformant producing the composition.
  • the enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose, or N-acetylcolcamine at the reducing end of the N-daricoside-linked complex sugar chain, position 6 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 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 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.
  • a host cell used for producing a recombinant glycoprotein pharmaceutical examples include embryonic stem cells or fertilized egg cells used to produce non-human transgenic animals that produce pesticides, and plant cells used to produce transgenic plants that produce recombinant glycoprotein drugs. It is done.
  • an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-glycyl-linked complex N-acetylyldarcosamine at the 6-position of N-acetylyldarcosamine 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.
  • 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.
  • a gene encoding a haptoglobin Hpl gene was introduced into a CHO cell into which a gene encoding ⁇ 1,6-fucosyltransferase was knocked out.
  • examples thereof include the HP1KO strain, which is a transformant, and the HP2KO strain, which is a transformant in which a gene encoding haptoglobin Hp2 is introduced into a CHO cell into which a gene encoding a 1,6-fucosyltransferase has been knocked out.
  • HP1KO and HP2KO strains were established on February 17, 2005 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Tsukuba Rinto, Tsukuba 1-chome, 1-Chuo 6th, Ibaraki, Japan). Deposited as FERM BP-10249 and FERM BP-10250, respectively.
  • the transformant of the present invention has a hemoglobin binding activity equivalent to that of the haptoglobin composition obtained from the parent cell line, and has a longer blood half-life than the haptoglobin composition obtained from the parent cell line.
  • a buttoglobin composition can be produced.
  • the binding activity of the complex to the receptor present in reticuloendothelial cells and the half-life in blood were determined by measuring the binding activity of the known hemoglobin and haptoglobin receptor in vitro tests, model animals such as mice and rats. It can be measured using an in vivo test used or a clinical test using humans (Clin. Chem., 42, 1589 (1996), Eur. J. Clin. Chem. Biochem., 35, 647 ( 1997), Nature, 409, 198 (2001), J. Biol. Chem., 279, 51561 (2004), Modern Medicine, 30, 656 (1975), Modern Medicine, 30,879 (1975), Res. Exp.
  • the host cell used for producing the haptoglobin thread and the composition of the present invention can be produced by the method described below.
  • the host cell used for the preparation of the haptoglobin composition of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or N-glycoside-linked complex sugar chain-reducing terminal N-acetylyldarcosamine 6
  • enzyme related to fucose modification By using the gene disruption method targeting the gene of the enzyme involved in the sugar chain modification in which position 1 of fucose binds to the position (hereinafter referred to as “enzyme related to fucose modification”) 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
  • enzymes involved in glycosylation in which the 1-position of fucose is a-linked to the 6-position of N-glycidyl darcosamine at the N-glycoside-bonded glycan reducing end examples include ⁇ 1,6-fucosyl Examples include transferase and a-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 haptoglobin composition of the present invention targets an enzyme gene related to fucose modification, and is described in Cell Engineering, 12, 239 (1993), Bio-Z Technology (BIO / TECHNOLOGY), 17, 1097 ( 1999), Human 'Molecular ⁇ ⁇ ⁇ Genetics (Hum.Mol. Genet.), 5, 1083 (1995), Cell engineering, 13, 255 (1994), Proceedings' o B. The National. Academia. Ob. Science ( proc . Natl. Acad. Sci. USA), 96, 1
  • 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 haptoglobin composition of the present invention By selecting a transformant using the activity of an enzyme related to fucose modification as an indicator, a host cell used for preparing the haptoglobin composition of the present invention can be obtained. It is also possible to obtain a host cell used for producing the haptoglobin composition of the present invention by selecting a transformant using the sugar chain structure of a glycoprotein on the cell membrane or the sugar chain structure of a produced glycoprotein molecule as an index. it can.
  • the host cell used for preparing the haptoglobin composition of the present invention has a gene for an enzyme related to target fucose modification, such as an enzyme, an animal cell, an insect cell, or a plant cell. Any material can be used. Specific examples include host cells described in 3 below.
  • 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.
  • 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.
  • a cDNA library can be screened to obtain DNA encoding an enzyme related to fucose modification.
  • V or human or non-human animal tissue or cell force may be prepared as follows.
  • 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 the method described in Molecular 'Crowing 2nd Edition, Current Protocols in Molecular Biology, A Laboratory Manual, 2nd Ed. (1989), etc., or a commercially available kit. For example, a method using Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies Neeri, ZAP—cDNA bynt hesis Kit (STRATAGENE)) can be mentioned.
  • Escherichia coli K12 strain is used as a cloning vector for preparing a cDNA library.
  • Any phage vector or plasmid vector can be used as long as it can replicate autonomously.
  • ZAP Express [STRATAGENE, Strategies,, 58
  • Escherichia coli is preferably used. Specifically, Escherichia coli XLl-Blue MRF '[STRATAGENE, Strategies, 5, 81 (1992)], Escherichia coli C600 "Genetics, 39, 440 (1954) 1, Escherichia coli Y108 8 [ Science, 222, 778 (1983) 1, Escherichia coli Yl 090 "Science, 222,778 (1983) 1, Escherichia coli NM522" Journal, Ob, Molecura ⁇ Bio Mouth 0.
  • the obtained gene fragment is DNA encoding an enzyme related to fucose modification.
  • an enzyme related to fucose modification for example, Sanger et al.'S dideoxy method [Proceedings of the National] ⁇ Academ ⁇ ⁇ ⁇ Ob ⁇ Science (Pro Natl. Ac ad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems) etc.
  • Sanger et al.'S dideoxy method [Proceedings of the National] ⁇ Academ ⁇ ⁇ ⁇ Ob ⁇ Science (Pro Natl. Ac ad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems) etc.
  • a colony hybridization or a plaque hybridization is synthesized 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. Amplify using the PCR method to repair fucose
  • the base sequence of the DNA encoding the enzyme related to the obtained fucose modification can be determined by a commonly used base sequence analysis method such as Sanger et al.'S dideoxy method [procedings ⁇ of ⁇ the 'national ⁇ academia ⁇ By analyzing using a base sequence analyzer such as ⁇ Ob Science (Pro Natl. Acad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems), The base sequence of the DNA can be determined.
  • a base sequence analyzer such as ⁇ Ob Science (Pro Natl. Acad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems).
  • a homology search program such as BLAST is used to search a base sequence database such as Genbank, EMBL, and DDBJ. It can also be confirmed that the gene encodes an enzyme related to fucose modification.
  • Examples of the base sequence of the gene encoding the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose obtained by the above method include the base sequence set forth in SEQ ID NO: 7 or 9.
  • N-glycoside-linked complex-type sugar chain-reducing terminal N-acetylcylcosamine obtained by the above 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: 11 or 12.
  • DNA synthesis using the phosphoramidite method is performed.
  • a DNA synthesizer such as model machine 392 (manufactured by Perkin Elmer)
  • cDNA of an enzyme related to fucose modification can also be obtained.
  • 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 (Genome Systems), Unigen GenomeWalker TM Kits (CLONTECH), or the like.
  • the obtained DNA base sequence encoding the enzyme related to fucose modification is usually used.
  • the base sequence of the 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 is also possible to confirm that the gene encodes an enzyme related to fucose modification.
  • nucleotide sequence of the genomic DNA of the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose obtained by the above method include the nucleotide sequences set forth in SEQ ID NOs: 15, 16, 17, and 18.
  • Examples thereof include the base sequence set forth in SEQ ID NO: 19.
  • the haptoglobin composition of the present invention is prepared by directly introducing into the host cell an antisense oligonucleotide or ribozyme designed based on the base sequence of the enzyme related to fucose modification without using an expression vector. The host cell used to do this is obtained.
  • 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.
  • oligonucleotide derivatives examples 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.
  • the host cell used for preparing the haptoglobin composition of the present invention can be 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. .
  • Genomic DNA of an enzyme related to fucose modification is prepared.
  • a target vector for homologous recombination of the target gene to be modified (for example, a structural gene of an enzyme related to fucose modification or a promoter gene) is prepared.
  • a host cell used for preparing the haptoglobin 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. Can be produced.
  • 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.
  • Examples thereof include a method for preparing genomic DNA described in (a).
  • 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 for example, the salts described in SEQ ID NOs: 15, 16, 17 and 18 Examples of the base sequence.
  • target vector for homologous exchange of target genes on chromosomes, Gene Targ eting, A Practical Approach, IRLPress at Oxford University Press (1993), noisyo-Muual 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.
  • the method for introducing a recombinant vector suitable for various host cells described in 3 below can be used.
  • Examples of methods for efficiently selecting homologous recombinants include Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Bio-European Series 8 Gene Targeting, Production of mutant mice using ES cells Methods such as positive selection, promoter selection, negative selection, poly A selection described in (Yodosha) (1995) and the like 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 preparing the haptoglobin composition of the present invention can be prepared as follows, for example, by targeting the gene of an enzyme related to fucose modification and using the RDO method.
  • cDNA or genomic DNA of an enzyme related to fucose modification is prepared using the method described in 1 (1) (a) above.
  • the portion encoding the enzyme related to fucose modification Based on the determined DNA sequence, the portion encoding the enzyme related to fucose modification, non- Design and synthesize an RDO construct of appropriate length including the translation region or intron.
  • the synthesized RDO is introduced into the host cell and related to the targeted enzyme, ie, fucose modification.
  • a host cell for preparing the haptoglobin composition of the present invention can be prepared.
  • 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.
  • the recombinant vector introduction method suitable for various host cells described in 3 below can be used.
  • 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 of a 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.
  • 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.
  • RDO constructs are described in Science, 273, 1386 (1996); Nichiya's Medicine (Nature Medicine), 4, 285 (1998); Hepatology, 25, 1462 (1997); Gene 'Therapies (Gene Therapy), 5, 1960 (1999); Gene' Therapies (Gene Therapy), 5, 1960 (1999); Journal 'Ob' Molekiyura 'Medellin (J. Mol. Med.), 75,829 (1997); Procedures' Ob The National 'Academy ⁇ ⁇ ⁇ Ob' Science (Proc. Natl. Acad. Sci. USA), 96, 8774 (1999); The National 'A Power Demi-Ob' Science (Proc. Natl. Acad. Sci.
  • the host cell used for preparing the haptoglobin composition of the present invention can be prepared as follows, for example, by targeting the gene of an enzyme related to fucose modification and using the RNAi method.
  • cDNA is prepared using the method described in (1) (a) of 1 above of an enzyme related to fucose modification.
  • RNAi gene construct of an appropriate length that includes the enzyme coding for fucose modification or the untranslated region is designed.
  • RNAi gene in order to express the RNAi gene in a cell, a prepared cDNA fragment or full length Is inserted downstream of the promoter of an appropriate expression vector to prepare a thread-replacement vector.
  • a transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • the haploglobin composition of the present invention by selecting a transformant using as an index the activity of the enzyme related to the introduced fucose modification, the glycoprotein molecule produced or the sugar chain structure of the glycoprotein on the cell surface.
  • the host cell to be used 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.
  • 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 include the method for preparing cDNA described in (a) of 1).
  • the host cell used for preparing the haptoglobin composition of the present invention can be obtained by directly introducing into the host cell the RNAi gene designed based on the base sequence of the enzyme related to fucose modification without using an expression vector. It can also be obtained.
  • RNAi gene can be prepared by a conventional method or using a DNA synthesizer.
  • NAi gene The construct of the NAi gene is [Nature, 391, 806 (1998); Gus Ob The National National Power Science (Proc.Natl. Acad. Sci. USA), 95, 15502 (1998); Nature, 395, 854 (1998); Proc Ding's 'Ob' The • National 'A Power Demi-Ob' Science (Proc. Natl. Acad. Sci. USA), 96, 5049 (19 99); Cell,, 1017 (1998); Proc Deings' Ob The National 'A Power Demi ⁇ Ob' Science (Proc. Natl. Acad. Sci.
  • the host cell used for producing the haptoglobin composition of the present invention is a transposon system described in Nature Genet., 25, 35 (2000), etc.
  • a host cell used for preparing the haptoglobin composition of the present invention can be prepared by selecting a mutant using as an index the activity or the glycoprotein structure of the glycoprotein molecule or glycoprotein on the cell membrane. .
  • 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 a mutation in the DNA of the host cell.
  • any yeast cell, animal cell, insect cell, plant cell or the like having an enzyme gene related to the target fucose modification can be used.
  • the host cells described in 3 below can be mentioned.
  • the method for introducing recombinant vectors suitable for various host cells described in 3 below is used. You can!
  • 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 (a) of (1) 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.
  • the host cell used to produce the haptoglobin composition of the present invention is related to fucose modification.
  • enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx.
  • N-glycoside bond As an enzyme involved in the sugar chain modification in which the 1-position of fucose is a-linked to the 6-position of the N-acetylyldarcosamine at the reducing end of the complex sugar chain, ⁇ ⁇ , 6— Examples include fucosyltransferase and ⁇ -L-fucosidase.
  • 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
  • GMD derived from CHO cells (SEQ ID NO: 8)
  • 155th threonine 157th glutamic acid
  • 179th tyrosine 183th
  • lysine a dominant negative
  • Genes with such amino acid substitutions were prepared using site-directed mutagenesis described in Molecular and Cloning 2nd Edition, Current Protocols' In Molecular ⁇ ⁇ Biology etc. Can be done.
  • the host cell used to produce the haptoglobin filamentous product of the present invention contains a gene encoding a dominant negative form of the target enzyme produced as described above (hereinafter abbreviated as a dominant negative form gene).
  • a dominant negative form gene a gene encoding a dominant negative form of the target enzyme produced as described above.
  • 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.
  • any yeast cell, animal cell, insect cell, plant cell, etc. having a gene for an enzyme related to the target fucose modification can be used.
  • the host cells described in 3 below can be mentioned.
  • an expression vector autonomous replication is possible in the above host cell or into the chromosome.
  • the one containing a promoter at a position where the DNA encoding the target dominant negative 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) 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 to prepare the haptoglobin composition of the present invention uses a technique in which a mutation is introduced into a gene of an enzyme related to fucose modification and a desired cell line in which the enzyme is mutated is selected. Can be produced.
  • 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 glucosamine at the N-glycoside-linked complex sugar chain reducing terminal include ⁇ ⁇ , 6-fucosyltransferase, Examples include ⁇ -L-fucosidase.
  • a method for introducing a mutation into an enzyme related to fucose modification 1) It is related to fucose modification from a mutant in which a parent strain was treated by a mutagenesis treatment or a naturally occurring mutant. 2) A method for selecting a desired cell line based on the activity of the enzyme to be produced, 2) a sugar chain of a production glycoprotein molecule from a mutant obtained by treating the parent strain by mutagenesis treatment or a spontaneously generated mutant. A method of selecting a desired cell line using the structure as an index; 3) a glycoprotein sugar on the cell membrane of a cell derived from a mutant obtained by treating a parent line by mutagenesis treatment or a naturally occurring mutant. Examples thereof include a method of selecting a desired cell line using the chain structure as an index. [0111] As the mutagenesis treatment, there are point mutations and deletions in the DNA of the parent cell line.
  • Any treatment that induces a mutation can be used.
  • mutagenic agent treatments 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.
  • Spontaneously occurring mutants include sudden mutants that occur spontaneously by continuing subculture under normal cell culture conditions without special mutagenesis. be able to.
  • 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 haptoglobin composition of the present invention targets the gene of an enzyme related to fucose modification, and antisense RNAZDNA technology [Bioscience and Industry, 50, 322 (1992), Chemistry, 681 (1991), Biotechnology, 9,358 (1992), Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology,! ⁇ , 152 (1992), Cell engineering, 16, 1463 (1997)], Triple helix technology [ Trends in Biotechnology, 10, 1 32 (1992)] or the like, and can be produced by suppressing transcription or translation of the target gene.
  • enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx.
  • an enzyme involved in the sugar chain modification in which the 1-position of fucose is ⁇ - bonded to the 6-position of N-glycidylcolcamine at the N-glycosidic complex sugar chain reducing terminal Specific examples include ⁇ , 6-fucosyltransferase, ⁇ -L-fucosidase, and the like.
  • the host cell used to prepare the haptoglobin composition of the present invention is a lectin that recognizes a sugar chain structure in which the N-glycidyl-linked sugar chain reducing terminal N-acetylylcosamine 6-position and fucose 1-position are linked. It can be produced by using a technique for selecting strains that are 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 derived from Lg Culinaris) Endumerme lectin PSA (Peum sativum derived Pe a Lectin), Broad bean lectin VFA (Agglutinin derived from Viciafaba), Hirochawantake lectin AAL ( Lectin from Aleuria aurantia).
  • 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 A strain resistant to a lectin that recognizes an a-linked sugar chain structure can be selected.
  • Transgenic non-human animals, plants, or their progeny whose genomic genes have been modified so that the enzyme activity involved in the modification of the sugar chain of the haptoglobin molecule is controlled can synthesize intracellular sugar nucleotides GDP-fucose.
  • Enzyme involved or N-glycoside bond complex The embryonic nature of the present invention produced using the above 1 targeting the gene of the enzyme involved in glycosylation in which the 1-position of fucose is ⁇ -linked to the 6-position of N-acetylyldarcosamine at the reducing end of the combined sugar chain
  • it can be prepared from stem cells, fertilized egg cells, and plant callus cells as follows.
  • target non-human animals such as ushi, hidge, goats, pigs, horses, mice, rats, and embryonic stem cells such as rabbits, monkeys, and rabbits.
  • target non-human animals such as ushi, hidge, goats, pigs, horses, mice, rats, and embryonic stem cells such as rabbits, monkeys, and rabbits.
  • embryonic stem cell clones and normal cell clones can be obtained by methods such as injection chimera or assembly chimera into fertilized eggs of animals.
  • a chimeric individual can be prepared. By combining this chimeric individual with a normal individual, the activity of the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose in whole body cells or the N-glycidyl-linked N-acetylyldarcosamine 6
  • Transgenic non-human animals can be obtained in which the activity of the enzyme involved in sugar chain modification in which position 1 of fucose is a- linked is reduced.
  • fertilized egg cells such as ushi, hidge, goat, pig, horse, mouse, rat, chicken, monkey, and rabbit
  • the activity of the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or the 1-position of fucose at the 6-position of N-glycyl-linked N-acetylyldarcosamine at the N-glycoside-linked complex type sugar chain reducing end A fertilized egg cell of the present invention in which the activity of an enzyme involved in modification of an oc-linked sugar chain is reduced can be produced.
  • the fertilized egg cells thus produced are transplanted into the oviduct or uterus of a pseudopregnant female using the embryo transfer method described in Mupureating 'Mouse' Embryo 2nd edition, etc. 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 intracellular sugar nucleotide GDP-fucose can be obtained by using the same method as described in 1. above on the target plant strength or cells.
  • To produce a callus of the present invention in which the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is a- linked to the 6-position of the N-glycidalcosamine at the reducing end of the active or N-glycoside-linked complex type sugar chain is reduced Can do.
  • the prepared callus was prepared by a known method [tissue culture, 20 (1994); tissue culture, 21 (1995);
  • the cells are cultured again in a medium containing auxin and cytokinin to synthesize intracellular sugar nucleotides GDP-fucose.
  • the haptoglobin composition of the present invention is a molecular 'cloning second edition, current' protocorores in molecular neurology, Antibodies, ALaboratory manual, Cold Spring Harbor Laboratory, 1988 (hereinafter abbreviated as antibodies), Monoclonal Antibod ies: pnnciples and practice, Third Edition, Acaa.Press, 1993 (below, Monochrome ⁇ Nanore Antibodys), Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996 (hereinafter referred to as Antibody Engineering) For example, it can be obtained by expressing in a host cell as follows.
  • a full-length cDNA of HP-1 type or HP-2 type haptoglobin molecule was prepared, and the haptoglobin was prepared. Prepare an appropriate length of DNA fragment containing the molecule-encoding portion.
  • a recombinant vector is prepared by inserting the DNA fragment or full-length cDNA into the downstream of the promoter of an appropriate expression vector.
  • a transformant producing a haptoglobin molecule can be obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • any yeast cell, animal cell, insect cell, plant cell, etc. that can express the target gene can be used.
  • Cells obtained by various artificial techniques shown in 1 can also be used as host cells.
  • an expression vector one that can replicate autonomously in the above host cell or can be integrated into a chromosome and contains a promoter at a position where the DNA encoding the target haptoglobin molecule can be transcribed is used. It is done.
  • the cDNA is obtained from a human or non-human animal thread or tissue or a cell-specific probe or primer. Etc. can be used.
  • yeast When yeast is used as a host cell, examples of expression vectors 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 Mrs, Trichosporon, Schusomyces, etc. You can power chwanniomvces alluvius.
  • a method for introducing the recombinant vector if the method is to introduce DNA into yeast, the deviation is also used.
  • an animal cell is used as a host, as an expression vector, for example, pcDNAU pcD M8
  • 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 method can be used as long as it is a method for introducing DNA into animal cells.
  • the electoral position method [Cytote chnology, 3, 133 (1990)]
  • Calcium phosphate method [Japanese Patent Laid-open No. 2-227075]
  • Lipofection method [Proceedings of the National Academy of Science (Pro Natl. Acad. Sci. USA), 84, 7413 (1987)]
  • injection method [Map of 'The Mouse' Embryo Laboratory Laboratory Manual]
  • method using particle gun (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 Invitorogen) and the like.
  • Autographa californica nu clear polyhedrosis virus can be used for the outgrafa 'Cali forum-force' Nuclea 1 'polyhedrosis' virus, which is a virus that infects the night stealing insects.
  • Insect cells include ovarian cells of Spodoptera frugiperda, S19, S1 1 [Current 'Pokoto Reno' In 'Molechu Fu'Noroshi 1 Baculovirus Expression Vectors, A La boratory Manual, WH Freeman and Company, New York (1992)], Trichoplusiani ovary cells such as 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 When plant cells are used as host cells, examples of expression vectors include Ti plus Examples include midos and tobacco mosaic virus vectors.
  • Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and the rice 1 promoter.
  • CaMV cauliflower mosaic virus
  • host cells include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat and barley.
  • any method can be used as long as it is a method for introducing DNA into plant cells.
  • Agrobacterium [April 59-140]
  • the haptoglobin composition can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating haptoglobin molecules in the culture, and collecting the haptoglobin from the culture.
  • the method of culturing the transformant in a medium can be performed according to a usual 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 as long as the organism can assimilate, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolyzate, acetic acid, propionic acid, etc. Alcohols such as organic acids, ethanol, and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, and ammonium acetate.
  • Ammonium salts of inorganic or organic acids such as humic and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, Soybean koji and soybean koji hydrolyzate, various fermented cells and digested products thereof 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.
  • 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 culture medium.
  • RPMI 1640 medium commonly used as a medium for culturing transformants obtained using animal cells as a host [The Journal of the American American Medical Association (The Journal of the American Medical Association), Plastic, 519 (1967)], Eagle's MEM medium [Science, 12 ⁇ , 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 ( 1959), 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], Whitten medium [Developmental engineering] Experiment Manual-Transgeneic • How to make mice (Kodansha) Motoya Katsaki (1987) Hota can use media such as fetal calf serum added to these media. [0144] Cultivation is usually carried out for 1 to 7 days under conditions of pH 6-8, 30-40 ° C, 5% CO, etc.
  • antibiotics such as kanamycin and penicillin may be added to the medium as needed during culture.
  • 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' Stag (MS) medium, White medium, or a plant hormone such as auxin or cytokinin is added to these mediums. It is possible to use the prepared medium.
  • the culture is usually carried out under conditions of pH 5-9 and 20-40 ° C for 3-60 days.
  • antibiotics such as kanamycin and hygromycin may be added to the medium as needed during the culture.
  • a transformant derived from a microorganism, animal cell, or plant cell having a thread-recombinant vector in which DNA encoding a haptoglobin molecule is incorporated is cultured according to a normal culture method, and a haptoglobin composition is obtained.
  • a haptoglobin composition can be produced by producing and accumulating products and collecting the haptoglobin composition from the culture.
  • the haptoglobin 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 haptoglobin molecule to be generated.
  • DNA encoding a haptoglobin molecule and DNA encoding a signal peptide suitable for expression of the haptoglobin molecule are inserted into an expression vector, and the expression vector is inserted into a host cell.
  • the target haptoglobin molecule can be actively secreted outside the host cell.
  • the production amount can also be increased by using a gene amplification system using.
  • an animal individual transgenic non-human animal
  • a plant individual transgenic plant
  • a haptoglobin composition can also be produced using.
  • the transformant is an animal individual or a plant individual, it is reared or cultivated according to a usual method, and a haptoglobin composition is produced and accumulated, and the haptoglobin composition is collected from the animal individual or plant individual.
  • the hubtoglobin composition can be produced.
  • a method for producing a haptoglobin composition using an animal individual for example, a known method [American Journal of Clinical Nutrition, 63, 639S (1996); American Introduced gene according to Journal of Clinical Nutrition, 63, 627S (1996); Bio / Technology, 9, 830 (1991)] Created
  • a method for producing a target haptoglobin composition in an animal In the case of an individual animal, for example, a transgenic non-human animal introduced with DNA encoding a haptoglobin molecule ⁇ ! Is generated and accumulated in the animal, and the haptoglobin composition is produced from the animal.
  • a haptoglobin composition can be produced by collecting the product. Examples of the production / accumulation location in the animal include milk of the animal (Japanese Patent Laid-Open No. 63-309192) and eggs.
  • Any promoter can be used as long as it can be expressed in animals. For example, a casein promoter, 13 casein promoter, j8 lactoglobulin promoter, whey acid, which are mammary cell specific promoters. (3) A protein promoter or the like is preferably used.
  • a method for producing a haptoglobin composition using an individual plant for example, a known method for transgenic plants into which DNA encoding a ptoglobin molecule has been introduced [tissue culture, (1994); tissue culture] , 21 (1995); cultivated according to Trends “In” biotechnology (Trends in Biotec hnology), 15, 45 (1997)], and a haptoglobin composition is produced and accumulated in the plant.
  • a method for producing a haptoglobin composition by collecting the composition can be mentioned.
  • a hub globin composition produced by a transformant introduced with a gene encoding a haptoglobin molecule is expressed in a dissolved state in cells
  • the cell is centrifuged after completion of the culture. Collected by separation, suspended in an aqueous buffer solution, and then disrupted with an ultrasonic crusher, French press, Manton Gaurin homogenizer, dynomill, 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 Jetylaminoethyl (DEAE) -Sepharose, DIA IONHPA-75 (Mitsubishi Chemical Corporation), 3 ⁇ 4- Sepharose F (Pharmacia
  • a purified preparation of the composition can be obtained. Specifically, a method combining PEG fractionation, ammonium sulfate fractionation, and ion-exchange chromatography can be mentioned (development of follow-up medicines 1
  • the haptoglobin composition When the haptoglobin composition is expressed by forming an insoluble substance in the cells, the cells are similarly collected, disrupted, and centrifuged to obtain an insoluble substance of the haptoglobin composition as a precipitate fraction. to recover.
  • the recovered insoluble material of the haptoglobin composition is solubilized with a protein denaturant. After diluting or dialyzing the solubilized solution, the hubtoglobin composition is returned to a normal three-dimensional structure, and then a purified sample of the hubtoglobin composition can be obtained by the same isolation and purification method as described above. .
  • the habutoglobin composition or a derivative thereof can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and the haptoglobin composition is obtained from the culture supernatant by using the same isolation and purification method as described above. It is possible to obtain a purified sample.
  • the haptoglobin composition of the present invention can be produced by purifying the haptoglobin composition.
  • the biological activity of the purified nobutoglobin composition can be measured using various known methods. Specifically, a method for measuring hemoglobin binding activity, a method for measuring CD163 binding activity which is a complex of hemoglobin and haptoglobin (hereinafter abbreviated as hemoglobin haptoglobin complex), a method for measuring intracellular signal from CD163, CD163
  • hemoglobin haptoglobin complex a method for measuring intracellular signal from CD163, CD163
  • In vitro tests such as interleukin and granulocyte-macrophage colony-stimulating factor production measurement method, plastaglandin synthesis inhibition activity measurement method, angiogenic activity measurement method, antibacterial activity measurement method, etc.
  • In vivo tests or clinical tests using humans Clin. Chem., 42, 1589 (1996), Eur. J. Clin • Chem.
  • Paokishidase activity of hemoglobin is suppressed at a low P H condition.
  • hemoglobin combined with haptoglobin can exhibit peroxidase activity even under low pH conditions.
  • a reagent kit for measuring the binding amount of haptoglobin and hemoglobin using this property is commercially available (manufactured by Tridelta). Hemoglobin binding activity can be quantified by carrying out this reagent kit according to the attached operation procedure manual.
  • Phosphate buffer solution pH7.0 manufactured by Invitrogen
  • PBS Phosphate buffer solution pH7.0
  • soluble CD163 J. Biol. Chem., 279, 51 561 (2004)
  • PBS Phosphate buffer solution pH7.0
  • the absorbance in the reaction solution was measured at a wavelength of 415, and the test substance haptoglobin was added, and the reaction solution in which the test substance or standard product at each dilution stage was added from the absorbance of the control reaction solution.
  • a value obtained by subtracting the absorbance of. This value is plotted on a semilogarithmic graph with the vertical axis representing the amount of haptoglobin bound to the neuroglobin receptor and the horizontal axis representing the dilution ratio of the test substance or standard.
  • the standard of the test substance By approximating the relationship between the amount of haptoglobin bound to the haptoglobin receptor and the dilution rate from the plotted measurement values, and comparing the approximate equations obtained as a result of measurement of the test substance and the standard product, the standard of the test substance The magnification for the product can be determined and its titer can be determined.
  • the binding activity between solubilized CD163 and hemoglobin haptoglobin complex should be measured using the surface plasmon resonance phenomenon by the method described in the literature [J. Biol. Chem., 279, 51561 (2004)]. You can also.
  • CD163 binding activity assay (method using CD163 expressing cells)
  • a purified haptoglobin composition as a test substance and a commercially available habutoglobin with a known concentration and specific activity as a standard product using PBS containing ushi serum albumin at a volume ratio of 1%, a certain amount Of hemoglobin.
  • This hemoglobin haptoglobin complex is added to cells expressing CD163 and incubated at 4 ° C for 1 hour or longer.
  • Rush serum albumin containing 1% volume ratio After rinsing cells several times with PBS to remove the hemoglobin haptoglobin complex that did not bind to the cell surface, it contained ushi serum albumin at a volume ratio of 1%.
  • the biological activity of haptoglobin is measured by measuring an increase in calcium concentration, an increase in inositol triphosphate concentration, or an increase in production of interleukin 1, interoral quinine 6, or granulocyte-macrophage colony-stimulating factor. (J. Leukoe. Biol, 66, 8 58 (1999); Circ. Res., 92, 1193 (2003)).
  • a plataglandin synthesis caused by hemoglobin is inhibited by binding a hemoglobin composition purified as a test substance to hemoglobin.
  • the biological activity of haptoglobin can be measured (Anthropol. Anz., 50, 281 (1992)).
  • mouse globin By adding a purified rabbit globin composition as a test substance to vascular epithelial cells, it is possible to observe angiogenic activities such as cell proliferation and cell proliferation, and to increase the strength of the vascular new life.
  • the biological activity of mouse globin can be measured by measuring (J. Clin. Invest "91, 977 (1993)).
  • a clearance test using a purified rabbit globin composition or the like can be examined using a model animal such as a rat.
  • a test substance that also has a haptoglobin composition or a haptoglobin-hemoglobin complex and a test substance that has been labeled with a radioisotope by a known method is administered to a rat by intravenous injection or subcutaneous injection.
  • Samples in rat blood can be collected by collecting blood at any time after administration and measuring the amount of the labeled compound or ELISA method that can specifically detect and quantify human hubtoglobin.
  • the concentration The amount of labeling of the test substance that has been labeled is measured on the label. Measurement is performed by a known analysis method. The obtained data is used to measure the pharmacokinetic parameters of each rat using, for example, PCNONLIN nonlinear regression analysis (Statistical Consultants, 1992).
  • the clearance test for haptoglobin compositions can also be evaluated using animal models such as primates that are more closely related to humans than rats.
  • the sugar chain structure of a haptoglobin molecule expressed in various cells can be performed according to the analysis of the sugar chain structure of a normal glycoprotein.
  • sugar chains bound to haptoglobin 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 carried out by using a method such as sugar chain structure analysis using a sugar chain map method.
  • neutral sugar or amino sugar can be liberated by 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 obtained by acid-branching decomposition according to a known method [Agric. Biol. Chem., 55 (1). 283-284 (1991)] -Fluorescent labeling by aminobilylation and composition analysis can be calculated by HPLC analysis.
  • the two-dimensional glycan mapping method is, for example, the retention time or the glycan retention time by reverse phase chromatography on the X axis. Is a method of estimating the sugar chain structure by plotting the elution position and the retention time or elution position of the sugar chain by normal phase chromatography on the Y axis and comparing them with the results of known sugar chains. is there.
  • the haptoglobin composition was decomposed with hydrazine, the haptoglobin molecular force also released the sugar chain, and fluorescent labeling of the sugar chain with 2-aminoviridine (hereinafter abbreviated as “PA”) [Journal After performing “Biochemistry” (J. Biochem.), 197 (1984)], the glycan is separated from excess PA reagent by gel filtration and subjected to reverse phase chromatography. Next, normal phase chromatography is performed for each peak of the collected sugar chain.
  • PA 2-aminoviridine
  • 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 map method.
  • the haptoglobin composition is composed of haptoglobin molecular forces having different sugar chain structures.
  • the haptoglobin composition of the present invention is characterized in that fucose is not bound to ⁇ -acetyldarcosamine at the reducing end of ⁇ -glycoside bond complex type sugar chain, and has a long blood half-life.
  • Such a haptoglobin composition can be identified by using the method for analyzing the sugar chain structure of the haptoglobin molecule described in 5. above. It can also be identified by using an immunological quantification method using lectins.
  • lectins used for identifying the sugar chain structure of haptoglobin molecules include WG A (wneat— germ agglutinin derived from T. vulgaris) onA (concanavali n A derived from C. ensiformis), RIC (derived from R. communis) ), L- PHA (leukoagglutinin from P. vulgaris), LC A (lentil agglutinin from L. culinaris), PSA (Pea lectin from P.
  • AAL Alpha-1 (Aleuanthria caustus Lectin) ) ⁇ BPL (Bauhinia purpurea Le ctin) 8 DSL (Datura stramonium Lectin) ⁇ DBA (Dolichos biflorus Agglutinin) ⁇ EBL (E1 derberry Balk Lectin) ⁇ ECL (Erythrina cristagalli Lectin) ⁇ EEL (Euthymus europaeu s Lectin), GNL (Galanth Lectin), GSL (Griffonia simphcifolia Lectin), HPA (Helix pomatia Agglutinin) ⁇ HHL (Hippeastrum Hybrid Lectin) ⁇ Jacalin, LTL (Lotus t etragonolobus Lectin), LEL (Lycopersicon esculentum Lectin), MAL (Maackia am
  • a lectin that specifically recognizes a sugar chain structure in which fucose is bound to N-acetylcolcamine at the N-darcoside-linked complex type 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 Viciafaba), An example is chiatake lectin AAL (Lectin derived from Aleuria aurantia).
  • the haptoglobin composition of the present invention has a half-life in blood when administered in vivo, compared to haptoglobin in which fucose is bound to N-glycidyl darcosamine at the N-glycoside-linked complex type sugar chain reducing end. It has the characteristic of being long. Therefore, the haptoglobi of the present invention
  • the composition can enable treatment with fewer doses and fewer doses in various diseases for which treatment with haptoglobin is indicated. This not only reduces the burden on patients and medical sites, but also contributes to the reduction of accidents and side effects resulting from current treatments that must be administered in large quantities.
  • haptoglobin preparations are produced from the plasma of multiple healthy individuals, various hepatitis viruses, AIDS viruses, human parvoviruses, mutant Creutzfeld's prions that cause Jacob disease, etc. The fear of infection cannot be completely denied. It is very useful to switch a currently marketed blood product to a product containing the haptoglobin composition of the present invention in order to prevent various infection accidents. Furthermore, polymorphisms and multimers exist in haptoglobin, and its physiological function has been reported to be different for each polymorphism or multimer. By using the haptoglobin composition of the present invention, it can be adapted to the indication disease. It is possible to prepare a therapeutic agent containing a suitable haptoglobin type and provide it to the patient.
  • Diseases using the haptoglobin composition of the present invention are also effective for the prevention and treatment of diseases involving hemolysis, diseases involving tissue damage caused by blood free hemoglobin, and bacterial infections.
  • hemolytic diseases Specifically, traumatic hemoglobinuria associated with burns and blood transfusions, open-heart surgery under extracorporeal circulation such as cardiopulmonary bypass, and functional damage to the liver, the organ that produces haptoglobin Alcoholic hepatitis or alcoholic cirrhosis, congenital deficiency, etc.
  • pathological symptoms such as hemoglobinuria and hemoglobinemia are exhibited, and anemia associated with the outflow of iron and renal damage due to adhesion of hemoglobin and iron to the kidney are observed.
  • Examples of diseases associated with yarn and tissue injury caused by blood free hemoglobin include diseases associated with oxidative vascular injury caused by blood free hemoglobin, diseases associated with renal tubule injury caused by blood free hemoglobin, and the like.
  • Diseases associated with acid-induced vascular injury due to blood free hemoglobin include diseases caused by the formation of oxidized LDL and oxidative damage of vascular endothelial cells, diseases exacerbated by free radical generation, and diseases caused by acid oxidation of nucleic acids. can give.
  • diseases caused by the formation of oxidized LDL and acid dysfunction of vascular endothelial cells include myocardial infarction, cerebral infarction, and arteriosclerosis (American Journal of Medicine, 105, 32S (1998)).
  • Diseases exacerbated by the generation of free radicals include emphysema, pulmonary fibrosis, acute respiratory distress syndrome, neurodegenerative diseases, and chronic inflammatory diseases.
  • diseases caused by acid oxidation of nucleic acids include carcinogenesis.
  • Examples of diseases associated with renal tubule injury caused by blood free hemoglobin include nephrotic syndrome, acute nephritis, and chronic nephritis.
  • Arteriosclerosis is a disease in which blood flow is impaired due to narrowing of the arterial vascular lumen, and arteries that have lost the elasticity of the arterial wall cause dilation, rupture, and the like.
  • Hpl type haptoglobin the effect of anti-acidic vitamins such as vitamins C and E has been observed to prevent arteriosclerosis (Diabetes Care, 27, 925 (2004)).
  • Myocardial infarction is a disease in which myocardial ischemia occurs due to vascular injury due to occlusion or stenosis of the coronary artery that sends blood to the heart, resulting in necrosis of the myocardium.
  • myocardial ischemia occurs due to vascular injury due to occlusion or stenosis of the coronary artery that sends blood to the heart, resulting in necrosis of the myocardium.
  • Hp2 haptoglobin gene type Circ. Res., 92, 1193 (2003).
  • Cerebral infarction is a disease in which cerebral ischemia occurs due to vascular injury caused by occlusion or stenosis of blood vessels that send blood to the brain, resulting in necrosis of brain cells.
  • Emphysema is a disease in which the alveolar wall is destroyed, and the adjacent alveoli are fused together to form cavities, reducing the surface area of the alveoli. A reduction in alveolar surface area leads to a decrease in lung function and induces significant dyspnea.
  • Pulmonary fibrosis is a pneumonia disease in which inflammation occurs in the lung stroma such as the alveolar wall.
  • the pharmaceutical containing the haptoglobin composition of the present invention can be administered alone as a prophylactic or therapeutic agent. Usually mixed with one or more pharmacologically acceptable carriers. However, it is desirable to provide it as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics.
  • the route of administration includes oral administration, where it is desirable to use the most effective treatment, or parenteral administration, such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous.
  • parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous.
  • intravenous administration can be desirable
  • the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes 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 water, sucrose, sorbitol, fructose, etc., Daricols such as polyethylene glycol, propylene glycol, oils such as sesame oil, olive oil, soybean oil, P-hydroxy Preservatives such as benzoates, strawberry
  • Flavors such as laver and peppermint can be used as additives.
  • Capsules, tablets, powders, granules and the like 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.
  • 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 lyophilizing the haptoglobin composition of the present invention according to a conventional method and adding sodium chloride salt thereto.
  • Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  • the propellant does not irritate the hubtoglobin composition itself, or a carrier that does not irritate the recipient's oral cavity and airway mucosa and facilitates absorption by dispersing the habutoglobin composition as fine particles. It is prepared using.
  • the carrier include lactose and glycerin.
  • the carrier include lactose and glycerin.
  • aerosols, dry powders and the like it is possible to prepare aerosols, dry powders and the like.
  • ingredients exemplified as additives for oral preparations to these parenteral preparations.
  • the dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but the amount of active ingredient is usually 4000 units per adult (1 unit is 1 mg The amount that can bind to hemoglobin of about 5.7 g).
  • the methods for examining the biological activity of the haptoglobin composition can be determined from the above-described hemoglobin binding activity measurement method, the CD163 binding activity measurement method for the hemoglobin haptoglobin complex receptor, and CD163. Intracellular signal measurement method through CD163
  • Examples include an in vitro test such as a measurement method for langine synthesis inhibitory activity, a method for measuring angiogenic activity, a method for measuring an antibacterial activity, or an in vivo test using a hemolyzed model animal.
  • an in vitro test such as a measurement method for langine synthesis inhibitory activity, a method for measuring angiogenic activity, a method for measuring an antibacterial activity, or an in vivo test using a hemolyzed model animal.
  • a FUT8 gene double knockout cell line producing human HP-1 haptoglobin was prepared by the method shown below.
  • the solution after the PCR reaction was subjected to 2% agarose gel electrophoresis, and an approximately 1067 bp PCR amplified fragment (containing a noptoglobin HP-1 type cDNA sequence) was purified using QIAquick Gel Extraction kit (manufactured by QIAGEN).
  • the obtained purified DNA fragment was dissolved in 17 L of water. Thereafter, 7.5 L of restriction enzyme EcoRI (manufactured by Takara Bio Inc.), 5 units of Bglll (manufactured by Takara Bio Inc.), and 2 L of 10 XH buffer were added to the solution to prepare a 20 L reaction solution. The extinguishing reaction was carried out at ° C for 16 hours.
  • pKANTEX93 (W097 / 10354) is dissolved in 17 ⁇ L of water, and 7.5 units of EcoRI and 2 ⁇ L of 10 XH buffer are added to the solution to prepare 20 ⁇ L of reaction solution.
  • the digestion reaction was performed at 37 ° C for 16 hours.
  • phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17 L of water.
  • 7.5 units of BamHI and 2 L of 10 XK buffer were added to the solution to prepare a 20 ⁇ L reaction solution, followed by digestion reaction at 37 ° C for 16 hours.
  • HP-1 haptoglobin DNA fragment (EcoRI-Bglll) and pKANTEX93 fragment (EcoRI-BamHI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and each of about 1060 bp and 3 kbp.
  • the DNA fragment was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
  • Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequence Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied) The base sequence was analyzed using Biosystems). As a result, plasmid pKAN-HP1 containing the cDNA sequence of HP-1 type haptoglobin was obtained (FIG. 3).
  • the plasmid pKAN-HPl prepared in the previous section 1 was introduced into FUT8 gene double knockout cells described in the literature (Biotechnology and Bioengineering 87, 614 (2004)). These genes were introduced by the following procedure according to a known electoral position method [Cytotechnology, 3, 133 (1990)].
  • the reaction was linearized by digestion with C for 16 hours. After the reaction, the reaction solution is subjected to phenol / chloroform extraction treatment and Purification was performed by ethanol precipitation, and the linear plasmid was recovered.
  • FUT8 gene double knockout cells described in the literature (Biotechnology and Bioengineering 87, 614 (2004)) were mixed with K-PBS buffer (137mmol / L KC1, 2.7mmol / L NaCl, 8.lmmol / L 8 x 10 7 cells suspended in Na HPO, 1.5mmol / L KH PO, 4.0mmol / L MgCl)
  • IMDM medium Life Technologies
  • Ushi fetal serum Life Technologies
  • 50 ⁇ g / mL gentamicin Nacalai Testa
  • the suspension was suspended in 10 mL, and seeded in an adherent cell culture T 75 flask (manufactured by Grainer). The culture was performed under conditions of 5% CO and 37 ° C.
  • IMDM medium 10 mL of IMDM medium supplemented with analysis serum, 50 ⁇ g / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Subsequently, the medium was replaced with IMDM medium supplemented with 10% urine fetal dialyzed serum, 50 g / mL gentamicin and 200 nM MTX, and cultured again every 3-4 days for 18 days. Obtained MTX resistant strain HP1KO
  • HP1KO is suspended in IMDM medium supplemented with 5 mL of 10% urine fetal dialyzed serum, 50 ⁇ g / mL gentamicin and 200 nM MTX at a concentration of 3.0 ⁇ 10 5 cells / mL, and seeded in a T25 flask. Then, static culture was performed for 3 days. The culture supernatant after 3 days of culture was collected, and the amount of haptoglobin contained in the supernatant was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited). It was confirmed that butoglobin was expressed.
  • Haptoglobin Kit manufactured by Tridelta Development Limited
  • the HP1KO strain is the name of HP1KO, and the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, February 17, 2005 (Tsukuba Ito, Ibaraki Pref. Has been deposited as FERMBP-10249.
  • the culture supernatant of the HP1KO strain prepared in this way is then used for general purification of haptoglobin (development of follow-up medicines HP-1 type haptoglobin obtained by purifying using the 20th store, page 215-219) has fucose produced by the normal CHO / DG44 strain and has a sugar chain! Increased blood half-life was observed compared to HP-1 haptoglobin.
  • a FUT8 gene double knockout cell line producing human HP-2 haptoglobin was prepared by the method described below.
  • the solution after the PCR reaction was subjected to 2% agarose gel electrophoresis, and an approximately 1244 bp PCR amplified fragment (containing the HP-2 haptoglobin cDNA sequence) was purified using QIAquick Gel Extraction kit (manufactured by QIAGEN). After the obtained purified DNA fragment was dissolved in 17 L of water, 7.5 units of restriction enzyme EcoRI (Takara Bio) and 5 units of Bglll (Takara Bio), 2 L of 10 XH buffer were added to the solution. A 20 L reaction solution was prepared and the quenching reaction was carried out at 37 ° C for 16 hours.
  • pKANTEX93 (described in W097 / 10354) is dissolved in 17 ⁇ L of water, and 7.5 units of EcoRI and 2 ⁇ L of 10 XH buffer are added to the solution to prepare a 20 ⁇ L reaction solution. After the preparation, digestion reaction was performed at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17 L of water. Furthermore, 7.5 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.
  • HP-2 haptoglobin DNA fragment (EcoRI-Bglll) and pKANTEX93 fragment (EcoRI-BamHI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and each about 1240 bp A 3 kbp DNA fragment was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
  • the ligation reaction was performed for 16 hours.
  • the resulting plasmid DNA was used to transform Escherichia coli DH5 strain (manufactured by Toyobo Co., Ltd.) by the heat shock method.
  • the base sequence was analyzed using Biosystems).
  • plasmid pKAN-HP2 containing the cDNA sequence of HP-2 haptoglobin was obtained (Fig. 4).
  • the plasmid pKAN-HPl prepared in Example 1 was introduced into double knockout cells. This
  • 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. lmmol / L 8 x 10 7 cells suspended in Na HPO, 1.5mmol / L KH PO, 4.0mmol / L MgCl)
  • IMDM medium 10 mL of IMDM medium supplemented with analysis serum, 50 ⁇ g / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. 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 dialyzed serum, g / m Lgentamicin and 200 nM MTX was similarly repeated every 3-4 days for 18 days, and 200 nM MTX The resistant strain HP2KO was obtained.
  • MTX methotrexate
  • the resulting HP2KO was suspended in IMDM medium supplemented with 5 mL of 10% urine fetal dialyzed serum, 50 ⁇ g / mL gentamicin and 200 nM MTX at a concentration of 3.0 X 10 5 cells / mL, and T25
  • the flask was inoculated and statically cultured for 3 days.
  • the culture supernatant after 3 days of culture was collected, and the amount of haptoglobin contained in the supernatant was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited). It was confirmed that butoglobin was expressed.
  • HP2KO strain is the name of HP2KO, and the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center on February 17, 2005 (Tsukuba Ibaraki 1-chome, 1st, 1st, 1st, 6th) Has been deposited as FERMBP-10250.
  • IMDM medium manufactured by Invitrogen
  • IMDM medium 1-fold concentration of IMDM medium (manufactured by Invitrogen)
  • PBS Dulbecco's PBS
  • the were detached cells were recovered by centrifugation operation performed by the conventional cell culture, 1 X 10 5 cells / mL IMDM-F BS (10 ) such that the density of - HT (1) medium was ⁇ Ka ⁇ After suspension, MNNG (manufactured by Sigma), which was not added or 0.1 g / mL alkylating agent, was added. 3 days at 37 ° C in CO incubator (TABAI)
  • the CHO / DG44 cell line was obtained.
  • 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. Subsequently, using a SUPER SCRIPT First-Strand synthesis system for RT-PCR (manufactured by Invitrogen), single-stranded cDNA was synthesized from 5 g of each RNA in a 20 L reaction solution according to the attached instruction manual.
  • the expression level of the GDP-mannose 4,6-dehydratase gene in each lectin resistant CHO / DG44 cell line obtained in (1) of this section was analyzed.
  • the CHO cell-derived ⁇ -actin cDNA sequence (NCBI accession number: U20114) has a salted salmon sequence represented by SEQ ID NO: 22
  • a 24-mer synthetic oligo DNA primer having a 24 mol synthetic oligo DNA primer having a salt-and-sugar sequence represented by SEQ ID NO: 23 was prepared.
  • reaction solution containing 0.5 ⁇ L of single-stranded cDNA derived from each cell line prepared in this section (1) as a cage [1 X EX Taq Buffer (Takara Shuzo), 0.2 mM dNTP Prepare a mixture of 0.5 units of Ex Taq polymerase (Takara Shuzo), 0.5 ⁇ of synthetic DNA primers of SEQ ID NOS: 24 and 25], and use a DNA thermal cycler 480 (Perkin Elma) to make 94 ° After heating at C for 5 minutes, 30 cycles of 94 ° C for 1 minute and 68 ° C for 2 minutes were performed.
  • DNA thermal cycler 480 Perkin Elma
  • the CHO SM strain is a lectin that recognizes the same sugar chain structure as that recognized by LCA, that is, the N-glycoside-linked sugar chain reducing terminal. It was also resistant to other lectins that recognize sugar chain structures in which the N-acetylyldarcosamine residue at position 6 and fucose at position 1 are attached by an a bond.
  • a medium supplemented with endumerme lectin (Pisumsativum Agglutinin; hereinafter referred to as PSA, manufactured by Vector) with a final concentration of 1 mg / mL, or a white bamboo lectin with a final concentration of 1 mg / mL (Aleuria) aurantia Lectin; hereinafter referred to as AAL, manufactured by Vector, Inc.) was also resistant.
  • CHO / DG44 cells and the CHO SM strain obtained in the previous section were cultured using IMDM-FBS (10) -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.
  • 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 of M buffer (Takara Shuzo), and restriction enzyme Hindlll (Takara Shuzo) was dissolved. 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, oligo DNA primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically bind to exon 5 were designed based on the publicly known human GMD genomic DNA sequence (NCBI accession number: NT_034880). This region corresponds to nucleotide numbers 349 to 538 of the human GMD cDNA sequence shown in SEQ ID NO: 7.
  • a reaction solution of 100 ⁇ L containing lOng of plasmid pAGE2 49GMD described in Example 15 of WO02 / 31140 [ExTaq buffer (Takara Shuzo), 0.2 mmol / L dNTPs, 2.5 / z mol / L above gene-specific Primers (SEQ ID NO: 26 and SEQ ID NO: 27)] were prepared and subjected to polymerase chain reaction (PCR). PCR was performed under the conditions of 30 cycles, with a reaction consisting of 94 ° C for 5 minutes, 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.
  • the obtained DNA fragment 25ng, [ «- 32 P] dCTP 1.75MBq and Megapr ime DNA labeling system, and radiation-labeled using dCTP (manufactured by Amersham Biosciences).
  • the probe was hybridized to 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 found in genomic DNA derived from CHOSM strain! Was not detected at all. From the above results, it was shown that the CHO SM strain is a GMD knockout cell lacking at least the region containing exon 5 in the genomic region encoding GMD.
  • GMD knockout cell line producing human HP-1 haptoglobin is prepared by the following method
  • plasmid pKAN-HP1 prepared in Example 1 was introduced into the CHO SM strain prepared in Example 3. These gene introductions were carried out by the following procedure according to a known electopore position method [Cytotechnology, 3, 133 (1990)].
  • plasmid pKAN-H Prepare a reaction solution of 30 ⁇ g PI (20 ⁇ L NE Buffer 3 (New England Biolabs) and 200 ⁇ L 200 ⁇ L restriction enzyme Mlul (New England Biolabs) at 37 ° C for 16 hours. A linear reaction was achieved by performing a digestion reaction. After the reaction, the reaction mixture was purified by phenol / chloroform extraction treatment and ethanol precipitation to recover the linear plasmid.
  • Example 3 the CHO SM strain obtained in Example 3 was added to K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8. lmmol / L Na HPO, 1.5 mmol / L KH PO, 4.0 mmol / L). 8 x 10 7 cells suspended in MgCl)
  • IMDM medium Life Technologies
  • Ushi fetal serum Life Technologies
  • 50 ⁇ g / mL gentamicin Nacalai Testa
  • the suspension was suspended in 10 mL, and seeded in an adherent cell culture T 75 flask (manufactured by Grainer). The culture was performed under conditions of 5% CO and 37 ° C.
  • IMDM medium 10 mL of IMDM medium supplemented with analysis serum, 50 ⁇ g / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Next, repeat the medium replacement using IMDM medium supplemented with 10% urine fetal dialyzed serum, 50 g / mL gentamicin and 20 OnM MTX in the same manner every 3-4 days, and culture for 18 days. A resistant strain was obtained.
  • MTX methotrexate
  • the amount of haptoglobin contained in the culture supernatant of the 200 nM MTX resistant strain was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited).
  • the culture supernatant of the 200nM MTX-resistant strain produced in this way was obtained by purifying using the general purification method for haptoglobin (development of follow-on pharmaceuticals, Yodogawa Shoten No. 20, pages 215-219).
  • HP-1 type haptoglobin has an increased blood half-life compared to HP-1 type haptoglobin, which has a sugar chain to which fucose is bound, produced by the normal CHO / DG44 strain.
  • GMD knockout cell line producing human HP-2 haptoglobin is prepared by the following method
  • the plasmid pKAN-HP2 prepared in Example 2 was introduced into the CHO SM strain prepared in Example 3. These gene introductions were carried out by the following procedure according to a known electopore position method [Cytotechnology, 3, 133 (1990)].
  • Example 3 the CHO SM strain obtained in Example 3 was added to K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8. lmmol / L Na HPO, 1.5 mmol / L KH PO, 4.0 mmol / L). 8 x 10 7 cells suspended in MgCl)
  • IMDM medium Life Technologies
  • Ushi fetal serum Life Technologies
  • 50 ⁇ g / mL gentamicin Nacalai Testa
  • the suspension was suspended in 10 mL, and seeded in an adherent cell culture T 75 flask (manufactured by Grainer). The culture was performed under conditions of 5% CO and 37 ° C.
  • IMDM medium 10 mL of IMDM medium supplemented with analysis serum, 50 ⁇ g / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Next, repeat the medium replacement using IMDM medium supplemented with 10% urine fetal dialyzed serum, 50 g / mL gentamicin and 200 nM MTX every 3-4 days. After culturing for 18 days, a 200 nM MTX resistant strain was obtained.
  • MTX methotrexate
  • the amount of haptoglobin contained in the culture supernatant of the 200 nM MTX resistant strain was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited).
  • the culture supernatant of the 200nM MTX-resistant strain produced in this way was obtained by purifying using the general purification method for haptoglobin (development of follow-on pharmaceuticals, Yodogawa Shoten No. 20, pages 215-219).
  • HP-2 haptoglobin was found to have an increased blood half-life compared to HP-2 haptoglobin, which has a sugar chain to which fucose is bound, produced by the normal CHO / DG44 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 at the branch of 1191 (2002)). Further, a high mannose type sugar chain having such a structure is also called a hypermannose type sugar chain.
  • the hybrid sugar chain which is an intermediate structure between the high-mannose sugar chain and the complex sugar chain, is mainly used as the structure of the N-linked sugar chain to be added.
  • the methods for preparing Pichia yeast strains and Saccharomyces yeast strains that express the HP-1 type or HP-2 type haptoglobin 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 PO vector (Invitrogen).
  • a vector such as 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 introduced yeast is cultured at room temperature using 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 was cultured at room temperature in YPD medium (Invitrogen) lacking uracil and grown. Colony strength also extracts genomic DNA.
  • 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 main N-linked sugar chain expressed in Pichia yeast has 2 residues of N-acetyl darcosamine in the core part on the reducing end side, and 8 structures on the non-reducing end side. It can be modified to a Man8 type high mannose type sugar chain having a structure in which a mannose residue is bound.
  • the cDNA encoding the active domain of is specifically amplified.
  • 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 Extract total RNA from yeast (Kluyveromyces lactis) using the RNeasy Mini Kit (Qiagen), and then prepare cDNA using the Superscript TM first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is used as a saddle type, and PCR is performed using specific primers and KOD polymerase (Toyobo) to encode the entire translation region of the yeast UDP-N-acetylyldarcosamine transporter. cDNA (Gen Bank accession number: AF106080) is specifically amplified.
  • the amplified cDNA of about 3700 bases in length is used as a restriction enzyme EcoRI cleavage site located downstream of the alcohol oxygenase promoter sequence of vectors such as the yeast expression vector pPIC3.5K (manufactured by Invitrogen).
  • vectors such as the yeast expression vector pPIC3.5K (manufactured by Invitrogen).
  • a vector that inserts between I cleavage sites and expresses UDP-N-acetyltilcosamine transporter in the Golgi apparatus of yeast is prepared.
  • this vector is stably introduced into the Pichia yeast strain introduced with the ⁇ -1,2-mannosidase gene described in the previous section by the electopore method.
  • the yeast after the 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 the expression of the recombinant UDP-N-acetylyldarcosamine transporter is observed is selected by PCR using the cDNA prepared from this total RNA as a saddle type.
  • 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 One is introduced into the Pichia yeast strain into which the UDP-N-acetylcyldarcosamine transporter gene described in the previous section has been introduced, by the lithium acetate method described in the manual attached to the expression vector pAUR123.
  • the yeast after gene transfer is cultured at room temperature in a YPD medium containing the drug mouthful brassin A (manufactured by Takara noisyo), and total RNA is extracted 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-type sugar chain, which is an intermediate structure between a no-, immannose-type sugar chain and a complex-type sugar chain, as an N-linked sugar chain has been described above.
  • 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.
  • this genomic DNA was used as a saddle-shaped PCR method to determine the sequence of the yeast OCH1 locus (GenBan k clone session number: AF540063) and MNN1 locus sequence (GenBank accession number: AF540063L23753) are amplified to select yeast clones in which both OCH1 locus and MNN1 locus are destroyed.
  • the structure of the main N-linked sugar chain expressed in Saccharomyces yeast has 2 residues N-acetylyldarcosamine in the core part on the reducing end side and 8 on the non-reducing end side. It can be modified into a Man8-type high mannose-type sugar chain having a structure in which the mannose residue is bound.
  • 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 full-length translation region of mold a-1,2-mannosidase (GenBank Accession picker 1: Amplify D49827) specifically.
  • a specific primer and KOD polymerase Toyobo Co., Ltd.
