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WO1997007670A1 - Vertebre presentant une tolerance immunologique et procede d'utilisation de ce vertebre - Google Patents

Vertebre presentant une tolerance immunologique et procede d'utilisation de ce vertebre Download PDF

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Publication number
WO1997007670A1
WO1997007670A1 PCT/JP1996/002312 JP9602312W WO9707670A1 WO 1997007670 A1 WO1997007670 A1 WO 1997007670A1 JP 9602312 W JP9602312 W JP 9602312W WO 9707670 A1 WO9707670 A1 WO 9707670A1
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thr
pro
ser
val
lie
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PCT/JP1996/002312
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English (en)
Japanese (ja)
Inventor
Hideyuki Matsushita
Ikunoshin Kato
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Takara Shuzo Co Ltd
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Takara Shuzo Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takara Shuzo Co Ltd filed Critical Takara Shuzo Co Ltd
Priority to AU67097/96A priority Critical patent/AU6709796A/en
Publication of WO1997007670A1 publication Critical patent/WO1997007670A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells

Definitions

  • the present invention relates to a heterologous protein-tolerant vertebrate useful for the production of a heterologous protein, a method of inducing tolerance to produce an immune-tolerant vertebrate, a method of producing a heterologous protein using the immune-tolerant vertebrate, and a method of producing an immunotolerant vertebrate. It relates to the tolerogenic agent used.
  • transgenic embryos that incorporate the substance into the chromosomes of the embryo.
  • This embryo can be grown by transplantation into a foster mother, and the resulting adult animal has incorporated the foreign DNA into its chromosome and can express it.
  • Transformed individuals are generally called transgenic animals [Science, Vol. 214, pp. 1244-1246 (1981)].
  • the incorporated exogenous DNA is called a transgene and generally comprises a promoter and a target gene such as cDNA.
  • the target gene is a gene encoding a heterologous protein
  • tolerance to the heterologous protein has been established, and the transgenic animal is used to produce the heterologous protein. I have. Purpose of the invention
  • the transgenic animal is produced as an animal having the target gene by transplanting the transformed embryo into a foster parent and developing and giving birth to the offspring, so that it takes a long time to produce the transgenic animal.
  • a large number of mother animals as foster mothers are required.
  • An object of the present invention is to provide a method for efficiently obtaining a heterologous protein-immune tolerant vertebrate without using a foster parent, a heterologous protein obtained by the method, an immunotolerant vertebrate, and the production of a heterologous protein by the immunotolerant vertebrate. It is an object of the present invention to provide a method and a tolerance-inducing agent for obtaining a vertebrate.
  • a first aspect of the present invention relates to a heterologous protein tolerant vertebrate, comprising the following steps (A), (B) and (C):
  • step (C) a step of introducing the recombinant hematopoietic stem cells obtained in the step (A) into the immunodeficient vertebrate obtained in the step (B);
  • a second aspect of the present invention relates to a method for inducing immune tolerance in a vertebrate, comprising the following steps (A), (B) and (C):
  • step (C) Recombinant hematopoietic stem cells obtained in step (A) are obtained in step (B). A step of introducing into the obtained immunodeficient vertebrate,
  • the third embodiment of the present invention comprises the following steps (A), (B), (C) and (D)
  • step (D) a step of expressing a heterologous protein in the animal obtained in the step (C), which provides a method for producing a heterologous protein.
  • a fourth aspect of the present invention is to provide a tolerogenic agent characterized by containing a vertebrate-derived hematopoietic stem cell into which a gene encoding a heterologous protein has been incorporated.
  • the immune-tolerant vertebrate into which the transgenic hematopoietic stem cells have been introduced according to the present invention can be efficiently prepared in a short period of time without using a foster parent, and can be used for the disease resistance, reproductivity, weight gain, milk yield, etc. of the animal itself. You can make improvements.
  • heterologous proteins can be efficiently produced, and can be used for the production of heterologous proteins useful as pharmaceuticals, diagnostics, or various reagents.
