JPH09249697A - Hepatocyte growth factor - Google Patents

Abstract

(57) Abstract: A large amount of hepatocyte growth factor (hHGF) is obtained by recombinant DNA technology. A hepatocyte growth factor represented by the amino acid sequences of FIGS. 1 and 2 and produced by gene recombination. [Effect] The fusion protein containing hepatocyte growth factor or hHGF may be a therapeutic agent for liver diseases such as a liver regeneration promoter, a liver function improving agent, a hepatitis therapeutic agent or a liver cirrhosis inhibitor.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hepatocyte growth factor obtained by gene recombination.

[0002]

2. Description of the Related Art The liver is the most highly differentiated organ in the living body. It mainly has various important functions such as processing (metabolism) of various nutrients (sugars, proteins, lipids, vitamins, hormones, etc.), storage, detoxification, decomposition, excretion, etc. Is known to play a central role in. Hepatic parenchymal cells responsible for these functions are placed under the control of various hormones in vivo, and in some cases, exhibit extremely vigorous proliferation. For example, if you excise approximately 2/3 of a rat's liver,
It is known that the size returns to the original size after about 10 days, and a treatment method by partial hepatectomy and subsequent regeneration is performed even in humans with liver cancer and the like. Many studies have been conducted on the mechanism of liver regeneration by proliferation of hepatocytes, and the presence of hepatocyte growth factor has been reported. In particular, some of the present inventors found that the plasma activity of hepatic parenchymal cells was extremely high in the plasma of human fulminant hepatitis patients.
(Biomed. Res., 6, 231 (1985) and Exp. Cell. Res. 16
6, 139 (1986)), and succeeded in purifying the factor having the activity as a single protein for the first time in the world (JP-A-63-22526 and J. Clin. Invest., 81, 414 (198).
8)).

This human hepatocyte growth factor (hereinafter abbreviated as “hHGF”) has an estimated molecular weight of about 76000-92000 by SDS-PAGE under non-reducing conditions, and a molecular weight of 56000-65000 by SDS-PAGE under reducing conditions. Divided into two bands of 32000-35000. Nakamura et al. Reported a factor with similar activity derived from rat platelets (Biochem. Biophys. Res. Comm.
un., 122, 1450 (1984)), but the estimated molecular weight was about 27,000 by SDS-PAGE (Proc. Natl. Ac
ad. Sci. USA, 83, 6489 (1986)), and then was purified as a single protein and reported to be a protein having a molecular weight of 82000 consisting of two polypeptides having a molecular weight of 69000 and 34000 (FEBS Letters, 224, 311 (1987)). Hepatocyte growth factor purified as a single protein other than hHGF and rat HGF has not been reported so far, and regarding hHGF and rat HGF, the primary structure and the nucleotide sequence of the corresponding cDNA were not determined. Has not been reported.

[0004]

A large amount of hHGF is required to examine in vitro the detailed function of hHGF in vivo or the effect on liver regeneration in the case of liver injury, but a large amount of hHGF is required from plasma of patients with fulminant hepatitis. It is not always easy to purify hHGF in terms of human, time, and cost, and it is extremely difficult to stably extract only hHGF from plasma containing an infectious agent. For this reason, stable and large-scale purification of hHGF from plasma of patients with fulminant hepatitis has not been performed.

[0005]

Means for Solving the Problems Accordingly, the present inventors have:
As a result of various studies to obtain a large amount of hHGF by recombinant DNA technology, a gene encoding hHGF useful for such a purpose was successfully cloned for the first time, and the present invention was completed.

That is, the gist of the present invention is hHG containing a signal sequence represented by the amino acid sequences shown in FIGS.
F, hHGF represented by the sequence from the 30th glutamic acid residue (Glu) to the last serine residue (Ser) in the amino acid sequences shown in FIGS. 1 and 2, 32 of the amino acid sequences shown in FIGS. The first glutamine (Gln) to the last serine residue
It exists in hHGF represented by the sequence up to (Ser).

