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WO1996009383A1 - Procede pour produire une substance de proliferation d'hepatocytes recombinee - Google Patents

Procede pour produire une substance de proliferation d'hepatocytes recombinee Download PDF

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Publication number
WO1996009383A1
WO1996009383A1 PCT/JP1995/001904 JP9501904W WO9609383A1 WO 1996009383 A1 WO1996009383 A1 WO 1996009383A1 JP 9501904 W JP9501904 W JP 9501904W WO 9609383 A1 WO9609383 A1 WO 9609383A1
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Prior art keywords
hepatocyte
substance
gene
cells
expression vector
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PCT/JP1995/001904
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English (en)
Japanese (ja)
Inventor
Hiroshi Hara
Hiromitsu Yoshimura
Yumiko Matsuki
Saeko Shindo
Kazunori Hanada
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Taisho Pharmaceutical Co., Ltd.
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Priority to AU35332/95A priority Critical patent/AU3533295A/en
Publication of WO1996009383A1 publication Critical patent/WO1996009383A1/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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II

Definitions

  • the present invention provides a novel expression vector, a eukaryotic cell line having the vector, and a cell line containing the vector, for producing a hepatocyte proliferating substance that promotes the proliferation of hepatocytes using a genetic recombination technique. And a method for producing a hepatocyte proliferating substance.
  • HGF hepatocyte growth factor
  • Both human HGF and rat HGF have a heterodimer structure in which an ⁇ chain consisting of 463 amino acid residues and a three chain consisting of 234 amino acid residues are crosslinked by one disulfide bond. In both cases, it has been clarified that two sugar chain binding sites exist. Also, cloning of a gene encoding rat-derived HGF and production by gene recombination have been reported (Nature, 342, 440. 1989).
  • the present inventors have found a hepatocyte proliferating substance having an activity of promoting proliferation of hepatocytes, which is completely different from the above-mentioned HGF, and succeeded in purifying this substance from rat liver. .
  • This hepatocyte proliferating substance had the following properties.
  • the estimated molecular weight by non-reducing SDS-PAGE is about 63,000 to about 69,000, and the estimated molecular weight by reducing SDS-PAGE is about 32,000 to about 36,
  • the estimated molecular weight by gel filtration is about 60-7 O Kd:
  • ⁇ ⁇ ⁇ Does not adsorb to heparin.
  • the present inventors have cloned the gene encoding the above substance from rat liver and human liver, and collected the cultured substance and the gene together in Japanese Patent Application Nos. 5-63905 and PCT / JP9. A patent application was filed for 2004.
  • hepatocyte proliferating substances form a homodimer structure in which two identical subunits composed of 290 amino acid residues are connected by disulfide bonds, and further, a disulfide bond is formed in each subunit. And has a structure completely different from that of the HGF, such as not being modified by an N-linked sugar chain.
  • hepatocyte proliferating substances are proteinaceous substances that specifically promote the proliferation of hepatocytes, and are used in many liver diseases such as hepatitis, cirrhosis, liver cancer, and partial liver transplantation. It has the effect of improving liver function by promoting proliferation of parenchymal cells.
  • Hepatocyte proliferating substance is a highly useful substance as described above, but it is present in a very small amount in the human body, and when extracted from the living body, a sufficient amount required for the purpose of medicine etc. can be obtained. It is difficult. Therefore, in order to utilize such proteinaceous factors present in minute amounts in living organisms as research reagents, diagnostic agents, and therapeutic agents, it is necessary to establish mass-production technologies. Due to the recent development of genetic engineering technology, mass production of useful proteins derived from higher animals represented by humans is often performed by genetic recombination.
  • An object of the present invention is to establish a method for constantly and efficiently producing a hepatocyte proliferating substance having a complex higher-order structure.
  • the present invention relates to a hepatocyte proliferating substance expression vector having a gene encoding a hepatocyte proliferating substance having the following physicochemical properties and physiological activities downstream of a promoter sequence that functions in eukaryotic cells.
  • Estimated molecular weight by SDS-PAGE under non-reducing is about 63,000 to about 69, The estimated molecular weight by SDS-PAGE under reduction is about 32,000 to about 36,000, and the estimated molecular weight by gel filtration is about 60 to 7 O Kd:
  • hepatocyte proliferating substance expression vector having a gene encoding a hepatocyte proliferating substance having the amino acid sequence shown in SEQ ID NO: 1 downstream of a promoter sequence that functions in eukaryotic cells.
