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WO1986004506A1 - Agent protecteur contre les infections contenant un facteur stimulant les colonies de granulocytes humains en tant qu'ingredient efficace - Google Patents

Agent protecteur contre les infections contenant un facteur stimulant les colonies de granulocytes humains en tant qu'ingredient efficace Download PDF

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Publication number
WO1986004506A1
WO1986004506A1 PCT/JP1986/000053 JP8600053W WO8604506A1 WO 1986004506 A1 WO1986004506 A1 WO 1986004506A1 JP 8600053 W JP8600053 W JP 8600053W WO 8604506 A1 WO8604506 A1 WO 8604506A1
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ctg
ser
cag
gcc
csf
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PCT/JP1986/000053
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English (en)
Japanese (ja)
Inventor
Masayoshi Ono
Hitoshi Nomura
Masahiko Tamura
Masahiko Matsumoto
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Chugai Seiyaku Kabushiki Kaisha
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Publication of WO1986004506A1 publication Critical patent/WO1986004506A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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 human granulocyte colony stimulating factor, and particularly to a specific stimulating factor, ie, a colony stimulating factor (hereinafter referred to as a "colony stimulating factor”), which is required mainly for colony formation of human granulocyte cells.
  • a colony stimulating factor a colony stimulating factor
  • CSF a gene encoding an active polypeptide, a recombinant vector incorporating the gene, a transformant containing the vector, and a product produced from the transformant
  • the present invention relates to a polypeptide or a glycoprotein having CSF activity and an infectious agent comprising CSF as an active ingredient.
  • CSF This factor is normally widely distributed in cells, such as T cells, monocyte macrophages, osteoblasts, It is known to be produced by endothelial cells and the like.
  • CSF has effects on granulocyte ⁇ monocyte macrophage stem cells, stimulates their proliferation and induces differentiation, and is composed of granulocytes and monocyte macrophages in soft agar.
  • Granulocyte-monocyte macrophage CSF (abbreviated as GH-CSF) that acts to form knees.Monomerocytes that mainly form colonies of monocyte macrophages.
  • M-CSF Sphere macrophage CSF
  • multi-CSF pluripotent CSF acting on more undifferentiated pluripotent stem cells
  • G-CSF granulocyte CSF
  • G-CSF granulocyte CSF
  • chemotherapeutic drugs in the field of infectious diseases have made it possible to treat conventional virulent substance producing bacteria having a specific pathogenicity. Due to the increased number of compromised hosts, infection with pathogens that are weakly pathogenic but resistant to drugs and disinfectants (opportunistic infections) poses new clinical problems. These opportunistic infections are caused by drug-resistant bacteria and fungi that are low in useful antibiotics, and their treatment involves activation of host defenses in addition to conventional chemotherapeutic agents. Although the use of drugs is desired, no effective drug has been found to date.
  • the phagocytic activity of leukocytes is thought to be the most influential among the host's defense functions, so it is necessary to enhance the proliferation and differentiation and maturation of neutrophils and macrophages.
  • the present inventors examined the protective effect of this human G-CSF using an infected animal model and found that the human G-CSF had a remarkable ability to mature neutrophils in vivo, and The present inventors have found that they are effective as a remedy for infectious diseases because they showed an infection protective effect, and then established the cell line CHU-2 derived from human oral floor carcinoma. From the culture supernatant of this cell line, G-CSF completely identical to that derived from CHU-1 was isolated from CNCM accession number I-483).
  • the method of isolating G-CSF from the culture supernatant using the cell culture method as described above is based on the fact that only a low concentration of G-CSF is produced and that a small amount of G-CSF can be obtained from a large amount of culture solution.
  • a large amount of uniform G-CSF has not yet been obtained. Therefore, there has been a demand for mass production of G-CSF using replacement DNA technology.
  • the present invention is to provide an anti-infective agent comprising human G-CSF as an active ingredient.
  • Human G-CSF which is the active ingredient of the infectious protective agent of the present invention, includes human G-CSF as well as those obtained by isolating from a culture supernatant obtained by culturing human G-CSF-producing cells.
  • Figure 1 shows the probe (IWQ), probe (A) and probe (LC) sequences.
  • Figure 2 shows the nucleotide sequence of the PHCS insert.
  • FIGS. 3 (B) and (I) show the amino acid sequences of the human G-CSF precursors deduced from PBRG4 CDNA.
  • FIGS. 3 (B) and (H) show the amino acid sequence of human mature G-CSF deduced from PBRG4 CDNA.
  • FIG. 4 (A) shows the nucleotide sequence of the cDNA insert of pBRV2.
  • FIGS. 4 (B) and (I) show the amino acid sequences of the human G-CSF precursors deduced from PBRV2 CDNA.
  • FIG. 4 (B) and (I) show the human maturation performed from PBRV2.CDNA.
  • FIG. 5 shows restriction enzyme cleavage sites of human G-CSF CDNA derived from PBRG4 or PBRV2.
  • Figure 6 shows part of the process for preparing the vector containing the tac promoter (+ VSE system).
  • Figure 7 shows the process for preparing a vector containing a synthetic PL motor (+ VSE system).
  • Figure 8 shows the process for preparing the vector containing the trp promoter (+ VSE system).
  • Figure 9 shows part of the process for preparing a vector containing the tac promoter (one VSE system).
  • Figure 10 shows the process for preparing a vector containing a synthetic PL motor. Indicates (— VSE series).
  • Figure 11 shows the process for preparing the vector containing the trp promoter (-VSF-system). '
  • Figure 12 shows the schematic structure of PHGA410.
  • FIG. 13 shows the process of constructing the expression vector PTN-G4.
  • Figures a and 14b show the construction process of pHGG4-dhfr, and Figure 15 shows the schematic structure of PHGV2.
  • FIG. 16 shows the process of constructing the expression vector PTN-V2.
  • FIG. 17a and FIG. 17b show the process of constructing the recombinant expression vector PHGV2-dhfr.
  • the gene encoding the polypeptide having human G-CSF activity according to the present invention has the human G-CSF activity obtained as a 15-17S fraction by sucrose density gradient centrifugation. It is a DNA (cDNA) that is complementary to a messenger RNA (mRNA) that encodes the polypeptide having it.
  • cDNA DNA
  • mRNA messenger RNA
  • One of the series of cDNAs has the gene encoding the polypeptide or ⁇ of FIG. 3 (B) or a part thereof, and more specifically, the cDNA of FIG. 3 (A). 5 'to base sequence from the end, from ATG at 32 to 34 nucleotides to CCC at 650 to 652 nucleotides, from 122 to "!! From ACC at 24 to CCC at 650 to 652
  • the sequence or the sequence described in Figure 3 ( ⁇ ) or Having a part of This series of cDNAs is referred to as CDNA (+ V3E).
  • the other series of cDNAs has the gene encoding polypeptide I or I of FIG. 4 (B) or a part thereof, and more specifically, has the nucleotide sequence of FIG. 4 (A).
  • 5 ' Sequence from ATG at nucleotide positions 31 to 33 to CCC at nucleotide positions 640 to 642, sequence from ACC at nucleotide positions 121 to 123 to CCC at positions 640 to 642 from terminal Or the sequence shown in FIG. 4 (A) or a part thereof.
  • This series of cDNAs is referred to as cDNA (-VSE).
  • the gene of the present invention is prepared, for example, by preparing G-CSF-encoding mRNA from a mammalian cell or the like having the ability to produce a polypeptide having G-CSF activity, and then preparing two mRNAs by a known method. It is converted to cDNA.
  • the above-mentioned mammalian cell serving as a source of mRNA is a cell line CHU-2 (Colection) derived from human oral floor cancer.
  • liiRNA is translated into a protein and examined for physiological activity or A method of determining the protein using a G-CSF antibody may be used.
  • mRNA is injected into the oocyte of the African frog (Xenopus laevis) and translated (Gurdon et al .;
  • a double-stranded cDNA is synthesized from the single-stranded cDNA, and the plasmid is exchanged with an appropriate vector DNA. Create This transforms Escherichia coli and the like,
  • a DNA group (hereinafter referred to as cDNA library) is obtained.
  • double-stranded cDNA from mRHA for example, use the 3'-end of fflRNA Oligos (dT) complementary to the poly-A-chain at the site are treated with reverse transcriptase as primers, or the oligos corresponding to part of the amino acid sequence of the G-CSF protein. It synthesizes a oligonucleotide and treats it with reverse transcriptase as a primer to synthesize cDNA complementary to mRNA. Double-stranded CDNA is degraded by alkaline treatment to degrade IBRNA.
  • dT fflRNA Oligos
  • the resulting single-stranded CDNA is treated with reverse transcriptase or DNA polymerase (eg, Kleenow fragment, etc.) and then treated with S1 It can be obtained by treating with nuclease or the like, or directly by treating with RNase H and DNA polymerase (for example, E. coli DNA polymerase I).
  • reverse transcriptase or DNA polymerase eg, Kleenow fragment, etc.
  • S1 S1 It can be obtained by treating with nuclease or the like, or directly by treating with RNase H and DNA polymerase (for example, E. coli DNA polymerase I).
  • the double-stranded cDNA obtained in this manner is used as an appropriate vector, for example, PSC101, pDF41, ColE1, pHB9, pBR322, pBR327,
  • E. coli After integration into EK-type plasmid vector represented by PACYC1 or phage vector represented by igt, ⁇ c, ⁇ gt10, ⁇ gtWES, etc., E. coli (X1776; HB101; DH1, C600 strains) Can be obtained to obtain a cDNA library (for example, see "Molecular cloning", supra).
