WO2001098347A1 - Facteur de croissance des cellules hepatiques canines - Google Patents
Facteur de croissance des cellules hepatiques canines Download PDFInfo
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- WO2001098347A1 WO2001098347A1 PCT/JP2001/005362 JP0105362W WO0198347A1 WO 2001098347 A1 WO2001098347 A1 WO 2001098347A1 JP 0105362 W JP0105362 W JP 0105362W WO 0198347 A1 WO0198347 A1 WO 0198347A1
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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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
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- 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 canine hepatocyte growth factor, a 5-amino acid-deleted canine hepatocyte growth factor thereof, and a gene encoding the same.
- Hepatocyte growth factor has been purified as a liver regeneration factor, the gene encoding it has been cloned, and its sequence has been determined. HGF was initially thought to function only for the proliferation of hepatocytes, but subsequent studies have shown that it not only protects the proliferation and regeneration of hepatocytes, but also protects lungs, kidneys, blood vessels, and heart tissue from strong damage and organs. It has been shown to have a regenerative effect. In addition, HGF has a wide variety of functions, including strong anticancer activity against certain types of cancer.
- HGF functions as a growth-promoting factor, a motility-promoting factor, a morphogenesis-promoting factor, and a tumor suppressor. HGF expression is also increased in organs such as the lung and kidney in response to liver damage, and liver regeneration is promoted by a blood-mediated mechanism. It has been confirmed that similar mechanisms promote regeneration in other organs such as kidney and lung.
- Each of these functions is a biological activity essential for the construction and maintenance of tissues and organs, and is expected to be applied clinically as a specific drug for intractable organ diseases for which a fundamental cure has not been established.
- gene therapy for chronic obstructive atherosclerosis in diabetic patients using the HGF gene is being attempted.
- HGF has been reported to have many variants due to alternative splicing. Above all, a mutant HGF lacking 15 base pairs in the first kringle domain corresponding to the receptor binding site, that is, a deletion of 5 amino acids, has a proliferative activity on epithelial cells of 2 compared to normal HGF. It is up to 3 times higher and has different physiological effects. This 15-base pair deletion form of HGF is a disease mainly caused by epithelial tissue damage. Therefore, a higher therapeutic effect is desired.
- HGF mRNA expression is rapidly induced in stromal cells such as liver cupper cells and sinusoidal endothelial cells with various liver disorders.
- HGF produced and secreted by stromal cells acts on epithelial cells such as hepatocytes and bile duct cells, and promotes liver regeneration.
- epithelial cells such as hepatocytes and bile duct cells
- tissue regeneration has been reported in many tissues such as liver (cirrhosis, hepatitis, hepatic fat), kidney (acute and chronic renal failure), lung, heart, and stomach.
- the total length of the human HGF gene is about 70 kb, and the transcript mRNA is about 6 kb, of which the protein-coding region is about 2.2 kb. It is.
- Human HGF is first synthesized as a single prepro-HGF consisting of 728 amino acids, and after cleavage of the N-terminal 31 amino acids, between the 494th Arg and the 495th Val Is cleaved by a protease to form a mature molecule in which the chain and the / 3 chain are connected by a single disulfide bond.
- HGF genes from humans, mice, and rats have been cloned and their nucleotide sequences have been determined.
- the present invention provides a canine hepatocyte growth factor and a fifteen base pair deleted form thereof, a canine hepatocyte growth factor and a gene encoding the fifteen base pair deleted hepatocyte growth factor. With the goal.
- the present inventors have conducted intensive studies to solve the above problems, and as a result, succeeded in determining the canine hepatocyte growth factor gene sequence using the RT-PCR method, and completed the present invention. Was.
- the present invention is the following recombinant protein (a) or (b).
- a protein comprising an amino acid sequence represented by SEQ ID NO: 2 or 4 in which one or several amino acids have been deleted, substituted or added, and having canine hepatocyte growth factor activity
- the present invention is a gene encoding the following protein (a) or (b).
- a protein comprising an amino acid sequence represented by SEQ ID NO: 2 or 4 in which one or several amino acids have been deleted, substituted or added, and having canine hepatocyte growth factor activity
- the present invention is a gene containing the following DNA (c) or (d).
- the present invention is a recombinant vector containing the above gene.
- the present invention is a transformant containing the above-mentioned recombinant vector.
- the present invention is a method for producing a canine hepatocyte growth factor, which comprises culturing the above transformant and collecting a canine hepatocyte growth factor from the obtained culture.
- the present invention is a reagent for detecting canine hepatocyte growth factor, which contains at least a part of the gene.
- the present invention is a pharmaceutical composition
- a pharmaceutical composition comprising the above-mentioned recombinant canine hepatocyte growth factor, wherein the above-mentioned pharmaceutical composition comprises a liver disease, a kidney disease, a lung disease, a bone disease, a digestive disease, and a heart circulation.
- the above-mentioned pharmaceutical composition comprises a liver disease, a kidney disease, a lung disease, a bone disease, a digestive disease, and a heart circulation.
- organ diseases or cerebral nervous system diseases for treating organ diseases or cerebral nervous system diseases.