  • a hepatocyte proliferating substance expression vector linked to a dihydrofolate reductase gene (hereinafter referred to as DHFR gene) that can be expressed in eukaryotic cells, and eukaryotic cells are expressed by the above-described expression vectors.
  • DHFR gene a dihydrofolate reductase gene
  • transformant obtained by transformation a method for producing a hepatocyte proliferating substance using the transformant, and the like.
  • FIG. 1 shows the structure of plasmid pcDLH—dhfr.
  • FIG. 2 shows the construction process of plasmid pcDLH—dhfr.
  • Figure 3 shows HP-041 produced in the medium when various concentrations of MTX-resistant strains obtained from Chinese hamster ovary-derived cells (dhfr-CHO) transformed with pc DLH-dhfr were cultured.
  • dhfr-CHO Chinese hamster ovary-derived cells
  • FIG. 4 shows an electropherogram of HP-041 produced in a medium by African green monkey kidney-derived cells (Verots cells) transformed with pc DLH.
  • FIG. 5 shows an electrophoretogram of HP-041 V produced in the culture medium when Chinese hamster ovary-derived cells (dhfr-CHO) transformed with pcDLHV-dhfr were cultured.
  • FIG. 6 shows the relative activities of hepatocyte proliferating substances of recombinant HP-041 and recombinant HP-041V.
  • FIG. 7 shows the relative activities of tyrosine transaminase (TAT) of recombinant HP-041 and recombinant HP-041V.
  • TAT tyrosine transaminase
  • a typical example of a gene encoding a hepatocyte proliferation substance is a gene encoding a hepatocyte proliferation substance (hereinafter referred to as HP-041) shown in SEQ ID NO: 1.
  • HP-041 a hepatocyte proliferation substance
  • the present invention has also been used for the cloning of a gene encoding a mutant human hepatocyte proliferating substance (hereinafter referred to as HP-04IV) in which amino acids are substituted at two positions in the amino acid sequence of the substance.
  • HP-04IV mutant human hepatocyte proliferating substance
  • the gene encoding HP-0441 is obtained by hybridizing the cDNA library prepared when cloning the HP-0441 gene with the HP-041 gene as the probe DNA. That is what I found.
  • HP-0441V The gene and amino acid sequence of HP-0441V are as shown in SEQ ID NO: 3.
  • the 50th isoleucine and the 83rd amino acid are counted from the N-terminal of the HP-0441 mature protein.
  • Leucinka ⁇ has a sequence substituted with palin and proline, and has a hepatocyte proliferation activity equivalent to that of HP-041 (Example 10 and FIG. 6)
  • the hepatocyte proliferating substance is synthesized as a precursor in which a signal peptide is present at the N-terminus of the mature protein. It is estimated to be cleaved between the 1st and + 1st amino acid residues.
  • the nucleotide sequence of the gene encoding the HP-041 precursor protein including the signal peptide consisting of 22 amino acid residues is shown in SEQ ID NO: 1.
  • the HP-041 mature protein deduced from this nucleotide sequence Is shown in SEQ ID NO: 2.
  • HP—04 1 V before including the signal peptide consisting of 22 amino acid residues The nucleotide sequence of the gene encoding the progenitor protein is shown in SEQ ID NO: 3, and the amino acid sequence of the mature HP-041V protein deduced from this nucleotide sequence is shown in SEQ ID NO: 4.
  • the hepatocyte proliferating substance expression vector is linked to a gene encoding a hepatocyte proliferating substance so that the expression is regulated by the promoter downstream of a promoter sequence having a transcription regulating function in eukaryotic cells. It was built.
  • a promoter having a transcriptional regulatory function in a eukaryotic cell is a promoter having a function of transcribing a gene sequence of a hepatocyte proliferative substance linked downstream of the promoter to a mRNA in a eukaryotic cell.
  • the promoters derived from the virus include SV40 early promoter, LTR derived from human T-cell leukemia virus, cytomegalovirus promoter, and Rous Zalcoma virus promoter, as promoters derived from eukaryotic cells.
  • Metallothionein gene promoter and casein gene promoter can be used. These promoters can be used in combination of two or more.
  • PcDL—SRa296 is known as an expression plasmid in eukaryotic cells (Y. Takebe et.al. Mol. Cel 1. Biol. 8.466.1988).
  • This plasmid has an SR40 early promoter and a promoter SR with high expression efficiency utilizing the LTR (long terminal repeat) of HTL V-1 (human T-cell leukemia virus).
