WO1994021809A1 - Facteur de migration des monocytes ou des macrophages - Google Patents

Facteur de migration des monocytes ou des macrophages Download PDF

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Publication number
WO1994021809A1
WO1994021809A1 PCT/JP1994/000397 JP9400397W WO9421809A1 WO 1994021809 A1 WO1994021809 A1 WO 1994021809A1 JP 9400397 W JP9400397 W JP 9400397W WO 9421809 A1 WO9421809 A1 WO 9421809A1
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protein
amino acid
seq
monocyte
cells
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PCT/JP1994/000397
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English (en)
Japanese (ja)
Inventor
Kazuo Kawamura
Kazuyoshi Watanabe
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Institute Of Cytosignal Research, Inc.
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Priority to JP52086594A priority Critical patent/JP3488459B2/ja
Priority to AU62201/94A priority patent/AU6220194A/en
Publication of WO1994021809A1 publication Critical patent/WO1994021809A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • 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.
  • phagocytes such as monocytes or macrophages or neutrophils at the site of infection
  • monocytes or macrophages break down bacterial constituents
  • B lymphocytes 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.
  • MCP-1 Macrophage Chemoattractant Protein-1
  • 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.
  • 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.
  • 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).
  • PDGF Platelet Derived Growth Factor
  • IGF Insulin-like growth factor
  • 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. .
  • LPS lipopolysaccharide
  • 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.
  • the present invention includes the following inventions.
  • 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.
  • 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.
  • 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.
  • 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.
  • culture means any of a culture solution, cultured cells / cells or a disrupted product thereof, and a culture supernatant.
  • 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.
  • 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.
  • preparation under stimulation with LPS, phorbol ester or the like is also performed.
  • the chemotactic factor can be obtained by a method usually used for protein separation and purification.
  • 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.
  • 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.
  • 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) ⁇
  • 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.
  • 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.
  • 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)]. .
  • the desired clone When the desired clone is obtained, its activity can be confirmed by reconnecting the cDNA to the expression vector and expressing it.
  • animal cells, silkworm cells, yeast, Escherichia coli and the like are mainly used as host cells.
  • the vector used depends on the host.
  • prokaryotic eg, Escherichia coli
  • eukaryotic eg, yeast, insect, or mammalian cells
  • mammalian cells include COS cells, Chinese Hamster Ovary cells, C-127 cells, BHK (Baby Hamster Kidney) cells, and the like.
  • yeast include baker's yeast (Saccharomyces cerevisiae) and a methanol-assimilating yeast (Pichia pastoris).
  • 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).
  • PKC30 Shiatake H. and M. Rosenberg, Nature, 292, 128-132, 1981
  • pTrc99A Mann E. et al., Gene, 69, 301-315, 1988.
  • p SV2-neo Southern and Berg; J. Mol. Appl. Genet., 1,327-341, 1982
  • p CAGGS Newa et al .; Gene, 108, 193-200, 1991
  • p cDL-SRa296 Takabe et al .; Mol. Cell. Biol., 8, 466-472, 1988
  • yeast pG-1 (Schena.
  • silkworm cells include a transfer vector pAc373 (Luckow et al .; Bio / Technology, 6, 47-55, 1988) for producing a recombinant virus. No.
  • 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.
  • vectors derived from SV40, adenovirus, dys-papilloma virus and the like can be used as mammalian cell vectors.
  • a vector for Escherichia coli those derived from ColEl, R factor, F factor and the like can be used.
  • yeast vector 2 ⁇ mDNA, ARS1-derived DNA, and the like can be used.
  • a vector for mammalian cells as a gene expression promoter, those derived from retrovirus, poliovirus, adenovirus, SV40 and the like can be used.
  • a vector derived from Pacteriophage, a trp, lpp, lac, tac promoter and the like can be used.
  • 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.
  • vectors for mammalian cells include neomycin (neo) resistance gene, thymidine kinase (TK) gene, dihydrofolate reductase (DHFR) gene, and Escherichia coli xanthinguanine phosphoribosyltransferase (Ecogpt).
  • neomycin resistance gene thymidine kinase (TK) gene
  • DHFR dihydrofolate reductase
  • Escherichia coli xanthinguanine phosphoribosyltransferase Escherichia coli xanthinguanine phosphoribosyltransferase
  • a messenger can be used.
  • a vector for Escherichia coli a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene and the like can be used.
  • yeast vector Leu2, Trpl, Ura3 genes and the like can be used.
  • the chemotactic factor can be produced by separating and purifying from the culture containing the chemotactic factor thus expressed.
  • 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.
  • the subject to which the factor is administered is not particularly limited.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition may contain either additives or preservatives.
  • 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.
  • additives such as a stabilizer, a buffer, a preservative, and a tonicity agent
  • 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.
  • 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.
  • Figure 1 shows the results of purification of HT-LCF using a C18 reverse-phase HP LC column.
  • 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
  • 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
  • the suspended mononuclear cell layer was collected, and RPMI 1640 medium (LIFE TECHNOLOGIES,
  • 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.
  • DMEM medium Dulbecco's modified Eagle minimum essential medium
  • Gibco a medium containing 5% fetal calf serum (manufactured by Gibco) in this medium was used.
  • C 0 2 incubator one was carried out at a temperature condition of 5% C0 2 concentration and 37 ° C.