- the present invention shows a strong regenerative ability for injured organs, and is expected to have clinical application for refractory organ diseases for which a radical treatment has not been established.
- a gene encoding the following, a recombinant cHGF, a recombinant vector containing the gene, a transformant containing the recombinant vector, a method for producing the cHGF, a method for detecting the cHGF, and a method for detecting the cHGF
- the present invention relates to a pharmaceutical composition containing
- its proliferative activity on epithelial cells is 2-3 times higher and is known to have different physiological effects. It is a mutant canine HGF (dcHGF) in which base pairs are deleted, that is, 5 amino acids are deleted.
- the present inventors extracted and purified RNA, designed several primers considered to be specific for HGF, and performed RT-PCR to obtain several DNA fragments.
- the obtained DNA fragments were cloned into a plasmid vector and the nucleotide sequence was determined. Based on the determined base sequence, the target canine HGF gene sequence was determined, excluding the overlapping portion of each DNA fragment.
- the gene of the present invention has been obtained by determining the target gene sequence by this method.
- Sources of mRNA include tissues such as dog liver, kidney, lung, brain, thymus, and leukocytes. Preparation of mRNA can be performed by a commonly used technique. For example, after extracting total RNA from the above tissues or cells by the guanidium thiosine-phenol method, etc., the affinity column method using oligo dT-cellulose, poly U-sepharose, etc. Alternatively, poly (A) + RNA (mRNA) can be obtained by the patch method. Further, poly (A) + RNA may be further fractionated by sucrose density gradient centrifugation or the like.
- oligo dT primer and reverse transcription A single-strand cDNA is synthesized using an enzyme.
- a degenerate sense primer and a degenerate antisense primer corresponding to the amino acid sequence of the hepatocyte growth factor protein family that have already been obtained are synthesized, and PCR is performed using this, and the obtained fragment is inserted into an appropriate cloning vector to prepare a recombinant vector.
- the recombinant vector is used to transform Escherichia coli and the like, and a transformant is selected by using tetracycline resistance, ampicillin resistance, etc. as an index to include a partial or full-length sequence of the cHGF and dcHGF genes.
- a clone can be obtained.
- the present invention is not limited to these primers.
- the transformation of E. coli was prepared by the method of Hanahan [Hanahan, D .: J. Mol. Biol. 166: 557-580 (1983)], that is, in the presence of calcium chloride, magnesium chloride or rubidium chloride. It can be carried out by a method of adding a recombinant vector to a competent cell.
- plasmid When plasmid is used as a vector, it is necessary to contain a drug resistance gene such as tetracycline or ampicillin.
- a cloning vector other than plasmid for example, I phage ( ⁇ gtll or the like) can be used.
- the nucleotide sequence of single or multiple isolated clones containing the above DNA fragment is determined using the PCR product as a template.
- the nucleotide sequence can be determined by a known method such as a chemical modification method of maxam-Gillpart or a dideoxynucleotide chain termination method using M13 phage.
- an automatic nucleotide sequencer for example, manufactured by Applied Biosystems
- the sequencing is performed using a Model 310 fluorescence sequencer.
- the purpose is to remove overlapping parts Determine the nucleotide sequence of cHGF or dcHGF.
- SEQ ID NO: 1 shows the nucleotide sequence of the canine hepatocyte growth factor gene of the present invention
- SEQ ID NO: 2 shows the amino acid sequence of the canine hepatocyte growth factor of the present invention
- SEQ ID NO: 3 shows the 15 base pair deletion of the present invention
- SEQ ID NO: 4 shows the nucleotide sequence of the canine hepatocyte growth factor gene
- SEQ ID NO: 4 shows the amino acid sequence of the 5-amino acid-deficient canine hepatocyte growth factor of the present invention.
- At least one, preferably one or several (e.g. 1 to 10, more preferably 1 to 5) amino acids of the amino acid sequence represented by SEQ ID NO: 2 or 4 may be deleted, At least one, preferably one or several (eg, 1 to 10, more preferably 1 to 5) amino acids may be added to the amino acid sequence represented by SEQ ID NO: 2 or 4, or At least one, preferably one or several (eg, 1 to 10, more preferably 1 to 5) amino acids of the amino acid sequence represented by No. 2 or 4 may be substituted with another amino acid.
- the gene of the present invention also includes a DNA capable of hybridizing with the above gene under the following conditions and encoding a protein having canine hepatocyte growth factor activity. That is, using a filter on which DNA is immobilized, 0.7-: hybridization is performed at 68 ° C. in the presence of L0M NaCl. This refers to conditions that can be identified by washing at 68 ° C using an SSC solution of ⁇ 2x concentration (1xSSC consists of 150mM NaCl and 15mM sodium citrate).
- the present invention also includes RNA against the above DNA, or RNA capable of hybridizing with the RNA under stringent conditions and encoding a protein having canine hepatocyte growth factor activity.
- a known method such as the Kunkel method or the Gapped duplex method or a method similar thereto, for example, a mutagenesis kit using a site-directed mutagenesis method (for example, Mutant-K (TAKARA) or Mutant-G (TAKARA)), or using the TAKARA LA PCR in vitro Mutagenesis series kit.
