JP2577091B2 - Hepatocyte growth factor and gene encoding the same - Google Patents

Description

The present invention relates to a hepatocyte growth factor obtained by genetic recombination and a gene encoding the same.

(Prior art) The liver is the most highly differentiated and largest organ in a living body. This is mainly due to various nutrients (sugar, protein,
Processing (metabolism), storage,
It has various important functions such as detoxification, decomposition, and excretion, and is known to play a central role in intermediary metabolism in vivo. 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, it is known that even if approximately 2/3 of the liver of a rat is resected, it will return to its original size after about 10 days, and even in humans with liver cancer, treatment by partial hepatectomy and subsequent regeneration The law is taking place. 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 have found that hepatic parenchymal cell proliferation activity is extremely high in human fulminant hepatitis plasma (Biomed. Re.
s., 6,231 (1958) and Exp. Cell. Res. 166,139 (198
6)) and succeeded in purifying the factor having the activity as a single protein for the first time in the world (JP-A-63-22).
No. 526 and J. Clin. Invest., 81, 414 (1988)).

This human hepatocyte growth factor (hereinafter abbreviated as "hHGF") has an estimated molecular weight of about 76000-92 by SDS-PAGE under non-reducing conditions.
000, and a molecular weight of 56000-6 by SDS-PAGE under reducing conditions.
5000 and 32000-35000. Nakamura et al. Reported a factor with similar activity derived from rat platelets (Biochem. Biophys. Res. Commun. 122, 1450
(1984)), the estimated molecular weight was about 270 by SDS-PAGE.
00, (Proc. Natl. Acad. Sci. USA, 83, 6
489 (1986)), which was subsequently purified as a single protein and reported to be a protein with a molecular weight of 82,000 consisting of two polypeptides with molecular weights of 69000 and 34000 (FEBS
Letters, 224, 311 (1987)). These hHGF and rat
Other than HGF, no hepatocyte growth factor purified as a single protein has been reported so far, and no report has been made on hHGF or rat HGF with respect to its primary structure or the corresponding nucleotide sequence of cDNA. Not.

(Problems to be solved by the invention) To examine the detailed function of hHGF in the living body or the effect on liver regeneration in the case of liver injury in vitro, a large amount of hHGF is required. HH
Purification of GF is not always easy from a human, time, and cost point of view, and it is extremely difficult to stably extract only hHGF from plasma containing an infectious source. For that reason
Stable and large-scale purification of hHGF from plasma of fulminant hepatitis patients has not been performed.

(Means for Solving the Problems) Therefore, the present inventors have conducted various studies to obtain a large amount of hHGF by recombinant DNA technology, and as a result, have cloned a gene encoding hHGF useful for this purpose for the first time. And succeeded in completing the present invention. That is,
The gist of the present invention is that hHGF containing the signal sequence represented by the amino acid sequence shown in FIG. 1 and glutamic acid residue (Glu) at the 30th position in the amino acid alignment shown in FIG. HHGF represented by the sequence up to), glutamine (Gln) at position 32 in the amino acid sequence shown in FIG.
HHG expressed in parallel from to the last serine residue (Ser)
F, a gene encoding each hHGF represented by the amino acid sequence, a gene encoding hHGF including a signal sequence represented by the nucleotide sequence shown in FIG. 2, and the 88th nucleotide in the nucleotide sequence shown in FIG. It is present in the gene represented by the sequence from G to the last G and the gene represented by the sequence from the 94th C to the last G in the base sequence shown in FIG.

Hereinafter, the present invention will be described. A gene (cDNA) encoding hHGF of the present invention has, for example, a nucleotide sequence as shown in FIG. In addition, other complementary base sequences were omitted from the base sequence, and only a single strand was described. Recombinant DNA from this gene
HH having the amino acid sequence shown in FIG.
GF can be expressed. At this time, the protein translated from the mRNA encoding hHGF contains a signal sequence, but when secreted from cells, the signal sequence is cleaved, resulting in a glutamic acid residue (Glu) at position 30 or a glutamine at position 32. HHG having amino acid sequence after residue (Glu)
F is produced. A signal sequence of another protein can be used as the signal sequence. When mature hHGF without a signal sequence is expressed in a host cell, 88% of the nucleotide sequence shown in FIG.
A gene having a nucleotide sequence subsequent to the Gth or 94th C may be used by connecting it to the ATG codon of the vector. Furthermore, in the present invention, a modification in which some amino acids or nucleic acids are removed, changed, or added within a range that does not impair the hepatic parenchymal cell proliferation activity is also included in the present invention.