  • the amplified cDNA with a length of about 1500 bases has a yeast endoplasmic reticulum localization signal peptide (Embo Journal 7, 913 (1988)), that is, histidine-aspartate, at the 3 'end from which the stop codon was deleted.
  • yeast endoplasmic reticulum localization signal peptide Embo Journal 7, 913 (1988)
  • histidine-aspartate at the 3 'end from which the stop codon was deleted.
  • this vector was stably added to the Saccharomyces yeast strain in which the a -1,6-mannose transferase gene and the a -1,3-mannose transferase gene described in the previous section were disrupted by the electopore method. Introduce.
  • 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 main N-linked sugar chain structure expressed by Saccharomyces yeast has 2 residues of N-acetyldarcosamine in the core of the reducing end, and the non-reducing end It can be modified into a Man5 type high mannose type sugar chain having a structure in which 5 mannose residues are bonded to the side.
  • RNA Extract total RNA from yeast (Kluyveromyces lactis) using the RNeasy Mini Kit (Qiagen), and then prepare cDNA using the Superscript TM first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is used as a saddle type, and PCR is performed using specific primers and KOD polymerase (Toyobo) to encode the entire translation region of the yeast UDP-N-acetylyldarcosamine transporter.
  • cDNA GenBank
  • the amplified cDNA having a length of about 3700 bases is combined with a restriction enzyme EcoRI cleavage site located downstream of a single alcohol oxygenase promoter sequence such as the yeast expression vector pPIC3.5K (manufactured by Invitrogen).
  • a vector that inserts between Not I cleavage sites and expresses UDP-N-acetyltilcosamine transporter in the yeast Golgi is prepared.
  • this vector is stably introduced into the Saccharomyces yeast strain into which the a-1,2-mannosidase gene has been introduced as described in the previous section by the electopore method.
  • the yeast after the 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 the expression of the recombinant UDP-N-acetylyldarcosamine transporter is observed is selected by PCR using the cDNA prepared from this total RNA as a saddle type.
  • N-acetylylcosamine transferase-1 (GenBank accession number) was obtained by performing PCR using human liver cDNA (Clontech) in a saddle shape and specific primers and KOD polymerase (Toyobo). : Amplify specifically the cDNA encoding the active domain of M55621).
  • the amplified cDNA contains the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752) at the 5 'end.
  • the cDNA sequence to be loaded After ligation of the cDNA sequence to be loaded, it is inserted between the restriction enzyme Kpnl cleavage site and the Xba I cleavage site located downstream of the alcohol dehydrogenase promoter sequence of vectors such as the yeast expression vector pAUR123 (Tacarano).
  • vectors such as the yeast expression vector pAUR123 (Tacarano).
  • a vector that expresses N-acetyltilcosamine transferase-1 in the Golgi apparatus of yeast is prepared.
  • this vector was introduced into the Saccharomyces 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. To do.
  • 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 end of the Man5 type high mannose type sugar chain having a structure in which one mannose residue is bound.
  • human liver cDNA (manufactured by Invitrogen) is used as a cage and Pyrobest DNA polymerase (manufactured by Takara Bio Inc.) is used as an amplification enzyme.
  • the cDNA encoding the full-length mature haptoglobin is specifically amplified by the PCR reaction.
  • the obtained HP-1 type or HP-2 type haptoglobin cDNA is used as a restriction enzyme located downstream of the alcohol oxygenase promoter sequence of a vector such as the yeast expression vector pPIC6a (manufactured by Invitrogen).
  • a vector PPIC6a / HP1 for expression and a vector pPIC6a / HP2 for secreting and expressing mature human HP-2 haptoglobin are prepared.
  • a Pichia yeast strain that mainly expresses a hybrid-type sugar chain as an N-linked sugar chain, or a Saccharomyces yeast that mainly expresses a hybrid-type sugar chain as an N-linked sugar chain described in Section 9 of this Example Introduced into the strain 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 a blasticidin-resistant colony.
  • the blasticidin-resistant colonies are transplanted into liquid YPD medium (Invitrogen) and cultured at 30 ° C for 24 hours or longer.
  • the culture supernatant obtained after the culture is analyzed using Haptoglobinkit (manufactured by Tridelta Development Limited) using a human plasma-derived haptoglobin preparation (manufactured by Mitsubishi Wellpharma) as a standard product.
  • Haptoglobinkit manufactured by Tridelta Development Limited
  • a human plasma-derived haptoglobin preparation manufactured by Mitsubishi Wellpharma
  • the recombinant HP-1 and HP-2 haptoglobins which have a hybrid sugar chain that does not contain fucose as an N-linked sugar chain, are secreted into the yeast culture supernatant, using the method of Krystal et al. (Bloodgl , 71 (1986)).
  • the purified HP-1 type and HP-2 type haptoglobin proteins can be analyzed for sugar chain structure according to the method of Skibeli et al. (Bloody, 3626 (2001)).
  • an N-linked glycan a Pichia that mainly expresses an ibubrid glycan with one N-acetyl darcosamine residue added to the non-reducing end of the Man5 type high mannose glycan.
  • Recombinant human HP-1 type and HP-2 type mainly using hybrid sugar chains that do not contain fucose as N-linked sugar chains, using yeast strains or similarly modified Saccharomyces yeast strains as hosts It was stated that haptoglobin can be prepared.
  • a method for producing a yeast strain expressing a recombinant human HP-1 type and HP-2 type haptoglobin mainly having a double-chain type sugar chain is described below.
  • the vector is inserted downstream of the promoter sequence of the vector to produce a vector that expresses ⁇ - mannosidase ⁇ in the yeast Golgi apparatus.
  • this vector expresses recombinant human HP-1 type or HP-2 type haptoglobin mainly having an glycan-linked glycan and an hybrid glycan as described in paragraph 11 of this Example. Stable introduction into yeast strains.
  • the fermented mother after gene transfer selects clones using auxotrophy and drug resistance as indicators, and then confirms the expression of chimeric ⁇ -mannosidase II by RT-PCR.
  • N-acetylyldarcosaminyltransferase is obtained by performing PCR using a specific primer and Pyrobest DNA polymerase (Takara Bio) from a human tissue (eg, liver-derived cDNA dnvitrogen). -Specific amplification of cDNA encoding the active domain of II (GenBank accession number: U15128). The amplified cDNA has a yeast mannose transferase (MNN9) gene (GenBank accession number: L23752) at the 5 'end.
  • MNN9 yeast mannose transferase
  • a vector for expressing transferase- ⁇ is prepared.
  • this vector was transformed into a chimeric a mannosidase described in the preceding paragraph in a yeast strain expressing recombinant human HP-1 or ⁇ ⁇ ⁇ ⁇ -2 haptoglobin mainly having a hybrid sugar chain as a ⁇ -linked sugar chain. Introduce stably to yeast strains that have stably introduced II. For the yeast after gene transfer, clones were selected using auxotrophy and drug resistance as indicators, and then the expression of chimeric N-acetylcolcamine transferase- ⁇ was confirmed by RT-PCR.
  • the sugar chain structure has two residues of N-acetyl darcosamine in the core portion on the reducing end side, and is linked to the non-reducing end side in a structure where three mannose residues are bifurcated. It can be modified into a complex double-stranded sugar chain containing fucose, with one N-acetylyldarcosamine residue added to each non-reducing end.
  • Amplify specific cDNA The amplified cDNA is inserted downstream of the expression motor sequence of an expression vector for yeast to produce a vector that expresses UDP-galactose-4-epimerase in the yeast cytosol.
  • yeast strain expressing recombinant human HP-1 type or HP-2 type haptoglobin mainly having an immature complex double chain type sugar chain as an N-linked type sugar chain as described in the previous section. In contrast, it is introduced stably. After the gene is introduced, clones are selected using auxotrophy and drug resistance as indicators, and then the expression of UDP-galactose-4-epimelase is confirmed by RT-PCR.
  • 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.
  • a vector is inserted downstream of the promoter sequence to express
  • this vector is used to express recombinant human HP-1 type or HP-2 type haptoglobin mainly having an immature complex double-stranded sugar chain as an N-linked sugar chain, as described in the previous section. Stable introduction into a yeast strain that has stably introduced chimeric ⁇ 1,4 galactosyltransferase into the yeast strain.
  • chimera 8 Major ⁇ -linked saccharides of recombinant HP-1 or ⁇ -2 haptoglobin expressed by yeast strains stably incorporating 1,4 galactosyltransferase
  • the chain structure has two residues ⁇ -acetyl darcosamine in the core part on the reducing end side, and the two non-reducing end side has three mannose residues in two branched structures. It can be modified into a complex double-stranded sugar chain in which one non-reducing end is added with one ⁇ -acetyldarcosamine residue and one galactose residue.
  • Recombinant HP-1 type or ⁇ -2 type haptoglobin which mainly has a complex double-stranded sugar chain with no fucose residue on the reducing end side and galactose added on the non-reducing end side, prepared in the previous section
  • the yeast strain to be expressed is seeded in a liquid YPD medium (manufactured by Invitrogen) and 24 hours at 30 ° C.
  • N-linked sugar chains secreted into the yeast culture supernatant Recombinant HP-1 type or HP-2 type haptoglobin with a complex double-stranded sugar chain containing fucose can be purified according to the method of Krystal et al. (Bloodgl, 71 (1986)) It is.
  • the purified HP-1 type or HP-2 type haptoglobin protein can be analyzed for the sugar chain structure according to the method of Skibeli et al. (Blood £ g, 3626 (2001)).
  • composition comprising a recombinant haptoglobin molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is N- It is possible to provide a haptoglobin composition which is a sugar chain in which fucose is bound to acetylyldarcosamine!

Abstract

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

Description

遺伝子組換えハプトグロビン組成物  Genetically modified haptoglobin composition
技術分野  Technical field
[0001] 本発明は、 N-グリコシド結合複合型糖鎖を有する遺伝子組換えハプトグロビン分子 力もなる組成物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末端の N-ァ セチルダルコサミンにフコースが結合して ヽな 、糖鎖であるハプトグロビン組成物及 びその用途に関する。  [0001] The present invention relates to a composition having a recombinant haptoglobin molecular force having an N-glycoside-linked complex sugar chain, wherein the N-glycoside-bonded complex sugar chain is N-acetyl at the reducing end of the sugar chain. The present invention relates to a haptoglobin composition which is a sugar chain and has its use, wherein fucose is bound to darcosamine.
背景技術  Background art
[0002] ハプトグロビンは、肝実質細胞やリンパ節などに存在する好酸球に代表される成熟 顆粒白血球で生産される分子量 8万 5千〜 40万の血漿糖蛋白質である。通常、 0.2〜 3g/Lの濃度で血中に存在しており、溶血によって血中にヘモグロビンが遊離されると 特異的に結合して、複合体を形成する (非特許文献 1)。この複合体は、肝臓へと運 ばれたのち、細網内皮細胞系の細胞に発現する CD 163等の受容体と結合することに よって細胞内に取り込まれ、ヘムォキシゲナーゼによってピリルビンにまで代謝される (非特許文献 2〜4)。ヒトハプトグロビンの血中半減期は 3.5〜5日である力 へモグロ ビンと結合した複合体の半減期は 10〜30分と極めて短ぐ速やかに取り込まれ分解 処理されていることが知られている。一方、熱傷や火傷、輸血、人工心肺などの体外 循環下開心術等の病的な状態の発生により溶血反応が生じ血中のヘモグロビン量 がハプトグロビン量を上回ると、腎糸球体よりヘモグロビンが漏出されることになり、体 内からの鉄の消耗や、遊離ヘモグロビンによる酸化的血管障害及び腎細尿管障害 などを引き起こす。ハプトグロビン製剤は、このようなヘモグロビン血症およびへモグ ロビン尿症の治療薬として使用されている。また、低ないし無ハプトグロビン血症患者 にハプトグロビンを内科的に補充することで、ヘモグロビンによる腎障害や貧血を防 止するという医療上の意義もある。ハプトグロビンの生理的機能の最も重要な役割は 、赤血球の溶血により生じた遊離ヘモグロビンと特異的にかつ強固に結合してへモ グロビンを肝臓に運び、生体が持つ代謝サイクルにのせることであり、ヘモグロビン代 謝における生体防御機構の中心的な役割を演じている因子であるということができる 。また、ハプトグロビンは急性期反応蛋白質としての特性も有しており、感染、炎症、 組織崩壊、悪性腫瘍などでは好酸球などの肝臓以外の異所性産生による血清ハプ トグロビンの著し 、増加が認められることが知られて 、る（非特許文献 5)。 [0002] Haptoglobin is a plasma glycoprotein with a molecular weight of 85,000 to 400,000 produced by mature granular leukocytes represented by eosinophils present in hepatocytes and lymph nodes. Usually, it exists in blood at a concentration of 0.2 to 3 g / L, and when hemoglobin is released into the blood by hemolysis, it specifically binds to form a complex (Non-patent Document 1). This complex is transported to the liver and then taken up into the cell by binding to receptors such as CD163 expressed in cells of the reticuloendothelial cell line, and to hepatoxylase by hemoxygenase. Metabolized (Non-Patent Documents 2 to 4). The blood half-life of human haptoglobin is 3.5 to 5 days. The half-life of the complex bound to hemoglobin is 10-30 minutes, and it is known that it is rapidly taken up and degraded. . On the other hand, if hemolysis occurs due to the occurrence of pathological conditions such as burns, burns, blood transfusions, open-heart surgery under extracorporeal circulation such as cardiopulmonary bypass, and hemoglobin in the blood exceeds haptoglobin, hemoglobin leaks from the glomeruli. This causes iron depletion from the body and oxidative vascular and renal tubule damage caused by free hemoglobin. Haptoglobin preparations are used as therapeutic agents for such hemoglobinemia and hemoglobinuria. In addition, there is a medical significance of preventing renal damage and anemia caused by hemoglobin by medically supplementing haptoglobin to patients with hypo- or non-haptoglobinemia. The most important role of the physiological function of haptoglobin is to specifically and firmly bind to free hemoglobin generated by hemolysis of erythrocytes, to transport hemoglobin to the liver, and to put it on the metabolic cycle of the living body. It can be said that it is a factor that plays a central role in the defense mechanism of hemoglobin . In addition, haptoglobin has characteristics as an acute phase reaction protein. In infection, inflammation, tissue disruption, malignant tumor, etc., serum haptoglobin is markedly increased due to ectopic production other than liver such as eosinophils. It is known to be accepted (Non-patent Document 5).
[0003] ハプトグロビンは、 α鎖と j8鎖からなる 2量体（図 1)がそれぞれの α鎖でジスルフィ ド結合することによって 4量体構造 (以下、 ( a |8 )2と表記する）をとり、この 4量体構造 を基本構造として多量体構造をとることができる糖蛋白質である（図 2) (非特許文献 6 )。ノ、プトグロビンの α鎖と j8鎖は 1つのオープンリーディングフレームにコードされた 単一遺伝子であり、ヒト第 6染色体長腕 22.1に存在する（非特許文献 7、 8)。この単一 遺伝子の翻訳産物であるハプトグロビン前駆体がハプトグロビンを発現させる細胞内 で 1箇所切断されることにより、 α鎖と j8鎖を生じる。ヒトハブトグロビン遺伝子には Hp 1と Hp2の 2種類があり遺伝的多型が存在するため（図 1)、各々のヒトは Hpl-1、 Hp2- 1、 Hp2-2の 3種類のハプトグロビンのいずれかを有している（図 2)。このハプトグロビ ンの遺伝型の分布には人種差があり、欧米人では Hp2-1が 45〜55%、 Hp2-2が 30〜 40%、 Hpl- 1が 10〜20%、 日本人では Hp2- 1が 58%、 Hp2- 2が 35%、 Hpl- 1が 7%、 アフリカ人では約半数が Hpl-1であることが知られている（非特許文献 9、 10)。また、 ハプトグロビン遺伝子座では、その頻度は低 、ものの分子内組換えによる Hp2遺伝 子から Hpl遺伝子への復帰突然変異が体細胞で恒常的に起こっており、キメラ状態 であることが報告されている（非特許文献 11)。さらに、ヒトハブトグロビン遺伝子のェ クソン 4にコードされる 52番目と 53番目のアミノ酸が Aspと Lysであるヒトと Asnと Gluであ るヒトが報告されており、 Hpl遺伝子についても 2種類の多型が存在する。また、 Hp2 遺伝子については後述する遺伝子重複の関係カゝら 4種類の多型が存在する（非特許 文献 12)。またさらに、ハプトグロビン製剤は数千人という多くの健常人の血漿を原料 として精製されている。したがって、均一な遺伝子型カゝらなるハプトグロビン血液製剤 を調製することは不可能な状況となって 、る。 [0003] Haptoglobin has a tetramer structure (hereinafter referred to as (a | 8) 2) by disulfide bonding of α-chain and j8-chain dimer (Fig. 1) at each α-chain. This is a glycoprotein that can take a multimeric structure based on this tetrameric structure (FIG. 2) (Non-patent Document 6). The alpha chain and the j8 chain of ptoglobin are single genes encoded in one open reading frame and exist in human chromosome 6 long arm 22.1 (Non-patent Documents 7 and 8). The haptoglobin precursor, which is the translation product of this single gene, is cleaved at one site in the cell that expresses haptoglobin, thereby generating an α chain and a j8 chain. There are two types of human habutoglobin genes, Hp 1 and Hp2, and there are genetic polymorphisms (Fig. 1), so each human has three types of haptoglobin, Hpl-1, Hp2-1 and Hp2-2. One of them (Figure 2). There is a racial difference in the genotype distribution of this haptoglobin. In Westerners, Hp2-1 is 45-55%, Hp2-2 is 30-40%, Hpl-1 is 10-20%, and Japanese is Hp2- It is known that 1 is 58%, Hp2-2 is 35%, Hpl-1 is 7%, and about half of Africans are Hpl-1 (Non-Patent Documents 9 and 10). In addition, at the haptoglobin locus, the frequency is low, but the back mutation from the Hp2 gene to the Hpl gene due to intramolecular recombination occurs constantly in somatic cells, and it has been reported that it is in a chimeric state. (Non-patent document 11). Furthermore, humans in which amino acids 52 and 53 encoded by exon 4 of the human habutoglobin gene are Asp and Lys and humans that are Asn and Glu have been reported. Two types of Hpl genes have also been reported. There is a polymorphism. As for the Hp2 gene, there are four types of polymorphisms such as the gene duplication relationship described later (Non-patent Document 12). Furthermore, haptoglobin preparations are purified from the plasma of many thousands of healthy people. Therefore, it is impossible to prepare a haptoglobin blood product having a uniform genotype.
[0004] ヒト Hp2遺伝子は、 Hpl遺伝子のシスティン残基が多く含まれる a鎖遺伝子のェクソ ン 3及び 4領域が遺伝子重複することで生じた遺伝子である（図 1) (非特許文献 12)。 そのため、 Hp2 a鎖を有する（ α β )2は重複で生じたシスティン残基の遊離 SH基を介 して多量体を形成することができる（図 2)。このことがハプトグロビン分子の更なる多 様性を規定しており、ヒト血漿において分子量 8万 5千〜 40万と幅の広い物理ィ匕学的 に不均一な分子種の混合物として存在する原因となっている。 [0004] The human Hp2 gene is a gene produced by gene duplication of the exon 3 and 4 regions of the a-chain gene containing many cysteine residues of the Hpl gene (Fig. 1) (Non-patent Document 12). Therefore, (αβ) 2 having an Hp2 a chain can form a multimer via the free SH group of the cysteine residue generated by duplication (Fig. 2). This further increases the number of haptoglobin molecules. It is a cause of existence as a mixture of molecular species with molecular weights ranging from 85,000 to 400,000 in human plasma, and a wide range of physical and physical heterogeneity.
[0005] Hpl a鎖を有する（ α β )2は、 Hp2 a鎖を有する（ α β )2や多量体に比べて単位蛋 白量当りのヘモグロビン結合量が高い（非特許文献 13、 10) 0ハプトグロビンとへモ グロビンの複合体が細網内皮細胞系細胞に発現する受容体と結合後細胞内に取り 込まれ結合したヘモグロビンを代謝させる活性に関しても、 Hplひ鎖を有する j8 )2 は Hp2 a鎖を有する（ α β )2に比べて優れていることが報告されている（非特許文献 1 4)。また、ハプトグロビンは、遊離のヘモグロビンと結合することで、ヘモグロビンが有 する鉄を介した活性酸素ラジカルの産生を抑え抗酸化剤として機能することができる 1S この抗酸化剤としての機能も Hp2 a鎖を有する（ α β )2や多量体に比べて Hpl a 鎖を有する（a j8 )2の方が高い（非特許文献 10)。事実、 Hp2-2型の遺伝子多型のヒ トでは血清中の鉄イオンの存在量が高ぐビタミン Cの安定性が悪いことが報告されて いる（非特許文献 15、 16)。組織における活性酸素による傷害を回避するためには、 その組織部位に存在する遊離ヘモグロビンを除去する必要があるが、多量体を形成 しゃす 、Hp2 a鎖を有するハプトグロブリンでは十分な糸且織移行性が期待できな 、。 したがって、均一な生物活性のハプトグロビン製剤を提供するためには、単位製剤蛋 白質当りのヘモグロビン結合量を調整するとともにその分子種の構成も一定にするこ とが望まれている。 [0005] (α β) 2 having an Hpl a chain has a higher amount of hemoglobin binding per unit protein than (α β) 2 having an Hp2 a chain or a multimer (Non-patent Documents 13 and 10) The haptoglobin-hemoglobin complex binds to the receptor expressed in reticuloendothelial cell line cells and is then taken up into the cell and metabolizes the bound hemoglobin. It has been reported that it is superior to (αβ) 2 having an a chain (Non-Patent Document 14). In addition, haptoglobin can function as an antioxidant by suppressing the production of active oxygen radicals via iron in hemoglobin by binding to free hemoglobin. 1S The function as an antioxidant is also Hp2 a chain. (A j8) 2 having an Hpl a chain is higher than (αβ) 2 having a phenotype and multimers (Non-patent Document 10). In fact, it has been reported that in humans of the Hp2-2 type gene polymorphism, vitamin C, which has a high abundance of iron ions in serum, has poor stability (Non-patent Documents 15 and 16). In order to avoid injury due to active oxygen in the tissue, it is necessary to remove free hemoglobin present in the tissue site. However, haptoglobulin having an Hp2 a chain is sufficient to transfer to yarn and tissue. I can not expect. Therefore, in order to provide a uniform bioactive haptoglobin preparation, it is desired to adjust the amount of hemoglobin binding per unit preparation protein and make the molecular species composition constant.
[0006] ヒトハプトグロビンの a鎖には多型が存在するが、 13鎖は単一の塩基配列からなる 遺伝子として存在している。 13鎖の Asn23、 Asn46、 Asn50、 Asn80の 4箇所には N-グリ コシド結合糖鎖付加部位が存在し、 N-ァセチルダルコサミン、シアル酸、ガラクトース 、マンノース、およびフコースカゝら構成される複合型糖鎖が結合している（非特許文 献 17〜20)。ハプトグロビンの糖鎖は、健常人においてはフコース修飾率が数％以 下と低 、ことが一つの特徴となって 、るが、癌化に伴って上昇することが報告されて いる（非特許文献 21、 22)。  [0006] Polymorphism exists in the a chain of human haptoglobin, but the 13 chain exists as a gene consisting of a single base sequence. There are N-glycoside-linked glycosylation sites at 13 positions of Asn23, Asn46, Asn50, and Asn80. A complex composed of N-acetylcylcosamine, sialic acid, galactose, mannose, and fucose Type sugar chains are attached (Non-patent Documents 17 to 20). It has been reported that the sugar chain of haptoglobin has a low fucose modification rate of a few percent or less in healthy individuals, but it increases with canceration (Non-patent Documents). 21, 22).
[0007] 現在、ハプトグロビン製剤は、数千人という多くの健常人の血漿を原料として製造さ れている。そのため、このよう製剤は各種肝炎ウィルス、 AIDSウィルス、ヒトパルボウイ ルス、変異型クロイツフェルト 'ヤコブ病の原因となるプリオンなどの感染の伝播の恐 れを完全には否定できない。また、不特定の異なる健常人力も得られる血漿を原料と せざるを得ないため、物理ィ匕学的な不均一性や生理活性的な不均一性を完全に克 服することは困難である。このような観点から、ハプトグロビン製剤の使用にあたって は、治療上の必要性を十分に検討した上で必要最小限の使用に留めることが指導さ れており、使用後も、投与を受けた患者の氏名及び住所、投与日、投与した製剤の 製造番号を記録し、少なくとも 20年間保存することが求められている。 [0007] Currently, haptoglobin preparations are produced from the plasma of many thousands of healthy individuals. For this reason, these preparations may cause transmission of infections such as various hepatitis viruses, AIDS viruses, human parvoviruses, and prions that cause mutant Creutzfeldt's Jacob disease. This cannot be completely denied. In addition, it is difficult to completely overcome the physical and physiological non-uniformity because it is necessary to use plasma that can be obtained from unspecified different healthy human power as a raw material. . From this point of view, it is instructed that the use of haptoglobin preparations should be kept to the minimum necessary after careful examination of the therapeutic need. The name and address, the date of administration, and the serial number of the administered product must be recorded and stored for at least 20 years.
このような状況下、現在まで、遺伝子組換え技術に基づく組換えハプトグロビンの 製造研究が試みられ、大腸菌や酵母、昆虫細胞を宿主としたノヽブトグロビンの生産 が報告されている (特許文献 1、非特許文献 23、 24)。し力しながら、これらの宿主細 胞で生産したハプトグロビンに結合した糖鎖構造は、ヒト血漿由来ハプトグロビンの糖 鎖構造とは大きく異なっており、ヒト血漿由来ハプトグロビンと同等の活性を有する遺 伝子組換え体の製造には成功して 、な 、。このように糖鎖構造が異なるハプトグロビ ンは、抗原性や血中動態、安定性の観点力も医薬品として好ましくない。また、既に 医薬品製造に用いられ、ヒトに投与しても問題となる糖鎖構造を有していないことが 実証されているチャイニーズハムスター卵巣由来 CHO細胞を宿主細胞として用い、 遺伝子組換えハプトグロビンを生産した例も報告されて 、る (非特許文献 25)。しかし ながら、その生産方法により得られたハプトグロビンに結合して 、る糖鎖の大部分に はフコースが結合しており、この構造は担癌患者の血漿中に観察される異常なハプト グロビンと同様の構造であることが知られている。ハプトグロビンの有する糖鎖へのフ コース修飾と癌化との因果関係については未だ不明である力 患者への治療におい て、ハプトグロビン製剤の 1回の投与量は約 5.7gと多量であるため、ハプトグロビンの 患者への投与には' 1真重にならざるをえない。また、ヘモグロビンゃノヽブトグロビンが 有する高い生理活性のゆえ、癌化との関わりのみならず、大幅な糖鎖構造の変化に よるハプトグロビン自身やヘモグロビンとの複合体の生体内分布などに及ぼす影響も 考慮されるべき課題として残されている。したがって、ヒト血漿由来ハプトグロビンと同 等あるいはそれ以上の薬理効果を有し、かつ現行血液製剤の欠点であるウィルス感 染の危険性ゃ不均一性を減らすことができる、遺伝子組換えハプトグロビンが求めら れている。 特許文献 1:日本公開公報 2001年第 46068号 Under such circumstances, production research of recombinant haptoglobin based on genetic recombination technology has been attempted, and production of rabbit globin using E. coli, yeast, and insect cells as a host has been reported (Patent Document 1, Non-patent Document 1). Patent Documents 23 and 24). However, the sugar chain structure bound to haptoglobin produced by these host cells is significantly different from the sugar chain structure of human plasma-derived haptoglobin, and has the same activity as human plasma-derived haptoglobin. Successful production of recombinants. Thus, haptoglobins having different sugar chain structures are not preferable as pharmaceuticals in terms of antigenicity, blood dynamics, and stability. In addition, CHO cells derived from Chinese hamster ovary that have already been used in pharmaceutical manufacturing and have been proven to have no sugar chain structure that causes problems when administered to humans are used as host cells to produce recombinant haptoglobin. An example has been reported (Non-patent Document 25). However, it is bound to haptoglobin obtained by the production method and fucose is bound to most of the sugar chains, and this structure is similar to the abnormal haptoglobin observed in the plasma of cancer-bearing patients. It is known that The causal relationship between fucose modification to sugar chains of haptoglobin and the carcinogenicity is still unknown. In the treatment of patients, the single dose of haptoglobin preparation is about 5.7 g, so haptoglobin In order to administer to patients, it must be serious. In addition, because of the high physiological activity of hemoglobin, buttoglobin, not only is it related to canceration, but also considers the effect on the biodistribution of haptoglobin itself and its complex with hemoglobin due to a significant change in sugar chain structure. It is left as an issue to be done. Therefore, there is a need for a recombinant haptoglobin that has a pharmacological effect equivalent to or higher than that of human plasma-derived haptoglobin, and that can reduce the risk of virus infection, which is a drawback of current blood products, and heterogeneity. It is. Patent Document 1: Japanese Patent Publication No. 46068 2001
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発明が解決しょうとする課題  Problems to be solved by the invention
[0009] 医療現場での安全性あるいは経済性の観点から、ヒトから採取された血液を原材料 として供給されて ヽる血液製剤から遺伝子組換え体への切り替えが求められて!/ヽる。 さらに、効率的な治療、医療事故や副作用の軽減、患者の負担軽減の観点から、現 行の製剤より物理ィ匕学的にも生理活性的にもより均一なハプトグロビン製剤が求めら れている。さら〖こ、そのような活性を満たす遺伝子組換え体で、かつ現行の遺伝子組 換え技術で製造されるハプトグロビン製剤よりも血中半減期が延長されたハプトグロ ビン製剤を提供することにある。  [0009] From the viewpoint of safety or economic efficiency in the medical field, there is a demand for switching from a blood product that is supplied from human blood as a raw material to a gene recombinant! Furthermore, from the viewpoint of efficient treatment, reduction of medical accidents and side effects, and reduction of burden on patients, there is a need for haptoglobin preparations that are more physically and physiologically uniform than current preparations. . Furthermore, it is an object of the present invention to provide a haptoglobin preparation which is a gene recombinant satisfying such an activity and has a longer half-life in blood than a haptoglobin preparation produced by the current gene recombination technology.
課題を解決するための手段  Means for solving the problem
[0010] 本発明は、以下の（1)〜（24)に関する。 The present invention relates to the following (1) to (24).
(1) N-グリコシド結合複合型糖鎖を有する遺伝子組換えハプトグロビン分子から なる組成物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末端の N-ァセチ ルダルコサミンにフコースが結合して 、な 、糖鎖であるハプトグロビン組成物。  (1) A composition comprising a recombinant haptoglobin molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain binds N-acetyldarcosamine at the reducing end of the sugar chain. A haptoglobin composition which is a sugar chain.
(2) N-グリコシド結合複合型糖鎖が、該糖鎖還元末端の N-ァセチルダルコサミン の 6位にフコースの 1位が α結合していない糖鎖である、上記（1)に記載のハプトグロ ビン組成物。  (2) The N-glycoside-linked complex type sugar chain is a sugar chain in which N-acetylyldarcosamine at the reducing end of the sugar chain is a sugar chain in which position 1 of fucose is not α-bonded. Haptoglobin composition.
[0011] (3) ハプトグロビン力 以下の (a)〜(i)力もなる群力 選ばれるポリペプチドと、以下 の (j)〜(l)力もなる群力も選ばれるポリペプチドとからなる蛋白質である、上記（1)また は（2)に記載のハプトグロビン組成物。  [0011] (3) Haptoglobin force A protein consisting of the following (a) to (i) group forces that also have forces, and the following (j) to (l) polypeptides that also have group forces that have selected forces The haptoglobin composition according to (1) or (2) above.
(a) 配列番号 4の 19〜101番目で表されるアミノ酸配列を有するポリペプチド； (b) 配列番号 5の 19〜101番目で表されるアミノ酸配列を有するポリペプチド；（c) 配 列番号 6の 19〜160番目で表されるアミノ酸配列を有するポリペプチド； (d) 配列番 号 4の 19〜101番目で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置 換、挿入および/または付加されたアミノ酸配列を有し、かつ φ〜(1)からなる群から 選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性を有するポリべプチ ド、；  (a) a polypeptide having an amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 4; (b) a polypeptide having an amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 5; (c) a sequence number (D) a polypeptide having an amino acid sequence represented by positions 19 to 160; (d) in the amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 4, wherein one or more amino acids are deleted, substituted, inserted, and A polypeptide having an amino acid sequence added and having hemoglobin metabolic activity in combination with a polypeptide selected from the group consisting of φ to (1);
(e) 配列番号 5の 19〜101番目で表されるアミノ酸配列において、 1以上のアミノ酸 が欠失、置換、挿入および Zまたは付加されたアミノ酸配列を有し、かつ (j)〜(i)から なる群力 選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性を有する ポリペプチド； (e) one or more amino acids in the amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 5 A polypeptide having an amino acid sequence deleted, substituted, inserted and Z or added, and having a hemoglobin metabolic activity in combination with a polypeptide selected from the group force consisting of (j) to (i);
(f) 配列番号 6の 19〜 160番目で表されるアミノ酸配列において、 1以上のアミノ酸 が欠失、置換、挿入および Zまたは付加されたアミノ酸配列を有し、かつ (j)〜(Dから なる群力 選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性を有する ポリペプチド；  (f) In the amino acid sequence represented by positions 19 to 160 of SEQ ID NO: 6, one or more amino acids have an amino acid sequence deleted, substituted, inserted and Z or added, and (from j) to (D A polypeptide having hemoglobin metabolic activity by combining with a selected polypeptide;
(g) 配列番号 4の 19〜 101番目で表されるアミノ酸配列と 80%以上の相同性を有 するアミノ酸配列を有し、かつ φ〜(1)力 なる群力も選ばれるポリペプチドと組合せる ことによりヘモグロビン代謝活性を有するポリペプチド；  (g) Combine with a polypeptide having an amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 4 and also having a group strength of φ to (1) A polypeptide having hemoglobin metabolic activity;
(h) 配列番号 5の 19〜101番目で表されるアミノ酸配列と 80%以上の相同性を有 するアミノ酸配列を有し、かつ φ〜(1)力 なる群力も選ばれるポリペプチドと組合せる ことによりヘモグロビン代謝活性を有するポリペプチド；  (h) Combine with a polypeptide that has an amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 5 and that also has a group strength of φ to (1) A polypeptide having hemoglobin metabolic activity;
(i) 配列番号 6の 19〜160番目で表されるアミノ酸配列と 80%以上の相同性を有 するアミノ酸配列を有し、かつ φ〜(1)力 なる群力も選ばれるポリペプチドと組合せる ことによりヘモグロビン代謝活性を有するポリペプチド。  (i) Combined with a polypeptide having an amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 19 to 160 of SEQ ID NO: 6 and also having a group strength of φ to (1) A polypeptide having hemoglobin metabolic activity.
(j) 配列番号 4の 103〜347番目で表されるアミノ酸配列を有するポリペプチド； (k) 配列番号 4の 103〜347番目で表されるアミノ酸配列において、 1以上のァミノ 酸が欠失、置換、挿入および/または付加されたアミノ酸配列を有し、かつ (a)〜(i)か らなる群力も選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性を有す るポリペプチド；  (j) a polypeptide having an amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4; (k) one or more amino acids deleted in the amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4, A polypeptide having a substituted, inserted and / or added amino acid sequence, and having a hemoglobin metabolic activity by combining with a polypeptide selected from the group forces of (a) to (i);
(1) 配列番号 4の 103〜347番目で表されるアミノ酸配列と 80%以上の相同性を有 するアミノ酸配列を有し、かつ (a)〜(i)力 なる群力 選ばれるポリペプチドと組合せる ことによりヘモグロビン代謝活性を有するポリペプチド；  (1) An amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4, and (a) to (i) a powerful group force A polypeptide having hemoglobin metabolic activity by combining;
(4)ハプトグロビン力 以下の (a)、（b)、（c)、（d)、（e)及び (1)力 なる群力 選ば れる DNAがコードする蛋白質である、上記（1)または（2)に記載のハプトグロビン組 成物。  (4) Haptoglobin force The following (a), (b), (c), (d), (e) and (1) Group force of force The above-mentioned (1) or ( The haptoglobin composition described in 2).
(a) 配列番号 1で表される塩基配列力 なる DNA; (b) 配列番号 2で表される塩基配列力 なる DNA； (a) DNA having a nucleotide sequence represented by SEQ ID NO: 1; (b) DNA having the nucleotide sequence represented by SEQ ID NO: 2;
(c) 配列番号 3で表される塩基配列力 なる DNA；  (c) DNA having a nucleotide sequence represented by SEQ ID NO: 3;
(d) 配列番号 1で表される塩基配列力 なる DNAとストリンジ ントな条件でノ、イブ リダィズし、かつヘモグロビン代謝活性を有する蛋白質をコードする DNA;  (d) DNA encoding a protein having a base sequence ability represented by SEQ ID NO: 1 and a protein that is hemoglobin-metabolizing and dyblyzed under stringent conditions;
(e) 配列番号 2で表される塩基配列からなる DNAとストリンジェントな条件でノ、イブ リダィズし、かつヘモグロビン代謝活性を有する蛋白質をコードする DNA;  (e) DNA encoding a protein having a hemoglobin metabolic activity, which has been or ibridized under stringent conditions with DNA comprising the base sequence represented by SEQ ID NO: 2;
(f) 配列番号 3で表される塩基配列力 なる DNAとストリンジ ントな条件でノ、イブ リダィズし、かつヘモグロビン代謝活性を有する蛋白質をコードする DNA;  (f) DNA encoding a protein having a base sequence ability represented by SEQ ID NO: 3 and a protein having a hemoglobin metabolic activity, stringent and conditioned under stringent conditions;
(5) ハプトグロビン分子をコードする DNAを宿主細胞に導入して得られる、上記（1) 〜 (4)の ヽずれか 1項に記載のハプトグロビン組成物を生産する形質転換体。  (5) A transformant producing the haptoglobin composition according to any one of (1) to (4) above, which is obtained by introducing DNA encoding a haptoglobin molecule into a host cell.
(6) 宿主細胞が、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、また は N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコース の 1位が α結合する糖鎖修飾に関与する酵素の活性が欠失するようにゲノムが改変 された細胞である、上記（5)に記載の形質転換体。  (6) The host cell is the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose, or the N-glycidyl-linked N-acetylyldarcosamine at the 6-position of the reducing glycan is located at position 1 of fucose α The transformant according to (5) above, which is a cell whose genome has been altered so that the activity of the enzyme involved in the modification of the sugar chain to be bound is lost.
(7) 宿主細胞が、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、また は Ν-グリコシド結合複合型糖鎖還元末端の Ν-ァセチルダルコサミンの 6位にフコース の 1位が a結合する糖鎖修飾に関与する酵素のゲノム上の対立遺伝子のすべてがノ ックアウトされた細胞である、上記（6)に記載の形質転換体。  (7) The host cell is the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose, or the first position of fucose at position 6 of 還 元 -acetylcylcosamine at the reducing end of Ν-glycosidic complex glycan The transformant according to (6) above, wherein the allele on the genome of the enzyme involved in the sugar chain modification to be bound is a knocked-out cell.
(8) 細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素力 GDP-マンノー ス 4,6-デヒドラターゼ及び GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼ 力もなる群力 選ばれる酵素である、上記（6)または（7)に記載の形質転換体。 (8) Enzymatic power involved in the synthesis of intracellular sugar nucleotides GDP-fucose GDP-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase The transformant according to (6) or (7) above, which is a selected enzyme.
(9) GDP-マンノース 4,6-デヒドラターゼカ 以下の（a)、（b)及び (c)力もなる群から 選ばれる蛋白質である、上記（8)に記載の形質転換体。 (9) The transformant according to the above (8), which is a protein selected from the group consisting of the following (a), (b), and (c) force (GDP-mannose 4,6-dehydratase).
(a)配列番号 8で表されるアミノ酸配列からなる蛋白質；  (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;
(b)配列番号 8で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ GDP-マンノース 4,6-デヒド ラターゼ活性を有する蛋白質；  (b) In the amino acid sequence represented by SEQ ID NO: 8, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have GDP-mannose 4,6-dehydratase activity protein;
(c)配列番号 8で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列から なり、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質； (c) From an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8 And a protein having GDP-mannose 4,6-dehydratase activity;
(10) GDP-マンノース 4,6-デヒドラターゼカ 以下の (a)及び (b)からなる群から選ば れる DNAがコードする蛋白質である、上記（8)に記載の形質転換体。  (10) The transformant according to (8) above, which is a protein encoded by DNA selected from the group consisting of the following (a) and (b): GDP-mannose 4,6-dehydratase
(a)配列番号 7で表される塩基配列からなる DNA；  (a) DNA consisting of the base sequence represented by SEQ ID NO: 7;
(b)配列番号 7で表される塩基配列力もなる DNAとストリンジェントな条件でハイブリダ ィズし、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質をコードする DN A。  (b) A DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 7 and encodes a protein having GDP-mannose 4,6-dehydratase activity.
(11) GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼが、以下の（a)、 (b) および（c)力もなる群力も選ばれる蛋白質である、上記（8)に記載の形質転換体。 (11) 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 (8) ).
(a)配列番号 10で表されるアミノ酸配列からなる蛋白質； (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 10;
(b)配列番号 10で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ GDP-4-ケト -6-デォキシ -D -マンノース- 3,5-ェピメラーゼ活性を有する蛋白質；  (b) In the amino acid sequence represented by SEQ ID NO: 10, one or more amino acids are deleted, substituted, inserted and Z or added, and the amino acid sequence power is GDP-4-keto-6-deoxy-D- A protein having mannose-3,5-epimerase activity;
(c)配列番号 10で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有する 蛋白質。  (c) It consists of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity protein.
(12) GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼが、以下の（a)及び (b)力もなる群力も選ばれる DNAがコードする蛋白質である、上記（8)に記載の細胞。 (12) The above-mentioned (8) GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is a protein encoded by DNA selected from the following (a) and (b) group forces that also include forces (8) ).
(a)配列番号 9で表される塩基配列からなる DNA； (a) DNA comprising the base sequence represented by SEQ ID NO: 9;
(b)配列番号 9で表される塩基配列力もなる DNAとストリンジェントな条件でハイブリダ ィズし、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3, 5-ェピメラーゼ活性を有する 蛋白質をコードする DNA。  (b) a protein that hybridizes under stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 9 and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimelase activity DNA encoding.
(13) N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフ コースの 1位が a結合する糖鎖修飾に関与する酵素が a 1,6-フコシルトランスフェラ ーゼである上記（6)または（7)に記載の形質転換体。  (13) N-glycoside-linked complex type sugar chain N-acetylyldarcosamine at the reducing end is an enzyme involved in sugar chain modification in which the 1-position of fucose is a-linked to a 1,6-fucosyltransferase The transformant according to (6) or (7) above, wherein
(14) a 1,6-フコシルトランスフェラーゼカ 以下の（a)、 (b)、（c)、（d)、（e)および (1)か らなる群力も選ばれる蛋白質である、上記（13)に記載の形質転換体。  (14) 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 (13 ).
(a)配列番号 13で表されるアミノ酸配列からなる蛋白質； (b)配列番号 14で表されるアミノ酸配列力もなる蛋白質； (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 13; (b) a protein having an amino acid sequence ability represented by SEQ ID NO: 14;
(c)配列番号 13で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ α 1 ,6-フコシルトランスフェラ ーゼ活性を有する蛋白質；  (c) In the amino acid sequence represented by SEQ ID NO: 13, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have α1,6-fucosyltransferase activity. A protein having;
(d)配列番号 14で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ α 1 ,6-フコシルトランスフェラ ーゼ活性を有する蛋白質；  (d) In the amino acid sequence represented by SEQ ID NO: 14, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have an α1,6-fucosyltransferase activity. A protein having;
(e)配列番号 13で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ a 1 ,6-フコシルトランスフェラーゼ活性を有する蛋白質；  (e) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 13, and having a 1,6-fucosyltransferase activity;
(1)配列番号 14で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ a 1 ,6-フコシルトランスフェラーゼ活性を有する蛋白質。  (1) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 14, and having a 1,6-fucosyltransferase activity.
( 15) a 1 ,6-フコシルトランスフェラーゼカ 以下の（a)、（b)、（c)及び (d)力もなる群か ら選ばれる DNAがコードする蛋白質である、上記（13)に記載の形質転換体。  (15) a 1, 6-fucosyltransferase is a protein encoded by DNA selected from the group consisting of the following (a), (b), (c) and (d) force: Transformant.
(a)配列番号 11で表される塩基配列力 なる DNA ;  (a) DNA having a nucleotide sequence represented by SEQ ID NO: 11;
(b)配列番号 12で表される塩基配列力もなる DNA；  (b) DNA having a nucleotide sequence represented by SEQ ID NO: 12;
(c)配列番号 11で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ α 1 ,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする DN Α ;  (c) a DN す る that encodes a protein that is hybridized under stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 11 and that has α 1, 6-fucosyltransferase activity;
(d)配列番号 12で表される塩基配列力もなる DNAとストリンジェントな条件でハイプリ ダイズし、かつ α 1 ,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする DN Α。  (d) a DN that encodes a protein that is hybridized with the DNA represented by SEQ ID NO: 12 under stringent conditions and that has α 1, 6-fucosyltransferase activity.
( 16) Ν-グリコシド結合複合型糖鎖還元末端の Ν-ァセチルダルコサミンの 6位とフコ ースの 1位が ex結合した糖鎖構造を認識するレクチンに耐性である、上記（5)〜（15 )の 、ずれか 1項に記載の形質転換体。  (16) Resistant to a lectin that recognizes a sugar chain structure in which the 6-position of 還 元 -acetyldarcosamine at the reducing end of Ν-glycoside-bonded glycan and the 1-position of fucos are ex-linked (5) The transformant according to any one of to (15).
( 17) レクチン耐性力 Ν-グリコシド結合複合型糖鎖還元末端の Ν-ァセチルダルコ サミンの 6位とフコースの 1位が a結合した糖鎖構造を認識するレクチンを含む培地 で培養した場合に、ゲノムが改変される以前の細胞よりも高 、生存率を示すことであ る、上記（16)に記載の形質転換体。 (18) 少なくとも、以下の (a)、（b)、（c)及び (d)力 なる群力 選ばれるレクチンの一つ に耐性である、上記（16)または（17)に記載の形質転換体。 (17) Resistance to lectin When cultured in a medium containing a lectin that recognizes a glycan structure in which the 6-position of ァ -acetyldarcosamine at the reducing end of Ν-glycoside-linked glycan and the 1-position of fucose are a-linked The transformant according to (16) above, which is higher in viability than a cell before being modified. (18) The transformation according to (16) or (17) above, which is resistant to at least one of the following (a), (b), (c) and (d) force group forces selected: body.
(a) レンズマメレクチン LCA (Lens Culinaris由来の Lentil Agglutinin)；  (a) Lentil lectin LCA (Lentil Agglutinin from Lens Culinaris);
(b) エンドゥマメレクチン PSA (Pisum sativum由来の Pea Lectin)；  (b) Endo bean lectin PSA (Peasum sativum-derived Pea Lectin);
(c) ソラマメレクチン VFA (Vicia faba由来の Agglutinin)：  (c) Broad bean lectin VFA (Agglutinin from Vicia faba):
(d) ヒィロチャワンタケレクチン AAL (Aleuria aurantia由来の Lectin)。  (d) Herochawantake lectin AAL (Lectin from Aleuria aurantia).
(19) 宿主細胞が、下記の (a)、（b)、（c)、（d)、（e)、（£)、（g)、（h)、（i)及び (j)力 なる群か ら選ばれる細胞である上記（5)〜（18)の 、ずれか 1項に記載の形質転換体。 (19) The host cell has the following (a), (b), (c), (d), (e), (£), (g), (h), (i) and (j) forces The transformant according to any one of (5) to (18) above, which is a cell selected from a group.
(a)チャイニーズノヽムスター卵巣組織由来 CHO細胞； (a) Chinese nomstar ovarian tissue-derived CHO cells;
(b)ラットミエローマ細胞株 YB2/3HL.P2.G11.16Ag.20細胞；  (b) rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cells;
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0- Agl4細胞；  (d) mouse myeloma cell line SP2 / 0-Agl4 cells;
(e)シリアンノヽムスター腎臓組織由来 BHK細胞；  (e) Syrian Nomster kidney tissue-derived BHK cells;
(1)ヒト白血病細胞株ナマルバ細胞； (1) human leukemia cell line Namalva cells;
(g)胚性幹細胞；  (g) embryonic stem cells;
(h)受精卵細胞；  (h) fertilized egg cells;
(0植物細胞； (0 plant cells;
(j)酵母。  (j) Yeast.
(20) 上記（5)〜（19)のいずれか 1項に記載の形質転換体を培地に培養し、培養 物中にハプトグロビン組成物を生成蓄積させ、該培養物からハプトグロビン組成物を 採取する工程を含む、ハプトグロビン組成物の製造方法。  (20) The transformant according to any one of (5) to (19) above is cultured in a medium, a haptoglobin composition is produced and accumulated in the culture, and the haptoglobin composition is collected from the culture The manufacturing method of a haptoglobin composition including a process.
(21) 上記（20)記載の製造方法で得られたハプトグロビン組成物。  (21) A haptoglobin composition obtained by the production method according to (20) above.
(22) 上記（1)〜（4)および（21)の 、ずれか 1項に記載のハプトグロビン組成物を 有効成分として含有する医薬。  (22) A medicament comprising the haptoglobin composition according to any one of (1) to (4) and (21) as an active ingredient.
(23) 上記（1)〜（4)および（21)の 、ずれか 1項に記載のハプトグロビン組成物を 有効成分として含有する、ヘモグロビン血症及びヘモグロビン尿症の治療剤。 (23) A therapeutic agent for hemoglobinemia and hemoglobinuria comprising the haptoglobin composition according to any one of (1) to (4) and (21) as an active ingredient.
(24) 上記（1)〜（4)および（21)の 、ずれ力 1項に記載のハプトグロビン組成物を 有効成分として含有する、血中遊離ヘモグロビンによる酸ィ匕的血管障害を伴う疾患 の治療剤。 (24) A disease associated with acid-induced vascular injury due to blood free hemoglobin, comprising as an active ingredient the haptoglobin composition according to item 1 of (1) to (4) and (21) above Therapeutic agent.
発明の効果  The invention's effect
[0012] 本発明により、 N-グリコシド結合複合型糖鎖を有する遺伝子組換えハプトグロビン 分子カゝらなる組成物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末端の N-ァセチルダルコサミンにフコースが結合して!/ヽな 、糖鎖であるハプトグロビン組成 物が提供される。  [0012] According to the present invention, there is provided a composition comprising a recombinant haptoglobin molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is N- A haptoglobin composition which is a sugar chain in which fucose is bound to acetylyldarcosamine is provided.
図面の簡単な説明  Brief Description of Drawings
[0013] [図 1]は、 HP-1型および HP-2型ハプトグロビンの構造を示す。 [0013] FIG. 1 shows the structure of HP-1 type and HP-2 type haptoglobin.
[図 2]は、 HP1-1型、 HP2-1型および HP2-2型におけるハプトグロビン複合体の構造を 示す。  [Fig. 2] shows the structure of the haptoglobin complex in HP1-1 type, HP2-1 type, and HP2-2 type.
[図 3]は、プラスミド pKAN- HP1の作製方法を示す。  [FIG. 3] shows a method for preparing plasmid pKAN-HP1.
[図 4]は、プラスミド pKAN- HP2の作製方法を示す。  [FIG. 4] shows a method for preparing plasmid pKAN-HP2.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0014] 本発明の、 N-グリコシド結合複合型糖鎖を有する遺伝子組換えハプトグロビン分子 力もなる組成物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末端の N-ァ セチルダルコサミンにフコースが結合して ヽな 、糖鎖であるハプトグロビン組成物（以 下、「本発明の組成物」と表記する）とは、 N-グリコシド結合複合型糖鎖還元末端の N -ァセチルダルコサミンにフコースが結合して!/、な!/、遺伝子組換えハプトグロビン分 子力もなるハプトグロビン組成物であればいかなる組成物も包含される。  [0014] The composition of the present invention also comprises a recombinant haptoglobin molecule having an N-glycoside-bonded complex sugar chain, wherein the N-glycoside-bonded complex sugar chain is N-acetyl at the reducing end of the sugar chain. A haptoglobin composition (hereinafter referred to as “the composition of the present invention”), which is a sugar chain in which fucose is bound to darcosamine, is an N-glycoside-linked complex sugar chain reducing end N-a. Any composition is included as long as it is a haptoglobin composition in which fucose is bound to cetyldarcosamine! /, Na! / And also has a recombinant haptoglobin molecular force.
[0015] 本発明にお!/、て、ハプトグロビンとは、ヘモグロビン及びハプトグロビン受容体に親 和性を有し、ヘモグロビン代謝活性を有する糖蛋白質であればいかなるものも包含さ れる。  [0015] In the present invention, haptoglobin includes any glycoprotein having affinity for hemoglobin and a haptoglobin receptor and having hemoglobin metabolic activity.
具体的なハプトグロビンの例としては、ハプトグロビン力 以下の (a)〜(i)からなる群 から選ばれるポリペプチドと、以下の (j)〜(l)からなる群から選ばれるポリペプチドとか らなる蛋白質などがあげられる。  Specific examples of haptoglobin include a polypeptide selected from the group consisting of (a) to (i) below the haptoglobin power and a polypeptide selected from the group consisting of (j) to (l) below: Examples include proteins.
(a) 配列番号 4の 19〜101番目で表されるアミノ酸配列を有するポリペプチド； (b) 配列番号 5の 19〜101番目で表されるアミノ酸配列を有するポリペプチド；（c) 配 列番号 6の 19〜160番目で表されるアミノ酸配列を有するポリペプチド； (d) 配列番 号 4の 19〜101番目で表されるアミノ酸配列において、 1以上のアミ (a) a polypeptide having an amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 4; (b) a polypeptide having an amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 5; (c) a sequence number A polypeptide having an amino acid sequence represented by positions 19 to 160 of 6; (d) a sequence number In the amino acid sequence represented by positions 19 to 101 of No. 4, one or more amino acids
ノ酸が欠失、置換、挿入および Zまたは付加されたアミノ酸配列を有し、かつ下記 G)No acid has amino acid sequence deleted, substituted, inserted and Z or added, and G)
〜(1)力もなる群力も選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性 を有するポリペプチド； -(1) a polypeptide having hemoglobin metabolic activity by combining with a polypeptide in which a group force is also selected;
(e) 配列番号 5の 19〜101番目で表されるアミノ酸配列において、 1以上のアミノ酸 が欠失、置換、挿入および Zまたは付加されたアミノ酸配列を有し、かつ下記 (j)〜(l) 力 なる群力も選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性を有 するポリペプチド；  (e) In the amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 5, one or more amino acids have an amino acid sequence deleted, substituted, inserted and Z or added, and the following (j) to (l ) A polypeptide having hemoglobin metabolic activity when combined with a polypeptide of which group power is also selected;
(f) 配列番号 6の 19〜 160番目で表されるアミノ酸配列において、 1以上のアミノ酸 が欠失、置換、挿入および Zまたは付加されたアミノ酸配列を有し、かつ下記 (j)〜(l) 力 なる群力も選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性を有 するポリペプチド；  (f) In the amino acid sequence represented by positions 19 to 160 of SEQ ID NO: 6, one or more amino acids have an amino acid sequence deleted, substituted, inserted and Z or added, and the following (j) to (l ) A polypeptide having hemoglobin metabolic activity when combined with a polypeptide of which group power is also selected;
(g) 配列番号 4の 19〜 101番目で表されるアミノ酸配列と 80%以上の相同性を有 するアミノ酸配列を有し、かつ下記 φ〜(1)力 なる群力 選ばれるポリペプチドと組合 せることによりヘモグロビン代謝活性を有するポリペプチド；  (g) an amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 4, and the following φ to (1) powerful group strength: combination with the selected polypeptide A polypeptide having hemoglobin metabolic activity;
(h) 配列番号 5の 19〜101番目で表されるアミノ酸配列と 80%以上の相同性を有 するアミノ酸配列を有し、かつ下記 φ〜(1)力 なる群力 選ばれるポリペプチドと組合 せることによりヘモグロビン代謝活性を有するポリペプチド；  (h) an amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 5, and the following φ to (1) powerful group strength: combination with a selected polypeptide A polypeptide having hemoglobin metabolic activity;
(i) 配列番号 6の 19〜160番目で表されるアミノ酸配列と 80%以上の相同性を有 するアミノ酸配列を有し、かつ下記 φ〜(1)力 なる群力 選ばれるポリペプチドと組合 せることによりヘモグロビン代謝活性を有するポリペプチド。  (i) an amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 19 to 160 of SEQ ID NO: 6, and the following φ to (1) powerful group strength: combination with a selected polypeptide A polypeptide having hemoglobin metabolic activity.
(j) 配列番号 4の 103〜347番目で表されるアミノ酸配列を有するポリペプチド； (k) 配列番号 4の 103〜347番目で表されるアミノ酸配列において、 1以上のァミノ 酸が欠失、置換、挿入および Zまたは付加されたアミノ酸配列を有し、かつ上記 (a)〜 (0力もなる群力も選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性 を有するポリペプチド；  (j) a polypeptide having an amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4; (k) one or more amino acids deleted in the amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4, A polypeptide having a substitution, insertion and Z or added amino acid sequence, and having hemoglobin metabolic activity in combination with the above-mentioned (a) to (polypeptides having a group strength of 0);
(1) 配列番号 4の 103〜347番目で表されるアミノ酸配列と 80%以上の相同性を有 するアミノ酸配列を有し、かつ上記 (a)〜(i)力 なる群力 選ばれるポリペプチドと組合 せることによりヘモグロビン代謝活性を有するポリペプチド； (1) A polypeptide having an amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4 and the above (a) to (i) And unions A polypeptide having hemoglobin metabolic activity;
また、ハプトグロビンのアミノ酸配列をコードする DNAとしては、配列番号 1、 2または 3で表される塩基配列を有する DNA、配列番号 1、 2または 3で表される塩基配列を 有する DNAとストリンジェントな条件でノ、イブリダィズし、かつヘモグロビン代謝活性を 有する蛋白質をコードする DNAなどがあげられる。  In addition, DNA encoding the amino acid sequence of haptoglobin is stringent with DNA having the base sequence represented by SEQ ID NO: 1, 2 or 3, and DNA having the base sequence represented by SEQ ID NO: 1, 2 or 3. Examples include DNA that encodes a protein that undergoes nobbreviation and hybridization under certain conditions and has hemoglobin metabolic activity.
