JPH07258293A - Novel protein and method for producing the same - Google Patents

Abstract

(57) [Summary] (Modified) [Objective] To provide a novel protein having an active oxygen production inhibitory action. [Structure] The present protein has the amino acid sequence shown below. The gene also has a nucleotide sequence encoding the amino acid sequence of the protein. This protein can be produced by cell culture technology or genetic engineering technology. [Effect] The present protein suppresses the production of active oxygen in eosinophils, and is effective in the prevention and treatment of diseases such as bronchial asthma.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel protein, and more particularly to a novel physiologically active substance purified from a culture supernatant obtained by culturing human-derived cells. The present invention also relates to an active oxygen production inhibitor, more specifically,
The present invention relates to a novel protein purified from a culture supernatant obtained by culturing human-derived cells and having a function of suppressing active oxygen production in granulocytes such as eosinophils, and a method for producing the same. The present invention also relates to a DNA probe synthesized from the amino acid sequence of the protein. The present invention also relates to the gene of the protein cloned by applying genetic engineering technology. The present invention also provides a gene for the protein and a vector DNA produced by applying a genetic engineering technique.
A recombinant DNA body consisting of The present invention also relates to a transformed cell expressing the protein, which is produced by applying a genetic engineering technique. Furthermore, the present invention relates to a method for producing the protein by applying the technology of genetic engineering. Furthermore, the present invention relates to an antibody characterized by specifically binding to the protein. The present invention also relates to an active oxygen production inhibitor containing the protein.

[0002]

2. Description of the Related Art A characteristic function of eosinophils in the biological defense mechanism is a strong parasite-damaging action during parasite infection. It is believed to be based on existing strong basic proteins. Maj as a basic protein in special granules
or basic protein (MBP), eos
inophyl cationic protein
(ECP), eosinophil derived
neurotoxin (EDN) and eosinop
hil peroxidase (EPO) is known. However, in recent years, it has been revealed that these tissue-damaging substances exert a strong damaging action not only on parasites and bacteria but also on their own cells at times.

In humans, eosinophils are known to have a higher ability to produce active oxygen than neutrophils ¹⁾ , and the produced active oxygen has bactericidal action and cytotoxic action. Among them, hydrogen peroxide serves as a substrate for EPO,
Halogenated oxidants (HOCl, HOBr, HO) exhibiting strong bactericidal activity in the presence of (Cl ⁻ , Br ⁻ , I ⁻ ).
I) is produced, and its action has been shown to have stronger bactericidal action and parasite-killing action than MBP and ECP in an in vitro comparison ^{2) 3)} . As described above, active oxygen and basic protein are attracting attention as eosinophil-derived tissue-damaging substances, but it is clear that active oxygen-dependent tissue damage plays a major role as a major cause of the expression of tissue damage. Has been done.

[0004] Bronchial asthma, atopic dermatitis, allergic rhinitis, etc. are examples of those in which eosinophilia is observed and eosinophils are considered to be involved in the pathological condition of the disease as the main immune cells. ⁴⁾ . [1] We
iss, S.S. J. et al. , Science (Scie
nce), 236, 200 (1984); 2) Kleb.
anoff, S.A. J. et al. , Journal of Immunology (J. Immunol), 143, 23
9 (1989); 3) Humann, K .; J. et a
l. , Journal of Immunology (J. Immu
Nol) 144, 3166 (1990); 4) "Eosinophils" Makinoso flat edition, 1991, International Medicine Publishing]

[0005]

As described above, it is becoming clear that the cause is inflammation caused by parasitic cell infection and tissue cell damage caused by active oxygen produced by granulocytes such as eosinophils. However, effective drugs against these diseases have not yet emerged.

[0006] The object of the present invention is to provide a novel active oxygen production inhibitory factor which suppresses the production of active oxygen produced from eosinophils as a tissue injury factor. is there. Another object of the present invention is a DNA synthesized based on the amino acid sequence of the active oxygen production inhibitory factor.
To provide a probe. Still another object of the present invention is to provide a gene for the active oxygen production inhibitor cloned from a cell producing the active oxygen production inhibitor. Still another object of the present invention is to provide a recombinant DNA body in which a base sequence containing the gene for the active oxygen production inhibitory factor is ligated to a vector DNA that can be expressed in a host cell. . Still another object of the present invention is to provide a cell transformed with the recombinant DNA body. Still another object of the present invention is to cultivate cells capable of producing the active oxygen production inhibitor in a medium, to produce the protein in the culture solution, and to collect and collect a culture supernatant from the culture solution. It is intended to provide a method for producing the protein, which comprises separating and purifying the protein from the culture supernatant. Still another object of the present invention is to specifically prepare the active oxygen production inhibitor, which is produced by using the active oxygen production inhibitor or a partial peptide of the active oxygen production inhibitor as an antigen. It is to provide an antibody characterized by binding. Still another object of the present invention is to provide a pharmaceutical composition containing a therapeutically effective amount of the active oxygen production inhibitor as an active ingredient, and the use of the same to exhibit eosinophilia and eosinophil pathology. It is intended to provide a method for preventing and treating diseases which are considered to be related to, for example, diseases such as bronchial asthma, atopic dermatitis and allergic rhinitis. Still another object of the present invention is to provide a research reagent containing an active oxygen production inhibitor as an active ingredient.

[0007]

[Means for Solving the Problems] The present inventors have conducted extensive studies to find out a novel active oxygen production inhibitor for the above-mentioned purpose, and as a result, a completely new active oxygen production in the culture supernatant of animal cells has been achieved. As a result of discovering a factor having an inhibitory ability and further conducting research, the following knowledge has been obtained, and the present invention has been completed.

That is, according to the present invention, SEQ ID NO: 4 in the sequence listing
A novel protein comprising the amino acid sequence shown by is provided. According to another aspect of the present invention, a DNA probe synthesized based on the amino acid sequence of the protein is provided. Furthermore, according to another aspect of the present invention, a gene for the protein is provided. Further, according to another aspect of the present invention, there is provided a recombinant DNA body in which a base sequence containing a gene of the protein is linked to a vector DNA that can be expressed in a host cell. Furthermore, according to another aspect of the present invention, there is provided a cell transformed with the recombinant DNA body.
Furthermore, according to another aspect of the present invention, cells capable of producing the protein are cultured in a medium, the protein is produced in the culture solution, and the culture supernatant is recovered from the culture solution and recovered. There is provided a method for producing the protein, which comprises separating and purifying the protein from the culture supernatant. Furthermore, according to another aspect of the present invention, there is provided an antibody that specifically binds to at least a part of the protein.

The above novel protein of the present invention can be obtained as follows. It can be obtained by culturing cells that produce the protein. That is, as the cells used in the method of the present invention, various cells capable of producing the protein can be used. Normal diploid cells can be used to advantage, for example in human kidney, intestine, lung, heart,
Urinary tract, skin, foreskin, tongue, thyroid, placenta, cells from uterus, preferably human fetal kidney, lungs, cells from foreskin,
Even more preferably, cells derived from human fetal lung can be used.

For example, human fetal lung cells (manufactured by Flow Laboratories) were added with 0.75% Proteose Peptone No.
3 (manufactured by Difco), which may or may not contain serum, was cultured in a serum-free 199 medium (manufactured by Gibco), the protein was produced in the culture supernatant, and the recovered culture was used. The protein can be obtained by separating and purifying the protein from the screen.

As a purification method, a method usually used in protein chemistry, for example, an adsorption method using a carrier, a salting-out method, an electrophoresis method, and ion exchange, gel filtration, reverse phase chromatography, affinity to an appropriate ligand is used. The various chromatographic methods and the like applying can be used alone or in combination. As the chromatography method, preferably, CM sepharose column chromatography using sepharose having a carboxymethyl group bonded thereto, gel filtration column chromatography using particles such as crosslinked dextran gel, and phenyl sepharose column using sepharose having a phenyl group are used. Chromatography, mono-S column chromatography which is a cation exchange prepacked column, reverse phase column chromatography using a packing material in which a C8 group is introduced into a hard polymer, and an antibody that specifically binds to the substance of the present invention is bound. Antibody affinity column chromatography can be used.

The protein thus produced can be easily measured and detected by an enzyme immunoassay (EIA) method using an antibody that specifically binds to the product of the present invention, Western blotting and the like. When the product has an active oxygen production inhibitory action, it can be measured using the activity as an index. The specific antibody used here is an antiserum if it recognizes at least a part of the protein,
It may be either a polyclonal antibody or a monoclonal antibody.

As the immunogen, a protein represented by the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing can be used. A partial peptide consisting of a continuous partial amino acid sequence in the amino acid sequence of the protein, For example, a peptide usually consisting of 6 or more amino acid sequences, more preferably 7 or more amino acid sequences can be used. The partial peptide can be produced by a known method of peptide synthesis. And, it may be either solid phase synthesis method or liquid phase synthesis method. As an example, "Peptide Synthesis" (Nobuo Izumiya et al.,
1975, Maruzen Co., Ltd.) or “Basics and Experiments of Peptide Synthesis” (Nobuo Izumiya et al., 1985, Maruzen Co., Ltd.).

The partial peptide may be produced by cleaving the protein with an appropriate enzyme. Examples of the method include "Biochemistry Experiment Course 1 Protein Chemistry".
II ”(edited by the Japanese Biochemical Society, 1976, Tokyo Kagaku Dojin), pp. 255-332. When the protein represented by the amino acid sequence set forth in SEQ ID NO: 4 of the Sequence Listing or its partial peptide is immunized, it may be used as a complex with a carrier protein for immunization.

As the carrier protein, for example, bovine serum albumin, bovine gamma globulin, hemocyanin and the like can be used. Coupling of the peptide and the carrier protein can be easily carried out using a known conventional means. Examples of the reagent used for binding include glutaraldehyde, water-soluble carbodiimide and the like. The ratio of peptide to carrier protein used is about 1: 1.
Or about 1 to 30 (weight ratio) is suitable, and especially 1 to 1
5 to 20 is preferable, and further about 1 to 1 to 1 to 4
Is preferred. The reaction pH often gives good results near neutral, especially around 7.3. The reaction time is often about 2 to 6 hours, but about 3 hours is particularly suitable. A composite can be produced in this manner.