  • This plasmid is opened downstream of the SRa promoter and upstream of the polyA addition signal sequence when the restriction enzymes Kpnl and Pstl are acted on, and if the target gene to be expressed is incorporated into this open site, It is an expression vector for eukaryotic cells constructed so that the expression of the target gene is regulated by the SRa promoter.
  • the hepatocyte proliferating substance expression vector pcDLH is a gene encoding a hepatocyte proliferating substance precursor containing the signal peptide shown in SEQ ID NO: 2, which is opened with Kpnl and Pstl described above. It was prepared by incorporating it into the plasmid pc DL-SR296. It encodes a precursor protein in which the signal peptide sequence of other eukaryotic cell-derived extracellular secretory proteins, such as rabies virus G protein and human growth hormone, is linked to the N-terminal of the mature protein of hepatocyte proliferative substance.
  • hepatic parenchymal cell proliferating substances extracellularly even by using a gene that binds. Furthermore, a gene encoding a sequence in which methionine is added to the N-terminus of a mature hepatocyte proliferating substance was prepared, and this gene was inserted into the plasmid pcDL_SRa296 in the same manner as described above. It is possible to produce parenchymal cell growth material. In this case, a hepatocyte proliferating substance in the form of methionine added to the N-terminus is produced intracellularly.
  • the present invention provides a method for culturing a eukaryotic cell line transformed with a gene encoding a hepatocyte proliferating substance and a DHFR gene in a medium containing methotrexate (hereinafter referred to as MTX),
  • the present invention also provides a method for obtaining a hepatic parenchymal cell proliferating substance-highly eukaryotic cell line by gradually increasing the MTX concentration in a medium, and a method for producing a hepatic parenchymal cell proliferating substance using the high-producing eukaryotic cell line.
  • Plasmid Mg4 (CS Gasser et al., Proc. Natl. Acad. Sci. USA., 79, p6522, 1982) in which the mouse-derived DHFR gene was incorporated into a bacterial-derived plasmid PBR327 opened with EcoRI-Pstl. ) Has been reported. Furthermore, the DHFR gene of 3.8 Kb of the EcoRI-Pstl region was extracted from the Mg4 force, and both ends were converted into BamHI-bonded ends, which were also incorporated into a plasmid pBR322, which was also opened with BamHI and was derived from bacteria. Plasmid pMg 4 is known. Any DHFR gene that can be expressed in eukaryotic cells, such as the DHFR gene on the above plasmid, can be used in the present invention.
  • a hepatocyte-proliferating substance-highly producing strain By utilizing the properties of the DHFR gene described above, a hepatocyte-proliferating substance-highly producing strain can be obtained. That is, by preparing an expression vector in which the DHFR gene has been incorporated into a hepatocyte proliferating substance expression vector and culturing eukaryotic cells transformed with the vector in the presence of MTX, amplification of the DHFR gene and The gene encoding hepatocyte proliferative substance is also amplified, so that MTX-resistant and hepatocyte proliferative substance-producing strain can be obtained.
  • the hepatocyte proliferating substance expression vector pcD LH is transferred to a region other than the replication region and other regions essential for the expression vector, for example, By cleaving at the restriction enzyme Clal recognition site or the like and incorporating the DHFR gene isolated from pMg4, the hepatic parenchymal cell growth substance expression vector pcDLH-dhfr can be prepared. By culturing eukaryotic cells transformed with this expression vector in the presence of MTX, an MTX-resistant high-producing hepatocyte proliferating substance-resistant strain can be obtained.
  • a DHFR gene that is incapable of replicating in a eukaryotic cell when introduced into a eukaryotic cell together with another foreign gene on a different molecule than the DHFR gene that cannot be replicated in the eukaryotic cell.
  • the gene is integrated into the host cell chromosome according to its proximity. It is known that when eukaryotic cells in which the DHFR gene and the foreign gene are integrated in close proximity on the chromosome are treated with MTX, the foreign gene is also amplified along with the amplification of the DHFR gene on the chromosome (Nunber et al. Proc. Natl. Acad. Sci., 75, P5553, 1978).
  • the DHFR gene and the gene encoding the hepatocyte proliferation substance do not need to be introduced into the host in a state of being integrated in a vector capable of replicating in the host eukaryotic cell.
  • the genes encoding the substances may be on different DNA molecules. For example, from a transformant obtained by co-transformation using a vector mixture of the above-described pMg4 and linear pcDLH, a high-producing liver parenchyma-producing substance can be obtained by MTX treatment.