[0016] 本発明にお!/、て、ストリンジヱントな条件下でハイブリダィズする DNAとは、例えば 配列番号 1、 2または 3で表される塩基配列を有する DNAなどの DNAまたはその一 部の断片をプローブとして、コ口-一'ハイブリダィゼーシヨン法、プラーク 'ハイブリダ ィゼーシヨン法ある 、はサザンブロットハイブリダィゼーシヨン法等を用いることにより 得られる DNAを意味し、具体的には、コロニーあるいはプラーク由来の DNAを固定 化したフィルターを用いて、 0. 7〜1. OMの塩化ナトリウム存在下、 65°Cでハイブリ ダイゼーシヨンを行った後、 0. 1〜2倍濃度の SSC溶液（1倍濃度の SSC溶液の組 成は、 150mM塩化ナトリウム、 15mMクェン酸ナトリウムよりなる）を用い、 65°C条件 下でフィルターを洗浄することにより同定できる DNAをあげることができる。ハイブリ ダィセ ~~ンヨン ί 、 Molecularし loning, A Laboratory Manual, Second Edition, Cold b pring Harbor Laboratory Press, (1989) (以下、モレキュラ^ ~ ·クロー-ング第 2版と略 す）、 Current Protocols in Molecular Biology, John Wiley & Sons,(1987- 1997) (以下 、カレント 'プロトコ一ルズ'イン'モレキュラ^ ~ ·バイオロジーと略す）、 DNA Cloning 1: し ore Techniques, A Practical Approacn, Second Edition, Oxford University (1995) 等に記載されて 、る方法に準じて行うことができる。ハイブリダィズ可能な DNAとして 具体的には、配列番号 1、 2または 3で表される塩基配列と少なくとも 60%以上の相 同性を有する DNA、好ましくは 70%以上、より好ましくは 80%以上、さらに好ましく は 90%以上、特に好ましくは 95%以上、最も好ましくは 98%以上の相同性を有する DNAをあげることができる。  [0016] In the present invention, the DNA that hybridizes under stringent conditions is, for example, DNA such as DNA having the base sequence represented by SEQ ID NO: 1, 2, or 3, or a fragment thereof. As a probe, Koguchi-1 'hybridization method, plaque' hybridization method is "means DNA obtained by using Southern blot hybridization method, etc., specifically, colony or Hybridization was performed at 65 ° C in the presence of 0.7 to 1. OM sodium chloride using a filter on which plaque-derived DNA was immobilized, and then an SSC solution (1 to 2 times in concentration) was added. The concentration of SSC solution is 150mM sodium chloride and 15mM sodium citrate), and DNA can be identified by washing the filter under 65 ° C conditions. . HYBRIDISE ~~ Nyon Yong, Molecular loning, A Laboratory Manual, Second Edition, Cold 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: Shise ore Techniques, A Practical Approacn, Second Edition, Oxford University (1995) etc. and can be carried out according to the method described above. Specifically, DNA capable of hybridizing is DNA having at least 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3, preferably 70% or more, more preferably 80% or more, and still more preferably May be DNA having a homology of 90% or more, particularly preferably 95% or more, and most preferably 98% or more.
[0017] 本発明において、配列番号 4、 5または 6で表されるアミノ酸配列において 1以上の ァ  In the present invention, in the amino acid sequence represented by SEQ ID NO: 4, 5, or 6, one or more amino acids
ミノ酸が欠失、置換、挿入および Zまたは付加されたアミノ酸配列力もなり、かつへモ グロビン代謝活性を有する蛋白質とは、モレキュラー 'クローユング第 2版、カレント' プロトコールズ.イン.モレキュラー.バイオロジー、 Nucleic Acids Research, 10, 6487 (1982)、 Proc. Natl. Acad. Sci. USA, 79, 6409 (1982)、 Gene, 34,315 (1985)、 Nucleic Acids Research, 13, 4431 (1985)、 Proc. Natl. Acad. Sci. USA, 82, 488 (1985)等に記 載の部位特異的変異導入法を用いて、例えば、配列番号 4、 5または 6で表されるァ ミノ酸配列を有する蛋白質をコードする DNAに部位特異的変異を導入することによ り取得することができる蛋白質を意味する。欠失、置換、挿入および Zまたは付加さ れるアミノ酸の数は 1個以上でありその数は特に限定されないが、上記の部位特異的 変異導入法等の周知の技術により、欠失、置換もしくは付加できる程度の数であり、 例えば、 1〜数十個、好ましくは 1〜20個、より好ましくは 1〜： L0個、さらに好ましくは 1〜5個である。 A protein that has amino acid sequence ability with deletion, substitution, insertion and Z or addition of mino acid, and also has a hemoglobin metabolic activity, is the molecular 'Clooung 2nd edition, current' Protocols in Molecular Biology, Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34,315 (1985), Nucleic Acids Research, 13 , 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc., for example, represented by SEQ ID NO: 4, 5, or 6 It means a protein that can be obtained by introducing a site-specific mutation into DNA encoding a protein having an amino acid sequence. 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. For example, it is 1 to several tens, preferably 1 to 20, more preferably 1 to: L0, and further preferably 1 to 5.
[0018] また、本発明にお 、て、配列番号 4、 5または 6で表されるアミノ酸配列と 80%以上 の相同性を有し、かつヘモグロビン代謝活性を有する蛋白質とは、 BLAST〔J.Mol. Biol, 215, 403 (1990)〕や FASTA [Methods in Enzymology, 183, 63(1990)〕等の解 析ソフトを用いて計算したときに、配列番号 4、 5または 6に記載のアミノ酸配列を有す る蛋白質と少なくとも 80%以上、好ましくは 85%以上、より好ましくは 90%以上、さら に好ましくは 95%以上、特に好ましくは 97%以上、最も好ましくは 99%以上である 蛋白質であることを意味する。  [0018] In the present invention, the protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4, 5, or 6 and having hemoglobin metabolic activity is BLAST [J. Mol. Biol, 215, 403 (1990)] and FASTA [Methods in Enzymology, 183, 63 (1990)], etc., and the amino acid sequence described in SEQ ID NO: 4, 5 or 6 At least 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more. Means that.
[0019] ヘモグロビン代謝活性とは、ハプトグロビン力 遊離ヘモグロビンとが結合して複合 体を形成し、該複合体が、肝臓の細網内皮細胞系細胞に発現するヘモグロビンーハ ブトグロビン複合体の受容体へ結合し、結合後細胞内に取り込まれ、細胞内でへモ グロビンの分解により生じるヘムを、ピリルビンにまで代謝させる活性を 、う。  [0019] Hemoglobin metabolic activity refers to the binding of haptoglobin force to free hemoglobin to form a complex, and the complex is expressed as a receptor for the hemoglobin-habu globin complex expressed in the reticuloendothelial cell line of the liver. It binds and is taken up into cells after binding, and has the activity of metabolizing heme generated by the degradation of hemoglobin into pyrilbin.
糖蛋白質に結合している N-グリコシド結合糖鎖は、様々な構造を有している力 い ずれの場合にも以下の構造式 (I)に示す共通のコア構造を有することが知られて 、 る。  N-glycoside-linked sugar chains bound to glycoproteins are known to have a common core structure represented by the following structural formula (I) in any of the various structures. .
[0020] [化 1]
Figure imgf000016_0001
[0020] [Chemical 1]
Figure imgf000016_0001
[0021] 構造式 (I)において、ァスパラギンと結合する糖鎖の末端が還元末端、反対側が非 還元末端と呼ばれている。 N-グリコシド結合糖鎖には、コア構造の非還元末端にマ ンノースのみが結合するハイマンノース型、コア構造の非還元末端側にガラクトース —N-ァセチルダルコサミン（以下、 Ga卜 GlcNAcと表記する）の枝を並行して 1な!、しは 複数本有し、更に Ga卜 GlcNAcの非還元末端側にシアル酸、ノイセクティングの N-ァ セチルダルコサミンなどの構造を有する複合型、コア構造の非還元末端側にハイマ ンノース型と複合型の両方の枝を持つハイブリッド型などがあることが知られている。 In Structural Formula (I), the end of the sugar chain that binds to asparagine is the reducing end, and the opposite side is non- It is called the reducing end. The N-glycoside-linked sugar chain is a high mannose type in which only mannose binds to the non-reducing end of the core structure, and galactose —N-acetyldarcosamine (hereinafter referred to as Ga 卜 GlcNAc) 1) in parallel, and more than one branch, and a complex type having a structure such as sialic acid, Neusecting N-acetylethylcosamine on the non-reducing end side of Ga 側 GlcNAc, It is known that there are hybrid types having both a hymannose type and a complex type branch on the non-reducing end side of the core structure.
[0022] 本発明の組成物を構成するハプトグロビン分子の j8鎖には 4箇所の N-グリコシド結 合糖鎖の付加配列が存在し、これら部位に N-グリコシド結合糖鎖が結合している。ハ ブトグロビンに結合する N-グリコシド結合糖鎖としては、具体的には、上述の N-グリコ シド結合複合型糖鎖を挙げることができる。ハプトグロビン分子に結合する N-グリコシ ド結合複合型糖鎖としては、前記構造式 (I)で示されるコア構造を含む ヽかなる糖鎖 も包含されるので、多数の糖鎖の組み合わせが存在することになる。  [0022] The j8 chain of the haptoglobin molecule constituting the composition of the present invention has four additional sequences of N-glycoside-linked sugar chains, and N-glycoside-linked sugar chains are bound to these sites. Specific examples of the N-glycoside-bonded sugar chain that binds to habtoglobin include the aforementioned N-glycoside-bonded complex sugar chains. The N-glycoside-bonded complex sugar chain that binds to the haptoglobin molecule includes a large number of sugar chains including the core structure represented by the structural formula (I), and therefore there are many combinations of sugar chains. It will be.
[0023] したがって、本発明の組成物は、 N-グリコシド結合複合型糖鎖を有する遺伝子組 換えハプトグロビン分子力もなる組成物であって、該 N-グリコシド結合複合型糖鎖が 該糖鎖還元末端の N-ァセチルダルコサミンにフコースが結合して ヽな 、糖鎖であれ ば、単一の糖鎖構造を有するハプトグロビン分子力も構成されていてもよいし、複数 の異なる糖鎖構造を有するハプトグロビン分子から構成されて 、てもよ 、。  [0023] Accordingly, the composition of the present invention is a composition having a genetically modified haptoglobin molecular force having an N-glycoside-bonded complex sugar chain, wherein the N-glycoside-bonded complex sugar chain is the sugar chain reducing end. As long as fucose is bound to N-acetyldarcosamine, a haptoglobin molecular force having a single sugar chain structure or a haptoglobin having a plurality of different sugar chain structures may be used. Consists of molecules.
[0024] N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンにフコースが結 合していない糖鎖とは、具体的には、フコースの 1位が N-グリコシド結合複合型糖鎖 の N-ァセチルダルコサミンの 6位に (X結合して!/ヽな ヽ糖鎖等があげられる。  [0024] An N-glycoside-linked complex type sugar chain is a sugar chain in which fucose is not bound to N-acetylyldarcosamine at the reducing end. Specifically, position 1 of fucose is an N-glycoside-linked complex type sugar. The 6-position of the N-acetylyldarcosamine in the chain (X-bonded!
N-グリコシド結合複合型糖鎖を有するハプトグロビン分子カゝらなる組成物中の糖鎖 構造の解析は、ハプトグロビン分子力 ヒドラジン分解や酵素消化などの公知の方法 [生物化学実験法 23—糖タンパク質糖鎖研究法 (学会出版センター)高橋禮子編 (19 89)]を用い、糖鎖を遊離させ、遊離させた糖鎖を蛍光標識又は同位元素標識し、標 識した糖鎖をクロマトグラフィー法にて分離することによって決定することができる。ま た、遊離させた糖鎖を HPAED- PAD法 [ジャーナル ·ォブ ·リキッド ·クロマトグラフィー（ J.Liq. Chromatogr.) , 6, 1577 (1983)]によって分析することで決定することもできる。  Analysis of the sugar chain structure in the composition consisting of a haptoglobin molecule having an N-glycoside-linked sugar chain can be carried out by known methods such as haptoglobin molecular force hydrazine degradation and enzymatic digestion [Biochemical Experimental Method 23-Glycoprotein Sugar Using the Chain Research Method (Academic Publishing Center) Takahashi Eiko (19 89)], the sugar chain is released, the released sugar chain is labeled with a fluorescent label or isotope, and the labeled sugar chain is chromatographed. It can be determined by separating. It can also be determined by analyzing the released sugar chain by the HPAED-PAD method [J. Liq. Chromatogr., 6, 1577 (1983)].
[0025] 本発明にお 、て、糖鎖還元末端の Ν-ァセチルダルコサミンにフコースが結合して いない糖鎖とは、糖鎖に、実質的にフコースが結合していないことをいい、好ましくは フコース含有率が 0%であることをいう。実質的にフコースが結合していないとは、具 体的には、後述の 4に記載の糖鎖分析において、フコースが実質的に検出できない ことをいう。実質的に検出できないとは、測定の検出限界以下であることを意味する。 本発明の組成物は、 N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミ ンにフコースが結合しているハプトグロビンに比べて、生体内に投与した場合に血中 半減期が長い。 [0025] In the present invention, fucose is bonded to 還 元 -acetyldarcosamine at the sugar chain reducing end. An unacceptable sugar chain means that fucose is not substantially bound to the sugar chain, and preferably means that the fucose content is 0%. The fact that fucose is not substantially bound means that fucose cannot be substantially detected in the sugar chain analysis described in 4 below. “Substantially undetectable” means below the detection limit of measurement. The composition of the present invention has a long half-life in blood when administered in vivo compared to haptoglobin in which fucose is bound to N-glycidyl darcosamine at the N-glycoside-linked complex type sugar chain reducing end. .
[0026] 本発明の形質転換体としては、本発明の組成物を生産することができる形質転換 体であれば、いかなる形質転換体でも包含される。具体的な例としては、ハプトグロビ ン分子をコードする DNAを、以下の (a)または (b)などの宿主細胞に導入して得られる 形質転換体があげられる。  [0026] The transformant of the present invention includes any transformant as long as it is capable of producing the composition of the present invention. A specific example is a transformant obtained by introducing a DNA encoding a haptoglobin molecule into a host cell such as (a) or (b) below.
(a)細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素の活性が欠失する ようにゲノムが改変された細胞；  (a) a cell whose genome has been modified to lack the activity of an enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose;
(b) N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコ ースの 1位が α結合する糖鎖修飾に関与する酵素の活性が欠失するようにゲノムが 改変された細胞。  (b) N-glycoside-linked complex-type sugar chain reducing N-acetylyldarcosamine at the 6-position of the genome so that the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked is deleted Is a modified cell.
[0027] 細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素としては、 GDP-マンノ ース 4,6-デヒドラターゼ（GMD)、 GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメ ラーゼ (FX)などがあげられる。  [0027] Enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose include: GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose-3,5 -Epimerase (FX).
本発明において、 GDP-マンノース 4,6-デヒドラターゼとしては、下記 (a)あるいは (b) の DNAがコードする蛋白質、または下記 (c)、（d)あるいは (e)の蛋白質などがあげられ る。  In the present invention, the GDP-mannose 4,6-dehydratase includes a protein encoded by the following DNA (a) or (b), or a protein (c), (d) or (e) below. .
[0028] (a)配列番号 7で表される塩基配列力 なる DNA;  [0028] (a) DNA having a nucleotide sequence represented by SEQ ID NO: 7;
(b)配列番号 7で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質をコードする DNA;  (b) a DNA that encodes a protein that has a base sequence ability represented by SEQ ID NO: 7 and hybridizes under stringent conditions and has a GDP-mannose 4,6-dehydratase activity;
(c)配列番号 8で表されるアミノ酸配列力 なる蛋白質；  (c) a protein having an amino acid sequence ability represented by SEQ ID NO: 8;
(d)配列番号 8で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ GDP-マンノース 4,6-デヒド ラターゼ活性を有する蛋白質； (d) In the amino acid sequence represented by SEQ ID NO: 8, one or more amino acids are deleted, substituted, or inserted A protein having amino acid sequence ability with added and Z or added, and having GDP-mannose 4,6-dehydratase activity;
(e)配列番号 8で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質。  (e) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, and having GDP-mannose 4,6-dehydratase activity.
[0029] 本発明において、 GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼとして は、 [0029] In the present invention, GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is:
下記 (a)あるいは (b)の DNAがコードする蛋白質、または下記 (c)、（d)あるいは (e)の蛋白 質などがあげられる。  Examples include the protein encoded by DNA (a) or (b) below, or the protein (c), (d) or (e) below.
(a)配列番号 9で表される塩基配列力 なる DNA；  (a) DNA having a nucleotide sequence represented by SEQ ID NO: 9;
(b)配列番号 9で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有す る蛋白質をコードする DNA;  (b) It is hybridized under stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 9, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimemerase activity. DNA encoding the protein
(c)配列番号 10で表されるアミノ酸配列からなる蛋白質；  (c) a protein comprising the amino acid sequence represented by SEQ ID NO: 10;
(d)配列番号 10で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、 挿入および Zまたは付加されたアミノ酸配列力もなり、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有する蛋白質；  (d) In the amino acid sequence represented by SEQ ID NO: 10, one or more amino acids are deleted, substituted, inserted and Z or added, and the amino acid sequence power is GDP-4-keto-6-deoxy-D-mannose. -A protein having 3,5-epimerase activity;
(e)配列番号 10で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有す る蛋白質。  (e) It consists of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity Protein.
[0030] N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコース の 1位が a結合する糖鎖修飾に関与する酵素としては、 a 1,6-フコシルトランスフェラ ーゼなどがあげられる。  [0030] N-glycoside-linked complex type sugar chain N-acetylyldarcosamine at the reducing end 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 For example.
本発明において、 α 1,6-フコシルトランスフェラーゼとしては、下記 (a)、（b)、（c)ある いは (d)の DNAがコードする蛋白質、または ( 、（£)、（g)、（h)、（0あるいは (j)の蛋白質な どがあげられる。  In the present invention, α 1,6-fucosyltransferase is a protein encoded by the following DNA (a), (b), (c) or (d), or (, (£), (g), (H), (0 or (j) protein, etc.).
[0031] (a)配列番号 11で表される塩基配列力 なる DNA; [0031] (a) DNA having a nucleotide sequence represented by SEQ ID NO: 11;
(b)配列番号 12で表される塩基配列力もなる DNA；  (b) DNA having a nucleotide sequence represented by SEQ ID NO: 12;
(c)配列番号 11で表される塩基配列からなる DNAとストリンジェントな条件でノ、イブ リダィズし、かつ (X 1,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする D NA; (c) DNA having the nucleotide sequence represented by SEQ ID NO: 11 and stringent conditions under stringent conditions Redis and (XNA encoding a protein having 1,6-fucosyltransferase activity;
(d)配列番号 12で表される塩基配列力もなる DNAとストリンジェントな条件でノ、イブ リダィズし、かつ a 1,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする D NA;  (d) DNA that encodes a protein that has a base sequence ability represented by SEQ ID NO: 12 and is stringent under a stringent condition and a protein having a 1,6-fucosyltransferase activity;
(e)配列番号 13で表されるアミノ酸配列力もなる蛋白質；  (e) a protein having an amino acid sequence ability represented by SEQ ID NO: 13;
(£)配列番号 14で表されるアミノ酸配列力もなる蛋白質；  (£) a protein having an amino acid sequence ability represented by SEQ ID NO: 14;
(g)配列番号 13で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、 挿入および Zまたは付加されたアミノ酸配列力もなり、かつ α 1,6-フコシルトランスフ エラーゼ活性を有する蛋白質；  (g) a protein having an amino acid sequence ability in which one or more amino acids are deleted, substituted, inserted and Z or added in the amino acid sequence represented by SEQ ID NO: 13 and having α1,6-fucosyltransferase activity;
(h)配列番号 14で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、 挿入および Zまたは付加されたアミノ酸配列力もなり、かつ α 1,6-フコシルトランスフ エラーゼ活性を有する蛋白質；  (h) a protein having an amino acid sequence ability in which one or more amino acids are deleted, substituted, inserted and Z or added in the amino acid sequence represented by SEQ ID NO: 14 and having α1,6-fucosyltransferase activity;
(0配列番号 13で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつ a 1,6-フコシルトランスフェラーゼ活性を有する蛋白質；  (0 a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 13, and having a 1,6-fucosyltransferase activity;
0)配列番号 14で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列 からなり、かつ a 1,6-フコシルトランスフェラーゼ活性を有する蛋白質。  0) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 14, and having a 1,6-fucosyltransferase activity.
本発明にお!/、て、ストリンジヱントな条件下でハイブリダィズする DNAとは、例えば配 列番号 7、 9、 11または 12で表される塩基配列力もなる DNAなどの DNAまたはその一 部の断片をプローブとして、コ口-一'ハイブリダィゼーシヨン法、プラーク 'ハイブリダ ィゼーシヨン法あるいはサザンノヽイブリダィゼーシヨン法等を用いることにより得られる DNAを意味し、具体的には、コロニーあるいはプラーク由来の DNAを固定化したフィ ルターを用いて、 0. 7〜1. OMの塩化ナトリウム存在下、 65°Cでハイブリダィゼーシ ヨンを行った後、 0. 1〜2倍濃度の SSC溶液（1倍濃度の SSC溶液の組成は、 150m M塩化ナトリウム、 15mMクェン酸ナトリウムよりなる）を用い、 65°C条件下でフィルタ 一を洗浄することにより同定できる DNAをあげることができる。ハイブリダィゼーシヨン 【ま、 Molecular Cloning, A Laboratory Manual, Second Edition,し old Spring HarborL aboratory Press, 1989 (以下、モレキュラ^ ~ ·クロー-ング第 2版と略す）、 Current Pro tocols in MolecularBiology, John Wiley & Sons, 1987— 1997 (以下、カレント 'プロトコ 一ルズ'イン'モレキュラ^ ~ ·バイオロジーと略す）、 DNA Cloning 1: Core Techniques, A PracticalApproach, Second edition, Oxford University (1995)等に己载されている 方法に準じて行うことができる。ストリンジェントな条件下でノ、イブリダィズ可能な DNA として具体的には、配列番号 7、 9、 11または 12で表される塩基配列と少なくとも 60 %以上の相同性を有する DNA、好ましくは 70%以上、より好ましくは 80%以上、さら に好ましくは 90%以上、特に好ましくは 95%以上、最も好ましくは 98%以上の相同 性を有する DNAをあげることができる。 In the present invention, DNA that hybridizes under stringent conditions is, for example, DNA such as DNA having a nucleotide sequence represented by SEQ ID NO: 7, 9, 11, or 12, or a fragment thereof. This means DNA obtained by using the Koguchi-ichi 'hybridization method, plaque' hybridization method, Southern hybridization method, etc. as a probe, specifically derived from colonies or plaques. After hybridization at 65 ° C in the presence of OM sodium chloride in the presence of 0.7 to 1. OM sodium chloride, 0.1 to 2 times the concentration of SSC solution (The composition of the SSC solution with a 1-fold concentration consists of 150 mM sodium chloride and 15 mM sodium quenate), and DNA can be identified by washing the filter under 65 ° C conditions. Hybridization [MA, Molecular Cloning, A Laboratory Manual, Second Edition, old Spring Harbor Laboratory Press, 1989 (hereinafter abbreviated as Molecular ^ ~ Cloning 2nd Edition), Current Pro tocols in MolecularBiology, John Wiley & Sons, 1987—1997 (hereinafter “Protocol Ones In” Molecular ^ ~ · Biology), DNA Cloning 1: Core Techniques, A PracticalApproach, Second edition, Oxford University (1995 ) Etc. can be carried out according to the method self-installed. Specifically, DNA that can be hybridized under stringent conditions is specifically DNA having at least 60% homology with the nucleotide sequence represented by SEQ ID NO: 7, 9, 11 or 12, 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.
[0033] 本発明において、配列番号 8で表されるアミノ酸配列において 1以上のアミノ酸が欠 失、置換、挿入および Zまたは付加されたアミノ酸配列からなり、かつ GDP-マンノー ス 4,6-デヒドラターゼ活性を有する蛋白質、配列番号 10で表されるアミノ酸配列にお いて 1以上のアミノ酸が欠失、置換、挿入および Zまたは付加されたアミノ酸配列から なり、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有する蛋 白質、または配列番号 13または 14で表されるアミノ酸配列にお 、て 1以上のアミノ酸 が欠失、置換、挿入および Zまたは付加されたアミノ酸配列力もなり、かつ a 1,6-フコ シルトランスフェラーゼ活性を有する蛋白質は、モレキュラー 'クローユング第 2版、力 レント.プロトコーノレズ 'イン'モレキュラー.バイオロジー、 Nucleic Acids Research, 10, 6487 (1982)、 Proc. Natl. Acad. Sci" USA, 79, 6409 (1982)、 Gene, 34,315 (1985)、 Nucleic Acids Research, 13, 4431 (1985)、 Proc. Natl. Acad. Sci USA.82, 488 (1985) 等に記載の部位特異的変異導入法を用いて、例えば、配列番号 8、 9、 13または 14 で表される塩基配列を有する DNAに部位特異的変異を導入することにより取得する ことができる。欠失、置換、挿入および Zまたは付加されるアミノ酸の数は 1個以上で ありその数は特に限定されないが、上記の部位特異的変異導入法等の周知の技術 により、欠失、置換もしくは付加できる程度の数であり、例えば、 1〜数十個、好ましく は 1〜20個、より好ましくは 1〜10個、さらに好ましくは 1〜5個である。  [0033] In the present invention, the amino acid sequence represented by SEQ ID NO: 8 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or added, and the GDP-mannose 4,6-dehydratase activity A protein having an amino acid sequence comprising one or more amino acid sequences deleted, substituted, inserted and Z or added in the amino acid sequence represented by SEQ ID NO: 10, and GDP-4-keto-6-deoxy-D -A protein having mannose-3,5-epimerase activity, or an 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: 13 or 14. And a protein with 1,6-fucosyltransferase activity is molecular 'Crowung 2nd edition, force lent.Protocorenoles' in' molecular. Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci "USA, 79, 6409 (1982), Gene, 34,315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl Using site-directed mutagenesis described in Acad. Sci USA.82, 488 (1985) etc., for example, site-specific mutagenesis is performed on DNA having the nucleotide sequence represented by SEQ ID NO: 8, 9, 13 or 14. 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, but the above-mentioned site-specific mutagenesis method, etc. The number is such that it can be deleted, substituted or added by, for example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5. is there.
[0034] また、本発明において、配列番号 8、 10、 13または 14で表されるアミノ酸配列と 80 %以上の相同性を有するアミノ酸配列からなり、かつ GDP-マンノース 4,6-デヒドラタ ーゼ活性、 GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性または α 1, 6-フコシルトランスフェラーゼ活性を有するためには、それぞれ配列番号 8、 10、 13 または 14で表されるアミノ酸配列と BLAST〔J. Mol. Biol, 215,403 (1990)〕や FAST A [Methods in Enzymology, 183, 63 (1990)〕等の解析ソフトを用いて計算したときに、 少なくとも 80%以上、好ましくは 85%以上、より好ましくは 90%以上、さらに好ましく は 95%以上、特に好ましくは 97%以上、最も好ましくは 99%以上の相同性を有する 蛋白質であることを意味する。 [0034] Further, in the present invention, it comprises an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, 10, 13 or 14, and has GDP-mannose 4,6-dehydratase activity. , GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity or α 1, In order to have 6-fucosyltransferase activity, the amino acid sequence represented by SEQ ID NO: 8, 10, 13 or 14 and BLAST [J. Mol. Biol, 215, 403 (1990)] or FAST A [Methods in Enzymology, 183 , 63 (1990)], etc., 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, it means a protein having a homology of 99% or more.
[0035] また、上述の酵素活性が欠失した宿主細胞、すなわち細胞内糖ヌクレオチド GDP- フコースの合成に関与する酵素、または N-グリコシド結合複合型糖鎖還元末端の N- ァセチルダルコサミンの 6位にフコースの 1位が a結合する糖鎖修飾に関与する酵素 の活性が欠失するようにゲノムが改変された宿主細胞に、ハプトグロビン分子をコー ドする DNAを導入することによって、本発明の組成物を生産する形質転換体を得るこ とがでさる。 [0035] Further, a host cell lacking the above-mentioned enzyme activity, that is, an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-acetylyldarcosamine at the N-glycoside-linked complex sugar chain reducing end By introducing DNA encoding a haptoglobin molecule into a host cell whose genome has been altered so that the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is linked to a at position 6 is a It is possible to obtain a transformant producing the composition.
[0036] ここで、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、または N-ダリ コシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が α結合する糖鎖修飾に関与する酵素の活性が欠失するようにゲノムが改変されたと は、該酵素の発現を消失させるように該遺伝子の発現調節領域に変異を導入したり 、あるいは該酵素の機能を消失させるように該遺伝子のアミノ酸配列に変異を導入す ることを意味する。変異を導入するとは、ゲノム上の塩基配列に欠失、置換、挿入お よび Ζまたは付加と 、つた塩基配列の改変を行うことを意味し、改変したゲノム遺伝 子の発現または機能を完全に抑制することをノックアウトすると 、う。ゲノム遺伝子をノ ックアウトする具体的な例としては、標的となる遺伝子のすべてまたは一部がゲノムか ら削除された例が挙げられる。標的となる遺伝子の開始コドンを含むエタソンのゲノム 領域を染色体上力 除くことでノックアウトすることができる。  [0036] Here, the enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose, or N-acetylcolcamine at the reducing end of the N-daricoside-linked complex sugar chain, position 6 of fucose is α-linked When the genome is modified so that the activity of the enzyme involved in the sugar chain modification is lost, a mutation is introduced into the expression regulatory region of the gene so as to eliminate the expression of the enzyme, or the function of the enzyme It means that a mutation is introduced into the amino acid sequence of the gene so as to disappear. Introducing mutation means that the base sequence on the genome is deleted, substituted, inserted, and deleted or added, and the base sequence is modified, completely suppressing the expression or function of the modified genomic gene. Knock out to do it. A specific example of knocking out a genomic gene is one in which all or part of the target gene has been deleted from the genome. It can be knocked out by removing the genomic region of the etason containing the start codon of the target gene.
[0037] このような細胞を取得する方法としては、目的とするゲノムの改変を行うことができれ ば、いずれの手法でも用いることができる。上述の酵素活性を欠失させる手法として  [0037] As a method for obtaining such cells, any method can be used as long as the target genome can be modified. As a method of deleting the above enzyme activity
(a)酵素の遺伝子を標的した遺伝子破壊の手法； (a) a gene disruption technique targeting an enzyme gene;
(b)酵素の遺伝子のドミナントネガティブ体を導入する手法； (c)酵素につ 、ての突然変異を導入する手法； (b) a method of introducing a dominant negative form of the enzyme gene; (c) a method of introducing all mutations in the enzyme;
(d)酵素の遺伝子の転写又は翻訳を抑制する手法；  (d) a method for suppressing transcription or translation of an enzyme gene;
(e) N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1 位が oc結合した糖鎖構造を認識するレクチンに耐性である株を選択する手法などが あげられる。  (e) A method of selecting a strain resistant to a lectin that recognizes a sugar chain structure in which the N-glycidyl glycosamine 6-position of the N-glycoside-linked sugar chain and the 1-position of fucose are oc-linked .
[0038] N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1位が  [0038] N-glycoside-linked sugar chain reducing terminal N-acetylcylcosamine position 6 and fucose position 1
a結合した糖鎖構造を認識するレクチンとしては、該糖鎖構造を認識できるレクチン であれば、いずれのレクチンでも用いることができる。その具体的な例としては、レン ズマメレクチン LCA (Lens Culinaris由来の Lentil Agglutinin)、エンドゥマメレクチン PS A (Pisum sativum由来の PeaLectin)、ソラマメレクチン VFA (Vicia faba由来の Agglutini n)、ヒィロチャワンタケレクチン AAL (Aleuria aurantia由来の Lectin)等を挙げることが できる。  As the lectin that recognizes the a-linked sugar chain structure, any lectin that can recognize the sugar chain structure can be used. Specific examples of this are: Lentil lectin LCA (Lentil Agglutinin from Lens Culinaris), Endumame lectin PS A (Peum sativum-derived PeaLectin), Broad bean lectin VFA (Agglutini n from Vicia faba), Hirochawantake lectin AAL ( Lectin from Aleuria aurantia).
[0039] レクチンに耐性な細胞とは、レクチンを有効濃度与えたときにも、生育が阻害されな い細胞をいう。有効濃度とは、ゲノム遺伝子が改変される以前の細胞（以下、「親株 細胞」とも称す)が正常に生育できない濃度以上であり、好ましくは、ゲノム遺伝子が 改変される以前の細胞が生育できない濃度と同濃度、より好ましくは 2〜5倍、さらに 好ましくは 10倍、最も好ましくは 20倍以上である。  [0039] 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.
[0040] 本発明において、生育が阻害されないレクチンの有効濃度は、細胞株に応じて適 宜定めればよいが、通常 10 /z g/ml〜10mg/ml、好ましくは 0.5mg/ml〜2.0mg/mlであ る。  [0040] In the present invention, the effective concentration of lectin whose growth is not inhibited may be appropriately determined depending on the cell line, but is usually 10 / zg / ml to 10mg / ml, preferably 0.5mg / ml to 2.0mg. / ml.
本発明の形質転換体としては、本発明の組成物を発現できる細胞であれば!/ヽかな る細胞でもよいが、酵母、動物細胞、昆虫細胞、植物細胞などがあげられ、これらの 細胞の具体的な例としては、後述の 3. に記載のものがあげられる。動物細胞の具体 例としては、チャイニーズノヽムスター卵巣組織由来の CHO細胞、ラットミエローマ細胞 株 YB2/3HL.P2.G11.16Ag.20細胞、マウスミエローマ細胞株 NS0細胞、マウスミエロー マ細胞株 SP2/0-Agl4細胞、シリアンハムスター腎臓組織由来 BHK細胞、ヒト白血病 細胞株ナマルバ細胞、胚性幹細胞、受精卵細胞などがあげられる。好ましくは、遺伝 子組換え糖蛋白質医薬品を製造するために用いる宿主細胞、遺伝子組換え糖蛋白 質医薬品を生産するヒト以外のトランスジエニック動物を製造するために用いる胚性 幹細胞または受精卵細胞、ならびに遺伝子組換え糖蛋白質医薬品を生産するトラン スジ ニック植物を製造するために用いる植物細胞などがあげられる。 As the transformant of the present invention, any cell that can express the composition of the present invention can be used. However, yeast, animal cells, insect cells, plant cells and the like can be mentioned. Specific examples include those described in 3. below. Specific examples of animal cells include CHO cells derived from Chinese omster ovary tissue, rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cell, mouse myeloma cell line NS0 cell, mouse myeloma cell line SP2 / 0- Examples include Agl4 cells, Syrian hamster kidney tissue-derived BHK cells, human leukemia cell lines Namalba cells, embryonic stem cells, and fertilized egg cells. Preferably, a host cell used for producing a recombinant glycoprotein pharmaceutical, a recombinant glycoprotein Examples include embryonic stem cells or fertilized egg cells used to produce non-human transgenic animals that produce pesticides, and plant cells used to produce transgenic plants that produce recombinant glycoprotein drugs. It is done.
[0041] 親株細胞としては、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、ま たは N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコ ースの 1位が OC結合する糖鎖修飾に関与する酵素のゲノム遺伝子を改変させるため の手法を施す前の細胞を包含する。例えば、以下の細胞が好適にあげられる。  [0041] As a parent cell line, an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-glycyl-linked complex N-acetylyldarcosamine at the 6-position of N-acetylyldarcosamine It includes cells prior to the application of a technique for altering the genomic gene of an enzyme involved in sugar chain modification in which position 1 is OC-linked. For example, the following cells are preferable.
NS0細胞の親株細胞としては、バイオ/テクノロジー (BIO/TECHNOLOGY), 10, 169 (1992)、バイオテクノロジ一'バイオエンジニアリング (Biotechnol.Bioeng.), 73, 261, (2 001)等の文献に記載されている NS0細胞があげられる。また、理化学研究所細胞開 発銀行に登録されている NS0細胞株（RCB0213)、あるいはこれら株を様々な無血清 培地に馴化させた亜株などもあげられる。  NS0 cell parent cell lines are described in the literature such as Bio / Technology (BIO / TECHNOLOGY), 10, 169 (1992), Biotechnology No. 1 Bioengineering (Biotechnol. Bioeng.), 73, 261, (2 001), etc. NS0 cells are listed. In addition, NS0 cell line (RCB0213) registered with the RIKEN Cell Development Bank, or sub-strains obtained by acclimatizing these strains to various serum-free media are also included.
[0042] SP2/0-Agl4細胞の親株細胞としては、ジャーナル'ォブ 'ィムノロジー (J. Immunol.)  [0042] As a parent cell of SP2 / 0-Agl4 cells, the journal 'Ob' Immunology (J. Immunol.)
126. 317, (1981)、ネイチヤー (Nature), 276, 269, (1978)、ヒューマン 'アンチイボディ ズ'アンド'ノ、イブリドーマズ (Human Antibodies and Hybridomas), 3, 129, (1992)等 の文献に記載されている SP2/0-Agl4細胞があげられる。また、 ATCCに登録されて V、る SP2/0-Agl4細胞（ATCC CRL-1581)ある!/、はこれら株を様々な無血清培地に 馴化させた亜株 (ATCC CRL-1581.1)などもあげられる。 126. 317, (1981), Nature, 276, 269, (1978), Human 'Antibodys' and' No, Human Antibodies and Hybridomas, 3, 129, (1992), etc. SP2 / 0-Agl4 cells described in 1. There are also V, SP2 / 0-Agl4 cells (ATCC CRL-1581) registered with ATCC! /, And sub-strains (ATCC CRL-1581.1) in which these strains are conditioned to various serum-free media. It is done.
[0043] チャイニーズノヽムスター卵巣組織由来 CHO細胞の親株細胞としては、 Journal of E xperimental Medicine, 108.945 (1958)、 Proc. Natl. Acad. Sci. USA, 60, 1275 (1968) 、 Genetics, 55,513 (1968)、 Chromosoma, 41, 129 (1973)、 Methods in Cell Science, 18,115 (1996)、 Radiation Research, 148, 260 (1997)、 Proc. Natl. Acad. Sci.USA, 77, 4216 (1980)、 Proc. Natl. Acad. Sci. 60, 1275 (1968)、 Cell,6, 121 (1975)、 Molecular Cell Genetics, Appendix 1,11 (p883- 900)等の文献に記載されている CHO細胞があげ られる。また、 ATCCに登録されている CHO- K1株（ATCC CCL-61)、 DUXB11株（A TCC CRL- 9096)、 Pro- 5株（ATCC CRL- 1781)や、市販の CHO- S株（Lifetechnologi es社製 Cat#l 1619)、あるいはこれら株を様々な無血清培地に馴化させた亜株なども あげられる。 [0043] As a parent cell line of CHO cells derived from Chinese nomstar ovary tissue, Journal of Xperimental Medicine, 108.945 (1958), Proc. Natl. Acad. Sci. USA, 60, 1275 (1968), Genetics, 55,513 (1968) ), Chromosoma, 41, 129 (1973), Methods in Cell Science, 18,115 (1996), Radiation Research, 148, 260 (1997), Proc. Natl. Acad. Sci. USA, 77, 4216 (1980), Proc. Natl. Acad. Sci. 60, 1275 (1968), Cell, 6, 121 (1975), Molecular Cell Genetics, Appendix 1, 11 (p883-900) and the like. In addition, CHO-K1 strain (ATCC CCL-61), DUXB11 strain (ATCC CRL-9096), Pro-5 strain (ATCC CRL-1781) registered in ATCC, and commercially available CHO-S strain (Lifetechnologi es Cat # l 1619), or sub-strains made by adapting these strains to various serum-free media can give.
[0044] ラットミエローマ細胞株 YB2/3HL.P2.G11.16Ag.20細胞の親株細胞としては、 Y3/Ag 1.2.3細胞 (ATCC CRL-1631)から樹立された株化細胞が包含される。その具体的な 例としては、 J. Cell. Biol., 93, 576 (1982)、 Methods Enzymol. 73B, 1 (1981)等の文 献に記載されている YB2/3HL.P2.G11.16Ag.20細胞があげられる。また、 ATCCに登 録されている YB2/3HL.P2.G11.16Ag.20細胞（ATCC CRL-1662)あるいはこれら株を 様々な無血清培地に馴化させた亜株などもあげられる。  [0044] 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.
[0045] 本発明の組成物を生産する細胞としては、具体的には、 α 1,6-フコシルトランスフエ ラーゼをコードする遺伝子がノックアウトされた CHO細胞にハプトグロビン Hpl遺伝子 をコードする遺伝子を導入した形質転 ·である HP1KO株、 a 1,6-フコシルトランス フェラーゼをコードする遺伝子がノックアウトされた CHO細胞にハプトグロビン Hp2をコ ードする遺伝子を導入した形質転 ·である HP2KO株等があげられる。  [0045] Specifically, as a cell producing the composition of the present invention, a gene encoding a haptoglobin Hpl gene was introduced into a CHO cell into which a gene encoding α1,6-fucosyltransferase was knocked out. Examples thereof include the HP1KO strain, which is a transformant, and the HP2KO strain, which is a transformant in which a gene encoding haptoglobin Hp2 is introduced into a CHO cell into which a gene encoding a 1,6-fucosyltransferase has been knocked out.
[0046] HP1KO株および HP2KO株は平成 17年 2月 17日付けで、独立行政法人産業技術総 合研究所特許生物寄託センター (日本国茨城県つくば巿東 1丁目 1番地 1中央第 6) に FERM BP-10249および FERM BP-10250としてそれぞれ寄託されている。  [0046] The HP1KO and HP2KO strains were established on February 17, 2005 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Tsukuba Rinto, Tsukuba 1-chome, 1-Chuo 6th, Ibaraki, Japan). Deposited as FERM BP-10249 and FERM BP-10250, respectively.
本発明の形質転換体は、親株細胞カゝら得られるハプトグロビン組成物と同等のへ モグロビンの結合活性を有し、かつ、該親株細胞から得られるハプトグロビン組成物 よりも血中半減期が延長されたノ、ブトグロビン組成物を製造することができる。  The transformant of the present invention has a hemoglobin binding activity equivalent to that of the haptoglobin composition obtained from the parent cell line, and has a longer blood half-life than the haptoglobin composition obtained from the parent cell line. A buttoglobin composition can be produced.
[0047] ハプトグロビン組成物のヘモグロビンとの結合活性、ヘモグロビンと結合して形成し た  [0047] Binding activity of haptoglobin composition to hemoglobin, formed by binding to hemoglobin
複合体の細網内皮系細胞に存在する受容体との結合活性および血中半減期は、既 に公知のヘモグロビンやハプトグロビン受容体との結合活性測定 in vitro試験、マウス やラットなどのモデル動物を用いた in vivo試験あるいはヒトを用いた臨床試験などを 用いて測定することができる（Clin. Chem., 42, 1589(1996)、 Eur. J. Clin. Chem. Bioc hem., 35, 647(1997)、 Nature,409, 198(2001)、 J. Biol. Chem., 279, 51561(2004)、最 新医学， 30, 656(1975)、最新医学， 30,879(1975)、 Res. Exp. Med. 177,1(1980)、新薬 と臨床， 38, 761(1989)、応用薬理， 33, 949(1987)、 Lab.lnvest., 14, 1506(1965)、基礎 と臨床， 18, 5913(1984)) o [0048] 以下、本発明を具体的に説明する。 The binding activity of the complex to the receptor present in reticuloendothelial cells and the half-life in blood were determined by measuring the binding activity of the known hemoglobin and haptoglobin receptor in vitro tests, model animals such as mice and rats. It can be measured using an in vivo test used or a clinical test using humans (Clin. Chem., 42, 1589 (1996), Eur. J. Clin. Chem. Biochem., 35, 647 ( 1997), Nature, 409, 198 (2001), J. Biol. Chem., 279, 51561 (2004), Modern Medicine, 30, 656 (1975), Modern Medicine, 30,879 (1975), Res. Exp. Med 177,1 (1980), new drugs and clinical, 38, 761 (1989), applied pharmacology, 33, 949 (1987), Lab.lnvest., 14, 1506 (1965), basic and clinical, 18, 5913 (1984) )) o [0048] The present invention will be specifically described below.
1.本発明のハプトグロビン組成物を生産するために用いる宿主細胞の作製 本発明のハプトグロビン糸且成物を生産するために用いる宿主細胞は、以下に述べ る手法により作製することができる。  1. Production of host cell used for producing haptoglobin composition of the present invention The host cell used for producing the haptoglobin thread and the composition of the present invention can be produced by the method described below.
(1)酵素の遺伝子を標的とした遺伝子破壊の手法  (1) Gene disruption methods targeting enzyme genes
本発明のハプトグロビン組成物作製のために用いる宿主細胞は、細胞内糖ヌクレ ォチド GDP-フコースの合成に関与する酵素または N-グリコシド結合複合型糖鎖還 元末端の N-ァセチルダルコサミンの 6位にフコースの 1位がひ結合する糖鎖修飾に 関与する酵素（以下、「フコース修飾に関連する酵素」と表記する）の遺伝子を標的と し、遺伝子破壊の方法を用いることにより作製することができる。細胞内糖ヌクレオチ ド GDP-フコースの合成に関与する酵素としては、具体的には、 GDP-マンノース 4,6- デヒドラターゼ（以下、「GMD」と表記する）、 GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ (以下、「Fx」と表記する）などがあげられる。 N-グリコシド結合複合 型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が a結合する糖 鎖修飾に関与する酵素としては、具体的には、 α 1,6-フコシルトランスフェラーゼ、 a -L-フコシダーゼなどがあげられる。  The host cell used for the preparation of the haptoglobin composition of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or N-glycoside-linked complex sugar chain-reducing terminal N-acetylyldarcosamine 6 By using the gene disruption method targeting the gene of the enzyme involved in the sugar chain modification in which position 1 of fucose binds to the position (hereinafter referred to as “enzyme related to fucose modification”) Can do. Specific examples of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GDP-mannose 4,6-dehydratase (hereinafter referred to as “GMD”), GDP-4-keto-6-deoxy -D-mannose-3,5-epimerase (hereinafter referred to as “Fx”). Specific examples of enzymes involved in glycosylation in which the 1-position of fucose is a-linked to the 6-position of N-glycidyl darcosamine at the N-glycoside-bonded glycan reducing end include α 1,6-fucosyl Examples include transferase and a-L-fucosidase.
[0049] ここでいう遺伝子とは、 DNAまたは RNAを含む。 [0049] The gene herein includes DNA or RNA.
遺伝子破壊の方法としては、標的とする酵素の遺伝子を破壊することができる方法 であればいかなる方法も包含される。その例としては、アンチセンス法、リボザィム法 、相同組換え法、 RNA-DNAオリゴヌクレオチド法（以下、「RDO法」と表記する）、 RNA インターフェアレンス法（以下、「RNAi法」と表記する）、レトロウイルスを用いた方法、 トランスポゾンを用いた方法等があげられる。以下これらを具体的に説明する。  Any method can be used as the gene disruption method as long as it can destroy the gene of the target enzyme. Examples include the antisense method, ribozyme method, homologous recombination method, RNA-DNA oligonucleotide method (hereinafter referred to as “RDO method”), RNA interference method (hereinafter referred to as “RNAi method”). ), A method using a retrovirus, a method using a transposon, and the like. These will be specifically described below.
[0050] (a)アンチセンス法又はリボザィム法による本発明のハプトグロビン組成物を作製す るための宿主細胞の作製 [0050] (a) Production of host cells for producing the haptoglobin composition of the present invention by antisense method or ribozyme method
本発明のハプトグロビン組成物を作製するために用いる宿主細胞は、フコース修飾 に関連する酵素遺伝子を標的とし、細胞工学， 12, 239(1993)、バイオ Zテクノロジー（ BIO/TECHNOLOGY), 17, 1097 (1999)、ヒューマン 'モレキュラ^ ~ ·ジエネテイクス (Hu m.Mol. Genet.), 5, 1083 (1995)、細胞工学， 13, 255 (1994)、プロシーディングス 'ォ ブ ·ザ.ナショナル.ァカデミ一.ォブ.サイエンス (proc. Natl. Acad. Sci. U.S.A.), 96, 1The host cell used to produce the haptoglobin composition of the present invention targets an enzyme gene related to fucose modification, and is described in Cell Engineering, 12, 239 (1993), Bio-Z Technology (BIO / TECHNOLOGY), 17, 1097 ( 1999), Human 'Molecular ^ ~ · Genetics (Hum.Mol. Genet.), 5, 1083 (1995), Cell engineering, 13, 255 (1994), Proceedings' o B. The National. Academia. Ob. Science ( proc . Natl. Acad. Sci. USA), 96, 1
886 (1999)等に記載されたアンチセンス法またはリボザィム法を用いて、例えば、以 下のように作製することができる。 For example, it can be prepared as follows using the antisense method or ribozyme method described in 886 (1999).
[0051] フコース修飾に関連する酵素をコードする cDNAあるいはゲノム DNAを調製する。 [0051] cDNA or genomic DNA encoding an enzyme related to fucose modification is prepared.
調製した cDNAあるいはゲノム DNAの塩基配列を決定する。  Determine the base sequence of the prepared cDNA or genomic DNA.
決定した DNAの配列に基づき、フコース修飾に関連する酵素をコードする DNA部 分、非翻訳領域の部分あるいはイントロン部分を含む適当な長さのアンチセンス遺伝 子またはリボザィムのコンストラクトを設計する。  Based on the determined DNA sequence, construct an antisense gene or ribozyme construct of appropriate length including the DNA part encoding the enzyme related to fucose modification, the part of the untranslated region or the intron part.
[0052] 該アンチセンス遺伝子、またはリボザィムを細胞内で発現させるために、調製した D[0052] D prepared in order to express the antisense gene or ribozyme in cells
NAの断片、または全長を適当な発現ベクターのプロモーターの下流に挿入すること により、組換えベクターを作製する。 A recombinant vector is prepared by inserting the NA fragment or full length downstream of the promoter of an appropriate expression vector.
該組換えベクターを、該発現べクタ一に適合した宿主細胞に導入することにより形 質転換体を得る。  A transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
[0053] フコース修飾に関連する酵素の活性を指標として形質転換体を選択することにより 、本発明のハプトグロビン組成物を作製するために用いる宿主細胞を得ることができ る。また、細胞膜上の糖蛋白質の糖鎖構造または産生糖蛋白質分子の糖鎖構造を 指標として形質転換体を選択することにより、本発明のハプトグロビン組成物を作製 するために用いる宿主細胞を得ることもできる。  [0053] By selecting a transformant using the activity of an enzyme related to fucose modification as an indicator, a host cell used for preparing the haptoglobin composition of the present invention can be obtained. It is also possible to obtain a host cell used for producing the haptoglobin composition of the present invention by selecting a transformant using the sugar chain structure of a glycoprotein on the cell membrane or the sugar chain structure of a produced glycoprotein molecule as an index. it can.
[0054] 本発明のハプトグロビン組成物を作製するために用いられる宿主細胞としては、酵 母、動物細胞、昆虫細胞、植物細胞など、標的とするフコース修飾に関連する酵素 の遺伝子を有しているものであればいずれも用いることができる。具体的には、後述 の 3に記載の宿主細胞があげられる。  [0054] The host cell used for preparing the haptoglobin composition of the present invention has a gene for an enzyme related to target fucose modification, such as an enzyme, an animal cell, an insect cell, or a plant cell. Any material can be used. Specific examples include host cells described in 3 below.
発現ベクターとしては、上記宿主細胞において自立複製が可能である力、ないしは 染色体中への組み込みが可能で、設計したアンチセンス遺伝子、またはリボザィムを 転写できる位置にプロモーターを含有しているものが用いられる。具体的には、後述 3に記載の発現ベクターがあげられる。  The expression vector is capable of autonomous replication in the above host cell, or can be integrated into the chromosome and contains a designed antisense gene or a promoter at a position where a ribozyme can be transcribed. . Specifically, the expression vector described in 3 below can be mentioned.
[0055] 各種宿主細胞への遺伝子の導入方法としては、後述 3に記載の各種宿主細胞に 適した組換えベクターの導入方法を用いることができる。 フコース修飾に関連する酵素の活性を指標として形質転換体を選択する方法とし ては、例えば、以下の方法があげられる。 [0055] As a method for introducing a gene into various host cells, the method for introducing a recombinant vector suitable for various host cells described in 3 below can be used. Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the following methods.
形皙転椽体を選択する方法  How to select a shape
フコース修飾に関連する酵素の活性が欠失した細胞を選択する方法としては、文 献 if生化学実験講座 3—糖質 I,糖タンパク質 (東京化学同人)日本生化学会編 (198 8)]、文献 [細胞工学,別冊，実験プロトコールシリーズ,グライコバイオロジー実験プロト コール,糖タンパク質 ·糖脂質 ·プロテオダリカン (秀潤社製)谷口直之 ·鈴木明美 ·古川 清.菅原一幸監修（1996)]、モレキュラー 'クローユング第 2版、カレント.プロトコール ズ'イン'モレキュラー 'バイオロジー等に記載された生化学的な方法あるいは遺伝子 工学的な方法などを用いて、フコース修飾に関連する酵素の活性を測定する方法が あげられる。生化学的な方法としては、例えば、酵素特異的な基質を用いて酵素活 性を評価する方法があげられる。遺伝子工学的な方法としては、例えば、酵素遺伝 子の mRNA量を測定するノーザン解析や RT-PCR法等があげられる。  As a method of selecting cells lacking the activity of the enzyme related to fucose modification, refer to the literature if biochemistry experiment course 3-carbohydrate I, glycoprotein (Tokyo Kagaku Dojin) edited by the Japanese Biochemical Society (198 8)], Literature [Cell engineering, separate volume, experimental protocol series, glycobiology experimental protocol, glycoproteins · glycolipids · proteodaricans (manufactured by Shujunsha) Naoyuki Taniguchi · Akemi Suzuki · Kiyoshi Furukawa (supervised by Kazuyuki Sugawara (1996)), Measure the activity of enzymes related to fucose modification using biochemical methods or genetic engineering methods described in Molecular 'Crowing 2nd Edition, Current Protocols' In'Molecular' Biology etc. There are methods. Examples of the biochemical method include a method for evaluating enzyme activity using an enzyme-specific substrate. Examples of genetic engineering methods include Northern analysis and RT-PCR method for measuring the amount of mRNA of an enzyme gene.
[0056] 細胞膜上の糖蛋白質の糖鎖構造を指標として形質転換体を選択する方法としては 、例えば、後述 1の（5)に記載の方法があげられる。産生糖蛋白質分子の糖鎖構造 を指標として形質転換体を選択する方法としては、例えば、後述 5または後述 6に記 載の方法があげられる。 [0056] Examples of a method for selecting a transformant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (1) below. Examples of the method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
フコース修飾に関連する酵素をコードする cDNAを調製する方法としては、例えば、 下記に記載の方法があげられる。 Examples of a method for preparing cDNA encoding an enzyme related to fucose modification include the methods described below.
[0057] cDNAの調製方法 [0057] Method for preparing cDNA
各種宿主細胞の組織又は細胞力ゝら全 RNA又は mRNAを調製する。  Total RNA or mRNA is prepared from the tissues or cell strength of various host cells.
調製した全 RNA又は mRNAから cDNAライブラリーを作製する。  A cDNA library is prepared from the prepared total RNA or mRNA.
フコース修飾に関連する酵素のアミノ酸配列に基づ 、て、デジエネレイティブプライ マーを作製し、作製した cDNAライブラリーを铸型として PCR法でフコース修飾に関連 する酵素をコードする遺伝子断片を取得する。  Based on the amino acid sequence of the enzyme related to fucose modification, a degenerative primer is prepared, and a gene fragment encoding the enzyme related to fucose modification is obtained by PCR using the prepared cDNA library as a saddle type To do.
[0058] 取得した遺伝子断片をプローブとして用い、 cDNAライブラリーをスクリーニングし、 フコース修飾に関連する酵素をコードする DNAを取得することができる。 [0058] Using the obtained gene fragment as a probe, a cDNA library can be screened to obtain DNA encoding an enzyme related to fucose modification.
ヒト又は非ヒト動物の糸且織又は細胞の mRNAは市販のもの (例えば Clontech社)を用 V、てもよ 、し、以下のようにしてヒト又は非ヒト動物の組織又は細胞力も調製してもよ い。 For human or non-human animal thread and tissue or cell mRNA, use commercially available products (for example, Clontech). V or human or non-human animal tissue or cell force may be prepared as follows.
ヒト又は非ヒト動物の組織又は細胞力も全 RNAを調製する方法としては、チォシアン 酸  As a method for preparing total RNA of human or non-human animal tissues or cells, thiocyanic acid can be used.
グァ-ジン-トリフルォロ酢酸セシウム法 [メソッズ ·イン ·ェンザィモロジ一 (Methods in Enzymology), 154, 3(1987)]、酸性チォシアン酸グァ-ジン'フエノール'クロ口ホルム (AGPC)法 [アナリティカル 'バイオケミストリー (Analytical Biochemistry), 162, 156 (1 987);実験医学、 9,1937 (1991)]などがあげられる。  Guazin-trifluoroacetate method [Methods in Enzymology, 154, 3 (1987)], Guazin acid thiocyanate 'Phenol' black mouth form (AGPC) method [Analytical Bio Chemistry (Analytical Biochemistry), 162, 156 (1 987); experimental medicine, 9,1937 (1991)].
[0059] また、全 RNA力 poly(A)+ RNAとして mRNAを調製する方法としては、オリゴ（dT)固 定ィ匕セルロースカラム法 (モレキュラー 'クローユング第 2版)等があげられる。 [0059] In addition, examples of a method for preparing mRNA as total RNA poly (A) + RNA include an oligo (dT) -fixed cellulose column method (Molecular 'Cloung 2nd edition).
さらに、 Fast Track mRNA Isolation Kit (Invitrogen社）、 Quick Prep mRNA Purification Kit (Pharmacia社）などの市販のキットを用いることにより mRNAを調製す ることがでさる。  Furthermore, mRNA can be prepared by using a commercially available kit such as Fast Track mRNA Isolation Kit (Invitrogen) or Quick Prep mRNA Purification Kit (Pharmacia).
[0060] 次に、調製したヒト又は非ヒト動物の組織又は細胞 mRNAから cDNAライブラリーを作 製する。 cDNAライブラリー作製法としては、モレキュラー 'クローユング第 2版、カレン ト.プロトコールズ.イン.モレキュラー.バイオロジー、 A Laboratory Manual, 2 nd Ed.( 1989)等に記載された方法、あるいは市販のキット、例えば Superscript Plasmid Syste m for cDNA Synthesis and Plasmid Cloning (Life Technologiesネエリ、 ZAP— cDNA bynt hesis Kit (STRATAGENE社）を用いる方法などがあげられる。  [0060] Next, a cDNA library is prepared from the prepared human or non-human animal tissue or cell mRNA. The cDNA library can be prepared by the method described in Molecular 'Crowing 2nd Edition, Current Protocols in Molecular Biology, A Laboratory Manual, 2nd Ed. (1989), etc., or a commercially available kit. For example, a method using Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies Neeri, ZAP—cDNA bynt hesis Kit (STRATAGENE)) can be mentioned.
[0061] cDNAライブラリーを作製するためのクローユングベクターとしては、大腸菌 K12株中 で  [0061] As a cloning vector for preparing a cDNA library, Escherichia coli K12 strain is used.
自立複製できるものであれば、ファージベクター、プラスミドベクター等いずれでも使 用できる。具体的には、 ZAP Express [STRATAGENE社、ストラテジーズ (Strategies), , 58  Any phage vector or plasmid vector can be used as long as it can replicate autonomously. Specifically, ZAP Express [STRATAGENE, Strategies,, 58
(1992)]、 pBluescript II SK(+) [ヌクレイック'アシッド'リサーチ (Nucleic Acids Research ), 17,9494 (1989)] , λ ZAP II (STRATAGENE社）、 gtl0、え gtl l [ディーェヌェ一' クロー-ング.ァ 'プラクティカル.アプローチ (DNA cloning, A Practical Approach),丄， 49 (1985)]、 TriplEx (Clontech社）、 λ ExCell (Pharmacia社）、 pT7T318U (Pharmaci a社）、 pcD2 [モレキユラ一'セルラ一'バイオロジー (Mol.Cell. Biol), 3, 280 (1983)]お よび pUC18 [ジーン (Gene), 33, 103 (1985)]等をあげることができる。 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17,9494 (1989)], λ ZAP II (STRATAGENE), gtl0, e gtl l 'DNA cloning, A Practical Approach, 丄, 49 (1985)], TriplEx (Clontech), λ ExCell (Pharmacia), pT7T318U (Pharmaci a)), pcD2 [Mole. Cell. Biol, 3, 280 (1983)] and pUC18 [Gene, 33, 103 (1985)]. it can.