- TAKARA Mutant-K
- TAKARA Mutant-G
- the gene of the present invention has the amino acid sequence of canine hepatocyte growth factor or the nucleotide sequence corresponding to the amino acid sequence of 15 base pair-deficient canine hepatocyte growth factor.
- the DNA is then synthesized by chemical synthesis, by PCR using cDNA as a type II, or by hybridizing with a DNA fragment having the nucleotide sequence as a probe. To obtain the gene of the invention Wear.
- the recombinant vector of the present invention can be obtained by ligating (inserting) the gene of the present invention into an appropriate vector.
- the vector for inserting the gene of the present invention is not particularly limited as long as it can be replicated in a host, and examples thereof include plasmid DNA and phage DNA.
- the plasmid DNA plasmid from E.coli (e.g. pBR322, P BR325, PUC118, pUC119 , pUC18, pUC19 etc.), Bacillus subtilis-derived plasmid (e.g. pUBllO, P TP5, etc.), plasmid derived from yeast (e.g. YEpl3, YE P 24, YCp50, etc.) and the like.
- the phage DNA phages (Charon4A, Charon21A, EMBL3, EMBL4 , X gtlO,; L gtll, ⁇ ⁇ etc.).
- retrovirus or animal virus vectors such as vaccinia virus and insect virus vectors such as Pacu-mouth vinoles can also be used.
- the purified DNA is digested with an appropriate restriction enzyme, and inserted into an appropriate vector DNA at a restriction enzyme site or a multiclonedin site to obtain a vector.
- a method of connection and the like are adopted.
- the vector of the present invention includes a vector containing a promoter, a gene of the present invention, and optionally a cis element such as an enhancer, a splicing signal, a polyaddition signal, a selection marker, a ribosome binding sequence (SD sequence), and the like.
- a selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like.
- the transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed.
- the host is not particularly limited as long as it can express the DNA of the present invention.
- the genus Escherichia such as Escherichia coli, or Bacillus' subtilis (Bacillus subtilis)
- bacteria belonging to the genus Pseudomonas such as Pseudomonas putida
- Saccharomyces' Saccharorayces cerevisiae Yeasts such as Schizosaccharomyces pombe
- animal cells such as COS cells and CH0 cells
- insect cells such as S121 or sf9.
- insects such as silkworms and gourds are used.
- the recombinant vector of the present invention is capable of autonomous replication in the bacterium, and is composed of a promoter, a ribosome binding sequence, the gene of the present invention, and a transcription termination sequence. Is preferred. In addition, a gene that controls a promoter may be included.
- Escherichia coli examples include Escherichia coli DH1 or JM109, and examples of Bacillus subtilis include Bacillus subtilis, but are not limited thereto.
- Any promoter can be used as long as it can be expressed in a host such as Escherichia coli.
- P L promoter such as [rho kappa promoter
- the promoter is used derived from Escherichia coli or phage.
- An artificially designed and modified promoter such as the tac promoter may be used.
- the method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria.
- a method using calcium ions [Cohen, S.N. et al .: Pro Natl. Acad. Sci., USA, 69: 2110 (1972)], an electroporation method and the like can be mentioned.
- yeast When yeast is used as a host, for example, Saccharomyces cerevisiae ⁇ schizosaccharomyces pomDe, Pichia pastoris (Pichiapastoris) and the like are used.
- the promoter is not particularly limited as long as it can be expressed in yeast.
- examples include gall promoter, gallO promoter, heat shock protein promoter, MFQ; 1 promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, A0X1 A promoter or the like can be used.
- the method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast.
- monkey cells COS-1 or COS-7, Vero, Chinese hamster ovary cells (CH0 cells), mouse L cells, rat GH3, human FL cells, etc. are used as the promoter.
- an SRa promoter, an SV40 promoter, an LTR promoter, a CMV promoter, or the like is used as the promoter, and an early gene promoter of human site megalovirus may be used.
- Methods for introducing the recombinant vector into animal cells include, for example, the electoporation method, the calcium phosphate method, and the lipofection method.
- S121 or sf9 cells are used.
- Methods for introducing the recombinant vector into insect cells include, for example, the calcium phosphate method, the ribofection method, and the electoral poration method.
- the protein of the present invention has an amino acid sequence encoded by the canine hepatocyte growth factor gene of the present invention or the 15 base pair deleted canine hepatocyte growth factor gene, or has at least one amino acid sequence in the amino acid sequence.
- the amino acid sequence has the amino acid sequence in which the above mutation has been introduced into each amino acid and has canine hepatocyte growth factor activity.
- the protein of the present invention is also referred to as a canine hepatocyte growth factor protein, and the 15 base pair deficient form thereof is also referred to as a 5-amino acid deficient canine hepatocyte growth factor protein.
- the canine hepatocyte growth factor protein of the present invention can be obtained by culturing the transformant and collecting from the culture.
- culture means any of a culture supernatant, a cultured cell or a cultured cell, and a cell or cell fragment.