  • an appropriate chemically synthesized DNA fragment is added in order to add a ligable end to the DNA end, and the vector-DNA and ATP, which have been cleaved with restriction enzymes in advance, are used. Can be performed by treating with T4 phage DNA ligase.
  • a vector previously cleaved with restriction enzymes -It is also possible to add dG, dC-chain (or dA, dT-chain) to each of DNA and double-stranded cDNA, and then gradually cool the solution containing both DHA. Yes (see Molecular Cloning above).
  • Transformation of a host cell with the recombinant DNA obtained in this way can be performed, for example, by the method described in detail by Hanahan when the host cell is E. coli (Bio to J.H0; —1 pp. 557 (1983) )), Ie, by adding the recombinant DNA to a recombinant cell prepared in the presence of CaCi 0, HgC 2 or RbC.
  • the fragment containing the gene encoding the polypeptide having the human G-CSF activity cloned in this manner can be re-incorporated into an appropriate vector DNA to obtain a fragment.
  • Other prokaryotic or eukaryotic host cells can be transformed.
  • the gene can be expressed in each host cell. Can be expressed.
  • Prokaryotic host cells include, for example, Escherichia COM, Bacillus subtiIis, Bacillus thermophiIus, etc.Compatible with the host to express the target gene in these host cells
  • the host cell may be transformed with a replicon from a possible species, a plasmid vector containing the origin of replication and regulatory sequences. It is also desirable that the vector has a sequence that can impart phenotypic (phenotypic) selectivity to the transformed cells.
  • E. col i is a vector that uses it as a host.
  • PBR322 Bactet al.
  • pBR322 contains genes for ampicillin and tetracycline resistance, and it is possible to identify transformed cells by using either of these resistances.
  • Promoters required for gene expression in prokaryotic hosts include the promoter for the 3-lactamase gene (Chang et al.; Nature _, Vol. 615 (1978)), the lacto-promoter (Goeddel). See Nature, Vol. 281, p. 544 (1979)) and Tributefan Promoter 1 (see Goeddel et al., Nuc Ieic Acid Res.), P. 4057 (1980)). Promoters can also be used to produce polypeptides having the activity of human G-CSF of the present invention.
  • host cells for eukaryotic microorganisms include, for example, Saccharomyces cerevisiae and the like, and plasmid YRp7 (Stinchcomb and the like; see Nature 282—39 (1979)) etc Can be used for transformation.
  • This plasmid has the TRP1 gene, which serves as a selection marker for yeast strains lacking the ability to produce tributaphan, and can be grown in the absence of tributaphan.
  • promoters that can be used for gene expression include acid phosphatase gene promoters (Hiyanohara et al .: Proc. Hat and Acad. USA, Vol. 80, p. 1 (1983)) and alcohol dehydrogenase gene promoter (Valenzuela et al., Nature, vol. 298, p. 347 (1982)).
  • mammalian cell-derived host cells examples include COS cells, Chinese hamster ovary (CH0) cells, C-127 cells, HeLa cells, and the like, and vectors for transforming these cells.
  • CH0 Chinese hamster ovary
  • C-127 cells examples include COS cells, Chinese hamster ovary (CH0) cells, C-127 cells, HeLa cells, and the like, and vectors for transforming these cells.
  • 'I mean, i) SV2—gpt (Muligan t Berg; Proc. Natl.
  • vectors contain an origin of replication, a selectable marker, a promoter located in front of the gene to be expressed, an RNA splice site polyadenylation signal, and the like.
  • promoters such as retrovirus, poliovirus, adenovirus, and simian virus 40 (SV40) may be used.
  • SV40 simian virus 40
  • the selectable markers include the phosphotransferase APH (3 ') II or I (neo) gene, the thymidine kinase (TK) gene, and Escherichia coli xanthine-guanine.
  • a phospholiposyltransferase (Ecogpt) gene, a dihydrofolate transporting enzyme (DHFR) gene and the like can be used.
  • a recombinant DNA having the gene incorporated into an appropriate site of the vector is used. After transforming the host cell and performing fc, the obtained transformant may be cultured. Further, the polypeptide can be separated and purified from the cells or from the culture solution by using known means.
  • eukaryotic genes are considered to exhibit polymorphism (polyfflorph'ysm), as in the case of human interferon genes. (Eg, Nishi et al .; J. Biochem. ._ L_, p. 153 (1985)). In some cases, one or more amino acids may be replaced by this polymorphism. Amino acids may not change at all.
  • polypeptides in which one or more of the amino acids have been replaced with one or more amino acids may also have G-CSF activity.
  • a polypeptide obtained by converting a base sequence corresponding to the cystine of the human interloinkin 2 (IL-2) gene into a base sequence corresponding to serine is an i-peptide. It is already known that it retains interloukin 2 activity (Wang et al .; Science, vol. 1431 (1984)). Therefore, as long as the naturally occurring or artificially synthesized polypeptide has human G-CSF activity, all the genes encoding such polypeptides are included in the present invention. .
  • a gene encoding a polypeptide having the activity of human G-CSF activity of the present invention, a recombinant vector having the gene, a transformant having the gene, and the transformant A method for obtaining a polypeptide or glycoprotein having the activity of human G-CSF expressed in the above is briefly described below.
  • the homogeneous human CSF ⁇ obtained from the culture supernatant of the tumor cell line CHU-2 was purified.
  • the amino acid sequence of the protein was determined from the N-terminus. After fragmentation, the amino acid sequence of the fragment was also determined.
  • the probe (A) is a mixed type probe consisting of 14 consecutive nucleotides.
  • Probes are used in the cloning of the human kinase kinin gene (Takahashi et al .; Proc. Natl. Acad. Sci., USA, ⁇ 1931 (1985). ) 30 consecutive nucleotides using dextrin inosine.
  • the probe (LC) was synthesized from the amino acid sequence shown in Example 3 (i), which corresponds to positions 32 to 39 from the N-terminal, based on the base sequence shown in Fig. 3. Done 24 It is a probe consisting of individual nucleotides.
  • Nucleotide chemical synthesis can be carried out by applying an improved phosphotriester method to the solid-phase method and is described in a review by Harang (Tetrahedron ⁇ Vol. 3-22 (1983)).
  • the probe used may be based on the amino acid sequence at a position other than the probe used in the present invention.
  • RNA from all the RAs using a cell line (dT) cellulose single-stranded cDNA was synthesized using reverse transcriptase, and RNase H and E. coli DN A The cDNA was added to obtain a double-stranded cDNA, which was added with an IIC chain and joined to a PBR322 vector having a dG chain added to the Pst I cleavage site.
  • E. coli strain X1776 was transformed to construct a PBR322 cDNA library (Examples 5 and 6).
  • a double-stranded cDNA was ligated to the ⁇ gt10 vector using an EcoRI linker to construct a phage-based cDNA library (Example 7).
  • a recombinant derived from the PBR322 cDNA library was immobilized on Pittman 541 filter paper, and colony hybridization was performed using a probe (IWQ) radiolabeled with 32 P. One clone was selected. This clone was applied to the Southern blotting method (Southern: J. Mol. Biol. 98: 503 (1975)). When used in more detail, the probe (A) was also hybridized.
  • the nucleotide sequence of this clone was obtained by the Sidge method.
  • FIG. 2 shows the nucleotide sequence of the obtained cDNA insert.
  • this cDNA insert consists of 308 base pairs including the probe (IWQ) and the probe (A), and contains the amino acid sequence shown in Example 3 (iii). It was found that it possessed the ability to code 83 amino acids-a open reading frame.
  • the PBR322-derived plasmid containing 308 base pairs is abbreviated as pHCS-1 below. (Defeat 8)
  • a DNA fragment containing 308 base pairs obtained from PHCS-1 was radiolabeled by the nickel-slanking method (see Hole cul- lar cloning described above), and this was used as a probe.
  • Plasmid cDNA library (from Benton)
  • this cDNA insert has one large open reading frame.
  • amino acid sequence encoded by this cDNA can perform as shown in FIG. 3 (A).
  • this cDNA was found to be 32 to 34 nucleotides from the 5'-end. Beginning with the ATG sequence at the nucleotide position and ending with the GCC sequence at positions 119-121, a signal peptide encoded by 90 base pairs and the ACC sequence at positions 122-124 650-652 It was found to contain the nucleotide sequence corresponding to the mature G-CSF polypeptide encoded by 531 base pairs ending with the CCC sequence at position 1. Therefore, the polypeptide of the amino acid sequence I shown in FIG. 3 (B) is composed of 207 amino acids, and has a molecular weight of 207.
  • polypeptide of amino acid sequence E consisted of 177 amino acids and had a molecular weight of 18986.74 daltons. (Example 9)
  • ATG at positions 32 to 34 or 68 to 70 can be considered in the same manner.
  • the E. coli X1776 strain harboring PBR322 with this CDNA (+ VSE) inserted into the EC0R1 cleavage site was deposited at the Research Institute of Microbial Industry and Technology of the Agency of Industrial Science and Technology. (FERM Deposit No. BP-954).
  • FIG. 5 shows the restriction enzyme cleavage sites of the obtained gene.
