WO2000009687A1

WO2000009687A1 - Novel gene and protein gmp30 encoded thereby

Info

Publication number: WO2000009687A1
Application number: PCT/JP1999/004351
Authority: WO
Inventors: Kiyoshi Takayamna; Makoto Yoshimoto
Original assignee: Taisho Pharmaceutical Co., Ltd.
Priority date: 1998-08-12
Filing date: 1999-08-11
Publication date: 2000-02-24
Also published as: AU5196599A; JP2000050879A

Abstract

[Object]: To provide a human monocyte-derived novel protein GMP30 having an inflammation regulatory activity and a gene gmp30 encoding the same. [Constitution]: A gene gmp30 encoding a novel protein GMP30 having an inflammation regulatory activity is obtained by cloning a human monocyte-derived cDNA library. Because of having the inflammation regulatory activity, this protein is usable in developing anti-inflammation drugs, etc.

Description

Specification

Novel genes and their encoded proteins GMP 30

The present invention relates to a novel protein GMP30 derived from monocytes having an inflammation regulating activity, and a gene gmp30 encoding the protein. Background art

Monocytes are mononuclear phagocytes derived from hematopoietic stem cells.Infections such as the incorporation of foreign microorganisms and the like into monocyte cells and melting by the action of lysosomes, which are numerous in monocytes. Plays an important role in the defense of the body.

In addition, monocytes have the property of migrating from the blood through cells of the vascular basement membrane toward cells that are undergoing an inflammatory reaction due to external stimuli, etc., and in the process, monocytes eventually become macrophages. It is known to differentiate. It is also known that monocytes and macrophages are activated by interferon expressed from cells undergoing an inflammatory response. In particular, since the inflammatory response caused by a foreign stimulus is controlled by the action of immune cells such as T cells and B cells, monocytes or macrophages are immune system cells (T cells, B cells, etc.) at the site of inflammation. It is supposed to have the function of controlling inflammation through some interaction with

However, there are still many unclear points concerning the inflammatory control function through the interaction with immune system cells, and in particular, what kind of molecule controls the inflammatory response. Therefore, it is presumed that elucidation of a molecule that regulates this inflammatory response makes it possible to use such a biomolecule directly as a drug or indirectly search for a drug compound.

An object of the present invention is to identify such a molecule and use it for the development of a medicine or the like. Disclosure of the invention

The present inventors aimed at identifying factors involved in the control of inflammation, and as a result of intensive studies to determine a desired protein from genes highly expressed in human monocytes, The present invention has succeeded in isolating the presence of GMP30 and the gene encoding it, gmp30, and completed the present invention.

That is, the present invention relates to (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1, or (b) an amino acid in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1. The present invention relates to a protein comprising a sequence and having an inflammation regulating function. Furthermore, the present invention relates to (c) a gene consisting of the DNA of SEQ ID NO: 2, or (d) a protein that hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and has an inflammatory regulatory function. It concerns the gene consisting of the encoding DNA.

GMP 30 of the present invention is a protein having a molecular weight of 30.3 kilodaltons (k d) consisting of a total of 266 amino acid residues as shown in SEQ ID NO: 1.

In addition, gmp 30 of the present invention is a gene consisting of 801 base pairs (bp) as shown in SEQ ID NO: 2.

The gene gmp30 can be isolated from a human monocyte-derived cDNA library as a cDNA fragment containing the gene. The cDNA library used by the present inventors was prepared based on mRNA extracted from healthy male peripheral blood monocytes according to a general method. Similarly, cDNA can be prepared based on mRNA of peripheral blood monocytes.

As a method for identifying genes highly expressed in human monocytes, a method for analyzing gene expression frequency by the method of Okubo et al. (Okubo et al., Nature Genet., 2, pl 73, 1992) was used. Can be used. Specifically, the following procedure is used.