- the method for culturing the transformant of the present invention is different from the usual method used for culturing a host. This is done accordingly.
- the medium for culturing the transformants obtained using microorganisms such as Escherichia coli and yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like, which can be used by the microorganisms, so that the cultivation of the transformants is efficient.
- a natural medium or a synthetic medium may be used as long as the medium can be performed in a controlled manner.
- Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol.
- Nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other nitrogen-containing compounds, as well as peptone, meat extract and corn steep liquor. And the like.
- examples of the inorganic substances include potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
- the cultivation is usually performed at 37 ° C under aerobic conditions such as shaking culture or aeration and stirring culture.
- the pH of the medium is adjusted using an inorganic or organic acid, an alkaline solution, or the like.
- an antibiotic such as ampicillin-tetracitalin may be added to the medium as needed.
- an inducer may be added to the medium as necessary.
- an inducer For example, when culturing a microorganism transformed with an expression vector using the Lac promoter, isopropyl-] 3-D-thiogalactovyranoside (IPTG) was transformed with an expression vector using a trp promoter.
- IPTG isopropyl-] 3-D-thiogalactovyranoside
- IAA indoleacetic acid
- a medium for culturing a transformant obtained using animal cells as a host commonly used RPMI1640 medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to such a medium is used.
- Culture is usually carried out 5% C0?. The presence of 1 to 30 days at 37 ° C. As needed during culture Antibiotics such as kanamycin and penicillin may be added to the medium.
- the cells or cells are disrupted to extract the canine hepatocyte growth factor protein.
- the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like.
- general biochemical methods used for the isolation and purification of proteins for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in combination as appropriate.
- the protein of the present invention can be isolated and purified from the culture.
- a probe that hybridizes with the above DNA or RNA and specifically detects the DNA or RNA is also provided as a reagent for detecting canine hepatocyte growth factor.
- the probe is typically a radioisotope used (e.g., 3, 3 3 ⁇ 4), (For example, digoxigenin, full O Roretsu fluorescein) enzyme, labeled with like, conventional flop port potting analysis, an In situ hybridization, etc.
- a radioisotope e.g., 3, 3 3 ⁇ 4
- digoxigenin, full O Roretsu fluorescein full O Roretsu fluorescein
- the DNA or RNA used as a probe in the present invention has at least a part of the nucleotide sequence of DNA or RNA described in SEQ ID NO: 2 or 3 and SEQ ID NO: 5 or 6.
- the length of the probe is 200 to 300 bases, but may have the entire sequence, and is not particularly limited.
- the recombinant canine hepatocyte growth factor of the present invention or the 15 base pair-deficient hepatocyte growth factor thereof is an extracted and purified recombinant canine hepatocyte growth factor or its 5 amino acid-deleted hepatocyte growth factor, Alternatively, it is a recombinant canine hepatocyte growth factor or a 5-amino acid-deleted hepatocyte growth factor that is inserted into a plasmid or the like and translated in the canine body, and is fulminant hepatitis, acute hepatitis, cirrhosis, pulmonary fibrosis, Liver diseases such as fatty liver and liver cancer, acute renal failure, chronic renal failure / nephrosclerosis, kidney transplantation, renal diseases such as diabetic nephropathy, lung diseases such as acute pneumonia, pulmonary fibrosis, osteoarthritis , Bone diseases such as rheumatoid arthritis, gastric ulcer, diabetes ( ⁇ ) Suppression of cell death of
- the pharmaceutical composition of the present invention is particularly useful for treating chronic diseases such as chronic renal failure in dogs, and can be used for a long time without antigenic problems.
- the pharmaceutical composition of the present invention is obtained by binding a cHGF gene or a dcHGF gene to a canine hepatocyte growth factor or a 15 base pair-deleted hepatocyte growth factor, a salt or a plasmid thereof, and the like. It contains the DNA fragment that is intended to be translated by a pharmaceutically acceptable carrier, diluent or excipient.
- the pharmaceutical composition of the present invention can be administered in various forms. Examples of such administration forms include oral administration using tablets, capsules, granules, powders, syrups, and the like, and parenteral administration using injections, drops, suppositories, and the like.
- Such a composition is produced by a known method and includes a carrier, a diluent, and an excipient that are commonly used in the field of formulation.
- a carrier for example, lactose, magnesium stearate and the like are used as carriers and excipients for tablets.
- An injection is prepared by dissolving, suspending or emulsifying canine hepatocyte growth factor or a salt thereof in a sterile aqueous or oily liquid commonly used for injections.
- aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents, for example, alcohols, polyalcohols such as propylene dalycol, and nonionic surfactants. You may use together with. Sesame oil, soybean oil, and the like are used as the oily liquid, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent.
- the dosage varies depending on symptoms, age, body weight, etc., but for oral administration, the dose is usually about 0.001 mg to 100 mg per animal per day, and is administered once or several times. Given. For parenteral administration, 0.001 mg to 100 mg per animal is administered by subcutaneous injection, intramuscular injection, or intravenous injection. In addition, recombinant cHGF or recombinant dcHGF inserted into a plasmid or the like to be translated in the body of a dog is 0.001 mg to 100 mg / animal once every few days, weeks or months. O mg is administered by subcutaneous, intramuscular, or intravenous injection. BRIEF DESCRIPTION OF THE FIGURES
- FIG. 1 shows the construction of recombinant cHGF and dcHGF vectors for protein expression used in COS-1 cells and CH0 cells in Example 2.