  • the heterologous protein-tolerant vertebrate of the present invention includes mammals (eg, mice, rats, puppies, higgins, goats, pigs, puppies, puppies, dogs, monkeys, chimpanzees, etc.), birds (eg, ⁇ Birds, turkeys, quails, ducks, ducks, etc.), reptiles (eg, snakes, dinosaurs, turtles, etc.), amphibians (eg, rickshaw, sanshowo, Mori), fishes (eg, horse mackerel, mackerel, sea bass, Thailand, grouper, puri, tuna, salmon, trout, carp, penis, flounder, shark, ray, sturgeon, etc.)
  • a hematopoietic stem cell is prepared from the vertebrate, a gene encoding a heterologous protein is incorporated into the hematopoietic stem cell, and then the immune function of the vertebrate is impaired, An animal can be obtained by introducing a recombinant hem
  • the heterologous protein in the present invention is a protein showing antigenicity to an untreated vertebrate, and is stably produced in the immunotolerant vertebrate of the present invention.
  • Such heterologous proteins include, for example, human SOD, inter-kinds (human, mouse, etc.), human interferon, human insulin, human colony stimulating factor, human tissue plasminogen activator, human —Properokinase, Perokinase, human—coagulation factor (IV, VII-XII), human erythropoietin, human nerve growth factor, human atrial natriuretic peptide, human secretory trypsin inhibitor, Growth hormones (humans, mice, birds, chickens, fish, etc.), growth hormone-releasing factors, antibodies and the like.
  • polypeptides having an amino acid sequence substantially identical to these heterologous proteins are also included in the heterologous proteins of the present invention.
  • a polypeptide having an amino acid sequence that is substantially identical to such a heterologous protein is defined as a polypeptide in which the amino acid of the heterologous protein differs from the other amino acid within a range that does not substantially impair the intrinsic activity of the heterologous protein. It means a polypeptide in which an amino acid has been replaced, a polypeptide in which an amino acid or amino acid sequence has been deleted, and a polypeptide in which an amino acid or amino acid sequence has been inserted.
  • Hematopoietic stem cells are multipotent and self-replicating cells. And can be obtained from vertebrate bone marrow cells. Hematopoietic stem cells, although only a frequency of 1 Z 1 0 about 5 in the bone marrow cells is a cell that does not exist, the cell surface receptor of the hematopoietic stem cells, stem Self actors: a receptor (stem cell factor SCF) c — It can be purified using an antibody against the kit receptor [Blood, Vol. 78, pp. 176-172 (1991)], which is used in the present invention. Can be.
  • kit receptor Kit receptor
  • the method for introducing a gene encoding a heterologous protein into hematopoietic stem cells is not particularly limited, but includes a known calcium phosphate method, a microphone injection method, an electroporation method, an adenovirus, and SV40.
  • a method using a virus, a simple virus, or the like can be used.
  • the use of a retroviral vector enables the stable insertion of a gene of interest into the chromosomes of dividing and proliferating cells.
  • a replication-defective system has been established so that a recombinant virus does not produce a new virus even if it infects target cells, and a high-titer and stable supply method has been reported [Hum. Gen. Ther. Vol. 5, pp. 19-28 (1994)].
  • the infection efficiency depends on the titer of the recombinant virus, co-culture of the recombinant virus-producing cell and the target cell is required for highly efficient gene transfer into hematopoietic stem cells [Nature, 31 Volume 0, pp. 476 (1994)].
  • fibronectin can be used as a method to introduce a virus supernatant, that is, virus particles only, into target cells and cultivate them to introduce genes with high efficiency.
  • the fibronectin fragment is represented by SEQ ID NO: 7 in the sequence listing described in JP-A-2-311498, and is described in Escheric hia coli HB101 / pHD102 (FERMP-10721).
  • a polypeptide produced by the method which is also represented by SEQ ID NO: 8 in the sequence listing, has Escherichia coli HB101 / pCH102 (FERM).