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION To explain the present invention below, the gene (cDNA) encoding hHGF of the present invention has a nucleotide sequence as shown in FIGS. 3 to 5, for example. In addition, other complementary base sequences were omitted from the base sequence, and only a single strand was described. From this gene, hHGF having the amino acid sequence shown in FIGS. 1 and 2 can be expressed by recombinant DNA technology. At this time, the protein translated from the mRNA encoding hHGF contains a signal sequence, but when secreted from the cell, the signal sequence is cleaved and the 30th glutamic acid residue (Glu) or 32nd glutamine is cleaved. residue
HHGF having an amino acid sequence after (Gln) is produced. A signal sequence of another protein can also be used as the signal sequence. When a mature hHGF without a signal sequence is expressed in a host cell, it has a nucleotide sequence starting from the 88th G or the 94th C in the nucleotide sequences shown in FIGS. 3 to 5 as a gene encoding hHGF. The gene may be used by connecting it to the ATG codon of the vector. Furthermore, in the present invention, those obtained by modifying such as removing, changing or adding a part of amino acids or nucleic acids are also included in the present invention within a range not impairing the hepatocyte proliferation promoting activity.

DNA of the gene encoding hHGF of the present invention
The fragment is obtained, for example, by the following method. From plasma of patients with fulminant hepatitis, for example, J. Clin. Invest, 81, 414 (198
The hHGF purified by the method described in 8) has a disulfide bond cleaved into two polypeptides under reducing conditions. A polypeptide having a molecular weight of 56000-65000 is an H chain, and a polypeptide having a molecular weight of 32000-35000 is an L chain. After the thiol group of the cysteine residue produced by reducing hHGF is carboxymethylated, the H chain and L chain are separated by reverse phase high performance liquid chromatography, or hHGF is electrophoresed under reducing conditions and the gel is used. To H
After extracting each of the chains and the L chains, the amino terminal amino acid sequences of both chains can be examined by analyzing with a gas phase protein sequencer manufactured by Applied Biosystems, for example. In addition, hHGF itself, or H
Chain, L chain after separation, suitable proteolytic enzyme such as achromobacter protease I (lysyl endopeptidase)
Then, the resulting peptide fragment is separated by, for example, reverse phase high performance liquid chromatography, and then each peptide is subjected to amino acid sequence analysis in the same manner as described above, whereby the amino acid sequence inside the polypeptide can be known. The DNA base sequence is deduced from these amino acid sequences, and a sequence that facilitates oligonucleotide production is selected.
For example, an oligonucleotide as shown in Examples described later is synthesized and used as a probe.

As a cDNA library for screening a gene encoding hHGF, human-derived liver cDNA is used.
A library, a spleen cDNA library, a placenta cDNA library, etc. can be used. These libraries are sold by Clonetech. Especially placenta cDNA
Library is preferred. In addition, a cDNA library may be prepared from a cell line expressing hHGF and tissue material according to a conventional method. The λ phage having such a cDNA incorporated therein is infected with Escherichia coli by the method of Maniatis et al. (“Molecular Cloning”, Cold Spring Harbor Laboratory, pp. 56-73 (1982)) and cultured. Plaque hybridization method using the oligonucleotides prepared from the nucleotide sequence deduced from the partial amino acid sequence of hHGF as a probe ("Molecular Cloning", Cold Spring Harbor Laboratory, pages 320-328 (1982)) The hHGF has the same amino acid sequence as a part of the amino acid sequence of the target hHGF, and
It is possible to obtain several different λ phage clones which also have a nucleotide sequence corresponding to a region other than the probe of the amino acid sequence of.

Further, phages are propagated from the above screening-positive plaques by the method of Maniatis et al. ("Molecular Cloning", Cold Spring Harbor Laboratory, pp. 76-79 (1982)), and the DNA is cultivated from the plaques according to the glycerol gradient method. Is purified and cleaved with an appropriate restriction enzyme such as EcoRI.
8, plasmid vector such as pUC 19 or M 13 mp
CD in single-stranded phage vectors such as 18 and M 13 mp 19
The NA was subcloned and the dideoxy method of Sanger et al. (Proc. Natl.
Acad. Sci. USA) 74 , 5463 (1977))
The base sequence of the DNA segment can be determined. By analyzing the nucleotide sequences of the obtained clones and integrating them, it is possible to obtain a gene corresponding to all the amino acid sequences of hHGF shown in FIGS. 1 and 2 by the cDNA clone group encoding a part of hHGF. it can.