- FIG. 2 shows the biological activity of cHGF and dcHGF produced in COS-1 cells in Example 2.
- FIG. 3 shows the biological activity of cHGF and dcHGF produced in CH0 cells in Example 2.
- FIG. 4 shows the construction of a recombinant cHGF virus vector for protein expression used in silkworm worm bodies and cultured insect cells in Example 3.
- FIG. 5 shows the results of eastern blot analysis of recombinant cHGF produced using the silkworm worm in Example 3.
- Lane 1 shows the results when the body fluid of the silkworm infected with the cHGF recombinant virus was used, and
- lane 2 shows the results when the body fluid of the silkworm infected with the non-recombinant virus was used.
- FIG. 6 shows the biological activity of the recombinant cHGF protein produced using the silkworm body in Example 3.
- FIG. 7 shows the results of the stamp lot analysis of the recombinant cHGF produced in the cultured insect cells in Example 3.
- Lane 1 shows the results obtained using the culture supernatant of the Sf9 cells infected with the recombinant cHGF virus
- lane 2 shows the results obtained using the culture supernatant of the Sf9 cells infected with the non-recombinant virus.
- FIG. 8 shows the biological activity of cHGF protein produced in cultured insect cells in Example 3.
- primers were initially used based on the previously reported base sequences corresponding to the 5 'and 3' protein untranslated regions of the human hepatocyte growth factor gene. Design and amplification by RT-PCR were attempted, but it was difficult to amplify the desired size product. Therefore, first, primers were designed using the nucleotide sequences relatively well conserved across the animal species of human, mouse and rat, and primers were designed.
- coli transformed and C, over ⁇ Yogo to purify the plasmid DNA (ultraclean mini plasmid DNA puricat ion kit, MOBIO) and nucleotide sequence analysis (Espec Oligoservice, AB I310 DNA Sequencer).
- the obtained base sequence was analyzed for homology using BLAST, an online homology analysis program, and showed high homology with HGF genes of human or other animal species. This sequence was suggested to be a partial sequence of the canine HGF gene.
- the composition of the PCR reaction solution is in accordance with the composition recommended by the kit.
- the PCR cycle consists of 94 ° C for 5 seconds, 68 ° C for 10 seconds, and 72 ° C for 3 minutes, and this cycle is repeated 25 times.
- the PCR cycle consists of 94 ° C for 5 seconds, 68 ° C for 10 seconds, and 72 ° C for 3 minutes, and this cycle is repeated 25 times.
- Nested primer 5'TCAGGACCATGTGAGGGAGATTATGGTGGC3, (SEQ ID NO: 8)
- the amplification product obtained was approximately 1.7 kbp, and the 3 'protein untranslated region predicted from the cDNAs of human and rat HGF reported previously. , About 3.6 kbp.
- this amplified fragment contained a stop codon, a polyA signal, and a polyA sequence, indicating that it was a variant due to alternative splicing within the 3 'protein untranslated region. It was suggested.
- RACE was performed using the following reaction solution and PCR cycle.
- the first PCR product was subjected to agarose gel electrophoresis and ethidium mouth staining, but no amplified fragment was observed.Therefore, nested PCR was performed, and the amplified fragment by 5'RACE was used. was gotten.
- the composition of the PCR reaction solution follows the composition recommended by the kit, and the PCR cycle consists of 94 ° C for 5 seconds, 68 ° C for 10 seconds, and 72 ° C for 2 minutes, and this cycle is repeated 25 times.
- the nested PCR product was subjected to agarose gel electrophoresis and ethidium bromide staining.As a result, multiple bands were observed.
- the longest amplified fragment was excised from the agarose gel in the same manner as described above, and cloned into the pGEM-T Easy vector. -And base sequence analysis. However, the resulting amplified fragment did not contain a protein translation initiation site.
- HGF gene is a relatively large gene with a total length of about 6 kbp, it is technically difficult to close the sequence at the 5 'end.
- canine HGF mRNA is expected to have a higher-order structure that makes it difficult for normal reverse transcription to occur due to the characteristics of its nucleotide sequence, and it was very difficult to obtain full-length cDNA.
- three types of reverse transcriptase, Superscriptll (Gibco-BRL), and SMART RACE cDNA Amplifi cat ion Kit (Clont ech) were used.
- a total of six types of cDNAs for 5′RACE were synthesized using Powerscr ipt ll (Shi lontech and Soshi ichi M-ML V Reverse Transcriptase RNase Minus (Promega), respectively.
- Nine types of primers were designed based on the above-mentioned canine HGF partial sequence and the nucleotide sequence of the canine HGF gene identified by the first 5'RACE, and numerous combinations of nested PCR reactions were performed.
