WO1994021809A1 - Monocyte or macrophage migration factor - Google Patents

Abstract

The invention relates to a protein having a monocyte or macrophage migration activity, a gene containing a DNA coding for the protein, a recombinant expression vector containing the gene, a transformant prepared by using the recombinant vector, a process for producing the protein by culturing the transformant and separating a protein having a monocyte or macrophage migration activity from the product of culture, and an immunopotentiator or wound remedy containing the protein as the active ingredient.

Description

 Specification

 Monocyte or macula phage migration factor

 Technical field

 The present invention relates to a novel monocyte or macrophage migration factor, a method for producing the same, and an immunostimulating agent or a wound healing agent. Background art

 The organism protects against infectious diseases caused by bacteria by infiltrating and accumulating phagocytes such as monocytes or macrophages or neutrophils at the site of infection, and killing and phagocytosing the invading bacteria. In addition, monocytes or macrophages break down bacterial constituents,

In addition to presenting T lymphocytes as antigens, B lymphocytes not only promote antigen-specific antibody production, but also themselves produce various cytokines to amplify inflammation and immune responses. Thus, monocytes or macrophages are cells that play a central role in biological defense against bacterial infection.

 The migration and factor of this monocyte or macula phage, which is a substance that attracts to the infected area, include Macrophage Chemoattractant Protein-1 (MCP-1: FEBS 1 et t., 244, 487-493, 1989), RANTES / Molecules such as SIS (Nature, 347, 669-671, 1990) have been elucidated so far, but in addition to these chemotactic factors, it has been shown that there are factors having chemotactic activity for monocytes or macrophages. I have.

 —On the other hand, the body naturally has natural healing power against wounds and burns, but severe burns, recurrent floor displacement, ulcers that occur as a complication of other diseases, and side effects of chemotherapy In many cases, the natural healing power of the living body cannot recover the mucosal damage, etc., and it is necessary to use a therapeutic agent for wounds, burns, ulcers and the like.

In particular, even among wounds, floor gaps are more likely to occur in bedridden elderly people, and this is becoming a major problem for the aging society. Among the ulcers, lower limb ulcers, which are likely to develop as a complication of diabetes, are a major problem in the lack of effective therapeutic agents as the number of patients with diabetes is increasing. In addition, mucosal damage, which occurs as a side effect of cancer chemotherapy and radiation therapy, is also a major problem in reducing patients' quality of life (QOL). Therefore, development of a therapeutic agent for early recovery from the above-mentioned various wounds, burns, and ulcers is desired.

 In addition, corneal damage and wounds after cataract surgery cannot be fully recovered by the natural healing power of the living body, and the development of therapeutic agents is awaited.

 Tocopherol derivatives (JP-B-49-25632) have been developed as wound treatment agents to date, and epidermal growth factor (Epidermal) has been developed as a wound treatment agent to date. Growth Factor: EGF; Carpenter, G. and Cohen, S., Annu. Rev. Biochem., 48, 193-216, 1979), Fibrob last Growth Factor: FGF; Gospodarowicz et al., Ol.Cell. Endocrinol., 46, 187-204, 1986), Platelet Derived Growth Factor (PDGF; Betshol tz et al., Nature, 320, 695-699, 1986). There is a need for the development of effective wound treatments.

 Insulin-like growth factor (IGF) -I and Π substances are also known (Froesch, ER et al .; Ann. Rev. Physiol. 47, 443-467, 1985), and effective wounds It is expected to be developed as a therapeutic agent. Disclosure of the invention

 An object of the present invention is to provide a novel factor having a novel monocyte or macrophage migration activity and a method for producing the same.

 Another object of the present invention is to provide an immunostimulant useful for infectious diseases caused by bacteria, etc., and to provide a new wound healing agent to the wound healing market where an effective drug has not yet been developed. .

Therefore, the present inventors conducted intensive studies on monocyte or macula phage migration factor and screened the culture supernatant of various cultured cells. As a result, HT treated with lipopolysaccharide (LPS), an endotoxin of Escherichia coli. — From a culture solution in which 1 376 cells were cultured for 1 to 3 days, a protein having a completely new monocyte or macrophage migration activity, which has never been reported, is detected and purified, and the protein amino acid is degraded by automated Edman degradation. The terminal amino acid sequence and the amino acid sequence of the partial peptide obtained by digestion with a protease were determined. Based on the amino acid sequence information, this protein The cDNA to be loaded was cloned and its full structure was determined. As a result of analyzing the cloned cDNA, it was predicted that the protein encoded by this cDNA would have the structure of a precursor protein containing the required migration factor protein in its N-terminal part. The entire cloned cDNA was ligated to an expression vector, expressed in Cos1 cells, and a method was developed to purify the N-terminal protein having migration activity secreted in the culture supernatant. In addition, only the cDNA encoding the N-terminal part of the precursor, which is secreted into HT-1376 cells or Cos 1 cell culture supernatant and thought to exhibit migration activity, is ligated to the expression vector. We have also developed a method to express and purify large amounts in E. coli using Escherichia coli, and established a method for producing this migration factor, HT-1376 cell derived Leukocyte Chemotactic Factor (LCF). As a result of examining the biological activity of the obtained transgenic HT-LCF using various test methods, it was found that the recombinant HT-LCF did not act on neutrophils, but on monocytes or macula phage. It has been revealed that these are chemotactic factors that act specifically on them. In addition, it was confirmed that the recombinant HT-LCF had a therapeutic effect on wounds and burns, and thus completed the present invention. That is, the present invention includes the following inventions.

(1): a protein substantially comprising the amino acid sequence represented by SEQ ID NO: 1 and having a monocyte or macaque phage migration activity; (2): a protein having the amino acid sequence represented by SEQ ID NO: 2 (3): a gene substantially containing DNA encoding the amino acid sequence represented by SEQ ID NO: 1, (4): SEQ ID NO: 2 (5): a gene containing a DNA substantially encoding the amino acid sequence represented by SEQ ID NO: 3; (6): a gene containing a DNA substantially encoding the amino acid sequence represented by SEQ ID NO: 3 (3) to (5) an expression recombinant vector containing the gene according to any one of (7): a transformant transformed by the expression recombinant vector according to (6), (8) ): The transformant according to (7) is cultured, and the protein according to (1) is obtained from the resulting culture. And (9): culturing the transformant according to (7), and culturing the transformant according to (7), wherein the protein according to (2) is obtained from the obtained culture. (10): an immunostimulant containing the protein of (1) as an active ingredient; and (11): a protein of (2). With active ingredients (12): a wound treating agent containing the protein of (2) as an active ingredient; (13): a protein having an active ingredient of monocyte or macrophage migration activity, and a pharmaceutically acceptable drug. A pharmaceutical composition containing an acceptable excipient, (14): the pharmaceutical composition according to the above (13), which is used for treating a wound, (15): a pharmaceutical composition according to the above (13), which is used for immunostimulation, 16): A method for treating a wound, which comprises administering an effective amount of a protein having a monocyte or macrophage migration activity to a human; (17): An effective amount of a protein having a monocyte or macrophage migration activity is administered to a human. (18): Use of an effective amount of a protein having monocyte or macrophage migration activity for human treatment for wound treatment, (19): for immunostimulation, Effective amount of monocytes or macrophages Use of a protein having a migration activity in humans.

 Here, in the present invention, the term “wound” broadly refers to a general term for tissue damage due to physical, biological, and chemical mechanisms, regardless of whether it occurs on the body surface or inside the body.

 Physical mechanisms include external force such as bruise, incision, scratch, rupture, and surgical operation, and other external energy such as heat, radiation, and electricity. Specific examples of wounds caused by physical mechanism Examples include cuts, bruises, burns, floor misalignment, mucosal damage, corneal damage, and cataract surgery as side effects of radiation therapy.

 Biological and chemical mechanisms include acids, alkalis, organic compounds, disruption of homeostasis, reduced healing power, stress, and enhanced immune function. Specific examples of wounds caused by biological and chemical mechanisms include: Ulcers, ulcers occurring as a complication with other diseases, dehydration and the like. More specifically, there may be mentioned gastrointestinal ulcer, diabetic skin; t ulcer, mucosal damage occurring as a side effect of chemotherapy, and the like.

 The term "substantially contains" or "encodes substantially" an amino acid sequence means that some of the amino acids have deletions, substitutions, additions, etc., as long as the peptide has monocyte or macrophage migration activity. Is also good.

 Furthermore, the term “culture” means any of a culture solution, cultured cells / cells or a disrupted product thereof, and a culture supernatant.

Hereinafter, the present invention will be described in detail. The migration activity of monocytes or macrophages can be determined by monitoring monocytes or macrophages derived from various animal species as needed, such as humans and rats, in the upper chamber of a chemotaxis chamber separated by a polycarbonate or cellulose micropore membrane filter. By placing a macrophage suspension in the lower chamber and testing it with a sample solution containing chemotactic factors, it is possible to measure how much monocytes or macrophages in the upper chamber are attracted to the lower chamber. It can be measured (J. Exp. Med., 115, 453-466, 1962).

 The monocyte or macrophage migration factor can be determined by measuring the presence or absence of migration activity in the culture supernatant of various cultured cells or primary cells, or in human urine, serum, or plasma using the above measurement method. Produced cultured cells and tissues can be selected, and natural sources for migrating factor purification can be specified.

 The cultured cells used here are not limited to human-derived cells. In addition, not only cells established but also cells in primary culture are included.

 Preparation of a cell culture supernatant sample is carried out in a medium containing 1 to 10% of fetal serum serum or 0.1 to 0.5% of serum albumin. In addition, preparation under stimulation with LPS, phorbol ester or the like is also performed.

 Using the thus obtained culture containing the chemotactic factor as a material, the chemotactic factor can be obtained by a method usually used for protein separation and purification. For example, purification is performed by appropriately combining salting out, centrifugation, various types of chromatography, electrophoresis, and the like. Various types of chromatography include hydrophobicity, gel filtration, ion exchange, reverse phase, and affinity chromatography. The purity and the approximate molecular weight of the purified product are confirmed using SDS (sodium lauryl sulfate) polyacrylamide gel electrophoresis, a mass spectrometer, or the like. 'The amino acid sequence of the purified protein is analyzed by an automatic Edman degradation method using a gas-phase protein sequencer. Analysis of the amino acid sequence is usually performed sequentially from the N-terminal side of the purified protein.However, after digesting the sample with a proteolytic enzyme such as tribsine, the purified peptide fragment can be sequentially analyzed from the N-terminal side. it can.

Based on the analyzed amino acid sequence, it is possible to predict the nucleotide sequence of DNA that is thought to encode that sequence. Based on the prediction, cDNA library From DNA degenerate oligonucleotide probes for screening of cDNA clones of interest or degenerate oligonucleotide primers used for PCR (polymerase-chain-reaction) using DNA synthesizers (Vu, H. and Hirschbein, B. L. et al .; Tetrahedron Lett., 32, 3005-3008, 1991) ο

 In order to clone cDNA, a cDNA library is prepared. The cDNA library is used to transfer cDNA synthesized based on mRNA purified from cells producing chemotactic factors into an appropriate vector having a microbial replicon, and then introduce it into a compatible host. It is produced by. As a method for synthesizing cDNA from purified mRNA, a method using oligo dT as a primer, a method using a random primer, a method using a synthetic primer having a specific nucleotide sequence, and the like are generally used. Plasmid vectors, phage vectors, cosmid vectors, phagemid vectors, YAC vectors, and the like are mainly used as the vectors used to construct the library. Escherichia coli, yeast, Bacillus subtilis and the like are mainly used as hosts (Molecular Cloning; Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).

 Synthetic cDNA may be used as it is in preparing the library, but if the size of the mRNA is known in advance, the corresponding size of the mRNA is subjected to density gradient centrifugation. Or the electrophoretic fractionation of cDNA can increase the frequency of containing the desired clone.