  • BP-2800 Genes encoding heterologous proteins can be introduced into target cells with high efficiency by using these polypeptides and the above method. This method is described in W095 26200 published October 5, 1995 internationally.
  • a gene encoding a heterologous protein to be incorporated into a retrovirus vector generally comprises a promoter and a target gene such as cDNA.
  • Recombinant hematopoietic stem cells into which the gene encoding the heterologous protein of interest has been incorporated are introduced into vertebrates with immunocompromised after collecting the hematopoietic stem cells.
  • a method for rendering the vertebrate immune-compromised for example, irradiation may be performed, or an immunocompromised drug may be administered.
  • a pure mouse is irradiated with about 1,000 rads of radiation. This dose is lethal to mice, after a short time blood cells are depleted, white blood cells are lost, immunity is compromised, infection occurs, and death occurs in about two weeks [Bad. Res., 14, 313-322 (1961)].
  • the mice can continue to survive due to hematopoietic cells reconstituted from the administered hematopoietic stem cells.
  • Immunodeficiency drugs include anti-lymphocyte serum, prednisolone, methylbu Corticosteroids such as redonizolone and dexamethasone, azathioprine,
  • Purine antagonists such as 6-MP, alkylating agents such as cyclophosphamide and busulfan, cyclosporine 8, FK506 and the like can be used.
  • anti-lymphocyte serum When using anti-lymphocyte serum, it may be administered intravenously or intramuscularly at a dose of 1-2 O mg / kg as agropurine, and 10-1 S mgZkg when using methylprednisolone as a corticosteroid. Large doses may be given.
  • azathioprine it may be administered at a dose of 2-3 mg / kg, and when cyclophosphamide is used, a large dose of 40-5 O mgZkg may be administered.
  • the use of these immunocompromised drugs impairs the vertebrate's immune competence. Similarly, immunocompromised vertebrates can survive by introducing recombinant hematopoietic stem cells.
  • the introduction of recombinant hematopoietic stem cells into immunodeficient vertebrates can be performed by intramuscular or subcutaneous administration, as long as the recombinant hematopoietic stem cells can reach the bloodstream, but intravascular, especially intravenous, preferable.
  • the foreign gene encoding the heterologous protein of interest integrated into the chromosome of the recombinant hematopoietic stem cell is expressed in the introduced vertebrate, and the expressed heterologous protein is recognized as self and undergoes rejection by the immune system None.
  • heterologous protein of interest can be recovered from a body fluid of an immune-tolerant vertebrate, such as blood, by a method known per se.
  • a disease model animal caused by the expressed protein can be provided, and the animal is useful, for example, for developing a therapeutic drug for human disease.
  • the immunological tolerance inducing agent of the present invention comprises a vertebrate-derived recombinant hematopoietic stem cell into which a gene encoding the above-described heterologous protein has been incorporated.
  • the inducer is not particularly limited, but may be, for example, a normal living cell preparation by a method known per se, and may be used for preparing a heterologous protein-tolerant animal.
  • the same method as in the introduction of recombinant hematopoietic stem cells into a deficient vertebrate can be employed, and the number of cells in the agent, the number of administrations, and the like can be appropriately determined according to the vertebrate to be applied.
  • hFN a polypeptide represented by SEQ ID NO: 1 in the sequence listing (hereinafter, referred to as hCH271) was prepared.
  • hCH271 is a polypeptide represented by SEQ ID NO: 2 in the sequence listing required for the cell adhesion activity of FN in the molecule and a polypeptide represented by SEQ ID NO: 3 in the sequence listing required for the heparin binding activity of FN which is prepared using Escherichia coli HB101 / pCH101 according to the method described in JP-A-2-311498. The strain has been deposited as FERM BP-2799 with the National Institute of Biotechnology and Industrial Technology. (2) Preparation of mouse recombinant fibronectin (mFN)
  • a polypeptide represented by SEQ ID NO: 4 in the sequence listing (hereinafter referred to as mCH271) was prepared as mFN.