  • eukaryotic cells such as DHFR-deficient Chinese hamster ovary-derived cells
  • pcDLH-dhfr eukaryotic cells after transformation using pcDLH-dhfr are selected twice a week in a selection medium containing 5 nM MTX (Nippon Regry). If the culture is continued for 2 to 3 weeks while replacing the medium, 5 nM MTX-resistant cells can be obtained.
  • a selection medium containing 5 nM MTX (Nippon Regry).
  • Examples of the host eukaryotic cell line used in the present invention include African green monkey kidney-derived cells (Vero or Verots cells), HeLa cells, Chinese hamster ovary-derived cells (CHO cells), human fetal lung-derived cells (W138 cells), Neonatal hamster kidney-derived cells (BHK cells), CV-1 derived cells (COS-7 cells), dog kidney derived cells (MDCK cells), mouse breast cancer derived cells (C127 cells), mouse glandular fibroblasts L cell) Examples include human leukemia cells (Namalvva cells) and human kidney-derived cells.
  • Chinese hamster ovary-derived cell K1 (CH0-K1 cell, registered as ATCC CCL61; the same applies hereinafter), DHFR-deficient Chinese hamster ovary-derived cell (dhfr-CHO cell ATCC CRL9096), neonate
  • DHFR-deficient Chinese hamster ovary-derived cell (dhfr-CHO cell ATCC CRL9096)
  • neonate There are hamster kidney-derived cells (BHK cell ATCC CCL10, ⁇ CV-1 derived cells (COS-7 cell ATCC CRL 1561), African green monkey kidney derived cells (Vero cell ATCC CCL-81), and the like.
  • each is most preferably a DHFR gene-deficient cell line, but is not particularly limited thereto.
  • Transformation of these host cells using a hepatocyte proliferating substance expression vector can be performed by a method known in the art, including a large number of published operation manuals and literatures. The method can be used, and no special operation is required.
  • Well-known transformation methods include the calcium phosphate method (Graham et al., Virology, 52, p456, 1973) or CelPhect from Pharmacia.
  • Examples include a method using a Transfection Kit.
  • dhfr-CHO cells can be transformed using the plasmid vector of the present invention, pcDLH-dhfr, by the calcium phosphate method.
  • Precipitate of pcDLH-dhfr and calcium phosphate was added to dhfr-CHO cells cultured at 37 ° C in the presence of ⁇ medium (Gibco), and the cells were collected at 37 ° C and recovered.
  • a-MEM selective medium
  • fetal serum Gibco
  • pcDLH When African green monkey kidney cells (Verots cell) cells are used, pcDLH can be introduced into a transforming method using a DOTAP reagent commercially available from Boehringer Mannheim.
  • the gene coding for the substance encoding hepatic parenchymal cell can function as a promoter located upstream thereof. And produce the target substance, a hepatic parenchymal cell culture substance.
  • transformed dhfr-CH0 cells the cells are transformed until confluent.
  • the cells are cultured on a plate and then transferred to a serum-free medium and cultured, hepatocyte proliferating substances are produced in the culture supernatant.
  • Transformed Verots cells produce hepatocyte proliferating substances in the culture supernatant when cultured in an appropriate medium, for example, DMEMZF12 (FBS-).
  • Hepatic parenchymal cell proliferating substances produced in the medium can be electrophoresed by SDS-PAGE (polyacrylamide gel electrophoresis) and then stained by silver staining to obtain about 63,000- Production can be confirmed as bands corresponding to about 69,000 and about 32,000 to 36,000 under reducing conditions.
  • the hepatocyte-proliferating substance produced in this manner exhibited a growth-promoting activity on egret hepatocytes (Experimental Example 10).
  • the abdominal skin I and peritoneum of the Sprague-Dawley rat (8-week-old os) were opened about 20 to 25 mm from the xiphoid process along the midline, and the right phrenic lobe of the liver 'left phrenic Leaves Three lobes of the left visceral lobe were pulled out of the body and excised. This means that about two-thirds of the entire liver has been removed.
  • laparotomy was performed again 12 hours after the excision at which RNA synthesis started, and the remaining liver was completely removed, and 10 volumes of 8 M guanidine hydrochloride / 2 OmM sodium acetate buffer (pH 5.5) was added.
  • the mixture was sufficiently stirred with a homogenizer and centrifuged at 180,000 X g to obtain a supernatant as a sample solution.
  • 5.7 M cesium chloride was put in an ultracentrifuge tube in advance, and 8 M guanidine hydrochloride-N 2 OmM sodium acetate buffer was overlaid, and a sample solution was further layered thereon.
  • Ultracentrifugation was performed at 25 ° C. for 20 hours at 20 ° C.