As a method for selecting a clone encoding the required migration factor cDNA from the prepared cDNA library, a method of screening a degenerate probe synthesized from an amino acid sequence after labeling it with ³² P or an enzyme, etc. DNA fragments obtained by PCR performed using degenerate primers can also be used as probes. If the activity evaluation system has high sensitivity and specificity, expression cloning can be performed using the activity in the culture supernatant expressing cDNA in animal cells as an indicator. If antibodies that detect migration factors are available, expression cloning using Escherichia coli as a host is also possible.

The nucleotide sequence of cDNA cloned in this manner can be either radiolabeled or It can be analyzed by the dideoxy method using a fluorescent label, the maxam-Gilbert method, or the like.

 The amino acid sequence encoded by the analyzed DNA can be predicted from the nucleotide sequence of the DNA. Whether the obtained cDNA clone encodes the required migration factor can be confirmed by determining whether the amino acid sequence of the purified protein is encoded by the clone.

 The DNA of the present invention can be obtained partially or entirely by chemical synthesis according to the method of Vu, H. and Hirschbein, BL et al. [Tetrahedron Lett. 32 (26), 3005-3008 (1991)]. .

 When the desired clone is obtained, its activity can be confirmed by reconnecting the cDNA to the expression vector and expressing it. In this case, animal cells, silkworm cells, yeast, Escherichia coli and the like are mainly used as host cells. The vector used depends on the host.

 In this case, prokaryotic (eg, Escherichia coli) and eukaryotic (eg, yeast, insect, or mammalian) cells can be used as host cells. Examples of mammalian cells include COS cells, Chinese Hamster Ovary cells, C-127 cells, BHK (Baby Hamster Kidney) cells, and the like. Examples of yeast include baker's yeast (Saccharomyces cerevisiae) and a methanol-assimilating yeast (Pichia pastoris). Examples of insect cells include silkworm cultured cells.

Vectors used to transform these host cells include PKC30 (Shimatake H. and M. Rosenberg, Nature, 292, 128-132, 1981), pTrc99A (Amann E. et al., Gene, 69, 301-315, 1988). For mammalian cells, p SV2-neo (Southern and Berg; J. Mol. Appl. Genet., 1,327-341, 1982), p CAGGS (Niwa et al .; Gene, 108, 193-200, 1991) or p cDL-SRa296 (Takabe et al .; Mol. Cell. Biol., 8, 466-472, 1988). For yeast, pG-1 (Schena. And Yamamoto KR; Science, 241, 965-967, 1988) and the like can be mentioned. Examples of silkworm cells include a transfer vector pAc373 (Luckow et al .; Bio / Technology, 6, 47-55, 1988) for producing a recombinant virus. No.

 These vectors may contain an origin of replication, a selectable marker, and a promoter, if necessary. Further, a vector for eukaryotic cells may be added with an RNA splice site, a polyadenylation signal, and the like, as necessary.

 As the origin of replication, vectors derived from SV40, adenovirus, dys-papilloma virus and the like can be used as mammalian cell vectors. As a vector for Escherichia coli, those derived from ColEl, R factor, F factor and the like can be used. As the yeast vector, 2〃mDNA, ARS1-derived DNA, and the like can be used.

 As a vector for mammalian cells as a gene expression promoter, those derived from retrovirus, poliovirus, adenovirus, SV40 and the like can be used. For Escherichia coli, a vector derived from Pacteriophage, a trp, lpp, lac, tac promoter and the like can be used. For example, ADH, PH05, GPD, PGK, and MAF promoters can be used for yeast vectors, and AOX1 promoter can be used for methanol-assimilating yeast. A vector derived from nuclear polyhedrosis virus can be used as a vector for silkworm cells.

 As selectable markers, vectors for mammalian cells include neomycin (neo) resistance gene, thymidine kinase (TK) gene, dihydrofolate reductase (DHFR) gene, and Escherichia coli xanthinguanine phosphoribosyltransferase (Ecogpt). ) A messenger can be used. As a vector for Escherichia coli, a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene and the like can be used. For the yeast vector, Leu2, Trpl, Ura3 genes and the like can be used. 'The above-described host-vector system can be appropriately combined to express the gene of the present invention to obtain a protein having a migration activity.

 The Boyden method (J. Exp. Med., 115, 453-466, 1962) is used to confirm migration activity. O

 The chemotactic factor can be produced by separating and purifying from the culture containing the chemotactic factor thus expressed.

In this case, a combination of methods used for normal protein separation and purification is used. Is done. For example, salting-out, centrifugation, and various types of chromatography are appropriately combined. Various types of chromatography include hydrophobicity, gel filtration, ion exchange, reversed phase, affinity chromatography, and the like. The purity of the purified product can be confirmed by SDS polyacrylamide gel electrophoresis.

 The amino acid sequence of the purified protein is confirmed by an automatic Edman degradation method using a gas-phase protein sequencer.

 The active ingredient of the chemotactic factor thus obtained can be used as it is as the immunostimulant or wound healing agent of the present invention or its active ingredient. Further, drying may be performed by spray drying, freeze drying, hot air drying, or the like.

 When the factor obtained as described above is administered as an immunostimulant or a wound treatment, the subject to which the factor is administered is not particularly limited. For example, it can be used for the specific purpose of preventing or treating individual diseases. The method of administration may be oral or parenteral, and oral administration includes sublingual administration. Parenteral administration includes injections, for example, subcutaneous, intramuscular, intravenous injections, infusions, suppositories, creams and the like. The dose varies depending on whether the animal or human, the age, the route of administration, and the number of administrations, and can be widely varied. In this case, the effective amount of the chemotactic factor of the present invention and the effective amount to be administered as a composition of a suitable diluent and a pharmacologically usable carrier are 1 to 1000 / z gZkg body weight Z day, and 1 day to 1 day. It is administered in several divided doses.

 When the immunopotentiator or wound healing agent of the present invention is orally administered, tablets, granules, fine granules, powders, capsules, and the like applied thereto are generally used in preparations in these compositions. It contains additives such as binders, inclusion agents, excipients, lubricants, disintegrants, and wetting agents. In addition, the liquid preparation for oral use may be in the form of a liquid solution for internal use, suspension, emulsion, syrup, etc., or may be a dry product that is redissolved when used. Good. Further, the composition may contain either additives or preservatives.

In the case of parenteral administration, it contains additives such as a stabilizer, a buffer, a preservative, and a tonicity agent, and is usually provided in the form of a unit-dose ampoule, a multi-dose container or a tube. The compositions described above are suitable for use as carriers, for example pyrogen-free The powder may be redissolved with sterilized water.

 In the case of a wound healing agent, one or more substances such as EGF, FGF, IGF-I, IGF-I, PDGF, and tocopherol derivative may be additionally contained in addition to the protein of the present invention. . BRIEF DESCRIPTION OF THE FIGURES

 Figure 1 shows the results of purification of HT-LCF using a C18 reverse-phase HP LC column.

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 FIG. 2 shows the results of SDS polyacrylamide gel electrophoresis of purified HT-LCF.

 FIG. 3 is a design drawing of degenerate primers 1 and 2.

 FIG. 4 is a diagram showing the results of SDS polyacrylamide gel electrophoresis of recombinant HT-LCF derived from C0s1 cells.

 FIG. 5 is a diagram showing the results of purification of recombinant HT-LCF derived from Cos1 cells by reverse-phase HPLC after digestion with V8 protease.

 FIG. 6 is a diagram showing the results of analysis of a recombinant HT-LCF derived from Cos 1 cells using a mass spectrometer.

 FIG. 7 is a diagram showing an analysis result of a peak A peptide obtained by V8 protease digestion using a mass spectrometer.

 FIG. 8 is a graph showing the results of the migration activity of the recombinant HT-LCF derived from Cos 1 cells on monocytes or macrophages.

 FIG. 9 is a graph showing the results of neutrophil migration activity of the recombinant gene-derived HT-LCF derived from Cos 1 cells.

 FIG. 10 is a diagram showing primers used for PCR.

 FIG. 11 is a graph showing the results of the migration activity of recombinant HT-LCF derived from Escherichia coli on human monocytes. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention These embodiments do not limit the technical scope.

[Example 1] Method for measuring monocyte or macrophage migration activity of HT-LCF. Human mononuclear cells were prepared from human peripheral blood as follows. PBS in 100 ml of blood

(Phosphate buffered saline) 100 ml were mixed, and FicoU-Paque (registered trademark; manufactured by Pharmacia LKB Biotechnology Inc .; hereinafter, referred to as "Pharmacia") was used. The layers were gently overlaid to avoid mixing, and centrifuged at 1,700 rpm for 20 minutes at room temperature.

 The suspended mononuclear cell layer was collected, and RPMI 1640 medium (LIFE TECHNOLOGIES,

Inc., hereinafter “Gibco”) and centrifuged at 1,700 rpm for 10 minutes at room temperature. The precipitate was diffused in RPMI 1640 medium, and further centrifuged at 1,200 rpm at room temperature for 3 minutes. The obtained precipitate was suspended in RPMI 1640 medium so that the concentration of monocytes was 1 to 1.5 million per ml. The cell suspension (50/1) was placed in the upper chamber of a 48-well microchemotaxis chamber 1 (manufactured by Neuroprobe), and the lower chamber was filled with 251 specimens which are thought to contain HT-LCF. A filter made of polycarbonate and having 5 im small holes was used. The device in C0 ₂ Inkyu beta in one, was incubated for 3 hours at 37 ° C. After the incubation, the liquid in the upper chamber was removed, and the upper chamber of the filter 1 was rubbed with a scraper to remove cells adhering to the upper chamber. The filters were air-dried and stained with Diff Quick Staining Solution (manufactured by Green Cross), and the total number of cells in five randomly selected visual fields was counted using a high-magnification objective lens (X40). Cells that spilled into the lower chamber were also counted.

[Example 2] Purification of HT-LCF from a culture solution of HT-1 376 cells Native HT-LCF was obtained from a human bladder transitional cell carcinoma cell line HT-1 376 cells (ATCC CRL-1 Purified from the culture solution of 4 7 2). The cytological properties of this cell have been reported by Rasheed et al. (J. Natl. Cancer Inst., 58, 881-890, 19977).

The HT-1 376 cells were cultured in a medium for culturing normal animal cells. That is, DMEM medium (Dulbecco's modified Eagle minimum essential medium; Gibco) was used. For normal culture, a medium containing 5% fetal calf serum (manufactured by Gibco) in this medium was used. Culture, using C 0 ₂ incubator one was carried out at a temperature condition of 5% C0 ₂ concentration and 37 ° C.

The following procedure was used to produce HT-LCF from HT-1 376 cells. © sheet at a concentration of fetal serum 5% by using a DMEM medium containing HT- 1 3 7 6 cells 3 X10 ⁵ cells Roh ml, seeded by 14ml petri dish with a diameter of 10 cm, 37 ° C in 5% C 0 ₂ concentration Cultured for 3 days. Next, the medium was replaced with a medium containing LPS (10 g / ml: SIGMA Chemical Company, hereinafter referred to as “Sigma”; No. L-3254) and fetal fetal serum (1%), followed by two more mediums. The cells were cultured under the same conditions for days.

 30 liters of the thus obtained culture solution was subjected to salting out with 80% saturated ammonium sulfate. The precipitated protein was dissolved in distilled water, dialyzed against distilled water (4 ° C, 2 days), and further diluted against 0.05M Tris-HCl (pH 7.0) buffer containing 0.05M NaCl. Dialysis (4 C, 30 hours) was performed. 110 ml of the dialyzed sample was passed through a CM-Sephadex® C-25 column (2.5 x 20 cm; manufactured by Pharmacia) equilibrated with the same buffer, and after washing, the salt concentration was linearly increased to 0.8 M. The protein was eluted by raising. Fractions containing chemoattractant activity are collected, ultrafiltrated using Amicon Dia Flow Membrane (YM-5), and concentrated 9-fold to 2 ml.

