JP3318323B2 - Recombinant hepatocyte growth factor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a polypeptide having a hepatocyte proliferation activity, and more particularly, to a polypeptide having in vitro activity.
The present invention relates to a novel polypeptide having a physiological activity enabling maintenance and proliferation of hepatocytes in (tro), a DNA encoding the polypeptide, a recombinant expression vector, a transformant, and a method for producing the polypeptide. . The polypeptide of the present invention is a liver parenchymal cell culture reagent, a liver regeneration promoter, a basic study of liver function, a study of the effects of various hormones and drugs on liver parenchymal cells, a study of liver carcinogenesis, and an antibody against the polypeptide. It can be expected to be used for clinical diagnostic reagents and therapeutic drugs for liver diseases.

[0002]

2. Description of the Related Art Conventionally, polypeptides having cell growth activity include epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve cell growth factor (NGF), platelet-derived growth factor (PDGF), Endothelial cell growth factor (ECGF) and the like are known. In addition to these cell growth factors, a polypeptide having hepatic parenchymal cell growth activity in vitro was partially purified from regenerated liver rat serum by Nakamura et al.
GF). Until the discovery of HGF, liver parenchymal cells did not show any proliferation of the cells even in the presence of mammalian serum in which various cell lines were actively growing, and usually fell off the wall of the culture vessel in about one week. As a result, long-term in vitro culture was not possible. However, hepatocytes grew very well in the presence of HGF, and the cells could be cultured (Biochem. Biophys. Res. Commun., 1) .
22 , 1450, 1984). Other researchers have suggested that this HGF
The activity was also confirmed to be present in the blood after partial hepatectomy and in the blood of fulminant hepatitis patients. After that, many researchers elucidated the purification method, chemical properties, and biological properties.However, it was not possible to identify the amino acid structure of this HGF or a polypeptide having hepatocyte proliferation activity similar to HGF. Did not. Under such circumstances, the present inventors have previously conducted studies by separating and purifying HGF from tissues such as rat platelets, and this platelet-derived HGF is composed of two types of subunits. It was found that hepatocytes proliferate very well in vitro and 27 amino acid sequences in a part of HGF were successfully identified (Japanese Patent Application No. 63-31866).
issue).

[0003]

Since in vivo HGF is a polypeptide secreted in a very small amount from organs such as liver, brain, lung, bone marrow, spleen, placenta and kidney, and blood cells such as platelets and leukocytes, Acquisition of raw material organization, HG
There are many problems such as the yield of F and stable supply. In order to use this HGF for hepatic parenchymal cell culture or hepatocyte research, its structure is clarified and HGF or a polypeptide having the same activity as HGF is supplied in large quantities by applying gene recombination technology. It is desired.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and as a result, have found that rat liver mRN
A, using an oligonucleotide synthesized based on the amino acid sequence of rat platelet-derived HGF from the cDNA library prepared from
Containing nucleotide sequence encoding long chain polypeptide
A was obtained. Further, using all or a part of the rat-derived cDNA as a probe,
It has been found that a cDNA containing a nucleotide sequence encoding a human HGF polypeptide can be obtained from a cDNA library prepared from human liver mRNA. It has also been found that human HGF cDNA can be obtained from cDNA libraries of organs other than human liver and blood cells. Furthermore, the present inventors have found that a transformant transformed by a recombinant expression vector containing the cDNA and that the transformant is cultured to express the human HGF gene, thereby completing the present invention. That is, the present invention provides a recombinant human hepatocyte growth factor, a DNA containing a nucleotide sequence encoding human hepatocyte growth factor, a recombinant expression vector capable of expressing the DNA, and a recombinant expression vector transformed with the recombinant expression vector. And a method of culturing the transformant and collecting and producing a recombinant human hepatocyte growth factor from the culture.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION A DNA encoding a human hepatocyte growth factor of the present invention, a recombinant expression vector, and a transformant are prepared, for example, as follows. That is,
(1) From animal tissues such as rat hepatocytes and rat megakaryocytes
RNA or chromosomal DNA is isolated and cD
To prepare an NA library or a chromosomal DNA library, and (2) use a synthetic oligonucleotide probe or an antibody to isolate an animal, for example, rat HGF cDNA or chromosomal DNA. A library or a chromosome library is screened, and a cDNA or chromosomal DNA of interest is extracted from the isolated clone, and a human organ or blood is probed using the cDNA of the animal or rat derived HGF or chromosomal DNA as a probe. A cDNA library prepared from mRNA of cells or the like is screened, and a cDNA of the target human-derived HGF is extracted from the isolated clone. In addition, an oligonucleotide synthesized based on the DNA sequence disclosed by the present invention or the amino acid sequence of human or animal HGF, or a human HGFcDN obtained by the present invention.
MRNA extracted directly from human organs or blood cells using A or human HGF chromosomal DNA as a probe, or using an antibody against human or animal HGF
The cDNA library prepared from the above was screened, and the desired human H
GF cDNA can also be extracted. (3) cD encoding human HGF from this human-derived HGF cDNA
The NA fragment was excised using a restriction enzyme and incorporated into an expression vector, (4) a host cell was transformed with the obtained recombinant expression vector to obtain a transformant, and (5) the transformed cell was cultured. The human HGF of the present invention can be collected and produced from the culture supernatant. Furthermore, a DNA containing the nucleotide sequence encoding the human HGF of the present invention can be obtained from the recombinant expression vector in the transformed cell by treatment with a restriction enzyme.

Hereinafter, each step of the present invention will be described in detail. (1) Isolation of mRNA and preparation of cDNA library: mR encoding HGF of animal, for example, rat or human
NA is an animal such as a rat or human liver, kidney, spleen, lung, brain, bone marrow, placenta and other organs or leukocytes,
It can be obtained from blood cells such as megakaryocytes and lymphocytes. For example, according to the method of JM Chirgvin et al. Described in Biochemistry, 18 , 5294, (1979), RNA obtained from a guanidine thiocyanate solution of an organ such as a rat or a human organ or blood cells is further subjected to oligo (dT) cellulose. The mRNA can be prepared by subjecting it to liquid chromatography using a column. In addition, mRN such as human liver, brain, placenta, leukocyte
Various mRNAs of animal cells and tissues such as A,
It can be purchased from Clontech and used as a commercial product.

[0007] Using these mRNAs as templates, reverse transcriptase or polymerase chain reaction (PCR)
Using the method of H. Okayama et al. (Mol. Cell. Bio.
l., 2 , 161, 1982, and Mol. Cell. Biol., 3 , 280,
1983) or the method of U. Gubler et al. (Gene, 25 , 263, 198).
3) Alternatively, the method of MA Frohman et al. (Proc. Natl. Acad. S.
ci. USA, 85 , 8998, 1988).
A cDNA library can be prepared by incorporating this cDNA into a plasmid or phage. As a plasmid vector incorporating the cDNA,
PBR322 (Toyobo), pUC18 and pUC19 (Toyobo) from Escherichia coli, pUB11 from Bacillus subtilis
0 (Sigma). Phage vectors incorporating cDNA include λgt10 and λgt11.
(Toyobo). These vectors are not limited to those exemplified here, as long as they are retained and replicated and amplified in host cells. As a method for preparing a cDNA library by incorporating cDNA synthesized using mRNA as a template into a plasmid or phage, the method of T. Maniatis (Molecular Cloning, Cold
Spring Harbor Laboratory, 1982, P. 239) or TV
Hyunh et al. (DNA Cloning: A Practical Approach,
1 , 49, 1985). Also, mR
As with NA, various cDNA libraries can be purchased as commercial products from Clontech or the like, and they can also be used.