[0062] cDNAライブラリーを作製するための宿主微生物としては、微生物であればいずれ でも用いることができるが、好ましくは大腸菌が用いられる。具体的には、 Escherichia coliXLl- Blue MRF' [STRATAGENE社、ストラテジーズ (Strategies), 5, 81 (1992)] , Es cherichia coliC600「ジエネテイクス (Genetics), 39, 440 (1954)1 , Escherichia coliY108 8 [サイエンス (Science) ,222, 778 (1983)1 , Escherichia coliYl 090「サイエンス (Science) , 222,778 (1983)1、 Escherichia coliNM522「ジャーナル，ォブ，モレキユラ ~·バイオ口 ジー 0. Mol.BioL), 166, 1 (1983)1、 Escherichia coli K802「ジャーナル ·ォブ ·モレキ ユラ ~·バイオロジー (J.Mol. Biol), 16, 118 (1966)Ίおよび Escherichia coli TM105「ジ ーン (Gene), 38,275 (1985)]等が用いられる。  [0062] As a host microorganism for preparing a cDNA library, any microorganism can be used, but Escherichia coli is preferably used. Specifically, Escherichia coli XLl-Blue MRF '[STRATAGENE, Strategies, 5, 81 (1992)], Es cherichia coli C600 "Genetics, 39, 440 (1954) 1, Escherichia coli Y108 8 [ Science, 222, 778 (1983) 1, Escherichia coli Yl 090 "Science, 222,778 (1983) 1, Escherichia coli NM522" Journal, Ob, Molecura ~ Bio Mouth 0. Mol.BioL), 166 , 1 (1983) 1, Escherichia coli K802 `` Journal of Mol. Biol, 16, 118 (1966) Ί and Escherichia coli TM 105 `` Gene, 38,275 (1985)] and the like are used.
[0063] この cDNAライブラリ一は、そのまま以降の解析に用いてもょ 、が、不完全長 cDNA の割合を下げ、なるべく完全長 cDNAを効率よく取得するために、菅野らが開発した オリゴキャップ法 [ジーン (Gene),12S, 171 (1994);ジーン (Gene), 200, 149 (1997);蛋 白質核酸酵素， 41,603 (1996);実験医学， U, 2491 (1993); cDNAクローユング (羊土 社) (1996);遺伝子ライブラリーの作製法 (羊土社）（1994)]を用いて調製して以下の解 祈に用いてもよい。  [0063] Although this cDNA library can be used as it is for subsequent analysis, the oligo cap method developed by Kanno et al. Has been developed to reduce the proportion of incomplete-length cDNA and to obtain full-length cDNA as efficiently as possible. [Gene, 12S, 171 (1994); Gene, 200, 149 (1997); Protein Nucleic Acid Enzyme, 41,603 (1996); Experimental Medicine, U, 2491 (1993); (1996); Gene library production method (Yodosha) (1994)] and may be used for the following prayers.
[0064] フコース修飾に関連する酵素のアミノ酸配列に基づいて、該アミノ酸配列をコード することが予測される塩基配列の 5'末端および 3'末端の塩基配列に特異的なデジ ネレイティブプライマーを作製し、作製した cDNAライブラリーを铸型として PCR法 [ピ 一シーア一ノレ'プロトコーノレズ (PCR Protocols), Academic Press (1990)]を用いて DN Aの増幅を行うことにより、フコース修飾に関連する酵素をコードする遺伝子断片を取 得することができる。  [0064] Based on the amino acid sequence of the enzyme related to the fucose modification, the creation of a degenerative primer specific to the 5'-end and 3'-end base sequences predicted to encode the amino acid sequence Using the prepared cDNA library as a cage, amplification of DNA was performed by PCR using the PCR method [PCR Protocols, Academic Press (1990)]. A gene fragment encoding the enzyme to be obtained can be obtained.
[0065] 取得した遺伝子断片がフコース修飾に関連する酵素をコードする DNAであることは 、通常用いられる塩基配列解析方法、例えばサンガー（Sanger)らのジデォキシ法 [ プロシーディングス ·ォブ ·ザ ·ナショナル ·アカデミ^ ~ ·ォブ ·サイエンス (Pro Natl. Ac ad. Sci. U.S.A.), 74, 5463 (1977)]あるいは ABI PRISM377DNAシークェンサ一（Appli ed Biosystems社製)等の塩基配列分析装置を用いて分析することにより、確認するこ とがでさる。 [0065] The obtained gene fragment is DNA encoding an enzyme related to fucose modification. For example, Sanger et al.'S dideoxy method [Proceedings of the National] · Academ ^ ~ · Ob · Science (Pro Natl. Ac ad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems) etc. To confirm Togashi.
[0066] 該遺伝子断片をプローブとして、ヒト又は非ヒト動物の組織又は細胞に含まれる mR NAから合成した cDNAあるいは cDNAライブラリーからコロニーハイブリダィゼーシヨン やプラークハイブリダィゼーシヨン (モレキュラー ·クロー-ング第 2版)等を用いて、フ コース修飾に関連する酵素の DNAを取得することができる。  [0066] Using the gene fragment as a probe, a colony hybridization or a plaque hybridization (molecular clone) is synthesized from cDNA or cDNA library synthesized from mRNA contained in tissues or cells of human or non-human animals. -Nu 2nd edition) etc. can be used to obtain DNA for enzymes related to fucose modification.
また、フコース修飾に関連する酵素をコードする遺伝子断片を取得するために用い たプライマーを用い、ヒト又は非ヒト動物の組織又は細胞に含まれる mRNAから合成し た cDNAあるいは cDNAライブラリーを铸型として、 PCR法を用いて増幅することにより 、フコース修  In addition, a cDNA or cDNA library synthesized from mRNA contained in human or non-human animal tissues or cells using the primers used to obtain a gene fragment encoding an enzyme related to fucose modification as a saddle type. Amplify using the PCR method to repair fucose
飾に関連する酵素の cDNAを取得することもできる。  It is also possible to obtain cDNA for enzymes related to decoration.
[0067] 取得したフコース修飾に関連する酵素をコードする DNAの塩基配列は、通常用い られる塩基配列解析方法、例えばサンガー（Sanger)らのジデォキシ法 [プロシーディ ングス ·ォブ ·ザ'ナショナル ·アカデミ^ ~·ォブ ·サイエンス (Pro Natl. Acad. Sci. U.S. A.), 74, 5463 (1977)]あるいは ABI PRISM377DNAシークェンサ一（Applied Biosyste ms社製)等の塩基配列分析装置を用いて分析することにより、該 DNAの塩基配列を 決定することができる。 [0067] The base sequence of the DNA encoding the enzyme related to the obtained fucose modification can be determined by a commonly used base sequence analysis method such as Sanger et al.'S dideoxy method [procedings · of · the 'national · academia ^ By analyzing using a base sequence analyzer such as ~ Ob Science (Pro Natl. Acad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems), The base sequence of the DNA can be determined.
[0068] 決定した cDNAの塩基配列をもとに、 BLAST等の相同性検索プログラムを用いて、 Genbank、 EMBLおよび DDBJなどの塩基配列データベースを検索することにより、取 得した DNAがデータベース中の遺伝子の中でフコース修飾に関連する酵素をコード して 、る遺伝子であることを確認することもできる。  [0068] Based on the determined cDNA base sequence, a homology search program such as BLAST is used to search a base sequence database such as Genbank, EMBL, and DDBJ. It can also be confirmed that the gene encodes an enzyme related to fucose modification.
上記の方法で得られる細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素 をコードする遺伝子の塩基配列としては、例えば、配列番号 7または 9に記載の塩基 配列があげられる。  Examples of the base sequence of the gene encoding the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose obtained by the above method include the base sequence set forth in SEQ ID NO: 7 or 9.
[0069] 上記の方法で得られる N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコ サミンの 6位にフコースの 1位が oc結合する糖鎖修飾に関与する酵素をコードする遺 伝子の塩基配列としては、例えば、配列番号 11または 12に記載の塩基配列があげ られる。  [0069] An N-glycoside-linked complex-type sugar chain-reducing terminal N-acetylcylcosamine obtained by the above 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: 11 or 12.
決定された DNAの塩基配列に基づ 、て、フォスフォアミダイト法を利用した DNA合 成機 model 392 (Perkin Elmer社製）等の DNA合成機で化学合成することにより、フコ ース修飾に関連する酵素の cDNAを取得することもできる。 Based on the determined DNA base sequence, DNA synthesis using the phosphoramidite method is performed. By chemically synthesizing with a DNA synthesizer such as model machine 392 (manufactured by Perkin Elmer), cDNA of an enzyme related to fucose modification can also be obtained.
[0070] フコース修飾に関連する酵素のゲノム DNAを調製する方法としては、例えば、以下 に記載の方法があげられる。 [0070] Examples of a method for preparing genomic DNA of an enzyme related to fucose modification include the methods described below.
ゲノム DNAの調製方法  Method for preparing genomic DNA
ゲノム DNAを調製する方法としては、モレキュラー ·クローユング第 2版やカレント · プロトコールズ ·イン.モレキュラー.バイオロジー等に記載された公知の方法があげら れる。また、ゲノム DNAライブラリースクリーニングシステム（Genome Systems社）や Uni versal GenomeWalker™ Kits (CLONTECH社）などを用いることにより、フコース修飾 に関連する酵素のゲノム DNAを取得することもできる。  Examples of methods for preparing genomic DNA include known methods described in Molecular Cloning 2nd Edition and Current Protocols in Molecular Biology. In addition, genomic DNA of an enzyme related to fucose modification can be obtained by using a genomic DNA library screening system (Genome Systems), Unigen GenomeWalker ™ Kits (CLONTECH), or the like.
[0071] 取得したフコース修飾に関連する酵素をコードする DNAの塩基配列は、通常用い られる [0071] The obtained DNA base sequence encoding the enzyme related to fucose modification is usually used.
塩基配列解析方法、例えばサンガー (Sanger)らのジデォキシ法 [プロシーディンダス •ォブ 'ザ'ナショナル 'ァ力デミ一'ォブ 'サイエンス (Proc.Natl. Acad. Sci. U.S.A.), 74 Nucleotide sequence analysis methods, such as Sanger et al.'S dideoxy method [Procedinas Abb. Scad. U.S.A., 74
, 5463 (1977)]あるいは ABI PRISM377DNAシークェンサ一（Applied Biosystems社製 )等の塩基配列分析装置を用いて分析することにより、該 DNAの塩基配列を決定す ることがでさる。 , 5463 (1977)] or by using a base sequence analyzer such as ABI PRISM377 DNA Sequencer (Applied Biosystems), the base sequence of the DNA can be determined.
[0072] 決定したゲノム DNAの塩基配列をもとに、 BLAST等の相同性検索プログラムを用い て、 Genbank、 EMBLおよび DDBJなどの塩基配列データベースを検索することにより 、取得した DNAがデータベース中の遺伝子の中でフコース修飾に関連する酵素をコ ードしている遺伝子であることを確認することもできる。  [0072] Based on the determined base sequence of genomic DNA, a homology search program such as BLAST is used to search base sequence databases such as Genbank, EMBL and DDBJ. It is also possible to confirm that the gene encodes an enzyme related to fucose modification.
決定された DNAの塩基配列に基づ 、て、フォスフォアミダイト法を利用した DNA合 成機 model 392 (Perkin Elmer社製）等の DNA合成機で化学合成することにより、フコ ース修飾に関連する酵素のゲノム DNAを取得することもできる。  Based on the determined DNA base sequence, it is related to fucose modification by chemical synthesis using a DNA synthesizer such as the DNA synthesizer model 392 (Perkin Elmer) using the phosphoramidite method. It is also possible to obtain genomic DNA of the enzyme.
[0073] 上記の方法で得られる細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素 のゲノム DNAの塩基配列としては、例えば配列番号 15、 16、 17および 18に記載の 塩基配列があげられる。  [0073] Examples of the nucleotide sequence of the genomic DNA of the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose obtained by the above method include the nucleotide sequences set forth in SEQ ID NOs: 15, 16, 17, and 18.
上記の方法で得られる N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコ サミンの 6位にフコースの 1位が α結合する糖鎖修飾に関与する酵素のゲノム DNAの 塩基配列と N-glycyl-bonded N-acetylyldarco at the reducing end obtained by the above method The base sequence of the genomic DNA of the enzyme involved in the sugar chain modification in which the position 1 of fucose is α-linked to position 6 of samine
しては、例えば配列番号 19に記載の塩基配列があげられる。  Examples thereof include the base sequence set forth in SEQ ID NO: 19.
[0074] また、発現ベクターを用いず、フコース修飾に関連する酵素の塩基配列に基づい て設計したアンチセンスオリゴヌクレオチドまたはリボザィムを、直接宿主細胞に導入 することで、本発明のハプトグロビン組成物を作製するために用いる宿主細胞を得る ことちでさる。 [0074] In addition, the haptoglobin composition of the present invention is prepared by directly introducing into the host cell an antisense oligonucleotide or ribozyme designed based on the base sequence of the enzyme related to fucose modification without using an expression vector. The host cell used to do this is obtained.
アンチセンスオリゴヌクレオチドまたはリボザィムは、常法または DNA合成機により調 製することができる。具体的には、フコース修飾に関連する酵素をコードする cDNAお よびゲノム DNAの塩基配列のうち、連続した 5〜150塩基、好ましくは 5〜60塩基、よ り好ましくは 10〜40塩基に相当する配列を有するオリゴヌクレオチドの配列情報に 基づき、該オリゴヌクレオチドと相補的な配列に相当するオリゴヌクレオチド (アンチセ ンスオリゴヌクレオチド)または該オリゴヌクレオチドの配列を含むリボザィムを合成し て調製することができる。  Antisense oligonucleotides or ribozymes can be prepared by conventional methods or DNA synthesizers. Specifically, it corresponds to a continuous 5 to 150 bases, preferably 5 to 60 bases, more preferably 10 to 40 bases in the base sequences of cDNA and genomic DNA encoding the enzyme related to fucose modification. Based on the sequence information of the oligonucleotide having the sequence, an oligonucleotide (antisense oligonucleotide) corresponding to a sequence complementary to the oligonucleotide or a ribozyme containing the sequence of the oligonucleotide can be synthesized and prepared.
[0075] オリゴヌクレオチドとしては、オリゴ RNAおよび該オリゴヌクレオチドの誘導体 (以下、 オリゴヌクレオチド誘導体と ヽぅ）等があげられる。 [0075] Examples of the oligonucleotide include oligo RNA and derivatives of the oligonucleotide (hereinafter referred to as oligonucleotide derivatives).
オリゴヌクレオチド誘導体としては、オリゴヌクレオチド中のリン酸ジエステル結合が ホスフォロチォエート結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド 中のリン酸ジエステル結合が Ν3'-Ρ5'ホスフォアミデート結合に変換されたオリゴヌク レオチド誘導体、オリゴヌクレオチド中のリボースとリン酸ジエステル結合がペプチド 核酸結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のゥラシルが C-5プロピ-ルゥラシルで置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中 のゥラシルが C-5チアゾールゥラシルで置換された誘導体オリゴヌクレオチド、オリゴ ヌクレオチド中のシトシンが C-5プロピ-ルシトシンで置換されたオリゴヌクレオチド誘 導体、オリゴヌクレオチド中のシトシンがフエノキサジン修飾シトシン（phenoxazine-mo dified cytosine)で置換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中のリボ ースが 2'-0-プロピルリボースで置換されたオリゴヌクレオチド誘導体、あるいはオリ ゴヌクレオチド中のリボースが 2しメトキシエトキシリボースで置換されたオリゴヌクレオ チド誘導体等があげられる [細胞工学， 16, 1463(1997)]。 Oligonucleotide derivatives include oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides are converted to に 3'-Ρ5 'phosphoramidate bonds. Oligonucleotide derivatives, oligonucleotide derivatives in which the ribose and phosphodiester bonds in the oligonucleotide are converted to peptide nucleic acid bonds, oligonucleotide derivatives in which the uracil in the oligonucleotide is replaced with C-5 propylene uracil, in the oligonucleotide Derivative uracil in which uracil is substituted with C-5 thiazoleuracil, cytosine in the oligonucleotide is substituted with C-5 propylcytosine, and the derivative is cytosine in the oligonucleotide. Oligonucleotide derivatives substituted with enoxazine-modified cytosine, oligonucleotide derivatives substituted with 2'-0-propylribose in the oligonucleotide, or ribose in the oligonucleotide 2 Oligonucleosides substituted with methoxyethoxyribose Examples include tide derivatives [Cell engineering, 16, 1463 (1997)].
[0076] (b)相同組換え法による本発明のハプトグロビン組成物を作製するために用いる宿 主細胞の作製 [0076] (b) Production of host cells used for producing the haptoglobin composition of the present invention by homologous recombination method
本発明のハプトグロビン組成物を作製するために用いる宿主細胞は、フコース修飾 に関連する酵素の遺伝子を標的とし、染色体上の標的遺伝子を相同組換え法を用 いて改変することによって作製することができる。  The host cell used for preparing the haptoglobin composition of the present invention can be 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. .
[0077] 染色体上の標的遺伝子の改変は、 Manipulating the Mouse Embryo A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1994) (以下、「マ-ピ ュレイティング 'ザ 'マウス'ェンブリオ 'ァ'ラボラトリ一'マニュアル」と略す）、 Gene Tar geting, A Practical Approach, IRL Press at Oxford University Press (1993)、ノヽィォマ -ュアルシリーズ 8ジーンターゲッティング， ES細胞を用いた変異マウスの作製,羊 土社（1995) (以下、「ES細胞を用いた変異マウスの作製」と略す)等に記載の方法を 用い、例えば以下のように行うことができる。 [0077] Modification of the target gene on the chromosome is described in Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (hereinafter referred to as "MuPurating 'The' Mouse 'Embryo' A 'Laboratory Laboratory"). Abbreviated 'manual'), Gene Tar geting, A Practical Approach, IRL Press at Oxford University Press (1993), Noyoma-8 Series Gene Targeting, Production of Mutant Mice Using ES Cells, Yodosha (1995) ( Hereinafter, the method described in “abbreviation of mutant mouse using ES cell”) can be used, for example, as follows.
[0078] フコース修飾に関連する酵素のゲノム DNAを調製する。 [0078] Genomic DNA of an enzyme related to fucose modification is prepared.
ゲノム DNAの塩基配列にも基づき、改変する標的遺伝子 (例えば、フコース修飾に 関連する酵素の構造遺伝子、あるいはプロモーター遺伝子）を相同組換えするため のターゲットベクターを作製する。  Based on the genomic DNA base sequence, a target vector for homologous recombination of the target gene to be modified (for example, a structural gene of an enzyme related to fucose modification or a promoter gene) is prepared.
作製したターゲットベクターを宿主細胞に導入し、染色体上の標的遺伝子とターゲ ットベクターの間で相同組換えを起こした細胞を選択することにより、本発明のハプト グロビン組成物を作製するために用いる宿主細胞を作製することができる。  A host cell used for preparing the haptoglobin 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. Can be produced.
[0079] 宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、標的とするフコース 修飾に関連する酵素の遺伝子を有して 、るものであれば 、ずれも用いることができる 。具体的には、後述 3に記載の宿主細胞があげられる。 [0079] As the host cell, any yeast cell, animal cell, insect cell, plant cell, etc. having an enzyme gene related to the target fucose modification can be used. Specifically, the host cells described in 3 below can be mentioned.
フコース修飾に関連する酵素のゲノム DNAを調製する方法としては、上記 1の（1) の  As a method for preparing genomic DNA of an enzyme related to fucose modification, the method described in (1) above is applicable.
(a)に記載のゲノム DNAの調製方法などがあげられる。  Examples thereof include a method for preparing genomic DNA described in (a).
[0080] 上記の方法で得られる細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素 のゲノム DNAの塩基配列として、例えば配列番号 15、 16、 17および 18に記載の塩 基配列があげられる。 [0080] As the nucleotide sequence of the genomic DNA of the enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose obtained by the above method, for example, the salts described in SEQ ID NOs: 15, 16, 17 and 18 Examples of the base sequence.
上記の方法で得られる N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコ サミンの 6位にフコースの 1位が α結合する糖鎖修飾に関与する酵素のゲノム DNAの 塩基配列として、例えば配列番号 19に記載の塩基配列があげられる。  As the base sequence of the genomic DNA of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetyldylcosamine at the N-glycoside-bonded sugar chain reducing end obtained by the above method, for example, the sequence The base sequence of number 19 is mentioned.
[0081] 染色体上の標的遺伝子を相同糸且換えするためのターゲットベクターは、 Gene Targ eting, A Practical Approach, IRLPress at Oxford University Press (1993)、ノヽィォマ -ュアルシリーズ 8ジーンターゲッティング， ES細胞を用いた変異マウスの作製 (羊 土社) (1995)等に記載の方法にしたがって作製することができる。ターゲットベクター は、リプレースメント型、インサーシヨン型いずれでも用いることができる。  [0081] As a target vector for homologous exchange of target genes on chromosomes, Gene Targ eting, A Practical Approach, IRLPress at Oxford University Press (1993), Noio-Muual 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.
[0082] 各種宿主細胞へのターゲットベクターの導入には、後述の 3に記載の各種宿主細 胞に適した組換えベクターの導入方法を用いることができる。  [0082] For introducing the target vector into various host cells, the method for introducing a recombinant vector suitable for various host cells described in 3 below can be used.
相同組換え体を効率的に選別する方法として、例えば、 Gene Targeting, A Practic al Approach, IRL Press at Oxford University Press (1993)、バイオマ-ユアノレシリー ズ 8ジーンターゲッティング， ES細胞を用いた変異マウスの作製 (羊土社) (1995)等に 記載のポジティブ選択、プロモーター選択、ネガティブ選択、ポリ A選択などの方法を 用いることができる。選別した細胞株の中から目的とする相同組換え体を選択する方 法としては、ゲノム DNAに対するサザンハイブリダィゼーシヨン法（モレキユラ一'クロ 一-ング第 2版）や PCR法 [ピーシーアール 'プロトコールズ (PCR Protocols), Academ ic Press (1990)]等があげられる。  Examples of methods for efficiently selecting homologous recombinants include Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Bio-European Series 8 Gene Targeting, Production of mutant mice using ES cells Methods such as positive selection, promoter selection, negative selection, poly A selection described in (Yodosha) (1995) and the like 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)].
[0083] (c) RDO方法による本発明のハプトグロビン組成物を作製するために用いる宿主細 胞の作製  [0083] (c) Preparation of host cells used for preparing the haptoglobin composition of the present invention by the RDO method
本発明のハプトグロビン組成物を作製するために用いる宿主細胞は、フコース修飾 に関連する酵素の遺伝子を標的とし、 RDO法を用い、例えば、以下のように作製する ことができる。  The host cell used for preparing the haptoglobin composition of the present invention can be prepared as follows, for example, by targeting the gene of an enzyme related to fucose modification and using the RDO method.
[0084] フコース修飾に関連する酵素の cDNAあるいはゲノム DNAを上記 1の（1)の（a)に記 載の方法を用い、調製する。  [0084] cDNA or genomic DNA of an enzyme related to fucose modification is prepared using the method described in 1 (1) (a) above.
調製した cDNAあるいはゲノム DNAの塩基配列を決定する。  Determine the base sequence of the prepared cDNA or genomic DNA.
決定した DNAの配列に基づき、フコース修飾に関連する酵素をコードする部分、非 翻訳領域の部分あるいはイントロン部分を含む適当な長さの RDOのコンストラクトを設 計し合成する。 Based on the determined DNA sequence, the portion encoding the enzyme related to fucose modification, non- Design and synthesize an RDO construct of appropriate length including the translation region or intron.
[0085] 合成した RDOを宿主細胞に導入し、標的とした酵素、すなわちフコース修飾に関連 する  [0085] The synthesized RDO is introduced into the host cell and related to the targeted enzyme, ie, fucose modification.
酵素に変異が生じた形質転換体を選択することにより、本発明のハプトグロビン組成 物作製のための宿主細胞を作製することができる。  By selecting a transformant having a mutation in the enzyme, a host cell for preparing the haptoglobin composition of the present invention can be prepared.
宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、標的とするフコース 修飾に関連する酵素の遺伝子を有して 、るものであれば 、ずれも用いることができる As a host cell, a yeast cell, an animal cell, an insect cell, a plant cell, etc. can be used as long as it has a gene for an enzyme related to the target fucose modification.
。具体的には、後述 3に記載の宿主細胞があげられる。 . Specifically, the host cells described in 3 below can be mentioned.
[0086] 各種宿主細胞への RDOの導入には、後述 3に記載の各種宿主細胞に適した組換 えベクターの導入方法を用いることができる。 フコース修飾に関連する酵素の cDNAを調製する方法としては、例えば、上記 1の（ 1)の（a)に記載の cDNAの調製方法などがあげられる。 [0086] For introduction of RDO into various host cells, the recombinant vector introduction method suitable for various host cells described in 3 below can be used. Examples of the method for preparing cDNA of the enzyme related to fucose modification include the method for preparing cDNA described in (1) (a) of 1 above.
フコース修飾に関連する酵素のゲノム DNAを調製する方法としては、例えば、上記 1の（1)の（a)に記載のゲノム DNAの調製方法などがあげられる。  Examples of a 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.
[0087] DNAの塩基配列は、適当な制限酵素などで切断後、 pBluescript SK (-) (Stratagene 社製)等のプラスミドにサブクローユングし、通常用いられる塩基配列解析方法、例え ば、サンガー（Sanger)らのジデォキシ法 [プロシーディングス ·ォブ ·ザ ·ナショナル · ァカデミ一'ォブ 'サイエンス (Proc.Natl. Acad. Sci.,U.S.A.), 74, 5463 (1977)]等の反 応を行い、塩基配列自動分析装置、例えば、 ABI PRISM377DNAシークェンサ一（Ap plied Biosystems社製)等の塩基配列分析装置を用いて分析することにより、確認す ることがでさる。 [0087] 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).
[0088] RDOは、常法または DNA合成機を用いることにより調製することができる。  [0088] RDO can be prepared by a conventional method or using a DNA synthesizer.
RDOを宿主細胞に導入し、標的とした酵素、フコース修飾に関連する酵素の遺伝 子に変異が生じた細胞を選択する方法としては、モレキュラー 'クローユング第 2版、 カレント.プロトコールズ.イン.モレキュラー.バイオロジー等に記載された染色体上の 遺伝子の変異を直接検出する方法があげられる。  As a method of introducing RDO into host cells and selecting cells in which the genes of the target enzyme and the enzyme related to fucose modification have been mutated, Molecular 'Crowing 2nd Edition, Current Protocols in Molecular A method for directly detecting gene mutations on chromosomes described in biology and the like can be mentioned.
[0089] また、前記 1の（1)の（a)に記載の、導入したフコース修飾に関連する酵素の活性 を指標として形質転換体を選択する方法、後述 1の（5)に記載の細胞膜上の糖蛋白 質の糖鎖構造を指標として形質転換体を選択する方法、あるいは、後述 5または後 述 6に記載の産生糖蛋白質分子の糖鎖構造を指標として形質転換体を選択する方 法も用 、ることができる。 [0089] The activity of the enzyme related to the introduced fucose modification described in (1) (a) of 1 above The method of selecting a transformant using as an index, the method of selecting a transformant using the sugar chain structure of a glycoprotein on a cell membrane as described in (5) of 1 below, or the method described in 5 or 6 below A method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule as an index can also be used.
[0090] RDOのコンストラクトは、サイエンス (Science), 273, 1386 (1996);ネィチヤ一'メディ シン (Nature Medicine) ,4, 285 (1998);へパトロジー (Hepatology), 25, 1462 (1997);ジ ーン'セラピー (Gene Therapy), 5, 1960 (1999);ジーン'セラピー (Gene Therapy), 5, 1960 (1999);ジャーナル'ォブ 'モレキユラ一'メデイシン (J. Mol. Med.), 75,829 (1997) ；プロシーデイングス'ォブ ·ザ'ナショナル ·アカデミ^ ~ ·ォブ 'サイエンス (Proc. Natl. Acad. Sci. USA), 96, 8774 (1999);プロシーデイングス'ォブ 'ザ ·ナショナル 'ァ力デミ ~ ·ォブ'サイエンス (Proc. Natl. Acad. Sci. USA), 96, 8768(1999);ヌクレイック 'ァシ ッド 'リサーチ (Nuc. Acids. Res.), 27, 1323 (1999);インべスティゲーシヨン'ォブ 'ダー マトロジー (Invest.Dematol.), m, 1172 (1998);ネイチヤ^ ~ ·バイオテクノロジー (Natur e Biotech.), 16,1343 (1998);ネイチヤ^ ~ ·バイオテクノロジー (Nature Biotech.), 18, 4 3 (2000);ネィチヤ一'バイオテクノロジー (Nature Biotech.), 18, 555 (2000)等の記載 に従って設計することができる。  [0090] RDO constructs are described in Science, 273, 1386 (1996); Nichiya's Medicine (Nature Medicine), 4, 285 (1998); Hepatology, 25, 1462 (1997); Gene 'Therapies (Gene Therapy), 5, 1960 (1999); Gene' Therapies (Gene Therapy), 5, 1960 (1999); Journal 'Ob' Molekiyura 'Medellin (J. Mol. Med.), 75,829 (1997); Procedures' Ob The National 'Academy ^ ~ · Ob' Science (Proc. Natl. Acad. Sci. USA), 96, 8774 (1999); The National 'A Power Demi-Ob' Science (Proc. Natl. Acad. Sci. USA), 96, 8768 (1999); Nuclidec 'Acid' Research (Nuc. Acids. Res.), 27 , 1323 (1999); Investigation 'Ob' Der Matology (Invest.Dematol.), M, 1172 (1998); Nature e Biotech., 16,1343 (1998) ; Nechiya ^ ~ · Biotechnology (Nature Biotech.), 18, 4 3 (2000); Nature's Biotech., 18, 555 (2000) and the like.
[0091] (d) RNAi法による本発明のハプトグロビン組成物を作製するために用いる宿主細 胞の作製  [0091] (d) Preparation of host cells used for preparing the haptoglobin composition of the present invention by RNAi method
本発明のハプトグロビン組成物を作製するために用いる宿主細胞は、フコース修飾 に関連する酵素の遺伝子を標的とし、 RNAi法を用い、例えば、以下のように作製す ることがでさる。  The host cell used for preparing the haptoglobin composition of the present invention can be prepared as follows, for example, by targeting the gene of an enzyme related to fucose modification and using the RNAi method.
[0092] フコース修飾に関連する酵素の上記 1の（1)の（a)に記載の方法を用い、 cDNAを 調製する。  [0092] cDNA is prepared using the method described in (1) (a) of 1 above of an enzyme related to fucose modification.
調製した cDNAの塩基配列を決定する。  Determine the base sequence of the prepared cDNA.
決定した cDNAの配列に基づき、フコース修飾に関連する酵素をコードする部分あ るいは非翻訳領域の部分を含む適当な長さの RNAi遺伝子のコンストラクトを設計す る。  Based on the determined cDNA sequence, an RNAi gene construct of an appropriate length that includes the enzyme coding for fucose modification or the untranslated region is designed.
[0093] 該 RNAi遺伝子を細胞内で発現させるために、調製した cDNAの断片、または全長 を適当な発現ベクターのプロモーターの下流に挿入することにより、糸且換えベクター を作製する。 [0093] In order to express the RNAi gene in a cell, a prepared cDNA fragment or full length Is inserted downstream of the promoter of an appropriate expression vector to prepare a thread-replacement vector.
該組換えベクターを、該発現べクタ一に適合した宿主細胞に導入することにより形 質転換体を得る。  A transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
導入したフコース修飾に関連する酵素の活性、あるいは産生糖蛋白質分子または 細胞表面上の糖蛋白質の糖鎖構造を指標に形質転換体を選択することで、本発明 のハプログロビン組成物を作製するために用いる宿主細胞を得ることができる。  In order to prepare the haploglobin composition of the present invention by selecting a transformant using as an index the activity of the enzyme related to the introduced fucose modification, the glycoprotein molecule produced or the sugar chain structure of the glycoprotein on the cell surface. The host cell to be used can be obtained.
[0094] 宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、標的とするフコース 修飾に関連する酵素の遺伝子を有して 、るものであれば 、ずれも用いることができる [0094] As the host cell, any yeast cell, animal cell, insect cell, plant cell, etc. having an enzyme gene related to the target fucose modification can be used.
。具体的には、後述 3に記載の宿主細胞があげられる。 . Specifically, the host cells described in 3 below can be mentioned.
発現ベクターとしては、上記宿主細胞において自立複製可能ないしは染色体への 組み込みが可能で、設計した RNAi遺伝子を転写できる位置にプロモーターを含有し ているものが用いられる。具体的には、後述 3に記載の発現ベクターがあげられる。  As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where the designed RNAi gene can be transcribed is used. Specifically, the expression vector described in 3 below can be mentioned.
[0095] 各種宿主細胞への遺伝子の導入には、後述 3に記載の各種宿主細胞に適した組 換えベクターの導入方法を用いることができる。 [0095] For introduction of genes into various host cells, the method for introducing recombinant vectors suitable for various host cells described in 3 below can be used.
フコース修飾に関連する酵素の活性を指標として形質転換体を選択する方法とし ては、例えば、本項 1の（1)の（a)に記載の方法があげられる。  Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the method described in (a) of (1) in this section 1.
細胞膜上の糖蛋白質の糖鎖構造を指標として形質転換体を選択する方法としては As a method of selecting transformants using the sugar chain structure of glycoprotein on the cell membrane as an index
、例えば、本項 1の（5)に記載の方法があげられる。産生糖蛋白質分子の糖鎖構造 を指標として形質転換体を選択する方法としては、例えば、後述 5または後述 6に記 載の方法があげられる。 For example, the method described in (5) of this item 1 can be mentioned. 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.
[0096] フコース修飾に関連する酵素の cDNAを調製する方法としては、例えば、本項 1の（[0096] As a method for preparing cDNA of an enzyme related to fucose modification, for example, (1) in this section
1)の（a)に記載された cDNAの調製方法などがあげられる。 Examples include the method for preparing cDNA described in (a) of 1).
また、発現ベクターを用いず、フコース修飾に関連する酵素の塩基配列に基づい て設計した RNAi遺伝子を、直接宿主細胞に導入することで、本発明のハプトグロビン 組成物を作製するために用いる宿主細胞を得ることもできる。  In addition, the host cell used for preparing the haptoglobin composition of the present invention can be obtained by directly introducing into the host cell the RNAi gene designed based on the base sequence of the enzyme related to fucose modification without using an expression vector. It can also be obtained.
[0097] RNAi遺伝子は、常法または DNA合成機を用いることにより調製することができる。 R[0097] The RNAi gene can be prepared by a conventional method or using a DNA synthesizer. R
NAi遺伝子のコンストラクトは、 [ネイチヤー (Nature), 391, 806 (1998);プロシーディン グス ·ォブ ·ザ'ナショナル 'ァ力デミ一 ·ォブ ·サイエンス (Proc.Natl. Acad. Sci. USA), 95, 15502 (1998);ネイチヤー (Nature), 395, 854(1998);プロシーディングス 'ォブ 'ザ •ナショナル 'ァ力デミ一 ·ォブ 'サイエンス (Proc. Natl. Acad. Sci. USA), 96, 5049 (19 99);セル (Cell), , 1017 (1998);プロシーデイングス'ォブ ·ザ'ナショナル 'ァ力デミ ~ ·ォブ'サイエンス (Proc. Natl. Acad. Sci. USA), 96, 1451 (1999);プロシーデイング ス'ォブ'ザ'ナショナル'ァカデミ一'ォブ 'サイエンス (Proc. Natl. Acad. Sci. USA), 9 5, 13959 (1998);ネィチヤ一'セル'バイオロジー (Nature Cell Biol), ^ 70 (2000)]等 の記載に従って設計することができる。 The construct of the NAi gene is [Nature, 391, 806 (1998); Gus Ob The National National Power Science (Proc.Natl. Acad. Sci. USA), 95, 15502 (1998); Nature, 395, 854 (1998); Proc Ding's 'Ob' The • National 'A Power Demi-Ob' Science (Proc. Natl. Acad. Sci. USA), 96, 5049 (19 99); Cell,, 1017 (1998); Proc Deings' Ob The National 'A Power Demi ~ Ob' Science (Proc. Natl. Acad. Sci. USA), 96, 1451 (1999); Proceedings' Ob 'The' National 'Academia Designed in accordance with the description of “One-Ob” Science (Proc. Natl. Acad. Sci. USA), 9 5, 13959 (1998); Nichiya “Cell” Biology (Nature Cell Biol), ^ 70 (2000)], etc. can do.
[0098] (e)トランスポゾンを用いた方法による、本発明のハプトグロビン組成物を作製する ために用いる宿主細胞の作製  [0098] (e) Production of host cells used for producing the haptoglobin composition of the present invention by a method using a transposon
本発明のハプトグロビン組成物を作製するために用いる宿主細胞は、ネィチャージエネテイク (Nature Genet.), 25, 35(2000)等に記載のトランスポゾンのシステムを用 い、フコース修飾に関連する酵素の活性、あるいは産生糖蛋白質分子または細胞膜 上の糖蛋白質の糖鎖構造を指標に突然変異体を選択することで、本発明のハプトグ ロビン組成物を作製するために用いる宿主細胞を作製することができる。  The host cell used for producing the haptoglobin composition of the present invention is a transposon system described in Nature Genet., 25, 35 (2000), etc. A host cell used for preparing the haptoglobin composition of the present invention can be prepared by selecting a mutant using as an index the activity or the glycoprotein structure of the glycoprotein molecule or glycoprotein on the cell membrane. .
[0099] トランスポゾンのシステムとは、外来遺伝子をランダムに染色体上に挿入させること で突然変異を誘発させるシステムであり、通常、トランスポゾンに挿まれた外来遺伝子 に突然変異を誘発させるベクターとして用い、この遺伝子を染色体上にランダムに挿 入させるためのトランスポゼースの発現ベクターを同時に細胞の中に導入する。 トランスポゼースは、用いるトランスポゾンの配列に適したものであれば!/、かなるもの ち用いることがでさる。  [0099] 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!
[0100] 外来遺伝子としては、宿主細胞の DNAに変異を誘起するものであればいかなる遺 伝子も  [0100] As a foreign gene, any gene can be used as long as it induces a mutation in the DNA of the host cell.
用!/、ることができる。  You can!
宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、標的とするフコース 修飾に関連する酵素の遺伝子を有して 、るものであれば 、ずれも用いることができる 。具体的には、後述 3に記載の宿主細胞があげられる。各種宿主細胞への遺伝子の 導入には、後述 3に記載の各種宿主細胞に適した組み換えベクターの導入方法を 用!/、ることができる。 As the host cell, any yeast cell, animal cell, insect cell, plant cell or the like having an enzyme gene related to the target fucose modification can be used. Specifically, the host cells described in 3 below can be mentioned. For the introduction of genes into various host cells, the method for introducing recombinant vectors suitable for various host cells described in 3 below is used. You can!
[0101] フコース修飾に関連する酵素の活性を指標として突然変異体を選択する方法とし ては、例えば、本項 1の（1)の（a)に記載の方法があげられる。  [0101] 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 (a) of (1) of this section 1.
細胞膜上の糖蛋白質の糖鎖構造を指標として突然変異体を選択する方法としては 、例えば、本項 1の（5)に記載の方法があげられる。産生糖蛋白質分子の糖鎖構造 を指標として突然変異体を選択する方法としては、例えば、後述 5または後述 6に記 載の方法があげられる。  Examples of a method for selecting a mutant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (5) of this section 1. Examples of the method for selecting a mutant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
[0102] (2)酵素の遺伝子のドミナントネガティブ体を導入する手法 [0102] (2) Techniques for introducing dominant negative forms of enzyme genes
本発明のハプトグロビン組成物を作製するために用いる宿主細胞は、フコース修飾 に関  The host cell used to produce the haptoglobin composition of the present invention is related to fucose modification.
連する酵素の遺伝子を標的とし、該酵素のドミナントネガティブ体を導入する手法を 用いることにより作製することができる。細胞内糖ヌクレオチド GDP-フコースの合成に 関与する酵素としては、具体的には、 GMD、 Fxなどがあげられる。 N-グリコシド結合 複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が a結合す る糖鎖修飾に関与する酵素としては、具体的には、 α ΐ, 6—フコシルトランスフェラ ーゼ、 α -L-フコシダーゼなどがあげられる。  It can be produced by using a method in which a gene of a linked enzyme is targeted and a dominant negative form of the enzyme is introduced. Specific examples of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx. N-glycoside bond As an enzyme involved in the sugar chain modification in which the 1-position of fucose is a-linked to the 6-position of the N-acetylyldarcosamine at the reducing end of the complex sugar chain, α α, 6— Examples include fucosyltransferase and α-L-fucosidase.
[0103] これらの酵素は、基質特異性を有したある特定の反応を触媒する酵素であり、この ような基質特異性を有した触媒作用を有する酵素の活性中心を破壊することで、これ らの酵素のドミナントネガティブ体を作製することができる。標的とする酵素のうち、 G[0103] These enzymes are enzymes that catalyze a specific reaction having substrate specificity, and these enzymes are destroyed by destroying the active center of the catalytic activity having substrate specificity. A dominant negative form of the enzyme can be prepared. G of target enzymes
MDを例として、そのドミナントネガティブ体に作製について具体的に以下に述べる。 大腸菌由来の GMDの立体構造を解析した結果、 4つのアミノ酸（133番目のトレオ- ン、 135番目のグルタミン酸、 157番目のチロシン、 161番目のリシン）が酵素活性に重 要な機能を担っていることが明らかにされている（Structure, 8, 2, 2000)。すなわち、 立体構造の情報にもとづきこれら 4つのアミノ酸を異なる他のアミノ酸に置換した変異 体を作製した結果、 、ずれの変異体にぉ ヽても有意に酵素活性が低下して 、たこと が示されている。一方、 GMDの補酵素 NADPや基質である GDP-マンノースとの結合 能に関しては、いずれの変異体においてもほとんど変化が観察されていない。従って 、 GMDの酵素活性を担うこれら 4つのアミノ酸を置換することによりドミナントネガティ ブ体を作製することができる。大腸菌由来の GMDのドミナントネガティブ体の作製の 結果に基づき、アミノ酸配列情 Taking MD as an example, the production of the dominant negative body will be specifically described below. As a result of analyzing the three-dimensional structure of GMD derived from E. coli, four amino acids (133th threonine, 135th glutamic acid, 157th tyrosine, 161th lysine) have important functions for enzyme activity. (Structure, 8, 2, 2000). That is, as a result of producing a mutant in which these four amino acids were substituted with other different amino acids based on the three-dimensional structure information, it was shown that the enzyme activity was significantly reduced even in the case of a deviation mutant. Has been. On the other hand, almost no change was observed in the GMD coenzyme NADP and its ability to bind to its substrate, GDP-mannose. Therefore, by substituting these four amino acids responsible for the enzyme activity of GMD, dominant negative Can be produced. Based on the results of the production of dominant negative GMD derived from E. coli, the amino acid sequence information
報をもとにした相同性比較や立体構造予測を行うことにより、例えば、 CHO細胞由来 の GMD (配列番号 8)では、 155番目のトレオニン、 157番目のグルタミン酸、 179番目 のチロシン、 183番目のリシンを他のアミノ酸に置換することによりドミナントネガティブ 体を作製することができる。このようなアミノ酸置換を導入した遺伝子の作製は、モレ キユラ ~ ·クロー-ング第 2版、カレント ·プロトコールズ 'イン ·モレキュラ^ ~ ·バイオロジ 一等に記載された部位特異的変異導入法を用いて行うことができる。  For example, in GMD derived from CHO cells (SEQ ID NO: 8), 155th threonine, 157th glutamic acid, 179th tyrosine, 183th By substituting lysine with another amino acid, a dominant negative can be prepared. Genes with such amino acid substitutions were prepared using site-directed mutagenesis described in Molecular and Cloning 2nd Edition, Current Protocols' In Molecular ^ ~ Biology etc. Can be done.
[0104] 本発明のハプトグロビン糸且成物を作製するために用いる宿主細胞は、上述のように 作製した標的酵素のドミナントネガティブ体をコードする遺伝子 (以下、ドミナントネガ ティブ体遺伝子と略記する）を用い、モレキュラー 'クローユング第 2版、カレント 'プロ トコーノレズ.イン .モレキュラー ·バイオロジー ·マ-ピュレーティング ·マウス ·ェンブリ ォ第 2版等に記載された遺伝子導入の方法に従って、例えば、以下のように作製す ることがでさる。 [0104] The host cell used to produce the haptoglobin filamentous product of the present invention contains a gene encoding a dominant negative form of the target enzyme produced as described above (hereinafter abbreviated as a dominant negative form gene). In accordance with the gene transfer method described in Molecular 'Crounging 2nd edition, Current' Protocoroles in.Molecular · Biology · Mapleating · Mouse · Embryo 2nd edition etc., for example, as follows: It can be made.
[0105] フコース修飾に関連する酵素のドミナントネガティブ体遺伝子を調製する。  [0105] A dominant negative gene of an enzyme related to fucose modification is prepared.
調製したドミナントネガティブ体遺伝子の全長 DNAをもとにして、必要に応じて、該 蛋白質をコードする部分を含む適当な長さの DNA断片を調製する。  Based on the prepared full length DNA of the dominant negative body gene, if necessary, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared.
該 DNA断片、または全長 DNAを適当な発現ベクターのプロモーターの下流に挿入 することにより、組換えベクターを作製する。  A recombinant vector is prepared by inserting the DNA fragment or full-length DNA downstream of the promoter of an appropriate expression vector.
[0106] 該組換えベクターを、該発現べクタ一に適合した宿主細胞に導入することにより、 形質転換体を得る。 [0106] A transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
フコース修飾に関連する酵素の活性、あるいは産生糖蛋白質分子または細胞膜上 の糖蛋白質の糖鎖構造を指標に形質転換体を選択することで、本発明のハプトグロ ビン組成物を作製するために用いる宿主細胞を作製することができる。  A host used for preparing the haptoglobin composition of the present invention by selecting a transformant using as an index the activity of an enzyme related to fucose modification, or the glycoprotein structure of a glycoprotein molecule or glycoprotein on a cell membrane. Cells can be made.
[0107] 宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、標的とするフコース 修飾に関連する酵素の遺伝子を有して 、るものであれば 、ずれも用いることができる 。具体的には、後述 3に記載の宿主細胞があげられる。 [0107] As the host cell, any yeast cell, animal cell, insect cell, plant cell, etc. having a gene for an enzyme related to the target fucose modification can be used. Specifically, the host cells described in 3 below can be mentioned.
発現ベクターとしては、上記宿主細胞において自立複製可能ないしは染色体中へ の^ aみ込みが可能で、目的とするドミナントネガティブ体をコードする DNAを転写でき る位置にプロモーターを含有しているものが用いられる。具体的には、後述 3に記載 の発現ベクターがあげられる。 As an expression vector, autonomous replication is possible in the above host cell or into the chromosome. The one containing a promoter at a position where the DNA encoding the target dominant negative can be transcribed is used. Specifically, the expression vector described in 3 below can be mentioned.
[0108] 各種宿主細胞への遺伝子の導入には、後述 3に記載の各種宿主細胞に適した組 換えベクターの導入方法を用いることができる。 [0108] For the introduction of genes into various host cells, the recombinant vector introduction method suitable for various host cells described in 3 below can be used.
フコース修飾に関連する酵素の活性を指標として形質転換体を選択する方法とし ては、例えば、後述 1の（1)の（a)に記載の方法があげられる。  Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the method described in (a) of (1) below.
細胞膜上の糖蛋白質の糖鎖構造を指標として形質転換体を選択する方法としては 、例えば、後述 1の（5)に記載の方法があげられる。産生糖蛋白質分子の糖鎖構造 を指標として形質転換体を選択する方法としては、例えば、後述 5または後述 6に記 載の方法があげられる。  Examples of the method for selecting a transformant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (1) below. Examples of the method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
[0109] (3)酵素に突然変異を導入する手法 [0109] (3) Methods for introducing mutations into enzymes
本発明のハプトグロビン組成物を作製するために用いる宿主細胞は、フコース修飾 に関連する酵素の遺伝子に突然変異を導入し、該酵素に突然変異を生じた所望の 細胞株を選択する手法を用いることにより作製できる。  The host cell used to prepare the haptoglobin composition of the present invention uses a technique in which a mutation is introduced into a gene of an enzyme related to fucose modification and a desired cell line in which the enzyme is mutated is selected. Can be produced.
細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素としては、具体的には、 GMD、 Fxなどがあげられる。 N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダ ルコサミンの 6位にフコースの 1位が α結合する糖鎖修飾に関与する酵素としては、 具体的には、 α ΐ, 6—フコシルトランスフェラーゼ、 α -L-フコシダーゼなどがあげら れる。 Specific examples of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx. Specific examples of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α- linked to the 6-position of N-glycidyl glucosamine at the N-glycoside-linked complex sugar chain reducing terminal include α ΐ, 6-fucosyltransferase, Examples include α-L-fucosidase.
[0110] フコース修飾に関連する酵素に突然変異を導入する方法としては、 1)突然変異誘 発処理で親株を処理した突然変異体あるいは自然発生的に生じた突然変異体から 、フコース修飾に関連する酵素の活性を指標として所望の細胞株を選択する方法、 2 )突然変異誘発処理で親株を処理した突然変異体あるいは自然発生的に生じた突 然変異体から、生産糖蛋白質分子の糖鎖構造を指標として所望の細胞株を選択す る方法、 3)突然変異誘発処理で親株を処理した突然変異体あるいは自然発生的に 生じた突然変異体から、該細胞の細胞膜上の糖蛋白質の糖鎖構造を指標として所 望の細胞株を選択する方法などがあげられる。 [0111] 突然変異誘発処理としては、親株の細胞の DNAに点突然変異、欠失ある!/、はフレ 一ムシ [0110] As a method for introducing a mutation into an enzyme related to fucose modification, 1) It is related to fucose modification from a mutant in which a parent strain was treated by a mutagenesis treatment or a naturally occurring mutant. 2) A method for selecting a desired cell line based on the activity of the enzyme to be produced, 2) a sugar chain of a production glycoprotein molecule from a mutant obtained by treating the parent strain by mutagenesis treatment or a spontaneously generated mutant. A method of selecting a desired cell line using the structure as an index; 3) a glycoprotein sugar on the cell membrane of a cell derived from a mutant obtained by treating a parent line by mutagenesis treatment or a naturally occurring mutant. Examples thereof include a method of selecting a desired cell line using the chain structure as an index. [0111] As the mutagenesis treatment, there are point mutations and deletions in the DNA of the parent cell line.
フト突然変異を誘起するものであればいかなる処理も用いることができる。  Any treatment that induces a mutation can be used.
具体的には、ェチルニトロソゥレア、ニトロソグァ-ジン、ベンゾピレン、アタリジン色 素による処理、放射線の照射などがあげられる。また、種々のアルキル化剤や発癌 物質も突然変異誘発物質として用いることができる。突然変異誘発物質を細胞に作 用させる方法としては、例えば、組織培養の技術第三版 (朝倉書店）日本組織培養 学会編 (1996)、ネィチヤ一'ジエネテイクス (Nature Genet.), 24, 314, (2000)等に記載 の方法を挙げることができる。  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.
[0112] 自然発生的に生じた突然変異体としては、特別な突然変異誘発処理を施さないで 、通常の細胞培養の条件で継代培養を続けることによって自然発生的に生じる突然 変異体を挙げることができる。 [0112] Spontaneously occurring mutants include sudden mutants that occur spontaneously by continuing subculture under normal cell culture conditions without special mutagenesis. be able to.
フコース修飾に関連する酵素の活性を測定する方法としては、例えば、本項 1の（1 As a method for measuring the activity of an enzyme related to fucose modification, for example, (1 in this section)
)の（a)に記載の方法があげられる。産生糖蛋白質分子の糖鎖構造を識別する方法 としては、例えば、後述 5または後述 6に記載の方法があげられる。細胞膜上の糖蛋 白質の糖鎖構造を識別する方法としては、例えば、本項の 1の（5)に記載の方法が あげられる。 ) Of (a). Examples of the method for identifying the sugar chain structure of the produced glycoprotein molecule include the methods described in 5 and 6 below. Examples of the method for identifying the sugar chain structure of a glycoprotein on the cell membrane include the method described in 1 (5) of this section.
[0113] (4)酵素の遺伝子の転写又は翻訳を抑制する手法  [0113] (4) Techniques for suppressing transcription or translation of enzyme genes
本発明のハプトグロビン組成物を作製するために用いる宿主細胞は、フコース修飾 に関連する酵素の遺伝子を標的とし、アンチセンス RNAZDNA技術 [バイオサイェン スとインダストリ一， 50, 322 (1992)、化学， 681 (1991)、 Biotechnology,9,358 (1992 )、 Trends in Biotechnology ,10, 87 (1992)、 Trends in Biotechnology,!^, 152 (1992)、 細胞工学， 16, 1463 (1997)]、トリプル 'ヘリックス技術 [Trends in Biotechnology ,10, 1 32 (1992)]等を用い、標的とする遺伝子の転写または翻訳を抑制することで作製する ことができる。  The host cell used to produce the haptoglobin composition of the present invention targets the gene of an enzyme related to fucose modification, and antisense RNAZDNA technology [Bioscience and Industry, 50, 322 (1992), Chemistry, 681 (1991), Biotechnology, 9,358 (1992), Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology,! ^, 152 (1992), Cell engineering, 16, 1463 (1997)], Triple helix technology [ Trends in Biotechnology, 10, 1 32 (1992)] or the like, and can be produced by suppressing transcription or translation of the target gene.
[0114] 細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素としては、具体的には、 GMD、 Fxなどがあげられる。 N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダ ルコサミンの 6位にフコースの 1位が α結合する糖鎖修飾に関与する酵素としては、 具体的には、 α ΐ , 6—フコシルトランスフェラーゼ、 α -L-フコシダーゼなどがあげら れる。 [0114] Specific examples of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx. As an enzyme involved in the sugar chain modification in which the 1-position of fucose is α- bonded to the 6-position of N-glycidylcolcamine at the N-glycosidic complex sugar chain reducing terminal Specific examples include αΐ, 6-fucosyltransferase, α-L-fucosidase, and the like.
(5) N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1 位が oc結合した糖鎖構造を認識するレクチンに耐性である株を選択する手法  (5) A method of selecting a strain resistant to a lectin that recognizes an oc-linked glycan structure in which the 6-position of N-glycidyl glycosamine at the N-glycoside-linked sugar chain reducing end and the 1-position of fucose are oc-linked
本発明のハプトグロビン組成物を作製するために用いる宿主細胞は、 N-グリコシド 結合糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1位がひ結合した糖 鎖構造を認識するレクチンに耐性である株を選択する手法を用いることにより作製す ることがでさる。  The host cell used to prepare the haptoglobin composition of the present invention is a lectin that recognizes a sugar chain structure in which the N-glycidyl-linked sugar chain reducing terminal N-acetylylcosamine 6-position and fucose 1-position are linked. It can be produced by using a technique for selecting strains that are resistant to.
[0115] N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1位が a結合した糖鎖構造を認識するレクチンに耐性である株を選択する手法としては、例 えば、ソマテイク'セル 'アンド'モレキュラ^ ~ ·ジエネテイクス（Somatic Cell Mol. Genet. ) , 12, 51 (1986)等に記載のレクチンを用いた方法があげられる。  [0115] As a technique for selecting a strain resistant to a lectin that recognizes a sugar chain structure in which the 6-position of N-glycidyl darcosamine at the reducing end of N-glycoside-linked sugar chain and the 1-position of fucose are a-linked For example, there is a method using a lectin described in Somatic Cell Mol. Genet., 12, 51 (1986).
レクチンとしては、 N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミンの 6位 とフコースの 1位が a結合した糖鎖構造を認識するレクチンであればいずれのレクチ ンでも用いることができる力 その具体的な例としては、レンズマメレクチン LCA (Lg Culinaris由来の Lentil Agglutinin)エンドゥマメレクチン PSA (Pisum sativum由来の Pe a Lectin)、ソラマメレクチン VFA (Viciafaba由来の Agglutinin)、ヒィロチャワンタケレク チン AAL (Aleuria aurantia由来の Lectin)等を挙げることができる。 As the lectin, any lectin can be used as long as it recognizes a sugar chain structure in which the N-glycidylcolcamine 6-position of the N-glycoside-linked sugar chain reducing end and the 1-position of fucose are a- linked. Specific examples include Lentil lectin LCA (Lentil Agglutinin derived from Lg Culinaris) Endumerme lectin PSA (Peum sativum derived Pe a Lectin), Broad bean lectin VFA (Agglutinin derived from Viciafaba), Hirochawantake lectin AAL ( Lectin from Aleuria aurantia).
[0116] 具体的には、 1 μ g/mL〜lmg/mLの濃度の上述のレクチンを含む培地で 1日〜2 週間、好ましくは 1日〜 1週間培養し、生存している細胞を継代培養あるいはコロニー をピックアップし別の培養容器に移し、さらに引き続きレクチンを含む培地で培養を 続けることによって、本発明の N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサ ミンの 6位とフコースの 1位が a結合した糖鎖構造を認識するレクチンに耐性である 株を選択することができる。  [0116] Specifically, the cells are cultured in a medium containing the above-mentioned lectin at a concentration of 1 μg / mL to 1 mg / mL for 1 to 2 weeks, preferably 1 to 1 week, and surviving cells are passed. Subculture or colonies are picked up, transferred to another culture vessel, and further cultured in a medium containing lectin, whereby the N-glycidyl-linked sugar chain reducing terminal N-acetylyldarcosamine of the present invention and position 1 of fucose A strain resistant to a lectin that recognizes an a-linked sugar chain structure can be selected.
[0117] 2.トランスジエニック非ヒト動物あるいは植物またはそれら子孫の作製  [0117] 2. Production of transgenic non-human animals or plants or their progeny
ハプトグロビン分子の糖鎖の修飾に係わる酵素の活性が制御されるようにゲノム遺 伝子が改変されたトランスジエニック非ヒト動物あるいは植物またはそれら子孫は、細 胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素または N-グリコシド結合複 合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が α結合する 糖鎖修飾に関与する酵素の遺伝子を標的として、前記 1を用いて作製した本発明の 胚性幹細胞、受精卵細胞、植物カルス細胞より、例えば以下のように作製することが できる。 Transgenic non-human animals, plants, or their progeny whose genomic genes have been modified so that the enzyme activity involved in the modification of the sugar chain of the haptoglobin molecule is controlled can synthesize intracellular sugar nucleotides GDP-fucose. Enzyme involved or N-glycoside bond complex The embryonic nature of the present invention produced using the above 1 targeting the gene of the enzyme involved in glycosylation in which the 1-position of fucose is α-linked to the 6-position of N-acetylyldarcosamine at the reducing end of the combined sugar chain For example, it can be prepared from stem cells, fertilized egg cells, and plant callus cells as follows.