Polyclonal antibodies can be prepared, for example, by "Newborn Chemistry Experiment Course 12 Molecular Immunology III" (ed.
It can be produced by the method described in 1992, Tokyo Kagaku Dojin). That is, in order to produce a polyclonal antibody, the warm-blooded animal is inoculated with the immunogen produced as described above. Examples of warm-blooded animals used for the production of antibodies include warm-blooded mammals (eg, sheep, goats, rabbits,
Cow, rat, mouse, guinea pig, horse, pig), birds (eg, chicken, pigeon, duck, goose, quail)
And so on. As a method of inoculating the warm-blooded animal with the immunogen, the immunogen inoculated to the animal may be in an amount effective for producing antibodies. In many cases, the antibody is produced by emulsifying FCA) and inoculating subcutaneously or intradermally at several sites every 4 weeks for 5 times.

In addition to FCA, Freund's incomplete adjuvant (FIA), aluminum hydroxide adjuvant, pertussis vaccine adjuvant, etc. can be used as the adjuvant. Thus, as a method for collecting the antibody formed in the warm-blooded animal, for example, in the case of rabbit, it is usually collected from the ear vein between 7 and 12 days after the final inoculation,
It is obtained by centrifugation as serum (antiserum).

Although the antiserum can be used as a reagent itself, a non-specific purification method in which an immunoglobulin class or a subclass is chemically separated from the antiserum by protein, or an antibody protein itself having antibody activity is isolated. The polyclonal antibody can be purified using the specific purification method described above. As a non-specific purification method, a purification method of separating and fractionating from each class of antibody of immunoglobulin (Ig) contained in antiserum based on the difference in molecular properties of each class is used. Since the IgG class generally contains the highest concentration of antibodies, the IgG purification method can be used.

Specifically, protein A and protein G
Using fractionation method, density gradient centrifugation method, ultrafiltration method, separation fractionation method using gel matrix, isoelectric focusing method, ion exchange chromatography method, salting out method with ammonium sulfate, sodium sulfate, etc. be able to.
As a specific purification method, for example, a method of purifying a polyclonal antibody by collecting a fraction adsorbed by affinity chromatography using a carrier holding an immunogen can be used.

The Koehler and Milstein method (N
A monoclonal antibody obtained by a method similar to that of Nature (1975) 256, 495) can also be used. That is, an immunized warm-blooded animal, for example, an individual with an antibody titer is selected from mice, similar to the method for preparing the above-mentioned polyclonal antibody, and 1 to 5 days after the final immunization, spleen or lymph nodes are collected and By fusing the antibody-producing cells contained therein with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The fusion operation is a known method, for example, the Koehler and Milstein method [Natur.
re (1975) 256, 495].
Examples of the fusing agent include polyethylene glycol (PEG) and Sendai virus, but PEG is preferable.
Is used.

To obtain the monoclonal antibody, it is preferable to use rat or mouse. When immunizing a mouse, for example, the method of immunization is preferably subcutaneous, intraperitoneal, or intravenous injection. Further, the degree of variability of the immunization interval, the immunization amount, etc. is high, and various methods are possible. A method using spleen cells extracted after 4 days is often used. The immunization amount is about 0.1 μg or more per mouse, preferably about 10 μg to 300 μg, as the amount of peptide at one time.
It is desirable to use g. In addition, it is desirable to perform partial blood sampling before removing the spleen, confirm the increase in antibody titer in blood, and then perform a fusion experiment using spleen cells.

The cell fusion of the above-mentioned spleen cells and myeloma cells is carried out, for example, by removing the extracted mouse spleen cells with hypoxanthine-guanine-phosphoribosyl transferase (h).
ypoxanthine guanine phosp
horibosyltransferase: HGPR
T) deficiency and thymidine kinase (thymidine k)
suitable allogeneic or xenogeneic myeloma cells (eg P3 / X63-Ag) with markers such as inase: TK) deficiency
8, P3 / NSI-1-Ag4-1, P3 / X63-A
g8. U1, Sp2 / O-Ag14). For example, myeloma cells and splenocytes may be suspended at a ratio of 1: 1 to 1:20 in, for example, RPMI1640 medium (manufactured by Flow Laboratories) and a fusion agent such as Sendai virus and polyethylene glycol (PEG) may be used. it can.
Of course, it is also possible to add dimethyl sulfoxide (DMSO) and other fusion promoters.

PEG usually has a degree of polymerization of about 1000-60.
No. 00 is added at a concentration of about 10 to 80%,
Cell fusion can be efficiently carried out by incubating at -40 ° C, preferably 30-37 ° C for 0.5-30 minutes. The fused cells are HAT (hypoxanthine: hypox
anthine, aminopterin: aminopter
in, thymidine: 10% F containing thymidine)
It can be selectively grown using CS-added RPMI1640 medium (HAT medium) or the like. The culture supernatant of the grown cells can be screened for the production of the desired antibody, and the antibody titer can be screened as follows. That is, the presence or absence of antibody production against the immunogen can be examined by a method such as a radioimmunoassay (RIA) method or an enzyme immunoassay (EIA) method, but these methods can be variously modified. Further, it can be detected in consideration of the molecular weight.

It is desirable to clone cells showing growth in a selective medium and showing antibody activity against the peptide used for immunization by the limiting dilution method or the like. By screening the supernatant of the cloned cells in the same manner as described above, a monoclonal antibody-producing hybridoma clone showing reactivity with the immunogen can be obtained.

The hybridoma cloned as described above is grown in liquid medium. Specifically, for example, by culturing in a liquid medium, for example, RPMI1640 medium (manufactured by Flow Laboratories) supplemented with about 0.1 to 40% fetal bovine serum for about 2 to 10 days, preferably about 3 to 5 days, The monoclonal antibody can be obtained from the culture supernatant. Further, the antibody can be obtained by inoculating the mammal intraperitoneally, growing the cells, and collecting ascites. For this purpose, for example, in the case of mouse, Pristan (2, 6, 10, 14-te)
Approximately 1 × 10 ⁴ to 1 × in BALB / c or the like mice previously inoculated with the
10 ⁷ hybridomas, preferably about 5 × 10 ⁵ to 2 × 10 ⁶ hybridomas, are intraperitoneally inoculated and about 7 to 20 days later, preferably about 10 to 14 days later, ascites fluid is collected. The antibody produced and accumulated in the ascites can be easily isolated as a pure immunoglobulin from the monoclonal antibody by, for example, ammonium sulfate fraction or DEAE-cellulose column chromatography.

Thus, a monoclonal antibody that specifically recognizes at least a part of the protein of the present invention can be obtained. The molecular weight of the protein purified by the above method was determined by SDS polyacrylamide gel electrophoresis (PAG).
According to E), it was as follows. Molecular weight 25000 ± 2000 (measured by non-reducing SDS-PAGE) 14000 ± 2000 (measured by reducing SDS-PAGE) Furthermore, digesting the purified protein or the protein with a digestive enzyme such as lysyl endopeptidase The partial amino acid sequence of the protein was clarified by analyzing the peptide fragmented by the method using an amino acid sequencer. The clarified partial amino acid sequence is shown below.

[0027] The amino acid sequence 1 Ala Arg Asn Gly Asp His X Pro Leu (however, X is unknown amino acid) the amino acid sequence 2 Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro X Y Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys (where X and Y are unknown amino acids) Amino acid sequence 3 Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys

Further, by applying a genetic engineering technique, the gene of the protein can be cloned from the gene library of cells producing the protein. That is,
For example, messenger (m) from cells producing the protein
RNA is separated, and single-stranded complementary DNA (c
DNA), then double-stranded DNA is synthesized, and the complementary DN
Incorporating A into a phage or plasmid, transforming a host with the obtained recombinant phage or plasmid, culturing the obtained transformant, and then transforming the transformant with a suitable method,
For example, a phage or plasmid containing the target DNA is isolated by hybridization with a DNA probe encoding a part of the protein or by an immunoassay method using a specific antibody that binds to the protein, and its recombinant is isolated. A cDNA library of a DNA containing a gene encoding the protein can be produced by cutting out the desired cloned DNA and ligating the cloned DNA or a part thereof downstream of a promoter in an expression vector. ..

MRNA encoding the protein can be obtained from various cells producing the protein, such as human fetal lung cells. As a method for preparing RNA from the protein-producing cells, the guanidine thiocyanate method (T. Ma
niatis et al. , Molecular c
longing 2nd edition, ColdSp
ring Harbor Lab. , 1989) and the like. Next, mRNA is obtained according to a conventional method. At this time, mRNA Purification Kit (manufactured by Pharmacia) can be used. Using the mRNA thus obtained as a template, the method of Okayama Berg (M
olecular and Cellular Bio
logy, 2, 161, 1982 and the same magazine, 3, 2
80, 1983) to synthesize cDNA and obtain c
The DNA is incorporated into the plasmid.

As a plasmid for incorporating cDNA,
For example, pBR322, pUC18, pUC derived from Escherichia coli
19, pUC118, pUC119 (all sold by Takara Shuzo Co., Ltd.) and the like can be used, but any other one can be used as long as it can be replicated and propagated in the host. Examples of the phage vector into which the cDNA is incorporated include λgt10 and λgt11, but other vectors can be used as long as they can be propagated in the host. The plasmid thus obtained may be used in a suitable host, such as Escherichia (Esch).
erichia), Bacillus
It is introduced into genus bacteria using the calcium chloride method or the like.

Examples of the above Escherichia bacterium include Escherichia coli K12HB101, MC1061 and LE.
392, JM105 and the like. Examples of the bacterium of the genus Bacillus include Bacillus subtilis MI114. In addition, the phage vector can be prepared by in vitro packaging (Proc.
atl. Acad. Sci. (1978) 71,244
2) can be introduced. By the above method, the cDNA of the protein-producing cell containing the protein cDNA
A library can be created.

A method for cloning the protein DNA from the protein cDNA library is, for example, the protein cD prepared by using the phage vector λgt10.
A plaque hybridization method using an NA library and an oligonucleotide encoding the protein as a probe can be used. The nucleotide sequence of the DNA thus obtained is determined, for example, by the method of Sanger et al.
er, F.E. , Et al. Proc. Natl. Aca
d. Sci. (1977) 74, 5463). As described above, the DNA encoding the protein is obtained. The nucleotide sequence of DNA containing the DNA encoding the protein obtained in Example 4 described later is shown in SEQ ID NO: 10 in the sequence listing.

In this DNA sequence, the 16th start codon (ATG) to the 942nd stop codon (T
Up to GA) there was an open reading frame which could encode a 308 amino acid sequence. The amino acid sequence translated from the open reading frame is shown in SEQ ID NO: 5 in the sequence listing. Furthermore, the N-terminal amino acid sequence of the protein obtained in Example 3 and the amino acid sequence of the protein determined with reference to the internal partial amino acid sequence are shown in SEQ ID NO: 4 of the sequence listing. Peak 1, peak 2, peak 3
The amino acid sequences (respectively SEQ ID NOS: 1, 2 and 3 in the sequence listing) of each peptide fragment of [Matches with N-terminal amino acid sequence (see Example 3)] are the same as those of the protein shown in SEQ ID NO: 4 in the sequence listing. The sequences were matched with the 92nd to 107th, 63rd to 91st, and 1st to 9th sequences, respectively.

