JP2779621B2 - Monoclonal antibodies, hybridomas, their production methods and their uses - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to monoclonal antibodies that specifically bind to basic fibroblast growth factor, hybridomas, methods for producing them, and uses thereof.

2. Description of the Related Art Basic fibroblast growth factor (herein, bFGF
May be abbreviated. ) Is a basic polypeptide hormone having a molecular weight of about 17,000, which is mainly secreted from the pituitary gland, and was initially isolated as a factor having a strong growth promoting effect on fibroblasts such as BALB / c3T3 cells [D. Gospodarowicz;
Nature 249 : 123 (1974)]. However, it was later found to show a growth-promoting effect on almost all cells derived from mesoderm [D. Gospodarowicz et al .; National Cancer Institute Monograph 48 ; 109 (197
8)]. Above all, the angiogenesis effect of bFGF, combined with the cell proliferation effect, shows potential as a remedy for damage and burns and as a prophylactic and remedy for thrombosis and arteriosclerosis.

Problems to be Solved by the Invention The amount of naturally occurring human bFGF is extremely small, and attempts to obtain it from human tissues have been extremely difficult due to various restrictions. Furthermore, a method that can be easily used as a method for quantifying bFGF has not been established so far.
BF indispensable for developing GF as a pharmaceutical
There are many unclear points about basic knowledge such as properties of GF.

Therefore, if a lot of basic knowledge on bFGF, for example, the distribution of bFGF in a living body and its production mode can be known, the development of bFGF as a pharmaceutical becomes easy.

It is also important to know the exact amount of bFGF when purifying this protein from a recombinant. further,
It is very important to monitor the blood FGF concentration of the animal to which bFGF has been administered, but it cannot be measured by the conventional method using 3T3 cells because serum is mixed in the sample. Usually bFG
The measurement of F is back-calculated based on how much the DNA synthesis ability is restored by adding bFGF to 3T3 cells in which the serum concentration has been reduced and cultured, and DNA synthesis has been reduced. However, this method has drawbacks that the operation is delicate and measurement error is large because cells are used, and it takes a long time to obtain a result. Therefore, it is desired to develop a simple and accurate means for measuring bFGF for the above purpose.

Means for Solving the Problems In view of the above circumstances, the present inventors have conducted various studies in order to find a practical means of measuring bFGF, and as a result, produced a monoclonal antibody that specifically binds to bFGF, which makes the measurement possible. As a result of further research based on this, the present invention was completed.

The present invention has the following properties: (1) a monoclonal antibody that specifically binds to bFGF; (a) molecular weight: about 140 to 160 kilodaltons (b) does not cross-react with acidic fibroblast growth factor.

(C) The immunoglobulin class belongs to IgG.

(D) Hybridoma HbF12 (IFO 50142), hybridoma HbF52 (IFO 50143), hybridoma HbF7, respectively
8 (IFO 50144) and hybridoma HbF98 (IFO 5014)
5) MoAb12, MoAb52, MoAb78 and M produced by
a monoclonal antibody selected from the group consisting of oAb98, (2), hybridoma HbF12 (IFO 50142), hybridoma HbF52 (IFO 50143), hybridoma HbF78 (IFO
Hybridoma HbF12 (50) and hybridoma HbF98 (IFO 50145); (3) hybridoma HbF12 (cell fusion of spleen cells of a mouse immunized with bFGF and mouse myeloma, followed by cloning). IFO 50142), hybridoma HbF52 (IFO 50143), hybridoma HbF78 (IFO
50144) and a method for producing a hybridoma selected from the group consisting of hybridoma HbF98 (IFO 50145), (4), a cloned hybridoma comprising spleen cells of a mouse immunized with bFGF and mouse myeloma
HbF12 (IFO 50142), hybridoma HbF52 (IFO 5014)
3) that a hybridoma selected from the group consisting of hybridoma HbF78 (IFO 50144) and hybridoma HbF98 (IFO 50145) is grown in a liquid medium or in the peritoneal cavity of a mouse to produce and accumulate monoclonal antibodies; A method for producing a monoclonal antibody which specifically binds to the factor, (5) a method for purifying bFGF using the monoclonal antibody of the above (1), and (6) a method for the above (1) A method for detecting and quantifying bFGF, characterized by using the monoclonal antibody described in (1).

The bFGF may be any bFGF of a warm-blooded mammal. Alternatively, the mutein may be used. Therefore, as used herein, “basic fibroblast growth factor (bFGF)” may also include its mutein unless otherwise specified.

Representative examples of the mammalian bFGF include, for example, bovine bFGF “Proceedings of the National
Academy of Sciences (Proc.Natl.Acad.Sc
i.) USA, Vol. 82, pp. 6507 (1985)], Human bFGF
[Japanese Patent Application No. 61-241053 (European Patent Application Publication No. 237,966), European Molecular Biology Organization (EMBO) J
ournal), Vol. 5, p. 2523 (1986)].

More specifically, Is preferred.

In addition, the general formula Wherein X represents Thr or Ser, and when X is Thr, Y is S
er, and when X is Ser, Y indicates Pro. Are preferred.

Human bFGF (hereinafter sometimes abbreviated as hbFGF)
For example, an expression type vector containing a DNA having a nucleotide sequence encoding the above-described hbFGF protein polypeptide can be obtained by, for example, (a) isolating RNA encoding hbFGF, and (b) simply separating the RNA from the RNA. (C) incorporating the complementary DNA into a plasmid, (d) transforming a host with the obtained recombinant plasmid, and (e) obtaining the complementary DNA (c). After culturing the transformant, a plasmid containing the desired DNA is isolated from the transformant by an appropriate method, for example, a colony hybridization method using a DNA probe, and (f) the desired cloning from the plasmid. DNA is cut out, and (g) the cloned DNA is ligated downstream of a promoter in a vehicle.

RNA encoding hbFGF can be obtained from various hbFGF-producing cells, for example, human pituitary-derived cells or human fibroblasts. The human fibroblasts include WI38 (AT
CC number CCL-75) or IMR90 (ATCC number CCL-186). The cells WI38 and IMR90 are used in the American Type Culture Collection (The Am
catalog of Cell Lines and Hybridomas (Cata, published by erican Type Culture Collection)
logue of Cell Lines & Hybridomas) 5th edition, 198
It is listed on 5.

The expression vector thus obtained is integrated into an appropriate host (eg, Escherichia coli, Bacillus subtilis, yeast, animal cells),
By culturing the obtained transformant in a medium, human bFGF can be produced.

The mutein in the present invention is originally a mutation of the amino acid sequence of the original peptide or protein. Therefore, the mutation includes addition of an amino acid, deletion of a constituent amino acid, and substitution with another amino acid.

Examples of the addition of the amino acid include those in which at least one amino acid is added.

The deletion of the constituent amino acids includes at least one bF
One in which a GF-constituting amino acid is deleted is mentioned.

The substitution with the other amino acid includes at least one
bFGF-constituting amino acids may be substituted with another amino acid.

The at least one amino acid in the mutein in which at least one amino acid has been added to bFGF does not include methionine caused by the initiation codon used in expressing the peptide and a signal peptide.

The number of added amino acids should be at least 1
The number may be any number as long as the characteristics of bFGF are not lost.
More preferably, a part or all of the amino acid sequence of a protein having homology (homology) with bFGF and exhibiting the same activity can be mentioned.

The number of deficient constituent amino acids in a mutein deficient in at least one bFGF constituent amino acid of bFGF may be any number as long as the characteristics of bFGF are not lost.

Examples of the missing constituent amino acids include human bFGF
Amino-terminal 10 residues of: 14 amino-terminal residues of human bFGF: And the like.

The number of at least one bFGF-constituting amino acid before substitution in a mutein in which at least one bFGF-constituting amino acid of bFGF is substituted with another amino acid is bFGF
Any number may be used as long as the characteristics of are not lost.

Examples of the constituent amino acids before substitution include cysteine and those other than cysteine. Cysteine is particularly preferred. Examples of constituent amino acids other than cysteine before substitution include aspartic acid, arginine, glycine, valine and the like.

When the constituent amino acid before substitution is cysteine, the substituted amino acid is preferably, for example, a neutral amino acid. Specific examples of the neutral amino acid include glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine, histidine,
Tryptophan, serine, threonine, methionine and the like. Particularly, serine and threonine are preferred.

When the constituent amino acid before substitution is other than cysteine, another substituted amino acid is, for example, an amino acid before substitution in terms of hydrophilicity, hydrophobicity or charge of the amino acid. Choose one with different properties. Specifically, when the amino acid before substitution is aspartic acid, the amino acids after substitution are asparagine, threonine, valine, phenylalanine,
Although arginine etc. are mentioned, especially asparagine and arginine are preferable.

When the amino acid before substitution is arginine, the amino acids after substitution are glutamine, threonine,
Leucine, phenylalanine and aspartic acid are exemplified, and glutamine is particularly preferred.

When the constituent amino acid before substitution is glycine, the amino acid after substitution is threonine,
Leucine, phenylalanine, serine, glutamic acid,
Arginine and the like can be mentioned, and threonine is particularly preferable.

When the constituent amino acid before substitution is serine, the amino acid after substitution is methionine,
Examples thereof include alanine, leucine, cysteine, glutamine, arginine, and aspartic acid, and methionine is particularly preferable.

When the constituent amino acid before substitution is valine, examples of the amino acid after substitution include serine, leucine, proline, glycine, lysine, and aspartic acid, with serine being particularly preferred.

As the original constituent amino acids before substitution, aspartic acid, arginine, glycine, serine, and valine are preferable.

Amino acids after substitution include asparagine,
Glutamine, arginine, sulfonin, methionine, serine, leucine are preferred.

The most preferred substituted muteins are those in which the constituent amino acid cysteine is replaced by serine.

In the above substitution, two or more substitutions may be performed simultaneously. In particular, it is preferred that two or three constituent amino acids are substituted.

The mutein of the present invention is characterized by the addition, deletion and substitution described above.
One or a combination of three may be used.

In order to produce the mutein, a site-directed mutagenesis technique is employed.
The technology is well known and is available from Lathe
r, RF) and Jay Lecoq (Lecoq, JP),
Genetic Engineer
ing), Academic Press (1983) pp. 31-50,
Is shown in Oligonucleotide directed mutagenesis was performed by M. Smith (M.) and S. Gillam (Gi.
llam, S.), Genetic Engineering: Principles and Methods, Plenum Press (1981), Vol. 3, pp. 1-32.

In order to produce the structural gene encoding the mutein, for example, (a) a single-stranded DNA consisting of a single strand of the structural gene of bFGF is hybridized with a mutant oligonucleotide primer (this single-stranded Cysteine codons to be replaced,
Alternatively, the primer is complementary to a region encompassing an antisense triplet that pairs with this codon. However, this does not apply to a mismatch between the codon and another codon for amino acid encoding, or in some cases, an antisense triplet. And (b) extending the primer with a DNA polymerase,
Forming a mutated heteroduplex, and (c) replicating the mutated heterodimer.

Next, the phage DN carrying the mutated gene
A is isolated and integrated into a plasmid.

Muteins can be produced by transforming a suitable host (as described above) with the thus obtained plasmid and culturing the resulting transformant in a medium.

When immunizing the bFGF, bFGF may be used as a complex with a carrier protein and then used for immunization.

Examples of the carrier protein include Freund's complete adjuvant (manufactured by Difco).

When a carrier protein complex is used, the coupling ratio between the carrier protein and bFGF is about 5 to 30 times (carrier / bFGF: weight ratio). Desirably about 15-20
Double is used.

Further, various condensing agents can be used for coupling the hapten and the carrier, and glutaraldehyde, carbodiimide and the like are conveniently used.

When immunizing with bFGF or a protein complex, mammals to be immunized include sheep, goats, rabbits, guinea pigs, rats,
Although experimental animals such as mice are used, rats and mice are preferred for obtaining monoclonal antibodies, and mice are particularly preferred. For example, when immunizing a mouse, subcutaneous, intraperitoneal, intravenous, intramuscular, intracutaneous, or any other route is possible.
Preferably, it is injected intravenously (especially subcutaneously). The immunity interval, immunity amount, etc. are also highly variable, and various methods are possible. For example, immunization is performed about 2 to 6 times at 2 week intervals, and about 1 to 5 times, preferably about 2 A method using spleen cells excised 4 days later is often used. The amount of immunization was about 0.1 μg or more per mouse as the amount of peptide at a time.
Preferably, about 10 μg to 300 μg is used.
Before removing the spleen, it is preferable to perform a fusion experiment using spleen cells after performing partial blood sampling to confirm an increase in the antibody titer in the blood.