The expression of the thus obtained cDNA can be carried out, for example, by connecting the DNA groups in such a manner that their nucleotide sequences are in the order of the amino acid sequence of hHGF and the total region of these hHGF is included.
As an NA fragment, this is connected to the downstream of the promoter of a plasmid such as pCDL-SRα296 in parallel with the translation initiation codon ATG to form a protein expression plasmid, which can be used in the host of animal cells transformed with the plasmid. It can be carried out. Then, the expressed protein can be recovered by a conventional method to obtain the HGF of the present invention.

[0012] For industrial production, it is desirable to construct a stable host-vector system as the above expression plasmid. Examples thereof include those described in Japanese Patent Application No. 1-115831. Specifically, when a microorganism such as Escherichia coli or Bacillus subtilis is used as a host, it preferably comprises a promoter, a ribosome binding sequence, an hHGF gene, a transcription termination factor, and a gene that controls the promoter. Examples of the promoter include tryptophan synthase operon (trp), lactose operon (lac), lipoprotein promoter (lpp), and the like.
Hybrid promoters such as tac (trp: lac), trc (trp: lac), pac (phage: E. coli), etc. may be used.
The hHGF gene is preferably one in which the portion corresponding to the signal sequence has been removed, but hHGF can be obtained by removing the signal sequence from the pre-body produced even if it contains the portion corresponding to the signal sequence. As a plasmid to be used, a plasmid having a large copy number in Escherichia coli or Bacillus subtilis, for example, pBR 322 series plasmid, pUB 110 series plasmid or the like is desirable. Escherichia coli, Bacillus subtilis, etc. transformed by a usual method can be used for 15-42
It may be cultured at ℃. When yeast is used as a host, a yeast-derived promoter such as pyruvate kinase (pYK),
Under the control of sequences such as phosphoglycerokinase (pGK), hHG
The F gene may be connected, introduced into yeast, and cultured at about 30 ° C.

However, considering that natural hHGF is a glycoprotein, an animal cell is preferable as a host. In addition, when an animal cell is used as a host, it is expected that the introduction of the hHGF gene containing a portion corresponding to the signal sequence will produce secreted hHGF without the signal sequence. As the signal sequence, a signal sequence of human serum albumin, interferon, human amylase, etc. may be used in addition to the original signal sequence of hHGF. The DNA fragment of the nucleotide sequence corresponding to the signal sequence of 1 may be substituted on the 5'side. When an animal cell is used as a host, the promoter includes SV40 late promoter, apolipoprotein E gene promoter, apolipoprotein A1 gene promoter,
Examples thereof include a heat shock protein gene promoter, a metallothionein gene promoter, an HSVTK promoter, an adenovirus promoter, and a retrovirus LTR, and the SV40 promoter and the metallothionein gene promoter are preferable. Expression vectors include:
A polyadenylation site is included downstream of the hHGF gene.
Specific examples of polyadenylation sites include SV40 DNA, β
-The one derived from the globin gene or the metallothionein gene.

Also, the polyadenylation site of β-globin gene and the polyadenylation site of SV40 DNA may be linked. The expression vector may have a transformant selection marker. Even without a selectable marker in the expression vector, transformed animal cells can be selected by double transformation. Such selectable markers include the DHFR gene, which confers methotrexate resistance,
Herpes simplex virus (HSV) tk ^- gene, 3'-, which enables selection of transformed tk strains in HAT medium
Examples include the aminoglycoside 3'-phosphotransferase gene from E. coli transposon Tn5, which confers resistance to the deoxystreptamine antibiotic G418, the bovine papillomavirus gene by multilayered growth, and the aprt gene. In addition, in order to select animal cells transformed with an expression vector by the double transformation method, a plasmid or other DNA containing a gene serving as the above-mentioned selection marker is transformed with the expression vector, and the selection marker Transformed cells can be selected by the expression phenotype described above.

It is advantageous that the expression vector has a plasmid fragment having a replication origin derived from bacteria such as Escherichia coli because it can be cloned in bacteria. Examples of such a plasmid fragment include plasmid fragments such as pBR322, pBR327 and pML. Specific examples of the plasmid vector used for the expression vector include SV40 early promoter, splice sequence DNA derived from rabbit β-globin gene, polyadenylation site from rabbit β-globin gene, polyadenylation from SV40 early region. Site and origin of replication from pBR322 and pKCR containing ampicillin resistance gene, pBR322 part of pKCR was replaced with pBR327, and Eco R1 site existing in exon 3 of rabbit β-globin gene was replaced with Hind III site. Examples include pBPV MT1 containing altered pKCR H2, BPV gene and metallothionein gene.