Since the amino acid sequence of the protein exists inside the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing, the protein represented by the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing corresponds to the protein. It is an example of a precursor protein to which a signal peptide consisting of the amino acid sequences 1 to 196 of SEQ ID NO: 5 in the Sequence Listing is added. The precursor protein is modified intracellularly or extracellularly,
It is expected that the protein of the present invention represented by SEQ ID NO: 4 in the sequence listing will mature when the signal peptide is cleaved off. Further, the precursor protein is present in the culture supernatant of cells transformed with an expression type plasmid having a DNA containing a gene encoding the precursor protein. The precursor protein can be detected by Western blotting, one example of which is shown in Reference Example 1.

SEQ ID No. 8 in the sequence listing, SEQ ID No. 9 in the sequence listing
The nucleotide sequence of is the DNA sequence of the protein obtained in the present invention. The base sequence shown in SEQ ID NO: 8 of the sequence listing is the same base sequence as the 589th to 927th base sequences of the base sequence of SEQ ID NO: 9 in the sequence listing. An example of the nucleotide sequence of the DNA encoding the amino acid sequence of the protein of the present invention is SEQ ID NO: 8 in the sequence listing. Further, as an example of the nucleotide sequence of the DNA encoding the amino acid sequence of the precursor protein shown in SEQ ID NO: 5 of the sequence listing, which is an example of the precursor protein of the protein, there is SEQ ID NO: 9 of the sequence listing.

The protein comprises a polypeptide having an amino acid sequence represented by SEQ ID NO: 4 in the sequence listing as a constituent.
The molecular weight of the protein measured by reducing SDS-PAGE is
It roughly corresponds to the molecular weight calculated from the polypeptide. The molecular weight of the protein measured by non-reducing SDS-PAGE is almost the same as the molecular weight calculated when the polypeptide forms a dimer. Therefore, the protein of the present invention is a polypeptide having the amino acid sequence of SEQ ID NO: 4 in the sequence listing, a dimer of the peptide, or a multimer of more than that depending on the case.

The polypeptide constituting the protein of the present invention may lack a part of the amino acid sequence of SEQ ID NO: 4 in the sequence listing, or may be partially mutated by the point mutation method. . Also,
It is possible that some amino acid residues or peptide residues are added to the N-terminal or C-terminal of the amino acid sequence. The homology of the amino acid sequence between the protein mutant and the protein is usually preferably 60% or more, and particularly 7
It is preferably 0% or more, more preferably 80% or more, and particularly preferably 90% or more. The protein of the present invention may comprise the polypeptide of the present invention as a constituent.

The DNA encoding the protein cloned as described above can be used as it is, or if desired after digestion with a restriction enzyme, depending on the purpose. An expression region can be obtained by cutting out a region to be expressed from the cloned DNA and ligating it downstream of a promoter in a vector suitable for expression.

D used to produce the protein of the invention
NA may be any DNA as long as it encodes the polypeptide represented by the amino acid sequence shown in SEQ ID NO: 4 of the Sequence Listing, and preferably the DNA shown in SEQ ID NO: 8 of the Sequence Listing is used. To be Furthermore, any D-containing DNA encoding a characteristic peptide such as a signal peptide is added to its 5'end or 3'end as long as it contains the DNA encoding the polypeptide.
It may be NA, but is preferably SEQ ID NO: 5 in the sequence listing.
Which encodes a precursor protein of the protein shown in
And more preferably the DNA represented by SEQ ID NO: 9 in the sequence listing. When the host is an animal cell, it is preferable to use the DNA shown in SEQ ID NO: 9 in the sequence listing.

The DNA may have a translation initiation codon at its 5'end and a translation termination codon at its 3'end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adaptor. Furthermore, in order to express the DNA, a promoter is connected upstream thereof. Examples of the vector include the above-mentioned Escherichia coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid, bacteriophage such as λ phage and retrovirus, and animal virus such as vaccinia virus. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression.

When the host to be transformed is Escherichia, the tac promoter, trp promoter, lac promoter, etc., and when the host is the Bacillus bacterium, SPO1 promoter, SPO2 promoter, etc., the host is yeast. In this case, PGK promoter, GAP promoter ADH promoter and the like are preferable. When the host is an animal cell, SV40-derived promoter, retrovirus promoter, metallothionein promoter, heat shock promoter and the like can be used.

The present inventors have also developed a technique for mass-producing a desired gene product in the process of expressing a DNA containing a gene encoding the protein of the present invention in a host cell and producing the protein. Marcos method (a method for obtaining a high-producing strain by cloning into a cosmid vector with a large number of desired gene expression units linked in the same direction and introducing the obtained recombinant DNA into an animal cell [eg, Ikeda et al .; GENE (198)
8) 71, 19-27]).

A transformant is produced using the expression plasmid containing the DNA encoding the protein thus constructed. Examples of the host include Escherichia, Bacillus, yeast, and animal cells.
Animal cells include, for example, monkey cells COS-1,
Vero, Chinese hamster cell CHO, silkworm cell SF9 and the like can be mentioned. Thus, a transformant transformed with the expression plasmid containing the DNA encoding the protein is obtained. As a preferred example of this transformant, the OSP-A1 (FE
RM BP-4562) strain.

The protein can be produced by culturing each transformant by a known method in an appropriate medium under appropriate culture conditions. Separation and purification of the protein from the above culture can be carried out, for example, by the following method. When the protein is extracted from the cultured bacterial cells or cells, the bacterial cells or cells are collected by a known method after culturing, suspended in an appropriate buffer solution, and then the ultrasonic cells, lysozyme, and / or freeze-thawing are used to suspend the bacterial cells. After destroying the body or cells, a method of obtaining a crude extract of the protein by centrifugation or filtration can be appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100.

When the protein is secreted into the culture medium, after the completion of the culture, the bacterial cells or cells and the supernatant are separated by a method known per se, and the supernatant is collected. The protein contained in the culture supernatant or the extract thus obtained can be separated and purified by the same method as for the protein which is a natural product. The N-terminal amino acid sequence of the recombinant protein thus produced coincided with the N-terminal amino acid sequence of the above-mentioned natural product, and the molecular weights of SDS-PAGE in the reduced state and the non-reduced state also coincided. Therefore, the recombinant protein is considered to be the same substance as the natural protein.

The protein can be measured by an enzyme immunoassay or the like using an antibody which specifically binds to the protein, which is a natural product. When the product has an active oxygen production inhibitory action, it can be measured using the activity as an index. The DNA encoding the protein of the present invention may be any DNA as long as it contains a nucleotide sequence encoding the amino acid sequence of the protein. For example, SEQ ID NO: 9 in the sequence listing, or The DNA containing the nucleotide sequence shown in SEQ ID NO: 8 or a part of the DNA is preferable.

The novel protein thus obtained has an activity of suppressing active oxygen production. The protein is in a suitable dosage form as a reagent for studying inhibition of active oxygen production in eosinophils, or by adding a carrier, diluent or excipient which can be administered as the protein alone or as at least one drug. It is also used as a medicine. The novel protein of the present invention can be used for the treatment and / or prevention of tissue damage caused by eosinophils, such as bronchial asthma, atopic dermatitis, and allergic rhinitis.

The novel protein of the present invention can also be used as an injection. In this case, a thickening agent such as sucrose, glycerin, methyl cellulose, carboxymethyl cellulose, a pH adjusting agent for various inorganic salts and the like can be added as additives. The dosage of the novel protein of the present invention per adult varies depending on age, sex, body weight, symptoms, etc., but is generally about 0.1 μg to 100 mg, and is administered once a day or several times as needed. can do.

The general operations necessary for handling the DNA described herein and Escherichia coli as a recombinant host are usually carried out by those skilled in the art, for example, Maniat.
is et al.'s experimental manual (T. Maniatis et a
l. , Molecular Cloning A Lab
oratory Manual, Cold Sprin
g Harbor Laboratory 1982
1989). The enzyme used,
All reagents can be commercial products, and unless otherwise specified, according to the usage conditions specified for the product,
You can achieve those goals perfectly.

[0050]

EXAMPLES In order to describe the present invention in more detail, examples will be described, but the scope of the present invention is not limited to these examples. Example 1 Production of Novel Protein by Human Cell Culture Method Commercially available human fetal lung normal diploid cells (manufactured by Flow Laboratories) were placed in a 100 l glass bottle at 10 ⁵ cells.
Implanted with Cytodex I (bead carrier for cell culture, manufactured by Pharmacia) at a density of 2.5 mg / ml at a density of s / ml and at 37 ° C. in air containing 5% CO 2 10% fetal bovine serum as a growth medium MEM medium (manufactured by Gibco) containing 60 l was added, and suspension culture was performed while stirring at a rotation speed of 60 rpm. After culturing for 8 days and allowing the cells to proliferate sufficiently, the bead carrier to which the cells adhered was washed with physiological saline, and 0.75% of proteose peptone No. Serum-free 199 medium (manufactured by Gibco) containing 3 (manufactured by Difco) was replaced with 60 l, and the cells were cultured with stirring at a rotation speed of 60 rpm. While changing this medium every 3rd day,
A conditioned medium containing this substance
ium; CM) was collected.

Example 2 Purification of Novel Protein Culture supernatant 1 of normal diploid human fetal lung obtained in Example 1
After adding about 1.1 liters of acetic acid to 70 liters to adjust the pH to 4, cell fragments and the resulting insoluble matter were removed by filtration. Adsorbed to a carboxymethyl sepharose (CM sepharose; Pharmacia) column (diameter 9 cm x height 23.5 cm) that had been sufficiently equilibrated with 20 mM acetate buffer of pH 4.0 containing 0.2 M sodium chloride in advance, and then equilibrated. PH 4.0 containing 13.5 liters buffer and 0.4M sodium chloride
After washing with 6 liters of 20 mM acetate buffer, the adsorbed protein was eluted with 12 liters of 20 mM acetate buffer of pH 4.0 containing 0.75 M sodium chloride and 10 mM lysine hydrochloride. By this operation, about 5 l of an eluate was obtained as a crude purified solution I containing the present protein. The proteose peptone component contained in a large amount in the culture supernatant was reduced to 1% or less.