  • RNA was separated as a pellet. Transfer this RNA to an appropriate amount of sterile water. was dissolved. Add an equal volume of 0.4 mM Na C 1 Z2 0 mM Tris-HCl buffer (pH 7.4) and equilibrate with 0.2 M Na C 1 Z1 OmM Tris-HCl buffer (pH 7.4) in advance. The mixture was added to the oligo (dT) cellulose column, washed with the same solution, and eluted with 1 OmM Tris-HCl buffer / 1 mM EDTA (pH 7.4).
  • the eluate was fractionated, and a part thereof was measured for absorbance at a wavelength of 260 nm using a spectrophotometer, and an elution peak was collected.
  • An equal volume of 4 M ammonium acetate was added thereto and stirred. Two times the volume of this stirred solution was added to ethanol, and the mixture was sufficiently stirred. After leaving at ⁇ 20 ° C. for 3 hours, the mixture was centrifuged at 4 ° C. at 15,00 rpm for 5 minutes, and the precipitate was used as mRNA for the following experiments. Double-stranded cDNA was synthesized from this mRNA according to the method of Land, H (Nucl. Acids Res., 9, p2251, 1981).
  • the double-stranded cDNA synthesized from the above mRNA was incorporated into E. coli plasmid pUC8 by the method of Maniatis, T (Molecular Cloning, p249, 1982), and this recombinant DNA was transformed into E. coli HB101. ) And transformed. About 68,000 kinds of such transformed clones were obtained. This was designated as a partial liver resection rat remaining liver cDNA library.
  • Each transformed clone of the partial liver resection rat remaining liver cDNA library was transferred to each of two filters.
  • pre-sterilized filter was added to ampinulin (50 g / m 1) LB agar medium (1% pact tryptone, 0.5% yeast extract, 1% NaCl, pH7 .5), and the transformed clone was transferred to a sterilized bamboo skewer or toothpick in the same manner for two filters. After overnight culture at 37 ° C, E. coli clones grew on the filters and formed colonies.
  • This filter was subjected to alkali treatment according to the method of Grunstein, M et al. (Proc. Natl. Acad. Sci.
  • the remaining liver from the partial hepatectomy rat c labeled with [ ⁇ —′- P] d CTP used here DNA is on In (1), double-stranded DNA is synthesized using a part of the mRNA obtained by the oligo (dT) cellulose column chromatography, and the [ ⁇ - : ' 2 P] d Obtained by labeling with CTP.
  • liver c DNA may laparotomy rats, liver sutured peritoneum 'skin without excision again laparotomy, the remaining liver was Zen ⁇ after 12 hours It is obtained by preparing mRNA in the same manner as in (1) above and labeling it with [ ⁇ - 32 P] d CTP by the nick translation method.
  • each filter was washed with 2 XS SC (0.3 MNa C 3 OmM sodium citrate) /0.1% SDS (Sodium Dodecyl Sulphate) at room temperature and further 0.1 L x SSC / 0 1% SDST Washed at 50 ° C and dried.
  • the dried filters were each superimposed on an X-ray film, exposed to light at 180 ° C in an X-ray film cassette, and subjected to autoradiography.
  • the clones exposed with the partial hepatectomy rat residual liver cDNA probe and the rat sham-operated liver cDNA probe were more sensitive to exposure when the partially hepatectomized rat residual liver cDNA was used than when the liver was used. Only 40 clones were selected.
  • the mRMA extracted from the remaining liver 12 hours after partial hepatectomy was further adsorbed to these filters in the presence of NaC1.
  • the obtained filter was washed with NaC1 and eluted with water, whereby mRNA having a sequence complementary to the previously adsorbed clone cDNA could be selectively obtained.
  • this mRNA was concentrated, microinjected into African oocytes, and cultured in Barth's modified culture at 20 ° C. After 48 hours, the culture was recovered, and the proliferation activity of hepatocytes was evaluated for this culture as follows.
  • Rat liver was allowed to flow through the primary culture method of hepatocytes according to the method of Nakamura et al. (Primary culture hepatocyte experiment method 6 and 161, 1987), and hepatocytes were separated and cultured for the experiment.
  • the liver of a rat (Wistar 8-week-old os) was flushed with Philiams E medium containing 0.05% collagenase, and the harbor cells were separated. Separated port cells were centrifuged at 50 X g for 1 minute to remove the supernatant, and the precipitated cells were gently suspended in Williams E medium, and the same centrifugation was repeated several times. By this operation, high-purity hepatic parenchymal cells could be obtained.
