JPH0832726B2 - Rat bFGF and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to rat basic fibroblast growth factor (hereinafter,
It may also be abbreviated as rbFGF. ) And its manufacturing method.

Conventional technology Basic fibroblast growth factor (bFGF) is a basic polypeptide hormone with a molecular weight of about 17,000 that is secreted mainly from the pituitary gland and initially shows a strong growth-promoting effect on fibroblasts such as BALB / c3T3 cells. Separated as a factor [D.Gosp
odarowicz; Nature 249 : 123 (1974)].
However, it was subsequently found to have a growth-promoting effect on almost all cells derived from mesoderm [D. Gospodarowicz.
Et al: National Cancer Institute Monograph 48;
109 (1978)]. Among them, the angiogenic effect of bFGF, combined with the cell growth promoting effect, shows potential as a therapeutic agent for damage and a preventive and therapeutic agent for thrombosis, arteriosclerosis and the like. However, the amount of naturally occurring rbFGF is extremely small, and the amino acid sequence of rbFGF and the base sequence of the gene have not yet been determined.

On the other hand, bovine bFGF, whose material is relatively easy to obtain, has already been purified and its amino acid sequence has been determined [F, Esch
Et al .; Proceedings of the Nationa [Proceedings of the Nationa]
l Academy of Sciences of the United States of Amer
ica (hereinafter sometimes abbreviated as Proc.Natl.Acad.Sci.USA.) 82, 6507 (1985 )]]. However, even in this case, mass production is very difficult.

Meanwhile, the production of human bFGF by recombinant DNA technology has been reported [EMBO (European Molecular
Biology Organization Journal, 5 ,
2523 (1986), PCT International Publication No. WO87 / 01728].

Problems to be Solved by the Invention As described above, there are many unclear points regarding the properties, amino acid sequence, and gene of rbFGF. Rat bFGF may also be used as a drug or a reagent. Therefore
A method of identifying a gene encoding rbFGF and mass-producing the protein by gene recombination technology has been desired.

Means for Solving the Problems In general, proteins of animals closer to humans have very high homology in their amino acid sequences, and most of the amino acid differences are in one-point mutati
It is guided by on. Therefore, the DAN sequence derived from the amino acid sequence of human bFGF described above is rbFGF
It is presumed to be very similar to the DNA sequence of a gene.

Therefore, the present inventors have based on such an idea, human bF.
A part of GF gene is used as a DNA probe, and rbFGF
The cDNA was cloned from rat cells. It was found that rbFGF is produced by constructing a recombinant DNA containing the cDNA and culturing a transformant transformed with the DNA.
The present inventors have completed the present invention as a result of further research based on these findings.

The present invention (1), amino acid sequence: Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Gly-Ala-Phe-Pro-Pro-Gly-His-Phe-Lys-Asp-Pro-Lys-. Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro-Asp-Cly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys- Ser-Asp-Pro-His-Val-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-Cys-Ala-Asn- Arg-Tyr-Leu-ala-Met-Lys-Glu- Asp-Gly-Arg-Leu-Leu-Ala-Ser- Lys-Cys-Val-Thr-Glu-Glu-Cys-Phe-Phe-Phe-Glu- Arg-Leu-Glu- Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-Thr- Gly-Gln-Tyr-Lys-Leu-Gly- Ser-Lys-Thr-Gly-Pro-Gly-Gln-Lys-Ala-Ile-Leu-Phe-Leu-pro-Met-Ser-Ala-Lys-Ser ( Rat base which is a polypeptide represented by I) Fibroblast growth factor, 2, transformed with a vector containing recombinant DNA containing a nucleotide sequence encoding rat basic fibroblast growth factor, a polypeptide represented by amino acid sequence (I) A method for producing a factor, which comprises culturing a bacterium in a medium, producing and accumulating rat basic fibroblast growth factor in the culture, and collecting the factor.

In addition, in the present specification, a recombinant DNA (III) containing a nucleotide sequence encoding rat basic fibroblast growth factor, which is a polypeptide represented by the amino acid sequence (I), and DNA (III) A transformant transformed with the vector containing is also disclosed.