  • the plasmid obtained by linking this cDNA with pBR327 [Soberon et al., Gene j, p. 287 (1980)] at the EcoRI site is referred to as PBRG4.
  • PBRG4 obtained in this manner is converted with the restriction enzyme EC0RI.
  • the DNA fragment containing about 1500 base pairs of cDNA obtained by the treatment is radiolabeled by the nickel tran- sition method (see Molecular Cloning, supra), and used as a probe again. From i gtio
  • the cDNA library was screened using a plaque hybridization procedure (see Benton and Davis, supra). This At the same time, two sheets of nitrocellulose arrest paper with immobilized sphage DNA were prepared at the same time, and Zhou-sama's plaque-hyperidization solution was probed using the probe (LC) mentioned earlier. And selected positive phages in both props. The clones considered to be full-length were selected, and the nucleotide sequence of the cDNA insert was determined by the didexy method, as shown in FIG. 4 (A).
  • This CDHA has one large open reading frame, and the encoded amino acid sequence is interrupted as shown in FIG. 4 (A).
  • this CDN A was 31-33 nucleotides ATG from the 5'-terminal. Starting with the sequence, ending with the GCC sequence at positions 118-120, the signal peptide encoded by 90 base pairs and starting with the ACC sequence at positions 121-123, ending with the CCC sequence at positions 640-642 Mature G-encoded by 522 base pairs
  • the polypeptide of the amino acid sequence I shown in FIG. 4 (B) is composed of 204 amino acids and has a molecular weight of 204 amino acids.
  • polypeptide of the amino acid sequence E was composed of 174 amino acids and had a molecular weight of 18671.42 daltons. (Example 10)
  • ATG at positions 58 to 60 or 67 to 69 in addition to positions 31 to 33 may be considered in a similar manner.
  • FIG. 5 shows the cleavage site of the ocular antioxidant enzyme obtained.
  • PBRV2 A plasmid obtained by binding this cDNA to PBR327 at an EcoRI site is referred to as PBRV2.
  • E. coli DH1, E. COM H4830 or E. COI .JH105 strains were transformed. (Examples 11, 12, 13 and 18)
  • the obtained transformant was first cultured in an ampicillin-containing luria (ria) medium, and then, if necessary, induced appropriately, followed by culturing. The expression was performed. (Examples 14 and 19)
  • the culture of the transformant is collected by centrifugation to collect the cells, and then treated with lysozyme, followed by freezing and thawing, and then lysing.
  • the cells are lysed to obtain the supernatant, or the supernatant is obtained by centrifugation after treatment with guanidine hydrochloride.
  • Example 16 and 19 Furthermore, the amino acid composition of the obtained G CSF polypeptide was determined using a special amino acid by using a Hitachi 835 amino acid automatic analyzer (manufactured by Hitachi, Ltd.). It was analyzed by acid analysis. N-terminal amino acid analysis was carried out by Edman degradation using a gas-phase sequencer, and using a high-speed liquid chromatograph and a Urasphere-0DS column. (Examples 17 and 20)
  • PHGA410 plasmid (Example 21) (FIG. 12)
  • Hindlinker followed by HindEI treatment and T4 DNA ligase treatment, and then the E.coli DHI strain was transformed using the rubidium chloride method (see above, Molecular cloning).
  • the resulting plasmid is named pHGA410 (H) ( Figure 13).
  • pHG a replacement vector for CH0 cells (+ VSE)
  • PHGV2 plasmid-(Example 27) This is partially digested with EcoRI, and the end is blunted. To the end (blunt end). HindDI linker was added to this DNA, followed by HiHI treatment and T40NA ligase treatment, and then the E. coli DHI strain was transformed using the rubidium chloride method (see above, Molecular cloning). Converted.
  • the obtained plasmid is named pHGV2 (H).
  • pHGV2 (H) was treated with SalI, then the ends were plant-ended and then treated again with HindDI to recover the HindH-SalI fragment.
  • pdBPV-1 plasmid is treated with Hindffl and PvuI to separate the larger DNA fragment, and the Hindi-SalI fragment is ligated to the larger DNA fragment.
  • the E. coli DHI strain is transformed to obtain pTN-V2, a plasmid having CSF-cDNA derived from PHGV2 (Fig. 16) (Example 28).
  • PHGV2-dhfr a recombinant vector (one VSE) for CH0 cells, was constructed using PHGV2 plasmid or pHGV2 (H) plasmid and pAdD26SVpA plasmid in the same manner as VSE (Figs. 17a and b).
  • Example 30 ).
  • pTN G4 plasmid or ⁇ -V2 plasmid has been treated with BamHI.
  • C-127 cells grown in culture are transformed by the calcium phosphate method.
  • the transformed cells are cultured and clones having high CSF-producing ability are selected.
  • the glycoprotein of the expressed G-CSF was recovered and purified from the culture of the transformed cells, and it was confirmed that the glycoprotein exhibited human G-CSF activity.
  • the target glycoprotein was also confirmed by amino acid analysis and sugar content analysis of the sample.
  • the CSF sample used for the amino acid analysis was used.
  • the quantification of sialic acid was carried out, respectively.
  • the quantification method is described in Chapter 13 of Biochemistry Experiment Course, Vol. 4, “Carbohydrate Chemistry (2nd volume)” (Tokyo Chemical Shujin).
  • the weight% was converted from each quantitative value.
  • the sugar content of the obtained G-CSF was distributed in the range of 1 to 20 (% by weight) depending on the host cell, expression vector, culture conditions, etc. Was .
  • the present invention also relates to an agent for preventing infection comprising human G CSF as an active ingredient. Also provide.
  • Human G-CSF which is an active ingredient of the present invention, can be obtained by the above-described gene recombination technique, and was disclosed, for example, in the aforementioned patent (Japanese Patent No. 59-153273).
  • a hybrid obtained from human CSF-producing cells (CHU-1 or CHU-2) according to the method, or a cell fusion of G-CSF-producing cells with self-proliferating malignant tumor cells Ma can also be obtained by culturing the yeast in the presence or absence of mitogen.
  • the human G-CSF obtained by these methods is all included in the present invention.
  • the obtained human G-CSF-containing solution may be further purified and concentrated by known means as necessary, and then frozen and stored.
  • the cells can be stored by removing water by means such as freeze-drying or vacuum drying, or, if desired, dissolving human G-CSF in an appropriate buffer, followed by milling. It can also be used as an injection by aseptically removing it through a pore filter or the like.
  • the infectious agent of the present invention comprises a pharmaceutical carrier and an excipient necessary for taking a form as a pharmaceutical preparation suitable for human or veterinary medicine, and further comprises a stabilizing agent and an adsorbent.
  • the dose and frequency of administration of the human G CSF contained in the infectious protective agent of the present invention can be determined in consideration of the condition of the target patient, but usually 0.1 to 500 ⁇ per adult.
  • ⁇ F A formulation with a human G-CSF, preferably between 5 and 100 ⁇ f, can be administered 1 to 7 times per week.
  • the present invention is not limited by the content of human G CSF.
  • Infection protective agent containing human G-CSF of the present invention as an active ingredient is used
  • target infectious bacteria include various bacteria, and are not particularly limited. Among them, Staphylococcus spp., Streptococcus spp., Etc. Gram-positive cocci, enterobacteria such as Escherichia, Serratia, and Klebsiella, and Gram-negative facultative anaerobic bacteria, including Hemofilus, seromonads, and alkaline Gram-negative aerobic bacteria such as Genes, Gram-negative anaerobic bacteria such as Pacteroides, fungi such as Candida and Aspergillus, and intracellular parasites such as Listeria.
  • a protective effect can be obtained against single infection or multiple infections caused by several bacteria.
  • CSA CSF activity
  • the 5A culture solution and the human bone marrow non-adherent cell suspension used in (a) were prepared as follows.
  • McCoy's 5A culture solution (GIBCO) 12 ⁇ f, MH amino acid vitamin medium (Nissui Pharmaceutical) 2.553, sodium bicarbonate 2,183, penicillin and calcium 50000 After dissolving the unit twice in 500 ⁇ distilled water, it was sterilized with a 0.22 ⁇ m Millipore filter.
  • Human G- 'CSF was isolated and purified from the culture supernatant of human G-CSF-producing cell Cffli-1 (CNCH accession number "I-315") by the method described in Example 2 below.
  • the human G-CSF obtained in this manner had the following physicochemical properties.
  • I Isoelectric point (PI) Isoelectric point
  • Amino acid composition It has the amino acid composition shown in Table 1 IT below.
  • Tumors from patients with oral floor cancer with marked neutrophil enrichment were transplanted into nu nu mice. Approximately 10 days after transplantation, the tumor showed marked tumor growth and increased neutrophils. The tumors were aseptically excised after transplantation 12 days, minced into 1 ⁇ 2 m 3 corners, and cultured as follows this.
  • the CSF activity of the obtained 11 clones was examined. As a result, a clone (CHU-2) having approximately 10 times the activity of the other clones was obtained.
  • Cells were collected from two 150 cm2 culture flasks in which the cells established as described above had grown completely densely, and transferred to 500 F-10 culture medium containing 10% fetal calf serum. After floating, it was transferred to a glass port of 1580cffl 2 (made by Belco).
  • Rotary culture was performed at a speed of 0.5 rpI.
  • the culture medium was replaced with serum-free RPHI 1640, and after culturing for 4 days, the culture supernatant was collected and the fetal serum was recovered. Add 10% F-10 and continue the culture. After culturing for 3 days, the solution was again changed to serum-free RPHI 1640, and the culture supernatant was recovered after 4 days.