(2) Cloning of cDNA library: The recombinant expression vector such as a plasmid or phage obtained as a cDNA library is maintained in a suitable host cell such as Escherichia coli. Examples of Escherichia coli which can be a host include, for example, Escherichia coli NM514, C60
0 (Stratagene), NM522, JM101 (Pharmacia) and the like. cDNA
When the vector is a plasmid, the calcium chloride method, the calcium chloride / rubidium chloride method, etc.
When the DNA vector is a phage, it can be retained in a host cell that has been grown in advance using an in vitro packaging method or the like (Molecular Cloning, Cold S.
pring Harbor Labratory, 1982, p. 249). From the transformant thus obtained, an oligonucleotide encoding the amino acid sequence of the portion of the hepatocyte growth factor of an animal such as a rat or human is synthesized, and the oligonucleotide is labeled with ³² P to be used as a probe. Colony hybridization method (Gene, 10 , 63, 1980), plaque hybridization method (Scienc, 196 , 180, 197)
7) The cDNA clone can be caught by the method described above. In addition, using an antibody against the target polypeptide, a labeled antibody method (DNA Cloning: A Practical Approach,
1 , 49, 1985), it is also possible to clone a cDNA clone. The transformant thus cloned contains a cDNA having a nucleotide sequence encoding the entire amino acid sequence of HGF derived from an animal such as a rat or a human, or a part thereof.

Next, from the transformant, a conventional method (Molecular Clo
ning, Cold Spring Harbor Laboratory, New Yor, 198
Isolate recombinant DNA such as plasmid or phage according to 2) and digest
The NA base sequence is determined. Using a cDNA derived from an animal such as the rat or a human obtained first, as a probe,
In a similar manner, a cDNA library prepared from mRNA derived from human organs or blood cells can be cloned. Hybridization methods and conditions used herein include, for example, Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor L
aboratory, 1st ed. (1982) and the like. The nucleotide sequence of the obtained HGF cDNA derived from animals such as rats or humans is determined by the chemical method of Maxam and Gilbert (Proc. Natl. Acad. Sci. USA,
74 , 560, 1977) and the Sanger dideoxy method (Proc. Nat.
l. Acad. Sci. USA, 74 , 5463, 1977). Further, if necessary, the described mRNA and a part of the cDNA whose base sequence has been determined or its cDNA
Primer extension method (Proc. Natl. Acad. Sci. US
A, 76 , 731, 1979).
In the same manner as described above, it is possible to clone a recombinant DNA such as a plasmid or a phage containing a second cDNA that can be ligated to the first cDNA already obtained from the cDNA library. This step of primer extension and cloning is repeated multiple times as necessary.

(3) Construction of human HGF recombinant expression vector:
CDNA is excised from recombinant vectors such as several plasmids and phages containing cDNAs encoding all or a part of the amino acid sequence of the cloned human HGF by a restriction enzyme, and a vector suitable for human HGF expression is obtained. A recombinant expression vector can be prepared by religating downstream of the promoter using a restriction enzyme and DNA ligase. More specifically, in order to efficiently express the human HGF of the present invention, the recombinant expression vector is required in order in the downstream direction of transcription as necessary (1) promoter, (2) ribosome binding site, (3) start codon, (4) DNA containing the nucleotide sequence encoding human HGF of the present invention, (5) stop codon,
(6) Constructed to include a terminator. Escherichia coli-derived plasmids pBR322 and pUC18 (Toyobo), Bacillus subtilis-derived plasmid pUB110 (Sigma), and yeast-derived plasmid pRB15 (ATCC37) can be used as DNA vectors in the present invention.
062), bacteriophages λgt10, λgt11
(Stratagene), virus SV40 (BRL)
), BPV (ATCC VR-703), retrovirus gene-derived vectors, and the like, but are not particularly limited as long as they can be replicated and amplified in a host.
In particular, in order to easily express the human HGF of the present invention, S
It is preferable to use a vector derived from the gene of a virus such as V40. For example, a recombinant expression vector in which the above-described DNA encoding the cloned human HGF is ligated to the late region of the SV40 vector is a COS cell (Cell, 2).
3 , 175, 1981).

[0011] The promoter and terminator are not particularly limited as long as they correspond to the host used for the expression of the desired base sequence encoding human HGF. For example, when the host is Escherichia coli, the promoter is trp promoter, 1ac promoter or the like. When the host is Bacillus subtilis, the promoter is SP01 promoter.
When the host is yeast, such as SP02 promoter,
When the host is an animal cell such as a mouse fibroblast or a Chinese hamster ovary cell, examples thereof include a virus-derived SV40 promoter, an HSV1 TK promoter, a metallothionein promoter, and a heat shock promoter. I can do it. When the host is Escherichia coli, the terminator is a trp terminator,
If the host is Bacillus subtilis, a
When the host is yeast, CY is used.
When the host is an animal cell, a C1 terminator is used.
V40 terminator, HSV1TK terminator and the like can be exemplified. These promoters and terminators are appropriately combined depending on the host used.

The DNA containing the nucleotide sequence encoding the human HGF of the present invention is not particularly limited as long as the polypeptide expressing the DNA has a hepatic parenchymal cell growth activity. The base sequence shown in FIGS. 5 to 8) is exemplified, and a DNA having a base sequence in which a part of the base sequence is substituted, deleted, inserted, or a combination thereof may be used. Similarly, with respect to the amino acid sequence shown in SEQ ID NO: 2 (FIGS. 5 to 8), one or more amino acids may be substituted, deleted and / or added in the amino acid sequence. The above-mentioned substitution, deletion and / or addition of the amino acid sequence can be carried out by a site-directed mutagenesis method or the like which is a well-known technique before the priority date of the present application. For example, Proc. Natl. Acad. Sci.
USA, 81 , 4662-5666, 1984; Nucleic Acid Res. 10 , 64
87-6500, 1982; WO85 / 00817; Nature 316 , 601-605, 19
85, etc. In addition,
Substitution, deletion and / or addition of one or more amino acids refers to substitution, deletion and / or addition of a number of amino acids that can be substituted, deleted and / or added by a known method such as site-directed mutagenesis. // means to be added. Those obtained by such substitution, deletion and / or addition can be easily evaluated, for example, by the activity measurement method described below. The DNA containing the nucleotide sequence encoding human HGF of the present invention may have ATG as a translation initiation codon and TAA, TGA, or TAG as a translation termination codon. Further, if necessary, one or more initiation codons or termination codons may be combined or arranged in combination with other codons, and these are not particularly limited. Further, it is preferable that one or more of ampicillin resistance gene, neomycin resistance gene, DHFR gene, and the like, which can serve as a selection marker for a host transformed with the recombinant expression vector, be contained at an appropriate position in the vector.