 Examples of the DNA (III), for example, nucleotide sequence: CCCGCACTGC CGGAGGACGG CGGCGGCGCC TTCCCACCCG GCCACTTCAA GGATCCCAAG CGGCTCTACT GCAAGAACGG CGGCTTCTTC CTGCGCATCC ATCCAGACGG CCGCGTGGAC GGCGTCCGGG AGAAGAGCGA CCCACACGTC AAACTACAGC TCCAAGCAGA AGAGAGAGGA GTTGTGTCCA TCAAGGGAGT GTGTGCGAAC CGGTACCTGG CTATGAAGGA AGATGGACGG CTGCTGGCTT CTAAGTGTGT TACAGAAGAG TGTTTCTTCT TTGAACGCCT GGAGTCCAAT AACTACAACA CTTACCGGTC ACGGAAATAC TCCAGTTGGT ATGTGGCACT GAAACGAACT GGGCAGTATA AACTCGGATC A base sequence (V) containing CAAAACGGGG CCTGGACAGA AGGCCATACT GTTTCTTCCA ATGTCTGCTA AGAGC (IV) is preferred.

The rbFGF in the DNA of the present invention and the method for producing the same is preferably the one represented by the above amino acid sequence (I).

Expression vectors containing a DNA having a nucleotide sequence encoding a polypeptide of the rbFGF protein in the method of the present invention include, for example, (a) isolation of RNA encoding rbFGF, and (b) single-stranded complementary DNA from the RNA. (CDNA) and then double-stranded DNA are synthesized, (c) the complementary DNA is incorporated into a plasmid, (d) the host is transformed with the obtained recombinant presmid, and (e) the obtained transformant is After culturing, a plasmid containing the target DNA is isolated from the transformant by an appropriate method, for example, a colony hybridization method using a DNA probe, and (f) the target cloned DNA is excised from the plasmid, (G) It can be produced by ligating the cloned DNA downstream of the promoter in the vehicle.

RNA encoding rbFGF can be obtained from various rat organs such as brain, pituitary gland, and liver.

Examples of the method for preparing RNA from rat organs include the guanidine thiocyanate method [JMChirgwin et al., Biochemistry, 18 , 5294 (1979)].

Using the RNA thus obtained as a template, cDNA is synthesized, for example, the method of Watson and Jackson (Watson, CJand Ja
ckson, JF, DNA Cloning, A Practical Approach IRL Pr
ess, Oxford, p79, 1985), for example, λ phage vector λg + 10 (Huynh, TV et al., DNA Cloning a Practical Approach, IRL Press, Oxford, p49, 198).
5) and incorporate this into E. coli, such as C600, HflA (Huyn
h, TV et al., ibid.) to create a cDNA library.

From the cDNA library thus obtained, a method known per se, for example, plaque hybridization method (Maniatis et al., Molecular cloning (Molecu
lar Cloning) Cold Spring Harbor Laboratory, p320, 19
82) and DNA sequencing method [Proc.Natl.Acad.Sci.US
A 74, 560 (1977), using New clay click Acids Research (Nucleic Acids Research) 9, 309 (1981)], selects the phage clones sought.

Next, the forge clones are collected and, for example, the method of Davis et al. (Davis et al., Advanced Bacterial Gentics), Cold Spring.
It is also convenient to extract the phage DNA by Harbor Laboratory 1980), excise the cDNA portion thereof with a restriction enzyme, reintegrate this into a plasmid such as pUC13, and use.

The plasmid having the DNA containing the cloned nucleotide sequence encoding rbFGF is cut out as it is or, if desired, with a restriction enzyme.

The cloned gene can be ligated downstream of a promoter in a vehicle (vector) suitable for expression to obtain an expression type vector.

Examples of the vector include a plasmid derived from Escherichia coli.
pBR322 [gene, 2 , 95 (1977)], pBR325 [gene, 4 , 121 (1978)], pUC12 [gene, 19,259 (198)
2)], pUC13 [Gene, 19 , 259 (1982)], pUB110 derived from Bacillus subtilis [Biochemical and Biophysica (Biochemical and Biophysica)
l Research Communication), 112 , 6678 (1983)], pT
P, pc194, etc.