Since the eluate usually contains a large amount of tissue plasminogen activator (tPA), it was specifically removed. That is, 5 M sodium hydroxide solution was added to adjust the pH to 7.0, and the monoclonal antibody against tPA, which had been preliminarily equilibrated with 20 mM Tris-hydrochloric acid buffer containing 0.5 M sodium chloride and having a pH of 7.5, was added to sepharose. The bound (3 mg / ml gel) was passed through an antibody column (9 cm diameter x 29 cm height). By this operation, about 6 l of the pass-through solution was obtained as the crude purified solution II, and the tPA activity contained in the crude purified solution I in a large amount was removed.

6 l of the crude purified liquid II was concentrated to 300 ml with an ultrafiltration module (manufactured by Asahi Kasei Co., Ltd., model SIP-0013), and pH 7.0 containing 10 volumes of about 0.05 M sodium chloride was added.
20 mM phosphate buffer solution was added and concentrated again to 300 ml, and the buffer solution was exchanged. The sample is 0.05
Adsorbed on a CM sepharose column (diameter 5 cm × height 15 cm) sufficiently equilibrated with 20 m phosphate buffer of pH 7.0 containing M sodium chloride, washed with 400 ml of the same buffer, and then washed with about 200 ml of 0.05M. Elution was performed with the same buffer solution containing E. coli (E1), about 200 ml of the same buffer solution containing 0.15 M sodium chloride (E2), and about 200 ml of the same buffer solution containing 0.5 M sodium chloride. Flow rate is 200 ml
/ Hour. The protein was mainly recovered as the E2 fraction, that is, about 200 ml of the crude purified liquid III at a recovery rate of 30 to 50%.

Crude purified solution III Approximately 200 ml was concentrated 10 times using the above ultrafiltration hollow fiber, and this was sufficiently equilibrated with PBS in advance on Sephacryl S-200 (Pharmacia) column (diameter 2.6 cm). Gel filtration at a height of 92 cm). The protein of the present invention is a fraction having a peak at an eluting molecular weight of about 25 kd, that is, about 100 ml of a crude purified liquid.
As IV, it was recovered at a recovery rate of 40 to 60%.

Ammonium sulfate was added to the crude purified solution IV so that the final concentration was 1 M, and then a phenyl sepharose (Pharmacia) column (diameter 1.6 cm × height 5 c
m), and the pH containing 1M ammonium sulfate in advance
After washing with 40 ml of 7.0 mM 10 mM phosphate buffer, 1
Elution was performed with a linear gradient decreasing the salt concentration of ˜0 M ammonium sulfate, followed by elution with 50% ethanol. The flow rate was 20 ml / hour. This protein contains 0.4 to 0 M ammonium sulfate concentration fraction and 50%
Eluted in the ethanol fraction, ie 20 ml of crude semen V
Was recovered at a recovery rate of 20 to 40%.

The crude semen V was dialyzed in advance against a 20 mM phosphate buffer solution having a pH of 6.0 to exchange the buffer solution, and this was adsorbed to a monoS HR5 / 5 (Pharmacia) column that had been sufficiently equilibrated with the same buffer solution in advance. Let After washing with 10 ml of the same buffer, elution was performed with a linear concentration gradient of a salt concentration of 0 to 1 M in the same buffer. The flow rate was 60 ml / hour. This protein has a salt concentration of 0.15 to 0.3
Fraction with a peak between M, ie about 7 ml of crude semen
The VI was recovered at a recovery rate of 10 to 30%. From the analysis of the same fraction by non-reducing SDS-PAGE, the substance of the present invention, which is a protein having a molecular weight of 25 ± 2 kd, was identified as the protein of the present invention contained in the same fraction.

Furthermore, a reduced sample obtained by boiling the same substance in the presence of β-mercaptoethanol for 3 minutes was subjected to SDS-P.
When analyzed by AGE, it showed a molecular weight of 14 ± 2 kd. Non-reduced SDS-PAGE is shown in Fig. 1, reduced SDS-P
The AGE is shown in FIG. The molecular weight is LMWkitE (94kd: phosphorylase b, 67k).
d: albumin, 43kd: ovalbumin, 30k
d: carbonic anhydrase, 20 kd: trypsin inhibitor, 14 kd: α-lactalbumin)
(Manufactured by Pharmacia).

The crude purified solution VI was preliminarily supplemented with 0% containing 0.1% TFA.
Adsorbed on Asahipak C8P-50 (manufactured by Asahi Kasei Co., Ltd.) (diameter 4.6 mm × height 15 cm) thoroughly washed with a linear concentration gradient of acetonitrile concentration of ˜100%,
Elution was performed with a linear concentration gradient of acetonitrile concentration of 0 to 100% containing 0.1% TFA.
A single eluting peak from 0 to 80% was freeze-dried to obtain about 10 μg of this substance.

Example 3 Determination of Amino Acid Sequence of Novel Protein 10 μg of the substance isolated by the method described in Example 2 was added to DEVELOSILN. P. ODS-2 (manufactured by Nomura Chemical Co., Ltd.) (diameter 4 mm x height 10 mm) is adsorbed on a column,
Elution was performed with a linear concentration gradient of acetonitrile-water containing 10% to 90% acetonitrile containing 0.05% TFA to obtain a single peak. Lyophilize this peak,
The substance was introduced into a gas phase sequencer (PSQ-1 system, manufactured by Shimadzu Corporation), and the N-terminal amino acid sequence of this substance was determined. The confirmed amino acid sequence is shown below.

Amino acid sequence Ala-Arg-Asn-Gly-Asp-His-X-Pro-Leu (where X is an unknown amino acid) Furthermore, 10 μg of this substance was added to 100 mM T containing 2M urea.
It was dissolved in a buffer solution of ris-HCl, pH 9.2, and lysyl endopeptidase (Lysyl Endopepti) was added.
Dase) (manufactured by Wako Pure Chemical Industries, Ltd.) was added and reacted at 37 ° C. overnight to fragment this substance. Reaction solution is D
EVELOSIL N.E. P. ODS-2 (manufactured by Nomura Chemical Co., Ltd.) (diameter 4 mm x height 10 mm) is adsorbed on a column,
Elution was performed with a linear concentration gradient of 10 to 90% acetonitrile containing 0.05% TFA to separate and elute the fragments. As a result, three elution peaks 1 to 3 were obtained in the range of 15 to 40% acetonitrile concentration. Each peak was freeze-dried and introduced into a gas phase sequencer (PSQ-1 system, Shimadzu Corp.) to determine the amino acid sequence.

The amino acid sequence of the fragment whose amino acid sequence was confirmed is shown below. Amino acid sequence of peak 1 Thr-Asp-Thr-Gly-Val-Ser-Leu-Gln-Thr-Tyr-Asp-Asp-Leu-Leu-Ala-Lys Peak 2 amino acid sequence Thr-Ser-Leu-His-Arg -Leu-Lys-Pro-Asp-Thr-Val-Pro-Ala-Pro-X-Y-Val-Pro-Ala-Ser-Tyr-Asn-Pro-Met-Val-Leu-Ile-Gln-Lys (however, , X and Y are unknown amino acids) Amino acid sequence of peak 3 Ala-Arg-Asn-Gly-Asp-His-X-Pro-Leu (where X is an unknown amino acid) The amino acid sequence obtained from peak 3 is It was in agreement with the N-terminal amino acid sequence of the protein determined in.

Example 4 Determination of Gene Sequence of Novel Protein (1) Antisense oligonucleotide M1 corresponding to the amino acid sequence of the active oxygen production inhibitor obtained in Example 3
a and M2a were synthesized as follows. Oligonucleotides were synthesized using a DNA synthesizer (Applied Biosystems: Applied Biosystems) according to the attached protocol. M1a: AGG (A) TCG (A) TCG (A) TANGTT (C) TG M2a: TTT (C) TGT (GA) ATNAGNACCAT (where N is an arbitrary nucleotide) Both oligonucleotides M1a and M2a are respectively Maniatis et al. Experimental book (T. Maniatis et a
l. , Molecular cloning A La
laboratory Manual, Cold Spri
ng Harbor Laboratory (1982)) according to the method described in T4 polynucleotide kinase and γ- ³² P-A.
It was labeled with TP and used as a probe for the hybridization experiment described below.

(2) 500 ml of human fetal lung cells (1.5 × 10 ⁸ cells) cultured by the method described in Example 1
PBS (phosphate buffered saline,
It was suspended in 10 mM phosphate buffer (pH 7.0) containing 0.15 M NaCl, washed twice by centrifugation, and then 24 ml of a guanidine solution (6 M Guani) was added.
dine isothiocyanate, 10 mM
Na-Citrate pH 7.0, 0.5% Mark
osyl, 0.1M β mercaptoethanol). Next, 1 g of cesium chloride was added to 2.5 ml of the cell suspension, and then 12 ml of a 5.7 M cesium chloride solution was added.
Was placed in a centrifuge tube. 27 ° C at 20 ° C
After centrifugation at pm for 17 hours, the precipitate was dissolved in sterile water and extracted with an equal amount of chloroform / butanol (4: 1). 2.5
A double volume of ethanol was added to precipitate RNA, which was then dissolved in sterile water. In this way, about 1 mg of RNA was obtained.

Next, the total amount of this RNA is transferred to mRNA Pur.
Purification was performed using an oligo dT cellulose column according to the attached protocol using the purification Kit (Pharmacia) to obtain 40 μg of mRNA.

(3) From 2 μg of the mRNA prepared in (2), Gubler, U. & Hoffmann, B.H. J.
(Gene 25, 263, 1983),
A double-stranded cDNA was prepared using cDNA Synthesis Kit (Boehringer Mannheim) according to the attached protocol. That is, single-stranded cDNA was synthesized by reverse transcriptase using mRNA as a template. Then this m
After forming a nick and a gap with RNA / cDNA hybrids by ribonuclease H, double-stranded cDNA was synthesized by replacing mRNA with DNA by Escherichia coli DNA polymerase I, and blunt-ended with T4 DNA polymerase. A cDNA was prepared.
0.4 μg of double-stranded cDNA was obtained.

(4) Double-stranded cDNA prepared in (3)
From 0.1 μg to cDNA using λ phage λgt10
Using the A cloning system (manufactured by Amersham), about 200,000 clones of λgt1 according to the attached protocol.
A cDNA library consisting of 0 recombinants was prepared.
That is, an EcoRI adapter was ligated to 0.1 μg of cDNA using T4 DNA ligase. After the excess adapter was removed using a SizeSep ^™ 400 span column (Pharmacia), the ends of the cDNA were phosphorylated with T4 polynucleotide kinase.