(4) Transformation of host cells and culture thereof: The recombinant human HGF expression vector thus constructed is
Competent cell method (J. Mol. Biol., 53 , 154, 1970),
Protoplast method (Proc. Natl. Acad. Sci. USA, 75 , 1
929, 1978), calcium phosphate method (Science, 221 , 551,
1983), DEAE dextran method (Science, 215 , 166, 1
982), electric pulse method (Proc. Natl. Acad. Sci. USA, 81 ,
7161, 1984), an in vitro packaging method (Proc. Nat.
Acad. Sci. USA, 72 , 581, 1975), viral vector method (Cell, 37 , 1053, 1984), or microinjection method (Exp. Cell. Res., 153 , 347, 1984). A transformant is produced. At this time, Bacillus subtilis, yeast, animal cells, and the like are used as hosts in addition to the aforementioned Escherichia coli. Especially mouse fibroblast C12
7 (J. Virol., 26 , 191, 1978) and Chinese hamster ovary cell CHO (Proc. Natl. Acad. Sci. USA, 77 , 4).
216, 1980).

The obtained transformant is cultured in an appropriate medium according to the host in order to produce the desired recombinant human HGF. The medium contains a carbon source, a nitrogen source, an inorganic substance, a vitamin, a serum, a drug and the like necessary for growth of the transformant. As an example of the medium, when the host of the transformant is Escherichia coli, LB medium (Nissui Pharmaceutical), M9 medium (J. Exp. Mol. Genet., Cold Spring Harbor Laborat)
or YYPD medium (Genetic Engineering, vol. 1, Plenu) when the host is yeast.
m Press, New York, 1979, p. 117), and when the host is an animal cell, ME containing 20% or less of fetal bovine serum.
M medium, DMEM medium, RPMI1640 medium (Nissui Pharmaceutical) and the like. Culture of the transformant
Usually, the reaction is carried out at a temperature of 20 ° C. to 45 ° C. and a pH of 5 to 8, and aeration and stirring are carried out as necessary. When the host is an adhesive animal cell, a carrier such as glass beads, collagen beads, or acetylcellulose follow fiber is used. The method can be carried out as long as the transformant grows under a medium composition or culture conditions other than those described above, and is not limited thereto.

(5) Purification of human HGF: The recombinant human HGF thus produced in the culture supernatant of the transformant or in the transformant can be purified by a known salting-out method, solvent precipitation method, dialysis method, or the like.
Separation and purification can be performed by ultrafiltration, gel electrophoresis, or a combination of gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, and affinity chromatography. In particular, a combination of ammonium sulfate salting out, S-Sepharose ion chromatography, heparin Sepharose affinity chromatography, and phenyl sepharose reverse phase chromatography, or a combination of ammonium sulfate salting out, S-Sepharose ion chromatography, and anti-HGF antibody Sepharose affinity chromatography Are preferred and effective purification methods.
Novel recombinant human HGF obtained by the method described above
Showed an activity to significantly promote the proliferation of rat liver parenchymal cells, similar to HGF derived from rat liver and rat platelets.

Measurement of HGF activity [0016] HGF activity was determined by the method described in Proc. Natl. Acad. Sci. USA, 80 , 7229.
(1983) according to the method described in the following. Hepatocytes were separated and purified from Wistar rats by collagenase reflux. The obtained rat liver parenchymal cells were subjected to 5% bovine serum, 2 × 10 ⁻⁹ M insulin and 2 × 10 ^9
The cells were suspended in a Williams E medium (Flow Laboratories) supplemented with ^-9 M dexamethasone, and seeded on a 24-well multiplate at a concentration of 1.25 × 10 ⁵ cells / well. 5
% CO ₂ and 30% O ₂ and 65% N ₂ ,
After culturing at 37 ° C. for 20 hours, the medium was replaced with a Williams E medium supplemented with 0.1 μg / ml aprotinin, and a predetermined amount of a test sample was added at the same time. After 15 hours, 15 μCi
Per ml of ¹²⁵ I deoxyuridine was added at 10 μl / well. The control group received 5 μg / ml aphidicolin 15 minutes before the addition of ¹²⁵ I-deoxyuridine. After further culturing for 6 hours, the ^cells were labeled with ¹²⁵ I. After washing the cells twice with PBS at pH 7.4, the cells were fixed with a cold 10% aqueous solution of trichloroacetic acid (TCA). The cells were solubilized with 0.5 ml of 1N aqueous sodium hydroxide solution per well, and the radioactivity was measured by a gamma counter. In addition, a part of the sample after radioactivity measurement was taken and the Lowry method (J. Bi
ol. Chem., 193 , 265, 1951).
¹²⁵ incorporated into liver parenchymal cells when the test sample is added
The amount of I was determined as the difference between the count and the control, and this was converted into 1 mg of rat hepatocyte protein,
DNA synthesis activity (dpm / mg protein). The HGF activity of the test sample was determined by comparing the D value of hepatocytes with 10 ng / ml of epidermal growth factor (EGF) in the same test.
The activity corresponding to 50% of the NA synthesis activity was defined as one unit and displayed.

[0017]

According to the present invention, there is provided a novel bioactive peptide which enables the proliferation of liver parenchymal cells in vitro. The recombinant human HGF of the present invention is useful as a clinical diagnostic reagent or a therapeutic agent for liver disease. Further, the recombinant human HG of the present invention
Hepatocytes proliferated and maintained by the action of F can be used, for example, for basic research on liver function, for studying the effects of various hormones and drugs on hepatocytes, for carcinogenesis research of liver cancer, or for in vitro culture of hepatitis virus. It is extremely useful as a host cell.

[0018]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples. Example 1 (1) Isolation of rat liver mRNA: rat liver mRNA
The guanidine thiocyanate method (Biochemistry, 18 , 529)
4, 1979), and oligo dT cellulose column chromatography (Proc. Natl. Acad. Sci. USA, 69 ,
1408, 1972). 20 ml of carbon tetrachloride diluted with commercially available edible vegetable oil was intraperitoneally administered at 1 ml per 100 g of SD rats. Ten hours after carbon tetrachloride administration, the liver was removed. 0.90 g of the obtained rat liver
Then, 16 ml of a 5.5 M guanidium solution (a solution of 5.5 M guanidine thiocyanic acid, 25 mM citric acid, 0.5% sodium lauryl sarcosine at pH 7.0) was added thereto, and homogenized. 16 ml of the above rat liver dispersion was overlaid on 17 ml of a cesium trifluoroacetic acid solution (1 g / ml) containing 0.1 M EDTA, and a Beckman ultracentrifuge was used.
85000g by L8-55 type, 20 hours, 20 ° C
Was centrifuged under the following conditions. After removing the DNA layer, the precipitated RNA layer was dissolved in 1 ml of sterile distilled water. 6.24 from this aqueous RNA solution by cold ethanol precipitation.
mg of RNA was obtained. The obtained RNA was subjected to 1 mM EDT.
A containing 10 mM Tris-HCl buffer (pH 7.5) (hereinafter TE
The mixture was dissolved in 0.5 ml of a buffer solution, and heated at 65 ° C. for 5 minutes, and then 0.5 ml of a 1M NaCl solution was added. After activation with 0.1 N NaOH, 0.5 M Na
Oligo dT equilibrated with 10 mM Tris-HCl buffer (STE buffer) containing Cl and 1 mM ETDA
0.5 ml of the RNA solution was injected into the cellulose column.
After washing with about 5 ml of STE buffer, the adsorbed poly (A) RNA was eluted with TE buffer. This poly (A) RNA
The poly (A) RNA obtained by cold ethanol precipitation from 500 μl of the solution was dissolved again in TE buffer, and 1 μg / μ
1 concentration.