  • mCH271 contains in its molecule a polypeptide represented by SEQ ID NO: 5 in the sequence listing required for cell adhesion activity of FN and a polypeptide represented by SEQ ID NO: 6 in the sequence listing required for heparin binding activity of FN.
  • the polypeptide was prepared using Escherichia coli HB101 / pMCH11 (FERM BP-5580) as described below.
  • poly (A) + mRNA was prepared using a kit “FastTrack” manufactured by Invitrodin.
  • a mouse liver cDNA was obtained from the prepared poly (A) + mRNA by using the Amersham kit “cDNA Synthesis System PI usj.”
  • PCR specific primer for the region encoding the cell adhesion domain and the heparin binding domain of mFN was devised from the rat fibronectin cDNA sequence (DNAS IS database), and the following PCR primer was synthesized. .
  • RC-6 5'TTGAAGGCAGCCACCTGACACT 3
  • RC-7 5'AGAGGAAGACAGAAAACAGGTCT 3
  • RC-10 5'GGCTCTCCTCTCTGCCATT 3 ' Primer for heparin binding domain code region:
  • RH-1 5'TACACAGTCAGTGTGGTTGCCT 3 'RH-2: 5'AGATCTCTGGTCCATGAAGATT 3'
  • RH-3 5'TACTCAAGCCCTGAGGATGGAA 3 'RH-4: 5'TGTCATACCCAGGGTTGGTGACGAA 3'
  • the nucleotide sequences of RC-5, RC-6, RC-7, and RC-10 are shown in SEQ ID NOs: 9 to 12 in the sequence listing.
  • the base sequences of RH-1, RH-2, RH-3, and RH-4 are shown in SEQ ID NOs: 13 to 16 in the sequence listing.
  • a plasmid containing the DNA fragment amplified by RC-5 / RC-10 pMC 35
  • NcoI site was introduced by in vitro mutagenesis into sites corresponding to the N-terminal and C-terminal of the cell adhesion domain of the plasmid pMC5 insert obtained in (2-2), and the DNA fragment cut out with Ncol.
  • Ncol site was introduced at the position corresponding to the N-terminus of the heparin-binding domain of the insulin of pMH5, and stop codons TAA TAG and EcoRI site were introduced at the position corresponding to the C-terminus.
  • Escherichia coli HB101 carrying this plasmid pMCHl1 is referred to as Escherichia coli HB101 / pCH11, and FERM BP-5580 deposit number at the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Tsukuba, 1-3-1, Higashi, Ibaraki Prefecture, Japan) (Original deposit date: 1995 August 25).
  • Escherichia coli / pM CH11 obtained in (2-3) was cultured with shaking at 37 ° C. overnight in 5 ml of L-broth supplemented with 0.1 mg of ampicillin. This was inoculated into 500 ml of the same medium, cultured for 20 hours, and collected. The whole cell pellet was suspended in 5 OmM Tris-HC1 (pH 7.5), 1 mM EDTA, 1 OmM mercaptoethanol, 0.5 mM PMSF (Phenylmethanesulfonylfluoride) solution and sonicated. I got it. After centrifugation at 12,000 rpm for 20 minutes, 25 ml of the supernatant was obtained.
  • 5 OmM Tris-HC1 pH 7.5
  • 1 mM EDTA 1 mM EDTA
  • 1 OmM mercaptoethanol 0.5 mM PMSF (Phenylmethanesulfonylfluoride) solution and sonicated. I got it.
  • BHK-21 baby hamster kidney cells
  • Dulbecco's minimum nutrient medium a concentration of 5 x 10 5 cells / ml
  • the BHK-21 cells used were obtained by subculturing a cryopreserved strain and then treating with trypsin (37 ° C, 5 minutes).
  • trypsin 37 ° C, 5 minutes
  • the cells were fixed on the plate with a 3% formalin solution. As a result of observing the extension of BHK-21 cells under a microscope, it was confirmed that the cells possess cell adhesion activity.