Next, 0.1 μg of the phosphorylated cDNA was ligated to the λgt10 EcoRI arm using T4 DNA ligase and packaged using the packaging extracts A and B to prepare a λgt10 recombinant.
SM buffer (0.1 M NaCl, 8 mM MgSO
_{4 · 7H 2 O, 50mM Tris} -HCl (pH7.
5), diluted to 500 μl with 0.01% gelatin) to obtain a λgt10 phage solution. A part of the phage solution was diluted with several kinds of dilutions and the diluted E. coli
A phage plating cell prepared from NM514 was infected and titrated by forming plaques on an agar plate of L medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl). It was confirmed to be x10 ⁵ pfu / ml, and it was confirmed that a cDNA library composed of about 2x10 ⁵ clones of λgt10 recombinant could be prepared.

(5) About 100,000 clones of the λgt10 recombinant obtained in (4) were screened by plaque hybridization using the oligonucleotide probes M1a and M2a synthesized in (1) to obtain 3 positive clones. It was That is, 250 μl of the phage solution obtained in (4) was added to E. 1 × 10 ⁵ plaques that had been infected with E. coli NM514 and formed on an agar plate of L medium were subjected to the method of Benton et al. (Benton et al.
l. , Science (1977) 196, 180-1.
82) and transfer to a nitrocellulose filter,
Alkali denaturation and neutralization were performed to fix the DNA.

Then, hybridization was carried out with the M1a probe prepared in (1) under the conditions described below. Hybridization is performed with 6 × SET (20 × S
ET: 3M NaCl, 0.4M Tris-HCl
(PH 7.8), 20 mM EDTA, 10 × Den Hearts (100 × Den Hearts: Ficoll 10 g, polyvinylpyrrolidone 10 g, bovine serum albumin 10 g, water 5)
(Dissolved in 00 ml), 0.1% SDS (SDS:
Sodium dodecyl sulfate), 100 μg / ml denatured salmon sperm DNA, and a solution containing 1 × 10 ⁶ cpm / ml of M1a probe, and the reaction was carried out at 40 ° C. for 2 hours.

Next, the filter is set to 6 × SSC (0.9M
NaCl, 90 mM sodium citrate) solution,
It was washed at room temperature for 15 minutes and then in the same solution at 39 ° C. for 15 minutes. Furthermore, in a 2 × SSC solution containing 0.1%, 3
It was washed at 9 ° C for 15 minutes. As a result of performing autoradiography for 24 hours at −80 ° C. using an intensifier screen, 5 positive spots were recognized.
Phages were extracted from the agar region corresponding to the positive spots, and plaque hybridization was performed again according to the above steps to obtain 5 types of purified λgt10 recombinants.

Furthermore, 5 kinds of λgt10 recombinants were
Plaque hybridization was performed according to the above steps using the 2a probe. As a result, M1a and M2a
Three λgt10 recombinants were obtained that hybridized to both probes. From these three recombinants, the method of Grossberger et al.
l. , Nucleic Acids Research
(1987) 15, 6737), λgt10 recombinant DNA was extracted and named OSP-C, D, and E, respectively.

(6) OSP-D, which is one of the λgt10 recombinant DNAs obtained in (5), was cleaved with EcoRI and then subjected to agarose gel electrophoresis.
The insert cDNA of b was recognized, and the gel fragment containing this cDNA fragment was cut out. DNA was extracted from the gel fragment using Gene Clean (GENE CLEAN ^™ , sold by Funakoshi Co., Ltd.) according to the attached protocol. The obtained DNA fragment of about 1.2 kb was subcloned into the EcoRI site of M13mp18 (Takara Shuzo) to obtain plasmid pKI5. A deletion mutant for determining the nucleotide sequence.
t) was prepared.

A dilation kit for kilosequence (manufactured by Takara Shuzo) was used for the production of mutants. The nucleotide sequence of each mutant is determined by the method of Sanger et al.
r, F. , Et al. Proc. Natl. Aca
d. Sci. (1977) 74, 5463), and the entire base sequence of the 1.2 kb cDNA fragment was determined. The determined nucleotide sequence is shown in SEQ ID NO: 10 in the sequence listing. A DNA fluorescent sequencer (manufactured by Applied Biosystems: Applied Biosy) is used to determine the nucleotide sequence.
It was performed according to the attached manual.

In this DNA sequence, the 16th start codon (ATG) to the 942nd stop codon (T
Up to GA) there was an open reading frame which could encode a 308 amino acid sequence. The amino acid sequence translated from the open reading frame is shown in SEQ ID NO: 5 in the sequence listing. Further, the amino acid sequence of the protein was determined with reference to the N-terminal amino acid sequence of the protein obtained in Example 3 and the internal partial amino acid sequence. The determined amino acid sequence is shown in SEQ ID NO: 4 in the sequence listing. The amino acid sequences of the respective peptide fragments of peak 1, peak 2 and peak 3 (sequence listings: SEQ ID NOS: 1, 2 and 3, respectively) are 92 to 107th amino acid sequences of the protein shown in SEQ ID NO: 4 of the sequence listing, respectively. It was revealed that the sequence matches the 63rd to 91st and 1st to 9th sequences.

Since the amino acid sequence of the protein corresponds to amino acid residues 197 to 308 of the amino acid sequence shown in SEQ ID NO: 5 of the sequence listing, the protein has the amino acid sequence shown in SEQ ID NO: 5 of the sequence listing. The precursor protein of the protein represented is produced by intracellular or extracellular modification by cleavage of the N-terminal 1 to 196 amino acid residues of SEQ ID NO: 5 in the sequence listing. It was suggested to be a protein.

Example 5 Construction of plasmid pKI9 Plasmid pKI9 obtained by the method described in Example 4 for introducing the gene of the protein into animal cells was prepared.
Marcos manufacturing method (IKEDA.H et al., GE
Constructed according to NE (1988) 71, 19-27). The steps are shown in FIGS. 3 to 5, and the steps will be described below. (1) [FIG. 3] First, the plasmid pSV2-dhfr (ATCC371
46) was digested with PvuII and BamHI and then subjected to agarose gel electrophoresis, using GeneClean to obtain about 2 kb.
A DNA fragment consisting of SV40 early promoter (Pe), dhfr gene, small T intron, and poly A addition signal was isolated and purified.

On the other hand, pUC18 (manufactured by Takara Shuzo) was Hin
After cutting with dIII, the cut surface was blunt-ended using a DNA blunting kit (Takara Shuzo) according to the attached protocol. Then Ba produced by cutting with BamHI
The above-mentioned DNA fragment containing the dhfr gene was inserted between mHI-HindIII (blunt-ended) to obtain a plasmid pmHB1. Then the plasmid pSV2-dhf
r was cleaved with PvuII and HindIII, and then subjected to agarose gel electrophoresis and 0.3 kb using Geneclean.
The DNA fragment containing the SV40 early promoter of was isolated and purified, and the fragment was blunt-ended with a DNA blunting kit. The SV40 early promoter DNA fragment was inserted into the SmaI site of plasmid pmHB1. A plasmid in which the SV40 early promoter was inserted in the same direction as the SV40 early promoter already present in pmHB1 was isolated and designated as an expression unit cassette plasmid pmHB2.

(2) [FIG. 4] Cosmid vector pLAFR1 (ATCC37167)
Is cleaved with BglII and has a COS site of about 1.6 kb.
Was isolated and purified. The DNA fragment and pmHB cleaved with BamHI and treated with alkaline phosphatase
1 (see (1) above) was ligated with T4 ligase to obtain pCD. Then the plasmid pSV2-n
eo (ATCC37150) was digested with SmaI and BglII to separate and purify a fragment of about 1 kb containing the neomycin resistance gene, and the digested surface was blunt-ended using a DNA blunting kit. The DNA fragment was replaced with Hind I
It was cut with II and BglII to remove the dhfr gene, and the cut surface was ligated with blunt-ended pCD and T4 ligase. A plasmid in which the neomycin resistance gene was inserted in the same direction as the SV40 early promoter was isolated and named cosmid vector pCN.

(3) [FIG. 5] A plasmid pKI having one of the DNAs encoding the protein, OSP-D, obtained by the method described in Example 4
After cutting 5 with Alw44I, the cut surface was blunt-ended. Next, after digestion with EcoRI, it was subjected to agarose gel electrophoresis, and a gene fragment of about 1 kb encoding the protein was separated and purified using Geneclean. The DNA fragment was EcoRI-Sm of M13mp19 (Takara Shuzo).
pKI6 was obtained by inserting into aI. Next, Eco
After digestion with RI, the end was digested with Bal31 to obtain λgt10EcoRI existing on the 5'end side of the protein gene.
The translation initiation codon ATG derived from the adapter was removed, the cross section was blunt-ended, and a HindIII linker (Takara Shuzo) was inserted for recircularization. PK obtained in this way
By digesting I7 with both HindIII and BamHI digestive enzymes, a DNA fragment encoding the protein having a HindIII cleavage site at the 5'end and a BamHI cleavage site at the 3'end was obtained.

The DNA fragment was inserted into HindIII and BglII sites of pmHB2 [see (1) above]. The plasmid obtained by this operation was designated as pKI8. A cosmid vector pCN having a neomycin resistance gene (see (2) above) was cleaved with SfiI, and a fragment of about 2.3 kb, which is an expression unit of the protein gene obtained by cleaving plasmid pKI8 with SfiI, was used. The mixture was mixed and connected at a ratio of about 1 to 20. Then, E. coli HB101 was transformed after packaging using an in vitro packaging kit Gigapak gold (sold by Toyobo Co., Ltd.). A plasmid was prepared from the obtained colonies, and the size of the plasmid was identified by agarose gel electrophoresis.
A 0 kb plasmid pKI9 was obtained.

Example 6 Transformation of CHO cells with plasmid CHO-DXB11 strain (Chasin et al.,
Proc. Natl. Acad. Sci. USA (19
80) 77, 4216) to Current Proto
cols in Molecular Biology
[Published by Current Pro
tocols (1987-1993)] UNIT 9.4
Transformation was performed according to the method described in 1. Specifically, DOT
Transformation was performed with AP (Boehringer Mannheim).

That is, the expression plasmids pKI9, 5 μg and pSV2-dhfr obtained by the method described in Example 5 were used.
(ATCC 37146) 0.5 μg was mixed and ethanol-precipitated. After air-drying, 100 μl of HBS (20 mM H 2
epes, 150 mM NaCl, pH 7.4). 30 μl of transfection-reag
ent (Boehringer Mannheim) 70 μl of H
After diluting with BS, it was mixed with the DNA-HBS solution. 1
After standing at room temperature for 0 minutes, 5.8 ml of 10% (v /
v) Mixed with Ham's F-12 medium (manufactured by Flow Laboratory) containing FCS. On the other hand, on the day before the transformation, CHO cells were seeded at 5 × 10 ⁵ cells / dish in a 6 cm diameter cell culture dish and cultured overnight. After aspiration of the medium, Ha containing the above-mentioned plasmid DNA
The m's F-12 medium was added, and the mixture was cultured at 37 ° C for 4 to 5 hours. The medium was replaced with a fresh medium and the cells were further cultured for 24 hours.