Human monocyte-derived mRNA was converted to type II, and cDNA synthesis was performed using a primer obtained by binding oligo dT to one end of a vector plasmid opened with an appropriate restriction enzyme as a primer, followed by the restriction enzyme MboI. Cleavage with restriction enzyme BamHI. Since this vector was prepared using dam methylase-positive E. coli as a host, the A residue of “GATC”, which is a recognition sequence of MboI, is methylated. Thus, MboI cuts only the newly synthesized cDNA portion. Since this vector has only one BamHI cleavage site near the end opposite to the end to which the oligo dT was ligated, the enzyme cuts the vector at one position, and C DN synthesized to If the BamHI recognition sequence is present in part A, the site is also cleaved. BamHI and Mbol are composed of the sequence “GATC” and generate the same cohesive end. Plasmids can be closed by cleaving with both enzymes and then reacting with DNA ligase. Using the plasmid thus prepared, Escherichia coli was transformed to construct a 3′-end cDNA library. Therefore, the library 1 includes a region from the poly A site at the 3 ′ end of each mRNA to the site where the base sequence of GATC first appears in the 5 ′ side thereof. An appropriate number of recombinants are randomly selected from the 3′-end cDNA library, and the entire nucleotide sequence of the cDNA fragment in each recombinant is determined. An organ-specific gene and a highly expressed gene can be identified based on how many cDNA fragments having the specific sequence determined in this manner are confirmed from randomly selected recombinants.

In the above-mentioned method for identifying a highly expressed gene, the total number of recombinants selected at random is suitably several hundreds to about 1,000, but if necessary, more recombinants may be processed.

The present inventors carried out the above method, determined all the nucleotide sequences of the cDNA fragments in the 117 recombinants, and found that the frequency of appearance as cDNA having the same sequence was 5Z11 The 74 cDNA fragment was selected as a candidate DNA fragment of a gene highly expressed in human monocytes.

As described above, the cDNA fragment contains only a part of the 3 'end of the mRNA. Therefore, the present inventors obtained a full-length cDNA based on the nucleotide sequence information of the region (hereinafter, 3 ′ fragment).

A human peripheral blood monocyte cDNA library commercially available from Clontech Co., Ltd. was designated as type 、, and an oligonucleotide of an appropriate length having a sequence in the above 3 ′ fragment and a similar sequence having a sequence in a vector were used. Oligonucleotides of each length were synthesized, and PCR was performed using these as primers. As a result, a DNA fragment of about 1. 1 kb could be amplified. At this time, it can also be carried out by using mRNA extracted from human monocytes as type I and using a 5 'RACE kit from Clonetech or Gibco. Furthermore, it also uses the above 3 ′ fragment as a probe to clone the above human peripheral blood monocyte cDNA library by colony hybridization or Screening can also be performed by plaque hybridization.

The cDNA fragment amplified by the above method is incorporated into a vector pTBlue Vector (Novagen), and transfected into E. coli to cut out the fragment containing the cDNA from the clone as a circular plasmid to obtain all bases. At this time _c, the sequence was determined, to obtain 2 clones of recombinant DNA independently, by determining the nucleotide sequence of each of c DNA fragment was confirmed sequence. One protein translation region (Open Reading Flame, ORF) was found in this sequence, and this gene was named gmp30, and the protein encoded by the gene was named GMP30.

The gene gmp30 can be made into a recombinant gene by a general gene recombination technique using an appropriate host vector system. Suitable vectors include E. coli-derived plasmids (eg, pBR322, pUC118, etc.), and Bacillus subtilis-derived plasmids (eg, pUB110, pC194, etc.) ), Yeast-derived plasmids (eg, PSH19 and others), and animal viruses such as Pacteriophage II retrovirus and vaccinia virus can be used. Upon recombination, a translation initiation codon and a translation termination codon can be added using an appropriate synthetic DNA adapter. In order to further express the gene, an appropriate expression promoter is connected upstream of the gene. The promoter to be used may be appropriately selected depending on the host. For example, when the host is Escherichia coli, T7 promoter, 1 ac promoter, trp promoter, λPL promoter, etc., and when the host is Bacillus, S S0 promoter, etc. If the host is yeast, use 5 promoters, GAP promoter, ADH promoter, etc.If the host is animal cells, use SV40-derived promoter, retrovirus promoter, etc. it can.

The gene can also be expressed as a fusion protein with another protein (eg, glutathione S transferase, protein A, etc.). The fused GMP30 expressed in this manner can be excised using an appropriate protease (eg, thrombin and others).

