WO2008018562A1 - Production d'une protéine utile telle qu'un anticorps ayant une chaîne glucidique modifiée dans le jaune d'oeuf d'un poulet transgénique - Google Patents

Production d'une protéine utile telle qu'un anticorps ayant une chaîne glucidique modifiée dans le jaune d'oeuf d'un poulet transgénique Download PDF

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Publication number
WO2008018562A1
WO2008018562A1 PCT/JP2007/065655 JP2007065655W WO2008018562A1 WO 2008018562 A1 WO2008018562 A1 WO 2008018562A1 JP 2007065655 W JP2007065655 W JP 2007065655W WO 2008018562 A1 WO2008018562 A1 WO 2008018562A1
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Prior art keywords
fusion protein
sugar chain
antibody
protein
egg yolk
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PCT/JP2007/065655
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English (en)
Japanese (ja)
Inventor
Shinji Iijima
Masamichi Kamihira
Kenichi Nishijima
Takashi Yamashita
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Kaneka Corporation
Nagoya University
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Priority to JP2008528887A priority Critical patent/JPWO2008018562A1/ja
Publication of WO2008018562A1 publication Critical patent/WO2008018562A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/30—Bird
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a glycosylated fusion protein comprising accumulating an exogenous fusion protein in egg yolk derived from a transgenic bird or a progeny thereof into which the exogenous fusion protein gene has been introduced, and separating it. It relates to the production method. More specifically, the present invention relates to a method for producing a glycosylated fusion protein, wherein the foreign fusion protein accumulated in egg yolk has a sugar chain structure that is equivalent to or similar to a human sugar chain structure.
  • sugar chains that modify glycoproteins are known to have species-specific effects on glycoproteins because their sugar chain structures differ depending on the species. Therefore, it is desired to develop a method for producing a useful protein optimal for each species at low cost and in large quantities. That is, in order to produce pharmaceuticals for humans, the sugar chain structure needs to be a human type.
  • Non-Patent Document 3 shows that sialic acid at the end of the modified sugar chain of purified human IgG and chicken IgG is composed only of N-acetylneuraminic acid, unlike IgG of other animals.
  • ADCC activity is high because fucose is not added to chicken IgG! /.
  • the sugar chain structure of the exogenous monoclonal antibody produced in chicken egg white does not contain sialic acid and has low blood stability! / (See Patent Document 4).
  • Non-patent literature l Trends Biotechnol. 1999 Sep; 17 (9): 367—74
  • Non-Patent Document 2 Chemical Immunology, 65, 88, 1997
  • Non-Patent Document 3 Glycobiology vol. 10 no. 5 pp477—486, 2000
  • Non-Patent Document 4 Nat. Biotechnol. 2005 Sep; 23 (9): 1159- 69
  • Non-Patent Document 5 Biochem. J. 1999, 338, 529-538
  • the sugar chain structure In order for the foreign protein to be produced to be a high-performance / pharmaceutical human pharmaceutical protein, the sugar chain structure must have a sugar chain equivalent or similar to the human sugar chain structure. Must.
  • an exogenous protein accumulated in the transgenic bird's egg yolk has a sugar chain that is equivalent to or similar to the human sugar chain structure.
  • the present invention provides the following.
  • the foreign fusion protein produced in egg yolk derived from a transgenic bird or a progeny thereof into which a foreign fusion protein gene has been introduced has a human sugar chain structure. It has the same or similar sugar chain.
  • the method for producing a transgenic bird used in the present invention has already been established by the present inventors (WO 04/016081 pamphlet), but is not limited to this, and any known method is used. be able to.
  • G1 transgenic chimera birds carrying foreign fusion protein genes in germ cells are crossed with wild-type birds, GO transgenic chimera birds or their descendants, and G1 transgenics are selected by selecting hatched chicks. Nick birds can be obtained. G1 transgenic birds can confirm gene transfer into somatic cells and germ cells by examining DNA, RNA, etc. derived from blood, somatic cells, sperm, or eggs by PCR. It can also be determined from the expression of the exogenous fusion protein introduced. The expression of the protein can be examined by ELISA, electrophoresis, activity measurement or the like. [0013] Transgenic birds of the G2 and subsequent generations are produced by crossing G1 transgenic birds.