The cells attached to the dish were peeled using a 0.25% trypsin, 0.02% EDTA solution, and diluted in a culture dish with a diameter of 10 cm to 10 to 100 times and cultured. After 24 hours, the medium was replaced with a selective medium. The composition of the selective medium was 40% in DMEM containing 10% dialyzed FCS.
Geneticin G-418 to be 0 μg / ml
(Manufactured by Gibco) is added. The transformed cells were cloned by culturing for about 2 weeks while changing the medium every 3 to 4 days to obtain 3 transformants. One of the three transformants was transformed with OSP-A1 (FERM
BP-4562).

Example 7 Production of Recombinant Protein: 8% bovine serum (hereinafter abbreviated as CBS) obtained by adding 4 g of Asahi Kasei Microcarriers (manufactured by Asahi Kasei) to a spinner flask having a volume of 3 liters (high clone) 1.2 liters of DMEM (manufactured by Gibco) medium containing 25% / min.
Stir on rotation. 3.5 × 10 ⁸ pieces of OSP-A1 (see Example 6) were seeded on this and adhered. After the completion of the adhesion, 0.8 liter of the same medium was added to make the culture solution amount to 2 liter, and the cells were cultured overnight, and then perfusion culture was started. Cell density is 5 × 10
^When it reached ⁶ cells / ml, it was replaced with a production medium in which the CBS concentration was reduced to 0.5%, and production culture was performed for 5 days.
0 l of culture was obtained.

Example 8 Purification of Recombinant Protein of the Present Invention To 10 liters of a culture solution containing the recombinant protein prepared by the method described in Example 7, one-tenth amount of 200 mM phosphate buffer (pH 6) was added. , Further adjusted to pH 6 with hydrochloric acid, and then treated with SP Sepharose F.I. F. (Made by Pharmacia)
It was subjected to column (diameter 5 cm × height 15 cm) chromatography. That is, SP Sepharose F. F. Through column chromatography, containing 0.1 M NaCl 20
After washing with mM phosphate buffer (pH 6), 0.4M NaC
It was eluted with a 20 mM phosphate buffer solution (pH 6) containing 1 to prepare a crude purified solution A.

Ammonium sulfate was added to the crude purified solution A so that the final concentration was 1 M, and then phenyl sepharose F.
F. (Pharmacia) column (diameter 5 cm x height 5
cm) It was subjected to chromatography. That is, after washing with a 20 mM phosphate buffer solution (pH 7) containing 1 M ammonium sulfate, elution was performed with a linear concentration gradient of 1 to 0 M ammonium sulfate. Each elution fraction had a polyacrylamide concentration of 1
SDS polyacrylamide gel electrophoresis using a 5-25% gradient was performed and stained with 2D-silver staining reagent II "Daiichi" (manufactured by Daiichi Pure Chemicals Co., Ltd.). Fractions in which the recombinant type protein of the present invention was observed by observing the bands were pooled to obtain a crude purified solution B.

The crude purified solution B was concentrated about 20 times using an ultrafiltration hollow fiber (manufactured by Asahi Kasei Corporation), and then Sephacryl S-
200 (Pharmacia) column (diameter 2.6 cm x
Gel filtration at a height of 90 cm). Observation of the eluted fraction by silver staining revealed a fraction containing the recombinant protein of the present invention in a single band.

Example 9 Preparation of anti-M2 antibody First, an M2 peptide represented by the following amino acid sequence having a partial peptide of the protein of the present invention represented by the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing was chemically synthesized. did. The synthesis was carried out using a peptide synthesizer (manufactured by Applied Biosystems) according to the attached protocol. Amino acid sequence Cys Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala The M2 peptide was combined with the carrier protein keyhole limpet hemocyanin (KLH) to form a complex. That is, the M2 peptide and KLH are in a weight ratio of about 1: 2.
A complex was prepared by mixing at 0 and reacting at pH 7.3 for 3 hours.

Next, an anti-M2 peptide antibody was obtained by immunizing a rabbit with the complex. That is, in a rabbit, 1 mg of the M2 peptide complex was emulsified in 1 ml of physiological saline and Freund's complete adjuvant (FCA) at a time,
The cells were subcutaneously or intradermally inoculated 5 times every 4 weeks.
Blood was collected from the ear vein 10 days after the final inoculation and centrifuged to obtain anti-M2 antibody. It was confirmed by Western blotting that the method was described in Reference Example 1 that the M2 antibody specifically binds to at least a part of the protein of the present invention.

Reference Example 1 Detection of Novel Protein Using Western Blotting The protein can be detected by Western blotting using an anti-protein antibody that specifically binds to the protein. Specifically, the ECL Western blotting analysis system (manufactured by Amersham) was used according to the attached protocol. The outline is shown below. The protein or a mixture containing the protein is boiled in the presence of β-mercaptoethanol for 3 minutes and then subjected to SDS-PAGE.
To separate by. The separated protein is transferred from the gel to a high bond ECL membrane (manufactured by Amersham) using a Western blotting system (manufactured by Bio-Rad) according to the attached protocol.

The membrane was treated with a Tris buffer solution containing 20% (w / v) skim milk (20 mM Tris, 137 mM sodium chloride, 0.2% (v / v) Tween-20 (BioRad), pH 7.6. ), Followed by 0.1% (v / v) anti-M2 antibody (see Example 9).
And the primary antibody is bound by reacting with Tris buffer containing 0.5% (w / v) skim milk. After the reaction, the reaction mixture was washed with Tris buffer and then reacted with Tris buffer containing 0.1% (v / v) horseradish peroxidase (HRP) -labeled anti-rabbit Ig antibody (manufactured by Amersham) to give a secondary antibody. To combine.

Next, after washing with Tris buffer, detection reagent (detection reagent) was detected.
By exposing the membrane reacted in ts) to Hyperfilm ECL (manufactured by Amersham) and developing the film, a blotting image of the protein can be obtained.
The precursor protein of the protein (see Example 4) is detected as a band around 30 kd, and the protein is detected as a band around 14 kd. Western blotting analysis of the culture supernatant of animal cells transformed with an expression plasmid containing the gene of the protein, the protein secreted in the culture supernatant,
In addition, FIG. 6 shows an example of detecting the precursor protein. In this way, the protein can be purified from various purification sources using Western blotting as an index.

Example 10 Active Oxygen Production Suppressing Activity The active oxygen production inhibiting activity of eosinophils of the recombinant protein of the present invention produced by the method described in Example 8 was compared with the active oxygen production inhibiting activity described in Reference Example 2. It was measured by an activity measuring method.
The result is shown in FIG. 7.

Reference Example 2 Method for Measuring Active Oxygen Production Suppressing Activity in Eosinophils The active oxygen production suppressing activity in eosinophils of the novel protein obtained by the present invention was measured by the following method. (1) Induction and purification of guinea pig eosinophils Guinea pig (Hartley system, male, 300 to 350 g)
(Sales by Charles River) 1 ml per animal per week
Was intraperitoneally administered to horse serum. The guinea pigs administered for 8 weeks or more were anesthetized with ether the day after the final administration, and 50 ml of peritoneal lavage fluid (0.85% NaCl, 0.4% sodium citrate) was injected into the abdominal cavity, and the intraperitoneal fluid was collected after massage. This operation was repeated 4 times to obtain about 200 ml of intraperitoneal fluid.

The collected intraperitoneal fluid was centrifuged at 200 × g for 10 minutes with a tabletop centrifuge (Kubota Co., Ltd .: type 8100).
The precipitate was mixed with 3 ml of 0.1% BSA in Hanks' balanced salt solution (Hank's Balanced Salt Sol).
solution: HBSS) (manufactured by Gibco Co.) and then hemolyzed by adding 20 ml of 0.2% NaCl solution. After standing at room temperature for 1 minute, an equal amount of 1.6% N
An aCl solution was added to make the solution isotonic, centrifuged, and washed. The obtained precipitate was made into Perc containing 10% FCS and having a specific gravity of 1.07.
Suspended in an ol solution, and has a specific gravity of 1.123 and 1.09 Per
It was layered on top of the coll liquid and centrifuged at 1700 xg for 20 minutes with a tabletop centrifuge (Kubota Co., Ltd.). The eosinophil band formed between the specific gravities 1.123 and 1.09 was collected, washed, and then suspended in HBSS containing an appropriate amount of 0.1% BSA.

(2) Measurement of active oxygen amount 160 μM dissolved in HBSS containing 0.1% BSA
Into a 96-well plate in which 100 μl of cytochrome C of 10 μl of
And 100 μ of eosinophil suspension with a concentration of 3 × 10 ⁶ cells / ml
1 was added. After allowing to stand at room temperature for 10 minutes, 20 μl of 10 ⁻⁴ M calcium ionophore A23187 (manufactured by Wako Pure Chemical Industries, Ltd.) was kept in a 37 ° C. incubator.
In addition, the reaction is started. 5 minutes after 10 minutes and 20 minutes
The absorbance at 50 nm was measured with a microplate reader (Biorad: Model 2550).

Example 11 COS-1 with plasmid
Transformation of cells Cured COS-1 cells (ATCC CRL1650)
rent Protocols in Molecule
ar Biology [published by Cu
current Protocols (1987-199)
3)] Transformation was performed according to the method described in UNIT 9.2.

That is, Dulbecco's minimum essential medium (10% (v / v) fetal calf serum (hereinafter abbreviated as FCS) (manufactured by Gibco) containing COS-1 cells in a plastic dish having a diameter of 10 cm) ( Hereinafter abbreviated as DMEM)
(Manufactured by Flow Laboratories) was used to culture at 37 ° C. in a 5% carbon dioxide gas incubator until the logarithmic growth phase. The day before transformation, 0.1% trypsin and 0.02
The cells that had adhered and proliferated on the dish were peeled off using% EDTA, and the cells were replated at 1 × 10 ⁶ cells / dish. The expression plasmid pKI9, obtained by the method described in Example 5, 5 to 10 μg was added to 60 μl of TB.
S [A solution (NaCl 80 g / l, KCl 3.8 g
/ L, Na ₂ HPO ₄ 2 g / l, Trisbase 30
g / l, pH 7.5) 10 ml, B solution (CaCl ₂ 1
5 g / l, MgCl ₂ 10 g / l) 1 ml, H ₂ O 8
9 ml] and then 120 μl of DEAE-dex
It was added dropwise to the tran solution (10 mg / ml) with stirring.