Animal cells transformed with the expression vector include CHO cells, COS cells, mouse L cells and mouse C cells.
127 cells, mouse FM3A cells and the like. The transfer of the expression vector into animal cells is performed by a transfection method, a microinjection method, etc. Among them, the calcium phosphate method is the most common. The culture of the animal cells transformed by the transfer can be performed by suspension culture or adherent culture by a conventional method. As the medium, MEM, RPMI 1640, etc. are generally used.

Separation and purification of the produced hHGF can be carried out by column chromatography using heparin / sepharose, hydroxyapatite or the like, as in the case of purification from plasma of patients with fulminant hepatitis.

[0018]

The gene encoding hHGF according to the present invention is introduced into an expression vector by a conventional method,
By using this as a template, hHGF or an hHGF-like substance or a fusion protein containing the same can be obtained. The obtained recombinant hHGF, hHGF-like substance or fusion protein containing hHGF may be a therapeutic agent for liver diseases such as liver regeneration promoter, liver function improving agent, hepatitis therapeutic agent or liver cirrhosis inhibitor.

[0019]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the scope of the invention. Example 1 [1] Determination of partial amino acid sequence of hHGF and preparation of probe From plasma of patients with fulminant hepatitis, J. Clin. Invest., 81, 414 (19
HHGF was purified according to the method described in 88). When subjected to SDS-PAGE, a slightly broad single band appeared at the position of molecular weight 76000-92000 under non-reducing conditions, and a slightly broad band with molecular weight 56000-65000 and molecular weight 32000-35000 under reducing conditions. Two bands appeared. 50 μg of this purified hHGF was dissolved in 100 μl of 50 mM Tris-HCl buffer at pH 9 containing 5 molar urea, and achromobacter protease I corresponding to 1/200 in molar ratio to hHGF was added thereto. In addition, the mixture was reacted at 37 ° C for 6 hours. The resulting peptide mixture was subjected to reductive carboxymethylation by a conventional method, and then J.
Each peptide was separated by reverse phase high performance liquid chromatography using Bakerbond WP Octyl Column manufactured by T. Baker.

When the six peptides were subjected to amino acid sequence analysis using a gas phase protein sequencer (Model 470A, Applied Biosystems), the sequences shown in Table 1 were found.

[Table 1]

[2] Screening of cDNA Encoding Part of hHGF (1) Plaque Hybridization Screening of 34-week-old human placenta-derived cDNA (Clontech) as a λ phage cDNA library for screening Went according to. Infect E. coli strain Y-1090 with 1 million clones of phage. 24.
NZY soft agar in a 5 cm x 24.5 cm dish [NZY medium; 1% NZ-amine, 0.5% yeast extract, 0.
5% sodium chloride, adjusted to pH 7.5 and 0.25% magnesium chloride added, NZY soft agar medium; 0.7% in NZY medium
Were autoclaved by adding agar so that the amount of each of them became 200,000, and 5 sheets were cultured overnight at 42 ° C at a rate of 200,000 clones per sheet.

Next, the λ phage clone in the medium was transferred onto a commercially available nylon membrane, Gene Screening Plus (DuPont), and subjected to plaque hybridization as described below. That is, phage particles were transferred at a ratio of 2 nylon membranes per 1 dish, and the nylon membrane thus prepared was washed with 0.1M sodium hydroxide-
Soaked with 1.5M sodium chloride, let it sit on the paper for 2 minutes, and remove the water on a separate dry filter paper. Then, in the same manner, use 2 x SSCP (2 times the concentration of SSCP solution, and so on. Take the notation: 10 x SSCP; 1.2M sodium chloride,
150mM sodium citrate, 130mM potassium dihydrogen phosphate, 1mM EDTA pH 7.2) -0.2M Tris-hydrochloric acid (pH 7.4) This nylon membrane was allowed to stand on a filter paper soaked and air-dried on a dry filter paper. The operation was repeated again. Nylon membranes treated in this way were 3 x SSC (20x concentrated SSC solution; 3M sodium chloride, 0.3M sodium citrate) -0.
Wash twice with 1% SDS for 15 minutes at 60 ℃, then add 5 ml of prehybridization solution [3 x SSC,
0.1% SDS, 10 × Denhardt's (50 times the concentration of Denhardt's solution; 1% BSA (bovine serum albumin) 1% polyvinylpyrrolidone, and 1% Ficoll 400), 20 μg / ml salmon sperm DNA]
It was immersed in the solution at 65 ° C for 3 hours.