- Nested primer 5 ACGGCGACGGGCAGCAGGAGGAGGTGC3, (SEQ ID NO: 12)
- the composition of the reaction solution and the conditions for the PCR cycle were the same as in the first 5'RACE. Although the resulting PCR product was not a single amplification product, It was recovered from the loin gel and cloned into the pGEM-T Easy vector. Then, 24 clones were selected at random and analyzed for their nucleotide sequences. As a result, 3 clones contained the translation start point.
- the nucleotide sequences of the gene fragments obtained in (a) were combined using Genetyx-win ver. 4 software (software development) to obtain the entire nucleotide sequence of the canine hepatocyte growth factor gene protein translation region.
- the sequence is shown in SEQ ID NO: 1.
- SEQ ID NO: 1 was composed of 2193 bp, and the nucleotide sequence described in SEQ ID NO: 1 was searched using GENBANK / EMBL DNA Data Base, but no identical sequence was found. Therefore, it was confirmed that the DNA having this nucleotide sequence was completely novel.
- a homology search was performed using BLAST, an online homology search program, and Genetyxiin ver. 4 software, the nucleotide sequence of SEQ ID NO: 1 was found to be human (92.2%), mouse (87.8%). %), And high homology with the rat (87.5 etc.) hepatocyte growth factor gene sequence.
- the amino acid sequence deduced from the nucleotide sequence of SEQ ID NO: 1 is described in SEQ ID NO: 2.
- the amino acid sequence of SEQ ID NO: 2 was found to be human (92.3%), mouse (92.1%), rat (92.0%) High homology was observed with the amino acid sequence of hepatocyte growth factor.
- a primer was set to amplify the canine HGF protein translation region, and PCR was performed using canine leukocyte-derived cDNA as type II.
- a reaction solution having the following composition was used. The reaction was performed at 94 ° C for 2 minutes, followed by one cycle of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 68 ° C for 2 minutes. The PCR was repeated for 5 minutes at 68 ° C and then stored at 4 ° C.
- Nested antisense primer 5'CTATGACTGTTGTATCTTATACGTTAA3, (SEQ ID NO: 16)
- the resulting PCR product was subjected to agarose gel electrophoresis in the presence of ethidium amide, to confirm the size of the product.
- Cloning into a plasmid vector and analysis of the nucleotide sequence were performed in the same manner as described above. Analysis revealed that the adenine and guanine clones were present at the 414th base from the translation start site, respectively. This is probably due to single nucleotide polymorphisms since multiple clones were obtained for each. However, the amino acids encoded by both were glycine, and no difference was found in the amino acid sequences. Other sequences were completely identical to those obtained in (a).
- a reaction solution having the following composition was used, and the reaction was first performed at 94 ° C for 2 minutes, followed by 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C This cycle was repeated 30 times with one second as one cycle. Finally, after performing the reaction at 72 ° C for 5 minutes, PCR was performed in a cycle of storing at 4 ° C.
- reaction solution IX PCR buffer, 0.2 mM dNTP, 0.005 units / ⁇ 1 Taq polymer (TaKaRa EX Taq), 0.5 ⁇ each primer
- the resulting dcHGF clone had a 15 base pair deletion at the site corresponding to the first kringle domain, resulting in a deletion of 5 amino acids.
- clones in which the 414th base from the translation initiation site was adenine and guanine were present, respectively. . No difference was found in other base sequences and amino acid sequences.
- Example 1 Plasmid g obtained in (c) and (d) was digested with 10 cut restriction enzymes Sail and Not I (TaKaRa) at 37 ° C for 2 hours, and subjected to agarose gel electrophoresis. About 2.2 kb of cHGF and dcHGF DNA fragments were recovered using RECOCHIP (TaKaRa).
- pCl-neo Mammalian Expression Vector (Promega) lg an expression vector for mammalian cells, is digested with 10 units of restriction enzymes Sail and Notl (TaKaRa) at 37 ° C for 2 hours, and phenol is digested in the usual manner.
- the number of cells was counted according to a conventional method. The number of cells was adjusted to 8 ⁇ 10 5 cells in 5 ml of medium, seeded on a 60 mm diameter dish (FALCON), and cultured overnight at 37 ° C. in the presence of 5% CO 2 .
- the plasmid DNA for expression of cHGF and dcHGF obtained in (a) was purified (Wizard SV Minipreps DNA purification system (Promega)) and adjusted to a concentration of 1 ⁇ g / ⁇ 1 in distilled water.
- CH0 cells Using Chinese Hamster CH0 cells, a cell line stably expressing recombinant cHGF and dcHGF protein was obtained. CH0 cells, 10% ⁇ Shi calf serum (Moregate), 0. 3% Tryptose Phosphate broth (DIFCO) E containing - in MEM (Nissui Pharmaceutical) medium, 37 ° C, 5% C0 2 splicing in the presence Teens. The day before the transformation, the CH0 cells grown to a saturated cell density were detached from the plate and suspended in a medium as described above, and the number of cells was counted.
- MEM Shi calf serum
- DIFCO Tryptose Phosphate broth
- the number of cells was adjusted to 1.2 ⁇ 10 5 cells in 500 ⁇ l of the medium, seeded in a 24-well petri dish (FALCON), and cultured overnight at 37 ° C. in the presence of 5% CO 2 .