Obtained DNA / DEAE-dextra
n was evenly added dropwise to the dish and cultured overnight in a 37 ° C. incubator. The next day, mixed cells of transformed cells and non-transformed cells could be obtained on the dish. Replace the medium with DMEM without fetal bovine serum and replace with 3-5
By culturing for a day, the protein of the present invention could be produced in the culture supernatant. The protein of the present invention produced in the culture supernatant was detected by subjecting the culture supernatant to Western blotting according to the method described in Reference Example 1. The transformed cells produced in this example died within a few days.

[0100]

[Application Examples] Application examples of the novel protein of the present invention will be described below with reference to application examples, but the present invention is not limited to these application examples. Application Example 1 Product of the present invention 1 mg Purified gelatin 20 mg Mannitol 100 mg Sodium chloride 7.8 mg Sodium phosphate 15.4 mg The above components are dissolved in 2 ml of distilled water for injection, put into a sterile vial, and vacuum degree is 0.075 Torr at -35 ° C. Primary drying for 35 hours, then 30 ° C, vacuum degree 0.03 Torr
The vial for injection was manufactured by secondary drying for 5 hours. The obtained composition is dissolved in 500 ml of physiological saline or glucose injection immediately before administration and used for intravenous infusion.

Application Example 2 Product of the Present Invention 10 μg Albumin 5 mg Mannitol 25 mg Sodium Chloride 1.95 mg Sodium Phosphate 3.85 mg Using the above components, a vial for injection was manufactured in substantially the same manner as in Application Example 1.

[0102]

INDUSTRIAL APPLICABILITY The novel protein of the present invention has active oxygen production inhibitory activity. The novel protein of the present invention can be effectively used for prevention and treatment of diseases such as bronchial asthma.

[Sequence list]

SEQ ID NO: 1 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys 1 5 10 15

SEQ ID NO: 2 Sequence length: 29 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Xaa Xaa 1 5 10 15 Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys 20 25

SEQ ID NO: 3 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Ala Arg Asn Gly Asp His Xaa Pro Leu 1 5

SEQ ID NO: 4 Sequence length: 112 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10 15 Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30 Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys 35 40 45 Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60 Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro 65 70 75 80 Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile 100 105 110

SEQ ID NO: 5 Sequence length: 308 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Pro Gly Gln Glu Leu Arg Thr Leu Asn Gly Ser Gln Met Leu Leu 1 5 10 15 Val Leu Leu Val Leu Ser Trp Leu Pro His Gly Gly Ala Leu Ser Leu 20 25 30 Ala Glu Ala Ser Arg Ala Ser Phe Pro Gly Pro Ser Glu Leu His Ser 35 40 45 Glu Asp Ser Arg Phe Arg Glu Leu Arg Lys Arg Tyr Glu Asp Leu Leu 50 55 60 Thr Arg Leu Arg Ala Asn Gln Ser Trp Glu Asp Ser Asn Thr Asp Leu 65 70 75 80 Val Pro Ala Pro Ala Val Arg Ile Leu Thr Pro Glu Val Arg Leu Gly 85 90 95 Ser Gly Gly His Leu His Leu Arg Ile Ser Arg Ala Ala Leu Pro Glu 100 105 110 Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe Arg Leu Ser 115 120 125 Pro Thr Ala Ser Arg Ser Trp Asp Val Thr Arg Pro Leu Arg Arg Gln 130 135 140 Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala Leu His Leu Arg Leu Ser 145 150 155 160 Pro Pro Pro Ser Gln Ser Asp Gln Leu Leu Ala Glu Ser Ser Ser Ala 165 170 175 Arg Pro Gln Leu Glu Leu His Leu Arg Pro Gln Ala Ala Arg Gly Arg 180 185 190 Arg Arg Ala Arg Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly 195 200 205 Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Asp Leu Gly 210 215 220 Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys 225 230 235 240 Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln 245 250 255 Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro 260 265 270 Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr 275 280 285 Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp 290 295 300 Cys His Cys Ile 305

SEQ ID NO: 6 Sequence Length: 17 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence AGRTCRTCRT ANGTYTG

SEQ ID NO: 7 Sequence Length: 17 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TTYTGDATNA GNACCAT

SEQ ID NO: 8 Sequence length: 339 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Human cell type: human embryonal run
g Sequence feature Characteristic symbol: peptide Location: 1. ． 339 Method of Characterizing: S Sequence GCG CGC AAC GGG GAC CAC TGT CCG CTC GGG CCC GGG CGT TGC TGC CGT 48 Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10 15 CTG CAC ACG GTC CGC GCG TCG CTG GAA GAC CTG GGC TGG GCC GAT TGG 96 Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30 GTG CTG TCG CCA CGG GAG GTG CAA GTG ACC ATG TGC ATC GGC GCG TGC 144 Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys 35 40 45 CCG AGC CAG TTC CGG GCG GCA AAC ATG CAC GCG CAG ATC AAG ACG AGC 192 Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60 CTG CAC CGC CTG AAG CCC GAC ACG GTG CCA GCG CCC TGC TGC GTG CCC 240 Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro 65 70 75 80 GCC AGC TAC AAT CCC ATG GTG CTC ATT CAA AAG ACC GAC ACC GGG GTG 288 Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val 85 90 95 TCG CTC CAG ACC TAT GAT GAC TTG TTA GCC AAA GAC TGC CAC TGC ATA 336 Ser Leu Gln Thr Tyr Asp Asp Leu L eu Ala Lys Asp Cys His Cys Ile 100 105 110 TGA 339

SEQ ID NO: 9 Sequence length: 927 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Human cell type: human embryonic luna
g Sequence feature Characteristic symbol: peptide Location: 1. ． 927 Characterized Method: S Sequence ATG CCC GGG CAA GAA CTC AGG ACG CTG AAT GGC TCT CAG ATG CTC CTG 48 Met Pro Gly Gln Glu Leu Arg Thr Leu Asn Gly Ser Gln Met Leu Leu -195 -190 -185 GTG TTG CTG GTG CTC TCG TGG CTG CCG CAT GGG GGC GCC CTG TCT CTG 96 Val Leu Leu Val Leu Ser Trp Leu Pro His Gly Gly Ala Leu Ser Leu -180 -175 -170 -165 GCC GAG GCG AGC CGC GCA AGT TTC CCG GGA CCC TCA GAG TTA CAC TCC 144 Ala Glu Ala Ser Arg Ala Ser Phe Pro Gly Pro Ser Glu Leu His Ser -160 -155 -150 GAA GAC TCC AGA TTC CGA GAG TTG CGG AAA CGC TAC GAG GAC CTG CTA 192 Glu Asp Ser Arg Phe Arg Glu Leu Arg Lys Arg Tyr Glu Asp Leu Leu -145 -140 -135 ACC AGG CTG CGG GCC AAC CAG AGC TGG GAA GAT TCG AAC ACC GAC CTC 240 Thr Arg Leu Arg Ala Asn Gln Ser Trp Glu Asp Ser Asn Thr Asp Leu -130 -125 -120 GTC CCG GCC CCT GCA GTC CGG ATA CTC ACG CCA GAA GTG CGG CTG GGA 288 Val Pro Ala Pro Ala Val Arg Ile Leu Thr Pro Glu Val Arg Leu Gly -115 -110 -105 TCC GGC GGC CAC CTG CAC CTG CGT ATC TCT CGG GCC GCC CTT CCT GAG 336 Ser Gly Gly His Leu His Leu Arg Ile Ser Arg Ala Ala Leu Pro Glu -100 -95 -90 -85 GGG CTC CCC GAG GCC TCC CGC CTT CAC CGG GCT CTG TTC CGG CTG TCC 384 Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe Arg Leu Ser -80 -75 -70 CCG ACG GCG TCA AGG TCG TGG GAC GTG ACA CGA CCG CTG CGG CGT CAG 432 Pro Thr Ala Ser Arg Ser Trp Asp Val Thr Arg Pro Leu Arg Arg Gln -65- 60 -55 CTC AGC CTT GCA AGA CCC CAG GCG CCC GCG CTG CAC CTG CGA CTG TCG 480 Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala Leu His Leu Arg Leu Ser -50 -45 -40 CCG CCG CCG TCG CAG TCG GAC CAA CTG CTG GCA GAA TCT TCG TCC GCA 528 Pro Pro Pro Ser Gln Ser Asp Gln Leu Leu Ala Glu Ser Ser Ser Ala -35 -30 -25 CGG CCC CAG CTG GAG TTG CAC TTG CGG CCG CAA GCC GCC AGG GGG CGC 576 Arg Pro Gln Leu Glu Leu His Leu Arg Pro Gln Ala Ala Arg Gly Arg -20 -15 -10 -5 CGC AGA GCG CGT GCG CGC AAC GGG GAC CAC TGT CCG CTC GGG CCC GGG 624 Arg Arg Ala Arg Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly 1 5 10 CGT TGC TGC CGT CTG CAC ACG GTC CGC GCG TCG CTG GAA G AC CTG GGC 672 Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly 15 20 25 TGG GCC GAT TGG GTG CTG TCG CCA CGG GAG GTG CAA GTG ACC ATG TGC 720 Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys 30 35 40 ATC GGC GCG TGC CCG AGC CAG TTC CGG GCG GCA AAC ATG CAC GCG CAG 768 Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln 45 50 55 60 ATC AAG ACG AGC CTG CAC CGC CTG AAG CCC GAC ACG GTG CCA GCG CCC 816 Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro 65 70 75 TGC TGC GTG CCC GCC AGC TAC AAT CCC ATG GTG CTC ATT CAA AAG ACC 864 Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr 80 85 90 GAC ACC GGG GTG TCG CTC CAG ACC TAT GAT GAC TTG TTA GCC AAA GAC 912 Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp 95 100 105 TGC CAC TGC ATA TGA 927 Cys His Cys Ile 110