  • Mating types include G1 transgenenics and wild females, G1 transgenic females and wild males, G1 transgenenic males and females, and backcrossing by offspring and their parents is also possible .
  • the cross between G1 male and wild-type female is preferable from the viewpoint of efficiency because it is possible to cross multiple wild-type females to one G1 male.
  • the birds used in the present invention are not particularly limited, but are preferably poultry birds that can be used as livestock.
  • poultry birds include chickens, turkeys, ducks, ostriches, quails and ducks.
  • chickens are easy to obtain, are prolific as spawning species, have large eggs, and have established large-scale breeding methods, and their safety has been proven in the production of vaccines, etc. ,.
  • the exogenous fusion protein of the present invention is not particularly limited, and examples thereof include antibodies useful in the gene industry such as human monoclonal antibodies, fusion proteins fused with enzymes, growth factors, cytosites, and the like. . Particularly preferred is a protein that is a glycoprotein and whose sugar chain is regarded as important for its activity.
  • TNFR-Fc which is a fusion protein of a TNF receptor and a human immunoglobulin constant region, is preferably a fusion protein with a constant region of human immunoglobulin.
  • a humanized antibody and a chimeric antibody are particularly preferable among the recombinant antibodies that can be produced by the recent development of genetic engineering, but are not limited thereto.
  • the term “humanized antibody” as used herein refers to an antibody having an antibody sequence usually produced by humans other than the amino acid sequence region that functions for antibody antigen recognition.
  • the chimeric antibody herein refers to an antibody in which a part of an antibody produced in a certain animal is replaced with a corresponding region of an antibody produced by a heterologous animal.
  • the region to be replaced is not particularly limited, but is preferably a constant region of an antibody normally produced by humans from the viewpoint of blood stability.
  • the force using an antibody gene having a constant region of human IgG class is not limited thereto.
  • Human Ig Examples of the antibody having a constant region of the Gl subclass include an anti-CD2 HgG antibody.
  • Immunoglobulin IgG has a domain consisting of VH and VL heterodimers called variable regions (Fv: Fragment of variable regions) that bind directly to antigens. This Fv domain is about one-fifth of IgG. It has a sufficient antigen-binding ability by itself even though it is small.
  • An artificially linked peptide linker between the VH and VL domains is a single-chain antibody (scFv: single chain Fv), which can be more stable than VH and VL alone. It was known.
  • the scFv-Fc antibody is also preferable as an exogenous fusion protein obtained by the production method of the present invention.
  • the glycosylated fusion protein that accumulates in egg yolk of the present invention is characterized by the sugar chain structure it has.
  • a glycoprotein modified with a sugar chain is used as a pharmaceutical product for humans, if the sugar chain structure is not equivalent to or similar to the human sugar chain structure, the effect decreases and / or disappears, and allergies, etc. It may cause side effects of medication.
  • the protein has a sugar chain structure that is the same as or similar to the human sugar chain structure, thereby improving blood stability, which is a problem for pharmaceuticals, and emphasizing signal action to target cells or targets.
  • the ability to promote uptake into organs, increase the physiological activity originally imparted, and impart new physiological activity can also be expected.
  • the present inventors are an exogenous fusion protein produced in an egg derived from the transgenic bird of the present invention or its progeny, and in particular, the exogenous fusion protein produced in the yolk contains sialic acid. It was found that the sugar chain was modified. In addition, it has already been reported that the sialic acid of the sugar chain added to chicken IgG is composed only of N-acetylneuraminic acid, similar to human sugar chains (Glycobiology vol. 10 no. 5 pp477-486, 2000) and the foreign fusion protein that accumulates in the yolk is a protein force expressed in all tissues of transgenic birds. In either of these, there is a sugar-modifying enzyme that performs a modification equivalent to that of human sugar chain, and it can be presumed that the sugar chain structural strength is equivalent to or similar to that of human.
  • the present inventors have further found that some proteins positively accumulate in egg yolk.
  • the antibody content in egg white is 1 ag / ml or less or 0.1 ag / ml or less
  • the antibody content in egg yolk is 0 ⁇ 1 ag / ml or more, or 1 ag / ml or more.