- the plasmid DNA for expression of cHGF and dcHGF obtained in (a) was purified (Wizard SV Minipreps DNA purification system (Promega)) and adjusted to a concentration of 1 ⁇ g / ⁇ 1 in distilled water.
- Gene transfer into CH0 cells was performed using Lipofectamine2000 Regent (GIBCO-BRL), and the gene transfer operation was performed according to the instructions. After gene transfer, 37 ° C, 5% C0 2 Culture overnight, remove cells as above, resuspend in 12 ml of the above growth medium containing 600 g / ml GENETIC IN (GIBCO BRL), 24-well Petri dish
- the culture supernatant was collected, and the biological activity was measured as described in Example.
- the motility of MDCK cells was observed to be increased, and it was confirmed that the recombinant cHGF and dcHGF protein produced by CH0 cells had biological activity.
- Example 3 Production of recombinant cHGF protein by Bombyx mori bodies and cultured insect cells
- a recombinant vaccinia virus recombinant with cHGF-encoding DNA was obtained by Katakura Industry Co., Ltd. Superworm Service (FIG. 4).
- the obtained virus solution of the recombinant virus was administered to the silkworm insect body, and after rearing for several days, the insect body fluid was collected.
- silkworm worms fluid sample SDS-after poly acrylamide electrophoresis, performed Western blot Tain grayed analysis to detect recombinant cHGF protein at a position corresponding to a molecular weight of about from 80,000 to 90,000 (FIG. 5)
- the biological activity was measured as described in Example 4, and an increase in MDCK cell motility was observed, confirming that the recombinant cHGF protein produced from Bombyx mori had biological activity (FIG. 6).
- the recombinant virus solution obtained above was added to the Sf9 cell culture supernatant, and after culturing for about one week, the culture supernatant was recovered.
- the culture supernatant sample was subjected to SDS-polyacrylamide electrophoresis according to a conventional method, and then subjected to stamplotting analysis to detect recombinant cHGF protein at a position of about 80,000 to 90,000 in molecular weight (FIG. 7).
- the biological activity was measured as described in Example 4, and an increase in MDCK cell motility was observed. It was confirmed that the vesicle-producing recombinant cHGF protein had biological activity (FIG. 8).
- Biological activities of the recombinant cHGF and dcHGF proteins were performed by observing hypermotility of canine renal epithelium (Madin-Darby Canine Kidney; MDCK) cells. MDCK cells were passaged in the growth medium described above. Separated from plates of the cells reached saturation cell density by the above method, cell numbers were force Unto was adjusted to 3Xl0 4 cells / ml. This cell suspension was dispensed at 100 1 / well in a 96-well plate (FALCON). Then, 501 culture supernatants of C0S-1 and CH0 cells into which the cHGF and dcHGF expression vectors obtained in Example 2 had been introduced were added.
- MDCK canine renal epithelium
- the body fluid of Bombyx mori obtained in Example 3 was diluted 2000-fold with the MDCK cell growth medium, and the supernatant of the cultured insect cells was similarly diluted 4-fold, and added 501 each. Twenty-four hours after the addition of the sample, 1/10 volume of 25% glutaraldehyde solution (Wako Pure Chemical Industries, Ltd.) was added to fix the cells, and Giemsa staining was performed to observe the motility and morphology of the cells under a microscope.
- a canine hepatocyte growth factor and its 5-amino acid-deficient canine hepatocyte growth factor, a gene encoding the canine hepatocyte growth factor and its 5-amino acid-deleted hepatocyte growth factor are provided.
- the recombinant canine hepatocyte growth factor of the present invention and the recombinant cat hepatocyte growth factor having a 5-amino acid deletion thereof are useful for treating chronic diseases such as liver disease and kidney disease in dogs.