  • a serum-free culture supernatant of 500 / ⁇ each week was obtained from one bottle per week, and for a longer period of time by this method. It was possible to maintain cells and recover the culture supernatant.
  • the mixture was collected and concentrated to about 5 ⁇ by an ultra-low temperature regulator using ⁇ -10 (manufactured by Amicon).
  • the sample was digested with Edman using a gas-phase sequencer (manufactured by Applied Dubai Saiki Systems), and the resulting PTH amino acid was subjected to a high-speed liquid chromatograph (Beckman Inn).
  • the analysis was performed by a conventional method using an IHtrasphere-0D8 column (manufactured by Storment) and an IHtrasphere-0D8 column (manufactured by Peckman * Instrument). Column (5 m, diameter 4 '.
  • the sample was dissolved in 70% formic acid, and 200 equivalents of sublimated purified cyanide were added and reacted at 37 ° C overnight.
  • the reaction product was freeze-dried, and fractionated by HPLC using a TSK G3000SW column (manufactured by Toyo Soda Co., Ltd.) to obtain four peaks.
  • the peaks were named CN-1, CN-2, CN-3, and -4 in descending order of molecular weight, and the amino acid sequence of -1 CN-2 was determined by the automatic gas phase sequencer (Applied Dubai). (Manufactured by Toshi System) under the same conditions as (().
  • -1 was an peptide from the N-terminus of the G-CSF protein.
  • CN-2 had the following amino acid sequence.
  • the inside of the system was replaced with nitrogen gas, and the mixture was shaken occasionally at room temperature for 45 minutes to condense the nucleotide resin and the dimer.
  • the column was washed with pyridine, and then the unreacted 0-group was acetylated with an excess of acetic anhydride-4-4-dimethylaminopyridin, followed by acetylation.
  • the column was washed again with pyridine.
  • DHTR equal mixture of GpAp (NHR 3) (DHTr) CpAp (NHR 3) (DHTr) equal mixture of ApAp and (NHR 3) and (DHTr) ApGp (NH 3) , (DHTr) GpCp (NH 3 ), (DHTr) TpGp (NH 3 ),
  • the synthesis was carried out in the same manner as in the case of the probe (IWQ), and (DHTr) CpAp (NHR 3 ) was added to the nucleotide resin AP-d (T) (Yamasa Corporation).
  • the cells were resuspended in 5 fflg of EIA-free saline, and centrifuged at 1500 ⁇ PM for 10 minutes to obtain a cell pellet (wet weight: about 0.83).
  • the cells thus obtained were Store frozen at -80 ⁇ until RNA extraction.
  • sucrose density gradient of 5% to 25% is created in a tube for the SW40Ti rotor (made by Bechman).
  • the sucrose solution was prepared by adding 5% and 25% of the RNase free solution to a solution of 0.1M NaCi, 10mM Tris-HCl, (t) H7.5), 1mH EDTA, 0.5% SD8, respectively. Contains sucrose (Schwarz-Hann).
  • the G-CSF activity of each fraction was measured at weekly intervals using an oocyte line of the African frog (Xenopus laevis). That is, the mRNA of each fraction was prepared in an aqueous solution of 1 / ⁇ ⁇ concentration, and injected into a single oocyte taken from a toad frog (about 1 year after birth) at a rate of 50 ng mRNA, followed by 96 wells.
  • the cDNA was obtained by taking into account the method of Gubler and Hoffman [Gene, vol. 263 (1983)]. 1) Synthesis of single-stranded cDNA
  • MgCi 50mM (H 4) 2 S0 4, 500mH KC), including 4 of 4 mM dNTP (dATP, dCTP, dGTP, each 4 mM of dTTP), 60 3t of 1 mM 3 - NAD, and 210 I distilled Water, 20
  • E. coli DNA polymerase (Takara Shuzo), 15 i E. coli DNA ligase (Takara Shuzo), 15 E. coli Nase H
  • the TE solution 10i in which the poly (A + )-NA12Sf is dissolved is placed in a reaction tube containing 10 actinomycin D (manufactured by Sigma), and then the reagents are added in the following order.
  • 20 ⁇ reverse transcription buffer 250mH Tris-HCl (PH8.3), 40mM HgCJl, 250mM KCil
  • 20u ⁇ of 5mM dNTP dATP, dGTP, dCTP, dTTP
  • 5mM dNTP dATP, dGTP, dCTP, dTTP
  • the CDHA obtained as described above was dissolved in a 29. ⁇ solution, and the reagents were added according to the following procedure to prepare a reaction solution; 25 ⁇ Polymerase 'buffer (400mH Hepes (pH7.6)) 16mH HgCi 2 ; 63-mH 3-mercaptoethanol: 270mM KCi): 10]! 5 mM dNTPs in:! 1.0 ⁇ of 15mM 3 - NAD; 1. ⁇ of a - [ "2 p] dATP (10 Z ⁇ / u S); 0.2 J E. co I i DNA ligase (60un Z Jl Takara Shuzo 5.0 ⁇ 11 E. coli DNA polymerase I (New England Biolabs, 10un /); 0.1% RNaseH (60un / Takara Shuzo): 28.7 zil of distilled water.
  • 25 ⁇ Polymerase 'buffer 400mH Hepes (pH7.6)
  • reaction solution was incubated at 14 for 1 hour, then returned to room temperature, and further incubated for 1 hour. Then of
  • the reaction was stopped by adding 0.5H EDTA and 1% of 20% SDS, and subjected to phenol-chloroform treatment and ethanol precipitation. Dissolve the obtained DNA in 0.5 mM ⁇ 0 ⁇ 20 ⁇ and add 3 W KI enow buffer (500mH tris-hydrochloric acid (pH 8.0), 50mM MgCJl 2 ) 3 ⁇ of 5mH dNTP and 4d of water were added to prepare a reaction solution, and then 1 DNA polymerase was prepared. (Kleenow fragment) (Takara Shuzo) was added, and the mixture was incubated at 30 for 15 minutes.
  • 3 W KI enow buffer 500mH tris-hydrochloric acid (pH 8.0), 50mM MgCJl 2 ) 3 ⁇ of 5mH dNTP and 4d of water were added to prepare a reaction solution, and then 1 DNA polymerase was prepared. (Kleenow fragment) (Takara Shuzo) was added,
  • the reaction solution was diluted with a 70 ⁇ z TE solution, and the reaction was terminated by adding 5 0.5H EDTA,> S 20% SDS.
  • the reaction solution was subjected to phenol-chloroform treatment, and ethanol precipitation was performed to obtain about 8 ⁇ ⁇ f double-stranded cDNA.
  • a ligase buffer 250 mM tris-hydrochloric acid (pH 7, 5), 100 mM HgCi 2 ) ⁇ St-pre-phosphorylated EcoRI solution was added to about 1.2 ⁇ of the methylated double-stranded DNA. Lanka made one 0.5 ⁇ Domer.
  • EcoRI (10unit / i) 3.5 / ⁇ was added and reacted at 37 ° C for 2 hours. Then, 0.5M ⁇ 2.5 ⁇ ; 20% 0.5S ⁇ was added, followed by phenol-chloroform treatment, and DNA was recovered by ethanol precipitation. After that, unreacted EcoR linker is removed by gel-gel method or agarose gel electrophoresis of Urogel AcA34 (manufactured by LKB), and a linker-added double-stranded CDNA of about 0.5 to 0 ⁇ 7 ⁇ ⁇ was recovered.
  • a gt10 vector (Vector Cloning Systems, Inc.) prepared by processing the above-mentioned linker-added double-stranded CDNA at 2.4 Z ⁇ f in advance with EcoRI, a ligase buffer (250 mM Hydrochloric acid, 100 mM WS 2 )
  • Watman 541 filter paper was placed on the agar medium on which the colonies had grown, and left at 37 ° C for 2 hours. Below, Taub and Thompson's method
  • the colonies were transferred to 541 paper, then transferred to an agar medium containing chloramphenicol (250 ⁇ 3Z), and left at 37 overnight.
  • Southern blotting was performed using the probe (A). The hybridization was carried out for 1 hour at 491; using the above-mentioned hybridization buffer for 1 hour, cooled slowly to 39, and then carried out at 39 for 1 hour. After the completion of the reaction, the cellulosic filter was washed twice in 30 minutes at room temperature with 6 ⁇ SSC containing 0.1% SDS, and then washed for 3 minutes at 39. A radiograph was performed.
  • nucleotide sequence was determined by the didexy method, as shown in Fig. 2, it was found to be a DNA consisting of 308 nucleotides including the probe (IWQ) and probe (A) portions. It was found and the plasmid derived from PBR322 containing this insert was named PHCS-.
  • Example 9 DNA probe derived from pHCS-1; (Screening of phage-based library)
  • the plaque-hyperidization was performed according to the method of Benton and Davids [Science 96, 180, (1977)].
  • the PHGS-1 obtained in Example 8 was treated with Sau3A and EcoRI to obtain a fragment of about 600 base pairs, and this DNA fragment was radiolabeled with a nickel translation by a conventional method.
  • the compost paper was dried and ripened at 80 ° C for 2 hours to fix the DNA.