Cell fusion between the above spleen cells and lymphoid cells is performed, for example, by removing the spleen cells of the excised mouse from hypoxanthine-guanine-phosphoribosyltransferase deficiency. Or thymidine kinase deficiency And a suitable homologous or heterologous (preferably homologous) lymphoid cell line with a marker such as As the lymphoid cell line, myeloma is preferable, and examples thereof include myeloma P3-X63-Ag / 8U1 (Ichimori et al., Journal of Immunological Methods 80 55 (198)
5)). For example, it is produced by fusing according to the method of Keller and Milstein et al. [Nature 256 : 495 (1975)]. For example, a myeloma cell and a spleen cell at a ratio of about 1: 5, for example, a 1: 1 mixture of Iscove's medium and Ham's F-12 medium (hereinafter I)
It is called H medium. ), And a fusion agent such as Sendai virus and polyethylene glycol (PEG) is used. Of course, it is also possible to add dimethyl sulfoxide (DMSO) or other fusion promoters. The degree of polymerization of PEG is
Usually about 1000-6000, time about 0.5-30 minutes, concentration about 10%
~ 80% or the like is used.
By treating PEG6000 at about 35 to 55% for about 4 to 10 minutes, efficient fusion can be achieved. The fused cells can be selectively grown using a hypoxanthine-aminopterin-thymidine medium [HAT medium; Nature, 256 , 495 (1975)] or the like.

The culture supernatant of the proliferating cells can be screened for the production of the desired antibody, and the antibody titer can be screened as follows. That is, in this case, first, as a first step, the presence or absence of production of an antibody against the immunized peptide is determined by a radioimmunoassay (RIA) method or an enzyme immunoassay (EI
A) The method can be examined by a method such as the method, but various modifications of these methods are also possible. As an example of a preferable measurement method, one method using EIA will be described.
For example, a rabbit anti-mouse immunoglobulin antibody is coupled to a carrier such as cellulose beads according to a conventional method, and a culture supernatant or mouse serum to be measured is added thereto, and a constant temperature (about 4 to 40 ° C. The same applies to the following.) Thereafter, the reaction product is thoroughly washed, and then a peptide labeled with an enzyme (purified after coupling the enzyme and the peptide according to a conventional method) is added, and the reaction is carried out at a constant temperature for a certain period of time. After thoroughly washing the reaction product, an enzyme substrate is added, and the reaction is allowed to proceed at a constant temperature for a certain period of time. Thereafter, the formed color product can be measured by absorbance or fluorescence.

It is desirable that the cells of the wells that show growth in the selective medium and exhibit only antibody activity against the peptide used for immunization be cloned by a limiting dilution method or the like. By screening the supernatant of the cloned cells in the same manner and increasing the number of well cells with a high antibody titer, a monoclonal antibody-producing hybridoma clone that is reactive with the immunized peptide can be obtained.

The hybridoma cloned in this way is
Grow in liquid medium. Specifically, for example, a liquid medium such as RPMI-1640 [Moore, GE, et.al.
Of American Medical Association (J.
Am.Med.Assoc.) 199, 549 (1967 )] of about between 0.1 to 40% bovine serum medium and the like at about 2-10 days plus, preferably about 3
By culturing for 5 days, the monoclonal antibody can be obtained from the culture solution. In addition, an antibody can be obtained by inoculating intraperitoneally into a mammal, growing cells, and collecting ascites. For this, for example, in the case of mice, BALB / c inoculated in advance with mineral oil, etc.
About 1 × 10 ⁴ to 1 × 10 ⁷ mice, preferably about 5 ×
10 ^{5 to} 2 × 10 ⁶ hybridomas are inoculated intraperitoneally, and the ascites fluid is collected after about 7 to 20 days, preferably about 10 to 14 days. Antibodies produced and accumulated in ascites fluid include, for example, ammonium sulfate fraction, DEAE
-Monoclonal antibodies can be easily isolated as pure immunoglobulin by cellulose column chromatography or the like.

Thus, a monoclonal antibody that specifically binds to bFGF is obtained.

The monoclonal antibody of the present invention specifically binds to the immunogenic peptide bFGF.

The monoclonal antibody of the present invention may bind to bFGF different from the peptide of the immunogen used at the time of production.

The monoclonal antibody of the present invention is a monoclonal antibody against the immunogenic peptide bFGF or its mutein.

The monoclonal antibody of the present invention has a property that the antibody binds only to bFGF (and its mutein).
Is a monoclonal antibody that specifically binds to

As shown in Example 3 below, when human bFGF is used as an antigen, a monoclonal antibody whose immunoglobulin class belongs to IgM may be obtained.

Since the monoclonal antibody of the present invention specifically binds to bFGF, it is extremely useful as a reagent for measuring bFGF. Furthermore, facilitating the measurement of bFGF in living organs and tissues is a basic knowledge of bFGF (eg, biodistribution).
It is extremely useful in obtaining. In living organs and tissues
For the detection of bFGF, quantification by enzyme immunoassay (EIA) or the like, or fluorescent antibody method or radioimmunoassay (RIA) is usually used. In addition, western blotting of proteins is effective for determining the size of bFGF present in these organs and tissues. In this method, a crude extract derived from an organ or tissue or a partially purified sample thereof is subjected to acrylamide electrophoresis, then transferred to a membrane filter, and detected with an HRP-conjugated anti-bFGF antibody.

Also, some cancer cells themselves produce bFGF,
It is conceivable that the growth may be continued by GF. When anti-bFGF antibody acts on such cancer, it promotes proliferation
bFGF is neutralized, and is expected to inhibit the growth of cancer cells, that is, to act as an anticancer substance. In the case of bFGF-producing cancer, bFGF can be quantified using an antibody, and the present invention can be applied to a diagnostic test for cancer. Further, utilizing the binding ability between the antibody and bFGF, an antibody affinity column was prepared to prepare bFGF
It can also be used as a reagent for purification of.

EIA or R used to detect and quantify bFGF
In the IA method, for example, the purified antibody is immobilized on a carrier such as a 0.1 to 10 μg / well 96-well plastic plate (for example, an immunoplate from Nunc, Denmark), glass beads, or plastic beads. The fixation is carried out by reacting the plastic at about 4 ° C. overnight or at room temperature for about 0.5 to 4 hours. For glass, for example, Pro
c. Natl. Acad. Sci. USA, Vol. 80, pp. 3513-3516 (1983
Year). In addition, commercially available various plates for immobilizing antibodies (described above, Nunc, etc.) can also be used.

A solution containing the antigen bFGF (or its mutein) is added to the plate or beads on which the antibody is immobilized as described above, and an adsorption reaction is performed. The adsorption reaction may be performed at room temperature for about 0.2 to 2 hours, but preferably at about 4 ° C. overnight.

After antigen-antibody binding reaction, enzyme labeling (for EIA)
Alternatively, a radiolabeled (in the case of RIA) antibody is added to perform an adsorption reaction. As the enzyme, horseradish peroxidase (HRP), alkaline phosphatase and the like are often used, and ¹²⁵ I is often used as the radiolabel. For enzyme labels, the reaction substrate, e.g., 2,2 'for HRP
-Azinodi- [3-ethylbenzothiazoline sulfonate (6)] (2,2'-Adino-di [3-ethylbenzoth
iazoline sulfonate (6)]) and the like, and the absorbance is measured. For those radiolabeled, unbound radioactivity is measured with a scintillation counter. It can be quantified by comparing the absorbance or radioactivity of the sample with a value for a known amount of bFGF.

Sandwich EI sandwiching the antigen with the above two antibodies
In addition to the method A, a publicly known competitive EIA method and an indirect EIA method are performed. In the competitive EIA method, an antibody is immobilized on a carrier, and an enzyme or a radiolabeled antigen bFGF is added to a sample, reacted, and quantified. The measurement of the reaction conditions and the amount of label are performed in the same manner as described above. As an indirect EIA method,
The sample is allowed to react with the unfixed antibody, and the unadsorbed antibody is quantified using an antigen-fixed plate and an anti-mouse-labeled antibody. The measurement of the reaction conditions and the amount of label are performed in the same manner as described above.

In order to purify bFGF, the purified antibody is coupled to a suitable carrier such as activated agarose gel beads according to a conventional method, and then filled into a column. The material containing the crude bFGF is applied to an antibody affinity column, adsorbed, washed, and then eluted with a chaotropic reagent such as KSCN (potassium thiocyanate) or a weak acidic condition that does not deactivate bFGF. Purification can be performed efficiently by a method or the like.

The antibody column can be prepared by the following method, for example, by coupling the monoclonal antibody of the present invention purified from ascites or the like inoculated with a hybridoma to an appropriate carrier.

The carrier to be used may be any carrier as long as bFGF is specifically and efficiently adsorbed after coupling, and then can be appropriately eluted. Examples of the carrier include agarose, cellulose or acrylamide polymers. As an example, polyacrylamide gel beads activated to easily bind a primary amine of a protein, for example, Affigel-10 (manufactured by Bio-Rad), are conveniently used in the method described below. The reaction between Affigel-10 and the antibody is carried out in a buffer such as about 0.001 to 1M, preferably about 0.1M bicarbonate. Reaction conditions are about 0 ° to 20 ° C., about 10 minutes to 24 hours, and various pHs are possible, but preferably about 4 ° C., about 4 hours, pH about 3 to 10 are used. The amount ratio of Affigel-10 and antibody to be mixed is such that the larger the amount of antibody per 1 ml of Affigel, the greater the amount of antibody up to about 50 mg, the more antibodies will be attached.Therefore, any amount within this range may be used, but the binding efficiency and affinity column chromatography Approximately 10 to 30 mg of antibody is conveniently used in view of purification efficiency in. Antibody made in this way-
The carrier-bound substance is washed well with the buffer solution used for the reaction, and then left for several days, or added with a compound having a primary amine such as ethanolamine / hydrochloric acid or glycine at a final concentration of about 0.05 to 0.10 M, and added at about 4 ° C. The remaining unreacted active groups are blocked by a method such as reacting with a protein such as bovine serum albumin (BSA) at 4 ° C. overnight for about 1 to 4 hours, or by reacting the protein with 1-5% bovine serum albumin (BSA) overnight. By filling, it can be used as an antibody column.

When purifying with the above antibody column, for example,
The material containing bFGF protein is dissolved in a buffer solution near neutral, for example, a phosphate buffer solution or a Tris / HCl buffer solution, and adsorbed on an antibody column. Next, after washing the column with the same buffer,
Elute bFGF. As the eluate, a weakly acidic solution such as an acetic acid solution, a solution containing polyethylene glycol, a solution containing a peptide that is more easily bound by an antibody than a sample, a high-concentration salt solution, and a combination thereof are used. Which do not significantly promote is preferred.

The column eluate is neutralized with a buffer in a conventional manner. If necessary, the purification operation using the above antibody column can be performed again.

In this way, a substantially pure bFGF substantially free of pyrodiene and endotoxin is obtained. The substantially pure bFGF of the present invention has a protein content of bF
GF of 90% (w / w) or more, more preferably hbF
Those having a GF of 95% (w / w) or more are mentioned.

The bFGF protein solution obtained here is subjected to dialysis, and if necessary, it can be lyophilized to a powder. During lyophilization, stabilizers such as sorbitol, mannitol, dextrose, maltose, and glycerol can be added.

The hbFGF thus obtained has an action of promoting the proliferation of fibroblasts, an action of promoting the proliferation of endothelial cells, and an action of generating blood vessels, and has a low toxicity. It can be used as a therapeutic accelerator or a therapeutic agent for thrombosis or arteriosclerosis due to angiogenic action. Further, it can be used as a reagent for promoting cell culture.

To use the hbFGF as a medicament, it may be used as it is as a powder or other pharmacologically acceptable carriers, excipients,
As a pharmaceutical composition (eg, injection, tablet, capsule, liquid, ointment) together with a diluent, a warm-blooded mammal (eg, human,
Parenterally or orally to mice, rats, hamsters, rabbits, dogs, cats).

Formulation of an injection is carried out, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants according to a conventional method. Pharmaceutical compositions such as tablets and capsules can also be prepared according to conventional methods.

When the hbFGF is used as the above-mentioned medicament, for example, it is administered to the above-mentioned warm-blooded mammal by selecting an appropriate amount from a daily dose of about 1 ng to 100 μg / kg in consideration of the administration route and symptoms. You.

When the hbFGF is used as a reagent for promoting cell culture, it is preferable to add the hbFGF to the medium at a concentration of about 0.01 to 10 μg, more preferably about 0.1 to 10 μg per medium.