The DNA fragment of the gene encoding hHGF of the present invention can be obtained, for example, by the following method. HHGF purified from plasma of a patient with fulminant hepatitis by, for example, the method described in J. Clin. Invest. 81, 414 (1988), is disulfide bond-cleaved under reducing conditions to be separated into two polypeptides. Polypeptide with a molecular weight of 56000-65000 is converted to H chain, molecular weight
The 32000-35000 polypeptide is designated as the light chain. After carboxymethylation of the thiol group of the cysteine residue generated by reduction treatment of hHGF, the H-chain and L-chain are separated by reversed-phase high-performance liquid chromatography or hHGF is electrophoresed under reducing conditions, and the After extracting each of the H chain and the L chain from, the amino terminal amino acid sequence of both chains can be examined by, for example, analyzing with a gas phase protein sequencer manufactured by Applied Biosystems. In addition, hHGF itself or after separation of the heavy and light chains, is subjected to appropriate proteolytic oxygen, such as Achromobacter protease I.
(Lysyl endopeptidase), the resulting peptide fragments are separated by, for example, reverse-phase high performance liquid chromatography, and the amino acid sequence inside the polypeptide can be known by analyzing the amino acid sequence of each peptide in the same manner as described above. . From these amino acid sequences, the DNA base sequence is deduced to select a sequence from which an oligonucleotide can be easily prepared, and the oligonucleotide, for example, an oligonucleotide as described in Examples described later is synthesized and used as a probe. As a cDNA library for screening a gene encoding hHGF, a human-derived liver cDNA library, a spleen cDNA library, a placenta cDNA library, and the like can be used. These libraries are sold by Clonetech. In particular, a placenta cDNA library is desirable. Alternatively, a cDNA library may be prepared from a cell line expressing hHGF and a tissue material according to a conventional method. The λ phage into which such cDNA was incorporated
niatis et al. (“Molecular Cloning”, Cold Spring Harbor Laboratory, pp. 56-73 (19
82)) Infect E. coli and culture. Plaque hybridization method ("Molecular cloning", Cold Spring Harbor Laboratory, pp. 320-328 (1982)) using the oligonucleotide formed from the nucleotide sequence deduced from the partial amino acid sequence of hHGF as a probe to form the plaque The number of different λ phage clones having the same amino acid sequence as a part of the target amino acid sequence of hHGF and also having a nucleotide sequence corresponding to a region other than the probe of the amino acid sequence of hHGF can be easily determined by selecting Or you can get. Further, phages were grown from the screening positive plaques by the method of Maniatis et al. ("Molecular Cloning", Cold Spring Harbor Laboratory, pp. 76-79 (1982)), and DNA was purified therefrom according to the glycerol gradient method. After digestion with an appropriate restriction oxygen such as EcoRI, the cDNA is subcloned into a plasmid vector such as pUC18 or pUC19 or a single-stranded phage vector such as M13mp18 or M13mp19, and the dideoxy method of Sanger et al. (Proceedings of National Academy)・ Science USA (Pro
cad. Natl. Acad. Sci. USA) 74 5463 (1977)) to determine the nucleotide sequence of the target cDNA segment.
By analyzing the nucleotide sequences of the obtained clones and integrating them (FIG. 3), a gene corresponding to all of the entire amino acid sequence of hHGF shown in FIG. 1 was obtained by a group of cDNA clones encoding a part of hHGF. Obtainable. Expression of the cDNA thus obtained can be performed, for example, by connecting the DNAs in a form in which the base sequence is in accordance with the amino acid sequence of hHGF.
A DNA fragment containing the entire region of hHGF was converted to a translation initiation codon AT downstream of a plasmid promoter such as pCDL-SRα296.
G and phase can be connected together to form a protein expression plasmid, which can be carried out in a host of an animal cell transformed with the plasmid. Next, the HGF of the present invention can be obtained by recovering the expressed protein according to a conventional method.