(2) Preparation of cDNA library derived from rat liver: 5 μl of poly (A) RNA obtained in (1) above
Using the cDNA synthesis system plus (Amersham) as a template (Gene, 25 , 263, 1983)
CDNA was synthesized according to The yield of single-stranded cDNA was 1018 ng, and the yield of double-stranded cDNA was 1729.
ng. This double-stranded cDNA is purified by phenol / chloroform (1: 1, v / v) extraction and ethanol precipitation, and then dissolved in STE buffer to give about 0.7 μl.
g / 20 μl until use before use
Stored at ° C. This cDNA was prepared using the method of Huynh et al. (DNA Cloning I, A Practical Approach, 1 , 49, 49) using a cDNA cloning system λgt10 (Amersham).
1982) and cloned into the EcoRI site of λgt10 as follows. After methylation of 20 μl of the above cDNA solution using EcoRI methylase, T4D
EcoRI linkers were added to both ends of the cDNA using NA ligase. Excess linker was digested with EcoRI to obtain about 100 μl of the reaction solution. The above reaction solution 10 was applied to a gel filtration column for cDNA purification equilibrated with STE buffer.
0 μl was injected. Eluting with STE buffer, 500 μl of cDNA fraction was collected. After ethanol precipitation was repeated twice by a conventional method, the mixture was dried under reduced pressure to obtain a linker-added cDNA.
I got Again, dissolve in STE buffer and add 50 ng / μl
26 μl of the linker-added cDNA was prepared. Linker addition c to 1 μg of λgt10 arm prepared in advance
0.1 μg of DNA was inserted using T4 DNA ligase. This reaction solution was treated with cold ethanol and then lightly dried, and the whole amount of the obtained recombinant DNA was dissolved in 5 μl of TE buffer. This recombinant DNA was subjected to an in vitro packaging reaction to obtain a λgt10 recombinant phage. The number of recombinant phages obtained from 1 μg of cDNA measured by titration using Escherichia coli for phage plating was 5.0 × 10 ⁶ . The cDNA library prepared in this manner was used in an SE buffer (100 mM NaCl, 10 mM
It was stored at 4 ° C. in 20 mM Tris-HCl buffer (pH 7.5) containing M MgSO ₄ and 0.01% gelatin.

(3) Synthesis of DNA probe: The following rat HGF β chain N-terminal amino acid sequence of 15 found by the present inventors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 V-V-N-G- IPTTQTTV-GWWMV (wherein V is valine, N is asparagine, G is glycine, I is isoleucine, P is proline, T is threonine, Q is Is glutamine, W is tryptophan, and M is methionine), and the oligonucleotide ⁵ ′ ACCATCCAICCIAIGIGIG was deduced.
TITGIGTIGGIATICCITTIACIAC
³ '(I represents inosine) was converted to DNA synthesizer 38.
It was synthesized by 1A (Applied Biosystems). The obtained oligonucleotide was labeled with [γ ³² P] ATP (Amersham) using T4 polynucleotide kinase (Toyobo) to prepare a DNA probe.

(4) Partial isolation of rat HGF gene DNA and determination of its nucleotide sequence: about 5 × 10 ⁵ obtained in the above (2)
After infecting about 8 × 10 ⁹ E. coli NM514 (Stratagene) at 37 ° C. for 15 minutes, the recombinant phage was added to 270 ml of LB medium containing 0.7% agar heated to about 50 ° C. The mixture was uniformly cast on six 23 cm × 23 cm LB agar plates. After culturing in air at 37 ° C. for 12 hours, a nitrocellulose filter was adhered to the plate on which the plaque was formed for about 30 seconds. The nitrocellulose filter was mixed with 1.5 M NaCl and 0.1
Immersed in an alkaline solution consisting of N NaOH for 5 minutes, and further neutralized solution consisting of 0.2 M Tris-HCl buffer (pH 7.5), 25 mM phosphate buffer (pH 7.5), 2 mM EDTA and 2 × SSC buffer For 15 minutes. 8 after air drying
After heat treatment at 0 ° C. for 2 hours, the DNA of each plaque was immobilized on a nitrocellulose filter. The obtained nitrocellulose filter was immersed in a hybridization solution consisting of 6 × SSC buffer, 5 × Denhardt's solution, 50 mM IPES, and 100 mM phosphate buffer, pH 7.0, and pretreated at 65 ° C. for 5 hours. The ³² P-labeled synthetic oligonucleotide (about 3 × 10 ⁸ cpm) probe (3) heat-treated at 100 ° C. for 5 minutes and salmon testis DNA (0.
1 mg / ml), and a hybridization reaction was carried out at 45 ° C. for 16 hours. After the reaction, the nitrocellulose filter was filtered at 6 ° C containing 0.1% SDS at 50 ° C.
Washed three times with SC buffer and air dried. The nitrocellulose filter was brought into close contact with an intensifying screen, Lightning Plus (DuPont), an X-ray film, and RX (Fuji Photo Film), and exposed at -80 ° C for 30 hours. The obtained three positive plaques were collected, subjected to secondary screening by the same method as described above, and one obtained positive clone was named RBC1. This RBC1 phage was propagated by a conventional method, and RBC1 cDNA was isolated and purified. The nucleotide sequence of the obtained cDNA was determined by the dideoxy method using Sequenase (United State Biochemical). FIG. 1 shows RBC1cDN.
1 shows the entire nucleotide sequence of A. RBC1 cDNA is derived from rat H
It contains the base sequence (1st to 699th) that encodes the GFβ chain.