Further, the mutein of hbFGF can be used in the same manner as the above-mentioned hbFGF.

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
This is based on an abbreviation by ochemical Nomenclature or an abbreviation commonly used in the art, and examples thereof are described below. In addition, when an amino acid can have an optical isomer, the L-isomer is indicated unless otherwise specified.

DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate dCTP: deoxy Cytidine triphosphate ATP: Adenosine triphosphate Tdr: Thymidine EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine 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 In the reference examples described below, human bFGF Number of constituent amino acids, in the amino acid sequence Met is added to the N-terminus of the peptide X is Thr Y in the amino acid sequence of the aforementioned (II) is Ser, it shall count the Met as the first.

Mouse HbF99 cells obtained in Example 2 (3) described below,
Mouse HbF161 cells and mouse HbF165 cells were obtained from the Fermentation Research Institute (IFO) on January 28, 1987, respectively.
Has been deposited as the next accession number.

Mouse HbF99 cells: IFO 50122 Mouse HbF161 cells: IFO 50123 Mouse HbF165 cells: IFO 50124 Further, the mouse hybridomas HbF12, HbF52, HbF78 and HbF98 obtained in Example 4 described below were used in Showa 62, respectively.
It has been deposited with the IFO as of August 17, 2016 as the following accession number.

Mouse HbF12 cell: IFO 50142 Mouse HbF52 cell: IFO 50143 Mouse HbF78 cell: IFO 50144 Mouse HbF98 cell: IFO 50145 Of the transformants produced in the following Reference Examples and Examples, those with accession numbers Has been deposited with the Institute of Fermentation (IFO) and the Institute of Microbial Technology (FRI) of the Ministry of International Trade and Industry. The accession numbers and accession dates are shown in Table 1 below. In Table 1, those with both the FERM P number and the FERM BP number are initially deposited with a deposit number indicated by the FERM P number, and the deposit is switched to a deposit based on the Budapest Treaty. It has been assigned an accession number, indicated by the FERM BP number, and is kept at the Institute (FRI).

Examples The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto.

Reference Example 1 (Construction of plasmid containing gene encoding hbFGF) (1) Isolation of cDNA-containing plasmid: cDNA synthesized from human foreskin-derived primary cultured cell mRNA was expressed in pC
D vector [Okayama et al., Molecular and Cellular Biolog
y), 3 , 280 (1983)]. A cDNA library using Escherichia coli x1776 as a host was prepared by National Inst.
itute of Child Health and Human Development, Bethes
Received from Dr. Okayama of da, USA. From this cDNA library, the alkaline method (Birnboim.HC & Doly.J. Nucleic Acids Research)
arch), 1 , 1513 (1979)].
This DNA was used to infect Escherichia coli DH1 to prepare a cDNA library comprising about 2 × 10 ⁶ clones and using Escherichia coli DH1 as a host.

Spread 10 pieces of the above cDNA library using Escherichia coli DH1 on a nitrocellulose filter (Millipore, HATF filter) so as to have about 5 × 10 ⁴ clones / filter,
A total of 20 replica filters were prepared, each set including two filters each having the filter as a master filter. Escherichia coli on this replica filter was dissolved in a 0.5N NaOH solution, and the exposed and denatured plasmid DNA was fixed on the filter [Grunstein, M. & Hogness, DS, Proc.
I. Acad. Sci. USA 72 , 3961 (1975)].

On the other hand, it has been reported by F. Esch et al. [Proc.
ad. Sci. USA 82 , 6507 (1985)] Amino acids Nos. 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 89-96 (Thr-Asp-Glu-Cys-Phe-Phe-Phe-Phe
-Glu), the nucleotide sequences corresponding to these amino acid sequences (some codons were arbitrarily fixed at the third letter. 5′GG A / GTC T / CTT A / G AAA, respectively) / 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, underlined bases were fixed. ) Was chemically synthesized. Using T4 polynucleotide kinase (Takara Shuzo) for this oligonucleotide
50μ reaction solution [oligonucleotide 0.1μg, 50mM Tris
−HCl pH8.0,10mM MgCl ₂ , 10mM mercaptoethanol,
^{50μCiγ- 32 P ATP (> 5000Ci /} mmole), 3 units T4
Polynucleotide kinase] at 37 ° C for 1 hour,
It was labeled at the 5 'end of the oligonucleotide in ³² P.

Using the two oligonucleotides labeled by the above method as probes, they were separately associated with a replica filter on which DNA was immobilized. The association reaction contains 10 μCi of probe 5
× SSPE [180 mM NaCl, 10 mM NaH ₂ PO ₄ , 1 mM EDTA (pH 7.
4)], in 10 ml of 5 × Denhardt's, 0.1% SDS, 100 μg / ml denatured salmon sperm DNA solution, at 35 ° C. for 16 hours. After the reaction, the filter was filtered with 5 × SSC [0.15 M NaCl, 0.015 M sodium citrate] 0.1
% SDS solution at room temperature three times for 30 minutes each and further at 45 ° C for 30 minutes 2
[T. Maniatis et al., “Molecular Cloning” Cold
Spling Harbor Laboratory, P.309 (1982)].

Take a radioautogram from the washed filter,
One set of strains that react to both of the two types of probes
It was searched by superimposing radio autograms of two replica filters. 5 × 10 ⁵ clon by this method
One strain reacting to two types of probes from ies [Esche
richia coli K12 DH1 / pTB627 (IFO 14494, FERM BP-128
0)].

(2) The strain (Escherichia coli K12) obtained in (1) above
DH1 / pTB627 (IFO 14494, FERM BP-1280)] and the plasmid DNA (pTB627) by the alkaline method [Nucleic Acids Research, 1 , 1513].
(1979)].

(3) The nucleotide sequence of cDNA encoding hbFGF was determined by the dideoxynucleotide synthesis chain termination method [J. Messing et al., Nucleic Acids Research.
9 , 309 (1981)] and the amino acid sequence deduced from the sequence is shown in FIG.

Reference Example 2 (Expression of hbFGF-encoding gene in Escherichia coli) Construction of hbFGF expression plasmid pTB669: Plasmid pTB627 containing hbFGF cDNA obtained in Reference Example 1 (2) was digested with restriction enzymes AvaI and BalI. , Hb
A 0.44 Kb DNA fragment containing a region encoding FGF was obtained. The Bal I cleavage site of this DNA fragment (blunt end), by joining Bgl II linker pCAGATCTG with T ₄ DNA ligase, 0.44 kb
Ava I-Bgl II DNA fragment was isolated. This 0.44Kb Ava
I-Bgl II DNA fragment by reacting T ₄ DNA ligase Bgl II
After binding the cleavage sites, a DNA polymerase (Klenow fragment) reaction was performed in the presence of dXTP to smooth the AvaI cleavage site. This DNA fragment is added to the synthetic oligonucleotide ^{5 '} AATTCTATGCCAGCATTGC after the phosphorylation reaction.
^{3 'and 5'} GCAATGCTGGCATAG ^{3 'was} cut with EcoR I-Bgl II was bound by T ₄ DNA ligase, about 0.46KbDNA
Fragments were prepared. On the other hand, a plasmid ptrp781 having a trp promoter [Kurokawa, T. et al., Nucleic Acids Res., 11 , 3077-3085]
(1983)] DNA was cut with Pst I, were smoothed by T ₄ DNA polymerase reaction. T4D with Bgl II linker pCAGATCTG
EcoR after ligation to blunt ends by NA ligase reaction
After digestion with I-BglII, an approximately 3.2 Kb DNA fragment containing the trp promoter, the tetracycline resistance gene and the plasmid replication initiation site was isolated. The 0.46 Kb EcoR I-Bgl II DNA fragment containing the gene region encoding hbFGF and the 3.2 Kb DNA fragment were ligated by a T4 DNA ligase reaction, and hb
A plasmid pTB669 for expressing FGF was constructed.

Escherichia coli DH1 was transformed with this plasmid pTB669 to obtain a strain Escheric containing the plasmid pTB669.
hia coli DH1 / pTB669 was obtained.

Furthermore, E. coli K12 MM29 was similarly prepared using pTB669.
4, or C600, and transform Escherichia coli K12
MM294 / pTB669 (IFO 14532, FERM BP-1281), E. coli C6
00 / pTB669 was obtained respectively.

Reference Example 3 (Human basic fibroblast growth factor (hbFGF)
Purification) Escherichia coli K12 MM294 / pTB obtained in Reference Example 2
669 (IFO 14532, FERM BP-1281) in 1% glucose, 0.4%
% Casamino acid, 8 μg / ml tetracycline in M9 medium [T. Maniatis et al., Molecular Cloning Laboratory Manual, (Maniat
is, T. et al Melecular Cloning (1982), A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring H
arbor NY.USA)], and when the Klett value is about 200,
3β Indolylacrylic acid was added to a concentration of 25 μg / ml, and the cells were further cultured for 4 hours. After culturing, cells were collected and 1 /
The suspension was suspended in 20 volumes of 20 mM Tris.HCl, pH 7.6, 10% sucrose solution. To this suspension was added phenylmethylsulfonyl fluoride (PMSF) at a concentration of 1 mM, EDTA at 10 mM, NaCl at 0.1 M, spermidine hydrochloride at 10 mM, and lysozyme at 100 μg / ml (all final concentrations). ℃, after standing for 45 minutes, 3
Sonication was applied for 0 seconds. This solution was centrifuged at 18000 rpm (Serval centrifuge, SS34 rotor) for 30 minutes to obtain a supernatant,
A cell extract was used.

25 ml of this extract (prepared from 500 ml of culture solution) was added to 20 mM Tris
-HCl pH 7.6, passed through a DEAE cellulose (DE52, Whatman) column (diameter 2 × 10 cm) parallelized with 0.2 M NaCl solution to remove nucleic acid components in the extract. The flow-through from the column and the column wash with 20 mM Tris-HCl, pH 7.6, 0.2 M NaCl solution were combined and collected (DEAE flow-through fraction 44 ml).

14 ml of this fraction is applied to a heparin column Shodex AF-pak HR-8.
The sample was applied to a high-performance liquid chromatograph (Gilson) equipped with 94 (8 mm ID × 5 cm, Showa Denko). The column is loaded with 20 mM Tri
s-HCl pH7.6 solution, then 20mM Tris-HCl pH7.6,0.5M
After washing with NaCl solution, a linear gradient elution of 0.5 M to 2 M NaCl in 20 mM Tris-HCl pH 7.6, buffer
ent elution, 60 ml, flow rate 1.0 ml / min).

The hbFGF eluted by this operation showed a single band by SDS-polyacrylamide gel electrophoresis, and was sufficiently purified, and proved to be suitable for use as an antigen. The test of hbFGF was performed under the following conditions.

Mouse BALB / c3T3 cells were seeded in a NMEM 96-well microtiter plate (flat bottom) at 2 × 10 ³ cells / well in 0.2 ml medium in DMEM medium containing 5% calf serum, and cultured.
The next day, the medium was replaced with DMEM medium containing 0.5% calf serum. After the medium was cultured for 3 days, 10 μl / well of a bacterial extract was serially diluted 5 times with a DME medium containing 0.5% BSA, and cultured. After 20 hours, ³ H-Tdr (5 Ci / mmol, 0.5 mCi / ml RCC Amer
sham) was added to each well at 2 μl. 0.2 hours after 6 hours
% Trypsin-phosphate buffer solution (PBS) containing 0.02% EDTA
After the treatment, the cells were collected on a glass filter using a titer tech cell harvester, and the amount of ³ H-thymidine incorporated in the cells was measured with a scintillation counter.

Reference Example 4 (Production of Recombinant DNA Having Base Sequence Encoding Mutein) (1) Cloning of M13 Vector of Human bFGF Gene: Plasmid pTB669 obtained in Reference Example 2 was subjected to restriction enzyme EcoR
Digestion with I and BamHI. Phage vector M13mp8
[J. Messing, Methods in Enzymology, 101 , 20-78 (1983)] Replica type (R
F) Digest the DNA with restriction enzymes EcoR I and BamH I
Human bF from pTB669 digested with oR I and BamHI
It was mixed with the GF DNA fragment. Next, the mixture was ligated with T4 DNA ligase, the ligated DNA was transformed into cells capable of infecting Escherichia coli-JM105, and seeded on a plate using Xgal as an indicator species (Jay Meshing et al., Newclay Acids).・ Research (Nucleic Acids Res.) (1981)
9, pp. 309-321], a plaque (white plaque) containing the recombinant phage was picked up, and the nucleotide sequence of the recombinant portion was determined by the dideoxynucleotide synthesis chain termination method (J.
Et al., Nucleic Acids Research
Res.) 9 , 309 (1981)), and human bFGF
It was confirmed that the DNA was inserted correctly.