The gene contains ATG at its 5'end as a translation initiation codon.
And TAA as a translation termination codon at the 3'end,
It may have TGA or TAG. Furthermore, in order to express the gene, a promoter is connected upstream thereof. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.

When the host for transformation is a bacterium belonging to the genus Escherichia, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, etc.
When the host is Bacillus, SPO1 promoter, S
PO2 promoter, pen P promoter and the like are preferable. Particularly, it is preferable that the host is Escherichia and the promoter is trp promoter or λPL promoter.

A transformant is produced using the vector containing the DNA (III) thus constructed.

Examples of the host include Escherichia and Bacillus.

Examples of the above Escherichia bacteria include Escherichia
Escherichia coli K12DH1 [Proc.Natl.Acad.Sc
i.USA, 60, 160 (1968) ], M103 [ Nucleic Acids Research, (Nucleic Acads Research) 9, 309 (198
1)], JA221 [Journal of Molecular Biology (Journal of Molecular Biology)] 120 , 517
(1978)], HB101 [Journal of Molecular
Biology 41, 459 (1969)], C600 [ Genetics (Genetics), 39, 440 ( 1954)] , and the like.

Examples of the bacterium belonging to the genus Bacillus include Bacillus subtilis MI114 [gene, 24 , 255 (19
83)], 207-21 [Journal of Biochemistry 95 , 87 (1984)] and the like.

To transform the above Escherichia bacterium, for example,
Proc.Natl.Acad.Sci.USA, 69 , 2110 (1972), Gene, 1
7 , 107 (1982) and the like.

Transformations of Bacillus include, for example, Molecular and General Genetics.
lar & General Genetics), 168 , 111 (1979) and the like.

Thus, a transformant transformed with the vector containing DNA (III) is obtained.

When culturing a transformant whose host is a bacterium of the genus Escherichia or a bacterium of the genus Bacillus, a liquid medium is suitable as a medium used for the culture, and among them, a carbon source and nitrogen necessary for the growth of the transformant are contained. Sources, inorganic substances, etc. are included.
Examples of carbon sources include glucose, dextrin,
Examples of nitrogen sources such as soluble starch and sucrose include ammonium salts, nitrates, corn steep liquor,
Inorganic or organic substances such as peptone, casein, meat extract, soybean meal, and potato extract, examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added.

 The pH of the medium is preferably about 6-8.

As a medium for culturing with Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Mole
cular Genetics), 431-433, Cold Spring Harbor Labor
atory, New York (1972)] is preferred. If necessary, in order to make the promoter work efficiently, for example, 3
β-Indolyl A drug such as acrylic acid can be added.

When the host is Escherichia, the culture is usually about 15-43.
It may be carried out at ℃ for about 3 to 24 hours, and if necessary, aeration and stirring may be added.

When the host is a bacterium of the genus Bacillus, the culture is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and agitation can be added if necessary.

The rbFGF protein can be separated and purified from the culture described above, for example, by the following method.

When extracting rbFGF protein from cultured cells or cells, after culturing, cells or cells are collected by a known method, and the cells are suspended in a buffer solution containing a protein denaturant such as guanidine hydrochloride and then extracellularly added. A method of eluting the target protein,
French press, ultrasound, lysozyme and / or
After destroying the cells or cells by freeze-thawing, a method of obtaining rbFGF protein by centrifugation may be appropriately used. Especially, the French press method or the method of using lysozyme and ultrasonic treatment in combination is preferable.

The rbFGF protein can be purified from the above-mentioned supernatant by appropriately combining separation / purification methods known per se. These known separation and purification methods are mainly methods such as salting out and solvent precipitation, which utilize solubility, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method that utilizes the difference in molecular weight,
Ion exchange chromatography and other methods that utilize differences in charge, affinity chromatography and other methods that utilize specific affinity, reverse phase high performance liquid chromatography and other methods that utilize hydrophobic differences, isoelectric focusing The method of utilizing the difference of isoelectric points, such as the method, is included.