For the expression plasmid, it is desirable to construct a stable host-vector system for industrial production. For example, those described in Japanese Patent Application No. 1-115831 can be mentioned. Specifically, when a microorganism such as Escherichia coli or Bacillus subtilis is used as a host, it is preferably composed of 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), and lipoprotein promoter (lpp).
lac), trc (trp: lac), pac (phage: E. coli) and the like. The hHGF gene preferably has a portion corresponding to the signal sequence removed, but hHGF can be obtained by removing the signal sequence from the preform produced even if the portion includes the portion corresponding to the signal sequence. As a plasmid to be used, a plasmid having a high copy number in E. coli or Bacillus subtilis, for example, pBR3
A 22 type plasmid, a pUB110 type plasmid and the like are desirable. Escherichia coli and Bacillus subtilis transformed by ordinary methods
What is necessary is just to culture | cultivate at 15-42 degreeC using a normal medium. When yeast is used as a host, yeast-derived promoters such as pyruvate kinase (pYK), phosphoglycerokinase (p
The hHGF gene may be connected under the control of a sequence such as GK), introduced into yeast, and cultured at about 30 ° C.

However, considering that natural hHGF is a glycoprotein, animal cells are desirable as hosts. When an animal cell is used as a host, the introduction of the hHGF gene containing a portion corresponding to the signal sequence is expected to have an advantage that hHGF from which the signal sequence is removed is secreted and produced. In addition to the original signal sequence of hHGF, the signal sequence of human serum albumin, interferon, human
A signal sequence such as amylase may be used. In such a case, a DNA fragment having a base sequence corresponding to the signal sequence may be substituted on the 5 'side in place of the DNA fragment encoding the original signal sequence. When an animal cell is used as a host, the promoter includes an SV40 late promoter, an apolipoprotein E gene promoter, an apolipoprotein A1 gene promoter, a heat shock protein gene promoter, a metallothionein gene promoter, an HSVTK promoter, an edenovirus promoter, and a retrovirus. Examples include the viral LTR, and the SV40 promoter and the metallothionein gene promoter are preferred. The expression vector contains a polyadenylation site downstream of the hHGF gene. Specific examples of the polyadenylation site include those derived from SV40 DNA, β-globin gene or metallothionein gene. In addition, the polyadenylation site of the β-globin gene and SV40
The polyadenylation site of 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. Examples of such selectable markers include the DHFR gene that confers methotrexate resistance, the tk gene of herpes simplex virus (HSV) that enables selection of transformed tk ^- strains in HAT medium, and 3'-deoxystreptamine antibiotic. Examples include the aminoglycoside 3'-phosphotransferase gene from Escherichia coli transposon Tn5 that confers resistance to G418, the bovine papillomau virus gene by a multilayer growth, and the aprt gene. Further, in order to select animal cells transformed with the expression vector by the double transformation method, a plasmid or other DNA containing the above-described gene serving as a selection marker is transformed together with the expression vector, and Transformed cells can be selected according to the phenotype described above by expression. When the expression vector has a plasmid fragment having a replication origin derived from bacteria such as Escherichia coli, cloning in bacteria can be advantageously performed. Examples of such a plasmid fragment include plasmid fragments such as pBR322, pBR327, and pML. Specific examples of the plasmid vector used in the expression vector include the SV40 early promoter, a splice sequence DNA derived from the rabbit β-globin gene,
Polyadenylation site from rabbit β-globin gene, polyadenylation site from SV40 early region and pBR3
PKCR containing the replication origin from 22 and the ampicillin resistance gene, the pBR322 part of pKCR was replaced with pBR327, and EcoR1 present in exon 3 of the rabbit β-globin gene
PKCR H2 in which the site is changed to a Hind III site, pBPVMT1 containing a BPV gene and a metallothionein gene, and the like. Animal cells transformed with the expression vector include CHO cells, COS cells, mouse L cells, mouse C127 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, RP
MI1640 and the like are common.

Separation and purification of the produced hHGF can be performed by column chromatography using heparin / sepharose, hydroxyapatite, or the like, similarly to the purification from fulminant hepatitis patient plasma.

(Effect of the Invention) The gene encoding hHGF according to the present invention can be introduced into an expression vector by a conventional method, and by using this as a template, hHGF or an hHGF-like substance or a fusion protein containing the same can be obtained. Obtained recombinant hHGF, hHG
F-like substance or fusion protein containing hHGF is a liver regeneration promoter,
It may be a therapeutic agent for liver patients such as a liver function improving agent, hepatitis therapeutic agent or cirrhosis inhibitor.

(Examples) Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples as long as the gist of the present invention is not exceeded.