  • the measurement of heparin binding activity was performed as follows.
  • hCH271 and mCH271 were dissolved in physiological saline to prepare lmg / ml sample solutions.
  • Six-week-old female ICR mice (5 mice per group) were intravenously administered 0.2 ml of each sample solution, and the same amount was intravenously administered on days 1, 2, 3, 4, 6, and 8 .
  • hCH271 and mCH271 were dissolved in physiological saline to prepare Img ⁇ sample solutions, respectively.
  • Six-week-old female ICR mice (5 mice per group) were intravenously administered 0.2 ml of each sample solution, and the same amount was intravenously administered at 1, 2, 3, 4, 6, and 8 .
  • mice blood was collected from the orbit of each group of mice, and the serum was separated by centrifugation at 37 rpm for 1 hour and 3000 rpm for 10 minutes. In addition, control sera were separated from untreated mice.
  • a phosphate buffer solution hereinafter abbreviated as PBS
  • Serum obtained from each sample-administered mouse was diluted with 1% BSABPBS to an appropriate dilution, and each of the above-mentioned polypeptides was dispensed into wells in which the above polypeptides were immobilized. Next, after incubating at 37 ° C. for 1 hour, the plate was washed four times with PBS in the same manner as above.
  • Table 1 shows the results of experiments using 10,000-fold diluted serum prepared from each of the polypeptide-treated mice and untreated mice. The experiments were performed in triplicate.
  • C3HZHeJ mice Six-week-old C3HZHeJ mice were given 15 OmgZkg of 5-fluoruracil in the tail vein, and the femur and rib bone marrow were collected 2 days later.
  • the bone marrow cells were then transformed in the presence of a recombinant rat stem self-actor (rrSCF, manufactured by Amdi K.K.) and recombinant human interleukin 6 (rhIL-6, manufactured by Pepco Tech).
  • rrSCF recombinant rat stem self-actor
  • rhIL-6 human interleukin 6
  • a retrovirus vector derived from mouse moloni monoleukemia virus (ATCC VR-190) incorporating the hCH271 gene prepared from pCH101 was transformed into a helper cell ⁇ [mouse 3T3ZN IH cell (ATCC
  • mice hematopoietic stem cells and the recombinant retrovirus-producing cells are co-cultured at 37 ° C for 48 hours in the presence of rrSCF and rhlL16.
  • a retrovirus vector having a gene encoding hCH271 incorporated therein was introduced into mouse hematopoietic stem cells.
  • hematopoietic stem cells were transfected from the tail vein of 6-week-old C3HZHeJ mice, which had been completely immunodeficient by irradiation with 1,20 OcGy to completely destroy hematopoietic cells.
  • mice usually die in about 2 days, but administration of retroviral-incorporated hematopoietic stem cells allows the mice to survive for a long period of time, and the self-proteinized expression protein of the introduced hFN gene (hCH271). And continued to produce hCH271 in blood cells.
  • Peripheral blood was collected 3 and 6 months after hematopoietic stem cell transplantation, and the amount of hCH271 in the blood cells was quantified using the human fibronectin EIA Kit (Takara Shuzo). Production was confirmed.
  • a heterologous protein-tolerant vertebrate can be efficiently provided without using a foster parent.
  • the immune-tolerant vertebrate is useful in the production of a heterologous protein, and can efficiently produce a bioactive protein or a disease-treating protein from the vertebrate according to the purpose.
  • a method for inducing tolerance to a heterologous protein that exhibits antigenicity to a host animal, and a tolerant inducer useful for producing a tolerant animal are provided, so that a vertebrate tolerant animal can be easily produced by genetic engineering. It became.
  • the heterologous protein-tolerant animal can be used as a model animal for a disease caused by an expressed protein, and the animal is useful, for example, for the development of human disease treatment.