Further, as a specific example, contaminating nucleic acids, acidic proteins and the like can be removed by subjecting the supernatant to ion exchange chromatography using DEAE cellulose as a carrier. For example, it is effective to apply the supernatant to a DEAE cellulose column equilibrated with a buffer solution such as Tris, which is near neutral, and collect the flow-through fraction. Further, by subjecting it to ion exchange chromatography using CM cellulose or the like, the rbFGF protein, which is a basic protein, is adsorbed on the carrier, and it can be purified by eluting it with a salt solution.

RbFGF can be directly purified from the bacterial cell extract by column chromatography using an acidic resin such as CM Sephadex. For example, the supernatant can be efficiently applied by applying it to a CM-cellulose column equilibrated with a weakly acidic buffer (eg, phosphate buffer).
After washing the column with the same buffer, wash the column with salt (eg NaC
The rbFGF can be eluted by eluting with a buffer solution further containing l). These eluates can be lyophilized after dialysis.

Further, it is convenient to apply the affinity chromatography method using heparin-sepharose as a carrier to the rbFGF protein in the Escherichia coli extract as a purification method of rbFGF. For example, purify the rbFGF protein by applying the above eluent to a heparin-Sepharose column equilibrated with a buffer such as Tris or phosphate near neutrality, washing thoroughly, and then performing linear gradient elution with NaCl or the like. You can

In particular, a heparin column developed for high performance liquid chromatography (for example, Shodex AF-pak HR.894 Showa Denko) is effective.

Similar to the above heparin sepharose column, if the sample is applied with a buffer solution near neutrality, washed thoroughly and then eluted with a linear gradient of NaCl or the like, rbFGF can be recovered as a substantially uniform preparation.

The thus obtained standard is dialyzed and lyophilized,
It can also be a dry powder. Furthermore, it is preferable to add serum albumin or the like as a carrier and store it, since it is possible to prevent the sample from adsorbing to the container.

Also, the coexistence of a small amount of a reducing agent in the purification process or the storage process is suitable for preventing the oxidation of the preparation. Examples of the reducing agent include β-mercaptoethanol, dithiothreitol and glutathione.

In this way, substantially pure rbFGF, which is substantially free of pyrodiene and endotoxin, is obtained. Examples of the substantially pure rbFGF of the present invention include those having a protein content of rbFGF of 95% (w / w) or more, more preferably rbFGF of 98% (w / w) or more.

RbFGF obtained by the gene recombination technique of the present invention
As an example of the protein, for example, the protein represented by the polypeptide represented by amino acid numbers 1-145 in the amino acid sequence in FIG. 2 can be mentioned. The polypeptide may have Met at its N-terminus.

The activity of the rbFGF thus produced is known to be known as BALBA / c3T3.
It can be measured by the cell growth promoting effect.

In cells that are genetically infected or transformed with the DNA of the present invention, a large amount of rbFGF can be produced even in various cells that originally synthesize only a small amount of rbFGF, or not at all, and rbFGF can be advantageously derived.

The expression-type plasmid containing the gene encoding the rbFGF protein of the present invention can be introduced into various cells to cause the cells to produce rbFGF.
You can get a large amount of GF.

Since rbFGF produced here has a cell proliferation promoting activity and low toxicity, it is used as a healing accelerator for burns, wounds, postoperative tissues, etc., or as a therapeutic agent for thrombosis and arteriosclerosis due to angiogenic action. Can be used. It can also be used as a reagent for promoting cell culture.

When the rbFGF of the present invention is used as a medicine, it may be used as a powder as it is, or together with other pharmacologically acceptable carriers, excipients and diluents, a pharmaceutical composition (eg, injections, tablets,
As a capsule, liquid, ointment, a warm-blooded mammal (eg,
It can be safely administered parenterally or orally to humans, mice, rats, hamsters, rabbits, dogs, cats).

The injection is formulated according to a conventional method using, for example, physiological saline or an aqueous solution containing glucose and other auxiliary agents. Pharmaceutical compositions such as tablets and capsules can also be prepared according to a conventional method. Furthermore, an injection as a pharmaceutical composition,
When manufacturing liquids, tablets, capsules, etc., it is performed under aseptic conditions.