(5) Isolation of human HGF gene DNA and determination of nucleotide sequence: Human normal liver mRNA (Clontech)
Using 5 μg as a template, cDNA derived from human liver was synthesized in the same manner as in (2) above. The yield of single-stranded cDNA was 110
0 ng. 200 ng of the obtained cDNA was subjected to agarose electrophoresis and fractionated 4-7 kb cDNA
After extracting with Gene Clean (Bio 101 companies),
A cDNA library (I) was prepared in the same manner as in the above (2). From 1 μg of cDNA, 2 × 10 ⁵ recombinant phages were obtained. RBC labeled with [α ³² P] dCTP using a multiprime DNA labeling system (Amersham)
Using 1 cDNA as a probe, the hybridization reaction temperature and the washing temperature were 60 ° C., and the washing solution was 0.1% S.
Primary screening and secondary screening of the human liver-derived cDNA library (I) were performed in the same manner as in (4) except that 2 × SSC buffer containing DS was used, and a positive clone HBC25 was obtained. The HBC25 cDNA isolated and purified from the HBC25 phage by a conventional method was subjected to nucleotide sequence analysis and restriction enzyme cleavage analysis. FIG. 2 (a) shows HBC2
5 cDNA restriction enzyme map, FIG. 3 shows HBC25 cDNA
Shows a part of the base sequence. Next, an oligonucleotide having a base sequence of ⁵ 'TCATAATCTTTCAAGTCT ³ ' contained in the HBC25 cDNA was synthesized by a DNA synthesizer 381A (Applied Biosystems). 0.75 μg of this synthetic DNA was used as a primer, and 20 μg of human liver mRNA was used as a template for cDNA amplification.
4 μg was synthesized, and a cDNA library (I
I) was prepared. From this cDNA library (II), a 0.7 kb EcoRI fragment of HBC25 cDNA labeled with [α ³² P] dCTP was probed to obtain a positive clone HAC19. FIG. 2 (b) shows a restriction map of HAC19 cDNA, and FIG. 4 shows a part of the nucleotide sequence of HAC19 cDNA. HBC25 cDNA thus obtained
And the entire nucleotide sequence of the human HGF coding region obtained by combining the nucleotide sequences of HAC19 cDNA and the amino acid sequence deduced from the nucleotide sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, and FIGS.

From the entire human HGF cDNA base sequence, the translation initiation codon of human HGF is assumed to be the first ATG and the termination codon is assumed to be the TAG at the second position. The cDNA sequence of human HGF between these start and stop codons encodes a polypeptide consisting of 728 amino acid residues, the amino acid sequence following Met 1 is rich in Leu, and up to Gly 31 is secreted by HGF. For the signal sequence. Similarly, the N-terminus of the human HGF β chain
It is estimated to be the 495th Val. The binding site of the sugar chain of human HGF is estimated to be Asn at positions 294, 402, 566, and 653 having the amino acid sequence of Asn-x-Ser / Thr. As a result of homology search of the amino acid sequence of human HGF shown in SEQ ID NO: 2 by computer, human HGF was found to be plasminogen, plasmin, calyculein,
It has been found to have homology with serine proteases such as II. That is, human HGF has four putative kringle structures in its α-chain, and its β-chain is similar to the protease region of the above-mentioned serine protease. However, Ser and His, which are presumed to be the active centers of serine proteases, are different from human HGF β-
In the chain, they are substituted with Tyr (673) and Gln (534), respectively.

(6) Construction of human HGF expression vector for monkey COS cells: FIG. 9 shows a construction diagram of a human HGF expression vector pEUK [hHGFI] for monkey COS cells. the above
Using the HAC19 phage DNA obtained in (5), restriction enzyme B
After digestion with amHI and ScaI, a 0.9 kb DNA fragment was separated and purified by agarose electrophoresis. Similarly H
BC25 phage DNA was ligated with restriction enzymes ScaI and SmaI.
And a 2.1 kb DNA fragment was separated and purified.
These DNA fragments were mixed with Bluescript KSM13 + (Stratagene) previously digested with restriction enzymes BamHI and SmaI, and ligated with T4 DNA ligase to obtain plasmid pBS [hHGFI]. The obtained pBS [hHGFI] was digested with restriction enzymes XbaI and SmaI. A COS cell expression vector pEUK-C1 (Clontech) previously digested with restriction enzymes XbaI and SmaI was mixed with a 3.0 kb DNA fragment, and T4D
Human HGF expression vector pEU ligated with NA ligase
K [hHGFI] was obtained.

(7) Transformation of monkey COS cells and human HG
Expression of F gene: The obtained pEUK [hHGFI] plasmid was precipitated with ethanol, and then dissolved in 10 mM PBS buffer to adjust to 20 μg / ml. Next, 10%
Exponentially growing COS-1 cells (AT) grown in DMEM medium (Nissui Pharmaceutical) containing fetal bovine serum (Gibco)
CC CRL-1650) in 10 mM PBS buffer
After washing twice, trypsinization was performed. After washing three times with the same buffer, the cells were resuspended in the same buffer to a cell concentration of 2 × 10 ⁷ cells / ml. Plasmid solution 250 previously prepared
μl and 250 μl of the cell suspension were mixed and allowed to stand under ice cooling for 10 minutes. A high voltage pulse was applied to the ice-cooled plasmid / cell mixture using a high voltage pulse gene transfer apparatus ZA-1200 (PDS) under the conditions of an applied voltage of 4 kV / cm and a pulse time of 20 ms. The obtained cells were diluted with the above medium and cultured at 37 ° C. in the presence of 5% CO _{2 for} 3 days. The HGF activity in the culture supernatant on day 3 of the culture was measured using the above-mentioned rat hepatocyte, and was 50 units / ml. On the other hand, an expression vector into which HGF cDNA was not inserted, pEUK-C1, was prepared by the same method.
The cells were introduced into S-1 cells and cultured, but no HGF activity was observed in the culture supernatant.

Example 2 (1) Construction of Human HGF Expression Vector for Mouse C127 Cell: Human HGF Expression Vector pB for Mouse C127 Cell
The construction diagram of PMT [hHGFII] is shown in FIG. The HAC19 phage DNA obtained in Example 1 was
After digestion with amHI and ScaI, a 0.9 kb DNA fragment was separated and purified by agarose electrophoresis. Similarly H
BC25 phage DNA was ligated with the restriction enzymes ScaI and PstI.
And a 2.1 kb DNA fragment was separated and purified.
These DNA fragments are preliminarily digested with restriction enzymes BamI and PamI.
The mixture was mixed with BlueScript KSII + (Stratagene) digested with stI, ligated with T4 DNA ligase, and ligated with plasmid pBS [hHGFII] (Microtechnical Laboratories No. 1105).
0). Plasmid pBPMT is replaced with the restriction enzyme Eco.
After digestion with RV, bacterial alkaline phosphatase (BA
Plasmid pBS
[HHGFII] (Microtechnical Laboratories No. 11050) was digested with restriction enzymes XbaI, SalI and NaeI, blunt-ended with T4 DNA polymerase, and then separated and purified by agarose gel electrophoresis to obtain a 3.0 kb DNA fragment.
Inserted by ligase. The obtained human HGF expression vector pBPMT [hHGFII] has a human HGF gene between the MT-1 promoter and the poly (A) addition signal of the SV40 early gene. Transformation of mouse C127 cells with this expression vector is as follows: It is performed by the bovine papillomavirus (BPV) gene. The selection of the transformed cells was performed by selecting the neo gene of transposon Tn5 (G
ene, 19 , 327, 1982) by a neo chimeric gene linked to a promoter derived from the herpes simplex virus type 1 thymidine kinase (HSV-1 TK) gene and a poly (A) addition signal.