[0118] トランスジヱニック非ヒト動物の場合、 目的とする非ヒト動物、例えばゥシ、ヒッジ、ャ ギ、ブタ、ゥマ、マウス、ラット、 -ヮトリ、サル、ゥサギ等の胚性幹細胞に、前記 1. に記 載の手法を用いることにより、細胞内糖ヌクレオチド GDP-フコースの合成に関与する 酵素の活性または Ν-グリコシド結合複合型糖鎖還元末端の Ν-ァセチルダルコサミン の 6位にフコースの 1位が a結合する糖鎖修飾に関与する酵素の活性が制御された 本発明の胚性幹細胞を作製することができる。  [0118] In the case of transgenic non-human animals, target non-human animals such as ushi, hidge, goats, pigs, horses, mice, rats, and embryonic stem cells such as rabbits, monkeys, and rabbits. By using the method described in 1. above, the activity of the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or the 6th position of 還 元 -acetylcyldarcosamine at the reducing end of Ν-glycoside-linked complex type sugar chain Thus, the embryonic stem cell of the present invention in which the activity of an enzyme involved in sugar chain modification in which position 1 of fucose is a-linked can be produced.
[0119] 具体的は、染色体上の細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素 の活性または N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6 位にフコースの 1位が a結合する糖鎖修飾に関与する酵素をコードする遺伝子を公 知の相同組換えの手法 [例えば、 Nature, 326, 6110, 295 (1987)、 Cell, 51, 3, 503 (1 987)等]により不活ィ匕または任意の配列と置換した変異クローンを作製する。作製し た胚性幹細胞 (例えば、該変異クローン）を用い、動物の受精卵の胚盤胞 (blastcyst) への注入キメラ法または集合キメラ法等の手法により、胚性幹細胞クローンと正常細 胞カ なるキメラ個体を調製することができる。このキメラ個体と正常個体の掛け合わ せにより、全身の細胞で細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素 の活性または N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6 位にフコースの 1位が a結合する糖鎖修飾に関与する酵素の活性が低下したトラン スジエニック非ヒト動物を得ることができる。 [0119] Specifically, the activity of the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose on the chromosome or N-acetylyldarcosamine at the 6th position of N-glycidyl-complexed sugar chain reducing terminal 1 of fucose Known methods for homologous recombination of genes encoding enzymes involved in sugar chain modification with a linkage [eg Nature, 326, 6110, 295 (1987), Cell, 51, 3, 503 (1 987) Etc.] to produce mutant clones in which inactive or arbitrary sequences are substituted. Using the prepared embryonic stem cells (for example, the mutant clones), embryonic stem cell clones and normal cell clones can be obtained by methods such as injection chimera or assembly chimera into fertilized eggs of animals. A chimeric individual can be prepared. By combining this chimeric individual with a normal individual, the activity of the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose in whole body cells or the N-glycidyl-linked N-acetylyldarcosamine 6 Transgenic non-human animals can be obtained in which the activity of the enzyme involved in sugar chain modification in which position 1 of fucose is a- linked is reduced.
[0120] また、 目的とする非ヒト動物、例えばゥシ、ヒッジ、ャギ、ブタ、ゥマ、マウス、ラット、二 ヮトリ、サル、ゥサギ等の受精卵細胞に、前記 1.に記載の手法と同様の手法を用いる ことにより、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素の活性または N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が oc結合する糖鎖修飾に関与する酵素の活性が低下した本発明の受精卵細胞 を作製することができる。 [0121] 作製した受精卵細胞を、マ-ピューレーティング'マウス'ェンブリオ第 2版等に記載 の胚移植の方法を用いて偽妊娠雌の卵管あるいは子宮に移植し出産させることで、 細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素の活性または N-グリコシ ド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位がひ 結合する糖鎖修飾に関与する酵素の活性が低下したトランスジエニック非ヒト動物を 作製することができる。 [0120] In addition, to the target non-human animal, for example, fertilized egg cells such as ushi, hidge, goat, pig, horse, mouse, rat, chicken, monkey, and rabbit, By using a similar method, the activity of the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or the 1-position of fucose at the 6-position of N-glycyl-linked N-acetylyldarcosamine at the N-glycoside-linked complex type sugar chain reducing end A fertilized egg cell of the present invention in which the activity of an enzyme involved in modification of an oc-linked sugar chain is reduced can be produced. [0121] The fertilized egg cells thus produced are transplanted into the oviduct or uterus of a pseudopregnant female using the embryo transfer method described in Mupureating 'Mouse' Embryo 2nd edition, etc. 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.
[0122] トランスジエニック植物の場合、 目的とする植物体力ルスまたは細胞に、前記 1.に 記載の手法と同様の手法を用いることにより、細胞内糖ヌクレオチド GDP-フコースの 合成に関与する酵素の活性または N-グリコシド結合複合型糖鎖還元末端の N-ァセ チルダルコサミンの 6位にフコースの 1位が a結合する糖鎖修飾に関与する酵素の 活性が低下した本発明のカルスを作製することができる。 [0122] In the case of a transgenic plant, the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose can be obtained by using the same method as described in 1. above on the target plant strength or cells. To produce a callus of the present invention in which the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is a- linked to the 6-position of the N-glycidalcosamine at the reducing end of the active or N-glycoside-linked complex type sugar chain is reduced Can do.
[0123] 作製したカルスを、公知の方法 [組織培養， 20 (1994);組織培養， 21 (1995);  [0123] The prepared callus was prepared by a known method [tissue culture, 20 (1994); tissue culture, 21 (1995);
トレンズ'イン'バイオテクノロジー (Trends in Biotechnology), 15, 45 (1997)]に準じて オーキシン及びサイトカイニンを含む培地で培養することで再分ィ匕させ、細胞内糖ヌ クレオチド GDP-フコースの合成に関与する酵素の活性または N-グリコシド結合複合 型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が a結合する糖 鎖修飾に関与する酵素の活性が低下したトランスジエニック植物を作製することがで きる。  According to Trends in Biotechnology, 15, 45 (1997)], the cells are cultured again in a medium containing auxin and cytokinin to synthesize intracellular sugar nucleotides GDP-fucose. Transgeneic with reduced activity of the enzyme involved or the activity of the N-glycoside-linked complex-type N-acetylyldarcosamine at the reducing end of the N-acetylyldarcosamine at the 6-position of the fucose and the enzyme involved in the glycosylation modification Plants can be produced.
[0124] 3.本発明のハプトグロビン組成物の製造方法  [0124] 3. Method for producing haptoglobin composition of the present invention
本発明のハプトグロビン組成物は、モレキュラー 'クローユング第 2版、カレント 'プロ トコーノレズ.イン.モレキュラー.ノィォロジ一、 Antibodies, ALaboratory manual, Cold Spring Harbor Laboratory, 1988 (以下、アンチボディズと略す）、 Monoclonal Antibod ies:pnnciples and practice, Third Edition, Acaa. Press, 1993 ( 下、モノクロ ~~ナノレ アンチボディズと略す)、 Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996 (以下、アンチボディエンジニアリングと略す）等に記 載された方法を用い、例えば、以下のように宿主細胞中で発現させて取得することが できる。  The haptoglobin composition of the present invention is a molecular 'cloning second edition, current' protocorores in molecular neurology, Antibodies, ALaboratory manual, Cold Spring Harbor Laboratory, 1988 (hereinafter abbreviated as antibodies), Monoclonal Antibod ies: pnnciples and practice, Third Edition, Acaa.Press, 1993 (below, Monochrome ~~ Nanore Antibodys), Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996 (hereinafter referred to as Antibody Engineering) For example, it can be obtained by expressing in a host cell as follows.
[0125] HP-1型または HP-2型ハプトグロビン分子の全長 cDNAを調製し、該ハプトグロビン 分子をコードする部分を含む適当な長さの DNA断片を調製する。 [0125] A full-length cDNA of HP-1 type or HP-2 type haptoglobin molecule was prepared, and the haptoglobin was prepared. Prepare an appropriate length of DNA fragment containing the molecule-encoding portion.
該 DNA断片、または全長 cDNAを適当な発現ベクターのプロモーターの下流に揷 入することにより、組換えベクターを作製する。  A recombinant vector is prepared by inserting the DNA fragment or full-length cDNA into the downstream of the promoter of an appropriate expression vector.
該組換えベクターを、該発現べクタ一に適合した宿主細胞に導入することにより、 ハプトグロビン分子を生産する形質転換体を得ることができる。  A transformant producing a haptoglobin molecule can be obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
[0126] 宿主細胞としては、酵母、動物細胞、昆虫細胞、植物細胞等、目的とする遺伝子を 発現できるものであれば 、ずれも用いることができる。 [0126] As a host cell, any yeast cell, animal cell, insect cell, plant cell, etc. that can express the target gene can be used.
ハプトグロビン組成物に結合する N-グリコシド結合糖鎖の修飾に係わる酵素、すな わちフコース修飾に関連する酵素の活性が欠失した細胞を選択するか、または前述 Select an enzyme involved in the modification of the N-glycoside-linked sugar chain that binds to the haptoglobin composition, that is, a cell lacking the activity of the enzyme related to fucose modification, or the aforementioned
1に示された種々の人為的手法により得られた細胞を宿主細胞として用いることもで きる。 Cells obtained by various artificial techniques shown in 1 can also be used as host cells.
[0127] 発現ベクターとしては、上記宿主細胞において自立複製可能ないしは染色体中へ の組込が可能で、目的とするハプトグロビン分子をコードする DNAを転写できる位置 にプロモーターを含有して 、るものが用いられる。  [0127] As an expression vector, one that can replicate autonomously in the above host cell or can be integrated into a chromosome and contains a promoter at a position where the DNA encoding the target haptoglobin molecule can be transcribed is used. It is done.
cDNAは、前記 1.の（1)の（a)に記載の cDNAの調製方法に従い、ヒト又は非ヒト動 物の糸且織又は細胞より、目的とするハプトグロビン分子に特異的なプローブやプライ マー等を用いて調製することができる。  According to the cDNA preparation method described in 1. (1) (a) above, the cDNA is obtained from a human or non-human animal thread or tissue or a cell-specific probe or primer. Etc. can be used.
[0128] 酵母を宿主細胞として用いる場合には、発現ベクターとして、例えば、 YEP13 (ATC C37115)、 YEp24 (ATCC37051)、 YCp50 (ATCC37419)等をあげることができる。 プロモーターとしては、酵母菌株中で発現できるものであればいずれのものを用い てもよく、例えば、へキソースキナーゼ等の解糖系の遺伝子のプロモーター、 PH05 プロモーター、 PGKプロモーター、 GAPプロモーター、 ADHプロモーター、 gallプロモ 一ター、 gal 10プロモーター、ヒートショックタンパク質プロモーター、 MF a lプロモー ター、 CUP 1プロモーター等をあげることができる。  [0128] When yeast is used as a host cell, examples of expression vectors 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.
[0129] 宿主細胞としては、サッカロミセス属、シゾサッカロミセス属、クリュイべ口ミセス属、ト リコスポロン属、シュヮ-ォミセス属等に属する微生物、例えば、 Saccharomvces cerev lsiae. Schizosaccharomvces pomoe. Kiuweromvces lactis. mchosporonpullulans、 ¾ chwanniomvces alluvius等をあげること力できる。 組換えベクターの導入方法としては、酵母に DNAを導入する方法であれば 、ずれ も用い [0129] Examples of host cells include microorganisms belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth Mrs, Trichosporon, Schusomyces, etc. You can power chwanniomvces alluvius. As a method for introducing the recombinant vector, if the method is to introduce DNA into yeast, the deviation is also used.
ることができ、例えば、エレクト口ポレーシヨン法 [メソッズ'イン'ェンザィモロジ一 (Met hods. Enzymol.),194, 182(1990)]、スフエロプラスト法 [プロシーディングス 'ォブ 'ザ' ナショナル 'ァ力デミ一 ·ォブ 'サイエンス (Proc. Natl. Acad. Sci. U.S.A), 84, 1929 (19 For example, the electo mouth position method [Met hods. Enzymol., 194, 182 (1990)], the spheroplast method [Proceedings 'Ob' The 'National' A Demiichi Obu 'Science (Proc. Natl. Acad. Sci. USA), 84, 1929 (19
78)]、
Figure imgf000048_0001
163 ( 1983)]、プロシーディングス ·ォブ ·ザ ·ナショナル ·ァカデミ一'ォブ ·サイエンス (Proc. Natl. Acad. Sci. U.S.A), 75, 1929 (1978)]に記載の方法等をあげることができる。 78)],
Figure imgf000048_0001
163 (1983)], and Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)]. Can do.
[0130] 動物細胞を宿主として用いる場合には、発現ベクターとして、例えば、 pcDNAU pcD M8 [0130] When an animal cell is used as a host, as an expression vector, for example, pcDNAU pcD M8
(フナコシ社より巿販）、 PAGE107 [特開平 3- 22979 ;サイトテクノロジー (Cytotechnolog y), 3, 133, (1990)]、 pAS3- 3 [特開平 2- 227075]、 pCDM8 [ネイチヤー (Nature), ， 8 40, (1987)]、 pcDNAI/Amp (Invitrogen社）、 pREP4 (Invitrogen社）、 pAGE103 [ジャー ナル ·ォブ 'バイオケミストリー 0. Biochemistry),皿， 1307 (1987)]、 pAGE210等をあ げることができる。 (Purchased from Funakoshi), P AGE107 [JP 3-22979; Cytotechnolog y, 3, 133, (1990)], pAS3-3 [JP 2-27075], pCDM8 [Nature ,, 8 40, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 [Journal of Biochemistry 0. Biochemistry), Dish, 1307 (1987)], pAGE210, etc. I can raise it.
[0131] プロモーターとしては、動物細胞中で発現できるものであればいずれも用いることが でき、例えば、サイトメガロウィルス（CMV)の IE (immediateearly)遺伝子のプロモータ 一、 SV40の初期プロモーター、レトロゥイノレスのプロモーター、メタ口チォネインプロモ 一ター、ヒートショックプロモーター、 SR aプロモーター等をあげることができる。また、 ヒト CMVの IE遺伝子のェンハンサーをプロモーターと共に用いてもよ!、。  [0131] Any promoter can be used as long as it can be expressed in animal cells. For example, a promoter of the cytomegalovirus (CMV) IE (immediateearly) gene, an early promoter of SV40, a retroinores promoter , Meta-mouthone promoter, heat shock promoter, SRa promoter, and the like. You can also use the human CMV IE gene enhancer with a promoter!
[0132] 宿主細胞としては、ヒトの細胞であるナマルバ（Namalwa)細胞、サルの細胞である C OS細胞、チャイニーズ'ノ、ムスターの細胞である CHO細胞、 HBT5637 (特開昭 63- 29 9)、ラットミエローマ細胞、マウスミエローマ細胞、シリアンノヽムスター腎臓由来細胞、 胚性幹細胞、受精卵細胞等をあげることができる。  [0132] As host cells, Namalwa cells, human cells, COS cells, monkey cells, CHO cells, Chinese cells, Muster cells, HBT5637 (Japanese Patent Laid-Open No. 63-299) Rat myeloma cells, mouse myeloma cells, Syrian omster kidney-derived cells, embryonic stem cells, fertilized egg cells, and the like.
組換えベクターの導入方法としては、動物細胞に DNAを導入する方法であれば ヽ ずれも用いることができ、例えば、エレクト口ポレーシヨン法 [サイトテクノロジー (Cytote chnology), 3, 133 (1990)]、リン酸カルシウム法 [特開平 2-227075]、リポフエクシヨン 法 [プロシーディングス ·ォブ ·ザ ·ナショナル ·ァカデミ一'ォブ ·サイエンス (Pro Natl. Acad. Sci. U.S.A.), 84, 7413 (1987)]、インジェクション法 [マ-ピュレイティング'ザ' マウス .ェンブリオ.ァ ·ラボラトリー ·マニュアル]、パーティクルガン (遺伝子銃）を用い る方法 [特許第 2606856、特許第 2517813]、 DEAE-デキストラン法 [バイオマ-ユア ルシリーズ 4一遺伝子導入と発現 ·解析法 (羊土社)横田崇 ·新井賢一編 (1994)]、ゥ ィルスべクタ一法 [マ-ピュレーティング ·マウス ·ェンブリオ第 2版]等をあげることがで きる。 As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into animal cells. For example, the electoral position method [Cytote chnology, 3, 133 (1990)], Calcium phosphate method [Japanese Patent Laid-open No. 2-227075], Lipofection method [Proceedings of the National Academy of Science (Pro Natl. Acad. Sci. USA), 84, 7413 (1987)], injection method [Map of 'The Mouse' Embryo Laboratory Laboratory Manual], method using particle gun (gene gun) [Patent No. 2606856 , Patent No. 2517813], DEAE-Dextran Method [Bio-Year Series 4 Gene Introduction and Expression Method (Yodosha) Takashi Yokota, Kenichi Arai (1994)], Virus Vector Method [M- Such as Pulating, Mouse, Embryo Second Edition].
[0133] 昆虫細胞を宿主として用いる場合には、例えばカレント 'プロトコールズ'イン'モレ =Τュフ ~~ 'ノヽィォロン ~~ Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992)、ノ ィォ /ァクノロン ~~ (Bio/ Technology) , 6, 47 (1988)等に記載された方法によって、タンパク質を発現することができる。  [0133] When insect cells are used as a host, for example, the current 'protocols' in 'mole = フ üuf ~~' noyonlon ~~ Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992 ), Nano / Acnolone ~ (Bio / Technology), 6, 47 (1988), etc., can be used to express the protein.
即ち、組換え遺伝子導入ベクターおよびバキュロウィルスを昆虫細胞に共導入して 昆虫細胞培養上清中に組換えウィルスを得た後、さらに組換えウィルスを昆虫細胞 に感染させ、タンパク質を発現させることができる。  That is, the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the protein. it can.
[0134] 該方法において用いられる遺伝子導入べクタ一としては、例えば、 pVL1392、 pVLl 393、 pBlueBacIII (ともに Invitorogen社）等をあげることができる。  [0134] Examples of the gene transfer vector used in the method include pVL1392, pVLl393, pBlueBacIII (both Invitorogen) and the like.
バキュロウィルスとしては、例えば、夜盗蛾科昆虫に感染するウィルスであるアウトグ ラファ 'カリフオル-力'ヌクレア一'ポリへドロシス'ウィルス (Autographa californica nu clear polyhedrosis virusノ等 用 ヽ oこと力できる。  As a baculovirus, for example, Autographa californica nu clear polyhedrosis virus can be used for the outgrafa 'Califorum-force' Nuclea 1 'polyhedrosis' virus, which is a virus that infects the night stealing insects.
[0135] 昆虫細胞としては、 Spodoptera frugiperdaの卵巣細胞である S19、 S1 1 [カレント'プ 口トコ一ノレズ 'イン'モレキュフー'ノ ィォロシ一 Baculovirus Expression Vectors, A La boratory Manual, W.H. Freeman and Company, New York (1992)]、 Trichoplusianiの 卵巣細胞である High 5 (Invitrogen社)等を用いることができる。  [0135] Insect cells include ovarian cells of Spodoptera frugiperda, S19, S1 1 [Current 'Pokoto Reno' In 'Molechu Fu'Noroshi 1 Baculovirus Expression Vectors, A La boratory Manual, WH Freeman and Company, New York (1992)], Trichoplusiani ovary cells such as High 5 (Invitrogen), etc. can be used.
組換えウィルスを調製するための、昆虫細胞への上記組換え遺伝子導入ベクター と上記バキュロウィルスの共導入方法としては、例えば、リン酸カルシウム法 (特開平 2 -227075)、リポフエクシヨン法 [プロシーディングス 'ォブ ·ザ'ナショナル'ァカデミ一' ォブ ·サイエンス (Pro Natl. Acad. Sci. U.S.A.), 84, 7413 (1987)]等をあげることがで きる。  Examples of methods for co-introducing the above recombinant gene transfer vector and the above baculovirus into insect cells for preparing recombinant viruses include, for example, the calcium phosphate method (JP-A-2-227075), the lipofusion method [Proceedings' · The 'National' Academia's Science (Pro Natl. Acad. Sci. USA), 84, 7413 (1987)].
[0136] 植物細胞を宿主細胞として用いる場合には、発現ベクターとして、例えば、 Tiプラス ミド、タバコモザイクウィルスベクター等をあげることができる。 [0136] When plant cells are used as host cells, examples of expression vectors include Ti plus Examples include midos and tobacco mosaic virus vectors.
プロモーターとしては、植物細胞中で発現できるものであればいずれのものを用い てもよく、例えば、カリフラワーモザイクウィルス（CaMV)の 35Sプロモーター、イネァク チン 1プロモーター等をあげることができる。  Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and the rice 1 promoter.
[0137] 宿主細胞としては、タバコ、ジャガイモ、トマト、ニンジン、ダイズ、アブラナ、アルファ ルファ、イネ、コムギ、ォォムギ等の植物細胞等をあげることができる。 [0137] Examples of host cells include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat and barley.
組換えベクターの導入方法としては、植物細胞に DNAを導入する方法であれば ヽ ずれも用いることができ、例えば、ァグロバタテリゥム (Agrobacterium) [特開昭 59- 140 As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into plant cells. For example, Agrobacterium [April 59-140]
885、特開昭 60-70080、 WO94/00977] ,エレクト口ポレーシヨン法 [特開昭 60-251887885, Japanese Patent Laid-Open No. 60-70080, WO94 / 00977, Elect Mouth Position Method [Japanese Patent Laid-Open No. 60-251887]
]、パーティクルガン (遺伝子銃）を用いる方法 [日本特許第 2606856、 日本特許第 25], Method using particle gun (gene gun) [Japanese Patent No. 2606856, Japanese Patent No. 25
17813]等をあげることができる。 17813] and the like.
[0138] 遺伝子の発現方法としては、直接発現以外に、モレキュラー ·クロー-ング第 2版に 記載されている方法等に準じて、分泌生産等を行うことができる。 [0138] As a gene expression method, in addition to direct expression, secretory production or the like can be performed according to the method described in Molecular Cloning 2nd edition.
糖鎖の合成に関与する遺伝子を導入した、酵母、動物細胞、昆虫細胞または植物 細胞により発現させた場合には、導入した遺伝子によって糖あるいは糖鎖が付加さ れたノ、ブトグロビン分子を得ることができる。  When expressed in yeast, animal cells, insect cells, or plant cells into which a gene involved in sugar chain synthesis has been introduced, obtain a butoglobin molecule to which a sugar or sugar chain has been added by the introduced gene. Can do.
[0139] 以上のようにして得られる形質転換体を培地に培養し、培養物中にハプトグロビン 分子を生成蓄積させ、該培養物から採取することにより、ハプトグロビン組成物を製 造することができる。形質転換体を培地に培養する方法は、宿主細胞の培養に用い られる通常の方法に従って行うことができる。 [0139] The haptoglobin composition can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating haptoglobin molecules in the culture, and collecting the haptoglobin from the culture. The method of culturing the transformant in a medium can be performed according to a usual method used for culturing host cells.
酵母等の真核生物を宿主として得られた形質転換体を培養する培地としては、該 生物が資化し得る炭素源、窒素源、無機塩類等を含有し、形質転換体の培養を効率 的に行える培地であれば天然培地、合成培地の ヽずれを用いてもょ ヽ。  As a medium for culturing a transformant obtained by using a eukaryote such as yeast as a host, it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism, so that the transformant can be cultured efficiently. If the medium can be used, the difference between natural and synthetic media can be used.
[0140] 炭素源としては、該生物が資化し得るものであればよぐグルコース、フラクトース、 スクロース、これらを含有する糖蜜、デンプンあるいはデンプン加水分解物等の炭水 化物、酢酸、プロピオン酸等の有機酸、エタノール、プロパノールなどのアルコール 類等を用いることができる。 [0140] As the carbon source, as long as the organism can assimilate, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolyzate, acetic acid, propionic acid, etc. Alcohols such as organic acids, ethanol, and propanol can be used.
窒素源としては、アンモニア、塩化アンモ-ゥム、硫酸アンモ-ゥム、酢酸アンモ- ゥム、リン酸アンモ-ゥム等の無機酸もしくは有機酸のアンモ-ゥム塩、その他の含窒 素化合物、ならびに、ペプトン、肉エキス、酵母エキス、コーンスチープリカー、カゼィ ン加水分解物、大豆粕および大豆粕加水分解物、各種発酵菌体およびその消化物 等を用いることができる。 Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, and ammonium acetate. Ammonium salts of inorganic or organic acids such as humic and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, Soybean koji and soybean koji hydrolyzate, various fermented cells and digested products thereof can be used.
[0141] 無機塩類としては、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫 酸マグネシウム、塩ィ匕ナトリウム、硫酸第一鉄、硫酸マン癌、硫酸銅、炭酸カルシウム 等を用いることができる。  [0141] As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride salt, ferrous sulfate, mangan sulfate, copper sulfate, calcium carbonate, etc. are used. be able to.
培養は、通常振盪培養または深部通気攪拌培養などの好気的条件下で行う。培養 温度は 15〜40°Cがよぐ培養時間は、通常 16時間〜 7日間である。培養中の pHは 3. 0〜9. 0に保持する。 pHの調製は、無機または有機の酸、アルカリ溶液、尿素、 炭酸カルシウム、アンモニアなどを用いて行う。  The culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is 15-40 ° C, and the culture time is usually 16 hours to 7 days. The pH during the culture is maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
[0142] また、培養中必要に応じて、アンピシリンやテトラサイクリン等の抗生物質を培地に 添カロしてちょい。  [0142] If necessary, supplement the medium with antibiotics such as ampicillin or tetracycline.
プロモーターとして誘導性のプロモーターを用いた糸且換えベクターで形質転換した 微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。 例えば、 lacプロモーターを用いた組換えベクターで形質転換した微生物を培養する ときにはイソプロピル- β -D-チォガラタトピラノシド等を、 trpプロモーターを用いた組 換えベクターで形質転換した微生物を培養するときにはインドールアクリル酸等を培 地に添カ卩してもよい。  When cultivating a microorganism transformed with a thread-replacement vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using the lac promoter, when cultivating a microorganism transformed with isopropyl-β-D-thiogalatatopyranoside or the like with a recombinant vector using the trp promoter. Indole acrylic acid or the like may be added to the culture medium.
[0143] 動物細胞を宿主として得られた形質転換体を培養する培地としては、一般に使用さ れて 、る RPMI 1640培地 [ザ ·ジャーナル ·ォブ ·ザ ·アメリカン'メディカル ·ァソシエイ シヨン (The Journal of the American Medical Association),塑， 519 (1967)]、 Eagleの MEM培地 [サイエンス (Science), 12^,501 (1952)]、ダルベッコ改変 MEM培地 [ヴユウ口 ロジー (Virology), 8, 396 (1959)]、 199培地 [プロシーデイング'ォブ 'ザ 'ソサイエティ 'フォア'ザ 'バイオロジカノレ'メディスン (Proceeding of the Society for the Biological Medicine), 73, 1 (1950)]、 Whitten培地 [発生工学実験マニュアル-トランスジエニック •マウスの作り方 (講談社)勝木元也編（1987)ほたはこれら培地に牛胎児血清等を 添加した培地等を用いることができる。 [0144] 培養は、通常 pH6〜8、 30〜40°C、 5%CO存在下等の条件下で 1〜7日間行う [0143] RPMI 1640 medium commonly used as a medium for culturing transformants obtained using animal cells as a host [The Journal of the American American Medical Association (The Journal of the American Medical Association), Plastic, 519 (1967)], Eagle's MEM medium [Science, 12 ^, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 ( 1959), 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], Whitten medium [Developmental engineering] Experiment Manual-Transgeneic • How to make mice (Kodansha) Motoya Katsaki (1987) Hota can use media such as fetal calf serum added to these media. [0144] Cultivation is usually carried out for 1 to 7 days under conditions of pH 6-8, 30-40 ° C, 5% CO, etc.
2 また、培養中必要に応じて、カナマイシン、ペニシリン等の抗生物質を培地に添カロ してちよい。  2 In addition, antibiotics such as kanamycin and penicillin may be added to the medium as needed during culture.
昆虫細胞を宿主として得られた形質転換体を培養する培地としては、一般に使用さ れている TNM-FH培地（Pharmingen社）、 Sf- 900 II SFM培地（Life Technologies社）、 ExCell400、 ExCell405 (いずれも JRH Biosciences社）、 Grace's Insect Medium [ネイチ ヤー (Nature),!^, 788 (1962)]等を用いることができる。  As a medium for culturing transformants obtained using insect cells as hosts, the commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCell400, ExCell405 (all JRH Biosciences), Grace's Insect Medium [Nature,! ^, 788 (1962)] and the like can also be used.
[0145] 培養は、通常 pH6〜7、 25〜30°C等の条件下で、 1〜5日間行う。 [0145] Cultivation is usually carried out under conditions of pH 6-7, 25-30 ° C, etc. for 1-5 days.
また、培養中必要に応じて、ゲンタマイシン等の抗生物質を培地に添加してもよい 植物細胞を宿主として得られた形質転換体は、細胞として、または植物の細胞や器 官に分化させて培養することができる。該形質転換体を培養する培地としては、一般 に使用されているムラシゲ'アンド'スターグ (MS)培地、ホワイト (White)培地、またはこ れら培地にオーキシン、サイトカイニン等、植物ホルモンを添カ卩した培地等を用いるこ とがでさる。  In addition, antibiotics such as gentamicin may be added to the medium as needed during the culture.Transformants obtained using plant cells as hosts are cultured as cells or differentiated into plant cells and organs. can do. As a medium for culturing the transformant, commonly used Murashige 'and' Stag (MS) medium, White medium, or a plant hormone such as auxin or cytokinin is added to these mediums. It is possible to use the prepared medium.
[0146] 培養は、通常 pH5〜9、 20〜40°Cの条件下で 3〜60日間行う。  [0146] The culture is usually carried out under conditions of pH 5-9 and 20-40 ° C for 3-60 days.
また、培養中必要に応じて、カナマイシン、ハイグロマイシン等の抗生物質を培地 に添カ卩してもよい。  In addition, antibiotics such as kanamycin and hygromycin may be added to the medium as needed during the culture.
上記のとおり、ハプトグロビン分子をコードする DNAを^ aみ込んだ糸且換え体ベクター を保有する微生物、動物細胞、あるいは植物細胞由来の形質転換体を、通常の培 養方法に従って培養し、ハプトグロビン組成物を生成蓄積させ、該培養物よりハプト グロビン組成物を採取することにより、ハプトグロビン組成物を製造することができる。  As described above, a transformant derived from a microorganism, animal cell, or plant cell having a thread-recombinant vector in which DNA encoding a haptoglobin molecule is incorporated is cultured according to a normal culture method, and a haptoglobin composition is obtained. A haptoglobin composition can be produced by producing and accumulating products and collecting the haptoglobin composition from the culture.
[0147] ハプトグロビン組成物の生産方法としては、宿主細胞内に生産させる方法、宿主細 胞外に分泌させる方法、あるいは宿主細胞外膜上に生産させる方法があり、使用す る宿主細胞や、生産させるハプトグロビン分子の構造を変えることにより、該方法を選 択することができる。 [0147] The haptoglobin 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 haptoglobin molecule to be generated.
ハプトグロビン組成物が宿主細胞内あるいは宿主細胞外膜上に生産される場合、 ポールソンらの方法 [ジャーナル ·ォブ 'バイオロジカル ·ケミストリー (J. Biol.Chem.), 2 64, 17619 (1989)]、ロウらの方法 [プロシーデイングス'ォブ 'ザ ·ナショナル 'ァ力デミ ~ ·ォブ'サイエンス (Proc. Natl. Acad. Sci. U.S.A.), 86, 8227 (1989);ジーン'デベロ ップメン KGenes Develop.), 4, 1288 (1990) ]、または特開平 05-336963、特開平 06-8 23021等に記載の方法を準用することにより、該ハブトグロビン組成物を宿主細胞外 に積極的に分泌させることができる。 When the haptoglobin composition is produced in or on the host cell outer membrane, Paulson et al. [Journal of 'Biological Chemistry, 2 64, 17619 (1989)], Law et al. [Procedures of' The National 'Demi-Ob'Science (Proc. Natl. Acad. Sci. USA), 86, 8227 (1989); Gene 'Development KGenes Develop.), 4, 1288 (1990)], or JP 05-336963, By applying the method described in JP-A-06-823021 etc., the hub globin composition can be actively secreted outside the host cell.
[0148] すなわち、遺伝子組換えの手法を用いて、発現ベクターに、ハプトグロビン分子を コードする DNA、およびハプトグロビン分子の発現に適切なシグナルペプチドをコー ドする DNAを挿入し、該発現ベクターを宿主細胞へ導入の後にハプトグロビン分子を 発現させること〖こより、 目的とするハプトグロビン分子を宿主細胞外に積極的に分泌さ せることができる。 [0148] That is, using genetic recombination techniques, DNA encoding a haptoglobin molecule and DNA encoding a signal peptide suitable for expression of the haptoglobin molecule are inserted into an expression vector, and the expression vector is inserted into a host cell. By expressing the haptoglobin molecule after introduction into the cell, the target haptoglobin molecule can be actively secreted outside the host cell.
[0149] また、特開平 2-227075に記載されている方法に準じて、ジヒドロ葉酸還元酵素遺伝 子等  [0149] Further, according to the method described in JP-A-2-27075, dihydrofolate reductase gene and the like
を用いた遺伝子増幅系を利用して生産量を上昇させることもできる。  The production amount can also be increased by using a gene amplification system using.
さらに、遺伝子導入した動物または植物の細胞を再分化させることにより、遺伝子 が導入された動物個体 (トランスジエニック非ヒト動物)または植物個体 (トランスジェ- ック植物）を造成し、これらの個体を用いてハプトグロビン組成物を製造することもでき る。  Furthermore, by redifferentiating the cells of the animal or plant into which the gene has been introduced, an animal individual (transgenic non-human animal) or a plant individual (transgenic plant) into which the gene has been introduced is created. A haptoglobin composition can also be produced using.
[0150] 形質転換体が動物個体または植物個体の場合は、通常の方法に従って、飼育また は栽培し、ハプトグロビン組成物を生成蓄積させ、該動物個体または植物個体より該 ハプトグロビン組成物を採取することにより、該ハブトグロビン組成物を製造すること ができる。  [0150] When the transformant is an animal individual or a plant individual, it is reared or cultivated according to a usual method, and a haptoglobin composition is produced and accumulated, and the haptoglobin composition is collected from the animal individual or plant individual. Thus, the hubtoglobin composition can be produced.
動物個体を用いてハプトグロビン組成物を製造する方法としては、例えば公知の方 法 [アメリカン 'ジャーナル'ォブ 'クリニカル'二ユートリシヨン (American Journal of Clin ical Nutrition), 63, 639S (1996);アメリカン ·ジャーナル ·ォブ 'タリ-カル ·二ユートリシ ヨン (American Journal of Clinical Nutrition), 63, 627S (1996);バイオ Zテクノロジー（ Bio/Technology), 9, 830 (1991)]に準じて遺伝子を導入して造成し  As a method for producing a haptoglobin composition using an animal individual, for example, a known method [American Journal of Clinical Nutrition, 63, 639S (1996); American Introduced gene according to Journal of Clinical Nutrition, 63, 627S (1996); Bio / Technology, 9, 830 (1991)] Created
た動物中に目的とするハプトグロビン組成物を生産させる方法があげられる。 [0151] 動物個体の場合は、例えば、ハプトグロビン分子をコードする DNAを導入したトラン スジエニック非ヒト動物^!司育し、ハプトグロビン組成物を該動物中に生成 ·蓄積させ 、該動物中よりハプトグロビン組成物を採取することにより、ハプトグロビン組成物を製 造することができる。該動物中の生成'蓄積場所としては、例えば、該動物のミルク（ 特開昭 63-309192)、卵等をあげることができる。この際に用いられるプロモーターとし ては、動物で発現できるものであればいずれも用いることができる力 例えば、乳腺 細胞特異的なプロモーターである aカゼインプロモーター、 13カゼインプロモーター 、 j8ラクトグロブリンプロモーター、ホエー酸 ¾プロテインプロモーター等が好適に用 いられる。 And a method for producing a target haptoglobin composition in an animal. [0151] In the case of an individual animal, for example, a transgenic non-human animal introduced with DNA encoding a haptoglobin molecule ^! Is generated and accumulated in the animal, and the haptoglobin composition is produced from the animal. A haptoglobin composition can be produced by collecting the product. Examples of the production / accumulation location in the animal include milk of the animal (Japanese Patent Laid-Open No. 63-309192) and eggs. Any promoter can be used as long as it can be expressed in animals. For example, a casein promoter, 13 casein promoter, j8 lactoglobulin promoter, whey acid, which are mammary cell specific promoters. (3) A protein promoter or the like is preferably used.
[0152] 植物個体を用いてハプトグロビン組成物を製造する方法としては、例えばノ、プトグ ロビン分子をコードする DNAを導入したトランスジエニック植物を公知の方法 [組織培 養, (1994);組織培養， 21(1995);トレンド'イン'バイオテクノロジー (Trends inBiotec hnology), 15, 45 (1997)]に準じて栽培し、ハプトグロビン組成物を該植物中に生成' 蓄積させ、該植物中より該ハブトグロビン組成物を採取することにより、ハプトグロビン 組成物を生産する方法があげられる。  [0152] As a method for producing a haptoglobin composition using an individual plant, for example, a known method for transgenic plants into which DNA encoding a ptoglobin molecule has been introduced [tissue culture, (1994); tissue culture] , 21 (1995); cultivated according to Trends “In” biotechnology (Trends in Biotec hnology), 15, 45 (1997)], and a haptoglobin composition is produced and accumulated in the plant. A method for producing a haptoglobin composition by collecting the composition can be mentioned.
[0153] ハプトグロビン分子をコードする遺伝子を導入した形質転換体により製造されたハ ブトグロビン組成物は、例えばノヽブトグロビン組成物力 細胞内に溶解状態で発現し た場合には、培養終了後、細胞を遠心分離により回収し、水系緩衝液にけん濁後、 超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー、ダイノミル等により 細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得ら れる上清から、通常の酵素の単離精製法、即ち、溶媒抽出法、硫安等による塩析法 、脱塩法、有機溶媒による沈殿法、ジェチルアミノエチル (DEAE) -セファロース、 DIA IONHPA-75 (三菱化学 (株)製)等レジンを用いた陰イオン交換クロマトグラフィー法、 ¾- Sepharose F (Pharmacia  [0153] For example, when a hub globin composition produced by a transformant introduced with a gene encoding a haptoglobin molecule is expressed in a dissolved state in cells, the cell is centrifuged after completion of the culture. Collected by separation, suspended in an aqueous buffer solution, and then disrupted with an ultrasonic crusher, French press, Manton Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resin such as Jetylaminoethyl (DEAE) -Sepharose, DIA IONHPA-75 (Mitsubishi Chemical Corporation), ¾- Sepharose F (Pharmacia
社)等のレジンを用いた陽イオン交換クロマトグラフィー法、ブチルセファロース、フエ 二ルセファロース等のレジンを用いた疎水性クロマトグラフィー法、分子篩を用いたゲ ルろ過法、ァフィユティークロマトグラフィー法、クロマトフォーカシング法、等電点電 気泳動等の電気泳動法等の手法を単独あるいは組み合わせて用い、ハプトグロビン 組成物の精製標品を得ることができる。具体的には、 PEG分画、硫安分画、イオン交 換クロマトグラフィー法を組み合わせた方法をあげることができる (続医薬品の開発 1Cation exchange chromatography using resins such as butyl sepharose, hydrophobic chromatography using resins such as phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, Use haptoglobin using methods such as chromatofocusing and electrophoresis such as isoelectric focusing alone or in combination. A purified preparation of the composition can be obtained. Specifically, a method combining PEG fractionation, ammonium sulfate fractionation, and ion-exchange chromatography can be mentioned (development of follow-up medicines 1
0,血液製剤， pp.215- 233.,廣川書店， 1992)。 0, Blood products, pp.215-233., Yodogawa Shoten, 1992).
[0154] また、ハプトグロビン組成物が細胞内に不溶体を形成して発現した場合は、同様に 細胞を回収後破砕し、遠心分離を行うことにより、沈殿画分としてハプトグロビン組成 物の不溶体を回収する。回収したハプトグロビン組成物の不溶体をタンパク質変性 剤で可溶化する。該可溶化液を希釈または透析することにより、該ハブトグロビン組 成物を正常な立体構造に戻した後、上記と同様の単離精製法により該ハブトグロビ ン組成物の精製標品を得ることができる。  [0154] When the haptoglobin composition is expressed by forming an insoluble substance in the cells, the cells are similarly collected, disrupted, and centrifuged to obtain an insoluble substance of the haptoglobin composition as a precipitate fraction. to recover. The recovered insoluble material of the haptoglobin composition is solubilized with a protein denaturant. After diluting or dialyzing the solubilized solution, the hubtoglobin composition is returned to a normal three-dimensional structure, and then a purified sample of the hubtoglobin composition can be obtained by the same isolation and purification method as described above. .
[0155] ハプトグロビン組成物が細胞外に分泌された場合には、培養上清に該ハブトグロビ ン組成物あるいはその誘導体を回収することができる。即ち、該培養物を上記と同様 の遠心分離等の手法により処理することにより培養上清を取得し、該培養上清から、 上記と同様の単離精製法を用いることにより、ハプトグロビン組成物の精製標品を得 ることがでさる。  [0155] When the haptoglobin composition is secreted extracellularly, the habutoglobin composition or a derivative thereof can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and the haptoglobin composition is obtained from the culture supernatant by using the same isolation and purification method as described above. It is possible to obtain a purified sample.
すでに宿主細胞がハプトグロビン分子を発現する能力を有する場合には、上記 1に 記載した方法を用いてハプトグロビン分子を発現する能力を有する細胞を調製した 後に、該細胞を培養し、該培養物から目的とするハプトグロビン組成物を精製するこ とにより、本発明のハプトグロビン組成物を製造することができる。  When the host cell already has the ability to express a haptoglobin molecule, after preparing the cell having the ability to express a haptoglobin molecule using the method described in 1 above, the cell is cultured, and the target cell is cultured from the culture. The haptoglobin composition of the present invention can be produced by purifying the haptoglobin composition.
[0156] 4.ハプトグロビン組成物の活性評価 [0156] 4. Activity evaluation of haptoglobin composition
精製したノ、ブトグロビン組成物の生物活性は、既に公知の各種方法を用いて測定 することができる。具体的には、ヘモグロビン結合活性測定法、ヘモグロビンとハプト グロビンとの複合体 (以下、ヘモグロビン ハプトグロビン複合体と略記する）受容体 である CD163結合活性測定法、 CD163からの細胞内シグナル測定法、 CD163を介し たインターロイキンや顆粒球-マクロファージコロニー刺激因子の生産測定法、プラス タグランジンの合成阻害活性測定法、血管新生活性測定法、抗菌活性測定法など の in vitro試験、各種溶血モデル動物を用いた in vivo試験、あるいはヒトを用いた臨 床試験などを用いて測定することができる（Clin. Chem., 42, 1589(1996)、 Eur. J. Clin • Chem. Biochem., 35, 647(1997)、 Circ.Res., 92, 1193(2003)、 Anthropol. Anz" 50, 281(1992)、 J. Leukoe. Biol, 66, 858(1999)、 Circ. Res., 92, 1193(2003)、 J. Clin. Inv est., 91, 977(1993)、 Clin. Chem. Lab. Med., 36, 691(1998)、 Nature, 409, 198(2001) 、 J. Biol. Chem., 279,51561(2004)、最新医学， 30, 656(1975)、最新医学， 30, 879(19 75)、 Res. Exp. Med. 177,1(1980)、新薬と臨床, 38, 761(1989)、応用薬理， 33, 949(19 87)、 Lab. Invest., 14, 1506(1965)、基礎と臨床， 18, 5913(1984))。また、精製ハプトグ ロビン組成物を用いて in vivo試験を行うことで、精製したハプトグロビン組成物の血 中半減期を調べることができる。以下に、その具体的な例を示す。 The biological activity of the purified nobutoglobin composition can be measured using various known methods. Specifically, a method for measuring hemoglobin binding activity, a method for measuring CD163 binding activity which is a complex of hemoglobin and haptoglobin (hereinafter abbreviated as hemoglobin haptoglobin complex), a method for measuring intracellular signal from CD163, CD163 In vitro tests such as interleukin and granulocyte-macrophage colony-stimulating factor production measurement method, plastaglandin synthesis inhibition activity measurement method, angiogenic activity measurement method, antibacterial activity measurement method, etc. In vivo tests or clinical tests using humans (Clin. Chem., 42, 1589 (1996), Eur. J. Clin • Chem. Biochem., 35, 647 ( 1997), Circ.Res., 92, 1193 (2003), Anthropol. Anz "50, 281 (1992), J. Leukoe. Biol, 66, 858 (1999), Circ. Res., 92, 1193 (2003), J. Clin. Inv est., 91, 977 (1993), Clin. Chem. Lab Med., 36, 691 (1998), Nature, 409, 198 (2001), J. Biol. Chem., 279,51561 (2004), Latest Medicine, 30, 656 (1975), Latest Medicine, 30, 879 (19 75), Res. Exp. Med. 177,1 (1980), new drugs and clinical, 38, 761 (1989), applied pharmacology, 33, 949 (19 87), Lab. Invest., 14, 1506 (1965) ), Basic and Clinical, 18, 5913 (1984)). In addition, by conducting an in vivo test using the purified haptoglobin composition, the blood half-life of the purified haptoglobin composition can be examined. Specific examples are shown below.
[0157] (1)ヘモグロビン結合活性測定法 [0157] (1) Method for measuring hemoglobin binding activity
ヘモグロビンのパーォキシダーセ活性は低 PH条件にて抑制される。しかしながら、 ハプトグロビンと結合したヘモグロビンは低 pH条件においてもパーォキシダーゼ活性 を発揮することができる。この性質を利用して、ハプトグロビンとヘモグロビンの結合 量を測定するための試薬キットが市販されている（トリデルタ社製)。この試薬キットを 添付された操作手順書に準じて行うことにより、ヘモグロビン結合活性を定量すること ができる。 Paokishidase activity of hemoglobin is suppressed at a low P H condition. However, hemoglobin combined with haptoglobin can exhibit peroxidase activity even under low pH conditions. A reagent kit for measuring the binding amount of haptoglobin and hemoglobin using this property is commercially available (manufactured by Tridelta). Hemoglobin binding activity can be quantified by carrying out this reagent kit according to the attached operation procedure manual.
[0158] (2)ヘモグロビン ハプトグロビン複合体受容体である CD163結合活性測定法 (In vitro法）  [0158] (2) Measurement of CD163 binding activity of hemoglobin haptoglobin complex receptor (in vitro method)
被検物質として精製したハプトグロビン組成物と、標準品として濃度及び比活性既 知の巿販ハブトグロビンとを、体積比 1%でゥシ血清アルブミンを含有するリン酸緩衝 液 pH7.0 (インビトロジェン社製。以下、 PBSと称す)を用いてそれぞれ段階希釈したの ち、一定量のヘモグロビンを添カ卩する。また、可溶型 CD163 (J. Biol. Chem., 279, 51 561 (2004))を濃度 lOOng/mLで PBSに溶解後、 96穴平底 ELISA用プレートに 100 /z L /ゥエルで分注し 4時間室温することにより、可溶型ハプトグロビン受容体が固相化さ れたプレートを作製する。このプレートを体積比 1%でゥシ血清アルブミンを含有する PBSを用いてブロッキング後、上記段階希釈した被検物質及び標準品を 100 L/ゥェ ルで分注し、室温にて 1時間インキュベートする。体積比 0.01%で Tween20を含有す る PBSで各ゥエルを数回洗浄しプレートに結合しな力つたハプトグロビンを除去後、体 積比 1%でゥシ血清アルブミンを含有する PBSに溶解した巿販 HRP標識ィ匕抗ノヽプトグ ロビン抗体 (バイオジェネシス社製もしくは EYラボラトリーズ社製）を、 100 μ L/ゥエル で分注する。室温にて 1時間インキュベート後、体積比 0.01%で Tween20を含有する P BSで各ゥエルを数回洗浄しプレートに結合しな力つた抗ノヽブトグロビン抗体を除去し 、各ゥヱルに 100 L/ゥヱルにて、 0.1% (ν/ν)過酸化水素水添加 2,2- Azino- bis(3- eth ylbenzothiazoline)-6-sulfonic acid試薬（和光純薬製）を添加し室温にて数分間発色 させる。適当な発色が得られた時点で 5% (w/v)ラウリル硫酸ナトリウム溶液を加えて反 応を停止させる。その後、反応液中の吸光度を 415應の波長で測定し、被検物質で あるハプトグロビンを加えて 、な 、対照反応溶液の吸光度から各希釈段階の被検物 質あるいは標準品を加えた反応溶液の吸光度を差し引いた値を得る。この値をノヽプ トグロビン受容体に結合したハプトグロビン量として縦軸に、被検物質あるいは標準 品の希釈率を横軸にして片対数グラフにプロットする。プロットした測定値よりハプトグ ロビン受容体に結合したハプトグロビン量と希釈率の関係を直線近似し、被検物質と 標準品の測定の結果得られた近似式を比較することで、被検物質の標準品に対する 倍率を求めることができ、その力価を決定することができる。 Phosphate buffer solution pH7.0 (manufactured by Invitrogen) containing purified haptoglobin composition as a test substance and commercially available habutoglobin with a known concentration and specific activity as a standard product and containing sushi serum albumin at a volume ratio of 1% (Hereafter referred to as PBS), add a certain amount of hemoglobin after serial dilution. Also, soluble CD163 (J. Biol. Chem., 279, 51 561 (2004)) was dissolved in PBS at a concentration of lOOng / mL, and then dispensed into a 96-well flat-bottom ELISA plate at 100 / z L / well. Prepare a plate on which soluble haptoglobin receptor is solid-phased by incubating at room temperature for 4 hours. After blocking this plate with PBS containing ushi serum albumin at a volume ratio of 1%, the test substance and standard diluted in the above steps were dispensed at 100 L / well and incubated at room temperature for 1 hour. To do. Wash each well several times with PBS containing Tween 20 at a volume ratio of 0.01%, remove the strong haptoglobin bound to the plate, and then dissolve in PBS containing ushi serum albumin at a volume ratio of 1%. 100 μL / well of HRP-labeled anti-noptoglobin antibody (Biogenesis or EY Laboratories) Dispense with. After 1 hour incubation at room temperature, wash each well several times with PBS containing 0.01% Tween20 at a volume ratio to remove the strong anti-nobuglobin antibody bound to the plate, and add 100 L / rule to each tool. Add 0.1% (ν / ν) hydrogen peroxide solution and add 2,2-Azino-bis (3-ethylbenzothiazoline) -6-sulfonic acid reagent (manufactured by Wako Pure Chemical Industries). When a suitable color is obtained, stop the reaction by adding 5% (w / v) sodium lauryl sulfate solution. Then, the absorbance in the reaction solution was measured at a wavelength of 415, and the test substance haptoglobin was added, and the reaction solution in which the test substance or standard product at each dilution stage was added from the absorbance of the control reaction solution. A value obtained by subtracting the absorbance of. This value is plotted on a semilogarithmic graph with the vertical axis representing the amount of haptoglobin bound to the neuroglobin receptor and the horizontal axis representing the dilution ratio of the test substance or standard. By approximating the relationship between the amount of haptoglobin bound to the haptoglobin receptor and the dilution rate from the plotted measurement values, and comparing the approximate equations obtained as a result of measurement of the test substance and the standard product, the standard of the test substance The magnification for the product can be determined and its titer can be determined.
[0159] また、文献 [J. Biol. Chem., 279, 51561 (2004)]に記載の方法により、可溶化 CD163 とヘモグロビン ハプトグロビン複合体との結合活性を表面プラズモン共鳴現象を用 いて測定することも出来る。  [0159] In addition, the binding activity between solubilized CD163 and hemoglobin haptoglobin complex should be measured using the surface plasmon resonance phenomenon by the method described in the literature [J. Biol. Chem., 279, 51561 (2004)]. You can also.
(3) CD163結合活性測定法 (CD163発現細胞を用いる方法）  (3) CD163 binding activity assay (method using CD163 expressing cells)
被検物質として精製したハプトグロビン組成物と、標準品として濃度及び比活性既 知の巿販ハブトグロビンを、体積比 1%でゥシ血清アルブミンを含有する PBSを用いて それぞれ段階希釈したのち、一定量のヘモグロビンを添加する。このヘモグロビン ハプトグロビン複合体を、 CD163を発現している細胞へ添カ卩し、 4°Cにて 1時間以上ィ ンキュペートする。体積比 1%でゥシ血清アルブミンを含有する PBSで細胞を数回リン スして細胞表面に結合しな力つたヘモグロビン ハプトグロビン複合体を除去後、体 積比 1%でゥシ血清アルブミンを含有する PBSに溶解した巿販 FITC標識抗ノヽブトグロ ビン抗体 (EYラボラトリーズ社製)などのヘモグロビン—ハプトグロビン複合体を特異 的に認識できる抗体を反応させることにより、フローサイトメーターにて CD163発現細 胞へのヘモグロビン ハプトグロビン複合体の結合活性を測定することができる。  After serially diluting a purified haptoglobin composition as a test substance and a commercially available habutoglobin with a known concentration and specific activity as a standard product using PBS containing ushi serum albumin at a volume ratio of 1%, a certain amount Of hemoglobin. This hemoglobin haptoglobin complex is added to cells expressing CD163 and incubated at 4 ° C for 1 hour or longer. Rush serum albumin containing 1% volume ratio After rinsing cells several times with PBS to remove the hemoglobin haptoglobin complex that did not bind to the cell surface, it contained ushi serum albumin at a volume ratio of 1%. Sell to a CD163 expression cell using a flow cytometer by reacting with an antibody that can specifically recognize a hemoglobin-haptoglobin complex such as FITC-labeled anti-nobutoglobin antibody (EY Laboratories) dissolved in PBS. It is possible to measure the binding activity of the hemoglobin haptoglobin complex.
[0160] また、 CD163発現細胞表面へのヘモグロビン ハプトグロビン複合体の結合量を経 時的に観察することにより、 CD 163発現細胞にヘモグロビン ハプトグロビン複合体 が取り込まれる速度やその取り込み量を測定することができる（Circ. Res., 92, 1193 ( 2003)) [0160] Furthermore, the amount of hemoglobin haptoglobin complex bound to the surface of CD163-expressing cells By observing temporally, it is possible to measure the rate and amount of hemoglobin haptoglobin complex taken up into CD163 expressing cells (Circ. Res., 92, 1193 (2003))
(4) CD163からの細胞内シグナル測定法 (4) Intracellular signal measurement from CD163
被検物質として精製したノヽブトグロビン組成物を、ヘモグロビンと混合したのち、 CD 163発現細胞へと添加することにより、ヘモグロビン ハプトグロビン複合体が CD163 と結合することにより細胞内で引き起こされる細胞内情報伝達、例えば、カルシウム 濃度の上昇やイノシトール三リン酸濃度の上昇、またはインターロイキン 1、インター口 ィキン 6、あるいは顆粒球-マクロファージコロニー刺激因子等の生産上昇を測定する ことにより、ハプトグロビンの生物活性を測定することができる（J. Leukoe. Biol, 66, 8 58 (1999); Circ. Res., 92, 1193 (2003))。  Intracellular signal transduction triggered by the binding of the hemoglobin haptoglobin complex to CD163 by adding the purified rabbit globin composition as a test substance to hemoglobin and then adding it to CD163 expressing cells. For example, the biological activity of haptoglobin is measured by measuring an increase in calcium concentration, an increase in inositol triphosphate concentration, or an increase in production of interleukin 1, interoral quinine 6, or granulocyte-macrophage colony-stimulating factor. (J. Leukoe. Biol, 66, 8 58 (1999); Circ. Res., 92, 1193 (2003)).
[0161] (5)プラスタグランジン合成阻害活性測定法  [0161] (5) Measuring method for inhibitory activity of plastaglandin synthesis
被検物質として精製したノヽブトグロビン組成物を、ヘモグロビンと結合させることによ り、ヘモグロビンが引き起こすプラスタグランジン合成が阻害されるかどうか測定する 。この阻害活性の強さを測定することによりハプトグロビンの生物活性を測定すること ができる（Anthropol.Anz., 50, 281 (1992))。  It is determined whether or not a plataglandin synthesis caused by hemoglobin is inhibited by binding a hemoglobin composition purified as a test substance to hemoglobin. By measuring the strength of this inhibitory activity, the biological activity of haptoglobin can be measured (Anthropol. Anz., 50, 281 (1992)).
[0162] (6)血管新生活性測定法  [0162] (6) Angiogenic activity measurement method
被検物質として精製したノヽブトグロビン組成物を血管上皮系細胞へ添加することに より、細胞増殖ゃ分ィ匕などの血管新生活性を観察することができ、その血管新生活 性の強さを測定することでノヽブトグロビンの生物活性を測定することができる (J.Clin. I nvest" 91, 977 (1993))。  By adding a purified rabbit globin composition as a test substance to vascular epithelial cells, it is possible to observe angiogenic activities such as cell proliferation and cell proliferation, and to increase the strength of the vascular new life. The biological activity of mouse globin can be measured by measuring (J. Clin. Invest "91, 977 (1993)).
[0163] (7)抗菌活性測定法  [0163] (7) Antibacterial activity measurement method
被検物質として精製したノヽブトグロビン組成物をバクテリアに添加することにより、バ クテリアの増殖阻害を観察することができ、その増殖阻害活性の強さを測定すること でハプトグロビンの抗菌活性を測定することができる（Clin.Chem. Lab. Med., 36, 691 (1998))。  By adding purified buttoglobin composition as a test substance to bacteria, inhibition of bacterial growth can be observed, and the antibacterial activity of haptoglobin can be measured by measuring the strength of the growth inhibition activity. (Clin. Chem. Lab. Med., 36, 691 (1998)).
[0164] (8)正常家兎での溶血モデルを用いた in vivo試験 正常家兎に家兎ヘモグロビンを単独投与した群と、正常家兎へ家兎ヘモグロビン およびノヽブトグロビンの混合液を投与した群とを比較することにより、溶血によって生 じるヘモグロビン尿症やヘモグロビン血症の諸症状 (血色素尿ゃ腎機能障害、腎へ の鉄沈着、腎尿細管細胞障害など)のハプトグロビン投与による改善効果を観察する ことができる。その効果の強さを測定することでノヽブトグロビンの生物活性を測定する ことができる（最新医学,^, 656 (1975)、最新医学， 30, 879 (1975)、 Res. Exp. Med., 177. 1(1980))。 [0164] (8) In vivo test using hemolysis model in normal rabbit By comparing the group in which normal rabbits were given rabbit hemoglobin alone with the group in which rabbits were given a mixture of rabbit hemoglobin and nobutoglobin, hemoglobinuria and hemoglobinemia caused by hemolysis were compared. It is possible to observe the improvement effect of haptoglobin administration on these symptoms (hemoglobinuria, renal dysfunction, renal iron deposition, renal tubular cell damage, etc.). By measuring the strength of the effect, biological activity of buttoglobin can be measured (latest medicine, ^, 656 (1975), latest medicine, 30, 879 (1975), Res. Exp. Med., 177 1 (1980)).