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Next, gel filtration was performed using a column packed with Sephadex (registered trademark) G-75 (manufactured by Pharmacia). PBS was used as a buffer. The active fraction collected at the position where the protein having a molecular weight of 6,000 to 20,000 eluted was collected and dialyzed against distilled water (4 ° C, 30 hours). After freeze-drying, the sample was finally purified using a TSK ODS 120T C18 reverse phase HPLC column (manufactured by Toso I; 4.6 × 200 mm). Using 0.05% trifluoroacetic acid (TFA) solution as eluent A and 80% acetonitrile solution containing 0.05% TFA as eluent B, the concentration of acetonitrile increases linearly from 0% to 50% in 60 minutes. To elute the protein. The eluate was monitored using a wavelength of 220 nm. The flow rate was 0.8 ml / min. The eluted protein was fractionated for each peak, and the fractions obtained were measured according to the method of Example 1. As a result, four adjacent peaks (A and B) indicating the activity of inducing monocyte migration were obtained. , C, D) (Fig. 1 ) o

[Example 3] S.DS polyacrylamide gel electrophoresis of HT-LCF purified from culture solution of HT-1 376 cells

 Using the separation gel (17.5% acrylamide) containing 0.1% SDS, the sample obtained in Example 2 was subjected to electrophoresis. A 375 mM Tris-HCl (pH 8.8) buffer containing 0.1% SDS was used as a buffer in the gel, and a 25 mM Tris-200 mM glycine (pH 6.8) buffer was used as a buffer in the electrophoresis tank. After electrophoresis at a constant current of 15 mA for 1 hour, the proteins in the gel were stained with a silver staining kit (Biorad).

 The result is shown in figure 2. The molecular weight (kDa) of the standard molecular weight marker is shown on the left. M.W. Std. In the upper lane corresponds to the standard molecular weight marker, and A, B, C and D correspond to the four peak fractions eluted with HPLC, respectively. As a result of calculation based on the relative position with respect to the standard molecular weight marker, protein bands were observed at the molecular weight of 12,000 to 15,000 for all four peaks.

[Example 4] Determination of amino acid sequence of HT-LCF purified from culture solution of HT-1 376 cells

 Approximately 20 µg of purified HT-LCF together with 0.5 µg of lysyl peptidase (manufactured by Sigma) in 0.05 M Tris-HCl (pH 8.5) buffer (100 1) at 37 ° C using a water bath. A time incubation was performed. After drying by an evaporator, partial peptides were separated using a TSK ODS 120TC 18 reverse phase HP LC column. Reversed-phase chromatography was performed under the same conditions as those in Example 2 except that a large-diameter column having a diameter of 24 MI was used. The purified HT-LCF and the amino acid sequence of the partial peptide were subjected to automatic Edman degradation using a PSQ-1 gas-phase port sequencer manufactured by Shimadzu Corporation. As a result, the amino acid sequence shown below was determined.

Amino terminus: amino acid sequence of SEQ ID NO: 5

Fr.26: Amino acid sequence of SEQ ID NO: 6

Fr.32: Amino acid sequence of SEQ ID NO: 7 Fr.38: Amino acid sequence of SEQ ID NO: 8

Amino terminus: Amino acid sequence of SEQ ID NO: 9

 Fr. refers to the fraction (fraction) eluted by reverse phase chromatography. In addition, the sequence starting from cysteine lacking the first residue of alanine represented by the amino acid sequence of SEQ ID NO: 9 was also analyzed in the amino terminal sequence. The abundance ratio was 1 for those starting with alanine and 3 for those starting with cysteine.

[Example 5] Design and preparation of a degenerate oligonucleotide primer

 Based on the amino acid sequences determined in Example 4, combinations of nucleotide sequences considered to encode them were expected. Furthermore, codons that were frequently used in humans were selected from those combinations, and degenerate primers 1 and 2 were designed (FIG. 3). That is, the primer 1 was designed with 20 nucleotides and 144 mixes from the amino terminal sequence, and the primer 2 was designed with 17 nucleotides and 32 mixes from the Fr.38 sequence. For codon usage in humans, see Wada et al., Nucleic Acids Res., 18, 2367-2411, 1990.

 The designed primers were synthesized using an automated DNA synthesizer model 380B manufactured by Applied Biosystems (ABI), and purified using a 0PC column for purification of synthetic DNA produced by the company. Synthesis and purification were performed according to the instructions. The purified DNA was dissolved in TE buffer (10 mM Tris, ImM EDTA, pH 7.4) to a concentration of 50 M, and stored frozen at 20 ° C until use.

Example 6 Preparation of mRNA from HT-1 376 cells and conversion to cDNA

HT-1376 cells were cultured in DMEM medium containing 5% fetal bovine serum. After culturing the cells (48 hours) until the cells cover the entire culture area of the culture flask CFALCON (registered trademark) flask, culture area 175 cm ² , No. 3028; manufactured by Becton Dickinson, Inc. The cells were cultured at a final concentration of 10 g / ml for 12 hours. After culturing, remove the medium and wash about 5 x 10 ⁸ cells once with PBS After purification, total RNA was extracted by the AGPC method of Chomczynski et al. (Acid Guanidium Phenol Chroloform Method: Anal. Biochem., 162: 156-159, 1987).

About 5 xlO ⁸ cells were lysed by adding 40 ml of denaturing solution (4 M guanidine thiosinate, 25 mM sodium citrate, 0.5% sarkosyl, 0.1 M 2-mercaptoethanol, pH 7.0) Thereafter, 4 ml of 2 M sodium acetate, 40 ml of a water-saturated phenol, and 8 ml of chloroform were added and stirred. After standing in ice for 15 minutes, the mixture was centrifuged at 10,000 xg for 20 minutes at 4 ° C, then the aqueous phase was recovered, and 40 ml of isopropanol was added, and the mixture was allowed to stand at -20 ° C for 1 hour. After centrifugation at 10,000 × g for 20 minutes at 4 ° C., the supernatant was discarded. The pellet was dissolved in 5 ml of a denaturing solution, 5 ml of isopropanol was added, and the mixture was allowed to stand at −20 ° C. for 1 hour. After centrifugation at 10,000 × g for 20 minutes at 4 ° C, the pellet was rinsed with 75% ethanol, air-dried, and dissolved in 1 ml of TE buffer. The amount of RNA thus obtained was about 5 mg. CDNA was synthesized using 1 g of this RNA. For cDNA synthesis, 200 M dNTP (four kinds of deoxynucleoside triphosphates composed of dATP, dCTP, dGTP, and dTTP; manufactured by Takara Shuzo Co., Ltd.) and a 20-unit RNase inhibitor (Boehringer Mannheim Using 1M random primer (Takara Shuzo) and 200 units of reverse transcriptase (MMLV-derived, BRL) (reaction volume 201), after incubating at 37 ° C for 90 minutes The enzyme was inactivated by heating at 70 ° C for 10 minutes and stored frozen at 120 ° C until use.

[Example 7] Implementation of PCR using HT-1376 cell cDNA as type III PCR was performed using cDNA 11 synthesized in Example 6 as type III. As the primer, an oligomer designed and synthesized in Example 5 was used at a final concentration of 2 M each. 'The reaction system was performed at 100/1. In addition to the above, 200/1 ^ [(1 ^^ ??, a reaction buffer for AmpliTaq (registered trademark) DNA polymerase [10 mM Tris-HCl (pH 8.3), 50 mM KC1, Containing 1.5 mM MgC, 0.001% gelatin) and 10 units of AmpliTaq DNA polymerase [These are Perkin-Elmer Cetus Instruments' (hereinafter referred to as "Perkin-Elmer Cetus") GeneAmp® PCR Reagent Kit. (Reagent Kit) was used.

The reaction was performed in the following order: 92 ° C for 1 minute, 40 ° C for 1 minute, 72 ° C for 2 minutes. This was performed for 30 cycles using a DNA thermal cycler (Thermal Cycler; manufactured by Perkin-Elmer Cetus).

[Example 8] Subcloning of PCR fragment into vector and determination of nucleotide sequence

As a result of electrophoresis of 10/1 out of 100/1 of the PCR reaction solution in Example 7 in a 2% agarose gel, and staining of DNA with ethidium orifice, 170 base pairs (hereinafter referred to as `` base pairs '') were obtained. Is referred to as “bp”). Then, the reaction solution was extracted once with an equal volume of black-mouthed form, and DNA was precipitated by adding 1/20 volume and 5 volumes of 5M NaCl ethanol. This DNA is dissolved in 20 zl of a solution having a composition of 50 mM Tris buffer (pH 7.6), lOmM magnesium chloride, 10 mM 2-mercaptoethanol, and 100 M ATP, and 10 U of T4 polynucleotide kinase (Takara Shuzo) is added. After that, the mixture was reacted at 37 ° C for 30 minutes. After heating at 70 ° C for 10 minutes, dNTP was added so that the final concentration was 250 / M, and 5 U of T4 DNA polymerase (manufactured by Takara Shuzo) was added, followed by reaction at 37 ° C for 30 minutes. After heating at 70 ° C for 10 minutes, DNA was extracted once with an equal amount of water-saturated phenol, and then DNA was recovered by ethanol precipitation. The recovered DNA was dissolved in 20/1 TE buffer, 2〃1 of which were cloned with 10 ng of blunt-ended cloning vector (pUC13 digested with the restriction enzyme Smal: Pharmacia) and ligated (Takara Shuzo DNA Ligation). Kit: Nucleic Acids Res., 14,7617-7631, 1986; was used as directed), and 1/10 amount was used for E. coli concomitant cell JM109 (Gene, 33, 103-119, 1985, ATCC). No.53323; Takara Shuzo Co., Ltd.), and cultured on an agar medium containing the antibiotic ampicillin (100 / zg / ml) for 16 hours to grow colonies. After randomly selecting three of these colonies and culturing (16 hours, 37 ° C), plasmid DNA was extracted by alkaline SDS method (Nucleic Acids Res., 7, 1513-1523, 1979), and its nucleotide sequence was extracted. Was determined by the dideoxy method of performing radiolabeling at ³² P (Sanger et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467, 1977). For the primer, forward (M 4) and reverse (RV) primers manufactured by Takara Shuzo were used. As a result, the sequences of the three plasmids were identical, had the primer sequences used for PCR at both ends, and were determined from the internally purified protein. The nucleotide sequence corresponding to the amino acid sequence was confirmed.

 The result is shown in SEQ ID NO: 4.

 The nucleotide sequence corresponding to the amino acid sequence was 1st to 8th, 41st to 47th and 50th to 55th of the amino acid sequence number of SEQ ID NO: 4 in the sequence listing.

 Therefore, the DNA fragment subcloned here was considered to be a DNA fragment encoding the-part of HT-LCF cDNA. Therefore, this DNA fragment was used as a probe for screening cDNA, and a large amount of plasmid DNA was prepared. The prepared DNA is dissolved in TE buffer, digested with restriction enzymes EcoR I and BamH I, and electrophoresed with 2% Sea Plaque Agarose (Takara Shuzo Co., Ltd .; registered trademark). After heating at 65 ° C. for 10 minutes to dissolve the gel, the mixture was extracted three times with an equal volume of water-saturated phenol, purified by ethanol precipitation, and used as a probe for screening in Example 10.