Next, peptide 4 in Table 1, namely Asn-M
et-Glu-Asp-Leu-His-Arg-His-Ile-Phe-Trp-Glu-Pro-Asp
-Asn-Met-Glu-Asp-Leu-His of Ala-Ser-Lys and
A synthetic oligonucleotide was prepared based on His-Ile-Phe-Trp-Glu-Pro. That is, 17 bases in the order of the amino acid sequence described above.
64 types of TH 23 (5'-TGT / C / A / GAA / GA / GTCT / CT
-CCATA / GTT-3 '), 17 bases 24 types of TH 24 (5'-GG
-T / CTCCCAA / GAAA / G / TATA / GTG-3 ') was created. The 5'ends of these were reacted with polynucleotide kinase according to a conventional method in a reaction solution [50 mM Tris-HCl pH 7.6
, 10 mM magnesium chloride, 10 mM mercaptoethanol, 100 μM (γ ³² P) ATP, substrate DNA] for ³² P labeling, and then a DEAE cellulose column was applied according to a conventional method to remove extra mononucleotides. The result is ³²
Hybridization solution containing P-labeled synthetic oligonucleotide probe [3 x SSC, 10 x Denhardt's, 50 μg / ml
Salmon sperm DNA, 1M sodium chloride, 1% SDS, 250 μg / ml salmon sperm DNA, 100,000 cpm / ml ³² P per synthetic probe
Labeled probe DNA] In the above-mentioned filter, A or T was changed to 2 ° C and G or C was changed to 4 ° C depending on the probe, and the total temperature of all the bases was actually 42 ° C depending on the probe.
(TH23) Incubated at 46 ℃ (TH24) for 36 hours. Then, take out the nylon membrane, wash it twice in 4 × SSC solution at room temperature for 30 minutes, and then wash it in 4 × SSC solution at the same temperature as hybridization for 30 minutes twice and then in 2 × SSC solution for 15 minutes at room temperature. It was washed twice and autoradiographed.

There were 6 cases where the signals on the autoradiography of one set of 2 sheets of nylon membranes matched. To isolate the clones corresponding to the signals obtained, plaques on soft agar that correspond to these signals were punched out with glass tubes in the presence of 50 μl chloroform and 1 ml T.
Phage particles were extracted overnight in MG buffer [50 mM Tris-hydrochloric acid pH 7.5, 100 mM sodium chloride, 10 mM magnesium chloride and 0.01% gelatin], infected again with Escherichia coli Y-1090 strain, and cultured in an appropriate amount in a 9 cm dish. Plaque hybridization was performed as described above. By repeating this series of operations, each clone corresponding to the signal could be isolated. As a result, 6 independent clones were obtained. Two of them, λ hHGF
Regarding 21 and λ hHGF 502, the nucleotide sequences of the contained cDNAs were analyzed.

(2) Subcloning of cDNA fragment and determination of nucleotide sequence DNA was extracted from these λ phage clones as follows and subcloned into plasmid vectors pUC18, pUC19 and single-stranded phage vectors M13mp18, M13mp19. That is, 200 ml N in a 500 ml Erlenmeyer flask.
In ZY medium, λ phage clone 2 × 10 ⁷ pfu (pfu; plaque forming unit) suspended in 200 μl of TMG solution and 40 μl of Escherichia coli Y-1090 strain 2 × 10 ⁸ were incubated at 37 ° C.
It was infected by leaving it for 15 minutes. 15 minutes later 1 ml 1 more
M calcium chloride was added, and the mixture was cultured overnight for about 14 hours. Next, add 2 ml of chloroform and leave for about 10 minutes,
Add 15.8 g of sodium chloride and melt, and mix them at 4 ℃
At Hitachi Cooled Centrifuge SCR20BB, rotor RPR 9-2
20 g of polyethylene glycol 6000 was added to the supernatant after centrifugation at 6000 rpm for 20 minutes, and the mixture was sufficiently dissolved and then allowed to stand on ice for 1 hour.