When the rbFGF of the present invention is used as the above-mentioned drug, for example, the above-mentioned warm-blooded animal is administered in an appropriate dose selected from the range of about 1 ng to 100 μg / kg per day in consideration of the administration route and symptoms. To be done.

Moreover, when the rbFGF of the present invention is used as a reagent for promoting cell culture, it is about 0.01-10 μm per medium 1.
g, and more preferably 0.1 to 10 μg of the medium is preferably added.

In the specification and the drawings of the present invention, when bases, amino acids and the like are indicated by abbreviations, IUPAC-IUB Commision on Bi
It is based on the abbreviations by ochemical Nomenclature or the abbreviations commonly used in this field, and examples are given below. When amino acids may have optical isomers, the L-form is shown unless otherwise specified.

DNA: Deoxyribonucleic acid cDNA: Complementary deoxyriboacid A: Adenine T: Thymine G: Guanine C: Cytosine RNA: Ribonucleic acid dATP: Deoxyadenosine triphosphate dTTP: Deoxythymidine triphosphate dGTP: Deoxyguanosine triphosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate Tdr: Thymidine EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine Escherichia coli K12 DHI obtained in Reference Example 1 below.
/ pTB627 was deposited at the Fermentation Research Institute (IFO) under the deposit number IFL 14494 from March 13, 1986, and this microorganism has been registered on the Ministry of International Trade and Industry since April 2, 1986. Contract number F to the Institute of Microbial Technology (FRI)
Deposited as ERM P-8726, which has been converted to a deposit under the Budapest Treaty and is deposited under accession number FERM BP-1280.
Is stored in the Institute (FRI).

Escherichia coli K12 DHI obtained in Example 1 described later
/ pTB784 has been deposited with IFO under the deposit number IFO 14616 from May 29, 1987, and this microorganism is
It has been deposited with FRI under the deposit number FERM P-9403 from June 6, 2013.

EXAMPLES The present invention will be described in more detail with reference to the following reference examples and examples, but the present invention is not limited thereto.

Reference Example 1 (Construction of plasmid containing cDNA encoding human bFGF) cDNA synthesized from human foreskin-derived primary culture cell mRNA was transformed into pC.
CD using E. coli Х1776 as a host, which was prepared by incorporating it into a D vector [Okayama et al., Molecular Cell Biology, 3 , 280 (1983)]
NA library to National Institute of Child Health
and Dr. Okayama of Human Development, Bethesda, USA. From this cDNA library, the alkaline method (Birnboim.HC & Doly.J. Nucleic Acids
Research (Nucleic Acids Research), 1 , 1513 (197
9)], the plasmid DNA was extracted, and this DNA was infected with Escherichia coli DHI to prepare a cDNA library using E. coli DHI as a host consisting of about 2 × 10 ⁶ clones.

Spread 10 cDNA libraries using E. coli DHI on a nitrocellulose filter (Millipore, HATF filter) at about 5 × 10 ⁴ clones / filter,
A total of 20 replica filters were prepared, each set consisting of two filters, each of which was used as a master filter. E. coli on this replica filter was dissolved in 0.5N NaOH solution, and the exposed denatured plasmid DNA was immobilized on the filter [Grunstein, M. & Hogness, DS, Proc.Nat.
l.Acad.Sci.USA 72 , 3961 (1975)].

On the other hand, [Proc.Natl.Ac] reported by F. Esch et al.
ad.Sci.USA 82 , 6507 (1985)] Amino acid No. 13-20 based on the amino acid sequence of bovine basic fibroblast growth factor
(Pro-Pro-Gly-His-Phe-Lys-Asp-Pro) and amino acids No. 89-96 (Thr-Asp-Glu-Cys-Phe-Phe-Phe-Phe.
-Glu), the base sequences corresponding to these amino acid sequences were chemically synthesized. For some codons, the third letter was arbitrarily fixed. 5'GC A / GT CT / C TT A / G respectively
AA A / G TG G CC A GG A GG, and 5'TC A / G AA A / G AA A / G
AA A / G CA T / C TC G TC G GT, and the underlined bases are fixed. Using T4 polynucleotide kinase (Takara Shuzo) against this oligonucleotide, 50
μl reaction solution [Oligonucleotide 0.1 μg, 50 mM Tris-
HCl pH8.0,10mM MgCl ₂ , 10mM mercaptoethanol, 50μ
Ciγ- ³² P ATP (> 5000 Ci / mmole), 3 units T4 polynucleotide kinase] and reacted at 37 ° C. for 1 hour, and the 5 ′ end of the oligonucleotide was labeled with ³² P.