Single-stranded phage DNA was purified from this M13PO clone and used as a template for site-directed mutagenesis using synthetic oligonucleotides.

(2) Site-directed mutagenesis 0.1 mM adenosine triphosphate (ATP), 50 mM hydroxymethylaminomethane hydrochloride (Tris-HCl) pH 8.0, 10 mM MgC
l _2, 5 mM in the presence of dithiothreitol (DTT) and T4 kinase 9 units, synthetic oligonucleotides in 50μ [Primer for changing Cys26 to Ser (restriction enzyme Rs
The recognition sequence for aI disappears. )] 40 picomoles were treated with T4 kinase for 1 hour at 37 ° C. The kinased primer (12 picomoles) was incubated for 5 minutes at 67 ° C and 42 ° C in a 50μ mixture containing 50 mM NaCl, 1.0 mM Tris-HCl, pH 8.0, 10 mM MgCl ₂ and 10 mM β-mercaptoethanol. Single-stranded (ss) M13-P
5 μg of O DNA was hybridized. The annealed mixture was then cooled on ice and 0.5 mM dideoxynucleotide triphosphate (dNTP), 80 mM Tris-HCl, pH 7.4, 8 mM MgCl ₂ , 1
00 mM NaCl, DNA polymerase I Klenow fragment 9 units, 0.5m
Reaction mixture 50 containing M ATP and 2 units of T4 DNA ligase
and reacted at 37 ° C for 3 hours and 25 ° C for 2 hours,
The reaction was stopped by adding 2 μM of 0.2 mM EDTA. Used to transform infectious JM 105 cells, the bacteria were grown overnight and ssDNA was isolated from the culture supernatant. Using this ssDNA as a template in the second cycle of primer extension, the gel-purified RF-type DNA is transformed into infectious JM105 cells, plated on agar plates, and cultured overnight to yield phage plaques. Was.

(3) Site-directed mutagenesis: The procedure of the above (2) is repeated, except that the synthetic oligonucleotide primer used encodes cysteine 70 to encode serine. And (A recognition sequence for the restriction enzyme Hae II is generated.) (4) Site-directed mutagenesis: The procedure of the above (2) is repeated, except that the synthetic oligonucleotide primer used encodes cysteine 88. It encodes serine from things And (The recognition sequence of the restriction enzyme Alu I is generated.) (5) Site-directed mutagenesis: The procedure of the above (2) is repeated, except that the synthetic oligonucleotide primer used encodes cysteine 93 It encodes serine from things And (The recognition sequence for the restriction enzyme Hinf I is generated.) (6) Screening and identification of mutagenized plaques: plates containing mutated M13-PO plaques (section (1) above); The two plates containing the unmutated M13-PO phage plaques were cooled to 4 ° C., and the plaques from each plate were placed on two nitrocellulose circular filters, in the case of the first filter the dry filter was placed on an agar plate. Transfer 5 minutes on top and 15 minutes for the second filter. Next, 0.2N NaOH, 1.5M NaCl and
Place filters on thick filter paper soaked in 0.2% Triton X-100 for 5 minutes, then 0.5M Tris-HCl, pH 7.5 and 1.5
Neutralization was carried out for another 5 minutes on filter paper soaked in M NaCl. Filter in the same way 2xSSC (standard citrate)
The filter was washed twice on a filter soaked in, dried and dried at 80 ° C. for 2 hours in a vacuum drying oven. Overlap filters with 10 ml of DNA hybridization buffer (5 x SSC) per filter, pH
Denatured salmon 7.0, 4x denhardt solution (polyvinylpyrrolidone, ficoll and bovine serum albumin, 1x = 0.02% each), 0.1% sodium dodecyl sulfate (SDS), 50 mM sodium phosphate buffer, pH 7.0 and 100 µg / ml 55 depending on sperm DNA
Pre-hybridized at 4 ° C. for 4 hours. Was 24 hours hybridization at 42 ° C. Oligonucleotide primer 10 ⁵ cpm / ml. The filters were washed at 50 ° C. for 30 minutes each in a washing buffer containing 0.1% SDS and a reduced amount of SSC. Filters were first washed with buffer containing 2xSSC and control filters containing unmutated M13-PO plaque were tested for the presence of radioactivity using a Geiger counter. The SSC concentration was gradually reduced, and the unmutated M13-
Filters were washed until no detectable radioactivity remained on control filters with PO plaques. The minimum working concentration of SSC was 0.1 × SSC. The filters were air-dried and exposed to -70 ° C for 2-3 days and autoradiographed. 10,000 mutated M13-PO plaques and 100 unmutated control plaques were screened with a kinased oligonucleotide probe. In control plaques, none of the hybridized with the probe was present, while 3-10 mutated M13-PO plaques hybridized with the probe.

One of the mutant M13-PO plaques was picked and inoculated into JM105 medium. SsDNA was prepared from the supernatant, and double-stranded (ds) DNA was prepared from the cell pellet. The nucleotide sequence was analyzed using an appropriate oligonucleotide primer and ssDNA.

As a result, the TGC (Cys26) codon was converted to a TCT (Ser) codon, the TGT (Cys70) codon was converted to an AGC (Ser) codon, and the TGT (Cys88) codon was changed to TCT (Se
r) Conversion to codon, TGT (Cys93) codon is TCT
It was confirmed that each of them was converted to (Ser) codon.

Of the mutated M13-PO phage, the codon Cys-26 had Se
M13-P2 when r became codon, M13-P2 when codon Cys-70 became Ser, and M13 when codon Cys-88 became Ser.
M13-P4 was obtained when Ser-P3 and codon Cys-93 became Ser.

Reference Example 5 Expression in Escherichia coli of a gene encoding a mutein of human bFGF: (1) Construction of plasmid pTB739 for expression of mutein of human bFGF The replicative form (RF) of M13-P1 obtained in Reference Example 4 was used as a restriction enzyme. Cut with EcoR I and Rst I,
Approximately 0.5 Kb DN including the region encoding the mutein of human bFGF
A fragment was obtained.

On the other hand, a plasmid ptrp781 having a trp promoter
[Kurokawa, T. et al. New Nucleic Acids Research (Nucleic Acids Res.) 11 , 3077-3085 (1983)] DNA
Was digested with EcoR I-Pst I to isolate an approximately 3.2 Kb DNA fragment containing the trp promoter, the tetracycline resistance gene and the plasmid replication initiation site. The 0.5 Kb EcoR I-Pst containing a gene region encoding a mutein of human bFGF
The I DNA fragment and the 3.2 Kb DNA fragment were ligated by a T4 DNA ligase reaction, and the plasmid pT for mutein expression of human bFGF was synthesized.
B739 was constructed.

By transforming Escherichia coli DH1 using this plasmid pTB739, a strain Escherichia coli DH1 / containing a plasmid pTB739 containing a gene encoding a mutein was obtained.
pTB739 (IFO 14575, FERM BP-1641) was obtained.

(2) Preparation of cell extract The transformant obtained in the above (1) contains 1% glucose, 0.4% casamino acid and 8 μg / ml tetracycline.
The cells were cultured in M9 medium, and when the Klett value was about 200, 3β indolylacrylic acid was added to 25 μg / ml, and the cells were further cultured for 4 hours. After culturing, collect the cells, and 1/20 volume of 20 mM
The cells were suspended in Tris-HCl, pH 7.6, 10% sucrose solution. To this suspension was added phenylmethylsulfonyl fluoride (PMSF) at a concentration of 1 mM, EDTA at 10 mM, NaCl at 0.1 M, spermidine hydrochloride at 10 mM, and lysozyme at 100 μg / ml (all final concentrations). After standing at 45 ° C. for 45 minutes, ultrasonic treatment was applied for 30 seconds. This solution was centrifuged at 18000 rpm (Serval centrifuge, SS34 rotor) for 30 minutes to obtain a supernatant, which was used as a cell extract.

(3) Human bFGF activity of bacterial cell extract Mouse BALB / c3T3 cells were placed in a NMEM 96-well microtiter plate (flat bottom) using DMEM medium containing 5% calf serum, and 2 × 10 ³ cells / well in 0.2 ml medium. To inoculate, culture,
The next day, the medium was replaced with DMEM medium containing 0.5% calf serum. After culturing for 3 days, 10 μl / well of a cell extract, which was serially diluted 5 times with a DME medium containing 0.5% BSA, was added and cultured. After 20 hours, ³ H-thymidine (5 Ci / mmol, 0.5 mCi / ml R
CCAmersham) was added to each well at 2 μl. Remove the cells after 6 hours in phosphate buffer (PBS) treated with 0.2% trypsin -0.02% EDTA, using a Titertek cell harvester, ³ H incorporated into the cells were collected cells onto glass filter -The amount of thymidine was measured with a scintillation counter.

As a result, the cell extract of E. coli DH1 / pTB739 showed FGF activity.

In this way, mutein CS1 in which Cys at position 26 of human bFGF was replaced with Ser was obtained.

Reference Example 6 (Expression of human bFGF mutein-encoding gene in Escherichia coli) (1) Construction of plasmid pTB742 for expression of human bFGF mutein The replicative form (RF) of M13-P2 obtained in Reference Example 4 was restricted. Cleavage with enzymes EcoR I and Pst I,
Approximately 0.5 Kb DN including the region encoding the mutein of human bFGF
A fragment was obtained.

On the other hand, a plasmid ptrp781DN having a trp promoter
A was cut with EcoR I-Pst I to isolate an approximately 3.2 Kb DNA fragment containing the trp promoter, the tetracycline resistance gene and the plasmid replication initiation site. The 0.5Kb EcoR I-Ps containing the gene region encoding the mutein of human bFGF
The tI DNA fragment and the 3.2 Kb DNA fragment were ligated by a T4 DNA ligase reaction, and a plasmid for human bFGF mutein expression was obtained.
pTB742 was constructed.

By transforming Escherichia coli DH1 using this plasmid pTB742, a strain Escherichia coli DH1 / containing a plasmid pTB742 containing a gene encoding a mutein was obtained.
pTB742 (IFO 14584, FERM BP-1642) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Reference Example 5
Human bFGF activity was measured in the same manner as in (3).

As a result, the bacterial cell extract of E. coli DH1 / pTB742 showed FGF activity.

Thus, mutein CS2 in which Cys at position 70 of human bFGF was replaced with Ser was obtained.

Reference Example 7 (Expression of human bFGF mutein-encoding gene in Escherichia coli) (1) Construction of plasmid pTB743 for human bFGF mutein expression M13-P3 replicative form (RF) obtained in Reference Example 4 was restricted. Cleavage with enzymes EcoR I and Pst I,
Approximately 0.5 Kb DN including the region encoding the mutein of human bFGF
A fragment was obtained.

On the other hand, a plasmid ptrp781DN having a trp promoter
A was cut with EcoR I-Pst I to isolate an approximately 3.2 Kb DNA fragment containing the trp promoter, the tetracycline resistance gene and the plasmid replication initiation site. The 0.5Kb EcoR I-Ps containing the gene region encoding the mutein of human bFGF
The tI DNA fragment and the 3.2 Kb DNA fragment were ligated by a T4 DNA ligase reaction, and a plasmid for human bFGF mutein expression was obtained.
pTB743 was constructed.

By transforming Escherichia coli DH1 using this plasmid pTB743, a strain Escherichia coli DH1 / containing a plasmid pTB743 containing a gene encoding a mutein is obtained.
pTB743 (IFO 14585, FERM BP-1643) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Reference Example 5
Human bFGF activity was measured in the same manner as in (3).

As a result, the bacterial cell extract of E. coli DH1 / pTB743 showed FGF activity.

In this way, mutein CS3 in which Cys at position 88 of human bFGF was replaced with Ser was obtained.

Reference Example 8 (Expression of a gene encoding a human bFGF mutein in Escherichia coli) (1) Construction of plasmid pTB744 for expression of human bFGF mutein The replicative form (RF) of M13-P4 obtained in Reference Example 4 was restricted. Cleavage with enzymes EcoR I and Pst I,
Approximately 0.5 Kb DN including the region encoding the mutein of human bFGF
A fragment was obtained.

On the other hand, a plasmid ptrp781DN having a trp promoter
A was cut with EcoR I-Pst I to isolate an approximately 3.2 Kb DNA fragment containing the trp promoter, the tetracycline resistance gene and the plasmid replication initiation site. The 0.5Kb EcoR I-Ps containing the gene region encoding the mutein of human bFGF
The tI DNA fragment and the 3.2 Kb DNA fragment were ligated by a T4 DNA ligase reaction, and a plasmid for human bFGF mutein expression was obtained.
pTB744 was constructed.