Example 1 [1] Determination of partial amino acid sequence of hHGF and preparation of probe J. Clin. Invest., 81, 414 (198)
HHGF was purified according to the method described in 8). This is S
When subjected to DS-PAGE, under non-reducing conditions, the molecular weight was 76.
A slightly broad single band appears at the position of 000-92000,
Under reducing conditions, two bands appeared, a slightly broad band with a molecular weight of 56000-65000 and a band with a molecular weight of 32000-35000. 50 μg of this purified hHGF was dissolved in 100 μl of 50 mM Tris-HCl buffer (pH 9) containing 5 molar urea, to which was added Achromobacter protease I corresponding to a molar ratio of 1/200 with respect to hHGF. The reaction was performed at 37 ° C. for 6 hours. The resulting peptide mixture was subjected to reductive carboxymethylation by an ordinary method, and then JTBaker's Bakerbond
Separation was performed by reversed-phase high-performance liquid chromatography using a WP Octyl Column to separate each peptide. Gas phase protein sequencer for 6 peptides (Appl
Amino acid sequence analysis was performed using ied Biosystems (Mode1470A), and the sequence shown in Table 1 was found.

Table 1 Amino acid sequence of peptide No. Sequence 1.PheLeuProGluArgTyr-RroAspLys 2.GluPheGlyHisGluPhe-AspLeuTyrGluAsnLys 3.AspTyrGluAlaTrpLeu-GlyIleHisAspValHis-GlyArgGl
yAspXXXLys 4.AsnMetGluAspLeuHis-ArgHisIlePheTrpGlu-ProAspAl
aSerLys 5.ArgArgAsnThrIleHis-GluPheLys 6.IleAspProAlaLeuLysXXX is an undetermined amino acid [2] Screening of cDNA encoding a part of hHGF (1) Plaque hybridization Performing screening As a λ phage cDNA library derived from a 34-week-old human placenta cDNA (Clonetech)
Was screened according to the manufacturer's instructions. Infecting E. coli strain Y-1090 with 1 million clones of phage
NZY soft agar medium in a 4.5 m petri dish [NZY medium; 1% NZ-amine, 0.5% yeast extract, 0.5% sodium chloride, adjusted to pH 7.5 and added with 0.25% magnesium chloride, NZY soft agar medium ; NZY medium with 0.7% agar and autoclaved]
Five clones were cultured overnight at 42 ° C. at a ratio of 10,000 clones. Next, the λ phage clone in the medium was transferred onto Gene Screening Plus (DuPont) which is a commercially available nylon membrane, and plaque hybridization described below was performed. That is, the phage particles were transferred at a rate of two nylon membranes per petri dish, and the nylon membranes thus formed were soaked with 0.1 M sodium hydroxide-1.5 M sodium chloride, and allowed to stand on paper for two times to prepare separately. After removing the moisture on the dried filter paper, 2xSSGP (2x SSGP)
SSCP solution, the same notation is used hereafter. 10x SSC
P; 1.2 M sodium chloride, 150 mM sodium citrate, 130
This nylon membrane is allowed to stand on a filter paper impregnated with mM potassium dihydrogen phosphate, 1 mM EDTA pH 7.2) -0.2 M Tris-hydrochloric acid (pH 7.4), air-dried on a dry filter paper, and the same operation is repeated again. Was. The nylon membrane treated in this way is 3xSSC (2
Washed twice with 0% concentration SSC solution; 3M sodium chloride, 0.3M sodium citrate) -0.1% SDS at 60 ° C. for 15 minutes, and then 5 ml of prehybridization solution [3X SSC , 0.1% SDS.10X Denhardt's (50 times the concentration of D
enhardt's solution; 1% BSA (bovine serum albumin) 1% polyvinylpyrrolidone, and 1% Ficoll 400), 20 μg / ml
Salmon sperm DNA] at 65 ° C for 3 hours. Next, peptide 4 in Table 1, ie, Asn-Met-Glu-Asp-Leu-His-Arg
-His-Ilu-Phe-Trp-Glu-Pro-Asp-Ala-Ser-Lys
Of Asn-Met-Glu-Asp-Leu-His and His-Ile
A synthetic oligonucleotide was prepared based on -Phe-Trp-Glu-Pro. That is, TH23 (5'-TGT-T / C / AlG-AA / G-AlG-T) consisting of 64 types of 17 bases in the order of the amino acid sequence described above.
-CT / C-T-C-C-C-A-T-A / G-T-T-
3 ′), 17 bases and 24 kinds of TH24 (5′-GGTT / CT)
-CCCA-A / G-AA-A / G / T-A-TA-A / G
-TG-3 '). The 5'-end of these was reacted with a polynucleotide [5
mM Tris-HCl pH 7.6, 10 mM magnesium chloride, 10 mM mercaptoethanol, 100 μM [γ ³² P] ATP, substrate DNA]
After labeling with ³² P in the gel, a DEAE cellulose column was applied according to a conventional method to remove excess mononucleotides. A hybridization solution containing a ³² P-labeled synthetic oligonucleotide probe [3X SSC, 10X Denhard
t's, 50μg / ml salmon sperm DNA 1M sodium chloride, 1% SDS, 2
50 μg / ml salmon sperm DNA, 100,000 cp per type of synthetic probe
m./ml ³² P-labeled probe DNA], A or T was replaced with 2 ° C. and G or C was replaced with 4 ° C. according to the probe, and the total temperature of all bases was changed. (TH23) Incubated at 46 ° C (TH24) for 36 hours. Then, remove the nylon membrane, and in a 4X SSC solution at room temperature for 30 minutes.
2X for 30 minutes, 2X for 30 minutes at the same temperature as the hybridization with 4X SSC solution, then 15X with 2X SSC solution at room temperature.
Washed twice for 2 minutes and autolandography was performed.