Hosts that can be used for expression of the gene gmp30 include Escherichia bacteria. Various strains of certain Escherichia coli, various strains of Bacillus subtilis belonging to the genus Bacillus, various strains of Saccharomvces cerevisiae as yeast, and COS-7 cells and CHO cells as animal cells can be used. As a method for transforming host cells using the above-described recombinant vector, a transformation method generally used for each host cell can be applied.

In the present invention, in addition to the nucleotide sequence shown in SEQ ID NO: 2, a DNA that hybridizes with the sequence and encodes a protein having an inflammation regulating function is also within the scope of the present invention.

That is, in the full-length sequence of the gene gmp30, various artificial treatments, such as site-directed mutagenesis, random mutation by treatment with a mutagen, and mutation, deletion, and ligation of DNA fragments by restriction enzyme digestion, partially Even if the DNA sequence is altered, if the DNA mutant hybridizes with the gene gmp30 under stringent conditions and is a DNA encoding a protein having an inflammatory regulatory function, the sequence may be changed. Despite differences from the DNA sequence shown in No. 2, they are within the scope of the present invention.

The degree of the above-mentioned DNA mutation is within an allowable range as long as it has 90% or more homology with the DNA sequence of the gene gmp30. In addition, the degree of hybridization with the gene gmp30 can be determined by using a DIG DNA labeling kit under normal conditions (for example, DIG DNA Labeling kit, Boehringer's Mannheim Cat No. 1175033). Conditions for hybridizing in Easy Hyb solution (Boehringer Mannheim Cat No. 1603558) and washing the membrane in 50 x 0.5 XSSC solution (containing 0.1% [w / v] SDS) ( (1 XSSC is 0.15M NaC 0.015M sodium citrate), and only needs to hybridize to the gene gmp30.

In addition, a protein encoded by a mutant gene having high homology to the gene gmp30 as described above, which has an inflammation regulating function, is also included in the scope of the present invention.

That is, even if a mutant in which one or more amino acids of the amino acid sequence of the novel protein GMP30 is deleted, substituted or added, the mutant has an inflammatory regulatory function. If the protein has the following, the mutant is within the scope of the present invention.

The side chains of the amino acids that are the constituents of proteins differ in hydrophobicity, charge, size, etc., but do not substantially affect the three-dimensional structure (also called three-dimensional structure) of the entire protein In this sense, some highly conservative relationships are known empirically and by physicochemical measurements. For example, for substitution of amino acid residues, glycine (G1y) and proline (Pro), G1y and alanine (Ala) or valine (Va1), leucine (Leu) and isoleucine (I 1e), dal permic acid (G1u) and glutamine (Gin), aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), Thr Serine (Ser) or A1a; lysine (Lys) and arginine (Arg);

Therefore, even if the mutant protein is a mutation due to substitution, insertion, deletion, etc. in the amino acid sequence of the novel protein GMP30 shown in SEQ ID NO: 1, the mutation is a mutation that is highly conserved in the three-dimensional structure of the GMP30 protein. If the mutant protein has a function of regulating inflammation like GMP30, these can be said to be within the scope of the present invention. The degree of mutation is within a range in which homology to the amino acid sequence shown in SEQ ID NO: 1 is 90% or more. Industrial applicability

Since GMP30 has an inflammation regulating function, it is presumed that abnormal expression of the gene gmp30 or abnormal expression of the activity of GMP30 affects the inflammation enhancement and sedation. Therefore, a substance that regulates the expression of the gene or a substance that regulates the function of GMP 30 can be expected as an anti-inflammatory agent, and the gene gmp 30 and the protein GMP 30 are used to search for such a physiologically active substance. Can be used for For example, the effect of a test substance on gene expression can be investigated by coexisting a test substance in the transcriptional expression system of the gene gmp 30 and detecting the expression level of the gene gmp 30 by an appropriate method such as PCR. it can. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to examples. Unless otherwise specified below, various experimental methods shown in the Examples, such as extraction of recombinant cDNA and determination of the nucleotide sequence of cDNA, are all methods known to those skilled in the art (Molecular Cloning , 2nd. Ed., ColdSpring Harbor Lab. Press, 1989, and other methods described in technical manuals that introduce standard methods for those skilled in the art.