By transforming Escherichia coli DH1 using the plasmid pTB744, a strain Escherichia coli DH1 / containing the plasmid pTB744 containing a gene encoding a mutein is obtained.
pTB744 (IFO 14586, FERM BP-1644) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Reference Example 5
Human bFGF activity was measured in the same manner as in (3).

As a result, the bacterial cell extract of E. coli DH1 / pTB744 showed FGF activity.

In this way, mutein CS4 in which Cys at position 93 of human bFGF was substituted with Ser was obtained.

Reference Example 9 (Sieving and identification of mutagenized plaques) Plates containing mutated M13-P2 phage plaques obtained in Reference Example 4 and non-mutated M13 obtained in Reference Example 4 Cool the two plates containing the P2 phage plaques to 4 ° C. and place the plaques from each plate onto two nitrocellulose circular filters, or in the case of the first filter, the dry filter onto an agar plate for 5 minutes. Overlap and 15 minutes transfer for the second filter.
Next, the filters were placed on a thick filter paper soaked in 0.2N NaOH, 1.5M NaCl and 0.2% Triton X-100 for 5 minutes.
Neutralized for another 5 minutes on filter paper soaked in 5M Tris-HCl, pH 7.5, and 1.5M NaCl. The filters were washed twice on filters soaked in 2 × SSC (standard citrate) in a similar manner, dried and dried in a vacuum drying oven at 80 ° C. for 2 hours. 10 ml of DNA per filter for duplicate filters
Hybridization buffer (5 × SSC), pH 7.0, 4 × Denhardt solution (polyvinylpyrrolidone, ficoll and bovine serum albumin, 1 × = 0.02% each), 0.1% sodium dodecyl sulfate (SDS), 50 mM sodium phosphate buffer , pH 7.0 and 100μg / m
One pre-hybridized with 1 denatured salmon sperm DNA at 55 ° C. for 4 hours. Next, 10 ⁵ c of the oligonucleotide primer
Hybridization was performed at 42 ° C. for 24 hours using pm. 30 minutes each at 50 ° C in wash buffer containing 0.1% SDS and reduced amount of SSC
The filters were washed with. First, filter 2x
M13-P2 not washed, washed with buffer containing SSC
Control filters containing plaques were tested for the presence of radioactivity using a Geiger counter. The SSC concentration was reduced stepwise and the filters were washed until no detectable radioactivity remained on control filters with unmutated M13-P2 plaques. The minimum working concentration of SSC was 0.1 × SSC. The filters were air-dried and exposed to -70 ° C for 2-3 days and autoradiographed. Mutated M13
-10,000 P2 plaques and 100 unmutated control plaques were sieved with kinased oligonucleotide probes. No plaque hybridized with the probe in control plaques, while mutated
3-10 M13-P2 plaques hybridized with the probe.

One of the mutant M13-P2 plaques was picked and inoculated into JM105 medium. SsDNA was prepared from the supernatant, and double-stranded (ds) DNA was prepared from the cell pellet. The nucleotide sequence was analyzed using an appropriate oligonucleotide primer and ssDNA.

As a result, the TGC (Cys26) codon was converted to a TCT (Ser) codon, the TGT (Cys88) codon was converted to a TCT (Ser) codon, and the TGT (Cys93) codon was TCT (Ser).
The conversion to codon was confirmed respectively.

Among the mutated M13-P2 phages, those in which codons Cys-26 and -70 became Ser were M13-P12, those in which codons Cys-70 and -88 became Ser were M13-P23 and codon Cys-70.
And those in which -93 became Ser were referred to as M13-P24, respectively.

Reference Example 10 (Expression of human bFGF mutein-encoding gene in Escherichia coli) (1) Construction of Human pFGF Mutein Expression Plasmid pTB762 The replicative form (RF) of M13-P23 obtained in Reference Example 9 was Treated in the same manner as in Reference Example 5 (1),
The plasmid pTB762 for mutein expression of bFGF was constructed.

Escherichia coli MM294 was transformed with this plasmid pTB762 to obtain a strain Escherichia coli MM containing plasmid pTB762 containing a gene encoding a mutein.
294 / pTB762 (IFO 14613, FERM BP-1645) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Reference Example 5
Human bFGF activity was measured in the same manner as in (3).

As a result, the cell extract of E. coli MM294 / pTB762 was
Showed activity.

Thus, Cys at positions 70 and 88 of human bFGF is S
The mutein CS23 substituted with er was obtained.

Reference Example 11 (Production of recombinant DNA having base sequence encoding mutein) (1) Cloning of M13 vector of human bFGF gene: Plasmid pTB669 obtained in Reference Example 2 was subjected to restriction enzyme EcoR
Digestion with I and BamHI. Phage vector M13mp8
[J. Messing, Methods in Enzymology, 101 , 20-78 (1983)] Replica type (R
F) Digest the DNA with restriction enzymes EcoR I and BamH I
Human bF from pTB669 digested with oR I and BamHI
It was mixed with the GF DNA fragment. Next, the mixture was ligated with T4 DNA ligase, the ligated DNA was transformed into cells capable of infecting Escherichia coli-JM105 strain, and seeded on a plate using Xgal as an indicator species (Jay Meshing et al., Newclay Acids, Inc.).・ Research (Nicleic Acids Res.) (1981)
9, pp. 309-321], a plaque (white plaque) containing the recombinant phage was picked up, and the nucleotide sequence of the recombinant portion was determined by the dideoxynucleotide synthesis chain termination method (J. Messing).
Et al., Nucleic Acids Research
Res.) 9 , 309 (1981)), and human bFGF
It was confirmed that the DNA was inserted correctly.

Single-stranded phage DNA was purified from this M13-PO clone and used as a template for site-directed mutagenesis using synthetic oligonucleotides.

(2) Site-directed mutagenesis 0.1 mM adenosine phosphate (ATP), 50 mM hydroxymethylaminomethane hydrochloride (Tris-HCl) pH 8.0, 10 mM MgC
l _2, 5 mM in the presence of dithiothreitol (DTT) and T4 kinase 9 units, synthetic oligonucleotides in 50μ [Primer for generating a recognition site for restriction enzyme EcoRI in the base sequence and further changing Prol4 to Met] 40 picomoles were treated with T4 kinase at 37 ° C for 1 hour. 50mM NaC
1, 1.0 mM Tris-HCl, pH 8.0, 10 mM MgCl ₂ and 10 mM β
-In 50μ of a mixture containing mercaptoethanol,
This kinased primer (12 pmol) was added to 67
5 μg of single-stranded (ss) M13-PO DNA was hybridized by heating at 5 ° C. for 5 minutes and at 42 ° C. for 25 minutes. The annealed mixture was then cooled on ice and 0.5 mM each deoxynucleotide triphosphate (dNTP), 80 mM Tris-HCl, pH 7.4,
Add to a 50 μl reaction mixture containing 8 mM MgCl ₂ , 100 mM NaCl, 9 units of DNA polymerase I Klenow fragment, 0.5 mM ATP and 2 units of T4 DNA ligase, add 3 hours at 37 ° C. and 2 hours at 25 ° C.
After reacting for 2 hours, 2 μm of 0.2 mM EDTA was added to stop the reaction.
Used to transform infectious JM 105 cells, the bacteria were grown overnight and ssDNA was isolated from the culture supernatant. Using this ssDNA as a template in the second cycle of primer extension, gel-purified RF-type DNA is
05 cells were transformed, plated on agar plates and cultured overnight to obtain phage plaques.

(3) Site-directed mutagenesis: The procedure of the above (2) is repeated, except that the synthetic oligonucleotide primer used has a restriction enzyme in the base sequence.
Generates a recognition site for Nco I, and simultaneously converts Gly9 to Thr, Ser10
To Met, And

(4) Site-directed mutagenesis: The procedure of the above (2) is repeated, except that the synthetic oligonucleotide primer used has a restriction enzyme in the base sequence.
Creates an Afl II recognition site and simultaneously changes the codon encoding Lys87 to a stop codon. And

(5) Site-directed mutagenesis: The procedure of the above (2) is repeated, except that the synthetic oligonucleotide primer used has a restriction enzyme in the base sequence.
Generates a recognition site for Hpa I and simultaneously changes Asp42 to Asn. And

(6) Site-directed mutagenesis: The procedure of the above (2) is repeated, except that the synthetic oligonucleotide primer used has a restriction enzyme in the base sequence.
Hpa II recognition site is lost and at the same time Arg45 is changed to Gln And

(7) Screening and identification of mutagenized plaques: plates containing mutated M13-PO plaques (section (1) above) and two plates containing unmutated M13-PO phage plaques The plates were cooled to 4 ° C. and the plaques from each plate were stacked on two nitrocellulose round filters, the dried filter on the first filter for 5 minutes on the agar plate and the second filter on the agar plate for 15 minutes. Transferred in layers. Next, 0.2N NaOH, 1.5M NaCl and
Place filters on thick filter paper soaked in 0.2% Triton X-100 for 5 minutes, then 0.5M Tris-HCl, pH 7.5 and 1.5
Neutralization was carried out for another 5 minutes on filter paper soaked in M NaCl. Filter in the same way 2xSSC (standard citrate)
The filter was washed twice on a filter soaked in, dried and dried at 80 ° C. for 2 hours in a vacuum drying oven. Overlap filters with 10 ml of DNA hybridization buffer (5 x SSC) per filter, pH
Denatured salmon 7.0, 4x denhardt solution (polyvinylpyrrolidone, ficoll and bovine serum albumin, 1x = 0.02% each), 0.1% sodium dodecyl sulfate (SDS), 50 mM sodium phosphate buffer, pH 7.0 and 100 µg / ml 55 depending on sperm DNA
Pre-hybridized at 4 ° C. for 4 hours. The oligonucleotide primers 10 ⁵ cpm were hybridized at 42 ° C. for 24 hours. 0.1
The filters were washed at 50 ° C. for 30 minutes each in a washing buffer containing% SDS and decreasing amounts of SSC. Filters were first washed with buffer containing 2xSSC and control filters containing unmutated M13-PO plaque were tested for the presence of radioactivity using a Geiger counter. The SSC concentration was reduced stepwise and the filters were washed until no detectable radioactivity remained on control filters with unmutated M13-PO plaque. The minimum working concentration of SSC was 0.1 × SSC. Air dry the filter,
After exposure at 70 ° C. for 2 to 3 days, an autoradiograph was taken.
10,000 mutated M13-PO plaques and 100 unmutated control plaques were sieved with 32P-γ-ATP kinased oligonucleotide probes. In the control plaques, none of the probes hybridized, whereas the mutated M13-PO plaques 3-1
0 formed hybrids with the probe.

One of the mutant M13-PO plaques was picked and inoculated into JM105 medium. SsDNA was prepared from the supernatant, and double-stranded (ds) DNA was prepared from the cell pellet. The nucleotide sequence was analyzed using an appropriate oligonucleotide primer and ssDNA.

As a result, the AGC (Ser10) codon was converted to ATG (Met) codon, the CCG (Prol4) codon was converted to ATG (Met) codon, and the AAA (Lys87) codon was TAA (stop).
The GAC (Asp42) codon was converted to AAC (A
sn42) codon conversion, CGG (Arg45) codon
It was confirmed that they were converted to CAG (Gln45) codon.

Of the mutated M13-PO phage, codon Ser10 was Met
M13-PN10 and M13-PN14 when codon Pro14 is Met, and M13-PC86 when codon Lys87 is a stop codon
The one in which the codon Asp42 became Asn was referred to as M13-PDN42, and the one in which the codon Arg45 became Gln was referred to as M13-PRQ45.

Reference Example 12 (Expression of human bFGF mutein-encoding gene in Escherichia coli) (1) Construction of Human pFGF Mutein Expression Plasmid pTB795 The replicative form (RF) of M13-PN14 obtained in Reference Example 11 was By treating in the same manner as in Reference Example 5 (1), a plasmid pTB795 for mutein expression of human bFGF was constructed.
Escherichia coli MM294 was transformed with this plasmid pTB795 to obtain a strain Escherichia coli MM containing plasmid pTB795 containing a gene encoding mutein.
294 / pTB795 (IFO 14700, FERM BP-1660) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Reference Example 5
Human bFGF activity was measured in the same manner as in (3). As a result, the bacterial cell extract of E. coli MM294 / pTB795 showed FGF activity. Thus, mutein N14 deficient in human bFGF from Pro2 to Pro14 was obtained.

Reference Example 13 (Expression of human bFGF mutein-encoding gene in Escherichia coli) (1) Construction of human bFGF mutein expression plasmid pTB796 The replicative form (RF) of M13-PC86 obtained in Reference Example 11 was By treating in the same manner as in Reference Example 5 (1), a plasmid pTB796 for mutein expression of human bFGF was constructed.
By transforming Escherichia coli MM294 using this plasmid pTB796, a strain Escherichia coli MM containing the plasmid pTB796 containing a gene encoding a mutein was obtained.
294 / pTB796 (IFO 14701, FERM BP-1661) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, and a bacterium containing mutein C86 lacking amino acids after Lys87 of human bFGF was obtained. A body extract was obtained.

Reference Example 14 (Expression of human bFGF mutein-encoding gene in Escherichia coli) (1) Construction of Human pFGF Mutein Expression Plasmid pTB797 The replicative form (RF) of M13-PDN42 obtained in Reference Example 11 was By treating in the same manner as in Reference Example 5 (1), a plasmid pTB797 for mutein expression of human bFGF was constructed.
By transforming Escherichia coli MM294 using this plasmid pTB797, a strain Escherichia coli MM containing the plasmid pTB797 containing a gene encoding a mutein was obtained.
294 / pTB797 (IFO 14702, FERM BP-1662) was obtained.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Reference Example 5
Human bFGF activity was measured in the same manner as in (3). As a result, the bacterial cell extract of E. coli MM294 / pTB797 showed FGF activity. In this way, mutein DN42 in which Asp of human bFGF was substituted with Asn was obtained.

Reference Example 15 (Expression of human bFGF mutein-encoding gene in Escherichia coli) (1) Construction of plasmid pTB855 for mutein expression of human bFGF The DNA of plasmid pTB669 obtained in Reference Example 2 was digested with restriction enzyme Hinc II. , EcoR I linker p (5'-C
ATGAATTCATG-3 ') was bound by a T4 DNA ligase reaction. Furthermore, digest with restriction enzymes EcoR I and Pst I,
A 0.35 kb DNA fragment was separated. This DNA fragment and plasmid
About 3.2 kb obtained by cutting ptrp 781 with EcoR I-Pst I
The DNA fragment (Reference Example 5 (1)) was ligated by a T4 DNA ligase reaction to construct a plasmid pTB855 for human bFGF mutein expression.

By transforming Escherichia coli MM294 with this plasmid pTB855, the strain E. coli MM294 / pTB85 containing the plasmid pTB855 containing the gene encoding mutein
Got 5

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2) to obtain a supernatant, which was used as a bacterial cell extract.

(3) Human bFGF activity of bacterial cell extract Reference Example 5
Human bFGF activity was measured in the same manner as in (3). As a result, the bacterial cell extract of E. coli MM294 / pTB855 showed FGF activity. In this way, mutein N41 in which human bFGF from Pro2 to Val41 was deleted was obtained.

Reference Example 16 (Expression of human bFGF mutein-encoding gene in Escherichia coli) (1) Construction of plasmid pTB856 for mutein expression of human bFGF The DNA of plasmid pTB669 obtained in Reference Example 2 was partially digested with restriction enzyme BamHI. Into the BaFGF gene
Only the mHI recognition site was cut. Cleavage site is dATP, dCTP, dG
In the presence of TP and dTTP, blunt-ended using E. coli DNA polymerase I, NheI linker p (5'-CTAGCTAGCTAG-
3 ′) was ligated by a T4 DNA ligase reaction. After treatment with the restriction enzyme NheI, the cleavage site was further linked by a T4 DNA ligase reaction to construct a plasmid pTB856 for expressing mutein of human bFGF.

By transforming Escherichia coli MM294 using this plasmid pTB856, a strain E. coli MM294 / pTB85 containing a plasmid pTB856 containing a gene encoding a mutein.
Got 6.

(2) Preparation of bacterial cell extract The transformant was cultured in the same manner as in Reference Example 5 (2), a supernatant was obtained, and a bacterium containing mutein C129 lacking amino acids after Lys130 of human bFGF was obtained. A body extract was obtained.

Reference Example 17 H-Leu-Pro-Met-Ser-Ala-Lys-Ser-OH (bFGF [1
40-146]) Production of Boc-Ser (Bzl) -resin from 696 mg (0.72 mmol / g resin) on an Applied Biosystems (USA) Model 430A fully automatic peptide synthesizer, the following amino acids were sequentially condensed and DeBoc reaction was performed.

Boc-Lys (Z) -OH, Boc-Ala-OH, Boc-Ser (Bzl) -O
H, Boc-Met-OH, Boc-Pro-OH, Boc-Leu-OH Bzl: benzyl Boc: t-butoxycarbonyl Z: benzyloxycarbonyl Thus Boc-Leu-Pro-Met-Sre (Bzl) -Ala −Lys
1.08 g of (Z) -Ser (Bzl) -resin was obtained. 400mg of this
Was removed from the resin by incubation in 5.0 ml of hydrogen fluoride containing 0.5 ml of anisole and 0.5 ml of dimethyl sulfide at 0 ° C. for 60 minutes. Excess hydrogen fluoride was distilled off under reduced pressure, and the residue was washed with Et ₂ O, extracted with 30 ml of water, and the crude product obtained by freeze-drying was dissolved in 5 ml of water, and Amberlite IRA-400 was used.
(Acetic acid type) Perform ion exchange with a resin (column 2 × 5 cm, eluent water), concentrate the eluate under reduced pressure, and use Sephadex LH-
Purification by gel filtration using 20 (Pharmacia, column 2.5 × 125 cm, eluent 1N acetic acid) gave the desired product. yield
118 mg (78.8%) Rf 0.22 (ethyl acetate: acetic acid: butanol: water = 1: 1: 1:
1) ▲ [α] ²⁵ _D ▼ -81 ゜ (c = 0.11, in 1N acetic acid) Amino acid analysis value: Ser2.08, Pro1.06, Ala1.00, Met0.98, L
eu1.03, Lys0.99 Example 1 (immunization) 10 μg of antigen human bFGF (obtained in Reference Example 3) dissolved in 0.4 ml of Freund's complete adjuvant (manufactured by Difco) for BALB / c mice (4 weeks old) Was injected intraperitoneally. Three weeks later, Freund's Incomplete Adjuvant 0.4ml
10 μg of the antigen hbFGF was re-administered intraperitoneally. Three weeks later, the same booster immunization was performed, and two weeks later, 10 μg of human bFGF dissolved in physiological saline was intraperitoneally inoculated.

Example 2 (1) Cell fusion From the immunized mouse shown in Example 1, 4
One day later, the spleen was removed to obtain cells to be used for cell fusion. These cells were suspended in a medium (hereinafter abbreviated as IH medium) in which Iskov medium and Ham F-12 medium were mixed at a ratio of 1: 1.

Mouse myeloma cells P3-X63-Ag · 8U1 were subcultured in RPMI1640 medium containing 10% fetal bovine serum under the conditions of 5% carbon dioxide and 95% air.

Cell fusion was performed according to the method established by Kohler and Milstein et al. [Kohler, G. and Milstein, C .; Nature 256 , 495 (1975)]. The above 2.9 × 10 ⁷ myeloma cells and 1.5 × 10 ⁸ immunized lymphocytes obtained by the above method were mixed, spun down, and 0.3 ml of I
45% polyethylene glycol 6000 (hereinafter PEG6000) dissolved in H medium was added dropwise. The PEG6000 solution was pre-warmed to 37 ° C. and slowly added dropwise. Five minutes later, 0.5 ml was added to the IH medium preheated to 37 ° C. for 1 minute to make 10 ml, and then 600 rpm at room temperature.
After centrifugation for 10 minutes, the supernatant was removed. This cell pellet was suspended in 200 ml of IH medium containing 20% calf serum, and 2 ml of the suspension was inoculated in a 24-well microplate (Limbro). One day later, IH medium (containing 20% calf serum) containing HAT (hypoxanthine 1 × 10 ⁻⁴ M, aminopterin 4 × 10 ⁻⁷ M, thymidine 1.6 × 10 ⁻⁵ M)
(Hereinafter referred to as HAT medium) was added to each well in an amount of 1 ml, and every third day, half of the medium was replaced with HAT medium. The cells grown in this way are hybrid cells.

(2) Search for antibody-producing cells A hybrid cell culture supernatant was added to a 96-well microtiter plate made of polystyrene to which human bFGF had been previously fixed, and the mixture was incubated at room temperature for 2 hours. After removing the culture supernatant and washing, a horseradish peroxidase (HRP) -labeled anti-mouse IgG goat antibody (Miles) was added as a secondary antibody, and the mixture was incubated at room temperature for 2 hours. Remove the secondary antibody,
After well washing, a color reaction was performed by adding a reaction substrate (EIA method). Strong valency was observed in three wells by this method.

(3) were seeded cells hybrid cloning in these wells of cells, so as to be 0.5 per 1 well to the murine thymocytes advance 10 ⁴ cells / well in 96-well microtiter plates which had been seeded as vegetative cells And cloning. As a result, three clones, mouse HbF99 cells (IFO 50122) and mouse HbF161
Cells (IFO 50123) and mouse HbF165 cells (IFO 50124) were obtained.

Table 2 shows the measurement results of the antibody titers in these cell supernatants.

The cloned cells were stored in liquid nitrogen by adding 10% dimethyl sulfoxide (DMSO) to an IH medium containing 20% calf serum.

Example 3 (Immunoglobulin class of monoclonal antibody) The mouse antibody subclass detection kit (Bio-Rad) obtained in Example 2 was reacted with various standard immunoglobulins. Table 3 shows the results.

Table 3 shows that MoAb99, MoAb161 and MoAb165 all belong to the immunoglobulin class IgM.

Example 4 Mouse BALB / c immunized by the method described in Example 1
Of the hybridoma HbF12 (IFO) by the method described in Example 2 (1), (2) and (3).
50142), HbF45, HbF47, HbF52 (IFO 50143), HbF78 (IFO
50144) and HbF98 (IFO 50145).

Hybridoma HbF12, HbF45, HbF47, HbF52, HbF78, HbF9
For 8 each, 2 × 10 ⁶ cells were inoculated into mice that had been intraperitoneally administered with 0.5 ml of mineral oil in advance. Ten days later, 2 to 4 ml of ascites was collected per animal, and the monoclonal antibody MoAb12, M was obtained from each hybridoma.
oAb45, MoAb47, MoAb52, MoAb78 and MoAb98 were obtained.

According to the method described in Example 3, the immunoglobulin class of these monoclonal antibodies was measured. The results were determined as shown in Table 4 below.

Example 5 (1) Preparation of radiolabeled hbFGF Escherichia coli MM294 / pTB669 (IFO 14532,
Hb radiolabeled with ³⁵ S using FERM BP-1281)
FGF was obtained by the following method.

The above E. coli MM294 / pTB669 was cultured in the medium described in Reference Example 3.
Culture was performed until the Klett value reached 200. This culture 1/5 amount

Is a medium in which amino acids having the following composition are added to M9 medium containing 1% glucose and 8 μg / ml tetracycline.

Amino acid composition L-alanine 25.0 mg / L-arginine hydrochloride 84.0 L-asparagine monohydrate 28.4 L-aspartic acid 30.0 L-cysteine disodium salt 82.8 L-glutamic acid 75.0 L-glutamine 584.0 L-glycine 30.0 L-histidine hydrochloride Salt monohydrate 42.0 L-isoleucine 105.0 L-leucine 105.0 L-lysine hydrochloride 146.0 L-phenylalanine 66.0 L-proline 40.0 L-serine 42.0 L-threonine 95.0 L-tyrosine 83.9 L-valine 94.0 The culture was continued until the Klett value reached 200, 3β-indoleacrylic acid was added to a concentration of 25 μg / ml, and the culture was continued for another 2 hours. Thereafter, 1 ml was taken and 10 μCi of ³⁵ S-Met was collected.
(Specific activity> 1000 Ci / mmol) and cultured for 30 minutes. After the culture, the cells were collected to obtain a bacterial cell extract according to the method described in Reference Example 3.

Further, the vector plasmid ptrp781 described in Reference Example 2 was used.
The same treatment was performed for Escherichia coli MM294 having the above, to obtain a labeled bacterial cell extract.

(2) Immunoprecipitation Protein A (manufactured by BRL) was prepared according to the instructions and used as a 10% solution. Unlabeled E. coli extract is E. coli
MM294 / ptrp781 was obtained by the method described in Reference Example 3.

1 ml of the ascites fluid obtained in Example 4 was used for unlabeled E. coli extract
, And left at 4 ° C for 1 hour, followed by labeled cell extract (MM294 /
ptrp781 or MM294 / pTB669) was added at 10 ⁶ cpm, and the mixture was left overnight at 4 ° C.

Unlabeled E. coli extract 10 in 100 μl of protein A 10% solution
Add 0μ, mix, leave at 4 ° C overnight, centrifuge, and
NETBN solution [150 mM NaCl, 5 mM EDTA, 50 mM Tris Cl (pH7.
5), 0.1% BSA, 0.05% NP-40 (Non-idet)
P-40)]. A mixture of the labeled extract and ascites treated as described above was added thereto, mixed, and allowed to stand at 4 ° C. overnight. This was centrifuged, and the precipitate was suspended in 500 μl of NETBN solution. This operation was repeated 5 times to remove the label substance that did not adsorb, and finally suspended in 50 μm of an electrophoresis sample buffer, and subjected to polyacrylamide gel electrophoresis. The electrophoresis followed the method of Laemmli, UK [Nature, 227 , 680 (1970)]. After the electrophoresis, the gel was immersed in 50% trichloroacetic acid (TCA) for 1 hour, washed with distilled water four times for 30 minutes each to remove TCA, and then immersed in dimethyl sulfoxide (DMSO) for 1 hour. After this, DM containing 10% 2.5 diphenyl oxazole (DPO)
Soaked in SO for 1 hour. After washing the gel three times with distilled water for 30 minutes each, the gel was dried. From this, a radioautogram was taken to observe the immunoprecipitation pattern. This is shown in FIG. FIG. 2 shows that the monoclonal antibodies MoAb12, MoAb52, Mo
Ab78 and MoAb98 were found to bind to hbFGF in the bacterial cell extract.

Example 6 Among the monoclonal antibodies obtained in Example 4, the antibody titers of the four antibodies that showed immunoprecipitation in Example 5, that is, the monoclonal antibodies MoAb12, MoAb52, MoAb78 and MoAb98, were measured by the limiting dilution method.

That is, the monoclonal antibody Mo obtained in Example 4
Example 2 Ascites fluid containing Ab12, MoAb52, MoAb78 or MoAb98 was diluted with 1H medium containing 10% fetal bovine serum, respectively.
The amount of antibody in the diluent was measured by the EIA method shown in (2). The result is shown in FIG. In FIG. Shown respectively.

From FIG. 3, it can be seen that the ascites containing these antibodies has a limiting dilution value of 1 × 10 ⁶ or more, and therefore these antibodies have very high antibody valencies.

Example 7 (Determination of Antigen Recognition Site) The antigen recognition sites of the four antibodies whose antibody valencies were measured in Example 6 were examined by a competitive binding inhibition experiment.

As competitors, hbFGF obtained in Reference Example 3, synthetic peptide Pepl: Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser
−Tyr (N-terminal amino acid 2-10 with Tyr added) [Regulatory Peptides, 1
0 , 309-317 (1985)], Pep2: Leu-Pro-Met-Ser-Ala
-Lys-Ser (corresponding to amino acids 142-147) (obtained in Reference Example 17), and deletion bFGF mutein, ie, mutein N14 (obtained in Reference Example 12), and mutein N
41 (obtained in Reference Example 15) was used.

Synthetic peptides were prepared at a concentration of 100 μg / ml and 10% FCS
Medium (Iskoff and Ham F-12 mixed medium (1:
1)]. Muteins N14 and N41 were used by diluting an extract obtained by the method shown in Reference Example 12 (2) or 15 (2) with an IH medium containing 10% FCS. The ascites fluid obtained in Example 4 was diluted 5 × 10 ⁵ times for MoAb12, MoAb52 and MoAb98 and 5 × 10 ⁴ times for MoAb78 so that the amount of antibody was 0.7 to 1.0 at A415. As a diluent, an IH medium containing 10% FCS was used. A diluted competitor was added to the diluted antibody solution, and the mixture was stirred and kept at 37 ° C. for 30 minutes. The amount of unbound antibody contained in this solution was determined in Example 2.
It was measured by the EIA method shown in (2).

FIGS. 4 and 5 show the results when the synthetic peptide was used.
This is shown in FIG. 6, FIG. 6 and FIG. FIG. 4 shows the results for the monoclonal antibody MoAb12, FIG. 5 shows the results for the monoclonal antibody MoAb52, FIG. 6 shows the results for the monoclonal antibody MoAb78, and FIG. 7 shows the results for the monoclonal antibody MoAb98. In these figures, Shows the results of using Pep2 as a competitor. In the figure, the vertical axis indicates the absorption of the coloring agent (at a wavelength of 415 nm).
Is shown.

4 and 6, the monoclonal antibody MoAb12
And MoAb78 underwent competitive binding inhibition by Pep1.
From this, the monoclonal antibodies MoAb12 and MoAb78
Recognizes the N-terminal amino acids 2 to 10 of hbFGF.

From FIGS. 5 and 7, it was found that MoAb52 and MoAb98 were not inhibited by the binding by these synthetic peptides.

To determine the recognition sites for these antibodies, refer to Reference Example 12.
A competitive inhibition experiment was performed in the same manner as described above using the mutein N14 obtained in the above and N41 obtained in the reference example 15.
FIG. 9 shows the results of MoAb52, and FIG. 9 shows the results of MoAb98. In these figures, Shows the results of using mutein N41 as a competitor. In the figure, the vertical axis indicates the absorption of the coloring agent (at a wavelength of 415 nm). The horizontal axis shows the total protein amount of the crude extract of E. coli obtained by the method of Reference Example 3.

From FIGS. 8 and 9, the monoclonal antibody MoAb5
2. MoAb98 was competitively inhibited by mutein N14 but not mutein N41.

From this, the monoclonal antibodies MoAb52 and MoAb
98 was found to recognize amino acids 15-41 of hbFGF.

Next, bovine acidic FGF (baFGF) [R & DS
ystems Inc., USA) and the hbFGF obtained in Reference Example 3 were used to conduct a competitive inhibition experiment in the same manner as described above, and the results shown in Table 5 below were obtained. Numerical values indicate the amount of unbound antibody and the absorbance due to the color development of EIA according to the method described in Example 2 (2).

From this, the monoclonal antibodies MoAb12, MoAb52, MoAb12
Ab78 and MoAb98 were found not to cross-react with bovine acidic FGF.

From the above, the recognition sites of the four types of monoclonal antibodies are summarized in Table 6 below.

Example 8 Purification of Antibody from Ascites Fluid Monoclonal antibodies MoAb12 and MoA by the method of Example 4.
b52, MoAb78 and MoAb98 were inoculated into 10 mice each to obtain 20 to 30 ml of ascites fluid. The ascites fluid was centrifuged at 2000 rpm (Hitachi cooling centrifuge) to remove cells and the like, and then centrifuged at 4 ° C. for 2 hours in a Spinco SW28 rotor (Beckman, USA) to remove insoluble proteins and fats. Ammonium sulfate was added to the supernatant so as to be 40% saturated, and the mixture was gently stirred in ice for 1 hour. The precipitate was centrifuged at 4 ° C., 15,000 rpm using a Serval SS34 rotor (DuPont, USA), and after collection, buffer 1 [20 mM Tris · HCl (pH 7.9), 40 mM N
aCl] to give a protein concentration of 10 to 15 mg / ml, and dialyzed against Buffer 1 at 4 ° C overnight. This solution was mixed with DEAE-cellulose equilibrated with buffer 1.
It passed through DE52 (Whatman, USA) and was adsorbed. Elution was performed by a concentration gradient between buffer 1 and buffer 1 containing 0.4 M NaCl. Collect the immunoglobulin fraction and
The precipitate was added to a buffer 2 (0.1 M NaHCO ₃ ) with a protein concentration of 10-20 m.
g / ml, and dialyzed against buffer 2 at 4 ° C for 2 nights. After dialysis, it was replaced every night.

Furthermore, it was further purified by a hydroxyapatite column (HCA-column). At this time, 10 mM sodium phosphate (pH 6.8) was used as a starting buffer, and a 500 mM sodium phosphate buffer (p
H6.8) was used. Elution was performed with a linear gradient from the starting buffer to the elution buffer. Fractions containing the antibody were stored at 4 ° C.

Example 9 (Purification of hbFGF mutein using an antibody column) 5 ml of Affigel 10 (manufactured by Bio-Rad, USA) was placed on a sintered filter, washed with 10 volumes of ice-cold isopropanol under suction, and then washed with 10 volumes of ice. Washed with cold distilled water and transferred to the reactor. 5 to 15 ml of 15 mg of the monoclonal antibody MoAb78 (dissolved in buffer 2 of Example 8 or phosphate buffer) was added, and the mixture was allowed to react overnight at 4 ° C. Add 0.
Monoethanolamine (pH 8.0) was added so that the concentration became 01M, and the mixture was reacted at room temperature for 1 hour to inactivate unreacted reactive groups. After that, the gel was mixed with buffer 2 of Example 8
Washed using double volume. Thereafter, 2 ml of the gel was packed in a column, and an initial buffer [20 mM Tris-HCl (pH 7.6), 1 mM EDT
A, 0.15 M NaCl, 0.05% NP40].

On the other hand, the extract of the culture solution of the invertebrate Escherichia coli DH1 / pTB744 containing mutein CS4 obtained in Reference Example 8 (2) was diluted 3-fold with the initial buffer, and the diluted solution was added to the column at a flow rate of 20 ml. / h and allowed to adsorb. After the adsorption, the column was washed with 20 ml of the initial buffer, and thereafter, a high salt buffer [20 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1 M NaC
1, 0.05% NP40] 20 ml, elution buffer A [0.2 M acetate buffer (pH 4.5), 0.2 M NaCl] 20 ml, elution buffer B
[0.2 M acetic acid (pH 2.5), 0.2 M NaCl] were sequentially passed. The flow rate was 20 ml / h and the whole process was performed at 4 ° C.

The resulting fractions per, were subjected to electrophoresis of 17.25% acrylamide [Laemmli, Nature (Nature) 277, 680
(1970)]. Staining was performed by silver staining. The results are shown in FIG. In FIG. 10, MK is a molecular weight marker,
A is the result of the crude extract, B is the flow-through fraction, C is the fraction eluted with the high salt concentration buffer, D is the fraction eluted with the elution buffer A, and E is the fraction eluted with the elution buffer B. Show. Immediately after elution from the column, the D and E fractions were adjusted to pH 7.5 by adding 1 M Tris · HCl (pH 9.5). The FGF activity of each fraction was measured by the method described in Reference Example 5 (3). The results are shown in Table 7. In Table 7, A to E are the same as those in FIG. 10 described above.

From the above, it was found that the antibody column using the monoclonal antibody MoAb78 efficiently adsorbs bFGF mutein CS4, and bFGF mutein CS4 was purified by the above operation.

Example 10 (Quantification of hbFGF mutein using monoclonal antibody by EIA method) (1) Monoclonal antibody 78 obtained in Example 4 was purified from ascites by the method of Example 8. The antibody thus obtained is concentrated to 2 mg / ml or more, and then
Dialysis was performed against 0.2 M sodium phosphate buffer (pH 7.0). To 1.4 ml of the 2.8 mg / ml monoclonal antibody MoAb78, 50 μl of S-acetylmercaptosuccinic anhydride (Aldrich, USA) dissolved in NN′-dimethylformamide (DMF) was added to a concentration of 11.5 mg / ml. The air in the reactor was replaced with nitrogen gas. After sealing, the mixture was stirred at room temperature for 1 hour to introduce SH groups. Unreacted S-acetylmercaptosuccinic anhydride was added to 130 μM of 0.2M
Tris-HCl (pH 7.0), 13 μM of 0.2MEDTA, 130 μM of 2M hydroxyamine (pH 7.0) were added, and the mixture was treated at room temperature for 10 minutes to inactivate. The monoclonal antibody MoAb78 was fractionated by a gel filtration column packed with Sephadex G-25 (diameter 1 cm × 80 cm, Pharmacia, Sweden) (flow rate 20 ml /
h).

(2) 1.4 mg of horseradish peroxidase (HRP, Boehringer Mannheim, West Germany, Grade I) 10 mg
Was dissolved in 0.1 M phosphate buffer (pH 6.8). N-
14 mg of N-hydroxysuccinimide ester of (4-carboxycyclohexylmethyl) maleimide
Was dissolved in DMF, of which 100 μl was added to the HRP solution. The air in the reactor was replaced with nitrogen, sealed, and stirred at room temperature for 1 hour. Thereafter, a maleimide group was introduced through a gel filtration column (described above) packed with Sephadex G-25.
The HRP fraction was collected.