(2) Transformation of mouse C127 cells and expression of human HGF gene: Human HGF expression vector pBPM
T [hHGFII] was obtained by the method of Wigler et al. (Cell, 11 , 223, 1).
977) into mouse C127 cells. 20 μg of the pBPMT [hHGFII] plasmid obtained in the above (1) was dissolved in 240 μl of 0.5 M calcium chloride, and
0 mM HEPES, 280 mM NaCl and 1.5
2 × HEPES buffer consisting of mM sodium phosphate
(pH 7.1), 240 μl was added with stirring. 3 at room temperature
Stirring was continued for 0 minutes to form a coprecipitate of the plasmid and calcium phosphate. Using a DMEM medium (Nissui Pharmaceutical) supplemented with 10% fetal bovine serum (Gibco) and 10 mM glutamine in advance, 5 × 10 ⁵ C127 cells were
The cells were cultured at 37 ° C. for 24 hours in the presence of CO ₂ . After replacing the medium, add the plasmid and calcium phosphate co-precipitate,
It was left at room temperature for 20 minutes. After further incubation at 37 ° C. for 4 hours, the medium was removed, 1 × HEPES buffer containing 15% glycerin was added, and the mixture was left at room temperature for 5 minutes. After washing the cells with the medium, the medium was changed and the cells
Incubated at 2 ° C for 2 days. The cells were diluted 10-fold and cultured in the same medium containing 1 mg / ml G418 (Sigma) at 37 ° C. for 7 days in the presence of 5% CO ₂ to obtain transformed cells. H in the culture supernatant was obtained from the obtained cell line.
Cells having high GF activity were screened by the limiting dilution method to obtain a human HGF high-producing strain BPH89. The HGF-producing ability of the culture supernatant of the cells was 23,000 units / l / day.

Example 3 (1) Construction of Human HGF Expression Vector for Chinese Hamster CHO Cells: Human HGF Expression Vector pEVSVE [hHGFI for Chinese Hamster CHO Cells
The construction diagram of [I] is shown in FIG. Plasmid pEVSV
After digestion of E with the restriction enzyme EcoRV, the site where the phosphate group was removed with bacterial alkaline phosphatase (BAP),
Plasmid pBS [hHGFII] obtained in Example 2
(Microbial Laboratories No. 11050) with restriction enzymes XbaI and Sba
After digestion with alI and NaeI and blunting with T4 DNA polymerase, a 3.0 kb DNA fragment separated and purified by agarose electrophoresis was inserted with T4 DNA ligase. Obtained human HGF expression vector pEV
SVE [hHGFII] has the human HGF gene between the SV40 early promoter and the poly (A) addition signal of the SV40 early gene. In addition, the selection of the transformed cells can be made by a DHFR chimera gene in which the SV40 early promoter and the poly (A) addition signal are linked to the mouse DHFR gene.

(2) Transformation of Chinese hamster CHO cells and expression of human HGF gene: The human HGF expression vector pEVSVE [hHGFII] is deficient in dihydrofolate reductase (DHFR) of Chinese hamster CHO cells in the same manner as in Example 2. CHO DUKX cells were introduced. The obtained cell line was cultured in an α-MEM medium (Flow Laboratories) containing 10% fetal bovine serum (Kibco), 1% glutamine and 50 nM methotrexate without ribonucleoside and deoxynucleoside, and containing dialyzed 10% fetal bovine serum (Kibco). . The colonies that developed were stable human HG
In order to obtain a high F producing strain, the cells were grown in the same medium up to 18 generations. The resulting cell line stably produced human HGF and was named CHO-1. The human HGF-producing ability of these cells was 31,000 units / l / day.

Example 4 Recombinant human HGF was purified from the culture supernatant of the human HGF-producing mouse C127 recombinant cell line BPH89 obtained in Example 2. (1) Cation exchange chromatography Tween 80 was added to a culture solution of BPH89 strain (400 ml) to a final concentration of 0.01%, followed by filtration through a Sterivex HV filter (Nippon Millipore Limited). Add 1/20 volume of 1 M Tris · HCl (pH 8.5) to the filtrate.
Add buffer and add buffer A (50 mM Tris · HC)
1, 10 mM HEPES, 2 mM CaCl ₂ , 150
It was added to S-Sepharose FF (Pharmacia, column size inner diameter 1.6 cm, height 5 cm) equilibrated with mM NaCl, 0.01% Tween 80, pH 8.5. After washing unadsorbed substances with buffer A, 0.15 M to 1.0 M NaCl
The adsorbed substance was eluted with a linear concentration gradient (100 ml in total) according to. FIG. 12 shows the chromatographic pattern. Fractions having HGF activity were collected and used as S-Sepharose eluate.

(2) Affinity chromatography: S
-Sepharose eluate adjusted to pH 7.5 with 1N acetic acid
Dilute with double volume of distilled water containing 0.01% Tween 80, and add Buffer B (10 mM Tris.HCl, 0.3 M NaCl,
It was added to Heparin Sepharose CL-6B (Pharmacia, column size ID 1 cm, height 3 cm) equilibrated with 0.01% Tween 80, pH 7.5. After washing the column with buffer B, the column was eluted with a linear gradient (30 ml in total) from 0.3 M to 2.0 M NaCl. FIG. 13 shows the chromatographic pattern. Collecting fractions having HGF activity,
Heparin eluate was used.

(3) Reversed phase HPLC: C equilibrated with distilled water containing 0.1% TFA (trifluoroacetic acid, v / v%)
4 RP-304 column (manufactured by Bio-Rad, 4.6 m inside diameter)
m, height 250 mm), add 0.1%
Elution was performed with a gradient of acetonitrile from 0% to 90% containing TFA. Recombinant human HGF was eluted at about 40% acetonitrile concentration. FIG. 14 shows the chromatogram. The yield of the purified recombinant human HGF is about 25 μg, and the activity recovery rate from the culture supernatant is about 2 μg.
5%.

(4) SDS-polyacrylamide electrophoresis: Recombinant human HGF purified by the above three-stage chromatography was subjected to SDS-polyacrylamide electrophoresis under reduction and non-reduction of 2-mercaptoethanol. FIG. 15 shows the results. Purified recombinant HGF was prepared under non-reducing conditions (2-
ME (-)) shows a single band having a molecular weight of 70,000 to 90,000, and under reducing conditions (2-ME (+)), a molecular weight of 60,000 to 90,000
It was divided into a 75,000 α chain and a β chain with a molecular weight of 30,000 to 40,000.
That is, it was shown that the recombinant HGF was a heterodimer consisting of an α chain and a β chain. The β chain was separated into two bands, indicating that there was a difference in the number of sugar chains added to the β chain.

(5) N-terminal amino acid sequence of α-chain and β-chain: The α-chain and β-chain of purified recombinant HGF were separated, and their N-terminal amino acid sequences were examined. The purified sample was treated at 60 ° C. for 30 minutes in the presence of 4M guanidine, and reduced with 2-mercaptoethanol. Next, sodium monoiodoacetate was added to alkylate the SH group, and the reaction solution was subjected to reverse phase HPLC using a Micro Bonder Sphere C4 column (Waters) to separate the α chain and the β chain. Elution was with water containing 0.1% TFA and acetonitrile.
The α-chain and β-chain thus obtained were subjected to a protein sequencer, and as a result, the following amino acid sequences were obtained. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 α chain His Lys Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala 16 17 18 19 20 Lys Thr Thr Leu Ile 1 2 3 4 5 6 7 β Chain Val Val Asn Gly Ile Pro Thr These sequences correspond to the 32nd to 51st positions and 495 to 501 of the amino acid sequence (SEQ ID NO: 2) deduced from the entire nucleotide sequence of the HGF coding region of SEQ ID NO: 1 (FIGS. 5 to 8). The second match.