  • G0 transgenic chimeric birds will hatch 21 days after gene transfer.
  • the female chicks mature in about 5 months and lay about 1 egg per day.
  • the foreign fusion protein accumulated in the egg yolk is preferably separated and purified using a protein column.
  • the fusion protein accumulated in egg yolk according to the present invention can be purified by, for example, protein A or protein G utilizing the characteristics of Fc.
  • it can be purified and recovered from egg yolk by any known method utilizing the characteristics of Fc. It is also possible to separate or recover the target protein and Fc by treating the fusion protein with protease.
  • a glycosylated fusion protein obtained by the above-described method for producing a glycosylated fusion protein is also one aspect of the present invention.
  • glycosylated fusion protein of the present invention it is preferred that 5% or more of the total sugar chain is a sugar chain having monosialic acid. More preferred! /, The proportion of monosialic acid is 5 to 20 percent, more preferably 5 to 10 percent.
  • an exogenous fusion protein having a sugar chain structure equivalent to or similar to a human sugar chain structure is provided.
  • the exogenous fusion protein accumulated in egg yolk can be used, the present invention is highly advantageous.
  • the present inventors have already disclosed a method for producing an scFv-Fc antibody expression vector construct, a method for preparing a viral vector, and a method for producing a GO transgene chimeric chimera (WO 2004/016081 pamphlet). According to the present invention, a series of operations were performed in accordance with this, and a G0 transgene chimeric library was produced.
  • a viral vector construct for the production of a transgenic directory expressing TNFR-Fc was prepared as follows.
  • the gene region of TNFR is sold by Better, Invitrogen Clone Collection ID: 518 1070 (Invitrogen) was purchased and PCR amplified with the following primers.
  • Underlined are restriction enzyme Xhol and restriction enzyme Seal. 5 '-AATCTCGAGACCATGGCGC CCGTCGC— 3' (SEQ ID NO: 1)
  • the constant region hC ⁇ 1 region of the human immunoglobulin gene was obtained in the literature by Hotta et al. (Journal of Bioscience and Bioengineering Vol. 98 (2004), No. 4, p. 298-303), and this was amplified as a cage with the following primers.
  • Underlined are restriction enzyme Seal, restriction enzyme BamHI and restriction enzyme Clal. SEQ ID NO: 3) No. 4)
  • the PCR-amplified TNFR gene fragment was digested with restriction enzymes Xhol and Seal, and the PCR-enhanced hC y 1 gene was digested with restriction enzymes Seal and BamHI, and the pETB lue-2 vector (Novagen) BamHI was digested.
  • the yarn was penetrated into the Xhol site by ligation of 3 fragments. This vector was designated as pET / TNFR—Fc.
  • This pET / TNFR—Fc was digested with restriction enzymes Xhol and Clal, and pMSCV / GAA described in Hotta et al. (Journal of Bioscience and Bioengineering Vol. 98 (2004), No. 4, p. 298 — 303).
  • the pMSCV / GAATNFR—Fc vector was constructed by ligating to the restriction enzyme SalT and the restriction enzyme Clal site.
  • a packaging cell line was established based on the virus vector described in Example 2 by the method described in International Publication No. 2004/016081. Based on this packaging cell, a transgenic directory that expresses TNFR-Fc was prepared according to WO 2004/016081.
  • # 0513, # 0608, # 0709, # 0711, # 0712 ⁇ were used to produce the first day, and the TNFR—Fc concentration in the egg was quantified. / V), egg white was diluted with PBS so as to be 10% (V / V), and cryopreserved to obtain a sample for measurement.
  • An anti-human IgG antibody (manufactured by Cosmo Bio) diluted with PBS was placed in an ELISA plate at 100 ⁇ g / well and allowed to stand at 4 ° C. Each well was washed 3 times with PBS—0.05% Tween20 solution in 200 1, and then 2% skim minolek in PBS—0.05% Tween20 solution was added in 1 SO 1 / well.
  • a standard calibration curve was prepared using purified TNFR-Fc.