The number of the signals on the autoradiography of the pair of two nylon membranes was six. In order to isolate clones corresponding to the obtained signals, plaques on soft agar plate corresponding to these signals were punched out with a glass tube, and 1 ml of TMG buffer [50 mM Tris-HCl pH 7.5, in the presence of 50 μl chloroform, was added. 100 mM sodium chloride, 10
Phage particles were extracted overnight in [mM magnesium chloride and 0.01% gelatin].
An appropriate amount of the cells was cultured in a Petri dish, and plaque hybridization was performed by the method described above. By repeating this series of operations, each clone corresponding to the signal could be isolated. As a result, six independent clones were obtained. Two clones, λhHGF21 and λhH
For GF50, the contained cDNA base sequence was 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 and pUC19 and single-stranded phage vectors M13mp18 and M13mp19. That is, λ phage clone 2 × 10 ⁷ pfu (pfu; plaque forming unit) suspended in 200 μl of TMG solution in 200 ml of NZY medium in a 500 ml Erlenmeyer flask
And 40 μl of Escherichia coli Y-1090 strain 2 × 10 ⁸ at 37 ° C. for 15 minutes for infection. After 15 minutes, 1 ml of 1M calcium chloride was further added, and the cells were cultured overnight, for about 14 hours. Next, 2 ml of chlorofilm was added and left for about 10 minutes, and 15.8 g of sodium chloride was added and dissolved, and the mixture was rotated at 4 ° C. with a Hitachi cooling centrifuge SCR20BB at 6000 rpm using a rotor RPR9-2.
20 g of polyethylene glycol 6000
Was added and dissolved sufficiently, and the mixture was allowed to stand in ice for 1 hour. This was centrifuged at 6000 rpm for 20 minutes in a rotor RPR9-2 using a Hitachi cooled centrifuge SCR20BB to precipitate 6 ml of A buffer [0.5%
NP40, 36 mM calcium chloride, 30 mM Tris-HCl pH 7.5
50mM magnesium chloride, 125mM potassium chloride, 0.5mMED
TA, 0.25% deoxycholic acid, 0.6 mM mercaptoethanol], 100 μl of 10 mg / ml deoxyribonuclease I and 10 μl of 10 mg / ml ribonuclease A are added thereto, and the mixture is incubated at 30 ° C. for 30 minutes. Was decomposed. Thereafter, an equal amount of chloroform was added to the above reaction solution, and the mixture was stirred well, and then, a Tommy centrifuge LC-0 was added.
6. Centrifugation was performed at 3000 rpm for 10 minutes in a rotor TS-7 to obtain a supernatant. On the other hand, 40 centrifuge tubes for the Hitachi Ultracentrifuge rotor RPS40T
% Glycerol solution [0.5% NP40, 30 mM Tris-HCl pH7
1 ml of 5,125 mM potassium chloride, 0.5 mM EDTA, 0.6 mM mercaptoethanol, 10% glycerol] is placed therein, and 3 ml of a 10% glycerol solution [0.5% NP40, 30 mM Tris-HCl pH 7.5, 125 mM potassium chloride, 0.5mMEDTA,
0.6mM mercaptoethanol, 40% glycerol] and a phage suspension treated with the nuclease as above, and a Hitachi ultracentrifuge 70P72, rotor R
Centrifuge at 35,000 rpm for 1 hour with PS40T. After centrifugation, the phage particles that have fallen out as precipitates are washed with 0.4 ml of 40 mM Tris-HCl pH7.
The suspension was suspended in 5, 10 mM EDTA and 2% SDS, and 4 μl of 10 mg / ml proteinase K was added thereto, followed by incubation at 55 ° C. for 1 hour. The solution was then transferred to an Eppendorf tube and an equal volume of phenol /
By extracting phage DNA with chloroform and performing ethanol precipitation, 200 μg of the target phage DNA could be obtained. This phage DNA was cleaved with a restriction enzyme EcoRI in a conventional manner, and analyzed by agarose electrophoresis.
As a result, 0.2kb, 0.85kb and 0.72Kb from clone λhHGF21
The three EcoRI fragments were obtained. On the other hand, the target cDNA fragment could be obtained by recovering the insert cDNA fragment from the agarose gel according to a conventional method. These cDNA fragments
100 ng of a plasmid vector pUC18, pUC19 and 200 ng of a single-chain phage vector M13mp18, M13mp19 previously cut with the restriction enzyme EcoRI in a usual manner and 10 μl of a reaction solution [66 mM Tris-HCl pH 7.6, 6.6 mM magnesium chloride,
In 10 mM dithiothreitol, 66 μMATP, substrate DNA], the E. coli host, which is compatible with each vector, is ligated with the unit T4 DNA ligase and transformed according to a conventional method, thereby obtaining a subsequence having a partial sequence of the HGF protein at the EcoR I insertion site. A clone was obtained.