  • the cells were plated at 2 xl 0 4 pieces / cn ⁇ density of the presence of 5% carbon dioxide, between hours 3-6 was cultured at 37 ° C Later, the medium was replaced with fresh serum-free medium.
  • the medium was removed, the cells were gently washed with PBS (phosphoric acid buffer solution Z saline), and the cells were treated at 50 ° C in the presence of 2 N NaOH, and the cells were removed from a petri dish. After dissolution, the mixture was neutralized and the 3 H content was measured using a liquid scintillation counter. The presence of hepatocyte proliferating substances in the test sample confirms an increase in the amount of 3 H taken up into cells.
  • PBS phosphoric acid buffer solution Z saline
  • the 40 clone cDNA obtained in the above (2) was transformed into type III and the complementary mRNA obtained in the above (3) was transformed into African Xenopus oocytes.
  • the 40 cultures (corresponding to 40 clones) obtained by injecting and culturing the cells one (clone) showed a high hepatocyte proliferation activity.
  • the nucleotide sequence of the clone cDNA coding for the gene product exhibiting hepatic parenchymal cell proliferation activity selected in (3) above was determined by the didequin method. This clone cDNA was considered to be missing the 5 'end, so the following procedure was performed to determine the 5' end.
  • the remaining liver was first removed from the partial hepatectomy rat 12 hours after the hepatectomy was performed, using the same method as in (1) above. From this, mRNA was obtained. This partial hepatectomy mRNA was heated at 70 ° C. for 10 minutes and quenched.
  • TGAATTACAGTCCATC CTC CTCC (3 ′ end) Using this as a primer, the above partial hepatectomy mRNA was reacted with type III to prepare 5, terminal cDNA.
  • the nucleotide sequence of the cDNA obtained here was determined by the didekin method, and the full length gene encoding a rat-derived hepatocyte proliferating substance was cloned.
  • the plaque generated by the recombinant phage obtained in (1) was transferred to a nitrocellulose membrane, dried and immobilized, and then prehybridized with a solution having the following composition at 55 ° C for 4 hours.
  • cDNA of the rat parenchymal hepatocyte proliferating substance derived from the rat lacking the 5 'terminal obtained in (4) of Example 1 was added by :
  • a hybridization solution containing 10 ng / ml of the labeled DNA was added by :
  • 4 XSSC buffer containing 0.1% SDS at 55 ° C for 10 minutes was treated with 4 XSSC buffer containing 0.1% SDS at 55 ° C for 10 minutes, and with 2 XSSC buffer containing 0.1% SDS at 55 ° C for 15 minutes, 0.1% Washing was performed at 55 ° C.
  • the nucleotide sequence of the cDNA on the phage vector obtained in (2) was determined by the dideoxy method. Using a kit for nucleotide sequence determination manufactured by Amersham, Inc., the operation was performed according to the handbook attached to the kit, and the nucleotide sequence of the HP-041 gene was determined.
  • Plaques generated by the recombinant phage (cDNA library) obtained in (1) of Example 2 were transferred to a nitrocellulose membrane, dried and immobilized, and then treated in the same manner as in (2) of Example 2. Prehybridization was performed at 4 ° C for 4 hours. Prepare a pre-hybridization solution containing 10 ng / ml of HP-041 gene DNA labeled with 2 P, and use this labeled DNA as a probe at 65 ° C for 12 hours. The phage DNA immobilized on a nitrocellulose membrane was hybridized. The membrane was washed with 2 x SSC buffer containing 0.1% SDS for 65 min and 10 min, and with 1 x SSC buffer containing 0.1% SDS at 65 ° C for 15 min.
  • the nucleotide sequence of cDNA on the phage vector obtained in (1) was determined by the dideoxy method. The operation was performed according to the handbook attached to the kit, using a kit for nucleotide sequence determination manufactured by Amersham.
  • PSVL a plasmid about 4.9 Kb in size containing the SV40 late promoter available from Pharmacia, was cleaved with the restriction enzyme Smal.
  • the HP-041 cDNA gene on the Igt10 phage vector was amplified by the polymerase. Reaction method (PCR method).
  • primer DNA Primer 1 having the following sequence was prepared using a DNA synthesizer manufactured by Applied Biosystems.