This was centrifuged at 6000 rpm for 20 minutes in a rotor RPR 9-2 with a Hitachi cooling centrifuge SCR 20BB to precipitate 6 ml of the precipitate.
A buffer [0.5% NP 40, 36 mM calcium chloride, 30 mM Tris-hydrochloric acid pH 7.5 50 mM magnesium chloride, 125 mM potassium chloride, 0.5 mM EDTA, 0.25% deoxycholic acid, 0.6 m
M mercaptoethanol] and suspend it here in 100 μl of 10
mg / ml of Deoxyribonuclease I and 10 μl of 10 mg /
Nucleic acid derived from Escherichia coli was decomposed by adding ml of ribonuclease A and incubating at 30 ° C. for 30 minutes. Then, an equal amount of chloroform was added to the above reaction solution, and the mixture was stirred well and then centrifuged at 3000 rpm for 10 minutes with a Tommy centrifuge LC-06 and a rotor TS-7 to obtain a supernatant. Meanwhile, Hitachi Ultracentrifuge Rotor RPS
Centrifuge tubes for 40T in 40% glycerol solution [0.5% NP 40, 30
mM Tris-HCl pH 7.5, 125 mM potassium chloride, 0.5 mM
Put 1 ml of EDTA, 0.6 mM mercaptoethanol, 10% glycerol] and add 3 ml of 10% glycerol solution [0.5% NP 40, 30 mM Tris-HCl pH 7.5, 125 mM
Layered with potassium chloride, 0.5 mM EDTA, 0.6 mM mercaptoethanol, 40% glycerol] and layered with the nuclease-treated phage suspension described above, Hitachi Ultracentrifuge 70P 72, rotor RPS 35,000 at 40T
Spin for 1 hour.

After centrifugation, the phage particles that had fallen as a precipitate were collected in 0.4 ml of 40 mM Tris-hydrochloric acid pH 7.5, 10 mM EDTA, 2% S.
Suspend in DS and add 4 μL of 10 mg / ml proteinase K
Incubation was carried out at 55 ° C for 1 hour. Then, the solution was transferred to an Eppendorf tube, and the phage DNA was extracted with an equal amount of phenol / chloroform and ethanol precipitation was performed to obtain 200 μg of the desired phage DNA.
The phage DNA was digested with a restriction enzyme EcoRI according to a conventional method and analyzed by agarose electrophoresis. As a result, three EcoRIs of 0.2 kb, 0.85 kb and 0.72 kb from clone λ hHGF 21 were cloned.
A fragment was obtained. On the other hand, the insert from agarose gel cDN
The target cDN is obtained by recovering the A fragment according to a conventional method.
We were able to obtain the A fragment. Reaction solution of plasmid vector pUC18, pUC19 and single-stranded phage vector M13mp18, M13mp19 200 ng and 10 μl, in which 100 ng of these cDNA fragments were previously cleaved with restriction enzyme EcoRI according to a conventional method.
[66 mM Tris-HCl pH 7.6, 6.6 mM magnesium chloride, 1
[0 mM dithiothreitol, 66 μM ATP, substrate DNA] is bound by the unit T4 DNA ligase and transformed into host Escherichia coli corresponding to each vector according to a conventional method to have a partial sequence of HGF protein at the EcoRI insertion site. Subclones were obtained.

The nucleotide sequence of the obtained cDNA subclone was determined by the dideoxy method of Sanger et al. The primers used corresponded to the commercially available M13 phage vector. As a result, clone λ with the longest cDNA
Translation of the nucleotide sequence of hHGF 21 into amino acids results in Figs. 1 and 2.
It was found that the clones contained some of the amino acid sequences that had already been clarified as shown in Fig. 5 and were in a region different from the amino acid sequence used for the probe design. It was found to be a cDNA containing the region. Also, λ hHG
Of clone λ hHGF 502 containing a cDNA fragment not present in F21
As a result of analyzing the nucleotide sequence of the cDNA according to the Sanger method, clone λ hHGF 502 has the same nucleotide sequence as clone λ hHGF 21 as shown in FIGS. It shares a length of 0.8 kb up to the cleavage site and is not present in λ hHGF 21 on the 3'side.
It was found to have a base sequence of 0.7 kb. λ hHGF 502
It was found that among the nucleotide sequences of λ hHGF 21 among the nucleotide sequences of, there is a nucleotide sequence corresponding to the amino acid sequence that has already been analyzed. It was found that the nucleotide sequences of these two clones were joined together in a partially overlapping manner to cover the entire amino acid sequence of hHGF.