  • a hybridization buffer containing 5X SSC, 5X Denhardt solution, 50mM phosphate buffer, 50% formamide, 0.25 fflg / denatured DNA ( ⁇ testis DNA), and 1% SDS Performed a pre-hybridization step at 42 in the same step, and a pHCS-1 probe radiolabeled with a nickel transfusion 4 x 10 5 cpni Hydration solution containing 5X SSC, 5X Denhardt solution, 20mM phosphate buffer (pH 6.0), 50% form Amide, 0.1% SDS, 10% dextran sulfate,
  • Nitrocellulosic paper was washed at room temperature with 2 ⁇ SSC containing 0.1% SDS for 20 minutes, then at 44, 30 minutes with 0.1 ⁇ SSC containing 0.1% SDS for 30 minutes and then at room temperature After washing with 0.1X SSC for 10 minutes, detection by heat radiography was performed.
  • Plaque-hybridization was performed according to the method of Benton and Davis used in Example 9 (see the above-mentioned literature). Place the nitrocellulose filter paper (manufactured by S & S) on the agar medium on which the phage plaque was generated, transfer the phage, denature the DNA with 0.5 M NaOH, and perform the following steps. Filter paper was processed. NaOH 1.5M NaCJl for 20 seconds, followed by 0.5H tris-hydrochloric acid (f) H7.5), 1.5 ⁇ NaCi for 20 seconds twice, and finally 120mM NaCil, 15mM Oxygen over Da, 13mH KH 2 P0 4, mH EDTA, and 20 Byosho sense at (pH 7.2). Next, the paper was dried and ripened at 80 ° C for 2 hours to fix the DNA. In this manner, two identical papers were produced,
  • PBRG4-derived DNA probe and probe were used for screening.
  • PBRG4 was treated with EcoRI to obtain a DNA fragment of about 1500 base pairs, and the DNA fragment was radiolabeled by a nickel translation method according to a conventional method.
  • the above filter paper was mixed with 5X SSC, 5X Denhardt solution, 50mH phosphate buffer, 50% formamide, 0.25 fflSP / fflg denatured DNA ( ⁇ testis DKA), and a hybridase buffer containing 0.1% SDS.
  • Pre-hybridization was performed overnight at 42 ° C in a prehybridization kit containing the above-mentioned radiolabeled DNA containing approximately 1500 base pairs of DNA probe (approximately 1 x-106 cpm Zid).
  • Ion buffer [5X SSC, 5X Denhardt solution, 20mM phosphate buffer (pH 6.0), 50% formamide, 0.1% SDS, 10% dextran sulfate, denatured DNA of O.lfflSfZ (Mixture of salmon testis DNA)] and subjected to hybridization for 20 hours.
  • the 2-cellulose filter paper is washed at room temperature with 2 ⁇ SSC containing 0.1% SDS for 20 minutes, then at 44 ° C. with 0.1 ⁇ SSC containing 0.1% SDS for 30 minutes, and then at room temperature. After washing with 0.1% SSC for 10 minutes at room temperature, detection was performed by radiography.
  • the paper was pre-treated with 3X SSC containing 0.1% SDS at 65 for 2 hours, then 6X NET, x Denhardt solution, 100 ⁇ denatured DNA ( ⁇ testis) DNA) Prehybridization was performed in the solution at 65 ° C for 2 hours.
  • Radiolabeled probe (LC) (2 X 10 6 cpm) pre-hybridization Dizeshi Yo down buffer containing [6 X NET, 1 X
  • Denhardt solution 100 gf ⁇ denatured DNA (salmon testes DNA)]
  • overnight hybridization at 63, containing 0.1% SDS at room temperature with two microcell mouths at room temperature 6
  • this washing was performed three times, and then at 63 ° C for 2 minutes with 6 XSSC containing 0.1% SDS.
  • tac Promoter-containing vector pKK223-3 (Pharmacia Co.) 5 3 3 ⁇ reaction solution (40mH Tris-HCil, 7mH)
  • the mixture was treated with 8 units of EcoRI (manufactured by Takara Shuzo Co., Ltd.) in MgCJl 2 , 100 mM NaCi, 7 mM 2-mercaptoethanol> for 2 hours at 37.
  • 3 of alkaline phosphatase (Takara Shuzo Co., Ltd.) was added, and the mixture was treated with P at 60 ° C. for 30 minutes, and subjected to phenol treatment three times, ether treatment and ethanol precipitation according to a conventional method. To recover DNA fragments.
  • Synthetic linker one Sai Li Gore j click with the sequence CGAATGACCCCCCTGGGCC and CAGGGGGGTCATTCG Reochi 3 3 50mM Tris -. HCi, -10mH MgCi 2, 10 mH 2 - main Rukapu preparative ethanolate Lumpur, consisting 1 mM ⁇ reaction liquid 40 U T 4 port I j click Reochi Dokinaze 4 units presence in I, tighten react 37 ° G, 60 minutes, and re-phosphorylation.
  • the phosphorylated oligonucleotide was dissolved in 20 3 of TE (10mH Tris-HCJI, pH 8.0, 1 mM ⁇ 0 ⁇ ) containing 100niH NaCi in an amount of 0.23, and treated at 65 ° C for 10 minutes. Thereafter, annealing was performed by gradually cooling to room temperature.
  • TE Tris-HCJI, pH 8.0, 1 mM ⁇ 0 ⁇
  • the PBRG4 60 gp containing the CDNA shown in FIG. 3 (A) obtained in Example 9 was reacted with 6 mM Tris-HCi, 6 mH HgCJl 2 , 6 mM 2 —mercaptanol-reaction solution 200 ⁇ ⁇ Medium, 100 units of restriction enzyme Apa I (New England Biolabs), Dra I (Takara Shuzo) The mixture was treated with 50 units at 37 ° C for 3 hours, and about 23 bp of a 590 bp Apa I-Dra I fragment was recovered by 1.2% agarose gel electrophoresis. - ⁇ Concatenation of the above fragments
  • E.col was determined by the rubidium chloride method (see T. Hani at is et al. (Molecular cloning), p. 252 (1982), supra) using 20 ⁇ of the reaction solution containing the replacement vector obtained in (1) above.
  • the i 105 strain was transformed. Plasmid was isolated from the ampicillin-resistant colony culture and treated with the restriction enzymes BamHI, AccI, and Apa. Was confirmed.
  • P Shipuguchi motor over vector containing te pPL-lambda (manufactured by full Arumashi ⁇ Co.) limit 100 ⁇ enzyme BamHI, with 50 units reaction (10mH Tri s - HCi, pH7.6 , 7 mH MgCi 2, (100mH NaCi, 10mH DTT)
  • the cells were treated in 100 jW i at 37 ° C overnight. From this, about 49 fragments of about 4 Kbi) and about 11 fragments of about 1.2 Kbp were recovered by 1% agarose-gel electrophoresis.
  • alkaline phosphatase manufactured by Takara Shuzo Co., Ltd.
  • 5 U Si and 60 for 60 minutes and dephosphorylated was reacted with 5 U Si and 60 for 60 minutes and dephosphorylated.
  • the remaining fragment of about 1.2 Kbp was dissolved in 20 ⁇ l of buffer (10 (ilM Tris-HCJl, 10mH MgCi 9 , 6 ⁇ KCi, 1 niM DTT) 20 ⁇ .Si) and the enzyme Hbo I (New England Biolabs) Manufactured at 37 ° C overnight with 20 units.
  • buffer 10 (ilM Tris-HCJl, 10mH MgCi 9 , 6 ⁇ KCi, 1 niM DTT) 20 ⁇ .Si) and the enzyme Hbo I (New England Biolabs) Manufactured at 37 ° C overnight with 20 units.
  • Synthetic SZD linker 0.13 annealed with the approximately 4 Kbp fragment 0.13 and the BaraHI-HboII fragment having the PL region and the HboK-BamHI fragment having the tL ⁇ region of 0.053, respectively.
  • the 40 reaction solution ⁇ (66mH Tris HCi, 6.6mH MgCJl 2, 0mH DTT, 1 mH ⁇ ) in, T 4 DNA rigger - (Takara Shuzo) 175 units presence 12 ° C, tighten react overnight was.
  • E. coli N99C I + strain (Pharmacia) was transformed by the CaCi 2 method (see “Molecular cloning” above).
  • the transformant was cultured, the plasmid was separated from the culture of the ampicillin-resistant colony, and treated with the restriction enzymes EcoRI, BamHI and SmaI to confirm that the desired plasmid was obtained. Was done.
  • Example 13 Construction and transformation of a recombinant vector for Escherichia coli (+ VSE) [Example using vector containing trp promoter]
  • HpaI-TaqI fragment containing the triple-fan promoter was introduced into the ClaI site of PBR322, and pOY1 plasmid 103, which was prepared by 10 mM Tris-HCi, 6 ⁇ HgCi, 50mH NaCi, was prepared.
  • the mixture was treated with 7 units of restriction enzyme Cla I and 8 units of Pvu I in 30 ml of the reaction solution at 37 ° C. for 3 hours.
  • Synthetic linker CGCGAATCGACCCCCCCTGGGCC and CAGGGGGGGTCCATTCG were phosphorylated and annealed in the same manner as in (11) of Example 11.
  • reaction solution 20 ⁇ in (3) above was transformed into the E. coli DHI strain by the rubidium chloride method of "Holecular cloning" described above.
  • Plasmids were obtained from ampicillin-resistant colonies in the same manner as in Example 11, and the desired transformants were obtained with the restriction enzymes Apal, Dral, NruI, and Pstl. It was confirmed.