[0165] (9)熱傷家兎での溶血モデルを用いた in vivo試験  [9] (9) In vivo test using hemolysis model in burned rabbits
第 III度熱傷家兎に対して、ハプトグロビンと乳酸リンゲル液とを投与することにより、 高 、延命効果ゃ腎障害予防効果を観察することができ、その効果の強さ測定するこ とでノヽブトグロビンの生物活性を測定することができる（新薬と臨床， 38, 761 (1989))  By administering haptoglobin and lactated Ringer's solution to third-degree burned rabbits, it is possible to observe the effects of prolonging life and preventing kidney damage, and measuring the strength of the effects of nobutoglobin Bioactivity can be measured (new drugs and clinical practice, 38, 761 (1989))
(10)正常ィヌでの溶血液とエンドトキシンによる溶血モデルを用いた in vivo試験 Hoffineisterらにより、ィヌにエンドトキシンを投与した後、続けてィヌ溶血液を投与 することによって典型的な急性腎不全を惹起できることが報告されている（Lab. Invest ., 14, 1506 (1965))。この溶血による腎機能障害モデルにハプトグロビンを投与する ことにより、急性腎不全に対する腎機能改善効果を観察することができ、その効果の 強さ測定することでノヽブトグロビンの生物活性を測定することができる (応用薬理, 33, 949 (1987))。 (10) In vivo test using hemolysis model of normal innu and hemolysis by endotoxin After endotoxin was administered to innu by Hoffineister et al. It has been reported that failure can be induced (Lab. Invest., 14, 1506 (1965)). By administering haptoglobin to this hemolysis-induced renal dysfunction model, the effect of improving renal function on acute renal failure can be observed, and the biological activity of mouse globin can be measured by measuring the strength of the effect. (Applied pharmacology, 33, 949 (1987)).
[0166] (11)モデル動物を用いた in vivoクリアランス試験（血中半減期の測定）  [11] (11) In vivo clearance test using model animals (measurement of blood half-life)
精製したノヽブトグロビン組成物などを用いたクリアランス試験は、ラットなどのモデル 動物を用いて調べることができる。ハプトグロビン組成物もしくはハプトグロビンとへモ グロビンの複合体力もなる被検物質および公知の方法によりラジオアイソトープなど の標識処理をおこなった被検物質を、静脈注射あるいは皮下注射によってラットに単 回投与する。投与後の任意の時点において血液を採取し、ヒトハブトグロビンを特異 的に検出定量できる ELISA法あるいは標識ィ匕合物の量を測定することによりラット血 液中の被検物質  A clearance test using a purified rabbit globin composition or the like can be examined using a model animal such as a rat. A test substance that also has a haptoglobin composition or a haptoglobin-hemoglobin complex and a test substance that has been labeled with a radioisotope by a known method is administered to a rat by intravenous injection or subcutaneous injection. Samples in rat blood can be collected by collecting blood at any time after administration and measuring the amount of the labeled compound or ELISA method that can specifically detect and quantify human hubtoglobin.
濃度を測定する。標識処理をおこなった被検物質の標識量の測定は、その標識体に 合わせた公知の解析方法で測定する。得られたデータは、たとえば PCNONLIN非線 形回帰分析（Statistical Consultants, 1992)を用いて各ラットの薬物動態パラメーター を測定する。なお、ハプトグロビン組成物のクリアランス試験は、ラットよりヒトにより近 縁の霊長類、たとえば力-クイザル等の動物種モデルを用いて評価することもできる Measure the concentration. The amount of labeling of the test substance that has been labeled is measured on the label. Measurement is performed by a known analysis method. The obtained data is used to measure the pharmacokinetic parameters of each rat using, for example, PCNONLIN nonlinear regression analysis (Statistical Consultants, 1992). The clearance test for haptoglobin compositions can also be evaluated using animal models such as primates that are more closely related to humans than rats.
[0167] 5.ハプトグロビン組成物の糖鎖の分析 [0167] 5. Analysis of sugar chain of haptoglobin composition
各種細胞で発現させたハプトグロビン分子の糖鎖構造は、通常の糖蛋白質の糖鎖 構造の解析に準じて行うことができる。例えば、ハプトグロビン分子に結合している糖 鎖はガラクトース、マンノースなどの中性糖、 N-ァセチルダルコサミンなどのアミノ糖、 シアル酸などの酸性糖から構成されており、糖組成分析および二次元糖鎖マップ法 などを用いた糖鎖構造解析等の手法を用いて行うことができる。  The sugar chain structure of a haptoglobin molecule expressed in various cells can be performed according to the analysis of the sugar chain structure of a normal glycoprotein. For example, sugar chains bound to haptoglobin 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 carried out by using a method such as sugar chain structure analysis using a sugar chain map method.
[0168] (1)中性糖'アミノ糖組成分析  [0168] (1) Neutral sugar 'amino sugar composition analysis
ハプトグロビン分子の糖鎖の組成分析は、トリフルォロ酢酸等で、糖鎖の酸加水分 解を行うことにより、中性糖またはアミノ糖を遊離し、その組成比を分析することができ る。  In the composition analysis of the sugar chain of the haptoglobin molecule, neutral sugar or amino sugar can be liberated by acid hydrolysis of the sugar chain with trifluoroacetic acid or the like, and the composition ratio can be analyzed.
具体的な方法として、 Dionex社製糖組成分析装置を用いる方法があげられる。 Bio LCま HPAEC— PAD (hign performanceanion— exchange chromatography— pulsed amper ometric detection)法 [ジャーナノレ ·ォブ ·リキッド ·クロマトグラフィー (j.Liq.Chromatog r.) ,6, 1577 (1983)]によって糖組成を分析する装置である。  As a specific method, there is a method using a sugar composition analyzer manufactured by Dionex. Bio LC or HPAEC—PAD (hign performanceanion—exchange chromatography—pulsed amperometric detection) method [j. Liq. Chromatog r., 6, 1577 (1983)] It is a device to analyze.
[0169] また、 2-アミノビリジンによる蛍光標識ィ匕法でも組成比を分析することができる。具体 的には、公知の方法 [ァグリカルチュラル 'アンド'バイオロジカル ·ケミストリー (Agric. Biol. Chem.), 55(1). 283-284 (1991)]に従って酸カ卩水分解した試料を 2-アミノビリジ ル化で蛍光ラベル化し、 HPLC分析して組成比を算出することができる。 [0169] The composition ratio can also be analyzed by a fluorescent labeling method using 2-aminoviridine. Specifically, a sample obtained by acid-branching decomposition according to a known method [Agric. Biol. Chem., 55 (1). 283-284 (1991)] -Fluorescent labeling by aminobilylation and composition analysis can be calculated by HPLC analysis.
(2)糖鎖構造解析  (2) Sugar chain structure analysis
ハプトグロビン分子の糖鎖の構造解析は、 2次元糖鎖マップ法 [アナリティカル 'バイ オケミストリー（Anal. Biochem.) , 171, 73 (1988)、生物化学実験法 23-糖タンパク質糖 鎖研究法 (学会出版センター)高橋禮子編（1989年) ]により行うことができる。 2次元 糖鎖マップ法は、例えば、 X軸には逆相クロマトグラフィーによる糖鎖の保持時間また は溶出位置を、 Y軸には順相クロマトグラフィーによる糖鎖の保持時間または溶出位 置を、それぞれプロットし、既知糖鎖のそれらの結果と比較することにより、糖鎖構造 を推定する方法である。 Structural analysis of glycans of haptoglobin molecules is based on two-dimensional glycan mapping method [Analytical Biochemistry., 171, 73 (1988), Biochemical Experimental Method 23-Glycoprotein Glycan Research Method ( It can be carried out by the Society Publishing Center) Takahashi Eiko (1989)]. The two-dimensional glycan mapping method is, for example, the retention time or the glycan retention time by reverse phase chromatography on the X axis. Is a method of estimating the sugar chain structure by plotting the elution position and the retention time or elution position of the sugar chain by normal phase chromatography on the Y axis and comparing them with the results of known sugar chains. is there.
[0170] 具体的には、ハプトグロビン組成物をヒドラジン分解して、ハプトグロビン分子力も糖 鎖を遊離し、 2-アミノビリジン (以下、「PA」と略記する）による糖鎖の蛍光標識 [ジャー ナル 'ォブ 'バイオケミストリー（J.Biochem.) , 197 (1984)]を行った後、ゲルろ過に より糖鎖を過剰の PAィ匕試薬などと分離し、逆相クロマトグラフィーを行う。次いで、分 取した糖鎖の各ピークについて順相クロマトグラフィーを行う。これらの結果をもとに、 2次元糖鎖マップ上にプロットし、糖鎖スタンダード (TaKaRa社製）、文献 [アナリティ カル'バイオケミストリー（Anal. Biochem.) , ΓΠ, 73 (1988)]とのスポットの比較より糖 鎖構造を推定することができる。  [0170] Specifically, the haptoglobin composition was decomposed with hydrazine, the haptoglobin molecular force also released the sugar chain, and fluorescent labeling of the sugar chain with 2-aminoviridine (hereinafter abbreviated as “PA”) [Journal After performing “Biochemistry” (J. Biochem.), 197 (1984)], the glycan is separated from excess PA reagent by gel filtration and subjected to reverse phase chromatography. Next, normal phase chromatography is performed for each peak of the collected sugar chain. Based on these results, a plot is made on a two-dimensional glycan map, with glycan standard (TaKaRa), literature [Analytical Biochem., Γ ', 73 (1988)]. The sugar chain structure can be estimated from the spot comparison.
[0171] さらに各糖鎖の MALDI-TOF-MSなどの質量分析を行い、 2次元糖鎖マップ法によ り推定される構造を確認することができる。  [0171] Further, 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 map method.
6.ハプトグロビン分子の糖鎖構造を識別する免疫学的定量方法  6. Immunological quantification method to identify sugar chain structure of haptoglobin molecule
ハプトグロビン組成物は、糖鎖構造が異なったハプトグロビン分子力 構成されて いる。本発明のハプトグロビン組成物は、 Ν-グリコシド結合複合型糖鎖還元末端の Ν -ァセチルダルコサミンにフコースが結合しておらず、長い血中半減期を示す特徴を 有している。このようなハプトグロビン組成物は、上記 5.に記載のハプトグロビン分子 の糖鎖構造の分析法を用いることにより識別できる。また、レクチンを用いた免疫学 的定量方法を用いることによつても識別できる。  The haptoglobin composition is composed of haptoglobin molecular forces having different sugar chain structures. The haptoglobin composition of the present invention is characterized in that fucose is not bound to Ν-acetyldarcosamine at the reducing end of Ν-glycoside bond complex type sugar chain, and has a long blood half-life. Such a haptoglobin composition can be identified by using the method for analyzing the sugar chain structure of the haptoglobin molecule described in 5. above. It can also be identified by using an immunological quantification method using lectins.
[0172] レクチンを用いた免疫学的定量方法を用いたハプトグロビン分子の糖鎖構造の識 別は、文献 [モノクローナル ·アンティボディズ：プリンシプルズ 'アンド'アプリケーショ ンズ (Monoclonal Antibodies: Principles and Applications), Wiley— Liss, Inc., (199o ; 酵素免疫測定法，第 3版，医学書院 (1987) ;改訂版，酵素抗体法，学際企画 (1985) ] 等に記載のウェスタン染色、 RIA (Radioimmunoassay)、 VIA (Viroimmunoassay)、 EIA (Enzymoimmunoassay)、 FIA (Fluoroimmunoassay)、 MIA (Metalloimmunoassayノなと の免疫学的定量方法に準じて、例えば、以下のように行うことができる。  [0172] Identification of the glycan structure of haptoglobin molecules using immunological quantification using lectins has been described in the literature [Monoclonal Antibodies: Principles and Applications, Wiley—Liss, Inc., (199o; Enzyme Immunoassay, 3rd Edition, School of Medicine (1987); Revised Edition, Enzyme Antibody Method, Interdisciplinary Planning (1985)], etc., RIA (Radioimmunoassay), According to an immunological quantification method such as VIA (Viroimmunoassay), EIA (Enzymoimmunoassay), FIA (Fluoroimmunoassay), or MIA (Metalloimmunoassay), for example, the following can be carried out.
[0173] ハプトグロビン組成物を構成するハプトグロビン分子の糖鎖構造を認識するレクチ ンを標識し、標識したレクチンと試料であるハプトグロビン組成物を反応させる。次に 、標識したレクチンとハプトグロビン分子の複合体の量を測定する。 [0173] A rectifier that recognizes the sugar chain structure of the haptoglobin molecule constituting the haptoglobin composition The labeled lectin and the sample haptoglobin composition are reacted. Next, the amount of the complex of labeled lectin and haptoglobin molecule is measured.
ハプトグロビン分子の糖鎖構造を識別に用いられるレクチンとしては、例えば、 WG A (T. vulgaris由来の wneat— germ agglutinin)ゝ し onA(C. ensiformis由来の concanavali n A)、 RIC (R. communis由来の毒素)、 L- PHA (P.vulgaris由来の leukoagglutinin)、 LC A(L. culinaris由来の lentil agglutinin), PSA (P. sativum由来の Pea lectin), AAL (Aleu riaaurantia Lectin入 ACL (Amaranthus caudatus Lectin) ^ BPL (Bauhinia purpurea Le ctin八 DSL (Datura stramonium Lectin) ^ DBA (Dolichos biflorus Agglutinin) ^ EBL(E1 derberry Balk Lectin) ^ ECL (Erythrina cristagalli Lectin) ^ EEL (Euonymus europaeu s Lectin)、 GNL (Galanthus nivalis Lectin)、 GSL (Griffonia simphcifolia Lectin)、 HPA (Helix pomatia Agglutinin) ^ HHL (Hippeastrum Hybrid Lectin) ^ Jacalin、 LTL(Lotus t etragonolobus Lectin)、 LEL (Lycopersicon esculentum Lectin)、 MAL (Maackia amur ensis Lectin) ^ MPL (Maclura pomifera Lectin) ^ NPL (Narcissus pseudonarcissus Lect in)、 PNA (Peanut Agglutinin)、 E- PHA (Phaseolus vulgaris Erythroagglutinin)、 PTL(P sophocarpus tetragonolobus Lectin入 RCA (Ricinus communis Agglutinin) ^ STL (Sola num tuberosum Lectin八 SJA (Sophora japonica Agglutinin)、 SBA (Soybean Agglutini n)、 UEA (Ulex europaeus Agglutinin)、 WL (Vicia villosa Lectin)、 WFA(Wisteria flori bunda Agglutinin)力めげられる。  Examples of lectins used for identifying the sugar chain structure of haptoglobin molecules include WG A (wneat— germ agglutinin derived from T. vulgaris) onA (concanavali n A derived from C. ensiformis), RIC (derived from R. communis) ), L- PHA (leukoagglutinin from P. vulgaris), LC A (lentil agglutinin from L. culinaris), PSA (Pea lectin from P. sativum), AAL (Aleuanthria caustus Lectin) ) ^ BPL (Bauhinia purpurea Le ctin) 8 DSL (Datura stramonium Lectin) ^ DBA (Dolichos biflorus Agglutinin) ^ EBL (E1 derberry Balk Lectin) ^ ECL (Erythrina cristagalli Lectin) ^ EEL (Euthymus europaeu s Lectin), GNL (Galanth Lectin), GSL (Griffonia simphcifolia Lectin), HPA (Helix pomatia Agglutinin) ^ HHL (Hippeastrum Hybrid Lectin) ^ Jacalin, LTL (Lotus t etragonolobus Lectin), LEL (Lycopersicon esculentum Lectin), MAL (Maackia amur ensis Lectin) ^ MP (Maclura pomifera Lectin) ^ NPL (Narcissus pseudonarcissus Lect in), PNA (Peanut Agglutinin), E-PHA (Phaseolus vulgaris Erythroagglutinin), PTL (P sophocarpus tetragonolobus Lectin) RCA (Ricinus communis Agglutinin) ^ STL (Sola num tuberosum Lectin 8 SJA (Sophora japonica Agglutinin), SBA (Soybean AgA (Ulex europaeus Agglutinin), WL (Vicia villosa Lectin), WFA (Wisteria flori bunda Agglutinin).
[0174] 本発明にお ヽては上記レクチンのなかでも N-ダルコシド結合複合型糖鎖還元末端 の N-ァセチルダルコサミンにフコースが結合している糖鎖構造を特異的に認識する レクチンを用いることが好ましぐその具体的な例としては、レンズマメレクチン LCA(L ens Culinaris由来の Lentil Agglutinin)エンドゥマメレクチン PSA (Pisum sativum由来 の Pea Lectin)、ソラマメレクチン VFA(Viciafaba由来の Agglutinin)、ヒイロチヤワンタケ レクチン AAL (Aleuria aurantia由来の Lectin)を挙げることができる。  [0174] In the present invention, among the above-mentioned lectins, a lectin that specifically recognizes a sugar chain structure in which fucose is bound to N-acetylcolcamine at the N-darcoside-linked complex type sugar chain reducing end. Specific examples that are preferred to be used include lentil lectin LCA (Lentil Agglutinin from Lens Culinaris), endangered lectin PSA (Peasum sativum-derived Pea Lectin), broad bean lectin VFA (Agglutinin from Viciafaba), An example is chiatake lectin AAL (Lectin derived from Aleuria aurantia).
[0175] 7.本発明のハプトグロビン組成物の利用  [0175] 7. Use of the haptoglobin composition of the present invention
本発明のハプトグロビン組成物は、 N-グリコシド結合複合型糖鎖還元末端の N-ァ セチルダルコサミンにフコースが結合しているハプトグロビンに比べて、生体内に投 与した場合に血中半減期が長いという特徴を有する。従って、本発明のハプトグロビ ン組成物は、ハプトグロビンによる治療が適応される各種疾患において、投与量およ び投与回数をより少なくする治療を可能にすることができる。このことは、患者や医療 現場での負担を軽減するとともに、大量に投与しなけばならない現行治療の結果生 じる事故や副作用の軽減に貢献できる。 The haptoglobin composition of the present invention has a half-life in blood when administered in vivo, compared to haptoglobin in which fucose is bound to N-glycidyl darcosamine at the N-glycoside-linked complex type sugar chain reducing end. It has the characteristic of being long. Therefore, the haptoglobi of the present invention The composition can enable treatment with fewer doses and fewer doses in various diseases for which treatment with haptoglobin is indicated. This not only reduces the burden on patients and medical sites, but also contributes to the reduction of accidents and side effects resulting from current treatments that must be administered in large quantities.
[0176] また、現行のハプトグロビン製剤は複数の健常人の血漿を原料として製造されてい るため、各種肝炎ウィルス、 AIDSウィルス、ヒトパルボウイルス、変異型クロイツフェル ト 'ヤコブ病の原因となるプリオンなどの感染の恐れを完全には否定できない。現行 市販されている血液製剤を本発明のハプトグロビン組成物を含有する製剤に切り替 えることは、様々な感染事故を防止する上で非常に有用である。さらに、ハプトグロビ ンには多型及び多量体が存在しその生理機能は個々の多型あるいは多量体ごとに 異なることが報告されている力 本発明のハプトグロビン組成物を用いることによって 、適応疾患に合わせた適切なハプトグロビンの型を含む治療剤を調製し患者に提供 することが可能となる。  [0176] In addition, since the current haptoglobin preparations are produced from the plasma of multiple healthy individuals, various hepatitis viruses, AIDS viruses, human parvoviruses, mutant Creutzfeld's prions that cause Jacob disease, etc. The fear of infection cannot be completely denied. It is very useful to switch a currently marketed blood product to a product containing the haptoglobin composition of the present invention in order to prevent various infection accidents. Furthermore, polymorphisms and multimers exist in haptoglobin, and its physiological function has been reported to be different for each polymorphism or multimer. By using the haptoglobin composition of the present invention, it can be adapted to the indication disease. It is possible to prepare a therapeutic agent containing a suitable haptoglobin type and provide it to the patient.
[0177] 本発明のハプトグロビン組成物を用いる疾患としては、溶血を伴う疾患、血中遊離 ヘモグロビンによる組織傷害を伴う疾患、細菌感染症の予防及び治療に関しても有 効である。  [0177] Diseases using the haptoglobin composition of the present invention are also effective for the prevention and treatment of diseases involving hemolysis, diseases involving tissue damage caused by blood free hemoglobin, and bacterial infections.
溶血を伴う疾患としては、溶血性疾患があげられるが、具体的には火傷や輸血に 伴う外傷性ヘモグロビン尿症、人工心肺などの体外循環下開心術、ハプトグロビンの 産生臓器である肝臓の機能傷害をもたらすアルコール性肝炎あるいはアルコール性 肝硬変、先天性の欠損症などが挙げられる。いずれの疾患においても、ヘモグロビン 尿症およびヘモグロビン血症といった病的症状を呈し、鉄の体外流出に伴う貧血や 、ヘモグロビンや鉄が腎臓に付着することによる腎障害が観察される。  Diseases that involve hemolysis include hemolytic diseases. Specifically, traumatic hemoglobinuria associated with burns and blood transfusions, open-heart surgery under extracorporeal circulation such as cardiopulmonary bypass, and functional damage to the liver, the organ that produces haptoglobin Alcoholic hepatitis or alcoholic cirrhosis, congenital deficiency, etc. In any of the diseases, pathological symptoms such as hemoglobinuria and hemoglobinemia are exhibited, and anemia associated with the outflow of iron and renal damage due to adhesion of hemoglobin and iron to the kidney are observed.
[0178] 血中遊離ヘモグロビンによる糸且織傷害を伴う疾患としては、血中遊離ヘモグロビン による酸化的血管障害を伴う疾患、血中遊離ヘモグロビンによる腎細尿管障害を伴 う疾患などがあげられる。 [0178] Examples of diseases associated with yarn and tissue injury caused by blood free hemoglobin include diseases associated with oxidative vascular injury caused by blood free hemoglobin, diseases associated with renal tubule injury caused by blood free hemoglobin, and the like.
血中遊離ヘモグロビンによる酸ィ匕的血管障害を伴う疾患としては、酸化 LDLの形成 や血管内皮細胞の酸化障害によって生じる疾患、フリーラジカルの発生により増悪 する疾患、核酸の酸ィ匕による疾患などがあげられる。 酸化 LDLの形成や血管内皮細胞の酸ィ匕障害によって生じる疾患としては、心筋梗塞 、脳梗塞あるいは動脈硬化（American Journal of Medicine, 105, 32S (1998))などが あげられる。、フリーラジカルの発生によって増悪する疾患としては、肺気腫、肺繊維 症、急性呼吸窮迫症候群、神経変性疾患、慢性炎症性疾患などがあげられる。核酸 の酸ィ匕による疾患としては、発癌などがあげられる。 Diseases associated with acid-induced vascular injury due to blood free hemoglobin include diseases caused by the formation of oxidized LDL and oxidative damage of vascular endothelial cells, diseases exacerbated by free radical generation, and diseases caused by acid oxidation of nucleic acids. can give. Examples of the diseases caused by the formation of oxidized LDL and acid dysfunction of vascular endothelial cells include myocardial infarction, cerebral infarction, and arteriosclerosis (American Journal of Medicine, 105, 32S (1998)). Diseases exacerbated by the generation of free radicals include emphysema, pulmonary fibrosis, acute respiratory distress syndrome, neurodegenerative diseases, and chronic inflammatory diseases. Examples of diseases caused by acid oxidation of nucleic acids include carcinogenesis.
[0179] 血中遊離ヘモグロビンによる腎細尿管障害を伴う疾患としては、ネフローゼ症候群 、急性腎炎、慢性腎炎などがあげられる。  [0179] Examples of diseases associated with renal tubule injury caused by blood free hemoglobin include nephrotic syndrome, acute nephritis, and chronic nephritis.
動脈硬化は、動脈の血管内腔が狭くなることにより血液の流れが障害され、動脈壁 の弾力が失われた動脈が拡張蛇行や破裂等を引き起こす疾患である。ハプトグロビ ン遺伝子の型が Hplのヒトにおいては、ビタミン Cや Eといった抗酸ィ匕ビタミンによる動 脈硬化予防の効果が観察されている（Diabetes Care, 27, 925 (2004))。  Arteriosclerosis is a disease in which blood flow is impaired due to narrowing of the arterial vascular lumen, and arteries that have lost the elasticity of the arterial wall cause dilation, rupture, and the like. In humans with Hpl type haptoglobin, the effect of anti-acidic vitamins such as vitamins C and E has been observed to prevent arteriosclerosis (Diabetes Care, 27, 925 (2004)).
[0180] 心筋梗塞は、心臓に血液を送る冠動脈の閉塞あるいは狭窄による血管障害によつ て心筋虚血がおこり、その結果として心筋が壊死する疾患である。特に、糖尿病患者 にお!/、ては、ハプトグロビン遺伝子の型が Hp2の患者で心臓血管系に関する病気が 高いことが知られている（Circ. Res., 92, 1193 (2003))。  [0180] Myocardial infarction is a disease in which myocardial ischemia occurs due to vascular injury due to occlusion or stenosis of the coronary artery that sends blood to the heart, resulting in necrosis of the myocardium. In particular, it is known that diabetic patients have high cardiovascular disease in patients with Hp2 haptoglobin gene type (Circ. Res., 92, 1193 (2003)).
脳梗塞は、脳に血液を送る血管の閉塞あるいは狭窄による血管障害によって脳虚 血がおこり、その結果として脳細胞が壊死する疾患である。  Cerebral infarction is a disease in which cerebral ischemia occurs due to vascular injury caused by occlusion or stenosis of blood vessels that send blood to the brain, resulting in necrosis of brain cells.
[0181] 肺気腫は、肺胞壁が破壊されることによって、隣あった肺胞同士が融合し空洞化が 起こり肺胞の表面積力 、さくなる疾患である。肺胞の表面積の低下は肺機能の低下 を招き著しい呼吸困難を誘起する。  [0181] Emphysema is a disease in which the alveolar wall is destroyed, and the adjacent alveoli are fused together to form cavities, reducing the surface area of the alveoli. A reduction in alveolar surface area leads to a decrease in lung function and induces significant dyspnea.
肺線維症は、肺胞壁などの肺の間質に炎症が生じる肺炎疾患である。 本発明のハプトグロビン組成物を含有する医薬は、予防薬あるいは治療薬として単 独で投与することも可能ではある力 通常は薬理学的に許容される一つあるいはそ れ以上の担体と一緒に混合し、製剤学の技術分野にお!、てよく知られる任意の方法 により製造した医薬製剤として提供するのが望ましい。  Pulmonary fibrosis is a pneumonia disease in which inflammation occurs in the lung stroma such as the alveolar wall. The pharmaceutical containing the haptoglobin composition of the present invention can be administered alone as a prophylactic or therapeutic agent. Usually mixed with one or more pharmacologically acceptable carriers. However, it is desirable to provide it as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics.
[0182] 投与経路は、治療に際して最も効果的なものを使用するのが望ましぐ経口投与、 または口腔内、気道内、直腸内、皮下、筋肉内および静脈内等の非経口投与をあげ ることができ、ハプトグロビン製剤の場合、望ましくは静脈内投与をあげることができる 投与形態としては、噴霧剤、カプセル剤、錠剤、顆粒剤、シロップ剤、乳剤、座剤、 注射剤、軟膏、テープ剤等があげられる。 [0182] The route of administration includes oral administration, where it is desirable to use the most effective treatment, or parenteral administration, such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous. In the case of haptoglobin preparations, intravenous administration can be desirable Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
[0183] 経口投与に適当な製剤としては、乳剤、シロップ剤、カプセル剤、錠剤、散剤、顆粒 剤等があげられる。  [0183] Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
乳剤およびシロップ剤のような液体調製物は、水、ショ糖、ソルビトール、果糖等の 類、ポリエチレングリコール、プロピレングリコール等のダリコール類、ごま油、オリー ブ油、大豆油等の油類、 P-ヒドロキシ安息香酸エステル類等の防腐剤、ストロベリー フ  Liquid preparations such as emulsions and syrups include water, sucrose, sorbitol, fructose, etc., Daricols such as polyethylene glycol, propylene glycol, oils such as sesame oil, olive oil, soybean oil, P-hydroxy Preservatives such as benzoates, strawberry
レーバー、ペパーミント等のフレーバー類等を添加剤として用いて製造できる。  Flavors such as laver and peppermint can be used as additives.
[0184] カプセル剤、錠剤、散剤、顆粒剤等は、乳糖、ブドウ糖、ショ糖、マン-トール等の 賦形剤、デンプン、アルギン酸ナトリウム等の崩壊剤、ステアリン酸マグネシウム、タル ク等の滑沢剤、ポリビュルアルコール、ヒドロキシプロピルセルロース、ゼラチン等の 結合剤、脂肪酸エステル等の界面活性剤、グリセリン等の可塑剤等を添加剤として 用いて製造できる。  [0184] Capsules, tablets, powders, granules and the like 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.
[0185] 非経口投与に適当な製剤としては、注射剤、座剤、噴霧剤等があげられる。  [0185] Suitable preparations for parenteral administration include injections, suppositories, sprays and the like.
注射剤は、塩溶液、ブドウ糖溶液、あるいは両者の混合物カゝらなる担体等を用いて 調製される。または、本発明のハプトグロビン組成物を常法に従って凍結乾燥し、こ れに塩ィ匕ナトリウムを加えることによって粉末注射剤を調製することもできる。  The injection is prepared using a carrier such as a salt solution, a glucose solution, or a mixture of both. Alternatively, a powder injection can be prepared by lyophilizing the haptoglobin composition of the present invention according to a conventional method and adding sodium chloride salt thereto.
座剤はカカオ脂、水素化脂肪またはカルボン酸等の担体を用いて調製される。  Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
[0186] また、噴霧剤は該ハブトグロビン組成物そのもの、な 、しは受容者の口腔および気 道粘膜を刺激せず、かつ該ハブトグロビン組成物を微細な粒子として分散させ吸収 を容易にさせる担体等を用いて調製される。 [0186] Further, the propellant does not irritate the hubtoglobin composition itself, or a carrier that does not irritate the recipient's oral cavity and airway mucosa and facilitates absorption by dispersing the habutoglobin composition as fine particles. It is prepared using.
担体として具体的には乳糖、グリセリン等が例示される。該ハブトグロビン組成物お よび用いる担体の性質により、エアロゾル、ドライパウダー等の製剤が可能である。ま た、これらの非経口剤にぉ ヽても経口剤で添加剤として例示した成分を添加すること ちでさる。 [0187] 投与量または投与回数は、 目的とする治療効果、投与方法、治療期間、年齢、体 重等により異なるが、有効成分の量として、通常成人 1回当たり 4000単位（1単位は 1 mgのヘモグロビンと結合し得る量。約 5.7g)である。 Specific examples of the carrier include lactose and glycerin. Depending on the nature of the hubtoglobin composition and the carrier used, it is possible to prepare aerosols, dry powders and the like. In addition, it is possible to add ingredients exemplified as additives for oral preparations to these parenteral preparations. [0187] The dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but the amount of active ingredient is usually 4000 units per adult (1 unit is 1 mg The amount that can bind to hemoglobin of about 5.7 g).
また、ハプトグロビン組成物の血中ヘモグロビン代謝活性をはじめとする生物活性 を検討する方法は、上記のヘモグロビン結合活性測定法、ヘモグロビンハプトグロビ ン複合体受容体である CD163結合活性測定法、 CD163からの細胞内シグナル測定 法、 CD163を介した  In addition, the methods for examining the biological activity of the haptoglobin composition, such as blood hemoglobin metabolic activity, can be determined from the above-described hemoglobin binding activity measurement method, the CD163 binding activity measurement method for the hemoglobin haptoglobin complex receptor, and CD163. Intracellular signal measurement method through CD163
インターロイキンや顆粒球-マクロファージコロニー刺激因子の生産測定法、プラスタ グ  Interleukin and granulocyte-macrophage colony-stimulating factor production measurement method, plastic
ランジンの合成阻害活性測定法、血管新生活性測定法、抗菌活性測定法などの in V itro試験あるいは溶血モデル動物を用いた in vivo試験等があげられる。  Examples include an in vitro test such as a measurement method for langine synthesis inhibitory activity, a method for measuring angiogenic activity, a method for measuring an antibacterial activity, or an in vivo test using a hemolyzed model animal.
[0188] 以下の実施例により、本発明をより具体的に説明する力 実施例は本発明の単なる 例示を示すものにすぎず、本発明の範囲を限定するものではない。 [0188] The following examples illustrate the power of the present invention more specifically. The examples are merely illustrative of the present invention and do not limit the scope of the present invention.
実施例 1  Example 1
[0189] FUT8遺伝子ダブルノックアウト細胞による HP-1型ハプトグロビンの発現  [0189] Expression of HP-1 haptoglobin by FUT8 gene double knockout cells
ヒト HP-1型ハプトグロビンを生産する FUT8遺伝子ダブルノックアウト細胞株を以下 に示す方法で作製した。  A FUT8 gene double knockout cell line producing human HP-1 haptoglobin was prepared by the method shown below.
1.プラスミド pKAN- HP1の作製  1. Construction of plasmid pKAN-HP1
ヒトハプトグロビン遺伝子配列 (UniGene: Hs.513711、配列番号 1)より制限酵素サイト (EcoRI、 Bglll)及びコザック配列を付カ卩した二種類のハプトグロビン遺伝子特異的プ ライマー (配列番号 20および配列番号 21)を作製し、以下の PCRを行なった。即ち、ヒ ト肝臓由来 cDNA(Invitrogen社製）をテンプレートとして含む 20 Lの反応液 [Pyrobe st DNA polymerase (タカラバイオ社製)、 10 X PCR buffer、 0.2mmol/L dNTP mixtureゝ 0.5 mol/L上記プライマー (配列番号 20および配列番号 21)]を調製し、 94°Cで 5分 間加熱した後、 94°Cで 1分間、 68°Cで 2分間を 1サイクルとした 30サイクルの反応で PC Rを行なった。 PCR反応後の溶液は 2%ァガロースゲル電気泳動に供し、約 1067bpの P CR増幅断片（ノヽプトグロビン HP- 1型 cDNA配列を含む）を QIAquick Gel Extraction ki t (QIAGEN社製)を用いて精製した。得られた精製 DNA断片を 17 Lの水に溶解した 後、該液に 7.5単位の制限酵素 EcoRI (タカラバイオ社製)及び 5単位の Bglll (タカラバ ィォ社製 )、 2 Lの 10 X H bufferをカ卩えて 20 Lの反応液を調製し、 37°Cで 16時間消 化反応を行なった。続いて、 pKANTEX93(W097/ 10354)3 μ gを 17 μ Lの水に溶解し、 該液に 7.5単位の EcoRI、 2 μ Lの 10 X H bufferをカ卩えて 20 μ Lの反応液を調製後、 37 °Cで 16時間消化反応を行なった。反応後、フエノール/クロ口ホルム抽出処理及びェ タノール沈殿を行い、回収したプラスミドを 17 Lの水に溶解した。さらに該液に 7.5単 位の BamHI、 2 Lの 10 X K bufferを加えて 20 μ Lの反応液を調製後、 37°Cで 16時間 消化反応を行なった。 Two haptoglobin gene-specific primers (SEQ ID NO: 20 and SEQ ID NO: 21) with restriction enzyme sites (EcoRI, Bglll) and Kozak sequences from human haptoglobin gene sequences (UniGene: Hs.513711, SEQ ID NO: 1) And the following PCR was performed. That is, 20 L reaction solution containing human liver-derived cDNA (Invitrogen) as a template [Pyrobe st DNA polymerase (Takara Bio), 10 X PCR buffer, 0.2 mmol / L dNTP mixturemmol 0.5 mol / L above Prepare primers (SEQ ID NO: 20 and SEQ ID NO: 21), heat at 94 ° C for 5 minutes, and then perform PC for 30 cycles with 94 ° C for 1 minute and 68 ° C for 2 minutes. R was done. The solution after the PCR reaction was subjected to 2% agarose gel electrophoresis, and an approximately 1067 bp PCR amplified fragment (containing a noptoglobin HP-1 type cDNA sequence) was purified using QIAquick Gel Extraction kit (manufactured by QIAGEN). The obtained purified DNA fragment was dissolved in 17 L of water. Thereafter, 7.5 L of restriction enzyme EcoRI (manufactured by Takara Bio Inc.), 5 units of Bglll (manufactured by Takara Bio Inc.), and 2 L of 10 XH buffer were added to the solution to prepare a 20 L reaction solution. The extinguishing reaction was carried out at ° C for 16 hours. Subsequently, 3 μg of pKANTEX93 (W097 / 10354) is dissolved in 17 μL of water, and 7.5 units of EcoRI and 2 μL of 10 XH buffer are added to the solution to prepare 20 μL of reaction solution. The 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 L of water. Further, 7.5 units of BamHI and 2 L of 10 XK buffer were added to the solution to prepare a 20 μL reaction solution, followed by digestion reaction at 37 ° C for 16 hours.
[0190] 上記で得られた HP- 1型ハプトグロビンの DNA断片 (EcoRI- Bglll)及び pKANTEX93 断片 (EcoRI- BamHI)を 1.5%(W/V)ァガロースゲル電気泳動に供し、それぞれ約 1060 bp、 3kbpの DNA断片を QIAquick Gel Extraction Kit(QIAGEN社製)を用いて精製し た。次いで HP- 1型ハプトグロビンの DNA断片 (EcoRI- BglII)20ng、 pKANTEX93断片（ EcoRI- BamHI)80ng、 Ligation High (東洋紡社製)を含む反応液 20 Lを調製し、 16°C で 16時間連結反応を行なった。得られたプラスミド DNAを用い、 heat shock法により大 腸菌 DH5 α株 (東洋紡社製)を形質転換した。形質転換株より QIAprep(R) Spin Minipr ep Kit(QIAGEN社製)を用いてプラスミド DNAを調製し、 BigDye Terminator Cycle Se quencing Ready Reaction Kit v2.0(QIAGEN社製)と DNAシーケンサ ABI PRISM 377( Applied Biosystems社製)を用いて塩基配列を解析した。その結果、 HP-1型ハプトグ ロビンの cDNA配列を含むプラスミド pKAN- HP1を得た（図 3)。  [0190] The HP-1 haptoglobin DNA fragment (EcoRI-Bglll) and pKANTEX93 fragment (EcoRI-BamHI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and each of about 1060 bp and 3 kbp. The DNA fragment was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, prepare 20 L of a reaction solution containing 20 ng of DNA fragment of HP-1 haptoglobin (EcoRI-BglII), 80 ng of pKANTEX93 fragment (EcoRI-BamHI) and Ligation High (Toyobo Co., Ltd.) at 16 ° C for 16 hours. Was done. Using the obtained plasmid DNA, E. coli DH5α strain (manufactured by Toyobo Co., Ltd.) was transformed by the heat shock method. Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequence Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied) The base sequence was analyzed using Biosystems). As a result, plasmid pKAN-HP1 containing the cDNA sequence of HP-1 type haptoglobin was obtained (FIG. 3).
[0191] 2. FUT8遺伝子ダブルノックアウト細胞への HP-1型ハプトグロビン発現プラスミドの 導入  [0191] 2. Introduction of HP-1 type haptoglobin expression plasmid into FUT8 double knockout cells
文献（Biotechnology and Bioengineering 87, 614 (2004))に記載された FUT8遺伝 子ダブルノックアウト細胞に、前項 1で作製したプラスミド pKAN-HPlを導入した。これ らの遺伝子導入は公知のエレクト口ポレーシヨン法 [サイトテクノロジー（Cytotechnolo gy),3, 133 (1990)]により以下の手順で行った。まず、プラスミド pKAN-HPl 30 gを N E Buffer 3 (New England Biolabs社製) 20 μ Lと 200単位の制限酵素 Mlul (New Englan d Biolabs社製）を含む 200 Lの反応液を調製し、 37°Cで 16時間消化反応を行うこと により線状ィ匕した。反応後、該反応液に対しフエノール/クロ口ホルム抽出処理および エタノール沈殿により精製を行い、線状ィ匕プラスミドを回収した。 The plasmid pKAN-HPl prepared in the previous section 1 was introduced into FUT8 gene double knockout cells described in the literature (Biotechnology and Bioengineering 87, 614 (2004)). These genes were introduced by the following procedure according to a known electoral position method [Cytotechnology, 3, 133 (1990)]. First, prepare a 200 L reaction solution containing 30 μg of plasmid pKAN-HPl and 20 μL of NE Buffer 3 (New England Biolabs) and 200 units of restriction enzyme Mlul (New England Biolabs) at 37 ° The reaction was linearized by digestion with C for 16 hours. After the reaction, the reaction solution is subjected to phenol / chloroform extraction treatment and Purification was performed by ethanol precipitation, and the linear plasmid was recovered.
[0192] 次に、文献（Biotechnology and Bioengineering 87, 614 (2004))に記載された FUT8 遺伝子ダブルノックアウト細胞を K- PBS緩衝液 (137mmol/L KC1、 2.7mmol/L NaCl、 8 .lmmol/L Na HPO、 1.5mmol/L KH PO、 4.0mmol/L MgCl )に懸濁して 8 X 107細胞 [0192] Next, FUT8 gene double knockout cells described in the literature (Biotechnology and Bioengineering 87, 614 (2004)) were mixed with K-PBS buffer (137mmol / L KC1, 2.7mmol / L NaCl, 8.lmmol / L 8 x 10 7 cells suspended in Na HPO, 1.5mmol / L KH PO, 4.0mmol / L MgCl)
2 4 2 4 2  2 4 2 4 2
/mLとした。細胞懸濁液 L(1.6 X 106個）と上記の線状ィ匕プラスミド 10 gを混和 した後、細胞- DNA混和液の全量を Gene Pulser Cuvette (電極間距離 2mm) (BIO-RA D社製)へ移し、 Gene Pulser (BIO- RAD社製）を用いてパルス電圧 350V、電気容量 2 50 Fの条件で遺伝子導入を行った。遺伝子導入を行ったのち、細胞懸濁液を 10% ゥシ胎児血清（Life Technologies社製)および 50 μ g/mL gentamicin (ナカライテスタ 社製）を添カ卩した IMDM培地 (Life Technologies社製) 10mLに懸濁し、接着細胞培養 T 75フラスコ (グライナ一社製)へ播種した。培養は 5%CO、 37°Cの条件下で行った。 / mL. After the cell suspension L (1.6 X 10 6 cells) was mixed linear I spoon plasmid 10 g of the cell - the total amount of DNA mixed solution Gene Pulser Cuvette (inter-electrode distance 2mm) (BIO-RA D Co. The gene was transferred using Gene Pulser (manufactured by BIO-RAD) under the conditions of a pulse voltage of 350 V and an electric capacity of 250 F. After gene transfer, IMDM medium (Life Technologies) supplemented with 10% Ushi fetal serum (Life Technologies) and 50 μg / mL gentamicin (Nacalai Testa) The suspension was suspended in 10 mL, and seeded in an adherent cell culture T 75 flask (manufactured by Grainer). The culture was performed under conditions of 5% CO and 37 ° C.
2  2
[0193] 3. 200nM MTXffif性株の取得  [0193] 3. Acquisition of 200nM MTXffif stock
前項で得た pKAN-HPl導入細胞を 6日間培養した後、培養上清を除去し、 10%ゥ シ胎児透  After culturing the pKAN-HPl-introduced cells obtained in the previous section for 6 days, the culture supernatant is removed and 10% cyst fetal permeation is removed.
析血清、 50 μ g/mL gentamicinおよび 50nM methotrexate(MTX)(シグマ社製)を添カロ した IMDM培地を 10mL添カ卩した。この培地交換作業を 3〜4日毎に繰り返しながら 9日 間の培養を行った。次いで、 10%ゥシ胎児透析血清、 50 g/mL gentamicinおよび 2 00nMの MTXを添カ卩した IMDM培地を用いた培地交換作業を同様に 3〜4日毎に繰り 返しながら 18日間培養し、 200nM MTX耐性株 HP1KOを得た  10 mL of IMDM medium supplemented with analysis serum, 50 μg / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Subsequently, the medium was replaced with IMDM medium supplemented with 10% urine fetal dialyzed serum, 50 g / mL gentamicin and 200 nM MTX, and cultured again every 3-4 days for 18 days. Obtained MTX resistant strain HP1KO
得られた HP1KOを 3.0 X 105cells/mLの濃度で 5mLの 10%ゥシ胎児透析血清、 50 μ g/mL gentamicinおよび 200nMの MTXを添カ卩した IMDM培地に懸濁し、 T25フラスコ へ播種して 3日間静置培養を行った。培養 3日後の培養上清を回収し、上清中に含 まれるハプトグロビン量を Haptoglobin Kit(Tridelta Development Limited社製)を用い て測定したところ、培養上清中に 60.7 g/mLの濃度でノヽブトグロビンが発現している ことを確認した。なお、 HP1KO株は、 HP1KOの株名で、平成 17年 2月 17日付けで独 立行政法人産業技術総合研究所特許生物寄託センター (茨城県つくば巿東 1丁目 1 番地 1号中央第 6)に FERMBP-10249として寄託されている。このようにして作製した H P1KO株の培養上清を、ハプトグロビンの一般的な精製法 (続医薬品の開発 廣川書 店 第 20卷、 215-219ページ)を用いて精製することによって得られた HP-1型ハプト グロビンは、通常の CHO/DG44株で生産したフコースが結合して!/、る糖鎖を有する H P-1型ハプトグロビンに比較して、血中半減期の延長が認められた。 The resulting HP1KO is suspended in IMDM medium supplemented with 5 mL of 10% urine fetal dialyzed serum, 50 μg / mL gentamicin and 200 nM MTX at a concentration of 3.0 × 10 5 cells / mL, and seeded in a T25 flask. Then, static culture was performed for 3 days. The culture supernatant after 3 days of culture was collected, and the amount of haptoglobin contained in the supernatant was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited). It was confirmed that butoglobin was expressed. The HP1KO strain is the name of HP1KO, and the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, February 17, 2005 (Tsukuba Ito, Ibaraki Pref. Has been deposited as FERMBP-10249. The culture supernatant of the HP1KO strain prepared in this way is then used for general purification of haptoglobin (development of follow-up medicines HP-1 type haptoglobin obtained by purifying using the 20th store, page 215-219) has fucose produced by the normal CHO / DG44 strain and has a sugar chain! Increased blood half-life was observed compared to HP-1 haptoglobin.
実施例 2  Example 2
[0194] FUT8遺伝子ダブルノックアウト細胞による HP-2型ハプトグロビンの発現  [0194] Expression of HP-2 type haptoglobin by FUT8 gene double knockout cells
ヒト HP-2型ハプトグロビンを生産する FUT8遺伝子ダブルノックアウト細胞株を以下 に示す方法で作製した。  A FUT8 gene double knockout cell line producing human HP-2 haptoglobin was prepared by the method described below.
1.プラスミド pKAN- HP2の作製  1. Construction of plasmid pKAN-HP2
ヒトハプトグロビン遺伝子配列 (UniGene: Hs.513711、配列番号 1)より制限酵素サイト (EcoRI、 Bglll)及びコザック配列を付カ卩した二種類のハプトグロビン遺伝子特異的プ ライマー (配列番号 20および配列番号 21)を作製し、以下の PCRを行なった。即ち、ヒ ト肝臓由来 cDNA(Invitrogen社製）をテンプレートとして含む 20 Lの反応液 [Pyrobe st DNA polymerase (タカラバイオ社製)、 10 X PCR buffer、 0.2mmol/L dNTP mixtureゝ 0.5 mol/L上記プライマー (配列番号 20および配列番号 21)]を調製し、 94°Cで 5分 間加熱した後、 94°Cで 1分間、 68°Cで 2分間を 1サイクルとした 30サイクルの反応で PC Rを行なった。 PCR反応後の溶液は 2%ァガロースゲル電気泳動に供し、約 1244bpの P CR増幅断片（HP- 2型ハプトグロビン cDNA配列を含む）を QIAquick Gel Extraction ki t (QIAGEN社製)を用いて精製した。得られた精製 DNA断片を 17 Lの水に溶解した 後、該液に 7.5単位の制限酵素 EcoRI (タカラバイオ社製)及び 5単位の Bglll (タカラバ ィォ社製 )、 2 Lの 10 X H bufferをカ卩えて 20 Lの反応液を調製し、 37°Cで 16時間消 化反応を行なった。続いて、 pKANTEX93(W097/10354に記載) 3 μ gを 17 μ Lの水に 溶解し、該液に 7.5単位の EcoRI、 2 μ Lの 10 X H bufferを加えて 20 μ Lの反応液を調 製後、 37°Cで 16時間消化反応を行なった。反応後、フエノール/クロ口ホルム抽出処 理及びエタノール沈殿を行い、回収したプラスミドを 17 Lの水に溶解した。さらに該 液に 7.5単位の BamHI、 2 μ Lの 10 X K bufferを加えて 20 μ Lの反応液を調製後、 37°C で 16時間消化反応を行なった。  Two haptoglobin gene-specific primers (SEQ ID NO: 20 and SEQ ID NO: 21) with restriction enzyme sites (EcoRI, Bglll) and Kozak sequences from human haptoglobin gene sequences (UniGene: Hs.513711, SEQ ID NO: 1) And the following PCR was performed. That is, 20 L reaction solution containing human liver-derived cDNA (Invitrogen) as a template [Pyrobe st DNA polymerase (Takara Bio), 10 X PCR buffer, 0.2 mmol / L dNTP mixturemmol 0.5 mol / L above Prepare primers (SEQ ID NO: 20 and SEQ ID NO: 21), heat at 94 ° C for 5 minutes, and then perform PC for 30 cycles with 94 ° C for 1 minute and 68 ° C for 2 minutes. R was done. The solution after the PCR reaction was subjected to 2% agarose gel electrophoresis, and an approximately 1244 bp PCR amplified fragment (containing the HP-2 haptoglobin cDNA sequence) was purified using QIAquick Gel Extraction kit (manufactured by QIAGEN). After the obtained purified DNA fragment was dissolved in 17 L of water, 7.5 units of restriction enzyme EcoRI (Takara Bio) and 5 units of Bglll (Takara Bio), 2 L of 10 XH buffer were added to the solution. A 20 L reaction solution was prepared and the quenching reaction was carried out at 37 ° C for 16 hours. Subsequently, 3 μg of pKANTEX93 (described in W097 / 10354) is dissolved in 17 μL of water, and 7.5 units of EcoRI and 2 μL of 10 XH buffer are added to the solution to prepare a 20 μL reaction solution. After the preparation, digestion reaction was performed at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17 L of water. Furthermore, 7.5 units of BamHI and 2 μL of 10 × K buffer were added to the solution to prepare a 20 μL reaction solution, followed by digestion reaction at 37 ° C. for 16 hours.
[0195] 上記で得られた HP-2型ハプトグロビン DNA断片 (EcoRI- Bglll)及び pKANTEX93断 片 (EcoRI- BamHI)を 1.5%(W/V)ァガロースゲル電気泳動に供し、それぞれ約 1240bp 、 3kbpの DNA断片を QIAquick Gel Extraction Kit(QIAGEN社製)を用いて精製した。 次!、で HP- 2型ハプトグロビンの DNA断片 (EcoRI- BglII)20ng、 pKANTEX93断片 (Eco RI- BamHI)80ng、 Ligation High (東洋紡社製)を含む反応液 20 Lを調製し、 16°Cで 1 6時間連結反応を行なった。得られたプラスミド DNAを用い、 heat shock法により大腸 菌 DH5ひ株 (東洋紡社製)を形質転換した。形質転換株より QIAprep(R) Spin Miniprep Kit(QIAGEN社製)を用いてプラスミド DNAを調製し、 BigDye Terminator Cycle Sequ encing Ready Reaction Kit v2.0(QIAGEN社製)と DNAシーケンサ ABI PRISM 377(Ap plied Biosystems社製)を用いて塩基配列を解析した。その結果、 HP-2型ハプトグロビ ンの cDNA配列を含むプラスミド pKAN- HP2を得た（図 4)。 [0195] The HP-2 haptoglobin DNA fragment (EcoRI-Bglll) and pKANTEX93 fragment (EcoRI-BamHI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and each about 1240 bp A 3 kbp DNA fragment was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, prepare 20 L of a reaction solution containing 20 ng of HP-2 type haptoglobin DNA fragment (EcoRI-BglII), 80 ng of pKANTEX93 fragment (EcoRI-BamHI) and Ligation High (Toyobo) at 16 ° C. The ligation reaction was performed for 16 hours. The resulting plasmid DNA was used to transform Escherichia coli DH5 strain (manufactured by Toyobo Co., Ltd.) by the heat shock method. Prepare plasmid DNA from the transformant using QIAprep (R) Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Ap plied) The base sequence was analyzed using Biosystems). As a result, plasmid pKAN-HP2 containing the cDNA sequence of HP-2 haptoglobin was obtained (Fig. 4).
[0196] 2. FUT8遺伝子ダブルノックアウト細胞への HP-1型ハプトグロビン発現プラスミドの 導 [0196] 2. Introduction of HP-1 haptoglobin expression plasmid into FUT8 gene double knockout cells
入  Enter
文献（Biotechnology and Bioengineering 87, 614 (2004))に記載された FUT8遺伝 子  FUT8 gene described in the literature (Biotechnology and Bioengineering 87, 614 (2004))
ダブルノックアウト細胞に、本実施例 1で作製したプラスミド pKAN-HPlを導入した。こ れ  The plasmid pKAN-HPl prepared in Example 1 was introduced into double knockout cells. this
らの遺伝子導入は公知のエレクト口ポレーシヨン法 [サイトテクノロジー（Cytotechnolo gy), 3, 133 (1990)]により以下の手順で行った。まず、プラスミド pKAN- HP230 gを N EBuffer 3 (New England Biolabs社製) 20 μ Lと 200単位の制限酵素 Mlul (New England Biolabs社製)を含む 200 Lの反応液を調製し、 37°Cで 16時間消化反応を行うことに より線状ィ匕した。反応後、該反応液に対しフエノール/クロ口ホルム抽出処理およびェ タノール沈殿により精製を行 、、線状ィ匕プラスミドを回収した。  These gene introductions were carried out by the following procedure according to a known electopore position method [Cytotechnology, 3, 133 (1990)]. First, prepare a reaction solution of 200 L containing 20 μL of NE buffer 3 (New England Biolabs) and 200 units of restriction enzyme Mlul (New England Biolabs) and plasmid pKAN-HP230 g at 37 ° C. A linear reaction occurred after digestion for 16 hours. After the reaction, the reaction mixture was purified by phenol / chloroform extraction treatment and ethanol precipitation, and the linear plasmid was recovered.
[0197] 次に、文献（Biotechnology and Bioengineering 87, 614 (2004))に記載された FUT8 遺伝子ダブルノックアウト細胞を K- PBS緩衝液 (137mmol/L KC1、 2.7mmol/L NaCl、 8 .lmmol/L Na HPO、 1.5mmol/L KH PO、 4.0mmol/L MgCl )に懸濁して 8 X 107細胞 [0197] Next, FUT8 gene double knockout cells described in the literature (Biotechnology and Bioengineering 87, 614 (2004)) were mixed with K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8. lmmol / L 8 x 10 7 cells suspended in Na HPO, 1.5mmol / L KH PO, 4.0mmol / L MgCl)
2 4 2 4 2  2 4 2 4 2
/mLとした。細胞懸濁液 L(1.6 X 106個）と上記の線状ィ匕プラスミド 10 gを混和 した後、細胞- DNA混和液の全量を Gene Pulser Cuvette (電極間距離 2mm) (BIO-RA D社製)へ移し、 Gene Pulser (BIO- RAD社製）を用いてパルス電圧 350V、電気容量 2 50 Fの条件で遺伝子導入を行った。遺伝子導入を行ったのち、細胞懸濁液を 10% ゥシ胎児血清（Life Technologies社製)および 50 μ g/mL gentamicin (ナカライテスタ 社製）を添カ卩した IMDM培地 (Life Technologies社製) 10mLに懸濁し、接着細胞培養 T 75フラスコ (グライナ一社製)へ播種した。培養は 5%CO、 37°Cの条件下で行った。 / mL. After the cell suspension L (1.6 X 10 6 cells) was mixed linear I spoon plasmid 10 g of the cell - the total amount of DNA mixed solution Gene Pulser Cuvette (inter-electrode distance 2mm) (BIO-RA D Co. And using Gene Pulser (manufactured by BIO-RAD), pulse voltage 350V, electric capacity 2 Gene transfer was performed under the condition of 50 F. After gene transfer, IMDM medium (Life Technologies) supplemented with 10% Ushi fetal serum (Life Technologies) and 50 μg / mL gentamicin (Nacalai Testa) The suspension was suspended in 10 mL, and seeded in an adherent cell culture T 75 flask (manufactured by Grainer). The culture was performed under conditions of 5% CO and 37 ° C.
2  2
[0198] 3. 200nM MTXffif性株の取得  [0198] 3. Acquisition of 200nM MTXffif stock
前項で得た PKAN-HP2導入細胞を 6日間培養した後、培養上清を除去し、 10%ゥ シ胎児透  After culturing the PKAN-HP2-introduced cells obtained in the previous section for 6 days, the culture supernatant is removed and 10% cyst fetal permeation is removed.
析血清、 50 μ g/mL gentamicinおよび 50nM methotrexate(MTX)(シグマ社製)を添カロ した IMDM培地を 10mL添カ卩した。この培地交換作業を 3〜4日毎に繰り返しながら 9日 間の培養を行った。次いで、 10%ゥシ胎児透析血清、 g/m Lgentamicinおよび 2 00nMの MTXを添カ卩した IMDM培地を用いた培地交換作業を同様に 3〜4日毎に繰り 返しながら 18日間培養し、 200nM MTX耐性株 HP2KOを得た。  10 mL of IMDM medium supplemented with analysis serum, 50 μg / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. 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 dialyzed serum, g / m Lgentamicin and 200 nM MTX was similarly repeated every 3-4 days for 18 days, and 200 nM MTX The resistant strain HP2KO was obtained.
[0199] 得られた HP2KOを 3.0 X 105cells/mLの濃度で 5mLの 10%ゥシ胎児透析血清、 50 μ g/mL gentamicinおよび 200nMの MTXを添カ卩した IMDM培地に懸濁し、 T25フラスコ へ播種して 3日間静置培養を行った。培養 3日後の培養上清を回収し、上清中に含 まれるハプトグロビン量を Haptoglobin Kit(Tridelta Development Limited社製)を用い て測定したところ、培養上清中に 15.1 g/mLの濃度でノヽブトグロビンが発現している ことを確認した。なお、 HP2KO株は、 HP2KOの株名で、平成 17年 2月 17日付けで独 立行政法人産業技術総合研究所特許生物寄託センター (茨城県つくば巿東 1丁目 1 番地 1号中央第 6)に FERMBP-10250として寄託されている。このようにして作製した H P2KO株の培養上清を、ハプトグロビンの一般的な精製法 (続医薬品の開発 廣川書 店 第 20卷、 215-219ページ)を用いて精製することによって得られた HP-2型ハプト グロビンは、通常の CHO/DG44株で生産したフコースが結合して!/、る糖鎖を有する H P-2型ハプトグロビンに比較して、血中半減期の延長が認められた。 [0199] The resulting HP2KO was suspended in IMDM medium supplemented with 5 mL of 10% urine fetal dialyzed serum, 50 μg / mL gentamicin and 200 nM MTX at a concentration of 3.0 X 10 5 cells / mL, and T25 The flask was inoculated and statically cultured for 3 days. The culture supernatant after 3 days of culture was collected, and the amount of haptoglobin contained in the supernatant was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited). It was confirmed that butoglobin was expressed. The HP2KO strain is the name of HP2KO, and the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center on February 17, 2005 (Tsukuba Ibaraki 1-chome, 1st, 1st, 1st, 6th) Has been deposited as FERMBP-10250. HP obtained by purifying the culture supernatant of the HP2KO strain prepared in this way using a general purification method for haptoglobin (development of follow-on pharmaceuticals, Yodogawa Shoten, No. 20, pages 215-219) -2 type haptoglobin has an increased half-life in blood compared to HP-2 type haptoglobin, which is linked to fucose produced by normal CHO / DG44 strain! .