  • HT-LCF 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.
  • 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.
  • Example 3 S.DS polyacrylamide gel electrophoresis of HT-LCF purified from culture solution of HT-1 376 cells
  • Example 2 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).
  • 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.
  • protein bands were observed at the molecular weight of 12,000 to 15,000 for all four peaks.
  • Amino terminus amino acid sequence of SEQ ID NO: 5
  • Amino terminus Amino acid sequence of SEQ ID NO: 9
  • Fr. refers to the fraction (fraction) eluted by reverse phase chromatography.
  • 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 4 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.
  • 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).
  • RNA was synthesized using 1 g of this RNA.
  • dNTP four kinds of deoxynucleoside triphosphates composed of dATP, dCTP, dGTP, and dTTP; manufactured by Takara Shuzo Co., Ltd.
  • 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)
  • 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.
  • 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).
  • 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.
  • T4 polynucleotide kinase T4 polynucleotide kinase
  • 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).
  • 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.
  • 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.
  • RNA was extracted from about 5 ⁇ 10 8 cells by the AGP C method. After dissolving 500 ⁇ g 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; I ⁇ arm (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.
  • 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.
  • 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.
  • Phage DNA was prepared by the method of Cloning, 2nd. Edition, 2.60-2.80, 1989.
  • 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.
  • 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.
  • Example 4 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. ,
  • SR 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").
  • SR a Not I linker
  • GCGGCCGCTGCA3 ' synthesized using an automatic DNA synthesizer manufactured by ABI
  • 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.
  • 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 ⁇ __” ⁇ .
  • 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 ⁇ g.
  • 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).
  • 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.
  • Peptide # 1 Amino acid sequence of SEQ ID NO: 11
  • Peptide # 2 Amino acid sequence of SEQ ID NO: 12
  • 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
  • FIG. 5 shows the elution pattern with 0-40% acetate nitrile in reverse phase HPLC.
  • 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)
  • 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.
  • no peptide of peptide # 7 or later located on the C-terminal side of peptide # 6 could be confirmed.
  • 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.
  • 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 ⁇ peptide were 10, 780.7 (FIG.
  • the migration activity of monocytes or Macguchi phages was measured for purified rHT-LCF.
  • a wide dose range (0.01 to 100 nM) caused monocyte or macrophage migration in a dose-dependent manner (FIG. 8).
  • 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 ⁇ l of the cell suspension was placed in the upper chamber of a 48-well microchemotaxis chamber, and 25 ⁇ l 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).
  • 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.
  • 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.
  • a methionine codon (ATG) was introduced before alanine, the first amino acid, and an N col site (CCATGG) was added as a restriction enzyme.
  • CCATGG N col site
  • TAA stop codon
  • 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.
  • BRANSON Sonifier model 250
  • 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).
  • TFA solution (Wako Pure Chemical Industries; No. 206-10731)
  • a solution of 80% acetonitrile (Wako Pure Chemical Industries, No. 0 19-08631) containing 0.05% TFA solution as syneresis B
  • 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.
  • guanidine hydrochloride 3.1 g 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.
  • E-rHT-LCF recombinant HT-LCF derived from Escherichia coli
  • FIG. 11 the horizontal axis represents the concentration of E-rHT-LCF, and the vertical axis represents the number of migrated cells.
  • E-rHT-LCF 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
  • 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.
  • 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.
  • a FD pickup (TB-612 T; Nihon Kohden)
  • the resulting values were tested using a Dunnett multiple comparison test at a 5% risk level.
  • test group has a significant ( ⁇ ⁇ 0.05) difference compared to the control group.
  • Example 21 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.
  • 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.
  • 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.
  • ELISA or EIA enzyme immunoassay
  • RIA radioisotope
  • 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.
  • 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.
  • Lys lie lie Gly Arg Val Asn Leu Glu Glu Cys Phe Arg Ser Ala Asp
  • Sequence type nucleic acid
  • 901 901 901 OOT sAo 0Jd 3 ⁇ oJd 3 ⁇ 4iv ⁇ 3JV 3jy sXq B
  • 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
  • OOLZ 111 OVO 110 300 IVO VV9 WO 0V3 OVV WO 19V 301 001 300 919101
  • Sequence type nucleic acid
  • Glu Ala Asp Lys lie lie Gly Arg Val Asn Leu Glu Glu Cys Phe Arg
  • Fragment type N-terminal fragment
  • Fragment type middle fragment
  • Fragment type middle fragment
  • Fragment type middle fragment
  • Fragment type N-terminal fragment
  • Cys Lys Lys Val lie Leu Asn Val Pro Ser Lys Leu Glu Ala Asp Lys
  • Fragment type N-terminal fragment
  • Cys Lys Lys Val lie Leu Asn Val Pro Ser Lys Leu Glu 13 1 5 10 SEQ ID NO: 1 2
  • Ala Asp Lys lie lie Gly Arg Val Asn Leu Glu 11 1 5 10 SEQ ID NO: 1 3
  • Sequence type peptide Origin: Homo S aiens
  • Fragment type C-terminal fragment
  • Fragment type middle part fragment
  • Ala Asp Lys lie lie Gly Arg Val Asn Leu Glu 11 1 5 10 SEQ ID NO: 1 9
  • Fragment type middle fragment
  • Fragment type N-terminal fragment
  • Cys Lys Lys Val lie Leu Asn Val Pro Ser Lys Leu Glu 13 1 5 10 SEQ ID NO: 2 1
  • Fragment type middle part fragment
  • Fragment type C-terminal fragment
  • Sequence type nucleic acid

