JPH08103276A - Recombination type human interleukin 8 receptor protein, its production and use - Google Patents

Abstract

PURPOSE: To obtain a new expression vector holding the DNA of human interleukin 8(IL8) receptor protein and capable of highly expressing the human IL8 receptor protein useful for the development of antitumor agents, antiviral agents, etc., with CHO cells, etc. CONSTITUTION: The new expression vector holds a DNA coding the human interleukin 8 receptor protein, and is expressed by pAKKO 1.11/hIL8RA or pAKKO 1.11/hIL8RB. The expression vector is introduced into a Chinese hamster ovarian cell (CHO cell) defective in a dihydrofolic acid reductase gene. The cell is cultured under a condition enabling the expression of the expression vector, and the human IL8 receptor agonist or antagonist is efficiently screened. Thus, the human IL8 receptor protein useful for the development of medicines such as anticancer agents, antibacterial agents, antiviral agents, antiinflammatory agent, etc., can highly be expressed.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an expression vector having a DNA encoding a human interleukin 8 receptor protein, CHO cells containing the expression vector or a cell membrane fraction thereof, and a group produced from the CHO cells. Recombinant human interleukin 8 receptor protein or its partial peptide, method for screening interleukin 8 receptor agonist / antagonist using said CHO cells or recombinant human interleukin 8 receptor protein, said screening kit, said screening method or The present invention relates to an agonist or antagonist obtained by using a screening kit and a pharmaceutical composition containing the agonist or antagonist.

[0002]

2. Description of the Related Art Interleukin 8 was introduced in 1987.
A cytokine with a molecular weight of about 8,000, isolated and purified as a neutrophil chemotactic factor from peripheral blood monocytes stimulated with lipopolysaccharide (LPS) (Yoshimura, T. et a.
l. Proceedings of the National Academy of Sciences USA
tl. Acad. Sci. USA), 84, 9233-9237, 1987). further,
In 1988, the cDNA was cloned (Matsus
hima, K. et al. Journal of Experimental Medicine (J. Exp. Med.) 167, 1883-1893 (198)
8)). After that, T lymphocyte chemotactic factor was isolated and purified, and it was revealed that it is the same substance as neutrophil chemotactic factor (Lasen, CG et al. Science (Science) 2
43, 1464-1466 (1989)). This factor acts not only on neutrophils and lymphocytes but also on basophils, and is produced by various cells upon stimulation by other inflammatory cytokines such as interleukin-1 and tumor necrosis factor, and is an important mediator of acute inflammation formation. Therefore, it was named Interleukin 8. Anti-human interleukin-8 monoclonal antibody was shown to suppress neutrophil infiltration and reduce inflammatory response in rabbit lung ischemia-reperfusion injury, an arthritis model, and LPS dermatitis as an experimental model of inflammation. Have revealed the essential role of interleukin 8 in inflammation (Sekido, N. et al. Nature 365,654).
-657 (1993)). In addition, when interleukin 8 was locally produced, infiltration and activation of neutrophils into tumor tissue was observed, growth of tumor cells was suppressed, and interleukin 8 was shown to have an antitumor effect (Hirose Et al., 15th Japan Inflammation Society (1994)).

Mass production of interleukin 8 by E. coli (Furuta, R. et al. Journal of Biochemistry (J. Biochem.) 106, 436-441 (1989)) facilitates the production of recombinant interleukin 8. Now available, research on interleukin 8 receptor protein has also advanced. In 1991, two human interleukin 8 receptor proteins (Holmes, WE et
al. Science 253, 1278-1280 (1991), Mu
rphy, PM & Tiffany, HL Science 2
53, 1280-1283 (1991)) and the cDNA cloning of the rabbit interleukin 8 receptor protein were reported one after another, and the structure thereof was clarified. A comparison of the amino acid sequences of the two human interleukin 8 receptor proteins, type A and type B, shows a very good agreement, but low homology to the N-terminal region that is thought to be involved in ligand binding. Type A specifically binds only to interleukin 8, while type B is a group of GRO (Growth Related Pr) that has a high amino acid sequence homology with interleukin 8 in addition to interleukin 8.
otein), MIP-2 (Macrophage Inflammatory Protei
n 2) and so on. When the N-terminal regions of interleukin 8 receptor protein type A and type B are exchanged, the binding specificity of the receptor protein is also exchanged, indicating that the N-terminal region of the receptor protein is involved in the binding to the ligand (LaRosa, GJ Journal of Biological Chemistry (J. Biol. Che
m.) 267, 25402-25406 (1992)).

These two types of interleukin-8 receptor proteins are expressed in neutrophils, and the second messenger system has been studied in detail, but local chemotaxis during inflammation,
Detailed analysis has not yet been conducted on the receptor protein function during inflammation, such as the difference in expression level and functionality of infiltrating neutrophils. Site-directed
Interleukin 8 ligand and receptor protein type A alanine scan using mutagenesis revealed that the important site for ligand-receptor protein binding was ligand (Herbert, CA et al. Journal of Biological Chemistry (J. Biol. Chem.) 266, 1
8989-18994 (1991)), receptor protein (Leong, SR
et al. Journal of Biological Chemistry (J. Biol. Chem.) 269, 19343-19348 (1994)),
Each has been revealed. Furthermore, a method for screening an interleukin-8 receptor agonist using a human interleukin-8 receptor polypeptide or a fragment thereof has been reported (JP-A-6-10).
0595). However, human interleukin 8
No specific CHO cell line capable of highly expressing the receptor DNA and a method for screening an agonist / antagonist of human interleukin 8 receptor using the cell line are described.

In recent years, research on interleukin 8 and its receptor protein has progressed, and it has been suggested that interleukin 8 is essentially involved in inflammation. The chemotaxis and infiltration of neutrophils into the inflammatory region are induced by the action of interleukin 8 produced in the inflammatory region on the interleukin 8 receptor protein on the neutrophil membrane. Antagonists are expected as anti-inflammatory agents. On the other hand, because of the neutrophil activation effect of interleukin 8, the interleukin 8 receptor agonist is expected as an anticancer agent, an antibacterial agent, an antifungal agent, an antiviral agent and the like. However, at present, there are no reports of interleukin-8 receptor antagonists for the purpose of anti-inflammatory or interleukin-8 receptor agonists for the purposes of anticancer agents, antibacterial agents, antifungal agents, antiviral agents, etc. .

[0006]

DISCLOSURE OF THE INVENTION The present invention provides an expression vector having a DNA encoding a human interleukin 8 receptor protein, and a CH containing the expression vector.
O cell or cell membrane fraction thereof, recombinant human interleukin 8 receptor protein or its partial peptide produced from said CHO cell, interleukin 8 receptor using said CHO cell or recombinant human interleukin 8 receptor protein Provided are an agonist / antagonist screening method, the screening kit, an agonist or antagonist obtained by using the screening method or the screening kit, and a pharmaceutical composition containing the agonist or antagonist.

[0007]

[Means for Solving the Problems] In view of the above-mentioned problems, the present inventors have earnestly studied a method for screening an effective interleukin-8 receptor agonist / antagonist in humans, and as a result, found that the human interleukin-8 receptor protein was identified. We have succeeded in producing a highly expressing CHO cell line, and have found that the CHO cell line can be used to efficiently and reliably screen for an interleukin-8 receptor agonist / antagonist. The present inventors have completed the present invention as a result of further research based on these findings.

That is, the present invention provides (1) pAKKO1.11 / hIL8, which is characterized in that it retains DNA encoding human interleukin 8 receptor protein.
RA or pAKKO1.11 / hIL8RB, an expression vector labeled with (2) the expression vector according to item (1), or CHO cells or a cell membrane fraction thereof, and (3) dihydrofolate. The CHO cell according to item (2), which is a CHO cell lacking a reductase gene, and the CHO cell according to item (4), wherein the expression of the DNA encoding the human interleukin 8 receptor protein is A method for producing a recombinant human interleukin 8 receptor protein, which comprises culturing under a possible condition. (5) The CHO cells described in (2) above encode the human interleukin 8 receptor protein. D
CHO cells containing recombinant human interleukin 8 receptor protein or a cell membrane fraction thereof, which can be produced by culturing under conditions capable of expressing NA

(6) Recombinant human interleukin 8 receptor protein isolated from CHO cells containing the recombinant human interleukin 8 receptor protein according to item (5), a partial peptide thereof or a salt thereof. ,
(7) Human interleukin 8 characterized by using the CHO cell or the cell membrane fraction thereof described in (5).
Method for screening receptor agonist or antagonist, (8) Recombinant human interleukin 8 receptor protein according to item (6), a partial peptide thereof or a salt thereof is used. Alternatively, a method for screening an antagonist, (9) C according to item (5)
HO cells or a cell membrane fraction thereof, or a human interleukin 8 receptor agonist comprising the recombinant human interleukin 8 receptor protein according to (6), a partial peptide thereof, or a salt thereof, or Kit for screening antagonist, human interleukin 8 receptor agonist or salt thereof obtained by using the screening method according to (10) (7) or (8) or the screening kit according to (9) (11) A human interleukin 8 receptor antagonist or a salt thereof obtained by using the screening method according to item (7) or (8) or the screening kit according to item (9), (12). Including the agonist or its salt described in (10). A neutrophil activation-promoting agent or neutrophil migration-promoting agent, and (13) the antagonist or the salt thereof according to (11), An activation inhibitor or a neutrophil migration inhibitor is provided.

More specifically, the present invention provides (14)
(I) The labeled interleukin 8 is replaced with C described in the item (5).
When contacted with HO cells or a cell membrane fraction thereof,
(Ii) C of labeled interleukin 8 when the labeled interleukin 8 and the test compound are contacted with the CHO cell or the cell membrane fraction thereof according to the item (5).
The amount of binding to HO cells or a cell membrane fraction thereof is measured and compared, and the human according to (7),
Interleukin 8 receptor agonist or antagonist screening method, (15) (i) when interleukin 8 is contacted with CHO cells or cell membrane fraction thereof according to (5), (ii) interleukin 8 and Recombinant human interleukin 8 receptor-mediated cell stimulating activity (eg, arachidonic acid release, acetylcholine release, cells when the test compound is contacted with the CHO cell or the cell membrane fraction thereof according to (5). Intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, p
H-decrease, activity promoting or suppressing cell migration activity, etc.) is measured and compared, The method for screening a human interleukin 8 receptor agonist or antagonist according to item (7),
(16) (i) The case where the recombinant human interleukin 8 receptor protein according to item (6), a partial peptide thereof or a salt thereof and interleukin 8 are contacted, and (ii) item (6). The recombinant human interleukin 8 receptor protein, its partial peptide or salts thereof, interleukin 8 and a test compound are contacted with each other, and the human. Interleukin 8 receptor agonist or antagonist screening method,

(17) (i) When the recombinant human interleukin 8 receptor protein according to item (6), a partial peptide thereof or a salt thereof and interleukin 8 are contacted, and (ii) (6) ) Recombinant human interleukin 8 receptor protein, a partial peptide thereof or a salt thereof, when interleukin 8 and a test compound are contacted, for example, the recombinant human interleukin 8 receptor protein,
A method for screening a human interleukin 8 receptor agonist or antagonist according to (8), characterized in that the amount of interleukin 8 bound to the partial peptide or salt thereof, cell stimulating activity, etc. are measured and compared. (18) When (i) the labeled interleukin 8 is contacted with the recombinant human interleukin 8 receptor protein according to (6), a partial peptide thereof or a salt thereof, and (ii) the labeled interleukin When the leukin 8 and the test compound are contacted with the recombinant human interleukin 8 receptor protein according to (6), a partial peptide thereof or a salt thereof, the labeled interleukin 8 protein, a partial peptide thereof, or It is characterized in that the amount of binding to the salt is measured and compared. ) Screening method of human interleukin 8 receptor agonist or antagonist according to claim anticancer agent characterized by containing an agonist or a salt thereof (19) A (10) above, wherein,
Antibacterial, antifungal or antiviral, and (20)
Inflammation, rheumatism, gout, neutrophilic dermatitis, asthma, adult respiratory distress syndrome, sepsis, primary characterized by containing an antagonist or a salt thereof according to item (11).
Provided is a prophylactic / therapeutic agent for a disease associated with neutrophil infiltration selected from the group consisting of fibrosis, Crohn's disease, pyelonephritis, myocardial infarction, multiple organ disorder, Behcet's disease, Vogt-Koyanagi disease and hepatitis.

In the present invention, when an expression vector carrying a DNA encoding the human interleukin 8 receptor protein is prepared, examples of the DNA encoding the human interleukin 8 receptor protein include:
Although cDNA encoding human interleukin 8 receptor protein has been used, it encodes human interleukin 8 receptor protein or a partial peptide having substantially the same ligand binding activity as human interleukin 8 receptor protein. As long as it has a nucleotide sequence, it is not necessarily limited to this. For example, genomic DNA or synthetic DNA may be used. Specifically, a cDNA having the nucleotide sequence represented by SEQ ID NO: 1 encoding human interleukin 8 receptor protein type A having the amino acid sequence represented by SEQ ID NO: 2 (FIG. 1), SEQ ID NO: 4 CDNA having the nucleotide sequence represented by SEQ ID NO: 3 encoding human interleukin 8 receptor protein type B having the represented amino acid sequence, and the like are used. These cDNAs are known ones, and are known as Science (Science) 253, 1278-1280 (1991), Science (S
cience) 253, 1280-1283 (1991) and the like.
These cDNAs can be cloned using a genetic engineering technique known per se, or can be produced using a nucleotide synthesizer. For example, cloning can be performed according to the methods of Reference Example 1 and Reference Example 2 described below or methods equivalent thereto.

As the vector, pAKKO1.11,
pXT1, pRc / CMV, pRc / RSV, pcDN
AINeo is used, among which pAKKO1.1 is used.
1 and the like are preferable. Any promoter may be used as long as it can be efficiently expressed in the host cell.
40 promoter, CMV promoter, HSV-TK
Examples thereof include promoters and SRα promoters, of which CMV promoters and SRα promoters are preferable, and SRα promoters are particularly preferable. It is preferable to use an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker and the like in addition to the above. Examples of the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as DHFR) gene [methotrexate (MTX) resistance], neo gene [G418 resistance], and the like, and among them, the DHFR gene is preferable. Especially, DHFR
When the DHFR gene is used as a selectable marker using CHO cells deficient in the gene, selection can also be performed using a thymidine-free medium. CDNA encoding the human interleukin 8 receptor protein of the present invention
As an expression vector that retains, specifically, human
CD encoding the interleukin 8 receptor protein
The above promoter (in particular, SRα promoter) is inserted upstream of NA, and preferably SV is inserted downstream of the DNA encoding human interleukin 8 receptor protein.
An early gene PolyA addition signal is inserted, and DHF is further provided downstream of the PolyA addition signal.
Those in which the R gene or (and) ampicillin resistance gene is inserted are preferable. More specifically, pAKK
The expression vector designated by O1.11 / hIL8RA [FIG. 5] and the expression vector designated by pAKKO1.11 / hIL8RB [FIG. 6] are suitable.

A cell capable of highly expressing the DNA encoding the human interleukin 8 receptor protein by introducing into the host cell the expression vector having the DNA encoding the human interleukin 8 receptor protein thus produced. Can be produced. Animal cells are preferable as the host cells, for example, Vero
Cells, CHO cells, L cells, myeloma cells, C127
Cells, BALB / c3T3 cells, Sp-2 / O cells, etc., among which C lacking the DHFR gene
HO cells (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), Sp-2 / O cells and the like are preferable, and CHO cells are particularly preferable.
(Dhfr ⁻ ) cells are preferred. As a method for introducing an expression vector into a host cell, a known method, for example, the calcium phosphate method [Graham, FL and van der Eb, AJ
Virology 52, 456-467 (1973)], electroporation [Neumann, E. et al. Embo Journal (EMBO
J.) 1, 841-845 (1982)] and the like are used. A cell capable of highly expressing the human interleukin 8 receptor protein can be obtained by clonally selecting a cell in which the above expression vector is integrated in the chromosome. Specifically, first, a transformant is selected using the above selection marker as an index. Further, by repeatedly carrying out clone selection on the transformant thus obtained using the selection marker, it is possible to obtain a stable cell line having high expression ability of human interleukin 8 receptor protein. it can. Moreover, when the DHFR gene is used as a selection marker, methotrexate (MT
By gradually increasing the concentration of X) and culturing to select resistant cells, the transgene can be propagated in the cells to obtain a cell line with higher expression.

Specific examples of cells that can highly express the DNA encoding the human interleukin 8 receptor protein include pAKKO1.
CHO (dhfr ⁻ ) cells containing the expression vector designated 11 / hIL8RA, pAKKO1.11 / h
CHO containing an expression vector designated IL8RB
(Dhfr ⁻ ) cells and the like. These CHO
(Dhfr ⁻ ) cells have about 10 to 30 times, preferably about 20 times as much receptor activity (eg, ligand binding activity) as that of cells containing natural interleukin 8 receptor protein (eg, human neutrophils). Etc.). Recombinant by culturing cells containing the expression vector carrying the DNA encoding the human interleukin 8 receptor protein of the present invention under conditions capable of expressing the DNA encoding the human interleukin 8 receptor protein. Type human interleukin 8
A receptor protein can be produced. When culturing cells containing an expression vector carrying DNA encoding human interleukin 8 receptor protein, EM containing about 0.5 to 20% fetal calf serum as a medium
EM medium, DMEM medium, RPMI1640 medium, α-
MEM medium etc. are mentioned. In particular, CHO (dhfr
^- ) When using cells and DHFR selectable marker gene, it is preferable to use DMEM medium or α-MEM medium containing dialyzed bovine serum without thymidine. pH
Is preferably about 6-8. Culture is usually about 30 ~
It is carried out at 40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring are added. In this way, cells containing the recombinant human interleukin 8 receptor protein can be produced from cells containing the expression vector carrying the DNA encoding the human interleukin 8 receptor protein.

The cell membrane fraction of cells containing the recombinant human interleukin 8 receptor protein is crushed by the method described above after crushing the cells containing the recombinant human interleukin 8 receptor protein. The obtained fraction contains a large amount of cell membrane. As a method for crushing cells, for example, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or Polytron (manufactured by Kinematica), crushing with ultrasonic waves, thin nozzles for pressing cells with a French press etc. Examples include crushing by ejecting from. For the fractionation of the cell membrane, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate is fed at a low speed (500 rpm to 3000 rp
m) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in expressed human interleukin 8 receptor protein and membrane components such as cell-derived phospholipids and membrane proteins. The amount of the human interleukin 8 receptor protein in the cell containing the recombinant human interleukin 8 receptor protein of the present invention or the cell membrane fraction thereof is preferably 10 ³ to 10 ⁸ molecules per cell, It is preferably 10 ⁵ to 10 ⁷ molecules.
It should be noted that the higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, making it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. .

The recombinant human interleukin-8 receptor protein can be isolated from the cells containing the recombinant human interleukin-8 receptor protein obtained as described above, for example, by the following method. When extracting the recombinant human interleukin 8 receptor protein from cultured cells or cells,
Cells or cells are collected by a known method, suspended in an appropriate buffer solution, disrupted by ultrasonic waves, lysozyme and / or freeze-thaw, etc., and then centrifuged or filtered to give recombinant humans.・ Interleukin 8
A method for obtaining a crude extract of the receptor protein is appropriately used. A protein denaturing agent such as urea or guanidine hydrochloride or Triton X-100 (registered trademark.
Sometimes abbreviated as TM. ) Or the like may be included. The recombinant human interleukin 8 receptor protein contained in the obtained extract was purified by
The separation / purification method known per se can be appropriately combined and performed. Known separation and purification methods for these are methods utilizing solubility such as salting out and solvent precipitation, dialysis,
Ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis and other methods that mainly utilize the difference in molecular weight, ion exchange chromatography and other methods that utilize the difference in charge, affinity chromatography and other specific methods A method utilizing the reciprocity, a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography, a method utilizing a difference in isoelectric point such as an isoelectric focusing method are used.

When the recombinant human interleukin 8 receptor protein thus obtained is obtained as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely it can be obtained with a salt. In this case, the free form or other salt can be converted by a method known per se or a method analogous thereto. The recombinant human interleukin 8 receptor protein produced by the recombinant is treated with an appropriate protein-modifying enzyme before or after purification to arbitrarily modify or partially remove the polypeptide. You can also do it. As the protein modifying enzyme, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase and the like are used. As described above, produced by culturing cells containing an expression vector carrying the DNA encoding the human interleukin 8 receptor protein under conditions capable of expressing the DNA encoding the human interleukin 8 receptor protein. The recombinant human interleukin 8 receptor protein thus obtained has substantially the same activity as the naturally occurring human interleukin 8 receptor protein. Examples of substantially the same activity include ligand binding activity and signal transduction. The ligand-binding activity includes interleukin-8,
The binding activity with an interleukin 8 receptor agonist and the like can be mentioned. "Substantially the same quality" means that the ligand binding activity and the like are qualitatively the same. Therefore,
Quantitative factors such as the strength of the ligand-binding activity and the molecular weight of the receptor protein may be different.

A specific example of the recombinant human interleukin 8 receptor protein of the present invention has the amino acid sequence represented by SEQ ID NO: 2 produced from the cDNA having the nucleotide sequence represented by SEQ ID NO: 1. Human interleukin 8 receptor protein type A,
In addition to human interleukin 8 receptor protein type B having the amino acid sequence represented by SEQ ID NO: 4 produced from the cDNA having the nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 4. One or two or more amino acids deleted in the amino acid sequence represented by, SEQ ID NO: 2 or one or more amino acids added to the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 4 in which one or more amino acids are substituted with other amino acids. Furthermore, in these recombinant human interleukin 8 receptor proteins, the N-terminal signal peptide may be cleaved, or the side chain of the amino acid in the molecule may have a suitable protecting group (eg, formyl group, acetyl group, etc.). C _1-6 acyl group), or a sugar chain or the like may be bound. As the salt of the recombinant human interleukin 8 receptor protein of the present invention, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,
Methanesulfonic acid, benzenesulfonic acid) and the like are used.

The partial peptide of the recombinant human interleukin 8 receptor protein of the present invention is, for example,
Of the recombinant human interleukin 8 receptor protein molecule of the present invention, a site exposed outside the cell membrane is used. Specifically, it is a partial peptide analyzed to be an extracellular region (hydrophilic region) in a hydrophobicity plot analysis. Also, hydrophobicity (Hydtop
Peptides partially containing a hobic) site can also be used. Furthermore, a peptide containing each domain individually may be used, but a partial peptide containing multiple domains simultaneously may be used. As the salt of the partial peptide of the recombinant human interleukin 8 receptor protein of the present invention, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

The partial peptide of the recombinant human interleukin 8 receptor protein of the present invention or a salt thereof can be prepared according to a method known per se for peptide synthesis, or by using the recombinant human interleukin 8 receptor protein of the present invention in a suitable form. It can be produced by cleaving with peptidase. The peptide synthesis method may be either a solid phase synthesis method or a liquid phase synthesis method. That is,
The target peptide can be produced by condensing a partial peptide or amino acid that can form the protein of the present invention with the remaining portion and removing the protecting group if the product has a protecting group. Examples of known condensation methods and elimination of protective groups include the methods described in the following (1) to (3). M. Bodanszky and MA Ondetti,
Peptide Synthesis, Interscie
nce Publishers, New York (1966) Schroeder and Luebke, The Peptide,
Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervision of Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis Hirokawa Shoten The partial peptide of the present invention can be purified and isolated by a combination of purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted into an applicable salt by a known method, and conversely, when it is obtained with a salt, it can be converted into a free form by a known method. You can

CHO cells containing the recombinant human interleukin 8 receptor protein of the present invention or a cell membrane fraction thereof, or an isolated recombinant human interleukin 8 receptor protein, its partial peptide or salts thereof are , Useful for screening human interleukin 8 receptor agonists or antagonists. That is, the present invention includes (1) (i) a case where the recombinant human interleukin 8 receptor protein of the present invention, a partial peptide thereof or a salt thereof and interleukin 8 are contacted, and (ii) a combination of the present invention Recombinant human interleukin 8 receptor protein, its partial peptide or salts thereof,
A method for screening a human interleukin 8 receptor agonist or antagonist, which comprises performing a comparison with a case where interleukin 8 and a test compound are contacted, and (2) (i) the recombinant human interleukin of the present invention CHO cells containing leukin 8 receptor protein or a cell membrane fraction thereof and interleukin 8 are contacted, and (ii) CHO cells containing the recombinant human interleukin 8 receptor protein of the present invention or a cell membrane fraction thereof The present invention provides a method for screening a human interleukin 8 receptor agonist or antagonist, which comprises performing a comparison with the case of contacting interleukin 8 with a test compound. Specifically, in the screening method of the present invention, in the cases (i) and (ii), for example, the binding of interleukin 8 to the recombinant human interleukin 8 receptor protein, its partial peptide or salts thereof is used. It is characterized in that the amount, cell stimulating activity, etc. are measured and compared.

More specifically, the present invention provides (1a) (i) contacting labeled interleukin 8 with the recombinant human interleukin 8 receptor protein of the present invention, a partial peptide thereof or a salt thereof. And (ii) the labeled interleukin 8 and the test compound were contacted with the recombinant human interleukin 8 receptor protein of the present invention, a partial peptide thereof or a salt thereof, A method for screening a human interleukin 8 receptor agonist or antagonist, which comprises measuring and comparing the amount of binding to the receptor protein, a partial peptide thereof or a salt thereof (2a) (i) labeled interleukin 8 Recombinant human interleukin 8 receptor protein of the present invention The CHO cell containing the cytoplasm or the cell membrane fraction thereof, and (ii) the labeled interleukin 8 and the test compound were added to the CHO cell containing the recombinant human interleukin 8 receptor protein of the present invention or A method for screening a human interleukin 8 receptor agonist or antagonist, which comprises measuring and comparing the amount of labeled interleukin 8 bound to the CHO cells or the cell membrane fraction when contacted with the cell membrane fraction. ,and

(2b) (i) when interleukin 8 is contacted with CHO cells containing the recombinant human interleukin 8 receptor protein of the present invention or a cell membrane fraction thereof, and (ii) interleukin 8 And a test compound, when contacted with a CHO cell containing the recombinant human interleukin 8 receptor protein of the present invention or a cell membrane fraction thereof, a cell stimulating activity mediated by human interleukin 8 receptor (for example, Arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release,
Intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, activity promoting or suppressing cell migration activity, etc. And the like) are measured and compared. A method for screening a human interleukin 8 receptor agonist or antagonist is provided. The above (1a) or (2
In the screening method of a), the compound that binds to the recombinant human interleukin 8 receptor protein and inhibits the binding between interleukin 8 and the recombinant human interleukin 8 receptor protein is human interleukin 8 receptor. It can be selected as an agonist or an antagonist. Furthermore, in the screening method of the above (2b), it binds to the human interleukin 8 receptor and mediated cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production) via the receptor. , Intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease,
A compound having an activity of promoting or suppressing a cell migration activity or the like) can be selected as a human interleukin 8 receptor agonist, while a compound having no cell stimulating activity can be selected as a human interleukin 8 receptor. It can be selected as an antagonist. Prior to obtaining CHO cells containing the recombinant human interleukin 8 receptor protein of the present invention,
Since there were no animal cells that could highly express the human interleukin 8 receptor protein, it was not possible to efficiently screen for human interleukin 8 receptor agonists or antagonists.

A detailed description of the screening method of the present invention is given below. When CHO cells containing the human interleukin 8 receptor protein are used in the screening method of the present invention, the CHO cells can be immobilized with glutaraldehyde, formalin or the like. The immobilization method can be performed according to a method known per se. As the interleukin 8 used as a ligand, a commercially available product or the like can be used. Also, instead of interleukin 8, a known interleukin 8 receptor agonist or the like can be used. For example, MGSA (Melano) known to bind to interleukin 8 receptor protein type B
ma Growth-Stimulating Activity) and MIP-2 (Macr
ophage InflammatoryProtein-2) etc. can also be used. As the labeled interleukin 8, for example,
Interleukin 8 labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc. can be used, and further, [ ³ H], [ ¹²⁵ I], [ ¹⁴ C] , [ ³⁵ S] and other known interleukin-8 agonists (eg,
The above-mentioned MGSA, MIP-2, etc.) can also be used. For example, [ ¹²⁵ I] -labeled interleukin 8 (Amersham and DuPont) and the like are preferable. Examples of test compounds include peptides, proteins,
Non-peptidic compounds, synthetic compounds, fermentation products, etc. may be mentioned, and these compounds may be novel compounds or known compounds.

Specifically, the above (1a) or (2
To carry out the screening method of a), first, a CHO cell containing the recombinant human interleukin 8 receptor protein of the present invention or a cell membrane fraction thereof, or a recombinant human interleukin 8 receptor protein or its A receptor preparation is prepared by suspending a partial peptide in a buffer suitable for screening. The buffer should have a pH of about 4-10 (preferably p
Any buffer may be used as long as it does not inhibit the binding between the interleukin 8 and the receptor, such as a phosphate buffer of H about 6 to 8) or a Tris-hydrochloric acid buffer. Moreover, in order to reduce non-specific binding, CHAPS, T
ween-80 ^™ (Kao-Atlas), digitonin,
A detergent such as deoxycholate can also be added to the buffer. Further, protease inhibitors such as PMSF, leupeptin, E-64 (manufactured by Peptide Laboratories), pepstatin and the like can be added for the purpose of suppressing decomposition of the receptor and ligand by protease. On the other hand, when the CHO cells are fixed cells, the interleukin 8 and the interleukin 8 can be prepared by using the cells fixed in the incubator, that is, the CHO cells grown or the cells fixed with glutaraldehyde or paraformaldehyde. 8 receptor proteins can be bound. In this case, a medium or Hank's solution is used as the buffer solution. Then, a fixed amount (about 5000 cpm to 500000 cp) is added to 0.01 ml to 10 ml of the receptor solution.
m) labeled interleukin 8 was added and at the same time 1
A test compound of 0 ⁻⁴ M to ^{10 −10} M is made to coexist. A reaction tube containing a large excess of unlabeled interleukin 8 is also prepared in order to know the amount of non-specific binding (NSB). The reaction is carried out at 0 ° C to 50 ° C, preferably 4 ° C to 37 ° C for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction, the mixture is filtered through a glass fiber filter paper or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a γ-counter. Count (B ₀ −) obtained by subtracting the amount of non-specific binding (NSB) from the count (B ₀ ) in the absence of an antagonist.
NSB) as 100%, the specific binding amount (B-NS
A test compound having B) of, for example, 50% or less can be selected as an agonist or antagonist candidate compound.

Further, in order to carry out the screening method of (2b) above, cell stimulating activity mediated by human interleukin 8 receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, phosphorylation of intracellular proteins,
c-fos activation, pH decrease, activity promoting or suppressing cell migration activity, etc.) can be measured using a known method or a commercially available measurement kit. Specifically, first, CHO cells containing the recombinant human interleukin 8 receptor protein are cultured in a multiwell plate or the like. For screening, the medium was exchanged with a fresh medium or an appropriate buffer that did not show toxicity to cells in advance, and after adding a test compound or the like and incubating for a certain period of time, cells were extracted or supernatant was collected to generate The product is quantified according to the respective method. When the production of a substance that serves as an index of cell stimulating activity (for example, arachidonic acid) is difficult to assay by the degrading enzyme contained in the cell, an inhibitor for the degrading enzyme may be added to the assay. Also, c
The activity of suppressing AMP production and the like can be detected as a production suppressing action on cells whose basic production amount has been increased by forskolin or the like.

The screening kit of the present invention comprises a CHO cell containing the recombinant human interleukin 8 receptor protein of the present invention or a cell membrane fraction thereof, or a recombinant human interleukin 8 receptor protein of the present invention, It contains a partial peptide or a salt thereof. Examples of the screening kit of the present invention include the following. [Reagent for screening] Add 0.1% to Hanks' Balanced Salt Solution (manufactured by Gibco) for measurement buffer and washing buffer.
To which 05% bovine serum albumin (manufactured by Sigma) was added. Sterilize by filtration with a 0.45μm filter at 4 ℃
It may be stored at or prepared at the time of use. Recombinant human interleukin 8 receptor protein preparation CHO cells containing recombinant human interleukin 8 receptor protein were subcultured at 5 × 10 ⁵ cells / well on a 12-well plate at 37 ° C., 5% Cultivated with CO ₂ 95% air for 2 days. Labeled interleukin 8 Commercially available solution of interleukin 8 labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc. is stored at 4 ° C or -20 ° C for use. Sometimes diluted to 1 μM with assay buffer. Interleukin 8 standard solution Interleukin 8 was dissolved in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) to a concentration of 1 mM,
Store at -20 ° C.

[Measurement Method] CH Containing Recombinant Human Interleukin 8 Receptor Protein Cultured in a 12-well Tissue Culture Plate
The O cells were washed twice with 1 ml of the measurement buffer, and then 49
Add 0 μl assay buffer to each well. After adding 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M, 5 μl of labeled interleukin 8 was added, and 1 at room temperature.
React for hours. In order to know the amount of non-specific binding, 5 μl of 10 ⁻³ M interleukin was added in place of the test compound. Remove the reaction solution and wash 3 times with 1 ml of wash buffer. 0.2N of labeled interleukin 8 bound to cells
It is dissolved in NaOH-1% SDS and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
Radioactivity was measured using a Percent Maximum Binding
(PMB) is calculated by the following equation [Equation 1].

[Equation 1] PMB: Percent Maximum Binding B: Value when a sample is added NSB: Non-specific Binding B ₀ : Maximum binding amount

The compound or its salt obtained by using the screening method or the screening kit of the present invention is a compound which inhibits the binding between interleukin 8 and the recombinant human interleukin 8 receptor protein of the present invention, Specifically, a compound having a cell stimulating activity via human interleukin 8 receptor or a salt thereof (so-called human interleukin 8 receptor agonist), or a compound having no such stimulating activity or a salt thereof (so-called human). -Interleukin 8 receptor antagonist). The human interleukin-8 receptor agonist has the same physiological activity as that of interleukin-8, and is therefore useful as a safe and low-toxic pharmaceutical composition depending on the physiological activity. For example, it is useful as a neutrophil activation promoter or a neutrophil migration promoter, and also as an anti-cancer agent, antibacterial agent, antifungal agent, antiviral agent and the like. On the other hand, human
Since the interleukin-8 receptor antagonist can suppress the physiological activity of interleukin-8, it is useful as a safe and low-toxic pharmaceutical composition for suppressing the physiological activity. For example, as a neutrophil activation inhibitor or a neutrophil migration inhibitor, etc., further inflammation, rheumatism, gout, neutrophilic dermatitis, asthma, adult respiratory distress syndrome, sepsis, primary fibrosis, Crohn's disease It is also useful as a prophylactic / therapeutic agent for diseases associated with neutrophil infiltration such as pyelonephritis, myocardial infarction, multiple organ disorder, Behcet's disease, Vogt-Koyanagi disease, and hepatitis.

When the agonist or antagonist obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition,
It can be carried out in a conventional manner. For example, orally as tablets, capsules, elixirs, microcapsules, etc., optionally sugar-coated, or aseptic solution with water or other pharmaceutically acceptable liquid, or suspension It can be used parenterally in the form of injections such as. For example, a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, or the like of the compound or a salt thereof,
It can be prepared by mixing with stabilizers, binders and the like in a unit dose form generally required for accepted pharmaceutical practice. The amount of active ingredient in these preparations is such that a suitable amount within the indicated range can be obtained. Additives that can be mixed with tablets, capsules, etc. include, for example, gelatin, corn starch, tragacanth, binders such as gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Puffing agents, lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. .

Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.), and suitable solubilizing agents. , Alcohols (eg ethanol), polyalcohols (eg propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbate 80 (T
You may use together with M), HCO-50), etc. Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg,
Human serum albumin, polyethylene glycol, etc.),
You may mix | blend with preservatives (for example, benzyl alcohol, phenol, etc.), antioxidants, etc. The adjusted injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, warm-blooded mammals (for example, rat, rabbit, sheep, pig,
Bovine, cat, dog, monkey, human, etc.). The dose of the compound or its salt varies depending on the symptoms and the like, but in the case of oral administration, it is generally about 0.1 per day for an adult (as 60 kg).
-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc., but in the form of an injection, it is usually about 1 day in an adult (as 60 kg). About 0.01 to 30 mg, preferably about 0.1
It is convenient to administer about -20 mg, more preferably about 0.1-10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

In this specification and the drawings, when an abbreviation is used for a base or amino acid, IUPAC-IUB is used.
Based on the abbreviations used in Commision on Biochemical Nomenclature or conventional abbreviations in this field,
An example is given below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dCTP: deoxycytidine triphosphate Gly: glycine Ala: alanine Valu: valine Le. Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid

Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine BSA: Bovine serum albumin Transformed in Example 1 described below. Body Escherichia
Escherichia coli DH5 / pAKKO1.1
1 / hIL8RA and transformant Escherichia coli DH5 / pAKKO1.1
1 / hIL8RB has been deposited with the deposit number FERM BP-4820 and the deposit number FERM BP-4821 in the Institute of Biotechnology, Institute of Biotechnology (NIBH), Ministry of International Trade and Industry since October 5, 1994, respectively. Transformant CHO / A21- obtained in Example 2 described later
8 and the transformant CHO / B4-4 are 10
Depositary number FERM BP-4 from 5th March to the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH), Ministry of International Trade and Industry
822 and deposit number FERM BP-4823.

[0035]

The present invention will be described in more detail below with reference to Reference Examples and Examples, but the present invention is not limited thereto. The gene manipulation method using Escherichia coli is based on molecular cloning [Molecular Clonin
g] was followed.

[0035]

[Reference Example 1] Cloning of human interleukin 8 receptor protein type A cDNA Known human interleukin 8 receptor protein type A cDNA (Sciense, 253, 1278-128)
0 (1991)), and DNA oligomers and were synthesized. Is 5'-GTGCGTCGACA
GATCTGGACATGTCAAATTATTACAG
ATCC-3 '(SEQ ID NO: 5), -4 to +20
It is an oligomer containing the sense sequence (translation initiation site is +1). Also, is 5'-CCGCAAGCTT
TGGTTTTCAGAGGTTGGAAGAGAC-
3 '(SEQ ID NO: 6) and +1036 to +1059.
Is an oligomer containing the antisense sequence of CDNA synthesis was performed from 5 μg of human spleen mRNA (CLONTECH) using reverse transcriptase. After degrading RNA, PCR (Polymerase Chain Reaction) was subsequently performed using the above DNA oligomer and Taq polymerase. The reaction conditions are 94 ° C for 5 minutes and then
One cycle of 94 ° C. for 1 minute, 55 ° C. for 2 minutes and 72 ° C. for 3 minutes was repeated for 30 cycles, and finally the reaction was carried out at 72 ° C. for 15 minutes. After the reaction, the specifically amplified DNA fragment was cut out, blunt-ended with T4 polymerase and pUC
119 vector (Takara Shuzo) was inserted into the HincII site to obtain plasmid pUC119 / hIL8RA. When the nucleotide sequence of the inserted human interleukin 8 receptor protein type A cDNA was confirmed using a DNA sequencer (Applied Biochemical),
It perfectly matched the known nucleotide sequence [Fig. 1]. As described above, human interleukin 8 receptor protein type A
A plasmid containing the cDNA fragment was obtained.

[0036]

[Reference Example 2] Cloning of human interleukin 8 receptor protein type B cDNA Known publicly known human interleukin 8 receptor protein type B cDNA (Sciense, 253, 1280-128)
3 (1991)), and DNA oligomers and were synthesized. Is 5'-GTGCGTCGACT
GCAGTTAACATGGAGAGGTGACAGCT
TTG-3 '(SEQ ID NO: 7), and -5 to +19.
It is an oligomer containing the sense sequence (translation initiation site is +1). Also, is 5'-CCGCGTCGAC
AAGCTTGGTCTTAGAGAGTAGTGGA
AGTGTG-3 '(SEQ ID NO: 8), +104
It is an oligomer containing an antisense sequence of 8 to +1072. PCR (Polymerase Chain Reaction) was performed using 2 ng of human spleen cDNA (CLONTECH), the above DNA oligomer, and Taq polymerase. The reaction conditions are 94 ° C after 5 minutes at 94 ° C.
1 cycle, 2 minutes at 55 ° C., 3 minutes at 72 ° C. as one cycle, and 30 cycles were repeated, and finally reaction was performed at 72 ° C. for 15 minutes. After the reaction, the specifically amplified DNA fragment was excised, blunt-ended with T4 polymerase and pUC119.
It was inserted into the HincII site of the vector (Takara Shuzo) to obtain the plasmid pUC119 / hIL8RB. Inserted human interleukin 8 receptor protein type Bc
The base sequence of DNA is [α- ³² P] dCTP (Amersham
It was confirmed to be completely in agreement with a known base sequence [Fig. 3]. As described above, a plasmid containing the human interleukin 8 receptor protein type B cDNA fragment was obtained.

[0037]

Example 1 Construction of Human Interleukin 8 Receptor Protein cDNA Expression Vector As an expression vector in CHO cells, pAKKO1.
11 was used. pAKKO1.11 was constructed as follows. PTB1 described in JP-A-5-076385
417 to S by HindIII and ClaI treatment
A 1.4 kb DNA fragment containing the Rα promoter and the polyA addition signal was obtained. Also, pTB348
(Naruo, K. et al. Biochemical and Biophysical Research Communications (Bioche
M. Biophys. Res. Commun.) 128, 256-264 (1985)) by treatment with ClaI and SalI to obtain a 4.5 kb DNA fragment containing the dihydrofolate reductase (dhfr) gene. These DNA fragments were treated with T4 polymerase to make the ends blunt-ended and then ligated with T4 ligase to construct a pAKKO1.11 plasmid. next,
PUC119 / hIL8 obtained in Reference Example 1 and Reference Example 2
Human interleukin 8 receptor protein type Ac from RA and pUC119 / hIL8RB with SalI
The DNA and human interleukin 8 receptor protein type B cDNA fragment were excised and inserted into the SalI site, which is the only cloning site of the expression vector pAKKO1.11, to obtain human interleukin 8
Expression plasmid p of receptor protein type A cDNA
An expression plasmid pAKKO1.11 / hIL8RB for AKKO1.11 / hIL8RA and human interleukin 8 receptor protein type B cDNA was obtained. p
AKKO1.11 / hIL8RA and pAKKO1.
11 / hIL8RB with E. coli DH by the calcium chloride method
5 into the expression plasmid pAKK for human interleukin 8 receptor protein type A cDNA.
Transformant harboring O1.11 / hIL8RA Escherichia coli DH5 / pAKK
Transformant Escherichia coli DH5 harboring expression plasmid pAKKO1.11 / hIL8RB for O1.11 / hIL8RA and human interleukin 8 receptor protein type B cDNA
/PAKKO1.11/hIL8RB was obtained.

[0038]

EXAMPLE 2 Human interleukin 8 receptor protein cDNA in CHO (dhfr ^-) expression in cells CHO (dhfr ^-) cells 1 × 10 ⁶ cells, diameter 10cm
Culture was performed for 24 hours in an α-MEM medium containing fetal bovine serum, and the cells obtained from the human
Interleukin 8 receptor protein type A cDNA
Expression plasmid pAKKO1.11 / hIL8RA and human interleukin 8 receptor protein type B cDNA expression plasmid pAKKO1.11 / hI
1 μg of L8RB was introduced by the calcium phosphate method. 48 hours after the introduction, the medium was replaced with an α-MEM medium containing 10% fetal bovine serum, and cells in which the plasmid had been integrated into the chromosome were selected. Further, the selected cells were cloned from a single cell by the limiting dilution method, and a cell line CHO / A21-8 stably expressing human interleukin 8 receptor protein A and human interleukin 8 receptor protein type B were expressed. A cell line CHO / B4-4 that stably and highly expresses was obtained.

[0039]

Example 3 Preparation of Immobilized Cells Expressing Human Interleukin 8 Receptor Protein The CHO cells expressing the human interleukin 8 receptor protein obtained in Example 2 were cultured in 24-well or 96-well plates. After that, the medium was removed, phosphate-buffered saline containing 0.01% glutaraldehyde was added, and
Let stand for 0 minutes. After washing twice, 2% BSA, 0.2% Na
A phosphate buffer containing N ₃ was added, and the mixture was stored at -4 ° C.

[0040]

Example 4 Preparation of CHO Cell Membrane Fraction Containing Human Interleukin 8 Receptor Protein C expressing human interleukin 8 receptor protein type A and type B obtained in Example 2 respectively
HO cells (10 ⁹ cells) were suspended in phosphate buffered saline (PBS-EDTA) supplemented with 0.53 mM EDTA and centrifuged at 1000 rpm for 5 minutes. The cell pellet was added to the cell homogenate buffer (50 mM Tris-HCl buffer, 0.1 mM PMSF, 1 mM EDTA, 2 mM).
0 μg / ml leupeptin, 4 μg / ml E-64,
20 μl of 1 μg / ml pepstatin A) was added and homogenized using a Polytron homogenizer. 10
Centrifuge at 00 rpm for 5 minutes, and add the supernatant to 38,000 xg for 2
After centrifugation for 0 minutes, a precipitate of the membrane fraction was obtained. 2 of this precipitate
It was suspended in a homogenate buffer containing 0.2 ml of 0.2% BSA, dispensed, stored at -80 ° C, and thawed at each use.

[0041]

[Example 5] Measurement of human interleukin 8 receptor protein activity Twenty-four CHO cells expressing human interleukin 8 receptor protein type A and type B, respectively.
The plate was cultured. The binding reaction is 25 as a ligand
pM [ ¹²⁵ I] Interleukin 8 (2000 Ci /
mmol; NEM, NEX277) or [ ¹²⁵ I] GRO /
MGSA (2000 Ci / mmol; NEM, NEX321)
Containing 1 ml of a measurement buffer (10% fetal bovine serum,
Α-MEM medium containing 0.2% NaN ₃ ) at 37 ° C., 1
I went on time. After completion of the reaction, the supernatant was removed and the wells were washed 3 times with 2 ml of phosphate buffered saline. The amount of remaining [ ¹²⁵ I] was measured with a gamma counter after peeling the cells with a 1% SDS-0.2N NaOH solution, and this was taken as the total binding amount. Further, 250 pM of unlabeled interleukin 8 was added to the binding reaction and the same operation was performed, and the amount of [ ¹²⁵ I] obtained was defined as the amount of non-specific binding. The amount obtained by subtracting the non-specific binding amount from the total binding amount was defined as the specific binding amount. The results are shown in [Table 1].

[0042]

[Table 1] Lee, J. et al. Journal of Biological Chemistry (J. Biol. Chem.) 267, 16283-162
87 (1992)), the interleukin 8 receptor protein type A binds to interleukin 8 but GRO
/ MGSA does not bind, and interleukin 8 receptor protein type B is interleukin 8 and GRO /
It has been reported to bind to MGSA. The binding specificity results in [Table 1] are consistent with the report of Lee et al.
It was confirmed that HO / A21-8 expresses human interleukin 8 receptor type A and CHO / B4-4 expresses human interleukin 8 receptor type B.

[0043]

Example 6 Measurement of Expression Level and Dissociation Constant of Human Interleukin 8 Receptor Protein Cells expressing human interleukin 8 receptor protein type A and type B were cultured in a 96-well plate until confluency was reached. did. The maximum binding amount and dissociation constant (Kd) of interleukin 8 per cell were measured by the following methods. 50 μl of measurement buffer prepared in several steps with a final concentration of [ ¹²⁵ I] interleukin of 0.4 to 25 nM was added to each well.
Incubated at ℃ for 1 hour. After incubation, cells were bound by the method described in Example 5 [ ¹²⁵ I].
The activity was measured. Trypsin for the number of cells
-Measured with a hemocytometer after peeling off with EDTA solution. As a result of performing Scatchard analysis based on this measurement result, the maximum binding amount of [ ¹²⁵ I] interleukin 8 was 384,000 per cell for interleukin 8 receptor protein type A, and its dissociation constant was 2.9 nM. , Interleukin 8 receptor protein type B has a dissociation constant of 660,000 per cell and 4.
It was 7 nM. The results are shown in FIGS. 7 and 8. From these results, human interleukin 8 of the present invention
It was found that the cells expressing the receptor proteins type A and type B each had a ligand binding activity (specific activity) about 20 times that of human neutrophils. Furthermore, the dissociation constant of interleukin 8 was also found to be similar to that of the natural human interleukin 8 receptor protein (Samanta, AK et al. Journal of Experimental Medicine (J. Ex.
p. Med.) 169, 1185-1189 (1989)).

[0044]

INDUSTRIAL APPLICABILITY CHO cells carrying the expression vector carrying the DNA encoding the human interleukin 8 receptor protein of the present invention are human interleukin 8
It can highly express the receptor protein. In addition, CHO cells containing the recombinant human interleukin 8 receptor protein of the present invention or a cell membrane fraction thereof or a recombinant human interleukin 8 receptor protein or its partial peptide are used to produce human interleukin 8 receptor. An agonist or an antagonist can be screened efficiently. According to the screening method of the present invention, an agonist or an antagonist can be advantageously selected, and therefore, for example, a preventive / therapeutic agent for inflammatory diseases involving neutrophil infiltration, or a drug such as an antitumor agent can be used. Can be developed early.

[0045]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 1053 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: cDNA Characterization method: S sequence ATGTCAAATA TTACAGATCC ACAGATGTGG GATTTTGATG ATCTAAATTT 50 CACTGGCATG CCACCTGCAG ATGAAGATTA CAGCCCCTGT ATGCTAGAAA 100 CTGAGACACT CAACAAGTAT GTTGTGATCA TCGCCTATGC CCTAGTGTTC 150 CTGCTGAGCC TGCTGGGAAA CTCCCTGGTG ATGCTGGTCA TCTTATACAG 200 CAGGGTCGGC CGCTCCGTCA CTGATGTCTA CCTGCTGAAC CTGGCCTTGG 250 CCGACCTACT CTTTGCCCTG ACCTTGCCCA TCTGGGCCGC CTCCAAGGTG 300 AATGGCTGGA TTTTTGGCAC ATTCCTGTGC AAGGTGGTCT CACTCCTGAA 350 GGAAGTCAAC TTCTACAGTG GCATCCTGCT GTTGGCCTGC ATCAGTGTGG 400 ACCGTTACCT GGCCATTGTC CATGCCACAC GCACACTGAC CCAGAAGCGT 450 CACTTGGTCA AGTTTGTTTG TCTTGGCTGC TGGGGACTGT CTATGAATCT 500 GTCCCTGCCC TTCTTCCTTT TCCGCCAGGC TTACCATCCA AACAATTCCA 550 GTCCAGTTTG CTATGAGGTC CTGGGAAATG ACACAGCAAA ATGGCGGATG 600 GTGTTGCGGA TCCTGCCTCA CACCTTTGGC TTCATCGTGC CGCTGTTTGT 650CATCGGTATTCTG CACTGTTT AAGGCCCACA 700 TGGGGCAGAA GCACCGAGCC ATGAGGGTCA TCTTTGCTGT CGTCCTCATC 750 TTCCTGCTTT GCTGGCTGCC CTACAACCTG GTCCTGCTGG CAGACACCCT 800 CATGAGGACC CAGGTGATCC AGGAGACCTG TGAGCGCCGC AACAACATCG 850 GCCGGGCCCT GGATGCCACT GAGATTCTGG GATTTCTCCA TAGCTGCCTC 900 AACCCCATCA TCTACGCCTT CATCGGCCAA AATTTTCGCC ATGGATTCCT 950 CAAGATCCTG GCTATGCATG GCCTGGTCAG CAAGGAGTTC TTGGCACGTC 1000 ATCGTGTTAC CTCCTACACT TCTTCGTCTG TCAATGTCTC TTCCAACCTC 1050 TGA 1053

[0046]

[SEQ ID NO: 2] Sequence length: 350 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Ser Asn Ile Thr Asp Pro Gln Met Trp Asp Phe Asp Asp Leu Asn 1 5 10 15 Phe Thr Gly Met Pro Pro Ala Asp Glu Asp Tyr Ser Pro Cys Met Leu 20 25 30 Glu Thr Glu Thr Leu Asn Lys Tyr Val Val Ile Ile Ala Tyr Ala Leu 35 40 45 Val Phe Leu Leu Ser Leu Leu Gly Asn Ser Leu Val Met Leu Val Ile 50 55 60 Leu Tyr Ser Arg Val Gly Arg Ser Val Thr Asp Val Tyr Leu Leu Asn 65 70 75 80 Leu Ala Leu Ala Asp Leu Leu Phe Ala Leu Thr Leu Pro Ile Trp Ala 85 90 95 Ala Ser Lys Val Asn Gly Trp Ile Phe Gly Thr Phe Leu Cys Lys Val 100 105 110 Val Ser Leu Leu Lys Glu Val Asn Phe Tyr Ser Gly Ile Leu Leu Leu 115 120 125 Ala Cys Ile Ser Val Asp Arg Tyr Leu Ala Ile Val His Ala Thr Arg 130 135 140 Thr Leu Thr Gln Lys Arg His Leu Val Lys Phe Val Cys Leu Gly Cys 145 150 155 160 Trp Gly Leu Ser Met Asn Leu Ser Leu Pro Phe Phe Leu Phe Arg Gln 165 170 175 Ala Tyr His Pro Asn Asn Ser Ser Pro Val Cys Tyr Glu Val Leu Gly 180 185 190 Asn Asp Thr Ala Lys Trp Arg Met Val Leu Arg Ile Leu Pro His Thr 195 200 205 Phe Gly Phe Ile Val Pro Leu Phe Val Met Leu Phe Cys Tyr Gly Phe 210 215 220 Thr Leu Arg Thr Leu Phe Lys Ala His Met Gly Gln Lys His Arg Ala 225 230 235 240 Met Arg Val Ile Phe Ala Val Val Leu Ile Phe Leu Leu Cys Trp Leu 245 250 255 Pro Tyr Asn Leu Val Leu Leu Ala Asp Thr Leu Met Arg Thr Gln Val 260 265 270 Ile Gln Glu Thr Cys Glu Arg Arg Asn Asn Ile Gly Arg Ala Leu Asp 275 280 285 Ala Thr Glu Ile Leu Gly Phe Leu His Ser Cys Leu Asn Pro Ile Ile 290 295 300 Tyr Ala Phe Ile Gly Gln Asn Phe Arg His Gly Phe Leu Lys Ile Leu 305 310 315 320 Ala Met His Gly Leu Val Ser Lys Glu Phe Leu Ala Arg His Arg Val 325 330 335 Thr Ser Tyr Thr Ser Ser Ser Val Asn Val Ser Ser Asn Leu 340 345 350

[0047]

[SEQ ID NO: 3] Sequence length: 1068 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Characterization method: S sequence ATGGAGAGTG ACAGCTTTGA AGATTTCTGG AAAGGTGAAG ATCTTAGTAA 50 TTACAGTTAC AGCTCTACCC TGCCCCCTTT TCTACTAGAT GCCGCCCCAT 100 GTGAACCAGA ATCCCTGGAA ATCAACAAGT ATTTTGTGGT CATTATCTAT 150 GCCCTGGTAT TCCTGCTGAG CCTGCTGGGA AACTCCCTCG TGATGCTGGT 200 CATCTTATAC AGCAGGGTCG GCCGCTCCGT CACTGATGTC TACCTGCTGA 250 ACCTAGCCTT GGCCGACCTA CTCTTTGCCC TGACCTTGCC CATCTGGGCC 300 GCCTCCAAGG TGAATGGCTG GATTTTTGGC ACATTCCTGT GCAAGGTGGT 350 CTCACTCCTG AAGGAAGTCA ACTTCTATAG TGGCATCCTG CTACTGGCCT 400 GCATCAGTGT GGACCGTTAC CTGGCCATTG TCCATGCCAC ACGCACACTG 450 ACCCAGAAGC GCTACTTGGT CAAATTCATA TGTCTCAGCA TCTGGGGTCT 500 GTCCTTGCTC CTGGCCCTGC CTGTCTTACT TTTCCGAAGG ACCGTCTACT 550 CATCCAATGT TAGCCCAGCC TGCTATGAGG ACATGGGCAA CAATACAGCA 600 AACTGGCGGA TGCTGTTACG GATCCTGCCC CAGTCCTTTG GCTTCATCGT 650 GCCACTGCTGCTCATGCT TCACCCTG CGTACGCTGT 700 TTAAGGCCCA CATGGGGCAG AAGCACCGGG CCATGCGGGT CATCTTTGCT 750 GTCGTCCTCA TCTTCCTGCT TTGCTGGCTG CCCTACAACC TGGTCCTGCT 800 GGCAGACACC CTCATGAGGA CCCAGGTGAT CCAGGAGACC TGTGAGCGCC 850 GCAATCACAT CGACCGGGCT CTGGATGCCA CCGAGATTCT GGGCATCCTT 900 CACAGCTGCC TCAACCCCCT CATCTACGCC TTCATTGGCC AGAAGTTTCG 950 CCATGGACTC CTCAAGATTC TAGCTATACA TGGCTTGATC AGCAAGGACT 1000 CCCTGCCCAA AGACAGCAGG CCTTCCTTTG TTGGCTCTTC TTCAGGGCAC 1050 ACTTCCACTA CTCTCTAA 1068

[0048]

[SEQ ID NO: 4] Sequence length: 355 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Glu Ser Asp Ser Phe Glu Asp Phe Trp Lys Gly Glu Asp Leu Ser 1 5 10 15 Asn Tyr Ser Tyr Ser Ser Thr Leu Pro Pro Phe Leu Leu Asp Ala Ala 20 25 30 Pro Cys Glu Pro Glu Ser Leu Glu Ile Asn Lys Tyr Phe Val Val Ile 35 40 45 Ile Tyr Ala Leu Val Phe Leu Leu Ser Leu Leu Gly Asn Ser Leu Val 50 55 60 Met Leu Val Ile Leu Tyr Ser Arg Val Gly Arg Ser Val Thr Asp Val 65 70 75 80 Tyr Leu Leu Asn Leu Ala Leu Ala Asp Leu Leu Phe Ala Leu Thr Leu 85 90 95 Pro Ile Trp Ala Ala Ser Lys Val Asn Gly Trp Ile Phe Gly Thr Phe 100 105 110 Leu Cys Lys Val Val Ser Leu Leu Lys Glu Val Asn Phe Tyr Ser Gly 115 120 125 Ile Leu Leu Leu Ala Cys Ile Ser Val Asp Arg Tyr Leu Ala Ile Val 130 135 140 His Ala Thr Arg Thr Leu Thr Gln Lys Arg Tyr Leu Val Lys Phe Ile 145 150 155 160 Cys Leu Ser Ile Trp Gly Leu Ser Leu Leu Leu Ala Leu Pro Val Leu 165 170 175 Leu Phe Arg Arg Thr Val Tyr Ser Ser Asn Val Ser Pro Ala Cys Tyr 180 185 190 Glu Asp Met Gly Asn Asn Thr Ala Asn Trp Arg Met Leu Leu Arg Ile 195 200 205 Leu Pro Gln Ser Phe Gly Phe Ile Val Pro Leu Leu Ile Met Leu Phe 210 215 220 Cys Tyr Gly Phe Thr Leu Arg Thr Leu Phe Lys Ala His Met Gly Gln 225 230 235 240 Lys His Arg Ala Met Arg Val Ile Phe Ala Val Val Leu Ile Phe Leu 245 250 255 Leu Cys Trp Leu Pro Tyr Asn Leu Val Leu Leu Ala Asp Thr Leu Met 260 265 270 Arg Thr Gln Val Ile Gln Glu Thr Cys Glu Arg Arg Asn His Ile Asp 275 280 285 Arg Ala Leu Asp Ala Thr Glu Ile Leu Gly Ile Leu His Ser Cys Leu 290 295 300 Asn Pro Leu Ile Tyr Ala Phe Ile Gly Gln Lys Phe Arg His Gly Leu 305 310 315 320 Leu Lys Ile Leu Ala Ile His Gly Leu Ile Ser Lys Asp Ser Leu Pro 325 330 335 Lys Asp Ser Arg Pro Ser Phe Val Gly Ser Ser Ser Gly His Thr Ser 340 345 350 Thr Thr Leu 355

[0049]

[SEQ ID NO: 5] Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GTGCGTCGAC AGATCTGGAC ATGTCAAATA TTACAGATCC 40

[0050]

[SEQ ID NO: 6] Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CCGCAAGCTT TGGTTTTCAG AGGTTGGAAG AGAC 34

[0051]

[SEQ ID NO: 7] Sequence length: 39 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GTGCGTCGAC TGCAGTTAAC ATGGAGAGTG ACAGCTTTG 39

[0052]

[SEQ ID NO: 8] Sequence length: 41 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CCGCGTCGAC AAGCTTGGTC TTAGAGAGTA GTGGAAGTGT G 41

[0053]

[Brief description of drawings]

1] Human interleukin 8 obtained in Reference Example 1
1 shows the entire base sequence of receptor protein type A cDNA.

[0054]

FIG. 2 Human interleukin 8 obtained in Reference Example 1
The amino acid sequence encoded by the receptor protein type A cDNA is shown.

[0055]

FIG. 3 Human interleukin 8 obtained in Reference Example 2
1 shows the entire base sequence of receptor protein type B cDNA.

[0056]

FIG. 4 Human interleukin 8 obtained in Reference Example 2
The amino acid sequence encoded by the receptor protein type B cDNA is shown.

[0057]

FIG. 5 shows a schematic diagram of the expression vector labeled with pAKKO1.11 / hIL8RA.

[0058]

FIG. 6 shows a schematic diagram of the expression vector labeled with pAKKO1.11 / hIL8RB.

[0059]

FIG. 7: Human interleukin 8 obtained in Example 6
A Scatchard plot (dissociation constant curve) of receptor protein type A is shown.

[0060]

FIG. 8: Human interleukin 8 obtained in Example 6
A Scatchard plot (dissociation constant curve) of receptor protein type B is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 5/10 C12P 21/02 C 9282-4B C12Q 1/02 6807-4B // (C12P 21 / 02 C12R 1:91) 7729-4B C12N 5/00 B

Claims (13)

[Claims] 1. A pA which retains a DNA encoding a human interleukin 8 receptor protein.
KKO1.11 / hIL8RA or pAKKO1.1
An expression vector designated 1 / hIL8RB. 2. A CHO cell containing the expression vector according to claim 1. 3. The CHO cell according to claim 2, which is a CHO cell lacking the dihydrofolate reductase gene.
cell. 4. Recombinant human interleukin 8 receptor protein, characterized in that the CHO cells according to claim 2 are cultured under conditions that allow expression of DNA encoding human interleukin 8 receptor protein. Manufacturing method. 5. A recombinant human interleukin 8 receptor protein which can be produced by culturing the CHO cell according to claim 2 under conditions that allow the expression of a DNA encoding the human interleukin 8 receptor protein. CHO cells or cell membrane fractions thereof. 6. A recombinant human interleukin 8 receptor protein isolated from CHO cells containing the recombinant human interleukin 8 receptor protein according to claim 5, a partial peptide thereof or a salt thereof. 7. A method for screening a human interleukin 8 receptor agonist or antagonist, which comprises using the CHO cell according to claim 5 or a cell membrane fraction thereof. 8. A method for screening a human interleukin 8 receptor agonist or antagonist, which comprises using the recombinant human interleukin 8 receptor protein according to claim 6 or a partial peptide thereof or a salt thereof. 9. A CHO cell according to claim 5 or a cell membrane fraction thereof, or a recombinant human interleukin 8 receptor protein according to claim 6, a partial peptide thereof or a salt thereof. A kit for screening a human interleukin 8 receptor agonist or antagonist. 10. A human interleukin 8 receptor agonist or a salt thereof obtained by using the screening method according to claim 7 or claim 8 or the screening kit according to claim 9. 11. A human interleukin 8 receptor antagonist or a salt thereof obtained by using the screening method according to claim 7 or claim 8 or the screening kit according to claim 9. 12. A neutrophil activation-promoting agent or neutrophil migration-promoting agent, which comprises the agonist according to claim 10 or a salt thereof. 13. A neutrophil activation inhibitor or a neutrophil migration inhibitor, which comprises the antagonist according to claim 11 or a salt thereof.