Two kinds of the oligonucleotides labeled by the above method were used as probes and separately associated with the replica filter having the DNA immobilized thereon. The association reaction includes 10 μCi of probe
× SSPE (180mM NaCl, 10mM NaH ₂ PO ₄ , 1mM EDTA (pH 7.
4)], 5 x Denhard's, 0.1% SDS, 100 μg / ml denatured salmon sperm
Perform the reaction in 10 ml of DNA solution at 35 ℃ for 16 hours, and after the reaction, filter 5 × SSC [0.15M NaCl, 0.015M Sodium citrate] 0.1%
It was washed with SDS solution at room temperature three times for 30 minutes each and further twice at 45 ° C for 30 minutes each [T. Maniatis et al., "Molecular Cloning" Cold S
pring Harbor Ladoratory, P.309 (1982)].

Take a radio autogram from the washed filter,
A set of 2 strains that react with two types of probes
It was searched by superimposing radio autograms of the replica filters. By this method 5 × 10 ⁵ cloni
One strain that reacts to two types of probes from es [Escher
ichia coli K12 DH1 / pTB627 (IFO14494, FERM BP-128
0)], and thereby the plasmid DNA (pTB627) was extracted by the alkaline method [HC Birmboim et al., Nucleic Acids Research 1 , 1513 (1979)].

Example 1 (1) Preparation of cDNA Library Derived from Rat Brain mRNA RAN was isolated from rat brain by the guanidine isothiocyanate method [Chirgvim et al., “Biochemistr.
y) ”, 18 , 5294, 1978]. Poly (A) RNA was purified from this RNA by oligo dT cellulose column chromatography [Aviv and Lader, Proc. Natl. Acad. Sci. USA 69 , 1408, (1972)]. Using this poly (A) RNA as a template, a cDNA library was prepared using Watso
n Jackson's method [Watson.C Jand Jackson, JF, DNA cloning a practical approach (DNA Cl
oning A Practical Approach) IRL Press, Oxford, p79,1
985] according to λ phage vector λgt10 (Huynh, T.
V. et al. DNA Cloning a Practical Approach, IRL Press, Oxford, p49, 1985). Ten
Starting from μg of poly (A) RNA, it was possible to obtain a cDNA library consisting of approximately 1.5 × 10 ⁶ clones using Escherichia coli C600, HflA (Huynh TV et al., supra) as a host.

(2) E. coli C600, Hfl with the above phage cDNA library
Using A as a host, spread about 1 x 10 ⁵ clones each on 10 plates on a soft agar plate, transfer this to a nitrocellulose filter (Millipore, HATF filter), then 0.5N
Phage DNA that had been exposed and denatured by combing with a NaOH solution was dried and fixed on a filter (Maniatis et al., “Moleculer Cloning” Cold Spring Harbor).
Laboratory, p320, 1982).

On the other hand, human obtained by digesting the presmid ptB627 containing human bFGF cDNA obtained in Reference Example 1 with the restriction enzyme BamHI
A 430 bp DNA fragment containing the bFGF coding region was labeled with ³² P by the nick translation method (Maniatis et al., supra, p109) and used as a probe.