  • the culture was centrifuged to collect the cells, and the cells were suspended in a mixed solution 5a of 20mH Tris-HCi (pH 7.5) and 30mH NaCi.
  • 0.2 M phenylmethylsulfonyl fluoride, 0.2H E0TA, and lysozyme were added to each at 1 mH, 10H, and 0.2 fflg /, and the mixture was allowed to stand at 0 ° C for 30 minutes.
  • freeze-thaw was repeated three times or sonication was performed to lyse the cells.
  • the supernatant is obtained by centrifugation of the obtained lysate, or finally converted into 6 M guanidine hydrochloride using 8 M guanidine hydrochloride, and then centrifuged at 30,000 rpm for 5 hours. And the supernatant was obtained.
  • n-propanol (Tokyo Kasei Co., Ltd. (For amino acid sequencing) and triflic acid, and adjust to final concentrations of 30% and 0.1%, respectively, and leave it on ice for about 15 minutes, then at 15,000 rpm for 10 minutes. The precipitate was removed by heart. Then, after adsorbing to a Bondapak C18 column (Waters, semi-preparative, 8 nm 30CJ ») equilibrated with the above aqueous solution containing n-propanol and trifluroacetic acid, 30-60% Second elution was performed with a 0.1% aqueous solution of trichloroacetic acid containing a linear concentration gradient of n-propanol.
  • Bondapak C18 column Waters, semi-preparative, 8 nm 30CJ »
  • the obtained protein was treated with 2-mercaptoethanol, subjected to SDS-polyacrylamide gel (15.0%) electrophoresis (15 mV, 6 hours), and stained with a coomassie gel. At this point, the target G-CSF polypeptide was confirmed as a single band.
  • Example 16 Assay of G-CSF activity of expressed substance (+ VSE) The CSF sample obtained in Example 15 was assayed according to the aforementioned ⁇ Reference example> CSF activity 'measurement method (a). The results are shown in Table 1.
  • the CSF sample purified in step 15 is hydrolyzed by a conventional method, and the crude amino acid in the protein portion is converted to a special amino acid using a Hitachi 835 amino acid automatic analyzer (manufactured by Hitachi, Ltd.). The analysis was performed by the acid analysis method. The results are shown in Table-2.
  • the hydrolysis conditions are as follows.
  • Samples were dissolved in a solution (1.5 ⁇ ) containing 40% n-propanol and 0.1% triflic acid, and each was taken and dried with dry nitrogen gas. Alternatively, the reagent of (1) was added, the tube was sealed in a vacuum, and subjected to hydrolysis.
  • the measured values are the average of four times of the 24-hour value of 1 and the 24, 48, 72-hour values of 2.
  • T hr, Ser, 1/2 Cys, Met, Val, II e, and T rp were calculated by the following methods (Biochemical Chemistry Laboratory Course, Protein Chemistry H (Tokyo Kagaku Dojin Press)). Reference].
  • T hr, Ser, V 2 Cys, and Met take the time-dependent changes of 24, 48, and 72 hours in 2, and extrapolate to zero hours.
  • V a and I le are 72 hour values of 2.
  • T rp is the average of 24, 48, and 72 hour values of 2.
  • Table 1 2 (Amino acid analysis table)
  • the sample was digested with Edman using a gas-phase sequencer (manufactured by Applied Biosystems), and the resulting PTH
  • the acid was routinely analyzed using a high-speed liquid chromatograph (Beckman ⁇ Instrument) and an intrasphere-0DS column (Beckman * Instrument). More analysis.
  • a column (5 m, diameter 4.6 mm, length 250 mm) was equilibrated with a starting buffer (aqueous solution containing 15 mH sodium acetate buffer PH4.5, 40% acetate 2 tol). Thereafter, a sample (dissolved in 20 starting buffer solutions) was injected, and separation was performed by isocratic elution with the starting buffer solution. The flow rate was kept at min, and the column temperature was kept at 40 ° C.
  • the detection of PTH amino acids utilized the UV absorption of 269 and 320.
  • 2 nmoi of standard PTH amino acid manufactured by Sigma
  • was separated in a single system the retention time was determined, and identification was performed based on the retention time of the test sample.
  • Example 12 was repeated using CDNA (--VSE), and it was confirmed that the obtained transformant was the target transformant (FIG. 10). 3) Example using trp port motor-containing vector
  • Example 13 was repeated using CONA (-VSE), and it was confirmed that the desired transformant was obtained (FIG. 11).
  • Example 19 Assay of G-CSF activity of expressed substance (—VSE) Each of the transformants obtained in Example 18 was cultured according to the method described in Example 14, and then the Escherichia coli cultured was described in Example 15 According to the method described above, G-CSF polypeptide was recovered and purified to obtain human G-CSF polypeptide as a single band.
  • the sample was subjected to N-terminal amino acid analysis according to the method described in Example 17-2). As a result, PTH-methine and ⁇ ⁇ ⁇ -threenine were detected.
  • the EcoRI fragment of the cDNA shown in Figure 3 (A) obtained in Experiment 9 was treated with the restriction enzyme Dral at 37 for 2 hours, and then the Klenow fragment of DNA polymerase I (Takara Shuzo) ) To make the ends smooth.
  • the Bglll linker (8mer: manufactured by Takara Shuzo Co., Ltd.) was phosphorylated using ATP, and then ligated to the DNA fragment mixture of about ⁇ obtained above. Then, it was treated with restriction enzyme B glH and subjected to agarose gel electrophoresis, and only the largest DNA fragment was recovered.
  • This DNA fragment corresponded to about 710 base pairs including the portion coding for the human G-CSF polypeptide as shown in FIG.
  • Vector-pdKCR Factor-pdKCR (Fukunaga; Proc. Natl. Acad. Sc, USA; 81, 5086 (1984)) was treated with the restriction enzyme BamHI, and then treated with Alfa Lifos Phatase (Takara Shuzo Co., Ltd.). the vectored one DNA obtained in manufacturing) was de-phosphate was obtained T 4 DNA ligase (Takara Shuzo Co., Ltd.) was added to PHGA 410 is combined with CDNA fragment ( Figure 12).
  • this plasmid is the promoter of the SV40 early gene, the replication initiation region of SV40, a portion of the egret 3-globin gene, the replication initiation region of PBR322, and
  • PHGA410 plasmid 20xz ⁇ f obtained in Example 21 was converted to 50mH Tris-HC (pH 7.5), 7 raM HgCi! 9 , 100mH NaCJl, 7 DIM 2 — mercaptoethanol, 0.01% ⁇ Dissolved in the reaction solution of serum alpmin (BSA), added the restriction enzyme EcoRI (Takara Shuzo Co., Ltd., 10 to 15 units), reacted for about 30 minutes and 37, and performed partial digestion with EcoRI. . Then, the DNA fragment was treated twice with ethanol-chloroform (11) treatment twice, followed by ether treatment and ethanol precipitation.
  • E. coli DNA polymerase-K-enow fragment (manufactured by Takara Shuzo Co., Ltd.) (5 ⁇ ⁇ ) was added, and the mixture was incubated at ° C for 2 hours to obtain a blunt end. .
  • fragment 6iSf of about 5.8 kb was recovered by 0.8% agarose gel electrophoresis.
  • the recovered DNA fragment (5 ⁇ f) was dissolved again in a reaction mixture 50 containing 50 mM Tpis-HCJl (pH 7.6), 10 mM MgCil 2 , 10 mM DTT, and 1 mM ATP, and H ⁇ nd Linker (Takara Shuzo) 2 adding m and T 4 DNA rigger over peptidase (Takara Shuzo) 100 units, it was reacted hands overnight 4.
  • PHGA410 obtained in (H) ⁇ (20 / ⁇ f ) a 10mH Tris-- HCJi ( ⁇ 7 ⁇ 5) , 7 mH HgCi 9, 175mH NaCi!, 0.2mH EDTA 7 mH 2- mercapto Bok ethanolate Solution, dissolved in 50 ⁇ . (1) consisting of 0.01% ⁇ serum alpmin, add 20 units of restriction enzyme Sail (Takara Shuzo), and incubate at 37 ° C for 5 hours.
  • the mixture was collected, then treated with phenol, precipitated with ethanol, and treated with a DNA polymerase tf Klenow fragment (manufactured by Takara Shuzo) at 14 ° C in the same manner as in the previous reaction. After incubating for about 2 hours to obtain a plant end, this was treated with the restriction enzyme Hind DI, and the ethanol-precipitated DNA fragment was treated with the restriction enzyme Hind DI without recovering with agarose. About 2.7 kb of Hind I-Sail fragment was recovered 5% by 1% agarose gel electrophoresis, while bovine papi loma virus (BPV) was recovered. Plasmid-1 (Sarver, n.,
  • the E. coli DHI strain was transformed by the rubidium chloride method described in "Molecular cloning", and the E. coli colony having a plasmid having cDHA of G-CSF derived from PHGA410 was selected.
  • the plasmid is named pTN-G4 ( Figure 13).
  • Example 23 Transformation of C127 cells and its expression (+ VSE)
  • the PTN-G4 obtained in Example 22 is treated with a restriction enzyme BamHI before transforming into mouse C127 cells. That is, pTN-G4 plasmid 20 gp was dissolved in 10 mM Tris-HCi (pH 8.0), 7 mH HgCi 2 , IOO 1H NaCil, 2 mH 2 -mercaptoethanol, 0.01% BSA100 St and dissolved in BamHI. Treated in 20 units (manufactured by Takara Shuzo), phenol treatment, ether treatment, and ethanol precipitation.