The nucleotide sequence of the obtained cDNA subclone was determined by Sang
This was done by the dideoxy method of Er et al. Primers used corresponded to a commercially available M13 phage vector. As a result, when the nucleotide sequence of clone λhHGF21 having the longest cDNA was translated into amino acids, the amino acid sequence of a region different from the amino acid sequence used for probe design among the amino acid sequences already clarified as shown in FIG. And the clone was found to be a cDNA containing at least a region of hHGF. In addition, as a result of analyzing the cDNA base sequence of clone λhHGF502 containing a cDNA fragment not present in λhHGF21 according to the Sanger method, clone λhHGF502 was clone λhHGF21.
Fig. 2 shows the same nucleotide sequence as the restriction site Nco I
It was found to have a base sequence of 0.7 kb shared by the 0.8 kb length from the vicinity of the 3′-end to the vicinity of the third EcoRI cleavage site counted from the 5 ′ upstream, and a 3′-side non-existent λhHGF21 base sequence. λhHGF5
Among the base sequences of 02, it was found that among the base sequences not having λhHGF21, there was a base sequence corresponding to the already analyzed amino acid sequence. It was found that when the nucleotide sequences of these two clones were joined together in a partially overlapping manner, the entire amino acid sequence of hHGF was covered.

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of hHGF of the present invention. In the figure,
The underline indicates the amino acid sequence that has already been elucidated. FIG. 2 shows the nucleotide sequence of cDNA containing the gene encoding hHGF of the present invention obtained in Example 1. The main restriction oxygen recognition sites are also shown in the figure. The underline indicates the portion corresponding to the amino acid sequence already revealed, and the double underline indicates the amino acid sequence corresponding to the probe used to obtain the first clone.

Continuation of the front page (72) Inventor Kazunobu Takahashi 1000 Kamoshita-cho, Midori-ku, Yokohama-shi, Kanagawa Prefecture Mitsubishi Chemical Corporation Research Laboratory (56) References JP-A-63-22526 (JP, A) JP-A-3-3 130091 (JP, A) Clin, Invest. , Vol. 81 (1988) p. 414-419

Claims (6)

(57) [Claims] A gene encoding a hepatocyte growth factor represented by the following amino acid sequence: 2. A gene encoding a hepatocyte growth factor represented by the sequence from glutamic acid at position 30 to serine at position 728 of the amino acid sequence according to claim 1. 3. A gene encoding a hepatocyte growth factor represented by a sequence from glutamine 32 to serine 728 in the amino acid sequence according to claim 1. 4. A gene represented by the following nucleotide sequence, which encodes hepatocyte growth factor. 5. The nucleotide sequence according to claim 4, which is represented by a sequence from guanine 88 to guanine 2187.
A gene encoding hepatocyte growth factor. 6. The nucleotide sequence according to claim 4, which is represented by a sequence from cytosine at position 94 to guanine at position 2187.
A gene encoding hepatocyte growth factor.