The labeled probe and the DNA-immobilized filter
5x SSPE [0.9M NaCl 50mM sodium phosphate buffer (pH7.4), 5mM EDTA] 50% formamide containing labeled probe, 5x Denhardt's, 0.1% SDS, 100μg / ml denatured salmon sperm DN
Perform the association reaction in 10 ml of A solution at 42 ° C for 16 hours, and after the reaction,
The filter was placed in 2xSSC (1xSSC = 0.15M NaCl, 0.015M sodium citrate), 0.1% SDS solution twice at room temperature for 30 minutes twice each in 1xSSC, 0.1% SDS solution at 55 ° C. It was washed twice for each minute. After the washed filter was dried, a radio-autogram was taken to search for a clone that reacts with the probe. Da from clone λRF-1 obtained by this method
vis et al. (Davis et al., “Advance Bacterial.
Genetics (Advanced Bacterial Genetics), Co
ld Spring Harbor Laboratory 1980) Phage DN
A was extracted, and a cDNA portion having a chain length of 820 bp was cut out from this by digestion with a restriction enzyme EcoRI. A restriction enzyme map of this cDNA portion is shown in FIG.

This cDNA portion was incorporated into the EcoRI portion of plasmid pCU13 to prepare ptB784.

Escherichia coli K12 DHl was transformed with this plasmid pTB784 to obtain a transformant Eschrichia coli K12 DHl / pTB784 (IFO 14616, FE containing the plasmid pTB784.
RMP-9403) was obtained.

(3) Next, the nucleotide sequence of the EcoR I 820 bp DNA fragment, which is the cDNA portion of pTB784 obtained in (2) above, was analyzed by the dideoxynucleotide synthetic chain termination method [Messing et al., "Nucleic Acids Research" 9 , 30
9, (1981)].

From this result, the entire amino acid sequence of rat bFGF could be determined.

The nucleotide sequence of cDNA and the amino acid sequence deduced from the sequence are shown in FIG. In Fig. 2, trm is terminater
Indicates codon.

The amino acid sequence shown in FIG. 2 is very similar to the reported amino acid sequence of bovine or human bFGF, indicating that this cDNA encodes rat bFGF.

Reference Example 2 Method for Detecting rbFGF Activity The rbFGF activity can be expressed by the weight of a purified bovine brain FGF preparation (Takara Shuzo Co., Ltd.) showing the same activity due to its growth promoting effect on BALB / c3T3 cells.

Mouse BALB / c3T3 cells were seeded in DMEA medium containing 5% fetal calf serum in a Nunc 96-well microtiter plate (flat bottom) at 2 × 10 ³ cells / well in 0.2 ml medium, and cultured.
The next day, change to DMEM medium containing 0.5% calf serum. After culturing for 3 days, add 10μ per well of the specimen diluted stepwise with DME medium containing 0.5% BSA, and culture.
After ³ hours, ³ H-Tdr (5 Ci / mmol, 0.5 mCi / ml RCC Amersham)
Add 2μ to each hole. After 6 hours, the cells were removed by treatment with a phosphate buffer solution (PBS) containing 0.2% trypsin-0.02% EDTA, and the cells were collected on a glass filter using a titer tech cell harvester to incorporate the cells ³
-Measure the Tdr amount with a scintillation counter.
Bovine brain FGF (Takara Shuzo) activity of known weight is measured by the same operation, and the amount of rbFGF in the sample can be calculated from the obtained calibration curve.

Example 2 Expression of rbFGF-encoding gene in E. coli: (1) Construction of rbFGF expression plasmid pTB786 The plasmid pTB784 containing the rbFGF cDNA obtained in Example 1 (2) was cleaved with EcoRI to give a 0.8 kb DNA fragment. To get
This DNA fragment is further cleaved with HaeII to obtain a 0.5 kb DNA fragment. Separately, synthetic oligonucleotide 5'AATTCTATGCCAGCATTGC, 5'CGGAAGATGGAGGTGGCGC, 5 'after phosphate reaction
TCCGGCAATGCTGGCATAG and 5'CACCTCCATCT are ligated with T4 DNA ligase, which is further ligated to the above 0.5 kb DNA fragment using T4 DNA ligase. The combined product is EcoR I
Digest with to prepare a 0.54 kb DNA fragment.