実施例 3  Example 3
[0200] GDP-マンノースを GDP- 4-ケト, 6-デォキシ- GDP-マンノースに変換する脱水反応を 触媒する酵素の遺伝子が発現して ゝな 、細胞株の取得  [0200] Acquisition of a cell line that expresses a gene for an enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy- GDP-mannose
1.レクチン耐性 CHO/DG44株の取得 CHO/DG44細胞（Proc. Natl. Acad. Sci. USA, 77, 4216 (1980))を、 IMDM— FBS(10 )- HT(1)培地 [ゥシ胎児血清 (FBS) (インビトロジ ン社製）を 10%、 HT supplement (ィ ンビ 1. Acquisition of lectin resistant CHO / DG44 strain CHO / DG44 cells (Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)), IMDM—FBS (10) -HT (1) medium [Ushi Fetal Serum (FBS) (Invitrogen) 10%, HT supplement
トロジェン社製)を 1倍濃度で含む IMDM培地 (インビトロジェン社製) ]にて接着培養用 フラスコ 75cm2 (グライナ一社製）中で培養し、コンフルェント直前まで増殖させた。 5m Lのダルベッコ PBS (以下、 PBSと表記する）（インビトロジェン社製）にて細胞を洗浄後 、 PBSで希釈した 0.05%トリプシン (インビトロジェン社製）を 1.5mL添カ卩して 37°Cにて 5 分間放置し、細胞を培養器底面から剥離させた。剥離させた細胞を通常の細胞培養 で行われる遠心分離操作により回収し、 1 X 105細胞/ mLの密度になるように IMDM-F BS(10)- HT(1)培地を添カ卩して懸濁後、未添加又は 0.1 g/mLのアルキル化剤である MNNG (シグマ社製）を添カ卩した。 COインキュベーター (TABAI製)内で 37°Cにて 3日 In an IMDM medium (manufactured by Invitrogen) containing 1-fold concentration of IMDM medium (manufactured by Invitrogen)], the cells were cultured in a 75 cm 2 flask for adhesion culture (manufactured by Grainer) and grown until just before confluence. After washing the cells with 5 ml Dulbecco's PBS (hereinafter referred to as PBS) (Invitrogen), add 1.5 mL of 0.05% trypsin (Invitrogen) diluted with PBS at 37 ° C. The cells were left for 5 minutes to detach the cells from the bottom of the incubator. The were detached cells were recovered by centrifugation operation performed by the conventional cell culture, 1 X 10 5 cells / mL IMDM-F BS (10 ) such that the density of - HT (1) medium was添Ka卩After suspension, MNNG (manufactured by Sigma), which was not added or 0.1 g / mL alkylating agent, was added. 3 days at 37 ° C in CO incubator (TABAI)
2  2
間放置後、培養上清を除き、再び上述した操作と同様の操作で細胞を洗浄、剥離、 回収し、 IMDM- FBS(IO)- HT(1)培地に懸濁後、接着培養用 96穴プレート (旭テクノグ ラス社製)に 1000細胞/ゥエルの密度で播種した。各ゥエルには培地中終濃度で lmg/ mLのレンズマメレクチン (Lens culinaris agglutinin;以下、 LCAと表記、 Vector社製)を 添加した。 COインキュベータ内で 37°Cにて 2週間培養後、出現したコロニーをレクチ  After standing for a while, the culture supernatant is removed, and the cells are washed, detached, and collected again in the same manner as described above, suspended in IMDM-FBS (IO) -HT (1) medium, and then 96-well for adhesion culture. Plates (Asahi Techno Glass) were seeded at a density of 1000 cells / well. Each well was supplemented with lmg / mL lentil lectin (Lens culinaris agglutinin; hereinafter referred to as LCA, manufactured by Vector) at a final concentration in the medium. After incubation for 2 weeks at 37 ° C in a CO incubator, the colonies that appeared
2  2
ン耐性 CHO/DG44細胞株として取得した。  The CHO / DG44 cell line was obtained.
[0201] 2.取得したレクチン而ォ性 CHO/DG44細胞株の GDP-マンノース 4,6-デヒドラターゼ mRNAの定量 [0201] 2. Quantification of GDP-mannose 4,6-dehydratase mRNA in the obtained lectin metabolite CHO / DG44 cell line
前項で取得した各レクチン耐性 CHO/DG44細胞株における、 GDP-マンノースを G DP-4-ケト, 6-デォキシ- GDP-マンノースに変換する脱水反応を触媒する酵素である GDP-マンノース 4, 6-デヒドラターゼの発現量を、 RT-PCR法を用いて以下の様に解 祈した。  GDP-mannose 4, 6-, an enzyme that catalyzes the dehydration reaction of converting GDP-mannose into GDP-4-keto, 6-deoxy- GDP-mannose in each lectin-resistant CHO / DG44 cell line obtained in the previous section The expression level of dehydratase was calculated using the RT-PCR method as follows.
[0202] (1)レクチン耐性 CHO/DG44細胞株からの RNA調製と一本鎖 cDNAの調製  [0202] (1) Preparation of RNA from lectin-resistant CHO / DG44 cell line and preparation of single-stranded cDNA
親株である CHO/DG44細胞、および本実施例の 1項で取得した各レクチン耐性 CH 0/DG44細  The parental CHO / DG44 cells and the lectin resistant CH 0 / DG44 cells obtained in section 1 of this example.
胞株それぞれ 1 X 107細胞より、 RNeasy Protect Mini kit (キアゲン社製)を用いて、添 付 の使用説明書に従って RNAを調製した。続いて、 SUPER SCRIPT First-Strand synth esis system for RT-PCR (インビトロジェン社製）を用い、添付の使用説明書に従って 各 RNA5 gより 20 Lの反応液中にて一本鎖 cDNAを合成した。 Each cell line is attached from 1 X 10 7 cells using the RNeasy Protect Mini kit (Qiagen). RNA was prepared according to the instructions for use. Subsequently, using a SUPER SCRIPT First-Strand synthesis system for RT-PCR (manufactured by Invitrogen), single-stranded cDNA was synthesized from 5 g of each RNA in a 20 L reaction solution according to the attached instruction manual.
[0203] (2) RT- PCR法を用いた β -ァクチン遺伝子の発現量解析 [0203] (2) Expression analysis of β-actin gene using RT-PCR
本項の (1)で作製した各細胞株由来の一本鎖 cDNAの品質を確かめる目的で、 /3 - ァクチン cDNA力 PCR法によって増幅されるかを、以下の様に検討した。  For the purpose of confirming the quality of single-stranded cDNA derived from each cell line prepared in (1) of this section, we examined whether it can be amplified by the / 3-actin cDNA force PCR method as follows.
本項 (1)で取得したそれぞれのレクチン耐性 CHO/DG44細胞株の GDP-マンノース 4,6-デヒドラターゼ遺伝子の発現量解析を行った。まず、 /3 -ァクチン遺伝子の cDNA を PCR法によって増幅するために、 CHO細胞由来の β -ァクチンの cDNA配列（NCBI ァクセッション番号： U20114)より、配列番号 22で示される塩某配列を有する 24merの 合成オリゴ DNAプライマー 、配列番号 23で示される塩某配列を有する 24merの合 成オリゴ DNAプライマーを作製した。続いて、本項の (1)で作製した各細胞株由来の 一本鎖 cDNAO.5 μ Lを铸型として含む 20 μ Lの反応液 [1 X EX Taq Buffer (宝酒造社 製）、 0.2mMの dNTPsゝ 0.5単位の EX Taqpolymerase (宝酒造社製）、 0.5 μ Μの配列 番号 22 23の合成オリゴ DNAプライマー Ίを調製し、 DNAサーマルサイクラ一 480 (パ 一キンエルマ一社製）を用いて、 94°Cにて 5分間加熱した後、 94°Cにて 1分間、 68°C にて 2分間のサイクルを 14サイクル行なった。上記の該 PCR反応液 10 Lをァガロー ス電気泳動した後、サイバーグリーン (BMA社製)を用いて DNA断片を染色し、予想 される約 800bpの DNA断片量を Fluorlmager SI (モレキュラーダイナミクス社製）を用い て測定した。その結果、調製したいずれの細胞株由来の一本鎖 cDNAを用いても、同 程度の β -ァクチンの発現を検出することができた。  The expression level of the GDP-mannose 4,6-dehydratase gene in each lectin resistant CHO / DG44 cell line obtained in (1) of this section was analyzed. First, in order to amplify the cDNA of the / 3-actin gene by PCR, the CHO cell-derived β-actin cDNA sequence (NCBI accession number: U20114) has a salted salmon sequence represented by SEQ ID NO: 22 A 24-mer synthetic oligo DNA primer having a 24 mol synthetic oligo DNA primer having a salt-and-sugar sequence represented by SEQ ID NO: 23 was prepared. Subsequently, a 20 μL reaction solution containing 1 μC of 5 μL of single-stranded cDNA derived from each cell line prepared in (1) of this section as a cage [1 X EX Taq Buffer (Takara Shuzo), 0.2 mM DNTPs ゝ 0.5 units EX Taqpolymerase (Takara Shuzo), 0.5 μΜ of synthetic oligo DNA primer の of SEQ ID NO: 22 23 was prepared, and DNA thermal cycler 480 (manufactured by Pakin Elma) was used. After heating at ° C for 5 minutes, 14 cycles of 94 ° C for 1 minute and 68 ° C for 2 minutes were performed. After 10 L of the above PCR reaction solution was subjected to agarose electrophoresis, DNA fragments were stained using Cyber Green (manufactured by BMA), and the expected DNA fragment amount of about 800 bp was obtained from Fluorlmager SI (manufactured by Molecular Dynamics). Measured using As a result, the same level of β-actin expression could be detected using the single-stranded cDNA derived from any cell line prepared.
[0204] (3) RT-PCR法を用いた GDP-マンノース 4,6-デヒドラターゼ遺伝子の発現量解析 次に、本項 (1)で取得したそれぞれのレクチン耐性 CHO/DG44細胞株の GDP-マン ノース 4,6-デヒドラターゼ遺伝子の発現量解析を行った。 00?-マンノース4,6-デヒド ラターゼ遺伝子の cDNAを PCR法によって増幅するために、配列番号 7で示される CH 0細胞由来の GDP-マンノース 4,6-デヒドラターゼの cDNA配列より、配列番号 24で 示される塩基配列を有する 26merの合成オリゴ DNAプライマーと、配列番号 25で示さ れる塩基配列を有する 28mer の合成オリゴ DNAプライマーを作製した。続いて、本項 (1)で作製した各細胞株由来 の一本鎖 cDNA 0.5 μ Lを铸型として含む 20 μ Lの反応液 [1 X EX Taq Buffer (宝酒造 社製）、 0.2mMの dNTP mixtureゝ 0.5単位の Ex Taq polymerase (宝酒造社製）、 0.5 μ Μの配列番号 24と 25の合成 DNAプライマー]を調製し、 DNAサーマルサイクラ一 480 (パーキンエルマ一社製）を用いて、 94°Cにて 5分間加熱した後、 94°Cにて 1分間、 68 °Cにて 2分間のサイクルを 30サイクル行なった。上記の該 PCR反応液 10 Lをァガロ ース電気泳動した後、サイバーグリーン (BMA社製)を用いて DNA断片を染色し、予 想される約 430bpの DNA断片量を Fluor Imager SI (モレキュラーダイナミクス社製）を 用いて測定した。その結果、取得したレクチン耐性 CHO/DG44細胞株の中に、 GDP- マンノース 4,6-デヒドラターゼ遺伝子の発現が観察されな 、細胞株が存在することを 確認した。この GDP-マンノース 4,6-デヒドラターゼ遺伝子の発現が観察されない株 を CHO SM株と名付けた。なお、取得した CHO SM株の各種レクチンに対する耐性を 調べたところ、 CHO SM株は、 LCAが認識する糖鎖構造と同じ糖鎖構造を認識する レクチン、すなわち、 N-グリコシド結合糖鎖還元末端の N-ァセチルダルコサミン残基 の 6位とフコースの 1位が a結合で付加された糖鎖構造を認識する他のレクチンに対 しても耐性を示した。具体的には、終濃度 lmg/mLのエンドゥマメレクチン (Pisumsativ um Agglutinin ;以下、 PSAと表記、 Vector社製）が添加された培地、あるいは終濃度 1 mg/mLのヒイロチヤワンタケレクチン（Aleuria aurantia Lectin ;以下、 AALと表記、 Vec tor社製）が添加された培地でも耐性を有して 、た。 [0204] (3) Analysis of the expression level of GDP-mannose 4,6-dehydratase gene using RT-PCR method Next, the GDP-Mann of each lectin-resistant CHO / DG44 cell line obtained in (1) above. The expression level of the north 4,6-dehydratase gene was analyzed. In order to amplify the cDNA of 00? -Mannose 4,6-dehydratase gene by PCR, SEQ ID NO: 24 from the cDNA sequence of GDP-mannose 4,6-dehydratase derived from CH 0 cell shown in SEQ ID NO: 7 26mer synthetic oligo DNA primer having the nucleotide sequence shown and 28mer having the nucleotide sequence shown in SEQ ID NO: 25 A synthetic oligo DNA primer was prepared. Next, 20 μL of reaction solution containing 0.5 μL of single-stranded cDNA derived from each cell line prepared in this section (1) as a cage [1 X EX Taq Buffer (Takara Shuzo), 0.2 mM dNTP Prepare a mixture of 0.5 units of Ex Taq polymerase (Takara Shuzo), 0.5 μΜ of synthetic DNA primers of SEQ ID NOS: 24 and 25], and use a DNA thermal cycler 480 (Perkin Elma) to make 94 ° After heating at C for 5 minutes, 30 cycles of 94 ° C for 1 minute and 68 ° C for 2 minutes were performed. After 10 L of the above PCR reaction solution was subjected to agarose electrophoresis, the DNA fragment was stained using Cyber Green (manufactured by BMA), and the expected amount of DNA fragment of about 430 bp was obtained from Fluor Imager SI (Molecular Dynamics). The measurement was performed using As a result, it was confirmed that the cell line was present in the obtained lectin-resistant CHO / DG44 cell line when no expression of the GDP-mannose 4,6-dehydratase gene was observed. The strain in which the expression of the GDP-mannose 4,6-dehydratase gene was not observed was named CHO SM strain. In addition, when the resistance of the obtained CHO SM strain to various lectins was examined, the CHO SM strain is a lectin that recognizes the same sugar chain structure as that recognized by LCA, that is, the N-glycoside-linked sugar chain reducing terminal. It was also resistant to other lectins that recognize sugar chain structures in which the N-acetylyldarcosamine residue at position 6 and fucose at position 1 are attached by an a bond. Specifically, a medium supplemented with endumerme lectin (Pisumsativum Agglutinin; hereinafter referred to as PSA, manufactured by Vector) with a final concentration of 1 mg / mL, or a white bamboo lectin with a final concentration of 1 mg / mL (Aleuria) aurantia Lectin; hereinafter referred to as AAL, manufactured by Vector, Inc.) was also resistant.
3. GDP-マンノースを GDP- 4-ケト, 6-デォキシ- GDP-マンノースに変換する脱水反 応を触媒する酵素の遺伝子が発現して!/ヽな ヽ細胞株のゲノム解析  3. Gene analysis of an enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy- GDP-mannose!
CHO/DG44細胞、および前項で取得した CHO SM株を IMDM- FBS(10)- HT(1)培地 を用いて接着細胞培養用 T75フラスコ（グライナ一社製)でコンフルェントに到達する 直前まで培養した後、文献 [ヌクレイック'アシッド'リサーチ (Nuccleic Acid Research), 3, 2303, (1976)]に記載の方法従ってゲノム DNAを調製し、取得したゲノム DNAを TE- RNase緩衝液（pH8.0) [10mmol/lTris— HC1、 lmmol/1 EDTA、 200 μ g/ml RNase A] 30 0 1に一晩溶解させた。上記で調製したゲノム DNA 12 gを、 3種の異なる制限酵素 、 EcoRI (宝酒造社製）、 Hind!II (宝酒造社製）、 Bglll (宝酒造社製)でそれぞれ消化し 、エタノール沈殿法を用いて DNA断片を回収した後、 TE緩衝液 (pH8.0) [10mmol/l T ris- HC1、 lmmol/1 EDTA ] 20 μ 1に溶解し、 0.8%(w/v)ァガロースゲル電気泳動に供 した。泳動後、文献 [プロシーディンダス 'ォブ ·ザ'ナショナル 'アカデミー'ォブ 'サイ エンス (Proc. Natl. Acad. Sci. USA), 76, 3683,(1979)]に記載の方法に従い、ナイロン 膜へゲノム DNAを転写した。転写終了後、ナイロン膜に対して 80°Cで 2時間の熱処理 を行った。次に、ナイロンメンブレンに転写されたゲノム DNAの品質を確認する目的 で、細胞株を問わずゲノム中に均等に存在すると考えられる a 1,6-フコシルトランスフ エラーゼ（FUT8)遺伝子をプローブとしたサザンハイブリダィゼーシヨンを行った。 FU T8遺伝子を検出するためのプローブは以下のように調製した。まず、 WO02/31140の 実施例 11に記載のマウス FUT8cDNAを含むプラスミド m!FUT8- pCR2.1 10 μ gを 50 μ しの M buffer (宝酒造社製）に溶解し、制限酵素 Hindlll (宝酒造社製)で一晩消化し た後、反応液を H buffer (宝酒造社製）に置換し、制限酵素 EcoRI (宝酒造社製)でさ らに一晩消化反応を行った。反応終了後、該反応液を 2%ァガロース電気泳動に供 し、 FUT8遺伝子ェクソン 2を含む 156bpの EcoRI-Hindlll断片を精製した。得られた DN A断片 25ngに対し、 [ a -32P]dCTP 1.75MBqおよび Megaprime DNA labelingsystem, dCTP (アマシャムバイオサイエンス社製)を用いて放射標識した。次に、ハイブリダィ ゼーシヨンを以下のように行った。まず、上記ナイロン膜をローラーボトルへ封入し、 ハイブリダィゼーシヨン液 [4 X SSPE、 5 X Denhaldt，s液、 0.5%(w/v)SDS、 0.1mg/mLサ ケ*** DNA] 15mLをカ卩えて 65°Cで 3時間のプレハイブリダィゼーシヨンを行った。次 に32 P標識したプローブ DNAを熱変性してボトルへ投入し、 65°Cでー晚加温した。ノヽ イブリダィゼーシヨン後、ナイロン膜を 2 X SSC- 0.1%(w/v) SDS 50mLに浸漬し、 65°Cで 15分間加温した。上記の洗浄操作を 2回繰り返した後、膜を 0.2 X SSC-0.1%(w/v) S DS 50mLに浸漬し、 65°Cで 15分間加温した。洗浄後、ナイロン膜を X線フィルムへ- 80 °Cでニ晚暴露し現像した。現像後、ナイロン膜をストリッピング液 [1%SDS、 0.1 X SSC ]中で煮沸することにより、プローブを剥離させ、再度異なるプローブでのハイブリダィ ゼーシヨンに供することとした。上記の方法により、 CHO/DG44株および CHO SM株 いずれのゲノム DNAにおいても、 FUT8遺伝子ェクソン 2に特異的な断片が検出され た。以上の結果よりナイロン膜上に転写された CHO SM株および CHO/DG44株由来 のゲノム DNAは等しい品質を有して!/、ることが示された。 CHO / DG44 cells and the CHO SM strain obtained in the previous section were cultured using IMDM-FBS (10) -HT (1) medium in a T75 flask for adherent cell culture (manufactured by Grainer) until just before reaching confluence. Later, genomic DNA was prepared according to the method described in the literature [Nuccleic Acid Research, 3, 2303, (1976)], and the obtained genomic DNA was added to TE-RNase buffer (pH 8.0) [ 10 mmol / l Tris—HC1, lmmol / 1 EDTA, 200 μg / ml RNase A] 30 0 1 was dissolved overnight. 12 g of genomic DNA prepared above was digested with 3 different restriction enzymes, EcoRI (Takara Shuzo), Hind! II (Takara Shuzo), and Bglll (Takara Shuzo), respectively. After recovering the DNA fragment using the ethanol precipitation method, dissolve it in TE buffer (pH 8.0) [10mmol / l Tris-HC1, lmmol / 1 EDTA] 20μ1, 0.8% (w / v) The sample was subjected to agarose gel electrophoresis. After electrophoresis, nylon was produced according to the method described in the literature [Procedinas 'Ob The National' Academy 'Ob' Science (Proc. Natl. Acad. Sci. USA), 76, 3683, (1979)]. Genomic DNA was transferred to the membrane. After the transfer, the nylon membrane was heat treated at 80 ° C for 2 hours. Next, for the purpose of confirming the quality of the genomic DNA transferred to the nylon membrane, it is considered to exist evenly in the genome regardless of the cell line. A Southern probe using the 1,6-fucosyltransferase (FUT8) gene as a probe. Hybridization was performed. A probe for detecting the FU T8 gene was prepared as follows. First, 10 μg of plasmid m! FUT8-pCR2.1 containing mouse FUT8 cDNA described in Example 11 of WO02 / 31140 was dissolved in 50 μl of M buffer (Takara Shuzo), and restriction enzyme Hindlll (Takara Shuzo) was dissolved. After overnight digestion, the reaction solution was replaced with H buffer (Takara Shuzo), and the digestion reaction was further performed overnight with the restriction enzyme EcoRI (Takara Shuzo). After completion of the reaction, the reaction solution was subjected to 2% agarose electrophoresis, and a 156 bp EcoRI-Hindlll fragment containing FUT8 gene exon 2 was purified. The obtained DNA fragment (25 ng) was radiolabeled using [a- 32 P] dCTP 1.75 MBq and Megaprime DNA labeling system, dCTP (Amersham Biosciences). Next, hybridization was performed as follows. First, the above nylon membrane is sealed in a roller bottle, and 15 mL of a hybridization solution [4 X SSPE, 5 X Denhaldt, s solution, 0.5% (w / v) SDS, 0.1 mg / mL salmon sperm DNA] A prehybridization was performed at 65 ° C for 3 hours. Next, the 32 P-labeled probe DNA was heat denatured, put into a bottle, and heated at 65 ° C. After the hybridization, the nylon membrane was immersed in 50 mL of 2 X SSC-0.1% (w / v) SDS and heated at 65 ° C. for 15 minutes. After the above washing operation was repeated twice, the membrane was immersed in 50 mL of 0.2 X SSC-0.1% (w / v) S DS and heated at 65 ° C for 15 minutes. After washing, the nylon membrane was exposed to X-ray film at -80 ° C and developed. After development, the nylon membrane was boiled in a stripping solution [1% SDS, 0.1 X SSC] to peel off the probe and again subjected to hybridization with a different probe. By the above method, a fragment specific to FUT8 gene exon 2 was detected in the genomic DNA of both CHO / DG44 strain and CHO SM strain. Based on the above results, derived from CHO SM and CHO / DG44 strains transferred onto nylon membrane Of genomic DNA has been shown to have equal quality! /.
[0206] 一方、 GMD遺伝子ェクソン 5に特異的なプローブを以下のように調製した。まず、公 知であるヒト GMDゲノム DNA配列（NCBIァクセッション番号： NT_034880)を基に、ェ クソン 5に特異的に結合するオリゴ DNAプライマー（配列番号 26および配列番号 27) を設計した。該領域は配列番号 7に記載のヒト GMD cDNA配列の塩基番号 349から 塩基番号 538に相当する。次に、 WO02/31140の実施例 15に記載のプラスミド pAGE2 49GMDを lOng含む 100 μ Lの反応液 [ExTaq buffer (宝酒造社製）、 0.2mmol/L dNTPs 、 2.5 /z mol/L上記遺伝子特異的プライマー（配列番号 26および配列番号 27)]を調 製し、ポリメラーゼ連鎖反応 (PCR)を行った。 PCRは、 94°Cで 5分間の加熱の後、 94 °Cで 1分間、 58°Cで 2分間、 72°Cで 3分間からなる反応を 1サイクルとした 30サイクルの 条件で行った。 PCR後、反応液を 2%ァガロース電気泳動に供し、約 200bpの DNA断 片を精製した。得られた DNA断片 25ngに対し、 [ « -32P]dCTP 1.75MBqおよび Megapr ime DNA labeling system, dCTP (アマシャムバイオサイエンス社製）を用いて放射標 識した。該プローブを上記で示したナイロン膜に対してハイブリダィゼーシヨンを行つ た。その結果、 CHO/DG44細胞由来のゲノム DNAでは GMD遺伝子ェクソン 5の特異 的断片が見出されたのに対し、 CHOSM株由来のゲノム DNAにお!/ヽては GMD遺伝子 ェクソン 5の特異的断片が全く検出されな力つた。以上の結果から CHO SM株は GM Dをコードするゲノム領域のうち、少なくともェクソン 5を含む領域を欠損した GMDノッ クアウト細胞であることが示された。 [0206] On the other hand, a probe specific for GMD gene exon 5 was prepared as follows. First, oligo DNA primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically bind to exon 5 were designed based on the publicly known human GMD genomic DNA sequence (NCBI accession number: NT_034880). This region corresponds to nucleotide numbers 349 to 538 of the human GMD cDNA sequence shown in SEQ ID NO: 7. Next, a reaction solution of 100 μL containing lOng of plasmid pAGE2 49GMD described in Example 15 of WO02 / 31140 [ExTaq buffer (Takara Shuzo), 0.2 mmol / L dNTPs, 2.5 / z mol / L above gene-specific Primers (SEQ ID NO: 26 and SEQ ID NO: 27)] were prepared and subjected to polymerase chain reaction (PCR). PCR was performed under the conditions of 30 cycles, with a reaction consisting of 94 ° C for 5 minutes, 94 ° C for 1 minute, 58 ° C for 2 minutes, and 72 ° C for 3 minutes. After PCR, the reaction solution was subjected to 2% agarose electrophoresis, and an approximately 200 bp DNA fragment was purified. The obtained DNA fragment 25ng, [ «- 32 P] dCTP 1.75MBq and Megapr ime DNA labeling system, and radiation-labeled using dCTP (manufactured by Amersham Biosciences). The probe was hybridized to the nylon membrane shown above. As a result, a specific fragment of GMD gene exon 5 was found in genomic DNA derived from CHO / DG44 cells, whereas a specific fragment of GMD gene exon 5 was found in genomic DNA derived from CHOSM strain! Was not detected at all. From the above results, it was shown that the CHO SM strain is a GMD knockout cell lacking at least the region containing exon 5 in the genomic region encoding GMD.
実施例 4  Example 4
[0207] GMDノックアウト細胞による HP-1型ハプトグロビンの発現  [0207] Expression of HP-1 haptoglobin by GMD knockout cells
ヒト HP-1型ハプトグロビンを産生する GMDノックアウト細胞株を以下に示す方法で 作製  GMD knockout cell line producing human HP-1 haptoglobin is prepared by the following method
した。  did.
l.CHO SM株への HP- 1型ハプトグロビン発現プラスミドの導入  l. Introduction of HP-1 type haptoglobin expression plasmid into CHO SM strain
実施例 3にて作製した CHO SM株に、実施例 1で作製したプラスミド pKAN- HP1を導 入した。これらの遺伝子導入は公知のエレクト口ポレーシヨン法 [サイトテクノロジー (C ytotechnology), 3, 133 (1990)]により以下の手順で行った。まず、プラスミド pKAN-H PI 30 μ gを NE Buffer 3 (New England Biolabs社製) 20 μ Lと 200単位の制限酵素 Mlul (New England Biolabs社製)を含む 200 μ Lの反応液を調整し、 37°Cで 16時間消化反 応を行うことにより線状ィ匕した。反応後、該反応液に対しフ ノール/クロ口ホルム抽出 処理およびエタノール沈殿により精製を行い、線状ィ匕プラスミドを回収した。次に、実 施例 3で取得した CHO SM株を K- PBS緩衝液 (137mmol/L KC1、 2.7mmol/L NaCl、 8. lmmol/L Na HPO、 1.5mmol/L KH PO、 4.0mmol/L MgCl )に懸濁して 8 X 107細胞 The plasmid pKAN-HP1 prepared in Example 1 was introduced into the CHO SM strain prepared in Example 3. These gene introductions were carried out by the following procedure according to a known electopore position method [Cytotechnology, 3, 133 (1990)]. First, plasmid pKAN-H Prepare a reaction solution of 30 μg PI (20 μL NE Buffer 3 (New England Biolabs) and 200 μL 200 μL restriction enzyme Mlul (New England Biolabs) at 37 ° C for 16 hours. A linear reaction was achieved by performing a digestion reaction. After the reaction, the reaction mixture was purified by phenol / chloroform extraction treatment and ethanol precipitation to recover the linear plasmid. Next, the CHO SM strain obtained in Example 3 was added to K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8. lmmol / L Na HPO, 1.5 mmol / L KH PO, 4.0 mmol / L). 8 x 10 7 cells suspended in MgCl)
2 4 2 4 2  2 4 2 4 2
/mLとした。細胞懸濁液 200 L (1.6 X 106個）と上記の線状ィ匕プラスミド 10 gを混和 した後、細胞- DNA混和液の全量を Gene Pulser Cuvette (電極間距離 2mm)(BIO- RA D社製)へ移し、 Gene Pulser (BIO- RAD社製）を用いてパルス電圧 350V、電気容量 2 50 Fの条件で遺伝子導入を行った。遺伝子導入を行ったのち、細胞懸濁液を 10% ゥシ胎児血清（Life Technologies社製)および 50 μ g/mL gentamicin (ナカライテスタ 社製）を添カ卩した IMDM培地 (Life Technologies社製) 10mLに懸濁し、接着細胞培養 T 75フラスコ (グライナ一社製)へ播種した。培養は 5%CO、 37°Cの条件下で行った。 / mL. After mixing 200 L of cell suspension (1.6 × 10 6 cells) and 10 g of the above-mentioned linear plasmid, the total amount of the cell-DNA mixture was added to the Gene Pulser Cuvette (distance between electrodes 2 mm) (BIO-RA D The gene was transferred using Gene Pulser (manufactured by BIO-RAD) under the conditions of a pulse voltage of 350 V and an electric capacity of 250 F. After gene transfer, IMDM medium (Life Technologies) supplemented with 10% Ushi fetal serum (Life Technologies) and 50 μg / mL gentamicin (Nacalai Testa) The suspension was suspended in 10 mL, and seeded in an adherent cell culture T 75 flask (manufactured by Grainer). The culture was performed under conditions of 5% CO and 37 ° C.
2  2
2. 200nM MTXffif性株の取得  2. Acquisition of 200nM MTXffif stock
前項で得た pKAN-HPl導入細胞を 6日間培養した後、培養上清を除去し、 10%ゥ シ胎児透  After culturing the pKAN-HPl-introduced cells obtained in the previous section for 6 days, the culture supernatant is removed and 10% cyst fetal permeation is removed.
析血清、 50 μ g/mL gentamicinおよび 50nM methotrexate(MTX)(シグマ社製)を添カロ した IMDM培地を 10mL添カ卩した。この培地交換作業を 3〜4日毎に繰り返しながら 9日 間の培養を行った。次いで、 10%ゥシ胎児透析血清、 50 g/mLgentamicinおよび 20 OnMの MTXを添カ卩した IMDM培地を用いた培地交換作業を同様に 3〜4日毎に繰り 返しながら 18日間培養し、 200nM MTX耐性株を得た。 200nM MTX耐性株の培養上 清中に含まれるハプトグロビン量は、 Haptoglobin Kit(Tridelta Development Limited 社製)を用いて測定した。このようにして作製した 200nM MTX耐性株の培養上清を、 ハプトグロビンの一般的な精製法 (続医薬品の開発 廣川書店 第 20卷、 215-219ぺ ージ）を用いて精製することによって得られた HP-1型ハプトグロビンは、通常の CHO/ DG44株で生産したフコースが結合している糖鎖を有する HP-1型ハプトグロビンに比 較して、血中半減期の延長が認められた。 10 mL of IMDM medium supplemented with analysis serum, 50 μg / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Next, repeat the medium replacement using IMDM medium supplemented with 10% urine fetal dialyzed serum, 50 g / mL gentamicin and 20 OnM MTX in the same manner every 3-4 days, and culture for 18 days. A resistant strain was obtained. The amount of haptoglobin contained in the culture supernatant of the 200 nM MTX resistant strain was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited). The culture supernatant of the 200nM MTX-resistant strain produced in this way was obtained by purifying using the general purification method for haptoglobin (development of follow-on pharmaceuticals, Yodogawa Shoten No. 20, pages 215-219). In addition, HP-1 type haptoglobin has an increased blood half-life compared to HP-1 type haptoglobin, which has a sugar chain to which fucose is bound, produced by the normal CHO / DG44 strain.
実施例 5 [0209] GMDノックアウト細胞による HP-2型ハプトグロビンの発現 Example 5 [0209] Expression of HP-2 haptoglobin by GMD knockout cells
ヒト HP-2型ハプトグロビンを産生する GMDノックアウト細胞株を以下に示す方法で 作製  GMD knockout cell line producing human HP-2 haptoglobin is prepared by the following method
した。  did.
l.CHO SM株への HP- 2型ハプトグロビン発現プラスミドの導入  l. Introduction of HP-2 haptoglobin expression plasmid into CHO SM strain
実施例 3にて作製した CHO SM株に、実施例 2で作製したプラスミド pKAN- HP2を導 入した。これらの遺伝子導入は公知のエレクト口ポレーシヨン法 [サイトテクノロジー (C ytotechnology), 3, 133 (1990)]により以下の手順で行った。まず、プラスミド pKAN-H P2 30 μ gを NE Buffer 3 (New England Biolabs社製) 20 μ Lと 200単位の制限酵素 Mlul (New England Biolabs社製)を含む 200 μ Lの反応液を調整し、 37°Cで 16時間消化反 応を行うことにより線状ィ匕した。反応後、該反応液に対しフ ノール/クロ口ホルム抽出 処理およびエタノール沈殿により精製を行い、線状ィ匕プラスミドを回収した。次に、実 施例 3で取得した CHO SM株を K- PBS緩衝液 (137mmol/L KC1、 2.7mmol/L NaCl、 8. lmmol/L Na HPO、 1.5mmol/L KH PO、 4.0mmol/L MgCl )に懸濁して 8 X 107細胞 The plasmid pKAN-HP2 prepared in Example 2 was introduced into the CHO SM strain prepared in Example 3. These gene introductions were carried out by the following procedure according to a known electopore position method [Cytotechnology, 3, 133 (1990)]. First, prepare a 200 μL reaction solution containing 30 μg of plasmid pKAN-H P2 and 20 μL of NE Buffer 3 (New England Biolabs) and 200 units of restriction enzyme Mlul (New England Biolabs). Linear digestion was performed by digestion reaction at 37 ° C for 16 hours. After the reaction, the reaction mixture was purified by phenol / chloroform extraction treatment and ethanol precipitation to recover the linear plasmid. Next, the CHO SM strain obtained in Example 3 was added to K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8. lmmol / L Na HPO, 1.5 mmol / L KH PO, 4.0 mmol / L). 8 x 10 7 cells suspended in MgCl)
2 4 2 4 2  2 4 2 4 2
/mLとした。細胞懸濁液 200 L (1.6 X 106個）と上記の線状ィ匕プラスミド 10 gを混和 した後、細胞- DNA混和液の全量を Gene Pulser Cuvette (電極間距離 2mm)(BIO- RA D社製)へ移し、 Gene Pulser (BIO- RAD社製）を用いてパルス電圧 350V、電気容量 2 50 Fの条件で遺伝子導入を行った。遺伝子導入を行ったのち、細胞懸濁液を 10% ゥシ胎児血清（Life Technologies社製)および 50 μ g/mL gentamicin (ナカライテスタ 社製）を添カ卩した IMDM培地 (Life Technologies社製) 10mLに懸濁し、接着細胞培養 T 75フラスコ (グライナ一社製)へ播種した。培養は 5%CO、 37°Cの条件下で行った。 / mL. After mixing 200 L of cell suspension (1.6 × 10 6 cells) and 10 g of the above-mentioned linear plasmid, the total amount of the cell-DNA mixture was added to the Gene Pulser Cuvette (distance between electrodes 2 mm) (BIO-RA D The gene was transferred using Gene Pulser (manufactured by BIO-RAD) under the conditions of a pulse voltage of 350 V and an electric capacity of 250 F. After gene transfer, IMDM medium (Life Technologies) supplemented with 10% Ushi fetal serum (Life Technologies) and 50 μg / mL gentamicin (Nacalai Testa) The suspension was suspended in 10 mL, and seeded in an adherent cell culture T 75 flask (manufactured by Grainer). The culture was performed under conditions of 5% CO and 37 ° C.
2  2
[0210] 2. 200nM MTXffif性株の取得  [0210] 2. Acquisition of 200nM MTXffif sex stock
前項で得た PKAN-HP2導入細胞を 6日間培養した後、培養上清を除去し、 10%ゥ シ胎児透  After culturing the PKAN-HP2-introduced cells obtained in the previous section for 6 days, the culture supernatant is removed and 10% cyst fetal permeation is removed.
析血清、 50 μ g/mL gentamicinおよび 50nM methotrexate(MTX)(シグマ社製)を添カロ した IMDM培地を 10mL添カ卩した。この培地交換作業を 3〜4日毎に繰り返しながら 9日 間の培養を行った。次いで、 10%ゥシ胎児透析血清、 50 g/mL gentamicinおよび 2 00nMの MTXを添カ卩した IMDM培地を用いた培地交換作業を同様に 3〜4日毎に繰り 返しながら 18日間培養し、 200nM MTX耐性株を得た。 200nM MTX耐性株の培養上 清中に含まれるハプトグロビン量は、 Haptoglobin Kit(Tridelta Development Limited 社製)を用いて測定した。このようにして作製した 200nM MTX耐性株の培養上清を、 ハプトグロビンの一般的な精製法 (続医薬品の開発 廣川書店 第 20卷、 215-219ぺ ージ）を用いて精製することによって得られた HP-2型ハプトグロビンは、通常の CHO/ DG44株で生産したフコースが結合している糖鎖を有する HP-2型ハプトグロビンに比 較して、血中半減期の延長が認められた。 10 mL of IMDM medium supplemented with analysis serum, 50 μg / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Next, repeat the medium replacement using IMDM medium supplemented with 10% urine fetal dialyzed serum, 50 g / mL gentamicin and 200 nM MTX every 3-4 days. After culturing for 18 days, a 200 nM MTX resistant strain was obtained. The amount of haptoglobin contained in the culture supernatant of the 200 nM MTX resistant strain was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited). The culture supernatant of the 200nM MTX-resistant strain produced in this way was obtained by purifying using the general purification method for haptoglobin (development of follow-on pharmaceuticals, Yodogawa Shoten No. 20, pages 215-219). HP-2 haptoglobin was found to have an increased blood half-life compared to HP-2 haptoglobin, which has a sugar chain to which fucose is bound, produced by the normal CHO / DG44 strain.
実施例 6  Example 6
[0211] 酵母による遺伝子組換えハプトグロビンの発現  [0211] Expression of recombinant haptoglobin by yeast
酵母には多くの種類が知られているが、組換え蛋白質を発現させる宿主としてしば しば用いられる代表的な酵母として、ピキア（Pichia)属とサッカロマイセス (Saccaromy ces)属の酵母が挙げられる。通常、これらの酵母が発現する組換え蛋白質に付加さ れる N-結合型糖鎖の主要な構造は、還元末端側のコア部分に 2残基の N-ァセチル ダルコサミンを有し、非還元末端側の分岐部分に 9個から数十個のマンノース残基と 、数個から十数個のマンノース 6-リン酸残基を有する、ノ、ィマンノース型糖鎖であるこ とが知られている（Yeasty, 1191 (2002))。また、このような構造を有するハイマンノー ス型糖鎖は、ハイパーマンノース型糖鎖とも呼ばれる。  Many types of yeast are known, but typical yeasts often used as hosts for expressing recombinant proteins include yeasts of the genera Pichia and Saccaromyces. . Normally, the main structure of N-linked sugar chains added to recombinant proteins expressed by these yeasts has a 2-residue N-acetyl darcosamine in the core part on the reducing end, and the non-reducing end side. It is known that this is a mannose-type sugar chain having 9 to several tens of mannose residues and several to several tens of mannose 6-phosphate residues at the branch of 1191 (2002)). Further, a high mannose type sugar chain having such a structure is also called a hypermannose type sugar chain.
[0212] 以下に記載する実施例ではまず、主に付加される N-結合型糖鎖の構造として、ハ ィマンノース型糖鎖と複合型糖鎖の中間の構造である、ハイブリッド型糖鎖が主に付 カロされた HP-1型もしくは HP-2型ハプトグロビンを発現するピキア酵母株とサッカロマ イセス酵母株の作製方法につ!、て記載する。 [0212] In the examples described below, the hybrid sugar chain, which is an intermediate structure between the high-mannose sugar chain and the complex sugar chain, is mainly used as the structure of the N-linked sugar chain to be added. The methods for preparing Pichia yeast strains and Saccharomyces yeast strains that express the HP-1 type or HP-2 type haptoglobin that have been carotenized are described below.
1.ゲノム上に存在する PN01酵素遺伝子を破壊したピキア酵母株の作製 ピキア酵母株、たとえば Pichia pastoris GTS115株（インビトロジェン社製）などのゲノ ム DNAを铸型とし、 PCR法によって、ピキア酵母の PNOKphosphomannosylationof N- linked oligosaccharides 1)遺伝子（GenBankァクセッションナンバー： AB099514)の翻 訳領域全長の配列を増幅させる。増幅させた約 3200塩基長の PN01遺伝子配列は、 その 5，末端側半分の配列を、酵母由来の orotidine- 5'- phosphate decarboxylase (UR A3)遺伝子（GenBankァクセッションナンバー： AF321098)に置換した後、 pCR2.1- TO POベクター（インビトロジェン社製）などのベクターに挿入することにより、 PN01遺伝 子破壊用のプラスミドを作製する。次に、このプラスミド 100 gを制限酵素で線状ィ匕し た後、 PichiaExpressionKit (インビトロジェン社製）記載のエレクト口ポレーシヨン法によ つて、たとえば GTS115株などのピキア酵母に安定的に遺伝子導入を行う。次に、遺 伝子導入した酵母を、ゥラシルを欠損させた YPD培地 (インビトロジェン社製)を用い て室温にて培養し、増殖してきた各コロニーカゝらゲノム DNAを抽出する。次に、このゲ ノム DNAを铸型とした PCR法によって、酵母 PN01遺伝子座の配列を増幅させること により、相同組換えによって PN01遺伝子座が破壊された酵母クローンを選択する。 上記の方法により、ピキア酵母が発現する主要な N-結合型糖鎖の構造を、還元末端 側のコア部分に 2残基の N-ァセチルダルコサミンを有し、非還元末端側に 9個から数 十個のマンノース残基が結合した構造を有したハイマンノース型糖鎖に改変すること ができる。 1. Preparation of Pichia yeast strains that have disrupted the PN01 enzyme gene present on the genome Pichia yeast strains such as Pichia pastoris GTS115 (manufactured by Invitrogen Corp.) are used as genomic DNA, and PCR is used to perform PNOKphosphomannosylationof Pichia yeast. N-linked oligosaccharides 1) Amplify the entire translation region of the gene (GenBank accession number: AB099514). The amplified PN01 gene sequence with a length of about 3200 bases was replaced with the yeast orotidine-5'-phosphate decarboxylase (UR A3) gene (GenBank accession number: AF321098). After, pCR2.1-TO A plasmid for PN01 gene disruption is prepared by inserting into a vector such as PO vector (Invitrogen). Next, 100 g of this plasmid is linearized with a restriction enzyme, and then the gene is stably introduced into a Pichia yeast such as GTS115 strain, for example, by the electoral position method described in PichiaExpressionKit (manufactured by Invitrogen). . Next, the introduced yeast is cultured at room temperature using YPD medium (Invitrogen) deficient in uracil, and genomic DNA is extracted from each of the grown colonies. Next, a yeast clone in which the PN01 locus is disrupted by homologous recombination is selected by amplifying the yeast PN01 locus sequence by PCR using this genomic DNA as a saddle type. By the above method, the structure of the main N-linked sugar chain expressed in Pichia yeast has 9 residues on the non-reducing end side, with 2 residues of N-acetylyldarcosamine in the core part on the reducing end side. To a high mannose sugar chain having a structure in which several tens of mannose residues are bonded.
2.ゲノム上に存在する a -1,6-マンノース転移酵素遺伝子を破壊したピキア酵母株 の  2. Pichia yeast strains that have disrupted the a-1,6-mannose transferase gene present on the genome
作製 Production
ピキア酵母株、たとえば Pichia pastoris X- 33株（インビトロジェン社製）などのゲノム DNAを铸型とし、 PCR法によって、ピキア酵母の α - 1,6-マンノース転移酵素（OCH1 )遺伝子（GenBankァクセッションナンバー： AF540063)を増幅させる。増幅させた約 2 800塩  Pichia yeast strains, such as Pichia pastoris X-33 (manufactured by Invitrogen), are used in a vertical form, and by PCR, Pichia yeast α-1,6-mannose transferase (OCH1) gene (GenBank accession) Number: AF540063) is amplified. Amplified about 2 800 salt
基長の OCH1遺伝子配列は、その 5 '末端側半分の配列を、酵母由来の orotidine-5' -phosphate decarboxylase (URA3)遺伝子（GenBankァクセッションナンバー： AF3210 98)に置換した後、 pCR2.1-TOPOベクター（インビトロジェン社製）などのベクターに 挿入することにより、 OCH1遺伝子破壊用ベクターが作製される。次に、このベクター 100 gを制限酵素 Sfil (ニューイングランドバイオラブズ社製)で線状ィ匕した後、 Pichia Expression Kit (インビトロジェン社製）記載のエレクト口ポレーシヨン法によって、ピキ ァ酵母株、例えば前項に記載した PN01遺伝子破壊株や、 Pichia pastorisJC308株な どに対し、安定的な遺伝子導入を行う。次に、遺伝子導入した酵母を、ゥラシルを欠 損させた YPD培地 (インビトロジェン社製)を用いて室温にて培養し、増殖してきた各 コロニー力もゲノム DNAを抽出する。次に、このゲノム DNAを铸型とした PCR法によつ て、酵母 OCH1遺伝子座の配列を増幅させることにより、相同組換えによって OCH1 遺伝子座が破壊された酵母クローン株を選択する。上記の方法により、ピキア酵母が 発現する主要な N-結合型糖鎖の構造を、還元末端側のコア部分に 2残基の N-ァセ チルダルコサミンを有し、非還元末端側に 8個のマンノース残基が結合した構造を有 した Man8型ハイマンノース型糖鎖に改変することができる。 The OCH1 gene sequence of the base length was replaced with the yeast's orotidine-5'-phosphate decarboxylase (URA3) gene (GenBank accession number: AF3210 98) after replacing the 5 'terminal half sequence with pCR2.1 -A vector for disrupting the OCH1 gene is prepared by inserting into a vector such as TOPO vector (Invitrogen). Next, 100 g of this vector was linearly digested with the restriction enzyme Sfil (manufactured by New England Biolabs), and then the yeast yeast strain, for example, the above-mentioned item was obtained by the electoral position method described in Pichia Expression Kit (manufactured by Invitrogen). Stable gene transfer is carried out to the PN01 gene disruption strain described in 1) or the Pichia pastoris JC308 strain. Next, the transfected yeast was cultured at room temperature in YPD medium (Invitrogen) lacking uracil and grown. Colony strength also extracts genomic DNA. Next, a yeast clonal strain in which the OCH1 locus is destroyed by homologous recombination is selected by amplifying the yeast OCH1 locus sequence by PCR using this genomic DNA as a saddle type. By the above method, the structure of the main N-linked sugar chain expressed in Pichia yeast has 2 residues of N-acetyl darcosamine in the core part on the reducing end side, and 8 structures on the non-reducing end side. It can be modified to a Man8 type high mannose type sugar chain having a structure in which a mannose residue is bound.
[0214] 3.組換えキメラ型 a -1,2-マンノシダーゼ遺伝子を導入したピキア酵母株の作製 線虫（Caenorhabditis elegans)から RNeasy Mini Kit (キアゲン社製）を用いて全 RNA を抽出し、次にこの RNAを铸型として Superscript™first- strandcDNA synthesis kit (ィ ンビトロジェン社製）を用いて first- strand cDNAを調製する。次に、この cDNAを铸型 とし、特異的プライマーと KODポリメラーゼ (東洋紡績社製)を用いた PCRを行うことに より、線虫 α - 1,2-マンノシダーゼ（GenBankァクセッションナンバー： NM_073594)の 活性ドメインをコードする cDNAを特異的に増幅させる。増幅させた cDNAは、その 5 ' 末端側に、酵母の αマンノシダーゼ（MNS1)遺伝子（GenBankァクセッションナンパ 一： M63598)のリーダーペプチドをコードする cDNA配列を連結した後に、酵母用の 発現ベクター pPICZ (インビトロジェン社製)などのベクターに挿入し、酵母の小胞体 内にひ -1,2-マンノシダーゼを発現させるベクターを作製する。次にこのベクターを、 前項に記載した PN01遺伝子と OCH1遺伝子の両方の遺伝子を相同組換えで破壊し たピキア酵母株に対し、エレクト口ポレーシヨン法により安定的に導入する。遺伝子導 入後の酵母は、ゥラシルを欠損しゼォシン (インビトロジェン社製)を含有する YPD培 地 (インビトロジェン社製)で室温にて培養し、増殖してきた各コロニーから全 RNAを 抽出する。次に、この全 RNA力も調製した first-strand cDNAを铸型とした PCR法によ つて、組換えキメラ型 α - 1,2-マンノシダーゼの発現が認められた酵母クローン株を選 択する。上記の方法により、ピキア酵母が発現する主要な Ν-結合型糖鎖の構造を、 還元末端側のコア部分に 2残基の Ν-ァセチルダルコサミンを有し、非還元末端側に 5 個のマンノース残基が結合した構造を有した Man5型ハイマンノース型糖鎖に改変で きる。 [0214] 3. Preparation of Pichia yeast strain with recombinant chimeric a-1,2-mannosidase gene introduced Total RNA was extracted from Caenorhabditis elegans using RNeasy Mini Kit (Qiagen). First, prepare the first-strand cDNA using the Superscript ™ first-strand cDNA synthesis kit (manufactured by Invitrogen) using this RNA as a cage. Next, this cDNA was used as a saddle and PCR was carried out using specific primers and KOD polymerase (Toyobo Co., Ltd.), resulting in a nematode α-1,2-mannosidase (GenBank accession number: NM_073594). The cDNA encoding the active domain of is specifically amplified. The amplified cDNA is ligated to the 5 'end of the cDNA sequence encoding the yeast α-mannosidase (MNS1) gene (GenBank accession number: M63598) leader peptide, and then the yeast expression vector pPICZ. (Invitrogen) and other vectors are inserted into the yeast endoplasmic reticulum for expression of -1,2-mannosidase. Next, this vector is stably introduced into the Pichia yeast strain in which both the PN01 gene and the OCH1 gene described in the previous section have been disrupted by homologous recombination by the electopore method. The yeast after gene transfer is cultured at room temperature in a YPD medium (Invitrogen) containing zeosin (Invitrogen) and lacking uracil, and total RNA is extracted from each of the grown colonies. Next, a yeast clonal strain in which expression of the recombinant chimeric α-1,2-mannosidase is observed is selected by PCR using the first-strand cDNA prepared with this total RNA strength as a saddle type. By the above method, the structure of the main Ν-linked glycan expressed in Pichia yeast has 2 residues Ν-acetildarcosamine in the core part on the reducing end side and 5 on the non-reducing end side. It can be modified to a Man5 type high mannose type sugar chain having a structure in which the mannose residues are bound.
[0215] 4.組換え UDP-N-ァセチルダルコサミントランスポーター遺伝子を導入したピキア 酵母株の作製 [0215] 4. Pichia into which recombinant UDP-N-acetylyldarcosamine transporter gene was introduced Preparation of yeast strain
酵母（Kluyveromyces lactis)から RNeasy Mini Kit (キアゲン社製）を用いて全 RNAを 抽出し、次にこの RNAを铸型として Superscript™first- strandcDNA synthesis kit (ィ ンビトロジェン社製)を用いて cDNAを調製する。次に、この cDNAを铸型とし、特異的 プライマーと KODポリメラーゼ (東洋紡績社製)を用いた PCRを行うことにより、酵母 U DP-N-ァセチルダルコサミントランスポーターの翻訳領域全長をコードする cDNA(Ge nBankァクセッションナンバー： AF106080)を特異的に増幅させる。次に、増幅させた 約 3700塩基長の cDNAを、酵母用の発現ベクター pPIC3.5K (インビトロジェン社製）な どのベクターのアルコールォキシゲナーゼプロモーター配列の下流に位置する制限 酵素 EcoRI切断部位と Not I切断部位の間に挿入し、酵母のゴルジ体内に UDP-N-ァ セチルダルコサミントランスポーターを発現させるベクターを作製する。次にこのべク ターを、前項に記載した、 α - 1,2-マンノシダーゼ遺伝子を導入したピキア酵母株に 対し、エレクト口ポレーシヨン法により安定的に導入する。遺伝子導入後の酵母は、薬 剤 G418 (ナカライテスタ社製)を含有する YPD培地で室温にて培養し、増殖してきた 各コロニー力ゝら全 RNAを抽出する。次に、この全 RNAから調製した cDNAを铸型とした PCR法によって、組換え UDP-N-ァセチルダルコサミントランスポーターの発現が認め られた酵母クローン株を選択する。  Extract total RNA from yeast (Kluyveromyces lactis) using the RNeasy Mini Kit (Qiagen), and then prepare cDNA using the Superscript ™ first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is used as a saddle type, and PCR is performed using specific primers and KOD polymerase (Toyobo) to encode the entire translation region of the yeast UDP-N-acetylyldarcosamine transporter. cDNA (Gen Bank accession number: AF106080) is specifically amplified. Next, the amplified cDNA of about 3700 bases in length is used as a restriction enzyme EcoRI cleavage site located downstream of the alcohol oxygenase promoter sequence of vectors such as the yeast expression vector pPIC3.5K (manufactured by Invitrogen). A vector that inserts between I cleavage sites and expresses UDP-N-acetyltilcosamine transporter in the Golgi apparatus of yeast is prepared. Next, this vector is stably introduced into the Pichia yeast strain introduced with the α-1,2-mannosidase gene described in the previous section by the electopore method. The yeast after the gene introduction is cultured at room temperature in a YPD medium containing the drug G418 (manufactured by Nacalai Testa), and total RNA is extracted from each of the grown colonies. Next, a yeast clonal strain in which the expression of the recombinant UDP-N-acetylyldarcosamine transporter is observed is selected by PCR using the cDNA prepared from this total RNA as a saddle type.
5.組換えキメラ型 N-ァセチルダルコサミン転移酵素- 1遺伝子を導入したピキア酵 母株の作製  5. Production of a Pichia fermentation mother strain into which a recombinant chimeric N-acetyldylcosamine transferase-1 gene has been introduced
ヒト肝臓 cDNA (クロンテック社製)を铸型とし、特異的プライマーと KODポリメラーゼ (東洋紡績社製)を用いた PCRを行うことにより、 N-ァセチルダルコサミン転移酵素- 1 ( GenBankァクセッションナンバー： M55621)の活性ドメインをコードする cDNAを特異 的に増幅させる。増幅させた cDNAは、その 5'末端側に、酵母のマンノース転移酵素 (MNN9)遺伝子（GenBankァクセッションナンバー： L23752)のリーダーペプチドをコ ードする cDNA配列を連結した後に、酵母用の発現ベクター pAUR123 (タカラノィォ 社製）などのベクターのアルコールデヒドロゲナーゼプロモーター配列の下流に位置 する制限酵素 Kpnl切断部位と Xba I切断部位の間に挿入し、酵母のゴルジ体内に N- ァセチルダルコサミン転移酵素- 1を発現させるベクターを作製する。次にこのべクタ 一を、前項に記載した、 UDP-N-ァセチルダルコサミントランスポーター遺伝子を導入 したピキア酵母株に対し、発現ベクター pAUR123に添付のマニュアルに掲載された 酢酸リチウム法により導入する。遺伝子導入後の酵母は、薬剤ォ一口ブラシジン A (タ カラノィォ社製)を含有する YPD培地で室温にて培養し、増殖してきた各コロニーか ら全 RNAを抽出する。次に、この全 RNAから調製した cDNAを铸型とした PCR法によつ て、組換え N-ァセチルダルコサミン転移酵素- 1の発現が認められた酵母クローン株 を選択する。上記の方法により、ピキア酵母が発現する主要な N-結合型糖鎖の構造 を、還元末端側のコア部分に 2残基の N-ァセチルダルコサミンを有し、非還元末端側 に 5個のマンノース残基が結合した Man5型ハイマンノース型糖鎖の非還元末端側に 、 N-ァセチルダルコサミン残基が 1個付加された構造を有する、ノヽイブリツド型糖鎖に 改変することができる。 N-Acetyldarcosaminyltransferase-1 (GenBank accession number) was obtained by performing PCR using human liver cDNA (Clontech) in a cage and using specific primers and KOD polymerase (Toyobo). : Amplify specifically the cDNA encoding the active domain of M55621). The amplified cDNA is linked to the 5 'end of the cDNA sequence encoding the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752), and then expressed for yeast. It is inserted between the restriction enzyme Kpnl cleavage site and the Xba I cleavage site located downstream of the alcohol dehydrogenase promoter sequence of vectors such as vector pAUR123 (manufactured by Tacarano), and N-acetylyldarcosamine transferase- is inserted into the yeast Golgi. A vector for expressing 1 is prepared. Next, this vector One is introduced into the Pichia yeast strain into which the UDP-N-acetylcyldarcosamine transporter gene described in the previous section has been introduced, by the lithium acetate method described in the manual attached to the expression vector pAUR123. The yeast after gene transfer is cultured at room temperature in a YPD medium containing the drug mouthful brassin A (manufactured by Takara Noio), and total RNA is extracted from each of the grown colonies. Next, a yeast clonal strain in which expression of recombinant N-acetylyldarcosamine transferase-1 is observed is selected by PCR using the cDNA prepared from this total RNA as a saddle type. By the above method, the structure of the main N-linked sugar chain expressed in Pichia yeast has 2 residues of N-acetyldarcosamine in the core at the reducing end and 5 at the non-reducing end. It can be modified to a noblebrid sugar chain with a structure in which one N-acetylyldarcosamine residue is added to the non-reducing end of the Man5 type high mannose sugar chain to which the mannose residue is attached. .
[0217] 以上、 N-結合型糖鎖として、ノ、ィマンノース型糖鎖と複合型糖鎖の中間の構造で ある、ハイブリッド型糖鎖を主要に発現するピキア酵母株の作製方法について記載し た。上述のピキア酵母以外に、組換え蛋白質を発現させる宿主としてしばしば用いら れる酵母として、サッカロマイセス（Saccharomyces)属の酵母が挙げられる。以下、 N- 結合型糖鎖としてノ、イブリツド型糖鎖を主要に発現するサッカロマイセス酵母株の作 製方法について述べる。  [0217] The method for producing a Pichia yeast strain that mainly expresses a hybrid-type sugar chain, which is an intermediate structure between a no-, immannose-type sugar chain and a complex-type sugar chain, as an N-linked sugar chain has been described above. . In addition to the above-mentioned Pichia yeast, yeasts of the genus Saccharomyces can be mentioned as yeasts that are often used as hosts for expressing recombinant proteins. A method for producing a Saccharomyces yeast strain that mainly expresses N-linked sugar chains and hybrid sugar chains as follows is described.