  • Mouse C127I cells contain 10% fetal calf serum (GIBC0)
  • C127I cells grown on a 5 era plate were plated with the above-prepared DNA at a ratio of 10 to the phosphate-calcium method (Haynes, JS Weissmann, C (1983) Nucleic Transformation was carried out according to Acid Res 11, 687-706), and the cells were incubated for 12 hours and 37 hours after the treatment with glycerol.
  • the cells were transferred to three new five-diameter plates, and the medium was exchanged twice a week.
  • the portions where Foci (agglomerates) were formed were each transferred to a new plate and subcultured in the above-mentioned medium, and clones having high G-CSF productivity were selected.
  • G-CSF production at ⁇ 1 «Sf / i level was observed.
  • ⁇ 3 ⁇ 3 cells can be used as host cells.
  • MgCi 9 is dissolved in a reaction solution 100 containing 10 mM DTT and 1 mH dATP, dCTP, dGTP, and TTP, and added with E. coli DNA polymerase—Klenow fragment (Takara Shuzo Co., Ltd.) and added to 14C, After reacting for 2 hours, perform ethanol treatment, ether washing, and ethanol precipitation.
  • An EcoRI linker is added to this DNA. That is, the above DNA was dissolved in a reaction mixture consisting of 50 ⁇ l of 50 mM Tris-HCi (pH 7.4), 10 mM DTT, 0.5 niM spermidine, 2 mM ATP, 2.5 mM hexamin chloride copartol, and 20 / flig BSA. , EcoR I linker scratch (Takara Shuzo) was added, in 4 added 200 units of T 4 DNA ligase (Takara Shuzo Co., Ltd.), was reacted for 12-16 hours.
  • the resultant was partially digested with EcoRI, and was subjected to about 2.7% by 1% agarose gel electrophoresis. 3 jw of the kb fragment was recovered.
  • pAdD26SVpA plasmid (Kaufman, RGS Sharp, PA (1982) Hoi. Cell BiO I, ___________________________ 1304-1319) is treated with EcoRI, and then treated with EcoRI. BAP) treatment and dephosphorylation. That, pAdD26SVpA 20 gf and EcoRI 20 units of the reaction solution 50mM Tris- HCi (pH7.5), 7 niH HgCi 2, 100 mM NaCJl, 7 mM 2 - main Rukapu Bok ethanolate Lumpur, in addition to the 0.01% BSA 100 The mixture was allowed to react at 37 ° C for 10 hours.
  • PHGG4 plasmid was treated with Sail and partially digested with EcoRI without addition of EcoRI linker to recover a fragment of about 2.7 kb.
  • PHGG4-dhfr can also be prepared.
  • Example 22 The PHGA410 (H) obtained in Example 22 was described in Example 22-2). After treatment with restriction enzymes HindHI and Sall as described,
  • the PHGG4-dhfr can also be obtained by ligating the Hindni-SalI fragment to the plant-end EcoRI fragment of pAdD26SVpA (Fig. 14b).
  • CH0 cells (strain dhfr, obtained from University of Columbia, Dr. L. Chasin) were used in a 9 C / B plate (Nunc) containing 10% calf serum. -HEH, adenosine, deoxyxy-adenosine, and thymidine) and proliferate by culture using the calcium phosphate method (Wigler et al., Cel I, Vol. 14, p. 725 (1978)). Converted.
  • PHGG4-dhfr plasmid prepared in 1), add an appropriate amount of carrier-DNA (calf thymus DNA), dissolve in 375 ⁇ solution, and add 1H CaCi 2 125 ⁇ . After cooling on water for 3-5 minutes, add 500i of 2X HBS (50mH Hepes, 280mM NaCil, 1.5mM phosphate buffer), cool on ice again, mix with CH0 cell culture solution 1 described above, and plate. transfer to, and culture between 9 o'clock in C0 2 Lee Nkyu beta foremost.
  • carrier-DNA calf thymus DNA
  • TBS Tris-Buffered Sine
  • ⁇ -HEM medium the selection medium
  • the cells were divided into 1:10 cells using a selection medium (without adding nucleotides), and the culture was continued every two days while changing the medium using the selection medium, resulting in the formation of foci. ) was selected and moved to a new plate. In the new plate, the cells were grown in the presence of 0.02 H metrexet (MTX) and then grown again in the presence of 0.1XH HTX for cloning.
  • MTX 0.02 H metrexet
  • Cotransformation of pAdD26SVpA can also be performed (see Scaffill et al., Proc. Nat., Acad. Sc, USA__, Volume 4654-4658 (1983)).
  • a recombinant vector can be constructed by a method using a so-called liposome nick gene, and this can be used to transform CH0 cells.
  • pA JD26SVpA is treated with PstI, two fragments are recovered, and these are combined with a PBRG4-derived CSF cDNA fragment to obtain the adenovirus promoter, CSFcDNA, DHF, and SV40.
  • a recombinant vector arranged in the order of the A site was constructed and inserted into CH0 cells.
  • Example 25 Assay of G-CSF activity of expressed substance (+ VSE)
  • the culture supernatants of C127 cells and CH0 cells obtained in Examples 23 and 24 were each adjusted to PH4 with 1N acetic acid, etc. After adding a volume of n-propanol, the resulting precipitate is removed by centrifugation, and an open column (1 ⁇ ) packed with C8 reversed-phase support (Yamamura Chemical Co., Ltd.) is used.
  • G-CSF was approximately 20-fold enriched from C127 and CH0 cell supernatants.
  • Example 25 The crude CSF sample obtained in Example 25 was further purified according to the method (Hi) of Example 2.
  • the purified CSF sample is hydrolyzed by a conventional method, and the amino acid composition of the protein portion is analyzed using a special amino acid composition using a Hitachi 835 amino acid automatic analyzer (manufactured by Hitachi, Ltd.). The analysis was performed by the analysis method. The results are shown in Table-6.
  • water The decomposition conditions are as follows.
  • the sample was dissolved in a solution (1.5 ⁇ ) containing 40% n-propanol and 0.1% trifluoro group acid, each was taken to 0.1 / ⁇ , and dried with dry nitrogen gas. Alternatively, the reagent of (1) was added, the tube was sealed in a vacuum, and subjected to hydrolysis.
  • the measured values are the average of four times of the 24-hour value of 1 and the 24, 48, 72-hour values of 2.
  • T hr, Ser, V 2 Cys, Met, Val, II e and rp rp were calculated by the following methods (Experimental Course in Biological Chemistry ', Protein Chemistry I (Tokyo Chemical Co., Ltd. Publishing)).
  • TlK, S er, V 2 C ys, Met is Ri DOO the time course of 24, 48, 72-hour value of 2, extrapolation to zero hours.
  • V a and I le are 72 hour values of 2.
  • T rp is the average of 24, 48, and 72 hour values of 2.
  • TMS-forming reagents (pyridin: hexamethyl disilazane trimethyl chlorosilane-5: 1: mixed with ⁇ ) were added, and the mixture was reacted at 40 ° C for 20 minutes. saved.
  • the same operation is performed by combining galactose (Gal), N-acetylgalactosamine (GalNAc), sialic acid, etc. with 50 n moi and inositol 25 n moi as standard. Was performed.
  • This sample was subjected to gas chromatographic analysis under the following conditions.
  • the CSF of the present invention showed that galactose and ⁇ -acetyl Lugalactosamine and sialic acid were confirmed.
  • this DNA fragment corresponded to about 710 base pairs including a portion coding for the human G-CSF polypeptide.
  • pdKCR Factor for phosphorylation
  • the enzyme was re-phosphorylated (Takara Shuzo).
  • the vectored one DNA obtained by de-phosphate was obtained PHGV 2 is combined with CDNA fragments by the addition of T 4 0 nA rigger one t (manufactured by Takara Shuzo Co., Ltd.) at a company Ltd.) ( Figure 15).
  • this plasmid contains the promoter of the SV40 early gene, the replication initiation region of SV40, a part of the egret i3—globin gene,
  • Example 29 Transformation of C127 cells and its expression (1 VSE) V2 obtained in Example 28 is treated with a restriction enzyme BamHI before transforming into mouse C127 cells.
  • mouse C1271 cells were transformed with the above-prepared DNA and expressed (see Example 23), and clones having high G-CSF-producing ability were selected. As a result, G-CSF production of ⁇ ⁇ level was observed.
  • ⁇ 3 ⁇ 3 cells can be used as host cells in addition to the above-mentioned C127I cells.
  • PHGV2 plasmid was treated with Sail and partially digested with EcoRI without addition of EcoRI linker to recover a fragment of about 2.7 kb. col i DNA polymerase
  • PHGV2-dhfr can also be prepared.
  • PHGV2 (H) obtained in Example 28 was treated with the restriction enzymes Hindll and SalI described in Example 28-2), and the Hind IE-Sal I fragment was ligated to the pAdD26SVpA plasmid.
  • PHGV2-dhfr can be obtained even when ligated to the endogenous EcoRI fragment (Fig. 17b) 2) Transformation and expression
  • CH0 cells were transformed and expressed by the method described in 2) of Example 24.
  • a replacement vector can be constructed by a method using a so-called nick of the lip of the liposome, and this can be used to transform CH0 cells.