On the other hand, a plasmid ptrp781 having a trp promoter
[Kurokawa, T., et al. New Clay Acids Research (Nucleic Acids Res.) 11 , 3077-3085 (1983)] DNA
Is cleaved with EcoR I, and the above 0.54 kb DNA is ligated to this DNA for circularization to obtain a plasmid. Of these plasmids, the one in which the rbFGF cDNA is inserted in the forward direction of the trp promoter is selected to obtain the rbFGF expression plasmid pTB786.

Transformant Es containing plasmid pTB786 by transforming Escherichia coli DHl with this plasmid pTB786
Obtain cherichia coli DHl / ptB786.

(2) Preparation of bacterial cell extract The above transformants were cultured in M9 medium containing 1% glucose, 0.4% casamino acid and 8 μg / ml tetracycline, respectively, and at the time when the Klett value was about 200, 3β indolyl acryl was obtained. Acid is added to 25 μg / ml and the culture is continued for 4 hours. After culturing, collect the microbial cells and 1/20 volume of 20 mM Tris ・ HC
Suspend in l, pH 7.6.10% sucrose solution. Phenylmethylsulfonyl fluoride (PMSF) was added to this suspension.
1mM, EDTA 10mM, NaCl 0.1M, spermidine hydrochloride 10m
M and lysozyme were added at 100 μg / ml (both final concentrations), left at 0 ° C. for 45 minutes, and then sonicated for 30 seconds. This solution is centrifuged at 1800 rpm (serval centrifuge, SS34-rotor) for 30 minutes to obtain a supernatant, which is used as a cell extract.

By using the method described in Reference Example 2, strong rbFGF activity can be detected in the cell extract.

Effects of the Invention By culturing a medium of the transformant of the present invention, rb
Since FGF can be produced in large quantities, a large amount of rbFGF useful as medicines, reagents, etc. can be supplied.

[Brief description of drawings]

FIG. 1 shows the restriction enzyme map of the cDNA portion obtained in Example 1. FIG. 2 shows the nucleotide sequence of the cDNA portion obtained in Example 1,
The amino acid sequence deduced from the sequence is shown.

Continued on the front page (51) Int.Cl. ⁶ Identification number Reference number in the agency FI Technical indication (C12P 21/02 C12R 1:19) (C12N 1/21 C12R 1:19)

Claims (2)

[Claims] 1. Amino acid sequence: Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Gly-Ala-Phe-Pro-Pro-Gly-His-Phe-Lys-Asp-Pro-Lys-Arg- Leu-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro- Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser- Asp-Pro-His-Val-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-Cys-Ala-Asn-Arg- Tyr-Leu-Ala-Met-Lys-Glu- Asp-Gly-Arg-Leu-Leu-Ala-Ser-Lys-Cys-Val-Thr-Glu-Glu-Cys-Phe-Phe-Phe-Glu-Arg- Leu-Glu- Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-Thr-Gly- Gln-Tyr-Lys-Leu-Gly- Ser-Lys-Thr-Gly-Pro-Gly-Gln- Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser Rat basic fibroblast, which is a polypeptide Cell growth factor. 2. Amino acid sequence: Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Gly-Ala-Phe-Pro-Pro-Gly-His-Phe-Lys-Asp-Pro-Lys-Arg- Leu-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-Phe-Leu-Arg-Ile-His-Pro- Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu-Lys-Ser- Asp-Pro-His-Val-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly-Val-Cys-Ala-Asn-Arg- Tyr-Leu-Ala-Met-Lys-Glu- Asp-Gly-Arg-Leu-Leu-Ala-Ser-Lys-Cys-Val-Thr-Glu-Glu-Cys-Phe-Phe-Phe-Glu-Arg- Leu-Glu- Ser-Asn-Asn-Tyr-Asn-Thr-Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-Thr-Gly- Gln-Tyr-Lys-Leu-Gly- Ser-Lys-Thr-Gly-Pro-Gly-Gln- Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser Rat basic fibroblast, which is a polypeptide Bacteria transformed with a vector containing a recombinant DNA containing a nucleotide sequence encoding a vesicle growth factor are cultured in a medium to produce and accumulate rat basic fibroblast growth factor in the culture, which is collected. A method for producing the factor, which comprises: