JPH10120700A - Histamine production increasing factor and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new factor, comprising a protein, having a specific amino acid sequence and capable of manifesting increasing actions on the histamine production and useful as an inflammation treating agent for treating or preventing rheumatism, asthma, etc., and search, etc., for a compound capable of increasing the histamine production. SOLUTION: This new protein has amino acid sequences described in formulas I to VI and is capable of manifesting increasing actions on the histamine production, or the new protein has the substantially same amino acid sequences as above and is capable of manifesting the increasing actions on the histamine production. The proteins are useful for screening a compound having the increasing actions on the histamine production and as an inflammatory therapeutic agent for treating or a preventing, etc., of symptoms such as rheumatism or asthma. The two proteins are obtained by administering an acetic acid azobenzenearsonate-bovine serum albumin complex which is an antigen to a rat, etc., then further administering the antigen thereto, causing an allergic reaction, collecting the inflammatory exudate and purifying the collected exudate according to an anion exchange chromatography, reversed phase high- performance liquid chromatography and gel filtration, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to a protein which has an effect of increasing histamine production and which is useful as an anti-inflammatory agent, rheumatism, and a preventive / therapeutic agent for asthma.

[0002]

2. Description of the Related Art Histamine is

Embedded image A physiologically active amine with the structure of: has many pharmacological effects such as telangiectasia, smooth muscle contraction, gastric acid secretion, and other roles in neurotransmitters, inflammation, wound healing, cell proliferation, etc. [Tokyo Kagaku Doujin, Biochemical Dictionary (2nd edition), p. 1037]. Histamine is released not only by mast cells and basophil degranulation, but also by increased histamine production based on the induction of histidine decarboxylase (HDC), a histamine synthase. It is recognized in active organizations. The present inventors have examined the role of histamine using an air-sac type allergic inflammation model in rats, but histamine is not only released by mast cell degranulation in the anaphylactic phase, but also persists for up to 24 hours thereafter. Histamine in the second phase (post-anaphylactic phase)
With the increase in HDC activity in inflamed tissues, it histamine production is the result of enhanced, and histamine anaphylaxis phase via the H ₁ receptor, whereas are involved in increased vascular permeability, post anaphylaxis It has been shown that histamine in the phase has an inhibitory effect on leukocyte infiltration via the H ₂ receptor. In addition, in studies of histamine production, the present inventors have found that there is a histamine enhancer that increases histamine production in post-anaphylaxis in a rat allergic air sac inflammation model, and that its molecular weight is about 25,000-40000. Reported [International Archives of Allergy and Applied Immunology (Internationa
l Archives of Allergy and Applied Immunology), No. 8
8, pp. 386-393, 1989].

[0003]

DISCLOSURE OF THE INVENTION The present invention relates to a protein having an effect of increasing histamine production which can be used for the prevention and treatment of rheumatism and asthma, a method for measuring the same, and a method for screening a compound having an effect of increasing histamine production. The present invention relates to compounds obtained, pharmaceutical compositions containing these compounds, and the like.

[0004]

Means for Solving the Problems The present inventors have reported that a histamine production enhancing factor that increases histamine production is present in a rat model of allergic air sac inflammation. This factor was collected and purified. Then, the partial amino acid sequence was determined. The present inventors have further studied based on these findings and completed the present invention.

The present invention relates to (1) SEQ ID NO: 1, 2, 3, 4, 5
Or a protein having an activity of increasing histamine production according to any one of (1) and (6), or a protein having substantially the same amino acid sequence and exhibiting an effect of increasing histamine production, (2) a substantially identical amino acid The protein according to the above (1), wherein the sequence is an amino acid sequence in which one or several amino acids have been deleted, substituted or added;
(4) a protein having a partial sequence of the protein of (1) and exhibiting an action of increasing histamine production; (4) culturing an animal cell that produces the protein of (1); (5) the method for producing the protein according to (1), wherein the animal cell according to (4) is a mast cell, (6) the method for producing protein according to (1), (2) or (3) an antibody against the protein described in (3), (7) a method for measuring a protein having an effect of increasing histamine production, characterized by using the antibody described in (6) above, (8) an antibody according to (6) above (9) a kit for measuring a protein exhibiting an action of increasing histamine production, (9) contacting a cell producing the protein according to the above (1) with a test compound, and contacting the test compound without contacting the test compound. )
(10) A method for screening a peptidic or non-peptidic compound having a histamine production-enhancing action or a salt thereof, which comprises selecting a compound that produces a large amount of the protein according to (9). Compound,

(11) The histamine-producing cell is brought into contact with the protein or the test compound described in (1) above, and histamine is produced more than when the protein described in (1) is brought into contact with the histamine-producing cell. A method for screening a compound or a salt thereof, which enhances the action of the protein according to the above (1),
(12) A compound or a salt thereof that enhances the action of the protein of (1) obtained by the screening method of (11), or (13) the protein and test compound of histamine and the protein of (1). Contacting the histamine-producing cell with the protein of (1) above, and selecting a compound that produces less histamine than that obtained by contacting the protein of (1) with histamine-producing cells. (14) a compound or a salt thereof that attenuates the action of the protein of (1) obtained by the screening method of (11), (15) a compound or a salt thereof, ),
(2) or (3) the pharmaceutical composition containing the protein, (16) the pharmaceutical composition according to the above (15), which is an anti-inflammatory agent, (17) for treating or preventing rheumatism or asthma. (18) a peptide or non-peptidic compound which produces more histamine than the protein according to (1) when brought into contact with a histamine-producing cell together with the protein according to (1); A pharmaceutical composition for the prevention or treatment of rheumatism or asthma containing a peptide compound; A pharmaceutical composition for preventing and / or treating ulcers or immunocompromised diseases, which contains a peptide or non-peptide compound which is produced only in a small amount.

[0007] The protein of the present invention comprises SEQ ID NOs: 1, 2, 3, 4,
A protein having the amino acid sequence described in 5 and 6 and exhibiting a histamine production increasing effect, or a protein having substantially the same amino acid sequence as the above and exhibiting a histamine production increasing effect. The protein of the present invention comprises the above-mentioned SEQ ID NO: 1,
The amino acid sequences described in 2, 3, 4, 5, and 6 may be included in that order, or may be included in a different order. Having the amino acid sequences described in SEQ ID NOs: 1, 2, 3, 4, 5, and 6 means having all or a part of these amino acid sequences as partial peptides. The phrase “having substantially the same amino acid sequence as the protein of the present invention” refers to any protein having a substantially identical amino acid sequence, in which one or more amino acids are deleted or added. Or a protein having a sequence that may be substituted. That is, the protein of the present invention which has an effect of increasing histamine production includes, in addition to the protein having the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5 and 6 and which has the effect of increasing histamine production, And about 85-99.
It contains an amino acid sequence having 9% homology, more preferably about 90-99.9% homology, and contains SEQ ID NOs: 1, 2,
Proteins having the amino acid sequence described in 3, 4, 5, and 6, and having substantially the same activity as the protein having an activity of increasing histamine production, and the like. Substantially the same
This shows that the histamine production increasing effect is the same in nature. Therefore, the strength of the effect of increasing histamine production and the like, and the quantitative factors such as the molecular weight of the protein may be different.

More specifically, the proteins of the present invention having the effect of increasing histamine production include SEQ ID NOs: 1, 2, 3, 4,
1 or 2 or more (preferably 2 or more and 20 or less, more preferably 2 or more and 10 or less) in the amino acid sequence of the protein having the amino acid sequence described in 5 and 6 and exhibiting an action of increasing histamine production. A) the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, and 6; and one or more amino acids in the amino acid sequence of a protein exhibiting an action of increasing histamine production. (Preferably, 2 or more and 20 or less, more preferably 2 or more and 1 or less
0 or less), one or two of the amino acid sequences of a protein having the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5 and 6 and exhibiting an effect of increasing histamine production. Or more (preferably 2 or more and 20
Or less, more preferably 2 or more and 10 or less) amino acids are substituted with other amino acids.

Furthermore, SEQ ID NOs: 1, 2, 3, 4, 5 of the present invention
In the proteins having the amino acid sequence described in (6) and (6) and exhibiting an action of increasing histamine production, the N-terminal Met is protected by a protecting group (for example, a C _1-6 acyl group such as a formyl group and an acetyl group). One in which the N-terminal side of Glu has been cleaved in vivo and the Glu has been pyroglutaminated;
A protein in which the side chain of an amino acid in the molecule is protected with an appropriate protecting group (for example, a C _1-6 acyl group such as a formyl group or an acetyl group), or a complex protein such as a so-called glycoprotein to which a sugar chain is bound. Also included.

The protein of the present invention having an effect of increasing histamine production is, for example, a complete adjuvant of physiological saline containing an antigen (eg, azobenzenearsonate acetate-bovine serum albumin complex) in an animal (eg, rat) and Freund. (Difco) and sensitized by intradermal injection of the emulsion, 2 to 15 days later, an air sac in which air was injected under the back of the back was prepared, and 10 to 48 hours later, the antigen was injected into the air sac. Cause an allergic reaction. 10 to 36 hours after inducing the allergic reaction, the fluid in the air sac is collected, and the supernatant obtained by removing wet leukocytes by centrifugation is dialyzed to obtain inflammatory exudate. This is then subjected to a purification step. In the purification step, purification is performed by various column chromatography methods. For example, in anion exchange chromatography such as DEAE-cellulose, a protein having an action of increasing histamine production is 10 mM Tris.
-DEAE equilibrated with HCl buffer (pH 7.2)-
It is adsorbed on cellulose and eluted with the same buffer containing 0.1 M NaCl. Further, serum components such as albumin mixed in the exudate can be removed by dye affinity chromatography. Further, a protein having an action of increasing histamine production is fractionated by a gel filtration method and detected as an activity peak at a molecular weight of about 30 kD. When reverse-phase HPLC is used, a protein exhibiting an effect of increasing histamine production is recovered when the acetonitrile concentration is around 36%.

[0011] The protein of the present invention having the effect of increasing histamine production can also be produced by culturing animal cells that produce the protein. Animal cells that produce a protein having the effect of increasing histamine production of the present invention include:
For example, mast cells (eg, peritoneal mast cells, RBL-2H3
Cells, etc.), macrophages (eg, peritoneal macrophages,
Alveolar macrophages, Kupffer cells, mesangial cells, spleen macrophages, etc., lymphocytes (eg, lymph node cells, spleen lymphocytes, etc.), vascular endothelial cells (eg, aortic vascular endothelial cells, pulmonary vein endothelial cells, capillary endothelial cells) Cells). The above RBL-2H3 cells can be obtained from the Human Science Research Resource Bank of the Human Science Foundation under the number JCRB0023. The above-mentioned culture of animal cells producing a protein having an effect of increasing histamine production of the present invention may be performed, for example, using 0.1 to 10 μM calcium ionophore A23.
5 to 15 with a cell culture solution containing 187 (manufactured by Wako Pure Chemical Industries)
After culturing for a time, the culture solution is collected and centrifuged to obtain a supernatant. Furthermore, for example, the supernatant dialyzed in Hank's balanced salt solution (Hank's balanced salt solution) can be used as a crude product.
Further, for example, as a culture medium for culturing animal cells that produce the above-mentioned protein having an effect of increasing histamine production of the present invention, for example, 0.1 to 10 μM calcium ionophore A23187, about 5 to 20% fetal bovine serum Medium containing Eagle (Eagles Minimum Essential Medium, manufactured by Nippon Pharmaceutical Co., Ltd.) [Science, 122
Volume, 501 (1952)], DMEM medium (Dulbecco's Modified Eagles Medium, manufactured by Nippon Pharmaceutical Co., Ltd.) [Virology, 8, 396 (1959)], RPMI 1
640 medium (manufactured by Nippon Pharmaceutical) [Journal of the American Medical Association (The Jounal of t
he American Medical Association), 199, 519 (196
7)], 199 medium [Proceedings of the Society for the Experimental Biology and Medicine (Proceedings of the Society for the
Biological Medicine), 73, 1 (1950)]. Preferably, the pH is between about 6-8. The cultivation is usually carried out at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration or stirring is added.

[0012] The protein or its partial peptide of the present invention having the effect of increasing histamine production may form a salt,
As the salt, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) And tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid). The protein or its partial peptide of the present invention that has an effect of increasing histamine production,
It can also be produced according to a peptide synthesis method known per se. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion and, when the product has a protecting group, removing the protecting group. Known condensation methods and elimination of protecting groups include, for example, the methods described in (1) to (4) below. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptide,
Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis Hirokawa Shoten

After the reaction, a conventional purification method, for example,
The protein of the present invention can be purified and isolated by a combination of solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and the like. When the protein obtained by the above method is a free form, it can be converted into an appropriate salt by a method known per se, and conversely, when the protein obtained by a salt is converted into a free form by a known method. be able to. The DNA encoding the protein having the histamine production increasing effect of the present invention includes a protein having the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, and 6 of the present invention and having the histamine production increasing effect. Any amino acid sequence may be used as long as it contains an amino acid sequence or a nucleotide sequence encoding a protein containing an amino acid sequence substantially identical thereto. Human genomic DNA, human genomic DNA library, human tissue / cell-derived c
DNA, human tissue / cell-derived cDNA library,
Any of synthetic DNAs may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, directly using mRNA fraction prepared from tissues / cells
Reverse Transcriptase Polymerase Chain Reaction
(Hereinafter, abbreviated as RT-PCR method).

As a means for cloning the DNA encoding the protein of the present invention, the DNA of the present invention is amplified by the PCR method using a synthetic DNA primer having a partial nucleotide sequence of the protein, or the DNA incorporated into an appropriate vector is used. Selection is performed by hybridization with a DNA fragment having a partial or entire region of the protein or labeled with a synthetic DNA. The hybridization method is, for example, Molecular Cloning 2nd (ed .;
J. Sambrook et al., Cold Spring Harbor Lab. Press,
1989). When a commercially available library is used, the procedure is performed according to the method described in the attached instruction manual. The cloned DNA encoding the protein of the present invention can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. The above-mentioned deletion, addition or substitution of amino acid residues can be performed by a site-specific mutagenesis technique known per se, for example, Genetic Engineering (Genet Engineering).
ic Engineering), Academic Press, 31-50
1983; Genetic Engineering (G
enetic Engineering), Volume 3, pages 1-32, Prenum Pr
ess, New York (1981), Nucleic Acid Research, Vol. 10, No. 2
0, 6487-6500 (1982) and the like.

The expression vector for the protein of the present invention may be prepared, for example, by (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (b) converting the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by connecting to. As a vector,
E. coli-derived plasmids (eg, pBR322, pBR3
25, pUC118, pUC119) and plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC19)
4), yeast-derived plasmids (eg, pSH19, pSH1
5), bacteriophage such as λ phage, animal viruses such as retrovirus, vaccinia virus, and baculovirus are used. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.

When the host for transformation is Escherichia, a trp promoter, a lac promoter,
The recA promoter, the λPL promoter, the lpp promoter, etc. are used when the host is Bacillus sp.
When the host is yeast, such as an O1 promoter, an SPO2 promoter, or a penP promoter, a PHO5 promoter, a PGK promoter, a GAP promoter,
ADH promoters and the like are preferred. When the host is an animal cell, an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, an SRα promoter, and the like can be used. The use of enhancers for expression is also effective. If necessary, a signal sequence suitable for the host is added to the N-terminal side of the gene encoding the protein having the effect of increasing histamine production of the present invention. When the host is Escherichia, alkaline phosphatase
Signal sequence, OmpA signal sequence, etc., when the host is a Bacillus genus, α-amylase signal sequence, subtilisin signal sequence, etc., when the host is yeast, mating factor α signal sequence, When the host is an animal cell, such as an invertase signal sequence, for example, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule,
A signal sequence or the like can be used. Using the vector containing the DNA encoding the protein of the present invention thus constructed, a transformant is produced.

As the host, for example, Escherichia, Bacillus, yeast, insect, animal cells and the like are used. Specific examples of the genus Escherichia and the genus Bacillus include Escherichia coli K12.
DH1 [Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 1
60 (1968)], JM103 [Nukuilec Acids Research, (Nucleic Acids Research), 9, 3
09 (1981)], JA221 [Journal of Molecular Biol.
ogy)], 120, 517 (1978)], HB101
[Journal of Molecular Biology, 4
1, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Bacillus spp.
Bacillus subtilis MI114 [Gene,
24, 255 (1983)], 207-21 [Journal of Biochemis
try), 95, 87 (1984)].

As the yeast, for example, Saccharomyces cerevisiae AH22, A
^{H22R -, NA87-11A, DKD-5D} , 20B
-12 or the like is used. As insects, for example, silkworm larvae are used [Maeda et al., Nature (Natur
e), 315, 592 (1985)]. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO, DHFR gene-deficient Chinese hamster cells CHO (dhfr ^- CHO cells),
Mouse L cells, mouse myeloma cells, human FL cells and the like are used. To transform Escherichia, for example, Procagings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69
Vol. 2110 (1972) and Gene, Vol. 17, 1
07 (1982).
For transformation of Bacillus sp., For example, Molecular and General Genetics (Molecula
r & General Genetics), 168, 111 (197
This is performed according to the method described in 9). To transform yeast, for example, the Procesings of the National Academy of Sciences of Science
The USA (Proc. Natl. Acad. Sci. USA),
75, 1929 (1978). In order to transform insect cells, for example, Bio / Technology, 6, 47-55 (1988))
And the like. To transform animal cells, for example, Virology, 5
2, 456 (1973). Thus, a transformant transformed with the expression vector containing the DNA encoding the protein of the present invention that exhibits the histamine production-enhancing effect is obtained.

When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium to be used for cultivation, and a liquid medium necessary for growth of the transformant is contained therein. A carbon source, a nitrogen source, an inorganic substance and the like are contained. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like. Alternatively, examples of the organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast, vitamins, growth promoting factors and the like may be added.
The pH of the medium is preferably about 5 to 8. As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal
Journal of Experiments in Molecular Getics
netics), 431-433, Cold Spring Harbor Labor
atory, New York 1972]. If necessary, an agent such as 3β-indolyl acrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, culturing is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary,
Aeration and stirring can also be added. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant in which the host is yeast, as a medium, for example, Burkholder's minimal medium [Bostian, KL et al., "Processings of the National Academy of Sciences of Science" The USA (Proc. Natl. Acad. Sci. US
A), Vol. 77, 4505 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, GA et al., "Processings of the National Academy of Sciences of the Sciences". USA (Proc. Na
Acad. Sci. USA), 81, 5330 (198).
4)]. Preferably, the pH of the medium is adjusted to about 5-8. Culture is usually performed at about 20 ° C to 35 ° C for about 24 to
Perform for 72 hours, adding aeration and stirring as needed.

When culturing a transformant whose host is an insect, Grace's Insect Medium (Grace, T .;
CC, Nature, 195, 788 (1962)) to which an immobilized additive such as 10% bovine serum is appropriately added. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration and / or agitation are added. When culturing a transformant in which the host is an animal cell, examples of the medium include MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], D
MEM medium [Virology, 8, 396
(1959)], RPMI 1640 medium [Journal
The Jounal of the American Medical Association
199, 519 (1967)], 199 medium [Proceeding of the Society for the biological medicine (Proceeding of the Society for the biological medicine)]
r the Biological Medicine), 73, 1 (195
0)]. Preferably, the pH is between about 6-8. The cultivation is usually carried out at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration or stirring is added.

The protein of the present invention can be separated and purified from each of the cultures described above, for example, by the following method. When extracting the protein of the present invention from cultured cells or cells, after culturing, cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method described above, a method of obtaining a crude extract of the protein of the present invention by centrifugation or filtration may be appropriately used. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (registered trademark, hereinafter sometimes abbreviated as TM). When the protein of the present invention is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected. The protein of the present invention contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods. These known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SD.
A method using mainly a difference in molecular weight such as S-polyacrylamide gel electrophoresis, a method using a difference in charge such as ion exchange chromatography, a method using specific affinity such as affinity chromatography,
A method using a difference in hydrophobicity such as reversed phase high performance liquid chromatography, a method using a difference in isoelectric point such as isoelectric focusing, and the like are used.

When the thus-obtained protein having the effect of increasing histamine production of the present invention (hereinafter sometimes referred to as the protein of the present invention) is obtained in a free form, it is converted into a salt by a method known per se or a method analogous thereto. It can be converted, and conversely, when it is obtained as a salt, it can be converted into a free form or another salt by a method known per se or a method analogous thereto. The protein of the present invention produced by the recombinant can be arbitrarily modified or partially removed by activating a suitable protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.

The protein of the present invention has an excellent histamine production increasing action and low toxicity. Therefore, the protein of the present invention can be used safely in the treatment and prevention of rheumatic and asthmatic diseases in mammals (eg, humans, monkeys, cows, horses, dogs, cats, rabbits, rats, mice, etc.). When the protein of the present invention is used as a prophylactic / therapeutic agent for the above-mentioned diseases, according to a method known per se, either oral administration or parenteral administration is possible, and mixed with a pharmaceutically acceptable carrier, Usually, it is orally administered as a solid preparation such as tablets, capsules, granules and powders, or parenterally administered as an injection, suppository or sublingual tablet intravenously, subcutaneously or intramuscularly. Further, it may be administered sublingually, subcutaneously or intramuscularly as a sustained release preparation such as sublingual tablets and microcapsules. Further, in the prevention and treatment of asthma, it may be sprayed directly into the throat as a spray or an aerosol. The daily dose varies depending on the degree of symptoms; age, sex, body weight, and difference in sensitivity of the administration subject; timing of administration, interval, properties of pharmaceutical preparation, preparation, type; type of active ingredient, and is not particularly limited. Usually, about 0.01 to 20 mg, preferably about 0.05 to 10 mg per kg body weight of a mammal.
mg, more preferably 0.1 to 5 mg, which is usually administered in 1 to 4 divided doses per day.

As the above-mentioned pharmaceutically acceptable carrier, various organic or inorganic carrier materials commonly used as a drug substance can be used, such as excipients, lubricants, binders, disintegrants in solid preparations, and solvents in liquid preparations. , Dissolution aids, suspending agents,
It is blended as a tonicity agent, a buffer, a soothing agent and the like. If necessary, formulation additives such as preservatives, antioxidants, coloring agents and sweeteners can also be used. Preferred examples of the above-mentioned excipients include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid, and the like. Preferable examples of the lubricant include, for example, magnesium stearate, calcium stearate, talc, colloidal silica and the like. Preferable examples of the binder include crystalline cellulose, sucrose, D-
Mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like can be mentioned. Preferable examples of the above disintegrant include starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, sodium carboxymethyl starch and the like. Preferred examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like. Preferred examples of the solubilizer include, for example, polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine,
Examples include sodium carbonate and sodium citrate. Preferable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; Examples include hydrophilic polymers such as alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. Preferred examples of the tonicity agent include sodium chloride, glycerin, D-mannitol and the like. Suitable examples of the buffer include, for example, phosphate,
Buffers such as acetate, carbonate, citrate and the like can be mentioned. Preferred examples of the soothing agent include benzyl alcohol and the like. Suitable examples of the preservative include, for example, p-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Suitable examples of the antioxidant include, for example, sulfite, ascorbic acid and the like.

The protein of the present invention may contain a suspending agent, a solubilizing agent,
Stabilizing agents, isotonic agents, preservatives, and the like are added to obtain intravenous, subcutaneous, or intramuscular injections by a method known per se. At that time, if necessary, a freeze-dried product can be obtained by a method known per se. When the protein of the present invention or a salt thereof is administered to a human, for example, it is mixed with itself or an appropriate pharmacologically acceptable carrier, excipient or diluent, and is orally or parenterally administered as a pharmaceutical composition. It can be administered safely. Examples of the pharmaceutical composition include oral preparations (eg, powders, granules, capsules, tablets), injections, drops, external preparations (eg, intranasal preparations, transdermal preparations, etc.), suppositories (eg, Rectal suppositories, vaginal suppositories), sprays and the like. These preparations can be produced by a method known per se, which is generally used in the preparation process. The protein of the present invention comprises a dispersant (eg, Tween 80 (manufactured by Atlas Powder Co., USA), HOC 60 (manufactured by Nikko Chemicals), polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), a preservative (eg, methylparaben, propylparaben) , Benzyl alcohol, etc.), tonicity agents (eg, sodium chloride, mannitol, sorbitol,
Such as glucose or the like), or by dissolving, suspending or emulsifying in vegetable oil such as olive oil, sesame oil, cottonseed oil, corn oil, propylene glycol or the like, and shaping into an oily injection to give an injection.

In order to prepare a preparation for oral administration, the protein of the present invention can be prepared by, for example, excipients (eg, lactose, sucrose, starch, etc.), disintegrants (eg, starch, calcium carbonate, etc.), binders according to a method known per se. (E.g. starch, gum arabic,
Carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, etc.) or lubricants (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.) are added and compression-molded, and if necessary, taste masking, enteric coating or For long-lasting purposes, the preparation can be made into an oral administration preparation by coating with a method known per se. Examples of the coating agent include hydroxypropylmethylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, Tween 80, Bruronic.
F 68, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit (manufactured by Rohm, Germany, copolymerization of methacrylic acid / acrylic acid) and coloring matter (eg, bengara, titanium dioxide, etc.)
Are used. When an enteric preparation is used, an intermediate phase is preferably provided between the enteric phase and the drug-containing phase by a method known per se for the purpose of separating the two phases. According to a method known per se, the protein of the present invention may be solid,
It can be a semi-solid or liquid external administration agent. For example, as the solid, the protein of the present invention can be used as it is, or as an excipient (eg, glycol, mannitol, starch, microcrystalline cellulose, etc.), a thickener (eg,
Natural gums, cellulose derivatives, acrylic acid polymers, etc.) are added and mixed to form a powdery composition. The liquid is almost the same as the injection, and is an oily or aqueous suspension. In the case of semi-solid, an aqueous or oily gel or ointment is preferred. In addition, all of these are pH adjusters (eg, carbonic acid, phosphoric acid, citric acid, hydrochloric acid, sodium hydroxide, etc.), preservatives (eg, paraoxybenzoates, chlorobutanol, benzalkonium chloride, etc.), etc. May be added.

For example, in order to prepare a suppository, the protein of the present invention can be converted into an oily or aqueous solid, semi-solid or liquid suppository according to a method known per se. Examples of the oily base used in the composition include glycerides of higher fatty acids (eg, cocoa butter, witepsols (manufactured by Dynamite Nobel, Germany), etc.) and intermediate fatty acids (eg, miglyols (manufactured by Dynamite Nobel, Germany)) Such〕,
Alternatively, vegetable oils (eg, sesame oil, soybean oil, cottonseed oil, etc.) and the like can be mentioned. Examples of the aqueous base include polyethylene glycols and propylene glycol, and examples of the aqueous gel base include natural gums, cellulose derivatives, vinyl polymers, and acrylic acid polymers. According to a method known per se, the protein of the present invention may be used as it is or dissolved in a buffer, sugar, deionized water (eg, 0.1 to 10 mg / ml), and administered with a nebulizer. To adjust. Examples of buffers that can be used are sodium acetate, sodium citrate and glycine. The buffer preferably has a composition and molarity suitable for adjusting the solution to a pH in the range of 5-7. Usually a buffer molarity of 2 mM to 50 mM is suitable for this purpose. Examples of sugars that may be used include lactose, maltose, mannitol, sorbitol, trehalose and xylose, usually in amounts ranging from 1 to 10% by weight of the composition. Nebulizer compositions are known to be surface induced agglutination of proteins caused by atomizing the solution when forming an aerosol.
surfactants to reduce or prevent aggregation. A variety of conventional surfactants can be used, such as polyoxyethylene fatty acid esters and fatty alcohols, and polyoxyethylene sorbitan fatty acid esters. Amounts usually range from 0.001 to 4% by weight of the composition. A particularly preferred surfactant for the present invention is polyoxyethylene sorbitan monooleate. As two specific examples of commercially available nebulizers suitable for practicing the present invention,
Ultravent nebulizer and Marquest Me manufactured by Mailinckrodt, Inc., St. Louis, Missouri
dical Products, Englewood, Colorado, manufactured by Acorn II nebulizer.

An antibody (eg, a polyclonal antibody or a monoclonal antibody) against a protein having an effect of increasing histamine production of the present invention or an antiserum containing the same is a protein having an effect of increasing histamine production of the present invention or an effect of increasing histamine production of the present invention. Can be produced by using a partial peptide of the protein represented by In particular, the use of a monoclonal antibody is advantageous for increasing the measurement sensitivity.
A monoclonal antibody can be produced, for example, according to the following method. (1) Preparation of Monoclonal Antibody-Producing Cell: The protein or its partial peptide of the present invention having the effect of increasing histamine production is itself or a carrier or a diluent at a site capable of producing an antibody upon administration to a warm-blooded animal. Is administered. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually once every 2 to 6 weeks, for a total of 2 doses.
It is performed about 10 times. The warm-blooded animals used include:
For example, monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens can be mentioned, and mice and rats are preferably used. When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. By fusing the contained antibody-producing cells with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The measurement of the antibody titer in the antiserum is performed, for example, by reacting a protein having an action of increasing the production of labeled histamine described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, and the like, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000-PEG6000) is 10-8.
Cell fusion can be carried out efficiently by adding at a concentration of about 0% and incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes.

Various methods can be used to screen for a protein antibody-producing hybridoma exhibiting an antihistamine production-enhancing action. For example, a solid phase (eg, microparticles) in which a protein antigen exhibiting a histamine-production-enhancing action is adsorbed directly or together with a carrier. Plate), and then add anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice) or protein A labeled with a radioactive substance or an enzyme. In addition, a method for detecting a protein monoclonal antibody exhibiting an action of increasing antihistamine production bound to a solid phase, adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, and adding a radioactive substance, an enzyme, or the like Adding a labeled protein having a histamine production increasing effect, A method of detecting a protein monoclonal antibody exhibiting anti-histamine production increased activity bound to the phase and the like. Selection of a protein monoclonal antibody exhibiting an antihistamine production-enhancing effect can be carried out according to a method known per se or a method analogous thereto. Normal HAT
(Hypoxanthine, aminopterin, thymidine) in an animal cell culture medium. As a selection and breeding medium, any medium can be used as long as hybridomas can grow. For example, RPMI 1 containing 1-20%, preferably 10-20% fetal calf serum
640 medium, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20 to 40 ° C,
Preferably it is about 37 ° C. Culture time is usually 5 days to 3 days.
Weeks, preferably one to two weeks. The cultivation is usually performed under 5% carbon dioxide. The antibody titer of the culture supernatant of the hybridoma can be measured in the same manner as in the measurement of the antibody titer of a protein exhibiting an antihistamine production-enhancing effect in the antiserum.

(2) Purification of Monoclonal Antibody Separation and purification of a protein monoclonal antibody exhibiting an antihistamine production-enhancing effect is carried out in the same manner as ordinary polyclonal antibody separation and purification of immunoglobulin (eg, salting-out method, alcohol precipitation). Antibody, isoelectric focusing, electrophoresis, adsorption / desorption with an ion exchanger (eg, DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase, or antibody with an active adsorbent such as protein A or protein G Only collected,
Specific Purification Method for Obtaining Antibody by Dissociating Bond].

Production of polyclonal antibody or antiserum: When the protein of the present invention having the effect of increasing histamine production is used as an antigen, it is conjugated to a carrier protein. Examples of the carrier protein include hemocyanin and Freund's complete adjuvant (manufactured by Difco). The binding between the protein as the antigen and the protein for carrier can be carried out by using known conventional means. Examples of the reagent used for the binding include glutaraldehyde and water-soluble carbodiimide. The ratio of the protein used as an antigen to the carrier protein is preferably about 1: 1 to about 1:10, and the pH of the reaction is preferably around neutral, especially about 6 to 8 to give good results. There are many. The time required for the reaction is usually about 1 to 12 hours, but about 2 to 6 hours is particularly suitable. The composite thus prepared may be dialyzed against water at about 0 to about 18 ° C. by conventional means, and may be frozen and stored, or may be freeze-dried and stored. To produce polyclonal antibodies, a warm-blooded animal is inoculated with the immunogen produced as described above. Examples of the warm-blooded animal used for the production of the antibody include warm-blooded mammals (eg, rabbits, sheep, goats, rats, mice, guinea pigs, cows, horses,
Pigs), birds (eg, chickens, pigeons, ducks, geese, quails) and the like. As a method of inoculating an immunogen into a warm-blooded animal, the immunogen to be inoculated into the animal may be an effective amount for producing an antibody.
Is often emulsified with 1 ml of saline and complete Freund's adjuvant, and inoculated back and hind limbs subcutaneously five times every four weeks to produce antibodies. As a method for collecting the antibody formed in the warm-blooded animal in this manner, for example, in rabbits, usually, 1 to 7 days after the final inoculation,
It is collected from the ear vein during two days and centrifuged to obtain serum. The polyclonal antibody can be purified from the obtained antiserum by collecting the fraction adsorbed by affinity chromatography using a carrier holding each antigen peptide.

The protein antibody of the present invention having an effect of increasing histamine production can specifically recognize a protein having an effect of increasing histamine production, and thus is used for quantifying a protein having an effect of increasing histamine production in a test solution. can do. That is, for example, (i) an antibody against a protein having an effect of increasing histamine production of the present invention is allowed to competitively react with a test solution and a protein having an effect of increasing histamine production, and the effect of increasing histamine production bound to the antibody is determined. By measuring the proportion of the protein shown, the amount of the protein having the effect of increasing histamine production in the test solution can be measured. Further, (2) simultaneously or continuously reacting the test solution with the antibody insolubilized on the carrier and the labeled antibody, and then measuring the activity of the labeling agent on the insolubilized carrier, the test solution is used. A method of quantifying a protein having an effect of increasing histamine production, wherein one of the antibodies is an antibody recognizing the N-terminal of the protein having the effect of increasing histamine production, and the other antibody is the C-terminal of the protein having an effect of increasing histamine production. The present invention provides a method for quantifying a protein having an effect of increasing histamine production in a test liquid, which is an antibody that reacts with a histamine.

In the above measurement, the antibody molecule itself may be used, or a fraction of the antibody molecule, for example, F (ab ′) ₂ , Fab ′, Fab ″ or Fab fraction may be used. The measurement method using the above antibody is not particularly limited, and detects the amount of the antibody corresponding to the amount of the antigen in the test solution, the amount of the antigen or the antibody-antigen complex by chemical or physical means, Any measurement method may be used as long as it is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, competition method, immunometric method and sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use the sandwich method described later. Examples of the labeling agent used in the measurement method using a labeling substance include a radioisotope, an enzyme, a fluorescent substance, and a luminescent substance. As the radioisotope, for example, [ ¹²⁵
I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are preferably stable and have a large specific activity as the above-mentioned enzymes. Malate dehydrogenase, etc., as fluorescent substances, fluorescamine, fluorescein isothiocyanate, etc., as luminescent substances, luminol, luminol derivatives, luciferin,
Lucigenin and the like. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent. For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

In the sandwich method, a test solution is reacted with an insolubilized antihistamine-producing protein antibody (primary reaction), followed by a protein antibody having a labeled histamine-producing increasing effect (secondary reaction). By measuring the activity of the labeling agent on the insolubilized carrier, the amount of the protein having the effect of increasing histamine production in the test solution can be determined. The primary and secondary reactions can be performed in reverse order,
It may be performed simultaneously or at a different time. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may. In the method for measuring a protein having an effect of increasing histamine production by the sandwich method of the present invention, a protein antibody having an effect of increasing histamine production used in a primary reaction and a secondary reaction is a site to which a protein having an effect of increasing histamine production binds. Are preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of a protein exhibiting an action of increasing histamine production, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.

The protein antibody of the present invention having an effect of increasing histamine production can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry. In the competition method, an antigen in a test solution and a labeled antigen are allowed to competitively react with an antibody, and then the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody, or A solid-phase method using a soluble first antibody and a solid-phased antibody as the second antibody is used. In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated. Is reacted with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution. In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunological measurement methods to the measurement method of the present invention, no special conditions, operations, and the like need to be set. What is necessary is just to construct the measuring system of the protein which shows the histamine production increasing effect by adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, etc.
"Radio Immunoassay" edited by Hiroshi (Kodansha, published in 1974), "Improvement Radioimmunoassay" edited by Hiroshi Irie (published in 1974), Eiji Ishikawa et al. "Enzyme immunoassay" (Medical Publishing, published in 1978) ), Eiji Ishikawa et al., "Enzyme Immunoassay" (2nd edition) (Medical Publishing, published in 1982), Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd edition) (Medical Publishing, published in 1987) ), "Methods in ENZYMOLOGY"
Vol. 70 (Immunochemical Techniques (Part A)), same book
Vol. 73 (Immunochemical Techniques (Part B)), same book
Vol. 74 (Immunochemical Techniques (Part C)), Ibid.
Vol. 84 (Immunochemical Techniques (Part D: Selected
Immunoassays)), Ibid.Vol. 92 (Immunochemical Tech)
niques (Part E: Monoclonal Antibodies and General Im
munoassay Methods)), Ibid.Vol. 121 (Immunochemical
Techniques (Part I: Hybridoma Technology and Monocl
onal Antibodies)) (published by Academic Press)
Etc.].

As described above, by using the protein antibody of the present invention having the action of increasing antihistamine production, the protein having the action of increasing histamine production can be quantified with high sensitivity. As a protein measuring kit showing such a histamine production increasing effect and a measuring method using the same, an example in which a polyclonal antibody is used will be described below. Reagent: BSA-VB solution (0.1% containing 1% bovine serum albumin)
05M Veronal buffer) Anti-HPIF (histamine production increasing factor) antibody solution (eg rabbit anti-HPIF)
Serum diluted 1: 10,000 with BSA-VB buffer containing 1% normal rabbit serum) ¹²⁵ I-HPIF solution Dextran-charcoal powder (Dextran T-700.
025% solution and 0.25% solution of activated carbon Norit A were mixed in equal amounts) Measurement method: ¹²⁵ IH diluted in a test tube with BSA-VB in 200 μl of BSA-VB solution and 100 μl of anti-HPIF antibody solution
Add 100 μl (5000 cpm) of PIF solution, mix, and keep cold for two nights. 0.5 ml of dextran-charcoal powder solution is added, further kept refrigerated overnight, and spun down at 2500 × g for 15 minutes. After centrifugation, a certain amount of the supernatant is collected and the radioactivity is measured.

When a monoclonal antibody is used, examples of a kit for measuring a protein exhibiting an effect of increasing histamine production and an assay method using the same are described below. Reagent: BSA-VB solution (0.1% containing 1% bovine serum albumin)
05M Veronal buffer) Anti-HPIF monoclonal antibody solution (eg, rabbit anti-HPIF monoclonal antibody diluted 1: 10,000 with BSA-VB buffer) ¹²⁵ I-HPIF solution Dextran-charcoal powder (Dextran T-700) .
025% solution and 0.25% solution of activated carbon Norit A were mixed in equal amounts) Measurement method: ¹²⁵ IH diluted in a test tube with BSA-VB in 200 μl of BSA-VB solution and 100 μl of anti-HPIF antibody solution
Add 100 μl (5000 cpm) of PIF solution, mix, and keep cold for two nights. 0.5 ml of dextran-charcoal powder solution is added, further kept refrigerated overnight, and spun down at 2500 × g for 15 minutes. After centrifugation, a certain amount of the supernatant is collected and the radioactivity is measured.

Examples of reagents and measuring methods in the case of the sandwich method are described below. Reagent: Enzyme-labeled anti-HPIF monoclonal antibody solution Antibody-sensitized microplate (having immobilized HPIF-binding antibody with a different antigenic determination site from the monoclonal antibody described in HPIF) 0-50 ng / ml buffer Solution A (0.1 pH 0.1 containing 0.15 M NaCl)
Buffer B (25% Block Ace, 0.15 M NaC)
l-0.02M phosphate buffer at pH 7.0) o-phenylenediamine Buffer used for dissolving o-phenylenediamine (0.0
0.1 M citrate buffer (pH 5.5 containing 02% hydrogen peroxide and 0.005% thimerosal) Enzyme reaction stop solution (2N-sulfuric acid) Measurement method: HPIF standard solution or buffer dissolved in buffer B 100 μl of the test sample solution diluted with B
Inject into each well of the antibody-sensitized microplate washed in and react at 4 ° C. for 24 hours. After washing each well with buffer A, 10 times of enzyme-labeled anti-HPIF monoclonal antibody diluted 100 times with buffer B was added.
Add 0 μl and react at 25 ° C. for another 2 hours. After washing each well with the buffer A, 100 μl of an o-phenylenediamine solution dissolved at 0.15% with an o-phenylenediamine solution is added, and the mixture is reacted at 25 ° C. for 30 minutes. The reaction is stopped by adding 100 μl of an enzyme reaction stop solution to each well, and the absorbance at 492 nm is measured using an automatic colorimeter for microplate (MTP-32, manufactured by Corona). A standard HPIF calibration curve is prepared, and the HPIF concentration is obtained from the absorbance obtained from the test sample.

In the screening method of the present invention,
A test compound is brought into contact with a cell that produces a protein exhibiting an effect of increasing histamine production, and a compound that produces more protein exhibiting an effect of increasing histamine production than when not contacting the test compound is selected, thereby increasing histamine production. A peptidic or non-peptidic compound having an action or a salt thereof can be screened. "Produce more" means that the amount of histamine produced is 1.1.
-1000 times (weight), more preferably 10-500
It means to produce twice as much (weight), more preferably 20 to 200 times as much (weight). More specifically, rat bone marrow RBL-2H3 cells are prepared in MEM medium containing 10% bovine serum at a concentration of 0.5 × 10 ⁶ cells / ml, and cultured overnight. After washing with a serum-free MEM medium (MEM (-)), 0.1 μM calcium ionophore A23187 is added simultaneously with various concentrations of test compounds, and the cells are cultured for 8 hours. After the culture, the amount of histamine in the supernatant is measured. As a control, 0.1 μM calcium ionophore A23 was added to the culture system of RBL-2H3 cells.
187 was added, and the amount of histamine in the culture supernatant after 8 hours was measured. When the amount of histamine when only the calcium ionophore A23187 was added was set to 1.0, the amount of histamine when the test compound was added simultaneously was 1.1 to 1.0.
When the amount is 1000 times larger, the compound is considered to have a histamine production increasing effect.

Examples of the screening kit and the measuring method in this case include the following. 1. Screening reagent A23187 solution Dissolve in DMSO (Dimethyl Sulfoxide) to 1 mM and store at -20 ° C. Histamine determination reagent IN and 5N NaOH aqueous solution, 0.1N and 3N
HCl solution, NaCl saturated 0.1N NaOH solution, n-
BuOH, n-heptane, methanol solution with 1% o-phthalaldehyde, histamine dihydrochloride, 0.8N H
1. ClO ₄ , EMEM medium Assay method 100 μl of rat RBL-2H3 cell suspension prepared at 0.5 × 10 ⁶ cells / ml is added to a 96-well tissue culture plate. A23187 is added to a concentration of 0.1 μM, and the test compound is added to a concentration of 10 ⁻³ to 10 ⁻⁸ M. After culturing at 37 ° C. for 8 hours in the presence of 5% CO ₂ , the culture solution is collected and the centrifuged supernatant is prepared. The amount of histamine in the centrifuged supernatant was determined by the method of Filho et al. [European Journal of Immunology (European Journal of Immunology).
al of Immunology), 13: 841-845, 1983].

That is, the sample was thawed and centrifuged (880 ×
g: 2,000 rpm, 4 ° C., 20 minutes).
Transfer the μl to a small test tube with a screw cap, and add 5N Na
OH 12 μl, NaCl about 0.04 g, n-BuOH
0.35 ml was added. In a test tube stand with a small test tube,
Shake 100 times with the box covered. After centrifugation (880 × g: 2,000 rpm, 4 ° C., 10 minutes), the lower layer (aqueous phase) was removed with a Pasteur pipette.
000 rpm, 4 ° C, 10 minutes) to remove the lower layer.
Add 0.2 ml of saturated 0.1 N NaOH, shake, and centrifuge (2,000 rpm, 4 ° C, 10 minutes). Upper layer (n-Bu
OH) 3 ml into a large test tube with screw cap,
0.2N ml of 0.1N HCl and 0.4 ml of n-heptane are added, shaken and allowed to stand. The upper layer was removed by suction using an aspirator, and 0.2 ml of the lower layer (aqueous phase) was transferred to a small test tube.
N NaOH 40 μl, 1% o-phthalaldehyde
10 μl of MeOH solution was added and 4 minutes later 3N HC
l 20 μl is added to stop the reaction, and the fluorescence intensity is measured. Histamine 2HCl (0,5,1
0,50,100,300ng / ml) 100μl, 0.8N
A solution prepared by adding 30 μl of HClO _{4 and} 40 μl of EMEM (−) is prepared, and the same operation as in the sample is performed to measure the amount of histamine. Measurement conditions of fluorescence intensity: Measuring instrument Shimadzu spectrofluorometer RF-540 Excitation wavelength 355 nm (spectrum width 5 nm) Fluorescence wavelength 450 nm (spectrum width 10 nm)

In the screening method of the present invention,
A protein having a histamine production-enhancing effect and a test compound are brought into contact with a histamine-producing cell, and a compound which produces more histamine than when the protein having a histamine-augmenting effect is brought into contact with the histamine-producing cell is selected. Thus, a compound or a salt thereof that enhances the action of a protein exhibiting an action of increasing histamine production can be screened. "Produce more" means that the amount of histamine to be produced is 1.1 to 100 as compared with a case where a protein having an action of increasing histamine production is brought into contact with a single protein.
It means that it is produced in an amount of 0 times (weight), preferably 10 to 500 times (weight), more preferably 20 to 200 times (weight). More specifically, using rat bone marrow cells as histamine-producing cells, a histamine production-enhancing factor and various concentrations of a test compound are simultaneously added to a rat bone marrow cell culture system, and histamine in the culture supernatant 24 hours later is added. Measure the amount. On the other hand, as a control, only a histamine production increasing factor is added to a culture system of rat bone marrow cells, and the amount of histamine in the culture supernatant after culturing for 24 hours is measured. When the amount of histamine when only the histamine production enhancing factor is added is set to 1.0, and when the amount of histamine when the test compound is added at the same time is 1.1 to 1000 times larger than that of the test compound, the compound has a histamine production enhancing factor. It is considered to have activity to enhance the action. Furthermore, in the screening method of the present invention, a histamine-producing cell is contacted with a protein having a histamine production-enhancing action and a test compound, and histamine is produced more than when the protein having the histamine-production-enhancing action is contacted with a histamine-producing cell. By selecting a compound that produces a small amount, it is possible to screen for a peptidic or non-peptidic compound or a salt thereof that attenuates the action of a protein exhibiting an action of increasing histamine production. "Produce only a small amount"
The amount of histamine to be produced is 1/10 to 1/10 of that in the case where the protein having the action of increasing histamine production is contacted alone.
000 times amount (weight), more preferably 1/10 to 1/1
000 times (weight), more preferably 1/100 to 1
/ 1000 times (weight) means that it is produced in a small amount.
More specifically, rat bone marrow cells are used as histamine-producing cells, and a histamine production-enhancing factor and various concentrations of a test compound are simultaneously added to a rat bone marrow cell culture system.
After a period of time, the amount of histamine in the culture supernatant is measured. On the other hand, as a control, only a histamine production increasing factor is added to a culture system of rat bone marrow cells, and the amount of histamine in the culture supernatant after culturing for 24 hours is measured. The amount of histamine when only the histamine production enhancing factor was added was 1.
0, when the amount of histamine when the test compound is added simultaneously is 1/10 to 1/10000 times (weight),
The compound is considered to have activity to attenuate the effect of histamine production enhancer. As a measuring method, the amount of histamine was determined by the method of Shore et al. [Journal of Pharmacology and Experimental Therapeutics (J. Pharmac. Exp. Ther.), 127, 182-186]
(1959)], extract from the culture supernatant of bone marrow cells, and measure by fluorescence method.

In this case, examples of the screening kit and the measuring method include the following. 1. Screening reagent histamine production enhancing factor sample The histamine production enhancing factor is dissolved in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) at 1000 U / ml and stored at -20 ° C. Histamine determination reagent 5N NaOH solution, n-BuOH, saturated NaCl 0.1
1. N NaOH solution, 0.1 N HCl solution, n-heptane, 1 N NaOH solution, 1% o-phthalaldehyde added methanol solution, 3 N HCl solution, histamine dihydrochloride, 0.8 N HClO ₄ , EMEM medium Assay method 400 μl of rat bone marrow cell suspension prepared at 1 × 10 ⁷ cells / ml is added to a 12-well tissue culture plate. A test compound is added to a culture solution containing 10 to 100 U / ml of a histamine production enhancing factor to a concentration of 10 ⁻³ to 10 ⁻¹⁰ M, and then 400 μl of the test compound is added to the plate. (The unit will be described later.) After culturing for 24 hours at 37 ° C. in the presence of 5% CO ₂ , the culture solution is collected and the centrifuged supernatant is prepared. The amount of histamine in the centrifuged supernatant is measured according to the method of Filho et al.

That is, the sample obtained in 3 was thawed and centrifuged (880 × g: 2,000 rpm, 4 ° C., 20 minutes),
0.4 ml of the supernatant is placed in a small test tube with a screwed cap, and 60 μl of 5N NaOH, about 0.2 g of NaCl, n
-1.25 ml of BuOH were added. The test tube stand on which the small test tube has been set is covered with a box so as to hold it, and shaken 100 times. Centrifugation (880 × g: 2,000 rpm, 4 ° C., 10 minutes)
Thereafter, the lower layer (aqueous phase) is removed with a Pasteur pipette, and NaC
1 ml of 1N saturated NaOH was added, shaken, and centrifuged (2,000 rpm, 4 ° C., 10 minutes) to remove the lower layer.
Add 1 ml of 0.1N NaOH saturated with Cl, shake, and centrifuge (2,000 rpm, 4 ° C., 10 minutes). Upper layer (n-B
(uOH) (1.5 ml) is transferred to a large test tube with a screw cap, 1.25 ml of 0.1N HCl and 2 ml of n-heptane are added, shaken, and allowed to stand. The upper layer was removed by suction using an aspirator, and 1 ml of the lower layer (aqueous phase) was transferred to a small test tube.
200 μl of NNaOH and 50 μl of a 1% o-phthalaldehyde-MeOH solution were added.
The reaction is stopped by adding 100 μl of Cl, and the fluorescence intensity is measured. Histamine ・ 2HCl (0,
5, 10, 50, 100, 300 ng / ml) 100 μl,
300 μl of 0.8N HClO ₄ , EMEM (−) 400
A sample to which μl has been added is prepared, and the same operation as in the sample is performed to measure the amount of histamine. Measurement conditions of fluorescence intensity: Measuring instrument Shimadzu spectrofluorometer RF-540 Excitation wavelength 355 nm (spectrum width 5 nm) Fluorescence wavelength 450 nm (spectrum width 10 nm)

The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound having an action of increasing histamine production, and these compounds may be compounds having a novel structure,
The structure of the compound may be known. Compounds that attenuate the effect of the protein of the present invention that has an effect of increasing histamine production can also be screened using the above-described screening method or screening kit of the present invention in the same manner as in the above-described assay method. The structure may be a novel compound or a compound having a known structure. The protein of the present invention which has an effect of increasing histamine production, and a compound or a salt thereof which is screened and selected (in the case of a large amount of production) using the screening method or screening kit of the present invention has an anti-inflammatory effect and is safe. And low toxicity. Therefore, the compound is formed into a pharmaceutical composition, and is used for the treatment and prevention of symptoms such as rheumatism and asthma in mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, humans, etc.). Can be used as medicine. In addition, the protein of the present invention which has an effect of increasing histamine production, and a compound or a salt thereof which has been screened using the screening method or screening kit of the present invention and selected (if the amount is low), have an anti-ulcer effect or an immunopotentiating effect. It is effective, safe and low toxic. Thus, the compounds can be formulated into pharmaceutical compositions, for example, in ulcers of mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, humans, etc.) (eg,
It can be used as an anti-ulcer agent and an immunopotentiator for the treatment and prevention of gastric ulcers) and immunocompromised diseases.

When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be carried out according to a conventional method as described above. For example,
Sugar-coated tablets, capsules, elixirs, microcapsules, and the like, as necessary, orally, or a sterile solution of water or physiological saline or other pharmaceutically acceptable liquid, or It can be used parenterally in the form of injections, such as suspensions. For example, the protein, the compound or a salt thereof can be a physiologically acceptable carrier, flavoring agent,
It can be prepared by mixing with excipients, vehicles, preservatives, stabilizers, binders and the like in the unit dosage form generally required for the practice of accepted formulations. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained. Examples of additives that can be incorporated into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Swelling agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry are used. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. .

Examples of aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). It may be used in combination with a solubilizing agent, for example, an alcohol (for example, ethanol), a polyalcohol (for example, propylene glycol, polyethylene glycol), or a nonionic surfactant (for example, polysorbate 80 (TM) or HCO-50). Good. The oily liquid includes sesame oil, soybean oil and the like, and may be used in combination with benzyl benzoate, benzyl alcohol and the like as a solubilizing agent. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage Agents (eg, benzyl alcohol,
Phenol, etc.), antioxidants and the like.
The prepared injection is usually filled in a suitable ampoule. The preparation thus obtained is safe and has low toxicity and can be safely administered to the above-mentioned mammals. The dose of the compound or a salt thereof varies depending on the symptoms and the like.
g) about 1 to 1000 m per day
g, preferably about 2 to 500 mg, more preferably about 5 mg
３００300 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method and the like. About 0.05
About 50 mg, preferably about 0.1 to 10 mg,
More preferably, about 0.5 to 5 mg is administered by intravenous injection. In the case of the other mammals described above, the amount converted per 60 kg can be administered.

[0049]

The present invention will be described more specifically with reference to the following examples, reference examples and experimental examples. In the following experimental examples and the like, the activity of increasing histamine production was determined in Reference Example 2
The measurement was carried out by the method described in the above section. The unit is defined as the activity of increasing histamine production by 100% as one unit, and the histamine production increase rate is calculated by the following formula. Increase rate = [(AB) / B] × 100 A: Amount of histamine released when rat bone marrow cells are cultured for 24 hours in a culture solution containing HPIF at each concentration. B: The amount of histamine released when rat bone marrow cells were cultured in a culture medium without HPIF for 24 hours.

Reference Example 1 Collection of Bone Marrow Cells: Untreated rats were bled to death by cutting the carotid artery under ether anesthesia, and the tibia and femur were collected. Both ends of the collected bone were cut, and 2 ml of MEM (-) was injected from one end of the collected bone using a syringe with a 1/1 vein needle to collect bone marrow cells. Bone marrow cells were 1 × 10 ⁷ cells / m using MEM (-)
l.

Reference Example 2 Measurement of histamine production increasing activity: The histamine production increasing activity was measured according to the method of Filho et al. Using a 12-well cluster dish,
400 μl of the bone marrow cell suspension prepared in Reference Example 1 was added to 400 μl of MEM (−) containing the measurement sample,
The cells were cultured in the presence of% CO ₂ for 24 hours. After completion of the culture, the culture solution was collected and centrifuged (220 × g, 3 min, 4 ° C.), the amount of histamine in the supernatant was measured, and used as an index of histamine production increasing activity.

Example 1 (1) 140-160 g of SD male rats (purchased from Charles River Japan) containing 5 mg of an antigen [azobenzenearsonate acetate-bovine serum albumin complex] in physiological saline and Freund's complete adjuvant (Difco Company)
Sensitized by intradermal injection of a 1: 1 emulsion with 9
A day later, an air sac was injected into the back of the back with 8 ml of air, and 24 hours later, 2% containing 2 mg of antigen in the air sac.
Allergic reaction was induced by injecting 4 ml of sodium carboxymethylcellulose-0.9% saline. Twenty-four hours after the allergic reaction was induced, the fluid in the air sac was collected, and wet leukocytes were removed by centrifugation to obtain a supernatant. This was designated as inflammatory exudate. (2) Anion exchange chromatography using DEAE-cellulose: the inflammatory exudate obtained in the above (1) was purified by adding deionized water and then 0.05 M NaCl to 0.0.
After dialysis against 1 M Tris-HCl buffer (pH 7.2), DEAE-cellulose equilibrated with 0.05 M NaCl-0.01 M Tris-HCl buffer (pH 7.2) is suspended, and protein in the exudate is suspended. Was adsorbed. After 24 hours, suction filtration was performed, and the obtained filtrate was used as a non-adsorbed fraction. DEAE-cellulose is 0.1 M NaCl-0.0
Suspend in 1 M Tris-HCl buffer (pH 7.2)
After 4 hours, the mixture was subjected to suction filtration in the same manner, and the filtrate was washed with 0.1 M NaCl.
Fractions. Further, the same operation was performed using a buffer containing 0.5 M and 0.8 M NaCl, and the same operation was carried out.
The NaCl fraction was obtained. The results (specific activities and the like) of the DEAE-cellulose treatment are shown in [Table 1]. Specific activity is 5.6U / mg
It was measured.

[0053]

[Table 1]

Each of the obtained fractions was prepared using Hanks' balanced salt.
It was dialyzed against solution (HBSS) (manufactured by Gibco). The results are shown in FIG. In FIG. 1, (1) is a control (H
BSS), (2) is the result of the pass-through fraction, and (3) is 0.1M N
The results of the aCl fraction, (4) shows the results of the 0.5M NaCl fraction, and (5) shows the results of the 0.8M NaCl fraction. In FIG. 1, three stars indicate p <0.01, and two stars indicate p <0.1. (3) Dye affinity chromatography: DEAE-cellulose 0.1M NaC obtained in the above (2)
1 fraction was added to a 0.02 M sodium phosphate buffer (pH 7.1)
Dialysed. This was loaded onto a Blue Cellulofine column (20 × 180 mm) equilibrated with a 0.02 M sodium phosphate buffer (pH 7.1). Remove the flow-through fraction,
Proteins were eluted by sequentially flowing a buffer containing 0.5 M and 1.0 M NaCl, and finally, all proteins adsorbed on the column were eluted with a buffer containing 1.5 M NaSCN (elution conditions: flow rate 20 ml / h). , 4 ° C). Each obtained fraction is Hanks' balanced salt solution (HBSS)
After dialysis, the histamine production increasing activity was measured. The results (specific activity and the like) are shown in the above [Table 1]. Specific activity is 21U /
It was measured as mg. (4) Concentration: The eluate obtained in the above (3) was subjected to diaflow membrane YM10 (fraction molecular weight 10,000, A
The resulting concentrate was subjected to an ultrafiltration method using Micon Corporation, followed by freeze-drying.

(5) Gel filtration: Equilibrated with Phosphate buffered saline (PBS) containing 0.01% Tween 80
The concentrated active fraction obtained in the above (4) was loaded onto a Sephacryl S-200 column (32 × 850 mm), and fractionation was performed using the same buffer (fractionation conditions: flow rate 5.0 ml /). 25
min, 5.0 ml / fraction, 8 ° C). Capacity of each fraction, 28
The absorbance at 0 nm and the activity of increasing histamine production were measured. The results (specific activity and the like) are shown in FIG. 2 and Table 1 above. The specific activity was measured as 140 U / mg. The molecular weights were estimated by using standard substances (bovine serum albumin: 66 kD, ovalbumin: 45 kD, myoglobin: 17.5 kD, cytochrome C: 12.5 kD, blue dextran: 2,000 kD).
D) was fractionated under the same conditions as above, and the elution volume was measured. In the re-chromatography for gel filtration, the active fractions for gel filtration were pooled, concentrated,
It was loaded on an ephacryl S-200 column and fractionated under the same conditions. The histamine production increasing activity was measured. The results (specific activity and the like) are shown in the above [Table 1]. The specific activity was 1,200 U / mg. (6) Lectin affinity chromatography: The concentrated active fraction obtained by gel filtration obtained in (5) above was subjected to 0.02M Tris-HCl containing 0.5M NaCl.
Dialyzed against a buffer (pH 7.4) and buffered with the same buffer.
Loaded on nA-Sepharose column (10 × 70 mm).
After removing the protein not adsorbed to the column as a flow-through fraction, the glycoprotein was eluted with the same buffer containing 350 mM α-methylglucoside (elution conditions: flow rate 5.
3-6.2 ml / h, 2.6-3.1 ml / fraction, 4 ° C). Each fraction was dialyzed against HBSS and measured for histamine production increasing activity.

(7) FPLC (Fast Protein Liquid Ch
RPLC System (Pharmacia) with RES
After setting a OURCE RPC 3 ml column (6.4 × 100 mm, manufactured by Pharmacia) and equilibrating with solvent A (0.1% (v / v) trifluoroacetic acid (TFA) aqueous solution), the above (5)
2 ml of the active fraction obtained from the re-chromatography of the gel filtration was loaded. The protein was eluted with a linear gradient of increasing the concentration of solvent B (0.1% TFA acetonitrile) from 30 to 50% in 25 minutes (flow rate 1 ml / min). The obtained fraction was freeze-dried. Histamine production increasing activity was measured by dissolving in PBS. The results (specific activity and the like) are shown in FIG. 3 and Table 1 above. The specific activity was 50,000 U / mg.