  • An expression vector pc DLH for animal cultured cells having the HP-401 gene was prepared as follows. Plasmid pcDL—SRa296 (Y. Takebe et.al. Mol. Cel 1. Biol. 8.466. 1988) 1 g was opened using restriction enzymes Kpnl and Pstl, and DNA was precipitated by ethanol precipitation. Was collected by precipitation. After blunting both ends of the opened plasmid with T4 DNA polymerase, the blunt-ended DNA was recovered by ethanol precipitation again. The recovered DNA was subjected to 1.0% agar gel electrophoresis, and then a gel fraction containing 3.4 Kb of pcDL—SRa 296 was cut out, and DNA was extracted from this gel slice by electroelution. Then, after the phenol treatment, the DNA was recovered by ethanol precipitation. The terminal of the recovered DNA fragment was dephosphorylated using CIAP, and after the treatment with phenol, the dephosphorylated DNA was recovered by ethanol precipitation.
  • the previously prepared dephosphorylated pc DL-SRa 296 fragment and blunt-ended HP-0441 gene fragment were mixed with T4 DNA ligase and reacted.
  • the reaction product was incorporated into calcium-treated Escherichia coli HB101 by a transformation method, and pcDLH was obtained from the resulting transformant.
  • 3.8 mg of the mouse DHFR gene was incorporated into bacterial pBR322, a plasmid pMg4 (described above). 3 ⁇ g of the plasmid was completely digested with BamHI, treated with phenol, and precipitated with ethanol. After blunting the ends by the method described in (1) and subjecting it to 1.0% agar gel electrophoresis, a gel fraction containing the 3.8 Kb DHFR gene region was cut out, and DNA was extracted by electroelution. After the phenol treatment, the DNA was recovered by ethanol precipitation. The linear pc DL H obtained by the above method and the DHFR gene were reacted by adding T4 ligase. The reaction calcium ⁇ untreated E.
  • FIG. 1 shows the structure of the above pcDLH-dhfr
  • FIG. 2 shows the construction scheme of the plasmid.
  • the HP-041V gene (EcoRl fragment) was cut out by the action of the restriction enzyme EcoRl. This was blunt-ended in the same manner as in (2) of Example 4, and the blunt-ended fragment was replaced with pcDL-SRa according to the method described in (2) and Example 5 of Example 4.
  • Pc-DLHV which is an HP-041V expression vector integrated into 296, and pcDLHV-dh fr into which 1) the HFR gene was integrated were prepared.
  • HP-041 expression vector pcDLH-dhfr was introduced into 0 ⁇ 11-deficient 1 ⁇ 0 cells (ATCC CRL9096) using the calcium phosphate method and CellPect Transfection K (Pharmacia).
  • dhfr-CH0 cells are seeded at 2.0 x 10 5 cells / 23.75 cm 2 , and 5% CO is present in ⁇ medium (Gibco) containing 10% fetal serum (Gibco) After culturing at 37 ° C for 24 hours, the medium was replaced. After incubating at 37 ° C for 4 hours, a precipitate of plasmid and calcium phosphate was added, the cells were cultured at 37 ° C for 16 hours, and then the medium was removed. After washing the cells with the medium, replace the medium and collect the cells at 37 ° C for 1 hour, collect the cells, spread the ⁇ 16, 1/12, and 1/24 volumes each on a 6-cm Petri dish, and use a 10% dialysis solution.
  • the cells were cultured twice a week in a-MEM (selective medium) containing (Gibco) until the appearance of transformed cell colonies. Each formed colony was transferred to a 96-well plate and further cultured, and the cells were scaled up to a 24-well plate.
  • a-MEM selective medium
  • Gibco selective medium
  • Verots cells were plated at a concentration of 5 XI 0 5/6 0, 3 7. After standing at 1 ⁇ C for 1 day and at 33 ° C for 1 day, 20 ng of pc DLH was introduced using DOTAP reagent (Boehringer Mannheim). 3 After standing at 33 ° C for 6 hours, washing with MEM medium, and culturing in MEM medium containing 5% FBS for 3 days. Then, after 6 days after replacing with DMEMZF12 (FBS-), the culture supernatant was collected, reduced by treatment with 95-95 for 3 minutes in the presence of 2-mercaptoethanol, and subjected to 12% polyacrylamide gel electrophoresis. He ran and confirmed the production of HP-041 by silver staining.
  • Figure 4 shows the results of the electrophoresis.
  • Example 6 Using pcDLHV-dhfr obtained in Example 6, dhfr-CH0 cells were transformed according to the method described in (1) of Example 7. The obtained transformed cells were cultured according to the methods of (2) and (3) in Example 7 to produce recombinant HP-04IV. Recombinant HP-04IV produced in the medium was confirmed by silver staining using 12% polyacrylamide gel electrophoresis under non-reducing conditions. Fig. 5 shows the electropherogram.