[0218] 6.ゲノム上に存在する α - 1,6-マンノース転移酵素遺伝子と α - 1,3-マンノース転 移酵素遺伝子を破壊したサッカロマイセス酵母株の作製  [0218] 6. Production of Saccharomyces yeast strains with disrupted α-1,6-mannose transferase and α-1,3-mannose transferase genes present on the genome
Nakayamaらの方法（EMBO Journal, 11, 2511 (1992))に従い、相同組換えによって OCH1遺伝子座が破壊された酵母クローンを選択する。得られた OCH1遺伝子が破 壊されたサッカロマイセス酵母株は、 Shermanらの方法 (メソッズ'イン'ェンザィモロジ 一 194, 21 (1991))に従い、半数体細胞を誘導した後、 α - 1,3-マンノース転移酵素（ MNN1)遺伝子が破壊された変異酵母株 LB1-10B (カリフォルニア大学 Yeast Genetic Stock Center)の半数体細胞と混合し、窒素欠乏条件で培養することにより、二倍体 の接合子を形成させる。次に、得られた接合子を、ゥラシルとロイシンを欠損させた Y PD培地で室温にて培養し、増殖してきた各コロニー力もゲノム DNAを抽出する。次に 、このゲノム DNAを铸型とした PCR法によって、酵母 OCH1遺伝子座の配列（GenBan kァクセッションナンバー： AF540063)と、 MNN1遺伝子座の配列（GenBankァクセッシ ヨンナンバー： AF540063L23753)をそれぞれ増幅させることにより、 OCH1遺伝子座と MNN1遺伝子座の両方が破壊された酵母クローン株を選択する。上記の方法により、 サッカロマイセス酵母が発現する主要な N-結合型糖鎖の構造を、還元末端側のコア 部分に 2残基の N-ァセチルダルコサミンを有し、非還元末端側に 8個のマンノース残 基が結合した構造を有する、 Man8型ハイマンノース型糖鎖に改変できる。 According to the method of Nakayama et al. (EMBO Journal, 11, 2511 (1992)), a yeast clone in which the OCH1 locus is destroyed by homologous recombination is selected. The obtained Saccharomyces yeast strain in which the OCH1 gene was disrupted was derived from haploid cells according to the method of Sherman et al. (Methods'In'Enzymology 1 194, 21 (1991)), and then α-1,3-mannose. A diploid zygote is formed by mixing with haploid cells of the mutant yeast strain LB1-10B (University of California Yeast Genetic Stock Center) in which the transferase (MNN1) gene is disrupted and culturing under nitrogen-deficient conditions . Next, the obtained zygote is cultured at room temperature in YPD medium lacking uracil and leucine, and genomic DNA is extracted from each colony force that has grown. Next, this genomic DNA was used as a saddle-shaped PCR method to determine the sequence of the yeast OCH1 locus (GenBan k clone session number: AF540063) and MNN1 locus sequence (GenBank accession number: AF540063L23753) are amplified to select yeast clones in which both OCH1 locus and MNN1 locus are destroyed. By the above method, the structure of the main N-linked sugar chain expressed in Saccharomyces yeast has 2 residues N-acetylyldarcosamine in the core part on the reducing end side and 8 on the non-reducing end side. It can be modified into a Man8-type high mannose-type sugar chain having a structure in which the mannose residue is bound.
7.組換えキメラ型 a -1,2-マンノシダーゼ遺伝子を導入したサッカロマイセス酵母 株  7. Saccharomyces yeast strain introduced with recombinant chimeric a-1,2-mannosidase gene
の作製 Making
カビ（Aspergillus saitoi)から RNeasy Mini Kit (キアゲン社製）を用いて全 RNAを抽出 し、次にこの RNAを铸型として Superscript™first- strandcDNA synthesis kit (イン ビトロジェン社製)を用いて cDNAを調製する。次に、この cDNAを铸型とし、特異的プ ライマーと KODポリメラーゼ (東洋紡績社製)を用いた PCRを行うことにより、カビ a -1, 2-マンノシダーゼの翻訳領域全長をコードする cDNA (GenBankァクセッションナンパ 一： D49827)を特異的に増幅させる。増幅させた約 1500塩基長の cDNAは、その翻訳 終止コドンを削除した 3 '末端側に、酵母の小胞体局在シグナルペプチド (ェンボジャ ーナル 7, 913 (1988))、すなわちヒスチジンーァスパラギン酸一グルタミン酸一口イシ ンをコードする cDNA配列と翻訳終止コドンを連結した後に、酵母用の発現ベクター p PICZ (インビトロジェン社製）などのベクターに挿入し、酵母の小胞体内に α -1,2-マ ンノシダーゼを発現させるベクターを作製する。次にこのベクターを、前項に記載した 、 a -1 ,6-マンノース転移酵素遺伝子と a -1 ,3-マンノース転移酵素遺伝子を破壊し たサッカロマイセス酵母株に対し、エレクト口ポレーシヨン法により安定的に導入する。 遺伝子導入後の酵母は、ゥラシルを欠損しゼォシン (インビトロジェン社製)を含有す る YPD培地 (インビトロジェン社製)で室温にて培養し、増殖してきた各コロニー力ゝら全 RNAを抽出する。次に、この全 RNA力も調製した cDNAを铸型とした PCR法によって、 組換えキメラ型 (X -1,2-マンノシダーゼの発現が認められた酵母クローン株を選択す る。上記の方法により、サッカロマイセス酵母が発現する主要な N-結合型糖鎖の構 造を、還元末端側のコア部分に 2残基の N-ァセチルダルコサミンを有し、非還元末端 側に 5個のマンノース残基が結合した構造を有する、 Man5型ハイマンノース型糖鎖に 改変することができる。 Extract total RNA from mold (Aspergillus saitoi) using RNeasy Mini Kit (Qiagen), and then prepare cDNA using Superscript ™ first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is converted into a saddle type, and PCR using a specific primer and KOD polymerase (Toyobo Co., Ltd.) is performed to obtain a cDNA encoding the full-length translation region of mold a-1,2-mannosidase (GenBank Accession picker 1: Amplify D49827) specifically. The amplified cDNA with a length of about 1500 bases has a yeast endoplasmic reticulum localization signal peptide (Embo Journal 7, 913 (1988)), that is, histidine-aspartate, at the 3 'end from which the stop codon was deleted. After ligating a cDNA sequence encoding a monoglutamate bite isine and a translation termination codon, it is inserted into a vector such as the yeast expression vector pPICZ (Invitrogen) and α-1,2- Create a vector to express mannosidase. Next, this vector was stably added to the Saccharomyces yeast strain in which the a -1,6-mannose transferase gene and the a -1,3-mannose transferase gene described in the previous section were disrupted by the electopore method. Introduce. The yeast after gene transfer is cultured at room temperature in a YPD medium (Invitrogen) containing zeocin (Invitrogen) and lacking uracil, and total RNA is extracted from each of the grown colonies. Next, a recombinant chimeric type (yeast clone strain in which the expression of X-1,2-mannosidase was observed was selected by PCR using this cDNA with the total RNA strength prepared as a saddle type. By the above method, The main N-linked sugar chain structure expressed by Saccharomyces yeast has 2 residues of N-acetyldarcosamine in the core of the reducing end, and the non-reducing end It can be modified into a Man5 type high mannose type sugar chain having a structure in which 5 mannose residues are bonded to the side.
[0220] 8.組換え UDP-N-ァセチルダルコサミントランスポーター遺伝子を導入したサッカロ マイセス酵母株の作製  [0220] 8. Production of Saccharomyces yeast strains with recombinant UDP-N-acetylcylcosamine transporter gene
酵母（Kluyveromyces lactis)から RNeasy Mini Kit (キアゲン社製）を用いて全 RNAを 抽出し、次にこの RNAを铸型として Superscript™first- strandcDNA synthesis kit (ィ ンビトロジェン社製)を用いて cDNAを調製する。次に、この cDNAを铸型とし、特異的 プライマーと KODポリメラーゼ (東洋紡績社製)を用いた PCRを行うことにより、酵母 U DP-N-ァセチルダルコサミントランスポーターの翻訳領域全長をコードする cDNA (Ge nBankァクセ  Extract total RNA from yeast (Kluyveromyces lactis) using the RNeasy Mini Kit (Qiagen), and then prepare cDNA using the Superscript ™ first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is used as a saddle type, and PCR is performed using specific primers and KOD polymerase (Toyobo) to encode the entire translation region of the yeast UDP-N-acetylyldarcosamine transporter. cDNA (GenBank
ッシヨンナンバー: AF106080)を特異的に増幅させる。次に、増幅させた約 3700塩基 長の cDNAを、酵母用の発現ベクター pPIC3.5K (インビトロジェン社製）などのベクタ 一のアルコールォキシゲナーゼプロモーター配列の下流に位置する制限酵素 EcoRI 切断部位と Not I切断部位の間に挿入し、酵母のゴルジ体内に UDP-N-ァセチルダ ルコサミントランスポーターを発現させるベクターを作製する。次にこのベクターを、前 項に記載した、 a -1,2-マンノシダーゼ遺伝子を導入したサッカロマイセス酵母株に 対し、エレクト口ポレーシヨン法により安定的に導入する。遺伝子導入後の酵母は、薬 剤 G418 (ナカライテスタ社製)を含有する YPD培地で室温にて培養し、増殖してきた 各コロニー力ゝら全 RNAを抽出する。次に、この全 RNAから調製した cDNAを铸型とした PCR法によって、組換え UDP-N-ァセチルダルコサミントランスポーターの発現が認め られた酵母クローン株を選択する。  Specific number (AF106080). Next, the amplified cDNA having a length of about 3700 bases is combined with a restriction enzyme EcoRI cleavage site located downstream of a single alcohol oxygenase promoter sequence such as the yeast expression vector pPIC3.5K (manufactured by Invitrogen). A vector that inserts between Not I cleavage sites and expresses UDP-N-acetyltilcosamine transporter in the yeast Golgi is prepared. Next, this vector is stably introduced into the Saccharomyces yeast strain into which the a-1,2-mannosidase gene has been introduced as described in the previous section by the electopore method. The yeast after the gene introduction is cultured at room temperature in a YPD medium containing the drug G418 (manufactured by Nacalai Testa), and total RNA is extracted from each of the grown colonies. Next, a yeast clonal strain in which the expression of the recombinant UDP-N-acetylyldarcosamine transporter is observed is selected by PCR using the cDNA prepared from this total RNA as a saddle type.
[0221] 9.組換えキメラ型 N-ァセチルダルコサミン転移酵素- 1遺伝子を導入したサッカロマ イセス酵母株の作製 [0221] 9. Production of Saccharomyces yeast strains into which recombinant chimeric N-acetylcylcosamine transferase-1 gene was introduced
ヒト肝臓 cDNA (クロンテック社製)を铸型とし、特異的プライマーと KODポリメラーゼ (東洋紡績社製)を用いた PCRを行うことにより、 N-ァセチルダルコサミン転移酵素- 1 ( GenBankァクセッションナンバー： M55621)の活性ドメインをコードする cDNAを特異 的に増幅させる。増幅させた cDNAは、その 5 '末端側に、酵母のマンノース転移酵素 (MNN9)遺伝子（GenBankァクセッションナンバー： L23752)のリーダーペプチドをコ ードする cDNA配列を連結した後に、酵母用の発現ベクター pAUR123 (タカラノィォ 社製）などのベクターのアルコールデヒドロゲナーゼプロモーター配列の下流に位置 する制限酵素 Kpnl切断部位と Xba I切断部位の間に挿入し、酵母のゴルジ体内に N- ァセチルダルコサミン転移酵素- 1を発現させるベクターを作製する。次にこのべクタ 一を、前項に記載した、 UDP-N-ァセチルダルコサミントランスポーター遺伝子を導入 したサッカロマイセス酵母株に対し、発現ベクター pAUR123に添付のマニュアルに掲 載された酢酸リチウム法により導入する。遺伝子導入後の酵母は、薬剤ォ一口ブラシ ジン A (タカラバイオ社製)を含有する YPD培地で室温にて培養し、増殖してきた各コ 口-一から全 RNAを抽出する。次に、この全 RNA力も調製した cDNAを铸型とした PCR 法によって、組換え N-ァセチルダルコサミン転移酵素- 1の発現が認められた酵母クロ 一ン株を選択する。上記の方法により、サッカロマイセス酵母が発現する主要な N-結 合型糖鎖の構造を、還元末端側のコア部分に 2残基の N-ァセチルダルコサミンを有 し、非還元末端側に 5個のマンノース残基が結合した構造を有する Man5型ハイマンノ 一ス型糖鎖の非還元末端側に、 N-ァセチルダルコサミン残基が 1個付加された、ノヽ イブリツド型糖鎖に改変できる。 N-acetylylcosamine transferase-1 (GenBank accession number) was obtained by performing PCR using human liver cDNA (Clontech) in a saddle shape and specific primers and KOD polymerase (Toyobo). : Amplify specifically the cDNA encoding the active domain of M55621). The amplified cDNA contains the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752) at the 5 'end. After ligation of the cDNA sequence to be loaded, it is inserted between the restriction enzyme Kpnl cleavage site and the Xba I cleavage site located downstream of the alcohol dehydrogenase promoter sequence of vectors such as the yeast expression vector pAUR123 (Tacarano). A vector that expresses N-acetyltilcosamine transferase-1 in the Golgi apparatus of yeast is prepared. Next, this vector was introduced into the Saccharomyces 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. To do. The yeast after the gene introduction is cultured at room temperature in a YPD medium containing a drug mouthful brassin A (manufactured by Takara Bio Inc.), and total RNA is extracted from each grown mouthpiece. Next, a yeast clone strain in which the expression of recombinant N-acetyl dalcosamine transferase-1 has been observed is selected by PCR using this cDNA, which has also been prepared for total RNA, in a vertical form. According to the above method, the structure of the main N-linked sugar chain expressed in Saccharomyces yeast has a 2-residue N-acetylyldarcosamine in the core portion on the reducing end side and 5 in the non-reducing end side. It can be modified to a nodule-type sugar chain in which one N-acetylyldarcosamine residue is added to the non-reducing end of the Man5 type high mannose type sugar chain having a structure in which one mannose residue is bound.
[0222] 以上の通り、 N-結合型糖鎖として Man5型ハイマンノース型糖鎖の非還元末端側に N-ァセチルダルコサミン残基が 1個付加された、ハイブリッド型糖鎖を主要に発現す るピキア酵母株、あるいはサッカロマイセス酵母株の作製方法について述べた。次に 、これらの酵母株を宿主として用い、 N-結合型糖鎖としてハイブリッド型糖鎖を主要 に有する組換えヒトハブトグロビンの調製方法について述べる。  [0222] As described above, mainly hybrid type glycans with one N-acetylyldarcosamine residue added to the non-reducing end of Man5 type high mannose type glycans as N-linked type glycans A method for producing the Pichia yeast strain or the Saccharomyces yeast strain was described. Next, a method for preparing recombinant human hub globin mainly using hybrid sugar chains as N-linked sugar chains using these yeast strains as hosts will be described.
[0223] 10.組換えヒトハプトグロビン発現ベクターの作製  [0223] 10. Construction of recombinant human haptoglobin expression vector
Yamauchiらの方法 (Bioscience, Biotechnology and Biochemistry 5り, 600 (1992))に 従い、ヒト肝臓 cDNA(Invitrogen社製）を铸型とし、増幅用酵素として Pyrobest DNA p olymerase (タカラバイオ社製)を用いた PCR反応により、成熟型ハプトグロビンの全長 をコードする cDNAを特異的に増幅させる。次に、得られた HP-1型もしくは HP-2型ハ プトグロビン cDNAを、酵母用の発現ベクター pPIC6 a (インビトロジェン社製）などの ベクターのアルコールォキシゲナーゼプロモーター配列の下流に位置する制限酵素 Clal切断部位と Xbal切断部位の間に挿入し、成熟型ヒト HP-1型ハプトグロビンを分泌 発現させるベクター PPIC6 a /HP1および成熟型ヒト HP-2型ハプトグロビンを分泌発 現させるベクター pPIC6 a /HP2を作製する。 In accordance with the method of Yamauchi et al. (Bioscience, Biotechnology and Biochemistry 5, 600 (1992)), human liver cDNA (manufactured by Invitrogen) is used as a cage and Pyrobest DNA polymerase (manufactured by Takara Bio Inc.) is used as an amplification enzyme. The cDNA encoding the full-length mature haptoglobin is specifically amplified by the PCR reaction. Next, the obtained HP-1 type or HP-2 type haptoglobin cDNA is used as a restriction enzyme located downstream of the alcohol oxygenase promoter sequence of a vector such as the yeast expression vector pPIC6a (manufactured by Invitrogen). Inserted between Clal cleavage site and Xbal cleavage site to secrete mature human HP-1 haptoglobin A vector PPIC6a / HP1 for expression and a vector pPIC6a / HP2 for secreting and expressing mature human HP-2 haptoglobin are prepared.
11.組換えヒトハブトグロビン遺伝子を導入した酵母株の作製  11. Production of yeast strain with recombinant human hub globin gene
上述の成熟型ヒトハプトグロビンを分泌発現させるベクター PPIC6 a /HP1もしくは pP IC6 a /HP2 100 μ gを、制限酵素 Sail (ニューイングランドバイオラブズ社製)で HIS4遺 伝子内を切断し、フエノールクロ口ホルム抽出とエタノール沈殿によって、線状化べク ターを調製する。次に Mochizukiらの方法（Protein Expressionand Purification 23, 55 (2001))に従い、この線状ィ匕した HP-1型もしくは HP-2型ハプトグロビン発現ベクター を、上述の本実施例第 5項に記載した、 N-結合型糖鎖として主にハイブリッド型糖鎖 を発現するピキア酵母株、もしくは本実施例第 9項に記載した、 N-結合型糖鎖として 主にハイブリッド型糖鎖を発現するサッカロマイセス酵母株に対し、酢酸リチウム法に より導入する。遺伝子導入後の酵母は、薬剤ブラストシジン (インビトロジェン社製)を 含有する YPD培地 (インビトロジェン社製)で室温にて培養し、ブラストシジン耐性コロ ニーを取得する。次に、ブラストシジン耐性コロニーを液体 YPD培地 (インビトロジェン 社製）に移植し、 30°Cにて 24時間以上の回分培養を行う。培養後に得られる培養上 清は、ヒト血漿由来ハプトグロビン製剤 (三菱ゥエルファーマ社製)などを標準品とし、 Haptoglobinkit (Tridelta Development Limited社製）を用いて分析する。この分析に より、培養上清中に含まれる組換えヒト HP-1型もしくは HP-2型ハプトグロビンを検出 し、その濃度を測定することが可能である。この酵母培養上清中に分泌された、 N-結 合型糖鎖としてフコースを含まないハイブリッド型糖鎖を有する遺伝子組換え HP- 1型 および HP-2型ハプトグロビンは、 Krystalらの方法（Bloodgl, 71 (1986))に順じて精製 が可能である。また、精製された HP-1型および HP-2型ハプトグロビン蛋白質は、 Skib eliらの方法 (Bloody, 3626 (2001))に順じて糖鎖構造の解析が可能である。以上の 通り、 N-結合型糖鎖として、 Man5型ハイマンノース型糖鎖の非還元末端側に N-ァセ チルダルコサミン残基が 1個付加されたノ、イブリツド型糖鎖を主要に発現するピキア 酵母株、あるいは同様に改変されたサッカロマイセス酵母株を宿主として用い、 N-結 合型糖鎖としてフコースを含まないハイブリッド型糖鎖を主要に有する遺伝子組換え ヒト HP-1型および HP-2型ハプトグロビンを調製できることを述べた。次に、この N-結 合型糖鎖としてノ、イブリツド型糖鎖を主要に有する遺伝子組換えヒト HP- 1型もしくは HP-2型ハプトグロビンを発現する酵母株を用いて、 N-結合型糖鎖としてフコースを 含まない複合二本鎖型糖鎖を主要に有する遺伝子組換えヒト HP-1型および HP-2型 ハプトグロビンを発現する酵母株を作製する方法について以下に記載する。 The above-mentioned vector PPIC6 a / HP1 or pP IC6 a / HP2 100 μg that secretes and expresses mature human haptoglobin is cleaved in the HIS4 gene with the restriction enzyme Sail (New England Biolabs) Prepare linearized vector by form extraction and ethanol precipitation. Next, according to the method of Mochizuki et al. (Protein Expression and Purification 23, 55 (2001)), this linear HP-1 type or HP-2 type haptoglobin expression vector was described in the above-mentioned paragraph 5 of this Example. A Pichia yeast strain that mainly expresses a hybrid-type sugar chain as an N-linked sugar chain, or a Saccharomyces yeast that mainly expresses a hybrid-type sugar chain as an N-linked sugar chain described in Section 9 of this Example Introduced into the strain 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 a blasticidin-resistant colony. Next, the blasticidin-resistant colonies are transplanted into liquid YPD medium (Invitrogen) and cultured at 30 ° C for 24 hours or longer. The culture supernatant obtained after the culture is analyzed using Haptoglobinkit (manufactured by Tridelta Development Limited) using a human plasma-derived haptoglobin preparation (manufactured by Mitsubishi Wellpharma) as a standard product. By this analysis, it is possible to detect recombinant human HP-1 type or HP-2 type haptoglobin contained in the culture supernatant and to measure its concentration. The recombinant HP-1 and HP-2 haptoglobins, which have a hybrid sugar chain that does not contain fucose as an N-linked sugar chain, are secreted into the yeast culture supernatant, using the method of Krystal et al. (Bloodgl , 71 (1986)). The purified HP-1 type and HP-2 type haptoglobin proteins can be analyzed for sugar chain structure according to the method of Skibeli et al. (Bloody, 3626 (2001)). As described above, as an N-linked glycan, a Pichia that mainly expresses an ibubrid glycan with one N-acetyl darcosamine residue added to the non-reducing end of the Man5 type high mannose glycan. Recombinant human HP-1 type and HP-2 type mainly using hybrid sugar chains that do not contain fucose as N-linked sugar chains, using yeast strains or similarly modified Saccharomyces yeast strains as hosts It was stated that haptoglobin can be prepared. Next, this N- A non-fucose complex as an N-linked sugar chain using a yeast strain that expresses recombinant human HP-1 or HP-2 haptoglobin, which mainly has a hybrid sugar chain and a hybrid sugar chain A method for producing a yeast strain expressing a recombinant human HP-1 type and HP-2 type haptoglobin mainly having a double-chain type sugar chain is described below.
[0225] 12.組換えキメラ型 aマンノシダーゼ II遺伝子を導入した酵母株の作製 [0225] 12. Production of yeast strain with recombinant chimeric a-mannosidase II gene
ヒト組織由来、たとえば肝臓由来の cDNA dnvitrogen社製)を铸型とし、特異的ブラ イマ一と Pyrobest DNA polymerase (東洋紡績社製）を用いた PCRを行うことにより、 a マンノシダーゼ II (GenBankァクセッションナンバー： U31520)の活性ドメインをコード する cDNAを特異的に増幅させる。増幅させた cDNAは、その 5 '末端側に、酵母のマ ンノース転移酵素（MNN9)遺伝子（GenBankァクセッションナンバー： L23752)のリー ダーペプチドをコードする cDNA配列を連結した後に、酵母用の発現ベクターのプロ モーター配列の下流に挿入し、酵母のゴルジ体内に αマンノシダーゼ Πを発現させる ベクターを作製する。次にこのベクターを、本実施例第 11項に記載した、 Ν-結合型 糖鎖としてノ、イブリツド型糖鎖を主要に有する遺伝子組換えヒト HP- 1型もしくは HP- 2 型ハプトグロビンを発現する酵母株に対し、安定的に導入する。遺伝子導入後の酵 母は、栄養要求性と薬剤耐性を指標にしてクローンを選抜した後、 RT- PCRによって 、キメラ型 αマンノシダーゼ IIの発現を確認する。 By performing PCR using a specific primer and Pyrobest DNA polymerase (manufactured by Toyobo Co., Ltd.) using human tissue-derived, eg liver-derived cDNA dnvitrogen (manufactured by dnvitrogen), a mannosidase II (GenBank succession) Number: U31520) The cDNA encoding the active domain is specifically amplified. The amplified cDNA is linked to the 5 'end of the cDNA sequence encoding the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752) leader peptide, and then expressed for yeast. The vector is inserted downstream of the promoter sequence of the vector to produce a vector that expresses α- mannosidase に in the yeast Golgi apparatus. Next, this vector expresses recombinant human HP-1 type or HP-2 type haptoglobin mainly having an glycan-linked glycan and an hybrid glycan as described in paragraph 11 of this Example. Stable introduction into yeast strains. The fermented mother after gene transfer selects clones using auxotrophy and drug resistance as indicators, and then confirms the expression of chimeric α-mannosidase II by RT-PCR.
[0226] 13.組換えキメラ型 Ν-ァセチルダルコサミン転移酵素- II遺伝子を導入した酵母株 の [0226] 13. Recombinant chimeric type Ν-acetylcylcosamine transferase- of yeast strain introduced with II gene
作製  Production
ヒト組織由来、たとえば肝臓由来の cDNA dnvitrogen社製)を铸型とし、特異的ブラ イマ一と Pyrobest DNA polymerase (タカラバイオ社製）を用いた PCRを行うことにより、 N-ァセチルダルコサミン転移酵素- II (GenBankァクセッションナンバー： U15128)の活 性ドメインをコードする cDNAを特異的に増幅させる。増幅させた cDNAは、その 5 '末 端側に、酵母のマンノース転移酵素（MNN9)遺伝子（GenBankァクセッションナンパ 一： L23752)  N-acetylyldarcosaminyltransferase is obtained by performing PCR using a specific primer and Pyrobest DNA polymerase (Takara Bio) from a human tissue (eg, liver-derived cDNA dnvitrogen). -Specific amplification of cDNA encoding the active domain of II (GenBank accession number: U15128). The amplified cDNA has a yeast mannose transferase (MNN9) gene (GenBank accession number: L23752) at the 5 'end.
のリーダーペプチドをコードする cDNA配列を連結した後に、酵母用の発現ベクター のプロモーター配列の下流に挿入し、酵母のゴルジ体内に N-ァセチルダルコサミン 転移酵素- πを発現させるベクターを作製する。次にこのベクターを、前項に記載した 、Ν-結合型糖鎖としてハイブリッド型糖鎖を主要に有する組換えヒト HP-1型もしくは Η Ρ-2型ハプトグロビンを発現する酵母株にキメラ型 aマンノシダーゼ IIを安定的に導 入した酵母株に対し、安定的に導入する。遺伝子導入後の酵母は、栄養要求性と薬 剤耐性を指標にしてクローンを選抜した後、 RT-PCRによって、キメラ型 N-ァセチルダ ルコサミン転移酵素- Πの発現を確 After ligating the cDNA sequence encoding the leader peptide, insert it downstream of the promoter sequence of the yeast expression vector, and insert N-acetylyldarcosamine into the yeast Golgi. A vector for expressing transferase-π is prepared. Next, this vector was transformed into a chimeric a mannosidase described in the preceding paragraph in a yeast strain expressing recombinant human HP-1 or 有 す る Η-2 haptoglobin mainly having a hybrid sugar chain as a Ν-linked sugar chain. Introduce stably to yeast strains that have stably introduced II. For the yeast after gene transfer, clones were selected using auxotrophy and drug resistance as indicators, and then the expression of chimeric N-acetylcolcamine transferase- 確 was confirmed by RT-PCR.
認する。上記の方法により、キメラ型 N-ァセチルダルコサミン転移酵素- IIが安定的に 組み込まれた酵母株が発現する遺伝子組換え HP-1型もしくは HP-2型ハプトグロビン が有する主要な N-結合型糖鎖の構造を、還元末端側のコア部分に 2残基の N-ァセ チルダルコサミンを有し、その非還元末端側に 3個のマンノース残基が二分岐する構 造で結合し、二つの非還元末端のそれぞれに N-ァセチルダルコサミン残基が 1個ず つ付加された、フコースを含まな ヽ複合二本鎖型糖鎖に改変できる。  Admit. By the above method, the major N-linked form of recombinant HP-1 or HP-2 haptoglobin expressed by a yeast strain stably incorporating chimeric N-acetylylcosamine transferase-II The sugar chain structure has two residues of N-acetyl darcosamine in the core portion on the reducing end side, and is linked to the non-reducing end side in a structure where three mannose residues are bifurcated. It can be modified into a complex double-stranded sugar chain containing fucose, with one N-acetylyldarcosamine residue added to each non-reducing end.
[0227] 14.組換え UDP-ガラクトース- 4-ェピメラーゼ遺伝子を導入した酵母株の作製 [0227] 14. Production of yeast strains with recombinant UDP-galactose-4-epimerase gene
ヒト組織由来、たとえば肝臓由来の cDNA dnvitrogen社製)を铸型とし、特異的ブラ イマ一と Pyrobest DNA polymerase (タカラバイオ社製）を用いた PCRを行うことにより、 UDP-ガラクトース- 4-ェピメラーゼ（UniGeneナンバー Hs.76057)の翻訳領域全長をコ ード  By performing PCR using a specific primer and Pyrobest DNA polymerase (Takara Bio) from a human tissue (eg, cDNA dnvitrogen derived from liver), UDP-galactose-4-epimerase ( Code the entire translation region of UniGene number Hs.76057)
する cDNAを特異的に増幅させる。増幅させた cDNAは、酵母用の発現ベクターのプ 口モーター配列の下流に挿入し、酵母のサイトゾル内に UDP-ガラクトース- 4-ェピメラ ーゼを発現させるベクターを作製する。次にこのベクターを、前項に記載した、 N-結 合型糖鎖として未熟な複合二本鎖型糖鎖を主要に有する組換えヒト HP-1型もしくは HP-2型ハプトグロビンを発現する酵母株に対し、安定的に導入する。遺伝子導入後 の酵母は、栄養要求性と薬剤耐性を指標にしてクローンを選抜した後、 RT-PCRによ つて、 UDP-ガラクトース- 4-ェピメラーゼの発現を確認する。  Amplify specific cDNA. The amplified cDNA is inserted downstream of the expression motor sequence of an expression vector for yeast to produce a vector that expresses UDP-galactose-4-epimerase in the yeast cytosol. Next, the yeast strain expressing recombinant human HP-1 type or HP-2 type haptoglobin mainly having an immature complex double chain type sugar chain as an N-linked type sugar chain as described in the previous section. In contrast, it is introduced stably. After the gene is introduced, clones are selected using auxotrophy and drug resistance as indicators, and then the expression of UDP-galactose-4-epimelase is confirmed by RT-PCR.
[0228] 15.組換えキメラ型 β 1 ,4ガラクトース転移酵素遺伝子を導入した酵母株の作製 ヒト組織由来、たとえば肝臓由来の cDNA dnvitrogen社製)を铸型とし、特異的ブラ イマ一と Pyrobest DNA polymerase (タカラバイオ社製）を用いた PCRを行うことにより、 β 1 ,4ガラクトース転移酵素（GenBankァクセッションナンバー： M22921)の活性ドメィ ンをコードする CDNAを特異的に増幅させる。増幅させた cDNAは、その 5'末端側に、 酵母のマンノース転移酵素（MNN9)遺伝子（GenBankァクセッションナンバー： L2375 2)のリーダーペプチドをコードする cDNA配列を連結した後に、酵母用の発現べクタ 一のプロモーター配列の下流に挿入し、酵母のゴルジ体内に |8 1,4ガラクトース転移 酵素を発現させるベクターを作製する。次にこのベクターを、上述の前項に記載した 、N-結合型糖鎖として未熟な複合二本鎖型糖鎖を主要に有する組換えヒト HP-1型も しくは HP-2型ハプトグロビンを発現する酵母株にキメラ型 β 1,4ガラクトース転移酵素 を安定的に導入した酵母株に対し、安定的に導入する。遺伝子導入後の酵母は、栄 養要求性と薬剤耐性を指標にしてクローンを選抜した後、 RT-PCRによって、キメラ型 β 1,4ガラクトース転移酵素の発現を確認する。以上の方法により、キメラ型 |8 1,4ガラ クトース転移酵素が安定的に組み込まれた酵母株が発現する遺伝子組換え HP-1型 もしくは ΗΡ-2型ハプトグロビンが有する主要な Ν-結合型糖鎖の構造を、還元末端側 のコア部分に 2残基の Ν-ァセチルダルコサミンを有し、その非還元末端側に 3個のマ ンノース残基が二分岐する構造で結合し、二つの非還元末端のそれぞれに Ν-ァセ チルダルコサミン残基とガラクトース残基が 1個ずつ付加された、複合二本鎖型糖鎖 に改変することができる。 [0228] 15. Production of Yeast Strain Introduced with Recombinant Chimeric β1,4 Galactosyltransferase Gene Human tissue-derived, for example, liver-derived cDNA dnvitrogen (manufactured by cDNA dnvitrogen)) is used as a specific type and Pyrobest DNA. By performing PCR using polymerase (Takara Bio), the active domain of β 1,4 galactosyltransferase (GenBank accession number: M22921) Specifically amplifies the C- DNA encoding. 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. A vector is inserted downstream of the promoter sequence to express | 8 1,4 galactose transferase in the yeast Golgi. Next, this vector is used to express recombinant human HP-1 type or HP-2 type haptoglobin mainly having an immature complex double-stranded sugar chain as an N-linked sugar chain, as described in the previous section. Stable introduction into a yeast strain that has stably introduced chimeric β 1,4 galactosyltransferase into the yeast strain. For the yeast after gene transfer, clones are selected using nutrient requirement and drug resistance as indicators, and then the expression of chimeric β 1,4 galactose transferase is confirmed by RT-PCR. By the above method, chimera | 8 Major Ν-linked saccharides of recombinant HP-1 or ΗΡ-2 haptoglobin expressed by yeast strains stably incorporating 1,4 galactosyltransferase The chain structure has two residues Ν-acetyl darcosamine in the core part on the reducing end side, and the two non-reducing end side has three mannose residues in two branched structures. It can be modified into a complex double-stranded sugar chain in which one non-reducing end is added with one Ν-acetyldarcosamine residue and one galactose residue.
16.酵母を用いた遺伝子組換えハプトグロビン蛋白質の調製  16. Preparation of recombinant haptoglobin protein using yeast
前項で作製した、還元末端側にフコース残基を持たず非還元末端側にガラクトース が付加された複合二本鎖型糖鎖を主に有する遺伝子組換え HP-1型もしくは ΗΡ-2型 ハプトグロビンを発現する酵母株は、液体 YPD培地 (インビトロジェン社製）に播種し、 30°Cにて 24時  Recombinant HP-1 type or ΗΡ-2 type haptoglobin, which mainly has a complex double-stranded sugar chain with no fucose residue on the reducing end side and galactose added on the non-reducing end side, prepared in the previous section The yeast strain to be expressed is seeded in a liquid YPD medium (manufactured by Invitrogen) and 24 hours at 30 ° C.
間以上の回分培養を行うことにより、培養上清中に遺伝子組換え HP-1型もしくは HP -2型ハプトグロビンを分泌させることが可能である。培養後に得られる培養上清は、ヒ ト血漿由来ハプトグロビン製剤（三菱ゥエルファーマ社製)などを標準品とし、 Haptogl obinkit (Tridelta Development Limited社製）を用いて分析する。この分析により、培 養上清中 It is possible to secrete recombinant HP-1 type or HP-2 type haptoglobin into the culture supernatant by carrying out batch culture for more than a period. The culture supernatant obtained after culturing is analyzed using Haptogl obinkit (manufactured by Tridelta Development Limited) using a human plasma-derived haptoglobin preparation (manufactured by Mitsubishi Wellpharma) as a standard product. By this analysis, in the culture supernatant
に含まれる組換えヒト HP-1型もしくは HP-2型ハプトグロビンを検出し、その濃度を測 定することが可能である。また、この酵母培養上清中に分泌された、 N-結合型糖鎖と してフコースを含まなヽ複合二本鎖型糖鎖を有する遺伝子組換え HP-1型もしくは HP -2型ハプトグロビンは、 Krystalらの方法（Bloodgl, 71 (1986))に順じて精製が可能で ある。また、精製された HP-1型もしくは HP-2型ハプトグロビン蛋白質は、 Skibeliらの 方法 (Blood£g, 3626 (2001))に準じて糖鎖構造を解析することができる。 It is possible to detect recombinant human HP-1 type or HP-2 type haptoglobin and to measure its concentration. In addition, N-linked sugar chains secreted into the yeast culture supernatant Recombinant HP-1 type or HP-2 type haptoglobin with a complex double-stranded sugar chain containing fucose can be purified according to the method of Krystal et al. (Bloodgl, 71 (1986)) It is. The purified HP-1 type or HP-2 type haptoglobin protein can be analyzed for the sugar chain structure according to the method of Skibeli et al. (Blood £ g, 3626 (2001)).
産業上の利用可能性  Industrial applicability
[0230] 本発明により、 N-グリコシド結合複合型糖鎖を有する遺伝子組換えハプトグロビン 分子カゝらなる組成物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末端の N-ァセチルダルコサミンにフコースが結合して!/ヽな 、糖鎖であるハプトグロビン組成 物を提供することができる。 [0230] According to the present invention, there is provided a composition comprising a recombinant haptoglobin molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is N- It is possible to provide a haptoglobin composition which is a sugar chain in which fucose is bound to acetylyldarcosamine!
配列表フリ一テキスト  Sequence table free text
[0231] 配列番号 20- -人工配列の説明 ：合成 DNA  [0231] SEQ ID NO: 20- -Description of artificial sequence: Synthetic DNA
配列番号 21- -人工配列の説明 ：合成 DNA  SEQ ID NO: 21- Description of artificial sequence: synthetic DNA
配列番号 22- -人工配列の説明 ：合成 DNA  SEQ ID NO: 22- Description of artificial sequence: synthetic DNA
配列番号 23- -人工配列の説明 ：合成 DNA  SEQ ID NO: 23--Description of artificial sequence: synthetic DNA
配列番号 24- -人工配列の説明 ：合成 DNA  SEQ ID NO: 24--Description of Artificial Sequence: Synthetic DNA
配列番号 25- -人工配列の説明 ：合成 DNA  SEQ ID NO: 25- Description of artificial sequence: synthetic DNA
配列番号 26- -人工配列の説明 ：合成 DNA  SEQ ID NO: 26--Description of artificial sequence: synthetic DNA
配列番号 27- -人工配列の説明 ：合成 DNA  SEQ ID NO: 27- Description of artificial sequence: synthetic DNA

Claims

請求の範囲 The scope of the claims
[1] N-グリコシド結合複合型糖鎖を有する遺伝子組換えハプトグロビン分子カゝらなる組成 物であって、 N-グリコシド結合複合型糖鎖が該糖鎖の還元末端の N-ァセチルダルコ サミンにフコースが結合して ヽな 、糖鎖であるハプトグロビン組成物。  [1] A composition comprising a recombinant haptoglobin molecule having an N-glycoside-linked complex sugar chain, wherein the N-glycoside-bonded complex sugar chain is fucose to N-acetylyldarcosamine at the reducing end of the sugar chain. A haptoglobin composition which is a sugar chain, which is bound to.
[2] N-グリコシド結合複合型糖鎖力 該糖鎖還元末端の N-ァセチルダルコサミンの 6位 にフコースの 1位が α結合していない糖鎖である、請求項 1に記載のハプトグロビン 組成物。 [2] The haptoglobin according to claim 1, which is a sugar chain in which the 1-position of fucose is not α- bonded to the 6-position of N-acetylyldarcosamine at the reducing end of the sugar chain. Composition.
[3] ハプトグロビン力 以下の (a)〜(i)からなる群から選ばれるポリペプチドと、以下の (j) 〜(1)力もなる群力も選ばれるポリペプチドとからなる蛋白質である、請求項 1または 2 に記載のハプトグロビン組成物。  [3] Haptoglobin power A protein consisting of a polypeptide selected from the group consisting of the following (a) to (i) and a polypeptide selected from the group (j) to (1) whose power is also selected: The haptoglobin composition according to 1 or 2.
(a) 配列番号 4の 19〜101番目で表されるアミノ酸配列を有するポリペプチド； (a) a polypeptide having an amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 4;
(b) 配列番号 5の 19〜101番目で表されるアミノ酸配列を有するポリペプチド；(b) a polypeptide having an amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 5;
(c) 配列番号 6の 19〜160番目で表されるアミノ酸配列を有するポリペプチド；(c) a polypeptide having an amino acid sequence represented by positions 19 to 160 of SEQ ID NO: 6;
(d) 配列番号 4の 19〜101番目で表されるアミノ酸配列において、 1以上のアミノ酸 が欠失、置換、挿入および Zまたは付加されたアミノ酸配列を有し、かつ (j)〜(Dから なる群力 選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性を有する ポリペプチド； (d) In the amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 4, one or more amino acids have an amino acid sequence deleted, substituted, inserted and Z or added, and (j) to (from D A polypeptide having hemoglobin metabolic activity by combining with a selected polypeptide;
(e) 配列番号 5の 19〜101番目で表されるアミノ酸配列において、 1以上のアミノ酸 が欠失、置換、挿入および Zまたは付加されたアミノ酸配列を有し、かつ (j)〜(Dから なる群力 選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性を有する ポリペプチド；  (e) In the amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 5, one or more amino acids have an amino acid sequence deleted, substituted, inserted and Z or added, and (j) to (from D A polypeptide having hemoglobin metabolic activity by combining with a selected polypeptide;
(D 配列番号 6の 19〜 160番目で表されるアミノ酸配列において、 1以上のァミノ 酸が欠失、置換、挿入および Zまたは付加されたアミノ酸配列を有し、かつ (j)〜(l)か らなる群力も選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性を有す るポリペプチド；  (D in the amino acid sequence represented by positions 19 to 160 of SEQ ID NO: 6, wherein one or more amino acids have an amino acid sequence deleted, substituted, inserted and Z or added, and (j) to (l) A polypeptide having hemoglobin metabolic activity when combined with a selected polypeptide,
(g) 配列番号 4の 19〜101番目で表されるアミノ酸配列と 80%以上の相同性を有す るアミノ酸配列を有し、かつ φ〜(1)力 なる群力も選ばれるポリペプチドと組合せること によりヘモグロビン代謝活性を有するポリペプチド； (h) 配列番号 5の 19〜101番目で表されるアミノ酸配列と 80%以上の相同性を有す るアミノ酸配列を有し、かつ φ〜(1)力 なる群力も選ばれるポリペプチドと組合せること によりヘモグロビン代謝活性を有するポリペプチド； (g) Combination with a polypeptide having an amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 4 and also having a force of φ to (1) A polypeptide having hemoglobin metabolic activity; (h) Combination with a polypeptide having an amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 19 to 101 of SEQ ID NO: 5 and also having a group strength of φ to (1) A polypeptide having hemoglobin metabolic activity;
(0 配列番号 6の 19〜160番目で表されるアミノ酸配列と 80%以上の相同性を有す るアミノ酸配列を有し、かつ φ〜(1)力 なる群力も選ばれるポリペプチドと組合せること によりヘモグロビン代謝活性を有するポリペプチド。  (0 Combined with a polypeptide having an amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 19 to 160 of SEQ ID NO: 6, and also having a force of φ to (1) Thus, a polypeptide having hemoglobin metabolic activity.
(j)配列番号 4の 103〜347番目で表されるアミノ酸配列を有するポリペプチド； (k)配列番号 4の 103〜347番目で表されるアミノ酸配列において、 1以上のアミノ酸 が欠失、置換、挿入および/または付加されたアミノ酸配列を有し、かつ (a)〜(i)から なる群力 選ばれるポリペプチドと組合せることによりヘモグロビン代謝活性を有する ポリペプチド；  (j) a polypeptide having the amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4; (k) one or more amino acids deleted or substituted in the amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4 A polypeptide having an amino acid sequence inserted and / or added and having hemoglobin metabolic activity in combination with a polypeptide selected from the group force consisting of (a) to (i);
(1)配列番号 4の 103〜347番目で表されるアミノ酸配列と 80%以上の相同性を有 するアミノ酸配列を有し、かつ (a)〜(i)力 なる群力 選ばれるポリペプチドと組合せる ことによりヘモグロビン代謝活性を有するポリペプチド；  (1) an amino acid sequence having 80% or more homology with the amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4, and (a) to (i) a powerful group force A polypeptide having hemoglobin metabolic activity by combining;
[4] ハプトグロビン力 以下の (a)、（b)、（c)、（d)、（e)及び (1)力 なる群力 選ばれる DNA 力 Sコードする蛋白質である、請求項 1または 2に記載のハプトグロビン組成物。 [4] Haptoglobin force The following (a), (b), (c), (d), (e), and (1) Group force that is selected DNA force selected S-encoded protein The haptoglobin composition described in 1.
(a)配列番号 1で表される塩基配列からなる DNA;  (a) DNA consisting of the base sequence represented by SEQ ID NO: 1;
(b)配列番号 2で表される塩基配列力 なる DNA；  (b) DNA having a nucleotide sequence represented by SEQ ID NO: 2;
(c)配列番号 3で表される塩基配列力 なる DNA；  (c) DNA having a nucleotide sequence represented by SEQ ID NO: 3;
(d)配列番号 1で表される塩基配列力もなる DNAとストリンジェントな条件でハイブリダ ィズし、かつヘモグロビン代謝活性を有する蛋白質をコードする DNA;  (d) DNA that hybridizes under stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 1 and encodes a protein having hemoglobin metabolic activity;
(e)配列番号 2で表される塩基配列からなる DNAとストリンジェントな条件でノ、イブリダ ィズし、かつヘモグロビン代謝活性を有する蛋白質をコードする DNA;  (e) DNA encoding a protein having a hemoglobin metabolic activity, which is hybridized under stringent conditions with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 2;
(D配列番号 3で表される塩基配列からなる DNAとストリンジェントな条件でノ、イブリダ ィズし、かつヘモグロビン代謝活性を有する蛋白質をコードする DNA;  (D, DNA that encodes a protein that has a hemoglobin metabolic activity and is hybridized under stringent conditions with DNA comprising the base sequence represented by SEQ ID NO: 3;
[5] ハプトグロビン分子をコードする DNAを宿主細胞に導入して得られる、請求項 1〜4の い [5] The method according to any one of claims 1 to 4, obtained by introducing DNA encoding a haptoglobin molecule into a host cell.
ずれか 1項に記載のハプトグロビン組成物を生産する形質転換体。 A transformant that produces the haptoglobin composition according to claim 1.
[6] 宿主細胞が、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、または N- グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1 位が oc結合する糖鎖修飾に関与する酵素の活性が欠失するようにゲノムが改変され た細胞である、請求項 5に記載の形質転換体。 [6] The host cell is oc-coupled to position 6 of fucose at position 6 of the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-glycidyl-linked N-acetylyldarcosamine at the N-glycidyl-linked complex 6. The transformant according to claim 5, which is a cell whose genome has been altered so that the activity of an enzyme involved in sugar chain modification is lost.
[7] 宿主細胞が、細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素、または N- グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1 位が a結合する糖鎖修飾に関与する酵素のゲノム上の対立遺伝子のすべてがノック アウトされた細胞である、請求項 6に記載の形質転換体。  [7] The host cell is linked to the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose, or N-glycyl-linked N-acetylyldarcosamine at the 6-position of the reducing end of the sugar chain. The transformant according to claim 6, wherein all of the alleles on the genome of an enzyme involved in glycosylation are knocked out.
[8] 細胞内糖ヌクレオチド GDP-フコースの合成に関与する酵素力 GDP-マンノース 4,6 -デヒ  [8] Intracellular sugar nucleotides GDP-enzyme power involved in the synthesis of fucose GDP-mannose 4,6-dehy
ドラターゼ及び GDP-4-ケト -6-デォキシ- D-マンノース- 3, 5-ェピメラーゼからなる群 から  From the group consisting of dratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase
選ばれる酵素である、請求項 6または 7に記載の形質転換体。  The transformant according to claim 6 or 7, which is an enzyme to be selected.
[9] GDP-マンノース 4,6-デヒドラターゼカ 以下の（a)、（b)及び (c)力 なる群から選ばれ る蛋白質である、請求項 8に記載の形質転換体。 [9] The transformant according to claim 8, which is a protein selected from the following groups (a), (b) and (c): GDP-mannose 4,6-dehydratase.
(a)配列番号 8で表されるアミノ酸配列からなる蛋白質；  (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;
(b)配列番号 8で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入  (b) In the amino acid sequence represented by SEQ ID NO: 8, one or more amino acids are deleted, substituted, or inserted
および Zまたは付加されたアミノ酸配列力もなり、かつ GDP-マンノース 4,6-デヒドラ タ  And Z or added amino acid sequence, and GDP-mannose 4,6-dehydrata
ーゼ活性を有する蛋白質；  A protein having lyase activity;
(c)配列番号 8で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列から なり、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質；  (c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, and having GDP-mannose 4,6-dehydratase activity;
[10] GDP-マンノース 4,6-デヒドラターゼカ 以下の (a)及び (b)力 なる群力 選ばれる DN Aがコードする蛋白質である、請求項 8に記載の形質転換体。  [10] The transformant according to claim 8, which is a protein encoded by GDP A selected from GDP-mannose 4,6-dehydratase (a) and (b) group power as follows.
(a)配列番号 7で表される塩基配列からなる DNA；  (a) DNA consisting of the base sequence represented by SEQ ID NO: 7;
(b)配列番号 7で表される塩基配列力もなる DNAとストリンジェントな条件でハイブリダ ィズし、かつ GDP-マンノース 4,6-デヒドラターゼ活性を有する蛋白質をコードする DN A。 (b) DN that hybridizes with DNA having the nucleotide sequence shown in SEQ ID NO: 7 under stringent conditions and encodes a protein having GDP-mannose 4,6-dehydratase activity A.
[11] GDP- 4-ケト- 6-デォキシ- D-マンノース- 3,5-ェピメラーゼカ 以下の（a)、 (b)および (c)力もなる群力 選ばれる蛋白質である、請求項 8に記載の形質転換体。  [11] GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase categorized by the following (a), (b) and (c): Transformant.
(a)配列番号 10で表されるアミノ酸配列からなる蛋白質；  (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 10;
(b)配列番号 10で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ GDP-4-ケト -6-デォキシ -D -マンノース- 3,5-ェピメラーゼ活性を有する蛋白質；  (b) In the amino acid sequence represented by SEQ ID NO: 10, one or more amino acids are deleted, substituted, inserted and Z or added, and the amino acid sequence power is GDP-4-keto-6-deoxy-D- A protein having mannose-3,5-epimerase activity;
(c)配列番号 10で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼ活性を有する 蛋白質  (c) It consists of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity protein
[12] GDP-4-ケト -6-デォキシ- D-マンノース- 3,5-ェピメラーゼが、以下の（a)及び (b)から な [12] GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase consists of the following (a) and (b)
る群力 選ばれる DNAがコードする蛋白質である、請求項 8に記載の細胞。  The cell according to claim 8, which is a protein encoded by the selected DNA.
(a)配列番号 9で表される塩基配列からなる DNA；  (a) DNA comprising the base sequence represented by SEQ ID NO: 9;
(b)配列番号 9で表される塩基配列力もなる DNAとストリンジェントな条件でハイブリダ ィズし、かつ GDP-4-ケト -6-デォキシ- D-マンノース- 3, 5-ェピメラーゼ活性を有する 蛋白  (b) A protein that hybridizes under stringent conditions with DNA having the nucleotide sequence shown by SEQ ID NO: 9 and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimelase activity
質をコードする DNA。  DNA that encodes quality.
[13] N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位にフコースの 1位が a結合する糖鎖修飾に関与する酵素が a 1,6-フコシルトランスフェラーゼであ る請求項 6または 7に記載の形質転換体。  [13] N-glycoside-linked complex type sugar chain N-acetylyldarcosamine at the reducing end is a 1,6-fucosyltransferase, which is involved in sugar chain modification in which the 1-position of fucose is a-linked to the 6-position The transformant according to claim 6 or 7.
[14] a 1,6—フコシルトランスフェラーゼカ 以下の（a)、 (b)、（c)、（d)、（e)および (1)力らなる 群から選ばれる蛋白質である、請求項 13に記載の形質転換体。  [14] a 13,6-fucosyltransferase is a protein selected from the group consisting of the following (a), (b), (c), (d), (e) and (1) force A transformant according to 1.
(a)配列番号 13で表されるアミノ酸配列からなる蛋白質；  (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 13;
(b)配列番号 14で表されるアミノ酸配列力もなる蛋白質；  (b) a protein having an amino acid sequence ability represented by SEQ ID NO: 14;
(c)配列番号 13で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ a 1,6-フコシルトランスフェラ ーゼ活性を有する蛋白質； (c) In the amino acid sequence represented by SEQ ID NO: 13, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and a 1,6-fucosyltransferase A protein having lyase activity;
(d)配列番号 14で表されるアミノ酸配列において、 1以上のアミノ酸が欠失、置換、挿 入および Zまたは付加されたアミノ酸配列力もなり、かつ α 1,6-フコシルトランスフェラ ーゼ活性を有する蛋白質；  (d) In the amino acid sequence represented by SEQ ID NO: 14, one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have α 1,6-fucosyltransferase activity. A protein having;
(e)配列番号 13で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ a 1,6-フコシルトランスフェラーゼ活性を有する蛋白質；  (e) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 13, and having a 1,6-fucosyltransferase activity;
(1)配列番号 14で表されるアミノ酸配列と 80%以上の相同性を有するアミノ酸配列か らなり、かつ a 1,6-フコシルトランスフェラーゼ活性を有する蛋白質。  (1) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 14, and having a 1,6-fucosyltransferase activity.
[15] a 1,6-フコシルトランスフェラーゼカ 以下の（a)、（b)、（c)及び (d)力 なる群力 選 ばれる DNAがコードする蛋白質である、請求項 13に記載の形質転換体。 [15] The transformation according to claim 13, which is a protein encoded by a DNA selected from the following (a), (b), (c) and (d) powerful group forces: a 1,6-fucosyltransferase body.
(a)配列番号 11で表される塩基配列力 なる DNA;  (a) DNA having a nucleotide sequence represented by SEQ ID NO: 11;
(b)配列番号 12で表される塩基配列力もなる DNA；  (b) DNA having a nucleotide sequence represented by SEQ ID NO: 12;
(c)配列番号 11で表される塩基配列力 なる DNAとストリンジェントな条件でノ、イブリ ダイズし、かつ α 1,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする DN A  (c) DNA encoding a protein that is hybridized under stringent conditions with DNA having the nucleotide sequence shown in SEQ ID NO: 11 and that has α1,6-fucosyltransferase activity
(d)配列番号 12で表される塩基配列力もなる DNAとストリンジェントな条件でハイプリ ダイズし、かつ α 1,6-フコシルトランスフェラーゼ活性を有する蛋白質をコードする DN A (d) DN A that is hybridized under stringent conditions with DNA having the nucleotide sequence shown by SEQ ID NO: 12 and that encodes a protein having α1,6-fucosyltransferase activity
[16] N-グリコシド結合複合型糖鎖還元末端の N-ァセチルダルコサミンの 6位とフコースの 1位が α結合した糖鎖構造を認識するレクチンに耐性である、請求項 5〜15のいず れか 1項 [16] The N-glycoside-linked complex type sugar chain reducing terminal of N-acetylyldarcosamine at position 6 and fucose 1-position are resistant to a lectin that recognizes an α-linked sugar chain structure. Either one
に記載の形質転換体。  A transformant according to 1.
[17] レクチン耐性力 Ν-グリコシド結合複合型糖鎖還元末端の Ν-ァセチルダルコサミン の 6位とフコースの 1位が a結合した糖鎖構造を認識するレクチンを含む培地で培養 した場合に、ゲノムが改変される以前の細胞よりも高い生存率を示すことである、請求 項 16に記載の形質転換体。 [17] Resistance to lectin When cultured in a medium containing a lectin that recognizes a sugar chain structure in which the 6-position of 還 元 -glycidylcolacamine at the reducing end of Ν-glycoside-linked complex and 1-position of fucose are a-linked The transformant according to claim 16, wherein the transformant exhibits a higher survival rate than a cell before the genome is modified.
[18] 少なくとも、以下の (a)、（b)、（c)及び (d)力 なる群力 選ばれるレクチンの一つに耐性 である、請求項 16または 17に記載の形質転換体。 [18] The transformant according to claim 16 or 17, which is resistant to at least one of the following (a), (b), (c) and (d) force group forces selected:
(a)レンズマメレクチン LCA (Lens Culinaris由来の Lentil Agglutinin)；  (a) Lentil lectin LCA (Lentil Agglutinin from Lens Culinaris);
(b)エンドゥマメレクチン PS A (Pisum sativum由来の Pea Lectin)；  (b) Endo bean lectin PS A (Peasum sativum-derived Pea Lectin);
(c) ソラマメレクチン VFA (Vicia faba由来の Agglutinin)：  (c) Broad bean lectin VFA (Agglutinin from Vicia faba):
(d) ヒィロチャワンタケレクチン AAL (Aleuria aurantia由来の Lectin) .  (d) Herochawantake lectin AAL (Lectin from Aleuria aurantia).
[19] 宿主細胞が、下記の (a)、（b)、（c)、（d)、（ 、（1)、（g)、（h)、（i)及び (j)力 なる群力 選ば れる細胞である請求項 5〜18のいずれ力 1項に記載の形質転換体。  [19] The host cell has the following group power (a), (b), (c), (d), (, (1), (g), (h), (i) and (j) The transformant according to any one of claims 5 to 18, which is a selected cell.
(a)チャイニーズノヽムスター卵巣組織由来 CHO細胞；  (a) Chinese nomstar ovarian tissue-derived CHO cells;
(b)ラットミエローマ細胞株 YB2/3HL.P2.G11.16Ag.20細胞；  (b) rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cells;
(c)マウスミエローマ細胞株 NS0細胞；  (c) mouse myeloma cell line NS0 cells;
(d)マウスミエローマ細胞株 SP2/0- Agl4細胞；  (d) mouse myeloma cell line SP2 / 0-Agl4 cells;
(e)シリアンノヽムスター腎臓組織由来 BHK細胞；  (e) Syrian Nomster kidney tissue-derived BHK cells;
(1)ヒト白血病細胞株ナマルバ細胞；  (1) human leukemia cell line Namalva cells;
(g)胚性幹細胞；  (g) embryonic stem cells;
(h)受精卵細胞；  (h) fertilized egg cells;
(0植物細胞；  (0 plant cells;
(j)酵母。  (j) Yeast.
[20] 請求項 5〜19のいずれか 1項に記載の形質転換体を培地に培養し、培養物中にハ ブトグロビン組成物を生成蓄積させ、該培養物力 ハプトグロビン組成物を採取する 工程を含む、ハプトグロビン組成物の製造方法。  [20] The method comprises culturing the transformant according to any one of claims 5 to 19 in a medium, producing and accumulating a habtoglobin composition in the culture, and collecting the culture force haptoglobin composition. And a method for producing a haptoglobin composition.
[21] 請求項 20記載の製造方法で得られたハプトグロビン組成物。  [21] A haptoglobin composition obtained by the production method according to claim 20.
[22] 請求項 1〜4および 21のいずれか 1項に記載のハプトグロビン組成物を有効成分とし て含有する医薬。  [22] A medicament comprising the haptoglobin composition according to any one of claims 1 to 4 and 21 as an active ingredient.
[23] 請求項 1〜4および 21のいずれか 1項に記載のハプトグロビン組成物を有効成分とし て含有する、ヘモグロビン血症及びヘモグロビン尿症の治療剤。  [23] A therapeutic agent for hemoglobinemia and hemoglobinuria comprising the haptoglobin composition according to any one of claims 1 to 4 and 21 as an active ingredient.
[24] 請求項 1〜4および 21のいずれか 1項に記載のハプトグロビン組成物を有効成分とし て含有する、血中遊離ヘモグロビンによる酸ィ匕的血管障害を伴う疾患の治療剤。  [24] A therapeutic agent for diseases associated with acid-induced vascular disorders caused by blood free hemoglobin, comprising the haptoglobin composition according to any one of claims 1 to 4 and 21 as an active ingredient.
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