Example 1. Cloning of gene gmp30

1) Determination of partial sequence of gene

Using a human male healthy monocyte-derived mRNA as type I, a 3′-terminal cDNA library was prepared according to the method of Okubo et al. (Okubo et al. 1. Nature Genet., 1992> 2> pl73). From the library, 1174 recombinants were randomly selected, and the nucleotide sequence of the cDNA portion was determined. For sequencing, DNA sequencing (PRISM377, manufactured by ABI) and a reaction kit manufactured by ABI were used.

As a result of analyzing the expression frequency of each DNA fragment in the 117 recombinants, the expression frequency of the gene having the following sequence (sequence 11) was 5/117.

Array 1

AGAAGACGGATTATCTAGAGAATAATCATATATATGCATACGTAAAAAT 3 '

2) Cloning of cDNA fragment containing sequence 11

The cDNA fragment containing sequence-1 was cloned by the following method. First, an oligonucleotide (Sequence No. 2) which is a reverse complementary strand to a part of Sequence No. 11 and an oligonucleotide (Sequence No. 13) having a sequence near the cDNA insertion site of lambda phage cloning vector (A gtll) were prepared. , DNA synthesizer (ABI 380B) Was synthesized.

Sequence 1 2

5 '-GAAAAGATAGTAAAGCCCTGTTTGC-3'

Sequence 1 3

5 '-GCTGAATATCGACGGTTTCCATATGG-3'

Human Monocyte cDNA 1 ibrary (manufactured by Clontech Laboratories) using λ gt11 as a cloning vector was type 、, and oligonucleotides of sequence 12 and 13 were used as primers. The following PCR operation was performed using PCR Kit Ver.2 and PCR Thermal Cycler MP (both from Takara Shuzo).

cDNA library (≥10 ⁸ pfu / ml) 5 1

10X PCR buffer (including 25mM Mg ⁺⁺ ) 5 1

2.5mM dNTP 1 1

IOMM array—2 2 1

10 / M sequence 1 3 In 1

water

LA Tag * ⁰ Limera-Se '0.5 1

Total 50 1

PCR cycle: After holding at 94 ° C for 2 minutes, incubate at 98 ° C for 20 seconds, cool to 68 ° C at a rate of 11 ° C / 2 seconds, and heat at 68 ° C for 3 minutes. The holding was further performed 30 times at 72 ° C. for 10 minutes.

By the above method, a DNA fragment having the sequence 11 (about 1.5 kb) was specifically amplified.

3) Subcloning into nucleotide sequence determination vector

The DNA fragment amplified in 2) was fractionated by agarose gel electrophoresis (gel concentration 1%). The gel was stained with ethidium bromide and irradiated with ultraviolet light to cut out the gel containing the target band. Extraction and purification of DNA fragments from agarose gels were performed using GENECLEAN II Kit (manufactured by Hi-101).

This extracted and purified DNA fragment was used as a base sequence determination vector pT7blue (Novagen). Subcloning). The ligation solution was reacted for 1.5 hours at 16 ° C with the following composition using Evening DNA Ligation Kit Ver.2 (Takara Shuzo).

Extracted and purified DNA fragment 1 ^ 1 (50ng)

pT7blue 1 1 (10ng)

Water 3 1

Ligat ion solution 5 I

Total IO

Using the solution after the above reaction, Escherichia coli K12 strain DH5 was transformed. Transformants were treated with ampicillin (Amp) 50 g / ml, 5-Bromo-4-Chloro-3-indolyl-β-D-galactoside 40 g / m1, 1 sopropy 1-5-D-Thio- G lactopyranoside 100; plated on LB agar medium containing uM, and cultured at 37 ° C. at 晚 ° C. The colonies that appeared on the above plate were inoculated into 10 ml of LB liquid medium containing 50 g / m1 Amp, cultured at 37 ° C overnight, and the cells were collected by centrifugation.Then, QIAprep Spin Plasmid Recombinant DNA purified using Miniprep Kit (Qiagen) <4) Determination of base sequence of DNA fragment

The nucleotide sequence was determined using DNA Sequencer 1 (PRISM377, manufactured by ABI) and using the Daiyuichi Minator method. Oligonucleotides were synthesized based on the determined base sequences, and the entire base sequences of both strands were determined by the primer-walking method. SEQ ID NO: 3 shows the entire nucleotide sequence of the same DNA of the clone. Since the base sequence contained the upstream region of sequence 12 of sequence 12 and sequence 11, it was confirmed that the desired gene gmp30 was cloned.