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Abstract

L'invention concerne une protéine présentant une activité de migration des monocytes ou des macrophages, un gène contenant un ADN codant pour la protéine, un vecteur d'expression de recombinaison contenant ledit gène, un transformant préparé à l'aide du vecteur de recombinaison, un procédé de production de ladite protéine par culture dudit transformant et par séparation d'une protéine présentant une activité de migration des monocytes ou des macrophaes du produit de culture, et un immunopotentialisateur ou un cicatrisant dans lesquels la protéine est utilisée en tant que principe actif.
PCT/JP1994/000397 1993-03-15 1994-03-11 Facteur de migration des monocytes ou des macrophages WO1994021809A1 (fr)

Priority Applications (2)

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JP52086594A JP3488459B2 (ja) 1993-03-15 1994-03-11 単球又はマクロファージ遊走因子
AU62201/94A AU6220194A (en) 1993-03-15 1994-03-11 Monocyte or macrophage migration factor

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JP5/54349 1993-03-15
JP5434993 1993-03-15

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7476509B2 (en) * 2002-11-14 2009-01-13 Adherex Technologies Inc. Compounds and methods for modulating functions of nonclassical cadherins

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992022664A1 (fr) * 1991-06-13 1992-12-23 Dompé S.p.A. Facteur chimiotactique
JPH05271093A (ja) * 1992-03-30 1993-10-19 Toray Ind Inc 血管内皮細胞増殖剤

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992022664A1 (fr) * 1991-06-13 1992-12-23 Dompé S.p.A. Facteur chimiotactique
JPH05271093A (ja) * 1992-03-30 1993-10-19 Toray Ind Inc 血管内皮細胞増殖剤

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7476509B2 (en) * 2002-11-14 2009-01-13 Adherex Technologies Inc. Compounds and methods for modulating functions of nonclassical cadherins

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