SEQ ID NO: 10 Sequence length: 1200 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Human cell type: human embryonic luna
g Sequence feature Characteristic symbol: peptide Location: 16. ． 942 Method for determining the feature: S Symbol representing the feature: polyA site Location: 1184. ． 1200 Method of characterization: S sequence GCAACCTGCA CAGCC ATG CCC GGG CAA GAA CTC AGG ACG CTG AAT GGC 48 Met Pro Gly Gln Glu Leu Arg Thr Leu Asn Gly -195 -190 TCT CAG ATG CTC CTG GTG TTG CTG GTG CTC TCG TGG CTG CCG CAT GGG 96 Ser Gln Met Leu Leu Val Leu Leu Val Leu Ser Trp Leu Pro His Gly -185 -180 -175 -170 GGC GCC CTG TCT CTG GCC GAG GCG AGC CGC GCA AGT TTC CCG GGA CCC 144 Gly Ala Leu Ser Leu Ala Glu Ala Ser Arg Ala Ser Phe Pro Gly Pro -165 -160 -155 TCA GAG TTA CAC TCC GAA GAC TCC AGA TTC CGA GAG TTG CGG AAA CGC 192 Ser Glu Leu His Ser Glu Asp Ser Arg Phe Arg Glu Leu Arg Lys Arg -150 -145 -140 TAC GAG GAC CTG CTA ACC AGG CTG CGG GCC AAC CAG AGC TGG GAA GAT 240 Tyr Glu Asp Leu Leu Thr Arg Leu Arg Ala Asn Gln Ser Trp Glu Asp -135 -130 -125 TCG AAC ACC GAC CTC GTC CCG GCC CCT GCA GTC CGG ATA CTC ACG CCA 288 Ser Asn Thr Asp Leu Val Pro Ala Pro Ala Val Arg Ile Leu Thr Pro -120 -115 -110 GAA GTG CGG CTG GGA TCC GGC GGC CAC CTG CAC CTG CGT ATC TCT CGG 336 Glu Val Arg Leu Gly S er Gly Gly His Leu His Leu Arg Ile Ser Arg -105 -100 -95 -90 GCC GCC CTT CCT GAG GGG CTC CCC GAG GCC TCC CGC CTT CAC CGG GCT 384 Ala Ala Leu Pro Glu Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala -85 -80 -75 CTG TTC CGG CTG TCC CCG ACG GCG TCA AGG TCG TGG GAC GTG ACA CGA 432 Leu Phe Arg Leu Ser Pro Thr Ala Ser Arg Ser Trp Asp Val Thr Arg -70 -65 -60 CCG CTG CGG CGT CAG CTC AGC CTT GCA AGA CCC CAG GCG CCC GCG CTG 480 Pro Leu Arg Arg Gln Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala Leu -55 -50 -45 CAC CTG CGA CTG TCG CCG CCG CCG TCG CAG TCG GAC CAA CTG CTG GCA 528 His Leu Arg Leu Ser Pro Pro Pro Ser Gln Ser Asp Gln Leu Leu Ala -40 -35 -30 GAA TCT TCG TCC GCA CGG CCC CAG CTG GAG TTG CAC TTG CGG CCG CAA 576 Glu Ser Ser Ser Ala Arg Pro Gln Leu Glu Leu His Leu Arg Pro Gln -25 -20 -15 -10 GCC GCC AGG GGG CGC CGC AGA GCG CGT GCG CGC AAC GGG GAC CAC TGT 624 Ala Ala Arg Gly Arg Arg Arg Ala Arg Ala Arg Asn Gly Asp His Cys- 5 1 5 CCG CTC GGG CCC GGG CGT TGC TGC CGT CTG CAC ACG GTC CGC GCG TCG 672 Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser 10 15 20 CTG GAA GAC CTG GGC TGG GCC GAT TGG GTG CTG TCG CCA CGG GAG GTG 720 Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val 25 30 35 CAA GTG ACC ATG TGC ATC GGC GCG TGC CCG AGC CAG TTC CGG GCG GCA 768 Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala 40 45 50 55 AAC ATG CAC GCG CAG ATC AAG ACG AGC CTG CAC CGC CTG AAG CCC GAC 816 Asn Met His Ala Gln Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp 60 65 70 ACG GTG CCA GCG CCC TGC TGC GTG CCC GCC AGC TAC AAT CCC ATG GTG 864 Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val 75 80 85 CTC ATT CAA AAG ACC GAC ACC GGG GTG TCG CTC CAG ACC TAT GAT GAC 912 Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp 90 95 100 TTG TTA GCC AAA GAC TGC CAC TGC ATA TGAGCAGTCC TGGTCCTTCC 959 Leu Leu Ala Lys Asp Cys His Cys Ile 105 110 ACTGTGCACC TGCGCGGGGGGGCGCAGCTC AGTTGTCCTG CCCTGTGGAA TGGGCTCAAG 1019 GTTCCTGAGA CACCCGATTC CTGCCCAAATA AGCT AGTCT GTTATTTATT 1079 ATTAATTTAT TGGGGTGACC TTCTTGGGGA CTCGGGGGCT GGTCTGATGG AACTGTGTATAT 1139 TTATTTAAAA CTCTGGTGAT AAAAATAAAG CTGTCTGAAC TGTTAAAAAA AAAAAAAAAA 1199 A 1200

[Brief description of drawings]

FIG. 1 shows the non-reduced SDS-of the product of the present invention purified in Example 2.
A schematic diagram of a PAGE pattern is shown, in which lane 1 is a molecular weight marker and lane 2 is the purified sample.

FIG. 2 is a schematic diagram showing the reduced SDS-PAGE pattern of the product of the present invention purified in Example 2 as in FIG.
Lane 1 is a marker and lane 2 is this purified preparation.

FIG. 3 shows the steps for constructing an expression plasmid for animal cells of the protein of the present invention prepared in Example 5.

FIG. 4 shows the steps for constructing an expression plasmid for animal cells of the protein of the present invention following FIG.

FIG. 5 shows the steps of constructing an expression plasmid for animal cells of the protein of the present invention following FIG.

FIG. 6 shows an example of a Western blotting image obtained by the method described in Reference Example 1 in the reduced state of the protein of the present invention secreted into the culture supernatant of animal cells and the precursor protein of the protein. The protein is detected as a band at about 14 kd, and the precursor protein of the protein is detected as a band at about 30 kd. The molecular weights shown next to each other represent the positions of the molecular weight markers electrophoresed on the same filter.

FIG. 7 shows the active oxygen production inhibitory effect of the product of the present invention on eosinophils measured in Example 10, in which the horizontal axis represents reaction time and the vertical axis represents absorbance. The concentration of the product of the present invention is 5 pg /
It is measured by setting 4 steps in the range of ml to 5 ng / ml.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical display location A61K 38/00 ABF ACD ADA C12N 5/10 15/09 ZNA C12P 21/02 C 9282-4B 21/08 9161- 4B // (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (C12P 21/08 C12R 1:91) A61K 37/02 ABF ACD ADA 7729-4B C12N 5/00 B 9281- 4B 15/00 ZNA A (C12N 5/00 B C12R 1:91)

Claims (12)

[Claims] 1. A protein composed of a polypeptide having the following amino acid sequence. Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile 2. The protein according to claim 1, wherein the protein has the following molecular weight. 25000 ± 2000 (measured by non-reducing SDS-PAGE) 14000 ± 2000 (measured by reducing SDS-PAGE) 3. The protein according to claim 1, wherein the protein has an activity of suppressing active oxygen production in eosinophils. 4. A gene encoding the protein according to claim 1. 5. A recombinant DNA body obtained by linking a nucleotide sequence containing the gene encoding the protein according to claim 1 and a vector DNA expressible in a host cell. 6. The recombinant DNA body according to claim 5, wherein the base sequence containing the gene encoding the protein has the following base sequence. GCGCGCAACG GGGACCACTG TCCGCTCGGG CCCGGGCGTT GCTGCCGTCT GCACACGGTC CGCGCGTCGC TGGAAGACCT GGGCTGGGCC GATTGGGTGC TGTCGCCACG GGAGGTGCAA GTGACCATGT GCATCGGCGC GTGCCCGAGC CAGTTCCGGG CGGCAAACAT GCACGCGCAG ATCAAGACGA GCCTGCACCG CCTGAAGCCC GACACGGTGC CAGCGCCCTG CTGCGTGCCC GCCAGCTACA ATCCCATGGT GCTCATTCAA AAGACCGACA CCGGGGTGTC GCTCCAGACC TATGATGACT TGTTAGCCAA AG CTGCCAC TGCATATGA 7. The recombinant DNA body according to claim 5, wherein the base sequence containing the gene encoding the protein has the following base sequence. ATGCCCGGGC AAGAACTCAG GACGCTGAAT GGCTCTCAGA TGCTCCTGGT GTTGCTGGTG CTCTCGTGGC TGCCGCATGG GGGCGCCCTG TCTCTGGCCG AGGCGAGCCG CGCAAGTTTC CCGGGACCCT CAGAGTTACA CTCCGAAGAC TCCAGATTCC GAGAGTTGCG GAAACGCTAC GAGGACCTGC TAACCAGGCT GCGGGCCAAC CAGAGCTGGG AAGATTCGAA CACCGACCTC GTCCCGGCCC CTGCAGTCCG GATACTCACG CCAGAAGTGC GGCTGGGATC CGGCGGCCAC CTGCACCTGC GTATCTCTCG GG CGCCCTT CCTGAGGGGC TCCCCGAGGC CTCCCGCCTT CACCGGGCTC TGTTCCGGCT GTCCCCGACG GCGTCAAGGT CGTGGGACGT GACACGACCG CTGCGGCGTC AGCTCAGCCT TGCAAGACCC CAGGCGCCCG CGCTGCACCT GCGACTGTCG CCGCCGCCGT CGCAGTCGGA CCAACTGCTG GCAGAATCTT CGTCCGCACG GCCCCAGCTG GAGTTGCACT TGCGGCCGCA AGCCGCCAGG GGGCGCCGCA GAGCGCGTGC GCGCAACGGG GACCACTGTC CGCTCGGGCC CGGGCGTTGC TGCCGTCTGC ACACG TCCG CGCGTCGCTG GAAGACCTGG GCTGGGCCGA TTGGGTGCTG TCGCCACGGG AGGTGCAAGT GACCATGTGC ATCGGCGCGT GCCCGAGCCA GTTCCGGGCG GCAAACATGC ACGCGCAGAT CAAGACGAGC CTGCACCGCC TGAAGCCCGA CACGGTGCCA GCGCCCTGCT GCGTGCCCGC CAGCTACAAT CCCATGGTGC TCATTCAAAA GACCGACACC GGGGTGTCGC TCCAGACCTA TGATGACTTG TTAGCCAAAG ACTGCCACTG CATATGA 8. A cell transformed with the recombinant DNA body according to claim 5. 9. A protein produced by the cell according to claim 8, wherein the protein is a protein. 10. A method for producing a protein, which comprises culturing cells capable of producing the protein according to claim 1 in a medium and collecting the produced protein. 11. An antibody which specifically binds to the protein according to claim 1. 12. A protein containing the protein according to claim 1.
A drug for treating tissue damage due to active oxygen derived from human eosinophils.