  • pAdD26SVpA is processed by Pstl, By recovering the fragments and combining them with the CSF cDNA fragment derived from PBRV2, the recombinant vector arranged in the direction of the poly A site of adenovirus promoter, CSF CDNA, DHFR and SV40 was obtained. Was constructed and put into CH0 cells.
  • Example 31 Assay of G-CSF activity of expressed substance (—VSE) From the culture supernatants of C127 cells and CH0 cells obtained in Examples 29 and 30, according to the method described in Example 25, Human G-CSF was obtained, and its human G-CSF activity was assayed. The results are shown in Table 7.
  • Example 31 The crude CSF sample obtained in Example 31 was further purified according to the method (Hi) of Example 2. This purified CSF sample was purified by the method described in Example 26-1) to obtain an amino acid. It was subjected to acid composition analysis. Table 8 shows the results. Table 1-8 (Amino acid analysis table)
  • mice After intraperitoneal administration of endoxane (trade name, manufactured by Shisai-nogi) / to ICR mice (male) weighing 8 to 9 weeks (body weight: 35.3, 1.38 g), the mice were divided into three groups, and G-CSF (25000U / mouse or 50000UZ mouse) containing a solvent ( ⁇ % propanol in saline, 0.596 (W / V) mouse serum albumin) and another group containing only the solvent. Each was subcutaneously administered 0.1 times every 24 hours for 4 times. Three hours after the fourth administration, each group was infected subcutaneously with Pseudomonas aeruginosa GNB-139 (3.9 ⁇ 10 5 CFU mouse). Twenty-one hours after the infection, a vehicle containing human G-CSF (250001 / mouse or 50000 UZ mouse) or the vehicle alone was subcutaneously administered again to the corresponding groups.
  • endoxane trade name, manufactured by Shisai-nogi
  • the effect of preventing infection was examined based on the number of surviving mice up to 10 days after infection.
  • U-50-1 (a urine isolate from a leukemia patient, provided by the Department of Bacteriology, Tohoku University) 5.6 ⁇ 10 5 CFU / mouse was intravenously administered for infection. The effect of preventing infection was examined based on the number of surviving mice up to 10 days after infection. (Preparation of bacterial solution)
  • Table 1 13 Infection protective effect against Candida albicans Group CSF strain (UZ mouse Z day) Surviving number Total solvent 0 0 10
  • Table 16 shows the results. Table 1.Effects on 16 Monaco aeruginosa
  • Example 26 As described above, the infection protective effect of the above Test 1 was examined using the same purified human G-CSF sample derived from C127 cells used in the amino acid composition analysis of Example 26. Almost the same result was obtained.
  • Table 17 shows the results. Table 1 17 Effects on pseudomonas aeruginosa
  • Example 34 using the purified human G-CSF sample derived from C127 cells used for the amino acid composition analysis in Example 32, the effect of the above-mentioned test 1 for preventing infection was examined. Similar results were obtained.
  • Example 34
  • Example 2 The freeze-dried preparation of human G-CSF obtained in Example 2 was dissolved in a solubilizer for injection and dispensed so as to have a desired number of dosage units to obtain an injection.
  • a freeze-dried preparation of human G-CSF polypeptide from Escherichia coli obtained in Example 15 was dissolved in a solubilizer for injection and dispensed to obtain a desired number of dosage units to obtain an injection. .

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Abstract

Le facteur stimulant les colonies de granulocytes humanins (G-CSF) se compose d'un facteur obtenu à partir du liquide surnageant d'une culture de cellules humaines produisant du G-CSF et d'un polypeptide ou d'une glycoprotéine présentant une activité de G-CSF humain, obtenus par culture d'un transformant préparé en transformant des cellules hôtes avec un vecteur recombinant incorporant un gène codant un polypeptide présentant une activité de G-CSF humain.
PCT/JP1986/000053 1985-02-08 1986-02-07 Agent protecteur contre les infections contenant un facteur stimulant les colonies de granulocytes humains en tant qu'ingredient efficace WO1986004506A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0217404A2 (fr) * 1985-10-04 1987-04-08 Chugai Seiyaku Kabushiki Kaisha Composition pharmaceutique contenant un facteur stimulant les colonies de granulocyte pour le traitement de leucopénies
US4810643A (en) * 1985-08-23 1989-03-07 Kirin- Amgen Inc. Production of pluripotent granulocyte colony-stimulating factor
WO1989010932A1 (fr) * 1988-05-13 1989-11-16 Amgen Inc. Compositions et procede de traitement ou de prevention d'infections chez des animaux
US5532341A (en) * 1985-03-28 1996-07-02 Sloan-Kettering Institute For Cancer Research Human pluripotent hematopoietic colony stimulating factor
US6004548A (en) * 1985-08-23 1999-12-21 Amgen, Inc. Analogs of pluripotent granulocyte colony-stimulating factor
US6979442B1 (en) 1998-08-17 2005-12-27 Pfizer Inc. Stabilized protein compositions
WO2010011735A2 (fr) 2008-07-23 2010-01-28 Ambrx, Inc. Polypeptides g-csf bovins modifiés et leurs utilisations
US11273202B2 (en) 2010-09-23 2022-03-15 Elanco Us Inc. Formulations for bovine granulocyte colony stimulating factor and variants thereof
US12138296B2 (en) 2022-02-02 2024-11-12 Elanco Us Inc. Formulations for bovine granulocyte colony stimulating factor and variants thereof

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0618781B2 (ja) * 1986-10-18 1994-03-16 中外製薬株式会社 感染症治療剤
FR2607344B1 (fr) * 1986-11-21 1994-04-29 Nexo Distribution Dispositif de traitement d'un signal electrique audiofrequence
CA1329119C (fr) * 1988-03-29 1994-05-03 Milton David Goldenberg Therapie faisant appel a des agents cytotoxiques

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6030655B2 (ja) * 1981-10-03 1985-07-17 科学技術庁 放射線医学総合研究所長 Ｃｓｆ産生腫瘍移植法を用いたｃｓｆの製造法
JPS59210027A (ja) * 1983-05-14 1984-11-28 Kagaku Gijutsucho Hoshasen Igaku Sogo Kenkyusho Ｃｓｆの製造法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
No relevant documents have been disclosed. *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5808008A (en) * 1985-03-28 1998-09-15 Sloan-Kettering Institute Of Cancer Research Method of producing human pluripotent hematopoietic colony stimulating factor
US5532341A (en) * 1985-03-28 1996-07-02 Sloan-Kettering Institute For Cancer Research Human pluripotent hematopoietic colony stimulating factor
US5670146A (en) * 1985-03-28 1997-09-23 Sloan-Kettering Institute Pharmacological preparations comprising human pluripotent hematopoietic colony stimulating factor
US5580755A (en) * 1985-08-23 1996-12-03 Amgen Inc. Human pluripotent granulocyte colony-stimulating factor
US5676941A (en) * 1985-08-23 1997-10-14 Amgen Inc. Methods of enhancing bone marrow transplantation and treating burn wounds comprising administering human pluripotent granulocyte colony-stimulating factor
US4810643A (en) * 1985-08-23 1989-03-07 Kirin- Amgen Inc. Production of pluripotent granulocyte colony-stimulating factor
US5830705A (en) * 1985-08-23 1998-11-03 Amgen Inc. Method for recombinant production of human pluripotent granulocyte colony-stimulating factor
US6379661B1 (en) 1985-08-23 2002-04-30 Amen Inc. Pharmaceutical compositions comprising pluripotent granulocyte colony-stimulating factor
US6004548A (en) * 1985-08-23 1999-12-21 Amgen, Inc. Analogs of pluripotent granulocyte colony-stimulating factor
US6716606B2 (en) 1985-08-23 2004-04-06 Amgen Inc. Production of pluripotent granulocyte colony-stimulating factor
EP0217404A3 (en) * 1985-10-04 1988-07-13 Chugai Seiyaku Kabushiki Kaisha Pharmaceutical composition containing a human granulocyte colony stimulating factor for the treatment of leukopenia
EP0217404A2 (fr) * 1985-10-04 1987-04-08 Chugai Seiyaku Kabushiki Kaisha Composition pharmaceutique contenant un facteur stimulant les colonies de granulocyte pour le traitement de leucopénies
WO1989010932A1 (fr) * 1988-05-13 1989-11-16 Amgen Inc. Compositions et procede de traitement ou de prevention d'infections chez des animaux
US6979442B1 (en) 1998-08-17 2005-12-27 Pfizer Inc. Stabilized protein compositions
WO2010011735A2 (fr) 2008-07-23 2010-01-28 Ambrx, Inc. Polypeptides g-csf bovins modifiés et leurs utilisations
EP3225248A1 (fr) 2008-07-23 2017-10-04 Ambrx, Inc. Polypeptides g-csf bovins modifiés et leurs utilisations
US10138283B2 (en) 2008-07-23 2018-11-27 Ambrx, Inc. Modified bovine G-CSF polypeptides and their uses
US11273202B2 (en) 2010-09-23 2022-03-15 Elanco Us Inc. Formulations for bovine granulocyte colony stimulating factor and variants thereof
US12138296B2 (en) 2022-02-02 2024-11-12 Elanco Us Inc. Formulations for bovine granulocyte colony stimulating factor and variants thereof

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JPH0615477B2 (ja) 1994-03-02

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