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Description
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01941181A EP1293511A4 (en) | 2000-06-22 | 2001-06-22 | GROWTH FACTOR FOR CANINE HEPATIC CELLS |
AU7459101A AU7459101A (en) | 2000-06-22 | 2001-06-22 | Canine liver cell growth factor |
AU2001274591A AU2001274591B2 (en) | 2000-06-22 | 2001-06-22 | Canine liver cell growth factor |
US10/311,776 US7129064B2 (en) | 2000-06-22 | 2001-06-22 | Canine hepatocyte growth factor |
CA2413754A CA2413754C (en) | 2000-06-22 | 2001-06-22 | Canine hepatocyte growth factor |
NZ523221A NZ523221A (en) | 2000-06-22 | 2001-06-22 | Canine hepatocyte growth factor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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JP2000187724 | 2000-06-22 | ||
JP2000-187724 | 2000-06-22 |
Publications (1)
Publication Number | Publication Date |
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WO2001098347A1 true WO2001098347A1 (fr) | 2001-12-27 |
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ID=18687655
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Application Number | Title | Priority Date | Filing Date |
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PCT/JP2001/005362 WO2001098347A1 (fr) | 2000-06-22 | 2001-06-22 | Facteur de croissance des cellules hepatiques canines |
Country Status (6)
Country | Link |
---|---|
US (1) | US7129064B2 (ja) |
EP (1) | EP1293511A4 (ja) |
AU (2) | AU7459101A (ja) |
CA (1) | CA2413754C (ja) |
NZ (1) | NZ523221A (ja) |
WO (1) | WO2001098347A1 (ja) |
Cited By (1)
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CN110438030A (zh) * | 2019-06-27 | 2019-11-12 | 中国科学院城市环境研究所 | 一种恶臭假单胞菌Pseudomonas putida WP07、制备方法及用途 |
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JP4716875B2 (ja) * | 2003-12-16 | 2011-07-06 | 敏一 中村 | 糖鎖欠損型肝細胞増殖因子 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0412557A1 (en) * | 1989-08-11 | 1991-02-13 | Mitsubishi Chemical Corporation | Hepatic parenchymal cell growth factor, gene encoding the same, process for producing the factor, and transformants producing the factor |
JPH03255096A (ja) * | 1990-03-01 | 1991-11-13 | Toyobo Co Ltd | 組換ラット肝実質細胞増殖因子 |
EP0461560A1 (en) * | 1990-06-11 | 1991-12-18 | Toshikazu Nakamura | Recombinant human hepatocyte growth factor and method for production thereof |
Family Cites Families (4)
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JP2577091B2 (ja) * | 1989-08-11 | 1997-01-29 | 三菱化学株式会社 | 肝実質細胞増殖因子及びそれをコードする遺伝子 |
JP3200609B2 (ja) * | 1990-12-28 | 2001-08-20 | 敏一 中村 | 上皮細胞増殖促進剤 |
ES2181689T3 (es) * | 1992-05-18 | 2003-03-01 | Genentech Inc | Variantes del factor de crecimiento de hepatocitos. |
WO2001092332A1 (fr) * | 2000-05-31 | 2001-12-06 | Nippon Zenyaku Kogyo Ltd. | Facteur de croissance d'hepatocyte felin |
-
2001
- 2001-06-22 US US10/311,776 patent/US7129064B2/en not_active Expired - Lifetime
- 2001-06-22 WO PCT/JP2001/005362 patent/WO2001098347A1/ja active IP Right Grant
- 2001-06-22 NZ NZ523221A patent/NZ523221A/en not_active IP Right Cessation
- 2001-06-22 CA CA2413754A patent/CA2413754C/en not_active Expired - Fee Related
- 2001-06-22 EP EP01941181A patent/EP1293511A4/en not_active Withdrawn
- 2001-06-22 AU AU7459101A patent/AU7459101A/xx active Pending
- 2001-06-22 AU AU2001274591A patent/AU2001274591B2/en not_active Ceased
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0412557A1 (en) * | 1989-08-11 | 1991-02-13 | Mitsubishi Chemical Corporation | Hepatic parenchymal cell growth factor, gene encoding the same, process for producing the factor, and transformants producing the factor |
JPH03255096A (ja) * | 1990-03-01 | 1991-11-13 | Toyobo Co Ltd | 組換ラット肝実質細胞増殖因子 |
EP0461560A1 (en) * | 1990-06-11 | 1991-12-18 | Toshikazu Nakamura | Recombinant human hepatocyte growth factor and method for production thereof |
Non-Patent Citations (6)
Title |
---|
AI OKAJIMA ET AL.: "Primary structure of rat hepatocyte growth factor and induction of its mRNA during liver regeneration following hepatic injury", EUR. J. BIOCHEM., vol. 193, 1990, pages 375 - 381, XP002946906 * |
KEIJI MIYAZAWA ET AL.: "Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 163, no. 2, September 1989 (1989-09-01), pages 967 - 973, XP002946907 * |
KOSUKE TASHIRO ET AL.: "Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues", PROC. NATL. ACAD. SCI. USA, vol. 87, April 1990 (1990-04-01), pages 3200 - 3204, XP002946905 * |
MASATO SASAKI ET AL.: "Identification of mouse mammary fibroblast-derived mammary growth factor as hepatocyte growth factor", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 199, no. 2, March 1994 (1994-03-01), pages 772 - 779, XP002946903 * |
See also references of EP1293511A4 * |
YOUHUA LIU ET AL.: "Molecular cloning and characterization of cDNA encoding mouse hapatocyte growth factor", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1216, 1993, pages 299 - 303, XP002946904 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110438030A (zh) * | 2019-06-27 | 2019-11-12 | 中国科学院城市环境研究所 | 一种恶臭假单胞菌Pseudomonas putida WP07、制备方法及用途 |
Also Published As
Publication number | Publication date |
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US20030170685A1 (en) | 2003-09-11 |
AU7459101A (en) | 2002-01-02 |
EP1293511A1 (en) | 2003-03-19 |
CA2413754C (en) | 2013-07-30 |
NZ523221A (en) | 2005-07-29 |
US7129064B2 (en) | 2006-10-31 |
CA2413754A1 (en) | 2002-12-18 |
EP1293511A4 (en) | 2005-11-30 |
AU2001274591B2 (en) | 2006-08-17 |
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