(6) Amino acid composition and sugar composition: Purified recombinant HGF was added to 6N HCl containing 0.2% phenol in 1%.
The mixture was hydrolyzed by treatment at 10 ° C. for 24 hours and subjected to an amino acid analyzer to examine the amino acid composition. As shown in the following table, the results showed that the amino acid composition value estimated from the DNA base sequence and the measured value obtained from the purified recombinant preparation were in excellent agreement.

数 Number of amino acids per 1 mol of protein 蛋白計算 Calculated amino acid DNA sequence measured value 実 ━━━━━━━━━ ━━━ Lys 4746 His 24 24 Arg 43 42 Asp 85 80 Thr 4138 Ser 39 38 Glu 60 56 Pro 45 43 Gly 57 57 Ala 22 22 Cys 40 () Val 3335 Met 15 17 Ile 40 37 Leu 38 38 Tyr 32 31 Phe 18 19 Trp 18 () ━━━━━━━━━━━━━━━━━━━ ━━━━━ (() of the measured value means that it cannot be measured by this analysis method.)

Further, purified recombinant HGF was added to 2.5N TFA.
In the medium, heat treatment was performed at 110 ° C. for 6 hours, and sugar analysis was performed by HPLC using an anion exchange resin. As a result, mannose, galactose, fucose, and N-acetylgalactosamine specific to asparagine glycoside were detected, and recombinant H
GF was confirmed to be a glycoprotein.

(7) Hepatocyte proliferation activity of recombinant human HGF: Rat primary cultured hepatocytes are in vitro
It is a system that has the liver function closest to in vivo among the systems.
When purified recombinant human HGF was added to rat liver parenchymal cells obtained according to the method described in "Measurement method of HGF activity", strong cell proliferation was induced at a concentration of 1 ng / ml. The effect has reached its maximum. Factors that show proliferative activity in this culture system include insulin and EGF
However, recombinant human HGF alone had a stronger activity than both, and showed an additive action in the presence of these three. Therefore, it can be said that recombinant human HGF is the substance having the strongest proliferative activity on ex vivo hepatocytes among the currently known factors.

[0039]

[Sequence List] SEQ ID NO: 1 Sequence length: 2187 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin: human liver cDNA library Sequence ATG TGG GTG ACC AAA CTC CTG CCA GCC CTG CTG CTG CAG CAT GTC CTC 48 Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu 5 10 15 CTG CAT CTC CTC CTG CTC CCC ATC GCC ATC CCC TAT GCA GAG GGA CAT 96 Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly His 20 25 30 AAG AAA AGA AGA AAT ACA ATT CAC GAA TTC AAA AAA TCA GCA AAG ACT 144 Lys Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45 ACC CTA ATC AAA ATA GAT CCA GCA CTG AAG ATA AAA ACC AAA AAA GTG 192 Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60 AAT ACT GCA GAC CAA TGT GCT AAT AGA TGT ACT AGG AAT AAT GGA CTT 240 Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Asn Gly Leu 65 70 75 80 CCA TTC ACT TGC AAG GCC TTT GTT TTT GAT AAA GCG AGA AAA CAA TGC 288 Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95 CTC TGG TTC CCC TTC AAT AGC ATG TCA AGT GGA GTG AAG AAA GAA TTT 336 Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 GGC CAT GAA TTT GAC CTC TAT GAA AAC AAA GAC TAC ATT AGA AAC TGC 384 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120 125 ATC ATC GGT AAA GGA CGC AGC TAC AAG GGA ACA GTA TCT ATC ACT AAG 432 Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135 140 AGT GGC ATC AAA TGT CAG CCC TGG AGT TCC ATG ATA CCA CAC GAA CAC 480 Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His 145 150 155 160 AGC TTT TTG CCT TCG AGC TAT CGG GGT AAA GAC CTA CAG GAA AAC TAC 528 Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr 165 170 175 TGT CGA AAT CCT CGA GGG GAA GAA GGG GGA CCC TGG TGT TTC ACA AGC 576 Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser 180 185 190 AAT CCA GAG GTA CGC TAC GAA GTC TGT GA C ATT CCT CAG TGT TCA GAA 624 Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu 195 200 205 GTT GAA TGC ATG ACC TGC AAT GGG GAG AGT TAT CGA GGT CTC ATG GAT 672 Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp 210 215 220 CAT ACA GAA TCA GGC AAG ATT TGT CAG CGC TGG GAT CAT CAG ACA CCA 720 His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro 225 230 235 240 CAC CGG CAC AAA TTC TTG CCT GAA AGA TAT CCC GAC AAG GGC TTT GAT 768 His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp 245 250 255 GAT AAT TAT TGC CGC AAT CCC GAT GGC CAG CCG AGG CCA TGG TGC TAT 816 Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr 260 265 270 ACT CTT GAC CCT CAC ACC CGC TGG GAG TAC TGT GCA ATT AAA ACA TGC 864 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys 275 280 285 GCT GAC AAT ACT GTA AAT GAT ACT GAT GTT CCT ATG GAA ACA ACT GAA 912 Ala Asp Asn Thr Val Asn Asp Thr Asp Val Pro Met Glu Thr Thr Glu 290 295 300 TGC ATC CAA GGT CAA GGA G AA GGC TAC AGG GGC ACT GCC AAT ACC ATT 960 Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Ala Asn Thr Ile 305 310 315 320 TGG AAT GGA ATT CCA TGT CAG CGT TGG GAT TCT CAG TAT CCT CAC AAG 1008 Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Lys 325 330 335 CAT GAC ATG ACT CCT GAA AAT TTC AAG TGC AAG GAC CTA CGA GAA AAT 1056 His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350 TAC TGC CGA AAT CCA GAT GGG TCT GAA TCA CCC TGG TGT TTT ACC ACT 1104 Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355 360 365 GAT CCA AAC ATC CGA GTT GGT TAC TGC TCC CAA ATT CCA AAC TGT GAT 1152 Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp 370 375 380 ATG TCA AAT GGA CAA GAT TGT TAT CGT GGG AAT GGC AAA AAT TAT ATG 1200 Met Ser Asn Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met 385 390 395 400 GGC AAC TTA TCC CAA ACA AGA TCT GGA CTA ACG TGT TCA ATG TGG AAC 1248 Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asn 405 410 415 AAG AAC ATG GAA GAC TTA CAC CGT CAT ATC TTC TGG GAA CCA GAT GCA 1296 Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala 420 425 430 AGT AAG CTG AAT GAG AAT TAC TGC CGA AAT CCA GAT GAT GAT GCT CAT 1344 Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His 435 440 445 GGA CCC TGG TGC TAC ACG GGA AAT CCA CTC ATT CCT TGG GAT TAT TGC 1392 Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys 450 455 460 CCT ATT TCT CGT TGT GAA GGT GAT ACC ACA CCT ACA ATA GTC AAT TTA 1440 Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu 465 470 475 475 480 GAC CAT CCT GTA ATA TCT TGC GCC AAA ACG AAA CAA CTG CGA GTT GTA 1488 Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val 485 490 495 AAT GGG ATT CCA ACA CGA ACA AAT GTA GGA TGG ATG ATT AGT TTG AGA 1536 Asn Gly Ile Pro Thr Arg Thr Asn Val Gly Trp Met Ile Ser Leu Arg 500 505 510 TAC AGA AAT AAA CAT ATC TGC GGA GGA TCA TTG ATA AAG GAA AGT TGG 1584 Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp 515 520 525 GTT CTT ACT GCA CGA CAG TGT TTC CCT TCT CGA GAC TTG AAA GAT TAT 1632 Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr 530 535 540 540 GAG GCT TGG CTT GGA ATT CAT GAT GTC CAT GGA AGA GGA GAG GAG AAA 1680 Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Glu Glu Lys 545 550 555 560 CGC AAA CAG GTT CTC AAT GTT TCC CAG CTG GTA TAT GGC CCT GAA GGA 1728 Arg Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly 565 570 575 TCA GAT CTG GTT TTA ATG AAG CTT GCC AGA CCT GCT GTC CTG GAT GAT 1776 Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp 580 585 590 590 TTT GTT AAT ACA ATT GAT TTA CCT AAT TAT GGA TGC ACA ATT CCT GAA 1824 Phe Val Asn Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu 595 600 605 AAG ACC AGT TGC AGT GTT TAT GGC TGG GGC TAC ACT GGA TTG ATC AAC 1872 Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn 610 615 620 TAT GAT GGT CTA TTA CGA GTG GCA CAT CTC TAT ATA ATG GGA AAT GAG 1920 Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu 625 630 635 640 AAA TGC AGC CAG CAT CAC CGA GGG AAG GTG ACT CTG AAT GAG TCT GAA 1968 Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu 645 650 655 655 ATA TGT GCT GGG GCT GAG AAG ATT GGA TCA GGA CCA TGT GAG GGG GAT 2016 Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp 660 665 670 TAT GGT GGC CCA CTT GTT TGT GAG CAA CAT AAA ATG AGA ATG GTT CTT 2064 Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu 675 680 685 GGT GTC ATT GTT CCC GGC CGT GGA TGC GCC ATT CCA AAT CGT CCT GGT 2112 Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly 690 695 700 ATT TTT GTC CGA GTA GCA TAT TAT GCA AAA TGG ATA CAC AAA ATT ATT 2160 Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile 705 710 715 715 720 TTA ACA TAT AAG GTA CCA CAG TCA TAG 2187 Leu Thr Tyr Lys Val Pro Gln Ser 725