[Brief description of drawings]

FIG. 1 is the first of the entire amino acid sequences of hHGF of the present invention.
The 360th amino acid (Met) to the 360th amino acid (Ser)
It is a figure showing the arrangement to. The underline in the figure indicates the amino acid sequence that has already been clarified.

FIG. 2 shows the 36th amino acid sequence of the entire hHGF amino acid sequence of the present invention.
From the 1st amino acid (Glu) to the 728th amino acid (Se
It is a figure showing the arrangement up to r). The underline in the figure is the same as in FIG.

FIG. 3 is a diagram showing the sequence from the 1st base to the 780th base in the entire base sequence of the cDNA containing the gene encoding hHGF of the present invention obtained in Example 1. The recognition sites for major restriction enzymes are also shown in the figure. The underline indicates the portion corresponding to the amino acid sequence that has already been clarified.

FIG. 4 is a diagram showing the sequence from the 781st base to the 1620th base in the entire base sequence of the cDNA containing the gene encoding hHGF of the present invention obtained in Example 1. The recognition sites for major restriction enzymes are also shown in the figure. The underline in the figure is the same as in FIG. 3, and the double underline represents the nucleotide sequence corresponding to the probe used to obtain the first clone.

FIG. 5 is a diagram showing the sequence from the 1621st base to the 2187th base in the entire base sequence of the cDNA containing the gene encoding hHGF of the present invention obtained in Example 1. The recognition sites for major restriction enzymes are also shown in the figure. The underline is the same as in FIG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 15/09 ZNA C12N 5/00 B C12P 21/02 9282-4B 15/00 ZNAA (C12N 1 / 21 C12R 1:19) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1:91) (72) Inventor Naka Daichi Kamoshida, Midori-ku, Yokohama-shi, Kanagawa Prefecture 1000 Sancho Kasei Co., Ltd. Research Institute (72) Inventor Kazuhashi Takahashi 1000 Kamoshida-cho, Midori-ku, Yokohama-shi, Kanagawa Sankyo Kasei Research Institute

Claims (3)

[Claims] 1. A hepatocyte growth factor characterized by being represented by the following amino acid sequence: Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu 1 5 10 15 Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30 Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45 Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60 Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu 65 70 75 80 Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95 Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120 125 Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135 140 Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His 145 150 155 160 Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr 165 170 175 Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser 180 185 190 Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu 195 200 205 Val Glu Cys Met Thr Cy s Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp 210 215 220 His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro 225 230 235 240 His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp 245 250 255 Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr 260 265 270 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys 275 280 285 Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu 290 295 300 Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile 305 310 315 320 Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu 325 330 335 His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350 Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355 360 365 Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp 370 375 380 Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met 385 390 395 400 Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp 405 410 415 Lys Asn Met Glu Asp Le u His Arg His Ile Phe Trp Glu Pro Asp Ala 420 425 430 Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His 435 440 445 Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys 450 455 460 Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu 465 470 475 480 Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val 485 490 495 Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg 500 505 510 Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp 515 520 525 Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr 530 535 540 Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys 545 550 555 560 Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly 565 570 575 Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp 580 585 590 Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu 595 600 605 Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn 610 615 620 Tyr Asp Gly Leu Leu Arg Va l Ala His Leu Tyr Ile Met Gly Asn Glu 625 630 635 640 Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu 645 650 655 Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp 660 665 670 Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu 675 680 685 Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly 690 695 700 Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile 705 710 715 720 Leu Thr Tyr Lys Val Pro Gln Ser 725 2. A hepatocyte growth factor characterized by being represented by the sequence from glutamic acid at position 30 to serine at position 728 in the amino acid sequence of claim 1. 3. A hepatocyte growth factor, which is represented by the sequence from glutamine at position 32 to serine at position 728 in the amino acid sequence of claim 1.