  • the hepatocyte-proliferating activity of the recombinant hepatocyte-proliferating substance obtained in Example 7 and Example 9 was measured using the rat (Wistar—8 weeks old) used in the activity measurement method described in Example 1, (3). Hessian-derived hepatocytes were changed to Egret (Japanese White Egret 1-month-old Oss) hepatocytes, and the other procedures were performed in the same manner as in Example 1, (3).
  • Egret Japanese White Egret 1-month-old Oss
  • liver function of recombinant hepatic parenchymal cell proliferative substance ⁇ Strong effect evaluation The hepatic function / strong activity of the recombinant hepatocyte proliferating substance obtained in Examples 7 and 9 was measured. Hepatic function-enhancing activity was determined by the method of Grana et al. (Granner, D et al., Methods in Enzymol., 17, 633, 1970) as a reference. The change in the expression level of tyrosine transaminase (TAT) activity after the action of was performed.
  • TAT tyrosine transaminase
  • Hepatocytes derived from rat (Wistar 8 weeks old os) used in the activity measurement method described in (3) of Example 1 were changed to hepatocytes derived from Japanese egret (Japanese white egos 1 month old os)
  • the procedure up to the separation of hepatocytes was performed in the same manner as in Example 1, (3).
  • the present invention it becomes possible to constantly produce hepatocyte proliferating substances with high production efficiency. Therefore, the use of hepatocyte proliferating substances as therapeutic agents for liver diseases It can be a very useful supply means. Furthermore, the hepatocyte proliferating substance produced according to the present invention is also useful as a basic study for hepatocyte culture and the like, and as a clinical diagnostic reagent for hepatitis and liver function tests.
  • Sequence number (SEQ ID NO): 1 Sequence length: 939 bases
  • Sequence type nucleic acid
  • Characteristic symbol sig peptide Location: 6 7. .. 936 Characteristic character: mat peptide
  • Sequence type nucleic acid
  • Lys lie Lys Pro Leu Gin Ser Pro Ala Glu Phe Ser Val Tyr Cys
  • Lys lie Asp Leu Ala Asp Phe Glu Lys Asn Ser Arg Tyr Ala Gin

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

On produit de façon très efficace une substance de prolifération d'hépatocytes, en construisant un plasmide vecteur pcDLH-dhfr pour exprimer une substance de prolifération d'hépatocytes, ce plasmide portant un gène codant pour un précurseur de substance de prolifération d'hépatocytes lié à un promoteur SRa, un gène codant pour une dihydroplate réductase, une région de réplication d'un vecteur pBR322 du plasmide E. coli et un gène résistant à l'ampicilline se trouvant sur une seule et même molécule de celle-ci, en transformant des souches de cellules eucaryotes à l'aide du vecteur d'expression ainsi construit, puis en cultivant le transformant ainsi obtenu.
PCT/JP1995/001904 1994-09-22 1995-09-21 Procede pour produire une substance de proliferation d'hepatocytes recombinee WO1996009383A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU35332/95A AU3533295A (en) 1994-09-22 1995-09-21 Process for producing recombinant hepatocyte proliferating substance

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP6/228234 1994-09-22
JP22823494 1994-09-22

Publications (1)

Publication Number Publication Date
WO1996009383A1 true WO1996009383A1 (fr) 1996-03-28

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PCT/JP1995/001904 WO1996009383A1 (fr) 1994-09-22 1995-09-21 Procede pour produire une substance de proliferation d'hepatocytes recombinee

Country Status (2)

Country Link
AU (1) AU3533295A (fr)
WO (1) WO1996009383A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6693186B2 (en) * 1998-09-01 2004-02-17 Antex Biologics Inc Neisseria meningitidis polypeptide, nucleic acid sequence and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021678A1 (fr) * 1993-03-23 1994-09-29 Taisho Pharmaceutical Co., Ltd. Substance de croissance des cellules du parenchyme hepatique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021678A1 (fr) * 1993-03-23 1994-09-29 Taisho Pharmaceutical Co., Ltd. Substance de croissance des cellules du parenchyme hepatique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. BIOPHYS. RES. COMMUN., Vol. 193, No. 2, (June 1993), T. YAMAMOTO et al., "Molecular Cloning and Initial Characterization of a Novel Fibrinogen-Related Gene, HFREP-1". *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6693186B2 (en) * 1998-09-01 2004-02-17 Antex Biologics Inc Neisseria meningitidis polypeptide, nucleic acid sequence and uses thereof

Also Published As

Publication number Publication date
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