(3) Antibody MoAb78 into which SH group was introduced in (1) above
6 ml of the fraction and H in which a maleimide group was introduced in the above (2)
2 ml of the RP fraction was mixed, concentrated to 1 ml under reduced pressure using a Collodion bag (manufactured by Sartorius, West Germany), and concentrated at 4 ° C.
The reaction was performed for 20 hours. After the reaction, HRP-introduced antibody was purified with Ultrogel AcA44 (LKB, Sweden, diameter 1 cm × 80 c
m) and separated (flow rate 10 ml / h). Among the eluted peak fractions, the fraction with the highest number of HRPs per antibody molecule was 2.4 HR
P / antibody. This was used for the following (4) EIA.

(4) MoAb52 was purified by the same method as in the above (1). PBS this MoAb52 to 10μg / ml or 20μg / ml
Diluted in an immunoplate (Nunc, Denmark)
100 μ / well was injected, and allowed to stand at 4 ° C. overnight for adsorption. After removing non-adsorbed antibodies, BPS
, And 200 μ / well of PBS containing 0.01% merthiolate and 1% bovine serum albumin (BSA) was added, and the mixture was allowed to stand at 4 ° C. overnight.

(5) Human bFGF obtained in Reference Example 3 contains 0.1% BSA
Diluted in PBS. BSA from the plate prepared in (4) above
Remove the solution, wash 4 times with PBS, and dilute human bFGF
Was added at 100 μ / well, and adsorption was performed at 4 ° C. overnight. After removing unreacted human bFGF, the plate was washed four times with PBS, and the above (3)
The HRP-conjugated antibody (HRP-MoAb78) prepared in 1) was diluted 1/300 with PBS containing 0.1% BSA, added at 100 μ / well, and reacted at room temperature for 4 hours. After removing the antibody, the plate was washed six times with PBS, and a peroxidase substrate (Bio Rad, USA) was added at 100 μ / well to perform color development and colorimetric quantification.

(6) Fig. 11 shows that the amount of the monoclonal antibody MoAb52 immobilized on the plate was The bFGF detection curve in the case of is shown. The horizontal axis represents the added bFGF concentration, and the vertical axis represents the absorbance developed by HRP-MoAb78. From this figure, it was found that when MoAb52 was adsorbed to the plate at 2 μg / well, bFGF at a concentration of 0.5 ng / ml could be detected.

(7) MoAb98 was added to the plate at 2 μg /
The wells were adsorbed, and bFGF was colorimetrically determined by the method (5) above. FIG. 12 shows the result. The horizontal axis and the vertical axis represent the same as in FIG. 11 of the above (6). From this figure, it was found that MoAb98 can detect bFGF at a concentration of at least 0.5 ng / ml, similarly to MoAb52.

Example 11 (Quantification of hbFGF Mutein Using Monoclonal Antibody by EIA Method) The extract containing human bFGF mutein C86 obtained in Reference Example 13 was treated in the same manner as in Example 9, and the expression level of mutein was examined. did. As a result, it was found that C86 was expressed in Escherichia coli in a small amount.

Example 12 (Quantification of hbFGF mutein using monoclonal antibody by EIA method) The extract containing human bFGF mutein C129 obtained in Reference Example 14 was treated and examined in the same manner as in Example 9. As a result, it was found that a small amount of mutein C129 was produced in E. coli.

Example 13 (Western blotting detection of hbFGF by) Reference Example 3 hbFGF a 17.25% acrylamide gel electrophoresis obtained by the method described (Laemmli, UK (1970) Nature 277, 6
80-685), followed by a Salto blot (Sartorius, Inc.).
[Kyshse-Anderson, J. Journal of Biochemical and Biophysical Methods (Jour)]
nal of Biochemical and Biophysical Methods), 10 , 2
03-209 (1984)]. This membrane was washed with TBS [20 mM Tris-HCl (pH
7.5), 0.5M NaCl] twice for 5 minutes each, containing 4% BSA
It was left at room temperature for 1 hour in TBS to block unreacted portions on the membrane. The plate was washed twice with TBS containing 0.05% Tween 20 (TTBS) for 5 minutes each. Monoclonal antibody Mo obtained in Example 4
Ab12 or MoAb78 with 3000% TTBS containing 1% gelatin
The nitrocellulose membrane described above was added to the double-diluted solution, and reacted at room temperature overnight. The reaction solution was removed, and the membrane was washed twice with TTBS for 5 minutes, and then washed with TTBS containing 1% gelatin for 3000 minutes.
Two-fold diluted secondary antibody and HRP-labeled anti-mouse IgG goat serum (Bio-Rad, USA) were added and reacted at room temperature for 1 hour. The membrane was washed three times for 5 minutes each with TTBS, and further twice twice for 5 minutes each with TBS. Thereafter, 0.05% 4-chloro-1-naphthol and 0.015% hydrogen peroxide were added as coloring agents, and the reaction was carried out for 15 minutes. .

FIG. 13 shows the results of Western blotting using the monoclonal antibody MoAb78 as the primary antibody. In lane 1, 1 μg of hbFGF was migrated, 300 ng of hbFGF was migrated in 2, and 100 ng of hbFGF was migrated in 3, respectively. M represents a marker, and the number on the left is its molecular weight.

When MoAb12 was used instead of MoAb78, bFGF could be detected with the same sensitivity.

Effect of the Invention Since the monoclonal antibody of the present invention specifically binds to bFGF, it can be advantageously used for purification of bFGF measurement reagent and bFGF.

Furthermore, among the monoclonal antibodies of the present invention, those having a high antibody valency have the advantage that when used as a reagent for measuring bFGF, the amount of other reagents used simultaneously, for example, antiserum, can be reduced. In addition, there is an advantage that bFGF can be purified at a higher level.

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence encoding human bFGF obtained in Reference Example 1 and the amino acid sequence deduced therefrom. FIG. 2 shows an immunoprecipitation pattern by polyacrylamide gel electrophoresis obtained in Example 5. FIG. 3 shows the antibody valencies obtained in Example 6. FIG. 4 shows MoAb12 of various peptides obtained in Example 7.
9 shows the results of a competitive binding inhibition experiment for. FIG. 5 shows MoAb52 of various peptides obtained in Example 7.
9 shows the results of a competitive binding inhibition experiment for. FIG. 6 shows MoAb78 of various peptides obtained in Example 7.
9 shows the results of a competitive binding inhibition experiment for. FIG. 7 shows the MoAb98 of various peptides obtained in Example 7.
9 shows the results of a competitive binding inhibition experiment for. FIG. 8 shows the MoAb52 of bFGF mutein obtained in Example 7.
3 shows the results of a competitive inhibition experiment. FIG. 9 shows the bFGF mutein MoAb98 obtained in Example 7.
3 shows the results of a competitive inhibition experiment. FIG. 10 shows the electrophoresis pattern obtained in Example 9. FIG. 11 shows the results of quantification of bFGF by EIA using MoAb52 and HRP-MoAb78, obtained in Example 10. FIG. 12 shows that MoAb98 and HRP-MoAb78 obtained in Example 10.
4 shows the results of quantification of bFGF using the method. FIG. 13 shows the monoclonal antibody Mo obtained in Example 12.
The result of western blotting using Ab78 is shown.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/53 G01N 33/577 33/577 C12N 5/00 B // (C12N 5/10 C12R 1:91) (C12P 21/08 C12R 1 : 91) (56) Reference The EMBO. Journal. 5 [10] (1986) p. 2523-2528 Proc. Natl. Acad. Sc i. USA 82 (1985) p. 6507-6511 (58) Field surveyed (Int. Cl. ⁶ , DB name) BIOSiS (DIALOG) WPI (DIALOG)

Claims (17)

(57) [Claims] 1. A monoclonal antibody having the following properties and specifically binding to basic fibroblast growth factor (bFGF): (a) molecular weight: about 140 to 160 kilodalton; (b) acidic fibroblast (C) immunoglobulin class belongs to IgG, (d) hybridoma HbF12 (IFO 50142), hybridoma HbF52 (IFO 50143), hybridoma HbF7, respectively
8 (IFO 50144) and hybridoma HbF98 (IFO 5014)
5) MoAb12, MoAb52, MoAb78 and M produced by
A monoclonal antibody selected from the group consisting of oAb98. 2. The bFGF has the amino acid sequence: The monoclonal antibody according to claim 1, which is a polypeptide comprising: 3. A monoclonal antibody MoAb12 produced by hybridoma HbF12 (IFO 50142) and specifically binding to basic fibroblast growth factor (bFGF). 4. A monoclonal antibody MoAb52, which is produced by hybridoma HbF52 (IFO 50143) and specifically binds to basic fibroblast growth factor (bFGF). 5. A monoclonal antibody MoAb78 produced by hybridoma HbF78 (IFO 50144) and specifically binding to basic fibroblast growth factor (bFGF). 6. A monoclonal antibody MoAb98 produced by hybridoma HbF98 (IFO 50145) and specifically binding to basic fibroblast growth factor (bFGF). 7. Hybridoma HbF12 (IFO 50142), hybridoma HbF52 (IFO 50143), hybridoma HbF78
(IFO 50144) and hybridoma HbF98 (IFO 5014)
A hybridoma that is selected from the group consisting of 5) and produces a monoclonal antibody that specifically binds to basic fibroblast growth factor (bFGF). 8. A hybridoma obtained by fusing and cloning mouse spleen cells immunized with basic fibroblast growth factor (bFGF) and mouse myeloma.
HbF12 (IFO 50142), hybridoma HbF52 (IFO 5014)
3) A method for producing a hybridoma that is selected from the group consisting of hybridoma HbF78 (IFO 50144) and hybridoma HbF98 (IFO 50145) and produces a monoclonal antibody that specifically binds to basic fibroblast growth factor (bFGF). 9. The amino acid sequence of bFGF is: 9. The method according to claim 8, which is a polypeptide comprising: 10. A cloned hybridoma HbF12 (IFO 50142) comprising mouse spleen cells immunized with basic fibroblast growth factor (bFGF) and mouse myeloma.
Hybridoma HbF52 (IFO 50143), Hybridoma Hb
F78 (IFO 50144) and hybridoma HbF98 (IFO 501
45) a hybridoma selected from the group consisting of: growing in a liquid medium or in the abdominal cavity of a mouse, producing and accumulating a monoclonal antibody, and collecting the monoclonal antibody; A method for producing a monoclonal antibody that specifically binds to: (a) molecular weight: about 140 to 160 kDa, (b) does not cross-react with acidic fibroblast growth factor, (c) the immunoglobulin class belongs to IgG, d) Hybridoma HbF12 (IFO 50142), hybridoma HbF52 (IFO 50143), hybridoma HbF7, respectively
8 (IFO 50144) and hybridoma HbF98 (IFO 5014)
5) MoAb12, MoAb52, MoAb78 and M produced by
A monoclonal antibody selected from the group consisting of oAb98. (11) the amino acid sequence of bFGF is: 11. The production method according to claim 10, which is a polypeptide comprising: 12. A monoclonal antibody having the following properties and specifically binding to basic fibroblast growth factor (bFGF): (a) molecular weight: about 140 to 160 kilodalton; (b) acidic fibroblast (C) immunoglobulin class belongs to IgG, (d) hybridoma HbF12 (IFO 50142), hybridoma HbF52 (IFO 50143), hybridoma HbF7, respectively
8 (IFO 50144) and hybridoma HbF98 (IFO 5014)
5) MoAb12, MoAb52, MoAb78 and M produced by
A monoclonal antibody selected from the group consisting of oAb98, a method for purifying bFGF. 13. The bFGF has the amino acid sequence: 13. The purification method according to claim 12, which is a polypeptide comprising: 14. The purification method according to claim 12, wherein affinity chromatography is performed. 15. A monoclonal antibody having the following properties and specifically binding to basic fibroblast growth factor (bFGF): (a) molecular weight: about 140 to 160 kilodalton; (b) acidic fibroblast (C) immunoglobulin class belongs to IgG, (d) hybridoma HbF12 (IFO 50142), hybridoma HbF52 (IFO 50143), hybridoma HbF7, respectively
8 (IFO 50144) and hybridoma HbF98 (IFO 5014)
5) MoAb12, MoAb52, MoAb78 and M produced by
A monoclonal antibody selected from the group consisting of oAb98, a method for detecting and quantifying bFGF. 16. The amino acid sequence of bFGF is: 16. The detection and quantification method according to claim 15, which is a polypeptide comprising: 17. The method according to claim 15, wherein the enzyme immunoassay is performed.