  • the TNFR-Fc fusion protein vector construct prepared in Example 2 was introduced into GP293 cells by the lipofection method, and the culture supernatant was centrifuged at 3000 rpm for 10 minutes at 4 ° C. to remove solids. The supernatant was stirred while cooling, and ammonium sulfate finely crushed to 50% saturation was gradually added (313 g ammonium sulfate / 1000 ml water) to precipitate the protein. After leaving this at 4 ° C, centrifuge at 15000rpm for 10 minutes at 4 ° C. The precipitate was completely settled and dissolved in a small amount of PBS. Ammonium sulfate was removed by dialyzing 3 times with 2 L of PBS.
  • Binding Buffer 10 ml in this order (flow rate 2 ml / min).
  • the protein solution dissolved in PBS was flowed at lml / min, and TNFR-Fc was adsorbed on the column. Unnecessary protein is removed by flowing 20 ml of Binding Buffer at 1.7 ml / min, and then Elution Buffer (glycine 7.507 g / l, adjusted to pH 2.5-3.0 with 2N HC1) at 1.5 ml / min.
  • TNFR-Fc was eluted by flushing.
  • the eluted fraction was dialyzed 3 times with PBS (2 U, purified TNFR-Fc, and the protein concentration was quantified from the absorbance at a wavelength of 280 nm.
  • Fig. 1 The quantitative results for # 0314 are shown in Fig. 1, and the quantitative results for # 0513, # 0608, # 0709, # 0711, and # 0712 are shown in Fig.2. From Fig. 1, it can be seen that there is more blur in the yolk than in the egg white, although there is some blurring depending on the egg collection date. In addition, Fig. 2 shows that accumulation in the yolk is not due to transgenic chickens.
  • the G0 transgenic chimera chicken born in Example 1 was sexually matured and the eggs laid were collected. 15 ml of egg yolk was collected from 3 eggs. To this, 60 ml of PBS was added and centrifuged at 15000 rpm at 4 ° C for 10 minutes, and the supernatant was collected. This process was repeated 4 times. To this was added lml of rProtein A Sepharose Fast Flow (trade name, manufactured by Amersham), and the mixture was stirred at 4 ° C for 2 hours. Thereafter, the mixture was centrifuged at 15000 rpm for 5 minutes at 4 ° C, the supernatant was removed, and the plate was washed 5 times with PBS.
  • rProtein A Sepharose Fast Flow trade name, manufactured by Amersham
  • FISHERbrand screening column (trade name, manufactured by Fischer 'Henore Care), washed with 20 ml of PBS, eluted with 0.1 M glycine—HC1 ( ⁇ 2 ⁇ 7), and tris—HCl (pH 8.0). The sample was neutralized and dialyzed against 10 mM ammonium acetate to prepare a sample for sugar chain analysis.
  • the sample prepared in Example 6 was subjected to sugar chain analysis.
  • Sample 2 Omg was dialyzed against 2 L of 10 mmol / l ammonium bicarbonate. This This was collected in 6 test tubes for hydrazine decomposition and freeze-dried. Thereafter, it was set in Hydra Club S-20 4 (manufactured by Seikagaku Corporation) and dried at 50 ° C. under reduced pressure for 12 hours. 2 ml of anhydrous hydrazine was injected under reduced pressure, and hydrazine decomposition was performed at 100 ° C. for 2 hours. After 2 hours, hydrazine was distilled off under reduced pressure. It returned to room temperature and the test tube for hydrazine decomposition
  • Ammonium acetate buffer solution 200 11 1 was added to each test tube after hydrazine decomposition and stirred. Trimethylpyridine 101 was added to this and stirred. Further, acetic anhydride 101 was added and stirred. All were allowed to react at room temperature for 20 minutes and then dried to dryness with a centrifugal evaporator. An initial equilibrium of a cellulose cartridge for purification (manufactured by Takara Bio Inc.) was performed.
  • the cellulose cartridge was washed 3 times with 10 ml of purified water. This was washed with 10 ml of solvent 2 for cellulose cartridge (50 vol% ethanol). Further, equilibration was performed with 10 ml of solvent 1 for cellulose cartridge (67 vol% butanol / 17 vol% ethanol).
  • Acetic acid 1001 was added to 1 vial (300 mg) of 2-aminominocine (PALSTATION Pyridylamination Reagent Kit for sugar chain analysis, manufactured by Takara Bio Inc.) and dissolved in an oven by heating.
  • 2-aminominocine PALSTATION Pyridylamination Reagent Kit for sugar chain analysis, manufactured by Takara Bio Inc.
  • the coupling reagent 201 prepared above was added to each tube dried by a centrifugal evaporator and stirred. This was placed in a 90 ° C oven and allowed to react for 60 minutes. To this, reducing reagent 201 was added and stirred. Place this in an 80 ° C oven and react for 60 minutes.
  • the present inventors have already disclosed a method for producing an anti-CD2 human IgG antibody expression vector construct, a method for preparing a viral vector, and a method for producing a GO transgenic chimeric chicken (International Publication No. 2004/016081). .
  • a series of operations were performed in accordance with the present invention, and a GO transgenic chimera catalog was produced.
  • a sample for analysis of sugar chains of anti-CD2 HgG antibody accumulated in egg yolk was prepared according to Example 6.
  • the sugar chain analysis of the anti-CD2 HgG antibody accumulated in the yolk was performed according to Example 7.
  • FIG. 1 shows the amount of TNFR-Fc accumulated in one egg of a GO transgenic chimeric chicken into which a TNFR-Fc vector construct was introduced as measured in Example 5. It can be seen that the amount accumulated in egg yolk is higher than in egg white regardless of the egg-laying date!
  • FIG. 2 shows the amount of TNFR-Fc accumulated in 5 eggs of GO transgene chimeric chimera in which the TNFR-Fc vector construct was introduced as measured in Example 5. It can be seen that the amount accumulated in egg yolk is higher than in egg white regardless of the individual.
  • FIG. 3 shows the results of sugar chain analysis of scFv-Fc accumulated in egg yolk performed in Example 7.
  • a small amount of monosialoglycan (6.7%) was detected, which was mainly composed of a cashmere glycan from which sialic acid at the non-reducing glycan was removed. From this, it was found that sialic acid was added to the sugar chain of scFv-Fc produced in egg yolk.

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Abstract

Cette invention concerne les procédés pour produire une protéine exogène. Jusqu'à présent, un effet original ne pouvait pas être obtenu en raison d'une différence dans la structure de la chaîne glucidique devant être modifiée dans certaines protéines. Afin de résoudre ce problème, un procédé de production d'une protéine exogène ayant une structure de chaîne glucidique équivalente ou similaire à celle présente chez l'homme est nécessaire. Comme une protéine exogène accumulée dans le jaune d'oeuf d'un oiseau transgénique, en particulier le poulet, possède une structure de chaîne glucidique équivalente ou similaire à celle présente chez l'homme, il devient possible de produire une grande quantité de protéines pharmaceutiques humaines avec une bioactivité élevée en employant ce procédé.
PCT/JP2007/065655 2006-08-10 2007-08-09 Production d'une protéine utile telle qu'un anticorps ayant une chaîne glucidique modifiée dans le jaune d'oeuf d'un poulet transgénique WO2008018562A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004016081A1 (fr) * 2002-08-13 2004-02-26 Kaneka Corporation Technique d'expression de gene dans des oiseaux transgeniques au moyen de vecteur retroviral et oiseaux transgeniques ainsi obtenus
WO2006035687A1 (fr) * 2004-09-28 2006-04-06 Kaneka Corporation Méthode d’élaboration d’un oiseau transgénique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004016081A1 (fr) * 2002-08-13 2004-02-26 Kaneka Corporation Technique d'expression de gene dans des oiseaux transgeniques au moyen de vecteur retroviral et oiseaux transgeniques ainsi obtenus
WO2006035687A1 (fr) * 2004-09-28 2006-04-06 Kaneka Corporation Méthode d’élaboration d’un oiseau transgénique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KAMIHIRA M. ET AL.: "High-Level Expression of Single-Chain Fv-Fc Fusion Protein in Serum and Egg White of Genetically Manipulated Chickens by Using a Retroviral Vector", JOURNAL OF VIROLOGY, vol. 79, no. 17, 2005, pages 10864 - 10874, XP003020744 *
RAJU T.S. ET AL.: "Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recomninant glycoprotein therapeutics", GLYCOBIOLOGY, vol. 10, no. 5, 2000, pages 477 - 486, XP002964086 *

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