The cDNA contains an ORF encoding a protein consisting of 266 residues (GMP30) (SEQ ID NO: 3). Since a stop codon appeared in the same reading frame in the upstream region of the methionine residue which is the start codon of the protein, the amino acid sequence of the protein encoded by the cDNA fragment is the only one shown in SEQ ID NO: 3. It was confirmed that there was.

Example 2. Expression of the gene gmp 30 in animal cells

1) Construction of animal cell expression vector

An oligonucleotide having a sequence upstream of the initiation codon of the protein of SEQ ID NO: 3 (SEQ ID NO: 4) and an oligonucleotide (Sequence-5) having a sequence of a part of the reverse complementary strand downstream of the stop codon were synthesized using a DNA synthesizer (380B manufactured by ABI).

Sequence 1 4

5'-TATCTGCAGAATTCAGGATCCCTACC-3 '

Sequence 1 5

5 '-TCAATAATGTTTTTCTTGACTGTTCAAG- 3'

The recombinant cDNA containing SEQ ID NO: 3 isolated in <1. Cloning of gene gmp30> was designated as type III, and the oligonucleotides of sequence-4 and sequence-15 were designated as primers. .2 and PCR Thermal Cycler MP (both from Takara Shuzo) were used to perform the following PCR procedures.

cDNA 5/1

10XPCR buffer (including 25mM Mg ⁺⁺ ) 5 / x1

2.5mM dNTP 1 \

lO iM array 1 4 l] i 1

10 // M array 1 5 2 {i \

Water 34.

LA Taq * ° Limera-se '_ —0

Total 50 1

The PCR cycle consists of holding at 94 ° C for 2 minutes, reacting at 98 ° C for 20 seconds, cooling to 68 ° C — cooling at 1 ° C for 2 seconds, and 3 minutes at 68 ° C. The holding was further performed 30 times at 72 ° C. for 10 minutes. A DNA fragment (about 1.5 kb) was specifically amplified by the above method.

The amplified DNA fragment (1.5 kb) is fractionated by agarose gel electrophoresis at a gel concentration of 1%, and the gel is stained with ethidium bromide and irradiated with ultraviolet light to contain the target band. The gel was cut out. Extraction and purification of DNA fragments from agarose gel were performed using GENECLEAN II Kit (Bio101).

The extracted and purified DNA fragment was subcloned into an animal cell expression vector pTARGET (promega). The ligation solution was reacted for 1.5 hours at 16 ° C with the following composition using Evening DNA Ligation Kit Ver.2 (Takara Shuzo). Extracted and purified DNA fragment 1/1 (50ng)

pTARGET 1111 (lOng)

Water 3 1

Li gat ion solution 5 1

Total 10 1

Using the solution after the above reaction, Escherichia coli K12 strain DH5 was transformed. Transformants were treated with ampicillin (Amp) 50 g / m 15-Bromo-4-Chloro-3-indolyl-β-D-galactoside 40 gZm1, Isopropyl-3-D-Thio-Galactopyranoside 100 / iM Was plated on an LB agar medium containing and cultured overnight at 37 ° C. The colonies appearing on the above plate were inoculated into 10 ml of LB liquid medium containing 50 g / m1 Amp, cultured at 37 ° C overnight, and the cells were collected by centrifugation. The recombinant DNA was purified using Plasmid Miniprep Kit (manufactured by Qiagen) to obtain pTARGET-gmp30. This recombinant plasmid has a CMV promoter upstream of the inserted fragment, and gmp30 can be expressed by introducing the recombinant plasmid into an appropriate animal cell.

2) Introduction of recombinant vector into CH〇k1 cells

Using a CHO k1 cell as a host, a collagen petri dish with a diameter of 60 mm was added to a collagen Petri dish, and HamF- containing 10% fetal bovine serum (Dainippon Pharmaceutical), 50 units of Zml ぺ nicillin, and 50 gZm of 1 streptomycin. (Gibco, hereinafter referred to as the growth medium) 12 medium was used to culture at 3 7 ° C, 5% C_〇 ₂ presence.