  • Ser lie Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
  • 200 205 210 lie Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin Glu Phe
  • Asp Tyr Lys lie Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser
  • Ser lie Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
  • 200 205 210 lie Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin Glu Phe
  • Ala lie Pro Ala Pro Thr Asp Leu Lys Phe Thr Gin Val Thr Pro 1 5 10 15
  • Lys Glu lie Asn Leu Ala Pro Asp Ser Ser Ser Val Val Val Ser
  • Ser lie Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
  • 200 205 210 lie Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin Glu Phe
  • Asp Tyr Lys lie His Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser
  • Ser lie Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
  • 200 205 210 lie Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin Glu Phe
  • Lys Glu lie Asn Leu Ser Pro Asp Ser Ser Ser Val lie Val Ser
  • Ala lie Pro Ala Pro Thr Asp Leu Lys Phe Thr Gin Val Thr Pro 1 5 10 15
  • Lys Glu lie Asn Leu Ala Pro Asp Ser Ser Ser Val Val Val Ser
  • Ser lie Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
  • 200 205 210 lie Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin Glu Phe
  • Asp Tyr Lys lie Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser
  • Glu Pro Leu lie Gly Arg Lys Lys Thr Asp Glu Leu Pro Gin Leu 545 550 555
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA)
  • Sequence type Other nucleic acid (synthetic DNA) Sequence:
  • Sequence type nucleic acid
  • Sequence type Other nucleic acid (synthetic DNA) Sequence:
  • Sequence type nucleic acid
  • Sequence type Other nucleic acid (synthetic DNA) Sequence:
  • Sequence type nucleic acid
  • Sequence type Other nucleic acid (synthetic DNA) Sequence:
  • Sequence type nucleic acid
  • Sequence type Other nucleic acid (synthetic DNA) Sequence:
  • Sequence type nucleic acid
  • Sequence type Other nucleic acid (synthetic DNA) Sequence: TGTCATACCC AGGGTTGGTG ACGAA 25

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  • Life Sciences & Earth Sciences (AREA)
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  • Cell Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
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  • Biodiversity & Conservation Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Cette invention concerne un gène codant des protéines étrangères, lequel gène est intégré dans des cellules somatiques hématopoïétiques provenant d'un vertébré. Les cellules souches hématopoïétiques et recombinantes ainsi obtenues sont ensuite introduites dans le vertébré qui a été rendu immunodéficient. Ce système permet d'obtenir de manière efficace un vertébré qui présente une tolérance immunologique envers des protéines étrangères, et qui pourra être utilisé dans la production de protéines étrangères, entre autres.
PCT/JP1996/002312 1995-08-30 1996-08-19 Vertebre presentant une tolerance immunologique et procede d'utilisation de ce vertebre Ceased WO1997007670A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU67097/96A AU6709796A (en) 1995-08-30 1996-08-19 Immunologically tolerant vertebrate and use of the same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP22158395 1995-08-30
JP7/221583 1995-08-30

Publications (1)

Publication Number Publication Date
WO1997007670A1 true WO1997007670A1 (fr) 1997-03-06

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001011060A (ja) * 1999-06-28 2001-01-16 Nippon Synthetic Chem Ind Co Ltd:The 新規オキサゾール化合物及びその製造方法
WO2001077371A1 (fr) * 2000-04-07 2001-10-18 Shionogi & Co., Ltd. Procede de dosage de preincubation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KEIZABURO TANI et al., ONCOLOGIA, 26(1), p. 124-126 (1993). *
SEIJI OKADA et al., EXPERIMENTAL MEDICINE, 12(2), p. 171-177 (1994). *
TERESA LIMJOCO et al., VIROLOGY, 208(1), p. 75-83 (1995). *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001011060A (ja) * 1999-06-28 2001-01-16 Nippon Synthetic Chem Ind Co Ltd:The 新規オキサゾール化合物及びその製造方法
WO2001077371A1 (fr) * 2000-04-07 2001-10-18 Shionogi & Co., Ltd. Procede de dosage de preincubation
US7018796B2 (en) 2000-04-07 2006-03-28 Shionogi & Co., Ltd. Preincubation assay methods

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