SEQ ID NO: 2 Sequence length: 728 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu 5 10 15 Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly His 20 25 30 Lys 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 Asn 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 Cys 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 Val Asn Asp Thr Asp Val Pro Met Glu Thr Thr Glu 290 295 300 Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Ala Asn Thr Ile 305 310 315 320 Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Lys 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 370 375 380 380 Met Ser Asn 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 Asn 405 410 415 Lys Asn Met Glu Asp Leu 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 465 470 475 480 480 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 Val Gly Trp Met Ile 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 Glu Glu Lys 545 550 555 560 Arg 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 Asn Thr Il e 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 610 615 620 620 Tyr Asp Gly Leu Leu Arg Val 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 705 710 715 715 720 Leu Thr Tyr Lys Val Pro Gln Ser 725

[Brief description of the drawings]

FIG. 1 is a view showing a base sequence of RBC1 cDNA.

FIG. 2 shows a restriction map of HBC25 cDNA (a) and a restriction map of HAC19 cDNA (b).

FIG. 3 is a diagram showing a part of the base sequence of HBC25 cDNA.

FIG. 4 is a diagram showing a part of the nucleotide sequence of HAC19 cDNA.

FIG. 5 shows the nucleotide sequence of the human HGF coding region (base number 1).
624) and its amino acid sequence.

FIG. 6 shows the nucleotide sequence of the human HGF coding region (base No. 6).
25 to 1248) and its amino acid sequence.

FIG. 7 shows the nucleotide sequence of the human HGF coding region (base number 1).
249-1872) and its amino acid sequence.

FIG. 8 shows the nucleotide sequence of the human HGF coding region (base number 1).
873-2187) and their amino acid sequences.

FIG. 9 is a construction diagram of a human HGF expression vector for monkey COS cells.

FIG. 10 is a construction diagram of a human HGF expression vector for mouse C127 cells.

FIG. 11: Human H for Chinese hamster CHO cells
It is a construction diagram of a GF expression vector.

FIG. 12 is a diagram showing the relationship between the fraction of S-Sepharose eluate, the absorbance of eluted components, and their DNA synthesis activity.

FIG. 13 is a diagram showing the relationship between the fraction of a heparin eluate, the absorbance of the eluted components, and their DNA synthesis activity.

FIG. 14 is a diagram showing the relationship between the concentration of acetonitrile passed through and the absorbance of eluted components in reverse phase HPLC.

FIG. 15 shows SDS-polyacrylamide electrophoresis patterns of purified recombinant HGF under reducing and non-reducing conditions.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12P 21/02 A61K 37/36 (72) Inventor Manabu Shimonishi Green Bell Nishi-Otsu A, 1-20-36 Sakuranocho, Otsu City, Shiga Prefecture −203 (72) Inventor Shin Shimizu No. 303, Building No. 27, 1-23 Takano Higashikai-cho, Sakyo-ku, Kyoto (56) Reference Nature Vol. 342 (Nov. 1989), p. 440-443 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/09 C07K 14/52 BIOSIS (DIALOG) WPI (DIALOG) GenBank / EMBL / DDBJ SwissProt / PIR

Claims (1)

(57) [Claims] (1) a DNA encoding a protein consisting of the amino acid sequence represented by the following (a) to (e): SEQ ID NO: 2; (b) one or more amino acids in the amino acid sequence (a) are substituted; DNA consisting of a deleted and / or added amino acid sequence and encoding a protein having hepatocyte proliferation activity (c) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 (d) (c) DNA consisting of 60 ° C and 6 × SSC
Buffer, 5 × Denhardt's solution, 50 mM PIPES,
100 mM phosphate buffer (pH 7.0) and salmon testes D
DNA that hybridizes under the condition of NA (0.1 mg / ml) and encodes a protein having hepatocyte proliferation activity. (E) Due to the degeneracy of gene codons, shown in (c) and (d). A recombinant expression vector containing a gene that does not hybridize to DNA, but which comprises a DNA encoding a protein having the same amino acid sequence as the protein encoded by the DNA shown in (c) or (d) , Mouse C127 cells, Chinese hamster CH
Mouse C127 cells, Chinese hamster CHO cells or monkeys obtained by culturing a transformant obtained by transforming O cells or monkey COS cells and isolating and purifying the expressed recombinant hepatocyte growth factor. COS
A recombinant hepatocyte growth factor different from a cell-derived hepatocyte growth factor.