[Example 9] Preparation of HT-1 376 cell cDNA library

c As a material for preparing mRNA for preparing a DNA library, HT-1376 cells 12 hours after LPS treatment were used. All RNAs were extracted from about 5 × 10 ⁸ cells by the AGP C method. After dissolving 500 µg of this RNA in 500 1 of TE buffer, poly-A ⁺ was added using Oligotex-dT30 (registered trademark; manufactured by Takara Shuzo Co., Ltd .; Nucleic Acids Res. Sympo. Ser. No. 19, 61-64, 1988). RNA was purified (method followed instructions). CDNA was synthesized using 5 g of the purified poly A + RNA. Oligo dT was used as a primer, and cDNA was synthesized using cDNA synthesis system Brass (Amersham; Gene, 25, 263-269, 1983). After the synthesized cDNA was blunt-ended, an adapter (manufactured by Pharmacia) containing sequences of restriction enzymes EcoRI and NotI was added. The adapter-added cDNA is phosphorylated at the end, introduced into an EcoRI digested phage vector; IΖΑΡΠarm (manufactured by Stratagene; registered trademark), packaged [GIGAPACK (registered trademark) II Gold Instruction Manual] Transform the host strain XL-IBlue strain (Stratagene; Biotechniques, 5, 376, 1987) to form phage plaques and use a ⁵ × 10 ⁵ cDNA library. Was prepared. [Example 10] Screening of cDNA library using PCR fragment as a probe

The cDNA library prepared in Example 9 was spread on an agar medium in a 15-cm diameter petri dish so as to reach about 20,000 cells, cultured at 37 ° C, and grown until plaques could be confirmed. I let it. After preparing 10 sheets of the above plates, they were transferred to a nylon filter (Colony / Plaque Screen: manufactured by DuPont; registered trademark). The transferred filter is 1.5M NaCl, 0.5M Tris-HC1 (pH 8.0) solution for 5 minutes, 1.5M NaCl, 0.5M NaOH solution for 2 minutes, 0.2M Tris-HCl (pH 7.5) , 2 x SSC solution [SSC refers to an aqueous solution (pH 7.0) containing 0.15M NaCl and 0.015M sodium citrate. 2 XSSC refers to one with twice the concentration. ] For 30 seconds, and then heated in a vacuum oven at 80 ° C for 2 hours. These filters were placed in a hybrid solution [0.8 M NaCl, 20 mM PiS (PH6.5), 50% formamide, 0.5% SDS, and 100 µg / ml salmon sperm-derived DNA] for 2 hours at 42 ° C. After incubation, use a random primer DNA labeling kit (registered trademark; manufactured by Takara Shuzo Co., Ltd .; Anal. Biochem., 132, 6-13, 1983). ³² Ρ labeled DNA probe (see Example 8) contains 10 ⁷ cpm The solution was kept at 42 ° C for 20 hours in the hybrid solution. Thereafter, washing was performed twice at 42 ° C for 15 minutes in a solution containing 2 XSSC and 0.1% SDS. The filter was subjected to autoradiography at 170 ° C. for 16 hours using an intensifying screen and an X-OMAT (registered trademark) AR film manufactured by EASTMAN KODAK COMPANY. As a result of this autoradiography, 23 positive signals were obtained. Regions containing positive signals were picked up from the master plate and moved to secondary screening. Secondary screening conditions were the same as for primary screening. As a result, three strong signals were obtained. These clones showed the same restriction enzyme pattern, but were partially different in length. The clone with the longest length was selected for sequencing.

[Example 11] HT— LCF c DNA base sequence

A large amount of the phage clone obtained in Example 10 was cultured, and Phage DNA was prepared by the method of Cloning, 2nd. Edition, 2.60-2.80, 1989. When this DNA was digested with the restriction enzyme NotI, it was found that there was no NotI recognition sequence inside the cDNA, and that the cDNA fragment was excised by NotI digestion. Therefore, only the cDNA fragment of about 3.5 kbp was recovered and introduced into the NotI site of Plasmid Vector pBluescript II (registered trademark; manufactured by Stratagene).

^The nucleotide sequence of both ends of the subcloned DNA fragment was analyzed by the dideoxy method using ³² P. Further, internal sequencing was similarly determined by the dideoxy method using primers synthesized based on the sequence of the analyzed portion. By comparing the sequences of the DNA phase captures, they also confirmed the nucleotide sequence analyzed.

 As a result, the entire nucleotide sequence of about 3,500 bp of the subcloned cDNA fragment was determined.

 The result is shown in SEQ ID NO: 3.

 This cDNA fragment encodes the only long open reading frame, and the encoded amino acid sequence is shown in SEQ ID NO: 3 in the sequence listing. The portions corresponding to the amino acid sequence of the purified peptide are the amino acid sequence numbers 1 to 21, 54 to 60, 63 to 69, 78, 79, 81 to 84 of the amino acid sequence numbers in the sequence listing. And the 86th to 87th.

 All the amino acid sequences determined in Example 4 were included in the sequence predicted from cDNA, and it was confirmed that the cDNA clone obtained here was the desired one. It was also confirmed that the PCR fragment used as a probe corresponded to bases 196 to 362 of the cloned cDNA. ,

[Example 12] Expression of HT-LCF in Cos 1 cells

Whole cDNA cloned into the highly efficient expression vector PcDL-SR296 for animal cells (Takebe et al., Mol. Cell. Biol., 8, 466-472, 1988; hereinafter referred to as "SR"). Was introduced. In order to introduce the cDNA fragment excised by the restriction enzyme Not I into SR a, a Not I linker (5, GCGGCCGCTGCA3 ': synthesized using an automatic DNA synthesizer manufactured by ABI) was introduced into the Pst I site of the SR. I was connected. This Not I site with the same restriction enzyme The digested cDNA fragment was introduced, and a plasmid clone connected in the same direction as the direction of the promoter was selected and subjected to an expression experiment. Preparation of DNA for introduction into Cos1 cells was performed in the same manner as in Example 8. As a carrier for introducing DNA, TRANSFECTA (registered trademark; IBF; Proc. Natl. Acac. Sci., USA, 86, 6982-6986, 1989) was used as described. That is, the culture liquid of 10ml per culture dish with a diameter of 10 cm (insulin 5〃G / nil, transferrin 5 g / ml, monoethanolamine § Min 10〃M, I MDM including acetone sodium Len acid 2.5X10- ⁸ M: A mixture of 10; g of plasmid DNA and 30/1 TRANSFECTAM solution (10% aqueous ethanol solution) was added thereto using Iscove's modified D MEM), and the mixture was cultured at 37 ° C. After 72 hours, the culture was recovered and used as a sample for HT-LCF purification. This transfer experiment was repeated, and 900 ml of the culture supernatant was pooled. The collected supernatant was centrifuged at 1,500 × g for 10 minutes at room temperature, and after removing the precipitate, it was frozen and stored at 170 ° C. until use. Hereinafter, “Cos 1 cell-derived transgenic recombinant HT-LCF” purified from a culture solution of Cos 1 cells is referred to as “rHT-LCF” and “r ι__” <©.

[Example 13] Purification of rHT-LCF

Approximately 900 ml of Cos 1 cell culture was diluted 2-fold with Milli-Q water (deionized water), the pH was lowered to 5.6 with hydrochloric acid, and equilibrated with 25 mM citrate-25 mM NaCl (pH 5.6) buffer CM—passed through a Sepharose® CL-16B column. After washing with the same buffer, one-step elution was performed with a buffer whose salt concentration had been raised to 0.8 M NaCl. The peak of the eluted protein was collected and dialyzed against distilled water. The sample after freeze-drying was passed through a Superdex (registered trademark) H75 gel filtration column (Pharmacia) equilibrated with PBS. Fractions that induce migration activity on monocytes or macrophages were collected and dialyzed against distilled water (4 ° C, 20 hours). After lyophilization, the sample was passed through a Phenyl 5 PW RP reverse phase HPLC column (manufactured by Toso I; 4.6 x 75 mm) for final purification. In other words, a 0.05% TFA solution was used as eluent A, and an 80% acetonitrile solution containing 0.05% TFA was used as eluent B. The acetate nitrile concentration was linearly increased from 0% to 50% in 60 minutes. The protein was eluted. The eluate was monitored using a wavelength of 220 nm. The flow rate is set to lmlZ minutes, The peak for inducing migration activity was collected.

 The final purified rHT-LCF was 100-200 μg.

[Example 14] rDS-LCF SDS polyacrylamide gel electrophoresis Electrophoresis of the sample obtained in Example 13 using a separation gel (17.5% acrylamide) containing 0.1% SDS Was done. A 375 mM Tris-HCl (pH 8.8) buffer containing 0.1% SDS was used as a buffer in the gel, and a 25 mM Tris-192 mM glycine (pH 8.3) buffer was used as a buffer in the electrophoresis tank. After running at a constant voltage of 200 V for 45 minutes, the protein was stained with Coomassie brilliant blue. The molecular weight of the marker is shown on the left. Calculated from the relative position with the molecular weight marker, a single band was confirmed at the position of molecular weight of 12,000 to 15,000 (Fig. 4).

[Example 15] Determination of amino acid sequence of rHT-LCF

 The amino acid sequence at the amino terminal was determined in the same manner as in Example 4. As a result, the amino acid sequence shown in SEQ ID NO: 10 was confirmed.

[Example 16] Determination of C-terminal partial amino acid sequence of rHT-LCF and disulfide bond formation site

 30 zg of purified product of rHT-LCF was incubated with V8 protease (0.8 / zg) in 0.05M ammonia acetate buffer (pH 4.0) at 37 ° C for 18 hours. This sample was subjected to reverse-phase HPLC using ODS120T C18 (manufactured by Toso One Co.) to separate the generated peptide. The protein was eluted by using a 0.05% TFA solution in solution A and a 0.05% TFA solution containing 80% acetonitrile in solution B, and increasing the concentration of acetonitrile linearly from 0% to 50% in 60 minutes. . Predicted limited cleavage sites of the amino acid sequence of rHT-LCF deduced from the nucleotide sequence of cDNA by V8 protease are shown below.

Peptide # 1: Amino acid sequence of SEQ ID NO: 11

Peptide # 2: Amino acid sequence of SEQ ID NO: 12

Peptide # 3: Glu

Peptide # 4: Amino acid sequence of SEQ ID NO: 13 Peptide # 5: amino acid sequence of SEQ ID NO: 14

Peptide # 6: amino acid sequence of SEQ ID NO: 15

Peptide # 7: amino acid sequence of SEQ ID NO: 16

 As a result, five partial peptide peaks were confirmed, including the undegraded rHT-LCF peak (Fig. 5). FIG. 5 shows the elution pattern with 0-40% acetate nitrile in reverse phase HPLC.

 The peaks A, B, C, D, and E (unresolved rHT-LCF) were determined in the order of elution, and the N-terminal amino acid sequence of each peak was determined in the same manner as in Example 4. The results are shown below. Shown in

Peak A: amino acid sequence of SEQ ID NO: 17 (part of peptide # 6)

Peak B: amino acid sequence of SEQ ID NO: 18 (peptide # 2)

Peak C: amino acid sequence of SEQ ID NO: 19 (peptide # 5)

Peak D (1): amino acid sequence of SEQ ID NO: 20 (peptide # 1)

Peak D (2): Amino acid sequence of SEQ ID NO: 21 (part of peptide # 3 and peptide # 4)

 In peak D, peptides # 1 and # 4 contained one residue of cysteine and eluted simultaneously due to the formation of disulfide bonds. The peak power of cystine degradation, which was not detected in the first cycle of Edman degradation, was detected in the second cycle, confirming the formation of disulfide bonds. Peptide # 3 was a single residue of glutamic acid, but was not released and remained bound to peptide # 4. In addition, no peptide of peptide # 7 or later located on the C-terminal side of peptide # 6 could be confirmed. -Therefore, the most C-terminal peptide among the identified partial peptides was the portion corresponding to peptide # 6 of peak A, but the expected two C-terminal residues, arginine and glutamic acid Could not be confirmed.

From the above, it was found that the C-terminal amino acid of rHT-LCF is the threonine of peptide # 6 (the 94th residue counted from the N-terminal cysteine as a whole). [Example 17] Determination of C-terminal part of rHT-LCF by mass spectrometry r HT-LCF and r HT-LCF were measured by an electrospray ionization method using a mass spectrometer (manufactured by Fini Gammat, TSQ-700). Mass analysis of peak A obtained in Example 16 and determination of the amino acid sequence of peak A were performed. The measured average masses of rHT-LCF and the peak Α peptide were 10, 780.7 (FIG. 6) and 1,349.1 (FIG. 7), respectively, consisting of 94 residues obtained in Example 16; The theoretical values almost agreed with the theoretical values of 10, 783.5 and 1,348.8, assuming that these had disulfide bonds. In addition, the primary structure of Peak A was as shown in SEQ ID NO: 22 and was consistent with the result obtained in Example 16. Therefore, rHT-LCF was found to have a structure starting from cysteine and ending with threonine at the 94th residue, similar to the result of Example 16.

[Example 18] Measurement of migration activity of rHT-LCF to monocytes or macrophages

 Using the method of Example 1, the migration activity of monocytes or Macguchi phages was measured for purified rHT-LCF. As a result, it was confirmed that a wide dose range (0.01 to 100 nM) caused monocyte or macrophage migration in a dose-dependent manner (FIG. 8). In the figure, the horizontal axis represents the concentration of purified rHT-LCF in the lower chamber of the Boyden Chamber, and the vertical axis represents the total number of monocytes or macrophages measured by observing the stained filter for 5 fields under a microscope. Show.

[Example 19] Measurement of migration activity of rHT-LCF to neutrophils-Human peripheral blood leukocytes were prepared as follows. To 100 ml of blood was added 100 ml of physiological saline containing 3.5% dextran T-500, and the mixture was allowed to stand at room temperature for 30 minutes. Was. The precipitate was suspended in 30 ml of 0.2% NaCl, 30 ml of 1.6% NaCl was added, and the mixture was stirred and centrifuged (1,200 rpm, 5 minutes, 4 ° C). The pellet is suspended in 20 ml of PBS, gently layered on 30 ml of Ficoll-Paque solution so that they do not mix, and centrifuged (1, 300 rpm, 15 minutes, room temperature). Collected as fractions. The collected cells were suspended in RPMI164 medium and centrifuged (1, 200 rpm, 5 minutes, room temperature). The obtained precipitate was suspended in a RPMI 1640 medium so as to have a cell density of 1 to 1.5 million cells per ml. 50 μl of the cell suspension was placed in the upper chamber of a 48-well microchemotaxis chamber, and 25 μl of the specimen containing purified rHT-LCF was placed in the lower chamber. The filter was made of polycarbonate and had a pore size of 3 im. This apparatus was incubated at 37 ° C. for 3 hours in a 5% CO ₂ incubator. After the incubation, the cells that migrated to the lower chamber were counted using a Coulter Counter 1 as cells having a particle diameter of 6.267 // m or more as neutrophils. As a result, the purified rHT-LCF did not migrate neutrophils at all concentrations (FIG. 9). In FIG. 9, the horizontal axis represents the concentration of purified rHT-LCF in the Boyden chamber and the lower chamber, and the vertical axis represents the total number of neutrophils that migrated to the lower chamber as a percentage (migration rate) of the cells in the upper chamber. displayed.

[Example 20] Expression and purification of HT-LCF in E. coli

 Escherichia coli was expressed in order to obtain a large amount of a protein portion (a protein substantially containing the amino acid sequence represented by SEQ ID NO: 2) which was thought to be responsible for the migration activity. A DNA fragment corresponding to this protein was inserted into an expression vector for Escherichia coli and used for expression.

PCR was used to obtain DNA fragments. The primer used for PCR is as shown in Fig. 10. Restriction enzyme sequences were added to the 5 and 5 ends of each primer for convenient integration into the vector. In the N-terminal primer, a methionine codon (ATG) was introduced before alanine, the first amino acid, and an N col site (CCATGG) was added as a restriction enzyme. In the C-terminal primer, a stop codon (TAA) was added immediately after the last amino acid, arginine, followed by a BamHI site (GGATCC). These primers were synthesized using an automatic DNA synthesizer manufactured by ABI as in Example 5, and used in experiments. For PCR type II, 10 ng of plasmid DNA containing the cDNA fragment represented by SEQ ID NO: 3 was used. The reaction conditions were the same as in Example 7, except that the annealing temperature was changed, and the reaction was performed at 45 ° C. The 330 bp band obtained here was collected, digested with the restriction enzymes Ncol and BamHI, and similarly digested with the restriction enzymes PT rc99 expression vector. A (manufactured by Pharmacia, Code No. 27-0971-01), and transformed into a host E. coli YA21 (Mutoh N. Ml. Biol., 166, 557-580, 1983) by the method of Hanahan et al. Et al., J. Bacterid., 136, 693-699, 1978). From the obtained transformants, a clone containing the DNA fragment of ΗΤ-LCF was selected, and the nucleotide sequence of the HT-LCF portion was determined.

 As a result, the clone Y s HT 21 obtained begins with methionine, contains amino acids 1 (aranine) to 105 (arginine) of HT-LCF shown in SEQ ID NO: 2, and stops immediately. It was confirmed that the target DNA having codons was incorporated into the expression vector (SEQ ID NO: 23). In addition, no base substitution or the like due to misreading or the like was found in the middle sequence.

 This clone YsHT21 was shake-cultured in 10 liters of LB medium (37 ° C), and when the absorbance at 600 nm became 0.8, Isopropyl beta-D-Thiogalactoside (IPTG: final concentration ImM ) Was added and the cells were further cultured for 5 hours. After the culture, the cells are collected by centrifugation and suspended in a 6 M guanidine hydrochloride (Wako Pure Chemicals; No. 070-01825) solution containing ImM phenylmethylsulfonyl fluoride (PMS F; Wako Pure Chemicals; No. 1 64-12181). It became cloudy. Each 20 ml of the suspension was dispensed into a plastic centrifuge tube, and the cells were sonicated. Attach a microchip to the Sonifier model 250 (BRANSON) ultrasonic oscillator, put it in an ice-cooled suspension, increase the output, treat it for 2 minutes, then ice-cool the suspension again for another 2 minutes Processed. A part of the suspension after the treatment was sampled and examined under a microscope, and after confirming the disruption of the cells, the suspension was centrifuged and the supernatant was dialyzed against distilled water.

 Further, after equilibration with 25 mM citrate—25 mM NaCK pH 5.6), insoluble substances precipitated during dialysis were removed by centrifugation. ,

Pass the sample through a column (25x200 mm) of CM—Sepharose—CL-16B (Pharmacia; No. 17-0720-01) equilibrated with 25 mM citrate—25 mM NaCK pH 5.6). The protein was eluted by linearly increasing the concentration to M. After the elution, 20 ml portions were collected, and a fraction showing absorption at a wavelength of 280 nm was collected and dialyzed against 10 mM acetic acid. After lyophilization, the sample was purified using a C0SM0SIL 5C18-AR-300 reverse phase HP LC column (Nacalai Tesque, 10 x 250 mm). That is, 0.05% TFA solution (Wako Pure Chemical Industries; No. 206-10731) Using a solution of 80% acetonitrile (Wako Pure Chemical Industries, No. 0 19-08631) containing 0.05% TFA solution as syneresis B, increase the concentration of acetonitrile linearly from 0% to 50% in 60 minutes to increase protein. Eluted. The eluate was monitored for absorbance at 220 nm. The flow rate was 3.5 ml / min. The eluted protein was fractionated for each peak, and the N-terminal amino acid sequence was analyzed using Shimadzu P SQ-1 protein sequencer. As a result, a peak corresponding to 10 residues of the N-terminal amino acid sequence of HT-LCF was obtained. That is, as shown in SEQ ID NO: 3, amino acids 1 (alanine) to 10 (proline) were detected, and this purified product was a protein described in SEQ ID NO: 2 intended for expression in E. coli. It was confirmed to have the same N-terminal sequence as

 No monocyte-Z macrophage migration activity was observed in this purified peak, but this was probably due to the fact that HT-LCF expressed in E. coli had a different conformation from that expressed in animal cells. Therefore, we performed refolding as follows to correct it.

3.1 g of guanidine hydrochloride was dissolved in 10 ml of distilled water, and the pH was adjusted to 8 by adding a sodium hydroxide solution. To this, 61.5 mg of reduced glutathione (Wako Pure Chemical Industries, Ltd .; No.073-02013) was added, stirred, and then adjusted to pH 8 again. Further, 1 ml of a 6 M guanidine hydrochloride solution containing 1 mg / ml of the purified product was added and stirred, and 12.3 mg of oxidized glutathione (Nacalai Tesque; No. 170-10) was added to adjust the pH to 8. Distilled water was added to make the final volume 20 ml, the mixture was allowed to stand at room temperature overnight, and fractionated again with a reverse-phase HPLC column to obtain a peak that induces the migration activity of monocyte Z macrophages. Finally, 6 to 9 mg of recombinant HT-LCF derived from Escherichia coli (hereinafter referred to as “E-rHT-LCF”) could be purified. Were tested for monocyte macrophage migration activity, the finally obtained E- r HT-L CF from 10- ¹ 'Micromax at a concentration of 10- ⁷ M was found to have dose-dependent active (FIG. 11). In FIG. 11, the horizontal axis represents the concentration of E-rHT-LCF, and the vertical axis represents the number of migrated cells.

The specific activity of E-rHT-LCF obtained here is almost the same as that of rHT-LCF expressed in Cos1 cells, and large-scale expression and purification of E-rHT-LCF It can be said that the method for obtaining the active form has been established. [Example 21] Wound healing effect of E-rHT-LCF

 In order to examine the effect of E—rHT—LCF on wound treatment, a test was conducted using a wound treatment method. The experiment followed the method of Schulte and Domenjoz et al. (Med. Phamacol. Expt. 16, 453-458, 1967).

 Ten male SD rats weighing 110 to 130 g were used per group. After removing the back coat with a hair clipper, a suspension of 4 volumes of barium sulfide and 1 volume of tragacanth powder (Wako Pure Chemicals; 206-02242) was applied, and after 3 minutes, it was washed and depilated. Then, under ether anesthesia, a left or right 1.5 cm skin incision was made along the midline. After suturing the center of the incision at one point, streptomycin was intramuscularly injected to prevent suppuration. For the first time, 100 ^ 1 of saline solution of E-rHT-LCF is injected into the incision, and thereafter, a cream containing E-rHT-LCF is applied once a day for 5 days using a microspatula at the incision site. 0.1 g was applied. In the control group, only physiological saline was injected at first, and then only cream base was injected or applied. The formulation of the cream base is as follows.

In 100 g of cream base

 Cetanol 8.0 g

 Stearyl alcohol 2.0 g

 Liquid paraffin 7.0 g

 MYS- 40 (Nikko Chemicals) 3.0 g

 Propyl parapen 0.05g

 80.0 g of purified water

 E-rHT-LCF was prepared in physiological saline or the above cream base at a concentration of 1 l or 10 // g /. The thread was removed on the fourth day, and after bruising on the head on the sixth day, the animal was killed by exsanguination and the skin was peeled off. Using a FD pickup (TB-612 T; Nihon Kohden), prepare a 30 x 10 bark deer slice so that the wound site is in the center, and pull the both ends to pull the wound healing part. The resulting values were tested using a Dunnett multiple comparison test at a 5% risk level.

As a result, at the concentration of 1 g / g with respect to the tensile strength of the control group, the treatment promotion tendency was observed but was not significant. However, at a concentration of 10 g / g, a significant treatment promoting effect was observed (see Table 1). table 1

 In Table 1, “*” means that the test group has a significant (ρ <0.05) difference compared to the control group.

[Example 22] Effect of burn treatment of E-r CF- L CF

 The following experiment was conducted to examine the therapeutic effect of E—rHT—LCF on burns. The method mainly followed the experimental burn treatment experiment of Kamoshida et al. (Clinical Medicine, Vol. 4, No. 7, 1245-1251, 1988).

 Ten male SD rats weighing 190-210 g were used per group. Hair was removed in the same manner as in Example 21. Thereafter, under anesthesia with sodium pentobarbital (40 mg / kg; intraperitoneal administration), burns were made in two places along the midline using a stainless steel cylinder (01 OmmX 5 mm) heated with a dry heater. Assuming severe burns, the stainless steel cylinder was heated to 300 ° C and compressed by the cylinder's own weight (6 g) for 10 seconds. The cream containing E-rHT-LCF was applied to the created burn site and the periphery once a day for 15 days using a micro spatula. The control group received only the cream base. The composition of the cream base is the same as that described in Example 21. '' To determine the effect, the degree of injury at the burn site was observed over time, and a score of 0 to 5 was applied to each area according to the following criteria for the degree of burn injury, and the average of the two areas of the burn area was calculated. . Further, the number of days required for shedding of the necrotic tissue or the epithelial sclerotic tissue was calculated. The obtained values were tested using a Dunnet's multiple comparison test at a 5% risk level.

Burn injury criteria

 0: Complete healing (only burn scars)

1: The ulcer at the burn site after the epithelial sclerotic tissue has fallen 2: A part of epithelial sclerotic tissue has fallen off

 3: Lost necrotic tissue at burn site

 4: Part of the necrotic tissue at the burn site

 5: Tissue necrosis at burn site

As a result, a significant therapeutic effect was observed on the degree of burn injury at a concentration of 1 / ig / g from day 6 of application and at 10 zg / g from day 5 of application (see Table 2).

S¾ in the eclectic 2 cream

 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

5.00 Sat 5.00 Sat 5.00 Sat 4.75 Sat 4.50 Sat 4.05 ± 3.65 Sat 3.50 Sat 3.30 Sat 90 Sat 2, 35 soil 1.80Tasu 1.60 Sat-click ¹ bet transliteration

 0.00 0.00 0.00 0.11 0.20 0.17 0.20 0.21 0.21 0.27 0.35 0.25 0.23

E-rHT 5.00 Sat 5.00 Sat 5.00 Sat 4.55 Sat 4.00 ± 3.60 + 3.45 ± 3.25 Sat 2.85 ± 2.8 Sat 60 Sat 2 25 Sat 2 05 Sat 1.59 ± 1.45

 1

 -LCP 0.00 0.00 0.00 0.17 0.ZZ 0. 00 遍 0 0.08 0.16 0.23 0.22 0.26 0.Z

E-rHT 5.00 Sat 5.00 + 5.00 Sat 4.45 ± 3.80 Sat 3.35 Sat 3.30 Sat 3.25 ± 90 Sat 280 80 2.40 ± 2.35 Sat 2 10 Sat 1.85 Sat 1.85 1.65

 Ten

-LCF 0.00 0.00 0.00 0.17 0. ※ 0.13 ・ 0.08¾ $ 0. ※ 0.15 0.16 0.20 0.22 0.21 0.2

In addition, the number of days of necrotic tissue shedding was significantly reduced at both concentrations. However, there was no difference in the number of days of epithelial sclerotic tissue shedding compared to the control group (see Table 3). Table 3

The meaning of “Factory *” described in Tables 2 and 3 is the same as in Table 1. Industrial applicability

 According to the present invention, it is possible to provide a novel monocyte or macrophage migration factor that has never been known before.

 The migration factor of the present invention can be used as a control substance when measuring the concentration of the migration factor contained in serum, plasma, urine and various body fluids. That is, the chemotactic factor of the present invention can be used as a standard substance when measuring by the enzyme immunoassay (ELISA or EIA) or the immunoassay using a radioisotope (RIA). In addition, it can be used as an antigen at the time of producing a specific antibody which is indispensable for producing the above-mentioned assay method. Therefore, it becomes possible to analyze the mechanism in which the migration factor is involved in the infection defense system.

 Further, by administering the chemotactic factor of the present invention to various infectious diseases, various immunodeficiencies, and various cancer diseases, a preventive effect and a therapeutic effect can be obtained.

This factor is also responsible for the healing of various wounds (cuts, surgical wounds, floor shifts, burns, ulcers, etc.). It can be used as a therapeutic agent. In addition, a prophylactic effect and a therapeutic effect can be obtained when used in combination with other therapeutic agents and treatments (diabetes, various ulcers, cancer chemotherapy, various operations, etc.) with the same effect expected.

Sequence listing

 SEQ ID NO: 1

Array length: 94

Sequence type: amino acid

 Topology: linear

Sequence type: protein

Origin: Homo Sapiens (Homo S apiens)

Cell line: HT—1 3 7 6

Array:

 Cys Lys Lys Val He Leu Asn Val Pro Ser Lys Leu Glu Ala Asp Lys

 1 5 10 15 lie He Gly Arg Val Asn Leu Glu Glu Glu Cys Phe Arg Ser Ala Asp Leu

 20 25 30 lie Arg Ser Ser Asp Pro Asp Phe Arg Val Leu Asn Asp Gly Ser Val

 35 40 45

 Tyr Thr Ala Arg Ala Val Ala Leu Ser Asp Lys Lys Arg Ser Phe Thr

 50 55 60

 lie Trp Leu Ser Asp Lys Arg Lys Gin Thr Gin Lys Glu Val Thr Val 65 70 75 80

Leu Leu Glu His Gin Lys Lys Val Ser Lys Thr Arg His Thr 94

 85 90 SEQ ID NO: 2

Array length: 1 0 5

Sequence type: amino acid

 Topology: linear

Sequence type: protein

Origin: Homo Sapiens (Homo S apiens)

Cell line: HT—1 3 7 6 Array:

 Ala Cys Lys Lys Val lie Leu Asn Val Pro Ser Lys Leu Glu Ala Asp

 1 5 10 15

 Lys lie lie Gly Arg Val Asn Leu Glu Glu Cys Phe Arg Ser Ala Asp

 20 25 30

Leu lie Arg Ser Ser Asp Pro Asp Phe Arg Val Leu Asn Asp Gly Ser

 35 40 45

 Val Tyr Thr Ala Arg Ala Val Ala Leu Ser Asp Lys Lys Arg Ser Phe

 50 55 60

 Thr lie Trp Leu Ser Asp Lys Arg Lys Gin Thr Gin Lys Glu Val Thr 65 70 75 80

Val Leu Leu Glu His Gin Lys Lys Val Ser Lys Thr Arg His Thr Arg

 85 90 95

 Glu Thr Val Leu Arg Arg Ala Lys Arg

 100 105 SEQ ID NO: 3

Array length: 3 5 5 1

Sequence type: nucleic acid

Number of chains: double strand

 Topology: linear

Sequence type: cDNA t o mRN A

Origin: Homo sapiens (Homo S a piens)

Cell line: HT—1 3 7 6

Array:

 GGCAGGCAGG TCTCGTCTCG GCACCCTCCC GGCGCCCGCG TTCTCCTGGC CCTGCCCGGC 60 ATCCCG ATG GCC GCC GCT GGG CCC CGG CGC TCC GTG CGC GGA GCC GTC 108

 Met Ala Ala Ala Gly Pro Arg Arg Ser Val Arg Gly Ala Val

-30 -25-20 -9 2-

J3S 311 JAl 3qd Ι¾Λ Jm J usv BIV BIV dsv J8 $ ni {) \ \

88S 10V VIV VOX OVl Oil 010 13V XVI OVV OVO VOO VOO IVO 101 VVO 110

 SSI 02Ϊ 9Π

 uio uj naq aq Π9ΐ ojd aqa ojd λιο ngq jas usy ni uiO J3S

0 S VVO VVO 113 III 0X1 VOO 3X1 XOO 300 Oil 031 IVV 9V0 VVO OX 131

 901 901 OOT sAo 0Jd 3Π oJd ¾iv 丄 3JV 3jy sXq B | V 3JV 3JV naq Ι¾Λ 丄 26 301 1 3 11V 130 VOO OOX VOV 09V OVV 330 100 90V 0X3 XIO 10V VVO

 96 06 98

 3JV Jqi S IH 3jy Jill sAq jas m sAq sAq uio s! Nnig naq naq Ι¾Λ m VOV 13V OVO VOV VOV OVV 001 VIO 9VV OVV OVO XVO VVO VIO 013 0X0 08 91 01 99 jqi ΪΒΛnio sAq uio sAq dsv J3S naq dJX d \ \ J¾

966 IOV 110 OVO VVV OVO VOV OVO VVV OOV VVV OVO 101 110 901 VIV ODV

 09 sg os

 3qd J9S 3JV sXq s q dsv nai ¾ΐν ΐ¾Λ ¾IV 3JV BI Jqi J eight 8 ^ 8 ILL VOX VOV VVV OVV IVO 131 013 030 110 100 OOV 000 VOV OVl 010

 St 'Of- 9S

 J9S Ai9 ds usy nai 3jy aqj ds OJJ dsy J3S J8S 3JV 9Π naq 008 VOX 009 XVO IVV VXO 110 VOV Oil iVO 130 IVO 10V V31 003 OIV 313

 08 92 OZ

 dsv BIV J3S 3JV 9Md SAQ nio "10 ^ usv Ϊ¾Λ 3JV ^ IO 911 ai l sAq 29Z DVO VDO XDI OOV Oil 001 OVO VVO Oil IVV 119 VOV 000 11V VIV VVV

 ST 01 9 ΐ dsv ¾IV nio Π3Ί sAq jas OJJ Ι¾Λ USV naq 3Π Ι¾Λ sAq sAq s ¾iv 0Z OVO VOO OVO VIO VVV 131 133 VIO IV 1X3 VXV 019 OVV VVV 301 030

 S- 01- 91- nio Ai9 Biv 3JV J3S 3Md 311 \ U nai Jqi noq naq naq S I H naq sAo 9SI VV9 109 130 103 XOV Oil OIV 019 313 30V 010 010 0X3 1V3 313 001

L6 £ Q0 / P6d £ llDd 6Q91ZIV6 OAV One 9 ε-

8901 VOO 1X1 111 OVO 300 IVO OIV OVO WO VIO VVV OXV VIV 9X1 VOX 3V1

 S8Z 082 912

 sXl dsy ΐ¾Λ 1¾Λ ni9 3JV dsy naq J S I R J9S Ι¾Λ J¾ Jqi 311 \ U

OZOT OVV OVO VI9 119 9V0 VOV 0V9 Oil IVl IVO 131 0X0 VOV OOV OIV VIO

 OLZ 99Z 09Z

AI9 JMl J3S OJd S I H \ U J3S 3qd ^ IO OJd J3S 3JV OJd jqi U I Q

216 390 VOV OOV 003 IVO 010 101 1X1 310 000 103 VOX OOV VOO VOV OVO

 9S2 092 S ^ Z

 uio naq d \ \ jas J ^ i sAq naq 3JV jqi S IH aw J¾ dsy OJd nio dsy

^ 6 OVO Oil 丄 丄 V OOV OVl VVV 010 300 OOV IVO OIV VOV OVO 900 VV9 丄 V3

 982 082 923

3JV dsv JMi BIV sAo m m Ho Ϊ¾Λ jqi jm HO ojd 3JV jas ^

912 VOV OVO V3V 300 101 110 010 000 010 VOV 13V 100 103 VOV 10V 丄 3V

 ZZ 912 012 njo ngq ΪΒΛ nio 3iw usv J 9Π ¾IV nio jqi aqj \ OJd s! H usy 828 WO 9X1 110 WO 丄 丄 丄 IVV 1VI IIV VOO VV9 VOV Oil 119 100 OVO OVV

 90Z OOZ 96T

 dsy usv "19 dsv ni Ι¾Λ 3JV 911 OJJ naq OJ ^ na OJd ng dsy ¾IV

08i OVO IVV WO XVO OVO VIO OOV OXV 330 VXO VOO 013 033 013 IVO VOO

 061 98ΐ 081

J8S J ^ 19 dsy BIV Jill J3S ¾IV J ¾1V 911 ngq dsy aqd ϊ¾Λ dsv

Z £ L VOX IVl VOO XVO VOO 13V VOX 009 IVl XOO IIV 911 IVO 111 110 IVO

 S 丄 ΐ 0 丄 I S9T

 JAinio "10 8JV dsv Ϊ¾Λ OJd 3jy J¾ s d \ d nai usy AlO JMI dsv 89 IVl WO WO 100 XVO 010 130 090 I3V 391 111 VXO XVV VOO 13V 3V0

09ΐ 99Ϊ 09Ϊ S

8JV nig an JAi aqd naq usy naq OJd nig sAq dsy Ι¾Λ ^ IO 3JV 3

989 VOV WO VIV XVX III Oil IVV VII 133 WO VVV IVO 110 V09 103 VOO

 on get οετ

L6i00I 6d £ llOd 608TZ / ^ 6 OM Tyr Ser Leu lie Met Lys Val Gin Asp Met Asp Gly Gin Phe Phe Gly

290 295 300

 TTG ATA GGC ACA TCA ACT TGT ATC ATA ACA GTA ACA GAT TCA AAT GAT 1116 Leu lie Gly Thr Ser Thr Cys lie lie Thr Val Thr Asp Ser Asn Asp 305 310 315 320

AAT GCA CCC ACT TTC AGA CAA AAT GCT TAT GAA GCA TTT GTA GAG GAA 1164 Asn Ala Pro Thr Phe Arg Gin Asn Ala Tyr Glu Ala Phe Val Glu Glu

 325 330 335

 AAT GCA TTC AAT GTG GAA ATC TTA CGA ATA CCT ATA GAA GAT AAG GAT 1212 Asn Ala Phe Asn Val Glu lie Leu Arg lie Pro lie Glu Asp Lys Asp

 340 345 350

TTA ATT AAC ACT GCC AAT TGG AGA GTC AAT TTT ACC ATT TTA AAG GGA 1260 Leu lie Asn Thr Ala Asn Trp Arg Val Asn Phe Thr lie Leu Lys Gly

 355 360 365

 AAT GAA AAT GGA CAT TTC AAA ATC AGC ACA GAC AAA GAA ACT AAT GAA 1308 Asn Glu Asn Gly His Phe Lys He Ser Thr Asp Lys Glu Thr Asn Glu

 370 375 380

 GGT GTT CTT TCT GTT GTA AAG CCA CTG AAT TAT GAA GAA AAC CGT CAA 1356 Gly Val Leu Ser Val Val Lys Pro Leu Asn Tyr Glu Glu Asn Arg Gin 385 390 395 395 400

GTG AAC CTG GAA ATT GGA GTA AAC AAT GAA GCG CCA TTT GCT AGA GAT 1404 Val Asn Leu Glu lie Gly Val Asn Asn Glu Ala Pro Phe Ala Arg Asp

 405 410 415

 ATT CCC AGA GTG ACA GCC TTG AAC AGA GCC TTG GTT ACA GTT CAT GTG 1452 He Pro Arg Val Thr Ala Leu Asn Arg Ala Leu Val Thr Val His Val

 420 425 430

AGG GAT CTG GAT GAG GGG CCT GAA TGC ACT CCT GCA GCC CAA TAT GTG 1500 Arg Asp Leu Asp Glu Gly Pro Glu Cys Thr Pro Ala Ala Gin Tyr Val

435 440 445 CGG ATT AAA GAA AAC TTA GCA GTG GGG TCA AAG ATC AAC GGC TAT AAG 1548

Arg lie Lys Glu Asn Leu Ala Val Gly Ser Lys He Asn Gly Tyr Lys

 450 455 460

 GCA TAT GAC CCC GAA AAT AGA AAT GGC AAT GGT TTA AGG TAC AAA AAA 1596

Ala Tyr Asp Pro Glu Asn Arg Asn Gly Asn Gly Leu Arg Tyr Lys Lys

465 470 475 480

TTG CAT GAT CCT AAA GGT TGG ATC ACC ATT GAT GAA ATT TCA GGG TCA 1644

Leu His Asp Pro Lys Gly Trp lie Thr lie Asp Glu lie Ser Gly Ser

 485 490 495

 ATC ATA ACT TCC AAA ATC CTG GAT AGG GAG GTT GAA ACT CCC AAA AAT 1692 l ie l ie Thr Ser Lys l ie Leu Asp Arg Glu Val Glu Thr Pro Lys Asn

 500 505 510

GAG TTG TAT AAT ATT ACA GTC CTG GCA ATA GAC AAA GAT GAT AGA TCA 1740

Glu Leu Tyr Asn l ie Thr Val Leu Ala lie Asp Lys Asp Asp Arg Ser

 515 520 525

 TGT ACT GGA ACA CTT GCT GTG AAC ATT GAA GAT GTA AAT GAT AAT CCA 1788

Cys Thr Gly Thr Leu Ala Val Asn lie Glu Asp Val Asn Asp Asn Pro

 530 535 540

 CCA GAA ATA CTT CAA GAA TAT GTA GTC ATT TGC AAA CCA AAA ATG GGG 1836

Pro Glu lie Leu Gin Glu Tyr Val Val lie Cys Lys Pro Lys Met Gly

545 550 555 560

TAT ACC GAC ATT TTA GCT GTT GAT CCT GAT GAA CCT GTC CAT GGA GCT 1884

Tyr Thr Asp lie Leu Ala Val Asp Pro Asp Glu Pro Val His Gly Ala

 565 570 575

 CCA TTT TAT TTC AGT TTG CCC AAT ACT TCT CCA GAA ATC AGT AGA CTG 1932

Pro Phe Tyr Phe Ser Leu Pro Asn Thr Ser Pro Glu lie Ser Arg Leu

 580 585 590

TGG AGC CTC ACC AAA GTT AAT GAT ACA GCT GCC CGT CTT TCA TAT CAG 1980

Trp Ser Leu Thr Lys Val Asn Asp Thr Ala Ala Arg Leu Ser Tyr Gin -6 8-

09 1V3 IVO 009100000000301001 WO Oil oov ovo ovv voo voo vvv

 OS 丄 Sf-i, L

Ϊ9Ν ¾8W nio 3Π Jqi niO UTO ^ IO ^ IO usv sAq jaw AiO J8S ^ IO? 9W

S S OIV OXV WO 丄 丄 V OOV WO OVO 000 VOO IVV VVV 91V VOO V01 VOO OiV

 ss οει 9zi

 jqi λιο sAQ 3Md ^ 19 uio J8S J9S usv usv J¾ J¾ UIQ J¾ jaw aqd

^ 982 IOV 190191111199 WO OOV 101 OVV OVV OOV 10V WO OOV OIV 111

022.9 OIL 901

AID usv BIV J9S sXQ Ι¾Λ 3JV dsy dsy λίθ ojj ¾ιν ηϊθ J¾ usy J9S 9Ϊ8Ζ VOO IVV 330131001910 VOV IVO 0V9 V99133 VOO WO VOV OVV VOX

 002, 969 069

311 3Π Π3Ί usy uig uig ¾iv ^nd l dsv "10 ^0J d aqd 3JV sAq Aig sA

29ZZ VIV XIV VIX OVV WO OVO VOO VII XVO WO 100 ILL 130 VVV 009 VVV

 289 089 9A9

 J¾ BIV ^ ΪΟ a d Ϊ Λ ^ ΐθ SAQ Ι¾Λ nai Jqi naq naq Ι¾Λ J9S aiy naq

OZZS XOV VOO 190 丄 丄 丄 110 VOO 101 VIO VII 10V VIO Oil VIO 101 Hi 313

019 S99 099 ngq BJV 311 H na naq d \\ ¾iv ^nd l ^d \\ ¾IV dJ 丄 sAq Aio naq an

22,120X0 VOO VXV XOO 010 VXX VIV VOO 110 OIV V09 OOX VVV V90110 VIV

 999 0S9 9 ^ 9

\ n ^ ΪΟ JM1 J3S 3jy J3S Jqi ¾IV 3JV ^s uiO OJd S! H J¾ SAQ

^ TZ VIO V09 VOV 13V 90V VOX 10V 900 100 101 OVO XOV VOO IVO 10V

0 ^ 9 S89 0S9 9Z9 nio sAo naq usy Ι¾Λ 3JV ° 91 nai sXq 丄 iv ¾iv uiO ^ 10 ¾IV 3 ^J V

9 OZ VV9101010 IVV XXO VOV Oil VII VVV VOV VOO 130 WO 300 300 OOV

 029 SI9 019 dsv sAq Ι¾Λ JM1 ail OJd 911 JMl J nio UIQ aqd AJQ Ήΐν usy sAq

OVO VVV VIO 10V 丄 丄 V 133 丄 丄 V 03V LU WO VV3 ILL VOO 130 IVV VVV, S09 009 96S £ 00 / Wdf / 丄: M 608 / M OAV 0

^ 862 VllVVOOXOV OOVOOIOVVV OlOXVVViOO 13VV0V0V3V VVVIIOIVVV VVOXOVVVVV

^ 262 30IUI0IV1100110I10V VOOVVVlXll IVIVXUVVO 3V010V1301 VllVVOllil 982 VV3131111111V01VOIV1 V191V19IV19 V0VVOXllVVOl IOVVVlOliV IVVlVVVVVO

 G98 3JV sAq f02Z 01I100V091 OXIVOVOVOX OlllOlOOVX IVVOVIOOIO V0V0191VV1 VOV OVV

 098 998 098 jqi s BIV "10 ¾IV nai jqi d \\ 9qd sXq OJd nio naq usy usy naq SfLZ VOV 001 VOO WO VOO VII VOV IXV 丄 丄 丄 VVV 033 WO Oil IVV IVV Vll

 9 ^ 8 0 8 988 di \ d dsv "31 Aio dsv nio nig uio sAq nio J8S s SAQ A \ O \ 2 \ jas

OOLZ 111 OVO 110 300 IVO VV9 WO 0V3 OVV WO 19V 301 001 300 919101

 088 9Z8 028

 ΑϊΟ BIV OJd J8S Ai 3JV ^ 19 "TO J usv J ^ X-" il naq Ι¾Λ 丄 dsv 2992 100 130 VOO 101 VOO VOV VOO 3V0 IVl OVV XVI 10V 013 310 IVX IVO

 9Ϊ8 018 208

UJO J3S OJd jaw 3JV dsv nfo usv ^u \ 0 usv ^s 3JV S IH n3q sAq nyg 092 WO 301 VOO 01V 000 0V9 VV9 IVV OVO XVV 101 VOO IVO Oil VVV WO 008 961 062.98i

ΑΪ9 nai 3jy OJd uio J¾ 9Md J3S S IH "10 J3S J-" il J 3JV

999Z 100 310 103 330 WO 10V 丄 丄 丄 10V OVO 001 OVO 001 3V113V OVi VOV

 081 LL OLL sAo usv dsv Ϊ¾Λ "TO JMI S IH AJO λΙ9 3JV s J9S dsy naq jqi S IH

80S2 391 OVV OVO 010 OVO 03V OVO V90 VDO OOV 301 001 OVO 013 33V 丄 V3

 S9i 092, ggi,

S IH S IH Ai9 BIV A19 3JV SAQ J3S nio "91 Jqi uiO usv ^ 19 ^ 10 sAq 6C00 / f6dT / XD <I 608 6 O AGGTCTCTAA AGCATCTGCT CTTTTTTTTT TTTACAGATA TTTTAGTAAT AAATATGCTG 3044 GATAAATATT AGTCCAACAA TAGCTAAGTT ATGCTAATAT CACATTATTA TGTATTCACT 3104 TTAAGTGATA GTTTAAAAAA TAAACAAGAA ATATTGAGTA TCACTATGTG AAGAAAGTTT 3164 TGGAAAAGAA ACAATGAAGA CTGAATTAAA TTAAAAATGT TGCAGCTCAT AAAGAATTGG 3224 ACTCACCCCT ACTGCACTAC CAAATTCATT TGACTTTGGA GGCAAAATGT GTTGAAGTGC 3284 CCTATGAAGT AGCAATTTTC TATAGGAATA TAGTTGGAAA TAAATGTGTG TGTGTATATT 3344 ATTATTAATC AATGCAATAT TTAAATGAAA TGAGAACAAA GAGGAAAATG GTAAAAACTT 3404 GAAATGAGGC TGGGGTATAG TTTGTCCTAC AATAGAAAAA AGAGAGAGCT TCCTAGGCCT 3464 GGGCTCTTAA ATGCTGCATT ATAACTGAGT CTATGAGGAA ATAGTTCCTG TCCAATTTGT 3524

GTAATTTGTT TAAAATTGTA AATAAAT 3551 SEQ ID NO: 4

Array length: 1 6 7

Sequence type: nucleic acid

Number of chains: double strand

 Topology: linear

Sequence type: cDNA t o mRNA

Origin: Homo sapiens (Homo S a piens)

Cell line: HT—1 3 7 6

Array: GAG GCA GAC AAA ATA ATT GGC AGA GTT AAT TTG GAA GAG TGC TTC AGG 48

Glu Ala Asp Lys lie lie Gly Arg Val Asn Leu Glu Glu Cys Phe Arg

 1 5 10 15

 TCT GCA GAC CTC ATC CGG TCA AGT GAT CCT GAT TTC AGA GTT CTA AAT 96

Ser Ala Asp Leu lie Arg Ser Ser Asp Pro Asp Phe Arg Val Leu Asn

 20 25 30

GAT GGG TCA GTG TAC ACA GCC AGG GCT GTT GCG CTG TCT GAT AAG AAA 144

Asp Gly Ser Val Tyr Thr Ala Arg Ala Val Ala Leu Ser Asp Lys Lys

 35 40 45

 AGA TCA TTT ACC ATA TGG CTT TC 167

Arg Ser Phe Thr lie Trp Leu

 50 55 SEQ ID NO: 5

Array length: 2 1

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Fragment type: N-terminal fragment

Origin: Homo sapiens (H 0 mo S apien s)

Cell line: HT—1 3 7 6

Array:

 Ala Cys Lys Lys Val lie Leu Asn Val Pro Ser Lys Leu Glu Ala Asp

 1 5 10 15

 Lys lie He Gly Arg 21

 20 SEQ ID NO: 6

Array length: Ί Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Fragment type: middle fragment

Origin: Homo Sapiens (Homo S apiens)

Cell line: HT—1 3 7 6

Array:

 Ala Val Ala Leu Ser Asp Lys 7 1 5 SEQ ID NO: 7

Array length: 1 0

Sequence type: amino acid

 Topology: linear

Sequence type: ぺ butide

Fragment type: middle fragment

Origin: Homo Sapiens (Homo S apiens)

Cell line: HT—1 3 7 6

Array:

 Glu Val Xaa Val Leu Leu Glu Xaa Gin Lys 10 1 5 10 SEQ ID NO: 8

Array length: 8

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Fragment type: middle fragment

Origin: Homo S apiens Cell line: HT—1 3 7 6

Array:

 Ala Ser Phe Thr lie Trp Leu Ser 8 1 5 SEQ ID NO: 9

Array length: 20

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Fragment type: N-terminal fragment

Origin: Homo Sapiens (Homo S apiens)

Cell line: HT—1 3 7 6

Array:

 Cys Lys Lys Val lie Leu Asn Val Pro Ser Lys Leu Glu Ala Asp Lys

 1 5 10 15 lie lie Gly Arg 20

 20 SEQ ID NO: 1 0

Array length: 1 9

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Fragment type: N-terminal fragment

Origin: Homo Sapiens (Homo S apiens)

Cell line: HT—1 3 7 6

Array:

Cys Lys Lys Val He Leu Asn Val Pro Ser Lys Leu Glu Ala Asp Lys 1 10 15 lie lie Gly 19 SEQ ID NO: 1 1

Array length: 1 3

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Origin: Homo Sapiens (Homo S apiens)

Cell line: HT—1 3 7 6

Array:

 Cys Lys Lys Val lie Leu Asn Val Pro Ser Lys Leu Glu 13 1 5 10 SEQ ID NO: 1 2

Array length: 1 1

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Origin: Homo Sapiens (Homo S apiens)

Cell line: HT—1 3 7 6

Array:

 Ala Asp Lys lie lie Gly Arg Val Asn Leu Glu 11 1 5 10 SEQ ID NO: 1 3

Array length: 5 2

Sequence type: amino acid

Topology: linear Sequence type: peptide

Origin: Homo Sapiens (Homo S apiens)

Cell line: HT—1 3 7 6

Array:

 Cys Phe Arg Ser Ala Asp Leu lie Arg Ser Ser Asp Pro Asp Phe Arg

1 5 10 15

 Val Leu Asn Asp Gly Ser Val Tyr Thr Ala Arg Ala Val Ala Leu Ser

 20 25 30

 Asp Lys Lys Arg Ser Phe Thr lie Trp Leu Ser Asp Lys Arg Lys Gin

 35 40 45

 Thr Gin Lys Glu 52 50 SEQ ID NO: 1 4

Array length: 6

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Origin: Homo Sapiens (Homo S apiens)

Cell line: HT—1 3 7 6

Array:

 Val Thr Val Leu Leu Glu 6 1 5 SEQ ID NO: 15

Array length: 1 3

Sequence type: amino acid

 Topology: linear

Sequence type: peptide Origin: Homo S aiens

Cell line: HT—1 3 7 6

Array:

 His Gin Lys Lys Val Ser Lys Thr Arg His Thr Arg Glu 13 1 5 10 SEQ ID NO: 16

Array length: 1 9

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Origin: Homo sapiens (Homo S a piens)

Cell line: HT—1 3 7 6

Array:

 Thr Val Leu Arg Arg Ala Lys Arg Arg Trp Ala Pro He Pro Cys Ser

1 5 10 15

 Met Gin Glu 19 SEQ ID NO: 1 7

Array length: 1 1

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Fragment type: C-terminal fragment

Origin: Homo sapiens (Homo S a piens)

Cell line: HT—1 3 7 6

Array:

His Gin Lys Lys Val Ser Lys Thr Arg His Thr 11 1 5 10 SEQ ID NO: 1 8

Array length: 1 1

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Fragment type: middle part fragment

Origin: Homo Sapiens

Cell line: HT—1 3 7 6

Array:

 Ala Asp Lys lie lie Gly Arg Val Asn Leu Glu 11 1 5 10 SEQ ID NO: 1 9

Array length: 6

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Fragment type: middle fragment

Origin: Homo Sapiens

Cell line: HT—1 3 7 6

Array:

 Val Thr Val Leu Leu Glu 6 1 5 SEQ ID NO: 20

Array length: 1 3

Sequence type: amino acid

Topology: linear Sequence type: peptide

Fragment type: N-terminal fragment

Origin: Homo Sapiens (Homo Sapiens)

Cell line: HT—1 3 7 6

Array:

 Cys Lys Lys Val lie Leu Asn Val Pro Ser Lys Leu Glu 13 1 5 10 SEQ ID NO: 2 1

Array length: 1 3

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Fragment type: middle part fragment

Origin: Homo Sapiens

Cell line: HT—1 3 7 6

Array:

 Glu Cys Phe Arg Ser Ala Asp Leulie Arg Ser Ser Asp 13 1 5 10 SEQ ID NO: 2 2

Array length: 1 1

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Fragment type: C-terminal fragment

Origin: Homo Sapiens

Cell line: HT—1 3 7 6

Array: His Gin Lys Lys Val Ser Lys Thr Arg His Thr 11 1 5 10 SEQ ID NO: 2 3

Array length: 3 2 1

Sequence type: cDNA t o mRNA

 Topology: linear

Sequence type: nucleic acid

Origin: Homo sapiens (Homo S a piens)

Cell line: HT—1 3 7 6

Array:

 ATG GCC TGC AAA AAG GTG ATA CTT AAT GTA CCT TCT AAA CTA GAG GCA 48 Met Ala Cys Lys Lys Val lie Leu Asn Val Pro Ser Lys Leu Glu Ala

 1 5 10 15

 GAC AAA ATA ATT GGC AGA GTT AAT TTG GAA GAG TGC TTC AGG TCT GCA 96 Asp Lys lie lie Gly Arg Val Asn Leu Glu Glu Cys Phe Arg Ser Ala

 20 25 30

GAC CTC ATC CGG TCA ACT GAT CCT GAT TTC AGA GTT CTA AAT GAT GGG 144 Asp Leulie Arg Ser Ser Asp Pro Asp Phe Arg Val Leu Asn Asp Gly

 35 40 45

 TCA GTG TAC ACA GCC AGG GCT GTT GCG CTG TCT GAT AAG AAA AGA TCA 192 Ser Val Tyr Thr Ala Arg Ala Val Ala Leu Ser Asp Lys Lys Arg Ser

 50 55 60

 TTT ACC ATA TGG CTT TCT GAC AAA AGG AAA CAG ACA CAG AAA GAG GTT 240 Phe Thrlie Trp Leu Ser Asp Lys Arg Lys Gin Thr Gin Lys Glu Val 65 70 75 80

ACT GTG CTG CTA GAA CAT CAG AAG AAG GTA TCG AAG ACA AGA CAC ACT 288 Thr Val Leu Leu Glu His Gin Lys Lys Val Ser Lys Thr Arg His Thr

85 90 95 I 9

90Ϊ 001

3JV Al ¾iv ^3JV 3JV ^η3 Ί Ι¾Λ J 41 ^ ID 3 -IV 128 VVl 03V OVV 330 103 90V 310 11013V VVO VOV

L6tQ / P6di / lDd 60SIZIV6 OAV

Claims

The scope of the claims

1. A protein substantially comprising the amino acid sequence represented by SEQ ID NO: 1 and having monocyte or macrophage migration activity.

 2. A protein substantially comprising the amino acid sequence represented by SEQ ID NO: 2 and having monocyte or macrophage migration activity.

 3. An isfe containing DNA that substantially encodes the amino acid sequence represented by SEQ ID NO: 1.

 4. isfe containing DNA which substantially encodes the amino acid sequence represented by SEQ ID NO: 2

 5. Is s: containing DNA which substantially encodes the amino acid sequence represented by SEQ ID NO: 3.

 6. A recombinant vector for expression containing the gene according to any one of claims 3 to 5.

7. A transformant transformed by the recombinant vector for expression according to claim 6.

 8. The method for producing a protein according to claim 1, wherein the transformant according to claim 7 is cultured, and the protein according to claim 1 is separated from the obtained culture.

 9. The method for producing a protein according to claim 2, wherein the transformant according to claim 7 is cultured, and the protein according to claim 2 is separated from the obtained culture.

 10. An immunostimulant comprising the protein according to claim 1 as an active ingredient.

 11. An immunostimulant comprising the protein according to claim 2 as an active ingredient.

 12. A wound treatment agent comprising the protein according to claim 2 as an active ingredient.

 13. A pharmaceutical composition comprising a protein having a monocyte or macula phage migration activity as an active ingredient and a pharmaceutically acceptable excipient.

 14. The pharmaceutical composition according to claim 13, which is used for treating a wound.

 15. The pharmaceutical composition according to claim 13, which is used for immunostimulation.

 16. A method for treating a wound, comprising administering to a human an effective amount of a protein having monocyte or macrophage migration activity.

17. An immunostimulatory method comprising administering to a human an effective amount of a protein having monocyte or macrophage migration activity.

18. Use of an effective amount of a protein having monocyte or macrophage migration activity in a human for treatment of a wound.

 19. Use of an effective amount of a protein having monocyte or macrophage migration activity in humans for immunostimulation.