When the cell density reached 50%, a LIPOFECTAMINE reagent (manufactured by Gibco) containing pTARGET-gmp30 was overlaid on the cells, cultured for 6 hours, then replaced with a growth medium, and cultured for 48 hours. After dispersing the cells with trypsin, the cell suspension was dispensed into a 60 mm-diameter plastic petri dish and cultured for another 24 hours.

After removing the medium, the medium was replaced with a growth medium containing G418 reagent (manufactured by Gibco, 500 gZmL final concentration), and the growth medium was replaced every three days and cultured for 2 weeks. When the colonies of the cells became visible to the naked eye, three colonies were isolated using a stainless steel cup. To use as a control, pTARGET vector (Promega) alone was introduced into CHO k1 cells in the same manner as above, and a stable transformant was isolated. Released.

3) Confirmation of gene expression in transformants

Each isolated transformant was cultured on a G418-supplemented medium (final concentration 500 / mL) in a 6-well plate. When the cell density reached 80% confluence, the medium was removed, and PBS was added. After washing, total RNA was purified from the cells using Trizol (manufactured by Gibco). Using 2 g of total RNA as type II, cDNA was synthesized using Superscript reverse transcriptase (manufactured by Gibco). Further, using this cDNA as type III, a PCR reaction was carried out using the same oligonucleotides (sequences 2 and 3) as in Example 2-1).

cDNA 5 ^ 1

10 X PCR buffer (including 25 mM Mg ⁺⁺ ) 5 ^ 1

2.5mM dNTP 1 1

Array one 2 lu \

ΙΟ ^ Μ Sequence 1 3 2 1

Water 34.5 t 1

LA Taa Limera-Se '0.5 ^ ί 1

Total volume 50 pi 1

In the PCR cycle, hold at 94 ° C for 2 minutes, react at 98 ° C for 20 seconds, cool to 68 ° C at a rate of 12 seconds, hold at 68 ° C for 3 minutes, Further, holding at 70 ° C. for 10 minutes was repeated 30 times.

The reaction product was fractionated by 1% agarose gel electrophoresis, stained with ethidium die, and irradiated with ultraviolet light to check whether the target band was amplified.

As a result, the band of interest was amplified only in CHO k1 cells into which pTARGET-gmp30 had been introduced, and no amplification could be confirmed in CHO k1 cells into which the control vector had been introduced.

Test Example. Expression response of gmp30 gene induced by interferona

100 000,000 monocytes isolated from healthy males were used in a 10% non-mobilized ゥ fetal serum (Dainippon Pharmaceutical Co., Ltd.) in RM 1640 medium containing 50 units ZmL penicillin and 50 g / mL streptomycin. To achieve a final concentration of 1000 units The cells were further cultured for 24 hours after the addition of the fermenter (IFN-τ). Monocytes were prepared in a medium without IFN-I as a control.

After the culture, the medium was removed, PBS was added and the cells were washed, and then total RNA was purified from the cells using Trizol (manufactured by Gibco). 2 g of all RNAs were converted into type II and cDNA was synthesized using Superscript reverse transcriptase (manufactured by Gibco). Further, this cDNA was made into type III, and a PCR reaction was carried out using SEQ ID NO: 2 and SEQ ID NO: 3.

cDNA 5 1

10XPCR buffer (including 25mM Mg ⁺⁺ ) 5 1

2.5mM dNTP 1 n 1

10 t M array—2 2 ^ 1

IO M array — 3 2fi l

Water 34

LA TaqT Limera-1 1 1 1

50 1

The PCR cycle consists of holding at 94 ° C for 2 minutes, reacting at 98 ° C for 20 seconds, cooling to 68 ° C at a rate of 1 ° C / 2 seconds, holding at 68X for 3 minutes, The holding at 10 ° C. for 10 minutes was repeated 30 times.

The reaction product was fractionated by 1% agarose gel electrophoresis, stained with ethidium amide, and irradiated with ultraviolet light to check whether the target band was amplified.

As a result, it was confirmed that the gene gmp30 was significantly highly expressed in monocytes stimulated with IFN-α.

Claims

The scope of the claims

1 The following (a) or (b) protein:

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 1;

(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and having an inflammatory regulatory activity

2 DNA of (a) or (b) below

(a) DNA comprising the nucleotide sequence of SEQ ID NO: 2

(b) a DNA that hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and encodes a protein having an inflammatory regulatory activity.