Example 2 (1) SMART (Sensitivity Manipulation and Reco
Reverse technology using very technology) System: μRPC was applied to SMART System (Pharmacia).
C2 / C18 SC 2.1 / 10 column (2.1 × 100
mm, manufactured by Pharmacia), equilibrated with solvent A, and loaded with 100 μl of the sample. The protein was eluted with a linear gradient of increasing the concentration of solvent B from 0 to 50% in 25 minutes (flow rate 150 μl / min).
The results are shown in FIG. As shown in FIG. 4, a single peak is shown in the fraction having an acetonitrile concentration of about 36% at the retention time. (2) SDS-polyacrylamide gel electrophoresis (SD
S-PAGE): SDS-PAGE was performed according to the method of Laemmli et al. [Nature, 227: 680, 1970]. The separation gel was a 12.5% (w / v) polyacrylamide gel, and the concentrated gel was a 4% (w / v) polyacrylamide gel, and electrophoresed at a constant current of 30 mA for 3 to 4 hours. After completion of the electrophoresis, proteins were detected by silver staining using a silver staining kit (Wako). The results are shown in FIG. As shown in FIG. 5, the protein obtained in Example 1 (7) was SD
Shows a single band on S-PAGE. SDS-PAG
From E, the molecular weight is considered to be about 30 kD. (3) Limited degradation of protein by enzyme: The sample obtained in the above (1) was lyophilized, dissolved in 100 μl of a 20 mM ammonium acetate solution (pH 7.8), and V8 protease was 1/20 to 1/1 of the sample. The mixture was added to a volume of 100, and allowed to stand at 37 ° C. overnight. The next day, the fragmented peptide was fractionated by reverse phase chromatography using a SMART System in the same manner as in (1) above. The results are shown in FIG. In FIG. 6, A is a partial peptide having the amino acid sequence of SEQ ID NO: 1, B is a partial peptide having the amino acid sequence of SEQ ID NO: 2, C is a partial peptide having the amino acid sequence of SEQ ID NO: 3, D indicates a partial peptide having the amino acid sequence of SEQ ID NO: 4, E indicates a partial peptide having the amino acid sequence of SEQ ID NO: 5, and F indicates a partial peptide having the amino acid sequence 5 of SEQ ID NO: 6.

Example 3 Analysis of Amino Acid Sequence: The protein having the effect of increasing histamine production obtained in the above-mentioned Example 1 (7) was analyzed using a 477A type protein sequencer (pulse liquid phase method, Applied Biosys.
Automated Edman degradation using PTH-
Amino acid analysis was performed using a 120A type online PTH analyzer (Applied Biosystem). as a result,
A partial peptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, or 6 was obtained. The amino acid sequence of the obtained partial peptide is shown in Sequence Listing: SEQ ID NOs: 1 to 6. Sequence Listing: In SEQ ID NOs: 1 to 6, Xaa indicates an amino acid that has not yet been determined.

Example 4 Lactose 70%, corn starch 30%, and histamine production-enhancing protein obtained in Example 1 (7)
Hydroxypropylcellulose is added in an amount of 5%, and wet granulation is performed to produce tablets.

Example 5 An injection is prepared by dissolving 10 mg of the protein having the activity of increasing purified histamine production obtained in Example 1 (7) in 2 ml of physiological saline.

Example 6 10 mg of the protein having the activity of increasing purified histamine production obtained in Example 1 (7) is dissolved in 2 ml of glucose injection to prepare an injection.

Example 7 Preparation of a screening reagent comprising the following reagents Calcium ionophore A23187 solution Dissolve in DMSO (Dimethyl Sulfoxide) to 1 mM and store at -20 ° C. Histamine determination reagent IN and 5N NaOH aqueous solution, 0.1N and 3N
HCl solution, NaCl saturated 0.1N NaOH solution, n-
BuOH, n-heptane, methanol solution with 1% o-phthalaldehyde, histamine dihydrochloride, 0.8N H
ClO ₄ , EMEM medium.

Example 8 Preparation of a screening reagent comprising the following reagents: Histamine production enhancing factor standard The histamine production enhancing factor was adjusted to 10 U / ml with PBS containing 0.1% bovine serum albumin (manufactured by Sigma). Dissolve and store at -20 ° C. Histamine determination reagent 5N NaOH solution, n-BuOH, saturated NaCl 0.1
N NaOH solution, 0.1N HCl solution, n-heptane, 1N NaOH solution, 1% o-phthalaldehyde added methanol solution, 3N HCl solution, histamine dihydrochloride, 0.8N HClO ₄ , EMEM medium

Test Example 1 Histamine increasing effect: Rat bone marrow cells were
３００300 g) were collected from the femur and tibia and adjusted to 1 × 10 ⁷ cells / ml in MEM medium. On the other hand, the histamine production enhancing factor used was purified from the air sac fluid of the air sac-type allergic inflammation-induced rat by various chromatography. The histamine-producing activity was measured using rat bone marrow cell suspension 0.
0.4 ml of various concentration HPIF aqueous solution is mixed with 4 ml,
Using a 12-well / dish, the cells were cultured at 5% CO ₂ at 37 ° C. for 24 hours.
After centrifugation for one minute, the amount of histamine in the supernatant was measured by the method of Shore et al. The biological activity (specific activity) of the purified HPIF was determined by the above-mentioned method, and it was found that the purified HPIF had an activity of 50,000 units / mg.

[0065]

EFFECT OF THE INVENTION The protein of the present invention having the effect of increasing histamine production, and the compound or its salt obtained by using the screening method or the screening kit of the present invention can be used as a therapeutic agent for inflammation. For example, it can be used as a therapeutic / prophylactic agent for symptoms such as rheumatism and asthma.

[0066]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

[0067]

[SEQ ID NO: 2] Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide

[0068]

[SEQ ID NO: 3] Sequence length: 11 Sequence type: amino acid Topology: linear Sequence type: peptide

[0069]

[SEQ ID NO: 4] Sequence length: 13 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Tyr Xaa Xaa Pro Ser Gly Phe Tyr Pro Tyr Pro Val Gln 1 5 10

[0070]

[SEQ ID NO: 5] Sequence length: 13 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Ile Lys Gly Gly Ser Phe Xaag Leu Leu Gln Xaa Xaa Gln 1510

[0071]

[SEQ ID NO: 6] Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

[Brief description of the drawings]

FIG. 1 shows the results of histamine production increasing activity of fractions obtained by anion exchange chromatography using DEAE-cellulose obtained in Example 1 (2).

FIG. 2 shows the results of gel filtration obtained in Example 1 (5).

FIG. 3 shows the FPLC System obtained in Example 1 (7).
2 shows the results of reversed-phase chromatography using the method shown in FIG.

FIG. 4 shows the SMART System obtained in Example 2 (1).
2 shows the results of reversed-phase chromatography using m.

FIG. 5 shows the results of SDS-polyacrylamide gel electrophoresis obtained in Example 2 (2).

FIG. 6 shows the results of limited protein degradation by the enzyme obtained in Example 2 (3).

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/00 ADS A61K 37/02 ABE C07K 7/04 ABG 7/08 ACD // C12P 21/08 ADS

Claims (19)

[Claims] 1. A protein having the amino acid sequence of SEQ ID NOs. A protein that has a production increasing effect. 2. The protein according to claim 1, wherein the substantially identical amino acid sequence is an amino acid sequence in which one or several amino acids have been deleted, substituted or added. 3. It has a partial sequence of the protein according to claim 1,
And a protein having an action of increasing histamine production. 4. The method for producing a protein according to claim 1, wherein the animal cell producing the protein according to claim 1 is cultured. 5. The method for producing a protein according to claim 1, wherein the animal cell according to claim 4 is a mast cell. 6. An antibody against the protein according to claim 1, 2 or 3. 7. A method for measuring a protein having an effect of increasing histamine production, which comprises using the antibody according to claim 6. [8] A kit for measuring a protein having an effect of increasing histamine production, which comprises the antibody according to [6]. 9. A compound that produces a larger amount of the protein according to claim 1 than when the test compound is brought into contact with a cell that produces the protein according to claim 1 and the test compound is not contacted. A method of screening for a compound having a histamine production increasing effect or a salt thereof. (10) A compound obtained by the scouring method according to (9). 11. A compound that produces more histamine than that obtained by contacting the protein or test compound of claim 1 with a histamine-producing cell and contacting the protein of claim 1 with a histamine-producing cell. A method for screening a compound or a salt thereof, which enhances the action of the protein according to claim 1. [12] A compound or a salt thereof which enhances the action of the protein according to [1], which is obtained by the screening method according to [11]. 13. A compound that produces less histamine than when the protein and test compound of claim 1 are brought into contact with histamine-producing cells and the protein of claim 1 is brought into contact with histamine-producing cells. A method for screening a compound or a salt thereof, which reduces the action of the protein according to claim 1. [14] a compound or a salt thereof, which reduces the action of the protein according to [1], obtained by the screening method according to [11]; 15. A pharmaceutical composition containing the protein according to claim 1, 2 or 3. 16. The pharmaceutical composition according to claim 15, which is an anti-inflammatory agent. 17. The pharmaceutical composition according to claim 16, which is used for treating or preventing rheumatism or asthma. 18. A method for preventing rheumatism or asthma containing a peptidic or non-peptidic compound that produces more histamine when contacted with a histamine-producing cell together with the protein of claim 1.
A therapeutic pharmaceutical composition. (19) An ulcer or an immunocompromised disease containing a peptide or non-peptidic compound which produces less histamine than the protein of (1) when brought into contact with a histamine-producing cell together with the protein of (1). Pharmaceutical composition for prevention and treatment. [0001]